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nasdaq:sgen
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Seattle Genetics
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Feb 18th, 2020 12:00AM
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Apr 11th, 2019 12:00AM
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https://www.uspto.gov?id=US10561739-20200218
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Targeted pyrrolobenzodiazapine conjugates
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Provided are Conjugate comprising PBDs conjugated to a targeting agent and methods of using such PBDs.
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10561739
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1. A method for preparing a Drug linker compound, or a salt thereof, the method comprising the steps of:
a) contacting a PBD compound having the formula of:
wherein —R2 has the formula of:
wherein A is a C5-7 aryl group and X is
wherein RN is selected from the group consisting of H and C1-4 alkyl;
the asterisk indicates the point of attachment to Q2, and either:
(i) Q1 is a single bond and Q2 is a single bond or —Z—(CH2)n—, wherein Z is selected from the group consisting of a single bond, O, S and NH; and subscript n is from 1 to 3, or
(ii) Q1 is —CH═CH— and Q2 is a single bond;
and
R12 is a C5-10 aryl group, substituted by a group selected from the group consisting of —OH, —CO2H, and —C2RO, where RO is C1-4 alkyl;
R6 and R9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo, wherein R and R′ are independently selected from the group consisting of optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups, wherein C3-20 heterocyclyl is a monovalent moiety derived from removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S;
R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo,
R″ is a C3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms selected from the group consisting of O, S, and NRN2, wherein RN2 is H or C1-4 alkyl, and/or by an aromatic ring;
Y and Y′ are independently selected from the group consisting of O, S, and NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9, respectively, with a peptide coupling agent and a compound of formula G1-L1, wherein
L1 is a dipeptide of formula —NH—X1—X2—CO2H, wherein —NH— is the amino group of X1, and CO2H is the carboxylic acid functional group of X2 for peptide coupling by the peptide coupling agent to the nitrogen atom of X of the PBD compound and wherein the peptide is cleavable by the action of an enzyme for release of the PBD compound; and
G1 is a Stretcher Unit for connection to an antibody or antigen-binding fragment thereof, wherein G1 is comprised of a maleimide group for reaction with a reactive thiol functional group provided by the antibody or antigen-binding fragment for said connection, and wherein G1 further comprises the functionality —CO— connected directly to the amino terminus of X1, thereby forming an amide link with —X1—,
wherein said contacting provides the Drug Linker compound having the formula of G1-L1-D, wherein G1 is the Stretcher Unit and L1 and D correspond in structure to the dipeptide and PBD compound, respectively.
2. The method of claim 1, wherein G1 is selected from the group consisting of:
wherein the asterisk indicates the point of attachment to the amino group of X1 and subscript n is an integer ranging from 0 to 6,
wherein the asterisk indicates the point of attachment to the amino group of X1, subscript n is 0 or 1, and subscript m is an integer ranging from 0 to 30,
wherein the asterisk indicates the point of attachment to the amino group of X1 and subscript n is an integer ranging from 0 to 6, and
wherein the asterisk indicates the point of attachment to the amino group of X1, subscript n is 0 or 1, and subscript m is an integer ranging from 0 to 30.
3. The method of claim 1, wherein R7 is selected from the group consisting of H, OH and OR.
4. The method of claim 2, wherein R7 is a C1-4 alkyloxy group.
5. The method of claim 2, wherein Y and Y′ are O.
6. The method of claim 5, wherein R″ is C3-7 alkylene.
7. The method of claim 6, wherein R9 is H.
8. The conjugate according to claim 7, wherein R6 is selected from the group consisting of H and halo.
9. The conjugate according to claim 1, wherein A is phenyl, X is —NH2, and Q1 is a single bond.
10. The conjugate according to claim 9, wherein Q1 is a single bond and Q2 is a single bond.
11. The method of claim 1, wherein R12 is a C5-7 aryl group optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, C1-7 alkoxy, C5-20 aryloxy, C3-20 heterocyclyoxy, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene, wherein the C1-7 alkoxy group is optionally substituted by an amino group, and if the C3-7 heterocyclyl group is a C6 nitrogen containing heterocyclyl group, it is optionally substituted by a C1-4 alkyl group.
12. The method of claim 11, wherein the C5-7 aryl group is an optionally substituted phenyl group.
13. The method of claim 12, wherein R12 bears one to three substituent groups.
14. The method of claim 1, wherein R6′, R7′, R9′, and Y′ are the same as R6, R7, R9, and Y, respectively.
15. The method of claim 1, wherein G is:
wherein the asterisk indicates the point of attachment to L1; and subscript n is an integer ranging from 0 to 6.
16. The method of claim 15, wherein subscript n is 5.
17. The method of claim 16, wherein the dipeptide is selected from the group consisting of valine-alanine, valine-citrulline and phenylalanine-lysine.
18. The conjugate of claim 1, wherein the PBD compound has the formula:
19. The method of claim 1, wherein G1-L1-D has the formula:
wherein subscript n is an integer ranging from 1 to 11; R′ is —CH3 and R″ is CH(CH3)2.
20. The method of claim 1, wherein G1-L1-D has the formula:
21. The method of claim 1, wherein the peptide coupling agent is N-ethoxylcarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).
22. The method of claim 21, wherein G1-L1-D has the formula of:
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of copending U.S. application Ser. No. 15/951,753, filed on Apr. 12, 2018, which is a continuation of U.S. application Ser. No. 15/422,000, filed on Feb. 1, 2017, now abandoned, which is a continuation of U.S. application Ser. No. 14/995,944, filed on Jan. 14, 2016, now U.S. Pat. No. 9,592,240, which is a continuation of U.S. application Ser. No. 13/641,219, filed on Oct. 15, 2012, now U.S. Pat. No. 9,242,013, which claims the benefit of U.S. Provisional Application No. 61/324,623, filed on Apr. 15, 2010, the entire contents of which are fully incorporated herein by reference.
The present invention relates to targeted pyrrolobenzodiazepine (PBD) conjugates, in particular pyrrolobenzodiazepine dimers that are conjugated to a targeting agent via the C2 position of one of the monomers.
BACKGROUND TO THE INVENTION
Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:
They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position, which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag. New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). The ability of PBDs to form an adduct in the minor groove enables them to interfere with DNA processing, hence their use as antitumour agents.
The biological activity of these molecules can be potentiated by joining two PBD units together through their C8/C′-hydroxyl functionalities via a flexible alkylene linker (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity. One example of a PBD dimer is SG2000 (SJG-136):
(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C., et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al., Cancer Research, 64, 6693-6699 (2004)).
Due to the manner in which these highly potent compounds act in cross-linking DNA, PBD dimers have been made symmetrically, i.e., both monomers of the dimer are the same. This synthetic route provides for straightforward synthesis, either by constructing the PBD dimer moiety simultaneously having already formed the dimer linkage, or by reacting already constructed PBD monomer moieties with the dimer linking group. These synthetic approaches have limited the options for preparation of targeted conjugates containing PBDs. Due to the observed potency of PBD dimers, however, there exists a need for PBD dimers that are conjugatable to targeting agents for use in targeted therapy.
DISCLOSURE OF THE INVENTION
The present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein a PBD monomer has a substituent in the C2 position that provides an anchor for linking the compound to the targeting agent. The present invention also relates to Conjugates comprising dimers of PBDs conjugated to a targeting agent, wherein the PBD monomers of the dimer are different. One of PBD monomers has a substituent in the C2 position that provides an anchor for linking the compound to the targeting agent. The Conjugates described herein have potent cytotoxic and/or cytostatic activity against cells expressing a target molecule, such as cancer cells or immune cells. These conjugates exhibit good potency with reduced toxicity, as compared with the corresponding PBD dimer free drug compounds.
In some embodiments, the Conjugates have the following formula I:
L-(LU-D)p (I)
wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linker unit and D is a Drug unit comprising a PBD dimer. The subscript p is an integer of from 1 to 20. Accordingly, the Conjugates comprise a Ligand unit covalently linked to at least one Drug unit by a Linker unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. Accordingly, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease. These methods encompass the use of the Conjugates wherein the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.
In a first aspect, the Conjugates comprise a Conjugate of formula I (supra), wherein the Drug unit comprises a PBD dimer of the following formula II:
wherein:
R2 is of formula III:
where A is a C5-7 aryl group, X is an activatable group for conjugation to the Linker unit, wherein X is selected from the group comprising: —O—, —S—, —C(O)O—, —C(O)—, —NHC(O)—, and —N(RN)—, wherein RN is selected from the group comprising H. C1-4 alkyl and (C2H4O)mCH3, where m is 1 to 3, and either:
(i) Q1 is a single bond, and Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q1 is —CH═CH—, and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene:
R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, optionally substituted C3-20 heterocyclyl and optionally substituted C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo; either:
(a) R10 is H, and R11 is OH or ORA, where RA is C1-4 alkyl;
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or
(c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y′ are selected from O, S, or NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and
R11′ are the same as R10 and R11, wherein if R11 and R11′ are SOzM, M may represent a divalent pharmaceutically acceptable cation.
In a second aspect, the use of the Conjugate of formula I is provided for the manufacture of a medicament for treating a proliferative disease or autoimmune disease. In a related third aspect, the use of the Conjugate of formula I is provided for the treatment of a proliferative disease or an autoimmune disease.
In another aspect there is provided the use of a Conjugate of formula I to provide a PBD dimer, or a salt or solvate thereof, at a target location.
One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.
The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism. Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.
In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.
In a fourth aspect of the present invention comprises a method of making the Conjugates formula I.
The dimeric PBD compounds for use in the present invention are made by different strategies to those previously employed in making symmetrical dimeric PBD compounds. In particular, the present inventors have developed a method which involves adding each C2 aryl substituent to a symmetrical PBD dimer core in separate method steps. Accordingly, a sixth aspect of the present invention provides a method of making a Conjugate of formula I, comprising at least one of the method steps described herein.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1 to 6 show the effect of conjugates of the present invention in tumours.
DEFINITIONS
When a trade name is used herein, reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.
Binding Agent and Targeting Agent
The terms “binding agent” and “targeting agent as used herein refer to a molecule, e.g., protein, polypeptide or peptide, that specifically binds to a target molecule. Examples can include a full length antibody, an antigen binding fragment of a full length antibody, other agent (protein, polypeptide or peptide) that includes an antibody heavy and/or light chain variable region that specifically bind to the target molecule, or an Fc fusion protein comprising an extracellular domain of a protein, peptide polypeptide that binds to the target molecule and that is joined to an Fc region, domain or portion thereof, of an antibody.
Specifically Binds
The terms “specifically binds” and “specific binding” refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1×107 M−1, and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
Pharmaceutically Acceptable Cations
Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.
The pharmaceutically acceptable cation may be inorganic or organic.
Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca2+ and Mg2+. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
Substituents
The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.
Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
Examples of substituents are described in more detail below.
C1-12 alkyl: The term “C1-12 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C7).
Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7).
Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).
C2-12 Alkenyl: The term “C2-12 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH═CH2), 1-propenyl (—CH═CH—CH3), 2-propenyl (allyl, —CH—CH═CH2), isopropenyl (1-methylvinyl, —C(CH3)═CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).
C2-12 alkynyl: The term “C2-12 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH2—C≡CH).
C3-12 cycloalkyl: The term “C3-12 cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
Examples of cycloalkyl groups include, but are not limited to, those derived from:
Saturated Monocyclic Hydrocarbon Compounds:
cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7);
Unsaturated Monocyclic Hydrocarbon Compounds:
cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and
Saturated Polycyclic Hydrocarbon Compounds:
norcarane (C7), norpinane (C7), norbomane (C7).
C3-20 heterocyclyl: The term “C3-20 heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
In this context, the prefixes (e.g. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:
N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);
S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3: trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);
N2O1: oxadiazine (C6);
O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1O1S1: oxathiazine (C6).
Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
C5-20 aryl: The term “C5-20 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.
In this context, the prefixes (e.g. C3-20, C5-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6 aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.
The ring atoms may be all carbon atoms, as in “carboaryl groups”.
Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).
Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (C16).
Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:
N1: pyrrole (azole) (C5), pyridine (azine) (C6);
O1: furan (oxole) (C5);
S1: thiophene (thiole) (C5);
N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);
N2O1: oxadiazole (furazan) (C5);
N3O1: oxatriazole (C5):
N1S1: thiazole (C5), isothiazole (C5);
N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6);
N3: triazole (C5), triazine (C6); and,
N4: tetrazole (C5).
Examples of heteroaryl which comprise fused rings, include, but are not limited to:
C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);
C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);
C11 (with 2 fused rings) derived from benzodiazepine (N2);
C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and,
C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxathiin (O1S), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.
Halo: —F, —Cl, —Br, and —I.
Hydroxy: —OH.
Ether: —OR, wherein R is an ether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7alkyl group.
Alkoxy: —OR, wherein R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, —OMe (methoxy), -OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).
Acetal: —CH(OR1)(OR2), wherein R1 and R2 are independently acetal substituents, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, or, in the case of a “cyclic” acetal group, R1 and R2, taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe)2, —CH(OEt)2, and —CH(OMe)(OEt).
Hemiacetal: —CH(OH)(OR1), wherein R1 is a hemiacetal substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).
Ketal: —CR(OR1)(OR2), where R1 and R2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe)2, —C(Me)(OEt)2, —C(Me)(OMe)(OEt), —C(Et)(OMe)2, —C(Et)(OEt)2, and —C(Et)(OMe)(OEt).
Hemiketal: —CR(OH)(OR1), where R1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).
Oxo (keto, -one): ═O.
Thione (thioketone): ═S.
Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, =NEt, and ═NPh.
Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.
Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C1-7 alkyl group (also referred to as C1-7 alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH3 (acetyl), —C(═O)CH2CH3 (propionyl), —C(═O)C(CH3)3 (t-butyryl), and —C(═O)Ph (benzoyl, phenone).
Carboxy (carboxylic acid): —C(═O)OH.
Thiocarboxy (thiocarboxylic acid): —C(═S)SH.
Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.
Thionocarboxy (thionocarboxylic acid): —C(═S)OH.
Imidic acid: —C(═NH)OH.
Hydroxamic acid: —C(═NOH)OH.
Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH3, —C(═O)OCH2CH3, —C(═O)OC(CH3)3, and —C(═O)OPh.
Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH3 (acetoxy), —OC(═O)CH2CH3, —OC(═O)C(CH3)3, —OC(═O)Ph, and —OC(═O)CH2Ph.
Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH3, —OC(═O)OCH2CH3, —OC(═O)OC(CHa)3, and —OC(═O)OPh.
Amino: —NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7 alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a “cyclic” amino group, R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH2), secondary (—NHR1), or tertiary (—NHR1R2), and in cationic form, may be quaternary (—+NR1R2R3). Examples of amino groups include, but are not limited to, —NH2, —NHCH3, —NHC(CH3)2, —N(CH3)2, —N(CH2CH3)2, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH2, —C(═O)NHCH3, —C(═O)N(CH3)2, —C(═O)NHCH2CH3, and —C(═O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
Thioamido (thiocarbamyl): —C(═S)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH2, —C(═S)NHCH3, —C(═S)N(CH3)2, and —C(═S)NHCH2CH3.
Acylamido (acylamino): —NR1C(═O)R2, wherein R1 is an amide substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group, and R2 is an acyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH3, —NHC(═O)CH2CH3, and —NHC(═O)Ph. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
Aminocarbonyloxy: —OC(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH2, —OC(═O)NHMe, —OC(═O)NMe2, and —OC(═O)NEt2.
Ureido: —N(R1)CONR2R3 wherein R2 and R3 are independently amino substituents, as defined for amino groups, and R1 is a ureido substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH2, —NHCONHMe, —NHCONHEt, —NHCONMe2, —NHCONEt2, —NMeCONH2, —NMeCONHMe, —NMeCONHEt, —NMeCONMe2, and —NMeCONEt2.
Guanidino: —NH—C(═NH)NH2.
Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,
Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and =NEt.
Amidine (amidino): —C(═NR)NR2, wherein each R is an amidine substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH2, —C(═NH)NMe2, and —C(═NMe)NMe2.
Nitro: —NO2.
Nitroso: —NO.
Azido: —N3.
Cyano (nitrile, carbonitrile): —CN.
Isocyano: —NC.
Cyanato: —OCN.
Isocyanato: —NCO.
Thiocyano (thiocyanato): —SCN.
Isothiocyano (isothiocyanato): —NCS.
Sulfhydryl (thiol, mercapto): —SH.
Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7alkylthio group), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of C1-7 alkylthio groups include, but are not limited to, —SCH3 and —SCH2CH3.
Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group (also referred to herein as C1-7 alkyl disulfide). Examples of C1-7 alkyl disulfide groups include, but are not limited to, —SSCH3 and —SSCH2CH3.
Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH3 and —S(═O)CH2CH3.
Sulfone (sulfonyl): —S(═O)2R, wherein R is a sulfone substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, including, for example, a fluorinated or perfluorinated C1-7 alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)2CH3 (methanesulfonyl, mesyl), —S(═O)2CF3 (triflyl), —S(═O)2CH2CH3 (esyl), —S(═O)2C4F9 (nonaflyl), —S(═O)2CH2CF3 (tresyl), —S(═O)2CH2CH2NH2 (tauryl), —S(═O)2Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).
Sulfinic acid (sulfino): —S(═O)OH, —SO2H.
Sulfonic acid (sulfo): —S(═O)2OH, —SO3H.
Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH3 (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).
Sulfonate (sulfonic acid ester): —S(═O)2OR, wherein R is a sulfonate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)2OCH3 (methoxysulfonyl; methyl sulfonate) and —S(═O)2OCH2CH3 (ethoxysulfonyl; ethyl sulfonate).
Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH3 and —OS(═O)CH2CH3.
Sulfonyloxy: —OS(═O)2R, wherein R is a sulfonyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)2CH3 (mesylate) and —OS(═O)2CH2CH3 (esylate).
Sulfate: —OS(═O)2OR; wherein R is a sulfate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)2OCH3 and —SO(═O)2OCH2CH3.
Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH2, —S(═O)NH(CH3), —S(═O)N(CH3)h, —S(═O)NH(CH2CH3), —S(═O)N(CH2CH3)2, and —S(═O)NHPh.
Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)2NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to. —S(═O)2NH2, —S(═O)2NH(CH3), —S(═O)2N(CH3)2, —S(═O)2NH(CH2CH3), —S(═O)2N(CH2CH3)2, and —S(═O)2NHPh.
Sulfamino: —NR1S(═O)2OH, wherein R1 is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)2OH and —N(CH3)S(═O)2OH.
Sulfonamino: —NR1S(═O)2R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)2CH3 and —N(CH3)S(═O)2C6H5.
Sulfinamino: —NR1S(═O)R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH3 and —N(CH3)S(═O)C6H5.
Phosphino (phosphine): —PR2, wherein R is a phosphino substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphino groups include, but are not limited to, —PH2, —P(CH3)2, —P(CH2CH3)2, —P(t-Bu)2, and —P(Ph)2.
Phospho: —P(═O)2.
Phosphinyl (phosphine oxide): —P(═O)R2, wherein R is a phosphinyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group or a C5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH3)2, —P(═O)(CH2CH3)2, —P(═O)(t-Bu)2, and —P(═O)(Ph)2.
Phosphonic acid (phosphono): —P(═O)(OH)2.
Phosphonate (phosphono ester): —P(═O)(OR)2, where R is a phosphonate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH3)2, —P(═O)(OCH2CH3)2, —P(═O)(O-t-Bu)2, and —P(═O)(OPh)2.
Phosphoric acid (phosphonooxy): —OP(═O)(OH)2.
Phosphate (phosphonooxy ester): —OP(═O)(OR)2, where R is a phosphate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH3)2, —OP(═O)(OCH2CH3)2, —OP(═OXO-t-Bu)2, and —OP(═O)(OPh)2.
Phosphorous acid: —OP(OH)2.
Phosphite: —OP(OR)2, where R is a phosphite substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH3)2, —OP(OCH2CH3)2, —OP(O-t-Bu)2, and —OP(OPh)2.
Phosphoramidite: —OP(OR1)—NR22, where R1 and R2 are phosphoramidite substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH2CH3)—N(CH3)2, —OP(OCH2CH3)—N(i-Pr)2, and —OP(OCH2CH2CN)—N(i-Pr)2.
Phosphoramidate: —OP(═O)(OR1)—NR22, where R1 and R2 are phosphoramidate substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH2CH3)—N(CH3)2, —OP(═O)(OCH2CH3)—N(i-Pr)2, and —OP(═O)(OCH2CH2CN)—N(i-Pr)2.
Alkylene
C3-12 alkylene: The term “C3-12 alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
Examples of linear saturated C3-12 alkylene groups include, but are not limited to, —(CH2)n—where n is an integer from 3 to 12, for example, —CH2CH2CH2— (propylene), —CH2CH2CH2CH2— (butylene), —CH2CH2CH2CH2CH2 (pentylene) and —CH2CH2CH2CH2CH2CH2CH2— (heptylene).
Examples of branched saturated C3-12 alkylene groups include, but are not limited to, —CH(CH3)CH—, —CH(CH3)CH2CH—, —CH(CH3)CH2CH2CH2—, —CH2CH(CH3)CH2—, —CH2CH(CH3)CH2CH2—, —CH(CH2CH3)—, —CH(CH2CH3)CH2—, and —CH2CH(CH2CH3)CH2.
Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH2—, —CH2—CH═CH2—, —CH═CH—CH2—CH2—, —CH═CH—CH2—CH2—CH2—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH2—, —CH═CH—CH═CH—CH2—CH2—, —CH═CH—CH2—CH═CH—, —CH═CH—CH2—CH2—CH═CH—, and —CH2—C≡C—CH2—.
Examples of branched partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, —C(CH3)═CH—, —C(CH3)═CH—CH2—, —CH═CH—CH(CH3)— and —C≡C—CH(CH3)—.
Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).
Examples of alicyclic partially unsaturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).
Oxygen protecting group: the term “oxygen protecting group” refers to a moiety which masks a hydroxy group, and these are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons. Inc., 1999, which is incorporated herein by reference. Classes of particular interest include silyl ethers (e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g. acetate).
Carbamate nitrogen protecting group: the term “carbamate nitrogen protecting group” pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogen protecting group” pertains to a group having the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides Conjugates comprising a PBD dimer connected to a Ligand unit via a Linker Unit. In one embodiment, the Linker unit includes a Stretcher unit (A), a Specificity unit (L1), and a Spacer unit (L2). The Linker unit is connected at one end to the Ligand unit and at the other end to the PBD dimer compound.
In one aspect, such a Conjugate is shown below in formula Ia:
L-(A1s-L1s-L2y-D)p (Ia)
wherein:
L is the Ligand unit; and
-A1a-L1s-L2y- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit,
a is 1 or 2.
L1- is a Specificity unit,
s is an integer ranging from 1 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is an PBD dimer; and
p is from 1 to 20.
The drug loading is represented by p, the number of drug molecules per Ligand unit (e.g., an antibody). Drug loading may range from 1 to 20 Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20.
In some embodiments, p is from about 1 to about 8 Drug units per Ligand unit. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is from about 2 to about 8 Drug units per Ligand unit. In some embodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4 Drug units per Ligand unit. In some embodiments, p is about 2, about 4, about 6 or about 8 Drug units per Ligand unit.
The average number of Drugs units per Ligand unit in a preparation from a conjugation reaction may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Conjugates, where p is a certain value, from Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
In another aspect, such a Conjugate is shown below in formula Ib:
Also illustrated as:
L-(A1a-L2y(-L1s)-D)p (Ib)
wherein:
L is the Ligand unit; and
-A1a-L1s(L2y)- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit linked to a Stretcher unit (L2),
a is 1 or 2,
L1- is a Specificity unit linked to a Stretcher unit (L2),
s is an integer ranging from 0 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is a PBD dimer; and
p is from 1 to 20.
Preferences
The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
In one embodiment, the Conjugate has the formula:
L-(A1a-L1s-L2y-D)p
wherein L, A1, a, L1, s, L2, D and p are as described above.
In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit, which is a covalent bond, a self-immolative group or together with —OC(═O)— forms a self-immolative group, and L2 optional.
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
CBA-A1a-L1s-L2y-*
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit which is a covalent bond or a self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is 0 or 1 or 2.
In the embodiments illustrated above, L1 can be a cleavable Specificity unit, and may be referred to as a “trigger” that when cleaved activates a self-immolative group (or self-immolative groups) L2, when a self-immolative group(s) is present. When the Specificity unit L1 is cleaved, or the linkage (i.e., the covalent bond) between L1 and L2 is cleaved, the self-immolative group releases the Drug unit (D).
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug (D), CBA is the Cell Binding Agent, L1 is a Specificity unit connected to L2, A1 is a Stretcher unit connecting L2 to the Cell Binding Agent, L2 is a self-immolative group, and a is 1 or 2, s is 1 or 2, and y is 1 or 2.
In the various embodiments discussed herein, the nature of L1 and L2 can vary widely. These groups are chosen on the basis of their characteristics, which may be dictated in part, by the conditions at the site to which the conjugate is delivered. Where the Specificity unit L1 is cleavable, the structure and/or sequence of L1 is selected such that it is cleaved by the action of enzymes present at the target site (e.g., the target cell). L1 units that are cleavable by changes in pH (e.g. acid or base labile), temperature or upon irradiation (e.g. photolabile) may also be used. L1 units that are cleavable under reducing or oxidising conditions may also find use in the Conjugates.
In some embodiments, L1 may comprise one amino acid or a contiguous sequence of amino acids. The amino acid sequence may be the target substrate for an enzyme.
In one embodiment, L1 is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L1 may be cleaved by a lysosomal protease, such as a cathepsin.
In one embodiment, L2 is present and together with —C(═O)O— forms a self-immolative group or self-immolative groups. In some embodiments, —C(═O)O— also is a self-immolative group.
In one embodiment, where L1 is cleavable by the action of an enzyme and L2 is present, the enzyme cleaves the bond between L1 and L2, whereby the self-immolative group(s) release the Drug unit.
L1 and L2, where present, may be connected by a bond selected from:
—C(═O)NH—.
—C(═O)O—,
—NHC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH, and
—O— (a glycosidic bond).
An amino group of L1 that connects to L2 may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain.
A carboxyl group of L1 that connects to L2 may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain.
A hydroxy group of L1 that connects to L2 may be derived from a hydroxy group of an amino acid side chain, for example a serine amino acid side chain.
In one embodiment, —C(═O)O— and L2 together form the group:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to the L1, Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The phenylene ring is optionally substituted with one, two or three substituents as described herein.
In one embodiment, Y is NH.
In one embodiment, n is 0 or 1. Preferably, n is 0.
Where Y is NH and n is 0, the self-immolative group may be referred to as a p-aminobenzylcarbonyl linker (PABC).
The self-immolative group will allow for release of the Drug unit (i.e., the asymmetric PBD) when a remote site in the linker is activated, proceeding along the lines shown below (for n=0):
where the asterisk indicates the attachment to the Drug, L* is the activated form of the remaining portion of the linker and the released Drug unit is not shown. These groups have the advantage of separating the site of activation from the Drug.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above. Each phenylene ring is optionally substituted with one, two or three substituents as described herein. In one embodiment, the phenylene ring having the Y substituent is optionally substituted and the phenylene ring not having the Y substituent is unsubstituted.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or CR.
In one embodiment, D is N.
In one embodiment, D is CH.
In one embodiment, E is O or S.
In one embodiment, F is CH.
In a preferred embodiment, the covalent bond between L1 and L2 is a cathepsin labile (e.g., cleavable) bond.
In one embodiment, L1 comprises a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2 in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is -Phe-Lys-, Val-Cit or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described by Dubowchik et al., which is incorporated herein by reference.
In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective groups in Organic Synthesis, Greene and Wuts.
Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:
Arg: Z. Mtr, Tos;
Asn: Trt, Xan;
Asp: Bzl, t-Bu;
Cys: Acm, BzI, Bzl-OMe, Bzl-Me, Trt;
Glu: Bzl, t-Bu;
Gin: Trt, Xan;
His: Boc, Dnp, Tos, Trt;
Lys: Boc, Z—Cl, Fmoc, Z;
Ser: Bzl, TBDMS, TBDPS;
Thr: Bz;
Trp: Boc;
Tyr: Bzl, Z, Z—Br.
In one embodiment, —X2— is connected indirectly to the Drug unit. In such an embodiment, the Spacer unit L2 is present.
In one embodiment, the dipeptide is used in combination with a self-immolative group(s) (the Spacer unit). The self-immolative group(s) may be connected to —X2—.
Where a self-immolative group is present, —X2— is connected directly to the self-immolative group. In one embodiment, —X2— is connected to the group Y of the self-immolative group. Preferably the group —X2—CO— is connected to Y, where Y is NH.
—X1— is connected directly to A1. In one embodiment, —X1— is connected directly to A1. Preferably the group NH—X1— (the amino terminus of X1) is connected to A1. A1 may comprise the functionality —CO— thereby to form an amide link with —X1—.
In one embodiment, L1 and L2 together with —OC(═O)— comprise the group —X1—X2— PABC-. The PABC group is connected directly to the Drug unit. In one example, the self-immolative group and the dipeptide together form the group -Phe-Lys-PABC-, which is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
Alternatively, the self-immolative group and the dipeptide together form the group -Val-Ala-PABC-, which is illustrated below:
where the asterisk and the wavy line are as defined above.
In another embodiment, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group, and E is a group that is susceptible to cleavage thereby to activate a self-immolative group.
E is selected such that the group is susceptible to cleavage, e.g., by light or by the action of an enzyme. E may be —NO2 or glucuronic acid (e.g., β-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a 3-glucuronidase.
The group Y may be a covalent bond.
The group Y may be a functional group selected from:
—C(═O)—
—NH—
—O—
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O),
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH.
—C(═O)NHC(═O)—,
SO2, and
—S—.
The group Y is preferably —NH—, —CH2—, —O—, and —S—.
In some embodiments, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—.
In some embodiments, L1 and L2 together represent:
where the asterisk indicates the point of attachment to the remainder of L2 or the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—, —CH2—, —O—, and —S—.
In some further embodiments, Y is a functional group as set forth above, the functional group is linked to an amino acid, and the amino acid is linked to the Stretcher unit A1. In some embodiments, amino acid is β-alanine. In such an embodiment, the amino acid is equivalently considered part of the Stretcher unit.
The Specificity unit L1 and the Ligand unit are indirectly connected via the Stretcher unit.
L1 and A1 may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the connection between the Ligand unit and A1 is through a thiol residue of the Ligand unit and a maleimide group of A1.
In one embodiment, the connection between the Ligand unit and A1 is:
where the asterisk indicates the point of attachment to the remaining portion of A1, L1, L2 or D, and the wavy line indicates the point of attachment to the remaining portion of the Ligand unit. In this embodiment, the S atom is typically derived from the Ligand unit.
In each of the embodiments above, an alternative functionality may be used in place of the malemide-derived group shown below:
where the wavy line indicates the point of attachment to the Ligand unit as before, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with the group:
where the wavy line indicates point of attachment to the Ligand unit, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with a Ligand unit (e.g., a Cell Binding Agent), is selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH,
—C(═O)NHC(═O)—,
—S—,
—S—S—,
—CH2C(═O)—
—C(═O)CH2—,
═N—NH—, and
—NH—N═.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the Ligand unit, is selected from:
where the wavy line indicates either the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group, and the asterisk indicates the other of the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group.
Other groups suitable for connecting L1 to the Cell Binding Agent are described in WO 2005/082023.
In one embodiment, the Stretcher unit A1 is present, the Specificity unit L1 is present and Spacer unit L2 is absent. Thus, L1 and the Drug unit are directly connected via a bond. Equivalently in this embodiment, L2 is a bond.
L1 and D may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O),
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, L1 and D are preferably connected by a bond selected from:
—C(═O)NH—, and
—NHC(═O)—.
In one embodiment, L1 comprises a dipeptide and one end of the dipeptide is linked to D. As described above, the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2 in dipeptide, —NH—X1—X2CO—, is -Phe-Lys- or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described above.
In one embodiment, L1-D is:
where —NH—X1—X2—CO is the dipeptide, —NH— is part of the Drug unit, the asterisk indicates the point of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
In one embodiment, the dipeptide is valine-alanine and L1-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is phenylalanine-lysine and L1-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is valine-citrulline.
In one embodiment, the groups A1-L2 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3 or 7.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 is:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the rest of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4 or 8. In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the Stretcher unit is an acetamide unit, having the formula:
where the asterisk indicates the point of attachment to the remainder of the Stretcher unit, L1 or D, and the wavy line indicates the point of attachment to the Ligand unit.
In other embodiments, Linker-Drug compounds are provided for conjugation to a Ligand unit. In one embodiment, the Linker-Drug compounds are designed for connection to a Cell Binding Agent.
In one embodiment, the Drug Linker compound has the formula:
where the asterisk indicates the point of attachment to the Drug unit, G1 is a Stretcher group (A′) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or together with —OC(═O)— forms a self-immolative group(s).
In another embodiment, the Drug Linker compound has the formula:
G1-L1-L2-
where the asterisk indicates the point of attachment to the Drug unit. G1 is a Stretcher unit (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or a self-immolative group(s).
L1 and L2 are as defined above. References to connection to A1 can be construed here as referring to a connection to G1.
In one embodiment, where L1 comprises an amino acid, the side chain of that amino acid may be protected. Any suitable protecting group may be used. In one embodiment, the side chain protecting groups are removable with other protecting groups in the compound, where present. In other embodiments, the protecting groups may be orthogonal to other protecting groups in the molecule, where present.
Suitable protecting groups for amino acid side chains include those groups described in the Novabiochem Catalog 2006/2007. Protecting groups for use in a cathepsin labile linker are also discussed in Dubowchik et al.
In certain embodiments of the invention, the group L1 includes a Lys amino acid residue. The side chain of this amino acid may be protected with a Boc or Alloc protected group. A Boc protecting group is most preferred.
The functional group G1 forms a connecting group upon reaction with a Ligand unit (e.g., a cell binding agent.
In one embodiment, the functional group G1 is or comprises an amino, carboxylic acid, hydroxy, thiol, or maleimide group for reaction with an appropriate group on the Ligand unit. In a preferred embodiment, G1 comprises a maleimide group.
In one embodiment, the group G1 is an alkyl maleimide group. This group is suitable for reaction with thiol groups, particularly cysteine thiol groups, present in the cell binding agent, for example present in an antibody.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In each of the embodiments above, an alternative functionality may be used in place of the malemide group shown below:
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide-derived group is replaced with the group:
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide group is replaced with a group selected from:
—C(═O)OH,
—OH,
—NH2,
—SH,
—C(═O)CH2X, where X is Cl, Br or I,
—CHO,
—NHNH2
—C≡CH, and
—N3 (azide).
In one embodiment, L1 is present, and G1 is —NH2, —NHMe, —COOH, —OH or —SH.
In one embodiment, where L1 is present, G1 is —NH2 or —NHMe. Either group may be the N-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is —NH2, and L1 is an amino acid sequence —X1—X2—, as defined above.
In one embodiment, L1 is present and G1 is COOH. This group may be the C-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is OH.
In one embodiment, L1 is present and G1 is SH.
The group G1 may be convertable from one functional group to another. In one embodiment, L1 is present and G1 is —NH2. This group is convertable to another group G1 comprising a maleimide group. For example, the group —NH2 may be reacted with an acids or an activated acid (e.g., N-succinimide forms) of those G1 groups comprising maleimide shown above.
The group G1 may therefore be converted to a functional group that is more appropriate for reaction with a Ligand unit.
As noted above, in one embodiment, L1 is present and G1 is —NH2, —NHMe, —COOH, —OH or —SH. In a further embodiment, these groups are provided in a chemically protected form. The chemically protected form is therefore a precursor to the linker that is provided with a functional group.
In one embodiment, G1 is —NH2 in a chemically protected form. The group may be protected with a carbamate protecting group. The carbamate protecting group may be selected from the group consisting of:
Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.
Preferably, where G1 is —NH2, it is protected with an Alloc or Fmoc group.
In one embodiment, where G1 is —NH2, it is protected with an Fmoc group.
In one embodiment, the protecting group is the same as the carbamate protecting group of the capping group.
In one embodiment, the protecting group is not the same as the carbamate protecting group of the capping group. In this embodiment, it is preferred that the protecting group is removable under conditions that do not remove the carbamate protecting group of the capping group.
The chemical protecting group may be removed to provide a functional group to form a connection to a Ligand unit. Optionally, this functional group may then be converted to another functional group as described above.
In one embodiment, the active group is an amine. This amine is preferably the N-terminal amine of a peptide, and may be the N-terminal amine of the preferred dipeptides of the invention.
The active group may be reacted to yield the functional group that is intended to form a connection to a Ligand unit.
In other embodiments, the Linker unit is a precursor to the Linker unit having an active group. In this embodiment, the Linker unit comprises the active group, which is protected by way of a protecting group. The protecting group may be removed to provide the Linker unit having an active group.
Where the active group is an amine, the protecting group may be an amine protecting group, such as those described in Green and Wuts.
The protecting group is preferably orthogonal to other protecting groups, where present, in the Linker unit.
In one embodiment, the protecting group is orthogonal to the capping group. Thus, the active group protecting group is removable whilst retaining the capping group. In other embodiments, the protecting group and the capping group is removable under the same conditions as those used to remove the capping group.
In one embodiment, the Linker unit is:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of the Linker unit, as applicable or the point of attachment to G1. Preferably, the wavy line indicates the point of attachment to G1.
In one embodiment, the Linker unit is:
where the asterisk and the wavy line are as defined above.
Other functional groups suitable for use in forming a connection between L1 and the Cell Binding Agent are described in WO 2005/082023.
Ligand Unit
The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non-peptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.
Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).
In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; fully human antibody; or a single chain antibody. One embodiment the antibody is a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab′, F(ab′)2 and Fv fragments.
The antibody may be a diabody, a domain antibody (DAB) or a single chain antibody.
In one embodiment, the antibody is a monoclonal antibody.
Antibodies for use in the present invention include those antibodies described in WO 2005/082023 which is incorporated herein. Particularly preferred are those antibodies for tumour-associated antigens. Examples of those antigens known in the art include, but are not limited to, those tumour-associated antigens set out in WO 2005/082023. See, for instance, pages 41-55.
In some embodiments, the conjugates are designed to target tumour cells via their cell surface antigens. The antigens may be cell surface antigens which are either over-expressed or expressed at abnormal times or cell types. Preferably, the target antigen is expressed only on proliferative cells (preferably tumour cells); however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue.
Antibodies have been raised to target specific tumour related antigens including:
Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2.
The Ligand unit is connected to the Linker unit. In one embodiment, the Ligand unit is connected to A, where present, of the Linker unit.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a thioether bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a disulfide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an amide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an ester bond.
In one embodiment, the connection between the Ligand unit and the Linker is formed between a thiol group of a cysteine residue of the Ligand unit and a maleimide group of the Linker unit.
The cysteine residues of the Ligand unit may be available for reaction with the functional group of the Linker unit to form a connection. In other embodiments, for example where the Ligand unit is an antibody, the thiol groups of the antibody may participate in interchain disulfide bonds. These interchain bonds may be converted to free thiol groups by e.g. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit.
In some embodiments, the cysteine residue is an introduced into the heavy or light chain of an antibody. Positions for cysteine insertion by substitution in antibody heavy or light chains include those described in Published U.S. Application No. 2007-0092940 and International Patent Publication WO2008070593, which are incorporated herein.
Methods of Treatment
The Conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a Conjugate of formula I. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount of a Conjugate administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose.
In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 1 mg/kg per dose.
A conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy).
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a Conjugate of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Includes Other Forms
Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO−), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N+HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O−), a salt or solvate thereof, as well as conventional protected forms.
Salts
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound (the Conjugate), for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).
For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO−), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations such as Al+3. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
If the Conjugate is cationic, or has a functional group which may be cationic (e.g. —NH2 may be —NH3+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
Solvates
It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the Conjugate(s). The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active Conjugate, salt of active Conjugate) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
Carbinolamines
The invention includes Conjugate where a solvent adds across the imine bond of the PBD moiety, which is illustrated below for a PBD monomer where the solvent is water or an alcohol (RAOH, where RA is C1-4 alkyl):
These forms can be called the carbinolamine and carbinolamine ether forms of the PBD.
The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.
These particular compounds may be isolated in solid form, for example, by lyophilisation.
Isomers
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).
Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like.
Unless otherwise specified, a reference to a particular compound or Conjugate includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
General Synthetic Routes
The synthesis of PBD dimer compounds is extensively discussed in the following references, which discussions are incorporated herein by reference:
a) WO 00/12508 (pages 14 to 30);
b) WO 2005/023814 (pages 3 to 10);
c) WO 2004/043963 (pages 28 to 29); and
d) WO 2005/085251 (pages 30 to 39).
Synthesis Route
The Conjugates of the present invention, where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, can be synthesised from a compound of Compound formula 2:
where R2, R6, R7, R9, R6′, R7′, R9′, R12, X, X′ and R11 are as defined for compounds of formula II, ProtN is a nitrogen protecting group for synthesis and ProtO is a protected oxygen group for synthesis or an oxo group, by deprotecting the imine bond by standard methods.
The compound produced may be in its carbinolamine or carbinolamine ether form depending on the solvents used. For example if ProtN is Alloc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried using palladium to remove the N10 protecting group, followed by the elimination of the oxygen protecting group for synthesis. If ProtN is Troc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried out using a Cd/Pb couple to yield the compound of formula (I). If ProtN is SEM, or an analogous group, and ProtO is an oxo group, then the oxo group can be removed by reduction, which leads to a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group, followed by the elimination of water. The reduction of the compound of Compound formula 2 can be accomplished by, for example, lithium tetraborohydride, whilst a suitable means for removing the SEM protecting group is treatment with silica gel.
Compounds of Compound formula 2 can be synthesised from a compound of Compound formula 3a:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling an organometallic derivative comprising R12, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of Compound formula 2 can be synthesised from a compound of Compound formula 3b:
where R12, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling an organometallic derivative comprising R2, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of Compound formulae 3a and 3b can be synthesised from a compound of formula 4:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallic derivative, such as an organoboron derivative, comprising R2 or R12.
The couplings described above are usually carried out in the presence of a palladium catalyst, for example Pd(PPh3)4, Pd(OCOCH3)2, PdCl2, or Pd2(dba)3. The coupling may be carried out under standard conditions, or may also be carried out under microwave conditions.
The two coupling steps are usually carried out sequentially. They may be carried out with or without purification between the two steps. If no purification is carried out, then the two steps may be carried out in the same reaction vessel. Purification is usually required after the second coupling step. Purification of the compound from the undesired by-products may be carried out by column chromatography or ion-exchange separation.
The synthesis of compounds of Compound formula 4 where ProtO is an oxo group and ProtN is SEM are described in detail in WO 00/12508, which is incorporated herein by reference. In particular, reference is made to scheme 7 on page 24, where the above compound is designated as intermediate P. This method of synthesis is also described in WO 2004/043963.
The synthesis of compounds of Compound formula 4 where ProtO is a protected oxygen group for synthesis are described in WO 2005/085251, which synthesis is herein incorporated by reference.
Compounds of formula I where R10 and R10′ are H and R11 and R11″ are SOzM, can be synthesised from compounds of formula I where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, by the addition of the appropriate bisulphite salt or sulphinate salt, followed by an appropriate purification step. Further methods are described in GB 2 053 894, which is herein incorporated by reference.
Nitrogen Protecting Groups for Synthesis
Nitrogen protecting groups for synthesis are well known in the art. In the present invention, the protecting groups of particular interest are carbamate nitrogen protecting groups and hemi-aminal nitrogen protecting groups.
Carbamate nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.
Other possible groups are nitrobenzyloxycarbonyl (e.g. 4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.
Those protecting groups which can be removed with palladium catalysis are not preferred, e.g. Alloc.
Hemi-aminal nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. The groups disclosed herein can be applied to compounds for use in the present invention. Such groups include, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, and Cl3CCH2OCH2—.
Protected Oxygen Group for Synthesis
Protected oxygen group for synthesis are well known in the art. A large number of suitable oxygen protecting groups are described on pages 23 to 200 of Greene. T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates.
Preferred oxygen protecting groups include acetates, TBS and THP.
Further Preferences
The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
In some embodiments, R6′, R7′, R9′, R10′, R11′ and Y′ are preferably the same as R6, R7, R9, R10, R11 and Y respectively.
Dimer Link
Y and Y′ are preferably O.
R″ is preferably a C3-7 alkylene group with no substituents. More preferably R″ is a C3, C5 or C7 alkylene.
R6 to R9
R9 is preferably H.
R6 is preferably selected from H, OH, OR, SH, NH2, nitro and halo, and is more preferably H or halo, and most preferably is H.
R7 is preferably selected from H, OH, OR, SH, SR, NH2, NHR, NRR′, and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C1-7 alkyl, C3-10 heterocyclyl and C5-10 aryl groups. R may be more preferably a C1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH2Ph.
These preferences apply to R9′, R6′, and R7′ respectively.
R2
A in R2 may be phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl. In other embodiments, A is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
X is a group selected from the list comprising: —O—, —S—, —C(O)O—, —C(O)—, —NH(C═O)— and —N(RN)—, wherein RN is selected from the group comprising H and C1-4 alkyl. X may preferably be: —O—, —S—, —C(O)O—, —NH(C═O)— or —NH—, and may more preferably be: —O—, —S—, or —NH—, and most preferably is —NH—.
Q2-X may be on any of the available ring atoms of the C5-7 aryl group, but is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group (A) is phenyl, the substituent (Q2-X) is preferably in the meta- or para-positions, and more preferably is in the para-position, 1
In some embodiments, Q1 is a single bond. In these embodiments, Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and is from 1 to 3. In some of these embodiments, Q2 is a single bond. In other embodiments, Q2 is —Z—(CH2)n—. In these embodiments. Z may be O or S and n may be 1 or n may be 2. In other of these embodiments, Z may be a single bond and n may be 1.
In other embodiments, Q1 is —CH═CH—.
In some embodiments, R2 may be -A-CH2—X and -A-X. In these embodiments, X may be —O—, —S—, —C(O)O—, —C(O)— and —NH—. In particularly preferred embodiments, X may be —NH—.
R12
R12 may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R12 is preferably phenyl. In other embodiments, R12 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
R12 may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
R12 may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
Where R12 is C5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para-positions, and more preferably is in the para-position.
Where R12 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R12 Substituents
If a substituent on R12 is halo, it is preferably F or Cl, more preferably Cl.
If a substituent on R12 is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g. phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
If a substituent on R12 is C1-7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propyl, butyl).
If a substituent on R12 is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
If a substituent on R12 is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
Particularly preferred substituents for R12 include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl. Another particularly preferred substituent for R12 is dimethylaminopropyloxy.
R12 Groups
Particularly preferred substituted R12 groups include, but are not limited to, 4-methoxyphenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R12 group is 4-nitrophenyl.
M and Z
It is preferred that M and M′ are monovalent pharmaceutically acceptable cations, and are more preferably Na+.
z is preferably 3.
EXAMPLES
General Experimental Methods
Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. 1H and 13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt (NovaBiochem) and solid-supported reagents (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied without further purification. Anhydrous solvents were prepared by distillation under a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C.
Compound 1b was synthesised as described in WO 00/012508 (compound 210), which is herein incorporated by reference.
General LC/MS conditions: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then 5% B to 95% B within 3 min. The composition was held for 0.5 min at 95% B, and then returned to 5% B in 0.3 minutes. Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm
LC/MS conditions specific for compounds protected by both a Troc and a TBDMs group: Chromatographic separation of Troc and TBDMS protected compounds was performed on a Waters Alliance 2695 HPLC system utilizing a Onyx Monolitic reversed-phase column (3 μm particles, 50×4.6 mm) from Phenomenex Corp. Mobile-phase A consisted of 5% acetonitrile—95% water containing 0.1% formic acid, and mobile phase B consisted of 95% acetonitrile—5% water containing 0.1% formic acid. After 1 min at 5% B, the proportion of B was raised to 95% B over the next 2.5 min and maintained at 95% B for a further 1 min, before returning to 95% A in 10 s and re-equilibration for a further 50 sec, giving a total run time of 5.0 min. The flow rate was maintained at 3.0 mL/min.
LC/MS conditions specific for compound 33: LC was run on a Waters 2767 sample Manager coupled with a Waters 2996 photodiode array detector and a Waters ZQ single quadruple mass Spectrometer. The column used was Luna Phenyl-Hexyl 150×4.60 mm, 5 μm, Part no. 00F-4257-E0 (Phenomenex). The mobile phases employed were: Mobile phase A: 100% of HPLC grade water (0.05% triethylamine), pH=7 Mobile phase B: 20% of HPLC grade water and 80% of HPLC grade acetonitrile (0.05% triethylamine), pH=7
The gradients used were:
Time
Flow Rate
(min)
(ml/mm)
% A
% B
Initial
1.50
90
10
1.0
1.50
90
10
16.0
1.50
64
36
30.0
1.50
5
95
31.0
1.50
90
10
32.0
1.50
90
10
Mass Spectrometry was carried out in positive ion mode and SIR (selective ion monitor) and the ion monitored was m/z=727.2.
(a) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2a)
Method A: A catalytic amount of DMF (2 drops) was added to a stirred solution of the nitro-acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride (0.95 mL, 1.36 g, 10.7 mmol) in dry THF (20 mL). The reaction mixture was allowed to stir for 16 hours at room temperature and the solvent was removed by evaporation in vacuo. The resulting residue was re-dissolved in dry THF (20 mL) and the acid chloride solution was added dropwise to a stirred mixture of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (859 mg, 4.73 mmol) and TEA (6.6 mL, 4.79 g, 47.3 mmol) in THF (10 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for a further 3 hours after which time TLC (95:5 v/v CHCl3/MeOH) and LC/MS (2.45 min (ES+) m/z (relative intensity) 721 ([M+H]+, 20)) revealed formation of product. Excess THF was removed by rotary evaporation and the resulting residue was dissolved in DCM (50 mL). The organic layer was washed with 1N HCl (2×15 mL), saturated NaHCO3 (2×15 mL), H2O (20 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent gave the crude product as a dark coloured oil. Purification by flash chromatography (gradient elution: 100% CHCl3a to 96:4 v/v CHCl3/MeOH) isolated the pure amide 2a as an orange coloured glass (840 mg, 54%).
Method B: Oxalyl chloride (9.75 mL, 14.2 g, 111 mmol) was added to a stirred suspension of the nitro-acid 1a (17.3 g, 37.1 mmol) and DMF (2 mL) in anhydrous DCM (200 mL). Following initial effervescence the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. Conversion to the acid chloride was confirmed by treating a sample of the reaction mixture with MeOH and the resulting bis-methyl ester was observed by LC/MS. The majority of solvent was removed by evaporation in vacuo, the resulting concentrated solution was re-dissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40° C. The solid acid chloride was added portionwise over a period of 25 minutes to a stirred suspension of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (15.2 g, 84.0 mmol) and TEA (25.7 mL, 18.7 g, 185 mmol) in DCM (150 mL) at −40° C. (dry ice/CH3CN). Immediately, the reaction was complete as judged by LC/MS (2.47 min (ES+) m/z (relative intensity) 721 ([M+H]+, 100)). The mixture was diluted with DCM (150 mL) and washed with 1N HCl (300 mL), saturated NaHCO3 (300 mL), brine (300 mL), filtered (through a phase separator) and the solvent evaporated in vacuo to give the pure product 2a as an orange solid (21.8 g, 82%).
Analytical Data: [α]22D=−46.1° (c=0.47, CHCl3); 1H NMR (400 MHz, CDCl3) (rotamers) δ 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H, J=11.1 Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); 13C NMR (100 MHz, CDCl3) (rotamers) δ 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7, 108.2, 69.7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR, CHCl3) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211, 1177, 1072, 1050, 1008, 871 cm−1; MS (ES+) m/z (relative intensity) 721 ([M+H]+, 47), 388 (80); HRMS [M+H]+ theoretical C31H36N4O16 m/z 721.2199, found (ES+) m/z 721.2227.
(a) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2b)
Preparation from 1b according to Method B gave the pure product as an orange foam (75.5 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 749 ([M+H]+, 100).
(b) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3a)
Method A: A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) was added to a solution of the nitro-ester 2a (75 g, 104 mmol) in DMF (360 mL). The suspension was hydrogenated in a Parr hydrogenation apparatus over 8 hours. Progress of the reaction was monitored by LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H]J, 100), (ES−) m/z (relative intensity) 595 ([M+H]+, 100) after the hydrogen uptake had stopped. Solid Pd/C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 10 mbar) at 40° C. to afford a dark oil containing traces of DMF and residual charcoal. The residue was digested in EtOH (500 mL) at 40° C. on a water bath (rotary evaporator bath) and the resulting suspension was filtered through celite and washed with ethanol (500 mL) to give a clear filtrate. Hydrazine hydrate (10 mL, 321 mmol) was added to the solution and the reaction mixture was heated at reflux. After 20 minutes the formation of a white precipitate was observed and reflux was allowed to continue for a further 30 minutes. The mixture was allowed to cool down to room temperature and the precipitate was retrieved by filtration, washed with diethyl ether (2*1 volume of precipitate) and dried in a vacuum desiccator to provide 3a (50 g, 81%).
Method B: A solution of the nitro-ester 2a (6.80 g, 9.44 mmol) in MeOH (300 mL) was added to Raney™ nickel (4 large spatula ends of a ˜50% slurry in H2O) and anti-bumping granules in a 3-neck round bottomed flask. The mixture was heated at reflux and then treated dropwise with a solution of hydrazine hydrate (5.88 mL, 6.05 g, 188 mmol) in MeOH (50 mL) at which point vigorous effervescence was observed. When the addition was complete (˜30 minutes) additional Raney™ nickel was added carefully until effervescence had ceased and the initial yellow colour of the reaction mixture was discharged. The mixture was heated at reflux for a further 30 minutes at which point the reaction was deemed complete by TLC (90:10 v/v CHCl3/MeOH) and LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H], 100)). The reaction mixture was allowed to cool to around 40° C. and then excess nickel removed by filtration through a sinter funnel without vacuum suction. The filtrate was reduced in volume by evaporation in vacuo at which point a colourless precipitate formed which was collected by filtration and dried in a vacuum desiccator to provide 3a (5.40 g, 96%).
Analytical Data: [α]27D=+404° (c=0.10, DMF); 1H NMR (400 MHz, DMSO-d6) δ 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H, J=3.98 Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H, J=12.1, 3.60 Hz), 3.43 (dd, 2H, J=12.0, 4.72 Hz), 2.67-2.57 (m, 2H), 2.26 (p, 2H, J=5.90 Hz), 1.99-1.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 164.0, 149.9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8, 54.4, 53.1, 33.5, 27.5; IR (ATR, neat) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm−1; MS (ES+) m/z (relative intensity) 619 ([M+Na]+, 10), 597 ([M+H]+, 52), 445 (12), 326 (11); HRMS [M+H]+ theoretical C29H32N4O10 m/z 597.2191, found (ES+) m/z 597.2205.
(b) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (36)
Preparation from 2b according to Method A gave the product as a white solid (22.1 g, 86%).
Analytical Data: MS (ES−) m/z (relative intensity) 623.3 ([M−H]−, 100);
(c) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4a)
TBSCl (317 mg, 2.1 mmol) and imidazole (342 mg, 5.03 mmol) were added to a cloudy solution of the tetralactam 3a (250 mg, 0.42 mmol) in anhydrous DMF (6 mL). The mixture was allowed to stir under a nitrogen atmosphere for 3 hours after which time the reaction was deemed complete as judged by LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M+H]+, 100)). The reaction mixture was poured onto ice (˜25 mL) and allowed to warm to room temperature with stirring. The resulting white precipitate was collected by vacuum filtration, washed with H2O, diethyl ether and dried in the vacuum desiccator to provide pure 4a (252 mg, 73%).
Analytical Data: [α]23D=+2340 (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H, J=5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H), 2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H), 0.08 (s, 12H); 13C NMR (100 MHz, CDCl3) δ 170.4, 165.7, 151.4, 146.6, 129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56.3, 55.7, 54.2, 35.2, 28.7, 25.7, 18.0, −4.82 and −4.86; IR (ATR, CHCl3) 3235, 2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099, 1033 cm−1; MS (ES+) n/z (relative intensity) 825 ([M+H]+, 62), 721 (14), 440 (38); HRMS [M+H]+ theoretical C41H60N4O10Si2 n/z 825.3921, found (ES+) m/z 825.3948.
(c) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4b)
Preparation from 3b according to the above method gave the product as a white solid (27.3 g, 93%).
Analytical Data: MS (ES+) m/z (relative intensity) 853.8 ([M+H]+, 100), (ES−) m/z (relative intensity) 851.6 ([M−H]−, 100.
(d) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5a)
A solution of n-BuLi (4.17 mL of a 1.6 M solution in hexane, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise to a stirred suspension of the tetralactam 4a (2.20 g, 2.67 mmol) in anhydrous THF (30 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to stir at this temperature for 1 hour (now a reddish orange colour) at which point a solution of SEMCl (1.18 mL, 1.11 g, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise. The reaction mixture was allowed to slowly warm to room temperature and was stirred for 16 hours under a nitrogen atmosphere. The reaction was deemed complete as judged by TLC (EtOAc) and LC/MS (4.77 min (ES+) m/z (relative intensity) 1085 ([M+H]+, 100)). The THF was removed by evaporation in vacuo and the resulting residue dissolved in EtOAc (60 mL), washed with H2O (20 mL), brine (20 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (80:20 v/v Hexane/EtOAc) gave the pure N10-SEM-protected tetralactam 5a as an oil (2.37 g, 82%).
Analytical Data: [α]23D=+163° (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98 Hz), 4.68 (d, 2H. J=9.99 Hz), 4.57 (p, 2H, J=5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s, 6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81 Hz), 2.03-1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, −1.24, −4.73; IR (ATR, CHCl3) 2951, 1685, 1640, 1606, 1517, 1462, 1433, 1360, 1247, 1127, 1065 cm−1; MS (ES+) m/z (relative intensity) 1113 ([M+Na]+, 48), 1085 ([M+H]+, 100), 1009 (5), 813 (6); HRMS [M+H]+ theoretical C53H88N4O12Si4 m/z 1085.5548, found (ES+) m/z 1085.5542.
(d) 1,1′-[[(Pentane1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5b)
Preparation from 4b according to the above method gave the product as a pale orange foam (46.9 g, 100%), used without further purification.
Analytical Data: MS (ES+) m/z (relative intensity) 1114 ([M+H]+, 90), (ES−) m/z (relative intensity) 1158 ([M+2Na]−, 100).
(e) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6a)
A solution of TBAF (5.24 mL of a 1.0 M solution in THF, 5.24 mmol) was added to a stirred solution of the bis-silyl ether 5a (2.58 g, 2.38 mmol) in THF (40 mL) at room temperature.
After stirring for 3.5 hours, analysis of the reaction mixture by TLC (95:5 v/v CHCl3/MeOH) revealed completion of reaction. The reaction mixture was poured into a solution of saturated NH4Cl (100 mL) and extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96:4 v/v CHCl3/MeOH) gave the pure tetralactam 6a as a white foam (1.78 g, 87%).
Analytical Data: [α]23D=+202° (c=0.34, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0 Hz), 4.72 (d, 2H, J=10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83 Hz), 4.20-4.16 (m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH), 2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77 Hz), 2.11-2.05 (m, 2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 169.5, 165.9, 151.3, 147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67.2, 65.2, 56.5, 56.2, 54.1, 35.2, 29.1, 18.4, −1.23; IR (ATR, CHCl3) 2956, 1684, 1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm−1; MS (ES+) m/z (relative intensity) 885 ([M+29]+, 70), 857 ([M+H]+, 100), 711 (8), 448 (17); HRMS [M+H]+ theoretical C41H60N4O12Si2 m/z 857.3819, found (ES+) m/z 857.3826.
(e) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6b)
Preparation from 5b according to the above method gave the product as a white foam (15.02 g).
Analytical Data: MS (ES+) m/z (relative intensity) 886 ([M+H]+, 10), 739.6 (100), (ES−) m/z (relative intensity) 884 ([M−H]−, 40).
(f) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[((11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (7a)
Method A: A 0.37 M sodium hypochlorite solution (142.5 mL, 52.71 mmol, 2.4 eq) was added dropwise to a vigorously stirred mixture of the diol 6a (18.8 g, 21.96 mmol, 1 eq), TEMPO (0.069 g, 0.44 mmol, 0.02 eq) and 0.5 M potassium bromide solution (8.9 mL, 4.4 mmol, 0.2 eq) in DCM (115 mL) at 0° C. The temperature was maintained between 0° C. and 5° C. by adjusting the rate of addition. The resultant yellow emulsion was stirred at 0° C. to 5° C. for 1 hour. TLC (EtOAc) and LC/MS [3.53 min. (ES+) m/z (relative intensity) 875 ([M+Na]+, 50), (ES−) m/z (relative intensity) 852 ([M−H]−, 100)] indicated that reaction was complete.
The reaction mixture was filtered, the organic layer separated and the aqueous layer was backwashed with DCM (×2). The combined organic portions were washed with brine (×1), dried (MgSO4) and evaporated to give a yellow foam. Purification by flash column chromatography (gradient elution 35/65 v/v n-hexane/EtOAC, 30/70 to 25/75 v/v n-hexane/EtOAC) afforded the bis-ketone 7a as a white foam (14.1 g, 75%).
Sodium hypochlorite solution, reagent grade, available at chlorine 10-13%, was used. This was assumed to be 10% (10 g NaClO in 100 g) and calculated to be 1.34 M in NaClO. A stock solution was prepared from this by diluting it to 0.37 M with water. This gave a solution of approximately pH 14. The pH was adjusted to 9.3 to 9.4 by the addition of solid NaHCO3. An aliquot of this stock was then used so as to give 2.4 mol eq. for the reaction. On addition of the bleach solution an initial increase in temperature was observed. The rate of addition was controlled, to maintain the temperature between 0° C. to 5° C. The reaction mixture formed a thick, lemon yellow coloured, emulsion.
The oxidation was an adaptation of the procedure described in Thomas Fey et al, J. Org. Chem., 2001, 66, 8154-8159.
Method B: Solid TCCA (10.6 g, 45.6 mmol) was added portionwise to a stirred solution of the alcohol 6a (18.05 g, 21.1 mmol) and TEMPO (123 mg, 0.78 mmol) in anhydrous DCM (700 mL) at 0° C. (ice/acetone). The reaction mixture was stirred at 0° C. under a nitrogen atmosphere for 15 minutes after which time TLC (EtOAc) and LC/MS [3.57 min (ES+) m/z (relative intensity) 875 ([M+Na]+, 50)] revealed completion of reaction. The reaction mixture was filtered through celite and the filtrate was washed with saturated aqueous NaHCO3 (400 mL), brine (400 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash column chromatography (80:20 v/v EtOAc/Hexane) afforded the bis-ketone 7a as a foam (11.7 g, 65%).
Method C: A solution of anhydrous DMSO (0.72 mL, 0.84 g, 10.5 mmol) in dry DCM (18 mL) was added dropwise over a period of 25 min to a stirred solution of oxalyl chloride (2.63 mL of a 2.0 M solution in DCM, 5.26 mmol) under a nitrogen atmosphere at −60° C. (liq N2/CHCl3). After stirring at −55° C. for 20 minutes, a slurry of the substrate 6a (1.5 g, 1.75 mmol) in dry DCM (36 mL) was added dropwise over a period of 30 min to the reaction mixture. After stirring for a further 50 minutes at −55° C., a solution of TEA (3.42 mL, 2.49 g; 24.6 mmol) in dry DCM (18 mL) was added dropwise over a period of 20 min to the reaction mixture. The stirred reaction mixture was allowed to warm to room temperature (˜1.5 h) and then diluted with DCM (50 mL). The organic solution was washed with 1 N HCl (2×25 mL), H2O (30 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (80:20 v/v EtOAc/Hexane) to afford bis-ketone 7a as a foam (835 mg, 56%)
Analytical Data: [α]20D=+291 (c=0.26, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d, 2H, J=10.1 Hz), 4.60 (dd, 2H, J=9.85, 3.07 Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85 Hz), 2.76 (dd, 2H, J=19.2, 9.90 Hz), 2.42 (p, 2H, J=5.77 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 206.8, 168.8, 165.9, 151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9, 52.4, 37.4, 29.0, 18.4, −1.24; IR (ATR, CHCl3) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm−1; MS (ES+) m/z (relative intensity) 881 ([M+29]+, 38), 853 ([M+H]+, 100), 707 (8), 542 (12); HRMS [M+H]+ theoretical C41H56N4O12Si2 m/z 853.3506, found (ES+) m/z 853.3502.
(f) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl) 1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (7b)
Preparation from 6b according to Method C gave the product as a white foam (10.5 g, 76%).
Analytical Data: MS (ES+) m/z (relative intensity) 882 ([M+H]+, 30), 735 (100), (ES−) m/z (relative intensity) 925 ([M+45]−, 100), 880 ([M−H]−, 70).
(g) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11 aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8a)
Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in one portion to a vigorously stirred solution of bis-ketone 7a (6.08 g, 7.1 mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile cooling bath) under a nitrogen atmosphere. Anhydrous triflic anhydride, taken from a freshly opened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injected rapidly dropwise, while maintaining the temperature at −40° C. or below. The reaction mixture was allowed to stir at −45° C. for 1 hour at which point TLC (50/50 v/v n-hexane/EtOAc) revealed the complete consumption of starting material. The cold reaction mixture was immediately diluted with DCM (200 mL) and, with vigorous shaking, washed with water (1×100 mL), 5% citric acid solution (1×200 mL) saturated NaHCO3 (200 mL), brine (100 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (gradient elution: 90:10 v/v n-hexane/EtOAc to 70:30 v/v n-hexane/EtOAc) to afford bis-enol triflate 8a as a yellow foam (5.5 g, 70%).
Analytical Data: [α]24D=+271° (c=0.18, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97 Hz), 5.51 (d, 2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.69 Hz), 4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J=16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J=5.85 Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 167.1, 162.7, 151.9, 148.0, 138.4, 133.6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56.3, 30.8, 29.0, 18.4, −1.25; IR (ATR, CHCl3) 2958, 1690, 1646, 1605, 1517, 1456, 1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm−1; MS (ES+) m/z (relative intensity) 1144 ([M+28]+, 100), 1117 ([M+H]+, 48), 1041 (40), 578 (8); HRMS [M+H]+ theoretical C43H54N4O16Si2S2F6 m/z 1117.2491, found (ES+) m/z 1117.2465.
(g) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8b)
Preparation from 7b according to the above method gave the bis-enol triflate as a pale yellow foam (6.14 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 1146 ([M+H]+, 85).
Example 1
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,1(10H,11aH)-dione (9)
Solid Pd(PPh3)4 (20.18 mg, 17.46 mmol) was added to a stirred solution of the triflate 8a (975 mg, 0.87 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralane-2-yl)aniline (172 mg, 0.79 mmol) and Na2CO3 (138 mg, 3.98 mol) in toluene (13 mL) EtOH (6.5 mL) and H2O (6.5 mL). The dark solution was allowed to stir under a nitrogen atmosphere for 24 hours, after which time analysis by TLC (EtOAc) and LC/MS revealed the formation of the desired mono-coupled product and as well as the presence of unreacted starting material. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL), after eventual separation of the layers the aqueous phase was extracted again with EtOAc (2×25 mL). The combined organic layers were washed with H2O (50 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude Suzuki product. The crude Suzuki product was subjected to flash chromatography (40% EtOAc/60% Hexane→70% EtOAc, 30% Hexane). Removal of the excess eluent by rotary evaporation under reduced pressure afforded the desired product 9 (399 mg) in 43% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J=8.5 Hz), 7.15 (t, 1H, J=2.0 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.52 (d, 2H. J=10.0 Hz), 4.77 (d, 1H, J=10.0 Hz), 4.76 (d, 1H, J=10.0 Hz), 4.62 (dd, 1H, J=3.7, 11.0 Hz), 4.58 (dd, 1H, J=3.4, 10.6 Hz), 4.29 (t, 4H, J=5.6 Hz), 4.00-3.85 (m, 8H), 3.80-3.60 (m, 4H), 3.16 (ddd, 1H, J=2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J=2.2, 10.5, 16.1 Hz), 2.43 (p, 2H, J=5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H). 13C-NMR: (CDCl3, 100 MHz) δ 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147.6, 139.6, 134.8, 134.5, 127.9 (methine), 127.5, 125.1, 123.21, 121.5, 120.5 (methine), 120.1 (methine), 116.4 (methine), 113.2 (methine), 108.7 (methine), 79.8 (methylene), 79.6 (methylene), 68.7 (methylene), 68.5 (methylene), 67.0 (methylene), 66.8 (methylene), 58.8 (methine), 58.0 (methine), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3 (methylene), 19.7 (methylene), 0.25 (methyl).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (10)
Solid Pd(PPh3)4 (10 mg, 8.69 μmol) was added to a stirred solution of the mono-triflate 9 (230 mg, 0.22 mmol) in toluene (3 mL), EtOH (10 mL), with 4-methoxyphenyl boronic acid (43 mg, 0.28 mmol), Na2CO3 (37 mg, 0.35 mmol), in H2O (1.5 mL) at room temperature. The reaction mixture was allowed to stir under a nitrogen atmosphere for 20 h, at which point the reaction was deemed complete as judged by LC/MS and TLC (EtOAc). The solvent was removed by rotary evaporation under reduced pressure in vacuo and the resulting residue partitioned between EtOAc (75 mL) and H2O (75 mL). The aqueous phase was extracted with EtOAc (3×30 mL) and the combined organic layers washed with H2O (30 mL), brine (40 mL), dried (MgSO4), filtered and evaporated to provide the crude product. The crude product was purified by flash chromatography (60% Hexane: 40% EtOAc→80% EtOAc: 20% Hexane) to provide the pure dimer as an orange foam. Removal of the excess eluent under reduced pressure afforded the desired product 10 (434 mg) in 74% yield.
1H-NMR: (CDCl3, 400 MHz) δ7.38 (s, 2H), 7.34 (d, 2H, J=8.8 Hz), 7.30 (bs, 1H), 7.26-7.24 (m, 3H), 7.22 (d, 2H, J=8.5 Hz), 6.86 (d, 2H, J=8.8 Hz), 6.63 (d, 2H, J=8.5 Hz), 5.50 (d, 2H, J=10.0 Hz), 4.75 (d, 1H, J=10.0 Hz), 4.74 (d, 1H, J=10.0 Hz), 4.56 (td, 2H, J=3.3, 10.1 Hz), 4.27 (t, 2H, J=5.7 Hz), 4.00-3.85 (m, 8H), 3.80 (s, 3H), 3.77-3.60 (m, 4H), 3.20-3.00 (m, 2H), 2.42 (p, 2H, J=5.7 Hz), 0.96 (t, 4H, J=8.3 Hz), 0.00 (s, 18H). 13C-NMR: (CDCl3, 100 MHz) δ 169.8, 169.7, 162.9, 162.7, 160.6, 152.7, 152.6, 149.0, 147.5, 134.8, 127.8 (methine), 127.4, 126.8, 125.1, 123.1, 123.0, 121.5 (methine), 120.4 (methine), 116.4 (methine), 115.5 (methine), 113.1 (methine), 108.6 (methine), 79.6 (methylene), 68.5 (methylene), 66.9 (methylene), 58.8 (methine), 57.6 (methoxy), 56.7 (methoxy), 32.8 (methylene), 30.3 (methylene), 19.7 (methylene), 0.0 (methyl).
(c) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (11)
Fresh LiBH4 (183 mg, 8.42 mmol) was added to a stirred solution of the SEM-dilactam 10 (428 mg, 0.42 mmol) in THF (5 mL) and EtOH (5 mL) at room temperature. After 10 minutes, delayed vigorous effervescence was observed requiring the reaction vessel to be placed in an ice bath. After removal of the ice bath the mixture was allowed to stir at room temperature for 1 hour. LC/MS analysis at this point revealed total consumption of starting material with very little mono-reduced product. The reaction mixture was poured onto ice (100 mL) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3×30 mL) and the combined organic layers washed with H2O (20 mL), brine (30 mL) and concentrated in vacuo. The resulting residue was treated with DCM (5 mL), EtOH (14 mL), H2O (7 mL) and silica gel (10 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered slowly through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was washed with H2O (50 mL), brine 60 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (97% CHCl3: 3% MeOH) to provide the pure C2/C2′ aryl PBD dimer 11 (185 mg) 61% yield.
1H-NMR: (CDCl3, 400 MHz) δ7.88 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.20 (d, 2H, J=8.5 Hz), 6.89 (d, 2H, J=8.8 Hz), 6.87 (s, 1H), 6.86 (s, 1H), 6.67 (d, 2H, J=8.5 Hz), 4.40-4.20 (m, 6H), 3.94 (s, 6H), 3.82 (s, 3H), 3.61-3.50 (m, 2H), 3.40-3.30 (m, 2H), 2.47-2.40 (m, 2H). 13C-NMR: (CDCl3, 100 MHz) δ 162.5 (imine methine), 161.3, 161.1, 159.3, 156.0, 151.1, 148.1, 146.2, 140.3, 126.2 (methine), 123.2, 122.0, 120.5 (methine), 119.4, 115.2 (methine), 114.3 (methine), 111.9 (methine), 111.2 (methine), 65.5 (methylene), 56.2 (methoxy), 55.4 (methoxy), 53.9 (methine), 35.6 (methylene), 28.9 (methylene).
Example 2
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1.4]benzodiazepin-8-yloxy)pentyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (12)
Solid Pd(PPh3)4 (32 mg, 27.7 μmol) was added to a stirred solution of the bis-triflate 8b (1.04 g, 0.91 mmol) in toluene (10 mL), EtOH (5 mL), with 4-methoxyphenyl boronic acid (0.202 g, 1.32 mmol), Na2CO3 (0.169 g, 1.6 mmol), in H2O (5 mL) at 30° C. The reaction mixture was allowed to stir under a nitrogen atmosphere for 20 hours. Additional solid 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (0.203 g, 0.93 mmol) and Na2CO3 (0.056 g, 0.53 mmol) were added followed by solid Pd(PPh3)4 (10 mg, 8.6 μmol). The reaction mixture was allowed to stir under a nitrogen atmosphere for a further 20 hours. LC/MS indicated the formation of desired product. EtOAc (100 mL) and H2O (100 mL) were added, the aqueous was separated and extracted with EtOAc (3×30 mL). The combined organic layers were washed with H2O (100 mL), brine (100 mL), dried (MgSO4), filtered and evaporated to provide a dark brown oil. The oil was dissolved in DCM and loaded onto a 10 g SCX-2 cartridge pre-equilibrated with DCM (1 vol). The cartridge was washed with DCM (3 vol), MeOH (3 vol) and the crude product eluted with 2M NH3 in MeOH (2 vol). Flash chromatography (50% n-hexane: 50% EtOAc—20% n-hexane: 80% EtOAc) provided the pure dimer 12 as a yellow foam (0.16 g, 34%).
Analytical Data: [α]23D=+3880 (C=0.22, CHCl3); 1H-NMR: (CDCl3, 400 MHz) δ 7.39 (s, 2H), 7.35 (d, 2H, J=12.8 Hz), 7.32 (bs, 1H), 7.26-7.23 (m, 5H), 6.89 (d, 2H, J=8.8 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.55 (d, 2H, J=10.0 Hz), 4.73 (d, 1H, J=10.0 Hz), 4.72 (d, 1H, J=10.0 Hz), 4.62 (td, 2H, J=3.2, 10.4 Hz), 4.15-4.05 (m, 4H), 4.00-3.85 (m, 8H), 3.82 (s, 3H), 3.77-3.63 (m, 4H), 3.20-3.05 (m, 2H), 2.05-1.95 (m, 4H), 1.75-1.67 (m, 2H) 1.01-0.95 (m, 4H), 0.03 (s, 18H); MS (ES+) m/z (relative intensity) 1047 ([M+H]+, 45).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (13)
Fresh LiBH4 (66 mg, 3.04 mmol) was added to a stirred solution of the SEM-dilactam 12 (428 mg, 0.42 mmol) in THF (3 mL) and EtOH (3 mL) at 0° C. (ice bath). The ice bath was removed and the reaction mixture was allowed to reach room temperature (vigorous effervescence). After 2 hours LC/MS analysis indicated the complete consumption of starting material. The reaction mixture was poured onto ice (50 mL) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3×50 mL) and the combined organic layers washed with H2O (50 mL), brine (50 mL), dried (MgSO4) and concentrated in vacuo. The resulting residue was treated with DCM (2 mL), EtOH (5 mL), H2O (2.5 mL) and silica gel (3.7 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was dried (MgSO4) filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (99.5% CHCl3: 0.5% MeOH to 97.5% CHCl3: 2.5% MeOH in 0.5% increments)) to provide the pure C2/C2′ aryl PBD dimer 13 (59 mg, 52%).
Analytical Data: [α]28D=+760° (c=0.14, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.89 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.22 (d, 2H, J=8.4 Hz), 6.91 (d, 2H, J=8.8 Hz), 6.815 (s, 1H), 6.81 (s, 1H), 6.68 (d, 2H, J=8.4 Hz), 4.45-4.35 (m, 2H), 4.2-4.0 (m, 4H), 3.94 (s, 6H), 3.85-3.7 (s, 3H), 3.65-3.50 (m, 2H), 3.45-3.3 (m, 2H), 2.05-1.9 (m, 4H), 1.75-1.65 (m, 2H); MS (ES+) (relative intensity) 754.6 ([M+H]+, 100), (ES+) (relative intensity) 752.5 ([M−H]−, 100).
Example 3
(a)(S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (14)
Solid Pd(PPh3)4 (41 mg, 0.036 mmol) was added to a stirred solution of the bis-triflate 8a (1 g, 0.9 mmol) in toluene (10 mL), EtOH (5 mL), with thien-2-yl boronic acid (149 mg, 1.16 mmol), Na2CO3 (152 mg, 1.43 mmol), in H2O (5 mL). The reaction mixture was allowed to stir under a nitrogen atmosphere overnight at room temperature. The solvent was removed by evaporation in vacuo and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL). The aqueous layer was extracted with EtOAc (2×30 mL) and the combined organic layers washed with H2O (50 mL), brine (50 mL) dried (MgSO4), filtered and evaporated in vacuo to provide the crude product which was purified by flash chromatography (80 hexane: 20 EtOAc→50 hexane: 50 EtOAc) to provide the dimer 14 (188 mg, 20%) yield
Analytical data: LC-MS RT 4.27 mins, 1051 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.36 (s, 1H), 7.31 (bs, 1H), 7.27 (bs, 1H), 7.26-7.23 (m, 2H), 7.22-7.17 (m, 1H), 7.12 (bs, 1H), 7.02-6.96 (m, 2H), 5.50 (d, J=10.0 Hz, 2H), 7.75 (d, J=10.0 Hz, 2H), 4.65-4.55 (m, 2H), 4.37-4.13 (m, 4H), 4.00-3.85 (m, 8H), 3.8-3.6 (m, 4H), 3.20-3.10 (m, 2H), 2.50-2.35 (m, 2H), 1.0-0.9 (m, 4H), 0 (s, 18H).
(b) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (15)
Solid Pd(PPh3)4 (7.66 mg, 6.63 μmol) was added to a stirred, cloudy solution of 14 (174 mg, 0.17 mmol), Na2CO3 (28 mg, 0.22 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (47 mg, 0.22 mmol) in toluene (2-5 mL), EtOH (1.25 mL) and H2O (125 mL) at room temperature. The reaction mixture was allowed to stir under a N2 atmosphere for 24 hours at which point the reaction was deemed complete by LC/MS major peak (@ 3.97 min, FW=1016, M+Na) and TLC (EtOAc). The solvent was removed by evaporation in vacuo and the resulting residue partitioned between EtOAc (60 mL) and H2O (30 mL). The layers were separated and the organic phase was washed with H2O) (20 mL), brine (30 mL) dried (MgSO4) filtered and evaporated in vacuo to provide the crude product 123 mg, 75% yield.
Analytical data: LC-MS RT 3.98 mins, 100% area, 994 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.40 (d, J=5.3 Hz, 2H), 7.30 (t, J=1.70 Hz, 1H), 7.29-7.27 (m, 3H), 7.25 (d, J=8.5 Hz, 2H), 7.21 (dd, J=1.4, 4.73 Hz, 1H), 7.03-6.97 (m, 2H), 6.66 (d, J=8.5 Hz, 2H), 5.52 (d, J=10.0 Hz, 2H), 4.78 (d, J=10.0 Hz, 1H), 4.77 (d, J=10.0 Hz, 1H), 4.62 (dd, J=3.4, 10.5 Hz, 1H), 4.59 (dd, J=3.40, 10.6 Hz, 1H), 4.30 (t, J=5.85 Hz, 4H), 3.85-4.03 (m, 8H), 3.84-3.64 (m, 6H), 3.18 (ddd, J=2.2, 10.5, 16.0 Hz, 1H), 3.11 (ddd, J=2.2, 10.5, 16.0 Hz, 1H), 2.44 (p, J=5.85 Hz, 2H), 0.98 (t, J=1.5 Hz, 4H), 0 (s, 18H).
(c) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-aminophenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (16)
Fresh LiBH4 (47 mg, 2.22 mmol) was added to a stirred solution of the SEM-dilactam 15 (110 mg, 0.11 mmol) in dry THF (3 mL) and EtOH (3 mL) at 0° C. (ice bath). The ice bath was removed and the reaction mixture stirred under a N2 atmosphere for 1 hour. Analysis of the reaction by LC/MS analysis revealed significant formation of the desired product (Pk @ 2.57 min) (1=69.32), FW=702, M+H) and half-imine. The reaction mixture was allowed to stir for a further 1 hour after which time no further reaction progress was observed by LC/MS. The reaction mixture was poured onto ice, stirred and allowed to warm to room temperature. Following partition between DCM (50 mL) and water (50 mL), the aqueous phase was extracted with DCM (3×20 mL). The combined organic layers were washed with H2O (50 mL), brine (50 mL) and the solvent removed by evaporation in vacuo under reduced pressure.
The resulting residue was dissolved in DCM (5 mL), EtOH (15 mL) and H2O (7 mL) then treated with silica gel (5 g). The reaction was allowed to stir at room temperature for 48 h. The silica was removed by filtration through a sinter funnel and the residue rinsed with 90:10 CHCl3: MeOH (100 mL). H2O (50 mL) was added to the filtrate and the layers were separated (after shaking). The aqueous layer was extracted with CHCl3 (2×30 mL) and H2O (50 mL), brine (50 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude product. Flash chromatography (CHCl3→98% CHCl3: 2% MeOH) afforded the product (41 mg, 53%).
Analytical data: LC-MS RT 2.55 mins, 702 (M+H)
Example 4
(a) (S)-2-(4-methoxyphenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulphonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (17)
Solid 4-methoxybenzeneboronic acid (0.388 g, 2.55 mmol) was added to a solution of the SEM protected bis triflate (8a)(3.0 g, 2.69 mmol), sodium carbonate (426 mg, 4.02 mmol) and palladium tetrakis triphenylphosphine (0.08 mmol) in toluene (54.8 mL), ethanol (27 mL) and water (27 mL). The reaction mixture was allowed to stir at room temperature for 3 hours. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 30/70→35/65→40/60→45/55) to remove unreacted bis-triflate (0.6 g). Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (1.27 g, 1.18 mmol, 41%).
LC-MS RT 4.30 mins, 1076 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.41 (s, 1H), 7.39 (d, J=8.8 Hz, 2H), 7.35 (s, 1H), 7.34 (bs, 1H), 7.29 (s, 1H), 7.16 (t, J=1.9 Hz, 1H), 6.90 (d, J=8.8 Hz, 2H), 5.53 (d, J=10.0 Hz, 2H), 4.79 (d, J=10.0 Hz, 1H), 4.78 (d, J=10.0 Hz, 1H), 4.66-4.60 (m, 2H), 4.30 (t, J=5.7 Hz, 4H), 4.0-3.94 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.84 (s, 3H), 3.83-3.60 (m, 4H), 3.22-3.10 (m, 2H), 2.45 (t, J=5.9 Hz, 2H), 1.05-0.94 (m, 4H), 0 (s, 18H).
(b) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1.4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (18)
Solid 3-aminobenzeneboronic acid (0.143 g, 0.92 mmol) was added to a solution of the mono triflate (17)(0.619 g, 0.58 mmol), sodium carbonate (195 mg, 1.84 mmol) and palladium tetrakis triphenylphosphine (26.6 mg, 0.023 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir at room temperature for overnight at 30° C. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 70/30→85/15). Removal of excess eluent from selected fractions afforded the desired product (0.502 g, 0.49 mmol, 85%).
LC-MS RT 4.02 mins, 1019 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.38-7.35 (m, 4H), 7.33 (bs, 1H), 7.30 (bs, 1H), 7.25 (s, 2H), 7.10 (t, J=7.8 Hz, 1H), 6.88-6.80 (m, 3H), 6.72 (bs, 1H), 6.57 (dd, J=7.9, 1.8 Hz, 1H), 5.50 (d, J=10.0 Hz, 2H), 4.75 (d, 10.0 Hz, 2H), 4.58 (dd, J=10.6, 3.3 Hz, 2H), 4.27 (t, J=5.8 Hz, 4H), 3.95-3.91 (m, 2H), 3.90 (s, 6H), 3.80 (s, 3H), 3.77-3.60 (m, 6H), 3.15-3.05 (m, 2H), 2.41 (p, J=5.8 Hz, 2H), 0.95 (t, =8.25 Hz, 4H), 0 (s, 18H).
(c) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (19)
A solution of superhydride (0.56 mL, 0.56 mmol, 1.0 M in THF) was added dropwise to a solution of the SEM dilactam (18)(0.271 g, 0.27 mmol) in dry THF (10 mL) at −78° C. under a nitrogen atmosphere. After 1 hr a further aliquot of superhydride solution (0.13 ml, 0.13 mmol) was added and the reaction mixture was allowed to stir for another 0.5 hr, at which time LC-MS indicated that reduction was complete. The reaction mixture was diluted with water and allowed to warm to room temperature. The reaction mixture was partitioned between chloroform and water, the layers were separated and the aqueous layer extracted with additional chloroform (emulsions). Finally the combined organic phase was washed with brine and dried over magnesium sulphate. The reduced product was dissolved in methanol, chloroform and water and allowed to stir in the presence of silica gel for 72 hours. The crude product was subjected to flash column chromatography (methanol/chloroform gradient) to afford the desired imine product (150 mg, 0.21 mmol, 77%) after removal of excess eluent from selected fractions.
LC-MS RT 2.63 mins, 97% area, 726 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.85 (d, J=3.9 Hz, 1H), 7.84 (d, J=3.9 Hz, 1H), 7.50 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 7.32 (d, J=7.3 Hz, 2H), 7.11 (t, (d, J=7.8 Hz, 1H), 6.90-6.80 (m, 4H), 6.77 (d, J=7.9 Hz, 1H), 4.40-4.20 (m, 6H), 3.92 (s, 6H), 3.80 (s, 3H), 3.60-3.27 (m, 6H), 2.48-2.29 (m, 2H)
Example 5
(a) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo [2,1-c][1,4] benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-2-(trifluoromethylsulfonyloxy)-11,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 21
Solid 4-methoxybenzeneboronic acid (59 mg, 0.39 mmol) was added to a solution of the Troc protected bis triflate (Compound 44, WO 2006/111759) (600 mg, 0.41 mmol), sodium carbonate (65 mg, 0.61 mmoml) and palladium tetrakis triphenylphosphine (0.012 mmol) in toluene (10.8 mL), ethanol (5.4 mL) and water (5.4 mL). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was then partitioned between ethylacetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 20/80→30/70→40/60→60/40) to remove unreacted bis-triflate. Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (261 mg, 0.18 mmol, 46%).
LC-MS RT 4.17 mins, 1427 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.38 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.25 (s, 1H), 7.20 (bs, 1H), 6.92 (d, J=8.6 Hz, 2H), 6.77 (d, J=8.7 Hz, 2H), 6.0-5.90 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.18-4.08 (m, 2H), 4.07-3.89 (m, 10H), 3.81 (s, 3H), 3.44-3.25 (m, 2H), 2.85 (d, J=16.6 Hz, 2H), 2.05-1.90 (m, 4H), 1.76-1.64 (m, 2H), 0.93 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.26 (s, 6H).
(b) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo [2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 22
The Suzuki coupling procedure described in step (a) was applied to the synthesis of Compound 21. Compound 20 (62.5 mg 0.044 mmol,) was treated with 1 equivalent of 4-hydroxybenzeneboronic acid (10 mg) at 30° C. overnight to afford the desired compound after filtration through a pad of silica gel. (40 mg, 0.029 mmol, 66% yield). The compound was used directly in the subsequent step LC-MS RT 4.27 mins, 1371 (M+H)
(c) (S)-2-(4-hydroxyphenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 23
Cadmium/lead couple (100 mg, Q Dong et al. Tetrahedron Letters vol 36, issue 32, 5681-5682, 1995) was added to a solution of 21 (40 mg, 0.029 mmol) in THF (1 mL) and ammonium acetate (1N, 1 mL) and the reaction mixture was allowed to stir for 1 hour. The reaction mixture was partitioned between chloroform and water, the phases separated and the aqueous phase extracted with chloroform. The combined organic layers were washed with brine and dried over magnesium sulphate. Rotary evaporation under reduced pressure yielded the crude product which was subjected to column chromatography (silica gel, 0→4% MeOH/CHCl3). Removal of excess eluent by rotary evaporation under reduced pressure afforded the desired imine product (17 mg 0.023 mmol 79%).
LC-MS RT 2.20 mins, 755 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.89 (d, J=3.94 Hz, 1H), 7.89 (d, J=4.00 Hz, 1H), 7.53 (s, 1H), 7.52 (s, 1H), 7.38 (d, J=8.7 Hz, 2H), 7.33 (d, J=8.6 Hz, 2H), 7.28 (s, 1H), 6.90 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 6.82 (s, 1H), 6.81 (s, 1H), 5.68 (bs, 1H), 4.50-4.30 (m, 2H), 4.22-4.00 (m, 4H), 3.93 (s, 6H), 3.82 (s, 3H), 3.69-3.45 (m, 2H), 3.44-3.28 (m, 2H), 2.64-1.88 (m, 4H), 1.77-1.62 (m, 2H).
Example 6
(a) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-formylphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4] benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 24
The Suzuki coupling procedure described in Example 5, step (a), was applied to the synthesis of Compound 24. Compound 21 (62.5 mg, 0.044 mmol) was treated with 1 equivalent of 4-formylbenzeneboronic acid (10.5 mg) at room temperature overnight to afford the desired compound after filtration through a pad of silica gel (45 mg, 0.033 mmol, 75% yield). The compound was used directly in the subsequent step.
LC-MS RT 4.42 mins, 1383 (M+H)
(b) 4-((S)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl)benzaldehyde 25
Compound 24 was deprotected by the method described in Example 5, step (c), to yield the desired compound (18 mg, 0.023 mmol, 79%).
LC-MS RT 3.18 mins, 768 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 9.98 (s, 1H), 7.91 (d, J=3.90 Hz, 1H), 7.90-7.80 (m, 3H), 7.68 (s, 1H), 7.60-7.45 (m, 4H), 7.39 (s, 1H), 7.33 (d, J=8.7 Hz, 1H), 6.90 (d, J=8.7 Hz, 2H), 6.83 (s, 1H), 6.82 (s, 1H), 4.55-4.44 (m, 1H), 4.43-4.36 (m, 1H), 4.23-4.00 (m, 4H), 3.95 (s, 3H), 3.94 (s, 3H), 3.82 (s, 3H), 3.66-3.51 (m, 2H), 3.50-3.34 (m, 2H), 2.05-1.87 (m, 4H), 1.76-164 (m, 2H).
Example 7
(a) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsiloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-2-(trifluoromethylsulphonyloxy)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 26
The Suzuki coupling procedure described in Example 5, step (a), was applied to the synthesis of Compound 26, using 3-aminobenzeneboronic acid to afford the desired compound in 41% yield (230 mg, 0.163 mmol)
LC-MS RT 4.28 mins, 1411 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.44 (bs, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 7.20 (s, 1H), 7.16 (t, J=7.9 Hz, 1H), 6.84-6.73 (m, 3H), 6.70 (bs, 1H), 6.62 (dd, J=7.9, 1.7 Hz, 1H), 6.66-6.58 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.17-4.07 (m, 2H), 4.08-3.89 (m, 10H), 3.43-3.28 (m, 2H), 2.85 (d, J=1.65 Hz, 2H), 2.07-1.90 (m, 4H), 1.78-1.63 (m, 2H), 0.94 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.27 (s, 6H).
(b) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 27
Solid 4-[3-(dimethylamino)propoxybenzeneboronic acid pinacol ester (25 mg, 0.082 mmol) was added to a solution of 26 (73 mg, 0.052 mmol), sodium carbonate (18 mg, 0.17 mmol) and palladium tetrakis triphenylphosphine (3 mg) in toluene (1 mL), ethanol (0.5 mL) and water (0.5 mL). The reaction mixture was allowed to stir at room temperature over night. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was eluted through a plug of silica gel with chloroform/methanol. Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (50 mg, 0.035 mmol, 67%).
LC-MS RT 4.12 mins, 1440 (M+H)
(c) (S)-2-(3-aminophenyl)-8-(5-((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 28
Compound 27 was deprotected by the method described in Example 5, step (c), to yield the desired compound. The reaction mixture was partitioned between DCM and aqueous sodium hydrogen carbonate (emulsion) and the crude product purified by gradient column chromatography on silica gel (5% methanol chloroform→35% methanol/chloroform) to afford the desired unsymmetrical PBD imine (50 mg, 0.018 mmol, 58%) LC-MS RT 2.55 mins, 826 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.92-7.82 (m, 2H), 7.52 (bs, 2H), 7.45 (bs, 1H), 7.39 (bs, 1H), 7.31 (d, J=8.6 Hz, 2H), 7.14 (t, J=7.8 Hz, 1H), 6.89 (d, J=8.6 Hz, 2H), 6.85-6.75 (m, 3H), 6.72 (bs, 1H), 6.60 (d, J=8.0 Hz, 1H), 4.46-4.33 (m, 2H), 4.21-3.98 (m, 6H), 3.94 (s, 6H), 3.63-3.50 (m, 2H), 3.43-3.29 (m, 2H), 2.64-2.48 (m, 2H), 2.34 (s, 6H), 2.10-1.89 (m, 6H), 1.57 (m, 2H).
Example 8
(a) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 29
The method of Example 7, step (b), was performed to afford the desired product (58 mg, 0.0.040 mmol, 78%) after filtration through a plug of silica gel (with 1/3 methanol/chloroform) and removal of excess solvent by rotary evaporation under reduced pressure.
LC-MS RT 4.08 mins, 1439 (M+H)
(b) (S)-2-(3-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 30
The method for Example 7, step (c) was used to deprotect compound 29. The crude product was purified by silica gel gradient chromatography (2% methanol chloroform-35% methanol/chloroform) to afford the desired unsymmetrical PBD imine (18 mg, 0.022 mmol, 59%)
LC-MS RT 2.52 mins, 823 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.80 (d, J=3.8 Hz, 2H), 7.45 (s, 2H), 7.38 (s, 1H), 7.30 (s, 1H), 7.23 (d, J=8.6 Hz, 2H), 7.07 (t, J=7.8 Hz, 1H), 6.83 (d, J=8.6 Hz, 2H), 6.79-6.89 (m, 3H), 6.65 (s, 1H), 6.54 (d, J=7.9 Hz, 1H), 4.40-4.24 (m, 2H), 4.15-3.93 (m, 4H), 3.87 (s, 6H), 3.56-3.42 (m, 2H), 3.37-3.23 (m, 2H), 3.22-3.08 (m, 4H), 2.61-2.41 (m, 4H), 2.29 (s, 3H), 1.98-1.80 (m, 4H), 1.67-1.54 (m, 2H).
Example 9
(a) (S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione 31
Solid 4-aminomethylbenzeneboronic acid hydrochloride (0.111 g, 0.59 mmol) was added to a solution of 17 (0.394 g, 0.37 mmol), sodium carbonate (175 mg, 1.654 mmol) and palladium tetrakis triphenylphosphine (28.0 mg, 0.024 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir overnight at 30° C. The following day the reaction mixture was heated for a further 3 hours at 70° C. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 2/98→15/85). Removal of excess eluent from selected fractions afforded the desired product (0.230 mg, 0.22 mmol, 61%).
LC-MS RT 3.63 mins, 1034 (M+2H); 1H-NMR (400 MHz, DMSO d6) δ 11.7 (s, 2H), 7.52 (d, J=8.2 Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 7.40 (s, 1H), 7.50 (d, J=8.1 Hz, 2H), 7.38-7.19 (m, 5H) 6.93 (d, J=8.7 Hz, 2H), 5.40 (d, J=2.13 Hz, 1H), 5.38 (d, J=2.12 Hz, 1H), 5.32 (d, J=10.6 Hz, 2H), 5.25 (d, J=10.6 Hz, 2H), 4.87-4.72 (m, 2H), 4.35-4.15 (m, 4H), 3.85 (s, 6H), 3.79 (s, 3H), 3.73-3.56 (m, 2H), 3.55-3.39 (m, 4H), 3.22-3.02 (m, 2H), 2.39-2.23 (m, 2H), 0.94-0.67 (m, 4H), −0.06 (s, 18H).
(b) (S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 32
Compound 31 was deprotected following the method of Example 1, step (c). The crude product was purified by gradient column chromatography (5/95→30/70 MeOH/CHCl3) to afford the product as a mixture of imine and carbinolamine methyl ethers.
LC-MS RT 2.58 mins, 740 (M+H).
Example 10
(S)-2-(4-aminophenyl)-7-methoxy-11(S)-sulpho-8-(3-((S)-7-methoxy-11(S)-sulpho-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one disodium salt 33
Sodium bisulphite (8.5 mg, 3.1 eq) was added to a stirred suspension of bis-imine 11 (20 mg, 0.036 mmol) in isopropanol (4 mL) and water (2 mL). The reaction mixture was allowed to stir vigorously and eventually became clear (c, 1 hour). The reaction mixture was transferred to a funnel and filtered through a cotton wall (and then washed with 2 mL water). The filtrate was flash frozen (liquid and to bath) and lyophilized to afford the desired product 33 in quantitative yield.
LC-MS RT 11.77 mins, 727.2 (M+H) (Mass of parent compound, bisulphite adducts unstable in mass spectrometer); 1H-NMR (400 MHz, CDCl3) δ 7.66-7.55 (m, 5H), 7.43 (s, 1H), 7.39 (d, J=8.66 Hz, 2H), 7.06 (m, 2H), 6.93 (d, J=8.84 Hz, 2H), 6.54 (m, 2H), 5.29-5.21 (m, 2H), 4.32-4.28 (m, 2H), 4.14-4.20 (m, 4H), 3.96-3.83 (m, 2H), 3.77 (s, 3H), 3.73 (m, 6H), 3.52-3.43 (m, 2H), 3.30-3.08 (m, 2H), 2.24-2.21 (m, 2H).
Example 11
(a) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (103)
A catalytic amount of tetrakistriphenylphosphinepalladium (0) (11.2 mg) was added to a mixture of the mono triflate 17 (380 mg), the pinnacol ester of 2-aminophenylboronic acid (124 mg) and sodium carbonate (120 mg) in ethanol (5 mL), toluene (5 mL) and water (5 mL). The reaction mixture was allowed to stir over night at room temperature and at 40° C. until the reaction was complete (c, 2 hr). The reaction mixture was diluted with ethyl acetate and the organic layer was washed with water and brine. The ethyl acetate solution was dried over magnesium sulphate and filtered under vacuum. Removal of ethyl acetate by rotary evaporation under reduced pressure afforded the crude product which was subjected to flash chromatography (silica gel, ethyl acetate/hexane). Pure fractions were collected and combined. Removal of excess eluent by rotary evaporation under reduced pressure afforded the pure product 103 (330 mg, 86% yield). LC/MS RT: 4.17 min, ES+1018.48.
(b) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one (104)
A solution of Superhydride in dry tetrahydrofuran (1.0 M, 4.4 eq.) was added to a solution of the 2-analino compound 103 (300 mg) in dry tetrahydrofuran (5 mL) at −78° C. under an inert atmosphere. As reduction was proceeding slowly an aliquot of lithium borohydride (20 eq.) was added and the reaction mixture was allowed to return to room temperature. Water/ice was added to the reaction mixture to quench unreacted hydrides and the reaction was diluted with dichloromethane. The organic layer was washed sequentially with water (twice), citric acid and brine. Excess dichloromethane was removed by rotary evaporation under reduced pressure and the residue was redissolve in ethanol and water and treated with silica gel for 96 hours. The reaction mixture was vacuum filtered and the filtrate evaporated to dryness. The residue was subjected to flash column chromatography (silica gel, gradient chloroform/methanol). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under educed pressure to afford the pure product 104 (30 mg, 14% yield). LC/MS RT: 2.90 min, ES+726.09.
Example 12: Determination of In Vitro Cytotoxicity of Representative PBD Compounds
K562 Assay
K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37° C. in a humidified atmosphere containing 5% CO2 and were incubated with a specified dose of drug for 1 hour or 96 hours at 37° C. in the dark. The incubation was terminated by centrifugation (5 min, 300 g) and the cells were washed once with drug-free medium. Following the appropriate drug treatment, the cells were transferred to 96-well microtiter plates (104 cells per well, 8 wells per sample). Plates were then kept in the dark at 37° C. in a humidified atmosphere containing 5% CO2. The assay is based on the ability of viable cells to reduce a yellow soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Aldrich-Sigma), to an insoluble purple formazan precipitate. Following incubation of the plates for 4 days (to allow control cells to increase in number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well and the plates further incubated for 5 h. The plates were then centrifuged for 5 min at 300 g and the bulk of the medium pipetted from the cell pellet leaving 10-20 μL per well. DMSO (200 μL) was added to each well and the samples agitated to ensure complete mixing. The optical density was then read at a wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and a dose-response curve was constructed. For each curve, an IC50 value was read as the dose required to reduce the final optical density to 50% of the control value.
Compound 13 has an IC50 of 30 pM in this assay.
A2780 Assay
The A2780 parental cell line was grown in Dulbecco's Modified Eagles' Media (DMEM) containing ˜10% Foetal Calf Serum (FCS) and ˜1% 200 mM L-Glutamine solution and grown in Corning Cellbind 75 cm2 flasks.
A 190 μl cell suspension was added (at 1×104) to each well of columns 2 to 11 of a 96 well plate (Nunc 96F flat bottom TC plate), 190 μl of media was added to each well of columns 1 and 12. The media was Dulbecco's Modified Eagles' Media (DMEM) (which included ˜10% Foetal Calf Serum (FCS) and ˜1% 200 mM L-Glutamine solution).
Plates were incubated overnight at 37° C. before addition of drug if cells were adherent, 200 μM of the test compound solutions (in 100% DMSO) were serially diluted across a 96 well plate. Each resulting point was then further diluted 1/10 into sterile distilled water (SDW).
To the cell negative blanks and compound negative control wells, 10% DMSO was added at 5% v/v. Assay plates were incubated for the following durations at 37° C. in 5% CO2 in a humidified incubator for 72 hours. Following incubation, MTT solution to a final concentration of 1.5 μM was added to each well. The plates were then incubated for a further 4 hours at 37° C. in 5% CO2 in a humidified incubator. The media was then removed, and the dye was solubilised in 200 μl DMSO (99.99%).
Plates were read at 540 nm absorbance using an Envision plate reader. Data was analysed using Microsoft Excel and GraphPad Prism and IC50 values obtained.
Compound 11 has an IC50 of 11.7 μM in this assay.
Renal Cell and AML Cell Lines Assays
The cytotoxicity of various free drug compounds was tested on a renal cell cancer cell line, 786-O, a Hodgkin lymphoma cell line, L428 and two AML cell lines, HL60 and HEL.
For a 96-hour assay, cells cultured in log-phase growth were seeded for 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of test article (i.e., free drug) in cell culture media were prepared at 4× working concentration; 50 μL of each dilution was added to the 96-well plates. Following addition of test article, the cells were incubated with test articles for 4 days at 37° C. Resazurin was then added to each well to achieve a 50 μM final concentration, and the plates were incubated for an additional 4 h at 37° C. The plates were then read for the extent of dye reduction on a Fusion HT plate reader (Packard Instruments, Meridien, Conn., USA) with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
Referring to the following Table 1, the para-aniline compound 11 showed markedly increased activity on these cell lines as compared to the meta-aniline compound 19 in this assay.
TABLE 1
IC50 Summary for Free Drugs [nM]
Free Drug
L428
786-O
HL60
HEL
Compound 11
<0.00001
<0.00001
<0.00001
<0.00001
Compound 19
1
0.5
0.6
0.2
Referring to the following Table 2, the activity of compounds 28, 30 and 32 is shown on L428, 786-O, HEL, HL-60 and MCF-7 cells, as well as the activity for compound 19 on MCF-7 cells.
TABLE 2
IC50 Summary for Free Drugs [nM]
Free
Drug
L428
786-O
HEL
HL-60
MCF-7
Com-
<0.00001
<0.00001
<0.00001
<0.00001
<0.00001
pound
28
Com-
<0.00001
<0.00001
<0.00001
<0.00001
0.01
pound
30
Com-
<0.00001
<0.00001
<0.00001
<0.00001
1.0
pound
32
Com-
5
pound
19
Referring to the following Table 3, the activities of compounds 23, 25, are compared to that of compound 11 on 786-O, Caki-1, MCF-7, HL-60, THP-1, HEL, and TF1 cells. Cells were plated in 150 μL growth media per well into black-sided clear-bottom 96-well plates (Costar, Corning) and allowed to settle for 1 hour in the biological cabinet before placing in the incubator at 37° C., 5% CO2. The following day, 4× concentration of drug stocks were prepared, and then titrated as 10-fold serial dilutions producing 8-point dose curves and added at 50 μl per well in duplicate. Cells were then incubated for 48 hours at 37° C., 5% CO2. Cytotoxicity was measure by incubating with 100 μL Cell Titer Glo (Promega) solution for 1 hour, and then luminescence was measured on a Fusion HT plate reader (Perkin Elmer). Data was processed with Excel (Microsoft) and GraphPad (Prism) to produce dose response curves and IC50 values were generated and data collected.
TABLE 3
IC50 Summary for Free Drugs [nM]
Free Drug
786-O
Caki-1
MCF-7
HL-60
THP-1
HEL
TF1a
Compound
0.4
0.2
1
0.01
1
0.03
1
11
Compound
0.06
0.02
0.7
0.005
0.4
0.009
0.2
23
Compound
0.09
0.06
0.8
0.01
0.9
0.02
0.9
25
In Examples 13 to 16, the following compounds are referred to by the compound numbers as show below:
Compound
Alternative Designation
11
37
13
57
19
42
25
95
28
50
30
49
104
66
Example 13: Synthesis of PBD Drug Linker Compounds
General Information. In the following examples, all commercially available anhydrous solvents were used without further purification. Analytical thin layer chromatography was performed on silica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, N.J.). Radial chromatography was performed on Chromatotron apparatus (Harris Research, Palo Alto, Calif.). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Samples were eluted over a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reverse-phase column. The acidic mobile phase consisted of acetonitrile and water both containing either 0.05% trifluoroacetic acid or 0.1% formic acid (denoted for each compound). Compounds were eluted with a linear gradient of acidic acetonitrile from 5% at 1 min post injection, to 95% at 11 min, followed by isocratic 95% acetonitrile to 15 min (flow rate=1.0 mL/min). LC-MS was performed on a ZMD Micromass mass spectrometer interfaced to an HP Agilent 1100 HPLC instrument equipped with a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reverse phase column. The acidic eluent consisted of a linear gradient of acetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min, followed by isocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min). Preparative HPLC was carried out on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Products were purified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm, 80 Å reverse phase column eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The purification method consisted of the following gradient of solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min with monitoring at 254 nm. NMR spectral data were collected on a Varian Mercury 400 MHz spectrometer. Coupling constants (J) are reported in hertz.
(S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (36)
To a solution of Val-Ala dipeptide 34 (200 mg, 1.06 mmol) dissolved in 10.6 mL anhydrous DMF was added maleimidocaproyl NHS ester 35 (327 mg, 1.06 mmol). Diisopropylethyamine (0.92 mL, 5.3 mmol) was then added and the reaction was stirred under nitrogen at an ambient temperature for 18 h, at which time TLC and analytical HPLC revealed consumption of the starting material. The reaction was diluted with 0.1 M HCl (100 mL), and the aqueous layer was extracted with ethyl acetate (100 mL, 3×). The combined organic layer was washed with water and brine, then dried over sodium sulfate, filtered and concentrated. The crude product was dissolved in minimal methylene chloride and purified by radial chromatography on a 2 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (95:5 to 90:10 CH2Cl2/MeOH) to provide 36 (158 mg, 39%) as an oily residue. TLC: Rt=0.26, 10% MeOH in CH2Cl2. 1H NMR (CDCl3) δ (ppm) 0.95 (d, J=17 Hz, 3H), 0.98 (d, J=17 Hz, 3H), 1.30 (m, 2H), 1.40 (d, J=17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1H), 2.25 (dt, J=4, 19 Hz, 2H), 3.35 (s, 1H), 3.49 (t, J=17 Hz, 2H), 4.20 (d, J=18 Hz, 1H), 4.38 (m, 1H), 6.80 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.05 min. LC-MS: tR 11.17 min, m/z (ES+) found 381.9 (M+H)+, m/z (ES−) found 379.9 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (38)
A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.5 μmol), EEDQ (2.8 mg, 11.4 μmol), and 0.33 mL anhydrous CH2Cl2. Methanol (four drops, ˜80 μL) was added to facilitate dissolution and the mixture was stirred under nitrogen for 1 h. PBD dimer 37 (5.7 mg, 7.9 μmol) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide the drug linker 38 (3.9 mg, 45%). TLC: Rf=0.06, 5% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.51 min. LC-MS: tR 12.73 min, m/z (ES+) found 1089.6 (M+H)+, m/z (ES−) found 1087.3 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-((S)-7-methoxy-8-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (40)
To a flame-dried 10 mL flask was added PBD dimer 37 (25 mg, 34.4 μmol), which was dissolved in 1.4 mL of a 10% MeOH in CHCl3 solvent mixture. Maleimidocaproic acid (39) was added (7.3 mg, 34.4 μmol), followed by EEDQ (10.2 mg, 41.3 μmol) and pyridine (6 μL, 68.8 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 14 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker 40 (14.1 mg, 45%). LC-MS: tR 12.81 min, m/z (ES+) found 918.9 (M+H)+, m/z (ES−) found 917.0 (M−H)−.
2-bromo-N-(4-((S)-7-methoxy-8-(3((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)acetamide (41)
To a flame-dried 10 mL flask was added PBD dimer 37 (16.5 mg, 22.7 μmol), which was dissolved in 0.9 mL of a 10% MeOH in CHCl3 solvent mixture. Bromoacetic acid was added (3.2 mg, 22.7 μmol), followed by EEDQ (6.8 mg, 27.2 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 4 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 95:5 CH2Cl2/MeOH) to provide drug linker 41 (9.9 mg, 52%). TLC: Rf=0.09, 5% MeOH in CH2Cl2. LC-MS: tR 12.44 min, m/z (ES+) found 848.1 (M+H)+, m/z (ES−) found 845.7 (M−H).
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (43)
A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.4 μmol), EEDQ (2.8 mg, 11.3 μmol), and 0.38 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 42 (6.8 mg, 9.4 μmol) was then added and the reaction was stirred at room temperature for 2 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker 43 (3.1 mg, 30%). TLC: Rf=0.31, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.49 min. LC-MS: tR 12.28 min, m/z (ES+) found 1089.5 (M+H)+, m/z (ES−) found 1087.3 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (44)
To a flame-dried 10 mL flask was added PBD dimer 42 (8.0 mg, 11 μmol), which was dissolved in 0.44 mL of a 10% MeOH in CH2Cl2 solvent mixture. Maleimidocaproic acid (39) was added (2.3 mg, 11 μmol), followed by EEDQ (3.3 mg, 13.2 μmol) and pyridine (1.8 μL, 22 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker compound 44 (1.2 mg, 12%). TLC: Rf=0.45, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.71 min. LC-MS: tR 12.63 min, m/z (ES+) found 919.1 (M+H)+, m/z (ES−) found 917.1 (M−H)−.
(2S,3R,4S,5R,6R)-2-(2-(3-(((99H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (46)
A flame-dried flask was charged with glucuronide linker intermediate 45 (reference: Jeffrey et al., Bioconjugate Chemistry, 2006, 17, 831-840) (15 mg, 20 μmol), 1.4 mL anhydrous CH2Cl2, pyridine (20 μL, 240 μmol), and then cooled to −78° C. under nitrogen. Diphosgene (3.0 μL, 24 μmol) was then added and the reaction was stirred for 2 h at −78° C., after which time a small aliquot was quenched with methanol and analyzed by LC-MS for formation of the methyl carbonate, which confirmed formation of the glucuronide chloroformate. PBD dimer 42 (15 mg, 20 μmol) was then dissolved in 0.7 mL anhydrous CH2Cl2 and added dropwise to the reaction vessel. The reaction was warmed to 0° C. over 2 h and then diluted with 50 mL CH2Cl2. The organic layer was washed with water (50 mL), brine (50 mL), dried over sodium sulfate, filtered and concentrated. The crude reaction product was purified by radial chromatography on a 1 mm chromatotron plate eluted 10% MeOH in CH2Cl2 to provide 46 (5.7 mg, 19%). TLC: Rf=0.47, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 12.09 min. LC-MS: tR 14.05 min, m/z (ES+) found 1500.3 (M+H)+,
(2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-(((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (47)
A flask containing 46 (5.7 mg, 3.8 μmol) dissolved in a solvent mixture of 0.2 mL each of MeOH, tetrahydrofuran, and water was cooled to 0° C. To the stirred solution was added lithium hydroxide monohydrate (0.8 mg, 19 μmol) and the reaction was stirred at room temperature for 4 h, at which time LC-MS indicated conversion to product. Glacial acetic acid (1.1 μL, 19 μmol) was added and the reaction was concentrated to provide 47, which was carried forward without further purification. LC-MS: tR 11.59 min, m/z (ES+) found 1138.4 (M+H)+.
(2S,3S,4S,5R,6S)-6-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-4-((((3-(S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (48)
To a solution of 47 (4.3 mg, 3.8 umol) dissolved in 0.38 mL anhydrous DMF was added maleimidocaproyl NHS ester 35 (1.2 mg, 3.8 umol), followed by diisopropylethylamine (4.0 uL, 22.8 umol). The reaction was stirred at room temperature under nitrogen for 2 h, at which time LC-MS revealed conversion to product. The reaction was diluted with a mixture of acetonitrile (0.5 mL), DMSO (1 mL), water (0.5 mL), and then purified by preparative HPLC. The mobile phase consisted of A=water and B=acetonitrile, both containing 0.1% formic acid. A linear elution gradient of 90:10 A:B to 10:90 A:B over 75 minutes was employed and fractions containing the desired product were lyophilized to provide drug linker compound 48 (1.2 mg, 24% over two steps). Analytical HPLC (0.1% formic acid): tR 10.85 min. LC-MS: tR 12.12 min, m/z (ES+) found 1331.4 (M+H)+, m/z (ES−) found 1329.5 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((3-(S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c]l[1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (51)
A flame-dried 10 mL flask was charged with acid 36 (2.7 mg, 7.1 μmol). EEDQ (2.1 mg, 8.5 μmol), and 0.28 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 49 (5.8 mg, 7.1 μmol) was then added and the reaction was stirred at room temperature for 20 h, at which time LC-MS revealed conversion to product. The reaction was concentrated then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug linker compound 51 (2.7 mg, 32%). Analytical HPLC (0.1% formic acid): tR9.17 min. LC-MS: tR 11.25 min, m/z (ES+) found 1185.3 (M+H)+, m/z (ES−) found 1182.9 (M−H)−.
N—((S)-1-(((S)-1((3-(S)-8-((5-((S)-2-(4-(3(dimethylamino)propoxy)phenyl)-7-meth oxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (52)
A flame-dried 10 mL flask was charged with acid 36 (3.7 mg, 9.7 μmol), EEDQ (2.9 mg, 11.6 μmol), and 0.4 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 50 (8.0 mg, 9.7 μmol) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS revealed the presence of product. The reaction was concentrated then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug linker compound 52 (3.1 mg, 25%). Analytical HPLC (0.1% formic acid): tR 9.45 min. LC-MS: tR 11.75 min, m/z (ES+) found 1188.4 (M+H)+, m/z (ES−) found 1186.0 (M−H)−.
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)benzamine (54)
To a flame-dried 10 mL flask was added linker fragment 53 (7.7 mg, 20 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.1 mg, 25 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (12 mg, 16.5 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide 54 (2.4 mg, 13%). TLC: Rf=0.44, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.53 min. LC-MS: tR12.61 min, m/z (ES+) found 1095.4 (M+H)+, m/z (ES−) found 1093.9 (M−H)−.
(S)-2-(2-iodoacetamido)-N—((S)-1-((4-((S)-7-methoxy-8(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (56)
A flame-dried flask was charged with linker 55 (7.8 mg, 22 μmol), which was dissolved in 0.37 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.8 mg, 27.5 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (13 mg, 18 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h, at which time LC-MS revealed conversion to product.
The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 56 (3.5 mg, 18%). Analytical HPLC (0.1% formic acid): tR 11.43 min. LC-MS: tR 12.49 min, m/z (ES+) found 1064.6 (M+H)+, m/z (ES−) found 1098.9 (M+2H2O—H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4] benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (58)
To a flame-dried 10 mL flask was added linker fragment 36 (19 mg, 50 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (12.4 mg, 50 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 57 (12.5 mg, 16.6 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 5 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 58 (2.1 mg, 11%). Analytical HPLC (0.1% formic acid): tR 12.19 min. LC-MS: tR 12.58 min, m/z (ES+) found 1117.8 (M+H)+, m/z (ES−) found 1133.7 (M+H2O—H)−.
(R)-2-((R)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoic acid (60)
A flame dried flask was charged with Fmoc-D-Valine (200 mg, 0.59 mmol) and 5.9 mL anhydrous THF. N-hydroxysuccinimide (75 mg, 0.65 mmol) was added, followed by diisopropylcarbodiimide (0.1 mL, 0.65 mmol), and the reaction was stirred at an ambient temperature overnight, at which time LC-MS revealed conversion to product. The reaction mixture was diluted with CH2Cl2 and washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to dryness. The material was carried forward without further purification. LC-MS: tR 13.89 min, m/z (ES+) found 437.0 (M+H)+, Crude Fmoc-D-Val-OSu (0.59 mmol) was dissolved in dimethoxyethane (1.5 mL) and THF (0.8 mL). D-alanine (73 mg, 0.89 mmol) was dissolved in 2.3 mL water and added to the reaction mixture, followed by sodium bicarbonate (99 mg, 1.2 mmol). The resulting slurry was stirred at room temperature overnight, at which time the reaction had clarified and LC-MS revealed completion. The reaction was poured into 50 mL CH2Cl2 and the organic layer was washed with 50 mL 0.1 M HCl and then brine, dried over sodium sulfate, and then concentrated to dryness. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2 to provide 60 (128 mg, 54%). TLC: Rf=0.18, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 9.47 min. LC-MS: tR 13.09 min, m/z (ES+) found 411.1 (M+H)+, m/z (ES−) found 409.2 (M−H)−.
(R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (61)
Protected dipeptide 60 (70 mg, 0.37 mmol) was suspended in 6 mL anhydrous CH2Cl2, cooled on ice under nitrogen, and 2 mL of diethylamine was added dropwise. The reaction was warmed to room temperature and stirred under nitrogen for 2 h, at which time HPLC revealed consumption of starting material. The reaction was diluted with 6 mL of chloroform and concentrated. The crude reaction residue was re-dissolved in 6 mL chloroform and concentrated twice, followed by drying on a vacuum line for 2 h. The deprotected dipeptide was then dissolved in 3.7 mL anhydrous DMF. MC-OSu (138 mg, 0.44 mmol) was then added, followed by diisopropylethylamine (0.32 mL, 1.9 mmol). The reaction was stirred under a nitrogen atmosphere at room temperature overnight. Workup was achieved by pouring the reaction in to 50 mL 0.1 M HCl and extracting with ethyl acetate (50 mL, 3×). The combined organic layer was washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and concentrated. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (99:1 to 95:5 CH2Cl2/MeOH) to provide 61 (14 mg, 22%). 1H NMR (CD3OD) δ (ppm) 0.94 (d, J=14 Hz, 3H), 0.98 (d, J=14 Hz, 3H), 1.29 (m, 2H), 1.39 (d, J=7.4 Hz, 3H), 1.61 (m, 4H), 2.05 (m, 1H), 2.25 (dt, J=1.2, 7.4 Hz, 2H), 3.48 (t, J=7 Hz, 2H), 4.19 (m, 1H), 4.37 (m, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 10.04 min. LC-MS: tR 11.22 min, m/z (ES+) found 382.1 (M+H)+, m/z (ES−) found 380.0 (M−H)−.
6-(2,5-d oxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((R)-1-(((R)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (62)
To a flame-dried 10 mL flask was added linker 61 (9.5 mg, 25 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (7.3 mg, 30 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (12 mg, 16.5 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 62 (2.8 mg, 16%). TLC: R=0.39, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.50 min. LC-MS: tR 12.50 min, m/z (ES+) found 1089.7 (M+H)+, m/z (ES−) found 1088.0 (M−H)−.
(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanoic acid (64)
L-alanine (58 mg, 0.65 mmol) was suspended in 6.5 mL anhydrous DMF and MC-OSu 35 (100 mg, 0.324 mmol) was then added. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added and the reaction was stirred overnight at room temperature under nitrogen. The reaction was then diluted with 50 mL 0.1 M HCl and the aqueous layer was then extracted with ethyl acetate (50 mL, 3×). The combined organic layer was then washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and then concentrated to dryness.
The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (97.5:2.5 to 90:10 CH2Cl2/MeOH) to provide 64 (25 mg, 27%). TLC: Rf=0.25, 10% MeOH in CH2Cl2. 1H NMR (CD3OD) δ (ppm) 1.30 (m, 2H), 1.37 (d, J=7.4 Hz, 3H), 1.60 (m, 4H), 2.21 (t, J=7.4 Hz, 2H), 3.48 (t, J=7 Hz, 2H), 4.35 (q, J=7.4 Hz, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.06 min.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-((4-((S)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)hexanamide (65)
To a flame-dried 10 mL flask was added linker 64 (14 mg, 50 μmol), which was dissolved in 0.66 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (15 mg, 60 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (24 mg, 33 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide 65 (3.5 mg, 11%). Analytical HPLC (0.1% formic acid): tR 11.40 min. LC-MS: tR 12.39 min, m/z (ES+) found 990.6 (M+H)+, m/z (ES−) found 989.0 (M−H)−.
PBD Dimer 57 Linked Directly Through Maleimidocaproyl Spacer (Scheme 14):
PBD dimer 57 is coupled to maleimidocaproic acid 39 employing the chemistry described in Scheme 2.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-hydro-1H-yl)-N-(2-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4] benzodiazepin-2-yl)phenyl)hexanamide (68)
To a mixture of the 66 (10 mg, 0.013 mmol) in CH2Cl2 (300 L) was added DIPEA and MC-Cl (67) (3 mg, 0.013 mmol). After 1 h, an additional 3 equiv. of DIPEA (7 μL) and 2 equiv. of the acid chloride (6 mg, 0.026 mmol) were added. After 1 h, an additional quantity of DIPEA (7 μL) and acid chloride (6 mg, 0.026 mmol) were added. After an additional 3 h, the reaction mixture was aspirated directly onto a 1 mm radial chromatotron plate and eluted with dichloromethane followed by a gradient of methanol (1% to 5%) in dichloromethane. Product containing fractions, as a mixture with the starting aniline, were concentrated to a residue and dissolved in a mixture of 0.5 mL DMSO, 0.5 mL acetonitrile and 0.5 mL deionized water and was further purified by preparative HPLC. The major peak was collected and the fractions were combined, frozen and lyophilized to give 2.1 mg (18%): MS (ES+) m/z 919.2 [M+H]+.
Note: Acid chloride 67 was prepared by dissolving 100 mg of 39 in oxalyl chloride (5 mL). A drop of DMF was added and the mixture was stirred at an ambient temperature for several hours before being concentrated under reduced pressure. Dichloromethane was added and the mixture was concentrated a second time to afford an off-white solid which was used directly: 1H-NMR (400 MHz, CDCl3) δ 6.70 (s, 2H), 3.46 (t, J=7 Hz, 2H), 2.82 (t, J=7.2 Hz, 2H), 1.72 (pent, J=7.6 Hz, 2H), 1.61 (pent, J=7.4 Hz, 2H), 1.35 (pent, J=7.6 Hz, 2H).
tert-butyl 2-(2-aminoacetamido)acetate (69)
To a mixture of the glycine tert-butyl ester hydrogen chloride salt (70) (484 mg, 2.9 mmol) in dichloromethane (25 mL) was added Fmoc-Gly-OH (71) (0.861 mg, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was stirred at an ambient temperature for 16 h and then poured into ethyl acetate and was washed with water (3×) and brine (1×). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified via radial chromatography on a 2 mm plate eluting with 5% methanol/dichloromethane. Product containing fractions were concentrated under reduced pressure and treated with 20% piperidine/dichloromethane (10 mL) for 1 h, before being concentrated under reduced pressure and then purified twice via radial chromatography on a 2 mm plate eluting with a gradient of 5 to 10% methanol/dichloromethane to provide (200 mg, 37%): 1H-NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 4.00 (s, 2H), 3.39 (s, 2H), 1.47 (s, 9H).
2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetamido)acetic acid (72)
To a solution of the amine 69 (200 mg, 0.11 mmol) in DMF (1 mL) was added 35 (350 mg, 0.11 mmol) and the reaction mixture was allowed to stir at an ambient temperature for 2 h.
The mixture was concentrated under reduced pressure and was purify by radial chromatography on a 1 mm plate eluting with dichloromethane and a gradient of methanol (1 to 5%) in dichloromethane. Product containing fractions were concentrated under reduced pressure, dissolved in dichloromethane (4 mL) and treated with trifluoroacetic acid (4 mL). After 40 min the mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated to give 22.5 mg (19%) of 72 as white solid: 1H-NMR (400 MHz, CD3OD) δ 6.79 (s, 2H), 3.93 (s, 2H), 3.89 (s, 2H), 3.49 (t, J=6.8 Hz, 2H), 2.26 (t, J=6.8 Hz, 2H), 1.61 (m, 4H), 1.34 (m, 2H); MS (ES+) m/z 326.21 [M+H]+.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(2-((4-(S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (73)
To a mixture of 72 (15 mg, 0.046 mmol) in 5% methanol/dichloromethane (0.5 mL) was added EEDQ (11 mg, 0.046 mmol) and the mixture was stirred for 30 min at an ambient temperature, at which time 37 (16 mg, 0.023 mmol) was added. The reaction mixture was stirred for 3 h and was purified directly on a 1 mm radial chromatotron plate eluting with a 1% to 4% methanol/dichloromethane gradient to give 6.8 mg (29%) of 73 as a yellow solid: MS (ES+) m/z 1033.57 [M+H]+.
(S)-tert-butyl 1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (74)
To a mixture of L-proline-tert-butyl ester hydrogen chloride salt 75 (0.5 g, 2.9 mmol) in dichloromethane (50 mL) was added 76 (0.98 g, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was allowed to stir at an ambient temperature for 16 h. The mixture was poured into ethyl acetate (100 mL) and was washed with 0.2 N HCl (50 mL), water (50 mL), brine (50 mL) and dried over MgSO4. Chromatography was conducted on a 2 mm radial chromatotron plate eluting with 10% ethyl acetate in hexanes. Product-containing fractions were concentrated under reduced pressure, dissolved in dichloromethane (8 mL) and treated with piperidine (2 mL). The mixture was stirred for 1 h, concentrated under reduced pressure and purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane. This gave 200 mg (26%) of the dipeptide 74: 1H-NMR (400 MHz, CDCl3) δ 4.41 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H), 3.57 (m, 4H), 3.2 (m, 1H), 2.82 (m, 1H), 2.83-1.65 (m, 5H), 1.44 (m, 9H).
(S)-tert-butyl 1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (77)
To a mixture of the amine 74 (200 mg, 0.75 mmol), 39 (190 mg, 0.9 mmol) and DIPEA (0.32 mL, 1.8 mmol) was added HATU (342 mg, 0.9 mmol) and the mixture was allowed to stir at an ambient temperature for 5 h. The mixture was poured into ethyl acetate (100 mL) and washed with water (3×100 mL) and brine (1×100 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated. The resulting residue was subjected to radial chromatography on a 2 mm radial chromatotron plate eluting with dichloromethane followed by an increasing gradient of 1 to 5% methanol in dichloromethane. Two additional purifications, both eluting with a gradient of 1 to 5% methanol in dichloromethane, first on a 2 mm plate and then on a 1 mm plate afforded 113 mg (33%) of 77 as an white solid: 1H-NMR (400 MHz, CDCl3) δ 4.63 (m, 1H), 4.41 (m, 1H), 3.82 (m, 1H), 3.63 (m, 1H), 3.55 (m, 1H), 3.45 (m, 3H), 2.38-1.83 (m, 10H), 1.70-1.50 (m, 5H), 1.45 (m, 9H), 1.35 (m, 2H); MS (ES+) m/z 462.33 [M+H]+.
(S)-1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid (78)
To a mixture of the tert-butyl ester 77 in dichloromethane (4 mL) was added trifluoroacetic acid (4 mL). After 40 min the reaction was determined to be complete by HPLC analysis. The mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated a second time to give 37 mg (100%) of 78 as a white solid: 1H-NMR (400 MHz, CDCl3) δ 6.68 (s, 2H), 4.62 (m, 2H), 3.81 (m, 1H), 3.70 (m, 1H), 3.57 (m, 2H), 3.45 (m, 2H), 2.40-1.91 (m, 10H), 1.70-1.45 (m, 4H), 1.33 (m, 2H); MS (ES+) m/z 406.2 [M+H]+.
1-(1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)-N-(4-((S)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)pyrrolidine-2-carboxamide (79)
To a mixture of the 78 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (0.4 mL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 15 min at an ambient temperature and then 37 (15 mg, 0.021 mmol) was added. The mixture was stirred for 4 h, the reaction mixture was diluted with dichloromethane (2 mL) and was aspirated directly onto a 1 mm radial chromatotron plate. The product was eluted with a gradient of 1 to 5% methanol in dichloromethane to provide 6.8 mg (29%) of 79 as a yellow solid: MS (ES+) m/z 1113.51 [M+H]+.
(S)-5-(allyloxy)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-oxopentanoic acid (80)
To a mixture of the 2-chlorotrityl resin (1.0 g, 1.01 mmol) suspended in dichloromethane (10 ml) was added Fmoc-Glu-(OAllyl)-OH (81) (409 mg, 1.0 mmol) and DIPEA (173 μL, 1.0 mmol). The reaction mixture was shaken for 5 min, and an additional portion of DIPEA (260 μL, 1.5 mmol) was added and the mixture was shaken for 1 h. Methanol (0.8 mL) was added and the mixture was shaken for 5 min, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure. The resulting resin was subjected to 20% piperidine in dichloromethane (10 mL) for 1 h, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure.
To a mixture of the Fmoc-Val-OH (82) (1.03 g, 3.30 mmol)) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After thorough mixing, the solution as aspirated into a 10 mL syringe containing the resin prepared above. The mixture was capped and shaken for 16 h. The resin was washed with DMF (6×), dichloromethane (6×) and ether (6×). A small portion (10 mg) was isolated and treated with 20% TFA/Dichloromethane and the resulting solution analyzed by LC-MS which revealed one high purity peak which displayed the correct mass (MS (ES+) m/z 509.28 [M+H]+). The remaining resin was then treated with 20% piperidine/DMF (8 mL) for 2 h, before being washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure.
A mixture of allyl chloroformate (529 μL, 5.05 mmol), DIPEA (1.7 mL, 10 mmol) in dichloromethane (10 mL) was prepared and aspirated into a syringe containing the resin above. The mixture was capped and shaken. After approximately 2 h, the reaction mixture was drained, and washed with dichloromethane (6×). A small portion of the resin (˜10 mg) was cleaved with 20% TFA/dichloromethane and analyzed by LC-MS for masses of starting material and product. The main component was still the unreacted amine, so the resin was again subjected to the conditions described above. After 4 h, the resin was washed with dichloromethane (6×), and then treated repeatedly with 5% TFA in dichloromethane (4×7 mL). The resulting solution was concentrated under reduced pressure. The mixture was purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 107 mg of 80: 1H-NMR (400 MHz, CDCl3) δ 7.05 (s, 1H), 5.90 (m, 2H), 5.57 (d, 1H), 5.29 (d, J=14.7 Hz, 2H), 5.22 (t, J=10.9 Hz, 2H), 4.59 (m, 5H), 4.02 (m, 1H), 2.60-2.40 (m, 2H), 2.37-2.18 (m, 1H), 2.17-2.02 (m, 2H), 0.96 (d, J=6.4 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H); MS (ES+) m/z 371.12 [M+H]+.
(S)-allyl 4-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoate (83)
To a mixture of the acid 80 (30, 0.04 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (20 mg, 0.082 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (30 mg, 0.04 mmol) was added and the mixture was stirred for approximately 5 h. Partially purification by aspirating directly onto a 1 mm radial chromatotron plate and eluting with a gradient of 1% to 5% methanol/dichloromethane afforded a mixture of desired product and 37 (26 mg; ˜3:1 respectively) which was carried forward without further purification.
(S)-4-((S)-2-amino-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoic acid (84)
To the mixture of 83 and 37 (26 mg) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 mmol), pyrrolidine (4 μL, 0.048 mmol) and tetrakis palladium (0.7 mg, 0.6 μmol). After 2 h, an additional quantity (0.7 mg, 0.6 μmol) of tetrakis palladium was added and the reaction was allowed to stir for an additional 1 hr before being concentrated under reduced pressure. The residue was dissolved in DMSO (1 mL), acetonitrile with 0.05% formic acid (1 mL) and water with 0.05% formic acid (1 mL) and purified by preparative reverse phase HPLC. A single fraction of product was collected and lyophilized to give 6 mg (14% for two steps) of 84: MS (ES+) m/z 1078.6 [M+H]+.
(S)-4-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-((4-((S)-7-methoxy-83-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoic acid (85)
To a mixture of the 84 (6 mg, 6 μmol), and 35 (2 mg, 6 μmol) in DMF (200 μL) was added DIPEA (3 μL, 18 μmol) and the reaction mixture was stirred at an ambient temperature. After 1 h, an additional equivalent of 35 (2 mg, 6 μmol) was added and the reaction was allowed to continue to stir at an ambient temperature for 3 h. A third equivalent of 35 (2 mg, 6 μmol) was added and the mixture was stirred for approximately 1 h, concentrated under reduced pressure, dissolved in dichloromethane and aspirated directly onto a 1 mm radial chromatotron plate and eluted with 5% methanol in dichloromethane. This gave 2.5 mg (36%) of high purity 85: MS (ES+) m/z 1147.49 [M+H]+.
(21 S,24S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-isopropyl-24-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)-3,19,22-trioxo-7,10,13,16-tetraoxa-4,20,23-triazaheptacosan-27-oic acid (86)
To a mixture of the 84 (8 mg, 8.4 μmol) and Mal-PEG4-NHS (87) (6.5 mg, 12.6 μmol) in DMF (200 μL) was added DIPEA (4.3 μL, 25 μmol). The reaction mixture was stirred at an ambient temperature for 2 h, and was concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane and aspirated onto a 1 mm radial chromatotron plate. The material was polar and did not chromatograph on the silica gel-based chromatotron plate. The plate was eluted with methanol to recover the mixture which was isolated under reduced pressure. The residual material was purified via preparative reverse phase HPLC. A single main peak eluted and the fractions were combined, frozen and lyophilized to a residue of 0.9 mg (8%) of 86: MS (ES+) m/z 1353.04 [M+H]+.
(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)hexanoic acid (88)
To a mixture of the 2-chlorotrityl resin (1 g, 1.01 mmol) in CH2Cl2 (10 ml) was added Fmoc-Lys(Me)2-OH (89) (432 mg, 1.0 mmol) and DIPEA (433 μL, 2.5 mmol). The reaction mixture was shaken for 1 h. Methanol (0.8 mL) was added and the mixture was shaken for an additional 5 min, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure. The dried resin was subjected to 20% piperidine in DMF (10 mL) for 1 h. before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×).
To a mixture of the 39 (3.0 mmol, 633 mg) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After thorough mixing, the solution as aspirated into a 10 mL syringe containing the resin above. The mixture was capped, shaken for 16 h, filtered and the resin washed with DMF (6×), dichloromethane (6×), and ethyl ether (6×). The resin was by repeatedly treating with 5% TFA/dichloromethane (6 mL×5), shaking for 1 min, and then filtering. The resulting solution was concentrated under reduced pressure and under high vacuum. The material was purified by preparatory reverse phase HPLC to give 208 mg of 88: 1H-NMR (400 MHz, CD3OH/CDCl3 1:1 mixture) δ 6.73 (s, 2H), 4.41 (m, 1H), 3.48 (t, 2H), 3.31 (s, 1H), 3.03 (m, 2H), 2.84 (s, 6H), 2.22 (m, 2H), 1.87 (m, 2H), 1.78-1.52 (m, 6H), 1.43 (m, 2H), 1.31 (pent, 2H); MS (ES+) m/z 386.28 [M+H]+.
(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (90)
To a mixture of the 88 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (400 μL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (15 mg, 0.021 mmol) was added. After 4 h, the mixture was concentrated under reduced pressure, dissolved in a mixture of DMSO (1 mL), acetonitrile (2 mL containing 0.05% formic acid) and water (1 mL containing 0.05% formic acid) and purified by reverse-phase HPLC (method A). Product containing fractions were contaminated with 37, so the fractions were lyophilized to a residue and repurified as described above to give 0.5 mg (2%) of pure 90: MS (ES+) m/z 537.46 [M+H]/2+.
Allyl ((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (91)
To a mixture of the 92 (45 mg, 0.123 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (30.4 mg, 0.123 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (30 mg, 0.041 mmol) was added. The reaction mixture was stirred for approximately 5 h and then purified on a 1 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 22 mg (55%) of 91 which was not characterized but carried on directly.
1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (93)
To a solution of the 91 (22 mg, 0.022 mmol) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 mmol), pyrrolidine (4 μL, 0.048 mmol) and tetrakis palladium (0.7 mg, 6 μmol). After approximately 2 h, the reaction mixture was purified on a 1 mm radial chromatotron plate eluting with 5% to 10% methanol/dichloromethane. The major band was collected and concentrated to a residue which was dissolved in DMF (0.2 mL) and reacted with NHS ester 87 (10 mg, 0.19 mmol). The reaction was allowed to stir for 30 min, concentrated and purified by radial chromatography on a 1 mm plate eluting with 5% methanol/dichloromethane to give 3.2 mg (11%) of 93: MS (ES+) m/z 1294.7 [M+H]+.
(E)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-4-((S)-7-methoxy-8-(5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)benzylidene)hexanohydrazide (94)
To a mixture of the aldehyde 95 (5.4 mg, 7 μmol) in 5% methanol/dichloromethane at 0° C. was added the hydrazide-TFA salt 96 (4.5 mg, 14 μmol). The reaction mixture was allowed to warm to an ambient temperature and stir for 5 h before being concentrated under reduced pressure and purified on a silica gel column eluting with 3% methanol/dichloromethane to give 2.2 mg (32%) of 94: MS (ES+) m/z 974.49 [M+H]+.
(S)-tert-butyl 2-((S)-2-amino-3-methylbutanamido)propanoate (97)
To a mixture of the alanine-O-tert-butyl ester hydrogen chloride salt (98) (500 mg, 2.76 mmol) in dichloromethane (5 mL) was added Fmoc-val-OSu (99) (1.09 g, 2.51 mmol). DIPEA (0.96 ml, 5.5 mmol) was added and the reaction mixture was allowed to stir at an ambient temperature for 16 h. The mixture was poured into dichloromethane (100 mL) and washed with 1N HCl (50 mL) and water (50 mL) before being dried over magnesium sulfate. The material was chromatographed on a 2 mm radial chromatotron plate eluting with 1 to 5% methanol/dichloromethane gradient and product containing fractions were combined and concentrated. The resulting residue was dissolved in dichloromethane (16 mL) and piperidine (4 mL) was added. The mixture was stirred for 10 min before being concentrated under reduced pressure. The resulting residue was chromatographed on a 2 mm plate eluting first with ammonia-saturated dichloromethane followed by 5% methanol in ammonia-saturated dichloromethane to give 494 mg (2.02 mmol, 81% for two steps) of 97: 1H-NMR (400 MHz, CDCl3) δ 7.78 (bs, 1H), 4.47 (m, 1H), 3.30 (d, 1H), 2.30 (m, 1H), 1.38 (d, 3H), 1.47 (s, 9H), 1.00 (d, J=7.0 Hz, 3H), 0.84 (d, J=6.9 Hz, 3H).
(S)-tert-butyl 2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoate (100)
To a mixture of the 97 (100 mg, 0.41 mmol) and 4-maleimidobenzoic acid (101) (98 mg, 0.45 mmol) was added dichloromethane (5 mL), followed by TBTU (157 mg, 0.49 mmol) and DIPEA (212 uL, 1.23 mmol). The mixture was stirred at an ambient temperature for 16 h and then purified on a 2 mm radial chromatotron plate eluting with 50% ethyl acetate in hexanes to give 95 mg (51%) of 100: 1H-NMR (400 MHz, CDCl3) δ 7.85 (d, J=6.6 Hz, 2H), 7.42 (d, J=6.6 Hz, 2H), 6.81 (s, 2H), 6.38 (bs, 1H), 4.43 (m, 2H), 2.14 (sept, J=6.6 Hz, 1H), 1.41 (s, 9H), 1.31 (d, J=7.0 Hz, 3H), 0.98 (m, 6H); MS (ES−) m/z 441.90 [M−H].
(R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic acid (53)
To a mixture of 100 (47 mg, 0.11 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was monitored by TLC (50% ethyl acetate in hexane, after pumping down the TLC plate under high vacuum for 5 min). After 75 min. no starting material could be detected by TLC. The reaction was performed a second time using the same conditions and material from both reactions were combined and purified on a 2 mm radial chromatotron plate eluting with a gradient from 5-10% methanol in dichloromethane. The yield was 42 mg (49%) of 53: 1H-NMR (400 MHz, CDCl3) δ 7.92 (d, J=6.6 Hz, 2H), 7.51 (d, J=6.6 Hz, 2H), 7.0 (m, 1H), 6.89 (s, 2H), 6.70 (s, 1H), 4.60 M, 1H), 2.22 (m, 1H), 1.18 (d, J=6.6 Hz, 3H), 1.04 (m, 6H); MS (ES+) m/z 388.02 [M+H]+.
(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (102)
To a mixture of the 97 (100 mg, 0.41 mmol) in dichloromethane was added iodoacetamide-NHS ester (103) (115 mg, 0.41 mmol) and the mixture was stirred at an ambient temperature. After 30 min, the mixture was aspirated onto a 1 mm chromototron plate and eluted with ethyl acetate in hexanes (1:1). A single band was collected and the structure was confirmed: 1H-NMR (400 MHz, CDCl3) δ 6.70 (d, J=7.8 Hz, 1H), 6.27 (d, J=7.0 Hz, 1H), 4.45 (m, 1H), 4.26 (dd, J=8.6, 6.3 Hz, 1H), 3.72 (quart, J=11.3 Hz, 2H), 2.13 (sept, J=6.5 Hz, 1H), 1.47 (s, 9H), 1.38 (d, J=7.1 Hz, 3H), 0.99 (m, 6H); MS (ES+) m/z 412.87 [M+H]+.
(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (55)
See procedure for the synthesis of (R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic acid (53). This gave 22 mg (15% for two steps): 1H-NMR (400 MHz, D6-DMSO) δ 8.27 (d, J=9.4 Hz, 1H), 4.24 (m, 2H), 3.97 (bs, 2H), 3.83 (d, J=9.4 Hz, 1H), 3.71 (d, J=9.6 Hz, 1H), 2.07 (m, 1H), 1.33 (d, J=7.3 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H), 0.89 (d, J=6.9 Hz, 3H); MS (ES−) m/z 354.84 [M−H].
PBD Dimers Linked Through Aliphatic Amines (Scheme 21).
PBD dimers containing aliphatic amines, such as a benzyl amine (Example 9), are synthesized with peptidic linkers, the glucuronide linker, and/or linkers dependent on mAb degradation for release (i.e., non-cleavable linkers). Drug linkers conjugated through a benzyl amine will include: (1) a cleavable peptide employing chemistry similar to Scheme 1; (2) direct attachment with a maleimidocaproyl group (a noncleavable linker) (Scheme 2); (3) a glucuronide linker, prepared as described in Scheme 6.
Generic Peptide Linked 2-, 3-, and 4-Aniline PBD Dimers (Scheme 22).
PBD dimers with anilines at the 2-, 3-, and 4-positions will be conjugated to peptide-based linkers, employing the chemistry described in Scheme 1, or attached directly with maleimidocaproic acid, as exemplified in Scheme 2.
Example 14: Preparation of PDB Dimer Conjugates
Antibody-drug conjugates were prepared as previously described (see Doronina et al., Nature Biotechnology, 21, 778-784 (2003)) or as described below. Briefly, for maleimide drug-linker the mAbs (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 were reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progress of the reaction, which reduces interchain disulfides, was monitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb was achieved. The reduced antibody was then cooled to 0° C. and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 h, the reaction was quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker was removed by gel filtration over a PD-10 column. The ADC was then sterile-filtered through a 0.22 μm syringe filter. Protein concentration was determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography was used to determine the extent of antibody aggregation and RP-HPLC confirmed the absence of remaining NAC-quenched drug-linker.
For halo acetamide-based drug linkers, conjugation was performed generally as follows: To a 10 mg/mL solution of reduced and reoxidized antibody (having introduced cysteines by substitution of S239C in the heavy chains (see infra)) in 10 mM Tris (pH 7.4), 50 mM NaCl, and 2 mM DTPA was added 0.5 volumes of propylene glycol. A 10 mM solution of acetamide-based drug linker in dimethylacetamide was prepared immediately prior to conjugation. An equivalent amount of propylene glycol as added to the antibody solution was added to a 6-fold molar excess of the drug linker. The dilute drug-linker solution was added to the antibody solution and the pH was adjusted to 8.0-8.5 using 1 M Tris (pH 9). The conjugation reaction was allowed to proceed for 45 minutes at 37° C. The conjugation was verified by reducing and denaturing reversed phase PLRP-S chromatography. Excess drug linker was removed with Quadrasil MP resin (Sigma Aldrich; Product #679526) and the buffer was exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl, and 5% propylene glycol using a PD-10 desalting column (GE Heathcare; Product #17-0851-01).
Engineered hlgG1 antibodies with introduced cysteines: CD70 antibodies containing a cysteine residue at position 239 of the heavy chain (h1F6d) were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). Following a 1 hour incubation at 37° C., the reaction was cooled to 22° C. and 30 equivalents of dehydroascorbic acid were added to selectively reoxidize the native disulfides, while leaving cysteine 239 in the reduced state. The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction was allowed to proceed for 1 hour at 22° C. The pH of the solution was then raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0), 3.5 equivalents of the PBD drug linker in DMSO were placed in a suitable container for dilution with propylene glycol prior to addition to the reaction. To maintain solubility of the PBD drug linker, the antibody itself was first diluted with propylene glycol to a final concentration of 33% (e.g., if the antibody solution was in a 60 mL reaction volume, 30 mL of propylene glycol was added). This same volume of propylene glycol (30 mL in this example) was then added to the PBD drug linker as a diluent. After mixing, the solution of PBD drug linker in propylene glycol was added to the antibody solution to effect the conjugation; the final concentration of propylene glycol is 50%. The reaction was allowed to proceed for 30 minutes and then quenched by addition of 5 equivalents of N-acetyl cysteine. The ADC was then purified by ultrafiltration through a 30 kD membrane. (Note that the concentration of propylene glycol used in the reaction can be reduced for any particular PBD, as its sole purpose is to maintain solubility of the drug linker in the aqueous media.)
Example 15: Determination of In Vitro Activity of Selected Conjugates
The in vitro cytotoxic activity of the selected antibody drug conjugates was assessed using a resazurin (Sigma, St. Louis, Mo., USA) reduction assay (reference: Doronina et al., Nature Biotechnology, 2003, 21, 778-784). The antibody drug conjugates were prepared as described above in Example 13.
For the 96-hour assay, cells cultured in log-phase growth were seeded for 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of ADC in cell culture media were prepared at 4× working concentration; 50 μL of each dilution was added to the 96-well plates. Following addition of ADC, the cells were incubated with test articles for 4 days at 37° C. Resazurin was then added to each well to achieve a 50 μM final concentration, and the plates were incubated for an additional 4 h at 37° C. The plates were then read for the extent of dye reduction on a Fusion HT plate reader (Packard Instruments, Meridien, Conn., USA) with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
Referring to Table 4 (infra), the in vitro cytotoxicity of ADCs having para-aniline PBD dimers using the 96 hour assay is shown. The ADCs were tested against CD70 CD30 cell lines and a control CD70 CD30 cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942), a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) had a lower IC50 than conjugates with a maleimidyl or acetamide-based linker (compounds 40 and 41, respectively).
In vitro cytotoxic activity of ADCs bearing drug linkers derived from para-aniline PBD dimer 37:
TABLE 4
In vitro cytotoxic activity on CD70+ cell lines (ng/mL), all ADCs
2 drugs/mAb
renal cell carcinoma
AML
CD70+/30−
CD70−/30−
786-O
Caki-1
769-P
ACHN
HEL9217
h1F6d-38
30
5
1378
h1F6-38
4
118
26
cAC10-38
1052
4005
508
h1F6-40
7113
1764
cAC10-40
2644
1264
h1F6-41
580
1243
cAC10-41
1153
1121
Referring to Table 5, the in vitro cytotoxicity of ADCs conjugate to PBD dimers on CD30+ cell lines using the 96 hour assay is shown. The ADCs were tested against CD30+CD70+ cell lines and a CD70+ CD30+ cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) generally had a lower IC50 than conjugates with a maleimidyl or acetamide-based linker (compounds 40 and 41, respectively).
TABLE 5
In vitro cytotoxic activity on CD30+ cell lines (ng/mL), all
ADCs 2 drugs/mAb
ALCL
Hodgkin lymphoma
CD70−/30+
CD70+/30+
Karpas 299
L428
L540cy
L1236
Hs445
h1F6-38
1165
59
4
>10,000
5
cAC10-38
0.8
7
3
2012
0.2
h1F6-40
2195
7867
2557
cAC10-40
621
3172
134
h1F6-41
1330
3549
755
cAC10-41
340
957
13
In vitro cytotoxic activity of ADCs bearing drug linkers derived from meta-aniline PBD dimer 42:
Referring to Table 6, the in vitro cytotoxicity of ADCs containing PBD dimers on CD30+ cell lines using the 96 hour assay is shown. The activity was tested against CD30+CD70+ cell lines and a CD70− CD30+ cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 43) and a glucuronide linker (48) generally had a lower IC50 than conjugates with a maleimidyl-based linker (compound 44).
TABLE 6
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Hodgkin
renal cell carcinoma
lymphoma
Caki-1
786-O
L428
h1F6d-43 (2 dr/mAb)
7
39
>10,000
IgG-43 (2 dr/mAb)
>10,000
>10,000
h1F6-44 (3.5 dr/mAb)
1124
2142
IgG-44 (3.5 dr/mAb)
1491
1242
h1F6d-48 (2 dr/mAb)
89
4093
IgG-48 (2 dr/mAb)
2939
6376
In vitro cytotoxic activity of ADCs bearing drug linkers derived from para- and meta-aniline PBD dimers 38 and 42 (respectively):
Referring to Table 7, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines L428 and 786O and a CD70 AML cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker with a meta-aniline (drug linker compound 43) were somewhat less active than those having a maleimidyl-peptide linker with a para-aniline (drug linker compound 38). Reducing the drug loading of the meta-aniline compound to 2 per antibody reduced the activity. Conjugates with a glucuronide linker of the para-aniline compound (48) generally had a lower IC50 than conjugates with a maleimidyl-based linker (compound 39). Further, an aryl maleimide of the para-aniline compound (54) has no activity on these cell lines. Further, a conjugate having a maleimidyl linker conjugated directly to compound 42 has reduced activity as compared with conjugate h1F6-43 (data not shown).
TABLE 7
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Hodgkin
Renal cell
lymphoma
carcinina
L428
786O
control
h1F6- 43(4 dr/rnAb)
404
11
1205
h1F6d- 43 (2 dr/mAb)
Max inhib. = 40%
200
1625
h1F6d-48 (2 dr/rnAb)
4093
89
1964
h1F6 54 (4 dr/rnAb)
No effect
No effect
No effect
h1F6- 38 (2 dr/rnAb)
230 (n = 2)
25 (n =3)
503
In Vitro Cytotoxic Activity of ADCs Bearing Drug Linkers Derived from Aniline-Linked PBD Dimers
Referring to Table 8, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines Caki-1 and L428 and a CD70− cell line. The antibody used was a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h F6d). Linkage of a PBD through an amine at the ortho position via a non-cleavable linker (compound 68) markedly reduced activity, as compared with an ADC linked via a para-aniline-linked cleavable linker (compound 54). Compounds 73 and 85, having a cleavable linker, showed comparable activity to compound 54; both of these compounds are linked via a para-aniline. Compounds with cleavable linkers requiring more stringeng cleavage, compounds 79 and 90, showed somewhat reduced activity, as compared to compound 54.
TABLE 8
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
renal cell carcinoma
Caki-1
786-0
Control
h1F6d- 68 (2 dr/mAb)
3236
3486
5501
h1F6d- 73 (2 dr/mAb)
2
7
482
h1F6d- 79 (2 dr/mAb)
24
348
5385
h1F6d- 54 (2 dr/mAb)
6
17
4665
h1F6d- 85 (2 dr/mAb)
3
5
4700
h1F6d- 90 (1.4 dr/mAb)
. . . 12
47
678
In Vitro Cytotoxic Activity of ADCs Bearing Drug Linkers Derived from Aniline-Linked PBD Dimers
Referring to Table 9, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines Caki-1 and L428 and two CD70− leukemia cell lines. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Compound 56, having a cleavable linker linked to the antibody via an acetamide showed comparable activity to compound 38. A glucuronide-linked version of the meta-aniline linked PBD dimer, compound 48, demonstrated little activity in this assay. Compound 58, having five methylene groups in the PBD bridge, demonstrated comparable activity to compound 38, having three methylene groups in the PBD bridge.
TABLE 9
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Renal Cell
Caki-1
786-O
Leukemia
(CD70
(CD70
CD70−
CD70−
ADCs
#135,000)
#190,000)
Line 1
Line 2
h1F6d-56
3
6
1672
Max Inh =
(1.8 dr/Ab)
50%
h1F6d-48
Max Inh =
Max Inh =
No Effect
No Effect
(0.6 dr/Ab)
45%
35%
h1F6d-58
0.5
2
1750
4847
(1.9 dr/Ab)
h1F6d-38
5
15
2082
7188
(2 dr/Ab)
(3-5, n = 4)
(5-30, n = 4)
Example 16: Determination of In Vivo Cytotoxicity of Selected Conjugates
All studies were conducted in concordance with the Animal Care and Use Committee in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. In vivo tolerability was first assessed to ensure that the conjugates were tolerated at clinically relevant doses. BALB/c mice were treated with escalating doses of ADC formulated in PBS with 0.01% Tween 20. Mice were monitored for weight loss following drug treatment; those that experienced 20% weight loss or other signs of morbidity were euthanized. The antibodies used were a CD70 antibody, humanized 1 F6 (see Published U.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289).
Referring to FIG. 1, the results of a weight loss study are shown using cAC10-val-ala-SG3132(2) (cAC10-compound 38). A single dose of the conjugate administered at 5 mg administered either IP or IV resulted in little weight loss. A higher dose of the conjugate (15 mg/kg) caused weight loss in the mice.
Referring to FIG. 2, the results of a weight loss study are shown using h1F6-val-ala-SG3132(2) (h1F6-compound 38). A single dose of the conjugate administered at 5 mg administered IP resulted in some weight loss. A higher dose of the conjugate (10 mg/kg) caused significant weight loss in the mice.
Treatment studies were conducted in two CD70+ renal cell carcinoma xenograft models. Tumor (786-O and Caki-1) fragments were implanted into the right flank of Nude mice. Mice were randomized to study groups (n=5) on day eight (786-O) or nine (Caki-1) with each group averaging around 100 mm3. The ADC or controls were dosed ip according to a q4dx4 schedule. Tumor volume as a function of time was determined using the formula (L×W2)/2. Animals were euthanized when tumor volumes reached 1000 mm3. Mice showing durable regressions were terminated around day 100 post implant.
Referring to FIG. 3, the results of a treatment study using an h1F6-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. A control conjugate, cAC10-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice administered doses of the h1F6 conjugate at 0.1 mg/kg exhibited some tumor reduction, while higher doses at 0.3 mg/kg and 1 mg/kg appeared to exhibit complete tumor reduction. The control conjugate (non-binding) was less active the h1F6 conjugates.
Referring to FIG. 4, the results of a treatment study using an h1F6-mc-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. A control conjugate, cAC10-mc-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice administered doses of the h1F6 conjugate at 1 mg/kg appeared to exhibit complete tumor reduction. Mice administered lower doses at 0.3 mg/kg and 0.1 mg/kg exhibited lesser tumor reduction, respectively. The control conjugate (non-binding) was less active the h1F6 conjugate administered at a similar dose, although it exhibited more activity than the h1F6 conjugate administered at lower doses. The h1F6 conjugate was also more active than an h1F6-vc-MMAE conjugate (Published U.S. Application No. 2009-0148942) administered at higher doses.
Referring to FIG. 5, the results of a treatment study using a two loaded antibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loaded non-binding control, H00d conjugated to the same compound (h00d-38). The model was a Caki subcutaneous model in Nude mice. Doses were 0.1, 0.3 and 1 mg/kg q7d×2. The highest two doses of the h1F6 conjugate demonstrated complete regressions as 1 mg/kg and substantial tumor delay at 0.3 mg/kg. The non-binding control demonstated tumor delay at the 1 mg/kg dose.
Referring to FIG. 6, the results of a treatment study using a two loaded antibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loaded non-binding control, H00d conjugated to the same compound (h00d-38). The model was a 786-O subcutaneous model in Nude mice. Doses were 0.1, 0.3 and 1 mg/kg q7d×2. All three doses of the h1F6 conjugate demonstrated complete regressions or tumor delay, while the non-binding control demonstated tumor delay.
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16381448
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Mar 14th, 2017 12:00AM
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Jan 14th, 2016 12:00AM
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https://www.uspto.gov?id=US09592240-20170314
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Targeted pyrrolobenzodiazapine conjugates
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Provided are Conjugate comprising PBDs conjugated to a targeting agent and methods of using such PBDs.
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9592240
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1. A Conjugate having formula I:
L-(LU-D)p (I)
or a pharmaceutically acceptable salt thereof;
wherein L is a Ligand Unit selected from the group consisting of a full length antibody and an antigen binding fragment of a full length antibody;
LU is a Linker unit of formula X:
wherein E is of formula:
the asterisk indicates the point of attachment to a Drug Unit and the wavy line indicates the point of attachment to the Ligand Unit, and
wherein:
-A1- is selected from the group consisting of:
wherein n is 0 to 6;
wherein n is 0 to 6;
wherein n is 0 or 1, and m is 0 to 30; and
wherein n is 0 or 1, and m is 0 to 30;
wherein the asterisk indicates the point of attachment to the nitrogen atom of Formula X, and the wavy line indicates the point of attachment to the Ligand unit;
p is 1 to 20; and
D is a Drug unit, wherein the Drug Unit is a PBD dimer of formula I:
wherein:
R2 is of formula II:
wherein A is a C5-7 aryl group, X is attached to the carbonyl carbon of Formula X and is selected from the group consisting of: —O—, —S—, —NH(C═O)—, and —N(RN)—, wherein RN is selected from the group consisting of H, C1-4 alkyl and (C2H4O)mCH3, wherein m is 1 to 3; and either:
(i) Q1 is a single bond and Q2 is selected from the group consisting of a single bond and —Z—(CH2)n—, wherein Z is selected from the group consisting of a single bond, O, S and NH and n is from 1 to 3, or
(ii) Q1 is —CH═CH— and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, C1-7 alkoxy, C1-7 alkyl, C3-7 heterocyclyl, dimethyl-aminopropyloxy, piperazinyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo, wherein R and R′ are independently selected from the group consisting of optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; and either:
(a) R10 is H, and R11 is OH or ORA, wherein RA is C1-4 alkyl,
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or
(c) R10 is H and R11 is SOzM, wherein z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms that are selected from the group consisting of O, S, and NH, or by an aromatic ring;
Y and Y′ are selected from the group consisting of O, S, and NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9, respectively, and R10′ and R11′ are the same as R10 and R11, respectively, wherein if R11 and R11′ are SOzM, then each M is a monovalent pharmaceutically acceptable cation or together is a divalent pharmaceutically acceptable cation;
wherein C3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S; and
wherein C3-7 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 7 ring atoms, of which 1 to 4 are heteroatoms selected from the group consisting of N, O and S.
2. The Conjugate according to claim 1 wherein -LU-D is
wherein the wavy line indicates covalent attachment to the Ligand unit.
3. The Conjugate according to claim 2 wherein the Ligand unit is an antibody or an antigen-binding fragment thereof.
4. The Conjugate according to claim 3 wherein the monoclonal antibody is a humanized 1F6 antibody.
5. A compound having formula:
G1-L1-L2-D,
or a pharmaceutically acceptable salt thereof,
wherein -L1-L2- has the structure of formula X′:
wherein E is of formula:
the asterisk indicates the point of attachment to a Drug Unit, and the way line indicates the point of attachment to G1, wherein:
G1- is selected from the group consisting of:
wherein n is 0 to 6;
wherein n is 0 to 6;
wherein n is 0 or 1, and m is 0 to 30;
wherein n is 0 or 1, and m is 0 to 30; and
wherein the asterisk indicates the point of attachment to the nitrogen atom of Formula X′, and D is a Drug unit, wherein the Drug Unit is a PBD dimer having formula I:
wherein:
R2 is of formula II:
wherein A is a C5-7 aryl group, X is attached to the carbonyl carbon of Formula X and is selected from the group consisting of: —O—, —S—, —NH(C═O)—, and —N(RN)—, wherein RN is selected from the group consisting of H, C1-4 alkyl and (C2H4O)mCH3, wherein m is 1 to 3; and either:
(i) Q1 is a single bond and Q2 is selected from the group consisting of a single bond and —Z—(CH2)n—, wherein Z is selected from the group consisting of a single bond, O, S and NH; and n is from 1 to 3, or
(ii) Q1 is —CH═CH— and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, ether, C1-7 alkoxy, C3-7 heterocyclyl, dimethyl-aminopropyloxy, piperazinyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo;
R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo, wherein R and R′ are independently selected from the group consisting of optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; and either:
(a) R10 is H, and R11 is OH or ORA, where RA is C1-4 alkyl,
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or
(c) R10 is H and R11 is SOzM, wherein z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms that are selected from the group consisting of O, S, and NH, and/or by an aromatic ring;
Y and Y′ are selected from the group consisting of O, S, and NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9, respectively, and R10′ and R11′ are the same as R10 and R11, respectively, wherein if R11 and R11′ are SOzM, then each M is a monovalent pharmaceutically acceptable cation, or together is a divalent pharmaceutically acceptable cation;
wherein C3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S; and
wherein C3-7 heterocyclyl is a monovalent moiet obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 7 ring atoms, of which 1 to 4 are heteroatoms selected from the group consisting of N, O and S.
6. The compound according to claim 5 wherein the compound is
7. A compound having the structure of
or a pharmaceutically acceptable salt thereof,
wherein L1 is a Specificity unit, wherein the Specificity unit is an amino acid sequence and is cleavable by the action of an enzyme;
D is a Drug unit, wherein the Drug unit is a PBD dimer of formula I:
wherein:
R2 is of formula II:
wherein A is a C5-7 aryl group, X is attached to the amino acid sequence of L1 through the carbonyl carbon of it C-terminal amino acid residue and is selected from the group consisting of —O—, —S—, —NH(C═O)—, and —N(RN)—, wherein RN is selected from the group consisting of H, C1-4 alkyl and (C2H4O)mCH3, wherein m is 1 to 3; and either:
(i) Q1 is a single bond and Q2 is selected from the group consisting of a single bond and —Z—(CH2)n—, wherein Z is selected from the group consisting of a single bond, O, S and NH and n is from 1 to 3, or
(ii) Q1 is —CH═CH— and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, C1-7 alkoxy, C1-7 alkyl, C3-7 heterocyclyl, dimethyl-aminopropyloxy, piperazinyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo, wherein R and R′ are independently selected from the group consisting of optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; and either:
(a) R10 is H, and R11 is OH or ORA, where RA is C1-4 alkyl,
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or
(c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms that are selected from the group consisting of O, S, and NH, and/or by an aromatic ring;
Y and Y′ are selected from the group consisting of O, S, and NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9, respectively, and R10′ and R11′ are the same as R10 and R11, respectively, wherein if R11 and R11′ are SOzM, then each M is a monovalent pharmaceutically acceptable cation, or together is a divalent pharmaceutically acceptable cation;
wherein C3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S; and
wherein C3-7 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 7 ring atoms, of which 1 to 4 are heteroatoms selected from the group consisting of N, O and S.
8. The compound according to claim 7, wherein L1 is —NH-X1-X2-CO— wherein -X1-X2- is selected from the group consisting of -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-, wherein —NH— of L1 is the amino group of X1 and CO of L1 is the carbonyl group of X2.
9. The compound according to claim 7, wherein L1 is —NH-X1-X2-CO— wherein -X1-X2- is selected from the group consisting of -Gly-Gly-, -Pro-Pro-, and -Val-Glu-, wherein —NH— of L1 is the amino group of X1 and CO of L1 is the carbonyl group of X2.
10. The compound according to claim 9, wherein the compound has the structure of:
11. A method of preparing a compound having the structure of
G1-L1-L2-D
wherein -L1- is a Specificity unit, wherein the Specificity unit is an amino acid sequence and is cleavable by the action of an enzyme;
L2 is a bond;
G1 is selected from the group consisting of:
wherein n is 0 to 6; and
wherein n is 0 or 1, and m is 0 to 30;
and wherein the asterisk indicates the point of attachment to L1;
D is a Drug unit, wherein the Drug unit is a PBD dimer of formula I:
wherein:
R2 is of formula II:
wherein A is a C5-7 aryl group, X is attached to the carbonyl carbon of Formula X and is selected from the group consisting of —O—, —S—, —NH(C═O)—, and —N(RN)—, wherein RN is selected from the group consisting of H, C1-4 alkyl and (C2H4O)mCH3, wherein m is 1 to 3; and either:
(i) Q1 is a single bond and Q2 is selected from the group consisting of a single bond and —Z—(CH2)n—, wherein Z is selected from the group consisting of a single bond, O, S and NH and n is from 1 to 3, or
(ii) Q1 is —CH═CH— and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, C1-7 alkoxy, C1-7 alkyl, C3-7 heterocyclyl, dimethyl-aminopropyloxy, piperazinyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo, wherein R and R′ are independently selected from the group consisting of optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; and either:
(a) R10 is H, and R11 is OH or ORA, wherein RA is C1-4 alkyl,
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or
(c) R10 is H and R11 is SOzM, wherein z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms that are selected from the group consisting of O, S, and NH, and/or by an aromatic ring;
Y and Y′ are selected from the group consisting of O, S, and NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9, respectively, and R10′ and R11′ are the same as R10 and R11, respectively, wherein if R11 and R11′ are SOzM, then each M is a monovalent pharmaceutically acceptable cation, or together is a divalent pharmaceutically acceptable cation,
said method comprising the step of condensing a compound of formula L1-L2-D, suitably protected, with L1, L2 and D as previously defined, with a precursor of G1 having the structure selected from the group consisting of:
wherein n is 0 to 6; and
wherein n is 0 or 1, and m is 0 to 30; and
wherein from said condensation G1 and L1 are attached to each other through an amide bond;
wherein C3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S; and
wherein C3-7 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 7 ring atoms, of which 1 to 4 are heteroatoms selected from the group consisting of N, O and S.
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11
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The present invention relates to targeted pyrrolobenzodiazepine (PBD) conjugates, in particular pyrrolobenzodiazepine dimers that are conjugated to a targeting agent via the C2 position of one of the monomers.
BACKGROUND TO THE INVENTION
Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:
They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position, which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). The ability of PBDs to form an adduct in the minor groove enables them to interfere with DNA processing, hence their use as antitumour agents.
The biological activity of these molecules can be potentiated by joining two PBD units together through their C8/C′-hydroxyl functionalities via a flexible alkylene linker (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity. One example of a PBD dimer is SG2000 (SJG-136):
(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C., et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al., Cancer Research, 64, 6693-6699 (2004)).
Due to the manner in which these highly potent compounds act in cross-linking DNA, PBD dimers have been made symmetrically, i.e., both monomers of the dimer are the same. This synthetic route provides for straightforward synthesis, either by constructing the PBD dimer moiety simultaneously having already formed the dimer linkage, or by reacting already constructed PBD monomer moieties with the dimer linking group. These synthetic approaches have limited the options for preparation of targeted conjugates containing PBDs. Due to the observed potency of PBD dimers, however, there exists a need for PBD dimers that are conjugatable to targeting agents for use in targeted therapy.
DISCLOSURE OF THE INVENTION
The present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein a PBD monomer has a substituent in the C2 position that provides an anchor for linking the compound to the targeting agent. The present invention also relates to Conjugates comprising dimers of PBDs conjugated to a targeting agent, wherein the PBD monomers of the dimer are different. One of PBD monomers has a substituent in the C2 position that provides an anchor for linking the compound to the targeting agent. The Conjugates described herein have potent cytotoxic and/or cytostatic activity against cells expressing a target molecule, such as cancer cells or immune cells. These conjugates exhibit good potency with reduced toxicity, as compared with the corresponding PBD dimer free drug compounds.
In some embodiments, the Conjugates have the following formula I:
L-(LU-D)p (I)
wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linker unit and D is a Drug unit comprising a PBD dimer. The subscript p is an integer of from 1 to 20. Accordingly, the Conjugates comprise a Ligand unit covalently linked to at least one Drug unit by a Linker unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. Accordingly, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease. These methods encompass the use of the Conjugates wherein the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.
In a first aspect, the Conjugates comprise a Conjugate of formula I (supra), wherein the Drug unit comprises a PBD dimer of the following formula II:
wherein:
R2 is of formula III:
where A is a C5-7 aryl group, X is an activatable group for conjugation to the Linker unit, wherein X is selected from the group comprising: —O—, —S—, —C(O)O—, —C(O)—, —NHC(O)—, and —N(RN)—, wherein RN is selected from the group comprising H, C1-4 alkyl and (C2H4O)mCH3, where m is 1 to 3, and either:
(i) Q1 is a single bond, and Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q1 is —CH═CH—, and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, optionally substituted C3-20 heterocyclyl and optionally substituted C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo; either:
(a) R10 is H, and R11 is OH or ORA, where RA is C1-4 alkyl;
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or
(c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y′ are selected from O, S, or NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11, wherein if R11 and R11′ are SOZM, M may represent a divalent pharmaceutically acceptable cation.
In a second aspect, the use of the Conjugate of formula I is provided for the manufacture of a medicament for treating a proliferative disease or autoimmune disease. In a related third aspect, the use of the Conjugate of formula I is provided for the treatment of a proliferative disease or an autoimmune disease.
In another aspect there is provided the use of a Conjugate of formula I to provide a PBD dimer, or a salt or solvate thereof, at a target location.
One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.
The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.
In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.
In a fourth aspect of the present invention comprises a method of making the Conjugates formula I.
The dimeric PBD compounds for use in the present invention are made by different strategies to those previously employed in making symmetrical dimeric PBD compounds. In particular, the present inventors have developed a method which involves adding each C2 aryl substituent to a symmetrical PBD dimer core in separate method steps.
Accordingly, a sixth aspect of the present invention provides a method of making a Conjugate of formula I, comprising at least one of the method steps described herein.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1 to 6 show the effect of conjugates of the present invention in tumours.
DEFINITIONS
When a trade name is used herein, reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.
Binding Agent and Targeting Agent
The terms “binding agent” and “targeting agent as used herein refer to a molecule, e.g., protein, polypeptide or peptide, that specifically binds to a target molecule. Examples can include a full length antibody, an antigen binding fragment of a full length antibody, other agent (protein, polypeptide or peptide) that includes an antibody heavy and/or light chain variable region that specifically bind to the target molecule, or an Fc fusion protein comprising an extracellular domain of a protein, peptide polypeptide that binds to the target molecule and that is joined to an Fc region, domain or portion thereof, of an antibody.
Specifically Binds
The terms “specifically binds” and “specific binding” refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1×107 M−1, and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
Pharmaceutically Acceptable Cations
Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.
The pharmaceutically acceptable cation may be inorganic or organic.
Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca2+ and Mg2+. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
Substituents
The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.
Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
Examples of substituents are described in more detail below.
C1-12 alkyl: The term “C1-12 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C7).
Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7).
Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).
C2-12 Alkenyl: The term “C2-12 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH═CH2), 1-propenyl (—CH═CH—CH3), 2-propenyl (allyl, —CH—CH═CH2), isopropenyl (1-methylvinyl, —C(CH3)═CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).
C2-12 alkynyl: The term “C2-12 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH2—C≡CH).
C3-12 cycloalkyl: The term “C3-12 cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
Examples of cycloalkyl groups include, but are not limited to, those derived from:
saturated monocyclic hydrocarbon compounds:
cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7);
unsaturated monocyclic hydrocarbon compounds:
cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and
saturated polycyclic hydrocarbon compounds:
norcarane (C7), norpinane (C7), norbornane (C7).
C3-20 heterocyclyl: The term “C3-20 heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
In this context, the prefixes (e.g. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:
N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);
S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3: trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);
N2O1: oxadiazine (C6);
O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1O1S1: oxathiazine (C6).
Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
C5-20 aryl: The term “C5-20 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.
In this context, the prefixes (e.g. C3-20, C5-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6 aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.
The ring atoms may be all carbon atoms, as in “carboaryl groups”.
Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).
Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (C16).
Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:
N1: pyrrole (azole) (C5), pyridine (azine) (C6);
O1: furan (oxole) (C5);
S1: thiophene (thiole) (C5);
N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);
N2O1: oxadiazole (furazan) (C5);
N3O1: oxatriazole (C5);
N1S1: thiazole (C5), isothiazole (C5);
N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); N3: triazole (C5), triazine (C6); and,
N4: tetrazole (C5).
Examples of heteroaryl which comprise fused rings, include, but are not limited to:
C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);
C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);
C11 (with 2 fused rings) derived from benzodiazepine (N2);
C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and,
C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxathiin (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.
Halo: —F, —Cl, —Br, and —I.
Hydroxy: —OH.
Ether: —OR, wherein R is an ether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7alkyl group.
Alkoxy: —OR, wherein R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).
Acetal: —CH(OR1)(OR2), wherein R1 and R2 are independently acetal substituents, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, or, in the case of a “cyclic” acetal group, R1 and R2, taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe)2, —CH(OEt)2, and —CH(OMe)(OEt).
Hemiacetal: —CH(OH)(OR1), wherein R1 is a hemiacetal substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).
Ketal: —CR(OR1)(OR2), where R1 and R2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe)2, —C(Me)(OEt)2, —C(Me)(OMe)(OEt), —C(Et)(OMe)2, —C(Et)(OEt)2, and —C(Et)(OMe)(OEt).
Hemiketal: —CR(OH)(OR1), where R1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).
Oxo (keto, -one): ═O.
Thione (thioketone): ═S.
Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.
Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.
Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C1-7 alkyl group (also referred to as C1-7 alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH3 (acetyl), —C(═O)CH2CH3 (propionyl), —C(═O)C(CH3)3 (t-butyryl), and —C(═O)Ph (benzoyl, phenone).
Carboxy (carboxylic acid): —C(═O)OH.
Thiocarboxy (thiocarboxylic acid): —C(═S)SH.
Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.
Thionocarboxy (thionocarboxylic acid): —C(═S)OH.
Imidic acid: —C(═NH)OH.
Hydroxamic acid: —C(═NOH)OH.
Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH3, —C(═O)OCH2CH3, —C(═O)OC(CH3)3, and —C(═O)OPh.
Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH3 (acetoxy), —OC(═O)CH2CH3, —OC(═O)C(CH3)3, —OC(═O)Ph, and —OC(═O)CH2Ph.
Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH3, —OC(═O)OCH2CH3, —OC(═O)OC(CH3)3, and —OC(═O)OPh.
Amino: —NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a “cyclic” amino group, R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH2), secondary (—NHR1), or tertiary (—NHR1R2), and in cationic form, may be quaternary (—+NR1R2R3). Examples of amino groups include, but are not limited to, —NH2, —NHCH3, —NHC(CH3)2, —N(CH3)2, —N(CH2CH3)2, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH2, —C(═O)NHCH3, —C(═O)N(CH3)2, —C(═O)NHCH2CH3, and —C(═O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
Thioamido (thiocarbamyl): —C(═S)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH2, —C(═S)NHCH3, —C(═S)N(CH3)2, and —C(═S)NHCH2CH3.
Acylamido (acylamino): —NR1C(═O)R2, wherein R1 is an amide substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group, and R2 is an acyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH3, —NHC(═O)CH2CH3, and —NHC(═O)Ph. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
Aminocarbonyloxy: —OC(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH2, —OC(═O)NHMe, —OC(═O)NMe2, and —OC(═O)NEt2.
Ureido: —N(R1)CONR2R3 wherein R2 and R3 are independently amino substituents, as defined for amino groups, and R1 is a ureido substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a c1-7 alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH2, —NHCONHMe, —NHCONHEt, —NHCONMe2, —NHCONEt2, —NMeCONH2, —NMeCONHMe, —NMeCONHEt, —NMeCONMe2, and —NMeCONEt2.
Guanidino: —NH—C(═NH)NH2.
Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,
Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt.
Amidine (amidino): —C(═NR)NR2, wherein each R is an amidine substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH2, —C(═NH)NMe2, and —C(═NMe)NMe2.
Nitro: —NO2.
Nitroso: —NO.
Azido: —N3.
Cyano (nitrile, carbonitrile): —CN.
Isocyano: —NC.
Cyanato: —OCN.
Isocyanato: —NCO.
Thiocyano (thiocyanato): —SCN.
Isothiocyano (isothiocyanato): —NCS.
Sulfhydryl (thiol, mercapto): —SH.
Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7 alkylthio group), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of C1-7 alkylthio groups include, but are not limited to, —SCH3 and —SCH2CH3.
Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group (also referred to herein as C1-7 alkyl disulfide). Examples of C1-7 alkyl disulfide groups include, but are not limited to, —SSCH3 and —SSCH2CH3.
Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH3 and —S(═O)CH2CH3.
Sulfone (sulfonyl): —S(═O)2R, wherein R is a sulfone substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, including, for example, a fluorinated or perfluorinated C1-7 alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)2CH3 (methanesulfonyl, mesyl), —S(═O)2CF3 (triflyl), —S(═O)2CH2CH3 (esyl), —S(═O)2C4F9 (nonaflyl), —S(═O)2CH2CF3 (tresyl), —S(═O)2CH2CH2NH2 (tauryl), —S(═O)2Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).
Sulfinic acid (sulfino): —S(═O)OH, —SO2H.
Sulfonic acid (sulfo): —S(═O)2OH, —SO3H.
Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH3 (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).
Sulfonate (sulfonic acid ester): —S(═O)2OR, wherein R is a sulfonate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)2OCH3 (methoxysulfonyl; methyl sulfonate) and —S(═O)2OCH2CH3 (ethoxysulfonyl; ethyl sulfonate).
Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH3 and —OS(═O)CH2CH3.
Sulfonyloxy: —OS(═O)2R, wherein R is a sulfonyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group.
Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)2CH3 (mesylate) and —OS(═O)2CH2CH3 (esylate).
Sulfate: —OS(═O)2OR; wherein R is a sulfate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)2OCH3 and —SO(═O)2OCH2CH3.
Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH2, —S(═O)NH(CH3), —S(═O)N(CH3)2, —S(═O)NH(CH2CH3), —S(═O)N(CH2CH3)2, and —S(═O)NHPh.
Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)2NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O)2NH2, —S(═O)2NH(CH3), —S(═O)2N(CH3)2, —S(═O)2NH(CH2CH3), —S(═O)2N(CH2CH3)2, and —S(═O)2NHPh.
Sulfamino: —NR1S(═O)2OH, wherein R1 is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)2OH and —N(CH3)S(═O)2OH.
Sulfonamino: —NR1S(═O)2R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)2CH3 and —N(CH3)S(═O)2C6H5.
Sulfinamino: —NR1S(═O)R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH3 and —N(CH3)S(═O)C6H5.
Phosphino (phosphine): —PR2, wherein R is a phosphino substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphino groups include, but are not limited to, —PH2, —P(CH3)2, —P(CH2CH3)2, —P(t-Bu)2, and —P(PH)2.
Phospho: —P(═O)2.
Phosphinyl (phosphine oxide): —P(═O)R2, wherein R is a phosphinyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group or a C5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH3)2, —P(═O)(CH2CH3)2, —P(═O)(t-Bu)2, and —P(═O)(Ph)2.
Phosphonic acid (phosphono): —P(═O)(OH)2.
Phosphonate (phosphono ester): —P(═O)(OR)2, where R is a phosphonate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH3)2, —P(═O)(OCH2CH3)2, —P(═O)(O-t-Bu)2, and —P(═O)(OPh)2.
Phosphoric acid (phosphonooxy): —OP(═O)(OH)2.
Phosphate (phosphonooxy ester): —OP(═O)(OR)2, where R is a phosphate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably-H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH3)2, —OP(═O)(OCH2CH3)2, —OP(═O)(O-t-Bu)2, and —OP(═O)(OPh)2.
Phosphorous acid: —OP(OH)2.
Phosphite: —OP(OR)2, where R is a phosphite substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH3)2, —OP(OCH2CH3)2, —OP(O-t-Bu)2, and —OP(OPh)2.
Phosphoramidite: —OP(OR1)—NR22, where R1 and R2 are phosphoramidite substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH2CH3)—N(CH3)2, —OP(OCH2CH3)—N(i-Pr)2, and —OP(OCH2CH2CN)—N(i-Pr)2.
Phosphoramidate: —OP(═O)(OR1)—NR22, where R1 and R2 are phosphoramidate substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH2CH3)—N(CH3)2, —OP(═O)(OCH2CH3)—N(i-Pr)2, and —OP(═O)(OCH2CH2CN)—N(i-Pr)2.
Alkylene
C3-12 alkylene: The term “C3-12 alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
Examples of linear saturated C3-12 alkylene groups include, but are not limited to, —(CH2)n— where n is an integer from 3 to 12, for example, —CH2CH2CH2— (propylene), —CH2CH2CH2CH2— (butylene), —CH2CH2CH2CH2CH2— (pentylene) and —CH2CH2CH2CH—2CH2CH2CH2— (heptylene).
Examples of branched saturated C3-12 alkylene groups include, but are not limited to, —CH(CH3)CH2—, —CH(CH3)CH2CH2—, —CH(CH3)CH2CH2CH2—, —CH2CH(CH3)CH2—, —CH2CH(CH3)CH2CH2—, —CH(CH2CH3)—, —CH(CH2CH3)CH2—, and —CH2CH(CH2CH3)CH2—.
Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH2—, —CH2—CH═CH2—, —CH═CH—CH2—CH2—, —CH═CH—CH2—CH2—CH2—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH2—, —CH═CH—CH═CH—CH2—CH2—, —CH═CH—CH2—CH═CH—, —CH═CH—CH2—CH2—CH═CH—, and —CH2—. C≡C—CH2—.
Examples of branched partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, —C(CH3)═CH—, —C(CH3)═CH—CH2—, —CH═CH—CH(CH3)— and —C≡C—CH(CH3)—.
Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).
Examples of alicyclic partially unsaturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).
Oxygen protecting group: the term “oxygen protecting group” refers to a moiety which masks a hydroxy group, and these are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. Classes of particular interest include silyl ethers (e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g. acetate).
Carbamate nitrogen protecting group: the term “carbamate nitrogen protecting group” pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogen protecting group” pertains to a group having the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides Conjugates comprising a PBD dimer connected to a Ligand unit via a Linker Unit. In one embodiment, the Linker unit includes a Stretcher unit (A), a Specificity unit (L1), and a Spacer unit (L2). The Linker unit is connected at one end to the Ligand unit and at the other end to the PBD dimer compound.
In one aspect, such a Conjugate is shown below in formula Ia:
L-(A1a-L1s-L2y-D)p (Ia)
wherein:
L is the Ligand unit; and
-A1a-L1s-L2y- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit,
a is 1 or 2,
L1- is a Specificity unit,
s is an integer ranging from 1 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is an PBD dimer; and
p is from 1 to 20.
The drug loading is represented by p, the number of drug molecules per Ligand unit (e.g., an antibody). Drug loading may range from 1 to 20 Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20.
In some embodiments, p is from about 1 to about 8 Drug units per Ligand unit. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is from about 2 to about 8 Drug units per Ligand unit. In some embodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4 Drug units per Ligand unit. In some embodiments, p is about 2, about 4, about 6 or about 8 Drug units per Ligand unit.
The average number of Drugs units per Ligand unit in a preparation from a conjugation reaction may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Conjugates, where p is a certain value, from Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
In another aspect, such a Conjugate is shown below in formula Ib:
Also illustrated as:
L-(A1a-L2y(-L1s)-D)p (Ib)
wherein:
L is the Ligand unit; and
-A1a-L1s(L2y)- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit linked to a Stretcher unit (L2),
a is 1 or 2,
L1- is a Specificity unit linked to a Stretcher unit (L2),
s is an integer ranging from 0 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is a PBD dimer; and
p is from 1 to 20.
Preferences
The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
In one embodiment, the Conjugate has the formula:
L-(A1a-L1s-L2y-D)p
wherein L, A1, a, L1, s, L2, D and p are as described above.
In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit, which is a covalent bond, a self-immolative group or together with —OC(═O)— forms a self-immolative group, and L2 optional.
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
CBA-A1aL1s-L2y-*
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit which is a covalent bond or a self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is 0 or 1 or 2.
In the embodiments illustrated above, L1 can be a cleavable Specificity unit, and may be referred to as a “trigger” that when cleaved activates a self-immolative group (or self-immolative groups) L2, when a self-immolative group(s) is present. When the Specificity unit L1 is cleaved, or the linkage (i.e., the covalent bond) between L1 and L2 is cleaved, the self-immolative group releases the Drug unit (D).
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug (D), CBA is the Cell Binding Agent, L1 is a Specificity unit connected to L2, A1 is a Stretcher unit connecting L2 to the Cell Binding Agent, L2 is a self-immolative group, and a is 1 or 2, s is 1 or 2, and y is 1 or 2.
In the various embodiments discussed herein, the nature of L1 and L2 can vary widely. These groups are chosen on the basis of their characteristics, which may be dictated in part, by the conditions at the site to which the conjugate is delivered. Where the Specificity unit L1 is cleavable, the structure and/or sequence of L1 is selected such that it is cleaved by the action of enzymes present at the target site (e.g., the target cell). L1 units that are cleavable by changes in pH (e.g. acid or base labile), temperature or upon irradiation (e.g. photolabile) may also be used. L1 units that are cleavable under reducing or oxidising conditions may also find use in the Conjugates.
In some embodiments, L1 may comprise one amino acid or a contiguous sequence of amino acids. The amino acid sequence may be the target substrate for an enzyme.
In one embodiment, L1 is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L1 may be cleaved by a lysosomal protease, such as a cathepsin.
In one embodiment, L2 is present and together with —C(═O)O— forms a self-immolative group or self-immolative groups. In some embodiments, —C(═O)O— also is a self-immolative group.
In one embodiment, where L1 is cleavable by the action of an enzyme and L2 is present, the enzyme cleaves the bond between L1 and L2, whereby the self-immolative group(s) release the Drug unit.
L1 and L2, where present, may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH, and
—O— (a glycosidic bond).
An amino group of L1 that connects to L2 may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain.
A carboxyl group of L1 that connects to L2 may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain.
A hydroxy group of L1 that connects to L2 may be derived from a hydroxy group of an amino acid side chain, for example a serine amino acid side chain.
In one embodiment, —C(═O)O— and L2 together form the group:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to the L1, Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The phenylene ring is optionally substituted with one, two or three substituents as described herein.
In one embodiment, Y is NH.
In one embodiment, n is 0 or 1. Preferably, n is 0.
Where Y is NH and n is 0, the self-immolative group may be referred to as a p-aminobenzylcarbonyl linker (PABC).
The self-immolative group will allow for release of the Drug unit (i.e., the asymmetric PBD) when a remote site in the linker is activated, proceeding along the lines shown below (for n=0):
where the asterisk indicates the attachment to the Drug, L* is the activated form of the remaining portion of the linker and the released Drug unit is not shown. These groups have the advantage of separating the site of activation from the Drug.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above. Each phenylene ring is optionally substituted with one, two or three substituents as described herein. In one embodiment, the phenylene ring having the Y substituent is optionally substituted and the phenylene ring not having the Y substituent is unsubstituted.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or CR.
In one embodiment, D is N.
In one embodiment, D is CH.
In one embodiment, E is O or S.
In one embodiment, F is CH.
In a preferred embodiment, the covalent bond between L1 and L2 is a cathepsin labile (e.g., cleavable) bond.
In one embodiment, L1 comprises a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is -Phe-Lys-, Val-Cit or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described by Dubowchik et al., which is incorporated herein by reference.
In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective groups in Organic Synthesis, Greene and Wuts.
Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:
Arg: Z, Mtr, Tos;
Asn: Trt, Xan;
Asp: Bzl, t-Bu;
Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;
Glu: Bzl, t-Bu;
Gln: Trt, Xan;
His: Boc, Dnp, Tos, Trt;
Lys: Boc, Z—Cl, Fmoc, Z;
Ser: Bzl, TBDMS, TBDPS;
Thr: Bz;
Trp: Boc;
Tyr: Bzl, Z, Z—Br.
In one embodiment, —X2— is connected indirectly to the Drug unit. In such an embodiment, the Spacer unit L2 is present.
In one embodiment, the dipeptide is used in combination with a self-immolative group(s) (the Spacer unit). The self-immolative group(s) may be connected to —X2—.
Where a self-immolative group is present, —X2— is connected directly to the self-immolative group. In one embodiment, —X2— is connected to the group Y of the self-immolative group. Preferably the group —X2—CO— is connected to Y, where Y is NH.
—X1— is connected directly to A1. In one embodiment, —X1— is connected directly to A1. Preferably the group NH—X1— (the amino terminus of X1) is connected to A1. A1 may comprise the functionality —CO— thereby to form an amide link with —X1—.
In one embodiment, L1 and L2 together with —OC(═O)— comprise the group —X1—X2-PABC-. The PABC group is connected directly to the Drug unit. In one example, the self-immolative group and the dipeptide together form the group -Phe-Lys-PABC-, which is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
Alternatively, the self-immolative group and the dipeptide together form the group -Val-Ala-PABC-, which is illustrated below:
where the asterisk and the wavy line are as defined above.
In another embodiment, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group, and E is a group that is susceptible to cleavage thereby to activate a self-immolative group.
E is selected such that the group is susceptible to cleavage, e.g., by light or by the action of an enzyme. E may be —NO2 or glucuronic acid (e.g., β-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a β-glucuronidase.
The group Y may be a covalent bond.
The group Y may be a functional group selected from:
—C(═O)—
—NH—
—O—
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH,
—C(═O)NHC(═O)—,
SO2, and
—S—.
The group Y is preferably —NH—, —CH2—, —O—, and —S—.
In some embodiments, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—.
In some embodiments, L1 and L2 together represent:
where the asterisk indicates the point of attachment to the remainder of L2 or the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—, —CH2—, —O—, and —S—.
In some further embodiments, Y is a functional group as set forth above, the functional group is linked to an amino acid, and the amino acid is linked to the Stretcher unit A1. In some embodiments, amino acid is β-alanine. In such an embodiment, the amino acid is equivalently considered part of the Stretcher unit.
The Specificity unit L1 and the Ligand unit are indirectly connected via the Stretcher unit.
L1 and A1 may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the connection between the Ligand unit and A1 is through a thiol residue of the Ligand unit and a maleimide group of A1.
In one embodiment, the connection between the Ligand unit and A1 is:
where the asterisk indicates the point of attachment to the remaining portion of A1, L1, L2 or D, and the wavy line indicates the point of attachment to the remaining portion of the Ligand unit. In this embodiment, the S atom is typically derived from the Ligand unit.
In each of the embodiments above, an alternative functionality may be used in place of the malemide-derived group shown below:
where the wavy line indicates the point of attachment to the Ligand unit as before, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with the group:
where the wavy line indicates point of attachment to the Ligand unit, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with a Ligand unit (e.g., a Cell Binding Agent), is selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH,
—C(═O)NHC(═O)—,
—S—,
—S—S—,
—CH2C(═O)—
—C(═O)CH2—,
═N—NH—, and
—NH—N═.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the Ligand unit, is selected from:
where the wavy line indicates either the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group, and the asterisk indicates the other of the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group.
Other groups suitable for connecting L1 to the Cell Binding Agent are described in WO 2005/082023.
In one embodiment, the Stretcher unit A1 is present, the Specificity unit L′ is present and Spacer unit L2 is absent. Thus, L1 and the Drug unit are directly connected via a bond. Equivalently in this embodiment, L2 is a bond.
L1 and D may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, L1 and D are preferably connected by a bond selected from:
—C(═O)NH—, and
—NHC(═O)—.
In one embodiment, L1 comprises a dipeptide and one end of the dipeptide is linked to D. As described above, the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is -Phe-Lys- or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described above.
In one embodiment, L1-D is:
where —NH—X1—X2—CO is the dipeptide, —NH— is part of the Drug unit, the asterisk indicates the point of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
In one embodiment, the dipeptide is valine-alanine and L′-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is phenylalanine-lysine and L′-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is valine-citrulline.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3 or 7.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 is:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the rest of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4 or 8. In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the Stretcher unit is an acetamide unit, having the formula:
where the asterisk indicates the point of attachment to the remainder of the Stretcher unit, L1 or D, and the wavy line indicates the point of attachment to the Ligand unit.
In other embodiments, Linker-Drug compounds are provided for conjugation to a Ligand unit. In one embodiment, the Linker-Drug compounds are designed for connection to a Cell Binding Agent.
In one embodiment, the Drug Linker compound has the formula:
where the asterisk indicates the point of attachment to the Drug unit, G1 is a Stretcher group (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or together with —OC(═O)— forms a self-immolative group(s).
In another embodiment, the Drug Linker compound has the formula:
G1-L1-L2-*
where the asterisk indicates the point of attachment to the Drug unit, G1 is a Stretcher unit (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or a self-immolative group(s).
L1 and L2 are as defined above. References to connection to A1 can be construed here as referring to a connection to G1.
In one embodiment, where L1 comprises an amino acid, the side chain of that amino acid may be protected. Any suitable protecting group may be used. In one embodiment, the side chain protecting groups are removable with other protecting groups in the compound, where present. In other embodiments, the protecting groups may be orthogonal to other protecting groups in the molecule, where present.
Suitable protecting groups for amino acid side chains include those groups described in the Novabiochem Catalog 2006/2007. Protecting groups for use in a cathepsin labile linker are also discussed in Dubowchik et al.
In certain embodiments of the invention, the group L1 includes a Lys amino acid residue. The side chain of this amino acid may be protected with a Boc or Alloc protected group. A Boc protecting group is most preferred.
The functional group G1 forms a connecting group upon reaction with a Ligand unit (e.g., a cell binding agent.
In one embodiment, the functional group G1 is or comprises an amino, carboxylic acid, hydroxy, thiol, or maleimide group for reaction with an appropriate group on the Ligand unit. In a preferred embodiment, G1 comprises a maleimide group.
In one embodiment, the group G1 is an alkyl maleimide group. This group is suitable for reaction with thiol groups, particularly cysteine thiol groups, present in the cell binding agent, for example present in an antibody.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In each of the embodiments above, an alternative functionality may be used in place of the malemide group shown below:
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide-derived group is replaced with the group:
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide group is replaced with a group selected from:
—C(═O)OH,
—OH,
—NH2,
—SH,
—C(═O)CH2X, where X is Cl, Br or I,
—CHO,
—NHNH2
—C≡CH, and
—N3 (azide).
In one embodiment, L1 is present, and G1 is —NH2, —NHMe, —COOH, —OH or —SH.
In one embodiment, where L1 is present, G1 is —NH2 or —NHMe. Either group may be the N-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is —NH2, and L1 is an amino acid sequence —X1—X2—, as defined above.
In one embodiment, L1 is present and G1 is COOH. This group may be the C-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is OH.
In one embodiment, L1 is present and G1 is SH.
The group G1 may be convertable from one functional group to another. In one embodiment, L1 is present and G1 is —NH2. This group is convertable to another group G1 comprising a maleimide group. For example, the group —NH2 may be reacted with an acids or an activated acid (e.g., N-succinimide forms) of those G1 groups comprising maleimide shown above.
The group G1 may therefore be converted to a functional group that is more appropriate for reaction with a Ligand unit.
As noted above, in one embodiment, L1 is present and G1 is —NH2, —NHMe, —COOH, —OH or —SH. In a further embodiment, these groups are provided in a chemically protected form. The chemically protected form is therefore a precursor to the linker that is provided with a functional group.
In one embodiment, G1 is —NH2 in a chemically protected form. The group may be protected with a carbamate protecting group. The carbamate protecting group may be selected from the group consisting of:
Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.
Preferably, where G1 is —NH2, it is protected with an Alloc or Fmoc group.
In one embodiment, where G1 is —NH2, it is protected with an Fmoc group.
In one embodiment, the protecting group is the same as the carbamate protecting group of the capping group.
In one embodiment, the protecting group is not the same as the carbamate protecting group of the capping group. In this embodiment, it is preferred that the protecting group is removable under conditions that do not remove the carbamate protecting group of the capping group.
The chemical protecting group may be removed to provide a functional group to form a connection to a Ligand unit. Optionally, this functional group may then be converted to another functional group as described above.
In one embodiment, the active group is an amine. This amine is preferably the N-terminal amine of a peptide, and may be the N-terminal amine of the preferred dipeptides of the invention.
The active group may be reacted to yield the functional group that is intended to form a connection to a Ligand unit.
In other embodiments, the Linker unit is a precursor to the Linker unit having an active group. In this embodiment, the Linker unit comprises the active group, which is protected by way of a protecting group. The protecting group may be removed to provide the Linker unit having an active group.
Where the active group is an amine, the protecting group may be an amine protecting group, such as those described in Green and Wuts.
The protecting group is preferably orthogonal to other protecting groups, where present, in the Linker unit.
In one embodiment, the protecting group is orthogonal to the capping group. Thus, the active group protecting group is removable whilst retaining the capping group. In other embodiments, the protecting group and the capping group is removable under the same conditions as those used to remove the capping group.
In one embodiment, the Linker unit is:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of the Linker unit, as applicable or the point of attachment to G1. Preferably, the wavy line indicates the point of attachment to G1.
In one embodiment, the Linker unit is:
where the asterisk and the wavy line are as defined above.
Other functional groups suitable for use in forming a connection between L1 and the Cell Binding Agent are described in WO 2005/082023.
Ligand Unit
The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non-peptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.
Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).
In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; fully human antibody; or a single chain antibody. One embodiment the antibody is a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab′, F(ab′)2 and Fv fragments.
The antibody may be a diabody, a domain antibody (DAB) or a single chain antibody.
In one embodiment, the antibody is a monoclonal antibody.
Antibodies for use in the present invention include those antibodies described in WO 2005/082023 which is incorporated herein. Particularly preferred are those antibodies for tumour-associated antigens. Examples of those antigens known in the art include, but are not limited to, those tumour-associated antigens set out in WO 2005/082023. See, for instance, pages 41-55.
In some embodiments, the conjugates are designed to target tumour cells via their cell surface antigens. The antigens may be cell surface antigens which are either over-expressed or expressed at abnormal times or cell types. Preferably, the target antigen is expressed only on proliferative cells (preferably tumour cells); however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue.
Antibodies have been raised to target specific tumour related antigens including:
Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2.
The Ligand unit is connected to the Linker unit. In one embodiment, the Ligand unit is connected to A, where present, of the Linker unit.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a thioether bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a disulfide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an amide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an ester bond.
In one embodiment, the connection between the Ligand unit and the Linker is formed between a thiol group of a cysteine residue of the Ligand unit and a maleimide group of the Linker unit.
The cysteine residues of the Ligand unit may be available for reaction with the functional group of the Linker unit to form a connection. In other embodiments, for example where the Ligand unit is an antibody, the thiol groups of the antibody may participate in interchain disulfide bonds. These interchain bonds may be converted to free thiol groups by e.g. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit.
In some embodiments, the cysteine residue is an introduced into the heavy or light chain of an antibody. Positions for cysteine insertion by substitution in antibody heavy or light chains include those described in Published U.S. Application No. 2007-0092940 and International Patent Publication WO2008070593, which are incorporated herein.
Methods of Treatment
The Conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a Conjugate of formula I. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount of a Conjugate administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose.
In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 1 mg/kg per dose.
A conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy).
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a Conjugate of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Includes Other Forms
Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO−), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N+HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O−), a salt or solvate thereof, as well as conventional protected forms.
Salts
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound (the Conjugate), for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).
For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO−), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations such as Al+3. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
If the Conjugate is cationic, or has a functional group which may be cationic (e.g. —NH2 may be —NH3+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
Solvates
It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the Conjugate(s). The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active Conjugate, salt of active Conjugate) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
Carbinolamines
The invention includes Conjugate where a solvent adds across the imine bond of the PBD moiety, which is illustrated below for a PBD monomer where the solvent is water or an alcohol (RAOH, where RA is C1-4 alkyl):
These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.
These particular compounds may be isolated in solid form, for example, by lyophilisation.
Isomers
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).
Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like.
Unless otherwise specified, a reference to a particular compound or Conjugate includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
General Synthetic Routes
The synthesis of PBD dimer compounds is extensively discussed in the following references, which discussions are incorporated herein by reference:
a) WO 00/12508 (pages 14 to 30);
b) WO 2005/023814 (pages 3 to 10);
c) WO 2004/043963 (pages 28 to 29); and
d) WO 2005/085251 (pages 30 to 39).
Synthesis Route
The Conjugates of the present invention, where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, can be synthesised from a compound of Compound formula 2:
where R2, R6, R7, R9, R6′, R7′, R9′, R12, X, X′ and R″ are as defined for compounds of formula II, ProtN is a nitrogen protecting group for synthesis and ProtO is a protected oxygen group for synthesis or an oxo group, by deprotecting the imine bond by standard methods.
The compound produced may be in its carbinolamine or carbinolamine ether form depending on the solvents used. For example if ProtN is Alloc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried using palladium to remove the N10 protecting group, followed by the elimination of the oxygen protecting group for synthesis. If ProtN is Troc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried out using a Cd/Pb couple to yield the compound of formula (I). If ProtN is SEM, or an analogous group, and ProtO is an oxo group, then the oxo group can be removed by reduction, which leads to a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group, followed by the elimination of water. The reduction of the compound of Compound formula 2 can be accomplished by, for example, lithium tetraborohydride, whilst a suitable means for removing the SEM protecting group is treatment with silica gel.
Compounds of Compound formula 2 can be synthesised from a compound of Compound formula 3a:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling an organometallic derivative comprising R12, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of Compound formula 2 can be synthesised from a compound of Compound formula 3b:
where R12, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling an organometallic derivative comprising R2, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of Compound formulae 3a and 3b can be synthesised from a compound of formula 4:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallic derivative, such as an organoboron derivative, comprising R2 or R12.
The couplings described above are usually carried out in the presence of a palladium catalyst, for example Pd(PPh3)4, Pd(OCOCH3)2, PdCl2, or Pd2(dba)3. The coupling may be carried out under standard conditions, or may also be carried out under microwave conditions.
The two coupling steps are usually carried out sequentially. They may be carried out with or without purification between the two steps. If no purification is carried out, then the two steps may be carried out in the same reaction vessel. Purification is usually required after the second coupling step. Purification of the compound from the undesired by-products may be carried out by column chromatography or ion-exchange separation.
The synthesis of compounds of Compound formula 4 where ProtO is an oxo group and ProtN is SEM are described in detail in WO 00/12508, which is incorporated herein by reference. In particular, reference is made to scheme 7 on page 24, where the above compound is designated as intermediate P. This method of synthesis is also described in WO 2004/043963.
The synthesis of compounds of Compound formula 4 where ProtO is a protected oxygen group for synthesis are described in WO 2005/085251, which synthesis is herein incorporated by reference.
Compounds of formula I where R10 and R10′ are H and R11 and R11′ are SON, can be synthesised from compounds of formula I where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, by the addition of the appropriate bisulphite salt or sulphinate salt, followed by an appropriate purification step. Further methods are described in GB 2 053 894, which is herein incorporated by reference.
Nitrogen Protecting Groups for Synthesis
Nitrogen protecting groups for synthesis are well known in the art. In the present invention, the protecting groups of particular interest are carbamate nitrogen protecting groups and hemi-aminal nitrogen protecting groups.
Carbamate nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.
Other possible groups are nitrobenzyloxycarbonyl (e.g. 4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.
Those protecting groups which can be removed with palladium catalysis are not preferred, e.g. Alloc.
Hemi-aminal nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. The groups disclosed herein can be applied to compounds for use in the present invention. Such groups include, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, and Cl3CCH2OCH2—.
Protected Oxygen Group for Synthesis
Protected oxygen group for synthesis are well known in the art. A large number of suitable oxygen protecting groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates.
Preferred oxygen protecting groups include acetates, TBS and THP.
Further Preferences
The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
In some embodiments, R6′, R7′, R9′, R10′, R11′ and Y′ are preferably the same as R6, R7, R9, R10, R11 and Y respectively.
Dimer Link
Y and Y′ are preferably O.
R″ is preferably a C3-7 alkylene group with no substituents. More preferably R″ is a C3, C5 or C7 alkylene.
R6 to R9
R9 is preferably H.
R6 is preferably selected from H, OH, OR, SH, NH2, nitro and halo, and is more preferably H or halo, and most preferably is H.
R7 is preferably selected from H, OH, OR, SH, SR, NH2, NHR, NRR′, and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C1-7 alkyl, C3-10 heterocyclyl and C5-10 aryl groups. R may be more preferably a C1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH2Ph.
These preferences apply to R9′, R6′ and R7′ respectively.
R2
A in R2 may be phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl. In other embodiments, A is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
X is a group selected from the list comprising: —O—, —S—, —C(O)O—, —C(O)—, —NH(C═O)— and N(RN)—, wherein RN is selected from the group comprising H and C1-4 alkyl. X may preferably be: —O—, —S—, —C(O)O—, —NH(C═O)— or —NH—, and may more preferably be: —O—, —S—, or —NH—, and most preferably is —NH—.
Q2-X may be on any of the available ring atoms of the C5-7 aryl group, but is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group (A) is phenyl, the substituent (Q2-X) is preferably in the meta- or para-positions, and more preferably is in the para-position. 1
In some embodiments, Q1 is a single bond. In these embodiments, Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and is from 1 to 3. In some of these embodiments, Q2 is a single bond. In other embodiments, Q2 is —Z—(CH2)n—. In these embodiments, Z may be O or S and n may be 1 or n may be 2.
In other of these embodiments, Z may be a single bond and n may be 1.
In other embodiments, Q1 is —CH═CH—.
In some embodiments, R2 may be -A-CH2—X and -A-X. In these embodiments, X may be —O—, —S—, —C(O)O—, —C(O)— and —NH—. In particularly preferred embodiments, X may be —NH—.
R12
R12 may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R12 is preferably phenyl. In other embodiments, R12 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
R12 may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
R12 may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
Where R12 is C5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para-positions, and more preferably is in the para-position.
Where R12 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R12 Substituents
If a substituent on R12 is halo, it is preferably F or Cl, more preferably Cl.
If a substituent on R12 is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g. phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
If a substituent on R12 is C1-7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propyl, butyl).
If a substituent on R12 is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
If a substituent on R12 is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
Particularly preferred substituents for R12 include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl. Another particularly preferred substituent for R12 is dimethylaminopropyloxy.
R12 Groups
Particularly preferred substituted R12 groups include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R12 group is 4-nitrophenyl.
M and z
It is preferred that M and M′ are monovalent pharmaceutically acceptable cations, and are more preferably Na+.
z is preferably 3.
EXAMPLES
General Experimental Methods
Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. 1H and 13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt (NovaBiochem) and solid-supported reagents (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied without further purification. Anhydrous solvents were prepared by distillation under a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C.
Compound 1b was synthesised as described in WO 00/012508 (compound 210), which is herein incorporated by reference.
General LC/MS conditions: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then 5% B to 95% B within 3 min. The composition was held for 0.5 min at 95% B, and then returned to 5% B in 0.3 minutes. Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm
LC/MS conditions specific for compounds protected by both a Troc and a TBDMs group: Chromatographic separation of Troc and TBDMS protected compounds was performed on a Waters Alliance 2695 HPLC system utilizing a Onyx Monolitic reversed-phase column (3 μm particles, 50×4.6 mm) from Phenomenex Corp. Mobile-phase A consisted of 5% acetonitrile-95% water containing 0.1% formic acid, and mobile phase B consisted of 95% acetonitrile-5% water containing 0.1% formic acid. After 1 min at 5% B, the proportion of B was raised to 95% B over the next 2.5 min and maintained at 95% B for a further 1 min, before returning to 95% A in 10 s and re-equilibration for a further 50 sec, giving a total run time of 5.0 min. The flow rate was maintained at 3.0 mL/min.
LC/MS conditions specific for compound 33: LC was run on a Waters 2767 sample Manager coupled with a Waters 2996 photodiode array detector and a Waters ZQ single quadruple mass Spectrometer. The column used was Luna Phenyl-Hexyl 150×4.60 mm, 5 μm, Part no. 00E-4257-E0 (Phenomenex). The mobile phases employed were:
Mobile phase A: 100% of HPLC grade water (0.05% triethylamine), pH=7
Mobile phase B: 20% of HPLC grade water and 80% of HPLC grade acetonitrile (0.05% triethylamine), pH=7
The gradients used were:
Time
Flow Rate
(min)
(ml/min)
% A
% B
Initial
1.50
90
10
1.0
1.50
90
10
16.0
1.50
64
36
30.0
1.50
5
95
31.0
1.50
90
10
32.0
1.50
90
10
Mass Spectrometry was carried out in positive ion mode and SIR (selective ion monitor) and the ion monitored was m/z=727.2.
Synthesis of Key Intermediates
(a) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2a)
Method A: A catalytic amount of DMF (2 drops) was added to a stirred solution of the nitro-acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride (0.95 mL, 1.36 g, 10.7 mmol) in dry THF (20 mL). The reaction mixture was allowed to stir for 16 hours at room temperature and the solvent was removed by evaporation in vacuo. The resulting residue was re-dissolved in dry THF (20 mL) and the acid chloride solution was added dropwise to a stirred mixture of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (859 mg, 4.73 mmol) and TEA (6.6 mL, 4.79 g, 47.3 mmol) in THF (10 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for a further 3 hours after which time TLC (95:5 v/v CHCl3/MeOH) and LC/MS (2.45 min (ES+) m/z (relative intensity) 721 ([M+H]+., 20)) revealed formation of product. Excess THF was removed by rotary evaporation and the resulting residue was dissolved in DCM (50 mL). The organic layer was washed with 1N HCl (2×15 mL), saturated NaHCO3 (2×15 mL), H2O (20 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent gave the crude product as a dark coloured oil. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96:4 v/v CHCl3/MeOH) isolated the pure amide 2a as an orange coloured glass (840 mg, 54%).
Method B: Oxalyl chloride (9.75 mL, 14.2 g, 111 mmol) was added to a stirred suspension of the nitro-acid 1a (17.3 g, 37.1 mmol) and DMF (2 mL) in anhydrous DCM (200 mL). Following initial effervescence the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. Conversion to the acid chloride was confirmed by treating a sample of the reaction mixture with MeOH and the resulting bis-methyl ester was observed by LC/MS. The majority of solvent was removed by evaporation in vacuo, the resulting concentrated solution was re-dissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40° C. The solid acid chloride was added portionwise over a period of 25 minutes to a stirred suspension of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (15.2 g, 84.0 mmol) and TEA (25.7 mL, 18.7 g, 185 mmol) in DCM (150 mL) at −40° C. (dry ice/CH3CN). Immediately, the reaction was complete as judged by LC/MS (2.47 min (ES+) m/z (relative intensity) 721 ([M+H]+., 100)). The mixture was diluted with DCM (150 mL) and washed with 1N HCl (300 mL), saturated NaHCO3 (300 mL), brine (300 mL), filtered (through a phase separator) and the solvent evaporated in vacuo to give the pure product 2a as an orange solid (21.8 g, 82%).
Analytical Data: [α]22D=−46.1° (c=0.47, CHCl3); 1H NMR (400 MHz, CDCl3) (rotamers) δ 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H, J=11.1 Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); 13C NMR (100 MHz, CDCl3) (rotamers) δ 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7, 108.2, 69.7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR, CHCl3) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211, 1177, 1072, 1050, 1008, 871 cm−1; MS (ES+) m/z (relative intensity) 721 ([M]+., 47), 388 (80); HRMS [M+H]+. theoretical C31H36N4O16 m/z 721.2199. found (ES+) m/z 721.2227.
(a) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2b)
Preparation from 1b according to Method B gave the pure product as an orange foam (75.5 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 749 ([M+H]+., 100).
(b) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3a)
Method A: A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) was added to a solution of the nitro-ester 2a (75 g, 104 mmol) in DMF (360 mL). The suspension was hydrogenated in a Parr hydrogenation apparatus over 8 hours. Progress of the reaction was monitored by LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H]+., 100), (ES−) m/z (relative intensity) 595 ([M+H]+., 100) after the hydrogen uptake had stopped. Solid Pd/C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 10 mbar) at 40° C. to afford a dark oil containing traces of DMF and residual charcoal. The residue was digested in EtOH (500 mL) at 40° C. on a water bath (rotary evaporator bath) and the resulting suspension was filtered through celite and washed with ethanol (500 mL) to give a clear filtrate. Hydrazine hydrate (10 mL, 321 mmol) was added to the solution and the reaction mixture was heated at reflux. After 20 minutes the formation of a white precipitate was observed and reflux was allowed to continue for a further 30 minutes. The mixture was allowed to cool down to room temperature and the precipitate was retrieved by filtration, washed with diethyl ether (2*1 volume of precipitate) and dried in a vacuum desiccator to provide 3a (50 g, 81%).
Method B: A solution of the nitro-ester 2a (6.80 g, 9.44 mmol) in MeOH (300 mL) was added to Raney™ nickel (4 large spatula ends of a ˜50% slurry in H2O) and anti-bumping granules in a 3-neck round bottomed flask. The mixture was heated at reflux and then treated dropwise with a solution of hydrazine hydrate (5.88 mL, 6.05 g, 188 mmol) in MeOH (50 mL) at which point vigorous effervescence was observed. When the addition was complete (˜30 minutes) additional Raney™ nickel was added carefully until effervescence had ceased and the initial yellow colour of the reaction mixture was discharged. The mixture was heated at reflux for a further 30 minutes at which point the reaction was deemed complete by TLC (90:10 v/v CHCl3/MeOH) and LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H]+., 100)). The reaction mixture was allowed to cool to around 40° C. and then excess nickel removed by filtration through a sinter funnel without vacuum suction. The filtrate was reduced in volume by evaporation in vacuo at which point a colourless precipitate formed which was collected by filtration and dried in a vacuum desiccator to provide 3a (5.40 g, 96%).
Analytical Data: [α]27D=+404° (c=0.10, DMF); 1H NMR (400 MHz, DMSO-d6) δ 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H, J=3.98 Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H, J=12.1, 3.60 Hz), 3.43 (dd, 2H, J=12.0, 4.72 Hz), 2.67-2.57 (m, 2H), 2.26 (p, 2H, J=5.90 Hz), 1.99-1.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 164.0, 149.9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8, 54.4, 53.1, 33.5, 27.5; IR (ATR, neat) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm−1; MS (ES+) m/z (relative intensity) 619 ([M+Na]+., 10), 597 ([M+H]+., 52), 445 (12), 326 (11); HRMS [M+H]+. theoretical C29H32N4O10 m/z 597.2191. found (ES+) m/z 597.2205.
(b) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3b)
Preparation from 2b according to Method A gave the product as a white solid (22.1 g, 86%).
Analytical Data: MS (ES−) m/z (relative intensity) 623.3 ([M−H]−., 100);
(c) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4a)
TBSCI (317 mg, 2.1 mmol) and imidazole (342 mg, 5.03 mmol) were added to a cloudy solution of the tetralactam 3a (250 mg, 0.42 mmol) in anhydrous DMF (6 mL). The mixture was allowed to stir under a nitrogen atmosphere for 3 hours after which time the reaction was deemed complete as judged by LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M++H]+., 100)). The reaction mixture was poured onto ice (˜25 mL) and allowed to warm to room temperature with stirring. The resulting white precipitate was collected by vacuum filtration, washed with H2O, diethyl ether and dried in the vacuum desiccator to provide pure 4a (252 mg, 73%).
Analytical Data: [α]23D=+234° (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H, J=5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H), 2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H), 0.08 (s, 12H); 13C NMR (100 MHz, CDCl3) δ 170.4, 165.7, 151.4, 146.6, 129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56.3, 55.7, 54.2, 35.2, 28.7, 25.7, 18.0, −4.82 and −4.86; IR (ATR, CHCl3) 3235, 2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099, 1033 cm−1; MS (ES+) m/z (relative intensity) 825 ([M+H]+., 62), 721 (14), 440 (38); HRMS [M+H]+. theoretical C41H60N4O10Si2 m/z 825.3921. found (ES+) m/z 825.3948.
(c) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4b)
Preparation from 3b according to the above method gave the product as a white solid (27.3 g, 93%).
Analytical Data: MS (ES+) m/z (relative intensity) 853.8 ([M+H]+., 100), (ES−) m/z (relative intensity) 851.6 ([M−H]−., 100.
(d) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5a)
A solution of n-BuLi (4.17 mL of a 1.6 M solution in hexane, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise to a stirred suspension of the tetralactam 4a (2.20 g, 2.67 mmol) in anhydrous THF (30 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to stir at this temperature for 1 hour (now a reddish orange colour) at which point a solution of SEMCI (1.18 mL, 1.11 g, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise. The reaction mixture was allowed to slowly warm to room temperature and was stirred for 16 hours under a nitrogen atmosphere. The reaction was deemed complete as judged by TLC (EtOAc) and LC/MS (4.77 min (ES+) m/z (relative intensity) 1085 ([M+H]+., 100)). The THF was removed by evaporation in vacuo and the resulting residue dissolved in EtOAc (60 mL), washed with H2O (20 mL), brine (20 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (80:20 v/v Hexane/EtOAc) gave the pure N10-SEM-protected tetralactam 5a as an oil (2.37 g, 82%).
Analytical Data: [α]23D=+163° (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98 Hz), 4.68 (d, 2H, J=9.99 Hz), 4.57 (p, 2H, J=5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s, 6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81 Hz), 2.03-1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, −1.24, −4.73; IR (ATR, CHCl3) 2951, 1685, 1640, 1606, 1517, 1462, 1433, 1360, 1247, 1127, 1065 cm−1; MS (ES+) m/z (relative intensity) 1113 ([M+Na]+., 48), 1085 ([M+H]+., 100), 1009 (5), 813 (6); HRMS [M+H]+.theoretical C53H88N4O12Si4 m/z 1085.5548. found (ES+) m/z 1085.5542.
(d) 1,1′-[[(Pentane 1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5b)
Preparation from 4b according to the above method gave the product as a pale orange foam (46.9 g, 100%), used without further purification.
Analytical Data: MS (ES+) m/z (relative intensity) 1114 ([M+H]+., 90), (ES−) m/z (relative intensity) 1158 ([M+2Na]−., 100).
(e) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6a)
A solution of TBAF (5.24 mL of a 1.0 M solution in THF, 5.24 mmol) was added to a stirred solution of the bis-silyl ether 5a (2.58 g, 2.38 mmol) in THF (40 mL) at room temperature. After stirring for 3.5 hours, analysis of the reaction mixture by TLC (95:5 v/v CHCl3/MeOH) revealed completion of reaction. The reaction mixture was poured into a solution of saturated NH4Cl (100 mL) and extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96:4 v/v CHCl3/MeOH) gave the pure tetralactam 6a as a white foam (1.78 g, 87%).
Analytical Data: [α]23D=+202° (c=0.34, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0 Hz), 4.72 (d, 2H, J=10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83 Hz), 4.20-4.16 (m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH), 2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77 Hz), 2.11-2.05 (m, 2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 169.5, 165.9, 151.3, 147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67.2, 65.2, 56.5, 56.2, 54.1, 35.2, 29.1, 18.4, −1.23; IR (ATR, CHCl3) 2956, 1684, 1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm−1; MS (ES+) m/z (relative intensity) 885 ([M+29]+., 70), 857 ([M+H]+., 100), 711 (8), 448 (17); HRMS [M+H]+. theoretical C41H60N4O12Si2 m/z 857.3819. found (ES+) m/z 857.3826.
(e) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6b)
Preparation from 5b according to the above method gave the product as a white foam (15.02 g).
Analytical Data: MS (ES+) m/z (relative intensity) 886 ([M+H]+., 10), 739.6 (100), (ES−) m/z (relative intensity) 884 ([M−H]−., 40).
(f) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (7a)
Method A: A 0.37 M sodium hypochlorite solution (142.5 mL, 52.71 mmol, 2.4 eq) was added dropwise to a vigorously stirred mixture of the diol 6a (18.8 g, 21.96 mmol, 1 eq), TEMPO (0.069 g, 0.44 mmol, 0.02 eq) and 0.5 M potassium bromide solution (8.9 mL, 4.4 mmol, 0.2 eq) in DCM (115 mL) at 0° C. The temperature was maintained between 0° C. and 5° C. by adjusting the rate of addition. The resultant yellow emulsion was stirred at 0° C. to 5° C. for 1 hour. TLC (EtOAc) and LC/MS [3.53 min. (ES+) m/z (relative intensity) 875 ([M+Na]+., 50), (ES−) m/z (relative intensity) 852 ([M−H]−., 100)] indicated that reaction was complete.
The reaction mixture was filtered, the organic layer separated and the aqueous layer was backwashed with DCM (×2). The combined organic portions were washed with brine (×1), dried (MgSO4) and evaporated to give a yellow foam. Purification by flash column chromatography (gradient elution 35/65 v/v n-hexane/EtOAC, 30/70 to 25/75 v/v n-hexane/EtOAC) afforded the bis-ketone 7a as a white foam (14.1 g, 75%).
Sodium hypochlorite solution, reagent grade, available at chlorine 10-13%, was used. This was assumed to be 10% (10 g NaClO in 100 g) and calculated to be 1.34 M in NaClO. A stock solution was prepared from this by diluting it to 0.37 M with water. This gave a solution of approximately pH 14. The pH was adjusted to 9.3 to 9.4 by the addition of solid NaHCO3. An aliquot of this stock was then used so as to give 2.4 mol eq. for the reaction.
On addition of the bleach solution an initial increase in temperature was observed. The rate of addition was controlled, to maintain the temperature between 0° C. to 5° C. The reaction mixture formed a thick, lemon yellow coloured, emulsion.
The oxidation was an adaptation of the procedure described in Thomas Fey et al, J. Org. Chem., 2001, 66, 8154-8159.
Method B: Solid TCCA (10.6 g, 45.6 mmol) was added portionwise to a stirred solution of the alcohol 6a (18.05 g, 21.1 mmol) and TEMPO (123 mg, 0.78 mmol) in anhydrous DCM (700 mL) at 0° C. (ice/acetone). The reaction mixture was stirred at 0° C. under a nitrogen atmosphere for 15 minutes after which time TLC (EtOAc) and LC/MS [3.57 min (ES+) m/z (relative intensity) 875 ([M+Na]+., 50)] revealed completion of reaction. The reaction mixture was filtered through celite and the filtrate was washed with saturated aqueous NaHCO3 (400 mL), brine (400 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash column chromatography (80:20 v/v EtOAc/Hexane) afforded the bis-ketone 7a as a foam (11.7 g, 65%).
Method C: A solution of anhydrous DMSO (0.72 mL, 0.84 g, 10.5 mmol) in dry DCM (18 mL) was added dropwise over a period of 25 min to a stirred solution of oxalyl chloride (2.63 mL of a 2.0 M solution in DCM, 5.26 mmol) under a nitrogen atmosphere at −60° C. (liq N2/CHCl3). After stirring at −55° C. for 20 minutes, a slurry of the substrate 6a (1.5 g, 1.75 mmol) in dry DCM (36 mL) was added dropwise over a period of 30 min to the reaction mixture. After stirring for a further 50 minutes at −55° C., a solution of TEA (3.42 mL, 2.49 g; 24.6 mmol) in dry DCM (18 mL) was added dropwise over a period of 20 min to the reaction mixture. The stirred reaction mixture was allowed to warm to room temperature (−1.5 h) and then diluted with DCM (50 mL). The organic solution was washed with 1 N HCl (2×25 mL), H2O (30 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (80:20 v/v EtOAc/Hexane) to afford bis-ketone 7a as a foam (835 mg, 56%)
Analytical Data: [α]20D=+291° (c=0.26, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d, 2H, J=10.1 Hz), 4.60 (dd, 2H, J=9.85, 3.07 Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85 Hz), 2.76 (dd, 2H, J=19.2, 9.90 Hz), 2.42 (p, 2H, J=5.77 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 206.8, 168.8, 165.9, 151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9, 52.4, 37.4, 29.0, 18.4, −1.24; IR (ATR, CHCl3) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm−1; MS (ES+) m/z (relative intensity) 881 ([M+29]+., 38), 853 ([M+H]+., 100), 707 (8), 542 (12); HRMS [M+H]+. theoretical C41H56N4O12Si2 m/z 853.3506. found (ES+) m/z 853.3502.
(f) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (7b)
Preparation from 6b according to Method C gave the product as a white foam (10.5 g, 76%).
Analytical Data: MS (ES+) m/z (relative intensity) 882 ([M+H]+., 30), 735 (100), (ES−) m/z (relative intensity) 925 ([M+45]−., 100), 880 ([M−H]+., 70).
(g) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8a)
Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in one portion to a vigorously stirred solution of bis-ketone 7a (6.08 g, 7.1 mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile cooling bath) under a nitrogen atmosphere. Anhydrous triflic anhydride, taken from a freshly opened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injected rapidly dropwise, while maintaining the temperature at −40° C. or below. The reaction mixture was allowed to stir at −45° C. for 1 hour at which point TLC (50/50 v/v n-hexane/EtOAc) revealed the complete consumption of starting material. The cold reaction mixture was immediately diluted with DCM (200 mL) and, with vigorous shaking, washed with water (1×100 mL), 5% citric acid solution (1×200 mL) saturated NaHCO3 (200 mL), brine (100 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (gradient elution: 90:10 v/v n-hexane/EtOAc to 70:30 v/v n-hexane/EtOAc) to afford bis-enol triflate 8a as a yellow foam (5.5 g, 70%).
Analytical Data: [α]24D=+271° (c=0.18, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97 Hz), 5.51 (d, 2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.69 Hz), 4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J=16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J=5.85 Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 167.1, 162.7, 151.9, 148.0, 138.4, 133.6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56.3, 30.8, 29.0, 18.4, −1.25; IR (ATR, CHCl3) 2958, 1690, 1646, 1605, 1517, 1456, 1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm−1; MS (ES+) m/z (relative intensity) 1144 ([M+28]+., 100), 1117 ([M+H]+., 48), 1041 (40), 578 (8); HRMS [M+H]+. theoretical C43H54N4O16Si2S2F6 m/z 1117.2491. found (ES+) m/z 1117.2465.
(g) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8b)
Preparation from 7b according to the above method gave the bis-enol triflate as a pale yellow foam (6.14 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 1146 ([M+H]+., 85).
Example 1
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (9)
Solid Pd(PPh3)4 (20.18 mg, 17.46 mmol) was added to a stirred solution of the triflate 8a (975 mg, 0.87 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralane-2-yl)aniline (172 mg, 0.79 mmol) and Na2CO3 (138 mg, 3.98 mol) in toluene (13 mL) EtOH (6.5 mL) and H2O (6.5 mL). The dark solution was allowed to stir under a nitrogen atmosphere for 24 hours, after which time analysis by TLC (EtOAc) and LC/MS revealed the formation of the desired mono-coupled product and as well as the presence of unreacted starting material. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL), after eventual separation of the layers the aqueous phase was extracted again with EtOAc (2×25 mL). The combined organic layers were washed with H2O (50 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude Suzuki product. The crude Suzuki product was subjected to flash chromatography (40% EtOAc/60% Hexane→10% EtOAc, 30% Hexane). Removal of the excess eluent by rotary evaporation under reduced pressure afforded the desired product 9 (399 mg) in 43% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J=8.5 Hz), 7.15 (t, 1H, J=2.0 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.52 (d, 2H, J=10.0 Hz), 4.77 (d, 1H, J=10.0 Hz), 4.76 (d, 1H, J=10.0 Hz), 4.62 (dd, 1H, J=3.7, 11.0 Hz), 4.58 (dd, 1H, J=3.4, 10.6 Hz), 4.29 (t, 4H, J=5.6 Hz), 4.00-3.85 (m, 8H), 3.80-3.60 (m, 4H), 3.16 (ddd, 1H, J=2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J=2.2, 10.5, 16.1 Hz), 2.43 (p, 2H, J=5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H). 13C-NMR: (CDCl3, 100 MHz) δ 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147.6, 139.6, 134.8, 134.5, 127.9 (methine), 127.5, 125.1, 123.21, 121.5, 120.5 (methine), 120.1 (methine), 116.4 (methine), 113.2 (methine), 108.7 (methine), 79.8 (methylene), 79.6 (methylene), 68.7 (methylene), 68.5 (methylene), 67.0 (methylene), 66.8 (methylene), 58.8 (methine), 58.0 (methine), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3 (methylene), 19.7 (methylene), 0.25 (methyl).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (10)
Solid Pd(PPh3)4 (10 mg, 8.69 μmol) was added to a stirred solution of the mono-triflate 9 (230 mg, 0.22 mmol) in toluene (3 mL), EtOH (10 mL), with 4-methoxyphenyl boronic acid (43 mg, 0.28 mmol), Na2CO3 (37 mg, 0.35 mmol), in H2O (1.5 mL) at room temperature. The reaction mixture was allowed to stir under a nitrogen atmosphere for 20 h, at which point the reaction was deemed complete as judged by LC/MS and TLC (EtOAc). The solvent was removed by rotary evaporation under reduced pressure in vacuo and the resulting residue partitioned between EtOAc (75 mL) and H2O (75 mL). The aqueous phase was extracted with EtOAc (3×30 mL) and the combined organic layers washed with H2O (30 mL), brine (40 mL), dried (MgSO4), filtered and evaporated to provide the crude product. The crude product was purified by flash chromatography (60% Hexane: 40% EtOAc→80% EtOAc: 20% Hexane) to provide the pure dimer as an orange foam. Removal of the excess eluent under reduced pressure afforded the desired product 10 (434 mg) in 74% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.38 (s, 2H), 7.34 (d, 2H, J=8.8 Hz), 7.30 (bs, 1H), 7.26-7.24 (m, 3H), 7.22 (d, 2H, J=8.5 Hz), 6.86 (d, 2H, J=8.8 Hz), 6.63 (d, 2H, J=8.5 Hz), 5.50 (d, 2H, J=10.0 Hz), 4.75 (d, 1H, J=10.0 Hz), 4.74 (d, 1H, J=10.0 Hz), 4.56 (td, 2 H, J=3.3, 10.1 Hz), 4.27 (t, 2H, J=5.7 Hz), 4.00-3.85 (m, 8H), 3.80 (s, 3H), 3.77-3.60 (m, 4H), 3.20-3.00 (m, 2H), 2.42 (p, 2H, J=5.7 Hz), 0.96 (t, 4H, J=8.3 Hz), 0.00 (s, 18H). 13C-NMR: (CDCl3, 100 MHz) δ 169.8, 169.7, 162.9, 162.7, 160.6, 152.7, 152.6, 149.0, 147.5, 134.8, 127.8 (methine), 127.4, 126.8, 125.1, 123.1, 123.0, 121.5 (methine), 120.4 (methine), 116.4 (methine), 115.5 (methine), 113.1 (methine), 108.6 (methine), 79.6 (methylene), 68.5 (methylene), 66.9 (methylene), 58.8 (methine), 57.6 (methoxy), 56.7 (methoxy), 32.8 (methylene), 30.3 (methylene), 19.7 (methylene), 0.0 (methyl).
(c) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (11)
Fresh LiBH4 (183 mg, 8.42 mmol) was added to a stirred solution of the SEM-dilactam 10 (428 mg, 0.42 mmol) in THF (5 mL) and EtOH (5 mL) at room temperature. After 10 minutes, delayed vigorous effervescence was observed requiring the reaction vessel to be placed in an ice bath. After removal of the ice bath the mixture was allowed to stir at room temperature for 1 hour. LC/MS analysis at this point revealed total consumption of starting material with very little mono-reduced product. The reaction mixture was poured onto ice (100 mL) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3×30 mL) and the combined organic layers washed with H2O (20 mL), brine (30 mL) and concentrated in vacuo. The resulting residue was treated with DCM (5 mL), EtOH (14 mL), H2O (7 mL) and silica gel (10 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered slowly through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was washed with H2O (50 mL), brine 60 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (97% CHCl3: 3% MeOH) to provide the pure C2/C2′aryl PBD dimer 11 (185 mg) 61% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.88 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.20 (d, 2H, J=8.5 Hz), 6.89 (d, 2H, J=8.8 Hz), 6.87 (s, 1H), 6.86 (s, 1H), 6.67 (d, 2H, J=8.5 Hz), 4.40-4.20 (m, 6H), 3.94 (s, 6H), 3.82 (s, 3H), 3.61-3.50 (m, 2H), 3.40-3.30 (m, 2H), 2.47-2.40 (m, 2H). 13C-NMR: (CDCl3, 100 MHz) δ 162.5 (imine methine), 161.3, 161.1, 159.3, 156.0, 151.1, 148.1, 146.2, 140.3, 126.2 (methine), 123.2, 122.0, 120.5 (methine), 119.4, 115.2 (methine), 114.3 (methine), 111.9 (methine), 111.2 (methine), 65.5 (methylene), 56.2 (methoxy), 55.4 (methoxy), 53.9 (methine), 35.6 (methylene), 28.9 (methylene).
Example 2
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (12)
Solid Pd(PPh3)4 (32 mg, 27.7 μmol) was added to a stirred solution of the bis-triflate 8b (1.04 g, 0.91 mmol) in toluene (10 mL), EtOH (5 mL), with 4-methoxyphenyl boronic acid (0.202 g, 1.32 mmol), Na2CO3 (0.169 g, 1.6 mmol), in H2O (5 mL) at 30° C. The reaction mixture was allowed to stir under a nitrogen atmosphere for 20 hours. Additional solid 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (0.203 g, 0.93 mmol) and Na2CO3 (0.056 g, 0.53 mmol) were added followed by solid Pd(PPh3)4 (10 mg, 8.6 μmol). The reaction mixture was allowed to stir under a nitrogen atmosphere for a further 20 hours. LC/MS indicated the formation of desired product. EtOAc (100 mL) and H2O (100 mL) were added, the aqueous was separated and extracted with EtOAc (3×30 mL). The combined organic layers were washed with H2O (100 mL), brine (100 mL), dried (MgSO4), filtered and evaporated to provide a dark brown oil. The oil was dissolved in DCM and loaded onto a 10 g SCX-2 cartridge pre-equilibrated with DCM (1 vol). The cartridge was washed with DCM (3 vol), MeOH (3 vol) and the crude product eluted with 2M NH3 in MeOH (2 vol). Flash chromatography (50% n-hexane: 50% EtOAc→20% n-hexane: 80% EtOAc) provided the pure dimer 12 as a yellow foam (0.16 g, 34%).
Analytical Data: [α]23D=+388° (c=0.22, CHCl3); 1H-NMR: (CDCl3, 400 MHz) δ 7.39 (s, 2H), 7.35 (d, 2H, J=12.8 Hz), 7.32 (bs, 1H), 7.26-7.23 (m, 5H), 6.89 (d, 2H, J=8.8 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.55 (d, 2H, J=10.0 Hz), 4.73 (d, 1H, J=10.0 Hz), 4.72 (d, 1H, J=10.0 Hz), 4.62 (td, 2 H, J=3.2, 10.4 Hz), 4.15-4.05 (m, 4H), 4.00-3.85 (m, 8H), 3.82 (s, 3H), 3.77-3.63 (m, 4H), 3.20-3.05 (m, 2H), 2.05-1.95 (m, 4H), 1.75-1.67 (m, 2H) 1.01-0.95 (m, 4H), 0.03 (s, 18H); MS (ES+) m/z (relative intensity) 1047 ([M+H]+., 45).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (13)
Fresh LiBH4 (66 mg, 3.04 mmol) was added to a stirred solution of the SEM-dilactam 12 (428 mg, 0.42 mmol) in THF (3 mL) and EtOH (3 mL) at 0° C. (ice bath). The ice bath was removed and the reaction mixture was allowed to reach room temperature (vigorous effervescence). After 2 hours LC/MS analysis indicated the complete consumption of starting material. The reaction mixture was poured onto ice (50 mL) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3×50 mL) and the combined organic layers washed with H2O (50 mL), brine (50 mL), dried (MgSO4) and concentrated in vacuo. The resulting residue was treated with DCM (2 mL), EtOH (5 mL), H2O (2.5 mL) and silica gel (3.7 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was dried (MgSO4) filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (99.5% CHCl3: 0.5% MeOH to 97.5% CHCl3: 2.5% MeOH in 0.5% increments)) to provide the pure C2/C2′aryl PBD dimer 13 (59 mg, 52%).
Analytical Data: [α]28D=+760° (c=0.14, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.89 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.22 (d, 2H, J=8.4 Hz), 6.91 (d, 2H, J=8.8 Hz), 6.815 (s, 1H), 6.81 (s, 1H), 6.68 (d, 2H, J=8.4 Hz), 4.45-4.35 (m, 2H), 4.2-4.0 (m, 4H), 3.94 (s, 6H), 3.85-3.7 (s, 3H), 3.65-3.50 (m, 2H), 3.45-3.3 (m, 2H), 2.05-1.9 (m, 4H), 1.75-1.65 (m, 2H); MS (ES+) (relative intensity) 754.6 ([M+H]+., 100), (ES−) (relative intensity) 752.5 ([M−H]−., 100).
Example 3
(a)(S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (14)
Solid Pd(PPh3)4 (41 mg, 0.036 mmol) was added to a stirred solution of the bis-triflate 8a (1 g, 0.9 mmol) in toluene (10 mL), EtOH (5 mL), with thien-2-yl boronic acid (149 mg, 1.16 mmol), Na2CO3 (152 mg, 1.43 mmol), in H2O (5 mL). The reaction mixture was allowed to stir under a nitrogen atmosphere overnight at room temperature. The solvent was removed by evaporation in vacuo and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL). The aqueous layer was extracted with EtOAc (2×30 mL) and the combined organic layers washed with H2O (50 mL), brine (50 mL) dried (MgSO4), filtered and evaporated in vacuo to provide the crude product which was purified by flash chromatography (80 hexane: 20 EtOAc→50 hexane: 50 EtOAc) to provide the dimer 14 (188 mg, 20%) yield
Analytical data: LC-MS RT 4.27 mins, 1051 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.36 (s, 1H), 7.31 (bs, 1H), 7.27 (bs, 1H), 7.26-7.23 (m, 2H), 7.22-7.17 (m, 1H), 7.12 (bs, 1H), 7.02-6.96 (m, 2H), 5.50 (d, J=10.0 Hz, 2H), 7.75 (d, J=10.0 Hz, 2H), 4.65-4.55 (m, 2H), 4.37-4.13 (m, 4H), 4.00-3.85 (m, 8H), 3.8-3.6 (m, 4H), 3.20-3.10 (m, 2H), 2.50-2.35 (m, 2H), 1.0-0.9 (m, 4H), 0 (s, 18H).
(b) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (15)
Solid Pd(PPh3)4 (7.66 mg, 6.63 μmol) was added to a stirred, cloudy solution of 14 (174 mg, 0.17 mmol), Na2CO3 (28 mg, 0.22 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (47 mg, 0.22 mmol) in toluene (2-5 mL), EtOH (1.25 mL) and H2O (125 mL) at room temperature. The reaction mixture was allowed to stir under a N2 atmosphere for 24 hours at which point the reaction was deemed complete by LC/MS major peak (@ 3.97 min, FW=1016, M+Na) and TLC (EtOAc). The solvent was removed by evaporation in vacuo and the resulting residue partitioned between EtOAc (60 mL) and H2O (30 mL). The layers were separated and the organic phase was washed with H2O) (20 mL), brine (30 mL) dried (MgSO4) filtered and evaporated in vacuo to provide the crude product 123 mg, 75% yield.
Analytical data: LC-MS RT 3.98 mins, 100% area, 994 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.40 (d, J=5.3 Hz, 2H), 7.30 (t, J=1.70 Hz, 1H), 7.29-7.27 (m, 3H), 7.25 (d, J=8.5 Hz, 2H), 7.21 (dd, J=1.4, 4.73 Hz, 1H), 7.03-6.97 (m, 2H), 6.66 (d, J=8.5 Hz, 2H), 5.52 (d, J=10.0 Hz, 2H), 4.78 (d, J=10.0 Hz, 1H), 4.77 (d, J=10.0 Hz, 1H), 4.62 (dd, J=3.4, 10.5 Hz, 1H), 4.59 (dd, J=3.40, 10.6 Hz, 1H), 4.30 (t, J=5.85 Hz, 4H), 3.85-4.03 (m, 8H), 3.84-3.64 (m, 6H), 3.18 (ddd, J=2.2, 10.5, 16.0 Hz, 1H), 3.11 (ddd, J=2.2, 10.5, 16.0 Hz, 1H), 2.44 (p, J=5.85 Hz, 2H), 0.98 (t, J=1.5 Hz, 4H), 0 (s, 18H).
(c) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-aminophenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (16)
Fresh LiBH4 (47 mg, 2.22 mmol) was added to a stirred solution of the SEM-dilactam 15 (110 mg, 0.11 mmol) in dry THF (3 mL) and EtOH (3 mL) at 0° C. (ice bath). The ice bath was removed and the reaction mixture stirred under a N2 atmosphere for 1 hour. Analysis of the reaction by LC/MS analysis revealed significant formation of the desired product (Pk @ 2.57 min) (I=69.32), FW=702, M+H) and half-imine. The reaction mixture was allowed to stir for a further 1 hour after which time no further reaction progress was observed by LC/MS. The reaction mixture was poured onto ice, stirred and allowed to warm to room temperature. Following partition between DCM (50 mL) and water (50 mL), the aqueous phase was extracted with DCM (3×20 mL). The combined organic layers were washed with H2O (50 mL), brine (50 mL) and the solvent removed by evaporation in vacuo under reduced pressure.
The resulting residue was dissolved in DCM (5 mL), EtOH (15 mL) and H2O (7 mL) then treated with silica gel (5 g). The reaction was allowed to stir at room temperature for 48 h. The silica was removed by filtration through a sinter funnel and the residue rinsed with 90:10 CHCl3: MeOH (100 mL). H2O (50 mL) was added to the filtrate and the layers were separated (after shaking). The aqueous layer was extracted with CHCl3 (2×30 mL) and H2O (50 mL), brine (50 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude product. Flash chromatography (CHCl3→98% CHCl3: 2% MeOH) afforded the product (41 mg, 53%).
Anayltical data: LC-MS RT 2.55 mins, 702 (M+H)
Example 4
(a) (S)-2-(4-methoxyphenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulphonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (17)
Solid 4-methoxybenzeneboronic acid (0.388 g, 2.55 mmol) was added to a solution of the SEM protected bis triflate (8a)(3.0 g, 2.69 mmol), sodium carbonate (426 mg, 4.02 mmol) and palladium tetrakis triphenylphosphine (0.08 mmol) in toluene (54.8 mL), ethanol (27 mL) and water (27 mL). The reaction mixture was allowed to stir at room temperature for 3 hours. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 30/70→35/65→40/60→45/55) to remove unreacted bis-triflate (0.6 g). Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (1.27 g, 1.18 mmol, 41%).
LC-MS RT 4.30 mins, 1076 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.41 (s, 1H), 7.39 (d, J=8.8 Hz, 2H), 7.35 (s, 1H), 7.34 (bs, 1H), 7.29 (s, 1H), 7.16 (t, J=1.9 Hz, 1H), 6.90 (d, J=8.8 Hz, 2H), 5.53 (d, J=10.0 Hz, 2H), 4.79 (d, J=10.0 Hz, 1H), 4.78 (d, J=10.0 Hz, 1H), 4.66-4.60 (m, 2H), 4.30 (t, J=5.7 Hz, 4H), 4.0-3.94 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.84 (s, 3H), 3.83-3.60 (m, 4H), 3.22-3.10 (m, 2H), 2.45 (t, J=5.9 Hz, 2H), 1.05-0.94 (m, 4H), 0 (s, 18H).
(b) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (18)
Solid 3-aminobenzeneboronic acid (0.143 g, 0.92 mmol) was added to a solution of the mono triflate (17)(0.619 g, 0.58 mmol), sodium carbonate (195 mg, 1.84 mmol) and palladium tetrakis triphenylphosphine (26.6 mg, 0.023 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir at room temperature for overnight at 30° C. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 70/30→85/15). Removal of excess eluent from selected fractions afforded the desired product (0.502 g, 0.49 mmol, 85%).
LC-MS RT 4.02 mins, 1019 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.38-7.35 (m, 4H), 7.33 (bs, 1H), 7.30 (bs, 1H), 7.25 (s, 2H), 7.10 (t, J=7.8 Hz, 1H), 6.88-6.80 (m, 3H), 6.72 (bs, 1H), 6.57 (dd, J=7.9, 1.8 Hz, 1H), 5.50 (d, J=10.0 Hz, 2H), 4.75 (d, 10.0 Hz, 2H), 4.58 (dd, J=10.6, 3.3 Hz, 2H), 4.27 (t, J=5.8 Hz, 4H), 3.95-3.91 (m, 2H), 3.90 (s, 6H), 3.80 (s, 3H), 3.77-3.60 (m. 6H), 3.15-3.05 (m, 2H), 2.41 (p, J=5.8 Hz, 2H), 0.95 (t, =8.25 Hz, 4H), 0 (s, 18H).
(c) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (19)
A solution of superhydride (0.56 mL, 0.56 mmol, 1.0 M in THF) was added dropwise to a solution of the SEM dilactam (18)(0.271 g, 0.27 mmol) in dry THF (10 mL) at −78° C. under a nitrogen atmosphere. After 1 hr a further aliquot of superhydride solution (0.13 ml, 0.13 mmol) was added and the reaction mixture was allowed to stir for another 0.5 hr, at which time LC-MS indicated that reduction was complete. The reaction mixture was diluted with water and allowed to warm to room temperature. The reaction mixture was partitioned between chloroform and water, the layers were separated and the aqueous layer extracted with additional chloroform (emulsions). Finally the combined organic phase was washed with brine and dried over magnesium sulphate. The reduced product was dissolved in methanol, chloroform and water and allowed to stir in the presence of silica gel for 72 hours The crude product was subjected to flash column chromatography (methanol/chloroform gradient) to afford the desired imine product (150 mg, 0.21 mmol, 77%) after removal of excess eluent from selected fractions.
LC-MS RT 2.63 mins, 97% area, 726 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.85 (d, J=3.9 Hz, 1H), 7.84 (d, J=3.9 Hz, 1H), 7.50 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 7.32 (d, J=7.3 Hz, 2H), 7.11 (t, (d, J=7.8 Hz, 1H), 6.90-6.80 (m, 4H), 6.77 (d, J=7.9 Hz, 1H), 4.40-4.20 (m, 6H), 3.92 (s, 6H), 3.80 (s, 3H), 3.60-3.27 (m, 6H), 2.48-2.29 (m, 2H)
Example 5
(a) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10((2, 2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-2-(trifluoromethylsulfonyloxy)-11,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 21
Solid 4-methoxybenzeneboronic acid (59 mg, 0.39 mmol) was added to a solution of the Troc protected bis triflate (Compound 44, WO 2006/111759) (600 mg, 0.41 mmol), sodium carbonate (65 mg, 0.61 mmoml) and palladium tetrakis triphenylphosphine (0.012 mmol) in toluene (10.8 mL), ethanol (5.4 mL) and water (5.4 mL). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was then partitioned between ethylacetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 20/80→30/70→40/60→60/40) to remove unreacted bis-triflate. Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (261 mg, 0.18 mmol, 46%).
LC-MS RT 4.17 mins, 1427 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.38 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.25 (s, 1H), 7.20 (bs, 1H), 6.92 (d, J=8.6 Hz, 2H), 6.77 (d, J=8.7 Hz, 2H), 6.0-5.90 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.18-4.08 (m, 2H), 4.07-3.89 (m, 10H), 3.81 (s, 3H), 3.44-3.25 (m, 2H), 2.85 (d, J=16.6 Hz, 2H), 2.05-1.90 (m, 4H), 1.76-1.64 (m, 2H), 0.93 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.26 (s, 6H).
(b) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 22
The Suzuki coupling procedure described in step (a) was applied to the synthesis of Compound 21. Compound 20 (62.5 mg 0.044 mmol) was treated with 1 equivalent of 4-hydroxybenzeneboronic acid (10 mg) at 30° C. overnight to afford the desired compound after filtration through a pad of silica gel. (40 mg, 0.029 mmol, 66% yield). The compound was used directly in the subsequent step LC-MS RT 4.27 mins, 1371 (M+H)
(c) (S)-2-(4-hydroxyphenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 23
Cadmium/lead couple (100 mg, Q Dong et al. Tetrahedron Letters vol 36, issue 32, 5681-5682, 1995) was added to a solution of 21 (40 mg, 0.029 mmol) in THF (1 mL) and ammonium acetate (1N, 1 mL) and the reaction mixture was allowed to stir for 1 hour. The reaction mixture was partitioned between chloroform and water, the phases separated and the aqueous phase extracted with chloroform. The combined organic layers were washed with brine and dried over magnesium sulphate. Rotary evaporation under reduced pressure yielded the crude product which was subjected to column chromatography (silica gel, 0→4% MeOH/CHCl3). Removal of excess eluent by rotary evaporation under reduced pressure afforded the desired imine product (17 mg 0.023 mmol 79%).
LC-MS RT 2.20 mins, 755 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.89 (d, J=3.94 Hz, 1H), 7.89 (d, J=4.00 Hz, 1H), 7.53 (s, 1H), 7.52 (s, 1H), 7.38 (d, J=8.7 Hz, 2H), 7.33 (d, J=8.6 Hz, 2H), 7.28 (s, 1H), 6.90 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 6.82 (s, 1H), 6.81 (s, 1H), 5.68 (bs, 1H), 4.50-4.30 (m, 2H), 4.22-4.00 (m, 4H), 3.93 (s, 6H), 3.82 (s, 3H), 3.69-3.45 (m, 2H), 3.44-3.28 (m, 2H), 2.64-1.88 (m, 4H), 1.77-1.62 (m, 2H).
Example 6
(a) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-formylphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 24
The Suzuki coupling procedure described in Example 5, step (a), was applied to the synthesis of Compound 24. Compound 21 (62.5 mg, 0.044 mmol) was treated with 1 equivalent of 4-formylbenzeneboronic acid (10.5 mg) at room temperature overnight to afford the desired compound after filtration through a pad of silica gel (45 mg, 0.033 mmol, 75% yield). The compound was used directly in the subsequent step.
LC-MS RT 4.42 mins, 1383 (M+H)
(b) 4-((S)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl)benzaldehyde 25
Compound 24 was deprotected by the method described in Example 5, step (c), to yield the desired compound (18 mg, 0.023 mmol, 79%).
LC-MS RT 3.18 mins, 768 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 9.98 (s, 1H), 7.91 (d, J=3.90 Hz, 1H), 7.90-7.80 (m, 3H), 7.68 (s, 1H), 7.60-7.45 (m, 4H), 7.39 (s, 1H), 7.33 (d, J=8.7 Hz, 1H), 6.90 (d, J=8.7 Hz, 2H), 6.83 (s, 1H), 6.82 (s, 1H), 4.55-4.44 (m, 1H), 4.43-4.36 (m, 1H), 4.23-4.00 (m, 4H), 3.95 (s, 3H), 3.94 (s, 3H), 3.82 (s, 3H), 3.66-3.51 (m, 2H), 3.50-3.34 (m, 2H), 2.05-1.87 (m, 4H), 1.76-164 (m, 2H).
Example 7
(a) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-2-(trifluoromethylsulphonyloxy)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 26
The Suzuki coupling procedure described in Example 5, step (a), was applied to the synthesis of Compound 26, using 3-aminobenzeneboronic acid to afford the desired compound in 41% yield (230 mg, 0.163 mmol)
LC-MS RT 4.28 mins, 1411 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.44 (bs, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 7.20 (s, 1H), 7.16 (t, J=7.9 Hz, 1H), 6.84-6.73 (m, 3H), 6.70 (bs, 1H), 6.62 (dd, J=7.9, 1.7 Hz, 1H), 6.66-6.58 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.17-4.07 (m, 2H), 4.08-3.89 (m, 10H), 3.43-3.28 (m, 2H), 2.85 (d, J=1.65 Hz, 2H), 2.07-1.90 (m, 4H), 1.78-1.63 (m, 2H), 0.94 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.27 (s, 6H).
(b) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-10-(2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 27
Solid 4-[3-(dimethylamino)propoxybenzeneboronic acid pinacol ester (25 mg, 0.082 mmol) was added to a solution of 26 (73 mg, 0.052 mmol mmol), sodium carbonate (18 mg, 0.17 mmol) and palladium tetrakis triphenylphosphine (3 mg) in toluene (1 mL), ethanol (0.5 mL) and water (0.5 mL). The reaction mixture was allowed to stir at room temperature over night. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was eluted through a plug of silica gel with chloroform/methanol. Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (50 mg, 0.035 mmol, 67%).
LC-MS RT 4.12 mins, 1440 (M+H)
(c) (S)-2-(3-aminophenyl)-8-(5-((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 28
Compound 27 was deprotected by the method described in Example 5, step (c), to yield the desired compound. The reaction mixture was partitioned between DCM and aqueous sodium hydrogen carbonate (emulsion) and the crude product purified by gradient column chromatography on silica gel (5% methanol chloroform→35% methanol/chloroform) to afford the desired unsymmetrical PBD imine (50 mg, 0.018 mmol, 58%)
LC-MS RT 2.55 mins, 826 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.92-7.82 (m, 2H), 7.52 (bs, 2H), 7.45 (bs, 1H), 7.39 (bs, 1H), 7.31 (d, J=8.6 Hz, 2H), 7.14 (t, J=7.8 Hz, 1H), 6.89 (d, J=8.6 Hz, 2H), 6.85-6.75 (m, 3H), 6.72 (bs, 1H), 6.60 (d, J=8.0 Hz, 1H), 4.46-4.33 (m, 2H), 4.21-3.98 (m, 6H), 3.94 (s, 6H), 3.63-3.50 (m, 2H), 3.43-3.29 (m, 2H), 2.64-2.48 (m, 2H), 2.34 (s, 6H), 2.10-1.89 (m, 6H), 1.57 (m, 2H).
Example 8
(a) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-10-(2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 29
The method of Example 7, step (b), was performed to afford the desired product (58 mg, 0.0.040 mmol, 78%) after filtration through a plug of silica gel (with 1/3 methanol/chloroform) and removal of excess solvent by rotary evaporation under reduced pressure.
LC-MS RT 4.08 mins, 1439 (M+H)
(b) (S)-2-(3-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 30
The method for Example 7, step (c) was used to deprotect compound 29. The crude product was purified by silica gel gradient chromatography (2% methanol chloroform→35% methanol/chloroform) to afford the desired unsymmetrical PBD imine (18 mg, 0.022 mmol, 59%)
LC-MS RT 2.52 mins, 823 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.80 (d, J=3.8 Hz, 2H), 7.45 (s, 2H), 7.38 (s, 1H), 7.30 (s, 1H), 7.23 (d, J=8.6 Hz, 2H), 7.07 (t, J=7.8 Hz, 1H), 6.83 (d, J=8.6 Hz, 2H), 6.79-6.89 (m, 3H), 6.65 (s, 1H), 6.54 (d, J=7.9 Hz, 1H), 4.40-4.24 (m, 2H), 4.15-3.93 (m, 4H), 3.87 (s, 6H), 3.56-3.42 (m, 2H), 3.37-3.23 (m, 2H), 3.22-3.08 (m, 4H), 2.61-2.41 (m, 4H), 2.29 (s, 3H), 1.98-1.80 (m, 4H), 1.67-1.54 (m, 2H).
Example 9
(a) (S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione 31
Solid 4-aminomethylbenzeneboronic acid hydrochloride (0.111 g, 0.59 mmol) was added to a solution of 17 (0.394 g, 0.37 mmol), sodium carbonate (175 mg, 1.654 mmol) and palladium tetrakis triphenylphosphine (28.0 mg, 0.024 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir overnight at 30° C. The following day the reaction mixture was heated for a further 3 hours at 70° C. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 2/98→15/85). Removal of excess eluent from selected fractions afforded the desired product (0.230 mg, 0.22 mmol, 61%).
LC-MS RT 3.63 mins, 1034 (M+2H); 1H-NMR (400 MHz, DMSO d6) δ 11.7 (s, 2H), 7.52 (d, J=8.2 Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 7.40 (s, 1H), 7.50 (d, J=8.1 Hz, 2H), 7.38-7.19 (m, 5H) 6.93 (d, J=8.7 Hz, 2H), 5.40 (d, J=2.13 Hz, 1H), 5.38 (d, J=2.12 Hz, 1H), 5.32 (d, J=10.6 Hz, 2H), 5.25 (d, J=10.6 Hz, 2H), 4.87-4.72 (m, 2H), 4.35-4.15 (m, 4H), 3.85 (s, 6H), 3.79 (s, 3H), 3.73-3.56 (m, 2H), 3.55-3.39 (m, 4H), 3.22-3.02 (m, 2H), 2.39-2.23 (m, 2H), 0.94-0.67 (m, 4H), −0.06 (s, 18H).
(b) (S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 32
Compound 31 was deprotected following the method of Example 1, step (c). The crude product was purified by gradient column chromatography (5/95→30/70 MeOH/CHCl3) to afford the product as a mixture of imine and carbinolamine methyl ethers.
LC-MS RT 2.58 mins, 740 (M+H).
Example 10
(S)-2-(4-aminophenyl)-7-methoxy-11(S)-sulpho-8-(3-((S)-7-methoxy-11(S)-sulpho-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one disodium salt 33
Sodium bisulphite (8.5 mg, 3.1 eq) was added to a stirred suspension of bis-imine 11 (20 mg, 0.036 mmol) in isopropanol (4 mL) and water (2 mL). The reaction mixture was allowed to stir vigorously and eventually became clear (c. 1 hour). The reaction mixture was transferred to a funnel and filtered through a cotton wall (and then washed with 2 mL water). The filtrate was flash frozen (liquid and to bath) and lyophilized to afford the desired product 33 in quantitative yield.
LC-MS RT 11.77 mins, 727.2 (M+H) (Mass of parent compound, bisulphite adducts unstable in mass spectrometer); 1H-NMR (400 MHz, CDCl3) δ 7.66-7.55 (m, 5H), 7.43 (s, 1H), 7.39 (d, J=8.66 Hz, 2H), 7.06 (m, 2H), 6.93 (d, J=8.84 Hz, 2H), 6.54 (m, 2H), 5.29-5.21 (m, 2H), 4.32-4.28 (m, 2H), 4.14-4.20 (m, 4H), 3.96-3.83 (m, 2H), 3.77 (s, 3H), 3.73 (m, 6H), 3.52-3.43 (m, 2H), 3.30-3.08 (m, 2H), 2.24-2.21 (m, 2H).
Example 11
(a) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (103)
A catalytic amount of tetrakistriphenylphosphinepalladium (0) (11.2 mg) was added to a mixture of the mono triflate 17 (380 mg), the pinnacol ester of 2-aminophenylboronic acid (124 mg) and sodium carbonate (120 mg) in ethanol (5 mL), toluene (5 mL) and water (5 mL). The reaction mixture was allowed to stir over night at room temperature and at 40° C. until the reaction was complete (c. 2 hr). The reaction mixture was diluted with ethyl acetate and the organic layer was washed with water and brine. The ethyl acetate solution was dried over magnesium sulphate and filtered under vacuum. Removal of ethyl acetate by rotary evaporation under reduced pressure afforded the crude product which was subjected to flash chromatography (silica gel, ethyl acetate/hexane). Pure fractions were collected and combined. Removal of excess eluent by rotary evaporation under reduced pressure afforded the pure product 103 (330 mg, 86% yield). LC/MS RT: 4.17 min, ES+1018.48.
(b) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one (104)
A solution of Superhydride in dry tetrahydrofuran (1.0 M, 4.4 eq.) was added to a solution of the 2-analino compound 103 (300 mg) in dry tetrahydrofuran (5 mL) at −78° C. under an inert atmosphere. As reduction was proceeding slowly an aliquot of lithium borohydride (20 eq.) was added and the reaction mixture was allowed to return to room temperature. Water/ice was added to the reaction mixture to quench unreacted hydrides and the reaction was diluted with dichloromethane. The organic layer was washed sequentially with water (twice), citric acid and brine. Excess dichloromethane was removed by rotary evaporation under reduced pressure and the residue was redissolve in ethanol and water and treated with silica gel for 96 hours. The reaction mixture was vacuum filtered and the filtrate evaporated to dryness. The residue was subjected to flash column chromatography (silica gel, gradient chloroform/methanol). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under educed pressure to afford the pure product 104 (30 mg, 14% yield). LC/MS RT: 2.90 min, ES+726.09.
Example 12
Determination of In Vitro Cytotoxicity of Representative PBD Compounds
K562 Assay
K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37° C. in a humidified atmosphere containing 5% CO2 and were incubated with a specified dose of drug for 1 hour or 96 hours at 37° C. in the dark. The incubation was terminated by centrifugation (5 min, 300 g) and the cells were washed once with drug-free medium. Following the appropriate drug treatment, the cells were transferred to 96-well microtiter plates (104 cells per well, 8 wells per sample). Plates were then kept in the dark at 37° C. in a humidified atmosphere containing 5% CO2. The assay is based on the ability of viable cells to reduce a yellow soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Aldrich-Sigma), to an insoluble purple formazan precipitate. Following incubation of the plates for 4 days (to allow control cells to increase in number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well and the plates further incubated for 5 h. The plates were then centrifuged for 5 min at 300 g and the bulk of the medium pipetted from the cell pellet leaving 10-20 μL per well. DMSO (200 μL) was added to each well and the samples agitated to ensure complete mixing. The optical density was then read at a wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and a dose-response curve was constructed. For each curve, an IC50 value was read as the dose required to reduce the final optical density to 50% of the control value.
Compound 13 has an IC50 of 30 pM in this assay.
A2780 Assay
The A2780 parental cell line was grown in Dulbecco's Modified Eagles' Media (DMEM) containing ˜10% Foetal Calf Serum (FCS) and ˜1% 200 mM L-Glutamine solution and grown in Corning Cellbind 75 cm2flasks.
A 190 μl cell suspension was added (at 1×104) to each well of columns 2 to 11 of a 96 well plate (Nunc 96F flat bottom TC plate). 190 μl of media was added to each well of columns 1 and 12. The media was Dulbecco's Modified Eagles' Media (DMEM) (which included ˜10% Foetal Calf Serum (FCS) and ˜1% 200 mM L-Glutamine solution).
Plates were incubated overnight at 37° C. before addition of drug if cells were adherent. 200 μM of the test compound solutions (in 100% DMSO) were serially diluted across a 96 well plate. Each resulting point was then further diluted 1/10 into sterile distilled water (SDW).
To the cell negative blanks and compound negative control wells, 10% DMSO was added at 5% v/v. Assay plates were incubated for the following durations at 37° C. in 5% CO2 in a humidified incubator for 72 hours. Following incubation, MTT solution to a final concentration of 1.5 μM was added to each well. The plates were then incubated for a further 4 hours at 37° C. in 5% CO2 in a humidified incubator. The media was then removed, and the dye was solubilised in 200 μl DMSO (99.99%).
Plates were read at 540 nm absorbance using an Envision plate reader. Data was analysed using Microsoft Excel and GraphPad Prism and IC50 values obtained.
Compound 11 has an IC50 of 11.7 pM in this assay.
Renal Cell and AML Cell Lines Assays
The cytotoxicity of various free drug compounds was tested on a renal cell cancer cell line, 786-O, a Hodgkin lymphoma cell line, L428 and two AML cell lines, HL60 and HEL. For a 96-hour assay, cells cultured in log-phase growth were seeded for 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of test article (i.e., free drug) in cell culture media were prepared at 4x working concentration; 50 μL of each dilution was added to the 96-well plates. Following addition of test article, the cells were incubated with test articles for 4 days at 37° C. Resazurin was then added to each well to achieve a 50 μM final concentration, and the plates were incubated for an additional 4 h at 37° C. The plates were then read for the extent of dye reduction on a Fusion HT plate reader (Packard Instruments, Meridien, Conn., USA) with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
Referring to the following Table 1, the para-aniline compound 11 showed markedly increased activity on these cell lines as compared to the meta-aniline compound 19 in this assay.
TABLE 1
IC50 Summary for Free Drugs [nM]
Free Drug
L428
786-O
HL60
HEL
Compound 11
<0.00001
<0.00001
<0.00001
<0.00001
Compound 19
1
0.5
0.6
0.2
Referring to the following Table 2, the activity of compounds 28, 30 and 32 is shown on L428, 786-O, HEL, HL-60 and MCF-7 cells, as well as the activity for compound 19 on MCF-7 cells.
TABLE 2
IC50 Summary for Free Drugs [nM]
Free Drug
L428
786-O
HEL
HL-60
MCF-7
Compound
<0.00001
<0.00001
<0.00001
<0.00001
<0.00001
28
Compound
<0.00001
<0.00001
<0.00001
<0.00001
0.01
30
Compound
<0.00001
<0.00001
<0.00001
<0.00001
1.0
32
Compound
5
19
Referring to the following Table 3, the activities of compounds 23, 25, are compared to that of compound 11 on 786-O, Caki-1, MCF-7, HL-60, THP-1, HEL, and TF1 cells. Cells were plated in 150 μL growth media per well into black-sided clear-bottom 96-well plates (Costar, Corning) and allowed to settle for 1 hour in the biological cabinet before placing in the incubator at 37° C., 5% CO2. The following day, 4× concentration of drug stocks were prepared, and then titrated as 10-fold serial dilutions producing 8-point dose curves and added at 50 μl per well in duplicate. Cells were then incubated for 48 hours at 37° C., 5% CO2. Cytotoxicity was measure by incubating with 100 μL Cell Titer Glo (Promega) solution for 1 hour, and then luminescence was measured on a Fusion HT plate reader (Perkin Elmer). Data was processed with Excel (Microsoft) and GraphPad (Prism) to produce dose response curves and IC50 values were generated and data collected.
TABLE 3
IC50 Summary for Free Drugs [nM]
Free Drug
786-
Caki-
MCF-
O
1
7
HL-60
THP-1
HEL
TF1a
Compound 11
0.4
0.2
1
0.01
1
0.03
1
Compound 23
0.06
0.02
0.7
0.005
0.4
0.009
0.2
Compound 25
0.09
0.06
0.8
0.01
0.9
0.02
0.9
In Examples 13 to 16, the following compounds are referred to by the compound numbers as show below:
Compound
Alternative Designation
11
37
13
57
19
42
25
95
28
50
30
49
104
66
Example 13
Synthesis of PBD Drug Linker Compounds
General Information. In the following examples, all commercially available anhydrous solvents were used without further purification. Analytical thin layer chromatography was performed on silica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, N.J.). Radial chromatography was performed on Chromatotron apparatus (Harris Research, Palo Alto, Calif.). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Samples were eluted over a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 reverse-phase column. The acidic mobile phase consisted of acetonitrile and water both containing either 0.05% trifluoroacetic acid or 0.1% formic acid (denoted for each compound). Compounds were eluted with a linear gradient of acidic acetonitrile from 5% at 1 min post injection, to 95% at 11 min, followed by isocratic 95% acetonitrile to 15 min (flow rate=1.0 mL/min). LC-MS was performed on a ZMD Micromass mass spectrometer interfaced to an HP Agilent 1100 HPLC instrument equipped with a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reverse phase column. The acidic eluent consisted of a linear gradient of acetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min, followed by isocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min). Preparative HPLC was carried out on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Products were purified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm, 80 Å reverse phase column eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The purification method consisted of the following gradient of solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min with monitoring at 254 nm. NMR spectral data were collected on a Varian Mercury 400 MHz spectrometer. Coupling constants (J) are reported in hertz.
(S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (36): To a solution of Val-Ala dipeptide 34 (200 mg, 1.06 mmol) dissolved in 10.6 mL anhydrous DMF was added maleimidocaproyl NHS ester 35 (327 mg, 1.06 mmol). Diisopropylethyamine (0.92 mL, 5.3 mmol) was then added and the reaction was stirred under nitrogen at an ambient temperature for 18 h, at which time TLC and analytical HPLC revealed consumption of the starting material. The reaction was diluted with 0.1 M HCl (100 mL), and the aqueous layer was extracted with ethyl acetate (100 mL, 3×). The combined organic layer was washed with water and brine, then dried over sodium sulfate, filtered and concentrated. The crude product was dissolved in minimal methylene chloride and purified by radial chromatography on a 2 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (95:5 to 90:10 CH2Cl2/MeOH) to provide 36 (158 mg, 39%) as an oily residue. TLC: Rf=0.26, 10% MeOH in CH2Cl2. 1H NMR (CDCl3) δ (ppm) 0.95 (d, J=17 Hz, 3H), 0.98 (d, J=17 Hz, 3H), 1.30 (m, 2H), 1.40 (d, J=17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1H), 2.25 (dt, J=4, 19 Hz, 2H), 3.35 (s, 1H), 3.49 (t, J=17 Hz, 2H), 4.20 (d, J=18 Hz, 1H), 4.38 (m, 1H), 6.80 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.05 min. LC-MS: tR 11.17 min, m/z (ES+) found 381.9 (M-FH)+, m/z (ES−) found 379.9 (m−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (38): A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.5 μmol), EEDQ (2.8 mg, 11.4 μmol), and 0.33 mL anhydrous CH2Cl2. Methanol (four drops, ˜80 μL) was added to facilitate dissolution and the mixture was stirred under nitrogen for 1 h. PBD dimer 37 (5.7 mg, 7.9 μmol) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide the drug linker 38 (3.9 mg, 45%). TLC: Rf=0.06, 5% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.51 min. LC-MS: tR 12.73 min, m/z (ES+) found 1089.6 (M+H)+, m/z (ES−) found 1087.3 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (40): To a flame-dried 10 mL flask was added PBD dimer 37 (25 mg, 34.4 μmol), which was dissolved in 1.4 mL of a 10% MeOH in CHCl3 solvent mixture. Maleimidocaproic acid (39) was added (7.3 mg, 34.4 μmol), followed by EEDQ (10.2 mg, 41.3 μmol) and pyridine (6 μL, 68.8 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 14 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker 40 (14.1 mg, 45%). LC-MS: tR 12.81 min, m/z (ES+) found 918.9 (M+H)+, m/z (ES−) found 917.0 (M−H)−.
2-bromo-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)acetamide (41): To a flame-dried 10 mL flask was added PBD dimer 37 (16.5 mg, 22.7 μmol), which was dissolved in 0.9 mL of a 10% MeOH in CHCl3 solvent mixture. Bromoacetic acid was added (3.2 mg, 22.7 μmol), followed by EEDQ (6.8 mg, 27.2 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 4 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 95:5 CH2Cl2/MeOH) to provide drug linker 41 (9.9 mg, 52%). TLC: Rf=0.09, 5% MeOH in CH2Cl2. LC-MS: tR 12.44 min, m/z (ES+) found 848.1 (M-FH)+, m/z (ES−) found 845.7 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (43): A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.4 μmol), EEDQ (2.8 mg, 11.3 μmol), and 0.38 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 42 (6.8 mg, 9.4 μmol) was then added and the reaction was stirred at room temperature for 2 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker 43 (3.1 mg, 30%). TLC: Rf=0.31, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.49 min. LC-MS: tR 12.28 min, m/z (ES+) found 1089.5 (M+H)+, m/z (ES−) found 1087.3 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (44): To a flame-dried 10 mL flask was added PBD dimer 42 (8.0 mg, 11 μmol), which was dissolved in 0.44 mL of a 10% MeOH in CH2Cl2 solvent mixture. Maleimidocaproic acid (39) was added (2.3 mg, 11 μmol), followed by EEDQ (3.3 mg, 13.2 μmol) and pyridine (1.8 μL, 22 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker compound 44 (1.2 mg, 12%). TLC: Rf=0.45, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.71 min. LC-MS: tR 12.63 min, m/z (ES+) found 919.1 (M+H)+, m/z (ES−) found 917.1 (M−H)−.
(2S,3R,4S,5R,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltriacetate (46): A flame-dried flask was charged with glucuronide linker intermediate 45 (reference: Jeffrey et al., Bioconjugate Chemistry, 2006, 17, 831-840) (15 mg, 20 μmol), 1.4 mL anhydrous CH2Cl2, pyridine (20 μL, 240 μmol), and then cooled to −78° C. under nitrogen. Diphosgene (3.0 μL, 24 μmol) was then added and the reaction was stirred for 2 h at −78° C., after which time a small aliquot was quenched with methanol and analyzed by LC-MS for formation of the methyl carbonate, which confirmed formation of the glucuronide chloroformate. PBD dimer 42 (15 mg, 20 μmol) was then dissolved in 0.7 mL anhydrous CH2Cl2 and added dropwise to the reaction vessel. The reaction was warmed to 0° C. over 2 h and then diluted with 50 mL CH2Cl2.
The organic layer was washed with water (50 mL), brine (50 mL), dried over sodium sulfate, filtered and concentrated. The crude reaction product was purified by radial chromatography on a 1 mm chromatotron plate eluted 10% MeOH in CH2Cl2 to provide 46 (5.7 mg, 19%). TLC: Rf=0.47, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 12.09 min. LC-MS: tR 14.05 min, m/z (ES+) found 1500.3 (M+H)+.
(2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (47): A flask containing 46 (5.7 mg, 3.8 μmol) dissolved in a solvent mixture of 0.2 mL each of MeOH, tetrahydrofuran, and water was cooled to 0° C. To the stirred solution was added lithium hydroxide monohydrate (0.8 mg, 19 μmol) and the reaction was stirred at room temperature for 4 h, at which time LC-MS indicated conversion to product. Glacial acetic acid (1.1 μL, 19 μmol) was added and the reaction was concentrated to provide 47, which was carried forward without further purification. LC-MS: tR 11.59 min, m/z (ES+) found 1138.4 (M+H)+.
(2S,3S,4S,5R,6S)-6-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (48): To a solution of 47 (4.3 mg, 3.8 umol) dissolved in 0.38 mL anhydrous DMF was added maleimidocaproyl NHS ester 35 (1.2 mg, 3.8 umol), followed by diisopropylethylamine (4.0 uL, 22.8 umol). The reaction was stirred at room temperature under nitrogen for 2 h, at which time LC-MS revealed conversion to product. The reaction was diluted with a mixture of acetonitrile (0.5 mL), DMSO (1 mL), water (0.5 mL), and then purified by preparative HPLC. The mobile phase consisted of A=water and B=acetonitrile, both containing 0.1% formic acid. A linear elution gradient of 90:10 A:B to 10:90 A:B over 75 minutes was employed and fractions containing the desired product were lyophilized to provide drug linker compound 48 (1.2 mg, 24% over two steps). Analytical HPLC (0.1% formic acid): tR 10.85 min. LC-MS: tR 12.12 min, m/z (ES+) found 1331.4 (M+H)+, m/z (ES−) found 1329.5 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((3-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (51): A flame-dried 10 mL flask was charged with acid 36 (2.7 mg, 7.1 μmol), EEDQ (2.1 mg, 8.5 μmol), and 0.28 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 49 (5.8 mg, 7.1 μmol) was then added and the reaction was stirred at room temperature for 20 h, at which time LC-MS revealed conversion to product. The reaction was concentrated then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug linker compound 51 (2.7 mg, 32%). Analytical HPLC (0.1% formic acid): tR 9.17 min. LC-MS: tR 11.25 min, m/z (ES+) found 1185.3 (M+H)+, m/z (ES−) found 1182.9 (M−H)−.
N—((S)-1-(((S)-1-((3-((S)-8-((5-(((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (52): A flame-dried 10 mL flask was charged with acid 36 (3.7 mg, 9.7 μmol), EEDQ (2.9 mg, 11.6 μmol), and 0.4 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 50 (8.0 mg, 9.7 μmol) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS revealed the presence of product. The reaction was concentrated then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug linker compound 52 (3.1 mg, 25%). Analytical HPLC (0.1% formic acid): tR 9.45 min. LC-MS: tR 11.75 min, m/z (ES+) found 1188.4 (M+H)+, m/z (ES−) found 1186.0 (M−H)−.
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)benzamide (54): To a flame-dried 10 mL flask was added linker fragment 53 (7.7 mg, 20 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.1 mg, 25 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (12 mg, 16.5 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide 54 (2.4 mg, 13%). TLC: Rf=0.44, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.53 min. LC-MS: tR 12.61 min, m/z (ES+) found 1095.4 (M+H)+, m/z (ES−) found 1093.9 (M−H)−.
(S)-2-(2-iodoacetamido)-N—((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (56): A flame-dried flask was charged with linker 55 (7.8 mg, 22 μmol), which was dissolved in 0.37 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.8 mg, 27.5 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (13 mg, 18 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 56 (3.5 mg, 18%). Analytical HPLC (0.1% formic acid): tR 11.43 min. LC-MS: tR 12.49 min, m/z (ES+) found 1064.6 (M+H)+, m/z (ES−) found 1098.9 (M+2H2O—H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (58): To a flame-dried 10 mL flask was added linker fragment 36 (19 mg, 50 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (12.4 mg, 50 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 57 (12.5 mg, 16.6 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 5 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 58 (2.1 mg, 11%). Analytical HPLC (0.1% formic acid): tR 12.19 min. LC-MS: tR 12.58 min, m/z (ES+) found 1117.8 (M+H)+, m/z (ES−) found 1133.7 (M+H2O—H)−.
(R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoic acid (60): A flame dried flask was charged with Fmoc-D-Valine (200 mg, 0.59 mmol) and 5.9 mL anhydrous THF. N-hydroxysuccinimide (75 mg, 0.65 mmol) was added, followed by diisopropylcarbodiimide (0.1 mL, 0.65 mmol), and the reaction was stirred at an ambient temperature overnight, at which time LC-MS revealed conversion to product. The reaction mixture was diluted with CH2Cl2 and washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to dryness. The material was carried forward without further purification. LC-MS: tR 13.89 min, m/z (ES+) found 437.0 (M+H)+. Crude Fmoc-D-Val-OSu (0.59 mmol) was dissolved in dimethoxyethane (1.5 mL) and THF (0.8 mL). D-alanine (73 mg, 0.89 mmol) was dissolved in 2.3 mL water and added to the reaction mixture, followed by sodium bicarbonate (99 mg, 1.2 mmol). The resulting slurry was stirred at room temperature overnight, at which time the reaction had clarified and LC-MS revealed completion. The reaction was poured into 50 mL CH2Cl2 and the organic layer was washed with 50 mL 0.1 M HCl and then brine, dried over sodium sulfate, and then concentrated to dryness. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2 to provide 60 (128 mg, 54%). TLC: Rf=0.18, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 9.47 min. LC-MS: tR 13.09 min, m/z (ES+) found 411.1 (M+H)+, m/z (ES−) found 409.2 (M−H)−.
(R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (61): Protected dipeptide 60 (70 mg, 0.37 mmol) was suspended in 6 mL anhydrous CH2Cl2, cooled on ice under nitrogen, and 2 mL of diethylamine was added dropwise. The reaction was warmed to room temperature and stirred under nitrogen for 2 h, at which time HPLC revealed consumption of starting material. The reaction was diluted with 6 mL of chloroform and concentrated. The crude reaction residue was re-dissolved in 6 mL chloroform and concentrated twice, followed by drying on a vacuum line for 2 h. The deprotected dipeptide was then dissolved in 3.7 mL anhydrous DMF. MC-OSu (138 mg, 0.44 mmol) was then added, followed by diisopropylethylamine (0.32 mL, 1.9 mmol). The reaction was stirred under a nitrogen atmosphere at room temperature overnight. Workup was achieved by pouring the reaction in to 50 mL 0.1 M HCl and extracting with ethyl acetate (50 mL, 3×). The combined organic layer was washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and concentrated. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (99:1 to 95:5 CH2Cl2/MeOH) to provide 61 (14 mg, 22%). 1H NMR (CD3OD) δ (ppm) 0.94 (d, J=14 Hz, 3H), 0.98 (d, J=14 Hz, 3H), 1.29 (m, 2H), 1.39 (d, J=7.4 Hz, 3H), 1.61 (m, 4H), 2.05 (m, 1H), 2.25 (dt, J=1.2, 7.4 Hz, 2H), 3.48 (t, J=7 Hz, 2H), 4.19 (m, 1H), 4.37 (m, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 10.04 min. LC-MS: tR 11.22 min, m/z (ES+) found 382.1 (M+H)+, m/z (ES−) found 380.0 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((R)-1-(((R)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (62): To a flame-dried 10 mL flask was added linker 61 (9.5 mg, 25 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (7.3 mg, 30 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (12 mg, 16.5 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 62 (2.8 mg, 16%). TLC: Rf=0.39, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.50 min. LC-MS: tR 12.50 min, m/z (ES+) found 1089.7 (M+H)+, m/z (ES−) found 1088.0 (M−H)−.
(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanoic acid (64): L-alanine (58 mg, 0.65 mmol) was suspended in 6.5 mL anhydrous DMF and MC-OSu 35 (100 mg, 0.324 mmol) was then added. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added and the reaction was stirred overnight at room temperature under nitrogen. The reaction was then diluted with 50 mL 0.1 M HCl and the aqueous layer was then extracted with ethyl acetate (50 mL, 3×). The combined organic layer was then washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and then concentrated to dryness. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (97.5:2.5 to 90:10 CH2Cl2/MeOH) to provide 64 (25 mg, 27%). TLC: Rf=0.25, 10% MeOH in CH2Cl2. 1H NMR (CD3OD) δ (ppm) 1.30 (m, 2H), 1.37 (d, J=7.4 Hz, 3H), 1.60 (m, 4H), 2.21 (t, J=7.4 Hz, 2H), 3.48 (t, J=7 Hz, 2H), 4.35 (q, J=7.4 Hz, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.06 min.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)hexanamide (65): To a flame-dried 10 mL flask was added linker 64 (14 mg, 50 μmol), which was dissolved in 0.66 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (15 mg, 60 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (24 mg, 33 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide 65 (3.5 mg, 11%). Analytical HPLC (0.1% formic acid): tR 11.40 min. LC-MS: tR 12.39 min, m/z (ES+) found 990.6 (M+H)+, m/z (ES−) found 989.0 (M−H)−.
PBD Dimer 57 Linked Directly Through Maleimidocaproyl Spacer (Scheme 14): PBD dimer 57 is coupled to maleimidocaproic acid 39 employing the chemistry described in Scheme 2.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (68): To a mixture of the 66 (10 mg, 0.013 mmol) in CH2Cl2 (300 μL) was added DIPEA and MC-Cl (67) (3 mg, 0.013 mmol). After 1 h, an additional 3 equiv. of DIPEA (7 μL) and 2 equiv. of the acid chloride (6 mg, 0.026 mmol) were added. After 1 h, an additional quantity of DIPEA (7 μL) and acid chloride (6 mg, 0.026 mmol) were added. After an additional 3 h, the reaction mixture was aspirated directly onto a 1 mm radial chromatotron plate and eluted with dichloromethane followed by a gradient of methanol (1% to 5%) in dichloromethane. Product containing fractions, as a mixture with the starting aniline, were concentrated to a residue and dissolved in a mixture of 0.5 mL DMSO, 0.5 mL acetonitrile and 0.5 mL deionized water and was further purified by preparative HPLC. The major peak was collected and the fractions were combined, frozen and lyophilized to give 2.1 mg (18%): MS (ES+) m/z 919.2 [M+H]+.
Note: Acid chloride 67 was prepared by dissolving 100 mg of 39 in oxalyl chloride (5 mL). A drop of DMF was added and the mixture was stirred at an ambient temperature for several hours before being concentrated under reduced pressure. Dichloromethane was added and the mixture was concentrated a second time to afford an off-white solid which was used directly: 1H-NMR (400 MHz, CDCl3) δ 6.70 (s, 2H), 3.46 (t, J=7 Hz, 2H), 2.82 (t, J=7.2 Hz, 2H), 1.72 (pent, J=7.6 Hz, 2H), 1.61 (pent, J=7.4 Hz, 2H), 1.35 (pent, J=7.6 Hz, 2H).
tert-butyl 2-(2-aminoacetamido)acetate (69): To a mixture of the glycine tert-butyl ester hydrogen chloride salt (70) (484 mg, 2.9 mmol) in dichloromethane (25 mL) was added Fmoc-Gly-OH (71) (0.861 mg, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was stirred at an ambient temperature for 16 h and then poured into ethyl acetate and was washed with water (3×) and brine (1×). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified via radial chromatography on a 2 mm plate eluting with 5% methanol/dichloromethane. Product containing fractions were concentrated under reduced pressure and treated with 20% piperidine/dichloromethane (10 mL) for 1 h, before being concentrated under reduced pressure and then purified twice via radial chromatography on a 2 mm plate eluting with a gradient of 5 to 10% methanol/dichloromethane to provide (200 mg, 37%): 1H-NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 4.00 (s, 2H), 3.39 (s, 2H), 1.47 (s, 9H).
2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetamido)acetic acid (72): To a solution of the amine 69 (200 mg, 0.11 mmol) in DMF (1 mL) was added 35 (350 mg, 0.11 mmol) and the reaction mixture was allowed to stir at an ambient temperature for 2 h. The mixture was concentrated under reduced pressure and was purify by radial chromatography on a 1 mm plate eluting with dichloromethane and a gradient of methanol (1 to 5%) in dichloromethane. Product containing fractions were concentrated under reduced pressure, dissolved in dichloromethane (4 mL) and treated with trifluoroacetic acid (4 mL). After 40 min the mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated to give 22.5 mg (19%) of 72 as white solid: 1H-NMR (400 MHz, CD3OD) δ 6.79 (s, 2H), 3.93 (s, 2H), 3.89 (s, 2H), 3.49 (t, J=6.8 Hz, 2H), 2.26 (t, J=6.8 Hz, 2H), 1.61 (m, 4H), 1.34 (m, 2H); MS (ES+) m/z 326.21 [M+H]+.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (73): To a mixture of 72 (15 mg, 0.046 mmol) in 5% methanol/dichloromethane (0.5 mL) was added EEDQ (11 mg, 0.046 mmol) and the mixture was stirred for 30 min at an ambient temperature, at which time 37 (16 mg, 0.023 mmol) was added. The reaction mixture was stirred for 3 h and was purified directly on a 1 mm radial chromatotron plate eluting with a 1% to 4% methanol/dichloromethane gradient to give 6.8 mg (29%) of 73 as a yellow solid: MS (ES+) m/z 1033.57 [M+H]+.
(S)-tert-butyl 1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (74): To a mixture of L-proline-tert-butyl ester hydrogen chloride salt 75 (0.5 g, 2.9 mmol) in dichloromethane (50 mL) was added 76 (0.98 g, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was allowed to stir at an ambient temperature for 16 h. The mixture was poured into ethyl acetate (100 mL) and was washed with 0.2 N HCl (50 mL), water (50 mL), brine (50 mL) and dried over MgSO4. Chromatography was conducted on a 2 mm radial chromatotron plate eluting with 10% ethyl acetate in hexanes. Product-containing fractions were concentrated under reduced pressure, dissolved in dichloromethane (8 mL) and treated with piperidine (2 mL). The mixture was stirred for 1 h, concentrated under reduced pressure and purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane. This gave 200 mg (26%) of the dipeptide 74: 1H-NMR (400 MHz, CDCl3) δ 4.41 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H), 3.57 (m, 4H), 3.2 (m, 1H), 2.82 (m, 1H), 2.83-1.65 (m, 5H), 1.44 (m, 9H).
(S)-tert-butyl 1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (77): To a mixture of the amine 74 (200 mg, 0.75 mmol), 39 (190 mg, 0.9 mmol) and DIPEA (0.32 mL, 1.8 mmol) was added HATU (342 mg, 0.9 mmol) and the mixture was allowed to stir at an ambient temperature for 5 h. The mixture was poured into ethyl acetate (100 mL) and washed with water (3×100 mL) and brine (1×100 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated. The resulting residue was subjected to radial chromatography on a 2 mm radial chromatotron plate eluting with dichloromethane followed by an increasing gradient of 1 to 5% methanol in dichloromethane. Two additional purifications, both eluting with a gradient of 1 to 5% methanol in dichloromethane, first on a 2 mm plate and then on a 1 mm plate afforded 113 mg (33%) of 77 as an white solid: 1H-NMR (400 MHz, CDCl3) δ 4.63 (m, 1H), 4.41 (m, 1H), 3.82 (m, 1H), 3.6 3 (m, 1H), 3.55 (m, 1H), 3.45 (m, 3H), 2.38-1.83 (m, 10H), 1.70-1.50 (m, 5H), 1.45 (m, 9H), 1.35 (m, 2H); MS (ES+) m/z 462.33 [M+H]+.
(S)-1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid (78): To a mixture of the tert-butyl ester 77 in dichloromethane (4 mL) was added trifluoroacetic acid (4 mL). After 40 min the reaction was determined to be complete by HPLC analysis. The mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated a second time to give 37 mg (100%) of 78 as a white solid: 1H-NMR (400 MHz, CDCl3) 6.68 (s, 2H), 4.62 (m, 2H), 3.81 (m, 1H), 3.70 (m, 1H), 3.57 (m, 2H), 3.45 (m, 2H), 2.40-1.91 (m, 10H), 1.70-1.45 (m, 4H), 1.33 (m, 2H); MS (ES+) m/z 406.2 [M+H]+.
1-(1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)pyrrolidine-2-carboxamide (79): To a mixture of the 78 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (0.4 mL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 15 min at an ambient temperature and then 37 (15 mg, 0.021 mmol) was added. The mixture was stirred for 4 h, the reaction mixture was diluted with dichloromethane (2 mL) and was aspirated directly onto a 1 mm radial chromatotron plate. The product was eluted with a gradient of 1 to 5% methanol in dichloromethane to provide 6.8 mg (29%) of 79 as a yellow solid: MS (ES+) m/z 1113.51 [M+H]+.
(S)-5-(allyloxy)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-oxopentanoic acid (80): To a mixture of the 2-chlorotrityl resin (1.0 g, 1.01 mmol) suspended in dichloromethane (10 ml) was added Fmoc-Glu-(OAllyl)-OH (81) (409 mg, 1.0 mmol) and DIPEA (173 μL, 1.0 mmol). The reaction mixture was shaken for 5 min, and an additional portion of DIPEA (260 μL, 1.5 mmol) was added and the mixture was shaken for 1 h. Methanol (0.8 mL) was added and the mixture was shaken for 5 min, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure. The resulting resin was subjected to 20% piperidine in dichloromethane (10 mL) for 1 h, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure.
To a mixture of the Fmoc-Val-OH (82) (1.03 g, 3.30 mmol)) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After thorough mixing, the solution as aspirated into a 10 mL syringe containing the resin prepared above. The mixture was capped and shaken for 16 h. The resin was washed with DMF (6×), dichloromethane (6×) and ether (6×). A small portion (10 mg) was isolated and treated with 20% TFA/Dichloromethane and the resulting solution analyzed by LC-MS which revealed one high purity peak which displayed the correct mass (MS (ES+) m/z 509.28 [M+H]+). The remaining resin was then treated with 20% piperidine/DMF (8 mL) for 2 h, before being washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure.
A mixture of allyl chloroformate (529 μL, 5.05 mmol), DIPEA (1.7 mL, 10 mmol) in dichloromethane (10 mL) was prepared and aspirated into a syringe containing the resin above. The mixture was capped and shaken. After approximately 2 h, the reaction mixture was drained, and washed with dichloromethane (6×). A small portion of the resin (˜10 mg) was cleaved with 20% TFA/dichloromethane and analyzed by LC-MS for masses of starting material and product. The main component was still the unreacted amine, so the resin was again subjected to the conditions described above. After 4 h, the resin was washed with dichloromethane (6×), and then treated repeatedly with 5% TFA in dichloromethane (4×7 mL). The resulting solution was concentrated under reduced pressure. The mixture was purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 107 mg of 80: 1H-NMR (400 MHz, CDCl3) δ 7.05 (s, 1H), 5.90 (m, 2H), 5.57 (d, 1H), 5.29 (d, J=14.7 Hz, 2H), 5.22 (t, J=10.9 Hz, 2H), 4.59 (m, 5H), 4.02 (m, 1H), 2.60-2.40 (m, 2H), 2.37-2.18 (m, 1H), 2.17-2.02 (m, 2H), 0.96 (d, J=6.4 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H); MS (ES+) m/z 371.12 [M+H]+.
(S)-allyl 4-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoate (83): To a mixture of the acid 80 (30, 0.04 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (20 mg, 0.082 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (30 mg, 0.04 mmol) was added and the mixture was stirred for approximately 5 h. Partially purification by aspirating directly onto a 1 mm radial chromatotron plate and eluting with a gradient of 1% to 5% methanol/dichloromethane afforded a mixture of desired product and 37 (26 mg; ˜3:1 respectively) which was carried forward without further purification.
(S)-4-((S)-2-amino-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoic acid (84): To the mixture of 83 and 37 (26 mg) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 mmol), pyrrolidine (4 μL, 0.048 mmol) and tetrakis palladium (0.7 mg, 0.6 μmol). After 2 h, an additional quantity (0.7 mg, 0.6 μmol) of tetrakis palladium was added and the reaction was allowed to stir for an additional 1 hr before being concentrated under reduced pressure. The residue was dissolved in DMSO (1 mL), acetonitrile with 0.05% formic acid (1 mL) and water with 0.05% formic acid (1 mL) and purified by preparative reverse phase HPLC. A single fraction of product was collected and lyophilized to give 6 mg (14% for two steps) of 84: MS (ES+) m/z 1078.6 [M+H]+.
(S)-4-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoic acid (85): To a mixture of the 84 (6 mg, 6 μmol), and 35 (2 mg, 6 μmol) in DMF (200 μL) was added DIPEA (3 μL, 18 μmol) and the reaction mixture was stirred at an ambient temperature. After 1 h, an additional equivalent of 35 (2 mg, 6 μmol) was added and the reaction was allowed to continue to stir at an ambient temperature for 3 h. A third equivalent of 35 (2 mg, 6 μmol) was added and the mixture was stirred for approximately 1 h, concentrated under reduced pressure, dissolved in dichloromethane and aspirated directly onto a 1 mm radial chromatotron plate and eluted with 5% methanol in dichloromethane. This gave 2.5 mg (36%) of high purity 85: MS (ES+) m/z 1147.49 [M+H]+.
(21S,24S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-isopropyl-24-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)-3,19,22-trioxo-7,10,13,16-tetraoxa-4,20,23-triazaheptacosan-27-oic acid (86): To a mixture of the 84 (8 mg, 8.4 μmol) and Mal-PEG4-NHS (87) (6.5 mg, 12.6 μmol) in DMF (200 μL) was added DIPEA (4.3 μL, 25 μmol). The reaction mixture was stirred at an ambient temperature for 2 h, and was concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane and aspirated onto a 1 mm radial chromatotron plate. The material was polar and did not chromatograph on the silica gel-based chromatotron plate. The plate was eluted with methanol to recover the mixture which was isolated under reduced pressure. The residual material was purified via preparative reverse phase HPLC. A single main peak eluted and the fractions were combined, frozen and lyophilized to a residue of 0.9 mg (8%) of 86: MS (ES+) m/z 1353.04 [M+H]+.
(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)hexanoic acid (88): To a mixture of the 2-chlorotrityl resin (1 g, 1.01 mmol) in CH2Cl2 (10 ml) was added Fmoc-Lys(Me)2-OH (89) (432 mg, 1.0 mmol) and DIPEA (433 μL, 2.5 mmol). The reaction mixture was shaken for 1 h. Methanol (0.8 mL) was added and the mixture was shaken for an additional 5 min, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure. The dried resin was subjected to 20% piperidine in DMF (10 mL) for 1 h, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×).
To a mixture of the 39 (3.0 mmol, 633 mg) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After thorough mixing, the solution as aspirated into a 10 mL syringe containing the resin above. The mixture was capped, shaken for 16 h, filtered and the resin washed with DMF (6×), dichloromethane (6×), and ethyl ether (6×). The resin was by repeatedly treating with 5% TFA/dichloromethane (6 mL×5), shaking for 1 min, and then filtering. The resulting solution was concentrated under reduced pressure and under high vacuum. The material was purified by preparatory reverse phase HPLC to give 208 mg of 88: 1H-NMR (400 MHz, CD3OH/CDCl3 1:1 mixture) 6.73 (s, 2H), 4.41 (m, 1H), 3.48 (t, 2H), 3.31 (s, 1H), 3.03 (m, 2H), 2.84 (s, 6H), 2.22 (m, 2H), 1.87 (m, 2H), 1.78-1.52 (m, 6H), 1.43 (m, 2H), 1.31 (pent, 2H); MS (ES+) m/z 386.28 [M+H]+.
(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (90): To a mixture of the 88 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (400 μL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (15 mg, 0.021 mmol) was added. After 4 h, the mixture was concentrated under reduced pressure, dissolved in a mixture of DMSO (1 mL), acetonitrile (2 mL containing 0.05% formic acid) and water (1 mL containing 0.05% formic acid) and purified by reverse-phase HPLC (method A). Product containing fractions were contaminated with 37, so the fractions were lyophilized to a residue and repurified as described above to give 0.5 mg (2%) of pure 90: MS (ES+) m/z 537.46 [M+H]/2+.
Allyl ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (91): To a mixture of the 92 (45 mg, 0.123 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (30.4 mg, 0.123 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (30 mg, 0.041 mmol) was added. The reaction mixture was stirred for approximately 5 h and then purified on a 1 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 22 mg (55%) of 91 which was not characterized but carried on directly.
1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (93): To a solution of the 91 (22 mg, 0.022 mmol) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 mmol), pyrrolidine (4 μL, 0.048 mmol) and tetrakis palladium (0.7 mg, 6 μmol). After approximately 2 h, the reaction mixture was purified on a 1 mm radial chromatotron plate eluting with 5% to 10% methanol/dichloromethane. The major band was collected and concentrated to a residue which was dissolved in DMF (0.2 mL) and reacted with NHS ester 87 (10 mg, 0.19 mmol). The reaction was allowed to stir for 30 min, concentrated and purified by radial chromatography on a 1 mm plate eluting with 5% methanol/dichloromethane to give 3.2 mg (11%) of 93: MS (ES+) m/z 1294.7 [M+H]+.
(E)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-(4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)benzylidene)hexanehydrazide (94): To a mixture of the aldehyde 95 (5.4 mg, 7 μmol) in 5% methanol/dichloromethane at 0° C. was added the hydrazide-TFA salt 96 (4.5 mg, 14 μmol). The reaction mixture was allowed to warm to an ambient temperature and stir for 5 h before being concentrated under reduced pressure and purified on a silica gel column eluting with 3% methanol/dichloromethane to give 2.2 mg (32%) of 94: MS (ES+) m/z 974.49 [M+H]+.
(S)-tert-butyl 2-((S)-2-amino-3-methylbutanamido)propanoate (97): To a mixture of the alanine-O-tert-butyl ester hydrogen chloride salt (98) (500 mg, 2.76 mmol) in dichloromethane (5 mL) was added Fmoc-val-OSu (99) (1.09 g, 2.51 mmol). DIPEA (0.96 ml, 5.5 mmol) was added and the reaction mixture was allowed to stir at an ambient temperature for 16 h. The mixture was poured into dichloromethane (100 mL) and washed with 1N HCl (50 mL) and water (50 mL) before being dried over magnesium sulfate. The material was chromatographed on a 2 mm radial chromatotron plate eluting with 1 to 5% methanol/dichloromethane gradient and product containing fractions were combined and concentrated. The resulting residue was dissolved in dichloromethane (16 mL) and piperidine (4 mL) was added. The mixture was stirred for 10 min before being concentrated under reduced pressure. The resulting residue was chromatographed on a 2 mm plate eluting first with ammonia-saturated dichloromethane followed by 5% methanol in ammonia-saturated dichloromethane to give 494 mg (2.02 mmol, 81% for two steps) of 97: 1H-NMR (400 MHz, CDCl3) 7.78 (bs, 1H), 4.47 (m, 1H), 3.30 (d, 1H), 2.30 (m, 1H), 1.38 (d, 3H), 1.47 (s, 9H), 1.00 (d, J=7.0 Hz, 3H), 0.84 (d, J=6.9 Hz, 3H).
(S)-tert-butyl 2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoate (100): To a mixture of the 97 (100 mg, 0.41 mmol) and 4-maleimidobenzoic acid (101) (98 mg, 0.45 mmol) was added dichloromethane (5 mL), followed by TBTU (157 mg, 0.49 mmol) and DIPEA (212 uL, 1.23 mmol). The mixture was stirred at an ambient temperature for 16 h and then purified on a 2 mm radial chromatotron plate eluting with 50% ethyl acetate in hexanes to give 95 mg (51%) of 100: 1H-NMR (400 MHz, CDCl3) 7.85 (d, J=6.6 Hz, 2H), 7.42 (d, J=6.6 Hz, 2H), 6.81 (s, 2H), 6.38 (bs, 1H), 4.43 (m, 2H), 2.14 (sept, J=6.6 Hz, 1H), 1.41 (s, 9H), 1.31 (d, J=7.0 Hz, 3H), 0.98 (m, 6H); MS (ES−) m/z 441.90 [M−H]−.
(R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic acid (53): To a mixture of 100 (47 mg, 0.11 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was monitored by TLC (50% ethyl acetate in hexane, after pumping down the TLC plate under high vacuum for 5 min). After 75 min, no starting material could be detected by TLC. The reaction was performed a second time using the same conditions and material from both reactions were combined and purified on a 2 mm radial chromatotron plate eluting with a gradient from 5-10% methanol in dichloromethane. The yield was 42 mg (49%) of 53: 1H-NMR (400 MHz, CDCl3) 7.92 (d, J=6.6 Hz, 2H), 7.51 (d, J=6.6 Hz, 2H), 7.0 (m, 1H), 6.89 (s, 2H), 6.70 (s, 1H), 4.60 M, 1H), 2.22 (m, 1H), 1.18 (d, J=6.6 Hz, 3H), 1.04 (m, 6H); MS (ES+) m/z 388.02 [M+H]+.
(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (102): To a mixture of the 97 (100 mg, 0.41 mmol) in dichloromethane was added iodoacetamide-NHS ester (103) (115 mg, 0.41 mmol) and the mixture was stirred at an ambient temperature. After 30 min, the mixture was aspirated onto a 1 mm chromototron plate and eluted with ethyl acetate in hexanes (1:1). A single band was collected and the structure was confirmed: 1H-NMR (400 MHz, CDCl3) 6.70 (d, J=7.8 Hz, 1H), 6.27 (d, J=7.0 Hz, 1H), 4.45 (m, 1H), 4.26 (dd, J=8.6, 6.3 Hz, 1H), 3.72 (quart, J=11.3 Hz, 2H), 2.13 (sept, J=6.5 Hz, 1H), 1.47 (s, 9H), 1.38 (d, J=7.1 Hz, 3H), 0.99 (m, 6H); MS (ES+) m/z 412.87 [M+H]+.
(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (55): See procedure for the synthesis of (R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic acid (53). This gave 22 mg (15% for two steps): 1H-NMR (400 MHz, D6-DMSO) 8.27 (d, J=9.4 Hz, 1H), 4.24 (m, 2H), 3.97 (bs, 2H), 3.83 (d, J=9.4 Hz, 1H), 3.71 (d, J=9.6 Hz, 1H), 2.07 (m, 1H), 1.33 (d, J=7.3 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H), 0.89 (d, J=6.9 Hz, 3H); MS (ES−) m/z 354.84 [M−H]−.
PBD dimers linked through aliphatic amines (Scheme 21). PBD dimers containing aliphatic amines, such as a benzyl amine (Example 9), are synthesized with peptidic linkers, the glucuronide linker, and/or linkers dependent on mAb degradation for release (i.e., non-cleavable linkers). Drug linkers conjugated through a benzyl amine will include: (1) a cleavable peptide employing chemistry similar to Scheme 1; (2) direct attachment with a maleimidocaproyl group (a noncleavable linker) (Scheme 2); (3) a glucuronide linker, prepared as described in Scheme 6.
Generic peptide linked 2-, 3-, and 4-aniline PBD dimers (Scheme 22). PBD dimers with anilines at the 2-, 3-, and 4-positions will be conjugated to peptide-based linkers, employing the chemistry described in Scheme 1, or attached directly with maleimidocaproic acid, as exemplified in Scheme 2.
Example 14
Preparation of PDB Dimer Conjugates
Antibody-drug conjugates were prepared as previously described (see Doronina et al., Nature Biotechnology, 21, 778-784 (2003)) or as described below. Briefly, for maleimide drug-linker the mAbs (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 were reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progress of the reaction, which reduces interchain disulfides, was monitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb was achieved. The reduced antibody was then cooled to 0° C. and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 h, the reaction was quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker was removed by gel filtration over a PD-10 column. The ADC was then sterile-filtered through a 0.22 μm syringe filter. Protein concentration was determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography was used to determine the extent of antibody aggregation and RP-HPLC confirmed the absence of remaining NAC-quenched drug-linker.
For halo acetamide-based drug linkers, conjugation was performed generally as follows: To a 10 mg/mL solution of reduced and reoxidized antibody (having introduced cysteines by substitution of S239C in the heavy chains (see infra)) in 10 mM Tris (pH 7.4), 50 mM NaCl, and 2 mM DTPA was added 0.5 volumes of propylene glycol. A 10 mM solution of acetamide-based drug linker in dimethylacetamide was prepared immediately prior to conjugation. An equivalent amount of propylene glycol as added to the antibody solution was added to a 6-fold molar excess of the drug linker. The dilute drug-linker solution was added to the antibody solution and the pH was adjusted to 8.0-8.5 using 1 M Tris (pH 9). The conjugation reaction was allowed to proceed for 45 minutes at 37° C. The conjugation was verified by reducing and denaturing reversed phase PLRP-S chromatography. Excess drug linker was removed with Quadrasil MP resin (Sigma Aldrich; Product #679526) and the buffer was exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl, and 5% propylene glycol using a PD-10 desalting column (GE Heathcare; Product #17-0851-01).
Engineered hlgG1 antibodies with introduced cysteines: CD70 antibodies containing a cysteine residue at position 239 of the heavy chain (h1F6d) were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). Following a 1 hour incubation at 37° C., the reaction was cooled to 22° C. and 30 equivalents of dehydroascorbic acid were added to selectively reoxidize the native disulfides, while leaving cysteine 239 in the reduced state. The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction was allowed to proceed for 1 hour at 22° C. The pH of the solution was then raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5 equivalents of the PBD drug linker in DMSO were placed in a suitable container for dilution with propylene glycol prior to addition to the reaction. To maintain solubility of the PBD drug linker, the antibody itself was first diluted with propylene glycol to a final concentration of 33% (e.g., if the antibody solution was in a 60 mL reaction volume, 30 mL of propylene glycol was added). This same volume of propylene glycol (30 mL in this example) was then added to the PBD drug linker as a diluent. After mixing, the solution of PBD drug linker in propylene glycol was added to the antibody solution to effect the conjugation; the final concentration of propylene glycol is 50%. The reaction was allowed to proceed for 30 minutes and then quenched by addition of 5 equivalents of N-acetyl cysteine. The ADC was then purified by ultrafiltration through a 30 kD membrane. (Note that the concentration of propylene glycol used in the reaction can be reduced for any particular PBD, as its sole purpose is to maintain solubility of the drug linker in the aqueous media.)
Example 15
Determination of In Vitro Activity of Selected Conjugates
The in vitro cytotoxic activity of the selected antibody drug conjugates was assessed using a resazurin (Sigma, St. Louis, Mo., USA) reduction assay (reference: Doronina et al., Nature Biotechnology, 2003, 21, 778-784). The antibody drug conjugates were prepared as described above in Example 13.
For the 96-hour assay, cells cultured in log-phase growth were seeded for 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of ADC in cell culture media were prepared at 4× working concentration; 50 μL of each dilution was added to the 96-well plates. Following addition of ADC, the cells were incubated with test articles for 4 days at 37° C. Resazurin was then added to each well to achieve a 50 μM final concentration, and the plates were incubated for an additional 4 h at 37° C. The plates were then read for the extent of dye reduction on a Fusion HT plate reader (Packard Instruments, Meridien, Conn., USA) with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
Referring to Table 4 (infra), the in vitro cytotoxicity of ADCs having para-aniline PBD dimers using the 96 hour assay is shown. The ADCs were tested against CD70+ CD30− cell lines and a control CD70− CD30− cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942), a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) had a lower IC50 than conjugates with a maleimidyl or acetamide-based linker (compounds 40 and 41, respectively).
In vitro cytotoxic activity of ADCs bearing drug linkers derived from para-aniline PBD dimer 37:
TABLE 4
In vitro cytotoxic activity on CD70+
cell lines (ng/mL), all ADCs 2 drugs/mAb
renal cell carcinoma
AML
CD70+/30−
CD70−/30−
786-O
Caki-1
769-P
ACHN
HEL9217
h1F6d-38
30
5
1378
h1F6-38
4
118
26
cAC10-38
1052
4005
508
h1F6-40
7113
1764
cAC10-40
2644
1264
h1F6-41
580
1243
cAC10-41
1153
1121
Referring to Table 5, the in vitro cytotoxicity of ADCs conjugate to PBD dimers on CD30+ cell lines using the 96 hour assay is shown. The ADCs were tested against CD30+ CD70+ cell lines and a CD70− CD30+ cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) generally had a lower IC50 than conjugates with a maleimidyl or acetamide-based linker (compounds 40 and 41, respectively).
TABLE 5
In vitro cytotoxic activity on CD30+
cell lines (ng/mL), all ADCs 2 drugs/mAb
ALCL
Hodgkin lymphoma
CD70−/30+
CD70+/30+
Karpas 299
L428
L540cy
L1236
Hs445
h1F6-38
1165
59
4
>10,000
5
cAC10-38
0.8
7
3
2012
0.2
h1F6-40
2195
7867
2557
cAC10-40
621
3172
134
h1F6-41
1330
3549
755
cAC10-41
340
957
13
In vitro cytotoxic activity of ADCs bearing drug linkers derived from meta-aniline PBD dimer 42:
Referring to Table 6, the in vitro cytotoxicity of ADCs containing PBD dimers on CD30+ cell lines using the 96 hour assay is shown. The activity was tested against CD30+ CD70+ cell lines and a CD70− CD30+ cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 43) and a glucuronide linker (48) generally had a lower IC50 than conjugates with a maleimidyl-based linker (compound 44).
TABLE 6
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Hodgkin
renal cell carcinoma
lymphoma
Caki-1
786-O
L428
h1F6d-43 (2 dr/mAb)
7
39
>10,000
IgG-43 (2 dr/mAb)
>10,000
>10,000
h1F6-44 (3.5 dr/mAb)
1124
2142
IgG-44 (3.5 dr/mAb)
1491
1242
h1F6d-48 (2 dr/mAb)
89
4093
IgG-48 (2 dr/mAb)
2939
6376
In vitro cytotoxic activity of ADCs bearing drug linkers derived from para- and meta-aniline PBD dimers 38 and 42 (respectively):
Referring to Table 7, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines L428 and 7860 and a CD70− AML cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker with a meta-aniline (drug linker compound 43) were somewhat less active than those having a maleimidyl-peptide linker with a para-aniline (drug linker compound 38). Reducing the drug loading of the meta-aniline compound to 2 per antibody reduced the activity. Conjugates with a glucuronide linker of the para-aniline compound (48) generally had a lower IC50 than conjugates with a maleimidyl-based linker (compound 39). Further, an aryl maleimide of the para-aniline compound (54) has no activity on these cell lines. Further, a conjugate having a maleimidyl linker conjugated directly to compound 42 has reduced activity as compared with conjugate h1F6-43 (data not shown).
TABLE 7
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Hodgkin
Renal cell
lymphoma
carcinina
L428
786O
control
h1F6- 43 (4 dr/mAb)
404
11
1205
h1F6d- 43 (2 dr/mAb)
Max inhib. = 40%
200
1625
h1F6d-48 (2 dr/mAb)
4093
89
1964
h1F6 54 (4 dr/mAb)
No effect
No effect
No effect
h1F6- 38 (2 dr/mAb)
230 (n = 2)
25 (n = 3)
503
In vitro cytotoxic activity of ADCs bearing drug linkers derived from aniline-linked PBD dimers
Referring to Table 8, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines Caki-1 and L428 and a CD70− cell line. The antibody used was a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Linkage of a PBD through an amine at the ortho position via a non-cleavable linker (compound 68) markedly reduced activity, as compared with an ADC linked via a para-aniline-linked cleavable linker (compound 54). Compounds 73 and 85, having a cleavable linker, showed comparable activity to compound 54; both of these compounds are linked via a para-aniline. Compounds with cleavable linkers requiring more stringeng cleavage, compounds 79 and 90, showed somewhat reduced activity, as compared to compound 54.
TABLE 8
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
renal cell carcinoma
Caki-1
786-O
Control
h1F6d- 68 (2 dr/mAb)
3236
3486
5501
h1F6d- 73 (2 dr/mAb)
2
7
482
h1F6d- 79 (2 dr/mAb)
24
348
5385
h1F6d- 54 (2 dr/mAb)
6
17
4665
h1F6d- 85 (2 dr/mAb)
3
5
4700
h1F6d- 90 (1.4 dr/mAb)
. . . 12
47
678
In vitro cytotoxic activity of ADCs bearing drug linkers derived from aniline-linked PBD dimers
Referring to Table 9, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines Caki-1 and L428 and two CD70− leukemia cell lines. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Compound 56, having a cleavable linker linked to the antibody via an acetamide showed comparable activity to compound 38. A glucuronide-linked version of the meta-aniline linked PBD dimer, compound 48, demonstrated little activity in this assay. Compound 58, having five methylene groups in the PBD bridge, demonstrated comparable activity to compound 38, having three methylene groups in the PBD bridge.
TABLE 9
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Renal Cell
Leukemia
Caki-1
786-O
CD70−
CD70−
ADCs
(CD70 #135,000)
(CD70 #190,000)
Line 1
Line 2
h1F6d-56
3
6
1672
Max Inh =
(1.8dr/Ab)
50%
h1F6d-48
Max Inh =
Max Inh =
No Effect
No Effect
(0.6dr/Ab)
45%
35%
h1F6d-58
0.5
2
1750
4847
(1.9dr/Ab)
h1F6d-38
5
15
2082
7188
(2dr/Ab)
(3-5, n = 4)
(5-30, n = 4)
Example 16
Determination of In Vivo Cytotoxicity of Selected Conjugates
All studies were conducted in concordance with the Animal Care and Use Committee in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. In vivo tolerability was first assessed to ensure that the conjugates were tolerated at clinically relevant doses. BALB/c mice were treated with escalating doses of ADC formulated in PBS with 0.01% Tween 20. Mice were monitored for weight loss following drug treatment; those that experienced 20% weight loss or other signs of morbidity were euthanized. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289).
Referring to FIG. 1, the results of a weight loss study are shown using cAC10-val-ala-SG3132(2) (cAC10-compound 38). A single dose of the conjugate administered at 5 mg administered either IP or IV resulted in little weight loss. A higher dose of the conjugate (15 mg/kg) caused weight loss in the mice.
Referring to FIG. 2, the results of a weight loss study are shown using h1F6-val-ala-SG3132(2) (h1F6-compound 38). A single dose of the conjugate administered at 5 mg administered IP resulted in some weight loss. A higher dose of the conjugate (10 mg/kg) caused significant weight loss in the mice.
Treatment studies were conducted in two CD70+ renal cell carcinoma xenograft models. Tumor (786-O and Caki-1) fragments were implanted into the right flank of Nude mice. Mice were randomized to study groups (n=5) on day eight (786-O) or nine (Caki-1) with each group averaging around 100 mm3. The ADC or controls were dosed ip according to a q4dx4 schedule. Tumor volume as a function of time was determined using the formula (L×W2)/2. Animals were euthanized when tumor volumes reached 1000 mm3. Mice showing durable regressions were terminated around day 100 post implant.
Referring to FIG. 3, the results of a treatment study using an h1F6-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. A control conjugate, cAC10-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice administered doses of the h1F6 conjugate at 0.1 mg/kg exhibited some tumor reduction, while higher doses at 0.3 mg/kg and 1 mg/kg appeared to exhibit complete tumor reduction. The control conjugate (non-binding) was less active the h1F6 conjugates.
Referring to FIG. 4, the results of a treatment study using an h1F6-mc-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. A control conjugate, cAC10-mc-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice administered doses of the h1F6 conjugate at 1 mg/kg appeared to exhibit complete tumor reduction. Mice administered lower doses at 0.3 mg/kg and 0.1 mg/kg exhibited lesser tumor reduction, respectively. The control conjugate (non-binding) was less active the h1F6 conjugate administered at a similar dose, although it exhibited more activity than the h1F6 conjugate administered at lower doses. The h1F6 conjugate was also more active than an h1F6-vc-MMAE conjugate (Published U.S. Application No. 2009-0148942) administered at higher doses.
Referring to FIG. 5, the results of a treatment study using a two loaded antibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loaded non-binding control, H00d conjugated to the same compound (h00d-38). The model was a Caki subcutaneous model in Nude mice. Doses were 0.1, 0.3 and 1 mg/kg q7dX2. The highest two doses of the h1F6 conjugate demonstrated complete regressions as 1 mg/kg and substantial tumor delay at 0.3 mg/kg. The non-binding control demonstated tumor delay at the 1 mg/kg dose.
Referring to FIG. 6, the results of a treatment study using a two loaded antibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loaded non-binding control, H00d conjugated to the same compound (h00d-38). The model was a 786-O subcutaneous model in Nude mice. Doses were 0.1, 0.3 and 1 mg/kg q7dX2. All three doses of the h1F6 conjugate demonstrated complete regressions or tumor delay, while the non-binding control demonstated tumor delay.
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14995944
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Sep 9th, 2008 12:00AM
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Jun 12th, 2006 12:00AM
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https://www.uspto.gov?id=US07423116-20080909
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Pentapeptide compounds and uses related thereto
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Pentapeptide compounds are disclosed. The compounds have biological activity, e.g., cytotoxicity. Prodrugs having targeting groups and pentapeptide moieties, as well as precursors thereof are also disclosed. For example, precursors having a reactive linker that can serve as a reaction site for joining to a targeting agent, e.g., an antibody, as disclosed.
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7423116
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1. A compound of the formula
2. A compound of the formula
or a pharmaceutically acceptable salt or solvate thereof,
wherein, independently at each location:
R1 is hydrogen;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl;
R11 is selected from hydrogen and lower alkyl; and
R18 is selected from a hydroxyl protecting group and a direct bond such that OR18 represents=O.
3. A compound of claim 2 wherein R3 is isopropyl.
4. A compound of claim 2 wherein R4 is selected from lower alkyl, aryl, and —CH2—C5-7 carbocycle and R5 is selected from H and methyl.
5. A compound of claim 2 wherein R4 is selected from lower alkyl, and R5 is selected from H and methyl.
6. A compound of claim 2 wherein R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6.
7. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 2 wherein R6 is lower alkyl.
8. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 2 wherein R8 is hydrogen.
9. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 2 wherein R11 is hydrogen.
10. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 2 wherein —OR18 is=O.
11. A compound of the formula
or a pharmaceutically acceptable salt or solvate thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl; and
R19 is selected from hydroxyl- and oxo-substituted lower alkyl.
12. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R1 is hydrogen.
13. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R1 and R2 are methyl.
14. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R3 is isopropyl.
15. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle and R5 is selected from H and methyl.
16. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R4 is selected from lower alkyl, and R5 is selected from H and methyl.
17. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6.
18. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R6 is lower alkyl.
19. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R8 is hydrogen.
20. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 11 wherein R19 is oxo-substituted lower alkyl.
21. A compound of claim 11 having the formula
or a pharmaceutically acceptable salt or solvate thereof.
22. A composition comprising a compound or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1, 10 or 20 and a pharmaceutically acceptable carrier, diluent or excipient.
23. A compound of the formula
or a pharmaceutically acceptable salt or solvate thereof,
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl; and
R11 is selected from hydrogen and lower alkyl.
24. A compound of claim 23, wherein R1 is hydrogen.
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24
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This application is a division of application Ser. No. 10/476,391, filed Mar. 29, 2004, now U.S. Pat. No. 7,256,257, which is a national stage entry of PCT/US02/13435, filed Apr. 30, 2002, which is a continuation-in-part of application Ser. No. 10/001,191, filed Nov. 1, 2001, now U.S. Pat. No. 6,884,869, which is a continuation-in-part of application Ser No. 09/845,786, filed Apr. 30, 2001, now abandoned, the disclosures of each of which are incorporated by reference herein in their entireties.
FIELD OF THE INVENTION
The present invention is directed to biologically active organic compounds and precursors thereto, more specifically to pentapeptides and compositions containing these pentapeptides, and to methods for their use, e.g., as cytotoxic agents.
BACKGROUND OF THE INVENTION
Several short peptidic compounds have been isolated from natural sources and found to have biological activity. Analogs of these compounds have also been prepared, and some were found to have biological activity. For example, Auristatin E (Pettit, G. R., Barkoczy, J. “Tumor inhibiting tetrapeptide bearing modified phenethyl amides” U.S. Pat. No. 5,635,483) is a synthetic analogue of the marine natural product, dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (P ettit, G. R. “The dolastatins” Prog. Chem. Org. Nat. Prod. 1997, 70, 1-79). Dolastatin 10, auristatin PE, and auristatin E are linear peptides comprised of four amino acids, three of which are unique to this class of compounds. Both dolastatin 10 and auristatin PE are in human clinical trials. The structural differences between the drugs reside in the C-terminal residue, in which the thiazolephenethyl amine of dolastatin 10 is replaced by a norephedrine unit in auristatin E.
The following references disclose dolastatin and auristatin compounds and analogs thereof.
“Preparation of peptide derivatives as dolastatin 10 analogs with antitumor effects” Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi; Ito, Takeshi; Sugimura, Akihiro; Kobayashi, Motohiro. (Teikoku Hormone Mfg. Co., Ltd., Japan; Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi; Ito, Takeshi; Sugimura, Akihiro; Kobayashi, Motohiro). PCT International Patent Publication No. WO 9633212 A1 (1996);
“Preparation of dolastatin 10 analogs as cancer inhibitory peptides” Pettit, George R.; Srirangam, Jayaram K. (Arizona Board of Regents, USA). PCT International Patent Publication No. WO 9614856 A1 (1996);
“Preparation of tetra- and pentapeptide dolastatin analogs as anticancer agents” Pettit, George R.; Srirangam, Jayaram K.; Williams, Michael D. (Arizona Board of Regents, USA). Eur. Pat. Appl. No. EP 695757 A2 (1996);
“Preparation of dolastatin 10 analog pentapeptide amides and esters as anticancer agents” Pettit, George R.; Srirangam, Jayaram K. (Arizona Board of Regents, USA). Eur. Pat. Appl. No. EP 695758 A2 (1996);
“Preparation of dolastatin analog pentapeptide methyl esters as anticancer agents” Pettit, George R.; Williams, Michael D.; Srirangam, Jayaram K. (Arizona Board of Regents, USA). Eur. Pat. Appl. No. EP 695759 A2 (1996);
“Preparation of novel peptide derivative as antitumor agent” Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi; Ito, Takeshi; Sugimura, Akihiro; Kobayashi, Motohiro. (Teikoku Hormone Mfg. Co., Ltd., Japan). PCT International Patent Publication No. WO 9509864 A1 (1995);
“Preparation of tetrapeptide derivatives as antitumor agents” Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi. (Teikoku Hormone Mfg. Co., Ltd., Japan). PCT International Patent Publication No. WO 9303054 A1 (1993);
“Antineoplastic agents 365. Dolastatin 10 SAR probes” Pettit, George R.; Srirangam, Jayaram K.; Barkoczy, Jozsef; Williams, Michael D.; Boyd, Michael R.; Hamel, Ernest; Pettit, Robin K.; Hogan, Fiona; Bai, Ruoli; Chapuis, Jean-Charles; McAllister, Shane C.; Schmidt, Jean M., Anti-Cancer Drug Des. (1998), 13(4), 243-277;
“Antineoplastic agents. 337. Synthesis of dolastatin 10 structural modifications” Pettit, George R.; Srirangam, Jayaram K.; Barkoczy, Jozsef; Williams, Michael D.; Durkin, Kieran P. M.; Boyd, Michael R.; Bai, Ruoli; Hamel, Emest; Schmidt, Jean M.; Chapius, Jean-Charles, Anti-Cancer Drug Des. (1995), 10(7), 529-44; and
“Synthesis and antitumor activity of novel dolastatin 10 analogs” Miyazaki, Koichi; Kobayashi, Motohiro; Natsume, Tsugitaka; Gondo, Masaaki; Mikami, Takashi; Sakakibara, Kyoichi; Tsukagoshi, Shigeru, Chem. Pharm. Bull. (1995), 43(10), 1706-18.
There is a need in the art for improved antitumor agents. Despite promising in vitro data for dolastatin 10 and its analogs, significant general toxicities at doses required for achieving a therapeutic affect compromise their efficacy in clinical studies. The present invention is directed to fulfilling this need and provides further advantages as disclosed herein.
The recitation of any reference in Section 2 of this application is not an admission that the reference is prior art to this application.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl; and
R9 is selected from
R10 is selected from
R11 is selected from hydrogen and lower alkyl;
R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence;
R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene;
R15 is selected from hydrogen, lower alkyl and aryl;
R16 is selected from arylene (lower alkylene), lower alkylene, arylene, and —(CH2OCH2)pCH2— where p is 1-5; and
R17 is selected from
where Y═O or S.
In another aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl;
R11 is selected from hydrogen and lower alkyl;
R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; and
R20 is a reactive linker group having a reactive site that allows R20 to be reacted with a targeting moiety, where R20 can be bonded to the carbon labeled “x” by either a single or double bond.
In another aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2-C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl;
R11 is selected from hydrogen and lower alkyl;
R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; and
R20 is a reactive linker group having a reactive site that allows R20 to be reacted with a targeting moiety, where R20 can be bonded to the carbon labeled “x” by either a single or double bond.
In another aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl; and
R20 is a reactive linker group comprising a reactive site that allows R20 to be reacted with a targeting moiety.
In another aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof.
In another aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl;
R11 is selected from hydrogen and lower alkyl; and
R18 is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR18 represents ═O.
In another aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl; and
R19 is selected from hydroxy- and oxo-substituted lower alkyl.
In another aspect, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from hydrogen and lower alkyl;
R2 is selected from hydrogen and lower alkyl;
R3 is lower alkyl;
R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from hydrogen and lower alkyl;
R7 is sec-butyl or iso-butyl;
R8 is selected from hydrogen and lower alkyl; and
R9 is selected from
R10 is selected from
R11 is selected from hydrogen and lower alkyl;
R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence;
R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene; and
R15 is selected from hydrogen, lower alkyl and aryl.
Optionally, R9 is
and R10 is
Optionally, R9 is
and R10 is
Optionally, R9 is
and R10 is
Optionally, R9 is
and R10 is
In another aspect, the present invention provides a composition comprising a biologically active compound as described above or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect, the present invention provides a method for killing a cell, the method comprising administering to the cell a lethal amount of a compound, a pharmaceutically acceptable salt and solvate thereof, or composition as described above.
In another aspect, the present invention provides a method of killing a cell comprising
a. delivering a compound, a pharmaceutically acceptable salt or solvate thereof, or composition as described above to a cell, where the compound or pharmaceutically acceptable salt or solvate thereof enters the cell;
b. cleaving mAb from the remainder of the compound or pharmaceutically acceptable salt or solvate thereof; and
c. killing the cell with the remainder of the compound or pharmaceutically acceptable salt or solvate thereof.
In another aspect, the present invention provides a method of killing or inhibiting the multiplication of tumor cells or cancer cells in a human or other animal, the method comprising administering to the human or animal a therapeutically effective amount of a compound, pharmaceutically acceptable salt or solvate thereof, or composition described above.
In still another aspect, the present invention provides methods for killing or inhibiting the multiplication of a cell comprising:
a. delivering a compound of the invention, a pharmaceutically acceptable salt or solvate thereof, or composition as described above to a cell, where the compound enters the cell;
b. cleaving the antibody from the remainder of the compound or pharmaceutically acceptable salt or solvate thereof; and
c. killing the cell with the remainder of the compound or pharmaceutically acceptable salt or solvate thereof.
In yet another aspect, the present invention provides a method of killing or inhibiting the multiplication of tumor cells or cancer cells in a human or other animal, the method comprising administering to a human or other animal in need thereof an effective amount of a compound, pharmaceutically acceptable salt of the compound or composition described above.
In another aspect, the invention provides methods for treating cancer, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition, described above.
In still another aspect, the invention provides a method of killing or inhibiting the multiplication of cells that produce auto-immune antibodies, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition, described above.
In yet another aspect, the invention provides methods for treating an autoimmune disease, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition, described above.
In still another aspect, the invention provides methods for treating an infectious disease, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition described above.
These and related aspects of the present invention are described in more detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a synthetic scheme illustrating the preparation of specific tripeptides useful in preparing compounds of the present invention.
FIG. 2 shows a synthetic scheme illustrating the preparation of specific dipeptides useful in preparing compound of the present invention.
FIGS. 3, 4 and 5 show synthetic schemes illustrating the preparation of pentapeptides from tripeptides and dipeptides
FIGS. 6, 7 and 8 show synthetic schemes for conjugating a pentapeptide to a heterobifunctional linker to provide ligand (specifically a mAb) reactive drug molecules.
FIGS. 9, 10, 11 and 12 show synthetic schemes for conjugating a ligand, and specifically a mAb, to a drug-reactive linker conjugate to provide prodrugs of the present invention.
FIG. 13 shows the synthetic scheme used for the preparation of compound 49.
FIG. 14 shows a synthetic scheme for conjugating a pentapeptide to a heterobifunctional linker to provide drug-reactive linker conjugate 51.
FIG. 15 shows the synthetic scheme used for the preparation of compound 53.
FIG. 16 shows a synthetic scheme illustrating the preparation of a specific dipeptide (57) useful in preparing compounds of the present invention.
FIG. 17 shows a synthetic scheme for conjugating a pentapeptide (58) to a heterobifunctional linker to provide drug-reactive linker conjugate 59.
FIGS. 18A, 18B and 18C show the in vitro cytotoxicity of drugs and mAb-drug conjugates on (A) and (B) L2987 human lung adenoma cells, and (A) and (C) Kato III human gastric carcinoma cells.
FIGS. 19A and 19B show the cytotoxic effects of auristatin E and auristatin E-containing conjugates on L2987 human lung adenocarcinoma cells (FIG. 19A) and various hematologic cell lines treated with AC10-S-40a (FIG. 19B).
FIGS. 20A and 20B show median tumor volume observed when nude mice with subcutaneous L2987 human lung adenocarcinoma or 3396 human breast carcinoma xenografts were injected with conjugates or drug according to the schedule shown in the two Figures.
FIG. 21 shows the cytotoxic effects of drug conjugates of the invention on H3396 human breast carcinoma cells.
DETAILED DESCRIPTION OF THE INVENTION
Among other aspects, the present invention provides short peptide-containing compounds having biological activity, e.g., cytotoxicity, and methods for their use, e.g., in cancer therapy. Before describing the compounds of the present invention, reference is made to the following definitions that are used herein.
Definitions
As used herein the following terms have the indicated meanings:
“Animal subjects” include humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, and cats. Birds, including fowl, are also a suitable animal subject.
“Aryl” refers to aromatic groups which have at least one ring having a conjugated pi electron system and includes carbocyclic aryl and heterocyclic aryl. In specific aspects of the invention, the aryl group (and the arylene group as discussed below) has 5-14, or 6-12, or 6-10 atoms as part of one or more aromatic rings. In one aspect the aryl or arylene is a carbocyclic. “Carbocyclic aryl” refers to aromatic groups wherein the ring atoms of the aromatic ring are all carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic groups, all of which may be optionally substituted. Exemplary carbocyclic aryl groups include phenyl, naphthyl and anthracenyl. Exemplary substituted carbocyclic aryl groups include indene and phenyl substituted by one or two substituents such as, or selected from, lower alkyl, hydroxy, lower alkoxy, lower alkoxycarbonyl, halogen, trifluoromethyl, nitro, and cyano. “Heterocyclic aryl”, which may also be called “heteroaryl” refers to aryl groups having from 1 to 9 carbon atoms and the remainder of the atoms are heteroatoms, and includes those heterocyclic systems described in “Handbook of Chemistry and Physics,” 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber Co., Cleveland, Ohio. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems. Suitable heteroatoms include oxygen, nitrogen, and S(O)i, wherein i is 0, 1 or 2. Suitable examples of heteroaryl groups include, without limitation, benzofuran, benzothiophene, indole, benzopyrazole, coumarin, isoquinoline, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, triazole, quinoline, pyrimidine, pyridine, pyridone, pyrazine, pyridazine, isothiazole, isoxazole and tetrazole. “Aryl groups” are bonded to one other group, while “arylene” refers to an aryl group that is bonded to two other groups. For example, phenyl
is an aryl group, while para-phenylene
is one example of an arylene group. Other arylene groups are ortho- or meta-phenylene and naphthalene.
“Arylene (lower alkylene)” refers to a group formed from an arylene group and an alkylene group, that together link two other groups. For example, in X-arylene (lower alkylene)-Y the arylenealkylene group links X and Y, and is formed from a lower alkylene, e.g., methylene (—CH2—) and an arylene
to provide the aryl (lower alkylene), e.g.
“Carbocycle” or “carbocyclic ring” refers to any saturated, unsaturated or aromatic monocyclic ring where the ring atoms are carbon. A C5-7carbocycle refers to any carbocyclic ring having 5, 6 or 7 carbon atoms that form the ring. Examples of such carbocycles include, but are not limited to, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, and phenyl.
“Compound”, as in the terms “compound of the formula”, “compound of the structure”, “compound of the invention”, and the like, shall refer to and encompass the chemical compound itself as well as, whether explicitly stated or not, and unless the context makes clear that the following are to be excluded: amorphous and crystalline forms of the compound, including polymorphic forms, where these forms may be part of a mixture or in isolation; free acid and free base forms of the compound, which are typically the forms shown in the structures provided herein; isomers of the compound, which refers to optical isomers, and tautomeric isomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and the optical isomers include isolated optical isomers as well as mixtures of optical isomers including racemic and non-racemic mixtures; where an isomer may be in isolated form or in admixture with one or more other isomers; isotopes of the compound, including deuterium- and tritium-containing compounds, and including compounds containing radioisotopes, including therapeutically- and diagnostically-effective radioisotopes; multimeric forms of the compound, including dimeric, trimeric, etc. forms; salts of the compound, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts having organic counterions and inorganic counterions, and including zwitterionic forms, where if a compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates of the compound, including hemisolvates, monosolvates, disolvates, etc., including organic solvates and inorganic solvates, said inorganic solvates including hydrates; where if a compound is associated with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some instances, reference made herein to a compound of the invention will include an explicit reference to one or of the above forms, e.g., salts and solvates, however, this reference is for emphasis only, and is not to be construed as excluding other of the above forms as identified above.
“Direct bond” refers to a pair of electrons that hold together two adjacent atoms. Thus, when two atoms are separated by a direct bond, those two atoms are bonded together by a covalent bond. If a direct bond is attached to only one atom (X) and X is bonded to an adjacent atom (Y), then the direct bond represents a second bond between X and Y, i.e., the direct bond option provides a double bond between X and Y, at the expense of a hydrogen that would otherwise be bonded to Y. For example C—O—R where R is a direct bond denotes C═O.
“Hydroxyl protecting group” includes, but is not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
“Independently at each location” and “independently at each occurrence” are synonymous terms which mean that any selection is independent of any other selection. For instance, if a variable is selected from two options, and the variable occurs twice, then the option selected at one occurrence of the variable does not impact in any way the choice of the option selected at the second occurrence of the variable.
“Leaving group” refers to a functional group that can be readily substituted by another functional group. A preferred leaving group is replaced by SN2 displacement by a nucleophile. Such leaving groups are well known in the art, and include halides (e.g., chloride, bromide, iodide), methanesulfonate (mesylate), p-toluenesulfonate (tosylate), trifluoromethane sulfonate and trifluoromethylsulfonate.
“Lower alkyl” refers to a straight or a branched chain, cyclic or acyclic, monovalent saturated hydrocarbon, halocarbon or hydrohalocarbon radical of one to ten carbon atoms. In various aspects, the lower alkyl group has 1-8,1-6, 1-5, or 1-4 carbons. Thus, the alkyl group may optionally have halogen substitution, where a halocarbon contains only carbon and halogen, and where a hydrohalocarbon contains carbon, halogen a n d hydrogen. Exemplary lower alkyl groups are, without limitation, methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. “Lower alkylene” refers to a lower alkyl group that is bonded to two groups, such as (—CH2—)1-8, (—CH2—)1-6, (—CH2—)1-5 or (—CH2—)1-4. For example, methyl (—CH3) is a lower alkyl group, while methylene (—CH2—) is a lower alkylene group. A hydroxy- or oxo-substituted lower alkyl refers to a lower alkyl group wherein a hydrogen on the lower alkyl group is replaced by —OH (for a hydroxy-substituted lower alkyl), or two hydrogens on a single carbon of the lower alkyl group are replaced by ═O (for an oxo-substituted lower alkyl).
The term “antibody,” as used herein, refers to an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce auto-immune antibodies associated with an autoimmune disease. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Antibodies used in the invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, sFvs, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens and microbial antigens.
The shorthand designation “mAb” is used in chemical structures herein to encompass any antibody, i.e., any immunoglobin molecule or any immunologically active portion of an immunoglobin molecule.
As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) which is capable of eliciting an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein saccharaide, polysaccharide, or lipid molecule (e.g., a bacterial, fingi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) which is capable of eliciting an immune response.
“Pharmaceutically acceptable salt” and “salts thereof” refers to organic or inorganic salts of the pharmaceutically important molecule. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically important organic molecule may have more than one charged atom in its structure. Situations where multiple charged atoms are part of the molecule may have multiple counterions. Hence, the molecule of a pharmaceutically acceptable salt may contain one or more than one charged atoms and may also contain, one or more than one counterion. The desired charge distribution is determined according to methods of drug administration. Examples of pharmaceutically acceptable salts are well known in the art but, without limiting the scope of the present invention, exemplary presentations can be found in the Physician's Desk Reference, The Merck Index, The Pharmacopoeia and Goodman & Gilman's The Pharmacological Basis of Therapeutics. The salt will typically be either an acid addition salt, i.e., a salt formed by adding an acid to a pentapeptide compound, or a base addition salt, i.e., a salt formed by adding a base to a pentapeptide compound. The pentapeptide compounds of the invention contain at least one amine group, and accordingly acid addition salts may be formed with this amine group, where acid addition salts refer to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, isonicotinic, lactic, salicylic, oleic, tannic, pantothenic, ascorbic, succinic, gentisinic, fumaric, gluconic, glucuronic, formic, glutamic, benzenesulfonic, pamoic acid, and the like.
Compounds included in the present compositions that include an amino moiety may form pharmaceutically or cosmetically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds, included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium lithium, zinc, potassium, and iron salts.
“Pharmaceutically acceptable solvate” refers to a solvate, i.e., an association of a solvent and a compound of the invention, that retains the biological effectiveness and properties of the biologically active pentapeptide compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. It will be appreciated by those skilled in the art that solvated forms are biologically equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Sykes, R A., Guidebook to Mechanism in Organic Chemistry, 6th Ed (1986, John Wiley & Sons, N.Y.) is an exemplary reference that describe solvates.
In the context of cancer, the term “treating” includes any or all of: inhibiting tumor growth, inhibiting replication of tumor cells, lessening of overall tumor burden and ameliorating of one or more symptoms associated with the disease.
In the context of an autoimmune disease, the term “treating” includes any or all of: inhibiting replication of cells capable of producing an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.
In the context of an infectious disease, the term “treating” includes any or all of: inhibiting replication of an infectious agent, lessening the infectious agent burden and ameliorating one or more symptoms of an infectious disease.
The following abbreviations may be used herein and have the indicated definitions: t-Boc is tert-butoxy carbonyl, DCC is dicyclohexylcarbodiimide, DIAD is diisopropylazodicarboxylate; Fmoc is 9-fluorenylmethoxycarbonyl, DEPC is diethyl phosphorocyanidate, DMAP is 4-(N,N-dimethylamino) pyridine, PyBrop is bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, TEAA is triethylammonium acetate, TFA is trifluoroacetic acid, and Z is benzyloxycarbonyl.
In the phrase “R20 is a reactive linker having a reactive site that allows R20 to be reacted with a targeting moiety”, a “reactive site” refers to a functional moiety that can undergo reaction with a ligand so as to provide a covalent bond between the ligand and the reactive linker. In one aspect of the invention, the reactive site is reactive with a functional group selected from thiol, amino and hydroxyl, preferably, thiol and amino. Both thiol and amino groups are present in proteins, where thiol groups are, e.g., produced by reduction of disulfide bonds in proteins, and amino groups are present in, e.g., a lysine moiety of a protein.
In a preferred aspect of the invention, the reactive linker has a reactive site that is reactive with a thiol group. For instance, the reactive site may be a maleimide of the formula
where the carbon-carbon double bond is reactive with nucleophiles, e.g., thiol groups. Alternatively, the reactive site may have the formulae
where X is a leaving group that may be displaced by a nucleophile, e.g., a thiol group as found on a protein.
In another preferred aspect of the invention, the reactive linker contains a reactive site that is reactive with an amine or amino group. Suitable amine reactive sites include, without limitation, activated esters such as N-hydroxysuccinimide esters, p-nitrophenyl esters, pentafluorophenyl esters, and other reactive functionalities, such as isothiocyanates, isocyanates, anhydrides, acid chlorides, and sulfonyl chlorides. Each of these functionalities provides a reactive site for a reactive linker of the present invention.
Many compounds of the invention have a norephedrine moiety of the following general formula:
In one embodiment, the compounds' norephedrine moiety has (1S, 2R) stereochemistry as depicted below:
In alternate embodiments, the compounds' norephedrine moiety has (1S, 2S), (1R, 2S) or (1R, 2R) stereochemistry.
Compounds
In one aspect, the present invention provides compounds of the general structure “drug-linker-targeting agent”, where the drug is a pentapeptide as disclosed herein and the targeting agent is an antibody. As is understood by those skilled in the art, such compounds are generally known as antibody conjugates or antibody therapeutic-agent conjugates. Such compounds have the following structure, and may also be referred to herein as prodrugs.
(and pharmaceutically acceptable salts and solvates thereof)
In this structure, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R5 is selected from hydrogen and lower alkyl; and R9 is selected from
wherein: R10 is selected from
R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; and R13 is
wherein: R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene; R15 is selected from hydrogen, lower alkyl and aryl; R16 is selected from arylene (lower alkylene), lower alkylene, arylene, and —(CH2OCH2)pCH2— where p is 1-5; and R17 is selected from
where Y═O or S.
In optional embodiments of the invention: R1 is hydrogen; R1 and R2 are methyl; R3 is isopropyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle and R5 is selected from H and methyl; R4 is selected from lower alkyl, and R5 is selected from H and methyl; R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is lower alkyl; R8 is hydrogen.
In another optional embodiment, R9 is
and R10 is selected from
In one embodiment R10 is
while in another embodiment R10 is
In a preferred embodiment, R14 is selected from arylene and lower alkylene; R15 is selected from lower alkyl and aryl; and R16 is lower alkylene.
In another optional embodiment, R9 is
and R10 is selected from
In a preferred embodiment, R14 is selected from arylene and lower alkylene; R15 is selected from lower alkyl and aryl; and R16 is lower alkylene.
In another optional embodiment, R9 is
and R13 is
In a preferred embodiment, R15 is lower alkyl; and R16 is lower alkylene.
In another optional embodiment, R9 is
In a preferred embodiment, R16 is lower alkylene.
In another optional embodiment, R9 is
In a preferred embodiment, R16 is selected from lower alkylene and arylene.
In one embodiment R17 is
In another embodiment, R17 is
In another embodiment, R17 is
where Y═O or S.
For example, the present invention provides a prodrug of the formula
and pharmaceutically acceptable salts and solvates thereof.
In addition, the present invention provides a prodrug of the formula:
In addition, the present invention provides a prodrug of the formula:
and pharmaceutically acceptable salts and solvates thereof.
In addition to prodrugs as described above, the present invention also provides intermediate compounds that can be used to prepare prodrugs. These intermediate compounds contain a reactive linker group R20 having a reactive site that allows R20 to be reacted with a targeting moiety, e.g., an mAb.
In one aspect, the invention provides intermediate compounds of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R1 is selected from hydrogen and lower alkyl; R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; and R20 is a reactive linker group having a reactive site that allows R20 to be reacted with a targeting moiety, where R20 can be bonded to the carbon labeled “x” by either a single or double bond.
In another aspect, the present invention provides intermediate compounds of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; and R20 is a reactive linker group having a reactive site that allows R20 to be reacted with a targeting moiety, where R20 can be bonded to the carbon labeled “x” by either a single or double bond.
In another aspect, the present invention provides intermediate compounds of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; and R20 is a reactive linker group comprising a reactive site that allows R20 to be reacted with a targeting moiety.
In any of the foregoing intermediate compounds, in further aspects R20 comprises a hydrazone of the formula
wherein: R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene; R15 is selected from hydrogen, lower alkyl and aryl; and R16 is selected from arylene (lower alkylene), lower alkylene, arylene, and —(CH2OCH2)pCH2— where p is 1-5.
Additionally, in any of the foregoing intermediate compounds, in further aspects R20 comprises a hydrazone of the formula
wherein: R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene; R15 is selected from hydrogen, lower alkyl, and aryl; and R16 is selected from arylene (lower alkylene), lower alkylene, arylene, and —(CH2OCH2)pCH2— where p is 1-5.
Additionally, in any of the foregoing intermediate compounds, in further aspects R20 comprises a hydrazone of the formula:
wherein: R16 is selected from lower alkylene, arylene, and —(CH2OCH2)pCH2— where p is 1-5, and x identifies the carbon also marked x in the intermediate structures shown above.
As mentioned previously, a reactive linker group R20 includes a reactive site that can be reacted with a targeting moiety so as to indirectly join the targeting moiety to the drug. The following are exemplary reactive sites that may be present as part of the R20 group. For example, any of the following reactive sites may be joined to any of the hydrazone structures set forth above in order to form a complete R20 structure. In one aspect, R20 comprises a reactive site having the formula
In another aspect, R20 comprises a reactive site having the formula
wherein X is a leaving group. In another aspect, R20 comprises a reactive site having the formula
wherein X is a leaving group.
For example, the present invention provides intermediate compounds of the formulae:
and pharmaceutically acceptable salts and solvates thereof
wherein R4 is selected from iso-propyl and sec-butyl, and R5 is hydrogen;
and pharmaceutically acceptable salts and solvates thereof.
In addition to prodrugs as described above, and direct precursors thereto as described above and referred to as intermediate compounds, the present invention also provides precursors to the intermediate compounds. In one aspect of the invention, these precursors to the intermediate compounds have a carbonyl group. As discussed in more detail below, a carbonyl group is conveniently reacted with hydrazine derivatives in order to prepare intermediate compounds having R20 groups and reactive sites as part of the R20 groups. Thus, in a further aspect, the present invention provides the following precursors to the intermediate compounds, where these precursors have either carbonyl groups or hydroxyl groups that could be readily oxidized to a carbonyl group.
In one aspect, the present invention provides compounds of the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; and R19 is selected from hydroxy- and oxo-substituted lower alkyl. In various optional embodiments: R1 is hydrogen; or R1 and R2 are methyl; and/or R3 is iso-propyl; and/or R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle and R5 is selected from H and methyl; or R4 is selected from lower alkyl, and R5 is selected from H and methyl; or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; and/or R6 is lower alkyl; and/or R8 is hydrogen; and/or R19 is oxo-substituted lower alkyl. An exemplary compound of this aspect of the invention is
and pharmaceutically acceptable salts and solvates thereof.
In another aspect the present invention provides compounds of the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R9 is selected from
R10 is selected from
R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene; and R15 is selected from hydrogen, lower alkyl and aryl. In various embodiments of this aspect of the invention: R10 is
or R10 is
Some specific compounds of this aspect of the invention have the structures
and pharmaceutically acceptable salts and solvates thereof
wherein R4 is iso-propyl or sec-butyl and R5 is hydrogen.
In a further aspect the present invention provides pentapeptide drugs of the following structures. In these structures the drug may contain a carbonyl group that could optionally be reacted directly with a hydrazine derivative or other reactive group so as to introduce an R20 group into the molecule. Thus, in one embodiment, the present invention provides a pentapeptide drug of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location: R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein R1 and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R11 is selected from hydrogen and lower alkyl; and R18 is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR18 represents ═O. For instance, the present invention provides compound of the formula
and pharmaceutically acceptable salts and solvates thereof.
In another embodiment, the present invention provides a pentapeptide drug of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower allyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl, R11 is selected from hydrogen and lower alkyl; and R18 is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR18 represents ═O. For instance, the present invention provides compounds of the formulae
and pharmaceutically acceptable salts and solvates thereof
wherein R4 iso-propyl and R5 is hydrogen.
In another embodiment, the present invention provides a pentapeptide drug of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; and R19 is selected from hydroxy- and oxo-substituted lower alkyl. For instance, the present invention provides a compound of the formula
and pharmaceutically acceptable salts and solvates thereof.
In various further embodiments, the pentapeptide drugs of the invention have one of the three general pentapeptide structure as shown above, wherein: R1 is hydrogen; or R1 and R2 are methyl; and/or R3 is isopropyl; and/or R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle and R5 is selected from H and methyl; or R4 is selected from lower alkyl, and R5 is selected from H and methyl; or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; and/or R6 is lower alkyl; and/or R8 is hydrogen; and/or R11 is hydrogen; and/or —R18 is ═O; and/or R19 is oxo-substituted lower alkyl.
General Synthesis
In various aspects as detailed above, the present invention provides drug-linker-ligand conjugates as well as precursors thereto. A drug-linker-ligand conjugate of the present invention is designed so that upon delivery of the conjugate to a cell, and particularly a tumor cell, the conjugate will partially degrade in a manner that frees the drug, from the ligand with perhaps some residue from the linker remaining attached to the drug, and typically with some residue of the linker remaining attached to the ligand. The drug (or drug-linker residue) will then act in a cytotoxic manner to kill the tumor cells, or at least to mitigate the multiplication of the tumor cells. The drug-linker-ligand conjugate may be referred to herein as a prodrug. The ligand is typically a targeting moiety that will localize the prodrug in the vicinity of a tumor cell.
The prodrug has the basic structure shown below:
and pharmaceutically acceptable salts and solvates thereof
where the —linker-ligand portion of the prodrug is joined to the drug at any of positions a, b, or c. As described in more detail below, such conjugates are conveniently prepared using a heterobifunctional linker. In a preferred aspect of the invention, the heterobifunctional linker includes, on one end, a thiol acceptor i.e., a chemical moiety that will react with a thiol group so that the sulfur atom of the thiol group becomes covalently bonded to the thiol acceptor. Thiol acceptors are well known in the art, where exemplary thio acceptor group are maleimide and haloacetamide groups. The thiol acceptor group provides a convenient site for ligand attachment. At the other end, in a preferred aspect the heterobifunctional linker has a hydrazide group. Hydrazide groups are useful endgroups for a heterobifunctional linker because they readily react with carbonyl groups, i.e., aldehyde and ketone groups, where carbonyl groups can be readily incorporated into a pentapeptide compound as disclosed herein, to provide a hydrazone linkage. Incorporation of a hydrazone into the conjugate is particularly preferred in order to impart desired pH-sensitivity to the conjugate. Thus, under low pH conditions, as in acidic intracellular compartments (e.g., lysosomes), a hydrazone group will cleave and release the drug free from the ligand. A preferred linker is an acid-labile linker, that is cleaved under low pH conditions, i.e., conditions where the pH is less than about 6, preferably less than about 5.
The drugs of the present invention may be generally described as pentapeptides. Typically, pentapeptide drugs of the present invention can be prepared by introduction of a peptide bond between selected amino acids and/or modified amino acids, and/or peptide fragments. Such peptide bonds may be prepared, for example, according to the liquid phase synthesis method (see E. Schröder and K. Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
A preferred method to prepare pentapeptide drugs of the present invention entails preparing a dipeptide and a tripeptide, and combining stoichiometric equivalents of these two fragments in a one-pot reaction under suitable condensation conditions. This approach is illustrated in the following Schemes 1-3. Thus, the tripeptide 6 may be prepared as shown in Scheme 1, and the dipeptide 9 may be prepared as shown in Scheme 2. The two fragments 6 and 9 may be condensed to provide a pentapeptide (10) as shown in Scheme 3.
As illustrated in Scheme 1, a PG-protected amino acid 1 (where PG represents an amine protecting group, e.g., benzyloxycarbonyl (which is a preferred PG and is represented herein by Z), R4 is selected from lower alkyl, aryl, and CH2-C5-7carbocyclic when R5 is selected from H and methyl, or R4 and R5 together form a carbocyclic group of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6) is coupled to t-butyl ester 2 (where R6 is selected from hydrogen and lower alkyl; and R7 is sec-butyl or iso-butyl) under suitable coupling conditions, e.g., in the presence of PyBrop and diisopropylethylamine, or using DCC (as accomplished, for example, by Miyazaki, K. et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).
Suitable protecting groups PG, and suitable synthetic methods to protect an amino group with a protecting group are well known in the art. See, e.g., Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley & Sons. Preferred protected amino acids 1 are PG-Ile and, particularly, PG-Val, while other suitable protected amino acids include, without limitation: PG-cyclohexylglycine, PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid, PG-α-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and PG-tert-butylglycine. Z, i.e., benzyloxycarbonyl, is a preferred protecting group. Fmoc is another preferred protecting group. A preferred t-butyl ester 2 is dolaisoleuine t-butyl ester.
After chromatographic purification, the dipeptide 3 is deprotected, e.g. using H2 and 10% Pd—C in ethanol when PG is benzyloxycarbonyl, or using diethylamine for removal of an Fmoc protecting group. The resulting amine 4 readily forms a peptide bond with an amino acid 5 (where R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; and R3 is lower alkyl). N,N-Dialkyl amino acids are preferred amino acids 5, such as commercially available N,N-dimethyl valine. Other N,N-dialkyl amino acids can be prepared by reductive bis-alkylation following known procedures (see, e.g., Bowman, R. E, Stroud, H. H J. Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two preferred amino acids 5 for the synthesis of N-monoalkyl derivatives. The amine 4 and the amino acid 5 are conveniently joined to provide the tripeptide 6 using coupling reagent DEPC with triethylamine as the base.
The dipeptide 9 can be readily prepared by condensation of the modified amino acid t-Boc-Dolaproine 7 (see, for example, Pettit, G. R., et al. Synthesis, 996, 719-725), with commercially available (1S,2R)-norephedrine, L- or D-phenylalaninol, or with synthetic p-acetylphenethylamine 8 (Shavel, J., Bobowski, G., 1969, U.S. Pat. No. 3,445,518) using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in Scheme 2.
Scheme 3 illustrates the joining together of the tripeptide 6 with the dipeptide 9 to form a pentapeptide 10. The joining together of these two fragments can be accomplished using, e.g., TFA to facilitate Boc and t-butyl ester cleavage, respectively, followed by condensation conditions, e.g., utilizing DEPC, or similar coupling reagent, in the presence of excess base (triethylamine or equivalent) to give the desired product in moderate but acceptable yields.
To obtain N-monoalkyl pentapeptide 10, the Fmoc group should be removed from the N-terminal amino acid after final coupling by treatment with diethylamine (see General Procedure E described below).
Upon proper selection of a, b, and c, the present invention provides pentapeptides of the formula
and pharmaceutically acceptable salts and solvates thereof
which are drugs of the present invention upon appropriate selection of a, b, and c, where these drugs may be prepared as described above. Thus, as referred to herein, the drugs of the present invention are not necessarily the same as the degradation product of the prodrugs of the present invention. Rather, the drugs of the present invention are pharmacologically active, cytotoxic chemicals of the above structure, which are conveniently utilized to prepare prodrugs of the present invention, where these prodrugs will release a pharmacalogically active agent upon partial degradation of the prodrug.
Thus, in one aspect, the present invention provides drugs having the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R11 is selected from hydrogen and lower alkyl; and R18 is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR18 represents ═O.
In another aspect, the present invention provides drugs having the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R11 is selected from hydrogen and lower alkyl; and R18 is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR18 represents ═O.
In another aspect, the present invention provides drugs having the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; and R19 is selected from hydroxy- and oxo-substituted lower alkyl.
In order to prepare a prodrug of the present invention, the drug is either directly reacted with a linker, typically a heterobifunctional linker, or else the drug is somewhat modified in order that it contains a reactive site that is suitably reactive with a reactive site on a heterobifunctional linker. In general, the heterobifunctional linker has the structure
In a preferred embodiment of the invention, Reactive Site No. 1 is reactive with a carbonyl group of the drug, Reactive Site No. 2 is reactive with functionality on the ligand, and Reactive Sites 1 and 2 are reactive with different functional groups.
Many suitable heterobifunctional linkers are known in the art. See, e.g., S. S. Wong, Chemistry of Protein Conjugation and Crosslinking, CRC Press Inc., Boston 1991. In one aspect of the invention, Reactive Site No. 1 is a hydrazide, (i.e., a group of the formula
In one aspect of the invention, Reactive Site No. 2 is a thiol-accepting group. Suitable thiol-accepting groups include haloacetamide groups (i.e., groups of the formula
where X represents a halide) and maleimide groups (i.e., a group of the formula
In the heterobifunctional linker of the present invention, R16 is selected from arylene (lower alkylene), lower alkylene, arylene, and —(CH2OCH2)pCH2— where p is 1-5.
In general, suitable heterobifunctional linkers include commercially available maleimido hydrazides (e.g., β-maleimido propionic acid hydrazide, ε-maleimidocaproic acid hydrazide, and SMCC hydrazide, available from Molecular Biosciences, Inc. Boulder Colo.). Alternatively, the heterobifunctional linker can be easily prepared from a maleimido acid, including polyoxoethylene-based maleimido acids (Frisch, B., et al., Bioconjugate Chem., 1996, 7, 180-186) according to known procedures (King, H. D., et al. Bioconjugate Chem., 1999, 10, 279-288). According to this procedure, maleimido acids are first converted to their N-hydroxysuccinimide esters, followed by reaction with tert-butylcarbazate. After purification and Boc removal the maleimido hydrazide are usually obtained in good yields. Haloacetamide hydrazide heterobifunctional linkers can be prepared from available intermediates, for example succinimidyl 3-(bromoacetamido)propionate, or [2-[2-(2-bromo-acetylamino)-ethoxy]-ethoxy]-acetic acid (Frisch, B., et al., Bioconjugate Chem., 1996, 7, 180-186) by the reaction with tert-butylcarbazate described above.
In another aspect of the invention, Reactive Site No. 2 is an amine-reactive group. Suitable amine reactive groups include activated esters such as N-hydroxysuccinimide esters, p-nitrophenyl esters, pentafluorophenyl esters, and other reactive functionalities such as isothiocyanates, isocyanates, anhydrides, acid chlorides, and sulfonyl chlorides.
As indicated previously, in a preferred embodiment, the synthesis of the drug should provide for the presence of a carbonyl group, or at least a group that can be readily converted to, or elaborated to form, a carbonyl group. A carbonyl group may be readily made part of, or added to, the pentapeptide drug by any of the following exemplary procedures:
(1) oxidation of a hydroxyl group of the drug to form a carbonyl;
(2) reaction of a hydroxyl group of the drug with a molecule that contains both a carbonyl or protected carbonyl and a hydroxyl-reactive group, e.g., a carboxylic acid or ester. For instance, the hydroxyl group of the drug may be reacted with a keto acid or a keto ester, so as to convert the hydroxyl group to an ester group, where the ester group is joined to the keto group of the keto acid/ester;
(3) introducing a carbonyl group to the drug during synthesis of the drug, e.g., during peptide synthesis using carbonyl-containing amino acids, amines or peptides.
For example, if the pentapeptide 10 includes a carbonyl group, then it can be reacted directly with a heterobifunctional linker containing a carbonyl-reactive end, e.g. a hydrazide group. If the pentapeptide 10 does not include a carbonyl group, then a carbonyl group may be introduced into the pentapeptide in order to impart hydrazide reactivity to the pentapeptide. For instance, when R9 is either of
then the pentapeptide 10 can be subjected to oxidizing conditions, e.g., pyridinium chlorochromate (PCC)/pyridine (see, e.g., Synthesis, 1982, 245, 881, review), in order to provide the corresponding oxidized compounds wherein R9 is
respectively.
Alternatively, if the pentapeptide 10 does not contain a carbonyl group but does contain a hydroxyl group, then this hydroxyl group can be elaborated to provide a carbonyl group. One way to provide carbonyl functionality from hydroxyl functionality is to react the hydroxyl group with a keto acid. The chemical literature describes many keto acids, and some keto acids are also available from commercial sources. Most keto acids, although not α and β aliphatic keto acids, can be readily condensed with a hydroxyl group using DCC/DMAP chemistry to provide keto esters 11 (Larock, R. C., Comprehensive Organic Transformations, Wiley-VCH, 1999, p. 1937). Exemplary keto acids include, without limitation: levulinic acid, 4-acetylbutyric acid, 7-oxooctanoic acid, 4-acetylbenzoic acid, 4-carboxyphenylacetone, N-methyl-4-acetyl-3,5-dimethyl-2-pyrrole carboxylic acid, 3-benzoyl-2-pyridinecarboxylic acid, and 2-acetylthiazol-4-carboxylic acid.
The use of an α-keto acid to provide a carbonyl group requires initial protection of the carbonyl group as, for example, a dimethyl ketal. Dimethyl ketals can be readily obtained by treatment of keto acids with an excess of dimethylorthoformate in the presence of concentrated sulfuric acid as described, for example, in LaMattina, J. L., Muse, D. E. J. Org. Chem. 1987, 52, 3479-3481. After protection, standard DCC/DMAP-mediated condensation of the carboxyl group of the keto acid with the hydroxyl group of the pentapeptide can be used to form the ester bond. After aqueous work-up and low vacuum drying, this material may be used without further purification. Once the ester bond is formed, the dimethyl ketal can be hydrolyzed under mild acidic conditions to give the desired α-keto ester 11. Exemplary α-keto acids include, without limitation: pyruvic acid, 2-ketobutyric acid, 2-thiophenglyoxylic acid, and benzoylformic acid.
As illustrated in Scheme 4, preparation of β-keto esters of pentapeptides may be performed via DMAP-promoted transesterification of ethyl β-keto esters (see, e.g., Otera, J. Chem. Rev., 1993, 93, 1449-1470). Commercially available β-keto esters that may be used in this transformation include, without limitation, ethyl acetoacetate, ethyl β-oxo-3-furanpropionate, and ethyl 3-(1-adamantyl)-3-oxopropionate. For instance, when R9 is selected from
the hydroxyl group of R9 may be reacted with a ketoacid to form an ester linkage and the desired carbonyl group. This approach to preparing carbonyl-containing drugs is shown in Scheme 4.
where, in compound 11, R9 is selected from
Once formed, the keto esters of pentapeptides can be isolated from the reaction mixture and purified by, for example, flash silica gel chromatography, and/or reversed phase high performance liquid chromatography.
Instead of utilizing an ester linkage to join the reactive carbonyl group (which may be reacted with a heterobifunctional linker) to the remainder of the drug, the reactive carbonyl group may be joined to the remainder of the drug via an ether linkage. That is, instead of providing a compound of the formula
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R9 is selected from
and R10 is
(i.e., an ester linkage in addition to R14 is used to link a reactive carbonyl group to the remainder of the molecule), the present invention also provides compounds wherein R10 is
(i.e., an ether linkage in addition to R14 is used to link a reactive carbonyl group to the remainder of the molecule) and, in either case (ether or ester linkage to R14) R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene; and R15 is selected from hydrogen, lower alkyl and aryl.
The preparation of pentapeptides having an ether linkage to R14 is readily accomplished using amino alcohols (analogs of amino acids) which in turn are available from many commercial suppliers and are described in the chemical literature. The amino alcohol having a protected amino group (of general formula (protecting group) HN—R—OH where R is an organic moiety), may be reacted with an alcohol of the formula HO—R14—C(═O)—R15, for example via Mitsunobu reaction when R14=aryl, in the presence of PPh3, DIAD and a suitable solvent, e.g., dioxane, to provide the corresponding amino ether (of the formula (protecting group) HN—R—O—R14—C(—O)—R15). Upon deprotection of the amino group, this amino group can be reacted with a carboxylic acid or a synthon thereof, to provide a peptide (amide) group as a step toward the preparation of a pentapeptide. This chemistry is illustrated in FIGS. 16 and 17, where an exemplary and preferred amino alcohol is compound 54.
After reaction with a heterobifunctional linker, the resulting product is a pentapeptide drug conjugated to a linker moiety, where the linker moiety is a reactive linker moiety. In other words, the pentapeptide-linker conjugate contains a group that is reactive with functionality present on the ligand.
Thus, in one aspect, the present invention provides a pentapeptide-linker conjugate of the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; and R20 is a reactive linker group having a reactive site that allows R20 to be reacted with a targeting moiety.
In another aspect, the present invention provides a pentapeptide-linker conjugate of the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; and R20 is a reactive linker group having a reactive site that allows R20 to be reacted with a targeting moiety.
In yet another aspect, the present invention provides a pentapeptide-linker conjugate of the formula
and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocycle when R5 is selected from H and methyl, or R4 and R5 together form a carbocycle of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; and R20 is a reactive linker group having a reactive site that allows R20 to be reacted with a targeting moiety.
As shown in Scheme 5, hydrazone bond formation between the heterobifunctional linker and the carbonyl-containing pentapeptide drug, followed by treatment with the ligand, will provide compounds of formula I. The reaction of the drug with the heterobifunctional linker may be performed in a suitable solvent, e.g., anhydrous MeOH or DMSO, preferably in the presence of a small amount of acid, e.g., 0.01% AcOH or TFA at room temperature. The relative amounts of hydrazide to be applied and the time required for complete, or near complete reaction depends on the nature of the carbonyl group. Aromatic and α-carbonyl groups generally require higher excess of hydrazide and longer reaction time. The procedures of King, H. D., et al. Bioconjugate Chem., 1999, 10, 279-288, as used to prepare Doxorubicin-mAb conjugates, may be utilized to prepare the conjugates of the present invention. Stable hydrazones may be purified by a reversed phase HPLC. Hydrazone stability typically depends on the nature of corresponding keto esters.
The ligand is preferably a targeting ligand that directs the conjugate to a desired location in the animal subject. A preferred targeting ligand is an antibody, e.g., a chimeric or humanized antibody, or a fragment thereof.
Polyclonal antibodies which may be used in the compositions and methods of the invention are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies which may be used in the compositions and methods of the invention are homogeneous populations of antibodies to a particular antigen (e.g., a cancer cell antigen, a viral antigen, a microbial antigen). A monoclonal antibody to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the more recent human B cell hybridoma technique (Kozbor et al., 1983, immunology Today 4: 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
The monoclonal antibodies which may be used in the compositions and methods of the invention include, but are not limited to, human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).
The invention further provides for the use of bispecific antibodies. Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Mulstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low.
Similar procedures are disclosed in PCT Publication No. WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991, EMBO J. 10:3655-3659.
According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in PCT Publication No. WO 94/04690 published Mar. 3, 1994, which is incorporated herein by reference in its entirety.
For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 1986, 121:210.
The invention provides for the use of functionally active fragments, derivatives or analogs of antibodies which immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens. Functionally active means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized. Specifically, in a preferred embodiment the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay)
Other embodiments of the invention include fragments of the antibodies suitable for use in the invention such as, but not limited to, F(ab′)2 fragments, which contain the variable region, the light chain constant region and the CH1 domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. The invention also provides heavy chain and light chain dimers of the antibodies suitable for use in the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody of interest.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference n its entirety.
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of interest. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806; each of which is incorporated herein by reference in its entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903).
The antibodies suitable for use in the invention include antibodies with modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, the antibodies suitable for use in the invention include antibodies with modifications in amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., PCT Publication No. WO 97/34631, which is incorporated herein by reference in its entirety).
Antibodies immunospecific for a cancer cell antigen can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
In a specific embodiment, known antibodies for the treatment or prevention of cancer are used in accordance with the compositions and methods of the invention. Examples of antibodies available for the treatment of cancer include, but are not limited to, Herceptin® (Trastuzumab; Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer (Stebbing, J., Copson, E., and O'Reilly, S. “Herceptin (trastuzamab) in advanced breast cancer” Cancer Treat Rev. 26, 287-90, 2000); Rituxan® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG2a antibody for the treatment of colorectal cancer; BEC2 (ImClone Systems Inc., NY) which is murine IgG antibody for the treatment of lung cancer; IMC-C225 (Imclone Systems Inc., NY) which is a chimeric IgG antibody for the treatment of head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosite, MA) which is a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA) which is a humanized IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which is a humanized IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a humanized antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA) which is a murine antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma; anti-VEGF (Genentech, Inc., CA) which is humanized antibody for the treatment of lung and colorectal cancers; CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer; IMC-1C11 (imClone Systems, NJ) which is an anti-KDR chimeric antibody for the treatment of colorectal cancer, lung cancers, and melanoma; and Cetuximab (ImClone, NJ) which is an anti-EGFR chimeric antibody for the treatment of epidermal growth factor positive cancers.
Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), MUC1 (carcinomas), placental alkaline phosphatase (carcinomas), prostate sepcific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 and MAGE 3 (carcinomas), anti-transferrin receptor (carcinomas), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), and CD40 (lymphoma). Some specific useful antibodies include BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellström, I., Hellström, K. E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64 (Trail, P A, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R. S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom, K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against the CD 40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res. 2000, 60, 3225-3231), and mAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functional effects of CD30 on a large granular lymphoma cell line YT” J. Immunol., 151, 5896-5906, 1993). Many other internalizing antibodies that bind to tumor associated antigens can be used in this invention, and have been reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy of acute myeloid leukemia: a review” Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).
Antibodies that are useful in this invention for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti Phospholipid Antibody IgM, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; Anti SCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody.
In a specific embodiment, known antibodies for the treatment or prevention of viral or microbial infection are used in accordance with the compositions and methods of the invention. Examples of antibodies available for the treatment of viral infection or microbial infection include, but are not limited to, Synagis® (MedImmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment of patients with RSV infection; PRO542 (Progenics) which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a human antibody for the treatment of hepatitis B virus; Protovir (Protein Design Labs, Inc., CA) which is a humanized IgG1 antibody for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.
Other antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).
Other antibodies useful in this invention for treatment of viral disease include antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis β virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.
The antibodies suitable for use in the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
Recombinant expression of the antibodies suitable for use in the invention, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a nucleic acid molecule encoding an antibody may be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g. an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
If an antibody that specifically recognizes a particular antigen is not available (or a source for a cDNA library for cloning a nucleic acid encoding such an immunoglobulin), antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
Once a nucleic acid sequence encoding at least the variable domain of the antibody is obtained, it may be introduced into a vector containing the nucleotide sequence encoding the constant regions of the antibody (see, e.g., PCT Publication No. WO 86/05807; PCT Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors containing the complete light or heavy chain that allow for the expression of a complete antibody molecule are available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g. humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
Once a nucleic acid sequence encoding an antibody of the invention has been obtained, the vector for the production of the antibody may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
An expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody of the invention. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.
The host cells used to express the recombinant antibody of the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
A variety of host-expression vector systems may be utilized to express the immunoglobulin molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of the antibody may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the immunoglobulin molecule of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free gluta-thione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express an antibody may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1; and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).
The expression levels of an antibody can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once the antibody has been recombinantly expressed, it may be purified by any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
Internalizing monoclonal antibodies, e.g., BR96, AC10, herceptin, and S2C6 are preferred antibodies of the present invention. The ligand is suitably joined to the linker through a free thiol group or a free amino group on the ligand. For instance, when the ligand is an antibody, the free thiol group may be generated by reduction of a disulfide group present in the antibody with dithiothreitol as previously described (Trail, P. A. et al., Science 1993, 261, 212-215, and Firestone, R. A. Willner, D., Hofstead, S. J., King, H. D., Kaneko, T, Braslawsky, G. R., Greenfield, R. S., Trail, P. A., Lasch, S. J., Henderson, A. J., Casazza, A. M., Hellström, I., and Hellström, K. E., “Synthesis and antitumor activity of the immunoconjugate BR96-Dox” J. Controlled Rel. 1996, 39, 251-259). Alternatively, a lysine of the antibody may be reacted with iminothiolane hydrochloride (Traut's reagent) to introduce free thiol groups. Alternatively, free amino groups of the antibody may be reacted directly with linker-drug bearing suitable functionality, e.g., activated ester, isothiocyanate, isocyanate, anhydride, or acyl chloride functionalities. The drug-linker-ligand conjugate of the invention retains both specificity and therapeutic drug activity for the treatment of cell population expressing specific antigen for the ligand. Stable in serum, the hydrazone bond of the conjugate will be cleaved in acidic intracellular compartments releasing free highly-potent drug. This approach may be used to provide conjugates with high selectivity between target and non-target cell lines for example with up to about 1000 fold selectivity.
Scheme 5 illustrates the conjugation of the carbonyl-containing drug to a ligand through a heterobifunctional linker to form a compound of formula I.
wherein, in the compound of formula I, and independently at each location: R1 is selected from hydrogen and lower alkyl; R2 is selected from hydrogen and lower alkyl; R3 is lower alkyl; R4 is selected from lower alkyl, aryl, and —CH2—C5-7carbocyclic when R5 is selected from H and methyl, or R4 and R5 together form a carbocyclic group of the partial formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R6 is selected from hydrogen and lower alkyl; R7 is sec-butyl or iso-butyl; R8 is selected from hydrogen and lower alkyl; R9 is selected from
R10 is selected from
R11 is selected from hydrogen and lower alkyl; R12 is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R12 is independently selected at each occurrence; R13 is
R14 is selected from a direct bond, arylene (lower alkylene), lower alkylene and arylene; R15 is selected from hydrogen, lower alkyl and aryl; R16 is selected from arylene (lower alkylene), lower alkylene, arylene, and —(CH2OCH2)pCH2— where p is 1-5; and R17 is selected from
where Y═O or S.
Compositions
In other aspects, the present invention provides pentapeptide drugs as described above, and prodrugs as also described above that are based on the pentapeptide drugs, in combination with a pharmaceutically acceptable carrier, excipient or diluent. For convenience, the pentapeptide drugs and the prodrugs of the invention will simply be referred to as compounds of the invention. Thus, the present invention provides a pharmaceutical or veterinary composition (hereinafter, simply referred to as a pharmaceutical composition) containing a compound of the invention as described above, in admixture with a pharmaceutically acceptable carrier. The invention further provides a composition, preferably a pharmaceutical composition, containing an effective amount of a compound as described above, in association with a pharmaceutically acceptable carrier.
The pharmaceutical compositions of the present invention may be in any form that allows for the composition to be administered to an animal subject. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Preferably, the compositions are administered parenterally. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to an animal subject. Compositions that will be administered to a subject take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units.
Materials used in preparing the pharmaceutical compositions should be pharmaceutically pure and non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the particular form of the active ingredient, the manner of administration, and the composition employed.
In general, the pharmaceutical composition includes an (where “a” and “an” refers here, and throughout this specification, to one or more) active compounds of the invention in admixture with one or more carriers. The carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) may be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.
When intended for oral administration, the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following adjuvants may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
When the composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
The composition may be in the form of a liquid, e.g. an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
The liquid pharmaceutical compositions of the invention, whether they are solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.
A liquid composition intended for either parenteral or oral administration should contain an amount of a compound of the present invention such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a compound of the invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1% and about 80% of the weight of the composition. Preferred oral compositions contain between about 4% and about 50% of the compound of the invention. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01% to 2% by weight of active compound.
The present compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g. encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a present compound or composition. In certain embodiments, more than one compound or composition is administered to a subject. Methods of administration include but are not limited to oral administration and parenteral administration; parenteral administration including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition (such as the site of cancer or autoimmune disease).
In a preferred embodiment, the present compounds or compositions are administered parenterelly.
In a more preferred embodiment, the present compounds or compositions are administered intravenously.
In specific embodiments, it may be desirable to administer one or more compounds or compositions locally to the area in need of treatment. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue. In another embodiment, administration can be by direct injection at the site (or former site) of an autoimmune disease.
In certain embodiments, it may be desirable to introduce one or more compounds or compositions into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the compounds or compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
In another embodiment, the compounds of the invention can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compounds or compositions can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the compounds or compositions, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) may be used.
The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound of the invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a subject, the present compounds or compositions and pharmaceutically acceptable carriers are preferably sterile. Water is a preferred carrier when the compounds are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
In a preferred embodiment, the present compounds are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, the compounds for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions may contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade.
The amount of the compound or pharmaceutically acceptable salt or solvate that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, dose ranges for a liquid composition intended for either parenteral or oral administration should contain an amount of a compound of the present invention such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a compound by weight in the composition. When intended for oral administration, this amount may be varied to a range from about 0.1% to about 80% of the weight of the composition. Preferred oral compositions contain from about 4% and about 50% of the compound by weight of the composition. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2%, preferrably from about 0.01% to about 2% of compound by weight of the parenteral dosage unit. For intravenous administration, the formulation preferably will be prepared so that the amount administered to the patient will be from about 1 to about 250 mg/kg of body weight of the desired conjugate. Preferably, the amount administered will be in the range of about 4 to about 25 mg/kg of body weight of the conjugate.
The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of a compound of the present invention of from about 0.1% to about 10% w/v (weight per unit volume).
The composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The composition may include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule.
The pharmaceutical composition of the present invention may consist of gaseous dosage units, e.g., it may be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, spacers and the like, which together may form a kit. Preferred aerosols may be determined by one skilled in the art, without undue experimentation.
Whether in solid, liquid or gaseous form, the pharmaceutical composition of the present invention may contain one or more known pharmacological agents used in the treatment of cancer.
The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a compound of the invention with water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a compound of the invention so as to facilitate dissolution or homogeneous suspension of the active compound in the aqueous delivery system.
Biological Activity and Utility of Compounds
The present invention provides biology-active compounds and pro-drugs (collectively, compounds, or compounds of the invention), methods of preparing compounds of the invention, pharmaceutical compositions comprising the compounds of the invention, and methods for treatment of cancers and other tumors in animal subjects. For instance, the invention provides compounds and compositions for use in a method for treating tumors wherein the animal subject is treated, in a pharmaceutically acceptable manner, with a pharmaceutically effective amount of a compound or composition of the present invention.
Treatment of Cancer
The compounds and compositions of the invention can be used in a variety of settings for the treatment of mammalian cancers. The mAb-pentapeptide conjugates can be used to deliver the cytotoxic drug to tumor cells. Without being bound by theory, once the antibody has bound to tumor associated antigens, it is taken up inside cells through receptor-mediated endocytosis into endosomes and lysosomes. These intracellular vesicles are acidic and can induce the hydrolysis of an acid-sensitive linker bond, e.g. a hydrazone bond, between the drugs and the mAbs. In addition, ester bonds can be cleaved by proteases and esterases, which are in abundance within lysosomes. The released drug is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the drug is cleaved from the conjugate outside the cell and subsequently penetrates the cell.
The specificity of the mAb for a particular tumor type will dictate which tumors will be treated with the immunoconjugates. For example, BR96-pentapeptide conjugates may be used to treat antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas. Anti-CD30-pentapeptide and anti-CD40-pentapeptide conjugates may be used for treating hematologic malignancies.
Other particular types of cancers that may be treated with the compounds, compositions, and methods of the invention include, but are not limited to those disclosed in Table 1.
TABLE 1
Solid tumors, including but not limited to:
fibrosarcoma
myxosarcoma
liposarcoma
chondrosarcoma
osteogenic sarcoma
chordoma
angiosarcoma
endotheliosarcoma
lymphangiosarcoma
lymphangioendotheliosarcoma
synovioma
mesothelioma
Ewing's tumor
leiomyosarcoma
rhabdomyosarcoma
colon cancer
colorectal cancer
kidney cancer
pancreatic cancer
bone cancer
breast cancer
ovarian cancer
prostate cancer
esophogeal cancer
stomach cancer
oral cancer
nasal cancer
throat cancer
squamous cell carcinoma
basal cell carcinoma
adenocarcinoma
sweat gland carcinoma
sebaceous gland carcinoma
papillary carcinoma
papillary adenocarcinomas
cystadenocarcinoma
medullary carcinoma
bronchogenic carcinoma
renal cell carcinoma
hepatoma
bile duct carcinoma
choriocarcinoma
seminoma
embryonal carcinoma
Wilms' tumor
cervical cancer
uterine cancer
testicular cancer
small cell lung carcinoma
bladder carcinoma
lung cancer
epithelial carcinoma
glioma
glioblastoma multiforme
astrocytoma
medulloblastoma
craniopharyngioma
ependymoma
pinealoma
hemangioblastoma
acoustic neuroma
oligodendroglioma
meningioma
skin cancer
melanoma
neuroblastoma
retinoblastoma
blood-born cancers, including but not limited to:
acute lymphoblastic leukemia “ALL”
acute lymphoblastic B-cell leukemia
acute lymphoblastic T-cell leukemia
acute myeloblastic leukemia “AML”
acute promyelocytic leukemia “APL”
acute monoblastic leukemia
acute erythroleukemic leukemia
acute megakaryoblastic leukemia
acute myelomonocytic leukemia
acute nonlymphocyctic leukemia
acute undifferentiated leukemia
chronic myelocytic leukemia “CML”
chronic lymphocytic leukemia “CLL”
hairy cell leukemia
multiple myeloma
acute and chronic leukemias:
lymphoblastic
myelogenous
lymphocytic
myelocytic leukemias.
Polycythemia vera
Lymphomas:
Hodgkin's disease
non-Hodgkin's Lymphoma
Multiple myeloma
Waldenström's macroglobulinemia
Heavy chain disease
The compounds and compositions of the invention may also be used as chemotherapeutics in the untargeted form. For example, the Pentapeptide drugs themselves, or the drug-linker conjugates minus the antibody moiety may be used against ovarian, CNS, renal, lung, colon, melanoma, and hematologic tumors.
Conjugation of highly potent drugs to monoclonal antibodies specific for tumor markers results in specific targeting, thus reducing general toxicity of these compounds. pH-Sensitive linkers provide stability of the conjugate in blood, yet are hydrolyzable in acidic intracellular compartments, liberating active drug.
Treatment of Autoimmune Diseases
The compounds pharmaceutically acceptable salts or solvates and compositions of the invention can also be used for the treatment of an autoimmune disease by killing or inhibiting the multiplication of cells which produce the auto-immune antibodies associated with said autoimmune disease.
Particular types of autoimmune diseases that may be treated with the compounds, compositions, and methods of the invention include, but are not limited to those disclosed in Table 2.
TABLE 2
Active Chronic Hepatitis
Addison's Disease
Ankylosing Spondylitis
Anti-phosholipid Syndrome
Arthritis
Atopic Allergy
Behcet's Disease
Cardiomyopathy
Celiac Disease
Cogan's Syndrome
Cold Agglutinin Disease
Crohn's Disease
Cushing's Syndrome
Dermatomyositis
Discoid Lupus
Erythematosis
Fibromyalgia
Glomerulonephritis
Goodpasture's Syndrome
Graft v. Host Disease
Grave's Disease
Guillain-Barre Disease
Hashimoto's Thyroiditis
Idiopathic Adrenal Atrophy
Idiopathic Pulmonary Fibritis
IgA Nephropathy
Inflammatory Bowel Diseases
Insulin-dependent Diabetes Mellitus
Juvenile Arthritis
Lambert-Eaton Syndrome
Lichen Planus
Lupoid Hepatitis
Lupus
Lymphopenia
Meniere's Disease
Mixed Connective Tissue Disease
Multiple Sclerosis
Myasthenia Gravis
Pernicious Anemia
Polyglandular Syndromes
Primary Agammaglobulinemia
Primary Biliary Cirrhosis
Psoriasis
Psoriatic Arthritis
Raynauds Phenomenon
Reiter's Syndrome
Rheumatoid Arthritis
Schmidt's Syndrome
Scleroderma
Sjorgen's Syndrome
Stiff-Man Syndrome
Sympathetic Ophthalmia
Systemic Lupus Erythematosis
Takayasu's Arteritis
Temporal Arteritis
Thyrotoxicosis
Type B Insulin Resistance
Type I Diabetes Mellitus
Ulcerative Colitis
Uveitis
Vitiligo
Wegener's Granulomatosis
Treatment of Cancer in Combination With Other Treatment Modalities
Cancer or a neoplastic disease, including, but not limited to, a neoplasm, tumor, metastasis, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of the compounds and compositions of the invention.
In other embodiments, a composition comprising one or more compounds of the invention or pharmaceutically acceptable salts or solvates thereof is administered along with radiation therapy and/or with one or a combination of chemotherapeutic agents, preferably with one or more chemotherapeutic agents with which treatment of the cancer has not been found to be refractory. The compounds of the invention or pharmaceutically acceptable salts or solvates thereof can be administered to a patient that has also undergone surgery as treatment for the cancer.
In another specific embodiment, the invention provides a method to treat or prevent cancer that has shown to be refractory to treatment with a chemotherapy and/or radiation therapy.
In a specific embodiment, a composition comprising one or more compounds of the invention or pharmaceutically acceptable salts or solvates thereof is administered concurrently with chemotherapy or radiation therapy. In another specific embodiment, chemotherapy or radiation therapy is administered prior or subsequent to administration of a present composition, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e.g., up to three months), subsequent to administration of a therapeutic of the invention.
The chemotherapy or radiation therapy administered concurrently with, or prior or subsequent to, the administration of a present composition can be accomplished by any method known in the art. The chemotherapeutic agents are preferably administered in a series of sessions, any one or a combination of the chemotherapeutic agents listed above can be administered. With respect to radiation therapy, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, may also be administered to expose tissues to radiation.
Additionally, the invention provides methods of treatment of cancer or neoplastic disease with a present composition as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or may prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The subject being treated with the present compositions may, optionally, be treated with other cancer treatments such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
The present compositions may also be used in an in vitro fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants. This involves a multi-step process in which the patient's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the patient's remaining bone marrow cell population is then eradicated via the administration of a high dose of a composition of the invention +/−high dose radiation therapy, the stem cell graft is infused back into the patient, and supportive care is provided while bone marrow function is restored and the patient recovers.
Combination Therapy
In certain embodiments of the present invention, the compounds and pharmaceutically acceptable salts or solvates thereof can be used in combination with at least one other therapeutic agent.
The compound(s) and the other therapeutic agent(s) can act additively or, more preferably, synergistically. In a preferred embodiment, a composition comprising a compound is administered concurrently with the administration of one or more additional therapeutic agent(s), which can be part of the same composition or in a different composition from that comprising the compound. In another embodiment, a composition comprising the compound is administered prior to or subsequent to administration of another therapeutic agent(s).
In certain embodiments, the compounds can be used in combination with one or more anticancer agents and pharmaceutically acceptable salts or solvates thereof. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyarea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred embodiment, one or more of the compositions of the invention is used to treat or prevent cancer or neoplastic disease in combination with one or more chemotherapeutic or other anti-cancer agents including, but not limited to those presented in Table 3.
TABLE 3
Alkylating agents
Nitrogen mustards:
cyclophosphamide
Ifosfamide
trofosfamide
Chlorambucil
Nitrosoureas:
carmustine (BCNU)
Lomustine (CCNU)
Alkylsulphonates
busulfan
Treosulfan
Triazenes:
Dacarbazine
Platinum containing compounds:
Cisplatin
carboplatin
Plant Alkaloids
Vinca alkaloids:
vincristine
Vinblastine
Vindesine
Vinorelbine
Taxoids:
paclitaxel
Docetaxol
DNA Topoisomerase Inhibitors
Epipodophyllins:
etoposide
Teniposide
Topotecan
9-aminocamptothecin
campto irinotecan
crisnatol
mitomycins:
Mitomycin C
Anti-metabolites
Anti-folates:
DHFR inhibitors:
methotrexate
Trimetrexate
IMP dehydrogenase Inhibitors:
mycophenolic acid
Tiazofurin
Ribavirin
EICAR
Ribonuclotide reductase Inhibitors:
hydroxyurea
deferoxamine
Pyrimidine analogs:
Uracil analogs
5-Fluorouracil
Floxuridine
Doxifluridine
Ratitrexed
Cytosine analogs
cytarabine (ara C)
Cytosine arabinoside
fludarabine
Purine analogs:
mercaptopurine
Thioguanine
Hormonal therapies:
Receptor antagonists:
Anti-estrogens
Tamoxifen
Raloxifene
megestrol
LHRH agonists:
goscrclin
Leuprolide acetate
Anti-androgens:
flutamide
bicalutamide
Retinoids/Deltoids
Vitamin D3 analogs:
EB 1089
CB 1093
KH 1060
Photodyamic therapies:
vertoporfin (BPD-MA)
Phthalocyanine
photosensitizer Pc4
Demethoxy-hypocrellin A
(2BA-2-DMHA)
Cytokines:
Interferon-α
Interferon-γ
Tumor necrosis factor
Others:
Isoprenylation inhibitors:
Lovastatin
Dopaminergic neurotoxins:
1-methyl-4-phenylpyridinium ion
Cell cycle inhibitors:
staurosporine
Actinomycins:
Actinomycin D
Dactinomycin
Bleomycins:
bleomycin A2
Bleomycin B2
Peplomycin
Anthracyclines:
daunorubicin
Doxorubicin (adriamycin)
Idarubicin
Epirubicin
Pirarubicin
Zorubicin
Mitoxantrone
MDR inhibitors:
verapamil
Ca2+ ATPase inhibitors:
thapsigargin
In another preferred embodiment, the compounds can be used in combination with one or more immunosuppressant agents and pharmaceutically acceptable salts or solvates thereof to treat an autoimmune disease. In a preferred embodiment, the immunosuppressant agent includes, but is not limited to, cyclosporine, cyclospoxine A, mycophenylate mofetil, sirolimus, tacrolimus, enanercept, prednisone and azathioprine.
In still another embodiment, the other therapeutic agent can also be an adjuvant to reduce any potential side effects of treatment, such as, for example, an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, grarusetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.
The present compounds and compositions may also be used alone or in combination with one or more antitumor agents and one or more immunosuppressant agents to simultaneously treat a combination of cancer and an autoimmune disease.
The free drugs of the invention may also be used as chemotherapeutics in the untargeted form. For example, the pentapeptides of the present invention may be used against ovarian, CNS, renal, lung, colon, melanoma, and hematologic tumors.
Conjugation of these highly potent drugs to monoclonal antibodies specific for tumor markers results in specific targeting, thus reducing general toxicity of these compounds. pH-Sensitive linkers should provide stability of the conjugate in blood, yet should be hydrolyzed in acidic intracellular compartments liberating active drug.
The following examples are provided by way of illustration and not limitation.
EXAMPLES
The chemistry described in Examples 1-4 is illustrated in the schemes shown in FIG. 1-3. The chemistry described in Examples 5 and 6 is illustrated in the scheme shown in FIG. 4. The chemistry described in Example 7 is illustrated in the scheme shown in FIG. 5. The chemistry described in Example 8 to prepare compound 36 is illustrated in the scheme shown in FIG. 6, while the conjugation of compound 36 to an antibody is illustrated in FIG. 10. The chemistry described in Example 9 to prepare compounds 38 is illustrated in the scheme shown in FIG. 7. Chemistries to make compounds 40a and 40b, as described in Examples 10 and 11, are shown in FIG. 8, while the conjugation of 40a to an antibody is illustrated in FIG. 11. The preparation of drug-linker-mAb 42 as described in Example 12 is illustrated in the scheme shown in FIG. 9. The reaction of pentapeptide 47 with a heterobifunctional linker as described in Example 13 is illustrated in the scheme of FIG. 12, where this scheme also shows the formation of the drug-linker-antibody conjugate mAb-S-48. The scheme of FIG. 13 shows the preparation of the pentapeptide 49 as described in Example 14. The synthetic descriptions provided in Examples 15, 16, 17 and 18 are illustrated in the schemes provided in FIGS. 14, 15, 16 and 17, respectively.
Coupling Procedure A: Peptide Synthesis Using DEPC
To a cooled (ice bath) solution of [2S-[2R*(αS*,βS*)]]-1-[(1,1-dimethylethoxy)carbonyl]-β-methoxy-α-methyl-2-pyrrolidinepropanoic acid (t-Boc-dolaproine (20) 0.27 g, 0.94 mmol) in anhydrous CH2Cl2 (10 mL) is added an identified amine (1.03 mmol, 1.1 eq.) followed by triethylamine (0.40 mL, 2.8 mmol, 3.0 eq.) and DEPC (0.17 mL, 90%, 1.03 mmol, 1.1 eq.). The resulting solution is stirred under argon for 12 hours. The solvent is removed under reduced pressure at room temperature, and the residue is chromatographed (silica gel column using 4:1 hexanes-acetone as eluent). After the evaporation of solvent from the fractions selected according to TLC analysis, the residue is dried under vacuum overnight to afford the amide.
Coupling Procedure B: Peptide Synthesis using PyBrop
The amino acid (1.0 eq.) containing a carboxyl protecting group, if necessary, was dissolved in anhydrous CH2Cl2 followed by the addition of diisopropylethylamine (1.5 eq.). Fmoc-, Z-, or dimethyl amino acid/pepide (1.1 eq.) was added as a solid in one portion and the dissolved mixture had added PyBrop (1.2 eq.). The reaction was monitored by TLC or HPLC.
General Procedure C: Pentapeptide Synthesis
A solution of dipeptide and tripeptide (1 eq. each) in CH2Cl2 (10 mL) and trufluoroacetic acid (5 mL) is stirred (ice bath under a N2 atmosphere) for two hours. The reaction may be monitored by TLC or, preferably, HPLC. The solvent is removed under reduced pressure and the residue dissolved in toluene. Solvent is again removed in vacuo and the process repeated. The residue is dried under high vacuum for 12 h and then dissolved in dry CH2Cl2 followed by the addition of diisopropylethylamine (1.5 eq.). Depending on the residues, the peptides may be coupled using either PyBrop or DEPC (1.2 eq.). The reaction mixture is monitored by either TLC or HPLC.
General procedure D: Z-Removal Via Hydrogenolysis
Using a large, heavy-walled flask, a solution of Z-protected amino acid or peptide was dissolved in ethanol. 10% palladium on carbon was added (1% w/w peptide) and the mixture was introduced to H2. Reaction progress was monitored by HPLC and typically found to be complete within 1-2 h. The flask contents are filtered through a pre-washed pad of celite and then the celite is washed with methanol. The eluent solution is evaporated to an oil, dissolved in toluene, and re-evaporated.
General Procedure E: Fmoc-Removal Using Diethylamine
The Fmoc-containing compound is dissolved in CH2Cl2 to which an equal amount of diethylamine is added. Reaction progress is monitored by TLC (or HPLC) and is usually complete within 2 h. Solvents are removed in vacuo and the residue taken up in toluene and concentrated again. The residue is dried under high vacuum for at least 1 h.
General Procedure F: Hydrazone Formation
Hydrazone formation is performed in anhydrous MeOH, 0.01% AcOH (typically 1 mL per 10 mg of a drug) at room temperature. Time of the reaction (6 h-5 days) and an amount of a hydrazide (2-30 eq.) may be varied depending on the specific ketone. The reaction progress is monitored by C18 RP-HPLC. Typically, a newly formed acylhydrazone has lower retention time compared to the parent ketone. After the reaction is complete, DMSO (1-2 mL) is added to the reaction mixture and methanol is removed under reduced pressure. The residue is directly loaded onto a C18 RP column for preparative HPLC purification (Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 100 mM TEAA buffer, pH 7.0, from 10 to 95% at flow rate 4 mL/min). The appropriate fractions are concentrated under reduced pressure, co-evaporated with acetonitrile (4×25 mL), and finally dried in deep vacuum for 2 days.
Example 1
Preparation of Compound 29a
Preparation of Compound 14a
Compound 14a was prepared by reacting compounds 12 and 13a according to coupling procedure B. After concentration of the reaction mixture, the residue was directly injected onto a reverse phase preparative-HPLC column (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using an isocratic run of 25% aqueous MeCN at 20 mL/min) in order to remove the unwanted diastereomer. The pure fractions were concentrated to give the product as a clear oil. Yield 14a: 0.67 g (55%); ES-MS m/z 493.4 [M+H]+; UV λmax 215, 256 nm.
Preparation of Compound 15a
Compound 14a was treated according to deprotection procedure D to provide compound 15a.
Preparation of Compound 18a
Compounds 15a and 16 were coupled and characterized as described in Pettit et. al. J. Chem. Soc. Perk I, 1996, 859.
Preparation of [(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidine-1-carboxylic acid-tert-butyl ester (25)
t-Boc-dolaproine 20 and (1S,2R)-norephedrine 21 were combined in the presence of DEPC and triethylamine according to General procedure A (see also U.S. Pat. No. 5,635,483 to Pettit et al.) to provide compound 25.
Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide, (29a, Auristatin E)
Compound 18a and 25 were combined in the presence of trifluoroacetic acid in methylene chloride (1:1), followed by treatment with DEPC and triethylamine, to provide compound 29a using procedure C. The synthesis of this compound is also reported in U.S. Pat. No. 5,635,483, and Pettit et al., Anti-Cancer Drug Des. 1998, 243.
Example 2
Preparation of compound 29b
Preparation of Compound 14b According to coupling procedure B, this peptide was prepared using compounds 12 and 13b. After concentration of the reaction mixture, the residue was purified by C18 preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using an isocratic run of 75% aqueous MeCN at 20 mL/min) in order to remove the unwanted diastereomer. The pure fractions were concentrated to a clear oil. Yield 14b: 0.67 g (55%); Rf 0.32 (4:1 hexanes-acetone); UV λmax 215, 256 nm; 1H NMR (CDCl3) δ 7.14-7.40 (5 H, m), 6.19 (1 H, d, J=9.3 Hz), 5.09 (2 H, s), 4.53 (1 H, dd, J=6.6, 9.3 Hz), 3.34 (3 H, s), 2.97 (3 H, s), 2.25-2.50 (2 H, m), 1.50-1.78 (3 H, m), 1.46 (9H, s), 0.99 (3 H, d, J=6.9 Hz), 0.96 (3 H, d, J=6.9 Hz), 0.89 (3 H, t, J=6.9 Hz), 0.83 (3 H, t, J=7.2 Hz).
Preparation of Compound 15b
Compound 14b was treated according to deprotection procedure D to provide compound 15b.
Preparation of Compound 18b
Compounds 15b and 16 were combined using procedure A to provide compound 18b: Yield 18b: 0.19 g (82%); ES-MS m/z 500.5 [M+H]+; Rf 0.12 (4:1 hexanes-acetone); UV λmax 215 nm.
Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2s)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-isoleucinamide, (29b)
Following general procedure C, compounds 18b and 25 were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and triethylamine. The reaction mixture was purified by preparative-HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 100 mM aqueous triethylammonium carbonate from 10 to 100% in 40 min followed by 20 min at 0% buffer, at flow rate 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 29b: 85 mg (60%); ES-MS m/z 746.5 [M+H]+; UV λmax 215 nm.
Example 3
Preparation of Compound 31a
Preparation of Compound 19a
Compounds 15a and 17 were combined using procedure A to provide compound 19a: 107 mg (50%); ES-MS m/z 694.7 [M+H]+; Rf 0.64 (1:1 hexanes-EtOAc); UV λmax 215, 265 nm.
Preparation of Compound 30a
Following general procedure C, compounds 19a and 25 (prepared as described in Example 1) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and triethylamine. The reaction mixture was monitored by HPLC and after 4 h water was added and the layers separated. After drying (MgSO4), the contents were filtered and solvent removed to give compound 30a that was used in the next step without further purification. Yield 30a: 127 mg (91%); ES-MS m/z 940.9 [M+H]+.
Preparation of N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R, 2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide, (31a)
The Fmoc-protected peptide was treated with diethylamine according to deprotection procedure E. A complete reaction was observed by HPLC after 12 h. The reaction mixture was concentrated to an oil and purified by preparative-HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 ml/min). The desired fractions were pooled and concentrated to give an off-white solid. Yield 31a: 37 mg (38%); ES-MS m/z 718.7 [M+H]+; UV λmax 215 nm.
Example 4
Preparation of Compound 31b
Preparation of Compound 19b
Compounds 15b and 17 were combined using procedure A to provide compound 19b. Yield 19b: 0.50 g (73%); UV λmax 215, 265 nm.
Preparation of Compound 30b
Following general procedure C, compounds 19b and 25 (prepared as described in Example 1) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and triethylamine. The reaction mixture was purified by preparative-HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 30b: 64 mg (53%); ES-MS m/z 955.2 [M+H]+; UV λmax 215, 265 nm.
Preparation of N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R, 2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide, (31b)
The Fmoc-protected peptide 30b was treated with diethylamine according to deprotection procedure E. A complete reaction was observed by HPLC after 2 h. The reaction mixture was concentrated to an oil. Excess ether was added resulting in a white precipitate and the contents were cooled to 0° C. for 3 h. The product was filtered and the solid dried under high vacuum. Yield 31b: 47 mg (95%); ES-MS m/z 732.8 [M+H]+; UV λmax 215 nm.
Example 5
Preparation of Compound 32
Preparation of [(2S)-2-[(1R,2R)-3-[[(1S)-hydroxymethyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidine-1-carboxylic acid-tert-butyl ester (26)
The dipeptide 26 was synthesized from t-Boc-dolaproine (20) and (S)-(−)-2-amino-3-phenyl-1-propanol (L-phenylalaninol, 22) according to General procedure A. Yield 26: 0.50 g (55%); ES-MS m/z 421.0 [M+H]+; Rf 0.24 (100% EtOAc); UV λmax 215, 256 nm. 1H NMR (CDCl3) δ 7.14-7.40 (5 H, m), 6.19 (1 H, d, J=7.8 Hz), 4.11-4.28 (1 H, m), 3.44 (3 H, s), 3.20-3.84 (5 H, m), 2.80-3.05 (2 H, m), 2.20-2.38 (2 H, m), 1.65-1.98 (4H, m), 1.48 (9H, s), 1.16 (3 H, d, J=5.7 Hz).
Preparation of Compound 32
Following general procedure C, compounds 26 (42 mg, 0.1 mmol, 1 eq.) and 18a (65 mg, 0.13 mmol, 1.3 eq.) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and diisopropylethylamine. The reaction mixture was purified by preparative-HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 32: 60 mg (80%); ES-MS m/z 732.2 [M+H]+; UV λmax 215, 265 nn.
Example 6
Preparation of Compound 33
[(2S)-2-[(1R,2R)-3-[[(1R)-hydroxymethyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidine-1-carboxylic acid-tert-butyl ester (27)
The dipeptide 27 was synthesized from t-Boc-dolaproine (20) and (R)-(+)-2-amino-3-phenyl-1-propanol (D-phenylalaninol, 23) according to General procedure A. Yield 27: 0.53 g (58%); ES-MS m/z 421.0 [M+H]+; Rf 0.24 (100% EtOAc); UV λmax 215, 256 nm. 1H NMR (CDCl3) δ 7.14-7.40 (5 H, m), 6.19 (0.5H, d, J=5.4 Hz), 5.75 (0.5H, d, J=5.4 Hz), 4.10-4.23 (1 H, m), 3.38 (3 H, s), 3.10-3.85 (4H, m), 2.82-2.96 (2 H, m), 2.10-2.36 (2 H, m), 1.66-1.87 (4H, m), 1.47 (9H, bs), 1.16 (1.5H, d, J=6.6 Hz), 1.08 (1.5H, d, J=5.7 Hz).
Compound 33
Following general procedure C, compounds 27 (42 mg, 0.1 mmol, 1 eq.) and 18a (65 mg, 0.13 mmol, 1.3 eq.) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and diisopropylethylamine. The reaction mixture was purified by preparative-HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 33: 34 mg (46%); ES-MS m/z 732.1 [M+H]+; UV λmax 215, 265 nm.
Example 7
Preparation of Compound 34
Preparation of Compound 28
Dipeptide 28 is prepared by reacting Boc-dolaproine 20 and p-acetylphenethylamine (U.S. Pat. No. 3,445,518, 1969) according to coupling procedure B. After concentration of the reaction mixture, the residue is purified by reverse phase preparative-HPLC or via SiO2 chromatography.
Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(4-acetylphenyl)ethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1s)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide, (34)
Dipeptide 28 and tripeptide 18a are combined in the presence of trifluoroacetic acid in methylene chloride, following a coupling reaction with DEPC and triethylamine as described in general procedure C. The reaction mixture is purified under usual preparative-HPLC measures as previously described for other peptide compounds.
Example 8
Preparation of Compound 36
Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R)-2-oxo-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide (35)
Pyridinium chlorochromate (PCC) (13.6 mg, 0.06 mmol, 4.5 eq.) was added to a solution of Auristatin E (29a) (10 mg, 0.014 mmol, 1 eq.) in CH2Cl2 (2 mL) and pyridine (50 μL). The reaction mixture was stirred at room temperature for 3 h. HPLC analysis of the reaction mixture showed complete conversion of the 10.2 min peak into a new 11.3 min peak with different UV spectrum (max 245 nm, shoulder 280 nm). The product was purified by flash chromatography on a silica gel column (120×12 mm) in a step gradient of MeOH in CH2Cl2 from 0 to 10%. After concentration in vacuum, the residue was triturated with hexane to give white solid. Yield 35: 6.4 mg (64%); Rf 0.3 (CH2Cl2/MeOH, 10/1); UV λmax 215, 245 nm. HRMS m/z: found 730.5119 [M+H]+. C40H68N5O7 requires 730.5108.
Preparation of Hydrazone 36
Compound 36 was prepared from compound 35 by reaction with 30 eq. of maleimidocaproylhydrazide for 3 days according to General Procedure F. Yield 36: 2.4 mg (43%) of colorless glass; UV λmax 215, 240 (shoulder) nm. HMS m/z: found 937.6131 [M+H]+. C50H81N8O9 requires 937.6127.
Example 9
Preparation of Compound 38
Preparation of Levulinic Ester of Auristatin E (37)
Levulinic acid (2.5 μL, 0.025 mmol, 5 eq.) was added to a solution of auristatin E (29a, 3.6 mg, 0.005 mmol, 1 eq.) in anhydrous CH2Cl2 (1 ml), followed by DCC (5 mg, 0.025 mmol, 5 eq.) and DMAP (1 mg, cat). After reacting for overnight at room temperature, analysis by C18 RP-HPLC revealed formation of a new, more hydrophobic product. Precipitated DCU was filtered off. The resulting keto ester was isolated using preparative chromatography on silica gel in a step gradient of MeOH in CH2Cl2 from 0 to 10%. Yield 37: 3.3 mg (80.5%) of colorless glass; Rf 0.35 (CH2Cl2/MeOH, 10/1); ES-MS m/z 830 [M+H]+; UV λmax 215 nm.
Preparation of Hydrazone 38
Compound 38 was prepared from compound 37 and maleimidocaproylhydrazide (2 eq.) according to General Procedure F, reaction time—6 h. Yield 38: 1.3 mg (46%) of white solid, ES-MS m/z 1037 [M+H]+. UV λmax 215 nm.
Example 10
Preparation of Compound 40a
Preparation of 4-acetyl-benzoic ester of auristatin E (39a)
4-Acetylbenzoic acid (23.5 mg, 0.14 mmol, 2 eq.) was added to a solution of auristatin E (29a, 50 mg, 0.07 mmol, 1 eq.) in anhydrous CH2Cl2 (5 ml), followed by DCC (30 mg, 0.14 mmol, 2 eq.) and DMAP (5 mg, cat). After reacting overnight at room temperature, analysis by C18 RP-HPLC revealed formation of a new, more hydrophobic product. Precipitated DCU was filtered off. The resulting keto ester was isolated using preparative chromatography on silica gel in a step gradient of MeOH in CH2Cl2 from 0 to 10%. The product was eluted with 5% MeOH in CH2Cl2. Yield 39a: 57 mg (90%) of white solid; Rf 0.43 (CH2Cl2/MeOH, 10/1); UV λmax 215, 250 nm. HRMS m/z: found 878.5669 [M+H]+. C49H76N5O9 requires 878.5643. Anal. calcd for C49H75N5O9×H2O: C, 65.67; H, 8.66; N, 7.81. Found: C, 66.05; H, 8.61; N, 7.80.
Preparation of Hydrazone 40a
Compound 40a was prepared from compound 39a and maleimidocaproylhydrazide (3 eq.) according to General Procedure F, reaction time—12 h.
Yield 40a: 3 mg (65%) of solid; UV λmax 215, 295 nm. HRMS m/z: found 1085.6665 [M+H]+. C59H89N8O11 requires 1085.6651. Anal. calcd for C59H88N8O11×H2O: C, 64.22; H, 8.22; N, 10.16. Found: C, 64.27; H, 8.21; N, 9.92.
Example 11
Preparation of Compound 40b
Preparation of 4-acetyl-benzoic ester of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-isoleucinamide, (39b)
Compound 39b was prepared from compound 29b by reaction with 4-acetobenzoic acid in the presence of DCC, DMAP in CH2Cl2 as described in the Example 10. Yield 39b: 10.7 mg (80%) of white solid; Rf 0.37 (CH2Cl2/MeOH, 10/1); UV λmax 215, 250 nm. HRMS m/z: found 892.5775 [M+H]+. C50H78N5O9 requires 892.5800.
Preparation of Hydrazone (40b)
Compound 40b was prepared from compound 39b and maleimidocaproylhydrazide (5 eq.) according to General Procedure F, reaction time—18 h. Yield 40b: 4.8 mg (50%) of solid; ES-MS m/z: 1099 [M+H]+; UV λmax 215, 295 nm.
Example 12
Preparation of Compound 42
Preparation of Compound 42
To a solution of 39a (5 mg, 0.0056 mmol) in DMSO (100 μL) anhydrous MeOH (0.9 mL) was added, followed by 1% AcOH/MeOH (10 μL). 3-Bromoacetamido)propyonyl hydrazide (41) (0.025 mmol, 4.5 eq.) was added to the solution. The reaction was left at room temperature for 24 h. C18-RP HPLC analysis revealed the formation of a new compound with a lower retention time. DMSO (1 mL) was added to the reaction mixture and methanol was removed under reduced pressure. The residue was directly loaded onto a C18-RP column for preparative HPLC purification (Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 100 mM TEAA buffer, pH 7.0, from 10 to 95% at flow rate 4 mL/min). The appropriate fractions are concentrated under reduced pressure, co-evaporated with acetonitrile (4×25 mL), and finally dried in deep vacuum. Yield 42: 4.0 mg (66%) of solid; ES-MS m/z: 1084 [M+H]+, 1086; UV λmax 215, 295 nm.
Example 13
Preparation of Compound 48
Compound 47
Compound 47, shown below, was prepared from compound 32 (8.8 mg, 0.012 mmol) by reaction with 4-acetobenzoic acid in the presence of DCC, DMAP in CH2Cl2 as described in the Example 10. Yield 47: 7.3 mg (70%) of white solid; Rf 0.4 (CH2Cl2/MeOH, 10/1); ES-MS m/z 878.9 [M+H]+; UV λmax 215, 250 nm.
Preparation of Hydrazone 48
Compound 48 was prepared from compound 47 (6.4 mg, 0.007 mmol) and maleimidocaproylhydrazide (4 eq.) according to General Procedure F, reaction time—4 h. Yield 48: 4.3 mg (57%) of colorless glass; ES-MS m/z: 1085.7 [M+H]+; UV λmax 215, 294 nm.
Example 14
Preparation of Compound 49
Compound 49 (structure shown above) was prepared from compound 33 (8.0 mg, 0.01 mmol) by reaction with 4-acetobenzoic acid in the presence of DCC, DMAP in CH2Cl2 as described in the Example 10. Yield 49: 8.1 mg (92%) of white solid; Rf 0.37 (CH2Cl2/MeOH, 10/1); UV λmax 215, 250 nm. HRMS m/z: found 878.5653 [M+H]+. C49H76N5O9 requires 878.5643.
Example 15
Preparation of Compound 51
Preparation of 5-benzoylvaleric ester of auristatin E (50)
Compound 50 (structure shown below) was prepared from auristatin E (29a, 10 mg, 0.014 mmol) by reaction with 5-benzoylvaleric acid in the presence of DCC, DMAP in CH2Cl2 as described in the Example 10. Yield 50: 10 mg (79%) of pale yellow oil; Rf 0.34 (CH2Cl2/MeOH, 10/1); UV λmax 215 nm. HRMS m/z: found 920.6139 [M+H]+. C52H82N5O9 requires 920.6113.
Preparation of Hydrazone 51
Compound 51 was prepared from compound 50 (5 mg, 0.0054 mmol) and maleimidocaproylhydrazide (5 eq.) according to General Procedure F, reaction time—12 h. Yield 51: 3.5 mg (57%) of white solid; UV max215, 280 nm. HRMS m/z: found 1127.7132 [M+H]+. C62H95N8O11, requires 127.7120.
Example 16
Preparation of Compound 53
Preparation of acetoacetic ester of auristatin E (52)
A solution of auristatin E (29a, 10 mg, 0.014 mmol, 1 eq.), ethyl acetoacetate (9 μL, 0.07 mmol, 5 eq.), and DMAP (3.5 mg, 0.029 mmol, 2 eq.) in anhydrous toluene (5 mL) was refluxed for 10 h. The reaction mixture was concentrated under reduced pressure to dryness. The residue was re-dissolved in 2 mL of CH2Cl2 and the product was purified by silica gel flash chromatography in 10% MeOH in CH2Cl2. Yield 52 (structure shown below): 7 mg (60%) of colorless glass; Rf 0.4 (CH2Cl2/MeOH, 10/1); ES-MS m/z: 816.9 [M+H]+; UV λmax 215 nm.
Preparation of Hydrazone 53
Compound 53 was prepared from compound 52 and maleimidocaproylhydrazide (10 eq.) according to General Procedure F, reaction time—12 h. Hydrazone 53 was used for kinetic studies without isolation. UV λmax 215 nm.
Example 17
Preparation of Compound 57
Preparation of Boc-Phenylalaninol-p-acetylphenoxy ether (56)
A solution (S)-(−)-Boc-phenylalaninol (54, 0.50 g, 2.0 mmol, 1.0 eq.), 4-hydroxyacetophenone (55, 0.30 g, 2.2 mmol, 1.1 eq.), and triphenylphosphine (0.80 g, 3.0 mmol, 1.5 mmol), in anhydrous dioxane (20 mL) was cooled to 0° C. Dropwise addition of diisopropylazodicarboxylate (0.62 mL, 3.0 mmol, 1.5 eq.) was performed over a 2 min period and the reaction was monitored by reverse phase-HPLC. After 24 h, solvent was removed in vacuo and the residue purified by preparative-HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 20 mL/min). The desired fractions were concentrated to give the product as a white solid. Yield 56: 0.54 g (74%); ES-MS m/z 370.2 [M+H]+; UV λmax 215, 275 nm. 1H NMR (CDCl3) δ 7.96 (2 H, d, J=6.9 Hz), 7.18-7.35 (5H, m), 6.94 (2 H, d, J=8.4 Hz), 4.92 (1 H, bd, J=8.1 Hz), 4.14-4.28 (1 H, m), 3.91-4.02 (2 H, m), 2.99-3.04 (2 H, m), 2.58 (3 H, s), 1.45 (9H, s).
Preparation of dipeptide 57
A solution of 56 (70 mg, 0.19 mmol, 1.0 eq.) in 10 mL CH2Cl2-TFA (2:1) stood over a N2 atmosphere for 2 h. HPLC indicated a complete reaction. The reaction mixture was concentrated to an oil that was taken up in a minimum amount of dichloromethane and precipitated with hexanes. The white solid was collected and dried under high vacuum for 20 h. The free amine and t-Boc-dolaproine (20) were combined in the presence of DEPC (1.5 eq.) and triethylamine (3.0 eq.) according to General procedure A. After 24 h, solvent was removed in vacuo and the residue purified by preparative-HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 20 mL/min). The desired fractions were concentrated to give the product as a white solid. Yield 57: 52 mg (51%); ES-MS m/z 538.9 [M+H]+; UV λmax 215, 275 nm. 1H NMR (CDCl3) δ 7.92 (2 H, d, J=7.2 Hz), 7.18-7.33 (5 H, m), 6.94 (2 H, d, J=7.5 Hz), 6.69 (½ H, bd, J=7.2 Hz), 5.98 (½ H, bd, J=7.5 Hz), 4.46-4.57 (1 H, m), 3.96-4.08 (2 H, m), 3.48-3.88 (3 H, m), 3.38 (3 H, s), 2.92-3.26 (3 H, m), 2.56 (3 H, s), 2.23-2.40 (1 H, m), 1.55-1.90 (4H, m), 1.49 (4.8H, s), 1.45 (4.2H, s), 1.18 (3 H, d, J=6.9 Hz).
Example 18
Preparation of Compound 59
Preparation of compound 58
Following general procedure C, compounds 57 (25 mg, 0.046 mmol, 1 eq.) and 18a (34 mg, 0.07 mmol, 1.5 eq.) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and diisopropylethylamine. The reaction mixture was separated on silica gel column in a step gradient of MeOH from 0 to 10% in CH2Cl2. The product was eluted by 5% MeOH and after concentration was obtained as a white solid. Yield 58: 33 mg (85%); Rf 0.35 (CH2Cl2/MeOH, 10/1); ES-MS m/z 850.7 [M+H]+; UV λmax 215, 271 nm.
Preparation of Hydrazone 59
Compound 59 was prepared from compound 58 (33 mg, 0.04 mmol) and maleimidocaproylhydrazide (5 eq.) according to General Procedure F, reaction time—17 h. Yield 59: 17.3 mg (40%) of colorless glass; ES-MS m/z: 1057.9 [M+H]+; UV λmax 215, 265 nm.
Example 19
Cytotoxicity Study: Compounds 29a and 39a
In vitro cytotoxicity experiments on several cell lines were performed to compare the activities of auristatin E (29a) with the ketoester 39a. The ester proved to be cytotoxic on a wide panel of cell lines, ranging from as cytotoxic as auristatin E (29a) on L2981 cells, to being approximately 17-times less cytotoxic on Daudi Burkitt's lymphoma cells (Table 4).
TABLE 4
CELL LINES USED FOR THE EVALUATION OF MAB-
AURISTATIN E CONJUGATES, AND THE RELATIVE
CYTOTOXIC EFFECTS OF AURISTATIN E (29a) AND
THE KETOESTER DERIVATIVE 39a
Antigen
Expression (MFI)
IC50(nM)
Cell Line
Tumor Type
LeY
CD40
CD30
29a
39a
L2987
lung
620
171
7
1.0
0.9
Kato III
gastric
94
neg.
neg.
0.7
5
SKBR3
breast
2985
20
88
1.5
6
AU565
breast
869
19
57
1.5
6
Daudi
Burkitt's lymphoma
37
72
neg.
0.9
15
L428
Hodgkin's lymphoma
74
16
76
1.5
13
L540
Hodgkin's lymphoma
99
0
223
1.5
16
IM-9
Multiple myeloma
132
47
40
3.5
50
Karpas
ALCL
59
1
246
1.2
13
To obtain the results shown in Table 4, the cells were exposed to drugs for 1-2 hours, and the cytotoxic effects were determined 72 hours later by the incorporation of 3H-thymidine into DNA. Antigen expression was determined by flow cytometry and is expressed in terms of mean fluorescence intensity (MFI).
Further studies were undertaken to explore the stability of 39a in human serum, and it was shown that there was no detectable hydrolysis after 120 hours incubation at 37° C. Furthermore, prolonged exposure of 39a to purified esterases from human, rhesus monkey, guinea pig and rabbit livers failed to demonstrate any conversion of 39a to auristatin E (29a). Thus it appears that 39a may not be an auristatin E prodrug, but instead exhibits activity as a benzylic ester.
Example 20
Cytotoxicity Study: Compounds 29a, 39a, 32, 33, 47, 49, 50, 52 and 58
In vitro cytotoxicity experiments were performed to compare the activities of auristatin E (29a) and drugs of this invention. 3396 and Karpas cells were exposed to the drug for 1 and 2 hr respectively. The cells were washed, and cytotoxicity was determined 96 hr later using XTT to measure mitochondrial integrity. The results are shown in Table 5.
TABLE 5
THE RELATIVE CYTOTOXIC EFFECTS OF AURISTATIN E (29a)
AND DERIVATIVES
Tumor
IC50 (nM)
Cell Line
Type
29a
39a
32
33
47
49
50
52
58
3396
breast
6.0
16
4.5
70
0.35
34
3.0
20
2.5
Karpas 299
ALCL
1.2
4
0.075
Example 21
Structural Effects on Hydrazone Hydrolysis
In order to analyze the structural requirements for efficient hydrazone hydrolysis under mildly acidic conditions, a series of norephedrine derivatives. (Table 6) related in structure to the C-terminus of a pentapeptide of the invention were prepared.
TABLE 6
COMPOUNDS TESTED FOR HYDRAZONE HYDROLYSIS KINETICS
Compound
R =
43 (n = 2) 44 (n = 3) 45 (n = 5)
46
Condensation of N-acetylnorephedrine with various ketoacids in the presence of DCC led to the formation of the corresponding ketoesters. Upon reaction with maleimidocaproylhydrazide, hydrazones were formed. HPLC was used for stability determination at pH 5 and 7.2 at 37° C. 2-Mercaptoethanol (2 eq.) was added to quench the reactive maleimide functionalities forming compounds 43-46 in situ. The mercaptoethanol adducts 43-45 were unstable at both pH 5 and 7.2 (Table 7). In contrast, the benzylic hydrazone 46 hydrolyzed quite slowly at pH 7.2, but underwent hydrolysis at pH 5 with a half-life of 5 hours. The product formed upon hydrolysis of 46 was the parent ketoester.
TABLE 7
HYDROLYSIS OF THIOETHER-MODIFIED HYDRAZONES
OF NOREPHEDRIN AT 37° C.
Hydrazone
t1/2 at pH 5.0
t1/2 at pH 7.2
43
<2
minutes
8
hours
44
<2
minutes
2
hours
45
<2
minutes
<10
minutes
46
5
hours
60
hours
Example 22
Preparation of mAb-Drug Conjugates
A solution of monoclonal antibody (mAb) (5-10 mg/mL) in phosphate buffered saline, pH 7.2, is reduced with dithiothreitol (65 eq.) at 37° C. for 45 minutes. Separation of low molecular weight agents is achieved by size exclusion chromatography on a Sephadex G25 column, and the sulfhydryl content in the mAb is determined using 5,5′-dithiobis(2-nitrobenzoic acid) as described previously (Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) “Ellman's reagent: 5,5′-dithiobis(2-nitrobenzoic acid)-a reexamination” Anal. Biochem. 94, 75-81). There are typically between 6-9 free sulhydryl groups per mAb.
To the reduced mAb is added acetonitrile (12% final vol) followed by drug-(reactive linker) 36, 38, 40a, 40b, 48, 51 or 59 in a 1 mM solution of 9:1 acetonitrile:DMSO so that the final drug concentration is 1.9-fold higher than that of the mAb sulfhydryl groups. After 60 min at room temperature, glutathione (1 mM final concentration) and oxidized glutathione (5 mM final concentration) are added, and the solution is allowed to stand for an additional 10 min. Unbound drug is removed by repeated ultrafiltration (Amicon, YM30 filter) until there is no evidence of low molecular weight agents in the filtrate. The concentrated protein is gel filtered on Sephadex G25, a small portion of polystyrene beads is added to the pooled protein fraction, and the solution is sterile filtered using 0.4 micron filters. The protein concentration is determined at 280 nm (E0.1% 1.4).
Free drug concentration is determined by reversed-phase chromatography against standard solutions of drug-(reactive linker) standards (C18 Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 5 mM ammonium phosphate buffer, pH 7.0, from 10 to 90% in 10 min followed by 90. % MeCN for 5 min at flow rate 1 mL/min). Typically, there is less than 0.5% free drug in the purified conjugate preparations. Bound drug is quantified by hydrolyzing the linked drugs from the mAb with 1 volume of pH 1.5 aqueous HCl buffer for 15 minutes at room temperature, neutralizing the solution with borate buffer at pH 8, and analyzing drug concentration using reversed phase HPLC as indicated above. There are typically between 4-7 drugs/mAb.
Example 23
Hydrolysis of Thioether-Modified Linkers and Immunoconjugates
Synthesis of Thioethers of compounds 36, 38, 40a, 40b, 48, 51, 53 and 59
Mercaptoethanol adducts of compounds 36, 38, 40a, 40b, 48, 51, 53 and 59 were prepared in situ by a reaction of the corresponding maleimido hydrazones (0.3 mM in 10% aqueous PBS, 10% DMSO) with 2 eq. of mercaptoethanol for 15 min at room temperature. HPLC analysis (C18 Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN from 10 to 90% in 5 mM ammonium phosphate buffer, pH 7.0, in 10 min, then 90% of MeCN for 5 min at flow rate 1 mL/min) showed new peaks with retention times of 0.3-1 min less then for parent hydrazones. Reaction mixtures were directly used for hydrolysis kinetics.
Kinetics of the hydrazone bond hydrolysis for thioethers of compounds 36, 38, 40a, 40b, 48, 51, 53 and 59
Thioethers solutions (0.3 mM in 10% aqueous PBS, 10% DMSO) were adjusted to a final salt concentration of 30 mM with either 100 mM NaOAc, pH 5.0, or 100 mM Na2HPO4, pH 7.2, then incubated at 37° C. in a temperature controlled autosampler of the HPLC system. Aliquots (10 μL) were taken at timed intervals and the disappearance of starting material was monitored by HPLC at 215 nm (C18 Varian Dynamax column, 5μ, 100 Å, linear gradient from 10 to 90% of MeCN in 5 mM ammonium phosphate buffer, pH 7.0, in 10 min, then 90% of MeCN for 5 min at flow rate 1 mL/min). The results were expressed as percents of starting hydrazones. Half-lives of hydrazones were calculated from these curves and are listed in Table 8.
Immunoconjugate Hydrolysis Kinetics
Conjugates (1 mg/mL) in 10% aqueous DMSO, 50 mM buffer (either NaOAc, pH 5.0, or 50 mM Na2HPO4, pH 7.2) were incubated at 37° C. in a temperature controlled autosampler of the HPLC system. Aliquots (100 μL) were taken at timed intervals and the formation of a drug (keto-ester) was monitored by HPLC at 250 nm (C18 Varian Dynamax column, 5μ, 100 Å, linear gradient from 10 to 90% of MeCN in 5 mM ammonium phosphate buffer, pH 7.0, in 10 min, then 90% of MeCN for 5 min at flow rate 1 mL/min). Free drug was quantitated by integrating the corresponding peak (positively identified by comparison to a standard). The results were presented as percents of total drug released. Half-lives of immunoconjugates were calculated from these curves and are listed in Table 8.
TABLE 8
HYDROLYSIS OF THIOETHER-MODIFIED LINKERS
AND IMMUNOCONJUGATES AT 37° C.
Conjugate
t1/2 at pH 5.0
t1/2 at pH 7.2
Thioether of 38
4
hours
9
hours
Thioether of 40a
8
hours
110
hours
Thioether of 48
3.5
hours
>50
hours
Thioether of 51
3
hours
>40
hours
Thioether of 53
2
hours
<30
hours
Thioether of 59
4.5
hours
>55
hours
BR96-S-36
50
hours
>500
hours
BR96-S-40a
15
hours
250
hours
BR96-S-48
15
hours
>150
hours
BR96-doxorubicin
3.5
hours
>120
hours
AC10-S-48
17
hours
>350
hours
Example 24
Preparation of hBR96-S42 Conjugate
To 450 μL of PBS, pH 7.2, containing 3.0 mg hBR96 was added 50 μL of 100 mM dithiothreitol (DTT) in water. The mixture was incubated at 37° C. for 30 min. Excess DTT was removed from the reduction reaction by elution through a PD10 column (Pharmacia) with PBS containing 1 mM DTPA. The number of free thiols per antibody was determined to be 8.5, by measuring the protein concentration (A280, assuming the absorbance of a 1 mg/mL solution=1.4) and the A412 of an aliquot of the protein treated with DTNB, assuming a molar extinction coefficient of 14,150.
The pH of the 1.2 mL reduced antibody solution was raised to 8.7 by the addition of 150 μL 500 mM Na-Borate/500 mM NaCl, pH 8.7. A solution containing a 12-fold excess of compound 42 over antibody was prepared in 150 μL DMSO. The solution of compound 42 was added to the reduced antibody solution with vigorous stirring and the reaction mixture incubated at room temperature for about 3 hours. The reaction mixture was quenched by the addition of sufficient 200 mM sodium tetrathionate to make the solution 1 mM.
The quenched reaction mixture was purified by immobilization on an ion exchange matrix, rinsing with a partial organic solvent solution, elution from the matrix and buffer exchange into PBS. Thus, the quenched reaction mixture was diluted to 35 mL in 25 mM Tris, pH 9 (“Eq. Buffer”), then loaded onto an EMD TMAE column equilibrated in Eq. Buffer. The immobilized conjugate was rinsed with a mixture of 20% acetonitrile/80% Eq buffer, then eluted with 0.5 M NaCl/19 mM Tris, pH 9. Fractions were analyzed by size exclusion chromatography and pooled. The eluted conjugate was concentrated by centrifugal ultrafiltration, then eluted through a PD 10 column in PBS, producing 1.2 mL of a 2.0 mg/mL solution (based on A280). Elution of conjugate through a C18 column indicated that any unconjugated drug was below the detection level (˜0.2 μM). Treatment of the conjugate with pH 1.7 buffer for 15 min, followed by C18 HPLC analysis indicated that the drug was covalently conjugated to the antibody. Comparison of the HPLC peak area from the hydrolyzed conjugate with an standard curve of 39a determined the level of the drug attached to a given quantity of conjugate; comparison with the antibody concentration determined by A280, gave a ratio of 3.2 drug/hBR96. Treatment of H3396 cells with this conjugate inhibited cell growth with an IC50 of about 1.5 μg/mL.
Example 25
In Vitro Cytotoxicity Data For mAb-S-36
The cytotoxic effects of the mAb-S-36 conjugates on L2987 human lung adenocarcinoma cells (strongly BR96 antigen positive, weakly S2C6 antigen positive) and on Kato III human gastric carcinoma cells, (strongly BR96 antigen positive, S2C6 antigen negative) are shown in FIGS. 18A, 18B and 18C. These figures show the in vitro cytotoxicity of drugs and mAb-drug conjugates on (FIG. 18A) and (FIG. 18B) L2987 human lung adenoma cells, and (FIG. 18A) and (FIG. 18C) Kato III human gastric carcinoma cells. L2987 cells are positive for the antigens recognized by BR96 (LeY) and S2C6 (CD40), but express the LeY antigen at higher levels. Kato III cells are positive for the LeY antigen and negative for CD40. Cells were exposed to the conjugates for 2 hours, washed, and the cytotoxic effects were determined 3 days later using a thymidine incorporation assay.
Auristatin E (29a) and the ketone derivative 35 were at least 100-fold more cytotoxic than doxorubicin on L2987 and Kato III cells (FIG. 18A). On both cell lines, the BR96-S-36 conjugate displayed increased potency compared to S2C6-S-36, suggesting some degree of immunological specificity. The low potency of BR96-S-36 is most likely due to its stability at pH 5 (Table 8), suggesting that only a small portion of the conjugated drug is released under the assay conditions during a two day period.
Example 26
In Vitro Cytotoxicity Data for mAb-S40a
The cytotoxic effects of specific conjugates on several cell lines are shown in FIGS. 19A and 19B. These figures show the cytotoxic effects of auristatin E and auristatin E-containing conjugates on L2987 human lung adenocarcinoma cells (FIG. 19A); and various hematologic cell lines treated with AC10-S-40a (FIG. 19B). In all assays, cells were exposed to the drugs for 2 hours, washed, and the cytotoxic activities were measured 72 hours later using 3H-thymidine incorporation.
On L2987 cells (FIG. 19A), BR96-S-40a was significantly more cytotoxic than S2C6-S40a, reflecting increased LeY antigen expression compared to CD40 (Table 4). The effects of the AC10-S40a conjugate on several hematologic cell lines are shown in FIG. 19B. The most sensitive cells were L540 Hodgkin's lymphoma and Karpas anaplastic large cell lymphoma, both of which strongly express the CD30 antigen. The LA28 cell line, which is intermediate in CD30 antigen expression, was affected to a lesser extent by the AC10-S-40a conjugate. The effects appeared to be immunologically specific, since the antigen-negative control cell line, Daudi, was quite insensitive to the conjugate. One of the interesting findings in this study was that IM-9, a strong expresser of the CD30 antigen (Table 4), was also insensitive to the AC10-S40a conjugate. Upon further analysis, it was found that unlike the other cell lines, IM-9 did not internalize bound conjugate. Thus, these results demonstrate that activity requires not only specific binding, but also antigen internalization.
Example 27
In Vivo Activities of mAb-S40a Conjugates
Nude mice with subcutaneous L2987 human lung adenocarcinoma or 3396 human breast carcinoma xenografts were injected with conjugates or drug according to the schedule shown in FIGS. 20A and 20B. The BR96-S-40a conjugates bound to the tumor lines, while the IgG-S-40a did not. All animals were monitored daily for general health, and every 3-8 days for weight and tumor growth. Tumor volumes were estimated using the formula: (longest dimension)×dimension perpendicular2/2. There was no toxicity associated with the mAb-S-40a conjugates. The effects were compared to those of auristatin E at the maximum tolerated dose. Estradiol implants were used to sustain the growth of 3396 tumors. The implants released estradiol over a period of 60 days, at which point the experiment was terminated.
Example 28
In Vitro Cytotoxicity Data for mAb-S-40a, mAb-S-48, mAb-S-51, AND mAb-S-59
The cytotoxic effects of the conjugates on H3396 human breast carcinoma cells (strongly BR96 antigen positive) are shown in FIG. 21. To obtain this data, L2987 cells in RPMI medium were plated into 96-well plates (5,000 cells/well), and after 24 hours at 37° C., various concentrations of the conjugates in medium (0.05 mL) were added to triplicate samples. After 1 hour at 37° C., the cells were washed, and incubation was continued for an additional 24 hours, at which time the cells were washed again. The cytotoxic effects were determined after an additional 3 days using XTT as an indicator of cell viability. The data show that while all the conjugates are highly cytotoxic to H3396 cells, the BR96 conjugates prepared from compounds 48, 51 and 59 are more potent then BR96-40a.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by reference. For example, the book in Comprehensive Organic Transformations, A Guide to Functional Group Preparations, Second Edition, Richard C. Larock, John Wiley and Sons, Inc., 1999, and particularly the references cited therein, is incorporated herein by reference for all purposes.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.
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11451147
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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530/329
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Jan 26th, 2016 12:00AM
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Apr 15th, 2011 12:00AM
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https://www.uspto.gov?id=US09242013-20160126
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Targeted pyrrolobenzodiazapine conjugates
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Provided are Conjugate comprising PBDs conjugated to a targeting agent and methods of using such PBDs.
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9242013
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1. A conjugate having formula I:
L-(LU-D)p (I)
or a pharmaceutically acceptable salt thereof;
wherein L is a Ligand unit selected from an antibody, an antigen-binding fragment of an antibody or a Fc fusion protein,
LU is a Linker unit which is of formula 1a:
-A1-L1-, (1a)
wherein:
-A1- is selected from the group consisting of:
wherein the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6;
wherein the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30; and
wherein the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30;
L1 is an amino acid sequence, and is cleavable by the action of an enzyme;
p is 1 to 20; and
D is a Drug unit wherein the Drug unit is a PBD dimer having the following formula II:
wherein:
R2 is of formula III:
wherein A is a C5-7 aryl group, X is connected to the Linker unit and is selected from the group consisting of —O—, —S—, —C(O)O—, —C(O)—, —NH(C═O)—, and —N(RN)—, wherein RN is selected from the group consisting of H, C1-4 alkyl and (C2H4O)mCH3, where m is 1 to 3, and either:
(i) Q1 is a single bond, and Q2 is selected from the group consisting of a single bond and —Z—(CH2)n—, wherein Z is selected from the group consisting of a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q1 is —CH═CH—, and Q2 is a single bond;
R12 is a C5-10 aryl group optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, C1-7 alkoxy, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo;
wherein R and R′ are independently selected from the group consisting of optionally substituted C1-12 alkyl, C3-20 heterocyclyl, and C5-20 aryl groups;
either:
(a) R10 is H, and R11 is OH, ORA, wherein RA is C1-4 alkyl, or
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or
(c) R10 is H and R11 is SOzM, wherein z is 2;
R″ is a C3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms selected from the group consisting of O, S, and NH, or an aromatic ring;
Y and Y′ are selected from the group consisting of O, S, and NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively, and R10′ and R11′ are the same as R10 and R11, and each M is a monovalent pharmaceutically acceptable cation or both M groups together are a divalent pharmaceutically acceptable cation;
wherein C3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S; and
wherein C3-7 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 7 ring atoms, of which 1 to 4 are heteroatoms selected from the group consisting of N, O and S.
2. The conjugate according to claim 1, wherein R7 is selected from the group consisting of H, OH and OR.
3. The conjugate according to claim 2, wherein R7 is a C1-4 alkyloxy group.
4. The conjugate according to claim 2, wherein Y and Y′ are O.
5. The conjugate according to claim 4, wherein R″ is C3-7 alkylene.
6. The conjugate according to claim 5, wherein R9 is H.
7. The conjugate according to claim 6, wherein R6 is selected from the group consisting of H and halo.
8. The conjugate according to claim 1, wherein A is phenyl, X is selected from the group consisting of —O—, —S—, and —NH—, and Q1 is a single bond.
9. The conjugate according to claim 8, wherein X is NH.
10. The conjugate according to claim 9, wherein Q1 is a single bond and Q2 is a single bond.
11. The conjugate according to claim 1, wherein R12 is a C5-7 aryl group optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, C1-7 alkoxy, C5-20 aryloxy, C3-20 heterocyclyoxy, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene wherein the C1-7 alkoxy group is optionally substituted by an amino group, and if the C3-7 heterocyclyl group is a C6 nitrogen containing heterocyclyl group, it is optionally substituted by a C1-4 alkyl group.
12. The conjugate according to claim 11, wherein the C5-7 aryl group is an optionally substituted phenyl group.
13. The conjugate according to claim 12, wherein R12 bears one to three substituent groups.
14. The conjugate according to claim 1, wherein R10 and R11 form a nitrogen-carbon double bond.
15. The conjugate according to claim 1, wherein R6′, R7′, R9′, and Y′ are the same as R6, R7, R9, and Y respectively.
16. The conjugate of claim 1, wherein A1 is:
wherein the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6.
17. The conjugate of claim 16, wherein n is 5.
18. The conjugate of claim 1, wherein L1 is a dipeptide.
19. The conjugate of claim 18, wherein L1 is selected from the group consisting of valine-alanine, valine-citrulline and phenylalanine-lysine.
20. The conjugate of claim 1 wherein D has the formula:
wherein the wavy line of D indicates covalent attachment to LU.
21. The conjugate of claim 1 wherein LU-D has the formula:
wherein the wavy line of LU-D indicates covalent attachment to the antibody.
22. The conjugate of claim 21 wherein the Ligand unit is an antibody or antigen binding fragment thereof that specifically binds to a target antigen on the surface of a target cell.
23. The conjugate of claim 22 wherein the antibody is a monoclonal antibody.
24. The conjugate of claim 22 wherein the connection between the antibody or antigen binding fragment thereof and the Linker is formed between a thiol group of a cysteine residue of the antibody or antigen binding fragment thereof and a maleimide group of the Linker unit.
25. The conjugate of claim 24 wherein the cysteine residue is an introduced into the heavy chain or light chain of the antibody or antigen binding fragment thereof.
26. The conjugate of claim 20 wherein D has the formula:
wherein the wavy line of D indicates covalent attachment to LU.
27. The conjugate of claim 20 wherein D has the formula:
wherein the wavy line of D indicates covalent attachment to LU.
28. The conjugate of claim 26 wherein the Ligand unit is an antibody.
29. The conjugate of claim 28 wherein the connection between the antibody and the Linker unit is formed between a thiol group of a cysteine residue of the antibody and a maleimide group of the Linker unit.
30. The conjugate of claim 29 wherein the cysteine residue is an introduced cysteine residue in the heavy chain or light chain of the antibody or antigen binding fragment thereof.
31. The conjugate of claim 30 wherein the antibody is attached to the Linker unit via an introduced cysteine at amino acid heavy chain position 239, according to the EU numbering system.
32. The conjugate of claim 23 wherein the antibody is attached to the Linker unit via a thiol group of a cysteine reside of the antibody wherein the cysteine residue is an introduced cysteine at amino acid heavy chain position 239, according to the EU numbering system and a maleimide group of the Linker unit.
33. The conjugate of claim 31 wherein the antibody is a humanized 1F6 antibody.
34. The conjugate of claim 32 wherein the antibody is a humanized 1F6 antibody.
35. A pharmaceutical composition comprising a conjugate having formula I:
L-(LU-D)p (I)
or a pharmaceutically acceptable salt thereof;
wherein L is an antibody,
LU-D has the formula:
and
wherein the wavy line indicates covalent attachment to the antibody and p is 1 to 20; and a pharmaceutically acceptable excipient, carrier, buffer, or stabiliser.
36. The pharmaceutical composition of claim 35 wherein p is about 2.
37. The pharmaceutical composition of claim 36 wherein the antibody is a humanized 1F6 antibody.
38. A Drug Linker having the formula:
G1-L1-D
or a pharmaceutically acceptable salt thereof;
wherein G1 is seletected from the group consisting of:
wherein the asterisk indicates the point of attachment to NH and n is 0 to 6;
wherein the asterisk indicates the point of attachment to NH, n is 0 or 1, and m is 0 to 30;
wherein the asterisk indicates the point of attachment to NH, n is 0 or 1, and m is 0 to 30;
L1 is an amino acid sequence, and is cleavable by the action of an enzyme;
and D is a Drug unit wherein the Drug unit is a PBD dimer having the following formula II:
wherein:
R2 is of formula III:
wherein A is a C5-7 aryl group, X is connected to the Linker unit and is selected from the group consisting of —O—, —S—, —C(O)O—, —C(O)—, —NH(C═O)—, and —N(RN)—, wherein RN is selected from the group consisting of H, C1-4 alkyl and (C2H4O)mCH3, where m is 1 to 3, and either:
(i) Q1 is a single bond, and Q2 is selected from the group consisting of a single bond and —Z—(CH2)n—, wherein Z is selected from the group consisting of a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q1 is —CH═CH—, and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group consisting of halo, nitro, cyano, C1-7 alkoxy, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
R7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo;
wherein R and R′ are independently selected from the group consisting of optionally substituted C1-12 alkyl, C3-20 heterocyclyl, and C5-20 aryl groups;
either:
(a) R10 is H, and R11 is OH, ORA, wherein RA is C1-4 alkyl, or
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or
(c) R10 is H and R11 is SOzM, wherein z is 2;
R″ is a C3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms, selected from the group consisting of O, S, and NH, or an aromatic ring;
Y and Y′ are selected from the group consisting of O, S, and NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively, and R10′ and R11′ are the same as R10 and R11, and each M is a monovalent pharmaceutically acceptable cation or both M groups together are a divalent pharmaceutically acceptable cation;
wherein C3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S; and
wherein C3-7 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom of a heterocyclic compound which has 3 to 7 ring atoms, of which 1 to 4 are heteroatoms selected from the group consisting of N, O and S.
39. The Drug Linker of claim 38 having the formula:
or a pharmaceutically acceptable salt thereof.
40. A method of treating a mammal having a proliferative disease wherein the cell surface antigen bound by the Ligand unit is expressed by proliferative cells of the proliferative disease comprising administering an effective amount of the conjugate of claim 1, wherein the proliferative disease treated is selected from the group consisting of renal cell carcinoma, Hodgkin Lymphoma, anaplastic large cell lymphoma and acute myeloid leukemia.
41. The Drug Linker of claim 38 having the formula:
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41
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CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2011/032664, filed on Apr. 15, 2011, which claims priority to U.S. Provisional Patent Application No. 61/324,623, filed on Apr. 15, 2010. These applications are incorporated herein by reference in their entireties.
The present invention relates to targeted pyrrolobenzodiazepine (PBD) conjugates, in particular pyrrolobenzodiazepine dimers that are conjugated to a targeting agent via the C2 position of one of the monomers.
BACKGROUND TO THE INVENTION
Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102) (Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:
They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position, which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). The ability of PBDs to form an adduct in the minor groove enables them to interfere with DNA processing, hence their use as antitumour agents.
The biological activity of these molecules can be potentiated by joining two PBD units together through their C8/C′-hydroxyl functionalities via a flexible alkylene linker (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity. One example of a PBD dimer is SG2000 (SJG-136):
(Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C., et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al., Cancer Research, 64, 6693-6699 (2004)).
Due to the manner in which these highly potent compounds act in cross-linking DNA, PBD dimers have been made symmetrically, i.e., both monomers of the dimer are the same. This synthetic route provides for straightforward synthesis, either by constructing the PBD dimer moiety simultaneously having already formed the dimer linkage, or by reacting already constructed PBD monomer moieties with the dimer linking group. These synthetic approaches have limited the options for preparation of targeted conjugates containing PBDs. Due to the observed potency of PBD dimers, however, there exists a need for PBD dimers that are conjugatable to targeting agents for use in targeted therapy.
DISCLOSURE OF THE INVENTION
The present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein a PBD monomer has a substituent in the C2 position that provides an anchor for linking the compound to the targeting agent. The present invention also relates to Conjugates comprising dimers of PBDs conjugated to a targeting agent, wherein the PBD monomers of the dimer are different. One of PBD monomers has a substituent in the C2 position that provides an anchor for linking the compound to the targeting agent. The Conjugates described herein have potent cytotoxic and/or cytostatic activity against cells expressing a target molecule, such as cancer cells or immune cells. These conjugates exhibit good potency with reduced toxicity, as compared with the corresponding PBD dimer free drug compounds.
In some embodiments, the Conjugates have the following formula I:
L-(LU-D)p (I)
wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linker unit and D is a Drug unit comprising a PBD dimer. The subscript p is an integer of from 1 to 20. Accordingly, the Conjugates comprise a Ligand unit covalently linked to at least one Drug unit by a Linker unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. Accordingly, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease. These methods encompass the use of the Conjugates wherein the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.
In a first aspect, the Conjugates comprise a Conjugate of formula I (supra), wherein the Drug unit comprises a PBD dimer of the following formula II:
wherein:
R2 is of formula III:
where A is a C5-7 aryl group, X is an activatable group for conjugation to the Linker unit, wherein X is selected from the group comprising: —O—, —S—, —C(O)O—, —C(O)—, —NHC(O)—, and —N(RN)—, wherein RN is selected from the group comprising H, C1-4 alkyl and (C2H4O)mCH3, where m is 1 to 3, and either:
(i) Q1 is a single bond, and Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q1 is —CH═CH—, and Q2 is a single bond;
R12 is a C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene;
R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, optionally substituted C3-20 heterocyclyl and optionally substituted C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo; either:
(a) R10 is H, and R11 is OH or ORA, where RA is C1-4 alkyl;
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or
(c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y′ are selected from O, S, or NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11, wherein if R11 and R11′ are SOzM, M may represent a divalent pharmaceutically acceptable cation.
In a second aspect, the use of the Conjugate of formula I is provided for the manufacture of a medicament for treating a proliferative disease or autoimmune disease. In a related third aspect, the use of the Conjugate of formula I is provided for the treatment of a proliferative disease or an autoimmune disease.
In another aspect there is provided the use of a Conjugate of formula I to provide a PBD dimer, or a salt or solvate thereof, at a target location.
One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.
The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.
In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.
In a fourth aspect of the present invention comprises a method of making the Conjugates formula I.
The dimeric PBD compounds for use in the present invention are made by different strategies to those previously employed in making symmetrical dimeric PBD compounds. In particular, the present inventors have developed a method which involves adding each C2 aryl substituent to a symmetrical PBD dimer core in separate method steps. Accordingly, a sixth aspect of the present invention provides a method of making a Conjugate of formula I, comprising at least one of the method steps described herein.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1 to 6 show the effect of conjugates of the present invention in tumours.
DEFINITIONS
When a trade name is used herein, reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.
Binding Agent and Targeting Agent
The terms “binding agent” and “targeting agent as used herein refer to a molecule, e.g., protein, polypeptide or peptide, that specifically binds to a target molecule. Examples can include a full length antibody, an antigen binding fragment of a full length antibody, other agent (protein, polypeptide or peptide) that includes an antibody heavy and/or light chain variable region that specifically bind to the target molecule, or an Fc fusion protein comprising an extracellular domain of a protein, peptide polypeptide that binds to the target molecule and that is joined to an Fc region, domain or portion thereof, of an antibody.
Specifically Binds
The terms “specifically binds” and “specific binding” refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1×107 M−1, and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
Pharmaceutically Acceptable Cations
Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.
The pharmaceutically acceptable cation may be inorganic or organic.
Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca2+ and Mg2+. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
Substituents
The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.
Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
Examples of substituents are described in more detail below.
C1-12 alkyl: The term “C1-12 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C7).
Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7).
Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).
C2-12 Alkenyl: The term “C2-12 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH═CH2), 1-propenyl (—CH═CH—CH3), 2-propenyl (allyl, —CH—CH═CH2), isopropenyl (1-methylvinyl, —C(CH3)═CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).
C2-12 alkynyl: The term “C2-12 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH2—C≡CH).
C3-12 cycloalkyl: The term “C3-12 cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
Examples of cycloalkyl groups include, but are not limited to, those derived from:
saturated monocyclic hydrocarbon compounds:
cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7);
unsaturated monocyclic hydrocarbon compounds:
cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and
saturated polycyclic hydrocarbon compounds:
norcarane (C7), norpinane (C7), norbornane (C7).
C3-20 heterocyclyl: The term “C3-20 heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
In this context, the prefixes (e.g. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:
N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);
S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3: trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);
N2O1: oxadiazine (C6);
O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1O1S1: oxathiazine (C6).
Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
C5-20 aryl: The term “C5-20 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.
In this context, the prefixes (e.g. C3-20, C5-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6 aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.
The ring atoms may be all carbon atoms, as in “carboaryl groups”.
Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).
Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (C16).
Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:
N1: pyrrole (azole) (C5), pyridine (azine) (C6);
O1: furan (oxole) (C5);
S1: thiophene (thiole) (C5);
N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);
N2O1: oxadiazole (furazan) (C5);
N3O1: oxatriazole (C5);
N1S1: thiazole (C5), isothiazole (C5);
N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6);
N3: triazole (C5), triazine (C6); and,
N4: tetrazole (C5).
Examples of heteroaryl which comprise fused rings, include, but are not limited to:
C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);
C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);
C11 (with 2 fused rings) derived from benzodiazepine (N2);
C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and,
C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxathiin (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.
Halo: —F, —Cl, —Br, and —I.
Hydroxy: —OH.
Ether: —OR, wherein R is an ether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7alkyl group.
Alkoxy: —OR, wherein R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).
Acetal: —CH(OR1)(OR2), wherein R1 and R2 are independently acetal substituents, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, or, in the case of a “cyclic” acetal group, R1 and R2, taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe)2, —CH(OEt)2, and —CH(OMe)(OEt).
Hemiacetal: —CH(OH)(OR1), wherein R1 is a hemiacetal substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).
Ketal: —CR(OR1)(OR2), where R1 and R2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe)2, —C(Me)(OEt)2, —C(Me)(OMe)(OEt), —C(Et)(OMe)2, —C(Et)(OEt)2, and —C(Et)(OMe)(OEt).
Hemiketal: —CR(OH)(OR1), where R1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).
Oxo (keto, -one): ═O.
Thione (thioketone): ═S.
Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.
Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.
Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C1-7 alkyl group (also referred to as C1-7alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH3 (acetyl), —C(═O)CH2CH3 (propionyl), —C(═O)C(CH3)3 (t-butyryl), and —C(═O)Ph (benzoyl, phenone).
Carboxy (carboxylic acid): —C(═O)OH.
Thiocarboxy (thiocarboxylic acid): —C(═S)SH.
Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.
Thionocarboxy (thionocarboxylic acid): —C(═S)OH.
Imidic acid: —C(═NH)OH.
Hydroxamic acid: —C(═NOH)OH.
Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH3, —C(═O)OCH2CH3, —C(═O)OC(CH3)3, and —C(═O)OPh.
Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH3 (acetoxy), —OC(═O)CH2CH3, —OC(═O)C(CH3)3, —OC(═O)Ph, and —OC(═O)CH2Ph.
Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH3, —OC(═O)OCH2CH3, —OC(═O)OC(CH3)3, and —OC(═O)OPh.
Amino: —NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a “cyclic” amino group, R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH2), secondary (—NHR1), or tertiary (—NHR1R2), and in cationic form, may be quaternary (—+NR1R2R3). Examples of amino groups include, but are not limited to, —NH2, —NHCH3, —NHC(CH3)2, —N(CH3)2, —N(CH2CH3)2, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH2, —C(═O)NHCH3, —C(═O)N(CH3)2, —C(═O)NHCH2CH3, and —C(═O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
Thioamido (thiocarbamyl): —C(═S)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH2, —C(═S)NHCH3, —C(═S)N(CH3)2, and —C(═S)NHCH2CH3.
Acylamido (acylamino): —NR1C(═O)R2, wherein R1 is an amide substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group, and R2 is an acyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH3, —NHC(═O)CH2CH3, and —NHC(═O)Ph. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
Aminocarbonyloxy: —OC(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH2, —OC(═O)NHMe, —OC(═O)NMe2, and —OC(═O)NEt2.
Ureido: —N(R1)CONR2R3 wherein R2 and R3 are independently amino substituents, as defined for amino groups, and R1 is a ureido substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH2, —NHCONHMe, —NHCONHEt, —NHCONMe2, —NHCONEt2, —NMeCONH2, —NMeCONHMe, —NMeCONHEt, —NMeCONMe2, and —NMeCONEt2.
Guanidino: —NH—C(═NH)NH2.
Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,
Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt.
Amidine (amidino): —C(═NR)NR2, wherein each R is an amidine substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH2, —C(═NH)NMe2, and —C(═NMe)NMe2.
Nitro: —NO2.
Nitroso: —NO.
Azido: —N3.
Cyano (nitrile, carbonitrile): —CN.
Isocyano: —NC.
Cyanato: —OCN.
Isocyanato: —NCO.
Thiocyano (thiocyanato): —SCN.
Isothiocyano (isothiocyanato): —NCS.
Sulfhydryl (thiol, mercapto): —SH.
Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7alkylthio group), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of C1-7 alkylthio groups include, but are not limited to, —SCH3 and —SCH2CH3.
Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group (also referred to herein as C1-7 alkyl disulfide). Examples of C1-7 alkyl disulfide groups include, but are not limited to, —SSCH3 and —SSCH2CH3.
Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH3 and —S(═O)CH2CH3.
Sulfone (sulfonyl): —S(═O)2R, wherein R is a sulfone substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, including, for example, a fluorinated or perfluorinated C1-7 alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)2CH3 (methanesulfonyl, mesyl), —S(═O)2CF3 (triflyl), —S(═O)2CH2CH3 (esyl), —S(═O)2C4F9 (nonaflyl), —S(═O)2CH2CF3 (tresyl), —S(═O)2CH2CH2NH2 (tauryl), —S(═O)2Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).
Sulfinic acid (sulfino): —S(═O)OH, —SO2H.
Sulfonic acid (sulfo): —S(═O)2OH, —SO3H.
Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH3 (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).
Sulfonate (sulfonic acid ester): —S(═O)2OR, wherein R is a sulfonate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)2OCH3 (methoxysulfonyl; methyl sulfonate) and —S(═O)2OCH2CH3 (ethoxysulfonyl; ethyl sulfonate).
Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH3 and —OS(═O)CH2CH3.
Sulfonyloxy: —OS(═O)2R, wherein R is a sulfonyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)2CH3 (mesylate) and —OS(═O)2CH2CH3 (esylate).
Sulfate: —OS(═O)2OR; wherein R is a sulfate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)2OCH3 and —SO(═O)2OCH2CH3.
Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH2, —S(═O)NH(CH3), —S(═O)N(CH3)2, —S(═O)NH(CH2CH3), —S(═O)N(CH2CH3)2, and —S(═O)NHPh.
Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)2NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O)2NH2, —S(═O)2NH(CH3), —S(═O)2N(CH3)2, —S(═O)2NH(CH2CH3), —S(═O)2N(CH2CH3)2, and —S(═O)2NHPh.
Sulfamino: —NR1S(═O)2OH, wherein R1 is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)2OH and —N(CH3)S(═O)2OH.
Sulfonamino: —NR1S(═O)2R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)2CH3 and —N(CH3)S(═O)2C6H5.
Sulfinamino: —NR1S(═O)R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH3 and —N(CH3)S(═O)C6H5.
Phosphino (phosphine): —PR2, wherein R is a phosphino substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphino groups include, but are not limited to, —PH2, —P(CH3)2, —P(CH2CH3)2, —P(t-Bu)2, and —P(Ph)2.
Phospho: —P(═O)2.
Phosphinyl (phosphine oxide): —P(═O)R2, wherein R is a phosphinyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group or a C5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH3)2, —P(═O)(CH2CH3)2, —P(═O)(t-Bu)2, and —P(═O)(Ph)2.
Phosphonic acid (phosphono): —P(═O)(OH)2.
Phosphonate (phosphono ester): —P(═O)(OR)2, where R is a phosphonate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH3)2, —P(═O)(OCH2CH3)2, —P(═O)(O-t-Bu)2, and —P(═O)(OPh)2.
Phosphoric acid (phosphonooxy): —OP(═O)(OH)2.
Phosphate (phosphonooxy ester): —OP(═O)(OR)2, where R is a phosphate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH3)2, —OP(═O)(OCH2CH3)2, —OP(═O)(O-t-Bu)2, and —OP(═O)(OPh)2.
Phosphorous acid: —OP(OH)2.
Phosphite: —OP(OR)2, where R is a phosphite substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH3)2, —OP(OCH2CH3)2, —OP(O-t-Bu)2, and —OP(OPh)2.
Phosphoramidite: —OP(OR1)—NR22, where R1 and R2 are phosphoramidite substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH2CH3)—N(CH3)2, —OP(OCH2CH3)—N(i-Pr)2, and —OP(OCH2CH2CN)—N(i-Pr)2.
Phosphoramidate: —OP(═O)(OR1)—NR22, where R1 and R2 are phosphoramidate substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH2CH3)—N(CH3)2, —OP(═O)(OCH2CH3)—N(i-Pr)2, and —OP(═O)(OCH2CH2CN)—N(i-Pr)2.
Alkylene
C3-12 alkylene: The term “C3-12 alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
Examples of linear saturated C3-12 alkylene groups include, but are not limited to, —(CH2)n— where n is an integer from 3 to 12, for example, —CH2CH2CH2— (propylene), —CH2CH2CH2CH2— (butylene), —CH2CH2CH2CH2CH2— (pentylene) and —CH2CH2CH2CH—2CH2CH2CH2— (heptylene).
Examples of branched saturated C3-12 alkylene groups include, but are not limited to, —CH(CH3)CH2—, —CH(CH3)CH2CH2—, —CH(CH3)CH2CH2CH2—, —CH2CH(CH3)CH2—, —CH2CH(CH3)CH2CH2—, —CH(CH2CH3)—, —CH(CH2CH3)CH2—, and —CH2CH(CH2CH3)CH2—.
Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH2—, —CH2—CH═CH2—, —CH═CH—CH2—CH2—, —CH═CH—CH2—CH2—CH2—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH2—, —CH═CH—CH═CH—CH2—CH2—, —CH═CH—CH2—CH═CH—, —CH═CH—CH2—CH2—CH═CH—, and —CH2—, C≡C—CH2—.
Examples of branched partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and alkynylene groups) include, but are not limited to, —C(CH3)═CH—, —C(CH3)═CH—CH2—, —CH═CH—CH(CH3)— and —C≡C—CH(CH3)—.
Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).
Examples of alicyclic partially unsaturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).
Oxygen protecting group: the term “oxygen protecting group” refers to a moiety which masks a hydroxy group, and these are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. Classes of particular interest include silyl ethers (e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g. acetate).
Carbamate nitrogen protecting group: the term “carbamate nitrogen protecting group” pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogen protecting group” pertains to a group having the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides Conjugates comprising a PBD dimer connected to a Ligand unit via a Linker Unit. In one embodiment, the Linker unit includes a Stretcher unit (A), a Specificity unit (L1), and a Spacer unit (L2). The Linker unit is connected at one end to the Ligand unit and at the other end to the PBD dimer compound.
In one aspect, such a Conjugate is shown below in formula Ia:
L-(A1a-L1s-L2y-D)p (Ia)
wherein:
L is the Ligand unit; and
-A1a-L1s-L2y- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit,
a is 1 or 2,
L1- is a Specificity unit,
s is an integer ranging from 1 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is an PBD dimer; and
p is from 1 to 20.
The drug loading is represented by p, the number of drug molecules per Ligand unit (e.g., an antibody). Drug loading may range from 1 to 20 Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, p represents the average drug loading of the Conjugates in the composition, and p ranges from 1 to 20.
In some embodiments, p is from about 1 to about 8 Drug units per Ligand unit. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is from about 2 to about 8 Drug units per Ligand unit. In some embodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4 Drug units per Ligand unit. In some embodiments, p is about 2, about 4, about 6 or about 8 Drug units per Ligand unit.
The average number of Drugs units per Ligand unit in a preparation from a conjugation reaction may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Conjugates, where p is a certain value, from Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
In another aspect, such a Conjugate is shown below in formula Ib:
Also illustrated as:
L-(A1a-L2y(-L1s)-D)p (Ib)
wherein:
L is the Ligand unit; and
-A1a-L1s(L2y)- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit linked to a Stretcher unit (L2),
a is 1 or 2,
L1- is a Specificity unit linked to a Stretcher unit (L2),
s is an integer ranging from 0 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is a PBD dimer; and
p is from 1 to 20.
Preferences
The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
In one embodiment, the Conjugate has the formula:
L-(A1a-L1s-L2y-D)p
wherein L, A1, a, L1, s, L2, D and p are as described above.
In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit, which is a covalent bond, a self-immolative group or together with —OC(═O)— forms a self-immolative group, and L2 optional.
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
CBA-A1a-L1s-L2y-*
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit which is a covalent bond or a self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is 0 or 1 or 2.
In the embodiments illustrated above, L1 can be a cleavable Specificity unit, and may be referred to as a “trigger” that when cleaved activates a self-immolative group (or self-immolative groups) L2, when a self-immolative group(s) is present. When the Specificity unit L1 is cleaved, or the linkage (i.e., the covalent bond) between L1 and L2 is cleaved, the self-immolative group releases the Drug unit (D).
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug (D), CBA is the Cell Binding Agent, L1 is a Specificity unit connected to L2, A1 is a Stretcher unit connecting L2 to the Cell Binding Agent, L2 is a self-immolative group, and a is 1 or 2, s is 1 or 2, and y is 1 or 2.
In the various embodiments discussed herein, the nature of L1 and L2 can vary widely. These groups are chosen on the basis of their characteristics, which may be dictated in part, by the conditions at the site to which the conjugate is delivered. Where the Specificity unit L1 is cleavable, the structure and/or sequence of L1 is selected such that it is cleaved by the action of enzymes present at the target site (e.g., the target cell). L1 units that are cleavable by changes in pH (e.g. acid or base labile), temperature or upon irradiation (e.g. photolabile) may also be used. L1 units that are cleavable under reducing or oxidising conditions may also find use in the Conjugates.
In some embodiments, L1 may comprise one amino acid or a contiguous sequence of amino acids. The amino acid sequence may be the target substrate for an enzyme.
In one embodiment, L1 is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L1 may be cleaved by a lysosomal protease, such as a cathepsin.
In one embodiment, L2 is present and together with —C(═O)O— forms a self-immolative group or self-immolative groups. In some embodiments, —C(═O)O— also is a self-immolative group.
In one embodiment, where L1 is cleavable by the action of an enzyme and L2 is present, the enzyme cleaves the bond between L1 and L2, whereby the self-immolative group(s) release the Drug unit.
L1 and L2, where present, may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH, and
—O— (a glycosidic bond).
An amino group of L1 that connects to L2 may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain.
A carboxyl group of L1 that connects to L2 may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain.
A hydroxy group of L1 that connects to L2 may be derived from a hydroxy group of an amino acid side chain, for example a serine amino acid side chain.
In one embodiment, —C(═O)O— and L2 together form the group:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to the L1, Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The phenylene ring is optionally substituted with one, two or three substituents as described herein.
In one embodiment, Y is NH.
In one embodiment, n is 0 or 1. Preferably, n is 0.
Where Y is NH and n is 0, the self-immolative group may be referred to as a p-aminobenzylcarbonyl linker (PABC).
The self-immolative group will allow for release of the Drug unit (i.e., the asymmetric PBD) when a remote site in the linker is activated, proceeding along the lines shown below (for n=0):
where the asterisk indicates the attachment to the Drug, L* is the activated form of the remaining portion of the linker and the released Drug unit is not shown. These groups have the advantage of separating the site of activation from the Drug.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above. Each phenylene ring is optionally substituted with one, two or three substituents as described herein. In one embodiment, the phenylene ring having the Y substituent is optionally substituted and the phenylene ring not having the Y substituent is unsubstituted.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or CR.
In one embodiment, D is N.
In one embodiment, D is CH.
In one embodiment, E is O or S.
In one embodiment, F is CH.
In a preferred embodiment, the covalent bond between L1 and L2 is a cathepsin labile (e.g., cleavable) bond.
In one embodiment, L1 comprises a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is -Phe-Lys-, Val-Cit or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described by Dubowchik et al., which is incorporated herein by reference.
In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective groups in Organic Synthesis, Greene and Wuts.
Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:
Arg: Z, Mtr, Tos;
Asn: Trt, Xan;
Asp: Bzl, t-Bu;
Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;
Glu: Bzl, t-Bu;
Gln: Trt, Xan;
His: Boc, Dnp, Tos, Trt;
Lys: Boc, Z—Cl, Fmoc, Z;
Ser: Bzl, TBDMS, TBDPS;
Thr: Bz;
Trp: Boc;
Tyr: Bzl, Z, Z—Br.
In one embodiment, —X2— is connected indirectly to the Drug unit. In such an embodiment, the Spacer unit L2 is present.
In one embodiment, the dipeptide is used in combination with a self-immolative group(s) (the Spacer unit). The self-immolative group(s) may be connected to —X2—.
Where a self-immolative group is present, —X2— is connected directly to the self-immolative group. In one embodiment, —X2— is connected to the group Y of the self-immolative group. Preferably the group —X2—CO— is connected to Y, where Y is NH.
—X1— is connected directly to A1. In one embodiment, —X1— is connected directly to A1. Preferably the group NH—X1— (the amino terminus of X1) is connected to A1. A1 may comprise the functionality —CO— thereby to form an amide link with —X1—.
In one embodiment, L1 and L2 together with —OC(═O)— comprise the group —X1—X2—PABC-. The PABC group is connected directly to the Drug unit. In one example, the self-immolative group and the dipeptide together form the group -Phe-Lys-PABC-, which is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
Alternatively, the self-immolative group and the dipeptide together form the group -Val-Ala-PABC-, which is illustrated below:
where the asterisk and the wavy line are as defined above.
In another embodiment, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group, and E is a group that is susceptible to cleavage thereby to activate a self-immolative group.
E is selected such that the group is susceptible to cleavage, e.g., by light or by the action of an enzyme. E may be —NO2 or glucuronic acid (e.g., β-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a β-glucuronidase.
The group Y may be a covalent bond.
The group Y may be a functional group selected from:
—C(═O)—
—NH—
—O—
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH,
—C(═O)NHC(═O)—,
SO2, and
—S—.
The group Y is preferably —NH—, —CH2—, —O—, and —S—.
In some embodiments, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—.
In some embodiments, L1 and L2 together represent:
where the asterisk indicates the point of attachment to the remainder of L2 or the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—, —CH2—, —O—, and —S—.
In some further embodiments, Y is a functional group as set forth above, the functional group is linked to an amino acid, and the amino acid is linked to the Stretcher unit A1. In some embodiments, amino acid is β-alanine. In such an embodiment, the amino acid is equivalently considered part of the Stretcher unit.
The Specificity unit L1 and the Ligand unit are indirectly connected via the Stretcher unit.
L1 and A1 may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the connection between the Ligand unit and A1 is through a thiol residue of the Ligand unit and a maleimide group of A1.
In one embodiment, the connection between the Ligand unit and A1 is:
where the asterisk indicates the point of attachment to the remaining portion of A1, L1, L2 or D, and the wavy line indicates the point of attachment to the remaining portion of the Ligand unit. In this embodiment, the S atom is typically derived from the Ligand unit.
In each of the embodiments above, an alternative functionality may be used in place of the malemide-derived group shown below:
where the wavy line indicates the point of attachment to the Ligand unit as before, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with the group:
where the wavy line indicates point of attachment to the Ligand unit, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with a Ligand unit (e.g., a Cell Binding Agent), is selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH,
—C(═O)NHC(═O)—,
—S—,
—S—S—,
—CH2C(═O)—
—C(═O)CH2—,
═N—NH—, and
—NH—N═.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the Ligand unit, is selected from:
where the wavy line indicates either the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group, and the asterisk indicates the other of the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group.
Other groups suitable for connecting L1 to the Cell Binding Agent are described in WO 2005/082023.
In one embodiment, the Stretcher unit A1 is present, the Specificity unit L1 is present and Spacer unit L2 is absent. Thus, L1 and the Drug unit are directly connected via a bond. Equivalently in this embodiment, L2 is a bond.
L1 and D may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, L1 and D are preferably connected by a bond selected from:
—C(═O)NH—, and
—NHC(═O)—.
In one embodiment, L1 comprises a dipeptide and one end of the dipeptide is linked to D. As described above, the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is -Phe-Lys- or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described above.
In one embodiment, L1-D is:
where —NH—X1—X2—CO is the dipeptide, —NH— is part of the Drug unit, the asterisk indicates the point of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
In one embodiment, the dipeptide is valine-alanine and L1-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is phenylalanine-lysine and L1-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is valine-citrulline.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3 or 7.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 is:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the rest of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulphur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4 or 8. In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the Stretcher unit is an acetamide unit, having the formula:
where the asterisk indicates the point of attachment to the remainder of the Stretcher unit, L1 or D, and the wavy line indicates the point of attachment to the Ligand unit.
In other embodiments, Linker-Drug compounds are provided for conjugation to a Ligand unit. In one embodiment, the Linker-Drug compounds are designed for connection to a Cell Binding Agent.
In one embodiment, the Drug Linker compound has the formula:
where the asterisk indicates the point of attachment to the Drug unit, G1 is a Stretcher group (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or together with —OC(═O)— forms a self-immolative group(s).
In another embodiment, the Drug Linker compound has the formula:
G1-L1-L2-*
where the asterisk indicates the point of attachment to the Drug unit, G1 is a Stretcher unit (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or a self-immolative group(s).
L1 and L2 are as defined above. References to connection to A1 can be construed here as referring to a connection to G1.
In one embodiment, where L1 comprises an amino acid, the side chain of that amino acid may be protected. Any suitable protecting group may be used. In one embodiment, the side chain protecting groups are removable with other protecting groups in the compound, where present. In other embodiments, the protecting groups may be orthogonal to other protecting groups in the molecule, where present.
Suitable protecting groups for amino acid side chains include those groups described in the Novabiochem Catalog 2006/2007. Protecting groups for use in a cathepsin labile linker are also discussed in Dubowchik et al.
In certain embodiments of the invention, the group L1 includes a Lys amino acid residue. The side chain of this amino acid may be protected with a Boc or Alloc protected group. A Boc protecting group is most preferred.
The functional group G1 forms a connecting group upon reaction with a Ligand unit (e.g., a cell binding agent.
In one embodiment, the functional group G1 is or comprises an amino, carboxylic acid, hydroxy, thiol, or maleimide group for reaction with an appropriate group on the Ligand unit. In a preferred embodiment, G1 comprises a maleimide group.
In one embodiment, the group G1 is an alkyl maleimide group. This group is suitable for reaction with thiol groups, particularly cysteine thiol groups, present in the cell binding agent, for example present in an antibody.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In each of the embodiments above, an alternative functionality may be used in place of the malemide group shown below:
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide-derived group is replaced with the group:
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide group is replaced with a group selected from:
—C(═O)OH,
—OH,
—NH2,
—SH,
—C(═O)CH2X, where X is Cl, Br or I,
—CHO,
—NHNH2
—C≡CH, and
—N3 (azide).
In one embodiment, L1 is present, and G1 is —NH2, —NHMe, —COOH, —OH or —SH.
In one embodiment, where L1 is present, G1 is —NH2 or —NHMe. Either group may be the N-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is —NH2, and L is an amino acid sequence —X1—X2—, as defined above.
In one embodiment, L1 is present and G1 is COOH. This group may be the C-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is OH.
In one embodiment, L1 is present and G1 is SH.
The group G1 may be convertable from one functional group to another. In one embodiment, L1 is present and G1 is —NH2. This group is convertable to another group G1 comprising a maleimide group. For example, the group —NH2 may be reacted with an acids or an activated acid (e.g., N-succinimide forms) of those G1 groups comprising maleimide shown above.
The group G1 may therefore be converted to a functional group that is more appropriate for reaction with a Ligand unit.
As noted above, in one embodiment, L1 is present and G1 is —NH2, —NHMe, —COOH, —OH or —SH. In a further embodiment, these groups are provided in a chemically protected form. The chemically protected form is therefore a precursor to the linker that is provided with a functional group.
In one embodiment, G1 is —NH2 in a chemically protected form. The group may be protected with a carbamate protecting group. The carbamate protecting group may be selected from the group consisting of:
Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.
Preferably, where G1 is —NH2, it is protected with an Alloc or Fmoc group.
In one embodiment, where G1 is —NH2, it is protected with an Fmoc group.
In one embodiment, the protecting group is the same as the carbamate protecting group of the capping group.
In one embodiment, the protecting group is not the same as the carbamate protecting group of the capping group. In this embodiment, it is preferred that the protecting group is removable under conditions that do not remove the carbamate protecting group of the capping group.
The chemical protecting group may be removed to provide a functional group to form a connection to a Ligand unit. Optionally, this functional group may then be converted to another functional group as described above.
In one embodiment, the active group is an amine. This amine is preferably the N-terminal amine of a peptide, and may be the N-terminal amine of the preferred dipeptides of the invention.
The active group may be reacted to yield the functional group that is intended to form a connection to a Ligand unit.
In other embodiments, the Linker unit is a precursor to the Linker unit having an active group. In this embodiment, the Linker unit comprises the active group, which is protected by way of a protecting group. The protecting group may be removed to provide the Linker unit having an active group.
Where the active group is an amine, the protecting group may be an amine protecting group, such as those described in Green and Wuts.
The protecting group is preferably orthogonal to other protecting groups, where present, in the Linker unit.
In one embodiment, the protecting group is orthogonal to the capping group. Thus, the active group protecting group is removable whilst retaining the capping group. In other embodiments, the protecting group and the capping group is removable under the same conditions as those used to remove the capping group.
In one embodiment, the Linker unit is:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of the Linker unit, as applicable or the point of attachment to G1. Preferably, the wavy line indicates the point of attachment to G1.
In one embodiment, the Linker unit is:
where the asterisk and the wavy line are as defined above.
Other functional groups suitable for use in forming a connection between L1 and the Cell Binding Agent are described in WO 2005/082023.
Ligand Unit
The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non-peptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.
Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).
In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; fully human antibody; or a single chain antibody. One embodiment the antibody is a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab′, F(ab′)2 and Fv fragments.
The antibody may be a diabody, a domain antibody (DAB) or a single chain antibody.
In one embodiment, the antibody is a monoclonal antibody.
Antibodies for use in the present invention include those antibodies described in WO 2005/082023 which is incorporated herein. Particularly preferred are those antibodies for tumour-associated antigens. Examples of those antigens known in the art include, but are not limited to, those tumour-associated antigens set out in WO 2005/082023. See, for instance, pages 41-55.
In some embodiments, the conjugates are designed to target tumour cells via their cell surface antigens. The antigens may be cell surface antigens which are either over-expressed or expressed at abnormal times or cell types. Preferably, the target antigen is expressed only on proliferative cells (preferably tumour cells); however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue.
Antibodies have been raised to target specific tumour related antigens including:
Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2.
The Ligand unit is connected to the Linker unit. In one embodiment, the Ligand unit is connected to A, where present, of the Linker unit.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a thioether bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a disulfide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an amide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an ester bond.
In one embodiment, the connection between the Ligand unit and the Linker is formed between a thiol group of a cysteine residue of the Ligand unit and a maleimide group of the Linker unit.
The cysteine residues of the Ligand unit may be available for reaction with the functional group of the Linker unit to form a connection. In other embodiments, for example where the Ligand unit is an antibody, the thiol groups of the antibody may participate in interchain disulfide bonds. These interchain bonds may be converted to free thiol groups by e.g. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit.
In some embodiments, the cysteine residue is an introduced into the heavy or light chain of an antibody. Positions for cysteine insertion by substitution in antibody heavy or light chains include those described in Published U.S. Application No. 2007-0092940 and International Patent Publication WO2008070593, which are incorporated herein.
Methods of Treatment
The Conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a Conjugate of formula I. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount of a Conjugate administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose.
In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 1 mg/kg per dose.
A conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy).
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a Conjugate of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Includes Other Forms
Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO−), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N+HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O−), a salt or solvate thereof, as well as conventional protected forms.
Salts
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound (the Conjugate), for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).
For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO−), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations such as Al+3. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
If the Conjugate is cationic, or has a functional group which may be cationic (e.g. —NH2 may be —NH3+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
Solvates
It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the Conjugate(s). The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active Conjugate, salt of active Conjugate) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
Carbinolamines
The invention includes Conjugate where a solvent adds across the imine bond of the PBD moiety, which is illustrated below for a PBD monomer where the solvent is water or an alcohol (RAOH, where RA is C1-4 alkyl):
These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.
These particular compounds may be isolated in solid form, for example, by lyophilisation.
Isomers
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).
Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like.
Unless otherwise specified, a reference to a particular compound or Conjugate includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
General Synthetic Routes
The synthesis of PBD dimer compounds is extensively discussed in the following references, which discussions are incorporated herein by reference:
a) WO 00/12508 (pages 14 to 30);
b) WO 2005/023814 (pages 3 to 10);
c) WO 2004/043963 (pages 28 to 29); and
d) WO 2005/085251 (pages 30 to 39).
Synthesis Route
The Conjugates of the present invention, where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, can be synthesised from a compound of Compound formula 2:
where R2, R6, R7, R9, R6′, R7′, R9′, R12, X, X′ and R″ are as defined for compounds of formula II, ProtN is a nitrogen protecting group for synthesis and ProtO is a protected oxygen group for synthesis or an oxo group, by deprotecting the imine bond by standard methods.
The compound produced may be in its carbinolamine or carbinolamine ether form depending on the solvents used. For example if ProtN is Alloc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried using palladium to remove the N10 protecting group, followed by the elimination of the oxygen protecting group for synthesis. If ProtN is Troc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried out using a Cd/Pb couple to yield the compound of formula (I). If ProtN is SEM, or an analogous group, and ProtO is an oxo group, then the oxo group can be removed by reduction, which leads to a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group, followed by the elimination of water. The reduction of the compound of Compound formula 2 can be accomplished by, for example, lithium tetraborohydride, whilst a suitable means for removing the SEM protecting group is treatment with silica gel.
Compounds of Compound formula 2 can be synthesised from a compound of Compound formula 3a:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling an organometallic derivative comprising R12, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of Compound formula 2 can be synthesised from a compound of Compound formula 3b:
where R12, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling an organometallic derivative comprising R2, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of Compound formulae 3a and 3b can be synthesised from a compound of formula 4:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of Compound formula 2, by coupling about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallic derivative, such as an organoboron derivative, comprising R2 or R12.
The couplings described above are usually carried out in the presence of a palladium catalyst, for example Pd(PPh3)4, Pd(OCOCH3)2, PdCl2, or Pd2(dba)3. The coupling may be carried out under standard conditions, or may also be carried out under microwave conditions.
The two coupling steps are usually carried out sequentially. They may be carried out with or without purification between the two steps. If no purification is carried out, then the two steps may be carried out in the same reaction vessel. Purification is usually required after the second coupling step. Purification of the compound from the undesired by-products may be carried out by column chromatography or ion-exchange separation.
The synthesis of compounds of Compound formula 4 where ProtO is an oxo group and ProtN is SEM are described in detail in WO 00/12508, which is incorporated herein by reference. In particular, reference is made to scheme 7 on page 24, where the above compound is designated as intermediate P. This method of synthesis is also described in WO 2004/043963.
The synthesis of compounds of Compound formula 4 where ProtO is a protected oxygen group for synthesis are described in WO 2005/085251, which synthesis is herein incorporated by reference.
Compounds of formula I where R10 and R10′ are H and R11 and R11′ are SOzM, can be synthesised from compounds of formula I where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, by the addition of the appropriate bisulphite salt or sulphinate salt, followed by an appropriate purification step. Further methods are described in GB 2 053 894, which is herein incorporated by reference.
Nitrogen Protecting Groups for Synthesis
Nitrogen protecting groups for synthesis are well known in the art. In the present invention, the protecting groups of particular interest are carbamate nitrogen protecting groups and hemi-aminal nitrogen protecting groups.
Carbamate nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.
Other possible groups are nitrobenzyloxycarbonyl (e.g. 4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.
Those protecting groups which can be removed with palladium catalysis are not preferred, e.g. Alloc.
Hemi-aminal nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. The groups disclosed herein can be applied to compounds for use in the present invention. Such groups include, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, and Cl3CCH2OCH2—.
Protected Oxygen Group for Synthesis
Protected oxygen group for synthesis are well known in the art. A large number of suitable oxygen protecting groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates.
Preferred oxygen protecting groups include acetates, TBS and THP.
Further Preferences
The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
In some embodiments, R6′, R7′, R9′, R10′, R11′ and Y′ are preferably the same as R6, R7, R9, R10, R11 and Y respectively.
Dimer Link
Y and Y′ are preferably O.
R″ is preferably a C3-7 alkylene group with no substituents. More preferably R″ is a C3, C5 or C7 alkylene.
R6 to R9
R9 is preferably H.
R6 is preferably selected from H, OH, OR, SH, NH2, nitro and halo, and is more preferably H or halo, and most preferably is H.
R7 is preferably selected from H, OH, OR, SH, SR, NH2, NHR, NRR′, and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C1-7 alkyl, C3-10 heterocyclyl and C5-10 aryl groups. R may be more preferably a C1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH2Ph.
These preferences apply to R9′, R6′ and R7′ respectively.
R2
A in R2 may be phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl. In other embodiments, A is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
X is a group selected from the list comprising: —O—, —S—, —C(O)O—, —C(O)—, —NH(C═O)— and —N(RN)—, wherein RN is selected from the group comprising H and C1-4 alkyl. X may preferably be: —O—, —S—, —C(O)O—, —NH(C═O)— or —NH—, and may more preferably be: —O—, —S—, or —NH—, and most preferably is —NH—.
Q2-X may be on any of the available ring atoms of the C5-7 aryl group, but is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group (A) is phenyl, the substituent (Q2-X) is preferably in the meta- or para-positions, and more preferably is in the para-position. 1
In some embodiments, Q1 is a single bond. In these embodiments, Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and is from 1 to 3. In some of these embodiments, Q2 is a single bond. In other embodiments, Q2 is —Z—(CH2)n—. In these embodiments, Z may be O or S and n may be 1 or n may be 2.
In other of these embodiments, Z may be a single bond and n may be 1.
In other embodiments, Q1 is —CH═CH—.
In some embodiments, R2 may be -A-CH2—X and -A-X. In these embodiments, X may be —O—, —S—, —C(O)O—, —C(O)— and —NH—. In particularly preferred embodiments, X may be —NH—.
R12
R12 may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R12 is preferably phenyl. In other embodiments, R12 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
R12 may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4-yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4-yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred. R12 may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
Where R12 is C5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably 6 or y to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para-positions, and more preferably is in the para-position.
Where R12 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
R12 Substituents
If a substituent on R12 is halo, it is preferably F or Cl, more preferably Cl.
If a substituent on R12 is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g. phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
If a substituent on R12 is C1-7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propyl, butyl).
If a substituent on R12 is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
If a substituent on R12 is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
Particularly preferred substituents for R12 include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl. Another particularly preferred substituent for R12 is dimethylaminopropyloxy.
R12 Groups
Particularly preferred substituted R12 groups include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R12 group is 4-nitrophenyl.
M and z
It is preferred that M and M′ are monovalent pharmaceutically acceptable cations, and are more preferably Na+.
z is preferably 3.
EXAMPLES
General Experimental Methods
Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. 1H and 13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt (NovaBiochem) and solid-supported reagents (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied without further purification. Anhydrous solvents were prepared by distillation under a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C.
Compound 1b was synthesised as described in WO 00/012508 (compound 210), which is herein incorporated by reference.
General LC/MS conditions: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then 5% B to 95% B within 3 min. The composition was held for 0.5 min at 95% B, and then returned to 5% B in 0.3 minutes. Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm
LC/MS conditions specific for compounds protected by both a Troc and a TBDMs group: Chromatographic separation of Troc and TBDMS protected compounds was performed on a Waters Alliance 2695 HPLC system utilizing a Onyx Monolitic reversed-phase column (3 μm particles, 50×4.6 mm) from Phenomenex Corp. Mobile-phase A consisted of 5% acetonitrile—95% water containing 0.1% formic acid, and mobile phase B consisted of 95% acetonitrile—5% water containing 0.1% formic acid. After 1 min at 5% B, the proportion of B was raised to 95% B over the next 2.5 min and maintained at 95% B for a further 1 min, before returning to 95% A in 10 s and re-equilibration for a further 50 sec, giving a total run time of 5.0 min. The flow rate was maintained at 3.0 mL/min.
LC/MS conditions specific for compound 33: LC was run on a Waters 2767 sample Manager coupled with a Waters 2996 photodiode array detector and a Waters ZQ single quadruple mass Spectrometer. The column used was Luna Phenyl-Hexyl 150×4.60 mm, 5 μm, Part no. 00F-4257-E0 (Phenomenex). The mobile phases employed were:
Mobile phase A: 100% of HPLC grade water (0.05% triethylamine), pH=7
Mobile phase B: 20% of HPLC grade water and 80% of HPLC grade acetonitrile (0.05% triethylamine), pH=7
The gradients used were:
Time
Flow Rate
(min)
(ml/min)
% A
% B
Initial
1.50
90
10
1.0
1.50
90
10
16.0
1.50
64
36
30.0
1.50
5
95
31.0
1.50
90
10
32.0
1.50
90
10
Mass Spectrometry was carried out in positive ion mode and SIR (selective ion monitor) and the ion monitored was m/z=727.2.
Synthesis of Key Intermediates
(a) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2a)
Method A: A catalytic amount of DMF (2 drops) was added to a stirred solution of the nitro-acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride (0.95 mL, 1.36 g, 10.7 mmol) in dry THF (20 mL). The reaction mixture was allowed to stir for 16 hours at room temperature and the solvent was removed by evaporation in vacuo. The resulting residue was re-dissolved in dry THF (20 mL) and the acid chloride solution was added dropwise to a stirred mixture of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (859 mg, 4.73 mmol) and TEA (6.6 mL, 4.79 g, 47.3 mmol) in THF (10 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for a further 3 hours after which time TLC (95:5 v/v CHCl3/MeOH) and LC/MS (2.45 min (ES+) m/z (relative intensity) 721 ([M+H]+, 20)) revealed formation of product. Excess THF was removed by rotary evaporation and the resulting residue was dissolved in DCM (50 mL). The organic layer was washed with 1N HCl (2×15 mL), saturated NaHCO3 (2×15 mL), H2O (20 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent gave the crude product as a dark coloured oil. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96:4 v/v CHCl3/MeOH) isolated the pure amide 2a as an orange coloured glass (840 mg, 54%).
Method B: Oxalyl chloride (9.75 mL, 14.2 g, 111 mmol) was added to a stirred suspension of the nitro-acid 1a (17.3 g, 37.1 mmol) and DMF (2 mL) in anhydrous DCM (200 mL). Following initial effervescence the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. Conversion to the acid chloride was confirmed by treating a sample of the reaction mixture with MeOH and the resulting bis-methyl ester was observed by LC/MS. The majority of solvent was removed by evaporation in vacuo, the resulting concentrated solution was re-dissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40° C. The solid acid chloride was added portionwise over a period of 25 minutes to a stirred suspension of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (15.2 g, 84.0 mmol) and TEA (25.7 mL, 18.7 g, 185 mmol) in DCM (150 mL) at −40° C. (dry ice/CH3CN). Immediately, the reaction was complete as judged by LC/MS (2.47 min (ES+) m/z (relative intensity) 721 ([M+H]+, 100)). The mixture was diluted with DCM (150 mL) and washed with 1N HCl (300 mL), saturated NaHCO3 (300 mL), brine (300 mL), filtered (through a phase separator) and the solvent evaporated in vacuo to give the pure product 2a as an orange solid (21.8 g, 82%).
Analytical Data: [α]22D=−46.1° (c=0.47, CHCl3); 1H NMR (400 MHz, CDCl3) (rotamers) δ 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H, J=11.1 Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); 13C NMR (100 MHz, CDCl3) (rotamers) δ 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7, 108.2, 69.7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR, CHCl3) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211, 1177, 1072, 1050, 1008, 871 cm−1; MS (ES+) m/z (relative intensity) 721 ([M+H]+, 47), 388 (80); HRMS [M+H]+ theoretical C31H36N4O16 m/z 721.2199, found (ES+) m/z 721.2227.
(a) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2b)
Preparation from 1b according to Method B gave the pure product as an orange foam (75.5 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 749 ([M+H]+, 100).
(b) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3a)
Method A: A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) was added to a solution of the nitro-ester 2a (75 g, 104 mmol) in DMF (360 mL). The suspension was hydrogenated in a Parr hydrogenation apparatus over 8 hours. Progress of the reaction was monitored by LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H]+, 100), (ES−) m/z (relative intensity) 595 ([M+H]+, 100) after the hydrogen uptake had stopped. Solid Pd/C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 10 mbar) at 40° C. to afford a dark oil containing traces of DMF and residual charcoal. The residue was digested in EtOH (500 mL) at 40° C. on a water bath (rotary evaporator bath) and the resulting suspension was filtered through celite and washed with ethanol (500 mL) to give a clear filtrate. Hydrazine hydrate (10 mL, 321 mmol) was added to the solution and the reaction mixture was heated at reflux. After 20 minutes the formation of a white precipitate was observed and reflux was allowed to continue for a further 30 minutes. The mixture was allowed to cool down to room temperature and the precipitate was retrieved by filtration, washed with diethyl ether (2*1 volume of precipitate) and dried in a vacuum desiccator to provide 3a (50 g, 81%).
Method B: A solution of the nitro-ester 2a (6.80 g, 9.44 mmol) in MeOH (300 mL) was added to Raney™ nickel (4 large spatula ends of a ˜50% slurry in H2O) and anti-bumping granules in a 3-neck round bottomed flask. The mixture was heated at reflux and then treated dropwise with a solution of hydrazine hydrate (5.88 mL, 6.05 g, 188 mmol) in MeOH (50 mL) at which point vigorous effervescence was observed. When the addition was complete (˜30 minutes) additional Raney™ nickel was added carefully until effervescence had ceased and the initial yellow colour of the reaction mixture was discharged. The mixture was heated at reflux for a further 30 minutes at which point the reaction was deemed complete by TLC (90:10 v/v CHCl3/MeOH) and LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H]+, 100)). The reaction mixture was allowed to cool to around 40° C. and then excess nickel removed by filtration through a sinter funnel without vacuum suction. The filtrate was reduced in volume by evaporation in vacuo at which point a colourless precipitate formed which was collected by filtration and dried in a vacuum desiccator to provide 3a (5.40 g, 96%).
Analytical Data: [α]27D=+404° (c=0.10, DMF); 1H NMR (400 MHz, DMSO-d6) δ 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H, J=3.98 Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H, J=12.1, 3.60 Hz), 3.43 (dd, 2H, J=12.0, 4.72 Hz), 2.67-2.57 (m, 2H), 2.26 (p, 2H, J=5.90 Hz), 1.99-1.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 164.0, 149.9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8, 54.4, 53.1, 33.5, 27.5; IR (ATR, neat) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm−1; MS (ES+) m/z (relative intensity) 619 ([M+Na]+, 10), 597 ([M+H]+, 52), 445 (12), 326 (11); HRMS [M+H]+ theoretical C29H32N4O10 m/z 597.2191, found (ES+) m/z 597.2205.
(b) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3b)
Preparation from 2b according to Method A gave the product as a white solid (22.1 g, 86%).
Analytical Data: MS (ES−) m/z (relative intensity) 623.3 ([M−H]−, 100);
(c) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4a)
TBSCl (317 mg, 2.1 mmol) and imidazole (342 mg, 5.03 mmol) were added to a cloudy solution of the tetralactam 3a (250 mg, 0.42 mmol) in anhydrous DMF (6 mL). The mixture was allowed to stir under a nitrogen atmosphere for 3 hours after which time the reaction was deemed complete as judged by LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M+H]+, 100)). The reaction mixture was poured onto ice (˜25 mL) and allowed to warm to room temperature with stirring. The resulting white precipitate was collected by vacuum filtration, washed with H2O, diethyl ether and dried in the vacuum desiccator to provide pure 4a (252 mg, 73%).
Analytical Data: [α]23D=+234° (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H, J=5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H), 2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H), 0.08 (s, 12H); 13C NMR (100 MHz, CDCl3) δ 170.4, 165.7, 151.4, 146.6, 129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56.3, 55.7, 54.2, 35.2, 28.7, 25.7, 18.0, −4.82 and −4.86; IR (ATR, CHCl3) 3235, 2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099, 1033 cm−1; MS (ES+) m/z (relative intensity) 825 ([M+H]+, 62), 721 (14), 440 (38); HRMS [M+H]+ theoretical C41H60N4O10Si2 m/z 825.3921, found (ES+) m/z 825.3948.
(c) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4b)
Preparation from 3b according to the above method gave the product as a white solid (27.3 g, 93%).
Analytical Data: MS (ES+) m/z (relative intensity) 853.8 ([M+H]+, 100), (ES−) m/z (relative intensity) 851.6 ([M−H]+, 100.
(d) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5a)
A solution of n-BuLi (4.17 mL of a 1.6 M solution in hexane, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise to a stirred suspension of the tetralactam 4a (2.20 g, 2.67 mmol) in anhydrous THF (30 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to stir at this temperature for 1 hour (now a reddish orange colour) at which point a solution of SEMCl (1.18 mL, 1.11 g, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise. The reaction mixture was allowed to slowly warm to room temperature and was stirred for 16 hours under a nitrogen atmosphere. The reaction was deemed complete as judged by TLC (EtOAc) and LC/MS (4.77 min (ES+) m/z (relative intensity) 1085 ([M+H]+, 100)). The THF was removed by evaporation in vacuo and the resulting residue dissolved in EtOAc (60 mL), washed with H2O (20 mL), brine (20 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (80:20 v/v Hexane/EtOAc) gave the pure N10-SEM-protected tetralactam 5a as an oil (2.37 g, 82%).
Analytical Data: [α]23D=+163° (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98 Hz), 4.68 (d, 2H, J=9.99 Hz), 4.57 (p, 2H, J=5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s, 6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81 Hz), 2.03-1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, −1.24, −4.73; IR (ATR, CHCl3) 2951, 1685, 1640, 1606, 1517, 1462, 1433, 1360, 1247, 1127, 1065 cm−1; MS (ES+) m/z (relative intensity) 1113 ([M+Na]+, 48), 1085 ([M+H]+, 100), 1009 (5), 813 (6); HRMS [M+H]+ theoretical C53H88N4O12Si4 m/z 1085.5548, found (ES+) m/z 1085.5542.
(d) 1,1′-[[(Pentane 1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5b)
Preparation from 4b according to the above method gave the product as a pale orange foam (46.9 g, 100%), used without further purification.
Analytical Data: MS (ES+) m/z (relative intensity) 1114 ([M+H]+, 90), (ES−) m/z (relative intensity) 1158 ([M+2Na]−, 100).
(e) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6a)
A solution of TBAF (5.24 mL of a 1.0 M solution in THF, 5.24 mmol) was added to a stirred solution of the bis-silyl ether 5a (2.58 g, 2.38 mmol) in THF (40 mL) at room temperature. After stirring for 3.5 hours, analysis of the reaction mixture by TLC (95:5 v/v CHCl3/MeOH) revealed completion of reaction. The reaction mixture was poured into a solution of saturated NH4Cl (100 mL) and extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96:4 v/v CHCl3/MeOH) gave the pure tetralactam 6a as a white foam (1.78 g, 87%).
Analytical Data: [α]23D=+202° (c=0.34, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0 Hz), 4.72 (d, 2H, J=10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83 Hz), 4.20-4.16 (m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH), 2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77 Hz), 2.11-2.05 (m, 2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 169.5, 165.9, 151.3, 147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67.2, 65.2, 56.5, 56.2, 54.1, 35.2, 29.1, 18.4, −1.23; IR (ATR, CHCl3) 2956, 1684, 1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm−1; MS (ES+) m/z (relative intensity) 885 ([M+29]+, 70), 857 ([M+H]+, 100), 711 (8), 448 (17); HRMS [M+H]+ theoretical C41H60N4O12Si2 m/z 857.3819, found (ES+) m/z 857.3826.
(e) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6b)
Preparation from 5b according to the above method gave the product as a white foam (15.02 g).
Analytical Data: MS (ES+) m/z (relative intensity) 886 ([M+H]+, 10), 739.6 (100), (ES−) m/z (relative intensity) 884 ([M−H]+, 40).
(f) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (7a)
Method A: A 0.37 M sodium hypochlorite solution (142.5 mL, 52.71 mmol, 2.4 eq) was added dropwise to a vigorously stirred mixture of the diol 6a (18.8 g, 21.96 mmol, 1 eq), TEMPO (0.069 g, 0.44 mmol, 0.02 eq) and 0.5 M potassium bromide solution (8.9 mL, 4.4 mmol, 0.2 eq) in DCM (115 mL) at 0° C. The temperature was maintained between 0° C. and 5° C. by adjusting the rate of addition. The resultant yellow emulsion was stirred at 0° C. to 5° C. for 1 hour. TLC (EtOAc) and LC/MS [3.53 min. (ES+) m/z (relative intensity) 875 ([M+Na]+, 50), (ES−) m/z (relative intensity) 852 ([M−H]−, 100)] indicated that reaction was complete.
The reaction mixture was filtered, the organic layer separated and the aqueous layer was backwashed with DCM (×2). The combined organic portions were washed with brine (×1), dried (MgSO4) and evaporated to give a yellow foam. Purification by flash column chromatography (gradient elution 35/65 v/v n-hexane/EtOAC, 30/70 to 25/75 v/v n-hexane/EtOAC) afforded the bis-ketone 7a as a white foam (14.1 g, 75%).
Sodium hypochlorite solution, reagent grade, available at chlorine 10-13%, was used. This was assumed to be 10% (10 g NaClO in 100 g) and calculated to be 1.34 M in NaClO. A stock solution was prepared from this by diluting it to 0.37 M with water. This gave a solution of approximately pH 14. The pH was adjusted to 9.3 to 9.4 by the addition of solid NaHCO3. An aliquot of this stock was then used so as to give 2.4 mol eq. for the reaction.
On addition of the bleach solution an initial increase in temperature was observed. The rate of addition was controlled, to maintain the temperature between 0° C. to 5° C. The reaction mixture formed a thick, lemon yellow coloured, emulsion.
The oxidation was an adaptation of the procedure described in Thomas Fey et al, J. Org. Chem., 2001, 66, 8154-8159.
Method B: Solid TCCA (10.6 g, 45.6 mmol) was added portionwise to a stirred solution of the alcohol 6a (18.05 g, 21.1 mmol) and TEMPO (123 mg, 0.78 mmol) in anhydrous DCM (700 mL) at 0° C. (ice/acetone). The reaction mixture was stirred at 0° C. under a nitrogen atmosphere for 15 minutes after which time TLC (EtOAc) and LC/MS [3.57 min (ES+) m/z (relative intensity) 875 ([M+Na]+, 50)] revealed completion of reaction. The reaction mixture was filtered through celite and the filtrate was washed with saturated aqueous NaHCO3 (400 mL), brine (400 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash column chromatography (80:20 v/v EtOAc/Hexane) afforded the bis-ketone 7a as a foam (11.7 g, 65%).
Method C: A solution of anhydrous DMSO (0.72 mL, 0.84 g, 10.5 mmol) in dry DCM (18 mL) was added dropwise over a period of 25 min to a stirred solution of oxalyl chloride (2.63 mL of a 2.0 M solution in DCM, 5.26 mmol) under a nitrogen atmosphere at −60° C. (liq N2/CHCl3). After stirring at −55° C. for 20 minutes, a slurry of the substrate 6a (1.5 g, 1.75 mmol) in dry DCM (36 mL) was added dropwise over a period of 30 min to the reaction mixture. After stirring for a further 50 minutes at −55° C., a solution of TEA (3.42 mL, 2.49 g; 24.6 mmol) in dry DCM (18 mL) was added dropwise over a period of 20 min to the reaction mixture. The stirred reaction mixture was allowed to warm to room temperature (˜1.5 h) and then diluted with DCM (50 mL). The organic solution was washed with 1 N HCl (2×25 mL), H2O (30 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (80:20 v/v EtOAc/Hexane) to afford bis-ketone 7a as a foam (835 mg, 56%)
Analytical Data: [α]20D=+291° (c=0.26, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d, 2H, J=10.1 Hz), 4.60 (dd, 2H, J=9.85, 3.07 Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85 Hz), 2.76 (dd, 2H, J=19.2, 9.90 Hz), 2.42 (p, 2H, J=5.77 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 206.8, 168.8, 165.9, 151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9, 52.4, 37.4, 29.0, 18.4, −1.24; IR (ATR, CHCl3) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm−1; MS (ES+) m/z (relative intensity) 881 ([M+29]+, 38), 853 ([M+H]+, 100), 707 (8), 542 (12); HRMS [M+H]+ theoretical C41H56N4O12Si2 m/z 853.3506, found (ES+) m/z 853.3502.
(f) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (7b)
Preparation from 6b according to Method C gave the product as a white foam (10.5 g, 76%).
Analytical Data: MS (ES+) m/z (relative intensity) 882 ([M+H]+, 30), 735 (100), (ES−) m/z (relative intensity) 925 ([M+45]−, 100), 880 ([M−H]−, 70).
(g) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8a)
Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in one portion to a vigorously stirred solution of bis-ketone 7a (6.08 g, 7.1 mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile cooling bath) under a nitrogen atmosphere. Anhydrous triflic anhydride, taken from a freshly opened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injected rapidly dropwise, while maintaining the temperature at −40° C. or below. The reaction mixture was allowed to stir at −45° C. for 1 hour at which point TLC (50/50 v/v n-hexane/EtOAc) revealed the complete consumption of starting material. The cold reaction mixture was immediately diluted with DCM (200 mL) and, with vigorous shaking, washed with water (1×100 mL), 5% citric acid solution (1×200 mL) saturated NaHCO3 (200 mL), brine (100 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (gradient elution: 90:10 v/v n-hexane/EtOAc to 70:30 v/v n-hexane/EtOAc) to afford bis-enol triflate 8a as a yellow foam (5.5 g, 70%).
Analytical Data: [α]24D=+271° (c=0.18, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97 Hz), 5.51 (d, 2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.69 Hz), 4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J=16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J=5.85 Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 167.1, 162.7, 151.9, 148.0, 138.4, 133.6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56.3, 30.8, 29.0, 18.4, −1.25; IR (ATR, CHCl3) 2958, 1690, 1646, 1605, 1517, 1456, 1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm−1; MS (ES+) m/z (relative intensity) 1144 ([M+28]+, 100), 1117 ([M+H]+, 48), 1041 (40), 578 (8); HRMS [M+H]+ theoretical C43H54N4O16Si2S2F6 m/z 1117.2491, found (ES+) m/z 1117.2465.
(g) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8b)
Preparation from 7b according to the above method gave the bis-enol triflate as a pale yellow foam (6.14 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 1146 ([M+H]+, 85).
Example 1
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (9)
Solid Pd(PPh3)4 (20.18 mg, 17.46 mmol) was added to a stirred solution of the triflate 8a (975 mg, 0.87 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralane-2-yl)aniline (172 mg, 0.79 mmol) and Na2CO3 (138 mg, 3.98 mol) in toluene (13 mL) EtOH (6.5 mL) and H2O (6.5 mL). The dark solution was allowed to stir under a nitrogen atmosphere for 24 hours, after which time analysis by TLC (EtOAc) and LC/MS revealed the formation of the desired mono-coupled product and as well as the presence of unreacted starting material. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL), after eventual separation of the layers the aqueous phase was extracted again with EtOAc (2×25 mL). The combined organic layers were washed with H2O (50 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude Suzuki product. The crude Suzuki product was subjected to flash chromatography (40% EtOAc/60% Hexane→70% EtOAc, 30% Hexane). Removal of the excess eluent by rotary evaporation under reduced pressure afforded the desired product 9 (399 mg) in 43% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J=8.5 Hz), 7.15 (t, 1H, J=2.0 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.52 (d, 2H, J=10.0 Hz), 4.77 (d, 1H, J=10.0 Hz), 4.76 (d, 1H, J=10.0 Hz), 4.62 (dd, 1H, J=3.7, 11.0 Hz), 4.58 (dd, 1H, J=3.4, 10.6 Hz), 4.29 (t, 4H, J=5.6 Hz), 4.00-3.85 (m, 8H), 3.80-3.60 (m, 4H), 3.16 (ddd, 1H, J=2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J=2.2, 10.5, 16.1 Hz), 2.43 (p, 2H, J=5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H). 13C-NMR: (CDCl3, 100 MHz) δ 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147.6, 139.6, 134.8, 134.5, 127.9 (methine), 127.5, 125.1, 123.21, 121.5, 120.5 (methine), 120.1 (methine), 116.4 (methine), 113.2 (methine), 108.7 (methine), 79.8 (methylene), 79.6 (methylene), 68.7 (methylene), 68.5 (methylene), 67.0 (methylene), 66.8 (methylene), 58.8 (methine), 58.0 (methine), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3 (methylene), 19.7 (methylene), 0.25 (methyl).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (10)
Solid Pd(PPh3)4 (10 mg, 8.69 μmol) was added to a stirred solution of the mono-triflate 9 (230 mg, 0.22 mmol) in toluene (3 mL), EtOH (10 mL), with 4-methoxyphenyl boronic acid (43 mg, 0.28 mmol), Na2CO3 (37 mg, 0.35 mmol), in H2O (1.5 mL) at room temperature. The reaction mixture was allowed to stir under a nitrogen atmosphere for 20 h, at which point the reaction was deemed complete as judged by LC/MS and TLC (EtOAc). The solvent was removed by rotary evaporation under reduced pressure in vacuo and the resulting residue partitioned between EtOAc (75 mL) and H2O (75 mL). The aqueous phase was extracted with EtOAc (3×30 mL) and the combined organic layers washed with H2O (30 mL), brine (40 mL), dried (MgSO4), filtered and evaporated to provide the crude product. The crude product was purified by flash chromatography (60% Hexane: 40% EtOAc→80% EtOAc: 20% Hexane) to provide the pure dimer as an orange foam. Removal of the excess eluent under reduced pressure afforded the desired product 10 (434 mg) in 74% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.38 (s, 2H), 7.34 (d, 2H, J=8.8 Hz), 7.30 (bs, 1H), 7.26-7.24 (m, 3H), 7.22 (d, 2H, J=8.5 Hz), 6.86 (d, 2H, J=8.8 Hz), 6.63 (d, 2H, J=8.5 Hz), 5.50 (d, 2H, J=10.0 Hz), 4.75 (d, 1H, J=10.0 Hz), 4.74 (d, 1H, J=10.0 Hz), 4.56 (td, 2H, J=3.3, 10.1 Hz), 4.27 (t, 2H, J=5.7 Hz), 4.00-3.85 (m, 8H), 3.80 (s, 3H), 3.77-3.60 (m, 4H), 3.20-3.00 (m, 2H), 2.42 (p, 2H, J=5.7 Hz), 0.96 (t, 4H, J=8.3 Hz), 0.00 (s, 18H). 13C-NMR: (CDCl3, 100 MHz) δ 169.8, 169.7, 162.9, 162.7, 160.6, 152.7, 152.6, 149.0, 147.5, 134.8, 127.8 (methine), 127.4, 126.8, 125.1, 123.1, 123.0, 121.5 (methine), 120.4 (methine), 116.4 (methine), 115.5 (methine), 113.1 (methine), 108.6 (methine), 79.6 (methylene), 68.5 (methylene), 66.9 (methylene), 58.8 (methine), 57.6 (methoxy), 56.7 (methoxy), 32.8 (methylene), 30.3 (methylene), 19.7 (methylene), 0.0 (methyl).
(c) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (11)
Fresh LiBH4 (183 mg, 8.42 mmol) was added to a stirred solution of the SEM-dilactam 10 (428 mg, 0.42 mmol) in THF (5 mL) and EtOH (5 mL) at room temperature. After 10 minutes, delayed vigorous effervescence was observed requiring the reaction vessel to be placed in an ice bath. After removal of the ice bath the mixture was allowed to stir at room temperature for 1 hour. LC/MS analysis at this point revealed total consumption of starting material with very little mono-reduced product. The reaction mixture was poured onto ice (100 mL) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3×30 mL) and the combined organic layers washed with H2O (20 mL), brine (30 mL) and concentrated in vacuo. The resulting residue was treated with DCM (5 mL), EtOH (14 mL), H2O (7 mL) and silica gel (10 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered slowly through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was washed with H2O (50 mL), brine 60 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (97% CHCl3: 3% MeOH) to provide the pure C2/C2′ aryl PBD dimer 11 (185 mg) 61% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.88 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.20 (d, 2H, J=8.5 Hz), 6.89 (d, 2H, J=8.8 Hz), 6.87 (s, 1H), 6.86 (s, 1H), 6.67 (d, 2H, J=8.5 Hz), 4.40-4.20 (m, 6H), 3.94 (s, 6H), 3.82 (s, 3H), 3.61-3.50 (m, 2H), 3.40-3.30 (m, 2H), 2.47-2.40 (m, 2H). 13C-NMR: (CDCl3, 100 MHz) δ 162.5 (imine methine), 161.3, 161.1, 159.3, 156.0, 151.1, 148.1, 146.2, 140.3, 126.2 (methine), 123.2, 122.0, 120.5 (methine), 119.4, 115.2 (methine), 114.3 (methine), 111.9 (methine), 111.2 (methine), 65.5 (methylene), 56.2 (methoxy), 55.4 (methoxy), 53.9 (methine), 35.6 (methylene), 28.9 (methylene).
Example 2
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (12)
Solid Pd(PPh3)4 (32 mg, 27.7 μmol) was added to a stirred solution of the bis-triflate 8b (1.04 g, 0.91 mmol) in toluene (10 mL), EtOH (5 mL), with 4-methoxyphenyl boronic acid (0.202 g, 1.32 mmol), Na2CO3 (0.169 g, 1.6 mmol), in H2O (5 mL) at 30° C. The reaction mixture was allowed to stir under a nitrogen atmosphere for 20 hours. Additional solid 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (0.203 g, 0.93 mmol) and Na2CO3 (0.056 g, 0.53 mmol) were added followed by solid Pd(PPh3)4 (10 mg, 8.6 μmol). The reaction mixture was allowed to stir under a nitrogen atmosphere for a further 20 hours. LC/MS indicated the formation of desired product. EtOAc (100 mL) and H2O (100 mL) were added, the aqueous was separated and extracted with EtOAc (3×30 mL). The combined organic layers were washed with H2O (100 mL), brine (100 mL), dried (MgSO4), filtered and evaporated to provide a dark brown oil. The oil was dissolved in DCM and loaded onto a 10 g SCX-2 cartridge pre-equilibrated with DCM (1 vol). The cartridge was washed with DCM (3 vol), MeOH (3 vol) and the crude product eluted with 2M NH3 in MeOH (2 vol). Flash chromatography (50% n-hexane: 50% EtOAc→20% n-hexane: 80% EtOAc) provided the pure dimer 12 as a yellow foam (0.16 g, 34%).
Analytical Data: [α]23D=+388° (c=0.22, CHCl3); 1H-NMR: (CDCl3, 400 MHz) δ 7.39 (s, 2H), 7.35 (d, 2H, J=12.8 Hz), 7.32 (bs, 1H), 7.26-7.23 (m, 5H), 6.89 (d, 2H, J=8.8 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.55 (d, 2H, J=10.0 Hz), 4.73 (d, 1H, J=10.0 Hz), 4.72 (d, 1H, J=10.0 Hz), 4.62 (td, 2H, J=3.2, 10.4 Hz), 4.15-4.05 (m, 4H), 4.00-3.85 (m, 8H), 3.82 (s, 3H), 3.77-3.63 (m, 4H), 3.20-3.05 (m, 2H), 2.05-1.95 (m, 4H), 1.75-1.67 (m, 2H) 1.01-0.95 (m, 4H), 0.03 (s, 18H); MS (ES+) m/z (relative intensity) 1047 ([M+H]+, 45).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (13)
Fresh LiBH4 (66 mg, 3.04 mmol) was added to a stirred solution of the SEM-dilactam 12 (428 mg, 0.42 mmol) in THF (3 mL) and EtOH (3 mL) at 0° C. (ice bath). The ice bath was removed and the reaction mixture was allowed to reach room temperature (vigorous effervescence). After 2 hours LC/MS analysis indicated the complete consumption of starting material. The reaction mixture was poured onto ice (50 mL) and allowed to warm to room temperature with stirring. The aqueous mixture was extracted with DCM (3×50 mL) and the combined organic layers washed with H2O (50 mL), brine (50 mL), dried (MgSO4) and concentrated in vacuo. The resulting residue was treated with DCM (2 mL), EtOH (5 mL), H2O (2.5 mL) and silica gel (3.7 g). The viscous mixture was allowed to stir at room temperature for 3 days. The mixture was filtered through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was dried (MgSO4) filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (99.5% CHCl3: 0.5% MeOH to 97.5% CHCl3: 2.5% MeOH in 0.5% increments)) to provide the pure C2/C2′ aryl PBD dimer 13 (59 mg, 52%).
Analytical Data: [α]28D=+760° (c=0.14, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.89 (d, 1H, J=4.0 Hz), 7.87 (d, 1H, J=4.0 Hz), 7.52 (s, 2H), 7.39 (bs, 1H), 7.37-7.28 (m, 3H), 7.22 (d, 2H, J=8.4 Hz), 6.91 (d, 2H, J=8.8 Hz), 6.815 (s, 1H), 6.81 (s, 1H), 6.68 (d, 2H, J=8.4 Hz), 4.45-4.35 (m, 2H), 4.2-4.0 (m, 4H), 3.94 (s, 6H), 3.85-3.7 (s, 3H), 3.65-3.50 (m, 2H), 3.45-3.3 (m, 2H), 2.05-1.9 (m, 4H), 1.75-1.65 (m, 2H); MS (ES+) (relative intensity) 754.6 ([M+H]+, 100), (ES−) (relative intensity) 752.5 ([M−H]−, 100).
Example 3
(a) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (14)
Solid Pd(PPh3)4 (41 mg, 0.036 mmol) was added to a stirred solution of the bis-triflate 8a (1 g, 0.9 mmol) in toluene (10 mL), EtOH (5 mL), with thien-2-yl boronic acid (149 mg, 1.16 mmol), Na2CO3 (152 mg, 1.43 mmol), in H2O (5 mL). The reaction mixture was allowed to stir under a nitrogen atmosphere overnight at room temperature. The solvent was removed by evaporation in vacuo and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL). The aqueous layer was extracted with EtOAc (2×30 mL) and the combined organic layers washed with H2O (50 mL), brine (50 mL) dried (MgSO4), filtered and evaporated in vacuo to provide the crude product which was purified by flash chromatography (80 hexane: 20 EtOAc→50 hexane: 50 EtOAc) to provide the dimer 14 (188 mg, 20%) yield
Analytical data: LC-MS RT 4.27 mins, 1051 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.36 (s, 1H), 7.31 (bs, 1H), 7.27 (bs, 1H), 7.26-7.23 (m, 2H), 7.22-7.17 (m, 1H), 7.12 (bs, 1H), 7.02-6.96 (m, 2H), 5.50 (d, J=10.0 Hz, 2H), 7.75 (d, J=10.0 Hz, 2H), 4.65-4.55 (m, 2H), 4.37-4.13 (m, 4H), 4.00-3.85 (m, 8H), 3.8-3.6 (m, 4H), 3.20-3.10 (m, 2H), 2.50-2.35 (m, 2H), 1.0-0.9 (m, 4H), 0 (s, 18H).
(b) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethanesulfonyloxy)-5,11-dioxo-10((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-10((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (15)
Solid Pd(PPh3)4 (7.66 mg, 6.63 μmol) was added to a stirred, cloudy solution of 14 (174 mg, 0.17 mmol), Na2CO3 (28 mg, 0.22 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)aniline (47 mg, 0.22 mmol) in toluene (2-5 mL), EtOH (1.25 mL) and H2O (125 mL) at room temperature. The reaction mixture was allowed to stir under a N2 atmosphere for 24 hours at which point the reaction was deemed complete by LC/MS major peak (@ 3.97 min, FW=1016, M+Na) and TLC (EtOAc). The solvent was removed by evaporation in vacuo and the resulting residue partitioned between EtOAc (60 mL) and H2O (30 mL). The layers were separated and the organic phase was washed with H2O) (20 mL), brine (30 mL) dried (MgSO4) filtered and evaporated in vacuo to provide the crude product 123 mg, 75% yield.
Analytical data: LC-MS RT 3.98 mins, 100% area, 994 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.40 (d, J=5.3 Hz, 2H), 7.30 (t, J=1.70 Hz, 1H), 7.29-7.27 (m, 3H), 7.25 (d, J=8.5 Hz, 2H), 7.21 (dd, J=1.4, 4.73 Hz, 1H), 7.03-6.97 (m, 2H), 6.66 (d, J=8.5 Hz, 2H), 5.52 (d, J=10.0 Hz, 2H), 4.78 (d, J=10.0 Hz, 1H), 4.77 (d, J=10.0 Hz, 1H), 4.62 (dd, J=3.4, 10.5 Hz, 1H), 4.59 (dd, J=3.40, 10.6 Hz, 1H), 4.30 (t, J=5.85 Hz, 4H), 3.85-4.03 (m, 8H), 3.84-3.64 (m, 6H), 3.18 (ddd, J=2.2, 10.5, 16.0 Hz, 1H), 3.11 (ddd, J=2.2, 10.5, 16.0 Hz, 1H), 2.44 (p, J=5.85 Hz, 2H), 0.98 (t, J=1.5 Hz, 4H), 0 (s, 18H).
(c) (S)-2-(thien-2-yl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-aminophenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (16)
Fresh LiBH4 (47 mg, 2.22 mmol) was added to a stirred solution of the SEM-dilactam 15 (110 mg, 0.11 mmol) in dry THF (3 mL) and EtOH (3 mL) at 0° C. (ice bath). The ice bath was removed and the reaction mixture stirred under a N2 atmosphere for 1 hour. Analysis of the reaction by LC/MS analysis revealed significant formation of the desired product (Pk @ 2.57 min) (I=69.32), FW=702, M+H) and half-imine. The reaction mixture was allowed to stir for a further 1 hour after which time no further reaction progress was observed by LC/MS. The reaction mixture was poured onto ice, stirred and allowed to warm to room temperature. Following partition between DCM (50 mL) and water (50 mL), the aqueous phase was extracted with DCM (3×20 mL). The combined organic layers were washed with H2O (50 mL), brine (50 mL) and the solvent removed by evaporation in vacuo under reduced pressure.
The resulting residue was dissolved in DCM (5 mL), EtOH (15 mL) and H2O (7 mL) then treated with silica gel (5 g). The reaction was allowed to stir at room temperature for 48 h. The silica was removed by filtration through a sinter funnel and the residue rinsed with 90:10 CHCl3: MeOH (100 mL). H2O (50 mL) was added to the filtrate and the layers were separated (after shaking). The aqueous layer was extracted with CHCl3 (2×30 mL) and H2O (50 mL), brine (50 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude product. Flash chromatography (CHCl3→98% CHCl3: 2% MeOH) afforded the product (41 mg, 53%).
Analytical data: LC-MS RT 2.55 mins, 702 (M+H)
Example 4
(a) (S)-2-(4-methoxyphenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulphonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (17)
Solid 4-methoxybenzeneboronic acid (0.388 g, 2.55 mmol) was added to a solution of the SEM protected bis triflate (8a) (3.0 g, 2.69 mmol), sodium carbonate (426 mg, 4.02 mmol) and palladium tetrakis triphenylphosphine (0.08 mmol) in toluene (54.8 mL), ethanol (27 mL) and water (27 mL). The reaction mixture was allowed to stir at room temperature for 3 hours. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 30/70→35/65→40/60→45/55) to remove unreacted bis-triflate (0.6 g). Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (1.27 g, 1.18 mmol, 41%).
LC-MS RT 4.30 mins, 1076 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.41 (s, 1H), 7.39 (d, J=8.8 Hz, 2H), 7.35 (s, 1H), 7.34 (bs, 1H), 7.29 (s, 1H), 7.16 (t, J=1.9 Hz, 1H), 6.90 (d, J=8.8 Hz, 2H), 5.53 (d, J=10.0 Hz, 2H), 4.79 (d, J=10.0 Hz, 1H), 4.78 (d, J=10.0 Hz, 1H), 4.66-4.60 (m, 2H), 4.30 (t, J=5.7 Hz, 4H), 4.0-3.94 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.84 (s, 3H), 3.83-3.60 (m, 4H), 3.22-3.10 (m, 2H), 2.45 (t, J=5.9 Hz, 2H), 1.05-0.94 (m, 4H), 0 (s, 18H).
(b) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (18)
Solid 3-aminobenzeneboronic acid (0.143 g, 0.92 mmol) was added to a solution of the mono triflate (17) (0.619 g, 0.58 mmol), sodium carbonate (195 mg, 1.84 mmol) and palladium tetrakis triphenylphosphine (26.6 mg, 0.023 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir at room temperature for overnight at 30° C. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 70/30→85/15). Removal of excess eluent from selected fractions afforded the desired product (0.502 g, 0.49 mmol, 85%).
LC-MS RT 4.02 mins, 1019 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.38-7.35 (m, 4H), 7.33 (bs, 1H), 7.30 (bs, 1H), 7.25 (s, 2H), 7.10 (t, J=7.8 Hz, 1H), 6.88-6.80 (m, 3H), 6.72 (bs, 1H), 6.57 (dd, J=7.9, 1.8 Hz, 1H), 5.50 (d, J=10.0 Hz, 2H), 4.75 (d, 10.0 Hz, 2H), 4.58 (dd, J=10.6, 3.3 Hz, 2H), 4.27 (t, J=5.8 Hz, 4H), 3.95-3.91 (m, 2H), 3.90 (s, 6H), 3.80 (s, 3H), 3.77-3.60 (m. 6H), 3.15-3.05 (m, 2H), 2.41 (p, J=5.8 Hz, 2H), 0.95 (t,=8.25 Hz, 4H), 0 (s, 18H).
(c) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (19)
A solution of superhydride (0.56 mL, 0.56 mmol, 1.0 M in THF) was added dropwise to a solution of the SEM dilactam (18) (0.271 g, 0.27 mmol) in dry THF (10 mL) at −78° C. under a nitrogen atmosphere. After 1 hr a further aliquot of superhydride solution (0.13 ml, 0.13 mmol) was added and the reaction mixture was allowed to stir for another 0.5 hr, at which time LC-MS indicated that reduction was complete. The reaction mixture was diluted with water and allowed to warm to room temperature. The reaction mixture was partitioned between chloroform and water, the layers were separated and the aqueous layer extracted with additional chloroform (emulsions). Finally the combined organic phase was washed with brine and dried over magnesium sulphate. The reduced product was dissolved in methanol, chloroform and water and allowed to stir in the presence of silica gel for 72 hours The crude product was subjected to flash column chromatography (methanol/chloroform gradient) to afford the desired imine product (150 mg, 0.21 mmol, 77%) after removal of excess eluent from selected fractions.
LC-MS RT 2.63 mins, 97% area, 726 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.85 (d, J=3.9 Hz, 1H), 7.84 (d, J=3.9 Hz, 1H), 7.50 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 7.32 (d, J=7.3 Hz, 2H), 7.11 (t, (d, J=7.8 Hz, 1H), 6.90-6.80 (m, 4H), 6.77 (d, J=7.9 Hz, 1H), 4.40-4.20 (m, 6H), 3.92 (s, 6H), 3.80 (s, 3H), 3.60-3.27 (m, 6H), 2.48-2.29 (m, 2H).
Example 5
(a) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-2-(trifluoromethylsulfonyloxy)-11,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 21
Solid 4-methoxybenzeneboronic acid (59 mg, 0.39 mmol) was added to a solution of the Troc protected bis triflate (Compound 44, WO 2006/111759) (600 mg, 0.41 mmol), sodium carbonate (65 mg, 0.61 mmol) and palladium tetrakis triphenylphosphine (0.012 mmol) in toluene (10.8 mL), ethanol (5.4 mL) and water (5.4 mL). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was then partitioned between ethylacetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 20/80→30/70→40/60→60/40) to remove unreacted bis-triflate. Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (261 mg, 0.18 mmol, 46%).
LC-MS RT 4.17 mins, 1427 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.38 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.25 (s, 1H), 7.20 (bs, 1H), 6.92 (d, J=8.6 Hz, 2H), 6.77 (d, J=8.7 Hz, 2H), 6.0-5.90 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.18-4.08 (m, 2H), 4.07-3.89 (m, 10H), 3.81 (s, 3H), 3.44-3.25 (m, 2H), 2.85 (d, J=16.6 Hz, 2H), 2.05-1.90 (m, 4H), 1.76-1.64 (m, 2H), 0.93 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.26 (s, 6H).
(b) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 22
The Suzuki coupling procedure described in step (a) was applied to the synthesis of Compound 21. Compound 20 (62.5 mg 0.044 mmol), was treated with 1 equivalent of 4-hydroxybenzeneboronic acid (10 mg) at 30° C. overnight to afford the desired compound after filtration through a pad of silica gel. (40 mg, 0.029 mmol, 66% yield). The compound was used directly in the subsequent step
LC-MS RT 4.27 mins, 1371 (M+H)
(c) (S)-2-(4-hydroxyphenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 23
Cadmium/lead couple (100 mg, Q Dong et al. Tetrahedron Letters vol 36, issue 32, 5681-5682, 1995) was added to a solution of 21 (40 mg, 0.029 mmol) in THF (1 mL) and ammonium acetate (1N, 1 mL) and the reaction mixture was allowed to stir for 1 hour. The reaction mixture was partitioned between chloroform and water, the phases separated and the aqueous phase extracted with chloroform. The combined organic layers were washed with brine and dried over magnesium sulphate. Rotary evaporation under reduced pressure yielded the crude product which was subjected to column chromatography (silica gel, 0→4% MeOH/CHCl3). Removal of excess eluent by rotary evaporation under reduced pressure afforded the desired imine product (17 mg 0.023 mmol 79%).
LC-MS RT 2.20 mins, 755 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.89 (d, J=3.94 Hz, 1H), 7.89 (d, J=4.00 Hz, 1H), 7.53 (s, 1H), 7.52 (s, 1H), 7.38 (d, J=8.7 Hz, 2H), 7.33 (d, J=8.6 Hz, 2H), 7.28 (s, 1H), 6.90 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 6.82 (s, 1H), 6.81 (s, 1H), 5.68 (bs, 1H), 4.50-4.30 (m, 2H), 4.22-4.00 (m, 4H), 3.93 (s, 6H), 3.82 (s, 3H), 3.69-3.45 (m, 2H), 3.44-3.28 (m, 2H), 2.64-1.88 (m, 4H), 1.77-1.62 (m, 2H).
Example 6
(a) (11S,11aS)-2,2,2-trichloroethyl 11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-formylphenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 24
The Suzuki coupling procedure described in Example 5, step (a), was applied to the synthesis of Compound 24. Compound 21 (62.5 mg, 0.044 mmol) was treated with 1 equivalent of 4-formylbenzeneboronic acid (10.5 mg) at room temperature overnight to afford the desired compound after filtration through a pad of silica gel (45 mg, 0.033 mmol, 75% yield). The compound was used directly in the subsequent step.
LC-MS RT 4.42 mins, 1383 (M+H)
(b) 4-((S)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-2-yl)benzaldehyde 25
Compound 24 was deprotected by the method described in Example 5, step (c), to yield the desired compound (18 mg, 0.023 mmol, 79%).
LC-MS RT 3.18 mins, 768 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 9.98 (s, 1H), 7.91 (d, J=3.90 Hz, 1H), 7.90-7.80 (m, 3H), 7.68 (s, 1H), 7.60-7.45 (m, 4H), 7.39 (s, 1H), 7.33 (d, J=8.7 Hz, 1H), 6.90 (d, J=8.7 Hz, 2H), 6.83 (s, 1H), 6.82 (s, 1H), 4.55-4.44 (m, 1H), 4.43-4.36 (m, 1H), 4.23-4.00 (m, 4H), 3.95 (s, 3H), 3.94 (s, 3H), 3.82 (s, 3H), 3.66-3.51 (m, 2H), 3.50-3.34 (m, 2H), 2.05-1.87 (m, 4H), 1.76-164 (m, 2H).
Example 7
(a) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-2-(trifluoromethylsulphonyloxy)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 26
The Suzuki coupling procedure described in Example 5, step (a), was applied to the synthesis of Compound 26, using 3-aminobenzeneboronic acid to afford the desired compound in 41% yield (230 mg, 0.163 mmol)
LC-MS RT 4.28 mins, 1411 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.44 (bs, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 7.20 (s, 1H), 7.16 (t, J=7.9 Hz, 1H), 6.84-6.73 (m, 3H), 6.70 (bs, 1H), 6.62 (dd, J=7.9, 1.7 Hz, 1H), 6.66-6.58 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.17-4.07 (m, 2H), 4.08-3.89 (m, 10H), 3.43-3.28 (m, 2H), 2.85 (d, J=1.65 Hz, 2H), 2.07-1.90 (m, 4H), 1.78-1.63 (m, 2H), 0.94 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.27 (s, 6H).
(b) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 27
Solid 4-[3-(dimethylamino)propoxybenzeneboronic acid pinacol ester (25 mg, 0.082 mmol) was added to a solution of 26 (73 mg, 0.052 mmol), sodium carbonate (18 mg, 0.17 mmol) and palladium tetrakis triphenylphosphine (3 mg) in toluene (1 mL), ethanol (0.5 mL) and water (0.5 mL). The reaction mixture was allowed to stir at room temperature over night. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was eluted through a plug of silica gel with chloroform/methanol. Removal of excess eluent from selected fractions afforded the 4-methoxyphenyl coupled product (50 mg, 0.035 mmol, 67%).
LC-MS RT 4.12 mins, 1440 (M+H)
(c) (S)-2-(3-aminophenyl)-8-(5-((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepine-8-yloxy)pentyloxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]-benzodiazepine-5(11aH)-one 28
Compound 27 was deprotected by the method described in Example 5, step (c), to yield the desired compound. The reaction mixture was partitioned between DCM and aqueous sodium hydrogen carbonate (emulsion) and the crude product purified by gradient column chromatography on silica gel (5% methanol chloroform→35% methanol/chloroform) to afford the desired unsymmetrical PBD imine (50 mg, 0.018 mmol, 58%)
LC-MS RT 2.55 mins, 826 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.92-7.82 (m, 2H), 7.52 (bs, 2H), 7.45 (bs, 1H), 7.39 (bs, 1H), 7.31 (d, J=8.6 Hz, 2H), 7.14 (t, J=7.8 Hz, 1H), 6.89 (d, J=8.6 Hz, 2H), 6.85-6.75 (m, 3H), 6.72 (bs, 1H), 6.60 (d, J=8.0 Hz, 1H), 4.46-4.33 (m, 2H), 4.21-3.98 (m, 6H), 3.94 (s, 6H), 3.63-3.50 (m, 2H), 3.43-3.29 (m, 2H), 2.64-2.48 (m, 2H), 2.34 (s, 6H), 2.10-1.89 (m, 6H), 1.57 (m, 2H).
Example 8
(a) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-11-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 29
The method of Example 7, step (b), was performed to afford the desired product (58 mg, 0.040 mmol, 78%) after filtration through a plug of silica gel (with ⅓ methanol/chloroform) and removal of excess solvent by rotary evaporation under reduced pressure.
LC-MS RT 4.08 mins, 1439 (M+H)
(b) (S)-2-(3-aminophenyl)-7-methoxy-8-(5-((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepin-8-yloxy)pentyloxy)-1H-pyrrolo[2,1-c][1,4]-benzodiazepine-5(11aH)-one 30
The method for Example 7, step (c) was used to deprotect compound 29. The crude product was purified by silica gel gradient chromatography (2% methanol chloroform→35% methanol/chloroform) to afford the desired unsymmetrical PBD imine (18 mg, 0.022 mmol, 59%)
LC-MS RT 2.52 mins, 823 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.80 (d, J=3.8 Hz, 2H), 7.45 (s, 2H), 7.38 (s, 1H), 7.30 (s, 1H), 7.23 (d, J=8.6 Hz, 2H), 7.07 (t, J=7.8 Hz, 1H), 6.83 (d, J=8.6 Hz, 2H), 6.79-6.89 (m, 3H), 6.65 (s, 1H), 6.54 (d, J=7.9 Hz, 1H), 4.40-4.24 (m, 2H), 4.15-3.93 (m, 4H), 3.87 (s, 6H), 3.56-3.42 (m, 2H), 3.37-3.23 (m, 2H), 3.22-3.08 (m, 4H), 2.61-2.41 (m, 4H), 2.29 (s, 3H), 1.98-1.80 (m, 4H), 1.67-1.54 (m, 2H).
Example 9
(a) (S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione 31
Solid 4-aminomethylbenzeneboronic acid hydrochloride (0.111 g, 0.59 mmol) was added to a solution of 17 (0.394 g, 0.37 mmol), sodium carbonate (175 mg, 1.654 mmol) and palladium tetrakis triphenylphosphine (28.0 mg, 0.024 mmol) in toluene (10 mL), ethanol (5 mL) and water (5 mL). The reaction mixture was allowed to stir overnight at 30° C. The following day the reaction mixture was heated for a further 3 hours at 70° C. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 2/98→15/85). Removal of excess eluent from selected fractions afforded the desired product (0.230 mg, 0.22 mmol, 61%).
LC-MS RT 3.63 mins, 1034 (M+2H); 1H-NMR (400 MHz, DMSO d6) δ 11.7 (s, 2H), 7.52 (d, J=8.2 Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 7.40 (s, 1H), 7.50 (d, J=8.1 Hz, 2H), 7.38-7.19 (m, 5H) 6.93 (d, J=8.7 Hz, 2H), 5.40 (d, J=2.13 Hz, 1H), 5.38 (d, J=2.12 Hz, 1H), 5.32 (d, J=10.6 Hz, 2H), 5.25 (d, J=10.6 Hz, 2H), 4.87-4.72 (m, 2H), 4.35-4.15 (m, 4H), 3.85 (s, 6H), 3.79 (s, 3H), 3.73-3.56 (m, 2H), 3.55-3.39 (m, 4H), 3.22-3.02 (m, 2H), 2.39-2.23 (m, 2H), 0.94-0.67 (m, 4H), −0.06 (s, 18H).
(b) (S)-2-(4-(aminomethyl)phenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 32
Compound 31 was deprotected following the method of Example 1, step (c). The crude product was purified by gradient column chromatography (5/95→30/70 MeOH/CHCl3) to afford the product as a mixture of imine and carbinolamine methyl ethers.
LC-MS RT 2.58 mins, 740 (M+H).
Example 10
(S)-2-(4-aminophenyl)-7-methoxy-11(S)-sulpho-8-(3-((S)-7-methoxy-11(S)-sulpho-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propyloxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one disodium salt 33
Sodium bisulphite (8.5 mg, 3.1 eq) was added to a stirred suspension of bis-imine 11 (20 mg, 0.036 mmol) in isopropanol (4 mL) and water (2 mL). The reaction mixture was allowed to stir vigorously and eventually became clear (c. 1 hour). The reaction mixture was transferred to a funnel and filtered through a cotton wall (and then washed with 2 mL water). The filtrate was flash frozen (liquid and to bath) and lyophilized to afford the desired product 33 in quantitative yield.
LC-MS RT 11.77 mins, 727.2 (M+H) (Mass of parent compound, bisulphite adducts unstable in mass spectrometer); 1H-NMR (400 MHz, CDCl3) δ 7.66-7.55 (m, 5H), 7.43 (s, 1H), 7.39 (d, J=8.66 Hz, 2H), 7.06 (m, 2H), 6.93 (d, J=8.84 Hz, 2H), 6.54 (m, 2H), 5.29-5.21 (m, 2H), 4.32-4.28 (m, 2H), 4.14-4.20 (m, 4H), 3.96-3.83 (m, 2H), 3.77 (s, 3H), 3.73 (m, 6H), 3.52-3.43 (m, 2H), 3.30-3.08 (m, 2H), 2.24-2.21 (m, 2H).
Example 11
(a) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5,11-dioxo-10((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[2,1-c][1,4]-benzodiazepine-5,11(10H,11aH)-dione (103)
A catalytic amount of tetrakistriphenylphosphinepalladium (0) (11.2 mg) was added to a mixture of the mono triflate 17 (380 mg), the pinnacol ester of 2-aminophenylboronic acid (124 mg) and sodium carbonate (120 mg) in ethanol (5 mL), toluene (5 mL) and water (5 mL). The reaction mixture was allowed to stir over night at room temperature and at 40° C. until the reaction was complete (c. 2 hr). The reaction mixture was diluted with ethyl acetate and the organic layer was washed with water and brine. The ethyl acetate solution was dried over magnesium sulphate and filtered under vacuum. Removal of ethyl acetate by rotary evaporation under reduced pressure afforded the crude product which was subjected to flash chromatography (silica gel, ethyl acetate/hexane). Pure fractions were collected and combined. Removal of excess eluent by rotary evaporation under reduced pressure afforded the pure product 103 (330 mg, 86% yield). LC/MS RT: 4.17 min, ES+1018.48.
(b) (S)-2-(2-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one (104)
A solution of Superhydride in dry tetrahydrofuran (1.0 M, 4.4 eq.) was added to a solution of the 2-analino compound 103 (300 mg) in dry tetrahydrofuran (5 mL) at −78° C. under an inert atmosphere. As reduction was proceeding slowly an aliquot of lithium borohydride (20 eq.) was added and the reaction mixture was allowed to return to room temperature. Water/ice was added to the reaction mixture to quench unreacted hydrides and the reaction was diluted with dichloromethane. The organic layer was washed sequentially with water (twice), citric acid and brine. Excess dichloromethane was removed by rotary evaporation under reduced pressure and the residue was redissolve in ethanol and water and treated with silica gel for 96 hours. The reaction mixture was vacuum filtered and the filtrate evaporated to dryness. The residue was subjected to flash column chromatography (silica gel, gradient chloroform/methanol). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under educed pressure to afford the pure product 104 (30 mg, 14% yield). LC/MS RT: 2.90 min, ES+726.09.
Example 12
Determination of In Vitro Cytotoxicity of Representative PBD Compounds
K562 Assay
K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37° C. in a humidified atmosphere containing 5% CO2 and were incubated with a specified dose of drug for 1 hour or 96 hours at 37° C. in the dark. The incubation was terminated by centrifugation (5 min, 300 g) and the cells were washed once with drug-free medium. Following the appropriate drug treatment, the cells were transferred to 96-well microtiter plates (104 cells per well, 8 wells per sample). Plates were then kept in the dark at 37° C. in a humidified atmosphere containing 5% CO2. The assay is based on the ability of viable cells to reduce a yellow soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Aldrich-Sigma), to an insoluble purple formazan precipitate. Following incubation of the plates for 4 days (to allow control cells to increase in number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well and the plates further incubated for 5 h. The plates were then centrifuged for 5 min at 300 g and the bulk of the medium pipetted from the cell pellet leaving 10-20 μL per well. DMSO (200 μL) was added to each well and the samples agitated to ensure complete mixing. The optical density was then read at a wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and a dose-response curve was constructed. For each curve, an IC50 value was read as the dose required to reduce the final optical density to 50% of the control value.
Compound 13 has an IC50 of 30 μM in this assay.
A2780 Assay
The A2780 parental cell line was grown in Dulbecco's Modified Eagles' Media (DMEM) containing ˜10% Foetal Calf Serum (FCS) and ˜1% 200 mM L-Glutamine solution and grown in Corning Cellbind 75 cm2 flasks.
A 190 μl cell suspension was added (at 1×104) to each well of columns 2 to 11 of a 96 well plate (Nunc 96F flat bottom TC plate). 190 μl of media was added to each well of columns 1 and 12. The media was Dulbecco's Modified Eagles' Media (DMEM) (which included ˜10% Foetal Calf Serum (FCS) and ˜1% 200 mM L-Glutamine solution).
Plates were incubated overnight at 37° C. before addition of drug if cells were adherent. 200 μM of the test compound solutions (in 100% DMSO) were serially diluted across a 96 well plate. Each resulting point was then further diluted 1/10 into sterile distilled water (SDW).
To the cell negative blanks and compound negative control wells, 10% DMSO was added at 5% v/v. Assay plates were incubated for the following durations at 37° C. in 5% CO2 in a humidified incubator for 72 hours. Following incubation, MTT solution to a final concentration of 1.5 μM was added to each well. The plates were then incubated for a further 4 hours at 37° C. in 5% CO2 in a humidified incubator. The media was then removed, and the dye was solubilised in 200 μl DMSO (99.99%).
Plates were read at 540 nm absorbance using an Envision plate reader. Data was analysed using Microsoft Excel and GraphPad Prism and IC50 values obtained.
Compound II has an IC50 of 11.7 pM in this assay.
Renal Cell and AML Cell Lines Assays
The cytotoxicity of various free drug compounds was tested on a renal cell cancer cell line, 786-O, a Hodgkin lymphoma cell line, L428 and two AML cell lines, HL60 and HEL. For a 96-hour assay, cells cultured in log-phase growth were seeded for 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of test article (i.e., free drug) in cell culture media were prepared at 4× working concentration; 50 μL of each dilution was added to the 96-well plates. Following addition of test article, the cells were incubated with test articles for 4 days at 37° C. Resazurin was then added to each well to achieve a 50 μM final concentration, and the plates were incubated for an additional 4 h at 37° C. The plates were then read for the extent of dye reduction on a Fusion HT plate reader (Packard Instruments, Meridien, Conn., USA) with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
Referring to the following Table 1, the para-aniline compound II showed markedly increased activity on these cell lines as compared to the meta-aniline compound 19 in this assay.
TABLE 1
IC50 Summary for Free Drugs [nM]
Free Drug
L428
786-O
HL60
HEL
Compound 11
<0.00001
<0.00001
<0.00001
<0.00001
Compound 19
1
0.5
0.6
0.2
Referring to the following Table 2, the activity of compounds 28, 30 and 32 is shown on L428, 786-O, HEL, HL-60 and MCF-7 cells, as well as the activity for compound 19 on MCF-7 cells.
TABLE 2
IC50 Summary for Free Drugs [nM]
Free Drug
L428
786-O
HEL
HL-60
MCF-7
Compound 28
<0.00001
<0.00001
<0.00001
<0.00001
<0.00001
Compound 30
<0.00001
<0.00001
<0.00001
<0.00001
0.01
Compound 32
<0.00001
<0.00001
<0.00001
<0.00001
1.0
Compound 19
5
Referring to the following Table 3, the activities of compounds 23, 25, are compared to that of compound II on 786-O, Caki-1, MCF-7, HL-60, THP-1, HEL, and TF1 cells. Cells were plated in 150 μL growth media per well into black-sided clear-bottom 96-well plates (Costar, Corning) and allowed to settle for 1 hour in the biological cabinet before placing in the incubator at 37° C., 5% CO2. The following day, 4× concentration of drug stocks were prepared, and then titrated as 10-fold serial dilutions producing 8-point dose curves and added at 50 μl per well in duplicate. Cells were then incubated for 48 hours at 37° C., 5% CO2. Cytotoxicity was measure by incubating with 100 μL Cell Titer Glo (Promega) solution for 1 hour, and then luminescence was measured on a Fusion HT plate reader (Perkin Elmer). Data was processed with Excel (Microsoft) and GraphPad (Prism) to produce dose response curves and IC50 values were generated and data collected.
TABLE 3
IC50 Summary for Free Drugs [nM]
Free
Drug
786-O
Caki-1
MCF-7
HL-60
THP-1
HEL
TF1a
Com-
0.4
0.2
1
0.01
1
0.03
1
pound
11
Com-
0.06
0.02
0.7
0.005
0.4
0.009
0.2
pound
23
Com-
0.09
0.06
0.8
0.01
0.9
0.02
0.9
pound
25
In Examples 13 to 16, the following compounds are referred to by the compound numbers as show below:
Alternative
Compound
Designation
11
37
13
57
19
42
25
95
28
50
30
49
104
66
Example 13
Synthesis of PBD Drug Linker Compounds
General Information. In the following examples, all commercially available anhydrous solvents were used without further purification. Analytical thin layer chromatography was performed on silica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, N.J.). Radial chromatography was performed on Chromatotron apparatus (Harris Research, Palo Alto, Calif.). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Samples were eluted over a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reverse-phase column. The acidic mobile phase consisted of acetonitrile and water both containing either 0.05% trifluoroacetic acid or 0.1% formic acid (denoted for each compound). Compounds were eluted with a linear gradient of acidic acetonitrile from 5% at 1 min post injection, to 95% at 11 min, followed by isocratic 95% acetonitrile to 15 min (flow rate=1.0 mL/min). LC-MS was performed on a ZMD Micromass mass spectrometer interfaced to an HP Agilent 1100 HPLC instrument equipped with a C12 Phenomenex Synergi 2.0×150 mm, 4 μm, 80 Å reverse phase column. The acidic eluent consisted of a linear gradient of acetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min, followed by isocratic 95% acetonitrile for 5 min (flow rate=0.4 mL/min). Preparative HPLC was carried out on a Varian ProStar 210 solvent delivery system configured with a Varian ProStar 330 PDA detector. Products were purified over a C12 Phenomenex Synergi 10.0×250 mm, 4 μm, 80 Å reverse phase column eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The purification method consisted of the following gradient of solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL/min with monitoring at 254 nm. NMR spectral data were collected on a Varian Mercury 400 MHz spectrometer. Coupling constants (J) are reported in hertz.
(S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (36)
To a solution of Val-Ala dipeptide 34 (200 mg, 1.06 mmol) dissolved in 10.6 mL anhydrous DMF was added maleimidocaproyl NHS ester 35 (327 mg, 1.06 mmol). Diisopropylethyamine (0.92 mL, 5.3 mmol) was then added and the reaction was stirred under nitrogen at an ambient temperature for 18 h, at which time TLC and analytical HPLC revealed consumption of the starting material. The reaction was diluted with 0.1 M HCl (100 mL), and the aqueous layer was extracted with ethyl acetate (100 mL, 3×). The combined organic layer was washed with water and brine, then dried over sodium sulfate, filtered and concentrated. The crude product was dissolved in minimal methylene chloride and purified by radial chromatography on a 2 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (95:5 to 90:10 CH2Cl2/MeOH) to provide 36 (158 mg, 39%) as an oily residue. TLC: Rf=0.26, 10% MeOH in CH2Cl2. 1H NMR (CDCl3) δ (ppm) 0.95 (d, J=17 Hz, 3H), 0.98 (d, J=17 Hz, 3H), 1.30 (m, 2H), 1.40 (d, J=17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1H), 2.25 (dt, J=4, 19 Hz, 2H), 3.35 (s, 1H), 3.49 (t, J=17 Hz, 2H), 4.20 (d, J=18 Hz, 1H), 4.38 (m, 1H), 6.80 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.05 min. LC-MS: tR 11.17 min, m/z (ES+) found 381.9 (M+H)+, m/z (ES−) found 379.9 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (38)
A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.5 μmol), EEDQ (2.8 mg, 11.4 μmol), and 0.33 mL anhydrous CH2Cl2. Methanol (four drops, ˜80 μL) was added to facilitate dissolution and the mixture was stirred under nitrogen for 1 h. PBD dimer 37 (5.7 mg, 7.9 μmol) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide the drug linker 38 (3.9 mg, 45%). TLC: Rf=0.06, 5% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.51 min. LC-MS: tR 12.73 min, m/z (ES+) found 1089.6 (M+H)+, m/z (ES−) found 1087.3 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (40)
To a flame-dried 10 mL flask was added PBD dimer 37 (25 mg, 34.4 μmol), which was dissolved in 1.4 mL of a 10% MeOH in CHCl3 solvent mixture. Maleimidocaproic acid (39) was added (7.3 mg, 34.4 μmol), followed by EEDQ (10.2 mg, 41.3 μmol) and pyridine (6 μL, 68.8 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 14 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker 40 (14.1 mg, 45%). LC-MS: tR 12.81 min, m/z (ES+) found 918.9 (M+H)+, m/z (ES−) found 917.0 (M−H)−.
2-bromo-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)acetamide (41)
To a flame-dried 10 mL flask was added PBD dimer 37 (16.5 mg, 22.7 μmol), which was dissolved in 0.9 mL of a 10% MeOH in CHCl3 solvent mixture. Bromoacetic acid was added (3.2 mg, 22.7 μmol), followed by EEDQ (6.8 mg, 27.2 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 4 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 95:5 CH2Cl2/MeOH) to provide drug linker 41 (9.9 mg, 52%). TLC: Rf=0.09, 5% MeOH in CH2Cl2. LC-MS: tR 12.44 min, m/z (ES+) found 848.1 (M+H)+, m/z (ES−) found 845.7 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (43)
A flame-dried 10 mL flask was charged with acid 36 (3.6 mg, 9.4 μmol), EEDQ (2.8 mg, 11.3 μmol), and 0.38 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 42 (6.8 mg, 9.4 μmol) was then added and the reaction was stirred at room temperature for 2 h, at which time LC-MS revealed conversion to product. The reaction was concentrated, dissolved in minimal CH2Cl2, and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker 43 (3.1 mg, 30%). TLC: Rf=0.31, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.49 min. LC-MS: tR 12.28 min, m/z (ES+) found 1089.5 (M+H)+, m/z (ES−) found 1087.3 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (44)
To a flame-dried 10 mL flask was added PBD dimer 42 (8.0 mg, 11 μmol), which was dissolved in 0.44 mL of a 10% MeOH in CH2Cl2 solvent mixture. Maleimidocaproic acid (39) was added (2.3 mg, 11 μmol), followed by EEDQ (3.3 mg, 13.2 μmol) and pyridine (1.8 μL, 22 μmol). The reaction was stirred at room temperature under a nitrogen atmosphere for 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide drug linker compound 44 (1.2 mg, 12%). TLC: Rf=0.45, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.71 min. LC-MS: tR 12.63 min, m/z (ES+) found 919.1 (M+H)+, m/z (ES−) found 917.1 (M−H)−.
(2S,3R,4S,5R,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltriacetate (46)
A flame-dried flask was charged with glucuronide linker intermediate 45 (reference: Jeffrey et al., Bioconjugate Chemistry, 2006, 17, 831-840) (15 mg, 20 μmol), 1.4 mL anhydrous CH2Cl2, pyridine (20 μL, 240 μmol), and then cooled to −78° C. under nitrogen. Diphosgene (3.0 μL, 24 μmol) was then added and the reaction was stirred for 2 h at −78° C., after which time a small aliquot was quenched with methanol and analyzed by LC-MS for formation of the methyl carbonate, which confirmed formation of the glucuronide chloroformate. PBD dimer 42 (15 mg, 20 μmol) was then dissolved in 0.7 mL anhydrous CH2Cl2 and added dropwise to the reaction vessel. The reaction was warmed to 0° C. over 2 h and then diluted with 50 mL CH2Cl2. The organic layer was washed with water (50 mL), brine (50 mL), dried over sodium sulfate, filtered and concentrated. The crude reaction product was purified by radial chromatography on a 1 mm chromatotron plate eluted 10% MeOH in CH2Cl2 to provide 46 (5.7 mg, 19%). TLC: Rf=0.47, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 12.09 min. LC-MS: tR 14.05 min, m/z (ES+) found 1500.3 (M+H)+.
(2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (47)
A flask containing 46 (5.7 mg, 3.8 μmol) dissolved in a solvent mixture of 0.2 mL each of MeOH, tetrahydrofuran, and water was cooled to 0° C. To the stirred solution was added lithium hydroxide monohydrate (0.8 mg, 19 μmol) and the reaction was stirred at room temperature for 4 h, at which time LC-MS indicated conversion to product. Glacial acetic acid (1.1 μL, 19 μmol) was added and the reaction was concentrated to provide 47, which was carried forward without further purification. LC-MS: tR 11.59 min, m/z (ES+) found 1138.4 (M+H)+.
(2S,3S,4S,5R,6S)-6-(2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)-4-((((3-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (48)
To a solution of 47 (4.3 mg, 3.8 umol) dissolved in 0.38 mL anhydrous DMF was added maleimidocaproyl NHS ester 35 (1.2 mg, 3.8 umol), followed by diisopropylethylamine (4.0 uL, 22.8 umol). The reaction was stirred at room temperature under nitrogen for 2 h, at which time LC-MS revealed conversion to product. The reaction was diluted with a mixture of acetonitrile (0.5 mL), DMSO (1 mL), water (0.5 mL), and then purified by preparative HPLC. The mobile phase consisted of A=water and B=acetonitrile, both containing 0.1% formic acid. A linear elution gradient of 90:10 A:B to 10:90 A:B over 75 minutes was employed and fractions containing the desired product were lyophilized to provide drug linker compound 48 (1.2 mg, 24% over two steps). Analytical HPLC (0.1% formic acid): tR 10.85 min. LC-MS: tR 12.12 min, m/z (ES+) found 1331.4 (M+H)+, m/z (ES−) found 1329.5 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((3-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (51)
A flame-dried 10 mL flask was charged with acid 36 (2.7 mg, 7.1 μmol), EEDQ (2.1 mg, 8.5 μmol), and 0.28 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 49 (5.8 mg, 7.1 μmol) was then added and the reaction was stirred at room temperature for 20 h, at which time LC-MS revealed conversion to product. The reaction was concentrated then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug linker compound 51 (2.7 mg, 32%). Analytical HPLC (0.1% formic acid): tR 9.17 min. LC-MS: tR 11.25 min, m/z (ES+) found 1185.3 (M+H)+, m/z (ES−) found 1182.9 (M−H)−.
N—((S)-1-(((S)-1-((3-((S)-8-((5-(((S)-2-(4-(3-(dimethylamino)propoxy)phenyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (52)
A flame-dried 10 mL flask was charged with acid 36 (3.7 mg, 9.7 μmol), EEDQ (2.9 mg, 11.6 μmol), and 0.4 mL anhydrous CH2Cl2 containing 1% methanol. The reaction was stirred under nitrogen for 1 h; PBD dimer 50 (8.0 mg, 9.7 μmol) was then added and the reaction was stirred at room temperature for 6 h, at which time LC-MS revealed the presence of product. The reaction was concentrated then purified by preparative HPLC and fractions containing the desired product were lyophilized to provide drug linker compound 52 (3.1 mg, 25%). Analytical HPLC (0.1% formic acid): tR 9.45 min. LC-MS: tR 11.75 min, m/z (ES+) found 1188.4 (M+H)+, m/z (ES−) found 1186.0 (M−H)−.
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)benzamide (54)
To a flame-dried 10 mL flask was added linker fragment 53 (7.7 mg, 20 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.1 mg, 25 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (12 mg, 16.5 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide 54 (2.4 mg, 13%). TLC: Rf=0.44, 10% MeOH in CH2Cl2. Analytical HPLC (0.05% trifluoroacetic acid): tR 11.53 min. LC-MS: tR 12.61 min, m/z (ES+) found 1095.4 (M+H)+, m/z (ES−) found 1093.9 (M−H)−.
(S)-2-(2-iodoacetamido)-N-((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (56)
A flame-dried flask was charged with linker 55 (7.8 mg, 22 μmol), which was dissolved in 0.37 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (6.8 mg, 27.5 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (13 mg, 18 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 56 (3.5 mg, 18%). Analytical HPLC (0.1% formic acid): tR 11.43 min. LC-MS: tR 12.49 min, m/z (ES+) found 1064.6 (M+H)+, m/z (ES−) found 1098.9 (M+2H2O—H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (58)
To a flame-dried 10 mL flask was added linker fragment 36 (19 mg, 50 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (12.4 mg, 50 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 57 (12.5 mg, 16.6 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 5 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 58 (2.1 mg, 11%). Analytical HPLC (0.1% formic acid): tR 12.19 min. LC-MS: tR 12.58 min, m/z (ES+) found 1117.8 (M+H)+, m/z (ES−) found 1133.7 (M+H2O—H)−.
(R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoic acid (60)
A flame dried flask was charged with Fmoc-D-Valine (200 mg, 0.59 mmol) and 5.9 mL anhydrous THF. N-hydroxysuccinimide (75 mg, 0.65 mmol) was added, followed by diisopropylcarbodiimide (0.1 mL, 0.65 mmol), and the reaction was stirred at an ambient temperature overnight, at which time LC-MS revealed conversion to product. The reaction mixture was diluted with CH2Cl2 and washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to dryness. The material was carried forward without further purification. LC-MS: tR 13.89 min, m/z (ES+) found 437.0 (M+H)+. Crude Fmoc-D-Val-OSu (0.59 mmol) was dissolved in dimethoxyethane (1.5 mL) and THF (0.8 mL). D-alanine (73 mg, 0.89 mmol) was dissolved in 2.3 mL water and added to the reaction mixture, followed by sodium bicarbonate (99 mg, 1.2 mmol). The resulting slurry was stirred at room temperature overnight, at which time the reaction had clarified and LC-MS revealed completion. The reaction was poured into 50 mL CH2Cl2 and the organic layer was washed with 50 mL 0.1 M HCl and then brine, dried over sodium sulfate, and then concentrated to dryness. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2 to provide 60 (128 mg, 54%). TLC: Rf=0.18, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 9.47 min. LC-MS: tR 13.09 min, m/z (ES+) found 411.1 (M+H)+, m/z (ES−) found 409.2 (M−H)−.
(R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (61)
Protected dipeptide 60 (70 mg, 0.37 mmol) was suspended in 6 mL anhydrous CH2Cl2, cooled on ice under nitrogen, and 2 mL of diethylamine was added dropwise. The reaction was warmed to room temperature and stirred under nitrogen for 2 h, at which time HPLC revealed consumption of starting material. The reaction was diluted with 6 mL of chloroform and concentrated. The crude reaction residue was re-dissolved in 6 mL chloroform and concentrated twice, followed by drying on a vacuum line for 2 h. The deprotected dipeptide was then dissolved in 3.7 mL anhydrous DMF. MC-OSu (138 mg, 0.44 mmol) was then added, followed by diisopropylethylamine (0.32 mL, 1.9 mmol). The reaction was stirred under a nitrogen atmosphere at room temperature overnight. Workup was achieved by pouring the reaction in to 50 mL 0.1 M HCl and extracting with ethyl acetate (50 mL, 3×). The combined organic layer was washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and concentrated. The crude product was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (99:1 to 95:5 CH2Cl2/MeOH) to provide 61 (14 mg, 22%). 1H NMR (CD3OD) δ (ppm) 0.94 (d, J=14 Hz, 3H), 0.98 (d, J=14 Hz, 3H), 1.29 (m, 2H), 1.39 (d, J=7.4 Hz, 3H), 1.61 (m, 4H), 2.05 (m, 1H), 2.25 (dt, J=1.2, 7.4 Hz, 2H), 3.48 (t, J=7 Hz, 2H), 4.19 (m, 1H), 4.37 (m, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 10.04 min. LC-MS: tR 11.22 min, m/z (ES+) found 382.1 (M+H)+, m/z (ES−) found 380.0 (M−H)−.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((R)-1-(((R)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (62)
To a flame-dried 10 mL flask was added linker 61 (9.5 mg, 25 μmol), which was dissolved in 0.33 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (7.3 mg, 30 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (12 mg, 16.5 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 3 h, at which time LC-MS revealed conversion to product. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide 62 (2.8 mg, 16%). TLC: Rf=0.39, 10% MeOH in CH2Cl2. Analytical HPLC (0.1% formic acid): tR 11.50 min. LC-MS: tR 12.50 min, m/z (ES+) found 1089.7 (M+H)+, m/z (ES−) found 1088.0 (M−H)−.
(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanoic acid (64)
L-alanine (58 mg, 0.65 mmol) was suspended in 6.5 mL anhydrous DMF and MC-OSu 35 (100 mg, 0.324 mmol) was then added. Diisopropylethylamine (0.28 mL, 1.6 mmol) was added and the reaction was stirred overnight at room temperature under nitrogen. The reaction was then diluted with 50 mL 0.1 M HCl and the aqueous layer was then extracted with ethyl acetate (50 mL, 3×). The combined organic layer was then washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and then concentrated to dryness. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (97.5:2.5 to 90:10 CH2Cl2/MeOH) to provide 64 (25 mg, 27%). TLC: Rf=0.25, 10% MeOH in CH2Cl2. 1H NMR (CD3OD) δ (ppm) 1.30 (m, 2H), 1.37 (d, J=7.4 Hz, 3H), 1.60 (m, 4H), 2.21 (t, J=7.4 Hz, 2H), 3.48 (t, J=7 Hz, 2H), 4.35 (q, J=7.4 Hz, 1H), 6.78 (s, 2H). Analytical HPLC (0.1% formic acid): tR 9.06 min.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)hexanamide (65)
To a flame-dried 10 mL flask was added linker 64 (14 mg, 50 μmol), which was dissolved in 0.66 mL of a 5% MeOH in CH2Cl2 solvent mixture. EEDQ (15 mg, 60 μmol) was added and the reaction was stirred at room temperature under nitrogen for 15 minutes, at which time PBD dimer 37 (24 mg, 33 μmol) was added. The reaction was stirred at room temperature under a nitrogen atmosphere for an additional 4 h. The reaction was purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide 65 (3.5 mg, 11%). Analytical HPLC (0.1% formic acid): tR 11.40 min. LC-MS: tR 12.39 min, m/z (ES+) found 990.6 (M+H)+, m/z (ES−) found 989.0 (M−H)−.
PBD dimer 57 linked directly through maleimidocaproyl spacer (Scheme 14): PBD dimer 57 is coupled to maleimidocaproic acid 39 employing the chemistry described in Scheme 2.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (68)
To a mixture of the 66 (10 mg, 0.013 mmol) in CH2Cl2 (300 μL) was added DIPEA and MC-Cl (67) (3 mg, 0.013 mmol). After 1 h, an additional 3 equiv. of DIPEA (7 μL) and 2 equiv. of the acid chloride (6 mg, 0.026 mmol) were added. After 1 h, an additional quantity of DIPEA (7 μL) and acid chloride (6 mg, 0.026 mmol) were added. After an additional 3 h, the reaction mixture was aspirated directly onto a 1 mm radial chromatotron plate and eluted with dichloromethane followed by a gradient of methanol (1% to 5%) in dichloromethane. Product containing fractions, as a mixture with the starting aniline, were concentrated to a residue and dissolved in a mixture of 0.5 mL DMSO, 0.5 mL acetonitrile and 0.5 mL deionized water and was further purified by preparative HPLC. The major peak was collected and the fractions were combined, frozen and lyophilized to give 2.1 mg (18%): MS (ES+) m/z 919.2 [M+H]+.
Note: Acid chloride 67 was prepared by dissolving 100 mg of 39 in oxalyl chloride (5 mL). A drop of DMF was added and the mixture was stirred at an ambient temperature for several hours before being concentrated under reduced pressure. Dichloromethane was added and the mixture was concentrated a second time to afford an off-white solid which was used directly: 1H-NMR (400 MHz, CDCl3) δ 6.70 (s, 2H), 3.46 (t, J=7 Hz, 2H), 2.82 (t, J=7.2 Hz, 2H), 1.72 (pent, J=7.6 Hz, 2H), 1.61 (pent, J=7.4 Hz, 2H), 1.35 (pent, J=7.6 Hz, 2H).
tert-butyl 2-(2-aminoacetamido)acetate (69)
To a mixture of the glycine tert-butyl ester hydrogen chloride salt (70) (484 mg, 2.9 mmol) in dichloromethane (25 mL) was added Fmoc-Gly-OH (71) (0.861 mg, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was stirred at an ambient temperature for 16 h and then poured into ethyl acetate and was washed with water (3×) and brine (1×). The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The resulting residue was purified via radial chromatography on a 2 mm plate eluting with 5% methanol/dichloromethane. Product containing fractions were concentrated under reduced pressure and treated with 20% piperidine/dichloromethane (10 mL) for 1 h, before being concentrated under reduced pressure and then purified twice via radial chromatography on a 2 mm plate eluting with a gradient of 5 to 10% methanol/dichloromethane to provide (200 mg, 37%): 1H-NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 4.00 (s, 2H), 3.39 (s, 2H), 1.47 (s, 9H).
2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetamido)acetic acid (72)
To a solution of the amine 69 (200 mg, 0.11 mmol) in DMF (1 mL) was added 35 (350 mg, 0.11 mmol) and the reaction mixture was allowed to stir at an ambient temperature for 2 h. The mixture was concentrated under reduced pressure and was purify by radial chromatography on a 1 mm plate eluting with dichloromethane and a gradient of methanol (1 to 5%) in dichloromethane. Product containing fractions were concentrated under reduced pressure, dissolved in dichloromethane (4 mL) and treated with trifluoroacetic acid (4 mL). After 40 min the mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated to give 22.5 mg (19%) of 72 as white solid: 1H-NMR (400 MHz, CD3OD) δ 6.79 (s, 2H), 3.93 (s, 2H), 3.89 (s, 2H), 3.49 (t, J=6.8 Hz, 2H), 2.26 (t, J=6.8 Hz, 2H), 1.61 (m, 4H), 1.34 (m, 2H); MS (ES+) m/z 326.21 [M+H]+.
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (73)
To a mixture of 72 (15 mg, 0.046 mmol) in 5% methanol/dichloromethane (0.5 mL) was added EEDQ (11 mg, 0.046 mmol) and the mixture was stirred for 30 min at an ambient temperature, at which time 37 (16 mg, 0.023 mmol) was added. The reaction mixture was stirred for 3 h and was purified directly on a 1 mm radial chromatotron plate eluting with a 1% to 4% methanol/dichloromethane gradient to give 6.8 mg (29%) of 73 as a yellow solid: MS (ES+) m/z 1033.57 [M+H]+.
(S)-tert-butyl 1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (74)
To a mixture of L-proline-tert-butyl ester hydrogen chloride salt 75 (0.5 g, 2.9 mmol) in dichloromethane (50 mL) was added 76 (0.98 g, 2.99 mmol), DIPEA (756 mg, 4.35 mmol) and HATU (1.3 g, 3.5 mmol). The reaction mixture was allowed to stir at an ambient temperature for 16 h. The mixture was poured into ethyl acetate (100 mL) and was washed with 0.2 N HCl (50 mL), water (50 mL), brine (50 mL) and dried over MgSO4. Chromatography was conducted on a 2 mm radial chromatotron plate eluting with 10% ethyl acetate in hexanes. Product-containing fractions were concentrated under reduced pressure, dissolved in dichloromethane (8 mL) and treated with piperidine (2 mL). The mixture was stirred for 1 h, concentrated under reduced pressure and purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane. This gave 200 mg (26%) of the dipeptide 74: 1H-NMR (400 MHz, CDCl3) δ 4.41 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H), 3.57 (m, 4H), 3.2 (m, 1H), 2.82 (m, 1H), 2.83-1.65 (m, 5H), 1.44 (m, 9H).
(S)-tert-butyl 1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylate (77)
To a mixture of the amine 74 (200 mg, 0.75 mmol), 39 (190 mg, 0.9 mmol) and DIPEA (0.32 mL, 1.8 mmol) was added HATU (342 mg, 0.9 mmol) and the mixture was allowed to stir at an ambient temperature for 5 h. The mixture was poured into ethyl acetate (100 mL) and washed with water (3×100 mL) and brine (1×100 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated. The resulting residue was subjected to radial chromatography on a 2 mm radial chromatotron plate eluting with dichloromethane followed by an increasing gradient of 1 to 5% methanol in dichloromethane. Two additional purifications, both eluting with a gradient of 1 to 5% methanol in dichloromethane, first on a 2 mm plate and then on a 1 mm plate afforded 113 mg (33%) of 77 as an white solid: 1H-NMR (400 MHz, CDCl3) δ 4.63 (m, 1H), 4.41 (m, 1H), 3.82 (m, 1H), 3.63 (m, 1H), 3.55 (m, 1H), 3.45 (m, 3H), 2.38-1.83 (m, 10H), 1.70-1.50 (m, 5H), 1.45 (m, 9H), 1.35 (m, 2H); MS (ES+) m/z 462.33 [M+H]+.
(S)-1-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)pyrrolidine-2-carboxylic acid (78)
To a mixture of the tert-butyl ester 77 in dichloromethane (4 mL) was added trifluoroacetic acid (4 mL). After 40 min the reaction was determined to be complete by HPLC analysis. The mixture was concentrated under reduced pressure and the resulting residue was dissolved in dichloromethane and concentrated a second time to give 37 mg (100%) of 78 as a white solid: 1H-NMR (400 MHz, CDCl3) δ 6.68 (s, 2H), 4.62 (m, 2H), 3.81 (m, 1H), 3.70 (m, 1H), 3.57 (m, 2H), 3.45 (m, 2H), 2.40-1.91 (m, 10H), 1.70-1.45 (m, 4H), 1.33 (m, 2H); MS (ES+) m/z 406.2 [M+H]+.
1-(1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)pyrrolidine-2-carbonyl)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)pyrrolidine-2-carboxamide (79)
To a mixture of the 78 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (0.4 mL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 15 min at an ambient temperature and then 37 (15 mg, 0.021 mmol) was added. The mixture was stirred for 4 h, the reaction mixture was diluted with dichloromethane (2 mL) and was aspirated directly onto a 1 mm radial chromatotron plate. The product was eluted with a gradient of 1 to 5% methanol in dichloromethane to provide 6.8 mg (29%) of 79 as a yellow solid: MS (ES+) m/z 1113.51 [M+H]+.
(S)-5-(allyloxy)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-oxopentanoic acid (80)
To a mixture of the 2-chlorotrityl resin (1.0 g, 1.01 mmol) suspended in dichloromethane (10 ml) was added Fmoc-Glu-(OAllyl)-OH (81) (409 mg, 1.0 mmol) and DIPEA (173 μL, 1.0 mmol). The reaction mixture was shaken for 5 min, and an additional portion of DIPEA (260 μL, 1.5 mmol) was added and the mixture was shaken for 1 h. Methanol (0.8 mL) was added and the mixture was shaken for 5 min, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure. The resulting resin was subjected to 20% piperidine in dichloromethane (10 mL) for 1 h, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure.
To a mixture of the Fmoc-Val-OH (82) (1.03 g, 3.30 mmol)) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After thorough mixing, the solution as aspirated into a 10 mL syringe containing the resin prepared above. The mixture was capped and shaken for 16 h. The resin was washed with DMF (6×), dichloromethane (6×) and ether (6×). A small portion (10 mg) was isolated and treated with 20% TFA/Dichloromethane and the resulting solution analyzed by LC-MS which revealed one high purity peak which displayed the correct mass (MS (ES+) m/z 509.28 [M+H]+). The remaining resin was then treated with 20% piperidine/DMF (8 mL) for 2 h, before being washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure.
A mixture of allyl chloroformate (529 μL, 5.05 mmol), DIPEA (1.7 mL, 10 mmol) in dichloromethane (10 mL) was prepared and aspirated into a syringe containing the resin above. The mixture was capped and shaken. After approximately 2 h, the reaction mixture was drained, and washed with dichloromethane (6×). A small portion of the resin (˜10 mg) was cleaved with 20% TFA/dichloromethane and analyzed by LC-MS for masses of starting material and product. The main component was still the unreacted amine, so the resin was again subjected to the conditions described above. After 4 h, the resin was washed with dichloromethane (6×), and then treated repeatedly with 5% TFA in dichloromethane (4×7 mL). The resulting solution was concentrated under reduced pressure. The mixture was purified on a 2 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 107 mg of 80: 1H-NMR (400 MHz, CDCl3) δ 7.05 (s, 1H), 5.90 (m, 2H), 5.57 (d, 1H), 5.29 (d, J=14.7 Hz, 2H), 5.22 (t, J=10.9 Hz, 2H), 4.59 (m, 5H), 4.02 (m, 1H), 2.60-2.40 (m, 2H), 2.37-2.18 (m, 1H), 2.17-2.02 (m, 2H), 0.96 (d, J=6.4 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H); MS (ES+) m/z 371.12 [M+H]+.
(S)-allyl 4-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-5-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoate (83)
To a mixture of the acid 80 (30, 0.04 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (20 mg, 0.082 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (30 mg, 0.04 mmol) was added and the mixture was stirred for approximately 5 h. Partially purification by aspirating directly onto a 1 mm radial chromatotron plate and eluting with a gradient of 1% to 5% methanol/dichloromethane afforded a mixture of desired product and 37 (26 mg; ˜3:1 respectively) which was carried forward without further purification.
(S)-4-(S)-2-amino-3-methylbutanamido)-5-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoic acid (84)
To the mixture of 83 and 37 (26 mg) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 μmol), pyrrolidine (4 μL, 0.048 μmol) and tetrakis palladium (0.7 mg, 0.6 μmol). After 2 h, an additional quantity (0.7 mg, 0.6 μmol) of tetrakis palladium was added and the reaction was allowed to stir for an additional 1 hr before being concentrated under reduced pressure. The residue was dissolved in DMSO (1 mL), acetonitrile with 0.05% formic acid (1 mL) and water with 0.05% formic acid (1 mL) and purified by preparative reverse phase HPLC. A single fraction of product was collected and lyophilized to give 6 mg (14% for two steps) of 84: MS (ES+) m/z 1078.6 [M+H]+.
(S)-4-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-5-oxopentanoic acid (85)
To a mixture of the 84 (6 mg, 6 μmol), and 35 (2 mg, 6 μmol) in DMF (200 μL) was added DIPEA (3 μL, 18 μmol) and the reaction mixture was stirred at an ambient temperature. After 1 h, an additional equivalent of 35 (2 mg, 6 μmol) was added and the reaction was allowed to continue to stir at an ambient temperature for 3 h. A third equivalent of 35 (2 mg, 6 μmol) was added and the mixture was stirred for approximately 1 h, concentrated under reduced pressure, dissolved in dichloromethane and aspirated directly onto a 1 mm radial chromatotron plate and eluted with 5% methanol in dichloromethane. This gave 2.5 mg (36%) of high purity 85: MS (ES+) m/z 1147.49 [M+H]+.
(21S,24S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-isopropyl-24-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)carbamoyl)-3,19,22-trioxo-7,10,13,16-tetraoxa-4,20,23-triazaheptacosan-27-oic acid (86)
To a mixture of the 84 (8 mg, 8.4 μmol) and Mal-PEG4-NHS (87) (6.5 mg, 12.6 μmol) in DMF (200 μL) was added DIPEA (4.3 μL, 25 μmol). The reaction mixture was stirred at an ambient temperature for 2 h, and was concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane and aspirated onto a 1 mm radial chromatotron plate. The material was polar and did not chromatograph on the silica gel-based chromatotron plate. The plate was eluted with methanol to recover the mixture which was isolated under reduced pressure. The residual material was purified via preparative reverse phase HPLC. A single main peak eluted and the fractions were combined, frozen and lyophilized to a residue of 0.9 mg (8%) of 86: MS (ES+) m/z 1353.04 [M+H]+.
(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)hexanoic acid (88)
To a mixture of the 2-chlorotrityl resin (1 g, 1.01 mmol) in CH2Cl2 (10 ml) was added Fmoc-Lys(Me)2-OH (89) (432 mg, 1.0 mmol) and DIPEA (433 μL, 2.5 mmol). The reaction mixture was shaken for 1 h. Methanol (0.8 mL) was added and the mixture was shaken for an additional 5 min, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×) and dried under reduced pressure. The dried resin was subjected to 20% piperidine in DMF (10 mL) for 1 h, before being filtered and washed with DMF (6×), dichloromethane (6×), diethyl ether (6×).
To a mixture of the 39 (3.0 mmol, 633 mg) in DMF (7 mL) was added DIPEA (1.0 mL) and HATU (1.1 g, 3.03 mmol). After thorough mixing, the solution as aspirated into a 10 mL syringe containing the resin above. The mixture was capped, shaken for 16 h, filtered and the resin washed with DMF (6×), dichloromethane (6×), and ethyl ether (6×). The resin was by repeatedly treating with 5% TFA/dichloromethane (6 mL×5), shaking for 1 min, and then filtering. The resulting solution was concentrated under reduced pressure and under high vacuum. The material was purified by preparatory reverse phase HPLC to give 208 mg of 88: 1H-NMR (400 MHz, CD3OH/CDCl3 1:1 mixture) δ 6.73 (s, 2H), 4.41 (m, 1H), 3.48 (t, 2H), 3.31 (s, 1H), 3.03 (m, 2H), 2.84 (s, 6H), 2.22 (m, 2H), 1.87 (m, 2H), 1.78-1.52 (m, 6H), 1.43 (m, 2H), 1.31 (pent, 2H); MS (ES+) m/z 386.28 [M+H]+.
(S)-6-(dimethylamino)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-N-(4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)hexanamide (90)
To a mixture of the 88 (9.3 mg, 0.023 mmol) in 5% methanol/dichloromethane (400 μL) was added EEDQ (7 mg, 0.027 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (15 mg, 0.021 mmol) was added. After 4 h, the mixture was concentrated under reduced pressure, dissolved in a mixture of DMSO (1 mL), acetonitrile (2 mL containing 0.05% formic acid) and water (1 mL containing 0.05% formic acid) and purified by reverse-phase HPLC (method A). Product containing fractions were contaminated with 37, so the fractions were lyophilized to a residue and repurified as described above to give 0.5 mg (2%) of pure 90: MS (ES+) m/z 537.46 [M+H]/2+.
Allyl ((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (91)
To a mixture of the 92 (45 mg, 0.123 mmol) in 5% methanol/dichloromethane (1 mL) was added EEDQ (30.4 mg, 0.123 mmol). The mixture was stirred for 30 min at an ambient temperature and then 37 (30 mg, 0.041 mmol) was added. The reaction mixture was stirred for approximately 5 h and then purified on a 1 mm radial chromatotron plate eluting with 5% methanol/dichloromethane to give 22 mg (55%) of 91 which was not characterized but carried on directly.
1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (93)
To a solution of the 91 (22 mg, 0.022 mmol) in anhydrous dichloromethane (3 mL) was added Ph3P (0.3 mg, 0.0012 mmol), pyrrolidine (4 μL, 0.048 mmol) and tetrakis palladium (0.7 mg, 6 μmol). After approximately 2 h, the reaction mixture was purified on a 1 mm radial chromatotron plate eluting with 5% to 10% methanol/dichloromethane. The major band was collected and concentrated to a residue which was dissolved in DMF (0.2 mL) and reacted with NHS ester 87 (10 mg, 0.19 mmol). The reaction was allowed to stir for 30 min, concentrated and purified by radial chromatography on a 1 mm plate eluting with 5% methanol/dichloromethane to give 3.2 mg (11%) of 93: MS (ES+) m/z 1294.7 [M+H]+.
(E)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-(4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)benzylidene)hexanehydrazide (94)
To a mixture of the aldehyde 95 (5.4 mg, 7 μmol) in 5% methanol/dichloromethane at 0° C. was added the hydrazide-TFA salt 96 (4.5 mg, 14 μmol). The reaction mixture was allowed to warm to an ambient temperature and stir for 5 h before being concentrated under reduced pressure and purified on a silica gel column eluting with 3% methanol/dichloromethane to give 2.2 mg (32%) of 94: MS (ES+) m/z 974.49 [M+H]+.
(S)-tert-butyl 2-((S)-2-amino-3-methylbutanamido)propanoate (97)
To a mixture of the alanine-O-tert-butyl ester hydrogen chloride salt (98) (500 mg, 2.76 mmol) in dichloromethane (5 mL) was added Fmoc-val-OSu (99) (1.09 g, 2.51 mmol). DIPEA (0.96 ml, 5.5 mmol) was added and the reaction mixture was allowed to stir at an ambient temperature for 16 h. The mixture was poured into dichloromethane (100 mL) and washed with 1N HCl (50 mL) and water (50 mL) before being dried over magnesium sulfate. The material was chromatographed on a 2 mm radial chromatotron plate eluting with 1 to 5% methanol/dichloromethane gradient and product containing fractions were combined and concentrated. The resulting residue was dissolved in dichloromethane (16 mL) and piperidine (4 mL) was added. The mixture was stirred for 10 min before being concentrated under reduced pressure. The resulting residue was chromatographed on a 2 mm plate eluting first with ammonia-saturated dichloromethane followed by 5% methanol in ammonia-saturated dichloromethane to give 494 mg (2.02 mmol, 81% for two steps) of 97: 1H-NMR (400 MHz, CDCl3) δ 7.78 (bs, 1H), 4.47 (m, 1H), 3.30 (d, 1H), 2.30 (m, 1H), 1.38 (d, 3H), 1.47 (s, 9H), 1.00 (d, J=7.0 Hz, 3H), 0.84 (d, J=6.9 Hz, 3H).
(S)-tert-butyl 2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoate (100)
To a mixture of the 97 (100 mg, 0.41 mmol) and 4-maleimidobenzoic acid (101) (98 mg, 0.45 mmol) was added dichloromethane (5 mL), followed by TBTU (157 mg, 0.49 mmol) and DIPEA (212 uL, 1.23 mmol). The mixture was stirred at an ambient temperature for 16 h and then purified on a 2 mm radial chromatotron plate eluting with 50% ethyl acetate in hexanes to give 95 mg (51%) of 100: 1H-NMR (400 MHz, CDCl3) δ 7.85 (d, J=6.6 Hz, 2H), 7.42 (d, J=6.6 Hz, 2H), 6.81 (s, 2H), 6.38 (bs, 1H), 4.43 (m, 2H), 2.14 (sept, J=6.6 Hz, 1H), 1.41 (s, 9H), 1.31 (d, J=7.0 Hz, 3H), 0.98 (m, 6H); MS (ES−) m/z 441.90 [M−H]−.
(R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic acid (53)
To a mixture of 100 (47 mg, 0.11 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (5 mL) and the reaction mixture was monitored by TLC (50% ethyl acetate in hexane, after pumping down the TLC plate under high vacuum for 5 min). After 75 min, no starting material could be detected by TLC. The reaction was performed a second time using the same conditions and material from both reactions were combined and purified on a 2 mm radial chromatotron plate eluting with a gradient from 5-10% methanol in dichloromethane. The yield was 42 mg (49%) of 53: 1H-NMR (400 MHz, CDCl3) δ 7.92 (d, J=6.6 Hz, 2H), 7.51 (d, J=6.6 Hz, 2H), 7.0 (m, 1H), 6.89 (s, 2H), 6.70 (s, 1H), 4.60 M, 1H), 2.22 (m, 1H), 1.18 (d, J=6.6 Hz, 3H), 1.04 (m, 6H); MS (ES+) m/z 388.02 [M+H]+.
(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (102)
To a mixture of the 97 (100 mg, 0.41 mmol) in dichloromethane was added iodoacetamide-NHS ester (103) (115 mg, 0.41 mmol) and the mixture was stirred at an ambient temperature. After 30 min, the mixture was aspirated onto a 1 mm chromototron plate and eluted with ethyl acetate in hexanes (1:1). A single band was collected and the structure was confirmed: 1H-NMR (400 MHz, CDCl3) δ 6.70 (d, J=7.8 Hz, 1H), 6.27 (d, J=7.0 Hz, 1H), 4.45 (m, 1H), 4.26 (dd, J=8.6, 6.3 Hz, 1H), 3.72 (quart, J=11.3 Hz, 2H), 2.13 (sept, J=6.5 Hz, 1H), 1.47 (s, 9H), 1.38 (d, J=7.1 Hz, 3H), 0.99 (m, 6H); MS (ES+) m/z 412.87 [M+H]+.
(S)-2-((S)-2-(2-iodoacetamido)-3-methylbutanamido)propanoic acid (55)
See procedure for the synthesis of (R)-2-((S)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamido)-3-methylbutanamido)propanoic acid (53). This gave 22 mg (15% for two steps): 1H-NMR (400 MHz, D6-DMSO) δ 8.27 (d, J=9.4 Hz, 1H), 4.24 (m, 2H), 3.97 (bs, 2H), 3.83 (d, J=9.4 Hz, 1H), 3.71 (d, J=9.6 Hz, 1H), 2.07 (m, 1H), 1.33 (d, J=7.3 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H), 0.89 (d, J=6.9 Hz, 3H); MS (ES−) m/z 354.84 [M−H]−.
PBD dimers linked through aliphatic amines (Scheme 21). PBD dimers containing aliphatic amines, such as a benzyl amine (Example 9), are synthesized with peptidic linkers, the glucuronide linker, and/or linkers dependent on mAb degradation for release (i.e., non-cleavable linkers). Drug linkers conjugated through a benzyl amine will include: (1) a cleavable peptide employing chemistry similar to Scheme 1; (2) direct attachment with a maleimidocaproyl group (a noncleavable linker) (Scheme 2); (3) a glucuronide linker, prepared as described in Scheme 6.
Generic peptide linked 2-, 3-, and 4-aniline PBD dimers (Scheme 22). PBD dimers with anilines at the 2-, 3-, and 4-positions will be conjugated to peptide-based linkers, employing the chemistry described in Scheme 1, or attached directly with maleimidocaproic acid, as exemplified in Scheme 2.
Example 14
Preparation of PDB Dimer Conjugates
Antibody-drug conjugates were prepared as previously described (see Doronina et al., Nature Biotechnology, 21, 778-784 (2003)) or as described below. Briefly, for maleimide drug-linker the mAbs (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 were reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progress of the reaction, which reduces interchain disulfides, was monitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb was achieved. The reduced antibody was then cooled to 0° C. and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 h, the reaction was quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker was removed by gel filtration over a PD-10 column. The ADC was then sterile-filtered through a 0.22 μm syringe filter. Protein concentration was determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography was used to determine the extent of antibody aggregation and RP-HPLC confirmed the absence of remaining NAC-quenched drug-linker.
For halo acetamide-based drug linkers, conjugation was performed generally as follows: To a 10 mg/mL solution of reduced and reoxidized antibody (having introduced cysteines by substitution of S239C in the heavy chains (see infra)) in 10 mM Tris (pH 7.4), 50 mM NaCl, and 2 mM DTPA was added 0.5 volumes of propylene glycol. A 10 mM solution of acetamide-based drug linker in dimethylacetamide was prepared immediately prior to conjugation. An equivalent amount of propylene glycol as added to the antibody solution was added to a 6-fold molar excess of the drug linker. The dilute drug-linker solution was added to the antibody solution and the pH was adjusted to 8.0-8.5 using 1 M Tris (pH 9). The conjugation reaction was allowed to proceed for 45 minutes at 37° C. The conjugation was verified by reducing and denaturing reversed phase PLRP-S chromatography. Excess drug linker was removed with Quadrasil MP resin (Sigma Aldrich; Product #679526) and the buffer was exchanged into 10 mM Tris (pH 7.4), 50 mM NaCl, and 5% propylene glycol using a PD-10 desalting column (GE Heathcare; Product #17-0851-01).
Engineered hIgG1 antibodies with introduced cysteines: CD70 antibodies containing a cysteine residue at position 239 of the heavy chain (h1F6d) were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). Following a 1 hour incubation at 37° C., the reaction was cooled to 22° C. and 30 equivalents of dehydroascorbic acid were added to selectively reoxidize the native disulfides, while leaving cysteine 239 in the reduced state. The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction was allowed to proceed for 1 hour at 22° C. The pH of the solution was then raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5 equivalents of the PBD drug linker in DMSO were placed in a suitable container for dilution with propylene glycol prior to addition to the reaction. To maintain solubility of the PBD drug linker, the antibody itself was first diluted with propylene glycol to a final concentration of 33% (e.g., if the antibody solution was in a 60 mL reaction volume, 30 mL of propylene glycol was added). This same volume of propylene glycol (30 mL in this example) was then added to the PBD drug linker as a diluent. After mixing, the solution of PBD drug linker in propylene glycol was added to the antibody solution to effect the conjugation; the final concentration of propylene glycol is 50%. The reaction was allowed to proceed for 30 minutes and then quenched by addition of 5 equivalents of N-acetyl cysteine. The ADC was then purified by ultrafiltration through a 30 kD membrane. (Note that the concentration of propylene glycol used in the reaction can be reduced for any particular PBD, as its sole purpose is to maintain solubility of the drug linker in the aqueous media.)
Example 15
Determination of In Vitro Activity of Selected Conjugates
The in vitro cytotoxic activity of the selected antibody drug conjugates was assessed using a resazurin (Sigma, St. Louis, Mo., USA) reduction assay (reference: Doronina et al., Nature Biotechnology, 2003, 21, 778-784). The antibody drug conjugates were prepared as described above in Example 13.
For the 96-hour assay, cells cultured in log-phase growth were seeded for 24 h in 96-well plates containing 150 μL RPMI 1640 supplemented with 20% FBS. Serial dilutions of ADC in cell culture media were prepared at 4× working concentration; 50 μL of each dilution was added to the 96-well plates. Following addition of ADC, the cells were incubated with test articles for 4 days at 37° C. Resazurin was then added to each well to achieve a 50 μM final concentration, and the plates were incubated for an additional 4 h at 37° C. The plates were then read for the extent of dye reduction on a Fusion HT plate reader (Packard Instruments, Meridien, Conn., USA) with excitation and emission wavelengths of 530 and 590 nm, respectively. The IC50 value, determined in triplicate, is defined here as the concentration that results in a 50% reduction in cell growth relative to untreated controls.
Referring to Table 4 (infra), the in vitro cytotoxicity of ADCs having para-aniline PBD dimers using the 96 hour assay is shown. The ADCs were tested against CD70+ CD30− cell lines and a control CD70− CD30− cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942), a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) had a lower IC50 than conjugates with a maleimidyl or acetamide-based linker (compounds 40 and 41, respectively).
In vitro cytotoxic activity of ADCs bearing drug linkers derived from para-aniline PBD dimer 37:
TABLE 4
In vitro cytotoxic activity on CD70+ cell lines
(ng/mL), all ADCs 2 drugs/mAb
renal cell carcinoma
AML
CD70+/30−
CD70−/30−
786-O
Caki-1
769-P
ACHN
HEL9217
h1F6d-38
30
5
1378
h1F6-38
4
118
26
cAC10-38
1052
4005
508
h1F6-40
7113
1764
cAC10-40
2644
1264
h1F6-41
580
1243
cAC10-41
1153
1121
Referring to Table 5, the in vitro cytotoxicity of ADCs conjugate to PBD dimers on CD30+ cell lines using the 96 hour assay is shown. The ADCs were tested against CD30+CD70+ cell lines and a CD70− CD30+ cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289). Conjugates having a maleimidyl-peptide linker (drug linker compound 38) generally had a lower IC50 than conjugates with a maleimidyl or acetamide-based linker (compounds 40 and 41, respectively).
TABLE 5
In vitro cytotoxic activity on CD30+ cell lines
(ng/mL), all ADCs 2 drugs/mAb
ALCL
Hodgkin lymphoma
CD70−/30+
CD70+/30+
Karpas 299
L428
L540cy
L1236
Hs445
h1F6-38
1165
59
4
>10,000
5
cAC10-38
0.8
7
3
2012
0.2
h1F6-40
2195
7867
2557
cAC10-40
621
3172
134
h1F6-41
1330
3549
755
cAC10-41
340
957
13
In vitro cytotoxic activity of ADCs bearing drug linkers derived from meta-aniline PBD dimer 42:
Referring to Table 6, the in vitro cytotoxicity of ADCs containing PBD dimers on CD30+ cell lines using the 96 hour assay is shown. The activity was tested against CD30+ CD70+ cell lines and a CD70− CD30+ cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker (drug linker compound 43) and a glucuronide linker (48) generally had a lower IC50 than conjugates with a maleimidyl-based linker (compound 44).
TABLE 6
In vitro cytotoxic activity on CD70+ cell
lines (ng/mL)
renal cell
Hodgkin
carcinoma
lymphoma
Caki-1
786-O
L428
h1F6d-43 (2 dr/mAb)
7
39
>10,000
IgG-43 (2 dr/mAb)
>10,000
>10,000
h1F6-44 (3.5 dr/mAb)
1124
2142
IgG-44 (3.5 dr/mAb)
1491
1242
h1F6d-48 (2 dr/mAb)
89
4093
IgG-48 (2 dr/mAb)
2939
6376
In vitro cytotoxic activity of ADCs bearing drug linkers derived from para- and meta-aniline PBD dimers 38 and 42 (respectively):
Referring to Table 7, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines L428 and 786O and a CD70− AML cell line. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Conjugates having a maleimidyl-peptide linker with a meta-aniline (drug linker compound 43) were somewhat less active than those having a maleimidyl-peptide linker with a para-aniline (drug linker compound 38). Reducing the drug loading of the meta-aniline compound to 2 per antibody reduced the activity. Conjugates with a glucuronide linker of the para-aniline compound (48) generally had a lower IC50 than conjugates with a maleimidyl-based linker (compound 39). Further, an aryl maleimide of the para-aniline compound (54) has no activity on these cell lines. Further, a conjugate having a maleimidyl linker conjugated directly to compound 42 has reduced activity as compared with conjugate h1F6-43 (data not shown).
TABLE 7
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Hodgkin
Renal cell
lymphoma
carcinina
L428
786O
control
h1F6-43 (4 dr/mAb)
404
11
1205
h1F6d-43 (2 dr/mAb)
Max inhib. =
200
1625
40%
h1F6d-48 (2 dr/mAb)
4093
89
1964
h1F6-54 (4 dr/mAb)
No effect
No effect
No effect
h1F6-38 (2 dr/mAb)
230 (n = 2)
25 (n = 3)
503
In vitro cytotoxic activity of ADCs bearing drug linkers derived from aniline-linked PBD dimers
Referring to Table 8, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines Caki-1 and L428 and a CD70− cell line. The antibody used was a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Linkage of a PBD through an amine at the ortho position via a non-cleavable linker (compound 68) markedly reduced activity, as compared with an ADC linked via a para-aniline-linked cleavable linker (compound 54). Compounds 73 and 85, having a cleavable linker, showed comparable activity to compound 54; both of these compounds are linked via a para-aniline. Compounds with cleavable linkers requiring more stringent cleavage, compounds 79 and 90, showed somewhat reduced activity, as compared to compound 54.
TABLE 8
In vitro cytotoxic activity on CD70+
cell lines (ng/mL)
renal cell
carcinoma
Caki-1
786-O
Control
h1F6d-68 (2 dr/mAb)
3236
3486
5501
h1F6d-73 (2 dr/mAb)
2
7
482
h1F6d-79 (2 dr/mAb)
24
348
5385
h1F6d-54 (2 dr/mAb)
6
17
4665
h1F6d-85 (2 dr/mAb)
3
5
47UU
h1F6d-90 (1.4 dr/mAb)
. . . 12
47
678
In vitro cytotoxic activity of ADCs bearing drug linkers derived from aniline-linked PBD dimers
Referring to Table 9, the in vitro cytotoxicity of ADCs containing PBD dimers on CD70+ cell lines using the 96 hour assay is shown. The activity was tested against CD70+ cell lines Caki-1 and L428 and two CD70− leukemia cell lines. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD70 antibody (humanized 1F6) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d). Compound 56, having a cleavable linker linked to the antibody via an acetamide showed comparable activity to compound 38. A glucuronide-linked version of the meta-aniline linked PBD dimer, compound 48, demonstrated little activity in this assay. Compound 58, having five methylene groups in the PBD bridge, demonstrated comparable activity to compound 38, having three methylene groups in the PBD bridge.
TABLE 9
In vitro cytotoxic activity on CD70+ cell lines (ng/mL)
Renal Cell
Caki-1
786-O
Leukemia
(CD70
(CD70
CD70−
CD70−
ADCs
#135,000)
#190,000)
Line 1
Line 2
h1F6d-56 (1.8 dr/Ab)
3
6
1672
Max Inh =
50%
h1F6d-48 (0.6 dr/Ab)
Max Inh =
Max Inh =
No Effect
No Effect
45%
35%
h1F6d-58 (1.9 dr/Ab)
0.5
2
1750
4847
5
15
h1F6d-38 (2 dr/Ab)
(3-5,
(5-30,
2082
7188
n = 4)
n = 4)
Example 16
Determination of In Vivo Cytotoxicity of Selected Conjugates
All studies were conducted in concordance with the Animal Care and Use Committee in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. In vivo tolerability was first assessed to ensure that the conjugates were tolerated at clinically relevant doses. BALB/c mice were treated with escalating doses of ADC formulated in PBS with 0.01% Tween 20. Mice were monitored for weight loss following drug treatment; those that experienced 20% weight loss or other signs of morbidity were euthanized. The antibodies used were a CD70 antibody, humanized 1F6 (see Published U.S. Application No. 2009-148942) and a CD30 antibody, chimeric AC10 (see Published U.S. Application No. 2008-0213289).
Referring to FIG. 1, the results of a weight loss study are shown using cAC10-val-ala-SG3132(2) (cAC10-compound 38). A single dose of the conjugate administered at 5 mg administered either IP or IV resulted in little weight loss. A higher dose of the conjugate (15 mg/kg) caused weight loss in the mice.
Referring to FIG. 2, the results of a weight loss study are shown using h1F6-val-ala-SG3132(2) (h1F6-compound 38). A single dose of the conjugate administered at 5 mg administered IP resulted in some weight loss. A higher dose of the conjugate (10 mg/kg) caused significant weight loss in the mice.
Treatment studies were conducted in two CD70+ renal cell carcinoma xenograft models. Tumor (786-O and Caki-1) fragments were implanted into the right flank of Nude mice. Mice were randomized to study groups (n=5) on day eight (786-0) or nine (Caki-1) with each group averaging around 100 mm3. The ADC or controls were dosed ip according to a q4dx4 schedule. Tumor volume as a function of time was determined using the formula (L×W2)/2. Animals were euthanized when tumor volumes reached 1000 mm3. Mice showing durable regressions were terminated around day 100 post implant.
Referring to FIG. 3, the results of a treatment study using an h1F6-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. A control conjugate, cAC10-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice administered doses of the h1F6 conjugate at 0.1 mg/kg exhibited some tumor reduction, while higher doses at 0.3 mg/kg and 1 mg/kg appeared to exhibit complete tumor reduction. The control conjugate (non-binding) was less active the h1F6 conjugates.
Referring to FIG. 4, the results of a treatment study using an h1F6-mc-val-ala-SG3132(2) (h1F6-compound 38) conjugate are shown. A control conjugate, cAC10-mc-val-ala-SG3132(2) (cAC10-compound 38), was also used. Mice administered doses of the h1F6 conjugate at 1 mg/kg appeared to exhibit complete tumor reduction. Mice administered lower doses at 0.3 mg/kg and 0.1 mg/kg exhibited lesser tumor reduction, respectively. The control conjugate (non-binding) was less active the h1F6 conjugate administered at a similar dose, although it exhibited more activity than the h1F6 conjugate administered at lower doses. The h1F6 conjugate was also more active than an h1F6-vc-MMAE conjugate (Published U.S. Application No. 2009-0148942) administered at higher doses.
Referring to FIG. 5, the results of a treatment study using a two loaded antibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loaded non-binding control, H00d conjugated to the same compound (h00d-38). The model was a Caki subcutaneous model in Nude mice. Doses were 0.1, 0.3 and 1 mg/kg q7d×2. The highest two doses of the h1F6 conjugate demonstrated complete regressions as 1 mg/kg and substantial tumor delay at 0.3 mg/kg. The non-binding control demonstrated tumor delay at the 1 mg/kg dose.
Referring to FIG. 6, the results of a treatment study using a two loaded antibody h1F6d-linked to compound 38 (h1F6d-38) compared to a two-loaded non-binding control, H00d conjugated to the same compound (h00d-38). The model was a 786-0 subcutaneous model in Nude mice. Doses were 0.1, 0.3 and 1 mg/kg q7d×2. All three doses of the h1F6 conjugate demonstrated complete regressions or tumor delay, while the non-binding control demonstrated tumor delay.
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13641219
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Dec 14th, 2010 12:00AM
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Nov 18th, 2009 12:00AM
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https://www.uspto.gov?id=US07851437-20101214
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Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
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Drug-Linker-Ligand Conjugates are disclosed in which a Drug is linked to a Ligand via a peptide-based Linker unit. In one embodiment, the Ligand is an Antibody. Drug-Linker compounds and Drug compounds are also disclosed. Methods for treating cancer, an autoimmune disease or an infectious disease using the compounds and compositions of the invention are also disclosed.
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7851437
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1. A drug-linker-antibody conjugate of Formula Ia:
or a pharmaceutically acceptable salt thereof, wherein,
L- is an antibody that binds to an antigen expressed on an activated human lymphocyte, wherein the activated human lymphocyte is associated with an autoimmune disease;
-Aa-Ww-Yy- is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each -W- is independently an Amino Acid unit;
-Y- is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12,
y is 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and —C1-C8 alkyl;
R3 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from the group consisting of —H and -methyl; or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and —C1-C8 alkyl;
R7 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from the group consisting of —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from the group consisting of —H and —C1-C8 alkyl;
R10 is selected from the group consisting of:
Z is —O—, —S—,—NH—or —N(R14)—;
R11 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from the group consisting of -aryl and —C3-C8 heterocycle;
R13 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
2. A drug-linker-antibody conjugate of the formula Ia:
or a pharmaceutically acceptable salt thereof, wherein,
L- is an antibody that binds to an antigen expressed on an activated human lymphocyte, wherein the activated human lymphocyte is associated with an autoimmune disease;
-Aa-Ww-Yy- is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each -W- is independently an Amino Acid unit;
-Y- is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit having the structure
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and -methyl;
R3 is selected from the group consisting of —H, -methyl, and -isopropyl;
R4 is selected from the group consisting of —H and -methyl;
R5 is selected from the group consisting of -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— where Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and -methyl;
each R8 is independently selected from the group consisting of —OH, -methoxy and -ethoxy;
R10 is selected from the group consisting of:
R24 is selected from the group consisting of H and —C(O)R25; wherein R25 is selected from the group consisting of —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
Z is —O—, —NH—, —OC(O)—, —NHC(O)—, or —NR28C(O)—; where R28 is selected from the group consisting of —H and —C1-C8 alkyl;
n is 0 or 1; and
R27 is selected from the group consisting of —H, —N3, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1- C8 alkyl-(C3-C8 heterocycle) when n is 0; and R27 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 1.
3. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein R10 is
4. The drug-linker-antibody conjugate of claim 2 or a pharmaceutically acceptable salt thereof wherein R10 is
5. The drug-linker-antibody conjugate of claim 3 or a pharmaceutically acceptable salt thereof wherein R2 is —C1-C8 alkyl.
6. The drug-linker-antibody conjugate of claim 4 or a pharmaceutically acceptable salt thereof wherein R2 is methyl.
7. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof where -D is a Drug unit having the structure
8. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein the antibody is a monoclonal antibody.
9. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein the antibody is a full length immunoglobulin molecule.
10. The drug-linker-antibody conjugate of claim 8 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a human immunoglobulin constant region.
11. The drug-linker-antibody conjugate of claim 10 or a pharmaceutically acceptable salt thereof wherein the antibody is an IgG 1.
12. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof where -Ww- is -valine-citrulline-, the amino terminus of -Ww- forming a bond with a Stretcher unit, and the C— terminus of -Ww- forming a bond with a Spacer unit.
13. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to about 5.
14. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to 10.
15. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein the linker unit is cleavable by cathepsin B.
16. The drug-linker-antibody conjugate of claim 1 having the formula below:
or a pharmaceutically acceptable salt thereof, where E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or from 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
17. The drug-linker-antibody conjugate of claim 16 or a pharmaceutically acceptable salt thereof wherein the antibody is a monoclonal antibody.
18. The drug-linker-antibody conjugate of claim 16 or a pharmaceutically acceptable salt thereof wherein the antibody is a full length immunoglobulin molecule.
19. The drug-linker-antibody conjugate of claim 17 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a human immunoglobulin constant region.
20. The drug-linker-antibody conjugate of claim 19 or a pharmaceutically acceptable salt thereof wherein the antibody is an IgG1.
21. The drug-linker-antibody conjugate of claim 16 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to about 5.
22. The drug-linker-antibody conjugate of claim 16 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to 10.
23. The drug-linker-antibody conjugate of claim 1 having the formula below:
or a pharmaceutically acceptable salt thereof.
24. The drug-linker-antibody conjugate of claim 23 or a pharmaceutically acceptable salt thereof wherein the antibody is a monoclonal antibody.
25. The drug-linker-antibody conjugate of claim 23 or a pharmaceutically acceptable salt thereof wherein the antibody is full length immunoglobulin molecule.
26. A composition comprising drug-linker-antibody conjugates having Formula Ia:
or a pharmaceutically acceptable salt thereof;
wherein,
L- is an antibody that binds to an antigen expressed on an activated human lymphocyte, wherein the activated human lymphocyte is associated with an autoimmune disease;
-Aa-Ww-Yy- is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each -W- is independently an Amino Acid unit;
-Y- is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to 10 and is the average number of -Aa-Ww-Yy-D units per antibody in the composition; and
-D is a Drug unit of the formula
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and —C1-C8 alkyl;
R3 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from the group consisting of —H and -methyl; or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and —C1-C8 alkyl;
R7 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from the group consisting of —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from the group consisting of —H and —C1-C8 alkyl;
R10 is selected from the group consisting of:
Z is —O—, —S—,—NH— or —N(R14)—;
R11 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from the group consisting of -aryl and —C3-C8 heterocycle;
R13 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl; and a pharmaceutically acceptable carrier or vehicle.
27. A composition comprising drug-linker-antibody conjugates having Formula Ia:
or a pharmaceutically acceptable salt thereof, wherein,
L- is an antibody that binds to an antigen expressed on an activated human lymphocyte, wherein the activated human lymphocyte is associated with an autoimmune disease;
-Aa-Ww-Yy- is an enzymatically cleavable linker unit that links the Drug unit and the antibody , wherein:
-A- is a Stretcher unit;
a is 1;
each -W- is independently an Amino Acid unit;
-Y- is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to 10 and is the average number of -Aa-Ww-Yy-D units per antibody in the composition; and
-D is a Drug unit having the structure
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and -methyl;
R3 is selected from the group consisting of —H, -methyl, and -isopropyl;
R4 is selected from the group consisting of —H and -methyl;
R5 is selected from the group consisting of -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— where Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and -methyl;
each R8 is independently selected from the group consisting of —OH, -methoxy and -ethoxy;
R10 is selected from the group consisting of:
R24 is selected from the group consisting of H and —C(O)R25—; wherein R25 is selected from the group consisting of —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1 -C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
Z is —O—, —NH—, —OC(O)—, —NHC(O)—, or —NR28C(O)—; where R28 is selected from the group consisting of —H and —C1-C8 alkyl;
n is 0 or 1; and
R27 is selected from the group consisting of —H, —N3, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8alkyl-(C3-C8 heterocycle) when n is 0; and
R27 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-( C3-C8 heterocycle) when n is 1; and a pharmaceutically acceptable carrier or vehicle.
28. The composition of claim 26 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, R2 is —C1-C8 alkyl.
29. The composition of claim 27 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, R2 is methyl.
30. The composition of claim 26 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, -D is a Drug unit having the structure
31. The composition of claim 26 wherein the drug-linker-antibody conjugates have the formula
or a pharmaceutically acceptable salt thereof, where E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or from 1-10 when E is —CH2CH2—O—; F is —CH2—; and f is 0 or 1.
32. The composition of claim 31 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a monoclonal antibody.
33. The composition of claim 31 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a full length immunoglobulin molecule.
34. The drug-linker-antibody conjugate of claim 32 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a human immunoglobulin constant region.
35. The drug-linker-antibody conjugate of claim 34 or a pharmaceutically acceptable salt thereof wherein the antibody is an IgG 1.
36. The composition of claim 31 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, p ranges from 1 to about 5.
37. The composition of claim 26 wherein the drug-linker-antibody conjugates have the formula
or a pharmaceutically acceptable salt thereof.
38. The composition of claim 37 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a monoclonal antibody.
39. The composition of claim 37 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a full length immunoglobulin molecule.
40. The drug-linker-antibody conjugate of claim 38 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a human immunoglobulin constant region.
41. The drug-linker-antibody conjugate of claim 40 or a pharmaceutically acceptable salt thereof wherein the antibody is an IgG1.
42. The composition of claim 37 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, p ranges from 1 to about 5.
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42
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CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/408,646, filed Mar. 20, 2009, which is a continuation of U.S. patent application Ser. No. 10/522,911, filed Jul. 7, 2005, which was filed under 35 U.S.C. §371 as a national stage application of International Application No. PCT/US2003/24209, filed Jul. 31, 2003; which further claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/400,403, filed Jul. 31, 2002. This application is also a continuation of U.S. patent application Ser. No. 10/522,911, filed Jul. 7, 2005, which was filed under 35 U.S.C. §371 as a national stage application of International Application No. PCT/US2003/24209, filed Jul. 31, 2003; which further claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/400,403, filed Jul. 31, 2002. The disclosures of each of the foregoing applications are hereby incorporated herein by reference.
FIELD OF THE INVENTION
The present invention is directed to Drug-Linker-Ligand Conjugates and to Drug-Linker Compounds, to compositions comprising a Drug-Linker-Ligand Conjugate or a Drug-Linker Compound, and to methods for using the same to treat cancer, an autoimmune disease or an infectious disease.
BACKGROUND OF THE INVENTION
Several short peptidic compounds have been isolated from natural sources and found to have biological activity. Analogs of these compounds have also been prepared, and some were found to have biological activity. For example, Auristatin E (U.S. Pat. No. 5,635,483 to Pettit et al.) is a synthetic analogue of the marine natural product Dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (G. R. Pettit, Prog. Chem. Org. Nat. Prod., 70: 1-79 (1997)). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds. Both dolastatin 10 and auristatin PE are presently being used in human clinical trials to treat cancer. The structural differences between dolastatin 10 and auristatin E reside in the C-terminal residue, in which the thiazolephenethyl amine group of dolastatin 10 is replaced by a norephedrine unit in auristatin E.
The following references disclose dolastatin and auristatin compounds and analogs thereof, and their use for treating cancer:
International Publication No. WO 96/33212 A1 to Teikoku Hormone Mfg. Co., Ltd.;
International Publication No. WO 96/14856 A1 to Arizona Board of Regents;
European Patent Publication No. EP 695757 A2 to Arizona Board of Regents;
European Patent Publication No. EP 695758 A2 to Arizona Board of Regents;
European Patent Publication No. EP 695759 A2 to Arizona Board of Regents;
International Publication No. WO 95/09864 A1 to Teikoku Hormone Mfg. Co., Ltd.;
International Publication No. WO 93/03054 A1 to Teikoku Hormone Mfg. Co., Ltd.;
U.S. Pat. No. 6,323,315 B1 to Pettit et al.;
G. R. Pettit et al., Anti-Cancer Drug Des. 13(4): 243-277 (1998);
G. R. Pettit et al., Anti-Cancer Drug Des. 10(7): 529-544 (1995); and
K. Miyazaki et al., Chem. Pharm. Bull. 43(10), 1706-18 (1995).
Despite in vitro data for compounds of the dolastatin class and its analogs, significant general toxicities at doses required for achieving a therapeutic effect compromise their efficacy in clinical studies. Accordingly, there is a clear need in the art for dolastatin derivatives having significantly lower toxicity, yet useful therapeutic efficiency, compared to current dolastatin drug therapies.
The recitation of any reference in Section 2 of this application is not an admission that the reference is prior art to this application.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compounds of general Formula Ia:
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R2 is selected from -hydrogen and —C1-C8 alkyl;
R3 is selected from -hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from -hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In another aspect, the present invention provides compounds of general formula Ib:
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to Y when y is 1 or 2, or X is bonded to W when y is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-.
In another aspect, the present invention provides compounds of general formula Ic:
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each -W- is independently an Amino Acid unit;
w is an integer ranging from 0 to 12;
each n is independently 0 or 1;
p ranges from 1 to about 20; and
each -D is independently:
(a) a Drug unit of the formula:
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, -C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to —C(O)— when y is 1 or 2, or X is bonded to —CH2— when n is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-; or
(b) a Drug unit of the formula:
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
A compound of formula Ia, formula Ib, formula Ic or a pharmaceutically acceptable salt or solvate thereof (a “Drug-Linker-Ligand Conjugate”) is useful for treating or preventing cancer, an autoimmune disease or an infectious disease in an animal.
In another aspect, the present invention provides compounds of the formula IIa:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl; and
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O—(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIb:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
R13 is selected from hydrogen, —OH, —NH2, —NHR14, —N(R14)2, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-; and
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIc:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IId:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl
In another aspect, the present invention provides compounds of the formula IIe:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIf:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIg:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIh:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIi:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
A compound of formula IIa-i or a pharmaceutically acceptable salt or solvate thereof (a “Drug-Linker Compound”) is useful for treating cancer, an autoimmune disease or an infectious disease in an animal or useful as an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.
In another aspect, the present invention provides compositions comprising an effective amount of a Drug-Linker-Ligand Conjugate and a pharmaceutically acceptable carrier or vehicle.
In still another aspect, the present invention provides compositions comprising an effective amount of a Drug-Linker Compound and a pharmaceutically acceptable carrier or vehicle.
In yet another aspect, the present invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the present invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for treating cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for treating an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In still another aspect, the invention provides methods for treating an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the present invention provides methods for preventing the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the present invention provides methods for preventing the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for preventing cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for preventing an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for preventing an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In still another aspect, the invention provides methods for preventing an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides a Drug-Linker Compound which can be used as an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.
The present invention may be understood more fully by reference to the following detailed description, Figures and illustrative examples, which are intended to exemplify non-limiting embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the cytotoxicity of Compound 49 and Compound 53 against the H3396 cell line. Line -Δ- represents Compound 49 and line -∘- represents Compound 53.
FIG. 2 shows the cytotoxicity of Compounds 64, 65, 68 and 69 against the H3396 cell line. Line -♦- represents Compound 64, line -▪- represents Compound 65, line -▴- represents Compound 68, and line -×- represents Compound 69.
FIG. 3 shows the cytotoxicity of Compounds 64, 65, 68 and 69 against the HCT-116 cell line. Line -♦- represents Compound 64, line -▪- represents Compound 65, line -▴- represents Compound 68, and line -×- represents Compound 69.
FIG. 4 shows the cytotoxicity of Compounds 66 and 68 against the H3396 cell line. Line -□- represents Compound 66 and line -*- represents Compound 68.
FIG. 5 shows the cytotoxicity of Compounds 66, 68 and 69 against the Karpas human colorectal cell line. Line -♦- represents Compound 66, line -▴- represents Compound 68, and line -×- represents Compound 69.
FIG. 6 shows the cytotoxicity of Compounds 66 and 67 against the H3396 cell line as a function of exposure length. The cells were either exposed to the conjugates for the entire duration of the assay without washing (96 hours), or were exposed to the conjugates for 2 hours, washed, and then incubated for an additional 94 hours. At the end of the 96 hour period, the cells were pulsed with Alamar Blue to determine cell viability. Line - - represents Compound 66 at 2 h exposure, line -∘- represents Compound 67 at 2 h exposure, line -•- represents Compound 66 at 96 h exposure, and line -⋄- represents Compound 67 at 96 h exposure.
FIG. 7 shows the effect of Compounds 66-69 on the growth of L2987 human lung adenocarcinoma xenograft tumors which were implanted in nude mice. Line -×- represents untreated tumor, line -▾- represents Compound 66, line -♦- represents Compound 68, line -∇- Compound 67, and line -⋄- represents Compound 69.
FIG. 8 shows the effects of Compounds 66-69 on the growth of Karpas human anaplastic large cell lymphoma xenograft tumors which were implanted in nude mice. Line -×- represents untreated tumor, line -Δ- represents Compound 67, line -•- represents Compound 69, line -Δ- represents Compound 66, and line -∘- represents Compound 68.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Examples of an “animal” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl.
“Aryl” refers to a carbocyclic aromatic group Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. A carbocyclic aromatic group or a heterocyclic aromatic group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, —halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
The term “C1-C8 alkyl,” as used herein refers to a straight chain or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative “C1-C8 alkyl” groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C1-C8 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C1-C8 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl.methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A C1-C8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “C3-C8 carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. A C3-C8 carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “C3-C8 carbocyclo” refers to a C3-C8 carbocycle group defined above wherein one of the carbocycle groups hydrogen atoms is replaced with a bond.
A “C1-C10 alkylene” is a straight chain, saturated hydrocarbon group of the formula —(CH2)1-10—. Examples of a C1-C10 alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene.
An “arylene” is an aryl group which has two covalent bonds and can be in the ortho, meta, or para configurations as shown in the following structures:
in which the phenyl group can be unsubstituted or substituted with up to four groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, —halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “C3-C8 heterocycle” refers to an aromatic or non-aromatic C3-C8 carbocycle in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. Representative examples of a C3-C8 heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl. A C3-C8 Heterocycle can be unsubstituted or substituted with up to seven groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
“C3-C8 heterocyclo” refers to a C3-C8 heterocycle group defined above wherein one of the heterocycle groups hydrogen atoms is replaced with a bond. A C3-C8 heterocyclo can be unsubstituted or substituted with up to six groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “Compound of the Invention” is a Drug-Linker Compound or a Drug-Linker-Ligand Conjugate.
In one embodiment, the Compounds of the Invention are in isolated or purified form. As used herein, “isolated” means separated from other components of (a) a natural source, such as a plant or animal cell or cell culture, or (b) a synthetic organic chemical reaction mixture. As used herein, “purified” means that when isolated, the isolate contains at least 95%, preferably at least 98%, of a Compound of the Invention by weight of the isolate.
Examples of a “Hydroxyl protecting group” include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
“Leaving group” refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.
The term “antibody,” as used herein, refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce auto-immune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. Antibodies useful in the invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F(ab′) fragments, F(ab′)2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens.
The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a Compound of the Invention. The Compounds of the Invention contain at least one amino group, and accordingly acid addition salts can be formed with this amino group. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
“Pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a Compound of the Invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
In the context of cancer, the term “treating” includes any or all of: preventing growth of tumor cells or cancer cells, preventing replication of tumor cells or cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.
In the context of an autoimmune disease, the term “treating” includes any or all of: preventing replication of cells associated with an autoimmune disease state including, but not limited to, cells capable of producing an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.
In the context of an infectious disease, the term “treating” includes any or all of: preventing the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease.
The following abbreviations are used herein and have the indicated definitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is diethylamine, DEAD is diethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine, dil is dolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high pressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is (1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS is phosphate-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, Ph is phenyl, phe is L-phenylalanine, PyBrop is bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, SEC is size-exclusion chromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC is thin layer chromatography, UV is ultraviolet, val is valine.
Drug-Linker-Ligand Conjugates
As stated above, the invention provides compounds of the formula Ia:
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In one embodiment R10 is selected from
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
Illustrative classes of compounds of formula Ia have the structures:
and pharmaceutically acceptable salts and solvates thereof,
where L- is a Ligand unit, E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or from 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In another embodiment L is cBR96, cAC10 or 1F6.
Illustrative compounds of formula Ia have the structure:
and pharmaceutically acceptable salts and solvates thereof,
where p ranges from about 7 to about 9.
In one embodiment p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In another embodiment, p is about 8.
In yet another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another aspect, the present invention provides compounds of general formula Ib:
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to Y when y is 1 or 2, or X is bonded to W when y is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-.
In one embodiment, when R1 is —H, R10 is selected from:
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
Illustrative classes of compounds of formula Ib have the structure:
pharmaceutically acceptable salts and solvates thereof,
where L- is Ligand unit, E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another embodiment L is cBR96, cAC10 or 1F6.
Illustrative compounds of formula Ib have the structure:
and pharmaceutically acceptable salts and solvates thereof,
where p ranges from about 7 to about 9.
In one embodiment p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In another embodiment, p is about 8.
In yet another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another aspect, the present invention provides compounds of general formula Ic:
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each -W- is independently an Amino Acid unit;
w is an integer ranging from 0 to 12;
each n is independently 0 or 1;
p ranges from 1 to about 20; and
each -D is independently:
(a) a Drug unit of the formula:
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to —C(O)— when y is 1 or 2, or X is bonded to —CH2— when n is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-; or
(b) a Drug unit of the formula:
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In one embodiment, when the drug unit has the formula:
and R1 is —H, R10 is selected from
In another embodiment, when the drug unit has the formula:
R10 is selected from
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
An illustrative compound of formula Ic has the structure:
wherein where L- is Ligand unit, E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another embodiment L is cBR96, cAC10 or 1F6.
The Drug-Linker-Ligand Conjugates are useful for treating or preventing cancer, an autoimmune disease or an infectious disease in an animal.
It is understood that p is the average number of -Aa-Ww-Yy-D units per ligand in a Drug-Linker-Ligand Conjugate of formulas Ia, Ib and Ic.
In one embodiment p ranges from 1 to 15.
In another embodiment p ranges from 1 to 10.
In another embodiment, p ranges from 1 to about 8.
In a further embodiment p ranges from 1 to about 5.
In another embodiment p ranges from 1 to about 3.
In one embodiment p ranges from about 3 to about 5.
In one embodiment p ranges from about 7 to about 9.
In another embodiment p is about 8.
In yet another embodiment p is about 4.
In still another embodiment p is about 2.
The Drug-Linker-Ligand Conjugates of formulas Ia, Ib and Ic may exist as mixtures, wherein each component of a mixture has a different p value. For example, a Drug-Linker-Ligand Conjugate may exist as a mixture of two separate Conjugates, one Conjugate component wherein p is 7 and the other Conjugate component wherein p is 8.
In one embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 1, 2, and 3, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 3, 4, and 5, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 5, 6, and 7, respectively.
In still another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 7, 8, and 9, respectively.
In yet another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 9, 10, and 11, respectively.
In still another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 11, 12, and 13, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 13, 14, and 15, respectively.
Drug-Linker Compounds
The present invention provides compounds of the formula IIa:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to Y when y is 1 or 2, or X is bonded to W when y is 0;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
An illustrative compound of formula IIa has the structure:
and pharmaceutically acceptable salts and solvates thereof.
In another aspect, the present invention provides compounds of the formula IIb:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
R13 is selected from hydrogen, —OH, —NH2, —NHR14, —N(R14)2, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIc:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IId:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIe:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIf:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In one embodiment R1 is selected from —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached
Illustrative compounds of formula IIf have the structure:
and pharmaceutically acceptable salts and solvates thereof
In another aspect, the present invention provides compounds of the formula IIg:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIh:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIi:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from hydrogen, —OH, —NH2, —NHR14, —N(R14)2, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each -W- is independently an Amino Acid unit;
-Y- is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
Illustrative compounds of formula IIi have the structures:
and pharmaceutically acceptable salts and solvates thereof
The compounds of formulas IIa-i are useful for treating or preventing cancer, an autoimmune disease or an infectious disease in an animal.
The Linker Unit
The Linker unit of the Drug-Linker-Ligand Conjugate links the Drug unit and the Ligand unit and has the formula:
-Aa-Ww-Yy-
wherein:
-A- is a Stretcher unit;
a is 0 or 1;
each -W- is independently an Amino Acid unit;
w is independently an integer ranging from 0 to 12;
-Y- is a Spacer unit; and
y is 0, 1 or 2.
The Stretcher Unit
The Stretcher unit (-A-), when present, links a Ligand unit to an amino acid unit (- W). In this regard a Ligand (L) has a functional group that can form a bond with a functional group of a Stretcher. Useful functional groups that can be present on a ligand, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of a carbohydrate, and carboxyl. Preferred Ligand functional groups are sulfhydryl and amino. Sulfhydryl groups can be generated by reduction of an intramolecular disulfide bond of a Ligand. Alternatively, sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of a Ligand using 2-iminothiolane (Traut's reagent) or another sulfhydryl generating reagent.
In one embodiment, the Stretcher unit forms a bond with a sulfur atom of the Ligand unit. The sulfur atom can be derived from a sulfhydryl group of a Ligand. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas (IIIa) and (IIIb), wherein L-, -W-, -Y-, -D, w and y are as defined above and R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; and r is an integer ranging from 1-10.
An illustrative Stretcher unit is that of formula (IIIa) where R17 is —(CH2)5—:
Another illustrative Stretcher unit is that of formula (IIIa) where R17 is —(CH2CH2O)r—CH2—; and r is 2:
Still another illustrative Stretcher unit is that of formula (IIIb) where R17 is —(CH2)5—:
In another embodiment, the Stretcher unit is linked to the Ligand unit via a disulfide bond between a sulfur atom of the Ligand unit and a sulfur atom of the Stretcher unit. A representative Stretcher unit of this embodiment is depicted within the square brackets of Formula (IV), wherein R17, L-, -W-, -Y-, -D, w and y are as defined above.
In yet another embodiment, the reactive group of the Stretcher contains a reactive site that can form a bond with a primary or secondary amino group of a Ligand. Example of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas (Va) and (Vb), wherein —R17—, L-, -W-, -Y-, -D, w and y are as defined above;
In yet another aspect of the invention, the reactive group of the Stretcher contains a reactive site that is reactive to a carbohydrate's (—CHO) group that can be present on a Ligand. For example, a carbohydrate can be mildly oxidized using a reagent such as sodium periodate and the resulting (—CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko, T. et al. Bioconjugate Chem 1991, 2, 133-41. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas (VIa)-(VIc), wherein —R17—, L-, -W-, -Y-, -D, w and y are as defined above.
The Amino Acid Unit
The Amino Acid unit (-W-), when present, links the Stretcher unit to the Spacer unit if the Spacer unit is present, links the Stretcher unit to the Drug unit if the Spacer unit is absent, and links the Ligand unit to the Drug unit if the Stretcher unit and Spacer unit are absent.
-Ww- is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each -W- unit independently has the formula denoted below in the square brackets, and w is an integer ranging from 0 to 12:
wherein R19 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH2OH, —CH(OH)CH3, —CH2CH2SCH3, —CH2CONH2, —CH2COOH, —CH2CH2CONH2, —CH2CH2COOH, —(CH2)3NHC(═NH)NH2, —(CH2)3NH2, —(CH2)3NHCOCH3, —(CH2)3NHCHO, —(CH2)4NHC(═NH)NH2, —(CH2)4NH2, —(CH2)4NHCOCH3, —(CH2)4NHCHO, —(CH2)3NHCONH2, —(CH2)4NHCONH2, —CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
The Amino Acid unit of the Compounds of the Invention can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, to liberate the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide a Drug (D).
Illustrative Ww units are represented by formulas (VII)-(IX):
wherein R20 and R21 are as follows:
R20
R21
benzyl
(CH2)4NH2;
methyl
(CH2)4NH2;
isopropyl
(CH2)4NH2;
isopropyl
(CH2)3NHCONH2;
benzyl
(CH2)3NHCONH2;
isobutyl
(CH2)3NHCONH2;
sec-butyl
(CH2)3NHCONH2;
(CH2)3NHCONH2;
benzyl
methyl; and
benzyl
(CH2)3NHC(═NH)NH2;
wherein R20, R21 and R22 are as follows:
R20
R21
R22
benzyl
benzyl
(CH2)4NH2;
isopropyl
benzyl
(CH2)4NH2; and
H
benzyl
(CH2)4NH2;
wherein R20, R21, R22 and R23 are as follows:
R20
R21
R22
R23
H
benzyl
isobutyl
H; and
methyl
isobutyl
methyl
isobutyl.
Preferred Amino Acid units include, but are not limited to, units of formula (VII) where: R20 is benzyl and R21 is —(CH2)4NH2, R20 isopropyl and R21 is —(CH2)4NH2; R20 isopropyl and R21 is —(CH2)3NHCONH2. Another preferred Amino Acid unit is a unit of formula (VIII) where R20 is benzyl, R21 is benzyl, and R22 is —(CH2)4NH2.
-Ww- units useful in the present invention can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease. In one embodiment, a -Ww- unit is that whose cleavage is catalyzed by cathepsin B, C and D, or a plasmin protease.
In one embodiment, -Ww- is a dipeptide, tripeptide or pentapeptide.
Where R19, R20, R21, R22 or R23 is other than hydrogen, the carbon atom to which R19, R20, R21, R22 or R23 is attached is chiral.
Each carbon atom to which R19, R20, R21, R22 or R23 is attached is independently in the (S) or (R) configuration.
The Spacer Unit
The Spacer unit (-Y-), when present, links an Amino Acid unit to the Drug unit when an Amino Acid unit is present. Alternately, the Spacer unit links the Stretcher unit to the Drug unit when the Amino Acid unit is absent. The Spacer unit also links the Drug unit to the ligand unit when both the Amino Acid unit and Stretcher unit are absent. Spacer units are of two general types: self-immolative and non self-immolative. A non self-immolative Spacer unit is one in which part or all of the Spacer unit remains bound to the Drug unit after cleavage, particularly enzymatic, of an Amino Acid unit from the Drug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of a non self-immolative Spacer unit include, but are not limited to a (glycine-glycine) Spacer unit and a glycine Spacer unit (both depicted in Scheme 1). When a Compound of the Invention containing a glycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymatic cleavage via a tumor-cell associated-protease, a cancer-cell-associated protease or a lymphocyte-associated protease, a glycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved from L-Aa-Ww-. In one embodiment, an independent hydrolysis reaction takes place within the target cell, cleaving the glycine-Drug unit bond and liberating the Drug.
In a preferred embodiment, -Yy- is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3) whose phenylene portion is substituted with Qm where Q is is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
In one embodiment, a non self-immolative Spacer unit (-Y-) is -Gly-Gly-.
In another embodiment, a non self-immolative the Spacer unit (-Y-) is -Gly-.
In one embodiment, the invention provides a Drug-Linker Compound or a Drug-Linker Ligand Conjugate in which the Spacer unit is absent (y=0), or a pharmaceutically acceptable salt or solvate thereof.
Alternatively, a Compound of the Invention containing a self-immolative Spacer unit can release -D without the need for a separate hydrolysis step. In this embodiment, -Y- is a PAB group that is linked to -Ww- via the amino nitrogen atom of the PAB group, and connected directly to -D via a carbonate, carbamate or ether group. Without being bound by theory, Scheme 2 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via a carbamate or carbonate group.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1 to about 20.
Without being bound by theory, Scheme 3 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via an ether or amine linkage.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1 to about 20.
Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazol-5-methanol derivatives (see Hay et al., Bioorg. Med. Chem. Lett., 1999, 9, 2237) and ortho or para-aminobenzylacetals. Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacer useful in the Compounds of the Invention.
In a preferred embodiment, the Spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as depicted in Scheme 4, which can be used to incorporate and release multiple drugs.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from 1 to about 20.
In one embodiment, the -D moieties are the same.
In another embodiment, the -D moieties are different.
Preferred Spacer units (-Yy-) are represented by Formulas (X)-(XII):
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4;
The Drug Unit
-D is a Drug unit having a nitrogen or oxygen atom that can form a bond with the Spacer unit when y=1 or 2 or with the C-terminal carbonyl group of an Amino Acid unit when y=0.
In one embodiment, -D is represented by the formula:
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In one embodiment, R10 is selected from
In a preferred embodiment, -D has the formula
or a pharmaceutically acceptable salt or solvate thereof,
wherein, independently at each location:
R2 is selected from —H and -methyl;
R3 is selected from —H, -methyl, and -isopropyl;
R4 is selected from —H and -methyl; R5 is selected from -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl; or R4 and R5 join, have the formula —(CRaRb)n— where Ra and Rb are independently selected from —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and -methyl;
each R8 is independently selected from —OH, -methoxy and -ethoxy;
R10 is selected from
R24 is selected from H and —C(O)R25; wherein R25 is selected from —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R26 is selected from —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
Z is —O—, —NH—, —OC(O)—, —NHC(O)—, —N(R28)C(O)—; where R28 is selected from —H and —C1-C8 alkyl;
n is 0 or 1; and
R27 is selected from —H, —N3, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 0; and R27 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 1.
In one embodiment, R10 is selected from
In another embodiment, -D is represented by the formula:
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X forms a bond with a Linker unit;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —O—(C1-C8 alkyl), —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-.
In one embodiment, when R1 is —H, R10 is selected from:
In a preferred embodiment, -D has the formula
or a pharmaceutically acceptable salt or solvate thereof,
wherein, independently at each location:
R1 is selected from —H and -methyl;
R2 is selected from —H and -methyl;
R3 is selected from —H, -methyl, and -isopropyl;
R4 is selected from —H and -methyl; R5 is selected from -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl; or R4 and R5 join, have the formula —(CRaRb)n— where Ra and Rb are independently selected from —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and N is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and -methyl;
each R8 is independently selected from —OH, -methoxy and -ethoxy;
R10 is selected from
where X is —O—, —NH— or —N(R14)— and forms a bond with Y when y is 1 or 2, with W when y is 0, and with A when w and y are both 0;
Z is —O—, —NH— or —N(R14)—;
R13 is —H or -methyl;
R14 is C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo or —C3-C8 heterocyclo-,
In one embodiment, when R1 is -methyl, R10 is selected from
where X is —O—, —NH— or —N(R14)— and forms a bond with Y when y is 1 or 2, and with W when y is 0;
Z is —O—, —NH— or —N(R14)—;
R13 is —H or -methyl;
R14 is C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo or —C3-C8 heterocyclo-.
In another embodiment, when R1 is —H, R10 is selected from:
where X is —O—, —NH— or —N(R14)— and forms a bond with Y when y is 1 or 2, and with W when y is 0;
Z is —O—, —NH— or —N(R14)—;
R13 is —H or -methyl;
R14 is C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo or —C3-C8 heterocyclo-.
A Drug unit can form a bond with a Linker unit via a nitrogen atom of a Drug's primary or secondary amino group, via an oxygen atom of a Drug's hydroxyl group, or via a sulfur atom of a Drug's sulfhydryl group to form a Drug-Linker Compound.
In a preferred embodiment, Drug units have the formula
The Ligand Unit
The Ligand unit (L-) includes within its scope any unit of a Ligand (L) that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. A Ligand can be any molecule that binds to, complexes with or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. The Ligand unit acts to deliver the Drug unit to the particular target cell population with which the Ligand unit reacts. Such Ligands include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies, antibody fragments, smaller molecular weight proteins, polypeptide or peptides, and lectins.
A Ligand unit can form a bond to either a Stretcher unit or an Amino Acid unit of a Linker. A Ligand unit can form a bond to a Linker unit via a heteroatom of the Ligand. Heteroatoms that may be present on a Ligand unit include sulfur (in one embodiment, from a sulfhydryl group of a Ligand), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of a Ligand) and nitrogen (in one embodiment, from a primary or secondary amino group of a Ligand). These heteroatoms can be present on the Ligand in the Ligand's natural state, for example a naturally occurring antibody, or can be introduced into the Ligand via chemical modification.
In a preferred embodiment, a Ligand has a sulfhydryl group and the Ligand bonds to the Linker unit via the sulfhydryl group's sulfur atom.
In another embodiment, the Ligand can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups. The Ligand unit bonds to the Stretcher unit via the sulfhydryl group's sulfur atom.
In yet another embodiment, the Ligand can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) group (see Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The corresponding aldehyde can form a bond with a Reactive Site on a Stretcher. Reactive sites on a Stretcher that can react with a carbonyl group on a Ligand include, but are not limited to, hydrazine and hydroxylamine.
Useful non-immunoreactive protein, polypeptide, or peptide Ligands include, but are not limited to, transferrin, epidermal growth factors (“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (“TGF”), such as TGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II, lectins and apoprotein from low density lipoprotein.
Useful Polyclonal antibody Ligands are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, and guinea pigs. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
Useful monoclonal antibody Ligands are homogeneous populations of antibodies to a particular antigen (e.g., a cancer cell antigen, a viral antigen, a microbial antigen covalently linked to a second molecule). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
Useful monoclonal antibody Ligands include, but are not limited to, human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).
The Ligand can also be a bispecific antibody. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually performed using affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in International Publication No. WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation (International Publication No. WO 94/04690) which is incorporated herein by reference in its entirety.
For further details for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 1986, 121:210. Using such techniques, bispecific antibody Ligands can be prepared for use in the treatment or prevention of disease as defined herein.
Bifunctional antibodies are also described, in European Patent Publication No. EPA 0 105 360. As disclosed in this reference, hybrid or bifunctional antibodies can be derived either biologically, i.e., by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide-bridge forming reagents, and may comprise whole antibodies or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed for example, in International Publication WO 83/03679, and European Patent Publication No. EPA 0 217 577, both of which are incorporated herein by reference.
The Ligand can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, or microbial antigens. In this regard, “Functionally active” means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized. Specifically, in a preferred embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay)
Other useful Ligands include fragments of antibodies such as, but not limited to, F(ab′)2 fragments, which contain the variable region, the light chain constant region and the CH1 domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Other useful Ligands are heavy chain and light chain dimers of antibodies, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful Ligands. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671; European Patent Publication No. 184,187; European Patent Publication No. 171,496; European Patent Publication No. 173,494; International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 125,023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference in its entirety.
Completely human antibodies are particularly desirable for Ligands. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806; each of which is incorporated herein by reference in its entirety. Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).
Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Biotechnology 12:899-903).
In other embodiments, the Ligand is a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the antibody. Preferably, the antibody or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.
The Ligand antibodies include analogs and derivatives that are either modified, i.e, by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, but not by way of limitation, the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular Ligand unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnaturalamino acids.
The Ligand antibodies include antibodies having modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, the Ligand antibodies include antibodies having modifications in amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety). Antibodies immunospecific for a cancer cell antigen can be obtained commercially, for example, from Genentech (San Francisco, Calif.) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
In a specific embodiment, known antibodies for the treatment or prevention of cancer are used in accordance with the compositions and methods of the invention. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, HERCEPTIN (Trastuzumab; Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer (Stebbing, J., Copson, E., and O'Reilly, S. “Herceptin (trastuzamab) in advanced breast cancer” Cancer Treat Rev. 26, 287-90, 2000); RITUXAN (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG2a antibody for the treatment of colorectal cancer; BEC2 (ImClone Systems Inc., NY) which is murine IgG antibody for the treatment of lung cancer; IMC-C225 (Imclone Systems Inc., NY) which is a chimeric IgG antibody for the treatment of head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosite, MA) which is a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA) which is a humanized IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which is a humanized IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a humanized antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA) which is a murine antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma; anti-VEGF (Genentech, Inc., CA) which is humanized antibody for the treatment of lung and colorectal cancers; CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer; IMC-1C11 (ImClone Systems, NJ) which is an anti-KDR chimeric antibody for the treatment of colorectal cancer, lung cancers, and melanoma; and Cetuximab (ImClone, NJ) which is an anti-EGFR chimeric antibody for the treatment of epidermal growth factor positive cancers.
Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogene product (carcinomas). Some specific useful antibodies include, but are not limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellström, I., Hellström, K. E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64 (Trail, P A, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R. S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom, K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res. 2000, 60, 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb, and mAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functional effects of CD30 on a large granular lymphoma cell line YT” J. Immunol., 151, 5896-5906, 1993). Many other internalizing antibodies that bind to tumor associated antigens can be used in this invention, and have been reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy of acute myeloid leukemia: a review” Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).
In another specific embodiment, known antibodies for the treatment or prevention of an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. In another embodiment, useful Ligand antibodies that are immunospecific for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti Phospholipid Antibody IgM, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; Anti SCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody.
In certain preferred embodiments, antibodies useful in the present methods, can bind to both a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitable lectins are C-type, S-type, and I-type lectin.
In one embodiment, the Ligand is an antibody that binds to an activated lymphocyte that is associated with an autoimmune disease.
In another specific embodiment, useful Ligand antibodies that are immunospecific for a viral or a microbial antigen are monoclonal antibodies. Preferably, Ligand antibodies that are immunospecific for a viral antigen or microbial antigen are humanized or human monoclonal antibodies. As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.
Antibodies immunospecific for a viral or microbial antigen can be obtained commercially, for example, from Genentech (San Francisco, Calif.) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
In a specific embodiment, useful Ligand antibodies are those that are useful for the treatment or prevention of viral or microbial infection in accordance with the methods of the invention. Examples of antibodies available useful for the treatment of viral infection or microbial infection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody useful for the treatment of patients with RSV infection; PRO542 (Progenics) which is a CD4 fusion antibody useful for the treatment of HIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; PROTOVIR (Protein Design Labs, Inc., CA) which is a humanized IgG1 antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.
Other antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).
Other antibodies useful in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.
The antibodies suitable for use in the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
Production of Recombinant Antibodies
Ligand antibodies of the invention can be produced using any method known in the art to be useful for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
Recombinant expression of the Ligand antibodies, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a nucleic acid molecule encoding an antibody can be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the antibody can be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
If an antibody that specifically recognizes a particular antigen is not commercially available (or a source for a cDNA library for cloning a nucleic acid encoding such an immunoglobulin), antibodies specific for a particular antigen can be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody can be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
Once a nucleic acid sequence encoding at least the variable domain of the antibody is obtained, it can be introduced into a vector containing the nucleotide sequence encoding the constant regions of the antibody (see, e.g., International Publication No. WO 86/05807; International Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors containing the complete light or heavy chain that allow for the expression of a complete antibody molecule are available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).
Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab′)2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
Once a nucleic acid sequence encoding a Ligand antibody has been obtained, the vector for the production of the antibody can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
An expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.
The host cells used to express the recombinant Ligand antibody can be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al., 1990, BioTechnology 8:2).
A variety of host-expression vector systems can be utilized to express the immunoglobulin Ligands. Such host-expression systems represent vehicles by which the coding sequences of the antibody can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express a Ligand immunoglobulin molecule in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, vectors that direct the expression of high levels of fusion protein products that are readily purified might be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) or the analogous virus from Drosophila Melanogaster is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
In addition, a host cell strain can be chosen to modulate the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express an antibody can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody Such engineered cell lines can be particularly useful in screening and evaluation of tumor antigens that interact directly or indirectly with the antibody Ligand.
A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).
The expression levels of an antibody can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
The host cell can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers that enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used to encode both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.
Once the antibody has been recombinantly expressed, it can be purified using any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
In a preferred embodiment, the Ligand is an antibody.
In a more preferred embodiment, the Ligand is a monoclonal antibody.
In any case, the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an autoimmune disease, an infectious organism, or other disease state.
Synthesis of the Compounds of the Invention
As described in more detail below, the Compounds of the Invention are conveniently prepared using a Linker having two or more Reactive Sites for binding to the Drug and Ligand. In one aspect of the invention, a Linker has a Reactive site which has an electrophilic group that is reactive to a nucleophilic group present on a Ligand. Useful nucleophilic groups on a Ligand include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of a Ligand is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for Ligand attachment.
In another embodiment, a Linker has a Reactive site which has a nucleophilic group that is reactive to an electrophilic group present on a Ligand. Useful electrophilic groups on a Ligand include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on a Ligand and form a covalent bond to a Ligand unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on a Ligand provides a convenient site for attachment to a Linker.
Carboxylic acid functional groups and chloroformate functional groups are also useful reactive sites for a Linker because they can react with primary or secondary amino groups of a Drug to form an amide linkage. Also useful as a reactive site is a carbonate functional group on a Linker which can react with an amino group or hydroxyl group of a Drug to form a carbamate linkage or carbonate linkage, respectively. Similarly, a Drug's phenol moiety can react with the Linker, existing as an alcohol, under Mitsunobu conditions.
Typically, peptide-based Drugs can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schröder and K. Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
In one embodiment, a Drug is prepared by combining about a stoichiometric equivalent of a dipeptide and a tripeptide, preferably in a one-pot reaction under suitable condensation conditions. This approach is illustrated in the following Schemes 5-7. Thus, the tripeptide 6 can be prepared as shown in Scheme 5, and the dipeptide 9 can be prepared as shown in Scheme 6. The two fragments 6 and 9 can be condensed to provide a Drug 10 as shown in Scheme 7.
The synthesis of an illustrative Stretcher having an electrophilic maleimide group is illustrated in Schemes 8-9. General synthetic methods useful for the synthesis of a Linker are described in Scheme 10. Scheme 11 shows the construction of a Linker unit having a val-cit group, an electrophilic maleimide group and a PAB self-immolative Spacer group. Scheme 12 depicts the synthesis of a Linker having a phe-lys group, an electrophilic maleimide group, with and without the PAB self-immolative Spacer group. Scheme 13 presents a general outline for the synthesis of a Drug-Linker Compound, while Scheme 14 presents an alternate route for preparing a Drug-Linker Compound. Scheme 15 depicts the synthesis of a branched linker containing a BHMS group. Scheme 16 outlines the attachment of a Ligand to a Drug-Linker Compound to form a Drug-Linker-Ligand Conjugate, and Scheme 17 illustrates the synthesis of Drug-Linker-Ligand Conjugates having 2 or 4 drugs per Ligand.
As illustrated in Scheme 5, a protected amino acid 1 (where PG represents an amine protecting group, R4 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle, alkyl-(C3-C8 heterocycle) wherein R5 is selected from H and methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached) is coupled to t-butyl ester 2 (where R6 is selected from —H and —C1-C8 alkyl; and R7 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle)) under suitable coupling conditions, e.g., in the presence of PyBrop and diisopropylethylamine, or using DCC (see, for example, Miyazaki, K. et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).
Suitable protecting groups PG, and suitable synthetic methods to protect an amino group with a protecting group are well known in the art. See, e.g., Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley & Sons. Preferred protected amino acids 1 are PG-Ile and, particularly, PG-Val, while other suitable protected amino acids include, without limitation: PG-cyclohexylglycine, PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid, PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and PG-tert-butylglycine. Z is a preferred protecting group. Fmoc is another preferred protecting group. A preferred t-butyl ester 2 is dolaisoleuine t-butyl ester.
The dipeptide 3 can be purified, e.g., using chromatography, and subsequently deprotected, e.g., using H2 and 10% Pd—C in ethanol when PG is benzyloxycarbonyl, or using diethylamine for removal of an Fmoc protecting group. The resulting amine 4 readily forms a peptide bond with an amino acid 5 (where R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached; and R3 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle)). N,N-Dialkyl amino acids are preferred amino acids 5, such as commercially available N,N-dimethyl valine. Other N,N-dialkyl amino acids can be prepared by reductive bis-alkylation using known procedures (see, e.g., Bowman, R. E, Stroud, H. H J. Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two preferred amino acids 5 useful for the synthesis of N-monoalkyl derivatives. The amine 4 and the amino acid 5 react to provide the tripeptide 6 using coupling reagent DEPC with triethylamine as the base.
Illustrative DEPC coupling methodology and the PyBrop coupling methodology shown in Scheme 5 are outlined below in General Procedure A and General Procedure B, respectively. Illustrative methodology for the deprotection of a Z-protected amine via catalytic hydrogenation is outlined below in General Procedure C.
General Procedure A: Peptide synthesis using DEPC. The N-protected or N,N-disubstituted amino acid or peptide 4 (1.0 eq.) and an amine 5 (1.1 eq.) are diluted with an aprotic organic solvent, such as dichloromethane (0.1 to 0.5 M). An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by DEPC (1.1 eq.). The resulting solution is stirred, preferably under argon, for up to 12 hours while being monitored by HPLC or TLC. The solvent is removed in vacuo at room temperature, and the crude product is purified using, for example, HPLC or flash column chromatography (silica gel column). Relevant fractions are combined and concentrated in vacuo to afford tripeptide 6 which is dried under vacuum overnight.
General procedure B: Peptide synthesis using PyBrop. The amino acid 2 (1.0 eq.), optionally having a carboxyl protecting group, is diluted with an aprotic organic solvent such as dichloromethane or DME to provide a solution of a concentration between 0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is added. Fmoc-, or Z-protected amino acid 1 (1.1 eq.) is added as a solid in one portion, then PyBrop (1.2 eq.) is added to the resulting mixture. The reaction is monitored by TLC or HPLC, followed by a workup procedure similar to that described in General Procedure A.
General procedure C: Z-removal via catalytic hydrogenation. Z-protected amino acid or peptide 3 is diluted with ethanol to provide a solution of a concentration between 0.5 and 1.0 mM in a suitable vessel, such as a thick-walled round bottom flask. 10% palladium on carbon is added (5-10% w/w) and the reaction mixture is placed under a hydrogen atmosphere. Reaction progress is monitored using HPLC and is generally complete within 1-2 h. The reaction mixture is filtered through a pre-washed pad of celite and the celite is again washed with a polar organic solvent, such as methanol after filtration. The eluent solution is concentrated in vacuo to afford a residue which is diluted with an organic solvent, preferably toluene. The organic solvent is then removed in vacuo to afford the deprotected amine 4.
Table 1 lists representative examples of tripeptide intermediates (compounds 39-43) that were prepared according to Scheme 5.
TABLE 1
Compound
X1
X2
39
Fmoc-N-Me-L-val
L-val
40
Fmoc-N-Me-L-val
L-ile
41
Fmoc-N-Me-gly
L-ile
42
dov
L-val
43
dov
L-ile
adov = N,N-dimethyl-L-valine
The dipeptide 9 can be readily prepared by condensation of the modified amino acid Boc-Dolaproine 7 (see, for example, Pettit, G. R., et al. Synthesis, 1996, 719-725), with (1S,2R)-norephedrine, L- or D-phenylalaninol, or with synthetic p-acetylphenethylamine 8 (U.S. Pat. No. 3,445,518 to Shavel et al.) using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in Scheme 6. Compound 7 may also be condensed with commercially available compounds in this manner to form dipeptides of formula 9. Examples of commercially available compounds useful for this purpose include, but are not limited to, norephedrine, ephedrine, and stereoisomers thereof (Sigma-Sigma-Aldrich), L- or D-phenylalaninol (Sigma-Aldrich), 2-phenylethylamine (Sigma-Aldrich), 2-(4-aminophenyl)ethylamine (Sigma-Aldrich), 1,2-ethanediamine-1,2-diphenyl (Sigma-Aldrich), or 4-(2-aminoethyl)phenol (Sigma-Aldrich), or with synthetically prepared p-acetylphenethylamine, aryl- and heterocyclo-amides of L-phenylalanine, 1-azidomethyl-2-phenylethylamine (prepared from phenylalaninol according to a general procedure described in J. Chem. Research (S), 1992, 391), and 1-(4-hydroxyphenyl)-2-phenylethylamine (European Patent Publication No. 0356035 A2) among others.
where R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl); R9 is selected from —H and —C1-C8 alkyl; and R10 is selected from:
where Z is —O—, —S—, —NH— or —N(R14)—; R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond; each R12 is independently selected from -aryl and —C3-C8 heterocycle; R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and each R14 is independently —H or —C1-C8 alkyl.
Table 2 lists representative examples of dipeptides (Compounds 44-48) that were prepared according to Scheme 6.
TABLE 2
Compound
Y
44
45
46
47
48
Scheme 7 illustrates a procedure useful for coupling tripeptide 6 and dipeptide 9 to form Drug 10. The coupling of 6 and 9 can be accomplished using a strong acid, e.g. TFA, to facilitate Boc and t-butyl ester cleavage, from dipeptide 9 and tripeptide 6, respectively, followed by condensation conditions, e.g., utilizing DEPC, or similar coupling reagent, in the presence of excess base (triethylamine or equivalent) to provide Drug 10.
An illustrative procedure for the synthesis of Drug 10 as depicted in Scheme 7 is outlined below in General Procedure D.
The R10 group of a Drug of general formula 10 can be further modified, if desired, to include a functional group that allows the drug to be attached to a Linker. Examples of useful modifications to the R10 group of a Drug 10, include, but are not limited to the chemical transformations described below.
When R10 is
the hydroxyl group of R10 can be reacted with commercially available or synthetically derived carboxylic acids or carboxylic acid derivatives, including but not limited to, carboxylic esters, acid chlorides, anhydrides and carbonates to provide the corresponding esters according to well known methods in the art. Coupling reagents, including, but not limited to DCC/DMAP and EDCI/HOBt, can be useful in such coupling reactions between alcohols and carboxylic acids or carboxylic acid derivatives. In a preferred embodiment carboxylic acids are substituted or unsubstituted aryl-carboxylic acids, for example, 4-aminobenzoic acid. Thus, condensation of a hydroxyl group of the R10 group shown above with carboxylic acids provides drugs of the general structure 10
where R10 is
and where R11, R12, R14 and R15 are as previously described herein and X is selected from —OH, —NH2 and —NHR14.
When R10 is
the azido group of the drug can be reduced (for an example see J. Chem. Research (S), 1992, 391) to provide the corresponding amino derivative wherein R10 is
the amino group of which can be reacted with the carboxyl group of a carboxylic acid under general peptide coupling conditions to provide drugs of general structure 10, where
R10 is
and where R11, R12, R14 and R15 are as previously described herein and X is selected from —OH, —NH2 and —NHR14.
Carboxylic acids useful in the above regard include, but are not limited to, 4-aminobenzoic acid, p-acetylbenzoic acid and 2-amino-4-thiazolecarboxylic acid (Tyger Scientific, Inc., Ewing, N.J.).
An Fmoc-protected amino group may be present on an amine-containing R10 group of Drug 10 (e.g., as depicted in Table 2). The Fmoc group is removable from the protected amine using diethylamine (see General Procedure E as an illustrative example described below).
General procedure D: Drug synthesis. A mixture of dipeptide 9 (1.0 eq.) and tripeptide 6 (1 eq.) is diluted with an aprotic organic solvent, such as dichloromethane, to form a 0.1M solution, then a strong acid, such as trifluoroacetic acid (½ v/v) is added and the resulting mixture is stirred under a nitrogen atmosphere for two hours at 0° C. The reaction can be monitored using TLC or, preferably, HPLC. The solvent is removed in vacuo and the resulting residue is azeotropically dried twice, preferably using toluene. The resulting residue is dried under high vacuum for 12 h and then diluted with and aprotic organic solvent, such as dichloromethane. An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by either PyBrop (1.2 eq.) or DEPC (1.2 eq.) depending on the chemical functionality on the residue. The reaction mixture is monitored by either TLC or HPLC and upon completion, the reaction is subjected to a workup procedure similar or identical to that described in General Procedure A.
General procedure E: Fmoc-removal using diethylamine. An Fmoc-protected Drug 10 is diluted with an aprotic organic solvent such as dichloromethane and to the resulting solution is added diethylamine (½ v/v). Reaction progress is monitored by TLC or HPLC and is typically complete within 2 h. The reaction mixture is concentrated in vacuo and the resulting residue is azeotropically dried, preferably using toluene, then dried under high vacuum to afford Drug 10 having a deprotected amino group.
Thus, the above methods are useful for making Drugs that can be used in the present invention.
To prepare a Drug-Linker Compound of the present invention, the Drug is reacted with a reactive site on the Linker. In general, the Linker can have the structure:
when both a Spacer unit (-Y-) and a Stretcher unit (-A-) are present. Alternately, the Linker can have the structure:
when the Spacer unit (-Y-) is absent.
The Linker can also have the structure:
when both the Stretcher unit (-A-) and the Spacer unit (-Y-) are absent.
In general, a suitable Linker has an Amino Acid unit linked to an optional Stretcher Unit and an optional Spacer Unit. Reactive Site 1 is present at the terminus of the Spacer and Reactive site 2 is present at the terminus of the Stretcher. If a Spacer unit is not present, then Reactive site 1 is present at the C-terminus of the Amino Acid unit.
In one embodiment of the invention, Reactive Site No. 1 is reactive to a nitrogen atom of the Drug, and Reactive Site No. 2 is reactive to a sulfhydryl group on the Ligand. Reactive Sites 1 and 2 can be reactive to different functional groups.
In one aspect of the invention, Reactive Site No. 1 is
In another aspect of the invention, Reactive Site No. 1 is
wherein R is —Br, —Cl, —O-Su or —O-(4-nitrophenyl).
In one embodiment, Reactive Site No. 1 is
wherein R is —Br, —Cl, —O-Su or —O-(4-nitrophenyl), when a Spacer unit (-Y-) is absent.
Linkers having
at Reactive Site No. 1 where R is —Br or —Cl can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —COOH group with PX3 or PX5, where X is —Br or —Cl. Alternatively, linkers having
at Reactive Site No. 1 can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —COOH group with thionyl chloride. For a general discussion of the conversion of carboxylic acids to acyl halides, see March, Advanced Organic Chemistry—Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 437-438.
In another aspect of the invention, Reactive Site No. 1 is
In still another aspect of the invention, Reactive Site No. 1 is
wherein R is —Cl, —O—CH(Cl)CCl3 or —O-(4-nitrophenyl).
Linkers having
at Reactive Site No. 1 can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OH group with phosgene or triphosgene to form the corresponding chloroformate. Linkers having
at Reactive Site No. 1 where R is —O—CH(Cl)CCl3 or —O-(4-nitrophenyl) can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OC(O)Cl group with HO—CH(Cl)CCl3 or HO-(4-nitrophenyl), respectively. For a discussion of this chemistry, see March, Advanced Organic Chemistry—Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 392.
In a further aspect of the invention, Reactive Site No. 1 is
wherein X is —F, —Cl, —Br, —I, or a leaving group such as —O-mesyl, —O-tosyl or —O-triflate.
Linkers having
at Reactive Site No. 1 where X is —O-mesyl, —O-tosyl and O-triflate can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OH group with various reagents, including HCl, SOCl2, PCl5, PCl3 and POCl3 (where X is Cl); HBr, PBr3, PBr5 and SOBr2 (where X is Br); HI (where X is I); and CH3CH2NSF3 (DAST), SF4, SeF4 and p-toluenesulfonyl fluoride (where X is F). For a general discussion on the conversion of alcohols to alkyl halides, see March, Advanced Organic Chemistry—Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 431-433.
Linkers having
at Reactive Site No. 1 where X is —O-mesyl, —O-tosyl and —O-triflate, can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OH group with various mesylating, tosylating and triflating reagents, respectively. Such reagents and methods for their use will be well known to one of ordinary skill in the art of organic synthesis. For a general discussion of mesyl, tosyl and triflates as leaving groups, see March, Advanced Organic Chemistry—Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 353-354.
In one embodiment, when a Spacer unit (-Y-) is present, Reactive Site No. 1 is
wherein R is —Cl, —O—CH(Cl)CCl3 or —O-(4-nitrophenyl) and X is —F, —Cl, —Br, —I, or a leaving group such as —O-mesyl, —O-tosyl or —O-triflate.
In another aspect of the invention, Reactive Site No. 1 is
In still another aspect of the invention, Reactive Site No. 1 is a p-nitrophenyl carbonate having the formula
In one aspect of the invention, Reactive Site No. 2 is a thiol-accepting group. Suitable thiol-accepting groups include haloacetamide groups having the formula
where X represents a leaving group, preferably O-mesyl, O-tosyl, —Cl, —Br, or —I; or a maleimide group having the formula
Useful Linkers can be obtained via commercial sources, such as Molecular Biosciences Inc. (Boulder, Colo.), or synthesized in accordance with procedures described in U.S. Pat. No. 6,214,345 to Firestone et al., summarized in Schemes 8-10 below.
where X is —CH2— or —CH2OCH2—; and n is an integer ranging either from 0-10 when X is —CH2—; or 1-10 when X is —CH2OCH2—.
The method shown in Scheme 9 combines maleimide with a glycol under Mitsunobu conditions to make a polyethylene glycol maleimide Stretcher (see for example, Walker, M. A. J. Org. Chem. 1995, 60, 5352-5), followed by installation of a p-nitrophenyl carbonate Reactive Site group.
where E is —CH2— or —CH2OCH2—; and e is an integer ranging from 0-8;
Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can be prepared as described by Frisch, et al., Bioconjugate Chem. 1996, 7, 180-186.
Scheme 10 illustrates a general synthesis of an illustrative Linker unit containing a maleimide Stretcher group and optionally a p-aminobenzyl ether self-immolative Spacer.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and n is an integer ranging from 0-10.
Useful Stretchers may be incorporated into a Linker using the commercially available intermediates from Molecular Biosciences (Boulder, Colo.) described below by utilizing known techniques of organic synthesis.
Stretchers of formula (IIIa) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit as depicted in Schemes 11 and 12:
where n is an integer ranging from 1-10 and T is —H or —SO3Na;
where n is an integer ranging from 0-3;
Stretcher units of formula (IIIb) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
where X is —Br or —I; and
Stretcher units of formula (IV) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
Stretcher units of formula (Va) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
Other Stretchers useful in the invention may be synthesized according to known procedures. Aminooxy Stretchers of the formula shown below can be prepared by treating alkyl halides with N-Boc-hydroxylamine according to procedures described in Jones, D. S. et al., Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon, C. et al., Tetrahedron, 1967, 23(11), 4441-4447.
where —R17— is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, —(CH2CH2O)r—CH2—; and r is an integer ranging from 1-10;
Isothiocyanate Stretchers of the formula shown below may be prepared from isothiocyanatocarboxylic acid chlorides as described in Angew. Chem., 1975, 87(14), 517.
where —R17— is as described herein.
Scheme 11 shows a method for obtaining of a val-cit dipeptide Linker having a maleimide Stretcher and optionally a p-aminobenzyl self-immolative Spacer.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
Scheme 12 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linker unit having a maleimide Stretcher unit and a p-aminobenzyl self-immolative Spacer unit. Starting material 23 (lys(Mtr)) is commercially available (Bachem, Torrance, Calif.) or can be prepared according to Dubowchik, et al. Tetrahedrom Letters 1997, 38, 5257-60.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
As shown in Scheme 13, a Linker can be reacted with an amino group of a Drug 10 to form a Drug-Linker Compound that contains an amide or carbamate group, linking the Drug unit to the Linker unit. When Reactive Site No. 1 is a carboxylic acid group, as in Linker 29, the coupling reaction can be performed using HATU or PyBrop and an appropriate amine base, resulting in a Drug-Linker Compound 30, containing a amide bond between the Drug unit and the Linker unit. When Reactive Site No. 1 is a carbonate, as in Linker 31, the Linker can be coupled to the Drug using HOBt in a mixture of DMF/pyridine to provide a Drug-Linker Compound 32, containing a carbamate bond between the Drug unit and the Linker unit.
When Reactive Site No. 1 is an hydroxyl group, such as Linker 33, the Linker can be coupled with a phenol group of a Drug using Mitsunobu chemistry to provide a Drug-Linker Compound 34 having an ether linkage between the Drug unit and the Linker unit.
Alternately, when Reactive Site No. 1 is a good leaving group, such as in Linker 70, the Linker can be coupled with a hydroxyl group or an amine group of a Drug via a nucleophilic substitution process to provide a Drug-Linker Compound having an ether linkage (34) or an amine linkage (71) between the Drug unit and the Linker unit.
Illustrative methods useful for linking a Drug to a Ligand to form a Drug-Linker Compound are depicted in Scheme 13 and are outlined in General Procedures G-J.
General Procedure G: Amide formation using HATU. A Drug 10 (1.0 eq.) and an N-protected Linker containing a carboxylic acid Reactive site (1.0 eq.) are diluted with a suitable organic solvent, such as dichloromethane, and the resulting solution is treated with HATU (1.5 eq.) and an organic base, preferably pyridine (1.5 eq.). The reaction mixture is allowed to stir under an inert atmosphere, preferably argon, for 6 h, during which time the reaction mixture is monitored using HPLC. The reaction mixture is concentrated and the resulting residue is purified using HPLC to yield the amide 30.
General Procedure H: Carbamate formation using HOBt. A mixture of a Linker 31 having a p-nitrophenyl carbonate Reactive site (1.1 eq.) and Drug 10 (1.0 eq.) are diluted with an aprotic organic solvent, such as DMF, to provide a solution having a concentration of 50-100 mM, and the resulting solution is treated with HOBt (2.0 eq.) and placed under an inert atmosphere, preferably argon. The reaction mixture is allowed to stir for 15 min, then an organic base, such as pyridine (¼ v/v), is added and the reaction progress is monitored using HPLC. The Linker is typically consumed within 16 h. The reaction mixture is then concentrated in vacuo and the resulting residue is purified using, for example, HPLC to yield the carbamate 32.
General Procedure I: Ether formation using Mitsunobu chemistry. A Drug of general formula 10, which contains a free hydroxyl group, is diluted with THF to make a 1.0 M solution and to this solution is added a Linker (1.0 eq) containing an hydroxy group at Reactive site No. 1 (33), followed by triphenylphosphine (1.5 eq.). The reaction mixture is put under an argon atmosphere and cooled to 0° C. DEAD (1.5 eq.) is then added dropwise via syringe and the reaction is allowed to stir at room temperature while being monitored using HPLC. The reaction is typically complete in 0.5-12 h, depending on the substrates. The reaction mixture is diluted with water (in volume equal to that of the THF) and the reaction mixture is extracted into EtOAc. The EtOAc layer is washed sequentially with water and brine, then dried over MgSO4 and concentrated. The resulting residue is purified via flash column chromatography using a suitable eluent to provide ether 34.
General Procedure J: Ether/amine Formation via Nucleophilic Substitution. A Drug of general formula 10, which contains a free hydroxyl group or a free amine group, is diluted with a polar aprotic solvent, such as THF, DMF or DMSO, to make a 1.0 M solution and to this solution is added a non-nucleophilic base (about 1.5 eq), such as pyridine, diisopropylethylamine or triethylamine. The reaction mixture is allowed to stir for about 1 hour, and to the resulting solution is added an approximately 1.0M solution of Linker 70 in a polar aprotic solvent, such as THF, DMF or DMSO. The resulting reaction is stirred under an inert atmosphere while being monitored using TLC or HPLC. The reaction is typically complete in 0.5-12 h, depending on the substrates. The reaction mixture is diluted with water (in volume equal to that of the reaction volume) and extracted into EtOAc. The EtOAc layer is washed sequentially with water, 1N HCl, water, and brine, then dried over MgSO4 and concentrated. The resulting residue is purified via flash column chromatography using a suitable eluent to provide an ether of formula 34 or an amine of formula 71, depending on whether the drug 10 contained a free hydroxyl group or a free amine group.
An alternate method of preparing Drug-Linker Compounds of the invention is outlined in Scheme 14. Using the method of Scheme 14, the Drug is attached to a partial Linker unit (19a, for example), which does not have a Stretcher unit attached. This provides intermediate 35, which has an Amino Acid unit having an Fmoc-protected N-terminus. The Fmoc group is then removed and the resulting amine intermediate 36 is then attached to a Stretcher unit via a coupling reaction catalyzed using PyBrop or DEPC. The construction of Drug-Linker Compounds containing either a bromoacetamide Stretcher 39 or a PEG maleimide Stretcher 38 is illustrated in Scheme 14.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
Methodology useful for the preparation of a Linker unit containing a branched spacer is shown in Scheme 15.
Scheme 15 illustrates the synthesis of a val-cit dipeptide linker having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS) unit. The synthesis of the BHMS intermediate (75) has been improved from previous literature procedures (see International Publication No, WO 9813059 to Firestone et al., and Crozet, M. P.; Archaimbault, G.; Vanelle, P.; Nouguier, R. Tetrahedron Lett. 1985, 26, 5133-5134) and utilizes as starting materials, commercially available diethyl(4-nitrobenzyl)phosphonate (72) and commercially available 2,2-dimethyl-1,3-dioxan-5-one (73). Linkers 77 and 79 can be prepared from intermediate 75 using the methodology described in Scheme 11.
Scheme 16 illustrates methodology useful for making Drug-Linker-Ligand conjugates of the invention having about 2 to about 4 drugs per antibody.
General Procedure K: Preparation of Conjugates having about 2 to about 4 drugs per antibody.
Partial Reduction of the Antibody
In general, to prepare conjugates having 2 drugs per antibody, the relevant antibody is reduced using a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) (about 1.8 equivalents) in PBS with 1 mM DTPA, adjusted to pH 8 with 50 mM borate. The solution is incubated at 37° C. for 1 hour, purified using a 50 ml G25 desalting column equilibrated in PBS/1 mM DTPA at 4° C. The thiol concentration can be determined according to General Procedure M, the protein concentration can be determined by dividing the A280 value by 1.58 extinction coefficient (mg/ml), and the ratio of thiol to antibody can be determined according to General Procedure N.
Conjugates having 4 drugs per antibody can be made using the same methodology, using about 4.2 equivalents of a suitable reducing agent to partially reduce the antibody.
Conjugation of Drug-Linker to Partially Reduced Antibody
The partially reduced antibody samples can be conjugated to a corresponding Drug-Linker compound using about 2.4 and about 4.6 molar equivalents of Drug-Linker compound per antibody to prepare the 2 and 4 drug per antibody conjugates, respectively. The conjugation reactions are incubated on ice for 1 hour, quenched with about 20-fold excess of cysteine to drug, and purified by elution over a G25 desalting column at about 4° C. The resulting Drug-Linker-Ligand conjugates are concentrated to about 3 mg/ml, sterile filtered, aliquoted and stored frozen.
Scheme 17 depicts the construction of a Drug-Linker-Ligand Conjugate by reacting the sulfhydryl group of a Ligand with a thiol-acceptor group on the Linker group of a Drug-Linker Compound.
Illustrative methods for attaching a Ligand antibody to a Drug-Linker Compound are outlined below in General Procedures L-R.
General Procedure L: Attachment of an Antibody Ligand to a Drug-Linker Compound. All reaction steps are typically carried out at 4° C. Where the Ligand is a monoclonal antibody having one or more disulfide bonds, solutions of the monoclonal antibody (5-20 mg/mL) in phosphate buffered saline, pH 7.2, are reduced with dithiothreitol (10 mM final) at 37° C. for 30 minutes (See General Procedure M) and separation of low molecular weight agents is achieved by size exclusion chromatography on Sephadex G25 columns in PBS containing 1 mM diethylenetriaminepentaacetic acid.
The sulfhydryl content in the Ligand can be determined using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as described in General Procedure M (see Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Anal. Biochem. 94, 75-81). To a PBS solution of Ligand reduced according to General Procedure L, a Drug-Linker Compound in MeCN is added so that the solution is 20% MeCN/PBS (vol/vol). The amount of Drug-Linker Compound is approximately 10% more than the total number of sulfhydryl groups on a Ligand. After 60 min at 4° C., cysteine is added (20-fold excess over concentration of the Drug-Linker Compound), the solution is concentrated by ultrafiltration, and any low molecular weight agents are removed by gel filtration. The number of Drug-Linker Compounds per antibody is determined by uv/vis spectroscopy using formulas derived from the relative extinction coefficients of the Ligands and Drug-Linker Compounds as described in General Procedure O. The amount of quenched Drug-Linker Compound is then determined as described in General Procedure P using reverse-phase HPLC. The aggregation state of the Ligand Antibodies of the Drug-Linker-Ligand Conjugates can be determined using size-exclusion HPLC as described in General Procedure R. The Drug-Linker-Ligand Conjugates can be used without further purification.
General Procedure M: Reduction of the interchain disulfide bonds of an Antibody. To a solution of 24 mg of an antibody (2.4 mL of 10 mg/mL solution) in suitable buffer is added 300 μL of Borate buffer (500 mM sodium borate/500 mM sodium chloride, pH 8.0) followed by 300 μL of Dithiothreitol (DTT, 100 mM solution in H2O). The reaction mixture is stirred using a vortex instrument and incubated at 37° C. for 30 min. Three PD10 columns are equilibrated with PBS containing 1 mM DTPA (in PBS) and the reduced antibody is eluted through the three PD10 columns and collected in 4.2 mL PBS/DTPA solution (1.4 mL per column). The reduced antibody is then stored on ice. The number of thiols per antibody and the antibody concentration are determined according to General Procedure N.
General Procedure N: Determination of Number of Thiols Per Ligand
A reference sample of a Ligand or a sample of an antibody reduced according to General Procedure L is diluted to about 1:40 (w/w) in PBS, and the uv absorbance of the solution is measured at 280 nm using standard uv spectroscopic methods. Preferably, the ratio of Ligand:PBS in the solution is such that the uv absorbance ranges from about 0.13-0.2 AU (absorbance units).
A test sample of a Ligand or a test sample of an antibody reduced according to General Procedure L is diluted to about 1:20 with a PBS solution containing about 15 μL DTNB stock solution/mL PBS. A blank sample containing DTNB at the same concentration as the test solution (i.e., 15 μL DTNB stock/mL PBS) is then prepared. The spectrophotometer is referenced at zero nm with the blank sample, then the absorbance of the test sample is measured at 412 nm.
The molar concentration of the antibody is then determined using the formula: [Ligand]=(OD280/2.24e5)×dilution factor.
The molar concentration of thiol is then determined using the formula: [—SH]=(OD412/1.415e4)×dilution factor.
The [SH]/[Ligand] ratio is then calculated. A reduced monoclonal antibody Ligand can have from 1 to about 20 sulfhydryl groups, but typically has between about 6 to about 9 sulfhydryl groups. In a preferred embodiment, the [SH]/[Ligand] ratio range is from about 7 to about 9.
It is understood that the [SH]/[Ligand] ratio is the average number of -Aa-Ww-Yy-D units per Ligand unit.
General Procedure O: Determination of the number of Drug molecules per Antibody in a Drug-Linker-Antibody Conjugate. The Drug:Antibody ratio for a Drug-Linker-Antibody Conjugate is determined by measuring the number of Dithiothreitol (DTT) reducible thiols that remain after conjugation, using the following method: A 200 mL sample of a Drug-Linker-Antibody conjugate is treated with DTT (100 mM solution in water) to bring the concentration to 10 mM DTT. The resulting solution is incubated at 37° C. for 30 min, then eluted through a PD10 column using PBS/DTPA as the eluent. The OD280 of the reduced conjugate is then measured and the molar concentration is measured according to General Procedure Q.
The molar concentration of thiol is determined using DTNB as described in General Procedure M. The ratio of thiol concentration to antibody concentration is then calculated and the Drug:Ligand ratio is the difference between the Thiol:Antibody ratio (determined using General Procedure N) and the Drug:Antibody ratio as determined in the previous paragraph.
General Procedure P: Determination of the amount of quenched Drug-Linker compound in a Drug-Linker-Antibody Conjugate. This assay provides a quantitative determination of the Drug-Linker in the Drug-Linker-Antibody conjugate that is not covalently bound to Antibody. Assuming that all maleimide groups of Drug-Linker in the reaction mixture have been quenched with Cysteine, the unbound drug is the Cysteine quenched adduct of the Drug-Linker Compound, i.e. Drug-Linker-Cys. The proteinaceous Drug-Linker-Antibody Conjugate is denatured, precipitated, and isolated by centrifugation under conditions in which the Drug-Linker-Cys is soluble. The unbound Drug-Linker-Cys is detected quantitatively by HPLC, and the resulting chromatogram is compared to a standard curve to determine the concentration of unbound Drug-Linker-Cys in the sample. This concentration is divided by the total concentration of Drug in the conjugate as determined using General Procedure 0 and General Procedure Q.
Specifically, 100 mL of a 100 μM Drug-Linker-Cys adduct “working solution” is prepared by adding 1 μL of 100 mM Cysteine in PBS/DTPA and an appropriate volume of stock solution of a Drug-Linker compound to 98 μL of 50% methanol/PBS. The “appropriate volume” in liters is calculated using the formula: V=1c-8/[Drug-Linker]. Six tubes are then labelled as follows: “0”, “0.5”, “1”, “2”, “3”, and “5”, and appropriate amounts of working solution are placed in each tube and diluted with 50% methanol/PBS to give a total volume of 100 mL in each tube. The labels indicate the μM concentration of the standards.
A 50 μL solution of a Drug-Linker-Antibody Conjugate and a 50 μL solution of the Cysteine quenched reaction mixture (“qrm”) are collected in separate test tubes and are each diluted with 50 μL, of methanol that has been cooled to −20° C. The samples are then cooled to −20° C. over 10 min.
The samples are then centrifuged at 13000 rpm in a desktop centrifuge for 10 min. The supernatants are transferred to HPLC vials, and 90 μL aliquots of each sample are separately analyzed using HPLC (C12 RP column (Phenomenex); monitored at the absorbance maximum of the Drug-Linker Compound using a flow rate of 1.0 mL/min. The eluent used is a linear gradient of MeCN ranging from 10 to 90% in aqueous 5 mM ammonium phosphate, pH 7.4, over 10 min; then 90% MeCN over 5 min.; then returning to initial conditions). The Drug-Linker-Cys adduct typically elutes between about 7 and about 10 minutes.
A standard curve is then prepared by plotting the Peak Area of the standards vs. their concentration (in μM). Linear regression analysis is performed to determine the equation and correlation coefficient of the standard curve. R2 values are typically >0.99. From the regression equation is determined the concentration of the Drug-Linker-Cys adduct in the HPLC sample and in the conjugate, using the formulas:
[Drug-Linker-Cys](HPLC spl)=(Peak area−intercept)/slope;
[Drug-Linker-Cys](conjugate)=2×[Drug-Linker-Cys](HPLC spl)
The percent of Drug-Linker-Cys adduct present can be determined using the formula:
% Drug-Linker-Cys=100×[Drug-Linker-Cys](conjugate)/[drug]
where [drug]=[Conjugate]×drug/Ab, [Conjugate] is determined using the conjugate concentration assay, and the Drug: Antibody ratio is determined using the Drug: Antibody ratio assay.
General Procedure Q: Determination of Drug-Linker-Antibody Conjugate concentration for drug linkers with minimal uv absorbance at 280 nm. The concentration of Drug-Linker-Antibody conjugate can be determined in the same manner for the concentration of the parent antibody, by measuring the absorbance at 280 nm of an appropriate dilution, using the following formula:
[Conjugate](mg/mL)=(OD280×dilution factor/1.4)×0.9
Determination of Drug-Linker-Antibody Conjugate concentration for drug linkers with substantial uv absorbance at 280 nm (e.g. Compounds 68 and 69). Because the absorbances of Compounds 68 and 69 overlap with the absorbances of an antibody, spectrophotometric determination of the conjugate concentration is most useful when the measurement is performed using the absorbances at both 270 nm and 280 nm. Using this data, the molar concentration of Drug-Linker-Ligand conjugate is given by the following formula:
[Conjugate]=(OD280×1.23 e−5−OD270×9.35e−6)×dilution factor where the 1.23e−5 and 9.35e−6 are calculated from the molar extinction coefficients of the drug and the antibody, which are estimated as:
e270 Drug=2.06e4 e270 Antibody=1.87e5
e280 Drug=1.57e4 e280 Antibody=2.24e5
Determination of Drug-Linker-Antibody Conjugate concentration for drug linkers with substantial uv absorbance at 280 nm (e.g. Compounds 68 and 69). Because the absorbances of Compounds 68 and 69 overlap with the absorbances of an antibody, spectrophotometric determination of the conjugate concentration is most useful when the measurement is performed using the absorbances at both 270 nm and 280 nm. Using this data, the molar concentration of Drug-Linker-Ligand conjugate is given by the following formula:
[Conjugate]=(OD280×1.23 e−5−OD270×9.35e−6)×dilution factor where the values 1.23e−5 and 9.35e−6 are calculated from the molar extinction coefficients of the drug and the antibody, which are estimated as:
e270 Drug=2.06e4 e270 Antibody=1.87e5
e280 Drug=1.57e4 e280 Antibody=2.24e5
General Procedure R: Determination of the aggregation state of The Antibody in a Drug-Linker-Antibody Conjugate. A suitable quantity (−10 μg) of a Drug-Linker-Antibody Conjugate is eluted through a size-exclusion chromatography (SEC) column (Tosoh Biosep SW3000 4.6 mm×30 cm eluted at 0.35 mL/min. with PBS) under standard conditions. Chromatograms are obtained at 220 nm and 280 nm and the OD280/OD220 ratio is calculated. The corresponding aggregate typically has a retention time of between about 5.5 and about 7 min, and has about the same OD280/OD220 ratio as the monomeric Drug-Linker-Antibody Conjugate.
Compositions
In other aspects, the present invention provides a composition comprising an effective amount of a Compound of the Invention and a pharmaceutically acceptable carrier or vehicle. For convenience, the Drug units, Drug-Linker Compounds and Drug-Linker-Ligand Conjugates of the invention can simply be referred to as compounds of the invention. The compositions are suitable for veterinary or human administration.
The compositions of the present invention can be in any form that allows for the composition to be administered to an animal. For example, the composition can be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Preferably, the compositions are administered parenterally. Pharmaceutical compositions of the invention can be formulated so as to allow a Compound of the Invention to be bioavailable upon administration of the composition to an animal. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of a Compound of the Invention in aerosol form can hold a plurality of dosage units.
Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the Compound of the Invention, the manner of administration, and the composition employed.
The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) can be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.
When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
When the composition is in the form of a capsule, e.g., a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
The composition can be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
The liquid compositions of the invention, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material. Physiological saline is a preferred adjuvant. An injectable composition is preferably sterile.
The amount of the Compound of the Invention that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
The compositions comprise an effective amount of a Compound of the Invention such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a Compound of the Invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the Compound of the Invention by weight of the composition. Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the Compound of the Invention.
For intravenous administration, the composition can comprise from about 1 to about 250 mg of a Compound of the Invention per kg of the animal's body weight. Preferably, the amount administered will be in the range from about 4 to about 25 mg/kg of body weight of the Compound of the Invention.
Generally, the dosage of Compound of the Invention administered to an animal is typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight. Preferably, the dosage administered to an animal is between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.
The Compounds of the Invention or compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a Compound of the Invention or composition. In certain embodiments, more than one Compound of the Invention or composition is administered to an animal. Methods of administration include, but are not limited to, oral administration and parenteral administration; parenteral administration including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition (such as the site of cancer or autoimmune disease).
In a preferred embodiment, the present Compounds of the Invention or compositions are administered parenterally.
In a more preferred embodiment, the present Compounds of the Invention or compositions are administered intravenously.
In specific embodiments, it can be desirable to administer one or more Compounds of the Invention or compositions locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue. In another embodiment, administration can be by direct injection at the site (or former site) of a manifestation of an autoimmune disease.
In certain embodiments, it can be desirable to introduce one or more Compounds of the Invention or compositions into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the Compounds of the Invention or compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
In another embodiment, the Compounds of the invention can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the Compounds of the Invention or compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the Compounds of the Invention or compositions, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can be used.
The term “carrier” refers to a diluent, adjuvant or excipient, with which a Compound of the Invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the Compounds of the Invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the Compounds of the Invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
In a preferred embodiment, the Compounds of the Invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly human beings. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where a Compound of the Invention is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the Compound of the Invention is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade.
The compositions can be intended for topical administration, in which case the carrier may be in the form of a solution, emulsion, ointment or gel base. The base, for example, can comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents can be present in a composition for topical administration. If intended for transdermal administration, the composition can be in the form of a transdermal patch or an iontophoresis device. Topical formulations can comprise a concentration of a Compound of the Invention of from about 0.1% to about 10% w/v (weight per unit volume of composition).
The composition can be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the Compound of the Invention. The composition for rectal administration can contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The composition can include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition can include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.
The compositions can consist of gaseous dosage units, e.g., it can be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery can be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of Compounds of the Invention can be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the Compound(s) of the Invention. Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, Spacers and the like, which together can form a kit. Preferred aerosols can be determined by one skilled in the art, without undue experimentation.
Whether in solid, liquid or gaseous form, the compositions of the present invention can comprise a pharmacological agent used in the treatment of cancer, an autoimmune disease or an infectious disease.
The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a Compound of the Invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a Compound of the Invention so as to facilitate dissolution or homogeneous suspension of the active compound in the aqueous delivery system.
Therapeutic Uses of the Compounds of the Invention
The Compounds of the Invention are useful for treating cancer, an autoimmune disease or an infectious disease in an animal.
Treatment of Cancer
The Compounds of the Invention are useful for inhibiting the multiplication of a tumor cell or cancer cell, or for treating cancer in an animal. The Compounds of the Invention can be used accordingly in a variety of settings for the treatment of animal cancers. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug or Drug unit to a tumor cell or cancer cell. Without being bound by theory, in one embodiment, the Ligand unit of a Compound of the Invention binds to or associates with a cancer-cell or a tumor-cell-associated antigen, and the Compound of the Invention can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell. Once inside the cell, one or more specific peptide sequences within the Linker unit are hydrolytically cleaved by one or more tumor-cell or cancer-cell-associated proteases, resulting in release of a Drug or a Drug-Linker Compound. The released Drug or Drug-Linker Compound is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the Drug or Drug unit is cleaved from the Compound of the Invention outside the tumor cell or cancer cell, and the Drug or Drug-Linker Compound subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to the tumor cell or cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is on the surface of the tumor cell or cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is an extracellular matrix protein associated with the tumor cell or cancer cell.
In one embodiment, the tumor cell or cancer cell is of the type of tumor or cancer that the animal needs treatment or prevention of.
The specificity of the Ligand unit for a particular tumor cell or cancer cell can be important for determining those tumors or cancers that are most effectively treated. For example, Compounds of the Invention having a BR96 Ligand unit can be useful for treating antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas. Compounds of the Invention having an Anti-CD30 or an anti-CD40 Ligand unit can be useful for treating hematologic malignancies.
Other particular types of cancers that can be treated with Compounds of the Invention include, but are not limited to, those disclosed in Table 3.
TABLE 3
Solid tumors, including but not limited to:
fibrosarcoma
myxosarcoma
liposarcoma
chondrosarcoma
osteogenic sarcoma
chordoma
angiosarcoma
endotheliosarcoma
lymphangiosarcoma
lymphangioendotheliosarcoma
synovioma
mesothelioma
Ewing's tumor
leiomyosarcoma
rhabdomyosarcoma
colon cancer
colorectal cancer
kidney cancer
pancreatic cancer
bone cancer
breast cancer
ovarian cancer
prostate cancer
esophogeal cancer
stomach cancer
oral cancer
nasal cancer
throat cancer
squamous cell carcinoma
basal cell carcinoma
adenocarcinoma
sweat gland carcinoma
sebaceous gland carcinoma
papillary carcinoma
papillary adenocarcinomas
cystadenocarcinoma
medullary carcinoma
bronchogenic carcinoma
renal cell carcinoma
hepatoma
bile duct carcinoma
choriocarcinoma
seminoma
embryonal carcinoma
Wilms' tumor
cervical cancer
uterine cancer
testicular cancer
small cell lung carcinoma
bladder carcinoma
lung cancer
epithelial carcinoma
glioma
glioblastoma multiforme
astrocytoma
medulloblastoma
craniopharyngioma
ependymoma
pinealoma
hemangioblastoma
acoustic neuroma
oligodendroglioma
meningioma
skin cancer
melanoma
neuroblastoma
retinoblastoma
blood-borne cancers, including but not limited to:
acute lymphoblastic leukemia “ALL”
acute lymphoblastic B-cell leukemia
acute lymphoblastic T-cell leukemia
acute myeloblastic leukemia “AML”
acute promyelocytic leukemia “APL”
acute monoblastic leukemia
acute erythroleukemic leukemia
acute megakaryoblastic leukemia
acute myelomonocytic leukemia
acute nonlymphocyctic leukemia
acute undifferentiated leukemia
chronic myelocytic leukemia “CML”
chronic lymphocytic leukemia “CLL”
hairy cell leukemia
multiple myeloma
acute and chronic leukemias:
lymphoblastic
myelogenous
lymphocytic
myelocytic leukemias
Lymphomas:
Hodgkin's disease
non-Hodgkin's Lymphoma
Multiple myeloma
Waldenström's macroglobulinemia
Heavy chain disease
Polycythemia vera
The Compounds of the Invention can also be used as chemotherapeutics in the untargeted form. For example, the Drugs themselves, or the Drug-Linker Compounds are useful for treating ovarian, CNS, renal, lung, colon, melanoma, or hematologic cancers or tumors.
The Compounds of the Invention provide Conjugation specific tumor or cancer targeting, thus reducing general toxicity of these compounds. The Linker units stabilize the Compounds of the Invention in blood, yet are cleavable by tumor-specific proteases within the cell, liberating a Drug.
Multi-Modality Therapy for Cancer
Cancer, including, but not limited to, a tumor, metastasis, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of a Compound of the Invention.
In other embodiments, the invention provides methods for treating or preventing cancer, comprising administering to an animal in need thereof an effective amount of a Compound of the Invention and a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory. The Compounds of the Invention can be administered to an animal that has also undergone surgery as treatment for the cancer.
In one embodiment, the additional method of treatment is radiation therapy.
In a specific embodiment, the Compound of the Invention is administered concurrently with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of a Compound of the Invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e.g., up to three months), prior or subsequent to administration of a Compound of the Invention.
A chemotherapeutic agent can be administered over a series of sessions, any one or a combination of the chemotherapeutic agents listed in Table 4 can be administered. With respect to radiation, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered.
Additionally, the invention provides methods of treatment of cancer with a Compound of the Invention as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The animal being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
The Compounds of the Invention can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants. This can involve a multi-step process in which the animal's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the patient's remaining bone-marrow cell population is then eradicated via the administration of a high dose of a Compound of the Invention with or without accompanying high dose radiation therapy, and the stem cell graft is infused back into the animal. Supportive care is then provided while bone marrow function is restored and the animal recovers.
Multi-Drug Therapy for Cancer
The present invention includes methods for treating cancer, comprising administering to an animal in need thereof an effective amount of a Compound of the Invention and another therapeutic agent that is an anti-cancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred embodiment, the anti-cancer agent includes, but is not limited to, a drug listed in Table 4.
TABLE 4
Alkylating agents
Nitrogen mustards:
cyclophosphamide
Ifosfamide
trofosfamide
Chlorambucil
Nitrosoureas:
carmustine (BCNU)
Lomustine (CCNU)
Alkylsulphonates
busulfan
Treosulfan
Triazenes:
Dacarbazine
Platinum containing compounds:
Cisplatin
carboplatin
Plant Alkaloids
Vinca alkaloids:
vincristine
Vinblastine
Vindesine
Vinorelbine
Taxoids:
paclitaxel
Docetaxol
DNA Topoisomerase Inhibitors
Epipodophyllins:
etoposide
Teniposide
Topotecan
9-aminocamptothecin
camptothecin
crisnatol
mitomycins:
Mitomycin C
Anti-metabolites
Anti-folates:
DHFR inhibitors:
methotrexate
Trimetrexate
IMP dehydrogenase Inhibitors:
mycophenolic acid
Tiazofurin
Ribavirin
EICAR
Ribonuclotide reductase Inhibitors:
hydroxyurea
deferoxamine
Pyrimidine analogs:
Uracil analogs
5-Fluorouracil
Floxuridine
Doxifluridine
Ratitrexed
Cytosine analogs
cytarabine (ara C)
Cytosine arabinoside
fludarabine
Purine analogs:
mercaptopurine
Thioguanine
Hormonal therapies:
Receptor antagonists:
Anti-estrogen
Tamoxifen
Raloxifene
megestrol
LHRH agonists:
goscrclin
Leuprolide acetate
Anti-androgens:
flutamide
bicalutamide
Retinoids/Deltoids
Vitamin D3 analogs:
EB 1089
CB 1093
KH 1060
Photodynamic therapies:
vertoporfin (BPD-MA)
Phthalocyanine
photosensitizer Pc4
Demethoxy-hypocrellin A
(2BA-2-DMHA)
Cytokines:
Interferon-α
Interferon-γ
Tumor necrosis factor
Others:
Isoprenylation inhibitors:
Lovastatin
Dopaminergic neurotoxins:
1-methyl-4-phenylpyridinium ion
Cell cycle inhibitors:
staurosporine
Actinomycins:
Actinomycin D
Dactinomycin
Bleomycins:
bleomycin A2
Bleomycin B2
Peplomycin
Anthracyclines:
daunorubicin
Doxorubicin (adriamycin)
Idarubicin
Epirubicin
Pirarubicin
Zorubicin
Mitoxantrone
MDR inhibitors:
verapamil
Ca2+ATPase inhibitors:
thapsigargin
Treatment of Autoimmune Diseases
The Compounds of the Invention are useful for killing or inhibiting the replication of a cell that produces an autoimmune disease or for treating an autoimmune disease. The Compounds of the Invention can be used accordingly in a variety of settings for the treatment of an autoimmune disease in an animal. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. Without being bound by theory, in one embodiment, the Drug-Linker-Ligand Conjugate associates with an antigen on the surface of a target cell, and the Compound of the Invention is then taken up inside a target-cell through receptor-mediated endocytosis. Once inside the cell, one or more specific peptide sequences within the Linker unit are enzymatically or hydrolytically cleaved, resulting in release of a Drug. The released Drug is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the Drug is cleaved from the Compound of the Invention outside the target cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to an autoimmune antigen.
In another embodiment, the Ligand unit binds to an autoimmune antigen which is on the surface of a cell.
In another embodiment, the target cell is of the type of cell that produces the autoimmune antigen which causes the disease the animal needs treatment or prevention of.
In a preferred embodiment, the Ligand binds to activated lympocytes that are associated with the autoimmune diesease state.
In a further embodiment, the Compounds of the Invention kill or inhibit the multiplication of cells that produce an auto-immune antibody associated with a particular autoimmune disease.
Particular types of autoimmune diseases that can be treated with the Compounds of the Invention include, but are not limited to, Th2-lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft versus host disease); Th1 lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); activated B lymphocyte-related disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes); and those disclosed in Table 5.
TABLE 5
Active Chronic Hepatitis
Addison's Disease
Allergic Alveolitis
Allergic Reaction
Allergic Rhinitis
Alport's Syndrome
Anaphlaxis
Ankylosing Spondylitis
Anti-phosholipid Syndrome
Arthritis
Ascariasis
Aspergillosis
Atopic Allergy
Atropic Dermatitis
Atropic Rhinitis
Behcet's Disease
Bird-Fancier's Lung
Bronchial Asthma
Caplan's Syndrome
Cardiomyopathy
Celiac Disease
Chagas' Disease
Chronic Glomerulonephritis
Cogan's Syndrome
Cold Agglutinin Disease
Congenital Rubella Infection
CREST Syndrome
Crohn's Disease
Cryoglobulinemia
Cushing's Syndrome
Dermatomyositis
Discoid Lupus
Dressler's Syndrome
Eaton-Lambert Syndrome
Echovirus Infection
Encephalomyelitis
Endocrine opthalmopathy
Epstein-Barr Virus Infection
Equine Heaves
Erythematosis
Evan's Syndrome
Felty's Syndrome
Fibromyalgia
Fuch's Cyclitis
Gastric Atrophy
Gastrointestinal Allergy
Giant Cell Arteritis
Glomerulonephritis
Goodpasture's Syndrome
Graft v. Host Disease
Graves' Disease
Guillain-Barre Disease
Hashimoto's Thyroiditis
Hemolytic Anemia
Henoch-Schonlein Purpura
Idiopathic Adrenal Atrophy
Idiopathic Pulmonary Fibritis
IgA Nephropathy
Inflammatory Bowel Diseases
Insulin-dependent Diabetes Mellitus
Juvenile Arthritis
Juvenile Diabetes Mellitus (Type I)
Lambert-Eaton Syndrome
Laminitis
Lichen Planus
Lupoid Hepatitis
Lupus
Lymphopenia
Meniere's Disease
Mixed Connective Tissue Disease
Multiple Sclerosis
Myasthenia Gravis
Pernicious Anemia
Polyglandular Syndromes
Presenile Dementia
Primary Agammaglobulinemia
Primary Biliary Cirrhosis
Psoriasis
Psoriatic Arthritis
Raynauds Phenomenon
Recurrent Abortion
Reiter's Syndrome
Rheumatic Fever
Rheumatoid Arthritis
Sampter's Syndrome
Schistosomiasis
Schmidt's Syndrome
Scleroderma
Shulman's Syndrome
Sjorgen's Syndrome
Stiff-Man Syndrome
Sympathetic Ophthalmia
Systemic Lupus Erythematosis
Takayasu's Arteritis
Temporal Arteritis
Thyroiditis
Thrombocytopenia
Thyrotoxicosis
Toxic Epidermal Necrolysis
Type B Insulin Resistance
Type I Diabetes Mellitus
Ulcerative Colitis
Uveitis
Vitiligo
Waldenstrom's Macroglobulemia
Wegener's Granulomatosis
Multi-Drug Therapy of Autoimmune Diseases
The present invention also provides methods for treating an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Compound of the Invention and another therapeutic agent that known for the treatment of an autoimmune disease. In one embodiment, the anti-autoimmune disease agent includes, but is not limited to, agents listed in Table 6.
TABLE 6
cyclosporine
cyclosporine A
mycophenylate mofetil
sirolimus
tacrolimus
enanercept
prednisone
azathioprine
methotrexate cyclophosphamide
prednisone
aminocaproic acid
chloroquine
hydroxychloroquine
hydrocortisone
dexamethasone
chlorambucil
DHEA
danazol
bromocriptine
meloxicam
infliximab
Treatment of Infectious Diseases
The Compounds of the Invention are useful for killing or inhibiting the multiplication of a cell that produces an infectious disease or for treating an infectious disease. The Compounds of the Invention can be used accordingly in a variety of settings for the treatment of an infectious disease in an animal. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. Without being bound by theory, in one embodiment, the Drug-Linker-Ligand Conjugate associates with an antigen on the surface of a target cell, and the Compound of the Invention is then taken up inside a target-cell through receptor-mediated endocytosis. Once inside the cell, one or more specific peptide sequences within the Linker unit are enzymatically or hydrolytically cleaved, resulting in release of a Drug. The released Drug is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the Drug is cleaved from the Compound of the Invention outside the target cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to the infectious disease cell.
In one embodiment, the infectious disease type of infectious disease that the animal needs treatment or prevention of.
In one embodiment, the Compounds of the Invention kill or inhibit the multiplication of cells that produce a particular infectious disease.
Particular types of infectious diseases that can be treated with the Compounds of the Invention include, but are not limited to, those disclosed in Table 7.
TABLE 7
Bacterial Diseases:
Diptheria
Pertussis
Occult Bacteremia
Urinary Tract Infection
Gastroenteritis
Cellulitis
Epiglottitis
Tracheitis
Adenoid Hypertrophy
Retropharyngeal Abcess
Impetigo
Ecthyma
Pneumonia
Endocarditis
Septic Arthritis
Pneumococcal
Peritonitis
Bactermia
Meningitis
Acute Purulent Meningitis
Urethritis
Cervicitis
Proctitis
Pharyngitis
Salpingitis
Epididymitis
Gonorrhea
Syphilis
Listeriosis
Anthrax
Nocardiosis
Salmonella
Typhoid Fever
Dysentery
Conjuntivitis
Sinusitis
Brucellosis
Tullaremia
Cholera
Bubonic Plague
Tetanus
Necrotizing Enteritis
Actinomycosis
Mixed Anaerobic Infections
Syphilis
Relapsing Fever
Leptospirosis
Lyme Disease
Rat Bite Fever
Tuberculosis
Lymphadenitis
Leprosy
Chlamydia
Chlamydial Pneumonia
Trachoma
Inclusion Conjunctivitis
Systemic Fungal Diseases:
Histoplamosis
Coccicidiodomycosis
Blastomycosis
Sporotrichosis
Cryptococcsis
Systemic Candidiasis
Aspergillosis
Mucormycosis
Mycetoma
Chromomycosis
Rickettsial Diseases:
Typhus
Rocky Mountain Spotted Fever
Ehrlichiosis
Eastern Tick-Borne Rickettsioses
Rickettsialpox
Q Fever
Bartonellosis
Parasitic Diseases:
Malaria
Babesiosis
African Sleeping Sickness
Chagas' Disease
Leishmaniasis
Dum-Dum Fever
Toxoplasmosis
Meningoencephalitis
Keratitis
Entamebiasis
Giardiasis
Cryptosporidiasis
Isosporiasis
Cyclosporiasis
Microsporidiosis
Ascariasis
Whipworm Infection
Hookworm Infection
Threadworm Infection
Ocular Larva Migrans
Trichinosis
Guinea Worm Disease
Lymphatic Filariasis
Loiasis
River Blindness
Canine Heartworm Infection
Schistosomiasis
Swimmer's Itch
Oriental Lung Fluke
Oriental Liver Fluke
Fascioliasis
Fasciolopsiasis
Opisthorchiasis
Tapeworm Infections
Hydatid Disease
Alveolar Hydatid Disease
Viral Diseases:
Measles
Subacute sclerosing panencephalitis
Common Cold
Mumps
Rubella
Roseola
Fifth Disease
Chickenpox
Respiratory syncytial virus infection
Croup
Bronchiolitis
Infectious Mononucleosis
Poliomyelitis
Herpangina
Hand-Foot-and-Mouth Disease
Bornholm Disease
Genital Herpes
Genital Warts
Aseptic Meningitis
Myocarditis
Pericarditis
Gastroenteritis
Acquired Immunodeficiency Syndrome (AIDS)
Reye's Syndrome
Kawasaki Syndrome
Influenza
Bronchitis
Viral “Walking” Pneumonia
Acute Febrile Respiratory Disease
Acute pharyngoconjunctival fever
Epidemic keratoconjunctivitis
Herpes Simplex Virus 1 (HSV-1)
Herpes Simples Virus 2 (HSV-2)
Shingles
Cytomegalic Inclusion Disease
Rabies
Progressive Multifocal Leukoencephalopathy
Kuru
Fatal Familial Insomnia
Creutzfeldt-Jakob Disease
Gerstmann-Straussler-Scheinker Disease
Tropical Spastic Paraparesis
Western Equine Encephalitis
California Encephalitis
St. Louis Encephalitis
Yellow Fever
Dengue
Lymphocytic choriomeningitis
Lassa Fever
Hemorrhagic Fever
Hantvirus Pulmonary Syndrome
Marburg Virus Infections
Ebola Virus Infections
Smallpox
Multi-Drug Therapy of Infectious Diseases
The present invention also provides methods for treating an infectious disease, comprising administering to an animal in need thereof a Compound of the Invention and another therapeutic agent that is an anti-infectious disease agent. In one embodiment, the anti-infectious disease agent is, but not limited to, agents listed in Table 8.
TABLE 8
Antibacterial Agents:
β-Lactam Antibiotics:
Penicillin G
Penicillin V
Cloxacilliin
Dicloxacillin
Methicillin
Nafcillin
Oxacillin
Ampicillin
Amoxicillin
Bacampicillin
Azlocillin
Carbenicillin
Mezlocillin
Piperacillin
Ticarcillin
Aminoglycosides:
Amikacin
Gentamicin
Kanamycin
Neomycin
Netilmicin
Streptomycin
Tobramycin
Macrolides:
Azithromycin
Clarithromycin
Erythromycin
Lincomycin
Clindamycin
Tetracyclines:
Demeclocycline
Doxycycline
Minocycline
Oxytetracycline
Tetracycline
Quinolones:
Cinoxacin
Nalidixic Acid
Fluoroquinolones:
Ciprofloxacin
Enoxacin
Grepafloxacin
Levofloxacin
Lomefloxacin
Norfloxacin
Ofloxacin
Sparfloxacin
Trovafloxicin
Polypeptides:
Bacitracin
Colistin
Polymyxin B
Sulfonamides:
Sulfisoxazole
Sulfamethoxazole
Sulfadiazine
Sulfamethizole
Sulfacetamide
Miscellaneous Antibacterial Agents:
Trimethoprim
Sulfamethazole
Chloramphenicol
Vancomycin
Metronidazole
Quinupristin
Dalfopristin
Rifampin
Spectinomycin
Nitrofurantoin
Antiviral Agents:
General Antiviral Agents:
Idoxuradine
Vidarabine
Trifluridine
Acyclovir
Famicyclovir
Pencicyclovir
Valacyclovir
Gancicyclovir
Foscarnet
Ribavirin
Amantadine
Rimantadine
Cidofovir
Antisense Oligonucleotides
Immunoglobulins
Inteferons
Drugs for HIV infection:
Zidovudine
Didanosine
Zalcitabine
Stavudine
Lamivudine
Nevirapine
Delavirdine
Saquinavir
Ritonavir
Indinavir
Nelfinavir
Other Therapeutic Agents
The present methods can further comprise the administration of a Compound of the Invention and an additional therapeutic agent or pharmaceutically acceptable salts or solvates thereof. The Compound of the Invention and the other therapeutic agent can act additively or, more preferably, synergistically. In a preferred embodiment, a composition comprising a Compound of the Invention is administered concurrently with the administration of one or more additional therapeutic agent(s), which can be part of the same composition or in a different composition from that comprising the Compound of the Invention. In another embodiment, a Compound of the Invention is administered prior to or subsequent to administration of another therapeutic agent(s).
In the present methods for treating cancer, an autoimmune disease or an infectious disease, the other therapeutic agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.
In another embodiment, the other therapeutic agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and erythropoietin alfa.
In still another embodiment, the other therapeutic agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
The following examples are provided by way of illustration and not limitation.
EXAMPLES
Materials and Methods. Commercially available reagents and solvents were obtained as follows: HPLC-grade solvents, Fisher Scientific (Atlanta, Ga.); anhydrous solvents, Aldrich (St. Louis, Mo.); diisopropylazodicarboxylate (DIAD, 95%), Lancaster (Lancashire, England); 4-aminobenzyl alcohol, Alfa Aesar (Ward Hill, Mass.); L-citrulline, Novabiochem (Laufelfingen, Switzerland); all other amino acids, Advanced ChemTech (Louisville, Ky.) or Novabiochem (Laufelfingen, Switzerland); (1S,2R)-(+)-norephedrine and other commercially available reagents, Aldrich or Acros; all coupling reagents were acquired from Novabiochem or Aldrich. All solvents used as reaction media are assumed to be anhydrous unless otherwise indicated. 1H-NMR spectra were recorded on either a Varian Gemini at 300 MHz or Varian Mercury 400 MHz spectrophotometer. Flash column chromatography was performed using 230-400 mesh ASTM silica gel from Fisher. Analtech silica gel GHLF plates were used for thin-layer chromatography. Analytical HPLC was performed using a Waters Alliance system using a photodiode array detector. Preparative HPLC purification was performed using a Varian Prostar system that had either a photodiode array or dual wavelength detector. Combustion analyses were determined by Quantitative Technologies, Inc., Whitehouse, N.J.
Examples 5-12 relate to Drugs that can be used as Drug units in the invention.
Example 1
Preparation of Compound 21
Fmoc-(L)-val-(L)-cit-PAB-OH (19) (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (about 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC—OSu (Willner et al., Bioconjugate Chem. 4, 521, 1993, 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide Compound 20 as an off-white solid. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M−H]−
Compound 20 (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and to the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and diisopropylethylamine (3.66 mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oil that was precipitated with EtOAc, and then triturated using EtOAc (about 25 mL). The solute was further precipitated with ether (about 200 mL) and triturated for 15 min. The solid was filtered and dried under high vacuum to provide Compound 21 which was 93% pure according to HPLC and used in the next step without further purification. Yield: 9.7 g (94%).
Example 2
Preparation of Compound 27
Compound 26 (2.0 g, 2.31 mmol, 1.0 eq.) was diluted with dichloromethane (30 mL), and to the resulting solution was added bis(4-nitrophenyl)carbonate (2.72 g, 8.94 mmol, 3.8 eq.) followed by diisopropylethylamine (1.04 mL, 5.97 mmol, 2.6 eq.). The reaction was complete in 3 d, according to HPLC. The reaction mixture was concentrated and the resulting residue was triturated using ether, then filtered and dried under high vacuum to provide Compound 27 as a yellow solid (2.37 g, 97%).
Example 3
Preparation of Compound 28
Fmoc-phe-lys(Mtr)-OH (24) (0.5 g, 0.63 mmol, U.S. Pat. No. 6,214,345 to Firestone et al.) was diluted with dichloromethane to a concentration of 0.5 M and to this solution was added diethylamine in an amount that was approximately one-third of the volume of the Compound 24/dichloromethane solution. The reaction was allowed to stir and was monitored using HPLC. It was shown to be complete by HPLC in 3 h. The reaction mixture was concentrated in vacuo, and the resulting residue was diluted with ethyl acetate and then reconcentrated. The resulting residue was triturated using ether and filtered. The residual solid was diluted with dichloromethane to a concentration of 0.2M, and to the resulting solution was added MC—OSu (0.20 g, 0.63 mmol, 1.0 eq.) and diisopropylethylamine (0.12 mL, 0.70 mmol, 1.1 eq.). The reaction mixture was allowed to stir under a nitrogen atmosphere for 16 h, after which time HPLC showed very little starting material. The reaction mixture was then concentrated and the resulting residue was triturated using ether to provide Compound 28 as a colored solid. Yield: 100 mg (21%); ES-MS m/z 757.9 [M−H]−.
Example 4
Preparation of Compound 19A
Compound 19 (1.0 g, 1.66 mmol) was diluted with DMF (10 mL) and to the resulting solution was added bis(4-nitrophenyl)carbonate (1.0 g, 3.3 mmol, 2.0 eq.).
The reaction mixture was immediately treated with diisopropylethylamine (0.43 mL, 2.5 mmol, 1.5 eq.) and the reaction was allowed to stir under an argon atmosphere. The reaction was complete in 2.5 h according to HPLC. The reaction mixture was concentrated to provide a light brown oil that was precipitated using ethyl acetate (5 mL), then precipitated again using ether (about 100 mL). The resulting precipitate was allowed to stand for 30 min, and was then filtered and dried under high vacuum to provide Compound 19a as an off-white powder. Yield: 1.05 g (83%); ES-MS m/z 767.2 [M+H]+; UV λmax 215, 256 nm.
Example 5
Preparation of Compound 49
Compound 49 was made according to General Procedure D using Fmoc-Me-val-val-dil-O-t-Bu 39 (0.40 g, 0.57 mmol) as the tripeptide and Boc-dap-nor 44 (0.26 g, 0.62 mmol, 1.1 eq.) as the dipeptide. The reaction mixture was purified using flash column chromatography (silica gel column, eluant—100% EtOAc). Two Fmoc-containing products eluted: the Fmoc derivative of Compound 49 (Rf 0.17 in 100% EtOAc) and what was believed to be the Fmoc derivative of the TFA acetate of Compound 49 (Rf 0.37). The products were combined to provide a white foam that was subjected to General Procedure E. Reaction was complete after 2 h. Solvents were removed to provide an oil that was purified using flash column chromatography (eluant—9:1 Dichloromethane-methanol) to provide Compound 49.
Example 6
Preparation of Compound 50
Compound 50 was prepared by reacting tripeptide 42 and dipeptide 48 according to General Procedure D using triethylamine (5.0 eq.) as the base. After concentration of the reaction mixture, the resulting residue was directly injected onto a reverse phase preparative-HPLC column (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1M TEA/CO2 at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated, and the resulting residue was diluted with 10 mL of dichloromethane-ether (1:1). The solution was cooled to 0° C. and 1.0M ethereal HCl was added dropwise (approx. 10 eq.). The precipitate, Compound 50, was filtered and dried and was substantially pure by HPLC. Yield: 71 mg (43%); ES-MS m/z 731.6 [M+H]+; UV λmax 215, 238, 290 nm. Anal. Calc. C40H70N6O6.4H2O.2HCl: C, 54.84; H, 9.20; N, 9.59. Found: C, 55.12; H, 9.41; N, 9.82.
Example 7
Preparation of Compound 51
Compound 51 was prepared by reacting Fmoc-tripeptide 41 and dipeptide 46 according to General Procedure D using triethylamine as the base. After concentration of the reaction mixture, the residue was directly injected onto a reverse phase preparative-HPLC column (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1M TEA/CO2 at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide a white solid intermediate that was used in the next step without further purification. ES-MS m/z 882.9 [M+NH4]+, 899.9 [M+Na]+; UV λmax 215, 256 nm.
Deprotection of the white solid intermediate was performed according to General Procedure E. The crude product was purified using preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1M TEA/CO2 at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide Compound 51 as a sticky solid. ES-MS m/z 660.1 [M+H]+, 682.5 [M+Na]+; UV λmax 215 nm.
Example 8
Preparation of Compound 52
Boc-dolaproine (0.33 g, 1.14 mmol) and (1S,2S)-(−)-1,2-diphenylethylenediamine (0.5 g, 2.28 mmol, 2.0 eq.) were diluted with dichloromethane, (10 mL) and to the resulting solution was added triethylamine (0.32 mL, 2.28 mmol, 2.0 eq.), then DEPC (0.39 mL, 2.28 mmol, 2.0 eq.). After 4 h, additional DEPC (0.39 mL) was added and the reaction was allowed to stir overnight. The reaction mixture was concentrated and the resulting residue was purified using preparative-HPLC (Varian Dynamax C18 column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and water at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide a yellow gummy solid peptide intermediate that was used without further purification. Rf 0.15 (100% EtOAc); ES-MS m/z 482.4 [M+H]+; UV λmax 215, 256 nm.
The yellow gummy peptide intermediate (0.24 g, 0.50 mmol) was diluted with dichloromethane, and to the resulting solution was added diisopropylethylamine (0.18 mL, 1.0 mmol, 2.0 eq.) and Fmoc-Cl (0.15 g, 0.55 mmol, 1.1 eq.). The reaction was allowed to stir for 3 h, after which time HPLC showed a complete reaction. The reaction mixture was concentrated to an oil, and the oil was diluted with EtOAc and extracted successively with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, and brine. The EtOAc layer was dried, filtered, and concentrated, and the resulting residue was purified using flash column chromatography (silica gel 230-400 mesh; eluant gradient 4:1 hexanes-EtOAc to 1:1 hexanes-EtOAc) to provide Compound 45 as a white solid. Yield: 0.37 g (46% overall); Rf 0.47 (1:1 hexanes-EtOAc); ES-MS m/z 704.5 [M+H]+, 721.4 [M+NH4]+; UV λmax 215, 256 nm.
Compound 52 was prepared by reacting tripeptide 42 (94 mg, 0.13 mmol) and dipeptide compound 45 (65 mg, 0.13 mmol) according to General Procedure D (using 3.6 eq. of diisopropylethylamine as the base). After concentration of the reaction mixture, the resulting residue was diluted with EtOAc and washed successively with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, and brine. The organic phase was dried, filtered and concentrated to provide a white solid residue which was diluted with dichloromethane and deprotected according to General Procedure E. According to HPLC, reaction was complete after 2 h. The reaction mixture was concentrated to an oil. The oil was diluted with DMSO, and the resulting solution was purified using a reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). Two products having similar UV spectra were isolated. The major product, Compound 52, was provided as an off-white solid. Overall yield: 24 mg (23%); ES-MS m/z 793.5 [M+H]+; UV λmax 215 nm.
Example 9
Preparation of Compound 53
Boc-phenylalanine (1.0 g, 3.8 mmol) was added to a suspension of 1,4-diaminobenzene.HCl (3.5 g, 19.0 mmol, 5.0 eq.) in triethylamine (10.7 mL, 76.0 mmol, 20 eq.) and dichloromethane (50 mL). To the resulting solution was added DEPC (3.2 mL, 19.0 mmol, 5.0 eq.) via syringe. HPLC showed no remaining Boc-phe after 24 h. The reaction mixture was filtered, and the filtrate was concentrated to provide a dark solid. The dark solid residue was partitioned between 1:1 EtOAc-water, and the EtOAc layer was washed sequentially with water and brine. The EtOAc layer was dried and concentrated to provide a dark brown/red residue that was purified using HPLC (Varian Dynamax column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and water at 45 mL/min form 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide a red-tan solid intermediate. Yield: 1.4 g (100%); ES-MS m/z 355.9 [M+H]+; UV λmax 215, 265 nm; 1H NMR (CDCl3) δ 7.48 (1H, br s), 7.22-7.37 (5H, m), 7.12 (2H, d, J=8.7 Hz), 7.61 (2H, d, J=8.7 Hz), 5.19 (1H, br s), 4.39-4.48 (1H, m), 3.49 (2H, s), 3.13 (2H, d, J=5.7 Hz), 1.43 (9H, s).
The red-tan solid intermediate (0.5 g, 1.41 mmol) and diisopropylethylamine (0.37 mL, 2.11 mmol, 1.5 eq.) were diluted with dichloromethane (10 mL), and to the resulting solution was added Fmoc-Cl (0.38 g, 1.41 mmol). The reaction was allowed to stir, and a white solid precipitate formed after a few minutes. Reaction was complete according to HPLC after 1 h. The reaction mixture was filtered, and the filtrate was concentrated to provide an oil. The oil was precipitated with EtOAc, resulting in a reddish-white intermediate product, which was collected by filtration and dried under vacuum. Yield: 0.75 g (93%); ES-MS m/z 578.1 [M+H]+, 595.6 [M+NH4]+.
The reddish-white intermediate (0.49 g, 0.85 mmol), was diluted with 10 mL of dichloromethane, and then treated with 5 mL of trifluoroacetic acid. Reaction was complete in 30 min according to reverse-phase HPLC. The reaction mixture was concentrated and the resulting residue was precipitated with ether to provide an off-white solid. The off-white solid was filtered and dried to provide an amorphous powder, which was added to a solution of Boc-dap (0.24 g, 0.85 mmol) in dichloromethane (10 mL). To this solution was added triethylamine (0.36 mL, 2.5 mmol, 3.0 eq.) and PyBrop (0.59 g, 1.3 mmol, 1.5 eq.). The reaction mixture was monitored using reverse-phase HPLC. Upon completion, the reaction mixture was concentrated, and the resulting residue was diluted with EtOAc, and sequentially washed with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, water, and brine. The EtOAc layer was dried (MgSO4), filtered, and concentrated. The resulting residue was purified using flash column chromatography (silica gel) to provide Compound 47 as an off-white powder. Yield: 0.57 g (88%); ES-MS m/z 764.7 [M+NH4]+; UV λmax 215, 265 nm; 1H NMR (DMSO-d6) δ 10.0-10.15 (1H, m), 9.63 (1H, br s), 8.42 (½H, d, J=8.4 Hz), 8.22 (½H, d, J=8.4 Hz), 7.89 (2H, d, J=7.2 Hz), 7.73 (2H, d, J=7.6 Hz), 7.11-7.55 (13H, m), 4.69-4.75 (1H, m) 4.46 (2H, d, J=6.8 Hz), 4.29 (1H, t, J=6.4 Hz), 3.29 (3H, s), 2.77-3.47 (7H, m), 2.48-2.50 (3H, m), 2.25 (⅔H, dd, J=9.6, 7.2 Hz), 1.41-1.96 (4H, m), 1.36 (9H, s), 1.07 (1H, d, J=6.4 Hz, rotational isomer), 1.00 (1H, d, J=6.4 Hz, rotational isomer).
Tripeptide compound 42 (55 mg, 0.11 mmol) and dipeptide compound 47 (85 mg, 0.11 mmol) were reacted according to General Procedure D (using 3.0 eq. of diisopropylethylamine). After concentration of the reaction mixture, the resulting residue was diluted with EtOAc, and washed sequentially with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, and brine. The EtOAc layer was dried, filtered and concentrated to provide a yellow oil. The yellow oil was diluted with dichloromethane (10 mL) and deprotected according to General Procedure E. According to HPLC, reaction was complete after 2 h. The reaction mixture was concentrated to provide an oil. The oil was diluted with DMSO, and the DMSO solution was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 53 as an off-white solid. Overall yield: 42 mg (44% overall); ES-MS m/z 837.8 [M+H]+, 858.5 [M+Na]+; UV λmax 215, 248 nm.
Example 10
Preparation of Compound 54
Compound 54 was prepared according to K. Miyazaki, et al. Chem. Pharm. Bull. 1995, 43(10), 1706-18.
Example 11
Preparation of Compound 55
Compound 55 was synthesized in the same manner as Compound 54, but by substituting FmocMeVal-Ile-Dil-tBu (40) for FmocMeVal-Val-Dil-tBu (39) as the starting material.
Example 12
Preparation of Compound 56
Carbamic acid [(1S)-1-(azidomethyl)-2-phenylethyl]-1,1-dimethylethyl ester (0.56 g, 2 mmol, prepared as described in J. Chem. Research (S), 1992, 391), was diluted with a 4 M solution of HCl in dioxane (10 mL) and the resulting solution allowed to stir for 2 hr at room temperature. Toluene (10 mL) was then added to the reaction, the reaction mixture was concentrated and the resulting residue was azeotropically dried under vacuum using toluene (3×15 mL), to provide a white solid intermediate. ES-MS m/z 177.1 [M+H]+.
The white solid intermediate was diluted with dichloromethane (5 mL) and to the resulting solution was added sequentially N-Boc-Dolaproine (0.58 g, 1 eq.), triethylamine (780 μL, 3 eq.) and DEPC (406 μL, 1.2 eq.), and the reaction mixture was allowed to stir for 2 h at room temperature. Reaction progress was monitored using reverse-phase HPLC. Upon completion of reaction as determined by HPLC, the reaction mixture was diluted with dichloromethane (30 mL), the dichloromethane layer was washed successively with 10% aqueous citric acid (20 mL), saturated aqueous NaHCO3 (20 mL), and water (20 mL). The dichloromethane layer was concentrated and the resulting residue was purified via flash column chromatography using a step gradient of 0-5% methanol in dichloromethane. The relevant fractions were combined and concentrated to provide a solid intermediate, 0.78 g (88%). ES-MS m/z 446.1 [M+H]+, 468.3 [M+Na]+.
The solid intermediate (450 mg, 1 mmol) and Tripeptide 42 (534 mg, 1.1 eq.) were diluted with a 50% solution of TFA in dichloromethane (10 mL), and the resulting reaction was allowed to stir for 2 h at room temperature. Toluene (10 mL) was added to the reaction and the reaction mixture was concentrated. The resulting amine intermediate was azeotropically dried using toluene (3×20 mL) and dried under vacuum overnight.
The resulting amine intermediate was diluted with dichloromethane (2 mL) and to the resulting solution was added triethylamine (557 μL, 4 eq.), followed by DEPC (203 μL, 1.4 eq.). The reaction mixture was allowed to stir for 4 h at room temperature and reaction progress was monitored using HPLC. Upon completion of reaction, the reaction mixture was diluted with dichloromethane (30 mL) and the dichloromethane layer was washed sequentially using saturated aqueous NaHCO3 (20 mL) and saturated aqueous NaCl (20 mL). The dichloromethane layer was concentrated and the resulting residue was purified using flash column chromatography in a step gradient of 0-5% methanol in dichloromethane. The relevant fractions were combined and concentrated and the resulting residue was dried using a dichloromethane:hexane (1:1) to provide a white solid intermediate, 0.64 g (84%). ES-MS m/z 757.5 [M+H]+.
The white solid intermediate (536 mg, 0.73 mmol) was diluted with methanol and to the resulting solution was added 10% Pd/C (100 mg). The reaction was placed under a hydrogen atmosphere and was allowed to stir at atmospheric pressure and room temperature for 2 h. Reaction progress was monitored by HPLC and was complete in 2 h. The reaction flask was purged with argon and the reaction mixture was filtered through a pad of Celite. The Celite pad was subsequently washed with methanol (30 mL) and the combined filtrates were were concentrated to yield a gray solid intermediate which was used without further purification. Yield=490 mg (91%). ES-MS m/z 731.6 [M+H]+, 366.6 [M+2H]2+/2.
The gray solid intermediate (100 mg, 0.136 mmol), N-Boc-4-aminobenzoic acid (39 mg, 1.2 eq.) and triethylamine (90 μL, 4 eq.) were diluted with dichloromethane (2 mL) and to the resulting solution was added DEPC (28 μL, 1.2 eq.). The reaction mixture was allowed to stir at room temperature for 2 h, then the reaction mixture was diluted with dichloromethane (30 mL). The dichloromethane layer was sequentially washed with saturated aqueous NaHCO3 (20 mL) and saturated aqueous NaCl (20 mL). The dichloromethane layer was then concentrated and the resulting residue was purified via flash column chromatography using a step gradient of 0-5% in dichlormethane. The relevant fractions were combined and concentrated and the resulting residue was dried using dichloromethane:hexane (1:1) to provide a white solid intermediate. ES-MS m/z 950.7 [M+H]+.
The white solid intermediate was diluted with a 50% solution of TFA in dichloromethane and allowed to stir for 2 h at room temperature. Toluene (10 mL) was added to the reaction and the reaction mixture was concentrated. The resulting residue was azeotropically dried using toluene (3×15 mL), to provide a yellow oil which was purified using preparative HPLC (C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN from 10 to 95% in 0.05 M Triethylammonium carbonate buffer, pH 7.0, in 30 min at a flow rate of 10 mL/min). The relevant fractions were combined and concentrated and the resulting residue was azeotropically dried using MeCN (3×20 mL), to provide Compound 56 as white solid: 101 mg (87% over 2 steps). ES-MS m/z 850.6 [M+H]+, 872.6 [M+Na]+.
Example 13
Preparation of Compound 57
Compound 49 (100 mg, 0.14 mmol), Compound 27 (160 mg, 0.15 mmol, 1.1 eq.), and HOBt (19 mg, 0.14 mmol, 1.0 eq.) were diluted with DMF (2 mL). After 2 min, pyridine (0.5 mL) was added and the reaction mixture was monitored using reverse-phase HPLC. Neither Compound 49 nor Compound 27 was detected after 24 h. The reaction mixture was concentrated, and the resulting residue was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and Et3N—CO2 (pH 7) at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide an off-white solid intermediate. ES-MS m/z 1608.7 [M+H]+
The off-white solid intermediate was diluted with MeCN/water/TFA in an 85:5:10 ratio, respectively. The reaction mixture was monitored using HPLC and was complete in 3 h. The reaction mixture was directly concentrated and the resulting residue was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 57 as an off-white powder. Yield: 46 mg (32% overall); ES-MS m/z 1334.8 [M+H]+; UV λmax 215, 256 nm.
Example 14
Preparation of Compound 58
Compound 49 (1.69 g, 2.35 mmol), Compound 21 (2.6 g, 3.52 mmol, 1.5 eq.), and HOBt (64 mg, 0.45 mmol, 0.2 eq.) were diluted with DMF (25 mL). After 2 min, pyridine (5 mL) was added and the reaction was monitored using reverse-phase HPLC. The reaction was shown to be complete in 24 h. The reaction mixture was concentrated to provide a dark oil, which was diluted with 3 mL of DMF. The DMF solution was purified using flash column chromatography (silica gel, eluant gradient:100% dichloromethane to 4:1 dichloromethane-methanol). The relevant fractions were combined and concentrated to provide an oil that solidified under high vacuum to provide a mixture of Compound 58 and unreacted Compound 49 as a dirty yellow solid (Rf 0.40 in 9:1 dichloromethane-methanol). The dirty yellow solid was diluted with DMF and purified using reverse-phase preparative-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 8 m, 100 Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min) to provide Compound 58 as an amorphous white powder (Rf 0.40 in 9:1 dichloromethane-methanol) which was >95% pure by HPLC and which contained less than 1% of Compound 49. Yield: 1.78 g (57%); ES-MS m/z 1316.7 [M+H]+; UV λmax 215, 248 nm.
Example 15
Preparation of Compound 59
The hydrochloride salt of Compound 51 (11 mg, 15.2 mmol) and Compound 21 (11 mg, 15.2 mmol) were diluted with 1-methyl-2-pyrollidinone (1 mL) and to the resulting solution was added diisopropylethylamine (5.3 mL, 30.3 mmol, 2.0 eq.). The mixture was allowed to stir under argon atmosphere for 3 d while being monitored using HPLC. After this time, much unreacted starting material still remained, HOBt (1.0 eq.) was added and the reaction mixture was allowed to stir for 24 h, after which time no starting material remained according to HPLC. The reaction mixture was concentrated and the resulting residue was purified using preparative-HPLC (Varian Dynamax C18 column 21.4 mm×25 cm, 5 m, 100 Å, using a gradient run of MeCN and water at 20 mL/min from 10% to 100% over 30 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 59 as a white solid. Yield: 13 mg (67%); ES-MS m/z 1287.2 [M+H]+, 1304.3 [M+NH4]+; UV λmax 215, 248 nm
Example 16
Preparation of Compound 60
Compound 53 (9 mg, 10.8 μmol) and Compound 28 (5.2 mg, 10.8 μmol) were diluted with dichloromethane (1 mL) and to the resulting solution was added HATU (6.3 mg, 16.1 μmol, 1.5 eq.), followed by pyridine (1.3 μL, 16.1 μmol, 1.5 eq.). The reaction mixture was allowed to stir under argon atmosphere while being monitored using HPLC. The reaction was complete after 6 h. The reaction mixture was concentrated and the resulting residue was diluted with DMSO. The DMSO solution was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min) and the relevant fractions were combined and concentrated to provide an an off-white solid intermediate which was >95% pure according to HPLC.
The off-white solid intermediate was diluted with dichloromethane (2 mL) and the resulting solution was treated with TFA (0.5 mL). The reaction was monitored using HPLC, and was complete in 2 h. The reaction mixture was concentrated, and the resulting residue was diluted with DMSO and purified under the same conditions as described in Example 13. The relevant fractions were combined and concentrated to provide Compound 60 as an off-white powder. Yield: 14.9 mg (90%); ES-MS m/z 1304.6 [M+H]+; UV λmax 215, 275 nm.
Example 17
Preparation of Compound 61
The trifluoroacetate salt of Compound 53 (0.37 g, 0.39 mmol, 1.0 eq.) and Compound 18 (0.30 g, 0.58 mmol, 1.5 eq.) were diluted with DMF (5 mL, 0.1 M), and to the resulting solution was added pyridine (95 μL, 1.2 mmol, 3.0 eq.). HATU (0.23 g, 0.58 mmol, 1.5 eq.) wa then added as a solid and the reaction mixture was allowed to stir under argon atmosphere while being monitored using HPLC. The reaction progressed slowly, and 4 h later, 1.0 eq. of diisopropylethylamine was added. Reaction was complete in 1 h. The reaction mixture was concentrated in vacuo and the resulting residue was purified using preparative-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min) to provide a faint pink solid intermediate.
The pink solid intermediate was diluted with DMF (30 mL) and to the resulting solution was added diethylamine (15 mL). Reaction was complete by HPLC in 2 h. The reaction mixture was concentrated and the resulting residue was washed twice with ether. The solid intermediate was dried under high vacuum and then used directly in the next step.
The solid intermediate was diluted with DMF (20 mL) and to the resulting solution was added MC—OSu (0.12 g, 0.39 mmol, 1.0 eq.). After 4 d, the reaction mixture was concentrated to provide an oil which was purified using preparative-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). Compound 61 was isolated as a white flaky solid. Yield: 0.21 g (38% overall); ES-MS m/z 1285.9 [M+H]+; 13.07.8 [M+Na]+; UV λmax 215, 266 nm.
Example 18
Preparation of Compound 62
Fmoc-val-cit-PAB-OCO-Pnp (19a) (0.65 g, 0.85 mmol, 1.1 eq.), Compound 49 (0.55 g, 0.77 mmol, 1.0 eq.), and HOBt (21 mg, 0.15 mmol, 2.0 eq.) were diluted with DMF (2.0 mL) and dissolved using sonication. To the resulting solution was added pyridine (0.5 mL) and the reaction was monitored using HPLC. After 24 h, diisopropylethylamine (1.0 eq.) was added and the reaction was allowed to stand without stirring for 24 h. The reaction mixture was concentrated to provide an oil residue. The oil residue was purified using reverse phase preparative-HPLC (Varian Dynamax column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min.) The desired fractions were pooled and concentrated to yield an oil that was precipitated with ether to provide an off-white solid intermediate. Yield: 0.77 g (74%); ES-MS m/z 1345.7 [M+H]+; UV λmax 215, 254 nm.
The off-white solid intermediate (about 85 mg) was deprotected using diethylamine (1 mL) in DMF (3 mL). After 1 h, the reaction was complete. The reaction mixture was concentrated, and the resulting residue was precipitated in 1 mL of EtOAc followed by addition of excess ether (about 20 mL). The amine intermediate was filtered and dried under high vacuum and used in the next step without further purification.
The amine intermediate (70 mg, 61 μmol, 1.0 eq.) was taken up in DMF (10 mL), and to the resulting solution was added sequentially, bromoacetamidocaproic acid (17 mg, 67 μmol, 1.1. eq.), PyBrop (32 mg, 67 μmol, 1.1 eq.), and diisopropylethylamine (16 μL, 92 μmol, 1.5 eq.). After 24 h, an additional 1.0 eq. of bromoacetamidocaproic acid was added. Reaction was stopped after 30 h. The reaction mixture was concentrated to an oil and the oil purified using reverse phase preparative-HPLC (Synergi MaxRP C12 column 21.4 mm×25 cm, 5μ, 80 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min.). The relevant fractions were combined and concentrated to provide Compound 62 as a white solid. Yield: 23 mg (27%); ES-MS m/z 1356.7 [M+H]+; UV λmax 215, 247 nm.
Example 19
Preparation of Compound 63
Fmoc-val-cit-PAB-OC(O)-Me-val-val-dil-dap-nor (about 48 mg, obtained according to Example 18) was subjected to Fmoc-removal by treating with diethylamine (1 mL) in DMF (3 mL). After 1 h, the reaction was complete. The reaction mixture was concentrated and the resulting residue was precipitated using 1 mL of EtOAc followed by addition of excess ether (about 20 mL). The amine intermediate was filtered and dried under high vacuum and used in the next step without further purification.
The amine intermediate (35 μmol, 1.1 eq.) was diluted with DMF (2 mL), and to the resulting solution was added sequentially maleimido-PEG acid (Frisch, B.; Boeckler, C.; Schuber, F. Bioconjugate Chem. 1996, 7, 180-6; 7.8 mg, 32 μmol, 1.0 eq.), DEPC (10.7 μL, 64 μmol, 2.0 eq.), and diisopropylethylamine (11.3 μL, 64 μmol, 2.0 eq.). The reaction was complete in 15 min according to HPLC. The reaction mixture was concentrated to provide an oil. The oil was diluted with 1 mL of DMSO and purified using reverse phase preparative-HPLC (Synergi MaxRP C12 column 21.4 mm×25 cm, 5μ, 80 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 63 as a white solid.
Yield: 16.2 mg (34%); ES-MS m/z 1348.6 [M+H]+; UV λmax 215, 247 nm.
Examples 20-25 describe the conjugation of the monoclonal antibodies cBR96 and cAC10 to a Drug-Linker Compound. These antibodies were obtained as described in Bowen, et al., J. Immunol. 1993, 151, 5896; and Trail, et al., Science 1993, 261, 212, respectively.
The number of Drug-Linker moities per Ligand in a Drug-Linker-Ligand Conjugate varies from conjugation reaction to conjugation reaction, but typically ranges from about 7 to about 9, particularly when the Ligand is cBR96 or cAC10.
Example 20
Preparation of Compound 64
cBR96 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively. Result: [Ab]=4.7 mg/mL=29.4 μM; [thiol]=265 μM; SH/Ab=9.0 (Typical SH/Ab range is from about 7.8 to about 9.5).
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, a 130.5 μL stock solution of Compound 57 (8.4 mM in DMSO, 8.5 mol Compound 57 per mol reduced antibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 57 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 218 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, three PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 64, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the three pre-chilled PD10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 4.2 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 64, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 64]=3.8 mg/mLg
% Aggregate=trace
Residual Thiol Titration:Residual thiols=1.7/Ab. Drug/Ab˜9.0−1.7=7.3
Quenched Drug-Linker: undetectable
Yield: 4.2 mL, 16 mg, 66%.
Example 21
Preparation of Compounds 65
cAC10 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Results: [Ab]=4.9 mg/mL=30.7 μM;
[thiol]=283 μM; 9.2 SH/Ab
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, 125 μL of a stock solution of Compound 57 (8.4 mM in DMSO, 8.5 mol Compound 57 per mol reduced antibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 57 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., then returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 218 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 65, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 65, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were then determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 65]=2.8 mg/mL
% Aggregate=trace
Residual Thiol Titration:Residual thiols=1.6/Ab. Drug/Ab˜9.2−1.6=7.6
Not covalently bound Drug-Linker: undetectable
Yield: 5.6 mL, 15.7 mg, 65%.
Example 22
Preparation of Compound 66
cBR96 Antibody (24 mg) was was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Result: [Ab]=3.7 mg/mL=23.1 μM; [thiol]=218 μM; 9.4 SH/Ab
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, 145.5 μL of a stock solution of Compound 58 (8.3 mM in DMSO, 9.0 mol Compound 58 per mol reduced antibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 58 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., then returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 249 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, three PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 66, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the three pre-chilled PD10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 4.2 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 66, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 66]=3.0 mg/mL
% Aggregate=trace
Residual Thiol Titration:Residual thiols=0.4/Ab. Drug/Ab˜9.5−0.4=9.1
Not covalently bound Drug-Linker: 0.3% of 57-Cys adduct
Yield: 5.3 mL, 15.9 mg, 66%.
Example 23
Preparation of Compound 67
cAC10 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Result: [Ab]=3.9 mg/mL=24.5 μM; [thiol]=227 μM; 9.3 SH/Ab
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, 154.4 μL of a stock solution of Compound 58 (8.3 mM in DMSO, 9.0 mol Compound 58 per mol reduced antibody) was diluted with MeCN (1.46 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 58 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., then returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 249 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 67, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 67, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 67]=3.0 mg/mL
% Aggregate=trace
Residual Thiol Titration:Residual thiols=0.5/Ab. Drug/Ab˜9.5−0.5=9.0
Quenched Drug-Linker: 1.1% of 58-Cys adduct
Yield: 5.3 mL, 15.9 mg, 66%.
Example 24
Preparation of Compound 68
cBR96 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, repectively.
Result: [Ab]=4.4 mg/mL=27.2 μM; [thiol]=277 μM; 10.2 SH/Ab
Conjugation:
The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to 0° C. in an ice bath) so that the resulting solution was 20% DMSO. The solution was allowed to stir for 10 min. at 0° C., then 127.8 μL of a stock solution of Compound 60 (7.6 mM solution in DMSO; 9 mol Compound 60 per mol antibody) was rapidly added. The reaction mixture was immediately stirred using a vortex instrument and return to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 213 μL, of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 68, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 68, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Because the absorbances of Compound 60 and antibody largely overlap, spectrophotometric determination of the conjugate concentration requires the measurement of absorbance at 270 and 280 nm. The molar concentration of conjugate is given by the following formula:
[Conjugate]=(OD280×1.08e−5−OD270×8.20e−6)×dilution factor,
where the values 1.08e−5 and 8.20e−6 are calculated from the molar extinction coefficients of the drug and the antibody, which are estimated as:
ε270 Compound 60=2.06e4 ε270 cBR96=1.87e5
ε280 Compound 60=1.57e4 ε280 cBR96=2.24e5
Assay Results:
[Compound 68]=3.2 mg/mL
% Aggregate=trace
Residual Thiol Titration:Residual thiols=1.0/Ab. Drug/Ab˜10.2−1.0=9.2
Quenched Drug-Linker: trace
Yield: 5.6 mL, 17.9 mg, 75%.
Example 25
Preparation of Compound 69
cAC10 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, repectively.
Result: [Ab]=4.8 mg/mL=29.8 μM; [thiol]=281 μM; 9.4 SH/Ab
Conjugation:
The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to 0° C. in an ice bath) so that the resulting solution was 20% DMSO. The solution was allowed to stir for 10 min. at 0° C., then 140 μL of a stock solution of Compound 60 (7.6 mM solution in DMSO; 8.5 mol Compound 60 per mol antibody) was rapidly added. The reaction mixture was immediately stirred using a vortex instrument and return to the ice bath and allowed to stir for 1 hr at 0° C., after which time 213 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 69, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 69, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Because the absorbances of Compound 60 and antibody largely overlap, spectrophotometric determination of the conjugate concentration requires the measurement of absorbance at 270 and 280 nm. The molar concentration of conjugate is given by the following formula:
[Conjugate]=(OD280×1.08e−5−OD270×8.20e−6)×dilution factor,
where the values 1.08e−5 and 8.20e−6 are calculated from the molar extinction coefficients of the drug and the antibody, which are estimated as:
ε270 Compound 60=2.06e4 ε270 cAC10=2.10e5
δ280 Compound 60=1.57e4 ε280 cAC10=2.53e5
Assay Results:
[Compound 69]=3.0 mg/mL
% Aggregate=trace
Residual Thiol Titration:Residual thiols=0.7/Ab. Drug/Ab˜9.4−0.7=8.7
Quenched Drug-Linker: trace
Yield: 5.6 mL, 16.8 mg, 70%.
Example 26
Preparation of Compound 75
Diethyl (4-nitrobenzyl)phosphonate (1.1 g, 4.02 mmol) was diluted in anhydrous THF (4 mL) and the resulting mixture was cooled to 0° C. Sodium hydride (0.17 g, 4.22 mmol, 1.05 eq., 60% dispersion in mineral oil) was added and the resulting reaction was allowed to stir for 5 min. At this time gas evolution from the reaction mixture had ceased. 2,2-Dimethyl-1,3-dioxan-5-one (0.52 g, 4.02 mmol) in 1 mL of anydrous THF was then added to the reaction mixture via syringe and the reaction was allowed to warm to room temperature with stirring. Additional THF (1 mL) was added after 30 min to help dilute the resulting precipitate and the resulting mixture was stirred for an additional 30 min., was transferred to a separatory funnel containing EtOAc (10 mL) and water (10 mL). The organic phase was collected, washed with brine, and the combined aqueous extracts were washed with ethyl acetate (2×). The combined organic extracts were dried over MgSO4, filtered, and concentrated to provide a dark red crude oil that was purified using flash chromatography on a silica gel column (300×25 mm) and eluting with 9:1 hexanes-EtOAc to provide a pale yellow solid intermediate. Yield: 0.57 g (57%); Rf 0.24 (9:1 hexanes-EtOAc); UV λmax 225, 320 nm. 1H NMR (CDCl3) δ 8.19 (2H, d, J=8.4 Hz), 7.24 (2H, d, J=8.4 Hz), 6.33 (1H, s), 4.62 (2H, s), 4.42 (2H, s), 1.45 (6H, s). 13C NMR (CDCl3) δ 146.6, 142.7, 141.3, 129.4, 123.9, 121.1, 99.9, 64.4, 60.8, 24.1.
The pale yellow solid intermediate (0.25 g, 1.0 mmol) was diluted using THF (20 mL), the resulting mixture was treated with 1 N HCl (10 mL) and allowed to stir for 5 min. To the reaction mixture was added diethyl ether (150 mL) and water and the resulting mixture was transferred to a separatory funnel. The organic layer was dried (MgSO4), filtered and concentrated to give an oil. The resulting diol was then taken up in THF-methanol (1:1, 4 mL each, 0.3 M) followed by the addition of Raney Nickel (100 μL, 100 μL/mmol nitro-group, 50% slurry in water) and hydrazine (74 μL, 1.5 eq.). Gas evolution occurred while the reaction mixture was heated to 50-60° C. After 30 min and 1 h, 1.5 eq. of hydrazine was added each time. The yellow mixture was filtered through celite and washed with methanol. The filtrated was concentrated to provide Compound 75 as an oil which later crystallized to a yellow solid. Yield: 0.14 g (78%); UV λmax 215, 260 nm. 1H NMR (DMSO) δ 7.00 (2H, d, J=8.4 Hz), 6.51 (2H, d, J=8.4 Hz), 6.33 (1H, s), 5.20 (2H, bs), 4.64 (2H, bd), 4.04 (2H, s). 13C NMR (DMSO) δ 147.2, 138.1, 129.6, 126.1, 124.6, 113.7, 63.6, 57.5.
Example 27
Preparation of Compound 79
To a mixture of Compound 75 (BHMS, 0.12 g, 0.67 mmol) in methanol-dichloromethane (1:2, 4.5 mL total) was added Fmoc-Val-Cit (0.33 g, 0.67 mmol) followed by EEDQ (0.25 g, 1.0 mmol, 1.5 eq.) and the resulting reaction was allowed to stir for 15 hours under inert atmosphere. Additional EEDQ (1.5 eq.) and Fmoc-Val-Cit (1.0 eq.) were then added due to the presence of unreacted BHMS and the resulting reaction was allowed to stir for 2 days and concentrated. The resulting residue was triturated using ether to provide a tan solid intermediate. ES-MS m/z 659 [M+H]+, 681 [M+Na]+; UV λmax 215, 270 nm. 1H NMR (DMSO) δ 10.04 (1H, s), 8.10 (1H, d, J=7.2 Hz), 7.87 (2H, d, J=7.6 Hz), 7.72 (2H, t, J=7.6 Hz), 7.55 (2H, d, J=8.4 Hz), 7.37-7.43 (3H, m), 7.30 (2H, t, J=7.2 Hz), 7.24 (2H, d, J=8.4 Hz), 6.47 (1H, s), 5.96 (1H, t, J=5.2 Hz), 5.39 (1H, s), 4.83 (1H, t, J=5.2 Hz), 4.78 (1H, t, J=5.2 Hz), 4.40 (1H, dd, J=5.2, 8.0 Hz), 4.20-4.30 (3H, m), 4.11 (2H, d, J=4.4 Hz), 4.04 (2H, d, J=5.2 Hz), 3.91 (1H, t, J=7.2 Hz), 2.84-3.06 (2H, m), 1.91-2.03 (1H, m), 1.29-1.74 (4H, m), 0.86 (3H, d, J=6.8 Hz), 0.84 (3H, J=6.8 Hz).
The tan solid intermediate was diluted with DMF (10 mL) and the resulting mixture was treated with diethylamine (5 mL), allowed to stir for 1 hour and concentrated to provide a tan solid which was dried under high vacuum for 3 days. The tan solid was triturated using EtOAc (10 mL) and further precipitated using ether (80 mL) to provide a crude residue which was filtered through a sintered glass funnel and dried in vacuo to afford a light tan intermediate. ES-MS m/z 436 [M+H]+, 458 [M+Na]+; UV λmax 215, 270 nm.
The light tan intermediate was diluted with DMF (10 mL) and treated with 6-maleimidocaproic acid hydroxysuccinimde ester (0.16 g, 0.53 mmol, 1 eq.). The reaction was allowed to stir for 18 h, additional diisopropylethylamine (1.0 eq) was added followed by additional 6-maleimidocaproic acid hydroxysuccinimde ester (0.5 eq.). The resulting reaction was allowed to stir for 4 hours, after which time, HPLC indicated that the starting material had been consumed. The reaction mixture was concentrated to provide a crude residue that was triturated using EtOAc (10 mL) and then further precipitated using ether (75 mL). The precipitate was and dried overnight to provide a tan/orange powdered intermediate. Overall yield: 0.42 g (quant.). ES-MS m/z 629 [M+H]+, 651 [M+Na]+; UV λmax 215, 270 nm.
The tan/orange powdered intermediate (0.40 g, 0.64 mmol) was partially dissolved in DMF (20 mL) and to the resulting mixture was added bis(4-nitrophenyl) carbonate (0.98 g, 3.2 mmol, 5.0 eq.) and diisopropylethylamine (0.45 mL, 2.5 mmol, 4.0 eq.). The resulting reaction was allowed to stir for about 4 hours, after which time, HPLC monitoring indicated that no starting material remained and that the reaction mixture contained 2 products in a 3:2 ratio (the desired bis-carbonate and the 1,3-dioxan-2-one, respectively). The reaction mixture was concentrated and the resulting residue was triturated using EtOAc (10 mL), then further precipitated using ether (80 mL) in a one-pot manner. The EtOAc-ether mixture was filtered and the solid was dried to provide Compound 79 as a tan powder which was used without further purification.
Example 28
Preparation of Compound 80
Compound 49 (202 mg, 0.22 mmol, 2.0 eq., 80% pure) and Compound 79 (180 mg, 0.11 mmol, 1.0 eq., 60% pure) were suspended in dry DMF (2 mL, 0.1 M) and to the resulting mixture was added HOBt (3 mg, 22 μmol, 0.2 eq.) followed by pyridine (400 μL, ¼ v/v DMF). The resulting reaction was allowed to stir for 16 h, diluted with DMSO (2 mL) and and the resulting mixture was purified using preparative HPLC (C18-RP column, 5μ, 100 Å, linear gradient of MeCN in water 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 50 mL/min) to provide Compound 80 as a white solid. Yield: 70 mg (18%). MALDI-TOF MS m/z 2138.9 [M+Na]+, 2154.9 [M+K]+.
Example 29
Preparation of Compound 81
Compound 81 was made using the method described in Example 1 and substituting Fmoc-(D)-val-(L)-cit-PAB-OH for Compound 19.
Example 30
Preparation of Compound 82
Compound 82 was made using the method described in Example 1 and substituting Fmoc-(L)-val-(D)-cit-PAB-OH for Compound 19.
Example 31
Preparation of Compound 83
Compound 83 was made using the method described in Example 1 and substituting Fmoc-(D)-val-(D)-cit-PAB-OH for Compound 19.
Example 32
Preparation of Compound 84
Compound 84 was made using the method described in Example 14 and substituting Compound 81 for Compound 21.
Example 33
Preparation of Compound 85
Compound 85 was made using the method described in Example 14 and substituting Compound 82 for Compound 21.
Example 34
Preparation of Compound 86
Compound 86 was made using the method described in Example 14 and substituting Compound 83 for Compound 21.
Example 35
Preparation of Compound 87
A mixture of 6-Maleimidocaproic acid (1.00 g, 4.52 mmol, 1.0 eq.), p-aminobenzyl alcohol (1.11 g, 9.04 mmol, 2.0 eq.) and EEDQ (2.24 g, 9.04 mmol, 2.0 eq.) were diluted in dichloromethane (13 mL). The resulting reaction was stirred about 16 hr., then concentrated and purified using flash column chromatography in a step gradient 25-100% EtOAc in hexanes to provide a solid intermediate. Yield: 1.38 g (96%); ES-MS m/z 317.22 [M+H]+, 339.13 [M+Na]+; UV λmax 215, 246 nm.
The solid intermediate (0.85 g, 2.69 mmol, 1.0 eq.) and bis(4-nitrophenyl)carbonate (2.45 g, 8.06 mmol, 3.0 eq.) were diluted in DMF (10 mL), and to the resulting mixture was added diisopropylethylamine (0.94 mL, 5.37 mmol, 2.0 eq.). The resulting reaction was stirred for about 1 hr, after which time RP-HPLC indicated that the reaction was complete. The reaction mixture was concentrated in vacuo, and the resulting crude residue was triturated using diethyl ether (about 250 mL) to provide a white solid intermediate upon filtration. Yield: 1.25 g (96%); UV λmax 215, 252 nm.
The white solid intermediate (259 mg, 0.0538 mmol, 1.0 eq.), MMAE (464 mg, 0.646 mmol, 1.2 eq.), and HOBt (14.5 mg, 0.108 mmol, 0.2 eq.) were diluted in pyridine/DMF (1:5, 6 mL), and the resulting reaction was stirred for about 10 h, after which time RP-HPLC indicated incomplete reaction. The reaction mixture was concentrated, the resulting crude residue was diluted using DMF (3 mL), and to the resulting mixture was added diisopropylethylamine (0.469 mL, 0.538 mmol, 1.0 eq.) and the resulting reaction was allowed to stir for about 16 hr. The reaction mixture was directly purified using Chromatotron® (radial thin-layer chromatography) with a step gradient (0-5% methanol in dichloromethane), to provide Compound 87 as a white solid. Yield: 217 mg (38%); ES-MS m/z 1082.64 [M+Na]+; UV λmax 215, 248 nm.
Example 36
Preparation of Compound 88
Fmoc-val-cit (U.S. Pat. No. 6,214,345 to Firestone et al.) was suspended in dichloromethane (50 mL) and the resulting mixture was treated with 33% HBr in HOAc (20 mL), which was added via pipette over about 5 minutes. After stirring for about 10 minutes, the reaction mixture was shown to be complete using HPLC. The reaction mixture was diluted with ice (about 500 mL) and saturated aqueous sodium bicarbonate was slowly added while stirring until gas evolution ceased. The resulting gelatinous mass was filtered and washed with distilled water to provide a solid which was dried under high vacuum in the presence of P2O5 for 24 h. The resulting tan powdered intermediate (Fmoc-val-cit-PAB-Br) was about 70% pure by HPLC and was used without further purification.
The tan powdered intermediate (30 mg, 40.6 μmol) and Compound 53 (34 mg, 40.6 μmol) were dissolved in DMF (1 mL), and to the resulting mixture was added diisopropylethylamine (21 μL, 0.12 mmol, 3.0 eq.). The resulting reaction was allowed to stir for 6 h, diluted with DMSO (1 mL) and immediately purified using preparative-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% formic acid) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min), to provide as a slight tan powdered intermediate. Yield: 5 mg (8%); ES-MS m/z 1420 [M+H]+, 1443 [M+Na]+; UV λmax 205, 258 nm.
The slight tan powdered intermediate (4 mg, 9.51 μmol) was diluted using DMF (1 mL) and the resulting mixture was treated with diethylamine (0.5 mL). The resulting reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oily solid residue which was triturated with ether (3×) to provide a crude residue. The crude residue was diluted with DMF (1 mL) and to the resulting mixture was added 6-maleimidocaproic acid hydroxysuccinimide ester (3 mg, 9.5 μmol). The resulting reaction was allowed to stir at room temperature for about 16 h. The reaction mixture was directly purified using preparative-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% formic acid) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min) to provide Compound 88 as a slight tan solid. Yield: 3.9 mg (quant); ES-MS m/z 1391 [M+H]+; UV λmax 205, 250 nm.
Example 37
Preparation of Compound 89
Preparation of Compound 89A
Compound 89A was prepared using the method described in Example 9 and substituting tripeptide Compound 43 for tripeptide Compound 42, intermediate
Preparation of Compound 89
Compound 89a (0.13 g, 0.15 μmol, 1.0 mmol), Compound 21 (0.12 g, 0.17 mmol, 1.1 eq.), and HOBt (4 mg, 31 μmol, 0.2 eq.) were suspended in DMF/pyridine (2 mL/0.5 mL, respectively). The resulting reaction was allowed to stir for about 4 h, then diisopropylethylamine (27 μL, 0.15 mmol, 1.0 eq.) was added and the resulting reaction was allowed to stirred for about 54 h and concentrated in vacuo. The resulting crude oil was diluted with DMSO and purified using preparative-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min) to provide to a yellow oil that was taken up in a minimum amount of dichloromethane and precipitated with excess ether to afford Compound 89 as a tan powder. Yield: 0.15 mg (68%). ES-MS m/z 1449.14 [M+H]+; UV λmax 215, 258 nm.
Example 38
Preparation of Compound 90
1,4-Phenylenediamine dihydrochloride (3.06 g, 17 mmoles) and di-t-butyl dicarbonate (3.69 g, 17 mmoles) were diluted with 30 mL of dichloromethane. To the resulting mixture was added diisopropylethylamine (8.83 ml, 50.7 mmoles, 3.0 eq.) and the resulting reaction was allowed to stirred for 1 hr. The reaction mixture was transferred to a seperatory funnel and the organic phase was washed water (3×10 ml). The organic layer was stored at 4° C. for about 15 h and and crystalization of the product occurred. The crystals were collected by filtration and washed with cold dichloromethane to provide Compound 90 as a crystalline solid. (1.2 g, 34%). UV λmax 215, 250 nm. 1H NMR (DMSO) δ 8.78 (1H, bs), 7.04 (2H, bd, J=7.2 Hz), 6.43 (2H, d, J=7.2 Hz), 4.72 (2H, s), 1.41 (9H, s).
Example 39
Preparation of Compound 91
A solution of cAC10 (10 mg/mL in 25 mM sodium citrate, 250 mM sodium chloride, 0.02% Tween 80, pH 6.5) was adjusted to pH 7.5 by addition of 0.3 M sodium phosphate, dibasic. To this pH-adjusted cAC10 solution, EDTA was added to a final concentration of 5 mM. The cAC10 solution was then pre-heated to 37° C. by incubation in a temperature-controlled oven. After the temperature of the cAC10 solution has equilibrated to 37° C., DTT (from a stock solution of 10 mM) was added to achieve a final DTT-to-cAC 10 molar ratio of about 3.0 in the reduction reaction (a molecular weight of 148,500 Da was used for cAC10). The reduction reaction was then allowed to proceed for 2 hours at 37° C.
At the end of the incubation, the reduction reaction was cooled to an internal temperature of 2 to 8° C. in an ice-water bath. The temperature of the solution was kept at 2 to 8° C. throughout the remaining conjugation steps. The chilled reduction reaction was subjected to constant-volume diafiltration to remove excess DTT using a 30 kDa membrane and the buffer was exchanged into phosphate buffered saline, pH 7.4 (PBS). Following diafiltration, the concentration of free thiol in the reduced and diafiltered cAC10 was determined using General Procedure M. Conjugation is then carried out by addition of a 15% molar excess of Compound 58 (from a stock solution of 13 mg/mL in DMSO) relative to the total thiols determined using General Procedure M. Additional DMSO was added to the conjugation reaction to achieve a final DMSO concentration of 15% (v/v). The conjugation reaction was allowed to proceed for a total of 30 min.
At the end of the conjugation reaction, any unreacted excess Drug-Linker compound was quenched by addition of excess Cysteine (2× molar excess relative to the total thiols determined using General Procedure M, performed on the reduced and diafiltered cAC10 to produce the quenched reaction mixture. The quenched reaction mixture is then purified free of small-molecule contaminants via constant-volume diafiltration using a 30 kDa membrane and the buffer was exchanged into PBS, pH 7.4. After diafiltration, the conjugate was sterile-filtered using a 0.22 micron filter to provide Compound 91 in a clear, colorless solution.
Example 40
Preparation of Compound 92
Compound 92 was prepared using the method described in Example 39 using an amount of DTT (from a stock solution of 10 mM) which provides a final DTT-to-cAC10 molar ratio of about 1.5 in the reduction reaction.
In Vitro Cytotoxicity Experiments
The cell lines used were H3396 human breast carcinoma (cBR96 antigen positive, cAC10 antigen negative), HCT-116 human colorectal carcinoma (cBR96 and cAC10 antigen negative), and Karpas human anaplastic large cell lymphoma (ALCL) (cBR96 antigen negative, cAC10 antigen positive). These cell lines are available from ATCC. CD30-positive Hodgkin's Disease (HD) cell line L540 and the ALCL cell line Karpas 299 were obtained from the Deutsche Sammlung von Mikroorganism and Zellkulturen GmbH (Braunschweig, Germany). L540cy, a derivative of the HD line L540 adapted to xenograft growth, was provided by Dr. Phil Thorpe (U of Texas Southwestern Medical Center, Dallas, Tex.). Cell lines were grown in RPMI-1640 media (Life Technologies Inc., Gaithersburg, Md.) supplemented with 10% fetal bovine serum.H3396 cells in RPMI containing 10% fetal bovine serum (referred to as medium) were plated in 96-well plates at approximately 3,000-10,000 cells/well and allowed to adhere overnight. The non-adherent Karpas cell line was plated out at approximately 10,000 cells/well at the initiation of the assay. Various concentrations of illustrative Compounds of the Invention in medium were added in triplicate, and after the times indicated IN FIGS. 1-7, the medium was removed, and the cells were washed with fresh medium three times. After a 96 hour incubation period at 37° C., Alamar Blue was added and cell viability was determined 4 hours later as described by Ahmed S A, Gogal R M Jr, Walsh J E., J. Immunol. Methods, 170, 211-224, 1994.
C.B.-17 SCID (Harlan, Indianapolis, Ind.) mice were used for in vivo experiments
Example 41
In Vitro Cytotoxicity Data
The cytotoxic effects of Compound 49 and Compound 53 on H3396 human breast carcinoma cells are shown in FIG. 1. The data show that after exposure for 1 hour, Compound 53 is more cytotoxic than Compound 49 at concentrations of up to 0.01 mM. The compounds show substantially equal cytotoxicity at concentrations between 0.01 mM and 1.0 mM.
Example 42
In Vitro Cytotoxicity Data
FIG. 2 shows the cytotoxic effects of Compounds 64, 65, 68 and 69 on H3396 human breast carcinoma cells (cBR96 antigen positive, cAC10 antigen negative). The data show that the Compounds 64 and 68 demonstrate similar and significant cytotoxicity, while Compounds 65 and 69 are less efficacious, but nevertheless cytotoxic against H3396 cells in this particular assay.
Example 43
In Vitro Cytotoxicity Data
FIG. 3 shows the cytotoxic effects of Compounds 64, 65, 68 and 69 on HCT-116 human colorectal carcinoma cells (cBR96 antigen negative, cAC10 antigen negative). The data illustrate that none of Compounds 64, 65, 68 and 69 is cytotoxic toward the antigen negative HCT-116 cells in this assay.
Example 44
In Vitro Cytotoxicity Data
FIG. 4 illustrates the cytotoxicity of Compounds 66 and 68 on H3396 human breast carcinoma cells (cBR96 antigen positive). The data show that both Compounds are highly cytotoxic at concentrations above 0.1 mM and that Compound 68 demonstrates greater cytotoxicity than Compound 66 at concentrations between 0.01 mg/mL and 0.4 mg/mL.
Example 45
In Vitro Cytotoxicity Data
FIG. 5 illustrates the cytotoxicity of Compounds 66, 68 and 69 on Karpas human anaplastic large cell lymphoma (cBR96 antigen negative, cAC10 antigen positive). The data show that Compound 69 was more cytotoxic toward Karpas cells than compared to Compounds 68 and 66 in this assay. Compound 69 demonstrated significant cytotoxicity at concentrations above 0.001 mM, while Compound 66 and Compound 68 were not cytotoxic at concentrations below 1.0 mg/mL.
Example 46
In Vitro Cytotoxicity Data
FIG. 6 illustrates the cytotoxicity of Compound 66 and 67 at 2 h and 96 h on H3396 human breast carcinoma cells (cBR96 antigen positive, cAC10 antigen negative). The data show that Compound 66 is highly cytotoxic at concentrations above 100 mg/mL at short-term exposure (2 h) mg/mL, and at concentrations above 100 mg/mL over long-term exposure (96 h). Compound 67 did not demonstrate cytotoxicity against H3396 cells in this assay at concentrations up to 1000 mg/mL.
General Procedure S: In Vivo Testing of Selected Drug-Linker-Antibody Conjugates. For the L2987 human adenocarcinoma cell line, Athymic nude mice (8-10 weeks old) were implanted with xenograft tumors or tumor cells. For the Karpas human anaplastic large cell lymphoma model, CB-17 SCID mice were implanted subcutaneously with 5×106 cells. In both tumor models, therapy was initiated once the tumors reached an average volume of 100 mm3. Groups of mice were injected with one of Compounds 66-69 in phosphate buffered saline intravenously every fours days for a total of 6 injections for L2987 animals and 2 injections for Karpas animals. Tumor volume was computed using the formula: 0.5 (longest dimension×perpendicular dimension2). Mice were removed from the study when their tumors were approximately 1000 mm3, at which point the average tumor sizes from the particular group were no longer plotted.
Example 47
In Vivo Therapeutic Efficacy on 12987 Tumors
FIG. 7 shows the therapeutic effects of Compounds 66-69 on L2987 human lung adenocarcinoma xenograft tumors (cBR96 antigen positive, cAC10 antigen negative) implanted in athymic nude mice. General Procedure S was followed using subcutaneous
L2987 human lung tumors (from in vivo passaging). Mice were administered by injection with one of Compounds 66, 67, 68 or 69 at four day intervals for a total of 6 injections. The first injection was given at 15 days post tumor-implant. The data illustrate that administration of Compound 66 and Compound 68 markedly reduced tumor volume and no additional growth was noted in treated mice for at approximately 25 days after the last injection. Compound 67 and Compound 69 were less efficacious but nevertheless inhibited tumor cell multiplication in the treated mice. Testing was stopped in animals receiving Compounds 67 and 69 when tumor volume exceeded 1000 mm3.
Example 48
In Vivo Therapeutic Efficacy on Karpas Tumors
FIG. 8 shows the therapeutic effects of compounds 66-69 on Karpas human anaplastic large cell lymphoma xenograft tumors (cAC10 antigen positive, cBR96 antigen negative) implanted in nude mice. General Procedure S was followed using Karpas human anaplastic large cell lymphoma model, CB-17 SCID mice were implanted subcutaneously with 5×106 cells. Mice were dosed intravenously with one of Compounds 66, 67, 68 or 69 at four day intervals for a total of 2 injections starting on day 8. The data illustrate that Compounds 67 and 69 induced complete regressions, and that the tumors progressed in animals that received substantially equivalent amounts of Compounds 66 and 68.
Example 49
Determination of Cytotoxicity of Selected Compounds in CD30− and CD30+ Cells
Following their physical characterization, the in vitro cytotoxicity of Compounds 67, 91 and 92 was evaluated in CD30+ Karpas 299 and CD30− Raji cells using the Alamar Blue assay as described above. The percent viable cells was plotted versus concentration for each molecule to determine the IC50 (defined as the mAb concentration that gave 50% cell kill).
Compound 67 demonstrated activity against Karpas 299 cells with an IC50 of 4 ng/mL. The IC50 was inversely proportional to drug loading as it increased from 4 ng/mL for Compound 67 to 7 ng/mL for Compound 91, to 40 ng/mL for Compound 92. Selectivity of the tested compounds was evaluated using the antigen-negative Raji cell line which were insensitive to all cAC10-containing Compounds with IC50 values >1000 ng/ml for Compounds 67, 91 and 92.
Example 50
Cytotoxicity of Selected Compounds in Xenograft Models of HD and ALCL
Cytotoxicity of Compounds 67, 91 and 92 was evaluated in subcutaneous Karpas 299 human anaplastic large cell lymphoma and L540cy Hodgkin's Disease xenograft models in C.B.-17 SCID mice. Evaluations were initiated when tumor volumes averaged 50-100 mm3. Cohorts of Karpas-299 bearing mice were injected q4dx4 with Compound 92, Compound 91, or Compound 67 at either 0.25 mg/kg or 0.5 mg/kg. None of the animals treated at 0.25 mg/kg had a regression, although there was a delay in tumor growth compared to untreated controls for the animals treated with Compound 91 and Compound 67. Treatment of Karpas tumors with Compound 91 and Compound 67 at 0.5 mg/kg given q4dx4 achieved 5/5 complete regressions and 4/5 complete regressions, respectively. A delay in tumor growth compared to untreated animals was observed for Compound 92 at 0.5 mg/kg given q4dx4, but no complete regressions were obtained.
Efficacy was also tested in a subcutaneous Karpas model with selected compounds administered as a single dose. Compound 91 and Compound 67 were injected at single doses of 0.25, 0.5 and 2.0 mg/kg. At the dose of 0.25 mg/kg there was no antitumor activity in either group and mean tumor volume did not deviate from the untreated controls. A delay in the tumor growth was demonstrated by both molecules at 0.5 mg/kg, but no complete regressions were obtained. Treating the mice with Compound 91 and Compound 67 at 2 mg/kg achieved 100% complete regressions in both groups.
Compound 91 and Compound 67 were also compared in mice bearing subcutaneous L540cy human HD tumors treated q4dx4 with Compound 91 and Compound 67 at 1 and 3 mg/kg. At 1 mg/kg, mice treated with Compound 91 and Compound 67 had significant delays in tumor growth compared to the untreated animals. Complete regressions were observed in mice administered with both Compound 91 and Compound 67 at 3 mg/kg.
The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the are and are intended to fall within the scope of the appended claims.
A number of references have been cited, the entire disclosures of which are incorporated herein by reference.
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12621406
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/2
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Nov 9th, 2010 12:00AM
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Mar 20th, 2009 12:00AM
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https://www.uspto.gov?id=US07829531-20101109
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Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
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Drug-Linker-Ligand Conjugates are disclosed in which a Drug is linked to a Ligand via a peptide-based Linker unit. In one embodiment, the Ligand is an Antibody. Drug-Linker compounds and Drug compounds are also disclosed. Methods for treating cancer, an autoimmune disease or an infectious disease using the compounds and compositions of the invention are also disclosed.
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7829531
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1. A drug-linker-antibody conjugate of Formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt thereof,
wherein,
L- is an antibody that immunospecifically binds to a cancer cell antigen which is on the surface of a cancer cell;
-Aa-Ww—Yy— is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each —W— is independently an Amino Acid unit;
—Y— is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12,
y is 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and —C1-C8 alkyl;
R3 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from the group consisting of —H and -methyl; or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and —C1-C8 alkyl;
R7 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from the group consisting of —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from the group consisting of —H and —C1-C8 alkyl;
R10 is selected from the group consisting of:
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle); —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from the group consisting of -aryl and —C3-C8 heterocycle;
R13 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
2. A drug-linker-antibody conjugate of the formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt thereof,
wherein,
L- is an antibody that immunospecifically binds to a cancer cell antigen which is on the surface of a cancer cell;
-Aa-Ww—Yy— is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each —W— is independently an Amino Acid unit;
—Y— is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit having the structure
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and -methyl;
R3 is selected from the group consisting of —H, -methyl, and -isopropyl;
R4 is selected from the group consisting of —H and -methyl;
R5 is selected from the group consisting of -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— where Ra and Rb) are independently selected from the group consisting of —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and -methyl;
each R8 is independently selected from the group consisting of —OH, -methoxy and -ethoxy;
R10 is selected from the group consisting of:
R24 is selected from the group consisting of H and —C(O)R25; wherein R25 is selected from the group consisting of —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
Z is —O—, —NH—, —OC(O)—, —NHC(O)—, or —NR28C(O)—; where R28 is selected from the group consisting of —H and —C1-C8 alkyl;
n is 0 or 1; and
R27 is selected from the group consisting of —H, —N3, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 0; and R27 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 1.
3. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein R10 is
4. The drug-linker-antibody conjugate of claim 2 or a pharmaceutically acceptable salt thereof wherein R10 is
5. The drug-linker-antibody conjugate of claim 3 or a pharmaceutically acceptable salt thereof wherein R2 is —C1-C8 alkyl.
6. The drug-linker-antibody conjugate of claim 4 or a pharmaceutically acceptable salt thereof wherein R2 is methyl.
7. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof where -D is a Drug unit having the structure
8. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein the antibody is a monoclonal antibody.
9. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein the antibody is a full length immunoglobulin molecule.
10. The drug-linker-antibody conjugate of claim 9 or a pharmaceutically acceptable salt thereof wherein the antibody is bound to the Stretcher unit via an interchain thiol of the antibody.
11. The drug-linker-antibody conjugate of claim 8 or a pharmaceutically acceptable salt thereof wherein the antibody is a chimeric antibody.
12. The drug-linker-antibody conjugate of claim 11 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
13. The drug-linker-antibody conjugate of claim 12 or a pharmaceutically acceptable salt thereof wherein the antibody immunospecifically binds human CD30 antigen.
14. The drug-linker-antibody conjugate of claim 2 or a pharmaceutically acceptable salt thereof wherein the antibody is a monoclonal antibody.
15. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof where —Ww— is represented by Formula VII:
wherein
R20 is benzyl, methyl, or isopropyl and R21 is (CH2)4NH2; or
R20 is isopropyl, benzyl, isobutyl, or sec-butyl and R21 is (CH2)3NHCONH2; or
R20 is benzyl and R21 is methyl or (CH2)3NHC(═NH)NH2; or
R20 is
and R21 is (CH2)3NHCONH2.
16. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof where —Ww— is -valine-citrulline-, the amino terminus of —Ww— forming a bond with a Stretcher unit, and the C-terminus of —Ww— forming a bond with a Spacer unit.
17. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to about 5.
18. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to 10.
19. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein the linker unit is cleavable by cathepsin B.
20. The drug-linker-antibody conjugate of claim 2 or a pharmaceutically acceptable salt thereof wherein the linker unit is cleavable by cathepsin B.
21. The drug-linker-antibody conjugate of claim 1 having the formula below:
or a pharmaceutically acceptable salt thereof, where E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or from 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
22. The drug-linker-antibody conjugate of claim 21 or a pharmaceutically acceptable salt thereof wherein the antibody is a monoclonal antibody.
23. The drug-linker-antibody conjugate of claim 21 or a pharmaceutically acceptable salt thereof wherein the antibody is a full length immunoglobulin molecule.
24. The drug-linker-antibody conjugate of claim 23 or a pharmaceutically acceptable salt thereof wherein the antibody is bound to the Stretcher unit via an interchain thiol of the antibody.
25. The drug-linker-antibody conjugate of claim 22 or a pharmaceutically acceptable salt thereof wherein the antibody is a chimeric antibody.
26. The drug-linker-antibody conjugate of claim 25 or a pharmaceutically acceptable salt thereof wherein the chimeric antibody comprises a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
27. The drug-linker-antibody conjugate of claim 21 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to about 5.
28. The drug-linker-antibody conjugate of claim 21 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to 10.
29. The drug-linker-antibody conjugate of claim 1 having the formula below:
or a pharmaceutically acceptable salt thereof.
30. The drug-linker-antibody conjugate of claim 29 or a pharmaceutically acceptable salt thereof wherein the antibody is a monoclonal antibody.
31. The drug-linker-antibody conjugate of claim 30 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
32. A composition comprising drug-linker-antibody conjugates having Formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt thereof;
wherein,
L- is an antibody that immunospecifically binds to a cancer cell antigen which is on the surface of a cancer cell;
-Aa-Ww—Yy— is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each —W— is independently an Amino Acid unit;
—Y— is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to 10 and is the average number of -Aa-Ww—Yy-D units per antibody in the composition; and
-D is a Drug unit of the formula
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and —C1-C8 alkyl;
R3 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from the group consisting of —H and -methyl; or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and —C1-C8 alkyl;
R7 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from the group consisting of —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from the group consisting of —H and —C1-C8 alkyl;
R10 is selected from the group consisting of:
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from the group consisting of -aryl and —C3-C8 heterocycle;
R13 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl; and a pharmaceutically acceptable carrier or vehicle.
33. A composition comprising drug-linker-antibody conjugates having Formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt thereof, wherein,
L- is an antibody that immunospecifically binds to a cancer cell antigen which is on the surface of a cancer cell;
-Aa-Ww—Yy— is an enzymatically cleavable linker unit that links the Drug unit and the antibody , wherein:
-A- is a Stretcher unit;
a is 1;
each —W— is independently an Amino Acid unit;
—Y— is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to 10 and is the average number of -Aa-Ww—Yy-D units per antibody in the composition; and
-D is a Drug unit having the structure
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is selected from the group consisting of —H and -methyl;
R3 is selected from the group consisting of —H, -methyl, and -isopropyl;
R4 is selected from the group consisting of —H and -methyl;
R5 is selected from the group consisting of -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl or R4 and R5 join and faun a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— where Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and -methyl;
each R8 is independently selected from the group consisting of —OH, -methoxy and -ethoxy;
R10 is selected from the group consisting of:
R24 is selected from the group consisting of H and —C(O)R25—; wherein R25 is selected from the group consisting of —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
Z is —O—, —NH—, —OC(O)—, —NHC(O)—, or —NR28C(O)—; where R28 is selected from the group consisting of —H and —C1-C8 alkyl;
n is 0 or 1; and
R27 is selected from the group consisting of —H, —N3, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 0; and
R27 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 1; and a pharmaceutically acceptable carrier or vehicle.
34. The composition of claim 32 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, R2 is —C1-C8 alkyl.
35. The composition of claim 33 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, R2 is methyl.
36. The composition of claim 32 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, -D is a Drug unit having the structure
37. The composition of claim 32 wherein the drug-linker-antibody conjugates have the formula
or a pharmaceutically acceptable salt thereof, where E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or from 1-10 when E is —CH2CH2—O—; F is —CH2—; and f is 0 or 1.
38. The composition of claim 37 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a monoclonal antibody.
39. The composition of claim 37 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a full length immunoglobulin molecule.
40. The composition of claim 37 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is bound to the Stretcher unit via an interchain thiol of the antibody.
41. The composition of claim 38 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a chimeric antibody.
42. The composition of claim 41 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the chimeric antibody comprises a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
43. The composition of claim 37 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, p ranges from 1 to about 5.
44. The composition of claim 32 wherein the drug-linker-antibody conjugates have the formula
or a pharmaceutically acceptable salt thereof.
45. The composition of claim 44 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a monoclonal antibody.
46. The composition of claim 45 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody comprises a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
47. A method for the treatment of Hodgkin's Disease or Anaplastic Large Cell Lymphoma in a human, the method comprising administering to the human an effective amount of a drug-linker-antibody conjugate wherein the drug-linker-antibody conjugate is taken up inside the cancer cell and, once inside the cancer cell, the linker unit is cleaved, resulting in release of drug; the drug-linker-antibody conjugate having formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt thereof
wherein,
L- is an antibody that immunospecifically binds to human CD30 antigen;
-Aa-Ww—Yy— is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each —W— is independently an Amino Acid unit;
—Y— is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit having the structure
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is -methyl;
R3 is selected from the group consisting of —H, -methyl, and -isopropyl;
R4 is selected from the group consisting of —H and -methyl;
R5 is selected from the group consisting of -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— where Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and -methyl;
each R8 is independently selected from the group consisting of —OH, -methoxy and -ethoxy;
R10 is selected from the group consisting of:
R24 is selected from the group consisting of H and —C(O)R25—; wherein R25 is selected from the group consisting of —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
Z is —O—, —NH—, —OC(O)—, —NHC(O)—, or —NR28C(O)—; where R28 is selected from the group consisting of —H and —C1-C8 alkyl;
n is 0 or 1; and
R27 is selected from the group consisting of —H, —N3, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 0; and R27 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 1.
48. The method of claim 47 wherein the method is for treating Hodgkin's Disease.
49. The method of claim 47 wherein the drug-linker-antibody conjugate has the structure
or a pharmaceutically acceptable salt thereof.
50. The method of claim 49 wherein the antibody is a monoclonal antibody.
51. The method of claim 49 wherein the antibody is a chimeric antibody.
52. The method of claim 49 wherein the antibody is a humanized antibody.
53. A method for the treatment of Hodgkin's Disease or Anaplastic Large Cell Lymphoma in a human, the method comprising administering to the human an effective amount of a composition comprising drug-linker-antibody conjugates and a pharmaceutically acceptable carrier wherein the drug-linker-antibody conjugates are taken up inside the cancer cell and, once inside the cancer cell, the linker units of the drug-linker-antibody conjugates are cleaved, resulting in release of drug; the drug-linker-antibody conjugates having Formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt thereof;
wherein,
L- is an antibody that immunospecifically binds to human CD30 antigen;
-Aa-Ww—Yy— is an enzymatically cleavable linker unit that links the Drug unit and the antibody, wherein:
-A- is a Stretcher unit;
a is 1;
each —W— is independently an Amino Acid unit;
—Y— is a self-immolative Spacer unit;
w is an integer ranging from 2 to 12;
y is 1 or 2;
p ranges from 1 to 10 and is the average number of -Aa-Ww—Yy-D units per antibody in the composition; and
-D is a Drug unit of the formula
wherein, the wavy line indicates the point of attachment to the Spacer unit, and for each D:
R2 is —C1-C8 alkyl;
R3 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from the group consisting of —H and -methyl; or R4 and R5 join and form a ring with the carbon atom to which they are attached and R4 and R5 have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from the group consisting of —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from the group consisting of 2, 3, 4, 5 and 6;
R6 is selected from the group consisting of —H and —C1-C8 alkyl;
R7 is selected from the group consisting of —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from the group consisting of —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from the group consisting of —H and —C1-C8 alkyl;
R10 is selected from the group consisting of:
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from the group consisting of -aryl and —C3-C8 heterocycle;
R13 is selected from the group consisting of —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
54. The method of claim 53 wherein the drug-linker-antibody conjugates have the structure
or a pharmaceutically acceptable salt thereof.
55. The method of claim 54 wherein the antibody is a monoclonal antibody.
56. The method of claim 55 wherein the antibody comprises a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
57. The method of claim 54 wherein the antibody is a full length immunoglobulin molecule.
58. The method of claim 55 wherein the antibody is a humanized antibody.
59. The drug-linker-antibody conjugate of claim 14 or a pharmaceutically acceptable salt thereof wherein the antibody is a chimeric antibody.
60. The drug-linker-antibody conjugate of claim 14 or a pharmaceutically acceptable salt thereof wherein the antibody is a humanized antibody.
61. The drug-linker-antibody conjugate of claim 14 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a human immunoglobulin constant region.
62. The drug-linker-antibody conjugate of claim 61 or a pharmaceutically acceptable salt thereof wherein the antibody is an IgG1.
63. The drug-linker-antibody conjugate of claim 59 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
64. The drug-linker-antibody conjugate of claim 63 or a pharmaceutically acceptable salt thereof wherein the antibody immunospecifically binds human CD30 antigen.
65. The drug-linker-antibody conjugate of claim 26 or a pharmaceutically acceptable salt thereof wherein the antibody immunospecifically binds human CD30 antigen.
66. The drug-linker-antibody conjugate of claim 22 or a pharmaceutically acceptable salt thereof wherein the antibody comprises a human immunoglobulin constant region.
67. The drug-linker-antibody conjugate of claim 66 or a pharmaceutically acceptable salt thereof wherein the antibody is an IgG1.
68. The drug-linker-antibody conjugate of claim 30 or a pharmaceutically acceptable salt thereof wherein the antibody immunospecifically binds human CD30 antigen.
69. The composition of claim 38 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a humanized antibody.
70. The composition of claim 38 wherein in the drug-linker-antibody conjugates or a pharmaceutically acceptable salt thereof, the antibody comprises a human immunoglobulin constant region.
71. The composition of claim 70 wherein in the drug-linker-antibody conjugates or a pharmaceutically acceptable salt thereof, the antibody is an IgG1.
72. The composition of claim 42 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody immunospecifically binds human CD30 antigen.
73. The composition of claim 45 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody is a humanized antibody.
74. The composition of claim 46 wherein in the drug-linker-antibody conjugates or pharmaceutically acceptable salt thereof, the antibody immunospecifically binds human CD30 antigen.
75. The drug-linker-antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof wherein p ranges from about 7 to about 9.
76. The drug-linker-antibody conjugate of claim 21 or a pharmaceutically acceptable salt thereof wherein p ranges from about 7 to about 9.
77. The drug-linker-antibody conjugate of claim 29 or a pharmaceutically acceptable salt thereof wherein p ranges from 1 to 10.
78. The composition of claim 37 wherein in the drug-linker antibody conjugates or pharmaceutically acceptable salt thereof, p ranges from about 7 to about 9.
79. The composition of claim 44 wherein in the drug-linker antibody conjugates or pharmaceutically acceptable salt thereof, p ranges from 1 to about 5.
80. The method of claim 53 wherein in the drug-linker antibody conjugates or pharmaceutically acceptable salt thereof, p ranges from 1 to about 5.
81. The method of claim 54 wherein in the drug-linker antibody conjugates or pharmaceutically acceptable salt thereof, p ranges from 1 to about 5.
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81
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CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 10/522,911, filed Jul. 7, 2005 now U.S. Pat. No. 7,659,241, which was filed under 35 U.S.C. §371 as a national stage application of International Application No. PCT/US2003/24209, filed Jul. 31, 2003; which further claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/400,403, filed Jul. 31, 2002. The disclosures of each of the foregoing applications are hereby incorporated herein by reference.
1. FIELD OF THE INVENTION
The present invention is directed to Drug-Linker-Ligand Conjugates and to Drug-Linker Compounds, to compositions comprising a Drug-Linker-Ligand Conjugate or a Drug-Linker Compound, and to methods for using the same to treat cancer, an autoimmune disease or an infectious disease.
2. BACKGROUND OF THE INVENTION
Several short peptidic compounds have been isolated from natural sources and found to have biological activity. Analogs of these compounds have also been prepared, and some were found to have biological activity. For example, Auristatin E (U.S. Pat. No. 5,635,483 to Pettit et al.) is a synthetic analogue of the marine natural product Dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (G. R. Pettit, Prog. Chem. Org. Nat. Prod., 70:1-79 (1997)). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds. Both dolastatin 10 and auristatin PE are presently being used in human clinical trials to treat cancer. The structural differences between dolastatin 10 and auristatin E reside in the C-terminal residue, in which the thiazolephenethyl amine group of dolastatin 10 is replaced by a norephedrine unit in auristatin E.
The following references disclose dolastatin and auristatin compounds and analogs thereof, and their use for treating cancer:
International Publication No. WO 96/33212 A1 to Teikoku Hormone Mfg. Co., Ltd.;
International Publication No. WO 96/14856 A1 to Arizona Board of Regents;
European Patent Publication No. EP 695757 A2 to Arizona Board of Regents;
European Patent Publication No. EP 695758 A2 to Arizona Board of Regents;
European Patent Publication No. EP 695759 A2 to Arizona Board of Regents;
International Publication No. WO 95/09864 A1 to Teikoku Hormone Mfg. Co., Ltd.;
International Publication No. WO 93/03054 A1 to Teikoku Hormone Mfg. Co., Ltd.;
U.S. Pat. No. 6,323,315 B1 to Pettit et al.;
G. R. Pettit et al., Anti-Cancer Drug Des. 13(4): 243-277 (1998);
G. R. Pettit et al., Anti-Cancer Drug Des. 10(7): 529-544 (1995); and
K. Miyazaki et al., Chem. Pharm. Bull. 43(10), 1706-18 (1995).
Despite in vitro data for compounds of the dolastatin class and its analogs, significant general toxicities at doses required for achieving a therapeutic effect compromise their efficacy in clinical studies. Accordingly, there is a clear need in the art for dolastatin derivatives having significantly lower toxicity, yet useful therapeutic efficiency, compared to current dolastatin drug therapies.
The recitation of any reference in Section 2 of this application is not an admission that the reference is prior art to this application.
3. SUMMARY OF THE INVENTION
In one aspect, the present invention provides compounds of general Formula Ia:
LAa-Ww—Yy-D)p Ia
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R2 is selected from -hydrogen and —C1-C8 alkyl;
R3 is selected from -hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from -hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In another aspect, the present invention provides compounds of general formula Ib:
LAa-Ww—Yy-D)p Ib
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to Y when y is 1 or 2, or X is bonded to W when y is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-.
In another aspect, the present invention provides compounds of general formula Ic:
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
w is an integer ranging from 0 to 12;
each n is independently 0 or 1;
p ranges from 1 to about 20; and
each -D is independently:
(a) a Drug unit of the formula:
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to —C(O)— when y is 1 or 2, or X is bonded to —CH2— when n is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-; or
(b) a Drug unit of the formula:
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
A compound of formula Ia, formula Ib, formula Ic or a pharmaceutically acceptable salt or solvate thereof (a “Drug-Linker-Ligand Conjugate”) is useful for treating or preventing cancer, an autoimmune disease or an infectious disease in an animal.
In another aspect, the present invention provides compounds of the formula IIa:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl; and
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIb:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
R13 is selected from hydrogen, —OH, —NH2, —NHR14, —N(R14)2, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-; and
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)n—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIc:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR1″;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IId:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl
In another aspect, the present invention provides compounds of the formula IIe:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)n—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIf:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C10 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C10 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIg:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIh:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)n—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIi:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)n—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
A compound of formula IIa-i or a pharmaceutically acceptable salt or solvate thereof (a “Drug-Linker Compound”) is useful for treating cancer, an autoimmune disease or an infectious disease in an animal or useful as an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.
In another aspect, the present invention provides compositions comprising an effective amount of a Drug-Linker-Ligand Conjugate and a pharmaceutically acceptable carrier or vehicle.
In still another aspect, the present invention provides compositions comprising an effective amount of a Drug-Linker Compound and a pharmaceutically acceptable carrier or vehicle.
In yet another aspect, the present invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the present invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for treating cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for treating an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In still another aspect, the invention provides methods for treating an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the present invention provides methods for preventing the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the present invention provides methods for preventing the multiplication of a tumor cell or cancer cell, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for preventing cancer, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an auto-immune antibody, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for preventing an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for preventing an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker Compound.
In still another aspect, the invention provides methods for preventing an infectious disease, comprising administering to an animal in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides a Drug-Linker Compound which can be used as an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.
The present invention may be understood more fully by reference to the following detailed description, Figures and illustrative examples, which are intended to exemplify non-limiting embodiments of the invention.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the cytotoxicity of Compound 49 and Compound 53 against the H3396 cell line. Line -Δ- represents Compound 49 and line -∘- represents Compound 53.
FIG. 2 shows the cytotoxicity of Compounds 64, 65, 68 and 69 against the H3396 cell line. Line -♦- represents Compound 64, line -▪- represents Compound 65, line -Δ- represents Compound 68, and line -X- represents Compound 69.
FIG. 3 shows the cytotoxicity of Compounds 64, 65, 68 and 69 against the HCT-116 cell line. Line -♦- represents Compound 64, line -▪- represents Compound 65, Line -Δ- represents Compound 68, and line -X- represents Compound 69.
FIG. 4 shows the cytotoxicity of Compounds 66 and 68 against the H3396 cell line. Line -□- represents Compound 66 and line -*- represents Compound 68.
FIG. 5 shows the cytotoxicity of Compounds 66, 68 and 69 against the Karpas human colorectal cell line. Line -♦- represents Compound 66, line -▴- represents Compound 68, and line -X- represents Compound 69.
FIG. 6 shows the cytotoxicity of Compounds 66 and 67 against the H3396 cell line as a function of exposure length. The cells were either exposed to the conjugates for the entire duration of the assay without washing (96 hours), or were exposed to the conjugates for 2 hours, washed, and then incubated for an additional 94 hours. At the end of the 96 hour period, the cells were pulsed with Alamar Blue to determine cell viability. Line - - represents Compound 66 at 2 h exposure, line -- represents Compound 67 at 2 h exposure, line -●- represents Compound 66 at 96 h exposure, and line - - represents Compound 67 at 96 h exposure.
FIG. 7 shows the effect of Compounds 66-69 on the growth of L2987 human lung adenocarcinoma xenograft tumors which were implanted in nude mice. Line -X- represents untreated tumor, line -▾- represents Compound 66, line -♦- represents Compound 68, line -∇- Compound 67, and line -⋄- represents Compound 69.
FIG. 8 shows the effects of Compounds 66-69 on the growth of Karpas human anaplastic large cell lymphoma xenograft tumors which were implanted in nude mice. Line -X- represents untreated tumor, line -▴- represents Compound 67, line represents Compound 69, line -Δ- represents Compound 66, and line -∘- represents Compound 68.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Definitions
Examples of an “animal” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl.
“Aryl” refers to a carbocyclic aromatic group Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. A carbocyclic aromatic group or a heterocyclic aromatic group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
The term “C1-C8 alkyl,” as used herein refers to a straight chain or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative “C1-C8 alkyl” groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonly and -n-decyl; while branched C1-C8 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C1-C8 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl. methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A C1-C8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “C3-C8 carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. A C3-C8 carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “C3-C8 carbocyclo” refers to a C3-C8 carbocycle group defined above wherein one of the carbocycle groups hydrogen atoms is replaced with a bond.
A “C1-C10 alkylene” is a straight chain, saturated hydrocarbon group of the formula —(CH2)1-10—. Examples of a C1-C10 alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene.
An “arylene” is an aryl group which has two covalent bonds and can be in the ortho, meta, or para configurations as shown in the following structures:
in which the phenyl group can be unsubstituted or substituted with up to four groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “C3-C8 heterocycle” refers to an aromatic or non-aromatic C3-C8 carbocycle in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. Representative examples of a C3-C8 heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl. A C3-C8 Heterocycle can be unsubstituted or substituted with up to seven groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
“C3-C8 heterocyclo” refers to a C3-C8 heterocycle group defined above wherein one of the heterocycle groups hydrogen atoms is replaced with a bond. A C3-C8 heterocyclo can be unsubstituted or substituted with up to six groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from —C1-C8 alkyl and aryl.
A “Compound of the Invention” is a Drug-Linker Compound or a Drug-Linker-Ligand Conjugate.
In one embodiment, the Compounds of the Invention are in isolated or purified form. As used herein, “isolated” means separated from other components of (a) a natural source, such as a plant or animal cell or cell culture, or (b) a synthetic organic chemical reaction mixture. As used herein, “purified” means that when isolated, the isolate contains at least 95%, preferably at least 98%, of a Compound of the Invention by weight of the isolate.
Examples of a “Hydroxyl protecting group” include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
“Leaving group” refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.
The term “antibody,” as used herein, refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce auto-immune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. Antibodies useful in the invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F(ab′) fragments, F(ab′)2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens.
The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a Compound of the Invention. The Compounds of the Invention contain at least one amino group, and accordingly acid addition salts can be formed with this amino group. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
“Pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a Compound of the Invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
In the context of cancer, the term “treating” includes any or all of: preventing growth of tumor cells or cancer cells, preventing replication of tumor cells or cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.
In the context of an autoimmune disease, the term “treating” includes any or all of: preventing replication of cells associated with an autoimmune disease state including, but not limited to, cells capable of producing an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.
In the context of an infectious disease, the term “treating” includes any or all of: preventing the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease.
The following abbreviations are used herein and have the indicated definitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is diethylamine, DEAD is diethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine, dil is dolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high pressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is (1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS is phosphate-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, Ph is phenyl, phe is L-phenylalanine, PyBrop is bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, SEC is size-exclusion chromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC is thin layer chromatography, UV is ultraviolet, val is valine.
5.2 Drug-Linker-Ligand Conjugates
As stated above, the invention provides compounds of the formula Ia:
LAa-Ww—Yy-D)p Ia
and pharmaceutically acceptable salts and solvates thereof
wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In one embodiment R10 is selected from
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
Illustrative classes of compounds of formula Ia have the structures:
and pharmaceutically acceptable salts and solvates thereof,
where L- is a Ligand unit, E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or from 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In another embodiment L is cBR96, cAC10 or 1F6.
Illustrative compounds of formula Ia have the structure:
and pharmaceutically acceptable salts and solvates thereof,
where p ranges from about 7 to about 9.
In one embodiment p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In another embodiment, p is about 8.
In yet another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another aspect, the present invention provides compounds of general formula Ib:
LAa-Ww—Yy-D) Ib
and pharmaceutically acceptable salts and solvates thereof wherein,
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit of the formula
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to Y when y is 1 or 2, or X is bonded to W when y is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-.
In one embodiment, when R1 is —H, R10 is selected from:
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
Illustrative classes of compounds of formula Ib have the structure:
pharmaceutically acceptable salts and solvates thereof,
where L- is Ligand unit, E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another embodiment L is cBR96, cAC10 or 1F6.
Illustrative compounds of formula Ib have the structure:
and pharmaceutically acceptable salts and solvates thereof,
where p ranges from about 7 to about 9.
In one embodiment p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In another embodiment, p is about 8.
In yet another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another aspect, the present invention provides compounds of general formula Ic:
L- is a Ligand unit;
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
w is an integer ranging from 0 to 12;
each n is independently 0 or 1;
p ranges from 1 to about 20; and
each -D is independently:
(a) a Drug unit of the formula:
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to —C(O)— when y is 1 or 2, or X is bonded to —CH2— when n is 0;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-; or
(b) a Drug unit of the formula:
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In one embodiment, when the drug unit has the formula:
and R1 is —H, R10 is selected from
In another embodiment, when the drug unit has the formula:
R10 is selected from
In another embodiment, w is an integer ranging from 2 to 12.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
An illustrative compound of formula Ic has the structure:
wherein where L- is Ligand unit, E is —CH2— or —CH2CH2O—; e is an integer ranging either from 0-10 when E is —CH2—, or 1-10 when E is —CH2CH2—O—; F is —CH2—; f is 0 or 1; and p ranges from 1 to about 20.
In another embodiment, p ranges from 1 to about 8.
In another embodiment, p ranges from 1 to about 3.
In another embodiment, p ranges from about 3 to about 5.
In still another embodiment, p ranges from about 7 to about 9.
In another embodiment, p is about 8.
In another embodiment, p is about 4.
In a further embodiment, p is about 2.
In another embodiment L is cBR96, cAC10 or 1F6.
The Drug-Linker-Ligand Conjugates are useful for treating or preventing cancer, an autoimmune disease or an infectious disease in an animal.
It is understood that p is the average number of -Aa-Ww—Yy-D units per ligand in a Drug-Linker-Ligand Conjugate of formulas Ia, Ib and Ic.
In one embodiment p ranges from 1 to 15.
In another embodiment p ranges from 1 to 10.
In another embodiment, p ranges from 1 to about 8.
In a further embodiment p ranges from 1 to about 5.
In another embodiment p ranges from 1 to about 3.
In one embodiment p ranges from about 3 to about 5.
In one embodiment p ranges from about 7 to about 9.
In another embodiment p is about 8.
In yet another embodiment p is about 4.
In still another embodiment p is about 2.
The Drug-Linker-Ligand Conjugates of formulas Ia, Ib and Ic may exist as mixtures, wherein each component of a mixture has a different p value. For example, a Drug-Linker-Ligand Conjugate may exist as a mixture of two separate Conjugates, one Conjugate component wherein p is 7 and the other Conjugate component wherein p is 8.
In one embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 1, 2, and 3, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 3, 4, and 5, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 5, 6, and 7, respectively.
In still another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 7, 8, and 9, respectively.
In yet another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 9, 10, and 11, respectively.
In still another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 11, 12, and 13, respectively.
In another embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture of three separate conjugates wherein p for the three separate conjugates is 13, 14, and 15, respectively.
5.3 Drug-Linker Compounds
The present invention provides compounds of the formula IIa:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—, where X is bonded to Y when y is 1 or 2, or
X is bonded to W when y is 0;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
An illustrative compound of formula IIa has the structure:
and pharmaceutically acceptable salts and solvates thereof.
In another aspect, the present invention provides compounds of the formula IIb:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
R13 is selected from hydrogen, —OH, —NH2, —NHR14, —N(R14)2, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIc:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is -Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IId:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is -Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIe:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIf:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
X is —O—, —S—, —NH— or —N(R14)—;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In one embodiment R1 is selected from —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached
Illustrative compounds of formula IIf have the structure:
and pharmaceutically acceptable salts and solvates thereof.
In another aspect, the present invention provides compounds of the formula IIg:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIh:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
each R14 is independently —H or —C1-C8 alkyl;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
In another aspect, the present invention provides compounds of the formula IIi:
and pharmaceutically acceptable salts and solvates thereof
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from hydrogen, —OH, —NH2, —NHR14, —N(R14)2, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl;
R16 is —Yy-Ww-A′
wherein
each —W— is independently an Amino Acid unit;
—Y— is a Spacer unit;
w is an integer ranging from 0 to 12;
y is 0, 1 or 2; and
-A′ is selected from
wherein
G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;
J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR18;
R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)n—, and —(CH2CH2O)r—CH2—; r is an integer ranging from 1-10; and
R18 is —C1-C8 alkyl or -aryl.
Illustrative compounds of formula III have the structures:
and pharmaceutically acceptable salts and solvates thereof.
The compounds of formulas IIa-i are useful for treating or preventing cancer, an autoimmune disease or an infectious disease in an animal.
5.4 The Linker Unit
The Linker unit of the Drug-Linker-Ligand Conjugate links the Drug unit and the Ligand unit and has the formula:
—Aa-Ww—Yy—
wherein:
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
w is independently an integer ranging from 0 to 12;
—Y— is a Spacer unit; and
y is 0, 1 or 2.
5.4.1 The Stretcher Unit
The Stretcher unit (-A-), when present, links a Ligand unit to an amino acid unit (—W—). In this regard a Ligand (L) has a functional group that can form a bond with a functional group of a Stretcher. Useful functional groups that can be present on a ligand, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of a carbohydrate, and carboxyl. Preferred Ligand functional groups are sulfhydryl and amino. Sulfhydryl groups can be generated by reduction of an intramolecular disulfide bond of a Ligand. Alternatively, sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of a Ligand using 2-iminothiolane (Traut's reagent) or another sulfhydryl generating reagent.
In one embodiment, the Stretcher unit forms a bond with a sulfur atom of the Ligand unit. The sulfur atom can be derived from a sulfhydryl group of a Ligand. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas (IIIa) and (IIIb), wherein L-, —W—, —Y—, -D, w and y are as defined above and R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; and r is an integer ranging from 1-10.
An illustrative Stretcher unit is that of formula (IIIa) where R17 is —(CH2)5—:
Another illustrative Stretcher unit is that of formula (IIIa) where R17 is —(CH2CH2O)r—CH2—; and r is 2:
Still another illustrative Stretcher unit is that of formula (IIIb) where R17 is —(CH2)5—:
In another embodiment, the Stretcher unit is linked to the Ligand unit via a disulfide bond between a sulfur atom of the Ligand unit and a sulfur atom of the Stretcher unit. A representative Stretcher unit of this embodiment is depicted within the square brackets of Formula (IV), wherein R17, L-, —W—, —Y—, -D, w and y are as defined above.
In yet another embodiment, the reactive group of the Stretcher contains a reactive site that can form a bond with a primary or secondary amino group of a Ligand. Example of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas (Va) and (Vb), wherein —R17—, L-, —W—, —Y—, -D, w and y are as defined above;
In yet another aspect of the invention, the reactive group of the Stretcher contains a reactive site that is reactive to a carbohydrate's (—CHO) group that can be present on a Ligand. For example, a carbohydrate can be mildly oxidized using a reagent such as sodium periodate and the resulting (—CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko, T. et al. Bioconjugate Chem 1991, 2, 133-41. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas (VIa)-(VIc), wherein —R17—, L-, —W—, —Y—, -D, w and y are as defined above.
5.4.2 The Amino Acid Unit
The Amino Acid unit (—W—), when present, links the Stretcher unit to the Spacer unit if the Spacer unit is present, links the Stretcher unit to the Drug unit if the Spacer unit is absent, and links the Ligand unit to the Drug unit if the Stretcher unit and Spacer unit are absent.
—Ww— is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each —W— unit independently has the formula denoted below in the square brackets, and w is an integer ranging from 0 to 12:
wherein R19 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH2OH, —CH(OH)CH3, —CH2CH2SCH3, —CH2CONH2, —CH2COOH, —CH2CH2CONH2, —CH2CH2COOH, —(CH2)3NHC(═NH)NH2, —(CH2)3NH2, —(CH2)3NHCOCH3, —(CH2)3NHCHO, —(CH2)4NHC(═NH)NH2, —(CH2)4NH2, —(CH2)4NHCOCH3, —(CH2)4NHCHO, —(CH2)3NHCONH2, —(CH2)4NHCONH2, —CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
The Amino Acid unit of the Compounds of the Invention can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, to liberate the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide a Drug (D).
Illustrative Ww units are represented by formulas (VII)-(IX):
wherein R20 and R21 are as follows:
R20
R21
benzyl
(CH2)4NH2;
methyl
(CH2)4NH2;
isopropyl
(CH2)4NH2;
isopropyl
(CH2)3NHCONH2;
benzyl
(CH2)3NHCONH2;
isobutyl
(CH2)3NHCONH2;
sec-butyl
(CH2)3NHCONH2;
(CH2)3NHCONH2;
benzyl
methyl; and
benzyl
(CH2)3NHC(═NH)NH2;
wherein R20, R21 and R22 are as follows:
R20
R21
R22
benzyl
benzyl
(CH2)4NH2;
isopropyl
benzyl
(CH2)4NH2; and
H
benzyl
(CH2)4NH2;
wherein R20, R21, R22 and R23 are as follows:
R20
R21
R22
R23
H
benzyl
isobutyl
H; and
methyl
isobutyl
methyl
isobutyl.
Preferred Amino Acid units include, but are not limited to, units of formula (VII) where: R20 is benzyl and R21 is —(CH2)4NH2; R20 isopropyl and R21 is —(CH2)4NH2; R20 isopropyl and R21 is —(CH2)3NHCONH2. Another preferred Amino Acid unit is a unit of formula (VIII) where R20 is benzyl, R21 is benzyl, and R22 is —(CH2)4NH2.
—Ww— units useful in the present invention can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease. In one embodiment, a —Ww— unit is that whose cleavage is catalyzed by cathepsin B, C and D, or a plasmin protease.
In one embodiment, —Ww— is a dipeptide, tripeptide or pentapeptide.
Where R19, R20, R21, R22 or R23 is other than hydrogen, the carbon atom to which R19, R20, R21, R22 or R23 is attached is chiral.
Each carbon atom to which R19, R20, R21, R22 or R23 is attached is independently in the (S) or (R) configuration.
5.4.3 The Spacer Unit
The Spacer unit (—Y—), when present, links an Amino Acid unit to the Drug unit when an Amino Acid unit is present. Alternately, the Spacer unit links the Stretcher unit to the Drug unit when the Amino Acid unit is absent. The Spacer unit also links the Drug unit to the ligand unit when both the Amino Acid unit and Stretcher unit are absent. Spacer units are of two general types: self-immolative and non self-immolative. A non self-immolative Spacer unit is one in which part or all of the Spacer unit remains bound to the Drug unit after cleavage, particularly enzymatic, of an Amino Acid unit from the Drug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of a non self-immolative Spacer unit include, but are not limited to a (glycine-glycine) Spacer unit and a glycine Spacer unit (both depicted in Scheme 1). When a Compound of the Invention containing a glycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymatic cleavage via a tumor-cell associated-protease, a cancer-cell-associated protease or a lymphocyte-associated protease, a glycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved from L-Aa-Ww—. In one embodiment, an independent hydrolysis reaction takes place within the target cell, cleaving the glycine-Drug unit bond and liberating the Drug.
In a preferred embodiment, —Yy— is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3) whose phenylene portion is substituted with Qm where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
In one embodiment, a non self-immolative Spacer unit (—Y—) is -Gly-Gly-.
In another embodiment, a non self-immolative the Spacer unit (—Y—) is -Gly-.
In one embodiment, the invention provides a Drug-Linker Compound or a Drug-Linker Ligand Conjugate in which the Spacer unit is absent (y=0), or a pharmaceutically acceptable salt or solvate thereof.
Alternatively, a Compound of the Invention containing a self-immolative Spacer unit can release -D without the need for a separate hydrolysis step. In this embodiment, —Y— is a PAB group that is linked to —Ww— via the amino nitrogen atom of the PAB group, and connected directly to -D via a carbonate, carbamate or ether group. Without being bound by theory, Scheme 2 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via a carbamate or carbonate group.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1 to about 20.
Without being bound by theory, Scheme 3 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via an ether or amine linkage.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1 to about 20.
Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazol-5-methanol derivatives (see Hay et al., Bioorg. Med. Chem. Lett., 1999, 9, 2237) and ortho or para-aminobenzylacetals. Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacer useful in the Compounds of the Invention.
In a preferred embodiment, the Spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as depicted in Scheme 4, which can be used to incorporate and release multiple drugs.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from 1 to about 20.
In one embodiment, the -D moieties are the same.
In another embodiment, the -D moieties are different.
Preferred Spacer units (—Yy—) are represented by Formulas (X)-(XII):
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4;
5.5 The Drug Unit
-D is a Drug unit having a nitrogen or oxygen atom that can form a bond with the Spacer unit when y=1 or 2 or with the C-terminal carbonyl group of an Amino Acid unit when y=0.
In one embodiment, -D is represented by the formula:
wherein, independently at each location:
R2 is selected from —H and —C1-C8 alkyl;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and
each R14 is independently —H or —C1-C8 alkyl.
In one embodiment, R10 is selected from
In a preferred embodiment, -D has the formula
or a pharmaceutically acceptable salt or solvate thereof,
wherein, independently at each location:
R2 is selected from —H and -methyl;
R3 is selected from —H, -methyl, and -isopropyl;
R4 is selected from —H and -methyl; R5 is selected from -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl; or R4 and R5 join, have the formula —(CRaRb)n— where Ra and Rb are independently selected from —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and -methyl;
each R5 is independently selected from —OH, -methoxy and -ethoxy;
R10 is selected from
R24 is selected from H and —C(O)R25; wherein R25 is selected from —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R26 is selected from —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
Z is —O—, —NH—, —OC(O)—, —NHC(O)—, —N(R28)C(O)—; where R2 is selected from —H and —C1-C8 alkyl;
n is 0 or 1; and
R27 is selected from —H, —N3, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 0; and R27 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) when n is 1.
In one embodiment, R10 is selected from
In another embodiment, -D is represented by the formula:
wherein, independently at each location:
R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached;
R3 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from
X is —O—, —S—, —NH— or —N(R14)—, where X forms a bond with a Linker unit;
Z is —O—, —S—, —NH— or —N(R14)—;
R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond;
each R12 is independently selected from -aryl and —C3-C8 heterocycle;
R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —O—(C1-C8 alkyl), —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R14 is independently —H or —C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo- or —C3-C8 heterocyclo-.
In one embodiment, when R1 is —H, R10 is selected from:
In a preferred embodiment, -D has the formula
or a pharmaceutically acceptable salt or solvate thereof,
wherein, independently at each location:
R1 is selected from —H and -methyl;
R2 is selected from —H and -methyl;
R3 is selected from —H, -methyl, and -isopropyl;
R4 is selected from —H and -methyl; R5 is selected from -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl; or R4 and R5 join, have the formula—(CRaRb), where Ra and Rb are independently selected from —H, —C1-C8 alkyl, and —C3-C8 carbocycle, and N is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and -methyl;
each R8 is independently selected from —OH, -methoxy and -ethoxy; R10 is selected from
where X is —O—, —NH— or —N(R14)— and forms a bond with Y when y is 1 or 2, with W when y is 0, and with A when w and y are both 0;
Z is —O—, —NH— or —N(R14)—;
R13 is —H or -methyl;
R14 is C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo or —C3-C8 heterocyclo-,
In one embodiment, when R1 is -methyl, R10 is selected from
where X is —O—, —NH— or —N(R14)— and forms a bond with Y when y is 1 or 2, and with W when y is 0;
Z is —O—, —NH— or —N(R14)—;
R13 is —H or -methyl;
R14 is C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo or —C3-C8 heterocyclo-.
In another embodiment, when R1 is —H, R10 is selected from:
where X is —O—, —NH— or —N(R14)— and forms a bond with Y when y is 1 or 2, and with W when y is 0;
Z is —O—, —NH— or —N(R14)—;
R13 is —H or -methyl;
R14 is C1-C8 alkyl; and
R15 is -arylene-, —C3-C8 carbocyclo or —C3-C8 heterocyclo-.
A Drug unit can form a bond with a Linker unit via a nitrogen atom of a Drug's primary or secondary amino group, via an oxygen atom of a Drug's hydroxyl group, or via a sulfur atom of a Drug's sulfhydryl group to form a Drug-Linker Compound.
In a preferred embodiment, Drug units have the formula
5.6 The Ligand Unit
The Ligand unit (L-) includes within its scope any unit of a Ligand (L) that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. A Ligand can be any molecule that binds to, complexes with or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. The Ligand unit acts to deliver the Drug unit to the particular target cell population with which the Ligand unit reacts. Such Ligands include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies, antibody fragments, smaller molecular weight proteins, polypeptide or peptides, and lectins.
A Ligand unit can form a bond to either a Stretcher unit or an Amino Acid unit of a Linker. A Ligand unit can form a bond to a Linker unit via a heteroatom of the Ligand. Heteroatoms that may be present on a Ligand unit include sulfur (in one embodiment, from a sulfhydryl group of a Ligand), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of a Ligand) and nitrogen (in one embodiment, from a primary or secondary amino group of a Ligand). These heteroatoms can be present on the Ligand in the Ligand's natural state, for example a naturally occurring antibody, or can be introduced into the Ligand via chemical modification.
In a preferred embodiment, a Ligand has a sulfhydryl group and the Ligand bonds to the Linker unit via the sulfhydryl group's sulfur atom.
In another embodiment, the Ligand can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups. The Ligand unit bonds to the Stretcher unit via the sulfhydryl group's sulfur atom.
In yet another embodiment, the Ligand can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) group (see Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The corresponding aldehyde can form a bond with a Reactive Site on a Stretcher. Reactive sites on a Stretcher that can react with a carbonyl group on a Ligand include, but are not limited to, hydrazine and hydroxylamine.
Useful non-immunoreactive protein, polypeptide, or peptide Ligands include, but are not limited to, transferrin, epidermal growth factors (“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (“TGF”), such as TGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II, lectins and apoprotein from low density lipoprotein.
Useful Polyclonal antibody Ligands are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, and guinea pigs. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
Useful monoclonal antibody Ligands are homogeneous populations of antibodies to a particular antigen (e.g., a cancer cell antigen, a viral antigen, a microbial antigen covalently linked to a second molecule). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
Useful monoclonal antibody Ligands include, but are not limited to, human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).
The Ligand can also be a bispecific antibody. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually performed using affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in International Publication No. WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation (International Publication No. WO 94/04690) which is incorporated herein by reference in its entirety.
For further details for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 1986, 121:210. Using such techniques, bispecific antibody Ligands can be prepared for use in the treatment or prevention of disease as defined herein.
Bifunctional antibodies are also described, in European Patent Publication No. EPA 0 105 360. As disclosed in this reference, hybrid or bifunctional antibodies can be derived either biologically, i.e., by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide-bridge forming reagents, and may comprise whole antibodies or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed for example, in International Publication WO 83/03679, and European Patent Publication No. EPA 0 217 577, both of which are incorporated herein by reference.
The Ligand can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, or microbial antigens. In this regard, “Functionally active” means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized. Specifically, in a preferred embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay)
Other useful Ligands include fragments of antibodies such as, but not limited to, F(ab′)2 fragments, which contain the variable region, the light chain constant region and the CH1 domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Other useful Ligands are heavy chain and light chain dimers of antibodies, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful Ligands. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671; European Patent Publication No. 184,187; European Patent Publication No. 171,496; European Patent Publication No. 173,494; International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 125,023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference in its entirety.
Completely human antibodies are particularly desirable for Ligands. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806; each of which is incorporated herein by reference in its entirety. Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).
Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Biotechnology 12:899-903).
In other embodiments, the Ligand is a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the antibody. Preferably, the antibody or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.
The Ligand antibodies include analogs and derivatives that are either modified, i.e, by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, but not by way of limitation, the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular Ligand unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnaturalamino acids.
The Ligand antibodies include antibodies having modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, the Ligand antibodies include antibodies having modifications in amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety). Antibodies immunospecific for a cancer cell antigen can be obtained commercially, for example, from Genentech (San Francisco, Calif.) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
In a specific embodiment, known antibodies for the treatment or prevention of cancer are used in accordance with the compositions and methods of the invention. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, HERCEPTIN (Trastuzumab; Genentech, Calif.) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer (Stebbing, J., Copson, E., and O'Reilly, S. “Herceptin (trastuzamab) in advanced breast cancer” Cancer Treat Rev. 26, 287-90, 2000); RITUXAN (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG2a antibody for the treatment of colorectal cancer; BEC2 (ImClone Systems Inc., NY) which is murine IgG antibody for the treatment of lung cancer; IMC-C225 (Imclone Systems Inc., NY) which is a chimeric IgG antibody for the treatment of head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosite, Mass.) which is a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart M195 (Protein Design Labs, Inc., CA) which is a humanized IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which is a humanized IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a humanized antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA) which is a murine antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma; anti-VEGF (Genentech, Inc., CA) which is humanized antibody for the treatment of lung and colorectal cancers; CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer; IMC-1C11 (ImClone Systems, NJ) which is an anti-KDR chimeric antibody for the treatment of colorectal cancer, lung cancers, and melanoma; and Cetuximab (ImClone, NJ) which is an anti-EGFR chimeric antibody for the treatment of epidermal growth factor positive cancers.
Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE -4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp 00 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogene product (carcinomas). Some specific useful antibodies include, but are not limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellström, I., Hellström, K. E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64 (Trail, P A, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R. S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom, K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res. 2000, 60, 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb, and mAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functional effects of CD30 on a large granular lymphoma cell line YT” J. Immunol., 151, 5896-5906, 1993). Many other internalizing antibodies that bind to tumor associated antigens can be used in this invention, and have been reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy of acute myeloid leukemia: areview” Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).
In another specific embodiment, known antibodies for the treatment or prevention of an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. In another embodiment, useful Ligand antibodies that are immunospecific for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti Phospholipid Antibody IgM, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; Anti SCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody.
In certain preferred embodiments, antibodies useful in the present methods, can bind to both a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-IBB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitable lectins are C-type, S-type, and I-type lectin.
In one embodiment, the Ligand is an antibody that binds to an activated lymphocyte that is associated with an autoimmune disease.
In another specific embodiment, useful Ligand antibodies that are immunospecific for a viral or a microbial antigen are monoclonal antibodies. Preferably, Ligand antibodies that are immunospecific for a viral antigen or microbial antigen are humanized or human monoclonal antibodies. As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide ⅝) that is capable of eliciting an immune response.
Antibodies immunospecific for a viral or microbial antigen can be obtained commercially, for example, from Genentech (San Francisco, Calif.) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
In a specific embodiment, useful Ligand antibodies are those that are useful for the treatment or prevention of viral or microbial infection in accordance with the methods of the invention. Examples of antibodies available useful for the treatment of viral infection or microbial infection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody useful for the treatment of patients with RSV infection; PRO542 (Progenics) which is a CD4 fusion antibody useful for the treatment of HIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; PROTOVIR (Protein Design Labs, Inc., CA) which is a humanized IgG1 antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.
Other antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).
Other antibodies useful in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.
The antibodies suitable for use in the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
5.6.1 Production of Recombinant Antibodies
Ligand antibodies of the invention can be produced using any method known in the art to be useful for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
Recombinant expression of the Ligand antibodies, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a nucleic acid molecule encoding an antibody can be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the antibody can be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
If an antibody that specifically recognizes a particular antigen is not commercially available (or a source for a cDNA library for cloning a nucleic acid encoding such an immunoglobulin), antibodies specific for a particular antigen can be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody can be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
Once a nucleic acid sequence encoding at least the variable domain of the antibody is obtained, it can be introduced into a vector containing the nucleotide sequence encoding the constant regions of the antibody (see, e.g., International Publication No. WO 86/05807; International Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors containing the complete light or heavy chain that allow for the expression of a complete antibody molecule are available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydryl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).
Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab′)2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
Once a nucleic acid sequence encoding a Ligand antibody has been obtained, the vector for the production of the antibody can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
An expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.
The host cells used to express the recombinant Ligand antibody can be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al., 1990, BioTechnology 8:2).
A variety of host-expression vector systems can be utilized to express the immunoglobulin Ligands. Such host-expression systems represent vehicles by which the coding sequences of the antibody can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express a Ligand immunoglobulin molecule in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, vectors that direct the expression of high levels of fusion protein products that are readily purified might be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) or the analogous virus from Drosophila Melanogaster is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
In addition, a host cell strain can be chosen to modulate the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express an antibody can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody Such engineered cell lines can be particularly useful in screening and evaluation of tumor antigens that interact directly or indirectly with the antibody Ligand.
A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).
The expression levels of an antibody can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
The host cell can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers that enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used to encode both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.
Once the antibody has been recombinantly expressed, it can be purified using any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
In a preferred embodiment, the Ligand is an antibody.
In a more preferred embodiment, the Ligand is a monoclonal antibody.
In any case, the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an autoimmune disease, an infectious organism, or other disease state.
5.7 Synthesis of the Compounds of the Invention
As described in more detail below, the Compounds of the Invention are conveniently prepared using a Linker having two or more Reactive Sites for binding to the Drug and Ligand. In one aspect of the invention, a Linker has a Reactive site which has an electrophilic group that is reactive to a nucleophilic group present on a Ligand. Useful nucleophilic groups on a Ligand include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of a Ligand is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for Ligand attachment.
In another embodiment, a Linker has a Reactive site which has a nucleophilic group that is reactive to an electrophilic group present on a Ligand. Useful electrophilic groups on a Ligand include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on a Ligand and form a covalent bond to a Ligand unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on a Ligand provides a convenient site for attachment to a Linker.
Carboxylic acid functional groups and chloroformate functional groups are also useful reactive sites for a Linker because they can react with primary or secondary amino groups of a Drug to form an amide linkage. Also useful as a reactive site is a carbonate functional group on a Linker which can react with an amino group or hydroxyl group of a Drug to form a carbamate linkage or carbonate linkage, respectively. Similarly, a Drug's phenol moiety can react with the Linker, existing as an alcohol, under Mitsunobu conditions.
Typically, peptide-based Drugs can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schröder and K. Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
In one embodiment, a Drug is prepared by combining about a stoichiometric equivalent of a dipeptide and a tripeptide, preferably in a one-pot reaction under suitable condensation conditions. This approach is illustrated in the following Schemes 5-7. Thus, the tripeptide 6 can be prepared as shown in Scheme 5, and the dipeptide 9 can be prepared as shown in Scheme 6. The two fragments 6 and 9 can be condensed to provide a Drug 10 as shown in Scheme 7.
The synthesis of an illustrative Stretcher having an electrophilic maleimide group is illustrated in Schemes 8-9. General synthetic methods useful for the synthesis of a Linker are described in Scheme 10. Scheme 11 shows the construction of a Linker unit having a val-cit group, an electrophilic maleimide group and a PAB self-immolative Spacer group. Scheme 12 depicts the synthesis of a Linker having a phe-lys group, an electrophilic maleimide group, with and without the PAB self-immolative Spacer group. Scheme 13 presents a general outline for the synthesis of a Drug-Linker Compound, while Scheme 14 presents an alternate route for preparing a Drug-Linker Compound. Scheme 15 depicts the synthesis of a branched linker containing a BHMS group. Scheme 16 outlines the attachment of a Ligand to a Drug-Linker Compound to form a Drug-Linker-Ligand Conjugate, and Scheme 17 illustrates the synthesis of Drug-Linker-Ligand Conjugates having 2 or 4 drugs per Ligand.
As illustrated in Scheme 5, a protected amino acid 1 (where PG represents an amine protecting group, R4 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle, alkyl-(C3-C8 heterocycle) wherein R5 is selected from H and methyl; or R4 and R5 join, have the formula (CRaRb)n— wherein Ra and Rb are independently selected from hydrogen, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached) is coupled to t-butyl ester 2 (where R6 is selected from —H and —C1-C8 alkyl; and R7 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle)) under suitable coupling conditions, e.g., in the presence of PyBrop and diisopropylethylamine, or using DCC (see, for example, Miyazaki, K. et. al. Chem. Pharm. BuIl 1995, 43(10), 1706-1718).
Suitable protecting groups PG, and suitable synthetic methods to protect an amino group with a protecting group are well known in the art. See, e.g., Greene, T. W. and Wuts, P.G.M., Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley & Sons. Preferred protected amino acids 1 are PG-Ile and, particularly, PG-Val, while other suitable protected amino acids include, without limitation: PG-cyclohexylglycine, PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid, PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and PG-tert-butylglycine. Z is a preferred protecting group. Fmoc is another preferred protecting group. A preferred t-butyl ester 2 is dolaisoleuine t-butyl ester.
The dipeptide 3 can be purified, e.g., using chromatography, and subsequently deprotected, e.g., using H2 and 10% Pd—C in ethanol when PG is benzyloxycarbonyl, or using diethylamine for removal of an Fmoc protecting group. The resulting amine 4 readily forms a peptide bond with an amino acid 5 (where R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached; and R3 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle)). N,N-Dialkyl amino acids are preferred amino acids 5, such as commercially available N,N-dimethyl valine. Other N,N-dialkyl amino acids can be prepared by reductive bis-alkylation using known procedures (see, e.g., Bowman, R. E, Stroud, H. H J. Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two preferred amino acids 5 useful for the synthesis of N-monoalkyl derivatives. The amine 4 and the amino acid 5 react to provide the tripeptide 6 using coupling reagent DEPC with triethylamine as the base.
Illustrative DEPC coupling methodology and the PyBrop coupling methodology shown in Scheme 5 are outlined below in General Procedure A and General Procedure B, respectively. Illustrative methodology for the deprotection of a Z-protected amine via catalytic hydrogenation is outlined below in General Procedure C.
General Procedure A: Peptide synthesis using DEPC. The N-protected or N,N-disubstituted amino acid or peptide 4 (1.0 eq.) and an amine 5 (1.1 eq.) are diluted with an aprotic organic solvent, such as dichloromethane (0.1 to 0.5 M). An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by DEPC (1.1 eq.). The resulting solution is stirred, preferably under argon, for up to 12 hours while being monitored by HPLC or TLC. The solvent is removed in vacuo at room temperature, and the crude product is purified using, for example, HPLC or flash column chromatography (silica gel column). Relevant fractions are combined and concentrated in vacuo to afford tripeptide 6 which is dried under vacuum overnight.
General procedure B: Peptide synthesis using PyBrop. The amino acid 2 (1.0 eq.), optionally having a carboxyl protecting group, is diluted with an aprotic organic solvent such as dichloromethane or DME to provide a solution of a concentration between 0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is added. Fmoc-, or Z-protected amino acid 1 (1.1 eq.) is added as a solid in one portion, then PyBrop (1.2 eq.) is added to the resulting mixture. The reaction is monitored by TLC or HPLC , followed by a workup procedure similar to that described in General Procedure A.
General procedure C: Z-removal via catalytic hydrogenation. Z-protected amino acid or peptide 3 is diluted with ethanol to provide a solution of a concentration between 0.5 and 1.0 mM in a suitable vessel, such as a thick-walled round bottom flask. 10% palladium on carbon is added (5-10% w/w) and the reaction mixture is placed under a hydrogen atmosphere. Reaction progress is monitored using HPLC and is generally complete within 1-2 h. The reaction mixture is filtered through a pre-washed pad of celite and the celite is again washed with a polar organic solvent, such as methanol after filtration. The eluent solution is concentrated in vacuo to afford a residue which is diluted with an organic solvent, preferably toluene. The organic solvent is then removed in vacuo to afford the deprotected amine 4.
Table 1 lists representative examples of tripeptide intermediates (compounds 39-43) that were prepared according to Scheme 5.
TABLE 1
Compound
X1
X2
39
Fmoc-N-Me-L-val
L-val
40
Fmoc-N-Me-L-val
L-ile
41
Fmoc-N-Me-gly
L-ile
42
dov
L-val
43
dov
L-ile
adov = N,N-dimethyl-L-valine
The dipeptide 9 can be readily prepared by condensation of the modified amino acid Boc-Dolaproine 7 (see, for example, Pettit, G. R., et al. Synthesis, 1996, 719-725), with (1S,2R)-norephedrine, L- or D-phenylalaminol, or with synthetic p-acetylphenethylamine 8 (U.S. Pat. No. 3,445,518 to Shavel et al.) using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in Scheme 6. Compound 7 may also be condensed with commercially available compounds in this manner to form dipeptides of formula 9. Examples of commercially available compounds useful for this purpose include, but are not limited to, norephedrine, ephedrine, and stereoisomers thereof (Sigma-Sigma-Aldrich), L- or D-phenylalaminol (Sigma-Aldrich), 2-phenylethylamine (Sigma-Aldrich), 2-(4-aminophenyl)ethylamine (Sigma-Aldrich), 1,2-ethanediamine-1,2-diphenyl (Sigma-Aldrich), or 4-(2-aminoethyl)phenol (Sigma-Aldrich), or with synthetically prepared p-acetylphenethylamine, aryl- and heterocyclo-amides of L-phenylalanine, 1-azidomethyl-2-phenylethylamine (prepared from phenylalaninol according to a general procedure described in J. Chem. Research (S), 1992, 391), and 1-(4-hydroxyphenyl)-2-phenylethylamine (European Patent Publication No. 0356035 A2) among others.
where R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl); R9 is selected from —H and —C1-C8 alkyl; and R10 is selected from:
where Z is —O—, —S—, —NH— or —N(R14)—; R11 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); or R11 is an oxygen atom which forms a carbonyl unit (C═O) with the carbon atom to which it is attached and a hydrogen atom on this carbon atom is replaced by one of the bonds in the (C═O) double bond; each R12 is independently selected from -aryl and —C3-C8 heterocycle; R13 is selected from —H, —OH, —NH2, —NHR14, —N(R14)2, —C1-C8 alkyl, —C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle); and each R14 is independently —H or —C1-C8 alkyl.
Table 2 lists representative examples of dipeptides (Compounds 44-48) that were prepared according to Scheme 6.
TABLE 2
Compound
Y
44
45
46
47
48
Scheme 7 illustrates a procedure useful for coupling tripeptide 6 and dipeptide 9 to form Drug 10. The coupling of 6 and 9 can be accomplished using a strong acid, e.g. TFA, to facilitate Boc and t-butyl ester cleavage, from dipeptide 9 and tripeptide 6, respectively, followed by condensation conditions, e.g., utilizing DEPC, or similar coupling reagent, in the presence of excess base (triethylamine or equivalent) to provide Drug 10.
An illustrative procedure for the synthesis of Drug 10 as depicted in Scheme 7 is outlined below in General Procedure D.
The R10 group of a Drug of general formula 10 can be further modified, if desired, to include a functional group that allows the drug to be attached to a Linker. Examples of useful modifications to the R10 group of a Drug 10, include, but are not limited to the chemical transformations described below.
When R10 is
the hydroxyl group of R10 can be reacted with commercially available or synthetically derived carboxylic acids or carboxylic acid derivatives, including but not limited to, carboxylic esters, acid chlorides, anhydrides and carbonates to provide the corresponding esters according to well known methods in the art. Coupling reagents, including, but not limited to DCC/DMAP and EDCI/HOBt, can be useful in such coupling reactions between alcohols and carboxylic acids or carboxylic acid derivatives. In a preferred embodiment carboxylic acids are substituted or unsubstituted aryl-carboxylic acids, for example, 4-aminobenzoic acid. Thus, condensation of a hydroxyl group of the R10 group shown above with carboxylic acids provides drugs of the general structure 10 where R10 is
and where R11, R12, R14 and R15 are as previously described herein and X is selected from —OH, —NH2 and —NHR14
When R10 is
the azido group of the drug can be reduced (for an example see J. Chem. Research (S), 1992, 391) to provide the corresponding amino derivative wherein R10 is
the amino group of which can be reacted with the carboxyl group of a carboxylic acid under general peptide coupling conditions to provide drugs of general structure 10, where R10 is
and where R11, R12, R14 and R15 are as previously described herein and X is selected from —OH, —NH2 and —NHR14Carboxylic acids useful in the above regard include, but are not limited to, 4-aminobenzoic acid, p-acetylbenzoic acid and 2-amino-4-thiazolecarboxylic acid (Tyger Scientific, Inc., Ewing, N.J.).
An Fmoc-protected amino group may be present on an amine-containing R10 group of Drug 10 (e.g., as depicted in Table 2). The Fmoc group is removable from the protected amine using diethylamine (see General Procedure E as an illustrative example described below).
General procedure D: Drug synthesis. A mixture of dipeptide 9 (1.0 eq.) and tripeptide 6 (1 eq.) is diluted with an aprotic organic solvent, such as dichloromethane, to form a 0.1M solution, then a strong acid, such as trifluoroacetic acid (½ v/v) is added and the resulting mixture is stirred under a nitrogen atmosphere for two hours at 0° C. The reaction can be monitored using TLC or, preferably, HPLC. The solvent is removed in vacuo and the resulting residue is azeotropically dried twice, preferably using toluene. The resulting residue is dried under high vacuum for 12 h and then diluted with and aprotic organic solvent, such as dichloromethane. An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by either PyBrop (1.2 eq.) or DEPC (1.2 eq.) depending on the chemical functionality on the residue. The reaction mixture is monitored by either TLC or HPLC and upon completion, the reaction is subjected to a workup procedure similar or identical to that described in General Procedure A.
General procedure E: Fmoc-removal using diethylamine. An Fmoc-protected Drug 10 is diluted with an aprotic organic solvent such as dichloromethane and to the resulting solution is added diethylamine (½ v/v). Reaction progress is monitored by TLC or HPLC and is typically complete within 2 h. The reaction mixture is concentrated in vacuo and the resulting residue is azeotropically dried, preferably using toluene, then dried under high vacuum to afford Drug 10 having a deprotected amino group.
Thus, the above methods are useful for making Drugs that can be used in the present invention.
To prepare a Drug-Linker Compound of the present invention, the Drug is reacted with a reactive site on the Linker. In general, the Linker can have the structure:
when both a Spacer unit (—Y—) and a Stretcher unit (-A-) are present. Alternately, the Linker can have the structure:
when the Spacer unit (—Y—) is absent.
The Linker can also have the structure:
when both the Stretcher unit (-A-) and the Spacer unit (—Y—) are absent.
In general, a suitable Linker has an Amino Acid unit linked to an optional Stretcher Unit and an optional Spacer Unit. Reactive Site 1 is present at the terminus of the Spacer and Reactive site 2 is present at the terminus of the Stretcher. If a Spacer unit is not present, then Reactive site 1 is present at the C-terminus of the Amino Acid unit.
In one embodiment of the invention, Reactive Site No. 1 is reactive to a nitrogen atom of the Drug, and Reactive Site No. 2 is reactive to a sulfhydryl group on the Ligand. Reactive Sites 1 and 2 can be reactive to different functional groups.
In one aspect of the invention, Reactive Site No. 1 is
In another aspect of the invention, Reactive Site No. 1 is
wherein R is —Br, —Cl, —O-Su or —O-(4-nitrophenyl).
In one embodiment, Reactive Site No. 1 is
wherein R is —Br, —Cl, —O-Su or —O-(4-nitrophenyl), when a Spacer unit (—Y—) is absent.
Linkers having
at Reactive Site No. 1 where R is —Br or —Cl can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —COOH group with PX3 or PX5, where X is —Br or —Cl. Alternatively, linkers having
at Reactive Site No. 1 can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —COOH group with thionyl chloride. For a general discussion of the conversion of carboxylic acids to acyl halides, see March, Advanced Organic Chemistry—Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 437-438.
In another aspect of the invention, Reactive Site No. 1 is
In still another aspect of the invention, Reactive Site No. 1 is
wherein R is —Cl, —O—CH(Cl)CCl3 or —O-(4-nitrophenyl).
Linkers having
at Reactive Site No. 1 can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OH group with phosgene or triphosgene to form the corresponding chloroformate. Linkers having
at Reactive Site No. 1 where R is —O—CH(Cl)CCl3 or —O-(4-nitrophenyl) can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OC(O)Cl group with HO—CH(Cl)CCl3 or HO-(4-nitrophenyl), respectively. For a discussion of this chemistry, see March, Advanced Organic Chemistry—Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 392.
In a further aspect of the invention, Reactive Site No. 1 is
wherein X is —F, —Cl, —Br, —I, or a leaving group such as —O-mesyl, —O-tosyl or —O-triflate.
Linkers having
at Reactive Site No. 1 where X is —O-mesyl, —O-tosyl and O-triflate can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OH group with various reagents, including HCl, SOCl2, PCl5, PCl3 and POCl3 (where X is Cl); HBr, PBr3, PBr5 and SOBr2 (where X is Br); HI (where X is I); and CH3CH2NSF3 (DAST), SF4, SeF4 and p-toluenesulfonyl fluoride (where X is F). For a general discussion on the conversion of alcohols to alkyl halides, see March, Advanced Organic Chemistry—Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 431-433.
Linkers having
at Reactive Site No. 1 where X is —O-mesyl, —O-tosyl and —O-triflate, can be prepared from Linkers having
at Reactive Site No. 1 by reacting the —OH group with various mesylating, tosylating and triflating reagents, respectively. Such reagents and methods for their use will be well known to one of ordinary skill in the art of organic synthesis. For a general discussion of mesyl, tosyl and triflates as leaving groups, see March, Advanced Organic Chemistry-Reactions, Mechanisms and Structure, 4th Ed., 1992, John Wiley and Sons, New York, p. 353-354.
In one embodiment, when a Spacer unit (—Y—) is present, Reactive Site No. 1 is
wherein R is —Cl, —O—CH(Cl)CCl3 or —O-(4-nitrophenyl) and X is —F, —Cl, —Br, —I, or a leaving group such as —O-mesyl, —O-tosyl or —O-triflate.
In another aspect of the invention, Reactive Site No. 1 is
In still another aspect of the invention, Reactive Site No. 1 is a p-nitrophenyl carbonate having the formula
In one aspect of the invention, Reactive Site No. 2 is a thiol-accepting group. Suitable thiol-accepting groups include haloacetamide groups having the formula
where X represents a leaving group, preferably O-mesyl, O-tosyl, —Cl, —Br, or —I; or a maleimide group having the formula
Useful Linkers can be obtained via commercial sources, such as Molecular Biosciences Inc. (Boulder, Colo.), or synthesized in accordance with procedures described in U.S. Pat. No. 6,214,345 to Firestone et al., summarized in Schemes 8-10 below.
where X is —CH2— or —CH2OCH2—; and n is an integer ranging either from 0-10 when X is —CH2—; or 1-10 when X is —CH2OCH2—.
The method shown in Scheme 9 combines maleimide with a glycol under Mitsunobu conditions to make a polyethylene glycol maleimide Stretcher (see for example, Walker, M. A. J. Org. Chem. 1995, 60, 5352-5), followed by installation of a p-nitrophenyl carbonate Reactive Site group.
where E is —CH2— or —CH2OCH2—; and e is an integer ranging from 0-8;
Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can be prepared as described by Frisch, et al., Bioconjugate Chem. 1996, 7, 180-186.
Scheme 10 illustrates a general synthesis of an illustrative Linker unit containing a maleimide Stretcher group and optionally a p-aminobenzyl ether self-immolative Spacer.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and n is an integer ranging from 0-10.
Useful Stretchers may be incorporated into a Linker using the commercially available intermediates from Molecular Biosciences (Boulder, Colo.) described below by utilizing known techniques of organic synthesis.
Stretchers of formula (IIIa) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit as depicted in Schemes 11 and 12:
where n is an integer ranging from 1-10 and T is —H or —SO3Na;
where n is an integer ranging from 0-3;
Stretcher units of formula (IIIb) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
where X is —Br or —I; and
Stretcher units of formula (IV) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
Stretcher units of formula (Va) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
Other Stretchers useful in the invention may be synthesized according to known procedures. Aminooxy Stretchers of the formula shown below can be prepared by treating alkyl halides with N-Boc-hydroxylamine according to procedures described in Jones, D. S. et al., Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon, C. et al., Tetrahedron, 1967, 23(11), 4441-4447.
where —R17— is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)n—, —(CH2CH2O)r—CH2—; and r is an integer ranging from 1-10;
Isothiocyanate Stretchers of the formula shown below may be prepared from isothiocyanatocarboxylic acid chlorides as described in Angew. Chem., 1975, 87(14), 517.
where —R17— is as described herein.
Scheme 11 shows a method for obtaining of a val-cit dipeptide Linker having a maleimide Stretcher and optionally a p-aminobenzyl self-immolative Spacer.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
Scheme 12 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linker unit having a maleimide Stretcher unit and a p-aminobenzyl self-immolative Spacer unit. Starting material 23 (lys(Mtr)) is commercially available (Bachem, Torrance, Calif.) or can be prepared according to Dubowchik, et al. Tetrahedrom Letters 1997, 38, 5257-60.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
As shown in Scheme 13, a Linker can be reacted with an amino group of a Drug 10 to form a Drug-Linker Compound that contains an amide or carbamate group, linking the Drug unit to the Linker unit. When Reactive Site No. 1 is a carboxylic acid group, as in Linker 29, the coupling reaction can be performed using HATU or PyBrop and an appropriate amine base, resulting in a Drug-Linker Compound 30, containing a amide bond between the Drug unit and the Linker unit. When Reactive Site No. 1 is a carbonate, as in Linker 31, the Linker can be coupled to the Drug using HOBt in a mixture of
DMF/pyridine to provide a Drug-Linker Compound 32, containing a carbamate bond between the Drug unit and the Linker unit.
When Reactive Site No. 1 is an hydroxyl group, such as Linker 33, the Linker can be coupled with a phenol group of a Drug using Mitsunobu chemistry to provide a Drug-Linker Compound 34 having an ether linkage between the Drug unit and the Linker unit.
Alternately, when Reactive Site No. 1 is a good leaving group, such as in Linker 70, the Linker can be coupled with a hydroxyl group or an amine group of a Drug via a nucleophilic substitution process to provide a Drug-Linker Compound having an ether linkage (34) or an amine linkage (71) between the Drug unit and the Linker unit.
Illustrative methods useful for linking a Drug to a Ligand to form a Drug-Linker Compound are depicted in Scheme 13 and are outlined in General Procedures G-J.
General Procedure G: Amide formation using HATU. A Drug 10 (1.0 eq.) and an N-protected Linker containing a carboxylic acid Reactive site (1.0 eq.) are diluted with a suitable organic solvent, such as dichloromethane, and the resulting solution is treated with HATU (1.5 eq.) and an organic base, preferably pyridine (1.5 eq.). The reaction mixture is allowed to stir under an inert atmosphere, preferably argon, for 6 h, during which time the reaction mixture is monitored using HPLC. The reaction mixture is concentrated and the resulting residue is purified using HPLC to yield the amide 30.
General Procedure H: Carbamate formation using HOBt. A mixture of a Linker 31 having a p-nitrophenyl carbonate Reactive site (1.1 eq.) and Drug 10 (1.0 eq.) are diluted with an aprotic organic solvent, such as DMF, to provide a solution having a concentration of 50-100 mM, and the resulting solution is treated with HOBt (2.0 eq.) and placed under an inert atmosphere, preferably argon. The reaction mixture is allowed to stir for 15 min, then an organic base, such as pyridine (¼ v/v), is added and the reaction progress is monitored using HPLC. The Linker is typically consumed within 16 h. The reaction mixture is then concentrated in vacuo and the resulting residue is purified using, for example, HPLC to yield the carbamate 32.
General Procedure I: Ether formation using Mitsunobu chemistry. A Drug of general formula 10, which contains a free hydroxyl group, is diluted with THF to make a 1.0 M solution and to this solution is added a Linker (1.0 eq) containing an hydroxy group at Reactive site No. 1 (33), followed by triphenylphosphine (1.5 eq.). The reaction mixture is put under an argon atmosphere and cooled to 0° C. DEAD (1.5 eq.) is then added dropwise via syringe and the reaction is allowed to stir at room temperature while being monitored using HPLC. The reaction is typically complete in 0.5-12 h, depending on the substrates. The reaction mixture is diluted with water (in volume equal to that of the THF) and the reaction mixture is extracted into EtOAc. The EtOAc layer is washed sequentially with water and brine, then dried over MgSO4 and concentrated. The resulting residue is purified via flash column chromatography using a suitable eluent to provide ether 34.
General Procedure J: Ether/amine Formation via Nucleophilic Substitution. A Drug of general formula 10, which contains a free hydroxyl group or a free amine group, is diluted with a polar aprotic solvent, such as THF, DMF or DMSO, to make a 1.0 M solution and to this solution is added a non-nucleophilic base (about 1.5 eq), such as pyridine, diisopropylethylamine or triethylamine. The reaction mixture is allowed to stir for about 1 hour, and to the resulting solution is added an approximately 1.0M solution of Linker 70 in a polar aprotic solvent, such as THF, DMF or DMSO. The resulting reaction is stirred under an inert atmosphere while being monitored using TLC or HPLC. The reaction is typically complete in 0.5-12 h, depending on the substrates. The reaction mixture is diluted with water (in volume equal to that of the reaction volume) and extracted into EtOAc. The EtOAc layer is washed sequentially with water, 1N HCl, water, and brine, then dried over MgSO4 and concentrated. The resulting residue is purified via flash column chromatography using a suitable eluent to provide an ether of formula 34 or an amine of formula 71, depending on whether the drug 10 contained a free hydroxyl group or a free amine group.
An alternate method of preparing Drug-Linker Compounds of the invention is outlined in Scheme 14. Using the method of Scheme 14, the Drug is attached to a partial Linker unit (19a, for example), which does not have a Stretcher unit attached. This provides intermediate 35, which has an Amino Acid unit having an Fmoc-protected N-terminus. The Fmoc group is then removed and the resulting amine intermediate 36 is then attached to a Stretcher unit via a coupling reaction catalyzed using PyBrop or DEPC. The construction of Drug-Linker Compounds containing either a bromoacetamide Stretcher 39 or a PEG maleimide Stretcher 38 is illustrated in Scheme 14.
where Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
Methodology useful for the preparation of a Linker unit containing a branched spacer is shown in Scheme 15.
Scheme 15 illustrates the synthesis of a val-cit dipeptide linker having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS) unit. The synthesis of the BHMS intermediate (75) has been improved from previous literature procedures (see International Publication No, WO 9813059 to Firestone et al., and Crozet, M. P.; Archaimbault, G.; Vanelle, P.; Nouguier, R. Tetrahedron Lett. 1985, 26, 5133-5134) and utilizes as starting materials, commercially available diethyl (4-nitrobenzyl)phosphonate (72) and commercially available 2,2-dimethyl-1,3-dioxan-5-one (73). Linkers 77 and 79 can be prepared from intermediate 75 using the methodology described in Scheme 11.
Scheme 16 illustrates methodology useful for making Drug-Linker-Ligand conjugates of the invention having about 2 to about 4 drugs per antibody.
General Procedure K: Preparation of Conjugates Having about 2 to about 4 Drugs Per Antibody.
Partial Reduction of the Antibody
In general, to prepare conjugates having 2 drugs per antibody, the relevant antibody is reduced using a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) (about 1.8 equivalents) in PBS with 1 mM DTPA, adjusted to pH 8 with 50 mM borate. The solution is incubated at 37° C. for 1 hour, purified using a 50 ml G25 desalting column equilibrated in PBS/1 mM DTPA at 4° C. The thiol concentration can be determined according to General Procedure M, the protein concentration can be determined by dividing the A280 value by 1.58 extinction coefficient (mg/ml), and the ratio of thiol to antibody can be determined according to General Procedure N.
Conjugates having 4 drugs per antibody can be made using the same methodology, using about 4.2 equivalents of a suitable reducing agent to partially reduce the antibody.
Conjugation of Drug-Linker to Partially Reduced Antibody
The partially reduced antibody samples can be conjugated to a corresponding Drug-Linker compound using about 2.4 and about 4.6 molar equivalents of Drug-Linker compound per antibody to prepare the 2 and 4 drug per antibody conjugates, respectively. The conjugation reactions are incubated on ice for 1 hour, quenched with about 20-fold excess of cysteine to drug, and purified by elution over a G25 desalting column at about 4° C. The resulting Drug-Linker-Ligand conjugates are concentrated to about 3 mg/ml, sterile filtered, aliquoted and stored frozen.
Scheme 17 depicts the construction of a Drug-Linker-Ligand Conjugate by reacting the sulfhydryl group of a Ligand with a thiol-acceptor group on the Linker group of a Drug-Linker Compound.
Illustrative methods for attaching a Ligand antibody to a Drug-Linker Compound are outlined below in General Procedures L-R.
General Procedure L: Attachment of an Antibody Ligand to a Drug-Linker Compound. All reaction steps are typically carried out at 4° C. Where the Ligand is a monoclonal antibody having one or more disulfide bonds, solutions of the monoclonal antibody (5-20 mg/mL) in phosphate buffered saline, pH 7.2, are reduced with dithiothreitol (10 mM final) at 37° C. for 30 minutes (See General Procedure M) and separation of low molecular weight agents is achieved by size exclusion chromatography on Sephadex G25 columns in PBS containing 1 mM diethylenetriaminepentaacetic acid.
The sulfhydryl content in the Ligand can be determined using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as described in General Procedure M (see Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Anal. Biochem. 94, 75-81). To a PBS solution of Ligand reduced according to General Procedure L, a Drug-Linker Compound in MeCN is added so that the solution is 20% MeCN/PBS (vol/vol). The amount of Drug-Linker Compound is approximately 10% more than the total number of sulfhydryl groups on a Ligand. After 60 min at 4° C., cysteine is added (20-fold excess over concentration of the Drug-Linker Compound), the solution is concentrated by ultrafiltration, and any low molecular weight agents are removed by gel filtration. The number of Drug-Linker Compounds per antibody is determined by uv/vis spectroscopy using formulas derived from the relative extinction coefficients of the Ligands and Drug-Linker Compounds as described in General Procedure O. The amount of quenched Drug-Linker Compound is then determined as described in General Procedure P using reverse-phase HPLC. The aggregation state of the Ligand Antibodies of the Drug-Linker-Ligand Conjugates can be determined using size-exclusion HPLC as described in General Procedure R. The Drug-Linker-Ligand Conjugates can be used without further purification.
General Procedure M: Reduction of the interchain disulfide bonds of an Antibody. To a solution of 24 mg of an antibody (2.4 mL of 10 mg/mL solution) in suitable buffer is added 300 μL of Borate buffer (500 mM sodium borate/500 mM sodium chloride, pH 8.0) followed by 300 μL of Dithiothreitol (DTT, 100 mM solution in H2O). The reaction mixture is stirred using a vortex instrument and incubated at 37° C. for 30 min. Three PD10 columns are equilibrated with PBS containing 1 mM DTPA (in PBS) and the reduced antibody is eluted through the three PD10 columns and collected in 4.2 mL PBS/DTPA solution (1.4 mL per column). The reduced antibody is then stored on ice. The number of thiols per antibody and the antibody concentration are determined according to General Procedure N.
General Procedure N: Determination of number of thiols per Ligand.
A reference sample of a Ligand or a sample of an antibody reduced according to General Procedure L is diluted to about 1:40 (w/w) in PBS, and the uv absorbance of the solution is measured at 280 nm using standard uv spectroscopic methods.
Preferably, the ratio of Ligand:PBS in the solution is such that the uv absorbance ranges from about 0.13-0.2 AU (absorbance units).
A test sample of a Ligand or a test sample of an antibody reduced according to General Procedure L is diluted to about 1:20 with a PBS solution containing about 15 μL DTNB stock solution/mL PBS. A blank sample containing DTNB at the same concentration as the test solution (i.e., 15 μL DTNB stock/mL PBS) is then prepared. The spectrophotometer is referenced at zero nm with the blank sample, then the absorbance of the test sample is measured at 412 nm.
The molar concentration of the antibody is then determined using the formula: [Ligand]=(OD280/2.24e5)×dilution factor.
The molar concentration of thiol is then determined using the formula: [—SH]=(OD412/1.415e4)×dilution factor.
The [SH]/[Ligand] ratio is then calculated. A reduced monoclonal antibody Ligand can have from 1 to about 20 sulfhydryl groups, but typically has between about 6 to about 9 sulfhydryl groups. In a preferred embodiment, the [SH]/[Ligand] ratio range is from about 7 to about 9.
It is understood that the [SH]/[Ligand] ratio is the average number of -Aa-Ww—Yy-D units per Ligand unit.
General Procedure O: Determination of the number of Drug molecules per Antibody in a Drug-Linker-Antibody Conjugate. The Drug:Antibody ratio for a Drug-Linker-Antibody Conjugate is determined by measuring the number of Dithiothreitol (DTT) reducible thiols that remain after conjugation, using the following method: A 200 mL sample of a Drug-Linker-Antibody conjugate is treated with DTT (100 mM solution in water) to bring the concentration to 10 mM DTT. The resulting solution is incubated at 37° C. for 30 min, then eluted through a PD10 column using PBS/DTPA as the eluent. The OD280 of the reduced conjugate is then measured and the molar concentration is measured according to General Procedure Q.
The molar concentration of thiol is determined using DTNB as described in General Procedure M. The ratio of thiol concentration to antibody concentration is then calculated and the Drug:Ligand ratio is the difference between the Thiol:Antibody ratio (determined using General Procedure N) and the Drug:Antibody ratio as determined in the previous paragraph.
General Procedure P: Determination of the amount of quenched Drug-Linker compound in a Drug-Linker-Antibody Conjugate. This assay provides a quantitative determination of the Drug-Linker in the Drug-Linker-Antibody conjugate that is not covalently bound to Antibody. Assuming that all maleimide groups of Drug-Linker in the reaction mixture have been quenched with Cysteine, the unbound drug is the Cysteine quenched adduct of the Drug-Linker Compound, i.e. Drug-Linker-Cys. The proteinaceous Drug-Linker-Antibody Conjugate is denatured, precipitated, and isolated by centrifugation under conditions in which the Drug-Linker-Cys is soluble. The unbound Drug-Linker-Cys is detected quantitatively by HPLC, and the resulting chromatogram is compared to a standard curve to determine the concentration of unbound Drug-Linker-Cys in the sample. This concentration is divided by the total concentration of Drug in the conjugate as determined using General Procedure O and General Procedure Q.
Specifically, 100 mL of a 100 μM Drug-Linker-Cys adduct “working solution” is prepared by adding 1 μL of 100 mM Cysteine in PBS/DTPA and an appropriate volume of stock solution of a Drug-Linker compound to 98 μL of 50% methanol/PBS. The “appropriate volume” in liters is calculated using the formula: V=1e-8/[Drug-Linker]. Six tubes are then labelled as follows: “0”, “0.5”, “1”, “2”, “3”, and “5”, and appropriate amounts of working solution are placed in each tube and diluted with 50% methanol/PBS to give a total volume of 100 mL in each tube. The labels indicate the μM concentration of the standards.
A 50 μL solution of a Drug-Linker-Antibody Conjugate and a 50 μL solution of the Cysteine quenched reaction mixture (“qrm”) are collected in separate test tubes and are each diluted with 50 μL of methanol that has been cooled to −20° C. The samples are then cooled to −20° C. over 10 min.
The samples are then centrifuged at 13000 rpm in a desktop centrifuge for 10 min. The supernatants are transferred to HPLC vials, and 90 μL aliquots of each sample are separately analyzed using HPLC(C12 RP column (Phenomenex); monitored at the absorbance maximum of the Drug-Linker Compound using a flow rate of 1.0 mL/min. The eluent used is a linear gradient of MeCN ranging from 10 to 90% in aqueous 5 mM ammonium phosphate, pH 7.4, over 10 min; then 90% MeCN over 5 min.; then returning to initial conditions). The Drug-Linker-Cys adduct typically elutes between about 7 and about 10 minutes.
A standard curve is then prepared by plotting the Peak Area of the standards vs. their concentration (in μM). Linear regression analysis is performed to determine the equation and correlation coefficient of the standard curve. R2 values are typically >0.99. From the regression equation is determined the concentration of the Drug-Linker-Cys adduct in the HPLC sample and in the conjugate, using the formulas:
[Drug-Linker-Cys](HPLC spl)=(Peak area−intercept)/slope;
[Drug-Linker-Cys](conjugate)=2×[Drug-Linker-Cys](HPLC spl)
The percent of Drug-Linker-Cys adduct present can be determined using the formula:
% Drug-Linker-Cys=100×[Drug-Linker-Cys](conjugate)/[drug]
where [drug]=[Conjugate]×drug/Ab, [Conjugate] is determined using the conjugate concentration assay, and the Drug: Antibody ratio is determined using the Drug: Antibody ratio assay.
General Procedure Q: Determination of Drug-Linker-Antibody Conjugate concentration for drug linkers with minimal uv absorbance at 280 nm. The concentration of Drug-Linker-Antibody conjugate can be determined in the same manner for the concentration of the parent antibody, by measuring the absorbance at 280 nm of an appropriate dilution, using the following formula:
[Conjugate](mg/mL)=(OD280×dilution factor/1.4)×0.9
Determination of Drug-Linker-Antibody Conjugate concentration for drug linkers with substantial uv absorbance at 280 nm (e.g. Compounds 68 and 69). Because the absorbances of Compounds 68 and 69 overlap with the absorbances of an antibody, spectrophotometric determination of the conjugate concentration is most useful when the measurement is performed using the absorbances at both 270 nm and 280 nm. Using this data, the molar concentration of Drug-Linker-Ligand conjugate is given by the following formula:
[Conjugate]=(OD280×1.23e−5−OD270×9.35e−6)×dilution factor
where the values 1.23e−5 and 9.35e−6 are calculated from the molar extinction coefficients of the drug and the antibody, which are estimated as:
e270 Drug=2.06e4 e270 Antibody=1.87e5
e280 Drug=1.57e4 e280 Antibody=2.24e5
General Procedure R: Determination of the aggregation state of The Antibody in a Drug-Linker-Antibody Conjugate. A suitable quantity (˜10 μg) of a Drug-Linker-Antibody Conjugate is eluted through a size-exclusion chromatography (SEC) column (Tosoh Biosep SW3000 4.6 mm×30 cm eluted at 0.35 mL/min. with PBS) under standard conditions. Chromatograms are obtained at 220 nm and 280 nm and the OD280/OD220 ratio is calculated. The corresponding aggregate typically has a retention time of between about 5.5 and about 7 min, and has about the same OD280/OD220 ratio as the monomeric Drug-Linker-Antibody Conjugate.
5.8 Compositions
In other aspects, the present invention provides a composition comprising an effective amount of a Compound of the Invention and a pharmaceutically acceptable carrier or vehicle. For convenience, the Drug units, Drug-Linker Compounds and Drug-Linker-Ligand Conjugates of the invention can simply be referred to as compounds of the invention. The compositions are suitable for veterinary or human administration.
The compositions of the present invention can be in any form that allows for the composition to be administered to an animal. For example, the composition can be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Preferably, the compositions are administered parenterally. Pharmaceutical compositions of the invention can be formulated so as to allow a Compound of the Invention to be bioavailable upon administration of the composition to an animal. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of a Compound of the Invention in aerosol form can hold a plurality of dosage units.
Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the Compound of the Invention, the manner of administration, and the composition employed.
The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) can be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.
When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
When the composition is in the form of a capsule, e.g., a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
The composition can be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
The liquid compositions of the invention, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material. Physiological saline is a preferred adjuvant. An injectable composition is preferably sterile.
The amount of the Compound of the Invention that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
The compositions comprise an effective amount of a Compound of the Invention such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a Compound of the Invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the Compound of the Invention by weight of the composition. Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the Compound of the Invention.
For intravenous administration, the composition can comprise from about 1 to about 250 mg of a Compound of the Invention per kg of the animal's body weight. Preferably, the amount administered will be in the range from about 4 to about 25 mg/kg of body weight of the Compound of the Invention.
Generally, the dosage of Compound of the Invention administered to an animal is typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight. Preferably, the dosage administered to an animal is between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.
The Compounds of the Invention or compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a Compound of the Invention or composition. In certain embodiments, more than one Compound of the Invention or composition is administered to an animal. Methods of administration include, but are not limited to, oral administration and parenteral administration; parenteral administration including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition (such as the site of cancer or autoimmune disease).
In a preferred embodiment, the present Compounds of the Invention or compositions are administered parenterally.
In a more preferred embodiment, the present Compounds of the Invention or compositions are administered intravenously.
In specific embodiments, it can be desirable to administer one or more Compounds of the Invention or compositions locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue.
In another embodiment, administration can be by direct injection at the site (or former site) of a manifestation of an autoimmune disease.
In certain embodiments, it can be desirable to introduce one or more Compounds of the Invention or compositions into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the Compounds of the Invention or compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
In another embodiment, the Compounds of the invention can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the Compounds of the Invention or compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the Compounds of the Invention or compositions, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can be used.
The term “carrier” refers to a diluent, adjuvant or excipient, with which a Compound of the Invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the Compounds of the Invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the Compounds of the Invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
In a preferred embodiment, the Compounds of the Invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly human beings. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where a Compound of the Invention is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the Compound of the Invention is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade.
The compositions can be intended for topical administration, in which case the carrier may be in the form of a solution, emulsion, ointment or gel base. The base, for example, can comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents can be present in a composition for topical administration. If intended for transdermal administration, the composition can be in the form of a transdermal patch or an iontophoresis device. Topical formulations can comprise a concentration of a Compound of the Invention of from about 0.1% to about 10% w/v (weight per unit volume of composition).
The composition can be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the Compound of the Invention. The composition for rectal administration can contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The composition can include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition can include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.
The compositions can consist of gaseous dosage units, e.g., it can be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery can be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of Compounds of the Invention can be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the Compound(s) of the Invention. Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, Spacers and the like, which together can form a kit. Preferred aerosols can be determined by one skilled in the art, without undue experimentation.
Whether in solid, liquid or gaseous form, the compositions of the present invention can comprise a pharmacological agent used in the treatment of cancer, an autoimmune disease or an infectious disease.
The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a Compound of the Invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a Compound of the Invention so as to facilitate dissolution or homogeneous suspension of the active compound in the aqueous delivery system.
5.9 Therapeutic Uses of the Compounds of the Invention
The Compounds of the Invention are useful for treating cancer, an autoimmune disease or an infectious disease in an animal.
5.10 Treatment of Cancer
The Compounds of the Invention are useful for inhibiting the multiplication of a tumor cell or cancer cell, or for treating cancer in an animal. The Compounds of the Invention can be used accordingly in a variety of settings for the treatment of animal cancers. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug or Drug unit to a tumor cell or cancer cell. Without being bound by theory, in one embodiment, the Ligand unit of a Compound of the Invention binds to or associates with a cancer-cell or a tumor-cell-associated antigen, and the Compound of the Invention can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell. Once inside the cell, one or more specific peptide sequences within the Linker unit are hydrolytically cleaved by one or more tumor-cell or cancer-cell-associated proteases, resulting in release of a Drug or a Drug-Linker Compound. The released Drug or Drug-Linker Compound is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the Drug or Drug unit is cleaved from the Compound of the Invention outside the tumor cell or cancer cell, and the Drug or Drug-Linker Compound subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to the tumor cell or cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is on the surface of the tumor cell or cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is an extracellular matrix protein associated with the tumor cell or cancer cell.
In one embodiment, the tumor cell or cancer cell is of the type of tumor or cancer that the animal needs treatment or prevention of.
The specificity of the Ligand unit for a particular tumor cell or cancer cell can be important for determining those tumors or cancers that are most effectively treated. For example, Compounds of the Invention having a BR96 Ligand unit can be useful for treating antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas. Compounds of the Invention having an Anti-CD30 or an anti-CD40 Ligand unit can be useful for treating hematologic malignancies.
Other particular types of cancers that can be treated with Compounds of the Invention include, but are not limited to, those disclosed in Table 3.
TABLE 3
Solid tumors, including but not limited to:
fibrosarcoma
myxosarcoma
liposarcoma
chondrosarcoma
osteogenic sarcoma
chordoma
angiosarcoma
endotheliosarcoma
lymphangiosarcoma
lymphangioendotheliosarcoma
synovioma
mesothelioma
Ewing's tumor
leiomyosarcoma
rhabdomyosarcoma
colon cancer
colorectal cancer
kidney cancer
pancreatic cancer
bone cancer
breast cancer
ovarian cancer
prostate cancer
esophogeal cancer
stomach cancer
oral cancer
nasal cancer
throat cancer
squamous cell carcinoma
basal cell carcinoma
adenocarcinoma
sweat gland carcinoma
sebaceous gland carcinoma
papillary carcinoma
papillary adenocarcinomas
cystadenocarcinoma
medullary carcinoma
bronchogenic carcinoma
renal cell carcinoma
hepatoma
bile duct carcinoma
choriocarcinoma
seminoma
embryonal carcinoma
Wilms' tumor
cervical cancer
uterine cancer
testicular cancer
small cell lung carcinoma
bladder carcinoma
lung cancer
epithelial carcinoma
glioma
glioblastoma multiforme
astrocytoma
medulloblastoma
craniopharyngioma
ependymoma
pinealoma
hemangioblastoma
acoustic neuroma
oligodendroglioma
meningioma
skin cancer
melanoma
neuroblastoma
retinoblastoma
blood-borne cancers, including but not limited to:
acute lymphoblastic leukemia “ALL”
acute lymphoblastic B-cell leukemia
acute lymphoblastic T-cell leukemia
acute myeloblastic leukemia “AML”
acute promyelocytic leukemia “APL”
acute monoblastic leukemia
acute erythroleukemic leukemia
acute megakaryoblastic leukemia
acute myelomonocytic leukemia
acute nonlymphocyctic leukemia
acute undifferentiated leukemia
chronic myelocytic leukemia “CML”
chronic lymphocytic leukemia “CLL”
hairy cell leukemia
multiple myeloma
acute and chronic leukemias:
lymphoblastic
myelogenous
lymphocytic
myelocytic leukemias
Lymphomas:
Hodgkin's disease
non-Hodgkin's Lymphoma
Multiple myeloma
Waldenström's macroglobulinemia
Heavy chain disease
Polycythemia vera
The Compounds of the Invention can also be used as chemotherapeutics in the untargeted form. For example, the Drugs themselves, or the Drug-Linker Compounds are useful for treating ovarian, CNS, renal, lung, colon, melanoma, or hematologic cancers or tumors.
The Compounds of the Invention provide Conjugation specific tumor or cancer targeting, thus reducing general toxicity of these compounds. The Linker units stabilize the Compounds of the Invention in blood, yet are cleavable by tumor-specific proteases within the cell, liberating a Drug.
5.10.1 Multi-Modality Therapy for Cancer
Cancer, including, but not limited to, a tumor, metastasis, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of a Compound of the Invention.
In other embodiments, the invention provides methods for treating or preventing cancer, comprising administering to an animal in need thereof an effective amount of a Compound of the Invention and a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory. The Compounds of the Invention can be administered to an animal that has also undergone surgery as treatment for the cancer.
In one embodiment, the additional method of treatment is radiation therapy.
In a specific embodiment, the Compound of the Invention is administered concurrently with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of a Compound of the Invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e.g., up to three months), prior or subsequent to administration of a Compound of the Invention.
A chemotherapeutic agent can be administered over a series of sessions, any one or a combination of the chemotherapeutic agents listed in Table 4 can be administered. With respect to radiation, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered.
Additionally, the invention provides methods of treatment of cancer with a Compound of the Invention as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The animal being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
The Compounds of the Invention can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants. This can involve a multi-step process in which the animal's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the patient's remaining bone-marrow cell population is then eradicated via the administration of a high dose of a Compound of the Invention with or without accompanying high dose radiation therapy, and the stem cell graft is infused back into the animal. Supportive care is then provided while bone marrow function is restored and the animal recovers.
5.10.2 Multi-Drug Therapy for Cancer
The present invention includes methods for treating cancer, comprising administering to an animal in need thereof an effective amount of a Compound of the Invention and another therapeutic agent that is an anti-cancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred embodiment, the anti-cancer agent includes, but is not limited to, a drug listed in Table 4.
TABLE 4
Alkylating agents
Nitrogen mustards:
cyclophosphamide
Ifosfamide
trofosfamide
Chlorambucil
Nitrosoureas:
carmustine (BCNU)
Lomustine (CCNU)
Alkylsulphonates
busulfan
Treosulfan
Triazenes:
Dacarbazine
Platinum containing compounds:
Cisplatin
carboplatin
Plant Alkaloids
Vinca alkaloids:
vincristine
Vinblastine
Vindesine
Vinorelbine
Taxoids:
paclitaxel
Docetaxol
DNA Topoisomerase Inhibitors
Epipodophyllins:
etoposide
Teniposide
Topotecan
9-aminocamptothecin
camptothecin
crisnatol
mitomycins:
Mitomycin C
Anti-metabolites
Anti-folates:
DHFR inhibitors:
methotrexate
Trimetrexate
IMP dehydrogenase Inhibitors:
mycophenolic acid
Tiazofurin
Ribavirin
EICAR
Ribonuclotide reductase Inhibitors:
hydroxyurea
deferoxamine
Pyrimidine analogs:
Uracil analogs
5-Fluorouracil
Floxuridine
Doxifluridine
Ratitrexed
Cytosine analogs
cytarabine (ara C)
Cytosine arabinoside
fludarabine
Purine analogs:
mercaptopurine
Thioguanine
Hormonal therapies:
Receptor antagonists:
Anti-estrogen
Tamoxifen
Raloxifene
megestrol
LHRH agonists:
goscrclin
Leuprolide acetate
Anti-androgens:
flutamide
bicalutamide
Retinoids/Deltoids
Vitamin D3 analogs:
EB 1089
CB 1093
KH 1060
Photodynamic therapies:
vertoporfin (BPD-MA)
Phthalocyanine
photosensitizer Pc4
Demethoxy-hypocrellin A
(2BA-2-DMHA)
Cytokines:
Interferon-α
Interferon-γ
Tumor necrosis factor
Others:
Isoprenylation inhibitors:
Lovastatin
Dopaminergic neurotoxins:
1-methyl-4-phenylpyridinium ion
Cell cycle inhibitors:
staurosporine
Actinomycins:
Actinomycin D
Dactinomycin
Bleomycins:
bleomycin A2
Bleomycin B2
Peplomycin
Anthracyclines:
daunorubicin
Doxorubicin (adriamycin)
Idarubicin
Epirubicin
Pirarubicin
Zorubicin
Mitoxantrone
MDR inhibitors:
verapamil
Ca2+ ATPase inhibitors:
thapsigargin
5.11 Treatment of Autoimmune Diseases
The Compounds of the Invention are useful for killing or inhibiting the replication of a cell that produces an autoimmune disease or for treating an autoimmune disease. The Compounds of the Invention can be used accordingly in a variety of settings for the treatment of an autoimmune disease in an animal. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. Without being bound by theory, in one embodiment, the Drug-Linker-Ligand Conjugate associates with an antigen on the surface of a target cell, and the Compound of the Invention is then taken up inside a target-cell through receptor-mediated endocytosis. Once inside the cell, one or more specific peptide sequences within the Linker unit are enzymatically or hydrolytically cleaved, resulting in release of a Drug. The released Drug is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the Drug is cleaved from the Compound of the Invention outside the target cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to an autoimmune antigen.
In another embodiment, the Ligand unit binds to an autoimmune antigen which is on the surface of a cell.
In another embodiment, the target cell is of the type of cell that produces the autoimmune antigen which causes the disease the animal needs treatment or prevention of.
In a preferred embodiment, the Ligand binds to activated lympocytes that are associated with the autoimmune disease state.
In a further embodiment, the Compounds of the Invention kill or inhibit the multiplication of cells that produce an auto-immune antibody associated with a particular autoimmune disease.
Particular types of autoimmune diseases that can be treated with the Compounds of the Invention include, but are not limited to, Th2-lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft versus host disease); Th1 lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); activated B lymphocyte-related disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes); and those disclosed in Table 5.
TABLE 5
Active Chronic Hepatitis
Addison's Disease
Allergic Alveolitis
Allergic Reaction
Allergic Rhinitis
Alport's Syndrome
Anaphlaxis
Ankylosing Spondylitis
Anti-phosholipid Syndrome
Arthritis
Ascariasis
Aspergillosis
Atopic Allergy
Atropic Dermatitis
Atropic Rhinitis
Behcet's Disease
Bird-Fancier's Lung
Bronchial Asthma
Caplan's Syndrome
Cardiomyopathy
Celiac Disease
Chagas' Disease
Chronic Glomerulonephritis
Cogan's Syndrome
Cold Agglutinin Disease
Congenital Rubella Infection
CREST Syndrome
Crohn's Disease
Cryoglobulinemia
Cushing's Syndrome
Dermatomyositis
Discoid Lupus
Dressler's Syndrome
Eaton-Lambert Syndrome
Echovirus Infection
Encephalomyelitis
Endocrine opthalmopathy
Epstein-Barr Virus Infection
Equine Heaves
Erythematosis
Evan's Syndrome
Felty's Syndrome
Fibromyalgia
Fuch's Cyclitis
Gastric Atrophy
Gastrointestinal Allergy
Giant Cell Arteritis
Glomerulonephritis
Goodpasture's Syndrome
Graft v. Host Disease
Graves' Disease
Guillain-Barre Disease
Hashimoto's Thyroiditis
Hemolytic Anemia
Henoch-Schonlein Purpura
Idiopathic Adrenal Atrophy
Idiopathic Pulmonary Fibritis
IgA Nephropathy
Inflammatory Bowel Diseases
Insulin-dependent Diabetes Mellitus
Juvenile Arthritis
Juvenile Diabetes Mellitus (Type I)
Lambert-Eaton Syndrome
Laminitis
Lichen Planus
Lupoid Hepatitis
Lupus
Lymphopenia
Meniere's Disease
Mixed Connective Tissue Disease
Multiple Sclerosis
Myasthenia Gravis
Pernicious Anemia
Polyglandular Syndromes
Presenile Dementia
Primary Agammaglobulinemia
Primary Biliary Cirrhosis
Psoriasis
Psoriatic Arthritis
Raynauds Phenomenon
Recurrent Abortion
Reiter's Syndrome
Rheumatic Fever
Rheumatoid Arthritis
Sampter's Syndrome
Schistosomiasis
Schmidt's Syndrome
Scleroderma
Shulman's Syndrome
Sjorgen's Syndrome
Stiff-Man Syndrome
Sympathetic Ophthalmia
Systemic Lupus Erythematosis
Takayasu's Arteritis
Temporal Arteritis
Thyroiditis
Thrombocytopenia
Thyrotoxicosis
Toxic Epidermal Necrolysis
Type B Insulin Resistance
Type I Diabetes Mellitus
Ulcerative Colitis
Uveitis
Vitiligo
Waldenstrom's Macroglobulemia
Wegener's Granulomatosis
5.11.1 Multi-Drug Therapy of Autoimmune Diseases
The present invention also provides methods for treating an autoimmune disease, comprising administering to an animal in need thereof an effective amount of a Compound of the Invention and another therapeutic agent that known for the treatment of an autoimmune disease. In one embodiment, the anti-autoimmune disease agent includes, but is not limited to, agents listed in Table 6.
TABLE 6
cyclosporine
cyclosporine A
mycophenylate mofetil
sirolimus
tacrolimus
enanercept
prednisone
azathioprine
methotrexate cyclophosphamide
prednisone
aminocaproic acid
chloroquine
hydroxychloroquine
hydrocortisone
dexamethasone
chlorambucil
DHEA
danazol
bromocriptine
meloxicam
infliximab
5.12 Treatment of Infectious Diseases
The Compounds of the Invention are useful for killing or inhibiting the multiplication of a cell that produces an infectious disease or for treating an infectious disease. The Compounds of the Invention can be used accordingly in a variety of settings for the treatment of an infectious disease in an animal. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. Without being bound by theory, in one embodiment, the Drug-Linker-Ligand Conjugate associates with an antigen on the surface of a target cell, and the Compound of the Invention is then taken up inside a target-cell through receptor-mediated endocytosis. Once inside the cell, one or more specific peptide sequences within the Linker unit are enzymatically or hydrolytically cleaved, resulting in release of a Drug. The released Drug is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the Drug is cleaved from the Compound of the Invention outside the target cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to the infectious disease cell.
In one embodiment, the infectious disease type of infectious disease that the animal needs treatment or prevention of.
In one embodiment, the Compounds of the Invention kill or inhibit the multiplication of cells that produce a particular infectious disease.
Particular types of infectious diseases that can be treated with the Compounds of the Invention include, but are not limited to, those disclosed in Table 7.
TABLE 7
Bacterial Diseases:
Diptheria
Pertussis
Occult Bacteremia
Urinary Tract Infection
Gastroenteritis
Cellulitis
Epiglottitis
Tracheitis
Adenoid Hypertrophy
Retropharyngeal Abcess
Impetigo
Ecthyma
Pneumonia
Endocarditis
Septic Arthritis
Pneumococcal
Peritonitis
Bactermia
Meningitis
Acute Purulent Meningitis
Urethritis
Cervicitis
Proctitis
Pharyngitis
Salpingitis
Epididymitis
Gonorrhea
Syphilis
Listeriosis
Anthrax
Nocardiosis
Salmonella
Typhoid Fever
Dysentery
Conjuntivitis
Sinusitis
Brucellosis
Tullaremia
Cholera
Bubonic Plague
Tetanus
Necrotizing Enteritis
Actinomycosis
Mixed Anaerobic Infections
Syphilis
Relapsing Fever
Leptospirosis
Lyme Disease
Rat Bite Fever
Tuberculosis
Lymphadenitis
Leprosy
Chlamydia
Chlamydial Pneumonia
Trachoma
Inclusion Conjunctivitis
Systemic Fungal Diseases:
Histoplamosis
Coccicidiodomycosis
Blastomycosis
Sporotrichosis
Cryptococcsis
Systemic Candidiasis
Aspergillosis
Mucormycosis
Mycetoma
Chromomycosis
Rickettsial Diseases:
Typhus
Rocky Mountain Spotted Fever
Ehrlichiosis
Eastern Tick-Borne Rickettsioses
Rickettsialpox
Q Fever
Bartonellosis
Parasitic Diseases:
Malaria
Babesiosis
African Sleeping Sickness
Chagas' Disease
Leishmaniasis
Dum-Dum Fever
Toxoplasmosis
Meningoencephalitis
Keratitis
Entamebiasis
Giardiasis
Cryptosporidiasis
Isosporiasis
Cyclosporiasis
Microsporidiosis
Ascariasis
Whipworm Infection
Hookworm Infection
Threadworm Infection
Ocular Larva Migrans
Trichinosis
Guinea Worm Disease
Lymphatic Filariasis
Loiasis
River Blindness
Canine Heartworm Infection
Schistosomiasis
Swimmer's Itch
Oriental Lung Fluke
Oriental Liver Fluke
Fascioliasis
Fasciolopsiasis
Opisthorchiasis
Tapeworm Infections
Hydatid Disease
Alveolar Hydatid Disease
Viral Diseases:
Measles
Subacute sclerosing panencephalitis
Common Cold
Mumps
Rubella
Roseola
Fifth Disease
Chickenpox
Respiratory syncytial virus infection
Croup
Bronchiolitis
Infectious Mononucleosis
Poliomyelitis
Herpangina
Hand-Foot-and-Mouth Disease
Bornholm Disease
Genital Herpes
Genital Warts
Aseptic Meningitis
Myocarditis
Pericarditis
Gastroenteritis
Acquired Immunodeficiency Syndrome (AIDS)
Reye's Syndrome
Kawasaki Syndrome
Influenza
Bronchitis
Viral “Walking” Pneumonia
Acute Febrile Respiratory Disease
Acute pharyngoconjunctival fever
Epidemic keratoconjunctivitis
Herpes Simplex Virus 1 (HSV-1)
Herpes Simples Virus 2 (HSV-2)
Shingles
Cytomegalic Inclusion Disease
Rabies
Progressive Multifocal Leukoencephalopathy
Kuru
Fatal Familial Insomnia
Creutzfeldt-Jakob Disease
Gerstmann-Straussler-Scheinker Disease
Tropical Spastic Paraparesis
Western Equine Encephalitis
California Encephalitis
St. Louis Encephalitis
Yellow Fever
Dengue
Lymphocytic choriomeningitis
Lassa Fever
Hemorrhagic Fever
Hantvirus Pulmonary Syndrome
Marburg Virus Infections
Ebola Virus Infections
Smallpox
5.12.1 Multi-Drug Therapy of Infectious Diseases
The present invention also provides methods for treating an infectious disease, comprising administering to an animal in need thereof a Compound of the Invention and another therapeutic agent that is an anti-infectious disease agent. In one embodiment, the anti-infectious disease agent is, but not limited to, agents listed in Table 8.
TABLE 8
Antibacterial Agents:
β-Lactam Antibiotics:
Penicillin G
Penicillin V
Cloxacilliin
Dicloxacillin
Methicillin
Nafcillin
Oxacillin
Ampicillin
Amoxicillin
Bacampicillin
Azlocillin
Carbenicillin
Mezlocillin
Piperacillin
Ticarcillin
Aminoglycosides:
Amikacin
Gentamicin
Kanamycin
Neomycin
Netilmicin
Streptomycin
Tobramycin
Macrolides:
Azithromycin
Clarithromycin
Erythromycin
Lincomycin
Clindamycin
Tetracyclines:
Demeclocycline
Doxycycline
Minocycline
Oxytetracycline
Tetracycline
Quinolones:
Cinoxacin
Nalidixic Acid
Fluoroquinolones:
Ciprofloxacin
Enoxacin
Grepafloxacin
Levofloxacin
Lomefloxacin
Norfloxacin
Ofloxacin
Sparfloxacin
Trovafloxicin
Polypeptides:
Bacitracin
Colistin
Polymyxin B
Sulfonamides:
Sulfisoxazole
Sulfamethoxazole
Sulfadiazine
Sulfamethizole
Sulfacetamide
Miscellaneous Antibacterial Agents:
Trimethoprim
Sulfamethazole
Chloramphenicol
Vancomycin
Metronidazole
Quinupristin
Dalfopristin
Rifampin
Spectinomycin
Nitrofurantoin
Antiviral Agents:
General Antiviral Agents:
Idoxuradine
Vidarabine
Trifluridine
Acyclovir
Famcicyclovir
Pencicyclovir
Valacyclovir
Gancicyclovir
Foscarnet
Ribavirin
Amantadine
Rimantadine
Cidofovir
Antisense Oligonucleotides
Immunoglobulins
Inteferons
Drugs for HIV infection:
Zidovudine
Didanosine
Zalcitabine
Stavudine
Lamivudine
Nevirapine
Delavirdine
Saquinavir
Ritonavir
Indinavir
Nelfinavir
5.13 Other Therapeutic Agents
The present methods can further comprise the administration of a Compound of the Invention and an additional therapeutic agent or pharmaceutically acceptable salts or solvates thereof. The Compound of the Invention and the other therapeutic agent can act additively or, more preferably, synergistically. In a preferred embodiment, a composition comprising a Compound of the Invention is administered concurrently with the administration of one or more additional therapeutic agent(s), which can be part of the same composition or in a different composition from that comprising the Compound of the Invention. In another embodiment, a Compound of the Invention is administered prior to or subsequent to administration of another therapeutic agent(s).
In the present methods for treating cancer, an autoimmune disease or an infectious disease, the other therapeutic agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, proclorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.
In another embodiment, the other therapeutic agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and erythropoietin alfa.
In still another embodiment, the other therapeutic agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, piroxicam and sulindac.
The following examples are provided by way of illustration and not limitation.
6. EXAMPLES
Materials and Methods. Commercially available reagents and solvents were obtained as follows: HPLC-grade solvents, Fisher Scientific (Atlanta, Ga.); anhydrous solvents, Aldrich (St. Louis, Mo.); diisopropylazodicarboxylate (DIAD, 95%), Lancaster (Lancashire, England); 4-aminobenzyl alcohol, Alfa Aesar (Ward Hill, Mass.); L-citrulline, Novabiochem (Laufelfingen, Switzerland); all other amino acids, Advanced ChemTech (Louisville, Ky.) or Novabiochem (Laufelfingen, Switzerland); (1S,2R)-(+)-norephedrine and other commercially available reagents, Aldrich or Acros; all coupling reagents were acquired from Novabiochem or Aldrich. All solvents used as reaction media are assumed to be anhydrous unless otherwise indicated. 1H-NMR spectra were recorded on either a Varian Gemini at 300 MHz or Varian Mercury 400 MHz spectrophotometer. Flash column chromatography was performed using 230-400 mesh ASTM silica gel from Fisher. Analtech silica gel GHLF plates were used for thin-layer chromatography. Analytical HPLC was performed using a Waters Alliance system using a photodiode array detector. Preparative HPLC purification was performed using a Varian Prostar system that had either a photodiode array or dual wavelength detector. Combustion analyses were determined by Quantitative Technologies, Inc., Whitehouse, N.J.
Examples 5-12 relate to Drugs that can be used as Drug units in the invention.
Example 1
Preparation of Compound 21
Fmoc-(L)-val-(L)-cit-PAB-OH (19)(14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (about 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (Willner et al., Bioconjugate Chem. 4, 521, 1993, 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide Compound 20 as an off-white solid. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M−H]−
Compound 20 (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and to the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and diisopropylethylamine (3.66 mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oil that was precipitated with EtOAc, and then triturated using EtOAc (about 25 mL). The solute was further precipitated with ether (about 200 mL) and triturated for 15 min. The solid was filtered and dried under high vacuum to provide Compound 21 which was 93% pure according to HPLC and used in the next step without further purification. Yield: 9.7 g (94%).
Example 2
Preparation of Compound 27
Compound 26 (2.0 g, 2.31 mmol, 1.0 eq.) was diluted with dichloromethane (30 mL), and to the resulting solution was added bis(4-nitrophenyl)carbonate (2.72 g, 8.94 mmol, 3.8 eq.) followed by diisopropylethylamine (1.04 mL, 5.97 mmol, 2.6 eq.). The reaction was complete in 3 d, according to HPLC. The reaction mixture was concentrated and the resulting residue was triturated using ether, then filtered and dried under high vacuum to provide Compound 27 as a yellow solid (2.37 g, 97%).
Example 3
Preparation of Compound 28
Fmoc-phe-lys(Mtr)-OH (24) (0.5 g, 0.63 mmol, U.S. Pat. No. 6,214,345 to Firestone et al.) was diluted with dichloromethane to a concentration of 0.5 M and to this solution was added diethylamine in an amount that was approximately one-third of the volume of the Compound 24/dichloromethane solution. The reaction was allowed to stir and was monitored using HPLC. It was shown to be complete by HPLC in 3 h. The reaction mixture was concentrated in vacuo, and the resulting residue was diluted with ethyl acetate and then reconcentrated. The resulting residue was triturated using ether and filtered. The residual solid was diluted with dichloromethane to a concentration of 0.2M, and to the resulting solution was added MC-OSu (0.20 g, 0.63 mmol, 1.0 eq.) and diisopropylethylamine (0.12 mL, 0.70 mmol, 1.1 eq.). The reaction mixture was allowed to stir under a nitrogen atmosphere for 16 h, after which time HPLC showed very little starting material. The reaction mixture was then concentrated and the resulting residue was triturated using ether to provide Compound 28 as a colored solid. Yield: 100 mg (21%); ES-MS m/z 757.9 [M−H]−.
Example 4
Preparation of Compound 19A
Compound 19 (1.0 g, 1.66 mmol) was diluted with DMF (10 mL) and to the resulting solution was added bis(4-nitrophenyl)carbonate (1.0 g, 3.3 mmol, 2.0 eq.).
The reaction mixture was immediately treated with diisopropylethylamine (0.43 mL, 2.5 mmol, 1.5 eq.) and the reaction was allowed to stir under an argon atmosphere. The reaction was complete in 2.5 h according to HPLC. The reaction mixture was concentrated to provide a light brown oil that was precipitated using ethyl acetate (5 mL), then precipitated again using ether (about 100 mL). The resulting precipitate was allowed to stand for 30 min, and was then filtered and dried under high vacuum to provide Compound 19a as an off-white powder. Yield: 1.05 g (83%); ES-MS m/z 767.2 [M+H]+; UV λmax 215, 256 nm.
Example 5
Preparation of Compound 49
Compound 49 was made according to General Procedure D using Fmoc-Me-val-val-dil-O-t-Bu 39 (0.40 g, 0.57 mmol) as the tripeptide and Boc-dap-nor 44 (0.26 g, 0.62 mmol, 1.1 eq.) as the dipeptide. The reaction mixture was purified using flash column chromatography (silica gel column, eluant -100% EtOAc). Two Fmoc-containing products eluted: the Fmoc derivative of Compound 49 (Rf 0.17 in 100% EtOAc) and what was believed to be the Fmoc derivative of the TFA acetate of Compound 49 (Rf 0.37). The products were combined to provide a white foam that was subjected to General Procedure E. Reaction was complete after 2 h. Solvents were removed to provide an oil that was purified using flash column chromatography (eluant -9:1 Dichloromethane-methanol) to provide Compound 49.
Example 6
Preparation of Compound 50
Compound 50 was prepared by reacting tripeptide 42 and dipeptide 48 according to General Procedure D using triethylamine (5.0 eq.) as the base. After concentration of the reaction mixture, the resulting residue was directly injected onto a reverse phase preparative-HPLC column (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1M TEA/CO2 at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated, and the resulting residue was diluted with 10 mL of dichloromethane-ether (1:1). The solution was cooled to 0° C. and 1.0M ethereal HCl was added dropwise (approx. 10 eq.). The precipitate, Compound 50, was filtered and dried and was substantially pure by HPLC. Yield: 71 mg (43%); ES-MS m/z 731.6 [M+H]+; UV λmax 215, 238, 290 nm. Anal. Calc. C40H70N6O6.4H2O.2HCl: C, 54.84; H, 9.20; N, 9.59. Found: C, 55.12; H, 9.41; N, 9.82.
Example 7
Preparation of Compound 51
Compound 51 was prepared by reacting Fmoc-tripeptide 41 and dipeptide 46 according to General Procedure D using triethylamine as the base. After concentration of the reaction mixture, the residue was directly injected onto a reverse phase preparative-HPLC column (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1M TEA/CO2 at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide a white solid intermediate that was used in the next step without further purification. ES-MS m/z 882.9 [M+NH4]+, 899.9 [M+Na]+; UV λmax 215, 256 nm.
Deprotection of the white solid intermediate was performed according to General Procedure E. The crude product was purified using preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1M TEA/CO2 at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide Compound 51 as a sticky solid. ES-MS m/z 660.1 [M+H]+, 682.5 [M+Na]+; UV λmax 215 nm.
Example 8
Preparation of Compound 52
Boc-dolaproine (0.33 g, 1.14 mmol) and (1S,2S)-(−)-1,2-diphenylethylenediamine (0.5 g, 2.28 mmol, 2.0 eq.) were diluted with dichloromethane, (10 mL) and to the resulting solution was added triethylamine (0.32 mL, 2.28 mmol, 2.0 eq.), then DEPC (0.39 mL, 2.28 mmol, 2.0 eq.). After 4 h, additional DEPC (0.39 mL) was added and the reaction was allowed to stir overnight. The reaction mixture was concentrated and the resulting residue was purified using preparative-HPLC (Varian Dynamax C18 column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and water at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide a yellow gummy solid peptide intermediate that was used without further purification. Rf 0.15 (100% EtOAc); ES-MS m/z 482.4 [M+H]+; UV λmax 215, 256 nm.
The yellow gummy peptide intermediate (0.24 g, 0.50 mmol) was diluted with dichloromethane, and to the resulting solution was added diisopropylethylamine (0.18 mL, 1.0 mmol, 2.0 eq.) and Fmoc-Cl (0.15 g, 0.55 mmol, 1.1 eq.). The reaction was allowed to stir for 3 h, after which time HPLC showed a complete reaction. The reaction mixture was concentrated to an oil, and the oil was diluted with EtOAc and extracted successively with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, and brine. The EtOAc layer was dried, filtered, and concentrated, and the resulting residue was purified using flash column chromatography (silica gel 230-400 mesh; eluant gradient 4:1 hexanes-EtOAc to 1:1 hexanes-EtOAc) to provide Compound 45 as a white solid. Yield: 0.37 g (46% overall); Rf 0.47 (1:1 hexanes-EtOAc); ES-MS m/z 704.5 [M+H]+, 721.4 [M+NH4]+; UV λmax 215, 256 nm.
Compound 52 was prepared by reacting tripeptide 42 (94 mg, 0.13 mmol) and dipeptide compound 45 (65 mg, 0.13 mmol) according to General Procedure D (using 3.6 eq. of diisopropylethylamine as the base). After concentration of the reaction mixture, the resulting residue was diluted with EtOAc and washed successively with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, and brine. The organic phase was dried, filtered and concentrated to provide a white solid residue which was diluted with dichloromethane and deprotected according to General Procedure E. According to HPLC, reaction was complete after 2 h. The reaction mixture was concentrated to an oil. The oil was diluted with DMSO, and the resulting solution was purified using a reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). Two products having similar UV spectra were isolated. The major product, Compound 52, was provided as an off-white solid. Overall yield: 24 mg (23%); ES-MS m/z 793.5 [M+H]+; UV λmax 215 nm.
Example 9
Preparation of Compound 53
Boc-phenylalanine (1.0 g, 3.8 mmol) was added to a suspension of 1,4-diaminobenzene-HCl (3.5 g, 19.0 mmol, 5.0 eq.) in triethylamine (10.7 mL, 76.0 mmol, 20 eq.) and dichloromethane (50 mL). To the resulting solution was added DEPC (3.2 mL, 19.0 mmol, 5.0 eq.) via syringe. HPLC showed no remaining Boc-phe after 24 h. The reaction mixture was filtered, and the filtrate was concentrated to provide a dark solid. The dark solid residue was partitioned between 1:1 EtOAc-water, and the EtOAc layer was washed sequentially with water and brine. The EtOAc layer was dried and concentrated to provide a dark brown/red residue that was purified using HPLC (Varian Dynamax column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and water at 45 mL/min form 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide a red-tan solid intermediate. Yield: 1.4 g (100%); ES-MS m/z 355.9 [M+H]+; UV λmax 215, 265 nm; 1H NMR (CDCl3) δ 7.48 (1H, br s), 7.22-7.37 (5H, m), 7.12 (2H, d, J=8.7 Hz), 7.61 (2H, d, J=8.7 Hz), 5.19 (1H, br s), 4.39-4.48 (1H, m), 3.49 (2H, s), 3.13 (2H, d, J=5.7 Hz), 1.43 (9H, s).
The red-tan solid intermediate (0.5 g, 1.41 mmol) and diisopropylethylamine (0.37 mL, 2.11 mmol, 1.5 eq.) were diluted with dichloromethane (10 mL), and to the resulting solution was added Fmoc-Cl (0.38 g, 1.41 mmol). The reaction was allowed to stir, and a white solid precipitate formed after a few minutes. Reaction was complete according to HPLC after 1 h. The reaction mixture was filtered, and the filtrate was concentrated to provide an oil. The oil was precipitated with EtOAc, resulting in a reddish-white intermediate product, which was collected by filtration and dried under vacuum. Yield: 0.75 g (93%); ES-MS m/z 578.1 [M+H]+, 595.6 [M+NH4]+.
The reddish-white intermediate (0.49 g, 0.85 mmol), was diluted with 10 mL of dichloromethane, and then treated with 5 mL of trifluoroacetic acid. Reaction was complete in 30 min according to reverse-phase HPLC. The reaction mixture was concentrated and the resulting residue was precipitated with ether to provide an off-white solid. The off-white solid was filtered and dried to provide an amorphous powder, which was added to a solution of Boc-dap (0.24 g, 0.85 mmol) in dichloromethane (10 mL). To this solution was added triethylamine (0.36 mL, 2.5 mmol, 3.0 eq.) and PyBrop (0.59 g, 1.3 mmol, 1.5 eq.). The reaction mixture was monitored using reverse-phase HPLC. Upon completion, the reaction mixture was concentrated, and the resulting residue was diluted with EtOAc, and sequentially washed with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, water, and brine. The EtOAc layer was dried (MgSO4), filtered, and concentrated. The resulting residue was purified using flash column chromatography (silica gel) to provide Compound 47 as an off-white powder. Yield: 0.57 g (88%); ES-MS m/z 764.7 [M+NH4]+; UV λmax 215, 265 nm; 1H NMR (DMSO-d6) δ 10.0-10.15 (1H, m), 9.63 (1H, br s), 8.42 (1/2H, d, J=8.4 Hz), 8.22 (1/2H, d, J=8.4 Hz), 7.89 (2H, d, J=7.2 Hz), 7.73 (2H, d, J=7.6 Hz), 7.11-7.55 (13H, m), 4.69-4.75 (1H, m), 4.46 (2H, d, J=6.8 Hz), 4.29 (1H, t, J=6.4 Hz), 3.29 (3H, s), 2.77-3.47 (7H, m), 2.48-2.50 (3H, m), 2.25 (2/3H, dd, J=9.6, 7.2 Hz), 1.41-1.96 (4H, m), 1.36 (9H, s), 1.07 (1H, d, J=6.4 Hz, rotational isomer), 1.00 (1H, d, J=6.4 Hz, rotational isomer).
Tripeptide compound 42 (55 mg, 0.11 mmol) and dipeptide compound 47 (85 mg, 0.11 mmol) were reacted according to General Procedure D (using 3.0 eq. of diisopropylethylamine). After concentration of the reaction mixture, the resulting residue was diluted with EtOAc, and washed sequentially with 10% aqueous citric acid, water, saturated aqueous sodium bicarbonate, and brine. The EtOAc layer was dried, filtered and concentrated to provide a yellow oil. The yellow oil was diluted with dichloromethane (10 mL) and deprotected according to General Procedure E. According to HPLC, reaction was complete after 2 h. The reaction mixture was concentrated to provide an oil. The oil was diluted with DMSO, and the DMSO solution was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 53 as an off-white solid. Overall yield: 42 mg (44% overall); ES-MS m/z 837.8 [M+H]+, 858.5 [M+Na]+; UV λmax 215, 248 nm.
Example 10
Preparation of Compound 54
Compound 54 was prepared according to K. Miyazaki, et al. Chem. Pharm. Bull. 1995, 43(10), 1706-18.
Example 11
Preparation of Compound 55
Compound 55 was synthesized in the same manner as Compound 54, but by substituting FmocMeVal-Ile-Dil-tBu (40) for FmocMeVal-Val-Dil-tBu (39) as the starting material.
Example 12
Preparation of Compound 56
Carbamic acid [(1S)-1-(azidomethyl)-2-phenylethyl]-1,1-dimethylethyl ester (0.56 g, 2 mmol, prepared as described in J. Chem. Research (S), 1992, 391), was diluted with a 4 M solution of HCl in dioxane (10 mL) and the resulting solution allowed to stir for 2 hr at room temperature. Toluene (10 mL) was then added to the reaction, the reaction mixture was concentrated and the resulting residue was azeotropically dried under vacuum using toluene (3×15 mL), to provide a white solid intermediate. ES-MS m/z 177.1 [M+H]+.
The white solid intermediate was diluted with dichloromethane (5 mL) and to the resulting solution was added sequentially N-Boc-Dolaproine (0.58 g, 1 eq.), triethylamine (780 μL, 3 eq.) and DEPC (406 μL, 1.2 eq.), and the reaction mixture was allowed to stir for 2 h at room temperature. Reaction progress was monitored using reverse-phase HPLC. Upon completion of reaction as determined by HPLC, the reaction mixture was diluted with dichloromethane (30 mL), the dichloromethane layer was washed successively with 10% aqueous citric acid (20 mL), saturated aqueous NaHCO3 (20 mL), and water (20 mL). The dichloromethane layer was concentrated and the resulting residue was purified via flash column chromatography using a step gradient of 0-5% methanol in dichloromethane. The relevant fractions were combined and concentrated to provide a solid intermediate, 0.78 g (88%). ES-MS m/z 446.1 [M+H]+, 468.3 [M+Na]+.
The solid intermediate (450 mg, 1 mmol) and Tripeptide 42 (534 mg, 1.1 eq.) were diluted with a 50% solution of TFA in dichloromethane (10 mL), and the resulting reaction was allowed to stir for 2 h at room temperature. Toluene (10 mL) was added to the reaction and the reaction mixture was concentrated. The resulting amine intermediate was azeotropically dried using toluene (3×20 mL) and dried under vacuum overnight.
The resulting amine intermediate was diluted with dichloromethane (2 mL) and to the resulting solution was added triethylamine (557 μL, 4 eq.), followed by DEPC (203 μL, 1.4 eq.). The reaction mixture was allowed to stir for 4 h at room temperature and reaction progress was monitored using HPLC. Upon completion of reaction, the reaction mixture was diluted with dichloromethane (30 mL) and the dichloromethane layer was washed sequentially using saturated aqueous NaHCO3 (20 mL) and saturated aqueous NaCl (20 mL). The dichloromethane layer was concentrated and the resulting residue was purified using flash column chromatography in a step gradient of 0-5% methanol in dichloromethane. The relevant fractions were combined and concentrated and the resulting residue was dried using a dichloromethane:hexane (1:1) to provide a white solid intermediate, 0.64 g (84%). ES-MS m/z 757.5 [M+H]+.
The white solid intermediate (536 mg, 0.73 mmol) was diluted with methanol and to the resulting solution was added 10% Pd/C (100 mg). The reaction was placed under a hydrogen atmosphere and was allowed to stir at atmospheric pressure and room temperature for 2 h. Reaction progress was monitored by HPLC and was complete in 2 h. The reaction flask was purged with argon and the reaction mixture was filtered through a pad of Celite. The Celite pad was subsequently washed with methanol (30 mL) and the combined filtrates were concentrated to yield a gray solid intermediate which was used without further purification. Yield=490 mg (91%). ES-MS m/z 731.6 [M+H]+, 366.6 [M+2H]2+/2.
The gray solid intermediate (100 mg, 0.136 mmol), N-Boc-4-aminobenzoic acid (39 mg, 1.2 eq.) and triethylamine (90 μL, 4 eq.) were diluted with dichloromethane (2 mL) and to the resulting solution was added DEPC (28 μL, 1.2 eq.). The reaction mixture was allowed to stir at room temperature for 2 h, then the reaction mixture was diluted with dichloromethane (30 mL). The dichloromethane layer was sequentially washed with saturated aqueous NaHCO3 (20 mL) and saturated aqueous NaCl (20 mL). The dichloromethane layer was then concentrated and the resulting residue was purified via flash column chromatography using a step gradient of 0-5% in dichlormethane. The relevant fractions were combined and concentrated and the resulting residue was dried using dichloromethane:hexane (1:1) to provide a white solid intermediate. ES-MS m/z 950.7 [M+H]+.
The white solid intermediate was diluted with a 50% solution of TFA in dichloromethane and allowed to stir for 2 h at room temperature. Toluene (10 mL) was added to the reaction and the reaction mixture was concentrated. The resulting residue was azeotropically dried using toluene (3×15 mL), to provide a yellow oil which was purified using preparative HPLC(C18-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN from 10 to 95% in 0.05 M Triethylammonium carbonate buffer, pH 7.0, in 30 min at a flow rate of 10 mL/min). The relevant fractions were combined and concentrated and the resulting residue was azeotropically dried using MeCN (3×20 mL), to provide Compound 56 as white solid: 101 mg (87% over 2 steps). ES-MS m/z 850.6 [M+H]+, 872.6 [M+Na]+.
Example 13
Preparation of Compound 57
Compound 49 (100 mg, 0.14 mmol), Compound 27 (160 mg, 0.15 mmol, 1.1 eq.), and HOBt (19 mg, 0.14 mmol, 1.0 eq.) were diluted with DMF (2 mL). After 2 min, pyridine (0.5 mL) was added and the reaction mixture was monitored using reverse-phase HPLC. Neither Compound 49 nor Compound 27 was detected after 24 h. The reaction mixture was concentrated, and the resulting residue was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and Et3N—CO2 (pH 7) at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were pooled and concentrated to provide an off-white solid intermediate. ES-MS m/z 1608.7 [M+H]+
The off-white solid intermediate was diluted with MeCN/water/TFA in an 85:5:10 ratio, respectively. The reaction mixture was monitored using HPLC and was complete in 3 h. The reaction mixture was directly concentrated and the resulting residue was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 57 as an off-white powder. Yield: 46 mg (32% overall); ES-MS m/z 1334.8 [M+H]+; UV λmax 215, 256 nm.
Example 14
Preparation of Compound 58
Compound 49 (1.69 g, 2.35 mmol), Compound 21 (2.6 g, 3.52 mmol, 1.5 eq.), and HOBt (64 mg, 0.45 mmol, 0.2 eq.) were diluted with DMF (25 mL). After 2 min, pyridine (5 mL) was added and the reaction was monitored using reverse-phase HPLC. The reaction was shown to be complete in 24 h. The reaction mixture was concentrated to provide a dark oil, which was diluted with 3 mL of DMF. The DMF solution was purified using flash column chromatography (silica gel, eluant gradient: 100% dichloromethane to 4:1 dichloromethane-methanol). The relevant fractions were combined and concentrated to provide an oil that solidified under high vacuum to provide a mixture of Compound 58 and unreacted Compound 49 as a dirty yellow solid (Rf 0.40 in 9:1 dichloromethane-methanol). The dirty yellow solid was diluted with DMF and purified using reverse-phase preparative-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 8 m, 100 Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min) to provide Compound 58 as an amorphous white powder (Rf 0.40 in 9:1 dichloromethane-methanol) which was >95% pure by HPLC and which contained less than 1% of Compound 49. Yield: 1.78 g (57%); ES-MS m/z 1316.7 [M+H]+; UV λmax 215, 248 nm.
Example 15
Preparation of Compound 59
The hydrochloride salt of Compound 51 (11 mg, 15.2 mmol) and Compound 21 (11 mg, 15.2 mmol) were diluted with 1-methyl-2-pyrollidinone (1 mL) and to the resulting solution was added diisopropylethylamine (5.3 mL, 30.3 mmol, 2.0 eq.). The mixture was allowed to stir under argon atmosphere for 3 d while being monitored using HPLC. After this time, much unreacted starting material still remained, HOBt (1.0 eq.) was added and the reaction mixture was allowed to stir for 24 h, after which time no starting material remained according to HPLC. The reaction mixture was concentrated and the resulting residue was purified using preparative-HPLC (Varian Dynamax C18 column 21.4 mm×25 cm, 5 m, 100 Å, using a gradient run of MeCN and water at 20 mL/min from 10% to 100% over 30 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 59 as a white solid. Yield: 13 mg (67%); ES-MS m/z 1287.2 [M+H]+, 1304.3 [M+NH4]+; UV λmax 215, 248 nm.
Example 16
Preparation of Compound 60
Compound 53 (9 mg, 10.8 μmol) and Compound 28 (5.2 mg, 10.8 μmol) were diluted with dichloromethane (1 mL) and to the resulting solution was added HATU (6.3 mg, 16.1 μmol, 1.5 eq.), followed by pyridine (1.3 μL, 16.1 μmol, 1.5 eq.). The reaction mixture was allowed to stir under argon atmosphere while being monitored using HPLC. The reaction was complete after 6 h. The reaction mixture was concentrated and the resulting residue was diluted with DMSO. The DMSO solution was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min) and the relevant fractions were combined and concentrated to provide an off-white solid intermediate which was >95% pure according to HPLC.
The off-white solid intermediate was diluted with dichloromethane (2 mL) and the resulting solution was treated with TFA (0.5 mL). The reaction was monitored using HPLC, and was complete in 2 h. The reaction mixture was concentrated, and the resulting residue was diluted with DMSO and purified under the same conditions as described in Example 13. The relevant fractions were combined and concentrated to provide Compound 60 as an off-white powder. Yield: 14.9 mg (90%); ES-MS m/z 1304.6 [M+H]+; UV λmax 215, 275 nm.
Example 17
Preparation of Compound 61
The trifluoroacetate salt of Compound 53 (0.37 g, 0.39 mmol, 1.0 eq.) and Compound 18 (0.30 g, 0.58 mmol, 1.5 eq.) were diluted with DMF (5 mL, 0.1 M), and to the resulting solution was added pyridine (95 μL, 1.2 mmol, 3.0 eq.). HATU (0.23 g, 0.58 mmol, 1.5 eq.) was then added as a solid and the reaction mixture was allowed to stir under argon atmosphere while being monitored using HPLC. The reaction progressed slowly, and 4 h later, 1.0 eq. of diisopropylethylamine was added. Reaction was complete in 1 h. The reaction mixture was concentrated in vacuo and the resulting residue was purified using preparative-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min) to provide a faint pink solid intermediate.
The pink solid intermediate was diluted with DMF (30 mL) and to the resulting solution was added diethylamine (15 mL). Reaction was complete by HPLC in 2 h. The reaction mixture was concentrated and the resulting residue was washed twice with ether. The solid intermediate was dried under high vacuum and then used directly in the next step.
The solid intermediate was diluted with DMF (20 mL) and to the resulting solution was added MC-OSu (0.12 g, 0.39 mmol, 1.0 eq.). After 4 d, the reaction mixture was concentrated to provide an oil which was purified using preparative-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). Compound 61 was isolated as a white flaky solid. Yield: 0.21 g (38% overall); ES-MS m/z 1285.9 [M+H]+; 13.07.8 [M+Na]+; UV λmax 215, 266 nm.
Example 18
Preparation of Compound 62
Fmoc-val-cit-PAB-OCO-Pnp (19a) (0.65 g, 0.85 mmol, 1.1 eq.), Compound 49 (0.55 g, 0.77 mmol, 1.0 eq.), and HOBt (21 mg, 0.15 mmol, 2.0 eq.) were diluted with DMF (2.0 mL) and dissolved using sonication. To the resulting solution was added pyridine (0.5 mL) and the reaction was monitored using HPLC. After 24 h, diisopropylethylamine (1.0 eq.) was added and the reaction was allowed to stand without stirring for 24 h. The reaction mixture was concentrated to provide an oil residue. The oil residue was purified using reverse phase preparative-HPLC (Varian Dynamax column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 45 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min.) The desired fractions were pooled and concentrated to yield an oil that was precipitated with ether to provide an off-white solid intermediate. Yield: 0.77 g (74%); ES-MS m/z 1345.7 [M+H]+; UV λmax 215, 254 nm.
The off-white solid intermediate (about 85 mg) was deprotected using diethylamine (1 mL) in DMF (3 mL). After 1 h, the reaction was complete. The reaction mixture was concentrated, and the resulting residue was precipitated in 1 mL of EtOAc followed by addition of excess ether (about 20 mL). The amine intermediate was filtered and dried under high vacuum and used in the next step without further purification.
The amine intermediate (70 mg, 61 μmol, 1.0 eq.) was taken up in DMF (10 mL), and to the resulting solution was added sequentially, bromoacetamidocaproic acid (17 mg, 67 μmol, 1.1. eq.), PyBrop (32 mg, 67 μmol, 1.1 eq.), and diisopropylethylamine (16 μL, 92 μmol, 1.5 eq.). After 24 h, an additional 1.0 eq. of bromoacetamidocaproic acid was added. Reaction was stopped after 30 h. The reaction mixture was concentrated to an oil and the oil purified using reverse phase preparative-HPLC (Synergi MaxRP C12 column 21.4 mm×25 cm, 5μ, 80 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min.). The relevant fractions were combined and concentrated to provide Compound 62 as a white solid. Yield: 23 mg (27%); ES-MS m/z 1356.7 [M+H]+; UV λmax 215, 247 nm.
Example 19
Preparation of Compound 63
Fmoc-val-cit-PAB-OC(O)-Me-val-val-dil-dap-nor (about 48 mg, obtained according to Example 18) was subjected to Fmoc-removal by treating with diethylamine (1 mL) in DMF (3 mL). After 1 h, the reaction was complete. The reaction mixture was concentrated and the resulting residue was precipitated using 1 mL of EtOAc followed by addition of excess ether (about 20 mL). The amine intermediate was filtered and dried under high vacuum and used in the next step without further purification.
The amine intermediate (35 μmol, 1.1 eq.) was diluted with DMF (2 mL), and to the resulting solution was added sequentially maleimido-PEG acid (Frisch, B.; Boeckler, C.; Schuber, F. Bioconjugate Chem. 1996, 7, 180-6; 7.8 mg, 32 μmol, 1.0 eq.), DEPC (10.7 μL, 64 μmol, 2.0 eq.), and diisopropylethylamine (11.3 μL, 64 μmol, 2.0 eq.). The reaction was complete in 15 min according to HPLC. The reaction mixture was concentrated to provide an oil. The oil was diluted with 1 mL of DMSO and purified using reverse phase preparative-HPLC (Synergi MaxRP C12 column 21.4 mm×25 cm, 5μ, 80 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min). The relevant fractions were combined and concentrated to provide Compound 63 as a white solid.
Yield: 16.2 mg (34%); ES-MS m/z 1348.6 [M+H]+; UV λmax 215, 247 nm.
Examples 20-25 describe the conjugation of the monoclonal antibodies cBR96 and cAC10 to a Drug-Linker Compound. These antibodies were obtained as described in Bowen, et al., J. Immunol. 1993, 151, 5896; and Trail, et al., Science 1993, 261, 212, respectively.
The number of Drug-Linker moities per Ligand in a Drug-Linker-Ligand Conjugate varies from conjugation reaction to conjugation reaction, but typically ranges from about 7 to about 9, particularly when the Ligand is cBR96 or cAC10.
Example 20
Preparation of Compound 64
cBR96 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Result: [Ab]=4.7 mg/mL=29.4 μM; [thiol]=265 μM; SH/Ab=9.0 (Typical SH/Ab range is from about 7.8 to about 9.5).
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, a 130.5 μL stock solution of Compound 57 (8.4 mM in DMSO, 8.5 mol Compound 57 per mol reduced antibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 57 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 218 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, three PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 64, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the three pre-chilled PD 10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 4.2 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 64, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 64]=3.8 mg/mLg
% Aggregate=trace
Residual Thiol Titration: Residual thiols=1.7/Ab. Drug/Ab˜9.0−1.7=7.3
Quenched Drug-Linker: undetectable
Yield: 4.2 mL, 16 mg, 66%.
Example 21
Preparation of Compound 65
cAC10 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Results: [Ab]=4.9 mg/mL=30.7 μM;
[thiol]=283 μM; 9.2 SH/Ab
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, 125 μL of a stock solution of Compound 57 (8.4 mM in DMSO, 8.5 mol Compound 57 per mol reduced antibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 57 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., then returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 218 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 65, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD 10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 65, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were then determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 65]=2.8 mg/mL
% Aggregate=trace
Residual Thiol Titration: Residual thiols=1.6/Ab. Drug/Ab˜9.2−1.6=7.6
Not covalently bound Drug-Linker: undetectable
Yield: 5.6 mL, 15.7 mg, 65%.
Example 22
Preparation of Compound 66
cBR96 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Result: [Ab]=3.7 mg/mL=23.1 μM; [thiol]=218 μM; 9.4 SH/Ab
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, 145.5 μL of a stock solution of Compound 58 (8.3 mM in DMSO, 9.0 mol Compound 58 per mol reduced antibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 58 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., then returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 249 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, three PD 10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 66, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the three pre-chilled PD 10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 4.2 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 66, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 66]=3.0 mg/mL
% Aggregate=trace
Residual Thiol Titration: Residual thiols=0.4/Ab. Drug/Ab˜9.5−0.4=9.1
Not covalently bound Drug-Linker: 0.3% of 57-Cys adduct
Yield: 5.3 mL, 15.9 mg, 66%.
Example 23
Preparation of Compound 67
cAC10 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Result: [Ab]=3.9 mg/mL=24.5 μM; [thiol]=227 μM; 9.3 SH/Ab
Conjugation:
A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to 4.2 mL of reduced antibody and the resulting solution was cooled to 0° C. using an ice bath. In a separate flask, 154.4 μL of a stock solution of Compound 58 (8.3 mM in DMSO, 9.0 mol Compound 58 per mol reduced antibody) was diluted with MeCN (1.46 mL, pre-chilled to 0° C. in an ice bath). The MeCN solution of Compound 58 was rapidly added to the antibody solution and the reaction mixture was stirred using a vortex instrument for 5-10 sec., then returned to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 249 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 67, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD 10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 67, the number of Drug molecules per
Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Assay Results:
[Compound 67]=3.0 mg/mL
% Aggregate=trace
Residual Thiol Titration: Residual thiols=0.5/Ab. Drug/Ab˜9.5˜0.5=9.0
Quenched Drug-Linker: 1.1% of 58-Cys adduct
Yield: 5.3 mL, 15.9 mg, 66%.
Example 24
Preparation of Compound 68
cBR96 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Result: [Ab]=4.4 mg/mL=27.2 μM; [thiol]=277 μM; 10.2 SH/Ab
Conjugation:
The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to 0° C. in an ice bath) so that the resulting solution was 20% DMSO. The solution was allowed to stir for 10 min. at 0° C., then 127.8 μL of a stock solution of Compound 60 (7.6 mM solution in DMSO; 9 mol Compound 60 per mol antibody) was rapidly added. The reaction mixture was immediately stirred using a vortex instrument and return to the ice bath and allowed to stir at 0° C. for 1 hr, after which time 213 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 68, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD 10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 68, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Because the absorbances of Compound 60 and antibody largely overlap, spectrophotometric determination of the conjugate concentration requires the measurement of absorbance at 270 and 280 nm. The molar concentration of conjugate is given by the following formula:
[Conjugate]=(OD280×1.08e−5−OD270×8.20e−6)×dilution factor,
where the values 1.08e−5 and 8.20e−6 are calculated from the molar extinction coefficients of the drug and the antibody, which are estimated as:
ε270 Compound 60=2.06e4 ε270 cBR96=1.87e5
ε280 Compound 60=1.57e4 ε280 cBR96=2.24e5
Assay Results:
[Compound 68]=3.2 mg/mL
% Aggregate=trace
Residual Thiol Titration: Residual thiols=1.0/Ab. Drug/Ab˜10.2−1.0=9.2
Quenched Drug-Linker: trace
Yield: 5.6 mL, 17.9 mg, 75%.
Example 25
Preparation of Compound 69
cAC10 Antibody (24 mg) was reduced using DTT as described in General Procedure L, then the number of thiols per antibody and the antibody concentration were determined as described in General Procedure M and General Procedure N, respectively.
Result: [Ab]=4.8 mg/mL=29.8 μM; [thiol]=281 μM; 9.4 SH/Ab
Conjugation:
The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to 0° C. in an ice bath) so that the resulting solution was 20% DMSO. The solution was allowed to stir for 10 min. at 0° C., then 140 μL of a stock solution of Compound 60 (7.6 mM solution in DMSO; 8.5 mol Compound 60 per mol antibody) was rapidly added. The reaction mixture was immediately stirred using a vortex instrument and return to the ice bath and allowed to stir for 1 hr at 0° C., after which time 213 μL of a cysteine solution (100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of the quenched reaction mixture was saved as a “qrm” sample.
While the reaction proceeded, four PD10 columns (Sephadex G25, available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room and equilibrated with PBS (which had been pre-cooled to 0° C. using an ice bath).
The quenched reaction mixture, which contained Compound 69, was concentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4 centrifuge filtering devices (30K molecular weight cutoff membrane; Millipore Corp.; Bedford, Mass.; used according to manufacturer's instructions) which were pre-cooled to 4° C. in a refrigerator and the concentrated reaction mixture was eluted through the four pre-chilled PD 10 columns using PBS as the eluent (1 mL for each column). The eluted conjugate was collected in a volume of 1.4 mL per column, for a total eluted volume of 5.6 mL. The eluted Conjugate solution was then filtered using a sterile 0.2 micron syringe-end filter, 250 μL of Conjugate solution was set aside for analysis, and the remainder of the Conjugate solution was frozen in sterile vials.
The concentration of Compound 69, the number of Drug molecules per Antibody, the amount of quenched Drug-Linker and the percent of aggregates were determined using General Procedures P, N, O and Q, respectively.
Because the absorbances of Compound 60 and antibody largely overlap, spectrophotometric determination of the conjugate concentration requires the measurement of absorbance at 270 and 280 nm. The molar concentration of conjugate is given by the following formula:
[Conjugate]=(OD280×1.08e−5−OD270×8.20e−6)×dilution factor,
where the values 1.08e−5 and 8.20e−6 are calculated from the molar extinction coefficients of the drug and the antibody, which are estimated as:
ε270 Compound 60=2.06e4 ε270 cAC10=2.10e5
ε280 Compound 60=1.57e4 ε280 cAC10=2.53e5
Assay Results:
[Compound 69]=3.0 mg/mL
% Aggregate=trace
Residual Thiol Titration: Residual thiols=0.7/Ab. Drug/Ab˜9.4−0.7=8.7
Quenched Drug-Linker: trace
Yield: 5.6 mL, 16.8 mg, 70%.
Example 26
Preparation of Compound 75
Diethyl (4-nitrobenzyl)phosphonate (1.1 g, 4.02 mmol) was diluted in anhydrous THF (4 mL) and the resulting mixture was cooled to 0° C. Sodium hydride (0.17 g, 4.22 mmol, 1.05 eq., 60% dispersion in mineral oil) was added and the resulting reaction was allowed to stir for 5 min. At this time gas evolution from the reaction mixture had ceased. 2,2-Dimethyl-1,3-dioxan-5-one (0.52 g, 4.02 mmol) in 1 mL of a hydrous THF was then added to the reaction mixture via syringe and the reaction was allowed to warm to room temperature with stirring. Additional THF (1 mL) was added after 30 min to help dilute the resulting precipitate and the resulting mixture was stirred for an additional 30 min., was transferred to a separatory funnel containing EtOAc (10 mL) and water (10 mL). The organic phase was collected, washed with brine, and the combined aqueous extracts were washed with ethyl acetate (2×). The combined organic extracts were dried over MgSO4, filtered, and concentrated to provide a dark red crude oil that was purified using flash chromatography on a silica gel column (300×25 mm) and eluting with 9:1 hexanes-EtOAc to provide a pale yellow solid intermediate. Yield: 0.57 g (57%); Rf 0.24 (9:1 hexanes-EtOAc); UV λmax 225, 320 nm. 1H NMR (CDCl3) δ 8.19 (2H, d, J=8.4 Hz), 7.24 (2H, d, J=8.4 Hz), 6.33 (1H, s), 4.62 (2H, s), 4.42 (2H, s), 1.45 (6H, s). 13C NMR (CDCl3) δ 146.6, 142.7, 141.3, 129.4, 123.9, 121.1, 99.9, 64.4, 60.8, 24.1.
The pale yellow solid intermediate (0.25 g, 1.0 mmol) was diluted using THF (20 mL), the resulting mixture was treated with 1 N HCl (10 mL) and allowed to stir for 5 min. To the reaction mixture was added diethyl ether (150 mL) and water and the resulting mixture was transferred to a separatory funnel. The organic layer was dried (MgSO4), filtered and concentrated to give an oil. The resulting diol was then taken up in THF-methanol (1:1, 4 mL each, 0.3 M) followed by the addition of Raney Nickel (100 μL, 100 μL/mmol nitro-group, 50% slurry in water) and hydrazine (74 μL, 1.5 eq.). Gas evolution occurred while the reaction mixture was heated to 50-60° C. After 30 min and 1 h, 1.5 eq. of hydrazine was added each time. The yellow mixture was filtered through celite and washed with methanol. The filtrated was concentrated to provide Compound 75 as an oil which later crystallized to a yellow solid. Yield: 0.14 g (78%); UV λmax 215, 260 nm. 1H NMR (DMSO) δ 7.00 (2H, d, J=8.4 Hz), 6.51 (2H, d, J=8.4 Hz), 6.33 (1H, s), 5.20 (2H, bs), 4.64 (2H, bd), 4.04 (2H, s). 13C NMR (DMSO) δ 147.2, 138.1, 129.6, 126.1, 124.6, 113.7, 63.6, 57.5.
Example 27
Preparation of Compound 79
To a mixture of Compound 75 (BHMS, 0.12 g, 0.67 mmol) in methanol-dichloromethane (1:2, 4.5 mL total) was added Fmoc-Val-Cit (0.33 g, 0.67 mmol) followed by EEDQ (0.25 g, 1.0 mmol, 1.5 eq.) and the resulting reaction was allowed to stir for 15 hours under inert atmosphere. Additional EEDQ (1.5 eq.) and Fmoc-Val-Cit (1.0 eq.) were then added due to the presence of unreacted BHMS and the resulting reaction was allowed to stir for 2 days and concentrated. The resulting residue was triturated using ether to provide a tan solid intermediate. ES-MS m/z 659 [M+H]+, 681 [M+Na]+; UV λmax 215, 270 nm. 1H NMR (DMSO) δ 10.04 (1H, s), 8.10 (1H, d, J=7.2 Hz), 7.87 (2H, d, J=7.6 Hz), 7.72 (2H, t, J=7.6 Hz), 7.55 (2H, d, J=8.4 Hz), 7.37-7.43 (3H, m), 7.30 (2H, t, J=7.2 Hz), 7.24 (2H, d, J=8.4 Hz), 6.47 (1H, s), 5.96 (1H, t, J=5.2 Hz), 5.39 (1H, s), 4.83 (1H, t, J=5.2 Hz), 4.78 (1H, t, J=5.2 Hz), 4.40 (1H, dd, J=5.2, 8.0 Hz), 4.20-4.30 (3H, m), 4.11 (2H, d, J=4.4 Hz), 4.04 (2H, d, J=5.2 Hz), 3.91 (1H, t, J=7.2 Hz), 2.84-3.06 (2H, m), 1.91-2.03 (1H, m), 1.29-1.74 (4H, m), 0.86 (3H, d, J=6.8 Hz), 0.84 (3H, d, J=6.8 Hz).
The tan solid intermediate was diluted with DMF (10 mL) and the resulting mixture was treated with diethylamine (5 mL), allowed to stir for 1 hour and concentrated to provide a tan solid which was dried under high vacuum for 3 days. The tan solid was triturated using EtOAc (10 mL) and further precipitated using ether (80 mL) to provide a crude residue which was filtered through a sintered glass funnel and dried in vacuo to afford a light tan intermediate. ES-MS m/z 436 [M+H]+, 458 [M+Na]+; UV λmax 215, 270 nm.
The light tan intermediate was diluted with DMF (10 mL) and treated with 6-maleimidocaproic acid hydroxysuccinimde ester (0.16 g, 0.53 mmol, 1 eq.). The reaction was allowed to stir for 18 h, additional diisopropylethylamine (1.0 eq) was added followed by additional 6-maleimidocaproic acid hydroxysuccinimde ester (0.5 eq.). The resulting reaction was allowed to stir for 4 hours, after which time, HPLC indicated that the starting material had been consumed. The reaction mixture was concentrated to provide a crude residue that was triturated using EtOAc (10 mL) and then further precipitated using ether (75 mL). The precipitate was and dried overnight to provide a tan/orange powdered intermediate. Overall yield: 0.42 g (quant.). ES-MS m/z 629 [M+H]+, 651 [M+Na]+; UV λmax 215, 270 nm.
The tan/orange powdered intermediate (0.40 g, 0.64 mmol) was partially dissolved in DMF (20 mL) and to the resulting mixture was added bis(4-nitrophenyl) carbonate (0.98 g, 3.2 mmol, 5.0 eq.) and diisopropylethylamine (0.45 mL, 2.5 mmol, 4.0 eq.). The resulting reaction was allowed to stir for about 4 hours, after which time, HPLC monitoring indicated that no starting material remained and that the reaction mixture contained 2 products in a 3:2 ratio (the desired bis-carbonate and the 1,3-dioxan-2-one, respectively). The reaction mixture was concentrated and the resulting residue was triturated using EtOAc (10 mL), then further precipitated using ether (80 mL) in a one-pot manner. The EtOAc-ether mixture was filtered and the solid was dried to provide Compound 79 as a tan powder which was used without further purification.
Example 28
Preparation of Compound 80
Compound 49 (202 mg, 0.22 mmol, 2.0 eq., 80% pure) and Compound 79 (180 mg, 0.11 mmol, 1.0 eq., 60% pure) were suspended in dry DMF (2 mL, 0.1 M) and to the resulting mixture was added HOBt (3 mg, 22 μmol, 0.2 eq.) followed by pyridine (400 μL, ¼ v/v DMF). The resulting reaction was allowed to stir for 16 h, diluted with DMSO (2 mL) and the resulting mixture was purified using preparative HPLC(C18-RP column, 5μ, 100 Å, linear gradient of MeCN in water 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 50 mL/min) to provide Compound 80 as a white solid. Yield: 70 mg (18%). MALDI-TOF MS m/z 2138.9 [M+Na]+, 2154.9 [M+K]+.
Example 29
Preparation of Compound 81
Compound 81 was made using the method described in Example 1 and substituting Fmoc-(D)-val-(L)-cit-PAB-OH for Compound 19.
Example 30
Preparation of Compound 82
Compound 82 was made using the method described in Example 1 and substituting Fmoc-(L)-val-(D)-cit-PAB-OH for Compound 19.
Example 31
Preparation of Compound 83
Compound 83 was made using the method described in Example 1 and substituting Fmoc-(D)-val-(D)-cit-PAB-OH for Compound 19.
Example 32
Preparation of Compound 84
Compound 84 was made using the method described in Example 14 and substituting Compound 81 for Compound 21.
Example 33
Preparation of Compound 85
Compound 85 was made using the method described in Example 14 and substituting Compound 82 for Compound 21.
Example 34
Preparation of Compound 86
Compound 86 was made using the method described in Example 14 and substituting Compound 83 for Compound 21.
Example 35
Preparation of Compound 87
A mixture of 6-Maleimidocaproic acid (1.00 g, 4.52 mmol, 1.0 eq.), p-aminobenzyl alcohol (1.11 g, 9.04 mmol, 2.0 eq.) and EEDQ (2.24 g, 9.04 mmol, 2.0 eq.) were diluted in dichloromethane (13 mL). The resulting reaction was stirred about 16 hr., then concentrated and purified using flash column chromatography in a step gradient 25-100% EtOAc in hexanes to provide a solid intermediate. Yield: 1.38 g (96%); ES-MS m/z 317.22 [M+H]+, 339.13 [M+Na]+; UV λmax 215, 246 nm.
The solid intermediate (0.85 g, 2.69 mmol, 1.0 eq.) and bis(4-nitrophenyl) carbonate (2.45 g, 8.06 mmol, 3.0 eq.) were diluted in DMF (10 mL), and to the resulting mixture was added diisopropylethylamine (0.94 mL, 5.37 mmol, 2.0 eq.). The resulting reaction was stirred for about 1 hr, after which time RP-HPLC indicated that the reaction was complete. The reaction mixture was concentrated in vacuo, and the resulting crude residue was triturated using diethyl ether (about 250 mL) to provide a white solid intermediate upon filtration. Yield: 1.25 g (96%); UV λmax 215, 252 nm.
The white solid intermediate (259 mg, 0.0538 mmol, 1.0 eq.), MMAE (464 mg, 0.646 mmol, 1.2 eq.), and HOBt (14.5 mg, 0.108 mmol, 0.2 eq.) were diluted in pyridine/DMF (1:5, 6 mL), and the resulting reaction was stirred for about 10 h, after which time RP-HPLC indicated incomplete reaction. The reaction mixture was concentrated, the resulting crude residue was diluted using DMF (3 mL), and to the resulting mixture was added diisopropylethylamine (0.469 mL, 0.538 mmol, 1.0 eq.) and the resulting reaction was allowed to stir for about 16 hr. The reaction mixture was directly purified using Chromatotron® (radial thin-layer chromatography) with a step gradient (0-5% methanol in dichloromethane), to provide Compound 87 as a white solid. Yield: 217 mg (38%); ES-MS m/z 1082.64 [M+Na]+; UV λmax 215, 248 nm.
Example 36
Preparation of Compound 88
Fmoc-val-cit (U.S. Pat. No. 6,214,345 to Firestone et al.) was suspended in dichloromethane (50 mL) and the resulting mixture was treated with 33% HBr in HOAc (20 mL), which was added via pipette over about 5 minutes. After stirring for about 10 minutes, the reaction mixture was shown to be complete using HPLC. The reaction mixture was diluted with ice (about 500 mL) and saturated aqueous sodium bicarbonate was slowly added while stirring until gas evolution ceased. The resulting gelatinous mass was filtered and washed with distilled water to provide a solid which was dried under high vacuum in the presence of P2O5 for 24 h. The resulting tan powdered intermediate (Fmoc-val-cit-PAB-Br) was about 70% pure by HPLC and was used without further purification.
The tan powdered intermediate (30 mg, 40.6 μmol) and Compound 53 (34 mg, 40.6 μmol) were dissolved in DMF (1 mL), and to the resulting mixture was added diisopropylethylamine (21 μL, 0.12 mmol, 3.0 eq.). The resulting reaction was allowed to stir for 6 h, diluted with DMSO (1 mL) and immediately purified using preparative-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% formic acid) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min), to provide as a slight tan powdered intermediate. Yield: 5 mg (8%); ES-MS m/z 1420 [M+H]+, 1443 [M+Na]+; UV λmax 205, 258 nm.
The slight tan powdered intermediate (4 mg, 9.5 μmol) was diluted using DMF (1 mL) and the resulting mixture was treated with diethylamine (0.5 mL). The resulting reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oily solid residue which was triturated with ether (3×) to provide a crude residue. The crude residue was diluted with DMF (1 mL) and to the resulting mixture was added 6-maleimidocaproic acid hydroxysuccinimide ester (3 mg, 9.5 μmol). The resulting reaction was allowed to stir at room temperature for about 16 h. The reaction mixture was directly purified using preparative-HPLC(C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% formic acid) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min) to provide Compound 88 as a slight tan solid. Yield: 3.9 mg (quant); ES-MS m/z 1391 [M+H]+; UV λmax 205, 250 nm.
Example 37
Preparation of Compound 89
Preparation of Compound 89A
Compound 89A was prepared using the method described in Example 9 and substituting tripeptide Compound 43 for tripeptide Compound 42, intermediate
Preparation of Compound 89
Compound 89a (0.13 g, 0.15 μmol, 1.0 mmol), Compound 21 (0.12 g, 0.17 mmol, 1.1 eq.), and HOBt (4 mg, 31 μmol, 0.2 eq.) were suspended in DMF/pyridine (2 mL/0.5 mL, respectively). The resulting reaction was allowed to stir for about 4 h, then diisopropylethylamine (27 μL, 0.15 mmol, 1.0 eq.) was added and the resulting reaction was allowed to stirred for about 54 h and concentrated in vacuo. The resulting crude oil was diluted with DMSO and purified using preparative-HPLC(C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min) to provide to a yellow oil that was taken up in a minimum amount of dichloromethane and precipitated with excess ether to afford Compound 89 as a tan powder. Yield: 0.15 mg (68%). ES-MS m/z 1449.14 [M+H]+; UV λmax 215, 258 nm.
Example 38
Preparation of Compound 90
1,4-Phenylenediamine dihydrochloride (3.06 g, 17 mmoles) and di-t-butyl dicarbonate (3.69 g, 17 mmoles) were diluted with 30 mL of dichloromethane. To the resulting mixture was added diisopropylethylamine (8.83 ml, 50.7 mmoles, 3.0 eq.) and the resulting reaction was allowed to stirred for 1 hr. The reaction mixture was transferred to a separatory funnel and the organic phase was washed water (3×10 ml). The organic layer was stored at 4° C. for about 15 h and crystallization of the product occurred. The crystals were collected by filtration and washed with cold dichloromethane to provide Compound 90 as a crystalline solid. (1.2 g, 34%). UV λmax 215, 250 nm. 1H NMR (DMSO) δ 8.78 (1H, bs), 7.04 (2H, bd, J=7.2 Hz), 6.43 (2H, d, J=7.2 Hz), 4.72 (2H, s), 1.41 (9H, s).
Example 39
Preparation of Compound 91
A solution of cAC10 (10 mg/mL in 25 mM sodium citrate, 250 mM sodium chloride, 0.02% Tween 80, pH 6.5) was adjusted to pH 7.5 by addition of 0.3 M sodium phosphate, dibasic. To this pH-adjusted cAC10 solution, EDTA was added to a final concentration of 5 mM. The cAC10 solution was then pre-heated to 37° C. by incubation in a temperature-controlled oven. After the temperature of the cAC10 solution has equilibrated to 37° C., DTT (from a stock solution of 10 mM) was added to achieve a final DTT-to-cAC10 molar ratio of about 3.0 in the reduction reaction (a molecular weight of 148,500 Da was used for cAC10). The reduction reaction was then allowed to proceed for 2 hours at 37° C.
At the end of the incubation, the reduction reaction was cooled to an internal temperature of 2 to 8° C. in an ice-water bath. The temperature of the solution was kept at 2 to 8° C. throughout the remaining conjugation steps. The chilled reduction reaction was subjected to constant-volume diafiltration to remove excess DTT using a 30 kDa membrane and the buffer was exchanged into phosphate buffered saline, pH 7.4 (PBS). Following diafiltration, the concentration of free thiol in the reduced and diafiltered cAC10 was determined using General Procedure M. Conjugation is then carried out by addition of a 15% molar excess of Compound 58 (from a stock solution of 13 mg/mL in DMSO) relative to the total thiols determined using General Procedure M. Additional DMSO was added to the conjugation reaction to achieve a final DMSO concentration of 15% (v/v). The conjugation reaction was allowed to proceed for a total of 30 min.
At the end of the conjugation reaction, any unreacted excess Drug-Linker compound was quenched by addition of excess Cysteine (2× molar excess relative to the total thiols determined using General Procedure M, performed on the reduced and diafiltered cAC10 to produce the quenched reaction mixture. The quenched reaction mixture is then purified free of small-molecule contaminants via constant-volume diafiltration using a 30 kDa membrane and the buffer was exchanged into PBS, pH 7.4. After diafiltration, the conjugate was sterile-filtered using a 0.22 micron filter to provide Compound 91 in a clear, colorless solution.
Example 40
Preparation of Compound 92
Compound 92 was prepared using the method described in Example 39 using an amount of DTT (from a stock solution of 10 mM) which provides a final DTT-to-cAC10 molar ratio of about 1.5 in the reduction reaction.
6.2 In Vitro Cytotoxicity Experiments
The cell lines used were H3396 human breast carcinoma (cBR96 antigen positive, cAC10 antigen negative), HCT-116 human colorectal carcinoma (cBR96 and cAC10 antigen negative), and Karpas human anaplastic large cell lymphoma (ALCL) (cBR96 antigen negative, cAC10 antigen positive). These cell lines are available from ATCC. CD30-positive Hodgkin's Disease (HD) cell line L540 and the ALCL cell line Karpas 299 were obtained from the Deutsche Sammlung von Mikroorganism und Zellkulturen GmbH (Braunschweig, Germany). L540cy, a derivative of the HD line L540 adapted to xenograft growth, was provided by Dr. Phil Thorpe (U of Texas Southwestern Medical Center, Dallas, Tex.). Cell lines were grown in RPMI-1640 media (Life Technologies Inc., Gaithersburg, Md.) supplemented with 10% fetal bovine serum. H3396 cells in RPMI containing 10% fetal bovine serum (referred to as medium) were plated in 96-well plates at approximately 3,000-10,000 cells/well and allowed to adhere overnight. The non-adherent Karpas cell line was plated out at approximately 10,000 cells/well at the initiation of the assay. Various concentrations of illustrative Compounds of the Invention in medium were added in triplicate, and after the times indicated IN FIGS. 1-7, the medium was removed, and the cells were washed with fresh medium three times. After a 96 hour incubation period at 37° C., Alamar Blue was added and cell viability was determined 4 hours later as described by Ahmed S A, Gogal R M Jr, Walsh J E., J. Immunol. Methods, 170, 211-224, 1994.
C.B.-17 SCID (Harlan, Indianapolis, Ind.) mice were used for in vivo experiments.
Example 41
In Vitro Cytotoxicity Data
The cytotoxic effects of Compound 49 and Compound 53 on H3396 human breast carcinoma cells are shown in FIG. 1. The data show that after exposure for 1 hour, Compound 53 is more cytotoxic than Compound 49 at concentrations of up to 0.01 mM. The compounds show substantially equal cytotoxicity at concentrations between 0.01 mM and 1.0 mM.
Example 42
In Vitro Cytotoxicity Data
FIG. 2 shows the cytotoxic effects of Compounds 64, 65, 68 and 69 on H3396 human breast carcinoma cells (cBR96 antigen positive, cAC10 antigen negative). The data show that the Compounds 64 and 68 demonstrate similar and significant cytotoxicity, while Compounds 65 and 69 are less efficacious, but nevertheless cytotoxic against H3396 cells in this particular assay.
Example 43
In Vitro Cytotoxicity Data
FIG. 3 shows the cytotoxic effects of Compounds 64, 65, 68 and 69 on HCT-116 human colorectal carcinoma cells (cBR96 antigen negative, cAC10 antigen negative). The data illustrate that none of Compounds 64, 65, 68 and 69 is cytotoxic toward the antigen negative HCT-116 cells in this assay.
Example 44
In Vitro Cytotoxicity Data
FIG. 4 illustrates the cytotoxicity of Compounds 66 and 68 on H3396 human breast carcinoma cells (cBR96 antigen positive). The data show that both Compounds are highly cytotoxic at concentrations above 0.1 mM and that Compound 68 demonstrates greater cytotoxicity than Compound 66 at concentrations between 0.01 mg/mL and 0.4 mg/mL.
Example 45
In Vitro Cytotoxicity Data
FIG. 5 illustrates the cytotoxicity of Compounds 66, 68 and 69 on Karpas human anaplastic large cell lymphoma (cBR96 antigen negative, cAC10 antigen positive). The data show that Compound 69 was more cytotoxic toward Karpas cells than compared to Compounds 68 and 66 in this assay. Compound 69 demonstrated significant cytotoxicity at concentrations above 0.001 mM, while Compound 66 and Compound 68 were not cytotoxic at concentrations below 1.0 mg/mL.
Example 46
In Vitro Cytotoxicity Data
FIG. 6 illustrates the cytotoxicity of Compound 66 and 67 at 2 h and 96 h on H3396 human breast carcinoma cells (cBR96 antigen positive, cAC10 antigen negative). The data show that Compound 66 is highly cytotoxic at concentrations above 100 mg/mL at short-term exposure (2 h) mg/mL, and at concentrations above 100 mg/mL over long-term exposure (96 h). Compound 67 did not demonstrate cytotoxicity against H3396 cells in this assay at concentrations up to 1000 mg/mL.
General Procedure S: In Vivo Testing of Selected Drug-Linker-Antibody Conjugates. For the L2987 human adenocarcinoma cell line, Athymic nude mice (8-10 weeks old) were implanted with xenograft tumors or tumor cells. For the Karpas human anaplastic large cell lymphoma model, CB-17 SCID mice were implanted subcutaneously with 5×106 cells. In both tumor models, therapy was initiated once the tumors reached an average volume of 100 mm3. Groups of mice were injected with one of Compounds 66-69 in phosphate buffered saline intravenously every fours days for a total of 6 injections for L2987 animals and 2 injections for Karpas animals. Tumor volume was computed using the formula: 0.5 (longest dimension×perpendicular dimension2). Mice were removed from the study when their tumors were approximately 1000 mm3, at which point the average tumor sizes from the particular group were no longer plotted.
Example 47
In Vivo Therapeutic Efficacy on L2987 Tumors
FIG. 7 shows the therapeutic effects of Compounds 66-69 on L2987 human lung adenocarcinoma xenograft tumors (cBR96 antigen positive, cAC10 antigen negative) implanted in athymic nude mice. General Procedure S was followed using subcutaneous L2987 human lung tumors (from in vivo passaging). Mice were administered by injection with one of Compounds 66, 67, 68 or 69 at four day intervals for a total of 6 injections. The first injection was given at 15 days post tumor-implant. The data illustrate that administration of Compound 66 and Compound 68 markedly reduced tumor volume and no additional growth was noted in treated mice for at approximately 25 days after the last injection. Compound 67 and Compound 69 were less efficacious but nevertheless inhibited tumor cell multiplication in the treated mice. Testing was stopped in animals receiving Compounds 67 and 69 when tumor volume exceeded 1000 mm3.
Example 48
In Vivo Therapeutic Efficacy on Karpas Tumors
FIG. 8 shows the therapeutic effects of compounds 66-69 on Karpas human anaplastic large cell lymphoma xenograft tumors (cAC10 antigen positive, cBR96 antigen negative) implanted in nude mice. General Procedure S was followed using Karpas human anaplastic large cell lymphoma model, CB-17 SCID mice were implanted subcutaneously with 5×106 cells. Mice were dosed intravenously with one of Compounds 66, 67, 68 or 69 at four day intervals for a total of 2 injections starting on day 8. The data illustrate that Compounds 67 and 69 induced complete regressions, and that the tumors progressed in animals that received substantially equivalent amounts of Compounds 66 and 68.
Example 49
Determination of Cytotoxicity of Selected Compounds in CD30− and Cd30+ Cells
Following their physical characterization, the in vitro cytotoxicity of Compounds 67, 91 and 92 was evaluated in CD30+ Karpas 299 and CD30− Raji cells using the Alamar Blue assay as described above. The percent viable cells was plotted versus concentration for each molecule to determine the IC50 (defined as the mAb concentration that gave 50% cell kill).
Compound 67 demonstrated activity against Karpas 299 cells with an IC50 of 4 ng/mL. The IC50 was inversely proportional to drug loading as it increased from 4 ng/mL for Compound 67 to 7 ng/mL for Compound 91, to 40 ng/mL for Compound 92. Selectivity of the tested compounds was evaluated using the antigen-negative Raji cell line which were insensitive to all cAC10-containing Compounds with IC50 values >1000 ng/ml for Compounds 67, 91 and 92.
Example 50
Cytotoxicity of Selected Compounds in Xenograft Models of HD and ALCL
Cytotoxicity of Compounds 67, 91 and 92 was evaluated in subcutaneous Karpas 299 human anaplastic large cell lymphoma and L540cy Hodgkin's Disease xenograft models in C.B.-17 SCID mice. Evaluations were initiated when tumor volumes averaged 50-100 mm3. Cohorts of Karpas-299 bearing mice were injected q4d×4 with Compound 92, Compound 91, or Compound 67 at either 0.25 mg/kg or 0.5 mg/kg. None of the animals treated at 0.25 mg/kg had a regression, although there was a delay in tumor growth compared to untreated controls for the animals treated with Compound 91 and Compound 67. Treatment of Karpas tumors with Compound 91 and Compound 67 at 0.5 mg/kg given q4d×4 achieved 5/5 complete regressions and ⅘ complete regressions, respectively. A delay in tumor growth compared to untreated animals was observed for Compound 92 at 0.5 mg/kg given q4d×4, but no complete regressions were obtained.
Efficacy was also tested in a subcutaneous Karpas model with selected compounds administered as a single dose. Compound 91 and Compound 67 were injected at single doses of 0.25, 0.5 and 2.0 mg/kg. At the dose of 0.25 mg/kg there was no antitumor activity in either group and mean tumor volume did not deviate from the untreated controls. A delay in the tumor growth was demonstrated by both molecules at 0.5 mg/kg, but no complete regressions were obtained. Treating the mice with Compound 91 and Compound 67 at 2 mg/kg achieved 100% complete regressions in both groups.
Compound 91 and Compound 67 were also compared in mice bearing subcutaneous L540cy human HD tumors treated q4d×4 with Compound 91 and Compound 67 at 1 and 3 mg/kg. At 1 mg/kg, mice treated with Compound 91 and Compound 67 had significant delays in tumor growth compared to the untreated animals. Complete regressions were observed in mice administered with both Compound 91 and Compound 67 at 3 mg/kg.
The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the are and are intended to fall within the scope of the appended claims.
A number of references have been cited, the entire disclosures of which are incorporated herein by reference.
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12408646
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/2
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Oct 20th, 2020 12:00AM
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Jul 10th, 2019 12:00AM
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https://www.uspto.gov?id=US10808039-20201020
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Monomethylvaline compounds capable of conjugation to ligands
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Auristatin peptides, including MeVal-Val-Dil-Dap-Norephedrine (MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), were prepared and attached to Ligands through various linkers, including maleimidocaproyl-val-cit-PAB. The resulting ligand drug conjugates were active in vitro and in vivo.
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10808039
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1. An antibody-drug conjugate having the formula:
or a pharmaceutically acceptable salt thereof, wherein:
Ab is an antibody,
S is sulfur,
each —Ww— unit is a tetrapeptide; wherein each —W— unit is independently an Amino Acid unit having the formula denoted below in the square bracket:
wherein R19 is hydrogen or benzyl,
Y is a Spacer unit,
y is 0, 1 or 2,
D is a drug moiety, and
p ranges from 1 to about 20,
wherein the S is a sulfur atom on a cysteine residue of the antibody, and
wherein the drug moiety is intracellularly cleaved in a patient from the antibody of the antibody-drug conjugate or an intracellular metabolite of the antibody-drug conjugate.
2. The antibody-drug conjugate of claim 1, wherein Y is a self-immolative spacer.
3. The antibody-drug conjugate of claim 2, wherein y is 1.
4. The antibody-drug conjugate of claim 3, wherein p is about 3 to about 8.
5. The antibody-drug conjugate of claim 4, wherein p is about 8.
6. The antibody-drug conjugate of claim 1, 2, 3, 4, or 5, wherein the bioavailability of the antibody-drug conjugate or an intracellular metabolite of the antibody-drug conjugate in a patient is improved when compared to a drug compound comprising the drug moiety of the antibody-drug conjugate.
7. The antibody-drug conjugate compound of claim 1, 2, 3, 4, or 5, wherein the bioavailability of the antibody-drug conjugate or an intracellular metabolite of the antibody-drug conjugate in a patient is improved when compared to an analog of the antibody-drug conjugate not having the drug moiety.
8. The antibody-drug conjugate compound of claim 1, 2, 3, 4, or 5, wherein the drug moiety is intracellularly cleaved in a patient from an intracellular metabolite of the antibody-drug conjugate.
9. The antibody-drug conjugate of claim 1, 2, 3, 4, or 5, wherein the antibody is a monoclonal antibody.
10. The antibody-drug conjugate of claim 9, wherein the antibody is a humanized monoclonal antibody.
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10
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1. CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 15/811,190 filed Nov. 13, 2017, which is a continuation of U.S. patent application Ser. No. 15/188,843 filed Jun. 21, 2016, which is a continuation of U.S. patent application Ser. No. 14/194,106 filed Feb. 28, 2014, which is a continuation of U.S. patent application Ser. No. 13/098,391 filed Apr. 29, 2011 (now U.S. Pat. No. 8,703,714), which is a continuation of U.S. patent application Ser. No. 11/833,954 filed Aug. 3, 2007 (now U.S. Pat. No. 7,994,135), which is a divisional of U.S. patent application Ser. No. 10/983,340 filed Nov. 5, 2004 (now U.S. Pat. No. 7,498,298), which is an application claiming the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60/518,534, filed Nov. 6, 2003, and U.S. Provisional Patent Application No. 60/557,116, filed Mar. 26, 2004, and U.S. Provisional Patent Application No. 60/598,899, filed Aug. 4, 2004, and U.S. Provisional Patent Application No. 60/622,455, filed Oct. 27, 2004, each of which is incorporated herein by reference in its entirety.
2. JOINT RESEARCH AGREEMENT
Some of the subject matter in this application was made by or on behalf of Seattle Genetics, Inc. and Genentech, Inc. as a result of activities undertaken within the scope of a joint research agreement effective on or before the date the claimed invention was made.
3. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
NOT APPLICABLE
4. REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK
The sequence information in the paper copy of the Sequence Listing filed herewith is identical to the sequence information in the only computer readable form which was filed on Apr. 29, 2011 in application Ser. No. 13/098,391 filed Apr. 29, 2011. A request for Transfer of a Computer Readable Form Under 37 C.F.R. § 1.821(e) accompanies this filing.
5. FIELD OF THE INVENTION
The present invention is directed to a Drug Compound and more particularly to Drug-Linker-Ligand Conjugates, Drug-Linker Compounds, and Drug-Ligand Conjugates, to compositions including the same, and to methods for using the same to treat cancer, an autoimmune disease or an infectious disease. The present invention is also directed to antibody-drug conjugates, to compositions including the same, and to methods for using the same to treat cancer, an autoimmune disease or an infectious disease. The invention also relates to methods of using antibody-drug conjugate compounds for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, or associated pathological conditions.
6. BACKGROUND OF THE INVENTION
Improving the delivery of drugs and other agents to target cells, tissues and tumors to achieve maximal efficacy and minimal toxicity has been the focus. of considerable research for many years. Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g., to neighboring cells, is often difficult or inefficient.
Most agents currently administered to a patient parenterally are not targeted, resulting in systemic delivery of the agent to cells and tissues of the body where it is unnecessary, and often undesirable. This may result in adverse drug side effects, and often limits the dose of a drug (e.g., chemotherapeutic (anti-cancer), cytotoxic, enzyme inhibitor agents and antiviral or antimicrobial drugs) that can be administered. By comparison, although oral administration of drugs is considered to be a convenient and economical mode of administration, it shares the same concerns of non-specific toxicity to unaffected cells once the drug has been absorbed into the systemic circulation. Further complications involve problems with oral bioavailability and residence of drug in the gut leading to additional exposure of gut to the drug and hence risk of gut toxicities. Accordingly, a major goal has been to develop methods for specifically targeting agents to cells and tissues. The benefits of such treatment include avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues, such as uninfected cells. Intracellular targeting may be achieved by methods, compounds and formulations which allow accumulation or retention of biologically active agents, i.e. active metabolites, inside cells.
Monoclonal antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders.
The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, e.g., drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) theoretically allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, while systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., 1986, Lancet pp. (Mar. 15, 1986):603-05; Thorpe, 1985, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby. Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., 1986, supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Kerr et al., 1997, Bioconjugate Chem. 8(6):781-784; Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342). The toxins may affect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition (Meyer, D. L. and Senter, P. D. “Recent Advances in Antibody Drug Conjugates for Cancer Therapy” in Annual Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229-237). Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate composed of a murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 111In or 90Y radioisotope bound by a thiourea linker-chelator (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DM1, is advancing into Phase II trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody drug conjugate composed of the anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1, is under development for the potential treatment of prostate tumors. The same maytansinoid drug moiety, DM1, was linked through a non-disulfide linker, SMCC, to a mouse murine monoclonal antibody, TA.1 (Chari et al. (1992) Cancer Research 52:127-131). This conjugate was reported to be 200-fold less potent than the corresponding disulfide linker conjugate. The SMCC linker was considered therein to be “noncleavable.”
Several short peptidic compounds have been isolated from the marine mollusc Dolabella auricularia and found to have biological activity (Pettit et al. (1993) Tetrahedron 49:9151; Nakamura et al. (1995) Tetrahedron Letters 36:5059-5062; Sone et al. (1995) Jour. Org Chem. 60:4474). Analogs of these compounds have also been prepared, and some were found to have biological activity (for a review, see Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277). For example, auristatin E (U.S. Pat. No. 5,635,483) is a synthetic analogue of the marine natural product Dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same domain on tubulin as the anticancer drug vincristine (G. R. Pettit, (1997) Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds, and a C-terminal amide.
The auristatin peptides, auristain E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to: (i) chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas); (ii) cAC10 which is specific to CD30 on hematological malignancies (Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation to Ligands”; Francisco et al. (2003) Blood 102(4):1458-1465; U.S. Publication 2004/0018194; (iii) anti-CD20 antibodies such as RITUXAN® (WO 04/032828) for the treatment of CD20-expressing cancers and immune disorders; (iv) anti-EphB2 antibodies 2H9 and anti-IL-8 for treatment of colorectal cancer (Mao, et al. (2004) Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskar et al. (2003) Cancer Res. 63:6387-6394); and (vi) other anti-CD30 antibodies (WO 03/043583).
Auristatin E conjugated to monoclonal antibodies are disclosed in Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004.
Despite in vitro data for compounds of the dolastatin class and its analogs, significant general toxicities at doses required for achieving a therapeutic effect compromise their efficacy in clinical studies. Accordingly, there is a clear need in the art for dolastatin/auristatin derivatives having significantly lower toxicity, yet useful therapeutic efficiency. These and other limitations and problems of the past are addressed by the present invention.
The ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel of anti-ErbB2 antibodies has been characterized using the human breast tumor cell line SKBR3 (Hudziak et al., (1989) Mol. Cell. Biol. 9(3):1165-1172. Maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize ErbB2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF-α (U.S. Pat. No. 5,677,171). The anti-ErbB2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al. (1990) Cancer Research 50:1550-1558; Kotts et al. (1990) In vitro 26(3):59A; Sarup et al. (1991) Growth Regulation 1:72-82; Shepard et al. J. (1991) Clin. Immunol. 11(3):117-127; Kumar et al. (1991) Mol. Cell. Biol. 11(2):979-986; Lewis et al. (1993) Cancer Immunol. Immunother. 37:255-263; Pietras et al. (1994) Oncogene 9:1829-1838; Vitetta et al. (1994) Cancer Research 54:5301-5309; Sliwkowski et al. (1994) J. Biol. Chem. 269(20):14661-14665; Scott et al. (1991) J. Biol. Chem. 266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994) 91:7202-7206; Lewis et al. (1996) Cancer Research 56:1457-1465; and Schaefer et al. (1997) Oncogene 15:1385-1394.
Other anti-ErbB2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. Proc. Natl. Acad. Sci. USA 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al. (1991) Cancer Res. 51:4575-4580; Shawver et al. (1994) Cancer Res. 54:1367-1373; Arteaga et al. (1994) Cancer Res. 54:3758-3765; Harwerth et al. (1992) J. Biol. Chem. 267:15160-15167; U.S. Pat. No. 5,783,186; and Klapper et al. (1997) Oncogene 14:2099-2109.
Homology screening has resulted in the identification of two other ErbB receptor family members; ErbB3 (U.S. Pat. Nos. 5,183,884; 5,480,968; Kra U. S. et al. (1989) Proc. Natl. Acad. Sci. USA 86:9193-9197) and ErbB4 (EP 599274; Plowman et al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750; and Plowman et al. (1993) Nature 366:473-475). Both of these receptors display increased expression on at least some breast cancer cell lines. HERCEPTIN® (Trastuzumab) is a recombinant DNA-derived humanized monoclonal antibody that selectively binds with high affinity in a cell-based assay (Kd=5 nM) to the extracellular domain of the human epidermal growth factor receptor2 protein, HER2 (ErbB2) (U.S. Pat. Nos. 5,821,337; 6,054,297; 6,407,213; 6,639,055; Coussens L, et al. (1985) Science 230:1132-9; Slamon D J, et al. (1989) Science 244:707-12). Trastuzumab is an IgG1 kappa antibody that contains human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen and thus. inhibits the growth of cancerous cells. Because Trastuzumab is a humanized antibody, it minimizes any HAMA response in patients. The humanized antibody against HER2 is produced by a mammalian cell (Chinese Hamster Ovary, CHO) suspension culture. The HER2 (or c-erbB2) proto-oncogene encodes a transmembrane receptor protein of 185 kDa, which is structurally related to the epidermal growth factor receptor. HER2 protein overexpression is observed in 25%-30% of primary breast cancers and can be determined using an immunohistochemistry based assessment of fixed tumor blocks (Press M F, et al. (1993) Cancer Res 53:4960-70. Trastuzumab has been shown, in both in vitro assays and in animals, to inhibit the proliferation of human tumor cells that overexpress HER2 (Hudziak R M, et al. (1989) Mol Cell Biol 9:1165-72; Lewis G D, et al. (1993) Cancer Immunol Immunother; 37:255-63; Baselga J, et al. (1998) Cancer Res. 58:2825-2831). Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, ADCC (Hotaling T E, et al. (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471; Pegram M D, et al. (1997) [abstract]. Proc Am Assoc Cancer Res; 38:602). In vitro, Trastuzumab mediated ADCC has been shown to be preferentially exerted on HER2 overexpressing cancer cells compared with cancer cells that do not overexpress HER2. HERCEPTIN® as a single agent is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have received one or more chemotherapy regimens for their metastatic disease. HERCEPTIN® in combination with paclitaxel is indicated for treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have not received chemotherapy for their metastatic disease. HERCEPTIN® is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al, (1996) J. Clin. Oncol. 14:737-744).
The murine monoclonal anti-HER2 antibody inhibits the growth of breast cancer cell lines that overexpress HER2 at the 2+ and 3+(1-2×106 HER2 receptors per cell) level, but has no activity on cells that express lower levels of HER2 (Lewis et al., (1993) Cancer Immunol. Immunother. 37:255-263). Based on this observation, antibody 4D5 was humanized (huMAb4D5-8, rhuMAb HER2, U.S. Pat. No. 5,821,337; Carter et al., (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289) and tested in breast cancer patients whose tumors overexpress HER2 but who had progressed after conventional chemotherapy (Cobleigh et al., (1999) J. Clin. Oncol. 17: 2639-2648).
Although HERCEPTIN is a breakthrough in treating patients with ErbB2-overexpressing breast cancers that have received extensive prior anti-cancer therapy, some patients in this population fail to respond or respond only poorly to HERCEPTIN treatment.
Therefore, there is a significant clinical need for developing further HER2-directed cancer therapies for those patients with HER2-overexpressing tumors or other diseases associated with HER2 expression that do not respond, or respond poorly, to HERCEPTIN treatment.
The recitation of any reference in this application is not an admission that the reference is prior art to this application.
7. SUMMARY OF THE INVENTION
In one aspect, the present invention provides Drug-Linker-Ligand compounds having the Formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt or solvate thereof
wherein,
L- is a Ligand unit;
-Aa-Ww—Yy— is a Linker unit (LU), wherein the Linker unit includes:
-A- is a Stretcher unit,
a is 0 or 1,
each —W— is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
—Y— is a Spacer unit, and
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit having the Formulas DE and DF:
wherein, independently at each location:
R2 is selected from H and C1-C8 alkyl;
R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R5 is selected from H and methyl;
or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from H and C1-C8 alkyl;
R7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O—(C1-C8 alkyl);
R9 is selected from H and C1-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or C1-C8 alkyl;
each occurrence of R15 is independently H, COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, C1-C8 alkyl, or —(CH2)n—COOH;
where; n is an integer ranging from 0 to 6; and
R18 is selected from —C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3-C8 heterocycle), and —C(R8)2—C(R8)2—(C3-C8 carbocycle).
In another aspect, Drug Compounds having the Formula Ib are provided:
or pharmaceutically acceptable salts or solvates thereof,
wherein:
R2 is selected from hydrogen and —C1-C8 alkyl;
R3 is selected from hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 jointly, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from H and —C1-C8 alkyl;
R7 is selected from H, —C1-C8 alkyl, —C3-C8 carbocycle, aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from H and —C1-C8 alkyl;
R10 is selected from aryl group or —C3-C8 heterocycle;
Z is —O—, —S—, —NH—, or —NR12—, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, —C3-C8 heterocycle, —(R13O)n—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is —C2-C8 alkyl; R14 is H or —C1-C8 alkyl;
each occurrence of R15 is independently H, —COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, —C1-C8 alkyl, or —(CH2)n—COOH; and
n is an integer ranging from 0 to 6.
The compounds of Formula (Ib) are useful for treating cancer, an autoimmune disease or an infectious disease in a patient or useful as an intermediate for the synthesis of a Drug-Linker, Drug-Linker-Ligand Conjugate, and Drug-Ligand Conjugate having a cleavable Drug unit.
In another aspect, compositions are provided including an effective amount of a Drug-Linker-Ligand Conjugate and a pharmaceutically acceptable carrier or vehicle.
In still another aspect, the invention provides pharmaceutical compositions comprising an effective amount of a Drug-Linker Compound and a pharmaceutically acceptable carrier or vehicle.
In still another aspect, the invention provides compositions comprising an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate and a pharmaceutically acceptable carrier or vehicle.
In yet another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for treating an infectious. Disease including administering to a patient in need thereof an effective amount of a Drug-Linker
Compound.
In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In yet another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand
Conjugate.
In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker Compound.
In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Ligand Conjugate.
In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate.
In another aspect, a Drug Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker Compound having a cleavable Drug unit from the Drug-Ligand
Conjugate.
In another aspect, a Drug-Linker Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.
In another aspect, compounds having Formula Ia′ are provided:
AbAa-Ww—Yy-D)p Ia′
or a pharmaceutically acceptable salt or solvate thereof, wherein:
Ab includes an antibody including one which binds to CD30, CD40, CD70, and Lewis Y antigen,
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 20, and
D is a Drug unit selected from Formulas DE and DF:
wherein, independently at each location:
R2 is selected from H and C1-C8 alkyl;
R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R5 is selected from H and methyl;
or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from H and C1-C8 alkyl;
R7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O—(C1-C8 alkyl);
R9 is selected from H and C1-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or C1-C8 alkyl;
each occurrence of R15 is independently H, COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, C1-C8 alkyl, or —(CH2)n—COOH;
R18 is selected from —C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3-C8 heterocycle), and —C(R8)2—C(R8)2—(C3-C8 carbocycle); and
n is an integer ranging from 0 to 6.
In one embodiment, Ab is not an antibody which binds to an ErbB receptor or which binds to one or more of receptors (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);
(7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain I and short cytoplasmic domain, (18emaphoring) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN Cdna 2700050C12, RIKEN Cdna 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763);
(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);
(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP 003203 or NM_003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792, Genbank accession no. M26004);
(15) CD79b (Igb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20Rα (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM_004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763);
(26) BAFF-R (Genbank accession no. NP_443177.1);
(27) CD22 (Genbank accession no. NP-001762.1);
(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation, Genbank accession No. NP_001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes, Genbank accession No. NP_002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accession No. NP_001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); or
(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1).
In still another aspect, the invention provides pharmaceutical compositions comprising an effective amount of a Drug-Linker-Antibody Conjugate and a pharmaceutically acceptable carrier or vehicle.
In still another aspect, the invention provides compositions comprising an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit (moiety) from the Drug-Antibody Conjugate and a pharmaceutically acceptable carrier or vehicle.
In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for killing or inhibiting the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In yet another aspect, the invention provides methods for treating cancer including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for killing or inhibiting the replication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In yet another aspect, the invention provides methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In still another aspect, the invention provides methods for treating an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In yet another aspect, the invention provides methods for preventing cancer including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In another aspect, the invention provides methods for preventing the multiplication of a cell that expresses an autoimmune antibody including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In yet another aspect, the invention provides methods for preventing an autoimmune disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Linker-Antibody Conjugate.
In still another aspect, the invention provides methods for preventing an infectious disease including administering to a patient in need thereof an effective amount of a Drug-Antibody Conjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, a Drug Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker Compound having a cleavable Drug unit from the Drug-Antibody Conjugate.
In another aspect, a Drug-Linker Compound is provided which can be used as an intermediate for the synthesis of a Drug-Linker-Antibody Conjugate.
In one aspect, the present invention provides Drug-Linker-Antibody Conjugates (also referred to as antibody-drug conjugates) having Formula Ic:
Ab Aa-Ww—Yy-D)p Ic
or a pharmaceutically acceptable salt or solvate thereof, wherein:
Ab is an antibody which binds to one or more of the antigens (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain I and short cytoplasmic domain, (24emaphoring) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN Cdna 2700050C12, RIKEN Cdna 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763);
(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);
(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792, Genbank accession no. M26004);
(15) CD79b (Igb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20Rα (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM_004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763);
(26) BAFF-R (Genbank accession no. NP_443177.1);
(27) CD22 (Genbank accession no. NP-001762.1);
(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation, Genbank accession No. NP_001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes, Genbank accession No. NP_002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accession No. NP_001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); or
(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1);
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 20, and
D is a Drug moiety selected from Formulas DE and DF:
wherein the wavy line of DE and DF indicates the covalent attachment site to A, W, or Y, and independently at each location:
R2 is selected from H and C1-C8 alkyl;
R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R5 is selected from H and methyl;
or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from H and C1-C8 alkyl;
R7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O—(C1-C8 alkyl);
R9 is selected from H and C1-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, —(R13O)m-R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or C1-C8 alkyl;
each occurrence of R15 is independently H, COOH, —(CH2)n-N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, C1-C8 alkyl, or —(CH2)n—COOH;
R18 is selected from —C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3-C8 heterocycle), and —C(R8)2—C(R8)2—(C3-C8 carbocycle); and
n is an integer ranging from 0 to 6.
In another aspect, the antibody of the antibody-drug conjugate (ADC) of the invention specifically binds to a receptor encoded by an ErbB2 gene.
In another aspect, the antibody of the antibody-drug conjugate is a humanized antibody selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (Trastuzumab).
In another aspect, the invention includes an article of manufacture comprising an antibody-drug conjugate compound of the invention; a container; and a package insert or label indicating that the compound can be used to treat cancer characterized by the overexpression of an ErbB2 receptor.
In another aspect, the invention includes a method for the treatment of cancer in a mammal, wherein the cancer is characterized by the overexpression of an ErbB2 receptor and does not respond, or responds poorly, to treatment with an anti-ErbB2 antibody, comprising administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound of the invention.
In another aspect, a substantial amount of the drug moiety is not cleaved from the antibody until the antibody-drug conjugate compound enters a cell with a cell-surface receptor specific for the antibody of the antibody-drug conjugate, and the drug moiety is cleaved from the antibody when the antibody-drug conjugate does enter the cell.
In another aspect, the bioavailability of the antibody-drug conjugate compound or an intracellular metabolite of the compound in a mammal is improved when compared to a drug compound comprising the drug moiety of the antibody-drug conjugate compound, or when compared to an analog of the compound not having the drug moiety.
In another aspect, the drug moiety is intracellularly cleaved in a mammal from the antibody of the compound, or an intracellular metabolite of the compound.
In another aspect, the invention includes a pharmaceutical composition comprising an effective amount of the antibody-drug conjugate compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient. The composition may further comprise a therapeutically effective amount of chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor, and a DNA binder.
In another aspect, the invention includes a method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating tumor cells or cancer cells with an amount of the antibody-drug conjugate compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.
In another aspect, the invention includes a method of inhibiting cellular proliferation comprising exposing mammalian cells in a cell culture medium to an antibody drug conjugate compound of the invention, wherein the antibody drug conjugate compound enters the cells and the drug is cleaved from the remainder of the antibody drug conjugate compound; whereby proliferation of the cells is inhibited.
In another aspect, the invention includes a method of treating cancer comprising administering to a patient a formulation of an antibody-drug conjugate compound of the invention and a pharmaceutically acceptable diluent, carrier or excipient.
In another aspect, the invention includes an assay for detecting cancer cells comprising:
(a) exposing cells to an antibody-drug conjugate compound of the invention; and
(b) determining the extent of binding of the antibody-drug conjugate compound to the cells.
The invention will best be understood by reference to the following detailed description of the exemplary embodiments, taken in conjunction with the accompanying drawings, figures, and schemes. The discussion below is descriptive, illustrative and exemplary and is not to be taken as limiting the scope defined by any appended claims.
8. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an in vivo, single dose, efficacy assay of cAC10-mcMMAF in subcutaneous Karpas-299 ALCL xenografts.
FIG. 2 shows an in vivo, single dose, efficacy assay of cAC10-mcMMAF in subcutaneous L540cy. For this study there were 4 mice in the untreated group and 10 in each of the treatment groups.
FIGS. 3a and 3b show in vivo efficacy of cBR96-mcMMAF in subcutaneous L2987. The filed triangles in FIG. 3a and arrows in FIG. 3b indicate the days of therapy.
FIGS. 4a and 4b show in vitro activity of cAC10-antibody-drug conjugates against CD30+ cell lines.
FIGS. 5a and 5b show in vitro activity of cBR96-antibody-drug conjugates against Ley+ cell lines.
FIGS. 6a and 6b show in vitro activity of c1F6-antibody-drug conjugates against CD70+ renal cell carcinoma cell lines.
FIG. 7 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with antibody drug conjugates (ADC): -●- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -∘- Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -Δ- Trastuzumab-MC-MMAF, 4.8 MMAF/Ab, measured in Relative Fluorescence Units (RLU) versus μg/ml concentration of ADC. H=Trastuzumab where H is linked via a cysteine [cys].
FIG. 8 shows an in vitro, cell proliferation assay with BT-474 cells treated with ADC: -●- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -∘- Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -Δ- Trastuzumab-MC-MMAF, 4.8 MMAF/Ab.
FIG. 9 shows an in vitro, cell proliferation assay with MCF-7 cells treated with ADC: -●- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -∘- Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab, and -Δ- Trastuzumab-MC-MMAF, 4.1 MMAF/Ab.
FIG. 10 shows an in vitro, cell proliferation assay with MDA-MB-468 cells treated with ADC: -●- Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab, -∘- Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab, and -Δ- Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.
FIG. 11 shows a plasma concentration clearance study after administration of H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF to Sprague-Dawley rats: The administered dose was 2 mg of ADC per kg of rat. Concentrations of total antibody and ADC were measured over time. (H=Trastuzumab).
FIG. 12 shows a plasma concentration clearance study after administration of H-MC-vc-MMAE to Cynomolgus monkeys at different doses: 0.5, 1.5, 2.5, and 3.0 mg/kg administered at day 1 and day 21. Concentrations of total antibody and ADC were measured over time. (H=Trastuzumab).
FIG. 13 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 μg/m2) and Trastuzumab-MC-vc-PAB-MMAF (555 μg/m2). (H=Trastuzumab).
FIG. 14 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10 mg/kg (660 μg/m2) of Trastuzumab-MC-MMAE and 1250 μg/m2 Trastuzumab-MC-vc-PAB-MMAE.
FIG. 15 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with Vehicle and 650 μg/m2 trastuzumab-MC-MMAF.
FIG. 16 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with Vehicle and 350 μg/m2 of four trastuzumab-MC-MMAF conjugates where the MMAF/trastuzumab (H) ratio is 2, 4, 5.9 and 6.
FIG. 17 shows the Group mean change, with error bars, in animal (rat) body weights (Mean±SD) after administration of Vehicle, trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PAB-MMAF.
FIG. 18 shows the Group mean change in animal (rat) body weights (Mean SD) after administration of 9.94 mg/kg H-MC-vc-MMAF, 24.90 mg/kg H-MC-vc-MMAF, 10.69 mg/kg H-MC(Me)-vc-PAB-MMAF, 26.78 mg/kg H-MC(Me)-vc-PAB-MMAF, 10.17 mg/kg H-MC-MMAF, 25.50 mg/kg H-MC-MMAF, and 21.85 mg/kg H-MC-vc-PAB-MMAF. H=trastuzumab. The MC linker is attached via a cysteine of trastuzumab for each conjugate.
FIG. 19 shows the Group mean change, with error bars, in Sprague Dawley rat body weights (Mean±SD) after administration of trastuzumab (H)-MC-MMAF at doses of 2105, 3158, and 4210 μg/m2. The MC linker is attached via a cysteine of trastuzumab for each conjugate.
FIG. 20 shows examples of compounds with a non self-immolative Spacer unit.
FIG. 21 shows a scheme of a possible mechanism of Drug release from a PAB group which is attached directly to -D via a carbamate or carbonate group.
FIG. 22 shows a scheme of a possible mechanism of Drug release from a PAB group which is attached directly to -D via an ether or amine linkage.
FIG. 23 shows an example of a branched spacer unit, bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate and release multiple drug.
FIG. 24 shows a scheme of the CellTiter-Glo® Assay.
FIG. 25 shows the synthesis of an N-terminal tripeptide unit F which is a useful intermediate for the synthesis of the drug compounds of Formula Ib.
FIG. 26 shows the synthesis of an N-terminal tripeptide unit F which is a useful intermediate for the synthesis of the drug compounds of Formula Ib.
FIG. 27 shows the synthesis of an N-terminal tripeptide unit F which is a useful intermediate for the synthesis of the drug compounds of Formula Ib.
FIG. 28 shows the synthesis of useful linkers.
FIG. 29 shows the synthesis of useful linkers.
FIG. 30 shows a general synthesis of an illustrative Linker unit containing a maleimide Stretcher group and optionally a p-aminobenzyl ether self-immolative Spacer.
FIG. 31 shows the synthesis of a val-cit dipeptide Linker having a maleimide Stretcher and optionally a p-aminobenzyl self-immolative Spacer.
FIG. 32 shows the synthesis of a phe-lys(Mtr) dipeptide Linker unit having a maleimide Stretcher unit and a p-aminobenzyl self-immolative Spacer unit.
FIG. 33 shows the synthesis of a Drug-Linker Compound that contains an amide or carbamate group, linking the Drug unit to the Linker unit.
FIG. 34 shows illustrative methods useful for linking a Drug to a Ligand to form a Drug-Linker Compound.
FIG. 35 shows the synthesis of a val-cit dipeptide linker having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS) unit.
FIG. 36 shows methodology useful for making Drug-Linker-Ligand conjugates having about 2 to about 4 drugs per antibody.
FIG. 37 shows the synthesis of MC-MMAF via t-butyl ester.
FIG. 38 shows the synthesis of MC-MMAF (11) via dimethoxybenzyl ester.
FIG. 39 shows the synthesis of analog of mc-MMAF.
9. DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
9.1 Definitions and Abbreviations
Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
When trade names are used herein, applicants intend to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product.
The term “antibody” herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multi specific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. Described in terms of its structure, an antibody typically has a Y-shaped protein consisting of four amino acid chains, two heavy and two light. Each antibody has primarily two regions: a variable region and a constant region. The variable region, located on the ends of the arms of the Y, binds to and interacts with the target antigen. This variable region includes a complementary determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region, located on the tail of the Y, is recognized by and interacts with the immune system (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
The term “antibody” as used herein, also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin. In another aspect, the antibodies are polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F(ab′) fragments, F(ab′)2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature, 352:624-628 and Marks et al. (1991) J. Mol. Biol., 222:581-597, for example.
The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855).
Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses the cells of various species, including mice (murine), hamsters, rats, and humans. Another method to prepare MAbs uses genetic engineering including recombinant DNA techniques. Monoclonal antibodies made from these techniques include, among others, chimeric antibodies and humanized antibodies. A chimeric antibody combines DNA encoding regions from more than one type of species. For example, a chimeric antibody may derive the variable region from a mouse and the constant region from a human. A humanized antibody comes predominantly from a human, even though it contains nonhuman portions. Like a chimeric antibody, a humanized antibody may contain a completely human constant region. But unlike a chimeric antibody, the variable region may be partially derived from a human. The nonhuman, synthetic portions of a humanized antibody often come from CDRs in murine antibodies. In any event, these regions are crucial to allow the antibody to recognize and bind to a specific antigen.
As noted, murine antibodies can be used. While useful for diagnostics and short-term therapies, murine antibodies cannot be administered to people long-term without increasing the risk of a deleterious immunogenic response. This response, called Human Anti-Mouse Antibody (HAMA), occurs when a human immune system recognizes the murine antibody as foreign and attacks it. A HAMA response can cause toxic shock or even death.
Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the nonhuman portions of administered antibodies. Furthermore, chimeric and humanized antibodies have the additional benefit of activating secondary human immune responses, such as antibody dependent cellular cytotoxicity.
“Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragment(s).
An “intact” antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHL CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes.” There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The expressions “ErbB2” and “HER2” are used interchangeably herein and refer to human HER2 protein described, for example, in Semba et al., Proc. Natl. Acad. Sci. USA, 82:6497-6501 (1985) and Yamamoto et al., (1986) Nature, 319:230-234 (Genebank accession number X03363). The term “erbB2” refers to the gene encoding human ErbB2 and “neu” refers to the gene encoding rat p185neu. Preferred ErbB2 is native sequence human ErbB2.
Antibodies to ErbB receptors are available commercially from a number of sources, including, for example, Santa Cruz Biotechnology, Inc., California, USA.
By “ErbB ligand” is meant a polypeptide which binds to and/or activates an ErbB receptor. The ErbB ligand may be a native sequence human ErbB ligand such as epidermal growth factor (EGF) (Savage et al. (1972) J Biol. Chem., 247:7612-7621); transforming growth factor alpha (TGF-α) (Marquardt et al. (1984) Science 223:1079-1082); amphiregulin also known as schwanoma or keratinocyte autocrine growth factor (Shoyab et al. (1989) Science 243:1074-1076; Kimura et al., Nature, 348:257-260 (1990); and Cook et al., Mol. Cell. Biol., 11:2547-2557 (1991)); betacellulin (Shing et al., Science, 259:1604-1607 (1993); and Sasada et al., Biochem. Biophys. Res. Commun., 190:1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al., Science, 251:936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem., 270:7495-7500 (1995); and Komurasaki et al., Oncogene, 15:2841-2848 (1997)); a heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al., Nature, 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Natl. Acad. Sci., 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et al., Oncogene, 18:2681-89 (1999)) or cripto (CR-1) (Kannan et al., J Biol. Chem., 272(6):3330-3335 (1997)). ErbB ligands which bind EGFR include EGF, TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin. ErbB ligands which bind ErbB3 include heregulins. ErbB ligands capable of binding ErbB4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulins. The ErbB ligand may also be a synthetic ErbB ligand.
The synthetic ligand may be specific for a particular ErbB receptor, or may recognize particular ErbB receptor complexes. An example of a synthetic ligand is the synthetic heregulin/EGF chimera biregulin (see, for example, Jones et al., (1999) FEBS Letters, 447:227-231, which is incorporated by reference).
“Heregulin” (HRG) refers to a polypeptide encoded by the heregulin gene product as disclosed in U.S. Pat. No. 5,641,869 or Marchionni et al., Nature, 362:312-318 (1993). Examples of heregulins include heregulin-α, heregulin-β1, heregulin-β2 and heregulin-β3 (Holmes et al., Science, 256:1205-1210 (1992); and U.S. Pat. No. 5,641,869); neu differentiation factor (NDF) (Peles et al., Cell 69: 205-216 (1992)); acetylcholine receptor-inducing activity (ARIA) (Falls et al. (1993) Cell 72:801-815); glial growth factors (GGFs) (Marchionni et al., Nature, 362:312-318 (1993)); sensory and motor neuron derived factor (SMDF) (Ho et al., J. Biol. Chem., 270:14523-14532 (1995)); γ-heregulin (Schaefer et al., Oncogene, 15:1385-1394 (1997)). The term includes biologically active fragments and/or amino acid sequence variants of a native sequence HRG polypeptide, such as an EGF-like domain fragment thereof (e.g., HRGβ1177-244).
“ErbB hetero-oligomer” is a noncovalently associated oligomer comprising at least two different ErbB receptors. An “ErbB dimer” is a noncovalently associated oligomer that comprises two different ErbB receptors. Such complexes may form when a cell expressing two or more ErbB receptors is exposed to an ErbB ligand. ErbB oligomers, such as ErbB dimers, can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described in Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994), for example. Examples of such ErbB hetero-oligomers include EGFR-ErbB2 (also referred to as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and ErbB3-ErbB4 (HER3/HER4) complexes. Moreover, the ErbB hetero-oligomer may comprise two or more ErbB2 receptors combined with a different ErbB receptor, such as ErbB3, ErbB4 or EGFR (ErbB1). Other proteins, such as a cytokine receptor subunit (e.g., gp130) may be included in the hetero-oligomer.
A “native sequence” polypeptide is one which has the same amino acid sequence as a polypeptide, e.g., tumor-associated antigen receptor, derived from nature. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of naturally-occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species.
The term “amino acid sequence variant” refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% homology with at least one receptor binding domain of a native ligand, or with at least one ligand binding domain of a native receptor, such as a tumor-associated antigen, and preferably, they will be at least about 80%, more preferably, at least about 90% homologous with such receptor or ligand binding domains. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.
“Sequence identity” is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for the alignment are well known in the art. One such computer program is “Align 2,” authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.
“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, (1991) Annu. Rev. Immunol, 9:457-92. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., Prco. Natl. Acad. Sci. USA, 95:652-656 (1998).
The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and Fcγ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See review M. in Daëron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus. (Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).
“Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may be performed.
The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. supra) and/or those residues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol., 196:901-917). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fe” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The “light chains” of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.
“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature, 321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.
Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8)(HERCEPTIN® as described in Table 3 of U.S. Pat. No. 5,821,337 expressly incorporated herein by reference; humanized 520C9 (WO 93/21319) and humanized 2C4 antibodies as described herein below.
An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
An antibody “which binds” an antigen of interest is one capable of binding that antigen with sufficient affinity such that the antibody is useful in targeting a cell expressing the antigen.
An antibody which “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). The cell is a tumor cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells.
A “disorder” is any condition that would benefit from treatment of the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include benign and malignant tumors; leukemia and lymphoid malignancies, in particular breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.
The term “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
The term “substantial amount” refers to a majority, i.e. >50% of a population, of a collection or a sample.
The term “intracellular metabolite” refers to a compound resulting from a metabolic process or reaction inside a cell on an antibody drug conjugate (ADC). The metabolic process or reaction may be an enzymatic process such as proteolytic cleavage of a peptide linker of the ADC, or hydrolysis of a functional group such as a hydrazone, ester, or amide. Intracellular metabolites include, but are not limited to, antibodies and free drug which have undergone intracellular cleavage after entry, diffusion, uptake or transport into a cell.
The terms “intracellularly cleaved” and “intracellular cleavage” refer to a metabolic process or reaction inside a cell on an Drug-Ligand Conjugate, a Drug-Linker-Ligand Conjugate, an antibody drug conjugate (ADC) or the like whereby the covalent attachment, e.g., the linker, between the drug moiety (D) and the antibody (Ab) is broken, resulting in the free drug dissociated from the antibody inside the cell. The cleaved moieties of the Drug-Ligand Conjugate, a Drug-Linker-Ligand Conjugate or ADC are thus intracellular metabolites.
The term “bioavailability” refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.
The term “cytotoxic activity” refers to a cell-killing, cytostatic or anti-proliferation effect of an antibody drug conjugate compound or an intracellular metabolite of an antibody drug conjugate compound. Cytotoxic activity may be expressed as the IC50 value which is the concentration (molar or mass) per unit volume at which half the cells survive.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
An “ErbB2-expressing cancer” is one which produces sufficient levels of ErbB2 at the surface of cells thereof, such that an anti-ErbB2 antibody can bind thereto and have a therapeutic effect with respect to the cancer.
A cancer “characterized by excessive activation” of an ErbB2 receptor is one in which the extent of ErbB2 receptor activation in cancer cells significantly exceeds the level of activation of that receptor in non-cancerous cells of the same tissue type. Such excessive activation may result from overexpression of the ErbB2 receptor and/or greater than normal levels of an ErbB2 ligand available for activating the ErbB2 receptor in the cancer cells. Such excessive activation may cause and/or be caused by the malignant state of a cancer cell. In some embodiments, the cancer will be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression of an ErbB2 receptor is occurring which results in such excessive activation of the ErbB2 receptor. Alternatively, or additionally, the cancer may be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression an ErbB2 ligand is occurring in the cancer which attributes to excessive activation of the receptor. In a subset of such cancers, excessive activation of the receptor may result from an autocrine stimulatory pathway.
A cancer which “overexpresses” an ErbB2 receptor is one which has significantly higher levels of an ErbB2 receptor at the cell surface thereof, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. ErbB2 receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the ErbB2 protein present on the surface of a cell (e.g., via an immunohistochemistry assay; IHC). Alternatively, or additionally, one may measure levels of ErbB2-encoding nucleic acid in the cell, e.g., via fluorescent in situ hybridization (FISH; see WO 98/45479), southern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). Overexpression of the ErbB2 ligand, may be determined diagnostically by evaluating levels of the ligand (or nucleic acid encoding it) in the patient, e.g., in a tumor biopsy or by various diagnostic assays such as the IHC, FISH, southern blotting, PCR or in vivo assays described above. One may also study ErbB2 receptor overexpression by measuring shed antigen (e.g., ErbB2 extracellular domain) in a biological fluid such as serum (see, e.g., U.S. Pat. No. 4,933,294; WO 91/05264; U.S. Pat. No. 5,401,638; and Sias et al., (1990) J. Immunol. Methods, 132: 73-80). Aside from the above assays, various other in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.
The tumors overexpressing HER2 are rated by immunohistochemical scores corresponding to the number of copies of HER2 molecules expressed per cell, and can been determined biochemically: 0=0-10,000 copies/cell, 1+=at least about 200,000 copies/cell, 2+=at least about 500,000 copies/cell, 3+=about 1-2×106 copies/cell. Overexpression of HER2 at the 3+ level, which leads to ligand-independent activation of the tyrosine kinase (Hudziak et al., (1987) Proc. Natl. Acad. Sci. USA, 84:7159-7163), occurs in approximately 30% of breast cancers, and in these patients, relapse-free survival and overall survival are diminished (Slamon et al., (1989) Science, 244:707-712; Slamon et al., (1987) Science, 235:177-182).
Conversely, a cancer which is “not characterized by overexpression of the ErbB2 receptor” is one which, in a diagnostic assay, does not express higher than normal levels of ErbB2 receptor compared to a noncancerous cell of the same tissue type.
The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 60C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof. In one aspect, the term is not intended to include radioactive isotopes.
A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan)(HYCAMTIN®, CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti-neoplastic agents such as ALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-1 and capecitabine, and thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX′, TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine)(GEMZAR®) ; 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine)(VELBAN®; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine)(ONCOVIN®; vinca alkaloid; vinorelbine)(NAVELBINE®; novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.
Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
As used herein, the term “EGFR-targeted drug” refers to a therapeutic agent that binds to EGFR and, optionally, inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBITUX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. Pat. No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF (see WO 98/50433, Abgenix). The anti-EGFR antibody may be conjugated with a cyotoxic agent, thus generating an immunoconjugate (see, e.g., EP 659,439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD1839 or Gefitinib (IRESSA™; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA™; Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen).
A “tyrosine kinase inhibitor” is a molecule which inhibits to some extent tyrosine kinase activity of a tyrosine kinase such as an ErbB receptor. Examples of such inhibitors include the EGFR-targeted drugs noted in the preceding paragraph as well as quinazolines such as PD 153035, 4-(3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); or as described in any of the following patent publications: U.S. Pat. No. 5,804,396; WO 99/09016 (American Cyanamid); WO 98/43960 (American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347 (Pfizer, Inc); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO 96/33980 (Zeneca).
An “anti-angiogenic agent” refers to a compound which blocks, or interferes with to some degree, the development of blood vessels. The anti-angiogenic factor may, for instance, be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In one embodiment, the anti-angiogenic factor is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).
The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
The term “prodrug” as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically or hydrolytically activated or converted into the more active parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as including the anti-CD30, CD40, CD70 or Lewis Y antibodies and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
An “isolated” nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
The expression “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional practice.
As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
An “autoimmune disease” herein is a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), psoriasis, dermatitis including atopic dermatitis; chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis), and IBD with co-segregate of pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, and/or episcleritis), respiratory distress syndrome, including adult respiratory distress syndrome (ARDS), meningitis, IgE-mediated diseases such as anaphylaxis and allergic rhinitis, encephalitis such as Rasmussen's encephalitis, uveitis, colitis such as microscopic colitis and collagenous colitis, glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN, membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), juvenile onset diabetes, multiple sclerosis (MS) such as spino-optical MS, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitis (including Large Vessel vasculitis (including Polymyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis), Medium Vessel vasculitis (including Kawasaki's Disease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman's syndrome, Goodpasture's Syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection (including pretreatment for high panel reactive antibody titers, IgA deposit in tissues, and rejection arising from renal transplantation, liver transplantation, intestinal transplantation, cardiac transplantation, etc.), graft versus host disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmune polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune complex nephritis, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), thrombocytopenia (as developed by myocardial infarction patients, for example), including autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (IDDM), including pediatric IDDM, and Sheehan's syndrome; autoimmune hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barré Syndrome, Berger's Disease (IgA nephropathy), primary biliary cirrhosis, celiac sprue (gluten enteropathy), refractory sprue with co-segregate dermatitis herpetiformis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS), polychondritis such as refractory polychondritis, pulmonary alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis, monoclonal gammopathy of uncertain/unknown significance (MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS; autism, inflammatory myopathy, and focal segmental glomerulosclerosis (FSGS).
“Alkyl” is C1-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. Examples are methyl (Me, —CH3), ethyl (Et, —CH2CH3), 1-propyl (n-Pr, n-propyl, —CH2CH2CH3), 2-propyl (i-Pr, i-propyl, —CH(CH3)2), 1-butyl (n-Bu, n-butyl, —CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, —CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, —CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH3)3), 1-pentyl (n-pentyl, —CH2CH2CH2CH2CH3), 2-pentyl (—CH(CH3)CH2CH2CH3), 3-pentyl (—CH(CH2CH3)2), 2-methyl-2-butyl (—C(CH3)2CH2CH3), 3-methyl-2-butyl (—CH(CH3)CH(CH3)2), 3-methyl-1-butyl (—CH2CH2CH(CH3)2), 2-methyl-1-butyl (—CH2CH(CH3)CH2CH3), 1-hexyl (—CH2CH2CH2CH2CH2CH3), 2-hexyl (—CH(CH3)CH2CH2CH2CH3), 3-hexyl (—CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (—C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (—CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (—CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (—C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (—CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (—C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (—CH(CH3)C(CH3)3.
“Alkenyl” is C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp2 double bond. Examples include, but are not limited to: ethylene or vinyl (—CH═CH2), allyl (—CH2CH═CH2), cyclopentenyl (—C5H7), and 5-hexenyl (—CH2CH2CH2CH2CH═CH2).
“Alkynyl” is C2-C18 hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp triple bond. Examples include, but are not limited to: acetylenic (—C≡CH) and propargyl (—CH2C≡CH).
“Alkylene” refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (—CH2—) 1,2-ethyl (—CH2CH2—), 1,3-propyl (—CH2CH2CH2—), 1,4-butyl (—CH2CH2CH2CH2—), and the like.
“Alkenylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (—CH═CH—).
“Alkynylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene (—C≡C—), propargyl (—CH2C≡C—), and 4-pentynyl (—CH2CH2CH2C≡CH—).
“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as “Ar”. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.
“Arylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. The arylalkyl group comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety of the heteroarylalkyl group may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.
“Substituted alkyl”, “substituted aryl”, and “substituted arylalkyl” mean alkyl, aryl, and arylalkyl respectively, in which one or more hydrogen atoms are each independently replaced with a substituent. Typical substituents include, but are not limited to, —X, —R, —OR, —SR, —S−, —NR2, —NR3, ═NR, —CX3, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO2, ═N2, —N3, NC(═O)R, —C(═O)R, —C(═O)NR2, —SO3H, —S(═O)2R, —OS(═O)2OR, —S(═O)2NR, —S(═O)R, —OP(═O)(OR)2, —P(═O)(OR)2, —PO−3, —PO3H2, —C(═O)R, —C(═O)X, —C(═S)R, —CO2R, —CO2− —C(═S)OR, —C(═O)SR, —C(═S)SR, —C(═O)NR2, —C(═S)NR2, —C(═NR)NR2, where each X is independently a halogen: F, Cl, Br, or I; and each R is independently —H, C2-C18 alkyl, C6-C20 aryl, C3-C14 heterocycle, protecting group or prodrug moiety. Alkylene, alkenylene, and alkynylene groups as described above may also be similarly substituted.
“Heteroaryl” and “Heterocycle” refer to a ring system in which one or more ring atoms is a heteroatom, e.g., nitrogen, oxygen, and sulfur. The heterocycle radical comprises 1 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. A heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.
Heterocycles are described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
Examples of heterocycles include by way of example and not limitation pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and isatinoyl.
By way of example and not limitation, carbon bonded heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.
By way of example and not limitation, nitrogen bonded heterocycles are bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or β-carboline. Still more typically, nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g., arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6] system. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, and cyclooctyl.
“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, a linker is specified as LU. Linkers include a divalent radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: —(CR2)nO(CR2)n—, repeating units of alkyloxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, Jeffamine™); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.
The term “chiral” refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
The term “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
“Diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
“Enantiomers” refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.
Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
Examples of a “patient” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human.
“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. A carbocyclic aromatic group or a heterocyclic aromatic group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; wherein each R′ is independently selected from H, —C1-C8 alkyl and aryl.
The term “C1-C8 alkyl,” as used herein refers to a straight chain or branched, saturated or unsaturated hydrocarbon having from 1 to 8 carbon atoms. Representative “C1-C8 alkyl” groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C1-C8 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C1-C8 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl. methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A C1-C8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —SO3R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from H, —C1-C8 alkyl and aryl.
A “C3-C8 carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. A C3-C8 carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from H, —C1-C8 alkyl and aryl.
A “C3-C8 carbocyclo” refers to a C3-C8 carbocycle group defined above wherein one of the carbocycle groups' hydrogen atoms is replaced with a bond.
A “C1-C10 alkylene” is a straight chain, saturated hydrocarbon group of the formula —(CH2)1-10—. Examples of a C1-C10 alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene.
An “arylene” is an aryl group which has two covalent bonds and can be in the ortho, meta, or para configurations as shown in the following structures:
in which the phenyl group can be unsubstituted or substituted with up to four groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; wherein each R′ is independently selected from H, —C1-C8 alkyl and aryl.
A “C3-C8 heterocycle” refers to an aromatic or non-aromatic C3-C8 carbocycle in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. Representative examples of a C3-C8 heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl. A C3-C8 heterocycle can be unsubstituted or substituted with up to seven groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; wherein each R′ is independently selected from H, —C1-C8 alkyl and aryl.
“C3-C8 heterocyclo” refers to a C3-C8 heterocycle group defined above wherein one of the heterocycle group's hydrogen atoms is replaced with a bond. A C3-C8 heterocyclo can be unsubstituted or substituted with up to six groups including, but not limited to, —C1-C8 alkyl, —O—(C1-C8 alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2—C(O)NHR′, —C(O)N(R′)2—NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; wherein each R′ is independently selected from H, —C1-C8 alkyl and aryl.
An “Exemplary Compound” is a Drug Compound or a Drug-Linker Compound.
An “Exemplary Conjugate” is a Drug-Ligand Conjugate having a cleavable Drug unit from the Drug-Ligand Conjugate or a Drug-Linker-Ligand Conjugate.
In some embodiments, the Exemplary Compounds and Exemplary Conjugates are in isolated or purified form. As used herein, “isolated” means separated from other components of (a) a natural source, such as a plant or animal cell or cell culture, or (b) a synthetic organic chemical reaction mixture. As used herein, “purified” means that when isolated, the isolate contains at least 95%, and in another aspect at least 98%, of Exemplary Compound or Exemplary Conjugate by weight of the isolate.
Examples of a “hydroxyl protecting group” include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.
“Leaving group” refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, but are not limited to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.
The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an Exemplary Compound or Exemplary Conjugate. The Exemplary Compounds and Exemplary Conjugates contain at least one amino group, and accordingly acid addition salts can be formed with this amino group. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
“Pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a compound of the invention, e.g., an Exemplary Compound or Exemplary Conjugate. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
The following abbreviations are used herein and have the indicated definitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is diethylamine, DEAD is diethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine, dil is dolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid), DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospray mass spectrometry, EtOAc is ethyl acetate, Fmoc is N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is high pressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN (CH3CN) is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is (IS, 2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS is phosphate-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusion chromatography, Su is succinimide, TBTU is 0-benzotriazol-1-yl-N,N,N,N-tetramethyluronium tetrafluoroborate, TFA is trifluoroacetic acid, TLC is thin layer chromatography, UV is ultraviolet, and val is valine.
The following linker abbreviations are used herein and have the indicated definitions: Val Cit is a valine-citrulline, dipeptide site in protease cleavable linker; PAB is p-aminobenzylcarbamoyl; (Me)vc is N-methyl-valine citrulline, where the linker peptide bond has been modified to prevent its cleavage by cathepsin B; MC(PEG)6-OH is maleimidocaproyl-polyethylene glycol; SPP is N-Succinimidyl 4-(2-pyridylthio) pentanoate; and SMCC is N-Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate.
The terms “treat” or “treatment,” unless otherwise indicated by context, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
In the context of cancer, the term “treating” includes any or all of: preventing growth of tumor cells, cancer cells, or of a tumor; preventing replication of tumor cells or cancer cells, lessening of overall tumor burden or decreasing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.
In the context of an autoimmune disease, the term “treating” includes any or all of: preventing replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.
In the context of an infectious disease, the term “treating” includes any or all of: preventing the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease.
The following cytotoxic drug abbreviations are used herein and have the indicated definitions: MNIAE is mono-methyl auristatin E (MW 718); MMAF is N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine (MW 731.5); MMAF-DMAEA is MMAF with DMAEA (dimethylaminoethylamine) in an amide linkage to the C-terminal phenylalanine (MW 801.5); MMAF-TEG is MMAF with tetraethylene glycol esterified to the phenylalanine; MMAF-NtBu is N-t-butyl, attached as an amide to C-terminus of MMAF; AEVB is auristatin E valeryl benzylhydrazone, acid labile linker through the C-terminus of AE (MW 732); and AFP is Monoamide of p-phenylene diamine with C-terminal Phenylalanine of Auristatin F (MW 732).
9.2 The Compounds of the Invention
9.2.1 The Compounds of Formula (Ia)
In one aspect, the invention provides Drug-Linker-Ligand Conjugates having Formula Ia:
LAa-Ww—Yy-D)p Ia
or a pharmaceutically acceptable salt or solvate thereof
wherein,
L- is a Ligand unit;
-Aa-Ww—Yy— is a Linker unit (LU), wherein the Linker unit includes:
-A- is a Stretcher unit,
a is 0 or 1,
each —W— is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
—Y— is a Spacer unit, and
y is 0, 1 or 2;
p ranges from 1 to about 20; and
-D is a Drug unit having the Formulas DE and DF:
wherein, independently at each location:
R2 is selected from H and C1-C8 alkyl;
R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R5 is selected from H and methyl;
or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from H and C1-C8 alkyl;
R7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O—(C1-C8 alkyl);
R9 is selected from H and C1-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or C1-C8 alkyl;
each occurrence of R15 is independently H, COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, C1-C8 alkyl, or —(CH2)n—COOH;
R18 is selected from —C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3-C8 heterocycle), and —C(R8)2—C(R8)2—(C3-C8 carbocycle); and
n is an integer ranging from 0 to 6.
In another embodiment, the present invention provides Drug Compounds having the Formula Ib:
or pharmaceutically acceptable salts or solvates thereof,
wherein:
R2 is selected from hydrogen and —C1-C8 alkyl;
R3 is selected from hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 jointly, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from H and —C1-C8 alkyl;
R7 is selected from H, —C1-C8 alkyl, —C3-C8 carbocycle, aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from H and —C1-C8 alkyl;
R10 is selected from aryl group or —C3-C8 heterocycle;
Z is —O—, —S—, —NH—, or —NR12—, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, —C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is —C2-C8 alkyl;
R14 is H or —C1-C8 alkyl;
each occurrence of R15 is independently H, —COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, —C1-C8 alkyl, or —(CH2)n—COOH; and
n is an integer ranging from 0 to 6.
In yet another embodiment, the invention provides Drug-Linker-Ligand Conjugates having the Formula Ia′:
AbAa-Ww—Yy-Dp Formula Ia′
or pharmaceutically acceptable salts or solvates thereof.
wherein:
Ab is an antibody,
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 20, and
D is a Drug moiety selected from Formulas DE and DF:
wherein, independently at each location:
R2 is selected from H and C1-C8 alkyl;
R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R5 is selected from H and methyl;
or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from H and C1-C8 alkyl;
R7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O—(C1-C8 alkyl);
R9 is selected from H and C1-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or C1-C8 alkyl;
each occurrence of R15 is independently H, COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, C1-C8 alkyl, or —(CH2)n—COOH;
R18 is selected from —C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3-C8 heterocycle), and —C(R8)2—C(R8)2—(C3-C8 carbocycle); and
n is an integer ranging from 0 to 6.
Ab is any antibody covalently attached to one or more drug units. Ab includes an antibody which binds to CD30, CD40, CD70, Lewis Y antigen. In another embodiment, Ab does not include an antibody which binds to an ErbB receptor or to one or more of receptors
(1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);
(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);
(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763);
(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792, Genbank accession no. M26004);
(15) CD79b (IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20Rα (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM_004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763);
(26) BAFF-R (Genbank accession no. NP_443177.1);
(27) CD22 (Genbank accession no. NP-001762.1);
(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation, Genbank accession No. NP_001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes, Genbank accession No. NP_002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accession No. NP_001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); and/or
(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1).
In one embodiment -Ww- is -Val-Cit-.
In another embodiment, R3, R4 and R7 are independently isopropyl or sec-butyl and R5 is —H. In an exemplary embodiment, R3 and R4 are each isopropyl, R5 is —H, and R7 is sec-butyl. In yet another embodiment, R2 and R6 are each methyl, and R9 is —H.
In still another embodiment, each occurrence of R8 is —OCH3.
In an exemplary embodiment, R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is —H, R7 is sec-butyl, each occurrence of R8 is —OCH3, and R9 is —H.
In one embodiment, Z is —O— or —NH—.
In one embodiment, R10 is aryl In an exemplary embodiment, R10 is -phenyl.
In an exemplary embodiment, when Z is —O—, R11 is —H, methyl or t-butyl.
In one embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—N(R16)2, and R16 is —C1-C8 alkyl or —(CH2)n—COOH.
In another embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—SO3H.
In one aspect, Ab is cAC10, cBR96, cS2C6, c1F6, c2F2, hAC10, hBR96, hS2C6, h1F6, and h2F2.
Exemplary embodiments of Formula Ia have the following structures:
wherein L is an antibody, Val is valine, and Cit is citrulline.
The drug loading is represented by p, the average number of drug molecules per antibody in a molecule (e.g., of Formula Ia, Ia′ and Ic). Drug loading may range from 1 to 20 drugs (D) per Ligand (e.g., Ab or mAb). Compositions of Formula Ia and Formula Ia′ include collections of antibodies conjugated with a range of drugs, from 1 to 20. The average number of drugs per antibody in preparation of conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of Ligand-Drug-Conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Ligand-Drug-conjugates where p is a certain value from Ligand-Drug-Conjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
9.2.2 The Drug Compounds of Formula (Ib)
In another aspect, the present invention provides Drug Compounds having the Formula (Ib):
or a pharmaceutically acceptable salt or solvate thereof,
wherein:
R2 is selected from -hydrogen and —C1-C8 alkyl;
R3 is selected from -hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from -hydrogen, —C1-C8 alkyl, —C3-C8 carbocycle, -aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle) wherein R5 is selected from —H and -methyl; or R4 and R5 jointly, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached;
R6 is selected from —H and —C1-C8 alkyl;
R7 is selected from —H, —C1-C8 alkyl, —C3-C8 carbocycle, aryl, —C1-C8 alkyl-aryl, —C1-C8 alkyl-(C3-C8 carbocycle), —C3-C8 heterocycle and —C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from —H, —OH, —C1-C8 alkyl, —C3-C8 carbocycle and —O—(C1-C8 alkyl);
R9 is selected from —H and —C1-C8 alkyl;
R10 is selected from aryl group or —C3-C8 heterocycle;
Z is —O—, —S—, —NH—, or —NR12—, wherein R12 is C1-C8 alkyl;
R11 is selected from —H, C1-C20 alkyl, aryl, —C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is —C2-C8 alkyl;
R14 is —H or —C1-C8 alkyl;
each occurrence of R15 is independently —H, —COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently —H, —C1-C8 alkyl, or —(CH2)n—COOH; and
n is an integer ranging from 0 to 6.
In one embodiment, R3, R4 and R7 are independently isopropyl or sec-butyl and R5 is —H. In an exemplary embodiment, R3 and R4 are each isopropyl, R5 is —H, and R7 is sec-butyl.
In another embodiment, R2 and R6 are each methyl, and R9 is —H.
In still another embodiment, each occurrence of R8 is —OCH3.
In an exemplary embodiment, R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is —H, R7 is sec-butyl, each occurrence of R8 is —OCH3, and R9 is —H.
In one embodiment, Z is —O— or —NH—.
In one embodiment, R10 is aryl
In an exemplary embodiment, R10 is -phenyl.
In an exemplary embodiment, when Z is —O—, R11 is —H, methyl or t-butyl.
In one embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—N(R16)2, and R16 is —C1-C8 alkyl or —(CH2)n—COOH.
In another embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—SO3H. Illustrative Compounds of Formula (Ib), each of which may be used as drug moieties (D) in ADC, include compounds having the following structures:
and pharmaceutically acceptable salts or solvates thereof.
9.2.3 The Compounds of Formula (Ic)
In another aspect, the invention provides antibody-drug conjugate compounds (ADC) having Formula Ic:
Ab Aa-Ww—Yy-D)p Ic
comprising an antibody covalently attached to one or more drug units (moieites). The antibody-drug conjugate compounds include pharmaceutically acceptable salts or solvates thereof. Formula Ic compounds are defined wherein:
Ab is an antibody which binds to one or more tumor-associated antigen receptors (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203);
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);
(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449);
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486);
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823);
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424);
(7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878);
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);
(10) MSG783 (RNF124, hypothetical protein F1120315, Genbank accession no. NM_017763);
(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138);
(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636);
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212);
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004);
(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626);
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764);
(17) HER2 (Genbank accession no. M11730);
(18) NCA (Genbank accession no. M18728);
(19) MDP (Genbank accession no. BC017023);
(20) IL20Rα (Genbank accession no. AF184971);
(21) Brevican (Genbank accession no. AF229053);
(22) Ephb2R (Genbank accession no. NM_004442);
(23) ASLG659 (Genbank accession no. AX092328);
(24) PSCA (Genbank accession no. AJ297436);
(25) GEDA (Genbank accession no. AY260763;
(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3, NP_443177.1);
(27) CD22 (B-cell receptor CD22-B isoform, NP-001762.1);
(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation, Genbank accession No. NP_001774.1);
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia, Genbank accession No. NP_001707.1);
(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes, Genbank accession No. NP_002111.1);
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability, Genbank accession No. NP_002552.2);
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank accession No. NP_001773.1);
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis, Genbank accession No. NP_005573.1);
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation, Genbank accession No. NP_443170.1); and
(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies, Genbank accession No. NP_112571.1).
A is a Stretcher unit,
a is 0 or 1,
each W is independently an Amino Acid unit,
w is an integer ranging from 0 to 12,
Y is a Spacer unit, and
y is 0, 1 or 2,
p ranges from 1 to about 8, and
D is a Drug moiety selected from Formulas DE and DF:
wherein the wavy line of DE and DF indicates the covalent attachment site to A, W, or Y, and independently at each location:
R2 is selected from H and C1-C8 alkyl;
R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R5 is selected from H and methyl;
or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from H and C1-C8 alkyl;
R7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O—(C1-C8 alkyl);
R9 is selected from H and C1-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or C1-C8 alkyl;
each occurrence of R15 is independently H, COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, C1-C8 alkyl, or —(CH2)n—COOH; R18 is selected from —C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3-C8 heterocycle), and —C(R8)2—C(R8)2—(C3-C8 carbocycle); and
n is an integer ranging from 0 to 6.
In one embodiment -Ww- is -Val-Cit-.
In another embodiment, R3, R4 and R7 are independently isopropyl or sec-butyl and R5 is —H. In an exemplary embodiment, R3 and R4 are each isopropyl, R5 is —H, and R7 is sec-butyl.
In yet another embodiment, R2 and R6 are each methyl, and R9 is —H.
In still another embodiment, each occurrence of R8 is —OCH3.
In an exemplary embodiment, R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is —H, R7 is sec-butyl, each occurrence of R8 is —OCH3, and R9 is —H.
In one embodiment, Z is —O— or —NH—.
In one embodiment, R10 is aryl.
In an exemplary embodiment, R10 is -phenyl.
In an exemplary embodiment, when Z is —O—, R11 is —H, methyl or t-butyl.
In one embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—N(R16)2, and R16 is —C1-C8 alkyl or —(CH2)n—COOH.
In another embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—SO3H.
Exemplary embodiments of Formula Ic ADC have the following structures:
wherein Ab is an antibody which binds to one or more tumor-associated antigen receptors (1)-(35); Val is valine; and Cit is citrulline.
The drug loading is represented by p, the average number of drugs per antibody in a molecule of Formula I. Drug loading may range from 1 to 20 drugs (D) per antibody (Ab or mAb). Compositions of ADC of Formula I include collections of antibodies conjugated with a range of drugs, from 1 to 20. The average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV/visible spectroscopy, mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of ADC in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
For some antibody drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in the exemplary embodiments above, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate or linker reagent. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds of the invention exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT). Additionally, the antibody must be subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.
It is to be understood that where more than one nucleophilic group reacts with a drug-linker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by dual ELISA antibody assay, specific for antibody and specific for the drug. Individual ADC molecules may be identified in the mixture by mass spectroscopy, and separated by HPLC, e.g., hydrophobic interaction chromatography (“Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate”, Hamblett, K. J., et al, Abstract No. 624, American Association for Cancer Research; 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; “Controlling the Location of Drug Attachment in Antibody-Drug Conjugates”, Alley, S. C., et al, Abstract No. 627, American Association for Cancer Research; 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). Thus, a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
9.3 The Linker Unit
A “Linker unit” (LU) is a bifunctional compound which can be used to link a Drug unit and an Ligand unit to form Drug-Linker-Ligand Conjugates, or which are useful in the formation of immunoconjugates directed against tumor associated antigens. Such immunoconjugates allow the selective delivery of toxic drugs to tumor cells.
In one embodiment, the Linker unit of the Drug-Linker Compound and Drug-Linker-Ligand Conjugate has the formula:
-Aa-Ww—Yy—
wherein:
-A- is a Stretcher unit;
a is 0 or 1;
each —W— is independently an Amino Acid unit;
w is independently an integer ranging from 0 to 12;
—Y— is a Spacer unit; and
y is 0, 1 or 2.
In the Drug-Linker-Ligand Conjugate, the Linker is capable of linking the Drug moiety and the Ligand unit.
9.3.1 The Stretcher Unit
The Stretcher unit (-A-), when present, is capable of linking a Ligand unit to an amino acid unit (—W—). In this regard a Ligand (L) has a functional group that can form a bond with a functional group of a Stretcher. Useful functional groups that can be present on a ligand, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of a carbohydrate, and carboxyl. In one aspect, the Ligand functional groups are sulfhydryl and amino. Sulfhydryl groups can be generated by reduction of an intramolecular disulfide bond of a Ligand. Alternatively, sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of a Ligand using 2-iminothiolane (Traut's reagent) or another sulfhydryl generating reagent.
In one embodiment, the Stretcher unit forms a bond with a sulfur atom of the Ligand unit. The sulfur atom can be derived from a sulfhydryl group of a Ligand. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas Ma and IIIb, wherein L-, —W—, —Y—, -D, w and y are as defined above, and R17 is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, —C1-C10 alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, and —(CH2CH2O)r—CH2—; and r is an integer ranging from 1-10. It is to be understood from all the exemplary embodiments of Formula Ia, such as that even where not denoted expressly, from 1 to 20 drug moieties are linked to a Ligand (p=1-20).
An illustrative Stretcher unit is that of Formula IIIa wherein R17 is —(CH2)5:
Another illustrative Stretcher unit is that of Formula IIIa wherein R17 is —(CH2CH2O)r—CH2—; and r is 2:
Still another illustrative Stretcher unit is that of Formula IIIb wherein R17 is —(CH2)5—:
In another embodiment, the Stretcher unit is linked to the Ligand unit via a disulfide bond between a sulfur atom of the Ligand unit and a sulfur atom of the Stretcher unit. A representative Stretcher unit of this embodiment is depicted within the square brackets of Formula IV, wherein R17, L-, —W—, —Y—, -D, w and y are as defined above.
In yet another embodiment, the reactive group of the Stretcher contains a reactive site that can form a bond with a primary or secondary amino group of a Ligand. Example of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas Va and Vb, wherein —R17—, L-, —W—, —Y—, -D, w and y are as defined above;
In yet another aspect, the reactive group of the Stretcher contains a reactive site that is reactive to a modified carbohydrate's (—CHO) group that can be present on a Ligand. For example, a carbohydrate can be mildly oxidized using a reagent such as sodium periodate and the resulting (—CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko, T. et al. (1991) Bioconjugate Chem 2:133-41. Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas Via, VIb, and VIc, wherein —R17—, L-, —W—, —Y—, -D, w and y are as defined above.
9.3.2 The Amino Acid Unit
The Amino Acid unit (—W—), when present, links the Stretcher unit to the Spacer unit if the Spacer unit is present, links the Stretcher unit to the Drug moiety if the Spacer unit is absent, and links the Ligand unit to the Drug unit if the Stretcher unit and Spacer unit are absent. Ww— is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each —W— unit independently has the formula denoted below in the square brackets, and w is an integer ranging from 0 to 12:
wherein R19 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH2OH, —CH(OH)CH3, —CH2CH2SCH3, —CH2CONH2, —CH2COOH, —CH2CH2CONH2, —CH2CH2COOH, —(CH2)3NHC(═NH)NH2, —(CH2)3NH2, —(CH2)3NHCOCH3, —(CH2)3NHCHO, —(CH2)4NHC(═NH)NH2, —(CH2)4NH2, —(CH2)4NHCOCH3, —(CH2)4NHCHO, —(CH2)3NHCONH2, —(CH2)4NHCONH2, —CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
The Amino Acid unit can be enzymatically cleaved by one or more enzymes, including a tumor-associated protease, to liberate the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide a Drug (D).
Illustrative Ww units are represented by formulas (VII)-(IX):
wherein R20 and R21 are as follows:
R20
R21
Benzyl
(CH2)4NH2;
Methyl
(CH2)4NH2;
isopropyl
(CH2)4NH2;
isopropyl
(CH2)3NHCONH2;
benzyl
(CH2)3NHCONH2;
isobutyl
(CH2)3NHCONH2;
sec-butyl
(CH2)3NHCONH2;
(CH2)3NHCONH2;
benzyl
methyl; and
benzyl
(CH2)3NHC(═NH)NH2;
wherein R20, R21 and R22 are as follows:
R20
R21
R22
benzyl
Benzyl
(CH2)4NH2;
isopropyl
Benzyl
(CH2)4NH2; and
H
Benzyl
(CH2)4NH2;
wherein R20, R21, R22 and R23 are as follows:
R20
R21
R22
R23
H
benzyl
isobutyl
H; and
methyl
isobutyl
methyl
isobutyl.
Exemplary Amino Acid units include, but are not limited to, units of formula (VII) where: R20 is benzyl and R21 is —(CH2)4NH2; R20 isopropyl and R21 is —(CH2)4NH2; R20 isopropyl and R21 is —(CH2)3NHCONH2. Another exemplary Amino Acid unit is a unit of formula (VIII) wherein R20 is benzyl, R21 is benzyl, and R22 is —(CH2)4NH2.
Useful —Ww— units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease. In one embodiment, a —Ww— unit is that whose cleavage is catalyzed by cathepsin B, C and D, or a plasmin protease.
In one embodiment, —Ww— is a dipeptide, tripeptide, tetrapeptide or pentapeptide.
When R19, R20, R21, R22 or R23 is other than hydrogen, the carbon atom to which R19, R20, R21, R22 or R23 is attached is chiral.
Each carbon atom to which R19, R20, R21, R22 or R23 is attached is independently in the (S) or (R) configuration.
In one aspect of the Amino Acid unit, the Amino Acid unit is valine-citrulline. In another aspect, the Amino Acid unit is phenylalanine-lysine (i.e. fk). In yet another aspect of the Amino Acid unit, the Amino Acid unit is N-methylvaline-citrulline. In yet another aspect, the Amino Acid unit is 5-aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine serine valine glutamine and isonepecotic acid.
In certain embodiments, the Amino Acid unit can comprise natural amino acids. In other embodiments, the Amino Acid unit can comprise non-natural amino acids.
9.3.3 The Spacer Unit
The Spacer unit (—Y—), when present, links an Amino Acid unit to the Drug moiety when an Amino Acid unit is present. Alternately, the Spacer unit links the Stretcher unit to the Drug moiety when the Amino Acid unit is absent. The Spacer unit also links the Drug moiety to the Ligand unit when both the Amino Acid unit and Stretcher unit are absent.
Spacer units are of two general types: self-immolative and non self-immolative. A non self-immolative Spacer unit is one in which part or all of the Spacer unit remains bound to the Drug moiety after cleavage, particularly enzymatic, of an Amino Acid unit from the Drug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of a non self-immolative Spacer unit include, but are not limited to a (glycine-glycine) Spacer unit and a glycine Spacer unit (both depicted in FIG. 20) (infra). When an Exemplary Compound containing a glycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymatic cleavage via a tumor-cell associated-protease, a cancer-cell-associated protease or a lymphocyte-associated protease, a glycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved from L-Aa-Ww-. In one embodiment, an independent hydrolysis reaction takes place within the target cell, cleaving the glycine—Drug moiety bond and liberating the Drug.
In another embodiment, —Yy— is a p-aminobenzyl alcohol (PAB) unit (see FIGS. 21 and 22) whose phenylene portion is substituted with Qm wherein Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
In one embodiment, a non self-immolative Spacer unit (—Y—) is -Gly-Gly-.
In another embodiment, a non self-immolative the Spacer unit (—Y—) is -Gly-.
In one embodiment, a Drug-Linker Compound or a Drug-Linker Ligand Conjugate is provided in which the Spacer unit is absent (y=0), or a pharmaceutically acceptable salt or solvate thereof.
Alternatively, an Exemplary Compound containing a self-immolative Spacer unit can release -D without the need for a separate hydrolysis step. In this embodiment, —Y— is a PAB group that is linked to —Ww— via the amino nitrogen atom of the PAB group, and connected directly to -D via a carbonate, carbamate or ether group. Without being bound by any particular theory or mechanism, FIG. 21 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via a carbamate or carbonate group espoused by Toki et al. (2002) J Org. Chem. 67:1866-1872.
In FIG. 21 Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1 to about 20.
Without being bound by any particular theory or mechanism, FIG. 22 depicts a possible mechanism of Drug release of a PAB group which is attached directly to -D via an ether or amine linkage.
In FIG. 22 Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1 to about 20.
Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals. Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacer useful in Exemplary Compounds.
In one embodiment, the Spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as depicted in FIG. 23, which can be used to incorporate and release multiple drugs.
In FIG. 23 Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from 1 to about 20.
In one embodiment, the -D moieties are the same. In yet another embodiment, the -D moieties are different.
In one aspect, Spacer units (—Yy—) are represented by Formulas (X)-(XII):
wherein Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4;
Embodiments of the Formula Ia′ and Ic antibody-drug conjugate compounds include:
wherein w and y are each 0,
9.4 The Drug Unit (Moiety)
The drug moiety (D) of the antibody drug conjugates (ADC) are of the dolastatin/auristatin type (U.S. Pat. Nos. 5,635,483; 5,780,588) which have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965)
D is a Drug unit (moiety) having a nitrogen atom that can form a bond with the Spacer unit when y=1 or 2, with the C-terminal carboxyl group of an Amino Acid unit when y=0, with the carboxyl group of a Stretcher unit when w and y=0, and with the carboxyl group of a Drug unit when a, w, and y=0. It is to be understood that the terms “drug unit” and “drug moiety” are synonymous and used interchangeably herein.
In one embodiment, -D is either formula DE or DF:
wherein, independently at each location:
R2 is selected from H and C1-C8 alkyl;
R3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
R5 is selected from H and methyl;
or R4 and R5 jointly form a carbocyclic ring and have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from H, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
R6 is selected from H and C1-C8 alkyl;
R7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);
each R8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O—(C1-C8 alkyl);
R9 is selected from H and C1-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is O, S, NH, or NR12, wherein R12 is C1-C8 alkyl;
R11 is selected from H, C1-C20 alkyl, aryl, C3-C8 heterocycle, —(R13O)m—R14, or —(R13O)m—CH(R15)2;
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or C1-C8 alkyl;
each occurrence of R15 is independently H, COOH, —(CH2)n—N(R16)2, —(CH2)n—SO3H, or —(CH2)n—SO3—C1-C8 alkyl;
each occurrence of R16 is independently H, C1-C8 alkyl, or —(CH2)n—COOH;
R18 is selected from —C(R8)2—C(R8)2-aryl, —C(R8)2—C(R8)2—(C3-C8 heterocycle), and —C(R8)2—C(R8)2—(C3-C8 carbocycle); and
n is an integer ranging from 0 to 6.
In one embodiment, R3, R4 and R7 are independently isopropyl or sec-butyl and R5 is —H. In an exemplary embodiment, R3 and R4 are each isopropyl, R5 is H, and R7 is sec-butyl.
In another embodiment, R2 and R6 are each methyl, and R9 is H.
In still another embodiment, each occurrence of R8 is —OCH3.
In an exemplary embodiment, R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is H, R7 is sec-butyl, each occurrence of R8 is —OCH3, and R9 is H.
In one embodiment, Z is —O— or —NH—.
In one embodiment, R10 is aryl In an exemplary embodiment, R10 is -phenyl.
In an exemplary embodiment, when Z is —O—, R11 is H, methyl or t-butyl.
In one embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—N(R16)2, and R16 is —C1-C8 alkyl or —(CH2)n—COOH.
In another embodiment, when Z is —NH, R11 is —CH(R15)2, wherein R15 is —(CH2)n—SO3H.
Illustrative Drug units (-D) include the drug units having the following structures:
and pharmaceutically acceptable salts or solvates thereof.
In one aspect, hydrophilic groups, such as but not limited to triethylene glycol esters (TEG), as shown above, can be attached to the Drug Unit at R11. Without being bound by theory, the hydrophilic groups assist in the internalization and non-agglomeration of the Drug Unit.
9.5 The Ligand Unit
The Ligand unit (L-) includes within its scope any unit of a Ligand (L) that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. A Ligand is a molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. In one aspect, the Ligand unit acts to deliver the Drug unit to the particular target cell population with which the Ligand unit reacts. Such Ligands include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies, antibody fragments, smaller molecular weight proteins, polypeptide or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrient-transport molecules (such as, but not limited to, transferrin), or any other cell binding molecule or substance.
A Ligand unit can form a bond to a Stretcher unit, an Amino Acid unit, a Spacer Unit, or a Drug Unit. A Ligand unit can form a bond to a Linker unit via a heteroatom of the Ligand. Heteroatoms that may be present on a Ligand unit include sulfur (in one embodiment, from a sulfhydryl group of a Ligand), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of a Ligand) and nitrogen (in one embodiment, from a primary or secondary amino group of a Ligand). These heteroatoms can be present on the Ligand in the Ligand's natural state, for example a naturally-occurring antibody, or can be introduced into the Ligand via chemical modification.
In one embodiment, a Ligand has a sulfhydryl group and the Ligand bonds to the Linker unit via the sulfhydryl group's sulfur atom.
In yet another aspect, the Ligand has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The Ligand unit bonds to the Linker unit via the sulfhydryl group's sulfur atom. The reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut's Reagent).
In another embodiment, the Ligand can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups. The Ligand unit bonds to the Linker Unit, such as the Stretcher Unit, via the sulfhydryl group's sulfur atom.
In yet another embodiment, the Ligand can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) group (see, for e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The corresponding aldehyde can form a bond with a Reactive Site on a Stretcher. Reactive sites on a Stretcher that can react with a carbonyl group on a Ligand include, but are not limited to, hydrazine and hydroxylamine. Other protocols for the modification of proteins for the attachment or association of Drug Units are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.
Useful non-immunoreactive protein, polypeptide, or peptide Ligands include, but are not limited to, transferrin, epidermal growth factors (“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (“TGF”), such as TGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II, lectins and apoprotein from low density lipoprotein.
Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, and guinea pigs. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Köhler and Milstein (1975, Nature 256, 495-497), the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).
The antibody can also be a bispecific antibody. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Similar procedures are disclosed in International Publication No. WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. Nucleic acids with sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
In an embodiment of this approach, the bispecific antibodies have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation (International Publication No. WO 94/04690) which is incorporated herein by reference in its entirety.
For further details for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992, Bio/Technology 10:163-167; Carter et al., 1995, 1 of Hematotherapy 4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681. Using such techniques, bispecific antibodies can be prepared for use in the treatment or prevention of disease as defined herein.
Bifunctional antibodies are also described, in European Patent Publication No. EPA 0 105 360. As disclosed in this reference, hybrid or bifunctional antibodies can be derived either biologically, i.e., by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide-bridge forming reagents, and may comprise whole antibodies or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed for example, in International Publication WO 83/03679, and European Patent Publication No. EPA 0 217 577, both of which are incorporated herein by reference.
The antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, or microbial antigens or other antibodies bound to tumor cells or matrix. In this regard, “functionally active” means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized. Specifically, in an exemplary embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (See, for e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. of Immunology 125(3):961-969).
Other useful antibodies include fragments of antibodies such as, but not limited to, F(ab′)2 fragments, which contain the variable region, the light chain constant region and the CH1 domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Other useful antibodies are heavy chain and light chain dimers of antibodies, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody.
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671; European Patent Publication No. 184,187; European Patent Publication No. 171496; European Patent Publication No. 173494; International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 12,023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference in its entirety.
Completely human antibodies are particularly desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies. See, e.g., U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which is incorporated herein by reference in its entirety. Other human antibodies can be obtained commercially from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).
Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Biotechnology 12:899-903). Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Quan, M. P. and Carter, P. 2002. The rise of monoclonal antibodies as therapeutics. In Anti-IgE and Allergic Disease, Jardieu, P. M. and Fick Jr., R. B, eds., Marcel Dekker, New York, N.Y., Chapter 20, pp. 427-469).
In other embodiments, the antibody is a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the antibody. Preferably, the antibody or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.
Antibodies include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, but not by way of limitation, the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids.
The antibodies include antibodies having modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies include antibodies having modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631, which is incorporated herein by reference in its entirety). Antibodies immunospecific for a cancer cell antigen can be obtained commercially, for example, from Genentech (San Francisco, Calif.) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
In a specific embodiment, known antibodies for the treatment or prevention of cancer can be used. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, humanized anti-HER2 monoclonal antibody, HERCEPTIN® (trastuzumab; Genentech) for the treatment of patients with metastatic breast cancer; RITUXAN® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY) which is an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers, such as head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath UH (Leukosite, MA) which is a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA) which is a humanized anti-CD33 IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which is a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA) which is a radiolabeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA) which is an anti-VEGF humanized antibody for the treatment of lung and colorectal cancers; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA) which is an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer.
Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogene product (carcinomas). Some specific, useful antibodies include, but are not limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellstrom, I., Hellstrom, K. E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64 (Trail, P A, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R. S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom, K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res. 2000, 60, 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb and 2F2 mAb, and mAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functional effects of CD30 on a large granular lymphoma cell line YT” J. Immunol., 151, 5896-5906, 1993: Wahl et al., 2002 Cancer Res. 62(13):3736-42). Many other internalizing antibodies that bind to tumor associated antigens can be used and have been reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy of acute myeloid leukemia: a review” Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).
In certain embodiments, the antibody is not Trastuzumab (full length, humanized anti-HER2 (MW 145167)), HerceptinF(ab′)2 (derived from anti-HER2 enzymatically (MW 100000)), 4D5 (full-length, murine antiHER2, from hybridoma), rhu4D5 (transiently expressed, full-length humanized antibody), rhuFab4D5 (recombinant humanized Fab (MW 47738)), 4D5Fc8 (full-length, murine antiHER2, with mutated FcRn binding domain), or Hg (“Hingeless” full-length humanized 4D5, with heavy chain hinge cysteines mutated to serines. Expressed in E. coli (therefore non-glycosylated)).
In another specific embodiment, known antibodies for the treatment or prevention of an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization (e.g., a university scientist or a company) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. In another embodiment, useful antibodies are immunospecific for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti-ds DNA; Anti-ss DNA, Anti-Cardiolipin Antibody IgM, IgG; Anti-Phospholipid Antibody IgM, IgG; Anti-SM Antibody; Anti-Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; Anti-SCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti-SSB; Anti-Perital Cells Antibody; Anti-Histones; Anti-RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti-GBM Antibody.
In certain embodiments, useful antibodies can bind to both a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/0X40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitable lectins are C-type, S-type, and I-type lectin.
In one embodiment, the Ligand binds to an activated lymphocyte that is associated with an autoimmune disease.
In another specific embodiment, useful Ligands immunospecific for a viral or a microbial antigen are monoclonal antibodies. The antibodies may be chimeric, humanized or human monoclonal antibodies. As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.
Antibodies immunospecific for a viral or microbial antigen can be obtained commercially, for example, from BD Biosciences (San Francisco, Calif.), Chemicon International, Inc. (Temecula, Calif.), or Vector Laboratories, Inc. (Burlingame, Calif.) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, e.g., from the GenBank database or a database like it, literature publications, or by routine cloning and sequencing.
In a specific embodiment, useful Ligands are those that are useful for the treatment or prevention of viral or microbial infection in accordance with the methods disclosed herein. Examples of antibodies available useful for the treatment of viral infection or microbial infection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody useful for the treatment of patients with RSV infection; PRO542 (Progenics) which is a CD4 fusion antibody useful for the treatment of HIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; PROTOVIR (Protein Design Labs, Inc., CA) which is a humanized IgG1 antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.
Other antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococc aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).
Other antibodies useful in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.
In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies.
Antibodies which comprise Ab in Formula Ic antibody drug conjugates (ADC) and which may be useful in the treatment of cancer include, but are not limited to, antibodies against tumor-associated antigens (TAA). Such tumor-associated antigens are known in the art, and can prepared for use in generating antibodies using methods and information which are well known in the art. Examples of TAA include (1)-(35), but are not limited to TAA (1)-(35) listed below. For convenience, information relating to these antigens, all of which are known in the art, is listed below and includes names, alternative names, Genbank accession numbers and primary reference(s). Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the corresponding sequences listed (SEQ ID NOS: 1-35) or the sequences identified in the cited references. In some embodiments, TAA having amino acid sequence variants exhibit substantially the same biological properties or characteristics as a TAA having the sequence found in the corresponding sequences listed (SEQ ID NOS: 1-35). For example, a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed. The sequences and disclosure specifically recited herein are expressly incorporated by reference.
Tumor-Associated Antigens (1)-(35):
(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203, ten Dijke, P., et al. Science 264 (5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790-A1 (Page 38-39); WO2002102235 (claim 13; Page 296); WO2003055443 (Page 91-92); WO200299122 (Example 2; Page 528-530); WO2003029421 (claim 6); WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; Page 183); WO200254940 (Page 100-101); WO200259377 (Page 349-350); WO200230268 (claim 27; Page 376); WO200148204 (Example; FIG. 4)
NP_001194 bone morphogenetic protein receptor, type D3/pid=NP_001194.1—
-Cross-references: MIM:603248; NP_001194.1; NM_001203_1
502 aa
(SEQ ID NO: 1)
MLLRSAGKLNVGIKKEDGESTAPTPRPKVLRCKCHHHCPEDSVNNICSTD
GYCFTMIEEDDSGLPVVTSGCLGLEGSDFQCRDTPIPHQRRSIECCTERN
ECNKDLHPTLPPLKNRDEVDGPIHHRALLISVTVCSLLLVLIILFCYFRY
KRQETRPRYSIGLEQDETYIPPGESLRDLIEQSQSSGSGSGLPLLVQRTI
AKQIQMVKQIGKGRYGEVWMGKWRGEKVAVKVFETTEEASWFRETEIYQT
VLMRHENILGFIAADIKGTGSWTQLYLITDYHENGSLYDYLKSTTLDAKS
MLKLAYSSVSGLCHLHTEIFSTQGKPAIAHRDLKSKNILVKKNGTCCIAD
LGLAVKFISDTNEVDIPPNTRVGTKRYMPPEVLDESLNRNHFQSYIMADM
YSFGLILWEVARRCVSGGIVEEYQLPYHDLVPSDPSYEDMREIVCIKKLR
PSFPNRWSSDECLRQMGKLMTECWAHNPASRLTALRVKKTLAKMSESQDI
KL
(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486);
Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998), Gaugitsch, H. W., et al. (1992) J. Biol. Chem. 267 (16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV); WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (Example 2); WO200299074 (claim 19; Page 127-129); WO200286443 (claim 27; Pages 222, 393); WO2003003906 (claim 10; Page 293); WO200264798 (claim 33; Page 93-95); WO200014228 (claim 5; Page 133-136); US2003224454 (FIG. 3); WO2003025138 (claim 12; Page 150);
NP_003477 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5/pid=NP_003477.3—Homo sapiens
Cross-references: MIM:600182; NP_003477.3; NM_015923; NM_003486_1
507 aa
(SEQ ID NO: 2)
MAGAGPKRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLQRNI
TLLNGVAIIVGTIIGSGIFVTPTGVLKEAGSPGLALVVWAACGVFSIVGA
LCYAELGTTISKSGGDYAYMLEVYGSLPAFLKLWIELLIIRPSSQYIVAL
VFATYLLKPLFPTCPVPEEAAKLVACLCVLLLTAVNCYSVKAATRVQDAF
AAAKLLALALIILLGFVQIGKGVVSNLDPNFSFEGTKLDVGNIVLALYSG
LFAYGGWNYLNFVTEEMINPYRNLPLAIIISLPIVTLVYVLTNLAYFTTL
STEQMLSSEAVAVDFGNYHLGVMSWIIPVFVGLSCFGSVNGSLFTSSRLF
FVGSREGHLPSILSMIHPQLLTPVPSLVFTCVMTLLYAFSKDIFSVINFF
SFFNWLCVALAIIGMIWLRHRKPELERPIKVNLALPVFFILACLFLIAVS
FWKTPVECGIGFTIILSGLPVYFFGVWWKNKPKWLLQGIFSTTVLCQKLM
QVVPQET
(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449
Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al. (1999) Proc. Natl. Acad. Sci. USA. 96 (25):14523-14528); WO2004065577 (claim 6); WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (claim 2); WO2003042661 (claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page 618-619); WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173, Example 2; FIG. 2A); NP_036581 six transmembrane epithelial antigen of the prostate
Cross-references: MIM:604415; NP_036581.1; NM_012449_1
339 aa
(SEQ ID NO 3)
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTA
HADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLAT
SHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKF
PHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQV
QQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREF
HYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIV
VLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL
(4) 0772P (CA125, MUC16, Genbank accession no. AF361486
J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004045553 (claim 14); WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; Page 116-121); US2003124140 (Example 16); US2003091580 (claim 6); WO200206317 (claim 6; Page 400-408);
Cross-references: GI:34501467; AAK74120.3; AF361486_1
6995 aa
(SEQ ID NO: 4)
PVTSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEAMQPSTHTAVT
NVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSL
TSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDIS
TEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTEWSGTHSTASPGFSHS
EMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFESTLPESISSSPHPVTALL
TLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVI
YKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREIS
TSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRIS
TPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSR
GPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKT
TDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYS
SYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIH
SATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTES
TEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGEVPWLS
HPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSE
PETSSPPNLSSTSAEILATTEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSM
GITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPT
GATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPS
GATSQGTFTLDSSSTASWPGTHSATTQRFPRSVVTTPMSRGPEDVSWPSPLSVEKNSPPS
SLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNI
TSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSS
IRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPE
VSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQ
VTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLP
LTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIP
ATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVEDTTV
FTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLFGVPTSATTEVSMTEI
MSSNRTHIPDSDQSTMSPDITTEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLA
TTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSPFVEKTSSSSSLLSLPVTTSPS
VSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTE
KIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFET
TGFEAEPFSHLTSGLRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDW
PPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETIS
TGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSS
STETTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSS
PILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQT
STSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASMQ
MASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVENRESETTASLVSR
SGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFEHSELDTVSSTATSHGA
DVSSAIPTNISPSELDALTPLVTISGTDTSTTEPTLTKSPHETETRTTWLTHPAETSSTI
PRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPT
LTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNS
PGEPATTVSLVTHSAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEV
PGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVV
TSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVAS
SRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNS
TSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIP
ILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTFLVTSSRAVTSTTIPTLTISS
DEPETTTSLVTHSEAKMISAIPTLGVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPET
IDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPS
TVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATEPTVPESPHESEATASWVTHP
AVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDEPTITVSPDVPDMVTSQVTSSGTD
TSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPDASKMLTSLVISSGTDSTTT
FPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKESHSKSDTTLPVAITSPGPEASSAVS
TTTISPDMSDLVTSLVPSSGTDTSTTEPTLSETPYEPETTATWLTHPAETSTTVSGTIPN
FSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPG
EPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPD
ATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALL
STHPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVD
LSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLT
VSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPP
KTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTENTLAGSLFTPLTTPGMSTLA
SESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTESPGISTSSIPSSTAATVPFMV
PFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPE
KDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHR
SSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGSRK
FNTMESVLQGLLKPLEKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLN
REQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSS
LSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKENTTERVLQGLLGPIEKNTSVG
PLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYT
LDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITN
LKYEEDMHRPGSRKFNTTERVLQTLVGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDA
ICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPG
TSTVDLGSGTPSSLPSPTSATAGPLLVPFTLNFTITNLKYEEDMHCPGSRKENTTERVLQ
SLLGPMEKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELS
QLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGP
LLVPFTLNFTITNLQYEEDMHHPGSRKENTTERVLQGLLGPMEKNTSVGLLYSGCRLTLL
RPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGF
THRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPG
SRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSP
GLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGT
PSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLQGLLSPIFKNSSV
GPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPY
SLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTENFTIT
NLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVD
TICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGENPWSSVPTTSTP
GTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKENTTERVLQ
GLLKPLEKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELS
QLTNSVTELGPYTLDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGP
LLVPFTLNFTITNLQYEEDMRHPGSRKENTTERVLQGLLKPLEKSTSVGPLYSGCRLTLL
RPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGF
THRNEVPITSTPGTSTVHLGTSETPSSLPRPIVPGPLLVPFTLNFTITNLQYEEAMRHPG
SRKENTTERVLQGLLRPLFKNTSIGPLYSSCRLTLLRPEKDKAATRVDAICTHHPDPQSP
GLNREQLYWELSQLTHGITELGPYTLDRDSLYVDGFTHWSPIPTTSTPGTSIVNLGTSGI
PPSLPETTATGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLEKSTSV
GPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPY
TLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTII
NLQYEEDMHRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVD
AVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTP
DTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKENTTERVLQ
GLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELS
QLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASP
LLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLEKSTSVGPLYSGCRLTLL
RPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLEVNGF
THRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGS
RKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPG
LDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTI
NNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRV
DLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPDEPPTT
PKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRP
LFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHG
VTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQD
KVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHW
LGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRN
IEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFL
RMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGLITCLIC
GVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ
(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823
Yamaguchi, N., et al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. USA. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. USA. 93 (1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; Page 287-288); WO2002101075 (claim 4; Page 308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);
Cross-references: MIM:601051; NP_005814.2; NM_005823_1
622 aa
(SEQ ID NO: 5)
MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDG
VLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQ
LRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVD
LLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAES
AEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRG
LLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT
ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLD
VLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLE
VNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELS
SVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFL
GGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGL
KAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGT
PCLLGPGPVLTVLALLLASTLA
(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424, J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284 (1999), Feild, J. A., et al. (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582); WO2004022778 (claim 2); EP1394274 (Example 11); WO2002102235 (claim 13; Page 326); EP875569 (claim 1; Page 17-19); WO200157188 (claim 20; Page 329); WO2004032842 (Example IV); WO200175177 (claim 24; Page 139-140);
Cross-references: MIM:604217; NP_006415.1; NM_006424_1
690 aa
(SEQ ID NO: 6)
MAPWPELGDAQPNPDKYLEGAAGQQPIAPDKSKEINKTDNTEAPVTKIEL
LPSYSTATLIDEPTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQGIGR
LILLLGFLYFFVCSLDILSSAFQLVGGKMAGQFFSNSSIMSNPLLGLVIG
VLVTVLVQSSSISTSIVVSMVSSSLLIVRAAIPIIMGANIGTSITNTIVA
LMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHYLEIITQLIVE
SFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAKNKSLVK
IWCKTFINKTQINVIVPSTANCTSPSLCWIDGIQNWTMKNVTYKENIAKC
QHIFVNFHLPDLAVGTILLILSLLVLCGCLIMIVKILGSVLKGQVATVIK
KTINTDFPFPFAWLIGYLAILVGAGMTFIVQSSSVFTSALTPLIGIGVIT
IERAYPLTLGSNIGTITTAILAALASPGNALRSSLQIALCHFFFNISGIL
LWYPIPFTRLPIRMAKGLGNISAKYRWFAVFYLIIFFFLIPLIVFGLSLA
GWRVLVGVGVPVVFIIILVLCLRLLQSRCPRVLPKKLQNWNFLPLWMRSL
KPWDAVVSKFTGCFQMRCCYCCRVCCRACCLLCGCPKCCRCSKCCEDLEE
AQEGQDVPVKAPETFDNITISREAQGEVPASDSKTECTAL
(7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878, Nagase T., et al. (2000) DNA Res. 7 (2):143-150); WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; Page 50); WO200188133 (claim 1; Page 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11);
Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737;
1093 aa
(SEQ ID NO: 7)
MVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCALSKHPTVAF
EDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQ
ATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMC
TSRQVGNLSRTTEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGR
DPAIYRSLGSGPPLRTAQYNSKWLNEPNEVAAYDIGLFAYFFLRENAVEH
DCGRTVYSRVARVCKNDVGGRELLEDTWTTFMKARLNCSRPGEVPFYYNE
LQSAFHLPEQDLIYGVETTNVNSIAASAVCAFNLSAISQAFNGPFRYQEN
PRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPV
TPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRS
LHGCYLEELHVLPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAA
YRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQNITACPVRNVTR
DGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHI
ANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSR
EERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGC
GVEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLAD
PHGLQFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAA
WGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYQDCNPQACP
VRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEAL
CATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSRP
CPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLS
CQHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIP
DDRANFYPLQQTNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (Example 5); WO2003105758 (claim 12); US2003206918 (Example 5); EP1347046 (claim 1); WO2003025148 (claim 20);
Cross-references: GI:37182378; AAQ88991.1; AY358628_1
141 aa
(SEQ ID NO: 8)
MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNV
QDMCQKEVMEQSAGIMYRKSCASSAACLIASAGYQSFCSPGKLNSVCISC
CNTPLCNGPRPKKRGSSASALRPGLRTTILFLKLALFSAHC
(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);
Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177, 34-39, 1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178, 248-255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al. J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al. Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al. J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, sl-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999; Strausberg R. L., et al. Proc. Natl. Acad. Sci. USA. 99, 16899-16903, 2002; Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij J. B., et al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al. Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al. Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995; Auricchio A., et al. Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al. Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al. Hum. Genet. 103, 145-148, 1998; Fuchs S., et al. Mol. Med. 7, 115-124, 2001; Pingault V., et al. (2002) Hum. Genet. 111, 198-206; WO2004045516 (claim 1); WO2004048938 (Example 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (FIG. 1); WO2003016494 (FIG. 6); WO2003025138 (claim 12; Page 144); WO200198351 (claim 1; Page 124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; Page 297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (claim 1a; Col 31-34); WO2004001004;
442 aa
(SEQ ID NO: 9)
MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPT
KILWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETF
KYINTVVSCLVFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLH
IVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSLCALSIDRY
RAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIGFDIITMDYKGS
YLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYILMICEM
LRKKSGMQIALNDHLKQRREVAKTVFCLVLVFALCWLPLHLSRILKLTLY
NQNDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSC
LCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNFRSSNKYSSS
(10) MSG783 (RNF124, hypothetical protein F1120315, Genbank accession no. NM_017763); WO2003104275 (claim 1); WO2004046342 (Example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; Page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; FIG. 93); WO200166689 (Example 6);
Cross-references: LocusID:54894: NP_060233.2; NM_017763_1
783 aa
(SEQ ID NO: 10)
MSGGHQLQLAALWPWLLMATLQAGFGRTGLVLAAAVESERSAEQKAIIRV
IPLKMDPTGKLNLTLEGVFAGVAEITPAEGKLMQSHPLYLCNASDDDNLE
PGFISIVKLESPRRAPRPCLSLASKARMAGERGASAVLFDITEDRAAAEQ
LQQPLGLTWPVVLIWGNDAEKLMEFVYKNQKAHVRIELKEPPAWPDYDVW
ILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQA
SCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVD
PWLHQHRTCPLCVFNITEGDSFSQSLGPSRSYQEPGRRLHLIRQHPGHAH
YHLPAAYLLGPSRSAVARPPRPGPFLPSQEPGMGPRHHRFPRAAHPRAPG
EQQRLAGAQHPYAQGWGMSHLQSTSQHPAACPVPLRRARPPDSSGSGESY
CTERSGYLADGPASDSSSGPCHGSSSDSVVNCTDISLQGVHGSSSTFCSS
LSSDFDPLVYCSPKGDPQRVDMQPSVTSRPRSLDSVVPTGETQVSSHVHY
HRHRHHHYKKRFQWHGRKPGPETGVPQSRPPIPRTQPQPEPPSPDQQVTG
SNSAAPSGRLSNPQCPRALPEPAPGPVDASSICPSTSSLFNLQKSSLSAR
HPQRKRRGGPSEPTPGSRPQDATVHPACQIFPHYTPSVAYPWSPEAHPLI
CGPPGLDKRLLPETPGPCYSNSQPVWLCLTPRQPLEPHPPGEGPSEWSSD
TAEGRPCPYPHCQVLSAQPGSEEELEELCEQAV
(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138, Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397 (claim 1; FIG. 1); WO200272596 (claim 13; Page 54-55); WO200172962 (claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; FIG. 10);
Cross-references: GI:22655488; AAN04080.1; AF455138_1
490 aa
(SEQ ID NO: 11)
MESISMMGSPKSLSETVLPNGINGIKDARKVIVGVIGSGDFAKSLTIRLI
RCGYHVVIGSRNPKFASEFFPHVVDVIHHEDALTKINIIFVAIHREHYTS
LWDLRHLLVGKILIDVSNNMRINQYPESNAEYLASLFPDSLIVKGFNVVS
AWALQLGPKDASRQVYICSNNIQARQQVIELARQLNFIPIDLGSLSSARE
IENLPLRLFTLWRGPVVVAISLATFFFLYSFVRDVIHPYARNQQSDFYKI
PIEIVNKTLPIVAITLLSLVYLAGLLAAAYQLYYGTKYRRFPPWLETWLQ
CRKQLGLLSFFFAMVHVAYSLCLPMRRSERYLFLNMAYQQVHANIENSWN
EEEVWRIEMYISFGIMSLGLLSLLAVISIPSVSNALNWREFSFIQSTLGY
VALLISTFHVLIYGWKRAFEEEYYRFYIPPNFVLALVLPSIVILGKIILF
LPCISQKLKRIKKGWEKSQFLEEGIGGTIPHVSPERVTVM
(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636 Xu, X. Z., et al. Proc. Natl. Acad. Sci. USA. 98 (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820 (2003)); US2003143557 (claim 4); WO200040614 (claim 14; Page 100-103); WO200210382 (claim 1; FIG. 9A); WO2003042661 (claim 12); WO200230268 (claim 27; Page 391); US2003219806 (claim 4); WO200162794 (claim 14; FIG. 1A-D);
Cross-references: MIM:606936; NP_060106.2; NM_017636_1
1214 aa
(SEQ ID NO: 12)
MVVPEKEQSWIPKIFKKKTCTTFIVDSTDPGGTLCQCGRPRTAHPAVAME
DAFGAAVVTVWDSDAHTTEKPTDAYGELDFTGAGRKHSNFLRLSDRTDPA
AVYSLVTRTWGFRAPNLVVSVLGGSGGPVLQTWLQDLLRRGLVRAAQSTG
AWIVTGGLHTGIGRHVGVAVRDHQMASTGGTKVVAMGVAPWGVVRNRDTL
INPKGSFPARYRWRGDPEDGVQFPLDYNYSAFFLVDDGTHGCLGGENRFR
LRLESYISQQKTGVGGTGIDIPVLLLLIDGDEKMLTRIENATQAQLPCLL
VAGSGGAADCLAETLEDTLAPGSGGARQGEARDRIRRFFPKGDLEVLQAQ
VERIMTRKELLTVYSSEDGSEEFETIVLKALVKACGSSEASAYLDELRLA
VAWNRVDIAQSELFRGDIQWRSFHLEASLMDALLNDRPEFVRLLISHGLS
LGHFLTPMRLAQLYSAAPSNSLIRNLLDQASHSAGTKAPALKGGAAELRP
PDVGHVLRMLLGKMCAPRYPSGGAWDPHPGQGFGESMYLLSDKATSPLSL
DAGLGQAPWSDLLLWALLLNRAQMAMYFWEMGSNAVSSALGACLLLRVMA
RLEPDAEEAARRKDLAFKFEGMGVDLFGECYRSSEVRAARLLLRRCPLWG
DATCLQLAMQADARAFFAQDGVQSLLTQKWWGDMASTTPIWALVLAFFCP
PLIYTRLITFRKSEEEPTREELEFDMDSVINGEGPVGTADPAEKTPLGVP
RQSGRPGCCGGRCGGRRCLRRWFHFWGAPVTIFMGNVVSYLLFLLLFSRV
LLVDFQPAPPGSLELLLYFWAFILLCEELRQGLSGGGGSLASGGPGPGHA
SLSQRLRLYLADSWNQCDLVALTCFLLGVGCRLIPGLYHLGRIVLCIDFM
VFIVRLLHIFTVNKQLGPKIVIVSKMMKDVFFFLFFLGVWLVAYGVATEG
LLRPRDSDFPSILRRVFYRPYLQIFGQIPQEDMDVALMEHSNCSSEPGFW
AHPPGAQAGICVSQYANWLVVLLLVIFLLVANILLVNLLIAMFSYTFGKV
QGNSDLYWKAQRYRLIREFHSRPALAPPFIVISHLRLLLRQLCRRPRSPQ
PSSPALEHFRVYLSKEAERKLLTWESVHKENFLLARARDKRESDSERLKR
ISQKVDLALKQLGHIREYEQRLKVLEREVQQCSRVLGWVAEALSRSALLP
PGGPPPPDLPGSKD
(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212,
Ciccodicola, A., et al. EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555-565 (1991)); US2003224411 (claim 1); WO2003083041 (Example 1); WO2003034984 (claim 12); WO200288170 (claim 2; Page 52-53); WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; Page 94-95, 105); WO200222808 (claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col 17-18); U.S. Pat. No. 5,792,616 (FIG. 2);
Cross-references: MIM:187395; NP_003203.1; NM_003212_1
188 aa
(SEQ ID NO: 13)
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS
IWPQEEPAIRPRSSQRVPPMGIQHSKELNRICCLNGGICMLGSFCACPPS
FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
GLVMDEHLVASRIPELPPSARITTFMLVGICLSIQSYY
(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004,
Fujisaku et al. (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl. Acad. Sci. USA. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35, 1025-1031, 1998; Weis J. J., et al. Proc. Natl. Acad. Sci. USA. 83, 5639-5643, 1986; Sinha S. K., et al. (1993) J. Immunol. 150, 5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (claim 9); WO2004045520 (Example 4); WO9102536 (FIGS. 9.1-9.9); WO2004020595 (claim 1);
Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.
1033 aa
(SEQ ID NO: 14)
MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYS
CSGTFRLIGEKSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGY
KIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCQANNMWGPTRLPTCVSVF
PLECPALPMIHNGHHTSENVGSIAPGLSVTYSCESGYLLVGEKIINCLSS
GKWSAVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANFFCDEGYRLQG
PPSSRCVIAGQGVAWTKMPVCEEIFCPSPPPILNGRHIGNSLANVSYGSI
VTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTWSGPAPRCELSTSAVQ
CPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWE
PSAPVCEKECQAPPNILNGQKEDRHMVRFDPGTSIKYSCNPGYVLVGEES
IQCTSEGVWTPPVPQCKVAACEATGRQLLTKPQHQFVRPDVNSSCGEGYK
LSGSVYQECQGTIPWFMEIRLCKEITCPPPPVIYNGAHTGSSLEDFPYGT
TVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLLAV
QCSHVHIANGYKISGKEAPYFYNDTVTFKCYSGFTLKGSSQIRCKADNTW
DPEIPVCEKETCQHVRQSLQELPAGSRVELVNTSCQDGYQLTGHAYQMCQ
DAENGIWFKKIPLCKVIHCHPPPVIVNGKHTGMMAENFLYGNEVSYECDQ
GFYLLGEKKLQCRSDSKGHGSWSGPSPQCLRSPPVTRCPNPEVKHGYKLN
KTHSAYSHNDIVYVDCNPGFIMNGSRVIRCHTDNTWVPGVPTCIKKAFIG
CPPPPKTPNGNHTGGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTW
SQPAPHCKEVNCSSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYMLEGS
PQSQCQSDHQWNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISK
HRERNYYTDTSQKEAFHLEAREVYSVDPYNPAS
(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626 or 11038674, Proc. Natl. Acad. Sci. USA. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Muller et al. (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (claim 2, FIG. 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15); U.S. Pat. No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO 99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B); WO200055351 (claim 11, pages 1145-1146);
Cross-references: MIM:147245; NP_000617.1. NM_000626_1
229 aa
(SEQ ID NO: 15)
MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSP
RFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQ
NESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQ
LKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHIYEGLD
IDQTATYEDIVILRTGEVKWSVGEHPGQE
(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764, Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. USA. 98 (17):9772-9777 (2001), Xu, M. J., et al. (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; FIG. 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25);
Cross-references: MIM:606509; NP_110391.2; NM_030764_1
508 aa
(SEQ ID NO: 16)
MLLWSLLVIFDAVTEQADSLTLVAPSSVFEGDSIVLKCQGEQNWKIQKMA
YHKDNKELSVFKKFSDFLIQSAVLSDSGNYFCSTKGQLFLWDKTSNIVKI
KVQELFQRPVLTASSFQPIEGGPVSLKCETRLSPQRLDVQLQFCFFRENQ
VLGSGWSSSPELQISAVWSEDTGSYWCKAETVTHRIRKQSLQSQIHVQRI
PISNVSLEIRAPGGQVTEGQKLILLCSVAGGTGNVTFSWYREATGTSMGK
KTQRSLSAELEIPAVKESDAGKYYCRADNGHVPIQSKVVNIPVRIPVSRP
VLTLRSPGAQAAVGDLLELHCEALRGSPPILYQFYHEDVTLGNSSAPSGG
GASFNLSLTAEHSGNYSCEANNGLGAQCSEAVPVSISGPDGYRRDLMTAG
VLWGLFGVLGFTGVALLLYALFHKISGESSATNEPRGASRPNPQEFTYSS
PTPDMEELQPVYVNVGSVDVDVVYSQVWSMQQPESSANIRTLLENKDSQV
IYSSVKKS
(17) HER2 (ErbB2, Genbank accession no. M11730, Coussens L., et al. Science (1985) 230(4730):1132-1139); Yamamoto T., et al. Nature 319, 230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci. USA. 82, 6497-6501, 1985; Swiercz J. M., et al. J. Cell Biol. 165, 869-880, 2004; Kuhns J. J., et al. J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., et al. Nature 421, 756-760, 2003; Ehsani A., et al. (1993) Genomics 15, 426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I); WO2004009622; WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; FIG. 1A-B); WO2003025228 (claim 37; FIG. 5C); WO200222636 (Example 13; Page 95-107); WO200212341 (claim 68; FIG. 7); WO200213847 (Page 71-74); WO200214503 (Page 114-117); WO200153463 (claim 2; Page 41-46); WO200141787 (Page 15); WO200044899 (claim 52; FIG. 7); WO200020579 (claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (claim 3; Col 31-38); WO9630514 (claim 2; Page 56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4);
Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.
1255 aa
(SEQ ID NO: 17)
MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLY
QGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLR
IVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILK
GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCK
GSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHS
DCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP
YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHL
REVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVF
ETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGI
SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRP
EDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGL
PREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC
PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASP
LTSIISAVVGILLVVVLGVVEGILIKRRQQKIRKYTMRRLLQETELVEPL
TPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPV
AIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQL
MPYGCLLDHVRENRGRLGSQDLLNWCMQTAKGMSYLEDVRLVHRDLAARN
VLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT
HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTID
VYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPL
DSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSS
STRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVEDGDLGMGAAKGLQS
LPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPP
SPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ
GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLG
LDVPV
(18) NCA (CEACAM6, Genbank accession no. M18728);
Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al. Proc. Natl. Acad. Sci. USA. 99:16899-16903, 2002; WO2004063709; EP1439393 (claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (Example 2); WO200286443 (claim 27; Page 427); WO200260317 (claim 2);
Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728;
344 aa
(SEQ ID NO: 18)
MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKE
VLLLAHNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRET
TYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSIS
SNNSNPVEDKDAVAFTCEPEVQNTTYLWWVNGQSLPVSPRLQLSNGNMTL
TLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVPTISPSKANYR
PGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQ
AHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIGVLARVALI
(19) MDP (DPEP1, Genbank accession no. BC017023,
Proc. Natl. Acad. Sci. USA. 99 (26):16899-16903 (2002)); WO2003016475 (claim 1); WO200264798 (claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284 (FIG. 9);
Cross-references: MIM:179780; AAH17023.1; BC017023_1
411 aa
(SEQ ID NO: 19)
MWSGWWLWPLVAVCTADFFRDEAERIMRDSPVIDGHNDLPWQLLDMENNR
LQDERANLTTLAGTHTNIPKLRAGFVGGQFWSVYTPCDTQNKDAVRRTLE
QMDVVHRMCRMYPETFLYVTSSAGIRQAFREGKVASLIGVEGGHSIDSSL
GVLRALYQLGMRYLTLTHSCNTPWADNWLVDTGDSEPQSQGLSPFGQRVV
KELNRLGVLIDLAHVSVATMKATLQLSRAPVIFSHSSAYSVCASRRNVPD
DVLRLVKQTDSLVMVNFYNNYISCTNKANLSQVADHLDHIKEVAGARAVG
FGGDFDGVPRVPEGLEDVSKYPDLIAELLRRNWTEAEVKGALADNLLRVF
EAVEQASNLTQAPEEEPIPLDQLGGSCRTHYGYSSGASSLHRHWGLLLAS
LAPLVLCLSLL
(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);
Clark H. F., et al. Genome Res. 13, 2265-2270, 2003; Mungall A. J., et al. Nature 425, 805-811, 2003; Blumberg H., et al. Cell 104, 9-19, 2001; Dumoutier L., et al. J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al. J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al. (2003) Biochemistry 42:12617-12624; Sheikh F., et al. (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (Page 74-75); WO2003002717 (claim 2; Page 63); WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59); WO200146232 (Page 63-65); WO9837193 (claim 1; Page 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.
553 aa
(SEQ ID NO: 20)
MRAPGRPALRPLPLPPLLLLLLAAPWGRAVPCVSGGLPKPANITFLSINM
KNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAE
TSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKS
ISVVLTAPEKWKRNPEDLPVSMQQTYSNLKYNVSVLNTKSNRTWSQCVTN
HTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEFKAK
IIFWYVLPISITVELFSVMGYSIYRYIHVGKEKHPANLILIYGNEFDKRF
FVPAEKIVINFITLNISDDSKISHQDMSLLGKSSDVSSLNDPQPSGNLRP
PQEEEEVKHLGYASHLMEIFCDSEENTEGTSFTQQESLSRTIPPDKTVIE
YEYDVRTTDICAGPEEQELSLQEEVSTQGTLLESQAALAVLGPQTLQYSY
TPQLQDLDPLAQEHTDSEEGPEEEPSTTLVDWDPQTGRLCIPSLSSFDQD
SEGCEPSEGDGLGEEGLLSRLYEEPAPDRPPGENETYLMQFMEEWGLYVQ
MEN
(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)
Gary S. C., et al. Gene 256, 139-147, 2000; Clark H. F., et al. Genome Res. 13, 2265-2270, 2003; Strausberg R. L., et al. Proc. Natl. Acad. Sci. USA. 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1; FIG. 52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1);
911 aa
(SEQ ID NO: 21)
MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLQGVLG
GALTIPCHVHYLRPPPSRRAVLGSPRVKWTFLSRGREAEVLVARGVRVKV
NEAYRFRVALPAYPASLTDVSLALSELRPNDSGIYRCEVQHGIDDSSDAV
EVKVKGVVFLYREGSARYAFSFSGAQEACARIGAHIATPEQLYAAYLGGY
EQCDAGWLSDQTVRYPIQTPREACYGDMDGFPGVRNYGVVDPDDLYDVYC
YAEDLNGELFLGDPPEKLTLEEARAYCQERGAEIATTGQLYAAWDGGLDH
CSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNV
YCFRDSAQPSAIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESE
SRGAIYSIPIMEDGGGGSSTPEDPAEAPRTLLEFETQSMVPPTGESEEEG
KALEEEEKYEDEEEKEEEEEEEEVEDEALWAWPSELSSPGPEASLPTEPA
AQEKSLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPTETLPTPRERN
LASPSPSTLVEAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRA
PEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAVVPASGDC
VPSPCHNGGTCLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACY
KHFSTRRSWEEAETQCRMYGAHLASISTPEEQDFINNRYREYQWIGLNDR
TIEGDFLWSDGVPLLYENWNPGQPDSYFLSGENCVVMVWHDQGQWSDVPC
NYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVLRYRCREGLAQ
RNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKAL
LIPPSSPMPGP
(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NM_004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (claim 12); WO200053216 (claim 1; Page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (Page 128-132); WO200053216 (claim 1; Page 42);
Cross-references: MIM:600997; NP_004433.2; NM_004442_1
987 aa
(SEQ ID NO: 22)
MALRRLGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYD
ENMNTIRTYQVCNVFESSQNNWLRTKFIRRRGAHRIHVEMKFSVRDCSSI
PSVPGSCKETFNLYYYEADFDSATKTFPNWMENPWVKVDTIAADESFSQV
DLGGRVMKINTEVRSFGPVSRSGFYLAFQDYGGCMSLIAVRVEYRKCPRI
IQNGAIFQETLSGAESTSLVAARGSCIANAEEVDVPIKLYCNGDGEWLVP
IGRCMCKAGFEAVENGTVCRGCPSGTFKANQGDEACTHCPINSRTTSEGA
TNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETSLMLEWTPPRDS
GGREDLVYNIICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLA
HTQYTFEIQAVNGVTDQSPFSPQFASVNITTNQAAPSAVSIMHQVSRTVD
SITLSWSQPDQPNGVILDYELQYYEKELSEYNATAIKSPTNTVTVQGLKA
GAIYVFQVRARTVAGYGRYSGKMYFQTMTEAEYQTSIQEKLPLIIGSSAA
GLVFLIAVVVIAIVCNRRRGFERADSEYTDKLQHYTSGHMTPGMKIYIDP
FTYEDPNEAVREFAKEIDISCVKIEQVIGAGEFGEVCSGHLKLPGKREIF
VAIKILKSGYTEKQRRDFLSEASIMGQFDHPNVIHLEGVVIKSTPVMIIT
EFMENGSLDSFLRQNDGQFTVIQLVGMLRGIAAGMKYLADMNYVHRDLAA
RNILVNSNLVCKVSDFGLSRFLEDDISDPTYISALGGKIPIRWTAPEAIQ
YRKFTSASDVWSYGIVMWEVMSYGERPYWDMINQDVINAIEQDYRLPPPM
DCPSALHQLMLDCWQKDRNHRPKFGQIVNILDKMIRNPNSLKAMAPLSSG
INLPLLDRTIPDYTSFNIVDEWLEAIKMGQYKESFANAGFTSFDVVSQMM
MEDILRVGVTLAGHQKKILNSIQVMRAQMNQIQSVEV
(23) ASLG659 (B7h, Genbank accession no. AX092328) US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (FIG. 3); US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (FIG. 60); WO2002102235 (claim 13; Page 299); US2003091580 (Example 2); WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG. 7b); WO200202624 (claim 13; FIG. 1A-1B); US2002034749 (claim 54; Page 45-46); WO200206317 (Example 2; Page 320-321, claim 34; Page 321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318;
282 aa
(SEQ ID NO: 23)
MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGE
DGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGR
TAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKKNANLEYKTGAF
SMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFE
LNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSH
LQLLNSKASLCVSSFFAISWALLPLSPYLMLK
(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no. AJ297436) Reiter R. E., et al. Proc. Natl. Acad. Sci. USA. 95, 1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274 (Example 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; Page 164); WO2003003906 (claim 10; Page 288); WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b); WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17; Page 97); WO9851824 (claim 10; Page 94); WO9840403 (claim 2; FIG. 1B);
Accession: 043653; EMBL; AF043498; AAC39607.1.
123 aa
(SEQ ID NO: 24)
MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWT
ARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHAL
QPAAAILALLPALGLLLWGPGQL
(25) GEDA (Genbank accession No. AY260763);
AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1—Homo sapiens Species: Homo sapiens (human)
WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example 3, claim 20); US2003194704 (claim 45);
Cross-references: GI:30102449; AAP14954.1; AY260763_1
236 aa
(SEQ ID NO: 25)
MPGAAAAAAAAAAAMLPAQEAAKLYHTNYVRNSPAIGVLWAIFTICFAIV
NVVCFIQPYWIGDGVDTPQAGYFGLFHYCIGNGFSRELTCRGSFTDFSTL
PSGAFKAASFFIGLSMMLIIACIICFTLFFFCNTATVYKICAWMQLTSAA
CLVLGCMIFPDGWDSDEVKRMCGEKTDKYTLGACSVRWAYILAIIGILDA
LILSFLAFVLGNRQDSLMAEELKAENKVLLSQYSLE
(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. NP_443177.1);
NP_443177 BAFF receptor/pid=NP_443177.1—Homo sapiens
Thompson, J. S., et al. Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33); WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; Page 615-616); WO200294852 (Col 136-137); WO200238766 (claim 3; Page 133); WO200224909 (Example 3; FIG. 3); Cross-references: MIM:606269; NP_443177.1; NM_052945_1
184 aa
(SEQ ID NO: 26)
MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASS
PAPRTALQPQESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVS
WRRRQRRLRGASSAEAPDGDKDAPEPLDKVIILSPGISDATAPAWPPPGE
DPGTTPPGHSVPVPATELGSTELVTTKTAGPEQQ
(27) CD22 (B-cell receptor CD22-B isoform, Genbank accession No. NP-001762.1); Stamenkovic, I. and Seed, B., Nature 345 (6270), 74-77 (1990); US2003157113; US2003118592; WO2003062401 (claim 9); WO2003072036 (claim 1; FIG. 1); WO200278524 (Example 2); Cross-references: MIM:107266; NP_001762.1; NM_001771_1
847 aa
(SEQ ID NO: 27)
MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALD
GDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNK
NCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQLPPE
IQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFT
RSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTP
SDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVT
KDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVE
FLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAEN
ILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPS
VTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACARCNSWCSWASPVALN
VQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLL
GKESQLNEDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM
SPGDQVMEGKSATLTCESDANPPVSHYTWEDWNNQSLPHHSQKLRLEPVK
VQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILI
LAICGLKLQRRWKRTQSQQGLQENSSGQSFEVRNKKVRRAPLSEGPHSLG
CYNPMMEDGISYTTLREPEMNIPRTGDAESSEMQRPPRTCDDTVTYSALH
KRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH
(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation) PROTEIN SEQUENCE Full mpggpgv . . . dvqlekp (1 . . . 226; 226 aa), pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank accession No. NP_001774.1;
WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No. 5,644,033; Ha et al. (1992) J. Immunol. 148(5):1526-1531; Mueller et al. (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al. (1994) Immunogenetics 40(4):287-295; Preud'homme et al. (1992) Clin. Exp. Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol. 148(2) 633-637; Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464;
226 aa
(SEQ ID NO: 28)
MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDA
HFQCPHNSSNNANVIWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSH
GGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGI
ILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYE
DISRGLQGTYQDVGSLNIGDVQLEKP
(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia) PROTEIN SEQUENCE Full mnypltl . . . atslttf (1 . . . 372; 372 aa), pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession No. NP_001707.1; WO2004040000; WO2004015426; US2003105292 (Example 2); U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188 (claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1, pages 152-153, Example 2, pages 254-256); WO9928468 (claim 1, page 38); U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, FIG. 5); Dobner et al. (1992) Eur. J. Immunol. 22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779;
372 aa
(SEQ ID NO: 29)
MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFK
AVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLL
VFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLA
IVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLP
RCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQR
RPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSL
PVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQ
LFPSWRRSSLSESENATSLTTF
(30) HLA-DOB (Beta subunit of MEW class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes) PROTEIN SEQUENCE Full mgsgwvp . . . vllpqsc (1 . . . 273; 273 aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP_002111.1;
Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903; Servenius et al. (1987) J. Biol. Chem. 262:8759-8766; Beck et al. (1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) Tissue Antigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No. 6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat. No. 6,011,146 (col 145-146); Kasahara et al. (1989) Immunogenetics 30(1):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119;
273 aa
(SEQ ID NO: 30)
MGSGWVPWVVALLVNLTRLDSSMTQGTDSPEDFVIQAKADCYFTNGTEKV
QFVVRFIFNLEEYVRFDSDVGMFVALTKLGQPDAEQWNSRLDLLERSRQA
VDGVCRHNYRLGAPFTVGRKVQPEVTVYPERTPLLHQHNLLHCSVTGFYP
GDIKIKWFLNGQEERAGVMSTGPIRNGDWTFQTVVMLEMTPELGHVYTCL
VDHSSLLSPVSVEWRAQSEYSWRKMLSGIAAFLLGLIFLLVGIVIQLRAQ
KGYVRTQMSGNEVSRAVLLPQSC
(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability) PROTEIN SEQUENCE Full mgqagck . . . lephrst (1 . . . 422; 422 aa), pI: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No. NP_002552.2; Le et al. (1997) FEBS Lett. 418(1-2):195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al. (2000) Genome Res. 10:165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82);
422 aa
(SEQ ID NO: 31)
MGQAGCKGLCLSLFDYKTEKYVIAKNKKVGLLYRLLQASILAYLVVWVFL
IKKGYQDVDTSLQSAVITKVKGVAFTNTSDLGQRIWDVADYVIPAQGENV
FFVVTNLIVTPNQRQNVCAENEGIPDGACSKDSDCHAGEAVTAGNGVKTG
RCLRRENLARGTCEIFAWCPLETSSRPEEPFLKEAEDFTIFIKNHIRFPK
FNFSKSNVMDVKDRSFLKSCHFGPKNHYCPIFRLGSVIRWAGSDFQDIAL
EGGVIGINIEWNCDLDKAASECHPHYSFSRLDNKLSKSVSSGYNFRFARY
YRDAAGVEFRTLMKAYGIRFDVMVNGKGAFFCDLVLIYLIKKREFYRDKK
YEEVRGLEDSSQEAEDEASGLGLSEQLTSGPGLLGMPEQQELQEPPEAKR
GSSSQKGNGSVCPQLLEPHRST
(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Full maeaity . . . tafrfpd (1 . . . 359; 359 aa), pI: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No. NP_001773.1;
WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903;
359 aa
(SEQ ID NO: 32)
MAEAITYADLRFVKAPLKKSISSRLGQDPGADDDGEITYENVQVPAVLGV
PSSLASSVLGDKAAVKSEQPTASWRAVTSPAVGRILPCRTTCLRYLLLGL
LLTCLLLGVTAICLGVRYLQVSQQLQQTNRVLEVTNSSLRQQLRLKITQL
GQSAEDLQGSRRELAQSQEALQVEQRAHQAAEGQLQACQADRQKTKETLQ
SEEQQRRALEQKLSNMENRLKPFFTCGSADTCCPSGWIMHQKSCFYISLT
SKNWQESQKQCETLSSKLATFSETYPQSHSYYFLNSLLPNGGSGNSYWTG
LSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYIC
EMTAFRFPD
(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis) PROTEIN SEQUENCE Full mafdvsc . . . rwkyqhi (1 . . . 661; 661 aa), pI: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No. NP_005573.1;
US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al. (1996) Genomics 38(3):299-304; Miura et al. (1998) Blood 92:2815-2822; WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24-26);
661 aa
(SEQ ID NO: 33)
MAFDVSCFFWVVLFSAGCKVITSWDQMCIEKEANKTYNCENLGLSEIPDT
LPNTTEFLEFSFNFLPTIHNRTFSRLMNLTFLDLTRCQINWIHEDTFQSH
HQLSTLVLTGNPLIFMAETSLNGPKSLKHLFLIQTGISNLEFIPVHNLEN
LESLYLGSNHISSIKFPKDFPARNLKVLDFQNNAIHYISREDMRSLEQAI
NLSLNFNGNNVKGIELGAFDSTVFQSLNFGGTPNLSVIFNGLQNSTTQSL
WLGTFEDIDDEDISSAMLKGLCEMSVESLNLQEHRFSDISSTTFQCFTQL
QELDLTATHLKGLPSGMKGLNLLKKLVLSVNHFDQLCQISAANFPSLTHL
YIRGNVKKLHLGVGCLEKLGNLQTLDLSHNDIEASDCCSLQLKNLSHLQT
LNLSHNEPLGLQSQAFKECPQLELLDLAFTRLHINAPQSPFQNLHFLQVL
NLTYCFLDISNQHLLAGLPVLRHLNLKGNHFQDGTITKINLLQTVGSLEV
LILSSCGLLSIDQQAFHSLGKMSHVDLSHNSLTCDSIDSLSHLKGIYLNL
AANSINIISPRLLPILSQQSTINLSHNPLDCTCSNIHFLTWYKENLHKLE
GSEETTCANPPSLRGVKLSDVKLSCGITAIGIFFLIVFLLLLAILLFFAV
KYLLRWKYQHI
(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation) PROTEIN SEQUENCE Full mlprlll . . . vdyedam (1 . . . 429; 429 aa), pI: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No. NP_443170.1; WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7);
429 aa
(SEQ ID NO: 34)
MLPRLLLLICAPLCEPAELFLIASPSHPTEGSPVTLTCKMPFLQSSDAQF
QFCFFRDTRALGPGWSSSPKLQIAAMWKEDTGSYWCEAQTMASKVLRSRR
SQINVHRVPVADVSLETQPPGGQVMEGDRLVLICSVAMGTGDITFLWYK
GAVGLNLQSKTQRSLTAEYEIPSVRESDAEQYYCVAENGYGPSPSGLVS
ITVRIPVSRPILMLRAPRAQAAVEDVLELHCEALRGSPPILYWFYHEDI
TLGSRSAPSGGGASFNLSLTEEHSGNYSCEANNGLGAQRSEAVTLNFTV
PTGARSNHLTSGVIEGLLSTLGPATVALLFCYGLKRKIGRRSARDPLRS
LPSPLPQEFTYLNSPTPGQLQPIYENVNVVSGDEVYSLAYYNQPEQESV
AAETLGTHMEDKVSLDIYSRLRKANITDVDYEDAM
(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies) PROTEIN SEQUENCE Full mllwvil . . . assaphr (1 . . . 977; 977 aa), pI: 6.88 MW: 106468 TM: 1 [P] Gene Chromosome: 1q21, Genbank accession No. NP_112571.1;
WO2003024392 (claim 2, FIG. 97); Nakayama et al. (2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, FIG. 18B-1-18B-2);
977 aa
(SEQ ID NO: 35)
MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFREY
SPQKTKWYHRYLGKEILRETPDNILEVQESGEYRCQAQGSPLSSPVHLDF
SSASLILQAPLSVFEGDSVVLRCRAKAEVTLNNTIYKNDNVLAFLNKRTD
FHIPHACLKDNGAYRCTGYKESCCPVSSNTVKIQVQEPFTRPVLRASSFQ
PISGNPVTLTCETQLSLERSDVPLRFRFFRDDQTLGLGWSLSPNFQITAM
WSKDSGFYWCKAATMPHSVISDSPRSWIQVQIPASHPVLTLSPEKALNFE
GTKVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENS
GNYYCTADNGLGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTLH
CEAQRGSLPILYQFHHEDAALERRSANSAGGVAISFSLTAEHSGNYYCTA
DNGFGPQRSKAVSLSITVPVSHPVLTLSSAEALTFEGATVTLHCEVQRGS
PQILYQFYHEDMPLWSSSTPSVGRVSFSFSLTEGHSGNYYCTADNGFGPQ
RSEVVSLFVTVPVSRPILTLRVPRAQAVVGDLLELHCEAPRGSPPILYWF
YHEDVTLGSSSAPSGGEASFNLSLTAEHSGNYSCEANNGLVAQHSDTISL
SVIVPVSRPILTFRAPRAQAVVGDLLELHCEALRGSSPILYWFYHEDVTL
GKISAPSGGGASFNLSLTTEHSGIYSCEADNGPEAQRSEMVTLKVAVPVS
RPVLTLRAPGTHAAVGDLLELHCEALRGSPLILYRFFHEDVTLGNRSSPS
GGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYITGLTANRSGPFAT
GVAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDPARSPPDSDSQEPTYH
NVPAWEELQPVYTNANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGS
PIIYSEVKVASTPVSGSLFLASSAPHR
See also: WO04/045516 (3 Jun. 2004); WO03/000113 (3 Jan. 2003); WO02/016429 (28 Feb. 2002); WO02/16581 (28 Feb. 2002); WO03/024392 (27 Mar. 2003); WO04/016225 (26 Feb. 2004); WO01/40309 (7 Jun. 2001), and U.S. Provisional patent application Ser. No. 60/520,842 “COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR OF HEMATOPOIETIC ORIGIN”, filed 17 Nov. 2003; all of which are incorporated herein by reference in their entirety.
In an embodiment, the Ligand-Linker-Drug Conjugate has Formula IIIa, where the Ligand is an antibody Ab including one that binds at least one of CD30, CD40, CD70, Lewis Y antigen, w=0, y=0, and D has Formula Ib. Exemplary Conjugates of Formula IIIa include where R17 is —(CH2)5—. Also included are such Conjugates of Formula IIIa in which D has the structure of Compound 2 in Example 3 and esters thereof. Also included are such Conjugates of Formula IIIa containing about 3 to about 8, in one aspect, about 3 to about 5 Drug moieties D, that is, Conjugates of Formula Ia wherein p is a value in the range about 3-8, for example about 3-5. Conjugates containing combinations of the structural features noted in this paragraph are also contemplated as within the scope of the compounds of the invention.
In another embodiment, the Ligand-Linker-Drug Conjugate has Formula IIIa, where Ligand is an Antibody Ab that binds one of CD30, CD40, CD70, Lewis Y antigen, w=1, y=0, and D has Formula Ib. Included are such Conjugates of Formula IIIa in which W is —(CH2)5—. Also included are such Conjugates of Formula IIIa in which W is -Val-Cit-, and/or where D has the structure of Compound 2 in Example 3 and esters thereof. Also included are such Conjugates of Formula IIIa containing about 3 to about 8, preferably about 3 to about 5 Drug moieties D, that is, Conjugates of Formula Ia wherein p is a value in the range of about 3-8, preferably about 3-5. Conjugates containing combinations of the structural features noted in this paragraph are also exemplary.
In an embodiment, the Ligand-Linker-Drug Conjugate has Formula IIIa, where the Ligand is an Antibody Ab that binds one of CD30, CD40, CD70, Lewis Y antigen, w=1, y=1, and D has Formula Ib. Included are Conjugates of Formula IIIa in which R17 is —(CH2)5—. Also included are such Conjugates of Formula IIIa where: W is -Val-Cit-; Y has Formula X; D has the structure of Compound 2 in Example 3 and esters thereof; p is about 3 to about 8, preferably about 3 to about 5 Drug moieties D. Conjugates containing combinations of the structural features noted in this paragraph are also contemplated within the scope of the compounds of the invention.
A further embodiment is an antibody drug conjugate (ADC), or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is an antibody that binds one of the tumor-associated antigens (1)-(35) noted above (the “TAA Compound”).
Another embodiment is the TAA Compound or pharmaceutically acceptable salt or solvate thereof that is in isolated and purified form.
Another embodiment is a method for killing or inhibiting the multiplication of a tumor cell or cancer cell comprising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the TAA Compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to kill or inhibit the multiplication of a tumor cell or cancer cell.
Another embodiment is a method for treating cancer comprising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the TAA Compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to treat cancer, alone or together with an effective amount of an additional anticancer agent.
Another embodiment is a method for treating an autoimmune disease, comprising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the TAA Compound or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to treat an autoimmune disease.
The antibodies suitable for use in the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
9.5.1 Production of Recombinant Antibodies
Antibodies of the invention can be produced using any method known in the art to be useful for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression.
Recombinant expression of antibodies, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides, e.g., by PCR.
Alternatively, a nucleic acid molecule encoding an antibody can be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the antibody can be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by, e.g., PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
If an antibody that specifically recognizes a particular antigen is not commercially available (or a source for a cDNA library for cloning a nucleic acid encoding such an immunoglobulin), antibodies specific for a particular antigen can be generated by any method known in the art, for example, by immunizing a patient, or suitable animal model such as a rabbit or mouse, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody can be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
Once a nucleic acid sequence encoding at least the variable domain of the antibody is obtained, it can be introduced into a vector containing the nucleotide sequence encoding the constant regions of the antibody (see, e.g., International Publication No. WO 86/05807; WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors containing the complete light or heavy chain that allow for the expression of a complete antibody molecule are available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, 1 Biol. Chem. 253:6551).
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).
Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to the F(ab′)2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
Once a nucleic acid sequence encoding an antibody has been obtained, the vector for the production of the antibody can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
An expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.
The host cells used to express the recombinant antibody can be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al., 1990, BioTechnology 8:2). A variety of host-expression vector systems can be utilized to express the immunoglobulin antibodies. Such host-expression systems represent vehicles by which the coding sequences of the antibody can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody immunoglobulin molecule in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, vectors that direct the expression of high levels of fusion protein products that are readily purified might be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J Biol. Chem. 24:5503-5509); and the like. pGEX Vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) or the analogous virus from Drosophila Melanogaster is used as a vector to express foreign genes. The virus grows in Spodoptera frupperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
In addition, a host cell strain can be chosen to modulate the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express an antibody can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody. Such engineered cell lines can be particularly useful in screening and evaluation of tumor antigens that interact directly or indirectly with the antibody.
A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: DHFR, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).
The expression levels of an antibody can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
The host cell can be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers that enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used to encode both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.
Once the antibody has been recombinantly expressed, it can be purified using any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
In yet another exemplary embodiment, the antibody is a monoclonal antibody.
In any case, the hybrid antibodies have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an autoimmune disease, an infectious organism, or other disease state.
9.5.2 Production of Antibodies
The production of antibodies will be illustrated with reference to anti-CD30 antibodies but it will be apparent for those skilled in the art that antibodies to other members of the TNF receptor family can be produced and modified in a similar manner. The use of CD30 for the production of antibodies is exemplary only and not intended to be limiting.
The CD30 antigen to be used for production of antibodies may be, e.g., a soluble form of the extracellular domain of CD30 or a portion thereof, containing the desired epitope. Alternatively, cells expressing CD30 at their cell surface (e.g., L540 (Hodgkin's lymphoma derived cell line with a T cell phenotype) and L428 (Hodgkin's lymphoma derived cell line with a B cell phenotype)) can be used to generate antibodies. Other forms of CD30 useful for generating antibodies will be apparent to those skilled in the art.
In another exemplary embodiment, the ErbB2 antigen to be used for production of antibodies may be, e.g., a soluble form of the extracellular domain of ErbB2 or a portion thereof, containing the desired epitope. Alternatively, cells expressing ErbB2 at their cell surface (e.g., NIH-3T3 cells transformed to overexpress ErbB2; or a carcinoma cell line such as SK-BR-3 cells, see Stancovski et al. Proc. Natl. Acad. Sci. USA 88:8691-8695 (1991)) can be used to generate antibodies. Other forms of ErbB2 useful for generating antibodies will be apparent to those skilled in the art.
(i) Polyclonal Antibodies
Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with ⅕ to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
(ii) Monoclonal Antibodies
Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (MA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).
In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
The DNA also may be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al. (1984) Proc. Natl Acad. Sci. USA 81:6851), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
(iii) Humanized Antibodies
A humanized antibody may have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
In another embodiment, the antibodies may be humanized with retention of high affinity for the antigen and other favorable biological properties. Humanized antibodies may be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
Various forms of the humanized antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.
The Examples describe production of an exemplary humanized anti-ErbB2 antibody. The humanized antibody may, for example, comprise nonhuman hypervariable region residues incorporated into a human variable heavy domain and may further comprise a framework region (FR) substitution at a position selected from the group consisting of 69H, 71H and 73H utilizing the variable domain numbering system set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). In one embodiment, the humanized antibody comprises FR substitutions at two or all of positions 69H, 71H and 73H. Another Example describes preparation of purified trastuzumab antibody from the HERCEPTIN® formulation.
(iv) Human Antibodies
As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JO gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905. As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275). Human anti-CD30 antibodies are described in U.S. patent application Ser. No. 10/338,366.
(v) Antibody Fragments
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
(vi) Bispecific Antibodies
Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the CD30 protein. Alternatively, an anti-CD30 arm may be combined with an arm which binds to a Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the CD30-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD30.
Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO 1, 10:3655-3659 (1991). According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Recent progress has facilitated the direct recovery of Fab′-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
(vii) Other Amino Acid Sequence Modifications
Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.
A useful method for identification of certain residues or regions of the antibody that are favored locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.
Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally-occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).
To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
(viii) Glycosylation Variants
Antibodies in the ADC of the invention may be glycosylated at conserved positions in their constant regions (Jefferis and Lund, (1997) Chem. Immunol. 65:111-128; Wright and Morrison, (1997) TibTECH 15:26-32). The oligosaccharide side chains of the immunoglobulins affect the protein's function (Boyd et al., (1996) Mol. Immunol. 32:1311-1318; Wittwe and Howard, (1990) Biochem. 29:4175-4180), and the intramolecular interaction between portions of the glycoprotein which can affect the conformation and presented three-dimensional surface of the glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, (1996) Current Opin. Biotech. 7:409-416). Oligosaccharides may also serve to target a given glycoprotein to certain molecules based upon specific recognition structures. For example, it has been reported that in agalactosylated IgG, the oligosaccharide moiety ‘flips’ out of the inter-CH2 space and terminal N-acetylglucosamine residues become available to bind mannose binding protein (Malhotra et al., (1995) Nature Med. 1:237-243). Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibody which recognizes the CDw52 antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO) cells resulted in a complete reduction in complement mediated lysis (CMCL) (Boyd et al., (1996) Mol. Immunol. 32:1311-1318), while selective removal of sialic acid residues using neuraminidase resulted in no loss of DMCL. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisecting GlcNAc, was reported to have improved ADCC activity (Umana et al. (1999) Mature Biotech. 17:176-180).
Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
Glycosylation variants of antibodies are variants in which the glycosylation pattern of an antibody is altered. By altering is meant deleting one or more carbohydrate moieties found in the antibody, adding one or more carbohydrate moieties to the antibody, changing the composition of glycosylation (glycosylation pattern), the extent of glycosylation, etc.
Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of the antibody.
The amino acid sequence is usually altered by altering the underlying nucleic acid sequence. These methods include, but are not limited to, isolation from a natural source (in the case of naturally-occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.
The glycosylation (including glycosylation pattern) of antibodies may also be altered without altering the amino acid sequence or the underlying nucleotide sequence. Glycosylation largely depends on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g., antibodies, as potential therapeutics is rarely the native cell, significant variations in the glycosylation pattern of the antibodies can be expected. See, e.g., Hse et al., (1997) J. Biol. Chem. 272:9062-9070. In addition to the choice of host cells, factors which affect glycosylation during recombinant production of antibodies include growth mode, media formulation, culture density, oxygenation, pH, purification schemes and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261; 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H). In addition, the recombinant host cell can be genetically engineered, e.g., make defective in processing certain types of polysaccharides. These and similar techniques are well known in the art.
The glycosylation structure of antibodies can be readily analyzed by conventional techniques of carbohydrate analysis, including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharide compositional analysis, sequential enzymatic digestion, and HPAEC-PAD, which uses high pH anion exchange chromatography to separate oligosaccharides based on charge. Methods for releasing oligosaccharides for analytical purposes are also known, and include, without limitation, enzymatic treatment (commonly performed using peptide-N-glycosidase F/endo-β-galactosidase), elimination using harsh alkaline environment to release mainly O-linked structures, and chemical methods using anhydrous hydrazine to release both N- and O-linked oligosaccharides.
9.5.2a Screening for Antibody-Drug Conjugates (Adc)
Transgenic animals and cell lines are particularly useful in screening antibody drug conjugates (ADC) that have potential as prophylactic or therapeutic treatments of diseases or disorders involving overexpression of proteins including Lewis Y, CD30, CD40, and CD70. Transgenic animals and cell lines are particularly useful in screening antibody drug conjugates (ADC) that have potential as prophylactic or therapeutic treatments of diseases or disorders involving overexpression of HER2 (U.S. Pat. No. 6,632,979). Screening for a useful ADC may involve administering candidate ADC over a range of doses to the transgenic animal, and assaying at various time points for the effect(s) of the ADC on the disease or disorder being evaluated. Alternatively, or additionally, the drug can be administered prior to or simultaneously with exposure to an inducer of the disease, if applicable. Candidate ADC may be screened serially and individually, or in parallel under medium or high-throughput screening format. The rate at which ADC may be screened for utility for prophylactic or therapeutic treatments of diseases or disorders is limited only by the rate of synthesis or screening methodology, including detecting/measuring/analysis of data.
One embodiment is a screening method comprising (a) transplanting cells from a stable renal cell cancer cell line into a non-human animal, (b) administering an ADC drug candidate to the non-human animal and (c) determining the ability of the candidate to inhibit the formation of tumors from the transplanted cell line.
Another embodiment is a screening method comprising (a) contacting cells from a stable Hodgkin's disease cell line with an ADC drug candidate and (b) evaluating the ability of the ADC candidate to block ligand activation of CD40.
Another embodiment is a screening method comprising (a) contacting cells from a stable Hodgkin's disease cell line with an ADC drug candidate and (b) evaluating the ability of the ADC candidate to induce cell death. In one embodiment the ability of the ADC candidate to induce apoptosis is evaluated.
One embodiment is a screening method comprising (a) transplanting cells from a stable cancer cell line into a non-human animal, (b) administering an ADC drug candidate to the non-human animal and (c) determining the ability of the candidate to inhibit the formation of tumors from the transplanted cell line. The invention also concerns a method of screening ADC candidates for the treatment of a disease or disorder characterized by the overexpression of HER2 comprising (a) contacting cells from a stable breast cancer cell line with a drug candidate and (b) evaluating the ability of the ADC candidate to inhibit the growth of the stable cell line.
Another embodiment is a screening method comprising (a) contacting cells from a stable cancer cell line with an ADC drug candidate and (b) evaluating the ability of the ADC candidate to block ligand activation of HER2. In one embodiment the ability of the ADC candidate to block heregulin binding is evaluated. In another embodiment the ability of the ADC candidate to block ligand-stimulated tyrosine phosphorylation is evaluated.
Another embodiment is a screening method comprising (a) contacting cells from a stable cancer cell line with an ADC drug candidate and (b) evaluating the ability of the ADC candidate to induce cell death. In one embodiment the ability of the ADC candidate to induce apoptosis is evaluated.
Another embodiment is a screening method comprising (a) administering an ADC drug candidate to a transgenic non-human mammal that overexpresses in its mammary gland cells a native human HER2 protein or a fragment thereof, wherein such transgenic mammal has stably integrated into its genome a nucleic acid sequence encoding a native human HER2 protein or a fragment thereof having the biological activity of native human HER2, operably linked to transcriptional regulatory sequences directing its expression to the mammary gland, and develops a mammary tumor not responding or poorly responding to anti-HER2 antibody treatment, or to a non-human mammal bearing a tumor transplanted from said transgenic non-human mammal; and (b) evaluating the effect of the ADC candidate on the target disease or disorder. Without limitations, the disease or disorder may be a HER2-overexpressing cancer, such as breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic and bladder cancer. The cancer preferably is breast cancer which expressed HER2 in at least about 500,000 copies per cell, more preferably at least about 2,000,000 copies per cell. ADC drug candidates may, for example, be evaluated for their ability to induce cell death and/or apoptosis, using assay methods well known in the art and described hereinafter.
In one embodiment, candidate ADC are screened by being administered to the transgenic animal over a range of doses, and evaluating the animal's physiological response to the compounds over time. Administration may be oral, or by suitable injection, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound. If cell lines derived from the subject transgenic animals are used to screen for compounds useful in treating various disorders, the test compounds are added to the cell culture medium at an appropriate time, and the cellular response to the compound is evaluated over time using the appropriate biochemical and/or histological assays. In some cases, it may be appropriate to apply the compound of interest to the culture medium in conjunction with co-factors that would enhance the efficacy of the compound.
Thus, provided herein are assays for identifying ADC which specifically target and bind a target protein, the presence of which is correlated with abnormal cellular function, and in the pathogenesis of cellular proliferation and/or differentiation that is causally related to the development of tumors.
To identify an ADC which blocks ligand activation of an ErbB (e.g., ErbB2) receptor, the ability of the compound to block ErbB ligand binding to cells expressing the ErbB (ErbB2) receptor (e.g., in conjugation with another ErbB receptor with which the ErbB receptor of interest forms an ErbB hetero-oligomer) may be determined. For example, cells isolated from the transgenic animal overexpressing HER2 and transfected to express another ErbB receptor (with which HER2 forms hetero-oligomer) may be incubated, i.e. culturing, with the ADC and then exposed to labeled ErbB ligand. The ability of the compound to block ligand binding to the ErbB receptor in the ErbB hetero-oligomer may then be evaluated.
For example, inhibition of heregulin (HRG) binding to breast tumor cell lines, overexpressing HER2 and established from the transgenic non-human mammals (e.g., mice) herein, by the candidate ADC may be performed using monolayer cultures on ice in a 24-well-plate format. Anti-ErbB2 monoclonal antibodies may be added to each well and incubated for 30 minutes. 125I-labeled rHRGβ1177-224 (25,000 cpm) may then be added, and the incubation may be continued for 4 to 16 hours. Dose response curves may be prepared and an IC50 value (cytotoxic activity) may be calculated for the compound of interest.
Alternatively, or additionally, the ability of an ADC to block ErbB ligand-stimulated tyrosine phosphorylation of an ErbB receptor present in an ErbB hetero-oligomer may be assessed. For example, cell lines established from the transgenic animals herein may be incubated with a test ADC and then assayed for ErbB ligand-dependent tyrosine phosphorylation activity using an anti-phosphotyrosine monoclonal antibody (which is optionally conjugated with a detectable label). The kinase receptor activation assay described in U.S. Pat. No. 5,766,863 is also available for determining ErbB receptor activation and blocking of that activity by the compound.
In one embodiment, one may screen for ADC which inhibit HRG stimulation of p180 tyrosine phosphorylation in MCF7 cells essentially as described below. For example, a cell line established from a HER2-transgenic animal may be plated in 24-well plates and the compound may be added to each well and incubated for 30 minutes at room temperature; then rHRGβ1177-244 may be added to each well to a final concentration of 0.2 nM, and the incubation may be continued for about 8 minutes. Media may be aspirated from each well, and reactions may be stopped by the addition of 100 μlof SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 μl) may be electrophoresed on a 4-12% gradient gel (Novex) and then electrophoretically transferred to polyvinylidene difluoride membrane. Antiphosphotyrosine (at 1 μg/ml) immunoblots may be developed, and the intensity of the predominant reactive band at Mr-180,000 may be quantified by reflectance densitometry. An alternate method to evaluate inhibition of receptor phosphorylation is the KIRA (kinase receptor activation) assay of Sadick et al. (1998) Jour. of Pharm. and Biomed. Anal. Some of the well established monoclonal antibodies against HER2 that are known to inhibit HRG stimulation of p180 tyrosine phosphorylation can be used as positive control in this assay. A dose-response curve for inhibition of HRG stimulation of p180 tyrosine phosphorylation as determined by reflectance densitometry may be prepared and an ICso for the compound of interest may be calculated.
One may also assess the growth inhibitory effects of a test ADC on cell lines derived from a HER2-transgenic animal, e.g., essentially as described in Schaefer et al. (1997) Oncogene 15:1385-1394. According to this assay, the cells may be treated with a test compound at various concentrations for 4 days and stained with crystal violet or the redox dye Alamar Blue. Incubation with the compound may show a growth inhibitory effect on this cell line similar to that displayed by monoclonal antibody 2C4 on MDA-MB-175 cells (Schaefer et al., supra). In a further embodiment, exogenous HRG will not significantly reverse this inhibition.
To identify growth inhibitory compounds that specifically target an antigen of interest, one may screen for compounds which inhibit the growth of cancer cells overexpressing antigen of interest derived from transgenic animals, the assay described in U.S. Pat. No. 5,677,171 can be performed. According to this assay, cancer cells overexpressing the antigen of interest are grown in a 1:1 mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum, glutamine and penicillin streptomycin. The cells are plated at 20,000 cells in a 35 mm cell culture dish (2 mls/35 mm dish) and the test compound is added at various concentrations. After six days, the number of cells, compared to untreated cells is counted using an electronic COULTER™ cell counter. Those compounds which inhibit cell growth by about 20-100% or about 50-100% may be selected as growth inhibitory compounds.
To select for compounds which induce cell death, loss of membrane integrity as indicated by, e.g., PI, trypan blue or 7AAD uptake may be assessed relative to control. The PI uptake assay uses cells isolated from the tumor tissue of interest of a transgenic animal. According to this assay, the cells are cultured in Dulbecco's Modified Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay is performed in the absence of complement and immune effector cells. The cells are seeded at a density of 3×106 per dish in 100×20 mm dishes and allowed to attach overnight. The medium is then removed and replaced with fresh medium alone or medium containing various concentrations of the compound. The cells are incubated for a 3-day time period. Following each treatment, monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged at 1200 rpm for 5 minutes at 4° C., the pellet resuspended in 3 ml cold Ca2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2)) and aliquoted into 35 mm strainer-capped 12×75 mm tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Those compounds which induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing compounds.
In order to select for compounds which induce apoptosis, an annexin binding assay using cells established from the tumor tissue of interest of the transgenic animal is performed. The cells are cultured and seeded in dishes as discussed in the preceding paragraph. The medium is then removed and replaced with fresh medium alone or medium containing 10 μg/ml of the antibody drug conjugate (ADC). Following a three-day incubation period, monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged, resuspended in Ca′ binding buffer and aliquoted into tubes as discussed above for the cell death assay. Tubes then receive labeled annexin (e.g., annexin V-FITC) (1 μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Those compounds which induce statistically significant levels of annexin binding relative to control are selected as apoptosis-inducing compounds.
9.5.3 In Vitro Cell Proliferation Assays
Generally, the cytotoxic or cytostatic activity of an antibody drug conjugate (ADC) is measured by: exposing mammalian cells having receptor proteins to the antibody of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays were used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC of the invention.
The in vitro potency of antibody drug conjugates was measured by a cell proliferation assay (Example 18, FIGS. 7-10). The CellTiter-Glo® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, Wis.), homogeneous assay method based on the recombinant expression of Coleoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and 5,700,670). This cell proliferation assay determines the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay was conducted in 96 well format, making it amenable to automated high-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6:398-404). The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing. The cells may be treated continuously with ADC, or they may be treated and separated from ADC. Generally, cells treated briefly, i.e. 3 hours, showed the same potency effects as continuously treated cells.
The homogeneous “add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. The CellTiterGlo® Assay generates a “glow-type” luminescent signal, produced by the luciferase reaction, which has a half-life generally greater than five hours, depending on cell type and medium used (FIG. 24). Viable cells are reflected in relative luminescence units (RLU). The substrate, Beetle Luciferin, is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to AMP and generation of photons. The extended half-life eliminates the need to use reagent injectors and provides flexibility for continuous or batch mode processing of multiple plates. This cell proliferation assay can be used with various multiwell formats, e.g., 96 or 384 well format. Data can be recorded by luminometer or CCD camera imaging device. The luminescence output is presented as relative light units (RLU), measured over time.
The anti-proliferative effects of antibody drug conjugates were measured by the cell proliferation, in vitro cell killing assay above against four different breast tumor cell lines (FIGS. 7-10). IC50 values were established for SK-BR-3 and BT-474 which are known to over express HER2 receptor protein. Table 2a shows the potency (IC50) measurements of exemplary antibody drug conjugates in the cell proliferation assay against SK-BR-3 cells. Table 2b shows the potency (IC50) measurements of exemplary antibody drug conjugates in the cell proliferation assay against BT-474 cells.
Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab; Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; Trastuzumab-MC-MMAF, 4.1 MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; and Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab did not inhibit the proliferation of MCF-7 cells (FIG. 9).
Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab; Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; and Trastuzumab-MC-MMAF, 4.1 MMAF/Ab did not inhibit the proliferation of MDA-MB-468 cells (FIG. 10).
MCF-7 and MDA-MB-468 cells do not overexpress HER2 receptor protein. The anti-HER2 antibody drug conjugates of the invention therefore show selectivity for inhibition of cells which express HER2.
TABLE 2a
SK-BR-3 cells
Antibody Drug Conjugate
H = trastuzumab linked via a cysteine [cys]
IC50
except where noted
(μg ADC/ml)
H-MC-MMAF, 4.1 MMAF/Ab
0.008
H-MC-MMAF, 4.8 MMAF/Ab
0.002
H-MC-vc-PAB-MMAE,
0.007
H-MC-vc-PAB-MMAE
0.015
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab
0.0035-0.01
H-MC-vc-PAB-MMAF, 4.4 MMAF/Ab
0.006-0.007
H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab
0.006
H-MC-(N—Me)vc-PAB-MMAF, 3.9 MMAF/Ab
0.0035
H-MC-MMAF, 4.1 MMAF/Ab
0.0035
H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab
0.010
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab
0.007
H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab
0.015
H-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.
0.010
H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab
0.0025
H-MC-MMAE, 8.8 MMAE/Ab
0.018
H-MC-MMAE, 4.6 MMAE/Ab
0.05
H-MC-(L)val-(L)cit-PAB-MMAE, 8.7
0.0003
MMAE/Ab
H-MC-(D)val-(D)cit-PAB-MMAE, 8.2
0.02
MMAE/Ab
H-MC-(D)val-(L)cit-PAB-MMAE, 8.4
0.0015
MMAE/Ab
H-MC-(D)val-(L)cit-PAB-MMAE, 3.2
0.003
MMAE/Ab
H-Trastuzumab
0.083
H-vc-MMAE, linked via a lysine [lys]
0.002
H-phe-lys-MMAE, linked via a lysine [lys]
0.0015
4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab
0.004
Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab
0.01
7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab
0.01
4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab
0.02
Anti-TF Fab-MC-vc-PAB-MMAE*
—
TABLE 2b
BT474 cells
Antibody Drug Conjugate
IC50
H = trastuzumab linked via a cysteine [cys]
(μg ADC/ml)
H-MC-MMAF, 4.1 MMAF/Ab
0.008
H-MC-MMAF, 4.8 MMAF/Ab
0.002
H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab
0.015
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab
0.02-0.05
H-MC-vc-PAB-MMAF, 4.4 MMAF/Ab
0.01
H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab
0.01
H-MC-vc-PAB-MMAE, 3.3 MMAE/Ab
0.02
H-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.
0.02
H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab
0.015
H-MC-(N—Me)vc-PAB-MMAF, 3.9 MMAF/Ab
0.010
H-MC-MMAF, 4.1 MMAF/Ab
0.00015
H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab
0.0025
H-MC-MMAE, 8.8 MMAE/Ab
0.04
H-MC-MMAE, 4.6 MMAE/Ab
0.07
4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab
0.008
Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab
0.01
7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab
0.015
4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab
0.04
Anti-TF Fab-MC-vc-PAB-MMAE*
—
H = trastuzumab
7C2 = anti-HER2 murine antibody which binds a different epitope than trastuzumab.
Fc8 = mutant that does not bind to FcRn
Hg = “Hingeless” full-length humanized 4D5, with heavy chain hinge cysteines mutated to serines. Expressed in E. coli (therefore non-glycosylated.)
Anti-TF Fab = anti-tissue factor antibody fragment
*activity against MDA-MB-468 cells
In a surprising and unexpected discovery, the in vitro cell proliferation activity results of the ADC in Tables 2a and 2b show generally that ADC with a low average number of drug moieties per antibody showed efficacy, e.g., IC50<0.1 μg ADC/ml. The results suggest that at least for trastuzumab ADC, the optimal ratio of drug moieties per antibody may be less than 8, and may be about 2 to about 5.
9.5.4 In Vivo Plasma Clearance and Stability
Pharmacokinetic plasma clearance and stability of ADC were investigated in rats and cynomolgus monkeys. Plasma concentration was measured over time. Table 2c shows pharmacokinetic data of antibody drug conjugates and other dosed samples in rats. Rats are a non-specific model for ErbB receptor antibodies, since the rat is not known to express HER2 receptor proteins.
TABLE 2c
Pharmacokinetics in Rats
H = trastuzumab linked via a cysteine
[cys] except where noted
2 mg/kg dose except where noted
AUCinf
T ½
Sample
day*
CL
Cmax
Term.
%
dose mg/kg
μg/mL
mL/day/kg
μg/mL
days
Conj.
H-MC-vc-PAB-MMAE
78.6
26.3
39.5
5.80
40.6
(Total Ab
H-MC-vc-PAB-MMAE
31.1
64.4
33.2
3.00
(Conj.)
H-MC-vc-PAB-MMAF
170
12.0
47.9
8.4
50.0
(Total Ab)
H-MC-vc-PAB-MMAF
83.9
24.0
44.7
4.01
(Conj.)
H-MC-MMAE (Total Ab)
279
18.9
79.6
7.65
33
H-MC-MMAE (Conj.)
90.6
62.9
62.9
4.46
5 mg/kg
H-MC-MMAF (Total Ab)
299
6.74
49.1
11.6
37
H-MC-MMAF (Conj.)
110
18.26
50.2
4.54
H-MC-vc-MMAF, wo/PAB,
306
6.6
78.7
11.9
19.6
(Total Ab)
H-MC-vc-MMAF, wo/PAB,
59.9
33.4
82.8
2.1
(Conj.)
H-Me-vc-PAB-MMAF
186
10.8
46.9
8.3
45.3
(Total Ab)
H-Me-vc-PAB-MMAF
84.0
23.8
49.6
4.3
(Conj.)
H-Me-vc-PAB-MMAE
135
15.0
44.9
11.2
23.8
(Total Ab)
H-Me-vc-PAB-MMAE
31.9
63.8
45.2
3.0
(Conj.)
H-MC-vc-MMAF, wo/PAB,
306
6.6
78.7
11.9
19.6
(Total Ab)
H-MC-vc-MMAF, wo/PAB,
59.9
33.4
82.8
2.1
(Conj.)
H-MC-(D)val-(L)cit-PAB-
107
19.2
30.6
9.6
38.1
MMAE (Total Ab)
H-MC-(D)val-(L)cit-PAB-
40
50.4
33.7
3.98
MMAE (Conj.)
H-MC-(Me)-vc-PAB-MMAE,
135.1
15.0
44.9
11.2
23.8
Total Ab
H-MC-(Me)-vc-PAB-MMAE,
31.9
63.8
45.2
2.96
Conj.
H-MC-(D)val-(D)cit-PAB-
88.2
22.8
33.8
10.5
38.3
MMAE, Total Ab
H-MC-(D)val-(D)cit-PAB-
33.6
59.8
36.0
4.43
MMAE, Conj.
H-MC-vc-PAB-MMAE,
78.6
26.3
39.5
5.8
40.6
Total Ab
H-MC-vc-PAB-MMAE, Conj.
31.1
64.4
33.2
3.00
H linked to MC by lysine
[lys]
MMAF
0.99
204
280
0.224
—
200 μg/kg
MMAE
3.71
62.6
649
0.743
—
206 μg/kg
HER F(ab′)2-MC-vc-MMAE,
9.3
217
34.4
0.35
95
Total Ab
HER F(ab′)2-MC-vc-MMAE,
8.8
227
36.9
0.29
Conj.
4D5-H-Fab-MC-vc-MMAF,
43.8
46.2
38.5
1.49
68
Total Ab
4D5-H-Fab-MC-vc-MMAF,
29.9
68.1
34.1
1.12
Conj.
4D5-H-Fab-MC-vc-MMAE,
71.5
70.3
108
1.18
59
Total Ab
4D5-H-Fab-MC-vc-MMAE,
42.2
118.9
114
0.74
Conj.
4D5-H-Fab
93.4
53.9
133
1.08
—
H-MC-vc-PAB-MMAF,
170
12.03
47.9
8.44
49.5
Total Ab
H-MC-vc-PAB-MMAF,
83.9
23.96
44.7
4.01
Conj.
H-MC-vc-PAB-MMAF-
211
9.8
39.8
8.53
34.3
DMAEA, Total Ab
H-MC-vc-PAB-MMAF-
71.5
28.2
38.8
3.64
DMAEA, Conj.
H-MC-vc-PAB-MMAF-TEG,
209
9.75
53.2
8.32
29.7
Total Ab
H-MC-vc-PAB-MMAF-TEG,
63.4
31.8
34.9
4.36
Conj.
AUC inf is the area under the plasma concentration-time curve from time of dosing to infinity and is a measure of the total exposure to the measured entity (drug, ADC). CL is defined as the volume of plasma cleared of the measured entity in unit time and is expressed by normalizing to body weight. T1/2 term is the half-life of the drug in the body measured during its elimination phase. The % Conj. term is the relative amount of ADC compared to total antibody detected, by separate ELISA immunoaffinity tests (“Analytical Methods for Biotechnology Products”, Ferraiolo et al, p 85-98 in Pharmacokinetics of Drugs (1994) P. G. Welling and L. P. Balant, Eds., Handbook of Experimental Pharmacology, Vol. 110, Springer-Verlag. The % Conj. calculation is simply AUCinf of ADC÷ AUCinf total Ab, and is a general indicator of linker stability, although other factors and mechanisms may be in effect.
FIG. 11 shows a graph of a plasma concentration clearance study after administration of the antibody drug conjugates: H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF to Sprague-Dawley rats. Concentrations of total antibody and ADC were measured over time.
FIG. 12 shows a graph of a two stage plasma concentration clearance study where ADC was administered at different dosages and concentrations of total antibody and ADC were measured over time.
9.5.4a In Vivo Efficacy
The in vivo efficacy of the ADC of the invention was measured by a high expressing HER2 transgenic explant mouse model. An allograft was propagated from the Fo5 mmtv transgenic mouse which does not respond to, or responds poorly to, HERCEPTIN® therapy. Subjects were treated once with ADC and monitored over 3-6 weeks to measure the time to tumor doubling, log cell kill, and tumor shrinkage. Follow up dose-response and multi-dose experiments were conducted.
Tumors arise readily in transgenic mice that express a mutationally activated form of neu, the rat homolog of HER2, but the HER2 that is overexpressed in breast cancers is not mutated and tumor formation is much less robust in transgenic mice that overexpress nonmutated HER2 (Webster et al. (1994) Semin. Cancer Biol. 5:69-76).
To improve tumor formation with nonmutated HER2, transgenic mice were produced using a HER2 cDNA plasmid in which an upstream ATG was deleted in order to prevent initiation of translation at such upstream ATG codons, which would otherwise reduce the frequency of translation initiation from the downstream authentic initiation codon of HER2 (for example, see Child et al. (1999) J. Biol. Chem. 274: 24335-24341). Additionally, a chimeric intron was added to the 5′ end, which should also enhance the level of expression as reported earlier (Neuberger and Williams (1988) Nucleic Acids Res. 16: 6713; Buchman and Berg (1988) Mol. Cell. Biol. 8:4395; Brinster et al. (1988) Proc. Natl. Acad. Sci. USA 85:836). The chimeric intron was derived from a Promega vector, pCI-neo mammalian expression vector (bp 890-1022). The cDNA 3′-end is flanked by human growth hormone exons 4 and 5, and polyadenylation sequences. Moreover, FVB mice were used because this strain is more susceptible to tumor development. The promoter from MMTV-LTR was used to ensure tissue-specific HER2 expression in the mammary gland. Animals were fed the AIN 76A diet in order to increase susceptibility to tumor formation (Rao et al. (1997) Breast Cancer Res. and Treatment 45:149-158).
TABLE 2d
Tumor measurements in allograft mouse model—MMTV-HER2
Fo5 Mammary Tumor, athymic nude mice
single dose at day 1 (T = 0) except where noted
H = trastuzumab linked via a cysteine [cys] except where noted
Tumor
doubl-
Mean
ing
log
Sample Drugs
time
cell
per anibody
Dose
Ti
PR
CR
(days)
kill
Vehicle
2-5
0
H-MC-vc-
1250
μg/m2
5/
4/7
0/7
18
1.5
PAB-MMAE
5
8.7 MMEA/Ab
H-MC-vc-
555
μg/m2
2/
2/7
5/7
69
6.6
PAB-MMAF
5
3.8 MMAF/Ab
H-MC(Me)-vc-
>50
6.4
PAB-MMAF
H-MC-MMAF
9.2
mg/kg
7/
6/7
0/7
63
9
4.8 MMAF/Ab
Ab
7
550
μg/m2
at 0, 7, 14
and 21 days
H-MC-MMAF
14
mg/kg Ab
5/
5/7
2/7
>63
4.8 MMAF/Ab
840
μg/m2
5
at 0, 7, 14
and 21 days
H-MC-vc-
3.5
mg/kg
5/
1/7
3/7
>36
PAB-MMAF
Ab
6
5.9 MMAF/Ab
300
μg/m2
at 0, 21, and
42 days
H-MC-vc-
4.9
mg/kg
4/
2/7
5/7
>90
PAB-MMAF
Ab
7
5.9 MMAF/Ab
425
μg/m2
at 0, 21, and
42 days
H-MC-vc-
6.4
mg/kg
3/
1/7
6/7
>90
PAB-MMAF
Ab
6
5.9 MMAF/Ab
550
μg/m2
at 0, 21, and
42 days
H-(L)val-
10
mg/kg
7/
1/7
0.7
15.2
1.1
(L)cit-MMAE
7
8.7 MMAE/Ab
H-MC-MMAE
10
mg/kg
7/
0/7
0/7
4
0.1
4.6 MMAE/Ab
7
H-(D)val-
10
mg/kg
7/
0/7
0/7
3
(D)cit-MMAE
7
4.2 MMAE/Ab
H-(D)val-
13
mg/kg
7/
0/7
0/7
9
0.6
(D)cit-MMAE
7
3.2 MMAE/Ab
H-MC(Me)-vc-
13
mg/kg
7/
3/7
0.7
17
1.2
MMAE
7
3.0 MMAE/Ab
H-(L)val-
12
mg/kg
7/
0/7
0/7
5
0.2
(D)cit-MMAE
7
3.5 MMAE/Ab
H-vc-MMAE
10
mg/kg
7/
17
8.7 MMAE/Ab
7
H-cys-vc-
1
mg/kg
7/
3
MMAF
7
3.8 MMAF/Ab
H-cys-vc
3
mg/kg
7/
>17
-MMAF
7
3.8 MMAF/Ab
H-cys-vc-
10
mg/kg
4/
4/7
3/7
>17
MMAF
7
3.8 MMAF/Ab
H-MC-vc-
10
mg/kg
3/
1/7
6/7
81
7.8
MMAF-TEG
6
4 MMAF/Ab
H-MC-vc-
10
mg/kg
0/
0/7
7/7
81
7.9
MMAF-TEG
q3wk x 3
5
4 MMAF/Ab
H-vc-MMAF
10
mg/kg
4/
2/8
5/8
(lot 1)
6
H-vc-MMAF
10
mg/kg
7/
1/8
1/8
(lot 2)
8
H-MC-
10
mg/kg
8/
1/8
0/8
18
MMAF
550
μg/m2
8
H-(Me)-vc-
10
mg/kg
3/
2/8
5/8
MMAF
7
H-vc-MMAE
3.7
mg/kg at
6/
0/7
1/7
17
2.3
7.5 MMAE/
0, 7, 14, 21,
6
Ab
28 days
H-vc-MMAE
7.5
mg/kg at
5/
3/7
3/7
69
10
7.5 MMAE/
0, 7, 14, 21,
7
Ab
28 days
anti IL8-vc-
7.5
mg/kg at
7/
0/7
0/7
5
0.5
MMAE
0, 7, 14, 21,
7
7.5 MMAE/Ab
28 days
anti IL8-vc-
3.7
mg/kg at
6/
0/7
0/7
3
0.2
MMAE
0, 7, 14, 21,
6
7.5 MMAE/Ab
28 days
H-fk-MMAE
7.5
mg/kg at
7/
1/7
0/7
31
4.4
7.5 MMAE/
0, 7, 14, 21,
7
Ab
28 days
H-fk-MMAE
3/7
mg/kg at
7/
0/7
0/7
8.3
0.9
7.5 MMAE/
0, 7, 14, 21,
7
Ab
28 days
anti IL8-fk
7.5
mg/kg at
7/
0/7
0/7
6
0.5
-MMAE
0, 7, 14, 21,
7
7.5 MMAE/Ab
28 days
anti IL8-fk-
3.7
mg/kg at
7/
0/7
0/7
3
0.1
MMAE
0, 7, 14, 21,
7
7.5 MMAE/Ab
28 days
Trastuzumab
7.5
mg/kg at
7/
0/7
0/7
5
0.4
0, 7, 14, 21,
7
28 days
H-vc-MMAE
10
mg/kg
6/
3/6
0/6
15
1.3
8.7 MMAE/Ab
1250
μg/m2
6
H-vc-MMAE
10
mg/kg
7/
5/7
>19
1250
μg/m2
7
at 0, 7, and
14 days
H-vc-MMAE
3
mg/kg at
7/
8
0, 7, and 14
7
days
H-vc-MMAE
1
mg/kg at
7/
7
0, 7, and 14
7
days
H-vc-MMAF
10
mg/kg
8/
5/8
>21
8
H-vc-MMAF
10
mg/kg at
4/
4/7
3/7
>21
0, 7, and 14
7
days
H-vc-MMAF
3
mg/kg at
7/
6
0, 7, and 14
7
days
Trastuzumab
10
mg/kg at
8/
3
0 and 7 days
8
Hg-MC-vc-
10
mk/kg at
6/
3/8
5/8
56
5.1
PAB-MMAF
0 days
7
4.1 MMAF/Ab
Fc8-MC-vc-
10
mg/kg at
7/
6/8
0/8
25
2.1
PAB-MMAF
0 days
7
4.4 MMAF/Ab
7C2-MC-vc-
10
mg/kg at
5/
6/8
1/8
41
3.7
PAB-MMAF
0 days
6
4 MMAF/Ab
H-MC-vc-
10
mg/kg at
3/
3/8
5/8
62
5.7
PAB-MMAF
0 days
8
5.9 MMAF/Ab
2H9-MC-vc-
9/
>14 days
PAB-MMAE
9
2H9-MC-vc-
9/
>14 days
PAB-MMAF
9
11D10-vc-
9/
>14 days
PAB-MMAE
9
11D10-vc-
9/
11 days
PAB-MMAF
9
7C2 = anti-HER2 murine antibody which binds a different epitope than trastuzumab.
Fc8 = mutant that does not bind to FcRn
Hg = “Hingeless” full-length humanized 4D5, with heavy chain hinge cysteines mutated to serines. Expressed in E. coli (therefore non-glycosylated.)
2H9 = Anti-EphB2R
11D10 = Anti-0772P
The term Ti is the number of animals in the study group with tumor at T=0÷total animals in group. The term PR is the number of animals attaining partial remission of tumor÷animals with tumor at T=0 in group. The term CR is the number of animals attaining complete remission of tumor÷animals with tumor at T=0 in group. The term Log cell kill is the time in days for the tumor volume to double—the time in days for the control tumor volume to double divided by 3.32×time for tumor volume to double in control animals (dosed with Vehicle). The log-cell-kill calculation takes into account tumor growth delay resulting from treatment and tumor volume doubling time of the control group. Anti-tumor activity of ADC is classified with log-cell-kill values of:
++++
≥3.4
(highly active)
+++ =
2.5-3.4
++ =
1.7-2.4
+ =
1.0-1.6
inactive =
0
FIG. 13 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with: Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 μg/m2) and Trastuzumab-MC-vc-PAB-MMAF (555 μg/m2). (H=Trastuzumab). The growth of tumors was retarded by treatment with ADC as compared to control (Vehicle) level of growth. FIG. 14 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10 mg/kg (660 μg/m2) of Trastuzumab-MC-MMAE and 1250 μg/m2 Trastuzumab-MC-vc-PAB-MMAE. FIG. 15 shows the mean tumor volume change over time in athymic nude mice with MMTV-HER2 Fo5 Mammary tumor allografts dosed with 650 μg/m2 Trastuzumab-MC-MMAF. Table 2d and FIGS. 13-15 show that the ADC have strong anti-tumor activity in the allograft of a HER2 positive tumor (Fo5) that originally arose in an MMTV-HER2 transgenic mouse. The antibody alone (e.g., Trastuzumab) does not have significant anti-tumor activity in this model (Erickson et al. U.S. Pat. No. 6,632,979). As illustrated in FIGS. 13-15, the growth of the tumors was retarded by treatment with ADC as compared to control (Vehicle) level of growth.
In a surprising and unexpected discovery, the in vivo anti-tumor activity results of the ADC in Table 2d show generally that ADC with a low average number of drug moieties per antibody showed efficacy, e.g., tumor doubling time >15 days and mean log cell kill >1.0. FIG. 16 shows that for the antibody drug conjugate, trastuzumab-MC-vc-PAB-MMAF, the mean tumor volume diminished and did not progress where the MMAF:trastuzumab ratio was 2 and 4, whereas tumor progressed at a ratio of 5.9 and 6, but at a rate lower than Vehicle (buffer). The rate of tumor progression in this mouse xenograft model was about the same, i.e. 3 days, for Vehicle and trastuzumab. The results suggest that at least for trastuzumab ADC, the optimal ratio of drug moieties per antibody may be less than about 8, and may be about 2 to about 4.
9.5.5 Rodent Toxicity
Antibody drug conjugates and an ADC-minus control, “Vehicle”, were evaluated in an acute toxicity rat model. Toxicity of ADC was investigated by treatment of male and female Sprague-Dawley rats with the ADC and subsequent inspection and analysis of the effects on various organs. Gross observations included changes in body weights and signs of lesions and bleeding. Clinical pathology parameters (serum chemistry and hematology), histopathology, and necropsy were conducted on dosed animals.
It is considered that weight loss, or weight change relative to animals dosed only with Vehicle, in animals after dosing with ADC is a gross and general indicator of systemic or localized toxicity. FIGS. 17-19 show the effects of various ADC and control (Vehicle) after dosing on rat body weight.
Hepatotoxicity was measured by elevated liver enzymes, increased numbers of mitotic and apoptotic figures and hepatocyte necrosis. Hematolymphoid toxicity was observed by depletion of leukocytes, primarily granuloctyes (neutrophils), and/or platelets, and lymphoid organ involvement, i.e. atrophy or apoptotic activity. Toxicity was also noted by gastrointestinal tract lesions such as increased numbers of mitotic and apoptotic figures and degenerative enterocolitis.
Enzymes indicative of liver injury that were studied include:
AST (aspartate aminotransferase)
Localization: cytoplasmic; liver, heart, skeletal muscle, kidney
Liver:Plasma ratio of 7000:1
T1/2: 17 hrs
ALT (alanine aminotransferase)
Localization: cytoplasmic; liver, kidney, heart, skeletal muscle
Liver:Plasma ratio of 3000:1
T1/2: 42 hrs; diurnal variation
GGT (g-glutamyl transferase)
Localization: plasma membrane of cells with high secretory or absorptive capacity; liver, kidney, intestine
Poor predictor of liver injury; commonly elevated in bile duct disorders
The toxicity profiles of trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PAB-MMAF were studied in female Sprague-Dawley rats (Example 19). The humanized trastuzumab antibody does not bind appreciably to rat tissue, and any toxicity would be considered non-specific. Variants at dose levels of 840 and 2105 ug/m2 MMAF were compared to trastuzumab-MC-val-cit-PAB-MMAF at 2105 ug/m2.
Animals in groups 1, 2, 3, 4, 6, and 7 (Vehicle, 9.94 & 24.90 mg/kg trastuzumab-MC-val-cit-MMAF, 10.69 mg/kg trastuzumab-MC(Me)-val-cit-PAB-MMAF, and 10.17 & 25.50 mg/kg trastuzumab-MC-MMAF, respectively) gained weight during the study. Animals in groups 5 and 8 (26.78 mg/kg trastuzumab-MC(Me)-val-cit-PAB-MMAF and 21.85 mg/kg trastuzumab-MC-val-cit-PAB-MMAF, respectively) lost weight during the study. On Study Day 5, the change in body weights of animals in groups 2, 6 and 7 were not significantly different from group 1 animals. The change in body weights of animals in groups 3, 4, 5 and 8 were statistically different from group 1 animals (Example 19).
Rats treated with trastuzumab-MC-MMAF (groups 6 and 7) were indistinguishable from vehicle-treated control animals at both dose levels; i.e. this conjugate showed a superior safety profile in this model. Rats treated with trastuzumab-MC-val-cit-MMAF (without the self-immolative PAB moiety; groups 2 and 3) showed dose-dependent changes typical for MMAF conjugates; the extent of the changes was less compared with a full length MC-val-cit-PAB-MMAF conjugate (group 8). The platelet counts on day 5 were at approximately 30% of baseline values in animals of group 3 (high dose trastuzumab-MC-val-cit-MMAF) compared with 15% in animals of group 8 (high dose trastuzumab-MC-val-cit-PAB-MMAF). Elevation of liver enzymes AST and ALT, of bilirubin and the extent of thrombocytopenia was most evident in animals treated with trastuzumab-MC(Me)-val-cit-PAB-MMAF (groups 4 and 5) in a dose-dependent fashion; animals of group 5 (high dose group) showed on day 5 levels of ALT of approximately 10× the baseline value and platelets were reduced by approximately 90% at the time of necropsy.
Female Sprague Dawley Rats were also dosed at high levels (Example 19, High Dose study: Groups 2, 3, 4) with trastuzumab-MC-MMAF, and Vehicle control (Group 1). Mild toxicity signals were observed, including a dose-dependent elevation of liver enzymes ALT, AST and GGT. On day 5 animals in the highest dose group showed a 2-fold elevation of ALT and a 5-fold elevation of AST; GGT is also elevated (6 U/L). Enzyme levels show a trend towards normalization on day 12. There was a mild granulocytosis in all three dose groups on day 5, the platelet count remained essentially unchanged in all animals. Morphological changes were mild; animals treated at the 4210 μg/m2 dose level (Group 2) showed unremarkable histology of liver, spleen, thymus, intestines and bone marrow. Mildly increased apoptotic and mitotic activity was observed in thymus and liver, respectively in animals treated at the 5500 μg/m2 dose level (Group 3). The bone marrow was normocellular, but showed evidence of granulocytic hyperplasia, which is consistent with the absolute granulocytosis observed in the peripheral blood counts in these animals. Animals at the highest dose in group 4 showed qualitatively the same features; the mitotic activity in the liver appears somewhat increased compared to animals in Group 3. Also, extramedullary hematopoiesis was seen in spleen and liver.
EphB2R is a type 1 TM tyrosine kinase receptor with close homology between mouse and human, and is over-expressed in colorectal cancer cells. 2H9 is an antibody against EphB2R. The naked antibody has no effect on tumor growth, but 2H9-val-cit-MMAE killed EphB2R expressing cells and showed efficacy in a mouse xenograft model using CXF1103 human colon tumors (Mao et al (2004) Cancer Res. 64:781-788). 2H9 and 7C2 are both mouse IgG1 anti-HER2 antibodies. The toxicity profiles of 2H9-MC-val-cit-PAB-MMAF (3.7 MMAF/Ab), 7C2-MC-val-cit-PAB-MMAF (4 MMAF/Ab), and trastuzumab-MC-val-cit-PAB-MMAF (5.9 MMAF/Ab) were compared. The differences in the structure of each immunoconjugate or the drug portion of the immunoconjugate may affect the pharmacokinetics and ultimately the safety profile. The humanized trastuzumab antibody does not bind appreciably to rat tissue, and any toxicity would be considered non-specific.
9.5.6 Cynomolgus Monkey Toxicity/Safety
Similar to the rat toxicity/safety study, cynomolgus monkeys were treated with ADC followed by liver enzyme measurements, and inspection and analysis of the effects on various organs. Gross observations included changes in body weights and signs of lesions and bleeding. Clinical pathology parameters (serum chemistry and hematology), histopathology, and necropsy were conducted on dosed animals (Example 19).
The antibody drug conjugate, H-MC-vc-PAB-MMAE (H=trastuzumab linked through cysteine) showed no evidence of liver toxicity at any of the dose levels tested. Peripheral blood granulocytes showed depletion after a single dose of 1100 mg/m2 with complete recovery 14 days post-dose. The antibody drug conjugate H-MC-vc-PAB-MMAF showed elevation of liver enzymes at 550 (transient) and 880 mg/m2 dose level, no evidence of granulocytopenia, and a dose-dependent, transient (groups 2 & 3) decline of platelets.
9.6 Synthesis of the Compounds of the Invention
The Exemplary Compounds and Exemplary Conjugates can be made using the synthetic procedures outlined below in FIGS. 25-36. As described in more detail below, the Exemplary Compounds or Exemplary Conjugates can be conveniently prepared using a Linker having a reactive site for binding to the Drug and Ligand. In one aspect, a Linker has a reactive site which has an electrophilic group that is reactive to a nucleophilic group present on a Ligand, such as but not limited to an antibody. Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for antibody attachment.
In another embodiment, a Linker has a reactive site which has a nucleophilic group that is reactive to an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for attachment to a Linker.
Carboxylic acid functional groups and chloroformate functional groups are also useful reactive sites for a Linker because they can react with secondary amino groups of a Drug to form an amide linkage. Also useful as a reactive site is a carbonate functional group on a Linker, such as but not limited to p-nitrophenyl carbonate, which can react with an amino group of a Drug, such as but not limited to N-methyl valine, to form a carbamate linkage. Typically, peptide-based Drugs can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
The synthesis of an illustrative Stretcher having an electrophilic maleimide group is illustrated below in FIGS. 28 and 29. General synthetic methods useful for the synthesis of a Linker are described in FIG. 30. FIG. 31 shows the construction of a Linker unit having a val-cit group, an electrophilic maleimide group and a PAB self-immolative Spacer group. FIG. 32 depicts the synthesis of a Linker having a phe-lys group, an electrophilic maleimide group, with and without the PAB self-immolative Spacer group. FIG. 33 presents a general outline for the synthesis of a Drug-Linker Compound, while FIG. 34 presents an alternate route for preparing a Drug-Linker Compound. FIG. 35 depicts the synthesis of a branched linker containing a BHMS group. FIG. 36 outlines the attachment of an antibody to a Drug-Linker Compound to form a Drug-Linker-Antibody Conjugate, and FIG. 34 illustrates the synthesis of Drug-Linker-Antibody Conjugates having, for example but not limited to, 2 or 4 drugs per Antibody.
As described in more detail below, the Exemplary Conjugates are conveniently prepared using a Linker having two or more Reactive Sites for binding to the Drug and a Ligand. In one aspect, a Linker has a Reactive site which has an electrophilic group that is reactive to a nucleophilic group present on a Ligand, such as an antibody. Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups. The heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for antibody attachment.
In another embodiment, a Linker has a Reactive site which has a nucleophilic group that is reactive to an electrophilic group present on a Ligand, such as an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for attachment to a Linker.
9.6.1 Drug Moiety Synthesis
Typically, peptide-based Drugs can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schröder and K. Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
The auristatin/dolastatin drug moieties may be prepared according to the general methods of: U.S. Pat. Nos. 5,635,483; 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863.
In one embodiment, a Drug is prepared by combining about a stoichiometric equivalent of a dipeptide and a tripeptide, preferably in a one-pot reaction under suitable condensation conditions. This approach is illustrated in FIGS. 25-27, below.
FIG. 25 illustrates the synthesis of an N-terminal tripeptide unit F which is a useful intermediate for the synthesis of the drug compounds of Formula Ib.
As illustrated in FIG. 25, a protected amino acid A (where PG represents an amine protecting group, R4 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle, alkyl-(C3-C8 heterocycle) wherein R5 is selected from H and methyl; or R4 and R5 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from hydrogen, C1-C8 alkyl and C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they are attached) is coupled to t-butyl ester B (where R6 is selected from —H and —C1-C8 alkyl; and IC is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle)) under suitable coupling conditions, e.g., in the presence of PyBrop and diisopropylethylamine, or using DCC (see, for example, Miyazaki, K. et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).
Suitable protecting groups PG, and suitable synthetic methods to protect an amino group with a protecting group are well known in the art. See, e.g., Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley & Sons. Exemplary protected amino acids A are PG-Ile and, particularly, PG-Val, while other suitable protected amino acids include, without limitation: PG-cyclohexylglycine, PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid, PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and PG-tert-butylglycine. Z is an exemplary protecting group. Fmoc is another exemplary protecting group. An exemplary t-butyl ester B is dolaisoleuine t-butyl ester.
The dipeptide C can be purified, e.g., using chromatography, and subsequently deprotected, e.g., using H2 and 10% Pd—C in ethanol when PG is benzyloxycarbonyl, or using diethylamine for removal of an Fmoc protecting group. The resulting amine D readily forms a peptide bond with an amino acid BB (wherein R1 is selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle; and R2 is selected from —H and —C1-C8 alkyl; or R1 and R2 join, have the formula —(CRaRb)n— wherein Ra and Rb are independently selected from —H, —C1-C8 alkyl and —C3-C8 carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogen atom to which they are attached; and R3 is selected from hydrogen, C1-C8 alkyl, C3-C8 carbocycle, —O—(C1-C8 alkyl), -aryl, alkyl-aryl, alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and alkyl-(C3-C8 heterocycle)). N,N-Dialkyl amino acids are exemplary amino acids for BB, such as commercially available N,N-dimethyl valine. Other N,N-dialkyl amino acids can be prepared by reductive bis-alkylation using known procedures (see, e.g., Bowman, R. E, Stroud, H. H J. Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two exemplary amino acids BB useful for the synthesis of N-monoalkyl derivatives. The amine D and the amino acid BB react to provide the tripeptide E using coupling reagent DEPC with triethylamine as the base. The C-terminus protecting group of E is subsequently deprotected using HCl to provide the tripeptide compound of formula F.
Illustrative DEPC coupling methodology and the PyBrop coupling methodology shown in FIG. 25 are outlined below in General Procedure A and General Procedure B, respectively. Illustrative methodology for the deprotection of a Z-protected amine via catalytic hydrogenation is outlined below in General Procedure C.
General Procedure A: Peptide Synthesis Using DEPC.
The N-protected or N, N-disubstituted amino acid or peptide D (1.0 eq.) and an amine BB (1.1 eq.) are diluted with an aprotic organic solvent, such as dichloromethane (0.1 to 0.5 M). An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by DEPC (1.1 eq.). The resulting solution is stirred, preferably under argon, for up to 12 hours while being monitored by HPLC or TLC. The solvent is removed in vacuo at room temperature, and the crude product is purified using, for example, HPLC or flash column chromatography (silica gel column). Relevant fractions are combined and concentrated in vacuo to afford tripeptide E which is dried under vacuum overnight.
General Procedure B: Peptide Synthesis Using PyBrop.
The amino acid B (1.0 eq.), optionally having a carboxyl protecting group, is diluted with an aprotic organic solvent such as dichloromethane or DME to provide a solution of a concentration between 0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is added. Fmoc-, or Z-protected amino acid A (1.1 eq.) is added as a solid in one portion, then PyBrop (1.2 eq.) is added to the resulting mixture. The reaction is monitored by TLC or HPLC, followed by a workup procedure similar to that described in General Procedure A.
General Procedure C: Z-Removal Via Catalytic Hydrogenation.
Z-protected amino acid or peptide C is diluted with ethanol to provide a solution of a concentration between 0.5 and 1.0 mM in a suitable vessel, such as a thick-walled round bottom flask. 10% palladium on carbon is added (5-10% w/w) and the reaction mixture is placed under a hydrogen atmosphere. Reaction progress is monitored using HPLC and is generally complete within 1-2 h. The reaction mixture is filtered through a pre-washed pad of celite and the celite is again washed with a polar organic solvent, such as methanol after filtration. The eluent solution is concentrated in vacuo to afford a residue which is diluted with an organic solvent, preferably toluene. The organic solvent is then removed in vacuo to afford the deprotected amine C.
FIG. 26 shows a method useful for making a C-terminal dipeptide of formula K and a method for coupling the dipeptide of formula K with the tripeptide of formula F to make drug compounds of Formula Ib.
The dipeptide K can be readily prepared by condensation of the modified amino acid Boc-Dolaproine G (see, for example, Pettit, G. R., et al. Synthesis, 1996, 719-725), with an amine of formula H using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in FIG. 25.
The dipeptide of formula K can then be coupled with a tripeptide of formula F using General Procedure D to make the Fmoc-protected drug compounds of formula L which can be subsequently deprotected using General Procedure E in order to provide the drug compounds of formula (Ib).
General procedure D: Drug Synthesis.
A mixture of dipeptide K (1.0 eq.) and tripeptide F (1 eq.) is diluted with an aprotic organic solvent, such as dichloromethane, to form a 0.1M solution, then a strong acid, such as trifluoroacetic acid (1/2 v/v) is added and the resulting mixture is stirred under a nitrogen atmosphere for two hours at 0° C. The reaction can be monitored using TLC or, preferably, HPLC. The solvent is removed in vacuo and the resulting residue is azeotropically dried twice, preferably using toluene. The resulting residue is dried under high vacuum for 12 h and then diluted with and aprotic organic solvent, such as dichloromethane. An organic base such as triethylamine or diisopropylethylamine (1.5 eq.) is then added, followed by either PyBrop (1.2 eq.) or DEPC (1.2 eq.) depending on the chemical functionality on the residue. The reaction mixture is monitored by either TLC or HPLC and upon completion, the reaction is subjected to a workup procedure similar or identical to that described in General Procedure A.
General Procedure E: Fmoc-Removal Using Diethylamine.
An Fmoc-protected Drug L is diluted with an aprotic organic solvent such as dichloromethane and to the resulting solution is added diethylamine (1/2 v/v). Reaction progress is monitored by TLC or HPLC and is typically complete within 2 h. The reaction mixture is concentrated in vacuo and the resulting residue is azeotropically dried, preferably using toluene, then dried under high vacuum to afford Drug Ib having a deprotected amino group.
FIG. 27 shows a method useful for making MMAF derivatives of Formula (Ib).
The dipeptide O can be readily prepared by condensation of the modified amino acid Boc-Dolaproine G (see, for example, Pettit, G. R., et al. Synthesis, 1996, 719-725), with a protected amino acid of formula III using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in FIGS. 25 and 26.
The dipeptide of formula O can then be coupled with a tripeptide of formula F using General Procedure D to make the Fmoc-protected MMAF compounds of formula P which can be subsequently deprotected using General Procedure E in order to provide the MMAF drug compounds of formula (Ib).
Thus, the above methods are useful for making Drugs that can be used in the present invention.
9.6.2 Drug Linker Synthesis
To prepare a Drug-Linker Compound of the present invention, the Drug is reacted with a reactive site on the Linker. In general, the Linker can have the structure:
when both a Spacer unit (—Y—) and a Stretcher unit (-A-) are present. Alternately, the Linker can have the structure:
when the Spacer unit (—Y—) is absent.
The Linker can also have the structure:
when both the Stretcher unit (-A-) and the Spacer unit (—Y—) are absent.
The Linker can also have the structure:
when both the Amino Acid unit (W) and the Spacer Unit (Y) are absent.
In general, a suitable Linker has an Amino Acid unit linked to an optional Stretcher Unit and an optional Spacer Unit. Reactive Site 1 is present at the terminus of the Spacer and Reactive site 2 is present at the terminus of the Stretcher. If a Spacer unit is not present, then Reactive site 1 is present at the C-terminus of the Amino Acid unit.
In an exemplary embodiment of the invention, Reactive Site No. 1 is reactive to a nitrogen atom of the Drug, and Reactive Site No. 2 is reactive to a sulfhydryl group on the Ligand. Reactive Sites 1 and 2 can be reactive to different functional groups.
In one aspect of the invention, Reactive Site No. 1 is
In another aspect of the invention, Reactive Site No. 1 is
In still another aspect of the invention, Reactive Site No. 1 is a p-nitrophenyl carbonate having the formula
In one aspect of the invention, Reactive Site No. 2 is a thiol-accepting group. Suitable thiol-accepting groups include haloacetamide groups having the formula
wherein X represents a leaving group, preferably O-mesyl, O-tosyl, —Cl, —Br, or —I; or a maleimide group having the formula
Useful Linkers can be obtained via commercial sources, such as Molecular Biosciences Inc. (Boulder, Colo.), or prepared as summarized in FIGS. 28-30.
In FIG. 28 X is —CH2— or —CH2OCH2—; and n is an integer ranging either from 0-10 when X is —CH2—; or 1-10 when X is —CH2OCH2—.
The method shown in FIG. 29 combines maleimide with a glycol under Mitsunobu conditions to make a polyethylene glycol maleimide Stretcher (see for example, Walker, M. A. J. Org. Chem. 1995, 60, 5352-5), followed by installation of a p-nitrophenyl carbonate Reactive Site group.
In FIG. 29 E is —CH2— or —CH2OCH2—; and e is an integer ranging from 0-8;
Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can be prepared as described by Frisch, et al., Bioconjugate Chem. 1996, 7, 180-186.
FIG. 30 illustrates a general synthesis of an illustrative Linker unit containing a maleimide Stretcher group and optionally a p-aminobenzyl ether self-immolative Spacer.
In FIG. 30 Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0-4; and n is an integer ranging from 0-10.
Useful Stretchers may be incorporated into a Linker using the commercially available intermediates from Molecular Biosciences (Boulder, Colo.) described below by utilizing known techniques of organic synthesis.
Stretchers of formula (Ma) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit as depicted in FIGS. 31 and 32:
where n is an integer ranging from 1-10 and T is —H or —SO3Na;
where n is an integer ranging from 0-3;
Stretcher units of formula (IIIb) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
where X is —Br or —I; and
Stretcher units of formula (IV) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
Stretcher units of formula (Va) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
Other useful Stretchers may be synthesized according to known procedures. Aminooxy Stretchers of the formula shown below can be prepared by treating alkyl halides with N-Boc-hydroxylamine according to procedures described in Jones, D. S. et al., Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon, C. et al., Tetrahedron, 1967, 23(11), 4441-4447.
wherein —R17— is selected from —C1-C10 alkylene-, —C3-C8 carbocyclo-, —O—(C1-C8 alkyl)-, -arylene-, —C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, alkylene-(C3-C8 carbocyclo)-, —(C3-C8 carbocyclo)-C1-C10 alkylene-, —C3-C8 heterocyclo-, —C1-C10 alkylene-(C3-C8 heterocyclo)-, —(C3-C8 heterocyclo)-C1-C10 alkylene-, —(CH2CH2O)r—, —(CH2CH2O)r—CH2—; and r is an integer ranging from 1-10;
Isothiocyanate Stretchers of the formula shown below may be prepared from isothiocyanatocarboxylic acid chlorides as described in Angew. Chem., 1975, 87(14):517.
wherein —R17— is as described herein.
FIG. 31 shows a method for obtaining of a val-cit dipeptide Linker having a maleimide Stretcher and optionally a p-aminobenzyl self-immolative Spacer.
In FIG. 31 Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
FIG. 32 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linker unit having a maleimide Stretcher unit and a p-aminobenzyl self-immolative Spacer unit. Starting material AD (lys(Mtr)) is commercially available (Bachem, Torrance, Calif.) or can be prepared according to Dubowchik, et al. Tetrahedron Letters (1997) 38:5257-60.
In FIG. 32 Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
As shown in FIG. 33, a Linker can be reacted with an amino group of a Drug Compound of Formula (Ib) to form a Drug-Linker Compound that contains an amide or carbamate group, linking the Drug unit to the Linker unit. When Reactive Site No. 1 is a carboxylic acid group, as in Linker AJ, the coupling reaction can be performed using HATU or PyBrop and an appropriate amine base, resulting in a Drug-Linker Compound AK, containing an amide bond between the Drug unit and the Linker unit. When Reactive Site No. 1 is a carbonate, as in Linker AL, the Linker can be coupled to the Drug using HOBt in a mixture of DMF/pyridine to provide a Drug-Linker Compound AM, containing a carbamate bond between the Drug unit and the Linker unit
Alternately, when Reactive Site No. 1 is a good leaving group, such as in Linker AN, the Linker can be coupled with an amine group of a Drug via a nucleophilic substitution process to provide a Drug-Linker Compound having an amine linkage (AO) between the Drug unit and the Linker unit.
Illustrative methods useful for linking a Drug to a Ligand to form a Drug-Linker Compound are depicted in FIG. 33 and are outlined in General Procedures G-H.
General Procedure G: Amide Formation Using HATU.
A Drug (Ib) (1.0 eq.) and an N-protected Linker containing a carboxylic acid Reactive site (1.0 eq.) are diluted with a suitable organic solvent, such as dichloromethane, and the resulting solution is treated with HATU (1.5 eq.) and an organic base, preferably pyridine (1.5 eq.). The reaction mixture is allowed to stir under an inert atmosphere, preferably argon, for 6 h, during which time the reaction mixture is monitored using HPLC. The reaction mixture is concentrated and the resulting residue is purified using HPLC to yield the amide of formula AK.
Procedure H: Carbamate Formation Using HOBt.
A mixture of a Linker AL having a p-nitrophenyl carbonate Reactive site (1.1 eq.) and Drug (Ib) (1.0 eq.) are diluted with an aprotic organic solvent, such as DMF, to provide a solution having a concentration of 50-100 mM, and the resulting solution is treated with HOBt (2.0 eq.) and placed under an inert atmosphere, preferably argon. The reaction mixture is allowed to stir for 15 min, then an organic base, such as pyridine (1/4 v/v), is added and the reaction progress is monitored using HPLC. The Linker is typically consumed within 16 h. The reaction mixture is then concentrated in vacuo and the resulting residue is purified using, for example, HPLC to yield the carbamate AM.
An alternate method of preparing Drug-Linker Compounds is outlined in FIG. 34. Using the method of FIG. 34, the Drug is attached to a partial Linker unit (ZA, for example), which does not have a Stretcher unit attached. This provides intermediate AP, which has an Amino Acid unit having an Fmoc-protected N-terminus. The Fmoc group is then removed and the resulting amine intermediate AQ is then attached to a Stretcher unit via a coupling reaction catalyzed using PyBrop or DEPC. The construction of Drug-Linker Compounds containing either a bromoacetamide Stretcher AR or a PEG maleimide Stretcher AS is illustrated in FIG. 34.
In FIG. 34 Q is —C1-C8 alkyl, —O—(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
Methodology useful for the preparation of a Linker unit containing a branched spacer is shown in FIG. 35.
FIG. 35 illustrates the synthesis of a val-cit dipeptide linker having a maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS) unit. The synthesis of the BHMS intermediate (AW) has been improved from previous literature procedures (see International Publication No, WO 9813059 to Firestone et al., and Crozet, M. P.; Archaimbault, G.; Vanelle, P.; Nouguier, R. Tetrahedron Lett. (1985) 26:5133-5134) and utilizes as starting materials, commercially available diethyl (4-nitrobenzyl)phosphonate (AT) and commercially available 2,2-dimethyl-1,3-dioxan-5-one (AU). Linkers AY and BA can be prepared from intermediate AW using the methodology described in FIG. 29.
9.6.3 Dendritic Linkers
The linker may be a dendritic type linker for covalent attachment of more than one drug moiety through a branching, multifunctional linker moiety to a Ligand, such as but not limited to an antibody (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of drug to antibody, i.e. loading, which is related to the potency of the Drug-Linker-Ligand Conjugate. Thus, where a cysteine engineered antibody bears only one reactive cytsteine thiol group, a multitude of drug moieties may be attached through a dendritic linker.
The following exemplary embodiments of dendritic linker reagents allow up to nine nucleophilic drug moiety reagents to be conjugated by reaction with the chloroethyl nitrogen mustard functional groups:
9.6.4 Conjugation of Drug Moieties to Antibodies
FIG. 36 illustrates methodology useful for making Drug-Linker-Ligand conjugates having about 2 to about 4 drugs per antibody. An antibody is treated with a reducing agent, such as dithiothreitol (DTT) to reduce some or all of the cysteine disulfide residues to form highly nucleophilic cysteine thiol groups (—CH2SH). The partially reduced antibody thus reacts with drug-linker compounds, or linker reagents, with electrophilic functional groups such as maleimide or α-halo carbonyl, according to the conjugation method at page 766 of Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773.
For example, an antibody, e.g., AC10, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37° C. for about 30 minutes, the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of the absorbance at 412 nm. The reduced antibody dissolved in PBS is chilled on ice. The drug linker, e.g., MC-val-cit-PAB-MMAE in DMSO, dissolved in acetonitrile and water at known concentration, is added to the chilled reduced antibody in PBS. After about one hour, an excess of maleimide is added to quench the reaction and cap any unreacted antibody thiol groups. The reaction mixture is concentrated by centrifugal ultrafiltration and the ADC, e.g., AC10-MC-vc-PAB-MMAE, is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 μm filters under sterile conditions, and frozen for storage.
A variety of antibody drug conjugates (ADC) were prepared, with a variety of linkers, and the drug moieties, MMAE and MMAF. The following table is an exemplary group of ADC which were prepared following the protocol of Example 27, and characterized by HPLC and drug loading assay.
isolated
Target
amount
drug/Ab
(antigen)
ADC
(mg)
ratio
0772P
16E12-MC-vc-PAB-MMAE
1.75
0772P
11D10-MC-vc-PAB-MMAE
46.8
.4
0772P
11D10-MC-vc-PAB-MMAF
54.5
.8
Brevican
Brevican-MC-MMAF
2
Brevican
Brevican-MC-vc-MMAF
2
Brevican
Brevican-MC-vc-PAB-MMAF
1.4
CD21
CD21-MC-vc-PAB-MMAE
38.1
.3
CD21
CD21-MC-vc-PAB-MMAF
43
.1
CRIPTO
11F4-MC-vc-PAB-MMAF
6
.8
CRIPTO
25G8-MC-vc-PAB-MMAF
7.4
.7
E16
12G12-MC-vc-PAB-MMAE
2.3
.6
E16
3B5-MC-vc-PAB-MMAE
2.9
.6
E16
12B9-MC-vc-PAB-MMAE
1.4
.8
E16
12B9-MC-vc-PAB-MMAE
5.1
E16
12G12-MC-vc-PAB-MMAE
3
.6
E16
3B5-MC-vc-PAB-MMAE
4.8
.1
E16
3B5-MC-vc-PAB-MMAF
24.7
.4
EphB2R
2H9-MC-vc-PAB-MMAE
29.9
.1
EphB2R
2H9-MC-fk-PAB-MMAE
25
.5
EphB2R
2H9-MC-vc-PAB-MMAE
175
.1
EphB2R
2H9-MC-vc-PAB-MMAF
150
.8
EphB2R
2H9-MC-vc-PAB-MMAF
120
.7
EphB2R
2H9-MC-vc-PAB-MMAE
10.7
.4
IL-20Ra
IL20Ra-vc-MMAE
26
.7
IL-20Ra
IL20Ra-vc-MMAE
27
.3
ePhB2
IL8-MC-vc-PAB-MMAE
251
.7
MDP
MDP-vc-MMAE
32
MPF
19C3-vc-MMAE
1.44
.5
MPF
7D9-vc-MMAE
4.3
.8
MPF
19C3-vc-MMAE
7.9
MPF
7D9-MC-vc-PAB-MMAF
5
.3
Napi3b
10H1-vc-MMAE
4.5
.6
Napi3b
4C9-vc-MMAE
3.0
.4
Napi3b
10H1-vc-MMAE
4.5
.8
Nap3b
10H1-vc-MMAE
6.5
NCA
3E6-MC-fk-PAB-MMAE
49.6
.4
NCA
3E6-MC-vc-PAB-MMAE
56.2
.4
PSCA
PSCA-fk-MMAE
51.7
.9
PSCA
PSCA-vc-MMAE
61.1
.6
Napi3b
10H1-MC-vc-PAB-MMAE
75
.2
Napi3b
10H1-MC-vc-PAB-MMAF
95
.4
Napi3b
10H1-MC-MMAF
92
EphB2R
2H9-MC-vc-PAB-MMAE
79
EphB2R
2H9-MC-MMAF
92
.9
0772P
11D10(Fc chimera)-MC-vc-PAB-MMAE
79
.3
0772P
11D10(Fc chimera)-MC-vc-PAB-MMAF
70
.5
0772P
11D10(Fc chimera)-MC-MMAF
23
.5
Brevican
6D2-MC-vc-PAB-MMAF
0.3
.3
Brevican
6D2-MC-MMAF
0.36
.5
EphB2R
2H9(Fc chimera)-MC-vc-PAB-MMAE
1983
.3
E16
12B9-MC-vc-PAB-MMAE
14.1
.6
E16
12B9-MC-vc-PAB-MMAF
16.4
.5
E16
12G12-MC-vc-PAB-MMAE
10.5
.1
E16
12G12-MC-vc-PAB-MMAF
10.2
.8
E16
3B5-MC-vc-PAB-MMAE
58.6
.8
E16
3B5-MC-vc-PAB-MMAF
8
.1
0772P
11D10(Fc chimera)-MC-vc-PAB-MMAE
340
.9
Steap1
(Steap1-92)-MC-vc-PAB-MMAE
3.5
Steap1
(Steap1-92)-MC-vc_PAB-MMAF
4.7
Steap1
(Steap1-120)-MC-vc-PAB-MMAE
2
Steap1
(Steap1-120)-MC-vc-PAB-MMAF
2.3
E16
3B5-MC-vc-PAB-MMAF
52.2
.5
9.7 Compositions and Methods of Administration
In other embodiments, described is a composition including an effective amount of an Exemplary Compound and/or Exemplary Conjugate and a pharmaceutically acceptable carrier or vehicle. For convenience, the Drug units and Drug-Linker Compounds can be referred to as Exemplary Compounds, while Drug-Ligand Conjugates and Drug-Linker-Ligand Conjugates can be referred to as Exemplary Conjugates. The compositions are suitable for veterinary or human administration.
The present compositions can be in any form that allows for the composition to be administered to a patient. For example, the composition can be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intra-tumor, and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In one aspect, the compositions are administered parenterally. In yet another aspect, the Exemplary Compounds and/or the Exemplary Conjugates or compositions are administered intravenously.
Pharmaceutical compositions can be formulated so as to allow an Exemplary Compound and/or Exemplary Conjugate to be bioavailable upon administration of the composition to a patient. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of an Exemplary Compound and/or Exemplary Conjugate in aerosol form can hold a plurality of dosage units.
Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the Exemplary Compound or Exemplary Conjugate, the manner of administration, and the composition employed.
The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) can be gaseous or particulate, so as to provide an aerosol composition useful in, e.g., inhalatory administration.
When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
When the composition is in the form of a capsule, e.g., a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
The composition can be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
The liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material. Physiological saline is an exemplary adjuvant. An injectable composition is preferably sterile.
The amount of the Exemplary Compound and/or Exemplary Conjugate that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
The compositions comprise an effective amount of an Exemplary Compound and/or Exemplary Conjugate such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of an Exemplary Compound and/or Exemplary Conjugate by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. In one aspect, oral compositions can comprise from about 4% to about 50% of the Exemplary Compound and/or Exemplary Conjugate by weight of the composition. In yet another aspect, present compositions are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the Exemplary Compound and/or Exemplary Conjugate.
For intravenous administration, the composition can comprise from about 0.01 to about 100 mg of an Exemplary Compound and/or Exemplary Conjugate per kg of the animal's body weight. In one aspect, the composition can include from about 1 to about 100 mg of an Exemplary Compound and/or Exemplary Conjugate per kg of the animal's body weight. In another aspect, the amount administered will be in the range from about 0.1 to about 25 mg/kg of body weight of the Exemplary Compound and/or Exemplary Conjugate.
Generally, the dosage of an Exemplary Compound and/or Exemplary Conjugate administered to a patient is typically about 0.01 mg/kg to about 2000 mg/kg of the animal's body weight. In one aspect, the dosage administered to a patient is between about 0.01 mg/kg to about 10 mg/kg of the animal's body weight, in another aspect, the dosage administered to a patient is between about 0.1 mg/kg and about 250 mg/kg of the animal's body weight, in yet another aspect, the dosage administered to a patient is between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, in yet another aspect the dosage administered is between about 0.1 mg/kg to about 10 mg/kg of the animal's body weight, and in yet another aspect, the dosage administered is between about 1 mg/kg to about 10 mg/kg of the animal's body weight.
The Exemplary Compounds and/or Exemplary Conjugate or compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer an Exemplary Compound and/or Exemplary Conjugate or composition. In certain embodiments, more than one Exemplary Compound and/or Exemplary Conjugate or composition is administered to a patient.
In specific embodiments, it can be desirable to administer one or more Exemplary Compounds and/or Exemplary Conjugate or compositions locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue. In another embodiment, administration can be by direct injection at the site (or former site) of a manifestation of an autoimmune disease.
In certain embodiments, it can be desirable to introduce one or more Exemplary Compounds and/or Exemplary Conjugate or compositions into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant.
In yet another embodiment, the Exemplary Compounds and/or Exemplary Conjugate or compositions can be delivered in a controlled release system, such as but not limited to, a pump or various polymeric materials can be used. In yet another embodiment, a controlled-release system can be placed in proximity of the target of the Exemplary Compounds and/or Exemplary Conjugate or compositions, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can be used.
The term “carrier” refers to a diluent, adjuvant or excipient, with which an Exemplary
Compound and/or Exemplary Conjugate is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a patient, the Exemplary Compound and/or Exemplary Conjugate or compositions and pharmaceutically acceptable carriers are sterile. Water is an exemplary carrier when the Exemplary Compounds and/or Exemplary Conjugates are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
In an embodiment, the Exemplary Compounds and/or Exemplary Conjugates are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly human beings. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where an Exemplary Compound and/or Exemplary Conjugate is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the Exemplary Compound and/or Exemplary Conjugate is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be used.
The compositions can be intended for topical administration, in which case the carrier may be in the form of a solution, emulsion, ointment or gel base. If intended for transdermal administration, the composition can be in the form of a transdermal patch or an iontophoresis device. Topical formulations can comprise a concentration of an Exemplary Compound and/or Exemplary Conjugate of from about 0.05% to about 50% w/v (weight per unit volume of composition), in another aspect, from 0.1% to 10% w/v.
The composition can be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the Exemplary Compound and/or Exemplary Conjugate.
The composition can include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition can include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.
The compositions can consist of gaseous dosage units, e.g., it can be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery can be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients.
Whether in solid, liquid or gaseous form, the present compositions can include a pharmacological agent used in the treatment of cancer, an autoimmune disease or an infectious disease.
9.8 Therapeutic Uses of the Exemplary Conjugates
The Exemplary Compounds and/or Exemplary Conjugates are useful for treating cancer, an autoimmune disease or an infectious disease in a patient.
9.8.1 Treatment of Cancer
The Exemplary Compounds and/or Exemplary Conjugates are useful for inhibiting the multiplication of a tumor cell or cancer cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The Exemplary Compounds and/or Exemplary Conjugates can be used accordingly in a variety of settings for the treatment of animal cancers. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug or Drug unit to a tumor cell or cancer cell. Without being bound by theory, in one embodiment, the Ligand unit of an Exemplary Conjugate binds to or associates with a cancer-cell or a tumor-cell-associated antigen, and the Exemplary Conjugate can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell. Once inside the cell, one or more specific peptide sequences within the Linker unit are hydrolytically cleaved by one or more tumor-cell or cancer-cell-associated proteases, resulting in release of a Drug or a Drug-Linker Compound. The released Drug or Drug-Linker Compound is then free to migrate within the cell and induce cytotoxic or cytostatic activities. In an alternative embodiment, the Drug or Drug unit is cleaved from the Exemplary Conjugate outside the tumor cell or cancer cell, and the Drug or Drug-Linker Compound subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to the tumor cell or cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is on the surface of the tumor cell or cancer cell.
In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is an extracellular matrix protein associated with the tumor cell or cancer cell.
The specificity of the Ligand unit for a particular tumor cell or cancer cell can be important for determining those tumors or cancers that are most effectively treated. For example, Exemplary Conjugates having a BR96 Ligand unit can be useful for treating antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas. Exemplary Conjugates having an Anti-CD30 or an anti-CD40 Ligand unit can be useful for treating hematologic malignancies.
Other particular types of cancers that can be treated with Exemplary Conjugates include, but are not limited to, those disclosed in Table 3.
TABLE 3
Solid tumors, including but not limited to:
fibrosarcoma
myxosarcoma
liposarcoma
chondrosarcoma
osteogenic sarcoma
chordoma
angiosarcoma
endotheliosarcoma
lymphangiosarcoma
lymphangioendotheliosarcoma
synovioma
mesothelioma
Ewing's tumor
leiomyosarcoma
rhabdomyosarcoma
colon cancer
colorectal cancer
kidney cancer
pancreatic cancer
bone cancer
breast cancer
ovarian cancer
prostate cancer
esophogeal cancer
stomach cancer
oral cancer
nasal cancer
throat cancer
squamous cell carcinoma
basal cell carcinoma
adenocarcinoma
sweat gland carcinoma
sebaceous gland carcinoma
papillary carcinoma
papillary adenocarcinomas
cystadenocarcinoma
medullary carcinoma
bronchogenic carcinoma
renal cell carcinoma
hepatoma
bile duct carcinoma
choriocarcinoma
seminoma
embryonal carcinoma
Wilms' tumor
cervical cancer
uterine cancer
testicular cancer
small cell lung carcinoma
bladder carcinoma
lung cancer
epithelial carcinoma
glioma
glioblastoma multiforme
astrocytoma
medulloblastoma
craniopharyngioma
ependymoma
pinealoma
hemangioblastoma
acoustic neuroma
oligodendroglioma
meningioma
skin cancer
melanoma
neuroblastoma
retinoblastoma
blood-borne cancers, including but not limited to:
acute lymphoblastic leukemia “ALL”
acute lymphoblastic B-cell leukemia
acute lymphoblastic T-cell leukemia
acute myeloblastic leukemia “AML”
acute promyelocytic leukemia “APL”
acute monoblastic leukemia
acute erythroleukemic leukemia
acute megakaryoblastic leukemia
acute myelomonocytic leukemia
acute nonlymphocyctic leukemia
acute undifferentiated leukemia
chronic myelocytic leukemia “CML”
chronic lymphocytic leukemia “CLL”
hairy cell leukemia
multiple myeloma
acute and chronic leukemias:
lymphoblastic
myelogenous
lymphocytic
myelocytic leukemias
Lymphomas:
Hodgkin's disease
non-Hodgkin's Lymphoma
Multiple myeloma
Waldenström's macroglobulinemia
Heavy chain disease
Polycythemia vera
The Exemplary Conjugates provide conjugation-specific tumor or cancer targeting, thus reducing general toxicity of these compounds. The Linker units stabilize the Exemplary Conjugates in blood, yet are cleavable by tumor-specific proteases within the cell, liberating a Drug.
9.8.2 Multi-Modality Therapy for Cancer
Cancers, including, but not limited to, a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of an Exemplary Conjugate and/or an Exemplary Compound.
In other embodiments, methods for treating or preventing cancer are provided, including administering to a patient in need thereof an effective amount of an Exemplary Conjugate and a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory. The Exemplary Conjugates can be administered to a patient that has also undergone surgery as treatment for the cancer.
In one embodiment, the additional method of treatment is radiation therapy.
In a specific embodiment, the Exemplary Conjugate is administered concurrently with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of an Exemplary Conjugates, in one aspect at least an hour, five hours, 12 hours, a day, a week, a month, in further aspects several months (e.g., up to three months), prior or subsequent to administration of an Exemplary Conjugate.
A chemotherapeutic agent can be administered over a series of sessions. Any one or a combination of the chemotherapeutic agents listed in Table 4 can be administered. With respect to radiation, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered.
Additionally, methods of treatment of cancer with an Exemplary Compound and/or Exemplary Conjugate are provided as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The animal being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
The Exemplary Compounds and/or Exemplary Conjugates can also be used in an in vitro or ex vivo fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants. This can involve a multi-step process in which the animal's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the animal's remaining bone-marrow cell population is then eradicated via the administration of a high dose of an Exemplary Compound and/or Exemplary Conjugate with or without accompanying high dose radiation therapy, and the stem cell graft is infused back into the animal. Supportive care is then provided while bone marrow function is restored and the animal recovers.
9.8.3 Multi-Drug Therapy for Cancer
Methods for treating cancer including administering to a patient in need thereof an effective amount of an Exemplary Conjugate and another therapeutic agent that is an anti-cancer agent are disclosed. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In one aspect, the anti-cancer agent includes, but is not limited to, a drug listed in Table 4.
TABLE 4
Alkylating agents
Nitrogen mustards:
cyclophosphamide
ifosfamide
trofosfamide
chlorambucil
melphalan
Nitrosoureas:
carmustine (BCNU)
lomustine (CCNU)
Alkylsulphonates
busulfan
treosulfan
Triazenes:
decarbazine
Platinum containing compounds:
cisplatin
carboplatin
Plant Alkaloids
Vinca alkaloids:
vincristine
vinblastine
vindesine
vinorelbine
Taxoids:
paclitaxel
docetaxol
DNA Topoisomerase Inhibitors
Epipodophyllins:
etoposide
teniposide
topotecan
9-aminocamptothecin
camptothecin
crisnatol
mitomycins:
mitomycin C
Anti-metabolites
Anti-folates:
DHFR inhibitors:
methotrexate
trimetrexate
IMP dehydrogenase Inhibitors:
mycophenolic acid
tiazofurin
ribavirin
EICAR
Ribonucleotide reductase Inhibitors:
hydroxyurea
deferoxamine
Pyrimidine analogs:
Uracil analogs
5-Fluorouracil
floxuridine
doxifluridine
ratitrexed
Cytosine analogs
cytarabine (ara C)
cytosine arabinoside
fludarabine
Purine analogs:
mercaptopurine
thioguanine
Hormonal therapies:
Receptor antagonists:
Anti-estrogen
tamoxifen
raloxifene
megestrol
LHRH agonists:
goscrclin
leuprolide acetate
Anti-androgens:
flutamide
bicalutamide
Retinoids/Deltoids
Vitamin D3 analogs:
EB 1089
CB 1093
KH 1060
Photodynamic therapies:
vertoporfin (BPD-MA)
phthalocyanine
photosensitizer Pc4
demethoxy-hypocrellin A
(2BA-2-DMHA)
Cytokines:
Interferon- α
Interferon- γ
tumor necrosis factor
Others:
Gemcitabine
Velcade
Revamid
Thalamid
Isoprenylation inhibitors:
Lovastatin
Dopaminergic neurotoxins:
1-methyl-4-phenylpyridinium ion
Cell cycle inhibitors:
staurosporine
Actinomycins:
Actinomycin D
dactinomycin
Bleomycins:
bleomycin A2
bleomycin B2
peplomycin
Anthracyclines:
daunorubicin
Doxorubicin (adriamycin)
idarubicin
epirubicin
pirarubicin
zorubicin
mtoxantrone
MDR inhibitors:
verapamil
Ca2+ ATPase inhibitors:
thapsigargin
9.8.4 Treatment of Autoimmune Diseases
The Exemplary Conjugates are useful for killing or inhibiting the replication of a cell that produces an autoimmune disease or for treating an autoimmune disease. The Exemplary Conjugates can be used accordingly in a variety of settings for the treatment of an autoimmune disease in a patient. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. Without being bound by theory, in one embodiment, the Drug-Linker-Ligand Conjugate associates with an antigen on the surface of a target cell, and the Exemplary Conjugate is then taken up inside a target-cell through receptor-mediated endocytosis. Once inside the cell, one or more specific peptide sequences within the Linker unit are enzymatically or hydrolytically cleaved, resulting in release of a Drug. The released Drug is then free to migrate in the cytosol and induce cytotoxic or cytostatic activities. In an alternative embodiment, the Drug is cleaved from the Exemplary Conjugate outside the target cell, and the Drug subsequently penetrates the cell.
In one embodiment, the Ligand unit binds to an autoimmune antigen. Inone aspect, the antigen is on the surface of a cell involved in an autoimmune condition.
In another embodiment, the Ligand unit binds to an autoimmune antigen which is on the surface of a cell.
In one embodiment, the Ligand binds to activated lymphocytes that are associated with the autoimmune disease state.
In a further embodiment, the Exemplary Conjugates kill or inhibit the multiplication of cells that produce an autoimmune antibody associated with a particular autoimmune disease.
Particular types of autoimmune diseases that can be treated with the Exemplary Conjugates include, but are not limited to, Th2 lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft versus host disease); Th1 lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); activated B lymphocyte-related disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes); and those disclosed in Table 5.
TABLE 5
Active Chronic Hepatitis
Addison's Disease
Allergic Alveolitis
Allergic Reaction
Allergic Rhinitis
Alport's Syndrome
Anaphlaxis
Ankylosing Spondylitis
Anti-phosholipid Syndrome
Arthritis
Ascariasis
Aspergillosis
Atopic Allergy
Atropic Dermatitis
Atropic Rhinitis
Behcet's Disease
Bird-Fancier's Lung
Bronchial Asthma
Caplan's Syndrome
Cardiomyopathy
Celiac Disease
Chagas' Disease
Chronic Glomerulonephritis
Cogan's Syndrome
Cold Agglutinin Disease
Congenital Rubella Infection
CREST Syndrome
Crohn's Disease
Cryoglobulinemia
Cushing's Syndrome
Dermatomyositis
Discoid Lupus
Dressler's Syndrome
Eaton-Lambert Syndrome
Echovirus Infection
Encephalomyelitis
Endocrine opthalmopathy
Epstein-Barr Virus Infection
Equine Heaves
Erythematosis
Evan's Syndrome
Felty's Syndrome
Fibromyalgia
Fuch's Cyclitis
Gastric Atrophy
Gastrointestinal Allergy
Giant Cell Arteritis
Glomerulonephritis
Goodpasture's Syndrome
Graft v. Host Disease
Graves' Disease
Guillain-Barre Disease
Hashimoto's Thyroiditis
Hemolytic Anemia
Henoch-Schonlein Purpura
Idiopathic Adrenal Atrophy
Idiopathic Pulmonary Fibritis
IgA Nephropathy
Inflammatory Bowel Diseases
Insulin-dependent Diabetes Mellitus
Juvenile Arthritis
Juvenile Diabetes Mellitus (Type I)
Lambert-Eaton Syndrome
Laminitis
Lichen Planus
Lupoid Hepatitis
Lupus
Lymphopenia
Meniere's Disease
Mixed Connective Tissue Disease
Multiple Sclerosis
Myasthenia Gravis
Pernicious Anemia
Polyglandular Syndromes
Presenile Dementia
Primary Agammaglobulinemia
Primary Biliary Cirrhosis
Psoriasis
Psoriatic Arthritis
Raynauds Phenomenon
Recurrent Abortion
Reiter's Syndrome
Rheumatic Fever
Rheumatoid Arthritis
Sampter's Syndrome
Schistosomiasis
Schmidt's Syndrome
Scleroderma
Shulman's Syndrome
Sjorgen's Syndrome
Stiff-Man Syndrome
Sympathetic Ophthalmia
Systemic Lupus Erythematosis
Takayasu's Arteritis
Temporal Arteritis
Thyroiditis
Thrombocytopenia
Thyrotoxicosis
Toxic Epidermal Necrolysis
Type B Insulin Resistance
Type I Diabetes Mellitus
Ulcerative Colitis
Uveitis
Vitiligo
Waldenstrom's Macroglobulemia
Wegener's Granulomatosis
9.8.5 Multi-Drug Therapy of Autoimmune Diseases
Methods for treating an autoimmune disease are also disclosed including administering to a patient in need thereof an effective amount of an Exemplary Conjugate and another therapeutic agent known for the treatment of an autoimmune disease. In one embodiment, the anti-autoimmune disease agent includes, but is not limited to, agents listed in Table 6.
TABLE 6
cyclosporine
cyclosporine A
mycophenylate mofetil
sirolimus
tacrolimus
enanercept
prednisone
azathioprine
methotrexate cyclophosphamide
prednisone
aminocaproic acid
chloroquine
hydroxychloroquine
hydrocortisone
dexamethasone
chlorambucil
DHEA
danazol
bromocriptine
meloxicam
infliximab
9.8.6 Treatment of Infectious Diseases
The Exemplary Conjugates are useful for killing or inhibiting the multiplication of a cell that produces an infectious disease or for treating an infectious disease. The Exemplary Conjugates can be used accordingly in a variety of settings for the treatment of an infectious disease in a patient. The Drug-Linker-Ligand Conjugates can be used to deliver a Drug to a target cell. In one embodiment, the Ligand unit binds to the infectious disease cell.
In one embodiment, the Conjugates kill or inhibit the multiplication of cells that produce a particular infectious disease.
Particular types of infectious diseases that can be treated with the Exemplary Conjugates include, but are not limited to, those disclosed in Table 7.
TABLE 7
Bacterial Diseases:
Diphtheria
Pertussis
Occult Bacteremia
Urinary Tract Infection
Gastroenteritis
Cellulitis
Epiglottitis
Tracheitis
Adenoid Hypertrophy
Retropharyngeal Abcess
Impetigo
Ecthyma
Pneumonia
Endocarditis
Septic Arthritis
Pneumococcal
Peritonitis
Bactermia
Meningitis
Acute Purulent Meningitis
Urethritis
Cervicitis
Proctitis
Pharyngitis
Salpingitis
Epididymitis
Gonorrhea
Syphilis
Listeriosis
Anthrax
Nocardiosis
Salmonella
Typhoid Fever
Dysentery
Conjunctivitis
Sinusitis
Brucellosis
Tullaremia
Cholera
Bubonic Plague
Tetanus
Necrotizing Enteritis
Actinomycosis
Mixed Anaerobic Infections
Syphilis
Relapsing Fever
Leptospirosis
Lyme Disease
Rat Bite Fever
Tuberculosis
Lymphadenitis
Leprosy
Chlamydia
Chlamydial Pneumonia
Trachoma
Inclusion Conjunctivitis
Systemic Fungal Diseases:
Histoplamosis
Coccidiodomycosis
Blastomycosis
Sporotrichosis
Cryptococcsis
Systemic Candidiasis
Aspergillosis
Mucormycosis
Mycetoma
Chromomycosis
Rickettsial Diseases:
Typhus
Rocky Mountain Spotted Fever
Ehrlichiosis
Eastern Tick-Borne Rickettsioses
Rickettsialpox
Q Fever
Bartonellosis
Parasitic Diseases:
Malaria
Babesiosis
African Sleeping Sickness
Chagas' Disease
Leishmaniasis
Dum-Dum Fever
Toxoplasmosis
Meningoencephalitis
Keratitis
Entamebiasis
Giardiasis
Cryptosporidiasis
Isosporiasis
Cyclosporiasis
Microsporidiosis
Ascariasis
Whipworm Infection
Hookworm Infection
Threadworm Infection
Ocular Larva Migrans
Trichinosis
Guinea Worm Disease
Lymphatic Filariasis
Loiasis
River Blindness
Canine Heartworm Infection
Schistosomiasis
Swimmer's Itch
Oriental Lung Fluke
Oriental Liver Fluke
Fascioliasis
Fasciolopsiasis
Opisthorchiasis
Tapeworm Infections
Hydatid Disease
Alveolar Hydatid Disease
Viral Diseases:
Measles
Subacute sclerosing panencephalitis
Common Cold
Mumps
Rubella
Roseola
Fifth Disease
Chickenpox
Respiratory syncytial virus infection
Croup
Bronchiolitis
Infectious Mononucleosis
Poliomyelitis
Herpangina
Hand-Foot-and-Mouth Disease
Bornholm Disease
Genital Herpes
Genital Warts
Aseptic Meningitis
Myocarditis
Pericarditis
Gastroenteritis
Acquired Immunodeficiency Syndrome (AIDS)
Human Immunodeficiency Virus (HIV)
Reye's Syndrome
Kawasaki Syndrome
Influenza
Bronchitis
Viral “Walking” Pneumonia
Acute Febrile Respiratory Disease
Acute pharyngoconjunctival fever
Epidemic keratoconjunctivitis
Herpes Simplex Virus 1 (HSV-1)
Herpes Simplex Virus 2 (HSV-2)
Shingles
Cytomegalic Inclusion Disease
Rabies
Progressive Multifocal Leukoencephalopathy
Kuru
Fatal Familial Insomnia
Creutzfeldt-Jakob Disease
Gerstmann-Straussler-Scheinker Disease
Tropical Spastic Paraparesis
Western Equine Encephalitis
California Encephalitis
St. Louis Encephalitis
Yellow Fever
Dengue
Lymphocytic choriomeningitis
Lassa Fever
Hemorrhagic Fever
Hantvirus Pulmonary Syndrome
Marburg Virus Infections
Ebola Virus Infections
Smallpox
9.8.7 Multi-Drug Therapy of Infectious Diseases
Methods for treating an infectious disease are disclosed including administering to a patient in need thereof an Exemplary Conjugate and another therapeutic agent that is an anti-infectious disease agent. In one embodiment, the anti-infectious disease agent is, but not limited to, agents listed in Table 8.
TABLE 8
β-Lactam Antibiotics:
Penicillin G
Penicillin V
Cloxacilliin
Dicloxacillin
Methicillin
Nafcillin
Oxacillin
Ampicillin
Amoxicillin
Bacampicillin
Azlocillin
Carbenicillin
Mezlocillin
Piperacillin
Ticarcillin
Aminoglycosides:
Amikacin
Gentamicin
Kanamycin
Neomycin
Netilmicin
Streptomycin
Tobramycin
Macrolides:
Azithromycin
Clarithromycin
Erythromycin
Lincomycin
Clindamycin
Tetracyclines:
Demeclocycline
Doxycycline
Minocycline
Oxytetracycline
Tetracycline
Quinolones:
Cinoxacin
Nalidixic Acid
Fluoroquinolones:
Ciprofloxacin
Enoxacin
Grepafloxacin
Levofloxacin
Lomefloxacin
Norfloxacin
Ofloxacin
Sparfloxacin
Trovafloxicin
Polypeptides:
Bacitracin
Colistin
Polymyxin B
Sulfonamides:
Sulfisoxazole
Sulfamethoxazole
Sulfadiazine
Sulfamethizole
Sulfacetamide
Miscellaneous Antibacterial Agents:
Trimethoprim
Sulfamethazole
Chloramphenicol
Vancomycin
Metronidazole
Quinupristin
Dalfopristin
Rifampin
Spectinomycin
Nitrofurantoin
Antiviral Agents:
General Antiviral Agents:
Idoxuradine
Vidarabine
Trifluridine
Acyclovir
Famcicyclovir
Pencicyclovir
Valacyclovir
Gancicyclovir
Foscarnet
Ribavirin
Amantadine
Rimantadine
Cidofovir
Antisense Oligonucleotides
Immunoglobulins
Inteferons
Drugs for HIV infection:
Tenofovir
Emtricitabine
Zidovudine
Didanosine
Zalcitabine
Stavudine
Lamivudine
Nevirapine
Delavirdine
Saquinavir
Ritonavir
Indinavir
Nelfinavir
EXAMPLES
Example 1—Preparation of Compound AB
Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL).
The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (Willner et al., (1993) Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M−H]
Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (Willner et al., (1993) Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M−H].
The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and to the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and DIEA (3.66 mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oil that was precipitated with EtOAc, and then triturated with EtOAc (ca. 25 mL). The solute was further precipitated with ether (ca. 200 mL) and triturated for 15 min. The solid was filtered and dried under high vacuum to provide Compound AB which was 93% pure according to HPLC and used in the next step without further purification. Yield: 9.7 g (94%).
Example 2—Preparation of Compound 1
Phenylalanine t-butyl ester HCl salt (868 mg, 3 mmol), N-Boc-Dolaproine (668 mg, 1 eq.), DEPC (820 μL, 1.5 eq.), and DIEA (1.2 mL) were diluted with dichloromethane (3 mL). After 2 hours (h) at room temperature (about 28 degrees Celsius), the reaction mixture was diluted with dichloromethane (20 mL), washed successively with saturated aqueous (aq.) NaHCO3 (2×10 mL), saturated aq. NaCl (2×10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in ethyl acetate and was purified via flash chromatography in ethyl acetate. The relevant fractions were combined and concentrated to provide the dipeptide as a white solid: 684 mg (46%). ES-MS m/z 491.3 [M+H]+.
For selective Boc cleavage in the presence of t-butyl ester, the above dipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2 mL). 4M HCl/dioxane (960 μL, 3 eq.) was added, and the reaction mixture was stirred overnight at room temperature. Almost complete Boc deprotection was observed by RP-HPLC with minimal amount of t-butyl ester cleavage. The mixture was cooled down on an ice bath, and triethylamine (500 μL) was added. After 10 min., the mixture was removed from the cooling bath, diluted with dichloromethane (20 mL), washed successively with saturated aq. NaHCO3 (2×10 mL), saturated aq. NaCl (2×10 mL). The organic layer was concentrated to give a yellow foam: 287 mg (57%). The intermediate was used without further purification.
The tripeptide Fmoc-Meval-val-dil-O-t-Bu (prepared as described in WO 02/088172, entitled “Pentapeptide Compounds and Uses Related Thereto”; 0.73 mmol) was treated with TFA (3 mL), dichloromethane (3 mL) for 2 h at room temperature. The mixture was concentrated to dryness, the residue was co-evaporated with toluene (3×20 mL), and dried in vacuum overnight. The residue was diluted with dichloromethane (5 mL) and added to the deprotected dipeptide (287 mg, 0.73 mmol), followed by DIEA (550 μL, 4 eq.), DEPC (201 μL, 1.1 eq.). After 2 h at room temperature the reaction mixture was diluted with ethyl acetate (50 mL), washed successively with 10% aq. citric acid (2×20 mL), saturated aq. NaHCO3 (2×10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in ethyl acetate and was purified via flash chromatography in ethyl acetate. The relevant fractions were combined and concentrated to provide Fmoc-Meval-val-dil-dap-phe-O-t-Bu as a white solid: 533 mg (71%). Rf 0.4 (EtOAc). ES-MS m/z 1010.6 [M+H]t
The product (200 mg, 0.2 mmol) was diluted with dichloromethane (3 mL), diethylamine (1 mL). The reaction mixture was stirred overnight at room temperature. Solvents were removed to provide an oil that was purified by flash silica gel chromatography in a step gradient 0-10% MeOH in dichloromethane to provide Compound 1 as a white solid: 137 mg (87%). Rf 0.3 (10% MeOH/CH2Cl2). ES-MS m/z 788.6 [M+H]+.
Example 3—Preparation of Compound 2
Compound 2 was prepared from compound 1 (30 mg, 0.038 mmol) by treatment with 4M HCl/dioxane (4 ml) for 7 h at room temperature. The solvent was removed, and the residue was dried in a vacuum overnight to give provide Compound 2 as a hydroscopic white solid: 35 mg (120% calculated for HCl salt). ES-MS m/z 732.56 [M+H]t
Example 4—Preparation of Compound 3
Fmoc-Meval-val-dil-dap-phe-O-t-Bu (Example 2, 50 mg) was treated with 4M HCl/dioxane (4 ml) for 16 h at room temperature. The solvent was removed, and the residue was dried in vacuum overnight to give 50 mg of a hydroscopic white solid intermediate
The white solid intermediate (20 mg, 0.02 mmol) was diluted with dichloromethane (1 mL); DEPC (5 μL, 0.03 mmol, 1.5 eq.) was added followed by DIEA (11 μL, 0.06 mmol, 3 eq.), and t-butylamine (3.2 μL, 0.03 mmol, 1.5 eq.). After 2 h at room temperature, the reaction was found to be uncompleted by RP-HPLC. More DEPC (10 μL) and t-butylamine (5 μL) were added and the reaction was stirred for additional 4 h. Reaction mixture was diluted with dichloromethane (15 mL), washed successively with water (5 mL), 0.1 M aq. HCl (10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was diluted with dichloromethane and purified via flash chromatography in a step gradient 0-5% MeOH in dichloromethane. The relevant fractions were combined and concentrated to provide the Fmoc protected intermediate as a white solid: 7.3 mg (36%). Rf 0.75 (10% MeOH/CH2Cl2).
Fmoc protected intermediate was diluted with dichloromethane (0.5 mL) and treated with diethylamine (0.5 mL) for 3 h at room temperature. The reaction mixture was concentrated to dryness. The product was isolated by flash silica gel chromatography in a step gradient 0-10% MeOH in dichloromethane to provide Compound 3 as a white solid: 4 mg (70%). Rf 0.2 (10% MeOH/CH2Cl2). ES-MS m/z 787 [M+H]+, 809 [M+Na]+.
Example 5—Preparation of Compound 4
Boc-L-Phenylalanine (265 mg, 1 mmol, 1 eq.) and triethyleneglycol monomethyl ether (164 μL, 1 mmol, 1 eq.) were diluted with dichloromethane (5 mL). Then, DCC (412 mg, 2 mmol, 2 eq.) was added, followed by DMAP (10 mg). The reaction mixture was stirred overnight at room temperature. The precipitate was filtered off. The solvent was removed in a vacuum, the residue was diluted with ethyl acetate, and purified by silica gel flash chromatography in ethyl acetate. The product containing fractions were pulled, concentrated, and dried in vacuum to give a white solid: 377 mg (91%). Rf 0.5 (EtOAc). ES-MS m/z 434 [M+Na]+.
Removal of Boc protecting group was performed by treatment of the above material in dioxane (10 mL) with 4M HCl/dioxane (6 mL) for 6 h at room temperature. The solvent was removed in a vacuum, the residue was dried in a vacuum to give a white solid.
The HCl salt of Phenylalanine-triethyleneglycol monomethyl ether ester (236 mg, 0.458 mmol, 1 eq.) and N-Boc-Dolaproine (158 mg, 0.55 mmol, 1.2 eq.) were diluted with dichloromethane (3 mL). DEPC (125 μL, 1.5 eq.) and added to the mixture followed by DIEA (250 μL, 3 eq.). After 2 h at room temperature the reaction mixture was diluted with ethyl acetate (30 mL), washed successively with saturated aq. NaHCO3 (2×10 mL), 10% aq. citric acid (2×10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in ethyl acetate and was purified via flash chromatography on silica gel in ethyl acetate. The relevant fractions were combined and concentrated to provide a white foam intermediate: 131 mg (50%). Rf 0.25 (EtOAc). ES-MS m/z 581.3 [M+H]+.
Boc deprotection was done in dichloromethane (2 mL), TFA (0.5 mL) at room temperature for 2 h. Solvent was removed in vacuum, and the residue was co-evaporated with toluene (3×25 mL), then dried in vacuum to give 138 mg of dipeptide TFA salt.
Fmoc-Meval-val-dil-OH (Example 2, 147 mg, 0.23 mmol, 1 eq.), and dipeptide TFA salt (138 mg) were diluted with dichloromethane (2 mL). To the mixture DEPC (63 μL, 1.5 eq.) was added, followed by DIEA (160 μL, 4 eq.). After 2 h at room temperature the reaction mixture was diluted with dichloromethane (30 mL), washed successively with 10% aq. citric acid (2×20 mL), saturated aq. NaCl (20 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in dichloromethane and was purified via flash chromatography on silica gel in a step gradient 0-5% MeOH in dichloromethane. The relevant fractions were combined and concentrated to provide white foam: 205 mg (81%). Rf 0.4 (10% MeOH/CH2Cl2). ES-MS m/z 1100.6 [M+H]+, 1122.4 [M+Na]t
Fmoc protecting group was removed by treatment with diethylamine (2 mL) in dichloromethane (6 mL). After 6 h at room temperature solvent was removed in vacuum, product was isolated by flash chromatography on silica gel in a step gradient 0-10% MeOH in dichloromethane. The relevant fractions were combined and concentrated. After evaporation from dichloromethane/hexane, 1:1, Compound 4 was obtained as a white foam: 133 mg (80%). Rf 0.15 (10% MeOH/CH2Cl2). ES-MS m/z 878.6 [M+H]+.
Example 6—Preparation of Compound 5
Fmoc-Meval-val-dil-OH (Example 2, 0.50 g, 0.78 mmol) and dap-phe-OMe.HCl (0.3 g, 0.78 mmol, prepared according to Pettit, G. R., et al. Anti-Cancer Drug Design 1998, 13, 243-277) were dissolved in CH2Cl2 (10 mL) followed by the addition of diisopropylethylamine (0.30 mL, 1.71 mmol, 2.2 eq.). DEPC (0.20 mL, 1.17, 1.5 eq.) was added and the contents stood over Ar. Reaction was complete according to HPLC in 1 h. The mixture was concentrated to an oil and purified by SiO2 chromatography (300×25 mm column) and eluting with 100% EtOAc. The product was isolated as a white foamy solid. Yield: 0.65 g (87%). ES-MS m/z 968.35 [M+H]+, 991.34 [M+Na]+; UV λmax 215, 265 nm.
The Fmoc-protected peptide (0.14 g, 0.14 mmol) in methylene chloride (5 mL) was treated with diethylamine (2 mL) and the contents stood at room temperature for 2 h. The reaction, complete by HPLC, was concentrated to an oil, taken up in 2 mL of DMSO and injected into a preparative-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min). Fractions containing the product were evaporated to afford a white powder for the trifluoroacetate salt. Yield: 0.126 g (98%). Rf 0.28 (100% EtOAc); ES-MS m/z 746.59 [M+H]+, 768.51 [M+Na]+; UV λmax 215 nm.
Example 7—Preparation of Compound 6
The trifluoroacetate salt of Compound 5 (0.11 g, 0.13 mmol), Compound AB (0.103 g, 0.14 mmol, 1.1 eq.) and HOBt (3.4 mg, 26 μmol, 0.2 eq.) were suspended in DMF/pyridine (2 mL/0.5 mL, respectively). Diisopropylethylamine (22.5 μL, 0.13 mmol, 1.0 eq.) was added and the yellow solution stirred while under argon. After 3 h, an additional 1.0 eq. of DIEA was added.
24 hours later, 0.5 eq. of the activated linker was included in the reaction mixture. After 40 h total, the reaction was complete. The contents were evaporated, taken up in DMSO and injected into a prep-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 50 mL/min). The desired fractions were evaporated to give the product as a yellow oil. Methylene chloride (ca. 2 mL) and excess ether were added to provide Compound 6 as a white precipitate that was filtered and dried. Yield: 90 mg (52%). ES-MS m/z 1344.32 [M+H]+, 1366.29 [M+Na]+; UV λmax 215, 248 nm.
Example 8—Preparation of Compound 7
Compound 4 (133 mg, 0.15 mmol, 1 eq.), Compound AB, (123 mg, 0.167 mmol, 1.1 eq.), and HOBt (4 mg, 0.2 eq.) were diluted with DMF (1.5 mL). After 2 min, pyridine (5 mL) was added and the reaction was monitored using RP-HPLC. The reaction was shown to be complete within 18 h. The reaction mixture was diluted with dichloromethane (20 mL), washed successively with 10% aq. citric acid (2×10 mL), water (10 mL), saturated aq. NaCl (10 mL). The organic layer was separated and concentrated. The resulting residue was re-suspended in dichloromethane and was purified via flash chromatography on silica gel in a step gradient 0-10% MeOH in dichloromethane. The relevant fractions were combined and concentrated to provide Compound 7 as a white foam: 46 mg (21%). Rf 0.15 (10% MeOH/CH2Cl2). ES-MS m/z 1476.94 [M+H]t
Example 9—Preparation of MC-Val-Cit-PAB-MMAF t-butyl ester 8
Compound 1 (83 mg, 0.11 mmol), Compound AB (85 mg, 0.12 mmol, 1.1 eq.), and HOBt (2.8 mg, 21 μmol, 0.2 eq.) were taken up in dry DMF (1.5 mL) and pyridine (0.3 mL) while under argon. After 30 h, the reaction was found to be essentially complete by HPLC. The mixture was evaporated, taken up in a minimal amount of DMSO and purified by prep-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min) to provide Compound 8 as a white solid. Yield: 103 mg (71%). ES-MS m/z 1387.06 [M+H]+, 1409.04 [M+Na]+; UV λmax 205, 248 nm.
Example 10—Preparation of MC-val-cit-PAB-MMAF 9
Compound 8 (45 mg, 32 μmol) was suspended in methylene chloride (6 mL) followed by the addition of TFA (3 mL). The resulting solution stood for 2 h. The reaction mixture was concentrated in vacuo and purified by prep-HPLC (C12-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min). The desired fractions were concentrated to provide maleimidocaproyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAF (MC-val-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%). ES-MS m/z 1330.29 [M+1-1]+, 1352.24 [M+Na]+; UV λmax 205, 248 nm.
Example 11—Preparation of MC-val-cit-PAB-MMAF tert-butyl amide 10
Compound 3 (217 mg, 0.276 mmol, 1.0 eq.), Compound AB (204 mg, 0.276 mmol, 1.0 eq.), and HOBt (11 mg, 0.0828 mmol, 0.3 eq.) were diluted with pyridine/DMF (6 mL). To this mixture was added DIEA (0.048 mL), and the mixture was stirred ca. 16 hr. Volatile organics were evaporated in vacuo. The crude residue was purified by Chromatotron® (radial thin-layer chromatography) with a step gradient (0-5-10% methanol in DCM) to provide MC-val-cit-PAB-MMAF tert-butyl amide 10. Yield: 172 mg (45%); ES-MS m/z 1386.33 [M+H]+, 1408.36 [M+Na]+; UV λmax 215, 248 nm.
Example 12—Preparation of AC10-MC-MMAE by Conjugation of AC10 and MC-MMAE
AC10, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37° C. for about 30 minutes, the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of the absorbance at 412 nm. The reduced antibody dissolved in PBS is chilled on ice.
The drug linker reagent, maleimidocaproyl-monomethyl auristatin E, i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and water at known concentration, and added to the chilled reduced antibody AC10 in PBS. After about one hour, an excess of maleimide is added to quench the reaction and cap any unreacted antibody thiol groups. The reaction mixture is concentrated by centrifugal ultrafiltration and AC10-MC-MMAE is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 μm filters under sterile conditions, and frozen for storage.
Example 13—Preparation of AC10-MC-MMAF by Conjugation of AC10 and MC-MMAF
AC10-MC-MMAF was prepared by conjugation of AC10 and MC-MMAF following the procedure of Example 12.
Example 14—Preparation of AC10-MC-val-cit-PAB-MMAE by Conjugation of AC10 and MC-val-cit-PAB-MMAE
AC10-MC-val-cit-PAB-MMAE was prepared by conjugation of AC10 and MC-val-cit-PAB-MMAE following the procedure of Example 12.
Example 15—Preparation of AC10-MC-val-cit-PAB-MMAF by Conjugation of AC10 and MC-val-cit-PAB-MMAF (9)
AC10-MC-val-cit-PAB-MMAF was prepared by conjugation of AC10 and MC-val-cit-PAB-MMAF (9) following the procedure of Example 12.
Example 16—Determination of Cytotoxicity of Selected Compounds
Cytotoxic activity of MMAF and Compounds 1-5 was evaluated on the Lewis Y positive cell lines OVCAR-3, H3396 breast carcinoma, L2987 lung carcinoma and LS174t colon carcinoma Lewis Y positive cell lines can be assayed for cytotoxicity. To evaluate the cytotoxicity of Compounds 1-5, cells can be seeded at approximately 5-10,000 per well in 150 pd of culture medium then treated with graded doses of Compounds 1-5 in quadruplicates at the initiation of assay. Cytotoxicity assays are usually carried out for 96 hours after addition of test compounds. Fifty μl of resazurin dye may be added to each well during the last 4 to 6 hours of the incubation to assess viable cells at the end of culture. Dye reduction can be determined by fluorescence spectrometry using the excitation and emission wavelengths of 535 nm and 590 nm, respectively. For analysis, the extent of resazurin reduction by the treated cells can be compared to that of the untreated control cells.
For 1 h exposure assays cells can be pulsed with the drug for 1 h and then washed; the cytotoxic effect can be determined after 96 h of incubation.
Example 17—In Vitro Cytotoxicity Cata for Selected Compounds
Table 10 shows cytotoxic effect of cAC10 Conjugates of Compounds 7-10, assayed as described in General Procedure I on a CD30+ cell line Karpas 299. Data of two separate experiments are presented. The cAC10 conjugates of Compounds 7 and 9 were found to be slightly more active than cAC10-val-cit-MMAE.
TABLE 10
Conjugate
IC50 (ng/mL)
cAC10-val-cit-MMAE
6
cAC10-7
1.0
cAC10-8
15
cAC10-9
0.5
cAC10-10
20
In other experiments, BR96-val-cit-MMAF was at least 250 fold more potent than the free MMAF.
General Procedure I—Cytotoxicity determination.
To evaluate the cytotoxicity of Exemplary Conjugates 7-10, cells were seeded at approximately 5-10,000 per well in 150 μl of culture medium then treated with graded doses of Exemplary Conjugates 7-10 in quadruplicates at the initiation of assay. Cytotoxicity assays were carried out for 96 hours after addition of test compounds. Fifty μl of the resazurin dye was added to each well during the last 4 to 6 hours of the incubation to assess viable cells at the end of culture. Dye reduction was determined by fluorescence spectrometry using the excitation and emission wavelengths of 535 nm and 590 nm, respectively. For analysis, the extent of resazurin reduction by the treated cells was compared to that of the untreated control cells.
Example 18—In Vitro Cell Proliferation Assay
Efficacy of ADC can be measured by a cell proliferation assay employing the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488):
1. An aliquot of 100 μl of cell culture containing about 104 cells (SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each well of a 96-well, opaque-walled plate.
2. Control wells were prepared containing medium and without cells.
3. ADC was added to the experimental wells and incubated for 3-5 days.
4. The plates were equilibrated to room temperature for approximately 30 minutes.
5. A volume of CellTiter-Glo Reagent equal to the volume of cell culture medium present in each well was added.
6. The contents were mixed for 2 minutes on an orbital shaker to induce cell lysis.
7. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.
8. Luminescence was recorded and reported in graphs as RLU=relative luminescence units.
Example 19—Plasma Clearance in Rat
Plasma clearance pharmacokinetics of antibody drug conjugates and total antibody was studied in Sprague-Dawley rats (Charles River Laboratories, 250-275 gms each). Animals were dosed by bolus tail vein injection (IV Push). Approximately 300 μl, whole blood was collected through jugular cannula, or by tail stick, into lithium/heparin anticoagulant vessels at each timepoint: 0 (predose), 10, and 30 minutes; 1, 2, 4, 8, 24 and 36 hours; and 2, 3, 4, 7, 14, 21, 28 days post dose. Total antibody was measured by ELISA—ECD/GxhuFc-HRP. Antibody drug conjugate was measured by ELISA—MMAE/MMAF/ECD-Bio/SA-HRP.
Example 20—Plasma Clearance in Monkey
Plasma clearance pharmacokinetics of antibody drug conjugates and total antibody can be studied in cynomolgus monkeys. FIG. 12 shows a two-stage plasma concentration clearance study after administration of H-MC-vc-MMAE to Cynomolgus monkeys at different doses: 0.5, 1.5, 2.5, and 3.0 mg/kg, administered at day 1 and day 21. Concentrations of total antibody and ADC were measured over time. (H=Trastuzumab).
Example 21—Tumor Volume In Vivo Efficacy in Transgenic Explant Mice
Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Taconic (Germantown, N.Y.). Many strains are suitable, but FVB female mice are preferred because of their higher susceptibility to tumor formation. FVB males can be used for mating and vasectomized CD.1 studs can be used to stimulate pseudopregnancy. Vasectomized mice can be obtained from any commercial supplier. Founders can be bred with either FVB mice or with 129/BL6×FVB p53 heterozygous mice. The mice with heterozygosity at p53 allele can be used to potentially increase tumor formation. Some F1 tumors are of mixed strain. Founder tumors can be FVB only.
Animals having tumors (allograft propagated from Fo5 mmtv transgenic mice) can be treated with a single or multiple dose by IV injection of ADC. Tumor volume can be assessed at various time points after injection.
Example 22—Synthesis of MC-MMAF Via t-Butyl Ester
MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 128.6 mg, 0.163 mmol) was suspended in CH2C12 (0.500 mL). 6-Maleimidocaproic acid (68.9 mg, 0.326 mmol) and 1,3-diisopropylcarbodiimide (0.0505 mL, 0.326 mmol) were added followed by pyridine (0.500 mL). Reaction mixture was allowed to stir for 1.0 hr. HPLC analysis indicated complete consumption of starting compound 1. Volatile organics were evaporated under reduced pressure. Product was isolated via flash column chromatography, using a step gradient from 0 to 5% Methanol in CH2C12. A total of 96 mg of pure MC-MeVal-Val-Dil-Dap-Phe-OtBu (12) (60% yield) was recovered. ES-MS m/z 981.26 [M+H]+; 1003.47 [M+Na]+; 979.65 [M−H]− See FIG. 37. MC-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 12, 74 mg, 0.0754 mmol) was suspended in CH2Cl2 (2.0 mL) and TFA (1 mL) at room temperature. After 2.5 hr, HPLC analysis indicated complete consumption of starting material. Volatile organics were evaporated under reduced pressure, and the product was isolated via preparatory RP-HPLC, using a Phenomenex C12 Synergi Max-RP 80A Column (250×21.20 mm). Eluent: linear gradient 10% to 90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90% MeCN/0.05% TFA (aq) for an additional 20 minutes. ES-MS m/z 925.33 [M+H]+; 947.30 [M+Na]+; 923.45 [M−H]−.
Example 23a—Synthesis of MC-MMAF (11) Via Dimethoxybenzyl Ester
Preparation of Fmoc-L-Phenylalanine-2,4-dimethoxybenzyl ester (Fmoc-Phe-ODMB) See FIG. 38.
A 3-neck, 5-L round-bottom flask was charged with Fmoc-L-Phenylalanine (200 g, 516 mmol Bachem), 2,4-dimethoxybenzyl alcohol (95.4 g, 567 mmol, Aldrich), and CH2Cl2 (2.0 L). N,N-dimethylformamide t-butyl acetal (155 mL, 586 mmol, Fluka) was added to the resulting suspension over 20 min under N2, which resulted in a clear solution. The reaction was then stirred at room temperature overnight, after which time TLC analysis (0.42, Heptane/EtOAc=2:1) indicated that the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a light yellow oil, which was redissolved in CH2Cl2 (200 mL) and purified through a short plug of silica gel (25 cm×25 cm, CH2Cl2) to give a colorless foam (250 g). MeCN (1 L) was added into the resulting foam, which totally dissolved the batch. It was then concentrated to dryness and redissolved in MeCN (1 L) and the resulting suspension was stirred for 1 h, filtered and the filter cake was rinsed with MeCN (2×200 mL) to give Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester as a white solid (113.58 g, 41%, 95.5% AUC by HPLC analysis). Data: HPLC.
Preparation L-Phenylalanine-2,4-Dimethoxybenzyl Ester (Phe-ODMB)
A 500-mL round-bottom flask was charged with Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester (26.00 g, 48.3 mmol), CH2Cl2 (150 mL) and diethylamine (75 mL, Acros). Mixture was stirred at room temperature and the completion monitored by HPLC. After 4 h, the mixture was concentrated (bath temp <30° C.). The residue was resuspended in CH2Cl2 (200 mL) and concentrated. This was repeated once. To the residue was added MeOH (20 mL), which caused the formation of a gel. This residue was diluted with CH2Cl2 (200 mL), concentrated and the cloudy oil left under vacuum overnight. The residue was suspended in CH2Cl2 (100 mL), then toluene (120 mL) was added. The mixture was concentrated and the residue left under vacuum overnight.
Data: HPLC, 1H NMR.
Preparation of Fmoc-Dolaproine (Fmoc-Dap)
Boc-Dolaproine (58.8 g, 0.205 mol) was suspended in 4 N HCl in 1,4-dioxane (256 mL, 1.02 mol, Aldrich). After stirring for 1.5 hours, TLC analysis indicated the reaction was complete (10% MeOH/CH2Cl2) and the mixture was concentrated to near-dryness. Additional 1,4-dioxane was charged (50 mL) and the mixture was concentrated to dryness and dried under vacuum overnight. The resulting white solid was dissolved in H2O (400 mL) and transferred to a 3-L, three-neck, round-bottom flask with a mechanical stirrer and temperature probe. N,N-diisopropylethylamine (214.3 mL, 1.23 mol, Acros) was added over one minute, causing an exotherm from 20.5 to 28.2° C. (internal). The mixture was cooled in an ice bath and 1,4-dioxane was added (400 mL). A solution of Fmoc-OSu (89.90 g, 0.267 mol, Advanced ChemTech) in 1,4-dioxane (400 mL) was added from an addition funnel over 15 minutes, maintaining the reaction temperature below 9° C. The mixture was allowed to warm to room temperature and stir for 19 hours, after which the mixture was concentrated by rotary evaporation to an aqueous slurry (390 g). The suspension was diluted with H2O (750 mL) and Et2O (750 mL), causing a copious white precipitate to form. The layers were separated, keeping the solids with the organic layer. The aqueous layer was acidified using conc. HCl (30 mL) and extracted with EtOAc (3×500 mL). The combined extracts were dried over MgSO4, filtered and concentrated to give 59.25 g of a yellow oil A. The Et2O extract was extracted once with sat. NaHCO3 (200 mL), keeping the solids with the aqueous layer. The aqueous suspension was acidified using conc. HCl (50 mL) and extracted with Et2O (50 mL) keeping the solids with the organic layer. The organic layer was filtered and concentrated to give 32.33 g of a yellow oil B. The two oils (A and B) were combined and purified by flash chromatography on silica gel eluting with CH2C12 (3.5 L), then 3% MeOH/CH2Cl2 (9 L) to give 68.23 g of Fmoc-dolaproine as a white foam (81%, 97.5% purity by HPLC (AUC)).
Preparation of Fmoc-Dap-Phe-ODMB
Crude Phe-ODMB (48.3 mmol) was suspended in anhydrous DMF (105 mL, Acros) for 5 minutes and Fmoc-Dap (19.80 g, 48.3 mmol) was added. The mixture was cooled in an ice bath and TBTU (17.08 g, 53.20 mmol, Matrix Innovations) was added. N,N-diisopropylethylamine (25.3 mL, 145.0 mmol, Acros) was added via syringe over 3 min. After 1 h, the ice bath was removed and the mixture was allowed to warm over 30 min. The mixture was poured into water (1 L) and extracted with ethyl acetate (300 mL). After separation, the aqueous layer was re-extracted with ethyl acetate (2×150 mL). The combined organic layers were washed with brine (150 mL), dried (MgSO4) and filtered (filter paper) to remove the insolubles (inorganics and some dibenzofulvene). After concentration, the residue (41 g) was adsorbed on silica (41 g) and purified by chromatography (22 cm×8 cm column; 65% Heptane/EtOAc (2.5 L); 33% Heptane/EtOAc (3.8 L), to give 29.4 g of product as a white foam (86%, 92% purity by HPLC).
Data: HPLC, 1H NMR, TLC (1:1 EtOAc/Heptane Rf=0.33, red in vanillin stain).
Preparation of Dap-Phe-ODMB
A 1-L round bottom flask was charged with Fmoc-Dap-Phe-ODMB (27.66 g), CH2Cl2 (122 mL) and diethylamine (61 mL, Acros). The solution was stirred at room temperature and the completion monitored by HPLC. After 7 h, the mixture was concentrated (bath temp. <30° C.). The residue was suspended in CH2Cl2 (300 mL) and concentrated. This was repeated twice. To the residue was added MeOH (20 mL) and CH2Cl2 (300 mL), and the solution was concentrated. The residue was suspended in CH2Cl2 (100 mL) and toluene (400 mL), concentrated, and the residue left under vacuum overnight to give a cream-like residue.
Data: HPLC, 1H NMR, MS.
Preparation of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB
Crude Dap-Phe-ODMB (39.1 mmol) was suspended in anhydrous DMF (135 mL, Acros) for 5 minutes and Fmoc-MeVal-Val-Dil-OH (24.94 g, 39.1 mmol, see Example 2 for preparation) was added. The mixture was cooled in an ice bath and TBTU (13.81 g, 43.0 mmol, Matrix Innovations) was added. N,N-Diisopropylethylamine (20.5 mL, 117.3 mmol, Acros) was added via syringe over 2 minutes. After 1 hour, the ice bath was removed and the mixture was allowed to warm over 30 min. The mixture was poured into water (1.5 L) and diluted with ethyl acetate (480 mL). After standing for 15 minutes, the layers were separated and the aqueous layer was extracted with ethyl acetate (300 mL). The combined organic layers were washed with brine (200 mL), dried (MgSO4) and filtered (filter paper) to remove insolubles (inorganics and some dibenzofulvene). After concentration, the residue (49 g) was scraped from the flask and adsorbed on silica (49 g) and purified by chromatography (15 cm×10 cm dia column; 2:1 EtOAc/Heptane (3 L), EtOAc (5 L); 250 mL fractions) to give 31.84 g of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (73%, 93% purity by HPLC (AUC)).
Data: HPLC, TLC (2:1 EtOAc/heptane, Rf=0.21, red in vanillin stain).
Preparation of MeVal-Val-Dil-Dap-Phe-ODMB
A 1-L, round-bottom flask was charged with Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB (28.50 g), CH2Cl2 (80 mL) and diethylamine (40 mL). Mixture was stirred at room temperature overnight and then was concentrated under reduced pressure. The residue was adsorbed on silica (30 g) and purified by flash chromatography (15 cm×8 cm dia column; 2% MeOH/DCM (2 L), 3% MeOH/DCM (1 L), 6% MeOH/DCM (4 L); 250 mL fractions) to give 15.88 g of MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (69%, 96% purity by HPLC (AUC)).
Data: HPLC, TLC (6% MeOH/DCM, Rf=0.24, red in vanillin stain).
Preparation of MC-MeVal-Val-Dil-Dap-Phe-ODMB
A 50-mL, round-bottom flask was charged with MeVal-Val-Dil-Dap-Phe-ODMB (750 mg, 0.85 mmol), anhydrous DMF (4 mL), maleimidocaproic acid (180 mg, 0.85 mmol), and TBTU (300 mg, 0.93 mmol, Matrix Innovations) at room temperature. N,N-Diisopropylethylamine (450 μL, 2.57 mmol) was added via syringe. After 1.5 hours, the mixture was poured in water (50 mL) and diluted with ethyl acetate (30 mL). NaCl was added to improve the separation. After separation of the layers, the aqueous layer was extracted with ethyl acetate (25 mL). The combined organic layers were dried (MgSO4), filtered and concentrated. The resulting oil (1 g) was purified by flash chromatography [100 mL silica; 25% Heptane/EtOAc (100 mL), 10% Heptane/EtOAc (200 mL), EtOAc (1.5 L)] to give MC-MeVal-Val-Dil-Dap-Phe-ODMB (13) as a white foam (521 mg, 57%, 94% purity by HPLC(AUC)).
Data: 1H NMR, HPLC.
Preparation of MC-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF) (11)
A 50-mL, round-bottom flask was charged with MC-MeVal-Val-Dil-Dap-Phe-ODMB (Compound 13, 428 mg, 0.39 mmol) and dissolved in 2.5% TFA/CH2Cl2 (20 mL). The solution turned pink-purple over 2 min. The completion was monitored by HPLC and TLC (6% MeOH/DCM, KMnO4 stain). After 40 min, three drops of water were added and the cloudy pink-purple mixture was concentrated to give 521 mg of a pink residue. Purification by chromatography (15% IPA/DCM) gave 270 mg of MC-MMAF (73%, 92% purity by HPLC) as a white solid.
Example 23b—Synthesis of Analog of Mc-MMAF
MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 35 mg, 0.044 mmol) was suspended in DMF (0.250 mL). 4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic acid (11 mg, 0.049 mmol) and HATU (17 mg, 0.044 mmol) were added followed by DIEA (0.031 mL, 0.17 mmol) See FIG. 39. This reaction mixture was allowed to stir for 2.0 hr. HPLC analysis indicated complete consumption of starting compound 1.
Product was isolated via preparatory RP-HPLC, using a Phenomenex C12 Synergi Max-RP 80A Column (250×21.20 mm). Eluent: linear gradient 10% to 80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80% MeCN/0.05% TFA (aq) for an additional 12 minutes. A total of 20 mg of pure product (14) was isolated (0.02 mmol, 46% yield). ES-MS m/z 987.85 [M+H]+; 1019.41 [M+Na]+; 985.54 [M−H]−.
MB-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 14, 38 mg, 0.0385 mmol) was suspended in CH2Cl2 (1 mL) and TFA (1 mL). Mixture was stirred for 2.0 hr, and then volatile organics were evaporated under reduced pressure. Product was purified by preparatory RP-HPLC, using a Phenomenex C12 Synergi Max-RP 80A Column (250×21.20 mm). Eluent: linear gradient 10% to 80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80% MeCN/0.05% TFA (aq) for an additional 12 minutes. A total of 14.4 mg of MB-MMAF product was isolated (0.015 mmol, 40% yield). ES-MS m/z 930.96 [M+H]+ 952.98 [M+Na]+; 929.37 [M−H]−.
Example 23c—Preparation of MC-MeVal-Cit-PAB-MMAF (16)
To a room temperature suspension of Fmoc-MeVal-OH (3.03 g, 8.57 mmol) and N,N′-disuccimidyl carbonate (3.29 g, 12.86 mmol) in CH2Cl2 (80 mL) was added DIEA (4.48 mL, 25.71 mmol). This reaction mixture was allowed to stir for 3.0 hr, and then poured into a separation funnel where the organic mixture was extracted with 0.1 M HCl (aq). The crude organic residue was concentrated under reduced pressure, and the product was isolated by flash column chromatography on silica gel using a 20-100% ethyl acetate/hexanes linear gradient. A total of 2.18 g of pure Fmoc-MeVal-OSu (4.80 mmoles, 56% yield) was recovered.
To a room temperature suspension of Fmoc-MeVal-OSu (2.18 g, 4.84 mmol) in DME (13 mL) and THF (6.5 mL) was added a solution of L-citrulline (0.85 g, 4.84 mmol) and NaHCO3 (0.41 g, 4.84 mmol) in H2O (13 mL). The suspension was allowed to stir at room temperature for 16 hr, then it was extracted into tert-BuOH/CHCl3/H2O, acidified to pH=2-3 with 1 M HCl. The organic phase was separated, dried and concentrated under reduced pressure. The residue was triturated with diethyl ether resulting in 2.01 g of Fmoc-MeVal-Cit-COOH which was used without further purification.
The crude Fmoc-MeVal-Cit-COOH was suspended in 2:1 CH2C12/MeOH (100 mL), and to it was addedp-aminobenzyl alcohol (0.97 g, 7.9 mmol) and EEDQ (1.95 g, 7.9 mmol). This suspension was allowed to stir for 125 hr, then the volatile organics were removed under reduced pressure, and the residue was purified by flash column chromatography on silica gel using a 10% MeOH/CH2Cl2. Pure Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol, 18.5% yield) was recovered. ES-MS m/z 616.48 [M+H]t
To a suspension of Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol) in CH2Cl2 (40 mL) was added STRATOSPHERES' (piperizine-resin-bound) (>5 mmol/g, 150 mg). After being stirred at room temperature for 16 hr the mixture was filtered through celite (pre-washed with MeOH), and concentrated under reduced pressure. Residue was triturated with diethyl ether and hexanes. Resulting solid material, MeVal-Cit-PAB-OH, was suspended in CH2Cl2 (20 mL), and to it was added MC-OSu (0.28 g, 0.896 mmol), DIEA (0.17 mL, 0.99 mmol), and DMF (15 mL). This suspension was stirred for 16 hr, but HPLC analysis of the reaction mixture indicated incomplete reaction, so the suspension was concentrated under reduced pressure to a volume of 6 mL, then a 10% NaHCO3 (aq) solution was added and the suspension stirred for an additional 16 hr.
Solvent was removed under reduced pressure, and the residue was purified by flash column chromatography on silica gel using a 0-10% MeOH/CH2Cl2 gradient, resulting in 42 mg (0.072 mmol, 8% yield) of MC-MeVal-Cit-PAB-OH.
To a suspension of MC-MeVal-Cit-PAB-OH (2.37 g, 4.04 mmol) and bis(nitrophenyl)carbonate (2.59 g, 8.52 mmol) in CH2Cl2 (10 mL) was added DIEA (1.06 mL, 6.06 mmol). This suspension was stirred for 5.5 hr, concentrated under reduced pressure and purified by trituration with diethyl ether. MC-MeVal-Cit-PAB-OCO-pNP (147 mg, 0.196 mmol) was suspended in a 1:5 pyridine/DMF solution (3 mL), and to it was added HOBt (5 mg, 0.039 mmol), DIEA (0.17 mL, 0.978 mmol) and MMAF (compound 2, 150 mg, 0.205 mmol). This reaction mixture was stirred for 16 hr at room temperature, and then purified by preparatory RP-HPLC (×3), using a Phenomenex C12 Synergi Max-RP 80A Column (250×21.20 mm). Eluent: linear gradient 10% to 90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90% MeCN/0.05% TFA (aq) for an additional 20 minutes. MC-MeVal-Cit-PAB-MMAF (16) was obtained as a yellowish solid (24.5 mg, 0.0182, 0.45% yield). ES-MS m/z 1344.95 [M+H]+; 1366.94 [M+Na]+.
Example 23c—Preparation of Succinimide Ester of Suberyl-Val-Cit-PAB-MMAF (17)
Compound 1 (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 0.57 mmol, 1.5 eq.) were suspended in anhydrous pyridine, 5 mL. HOBt (10 mg, 0.076 mmol, 0.2 eq.) was added followed by DIEA (199 μl, 1.14 mmol, 3 eq.). Reaction mixture was sonicated for 10 min, and then stirred overnight at room temperature. Pyridine was removed under reduced pressure, residue was re-suspended in CH2C12. Mixture was separated by silica gel flash chromatography in a step gradient of MeOH, from 0 to 10%, in CH2C12. Product containing fractions were pulled, concentrated, dried in vacuum overnight to give 317 mg (59% yield) of Fmoc-Val-Cit-PAB-MMAF-OtBu. ES-MS m/z 1415.8 [M+H]+.
Fmoc-Val-Cit-PAB-MMAF-OtBu (100 mg) was stirred in 20% TFA/CH2Cl2 (10 mL), for 2 hrs. Mixture was diluted with CH2Cl2 (50 mL). Organic layer was washed successively with water (2×30 mL) and brine (1×30 mL). Organic phase was concentrated, loaded onto pad of silica gel in 10% MeOH/CH2Cl2. Product was eluted with 30% MeOH/CH2Cl2. After drying in vacuum overnight, Fmoc-Val-Cit-PAB-MMAF was obtained as a white solid, 38 mg, 40% yield. ES-MS m/z 1357.7 [M−H]−.
Fmoc-Val-Cit-PAB-MMAF, 67 mg, was suspended in CH2Cl2 (2 mL) diethylamine (2 mL) and DMF (2 mL). Mixture was stirred for 2 hrs at room temperature. Solvent was removed under reduced pressure. Residue was co-evaporated with pyridine (2 mL), then with toluene (2×5 mL), dried in vacuum. Val-Cit-PAB-MMAF was obtained as brownish oil, and used without further purification.
All Val-Cit-PAB-MMAF prepared from 67 mg of Fmoc-Val-Cit-PAB-MMAF, was suspended in pyridine (2 mL), and added to a solution of disuccinimidyl suberate (74 mg, 0.2 mmol, 4 eq.), in pyridine (1 mL). Reaction mixture was stirred at room temperature. After 3 hrs ether (20 mL) was added. Precipitate was collected, washed with additional amount of ether. Reddish solid was suspended in 30% MeOH/CH2Cl2, filtered trough a pad of silica gel with 30% MeOH/CH2Cl2 as an eluent. Compound 17 was obtained as white solid, 20 mg (29% yield). ES-MS m/z 1388.5 [M−H]−
Example 24—In Vivo Efficacy of mcMMAF Antibody-Drug Conjugates
Efficacy of cAC10-mcMMAF in Karpas-299 ALCL Xenografts:
To evaluate the in vivo efficacy of cAC10-mcMMAF with an average of 4 drug moieties per antibody (cAC10-mcF4), Karpas-299 human ALCL cells were implanted subcutaneously into immunodeficient C.B-17 SCID mice (5×106 cells per mouse). Tumor volumes were calculated using the formula (0.5×L×W2) where L and W are the longer and shorter of two bidirectional measurements. When the average tumor volume in the study animals reached approximately 100 mm3 (range 48-162) the mice were divided into 3 groups (5 mice per group) and were either left untreated or were given a single intravenous injection through the tail vein of either 1 or 2 mg/kg cAC10-mcF4 (FIG. 1). The tumors in the untreated mice grew rapidly to an average volume of >1,000 mm3 within 7 days of the start of therapy. In contrast, all of the cAC10-mcF4 treated tumor showed rapid regression with ⅗ in the 1 mg/kg group and 5/5 in the 2 mg/kg group obtaining complete tumor response. While the tumor in one of the complete responders in the 2 mg/kg group did recur approximately 4 weeks later, there were no detectable tumors in the remaining ⅘ responders in this group and in the 3 complete responders in the 1 mg/kg group at 10 weeks post therapy.
Efficacy of cBR96-mcMMAF in L2987 NSCLC Xenografts:
cBR96 is a chimeric antibody that recognizes the LeY antigen. To evaluate the in vivo efficacy of cBR96-mcMMAF with 4 drugs per antibody (cBR96-mcF4) L2987 non-small cell lung cancer (NSCLC) tumor fragments were implanted into athymic nude mice. When the tumors averaged approximately 100 mm3 the mice were divided into 3 groups: untreated and 2 therapy groups. For therapy, as shown in FIG. 3a, mice were administered cBR96-mcF4 at either 3 or 10 mg/kg/injection every 4 days for a total of 4 injections (q4dx4). As shown in FIG. 3b, mice were administered cBR96-mcF4 or a non-binding control conjugate, cAC10-mcF4, at 10 mg/kg/injection every 4 days for a total of 4 injections (q4dx4). As shown in FIGS. 3a and 3b, BR96-mcF4 produced pronounced tumor growth delay compared to the controls.
FIG. 2 shows an in vivo, single dose, efficacy assay of cAC10-mcMMAF in subcutaneous L540CY. For this study there were 4 mice in the untreated group and 10 in each of the treatment groups.
Example 25—In Vitro Efficacy of MC-MMAF Antibody-Drug Conjugates
Activity of cAC10-Antibody-Drug Conjugates Against CD30+ Cell Lines.
FIGS. 4a and 16b show dose-response curves from a representative experiment where cultures of Karpas 299 (anaplastic large cell lymphoma) and L428 (Hodgkin's Lymphoma) were incubated with serially diluted cAC10-mcMMAF (FIG. 4a) or cAC10-vcMMAF (FIG. 4b) for 96 hours. The cultures were labeled for 4 hours with 50 μM resazurin [7-hydroxy-3H-phenoxazin-3-one 10-oxide] and the fluorescence measured. The data were reduced in GraphPad Prism version 4.00 using the 4-parameter dose-response curve fit procedure. IC50 values are defined as the concentration where growth is reduced 50% compared with untreated control cultures. Each concentration was tested in quadruplicate.
Activity of cBR96-Antibody-Drug Conjugates Against Ley+ Cell Lines.
FIGS. 5a and 5b show dose-response curves from a representative experiment where cultures of H3396 (breast carcinoma) and L2987 (non small cell lung carcinoma) were incubated with serially diluted cBR96-mcMMAF (FIG. 5a) or -vcMMAF (FIG. 5b) for 96 hours. The cultures were labeled for 4 hours with 50 μM resazurin and the fluorescence measured. The data were reduced in GraphPad Prism version 4.00 using the 4-parameter dose-response curve fit procedure. IC50 values are defined as the concentration where growth is reduced 50% compared with untreated control cultures. Each concentration is tested in quadruplicate.
Activity of c1F6-Antibody-Drug Conjugates Against CD70+ Renal Cell Carcinoma Cell Lines.
FIGS. 6a and 6b show dose-response curves from a representative experiment where cultures of Caki-1 and 786-0 cells were incubated with serially diluted c1F6-mcMMAF (FIG. 6a) or -vcMMAF (FIG. 6b) for 96 hours. The cultures were labeled for 4 hours with 50 μM resazurin and the fluorescence measured. The data were reduced in GraphPad Prism version 4.00 using the 4-parameter dose-response curve fit procedure. IC50 values are defined as the concentration where growth is reduced 50% compared with untreated control cultures. Each concentration is tested in quadruplicate.
Example 26—Purification of Trastuzumab
One vial containing 440 mg HERCEPTIN® (huMAb4D5-8, rhuMAb HER2, U.S. Pat. No. 5,821,337) antibody) was dissolved in 50 mL MES buffer (25 mM MES, 50 mM NaCl, pH 5.6) and loaded on a cation exchange column (Sepharose S, 15 cm×1.7 cm) that had been equilibrated in the same buffer. The column was then washed with the same buffer (5 column volumes). Trastuzumab was eluted by raising the NaCl concentration of the buffer to 200 mM. Fractions containing the antibody were pooled, diluted to 10 mg/mL, and dialyzed into a buffer containing 50 mm potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5.
Example 27—Preparation of Trastuzumab-MC-MMAE by Conjugation of Trastuzumab and MC-MMAE
Trastuzumab, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT). After incubation at 37° C. for about 30 minutes, the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of the absorbance at 412 nm. The reduced antibody dissolved in PBS is chilled on ice.
The drug linker reagent, maleimidocaproyl-monomethyl auristatin E (MMAE), i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and water at known concentration, and added to the chilled reduced antibody trastuzumab in PBS. After about one hour, an excess of maleimide is added to quench the reaction and cap any unreacted antibody thiol groups. The reaction mixture is concentrated by centrifugal ultrafiltration and trastuzumab-MC-MMAE is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 μm filters under sterile conditions, and frozen for storage.
Example 28—Preparation of Trastuzumab-MC-MMAF by Conjugation of Trastuzumab and MC-MMAF
Trastuzumab-MC-MMAF was prepared by conjugation of trastuzumab and MC-MMAF following the procedure of Example 27.
Example 29—Preparation of Trastuzumab-MC-Val-Cit-PAB-MMAE by Conjugation of Trastuzumab and MC-Val-Cit-PAB-MMAE
Trastuzumab-MC-val-cit-PAB-MMAE was prepared by conjugation of trastuzumab and MC-val-cit-PAB-MMAE following the procedure of Example 27.
Example 30—Preparation of Trastuzumab-MC-Val-Cit-PAB-MMAF by Conjugation of Trastuzumab and MC-Val-Cit-PAB-MMAF 9
Trastuzumab-MC-val-cit-PAB-MMAF was prepared by conjugation of trastuzumab and MC-val-cit-PAB-MMAF 9 following the procedure of Example 27.
Example 31—Rat Toxicity
The acute toxicity profile of free drugs and ADC was evaluated in adolescent Sprague-Dawley rats (75-125 gms each, Charles River Laboratories (Hollister, Calif.). Animals were injected on day 1, complete chemistry and hematology profiles were obtained at baseline, day 3 and day 5 and a complete necropsy was performed on day 5. Liver enzyme measurements was done on all animals and routine histology as performed on three random animals for each group for the following tissues: sternum, liver, kidney, thymus, spleen, large and small intestine. The experimental groups were as follows:
μg
MMAF/
MMAF/
N/
Group
Administered
mg/kg
m2
MAb
Sex
1
Vehicle
0
0
0
2/F
2
trastuzumab-MC-val-cit-
9.94
840
4.2
6/F
MMAF
3
trastuzumab-MC-val-cit-
24.90
2105
4.2
6/F
MMAF
4
trastuzumab-MC(Me)-val-
10.69
840
3.9
6/F
cit-PAB-MMAF
5
trastuzumab-MC(Me)-val-
26.78
2105
3.9
6/F
cit-PAB-MMAF
6
trastuzumab-MC-MMAF
10.17
840
4.1
6/F
7
trastuzumab-MC-MMAF
25.50
2105
4.1
6/F
8
trastuzumab-MC-val-cit-
21.85
2105
4.8
6/F
PAB-MMAF
For trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PAB-MMAF, the μg MMAF/m2 was calculated using 731.5 as the MW of MMAF and 145167 as the MW of Herceptin.
The body surface area was calculated as follows: [{(body weight in grams to 0.667 power)×11.8}/10000]. (Guidance for Industry and Reviewers, 2002).
The dose solutions were administered by a single intravenous bolus tail-vein injection on Study Day 1 at a dose volume of 10 mL/kg. Body weights of the animals were measured pre-dose on Study Day 1 and daily thereafter. Whole blood was collected into EDTA containing tubes for hematology analysis. Whole blood was collected into serum separator tubes for clinical chemistry analysis. Blood samples were collected pre-dose on Study Day −4, Study Day 3 and Study Day 5. Whole blood was also collected into sodium heparin containing tubes at necropsy and the plasma was frozen at −70° C. for possible later analysis. The following tissues were collected and placed in neutral buffered formalin at necropsy: liver, kidneys, heart, thymus, spleen, brain, sternum and sections of the GI tract, including stomach, large and small intestine. Sternum, small intestine, large intestine, liver, thymus, spleen and kidney were examined.
Liver associated serum enzyme levels at each timepoint were compared to a range (5th and 95th percentile) from normal female Sprague-Dawley rats. White blood cell and platelet counts at each timepoint were compared to a range (5th and 95th percentile) from normal female Sprague-Dawley rats.
High Dose Study in Normal Female Sprague-Dawley Rats:
Group 1:
Vehicle
Group 2:
trastuzumab-MC-MMAF, 52.24 mg/kg, 4210 μg/m2
Group 3:
trastuzumab-MC-MMAF, 68.25 mg/kg, 5500 μg/m2
Group 4:
trastuzumab-MC-MMAF, 86.00 mg/kg, 6930 μg/m2
Tissues from 11 animals were submitted for routine histology. These animals had been part of an acute dose-ranging toxicity study using a trastuzumab-MC-MMAF immunoconjugate. Animals were followed for 12 days following dosing.
Example 32—Cynomolgus Monkey Toxicity/Safety
Three groups of four (2 male, 2 female) naive Macaca fascicularis (cynomolgus monkey) were studied for trastuzumab-MC-vc-PAB-MMAE and trastuzumab-MC-vc-PAB-MMAF. Intravenous administration was conducted at days 1 and 22 of the studies.
Sample
Group
Dose
Vehicle
1
day 1
1M/1F
day 22
H-MC-vc-PAB-MMAE
2
180 μg/m2 (0.5 mg/kg) at day 1
2M/2F
1100 μg/m2 (3.0 mg/kg) at day 22
H-MC-vc-PAB-MMAE
3
550 μg/m2 (1.5 mg/kg) at day 8
2M/2F
550 μg/m2 (1.5 mg/kg) at day 29
H-MC-vc-PAB-MMAE
4
880 μg/m2 (2.5 mg/kg) at day 15
2M/2F
880 μg/m2 (2.5 mg/kg) at day 36
Vehicle
1
day 1
1M/1F
day 22
H-MC-vc-PAB-MMAF
2
180 μg/m2 (0.5 mg/kg) at day 1
2M/2F
1100 μg/m2 (3.0 mg/kg) at day 22
H-MC-vc-PAB-MMAF
3
550 μg/m2 (1.5 mg/kg) at day 1
2M/2F
550 μg/m2 (1.5 mg/kg) at day 22
H-MC-vc-PAB-MMAF
4
880 μg/m2 (2.5 mg/kg) at day 1
2M/2F
880 μg/m2 (2.5 mg/kg) at day 22
H = trastuzumab
Dosing is expressed in surface area of an animal so as to be relevant to other species, i.e. dosage at μg/m2 is independent of species and thus comparable between species. Formulations of ADC contained PBS, 5.4 mM sodium phosphate, 4.2 mM potassium phosphate, 140 mM sodium chloride, pH 6.5.
Blood was collected for hematology analysis predose, and at 5 min., 6 hr, 10 hr, and 1, 3, 5, 7, 14, 21 days after each dose. Erythrocyte (RBC) and platelet (PLT) counts were measured by the light scattering method. Leukocyte (WBC) count was measured by the peroxidase/basophil method. Reticulocyte count was measured by the light scattering method with cationic dye. Cell counts were measured on an Advia 120 apparatus. ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were measured in U/L by UV/NADH; IFCC methodology on an Olympus AU400 apparatus, and using Total Ab ELISA—ECD/GxhuFc-HRP. Conj. Ab ELISA—MMAE/MMAFWECD-Bio/SA-HRP tests.
Example 33—Production, Characterization and Humanization of Anti-ErbB2 Monoclonal Antibody 4D5
The murine monoclonal antibody 4D5 which specifically binds the extracellular domain of ErbB2 was produced as described in Fendly et al. (1990) Cancer Research 50:1550-1558. Briefly, NIH 3T3/HER2-3400 cells (expressing approximately 1×105 ErbB2 molecules/cell) produced as described in Hudziak et al. Proc. Natl. Acad. Sci. (USA) 84:7158-7163 (1987) were harvested with phosphate buffered saline (PBS) containing 25 mM EDTA and used to immunize BALB/c mice. The mice were given injections i.p. of 107 cells in 0.5 ml PBS on weeks 0, 2, 5 and 7. The mice with antisera that immunoprecipitated 32P-labeled ErbB2 were given i.p. injections of a wheat germ agglutinin-Sepharose (WGA) purified ErbB2 membrane extract on weeks 9 and 13. This was followed by an i.v. injection of 0.1 ml of the ErbB2 preparation and the splenocytes were fused with mouse myeloma line X63-Ag8.653. Hybridoma supernatants were screened for ErbB2-binding by ELISA and radioimmunoprecipitation.
Epitope Mapping and Characterization
The ErbB2 epitope bound by monoclonal antibody 4D5 was determined by competitive binding analysis (Fendly et al. Cancer Research 50:1550-1558 (1990)). Cross-blocking studies were done by direct fluorescence on intact cells using the PANDEX™ Screen Machine to quantitate fluorescence. The monoclonal antibody was conjugated with fluorescein isothiocyanate (FITC), using established procedures (Wofsy et al. Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi (eds.) San Francisco: W. J. Freeman Co. (1980)). Confluent monolayers of NIH 3T3/HER2-3400 cells were trypsinized, washed once, and resuspended at 1.75×106 cell/ml in cold PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN3. A final concentration of 1% latex particles (IDC, Portland, Oreg.) was added to reduce clogging of the PANDEX™ plate membranes. Cells in suspension, 20 and 20 μl of purified monoclonal antibodies (100 μg/ml to 0.1 μg/ml) were added to the PANDEX™ plate wells and incubated on ice for 30 minutes. A predetermined dilution of the FITC-labeled monoclonal antibody in 20 μl was added to each well, incubated for 30 minutes, washed, and the fluorescence was quantitated by the PANDEX™. Monoclonal antibodies were considered to share an epitope if each blocked binding of the other by 50% or greater in comparison to an irrelevant monoclonal antibody control. In this experiment, monoclonal antibody 4D5 was assigned epitope I (amino acid residues from about 529 to about 625, inclusive within the ErbB2 extracellular domain.
The growth inhibitory characteristics of monoclonal antibody 4D5 were evaluated using the breast tumor cell line, SK-BR-3 (see Hudziak et al. (1989)Molec. Cell. Biol. 9(3):1165-1172). Briefly, SK-BR-3 cells were detached by using 0.25% (vol/vol) trypsin and suspended in complete medium at a density of 4×105 cells per ml. Aliquots of 100 μl (4×104 cells) were plated into 96-well microdilution plates, the cells were allowed to adhere, and 100 μl of media alone or media containing monoclonal antibody (final concentration 5 μgimp was then added. After 72 hours, plates were washed twice with PBS (pH 7.5), stained with crystal violet (0.5% in methanol), and analyzed for relative cell proliferation as described in Sugarman et al. (1985) Science 230:943-945. Monoclonal antibody 4D5 inhibited SK-BR-3 relative cell proliferation by about 56%.
Monoclonal antibody 4D5 was also evaluated for its ability to inhibit HRG-stimulated tyrosine phosphorylation of proteins in the Mr 180,000 range from whole-cell lysates of MCF7 cells (Lewis et al. (1996) Cancer Research 56:1457-1465). MCF7 cells are reported to express all known ErbB receptors, but at relatively low levels. Since ErbB2, ErbB3, and ErbB4 have nearly identical molecular sizes, it is not possible to discern which protein is becoming tyrosine phosphorylated when whole-cell lysates are evaluated by Western blot analysis. However, these cells are ideal for HRG tyrosine phosphorylation assays because under the assay conditions used, in the absence of exogenously added HRG, they exhibit low to undetectable levels of tyrosine phosphorylation proteins in the Mr 180,000 range.
MCF7 cells were plated in 24-well plates and monoclonal antibodies to ErbB2 were added to each well and incubated for 30 minutes at room temperature; then rHRGβ1177-244 was added to each well to a final concentration of 0.2 nM, and the incubation was continued for 8 minutes. Media was carefully aspirated from each well, and reactions were stopped by the addition of 100 μl of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 μl) was electrophoresed on a 4-12% gradient gel (Novex) and then electrophoretically transferred to polyvinylidene difluoride membrane. Antiphosphotyrosine (4G10, from UBI, used at 1 μgimp immunoblots were developed, and the intensity of the predominant reactive band at Mr □180,000 was quantified by reflectance densitometry, as described previously (Holmes et al. (1992) Science 256:1205-1210; Sliwkowski et al. J. Biol. Chem. 269:14661-14665 (1994)).
Monoclonal antibody 4D5 significantly inhibited the generation of a HRG-induced tyrosine phosphorylation signal at Mr 180,000. In the absence of HRG, but was unable to stimulate tyrosine phosphorylation of proteins in the Mr 180,000 range. Also, this antibody does not cross-react with EGFR (Fendly et al. Cancer Research 50:1550-1558 (1990)), ErbB3, or ErbB4. Monoclonal antibody 4D5 was able to block HRG stimulation of tyrosine phosphorylation by □50%.
The growth inhibitory effect of monoclonal antibody 4D5 on MDA-MB-175 and SK-BR-3 cells in the presence or absence of exogenous rHRGβ1 was assessed (Schaefer et al. Oncogene 15:1385-1394 (1997)). ErbB2 levels in MDA-MB-175 cells are 4-6 times higher than the level found in normal breast epithelial cells and the ErbB2-ErbB4 receptor is constitutively tyrosine phosphorylated in MDA-MB-175 cells. Monoclonal antibody 4D5 was able to inhibit cell proliferation of MDA-MB-175 cells, both in the presence and absence of exogenous HRG. Inhibition of cell proliferation by 4D5 is dependent on the ErbB2 expression level (Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993)). A maximum inhibition of 66% in SK-BR-3 cells could be detected. However this effect could be overcome by exogenous HRG.
The murine monoclonal antibody 4D5 was humanized, using a “gene conversion mutagenesis” strategy, as described in U.S. Pat. No. 5,821,337, the entire disclosure of which is hereby expressly incorporated by reference. The humanized monoclonal antibody 4D5 used in the following experiments is designated huMAb4D5-8. This antibody is of IgG1 isotype.
REFERENCES CITED
The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.
All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
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16507839
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Apr 15th, 2014 12:00AM
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Apr 15th, 2011 12:00AM
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https://www.uspto.gov?id=US08697688-20140415
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Pyrrolobenzodiazepines used to treat proliferative diseases
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Pyrrolobenzodiazepine dimers I having a C2-C3 double bond and an aryl group at the C2 position on one monomer unit, and a C2-C3 double bond and either a conjugated double or triple bond at the C2 position or an alkyl group at the C2 position on the other monomer unit, and conjugates of these compounds.
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8697688
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1. A compound with the formula I:
wherein:
R2 is of formula III:
Where A is —C6H4—, X is NHRN, wherein RN is H, and Q1 is a single bond, and Q2 is a single bond;
R12 is selected from:
(iia) methyl;
(iib) cyclopropyl;
(iic)
wherein R21 and R22 are hydrogen and R23 is methyl;
(iid)
wherein R25b is H and R25a is phenyl; and
(iie)
wherein R24 is H;
R6 and R9 are H;
Where R is independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is OH or OR;
R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound;
R″ os a C3 alkylene group;
Y and Y′ are O;
R6, R7′, R9′ are selected from the same groups as R6, R7, and R9 respectively and R10′ and R11′ are the same as R10 and R11.
2. A compound according to claim 1, with the structure:
3. A compound according to claim 1, wherein R12 is methyl.
4. A compound according to claim 1, wherein R12 is cyclopropyl.
5. A compound according to claim 1, wherein R12 is
and R21 and R22 are H and R23 is methyl.
6. A compound according to claim 1, wherein R12 is
and R25a is phenyl and R25b is H.
7. A compound according to claim 1, wherein R12 is
and R24 is H.
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7
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CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2011/032668, filed on Apr. 15, 2011, which claims priority to U.S. Provisional Patent Application No. 61/324,453, filed on Apr. 15, 2010. These applications are incorporated herein by reference in their entireties.
The present invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepine dimers having a C2-C3 double bond and an aryl group at the C2 position on one monomer unit, and a C2-C3 double bond and either a conjugated double or triple bond at the C2 position or an alkyl group at the C2 position on the other monomer unit.
BACKGROUND TO THE INVENTION
Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:
They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDeventer, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents.
It has been previously disclosed that the biological activity of this molecules can be potentiated by joining two PBD units together through their C8/C′-hydroxyl functionalities via a flexible alkylene linker (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, D. E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity. One example of a PBD dimmer, SG2000 (SJG-136):
has recently completed Phase I clinical trials in the oncology area and is about to enter Phase II (Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); Alley, M. C., et al., Cancer Research, 64, 6700-6706 (2004); Hartley, J. A., et al., Cancer Research, 64, 6693-6699 (2004)).
More recently, the present inventors have previously disclosed in WO 2005/085251, dimeric PBD compounds bearing C2 aryl substituents, such as SG2202 (ZC-207):
and in WO2006/111759, bisulphites of such PBD compounds, for example SG2285 (ZC-423):
These compounds have been shown to be highly useful cytotoxic agents (Howard, P. W., et al., Bioorg. Med. Chem. (2009), doi: 10.1016/j.bmc1.2009.09.012).
Due to the manner in which these highly potent compounds act in cross-linking DNA, these molecules have been made symmetrically. This provides for straightforward synthesis, either by constructing the PBD moieties simultaneously having already formed the dimer linkage, or by reacting already constructed PBD moieties with the dimer linking group.
Co-pending International Application PCT/GB2009/002498, filed 16 Oct. 2009, discloses unsymmetrical dimeric PBD compound bearing aryl groups in the C2 position of each monomer, where one of these groups bears a substituent designed to provide an anchor for linking the compound to another moiety.
DISCLOSURE OF THE INVENTION
The present inventors have developed further unsymmetrical dimeric PBD compounds bearing an aryl group in the C2 position of one monomer, said aryl group bearing a substituent designed to provide an anchor for linking the compound to another moiety, and either a unsaturated bond conjugated to the C2-C3 double bond or an alkyl group in the other monomer unit.
The present invention comprises a compound with the formula I:
wherein:
R2 is of formula II:
where A is a C5-7 aryl group, X is selected from the group comprising: OH, SH, CO2H, COH, N═C═O, NHNH2, CONHNH2,
NHRN, wherein RN is selected from the group comprising H and C1-4 alkyl, and either:
(i) Q1 is a single bond, and Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q1 is —CH═CH—, and Q2 is a single bond;
R12 is selected from:
(iia) C1-5 saturated aliphatic alkyl;
(iiib) C3-6 saturated cycloalkyl;
(iic)
wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;
(iid)
wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(iie)
where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo; either:
(a) R10 is H, and R11 is OH, ORA, where RA is C1-4 alkyl;
(b) R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or
(c) R10 is H and R11 is SOzM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;
R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y′ are selected from O, S, or NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and
R11′ are the same as R10 and R11, wherein if R11 and R11′ are SOzM, M may represent a divalent pharmaceutically acceptable cation.
A second aspect of the present invention provides the use of a compound of the first aspect of the invention in the manufacture of a medicament for treating a proliferative disease. The second aspect also provides a compound of the first aspect of the invention for use in the treatment of a proliferative disease.
One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.
A third aspect of the present invention comprises a compound of formula II:
wherein:
R2 is of formula II:
where A is a C5-7 aryl group, X is selected from the group comprising: OH, SH, CO2H, COH, N═C═O, NHNH2, CONHNH2,
NHRN, wherein RN is selected from the group comprising H and C1-4 alkyl, and either:
(i) Q1 is a single bond, and Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or
(ii) Q1 is —CH═CH—, and Q2 is a single bond;
R12 is selected from:
(iia) C1-5 saturated aliphatic alkyl;
(iib) C3-6 saturated cycloalkyl;
(iic)
10 wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;
(iid)
wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
(iie)
where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl: cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR′, nitro, Me3Sn and halo;
where R and R′ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C6-20 aryl groups;
R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR′, nitro, Me3Sn and halo; either:
(a) R10 is carbamate nitrogen protecting group, and R11 is O-ProtO, wherein ProtO is an oxygen protecting group;
(b) R10 is a hemi-aminal nitrogen protecting group and R11 is an oxo group;
R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;
Y and Y′ are selected from O, S, or NH;
R6′, R7′, R9′ are selected from the same groups as R6, R7 and R9 respectively and R10′ and R11′ are the same as R10 and R11.
A fourth aspect of the present invention comprises a method of making a compound of formula I from a compound of formula II by deprotection of the imine bond.
The unsymmetrical dimeric PBD compounds of the present invention are made by different strategies to those previously employed in making symmetrical dimeric PBD compounds. In particular, the present inventors have developed a method which involves adding each each C2 substituent to a symmetrical PBD dimer core in separate method steps. Accordingly, a fifth aspect of the present invention provides a method of making a compound of the first or third aspect of the invention, comprising at least one of the method steps set out below.
In a sixth aspect, the present invention relates to Conjugates comprising dimers of PBDs linked to a targeting agent, wherein a PBD is a dimer of formula I (supra).
In some embodiments, the Conjugates have the following formula III:
L-(LU-D)p (III)
wherein L is a Ligand unit (i.e., a targeting agent), LU is a Linker unit and D is a Drug unit comprising a PBD dimer. The subscript p is an integer of from 1 to 20. Accordingly, the Conjugates comprise a Ligand unit covalently linked to at least one Drug unit by a Linker unit. The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. Accordingly, the present invention also provides methods for the treatment of, for example, various cancers and autoimmune disease. These methods encompass the use of the Conjugates wherein the Ligand unit is a targeting agent that specifically binds to a target molecule. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.
The PBD dimer D is of formula I, except that X is selected from the group comprising: O, S, C(═O), C═, NH(C═O), NHNH, CONHNH,
NRN, wherein RN is selected from the group comprising H and C1-4 alkyl.
BRIEF DESCRIPTION OF FIGURE
FIG. 1 shows the effect of a conjugate of the invention on a tumour.
DEFINITIONS
Pharmaceutically Acceptable Cations
Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.
The pharmaceutically acceptable cation may be inorganic or organic.
Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca2+ and Mg2+. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
Substituents
The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.
Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
Examples of substituents are described in more detail below.
C1-12 alkyl: The term “C1-12 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C7).
Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7).
Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5).
C2-12 Alkenyl: The term “C2-12 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH═CH2), 1-propenyl (—CH═CH—CH3), 2-propenyl (allyl, —CH—CH═CH2), isopropenyl (1-methylvinyl, —C(CH3)═CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).
C2-12 alkynyl: The term “C2-12 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH2—C≡CH).
C3-12 cycloalkyl: The term “C3-12 cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
Examples of cycloalkyl groups include, but are not limited to, those derived from:
saturated monocyclic hydrocarbon compounds:
cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7);
unsaturated monocyclic hydrocarbon compounds:
cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and
saturated polycyclic hydrocarbon compounds:
norcarane (C7), norpinane (C7), norbornane (C7).
C3-20 heterocyclyl: The term “C3-20 heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
In this context, the prefixes (e.g. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:
N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);
S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3: trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);
N2O1: oxadiazine (C6);
O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1O1S1: oxathiazine (C6).
Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
C6-20 aryl: The term “C5-20 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.
In this context, the prefixes (e.g. C3-20, C5-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6 aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.
The ring atoms may be all carbon atoms, as in “carboaryl groups”.
Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).
Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (C16).
Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:
N1: pyrrole (azole) (C5), pyridine (azine) (C6);
O1: furan (oxole) (C5);
S1: thiophene (thiole) (C5);
N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);
N2O1: oxadiazole (furazan) (C5);
N3O1: oxatriazole (C5);
N1S1: thiazole (C5), isothiazole (C5);
N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6);
N3: triazole (C5), triazine (C6); and,
N4: tetrazole (C5).
Examples of heteroaryl which comprise fused rings, include, but are not limited to:
C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);
C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);
C11 (with 2 fused rings) derived from benzodiazepine (N2);
C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and,
C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene (O2), phenoxathiin (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).
The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.
Halo: —F, —Cl, —Br, and —I.
Hydroxy: —OH.
Ether: —OR, wherein R is an ether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7alkyl group.
Alkoxy: —OR, wherein R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).
Acetal: —CH(OR1)(OR2), wherein R1 and R2 are independently acetal substituents, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, or, in the case of a “cyclic” acetal group, R1 and R2, taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe)2, —CH(OEt)2, and —CH(OMe)(OEt).
Hemiacetal: —CH(OH)(OR1), wherein R1 is a hemiacetal substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).
Ketal: —CR(OR1)(OR2), where R1 and R2 are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe)2, —C(Me)(OEt)2, —C(Me)(OMe)(OEt), —C(Et)(OMe)2, —C(Et)(OEt)2, and —C(Et)(OMe)(OEt).
Hemiketal: —CR(OH)(OR1), where R1 is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).
Oxo (keto, -one): ═O.
Thione (thioketone): ═S.
Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.
Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.
Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C1-7 alkyl group (also referred to as C1-7alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH3 (acetyl), —C(═O)CH2CH3 (propionyl), —C(═O)C(CH3)3 (t-butyryl), and —C(═O)Ph (benzoyl, phenone).
Carboxy (carboxylic acid): —C(═O)OH.
Thiocarboxy (thiocarboxylic acid): —C(═S)SH.
Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.
Thionocarboxy (thionocarboxylic acid): —C(═S)OH.
Imidic acid: —C(═NH)OH.
Hydroxamic acid: —C(═NOH)OH.
Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH3, —C(═O)OCH2CH3, —C(═O)OC(CH3)3, and —C(═O)OPh.
Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH3 (acetoxy), —OC(═O)CH2CH3, —OC(═O)C(CH3)3, —OC(═O)Ph, and —OC(═O)CH2Ph.
Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH3, —OC(═O)CH2CH3, —OC(═O)OC(CH3)3, and —OC(═O)OPh.
Amino: —NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the case of a “cyclic” amino group, R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH2), secondary (—NHR1), or tertiary (—NHR1R2), and in cationic form, may be quaternary (—+NR1R2R3). Examples of amino groups include, but are not limited to, —NH2, —NHCH3, —NHC(CH3)2, —N(CH3)2, —N(CH2CH3)2, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH2, —C(═O)NHCH3, —C(═O)N(CH3)2, —C(═O)NHCH2CH3, and —C(═O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
Thioamido (thiocarbamyl): —C(═S)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH2, —C(═S)NHCH3, —C(═S)N(CH3)2, and —C(═S)NHCH2CH3.
Acylamido (acylamino): —NR1C(═O)R2, wherein R1 is an amide substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group, and R2 is an acyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH3, —NHC(═O)CH2CH3, and —NHC(═O)Ph. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
Aminocarbonyloxy: —OC(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH2, —OC(═O)NHMe, —OC(═O)NMe2, and —OC(═O)NEt2.
Ureido: —N(R1)CONR2R3 wherein R2 and R3 are independently amino substituents, as defined for amino groups, and R1 is a ureido substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH2, —NHCONHMe, —NHCONHEt, —NHCONMe2, —NHCONEt2, —NMeCONH2, —NMeCONHMe, —NMeCONHEt, —NMeCONMe2, and —NMeCONEt2.
Guanidino: —NH—C(═NH)NH2.
Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,
Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt.
Amidine (amidino): —C(═NR)NR2, wherein each R is an amidine substituent, for example, hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH2, —C(═NH)NMe2, and —C(═NMe)NMe2.
Nitro: —NO2.
Nitroso: —NO.
Azido: —N3.
Cyano (nitrile, carbonitrile): —CN.
Isocyano: —NC.
Cyanato: —OCN.
Isocyanato: —NCO.
Thiocyano (thiocyanato): —SCN.
Isothiocyano (isothiocyanato): —NCS.
Sulfhydryl (thiol, mercapto): —SH.
Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7alkylthio group), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of C1-7 alkylthio groups include, but are not limited to, —SCH3 and —SCH2CH3.
Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group (also referred to herein as C1-7 alkyl disulfide). Examples of C1-7 alkyl disulfide groups include, but are not limited to, —SSCH3 and —SSCH2CH3.
Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH3 and —S(═O)CH2CH3.
Sulfone (sulfonyl): —S(═O)2R, wherein R is a sulfone substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group, including, for example, a fluorinated or perfluorinated C1-7 alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)2CH3 (methanesulfonyl, mesyl), —S(═O)2CF3 (triflyl), —S(═O)2CH2CH3 (esyl), —S(═O)2C4F9 (nonaflyl), —S(═O)2CH2CF3 (tresyl), —S(═O)2CH2CH2NH2 (tauryl), —S(═O)2Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).
Sulfinic acid (sulfino): —S(═O)OH, —SO2H.
Sulfonic acid (sulfo): —S(═O)2OH, —SO3H.
Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH3 (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH2CH3 (ethoxysulfinyl; ethyl sulfinate).
Sulfonate (sulfonic acid ester): —S(═O)2OR, wherein R is a sulfonate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)2OCH3 (methoxysulfonyl; methyl sulfonate) and —S(═O)2OCH2CH3 (ethoxysulfonyl; ethyl sulfonate).
Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group.
Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH3 and —OS(═O)CH2CH3.
Sulfonyloxy: —OS(═O)2R, wherein R is a sulfonyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group.
Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)2CH3 (mesylate) and —OS(═O)2CH2CH3 (esylate).
Sulfate: —OS(═O)2OR; wherein R is a sulfate substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)2OCH3 and —SO(═O)2OCH2CH3.
Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH2, —S(═O)NH(CH3), —S(═O)N(CH3)2, —S(═O)NH(CH2CH3), —S(═O)N(CH2CH3)2, and —S(═O)NHPh.
Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)2NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O)2NH2, —S(═O)2NH(CH3), —S(═O)2N(CH3)2, —S(═O)2NH(CH2CH3), —S(═O)2N(CH2CH3)2, and —S(═O)2NHPh.
Sulfamino: —NR1S(═O)2OH, wherein R1 is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)2OH and —N(CH3)S(═O)2OH.
Sulfonamino: —NR1S(═O)2R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)2CH3 and —N(CH3)S(═O)2C6H5.
Sulfinamino: —NR1S(═O)R, wherein R1 is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH3 and —N(CH3)S(═O)C6H5.
Phosphino (phosphine): —PR2, wherein R is a phosphino substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphino groups include, but are not limited to, —PH2, —P(CH3)2, —P(CH2CH3)2, —P(t-Bu)2, and —P(Ph)2.
Phospho: —P(═O)2.
Phosphinyl (phosphine oxide): —P(═O)R2, wherein R is a phosphinyl substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group or a C5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH3)2, —P(═O)(CH2CH3)2, —P(═O)(t-Bu)2, and —P(═O)(Ph)2.
Phosphonic acid (phosphono): —P(═O)(OH)2.
Phosphonate (phosphono ester): —P(═O)(OR)2, where R is a phosphonate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH3)2, —P(═O)(OCH2CH3)2, —P(═O)(O-t-Bu)2, and —P(═O)(OPh)2.
Phosphoric acid (phosphonooxy): —OP(═O)(OH)2.
Phosphate (phosphonooxy ester): —OP(═O)(OR)2, where R is a phosphate substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH3)2, —OP(═O)(OCH2CH3)2, —OP(═O)(O-t-Bu)2, and —OP(═O)(OPh)2.
Phosphorous acid: —OP(OH)2.
Phosphite: —OP(OR)2, where R is a phosphite substituent, for example, —H, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH3)2, —OP(OCH2CH3)2, —OP(O-t-Bu)2, and —OP(OPh)2.
Phosphoramidite: —OP(OR1)—NR22, where R1 and R2 are phosphoramidite substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH2CH3)—N(CH3)2, —OP(OCH2CH3)—N(i-Pr)2, and —OP(OCH2CH2CN)—N(i-Pr)2.
Phosphoramidate: —OP(═O)(OR1)—NR22, where R1 and R2 are phosphoramidate substituents, for example, —H, a (optionally substituted) C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably —H, a C1-7 alkyl group, or a C5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH2CH3)—N(CH3)2, —OP(═O)(OCH2CH3)—N(i-Pr)2, and —OP(═O)(OCH2CH2CN)—N(i-Pr)2.
Alkylene
C3-12 alkylene: The term “C3-12 alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
Examples of linear saturated C3-12 alkylene groups include, but are not limited to, —(CH2)n— where n is an integer from 3 to 12, for example, —CH2CH2CH2— (propylene), —CH2CH2CH2CH2— (butylene), —CH2CH2CH2CH2CH2— (pentylene) and —CH2CH2CH2CH—2CH2CH2CH2— (heptylene).
Examples of branched saturated C3-12 alkylene groups include, but are not limited to, —CH(CH3)CH2—, —CH(CH3)CH2CH2—, —CH(CH3)CH2CH2CH2—, —CH2CH(CH3)CH2—, —CH2CH(CH3)CH2CH2—, —CH(CH2CH3)—, —CH(CH2CH3)CH2—, and —CH2CH(CH2CH3)CH2—.
Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH2—, —CH2—CH═CH2—, —CH═CH—CH2—CH2—, —CH═CH—CH2—CH2—CH2—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH2—, —CH═CH—CH═CH—CH2—CH2—, —CH═CH—CH2—CH═CH—, —CH═CH—CH2—CH2—CH═CH—, and —CH2—C≡C—CH2—.
Examples of branched partially unsaturated C3-12 alkylene groups (C3-12alkenylene and alkynylene groups) include, but are not limited to, —C(CH3)═CH—, —C(CH3)═CH—CH2—, —CH═CH—CH(CH3)— and —C≡C—CH(CH3)—.
Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).
Examples of alicyclic partially unsaturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).
Oxygen protecting group: the term “oxygen protecting group” refers to a moiety which masks a hydroxy group, and these are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. Classes of particular interest include silyl ethers (e.g. TMS, TBDMS), substituted methyl ethers (e.g. THP) and esters (e.g. acetate).
Carbamate nitrogen protecting group: the term “carbamate nitrogen protecting group” pertains to a moiety which masks the nitrogen in the imine bond, and these are well known in the art. These groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogen protecting group” pertains to a group having the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Conjugates
The present invention provides Conjugates comprising a PBD dimer connected to a Ligand unit via a Linker Unit. In one embodiment, the Linker unit includes a Stretcher unit (A), a Specificity unit (L1), and a Spacer unit (L2). The Linker unit is connected at one end to the Ligand unit and at the other end to the PBD dimer compound.
In one aspect, such a Conjugate is shown below in formula IIIa:
L-(A1a-L1s-L2y-D)p (IIIa)
wherein:
L is the Ligand unit; and
-A1a-L1s-L2y- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit,
a is 1 or 2,
L1- is a Specificity unit,
s is an integer ranging from 1 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is an PBD dimer; and
p is from 1 to 20.
In another aspect, such a Conjugate is shown below in formula IIIb:
Also illustrated as:
L-(A1a-L2y(-L1s)-D)p (Ib)
wherein:
L is the Ligand unit; and
-A1a-L1s(L2y)- is a Linker unit (LU), wherein:
-A1- is a Stretcher unit linked to a Stretcher unit (L2),
a is 1 or 2,
L1- is a Specificity unit linked to a Stretcher unit (L2),
s is an integer ranging from 0 to 12,
-L2- is a Spacer unit,
y is 0, 1 or 2;
-D is a PBD dimer; and
p is from 1 to 20.
Preferences
The following preferences may apply to all aspects of the invention as described above, or
may relate to a single aspect. The preferences may be combined together in any combination.
In one embodiment, the Conjugate has the formula:
L-(A1a-L1s-L2y-D)p
wherein L, A1, a, L1, s, L2, D and p are as described above.
In one embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit, which is a covalent bond, a self-immolative group or together with —OC(═O)— forms a self-immolative group, and L2 optional.
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
CBA-A1a-L1s-L2y-*
where the asterisk indicates the point of attachment to the Drug unit (D), CBA is the Cell Binding Agent, L1 is a Specificity unit, A1 is a Stretcher unit connecting L1 to the Cell Binding Agent, L2 is a Spacer unit which is a covalent bond or a self-immolative group, and a is 1 or 2, s is 0, 1 or 2, and y is 0 or 1 or 2.
In the embodiments illustrated above, L1 can be a cleavable Specificity unit, and may be referred to as a “trigger” that when cleaved activates a self-immolative group (or self-immolative groups) L2, when a self-immolative group(s) is present. When the Specificity unit L1 is cleaved, or the linkage (i.e., the covalent bond) between L1 and L2 is cleaved, the self-immolative group releases the Drug unit (D).
In another embodiment, the Ligand unit (L) is a Cell Binding Agent (CBA) that specifically binds to a target molecule on the surface of a target cell. An exemplary formula is illustrated below:
where the asterisk indicates the point of attachment to the Drug (D), CBA is the Cell Binding Agent, L1 is a Specificity unit connected to L2, A1 is a Stretcher unit connecting L2 to the Cell Binding Agent, L2 is a self-immolative group, and a is 1 or 2, s is 1 or 2, and y is 1 or 2.
In the various embodiments discussed herein, the nature of L1 and L2 can vary widely. These groups are chosen on the basis of their characteristics, which may be dictated in part, by the conditions at the site to which the conjugate is delivered. Where the Specificity unit L1 is cleavable, the structure and/or sequence of L1 is selected such that it is cleaved by the action of enzymes present at the target site (e.g., the target cell). L1 units that are cleavable by changes in pH (e.g. acid or base labile), temperature or upon irradiation (e.g. photolabile) may also be used. L1 units that are cleavable under reducing or oxidising conditions may also find use in the Conjugates.
In some embodiments, L1 may comprise one amino acid or a contiguous sequence of amino acids. The amino acid sequence may be the target substrate for an enzyme.
In one embodiment, L1 is cleavable by the action of an enzyme. In one embodiment, the enzyme is an esterase or a peptidase. For example, L1 may be cleaved by a lysosomal protease, such as a cathepsin.
In one embodiment, L2 is present and together with —C(═O)O— forms a self-immolative group or self-immolative groups. In some embodiments, —C(═O)O— also is a self-immolative group.
In one embodiment, where L1 is cleavable by the action of an enzyme and L2 is present, the enzyme cleaves the bond between L1 and L2, whereby the self-immolative group(s) release the Drug unit.
L1 and L2, where present, may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH, and
—O— (a glycosidic bond).
An amino group of L1 that connects to L2 may be the N-terminus of an amino acid or may be derived from an amino group of an amino acid side chain, for example a lysine amino acid side chain.
A carboxyl group of L1 that connects to L2 may be the C-terminus of an amino acid or may be derived from a carboxyl group of an amino acid side chain, for example a glutamic acid amino acid side chain.
A hydroxy group of L1 that connects to L2 may be derived from a hydroxy group of an amino acid side chain, for example a serine amino acid side chain.
In one embodiment, —C(═O)O— and L2 together form the group:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to the L1, Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0 to 3. The phenylene ring is optionally substituted with one, two or three substituents as described herein.
In one embodiment, Y is NH.
In one embodiment, n is 0 or 1. Preferably, n is 0.
Where Y is NH and n is 0, the self-immolative group may be referred to as a p-aminobenzylcarbonyl linker (PABC).
The self-immolative group will allow for release of the Drug unit (i.e., the asymmetric PBD) when a remote site in the linker is activated, proceeding along the lines shown below (for n=0):
where the asterisk indicates the attachment to the Drug, L* is the activated form of the remaining portion of the linker and the released Drug unit is not shown. These groups have the advantage of separating the site of activation from the Drug.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above. Each phenylene ring is optionally substituted with one, two or three substituents as described herein. In one embodiment, the phenylene ring having the Y substituent is optionally substituted and the phenylene ring not having the Y substituent is unsubstituted.
In another embodiment, —C(═O)O— and L2 together form a group selected from:
where the asterisk, the wavy line, Y, and n are as defined above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or CR.
In one embodiment, D is N.
In one embodiment, D is CH.
In one embodiment, E is O or S.
In one embodiment, F is CH.
In a preferred embodiment, the covalent bond between L1 and L2 is a cathepsin labile (e.g., cleavable) bond.
In one embodiment, L1 comprises a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is -Phe-Lys-, Val-Cit or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described by Dubowchik et al., which is incorporated herein by reference.
In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective groups in Organic Synthesis, Greene and Wuts.
Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:
Arg: Z, Mtr, Tos;
Asn: Trt, Xan;
Asp: Bzl, t-Bu;
Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;
Glu: Bzl, t-Bu;
Gln: Trt, Xan;
His: Boc, Dnp, Tos, Trt;
Lys: Boc, Z—Cl, Fmoc, Z;
Ser: Bzl, TBDMS, TBDPS;
Thr: Bz;
Trp: Boc;
Tyr: Bzl, Z, Z—Br.
In one embodiment, —X2— is connected indirectly to the Drug unit. In such an embodiment, the Spacer unit L2 is present.
In one embodiment, the dipeptide is used in combination with a self-immolative group(s) (the Spacer unit). The self-immolative group(s) may be connected to —X2—.
Where a self-immolative group is present, —X2— is connected directly to the self-immolative group. In one embodiment, —X2— is connected to the group Y of the self-immolative group. Preferably the group —X2—CO— is connected to Y, where Y is NH.
—X1— is connected directly to A1. In one embodiment, —X1— is connected directly to A1. Preferably the group NH—X1— (the amino terminus of X1) is connected to A1. A1 may comprise the functionality —CO— thereby to form an amide link with —X1—.
In one embodiment, L1 and L2 together with —OC(═O)— comprise the group —X1—X2-PABC-. The PABC group is connected directly to the Drug unit. In one example, the self-immolative group and the dipeptide together form the group -Phe-Lys-PABC-, which is illustrated below:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
Alternatively, the self-immolative group and the dipeptide together form the group -Val-Ala-PABC-, which is illustrated below:
where the asterisk and the wavy line are as defined above.
In another embodiment, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group, and E is a group that is susceptible to cleavage thereby to activate a self-immolative group.
E is selected such that the group is susceptible to cleavage, e.g., by light or by the action of an enzyme. E may be —NO2 or glucuronic acid (e.g., β-glucuronic acid). The former may be susceptible to the action of a nitroreductase, the latter to the action of a β-glucuronidase.
The group Y may be a covalent bond.
The group Y may be a functional group selected from:
—C(═O)—
—NH—
—O—
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH,
—C(═O)NHC(═O)—,
SO2, and
—S—.
The group Y is preferably —NH—, —CH2—, —O—, and —S—.
In some embodiments, L1 and L2 together with —OC(═O)— represent:
where the asterisk indicates the point of attachment to the Drug unit, the wavy line indicates the point of attachment to A, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—.
In some embodiments, L1 and L2 together represent:
where the asterisk indicates the point of attachment to the remainder of L2 or the Drug unit, the wavy line indicates the point of attachment to A1, Y is a covalent bond or a functional group and E is glucuronic acid (e.g., β-glucuronic acid). Y is preferably a functional group selected from —NH—, —CH2—, —O—, and —S—.
In some further embodiments, Y is a functional group as set forth above, the functional group is linked to an amino acid, and the amino acid is linked to the Stretcher unit A1. In some embodiments, amino acid is β-alanine. In such an embodiment, the amino acid is equivalently considered part of the Stretcher unit.
The Specificity unit L1 and the Ligand unit are indirectly connected via the Stretcher unit.
L1 and A1 may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the group A1 is:
where the asterisk indicates the point of attachment to L1, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the connection between the Ligand unit and A1 is through a thiol residue of the Ligand unit and a maleimide group of A1.
In one embodiment, the connection between the Ligand unit and A1 is:
where the asterisk indicates the point of attachment to the remaining portion of A1, L1, L2 or D, and the wavy line indicates the point of attachment to the remaining portion of the Ligand unit. In this embodiment, the S atom is typically derived from the Ligand unit.
In each of the embodiments above, an alternative functionality may be used in place of the malemide-derived group shown below:
where the wavy line indicates the point of attachment to the Ligand unit as before, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with the group:
where the wavy line indicates point of attachment to the Ligand unit, and the asterisk indicates the bond to the remaining portion of the A1 group, or to L1, L2 or D.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with a Ligand unit (e.g., a Cell Binding Agent), is selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—,
—NHC(═O)NH—,
—NHC(═O)NH,
—C(═O)NHC(═O)—,
—S—,
—S—S—,
—CH2C(═O)—
—C(═O)CH2—,
═N—NH—, and
—NH—N═.
In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the Ligand unit, is selected from:
where the wavy line indicates either the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group, and the asterisk indicates the other of the point of attachment to the Ligand unit or the bond to the remaining portion of the A1 group.
Other groups suitable for connecting L1 to the Cell Binding Agent are described in WO 2005/082023.
In one embodiment, the Stretcher unit A1 is present, the Specificity unit L1 is present and Spacer unit L2 is absent. Thus, L1 and the Drug unit are directly connected via a bond. Equivalently in this embodiment, L2 is a bond.
L1 and D may be connected by a bond selected from:
—C(═O)NH—,
—C(═O)O—,
—NHC(═O)—,
—OC(═O)—,
—OC(═O)O—,
—NHC(═O)O—,
—OC(═O)NH—, and
—NHC(═O)NH—.
In one embodiment, L1 and D are preferably connected by a bond selected from:
—C(═O)NH—, and
—NHC(═O)—.
In one embodiment, L1 comprises a dipeptide and one end of the dipeptide is linked to D. As described above, the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
In one embodiment, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-,
-Val-Cit-,
-Phe-Cit-,
-Leu-Cit-,
-Ile-Cit-,
-Phe-Arg-, and
-Trp-Cit-;
where Cit is citrulline. In such a dipeptide, —NH— is the amino group of X1, and CO is the carbonyl group of X2.
Preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is selected from:
-Phe-Lys-,
-Val-Ala-,
-Val-Lys-,
-Ala-Lys-, and
-Val-Cit-.
Most preferably, the group —X1—X2— in dipeptide, —NH—X1—X2—CO—, is -Phe-Lys- or -Val-Ala-.
Other dipeptide combinations of interest include:
-Gly-Gly-,
-Pro-Pro-, and
-Val-Glu-.
Other dipeptide combinations may be used, including those described above.
In one embodiment, L1-D is:
where —NH—X1—X2—CO is the dipeptide, —NH— is part of the Drug unit, the asterisk indicates the point of attachment to the remainder of the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of L1 or the point of attachment to A1. Preferably, the wavy line indicates the point of attachment to A1.
In one embodiment, the dipeptide is valine-alanine and L1-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is phenylalnine-lysine and L1-D is:
where the asterisk, —NH— and the wavy line are as defined above.
In one embodiment, the dipeptide is valine-citrulline.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most preferably 3 or 7.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups A1-L1 is:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the rest of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the group L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, S is a sulfur group of the Ligand unit, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to D, the wavy line indicates the point of attachment to the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the groups L-A1-L1 are:
where the asterisk indicates the point of attachment to L2 or D, the wavy line indicates the point of attachment to the remainder of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, the Stretcher unit is an acetamide unit, having the formula:
where the asterisk indicates the point of attachment to the remainder of the Stretcher unit, L1 or D, and the wavy line indicates the point of attachment to the Ligand unit.
In other embodiments, Linker-Drug compounds are provided for conjugation to a Ligand unit. In one embodiment, the Linker-Drug compounds are designed for connection to a Cell Binding Agent.
In one embodiment, the Drug Linker compound has the formula:
where the asterisk indicates the point of attachment to the Drug unit, G1 is a Stretcher group (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or together with —OC(═O)— forms a self-immolative group(s).
In another embodiment, the Drug Linker compound has the formula:
G1-L1-L2-*
where the asterisk indicates the point of attachment to the Drug unit, G1 is a Stretcher unit (A1) to form a connection to a Ligand unit, L1 is a Specificity unit, L2 (a Spacer unit) is a covalent bond or a self-immolative group(s).
L1 and L2 are as defined above. References to connection to A1 can be construed here as referring to a connection to G1.
In one embodiment, where L1 comprises an amino acid, the side chain of that amino acid may be protected. Any suitable protecting group may be used. In one embodiment, the side chain protecting groups are removable with other protecting groups in the compound, where present. In other embodiments, the protecting groups may be orthogonal to other protecting groups in the molecule, where present.
Suitable protecting groups for amino acid side chains include those groups described in the Novabiochem Catalog 2006/2007. Protecting groups for use in a cathepsin labile linker are also discussed in Dubowchik et al.
In certain embodiments of the invention, the group L1 includes a Lys amino acid residue.
The side chain of this amino acid may be protected with a Boc or Alloc protected group. A Boc protecting group is most preferred.
The functional group G1 forms a connecting group upon reaction with a Ligand unit (e.g., a cell binding agent.
In one embodiment, the functional group G1 is or comprises an amino, carboxylic acid, hydroxy, thiol, or maleimide group for reaction with an appropriate group on the Ligand unit. In a preferred embodiment, G1 comprises a maleimide group.
In one embodiment, the group G1 is an alkyl maleimide group. This group is suitable for reaction with thiol groups, particularly cysteine thiol groups, present in the cell binding agent, for example present in an antibody.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, L2 or D, and n is 0 to 6.
In one embodiment, n is 5.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably 4 or 8.
In one embodiment, the group G1 is:
where the asterisk indicates the point of attachment to L1, n is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8.
In each of the embodiments above, an alternative functionality may be used in place of the
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide-derived group is replaced with the group:
where the asterisk indicates the bond to the remaining portion of the G group.
In one embodiment, the maleimide group is replaced with a group selected from:
—C(═O)OH,
—OH,
—NH2,
—SH,
—C(═O)CH2X, where X is Cl, Br or I,
—CHO,
—NHNH2
—C≡CH, and
—N3 (azide).
In one embodiment, L1 is present, and G1 is —NH2, —NHMe, —COOH, —OH or —SH.
In one embodiment, where L1 is present, G1 is —NH2 or —NHMe. Either group may be the N-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is —NH2, and C is an amino acid sequence —X1—X2—, as defined above.
In one embodiment, L1 is present and G1 is COOH. This group may be the C-terminal of an L1 amino acid sequence.
In one embodiment, L1 is present and G1 is OH.
In one embodiment, L1 is present and G1 is SH.
The group G1 may be convertable from one functional group to another. In one embodiment, L1 is present and G1 is —NH2. This group is convertable to another group G1 comprising a maleimide group. For example, the group —NH2 may be reacted with an acids or an activated acid (e.g., N-succinimide forms) of those G1 groups comprising maleimide shown above.
The group G1 may therefore be converted to a functional group that is more appropriate for reaction with a Ligand unit.
As noted above, in one embodiment, L1 is present and G1 is —NH2, —NHMe, —COOH, —OH or —SH. In a further embodiment, these groups are provided in a chemically protected form. The chemically protected form is therefore a precursor to the linker that is provided with a functional group.
In one embodiment, G1 is —NH2 in a chemically protected form. The group may be protected with a carbamate protecting group. The carbamate protecting group may be selected from the group consisting of:
Alloc, Fmoc, Boc, Troc, Teoc, Cbz and PNZ.
Preferably, where G1 is —NH2, it is protected with an Alloc or Fmoc group.
In one embodiment, where G1 is —NH2, it is protected with an Fmoc group.
In one embodiment, the protecting group is the same as the carbamate protecting group of the capping group.
In one embodiment, the protecting group is not the same as the carbamate protecting group of the capping group. In this embodiment, it is preferred that the protecting group is removable under conditions that do not remove the carbamate protecting group of the capping group.
The chemical protecting group may be removed to provide a functional group to form a connection to a Ligand unit. Optionally, this functional group may then be converted to another functional group as described above.
In one embodiment, the active group is an amine. This amine is preferably the N-terminal amine of a peptide, and may be the N-terminal amine of the preferred dipeptides of the invention.
The active group may be reacted to yield the functional group that is intended to form a connection to a Ligand unit.
In other embodiments, the Linker unit is a precursor to the Linker uit having an active group. In this embodiment, the Linker unit comprises the active group, which is protected by way of a protecting group. The protecting group may be removed to provide the Linker unit having an active group.
Where the active group is an amine, the protecting group may be an amine protecting group, such as those described in Green and Wuts.
The protecting group is preferably orthogonal to other protecting groups, where present, in the Linker unit.
In one embodiment, the protecting group is orthogonal to the capping group. Thus, the active group protecting group is removable whilst retaining the capping group. In other embodiments, the protecting group and the capping group is removable under the same conditions as those used to remove the capping group.
In one embodiment, the Linker unit is:
where the asterisk indicates the point of attachment to the Drug unit, and the wavy line indicates the point of attachment to the remaining portion of the Linker unit, as applicable or the point of attachment to G1. Preferably, the wavy line indicates the point of attachment to G1.
In one embodiment, the Linker unit is:
where the asterisk and the wavy line are as defined above.
Other functional groups suitable for use in forming a connection between L1 and the Cell Binding Agent are described in WO 2005/082023.
Ligand Unit
The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non-peptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.
The terms “specifically binds” and “specific binding” refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1×107 M−1, and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non-peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).
In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; fully human antibody; or a single chain antibody. One embodiment the antibody is a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab′, F(ab′)2 and Fv fragments.
The antibody may be a diabody, a domain antibody (DAB) or a single chain antibody.
In one embodiment, the antibody is a monoclonal antibody.
Antibodies for use in the present invention include those antibodies described in WO 2005/082023 which is incorporated herein. Particularly preferred are those antibodies for tumour-associated antigens. Examples of those antigens known in the art include, but are not limited to, those tumour-associated antigens set out in WO 2005/082023. See, for instance, pages 41-55.
In some embodiments, the conjugates are designed to target tumour cells via their cell surface antigens. The antigens may be cell surface antigens which are either over-expressed or expressed at abnormal times or cell types. Preferably, the target antigen is expressed only on proliferative cells (preferably tumour cells); however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue.
Antibodies have been raised to target specific tumour related antigens including:
Cripto, CD19, CD20, CD22, CD30, CD33, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD56 (NCAM), CD70, CD79, CD138, PSCA, PSMA (prostate specific membrane antigen), BCMA, E-selectin, EphB2, Melanotransferin, Muc16 and TMEFF2.
The Ligand unit is connected to the Linker unit. In one embodiment, the Ligand unit is connected to A, where present, of the Linker unit.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a thioether bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through a disulfide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an amide bond.
In one embodiment, the connection between the Ligand unit and the Linker unit is through an ester bond.
In one embodiment, the connection between the Ligand unit and the Linker is formed between a thiol group of a cysteine residue of the Ligand unit and a maleimide group of the Linker unit.
The cysteine residues of the Ligand unit may be available for reaction with the functional group of the Linker unit to form a connection. In other embodiments, for example where the Ligand unit is an antibody, the thiol groups of the antibody may participate in interchain disulfide bonds. These interchain bonds may be converted to free thiol groups by e.g. treatment of the antibody with DTT prior to reaction with the functional group of the Linker unit.
In some embodiments, the cysteine residue is an introduced into the heavy or light chain of an antibody. Positions for cysteine insertion by substitution in antibody heavy or light chains include those described in Published U.S. Application No. 2007-0092940 and International Patent Publication WO2008070593, which are incorporated herein.
Methods of Treatment
The compounds of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a compound of formula I. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
A compound may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a compound of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
The Compounds and Conjugates can be used to treat proliferative disease and autoimmune disease. The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.
In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmunie disorder is a T cell-mediated immunological disorder.
In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose.
Includes Other Forms
Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO−), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N+HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O−), a salt or solvate thereof, as well as conventional protected forms.
Salts
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).
For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO−), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations such as Al+3. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
If the compound is cationic, or has a functional group which may be cationic (e.g. —NH2 may be —NH3+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
Solvates
It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
Carbinolamines
The invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (RAOH, where RA is C1-4 alkyl):
These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.
These particular compounds may be isolated in solid form, for example, by lyophilisation.
Isomers
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R—, S—, and meso-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).
Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like.
Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
General Synthetic Routes
The synthesis of PBD compounds is extensively discussed in the following references, which discussions are incorporated herein by reference:
a) WO 00/12508 (pages 14 to 30);
b) WO 2005/023814 (pages 3 to 10);
c) WO 2004/043963 (pages 28 to 29); and
d) WO 2005/085251 (pages 30 to 39).
Synthesis Route
The compounds of the present invention, where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, can be synthesised from a compound of Formula 2:
where R2, R6, R7, R9, R6′, R7′, R9′, R12, X, X′ and R″ are as defined for compounds of formula I, ProtN is a nitrogen protecting group for synthesis and ProtO is a protected oxygen group for synthesis or an oxo group, by deprotecting the imine bond by standard methods.
The compound produced may be in its carbinolamine or carbinolamine ether form depending on the solvents used. For example if ProtN is Alloc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried using palladium to remove the N10 protecting group, followed by the elimination of the oxygen protecting group for synthesis. If ProtN is Troc and ProtO is an oxygen protecting group for synthesis, then the deprotection is carried out using a Cd/Pb couple to yield the compound of formula (I). If ProtN is SEM, or an analogous group, and ProtO is an oxo group, then the oxo group can be removed by reduction, which leads to a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group, followed by the elimination of water. The reduction of the compound of Formula 2 can be accomplished by, for example, lithium tetraborohydride, whilst a suitable means for removing the SEM protecting group is treatment with silica gel.
Compounds of formula 2 can be synthesised from a compound of formula 3a:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of formula 2, by coupling an organometallic derivative comprising R12, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of formula 2 can be synthesised from a compound of formula 3b:
where R12, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of formula 2, by coupling an organometallic derivative comprising R2, such as an organoboron derivative. The organoboron derivative may be a boronate or boronic acid.
Compounds of formulae 3a and 3b can be synthesised from a compound of formula 4:
where R2, R6, R7, R9, R6′, R7′, R9′, X, X′ and R″ are as defined for compounds of formula 2, by coupling about a single equivalent (e.g. 0.9 or 1 to 1.1 or 1.2) of an organometallic derivative, such as an organoboron derivative, comprising R2 or R12.
The couplings described above are usually carried out in the presence of a palladium catalyst, for example Pd(PPh3)4, Pd(OCOCH3)2, PdCl2, Pd2(dba)3. The coupling may be carried out under standard conditions, or may also be carried out under microwave conditions.
The two coupling steps are usually carried out sequentially. They may be carried out with or without purification between the two steps. If no purification is carried out, then the two steps may be carried out in the same reaction vessel. Purification is usually required after the second coupling step. Purification of the compound from the undesired by-products may be carried out by column chromatography or ion-exchange separation.
The synthesis of compounds of formula 4 where ProtO is an oxo group and ProtN is SEM are described in detail in WO 00/12508, which is incorporated herein by reference. In particular, reference is made to scheme 7 on page 24, where the above compound is designated as intermediate P. This method of synthesis is also described in WO 2004/043963.
The synthesis of compounds of formula 4 where ProtO is a protected oxygen group for synthesis are described in WO 2005/085251, which synthesis is herein incorporated by reference.
Compounds of formula I where R10 and R10′ are H and R11 and R11′ are SOzM, can be synthesised from compounds of formula I where R10 and R11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, by the addition of the appropriate bisulphite salt or sulphinate salt, followed by an appropriate purification step. Further methods are described in GB 2 053 894, which is herein incorporated by reference.
Nitrogen Protecting Groups for Synthesis
Nitrogen protecting groups for synthesis are well known in the art. In the present invention, the protecting groups of particular interest are carbamate nitrogen protecting groups and hemi-aminal nitrogen protecting groups.
Carbamate nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Particularly preferred protecting groups include Troc, Teoc, Fmoc, BOC, Doc, Hoc, TcBOC, 1-Adoc and 2-Adoc.
Other possible groups are nitrobenzyloxycarbonyl (e.g. 4-nitrobenzyloxycarbonyl) and 2-(phenylsulphonyl)ethoxycarbonyl.
Those protecting groups which can be removed with palladium catalysis are not preferred, e.g. Alloc.
Hemi-aminal nitrogen protecting groups have the following structure:
wherein R′10 is R as defined above. A large number of suitable groups are described on pages 633 to 647 as amide protecting groups of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference. The groups disclosed herein can be applied to compounds of the present invention. Such groups include, but are not limited to, SEM, MOM, MTM, MEM, BOM, nitro or methoxy substituted BOM, Cl3CCH2OCH2—.
Protected Oxygen Group for Synthesis
Protected oxygen group for synthesis are well known in the art. A large number of suitable oxygen protecting groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.
Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, acetates, benzoates, carbonates, and sulfonates.
Preferred oxygen protecting groups include acetates, TBS and THP.
Synthesis of Drug Conjugates
Conjugates can be prepared as previously described. Linkers having a maleimidyl group (A), a peptide group (L1) and self-immolative group (L2) can be prepared as described in U.S. Pat. No. 6,214,345. Linkers having a maleimidyl group (A) and a peptide group (L1) can be prepared as described in WO 2009-0117531. Other linkers can be prepared according to the references cited herein or as known to the skilled artisan.
Linker-Drug compounds can be prepared according to methods known in the art. Linkage of amine-based X substituents (of the PDB dimer Drug unit) to active groups of the Linker units can be performed according to methods generally described in U.S. Pat. Nos. 6,214,345 and 7,498,298; and WO 2009-0117531, or as otherwise known to the skilled artisan.
Antibodies can be conjugated to Linker-Drug compounds as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37° C. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved. The reduced antibody is then cooled to 0° C. and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 hour, the reaction is quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the levels of remaining NAC-quenched drug-linker.
Further Preferences
The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.
In some embodiments, R6′, R7′, R9′, R10′, R11′ and Y′ are preferably the same as R6, R7, R9, R10, R11 and Y respectively.
Dimer Link
Y and Y′ are preferably O.
R″ is preferably a C3-7 alkylene group with no substituents. More preferably R″ is a C3, C5 or C7 alkylene. Most preferably, R″ is a C3 or C5 alkylene.
R6 to R9
R9 is preferably H.
R6 is preferably selected from H, OH, OR, SH, NH2, nitro and halo, and is more preferably H or halo, and most preferably is H.
R7 is preferably selected from H, OH, OR, SH, SR, NH2, NHR, NRR′, and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C1-7 alkyl, C3-10 heterocyclyl and C5-10 aryl groups. R may be more preferably a C1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH2Ph. Other substituents of particular interest are dimethylamino (i.e. —NMe2); —(OC2H4)qOMe, where q is from 0 to 2; nitrogen-containing C6 heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl.
These preferences apply to R9′, R6′ and R7′ respectively.
R2
A in R2 may be phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, A is preferably phenyl.
X is a group selected from the list comprising: OH, SH, CO2H, COH, N═C═O, NHNH2, CONHNH2,
and NHRN, wherein RN is selected from the group comprising H and C1 alkyl. X may preferably be: OH, SH, CO2H, —N═C═O or NHRN, and may more preferably be: OH, SH, CO2H, —N═C═O or NH2. Particularly preferred groups include: OH, SH and NH2, with NH2 being the most preferred group.
Q2-X may be on any of the available ring atoms of the C5-7 aryl group, but is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group (A) is phenyl, the substituent (Q2-X) is preferably in the meta- or para-positions, and more preferably is in the para-position.
In some embodiments, Q1 is a single bond. In these embodiments, Q2 is selected from a single bond and —Z—(CH2)n—, where Z is selected from a single bond, O, S and NH and is from 1 to 3. In some of these embodiments, Q2 is a single bond. In other embodiments, Q2 is —Z—(CH2)n—. In these embodiments, Z may be O or S and n may be 1 or n may be 2. In other of these embodiments, Z may be a single bond and n may be 1.
In other embodiments, Q1 is —CH═CH—.
In some embodiments, R2 may be -A-CH2—X and -A-X. In these embodiments, X may be OH, SH, CO2H, COH and NH2. In particularly preferred embodiments, X may be NH2.
R12
R12 is selected from:
(a) C1-5 saturated aliphatic alkyl;
(b) C3-6 saturated cycloalkyl;
(c)
wherein each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5;
(d)
wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and
(e)
where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
When R12 is C1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
When R12 is C3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
When R12 is
each of R21, R22 and R23 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R12 group is no more than 5. In some embodiments, the total number of carbon atoms in the R12 group is no more than 4 or no more than 3.
In some embodiments, one of R21, R22 and R23 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
In other embodiments, two of R21, R22 and R23 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that re not H are methyl.
In some embodiments, R21 is H.
In some embodiments, R22 is H.
In some embodiments, R23 is H.
In some embodiments, R21 and R22 are H.
In some embodiments, R21 and R23 are H.
In some embodiments, R22 and R23 are H.
When R12 is
one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
When R12 is
R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
In some embodiments, R24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R24 is selected from H and methyl.
M and z
It is preferred that M and M′ are monovalent pharmaceutically acceptable cations, and are more preferably Na+.
z is preferably 3.
Particularly preferred compounds of the present invention are of formula Ia:
where R12a is selected from:
and
the amino group is at either the meta or para positions of the phenyl group.
3rd Aspect
The preferences expressed above for the first aspect may apply to the compounds of this aspect, where appropriate.
When R10 is carbamate nitrogen protecting group, it may preferably be Teoc, Fmoc and Troc, and may more preferably be Troc.
When R11 is O-ProtO, wherein ProtO is an oxygen protecting group, ProtO may preferably be TBS or THP, and may more preferably be TBS.
When R10 is a hemi-aminal nitrogen protecting group, it may preferably be MOM, BOM or SEM, and may more preferably be SEM.
The preferences for compounds of formula I apply as appropriate to D in the sixth aspect of the invention.
EXAMPLES
General Experimental Methods
Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. 1H and 13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt (NovaBiochem) and solid-supported reagents (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied without further purification. Anhydrous solvents were prepared by distillation under a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C.
Compound 1b was synthesised as described in WO 00/012508 (compound 210), which is herein incorporated by reference.
General LC/MS conditions: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then 5% B to 95% B within 3 min. The composition was held for 0.5 min at 95% B, and then returned to 5% B in 0.3 minutes. Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm
LC/MS conditions specific for compounds protected by both a Troc and a TBDMs group: Chromatographic separation of Troc and TBDMS protected compounds was performed on a Waters Alliance 2695 HPLC system utilizing a Onyx Monolitic reversed-phase column (3 μm particles, 50×4.6 mm) from Phenomenex Corp. Mobile-phase A consisted of 5% acetonitrile—95% water containing 0.1% formic acid, and mobile phase B consisted of 95% acetonitrile—5% water containing 0.1% formic acid. After 1 min at 5% B, the proportion of B was raised to 95% B over the next 2.5 min and maintained at 95% B for a further 1 min, before returning to 95% A in 10 s and re-equilibration for a further 50 sec, giving a total run time of 5.0 min. The flow rate was maintained at 3.0 mL/min.
LC/MS conditions for Example 4: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B for 2.0 min rising to 50% B over 3 min. The composition was held for 1 min at 50% B, before rising to 95% B over 1 minute. The gradient composition then dropped to 5% B over 2.5 minutes and was held at this percentage for 0.5 minutes. Total gradient run time equals 10 min. Flow rate 1.5 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm
Synthesis of Key Intermediates
(a) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2a)
Method A: A catalytic amount of DMF (2 drops) was added to a stirred solution of the nitro-acid 1a (1.0 g, 2.15 mmol) and oxalyl chloride (0.95 mL, 1.36 g, 10.7 mmol) in dry THF (20 mL). The reaction mixture was allowed to stir for 16 hours at room temperature and the solvent was removed by evaporation in vacuo. The resulting residue was re-dissolved in dry THF (20 mL) and the acid chloride solution was added dropwise to a stirred mixture of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (859 mg, 4.73 mmol) and TEA (6.6 mL, 4.79 g, 47.3 mmol) in THF (10 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for a further 3 hours after which time TLC (95:5 v/v CHCl3/MeOH) and LC/MS (2.45 min (ES+) m/z (relative intensity) 721 ([M+H]+., 20)) revealed formation of product. Excess THF was removed by rotary evaporation and the resulting residue was dissolved in DCM (50 mL). The organic layer was washed with 1N HCl (2×15 mL), saturated NaHCO3 (2×15 mL), H2O (20 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent gave the crude product as a dark coloured oil. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96:4 v/v CHCl3/MeOH) isolated the pure amide 2a as an orange coloured glass (840 mg, 54%).
Method B: Oxalyl chloride (9.75 mL, 14.2 g, 111 mmol) was added to a stirred suspension of the nitro-acid 1a (17.3 g, 37.1 mmol) and DMF (2 mL) in anhydrous DCM (200 mL). Following initial effervescence the reaction suspension became a solution and the mixture was allowed to stir at room temperature for 16 hours. Conversion to the acid chloride was confirmed by treating a sample of the reaction mixture with MeOH and the resulting bis-methyl ester was observed by LC/MS. The majority of solvent was removed by evaporation in vacuo, the resulting concentrated solution was re-dissolved in a minimum amount of dry DCM and triturated with diethyl ether. The resulting yellow precipitate was collected by filtration, washed with cold diethyl ether and dried for 1 hour in a vacuum oven at 40° C. The solid acid chloride was added portionwise over a period of 25 minutes to a stirred suspension of (2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (15.2 g, 84.0 mmol) and TEA (25.7 mL, 18.7 g, 185 mmol) in DCM (150 mL) at −40° C. (dry ice/CH3CN). Immediately, the reaction was complete as judged by LC/MS (2.47 min (ES+) m/z (relative intensity) 721 ([M+H]+., 100)), the mixture was diluted with DCM (150 mL) and washed with 1N HCl (300 mL), saturated NaHCO3 (300 mL), brine (300 mL), filtered (through a phase separator) and the solvent evaporated in vacuo to give the pure product 2a as an orange solid (21.8 g, 82%).
Analytical Data: [α]22D=−46.1° (c=0.47, CHCl3); 1H NMR (400 MHz, CDCl3) (rotamers) δ 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H, J=11.1 Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); 13C NMR (100 MHz, CDCl3) (rotamers) δ 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7, 108.2, 69.7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR, CHCl3) 3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274, 1211, 1177, 1072, 1050, 1008, 871 cm−1; MS (ES+) m/z (relative intensity) 721 ([M+H]+., 47), 388 (80); HRMS [M+H]+. theoretical C31H36N4O16 m/z 721.2199. found (ES+) m/z 721.2227.
(a) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate] (2b)
Preparation from 1b according to Method B gave the pure product as an orange foam (75.5 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 749 ([M+H]+., 100).
(b) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (3a)
Method A: A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) was added to a solution of the nitro-ester 2a (75 g, 104 mmol) in DMF (360 mL). The suspension was hydrogenated in a Parr hydrogenation apparatus over 8 hours. Progress of the reaction was monitored by LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H]+., 100), (ES−) m/z (relative intensity) 595 ([M+H]+., 100) after the hydrogen uptake had stopped. Solid Pd/C was removed by filtration and the filtrate was concentrated by rotary evaporation under vacuum (below 10 mbar) at 40° C. to afford a dark oil containing traces of DMF and residual charcoal. The residue was digested in EtOH (500 mL) at 40° C. on a water bath (rotary evaporator bath) and the resulting suspension was filtered through celite and washed with ethanol (500 mL) to give a clear filtrate. Hydrazine hydrate (10 mL, 321 mmol) was added to the solution and the reaction mixture was heated at reflux. After 20 minutes the formation of a white precipitate was observed and reflux was allowed to continue for a further 30 minutes. The mixture was allowed to cool down to room temperature and the precipitate was retrieved by filtration, washed with diethyl ether (2*1 volume of precipitate) and dried in a vacuum desiccator to provide 3a (50 g, 81%).
Method B: A solution of the nitro-ester 2a (6.80 g, 9.44 mmol) in MeOH (300 mL) was added to Raney™ nickel (4 large spatula ends of a ˜50% slurry in H2O) and anti-bumping granules in a 3-neck round bottomed flask. The mixture was heated at reflux and then treated dropwise with a solution of hydrazine hydrate (5.88 mL, 6.05 g, 188 mmol) in MeOH (50 mL) at which point vigorous effervescence was observed. When the addition was complete (˜30 minutes) additional Raney™ nickel was added carefully until effervescence had ceased and the initial yellow colour of the reaction mixture was discharged. The mixture was heated at reflux for a further 30 minutes at which point the reaction was deemed complete by TLC (90:10 v/v CHCl3/MeOH) and LC/MS (2.12 min (ES+) m/z (relative intensity) 597 ([M+H]+., 100)). The reaction mixture was allowed to cool to around 40° C. and then excess nickel removed by filtration through a sinter funnel without vacuum suction. The filtrate was reduced in volume by evaporation in vacuo at which point a colourless precipitate formed which was collected by filtration and dried in a vacuum desiccator to provide 3a (5.40 g, 96%).
Analytical Data: [α]27D=+404° (c=0.10, DMF); 1H NMR (400 MHz, DMSO-d6) δ 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H, J=3.98 Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H, J=12.1, 3.60 Hz), 3.43 (dd, 2H, J=12.0, 4.72 Hz), 2.67-2.57 (m, 2H), 2.26 (p, 2H, J=5.90 Hz), 1.99-1.89 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 164.0, 149.9, 144.5, 129.8, 117.1, 111.3, 104.5, 54.8, 54.4, 53.1, 33.5, 27.5; IR (ATR, neat) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263, 1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm−1; MS (ES+) m/z (relative intensity) 619 ([M+Na]+., 10), 597 ([M+H]+., 52), 445 (12), 326 (11); HRMS [M+H]+. theoretical C29H32N4O10 m/z 597.2191. found (ES+) m/z 597.2205.
(b) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4′-benzodiazepin-5,11-dione] (3b)
Preparation from 2b according to Method A gave the product as a white solid (22.1 g, 86%).
Analytical Data: MS (ES−) m/z (relative intensity) 623.3 ([M−H]−, 100);
(c) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4a)
TBSCl (317 mg, 2.1 mmol) and imidazole (342 mg, 5.03 mmol) were added to a cloudy solution of the tetralactam 3a (250 mg, 0.42 mmol) in anhydrous DMF (6 mL). The mixture was allowed to stir under a nitrogen atmosphere for 3 hours after which time the reaction was deemed complete as judged by LC/MS (3.90 min (ES+) m/z (relative intensity) 825 ([M+H]+., 100)). The reaction mixture was poured onto ice (˜25 mL) and allowed to warm to room temperature with stirring. The resulting white precipitate was collected by vacuum filtration, washed with H2O, diethyl ether and dried in the vacuum desiccator to provide pure 4a (252 mg, 73%).
Analytical Data: [α]23D=+234° (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H, J=5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73-3.63 (m, 4H), 2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H), 0.08 (s, 12H); 13C NMR (100 MHz, CDCl3) δ 170.4, 165.7, 151.4, 146.6, 129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56.3, 55.7, 54.2, 35.2, 28.7, 25.7, 18.0, −4.82 and −4.86; IR (ATR, CHCl3) 3235, 2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251, 1220, 1120, 1099, 1033 cm−1; MS (ES+) m/z (relative intensity) 825 ([M+H]+., 62), 721 (14), 440 (38); HRMS [M+H]+. theoretical C41H60N4O10Si2 m/z 825.3921. found (ES+) m/z 825.3948.
(c) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (4b)
Preparation from 3b according to the above method gave the product as a white solid (27.3 g, 93%).
Analytical Data: MS (ES+) m/z (relative intensity) 853.8 ([M+H]+., 100), (ES−) m/z (relative intensity) 851.6 ([M−H]−., 100.
(d) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (5a)
A solution of n-BuLi (4.17 mL of a 1.6 M solution in hexane, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise to a stirred suspension of the tetralactam 4a (2.20 g, 2.67 mmol) in anhydrous THF (30 mL) at −30° C. (dry ice/ethylene glycol) under a nitrogen atmosphere. The reaction mixture was allowed to stir at this temperature for 1 hour (now a reddish orange colour) at which point a solution of SEMCl (1.18 mL, 1.11 g, 6.67 mmol) in anhydrous THF (10 mL) was added dropwise. The reaction mixture was allowed to slowly warm to room temperature and was stirred for 16 hours under a nitrogen atmosphere. The reaction was deemed complete as judged by TLC (EtOAc) and LC/MS (4.77 min (ES+) m/z (relative intensity) 1085 ([M+H]+., 100)). The THF was removed by evaporation in vacuo and the resulting residue dissolved in EtOAc (60 mL), washed with H2O (20 mL), brine (20 mL), dried (MgSO4) filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (80:20 v/v Hexane/EtOAc) gave the pure N10-SEM-protected tetralactam 5a as an oil (2.37 g, 82%).
Analytical Data: [α]23D=+163° (c=0.41, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98 Hz), 4.68 (d, 2H, J=9.99 Hz), 4.57 (p, 2H, J=5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s, 6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81 Hz), 2.03-1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9, 78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1, −1.24, −4.73; IR (ATR, CHCl3) 2951, 1685, 1640, 1606, 1517, 1462, 1433, 1360, 1247, 1127, 1065 cm−1; MS (ES+) m/z (relative intensity) 1113 ([M+Na]+., 48), 1085 ([M+H]+., 100), 1009 (5), 813 (6); HRMS [M+H]+. theoretical C53H88N4O12Si4 m/z 1085.5548. found (ES+) m/z 1085.5542.
(d) 1,1′-[[(Pentane 1,5-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1, 4]-benzodiazepin-5,11-dione] (5b)
Preparation from 4b according to the above method gave the product as a pale orange foam (46.9 g, 100%), used without further purification.
Analytical Data: MS (ES+) m/z (relative intensity) 1114 ([M+H]+., 90), (ES−) m/z (relative intensity) 1158 ([M+2Na]−, 100).
(e) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6a)
A solution of TBAF (5.24 mL of a 1.0 M solution in THF, 5.24 mmol) was added to a stirred solution of the bis-silyl ether 5a (2.58 g, 2.38 mmol) in THF (40 mL) at room temperature. After stirring for 3.5 hours, analysis of the reaction mixture by TLC (95:5 v/v CHCl3/MeOH) revealed completion of reaction. The reaction mixture was poured into a solution of saturated NH4Cl (100 mL) and extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCl3 to 96:4 v/v CHCl3/MeOH) gave the pure tetralactam 6a as a white foam (1.78 g, 87%).
Analytical Data: [α]23D=+202° (c=0.34, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0 Hz), 4.72 (d, 2H, J=10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83 Hz), 4.20-4.16 (m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH), 2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77 Hz), 2.11-2.05 (m, 2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 169.5, 165.9, 151.3, 147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67.2, 65.2, 56.5, 56.2, 54.1, 35.2, 29.1, 18.4, −1.23; IR (ATR, CHCl3) 2956, 1684, 1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm−1; MS (ES+) m/z (relative intensity) 885 ([M+29]+., 70), 857 ([M+H]+., 100), 711 (8), 448 (17); HRMS [M+H]+. theoretical C41H60N4O12Si2 m/z 857.3819. found (ES+) m/z 857.3826.
(e) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (6b)
Preparation from 5b according to the above method gave the product as a white foam (15.02 g).
Analytical Data: MS (ES+) m/z (relative intensity) 886 ([M+H]+., 10), 739.6 (100), (ES−) m/z (relative intensity) 884 ([M−H]−., 40).
(f) 1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-[1-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione]] (7a)
Method A: A 0.37 M sodium hypochlorite solution (142.5 mL, 52.71 mmol, 2.4 eq) was added dropwise to a vigorously stirred mixture of the diol 6a (18.8 g, 21.96 mmol, 1 eq), TEMPO (0.069 g, 0.44 mmol, 0.02 eq) and 0.5 M potassium bromide solution (8.9 mL, 4.4 mmol, 0.2 eq) in DCM (115 mL) at 0° C. The temperature was maintained between 0° C. and 5° C. by adjusting the rate of addition. The resultant yellow emulsion was stirred at 0° C. to 5° C. for 1 hour. TLC (EtOAc) and LC/MS [3.53 min. (ES+) m/z (relative intensity) 875 ([M+Na]+., 50), (ES−) m/z (relative intensity) 852 ([M−H]−., 100)] indicated that reaction was complete.
The reaction mixture was filtered, the organic layer separated and the aqueous layer was backwashed with DCM (×2). The combined organic portions were washed with brine (×1), dried (MgSO4) and evaporated to give a yellow foam. Purification by flash column chromatography (gradient elution 35/65 v/v n-hexane/EtOAC, 30/70 to 25/75 v/v n-hexane/EtOAC) afforded the bis-ketone 7a as a white foam (14.1 g, 75%).
Sodium hypochlorite solution, reagent grade, available at chlorine 10-13%, was used. This was assumed to be 10% (10 g NaClO in 100 g) and calculated to be 1.34 M in NaClO. A stock solution was prepared from this by diluting it to 0.37 M with water. This gave a solution of approximately pH 14. The pH was adjusted to 9.3 to 9.4 by the addition of solid NaHCO3. An aliquot of this stock was then used so as to give 2.4 mol eq. for the reaction.
On addition of the bleach solution an initial increase in temperature was observed. The rate of addition was controlled, to maintain the temperature between 0° C. to 5° C. The reaction mixture formed a thick, lemon yellow coloured, emulsion.
The oxidation was an adaptation of the procedure described in Thomas Fey et al, J. Org. Chem., 2001, 66, 8154-8159.
Method B: Solid TCCA (10.6 g, 45.6 mmol) was added portionwise to a stirred solution of the alcohol 6a (18.05 g, 21.1 mmol) and TEMPO (123 mg, 0.78 mmol) in anhydrous DCM (700 mL) at 0° C. (ice/acetone). The reaction mixture was stirred at 0° C. under a nitrogen atmosphere for 15 minutes after which time TLC (EtOAc) and LC/MS [3.57 min (ES+) m/z (relative intensity) 875 ([M+Na]+., 50)] revealed completion of reaction. The reaction mixture was filtered through celite and the filtrate was washed with saturated aqueous NaHCO3 (400 mL), brine (400 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash column chromatography (80:20 v/v EtOAc/Hexane) afforded the bis-ketone 7a as a foam (11.7 g, 65%).
Method C: A solution of anhydrous DMSO (0.72 mL, 0.84 g, 10.5 mmol) in dry DCM (18 mL) was added dropwise over a period of 25 min to a stirred solution of oxalyl chloride (2.63 mL of a 2.0 M solution in DCM, 5.26 mmol) under a nitrogen atmosphere at −60° C. (liq N2/CHCl3). After stirring at −55° C. for 20 minutes, a slurry of the substrate 6a (1.5 g, 1.75 mmol) in dry DCM (36 mL) was added dropwise over a period of 30 min to the reaction mixture. After stirring for a further 50 minutes at −55° C., a solution of TEA (3.42 mL, 2.49 g; 24.6 mmol) in dry DCM (18 mL) was added dropwise over a period of 20 min to the reaction mixture. The stirred reaction mixture was allowed to warm to room temperature (˜1.5 h) and then diluted with DCM (50 mL). The organic solution was washed with 1 N HCl (2×25 mL), H2O (30 mL), brine (30 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (80:20 v/v EtOAc/Hexane) to afford bis-ketone 7a as a foam (835 mg, 56%)
Analytical Data: [α]20D=+291° (c=0.26, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d, 2H, J=10.1 Hz), 4.60 (dd, 2H, J=9.85, 3.07 Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85 Hz), 2.76 (dd, 2H, J=19.2, 9.90 Hz), 2.42 (p, 2H, J=5.77 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 206.8, 168.8, 165.9, 151.8, 148.0, 133.9, 120.9, 111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9, 52.4, 37.4, 29.0, 18.4, −1.24; IR (ATR, CHCl3) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434, 1360, 1247, 1209, 1098, 1066, 1023 cm−1; MS (ES+) m/z (relative intensity) 881 ([M+29]+., 38), 853 ([M+H]+., 100), 707 (8), 542 (12); HRMS [M+H]+. theoretical C41H56N4O12Si2 m/z 853.3506. found (ES+) m/z 853.3502.
(f) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11-dione]] (7b)
Preparation from 6b according to Method C gave the product as a white foam (10.5 g, 76%).
Analytical Data: MS (ES+) m/z (relative intensity) 882 ([M+H]+., 30), 735 (100), (ES−) m/z (relative intensity) 925 ([M+45]−., 100), 880 ([M−H]−., 70).
(g) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1, 4]-benzodiazepin-5,11-dione] (8a)
Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in one portion to a vigorously stirred solution of bis-ketone 7a (6.08 g, 7.1 mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile cooling bath) under a nitrogen atmosphere. Anhydrous triflic anhydride, taken from a freshly opened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injected rapidly dropwise, while maintaining the temperature at −40° C. or below. The reaction mixture was allowed to stir at −45° C. for 1 hour at which point TLC (50/50 v/v n-hexane/EtOAc) revealed the complete consumption of starting material. The cold reaction mixture was immediately diluted with DCM (200 mL) and, with vigorous shaking, washed with water (1×100 mL), 5% citric acid solution (1×200 mL) saturated NaHCO3 (200 mL), brine (100 mL) and dried (MgSO4). Filtration and evaporation of the solvent in vacuo afforded the crude product which was purified by flash column chromatography (gradient elution: 90:10 v/v n-hexane/EtOAc to 70:30 v/v n-hexane/EtOAc) to afford bis-enol triflate 8a as a yellow foam (5.5 g, 70%).
Analytical Data: [α]24D=+271° (c=0.18, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97 Hz), 5.51 (d, 2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.69 Hz), 4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J=16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J=5.85 Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 167.1, 162.7, 151.9, 148.0, 138.4, 133.6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56.3, 30.8, 29.0, 18.4, −1.25; IR (ATR, CHCl3) 2958, 1690, 1646, 1605, 1517, 1456, 1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm−1; MS (ES+) m/z (relative intensity) 1144 ([M+28]+., 100), 1117 ([M+H]+., 48), 1041 (40), 578 (8); HRMS [M+H]+. theoretical C43H54N4O16Si2S2F6 m/z 1117.2491. found (ES+) m/z 1117.2465.
(g) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] (8b)
Preparation from 7b according to the above method gave the bis-enol triflate as a pale yellow foam (6.14 g, 82%).
Analytical Data: (ES+) m/z (relative intensity) 1146 ([M+H]+., 85).
Example 1
(a) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (9)
Solid Pd(PPh3)4 (20.18 mg, 17.46 μmol) was added to a stirred solution of the triflate 8a (975 mg, 0.87 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)aniline (172 mg, 0.79 mmol) and Na2CO3 (138 mg, 1.30 mmol) in toluene (13 mL) EtOH (6.5 mL) and H2O (6.5 mL). The dark solution was allowed to stir under a nitrogen atmosphere for 24 hours, after which time analysis by TLC (EtOAc) and LC/MS revealed the formation of the desired mono-coupled product and as well as the presence of unreacted starting material. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL), after eventual separation of the layers the aqueous phase was extracted again with EtOAc (2×25 mL). The combined organic layers were washed with H2O (50 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude Suzuki product. The crude Suzuki product was subjected to flash chromatography (40% EtOAc/60% Hexane→70% EtOAc, 30% Hexane). Removal of the excess eluent by rotary evaporation under reduced pressure afforded the desired product 9 (399 mg) in 43% yield.
1H-NMR: (CDCl3, 400 MHz) δ 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J=8.5 Hz), 7.15 (t, 1H, J=2.0 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.52 (d, 2H, J=10.0 Hz), 4.77 (d, 1H, J=10.0 Hz), 4.76 (d, 1H, J=10.0 Hz), 4.62 (dd, 1H, J=3.7, 11.0 Hz), 4.58 (dd, 1H, J=3.4, 10.6 Hz), 4.29 (t, 4H, J=5.6 Hz), 4.00-3.85 (m, 8H), 3.80-3.60 (m, 4H), 3.16 (ddd, 1H, J=2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H, J=2.2, 10.5, 16.1 Hz), 2.43 (p, 2H, J=5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H). 13C-NMR: (CDCl3, 100 MHz) δ 169.8, 168.3, 164.0, 162.7, 153.3, 152.6, 149.28, 149.0, 147.6, 139.6, 134.8, 134.5, 127.9 (methine), 127.5, 125.1, 123.21, 121.5, 120.5 (methine), 120.1 (methine), 116.4 (methine), 113.2 (methine), 108.7 (methine), 79.8 (methylene), 79.6 (methylene), 68.7 (methylene), 68.5 (methylene), 67.0 (methylene), 66.8 (methylene), 58.8 (methine), 58.0 (methine), 57.6 (methoxy), 32.8 (methylene), 32.0 (methylene), 30.3 (methylene), 19.7 (methylene), 0.25 (methyl).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (10)
A suspension of the 4-anilino triflate [see Patent 33], (210 mg, 0.198 mmol), methylboronic acid (50 mg, 0.835 mmol, 4.2 eq.), silver I oxide (139 mg, 0.600 mmol., 3 eq.), potassium phosphate tribasic (252 mg, 1.2 eq w/w), triphenylarsine (36.7 mg, 0.12 mmol, 0.6 eq.) and bis(benzonitrile)dichloro-palladium II (11.5 mg, 0.030 mmol, 0.15 eq.) was heated at 75° C. in dry dioxane (8 mL) in a sealed tube under an inert atmosphere for 1.5 hrs. The reaction mixture was filtered through cotton-wool and the filter pad rinsed with ethylacetate and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with 80% EtOAc: 20% Hexane. Removal of excess eluent by rotary evaporation under reduced pressure gave the product as an off-white foam (100 mg, 0.11 mmol, 54% yield).
LC-MS RT 3.87 mins, 926 (M+H)
(c) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (11)
Fresh LiBH4 (44 mg, 2.0 mmol, 20 eq.) was added to a stirred solution of the SEM-dilactam (90 mg, 0.1 mmol) in THF (8 mL) at room temperature. The reaction mixture was allowed to stir for 0.5 hr, at which time LC-MS revealed complete reaction. The reaction mixture was partitioned between water (50 mL) and chloroform (100 mL). The organic phase was washed with brine (50 mL), dried over magnesium sulphate and concentrated in vacuo. The resulting residue was treated with DCM (5 mL), EtOH (14 mL), H2O (7 mL) and silica gel (10 g). The viscous mixture was allowed to stir at room temperature for 5 days. The mixture was filtered slowly through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was washed with H2O (50 mL), brine 60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (gradient from 100% CHCl3: 0% MeOH to 96% CHCl3: 4% MeOH) to provide the PBD dimer (5 mg 8% yield).
LC-MS RT 2.30 mins, 634 (M+H)
1H-NMR (400 MHZ, CDCl3) δ 7.80 (d, J=4.0 Hz, 1H), 7.73 (d, J=4.0 Hz, 1H), 7.45 (s, 1H), 7.43 (s, 1H), 7.26 (bs, 1H), 7.14 (d, J=8.5 Hz, 1H), 6.79 (s, 1H), 6.77 (s, 1H), 6.71-6.64 (m, 1H), 4.34-4.03 (m, 6H), 3.86 (s, 3H), 3.85 (s 3H), 3.55-3.37 (m, 1H), 3.36-3.19 (m, 1H), 3.17-3.00 (m, 1H), 2.96-2.80 (m, 1H), 1.75 (s 3H).
Example 2
(a) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-1′-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-1′-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-2-(trifluoromethylsulphonyloxy)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4] benzodiazepindiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 13
Solid 3-aminobenzeneboronic acid (60.3 mg) was added to a solution of the Troc protected bis triflate 12(Compound 44, WO 2006/111759) (600 mg, 0.41 mmol), sodium carbonate (65 mg, 0.61 mmoml) and palladium tetrakis triphenylphosphine (0.012 mmol) in toluene (10.8 mL), ethanol (5.4 mL) and water (5.4 mL). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was then partitioned between ethylacetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; gradient elution EtOAc/hexane 20/80→30/70→40/60→60/40) to remove unreacted bis-triflate. Removal of excess eluent from selected fractions to afford the desired compound in 41% yield (230 mg, 0.163 mmol)
LC-MS RT 4.28 mins, 1411 (M+H); 1H-NMR (400 MHZ, CDCl3) δ 7.44 (bs, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 7.20 (s, 1H), 7.16 (t, J=7.9 Hz, 1H), 6.84-6.73 (m, 3H), 6.70 (bs, 1H), 6.62 (dd, J=7.9, 1.7 Hz, 1H), 6.66-6.58 (m, 2H), 5.25 (d, J=12.0 Hz, 1H), 5.24 (d, J=12.0 Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.17-4.07 (m, 2H), 4.08-3.89 (m, 10H), 3.43-3.28 (m, 2H), 2.85 (d, J=1.65 Hz, 2H), 2.07-1.90 (m, 4H), 1.78-1.63 (m, 2H), 0.94 (s, 9H), 0.90 (s, 9H), 0.30 (s, 6H), 0.27 (s, 6H).
(b) (11S,11aS)-2,2,2-trichloroethyl 2-(3-aminophenyl)-1′-(tert-butyldimethylsilyloxy)-8-(5-((11S,11aS)-1′-(tert-butyldimethylsilyloxy)-2-propenyl-7-methoxy-5-oxo-10-((2,2,2-trichloroethoxy)carbonyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4] benzodiazepindiazepin-8-yloxy)pentyloxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate 14
Solid 1-propenyl boronic acid (7.1 mg, 0.084 mmol) was added to a solution of the Troc protected triflate 13 (73 mg, 0.052 mmol), sodium carbonate (18 mg, 0.17 mmol) and palladium tetrakis triphenylphosphine (3 mg) in toluene (1 mL), ethanol (0.5 mL) and water (0.5 mL). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water and brine and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure and the resulting residue was eluted through a plug of silica gel with ethylacetate. Removal of excess eluent from selected fractions afforded the coupled product 14 (40 mg, 0.031 mmol, 59%).
LC-MS RT 4.38 mins, (1301, 1305, 1307, 1308, 1310 multiple masses due to chlorine isotopes)
(c) (S)-2-(3-aminophenyl)-8-(5-((S)-2-propenyl-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepine-8-yloxy)pentyloxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11 aH)-one 15
Cadmium/lead couple (100 mg, Q Dong et al. Tetrahedron Letters vol 36, issue 32, 5681-5682, 1995) was added to a solution of the Suzuki product 14 (40 mg, 0.029 mmol) in THF (1 mL) and ammonium acetate (1N, 1 mL) and the reaction mixture was allowed to stir for 1 hour. The reaction was filtered through cotton wool to remove particulates and break-up the emulsion. The reaction mixture was partitioned between chloroform and water, the phases separated and the aqueous phase extracted with chloroform. The combined organic layers were washed with brine and dried over magnesium sulphate. Rotary evaporation under reduced pressure yielded the crude product which was subjected to column chromatography (silica gel, 1→5% MeOH/CHCl3). Removal of excess eluent by rotary evaporation under reduced pressure afforded the desired imine product 15 (9 mg 0.013 mmol 43%)
LC-MS RT 2.80 mins, 689 (M+H)
1H-NMR (400 MHZ, CDCl3) δ 7.88 (d, J=3.9 Hz, 1H), 7.82 (d, J=3.9 Hz, 1H), 7.52 (s, 1H), 7.49 (s, 1H), 7.45 (bs, 1H), 7.15 (t, J=7.8 Hz, 1H), 6.92 (bs, 1H), 6.84-6.76 (m, 3H), 6.72 (bs, 1H), 6.60 (dd, J=7.9, 1.9 Hz, 1H), 6.26 (d, J=15.3 Hz, 1H), 5.67-5.51 (m, 1H), 4.46-4.35 (m, 1H), 4.34-4.24 (m, 1H), 4.20-4.00 (m, 4H), 3.94 (s, 3H), 3.93 (s 3H), 3.62-3.44 (m, 1H), 3.43-3.23 (m, 2H), 3.19-3.02 (m, 1H), 2.06-1.89 (m, 4H), 1.84 (d, J=6.5 Hz, 3H), 1.76-1.62 (m, 2H).
Example 3
(a) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione 16
Solid Pd(PPh3)4 (20 mg, 17.8 μmol) was added to a stirred solution of the triflate 8a (2.5 g, 2.24 mmol), 3-aminobenzeneboronic acid (291 mg, 2.12 mmol) and Na2CO3 (356 mg, 3.35 mmol) in toluene (20 mL), EtOH (10 mL) and H2O (10 mL). The solution was allowed to stir under a nitrogen atmosphere for 3 hours at room temperature, after which time analysis by TLC (EtOAc) and LC/MS revealed the formation of the desired mono-coupled product and as well as the presence of unreacted starting material. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue partitioned between H2O (100 mL) and EtOAc (100 mL), after eventual separation of the layers the aqueous phase was extracted again with EtOAc (2×25 mL). The combined organic layers were washed with H2O (50 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude Suzuki product. The crude Suzuki product was subjected to flash chromatography (30% EtOAc/70% Hexane→80% EtOAc, 20% Hexane). Removal of the excess eluent by rotary evaporation under reduced pressure afforded the desired product (1 g) in 42% yield.
LC-MS, 4.17 minutes, ES+1060.19.
(b) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11 aH)-dione 17
A suspension of the 3-anilino triflate, (50 mg, 47.2 μmol), methylboronic acid (8.47 mg, 141 μmol, 3 eq.), silver(I)oxide (21.8 mg, 94.3 μmol., 2 eq.), potassium phosphate tribasic (60 mg, 1.2 eq w/w), triphenylarsine (5.78 mg, 18.9 μmol, 0.4 eq.) and bis(benzonitrile)dichloro-palladium II (1.81 mg, 4.7 μmol, 0.1 eq.) was heated at 67° C. in dry dioxane (2 mL) in a sealed tube under an inert atmosphere for 3 hrs. The reaction mixture was filtered through cotton-wool and the filter pad rinsed with ethylacetate and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with 80% EtOAc: 20% Hexane. Removal of excess eluent by rotary evaporation under reduced pressure gave the product as an off-white foam (18 mg, 19.4 μmol, 41% yield). The reaction was subsequently repeated on a larger scale to afford 250 mg of the 2-methyl product.
LC-MS 3.88 mins, 925.86 (M+H)
(c) (S)-2-(3-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one 18
Fresh LiBH4 (20.6 mg, 0.95 mmol, 3.5 eq.) was added to a stirred solution of the SEM-dilactam (250 mg, 0.27 mmol) in THF (4 mL) at room temperature. The reaction mixture was allowed to stir for 1.0 hr, at which time LC-MS revealed complete reaction. Excess LiBH4 was quenched with acetone (c. 1 mL) at 0° C. (ice bath). The reaction mixture was partitioned between water (50 mL) and 10% methanol in chloroform (100 mL). The organic phase was washed with brine (50 mL), dried over magnesium sulphate and concentrated in vacuo.
The resulting residue was treated with 10% methanol in chloroform (c. 50 mL) and silica gel (20 g). The viscous mixture was allowed to stir at room temperature for 5 days. The mixture was filtered slowly through a sinter funnel and the silica residue washed with 90% CHCl3: 10% MeOH (˜250 mL) until UV activity faded completely from the eluent. The organic phase was washed with H2O (50 mL), brine 60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude material. The crude product was purified by flash chromatography (gradient from 100% CHCl3: 0% MeOH to 96% CHCl3: 4% MeOH) to provide the PBD dimer 18.
Example 4
Part (i)
(a) Alternate Synthesis of (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(trifluoromethylsulfonyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (9)
Tetrakis(triphenylphosphine)palladium(0) (208 mg) was added to triflate (8a)(5 g), 4-anilineboronic acid (0.93 g) and sodium carbonate (0.62 g) in a mixture of toluene (60 mL), ethanol (30 mL) and water (10 mL). The reaction mixture was allowed to stir for 3 days at room temperature. The reaction mixture was washed with water, brine and dried over magnesium sulphate. After filtration excess solvent was removed by rotary evaporation under reduced pressure. The crude coupling product was purified by flash column chromatography (silica gel; gradient: 100% hexane to 100% ethyl acetate). Pure fractions were combined and removal of excess eluent afforded the pure product as a solid (2.2 g, 93% yield, LC/MS 8.05 mins, m/z ES+1060).
(b) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(phenyl-vinyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (20)
A mixture of triflate 9 (0.5 g), trans-2-phenylvinylboronic acid (0.091 g), triethylamine (0.38 g) and tetrakis(triphenylphosphine)palladium(0) (30 mL) in ethanol (3 mL), toluene (6 mL) and water (1 mL) was irradiated with microwaves for 8 minutes at 80° C. in a sealed microwave vial. The reaction mixture was diluted with dichloromethane washed with water and dried over magnesium sulphate. Excess solvent was removed by rotary evaporation under reduced pressure to afford the crude product which was used without further purification in the next reaction. Retention time 8.13 mins, ES+1014.13.
(c) (S)-2-(4-aminophenyl)-7-methoxy-8-(3-((S)-7-methoxy-2-(phenyl-vinyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propoxy)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5(11aH)-one (21)
A solution of superhydride in THF (1 M, 1.2 mL) was added by syringe to a solution of the crude Suzuki product (0.477 g) in THF (10 mL) at −78° C. (acetone/dry ice bath). The reaction mixture was allowed to stir at −78° C. for 20 minutes, after which time the reaction was quenched with water. The reaction mixture was extracted with ethyl acetate and the organic layer washed with brine and dried over magnesium sulphate. Removal of excess solvent by rotary evaporation under reduced pressure afforded the crude SEM-carbinolamine which was dissolved in dichloromethane (3 mL), ethanol (6 mL) and water (1 mL) and stirred with silica gel for 2 days. The reaction mixture was filtered excess solvent evaporated by rotary evaporation under reduced pressure and the residue subjected to flash column chromatography (3% methanol in chloroform). Pure fractions were combined and excess eluent removed by rotary evaporation under reduced pressure to afford compound 21 (0.75 mg, 22% yield over 3 steps). Retention time 5.53 mins, ES+721.99.
Part (ii)
(a) (S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanoic acid (23)
A suspension of dipeptide (22) (0.1 g, 0.54 mmol, 1 eq.) and 6-maleimidohexanoic acid succinimide ester (0.165 g, 0.54 mmol, 1 eq.) in anhydrous DMF (5 mL) was stirred at room temperature for 24 hours at which time LCMS indicated 50% conversion to a new product. The reaction mixture was diluted with anhydrous DMF (5 mL) and the reaction was allowed to continue for a further 24 hours. The solvent was evaporated under reduced pressure to give a colourless residue. Diethyl ether (60 mL) was added and the mixture was sonicated for 5 min, the ether was decanted and the process was repeated (×2). The final ethereal portion was filtered to isolate the product (23) as a white powder which was dried under vacuum (0.105 g, 52%). Analytical Data: RT 2.28 min; MS (ES+) m/z (relative intensity) 382 ([M+H]+., 90), MS (ES−) m/z (relative intensity) 380 ([M−H])−., 100).
(b) 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-styryl)-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (24)
The unsymmetrical PBD dimer (21) (0.019 g, 26 μmol, 1 eq.) was added to a solution of the linker (23) (0.0121 g, 31.6 μmol, 1.2 eq.) and EEDQ (0.0098 g, 39.6 μmol, 1.5 eq.) in a mixture of anhydrous DCM/MeOH (3 mL/0.5 mL) under an argon atmosphere. The resultant solution was stirred at room temperature for 5 hours at which time LCMS indicated 50% conversion to a new product. The reaction mixture was diluted with anhydrous DCM (2 mL) and the reaction was allowed to continue for a further 18 hours. The solvent was evaporated under reduced pressure and the residue purified by flash column chromatography [DCM 100% to DCM 94%/MeOH 6% in 1% increments] to give the product as a yellow solid (5.2 mg, 18%). Analytical Data: RT 3.10 min; MS (ES+) m/z (relative intensity) 1085 ([M+H]+., 90).
Example 5
(a) (S)-2-(4-aminophenyl)-8-(3-((S)-2-cyclopropyl-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (25)
A suspension of silver (I) oxide (0.441 g), potassium phosphate tribasic (1.187 g), triphenylarsine (0.116 g), cyclopropylboronic acid (0.206 g) and starting material 9 (0.5 g) in dioxane (15 mL) under and an argon atmosphere was heated to 71° C. A catalytic amount of palladium (II) bis(benzonitrile chloride) (0.036 g) was added and the reaction mixture was allowed to stir for 2 hours and 10 mins at 71° C. The reaction mixture was filtered through celite and the filter pad washed with ethyl acetate (400 mL). The organic solution was extracted with water (2×600 mL) and brine (600 mL) and dried over magnesium sulphate. Removal of organic solvent by rotary evaporation under reduced pressure afforded the crude product which was purified via gravity silica gel chromatography (ethyl acetate only as eluent). Removal of excess eluent by rotary evaporation under reduced pressure afforded the product 25 as a yellow solid (145 mg, 32% yield). LCMS RT 3.92 mins, ES+952.06
(b) (S)-2-(4-aminophenyl)-8-(3-((S)-2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one (26)
A solution of super hydride (0.361 mL, 1M in THF) was added drop wise over 5 minutes to a solution of the SEM dilictam 25 (0.137 g) in anhydrous tetrahydrofuran (5 mL) under an argon atmosphere at −78° C. LCMS after 35 minutes revealed that the reaction was complete and excess super hydride was quenched with water (4 mL) followed by brine (4 mL). The aqueous solution was extracted with a mixture of dichloromethane/methanol (9:1, 2×16 mL) and the organic layer dried over magnesium sulphate. Solvent was removed by rotary evaporation under reduced pressure and the crude product was taken up in a mixture of ethanol, dichloromethane and water (8:3:1, 15 mL) and treated with silica gel. The thick suspension was allowed to stir for 4 days. The mixture was filtered through a sinter, washing with dichloromethane/methanol (9:1, 140 mL) until product ceased to be eluted. The organic layer was washed with brine (2×250 mL) and then dried over magnesium sulphate. Rotary evaporation under reduced pressure afforded the crude product which was subjected to flash column chromatography (silica gel; gradient 100% to 5% methanol/dichloromethane). Removal of excess eluent afforded the product 26 (23 mg, 25% yield). LCMS RT 2.42, ES+659.92
Example 6
(S)-2-(4-aminophenyl)-7-methoxy-8-(3-(((S)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-2-((trimethylsilyl)-ethynyl)-5,10,11,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (27)
A mixture of 9 (0.150 g, 0.14 mmol), CuI (0.003 g, 0.014 mmol, 0.1 eq), Pd(PPh3)4 (0.0162 g, 0.014 mmol, 0.1 eq) and PPh3 (0.007 g, 0.028 mmol, 0.2 eq) was dissolved in piperidine (9 mL) in presence of molecular sieves under an argon atmosphere. Ethynyltrimethylsilane (0.06 ml, 0.42 mmol, 3 eq) was added to the mixture at 70° C. and the reaction mixture was allowed to stir overnight. The solvent was removed by rotary evaporation under reduced pressure and the resulting brown solid purified by flash column chromatography (silica gel, 90% EtOAc, 10% hexane). Compound 27 was obtained as an orange solid (0.043 g, 30%); Rf 0.69 [EtOAc]; LC-MS (5 min) 4.28 min, ES+1008.28.
Example 7
(a)
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]-benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (30)
To a mixture of carboxylic acid 23 (8 mg, 21 umol) in 5% methanol/dichloromethane was added EEDQ (6.1 mg, 24.6 umol) and the mixture was stirred for 15 minutes under nitrogen at an ambient temperature. The resulting mixture was added to 11 (12 mg, 18.9 umol) and stirred for 3 hours under nitrogen. The reaction mixture was aspirated directly onto a 1 mm radial chromatotron plate and eluted with a gradient of 1 to 4% methanol in dichloromethane. Product containing fractions were concentrated under reduced pressure to give 9.4 mg (50%) of 30 as a yellow solid: MS (ES−) m/z 997.18 (M+H)+.
(b)
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-(3-((S)-7-methoxy-8-(3-((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzo diazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-amino)-3-methyl-1-oxobutan-2-yl)hexanamide (31)
Compound 31 was synthesised from compound 18 using the same method as in part (a) with a yield of 25%.
Example 8
Determination of Free Drug In Vitro Cytotoxicity
Cells as detailed below were collected and plated in 96 well black-sided plates at a density of 10,000 cells/well in 150 μL of medium. Serial dilutions of the test article (50 μL) were added, and incubation was carried out for 92 hours at 37° C. After addition of test compound, cultures were incubated to 96 hours at 37° C. Resazurin (0.25 mM, 50 μL, Sigma, St. Louis, Mo.) in medium was added and incubation was continued for 4 h. The plates were read on a Fusion HT microplate reader (Packard, Meriden, Conn.) using an excitation wavelength of 525 nm and an emission wavelength of 590 nm. Data from all assays were reduced using GraphPad Prism Version 4 for Windows (GraphPad Software, San Diego, Calif.). The IC50 concentrations compared to untreated control cells were determined using a 4 parameter curve fits.
The IC50 (nM) values for compound 15 are:
L428
786-O
HEL
HL-60
MCF-7
IC50 (nm)
<0.00001
<0.00001
<0.00001
<0.00001
0.03
The same method was also used to determine the activity of compounds 11 and 18:
IC50 (nM)
Caki-1
786-O
TF1a
MCF-7
11
0.06
0.1
0.07
0.2
18
0.6
1
0.7
2
Alternative Cell Assay
Cells were plated in 150 μL growth media per well into black-sided clear-bottom 96-well plates (Costar, Corning) and allowed to settle for 1 hour in the biological cabinet before placing in the incubator at 37° C., 5% CO2. The following day, 4× concentration of drug stocks were prepared, and then titrated as 10-fold serial dilutions producing 8-point dose curves and added at 50 μl per well in duplicate. Cells were then incubated for 48 hours at 37° C., 5% CO2 Cytotoxicity was measure by incubating with 100 μL Cell Titer Glo (Promega) solution for 1 hour, and then luminescence was measured on a Fusion HT plate reader (Perkin Elmer). Data was processed with Excel (Microsoft) and GraphPad (Prism) to produce dose response curves and IC50 values were generated and data collected.
IC50 (nM)
786-O
Caki-1
MCF-7
BxPC-3
HL-60
HEL
11
0.85
0.4
7
3
0.1
0.06
Example 9
Preparation of PDB Dimer Conjugates
Antibody-drug conjugates were prepared as previously described (see Doronina et al., Nature Biotechnology, 21, 778-784 (2003)) or as described below.
Engineered hlgG1 antibodies with introduced cysteines: CD70 antibodies containing a cysteine residue at position 239 of the heavy chain (h1F6d) were fully reduced by adding 10 equivalents of TCEP and 1 mM EDTA and adjusting the pH to 7.4 with 1M Tris buffer (pH 9.0). Following a 1 hour incubation at 37° C., the reaction was cooled to 22° C. and 30 equivalents of dehydroascorbic acid were added to selectively reoxidize the native disulfides, while leaving cysteine 239 in the reduced state. The pH was adjusted to 6.5 with 1M Tris buffer (pH 3.7) and the reaction was allowed to proceed for 1 hour at 22° C. The pH of the solution was then raised again to 7.4 by addition of 1 M Tris buffer (pH 9.0). 3.5 equivalents of the PBD drug linker in DMSO were placed in a suitable container for dilution with propylene glycol prior to addition to the reaction. To maintain solubility of the PBD drug linker, the antibody itself was first diluted with propylene glycol to a final concentration of 33% (e.g., if the antibody solution was in a 60 mL reaction volume, 30 mL of propylene glycol was added). This same volume of propylene glycol (30 mL in this example) was then added to the PBD drug linker as a diluent. After mixing, the solution of PBD drug linker in propylene glycol was added to the antibody solution to effect the conjugation; the final concentration of propylene glycol is 50%. The reaction was allowed to proceed for 30 minutes and then quenched by addition of 5 equivalents of N-acetyl cysteine. The ADC was then purified by ultrafiltration through a 30 kD membrane. (Note that the concentration of propylene glycol used in the reaction can be reduced for any particular PBD, as its sole purpose is to maintain solubility of the drug linker in the aqueous media.)
Example 10
Determination of Conjugate In Vitro Cytotoxicity
Cells as detailed below were collected and plated in 96 well black-sided plates at a density of 10,000 cells/well in 150 μL of medium. Serial dilutions of the test article (50 μL) were added, and incubation was carried out for 92 hours at 37° C. After addition of test compound, cultures were incubated to 96 hours at 37° C. Resazurin (0.25 mM, 50 μL, Sigma, St. Louis, Mo.) in medium was added and incubation was continued for 4 h. The plates were read on a Fusion HT microplate reader (Packard, Meriden, Conn.) using an excitation wavelength of 525 nm and an emission wavelength of 590 nm. Data from all assays were reduced using GraphPad Prism Version 4 for Windows (GraphPad Software, San Diego, Calif.). The IC50 concentrations compared to untreated control cells were determined using a 4 parameter curve fits. The antibody used was a CD70 antibody (humanized 1F6; see Published U.S. Application No. 2009-148942) having introduced cysteine residues at amino acid heavy chain position 239 (according to the EU numbering system) (indicated as h1F6d).
The IC50 (nM) values for ADCs of compound 31 are:
ADCs
Caki-1
786-O
HL60
HEL
TF1a
h1F6d-31
7
36
5371
Max Inh = 50%
Max Inh =
(2dr/Ab)
40%
Note:
Maximum inhibition (Max Inhb) = % inhibition at top concentration out of 100% untreated.
Example 11
Determination of In Vivo Cytotoxicity of Selected Conjugates
The following study was conducted in concordance with the Animal Care and Use Committee in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The antibodies used were an antibody having introduced cysteine residues at position 239 (S239C) in the heavy chains and conjugated to compound 31, and a nonbinding control conjugated to the same compound 31.
Treatment studies were conducted in an antigen+xenograft model. Tumor cells were implanted subcutaneously into scid mice. Mice were randomized to study groups (n=6). The ADC-compound 31 or control ADCs were dosed ip according to a q4dx4 schedule (as shown by the triangles on the x axis). Tumor volume as a function of time was determined using the formula (L×W2)/2. Animals were euthanized when tumor volumes reached 1000 mm3.
Referring to FIG. 1, the ADC of compound 31 was dosed at 0.1 (□), 0.3 () and 1 (▪) mg/kg. A nonbinding control, conjugated to compound 31, was administered at the same doses (0.1 (Δ), 0.3 () and 1(▴) mg/kg). All three doses of the Ab-compound 31 conjugate had better activity than the nonbinding control conjugate. Untreated tumours are shown by *.
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13641180
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seattle genetics inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/220
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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May 12th, 2015 12:00AM
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Mar 12th, 2012 12:00AM
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https://www.uspto.gov?id=US09029406-20150512
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N-carboxyalkylauristatins and use thereof
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The present application relates to new derivatives, substituted on the N terminus by a carboxyalkyl group, of monomethylauristatin E and monomethylauristatin F, to processes for preparing these derivatives, to the use of these derivatives for treating and/or preventing diseases, and to the use of these derivatives for producing medicaments for treating and/or preventing diseases, more particularly hyperproliferative and/or angiogenic disorders such as cancer disorders, for example. Such treatments may be applied as a monotherapy or else in combination with other medicaments or further therapeutic measures.
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9029406
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1. A compound of formula (I)
or a salt or solvate thereof, in which
L is linear (C1-C12)-alkanediyl, which may be substituted with methyl up to four times and in which (a) two carbon atoms in 1,2-, 1,3- or 1,4-relation to one another are optionally bridged by including the carbon atoms optionally between them to form a (C3-C6)-cycloalkyl ring or a phenyl ring, or (b) up to three CH2 groups not vicinal to one another are optionally replaced by —O—,
and
T is a group of the formula
wherein
* denotes the linkage site to the nitrogen atom,
R1 is phenyl or 1H-indol-3-yl,
and
R2 is hydrogen or a group of the formula
wherein
** denotes the respective linkage site to the radical of the respective group T,
A is linear (C1-C4)-alkanediyl or linear (C2-C4)-alkenediyl,
R3 is phenyl that is optionally substituted with (C1-C4)-alkoxycarbonyl or carboxyl,
n is the number 0, 1 or 2,
R4 is phenyl, benzyl or 2-phenylethyl which is optionally substituted with (C1-C4)-alkoxycarbonyl or carboxyl in the phenyl group
Het is a divalent 5-membered heteroaryl ring with up to three ring heteroatoms from the series N, O and/or S,
and
R5 is (C3-C6)-cycloalkyl, phenyl or (C1-C4)-alkyl, which is optionally substituted with phenyl, wherein the aforementioned phenyl groups are optionally substituted with (C1-C4)-alkoxycarbonyl or carboxyl.
2. The compound of claim 1 or a salt or solvate thereof, wherein
L is linear (C1-C8)-alkanediyl, in which (a) two carbon atoms in 1,3- or 1,4-relation to one another are optionally bridged by including one or two of the carbon atoms between them to form a phenyl ring, or (b) up to two CH2 groups not vicinal to one another are optionally replaced by —O—,
and
T is a group of the formula
wherein
* denotes the linkage site to the nitrogen atom,
R1 is phenyl or 1H-indol-3-yl,
and
R2 is hydrogen or a group of the formula
wherein
** denotes the linkage site to the radical of the respective group T,
A is ethene-1,2-diyl or propene-1,3-diyl,
R3 is phenyl, which is optionally substituted with (C1-C4)-alkoxycarbonyl or carboxyl,
Het is a divalent 5-membered heteroaryl ring selected from the series of pyrazolyl, imidazolyl, 1,3-oxazolyl, 1,3-thiazolyl, 1,2,4-oxadiazolyl and 1,3,4-oxadiazolyl,
and
R5 is a phenyl, which is optionally may be substituted with (C1-C4)-alkoxycarbonyl or carboxyl.
3. The compound of claim 1 or a salt or solvate thereof, wherein
L is linear (C1-C6)-alkanediyl,
and
T is a group of the formula
wherein
* denotes the linkage site to the nitrogen atom,
and
R2 is hydrogen or a group of the formula
wherein
** denotes the linkage site to the radical of the respective group T,
A is ethene-1,2-diyl,
R3 is phenyl, which is optionally substituted with methoxycarbonyl or carboxyl,
Het is 1,3,4-oxadiazol-2,5-yl,
and
R5 is a phenyl, which is optionally substituted with methoxycarbonyl or carboxyl.
4. A method for preparing a compound of claim 1 or a salt or solvate thereof, the method comprising providing a compound of formula (II)
in which T has the meaning given in claim 1,
and reacting the compound of formula (II) in an inert solvent, either
[A] by base-induced alkylation with a compound of formula (III)
in which L has the meaning given in claim 1,
E1 is hydrogen, (C1-C4)-alkyl or benzyl,
and
X is a leaving group selected from the group consisting of chloride, bromide, iodide, mesylate, triflate, and tosylate,
to form a compound of formula (IV)
in which L and T have the meanings given in claim 1,
and E1 is hydrogen, (C1-C4)-alkyl or benzyl, wherein, when E1 is (C1-C4)-alkyl or benzyl, the ester radical is removed, thereby producing a hydrogen at E1 in formula (III), thus producing the carboxylic acid of formula (I);
or
[B] with a compound of formula (V)
wherein
E1 is hydrogen, (C1-C4)-alkyl or benzyl,
and
LA has the meaning of L given in claim 1, but is shortened by one CH2 unit in the alkyl chain length,
in the presence of a suitable reducing agent to produce a compound of formula (VI)
in which LA has the meaning of L given in claim 1, but is shortened by one CH2 unit in the alkyl chain length,
T has the meaning given in claim 1, and
E1 is hydrogen, (C1-C4)-alkyl or benzyl, wherein, when E1 stands for (C1-C4)-alkyl or benzyl, the ester radical is removed, thereby producing a hydrogen at E1 in formula (V),
thus producing the carboxylic acid of formula (I-A)
in which LA has the meaning of L given in claim 1, but is shortened by one CH2 unit in the alkyl chain length, and T has the meaning given in claim 1.
5. A pharmaceutical composition comprising a compound as defined in claim 1 or a salt or solvate thereof, and further comprising one or more inert, non-toxic, pharmaceutically suitable excipients.
6. The pharmaceutical composition of claim 5, further comprising one or more additional active ingredients.
7. A method for the treatment of cancer or tumor conditions in humans or animals, said method comprising administering to a subject an effective amount of at least one compound as defined in claim 1.
8. A method for treatment of cancer or tumor diseases in humans or animals, said method comprising administering to a subject an effective amount of at least one pharmaceutical composition of claim 5.
9. The compound of claim 1, wherein said compound is selected from the group consisting of:
10. An antiproliferative conjugate in which a compound of claim 1 is connected with a protein.
11. The antiproliferative conjugate according to claim 10, wherein the protein is an antibody.
12. An antiproliferative conjugate in which a compound of claim 8 is connected with a protein.
13. The antiproliferative conjugate according to claim 12, wherein the protein is an antibody.
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13
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The present patent application relates to novel derivatives of monomethylauristatin E and monomethylauristatin F, substituted with a carboxyalkyl group on the N terminus, methods of synthesis of these derivatives, use of these derivatives for treatment and/or prevention of diseases and use of these derivatives for production of pharmaceutical drugs for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic diseases, such as the various forms of cancer, for example. Such treatments may be in the form of monotherapy or in combination with other drugs or other therapeutic measures.
Cancer is the result of uncontrolled cell growth of a wide variety of tissues. In many cases, the new cells grow into existing tissue (invasive growth) or metastasize to remote organs. Cancer occurs in a wide variety of organs and the pathology often has a tissue-specific course. The term cancer is therefore a generic term that describes a large group of specific diseases of various organs, tissues and types of cells.
Early-stage tumors can in some cases be removed by surgical and radiotherapeutic measures. Metastatic tumors can usually be treated only palliatively by chemotherapeutic agents. The goal here is to find the optimum combination of prolonging life and improving the quality of life.
Most chemotherapeutic agents administered parenterally today are not distributed to the tumor tissue or tumor cells in a targeted manner but instead are nonspecifically distributed throughout the patient's body through systemic administration, i.e., at sites where exposure to the drug is often undesirable, such as in healthy cells, tissues and organs, for example. This may lead to adverse effects or even serious general toxic effects, which then often severely limit the therapeutically usable drug dosage range or necessitate complete cessation of the medication.
The improved and selective availability of these chemotherapeutic agents in the tumor cell or the immediate surrounding tissue and the associated increase in effect, on the one hand, and minimization of toxic side effects, on the other hand, have therefore for many years been the focus of work in developing new chemotherapeutic drugs. There have been numerous attempts so far to develop efficient methods for introducing drugs into the target cell. However, it is still a difficult task to optimize the association between the drug and the intracellular target and to minimize the intercellular distribution of the drug, e.g., to neighboring cells.
Monoclonal antibodies, for example, are suitable for targeted addressing of tumor tissue and tumor cells. The importance of such antibodies for clinical treatment of cancer has grown enormously in recent years based on the efficacy of such agents as trastuzumab (Herceptin), rituximab (Rituxan), cetuximab (Erbitux) and bevacizumab (Avastin) which have been approved in the meantime for treatment of individual specific tumor conditions (see, for example, G. P. Adams and L. M. Weiner, Nat. Biotechnol. 23, 1147-1157 (2005)). As a result, there has been a significant increase in interest in so-called immunoconjugates, in which an internalizing antibody directed against a tumor-associated antigen is bound covalently to a cytotoxic agent by a linking unit (“linker”). After introducing the conjugate into the tumor cell and then it splitting it off, the cytotoxic agent is released inside the tumor cell, where it can manifest its effect directly and selectively. In this way, the damage to normal tissue can be kept within significantly narrower limits in comparison with conventional chemotherapy for cancer (see, for example, J. M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); A. M. Wu and P. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005); P. D. Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B. Stump, Bioconjugate Chem. 21, 5-13 (2010)).
Instead of antibodies, binders from the field of small drug molecules may be used as binders to selectively bind to a specific “target” such as, for example, to a receptor (see, e.g., E. Ruoslahti et al., Science, 279, 377-380 (1998); D. Karkan et al., PLoS ONE 3 (6), e2469 (Jun. 25, 2008)). Conjugates of a cytotoxic drug and an addressing ligand having a defined cleavage site between the ligand and the drug for release of the drug are also known. One such “intended breaking point” may consist of a peptide chain, for example, which can be cleaved selectively at a certain site by a specific enzyme at the site of action (see, for example, R. A. Firestone and L. A. Telan, US Patent Application US 2002/0147138).
Auristatin E (AE) and monomethylauristatin E (MMAE) are synthetic analogs of the dolastatins, a special group of linear pseudopeptides, which were originally isolated from marine sources, and some of which have a very potent cytotoxic activity with respect to tumor cells (for an overview, see, for example, G. R. Pettit, Prog. Chem. Org. Nat. Prod. 70, 1-79 (1997); G. R. Pettit et al., Anti-Cancer Drug Design 10, 529-544 (1995); G. R. Pettit et al., Anti-Cancer Drug Design 13, 243-277 (1998)).
However, MMAE has the disadvantage of a comparatively high systemic toxicity. Furthermore, when used in the form of antibody-drug conjugates (immunoconjugates), this compound is not compatible with linking units (linkers) between antibody and active ingredient/drug, which do not have any enzymatically cleavable intended breaking points (S. O. Doronima et al., Bioconjugate Chem. 17, 114-124 (2006)).
Monomethylauristatin F (MMAF) is an auristatin derivative with a C-terminal phenylalanine unit having only a moderate antiproliferative effect in comparison with MMAE. This can very likely be attributed to the free carboxyl group, which has a negative effect on the cell viability of this compound because of its polarity and charge. In this context, the methyl ester of MMAF (MMAF-OMe) has been described as a prodrug derivative, which has a neutral charge and can pass through the cell membrane; it also has an increased in vitro cytotoxicity, which is greater by several orders of magnitude in comparison with MMAF with respect to various carcinoma cell lines (S. O. Doronina et al., Bioconjugate Chem. 17, 114-124 (2006)). It may be assumed that this effect is caused by the MMAF itself, which is rapidly released by intracellular ester hydrolysis after the prodrug has been incorporated into the cells.
However, drug compounds based on simple ester derivatives are generally at risk of chemical instability due to a nonspecific ester hydrolysis, which is independent of the intended site of action, for example, due to esterases present in blood plasma. This can greatly restrict the usability of such compounds in treatment. In addition, auristatin derivatives such as MMAE and MMAF are also substrates for transporter proteins that are expressed by tumor cells, which can lead to the development of a resistance to these active ingredients.
The object of the present invention was therefore to identify novel auristatin compounds and supply them for the treatment of cancer in particular, such that these auristatin compounds have a stronger cytotoxic activity in whole-cell assays in comparison with monomethylauristatin F (MMAF), which has only a moderate efficacy, and/or have less pronounced substrate properties for transporter proteins. Such substances could also be especially suitable as toxophores for linking to proteins, such as antibodies in particular, or to low-molecular ligands to form (immuno-)conjugates having antiproliferative effects.
Monomethylauristatin F (MMAF) as well as various ester and amide derivatives thereof were disclosed in WO 2005/081711 A2. Additional auristatin analogs having a C-terminal amide-substituted phenylalanine unit are described in WO 01/18032 A2. MMAF analogs involving side chain modifications of phenylalanine are claimed in WO 02/088172 A2 and WO 2007/008603 A1, and WO 2007/008848 A2 describes those in which the carboxyl group of phenylalanine is modified. Additional auristatin conjugates linked via the N- or C-terminus are described in WO 2009/117531 A1 (see also S. O. Doronina et al., Bioconjugate Chem. 19, 1960-1963 (2008)).
The subject matter of the present invention is compounds of general formula (I):
in which
L stands for linear (C1-C12)-alkanediyl, which may be substituted with methyl up to four times and in which (a) two carbon atoms in 1,2-, 1,3- or 1,4-relation to one another may be bridged by including the carbon atoms optionally between them to form a (C3-C6)-cycloalkyl ring or a phenyl ring, or (b) up to three CH2 groups not vicinal to one another may be replaced by —O—,
and
T stands for a group of the formula
wherein
* denotes the linkage site to the nitrogen atom,
R1 stands for phenyl or 1H-indol-3-yl,
and
R2 stands for hydrogen or a group of the formula
wherein
** denotes the respective linkage site to the radical of the respective group T,
A stands for linear (C1-C4)-alkanediyl or linear (C2-C4)-alkenediyl,
R3 stands for phenyl that may be substituted with (C1-C4)-alkoxycarbonyl or carboxyl,
n stands for the number 0, 1 or 2,
R4 stands for phenyl, benzyl or 2-phenylethyl which may be substituted with (C1-C4)-alkoxycarbonyl or carboxyl in the phenyl group
Het stands for a divalent 5-membered heteroaryl ring with up to three ring heteroatoms from the series N, O and/or S,
and
R5 stands for (C3-C6)-cycloalkyl, phenyl or (C1-C4)-alkyl, which may be substituted with phenyl,
wherein the aforementioned phenyl groups may in turn be substituted with (C1-C4)-alkoxycarbonyl or carboxyl,
as well as their salts and solvates and the solvates of the salts.
Compounds according to the invention include the compounds of formula (I) and their salts and solvates as well as the solvates of the salts, the compounds of the formulas given below that are covered by formula (I) and their salts and solvates as well as the solvates of the salts and the compounds covered by formula (I) and referred to below as exemplary embodiments as well as their salts and solvates as well as the solvates of the salts, as long as the compounds covered by formula (I) and listed below are not already the salts and solvates as well as the solvates of the salts.
The compounds according to the invention may exist in different stereoisomeric forms depending on their structure, i.e., in the form of configurational isomers or optionally also as conformational isomers (enantiomers and/or diastereomers, including those in atropisomers). The present invention therefore includes the enantiomers and diastereomers and their respective mixtures. The stereoisomerically uniform components can be isolated in a known way from such mixtures of enantiomers and/or diastereomers. Chromatographic methods, in particular HPLC chromatography on a chiral or achiral phase, are preferably used for this purpose.
If the compounds according to the invention can occur in tautomeric forms, then the present invention also includes all the tautomeric forms.
Within the scope of the present invention, the preferred salts are the physiologically safe salts of the compounds according to the invention. This also includes salts that are not suitable for pharmaceutical applications per se but may be used for isolating or purifying the compounds according to the invention, for example.
Physiologically safe salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methane sulfonic acid, ethane sulfonic acid, benzene sulfonic acid, toluene sulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.
Physiologically safe salts of the compounds according to the invention also include the salts of conventional bases such as preferably and for example, alkali metal salts (e.g., sodium and potassium salts), alkaline earth salts (e.g., calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines with 1 to 16 carbon atoms, such as preferably and for example, ethylamine, diethylamine, triethylamine, N,N-diisopropylethyl amine, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, procaine, dicyclohexylamine, dibenzyl-amine, N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylene-diamine.
Within the scope of the invention, solvates refer to forms of the compounds according to the invention which form a complex in a solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates in which molecules are coordinated with water. Hydrates are the preferred solvates within the scope of the present invention.
Furthermore, the present invention also includes prodrugs of the compounds according to the invention. The term “prodrugs” here refers to compounds which may be biologically active or inactive themselves but are converted (e.g., metabolically or hydrolytically) to the compounds according to the invention during their dwell time in the body.
Within the scope of the present invention, the substituents have the following meanings, unless otherwise specified:
(C1-C4)-Alkyl within the scope of the invention stands for a linear or branched alkyl radical with 1 to 4 carbon atoms. The following can be mentioned, preferably and for example: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
(C1-C12)-Alkanediyl, (C1-C8)-alkanediyl and (C1-C6)-alkanediyl, within the scope of the invention, stand for a linear α,ω-divalent alkyl radical having 1 to 12, 1 to 8 or 1 to 6 carbon atoms. A linear alkanediyl group having 1 to 8, especially preferably 1 to 6 carbon atoms is preferred. The following can be mentioned preferably and for example: methylene, ethane-1,2-diyl (1,2-ethylene), propane-1,3-diyl (1,3-propylene), butane-1,4-diyl (1,4-butylene), pentane-1,5-diyl (1,5-pentylene), hexane-1,6-diyl (1,6-hexylene), heptane-1,7-diyl (1,7-hexylene), octane-1,8-diyl (1,8-octylene), nonane-1,9-diyl (1,9-nonylene), decane-1,10-diyl (1,10-decylene), undecane-1,11-diyl (1,11-undecylene) and dodecane-1,12-diyl (1,12-dodecylene).
(C1-C4)-Alkanediyl, within the scope of the invention, stands for a linear α,ω-divalent alkyl radical having 1 to 4 carbon atoms. Preferred examples include: methylene, ethane-1,2-diyl (1,2-ethylene), propane-1,3-diyl (1,3-propylene) and butane-1,4-diyl (1,4-butylene).
(C2-C4)-Alkenediyl, within the scope of the invention, stands for a linear α,ω-divalent alkenyl radical having 2 to 4 carbon atoms and a double bond. Preferred examples include: ethene-1,2-diyl, propene-1,3-diyl, but-1-ene-1,4-diyl and but-2-ene-1,4-diyl. The double bond here may be in a cis- or trans-configuration.
(C1-C12)-Alkoxy, within the scope of the invention, stands for a linear or branched alkoxy radical having 1 to 4 carbon atoms. Preferred examples include: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and tert-butoxy.
(C1-C4)-Alkoxycarbonyl, within the scope of the invention, stands for a linear or branched alkoxy radical having 1 to 4 carbon atoms, linked to the oxygen atom via a carbonyl group [—C(═O)—]. Preferred examples include: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl and tert-butoxycarbonyl.
(C3-C6)-Cycloalkyl, within the scope of the invention, stands for a monocyclic, saturated cycloalkyl group having 3 to 6 carbon atoms. Preferred examples include: cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
A 5-membered heteroaryl in the definition of the ring Het, stands for a divalent aromatic heterocycle (heteroaromatic) having a total of five ring atoms, containing up to three ring heteroatoms, which may be the same or different, from the series of N, O and/or S, and linked via two ring carbon atoms or optionally one ring nitrogen atom and one ring carbon atom. Examples include: furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, 1,2-oxazolyl, 1,3-oxazolyl, 1,2-thiazolyl, 1,3-thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl and 1,3,4-thiadiazolyl. A 5-membered heteroaryl having two or three heteroatoms, which may be the same or different, from the series of N, O and/or S, such as in particular pyrazolyl, imidazolyl, 1,3-oxazolyl, 1,3-thiazolyl, 1,2,4-triazolyl, 1,2,4-oxadiazolyl and 1,3,4-oxadiazolyl.
Within the scope of the present invention, it is true that for all radicals that occur several times, their meanings are independent of one another. If radicals are substituted in the compounds according to the invention, then the radicals may be substituted one or more times, unless otherwise specified. Substitution with one or two substituents that are the same or different is preferred. Substitution with one substituent is especially preferred.
Preferred within the scope of the present invention are compounds of formula (I) in which:
L stands for linear (C1-C8)-alkanediyl, in which (a) two carbon atoms in 1,3- or 1,4-relation to one another may be bridged by including one or two of the carbon atoms between them to form a phenyl ring, or (b) up to two CH2 groups not vicinal to one another may be replaced by —O—,
and
T stands for a group of the formula
wherein
* denotes the linkage site to the nitrogen atom,
R1 denotes phenyl or 1H-indol-3-yl,
and
R2 denotes hydrogen or a group of the formula
wherein
** denotes the linkage site to the radical of the respective group T,
A denotes ethene-1,2-diyl or propene-1,3-diyl,
R3 stands for phenyl, which may be substituted with (C1-C4)-alkoxycarbonyl or carboxyl,
Het is a divalent 5-membered heteroaryl ring selected from the series of pyrazolyl, imidazolyl, 1,3-oxazolyl, 1,3-thiazolyl, 1,2,4-oxadiazolyl and 1,3,4-oxadiazolyl,
and
R5 denotes a phenyl, which may be substituted with (C1-C4)-alkoxycarbonyl or carboxyl,
as well as their salts and solvates and the solvates of the salts.
Especially preferred within the scope of the present invention are compounds of formula (I) in which:
L stands for linear (C1-C6)-alkanediyl,
and
T stands for a group of the formula
wherein
* denotes the linkage site to the nitrogen atom,
and
R2 denotes hydrogen or a group of the formula
wherein
** denotes the linkage site to the radical of the respective group T,
A denotes ethene-1,2-diyl,
R3 denotes phenyl, which may be substituted with methoxycarbonyl or carboxyl,
Het is 1,3,4-oxadiazol-2,5-yl,
and
R5 is a phenyl, which may be substituted with methoxycarbonyl or carboxyl,
as well as their salts and solvates and the solvates of the salts.
Especially important within the scope of the present invention are compounds of formula (I), in which:
L stands for propane-1,3-diyl,
as well as their salts and solvates and the solvates of the salts.
Especially important within the scope of the present invention are compounds of formula (I), in which:
T stands for a group of the formula
in which
* denotes the linkage site to the nitrogen atom,
and
R2A has the meanings of R2 defined above, but does not stand for hydrogen,
as well as their salts and solvates and the solvates of the salts.
The definitions of the radicals given in detail in the respective combinations and/or preferred combinations of radicals are also replaced by definitions of any radicals in other combinations, regardless of the respective combinations indicated. Most especially preferred are combinations of two or more of the preferred ranges defined above.
An additional subject matter of the present method is a method for preparing the compounds of formula (I) according to the invention, characterized in that a compound of formula (II)
in which T has the meanings given above,
is reacted in an inert solvent, either
[A] by base-induced alkylation with a compound of formula (III)
in which L has the meaning given above,
E1 stands for hydrogen, (C1-C4)-alkyl or benzyl,
and
X stands for a leaving group, such as chloride, bromide, iodide, mesylate, triflate or tosylate,
to form a compound of formula (IV)
in which E1, L and T have the meanings given above,
and then in the case when E1 stands for (C1-C4)-alkyl or benzyl, this ester radical is split off by conventional methods, so that, just as in the case when E1 in formula (III) stands for hydrogen, the carboxylic acid of formula (I)
in which L and T have the meanings given above,
is obtained,
or
[B] by reacting with a compound of formula (V)
E1 stands for hydrogen, (C1-C4)-alkyl or benzyl,
and
LA has the meaning of L given above, but is shortened by one CH2 unit in the alkyl chain length,
in the presence of a suitable reducing agent is converted to a compound of formula (VI)
in which E1, LA and T have the meanings given above,
and then in the case when E1 stands for (C1-C4)-alkyl or benzyl, this ester radical is split off by conventional methods, so that, just as in the case when E1 in formula (V) stands for hydrogen, the carboxylic acid of formula (I-A)
in which LA and T have the meanings given above,
is obtained,
and the resulting compounds of formulas (I) and/or (I-A) are optionally separated into their enantiomers and/or diastereomers and/or reacted with the corresponding (i) solvents and/or (ii) bases or acids to form their solvates, salts and/or solvates of the salts.
Examples of suitable inert solvents for the reaction of (II)+(III)→(IV) include ethers such as diethyl ether, diisopropyl ether, methyl-tert-butylmethyl ether, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether, hydrocarbons such as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane or petroleum fractions or dipolar aprotic solvents such as acetone, methyl ethyl ketone, acetonitrile, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N,N′-dimethylpropylene urea (DMPU), N-methylpyrrolidinone (NMP) or pyridine. It is also possible to use mixtures of such solvents. Acetone or N,N-dimethylformamide is preferred.
Suitable bases for these alkylation reactions include in particular alkali hydroxides, such as lithium hydroxide, sodium hydroxide or potassium hydroxide, alkali carbonates or alkaline earth carbonates, such as lithium carbonate, sodium carbonate, potassium carbonate, calcium carbonate or cesium carbonate or the usual organic amines such as triethylamine, N-methylmorpholine, N-methylpiperidine, N,N-diisopropylethylamine, pyridine or 4-N,N-dimethylaminopyridine. Potassium or cesium carbonate is preferably used. It is optionally advantageous to add an alkylating catalyst, such as lithium bromide or iodide, sodium or potassium iodide, tetra-n-butylammonium bromide or iodide or benzyltriethylammonium bromide, for example.
The reaction (II)+(III)→(IV) is generally carried out in a temperature range from −20° C. to +100° C., preferably at 0° C. to +50° C. The reaction may take place at normal, elevated or reduced pressure (e.g., from 0.5 bar to 5 bar). It is usually carried out under normal pressure.
The reaction (II)+(V)→(VI) takes place in solvents that are inert under the reaction conditions and are typically used for reductive amination, optionally in the presence of an acid and/or a water-withdrawing agent as the catalyst. Such solvents include, for example, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol or tert-butanol, ethers such as tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether or other solvents such as dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide or even water. Likewise, it is possible to use mixtures of these solvents. The preferred solvent for use here is a 1,4-dioxane/water mixture to which acetic acid or dilute hydrochloric acid is added as a catalyst.
Suitable reducing agents for this reaction include in particular complex borohydrides, such as sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride or tetra-n-butylammonium borohydride. Sodium cyanoborohydride is preferred.
The reaction (II)+(V)→(VI) is generally carried out in a temperature range from 0° C. to +120° C., preferably at +50° C. to +100° C. The reaction may take place at normal, elevated or reduced pressure (e.g., from 0.5 bar to 5 bar). It is usually carried out under normal pressure.
An ester radical E1 is split off by the usual methods in the process steps (IV)→(I) and (VI) [E1=(C1-C4)-alkyl or benzyl] according to the usual methods by treating the ester with an acid or a base in an inert solvent, whereby in the last variant, the carboxylate salt obtained first is converted to the free carboxylic acid by subsequent addition of an acid. In the case of a tert-butyl ester, the cleavage is preferably performed by using an acid. In the case of a benzyl ester, the cleavage may also take place by hydrogenolysis in the presence of a suitable palladium catalyst, such as palladium on activated carbon, for example.
The ester radical E1 originating from compound (III) and/or (V) is selected here so that the conditions of its cleavage are compatible with the respective group T in compounds (IV) and (VI).
The usual inorganic bases are suitable as the bases for ester hydrolysis. These include in particular alkali hydroxides or alkaline earth hydroxides such as lithium, sodium, potassium or barium hydroxide, or alkali carbonates or alkaline earth carbonates such as sodium, potassium or calcium carbonates. Lithium, sodium or potassium hydroxide is preferred.
Suitable acids for the ester cleavage reaction include in general sulfuric acid, hydrochloric acid/hydrogen chloride, hydrobromic acid/hydrogen bromide, phosphoric acid, acetic acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid or trifluoromethanesulfonic acid or mixtures thereof, optionally with the addition of water. Hydrochloric acid or trifluoroacetic acid are preferred in the case of a tert-butyl ester and hydrochloric acid is preferred in the case of a methyl ester.
Suitable inert solvents for these reactions include water or the organic solvents typically used for ester cleavage. These preferably include low alcohols such as methanol, ethanol, n-propanol or isopropanol, ethers such as diethyl ether, tetrahydrofuran, 1,4-dioxane or 1,2-dimethoxyethane, or other solvents such as dichloromethane, acetone, methyl ethyl ketone, N,N-dimethylformamide or dimethyl sulfoxide. It is also possible to use mixtures of these solvents. In the case of a basic ester hydrolysis, mixtures of water with 1,4-dioxane, tetrahydrofuran, methanol, ethanol and/or dimethylformamide are preferred for use here. In the case of the reaction with trifluoroacetic acid, dichloromethane is preferred, and in the case of the reaction with hydrochloric acid, tetrahydrofuran, diethyl ether, 1,4-dioxane or water is preferred.
The ester cleavage generally takes place in a temperature range of −20° C. to +100° C., preferably at 0° C. to +50° C.
The compounds of formula (II) can be synthesized by the usual methods of peptide chemistry by coupling a compound of formula (VII)
in which
PG stands for an amino protective group such as (9H-fluoren-9-ylmethoxy)carbonyl, tert-butoxycarbonyl or benzyloxycarbonyl,
in an inert solvent with activation of the carboxyl function in (VII), either
[C] first with a compound of formula (VIII)
in which
E2 stands for hydrogen, (C1-C4)-alkyl or benzyl,
or a salt of this compound to form a compound of formula (IX)
in which E2 and PG have the meanings given above,
then in the event that E2 stands for (C1-C4)-alkyl or benzyl, this ester radical is split off by the usual methods and the resulting carboxylic acid of formula (X)
in which PG has the meaning given above,
then in an inert solvent with activation of the carboxyl function with a compound of formula (XI):
H2N-T (XI),
in which T has the meanings given above,
or with a salt of this compound to form a compound of formula (XII)
in which PG and T have the meanings given above,
or
[D] with a compound of formula (XIII)
in which T has the meanings given above,
or with a salt of this compound, likewise to form the compound of formula (XII)
in which PG and T have the meanings given above,
and the compound of formula (XII) is then deprotected in the usual way to form a compound of formula (II)
in which T has the meanings given above.
The coupling reactions described above (formation of amide from the respective amine and carboxylic acid components) are performed according to the standard methods of peptide chemistry (see, for example, M. Bodanszky, Principles of Peptide Synthesis, Springer Verlag, Berlin, 1993; M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, Springer Verlag, Berlin, 1984; H. D. Jakubke and H. Jeschkeit, Aminosäuren, Peptide, Proteine [Amino Acids, Peptides, Proteins], Verlag Chemie, Weinheim, 1982).
Inert solvents for these coupling reactions (VII)+(VIII)→(IX), (X)+(XI)→(XII) and (VII), (XIII)→(XII) include, for example, ethers such as diethyl ether, diisopropyl ether, tert-butylmethyl ether, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether, hydrocarbons such as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane or petroleum fractions, halohydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, 1,2-dichloroethane, trichloroethylene or chlorobenzene or dipolar aprotic solvents such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N,N′-dimethylpropylene urea (DMPU) or N-methylpyrrolidinone (NMP). It is also possible to use mixtures of such solvents. N,N-Dimethylformamide is preferred.
Suitable activation/condensation agents for these coupling reactions include, for example, carbodiimides such as N,N′-diethyl, N,N′-dipropyl, N,N′-diisopropyl, N,N′-dicyclohexylcarbodiimide (DCC) or N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), phosgene derivatives such as N,N′-carbonyldiimidazole (CDI) or isobutyl chloroformate, 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulfate or 2-tert-butyl-5-methylisoxazolium perchlorate, acylamino compounds, such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, α-chlorenamines such as 1-chloror-2-methyl-1-dimethylamino-1-propene, phosphorus compounds such as propane phosphonic acid anhydride, cyanophosphonic acid diethyl ester, bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride, benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yloxy-tris-(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), or uronium compounds such as O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), 2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) or O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), optionally in combination with additional excipients, such as 1-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (HOSu) as well as bases, such as alkali carbonates, e.g., sodium or potassium carbonate or tertiary amine bases, such as triethylamine, N-methylmorpholine, N-methylpiperidine, N,N-diisopropylethylamine, pyridine or 4-N,N-dimethylaminopyridine.
Within the context of the present invention, the preferred activation/condensation agents for such coupling reactions include N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) in combination with 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), likewise in combination with N,N-diisopropylethylamine.
The coupling reactions (VII)+(VIII)→(IX), (X)+(XI)→(XII) and (VII)+(XIII)→(XII) are usually performed in a temperature range from −20° C. to +60° C., preferably at 0° C. to +40° C. The reactions may be performed under normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is customary to work under normal pressure.
The functional groups optionally present in the compounds—such as amino, hydroxyl and carboxyl groups in particular—may also, if expedient or necessary, be present in a temporarily protected form in the process steps described above. Such protective groups are introduced and removed according to the standard methods of peptide chemistry (see, for example, T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Wiley, New York, 1999; M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, Springer Verlag, Berlin, 1984). In the presence of several protected groups, they may optionally be released again simultaneously in either a one-pot reaction or in separate reaction steps.
The preferred amino protective group is tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or (9H-fluoren-9-ylmethoxy)carbonyl (Fmoc); tert-butyl or benzyl is preferably used as the protective group PG2 for a hydroxyl or carboxyl function. A tert-butyl or tert-butoxycarbonyl group is usually split off by treating it with a strong acid such as hydrochloric acid, hydrobromic acid or trifluoroacetic acid in an inert solvent such as diethyl ether, 1,4-dioxane, dichloromethane or acetic acid. This reaction may optionally also be performed without adding an inert solvent. In the case of benzyl or benzyloxycarbonyl as the protective group, such a protective group is preferably removed by hydrogenolysis in the presence of a suitable palladium catalyst, such as palladium on activated carbon, for example. The (9H-fluoren-9-ylmethoxy)carbonyl group is generally split off with the help of a secondary amine base, such as diethylamine or piperidine.
An ester radical E2 in compound (VIII) [E2=(C1-C4)-alkyl or benzyl] here is selected so that the conditions of its being split off are compatible with the respective protective group PG from compound (VII).
The compounds of formula (VII) can be synthesized by a similar method, for example, by first coupling N-(benzyloxycarbonyl)-L-valine of formula (XIV)
in which Z stands for the benzyloxycarbonyl protective group,
with a compound of formula (XV) with the help of a condensation agent:
in which E3 stands for (C1-C4)-alkyl,
or with a salt of this compound to form a compound of formula (XVI)
in which E3 and Z have the meanings given above,
then, after hydrogenolytic removal of the Z-protective group, this compound is then coupled with N-protected N-methyl-L-valine of the formula (XVII) in the presence of a condensation agent:
in which
PG stands for an amino protective group, such as (9H-fluoren-9-ylmethoxy)carbonyl, tert-butoxycarbonyl or benzyloxycarbonyl,
to form a compound of the formula (XVIII)
in which E3 and PG have the meanings given above,
and then the ester group —C(O)O-E3 in (XVIII) is reacted by the usual methods to form the free carboxylic acid (VII).
The coupling reactions (XIV)+(XV)→(XVI) and Z-deprotected (XVI)+(XVII)→(XVIII) are performed under reaction conditions similar to those described above for the coupling steps shown in methods [C] and [D].
The ester group —C(O)O-E3 is hydrolyzed in reaction step (XVIII)→(VII) in a process similar to that described above as part of the process sequences [A] and [B] for the ester radical E1. The alkyl group E3 in compound (XV) is selected here so that the conditions of their cleavage are compatible with the respective protective group PG from compound (XVII).
The compounds of formula (XIII) are in turn accessible by coupling the compound (XI) described above with the compound (XIX):
in which Boc stands for the tert-butoxycarbonyl protective group,
to yield a compound of formula (XX)
in which Boc and T have the meanings given above,
and then splitting off the Boc protective group.
The coupling reaction (XI)+(XIX)→(XX) is in turn performed under similar conditions like those described above for the coupling steps in methods [C] and [D].
The compounds of formulas (III), (V), (VIII), (XI), (XIV), (XV), (XVII) and (XIX), including chiral or diastereomeric forms thereof, if applicable, are available commercially or have been described as such in the literature or they can be synthesized by methods like those published in the literature in a manner that would be self-evident for those skilled in the art. Numerous detailed publications and specifications in the literature concerning the synthesis of the starting materials can also be found in the Experimental Part in the section on the synthesis of the starting compounds and intermediates.
If corresponding isomer-pure starting materials are not available, then the compounds according to the invention can expediently be separated into the corresponding enantiomers and/or diastereomers already at the stage of the compounds (II), (IV), (VI), (XI), (XII), (XIII) and (XX), which are then reacted further in isolated form according to the reaction steps described above. Such a separation of the stereoisomers can be performed according to the usual methods familiar to those skilled in the art. Chromatographic methods on chiral and/or achiral separation phases are preferably used. In the case of free carboxylic acids as the intermediates, separation via diastereomeric salts with the help of chiral bases may also be performed as an alternative.
Synthesis of the compounds according to the invention can be illustrated by the following reaction schemes as an example:
These compounds have valuable pharmacological properties and can be used for preventing and treating diseases humans and animals.
In comparison with other auristatin derivatives known from the prior art, the N-terminal carboxyalkyl group [HOOC-L- in formula (I)] present in the compounds according to the present invention does not have the mere function of a linker for the potential linkage to antibody proteins or other ligands, but instead is a constituent structural element for the surprisingly advantageous profile of properties of these compounds.
These compounds according to the invention have a stronger cytotoxic activity in comparison with monomethylauristatin F (MMAF), for example, or have a reduced potential, while at the same time also being substrates for cellular transporter proteins.
The compounds according to the invention are therefore particularly suitable for treatment of hyperproliferative diseases in humans and mammals in general. These compounds can on the one hand inhibit, block, reduce or restrict cell proliferation and cell division while increasing apoptosis on the other hand.
The hyperproliferative diseases for treatment of which the compounds according to the invention may be used include in particular the group of cancers and tumor diseases. These are understood to include in particular the following diseases within the scope of the present invention without being limited to these: breast cancer and breast tumors (ductile and lobular forms, also in situ), respiratory tract tumors (small cell and non-small-cell carcinomas, bronchial carcinoma), brain tumors (e.g., of the brain stem and the hypothalamus, astrocytoma, medulloblastoma, ependymoma and neuroectodermal and pineal tumors), tumors of the digestive tract (esophagus, stomach, gallbladder, small intestine, large intestine, rectum), liver tumors (including hepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma), tumors of the head and neck area (larynx, hypopharynx, nasopharynx, oropharynx, lips and oral cavity), skin tumors (squamous epithelial carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer and non-melanoma type skin cancer), tumors of the soft tissues (including soft tissue sarcomas, malignant fibrous histiocytoma, lymphosarcoma and rhabdomyosarcoma), tumors of the eyes (including intraocular melanoma and retinoblastoma), tumors of the endocrine and exocrine glands (e.g., thyroid and parathyroid glands, pancreatic gland and esophageal gland), tumors of the urinary tract (bladder, penis, kidney, renal pelvis and urethral tumors) as well as tumors of the reproductive organs (endometrium, cervical, ovarian, vaginal, vulval and uterine carcinomas in the woman and prostatic and testicular carcinomas in males). These also include proliferative blood diseases in solid form and as circulating blood cells such as lymphomas, leukemias and myeloproliferative diseases, e.g., acute myeloid leukemia, acute lymphoblastic, chronic lymphocytic leukemia, chronic myelogenous leukemia and hairy cell leukemia as well as AIDS-related lymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Burkitt's lymphomas and lymphomas of the central nervous system.
These human diseases, which have been characterized well, may also occur with a comparable etiology in other mammals and can also be treated with the compounds according to the present invention in those cases.
Treatment of the types of cancer mentioned above by means of the compounds according to the invention includes treatment of such tumors as well as treatment of metastatic or circulating forms thereof.
The terms “treatment” or “to treat” are used in the conventional sense within the scope of this invention and refers to the care, treatment and consultation of a patient with the goal of combatting, reducing, diminishing or ameliorating a disease or health deviation and improving the quality of life, which is impaired by this disease, such as in a cancer, for example.
An additional subject matter of the present invention thus relates to the use of the compounds according to the invention for treatment and/or prevention of diseases, in particular the diseases cited above.
An additional subject matter of the present invention is the use of the compounds according to the invention for producing a pharmaceutical drug for treatment and/or prevention of diseases, in particular the diseases cited above.
An additional subject matter of the present invention is the use of the compounds according to the invention in a method for treatment and/or prevention of diseases, in particular the diseases cited above.
An additional subject matter of the present invention is a method for treatment and/or prevention of diseases, in particular the diseases cited above, using an effective amount of at least one of the compounds according to the invention.
The compounds according to the invention may be used alone or, if necessary, in combination with one or more other pharmacologically active substances, as long as this combination does not lead to adverse effects and unacceptable side effects. Another subject matter of the present invention therefore relates to pharmaceutical drugs containing at least one of the compounds according to the invention and one or more additional active ingredients, in particular for treating and/or preventing the diseases listed above.
The compounds according to the invention may be combined with known antihyperproliferative, cytostatic or cytotoxic substances, for example, for treatment of cancer. Examples of suitable combination drugs include the following:
aldesleukin, alendronic acid, alfaferone, alitretinoin, allopurinol, aloprim, aloxi, altretamine, amino glutethimide, amifostine, amrubicin, amsacrine, anastrozol, anzmet, aranesp, arglabin, arsentrioxide, aromasine, 5-azacytidine, azathioprine, BCG or tice-BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, broxuridine, bortezomib, busulfan, calcitonin, campath, capecitabine, carboplatin, casodex, cefesone, celmoleukin, cerubidine, chlorambucil, cisplatin, cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome, decadrone, decadrone phosphate, delestrogen, denileukin diftitox, depo medrol, desloreline, dexrazoxane, diethylstilbestrol, diflucan, docetaxel, doxifluridine, doxorubicin, dronabinol, DW-166HC, eligard, elitek, ellence, emend, epirubicin, epoetin alfa, epogen, eptaplatin, ergamisole, estrace, estradiol, estramustine sodium phosphate, ethinyl estradiol, ethyol, etidronic acid, etopophos, etoposide, fadrozole, farstone, filgrastim, finasteride, fligrastim, floxuridine, fluconazole, fludarabin, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, formestane, fosteabine, fotemustine, fulvestrant, gammagard, gemcitabine, gemtuzumab, gleevec, gliadel, gosereline, granisetrone hydrochloride, histreline, hycamtine, hydrocortone, erythrohydroxynonyladenine, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, interferon-alpha, interferon-alpha-2, interferon-alpha-2α, interferon-alpha-2β, interferon-alpha-n1, interferon-alpha-n3, interferon-beta, interferon-gamma-1α, interleukin-2, intron A, iressa, irinotecan, kytril, lentinane sulfate, letrozole, leucovorine, leuprolide, leuprolide acetate, levamisole, levofolic acid calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamine, medroxyprogesterone acetate, megestrole acetate, melphalane, menest, 6-mercaptopurine, mesna, methotrexate, metvix, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myocet, nedaplatin, neulasta, neumega, neupogen, nilutamide, nolvadex, NSC-631570, OCT-43, octreotide, ondansetrone hydrochloride, orapred, oxaliplatin, paclitaxel, pediapred, pegaspargase, pegasys, pentostatin, picibanil, pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustin, prednisolone, prednisone, premarin, procarbazine, procrit, raltitrexed, rebif, rhenium 186 etidronate, rituximab, roferone A, romurtide, salagen, sandostatin, sargramostim, semustine, sizofiran, sobuzoxane, solu-medrol, streptozocin, strontium-89 chloride, synthroid, tamoxifen, tamsulosine, tasonermine, tastolactone, taxoter, teceleukin, temozolomide, teniposide, testosterone propionate, testred, thioguanine, thiotepa, thyro-tropin, tiludronic acid, topotecan, toremifen, tositumomab, tastuzumab, teosulfane, tre-tinoin, trexall, trimethylmelamine, trimetrexate, triptoreline acetate, triptoreline pamoate, uft, uridine, valrubicin, vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, virulizine, zinecard, zinostatin stimalamer, zofran; ABI-007, acolbifene, actimmune, affinitak, aminopterine, arzoxifene, asoprisnil, atamestane, atrasentane, avastin, BAY 43-9006 (sorafenib), CCI-779, CDC-501, celebrex, cetuximab, crisnatol, cyproterone acetate, decitabin, DN-101, doxorubicin MTC, dSLIM, dutasteride, edotecarin, eflornithine, exatecane, fenretinide, histamine dihydrochloride, histreline-hydrogel implant, holmium-166-DOTMP, ibandronic acid, interferon-gamma, intron PEG, ixabepilone, keyhole limpet hemocyanine, L-651582, lanreotide, lasofoxifen, libra, lona-farnib, miproxifen, minodronate, MS-209, liposomales MTP-PE, MX-6, nafareline, nemorubicin, neovastat, nolatrexed, oblimersen, onko-TCS, osidem, paclitaxel polyglutamate, pamidronate disodium, PN-401, QS-21, quazepam, R-1549, raloxifene, ranpirnase, 13-cis-retinic acid, satraplatin, seocalcitol, T-138067, tarceva, taxoprexine, thymosine-alpha-1, tiazofurine, tipifarnib, tirapazamine, TLK-286, toremifene, transmid 107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acid as well as combinations thereof.
In a preferred embodiment, the compounds according to the present invention may be combined with antihyperproliferative agents, which may include the following, for example, although this list is not conclusive:
aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, bleomycin, busulfan, carboplatin, carmustin, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, 2′,2′-difluorodeoxycytidine, docetaxel, doxorubicin (adriamycin), epirubicin, epothilone and seine derivate, erythrohydroxynonyladenine, ethinyl estradiol, etoposide, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluordeoxyuridine monophosphate, 5-fluoruracil, fluoxymesterone, flutamide, hexamethyl melamine, hydroxyurea, hydroxyprogesterone caproate, idarubicin, ifosfamide, interferon, irinotecan, leucovorin, lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, paclitaxel, pentostatin, n-phosphonoacetyl L-aspartate (PALA), plicamycin, prednisolone, prednisone, procarbazine, raloxifene, semustine, streptozocin, tamoxifen, teniposide, testosterone propionate, thioguanine, thiotepa, topotecan, trimethylmelamine, uridine, vinblastine, vincristine, vindesine and vinorelbine.
According to one very promising feature, the compounds according to the invention can also be combined with biological therapeutic agents such as antibodies (e.g., Avastin, Rituxan, Erbitux, Herceptin). The compounds according to the invention may also achieve positive effects in combination with treatments directed against angiogenesis, for example, with Avastin, axitinib, recentin, regorafenib, sorafenib or sunitinib. Combinations with inhibitors of the proteasome and of mTOR as well as combinations with antihormones and steroidal metabolic enzyme inhibitors are also especially suitable because of their favorable profile of side effects.
In general, the following goals can be pursued with the combination of compounds of the present invention with other active cytostatic or cytotoxic agents:
improved efficacy in retarding the growth of a tumor, reducing its size or even completely eliminating it in comparison with treatment with a single drug;
the possibility of using the chemotherapeutic drugs in a lower dose than in monotherapy;
the possibility of a tolerable therapy with few adverse effects in comparison with a single dose;
the possibility of treatment of a broader spectrum of tumors;
achieving a higher response rate to the treatment;
longer survival time for patients in comparison with today's standard therapy.
In addition, the compounds according to the invention may also be used in combination with radiation therapy and/or a surgical intervention.
Another subject matter of the present invention relates to pharmaceutical drugs containing at least one compound according to the invention, usually together with one or more inert, non-toxic, pharmaceutically suitable excipients as well as their use for the purposes indicated above.
The compounds according to the invention may can systemically and/or topically. To this end, they are administered by a suitable route, for example, by an oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, transdermal, conjunctival or otic route or as an implant and/or a stent.
For these methods of administration, the compounds according to the invention may be administered in suitable dosage forms.
For oral administration, the suitable dosage forms that function according to the state of the art and deliver the compounds according to the invention rapidly and/or in a modified form contain the compounds according to the invention in a crystalline and/or amorphized and/or dissolved form, e.g., tablets (coated or uncoated tablets, for example, with enteric coatings or insoluble coatings or those with a delayed release that control the release of the compound according to the invention), tablets or films/oblates that disintegrate rapidly in the mouth, films/lyophilisates, capsules (for example, hard or soft gelatin capsules), coated pills, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
Parenteral administration may be used to bypass a resorption step (e.g., intravenous, intra-arterial, intracardiac, intraspinal or intralumbar) or with the inclusion of resorption (e.g., intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Suitable dosage forms for parenteral administration include infusion and injection preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
For the other routes of administration, it is suitable to use inhalation dosage forms (including powder inhalers, nebulizers), nose drops, solutions or sprays, tablets to be administered lingually, sublingually or buccally, films, oblates or capsules, suppositories, ear or eye preparations, vaginal suppositories, aqueous suspensions (lotions, shake mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (e.g., patches), milks, pastes, foams, dusting powders, implants or stents.
Oral or parenteral administration is preferred, in particular oral and intravenous administration.
The compounds according to the invention may be converted to the dosage forms indicated. This may be done in a known way by mixing with inert, non-toxic, pharmaceutically suitable excipients. These excipients include, among others, vehicles (for example, microcrystalline cellulose, lactose, mannitol), solvents (e.g., liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (for example, sodium dodecyl sulfate, polyoxysorbitan oleate), binders (for example, polyvinyl pyrrolidone), synthetic and natural polymers (for example, albumin), stabilizers (e.g., antioxidants such as ascorbic acid), coloring agents (e.g., inorganic pigments such as iron oxides, for example) and taste and/or odor correcting substances).
For parenteral administration in general, it has proven advantageous to administer doses of approx. 0.001 to 1 mg/kg, preferably approx. 0.01 to 0.5 mg/kg body weight to achieve effective results. In oral administration, the dose is approx. 0.01 to 100 mg/kg, preferably approx. 0.01 to 20 mg/kg and most especially preferably 0.1 to 10 mg/kg body weight.
Nevertheless, it may optionally be necessary to deviate from the stated amounts, namely depending on body weight, route of administration, individual response to the active ingredient, type of administration and time or interval in which the substance is administered. This in some cases it may be sufficient to use less than the aforementioned minimum dose, whereas in other cases the aforementioned upper limit must be exceeded. In the case of administration of larger doses, it may be advisable to divide them into multiple individual doses distributed throughout the day.
The following exemplary embodiments illustrate the invention. The invention is not limited to these examples.
The percentage amounts in the following tests and examples are percent by weight, unless otherwise indicated. Parts are parts by weight. Solvent ratios, dilution ratios and concentration data on liquid/liquid solutions are each based on volume.
A. Examples
Abbreviations and Acronyms
abs. absolute
Ac acetyl
aq. aqueous, aqueous solution
Boc tert-butoxycarbonyl
br. wide (in NMR)
sp. example
ca. circa, approx.
CI chemical ionization (in MS)
d doublet (in NMR)
d day(s)
TLC thin-layer chromatography
DCI direct chemical ionization (in MS)
dd doublet of doublet (in NMR)
DMAP 4-N,N-dimethylaminopyridine
DME 1,2-dimethoxyethane
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide
DPBS Dulbecco's phosphate-buffered saline solution
dt doublet of triplet (in NMR)
theor. of the theoretical
EDC N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
EI electron collision ionization (in MS)
eq. equivalent(s)
ESI electron spray ionization (in MS)
FCS fetal calf serum
Fmoc (9H-fluoren-9-ylmethoxy)carbonyl
GC-MS gas chromatography-linked mass spectrometry
sat. saturated
GTP guanosine 5′-triphosphate
h hour(s)
HATU O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate
HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
HOAc acetic acid
HOBt 1-hydroxy-1H-benzotriazole hydrate
HOSu N-hydroxysuccinimide
HPLC high-pressure, high-performance liquid chromatography
HR-MS high-resolution mass spectrometry
conc. concentrated
LC-MS liquid chromatography-linked mass spectrometry
m multiplet (in NMR)
min minute(s)
MS mass spectrometry
MTBE methyl-tert-butyl ether
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
NMM N-methylmorpholine
NMP N-methyl-2-pyrrolidinone
NMR nuclear magnetic resonance spectrometry
PBS phosphate-buffered saline solution
Pd/C palladium on activated carbon
quant. quantitative (in yield)
quart quartet (in NMR)
quint quintet (in NMR)
Rf retention index (in TLC)
RT room temperature
Rt retention time (in HPLC)
singled (in NMR)
t triplet (in NMR)
tert tertiary
TFA trifluoroacetic acid
THF tetrahydrofuran
UV ultraviolet spectrometry
v/v volume to volume ratio (of a solution)
Z benzoxycarbonyl
tog. together
HPLC, LC-MS and GC-MS Methods:
Method 1 (LC-MS)
Instrument: Waters Acquity SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8μ 50 mm×1 mm; eluent A: 1 liter water+0.25 mL 99% formic acid; eluent B: 1 liter acetonitrile+0.25 mL 99% formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A; flow rate: 0.40 mL/min; oven: 50° C.; UV detection: 210-400 nm.
Method 2 (LC-MS)
Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column: Thermo Hypersil GOLD 1.9μ 50 mm×1 mm; eluent A: 1 liter water+0.5 mL 50% formic acid; eluent B: 1 liter acetonitrile+0.5 mL 50% formic acid; gradient: 0.0 min 90% A→0.1 min 90% A→1.5 min 10% A→2.2 min 10% A; flow rate: 0.33 mL/min; oven: 50° C.; UV detection: 210 nm.
Method 3 (LC-MS)
Instrument: Micromass Quattro Micro MS with HPLC Agilent series 1100; column: Thermo Hypersil GOLD 3μ 20 mm×4 mm; eluent A: 1 liter water+0.5 mL 50% formic acid; eluent B: 1 liter acetonitrile+0.5 mL 50% formic acid; gradient: 0.0 min 100% A→3.0 min 10% A→4.0 min 10% A→4.01 min 100% A (flow rate: 2.5 mL/min)→5.00 min 100% A; oven: 50° C.; flow rate: 2 mL/min; UV detection: 210 nm.
Method 4 (LC-MS)
Instrument type MS: Micromass ZQ; instrument type HPLC: HP 1100 series; UV DAD; column: Phenomenex Gemini 3μ 30 mm×3.00 mm; eluent A: 1 liter water+0.5 mL 50% formic acid; eluent B: 1 liter acetonitrile+0.5 mL 50% formic acid; gradient: 0.0 min 90% A→2.5 min 30% A→3.0 min 5% A→4.5 min 5% A; flow rate: 0.0 min 1 mL/min)→2.5 min/3.0 min/4.5 min 2 mL/min; oven: 50° C.; UV detection: 210 nm.
Method 5 (HPLC)
Instrument: HP 1090 series II; column: Merck Chromolith Speed ROD RP-18e, 50 mm×4.6 mm; precolumn: Merck Chromolith guard Cartridge Kit RP-18e, 5 mm×4.6 mm; injection volume: 5 μL; eluent A: 70% HClO4 in water (4 mL/L); eluent B: acetonitrile; gradient: 0.00 min 20% B→4.00 min 20% B; flow rate: 5 mL/min; column temperature: 40° C.
Method 6 (HPLC)
Instrument: Waters 2695 with DAD 996; column: Merck Chromolith Speed ROD RP-18e, 50 mm×4.6 mm; precolumn: Merck Chromolith Guard Cartridge Kit RP-18c, 5 mm×4.6 mm; eluent A: 70% HCLO4 in water (4 mL/L); eluent B: acetonitrile; gradient: 0.00 min 5% B→3.00 min 95% B→4.00 min 95% B; flow rate: 5 mL/min.
Method 7 (LC-MS)
Instrument type: Waters ZQ; instrument type HPLC: Agilent 1100 series; UV DAD; column: Thermo Hypersil GOLD 3μ 20 mm×4 mm; eluent A: 1 liter water+0.5 mL 50% formic acid; eluent B: 1 liter acetonitrile+0.5 mL 50% formic acid; gradient: 0.0 min 100% A→3.0 min 10% A→4.0 min 10% A→4.1 min 100% A (flow rate: 2.5 mL/min); oven: 55° C.; flow rate: 2 mL/min; UV detection: 210 nm.
Method 8 (LC-MS)
Instrument type MS: Waters ZQ; instrument type HPLC: Agilent 1100 series; UV DAD; column: Thermo Hypersil GOLD 3μ 20 mm×4 mm; eluent A: 1 liter water+0.5 mL 50% formic acid; eluent B: 1 liter acetonitrile+0.5 mL 50% formic acid; gradient: 0.0 min 100% A→2.0 min 60% A→2.3 min 40% A→3.0 min 20% A→4.0 min 10% A→4.2 min 100% A (flow rate: 2.5 mL/min); oven: 55° C.; flow rate: 2 mL/min; UV detection: 210 nm.
Method 9 (LC-MS)
Instrument: Waters Acquity SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8μ 50 mm×1 mm; eluent A: 1 liter water+0.25 mL 99% formic acid; eluent B: 1 liter acetonitrile+0.25 mL 99% formic acid; gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% A; oven: 50° C.; flow rate: 0.35 mL/min; UV detection: 210-400 nm.
Method 10 (HPLC)
Instrument: Agilent 1200 series; column: Agilent Eclipse XDB-C18 5μ 4.6 mm×150 mm; pecolumn: Phenomenex KrudKatcher disposable precolumn; injection volume: 5 μL; eluent A: 1 liter water+0.01% trifluoroacetic acid; eluent B: 1 liter acetonitrile; gradient: 0.0 min 10% B→1.00 min 10% B→1.50 min 90% B→5.5 min 10% B; flow rate: 2 mL/min; column temperature: 30° C.
Method 11 (LC-MS)
Instrument: Waters Acquity SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8μ 30 mm×2 mm; eluent A: 1 liter water+0.25 mL 99% formic acid; eluent B: 1 liter acetonitrile+0.25 mL 99% formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A; flow rate: 0.60 mL/min; oven: 50° C.; UV detection: 208-400 nm.
Method 12 (GC-MS)
Instrument: Micromass GCT, GC 6890; column: Restek RTX-35, 15 m×200 μm×0.33 μm; constant flow rate of helium: 0.88 mL/min; oven: 70° C.; inlet: 250° C.; gradient: 70° C., 30° C./min→310° C. (3 min hold).
Method 13 (HR-MS)
Instrument: Thermo Scientific LTQ Orbitrap XL; FTMS ESI Positive
All reactants and reagents whose preparation procedures are not described explicitly below can be acquired commercially from generally accessible sources. For all other reactants and reagents, whose preparation procedures are also not described below and which were not available commercially or which were acquired from sources not generally available, the published literature describing their preparation has been referenced.
Starting Compounds and Intermediates
Starting Compound 1
(2R,3R)-3-[(2S)-1-(tert-Butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Boc-Dolaproin) dicyclohexylamine salt
The title compound can be synthesized by various methods according to procedures described in the literature; see, e.g., Pettit et al., Synthesis 1996, 719; Shioiri et al., Tetrahedron Lett. 1991, 32, 931; Shioiri et al., Tetrahedron 1993, 49, 1913; Koga et al., Tetrahydron Lett. 1991, 32, 2395; Vidal et al., Tetrahedron 2004, 60, 9715; Poncet et al., Tetrahedron 1994, 50, 5345. It was synthesized here according to the procedure by Shioiri et al. (Tetrahedron Lett. 1991, 32, 931).
Starting Compound 2
tert-Butyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino) heptanoate hydrochloride (Dolaisoleucin OtBu×HCl)
The title compound can be synthesized by various methods described in the literature; see, e.g., Pettit et al., J. Org. Chem. 1994, 59, 1796; Koga et al., Tetrahedron Lett. 1991, 32, 2395; Shioiri et al., Tetrahedron Lett. 1991, 32, 931; Shioiri et al., Tetrahedron 1993, 49, 1913. It was synthesized here according to the procedure by Koga et al. (Tetrahedron Lett. 1991, 32, 2395).
Intermediate 1
tert-Butyl-(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methyl heptanoate
425 mg (1.7 mmol) N-[(benzyloxy)carbonyl]-L-valine was dissolved in 50 mL DMF and mixed in succession with 500 mg (1.7 mmol) tert-butyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino) heptanoate hydrochloride (starting compound 2), 356 mg (1.9 mmol) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 285 mg (1.9 mmol) 1-hydroxy-1H-benzotriazole hydrate and 655 mg (5.1 mmol) N,N-diisopropylethylamine. The mixture was stirred for 20 hours at RT. Then 142 mg (0.5 mmol) N-[(benzyloxy)-carbonyl]-L-valine, 119 mg (0.6 mmol) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 95 mg (0.6 mmol) 1-hydroxy-1H-benzotriazole hydrate and 218 mg (1.7 mmol) N,N-diisopropylemethylamine were also added, and the mixture was treated ultrasonically for 90 min. The batch was then poured into a mixture of 50% saturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was separated, then washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated. The residue was then purified by preparative HPLC, yielding 329 mg (40% of the theoretical) of the title compound as a colorless oil.
HPLC (method 5): Rt=2.5 min;
LC-MS (method 1): Rt=1.45 min; MS (ESIpos): m/z=493 (M+H)+.
Intermediate 2
tert-Butyl-(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino)heptanoate
500 mg (1 mmol) tert-Butyl-(3R,4S,5S)-4[{N-[(benzyloxy)carbonyl]-(L-valyl}(methyl)-amino]-3-methoxy-5-methylheptanoate (intermediate 1) was dissolved in 50 mL methanol and hydrogenated for one hour at RT under normal pressure after adding 100 mg 10% palladium on activated carbon. The catalyst was then filtered out and the solvent was removed in vacuo, yielding 370 mg (quantitative) of the title compound as an almost colorless oil.
HPLC (method 5): Rt=1.59 min;
LC-MS (method 1): Rt=0.74 min; MS (ESIpos): m/z=359 (M+H)+.
Intermediate 3
N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
396 mg (1.1 mmol) N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valine was dissolved in DMF and then mixed in succession with 365 mg (1 mmol) tert-butyl-(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoate (intermediate 2), 234 mg (1.2 mmol) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 187 mg (1.2 mmol) 1-hydroxy-1H-benzotriazole hydrate. The mixture was stirred over night at RT. The batch was then poured into a mixture of 50% saturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was separated, washed in succession with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated. The residue was used directly in the next step without further purification.
Yield: 660 mg (68% of the theoretical)
HPLC (method 5): Rt=3.0 min;
LC-MS (method 1): Rt=1.61 min; MS (ESIpos): m/z=694 (M+H)+.
Intermediate 4
N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
650 mg (0.94 mmol) N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 3) was dissolved in 5 mL dichloromethane, mixed with 5 mL trifluoroacetic acid and stirred over night at RT. Then the mixture was concentrated in vacuo, and the remaining residue was purified by preparative HPLC, yielding 430 mg (72% of the theoretical) of the title compound as a colorless foam.
HPLC (method 5): Rt=2.4 min;
LC-MS (method 2): Rt=1.51 min; MS (ESIpos): m/z=638 (M+H)+.
Intermediate 5
N-tert-Butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methyl-hexan-3-yl]-N-methyl-L-valinamide
51 mg (0.08 mmol) N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (intermediate 4) was dissolved in 10 mL DMF and mixed with 0.5 mL piperidine. After stirring for 10 min at RT, the batch was concentrated in vacuo and the residue was stirred with diethyl ether. The insoluble ingredients were filtered out and washed several times with diethyl ether. Then the filter residue was dissolved in 5 mL dioxane/water (1:1) and the solution was adjusted to pH 11 with 1N sodium hydroxide solution. While treating with ultrasound, a total of 349 mg (1.6 mmol) di-tert-butyl dicarbonate was added in several portions, while the pH of the solution was kept at 11. After the end of the reaction, the dioxane was evaporated and the pH of the aqueous solution was adjusted to 2-3 with citric acid. Extraction was performed twice with 50 mL ethyl acetate each time. The organic phases were combined, dried over magnesium sulfate and concentrated in vacuo. The residue was dissolved in diethyl ether and the product was precipitated with pentane. The solvent was separated by decanting. The residue was digested again with pentane and finally dried in a high vacuum, yielding 31 mg (93% of the theoretical) of the title compound.
HPLC (method 6): Rt=2.2 min;
LC-MS (method 2): Rt=1.32 min; MS (ESIpos): m/z=516 (M+H)+.
Intermediate 6
Benzyl-(2R,3S)-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoate trifluoroacetic acid salt
First, (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid was released from 1.82 g (3.88 mmol) of the dicyclohexylamine salt (starting compound 1) by dissolving it in 150 mL ethyl acetate and extracting with 100 mL 0.5% aqueous sulfuric acid. The organic phase was dried over magnesium sulfate, filtered and concentrated. The residue was dissolved in 10 mL dioxane and 10 mL water, mixed with 1517 mg (4.66 mmol) cesium carbonate and treated for 5 min in an ultrasonic bath. It was then concentrated in vacuo and the residue was co-distilled once with DMF. The residue was then dissolved in 15 mL DMF and mixed with 1990 mg (11.64 mmol) benzyl bromide. The mixture was treated in an ultrasonic bath for 15 min and then concentrated in vacuo. The residue was distributed between ethyl acetate and water. The organic phase was separated, washed with sodium chloride solution and then concentrated. The residue was finally purified by preparative HPLC, thereby yielding 1170 mg (80% of the theoretical) of the Boc-protected intermediate tert-butyl-(2S)-2-[(1R,2R)-3-(benzyloxy)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate.
This 1170 mg of the intermediate was immediately dissolved in 15 mL dichloromethane and mixed with 5 mL trifluoroacetic acid. After stirring for 15 min at RT, the batch was concentrated in vacuo and the residue was lyophilized from dioxane. After drying in a high vacuum, there remained 1333 mg (84% of the theoretical) of the title compound as a yellow oil.
HPLC (method 5): Rt=1.5 min;
LC-MS (method 1): Rt=0.59 min; MS (ESIpos): m/z=278 (M+H)+.
Intermediate 7
N-(tert-Butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
1200 mg (2.33 mmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methyl-hexan-3-yl]-N-methyl-L-valinamide (intermediate 5) was combined with 910.8 mg (2.33 mmol) benzyl-(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoate trifluoroacetic acid salt (intermediate 6), 1327 mg (3.49 mmol) O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 2027 μL N,N-diisopropylethylamine in 50 mL DMF and stirred for 5 min at RT. Next the solvent was removed in vacuo. The remaining residue was dissolved in ethyl acetate and then extracted with 5% aqueous citric acid solution and saturated sodium bicarbonate solution in succession. The organic phase was separated and concentrated. The residue was purified by preparative HPLC. The product fractions were combined, concentrated and the residue was dried in a high vacuum, yielding 1000 mg (55% of the theoretical) of the benzyl ester intermediate N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-benzyloxy)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide as a resin.
LC-MS (method 1): Rt=1.56 min; MS (ESIpos): m/z=775 (M+H)+.
The total amount of the intermediate obtained was dissolved in 25 mL of a mixture of methanol and dichloromethane (20:1) and the benzyl ester group was removed by hydrogenation under normal pressure with 10% palladium on activated carbon as the catalyst. After stirring for 30 min at RT, the catalyst was filtered out and the filtrate was concentrated in vacuo, yielding 803 mg (91% of the theoretical) of the title compound as a white solid.
HPLC (method 5): Rt=2.1 min;
LC-MS (method 1): Rt=1.24 min; MS (ESIpos): m/z=685 (M+H)+.
Intermediate 8
Nα-(tert-Butoxycarbonyl)-N-methoxy-N-methyl-L-phenylalaninamide
1000 mg (3.77 mmol) N-(tert-butoxycarbonyl)-L-phenylalanine was placed in 10 mL dichloromethane and mixed with 733 mg (4.52 mmol) 1,1′-carbonyldiimidazole. The batch was stirred for 15 min until the evolution of gas had stopped. Next the mixture was mixed with 441 mg (4.52 mmol) N,O-dimethylhydroxylamine hydrochloride and 657 μL (3.77 mmol) N,N-diisopropylethylamine and stirred for 1 hour at RT. Next the batch was diluted with dichloromethane and washed with distilled water, 0.5N hydrochloric acid and saturated sodium chloride solution in succession. The organic phase was separated and the combined aqueous phases were re-extracted with ethyl acetate. The combined organic phases were dried over magnesium sulfate and concentrated in vacuo, yielding 1090 mg (93% of the theoretical) of the title compound.
LC-MS (method 1): Rt=1.02 min; MS (ESIpos): m/z=309 (M+H)+.
Intermediate 9
tert-Butyl-[(2S)-1-oxo-3-phenylpropan-2-yl]carbamate
1090 mg (3.5 mmol) Nα-(tert-butoxycarbonyl)-N-methoxy-N-methyl-L-phenylalanine-amide was dissolved in 20 mL 2-methyltetrahydrofuran and cooled to 0° C. Then 4.2 mL (4.2 mmol) of a 1M lithium aluminum hydride solution was added slowly to THF, and the reaction mixture was stirred for 30 min at 0° C. Next 5% aqueous potassium hydrogen sulfate solution was added cautiously. The batch was then diluted with water and extracted with MTBE. The organic phase was dried over magnesium sulfate and concentrated in vacuo, yielding 820 mg (94% of the theoretical) of the title compound.
GC-MS (method 12): Rt=5.61 mm; MS (ESIpos): m/z=220 (M−29)+
1H-NMR (500 MHz, DMSO-d6): δ [ppm]=1.15-1.42 (m, 9H), 7.11-7.39 (m, 5H), 9.52 (s, 1H) [additional signals concealed beneath solvent peaks]
Intermediate 10
(2S,3Z)-1,5-Diphenylpent-3-en-2-amine trifluoroacetic acid salt
Under argon, 842 μL (2.1 mmol) 2.5 M n-butyllithium solution in hexane was added to a suspension of 986 mg (2.2 mmol) triphenyl-(2-phenylethyl)phosphonium bromide [can be synthesized, e.g., according to R. W. Hartmann, M. Reichert, Archiv der Pharmazie 333, 145 (2000); K. C. Nicolaou et al., European J. Chem. 1, 467 (1995)] in 125 mL THF at −78° C., and the mixture was then stirred for one hour at 0° C. The reaction mixture was then cooled back to −78° C. and a solution of 500 mg (2.0 mmol) tert-butyl-[(2S)-1-oxo-3-phenylpropan-2-yl]carbamate in 5 mL dry THF was added. The batch was heated to 0° C. and stirred further for three hours at this temperature. The reaction was then terminated by adding saturated aqueous ammonium chloride solution. The mixture was diluted with MTBE, the phases were separated, and the organic phase was dried over magnesium sulfate and concentrated in vacuo. The residue was purified over a silica gel column using cyclohexane/ethyl acetate 5:1 as the mobile phase. After concentrating the corresponding fractions, 173 mg (25.6% of the theoretical) of the Boc-protected intermediate, tert-butyl-[(2S,3Z)-1,5-diphenylpent-3-en-2-yl]carbamate was obtained.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=1.16-1.46 (m, 9H), 2.62 (dd, J=13.20 Hz, 7.34 Hz, 1H), 2.73-3.18 (m, 1H), 4.56 (t, J=7.46 Hz, 1H), 5.27-5.57 (m, 1H), 6.98-7.32 (m, 10H) [additional signals concealed beneath solvent peaks].
173 mg (512 μmol) of the intermediate tert-butyl[2S,3Z)-1,5-diphenylpent-3-en-2-yl]carbamate was placed in 16 mL dichloromethane, mixed with 4 mL trifluoroacetic acid and left to stand for 30 min at RT. Next the reaction mixture was concentrated and the residue was dried in vacuo, yielding 180 mg (99% of the theoretical) of the title compound.
LC-MS (method 1): Rt=0.74 min; MS (ESIpos): m/z=238 (M+H)+.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.64-2.83 (m, 1H), 2.88-3.22 (m, 2H), 4.00-4.55 (m, 1H), 5.16-5.46 (m, 1H), 5.48-5.78 (m, 1H), 6.60-6.89 (m, 2H), 7.14 (s, 3H), 7.22-7.36 (m, 5H), 7.89-8.27 (m, 2H).
Intermediate 11
(2S)-1-(Benzylsulfonyl)-3-phenylpropan-2-amine
200 mg (1.13 mmol) (4S)-4-benzyl-1,3-benzyl-1,3-oxazolidin-2-one was placed in 3 mL tert-butanol and mixed with 280 mg (2.26 mmol) benzylmercaptan. The mixture was then heated at reflux for two days. Then the batch was concentrated on the rotary evaporator and the resulting intermediate (2S)-1-(benzylsulfanyl)-3-phenylpropan-2-amine was reacted further without workup.
HPLC (method 10): Rt=2.63 min;
LC-MS (method 1): Rt=0.67 min; MS (ESIpos): m/z=258 (M+H)+.
The crude intermediate obtained above was dissolved in a solution of 2 mL 30% hydrogen peroxide and 5 mL formic acid and stirred for 12 h at RT. Then the reaction mixture was poured into saturated aqueous sodium sulfate solution and extracted three times with ethyl acetate. The organic phase was dried over magnesium sulfate and concentrated in vacuo. The resulting raw product was purified by preparative HPLC, thus yielding 343 mg (61% of the theoretical) of the title compound.
HPLC (method 10): Rt=2.40 min;
LC-MS (method 1): Rt=0.65 min; MS (ESIpos): m/z=290 (M+H)+.
Intermediate 12
(2S,3Z)-1,4-Diphenylbut-3-en-2-amine
552.7 mg (9.85 mmol) potassium hydroxide was dissolved in methanol, absorbed onto 1.1 g aluminum oxide and then dried in a high vacuum. At 5-10° C., 307 μL (3.3 mmol) dibromodifluoromethane was added by drops to a solution of 240 mg (0.82 mmol) (2S)-1-benzylsulfonyl)-3-phenylpropan-2-amine and 1.56 g of the potassium hydroxide prepared in this way on aluminum oxide in 6.2 mL n-butanol. The reaction mixture was stirred for two hours at RT, then filtered through Celite, and the residue was rewashed well with dichloromethane. The filtrate was concentrated and the resulting residue was dried in vacuo. The resulting raw produce was purified by preparative HPLC, yielding 98 mg (35% of the theoretical) of the title compound with an E/Z diasteromer ratio of 4:1.
HPLC (method 10): Rt=2.46 min;
LC-MS (method 1): Rt=0.75 min; MS (ESIpos): m/z=224 (M+H)+.
The E/Z diastereomer mixture obtained above was dissolved in 2 mL ethanol and 0.2 mL N,N-diisopropylethylamine and separated over HPLC on a chiral phase [column: Daicel Chiralpak AD-H, 5 μm, 250 mm×20 mm; eluent: hexane/(ethanol+0.2% diethylamine) 50:50 v/v; UV detection: 220 nm; temperature: 30° C.]. The corresponding fractions were concentrated on a rotary evaporator and the residue was dried in vacuo, yielding 10 mg of the title compound as a pure Z isomer.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.71 (d, J=6.60 Hz, 2H), 3.73-3.95 (m, 1H), 5.42-5.67 (m, 1H), 6.21-6.50 (m, 1H), 7.08-7.38 (m, 10H) [additional signals concealed beneath solvent peaks].
Intermediate 13
(2S,3E)-1,4-Diphenylbut-3-en-2-amine
The title compound (pure E isomer) was obtained in a yield of 45 mg in the course of chromatographic diastereomer separation on a chiral phase, as described for intermediate 12.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.62-2.83 (m, 2H), 3.52-3.71 (m, 1H), 6.18-6.30 (m, 1H), 6.34-6.46 (m, 1H), 6.98-7.57 (m, 10H) [additional signals concealed beneath solvent peaks].
Intermediate 14
(1S)-2-Phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetic acid salt
200 mg (0.75 mmol) N-(tert-butoxycarbonyl)-L-phenylalanine was placed in 5.5 mL dichloromethane at 0° C. and mixed with 128 mg (0.79 mmol) 1,1′-carbonyldiimidazole. After 30 min, 103 mg (0.75 mmol) benzoyl hydrazide was added. Finally, after 45 min more at 0° C., 500 mg (1.5 mmol) carbon tetrabromide and 395 mg (1.5 mmol) triphenylphosphine were added. The batch was first stirred for 2 h at 0° C. and then stirred over night at RT. The mixture was then concentrated on a rotary evaporator and the residue was dried in a high vacuum. The resulting raw product was purified by preparative HPLC, yielding 217 mg (78% of the theoretical) of the Boc-protected intermediate tert-butyl-[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]carbamate.
HPLC (method 10): Rt=3.01 min;
LC-MS (method 1): Rt=1.15 min; MS (ESIpos): m/z=366 (M+H)+.
217 mg (0.59 mmol) of this intermediate was dissolved in 3 mL dichloromethane, mixed with 0.6 mL trifluoroacetic acid and stirred for 30 min at RT. Then the mixture was concentrated in vacuo. The remaining residue was dried further in vacuo and then lyophilized from dioxane, thereby yielding 214 mg (90% of the theoretical) of the title compound as a white solid.
HPLC (method 10): Rt=2.43 min;
LC-MS (method 1): Rt=0.62 min; MS (ESIpos): m/z=266 (M+H)+.
Intermediate 15
(1R)-2-Phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetic acid salt
200 mg (0.75 mmol) N-(tert-butoxycarbonyl)-D-phenylalanine was placed in 5.5 mL dichloromethane at 0° C. and mixed with 128.3 mg (0.79 mmol) 1,1′-carbonyldiimidazole. After 30 min, 103 mg (0.75 mmol) benzoyl hydrazide was added. Finally, after 45 min more at 0° C., 500 mg (1.5 mmol) carbon tetrabromide and 395 mg (1.5 mmol) triphenylphosphine were added. The batch was first stirred for 2 h at 0° C. and then stirred over night at RT. The mixture was then concentrated on a rotary evaporator and the residue was dried in a high vacuum. The resulting raw product was purified by preparative HPLC, yielding 219 mg (80% of the theoretical) of the Boc-protected intermediate tert-butyl-[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]carbamate.
HPLC (method 10): Rt=3.01 min;
LC-MS (method 2): Rt=1.36 min; MS (ESIpos): m/z=366 (M+H)+.
219 mg (0.6 mmol) of this intermediate was dissolved in 3 mL dichloromethane, mixed with 0.6 mL trifluoroacetic acid and stirred for 30 min at RT. Next the mixture was concentrated in vacuo. The remaining residue was dried further in vacuo and then lyophilized from dioxane, thus yielding 196 mg (86% of the theoretical) of the title compound as a white solid.
HPLC (method 10): Rt=2.41 min.
Intermediate 16
Methyl-4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl)benzoate trifluoroacetic acid salt
0.9 mg (4 μmol) palladium acetate was placed in 5 mL DMF and then mixed in succession with 20.8 mg (97 μmol) methyl-4-bromobenzoate, 20 mg (81 μmol) (S)-tert-butyl-1-phenylbut-3-en-2-ylcarbamate, 1.1 mg (8 μmol) phenylurea and 11.2 mg (81 μmol) potassium carbonate. The reaction mixture was then stirred for 15 min at 160° C. in a microwave apparatus (Emrys™ Optimizer). The mixture was then filtered and the filtrate was separated into its components by preparative HPLC (eluent: methanol/water gradient with 0.1% TFA), yielding 21.3 mg (68% of the theoretical) of the title compound.
HPLC (method 10): Rt=3.23 min;
LC-MS (method 11): Rt=1.32 min; MS (ESIpos): m/z=382 (M+H)+.
Intermediate 17
N-Methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,5-diphenylpent-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
15 mg (22 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) was placed in 750 μL DMF and mixed with 11.44 μL (66 μmol) N,N-diisopropylethylamine and 10 mg (26 μmol HATU. The batch was stirred for 30 min at RT. Then 8.5 mg (24 μmol) (2S,3Z)-1,5-diphenylpent-3-en-2-amine trifluoroacetic acid salt (intermediate 10) was added and the batch was stirred over night at RT. The reaction mixture was then immediately separated into its components by preparative HPLC, yielding 18.1 mg (91% of the theoretical) of the Boc-protected intermediate N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,5-diphenylpent-3-en-2-yl]amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in the form of a white solid.
HPLC (method 10): Rt=4.74 min;
LC-MS (method 11): Rt=1.58 min; MS (ESIpos): m/z=905 (M+H)+.
16 mg (18 μmol) of this intermediate was dissolved in 1 mL dichloromethane, mixed with 0.2 mL trifluoroacetic acid and stirred for 30 min at RT. Then the mixture was concentrated in vacuo. The remaining residue was dried further in vacuo and then the dioxane was lyophilized, thus yielding 15.8 mg (97% of the theoretical) of the title compound.
HPLC (method 10): Rt=2.66 min;
LC-MS (method 1): Rt=1.03 min; MS (ESIpos): m/z=805 (M+H)+.
Intermediate 18
N-Methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
First, N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was synthesized by analogy with the synthesis of intermediate 17 by reacting 20 mg (29 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) with 7.1 mg (32 μmol) (2S,3Z)-1,4-diphenylbut-3-en-2-amine (intermediate 12).
Yield: 9.2 mg (35% of the theoretical)
HPLC (method 10): Rt=4.52 min;
LC-MS (method 1): Rt=1.54 min; MS (ESIpos): m/z=891 (M+H)+.
Then 9.5 mg (99% of the theoretical) of the title compound was obtained by subsequent cleavage of the Boc protective group with trifluoroacetic acid.
HPLC (method 10): Rt=2.58 min;
LC-MS (method 1): Rt=0.97 min; MS (ESIpos): m/z=791 (M+H)+.
Intermediate 19
N-Methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
First, N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was synthesized by analogy with the synthesis of intermediate 17 by reacting 20 mg (29 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) with 7.1 mg (32 μmol) (2S,3E)-1,4-diphenylbut-3-en-2-amine (intermediate 13).
Yield: 15.1 mg (58% of the theoretical)
HPLC (method 10): Rt=4.2 min;
LC-MS (method 1): Rt=1.51 min; MS (ESIpos): m/z=891 (M+H)+.
Then 15.7 mg (99% of the theoretical) of the title compound was obtained by subsequent cleavage of the Boc protective group with trifluoroacetic acid.
HPLC (method 10): Rt=2.62 min;
LC-MS (method 1): Rt=0.97 min; MS (ESIpos): m/z=791 (M+H)+.
Intermediate 20
N-Methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
First, N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was synthesized by analogy with the synthesis of intermediate 17 by reacting 20 mg (29 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) with 9.3 mg (20 μmol) (2S)-1-(benzylsulfanyl)-3-phenylpropan-2-amine (intermediate 11).
Yield: 19.2 mg (68% of the theoretical)
HPLC (method 10): Rt=3.5 min;
LC-MS (method 1): Rt=1.41 min; MS (ESIpos): m/z=957 (M+H)+.
Then 19.3 mg (99% of the theoretical) of the title compound was obtained by subsequently splitting off the Boc protective group with trifluoroacetic acid.
HPLC (method 10): Rt=2.52 min;
LC-MS (method 1): Rt=0.86 min; MS (ESIpos): m/z=857 (M+H)+.
Intermediate 21
N-Methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
First, N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was synthesized by analogy with the synthesis of intermediate 17 by reacting 20 mg (29 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) with 12.2 mg (32 μmol) (1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetic acid salt (intermediate 14).
Yield: 22 mg (81% of the theoretical)
HPLC (method 10): Rt=3.74 min;
LC-MS (method 1): Rt=1.45 min; MS (ESIpos): m/z=933 (M+H)+.
Then 22.4 mg (98% of the theoretical) of the title compound was obtained by subsequently splitting off the Boc protective group with trifluoroacetic acid.
HPLC (method 10): Rt=2.52 min;
LC-MS (method 1): Rt=0.85 min; MS (ESIpos): m/z=833 (M+H)+.
Intermediate 22
N-Methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
First, N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was synthesized by analogy with the synthesis of intermediate 17 by reacting 20 mg (29 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) with 12.2 mg (32 μmol) (1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetic acid salt (intermediate 15).
Yield: 17 mg (64% of the theoretical)
HPLC (method 10): Rt=3.74 min;
LC-MS (method 1): Rt=1.45 min; MS (ESIpos): m/z=933 (M+H)+.
Then 17.1 mg (99% of the theoretical) of the title compound was obtained by subsequently splitting off the Boc protective group with trifluoroacetic acid.
HPLC (method 10): Rt=2.55 min;
LC-MS (method 11): Rt=0.85 min; MS (ESIpos): m/z=833 (M+H)+.
Intermediate 23
N-Methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S,3E)-4-[4-(methoxycarbonyl)phenyl]-1-phenylbut-3-en-2-yl}amino)-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
First, N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S,3E)-4-[4-(methoxycarbonyl)phenyl]-1-phenylbut-3-en-2-yl}amino)-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was synthesized by analogy with the synthesis of intermediate 17 by reacting 20 mg (29 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) with 12.7 mg (32 μmol) methyl-4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzoate trifluoroacetic acid salt (intermediate 16).
Yield: 8.8 mg (32% of the theoretical)
LC-MS (method 1): Rt=1.53 min; MS (ESIpos): m/z=949 (M+H)+.
Then 8 mg (90% of the theoretical) of the title compound was obtained by subsequently splitting off the Boc protective group with trifluoroacetic acid.
LC-MS (method 1): Rt=1.00 min; MS (ESIpos): m/z=849 (M+H)+.
Intermediate 24
N-Methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt
76 μL (438 μmol) N,N-diisopropylethylamine, 83 mg (219 μmol HATU and 26 mg (161 μmol) 2-(1H-indol-3-yl)ethanamine were added to a solution of 100 mg (146 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (intermediate 7) in 30 mL DMF at RT. The mixture was stirred for 15 min at RT. Then the reaction mixture was concentrated in vacuo and the residue was separated into its components by preparative HPLC, yielding 101 mg (83% of the theoretical) of the Boc-protected intermediate, N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-indoll-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
LC-MS (method 1): Rt=1.32 min; m/z=828 (M+H)+.
101 mg (122 μmol) of this intermediate was dissolved in 15 mL dichloromethane, mixed with 1 mL trifluoroacetic acid and stirred for 30 min at RT. Then the mixture was concentrated in vacuo and the remaining residue was lyophilized from water/acetonitrile, yielding 108 mg of the title compound in a quantitative yield as an almost colorless foam.
LC-MS (method 1): Rt=0.84 min; MS (ESIpos): m/z=728 (M+H)+.
Intermediate 25
N-Methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide trifluoroacetic acid salt
The title compound was obtained by analogy with the synthesis of intermediate 24 in two steps, starting with 60 mg (88 μmol) of intermediate 7 by coupling with 10 mg (88 μmol) 2-phenylethanamine and then splitting off Boc using trifluoroacetic acid. this yielded 34 mg (97% of the theoretical) of the title compound.
HPLC (method 5): Rt=2.71 min;
LC-MS (method 1): Rt=0.80 min; MS (ESIpos): m/z=689 (M+H)±.
EXEMPLARY EMBODIMENTS
Example 1
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,5-diphenylpent-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
14.5 mg (16 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,5-diphenylpent-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 17) was dissolved in 1 mL dioxane/water (1:1) and mixed with 20.4 μL (32 μmol) of a 15% aqueous solution of 4-oxobutanoic acid. The batch was then stirred for one hour at 100° C. After cooling to RT, 1.1 mg (17 μmol) sodium cyanoborohydride was added and the mixture was adjusted to a pH of 3 by adding approx. 150 μL 0.1N hydrochloric acid. The batch was then stirred for two hours more at 100° C. Then 1.1 mg (17 μmol) sodium cyanoborohydride was added again and the mixture was next adjusted to a pH of 3 by adding approx. 300 μL 0.1N hydrochloric acid. The batch was then stirred again for two hours at 100° C. If the reaction was still incomplete, this procedure was repeated once more. Finally, the batch was concentrated and the raw product was purified by preparative HPLC and lyophilized from dioxane, thereby yielding 13.1 mg (93% of the theoretical) of the title compound in the form of a white solid.
HPLC (method 10): Rt=2.63 min;
LC-MS (method 1): Rt=1.01 min; MS (ESIpos): m/z=891 (M+H)+.
Example 2
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
9 mg (10 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3Z)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 18) was dissolved in 0.6 mL dioxane/water (1:1) and reacted with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride in a process similar to the synthesis in Example 1. After lyophilization from dioxane, 5.6 mg (64% of the theoretical) of the title compound was obtained in the form of a white solid.
HPLC (method 10): Rt=2.61 min;
LC-MS (method 11): Rt=0.94 min; MS (ESIpos): m/z=877 (M+H)+.
HR-MS (method 13): m/z=876.5.
Example 3
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
15.5 mg (10 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 19) was dissolved in 1.0 mL dioxane/water (1:1) and reacted with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride, by analogy with the synthesis in Example 1. After lyophilization from dioxane, 10.3 mg (68% of the theoretical) of the title compound was obtained in the form of a white solid.
HPLC (method 10): Rt=2.59 min;
LC-MS (method 11): Rt=0.94 min; MS (ESIpos): m/z=877 (M+H)+.
HR-MS (method 13): m/z=876.6;
1H-NMR (500 MHz, dichloromethane-d2): δ [ppm]=0.72-1.21 (m, 18H), 1.23-1.47 (m, 3H), 1.51-2.22 (m, 8H), 2.25-2.54 (m, 5H), 2.65-2.86 (m, 2H), 2.90-3.47 (m, 16H), 3.53-4.46 (m, 6H), 4.71-5.27 (m, 4H), 5.46-5.72 (m, 1H), 6.10-6.36 (m, 1H), 6.44-6.67 (m, 2H), 7.03-7.67 (m, 10H), 9.13 (br. s, 1H)
Example 4
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulfanyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
19.3 mg (20 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulfanyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 20) was dissolved in 1.2 mL dioxane/water (1:1) and reacted with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride, by analogy with the synthesis in Example 1. After lyophilization from dioxane, 8.6 mg (45% of the theoretical) of the title compound was obtained in the form of a white solid.
LC-MS (method 11): Rt=0.85 min; MS (ESIpos): m/z=943 (M+H)+.
HR-MS (method 13): m/z=942.6;
1H-NMR (500 MHz, dichloromethane-d2): δ [ppm]=0.72-1.23 (m, 18H), 1.26-1.56 (m, 2H), 1.60-1.94 (m, 4H), 1.95-2.17 (m, 3H), 2.22-2.54 (m, 5H), 2.69-2.87 (m, 2H), 2.90-3.27 (m, 11H), 3.31-3.53 (m, 8H), 3.58-4.20 (m, 7H), 4.25-4.54 (m, 3H), 4.59-5.15 (m, 4H), 6.22 (br. s, 1H), 6.97-8.00 (m, 10H), 9.13 (br. s, 1H)
Example 5
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
22.4 mg (24 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]-amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 21) was dissolved in 1.4 mL dioxane/water (1:1) and reacted with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride by analogy with the synthesis according to Example 1. After lyophilization from dioxane, 8.2 mg (38% of the theoretical) of the title compound was obtained in the form of a white solid.
HPLC (method 10): Rt=2.54 min;
LC-MS (method 1): Rt=0.94 min; MS (ESIpos): m/z=919 (M+H)+.
HR-MS (method 13): m/z=918.6;
1H-NMR (500 MHz, dichloromethane-d2): δ [ppm]=0.58-1.21 (m, 20H), 1.25-1.52 (m, 2H), 1.62-2.19 (m, 8H), 2.28-2.50 (m, 5H), 2.64-2.84 (m, 2H), 2.89-3.16 (m, 6H), 3.19-3.52 (m, 10H), 3.59-4.00 (m, 4H), 4.02-4.40 (m, 3H), 4.66-5.13 (m, 3H), 5.61 (d, 1H), 7.32 (d, 5H), 7.49-7.69 (m, 3H), 7.93-8.16 (m, 2H), 9.07 (br. s, 1H).
Example 6
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
17.1 mg (18 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]-amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 22) was dissolved in 0.6 mL dioxane/water (1:1) and reacted with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride in a process similar to the synthesis process in Example 1. After lyophilization from dioxane, 14.8 mg (89% of the theoretical) of the title compound was obtained in the form of a white solid.
HPLC (method 10): Rt=2.54 min;
LC-MS (method 1): Rt=0.92 min; MS (ESIpos): m/z=919 (M+H)+.
Example 7
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
The title compound was synthesized by analogy with the synthesis process of Example 1 by reacting 100 mg (119 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 24) with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride.
Yield: 50 mg (49% of the theoretical)
LC-MS (method 1): Rt=0.87 min; MS (ESIpos): m/z=814 (M+H)+.
Example 8
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
The title compound was synthesized by analogy with the synthesis process of Example 1 by reacting 57 mg (71 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 25) with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride.
Yield: 10 mg (19% of the theoretical)
LC-MS (method 1): Rt=0.85 min; MS (ESIpos): m/z=775 (M+H)+.
Example 9
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
The title compound was synthesized by analogy with the synthesis process of Example 1 by reacting 57 mg (71 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt [synthesized like intermediate 17 by coupling intermediate 7 with (1S,2R)-(+)-norephedrine and then deprotecting it with trifluoroacetic acid] with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride.
Yield: 94 mg (84% of the theoretical)
LC-MS (method 1): Rt=0.79 min; MS (ESIpos): m/z=805 (M+H)+.
Example 10
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-({(2S,3E)-4-[4-(methoxycarbonyl)phenyl]-1-phenylbut-3-en-2-yl}amino)-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
The title compound was synthesized by analogy with the synthesis process of Example 1 by reacting 45 mg (47 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S,3E)-4-[4-(methoxycarbonyl)phenyl]-1-phenylbut-3-en-2-yl}amino)-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid salt (intermediate 23) with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride.
Yield: 33.9 mg (78% of the theoretical)
LC-MS (method 1): Rt=1.02 min; MS (ESIpos): m/z=933 (M+H)+.
Example 11
N-(3-Carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1{(2S)-2-[(1R,2R)-3-{[2S,3E)-4-(4-carboxyphenyl)-1-phenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
33.9 mg (36 μmol) N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-({(2S,3E)-4-[4-(methoxycarbonyl)phenyl]-1-phenylbut-3-en-2-yl}amino)-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Example 10) was placed in 1.1 mL THF/water (1:1) and mixed with 3.5 mg (145 μmol) lithium hydroxide. The reaction mixture was stirred over night at RT, then acidified by adding 1N hydrochloric acid and extracted twice with 10 mL each time. The combined organic phases were dried over magnesium sulfate and concentrated, yielding 18.3 mg (84% purity, 46% of the theoretical) of the title compound.
LC-MS (method 9): Rt=4.98 min; MS (ESIpos): m/z=919 (M+H)+.
B. Evaluation of Biological Efficacy
The biological activity of the compounds according to the invention can be demonstrated by in vitro and in vivo investigations, such as those with which those skilled in the art are familiar. For example, the pharmacological and pharmacokinetic properties of the compounds can according to the invention be determined with the help of the assays described below.
B-1. Determination of the Antiproliferative Effect on the HT29 Wt Cell Line:
A defined cell count of the human colon carcinoma cell line HT29 wt (wild type) was sown in a 96-well microtiter plate in whole medium (10% FCS-RPMI) (2500 cells/well) and incubated overnight at 37° C./5% CO2. After 18 hours, the inoculation medium was replaced by fresh medium with 10% FCS. The treatment started with addition of the respective test substance. Of the substances to be investigated, dose-effect curves were determined in a concentration range of 10−5 M to 10−14 M (1:10 dilution series). Incubation times of 48 h to 96 h were selected. Proliferation was detected with the help of the MTT assay (ATCC, Manassas, Va., USA, catalog no. 30-1010K). After the end of the selected incubation time, the MTT reagent was incubated with the cells for 4 h before lysis of the cells was performed overnight by adding the detergent. The dye that was formed was detected at 570 nm. The proliferation with otherwise identically treated cells but not with the test substance was defined as the 100% value. The data obtained from this test represents triple determinations, and at least two independent experiments were performed.
The IC50 values of representative exemplary embodiments from this assay are listed in Table 1 below:
TABLE 1
Exemplary embodiment
IC50 [nM]
1
15
2
3.3
3
0.3
4
1.1
5
0.1
6
0.1
8
0.5
9
6
11
4.5
In comparison with this, monomethylauristatin F (MMAF) had an IC50 value of 10 nM in this test.
B-2. Determination of the Influence on Tubulin Polymerization
Cancer cells are degenerate or neoplastic cells, which often lead to development of a tumor through increased cell division. Microtubules form the spindle fibers of the spindle apparatus and are an essential component of the cell cycle. Regulated buildup and breakdown of microtubule permit an accurate distribution of chromosomes to the daughter cells and represent a continuous dynamic process. A disturbance in these dynamics leads to faulty cell division and ultimately to cell death. However, the increased cell division of cancer cells makes them especially susceptible to spindle fiber toxins, which are a fixed component of chemotherapy. Spindle fiber toxins such as paclitaxel or epothilone lead to a greatly increased rate of polymerization of the microtubules, whereas vinca alkaloids or monomethylauristatin E (MMAE) will lead to a greatly reduced rate of polymerization of the microtubules. In both cases, the necessary dynamics of the cell cycle are sensitive to disturbance. The compounds investigated in the context of the present invention lead to a reduced rate of polymerization of the microtubules.
The “Fluorescence-based Microtubule Polymerization Assay Kit” from the company Cytoskeleton (Denver, Colo., USA; order no. BK011) was used to investigate tubulin polymerization. In this assay, GTP is added to unpolymerized tubulin, so the polymerization can take place spontaneously. The assay is based on the binding of the fluorophore 4′,6-diamidino-2-phenylindole (DAPI) to tubulin. Free and bound DAPI can be differentiated on the basis of different emission spectra. DAPI has a much higher affinity for polymerized tubulin in comparison with unpolymerized tubulin, so tubulin polymerization can be tracked on the basis of the increase in the fluorescence of bound DAPI fluorophores.
To perform this assay, the test substances dissolved in DMSO were diluted in water from their initial concentration of 10 mM to 1 μM. In addition to the buffer controls, polymerization-increasing paclitaxel and polymerization-inhibiting vinblastine were also included as assay controls. For the measurement, 96-well plates with a half bottom area were used, tracking the kinetics of the tubulin polymerization for one hour at 37° C. in a fluorimeter. The excitation wavelength was 355 nm, and the emission was tracked at 460 nm. For the range of the linear rise within the first 10 minutes, the change in fluorescence per minute (ΔF/min) was calculated, representing the rate of polymerization of the microtubules. The potency of the test substances was quantified on the basis of the respective reduction in the rate of polymerization.
B-3. Determination of the Plasma Stability In Vitro
Method A:
Of the respective test substance, 1 mg was dissolved in 0.5 mL acetonitrile/DMSO (9:1). Of this solution, 20 μL was removed and added to 1 mL rat plasma and human plasma at 37° C. (plasma of male Wistar rats with Li heparin, Harlan & Winkelmann and/or human leukocyte-depleted fresh plasma from whole blood specimens). Immediately after adding the specimen (initial value as reference variable) and then after 5, 10, 30, 60, 120, 180 and 240 minutes and optionally also after 24 hours, 100 μL aliquots were taken and added to 300 μL acetonitrile. The precipitated plasma proteins were centrifuged for 10 minutes at 5000 rpm, and then 30 μL of the supernatant was analyzed by HPLC to determine its unchanged test substance content. The results were quantified based on area percent of the corresponding peaks.
HPLC Method on Rat Plasma:
Instrument: Agilent 1200 with DAD, binary pump, autosampler, column oven and thermostat; column: Kromasil 100 C18, 250 mm×4 mm, 5 μm; column temperature: 45° C.; eluent A: 5 mL perchloric acid/L water; eluent B: acetonitrile; gradient: 0-8 min 98% A, 2% B: 8-15 min 56% A, 44% B; 15-20 min 10% A, 90% B; 20-21 min 10% A, 90% B; 21-23 min 98% A, 2% B; 23-25 min 98% A, 2% B; flow rate: 2 mL/min; UV detection: 220 nm.
HPLC Method on Human Plasma:
Instrument: Agilent 1100 with DAD, binary pump, autosampler, column over and thermostat; column: Kromasil 100 C18, 250 mm×4 mm, 5 μm; column temperature: 45° C.; eluent A: 5 mL perchloric acid/L water; eluent B: acetonitrile; gradient: 0-3 min 98% A, 2% B; 3-10 min 65% A, 35% B; 10-15 min 40% A, 60% B; 15-21 min 10% A, 90% B; 21-22 min 10% A, 90% B; 22-24 min 98% A, 2% B; 24-26 min 98% A, 2% B; flow rate 2 mL/min; UV detection: 220 nm.
Method B:
The respective test substance was incubated in rat plasma and/or human plasma at 37° C. for a period of 5 h while stirring lightly. At various points in time (0, 2, 5, 10, 20, 30, 60, 120, 180 and 300 minutes), a 100 μL aliquot was taken. After adding an internal standard (10 μL), the proteins were precipitated by adding 200 μL and the mixture was centrifuged for 5 minutes in an Eppendorf centrifuge. After adding 150 μL ammonium acetate buffer, pH 3, to 150 μL of the supernatant, the unchanged test substance content was analyzed by LC/MSMS.
B-4. Determination of Cell Permeability:
The cell permeability of a substance can be analyzed by in vitro testing in a flux assay using Caco-2 cells [M. D. Troutman and D. R. Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. To do so, the cells were cultured for 15-16 days on 24-hole filter plates. To determine the permeation, the respective test substance was applied to the cells either apically (A) or basally (B) in a HEPES buffer and incubated for 2 h. After 0 h and after 2 h, samples were taken from the cis- and trans-compartments. The samples were separated by HPLC (Agilent 1200, Böblingen, Germany) using reverse phase columns. The HPLC system was coupled via a turbo ion spray interface to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems Applera, Darmstadt, Germany). The permeability was evaluated on the basis of a Papp value, which was calculated using the equation published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1717-1725 (2003)]. A substance was classified as being actively transported if the ratio of Papp (B−A) to Papp (A−B) was >2 or <0.5.
The permeability of B to A [Papp (B−A)] is of crucial importance for toxophores that are released intracellularly. The lower this permeability, the longer is the dwell time of the substance in the cell after intracellular release and thus also the time available for an interaction with the biochemical target (here: tubulin).
Table 2 below shows permeability data for representative exemplary embodiments from this assay:
TABLE 2
Exemplary embodiment
Papp (B-A) [nm/s]
2
157
3
179
4
19
5
29
6
45
7
11
8
10
9
4.5
11
2
In comparison with this, monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF) had a Papp value of 89 nm/s or 73 nm/s, respectively, in this test.
B-5. Determination of the Substance Properties for P-Glycoprotein (P-gp):
Many tumor cells express transporter proteins for active ingredients and drugs, which is often associated with development of a resistance to cytostatics. Substances that are not substrates of such transporter proteins, such as P-glycoprotein (P-gp) or BCRP could thus have an improved profile of effect.
The substrate properties of a substance for P-gp (ABCB1) were determined by means of a flux assay using LLC-PK1 cells which overexpress P-gp (L-MDR1 cells) [A. H. Schinkel et al., J. Clin. Invest. 96, 1698-1705 (1995)]. To do so, the LLC-PK1 cells or L-MDR1 cells were cultured for 3-4 days on 96-well filter plates. To determine the permeation, the respective test substance, either alone or in the presence of an inhibitor (e.g., ivermectin or verapamil) in a HEPES buffer was applied to the cells either apically (A) or basally (B) and incubated for 2 h. After 0 h and after 2 h, samples were taken from the cis- and trans-compartments. The samples were separated by HPLC using reverse phase columns. The HPLC system was coupled via a turbo ion spray interface to an API 3000 triple quadrupole mass spectrometer (Applied Biosystems Applera, Darmstadt, Germany). The permeability was evaluated on the basis of a Papp value, which was calculated using the equation published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. A substance was classified as being a P-gp substrate if the efflux ratio Papp (B−A) to Papp (A−B) was >2.
The efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in the presence or absence of an initiator can be compared with one another as additional criteria for evaluating the P-gp substrate properties. If these values differ by more than a factor of 2, then the respective substance is a P-gp substrate.
C. Exemplary Embodiments for Pharmaceutical Compositions
The compounds according to the invention may be converted to pharmaceutical preparations by the following method:
Tablet:
Composition:
100 mg of the compound according to the invention, 50 mg lactose (monohydrate), 50 mg cornstarch (native), 10 mg polyvinylpyrrolidone (PVP 25) (BASF, Ludwigshafen, Germany) and 2 mg magnesium stearate
Tablet weight 212 mg; diameter 8 mm; radius of curvature 12 mm.
Preparation:
The mixture of the compound according to the invention, lactose and starch is granulated with a 5% solution (w/w) of the PVP in water. The granules are mixed with the magnesium stearate for five minutes after drying. This mixture is pressed with a conventional tablet press (see above for the format of the tablet). A pressing force of 15 kN was used as the guideline value for pressing the tablets.
Suspension for Oral Administration:
Composition:
1000 mg of the compound according to the invention, 1000 mg ethanol (96%), 400 mg Rhodigel® (xanthan gum from FMC, Pennsylvania, USA) and 99 g water.
10 mL oral suspension corresponds to a single dose of 100 mg of the compound according to the invention.
Solution for Oral Administration:
Composition:
500 mg of the compound according to the invention, 2.5 g polysorbate and 97 g polyethylene glycol 400.20 g of the oral solution corresponds to a single dose of 100 mg of the compound according to the invention.
Preparation:
The compound according to the invention is suspended in the mixture of polyethylene glycol and polysorbate while stirring. The stirring process is continued until the compound according to the invention is completely dissolved.
i.v. Solution:
The compound according to the invention is dissolved in a concentration below the saturation solubility in a physiologically tolerable solvent (e.g., isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%). The solution is sterile filtered and bottled in sterile and pyrogen-free injection vials.
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14004699
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seattle genetics, inc
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/364
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:sgen
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Seattle Genetics
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Jul 9th, 2019 12:00AM
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Oct 21st, 2016 12:00AM
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https://www.uspto.gov?id=US10342811-20190709
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Methods of inhibition of protein fucosylation in vivo using fucose analogs
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The invention provides methods and compositions for the inhibition of fucosylation of proteins, including antibodies, in vivo by administration of a fucose analog.
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10342811
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1. A method for the treatment of cancer having protein fucosylation in a human in need thereof, comprising administering to the human a therapeutically effective amount of a fucose analog or a biologically acceptable salt thereof, or a composition thereof, wherein each fucose analog has formulae (III) or (IV):
wherein each fucose analog of formula (III) or (IV) can be the alpha or beta anomer or the corresponding aldose form;
wherein each of R1, R3, and R4 is independently selected from the group consisting of —OH and —OC(O)C1-C10 alkyl, R2 is F, R2a and R3a are each H, and R5 is —CH3.
2. The method of claim 1, wherein each of R1, R3, and R4 is independently selected from the group consisting of —OH and —OAc, R2 is F, R2a and R3a are each H, and R5 is —CH3.
3. The method of claim 1 wherein the fucose analog is 2-deoxy-2-fluorofucose.
4. The method of claim 1 wherein the fucose analog is 2-deoxy-2-fluorofucose peracetate.
5. The method of claim 1 further comprising administering a tumor-associated antigen or an antigenic fragment thereof as an immunogen.
6. The method of claim 1 further comprising administering a chemotherapeutic agent.
7. The method of claim 1 wherein administration is oral administration.
8. The method of claim 1 wherein administration of the therapeutically effective amount is in the form of one or more dosage units.
9. The method of claim 1 wherein protein fucosylation in the human is reduced by at least 10% relative to the amount of protein fucosylation in the absence of administration of said fucose analog.
10. The method of claim 9 wherein protein fucosylation in the human is reduced by about 20% relative to the amount of protein fucosylation in the absence of administration of said fucose analog and wherein the mammal is a human.
11. The method of claim 9 wherein protein fucosylation in the human is reduced by about 30% relative to the amount of protein fucosylation in the absence of administration of said fucose analog.
12. The method of claim 9 wherein protein fucosylation in the human is reduced by about 40% relative to the amount of protein fucosylation in the absence of administration of said fucose analog.
13. The method of claim 9 wherein protein fucosylation in the human is reduced by about 50% relative to the amount of protein fucosylation in the absence of administration of said fucose analog.
14. The method of claim 9 wherein protein fucosylation in the human is reduced by about 60% relative to the amount of protein fucosylation in the absence of administration of said fucose analog.
15. The method of claim 1 wherein fucosylation of white blood cells in the serum of the human is reduced by at least about 10% relative to the amount of fucosylation of white blood cells in the serum of a mammal in the absence of administration of said fucose analog.
16. The method of claim 15 wherein fucosylation of white blood cells in the serum of the human is reduced by at least about 20% relative to the amount of fucosylation of white blood cells in the serum of a mammal in the absence of administration of said fucose analog.
17. The method of claim 16 wherein fucosylation of white blood cells in the serum of the human is reduced by at least about 60% relative to the amount of fucosylation of white blood cells in the serum of a mammal in the absence of administration of said fucose analog.
18. The method of claim 1 wherein the cancer is a hematologic malignancy.
19. The method of claim 18 wherein the hematologic malignancy is lymphoma.
20. The method of claim 18 wherein the hematologic malignancy is leukemia.
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20
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CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/814,083 filed on Feb. 4, 2013, which is the national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2011/046857 filed Aug. 5, 2011, which claims the benefit of U.S. Provisional Application No. 61/371,116, filed Aug. 5, 2010, the disclosures of each are incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
L-fucose, also referred to as 6-deoxy-L-galactose, is a monosaccharide that is a component of some N- and O-linked glycans and glycolipids in animals. (See Becker and Lowe, Glycobiology 13:41R-51R (2003).) Fucose is typically added as a terminal modification to glycans, including glycans attached to blood group antigens, selectins and antibodies. Fucose can be attached to glycans via α(1,2)-, α(1,3)-, α(1,4)- and α(1,6)-linkages by specific fucosyltransferases. α(1,2)-fucose linkages are typically associated with the H-blood group antigens. α(1,3)- and α(1,4)-fucose linkages are associated with modification of LewisX antigens. α(1,6)-fucose linkages are associated with N-linked GlcNAc molecules, such as those on antibodies.
Fucosylation of proteins is believed to play a role in mammalian development. Mice homozygous for a targeted mutation of the FX gene exhibit pleiotropic abnormalities including a lethal phenotype. Reduced recovery of mice from heterozygous crosses was also reported. (Becker et al., Mammalian Genome 14:130-139 (2003). Aberrant protein fucosylation has been proposed to be associated with human disease, including up-regulation of sialyl LewisX and sialyl Lewisy in cancers. These glycans are ligands for E- and P-selectin molecules. In it speculated that increases in sialyl LewisX and sialyl Lewisy glycans on cancer cells increases metastases through interaction with E- and P-selectins on endothelium. Increased fucosylated glycans have also been observed in patients with rheumatoid arthritis. Currently, however, there are no approved therapeutic approaches targeting protein fucosylation levels.
SUMMARY OF THE INVENTION
The methods and compositions described herein are premised in part on the unexpected results presented in the Examples, showing that animals administered a fucose analog have reduced protein fucosylation. Fucosylation of antibodies and other proteins can be modulated using the fucose analogs described herein.
In one aspect, methods and compositions for the in vivo production of defucosylated proteins are provided. Animals, such as mammals, administered a fucose analog (having formula I, II, III, IV, V or VI) produce proteins, such as cell surface proteins, having reduced fucosylation. The reduction in fucosylation is relative to animals untreated with the fucose analogs having formula I, II, III, IV, V or VI, respectively.
In a related aspect, methods and compositions for the in vivo production of antibodies and antibody derivatives with reduced core fucosylation are provided. Animals administered a fucose analog (having formula I, II, III, IV, V or VI) produce antibodies and antibody derivatives having reduced core fucosylation (i.e., reduced fucosylation of N-acetylglucosamine of the complex N-glycoside-linked sugar chains bound to the Fc region through the N-acetylglucosamine of the reducing terminal of the sugar chains). The reduction in core fucosylation is relative to animals untreated with the fucose analogs of having formula I, II, III, IV, V or VI, respectively.
In another aspect, pharmaceutical compositions containing fucose analogs and formulated for administration to a target animal are provided. The fucose analogs can be formulated for administration to an animal to inhibit or reduce fucosylation in vivo.
These and other aspects of the present invention may be more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments, and the appended figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of administration of fucose analogs (via ip injection) on antibody fucosylation. Dot blots are shown on the left panel and a graph is shown on the right panel. The dot blot protein loading levels (upper left) and fucose-specific bioluminescence (lower left) for antibody cAC10 standards (lower dot blot, left most dashed rectangle and corresponding columns of upper dot blot), untreated control (lower dot blot, second dashed rectangle from the left and corresponding column of upper dot blot), and alkynyl fucose (SGD-1887; lower dot blot, middle dashed rectangle and corresponding column of upper dot blot), alkynyl fucose peracetate (SGD-1890; lower dot blot, second dashed rectangle from the right and corresponding column of upper dot blot), and 2-fluorofucose (SGD-2083; lower dot blot, right most rectangle and corresponding column of upper dot blot). After correcting for loading level, the % fucosylation is shown on the graph at the right.
FIG. 2 shows the effects on antibody core fucosylation of administration of fucose analogs via drinking water. The graphs show % fucosylation of antibodies as a determined by gas chromatograph (GC): panels A and B show fucosylation levels of the anti-KLH antibodies (Abs) isolated from the treated groups while panels C and D show the fucosylation levels of the remaining (non-KLH-specific) IgG antibodies. Panels A and C show the percent fucosylation of each animal determined using a purified antibody standard curve (0-100% fucosylation). Panels B and D show the fucosylation level of the treated groups as a percentage of the average untreated control group value.
FIG. 3 shows the effects on antibody core fucosylation of administration of fucose analogs via drinking water. In this figure, fucosylation levels of the non-KLH-specific antibodies are shown. Dot blots of protein loading levels (upper left) and fucose specific bioluminescence (lower left) are shown for cAC10 standards (upper and lower dot blots, left most rectangle), untreated control (upper and lower dot blots, second from the left (upper) and right rectangles), and 2-fluorofucose (upper and lower dot blots, second from the left (lower) and second from the right rectangles (upper and lower)). After correcting for loading level, the % fucosylation is shown in the graph on the right.
FIG. 4 shows the effects of different doses of 2-fluorofucose, administered via drinking water, on antibody core fucosylation. The dot blots show protein loading levels (left) and fucose specific bioluminescence (middle) for untreated control and 1, 10, and 100 mM SGD-2083 (as indicated). The % fucosylation compared to untreated is shown in the graph on the right.
FIG. 5 shows the effects of administration of 2-fluorofucose) on circulating white blood cells and neutrophils. Panel A. Blood samples were collected from individual mice, and the white cell count was determined by counting on a hemacytometer using Turk's solution of exclude red blood cells. Panel B. To determine neutrophil counts, the percentage of white blood cells that were Gr-1+ was determined by flow cytometry and applied to the total cell count determined in (A). Panel C. A pool of lymph nodes was collected from individual mice, single cell suspensions were prepared and cells were counted on a hemacytometer. Symbols represent individual mice (n=3 per group; diamonds, untreated; squares, 1 mM 2-fluorofucose (SGD-2083); triangles, 10 mM 2-fluorofucose; circles, 100 mM 2-fluorofucose).
FIG. 6 shows the effects of administration of 2-fluorofucose on E-selectin binding to neutrophils. Panel A. An example of neutrophil identification by flow cytometry. Cells were gated on forward and side scatter to include live white blood cells and then applied to the histogram depicting Gr-1 staining to identify neutrophils. The positive cells were gated, the percentage positive cells determined (used for cell counts in FIG. 5B), and the gate was applied to the histograms in (B). Panel B. Examples of E-selectin binding to neutrophils from an untreated animal (left) and an animal treated with orally administered 2-fluorofucose (SGD-2083) at 100 mM (right). Grey histograms show E-selectin binding and the dotted lines show binding of the secondary reagent alone. The geometric mean fluorescent intensity was determined for E-selectin binding. Panel C. Geometric mean fluorescent intensity of E-selectin binding was determined for each animal as in (B) and compared between groups (n=3, per group; error bars represent standard deviation).
FIG. 7 shows the effects on protein fucosylation for cell lines cultured with certain fucose analogs. The LS174T, PC-3, Ramos, HL-60cy and Caki-1 cell lines were examined. Effects on LCA (lectin), Anti-SSEAI (Lewis X), cBR96 (Lewis Y), and P-Selectin (Selectin Ligand) of these cell lines, are shown in panels A-D, respectively.
FIG. 8 shows the effects of administration of fucose analogs to mouse xenograft cancer models. The results of mouse xenograft models with L5174T, PC-3, Ramos, HL-60 and Caki-1 cell lines (pre-treated with 2-flurofucose (SGD-2083)), are shown in panels A-E, respectively. The results of a mouse xenograft model with an untreated LS174Tcell lines are shown in panel F.
FIG. 9 shows the study design (panel A) and results (panel B) of a tumor vaccine model based on preimmunization with killed A20 murine lymphoma cells, followed by challenge with live A20 cells with or without administration of a fucose analog, (2-fluorofucose).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “antibody” refers to (a) immunoglobulin polypeptides and immunologically active portions of immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family, or fragments thereof, that contain an antigen binding site(s) that immunospecifically binds to a specific antigen and have an Fc domain comprising a complex N-glycoside-linked sugar chain(s), or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments that immunospecific ally bind to the antigen. Antibodies are generally described in, for example, Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).
An “antibody derivative” means an antibody, as defined above (including an antibody fragment), or Fc domain or region of an antibody comprising a complex N-glycoside linked sugar chain, that is modified by covalent attachment of a heterologous molecule such as, e.g., by attachment of a heterologous polypeptide (e.g., a ligand binding domain of heterologous protein), or by glycosylation (other than core fucosylation), deglycosylation (other than non-core fucosylation), acetylation, phosphorylation or other modification not normally associated with the antibody or Fc domain or region.
The term “monoclonal antibody” refers to an antibody that is derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone, and not the method by which it is produced. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
The term “Fc region” refers to the constant region of an antibody, e.g., a CH1-hinge-CH2-CH3 domain, optionally having a CH4 domain, or a conservatively substituted derivative of such an Fc region.
The term “Fc domain” refers to the constant region domain of an antibody, e.g., a CH1 hinge, CH2, CH3 or CH4 domain, or a conservatively substituted derivative of such an Fc domain.
An “antigen” is a molecule to which an antibody or antibody derivative specifically binds.
The terms “specific binding” and “specifically binds” mean that the antibody or antibody derivative will bind, in a highly selective manner, with its corresponding target antigen and not with the multitude of other antigens. Typically, the antibody or antibody derivative binds with an affinity of at least about 1×10−7 M, and preferably 10−8 M to 10−9 M, 10−10 M, 10−11 M, or 10−12 M and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
The terms “inhibit” or “inhibition of” means to reduce by a measurable amount, or to prevent entirely.
As used herein, “alkynyl fucose peracetate” refers to any or all forms of alkynyl fucose (5-ethynylarabinose) with acetate groups on positions R1-4 (see formula I and II, infra), including 6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate, including the (2S,3S,4R,5R,6S) and (2R,3S,4R,5R,6S) isomers, and 5-((S)-1-hydroxyprop-2-ynyl)-tetrahydrofuran-2,3,4-triyl tetraacetate, including the (2S,3S,4R,5R) and (2R,3S,4R,5R) isomers, and the aldose form, unless otherwise indicated by context. The terms “alkynyl fucose triacetate”, “alkynyl fucose diacetate” and “alkynyl fucose monoacetate” refer to the indicated tri-, di- and mono-acetate forms of alkynyl fucose, respectively.
Unless otherwise indicated by context, the term “alkyl” refers to an unsubstituted saturated straight or branched hydrocarbon having from 1 to 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), unless otherwise specified. An alkyl group of 1 to 3, 1 to 8 or 1 to 10 carbon atoms is preferred. Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.
Alkyl groups, whether alone or as part of another group, when substituted can be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including, but not limited to: halogen, —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —SR′, —SO3R′, —S(O)2R′, —S(O)R′, —OH, ═O, —NH2, —NH(R′), —N(R′)2 and CN; where each R′ is independently selected from —H, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, or aryl.
Unless otherwise indicated by context, the terms “alkenyl” and “alkynyl” refer to unsubstituted or optionally substituted (were indicated) straight and branched carbon chains having from 2 to 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from 2 to 3, 2 to 4, 2 to 8 or 2 to 10 carbon atoms being preferred. An alkenyl chain has at least one double bond in the chain and an alkynyl chain has at least one triple bond in the chain. Examples of alkenyl groups include, but are not limited to, ethylene or vinyl, allyl, -1 butenyl, -2 butenyl, -isobutylenyl, -1 pentenyl, -2 pentenyl, 3-methyl-1-butenyl, -2 methyl 2 butenyl, and -2,3 dimethyl 2 butenyl. Examples of alkynyl groups include, but are not limited to, acetylenic, propargyl, acetylenyl, propynyl, -1 butynyl, -2 butynyl, -1 pentynyl, -2 pentynyl, and -3 methyl 1 butynyl.
Alkenyl and alkynyl groups, whether alone or as part of another group, when substituted can be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to: halogen, —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —SR′, —SO3R′, —S(O)2R′, —S(O)R′, —OH, ═O, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from H, —C1-C8 alkyl, —C2—C alkenyl, —C2-C8 alkynyl, or aryl.
Unless otherwise indicated by context, the term “alkylene” refers to an unsubstituted saturated branched or straight chain hydrocarbon radical having from 1 to 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from 1 to 8 or 1 to 10 carbon atoms being preferred and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylenes include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene, decalene, 1,4-cyclohexylene, and the like.
Alkylene groups, whether alone or as part of another group, when substituted can be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including, but not limited to: halogen, —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —SR′, —SO3R′, —S(O)2R′, —S(O)R′, —OH, ═O, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from H, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, or aryl.
“Alkenylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of an alkenyl group (as described above), and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. An “alkenylene” group can be unsubstituted or optionally substituted (were indicated), as described above for alkenyl groups. In some embodiments, an “alkenylene” group is not substituted.
“Alkynylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of an alkynyl group (as described above), and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. An “alkynylene” group can be unsubstituted or optionally substituted (were indicated), as described above for alkynyl groups. In some embodiments, an “alkynylene” group is not substituted.
Unless otherwise indicated by context, the term “aryl” refers to a substituted or unsubstituted monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as “Ar”. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and the like.
An aryl group, whether alone or as part of another group, can be optionally substituted with one or more, preferably 1 to 5, or even 1 to 2 groups including, but not limited to: halogen, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —SR′, —SO3R′, —S(O)2R′, —S(O)R′, —OH, —NO2, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from H, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, or aryl.
Unless otherwise indicated by context, the term “heterocycle” refers to a substituted or unsubstituted monocyclic ring system having from 3 to 7, or 3 to 10, ring atoms (also referred to as ring members) wherein at least one ring atom is a heteroatom selected from N, O, P, or S (and all combinations and subcombinations of ranges and specific numbers of carbon atoms and heteroatoms therein). The heterocycle can have from 1 to 4 ring heteroatoms independently selected from N, O, P, or S. One or more N, C, or S atoms in a heterocycle can be oxidized. A monocyclic heterocycle preferably has 3 to 7 ring members (e.g., 2 to 6 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S). The ring that includes the heteroatom can be aromatic or non-aromatic. Unless otherwise noted, the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.
Heterocycles are described in Paquette, “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 82:5566 (1960). Examples of “heterocycle” groups include by way of example and not limitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, fucosyl, azirdinyl, azetidinyl, oxiranyl, oxetanyl, and tetrahydrofuranyl.
A heterocycle group, whether alone or as part of another group, when substituted can be substituted with one or more groups, preferably 1 to 2 groups, including but not limited to: —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, halogen, —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —SR′, —SO3R′, —S(O)2R′, —S(O)R′, —OH, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from H, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, or -aryl.
By way of example and not limitation, carbon-bonded heterocycles can be bonded at the following positions: position 2, 3, 4, 5, or 6 of a pyridine; position 3, 4, 5, or 6 of a pyridazine; position 2, 4, 5, or 6 of a pyrimidine; position 2, 3, 5, or 6 of a pyrazine; position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole or thiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole; position 2 or 3 of an aziridine; or position 2, 3, or 4 of an azetidine. Exemplary carbon bonded heterocycles can include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.
By way of example and not limitation, nitrogen bonded heterocycles can be bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole; position 2 of a isoindole, or isoindoline; and position 4 of a morpholine. Still more typically, nitrogen bonded heterocycles include 1-aziridyl, 1-azetidyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
Unless otherwise noted, the term “carbocycle,” refers to a substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic ring system having from 3 to 6 ring atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein) wherein all of the ring atoms are carbon atoms.
Carbocycle groups, whether alone or as part of another group, when substituted can be substituted with, for example, one or more groups, preferably 1 or 2 groups (and any additional substituents selected from halogen), including, but not limited to: halogen, C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —SR′, —SO3R′, —S(O)2R′, —S(O)R′, —OH, ═O, —NH2, —NH(R′), —N(R′)2 and —CN; where each R′ is independently selected from H, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, or aryl.
Examples of monocyclic carbocylic substituents include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, cyclooctyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, and -cyclooctadienyl.
When any variable occurs more than one time in any constituent or in any formula, its definition in each occurrence is independent of its definition at every other. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
Unless otherwise indicated by context, a hyphen (-) designates the point of attachment to the pendant molecule. Accordingly, the term “—(C1-C10 alkylene)aryl” or “—C1-C10 alkylene(aryl)” refers to a C1-C10 alkylene radical as defined herein wherein the alkylene radical is attached to the pendant molecule at any of the carbon atoms of the alkylene radical and one of the hydrogen atom bonded to a carbon atom of the alkylene radical is replaced with an aryl radical as defined herein.
When a particular group is “substituted”, that group may have one or more substituents, preferably from one to five substituents, more preferably from one to three substituents, most preferably from one to two substituents, independently selected from the list of substituents. The group can, however, generally have any number of substituents selected from halogen.
It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. It is understood that substituents and substitution patterns on the compounds of this invention can be selected by one of ordinary skill in the art to provide compounds that are active and chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein.
The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “pharmaceutically compatible ingredient” refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or vehicle with which the fucose analog is administered.
“Small electron-withdrawing groups” refers to any substituent that has greater electronegativity at the site of substituent attachment than, e.g., a hydrogen atom or hydroxy group or relative to the substituent present in fucose at that site. Generally, the small electron-withdrawing group has 10 or fewer atoms (other than hydrogen) and includes groups such as nitro; cyano and cyanoalkyl (e.g., —CH2CH2CN); halogens; acetylene or other alkynes or halo alkynes (e.g., —C≡CCF3); alkenes or halo alkenes; allenes; carboxylic acids, ester, amides and halo substituted forms thereof; sulfonic and phosphonic acids, esters and amides, and halo substituted forms thereof; haloalkyl groups (e.g., —CF3, —CHF2, —CH2CF3), acyl and haloacyl groups (e.g., —C(O)CH3 and —C(O)CF3); alkylsulfonyl and haloalkylsulfonyl (e.g., —S(O)2alkyl and —S(O)2haloalkyl); aryloxy (e.g., phenoxy and substituted phenoxy); aralkyloxy (e.g, benzyloxy and substituted benzyloxy); and oxiranes. Preferred small electron-withdrawing groups are those having 8, 7 or 6 or fewer atoms (other than hydrogen).
The fucose analogs are typically substantially pure from undesired contaminant. This means that the analog is typically at least about 50% w/w (weight/weight) purity, as well as being substantially free from interfering proteins and other contaminants. Sometimes the agents are at least about 80% w/w and, more preferably at least 90% or about 95% w/w purity. Using conventional purification techniques, homogeneous product of at least 99% w/w can be obtained.
General
The invention provides methods and compositions for reducing protein fucosylation in an animal. The methods are premised in part on the unexpected results presented in the Examples showing that administering a fucose analog to a subject (e.g., a mammal) results in an antibody or antibody derivative having reduced core fucosylation, and other proteins also having reduced fucosylation. “Reduced fucosylation” in the context of proteins generally refers to reduced addition of fucose to glycans via α(1,2)-, α(1,3)-, α(1,4)- and α(1,6)-linkages. “Core fucosylation” in the context of an antibody refers to addition of fucose (“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at the reducing terminal of an N-linked glycan of an antibody. “Reduced core fucosylation” in the context of an antibody refers to a reduction of fucose linked to N-acetylglucosamine (“GlcNAc”) at the reducing terminal of an N-linked glycan of an antibody, as compared to an untreated animal.
In the various aspects described herein, the animal to which the fucose analog is administered is typically a mammal and is preferably human. The invention therefore further provides methods and compositions for reducing protein fucosylation in a mammal, such as a human.
In other aspects, pharmaceutical compositions of fucose analogs and pharmaceutical excipients are provided in which an effective amount of a fucose analog(s) is in admixture with the excipients, suitable for administration to a animal. In some embodiments, the fucose analog is in dry form (e.g., lyophilized), optionally with stabilizers that enhance the composition stability for longer term storage. In some embodiments, a pharmaceutical composition of a fucose analogs and pharmaceutical excipients is formulated for administration to a mammal. In some further embodiments, a pharmaceutical composition of a fucose analogs and pharmaceutical excipients is formulated for administration to a human.
In some embodiments, fucosylation of complex N-glycoside-linked sugar chains bound to the Fc region (or domain) of an antibody is reduced. As used herein, a “complex N-glycoside-linked sugar chain” is typically bound to asparagine 297 (according to the numbering system of Kabat), although a complex N-glycoside linked sugar chain can also be linked to other asparagine residues. As used herein, the complex N-glycoside-linked sugar chain has a bianntennary composite sugar chain, mainly having the following structure:
where ± indicates the sugar molecule can be present or absent, and the numbers indicate the position of linkages between the sugar molecules. In the above structure, the sugar chain terminal which binds to asparagine is called a reducing terminal (at right), and the opposite side is called a non-reducing terminal. Fucose is usually bound to N-acetylglucosamine (“GlcNAc”) of the reducing terminal, typically by an α1,6 bond (the 6-position of GlcNAc is linked to the 1-position of fucose). “Gal” refers to galactose, and “Man” refers to mannose.
A “complex N-glycoside-linked sugar chain” excludes a high mannose type of sugar chain, in which only mannose is incorporated at the non-reducing terminal of the core structure, but includes 1) a complex type, in which the non-reducing terminal side of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally has a sialic acid, bisecting N-acetylglucosamine or the like; or 2) a hybrid type, in which the non-reducing terminal side of the core structure has both branches of the high mannose N-glycoside-linked sugar chain and complex N-glycoside-linked sugar chain.
In some embodiments, the “complex N-glycoside-linked sugar chain” includes a complex type in which the non-reducing terminal side of the core structure has zero, one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally further has a structure such as a sialic acid, bisecting N-acetylglucosamine or the like, but excludes chains with a high mannose component.
According to the present methods, typically only a minor amount of fucose is incorporated into the sugar chain(s) (e.g., a glycan or complex N-glycoside-linked sugar chains) after administering a fucose analog. For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the antibodies in the serum of the animal (e.g., a mammal, such as a human) are core fucosylated, as compared to an animal not receiving the fucose analog. In some embodiments, substantially none (i.e., less than 0.5%) of the antibodies in the serum of the animal are not core fucosylated, as compared to an animal not receiving the fucose analog.
In some embodiments, protein fucosylation is reduced by about 60%, by about 50%, by about 40%, by about 30%, by about 20%, by about 15%, by about 10%, by about 5%, or by about 1% for cell surface proteins in the animal (e.g., a mammal, such as a human) are fucosylated, as compared to an animal not receiving the fucose analog. In some embodiments, protein fucosylation via α(1,2)-linkage is reduced by about 60%, by about 50%, by about 40%, by about 30%, by about 20%, by about 15%, by about 10%, by about 5%, or by about 1% for cell surface proteins in the animal (e.g., a mammal, such as a human) are fucosylated, as compared to an animal not receiving the fucose analog.
In some embodiments, protein fucosylation via α(1,3)-linkage is reduced by about 60%, by about 50%, by about 40%, by about 30%, by about 20%, by about 15%, by about 10%, by about 5%, or by about 1% for cell surface proteins in the animal (e.g., a mammal, such as a human) are fucosylated, as compared to an animal not receiving the fucose analog. In some embodiments, protein fucosylation via α(1,4)-linkage is reduced by about 60%, by about 50%, by about 40%, by about 30%, by about 20%, by about 15%, by about 10%, by about 5%, or by about 1% for cell surface proteins in the animal (e.g., a mammal, such as a human) are fucosylated, as compared to an animal not receiving the fucose analog.
In some embodiments, protein fucosylation via α(1,6)-linkage is reduced by about 60%, by about 50%, by about 40%, by about 30%, by about 20%, by about 15%, by about 10%, by about 5%, or by about 1% for cell surface proteins in the animal (e.g., a mammal, such as a human) are fucosylated, as compared to an animal not receiving the fucose analog.
In some embodiments, fucosylation of white blood cells in the serum of the animal (e.g., a mammal, such as a human) is reduced by at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, as compared to an animal not receiving the fucose analog. In some embodiments, fucosylation via α(1,3) linkages of white blood cells in the serum of the animal (e.g., a mammal, such as a human) is reduced by at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, as compared to an animal not receiving the fucose analog. In some embodiments, fucosylation via α(1,4) linkages of white blood cells in the serum of the animal (e.g., a mammal, such as a human) is reduced by at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, as compared to an animal not receiving the fucose analog.
In some embodiments, fucosylation of antibodies in the serum of the animal (e.g., a mammal, such as a human) is reduced by at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, as compared to an animal not receiving the fucose analog.
In certain embodiments, only a minor amount of a fucose analog (or a metabolite or product of the fucose analog) is incorporated into glycans (e.g., the complex N-glycoside-linked sugar chain(s)) of the antibody, antibody derivative or other glycans of proteins. For example, in various embodiments, less than about 60%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the fucose analog (or a metabolite or product of the fucose analog) is incorporated into glycans of the antibodies in the serum of the animal, as compared to an animal not receiving the fucose analog. In some embodiments, less than about 60%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the fucose analog (or a metabolite or product of the fucose analog) is incorporated into glycans of cell surface proteins of the animal, as compared to an animal not receiving the fucose analog.
In some embodiments, less than about 60%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the fucose analog (or a metabolite or product of the fucose analog) is incorporated into glycans of white blood cells in the serum of the animal, as compared to an animal not receiving the fucose analog.
Fucose Analogs
Suitable fucose analogs for the methods of the present invention (identified below as Formula I, II, III, IV, V and VI) are those that can be safely administered to a mammal in an amount effective to inhibit core fucosylation of complex N-glycoside-linked sugar chains of antibodies or antibody derivatives. Fucose analogs are described in Published US Patent Application 2009-0317869 that reduce the incorporation of fucose into complex N-glycoside-linked sugar chains of antibodies or antibody derivatives produced by host cells in vitro. The fucose analog can be given to a subject animal (e.g., a mammal) by parental, orally or other suitable mode of administration.
In some embodiments, a fucose analog (or an intracellular metabolite or product of the fucose analog) inhibits an enzyme(s) in the fucose salvage pathway. (As used herein, an intracellular metabolite can be, for example, a GDP-modified analog or a fully or partially de-esterified analog. A product can be, for example, a fully or partially de-esterified analog.) For example, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of fucokinase, or GDP-fucose-pyrophosphorylase. In some embodiments, a fucose analog (or an intracellular metabolite or product of the fucose analog) inhibits fucosyltransferase (such as a 1,2-fucosyltransferase, 1,3-fucosyltransferase, 1,4-fucosyltransferase, or 1,6-fucosyltransferase (e.g., the FUT8 protein)). In some embodiments, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of an enzyme in the de novo synthetic pathway for fucose. For example, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of GDP-mannose 4,6-dehydratase or/or GDP-fucose synthetase. In some embodiments, the fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit a fucose transporter (e.g., GDP-fucose transporter).
In some embodiments, the fucose analog has the following formula (I) or (II):
or a biologically acceptable salt or solvate of the analog, wherein each of formula (I) or (II) can be the alpha or beta anomer or the corresponding aldose form. In the above formulae, each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), —OC(O)C2-C10 alkynylene(heterocycle), —OC(O)CH2O(CH2CH2O)nCH3, —OC(O)CH2CH2O(CH2CH2O)nCH3, —O-tri-C1-C3 alkyl silyl, —OC1-C10 alkyl, —OCH2OC(O) alkyl, —OCH2OC(O) alkenyl, —OCH2OC(O) alkynyl, —OCH2OC(O) aryl, —OCH2OC(O) heterocycle, —OCH2OC(O)O alkyl, —OCH2OC(O)O alkenyl, —OCH2OC(O)O alkynyl, —OCH2OC(O)O aryl and —OCH2OC(O)O heterocycle, wherein each n is an integer independently selected from 0-5; and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is F, Br, Cl or I), —CHX2 (wherein each X is F, Br or Cl) and methoxiran.
In some embodiments, the fucose analog has formula (I) or (II), wherein: each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)CH2O(CH2CH2O)nCH3, —OC(O)CH2CH2O(CH2CH2O)nCH3, —O-tri-C1-C3 silyl, —OC1-C10 alkyl, —OCH2OC(O) alkyl, —OCH2OC(O)O alkyl, —OCH2OC(O) aryl, and —OCH2OC(O)O aryl, wherein each n is an integer independently selected from 0-5; and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is F, Br, Cl or I), —CHX2 (wherein each X is F, Br or Cl), and methoxiran.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is F, Br, Cl or I), —CHX2 (wherein each X is F, Br or Cl), and methoxiran.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —O-tri-C1-C3 silyl and —OC1-C10 alkyl; and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is Br, Cl or I), and methoxiran.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OCH2OC(O) alkyl, —OCH2OC(O) alkenyl, —OCH2OC(O) alkynyl, —OCH2OC(O) aryl, —OCH2OC(O) heterocycle, —OCH2OC(O)O alkyl, —OCH2OC(O)O alkenyl, —OCH2OC(O)O alkynyl, —OCH2OC(O)O aryl, and —OCH2OC(O)O heterocycle; and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is F, Br, Cl or I), —CHX2 (wherein each X is F, Br or Cl), and methoxiran.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, and methoxiran.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —CH2F, —CH2Br, and —CH2Cl.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —CHF2, —CHBr2, and —CHCl2.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —C≡CH, —C≡CCH3 and —CH2C≡CH.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —(CH2)n(CN) (where n=0 or 1) and —CO(O)CH3.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2CN and —CO(O)CH3.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), and —OC(O)C2-C10 alkynylene(heterocycle); and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH(OAc)CH3, —CH2CN, and —CO(O)CH3.
In some embodiments, the fucose analog has formula (I) or (II), wherein R5 is as defined herein, and each of R1-R4 is hydroxyl or —OC(O)C1-C10 alkyl.
In some embodiments, the fucose analog has formula (I) or (II), wherein R5 is as defined herein, and each of R1-R4 is hydroxyl or —OAc.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and OC(O)C1-C10 alkyl; and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH(OAc)CH3, —CH2CN, —CO(O)CH3, —CH2F and —CHF2
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OAc; and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH(OAc)CH3, —CH2CN, —CO(O)CH3, —CH2F and —CHF2
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OC(O)C1-C10 alkyl; and R5 is selected from the group consisting of —C≡CH, —CH2F and —CHF2.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OAc; and R5 is selected from the group consisting of —C≡CH, —CH2F and —CHF2.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OC(O)C1-C10 alkyl; and R5 is —C≡CH.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OAc; and R5 is —C≡CH.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OC(O)C1-C10 alkyl; and R5 is —CHF2.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OAc; and R5 is —CHF2.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is —OH or an ester selected from the group consisting of —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), —OC(O)C2-C10 alkynylene(heterocycle), —OC(O)CH2O(CH2CH2O)nCH3 (where n is 0-5), and —OC(O)CH2CH2O(CH2CH2O)nCH3 (where n is 0-5); and R5 is selected from the group consisting of —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is F, Br, Cl or I), —CHX2 (wherein each X is F, Br or Cl), and methoxiran.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OC(O)C1-C10 alkyl; and R5 is —CH2X (wherein X is F, Br, Cl or I).
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OAc; and R5 is —CH2X (wherein X is F, Br, Cl or I).
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OC(O)C1-C10 alkyl; and R5 is —CH2Br.
In some embodiments, the fucose analog has formula (I) or (II), wherein each of R1-R4 is independently selected from the group consisting of —OH, and —OAc; and R5 is —CH2Br.
In some embodiments, the fucose analog has a molecular weight of less than 2000 daltons. In some embodiments, the fucose analog has a molecular weight of less than 1000 daltons.
In some embodiments, R5 is not substituted.
In some embodiments, each of R1-R4 is not substituted.
In some embodiments, R5 is not a ketone (—C(O)alkyl).
In some embodiments, R5 is not —CH(CH3)OAc.
In some embodiments, R5 is not —CH(CH3)OAc, when each of R1-R4 is —OAc.
In some embodiments, R5 is not —C≡CH.
In some embodiments, R5 is not —C≡CH, when any of R1-R4 is —OAc.
In some embodiments, R5 is not —C≡CH, when any of R1-R4 is —OC(O)alkyl.
In some embodiments, R5 is not —C≡CH, when each of R1-R4 is —OC(O)alkyl.
In some embodiments, R5 is not —C≡CH3 when each of R1-R4 is OH.
In some embodiments, the fucose analog is alkynyl fucose peracetate. In some embodiments, the fucose analog is alkynyl fucose triacetate. In some embodiments, the fucose analog is alkynyl fucose diacetate. In some embodiments, the fucose analog is mixture of alkynyl fucose peracetate, alkynyl fucose triacetate and alkynyl fucose diacetate.
In some embodiments, the fucose analog is mixture of alkynyl fucose peracetate, alkynyl fucose triacetate, alkynyl fucose diacetate and alkynyl fucose monoacetate.
In any of the various embodiments, the fucose analog is not fucose. In some embodiments, the fucose analog is not alkynyl fucose peracetate. In some embodiments, the fucose analog is not galactose or L-galactose.
In another group of embodiments, the fucose analog has the following formula (III) or (IV):
or a biologically acceptable salt or solvate thereof, wherein each of formula (III) or (IV) can be the alpha or beta anomer or the corresponding aldose form; and wherein,
each of R1-R4 is independently selected from the group consisting of fluoro, chloro, —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynyl(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), —OC(O)C2-C10 alkynylene(heterocycle), —OCH2OC(O) alkyl, —OCH2OC(O)O alkyl —OCH2OC(O) aryl, —OCH2OC(O)O aryl, —OC(O)CH2O(CH2CH2O)nCH3, —OC(O)CH2CH2O(CH2CH2O)nCH3, —O-tri-C1-C3 alkylsilyl and —OC1-C10 alkyl, wherein each n is an integer independently selected from 0-5; and
each of R2a and R3a is independently selected from the group consisting of H, F and Cl;
R5 is selected from the group consisting of —CH3, —CHF2, —CH═C═CH2, —C≡CH, —C≡CCH3, —CH2C≡CH, —C(O)OCH3, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is F, Br, Cl or I), and methoxiran;
wherein when R5 is other than —CH═C═CH2, —CH2F or —CHF2, at least one of R1, R2, R3, R2a and R3a is fluoro or chloro.
In some embodiments of formulae (III) or (IV), R1 is F.
In some embodiments of formulae (III) or (IV), R2 is F.
In some embodiments of formulae (III) or (IV), R3 is F.
In some embodiments of formulae (III) or (IV), R1 and R2 are each F.
In some embodiments of formulae (III) or (IV), R2 and R2a are each F.
In some embodiments of formulae (III) or (IV), R1, R3 and R4 are each independently selected from —OH and —OC(O)C1-C10 alkyl; R2 is F; and R5 is —CH3.
In some embodiments of formulae (III) or (IV), R1, R3 and R4 are each independently selected from —OH and —OAc; R2 is F; and R5 is —CH3.
In some embodiments of formulae (III) or (IV), R1, R3 and R4 are each independently selected from —OH and —OC(O)C1-C10 alkyl; R2 is F; R2a and R3a are each H; and R5 is —CH3.
In some embodiments of formulae (III) or (IV), R1, R3 and R4 are each independently selected from —OH and —OAc; R2 is F; R2a and R3a are each H; and R5 is —CH3.
In some embodiments of formulae (III) or (IV), R1, R2, R3 and R4 are each independently selected from —OH and —OC(O)C1-C10 alkyl; R2a and R3a are each H; and R5 is —CHF2.
In some embodiments of formulae (III) or (IV), R1, R2, R3 and R4 are each independently selected from —OH and —OAc; R2a and R3a are each H; and R5 is —CHF2.
In some embodiments of formulae (III) or (IV), R1, R2, R3 and R4 are each independently selected from —OH and —OC(O)C1-C10 alkyl; R2a and R3a are each H; and R5 is —CH2F.
In some embodiments of formulae (III) or (IV), R1, R2, R3 and R4 are each independently selected from —OH and —OAc; R2a and R3a are each H; and R5 is —CH2F.
In another group of embodiments, the fucose analog has the following formula (V) or (VI):
or a biologically acceptable salt or solvate thereof, wherein each of formula (V) or (VI) can be the alpha or beta anomer or the corresponding aldose form; and wherein,
each of R1, R2, R2a, R3, R3a and R4 is independently selected from the group consisting of —OH, —OC(O)H, —OC(O)C1-C10 alkyl, —OC(O)C2-C10 alkenyl, —OC(O)C2-C10 alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C1-C10 alkylene(aryl), —OC(O)C2-C10 alkenylene(aryl), —OC(O)C2-C10 alkynylene(aryl), —OC(O)C1-C10 alkylene(heterocycle), —OC(O)C2-C10 alkenylene(heterocycle), —OC(O)C2-C10 alkynylene(heterocycle), —OCH2OC(O) alkyl, —OCH2OC(O)O alkyl, —OCH2OC(O) aryl, —OCH2OC(O)O aryl, —OC(O)CH2O(CH2CH2O)nCH3, —OC(O)CH2CH2O(CH2CH2O)nCH3, —O-tri-C1-C3 alkylsilyl, —OC1-C10 alkyl, and a small electron withdrawing group, wherein each n is an integer independently selected from 0-5;
R5 is a member selected from the group consisting of —CH3, —CHX2, —CH2X, —CH(X′)—C1-C4 alkyl unsubstituted or substituted with halogen, —CH(X′)—C2-C4 alkene unsubstituted or substituted with halogen, —CH(X′)—C2-C4 alkyne unsubstituted or substituted with halogen, —CH═C(R10)(R11), —C(CH3)═C(R12)(R13), —C(R14)═C═C(R15)(R16), —C3 carbocycle unsubstituted or substituted with methyl or halogen, —CH(X′)—C3 carbocycle unsubstituted or substituted with methyl or halogen, C3 heterocycle unsubstituted or substituted with methyl or halogen, —CH(X′)—C3 heterocycle unsubstituted or substituted with methyl or halogen, —CH2N3, —CH2CH2N3, and benzyloxymethyl, or R5 is a small electron withdrawing group; wherein R10 is hydrogen or C1-C3 alkyl unsubstituted or substituted with halogen; R11 is C1-C3 alkyl unsubstituted or substituted with halogen; R12 is hydrogen, halogen or C1-C3 alkyl unsubstituted or substituted with halogen; R13 is hydrogen, or C1-C3 alkyl unsubstituted or substituted with halogen; R14 is hydrogen or methyl; R15 and R16 are independently selected from hydrogen, methyl and halogen; X is halogen; X′ is halogen or hydrogen; and
additionally, each of R1, R2, R2a, R3 and R3a are optionally hydrogen; optionally two R1, R2, R2a, R3 and R3a on adjacent carbon atoms are combined to form a double bond between said adjacent carbon atoms; and
provided that at least one of R1, R2, R2a, R3, R3a, R4 and R5 is a small electron withdrawing group, or R5 comprises a halogen, site of unsaturation, carbocycle, heterocycle or azide, except when (i) R2 and R2a are both hydrogen, (ii) R3 and R3a are both hydrogen, (iii) R1 is hydrogen, (iv) a double bond is present between said adjacent carbon atoms, or (v) R5 is benzyloxymethyl; and
wherein protein, antibody or antibody derivative produced in vivo has reduced fucosylation compared to the protein, antibody or antibody derivative produced in vivo in the absence of the fucose analog.
In some embodiments of formulae (V) and (VI), R2a and R3a are each hydrogen.
In some embodiments of formulae (V) and (VI), R5 is selected from the group consisting of —CH3, —CH2CH3, —CH2C≡CH, —CH═CHCH3, -cyclopropyl, -oxirane, -oxirane substituted with methyl, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CHF2, —CH═C═CH2, —CH2N3 and —CH2CH2N3.
In some embodiments of formulae (V) and (VI), the small electron withdrawing group is selected from fluoro, chloro, bromo, —CHF2, —CH═C═CH2, —C≡CH, —C≡CCH3, —CH2C≡CH, —CO2H, —C(O)OC1-C4 alkyl, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is Br, Cl or I), and methoxiran.
In some embodiments of formulae (V) and (VI), R5 is selected from the group consisting of —CH3, —C≡CH, —CH2F, —CH2Br, and —CHF2. In some further embodiments, each of R1, R2, R2a, R3, R3a and R4 is independently selected from the group consisting of —OH, —OC(O)H, and —OC(O)C1-C10 alkyl.
In some embodiments of formulae (V) and (VI), the small electron withdrawing group is selected from fluoro, chloro, bromo, —CHF2, —CH═C═CH2, —C≡CH, —C≡CCH3, —CH2C≡CH, —CO2H, —C(O)OC1-C4 alkyl, —CH(OAc)CH3, —CN, —CH2CN, —CH2X (wherein X is Br, Cl or I), and methoxiran.
In some embodiments of formulae (V) and (VI), at least two of R1, R2, R2a, R3, R3a and R4 are independently selected small electron withdrawing groups.
In some embodiments of formulae (V) and (VI), the fucose analog is selected from compounds of Tables 1, 2 or 3.
Pharmaceutical Compositions
Fucose analogs of formulae I, II, III, IV, V and VI (hereinafter ‘fucose analogs’) can be formulated for therapeutic applications. The fucose analogs can be formulated as pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of the fucose analog and one or more pharmaceutically compatible (acceptable) ingredients. For example, a pharmaceutical or non-pharmaceutical composition typically includes one or more carriers (e.g., sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include, for example, amino acids, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will typically contain a therapeutically effective amount of the fucose analog, typically in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulations correspond to the mode of administration.
The pharmaceutical compositions described herein can be in any form that allows for the composition to be administered to an animal (e.g., a mammal). The pharmaceutical compositions described herein can be in any form that allows for the composition to be administered to a mammal. The pharmaceutical compositions described herein can be in any form that allows for the composition to be administered to a human.
The compositions can be in the form of a solid or liquid. Typical routes of administration include, without limitation, oral, parenteral, sublingual, and ocular. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Preferably, the compositions are administered parenterally or orally. These pharmaceutical compositions can be formulated so as to allow a fucose analog to be bioavailable upon administration of the composition to an animal. Compositions can also take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of a fucose analog in solid form can hold a plurality of dosage units.
Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the fucose analog, the manner of administration, the composition employed, and the severity of the disease or condition being treated.
The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid.
When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
When the composition is in the form of a capsule, e.g., a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
The composition can be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection (as described above), one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
Liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. Physiological saline is a preferred adjuvant. An injectable composition is preferably sterile.
As noted above, the amount of the fucose analog that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
The compositions comprise an effective amount of a fucose analog such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a fucose analog by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the fucose analog by weight of the composition. Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the fucose analog.
For intravenous administration, the composition can comprise from about 1 to about 250 mg of a fucose analog per kg of the animal's body weight. In some embodiments, the amount administered will be in the range from about 1 to about 25 mg/kg of body weight of the fucose analog. Preferably, the amount administered will be in the range from about 4 to about 25 mg/kg of body weight of the fucose analog.
Generally, the dosage of fucose analog administered to an animal is typically about 0.1 mg/kg to about 250 mg/kg of the animals body weight. Preferably, the dosage administered to an animal is between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.
The compositions comprise an effective amount of a fucose analog such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a fucose analog by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the fucose analog by weight of the composition. Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the fucose analog.
For intravenous administration, the composition can comprise from about 1 to about 250 mg of a fucose analog per kg of the animal's body weight. In some embodiments, the amount administered will be in the range from about 1 to about 25 mg/kg of body weight of the fucose analog. Preferably, the amount administered will be in the range from about 4 to about 25 mg/kg of body weight of the fucose analog.
Generally, a fucose analog or a pharmaceutical composition thereof can be administered on a daily, weekly, biweekly or monthly schedule, according to the desired effect. A fucose analog or a pharmaceutical composition thereof can be administered from about 1 to 5, about 1 to about 10, about 1 to about 15, or more cycles, wherein each cycle is a month in duration. The doses within each cycle can be given on daily, every other day, twice weekly, weekly, bi-weekly, once every three weeks or monthly. A cycle may optionally include a resting period, during which fucosylation of proteins (e.g., antibodies or other proteins) increases. Alternatively, a resting period can be included between cycles. Such a resting period can allow restoration of fucosylation of proteins involved in essential functions.
The preferred mode of administration of a fucose analog, or a pharmaceutical composition thereof, is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition (such as the site of cancer or autoimmune disease). In one embodiment, the fucose analog or compositions are administered parenterally. In another embodiment, the fucose analog or compositions are administered orally.
In specific embodiments, it can be desirable to administer one or more fucose analogs or compositions locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery; topical application; by injection; or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue.
In another embodiment, administration can be by direct injection at the site (or former site) of a manifestation of an autoimmune disease.
In another embodiment, the fucose analogs can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).
In yet another embodiment, the fucose analogs or compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) can be used.
The term “carrier” refers to a diluent, adjuvant or excipient, with which a fucose analog is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the fucose analogs or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the fucose analogs are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
Therapeutic Methods Using Fucose Analogs to Reduce Antibody and Other Protein Fucosylation In Vivo
The fucose analogs of formulae I, II, III, IV, V and VI (hereinafter ‘the fucose analogs’) as provided herein are useful for treating cancer, an autoimmune disease or an infectious disease in an animal.
Treatment of Cancer
The fucose analogs are useful for treating cancer in patients. Administration of a fucose analog to an animal (e.g., a mammal, such as a human) in need thereof can result in inhibition of the multiplication of a tumor cell(s) or cancer cell(s), or treatment of cancer in an animal (e.g., a human patient). The fucose analogs can be used accordingly in a variety of settings for the treatment of animal cancers.
The fucose analogs are also useful for enhancing the in vivo production of antibodies lacking core fucosylation. Increasing the proportion of such antibodies against cancer targets in a patient can result in inhibition of the multiplication of a tumor cell(s) or cancer cell(s), or treatment of cancer in an animal (e.g., a human patient). The fucose analogs can be used accordingly in a variety of settings for the treatment of animal cancers.
Particular types of cancers that can be treated with the fucose analogs include, solid tumors and hematologic malignancies. Such cancers include, but are not limited to: (1) solid tumors, including but not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma, multiforme astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma; (2) blood-borne cancers, including but not limited to acute lymphoblastic leukemia “ALL”, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairy cell leukemia, multiple myeloma, acute and chronic leukemias, e.g., lymphoblastic myelogenous and lymphocytic myelocytic leukemias, and (3) lymphomas such as Hodgkin's disease, non-Hodgkin's Lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, and Polycythemia vera.
Multi-Modality Therapy for Cancer
Cancer, including, but not limited to, a tumor, a metastasis, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of a fucose analog of any of formulae I, II, III, IV, V or VI as provided above, to an animal (e.g., a mammal, such as a human) in need thereof. In some embodiments, the invention provides methods for treating or preventing cancer, comprising administering to an animal in need thereof an effective amount of a fucose analog and optionally a chemotherapeutic agent. In one embodiment the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory. In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory. The fucose analogs can be administered to an animal that has also undergone surgery as treatment for the cancer.
In one embodiment, the additional method of treatment is radiation therapy.
In a specific embodiment, the fucose analog is administered concurrently with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of a fucose analog, preferably at least an hour, five hours, 12 hours, a day, a week, two weeks, three weeks, a month, or several months (e.g., up to three months), prior or subsequent to administration of a fucose analog.
A chemotherapeutic agent can be administered over a series of sessions, and can be any one or a combination of the chemotherapeutic agents provided herein. With respect to radiation, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, can also be administered.
Additionally, the invention provides methods of treatment of cancer with a fucose analog as an alternative to chemotherapy or radiation therapy, where the chemotherapy or the radiation therapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The animal being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
Multi-Drug Therapy for Cancer
The present invention includes methods for treating cancer, comprising administering to an animal in need thereof an effective amount of a fucose analog and a therapeutic agent that is an anti-cancer agent. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, a camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred embodiment, the anti-cancer agent includes, but is not limited to: alkylating agents, nitrogen mustards (cyclophosphamide, Ifosfamide, trofosfamide, Chlorambucil), nitrosoureas (carmustine (BCNU), Lomustine (CCNU)), alkylsulphonates (busulfan, Treosulfan), triazenes (Dacarbazine), platinum containing compounds (cisplatin, oxaliplatin, carboplatin), plant alkaloids (vinca alkaloids vincristine, Vinblastine, Vindesine, Vinorelbine), taxoids (paclitaxel, Docetaxol), DNA Topoisomerase Inhibitors, Epipodophyllins (etoposide, Teniposide, Topotecan, 9-aminocamptothecin, camptothecin), crisnatol, mitomycins (Mitomycin C); Anti-metabolites such as Anti-folates: DHFR inhibitors: methotrexate, Trimetrexate; IMP dehydrogenase Inhibitors: mycophenolic acid, Tiazofurin, Ribavirin, EICAR; Ribonucleotide reductase Inhibitors: hydroxyurea deferoxamine; Pyrimidine analogs: Uracil analogs: 5-Fluorouracil, Floxuridine, Doxifluridine, Ratitrexed; Cytosine analogs: cytarabine (ara C), Cytosine arabinoside, fludarabine; Purine analogs: mercaptopurine, Thioguanine; Hormonal therapies: Receptor antagonists: Anti-estrogen: Tamoxifen, Raloxifene, megestrol; LHRH agonists: goscrclin, Leuprolide acetate; Anti-androgens: flutamide, bicalutamide; Retinoids/Deltoids: Vitamin D3 analogs: EB 1089, CB 1093, KH 1060; Photodynamic therapies: vertoporfin (BPD-MA), Phthalocyanine, photosensitizer Pc4, Demethoxy-hypocrellin A (2BA-2-DMHA); Cytokines: Interferon-alpha, Interferon-gamma; Tumor necrosis factor: Others: Isoprenylation inhibitors: Lovastatin; Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion; Cell cycle inhibitors: staurosporine; Actinomycins: Actinomycin D, Dactinomycin; Bleomycins: bleomycin A2, Bleomycin B2, Peplomycin; Anthracyclines: daunorubicin, Doxorubicin (adriamycin), Idarubicin, Epirubicin, Pirarubicin, Zorubicin, Mitoxantrone; MDR inhibitors: verapamil; and Ca2+ ATPase inhibitors: thapsigargin.
Adjuvant Therapy for Cancer
The fucose analogs can be used as an adjuvant, in combination with a cancer vaccine. The term “cancer vaccine” as used herein means a compound that selectively damages tumor cells by inducing and/or enhancing a specific immune response against the tumor cells. A cancer vaccine can be, for example, a medicament comprising a peptide, polypeptide or protein of a TAA or TSA, and pharmaceutical compositions containing a peptide, polypeptide or protein of a TAA or TSA. As used herein, TSA refers to a “tumor-specific antigen” and TAA refers to a tumor-associated antigen. TSAs are molecules unique to cancer cells. TAAs are molecules shared, but differently expressed, by cancer cells and normal cells.
The dosage of the cancer vaccine can be determined with appropriate modifications according to the extent of stimulation of an immune response against the vaccine. In general, it is between 0.01 and 100 mg/day/adult human, or preferably between 0.1 and 10 mg/day/adult human as an active principle. The cancer vaccine can be administered from once every few days to every few months. Administration can be carried out according to well-known methods for administrating a peptide, polypeptide or protein for medical use, such as subcutaneously, intravenously, or intramuscularly. In order to induce and/or enhance the immune response during administration, the peptide, polypeptide or protein can be used, in the presence or absence of an appropriate adjuvant, with or without linking to a carrier. The carrier is not limited as long as it exerts no harmful effect by itself onto the human body and is capable of enhancing antigenicity; cellulose, polymeric amino acids, albumin, and the like can be given as examples of carriers. Adjuvants can be those used in general for peptide vaccine inoculation, and a Freund incomplete adjuvant (HA), aluminum adjuvant (ALUM), Bordetella pertussis vaccine, mineral oil, and the like can be given as examples. In addition, the formulation can be suitably selected by applying a suitable well-known method for formulating a peptide, polypeptide or protein.
Otherwise, an effective cancer vaccine effect can be obtained also by collecting a fraction of mononuclear cells from the peripheral blood of a patient, incubating them with the peptide, polypeptide or protein of the present invention, and then returning the fraction of mononuclear cells in which induction of CTL and/or activation of CTL was observed, into the blood of the patient. A fucose analog can be co-administered during or after re-administration of the mononuclear cells. Culture conditions, such as mononuclear cell concentration, concentration of the peptide, polypeptide or proteins, culture time, and the like, can be determined by simply repeating studies. A substance having a capability to enhance the growth of lymphocytes, such as interleukin-2, may be added during culturing.
Treatment of Autoimmune Diseases
The fucose analogs are useful for modulating an autoimmune disease or for treating an autoimmune disease, so as to decrease symptoms and/or the autoimmune response. The fucose analogs can be used accordingly in a variety of settings for the treatment of an autoimmune disease in an animal.
In one embodiment, the fucose analogs down-regulate or down-modulate an auto-immune antibody associated with a particular autoimmune disease.
Particular types of autoimmune diseases that can be treated with the fucose analogs include, but are not limited to, Th2-lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft versus host disease); Th1 lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); activated B lymphocyte-related disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes); and those disclosed below.
Active Chronic Hepatitis, Addison's Disease, Allergic Alveolitis, Allergic Reaction, Allergic Rhinitis, Alport's Syndrome, Anaphlaxis, Ankylosing Spondylitis, Anti-phosholipid Syndrome, Arthritis, Ascariasis, Aspergillosis, Atopic Allergy, Atropic Dermatitis, Atropic Rhinitis, Behcet's Disease, Bird-Fancier's Lung, Bronchial Asthma, Caplan's Syndrome, Cardiomyopathy, Celiac Disease, Chagas' Disease, Chronic Glomerulonephritis, Cogan's Syndrome, Cold Agglutinin Disease, Congenital Rubella Infection, CREST Syndrome, Crohn's Disease, Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis, Discoid Lupus, Dressler's Syndrome, Eaton-Lambert Syndrome, Echovirus Infection, Encephalomyelitis, Endocrine opthalmopathy, Epstein-Barr Virus Infection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty's Syndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy, Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis, Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease, Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia, Henoch-Schonlein, Purpura Idiopathic Adrenal Atrophy, Idiopathic Pulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases, Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, Juvenile Diabetes Mellitus (Type I), Lambert-Eaton Syndrome, Laminitis, Lichen Planus, Lupoid Hepatitis, Lupus Lymphopenia, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Psoriatic Arthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome, Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome, Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, Systemic Lupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis, Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic Epidermal Necrolysis Type B, Insulin Resistance Type I Diabetes Mellitus, Ulcerative Colitis, Uveitis Vitiligo, Waldenstrom's Macroglobulemia, and Wegener's Granulomatosis.
Multi-Drug Therapy of Autoimmune Diseases
The present invention also provides methods for treating an autoimmune disease, comprising administering to an animal (e.g., a mammal) in need thereof an effective amount of a fucose analog and optionally a therapeutic agent that known for the treatment of an autoimmune disease. In one embodiment, the anti-autoimmune disease agent includes, but is not limited to cyclosporine, cyclosporine A, mycophenylate, mofetil, sirolimus, tacrolimus, enanercept, prednisone, azathioprine, methotrexate, cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam or infliximab.
Treatment of Infectious Diseases
The fucose analogs are useful for enhancing an immune response that results in increased killing or inhibition of the multiplication of a cell that produces an infectious disease or for treating an infectious disease. The fucose analogs can be used accordingly in a variety of settings for the treatment of an infectious disease in an animal.
In one embodiment, the fucose analogs enhance an immune response, resulting in kill or inhibit, or increased killing or inhibition, of the multiplication of cells that produce a particular infectious disease.
Particular types of infectious diseases that can be treated with the fucose analogs include, but are not limited to, (1) Bacterial Diseases: Diptheria, Pertussis, Occult Bacteremia, Urinary Tract Infection, Gastroenteritis, Cellulitis, Epiglottitis, Tracheitis, Adenoid Hypertrophy, Retropharyngeal Abcess, Impetigo, Ecthyma, Pneumonia, Endocarditis, Septic Arthritis, Pneumococcal, Peritonitis, Bactermia Meningitis, Acute Purulent Meningitis, Urethritis, Cervicitis, Proctitis, Pharyngitis, Salpingitis, Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax, Nocardiosis, Salmonella, Typhoid Fever, Dysentery, Conjuntivitis, Sinusitis, Brucellosis, Tullaremia, Cholera, Bubonic Plague, Tetanus, Necrotizing Enteritis, Actinomycosis Mixed Anaerobic Infections, Syphilis, Relapsing Fever, Leptospirosis, Lyme Disease, Rat Bite Fever, Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, Chlamydial Pneumonia, Trachoma, Inclusion Conjunctivitis, Systemic; (2) Fungal Diseases: Histoplamosis, Coccicidiodomycosis, Blastomycosis, Sporotrichosis, Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis, Mycetoma, Chromomycosis; (3) Rickettsial Diseases: Typhus, Rocky Mountain Spotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses, Rickettsialpox, Q Fever, and Bartonellosis; (4) Parasitic Diseases: Malaria, Babesiosis, African Sleeping Sickness, Chagas Disease, Leishmaniasis, Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis, Keratitis, Entamebiasis, Giardiasis, Cryptosporidiasis, Isosporiasis, Cyclosporiasis, Microsporidiosis, Ascariasis, Whipworm Infection, Hookworm Infection, Threadworm Infection, Ocular Larva Migrans, Trichinosis, Guinea Worm Disease, Lymphatic Filariasis, Loiasis, River Blindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch, Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis, Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Disease, Alveolar Hydatid Disease; (5) Viral Diseases: Measles, Subacute sclerosing panencephalitis, Common Cold, Mumps, Rubella, Roseola, Fifth Disease, Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis, Infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot-and-Mouth Disease, Bornholm Disease, Genital Herpes, Genital Warts, Aseptic Meningitis, Myocarditis Pericarditis, Gastroenteritis, Acquired Immunodeficiency Syndrome (AIDS), Reye's Syndrome, Kawasaki Syndrome, Influenza, Bronchitis, Viral “Walking” Pneumonia, Acute Febrile Respiratory Disease, Acute pharyngoconjunctival fever, Epidemic keratoconjunctivitis, Herpes Simplex Virus 1 (HSV-1), Herpes Simples Virus 2 (HSV-2), Shingles, Cytomegalic Inclusion Disease, Rabies, Progressive Multifocal Leukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt-Jakob Disease, Gerstmann-Straussler-Scheinker Disease, Tropical Spastic Paraparesis, Western Equine Encephalitis, California Encephalitis, St. Louis Encephalitis, Yellow Fever, Dengue Lymphocytic choriomeningitis, Lassa Fever, Hemorrhagic Fever, Hantvirus, Pulmonary Syndrome, Marburg Virus Infections, Ebola Virus Infections and Smallpox.
Multi-Drug Therapy of Infectious Diseases
The present invention also provides methods for treating an infectious disease, comprising administering to an animal (e.g., a mammal) in need thereof a fucose analog and optionally a therapeutic agent that is an anti-infectious disease agent. In one embodiment, the anti-infectious disease agent is, but not limited to: (1) Antibacterial Agents: β-Lactam Antibiotics: Penicillin G, Penicillin V, Cloxacilliin, Dicloxacillin, Methicillin, Nafcillin, Oxacillin, Ampicillin, Amoxicillin, Bacampicillin, Azlocillin, Carbenicillin, Mezlocillin, Piperacillin, Ticarcillin; Aminoglycosides: Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin; Macrolides: Azithromycin, Clarithromycin, Erythromycin, Lincomycin, Clindamycin; Tetracyclines: Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline; Quinolones: Cinoxacin, Nalidixic Acid, Fluoroquinolones: Ciprofloxacin, Enoxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxicin; Polypeptides: Bacitracin, Colistin, Polymyxin B; Sulfonamides: Sulfisoxazole, Sulfamethoxazole, Sulfadiazine, Sulfamethizole, Sulfacetamide; Miscellaneous Antibacterial Agents: Trimethoprim, Sulfamethazole, Chloramphenicol, Vancomycin, Metronidazole, Quinupristin, Dalfopristin, Rifampin, Spectinomycin, Nitrofurantoin; Antiviral Agents: General Antiviral Agents: Idoxuradine, Vidarabine, Trifluridine, Acyclovir, Famcicyclovir, Pencicyclovir, Valacyclovir, Gancicyclovir, Foscarnet, Ribavirin, Amantadine, Rimantadine, Cidofovir; Antisense Oligonucleotides; Immunoglobulins; Inteferons; Drugs for HIV infection: Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Nevirapine, Delavirdine, Saquinavir, Ritonavir, Indinavir and Nelfinavir.
Other Therapeutic Agents
The present methods can further comprise the administration of a fucose analog and a therapeutic agent or pharmaceutically acceptable salts or solvates thereof. The fucose analog and the therapeutic agent can act additively or, more preferably, synergistically. In a preferred embodiment, a composition comprising a fucose analog is administered concurrently with the administration of one or more therapeutic agent(s), which can be part of the same composition or in a different composition from that comprising the fucose analog. In another embodiment, a fucose analog is administered prior to or subsequent to administration of the therapeutic agent(s).
In the present methods for treating cancer, an autoimmune disease or an infectious disease, the therapeutic agent also can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, proclorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.
In another embodiment, the therapeutic agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and erythropoietin alfa.
In still another embodiment, the therapeutic agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, piroxicam and sulindac.
The invention is further described in the following examples, which are not intended to limit the scope of the invention.
EXAMPLES
Example 1
In Vivo Production of Non-Fucosylated Antibodies
Methods:
Female BALB/c mice were immunized with Keyhole Limpet Hemocyanin (KLH) 30-60 days prior to starting this study. For the first study, mice were dosed ip with 150 mg/kg of alkynyl fucose (SGD-1887), alkynyl fucose peracetate (SGD-1890), 2-fluorofucose (SGD-2083), or 2-fluorofucose triacetate (SGD-2084) daily for 7 days or were untreated. On day 2, mice were also boosted with KLH. On the day after the last ip dose, mice were terminally bled and serum obtained. For the second study, mice were given 100 mM of 2-fluorofucose (SGD-2083) in their drinking water or given untreated water for 7 days, boosted with KLH, and continued with 100 mM 2-fluorofucose containing drinking water or untreated water for 7 more days before being terminally bled. Serum was then obtained. No mice died in either study. For both studies, serum was passed over a commercial anti-KLH affinity column to obtain KLH-specific polyclonal antibodies. The flow through from the anti-KLH affinity column was passed over a commercial protein A column to obtain the remaining antibodies.
Dot blots: 0.5 μg each of antibodies from untreated and treated animals, as well as standards of antibody cAC10 having known amounts of core fucosylation (0 to 100% fucose), were blotted onto a nitrocellulose membrane. The proteins levels were visualized by Ponceau staining. The blot was probed with biotinylated Aspergillus oryzae L-fucose-specific lectin (AOL) (which binds to fucosylated antibodies) and developed with streptavidin HRP and ECL. Gel loading (visible) and fucose signals (bioluminescence) were measured with an Alpha Innotech camera and quantitated with the machine software.
Gas chromatography (GC): 40 μg each of the antibodies from untreated and treated animals that had been dialyzed against water, were subjected to methanolysis in methanolic HCl. Control samples of antibody cAC10 with 0 to 100% fucose were similarly treated. The resulting methylglycosides were derivatized by trimethylsilylation of the monosaccharide alcohols using a commercially available cocktail, Tri-Sil. The resulting trimethylsilyl methylglycosides were examined on a Hewlet Packard gas chromatograph with flame ionization detection using a temperature gradient on an Agilent J&W DB-1 column. The relevant peaks were identified by retention time comparison to sugar standards derivatized in parallel with the Ab samples. Peaks were integrated using the GC software. The fucose/mannose peak area ratios were used to determine the fucosylation state of the antibodies.
Results:
Study 1: FIG. 1 shows the dot blot (left side) from antibodies that did not bind to KLH, but were recovered from protein A column. Results are shown for cAC10 standards (lower dot blot, left most dashed rectangle and corresponding columns of upper dot blot), untreated control (lower dot blot, second dashed rectangle from the left and corresponding column of upper dot blot), and alkynyl fucose (SGD-1887; lower dot blot, middle dashed rectangle and corresponding column of upper dot blot), alkynyl fucose peracetate (SGD-1890, lower dot blot, second dashed rectangle from the right and corresponding column of upper dot blot), and 2-fluorofucose (SGD-2083; lower dot blot, right most rectangle and corresponding column of upper dot blot). Normalizing for loading level, the percent fucosylation is also shown in the graph on the right. On average, the fucosylation levels of the antibodies were reduced by about one third, as compared to the untreated controls.
FIG. 2 shows the fucosylation levels of both the anti-KLH antibodies (panels A and B) and the remaining serum IgG molecules isolated from the treated groups (panels C and D). Fucosylation levels are shown both as percent fucosylation based on the cAC10 antibody standards (panels A and C) and the average value for the treated groups as a percentage of the average value for the untreated control group (panels B and D). On average, the fucosylation levels of the anti-KLH antibodies were reduced by about one half by treatment with three of the fucose analogs. The remaining collected antibodies also exhibited a reduction in core fucosylation of about one quarter. In this study, overall antibody levels (KLH-specific and non-specific) increased in the mice after exposure to KLH. As a result, most antibodies were newly synthesized during the treatment periods. These observations indicate that newly produced antibodies can exhibit reduced core fucosylation following administration of a fucose analog.
Study 2: In this study, the effect of oral administration of fucose analogs was examined FIG. 3 shows the results of treatment of mice by oral administration of fucose analogs. Antibody fucosylation levels examined were those of the antibodies that did not bind to KLH, but were recovered from the protein A column. Results are shown for cAC10 standards (upper and lower dot blots, left most rectangle), untreated control (upper and lower dot blots, second from the left (upper) and right rectangles), and 2-fluorofucose (upper and lower dot blots, second from the left (lower) and second from the right rectangles (upper and lower)). Normalizing for loading level, the percent fucosylation is also shown in the graph at the right. Core fucosylation levels of antibodies from the treated animals were nearly eliminated: on average, fucosylation levels were 7% for treated and 81% for untreated animals. These observations indicate that oral administration of fucose analogs is an effective means to decrease antibody fucosylation levels.
Example 2
Activity of Fucose Analogs In Vitro in Cell Culture
Fucose analogs have been evaluated for their effect on antibody core fucosylation at concentrations of 50 μM and 1 mM generally as described in Published US Patent Application 2009-0317869. Briefly, the protocol was as follows: A CHO DG44 cell line producing a humanized IgG1 anti-CD70 monoclonal antibody, h1F6 (see International Patent Publication WO 06/113909) was cultured at 7.5×105 cells per mL in 2 mLs of CHO culture media at 37°, 5% CO2 and shaking at 100 RPM in a 6 well tissue culture plate. Media was supplemented with insulin like growth factor (IGF), penicillin, streptomycin and either 1 mM or 50 μM of the fucose analog. On day 5 post inoculation, the culture was centrifuged at 13000 RPM for 5 minutes to pellet the cells; antibodies were then purified from supernatant.
Antibody purification was performed by applying the conditioned media to protein A resin pre-equilibrated with 1× phosphate buffered saline (PBS), pH 7.4. After washing resin with 20 resin bed volumes of 1× PBS, antibodies were eluted with 5 resin bed volumes of Immunopure IgG elution buffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of 1M Tris pH 8.0 was added to neutralize the eluted fraction. The results are shown in the following tables.
TABLE 1
Name
Inhibition at
Inhibition
(Chemical name)
R5
R1-R4
50 μM
at 1 mM
Alkynyl fucose
—C≡CH
—OH
>80%
ND
(5-ethynylarabinose)
Alkynyl fucose peracetate
—C≡CH
—OAc
>80%
>80%
Alkynyl fucose tetraacetate
(5-ethynylarabinose tetraacetate)
5-propynyl fucose tetraacetate
—C≡CCH3
—OAc
50%
>80%
(5-propynylarabinose
tetraacetate)
propargyl fucose tetraacetate
—CH2C≡CH
—OAc
~10%
~10-20%
((3S,4R,5R,6S)-6-(prop-2-
ynyl)-tetrahydro-2H-pyran-
2,3,4,5-tetrayl tetraacetate)
Peracetyl galactose
—OAc
—OAc
~0%
~0%
(galactose pentaacetate)
5-vinyl fucose tetraacetate
—CHCH2
—OAc
~0%
~4%
(5-ethylenylarabinose
tetraacetate)
6-cyano fucose tetraacetate
—CH2CN
—OAc
30%
>80%
(6-cyanofucose tetraacetate)
5-cyano fucose tetraacetate
—CN
—OAc
20%
ND
(pyranose form)
(5-cyanoarabinopyranose
tetraacetate)
5-cyano fucose tetraacetate
—CN
—OAc
5-10%
ND
(furanose form)
(5-cyanoarabinofuranose
tetraacetate)
5-methylester fucose
—C(O)OCH3
—OAc
30%
>80%
tetraacetate
(5-carboxymethyl arabinose
tetraacetate)
5-(CH(OAc)CH3) peracetyl
—CH(OAc)CH3
—OAc
~0%
40%
fucose
(6-methylgalactose
pentaacetate)
5-methyloxiran-arabinose tetraacetate ((3S,4R,5S,6R)-6-((S)-2- methyloxiran-2-yl)-tetrahydro- 2H-pyran-2,3,4,5-tetrayl tetraacetate)
—OAc
~0%
~35-40%
6-iodo-fucose tetraacetate
—CH2I
—OAc
3%
30%
(6-iodofucose tetraacetate)
6-chloro-fucose tetraacetate
—CH2Cl
—OAc
20%
20-30%
(6-chlorofucose tetraacetate
6-bromo-fucose tetraacetate
—CH2Br
—OAc
50%
80%
(6-bromofucose tetraacetate)
Alkynyl fucose tetrapropanonate
—C≡CH
—OC(O)CH2—CH3
>80%
>80%
(5-ethynylarabinose
tetrapropropanoate)
Alkynyl fucose tetra-
—C≡CH
—OC(O)(CH2)4—CH3
>80%
>80%
n-hexanoate
(5-ethynylarabinose
tetrahexanoate)
Alkynyl fucose
—C≡CH
—OC(O)C(CH3)3
20%
60%
tetrakis(trimethylacetate)
(5-ethynylarabinose
tetra(trimethylacetate))
Alkynyl fucose
—C≡CH
—OC(O)C(CH3)3
5%
10%
tetrakis(trimethylacetate)
(5-ethynylarabinose
tetra(trimethylacetate))
Alkynyl fucose 1,2,3-
—C≡CH
—OC(O)C(CH3)3
~0%
ND
(trimethylacetate)
and —OH
(5-ethynylarabinose 1,2,3-
(trimethylacetate))
Alkynyl fucose
—C≡CH
—OC(O)C(CH3)3
>80%
ND
di(trimethylacetate)
and —OH
(5-ethynylarabinose 1,3-
(trimethylacetate))
Alkynyl fucose pernicotinate
—C≡CH
—C(O)-3-pyridyl
>80%
>80%
Alkynyl fucose perisonicotinate
—C≡CH
—C(O)-4-pyridyl
>80%
>80%
Alkynyl fucose per-PEG ester
—C≡CH
—C(O)—(CH2CH2O)2—OCH3
>80%
>80%
1-methyl-2,3,4-triacetyl alkynyl
—C≡CH
R1 = OCH3
68%
>80%
fucose
R2, R3, R4 = OAc
Alkynyl fucose perisobutanoate
—C≡CH
—OC(O)CH(CH3)2
>80%
>80%
“ND” means not detected due to poor antibody production or inhibition of cell growth in the presence of the fucose analog.
TABLE 2
Name
Inhibition
Inhibition
(Chemical name)
R5
R1
R2/R2a
R3/R3a
at 50 μM
at 1 mM
2-deoxy-2-fluorofucose
—CH3
—OH
—F/—H
—OAc/
>80%
>80%
diacetate
—H
(R4 = OAc)
2-deoxy-2-chlorofucose
—CH3
—OAc
—Cl/
—OAc/
17%
>80%
triacetate
—H
—H
(R4 = OAc)
Allene
—CH═C═CH2
—OAc
—OAc/
—OAc/
23%
34%
(R4 = OAc)
—H
—H
2-deoxy-2-fluorofucose
—CH3
—OH
—F/—H
—OH/
>80%
>80%
(R4 = OH)
—H
2-deoxy-2-fluorofucose
—CH3
—OAc
—F/—H
—OAc/
>80%
>80%
peracetate
—H
(R4 = OAc)
1,2-difluoro-1,2-didexoy
—CH3
—F
—F/—H
—OAc/
>80%
>80%
fucose peracetate
—H
(R4 = OAc)
6,6-difluorofucose
—CHF2
—OAc
—OAc/
—OAc/
>80%
>80%
tetraacetate
—H
—H
(R4 = OAc)
2-deoxy-2,2-
—CH3
—OAc
—F/—F
—OAc/
0
64%
difluorofucopyranose
—H
triacetate (alpha)
(R4 = OAc)
2-deoxy-2,2-
—CH3
—OAc
—F/—F
—OAc/
0
75%
difluorofucopyranose
—H
triacetate (beta)
(R4 = OAc)
6-methyl-tetrahydro-2H-
—CH3
—OAc
—H/—H
—OAc/
0
36%
pyran-2,4,5-triyl triacetate
—H
(R4 = OAc)
5-Benzyloxy fucose
—CH2OCH2Ph
—OAc
—OAc/
—OAc/
0
75%
peracetate
—H
—H
(R4 = OAc)
“ND” not detected due to poor antibody production or inhibition of cell growth in the presence of the fucose analog.
Certain other fucose analogs were tested for their ability to be incorporated into antibodies. These fucose analogs were tested at concentrations of 50 μM and 1 mM using the methodology as described above. The results are shown in the following table.
TABLE 3
Name
(Chemical name)
R5
R1-R4
% Incorporation
Propargyl fucose or (3S,4R,5R)-6-(prop-2- ynyl)tetrahydro-2H-pyran- 2,3,4,5-tetrayl tetraacetate
—OAc
80% (1 mM)
5-(Z)-propenyl fucose peracetate
—OAc
~30%
Isopropenyl peracetyl fucose or (3S,4R,5R,6S)-6- (prop-1-en-2- yl)-tetrahydro-2H- pyran-2,3,4,5- tetrayl tetraacetate
—OAc
>80% (1 mM and 50 uM)
5-ethyl fucose
—CH2CH3
—OH
>80% (1 mM and
or
50 uM)
(3S,4R,5S,6S)-6-ethyl-
tetrahydro-2H-pyran-
2,3,4,5-tetraol
5-ethyl fucose
—CH2CH3
—OAc
>90% (1 mM and
peracetate or
50 uM)
(3S,4R,5S,6S)-6-ethyl-
tetrahydro-2H-pyran-
2,3,4,5-tetrayl tetraacetate
5-cyclopropyl fucose or (3S,4R,5S,6S)-6- cyclopropyltetrahydro- 2H-pyran- 2,3,4,5-tetraol
—OH
~80%
5-cyclopropyl fucose peracetate or (3S,4R,5R,6S)-6- cyclopropyltetrahydro- 2H-pyran-2,3,4,5- tetrayl tetraacetate
—OAc
~80%
5-propyloxyarabinose tetraacetate or (3S,4R,5S,6R)-6-((S)-2- methyloxiran-2-yl) tetrahydro-2H-pyran- 2,3,4,5-tetrayl tetraacetate
—OAc
~60%
Fluoromethylene fucose
—CH2F
—OAc
>90% (1 mM and
or
50 μM)
(3S,4R,5S)-6-
(fluoromethyl)tetrahydro-
2H-pyran-2,3,4,5-tetrayl
tetraacetate
5-chloromethylene
—CH2Cl
—OAc
~80%
peracetyl fucose
or
(3S,4R,5S)-6-
(chloromethyl)tetrahydro-
2H-pyran-2,3,4,5-tetrayl
tetraacetate
5-bromomethylene
—CH2Br
—OAc
~50%
peracetyl fucose
(50 uM; 20% at
or
1 mM)
(3S,4R,5S)-6-
(bromomethyl)tetrahydro-
2H-pyran-2,3,4,5-
tetrayl tetraacetate
5-iodomethylene-
—CH2I
—OAc
~30%
peracetyl fucose
or
(3S,4R,5S)-6-
(iodomethyl)tetrahydro-
2H-pyran-2,3,4,5-
tetrayl tetraacetate
Azido peracetyl fucose
—CH2N3
—OAc
60%
or
(3S,4R,5R)-6-
(azidomethyl)tetrahydro-
2H-pyran-2,3,4,5-
tetrayl tetraacetate
5-(2-azidoethyl)
—CH2CH2N3
—OAc
20%
arabinose tetraacetate
or
(3S,4R,5R,6S)-6-(2-
azidoethyl)tetrahydro-
2H-pyran-2,3,4,5-
tetrayl tetraacetate
—CH═C═CH2
—OAc
~30%
Isopropyl peracetyl
Isopropyl
—OAc
Not detected
fucose or
(3S,4R,5R,6S)-6-
isopropyltetrahydro-
2H-pyran-2,3,4,5-
tetrayl tetraacetate
These assays identified candidate compounds for inhibition of core antibody fucosylation in mammals.
Example 3
Production of Non-fucosylated Antibodies In Vivo Following Oral Administration
In this study, the effects of oral administration of the fucose analog 2-fluorofucose (SGD-2083) were further examined Female BALC/c mice were offered 1, 10 and 100 mM 2-fluorofucose in their drinking water for 14 days. Mice were immunized with TiterMAX Classic and offered 1, 10 and 100 mM 2-fluorofucose in their drinking water for an additional 7 days. Mice were then terminally bled and serum obtained. Endogenous antibodies were purified by passing the serum over a protein A column.
The collected antibodies were evaluated for fucosylation levels by dot blot as follows. Antibodies from untreated and treated animals (0.5 μg each), as well as standards of cAC10 with 0 to 100% fucose (only study 1), were blotted onto a nitrocellulose membrane. The proteins levels were visualized with Ponceau S. The blot was probed with biotinylated AOL lectin and developed with streptavidin HRP and ECL (as described above). Gel loading (visible) and fucose signals (bioluminescence) were measured with an Alpha Innotech camera and quantitated with the machine software.
Results:
There was a dose-dependent decrease in fucosylation levels of antibodies over the three concentrations of 2-fluorofucose (SGD-2083). Referring to FIG. 4, antibody fucosylation levels were highest in the untreated control and 1 mM 2-fluorofucose groups (left and middle panels, upper two rectangles). Antibodies from the intermediate concentration of 2-fluorofucose were nearly as depleted of fucose as the high concentration of 100 mM 2-fluorofucose. These results confirm that administration of 2-fluorofucose to mice can inhibit core antibody fucosylation.
Example 4
Affects of Fucose Analogs on Human Cells
The ability of different fucose analogs to inhibit the fucosylation of IgG antibodies produced by human myeloma cells as well as the fucosylation of surface proteins of human cancer cell lines was investigated. In a first study, the ability of fucose analogs to inhibit fucosylation of IgG produced by the cell line LP-1, a human multiple myeloma cell line, was investigated. The antibodies produced by, and found in the culture medium of, untreated LP-1 cells were confirmed to be of the IgG type by western blot using anti-human IgG detection (data not shown). This was accomplished by growing 20 mL of LP-1 cells in a T-75 culture flask (250,000 cells/mL) for 5 days at 37° C. in a humidified atmosphere of 5% CO2 in IgG-depleted tissue culture media (90% RPMI with 10% IgG-depleted heat inactivated FBS). Harvest of the cells was by centrifugation (200×g, 4° C., 5 min), and the culture medium was collected. The medium was filtered through a 0.22 μm filter and then incubated with 1 mL of a 50% MabSelect™ protein A resin slurry in PBS at 4° C. with rotation overnight to capture the IgG. The resin slurry was allowed to settle and most of the medium was removed. The resin slurry was transferred with ˜0.5 mL media to two cellulose acetate filter spin cups and was centrifuged at 5000×g for 1 mm. The resin bed was then washed 3 times with 0.5 mL PBS. The IgG was eluted with 700 μL of Pierce IgG elution buffer (into with 52 μL of 9 M Tris buffer, pH 9.5 to adjust the pH after elution). The resulting elution was transferred to a 10,000 MW cutoff centrifugal concentrator and the sample was concentrated to approximately 20 μL. 1 μL of the concentrated sample was loaded on an SDS polyacrylamide gel for separation followed by blotting onto nitrocellulose membrane. Staining of the blot for total protein was with Ponceau S and for identification of isotype with anti-human IgG antibody. The total protein stain showed bands consistent with molecular weights of IgG heavy and light chains and the anti-human IgG stain showed reaction with the protein band consistent with the molecular weight of heavy chain as expected.
Antibody fucosylation can also be determined using a biotin-labeled Aspergillus oryzae L-fucose-specific lectin (AOL), which binds specifically to the α-1,6-linked fucose of the antibody. This method for fucose detection works for both blotted protein that has either been separated by SDS-PAGE or for protein that has been applied to nitrocellulose without separation. The fluorescent signal generated using the AOL-biotin conjugate with streptavidin-HRP binding and ECL detection can be quantitated using an Alpha Innotech FlourChem® Q system. The IgG isolated from LP-1 culture displayed an AOL-dependent signal in the band corresponding to the MW of the heavy chain as expected (data not shown). The analogs 2-fluorofucose (SGD-2083) and 2-fluorofucose peracetate (SGD-2084) did not inhibit fucosylation of antibody, but alkynyl fucose peracetate (SGD-1890) did.
To further evaluate the activities of different fucose analogs, 48 different fucose analogs and other four glycosylation inhibitors were tested for their ability to affect the fucosylation the LP-1-generated IgG. LP-1 cells (250,000 cells/mL, 3 mL per compound in 6-well plates) were incubated with 100 μM of each fucose analog for 5 days at 37° C. with a humidified atmosphere of 5% CO2 in IgG-depleted tissue culture media (90% RPMI with 10% IgG-depleted heat inactivated FBS). The IgG was isolated as described above using only 0.5 mL of MabSelect™ protein A resin slurry and one spin cup per sample with elution in 400 μL of IgG elution buffer into 25 μL of 9 M Tris buffer, pH 9. The eluates were concentrated to 10-20 μL per sample and 2 μL of each of the concentrated eluates were dotted onto a nitrocellulose membrane and stained with Ponceau S to estimate and adjust the sample loading for AOL staining. From this estimation of total protein in each sample, approximately 0.5 μg of each sample was dotted onto the membrane, air-dried, and stained with Ponceau S. An image of this stained membrane was captured using an Alpha Innotech FlourChem® Q system. The membrane was then blocked with 5% Bovine Serum Albumin (BSA) in Tris Buffered Saline (TBS) for 1 hr, washed with TBST (TBS with Triton) 3 times and then incubated with 5 μg/ml biotinylated-AOL for 1 hr. The membrane was washed again with TBST 3 times, followed by Streptavidin-HRP incubation for 30 mm and final washes with TBST 3 times. The bioluminescent signal was revealed using chemiluminescence reagents (ECL) and was analyzed using an Alpha Innotech FlourChem® Q system and Alphaview® software. The results are shown in the following table. For some analogs, multiple samples were analyzed, as indicated in the table.
TABLE 4
% of control
fucosylated
Molecule Namee
SGD number
IgG value
alkynyl fucose
1887
3
alkynyl fucose peracetate
1890
0; 0.08; 2
5-vinyl fucose tetraacetate
1922
2
5-cyanomethylene fucose tetraacetate
1924
96
L-galactono-1,4-lactone
1931
81
Methyl a-L-fucopyranoside
1932
87
5-propynyl fucose tetraacetate
1937
315
5-(Z)-propenyl fucose peracetate
1944
72
6-propargylamino fucose
1950
40
5-methyl ester fucose tetraacetate
1959
94
castanospermine
1960
300
5-methylketo fucose tetraacetate
1964
5
6-bromo fucose tetraacetate
1969
1
5-isopropyl fucose tetraaceate
1977
79
Kifunensine
1978
0.44; 2
propargyl fucose tetraacetate
1987
38
6-fluoro fucose tetraacetate
1988
15
5-ethyl fucose tetraacetate
1989
11
5-carboxamido fucose tetraacetate
1995
79
6-alkyne-6-acetoxy fucose tetraacetate
2004
29
alkynyl fucose tetrapropionate
2010
1.5
alkynyl fucose tetrahexanoate
2012
67
5-epoxy fucose tetraacetate
2020
5
6-thio galactose pentaacetate
2025
44
1-methyl fucose triacetate
2039
1
alkynyl fucose tetraisobutanoate
2043
34
6-formyl fucose tetraacetate
2045
70
6′6-difluoro fucose tetraacetate
2046
2
alkynyl fucose tetranicotinate
2047
50
benzyloxy fucose tetraacetate
2048
114
alkynyl fucose tetra PEG ester
2057
64
alkynyl fucose tetraisonicotinate
2058
34
1-methyl alkynyl fucose triacetate
2059
89
6-carboxymethyl ester fucose tetraacetate
2061
71
6-keto-6-ethyl fucose tetraacetate
2067
3
5-(2-cyanoethyl)arabinose tetraacetate
2070
51
D-galactose pentaacetate
2074
118
1,2-dideoxy-1,2-dehydro fucose diacetate
2080
159
1-deoxy fucose triacetate
2081
70
1,2-difuloro fucose diacetate
2082
87
2-fluoro-2-deoxy fucose
2083
66
2-fluoro-2-deoxy fucose tetraacetate
2084
52
6-allene fucose tetraacetate
2097
45
2-chloro-2-deoxy fucose tetraacetate
2099
146
2-deoxy fucose triacetate
2108
104
3-thio fucose tetraacetate
2112
64
6-deoxy-L-talose
2113
93
4-deoxy fucose triacetate
2134
49
Three fucose analogs were chosen for a full SDS-PAGE/Western blot analysis to show that changes in the fucose signal on the heavy chain can be detected by this technique. The three analogs chosen were used at a concentration of 50 μM. These analyses compared the activity of 2-fluorofucose (SGD-2083), 2-fluorofucose peracetate (SGD-2084), and alkynyl fucose peracetate (SGD-1890) with antibody from untreated cells. Use of alkynyl fucose peracetate produced IgG that did not show reactivity with the biotinylated AOL, confirming that changes in AOL signal can be detected by this method while 2-fluorofucose (SGD-2083) and 2-fluorofucose peracetate (SGD-2084) showed no apparent change in the AOL signal. These results are generally consistent with the results for these compounds in Table 4.
Many of the fucose analogs tested in Table 4 appeared to decrease fucosylation of antibody produced by human myeloma cells. The dot blots of the compounds showed that 10 of them were potentially strong inhibitors of IgG fucosylation in human cells, using decrease in AOL signal as an indication of inhibition. These fucose analogs are alkynyl fucose peracetate (SGD-1890), alkynyl fucose tetrapropionate (SGD-2010), 1-methyl fucose triacetate (SGD-2039), 5-ethyl fucose tetraacetate (SGD-1989), 6-fluoro fucose tetraacetate (SGD-1988), 6-bromo fucose tetraacetate (SGD-1969), 6′6-difluoro fucose tetraacetate (SGD-2046), 6-keto-6-ethyl fucose tetraacetate (SGD-2067), 5-epoxy fucose tetraacetate (SGD-2020), and 5-methylketo fucose tetraacetate (SGD-1964).
To further define the results of the AOL dot blot, samples of the IgGs produced by cells treated with the following fucose analogs (that gave moderate to strong decreases in the dot blot AOL signal) were isolated and examined by reducing PLRP-MS to verify the fucosylation status using the MW of the heavy chain: alkynyl fucose peracetate; 5-vinylfucose tetraaceate; 5-methylketofucose tetraacetate; 6-bromofucose tetraacetate; 6-fluorofucose tetraacetate; 5-ethylfucose tetraacetate; 5-epoxyfucose tetraacetate; 6′6-difluorofucose tetraacetate; 6-keto-6-ethyl fucose tetraacetate; and 2-fluorofucose peracetate.
40 mL samples of LP-1 cells (250,000 cells/mL) were treated with 100 μM of a fucose analog for 5 days as described above, and the IgGs were purified as described using protein A resin. The yields were estimated by UV spectroscopy assuming an extinction coefficient of 1.4 AU/(mg/mL). Seven of the ten compounds yielded enough IgG to perform the analysis (use of SGD-2067, SGD-1964, and SGD-2020 yielded <10 μg of IgG, likely due to toxicity of the analogs to the cells). The remaining IgGs were reduced with 10 mM DTT at 37° C. for 15 mm and were separated on PLRP followed by MS analysis using a QTOF mass spectrometer. The resulting heavy chain peaks were examined and compared to the IgG generated by untreated cells.
The mass spectrometry results are shown in Table 5 (below). The mass spectrometry signals were evaluated by comparing the peak height of the heavy chain versus heavy chain minus fucose and heavy chain minus fucose plus the mass of the fucose analog (which would arise if there was incorporation of the analog into the antibody carbohydrate). Four of the ten compounds tested were partial or full inhibitors of 1,6-fucosylation on the antibody. Alkynyl fucose peracetate (SGD-1890) provided complete inhibition while 2-fluorofucose peracetate (SGD-2084) was next best with 70% inhibition followed by 6′6-difluorofucose tetraacetate (SGD-2046) and 6-bromofucose tetraacetate (SGD-1969) with 33 and 20% inhibition mixed with incorporation of the analog into the carbohydrate.
TABLE 5
Results of PLRP-MS vs. dot blot for LP-1-generated IgG
PLRP/MS (inhibition
dot blot results
SGD number
or incorporation)
(% fucose signal of control)
Untreated
Control
100
SGD-1890
Full inhibitor
~1
SGD-1922
Full incorporator
2
SGD-1964
Not determined
5
SGD-1969
Partial incorporator and
1
20% fucose inhibitor
SGD-1988
Full incorporator
15
SGD-1989
Fully incorporated
11
SGD-2020
Not determined
5
SGD-2046
Partial incorporator and
2
33% fucose inhibitor
SGD-2067
Not determined
3
SGD-2084
70% inhibitor
52
Example 5
Affects of Fucose Analogs on Protein Fucosylation
The effects of the four partial to full inhibitors, alkynyl fucose peracetate (SGD-1890), 2-fluorofucose peracetate (SGD-2084), 6′6-difluorofucose tetraacetate (SGD-2046), and 6-bromofucose tetraacetate (SGD-1969), on protein cell surface fucosylation was tested for human cancer cells by incubation of five different human-derived cancer cell lines (Caki-1, PC-3, Ramos, LS174t, and HL60cy). 100 μM of each inhibitor was used under standard culture conditions for approximately 1-2 weeks with regular changes of culture medium including fresh inhibitor. After the incubation period, the cells were analyzed by FACS using four different detection reagents: biotinylated-Lens culimaris agglutinin-A (LCA), anti-Lewisx antibody (anti-SSEA1), an anti-Lewisy antibody (cBR96), and a Recombinant Human P-Selectin/CD62P/Fc Fusion protein. The procedure involved washing of the cells with FACS buffer (PBS+10% bovine serum albumin+0.02% sodium azide) 3 times followed by incubation with the primary detection reagent for 1 hr at 4° C., followed by 3 washes with FACS buffer and then incubation with the secondary detection reagent for 1 hr at 4° C. The cells were finally washed with FACS buffer 3 times and resuspended in FACS buffer and examined using a BD FACScan instrument. The LCA reagent recognizes sequences containing α-linked mannose residues and its affinity is markedly enhanced by α-linked fucose residues attached to the N-acetylchitobiose portion of the core oligosaccharide. The P-selectin fusion protein detects P-selectin ligand present on the surface of cells, an interaction which involves the sialyl Lewisx epitope present of the P-selectin ligand.
All of the cell lines examined showed staining with the LCA reagent, which recognizes sequences containing α-linked mannose residues, the affinity of which is markedly enhanced by α-linked fucose residues attached to the N-acetylchitobiose portion of the core oligosaccharide. The LCA detection of this sugar epitope was decreased upon treatment of the cells with all of the inhibitors (100 μM). This suggests that the overall presence of fucose on the cell surface is affected by treatment with the six fucose analogs examined.
FIG. 7 shows the results of these studies. For LewisX, of the cell lines examined only untreated LS1745t and HL60cy had significant LewisX detected on the cell surface (anti-SSEAI staining) (FIG. 7A). The anti-SSEAI detection of this structure was significantly decreased upon treatment of the cells with all of the fucose analogs (100 μM).
For LewisY, of the cell lines examined, only untreated LS1745t and HL60cy had significant Lewis Y detected on the cell surface (cBR96 staining) (FIG. 7B). The cBR96 detection of this structure was significantly decreased upon treatment of the cells with all of the fucose analogs (100 μM).
For P-selectin, of the cell lines examined, only untreated HL60cy had significant P-selectin ligand detected on the cell surface. The detection of this ligand was decreased somewhat by treatment of the cells with all fucose analogs, except for alkynyl furocse peracetate (SGD-1890) (100 μM) (FIG. 7C). Untreated Ramos cells showed little P-selectin ligand; however, upon treatment with the fucose analogs the signal for this ligand increased. This is unusual and was not observed with previous treatment of these cells with 2-fluorofucose (SGD-2083) or alkylnyl fucose (SGD-1887).
The results suggest that treatment with these fucose analogs can affect the presence of fucose on the cell surface in general and also specifically the fucosylation of Lewis X and Lewis Y modifications on the cell surface and sialyl LewisX present on the P-selectin ligand.
Example 6
Leukocytosis and Decreased E-selectin Binding Following Oral Dosing of 2-fluorofucose
The effects of a fucose analog on leukocytosis and E-selectin binding were examined in mice. Female Balb/c mice were given oral 2-fluorofucose (SGD-2083) in the drinking water or left untreated. Mice were bled prior to dosing and then weekly for three weeks to assess circulating cell numbers and their ability to bind E-selectin. In one study, 2-fluorofucose was formulated at 1 mM, 10 mM or 100 mM in the drinking water (n=3 per group). At day 14, mice were treated with TiterMAX® Classic adjuvant (Sigma) to stimulate polyclonal, antigen non-specific antibody production by B cells, and remained on the 2-fluorofucose-containing water through day 21. In a second study, mice were given oral 2-fluorofucose formulated at 10 mM and 100 mM in the drinking water for three weeks without any other treatments (n=6). On day 21, a pool of lymph nodes (axillary, brachial, superficial inguinal, and mesenteric) from each of three animals was assessed in addition to blood. Lymph nodes were homogenized into single cell suspensions, and total cell numbers were determined by counting on a hemcytometer, using Trypan Blue for dead cell exclusion. To determine total white cell numbers/μL blood, samples of blood from individual animals were counted on a hemacytometer, using Turk's solution (0.01% gentian violet in 3% acetic acid) to exclude red blood cells (RBCs). RBCs were eliminated from the remainder of the blood by osmotic lysis for flow cytometric analysis. Cells were incubated with anti-Gr-1-FITC antibodies (BD Biosciences) to identify neutrophils, and a recombinant E-selectin-human Fc fusion protein (R&D Systems). Cells were washed and then incubated with a PE-labeled goat anti-human IgG-Fc secondary antibody (Jackson Immunoresearch) to detect bound E-selectin. Samples were collected on a FACSCalibur flow cytometer and analyzed using CellQuest software. The percentage of Gr-1+ cells was determined and absolute number of neutrophils was calculated using the total white cell number from the hemacytometer count. In addition, flow samples were gated for Gr-1+ cells to assess E-selectin binding to neutrophils by histogram analysis. The geometric mean of the E-selectin fluorescent signal was determined from the histogram.
Results
The results in FIGS. 5A and 5B show that oral administration of 2-fluorofucose (SGD-2083) resulted in an increase in circulating white blood cells and neutrophils, in a dose-dependent manner 2-fluorofucose given at 1 mM had very little effect, whereas increasing effect was observed with increasing doses of 10 mM and 100 mM 2-fluorofucose. The data shown in FIGS. 5A and 5B are from the first study, day 14. Similar results were obtained at days 7 and 21 in the first study as well as days 7, 14, and 21 in the second study (data not shown). Lymph nodes were also assessed at day 21 in the second study and FIG. 5C shows that oral administration of 2-fluorofucose results in a marked decreased in cellularity in the lymph nodes. The effect was more severe at 100 mM compared to 10 mM.
Oral administration of 2-fluorofucose also results in decreased in E-selectin binding to neutrophils (FIG. 6). The effects of the fucoses analogs was also dose-dependent, with 1 mM having little effect and 10 mM and 100 mM having increasing effects (FIGS. 5B and 5C).
The observed increases in circulating white blood cells and neutrophils (leukocytosis) is consistent with the inhibition of E-selectin binding by neutrophils. E-selectin mediates extravization of white blood cells into the periphery and lymph nodes, and inhibition of E-selectin binding (by inhibiting fucosylation) would also reduce extravization and result in accumulation of white blood cells in the blood. These results suggest that fucose analogs that inhibit protein fucosylation, and E-selecin fucosylation in particular, can act to inhibit autoimmunity.
Example 7
Tumor Growth Inhibition by Administration of Fucose Analogs
Study 1
Human-derived cell lines were evaluated for their susceptibility to the fucose analog 2-fluorofucose in vitro. The cells lines were: LS174T colon adenocarcinoma, PC-3 colon adenocarcinoma, HL-60 acute mylogenous leukemia, Ramos Burkitt lymphoma, and Caki-1 renal cell carcinoma. The cell lines were cultured in the presence of 100 μM 2-fluorofucose (SGD-2083) in growth media, 100 μM alkynylfucose (SGD-1887) in growth media, or control growth media (without a fucose analog) for two weeks. The growth media were MEM Eagle with 10% FBS (LS174T), 50:50 F12 and RPMI with 10% FBS (PC-3), RPMI with 10% FBS (HL-60), IMDM with 10% FBS (Ramos), and McCoy with 10% 1-BS (PC-3). The cells were evaluated for cell surface fucosylation by FACS using antibody cBR96 to detect LewisY, antibody SSEA-1 to detect LewisX, P-selectin ligand to detect P-selectin, and AOL lectin to detect the general level of fucosylation.
Results:
The results of the FACS evaluation revealed variable levels of fucosylated cell surface proteins on the different cell lines (data not shown). 2-fluorofucose (SGD-2083) was generally a better inhibitor of protein fucosylation than alkynyl fucose (SGD-1887).
Study 2
To further evaluate the activity of these fucose analogs, further studies were performed in vivo using tumor cells that had been pre-treated by culturing in the presence of a fucose analog, or using untreated tumor cells. Tumor cells were implanted into 10 mice per group as follows. For the LS174T, PC-3, and Caki-1 cell lines, 5×105 cells in 25% Matrigel were implanted subcutaneously into female nude mice. For HL-60 and Ramos cell lines, 5×106 cells were implanted subcutaneously into female SCID mice. For mice implanted with untreated tumor cells, mice were provided regular drinking water. For mice implanted with tumor cells pre-treated with 2-fluorofucose (SGD-2083), the mice were provided drinking water supplemented with 20 mM 2-fluorofucose (SGD-2083). For mice implanted with tumor cells pre-treated with alkylnyl fucose (SGD-1887), the mice were provided with regular drinking water. The mice did not drink water containing alkynyl fucose.
After 3 weeks of receiving 2-fluorofucose-containing drinking water, mice were returned to regular drinking water, except for mice with Caki-1 tumors. The latter mice were returned to regular drinking water for one week. After the week of receiving regular water, mice were randomized to two groups of 5 each to receive drinking water supplemented with 20 mM 2-fluorofucose or regular drinking water. Mice were sacrificed when tumors reached about 1000 mm3.
Referring to FIG. 8A-E, tumor growth inhibition in vivo was seen for LS174T, PC-3, and Caki-1 cells treated with 2-flurofucose (SGD-2083). No change in tumor growth was observed for HL-60 and Ramos cells. For Caki-1, tumor growth inhibition was not observed during the first treatment period, but was observed after the mice were returned to 2-fluorofucose treatment. For the other cell lines, tumor growth inhibition appeared to start when tumor size had reached about 150 mm3. The slower growing Caki-1 tumors did not reach this point until the second treatment period with 2-fluorofucose (SGD-2083). These results indicate that treatment with fucose analogs can inhibit tumor growth.
Study 3
In a third study, tumor cells were implanted without prior treatment with a fucose analog. LS174T colon adenocarcinoma cells (5×105 cells in 25% Matrigel) were implanted subcutaneously into female nude mice. Mice were supplied with 50 mM 2-fluorofucose (SGD-2083) in their drinking water from 7 days before implant until 21 days after implant, or were supplied with regular drinking water.
Results
Referring to FIG. 8F, Mice given 50 mM 2-fluorofucose (SGD-2083) in their drinking water showed a substantial inhibition of tumor growth, achieving an average tumor size of 110 mm3 versus 734 mm3 for mice supplied with regular drinking water. Collectively, these results suggest that administration of a fucose analog can inhibit tumor growth.
Example 8
Tumor Vaccine Model
Female Balb/c mice were immunized by subcutaneous implantation of 1 million A20 murine lymphoma cells (killed by irradiation) on day −21 and day −7. Another group of mice were not given any immunization. On day 0, all mice were inoculated iv with 1.5 or 5 million live A20 cells. On days −14 through +21, mice were provided with 50 mM 2-fluorofucose (SGD-2083) in their drinking water or given regular drinking water. The 8 treatment groups were as follows:
1. No immunization, 1.5 million live A20 cells, regular drinking water
2. No immunization, 5 million live A20 cells, regular drinking water
3. No immunization, 1.5 million live A20 cells, 50 mM SGD-2083 in drinking water
4. No immunization, 5 million live A20 cells, 50 mM SGD-2083 in drinking water
5. Immunized, 1.5 million live A20 cells, regular drinking water
6. Immunized, 5 million live A20 cells, regular drinking water
7. Immunized, 1.5 million live A20 cells, 50 mM SGD-2083 in drinking water
8. Immunized, 5 million live A20 cells, 50 mM SGD-2083 in drinking water
Results
Referring to FIG. 9A, the study design is shown. Referring to FIG. 9B, mice that did not receive any immunization succumbed to the live A20 challenge from days 22-35. Mice receiving 2-fluorofucse (SGD-2083) survived a few days longer than those receiving regular drinking water. Two mice immunized with 5 million killed A20 cells and receiving regular drinking water succumbed to the live A20 challenge. All mice receiving immunization and 2-fluorofucose (SGD-2083) in their drinking water were still alive at data collection.
The present invention is not limited in scope by the specific embodiments described herein. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Unless otherwise apparent from the context any step, element, embodiment, feature or aspect of the invention can be used in combination with any other. All patent filings, and scientific publications, accession numbers and the like referred to in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if so individually denoted.
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15299894
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seattle genetics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:03PM
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Apr 1st, 2022 06:03PM
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Seattle Genetics
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Health Care
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Pharmaceuticals & Biotechnology
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