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nyse:pfe
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Pfizer
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Apr 26th, 2022 12:00AM
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Apr 15th, 2019 12:00AM
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https://www.uspto.gov?id=US11312782-20220426
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Chimeric antigen receptors targeting B-cell maturation antigen
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The invention provides CARs (CARs) that specifically bind to BCMA (B-Cell Maturation Antigen). The invention further relates to engineered immune cells comprising such CARs, CAR-encoding nucleic acids, and methods of making such CARs, engineered immune cells, and nucleic acids. The invention further relates to therapeutic methods for use of these CARs and engineered immune cells for the treatment of a condition associated with malignant cells expressing BCMA (e.g., cancer).
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11312782
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1. An engineered immune cell expressing at its cell surface membrane a B-cell maturation antigen (BCMA) specific chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain Fv fragment (scFv) comprising a heavy chain variable (VH) region comprising three complementarity determining regions (CDRs) comprising the sequences shown in SEQ ID NO: 33, 72, 39, 76, 83, 92, 25, 112, or 8 of Table 1; and a light chain variable (VL) region comprising three CDRs comprising the sequences shown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, 38, or 80 of Table 1, wherein the first transmembrane domain comprises a CD8α chain transmembrane domain, and wherein the intracellular signaling domain comprises a CD3ζ signaling domain and/or a 4-1BB signaling domain.
2. The engineered immune cell of claim 1, wherein the VH region of the BCMA specific CAR comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 150, 151, or 152; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 153 or 154; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 155; and the VL region of the BCMA specific CAR comprises a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 209; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 221; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 222.
3. The engineered immune cell of claim 2, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 33 and the VL region comprises the amino acid sequence shown in SEQ ID NO: 34.
4. The engineered immune cell of claim 1, wherein the VH region of the BCMA specific CAR comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 151, 156, or 157; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 158 or 159; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 155; and wherein the VL region of the BCMA specific CAR comprises a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 209; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 221; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 225.
5. The engineered immune cell of claim 4, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 112 and the VL region comprises the amino acid sequence shown in SEQ ID NO: 38.
6. The engineered immune cell of claim 1, wherein the BCMA specific CAR comprises the amino acid sequence shown in SEQ ID NO: 344.
7. The engineered immune cell of claim 6, wherein the BCMA specific CAR comprises a CD20 epitope.
8. The engineered immune cell of claim 7, wherein the CD20 epitope comprises the amino acid sequence shown in SEQ ID NO: 397 or SEQ ID NO: 398.
9. The engineered immune cell of claim 1, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 33; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 34; a CD20 epitope having the sequence of SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
10. The engineered immune cell of claim 1, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 112; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 38; a CD20 epitope having the sequence of SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
11. The engineered immune cell of claim 1, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 112; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 38; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
12. The engineered immune cell of claim 1, wherein the BCMA specific CAR further comprises a stalk domain between the extracellular ligand-binding domain and the first transmembrane domain.
13. The engineered immune cell of claim 12, wherein the stalk domain is selected from the group consisting of: a human CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge.
14. The engineered immune cell of claim 1, wherein the BCMA specific CAR further comprises a CD20 epitope.
15. The engineered immune cell of claim 14, wherein the CD20 epitope comprises the amino acid sequence shown in SEQ ID NO: 397 or SEQ ID NO: 398.
16. The engineered immune cell of claim 1, wherein the BCMA specific CAR further comprises another extracellular ligand-binding domain that is not specific for BCMA binding.
17. The engineered immune cell of claim 1, further comprising another CAR that is not specific for BCMA.
18. The engineered immune cell of claim 1, further comprising a polynucleotide encoding a suicide polypeptide.
19. The engineered immune cell of claim 1, further comprising a disruption in one or more endogenous genes, wherein the endogenous gene encodes TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or an immune checkpoint protein.
20. The engineered immune cell of claim 19, wherein the engineered immune cell comprises a disruption in an endogenous gene encoding an immune checkpoint protein, wherein the immune checkpoint protein is selected from the group consisting of PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10 and 2B4.
21. The engineered immune cell of claim 1, wherein the immune cell is selected from the group consisting of: a T cell, a dendritic cell, a killer dendritic cell, a mast cell, an NK-cell, and a B cell.
22. The engineered immune cell of claim 1, wherein the immune cell is an inflammatory T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or a helper T-lymphocyte.
23. The engineered immune cell of claim 1, wherein the immune cell is an autologous immune cell or an allogeneic immune cell.
24. A pharmaceutical composition comprising a population of the engineered immune cell of claim 1.
25. The pharmaceutical composition of claim 24, wherein the VH region of the BCMA specific CAR comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 150, 151, or 152; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 153 or 154; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 155; and the VL region of the BCMA specific CAR comprises a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 209; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 221; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 222.
26. The pharmaceutical composition of claim 25, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 33 and the VL region comprises the amino acid sequence shown in SEQ ID NO: 34.
27. The pharmaceutical composition of claim 24, wherein the VH region of the BCMA specific CAR comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 151, 156, or 157; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 158 or 159; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 155; and wherein the VL region of the BCMA specific CAR comprises a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 209; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 221; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 225.
28. The pharmaceutical composition of claim 27, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 112 and the VL region comprises the amino acid sequence shown in SEQ ID NO: 38.
29. The pharmaceutical composition of claim 24, wherein the BCMA specific CAR comprises the amino acid sequence shown in SEQ ID NO: 344.
30. The pharmaceutical composition of claim 29, wherein the BCMA specific CAR comprises a CD20 epitope.
31. The pharmaceutical composition of claim 30, wherein the CD20 epitope comprises the amino acid sequence shown in SEQ ID NO: 397 or SEQ ID NO: 398.
32. The pharmaceutical composition of claim 24, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 33; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 34; a CD20 epitope having the sequence of SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
33. The pharmaceutical composition of claim 24, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 112; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 38; a CD20 epitope having the sequence of SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
34. The pharmaceutical composition of claim 24, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 112; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 38; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
35. A method of engineering an immune cell, the method comprising:
a. providing an immune cell; and
b. introducing into the cell at least one polynucleotide encoding a B-cell maturation antigen (BCMA) specific chimeric antigen receptor (CAR) comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain Fv fragment (scFv) comprising a heavy chain variable (VH) region comprising three complementarity determining regions (CDRs) comprising the sequences shown in SEQ ID NO: 33, 72, 39, 76, 83, 92, 25, 112, or 8 of Table 1; and a light chain variable (VL) region comprising three CDRs comprising the sequences shown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, 38, or 80 of Table 1, wherein the first transmembrane domain comprises a CD8α chain transmembrane domain, and wherein the intracellular signaling domain comprises a CD3ζ signaling domain and/or a 4-1BB signaling domain.
36. The method of claim 35, comprising:
c. introducing into the cell at least one polynucleotide encoding at least one other CAR which is not specific for BCMA.
37. The method of claim 35, wherein the VH region of the BCMA specific CAR comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 150, 151, or 152; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 153 or 154; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 155; and the VL region of the BCMA specific CAR comprises a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 209; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 221; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 222.
38. The method of claim 37, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 33 and the VL region comprises the amino acid sequence shown in SEQ ID NO: 34.
39. The method of claim 35, wherein the VH region of the BCMA specific CAR comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 151, 156, or 157; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 158 or 159; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 155; and wherein the VL region of the BCMA specific CAR comprises a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 209; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 221; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 225.
40. The method of claim 39, wherein the VH region comprises the amino acid sequence shown in SEQ ID NO: 112 and the VL region comprises the amino acid sequence shown in SEQ ID NO: 38.
41. The method of claim 35, wherein the BCMA specific CAR comprises the amino acid sequence shown in SEQ ID NO: 344.
42. The method of claim 41, wherein the BCMA specific CAR comprises a CD20 epitope.
43. The method of claim 42, wherein the CD20 epitope comprises the amino acid sequence shown in SEQ ID NO: 397 or SEQ ID NO: 398.
44. The method of claim 35, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 33; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 34; a CD20 epitope having the sequence of SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
45. The method of claim 35, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 112; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 38; a CD20 epitope having the sequence of SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
46. The method of claim 35, wherein the BCMA specific CAR comprises a CD8α signal peptide having the sequence of SEQ ID NO: 318; a VH region having the sequence of SEQ ID NO: 112; a GS linker having the sequence of SEQ ID NO: 333; a VL region having the sequence of SEQ ID NO: 38; a CD8α hinge having the sequence of SEQ ID NO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 323; and a CD3ζ intracellular signaling domain having the sequence of SEQ ID NO: 324.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 15/085,317, filed on Mar. 30, 2016, now issued as U.S. Pat. No. 10,294,304 on May 21, 2019, which claims the benefit of U.S. Provisional Application No. 62/146,825 filed Apr. 13, 2015, U.S. Provisional Application No. 62/286,473 filed Jan. 25, 2016, and U.S. Provisional Application No. 62/301,177 filed Feb. 29, 2016, all of which are hereby incorporated by reference in their entireties.
REFERENCE TO SEQUENCE LISTING
This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “ALGN_003_04US_SeqList_ST25.txt” created on Jan. 29, 2019, and having a size of ˜372,605 bytes. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.
FIELD
The invention relates to chimeric antigen receptors (CAR). CARs are able to redirect immune cell specificity and reactivity toward a selected target exploiting the ligand-binding domain properties. In particular, the invention relates to CARs that specifically bind to B-Cell Maturation Antigen (BCMA specific CARs). The invention further relates to polynucleotides encoding BCMA specific CAR and isolated cells expressing BCMA specific CARs at their surface. The invention further relates to methods for engineering immune cells expressing BCMA specific CARs at their surface. The invention is particularly useful for the treatment of B-cell lymphomas and leukemia. The invention further relates to immune cells comprising the BCMA specific CARs (BCMA specific CAR-T cells), compositions comprising the BCMA specific CAR-T cells, and methods of using the BCMA specific CAR-T cells for treating conditions associated with malignant cells expressing BCMA (e.g., cancer).
BACKGROUND
Multiple myeloma is a malignancy characterized by an accumulation of clonal plasma cells (see, e.g., Lonial et al., Clinical Cancer Res., 77(6): 1264-1277 (2011)). Current therapies for MM often cause remissions, but nearly all patients eventually relapse and die (see, e.g., Rajkumar, Nature Rev. Clinical Oncol, 5(8): 479-491 (2011)).
Adoptive transfer of T cells genetically modified to recognize malignancy-associated antigens is showing promise as a new approach to treating cancer (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2): 251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4): 299-308 (2008)). T cells can be genetically modified to express chimeric antigen receptors (CARs), which are fusion proteins comprised of an antigen recognition moiety and T cell activation domains (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993), and Sadelain et al., Curr. Opin. Immunol, 21(2): 215-223 (2009)).
B-cell maturation antigen (BCMA, CD269, or TNFRSF17) is a member of the tumor necrosis factor receptor (TNFR) superfamily. BCMA was identified in a malignant human T cell lymphoma containing a t(4;16) translocation. The gene is selectively expressed in the B-cell lineage with the highest expression in plasmablasts and plasma cells, antibody secreting cells. BCMA binds two ligands, B-cell activation factor (BAFF) (also called B-lymphocyte stimulator (BLyS) and APOL-related leukocyte expressed ligand (TALL-1)) and a proliferation-inducing ligand (APRIL) with affinity of 1 uM and 16 nM, respectively. Binding of APRIL or BAFF to BCMA promotes a signaling cascade involving NF-kappa B, Elk-1, c-Jun N-terminal kinase and the p38 mitogen-activated protein kinase, which produce signals for cell survival and proliferation. BCMA is also expressed on malignant B cells and several cancers that involve B lymphocytes including multiple myeloma, plasmacytoma, Hodgkin's Lymphoma, and chronic lymphocytic leukemia. In autoimmune diseases where plasmablasts are involved such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, BCMA expressing antibody-producing cells secrete autoantibodies that attack self.
In the case of multiple myeloma, about 24,000 new cases are newly diagnosed in the United States each year, and this number represents about 15% of the newly diagnosed hematological cancers in the United States. An average of 11,000 deaths result from multiple myeloma each year, and the average 5-year survival rate is about 44%, with median survival of 50-55 months. Current treatment for multiple myeloma is focused on plasma cells apoptosis and/or decreasing osteoclast activity (e.g., chemotherapy, thalidomide, lenalidomide, bisphosphonates, and/or proteasome inhibitors such as bortezomib (VELCADE®) or carfilzomib). However, multiple myeloma remains an incurable disease, and almost all patients have developed resistance to these agents and eventually relapse. Accordingly, an alternative treatment to multiple myeloma, such as using an anti-BCMA antagonist including BCMA specific CARs and BCMA specific CAR-T cells, would make a superior therapeutic agent.
SUMMARY
Chimeric antigen receptors (CARs) that bind to BCMA are provided. It is demonstrated that certain BCMA specific CARs are effective when expressed in T cells to activate T cells upon contact with BCMA. Advantageously, the BCMA specific CARs provided herein bind human and cynomolgous monkey BCMA. Also advantageously, the BCMA specific CAR-T cells provided herein exhibit degranulation activity, increased interferon gamma production, and/or cytotoxic activity upon contact with BCMA-expressing cells.
In one aspect, the invention provides a BCMA specific CAR comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular ligand-binding domain comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence SYX1MX2, wherein X1 is A or P; and X2 is T, N, or S (SEQ ID NO: 301), GFTFX1SY, wherein X1 is G or S (SEQ ID NO: 302), or GFTFX1SYX2MX3, wherein X1 is G or S, X2 is A or P; and X3 is T, N, or S (SEQ ID NO: 303); (ii) a VH CDR2 comprising the sequence AX1X2X3X4GX5X6X7X8YADX9X10KG, wherein X1 is I, V, T, H, L, A, or C; X2 is S, D, G, T, I, L, F, M, or V; X3 is G, Y, L, H, D, A, S, or M; X4 is S, Q, T, A, F, or W; X5 is G or T; X6 is N, S, P, Y, W, or F; X7 is S, T, I, L, T, A, R, V, K, G, or C; X8 is F, Y, P, W, H, or G; X9 is V, R, or L; and X10 is G or T (SEQ ID NO: 305), or X1X2X3X4X5X6, wherein X1 is S, V, I, D, G, T, L, F, or M; X2 is G, Y, L, H, D, A, S, or M; X3 is S, G, F, or W; X4 is G or S; X5 is G or T; and X6 is N, S, P, Y, or W (SEQ ID NO: 306); and iii) a VH CDR3 comprising the sequence VSPIX1X2X3X4, wherein X1 is A or Y; X2 is A or S; and X3 is G, Q, L, P, or E (SEQ ID NO: 307), or YWPMX1X2, wherein X1 is D, S, T, or A; and X2 is I, S, L, P, or D (SEQ ID NO: 308); and/or (b) a light chain variable (VL) region comprising (i) a VL CDR1 comprising the sequence X1X2X3X4X5X6X7X8X9X10X11X12, wherein X1 is R, G, W, A, or C; X2 is A, P, G, L, C, or S; X3 is S, G, or R; X4 is Q, C, E, V, or I; X5 is S, L, P, G, A, R, or D; X6 is V, G, or I; X7 is S, E, D, or P; X8 is S, P, F, A, M, E, V, N, D, or Y; X9 is I, T, V, E, S, A, M, Q, Y, H, or R; X10 is Y or F; X11 is L, W, or P; and X12 is A, S, or G (SEQ ID NO: 309); (ii) a VL CDR2 comprising the sequence X1ASX2RAX3, wherein X1 is G or D; X2 is S or I; and X3 is T or P (SEQ ID NO: 310); and (iii) a VL CDR3 comprising the sequence QQYX1X2X3PX4T, wherein X1 is G, Q, E, L, F, A, S, M, K, R, or Y; X2 is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C; X3 is W, F, or S; and X4 is L or I (SEQ ID NO: 311), or QQYX1X2X3PX4, wherein X1 is G, Q, E, L, F, A, S, M, R, K, or Y; X2 is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y; X3 is W, S, or F; and X4 is L or I (SEQ ID NO: 312).
In another aspect, the invention provides a BCMA specific CAR comprising an extracellular ligand-binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises a single chain Fv fragment (scFv) comprising a heavy chain variable (VH) region comprising three CDRs from the VH region comprising the sequence shown in SEQ ID NO: 33, 72, 39, 76, 83, 92, 25, or 8; and a light chain variable (VL) region comprising three CDRs from the VL region shown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, or 80. In some embodiments, the VH region can comprise the sequence shown in SEQ ID NO: 33, 72, 39, 76, 83, 92, 25, or 8, or a variant thereof with one or several conservative amino acid substitutions in residues that are not within a CDR and/or the VL region can comprise the amino acid sequence shown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, or 80, or a variant thereof with one or several amino acid substitutions in amino acids that are not within a CDR.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, 152, 156, 157, 129, 130, or 131; (ii) a VH CDR2 comprising the sequence shown in 153, 154, 187, 188, 165, 166, 162, 159, 190, 191, 169, 154, 139, 140, 132, or 133; and (iii) a VH CD3 comprising the sequence shown in 155, 161, 134, or 137; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 209, 249, 226, 251, 262, 271, 217, or 377; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221, 252, or 210; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 222, 225, 227, 253, 263, 272, 216, or 214.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152; (ii) a VH CDR2 comprising the sequence shown in 153 or 154; and (iii) a VH CD3 comprising the sequence shown in 155; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221, and (iii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 222.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152; (ii) a VH CDR2 comprising the sequence shown in 187 or 188; and (iii) a VH CD3 comprising the sequence shown in 155; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 249; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 225.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152; (ii) a VH CDR2 comprising the sequence shown in 165 or 166; and (iii) a VH CD3 comprising the sequence shown in 155; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 226; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 227.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 156, 151, or 157; (ii) a VH CDR2 comprising the sequence shown in 162 or 159; and (iii) a VH CD3 comprising the sequence shown in 161; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 251; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 252, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 253.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 156, 151, or 157; (ii) a VH CDR2 comprising the sequence shown in 190 or 191; and (iii) a VH CD3 comprising the sequence shown in 161; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 262; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 252, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 263.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152; (ii) a VH CDR2 comprising the sequence shown in 169 or 154; and (iii) a VH CD3 comprising the sequence shown in 155; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 271; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 272.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 129, 130, or 131; (ii) a VH CDR2 comprising the sequence shown in 139 or 140; and (iii) a VH CD3 comprising the sequence shown in 134; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 217; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 210, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 216.
In some embodiments, the extracellular ligand-binding domain of a BCMA specific CAR provided herein comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence shown in SEQ ID NO: 129, 130, or 131; (ii) a VH CDR2 comprising the sequence shown in 132 or 133; and (iii) a VH CD3 comprising the sequence shown in 137; and/or (b) a light chain variable region (VL) comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 377; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 210, and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 214.
In some embodiments, the intracellular signaling domain comprises a CD3ζ signalling domain. In some embodiments, the intracellular signaling domain comprises a 4-1BB domain. In some embodiments, the CAR can further comprise another intracellular signaling domain. In some embodiments, the additional intracellular signaling domain can comprise a 4-1BB domain.
In some embodiments, the CAR can comprise a stalk domain between the extracellular ligand-binding domain and the first transmembrane domain. In some embodiments, the stalk domain can be selected from the group consisting of: a human CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge.
In some embodiments, the first transmembrane domain can comprise a CD8α chain transmembrane domain.
In some embodiments, the CAR can comprise a CD20 epitope.
In some embodiments, the CAR can comprise another extracellular ligand-binding domain which is not specific for BCMA.
In some embodiments, the BCMA specific CAR can comprise the amino acid sequence shown in SEQ ID NO: 396.
In some embodiments of a CAR, the extracellular ligand-binding domain(s), the first transmembrane domain, and intracellular signaling domain(s) are on a single polypeptide.
In some embodiments, the CAR can comprise a second transmembrane domain, wherein the first transmembrane domain and the extracellular ligand-binding domain(s) are on a first polypeptide, and wherein the second transmembrane domain and the intracellular signaling domain(s) are on a second polypeptide, wherein the first transmembrane domain comprises a transmembrane domain from the a chain of the high-affinity IgE receptor (FcεRI) and the second transmembrane domain comprises a transmembrane domain from the γ or β chain of FcεRI. In some embodiments, the CAR can comprise a third polypeptide comprising a third transmembrane domain fused to an intracellular signaling domain from a co-stimulatory molecule, wherein the third transmembrane domain comprises a transmembrane domain from the γ or β chain of FcεRI.
In another aspect, the invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a BCMA specific CAR as described herein.
In another aspect, the invention provides an expression vector comprising a nucleic acid sequence encoding a BCMA specific CAR antibody as described herein.
In another aspect, the invention provides engineered immune cell expressing at its cell surface membrane a BCMA specific CAR as described herein. In some embodiments, the engineered immunce cell can comprise another CAR which is not specific for BCMA. In some embodiments, the engineered immunce cell can comprise a polynucleotide encoding a suicide polypeptide. In some embodiments, the suicide polypeptide is RQR8.
In some embodiments, the immune cell can be derived from an inflammatory T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or a helper T-lymphocyte.
In some embodiments, the engineered immune cell can comprise a disruption one or more endogenous genes, wherein the endogenous gene encodes TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (DCK), or an immune checkpoint protein such as for example programmed death-1 (PD-1).
In some embodiments, immune cell is obtained from a healthy donor. In some embodiments, the immune cell is obtained from a patient.
In another aspect, the invention provides an engineered immune cell expressing at its cell surface membrane a BCMA specific CAR as described herein for use as a medicament. In some embodiments, the medicament is for use in treatment of a B-cell related cancer selecting from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma, Kahler's disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenström macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell related lymphoma.
In another aspect, the invention provides a method of engineering an immune cell comprising: providing an immune cell; and expressing at the surface of the cell at least one BCMA specific CAR as described herein.
In some embodiments, the method comprises: providing an immune cell; introducing into the cell at least one polynucleotide encoding said BCMA specific CAR; and expressing said polynucleotide into the cell.
In some embodiments, the method comprises providing an immune cell; introducing into the cell at least one polynucleotide encoding said BCMA specific CAR; and introducing at least one other CAR which is not specific for BCMA.
In another aspect, the invention provides a method of treating a subject suffering from a condition associated with malignant cells, the method comprising: providing a immune cell expressing at the surface a BCMA specific CAR as described herein; and administering said immune cells to said patient.
In another aspect, the invention provides a pharmaceutical composition comprising an engineered immune cell as described herein.
In another aspect, the invention provides a method of treating a condition associated with malignant cells expressing BCMA in a subject comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of claim comprising an engineered immune cell as described herein. In some embodiments, the condition is a cancer. In some embodiments, the cancer is a B-cell related cancer selecting from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma, Kahler's disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenström macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell related lymphoma.
In another aspect, the invention provides a method of inhibiting tumor growth or progression in a subject who has malignant cells expressing BCMA, comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein.
In another aspect, the invention provides a method inhibiting metastasis of malignant cells expressing BCMA in a subject, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition comprising an engineered immune cell as described herein.
In another aspect, the invention provides a method inducing tumor regression in a subject who has malignant cells expressing BCMA, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of a pharmaceutical composition comprising an engineered immune cell as described herein.
In some embodiments, any of the above methods further comprises administering one or more additional therapies, such as for example, a monoclonal antibody and/or a chemotherapeutic. In some embodiments, the monoclonal antibody can be, for example, an antibody that binds to a checkpoint inhibitor such as, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, any of the above methods further comprises administering a nucleoside analog therapy, such as for example fludarabine or clofarabine, to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a graph summarizing the results of treatment with BCMA specific CAR-T cells in the MM1.S tumor model.
FIG. 2 depicts a graph summarizing the results of treatment with BCMA specific CAR-T cells in the Molp8 tumor model.
DETAILED DESCRIPTION
The invention disclosed herein provides chimeric antigen receptors (CARs) and immune cells comprising CARs (CAR-T cells) that specifically bind to BCMA (e.g., human BCMA). The invention also provides polynucleotides encoding these CARs, compositions comprising these CAR-T cells, and methods of making and using these CARs and CAR-T cells. The invention also provides methods for treating a condition associated with malignant BCMA expression in a subject, such as cancer.
General Techniques
The practice of the invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).
Definitions
The term “extracellular ligand-binding domain” as used herein refers to an oligo- or polypeptide that is capable of binding a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
The term “stalk domain” or “hinge domain” are used interchangeably herein to refer to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, stalk domains are used to provide more flexibility and accessibility for the extracellular ligand-binding domain.
The term “intracellular signaling domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.
A “co-stimulatory molecule” as used herein refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.
A “co-stimulatory ligand” refers to a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory signal molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation activation, differentiation and the like. A co-stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The term “antigen binding fragment” or “antigen binding portion” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., BCMA). Antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term “antigen binding fragment” of an antibody include Fab; Fab′; F(ab′)2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989), and an isolated complementarity determining region (CDR).
An antibody, an antibody conjugate, or a polypeptide that “preferentially binds” or “specifically binds” (used interchangeably herein) to a target (e.g., BCMA protein) is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a BCMA epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other BCMA epitopes or non-BCMA epitopes. It is also understood that by reading this definition, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.
A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.
A “CDR” of a variable domain are amino acid residues within the variable region that are identified in accordance with the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, and/or conformational definitions or any method of CDR determination well known in the art. Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include the “AbM definition,” which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the “contact definition” of CDRs based on observed antigen contacts, set forth in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.
As used herein, “monoclonal antibody” 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 typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. 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 invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554, 1990, for example.
As used herein, “humanized” antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize 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 CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Preferred are antibodies having Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.
As used herein, “human antibody” means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or which has been made using any of the techniques for making human antibodies known to those skilled in the art or disclosed herein. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be made by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of the endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or from single cell cloning of the cDNA, or may have been immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147 (1):86-95, 1991; and U.S. Pat. No. 5,750,373.
The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length, preferably, relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.
A “monovalent antibody” comprises one antigen binding site per molecule (e.g., IgG or Fab). In some instances, a monovalent antibody can have more than one antigen binding sites, but the binding sites are from different antigens.
A “bivalent antibody” comprises two antigen binding sites per molecule (e.g., IgG). In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific.
A “bispecific,” “dual-specific” or “bifunctional” antibody is a hybrid antibody having two different antigen binding sites. The two antigen binding sites of a bispecific antibody bind to two different epitopes, which may reside on the same or different protein targets.
Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S. D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F. A., et al, J. Mol. Biol., 373(4):924-40, 2007).
As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
As known in the art a “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.
As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), more preferably, at least 90% pure, more preferably, at least 95% pure, yet more preferably, at least 98% pure, and most preferably, at least 99% pure.
A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.
As used herein, “immune cell” refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptive immune response.
As known in the art, the term “Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The numbering of the residues in the Fc region is that of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant regions, CH2 and CH3.
As used in the art, “Fc receptor” and “FcR” 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 FcγRIII 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. FcRs are reviewed in Ravetch and Kinet, Ann. 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. “FcR” 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).
The term “compete”, as used herein with regard to an antibody, means that a first antibody, or an antigen binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen binding portion thereof, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.
As used herein “autologous” means that cells, a cell line, or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor.
As used herein “allogeneic” means that cells or population of cells used for treating patients are not originating from said patient but from a donor.
As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of BCMA expressing tumor, remission of a BCMA associated disease (e.g., cancer), decreasing symptoms resulting from a BCMA associated disease (e.g., cancer), increasing the quality of life of those suffering from a BCMA associated disease (e.g., cancer), decreasing the dose of other medications required to treat a BCMA associated disease (e.g., cancer), delaying the progression of a BCMA associated disease (e.g., cancer), curing a BCMA associated disease (e.g., cancer), and/or prolong survival of patients having a BCMA associated disease (e.g., cancer).
“Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering a BCMA antibody or a BCMA antibody conjugate. “Ameliorating” also includes shortening or reduction in duration of a symptom.
As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing incidence or amelioration of one or more symptoms of various BCMA associated diseases or conditions (such as for example multiple myeloma), decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the BCMA associated disease of patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
An “individual” or a “subject” is a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
As used herein, “vector” means a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
As used herein, “expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.
As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).
The term “kon”, as used herein, refers to the rate constant for association of an antibody to an antigen.
The term “koff”, as used herein, refers to the rate constant for dissociation of an antibody from the antibody/antigen complex.
The term “KD”, as used herein, refers to the equilibrium dissociation constant of an antibody-antigen interaction.
Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range.
It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.
Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the invention. The materials, methods, and examples are illustrative only and not intended to be limiting.
BCMA Specific CARs and Methods of Making Thereof
The invention provides CARs that bind to BCMA (e.g., human BCMA (e.g., SEQ ID NO: 354 or accession number: Q02223-2). BCMA specific CARs provided herein include single chain CARS and multichain CARs. The CARs have the ability to redirect T cell specificity and reactivity toward BCMA in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.
In some embodiments, CARs provided herein comprise an extracellular ligand-binding domain (e.g., a single chain variable fragment (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand-binding domain, transmembrane domain, and intracellular signaling domain are in one polypeptide, i.e., in a single chain. Multichain CARs and polypeptides are also provided herein. In some embodiments, the multichain CARs comprise: a first polypeptide comprising a transmembrane domain and at least one extracellular ligand-binding domain, and a second polypeptide comprising a transmembrane domain and at least one intracellular signaling domain, wherein the polypeptides assemble together to form a multichain CAR.
In some embodiments, a BCMA specific multichain CAR is based on the high affinity receptor for IgE (FcεRI). The FcεRI expressed on mast cells and basophiles triggers allergic reactions. FcεRI is a tetrameric complex composed of a single a subunit, a single β subunit, and two disulfide-linked γ subunits. The α subunit contains the IgE-binding domain. The β and γ subunits contain ITAMs that mediate signal transduction. In some embodiments, the extracellular domain of the FcRα chain is deleted and replaced by a BCMA specific extracellular ligand-binding domain. In some embodiments, the multichain BCMA specific CAR comprises an scFv that binds specifically to BCMA, the CD8α hinge, and the ITAM of the FcRβ chain. In some embodiments, the CAR may or may not comprise the FcRγ chain.
In some embodiments, the extracellular ligand-binding domain comprises an scFv comprising the light chain variable (VL) region and the heavy chain variable (VH) region of a target antigen specific monoclonal antibody joined by a flexible linker. Single chain variable region fragments are made by linking light and/or heavy chain variable regions by using a short linking peptide (Bird et al., Science 242:423-426, 1988). An example of a linking peptide is the GS linker having the amino acid sequence (GGGGS)3 (SEQ ID NO: 333), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). In general, linkers can be short, flexible polypeptides and preferably comprised of about 20 or fewer amino acid residues. Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports. The single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resultant scFv can be isolated using standard protein purification techniques known in the art.
In some embodiments, the extracellular ligand-binding domain comprises (a) a VH region comprising (i) a VH complementarity determining region one (CDR1) comprising the sequence SYX1MX2, wherein X1 is A or P; and X2 is T, N, or S (SEQ ID NO: 301), GFTFX1SY, wherein X1 is G or S (SEQ ID NO: 302), or GFTFX1SYX2MX3, wherein X1 is G or S, X2 is A or P; and X3 is T, N, or S (SEQ ID NO: 303); (ii) a VH CDR2 comprising the sequence AX1X2X3X4GX5X6X7X8YADX9X10KG, wherein X1 is I, V, T, H, L, A, or C; X2 is S, D, G, T, I, L, F, M, or V; X3 is G, Y, L, H, D, A, S, or M; X4 is S, Q, T, A, F, or W; X5 is G or T; X6 is N, S, P, Y, W, or F; X7 is S, T, I, L, T, A, R, V, K, G, or C; X8 is F, Y, P, W, H, or G; X9 is V, R, or L; and X10 is G or T (SEQ ID NO: 305), or X1X2X3X4X5X6, wherein X1 is S, V, I, D, G, T, L, F, or M; X2 is G, Y, L, H, D, A, S, or M; X3 is S, G, F, or W; X4 is G or S; X5 is G or T; and X6 is N, S, P, Y, or W (SEQ ID NO: 306); and iii) a VH CDR3 comprising the sequence VSPIX1X2X3X4, wherein X1 is A or Y; X2 is A or S; and X3 is G, Q, L, P, or E (SEQ ID NO: 307), or YWPMX1X2, wherein X1 is D, S, T, or A; and X2 is I, S, L, P, or D (SEQ ID NO: 308); and a VL region comprising (i) a VL CDR1 comprising the sequence X1X2X3X4X5X6X7X8X9X10X11X12, wherein X1 is R, G, W, A, or C; X2 is A, P, G, L, C, or S; X3 is S, G, or R; X4 is Q, C, E, V, or I; X5 is S, L, P, G, A, R, or D; X6 is V, G, or I; X7 is S, E, D, or P; X8 is S, P, F, A, M, E, V, N, D, or Y; X9 is I, T, V, E, S, A, M, Q, Y, H, or R; X10 is Y or F; X11 is L, W, or P; and X12 is A, S, or G (SEQ ID NO: 309); (ii) a VL CDR2 comprising the sequence X1ASX2RAX3, wherein X1 is G or D; X2 is S or I; and X3 is T or P (SEQ ID NO: 310); and (iii) a VL CDR3 comprising the sequence QQYX1X2X3PX4T, wherein X1 is G, Q, E, L, F, A, S, M, K, R, or Y; X2 is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C; X3 is W, F, or S; and X4 is L or I (SEQ ID NO: 311), or QQYX1X2X3PX4, wherein X1 is G, Q, E, L, F, A, S, M, R, K, or Y; X2 is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y; X3 is W, S, or F; and X4 is L or I (SEQ ID NO: 312). In some embodiments, the VH and VL are linked together by a flexible linker. In some embodiments a flexible linker comprises the amino acid sequence shown in SEQ ID NO: 333.
In another aspect, provided is CAR, which specifically binds to BCMA, wherein the CAR comprises an extracellular ligand-binding domain comprising: a VH region comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 78, 95, 97, 99, 101, 104, 106, 110, 112, 114, 76, 118, 120, 122, 112, 125, 127, 313, or 314; and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 317, 81, 82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315, or 316. In some embodiments, the VH and VL are linked together by a flexible linker. In some embodiments a flexible linker comprises the amino acid sequence shown in SEQ ID NO: 333.
In some embodiments, a CAR of the invention comprises an extracellular ligand-binding domain having any one of partial light chain sequence as listed in Table 1 and/or any one of partial heavy chain sequence as listed in Table 1. In Table 1, the underlined sequences are CDR sequences according to Kabat and in bold according to Chothia, except for the following heavy chain CDR2 sequences, in which the Chothia CDR sequences are underlined and the Kabat CDR sequences are in bold: P5A2_VHVL, A02_Rd4_0.6nM_C06, A02_Rd4_0.6nM_C09 A02_Rd4_6nM_C16, A02_Rd4_6nM_C03, A02_Rd4_6nM_C01, A02_Rd4_6nM_C26 A02_Rd4_6nM_C25, A02_Rd4_6nM_C22, A02_Rd4_6nM_C19, A02_Rd4_0.6nM_C03 A02_Rd4_6nM_C07, A02_Rd4_6nM_C23, A02_Rd4_0.6nM_C18, A02_Rd4_6nM_C10, A02_Rd4_6nM_C05, A02_Rd4_0.6nM_C10, A02_Rd4_6nM_C04, A02_Rd4_0.6nM_C26, A02_Rd4_0.6nM_C13, A02_Rd4_0.6nM_C01, A02_Rd4_6nM_C08, P5C1_VHVL, C01_Rd4_6nM_C24, C01_Rd4_6nM_C26, C01_Rd4_6nM_C10, C01_Rd4_0.6nM_C27, C01_Rd4_6nM_C20, C01_Rd4_6nM_C12, C01_Rd4_0.6nM_C16, C01_Rd4_0.6nM_C09, C01_Rd4_6nM_C09, C01_Rd4_0.6nM_C03, C01_Rd4_0.6nM_C06, C01_Rd4_6nM_C04, COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10 COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_C25, COMBO_Rd4_0.6nM_C21, COMBO_Rd4_6nM_C11, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09, COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02, COMBO_Rd4_0.6nM_C23, COMBO_Rd4_0.6nM_C29, COMBO_Rd4_0.6nM_C09, COMBO_Rd4_6nM_C12, COMBO_Rd4_0.6nM_C30, COMBO_Rd4_0.6nM_C14, COMBO_Rd4_6nM_C07, COMBO_Rd4_6nM_C02, COMBO_Rd4_0.6nM_C05, COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, and COMBO_Rd4_0.6nM_C11.
TABLE 1
mAb
Light Chain
Heavy Chain
P6E01/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
P6E01
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YGSPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 1)
VSS (SEQ ID NO: 2)
P6E01/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H3.AQ
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YGSPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 1)
VSS (SEQ ID NO: 3)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSC
EVQLLESGGGLVQPGGSLRLSCA
L3.KW/
RASQSLGSFYLAWYQQKPGQA
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
PRLLIYGASSRATGIPDRFSGSG
WVSAISGSGGNTFYADSVKGRFTI
SGTDFTLTISRLEPEDFAVYYCKH
SRDNSKNTLYLQMNSLRAEDTAV
YGWPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 4)
VSS (SEQ ID NO: 2)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 5)
VSS (SEQ ID NO: 2)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSVGDFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 6)
VSS (SEQ ID NO: 2)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KW/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKHY
SRDNSKNTLYLQMNSLRAEDTAV
GWPPSFTFGQGTKVEIK (SEQ ID
YYCARARVSPIAALMDYWGQGTL
NO: 4)
VTVSS (SEQ ID NO: 7)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KW/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AP
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKHY
SRDNSKNTLYLQMNSLRAEDTAV
GWPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAPMDYWGQGTLVT
NO: 4)
VSS (SEQ ID NO: 8)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KW/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKHY
SRDNSKNTLYLQMNSLRAEDTAV
GWPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAQMDYWGQGTLVT
NO: 4)
VSS (SEQ ID NO: 3)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.PY/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AP
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAPMDYWGQGTLVT
ID NO: 9)
VSS (SEQ ID NO: 8)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.PY/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 9)
VSS (SEQ ID NO: 3)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAALMDYWGQGTLVT
ID NO: 10)
VSS (SEQ ID NO: 7)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AP
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAPMDYWGQGTLVT
ID NO: 10)
VSS (SEQ ID NO: 8)
L1.LGF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSLGSFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 10)
VSS (SEQ ID NO: 3)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KW/
ASQSVGDFYLAPWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKHY
SRDNSKNTLYLQMNSLRAEDTAV
GWPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAALMDYWGQGTLVT
NO: 11)
VSS (SEQ ID NO: 7)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KW/
ASQSVGDFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AP
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKHY
SRDNSKNTLYLQMNSLRAEDTAV
GWPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAPMDYWGQGTLVT
NO: 11)
VSS (SEQ ID NO: 8)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KW/
ASQSVGDFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKHY
SRDNSKNTLYLQMNSLRAEDTAV
GWPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAQMDYWGQGTLVT
NO: 11)
VSS (SEQ ID NO: 3)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.PY/
ASQSVGDFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 12)
VSS (SEQ ID NO: 3)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSVGDFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAALMDYWGQGTLVT
ID NO: 13)
VSS (SEQ ID NO: 7)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSVGDFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AP
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAPMDYWGQGTLVT
ID NO: 13)
VSS (SEQ ID NO: 8)
L1.GDF/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.NY/
ASQSVGDFYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 14)
VSS (SEQ ID NO: 3)
L3.KW/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
P6E01
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKHY
SRDNSKNTLYLQMNSLRAEDTAV
GWPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 15)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
P6E01
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 2)
L3.NY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
P6E01
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YNYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 17)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
LLIYGASSRATGIPDRFSGSGSG
WVSAISGSGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PH/
ASQSVSPHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 21)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
P6E01
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H2.QR
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQRKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 24)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H2.DY
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAIDYSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 25)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H2.YQ
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISYQGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 26)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H2.LT
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISLTGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 27)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H2.HA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISHAGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 28)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H2.QL
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQLKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 29)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H3.YA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIYAGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 30)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H3.AE
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAEMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 31)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H3.AQ
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 3)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
H3.TAQ
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCTRVSPIAAQMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 32)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
P6E01
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 16)
VSS (SEQ ID NO: 2)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QR
RLLIYGASSRATGIPDRFSGSGS
WVSAIVSGSGGNTFYADQRKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 24)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.DY
RLLIYGASSRATGIPDRFSGSGS
WVSAIDYSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 25)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.YQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISYQGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 26)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.LT
RLLIYGASSRATGIPDRFSGSGS
WVSAISLTGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 27)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.HA
RLLIYGASSRATGIPDRFSGSGS
WVSAISHAGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 28)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQLKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 29)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.YA
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIYAGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 30)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAEMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 31)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 3)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PS/
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.TAQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCTRVSPIAAQMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 32)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QR
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQRKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 24)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.DY
RLLIYGASSRATGIPDRFSGSGS
WVSAIDYSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 25)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.YQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISYQGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 26)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.LT
RLLIYGASSRATGIPDRFSGSGS
WVSAISLTGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 27)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.HA
RLLIYGASSRATGIPDRFSGSGS
WVSAISHAGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 28)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQLKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 29)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.YA
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIYAGMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 30)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAEMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 31)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 3)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.AH/
ASQSVSAHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.TAQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCTRVSPIAAQMDYWGQGTLVT
ID NO: 19)
VSS (SEQ ID NO: 32)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H2.QR
LLIYGASSRATGIPDRFSGSGSG
WVSAISGSGGNTFYADQRKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 24)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H2.DY
LLIYGASSRATGIPDRFSGSGSG
WVSAIDYSGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 25)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H2.YQ
LLIYGASSRATGIPDRFSGSGSG
WVSAISYQGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ
ID YYCARVSPIASGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 26)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H2.LT
LLIYGASSRATGIPDRFSGSGSG
WVSAISLTGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 27)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H2.HA
LLIYGASSRATGIPDRFSGSGSG
WVSAISHAGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 28)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H2.QL
LLIYGASSRATGIPDRFSGSGSG
WVSAISGSGGNTFYADQLKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 29)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H3.YA
LLIYGASSRATGIPDRFSGSGSG
WVSAISGSGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIYAGMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 30)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H3.AE
LLIYGASSRATGIPDRFSGSGSG
WVSAISGSGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAEMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 31)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
LLIYGASSRATGIPDRFSGSGSG
WVSAISGSGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAQMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 3)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.FF/
ASQSVSSFFLAWYQQKPGQAPR
ASGFTFGSYAMTWVRQAPGKGLE
H3.TAQ
LLIYGASSRATGIPDRFSGSGSG
WVSAISGSGGNTFYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQHYP
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCTRVSPIAAQMDYWGQGTLVT
NO: 20)
VSS (SEQ ID NO: 32)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PH/
ASQSVSPHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QR
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQRKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 21)
VSS (SEQ ID NO: 24)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PH/
ASQSVSPHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.HA
RLLIYGASSRATGIPDRFSGSGS
WVSAISHAGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 21)
VSS (SEQ ID NO: 28)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PH/
ASQSVSPHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAEMDYWGQGTLVT
ID NO: 21)
VSS (SEQ ID NO: 31)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PH/
ASQSVSPHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIAAQMDYWGQGTLVT
ID NO: 21)
VSS (SEQ ID NO: 3)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L1.PH/
ASQSVSPHYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.TAQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCTRVSPIAAQMDYWGQGTLVT
ID NO: 21)
VSS (SEQ ID NO: 32)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QR
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQRKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 24)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.DY
RLLIYGASSRATGIPDRFSGSGS
WVSAIDYSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 25)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.YQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISYQGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 26)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.LT
RLLIYGASSRATGIPDRFSGSGS
WVSAISLTGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 27)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.HA
RLLIYGASSRATGIPDRFSGSGS
WVSAISHAGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 28)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQLKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 29)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.YA
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIYAGMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 30)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KY/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.TAQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKYY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCTRVSPIAAQMDYWGQGTLVT
NO: 22)
VSS (SEQ ID NO: 32)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.DY
RLLIYGASSRATGIPDRFSGSGS
WVSAIDYSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 25)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.YQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISYQGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 26)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.LT
RLLIYGASSRATGIPDRFSGSGS
WVSAISLTGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 27)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H2.QL
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADQLKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIASGMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 29)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.YA
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIYAGMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 30)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AE
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAEMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 31)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.AQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAQMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 3)
L3.PY/
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
L3.KF/
ASQSVSSSYLAWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
H3.TAQ
RLLIYGASSRATGIPDRFSGSGS
WVSAISGSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCKFY
SRDNSKNTLYLQMNSLRAEDTAV
PYPPSFTFGQGTKVEIK (SEQ ID
YYCTRVSPIAAQMDYWGQGTLVT
NO: 23)
VSS (SEQ ID NO: 32)
P5A2_VHVL
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISDSGGSTYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YGSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 34)
(SEQ ID NO: 33)
A02_Rd4_0.6nM_C06
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSVIYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAISDSGGSAWYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
QRWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 36)
(SEQ ID NO: 35)
A02_Rd4_0.6nM_C09
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISDSGGSMWYADSVKGRF
GTDFTLTISRLEPEDFAVYYCQQ
TISRDNSKNTLYLQMNSLRAEDTA
YQSWPLTFGQGTKVEIK (SEQ ID
VYYCARYWPMSLWGQGTLVTVS
NO: 38)
S (SEQ ID NO: 37)
A02_Rd4_6nM_C16
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
(P5AC16)
ASQSVSDIYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAISdFGGSTYYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
QTWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 40)
(SEQ ID NO: 39)
A02_Rd4_6nM_C03
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSNLYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISDSGGSTYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YQGWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 41)
(SEQ ID NO: 33)
A02_Rd4_6nM_C01
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSAYYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAITASGGSTYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YERWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 43)
(SEQ ID NO: 42)
A02_Rd4_6nM_C26
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSLYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAISDSGGSTYYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
QVWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 45)
(SEQ ID NO: 44)
A02_Rd4_6nM_C25
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISdSGGSRWYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YLDWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMTPWGQGTLVTVSS
NO: 47)
(SEQ ID NO: 46)
A02_Rd4_6nM_C22
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAVLdSGGSTYYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YQVWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMTPWGQGTLVTVSS
NO: 49)
(SEQ ID NO: 48)
A02_Rd4_6nM_C19
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSVIYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAISdSGGSRWYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQYL
ISRDNSKNTLYLQMNSLRAEDTAV
AWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSDWGQGTLVTVSS
NO: 51)
(SEQ ID NO: 50)
A02_Rd4_0.6nM_C03
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISdSGGSKWYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YFTWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 53)
(SEQ ID NO: 52)
A02_Rd4_6nM_C07
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSPyYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSLPYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQYE
ISRDNSKNTLYLQMNSLRAEDTAV
RWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 55)
(SEQ ID NO: 54)
A02_Rd4_6nM_C23
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSVEYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISdSGGSGWYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YARWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 57)
(SEQ ID NO: 56)
A02_Rd4_0.6nM_C18
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSEIYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAVLdSGGSTYYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQYF
ISRDNSKNTLYLQMNSLRAEDTAV
GWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 59)
(SEQ ID NO: 58)
A02_Rd4_6nM_C10
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVEMSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISdSGGSCWYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YAHWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMTPWGQGTLVTVSS
NO: 61)
(SEQ ID NO: 60)
A02_Rd4_6nM_C05
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIFaSGGSTYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YQRWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMTPWGQGTLVTVSS
NO: 63)
(SEQ ID NO: 62)
A02_Rd4_0.6nM_C10
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSAQYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISgWGGSLPYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YQRWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 65)
(SEQ ID NO: 64)
A02_Rd4_6nM_C04
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSAIYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIMsSGGPLYYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
QVWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMALWGQGTLVTVSS
NO: 67)
(SEQ ID NO: 66)
A02_Rd4_0.6nM_C26
EIVLTQSPGTLSLSPGERATLSCG
EVQLLESGGGLVQPGGSLRLSCA
PSQSVSSSYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAILmSGGSTYYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
QSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 69)
(SEQ ID NO: 68)
A02_Rd4_0.6nM_C13
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYWAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISdSGGYRYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YESWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 71)
(SEQ ID NO: 70)
A02_Rd4_0.6nM_C01
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
(P5AC1)
GGQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAILsSGGSTYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YQSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 73)
(SEQ ID NO: 72)
A02_Rd4_6nM_C08
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSFIYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAILdSGGSTYYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
GSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSPWGQGTLVTVSS
NO: 75)
(SEQ ID NO: 74)
P5C1_VHVL
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
(PC1)
ASQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGGSGGSTYYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQYS
ISRDNSKNTLYLQMNSLRAEDTAV
TSPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 77)
(SEQ ID NO: 76)
C01_Rd4_6nM_C24
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSPEYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLIYDASSRAPGIPDRFSGSGS
WVSAIGGSGGSLPYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YSVWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 79)
(SEQ ID NO: 78)
C01_Rd4_6nM_C26
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSAIYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGGSGGSLPYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQYS
ISRDNSKNTLYLQMNSLRAEDTAV
AWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 317)
(SEQ ID NO: 78)
C01_Rd4_6nM_C10
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSvYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQYS
SRDNSKNTLYLQMNSLRAEDTAV
TWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 79)
(SEQ ID NO: 78)
C01_Rd4_0.6nM_C27
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQYS
SRDNSKNTLYLQMNSLRAEDTAV
RWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 81)
(SEQ ID NO: 78)
C01_Rd4_6nM_C20
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSPIYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQYS
SRDNSKNTLYLQMNSLRAEDTAV
AFPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 82)
(SEQ ID NO: 78)
C01_Rd4_6nM_C12
EIVLTQSPGTLSLSPGERATLSC
EVQLLESGGGLVQPGGSLRLSCA
(PC1C12)
WLSQSVSSTYLAWYQQKPGQA
ASGFTFSSYPMSWVRQAPGKGLE
PRLLIYDASSRAPGIPDRFSGSG
WVSAIGgSGGWSYYADSVKGRFT
SGTDFTLTISRLEPEDFAVYYCQ
ISRDNSKNTLYLQMNSLRAEDTAV
QYSEWPLTFGQGTKVEIK (SEQ
YYCARYWPMDSWGQGTLVTVSS
ID NO: 84)
(SEQ ID NO: 83)
C01_Rd4_0.6nM_C16
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQYS
SRDNSKNTLYLQMNSLRAEDTAV
SWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 85)
(SEQ ID NO: 78)
C01_Rd4_0.6nM_C09
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSIFLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQYS
SRDNSKNTLYLQMNSLRAEDTAV
AWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 86)
(SEQ ID NO: 78)
C01_Rd4_6nM_C09
EIVLTQSPGTLSLSPGERATLSCA
EVQLLESGGGLVQPGGSLRLSCA
CSQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSATVgSGGSIGYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQYS
SRDNSKNTLYLQMNSLRAEDTAV
AWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 88)
(SEQ ID NO: 87)
C01_Rd4_0.6nM_C03
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASCDVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
MRSPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 89)
(SEQ ID NO: 78)
C01_Rd4_0.6nM_C06
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASEAVPSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGTISR
TDFTLTISRLEPEDFAVYYCQQYS
DNSKNTLYLQMNSLRAEDTAVYY
AFPLTFGQGTKVEIK (SEQ ID
CARYWPMDSWGQGTLVTVSS
NO: 90)
(SEQ ID NO: 78)
C01_Rd4_6nM_C04
EIVLTQSPGTLSLSPGERATLSCC
EVQLLESGGGLVQPGGSLRLSCA
SSQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLIYDASSRAPGIPDRFSGSGSG
WVSAIGgSGGSLPYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQYS
SRDNSKNTLYLQMNSLRAEDTAV
AFPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 91)
(SEQ ID NO: 78)
COMBO_Rd4_0.6nM_C22
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
(COM22)
ASVRVSSTYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAISdSGGSRWYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
MKWPLTFGQGTKVEIK (SEQ ID
YYCTRYWPMDIWGQGTLVTVSS
NO: 93)
(SEQ ID NO: 92)
COMBO_Rd4_6nM_C21
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSAAYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIGgSGGSLPYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YMCWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 94)
(SEQ ID NO: 78)
COMBO_Rd4_6nM_C10
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYWGWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIGgSGGSIHYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YQCWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 96)
(SEQ ID NO: 95)
COMBO_Rd4_0.6nM_C04
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAHIgSGGSTYYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
QSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 98)
(SEQ ID NO: 97)
COMBO_Rd4_6nM_C25
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSpYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGgSGGSTYYADSVKGRFTI
TDFTLTISRLEPEDFAVYYCQQY
SRDNSKNTLYLQMNSLRAEDTAV
QSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDPWGQGTLVTVSS
NO: 100)
(SEQ ID NO: 99)
COMBO_Rd4_0.6nM_C21
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIGgSGGSLPYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YQSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 38)
(SEQ ID NO: 78)
COMBO_Rd4_6nM_C11
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSPIYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSLGYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
KAWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 102)
(SEQ ID NO: 101)
COMBO_Rd4_0.6nM_C20
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSYLYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSLPYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
MEWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 103)
(SEQ ID NO: 78)
COMBO_Rd4_6nM_C09
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSAQYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIFASGGSTYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YQAWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 105)
(SEQ ID NO: 104)
COMBO_Rd4_6nM_C08
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIGGSGTWTYYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YQKWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 107)
(SEQ ID NO: 106)
COMBO_Rd4_0.6nM_C19
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSAVYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIGGSGGSLPYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YRAWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 108)
(SEQ ID NO: 78)
COMBO_Rd4_0.6nM_C02
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASIAVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSLPYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
MVWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 109)
(SEQ ID NO: 78)
COMBO_Rd4_0.6nM_C23
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
PRQSVSSSYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSALFGSGGSTYYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YQDWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 111)
(SEQ ID NO: 110)
COMBO_Rd4_0.6nM_C29
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIGGSGGSLPYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YQSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 38)
(SEQ ID NO: 112)
COMBO_Rd4_0.6nM_C09
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSLPYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
QEWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 113)
(SEQ ID NO: 112)
COMBO_Rd4_6nM_C12
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSASYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAALGSGGSTYYADSVKGRF
GTDFTLTISRLEPEDFAVYYCQQ
TISRDNSKNTLYLQMNSLRAEDTA
YMSWPLTFGQGTKVEIK (SEQ ID
VYYCARYWPMDSWGQGTLVTVS
NO: 115)
S (SEQ ID NO: 114)
COMBO_Rd4_0.6nM_C30
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSYMYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLIYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSTYYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
KSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 116)
(SEQ ID NO: 76)
COMBO_Rd4_0.6nM_C14
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSALYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAIGGSGGSLPYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YYGWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 117)
(SEQ ID NO: 112)
COMBO_Rd4_6nM_C07
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQPISSSYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSLPYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
QGWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMADWGQGTLVTVSS
NO: 119)
(SEQ ID NO: 118)
COMBO_Rd4_6nM_C02
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAISDSGGFVYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQQ
SRDNSKNTLYLQMNSLRAEDTAV
YEFWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 121)
(SEQ ID NO: 120)
COMBO_Rd4_0.6nM_C05
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSTYLAWYQQKPGQAPR
ASGFTFSSYAMNWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSTYYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
MSWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMSLWGQGTLVTVSS
NO: 123)
(SEQ ID NO: 122)
COMBO_Rd4_0.6nM_C17
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQGISSTYLAWYQQKPGQAPR
ASGFTFSSYPMSWVRQAPGKGLE
LLMYDASIRATGIPDRFSGSGSG
WVSAIGGSGGSLPYADSVKGRFT
TDFTLTISRLEPEDFAVYYCQQY
ISRDNSKNTLYLQMNSLRAEDTAV
AYWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDIWGQGTLVTVSS
NO: 124)
(SEQ ID NO: 112)
COMBO_Rd4_6nM_C22
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMNWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSACLDSGGSTYYADSVKGRFT
GTDFTLTISRLEPEDFAVYYCQQ
ISRDNSKNTLYLQMNSLRAEDTAV
YQGWPLTFGQGTKVEIK (SEQ ID
YYCARYWPMDSWGQGTLVTVSS
NO: 126)
(SEQ ID NO: 125)
COMBO_Rd4_0.6nM_C11
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSVRYLAWYQQKPGQAP
ASGFTFSSYPMSWVRQAPGKGLE
RLLMYDASIRATGIPDRFSGSGS
WVSAALGSGGSTYYADSVKGRF
GTDFTLTISRLEPEDFAVYYCQQ
TISRDNSKNTLYLQMNSLRAEDTA
YGSWPITFGQGTKVEIK (SEQ ID
VYYCARYWPMSLWGQGTLVTVS
NO: 128)
S (SEQ ID NO: 127)
P6DY
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYPSWYQQKPGQAP
ASGFTFGSYAMTWVRQAPGKGLE
RLLIYGASSRATGIPDRFSGSGS
WVSAIDYSGGNTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YPYPPSFTFGQGTKVEIK (SEQ
YYCARVSPIASGMDYWGQGTLVT
ID NO: 18)
VSS (SEQ ID NO: 25)
P6AP
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQLGSFYLAWYQQKPGQAPRL
ASGFTFGSYAMTWVRQAPGKGLE
LIYGASSRATGIPDRFSGSGSGT
WVSAISGSGGNTFYADSVKGRFTI
DFTLTISRLEPEDFAVYYCQHYN
SRDNSKNTLYLQMNSLRAEDTAV
YPPSFTFGQGTKVEIK (SEQ ID
YYCARVSPIAAPMDYWGQGTLVT
NO: 80)
VSS (SEQ ID NO: 8)
Consensus
EIVLTQSPGTLSLSPGERATLSC
EVQLLESGGGLVQPGGSLRLSCA
X1X2X3X4X5X6X7X8X9X10X11X12WY
ASGFTFX1SYX2MX3WVRQAPGKG
QQKPGQAPRLLMYX13ASX14RAX15
LEWVSAX4X5X6X7GX8X9X10X11YAD
GIPDRFSGSGSGTDFTLTISRLE
X12X13KGRFTISRDNSKNTLYLQMN
PEDFAVYYCX16X17YX18X19PPSF
SLRAEDTAVYYCARVSPIX14X15X16
TFGQGTKVEIK, wherein X1 is R,
MDYWGQGTLVTVSS, wherein X1
G, W, A, or C; X2 is A, P, G, L, C,
is G or S, X2 is A or P; X3 is T, N, or
or S; X3 is S, G, or R; X4 is Q, C,
S; X4 is I, V, T, H, L, A, or C; X5 is S,
E, V, or I; X5 is S, P, G, A, R, or D;
D, G, T, I, L, F, M, or V; X6 is G, Y,
X6 is V, G, I, or L; X7 is S, E, D, P,
L, H, D, A, S, or M; X7 is S, Q, T, A,
or G; X8 is S, P, F, A, M, E, V, N,
F, or W; X8 is G or T; X9 is N, S, P,
D, or Y; X9 is I, T, V, E, S, A, M, Q,
Y, W, or F; X10 is S, T, I, L, T, A, R,
Y, H, R, or F; X10 is Y or F; X11 is
V, K, G, or C; X11 is F, Y, P, W, H,
L, W, or P; X12 is A, S, or G, X13 is
or G; X12 is V, R, or L; X13 is G or T;
G or D; X14 is S or I; X15 is T or P;
X14 is A or Y; X15 is A or S; and X16 is
X16 is Q or K; X17 is H or Y; X18 is
G, Q, L, P, or E (SEQ ID NO: 313);
G, N, or P; and X19 is S, W, or Y
or
(SEQ ID NO: 315); or
EVQLLESGGGLVQPGGSLRLSCA
EIVLTQSPGTLSLSPGERATLSC
ASGFTFX1SYX2MX3WVRQAPGKG
X1X2X3X4X5X6X7X8X9X10X11X12WY
LEWVSAX4X5X6X7GX8X9X10X11YAD
QQKPGQAPRLLMYX13ASX14RAX15
X12X13KGRFTISRDNSKNTLYLQMN
GIPDRFSGSGSGTDFTLTISRLE
SLRAEDTAVYYCARYWPMX14X15
PEDFAVYYCQQYX16X17X18PX19F
WGQGTLVTVSS, wherein X1 is G
GQGTKVEIK, wherein X1 is R, G,
or S, X2 is A or P; X3 is T, N, or S;
W, A, or C; X2 is A, P, G, L, C, or
X4 is I, V, T, H, L, A, or C; X5 is S, D,
S; X3 is S, G, or R; X4 is Q, C, E,
G, T, I, L, F, M, or V; X6 is G, Y, L,
V, or I; X5 is S, L, P, G, A, R, or D;
H, D, A, S, or M; X7 is S, Q, T, A, F,
X6 is V, G, or I; X7 is S, E, D, or P;
or W; X8 is G or T; X9 is N, S, P, Y,
X8 is S, P, F, A, M, E, V, N, D, or
W, or F; X10 is S, T, I, L, T, A, R, V,
Y; X9 is I, T, V, E, S, A, M, Q, Y, H,
K, G, or C; X11 is F, Y, P, W, H, or
or R; X10 is Y or F; X11 is L, W, or
G; X12 is V, R, or L; X13 is G or T;
P; X12 is A, S, or G, X13 is G or D;
X14 is D, S, T, or A; and X15 is I, S, L,
X14 is S or I; X15 is T or P; X16 is G,
P, or D (SEQ ID NO: 314)
Q, E, L, F, A, S, M, R, K, or Y; X17
is S, R, T, G, R, V, D, A, H, E, K,
C, F, or Y; X18 is W, S, or F; and
X19 is L or I (SEQ ID NO: 316)
P4G4
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQSVSSSYLAWYQQKPGQAP
ASGFTFSSYAMSWVRQAPGKGLE
RLLIYGASSRAYGIPDRFSGSGS
WVSAISASGGSTYYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YGSPPLFTFGQGTKVEIK (SEQ
YYCARLSWSGAFDNWGQGTLVT
ID NO: 401)
VSS (SEQ ID NO: 378)
P1A11
EIVLTQSPGTLSLSPGERATLSCR
EVQLLESGGGLVQPGGSLRLSCA
ASQNVSSSYLAWYQQKPGQAP
ASGFTFRSYAMSWVRQAPGKGLE
RLLIYGASYRATGIPDRFSGSGS
WVSAISGSGGSTFYADSVKGRFTI
GTDFTLTISRLEPEDFAVYYCQH
SRDNSKNTLYLQMNSLRAEDTAV
YGSPPSFTFGQGTKVEIK (SEQ
YYCATVGTSGAFGIWGQGTLVTV
ID NO: 379)
SS (SEQ ID NO: 380)
Also provided herein are CDR portions of extracellular ligand-binding domains of CARs to BCMA (including Chothia, Kabat CDRs, and CDR contact regions). Determination of CDR regions is well within the skill of the art. It is understood that in some embodiments, CDRs can be a combination of the Kabat and Chothia CDR (also termed “combined CRs” or “extended CDRs”). In some embodiments, the CDRs are the Kabat CDRs. In other embodiments, the CDRs are the Chothia CDRs. In other words, in embodiments with more than one CDR, the CDRs may be any of Kabat, Chothia, combination CDRs, or combinations thereof. Table 2 provides examples of CDR sequences provided herein.
TABLE 2
Heavy Chain
mAb
CDRH1
CDRH2
CDRH3
P6E01
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIASGMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
P6E01/P6E01; L1.LGF/L3.KW/
GFTFGSY (SEQ ID
NO: 132) (Kabat)
NO: 134)
P6EO1;
NO: 130) (Chothia);
SGSGGN (SEQ
L1.LGF/L3.NY/P6E01;
GFTFGSYAMT (SEQ
ID NO: 133)
L1.GDF/L3.NY/P6E01;
ID NO: 131)
(Chothia)
L3.KW/P6E01;
(extended)
L3.PY/P6E01;
L3.NY/P6E01;
L3.PY/L1.PS/P6E01;
L3.PY/L1.AH/P6E01;
L3.PY/L1.FF/P6E01;
L3.PY/L1.PH/P6E01;
L3.PY/L3.KY/P6E01;
L3.PY/L3.KF/P6E01;
and L3.PY/P6E01.
H3.AQ
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIAAQMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
P6E01/H3.AQ;
GFTFGSY (SEQ ID
NO: 132) (Kabat)
NO: 135)
L1.LGF/L3.KW/H3.AQ;
NO: 130) (Chothia);
SGSGGN (SEQ
L1.LGF/L3.PY/H3.AQ
GFTFGSYAMT (SEQ
ID NO: 133)
ID NO: 131)
(Chothia)
(extended)
H3.AL
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIAALMDY
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
(SEQ ID NO:
L1.LGF/L3.KW/H3.AL;
GFTFGSY (SEQ ID
NO: 132) (Kabat)
136)
L1.LGF/L3.NY/H3.AL;
NO: 130) (Chothia);
SGSGGN (SEQ
and
GFTFGSYAMT (SEQ
ID NO: 133)
L1.GDF/L3.NY/H3.AL.
ID NO: 131)
(Chothia)
(extended)
H3.AP
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIAAPMDY
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
(SEQ ID NO:
L1.LGF/L3.KW/H3.AP;
GFTFGSY (SEQ ID
NO: 132) (Kabat)
137)
L1.LGF/L3.PY/H3.AP;
NO: 130) (Chothia);
SGSGGN (SEQ
L1.LGF/L3NY/H3.AP;
GFTFGSYAMT (SEQ
ID NO: 133)
L1.GDF/L3.KW/H3.AP;
ID NO: 131)
(Chothia)
L1.GDF/L3NY/H3.AP;
(extended)
P6AP.
H2.QR
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIASGMD
For the following mAbs:
129) (Kabat);
DQRKG (SEQ ID
Y (SEQ ID
L3.PY/H2.QR;
GFTFGSY (SEQ ID
NO: 138) (Kabat)
NO: 134)
L3.PY/L1.PS/H2.QR;
NO: 130) (Chothia);
SGSGGN (SEQ
L3.PY/L1.AH/H2.QR;
GFTFGSYAMT (SEQ
ID NO: 133)
L3.PY/L1.FF/H2.QR;
ID NO: 131)
(Chothia)
L3.PY/L1.PH/H2.QR;
(extended)
and
L3.PY/L3.KY/H2.QR.
H2.DY
SYAMT (SEQ ID NO:
AIDYSGGNTFYA
VSPIASGMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
L3.PY/H2.DY; P6DY;
GFTFGSY (SEQ ID
NO: 139) (Kabat)
NO: 134)
L3.PY/L1.PS/H2.DY;
NO: 130) (Chothia);
DYSSGN (SEQ
L3.PY/L1.AH/H2.DY;
GFTFGSYAMT (SEQ
ID NO: 140)
L3.PY/L1.FF/H2.DY;
ID NO: 131)
(Chothia)
L3.PY/L3.KY/H2.DY;
(extended)
and
L3.PY/L3.KF/H2.DY.
H2.YQ
SYAMT (SEQ ID NO:
AISYQGGNTFYA
VSPIASGMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
L3.PY/H2.YQ;
GFTFGSY (SEQ ID
NO: 141) (Kabat)
NO: 134)
L3.PY/L1.PS/H2.YQ;
NO: 130) (Chothia);
SYQGGN (SEQ
L3.PY/L1.AH/H2.YQ;
GFTFGSYAMT (SEQ
ID NO: 142)
L3.PY/L1.FF/H2.YQ;
ID NO: 131)
(Chothia)
L3.PY/L3.KY/H2.YQ;
(extended)
and
L3.PY/L3.KF/H2.YQ.
H2.LT
SYAMT (SEQ ID NO:
AISLTGGNTFYA
VSPIASGMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
L3.PY/H2.LT;
GFTFGSY (SEQ ID
NO: 143) (Kabat)
NO: 134)
L3.PY/L1.PS/H2.LT;
NO: 130) (Chothia);
SLTGGN (SEQ
L3.PY/L1.AH/H2.LT;
GFTFGSYAMT (SEQ
ID NO: 144)
L3.PY/L1.FF/H2.LT;
ID NO: 131)
(Chothia)
L3.PY/L3.KY/H2.LT;
(extended)
and
L3.PY/L3.KF/H2.LT.
H2.HA
SYAMT (SEQ ID NO:
AISHAGGNTFYA
VSPIASGMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
L3.PY/H2.HA;
GFTFGSY (SEQ ID
NO: 145) (Kabat)
NO: 134)
L3.PY/L1.AH/H2.HA;
NO: 130) (Chothia);
SHAGGN (SEQ
L3.PY/L1.FF/H2.HA;
GFTFGSYAMT (SEQ
ID NO: 146)
L3.PY/L1.PH/H2.HA;
ID NO: 131)
(Chothia)
and
(extended)
L3.PY/L3.KY/H2.HA.
H2.QL
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIASGMD
For the following mAbs:
129) (Kabat);
DQLKG (SEQ ID
Y (SEQ ID
L3.PY/H2.QL;
GFTFGSY (SEQ ID
NO: 147) (Kabat)
NO: 134)
L3.PY/L1.PS/H2.QL;
NO: 130) (Chothia);
SGSGGN (SEQ
L3.PY/L1.AH/H2.QL;
GFTFGSYAMT (SEQ
ID NO: 133)
L3.PY/L1.FF/H2.QL;
ID NO: 131)
(Chothia)
L3.PY/L3.KY/H2.QL;
(extended)
and
L3.PY/L3.KF/H2.QL.
H3.YA
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIYAGMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
L3.PY/H3.YA;
GFTFGSY (SEQ ID
NO: 132) (Kabat)
NO: 148)
L3.PY/L1.PS/H3.YA;
NO: 130) (Chothia);
SGSGGN (SEQ
L3.PY/L1.AH/H3.YA;
GFTFGSYAMT (SEQ
ID NO: 133)
L3.PY/L1.FF/H3.YA;
ID NO: 131)
(Chothia)
L3.PY/L3.KY/H3.YA;
(extended)
and
L3.PY/L3.KF/H3.YA.
H3.AE
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIAAEMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
L3.PY/H3.AE;
GFTFGSY (SEQ ID
NO: 132) (Kabat)
NO: 149)
L3.PY/L1.AH/H3.AE;
NO: 130) (Chothia);
SGSGGN (SEQ
L3.PY/L1.FF/H3.AE;
GFTFGSYAMT (SEQ
ID NO: 133)
L3.PY/L1.PH/H3.AE;
ID NO: 131)
(Chothia)
and
(extended)
L3.PY/L3.KF/H3.AE.
H3.TAQ
SYAMT (SEQ ID NO:
AISGSGGNTFYA
VSPIAAQMD
For the following mAbs:
129) (Kabat);
DSVKG (SEQ ID
Y (SEQ ID
L3.PY/H3.TAQ;
GFTFGSY (SEQ ID
NO: 132) (Kabat)
NO: 135)
L3.PY/L1.PS/H3.TAQ;
NO: 130) (Chothia);
SGSGGN (SEQ
L3.PY/L1.AH/H3.TAQ;
GFTFGSYAMT (SEQ
ID NO: 133)
L3.PY/L1.FF/H3.TAQ;
ID NO: 131)
(Chothia)
L3.PY/L1.PH/H3.TAQ;
(extended)
and
L3.PY/L3.KF/H3.TAQ.
P5A2_VHVL and
SYAMN (SEQ ID NO:
AISDSGGSTYYA
YWPMDI
A02_Rd4_6nM_C03
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 153)
155)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
COMBO_Rd4_0.6nM_C17;
SYPMS (SEQ ID NO:
AIGGSGGSLPYA
YWPMDI
COMBO_Rd4_0.6nM_C14;
156) (Kabat);
DSVKG
(SEQ ID NO:
COMBO_Rd4_0.6nM_C29;
GFTFSSY (SEQ ID
(SEQ ID NO: 158)
155)
and
NO: 151) (Chothia);
(Kabat)
COMBO_Rd4_0.6nM_C09
GFTFSSYPMS (SEQ
GGSGGS (SEQ
ID NO: 157)
ID NO: 159)
(extended)
(Chothia)
C01_Rd4_6nM_C04;
SYPMS (SEQ ID NO:
AIGGSGGSLPYA
YWPMDS
C01_Rd4_0.6nM_C03;
156) (Kabat);
DSVKG
(SEQ ID NO:
C01_Rd4_0.6nM_C06;
GFTFSSY (SEQ ID
(SEQ ID NO: 158)
161)
COMBO_Rd4_0.6nM_C02;
NO: 151) (Chothia);
(Kabat)
COMBO_Rd4_6nM_C21;
GFTFSSYPMS (SEQ
GGSGGS (SEQ
C01_Rd4_6nM_C26;
ID NO: 157)
ID NO: 159)
COMBO_Rd4_0.6nM_C19;
(extended)
(Chothia)
C01_Rd4_6nM_C24;
C01_Rd4_6nM_C20;
C01_Rd4_0.6nM_C09;
COMBO_Rd4_0.6nM_C21;
C01_Rd4_0.6nM_C04_C27;
C01_Rd4_0.6nM_C16;
C01_Rd4_6nM_C10;
COMBO_Rd4_0.6nM_C20
P5C1_VHVL (PC1) and
SYPMS (SEQ ID NO:
AIGGSGGSTYYA
YWPMDS
COMBO_Rd4_0.6nM_C30
156) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 162)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
GGSGGS (SEQ
ID NO: 157)
ID NO: 159)
(extended)
(Chothia)
A02_Rd4_0.6nM_C06
SYAMN (SEQ ID NO:
AISDSGGSAWY
YWPMSL
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 163)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_0.6nM_C09
SYAMN (SEQ ID NO:
AISDSGGSAWY
YWPMSL
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 163)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_0.6nM_C16;
SYAMN (SEQ ID NO:
AISDFGGSTYYA
YWPMDI
A02_Rd4_6nM_C16
150) (Kabat);
DSVKG
(SEQ ID NO:
(P5A16)
GFTFSSY (SEQ ID
(SEQ ID NO: 165)
155)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDFGGS (SEQ
ID NO: 152)
ID NO: 166)
(extended)
(Chothia)
A02_Rd4_6nM_C01
SYAMN (SEQ ID NO:
AITASGGSTYYA
YWPMSL
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 167)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
TASGGS (SEQ
ID NO: 152)
ID NO: 168)
(extended)
(Chothia)
A02_Rd4_6nM_C26
SYAMN (SEQ ID NO:
AISDSGGSTYYA
YWPMSL
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 153)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_6nM_C25
SYAMN (SEQ ID NO:
AISDSGGSRWY
YWPMTP
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 169)
170)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_6nM_C22
SYAMN (SEQ ID NO:
AVLDSGGSTYY
YWPMTP
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 171)
170)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
LDSGGS (SEQ
ID NO: 152)
ID NO: 172)
(extended)
(Chothia)
A02_Rd4_6nM_C19
SYAMN (SEQ ID NO:
AISDSGGSRWY
YWPMSD
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 169)
173)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_0.6nM_C03
SYAMN (SEQ ID NO:
AISDSGGSKWY
YWPMSL
150) (Kabat);
ADSVKG (SEQ
(SEQ ID NO:
GFTFSSY (SEQ ID
ID NO: 174)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_6nM_C07
SYAMN (SEQ ID NO:
AIGGSGGSLPYA
YWPMDS
150) (Kabat);
DSVKG(SEQ ID
(SEQ ID NO:
GFTFSSY (SEQ ID
NO: 158) (Kabat)
161)
NO: 151) (Chothia);
GGSGGS (SEQ
GFTFSSYAMN (SEQ
ID NO: 159)
ID NO: 152)
(Chothia)
(extended)
A02_Rd4_6nM_C23
SYAMN (SEQ ID NO:
AISDSGGSGWY
YWPMSL
150) (Kabat);
ADSVKG (SEQ
(SEQ ID NO:
GFTFSSY (SEQ ID
ID NO: 175)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_0.6nM_C18
SYAMN (SEQ ID NO:
AVLDSGGSTYY
YWPMSL
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 171)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
LDSGGS (SEQ
ID NO: 152)
ID NO: 172)
(extended)
(Chothia)
A02_Rd4_6nM_C10
SYAMN (SEQ ID NO:
AISDSGGSCWY
YWPMTP
150) (Kabat);
ADSVKG (SEQ
(SEQ ID NO:
GFTFSSY (SEQ ID
ID NO: 176)
170)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
A02_Rd4_6nM_C05
SYAMN (SEQ ID NO:
AIFASGGSTYYA
YWPMTP
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 177)
170)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
FASGGS (SEQ
ID NO: 152)
ID NO: 178)
(extended)
(Chothia)
A02_Rd4_0.6nM_C10
SYAMN (SEQ ID NO:
AISGWGGSLPY
YWPMDS
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 304)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SGWGGS (SEQ
ID NO: 152)
ID NO: 179)
(extended)
(Chothia)
A02_Rd4_6nM_C04
SYAMN (SEQ ID NO:
AIMSSGGPLYYA
YWPMAL
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 180)
182)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
MSSGGP (SEQ
ID NO: 152)
ID NO: 181)
(extended)
(Chothia)
A02_Rd4_0.6nM_C26
SYAMN (SEQ ID NO:
AILMSGGSTYYA
YWPMSL
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 183)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
LMSGGS (SEQ
ID NO: 152)
ID NO: 184)
(extended)
(Chothia)
A02_Rd4_0.6nM_C13
SYAMN (SEQ ID NO:
AISDSGGYRYYA
YWPMSL
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 185)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGY (SEQ
ID NO: 152)
ID NO: 186)
(extended)
(Chothia)
A02_Rd4_0.6nM_C01
SYAMN (SEQ ID NO:
AILSSGGSTYYA
YWPMDI
(P5AC1)
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 187)
155)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
LSSGGS (SEQ
ID NO: 152)
ID NO: 188)
(extended)
(Chothia)
A02_Rd4_6nM_C08
SYAMN (SEQ ID NO:
AILDSGGSTYYA
YWPMSP
150) (Kabat);
DSVKG (SEQ ID
(SEQ ID NO:
GFTFSSY (SEQ ID
NO: 160) (Kabat)
189)
NO: 151) (Chothia);
LDSGGS (SEQ
GFTFSSYAMN (SEQ
ID NO: 172)
ID NO: 152)
(Chothia)
(extended)
C01_Rd4_6nM_C12
SYPMS (SEQ ID NO:
AIGGSGGWSYY
YWPMDS
(PC1C12)
156) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 190)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
GGSGGW (SEQ
ID NO: 157)
ID NO: 191)
(extended)
(Chothia)
C01_Rd4_6nM_C09
SYPMS (SEQ ID NO:
ATVGSGGSIGYA
YWPMDS
156) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO:
161)
NO: 151) (Chothia);
192) (Kabat)
GFTFSSYPMS (SEQ
VGSGGS (SEQ
ID NO: 157)
ID NO: 193)
(extended)
(Chothia)
COMBO_Rd4_0.6nM_C22
SYAMN (SEQ ID NO:
AISDSGGSRWY
YWPMDI
(COM22)
150) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 169)
155)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGS (SEQ
ID NO: 152)
ID NO: 154)
(extended)
(Chothia)
COMBO_Rd4_0.6nM_C10
SYPMS (SEQ ID NO:
AIGGSGGSIHYA
YWPMDS
156) (Kabat);
DSVKG (SEQ ID
(SEQ ID NO:
GFTFSSY (SEQ ID
NO: 194) (Kabat)
161)
NO: 151) (Chothia);
GGSGGS (SEQ
GFTFSSYPMS (SEQ
ID NO: 159)
ID NO: 157)
(Chothia)
(extended)
COMBO_Rd4_0.6nM_C04
SYPMS (SEQ ID NO:
AHIGSGGSTYYA
YWPMDS
156) (Kabat);
DSVKG (SEQ ID
(SEQ ID NO:
GFTFSSY (SEQ ID
NO: 195) (Kabat)
161)
NO: 151) (Chothia);
IGSGGS (SEQ ID
GFTFSSYPMS (SEQ
NO: 196)
ID NO: 157)
(Chothia)
(extended)
COMBO_Rd4_0.6nM_C25
SYPMS (SEQ ID NO:
AIGGSGGSTYYA
YWPMDP
156) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 162)
197)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
GGSGGS (SEQ
ID NO: 157)
ID NO: 159)
(extended)
(Chothia)
COMBO_Rd4_6nM_C21
SYPMS (SEQ ID NO:
AIGGSGGSLPYA
YWPMDS
156) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 158)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
GGSGGS (SEQ
ID NO: 157)
ID NO: 159)
(extended)
(Chothia)
COMBO_Rd4_6nM_C11
SYPMS (SEQ ID NO:
AIGGSGGSLGYA
YWPMDS
156) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO:
161)
NO: 151) (Chothia);
198)(Kabat)
GFTFSSYPMS (SEQ
GGSGGS (SEQ
ID NO: 157)
ID NO: 159)
(extended)
(Chothia)
COMBO_Rd4_6nM_C09
SYPMS (SEQ ID NO:
AIFASGGSTYYA
YWPMDS
156) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 177)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
FASGGS (SEQ
ID NO: 157)
ID NO: 178)
(extended)
(Chothia)
COMBO_Rd4_6nM_C08
SYPMS (SEQ ID NO:
AIGGSGTWTYY
YWPMDS
156) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 199)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
GGSGTW (SEQ
ID NO: 157)
ID NO: 200)
(extended)
(Chothia)
COMBO_Rd4_0.6nM_C23
SYPMS (SEQ ID NO:
ALFGSGGSTYY
YWPMDS
156) (Kabat);
ADSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 201)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
FGSGGS
ID NO: 157)
(SEQ ID NO: 202)
(extended)
(Chothia)
COMBO_Rd4_0.6nM_C12
SYPMS (SEQ ID NO:
AALGSGGSTYY
YWPMDS
156) (Kabat);
ADSVKG (SEQ
(SEQ ID NO:
GFTFSSY (SEQ ID
ID NO: 203)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
LGSGGS (SEQ
ID NO: 157)
ID NO: 204)
(extended)
(Chothia)
COMBO_Rd4_6nM_C07
SYPMS (SEQ ID NO:
AIGGSGGSLPYA
YWPMAD
156) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 158)
205)
NO: 151) (Chothia);
(Kabat)
GFTFSSYPMS (SEQ
GGSGGS (SEQ
ID NO: 157)
ID NO: 159)
(extended)
(Chothia)
COMBO_Rd4_6nM_C02
SYAMN (SEQ ID NO:
AISDSGGFVYYA
YWPMDS
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 206)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
SDSGGF (SEQ
ID NO: 152)
ID NO: 207)
(extended)
(Chothia)
COMBO_Rd4_6nM_C05
SYAMN (SEQ ID NO:
AIGGSGGSTYYA
YWPMSL
150) (Kabat);
DSVKG
(SEQ ID NO:
GFTFSSY (SEQ ID
(SEQ ID NO: 162)
164)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
GGSGGS (SEQ
ID NO: 152)
ID NO: 159)
(extended)
(Chothia)
COMBO_Rd4_6nM_C22
SYAMN (SEQ ID NO:
ACLDSGGSTYY
YWPMDS
150) (Kabat);
ADSVKG (SEQ
(SEQ ID NO:
GFTFSSY (SEQ ID
ID NO: 208)
161)
NO: 151) (Chothia);
(Kabat)
GFTFSSYAMN (SEQ
LDSGGS (SEQ
ID NO: 152)
ID NO: 172)
(extended)
(Chothia)
COMBO_Rd4_6nM_C11
SYPMS (SEQ ID NO:
AALGSGGSTYY
YWPMSL
156) (Kabat);
ADSVKG (SEQ
(SEQ ID NO:
GFTFSSY (SEQ ID
ID NO: 203)
164)
NO: 151) (Chothia);
(Chothia)
GFTFSSYPMS (SEQ
LGSGGS (SEQ
ID NO: 157)
ID NO: 204)
(extended)
(Chothia)
Heavy chain consensus
SYX1MX2, wherein X1
AX1X2X3X4GX5X6
VSPIX1X2X3
is A or P; and X2 is T,
X7X8YADX9X10KG,
MDY, wherein
N, or S (Kabat) (SEQ
wherein X1 is I,
X1 is A or Y;
ID NO: 301)
V, T, H, L, A, or
X2 is A or S;
GFTFX1SY, wherein
C; X2 is S, D, G,
and X3 is G,
X1 is G or S (Chothia)
T, I, L, F, M, or V;
Q, L, P, or E
(SEQ ID NO: 302)
X3 is G, Y, L, H,
(SEQ ID NO:
GFTFX1SYX2MX3,
D, A, S, or M; X4
307)
wherein X1 is G or S,
is S, Q, T, A, F, or
YWPMX1X2,
X2 is A or P; and X3 is
W; X5 is G or T; X6
wherein X1 is
T, N, or S (SEQ ID
is N, S, P, Y, W,
D, S, T, or A;
NO: 303) (extended)
or F; X7 is S, T, I,
and X2 is I, S,
L, T, A, R, V, K,
L, P, or D
G, or C; X8 is F,
(SEQ ID NO:
Y, P, W, H, or G;
308)
X9 is V, R, or L;
and X10 is G or T
(Kabat)
(SEQ ID NO:
305)
X1X2X3X4X5X6,
wherein X1 is S,
V, I, D, G, T, L, F,
or M; X2 is G, Y,
L, H, D, A, S, or
M; X3 is S, G, F,
or W; X4 is G or
S; X5 is G or T;
and X6 is N, S, P,
Y, or W (Chothia)
(SEQ ID NO:
306)
P4G4
SYAMS (SEQ ID NO:
SASGGS (SEQ
LSWSGAFD
381) (Kabat);
ID NO: 383)
N (SEQ ID
GFTFSSY (SEQ ID
(Chothia)
NO: 385)
NO: 151) (Chothia);
AISASGGSTYYA
GFTFSSYAMS (SEQ
DSVKG (SEQ ID
ID NO: 382)
NO: 384) (Kabat)
(extended)
P1A11
SYAMS (SEQ ID NO:
SGSGGS (SEQ
VGTSGAFGI
386) (Kabat);
ID NO: 389)
(SEQ ID NO:
GFTFRSY (SEQ ID
(Chothia)
391)
NO: 387)
AISGSGGSTFYA
GFTFRSYAMS (SEQ
DSVKG (SEQ ID
ID NO: 388)
NO: 390) (Kabat)
Light Chain
mAb
CDRL1
CDRL2
CDRL3
P6E01
RASQSVSSSYLA
GASSRAT (SEQ
QHYGSPPSF
For the following mAbs:
(SEQ ID NO: 209)
ID NO: 210)
T (SEQ ID
P6E01/P6E01; and
NO: 211)
P6E01/H3.AQ.
L1.LGF/L3.KW
RASQSLGSFYLA
GASSRAT (SEQ
KHYGWPPS
For the following mAbs:
(SEQ ID NO: 212)
ID NO: 210)
FT (SEQ ID
L1.LGF/L3.KW/P6E01;
NO: 213)
L1.LGF/L3.KW/H3.AL;
L1.LGF/L3.KW/H3.AP;
and
L1.LGF/L3.KW/H3.AQ
L1.LGF/L3.NY
RASQSLGSFYLA
GASSRAT (SEQ
QHYNYPPSF
For the following mAbs:
(SEQ ID NO: 212)
ID NO: 210)
T (SEQ ID
L1.LGF/L3.NY/P6E01;
NO: 214)
L1.LGF/L3.NY/H3.AL;
L1.LGF/L3.NY/H3.AP;
and
L1.LGF/L3.NY/H3AQ
L1.GDF/L3.NY
RASQSVGDFYLA
GASSRAT (SEQ
QHYNYPPSF
For the following mAbs:
(SEQ ID NO: 215)
ID NO: 210)
T (SEQ ID
L1.GDF/L3.NY/P6E01;
NO: 214)
L1.GDF/L3.NY/H3.AL;
L1.GDF/L3.NY/H3.AP;
and
L1.GDF/L3.NY/H3.AQ
L1.LGF/L3.PY
RASQSLGSFYLA
GASSRAT (SEQ
QHYPYPPSFT
For the following mAbs:
(SEQ ID NO: 212)
ID NO: 210)
(SEQ ID NO:
L1.LGF/L3.PY/H3.AP;
216)
and
L1.LGF/L3.PY/H3.AQ
L1.GDF/L3.KW
RASQSVGDFYLA
GASSRAT (SEQ
KHYGWPPS
For the following mAbs:
(SEQ ID NO: 215)
ID NO: 210)
FT (SEQ ID
L1.GDF/L3.KW/H3.AL;
NO: 213)
L1.GDF/L3.KW/H3.AP;
and L1.GDF/
L3.KW/H3.AQ
L1.GDF/L3.PY/H3.AQ
RASQSVGDFYLA
GASSRAT (SEQ
QHYPYPPSF
(SEQ ID NO: 215)
ID NO: 210)
T (SEQ ID
NO: 216)
L3.KW/P6E01
RASQSVSSSYLA
GASSRAT (SEQ
KHYGWPPS
(SEQ ID NO: 209)
ID NO: 210)
FT (SEQ ID
NO: 213)
L3.PY
RASQSVSSSYLA
GASSRAT (SEQ
QHYPYPPSFT
For the following mAbs:
(SEQ ID NO: 209)
ID NO: 210)
(SEQ ID NO:
L3.PY/P6E01;
216)
L3.PY/H2.QR;
L3.PY/H2.DY;
L3.PY/H2.YQ;
L3.PY/H2.LT;
L3.PY/H2.HA;
L3.PY/H2.QL;
L3.PY/H3.YA;
L3.PY/H3.AE;
L3.PY/H3.AQ;
L3.PY/H3.TAQ
L3.NY/P6E01
RASQSVSSSYLA
GASSRAT (SEQ
QHYNYPPSFT
(SEQ ID NO: 209)
ID NO: 210)
(SEQ ID NO:
214)
L3.PY/L1.PS
RASQSVSSSYPS
GASSRAT (SEQ
QHYPYPPSFT
For the following mAbs:
(SEQ ID NO: 217)
ID NO: 210)
(SEQ ID NO:
L3.PY/L1.PS/P6E01;
216)
P6DY;
L3.PY/L1.PS/H2.Q R;
L3.PY/L1.PS/H2.DY;
L3.PY/L1.PS/H2.YQ;
L3.PY/L1.PS/H2.LT;
L3.PY/L1.PS/H2.HA;
L3.PY/L1.PS/H2.QL;
L3.PY/L1.PS/H3.YA;
L3.PY/L1.PS/H3.AE;
L3.PY/L1.PS/H3.AQ;
L3.PY/L1.PS/H3.TAQ;
L3.PY/L1.AH
RASQSVSAHYLA
GASSRAT (SEQ
QHYPYPPSFT
For the following mAbs:
(SEQ ID NO: 218)
ID NO: 210)
(SEQ ID NO:
L3.PY/L1.AH/P6E01;
216)
L3.PY/L1.AH/H2.QR;
L3.PY/L1.AH/H2.DY;
L3.PY/L1.AH/H2.YQ;
L3.PY/L1.AH/H2.LT;
L3.PY/L1.AH/H2.HA;
L3.PY/L1.AH/H2.QL;
L3.PY/L1.AH/H3.YA;
L3.PY/L1.AH/H3.AE;
L3.PY/L1.AH/H3.AQ;
L3.PY/L1.AH/H3.TAQ
L3.PY/L1.FF
RASQSVSSFFLA
GASSRAT (SEQ
QHYPYPPSFT
For the following mAbs:
(SEQ ID NO: 219)
ID NO: 210)
(SEQ ID NO:
L3.PY/L1.FF/P6E01;
216)
L3.PY/L1.FF/H2.QR;
L3.PY/L1.FF/H2.DY;
L3.PY/L1.FF/H2.YQ;
L3.PY/L1.FF/H2.LT;
L3.PY/L1.FF/H2.HA;
L3.PY/L1.FF/H2.QL;
L3.PY/L1.FF/H3.YA;
L3.PY/L1.FF/H3.AE;
L3.PY/L1.FF/H3.AQ;
and
L3.PY/L1.FF/H3.TAQ
L3.PY/L1.PH
RASQSVSPHYLA
GASSRAT (SEQ
QHYPYPPSFT
For the following mAbs:
(SEQ ID NO: 219)
ID NO: 210)
(SEQ ID NO:
L3.PY/L1.PH/P6E01;
216)
L3.PY/L1.PH/H2.QR;
L3.PY/L1.PH/H2.HA;
L3.PY/L1.PH/H3.AE;
L3.PY/L1.PH/H3.AQ;
and
L3.PY/L1.PH/H3.TAQ
L3.PY/L3.KY
RASQSVSSSYLA
GASSRAT (SEQ
KYYPYPPSFT
For the following mAbs:
(SEQ ID NO: 209)
ID NO: 210)
(SEQ ID NO:
L3.PY/L3.KY/P6E01;
220)
L3.PY/L3.KY/H2.QR;
L3.PY/L3.KY/H2.DY;
L3.PY/L3.KY/H2.YQ;
L3.PY/L3.KY/H2.LT;
L3.PY/L3.KY/H2.HA;
L3.PY/L3.KY/H2.QL;
L3.PY/L3.KY/H3.YA;
and
L3.PY/L3.KY/H3.TAQ
L3.PY/L3.KF
RASQSVSSSYLA
GASSRAT (SEQ
KFYPYPPSF
For the following mAbs:
(SEQ ID NO: 209)
ID NO: 210)
T (SEQ ID
L3.PY/L3.KF/H2.DY;
NO: 220)
L3.PY/L3.KF/H2.YQ;
L3.PY/L3.KF/H2.LT;
L3.PY/L3.KF/H2.QL;
L3.PY/L3.KF/H3.YA;
L3.PY/L3.KF/H3.AE;
L3.PY/L3.KF/H3.AQ;
and
L3.PY/L3.KF/H3.TAQ
P5A2_VHVL (P5A)
RASQSVSSSYLA
DASIRAT
QQYGSWPL
(SEQ ID NO: 209)
(SEQ ID NO: 221)
T (SEQ ID
NO: 222)
A02_Rd4_0.6nM_C06
RASQSVSVIYLA
DASIRAT
QQYQRWPLT
(SEQ ID NO: 223)
(SEQ ID NO: 221)
(SEQ ID NO:
224)
A02_Rd4_0.6nM_C09;
RASQSVSSSYLA
DASIRAT
QQYQSWPLT
COMBO_Rd_0.6nM_C29;
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
and
225)
COMBO_Rd4_0.6nM_C21
A02_Rd4_6nM_C16
RASQSVSDIYLA
DASIRAT
QQYQTWPL
(P5AC16)
(SEQ ID NO: 226)
(SEQ ID NO: 221)
T (SEQ ID
NO: 227)
A02_Rd4_6nM_C03
RASQSVSNIYLA
DASIRAT
QQYQGWPL
(SEQ ID NO: 228)
(SEQ ID NO: 221)
T (SEQ ID
NO: 229)
A02_Rd4_6nM_C01
RASQSVSAYYLA
DASIRAT
QQYERWPLT
(SEQ ID NO: 230)
(SEQ ID NO: 221)
(SEQ ID NO:
231)
A02_Rd4_6nM_C26
RASQSVSSIYLA
DASIRAT
QQYQVWPLT
(SEQ ID NO: 232)
(SEQ ID NO: 221)
(SEQ ID NO:
233)
A02_Rd4_6nM_C25
RASQSVSSSYLA
DASIRAT
QQYLDWPLT
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
234)
A02_Rd4_6nM_C22
RASQSVSSSYLA
DASIRAT
QQYQVWPLT
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
233)
A02_Rd4_6nM_C19
RASQSVSVIYLA
DASIRAT
QQYLAWPLT
(SEQ ID NO: 223)
(SEQ ID NO: 221)
(SEQ ID NO:
236)
A02_Rd4_0.6nM_C03
RASQSVSSSYLA
DASIRAT
QQYFTWPLT
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
237)
A02_Rd4_6nM_C07
RASQSVSPYYLA
DASIRAT
QQYERWPLT
(SEQ ID NO: 238)
(SEQ ID NO: 221)
(SEQ ID NO:
231)
A02_Rd4_6nM_C23
RASQSVSVEYLA
DASIRAT
QQYARWPLT
(SEQ ID NO: 239)
(SEQ ID NO: 221)
(SEQ ID NO:
240)
A02_Rd4_0.6nM_C18
RASQSVSEIYLA
DASIRAT
QQYFGWPLT
(SEQ ID NO: 241)
(SEQ ID NO: 221)
(SEQ ID NO:
242)
A02_Rd4_6nM_C10
RASQSVEMSYLA
DASIRAT
QQYAHWPLT
(SEQ ID NO: 243)
(SEQ ID NO: 221)
(SEQ ID NO:
244)
A02_Rd4_6nM_C05
RASQSVSSSYLA
DASIRAT
QQYQRWPLT
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
224)
A02_Rd4_0.6nM_C10
RASQSVSAQYLA
DASIRAT
QQYQRWPLT
(SEQ ID NO: 245)
(SEQ ID NO: 221)
(SEQ ID NO:
224)
A02_Rd4_6nM_C04
RASQSVSAIYLA
DASIRAT
QQYQVWPLT
(SEQ ID NO: 235)
(SEQ ID NO: 221)
(SEQ ID NO:
233)
A02_Rd4_0.6nM_C26
GPSQSVSSSYLA
DASIRAT
QQYQSWPLT
(SEQ ID NO: 246)
(SEQ ID NO: 221)
(SEQ ID NO:
225)
A02_Rd4_0.6nM_C13
RASQSVSSSYWA
DASIRAT
QQYESWPLT
(SEQ ID NO: 247)
(SEQ ID NO: 221)
(SEQ ID NO:
248)
A02_Rd4_0.6nM_C01
RGGQSVSSSYLA
DASIRAT
QQYQSWPLT
(P5AC1)
(SEQ ID NO: 249)
(SEQ ID NO: 221)
(SEQ ID NO:
225)
A02_Rd4_6nM_C08
RASQSVSFIYLA
DASIRAT
QQYGSWPL
(SEQ ID NO: 250)
(SEQ ID NO: 221)
T (SEQ ID
NO: 222)
P5C1_VHVL (PC1)
RASQSVSSTYLA
DASSRAP
QQYSTSPLT
(SEQ ID NO: 251)
(SEQ ID NO: 252)
(SEQ ID NO:
253)
C01_Rd4_6nM_C24
RASQSVSPEYLA
DASSRAP
QQYSVWPLT
(SEQ ID NO: 254)
(SEQ ID NO: 252)
(SEQ ID NO:
255)
C01_Rd4_6nM_C26
RASQSVSAIYLA
DASSRAP
QQYSAWPLT
(SEQ ID NO: 235)
(SEQ ID NO: 252)
(SEQ ID NO:
256)
C01_Rd4_6nM_C10
RASQSVSSVYLA
DASSRAP
QQYSTWPLT
(SEQ ID NO: 257)
(SEQ ID NO: 252)
(SEQ ID NO:
258)
C01_Rd4_0.6nM_C27
RASQSVSSTYLA
DASSRAP
QQYSRWPLT
(SEQ ID NO: 251)
(SEQ ID NO: 252)
(SEQ ID NO:
259)
C01_Rd4_6nM_C20
RASQSVSPIYLA
DASSRAP
QQYSAFPLT
(SEQ ID NO: 260)
(SEQ ID NO: 252)
(SEQ ID NO:
261)
C01_Rd4_6nM_C12
WLSQSVSSTYLA
DASSRAP
QQYSEWPLT
(PC1C12)
(SEQ ID NO: 262)
(SEQ ID NO: 252)
(SEQ ID NO:
263)
C01_Rd4_0.6nM_C16
RASQSVSSTYLA
DASSRAP
QQYSSWPLT
(SEQ ID NO: 251)
(SEQ ID NO: 252)
(SEQ ID NO:
264)
C01_Rd4_0.6nM_C09
RASQSVSSIFLA
DASSRAP
QQYSAWPLT
(SEQ ID NO: 265)
(SEQ ID NO: 252)
(SEQ ID NO:
256)
C01_Rd4_6nM_C09
ACSQSVSSTYLA
DASSRAP
QQYSAWPLT
(SEQ ID NO: 266)
(SEQ ID NO: 252)
(SEQ ID NO:
256)
C01_Rd4_0.6nM_C03
RASCDVSSTYLA
DASSRAP
QQYMRSPLT
(SEQ ID NO: 267)
(SEQ ID NO: 252)
(SEQ ID NO:
268)
C01_Rd4_0.6nM_C06
RASEAVPSTYLA
DASSRAP
QQYSAFPLT
(SEQ ID NO: 269)
(SEQ ID NO: 252)
(SEQ ID NO:
261)
C01_Rd4_0.6nM_C04
CSSQSVSSTYLA
DASSRAP
QQYSAFPLT
(SEQ ID NO: 270)
(SEQ ID NO: 252)
(SEQ ID NO:
261)
COMBO_Rd4_0.6nM_C22
RASVRVSSTYLA
DASIRAT
QQYMKWPLT
(COM22)
(SEQ ID NO: 271)
(SEQ ID NO: 221)
(SEQ ID NO:
272)
COMBO_Rd4_6nM_C21
RASQSVSAAYLA
DASIRAT
QQYMCWPLT
(SEQ ID NO: 273)
(SEQ ID NO: 221)
(SEQ ID NO:
274)
COMBO_Rd4_6nM_C10
RASQSVSSSYWG
DASIRAT
QQYQCWPLT
(SEQ ID NO: 275)
(SEQ ID NO: 221)
(SEQ ID NO:
276)
COMBO_Rd4_0.6nM_C04
RASQSVSSTYLA
DASIRAT
QQYQSWPLT
(SEQ ID NO: 251)
(SEQ ID NO: 221)
(SEQ ID NO:
225)
COMBO_Rd4_6nM_C25
RASQSVSSPYLA
DASIRAT
QQYQSWPLT
(SEQ ID NO: 277)
(SEQ ID NO: 221)
(SEQ ID NO:
225)
COMBO_Rd4_6nM_C11
RASQSVSPIYLA
DASIRAT
QQYKAWPLT
(SEQ ID NO: 260)
(SEQ ID NO: 221)
(SEQ ID NO:
278)
COMBO_Rd4_0.6nM_C20
RASQSVSYLYLA
DASIRAT
QQYMEWPLT
(SEQ ID NO: 279)
(SEQ ID NO: 221)
(SEQ ID NO:
280)
COMBO_Rd4_6nM_C09
RASQSVSAQYLA
DASIRAT
QQYQAWPLT
(SEQ ID NO: 245)
(SEQ ID NO: 221)
(SEQ ID NO:
281)
COMBO_Rd4_6nM_C08
RASQSVSSSYLA
DASIRAT
QQYQKWPLT
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
282)
COMBO_Rd4_0.6nM_C19
RASQSVSAVYLA
DASIRAT
QQYRAWPLT
(SEQ ID NO: 283)
(SEQ ID NO: 221)
(SEQ ID NO:
284)
COMBO_Rd4_0.6nM_C02
RASIAVSSTYLA
DASIRAT
QQYMVWPLT
(SEQ ID NO: 285)
(SEQ ID NO: 221)
(SEQ ID NO:
286)
COMBO_Rd4_0.6nM_C23
RPRQSVSSSYLA
DASIRAT
QQYQDWPLT
(SEQ ID NO: 287)
(SEQ ID NO: 221)
(SEQ ID NO:
288)
COMBO_Rd4_0.6nM_C09
RASQSVSSTYLA
DASIRAT
QQYQEWPLT
(SEQ ID NO: 251)
(SEQ ID NO: 221)
(SEQ ID NO:
289)
COMBO_Rd4_6nM_C12
RASQSVSASYLA
DASIRAT
QQYMSWPLT
(SEQ ID NO: 290)
(SEQ ID NO: 221)
(SEQ ID NO:
291)
COMBO_Rd4_0.6nM_C30
RASQSVSYMYLA
DASIRAT
QQYKSWPLT
(SEQ ID NO: 292)
(SEQ ID NO: 221)
(SEQ ID NO:
293)
COMBO_Rd4_0.6nM_C14
RASQSVSAIYLA
DASIRAT
QQYYGWPLT
(SEQ ID NO: 235)
(SEQ ID NO: 221)
(SEQ ID NO:
294)
COMBO_Rd4_6nM_C07
RASQPISSSYLA
DASIRAT
QQYQGWPLT
(SEQ ID NO: 295)
(SEQ ID NO: 221)
(SEQ ID NO:
229)
COMBO_Rd4_6nM_C02
RASQSVSSSYLA
DASIRAT
QQYEFWPLT
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
296)
COMBO_Rd4_0.6nM_C05
RASQSVSSTYLA
DASIRAT
QQYMSWPLT
(SEQ ID NO: 251)
(SEQ ID NO: 221)
(SEQ ID NO:
291)
COMBO_Rd4_0.6nM_C17
RASQGISSTYLA
DASIRAT
QQYAYWPLT
(SEQ ID NO: 297)
(SEQ ID NO: 221)
(SEQ ID NO:
298)
COMBO_Rd4_6nM_C22
RASQSVSSSYLA
DASIRAT
QQYQGWPLT
(SEQ ID NO: 209)
(SEQ ID NO: 221)
(SEQ ID NO:
229)
COMBO_Rd4_0.6nM_C11
RASQSVSVRYLA
DASIRAT
QQYGSWPIT
(SEQ ID NO: 299)
(SEQ ID NO: 221)
(SEQ ID NO:
300)
Light chain consensus
X1X2X3X4X5X6X7X8X9
X1ASX2RAX3,
X1X2YX3X4PP
X10X11X12, wherein X1
wherein X1 is G or
SFT, wherein
is R, G, W, A, or C; X2
D; X2 is S or I;
X1 is Q or K;
is A, P, G, L, C, or S;
and X3 is T or P
X2 is H or Y;
X3 is S, G, or R; X4 is
(SEQ ID NO: 310)
X3 is G, N, or
Q, C, E, V, or I; X5 is
P; and X4 is
S, P, G, A, R, or D; X6
S, W, or Y
is V, G, I, or L; X7 is S,
(SEQ ID NO:
E, D, P, or G; X8 is S,
311)
P, F, A, M, E, V, N, D,
QQYX1X2X3P
or Y; X9 is I, T, V, E, F
X4T, wherein
S, A, M, Q, Y, H, or R;
X1 is G, Q, E,
X10 is Y or F; X11 is L,
L, F, A, S, M,
W, or P; and X12 is A,
K, R, or Y; X2
S, or G (SEQ ID NO:
is S, R, T, G,
309)
V, F, Y, D, A,
H, V, E, K, or
C; X3 is W, F,
or S; and X4
is L or I (SEQ
ID NO: 312)
P4G4
RASQSVSSSYLA
GASSRAY (SEQ
QHYGSPPLF
(SEQ ID NO: 209)
ID NO: 392)
T (SEQ ID
NO: 393)
P1A11
RASQNVSSSYLA
GASYRAT (SEQ
QHYGSPPSF
(SEQ ID NO: 379)
ID NO: 395)
T (SEQ ID
NO: 211)
P6AP
RASQLGSFYLA
GASSRAT (SEQ
QHYNYPPSF
(SEQ ID NO: 377)
ID NO: 210)
T (SEQ ID
NO: 214)
The invention encompasses modifications to the CARs and polypeptides of the invention variants shown in Table 1, including functionally equivalent CARs having modifications which do not significantly affect their properties and variants which have enhanced or decreased activity and/or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with the desired binding affinity to BCMA. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or which mature (enhance) the affinity of the polypeptide for its ligand, or use of chemical analogs.
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 an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.
Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 2.1 under the heading of “conservative substitutions.” If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table 2.1, or as further described below in reference to amino acid classes, may be introduced and the products screened.
TABLE 2.1
Amino Acid Substitutions
Original Residue
(naturally
occurring amino
Conservative
acid)
Substitutions
Exemplary Substitutions
Ala (A)
Val
Val; Leu; Ile
Arg (R)
Lys
Lys; Gln; Asn
Asn (N)
Gln
Gln; His; Asp, Lys; Arg
Asp (D)
Glu
Glu; Asn
Cys (C)
Ser
Ser; Ala
Gln (Q)
Asn
Asn; Glu
Glu (E)
Asp
Asp; Gln
Gly (G)
Ala
Ala
His (H)
Arg
Asn; Gln; Lys; Arg
Ile (I)
Leu
Leu; Val; Met; Ala; Phe;
Norleucine
Leu (L)
Ile
Norleucine; Ile; Val; Met;
Ala; Phe
Lys (K)
Arg
Arg; Gln; Asn
Met (M)
Leu
Leu; Phe; Ile
Phe (F)
Tyr
Leu; Val; Ile; Ala; Tyr
Pro (P)
Ala
Ala
Ser (S)
Thr
Thr
Thr (T)
Ser
Ser
Trp (W)
Tyr
Tyr; Phe
Tyr (Y)
Phe
Trp; Phe; Thr; Ser
Val (V)
Leu
Ile; Leu; Met; Phe; Ala;
Norleucine
In some embodiments, the invention provides a CAR comprising an extracellular ligand-binding domain that binds to BCMA and competes for binding to BCMA with a CAR described herein, including P6E01/P6E01, P6E01/H3.AQ, L1.LGF/L3.KW/P6E01; L1.LGF/L3.NY/P6E01, L1.GDF/L3.NY/P6E01, L1.LGF/L3.KW/H3.AL, L1.LGF/L3.KW/H3.AP, L1.LGF/L3.KW/H3.AQ, L1.LGF/L3.PY/H3.AP, L1.LGF/L3.PY/H3.AQ, L1.LGF/L3.NY/H3.AL, L1.LGF/L3.NY/H3.AP, L1.LGF/L3.NY/H3.AQ, L1.GDF/L3.KW/H3.AL, L1.GDF/L3.KW/H3.AP, L1.GDF/L3.KW/H3.AQ, L1.GDF/L3.PY/H3.AQ, L1.GDF/L3.NY/H3.AL, L1.GDF/L3.NY/H3.AP, L1.GDF/L3.NY/H3.AQ, L3.KW/P6E01, L3.PY/P6E01, L3.NY/P6E01, L3.PY/L1.PS/P6E01, L3.PY/L1.AH/P6E01, L3.PY/L1.FF/P6E01, L3.PY/L1.PH/P6E01, L3.PY/L3.KY/P6E01, L3.PY/L3.KF/P6E01, L3.PY/H2.QR, L3.PY/H2.DY, L3.PY/H2.YQ, L3.PY/H2.LT, L3.PY/H2.HA, L3.PY/H2.QL, L3.PY/H3.YA, L3.PY/H3.AE, L3.PY/H3.AQ, L3.PY/H3.TAQ, L3.PY/P6E01, L3.PY/L1.PS/H2.QR, L3.PY/L1.PS/H2.DY, L3.PY/L1.PS/H2.YQ, L3.PY/L1.PS/H2.LT, L3.PY/L1.PS/H2.HA, L3.PY/L1.PS/H2.QL, L3.PY/L1.PS/H3.YA, L3.PY/L1.PS/H3.AE, L3.PY/L1.PS/H3.AQ, L3.PY/L1.PS/H3.TAQ, L3.PY/L1.AH/H2.QR, L3.PY/L1.AH/H2.DY, L3.PY/L1.AH/H2.YQ, L3.PY/L1.AH/H2.LT, L3.PY/L1.AH/H2.HA, L3.PY/L1.AH/H2.QL, L3.PY/L1.AH/H3.YA, L3.PY/L1.AH/H3.AE, L3.PY/L1.AH/H3.AQ, L3.PY/L1.AH/H3.TAQ, L3.PY/L1.FF/H2.QR, L3.PY/L1.FF/H2.DY, L3.PY/L1.FF/H2.YQ, L3.PY/L1.FF/H2.LT, L3.PY/L1.FF/H2.HA, L3.PY/L1.FF/H2.QL, L3.PY/L1.FF/H3.YA, L3.PY/L1.FF/H3.AE, L3.PY/L1.FF/H3.AQ, L3.PY/L1.FF/H3.TAQ, L3.PY/L1.PH/H2.QR, L3.PY/L1.PH/H2.HA, L3.PY/L1.PH/H3.AE, L3.PY/L1.PH/H3.AQ, L3.PY/L1.PH/H3.TAQ, L3.PY/L3.KY/H2.QR, L3.PY/L3.KY/H2.DY, L3.PY/L3.KY/H2.YQ L3.PY/L3.KY/H2.LT, L3.PY/L3.KY/H2.HA, L3.PY/L3.KY/H2.QL, L3.PY/L3.KY/H3.YA L3.PY/L3.KY/H3.TAQ, L3.PY/L3.KF/H2.DY, L3.PY/L3.KF/H2.YQ, L3.PY/L3.KF/H2.LT L3.PY/L3.KF/H2.QL, L3.PY/L3.KF/H3.YA, L3.PY/L3.KF/H3.AE, L3.PY/L3.KF/H3.AQ L3.PY/L3.KF/H3.TAQ, P5A2_VHVL, A02_Rd4_0.6nM_C06, A02_Rd4_0.6nM_C09 A02_Rd4_6nM_C16, A02_Rd4_6nM_C03, A02_Rd4_6nM_C01, A02_Rd4_6nM_C26 A02_Rd4_6nM_C25, A02_Rd4_6nM_C22, A02_Rd4_6nM_C19, A02_Rd4_0.6nM_C03 A02_Rd4_6nM_C07, A02_Rd4_6nM_C23, A02_Rd4_0.6nM_C18, A02_Rd4_6nM_C10 A02_Rd4_6nM_C05, A02_Rd4_0.6nM_C10, A02_Rd4_6nM_C04, A02_Rd4_0.6nM_C26 A02_Rd4_0.6nM_C13, A02_Rd4_0.6nM_C01, A02_Rd4_6nM_C08, P5C1_VHVL, C01_Rd4_6nM_C24, C01_Rd4_6nM_C26, C01_Rd4_6nM_C10, C01_Rd4_0.6nM_C27 C01_Rd4_6nM_C20, C01_Rd4_6nM_C12, C01_Rd4_0.6nM_C16, C01_Rd4_0.6nM_C09 C01_Rd4_6nM_C09, C01_Rd4_0.6nM_C03, C01_Rd4_0.6nM_C06, C01_Rd4_6nM_C04 COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10, COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_C25, COMBO_Rd4_0.6nM_C21, COMBO_Rd4_6nM_C11, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09, COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02, COMBO_Rd4_0.6nM_C23, COMBO_Rd4_0.6nM_C29, COMBO_Rd4_0.6nM_C09, COMBO_Rd4_6nM_C12, COMBO_Rd4_0.6nM_C30, COMBO_Rd4_0.6nM_C14, COMBO_Rd4_6nM_C07, COMBO_Rd4_6nM_C02, COMBO_Rd4_0.6nM_C05, COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, COMBO_Rd4_0.6nM_C11, or COMBO_Rd4_0.6nM_C29.
In some embodiments, the invention provides a CAR, which specifically binds to BCMA, wherein the CAR comprises a VH region comprising a sequence shown in SEQ ID NO: 33; and/or a VL region comprising a sequence shown in SEQ ID NO: 34. In some embodiments, the invention provides a CAR, which specifically binds to BCMA, wherein the CAR comprises a VH region comprising a sequence shown in SEQ ID NO: 33, 72, 39, 76, 83, 92, 25, or 8; and/or a VL region comprising a sequence shown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, or 80. In some embodiments, the invention also provides CARs comprising CDR portions of antibodies to BCMA antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that imbue specificity to the antibody for an antigen. In general, CDR contact regions include the residue positions in the CDRs and Vernier zones which are constrained in order to maintain proper loop structure for the antibody to bind a specific antigen. See, e.g., Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of CDR contact regions is well within the skill of the art. The binding affinity (KD) of the BCMA specific CAR as described herein to BCMA (such as human BCMA (e.g., (SEQ ID NO: 354) can be about 0.002 to about 6500 nM. In some embodiments, the binding affinity is about any of 6500 nm, 6000 nm, 5986 nm, 5567 nm, 5500 nm, 4500 nm, 4000 nm, 3500 nm, 3000 nm, 2500 nm, 2134 nm, 2000 nm, 1500 nm, 1000 nm, 750 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nM, 193 nM, 100 nM, 90 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 19 nm, 18 nm, 17 nm, 16 nm, 15 nM, 10 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM, or 0.002 nM. In some embodiments, the binding affinity is less than about any of 6500 nm, 6000 nm, 5500 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, 1000 nm, 900 nm, 800 nm, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.
The intracellular signaling domain of a CAR according to the invention is responsible for intracellular signaling following the binding of extracellular ligand-binding domain to the target resulting in the activation of the immune cell and immune response. The intracellular signaling domain has the ability to activate of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines.
In some embodiments, an intracellular signaling domain for use in a CAR can be the cytoplasmic sequences of, for example without limitation, the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Intracellular signaling domains comprise two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic signaling sequences can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAM used in the invention can include as non limiting examples those derived from TCRζ, FcRγ, FcRβ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, the intracellular signaling domain of the CAR can comprise the CD3ζ signaling domain which has amino acid sequence with at least about 70%, preferably at least 80%, more preferably at least 90%, 95% 97%, or 99% sequence identity with an amino acid sequence shown in SEQ. ID NO: 324. In some embodiments the intracellular signaling domain of the CAR of the invention comprises a domain of a co-stimulatory molecule.
In some embodiments, the intracellular signaling domain of a CAR of the invention comprises a part of co-stimulatory molecule selected from the group consisting of fragment of 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of the CAR of the invention comprises amino acid sequence which comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with an amino acid sequence shown in SEQ. ID NO: 323 and SEQ. ID NO: 327.
CARs are expressed on the surface membrane of the cell. Thus, the CAR can comprise a transmembrane domain. Suitable transmembrane domains for a CAR disclosed herein have the ability to (a) be expressed at the surface of a cell, preferably an immune cell such as, for example without limitation, lymphocyte cells or Natural killer (NK) cells, and (b) interact with the ligand-binding domain and intracellular signaling domain for directing cellular response of immune cell against a predefined target cell. The transmembrane domain can be derived either from a natural or from a synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a subunit of the T cell receptor such as α, β, γ or δ, polypeptide constituting CD3 complex, IL-2 receptor p55 (a chain), p75 (β chain) or γ chain, subunit chain of Fc receptors, in particular Fcγ receptor III or CD proteins. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments said transmembrane domain is derived from the human CD8α chain (e.g., NP_001139345.1). The transmembrane domain can further comprise a stalk domain between the extracellular ligand-binding domain and said transmembrane domain. A stalk domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Stalk region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of an antibody constant region. Alternatively the stalk domain may be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence. In some embodiments said stalk domain is a part of human CD8α chain (e.g., NP_001139345.1). In another particular embodiment, said transmembrane and hinge domains comprise a part of human CD8α chain, preferably which comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95% 97%, or 99% sequence identity with amino acid sequence selected from the group consisting of SEQ ID NO: 318. In some embodiments, CARs disclosed herein can comprise an extracellular ligand-binding domain that specifically binds BCMA, CD8α human hinge and transmembrane domains, the CD3ζ signaling domain, and 4-1BB signaling domain.
Table 3 provides exemplary sequences of domains which can be used in the CARs disclosed herein.
TABLE 3
Exemplary sequences of CAR Components
SEQ ID
Domain
Amino Acid Sequence
NO:
CD8α signal peptide
MALPVTALLLPLALLLHAARP
318
FcγRIIIα hinge
GLAVSTISSFFPPGYQ
319
CD8α hinge
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
320
IgG1 hinge
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDV
321
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
CD8α
IYIWAPLAGTCGVLLLSLVITLYC
322
transmembrane
(TM) domain
41BB intracellular
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
323
signaling domain
(ISD)
CD3ζ intracellular
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG
324
signaling domain
KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST
(ISD)
ATKDTYDALHMQALPPR
FcεRI α-TM-IC (FcεRI
FFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRTRKGFRLLNPHPKPNPKNN
325
α chain
transmembrane and
intracellular domain)
FcεRIβ-ΔITAM (FcεRI
MDTESNRRANLALPQEPSSVPAFEVLEISPQEVSSGRLLKSASSPPLHTWL
326
β chain without
TVLKKEQEFLGVTQILTAMICLCFGTVVCSVLDISHIEGDIFSSFKAGYPFW
ITAM)
GAIFFSISGMLSIISERRNATYLVRGSLGANTASSIAGGTGITILIINLKKSLAY
IHIHSCQKFFETKCFMASFSTEIVVMMLFLTILGLGSAVSLTICGAGEELKG
NKVPE
41BB-IC (41BB co-
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
327
stimulatory domain)
CD28-IC (CD28 co-
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
328
stimulatory domain)
FcεRIγ-SP (signal
MIPAVVLLLLLLVEQAAA
329
peptide)
FcεRI γ-ΔITAM (FcεRI
LGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKS
330
γ chain without
ITAM)
GSG-P2A (GSG-P2A
GSGATNFSLLKQAGDVEENPGP
331
ribosomal skip
polypeptide)
GSG-T2A (GSG-T2A
GSGEGRGSLLTCGDVEENPGP
332
ribosomal skip
polypeptide)
Downregulation or mutation of target antigens is commonly observed in cancer cells, creating antigen-loss escape variants. Thus, to offset tumor escape and render immune cell more specific to target, the BCMA specific CAR can comprise one or more additional extracellular ligand-binding domains, to simultaneously bind different elements in target thereby augmenting immune cell activation and function. In one embodiment, the extracellular ligand-binding domains can be placed in tandem on the same transmembrane polypeptide, and optionally can be separated by a linker. In some embodiments, said different extracellular ligand-binding domains can be placed on different transmembrane polypeptides composing the CAR. In some embodiments, the invention relates to a population of CARs, each CAR comprising a different extracellular ligand-binding domain. In a particular, the invention relates to a method of engineering immune cells comprising providing an immune cell and expressing at the surface of the cell a population of CARs, each CAR comprising different extracellular ligand-binding domains. In another particular embodiment, the invention relates to a method of engineering an immune cell comprising providing an immune cell and introducing into the cell polynucleotides encoding polypeptides composing a population of CAR each one comprising different extracellular ligand-binding domains. By population of CARs, it is meant at least two, three, four, five, six or more CARs each one comprising different extracellular ligand-binding domains. The different extracellular ligand-binding domains according to the invention can preferably simultaneously bind different elements in target thereby augmenting immune cell activation and function. The invention also relates to an isolated immune cell which comprises a population of CARs each one comprising different extracellular ligand-binding domains.
In another aspect, the invention provides polynucleotides encoding any of the CARs and polypeptides described herein. Polynucleotides can be made and expressed by procedures known in the art.
In another aspect, the invention provides compositions (such as a pharmaceutical compositions) comprising any of the cells of the invention. In some embodiments, the composition comprises a cell comprising a polynucleotide encoding any of the CARs described herein. In still other embodiments, the composition comprises either or both of the polynucleotides shown in SEQ ID NO: 367 and SEQ ID NO:368 below:
P5A Heavy Chain Variable Region
(SEQ ID NO: 367)
GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTC
TCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCA
TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCGCC
ATCAGCGATAGCGGCGGCAGCACCTACTACGCCGATAGCGTGAAGGGCCG
GTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGA
ACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCANATACTGG
CCCATGGACATCTGGGGCCAGGGAACCTTGGTCACCGTCTCCTCA
P5A Light Chain Variable Region
(SEQ ID NO: 368)
GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCAGGCGA
AAGAGCCACCCTGTCCTGCAAAGCCAGCCAGAGCGTGTCCAGCAGCTACC
TGGCCTGGTATCAGCAAAAGCCCGGCCAGGCTCCCCGGCTGCTGATGTAC
GATGCCAGCATCAGAGCCACCGGCATCCCCGACAGATTTTCCGGCTCTGG
CAGCGGCACCGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACT
TCGCCGTGTACTACTGCCAGCAGTACGGCAGCTGGCCCCTGACATTTGGC
CAGGGCACAAAGGTGGAGATCAAA
In other embodiments, the composition comprises either or both of the polynucleotides shown in SEQ ID NO: 369 and SEQ ID NO: 370 below:
P5AC1 Heavy Chain Variable Region
(SEQ ID NO: 369)
GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTC
TCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCA
TGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCT
ATTCTGTCGTCTGGTGGTTCTACTTACTATGCCGATTCTGTTAAGGGTAG
ATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGA
ACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGG
CCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA
P5AC1 Light Chain Variable Region
(SEQ ID NO: 370)
GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGA
AAGAGCTACTTTGTCTTGTAGAGGGGGTCAATCCGTTTCCTCTTCTTATT
TGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTAC
GATGCTTCTATTAGAGCCACCGGTATTCCAGATAGATTTTCTGGTTCTGG
TTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATT
TCGCTGTTTACTACTGTCAACAATATCAGTCTTGGCCATTGACTTTTGGT
CAAGGTACAAAGGTTGAAATCAAA
In other embodiments, the composition comprises either or both of the polynucleotides shown in SEQ ID NO: 371 and SEQ ID NO: 372 below:
PC1 Heavy Chain Variable Region
(SEQ ID NO: 371)
GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTC
TCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACCCTA
TGAGCTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCGCC
ATCGGAGGCTCTGGCGGCAGCACCTACTACGCCGATAGCGTGAAGGGCCG
GTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAAATGA
ACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATACTGG
CCCATGGACAGCTGGGGCCAGGGAACTTTGGTCACCGTCTCCTCA
PC1 Light Chain Variable Region
(SEQ ID NO: 372)
GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCAGGCGA
AAGAGCCACCCTGTCCTGCAAAGCCAGCCAGAGCGTGTCCAGCACATACC
TGGCCTGGTATCAGCAAAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTAC
GATGCCTCTTCTAGAGCCCCTGGCATCCCCGACAGATTCAGCGGCTCTGG
CAGCGGCACCGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACT
TCGCCGTGTACTACTGCCAGCAGTACAGCACCAGCCCCCTGACCTTTGGC
CAGGGCACAAAGGTGGAGATCAAA.
In other embodiments, the composition comprises either or both of the polynucleotides shown in SEQ ID NO: 373 and SEQ ID NO: 374 below:
PC1C12 Heavy Chain Variable Region
(SEQ ID NO: 373)
GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTC
TCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACCCTA
TGAGCTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCT
ATTGGTGGTTCAGGTGGTTGGAGTTATTATGCCGATTCTGTTAAGGGTAG
ATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGA
ACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGG
CCAATGGATTCTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA
PC1C12 Light Chain Variable Region
(SEQ ID NO: 374)
GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGA
AAGAGCTACTTTGTCTTGTTGGTTGTCTCAATCTGTTTCCTCTACTTACT
TGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATCTAC
GATGCTTCTTCTAGAGCACCAGGTATTCCAGATAGATTTTCTGGTTCTGG
TTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATT
TCGCTGTTTACTACTGCCAACAATACTCTGAGTGGCCATTGACTTTTGGT
CAAGGTACAAAGGTTGAAATCAAA.
In other embodiments, the composition comprises either or both of the polynucleotides shown in SEQ ID NO: 375 and SEQ ID NO: 376 below:
COM22 Heavy Chain Variable Region
(SEQ ID NO: 375)
GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTC
TCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCA
TGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCT
ATTTCTGATTCTGGTGGTTCTAGGTGGTATGCCGATTCTGTTAAGGGTAG
ATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGA
ACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTACGCGGTACTGG
CCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA
COM22 Light Chain Variable Region
(SEQ ID NO: 376)
GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGA
AAGAGCTACTTTGTCTTGTTGGTTGTCTCAATCTGTTTCCTCTACTTACT
TGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATCTAC
GATGCTTCTTCTAGAGCACCAGGTATTCCAGATAGATTTTCTGGTTCTGG
TTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATT
TCGCTGTTTACTACTGCCAACAATACTCTGAGTGGCCATTGACTTTTGGT
CAAGGTACAAAGGTTGAAATCAAA.
Expression vectors, and administration of polynucleotide compositions are further described herein.
In another aspect, the invention provides a method of making any of the polynucleotides described herein.
Polynucleotides complementary to any such sequences are also encompassed by the invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes an antibody or a portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished, relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 90% identity, and most preferably, at least about 95% identity to a polynucleotide sequence that encodes a native antibody or a portion thereof.
Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O., 1978, A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.
Preferably, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).
Suitable “moderately stringent conditions” include prewashing in a solution of 5× SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.
As used herein, “highly stringent conditions” or “high stringency conditions” are those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
The polynucleotides of this invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.
For preparing polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known within the art. See, e.g., Sambrook et al., 1989.
Alternatively, PCR allows reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.
RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those of skill in the art, as set forth in Sambrook et al., 1989, supra, for example.
Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.
Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the invention. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.
A polynucleotide encoding a BCMA specific CAR disclosed herein may exist in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for transfection of a mammalian host cell). In some embodiments, a polynucleotide or vector can include a nucleic acid sequence encoding ribosomal skip sequences such as, for example without limitation, a sequence encoding a 2A peptide. 2A peptides, which were identified in the Aphthovirus subgroup of picornaviruses, causes a ribosomal “skip” from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). By “codon” is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an imRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.
To direct transmembrane polypeptides into the secretory pathway of a host cell, in some embodiments, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in a polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5′ to the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In some embodiments the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 318 or 329. Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. In some embodiments, nucleic acid sequences of the invention are codon-optimized for expression in mammalian cells, preferably for expression in human cells. Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the amino acids as the codons that are being exchanged.
In some embodiments, a polynucleotide according to the invention comprises the nucleic acid sequence selected from the group consisting of: SEQ. ID NO: 1397. The invention relates to polynucleotides comprising a nucleic acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with nucleic acid sequence selected from the group consisting of SEQ ID NO: 1397.
Methods of Engineering an Immune Cell
Methods of preparing immune cells for use in immunotherapy are provided herein. In some embodiments, the methods comprise introducing a CAR according to the invention into immune cells, and expanding the cells. In some embodiments, the invention relates to a method of engineering an immune cell comprising: providing a cell and expressing at the surface of the cell at least one CAR as described above. Methods for engineering immune cells are described in, for example, PCT Patent Application Publication Nos. WO/2014/039523, WO/2014/184741, WO/2014/191128, WO/2014/184744, and WO/2014/184143, each of which is incorporated herein by reference in its entirety. In some embodiments, the method comprises: transfecting the cell with at least one polynucleotide encoding CAR as described above, and expressing the polynucleotides in the cell.
In some embodiments, the polynucleotides are present in lentiviral vectors for stable expression in the cells.
In some embodiments, the method can further comprise a step of genetically modifying a cell by inactivating at least one gene expressing, for example without limitation, a component of the TCR, a target for an immunosuppressive agent, an HLA gene, and/or an immune checkpoint protein such as, for example, PDCD1 or CTLA-4. By inactivating a gene it is intended that the gene of interest is not expressed in a functional protein form. In some embodiments, the gene to be inactivated is selected from the group consisting of, for example without limitation, TCRα, TCRβ, CD52, GR, PD-1, and CTLA-4. In some embodiments the method comprises inactivating one or more genes by introducing into the cells a rare-cutting endonuclease able to selectively inactivate a gene by selective DNA cleavage. In some embodiments the rare-cutting endonuclease can be, for example, a transcription activator-like effector nuclease (TALE-nuclease) or Cas9 endonuclease.
In some embodiments, an additional catalytic domain is used with a rare-cutting endonuclease to enhance its capacity to inactivate targeted genes. For example, an additional catalytic domain can be a DNA end-processing enzyme. Non-limiting examples of DNA end-processing enzymes include 5-3′ exonucleases, 3-5′ exonucleases, 5-3′ alkaline exonucleases, 5′ flap endonucleases, helicases, phosphatase, hydrolases and template-independent DNA polymerases. Non-limiting examples of such catalytic domain comprise of a protein domain or catalytically active derivate of the protein domain selected from the group consisting of hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase) Human DNA2, Yeast DNA2 (DNA2_YEAST). In some embodiments, an additional catalytic domain can have a 3′-5′-exonuclease activity, and In some embodiments, said additional catalytic domain is TREX, more preferably TREX2 catalytic domain (WO2012/058458). In some embodiments, said catalytic domain is encoded by a single chain TREX polypeptide. The additional catalytic domain may be fused to a nuclease fusion protein or chimeric protein. In some embodiments, the additional catalytic domain is fused using, for example, a peptide linker.
In some embodiments, the method further comprises a step of introducing into cells an exogeneous nucleic acid comprising at least a sequence homologous to a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target nucleic acid sequence and the exogeneous nucleic acid. In some embodiments, said exogenous nucleic acid comprises first and second portions which are homologous to region 5′ and 3′ of the target nucleic acid sequence, respectively. The exogenous nucleic acid may also comprise a third portion positioned between the first and the second portion which comprises no homology with the regions 5′ and 3′ of the target nucleic acid sequence. Following cleavage of the target nucleic acid sequence, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. In some embodiments, homologous sequences of at least about 50 bp, greater than about 100 bp, or greater than about 200 bp can be used within the donor matrix. The exogenous nucleic acid can be, for example without limitation, from about 200 bp to about 6000 bp, more preferably from about 1000 bp to about 2000 bp. Shared nucleic acid homologies are located in regions flanking upstream and downstream the site of the break, and the nucleic acid sequence to be introduced is located between the two arms.
In some embodiments, a nucleic acid successively comprises a first region of homology to sequences upstream of said cleavage; a sequence to inactivate a targeted gene selected from the group consisting of TCRα, TCRβ3, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (DCK), and an immune checkpoint protein such as for example programmed death-1 (PD-1); and a second region of homology to sequences downstream of the cleavage. The polynucleotide introduction step can be simultaneous, before or after the introduction or expression of the rare-cutting endonuclease. Depending on the location of the target nucleic acid sequence wherein break event has occurred, such exogenous nucleic acid can be used to knock-out a gene, e.g. when exogenous nucleic acid is located within the open reading frame of the gene, or to introduce new sequences or genes of interest. Sequence insertions by using such exogenous nucleic acid can be used to modify a targeted existing gene, by correction or replacement of the gene (allele swap as a non-limiting example), or to up- or down-regulate the expression of the targeted gene (promoter swap as non-limiting example), the targeted gene correction or replacement. In some embodiments, inactivation of a genes selected from the group consisting of TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint proteins, can be done at a precise genomic location targeted by a specific TALE-nuclease, wherein said specific TALE-nuclease catalyzes a cleavage and wherein the exogenous nucleic acid successively comprising at least a region of homology and a sequence to inactivate one targeted gene selected from the group consisting of TCRα, TCRβ, CD52, GR, DCK, immune checkpoint proteins which is integrated by homologous recombination. In some embodiments, several genes can be, successively or at the same time, inactivated by using several TALE-nucleases respectively and specifically targeting one defined gene and several specific polynucleotides for specific gene inactivation.
In some embodiments, the method comprises inactivation of one or more additional genes selected from the group consisting of TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint proteins. In some embodiments, inactivation of a gene can be accomplished by introducing into the cells at least one rare-cutting endonuclease such that the rare-cutting endonuclease specifically catalyzes cleavage in a targeted sequence of the cell genome; and optionally, introducing into the cells an exogenous nucleic acid successively comprising a first region of homology to sequences upstream of the cleavage, a sequence to be inserted in the genome of the cell, and a second region of homology to sequences downstream of the cleavage; wherein the introduced exogenous nucleic acid inactivates a gene and integrates at least one exogenous polynucleotide sequence encoding at least one recombinant protein of interest. In some embodiments, the exogenous polynucleotide sequence is integrated within a gene encoding a protein selected from the group consisting of TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint protein.
In another aspect, a step of genetically modifying cells can comprise: modifying T cells by inactivating at least one gene expressing a target for an immunosuppressive agent, and; expanding the cells, optionally in presence of the immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. An immunosuppressive agent can diminish the extent and/or voracity of an immune response. Non-limiting examples of immunosuppressive agents include calcineurin inhibitors, targets of rapamycin, interleukin-2 α-chain blockers, inhibitors of inosine monophosphate dehydrogenase, inhibitors of dihydrofolic acid reductase, corticosteroids, and immunosuppressive antimetabolites. Some cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act through activation of T cells or by inhibiting the activation of helper cells. The methods according to the invention allow conferring immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in T cells. As non-limiting examples, targets for immunosuppressive agent can be a receptor for an immunosuppressive agent such as for example without limitation CD52, glucocorticoid receptor (GR), FKBP family gene members, and cyclophilin family gene members.
In some embodiments, the genetic modification of the method involves expression, in provided cells to engineer, of one rare-cutting endonuclease such that the rare-cutting endonuclease specifically catalyzes cleavage in one targeted gene, thereby inactivating the targeted gene. In some embodiments, a method of engineering cells comprises at least one of the following steps: providing a T cell, such as from a cell culture or from a blood sample; selecting a gene in the T cell expressing a target for an immunosuppressive agent; introducing into the T cell a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break the gene encoding a target for the immunosuppressive agent, and expanding the cells, optionally in presence of the immunosuppressive agent.
In some embodiments, the method comprises: providing a T cell, such as from a cell culture or from a blood sample; selecting a gene in the T cell wherein the gene expresses a target for an immunosuppressive agent; transfecting the T cell with nucleic acid encoding a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break the gene encoding a target for the immunosuppressive agent, and expressing the rare-cutting endonucleases into the T cells; and expanding the cells, optionally in presence of the immunosuppressive agent.
In some embodiments, the rare-cutting endonuclease specifically targets CD52 or GR. In some embodiments, the gene selected for inactivation encodes CD52, and the immunosuppressive treatment comprises a humanized antibody targeting CD52 antigen. In some embodiments, the gene selected for inactivation encodes GR, and the immunosuppressive treatment comprises a corticosteroid such as dexamethasone. In some embodiments, the gene selected for inactivation is a FKBP family gene member or a variant thereof and the immunosuppressive treatment comprises FK506, also known as Tacrolimus or fujimycin. In some embodiments, the FKBP family gene member is FKBP12 or a variant thereof. In some embodiments, gene selected for inactivation is a cyclophilin family gene member or a variant thereof and the immunosuppressive treatment comprises cyclosporine.
In some embodiments, the rare-cutting endonuclease can be, for example, a meganuclease, a zinc finger nuclease, or a TALE-nuclease (TALEN). In some embodiments, the rare-cutting endonuclease is a TALE-nuclease.
Also provided herein are methods of engineering T cells, suitable for immunotherapy, wherein the methods comprise: genetically modifying T cells by inactivating at least immune checkpoint protein. In some embodiments the immune checkpoint protein is, for example, PD-1 and/or CTLA-4.In some embodiments, methods of genetically modifying a cell comprises: modifying T cells by inactivating at least one immune checkpoint protein; and expanding the cells. Immune checkpoint proteins include, but are not limited to Programmed Death 1 (PD-1, also known as PDCD1 or CD279, accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, GenBank accession number AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_181780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as VSTM3, accession number: NM_173799), B7H5 (also known as C10orf54, homolog of mouse vista gene, accession number: NM_022153.1), LAIR1 (also known as CD305, GenBank accession number: CR542051.1), SIGLEC10 (GeneBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM_001166664.1), which directly inhibit immune cells. For example, CTLA-4 is a cell-surface protein expressed on certain CD4 and CD8 T cells; when engaged by its ligands (B7-1 and B7-2) on antigen presenting cells, T cell activation and effector function are inhibited.
In some embodiments, said method to engineer cells comprises at least one of the following steps: providing a T cell, such as from a cell culture or from a blood sample; introducing into the T cell a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break one gene encoding a immune checkpoint protein; and expanding the cells. In some embodiments, the method comprises: providing a T cell, such as from a cell culture or from a blood sample; transfecting said T cell with nucleic acid encoding a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break a gene encoding a immune checkpoint protein; expressing the rare-cutting endonucleases into the T cells; expanding the cells. In some embodiments, the rare-cutting endonuclease specifically targets a gene selected from the group consisting of: PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCRα, and TCRβ. In some embodiments, the rare-cutting endonuclease can be a meganuclease, a zinc finger nuclease or a TALE-nuclease. In some embodiments, the rare-cutting endonuclease is a TALE-nuclease.
In some embodiments, the present invention can be particularly suitable for allogeneic immunotherapy. In such embodiments, cells may be modified by a method comprising: inactivating at least one gene encoding a component of the T cell receptor (TCR) in T cells; and expanding the T cells. In some embodiments, the genetic modification of the method relies on the expression, in provided cells to engineer, of one rare-cutting endonuclease such that the rare-cutting endonuclease specifically catalyzes cleavage in one targeted gene thereby inactivating the targeted gene. In some embodiments, said method to engineer cells comprises at least one of the following steps: providing a T cell, such as from a cell culture or from a blood sample; introducing into the T cell a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break at least one gene encoding a component of the T cell receptor (TCR), and expanding the cells.
In some embodiments, the method comprises: providing a T cell, such as from a cell culture or from a blood sample; transfecting said T cell with nucleic acid encoding a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break at least one gene encoding a component of the T cell receptor (TCR); expressing the rare-cutting endonucleases into the T cells; sorting the transformed T cells, which do not express TCR on their cell surface; and expanding the cells.
In some embodiments, the rare-cutting endonuclease can be a meganuclease, a zinc finger nuclease or a TALE-nuclease. In some embodiments, the rare-cutting endonuclease is a TALE-nuclease. In some embodiments the TALE-nucleases recognize and cleave a sequence encoding TCRα or TCRβ. In some embodiments a TALE-nuclease comprises a polypeptide sequence selected from the amino acid sequence shown in SEQ ID NO: 334, 335, 336, 337, 338, 339, 340, or 341
TALE-nuclease polypeptide sequences:
Repeat TRAC_T01-L
(SEQ ID NO: 334)
LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK
QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDG
GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASH
DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPV
LCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIA
SNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVA
IASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR
LLPVLCQAHGLTPQQVVAIASNGGGRPALE
Repeat TRAC_T01-R
(SEQ ID NO: 335)
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGK
QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNG
GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC
QAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN
NGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPV
LCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIA
SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVA
IASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQR
LLPVLCQAHGLTPQQVVAIASNGGGRPALE
Repeat TRBC_T01-L
(SEQ ID NO: 336)
LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGK
QALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA
HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGG
GKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC
QAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN
IGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPV
LCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIA
SHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALL
PVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQA
LLPVLCQAHGLTPQQVVAIASNGGGRPALE
Repeat TRBC_T01-R
(SEQ ID NO: 337)
NPQRSTVVVYLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQV
VAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETV
QRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPE
QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALE
TVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLT
PQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQA
LETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHG
LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK
QALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA
HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNG
GKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE
Repeat TRBC_T02-L
(SEQ ID NO: 338)
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK
QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDG
GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN
NGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPV
LCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIA
SNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVA
IASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR
LLPVLCQAHGLTPQQVVAIASNGGGRPALE
Repeat TRBC_T02-R
(SEQ ID NO: 339)
LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK
QALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNG
GKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLC
QAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH
DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPV
LCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIA
SNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLL
PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVA
IASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR
LLPVLCQAHGLTPQQVVAIASNGGGRPALE
Repeat CD52_T02-L
(SEQ ID NO: 340)
LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK
QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA
HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDG
GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC
QAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN
IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPV
LCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIA
SHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALL
PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQA
LLPVLCQAHGLTPQQVVAIASNGGGRPALE
Repeat CD52_T02-R
(SEQ ID NO: 341)
LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK
QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA
HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDG
GKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC
QAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN
IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPV
LCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIA
SNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLL
PVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVA
IASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR
LLPVLCQAHGLTPQQVVAIASNGGGRPALE
In another aspect, one another step of genetically modifying cell can be a method of expanding TCRα deficient T cells comprising introducing into the T cell pTα (also known as preTCRα) or a functional variant thereof and expanding the cells, optionally through stimulation of the CD3 complex. In some embodiments, the method comprises: a) transfecting the cells with nucleic acid encoding at least a fragment of pTα to support CD3 surface expression; b) expressing said pTα into the cells; and c) expanding the cells, optionally through stimulation of the CD3 complex.
Also provided are methods of preparing T cells for immunotherapy comprising steps of the method for expansion for T cell. In some embodiments, the pTα polynucleotide sequence can be introduced randomly or by homologous recombination. In some embodiments, the insertion can be associated with the inactivation of the TCRα gene. Different functional variants of pTα can be used. A “functional variant” of the peptide refers to a molecule substantially similar to either the entire peptide or a fragment thereof. A “fragment” of the pTα or functional variant thereof refers to any subset of the molecule, that is, a shorter peptide than the full-length pTα. In some embodiments, pTα or functional variants can be, for example, full-length pTα or a C-terminal truncated pTα version. C-terminal truncated pTα lacks in C-terminal end one or more residues. As non limiting examples, C-terminal truncated pTα version lacks 18, 48, 62, 78, 92, 110 or 114 residues from the C-terminus of the protein. Amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the peptide. Such functional variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity, in particular the restoration of a functional CD3 complex. In preferred embodiment, at least one mutation is introduced in the different pTα versions as described above to affect dimerization. As non limiting example, mutated residue can be at least W46R, D22A, K24A, R102A or R117A of the human pTα protein or aligned positions using CLUSTALW method on pTα family or homologue member. Preferably pTα or variant thereof as described above comprise the mutated residue W46R or the mutated residues D22A, K24A, R102A and R117A. In some embodiments, said pTα or variants are also fused to a signal-transducing domain such as CD28, OX40, ICOS, CD27, CD137 (4-1BB) and CD8 as non limiting examples. The extracellular domain of pTα or variants as described above can be fused to a fragment of the TCRα protein, particularly the transmembrane and intracellular domain of TCRα. pTα variants can also be fused to the intracellular domain of TCRα.
In some embodiments, pTα versions can be fused to an extracellular ligand-binding domain. In some embodiments, pTα or functional variant thereof is fused to a single chain antibody fragment (scFv) comprising the light and the heavy variable fragment of a target antigen specific monoclonal antibody joined by a flexible linker.
The term “TCRα deficient T cell” refers to an isolated T cell that lacks expression of a functional TCRα chain. This may be accomplished by different means, as non limiting examples, by engineering a T cell such that it does not express any functional TCRα on its cell surface or by engineering a T cell such that it produces very little functional TCRα chain on its surface or by engineering a T cell to express mutated or truncated form of TCRα chain. TCRα deficient cells can no longer be expanded through CD3 complex. Thus, to overcome this problem and to allow proliferation of TCRα deficient cells, pTα or functional variant thereof is introduced into the cells, thus restoring a functional CD3 complex. In some embodiments, the method further comprises introducing into said T cells rare-cutting endonucleases able to selectively inactivate by DNA cleavage one gene encoding one component of the T cell receptor (TCR). In some embodiments, the rare-cutting endonuclease is a TALE-nuclease.
In another aspect, engineered T cells obtained by the methods described herein can be contacted with bispecific antibodies. For example, the T cells can be contacted with bispecific antibodies ex vivo prior to administration to a patient, or in vivo following administration to a patient. Bispecific antibodies comprise two variable regions with distinct antigen properties that facilitate bringing the engineered cells into proximity to a target antigen. As a non-limiting example, a bispecific antibody can be directed against a tumor marker and lymphocyte antigen, such as for example without limitation CD3, and has the potential to redirect and activate any circulating T cells against tumors.
In some embodiments, polynucleotides encoding polypeptides according to the present invention can be mRNA which is introduced directly into the cells, for example by electroporation. In some embodiments, cytoPulse technology can be used to transiently permeabilize living cells for delivery of material into the cells. Parameters can be modified in order to determine conditions for high transfection efficiency with minimal mortality.
Also provided herein are methods of transfecting T cell. In some embodiments, the method comprises: contacting a T cell with RNA and applying to T cell an agile pulse sequence consisting of: (a) an electrical pulse with a voltage range from about 2250 to 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval of about 0.2 to 10 ms between the electrical pulses of step (a) and (b); (d) an electrical pulse with a voltage range from about 2250 to 3000 V with a pulse width of about 100 ms and a pulse interval of about 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (e) four electrical pulses with a voltage of about 325 V with a pulse width of about 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses. In some embodiments, a method of transfecting T cell comprising contacting said T cell with RNA and applying to T cell an agile pulse sequence comprising: (a) an electrical pulse with a voltage of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V per centimeter; (b) a pulse width of 0.1 ms; (c) and a pulse interval of about 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms between the electrical pulses of step (a) and (b); (d) one electrical pulse with a voltage range from about 2250, of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (e) 4 electrical pulses with a voltage of about 325 V with a pulse width of about 0.2 ms and a pulse interval of about 2 ms between each of 4 electrical pulses. Any values included in the value range described above are disclosed in the present application. Electroporation medium can be any suitable medium known in the art. In some embodiments, the electroporation medium has conductivity in a range spanning about 0.01 to about 1.0 milliSiemens.
In some embodiments, as non limiting examples, an RNA encodes a rare-cutting endonuclease, one monomer of the rare-cutting endonuclease such as half-TALE-nuclease, a CAR, at least one component of the multi-chain chimeric antigen receptor, a pTα or functional variant thereof, an exogenous nucleic acid, and/or one additional catalytic domain.
Engineered Immune Cells
The invention also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, a CAR can be introduced into an immune cell as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also contain, for example, a selection marker which provides for identification and/or selection of cells which received the vector.
CAR polypeptides may be synthesized in situ in the cell after introduction of polynucleotides encoding the CAR polypeptides into the cell. Alternatively, CAR polypeptides may be produced outside of cells, and then introduced into cells. Methods for introducing a polynucleotide construct into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct not integrated into the genome of the cell. In other embodiments, virus-mediated methods can be used. The polynucleotides may be introduced into a cell by any suitable means such as for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomes, and the like. Transient transformation methods include, for example without limitation, microinjection, electroporation or particle bombardment. Polynucleotides may be included in vectors, such as for example plasmid vectors or viral vectors.
Also provided herein are isolated cells and cell lines obtained by the above-described methods of engineering cells provided herein. In some embodiments, an isolated cell comprises at least one CAR as described above. In some embodiments, an isolated cell comprises a population of CARs, each CAR comprising different extracellular ligand-binding domains.
Also provided herein are isolated immune cells obtained according to any one of the methods described above. Any immune cell capable of expressing heterologous DNAs can be used for the purpose of expressing the CAR of interest. In some embodiments, the immune cell is a T cell. In some embodiments, an immune cell can be derived from, for example without limitation, a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. The isolated cell can also be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. I n some embodiments, the cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes.
Prior to expansion and genetic modification, a source of cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art, may be used. In some embodiments, cells can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In some embodiments, cells can be part of a mixed population of cells which present different phenotypic characteristics.
Also provided herein are cell lines obtained from a transformed T cell according to any of the above-described methods. Also provided herein are modified cells resistant to an immunosuppressive treatment. In some embodiments, an isolated cell according to the invention comprises a polynucleotide encoding a CAR.
The immune cells of the invention can be activated and expanded, either prior to or after genetic modification of the T cells, using methods as generally described, for example without limitation, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo. Generally, the T cells of the invention can be expanded, for example, by contact with an agent that stimulates a CD3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.
In some embodiments, T cell populations may be stimulated in vitro by contact with, for example, an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF, or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to varied stimulation times may exhibit different characteristics
In some embodiments, the cells of the invention can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example in the subject's blood after administrating the cell into the subject.
In some embodiments, an isolated cell according to the present invention comprises one inactivated gene selected from the group consisting of CD52, GR, PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα and TCRβ and/or expresses a CAR, a multi-chain CAR and/or a pTα transgene. In some embodiments, an isolated cell comprises polynucleotides encoding polypeptides comprising a multi-chain CAR. In some embodiments, the isolated cell according to the present invention comprises two inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ and/or expresses a CAR, a multi-chain CAR and a pTα transgene.
In some embodiments, TCR is rendered not functional in the cells according to the invention by inactivating TCRα gene and/or TCRβ gene(s). In some embodiments, a method to obtain modified cells derived from an individual is provided, wherein the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells, which can proliferate independently of the MHC signaling pathway, susceptible to be obtained by this method are encompassed in the scope of the present invention. Modified cells disclosed herein can be used in for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present invention is a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said patient by administering to said patient an effective amount of modified cells comprising inactivated TCRα and/or TCRβ genes.
In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapy drugs. The chemotherapy drug can be, for example, a purine nucleotide analogue (PNA), thus making the immune cell suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) into mono-, di-, and tri-phosphate PNA. Their tri-phosphate forms compete with ATP for DNA synthesis, act as pro-apoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production. Provided herein are BCMA specific CAR-T cells comprising an inactivated dCK gene. In some embodiments, the dCK knockout cells are made by transfection of T cells using polynucleotides encoding specific TAL-nuclease directed against dCK genes by, for example, electroporation of mRNA. The dCK knockout BCMA specific CAR-T cells are resistant to PNAs, including for example clofarabine and/or fludarabine, and maintain T cell cytotoxic activity toward BCMA-expressing cells.
In some embodiments, isolated cells or cell lines of the invention can comprise a pTα or a functional variant thereof. In some embodiments, an isolated cell or cell line can be further genetically modified by inactivating the TCRα gene.
In some embodiments, the CAR-T cell comprises a polynucleotide encoding a suicide polypeptide, such as for example RQR8. See, e.g., WO2013153391A, which is hereby incorporated by reference in its entirety. In CAR-T cells comprising the polynucleotide, the suicide polypeptide is expressed at the surface of a CAR-T cell. In some embodiments, the suicide polypeptide comprises the amino acid sequence shown in SEQ ID NO: 342.
(SEQ ID NO: 342)
CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSG
GGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW
APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV
The suicide polypeptide may also comprise a signal peptide at the amino terminus. In some embodiments, the suicide polypeptide comprises the amino acid sequence shown in SEQ ID NO: 400.
(SEQ ID NO: 400)
MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTN
VSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACR
PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVC
KCPRPVV
When the suicide polypeptide is expressed at the surface of a CAR-T cell, binding of rituximab to the R epitopes of the polypeptide causes lysis of the cell. More than one molecule of rituximab may bind per polypeptide expressed at the cell surface. Each R epitope of the polypeptide may bind a separate molecule of rituximab. Deletion of BCMA specific CAR-T cells may occur in vivo, for example by administering rituximab to a patient. The decision to delete the transferred cells may arise from undesirable effects being detected in the patient which are attributable to the transferred cells, such as for example, when unacceptable levels of toxicity are detected.
In some embodiments, the CAR-T cell comprises a selected epitope within the scFv having a specificity to be recognized by a specific antibody. See, e.g., PCT application “mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS,” filed on Jan. 25, 2016, which is hereby incorporated by reference in its entirety. Such an epitope facilitates sorting and/or depleting the CAR-T cells. The epitope can be selected from any number of epitopes known in the art. In some embodiments, the epitope can be a target of a monoclonal antibody approved for medical use, such as, for example without limitation, the CD20 epitope recognized by rituximab. In some embodiments, the epitope comprises the amino acid sequence shown in SEQ ID NO: 397.
(SEQ ID NO: 397)
CPYSNPSLC
In some embodiments, the epitope is located within the CAR. For example without limitation, the epitope can be located between the scFv and the hinge of a CAR. In some embodiments, two instances of the same epitope, separate by linkers, may be used in the CAR. For example, the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 398 can be used within a CAR, located between the light chain variable region and the hinge.
(SEQ ID NO: 398)
GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGS
In some embodiments, the epitope-specific antibody may be conjugated with a cytotoxic drug. It is also possible to promote CDC cytotoxicity by using engineered antibodies on which are grafted component(s) of the complement system. In some embodiments, activation of the CAR-T cells can be modulated by depleting the cells using an antibody which recognizes the epitope.
Therapeutic Applications
Isolated cells obtained by the methods described above, or cell lines derived from such isolated cells, can be used as a medicament. In some embodiments, such a medicament can be used for treating cancer. In some embodiments, the cancer is multiple myeloma malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma, Kahler's disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenström macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, or another B-cell related lymphomas.
In some embodiments, an isolated cell according to the invention, or cell line derived from the isolated cells, can be used in the manufacture of a medicament for treatment of a cancer in a patient in need thereof.
Also provided herein are methods for treating patients. In some embodiments the method comprises. providing an immune cell of the invention to a patient in need thereof. In some embodiments, the method comprises a step of administrating transformed immune cells of the invention to a patient in need thereof.
In some embodiments, T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.
Methods of treatment of the invention can be ameliorating, curative or prophylactic. The method of the invention may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. The invention is particularly suitable for allogeneic immunotherapy. T cells from donors can be transformed into non-alloreactive cells using standard protocols and reproduced as needed, thereby producing CAR-T cells which may be administered to one or several patients. Such CAR-T cell therapy can be made available as an “off the shelf” therapeutic product.
Cells that can be used with the disclosed methods are described in the previous section. Treatment can be used to treat patients diagnosed with, for example, cancer. Cancers that may be treated include, for example without limitation, cancers that involve B lymphocytes, including any of the above-listed cancers. Types of cancers to be treated with the CARs and CAR-T cells of the invention include, but are not limited to certain leukemia or lymphoid malignancies. Adult tumors/cancers and pediatric tumors/cancers are also included. In some embodiments, the treatment can be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
In some embodiments, treatment can be administrated into patients undergoing an immunosuppressive treatment. Indeed, the invention preferably relies on cells or population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In this aspect, the immunosuppressive treatment should help the selection and expansion of the T cells according to the invention within the patient. The administration of the cells or population of cells according to the invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the invention are preferably administered by intravenous injection.
In some embodiments the administration of the cells or population of cells can comprise administration of, for example, about 104 to about 109 cells per kg body weight including all integer values of cell numbers within those ranges. In some embodiments the administration of the cells or population of cells can comprise administration of about 105 to 106 cells per kg body weight including all integer values of cell numbers within those ranges. The cells or population of cells can be administrated in one or more doses. In some embodiments, said effective amount of cells can be administrated as a single dose. In some embodiments, said effective amount of cells can be administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In some embodiments, an effective amount of cells or composition comprising those cells are administrated parenterally. In some embodiments, administration can be an intravenous administration. In some embodiments, administration can be directly done by injection within a tumor.
In some embodiments of the invention, cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as monoclonal antibody therapy, CCR2 antagonist (e.g., INC-8761), antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients. In some embodiments, BCMA specific CAR-T cells are administered to a patient in conjunction with one or more of the following: an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or PF-06801591), an anti-PD-L1 antibody (e.g., avelumab, atezolizumab, or durvalumab), an anti-OX40 antibody (e.g., PF-04518600), an anti-4-1BB antibody (e.g., PF-05082566), an anti-MCSF antibody (e.g., PD-0360324), an anti-GITR antibody, and/or an anti-TIGIT antibody. In some embodiments, a BCMA specific CAR comprising the amino acid sequence shown in SEQ ID NO: 396 is administered to a patient in conjunction with anti-PD-L1 antibody avelumab. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and/or irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In a further embodiment, the cell compositions of the invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In some embodiments, the cell compositions of the invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the invention. In some embodiments, expanded cells are administered before or following surgery.
Kits
The invention also provides kits for use in the instant methods. Kits of the invention include one or more containers comprising a polynucleotide encoding a BCMA specific CAR, or an engineered immune cell comprising a polynucleotide encoding a BCMA specific CAR as described herein, and instructions for use in accordance with any of the methods of the invention described herein. Generally, these instructions comprise a description of administration of the engineered immune cell for the above described therapeutic treatments.
The instructions relating to the use of the engineered immune cells as described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a BCMA antibody. The container may further comprise a second pharmaceutically active agent.
Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
Representative materials of the present invention were deposited in the American Type Culture Collection (ATCC) on Feb. 9, 2016. The biological deposit having ATCC Accession No. PTA-122834 is a vector comprising a polynucleotide encoding a BCMA specific CAR. The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Pfizer, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. Section 122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. Section 1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
EXAMPLES
Example 1
Determination of Kinetics and Affinity of BCMA/Human IgG Interactions at 25° C. and/or 37° C.
This example determines the kinetics and affinity of various anti-BCMA antibodies at 25° C. and 37° C.
All experiments were performed on a Bio-Rad Proteon XPR36 surface Plasmon resonance biosensor (Bio-Rad, Hercules, Calif.). An array of anti-BCMA antibodies was prepared using an amine-coupling method on a Bio-Rad GLC Sensor Chip similar to that described in Abdiche, et al., Anal. Biochem. 411, 139-151 (2011). The analysis temperature for the immobilization was 25° C. and the running buffer was HBS-T+ (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). Channels were activated in the analyte (horizontal) direction by injecting a mixture of 1 mM ECD and 0.25 mM NHS for 3 minutes at a flow rate of 30 μL/min. IgGs were immobilized on the activated spots by injecting them in the ligand (vertical) direction at 20 μg/mL in 10 mM Acetate pH 4.5 buffer for 1.5 minutes at 30 μg/m L. The activated surfaces were blocked by injecting 1M ethanolamine, pH 8.5 in the analyte direction for 3 minutes at 30 μL/min.
The analysis temperature for the BCMA binding analysis was 37° C. or 25° C. in a running buffer of HBS-T+, supplemented with 1 mg/mL BSA. A kinetic titration method was employed for the interaction analysis as described in Abdiche, et al. Human BCMA (huBCMA) or cynomolgus monkey BCMA (cyBCMA) analyte was injected in the analyte direction using a series of injections from low to high concentration. The concentrations used were 0.08 nM, 0.4 nM, 2 nM, 10 nM and 50 nM (a 5-membered series, with a 5-fold dilution factor and top concentration of 50 nM). The association time for a given analyte dilution was two minutes. Immediately after the 50 nM BCMA injection, dissociation was monitored for 2 hours. Prior to the BCMA analyte injections, buffer was injected 5 times using the same association and dissociation times at the BCMA analyte cycles to prepare a buffer blank sensorgram for double-referencing purposes (double referencing as described in Myszka, J. Mol. Recognit. 12, 279-284 (1999).
The sensorgrams were double-referenced and fit to a 1:1 Langmuir with mass transport kinetic titration model in BIAevaluation Software version 4.1.1 (GE Lifesciences, Piscataway, N.J.). The kinetics and affinity parameters for various anti-BCMA antibodies of the invention are shown in Tables 4A-4C. The antibodies shown in Tables 4A-4C share the same VH and VL regions as the CARs shown in Table 1 having the same name.
TABLE 4A
KD
Sample
ka (1/Ms)
kd (1/s)
t1/2 (min)
(pM)
A02_Rd4_6nM_C01
1.2E+06
2.8E−05
411
24
A02_Rd4_6nM_C16
1.1E+06
6.2E−05
187
59
Combo_Rd4_0.6nM_C29
6.6E+06
1.4E−04
83
21
L3PY/H3TAQ
2.6E+06
1.4E−04
84
53
TABLE 4B
ka (1/Ms) huBCMA
kd (1/s) huBCMA
T½ (min) to
KD (nM) to
Antibody
@ 25° C.
@ 25° C.
huBCMA @ 25° C.
huBCMA @ 25° C.
P6E01/P6E01
1.04E+06
4.15E−03
2.8
4.0
P6E01/H3.AQ
8.35E+05
3.45E−04
33.53
0.41
L1.LGF/L3.KW/P6E01
8.31E+05
7.55E−03
1.53
9.08
L1.LGF/L3.NY/P6E01
1.33E+06
4.40E−03
2.63
3.32
L1.GDF/L3.NY/P6E01
1.60E+06
5.92E−03
1.95
3.70
L1.LGF/L3.KW/H3.AL
4.28E+05
1.23E−03
9.40
2.87
L1.LGF/L3.KW/H3.AP
9.28E+05
2.27E−03
5.10
2.44
L1.LGF/L3.KW/H3.AQ
5.24E+05
9.56E−04
12.09
1.82
L1.LGF/L3.PY/H3.AP
4.57E+05
9.69E−04
11.92
2.12
L1.LGF/L3.PY/H3.AQ
9.31E+05
8.86E−04
13.04
0.95
L1.LGF/L3.NY/H3.AL
7.63E+05
9.70E−04
11.91
1.27
L1.LGF/L3.NY/H3.AP
9.36E+05
5.33E−04
21.67
0.57
L1.LGF/L3.NY/H3.AQ
6.66E+05
2.99E−04
38.61
0.45
L1.GDF/L3.KW/H3.AL
4.45E+05
3.90E−03
2.96
8.76
L1.GDF/L3.KW/H3.AP
1.17E+06
4.61E−03
2.51
3.93
L1.GDF/L3.KW/H3.AQ
7.97E+05
3.48E−03
3.32
4.37
L1.GDF/L3.PY/H3.AQ
1.42E+06
1.35E−02
0.86
9.49
L1.GDF/L3.NY/H3.AL
9.07E+05
4.03E−03
2.87
4.44
L1.GDF/L3.NY/H3.AP
1.41E+06
1.41E−03
8.21
1.00
L1.GDF/L3.NY/H3.AQ
9.84E+05
7.22E−04
16.00
0.73
L3.KW/P6E01
7.40E+05
3.15E−04
36.66
0.43
L3.PY/P6E01
7.12E+05
2.28E−04
50.74
0.32
L3.NY/P6E01
8.76E+05
3.84E−04
30.08
0.44
ka (1/Ms) huBCMA
kd (1/s) huBCMA
T½ (min) to
KD (nM) to
Antibody
@ 37° C.
@ 37° C.
huBCMA @ 37° C.
huBCMA @ 37° C.
L3.PY/L1.PS/P6E01
2.49E+06
1.13E−03
10.21
0.45
L3.PY/L1.AH/P6E01
2.55E+06
1.26E−03
9.19
0.49
L3.PY/L1.FF/P6E01
2.39E+06
1.41E−03
8.18
0.59
L3.PY/L1.PH/P6E01
2.81E+06
9.13E−04
12.65
0.32
L3.PY/L3.KY/P6E01
3.18E+06
1.09E−03
10.65
0.34
L3.PY/L3.KF/P6E01
2.88E+06
2.08E−03
5.56
0.72
L3.PY/H2.QR
2.56E+06
1.19E−03
9.75
0.46
L3.PY/H2.DY
2.60E+06
1.38E−03
8.37
0.53
L3.PY/H2.YQ
2.58E+06
1.56E−03
7.41
0.60
L3.PY/H2.LT
2.40E+06
1.29E−03
8.95
0.54
L3.PY/H2.HA
2.43E+06
1.47E−03
7.89
0.60
L3.PY/H2.QL
2.64E+06
2.18E−03
5.31
0.82
L3.PY/H3.YA
3.15E+06
1.18E−03
9.82
0.37
L3.PY/H3.AE
3.29E+06
1.39E−03
8.32
0.42
L3.PY/H3.AQ
3.08E+06
1.73E−03
6.69
0.56
L3.PY/H3.TAQ
3.08E+06
1.14E−03
10.13
0.37
L3.PY/P6E01
2.65E+06
1.96E−03
5.91
0.74
L3.PY/L1.PS/H2.QR
3.97E+06
1.03E−01
0.11
25.85
L3.PY/L1.PS/H2.DY
3.22E+06
3.61E−03
3.20
1.12
L3.PY/L1.PS/H2.YQ
3.35E+06
4.30E−03
2.69
1.28
L3.PY/L1.PS/H2.LT
3.40E+06
4.65E−03
2.49
1.37
L3.PY/L1.PS/H2.HA
3.30E+06
1.06E−02
1.09
3.21
L3.PY/L1.PS/H2.QL
1.52E+07
3.14E−01
0.04
20.64
L3.PY/L1.PS/H3.YA
3.07E+06
9.05E−03
1.28
2.95
L3.PY/L1.PS/H3.AE
3.14E+06
1.46E−03
7.93
0.46
L3.PY/L1.PS/H3.AQ
3.26E+06
1.79E−03
6.46
0.55
L3.PY/L1.PS/H3.TAQ
3.25E+06
2.46E−03
4.70
0.76
L3.PY/L1.AH/H2.QR
3.13E+06
1.81E−03
6.39
0.58
L3.PY/L1.AH/H2.DY
3.05E+06
1.52E−03
7.62
0.50
L3.PY/L1.AH/H2.YQ
2.42E+06
1.93E−03
6.00
0.80
L3.PY/L1.AH/H2.LT
3.16E+06
1.23E−03
9.38
0.39
L3.PY/L1.AH/H2.HA
3.33E+06
1.81E−03
6.37
0.54
L3.PY/L1.AH/H2.QL
3.04E+06
1.60E−03
7.22
0.53
L3.PY/L1.AH/H3.YA
3.00E+06
1.50E−03
7.73
0.50
L3.PY/L1.AH/H3.AE
3.32E+06
1.73E−03
6.70
0.52
L3.PY/L1.AH/H3.AQ
3.03E+06
1.97E−03
5.85
0.65
L3.PY/L1.AH/H3.TAQ
3.27E+06
1.19E−03
9.68
0.37
L3.PY/L1.FF/H2.QR
3.47E+06
1.77E−03
6.54
0.51
L3.PY/L1.FF/H2.DY
4.14E+06
2.71E−03
4.27
0.65
L3.PY/L1.FF/H2.YQ
3.32E+06
1.52E−03
7.61
0.46
L3.PY/L1.FF/H2.LT
3.30E+06
1.67E−03
6.92
0.51
L3.PY/L1.FF/H2.HA
3.49E+06
2.19E−03
5.29
0.63
L3.PY/L1.FF/H2.QL
3.48E+06
1.40E−03
8.28
0.40
L3.PY/L1.FF/H3.YA
3.50E+06
1.80E−03
6.41
0.51
L3.PY/L1.FF/H3.AE
3.82E+06
2.63E−03
4.39
0.69
L3.PY/L1.FF/H3.AQ
3.32E+06
1.54E−03
7.51
0.46
L3.PY/L1.FF/H3.TAQ
3.52E+06
1.89E−03
6.12
0.54
L3.PY/L1.PH/H2.QR
3.69E+06
2.36E−03
4.89
0.64
L3.PY/L1.PH/H2.HA
2.37E+06
1.16E−03
9.99
0.49
L3.PY/L1.PH/H3.AE
3.68E+06
1.34E−03
8.61
0.36
L3.PY/L1.PH/H3.AQ
3.08E+06
1.59E−03
7.27
0.52
L3.PY/L1.PH/H3.TAQ
3.58E+06
2.13E−03
5.43
0.59
L3.PY/L3.KY/H2.QR
2.95E+06
9.90E−04
11.67
0.34
L3.PY/L3.KY/H2.DY
3.19E+06
6.42E−04
18.00
0.20
L3.PY/L3.KY/H2.YQ
2.14E+06
1.65E−03
7.02
0.77
L3.PY/L3.KY/H2.LT
2.92E+06
9.06E−04
12.75
0.31
L3.PY/L3.KY/H2.HA
3.29E+06
1.63E−03
7.10
0.49
L3.PY/L3.KY/H2.QL
3.65E+06
2.08E−03
5.56
0.57
L3.PY/L3.KY/H3.YA
3.30E+06
9.12E−04
12.67
0.28
L3.PY/L3.KY/H3.TAQ
2.79E+06
6.49E−04
17.79
0.23
L3.PY/L3.KF/H2.DY
2.74E+06
1.82E−03
6.35
0.67
L3.PY/L3.KF/H2.YQ
1.96E+06
2.23E−03
5.18
1.14
L3.PY/L3.KF/H2.LT
2.75E+06
1.91E−03
6.05
0.69
L3.PY/L3.KF/H2.QL
2.07E+06
1.25E−03
9.26
0.60
L3.PY/L3.KF/H3.YA
3.12E+06
1.47E−03
7.85
0.47
L3.PY/L3.KF/H3.AE
3.07E+06
1.55E−03
7.44
0.51
L3.PY/L3.KF/H3.AQ
3.48E+06
2.27E−03
5.09
0.65
L3.PY/L3.KF/H3.TAQ
2.82E+06
1.62E−03
7.12
0.58
ka (1/Ms) cyBCMA
kd (1/s) cyBCMA
T½ (min) to
KD (nM) to
Antibody
@ 25° C.
@ 25° C.
cyBCMA @ 25° C.
cyBCMA @ 25° C.
P6E01/P6E01
7.02E−02
0.16
115.4
P6E01/H3.AQ
1.08E+06
7.40E−03
1.6
6.9
L1.LGF/L3.KW/P6E01
4.55E+05
1.95E−02
0.6
42.8
L1.LGF/L3.NY/P6E01
9.20E+05
1.05E−02
1.1
11.4
L1.GDF/L3.NY/P6E01
1.20E+06
7.67E−03
1.5
6.4
L1.LGF/L3.KW/H3.AL
2.90E+05
1.21E−02
1.0
41.8
L1.LGF/L3.KW/H3.AP
5.54E+05
1.54E−02
0.7
27.8
L1.LGF/L3.KW/H3.AQ
5.27E+05
3.55E−03
3.3
6.7
L1.LGF/L3.PY/H3.AP
3.64E+05
1.30E−02
0.9
35.8
L1.LGF/L3.PY/H3.AQ
1.00E+06
4.77E−03
2.4
4.8
L1.LGF/L3.NY/H3.AL
6.35E+05
1.48E−02
0.8
23.2
L1.LGF/L3.NY/H3.AP
8.30E+05
5.57E−03
2.1
6.7
L1.LGF/L3.NY/H3.AQ
7.51E+05
1.48E−03
7.8
2.0
L1.GDF/L3.KW/H3.AL
3.18E+05
1.80E−02
0.6
56.7
L1.GDF/L3.KW/H3.AP
8.14E+05
2.03E−02
0.6
24.9
L1.GDF/L3.KW/H3.AQ
8.02E+05
5.65E−03
2.0
7.0
L1.GDF/L3.PY/H3.AQ
1.55E+06
1.66E−02
0.7
10.7
L1.GDF/L3.NY/H3.AL
9.00E+05
2.19E−02
0.5
24.3
L1.GDF/L3.NY/H3.AP
1.36E+06
7.02E−03
1.6
5.2
L1.GDF/L3.NY/H3.AQ
1.18E+06
1.36E−03
8.5
1.2
L3.KW/P6E01
7.63E+05
2.57E−03
4.5
3.4
L3.PY/P6E01
8.55E+05
2.93E−03
3.9
3.4
L3.NY/P6E01
1.01E+06
2.87E−03
4.0
2.8
ka (1/Ms) cyBCMA
kd (1/s) cyBCMA
T½ (min) to
KD (nM) to
Antibody
@ 37° C.
@ 37° C.
cyBCMA @ 37° C.
cyBCMA @ 37° C.
L3.PY/L1.PS/P6E01
2.17E+06
6.06E−03
1.91
2.79
L3.PY/L1.AH/P6E01
2.16E+06
5.72E−03
2.02
2.65
L3.PY/L1.FF/P6E01
2.45E+06
5.91E−03
1.96
2.41
L3.PY/L1.PH/P6E01
2.17E+06
7.89E−03
1.46
3.63
L3.PY/L3.KY/P6E01
2.27E+06
5.02E−03
2.30
2.21
L3.PY/L3.KF/P6E01
2.39E+06
8.30E−03
1.39
3.48
L3.PY/H2.QR
2.18E+06
6.58E−03
1.76
3.02
L3.PY/H2.DY
2.24E+06
6.18E−03
1.87
2.76
L3.PY/H2.YQ
2.46E+06
6.21E−03
1.86
2.53
L3.PY/H2.LT
2.09E+06
7.57E−03
1.53
3.63
L3.PY/H2.HA
1.99E+06
7.55E−03
1.53
3.79
L3.PY/H2.QL
2.05E+06
1.26E−02
0.91
6.16
L3.PY/H3.YA
2.87E+06
5.40E−03
2.14
1.88
L3.PY/H3.AE
2.82E+06
5.04E−03
2.29
1.79
L3.PY/H3.AQ
2.77E+06
5.39E−03
2.14
1.94
L3.PY/H3.TAQ
2.57E+06
4.37E−03
2.64
1.70
L3.PY/P6E01
2.20E+06
1.31E−02
0.88
5.96
L3.PY/L1.PS/H2.QR
5.25E+05
6.70E−04
17.23
1.28
L3.PY/L1.PS/H2.DY
1.90E+06
3.78E−03
3.06
1.99
L3.PY/L1.PS/H2.YQ
2.00E+06
3.74E−03
3.09
1.87
L3.PY/L1.PS/H2.LT
2.17E+06
4.11E−03
2.81
1.89
L3.PY/L1.PS/H2.HA
1.45E+06
2.69E−03
4.30
1.86
L3.PY/L1.PS/H2.QL
6.57E+05
6.36E−04
18.17
0.97
L3.PY/L1.PS/H3.YA
1.77E+06
9.98E−03
1.16
5.65
L3.PY/L1.PS/H3.AE
2.46E+06
4.13E−03
2.80
1.68
L3.PY/L1.PS/H3.AQ
2.52E+06
4.33E−03
2.67
1.72
L3.PY/L1.PS/H3.TAQ
2.58E+06
5.52E−03
2.09
2.14
L3.PY/L1.AH/H2.QR
2.20E+06
4.91E−03
2.35
2.23
L3.PY/L1.AH/H2.DY
2.32E+06
4.51E−03
2.56
1.95
L3.PY/L1.AH/H2.YQ
1.58E+06
4.31E−03
2.68
2.74
L3.PY/L1.AH/H2.LT
2.19E+06
2.96E−03
3.91
1.35
L3.PY/L1.AH/H2.HA
2.58E+06
4.39E−03
2.63
1.70
L3.PY/L1.AH/H2.QL
2.62E+06
9.55E−03
1.21
3.65
L3.PY/L1.AH/H3.YA
2.37E+06
5.26E−03
2.20
2.22
L3.PY/L1.AH/H3.AE
2.25E+06
3.56E−03
3.25
1.58
L3.PY/L1.AH/H3.AQ
2.24E+06
3.99E−03
2.90
1.78
L3.PY/L1.AH/H3.TAQ
2.28E+06
3.02E−03
3.83
1.32
L3.PY/L1.FF/H2.QR
2.55E+06
4.21E−03
2.75
1.65
L3.PY/L1.FF/H2.DY
2.66E+06
5.00E−03
2.31
1.88
L3.PY/L1.FF/H2.YQ
2.19E+06
3.26E−03
3.55
1.49
L3.PY/L1.FF/H2.LT
2.19E+06
3.41E−03
3.38
1.56
L3.PY/L1.FF/H2.HA
2.33E+06
4.17E−03
2.77
1.79
L3.PY/L1.FF/H2.QL
2.36E+06
4.49E−03
2.57
1.91
L3.PY/L1.FF/H3.YA
2.46E+06
4.16E−03
2.77
1.69
L3.PY/L1.FF/H3.AE
2.85E+06
5.01E−03
2.31
1.76
L3.PY/L1.FF/H3.AQ
2.18E+06
3.29E−03
3.51
1.51
L3.PY/L1.FF/H3.TAQ
2.32E+06
3.76E−03
3.07
1.62
L3.PY/L1.PH/H2.QR
2.42E+06
4.36E−03
2.65
1.80
L3.PY/L1.PH/H2.HA
1.61E+06
5.53E−03
2.09
3.44
L3.PY/L1.PH/H3.AE
2.61E+06
2.02E−03
5.72
0.77
L3.PY/L1.PH/H3.AQ
2.28E+06
3.41E−03
3.39
1.50
L3.PY/L1.PH/H3.TAQ
2.51E+06
3.20E−03
3.61
1.28
L3.PY/L3.KY/H2.QR
2.05E+06
7.74E−03
1.49
3.78
L3.PY/L3.KY/H2.DY
1.96E+06
2.43E−03
4.75
1.24
L3.PY/L3.KY/H2.YQ
1.27E+06
2.58E−03
4.47
2.04
L3.PY/L3.KY/H2.LT
1.82E+06
2.32E−03
4.98
1.27
L3.PY/L3.KY/H2.HA
2.28E+06
3.18E−03
3.63
1.40
L3.PY/L3.KY/H2.QL
2.75E+06
4.09E−03
2.83
1.49
L3.PY/L3.KY/H3.YA
1.84E+06
4.28E−03
2.70
2.33
L3.PY/L3.KY/H3.TAQ
1.81E+06
1.92E−03
6.03
1.06
L3.PY/L3.KF/H2.DY
2.08E+06
3.68E−03
3.14
1.77
L3.PY/L3.KF/H2.YQ
1.41E+06
5.01E−03
2.30
3.55
L3.PY/L3.KF/H2.LT
1.91E+06
4.13E−03
2.80
2.16
L3.PY/L3.KF/H2.QL
1.42E+06
3.10E−03
3.73
2.18
L3.PY/L3.KF/H3.YA
2.10E+06
7.96E−03
1.45
3.78
L3.PY/L3.KF/H3.AE
1.85E+06
5.64E−03
2.05
3.05
L3.PY/L3.KF/H3.AQ
2.55E+06
2.38E−03
4.85
0.93
L3.PY/L3.KF/H3.TAQ
2.01E+06
1.91E−03
6.05
0.95
TABLE 4C
Human BCMA
Cyno BCMA
ka
kd
KD
ka
kd
KD
Antibody
(1/Ms)
(1/s)
(pM)
(1/Ms)
(1/s)
(pM)
P5A2_VHVL (P5A)
6.96E+06
3.87E−02
5567
1.61E+06
1.64E−02
10230
A02_Rd4_0.6nM_C06
3.49E+06
7.37E−05
21
1.81E+06
1.05E−04
58
A02_Rd4_0.6nM_C09
5.50E+06
9.75E−05
18
2.13E+06
1.74E−04
82
A02_Rd4_6nM_C16 (P5AC16)
1.56E+06
1.41E−04
90
1.34E+06
1.58E−04
118
A02_Rd4_6nM_C03
1.69E+06
1.26E−04
75
1.17E+06
1.85E−04
158
A02_Rd4_6nM_C01
3.11E+06
9.20E−05
30
1.45E+06
5.83E−04
401
A02_Rd4_6nM_C26
4.26E+06
1.39E−04
33
2.21E+06
4.48E−04
203
A02_Rd4_6nM_C25
2.75E+06
1.80E−04
65
1.50E+06
3.30E−04
220
A02_Rd4_6nM_C22
3.38E+06
1.82E−04
54
1.84E+06
3.24E−04
176
A02_Rd4_6nM_C19
3.00E+06
1.48E−04
49
2.54E+06
6.61E−04
260
A02_Rd4_0.6nM_C03
4.27E+06
1.82E−04
43
2.12E+06
4.26E−04
201
A02_Rd4_6nM_C07
1.48E+06
1.89E−04
128
6.91E+05
7.86E−04
1138
A02_Rd4_6nM_C23
1.22E+07
2.55E−04
21
2.63E+06
4.14E−04
157
A02_Rd4_0.6nM_C18
4.73E+06
2.29E−04
48
3.24E+06
6.39E−04
197
A02_Rd4_6nM_C10
4.51E+06
3.15E−04
70
1.90E+06
8.98E−04
472
A02_Rd4_6nM_C05
3.10E+06
3.08E−04
99
1.36E+06
1.29E−03
950
A02_Rd4_0.6nM_C10
2.30E+06
2.96E−04
129
8.83E+05
1.63E−03
1842
A02_Rd4_6nM_C04
4.47E+06
6.03E−04
135
2.18E+06
8.31E−04
381
A02_Rd4_0.6nM_C26
7.26E+06
4.43E−04
61
2.71E+06
2.56E−03
941
A02_Rd4_0.6nM_C13
8.53E+06
5.66E−04
66
2.29E+06
1.28E−03
560
A02_Rd4_0.6nM_C01 (P5AC1)
4.74E+06
9.15E−04
193
2.39E+06
1.57E−03
655
A02_Rd4_6nM_C08
3.92E+06
7.38E−04
188
2.23E+06
1.13E−02
5072
P5C1_VHVL (PC1)
1.16E+07
6.92E−02
5986
3.53E+06
5.38E−02
15231
C01_Rd4_6nM_C24
7.47E+06
3.48E−03
467
3.17E+06
8.91E−04
281
C01_Rd4_6nM_C26
1.50E+07
1.36E−03
90
4.75E+06
1.99E−03
419
C01_Rd4_6nM_C02
1.61E+07
1.44E−03
89
5.12E+06
2.18E−03
426
C01_Rd4_6nM_C10
1.31E+07
2.12E−03
162
4.44E+06
2.19E−03
493
C01_Rd4_0.6nM_C27
1.23E+07
3.74E−03
303
3.34E+06
2.85E−03
852
C01_Rd4_6nM_C20
6.02E+06
2.76E−03
459
3.60E+06
6.25E−03
1737
C01_Rd4_6nM_C12
1.21E+07
6.49E−03
535
4.51E+06
3.70E−03
820
C01_Rd4_0.6nM_C16
1.55E+07
6.30E−03
407
4.95E+06
4.64E−03
939
C01_Rd4_0.6nM_C09
1.51E+07
8.25E−03
545
5.28E+06
9.36E−03
1773
C01_Rd4_6nM_C09
1.58E+07
1.28E−02
811
3.73E+06
8.68E−03
2328
C01_Rd4_0.6nM_C03
1.55E+07
1.50E−02
964
4.72E+06
1.19E−02
2528
C01_Rd4_0.6nM_C06
1.82E+07
1.54E−02
847
6.22E+06
1.21E−02
1948
C01_Rd4_6nM_C04
2.33E+07
4.97E−02
2134
6.34E+06
3.27E−02
5156
COMBO_Rd4_0.6nM_C22
1.97E+06
7.15E−05
36
1.34E+06
6.66E−05
50
COMBO_Rd4_6nM_C21
1.17E+07
7.34E−05
6
3.17E+06
2.48E−04
78
COMBO_Rd4_6nM_C10
5.47E+06
9.72E−05
18
1.52E+06
1.60E−04
105
COMBO_Rd4_0.6nM_C04
1.07E+07
1.58E−04
15
3.52E+06
1.37E−04
39
COMBO_Rd4_6nM_C25
7.98E+06
1.13E−04
14
2.85E+06
2.26E−04
79
COMBO_Rd4_0.6nM_C21
1.34E+07
1.15E−04
9
3.63E+06
3.04E−04
84
COMBO_Rd4_6nM_C11
6.74E+06
1.24E−04
18
2.64E+06
4.12E−04
156
COMBO_Rd4_0.6nM_C20
7.65E+06
1.46E−04
19
3.09E+06
2.84E−04
92
COMBO_Rd4_6nM_C09
8.85E+06
1.43E−04
16
2.37E+06
3.18E−04
134
COMBO_Rd4_6nM_C08
8.99E+06
1.69E−04
19
3.06E+06
4.28E−04
140
COMBO_Rd4_0.6nM_C19
7.86E+06
1.55E−04
20
2.92E+06
9.79E−04
336
COMBO_Rd4_0.6nM_C02
8.57E+06
1.85E−04
22
3.01E+06
4.94E−04
164
COMBO_Rd4_0.6nM_C23
7.39E+06
2.10E−04
28
2.81E+06
5.31E−04
189
COMBO_Rd4_0.6nM_C29
1.47E+07
2.77E−04
19
4.00E+06
3.36E−04
84
COMBO_Rd4_0.6nM_C09
1.04E+07
3.19E−04
31
3.77E+06
3.46E−04
92
COMBO_Rd4_6nM_C12 (PC1C12)
1.38E+07
2.70E−04
20
3.29E+06
4.86E−04
148
COMBO_Rd4_0.6nM_C30
4.35E+06
2.82E−04
65
1.68E+06
8.08E−04
481
COMBO_Rd4_0.6nM_C14
8.66E+06
3.28E−04
38
3.48E+06
6.45E−04
185
COMBO_Rd4_6nM_C07
1.05E+07
3.71E−04
35
3.94E+06
9.34E−04
237
COMBO_Rd4_6nM_C02
1.05E+06
4.43E−04
422
7.95E+05
1.36E−03
1714
COMBO_Rd4_0.6nM_C05
4.32E+06
4.97E−04
115
1.94E+06
1.72E−03
886
COMBO_Rd4_0.6nM_C17
8.68E+06
8.01E−04
92
3.06E+06
1.01E−03
330
COMBO_Rd4_6nM_C22 (COM22)
3.03E+06
7.75E−04
256
1.70E+06
1.65E−03
972
COMBO_Rd4_0.6nM_C11
5.11E+06
1.06E−03
207
2.20E+06
4.23E−03
1924
Example 2
BCMA Specific CAR-T Cells
This example demonstrates functional activity of BCMA specific CAR-T cells against BCMA positive (BCMA+) tumor cells.
Among all the BCMA specific CAR molecules generated, eight were selected for further activity tests based on affinity to BCMA, cross-reactivity to human BCMA and cyno BCMA, and epitope. The CAR molecules tested included: P5A, P5AC1, P5AC16, PC1, PC1C12, COM22, P6DY, and P6AP. Three different architectures were designed: version 1 (v1) comprises an FcγRIIIα hinge, version 2 (v2) comprises a CD8α hinge, and version 3 (v3) comprises and IgG1 hinge. The chimeric antigen receptors (CARs) shown in Table 5 were prepared and used and assessed for their degranulation activity towards BCMA+ cells. Degranulation activity was determined upon transient expression of each CAR in human T cells.
TABLE 5
Exemplary BCMA specific CARs
CAR
CAR Amino Acid Sequence
Components
P5A-V1
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFSSYAMNWVRQAPGKGLEWVSAISDSGGSTYYADSVK
P5A2_VHVL VH (Table
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
1 SEQ ID NO: 33);
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
P5A2_VHVL VL (SEQ ID
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSWPLTFGQGTKVEIK
NO: 34);
GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
FcγRIIIα hinge;
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
CD8α TM domain;
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
41BB ISD;
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
CD3ζ ISD
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 343)
P5A-V2
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFSSYAMNWVRQAPGKGLEWVSAISDSGGSTYYADSVK
P5A2_VHVL VH;
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
GS linker;
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
P5A2_VHVL VL;
SCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
CD8α hinge;
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSWPLTFGQGTKVEIK
CD8α TM domain;
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC
41BB ISD;
DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
CD3ζ ISD
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH
MQALPPR(SEQ ID NO: 344)
P5A-V3
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFSSYAMNWVRQAPGKGLEWVSAISDSGGSTYYADSVK
P5A2_VHVL VH;
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
GS linker;
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
P5A2_VHVL VL;
SCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
IgG1 hinge;
GSGSGTDFTLTISRLEPEDFAVYYCQQYGSwpriFGQGTKVEIK
CD8α TM domain;
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT
41BB ISD;
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
CD3ζ ISD
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR(SEQ ID NO: 345)
P5AC1-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V1
ASGFTFSSYAMNWVRQAPGKGLEWVSAILsSGGSTYYADSVK
A02_Rd4_0.6nM_C01
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
VH (SEQ ID NO: 72);
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
A02_Rd4_0.6nM_C01
GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK
VL (SEQ ID NO: 73);
GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
FcγRIIIα hinge;
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRvkFSRSAD
CD8α TM domain;
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
41BB ISD;
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
CD3ζ ISD
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 346)
P5AC1-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V2
ASGFTFSSYAMNWVRQAPGKGLEWVSAILsSGGSTYYADSVK
A02_Rd4_0.6nM_C01
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
VH;
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
A02_Rd4_0.6nM_C01
GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK
VL;
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC
CD8α hinge;
DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
CD8α TM domain;
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY
41BB ISD;
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
CD3ζ ISD
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH
MQALPPR (SEQ ID NO: 347)
P5AC1-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V2.1
ASGFTFSSYAMNWVRQAPGKGLEWVSAILSSGGSTYYADSVK
A02_Rd4_0.6nM_C01
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
VH;
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
A02_Rd4_0.6nM_C01
GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK
VL; rituximab epitope;
GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGSTTTPAP
CD8α hinge;
RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA
CD8α TM domain;
PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED
41BB ISD;
GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG
CD3ζ ISD
RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA
EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR (SEQ ID NO: 396)
P5AC1-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V3
ASGFTFSSYAMNWVRQAPGKGLEWVSAILsSGGSTYYADSVK
A02_Rd4_0.6nM_C01
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
VH;
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
A02_Rd4_0.6nM_C01
GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK
VL;
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT
IgG1 hinge;
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
CD8α TM domain;
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
41BB ISD;
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
CD3ζ ISD
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 348)
P5AC16-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V1
ASGFTFSSYAMNWVRQAPGKGLEWVSAISdFGGSTYYADSVK
A02_Rd4_6nM_C16 VH
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
(SEQ ID NO: 39);
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRASQSVSDIYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
A02_Rd4_6nM_C16 VL
GSGSGTDFTLTISRLEPEDFAVYYCQQYQTWPLTFGQGTKVEIK
(SEQ ID NO: 40);
GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
FcγRIIIα hinge;
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVkFSRSAD
CD8α TM domain;
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
41BB ISD;
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
CD3ζ ISD;
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 349)
P5AC16-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V2
ASGFTFSSYAMNWVRQAPGKGLEWVSAISdFGGSTYYADSVK
A02_Rd4_6nM_C16
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
VH;
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRASQSVSDIYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
A02_Rd4_6nM_C16 VL;
GSGSGTDFTLTISRLEPEDFAVYYCQQYQTWPLTFGQGTKVEIK
CD8α hinge;
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGANHTRGLDFAC
CD8α TM domain;
DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
41BB ISD;
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY
CD3ζ ISD
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH
MQALPPR (SEQ ID NO: 350)
P5AC16-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V3
ASGFTFSSYAMNWVRQAPGKGLEWVSAISdFGGSTYYADSVK
A02_Rd4_6nM_C16
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ
VH;
GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL
GS linker;
SCRASQSVSDIYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS
A02_Rd4_6nM_C16 VL;
GSGSGTDFTLTISRLEPEDFAVYYCQQYQTWPLTFGQGTKVEIK
IgG1 hinge;
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT
CD8α TM domain;
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
41BB ISD;
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
CD3ζ ISD
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 351)
PC1-V1
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSTYYADSVK
P5C1_VHVL VH (SEQ ID
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG
NO: 76);
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
GS linker;
TLSCRASQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF
P5C1_VHVL VL (SEQ ID
SGSGSGTDFTLTISRLEPEDFAVYYCQQYSTSPLTFGQGTKVEIK
NO: 77);
GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
FcγRIIIα hinge;
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRvKFSRSAD
CD8α TM domain;
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
41BB ISD;
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
CD3ζ ISD
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 352)
PC1-V2
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSTYYADSVK
P5C1_VHVL VH;
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG
GS linker;
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
P5C1_VHVL VL;
TLSCRASQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF
CD8α hinge;
SGSGSGTDFTLTISRLEPEDFAVYYCQQYSTSPLTFGQGTKVEIK
CD8α TM domain;
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC
41BB ISD;
DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
CD3ζ ISD
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH
MQALPPR (SEQ ID NO: 353)
PC1-V3
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSTYYADSVK
P5C1_VHVL VH;
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG
GS linker;
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
P5C1_VHVL VL;
TLSCRASQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF
IgG1 hinge;
SGSGSGTDFTLTISRLEPEDFAVYYCQQYSTSPLTFGQGTKVEIK
CD8α TM domain;
EPKSPDKTHTCPPCPAPPVAGPSVFLFRRKRKDTLNAIARTPEVT
41BB ISD;
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
CD3ζ ISD
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 354)
PC1C12-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V1
ASGFTFSSYPMSWVRQAPGKGLEWVSAIGgSGGWSYYADSVK
C01_Rd4_6nM_C12 VH
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG
(SEQ ID NO: 83);
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
GS linker;
TLSCWLSQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF
C01_Rd4_6nM_C12 VL
SGSGSGTDFTLTISRLEPEDFAVYYCQQYSEWPLTFGQGTKVEIK
(SEQ ID NO: 84);
GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
FcγRIIIα hinge;
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
CD8α TM domain;
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
41BB ISD;
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
CD3ζ ISD
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 355)
PC1C12-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V2
ASGFTFSSYPMSWVRQAPGKGLEWVSAIGgSGGWSYYADSVK
C01_Rd4_6nM_C12
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG
VH;
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
GS linker;
TLSCWLSQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF
C01_Rd4_6nM_C12 VL;
SGSGSGTDFTLTISRLEPEDFAVYYCQQYSEWPLTFGQGTKVEIK
CD8α hinge;
TTTPAPRPPTPAPTIASQPLSLRPEACRpAAGGAVHTRGLDFAC
CD8α TM domain;
DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
41BB ISD;
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY
CD3ζ ISD
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH
MQALPPR (SEQ ID NO: 356)
PC1C12-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V3
ASGFTFSSYPMSWVRQAPGKGLEWVSAIGgSGGWSYYADSVK
C01_Rd4_6nM_C12
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG
VH;
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
GS linker;
TLSCWLSQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF
C01_Rd4_6nM_C12 VL;
SGSGSGTDFTLTISRLEPEDFAVYYCQQYSEWPLTFGQGTKVEIK
IgG1 hinge;
EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT
CD8α TM domain;
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
41BB ISD;
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
CD3ζ ISD
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR (SEQ ID NO: 357)
COM22-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V1
ASGFTFSSYAMNWVRQAPGKGLEWVSAISdSGGSRWYADSV
COMBO_Rd4_0.6nM_C22
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRYWPMDIWG
VH (SEQ ID NO: 92);
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
GS linker;
TLSCRASVRVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRF
COMBO_Rd4_0.6nM_C22
SGSGSGTDFTLTISRLEPEDFAVYYCQQYMKWPLTFGQGTKVEI
VL (SEQ ID NO: 93);
KGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRK
FcγRIIIα hinge;
KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
CD8α TM domain;
ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
41BB ISD;
PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY
CD3ζ ISD
QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 358)
COM22-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V2
ASGFTFSSYAMNWVRQAPGKGLEWVSAISdSGGSRWYADSV
COMBO_Rd4_0.6nM_C22
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRYWPMDIWG
VH;
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
GS linker;
TLSCRASVRVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRF
COMBO_Rd4_0.6nM_C22
SGSGSGTDFTLTISRLEPEDFAVYYCQQYMKWPLTFGQGTKVEI
VL;
KTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA
CD8α hinge;
CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV
CD8α TM domain;
QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ
41BB ISD;
LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE
CD3ζ ISD
LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA
LHMQALPPR (SEQ ID NO: 359)
COM22-
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
V3
ASGFTFSSYAMNWVRQAPGKGLEWVSAISdSGGSRWYADSV
COMBO_Rd4_0.6nM_C22
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRYWPMDIWG
VH;
QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA
GS linker;
TLSCRASVRVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRF
COMBO_Rd4_0.6nM_C22
SGSGSGTDFTLTISRLEPEDFAVYYCQQYMKWPLTFGQGTKVEI
VL;
KEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEV
IgG1 hinge;
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
CD8α TM domain;
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
41BB ISD;
PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
CD3ζ ISD
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRK
KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY
QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 360)
P6DY-V1
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFGSYAMTWVRQAPGKGLEWVSAIDYSGGNTFYADSVK
L3.PY/H2.DY VH (SEQ
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIASGMDY
ID NO: 25);
WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG
GS linker;
ERATLSCRASQSVSSSYPSWYQQKPGQAPRLLIYGASSRATGIP
L3.PY/L1.PS/P6E01
DRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYPYPPSFTFGQGTK
VL (SEQ ID NO: 18);
VEIKGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRG
FcγRIIIα hinge;
RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS
CD8α TM domain;
RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG
41BB ISD;
GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD
CD3ζ ISD
GLYQGLSTATKDTYDALHMQ (SEQ ID NO: 361)
P6DY-V2
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFGSYAMTWVRQAPGKGLEWVSAIDYSGGNTFYADSVK
L3.PY/H2.DY VH;
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIASGMDY
GS linker;
WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG
L3.PY/L1.PS/P6E01
ERATLSCRASQSVSSSYPSWYQQKPGQAPRLLIYGASSRATGIP
VL;
DRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYPYPPSFTFGQGTK
CD8α hinge;
VEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD
CD8α TM domain;
FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP
41BB ISD;
VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN
CD3ζ ISD
QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN
ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD
ALHMQALPPR (SEQ ID NO: 362)
P6DY-V3
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFGSYAMTWVRQAPGKGLEWVSAIDYSGGNTFYADSVK
L3.PY/H2.DY VH;
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIASGMDY
GS linker;
WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG
L3.PY/L1.PS/P6E01
ERATLSCRASQSVSSSYPSWYQQKPGQAPRLLIYGASSRATGIP
VL;
DRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYPYPPSFTFGQGTK
IgG1 hinge;
VEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP
CD8α TM domain;
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
41BB ISD;
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
CD3ζ ISD
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGR
KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR
SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG
KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
YQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 363)
P6AP-V1
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFGSYAMTWVRQAPGKGLEWVSAISGSGGNTFYADSVK
P6AP-V1 VH (SEQ ID
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIAAPMDY
NO: 8);
WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG
GS linker;
ERATLSCRASQLGSFYLAWYQQKPGQAPRLLIYGASSRATGIPD
P6AP-V1 VL (SEQ ID
RFSGSGSGTDFTLTISRLEPEDFAVYYCQHYNYPPSFTFGQGTKV
NO: 80)
EIKGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGR
FcγRIIIα hinge;
KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGcELRVKFSR
CD8α TM domain;
SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG
41BB ISD;
KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
CD3ζ ISD
YQGLSTATKDTYDALHMQA (SEQ ID NO: 364)
P6AP-V2
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFGSYAMTWVRQAPGKGLEWVSAISGSGGNTFYADSVK
L1.LGF/L3.KW/H3.AP
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIAAPMDY
VH;
WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG
GS linker;
ERATLSCRASQLGSFYLAWYQQKPGQAPRLLIYGASSRATGIPD
P6AP-V1 VL;
RFSGSGSGTDFTLTISRLEPEDFAVYYCQHYNYPPSFTFGQGTKV
CD8α hinge;
EIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF
CD8α TM domain;
ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV
41BB ISD;
QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ
CD3ζ ISD
LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE
LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA
LHMQALPPR (SEQ ID NO: 365)
P6AP-V3
MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA
CD8α signal peptide;
ASGFTFGSYAMTWVRQAPGKGLEWVSAISGSGGNTFYADSVK
L1.LGF/L3.KW/H3.AP
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIAAPMDY
VH;
WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG
GS linker;
ERATLSCRASQLGSFYLAWYQQKPGQAPRLLIYGASSRATGIPD
P6AP-V1 VL;
RFSGSGSGTDFTLTISRLEPEDFAVYYCQHYNYPPSFTFGQGTKV
IgG1 hinge;
EIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPE
CD8α TM domain;
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
41BB ISD;
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
CD3ζ ISD
EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRK
KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY
QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 366)
For the activity assays, T cells from thirteen healthy donors (Donors 1-13) were obtained. Briefly, the T cells were purified from buffy-coat samples and activated using CD3/CD28 beads. Cells were transiently transfected with mRNAs encoding the different CAR molecules at D11/12 after activation. CAR activity was assessed by measuring their degranulation capacity, the interferon-γ (IFNγ) release, and the cytotoxic activity when co-cultured with (a) cells expressing BCMA (MM1S, KMS12BM, and L363), or (b) cells that do not express the BCMA protein (K562). Also included for each assay were mock transfected T cells (T cells in in buffer) to determine baseline activity of T cells that do not express a CAR.
CAR detection was done using a fusion protein in which the extracellular domain of the human BCMA protein was fused to a mouse IgG1 derived Fc fragment. Binding of the CAR at the cell surface with the BCMA portion of the fusion protein was detected with anti-Fc PE-conjugated antibody and analyzed by flow cytometry.
Materials and Methods
Primary T Cell Cultures
T cells were purified from Buffy coat samples provided by EFS (Etablissement Français du Sang, Paris, France) using Ficoll gradient density medium (Ficoll Paque PLUS/GE Healthcare Life Sciences). The PBMC layer was recovered and T cells were purified using a commercially available T cell enrichment kit (Stem Cell Technologies). Purified T cells were activated in X-Vivo™-15 medium (Lonza) supplemented with 20 ng/mL Human IL-2 (Miltenyi Biotech), 5% Human Serum (Sera Laboratories), and Dynabeads Human T activator CD3/CD28 at a bead:cell ratio 1:1 (Life Technologies). After activation cells were grown and maintained in X-Vivo™-15 medium (Lonza) supplemented with 20 ng/mL Human IL-2 (Miltenyi Biotec) and 5% Human Serum (Sera Laboratories)
CAR mRNA Transfection
Transfections were done at Day 4/5 or Day 11/12 after T cell purification and activation. 5 millions of cells were transfected with 15 μg of mRNA encoding the different CAR constructs. CAR mRNAs were produced using the mMESSAGE mMACHINE T7 Kit (Life Technologies) and purified using RNeasy Mini Spin Columns (Qiagen). Transfections were done using PulseAgile™ Cytopulse technology, by applying two 0.1 mS pulses at 3000V/cm followed by four 0.2 mS pulses at 325V/cm in 0.4 cm gap cuvettes in a final volume of 200 μl of “Cytoporation buffer T” (BTX Harvard Apparatus). Cells were immediately diluted in X-Vivo™-15 media (Lonza) and incubated at 37° C. with 5% CO2. IL-2 (from Miltenyi Biotec was added 2 h after electroporation at 20 ng/mL.
Degranulation Assay (CD107a Mobilization)
T cells were incubated in 96-well plates (50,000 cells/well), together with an equal amount of cells expressing or not the BCMA protein. Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15 medium (Lonza) for 6 hours at 37° C. with 5% CO2. CD107a staining was done during cell stimulation, by the addition of a fluorescent anti-CD107a antibody (APC conjugated, from Miltenyi Biotec) at the beginning of the co-culture, together with 1 μg/ml of anti-CD49d (BD Pharmingen), 1 μg/ml of anti-CD28 (Miltenyi Biotec), and 1× Monensin solution (eBioscience). After the 6h incubation period, cells were stained with a fixable viability dye (eFluor 780, from eBioscience) and fluorochrome-conjugated anti-CD8 (PE conjugated Miltenyi Biotec) and analyzed by flow cytometry. The degranulation activity was determined as the % of CD8+/CD107a+ cells, and by determining the mean fluorescence intensity signal (MFI) for CD107a staining among CD8+ cells. Degranulation assays were carried out 24 h after mRNA transfection. Results are summarized in the Tables 6A-9H and 9A-9C below. In the tables, the second column (labeled “CAR-T cell”) indicates the BCMA specific CAR being expressed in the transfected T cells.
CD107a expression on cells is a marker of antigen specific activation. The percent and MFI of CD107a on CD8 T cells expressing BCMA specific CARs increase when incubated with BCMA high (H929), medium (MM1S) and low (KMS12BM, L363) expressing cells but not BCMA negative cells (K562 and Daudi) (Tables 6A-9H and 9A-9C). CD107a expression levels did not increase on mock transfected T cells contacted with BCMA. Thus, the BCMA specific CAR-T cells are activated in the presence of BCMA-expressing cells but not in the presence of cells that do not express BCMA.
These results demonstrate that T cells expressing BCMA specific CARs are activated when incubated with BCMA expressing cells, and that the activation is antigen-specific.
IFN γ Release Assay
T cells were incubated in 96-well plates (50,000 cells/well), together with (a) cells expressing BCMA (MM1S, KMS12BM, and L363) or (b) cells that do not express the BCMA protein (K562). Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15 medium (Lonza) for 24 hours at 37° C. with 5% CO2. After this incubation period the plates were centrifuged at 1500 rpm for 5 minutes and the supernatants were recovered in a new plate. IFNγ detection in the cell culture supernatants was done by ELISA assay (Human IFNγ Quantikine ELISA Kit, from R&D Systems). The IFNγ release assays were carried by starting the cell co-cultures 24 h after mRNA transfection. Results are summarized in the Tables 8A-8D and 10 below.
As shown in Tables 8A-8D and 10, CD8 T cells expressing BCMA specific CARs produce IFNγ when incubated with either medium BCMA-expressing cells (MM1S) or low BCMA-expressing cells (KMS12BM, L363). In contrast, CD8 T cells expressing BCMA specific CARs produce negligible IFNγ when incubated with BCMA negative cells (K562).
These results demonstrate that T cells expressing BCMA specific CARs are activated when incubated with BCMA expressing cells, and that the activation is antigen-specific.
Cytotoxicity Assay
T cells were incubated in 96-well plates (100,000 cells/well), together with 10,000 target cells (expressing BCMA) and 10,000 control (BCMAneg) cells in the same well. Target and control cells were labelled with fluorescent intracellular dyes (CFSE or Cell Trace Violet, from Life Technologies) before co-culturing them with CAR+ T cells. The co-cultures were incubated for 4 hours at 37° C. with 5% CO2. After this incubation period, cells were labelled with a fixable viability dye (eFluor 780, from eBioscience) and analyzed by flow cytometry. Viability of each cellular population (target cells or BCMAneg control cells) was determined and the % of specific cell lysis was calculated. Cytotoxicity assays were carried out 48h after mRNA transfection. Results are summarized in the Tables 7A-7H below. In the tables, the cytotoxicity data are shown as percent viable cells, then calculated as a ratio of live BCMA positive cells/live BCMA negative cells. Cell lysis is calculated as 100—mock transfected T cells.
As shown in Tables 7A-7H, T cells expressing BCMA specific CARs exhibit killing activity when incubated with either medium BCMA-expressing cells (MM1S) or low BCMA-expressing cells (L363). In contrast, CD8 T cells expressing BCMA specific CARs do not exhibit killing activity when incubated with BCMA negative cells (K562).
In summary, T cells expressing the selected BCMA specific CARs shown in Table 5 are selectively activated upon contact with BCMA-expressing cells. While all versions of the BCMA specific CARs exhibited BCMA-specific activation, BCMA specific CARs comprising a CD8α hinge (v2) exhibited increased activation levels compared to BCMA specific CARs comprising a FcγRIIIα (v1) hinge or IgG1 (v3) hinge.
TABLE 6A
Degranulation Assay Results, Donor 1
%
MFI
CD107a+
CD107a+
(in CD8+)
Donor 1
mock
T cells
410
2.45
transfected
PMA/Iono
4038
76.1
T cells
MM1S
547
6.78
K562
610
7.55
P6DY
v1
T cells
588
5.19
PMA/Iono
3758
75.1
MM1S
850
14.9
K562
829
9.76
v2
T cells
756
6.86
PMA/Iono
4103
75.5
MM1S
3872
75.4
K562
1130
20.7
v3
T cells
707
7.71
PMA/Iono
4336
78.7
MM1S
3665
72.6
K562
612
7.7
P6AP
v1
T cells
604
4.61
PMA/Iono
3526
72.8
MM1S
1847
46.4
K562
503
4.28
v2
T cells
1380
27.8
PMA/Iono
2504
58
MM1S
5299
83.9
K562
949
14.6
v3
T cells
856
12.6
PMA/Iono
2500
58.9
MM1S
3638
73
K562
718
9.15
TABLE 6B
Degranulation Assay Results, Donor 2
%
MFI
CD107a+
CD107a+
(in CD8+)
Donor 2
mock
T cells
270
1.66
transfected T
PMA/Iono
3872
88.3
cells
MM1S
499
11
K562
492
8.78
P5A
v1
T cells
423
7.2
PMA/Iono
6034
96.3
MM1S
2670
77.6
K562
648
16.6
v2
T cells
428
7.14
PMA/Iono
4420
90.7
MM1S
5019
91.8
K562
620
13.8
v3
T cells
451
8.87
PMA/Iono
4835
93.2
MM1S
4191
88.5
K562
607
14.1
P5A_C1
v1
T cells
315
4.12
PMA/Iono
3567
85.8
MM1S
2193
68.6
K562
537
10.1
v2
T cells
413
7.46
PMA/Iono
4423
91.1
MM1S
4575
90.6
K562
660
17.2
v3
T cells
429
7.82
PMA/Iono
4442
93.5
MM1S
3710
84.4
K562
597
13.9
P5A_C16
v1
T cells
424
7.95
PMA/Iono
4325
91.1
MM1S
1858
61.6
K562
636
14.9
v2
T cells
401
5.69
PMA/Iono
3007
80
MM1S
4228
87.9
K562
696
17.6
v3
T cells
372
5.25
PMA/Iono
3611
86.6
MM1S
3372
83.6
K562
476
7.72
TABLE 6C
Degranulation Assay Results, Donor 3
%
MFI
CD107a+
CD107a+
(in CD8+)
Donor 3
mock
T cells
338
3.61
transfected T
PMA/Iono
7111
98.1
cells
MM1S
464
9.44
K562
533
9.73
PC1
v1
T cells
454
6.67
PMA/Iono
5226
96.5
MM1S
2178
75.6
K562
753
22.3
v2
T cells
507
13
PMA/Iono
4743
95.2
MM1S
759
25.5
K562
649
15.5
v3
T cells
463
6.84
PMA/Iono
7092
98.1
MM1S
2857
87.2
K562
665
15
PC1C12
v1
T cells
373
3.35
PMA/Iono
6214
97.2
MM1S
1960
68.2
K562
513
7.61
v2
T cells
579
11.5
PMA/Iono
6341
97.5
MM1S
4478
95.1
K562
680
15
v3
T cells
533
10.1
PMA/Iono
5785
97.4
MM1S
3739
91
K562
648
13.2
COM22
v1
T cells
354
2.74
PMA/Iono
5894
96.7
MM1S
2219
76.1
K562
445
5.62
v2
T cells
401
6.52
PMA/Iono
5802
94.6
MM1S
2372
79.2
K562
534
8.9
v3
T cells
501
10.4
PMA/Iono
6387
97.6
MM1S
2780
85.9
K562
648
13.8
TABLE 6D
Degranulation Assay Results, Donor 4
%
MFI
CD107a+
CD107a+
(in CD8+)
Donor
mock
T cells
248
2.64
4 (v3
transfected
PMA/Iono
5750
94.9
only)
T cells
MM1S
363
8.89
K562
368
6.86
P5A
T cells
335
3.82
PMA/Iono
6025
93
MM1S
3150
86.7
K562
418
9.91
P5AC1
T cells
505
22.1
PMA/Iono
6950
98.3
MM1S
2975
84.7
K562
575
23.3
P5AC16
T cells
368
6.2
PMA/Iono
5775
97.7
MM1S
3675
86.8
K562
420
9.73
PC1
T cells
403
9.05
PMA/Iono
6975
97.8
MM1S
4625
93
K562
543
15.8
PC1C12
T cells
485
12.9
PMA/Iono
6400
96.5
MM1S
3575
90.4
K562
585
18.9
COM22
T cells
535
20.5
PMA/Iono
7250
98.3
MM1S
3725
91.4
K562
533
16.9
P6DY
T cells
313
3.08
PMA/Iono
5125
94.3
MM1S
2435
79.9
K562
438
10.4
P6AP
T cells
430
10.4
PMA/Iono
6100
94.2
MM1S
3800
91.7
K562
478
14.6
TABLE 6E
Degranulation Assay Results, Donor 5
%
CD107a+
MFI
(in CD8+)
CD107a+
Donor
CAR-
L363
47
917
5 (v3
BCMA-P5A
MM1S
65.3
1713
only)
K562
3.65
247
T cells
1.71
199
PMA/iono
98.6
4797
CAR-
L363
50.6
1117
BCMA-
MM1S
65.5
1753
P5AC1
K562
5.29
265
T cells
1.93
213
PMA/iono
99.1
5755
CAR-
L363
57.2
1392
BCMA-
MM1S
73.9
2520
P5AC16
K562
4.13
273
T cells
2.57
232
PMA/iono
98.1
5120
CAR-
L363
71.9
2167
BCMA-
MM1S
82.9
2987
PC1
K562
4.5
316
T cells
2.47
273
PMA/iono
98.5
5556
CAR-
L363
57.8
1492
BCMA-
MM1S
71.5
2094
PC1C12
K562
3.72
313
T cells
2.53
272
PMA/iono
98.2
4480
CAR-
L363
61.3
1574
BCMA-
MM1S
78.1
2602
COM22
K562
5.84
296
T cells
5.26
284
PMA/iono
98.3
4434
CAR-
L363
43.4
859
BCMA-
MM1S
63.6
1624
P6DY
K562
3.99
256
T cells
1.95
228
PMA/iono
98.1
4075
CAR-
L363
63.4
1745
BCMA-
MM1S
77.8
2461
P6AP
K562
4.81
310
T cells
4.74
300
PMA/iono
98.9
32
mock
L363
2.54
200
transfected
MM1S
5.19
233
T cells
K562
4.02
201
T cells
1.95
192
PMA/iono
97.7
3216
TABLE 6F
Degranulation Assay Results, Donor 6
%
MFI
CD107a+
CD107a+
(in CD8+)
Donor 6
BCMA_BC30_v3 (18)
Tcells alone
121
1.04
T cells PMA
5253
87.4
IONO
T cells K562
230
3.21
T cells MM1S
1321
50.4
T cells L363
986
41.8
CAR_BCMA_P5AC1_v2
Tcells alone
150
1.07
T cells PMA
4701
83.2
IONO
T cells K562
256
5.5
T cells MM1S
2193
63.8
T cells L363
1400
50.9
CAR_BCMA_P5AC1_v3
Tcells alone
166
0.96
T cells PMA
4518
80.2
IONO
T cells K562
301
6.87
T cells MM1S
1101
40.7
T cells L363
728
29.8
CAR_BCMA_PC1_v3
Tcells alone
217
1.63
T cells PMA
4711
82.4
IONO
T cells K562
329
6.36
T cells MM1S
2083
60.3
T cells L363
1500
52.1
CAR_BCMA_PC1C12_v2
Tcells alone
209
2.01
T cells PMA
5401
87.8
IONO
T cells K562
332
7.7
T cells MM1S
2588
68.4
T cells L363
1976
59.5
CAR_BCMA_PC1C12_v3
Tcells alone
162
1.72
T cells PMA
5299
85.3
IONO
T cells K562
266
6.25
T cells MM1S
669
28.8
T cells L363
414
18.6
CAR_BCMA_COM22_v3
Tcells alone
193
3.23
T cells PMA
4750
82.7
IONO
T cells K562
288
5.13
T cells MM1S
814
35.7
T cells L363
606
26.8
CAR_BCMA_P6AP_v2
Tcells alone
359
9.69
T cells PMA
5521
87.4
IONO
T cells K562
327
7.69
T cells MM1S
2289
63.8
T cells L363
1876
56.9
CAR_BCMA_P6AP_v3
Tcells alone
284
4.87
T cells PMA
4480
82.7
IONO
T cells K562
331
5.9
T cells MM1S
1409
46.9
T cells L363
926
35.3
mock transfected T cells
Tcells alone
184
0.92
T cells PMA
3955
78.6
IONO
T cells K562
278
3.58
T cells MM1S
393
4.7
T cells L363
190
1.12
TABLE 6G
Degranulation Assay Results, Donor 7
%
MFI
CD107a+
CD107a+
(in CD8+)
Donor 7
mock transfected T
Tcells alone
68.3
1.55
cells
T cells PMA
3097
94.6
IONO
T cells MM1S
118
7.15
T cells L363
90.3
2.63
T cells K562
144
3.4
T cells Daudi
117
1.93
BCMA_BC30_v3
Tcells alone
69.7
2.69
(18)
T cells PMA
2864
94.9
IONO
T cells MM1S
1630
68.9
T cells L363
529
43.8
T cells K562
125
3.85
T cells Daudi
426
38.5
P5AC1_v2
Tcells alone
111
3.67
T cells PMA
2859
95.6
IONO
T cells MM1S
2305
71.5
T cells L363
877
53.1
T cells K562
166
8.54
T cells Daudi
770
51.5
P5AC1_v3
Tcells alone
70.8
1.04
T cells PMA
2740
94.6
IONO
T cells MM1S
526
43.3
T cells L363
209
20.4
T cells K562
118
8.32
T cells Daudi
450
35.9
PC1_v3
Tcells alone
61
1.37
T cells PMA
2786
94.6
IONO
T cells MM1S
1027
56.3
T cells L363
314
29.9
T cells K562
140
12.1
T cells Daudi
536
39.6
PC1C12_v2
Tcells alone
98
5.95
T cells PMA
3493
95.3
IONO
T cells MM1S
1917
73.7
T cells L363
939
56.2
T cells K562
192
11.5
T cells Daudi
1485
64.6
PC1C12_v3
Tcells alone
84.2
2.28
T cells PMA
3017
95.2
IONO
T cells MM1S
342
28.2
T cells L363
145
8.72
T cells K562
186
7.53
T cells Daudi
223
11.8
COM22_v3
Tcells alone
93.6
5.32
T cells PMA
2989
96.3
IONO
T cells MM1S
540
40
T cells L363
154
12.5
T cells K562
138
8.29
T cells Daudi
93.5
3.99
P6AP_v2
Tcells alone
164
13.7
T cells PMA
3303
95.9
IONO
T cells MM1S
2755
76
T cells L363
859
50.3
T cells K562
287
15.8
T cells Daudi
1263
58.2
P6AP_v3
Tcells alone
114
10.5
T cells PMA
3084
94.5
IONO
T cells MM1S
849
51.6
T cells L363
380
30.9
T cells K562
211
8.46
T cells Daudi
678
42.7
TABLE 6H
Degranulation Assay Results, Donor 8
%
MFI
CD107a+
CD107a+
(in CD8+)
Donor 8
mock transfected T
Tcells alone
154
0.67
cells
T cells PMA
3777
66.2
IONO
T cells MM1S
229
2.16
T cells L363
166
1.51
T cells K562
220
2.08
BCMA_BC30_v3
Tcells alone
210
1.05
(18)
T cells PMA
4302
70.6
IONO
T cells MM1S
1661
42
T cells L363
1049
26.5
T cells K562
262
3.46
P5AC1_v2
Tcells alone
207
0.86
T cells PMA
4298
71.5
IONO
T cells MM1S
1648
40.8
T cells L363
1099
26.5
T cells K562
232
1.72
P5AC1_v3
Tcells alone
187
0.84
T cells PMA
3989
68.8
IONO
T cells MM1S
766
21.2
T cells L363
521
14.2
T cells K562
258
2.05
PC1_v3
Tcells alone
242
1.23
T cells PMA
4256
70.6
IONO
T cells MM1S
1046
23.1
T cells L363
1183
27.4
T cells K562
283
2.97
PC1C12_v2
Tcells alone
257
1.87
T cells PMA
3487
60.2
IONO
T cells MM1S
2463
51.2
T cells L363
1657
35.4
T cells K562
314
4.05
PC1C12_v3
Tcells alone
166
0.86
T cells PMA
4238
69.1
IONO
T cells MM1S
641
17.3
T cells L363
507
14.2
T cells K562
296
3.52
COM22_v3
Tcells alone
283
2.55
T cells PMA
4800
75.9
IONO
T cells MM1S
1035
27.9
T cells L363
704
22.7
T cells K562
334
4.82
P6AP_v2
Tcells alone
545
8.33
T cells PMA
4362
68.6
IONO
T cells MM1S
2273
46.7
T cells L363
1671
34.7
T cells K562
629
9.71
P6AP_v3
Tcells alone
360
3.87
T cells PMA
3584
61.5
IONO
T cells MM1S
1553
34.5
T cells L363
1045
23
T cells K562
595
7.4
TABLE 7A
Cytotoxicity Data, Donor 6
Viability (mean)
CAR
L363
K562
MM1S
K562
Donor 6
BC30_v3
22.93
89.90
16.30
88.43
P5AC1_v2
27.27
90.07
21.47
90.17
P5AC1_v3
36.03
89.30
19.80
88.50
PC1_v3
19.03
88.23
13.57
87.50
PC1C12_v2
19.60
86.13
14.67
84.67
PC1C12_v3
55.50
89.33
41.33
88.67
COM22_v3
42.00
90.33
25.67
88.30
P6AP_v2
29.40
80.27
21.07
82.10
P6AP_v3
48.53
85.20
25.57
81.30
mock
90.90
88.20
91.77
86.30
transfected T
cells
TABLE 7B
Cytotoxicity Data, Donor 6
Ratio to Mock
BCMA+/BCMA−
transfected T cells
Cell lysis
CAR
L363
MM1S
L363
MM1S
L363
MM1S
Donor 6
BC30_v3
25.51
18.43
0.24752108
0.17333946
75.2
82.7
P5AC1_v2
30.27
23.81
0.29374647
0.22389502
70.6
77.6
P5AC1_v3
40.35
22.37
0.39152336
0.21040098
60.8
79.0
PC1_v3
21.57
15.50
0.2093085
0.14581122
79.1
85.4
PC1C12_v2
22.76
17.32
0.22079514
0.1629089
77.9
83.7
PC1C12_v3
62.13
46.62
0.60281513
0.4383953
39.7
56.2
COM22_v3
46.49
29.07
0.45113441
0.27335977
54.9
72.7
P6AP_v2
36.63
25.66
0.35539949
0.24131177
64.5
75.9
P6AP_v3
56.96
31.45
0.55272006
0.29573955
44.7
70.4
mock transfected T cells
103.06
106.33
1
1
0.0
0.0
TABLE 7C
Cytotoxicity Data, Donor 7
Viability (mean)
CAR
L363
K562
MM1S
K562
Donor 7
mock
92.53
92.80
90.70
92.33
transfected T
cells
BC30_v3
46.00
90.40
34.00
89.83
P5AC1_v2
50.50
90.73
35.17
89.40
P5AC1_v3
60.20
89.97
43.03
89.53
PC1_v3
49.43
89.67
37.33
88.97
PC1C12_v2
40.23
88.50
22.53
87.53
PC1C12_v3
81.03
91.30
71.70
89.83
COM22_v3
67.87
90.00
52.97
89.20
P6AP_v2
57.33
89.93
32.87
87.10
P6AP_v3
66.37
91.60
46.35
94.00
TABLE 7D
Cytotoxicity Data, Donor 7
Ratio to Mock
BCMA+/BCMA−
transfected T cells
Cell lysis
CAR
L363
MM1S
L363
MM1S
L363
MM1S
Donor 7
mock transfected T cells
99.71
98.23
1
1
0.0
0.0
BC30_v3
50.88
37.85
0.51031598
0.38529434
49.0
61.5
P5AC1_v2
55.66
39.34
0.55818001
0.40044688
44.2
60.0
P5AC1_v3
66.91
48.06
0.67106507
0.48929577
32.9
51.1
PC1_v3
55.13
41.96
0.55288988
0.4271896
44.7
57.3
PC1C12_v2
45.46
25.74
0.45592406
0.26206149
54.4
73.8
PC1C12_v3
88.76
79.81
0.89010798
0.81251777
11.0
18.7
COM22_v3
75.41
59.38
0.7562472
0.60448985
24.4
39.6
P6AP_v2
63.75
37.73
0.63934647
0.38413929
36.1
61.6
P6AP_v3
72.45
49.31
0.7266149
0.50196463
27.3
49.8
TABLE 7E
Cytotoxicity Data, Donor 8
Viability (mean)
CAR
L363
K562
MM1S
K562
Donor 8
mock transfected
93.97
91.13
95.97
88.07
T cells
BC30_v3
67.97
86.80
46.40
78.87
P5AC1_v2
69.80
85.37
47.13
79.17
P5AC1_v3
77.90
88.77
62.70
84.40
PC1_v3
61.67
86.60
41.67
78.97
PC1C12_v2
62.43
85.27
35.27
78.20
PC1C12_v3
85.17
85.27
78.87
77.77
COM22_v3
76.70
87.87
56.40
84.50
P6AP_v2
77.23
84.90
61.47
83.47
P6AP_v3
83.23
85.67
72.57
84.63
cell lines
95.20
94.97
96.97
94.20
TABLE 7F
Cytotoxicity Data, Donor 8
Ratio to Mock
BCMA+/BCMA−
transfected T cells
Cell lysis
CAR
L363
MM1S
L363
MM1S
L363
MM1S
Donor 8
mock transfected T cells
1.03
0.95
1
1
0.0
0.0
BC30_v3
0.78
0.59
0.75941589
0.61953757
24.1
38.0
P5AC1_v2
0.82
0.60
0.79299515
0.62694429
20.7
37.3
P5AC1_v3
0.88
0.74
0.85112036
0.78229085
14.9
21.8
PC1_v3
0.71
0.53
0.69061501
0.5556331
30.9
44.4
PC1C12_v2
0.73
0.45
0.7101346
0.47489852
29.0
52.5
PC1C12_v3
1.00
1.01
0.96871004
1.06793091
3.1
−6.8
COM22_v3
0.87
0.67
0.84659295
0.70285469
15.3
29.7
P6AP_v2
0.91
0.74
0.88226799
0.77547847
11.8
22.5
P6AP_v3
0.97
0.86
0.94229927
0.9028984
5.8
9.7
cell lines
1.00
1.03
0.97223038
1.08396365
2.8
−8.4
TABLE 7G
Cytotoxicity Data, Donor 9
Viability (mean)
CAR
L363
K562
MM1S
K562
Donor 9
mock transfected
86.3
87.8
69.6
86.5
T cells
BC30_v3
27.1
86.6
16.0
86.6
P5AC1_v2
31.9
87.9
21.0
87.2
P5AC1_v3
46.9
85.1
36.3
84.0
PC1_v3
27.8
85.3
25.4
85.0
PC1C12_v2
29.3
88.7
15.0
86.0
COM22_v3
49.0
88.8
35.7
87.5
P6AP_v2
41.4
85.7
22.8
84.0
P6AP_v3
56.4
84.3
44.9
84.4
Cell lines
92.3
91.7
83.5
91.8
TABLE 7H
Cytotoxicity Data, Donor 9
Ratio to Mock
BCMA+/BCMA−
transfected T cells
Cell lysis
CAR
L363
MM1S
L363
MM1S
L363
MM1S
Donor 9
mock transfected T cells
0.98216319
0.80469954
1
1
0
0
BC30_v3
0.31331794
0.18444359
0.31900802
0.22920802
68.10
77.08
P5AC1_v2
0.3631539
0.24111578
0.36974905
0.29963455
63.03
70.04
P5AC1_v3
0.55133229
0.43231441
0.56134489
0.53723706
43.87
46.28
PC1_v3
0.32551778
0.29831439
0.33142942
0.37071525
66.86
62.93
PC1C12_v2
0.3298272
0.17473847
0.3358171
0.21714748
66.42
78.29
COM22_v3
0.55159475
0.40746382
0.56161212
0.50635523
43.84
49.36
P6AP_v2
0.48289269
0.27092424
0.49166238
0.33667751
50.83
66.33
P6AP_v3
0.66903915
0.53199052
0.68118939
0.66110454
31.88
33.89
Cell lines
1.00690909
0.90889292
1.02519531
1.1294811
−2.52
−12.95
TABLE 8A
IFNγ Production (pg/mL), Donor 6
Donor 6
CAR
pg/ml
mock transfected T cells
Tcells alone
155.1
BCMA_BC30_v3 (18)
654.71
P5AC1_v2
174.035
P5AC1_v3
61.215
PC1_v3
255.045
PC1C12_v2
481.595
PC1C12_v3
463.08
COM22_v3
2996.305
P6AP_v2
1294.055
P6AP_v3
500.435
mock transfected T cells
T cells PMA IONO
81654.2
BCMA_BC30_v3 (18)
49368.7
P5AC1_v2
49102.7
P5AC1_v3
66837.7
PC1_v3
70798.2
PC1C12_v2
56402.2
PC1C12_v3
121954.7
COM22_v3
125878.7
P6AP_v2
73577.2
P6AP_v3
51242.7
mock transfected T cells
T cells K562
−83.215
BCMA_BC30_v3 (18)
265.565
P5AC1_v2
−10.05
P5AC1_v3
36.475
PC1_v3
−74.04
PC1C12_v2
344.72
PC1C12_v3
583.99
COM22_v3
610.97
P6AP_v2
40.66
P6AP_v3
36.775
mock transfected T cells
T cells MM1S
660.33
BCMA_BC30_v3 (18)
8004.42
P5AC1_v2
5667.72
P5AC1_v3
2619.735
PC1_v3
6152.67
PC1C12_v2
8526.27
PC1C12_v3
1405.945
COM22_v3
3330.27
P6AP_v2
5436.27
P6AP_v3
3881.115
mock transfected T cells
T cells L363
1287.38
BCMA_BC30_v3 (18)
6363.72
P5AC1_v2
3116.725
P5AC1_v3
2720.52
PC1_v3
6661.97
PC1C12_v2
9478.72
PC1C12_v3
1707.885
COM22_v3
2397.83
P6AP_v2
5911.97
P6AP_v3
3470.38
TABLE 8B
IFNγ Production (pg/mL), Donor 7
Donor 7
CAR
pg/ml
mock transfected T cells
Tcells alone
−3.1
BCMA_BC30_v3 (18)
64.1
P5AC1_v2
−18.0
P5AC1_v3
−73.0
PC1_v3
6.1
PC1C12_v2
156.5
PC1C12_v3
100.1
COM22_v3
182.9
P6AP_v2
564.7
P6AP_v3
107.0
mock transfected T cells
T cells PMA IONO
44970.8
BCMA_BC30_v3 (18)
32725.3
P5AC1_v2
27476.6
P5AC1_v3
13100.5
PC1_v3
40824.4
PC1C12_v2
39884.0
PC1C12_v3
30245.2
COM22_v3
62690.4
P6AP_v2
69923.2
P6AP_v3
88578.4
mock transfected T cells
T cells MM1S
29.9
BCMA_BC30_v3 (18)
4662.6
P5AC1_v2
3420.3
P5AC1_v3
1173.7
PC1_v3
2478.5
PC1C12_v2
5314.6
PC1C12_v3
809.9
COM22_v3
1344.6
P6AP_v2
3020.3
P6AP_v3
2166.7
mock transfected T cells
T cells L363
15.6
BCMA_BC30_v3 (18)
2360.2
P5AC1_v2
2576.3
P5AC1_v3
582.7
PC1_v3
1723.3
PC1C12_v2
2962.9
PC1C12_v3
136.6
COM22_v3
467.4
P6AP_v2
2081.4
P6AP_v3
1119.0
mock transfected T cells
T cells K562
−80.5
BCMA_BC30_v3 (18)
−127.2
P5AC1_v2
−124.4
P5AC1_v3
−47.9
PC1_v3
−93.6
PC1C12_v2
21.8
PC1C12_v3
−55.4
COM22_v3
−36.1
P6AP_v2
83.8
P6AP_v3
83.8
mock transfected T cells
335.1
BCMA_BC30_v3 (18)
T cells Daudi
7794.8
P5AC1_v2
8093.7
P5AC1_v3
3870.6
PC1_v3
6068.9
PC1C12_v2
10190.2
PC1C12_v3
1638.8
COM22_v3
4287.6
P6AP_v2
6971.6
P6AP_v3
5280.0
TABLE 8C
IFN-γ Production (pg/mL), Donor 8
Donor 8
CAR
pg/ml
mock transfected T cells
Tcells alone
−697.44
BCMA_BC30_v3 (18)
−660.92
P5AC1_v2
−603.38
P5AC1_v3
−543.44
PC1_v3
−552.22
PC1C12_v2
−399.26
PC1C12_v3
−652.73
COM22_v3
−530.09
P6AP_v2
17.24
P6AP_v3
−289.82
mock transfected T cells
T cells PMA IONO
37206.73
BCMA_BC30_v3 (18)
53311.73
P5AC1_v2
57732.14
P5AC1_v3
52577.56
PC1_v3
48925.48
PC1C12_v2
38310.06
PC1C12_v3
71881.73
COM22_v3
61941.73
P6AP_v2
82339.64
P6AP_v3
63337.14
mock transfected T cells
T cells MM1S
−684.65
BCMA_BC30_v3 (18)
2976.34
P5AC1_v2
2727.71
P5AC1_v3
769.05
PC1_v3
2682.98
PC1C12_v2
5019.05
PC1C12_v3
−198.04
COM22_v3
1155.19
P6AP_v2
2945.65
P6AP_v3
671.21
mock transfected T cells
T cells L363
−664.74
BCMA_BC30_v3 (18)
2934.77
P5AC1_v2
2342.50
P5AC1_v3
579.85
PC1_v3
2232.65
PC1C12_v2
3676.59
PC1C12_v3
−303.86
COM22_v3
695.72
P6AP_v2
1612.74
P6AP_v3
311.07
mock transfected T cells
T cells K562
−672.42
BCMA_BC30_v3 (18)
−583.71
P5AC1_v2
−631.02
P5AC1_v3
−650.83
PC1_v3
−615.50
PC1C12_v2
−501.18
PC1C12_v3
−615.17
COM22_v3
−596.02
P6AP_v2
−393.94
P6AP_v3
−476.71
TABLE 8D
IFN-γ Production (pg/mL), Donor 9
Donor 9
CAR
pg/ml
mock transfected T cells
Tcells alone
93.2
BCMA_BC30_v3 (18)
1225.2
P5AC1_v2
1344.5
P5AC1_v3
632.3
PC1_v3
2745.7
PC1C12_v2
48.1
COM22_v3
2656.5
P6AP_v2
566.5
P6AP_v3
−335.8
mock transfected T cells
T cells PMA IONO
12505.8
BCMA_BC30_v3 (18)
12312.2
P5AC1_v2
10607.5
P5AC1_v3
12014.7
PC1_v3
12829.9
PC1C12_v2
13829.5
COM22_v3
13489.5
P6AP_v2
13182.1
P6AP_v3
13506.3
mock transfected T cells
T cells MM1S
1006.4
BCMA_BC30_v3 (18)
2376.8
P5AC1_v2
−359.5
P5AC1_v3
97.8
PC1_v3
290.1
PC1C12_v2
752.7
COM22_v3
−601.0
P6AP_v2
−304.1
P6AP_v3
−394.9
mock transfected T cells
T cells L363
−228.2
BCMA_BC30_v3 (18)
3000.2
P5AC1_v2
2314.0
P5AC1_v3
1646.4
PC1_v3
−15.4
PC1C12_v2
2796.5
COM22_v3
320.6
P6AP_v2
−163.0
P6AP_v3
−233.9
mock transfected T cells
T cells K562
−227.9
BCMA_BC30_v3 (18)
2027.5
P5AC1_v2
3928.4
P5AC1_v3
300.2
PC1_v3
74.9
PC1C12_v2
1835.7
COM22_v3
45.0
P6AP_v2
51.4
P6AP_v3
158.3
TABLE 9A
Degranulation Assay Results, Donor 10
%
MFI
CD107a+
Donor 10
CD107a+
(in CD8+)
LT alone
mock transfected T cells
82.2
1.95
26859 P5AC1-V2
83.8
1.47
26868 PC1C12-V2
94.2
3.21
26871 COM22-V2
107
5.96
PMA Iono
mock transfected T cells
5933
99
26859 P5AC1-V2
5863
99
26868 PC1C12-V2
6366
99.4
26871 COM22-V2
6149
99
MM1S
mock transfected T cells
211
16.5
26859 P5AC1-V2
1377
74.4
26868 PC1C12-V2
1760
79.1
26871 COM22-V2
1470
76.5
H929
mock transfected T cells
141
6.09
26859 P5AC1-V2
1026
65.4
26868 PC1C12-V2
1262
71.1
26871 COM22-V2
784
59.2
L363
mock transfected T cells
153
6.48
26859 P5AC1-V2
793
60.1
26868 PC1C12-V2
1054
67.3
26871 COM22-V2
827
61.7
MM1S GFP LUC
mock transfected T cells
187
9.88
26859 P5AC1-V2
1228
70.5
26868 PC1C12-V2
1476
74.9
26871 COM22-V2
1095
68.5
H929 GFP LUC
mock transfected T cells
153
9.48
26859 P5AC1-V2
1648
77.8
26868 PC1C12-V2
1960
84
26871 COM22-V2
1029
69.4
L363 GFP LUC
mock transfected T cells
104
3.06
26859 P5AC1-V2
753
60.7
26868 PC1C12-V2
873
64.6
26871 COM22-V2
766
61.1
KMS12BM GFP LUC
mock transfected T cells
91.3
2.67
26859 P5AC1-V2
945
67.2
26868 PC1C12-V2
1192
71.2
26871 COM22-V2
961
67.2
K562
mock transfected T cells
127
6.06
26859 P5AC1-V2
136
9.1
26868 PC1C12-V2
119
9.49
26871 COM22-V2
135
9.55
TABLE 9B
Degranulation Assay Results, Donor 11
%
MFI
CD107a+
Donor 11
CD107a+
(in CD8+)
LT alone
mock transfected T cells
69.9
0.57
26859 P5AC1-V2
68.3
0.62
26868 PC1C12-V2
67.2
0.88
26871 COM22-V2
80.9
3.95
PMA Iono
mock transfected T cells
5511
91.7
26859 P5AC1-V2
5360
97.4
26868 PC1C12-V2
4741
96.1
26871 COM22-V2
5066
95.7
KMS12BM GFP LUC
mock transfected T cells
77.8
1.81
26859 P5AC1-V2
1304
68.3
26868 PC1C12-V2
650
45.5
26871 COM22-V2
986
62.5
H929 GFP LUC
mock transfected T cells
73
1.04
26859 P5AC1-V2
738
49.6
26868 PC1C12-V2
428
30.9
26871 COM22-V2
468
35.5
MM1S
mock transfected T cells
121
2.67
26859 P5AC1-V2
854
52
26868 PC1C12-V2
399
26.4
26871 COM22-V2
486
33.4
K562
mock transfected T cells
125
3.08
26859 P5AC1-V2
140
3.35
26868 PC1C12-V2
123
1.84
26871 COM22-V2
161
4.11
TABLE 9C
Degranulation Assay Results, Donor 12
%
MFI
CD107a+
Donor 11
CD107a+
(in CD8+)
LT alone
mock transfected T cells
69.9
0.57
26859 P5AC1-V2
68.3
0.62
26868 PC1C12-V2
67.2
0.88
26871 COM22-V2
80.9
3.95
PMA Iono
mock transfected T cells
5511
91.7
26859 P5AC1-V2
5360
97.4
26868 PC1C12-V2
4741
96.1
26871 COM22-V2
5066
95.7
KMS12BM GFP LUC
mock transfected T cells
77.8
1.81
26859 P5AC1-V2
1304
68.3
26868 PC1C12-V2
650
45.5
26871 COM22-V2
986
62.5
H929 GFP LUC
mock transfected T cells
73
1.04
26859 P5AC1-V2
738
49.6
26868 PC1C12-V2
428
30.9
26871 COM22-V2
468
35.5
MM1S
mock transfected T cells
121
2.67
26859 P5AC1-V2
854
52
26868 PC1C12-V2
399
26.4
26871 COM22-V2
486
33.4
K562
mock transfected T cells
125
3.08
26859 P5AC1-V2
140
3.35
26868 PC1C12-V2
123
1.84
26871 COM22-V2
161
4.11
TABLE 10
IFN gamma release assay results, Donor 10
Donor 10
mock transfected T cells
T cells alone
871.8
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
1466.2
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
1172.2
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
1873.1
pg/mL
mock transfected T cells
MM1S LucGFP
1436.5
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
12208.4
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
13695.3
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
10784.1
pg/mL
mock transfected T cells
MM1S
5329.0
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
6060.3
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
6776.1
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
7827.0
pg/mL
mock transfected T cells
H929 LucGFP
754.2
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
16589.9
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
15989.7
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
14410.4
pg/mL
mock transfected T cells
H929
809.8
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
18072.7
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
17948.1
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
14437.3
pg/mL
mock transfected T cells
L363 LucGFP
1184.5
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
11556.9
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
13254.5
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
11384.1
pg/mL
mock transfected T cells
L363
1777.3
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
15685.1
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
14929.1
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
14995.7
pg/mL
mock transfected T cells
L363 LucGFP
1184.5
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
11556.9
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
13254.5
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
11384.1
pg/mL
mock transfected T cells
KMS12BM LucGFP
1283.2
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
9073.3
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
10060.6
pg/mL
pCLS26871 CAR_BCMA_COM22_v2
10687.2
pg/mL
mock transfected T cells
K562
691.6
pg/mL
pCLS26859 CAR_BCMA_P5AC1_v2
684.1
pg/mL
pCLS26868 CAR_BCMA_PC1C12_v2
904.2
pg/mL
pCLS26871 CAR BCMA COM22 v2
969.0
pg/mL
Example 3
BCMA Specific CAR-T Cells Induce Tumor Regression in MM1.S Tumor Model
This example illustrates treatment of tumors with BCMA specific CAR-T cells using the MM1.S tumor model.
In vivo efficacy study of BCMA specific CAR-T cells was performed with MM1.S, expressing luciferase and GFP, orthotopic model. Five million MM1.S Luc2AGFP cells were injected intravenously through the tail vein into 6-8 weeks old female Nod/Scid/IL2Rg−/−(NSG) animals. Intraperitoneal injection of D-luciferin (Regis Technologies, Morton Grove, Ill.) (200 uL per animal at 15 mg/mL), followed by anesthesia with isofluorane and subsequent whole body bioluminescence imaging (BLI) enable monitoring of tumor burden. Bioluminescent signals emitted by the interaction between luciferase expressed by the tumor cells and luciferin were captured by imaging using an IVIS Spectrum CT (Perkin Elmer, Mass.) and quantified as total flux (photons/sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, Calif.).
Three different BCMA specific CAR-T cells were used in this study: T cells expressing the BCMA specific CAR contructs P5AC1-V2, PC1C12-V2, or COM22-V2 (see, Table 5 above). Non-transduced control T cells were used as the negative control. All T cells were engineered to be TCRα deficient.
When the total flux reached an average of 45E6 for all animals (day 20 post tumor implant), the animals were randomized into four groups. A single dose of human either BCMA specific CAR-T cells or non-transduced control T cells was administered through bolus tail vein injection. Animals were terminated when they exhibit hindlimb paralysis or a 20% loss of body weight, an endpoint for MM1.S orthotopic models.
Results of this study are summarized in FIG. 1. In FIG. 1, total flux [p/s] represents tumor progression. Treatment with BCMA specific CAR-T cells (triangles, diamonds, squares) resulted in lower total flux as compared to the negative control (circles). Thus, treatment with BCMA specific CAR-T cells inhibited tumor progression as compared to the negative control.
These results demonstrate BCMA specific CAR-T cells are effective to induce tumor regression.
Example 4
Treatment of Multiple Myeloma with BCMA Specific CAR-T Cells
This example illustrates treatment of multiple myeloma with BCMA specific CAR-T cells using the Molp8 orthotopic model.
In vivo efficacy study of BCMA specific CAR-T cells was performed with Molp8, expressing luciferase and GFP, orthotopic model. Two million Molp8 Luc2AGFP cells were injected intravenously through the tail vein into 6-8 weeks old female NSG animals. Intraperitoneal injection of D-luciferin (Regis Technologies, Morton Grove, Ill.) (200 uL per animal at 15 mg/mL), followed by anesthesia with isofluorane and subsequent whole body bioluminescence imaging (BLI) enable monitoring of tumor burden. Bioluminescent signals emitted by the interaction between luciferase expressed by the tumor cells and luciferin were captured by imaging using an IVIS Spectrum CT (Perkin Elmer, Mass.) and quantified as total flux (photons/sec) using Living Image 4.4 (Caliper Life Sciences, Alameda, Calif.).
When the total flux reached an average of 30E6 for all animals (day 8 post tumor implant), the animals were randomized into three groups. Each group was administered one of the following cells: 1) non-transduced T cells TCR KO (“TCR KO”) used as a control, 2) BCMA specific CAR-T cells expressing P5AC1-V2.1 (“P5AC1 V2 R2 TCR KO”), or 3) BCMA specific CAR-T cells expressing P5AC1-V2 and the RQR8 suicide polypeptide (“P5AC1 V2 RQR8 TCR KO”). All of cells 1-3 are TCRα deficient. The BCMA specific CAR-T cells were prepared as described in example above. BCMA specific CAR constructs P5AC1-V2.1 and P5AC1-V2 are shown in Table 5 above. A single dose of 3 million control (TCR KO) or BCMA specific CAR-T (P5AC1 V2 R2 TCR KO or P5AC1 V2 RQR8 TCR KO) cells were administered through bolus tail vein injection. Animals were terminated when they lose more than 15% of total body weight, an endpoint for Molp8 orthotopic models.
Results from the study are summarized in FIG. 2. A single dose of 3 million P5AC1 R2 TCRKO BCMA specific CAR-T cells (squares) or P5AC1 RQR8 TCRKO CAR-T cells (triangles) BCMA specific CAR-T cells resulted in lower total flux from days 10-35 post tumor implant as compared to the negative control (circles) (FIG. 2). Thus, treatment with BCMA specific CAR-T cells inhibited tumor progression as compared to the negative control.
These results demonstrate BCMA specific CAR-T cells are effective to inhibit tumor progression.
Example 5
Treatment of Multiple Myeloma with BCMA Specific CAR-T Cells
This example illustrates the therapeutic activity of BCMA specific CAR-T cells in orthotopic mouse models of multiple myeloma.
Two humanized mouse models were used to evaluate the efficacy of BCMA specific CAR-T cells against human myeloma cell lines expressing BCMA. Six (6) to eight (8) week old female Nod/Scid IL2rg−/− (NSG) mice were purchased from the Jackson Laboratories. All animals were housed in a pathogen free vivarium facility at Rinat and experiments were conducted according to the protocols in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
The MM1.S and Molp-8 cell lines were purchased from the American Type Culture Collection (ATCC.org) and the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ.de). Cell lines were engineered to express a Luc-GFP fusion protein using lentiviral particles (amsbio). Cells were cultured in RPMI 1640 medium with L-glutamine supplemented with either 10% fetal calf serum for MM1.S or with 20% FCS for Molp-8 cells at 37° C. in 5% carbon dioxide (CO2). Cells growing in an exponential growth phase were harvested and used for tumor inoculation.
Therapeutic BCMA specific CAR-T cells were produced as described. Healthy human donor cells, peripheral blood mononuclear cells (PBMC) or purified pan-T cells, are activated and transduced with lentiviral particles encoding a BCMA specific CAR and RQR8 driven by EF-1a promoter. Three different BCMA specific CARs were used in this study: P5AC1-V2, PC1C12-V2 and COM22-V2 (see, Table 5 above). T cells were gene edited for deletion of the TCRα gene. Cells were cultured for 14 to 17 days and then cryopreserved in 90% FCS/10% DMSO. For T cell injection, T cells were rapidly thawed in a 37° C. waterbath and washed twice with RPMI 1640 medium containing 25 mM Hepes. Cells were injected in 0.2 ml RPMI 1640 with 25 mM Hepes into the tail vein of tumor-bearing animals.
NSG mice were irradiated with 1 Gy total body irradiation (RAD Source Technologies) one day prior to tumor cell inoculation. 5×106 MM1.S/Luc2-EGFP cells or 2×106 Molp-8/Luc2-EGFP cells were injected into the tail vein in 0.1 ml of phosphate-buffered saline (PBS). Tumor burden was measured twice weekly using bioluminescence imaging. Mice were injected with 3 ug D-Luciferin dissolved in 0.2 ml PBS and anesthetized using isofluorane. 7 minutes after injection animals were imaged using a Perkin Elmer IVIS Spectrum camera system. The total body luminescence with the exception of the mouse tail was measured and tumor burden is reported as total flux (photons per second). Tumors were allowed to establish until exponential growth occurred. Animals were randomized into treatment groups based on total flux and treated with BCMA specific CAR-T cells or untransduced control T cells from the same donor. The effect of CAR-T treatment was assessed twice weekly using bioluminescence imaging and body weight measurements. The study endpoint was reached when the first animal exhibited end-stage disease as indicated by body weight loss (>20% of initial body weight), hindleg paralysis, or other signs of animal distress. Statistical analysis was performed using GraphPad Prism 6. Repeated measures one-way ANOVA with Tukey's correction was used to compare anti-tumor efficacy between all groups. P<0.05 was considered significant.
Results are summarized in Table 11 (MM1.S) and Table 12 (Molp-8) below (log10 values of total flux in photons per second+/−SEM). A suboptimal CAR-T cell dose was used to compare BCMA specific CAR-T cells having different scFvs. The BCMA specific CAR-T cell groups are P5AC1-V2, PC1C12-V2 and COM22-V2 (see, Table 5 above). In the MM1.S model, 3.5×106 CAR-expressing T cells were injected on day 17 after tumor implantation. In the Molp8 model, 4×106 CAR-expressing T cells were injected on day 7 after tumor implantation. Transduction efficiencies ranged from 19% to 29% for BCMA specific CAR-T cells dosed in MM1.S mouse model and 31% to 36% for BCMA specific CAR-T cells dosed in Molp8 mouse model. An equivalent total dose of untransduced T cells was used for the control group. The control T cell-treated group exhibited progressive tumor growth until the study endpoint was reached at day 35 for MM1.S and day 23 for Molp8. Statistical analysis of the tumor burden using the RM-ANOVA test with Dunnets correction showed that in all three BCMA specific CAR-T treated groups, tumor burden was significantly lower compared to the tumor burden in the control group (p<0.01) (Tables 11 and 12). For example, in the MM1.S tumor model, mean total flux in animals treated with P5AC1-V2 BCMA specific CAR-T cells was 6.44 log 10 photons/s at day 25, compared to 9.22 log 10 photons/s in animals given control T cells (Table 11). At day 35 post tumor implantation, mean total flux in animals treated with P5AC1-V2 BCMA specific CAR-T cells was 6.82 log 10 photons/s, compared to 10.18 log 10 photons/s in animals given control T cells (Table 11). In the Molp8 tumor model, mean total flux in animals treated with P5AC1-V2 BCMA specific CAR-T cells was 7.88 log 10 photons/s at day 14, compared to 9.39 log 10 photons/s in animals given control T cells (Table 12). At day 23 post tumor implantation, mean total flux in animals treated with P5AC1-V2 BCMA specific CAR-T cells was 9.29 log 10 photons/s, compared 10.37 log 10 photons/s in animals given control T cells (Table 12).
These results demonstrate that treatments with BCMA specific CAR-T cells are effective to induce tumor regression.
TABLE 11
Tumor bioluminescence measurements
of orthotopic MM1.S tumor model
Group 1: Control T cells
Days after tumor
Mean total flux
implantation
(log10 photons/s)
SEM
N
17
7.84
0.04
10
21
8.16
0.19
10
25
9.22
0.02
10
28
9.53
0.02
10
32
9.96
0.05
10
35
10.18
0.07
10
Days after tumor
Mean total flux
implantation
(log10)
SEM
N
Group 2: P5AC1-V2 BCMA specific CAR-T cells
17
7.84
0.03
10
21
8.14
0.11
10
25
6.44
0.16
10
28
6.51
0.09
10
32
6.72
0.10
10
35
6.82
0.09
10
Group 3: PC1C12-V2 BCMA specific CAR-T cells
17
7.86
0.04
10
21
8.56
0.15
10
25
6.85
0.26
10
28
6.41
0.30
10
32
6.64
0.29
10
35
6.62
0.30
10
Group 4: COM22-V2 BCMA specific CAR-T cells
17
7.84
0.04
10
21
8.49
0.10
10
25
6.55
0.08
10
28
6.40
0.09
10
32
6.98
0.14
10
35
6.87
0.22
10
TABLE 12
Tumor bioluminescence measurements
of orthotopic Molp-8 tumor model
Days after tumor
Mean total flux
implantation
(log10)
SEM
N
Group 1: T cell only control
0
5.80
0.02
10
7
7.48
0.04
10
10
8.24
0.06
10
14
9.39
0.04
10
17
9.88
0.03
10
21
10.12
0.04
10
23
10.37
0.03
10
Group 2: P5AC1-V2 BCMA specific CAR-T cells
0
5.80
0.02
10
7
7.48
0.04
10
10
8.41
0.05
10
14
7.88
0.18
10
17
7.39
0.21
10
21
7.98
0.12
10
23
8.29
0.11
10
Group 3: PC1C12-V2 BCMA specific CAR-T cells
0
5.80
0.02
10
7
7.51
0.04
10
10
8.31
0.07
10
14
7.07
0.21
10
17
6.51
0.15
10
21
7.37
0.13
10
23
7.75
0.13
10
Group 4: COM22-V2 BCMA specific CAR-T cells
Days after tumor
Mean total flux
implantation
(log10
SEM
N
0
5.80
0.02
10
7
7.49
0.04
10
10
8.39
0.07
10
14
7.78
0.16
10
17
7.51
0.21
10
21
7.89
0.17
10
23
8.32
0.14
10
Example 6
Treatment of Multiple Myeloma with TCRα/dCK Knockout BCMA Specific CAR-T Cells
This example illustrates the therapeutic activity of BCMA specific CAR-T cells in orthotopic mouse models of multiple myeloma.
A humanized mouse model was used to evaluate the efficacy of BCMA CAR-T cells against human myeloma cell lines expressing BCMA. 6 to 8 week old female Nod/Scid IL2rg−/− (NSG) mice were purchased from the Jackson Laboratories. All animals were housed in a pathogen free vivarium facility at Rinat and experiments were conducted according to the protocols in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
The MM1.S cell lines was purchased from the American Type Culture Collection (ATCC.org). The Cell line was engineered to express a Luc-GFP fusion protein using lentiviral particles (amsbio) and gene edited using TALEN nucleases to disable the deoxycytidine (dCK) gene. Cells were cultured in RPMI 1640 medium with L-glutamine supplemented with 10% fetal calf serum at 37° C. in 5% carbon dioxide (CO2). Cells growing in an exponential growth phase were harvested and used for tumor inoculation.
Therapeutic CAR-T cells were produced as described. Healthy human donor cells, peripheral blood mononuclear cells (PBMC) or purified pan-T cells, are activated and transduced with lentiviral particles encoding for BCMA scFV, CD8 hinge, CD8 transmembrane, 41BB and CD3ζ with RQR8 genes under the control of the EF-1a promoter. The BCMA specific CAR-T cells were gene edited to delete the TCRα and/or the dCK gene using a combination of TCRα and dCK TALEN, or TCRα TALEN alone. Transduction efficiency for all T cells was 70%. TCRα knockout T cells were purified using magnetic selection kits for CD3-positive cells (Miltenyi); dCK knockout T cells were purified by expansion in the presence of 0.5 μM clofarabine. Cells were cultured for 14 to 17 days and then cryopreserved in 90% FCS/10% DMSO. For T cell injection, T cells were rapidly thawed in a 37° C. water bath and washed twice with RPMI 1640 medium containing 25 mM Hepes. For treatment, T cells were injected in 0.2 ml RPMI 1640 with 25 mM Hepes into the tail vein of tumor-bearing animals.
For the mouse tumor model, animals were injected with MM1.S/dCK KO tumor cells. Mice were then treated with 2.5×106 BCMA specific CAR-T cells on day 18 post tumor cell implantation. An equivalent dose of untransduced T cells that received TCRα and dCK TALEN was used as control. Animals were treated with clofarabine or vehicle for five days after T cell injection.
Results: the control T cell-treated group exhibited progressive tumor growth until the study endpoint was reached at day 35 (Table 13, Group 1). Compared against control, groups treated with TCRα knockout BCMA specific CAR-T cells and vehicle exhibited a significant decrease in tumor burden (p<0.05) that was diminished upon coadministration of clofarabine (p<0.05) (Table 13, Groups 2 and 3). Tumor burden was significantly reduced in animals treated with TCRα/dCK double knockout CAR-T cells, irrespective of whether the animals received vehicle or clofarabine (p<0.05) (Table 13, Groups 4 and 5). Reduction of tumor burden in the groups receiving TCRα/dCK double knockout T cells did not differ from the group receiving TCRα single knockout T cells and vehicle (p>0.1) (Table 13, Groups 2, 4, and 5).
These results demonstrate that treatments with TCRα/dCK double knockout BCMA CAR-T cells are effective to induce tumor regression in the presence of nucleoside analog therapies such as fludarabine and clofarabine.
TABLE 13
Tumor bioluminescence measurements of nucleoside analog
therapy-resistant orthotopic MM1.S tumor model.
Group 1: TCRα/dCK KO control T cells + clofarabine
Days after T cell
Mean total flux
administration
(log10 photons/s)
SEM
N
0
7.87
0.04
10
4
8.94
0.08
10
8
9.22
0.05
10
11
9.52
0.04
10
15
10.00
0.04
10
18
10.38
0.04
10
Days after T cell
Mean total flux
administration
(log10)
SEM
N
Group 2: TCRα KO BCMA specific CAR-T cells + vehicle
0
7.86
0.04
10
4
9.28
0.07
10
8
8.58
0.12
10
11
8.04
0.14
10
15
8.14
0.15
10
18
8.24
0.15
10
Group 3: TCRα KO BCMA specific CAR-T cells + clofarabine
0
7.87
0.04
10
4
9.33
0.07
10
8
9.17
0.07
10
11
8.95
0.14
10
15
9.36
0.08
10
18
9.50
0.07
10
Group 4: TCRα/dCK KO BCMA specific CAR-T cells + vehicle
0
7.86
0.04
10
4
9.19
0.08
10
8
9.08
0.12
10
11
8.59
0.18
10
15
8.60
0.21
10
18
8.69
0.18
10
Group 5: TCRα/dCK KO BCMA specific CAR-T cells + clofarabine
0
7.87
0.04
10
4
9.26
0.09
10
8
9.07
0.10
10
11
8.51
0.14
10
15
8.42
0.21
10
18
8.49
0.18
10
Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.
All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.
The foregoing description and Examples detail certain specific embodiments of the invention and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.
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16384719
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pfizer 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.
|
Open
|
|
|
Apr 27th, 2022 09:01AM
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Apr 27th, 2022 09:01AM
|
Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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|
nyse:pfe
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Pfizer
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Apr 26th, 2022 12:00AM
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Mar 6th, 2018 12:00AM
|
https://www.uspto.gov?id=US11312713-20220426
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Imidazo[4,5-C]quinoline derivatives as LRRK2 inhibitors
|
The present invention provides novel imidazo[4,5-c]quinoline derivatives of Formula (I), and the pharmaceutically acceptable salts thereof I wherein R1, R2 and R3 are as defined in the specification. The invention is also directed to pharmaceutical compositions comprising the compounds of Formula I and to use of the compounds in the treatment of diseases associated with LRRK2, such as neurodegenerative diseases including Parkinson's disease or Alzheimer's disease, cancer, Crohn's disease or leprosy.
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11312713
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1. A compound of Formula I
wherein
R1 is selected from the group consisting of methyl, ethyl, cyclobutyl, cyclopentyl,
R2 is selected from the group consisting of 2,2-difluoropropyl,
and
R3 is selected from the group consisting of fluoro, chloro, cyano, difluoromethyl and trifluoromethyl;
or a pharmaceutically acceptable salt, isotopically labeled derivative, or isotopically labeled derivative of the pharmaceutically acceptable salt thereof.
2. A compound selected from the group consisting of:
[(2S,4R)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile;
[(2R,4S)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2;
8-chloro-1-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline;
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(1-methyl-1H-1,2,3-triazol-4-yl) methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
[cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 1;
[cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 2;
8-(difluoromethyl)-2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyl tetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-(difluoromethyl)-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c] quinolin-2-yl}(5-methylpyrazin-2-yl)methanol, DIAST 1;
{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-methylpyrazin-2-yl)methanol, DIAST 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methoxy-1H-pyrazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methoxy-1H-pyrazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-fluoro-2-[(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
2-cyclopentyl-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c] quinoline-8-carbonitrile;
[cis-4-(8-chloro-2-methyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1;
2-[(5-methylpyrazin-2-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-2H-tetrazol-2-yl)methyl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
2-[(3-methyl-1,2-oxazol-5-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
2-[(5-methyl-1,3-oxazol-2-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-{[5-(trifluoromethyl)pyrazin-2-yl]methyl}-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline;
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-yl)methyl]-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-2H-tetrazol-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2-oxazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2-oxazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 1;
2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1-[(3R)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-[cis-2-(difluoromethyl)tetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,2-oxazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,2,3-thiadiazol-4-ylmethyl)-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-fluoro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-imidazo[4,5-c]quinoline;
2-(1,3-benzoxazol-2-ylmethyl)-1-[cis-3-fluorocyclopentyl]-1H-imidazo[4,5-c] quinoline-8-carbonitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1H-1,2,4-triazol-1-ylmethyl)-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-2-[(5-methylpyrazin-2-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline;
1-[cis-3-fluorocyclopentyl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-4-ylmethyl)-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
2-[(5-methyl-1,3,4-oxadiazol-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(2,2-difluoropropyl)-2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline;
8-fluoro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-{[5-(trifluoromethyl)pyrazin-2-yl]methyl}-1H-imidazo[4,5-c]quinoline;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,3,4-thiadiazol-2-yl) methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-[cis-3-fluorocyclopentyl]-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline;
3-{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}-2-methylpropanenitrile, DIAST 2;
8-fluoro-1-[cis-3-fluorocyclopentyl]-2-(1,2,3-thiadiazol-4-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
3-{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}propanenitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(5-methyl-2H-tetrazol-2-yl)methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-tetrazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(1-methyl-1H-1,2,4-triazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-tetrazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,3,4-thiadiazol-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-(difluoromethyl)-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline;
8-(difluoromethyl)-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-yl)methyl]-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinoline; and
[5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl} methyl)pyrazin-2-yl]methanol
or a pharmaceutically acceptable salt thereof.
3. The compound of claim 2 which is selected from the group consisting of
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
[(2S,4R)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile;
8-(difluoromethyl)-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1;
8-fluoro-2-[(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
2-[(5-methylpyrazin-2-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-2H-tetrazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
[cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2; and
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
or a pharmaceutically acceptable salt thereof.
4. The compound of claim 3 which is selected from the group consisting of
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
[(2S,4R)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile;
8-(difluoromethyl)-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline; and
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1;
or a pharmaceutically acceptable salt thereof.
5. The compound of claim 4 which is selected from the group consisting of
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2; and
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2;
or a pharmaceutically acceptable salt thereof.
6. The compound according to claim 2, wherein the compound is 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
or a pharmaceutically acceptable salt thereof.
7. The compound according to claim 2, wherein the compound is 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
or a pharmaceutically acceptable salt thereof.
8. The compound according to claim 2, wherein the compound is 2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methyl pyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
or a pharmaceutically acceptable salt thereof.
9. The compound according to claim 2, wherein the compound is 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl pyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
or a pharmaceutically acceptable salt thereof.
10. The compound according to claim 2, wherein the compound is 1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2;
or a pharmaceutically acceptable salt thereof.
11. The compound according to claim 1, wherein
R1 is ethyl,
R2 is
and
R3 is chloro, cyano, difluoromethyl, or trifluoromethyl,
or a pharmaceutically acceptable salt, isotopically labeled derivative, or isotopically labeled derivative of the pharmaceutically acceptable salt thereof.
12. The compound according to claim 1, wherein
R1 is
R2 is
and
R3 is chloro or cyano,
or a pharmaceutically acceptable salt, isotopically labeled derivative, or isotopically labeled derivative of the pharmaceutically acceptable salt thereof.
13. The compound of claim 11, wherein
R1 is
R2 is
and
R3 is chloro,
or a pharmaceutically acceptable salt thereof.
14. The compound according to claim 2, wherein the compound is [5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl]methanol; or a pharmaceutically acceptable salt thereof.
15. The compound according to claim 1, wherein the compound is 8-chloro-2-{[5-(2H3)methylpyrazin-2-yl]methyl}-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline; or a pharmaceutically acceptable salt thereof.
16. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1, 2 or 15, or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.
17. A method of treating a LRRK2-mediated (or LRRK2-associated) disease or disorder selected from the group consisting of Crohn's disease, Parkinson's disease, Lewy body dementia, frontotemporal dementia, corticobasal dementia, progressive supranuclear palsy, leprosy, Alzheimer's disease, tauopathy disease and Alpha-synucleinopathy in a patient, the method comprising administering to a patient in need of treatment thereof a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof according to claim 1, 2 or 15, or a pharmaceutical composition of claim 16.
18. A method of treating a LRRK2-mediated (or LRRK2-associated) disease or disorder selected from the group consisting of Crohn's disease, Parkinson's disease, Lewy body dementia, frontotemporal dementia, corticobasal dementia, progressive supranuclear palsy, leprosy, Alzheimer's disease, tauopathy disease and Alpha-synucleinopathy in a patient, the method comprising administering to a patient in need of treatment thereof a pharmaceutical composition of claim 16.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a United States National Phase Application filed under 35 U.S.C. § 371 from International Patent Application No. PCT/IB2018/051439, filed Mar. 6, 2018, which claims the benefit of priority to U.S. Provisional Patent Application Ser. Nos. 62/629,152, Feb. 12, 2018, and 62/469,756, filed Mar. 10, 2017, the disclosures of which are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
The present invention relates to small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2). This invention also relates to methods of inhibiting, in mammals, including humans, LRRK2 by administration of the small molecule LRRK2 inhibitors. The present invention also relates to the treatment of Parkinson's disease (PD) and other neurodegenerative and/or neurological disorders in mammals, including humans, with the LRRK2 inhibitors. More particularly, this invention relates to novel imidazo[4,5-c]quinoline compounds useful for the treatment of neurodegenerative and/or neurological disorders, such as PD, Alzheimer's disease (AD) and other LRRK2 associated disorders.
BACKGROUND OF THE INVENTION
LRRK2 is a 286 kDa protein in the ROCO protein family with a complex multidomain structure. Protein motifs that have been established for LRRK2 include an armadillo-like (ARM) domain, an ankyrin-like (ANK) domain, a leucine-rich repeat (LRR) domain, a Ras (renin-angiotensin system) of complex (ROC) domain, a C-terminal of ROC (COR) domain, a kinase domain, and a C-terminal WD40 domain. The ROC domain binds guanosine triphosphate (GTP) and the COR domain may be a regulator of the ROC domain's GTPase activity. The kinase domain has structural homology to the MAP kinase kinase kinases (MAPKKK) and has been shown to phosphorylate a number of cellular proteins in vitro, but the endogenous substrate has yet to be determined. LRRK2 has been found in various regions of the brain as well as in a number of peripheral tissues including heart, lung, spleen, and kidney.
LRRK2 has the ability to potentially play a complex role in multiple cellular processes as a consequence of its multi-domain construct, each associated with putative protein-protein interactions, guanosine triphosphatase (GTPase) activity, and kinase activity. For example, LRRK2 has been associated with NFAT inhibition in the immune system and has been linked to vesicle trafficking, presynaptic homeostasis, mammalian target of rapamycin (mTOR) signaling, signaling through the receptor tyrosine kinase MET in papillary renal and thyroid carcinomas, cytoskeletal dynamics, the mitogen-activated protein kinase (MAPK) pathway, the tumor necrosis factor-α (TNF-α) pathway, the Wnt pathway and autophagy. Recent genome-wide association (GWA) genetic studies have implicated LRRK2 in the pathogenesis of various human diseases such as PD, inflammatory bowel disease (Crohn's disease), cancer and leprosy (Lewis, P. A. and Manzoni, C. Science Signaling 2012, 5(207), pe2).
Parkinson's disease (PD) is a relatively common age-related neurodegenerative disorder resulting from the progressive loss of dopamine-producing neurons and which affects up to 4% of the population over age 80. PD is characterized by both motor symptoms, such as tremor at rest, rigidity, akinesia and postural instability as well as non-motor symptoms such as impairment of cognition, sleep and sense of smell. GWA studies have linked LRRK2 to PD and many patients with point mutations in LRRK2 present symptoms that are indistinguishable from those with idiopathic PD. Over 20 LRRK2 mutations have been associated with autosomal-dominant Parkinsonism, and the R1441C, R1441G, R1441H, Y1699C, G2019S, I2020T and N1437H missense mutations are considered to be pathogenic. The LRRK2 R1441G mutation has been shown to increase the release of proinflammatory cytokines (higher levels of TNF-α, IL-1β, IL-12 and lower levels of IL-10) in microglial cells from transgenic mice and thus may result in direct toxicity to neurons (Gillardon, F. et al. Neuroscience 2012, 208, 41-48). In a murine model of neuroinflammation, induction of LRRK2 in microglia was observed and inhibition of LRRK2 kinase activity with small molecule LRRK2 inhibitors (LRRK2-IN-1 or sunitinib) or LRRK2 knockout resulted in attenuation of TNF-α secretion and nitric oxide synthase (iNOS) induction (Moehle, M. et al. J. Neurosci. 2012, 32(5), 1602-1611). The most common of the LRRK2 mutations, G2019S, is present in more than 85% of PD patients carrying LRRK2 mutations. This mutation, which is present in the LRRK2 kinase domain, leads to an enhancement of LRRK2 kinase activity. In the human brain LRRK2 expression is highest in the same regions of the brain that are impacted by PD, and LRRK2 is found in Lewy Bodies, a hallmark of PD. Recent studies indicate that a potent, selective, brain-penetrant kinase inhibitor for LRRK2 could be a therapeutic treatment for PD.
Dementia results from a wide variety of distinctive pathological processes. The most common pathological processes causing dementia are AD, cerebral amyloid angiopathy (CM) and prion-mediated diseases (see, e.g., Haan et al., Clin. Neurol. Neurosurg. 1990, 92(4):305-310; Glenner et al., J. Neurol. Sci. 1989, 94:1-28). AD is a progressive, neurodegenerative disorder characterized by memory impairment and cognitive dysfunction. AD affects nearly half of all people past the age of 85, the most rapidly growing portion of the United States population. As such, the number of AD patients in the United States is expected to increase from about 4 million to about 14 million by 2050. LRRK2 mutations have been associated with AD-like pathology, which suggests that there may be a partial overlap between the neurodegenerative pathways in both AD and PD (Zimprach, A. et al. Neuron 2004, 44, 601-607). In addition, the LRRK2 R1628P variant (COR domain) has been associated with an increased incidence of AD in a certain population, perhaps resulting from increased apoptosis and cell death (Zhao, Y. et al.; Neurobiology of Aging 2011, 32, 1990-1993).
An increased incidence of certain non-skin cancers such as renal, breast, lung and prostate cancers, as well as acute myelogenous leukemia (AML), has been reported in Parkinson's disease patients with the LRRK2 G2019S mutation (Saunders-Pullman, R. et al.; Movement Disorders, 2010, 25(15), 2536-2541). Since the G2019S mutation is associated with increased LRRK2 kinase activity, inhibition of this activity may be useful in the treatment of cancer, such as kidney, breast, lung, prostate and blood cancers.
Inflammatory bowel disease (IBD) or Crohn's disease (CD) is a complex disease and is believed to result from an inappropriate immune response to microbiota in the intestinal tract. GWA studies have recently identified LRRK2 as a major susceptibility gene for Crohn's disease, particularly the M2397T polymorphism in the WD40 domain (Liu, Z. et al. Nat. Immunol. 2011, 12, 1063-1070). In a recent study LRRK2 deficient mice were found to be more susceptible to dextran sodium sulfate induced colitis than their wild-type counterparts, indicating that LRRK2 may play a role in the pathogenesis of IBD (Liu, Z. and Lenardo, M.; Cell Research 2012, 1-3).
Both non-selective and selective small molecule compounds with LRRK2 inhibitory activity such as staurosporine, sunitinib, LRRK2-IN-1, CZC-25146, TAE684 and those in WO 2011/141756, WO 2012/028629 and WO 2012/058193 have been described. It is desirable to provide compounds which are potent and selective inhibitors of LRRK2 with a favorable pharmacokinetic profile and the ability to traverse the blood-brain barrier. Accordingly, the present invention is directed to novel imidazo[4,5-c]quinoline compounds with LRRK2 inhibitory activity and the use of these compounds in the treatment of diseases associated with LRRK2, such as neurodegenerative diseases, including PD.
SUMMARY OF THE INVENTION
The present invention is directed at compounds of Formula I
wherein
R1 is selected from the group consisting of methyl, ethyl, cyclobutyl, cyclopentyl,
R2 is selected from the group consisting of 2,2-difluoropropyl,
and R3 is selected from the group consisting of fluoro, chloro, cyano, difluoromethyl and trifluoromethyl; or a pharmaceutically acceptable salt thereof.
The present invention is also directed at pharmaceutical compositions which include a pharmaceutically acceptable carrier and a compound of Formula I or a pharmaceutically acceptable salt thereof, present in a therapeutically effective amount.
The present invention is also directed at a method for the treatment of disorder or condition selected from Parkinson's disease (but also including other neurological diseases which may include migraine; epilepsy; Alzheimer's disease; brain injury; stroke; cerebrovascular diseases (including cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, and brain hypoxia-ischemia); cognitive disorders (including amnesia, senile dementia, HIV-associated dementia, Alzheimer's disease, Huntington's disease, Lewy body dementia, vascular dementia, drug-related dementia, tardive dyskinesia, myoclonus, dystonia, delirium, Pick's disease, Creutzfeldt-Jacob disease, HIV disease, Gilles de la Tourette's syndrome, epilepsy, muscular spasms and disorders associated with muscular spasticity or weakness including tremors, and mild cognitive impairment); mental deficiency (including spasticity, Down syndrome and fragile X syndrome); sleep disorders (including hypersomnia, circadian rhythm sleep disorder, insomnia, parasomnia, and sleep deprivation) and psychiatric disorders such as anxiety (including acute stress disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, post-traumatic stress disorder, agoraphobia, and obsessive-compulsive disorder); factitious disorder (including acute hallucinatory mania); impulse control disorders (including compulsive gambling and intermittent explosive disorder); mood disorders (including bipolar I disorder, bipolar II disorder, mania, mixed affective state, major depression, chronic depression, seasonal depression, psychotic depression, seasonal depression, premenstrual syndrome (PMS) premenstrual dysphoric disorder (PDD), and postpartum depression); psychomotor disorder; psychotic disorders (including schizophrenia, schizoaffective disorder, schizophreniform, and delusional disorder); drug dependence (including narcotic dependence, alcoholism, amphetamine dependence, cocaine addiction, nicotine dependence, and drug withdrawal syndrome); eating disorders (including anorexia, bulimia, binge eating disorder, hyperphagia, obesity, compulsive eating disorders and pagophagia); sexual dysfunction disorders; urinary incontinence; neuronal damage disorders (including ocular damage, retinopathy or macular degeneration of the eye, tinnitus, hearing impairment and loss, and brain edema) and pediatric psychiatric disorders (including attention deficit disorder, attention deficit/hyperactive disorder, conduct disorder, and autism) in a mammal, preferably a human, comprising administering to a subject a therapeutically effective amount of a composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
DETAILED DESCRIPTION OF THE INVENTION
The present invention may be understood more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein.
It is to be understood that this invention is not limited to specific synthetic methods of making that may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
As used herein in the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.
The term “about” refers to a relative term denoting an approximation of plus or minus 10% of the nominal value it refers, in one embodiment, to plus or minus 5%, in another embodiment, to plus or minus 2%. For the field of this disclosure, this level of approximation is appropriate unless the value is specifically stated to require a tighter range.
The term “treating”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term “treatment”, as used herein, unless otherwise indicated, refers to the act of treating as “treating” is defined immediately above. The term “treating” also includes adjuvant and neo-adjuvant treatment of a subject.
“Therapeutically effective amount” means an amount of a compound of the present invention that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. By “pharmaceutically acceptable” is meant that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
As used herein, the expressions “reaction-inert solvent” and “inert solvent” refer to a solvent or a mixture thereof which does not interact with starting materials, reagents, intermediates or products in a manner which adversely affects the yield of the desired product.
The term “neurological” refers to the central nervous system. The treatment of neurological conditions refers to the treatment of a condition, disease, ailment, etc. impacting the central nervous system (“CNS”). Such diseases can impact tissues in the periphery as well as the central nervous system.
If substituents are described as being “independently selected” from a group, each substituent is selected independent of the other. Each substituent therefore may be identical to or different from the other substituent(s).
As used herein the terms “formula I”, “Formula I”, “formula (I)” or “Formula (I)” may be referred to as a “compound(s) of the invention.” Such terms are also defined to include all forms of the compound of formula I, including hydrates, solvates, isomers, crystalline and non-crystalline forms, isomorphs, polymorphs, and metabolites thereof. For example, the compounds of the invention, or pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.
The compounds of the invention may exist as clathrates or other complexes. Included within the scope of the invention are complexes such as clathrates, drug-host inclusion complexes wherein the drug and host are present in stoichiometric or non-stoichiometric amounts. Also included are complexes of the compounds of the invention containing two or more organic and/or inorganic components which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of such complexes, see J. Pharm. Sci., 64 (8), 1269-1288 by Haleblian (August 1975).
The compounds of the invention may have asymmetric carbon atoms. The carbon-carbon bonds of the compounds of the invention may be depicted herein using a solid line () a solid wedge (), or a dotted wedge (). The use of a solid line to depict bonds to asymmetric carbon atoms is meant to indicate that all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom are included. The use of either a solid or dotted wedge to depict bonds to asymmetric carbon atoms is meant to indicate that only the stereoisomer shown is meant to be included. It is possible that compounds of Formula (I) may contain more than one asymmetric carbon atom. In those compounds, the use of a solid line to depict bonds to asymmetric carbon atoms is meant to indicate that all possible stereoisomers are meant to be included. For example, unless stated otherwise, it is intended that the compounds of Formula (I) can exist as enantiomers and diastereomers or as racemates and mixtures thereof. The use of a solid line to depict bonds to one or more asymmetric carbon atoms in a compound of Formula (I) and the use of a solid or dotted wedge to depict bonds to other asymmetric carbon atoms in the same compound is meant to indicate that a mixture of diastereomers is present.
Stereoisomers of Formula I include cis and trans isomers, optical isomers such as R and S enantiomers, diastereomers, geometric isomers, rotational isomers, conformational isomers, and tautomers of the compounds of the invention, including compounds exhibiting more than one type of isomerism; and mixtures thereof (such as racemates and diastereomeric pairs). Also included are acid addition or base addition salts wherein the counterion is optically active, for example, D-lactate or L-lysine, or racemic, for example, DL-tartrate or DL-arginine.
When any racemate crystallizes, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer.
Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically high pressure liquid chromatography (HPLC) or supercritical fluid chromatography (SFC), on a resin with an asymmetric stationary phase and with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% isopropanol, typically from 2 to 20%, and from 0 to 5% of an alkylamine, typically 0.1% diethylamine (DEA) or isopropylamine. Concentration of the eluent affords the enriched mixture.
Diastereomeric mixtures can be separated into their individual diastereoisomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g. chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereoisomers and converting (e.g. hydrolyzing) the individual diastereoisomers to the corresponding pure enantiomers. Enantiomers can also be separated by use of a chiral HPLC column. Alternatively, the specific stereoisomers may be synthesized by using an optically active starting material, by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one stereoisomer into the other by asymmetric transformation. The present invention comprises the tautomeric forms of compounds of the invention. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism (‘tautomerism’) can occur. This can take the form of proton tautomerism in compounds of the invention containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism. The various ratios of the tautomers in solid and liquid form are dependent on the various substituents on the molecule as well as the particular crystallization technique used to isolate a compound.
The compounds of this invention may be used in the form of salts derived from inorganic or organic acids. Depending on the particular compound, a salt of the compound may be advantageous due to one or more of the salt's physical properties, such as enhanced pharmaceutical stability in differing temperatures and humidities, or a desirable solubility in water or oil. In some instances, a salt of a compound also may be used as an aid in the isolation, purification, and/or resolution of the compound.
Where a salt is intended to be administered to a patient (as opposed to, for example, being used in an in vitro context), the salt preferably is pharmaceutically acceptable. The term “pharmaceutically acceptable salt” refers to a salt prepared by combining a compound of Formula I with an acid whose anion, or a base whose cation, is generally considered suitable for human consumption. Pharmaceutically acceptable salts are particularly useful as products of the methods of the present invention because of their greater aqueous solubility relative to the parent compound. For use in medicine, the salts of the compounds of this invention are non-toxic “pharmaceutically acceptable salts.” Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.
Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention when possible include those derived from inorganic acids, such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids. Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids.
Specific examples of suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate (pamoate), methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate, β-hydroxybutyrate, galactarate, galacturonate, adipate, alginate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate, glycerophosphate, heptanoate, hexanoate, nicotinate, 2-naphthalesulfonate, oxalate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, and undecanoate.
Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, i.e., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. In another embodiment, base salts are formed from bases which form non-toxic salts, including aluminum, arginine, benzathine, choline, diethylamine, diolamine, glycine, lysine, meglumine, olamine, tromethamine and zinc salts.
Organic salts may be made from secondary, tertiary or quaternary amine salts, such as tromethamine, diethylamine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl (C1-C6) halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (i.e., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others.
In one embodiment, hemisalts of acids and bases may also be formed, for example, hemisulfate and hemicalcium salts.
Also within the scope of the present invention are so-called “prodrugs” of the compound of the invention. Thus, certain derivatives of the compound of the invention which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into the compound of the invention having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as “prodrugs.” Further information on the use of prodrugs may be found in “Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press, 1987 (ed. E. B. Roche, American Pharmaceutical Association). Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of any of Formula (I) with certain moieties known to those skilled in the art as “pro-moieties” as described, for example, in “Design of Prodrugs” by H. Bundgaard (Elsevier, 1985).
The present invention also includes isotopically labeled compounds, which are identical to those recited in Formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 11C, 14C, 15N, 18O, 17O, 32P, 35S, 18F, and 36Cl, respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of Formula I of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples and Preparations below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
Typically, a compound of the invention is administered in an amount effective to treat a condition as described herein. The compounds of the invention are administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. Therapeutically effective doses of the compounds required to treat the progress of the medical condition are readily ascertained by one of ordinary skill in the art using preclinical and clinical approaches familiar to the medicinal arts.
Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compound into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the instant invention. Such excipients and carriers are described, for example, in “Remington's Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991). The formulations of the invention can be designed to be short-acting, fast-releasing, long-acting, and sustained-releasing. Thus, the pharmaceutical formulations can also be formulated for controlled release or for slow release.
The pharmaceutical composition comprises a compound of the invention or a combination in an amount generally in the range of from about 1% to about 75%, 80%, 85%, 90% or even 95% (by weight) of the composition, usually in the range of about 1%, 2% or 3% to about 50%, 60% or 70%, more frequently in the range of about 1%, 2% or 3% to less than 50% such as about 25%, 30% or 35%.
The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed, by which the compound enters the blood stream directly from the mouth.
In another embodiment, the compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
In another embodiment, the compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. In another embodiment, the compounds of the invention can also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention may be administered rectally or vaginally. In another embodiment, the compounds of the invention may also be administered directly to the eye or ear.
The dosage regimen for the compounds and/or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus the dosage regimen may vary widely. Dosage levels of the order from about 0.01 mg to about 100 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions. In one embodiment, the total daily dose of a compound of the invention (administered in single or divided doses) is typically from about 0.01 to about 100 mg/kg. In another embodiment, the total daily dose of the compound of the invention is from about 0.1 to about 50 mg/kg, and in another embodiment, from about 0.5 to about 30 mg/kg (i.e., mg compound of the invention per kg body weight). In one embodiment, dosing is from 0.01 to 10 mg/kg/day. In another embodiment, dosing is from 0.1 to 1.0 mg/kg/day. Dosage unit compositions may contain such amounts or submultiples thereof to make up the daily dose. In many instances, the administration of the compound will be repeated a plurality of times in a day (typically no greater than 4 times). Multiple doses per day typically may be used to increase the total daily dose, if desired.
For oral administration, the compositions may be provided in the form of tablets containing from about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, from about 1 mg to about 100 mg of active ingredient. Intravenously, doses may range from about 0.1 to about 10 mg/kg/minute during a constant rate infusion.
Suitable subjects according to the present invention include mammalian subjects. Mammals according to the present invention include, but are not limited to, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like, and encompass mammals in utero. In one embodiment, humans are suitable subjects. Human subjects may be of either gender and at any stage of development.
In another embodiment, the invention comprises the use of one or more compounds of the invention for the preparation of a medicament for the treatment of the conditions recited herein.
For the treatment of the conditions referred to above, the compound of the invention can be administered as compound per se. Alternatively, pharmaceutically acceptable salts are suitable for medical applications because of their greater aqueous solubility relative to the parent compound.
In another embodiment, the present invention comprises pharmaceutical compositions. Such pharmaceutical compositions comprise a compound of the invention presented with a pharmaceutically acceptable carrier. The carrier can be a solid, a liquid, or both, and may be formulated with the compound as a unit-dose composition, for example, a tablet, which can contain from 0.05% to 95% by weight of the active compounds. A compound of the invention may be coupled with suitable polymers as targetable drug carriers. Other pharmacologically active substances can also be present.
The compounds of the present invention may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The active compounds and compositions, for example, may be administered orally, rectally, parenterally, or topically.
Oral administration of a solid dose form may be, for example, presented in discrete units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present invention. In such solid dos-age forms, a compound of the present invention or a combination is admixed with at least one inert excipient, diluent or carrier. Suitable excipients, diluents or carriers include materials such as sodium citrate or dicalcium phosphate and/or (a) one or more fillers or extenders (e.g., microcrystalline cellulose (available as Avicel™ from FMC Corp.) starches, lactose, sucrose, mannitol, silicic acid, xylitol, sorbitol, dextrose, calcium hydrogen phosphate, dextrin, alpha-cyclodextrin, beta-cyclodextrin, polyethylene glycol, medium chain fatty acids, titanium oxide, magnesium oxide, aluminum oxide and the like); (b) one or more binders (e.g., carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gelatin, gum arabic, ethyl cellulose, polyvinyl alcohol, pullulan, pregelatinized starch, agar, tragacanth, alginates, gelatin, polyvinylpyrrolidone, sucrose, acacia and the like); (c) one or more humectants (e.g., glycerol and the like); (d) one or more disintegrating agents (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, sodium carbonate, sodium lauryl sulphate, sodium starch glycolate (available as Explotab™ from Edward Mendell Co.), cross-linked polyvinyl pyrrolidone, croscarmellose sodium A-type (available as Ac-di-sol™), polyacrilin potassium (an ion exchange resin) and the like); (e) one or more solution retarders (e.g., paraffin and the like); (f) one or more absorption accelerators (e.g., quaternary ammonium compounds and the like); (g) one or more wetting agents (e.g., cetyl alcohol, glycerol monostearate and the like); (h) one or more adsorbents (e.g., kaolin, bentonite and the like); and/or (i) one or more lubricants (e.g., talc, calcium stearate, magnesium stearate, stearic acid, polyoxyl stearate, cetanol, talc, hydrogenated caster oil, sucrose esters of fatty acid, dimethylpolysiloxane, microcrystalline wax, yellow beeswax, white beeswax, solid polyethylene glycols, sodium lauryl sulfate and the like). In the case of capsules and tablets, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be used as fillers in soft or hard filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, and granules may be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may also contain opacifying agents, and can also be of such composition that they release the compound of the present invention and/or the additional pharmaceutical agent in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The drug may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
For tablets, the active agent will typically comprise less than 50% (by weight) of the formulation, for example less than about 10% such as 5% or 2.5% by weight. The predominant portion of the formulation comprises fillers, diluents, disintegrants, lubricants and optionally, flavors. The composition of these excipients is well known in the art. Frequently, the fillers/diluents will comprise mixtures of two or more of the following components: microcrystalline cellulose, mannitol, lactose (all types), starch, and dicalcium phosphate. The filler/diluent mixtures typically comprise less than 98% of the formulation and preferably less than 95%, for example 93.5%. Preferred disintegrants include Ac-di-sol™, Explotab™, starch and sodium lauryl sulphate. When present a disintegrant will usually comprise less than 10% of the formulation or less than 5%, for example about 3%. A preferred lubricant is magnesium stearate. When present a lubricant will usually comprise less than 5% of the formulation or less than 3%, for example about 1%.
Tablets may be manufactured by standard tabletting processes, for example, direct compression or a wet, dry or melt granulation, melt congealing process and extrusion. The tablet cores may be mono or multi-layer(s) and can be coated with appropriate overcoats known in the art.
In another embodiment, oral administration may be in a liquid dose form. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the compound of the present invention or the combination, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame seed oil and the like), Miglyole® (available from CONDEA Vista Co., Cranford, N.J.), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
Besides such inert diluents, the composition may also include excipients, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Oral liquid forms of the compounds of the invention or combinations include solutions, wherein the active compound is fully dissolved. Examples of solvents include all pharmaceutically precedented solvents suitable for oral administration, particularly those in which the compounds of the invention show good solubility, e.g., polyethylene glycol, polypropylene glycol, edible oils and glyceryl- and glyceride-based systems. Glyceryl- and glyceride-based systems may include, for example, the following branded products (and corresponding generic products): Captex™ 355 EP (glyceryl tricaprylate/caprate, from Abitec, Columbus Ohio), Crodamol™ GTC/C (medium chain triglyceride, from Croda, Cowick Hall, UK) or Labrafac™ CC (medium chain triglyides, from Gattefosse), Captex™ 500P (glyceryl triacetate i.e. triacetin, from Abitec), Capmul™ MCM (medium chain mono- and diglycerides, fromAbitec), Migyol™ 812 (caprylic/capric triglyceride, from Condea, Cranford N.J.), Migyol™ 829 (caprylic/capric/succinic triglyceride, from Condea), Migyol™ 840 (propylene glycol dicaprylate/dicaprate, from Condea), Labrafil™ M1944CS (oleoyl macrogol-6 glycerides, from Gattefosse), Peceol™ (glyceryl monooleate, from Gattefosse) and Maisine™ 35-1 (glyceryl monooleate, from Gattefosse). Of particular interest are the medium chain (about C.sub.8 to C.sub.10) triglyceride oils. These solvents frequently make up the predominant portion of the composition, i.e., greater than about 50%, usually greater than about 80%, for example about 95% or 99%. Adjuvants and additives may also be included with the solvents principally as taste-mask agents, palatability and flavoring agents, antioxidants, stabilizers, texture and viscosity modifiers and solubilizers.
Suspensions, in addition to the compound of the present invention or the combination, may further comprise carriers such as suspending agents, e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, or mixtures of these substances, and the like.
In another embodiment, the present invention comprises a parenteral dose form. “Parenteral administration” includes, for example, subcutaneous injections, intravenous injections, intraperitoneal injections, intramuscular injections, intrasternal injections, and infusion. Injectable preparations (e.g., sterile injectable aqueous or oleaginous suspensions) may be formulated according to the known art using suitable dispersing, wetting agents, and/or suspending agents. Compositions suitable for parenteral injection generally include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers or diluents (including solvents and vehicles) include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, triglycerides including vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. A preferred carrier is Miglyol® brand caprylic/capric acid ester with glycerine or propylene glycol (e.g., Miglyol® 812, Miglyol® 829, Miglyol® 840) available from Condea Vista Co., Cranford, N.J. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions for parenteral injection may also contain excipients such as preserving, wetting, emulsifying, and dispersing agents. Prevention of microorganism contamination of the compositions can be accomplished with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents capable of delaying absorption, for example, aluminum monostearate and gelatin. In another embodiment, the present invention comprises a topical dose form. “Topical administration” includes, for example, transdermal administration, such as via transdermal patches or iontophoresis devices, intraocular administration, or intranasal or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments, and creams. A topical formulation may include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of this invention are administered by a transdermal device, administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated; see, for example, J. Pharm. Sci., 88 (10), 955-958, by Finnin and Morgan (October 1999).
Formulations suitable for topical administration to the eye include, for example, eye drops wherein the compound of this invention is dissolved or suspended in a suitable carrier. A typical formulation suitable for ocular or aural administration may be in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and nonbiodegradable (e.g., silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulosic polymer, for example, (hydroxypropyl)methyl cellulose, hydroxyethyl cellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.
For intranasal administration or administration by inhalation, the active compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant. Formulations suitable for intranasal administration are typically administered in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
In another embodiment, the present invention comprises a rectal or vaginal dose form. Such rectal dose form may be in the form of, for example, a suppository. Cocoa butter, polyethylene glycol and suppository wax are traditional suppository bases, but various alternatives may be used as appropriate. These bases are solid at ordinary room temperature, but liquid at body temperature, and therefore, melt in the rectum or vaginal cavity thereby re-leasing the active component(s).
Many of the present compounds are poorly soluble in water, e.g., less than about 1 μg/mL. Therefore, liquid compositions in solubilizing, non-aqueous solvents such as the medium chain triglyceride oils discussed above are a preferred dosage form for these compounds.
Solid amorphous dispersions, including dispersions formed by a spray-drying process, are also a preferred dosage form for the poorly soluble compounds of the invention. By “solid amorphous dispersion” is meant a solid material in which at least a portion of the poorly soluble compound is in the amorphous form and dispersed in a water-soluble polymer. By “amorphous” is meant that the poorly soluble compound is not crystalline. By “crystalline” is meant that the compound exhibits long-range order in three dimensions of at least 100 repeat units in each dimension. Thus, the term amorphous is intended to include not only material which has essentially no order, but also material which may have some small degree of order, but the order is in less than three dimensions and/or is only over short distances. Amorphous material may be characterized by techniques known in the art such as powder x-ray diffraction (PXRD) crystallography, solid state NMR, or thermal techniques such as differential scanning calorimetry (DSC).
Preferably, at least a major portion (i.e., at least about 60 wt %) of the poorly soluble compound in the solid amorphous dispersion is amorphous. The compound can exist within the solid amorphous dispersion in relatively pure amorphous domains or regions, as a solid solution of the compound homogeneously distributed throughout the polymer or any combination of these states or those states that lie intermediate between them. Preferably, the solid amorphous dispersion is substantially homogeneous so that the amorphous compound is dispersed as homogeneously as possible throughout the polymer. As used herein, “substantially homogeneous” means that the fraction of the compound that is present in relatively pure amorphous domains or regions within the solid amorphous dispersion is relatively small, on the order of less than 20 wt %, and preferably less than 10 wt % of the total amount of drug.
Water-soluble polymers suitable for use in the solid amorphous dispersions should be inert, in the sense that they do not chemically react with the poorly soluble compound in an adverse manner, are pharmaceutically acceptable, and have at least some solubility in aqueous solution at physiologically relevant pHs (e.g. 1-8). The polymer can be neutral or ionizable, and should have an aqueous-solubility of at least 0.1 mg/mL over at least a portion of the pH range of 1-8.
Water-soluble polymers suitable for use with the present invention may be cellulosic or non-cellulosic. The polymers may be neutral or ionizable in aqueous solution. Of these, ionizable and cellulosic polymers are preferred, with ionizable cellulosic polymers being more preferred.
Exemplary water-soluble polymers include hydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl methyl cellulose phthalate (HPMCP), carboxy methyl ethyl cellulose (CMEC), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), polyvinylpyrrolidone (PVP), hydroxypropyl cellulose (HPC), methyl cellulose (MC), block copolymers of ethylene oxide and propylene oxide (PEO/PPO, also known as poloxamers), and mixtures thereof. Especially preferred polymers include HPMCAS, HPMC, HPMCP, CMEC, CAP, CAT, PVP, poloxamers, and mixtures thereof. Most preferred is HPMCAS. See European Patent Application Publication No. 0 901 786 A2, the disclosure of which is incorporated herein by reference.
The solid amorphous dispersions may be prepared according to any process for forming solid amorphous dispersions that results in at least a major portion (at least 60%) of the poorly soluble compound being in the amorphous state. Such processes include mechanical, thermal and solvent processes. Exemplary mechanical processes include milling and extrusion; melt processes including high temperature fusion, solvent-modified fusion and melt-congeal processes; and solvent processes including non-solvent precipitation, spray coating and spray drying. See, for example, the following U.S. Patents, the pertinent disclosures of which are incorporated herein by reference: U.S. Pat. Nos. 5,456,923 and 5,939,099, which describe forming dispersions by extrusion processes; U.S. Pat. Nos. 5,340,591 and 4,673,564, which describe forming dispersions by milling processes; and U.S. Pat. Nos. 5,707,646 and 4,894,235, which describe forming dispersions by melt congeal processes. In a preferred process, the solid amorphous dispersion is formed by spray drying, as disclosed in European Patent Application Publication No. 0 901 786 A2. In this process, the compound and polymer are dissolved in a solvent, such as acetone or methanol, and the solvent is then rapidly removed from the solution by spray drying to form the solid amorphous dispersion. The solid amorphous dispersions may be prepared to contain up to about 99 wt % of the compound, e.g., 1 wt %, 5 wt 10 wt %, 25 wt 50 wt %, 75 wt %, 95 wt %, or 98 wt % as desired.
The solid dispersion may be used as the dosage form itself or it may serve as a manufacturing-use-product (MUP) in the preparation of other dosage forms such as capsules, tablets, solutions or suspensions. An example of an aqueous suspension is an aqueous suspension of a 1:1 (w/w) compound/HPMCAS-HF spray-dried dispersion containing 2.5 mg/mL of compound in 2% polysorbate-80. Solid dispersions for use in a tablet or capsule will generally be mixed with other excipients or adjuvants typically found in such dosage forms. For example, an exemplary filler for capsules contains a 2:1 (w/w) compound/HPMCAS-MF spray-dried dispersion (60%), lactose (fast flow) (15%), microcrystalline cellulose (e.g., Avicel.sup.(R0-102) (15.8%), sodium starch (7%), sodium lauryl sulfate (2%) and magnesium stearate (1%).
The HPMCAS polymers are available in low, medium and high grades as Aqoa.sup.(R)-LF, Aqoat.sup.(R)-MF and Aqoat.sup.(R)-HF respectively from Shin-Etsu Chemical Co., LTD, Tokyo, Japan. The higher MF and HF grades are generally preferred.
Other carrier materials and modes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions of the invention may be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures. The above considerations in regard to effective formulations and administration procedures are well known in the art and are described in standard textbooks. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.
The compounds of the present invention can be used, alone or in combination with other therapeutic agents, in the treatment of various conditions or disease states. The compound(s) of the present invention and other therapeutic agent(s) may be may be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially.
Two or more compounds may be administered simultaneously, concurrently or sequentially. Additionally, simultaneous administration may be carried out by mixing the compounds prior to administration or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration.
The phrases “concurrent administration,” “co-administration,” “simultaneous administration,” and “administered simultaneously” mean that the compounds are administered in combination. The present invention includes the use of a combination of a LRRK2 inhibitor compound as provided in Formula (I) and one or more additional pharmaceutically active agent(s). If a combination of active agents is administered, then they may be administered sequentially or simultaneously, in separate dosage forms or combined in a single dosage form. Accordingly, the present invention also includes pharmaceutical compositions comprising an amount of: (a) a first agent comprising a compound of Formula I or a pharmaceutically acceptable salt of the compound; (b) a second pharmaceutically active agent; and (c) a pharmaceutically acceptable carrier, vehicle or diluent.
Various pharmaceutically active agents may be selected for use in conjunction with the compounds of Formula (I), depending on the disease, disorder, or condition to be treated. For example, a pharmaceutical composition for use in treating Parkinson's disease may comprise a compound of Formula (I) or a pharmaceutically acceptable salt thereof together with another agent such as a dopamine (levodopa, either alone or with a DOPA decarboxylase inhibitor), a monoamine oxidase (MAO) inhibitor, a catechol O-methyltransferase (COMT) inhibitor or an anticholinergic agent, or any combination thereof. Particularly preferred agents to combine with the compounds of Formula (I) for use in treating Parkinson's disease include levodopa, carbidopa, tolcapone, entacapone, selegiline, benztropine and trihexyphenidyl, or any combination thereof. Pharmaceutically active agents that may be used in combination with the compounds of Formula (I) and compositions thereof include, without limitation:
(i) levodopa (or its methyl or ethyl ester), alone or in combination with a DOPA decarboxylase inhibitor (e.g., carbidopa (SINEMET, CARBILEV, PARCOPA), benserazide (MADOPAR), α-methyldopa, monofluoromethyldopa, difluoromethyldopa, brocresine, or m-hydroxybenzylhydrazine);
(ii) anticholinergics, such as amitriptyline (ELAVIL, ENDEP), butriptyline, benztropine mesylate (COGENTIN), trihexyphenidyl (ARTANE), diphenhydramine (BENADRYL), orphenadrine (NORFLEX), hyoscyamine, atropine (ATROPEN), scopolamine (TRANSDERM-SCOP), scopolamine methylbromide (PARMINE), dicycloverine (BENTYL, BYCLOMINE, DIBENT, DILOMINE), tolterodine (DETROL), oxybutynin (DITROPAN, LYRINEL XL, OXYTROL), penthienate bromide, propantheline (PRO-BANTHINE), cyclizine, imipramine hydrochloride (TOFRANIL), imipramine maleate (SURMONTIL), lofepramine, desipramine (NORPRAMIN), doxepin (SINEQUAN, ZONALON), trimipramine (SURMONTIL), and glycopyrrolate (ROBINUL);
(iii) catechol O-methyltransferase (COMT) inhibitors, such as nitecapone, tolcapone (TASMAR), entacapone (COMTAN), and tropolone;
(iv) monoamine oxidase (MAO) inhibitors, such as selegiline (EMSAM), selegiline hydrochloride (I-deprenyl, ELDEPRYL, ZELAPAR), dimethylselegiline, brofaromine, phenelzine (NARDIL), tranylcypromine (PARNATE), moclobemide (AURORIX, MANERIX), befloxatone, safinamide, isocarboxazid (MARPLAN), nialamide (NIAMID), rasagiline (AZILECT), iproniazide (MARSILID, IPROZID, IPRONID), iproclozide, toloxatone (HUMORYL, PERENUM), bifemelane, desoxypeganine, harmine (also known as telepathine or banasterine), harmaline, linezolid (ZYVOX, ZYVOXID), and pargyline (EUDATIN, SUPIRDYL);
(v) acetylcholinesterase inhibitors, such as donepezil hydrochloride (ARICEPT®, MEMAC), physostigmine salicylate (ANTILIRIUM®), physostigmine sulfate (ESERINE), ganstigmine, rivastigmine (EXELON®), ladostigil, NP-0361, galantamine hydrobromide (RAZADYNE®, REMINYL®, NIVALIN®), tacrine (COGNEX®), tolserine, memoquin, huperzine A (HUP-A; Neuro-Hitech), phenserine, bisnorcymserine (also known as BNC), and INM-176;
(vi) amyloid-β (or fragments thereof), such as Aβ1-15 to pan HLA DRbinding epitope (PADRE®), ACC-001 (Elan/Wyeth), and Affitope;
(vii) antibodies to amyloid-β (or fragments thereof), such as ponezumab, solanezumab, bapineuzumab (also known as AAB-001), AAB-002 (Wyeth/Elan), Gantenerumab, intravenous Ig (GAMMAGARD®), LY2062430 (humanized m266; Lilly), and those disclosed in International Patent Publication Nos WO04/032868, WO05/025616, WO06/036291, WO06/069081, WO06/118959, in US Patent Publication Nos US2003/0073655, US2004/0192898, US2005/0048049, US2005/0019328, in European Patent Publication Nos EP0994728 and 1257584, and in U.S. Pat. No. 5,750,349;
(viii) amyloid-lowering or -inhibiting agents (including those that reduce amyloid production, accumulation and fibrillization) such as eprodisate, celecoxib, lovastatin, anapsos, colostrinin, pioglitazone, clioquinol (also known as PBT1), PBT2 (Prana Biotechnology), flurbiprofen (ANSAID®, FROBEN®) and its Renantiomer tarenflurbil (FLURIZAN®), nitroflurbiprofen, fenoprofen (FENOPRON, NALFON®), ibuprofen (ADVIL®, MOTRIN®, NUROFEN®), ibuprofen lysinate, meclofenamic acid, meclofenamate sodium (MECLOMEN®), indomethacin (INDOCIN®), diclofenac sodium (VOLTAREN®), diclofenac potassium, sulindac (CLINORIL®), sulindac sulfide, diflunisal (DOLOBID®), naproxen (NAPROSYN®), naproxen sodium (ANAPROX®, ALEVE®), insulindegrading enzyme (also known as insulysin), the Gingko biloba extract EGb-761 (ROKAN®, TEBONIN®), tramiprosate (CEREBRIL®, ALZHEMED®), KIACTA®), neprilysin (also known as neutral endopeptidase (NEP)), scyllo-inositol (also known as scyllitol), atorvastatin (LIPITOR®), simvastatin (ZOCOR®), ibutamoren mesylate, BACE inhibitors such as LY450139 (Lilly), BMS-782450, GSK-188909; gamma secretase modulators and inhibitors such as ELND-007, BMS-708163 (Avagacestat), and DSP8658 (Dainippon); and RAGE (receptor for advanced glycation end-products) inhibitors, such as TTP488 (Transtech) and TTP4000 (Transtech), and those disclosed in U.S. Pat. No. 7,285,293, including PTI-777;
(ix) alpha-adrenergic receptor agonists, and beta-adrenergic receptor blocking agents (beta blockers); anticholinergics; anticonvulsants; antipsychotics; calcium channel blockers; catechol O-methyltransferase (COMT) inhibitors; central nervous system stimulants; corticosteroids; dopamine receptor agonists and antagonists; dopamine reuptake inhibitors; gamma-am inobutyric acid (GABA) receptor agonists; immunosuppressants; interferons; muscarinic receptor agonists; neuroprotective drugs; nicotinic receptor agonists; norepinephrine (noradrenaline) reuptake inhibitors; quinolines; and trophic factors;
(x) histamine 3 (H3) antagonists, such as PF-3654746 and those disclosed in US Patent Publication Nos US2005-0043354, US2005-0267095, US2005-0256135, US2008-0096955, US2007-1079175, and US2008-0176925; International Patent Publication Nos WO2006/136924, WO2007/063385, WO2007/069053, WO2007/088450, WO2007/099423, WO2007/105053, WO2007/138431, and WO2007/088462; and U.S. Pat. No. 7,115,600);
(xi) N-methyl-D-aspartate (NMDA) receptor antagonists, such as memantine (NAMENDA, AXURA, EBIXA), amantadine (SYMMETREL), acamprosate (CAMPRAL), besonprodil, ketamine (KETALAR), delucemine, dexanabinol, dexefaroxan, dextromethorphan, dextrorphan, traxoprodil, CP-283097, himantane, idantadol, ipenoxazone, L-701252 (Merck), lancicemine, levorphanol (DROMORAN), methadone, (DOLOPHINE), neramexane, perzinfotel, phencyclidine, tianeptine (STABLON), dizocilpine (also known as MK-801), ibogaine, voacangine, tiletamine, riluzole (RILUTEK), aptiganel (CERESTAT), gavestinel, and remacimide;
(xii) phosphodiesterase (PDE) inhibitors, including (a) PDE1 inhibitors; (b) PDE2 inhibitors; (c) PDE3 inhibitors; (d) PDE4 inhibitors; (e) PDE5 inhibitors; (f) PDE9 inhibitors (e.g., PF-04447943, BAY 73-6691 (Bayer AG) and those disclosed in US Patent Publication Nos US2003/0195205, US2004/0220186, US2006/0111372, US2006/0106035, and U.S. Ser. No. 12/118,062 (filed May 9, 2008)); and (g) PDE10 inhibitors such as 2-({4-[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]phenoxy}methyl)quinoline (PF-2545920);
(xiii) serotonin (5-hydroxytryptamine) 1A (5-HT1A) receptor antagonists, such as spiperone, levo-pindolol, lecozotan;
(xiv) serotonin (5-hydroxytryptamine) 2C (5-HT2c) receptor agonists, such as vabicaserin, and zicronapine; serotonin (5-hydroxytryptamine) 4 (5-HT4) receptor agonists/antagonists, such as PRX-03140 (Epix) and PF-04995274;
(xv) serotonin (5-hydroxytryptamine) 3C (5-HT3c) receptor antagonists, such as Ondansetron (Zofran);
(xvi) serotonin (5-hydroxytryptamine) 6 (5-HT6) receptor antagonists, such as mianserin (TOLVON, BOLVIDON, NORVAL), methiothepin (also known as metitepine), ritanserin, SB-271046, SB-742457 (GlaxoSmithKline), Lu AE58054 (Lundbeck NS), SAM-760, and PRX-07034 (Epix);
(xvii) serotonin (5-HT) reuptake inhibitors such as alaproclate, citalopram (CELEXA, CIPRAMIL), escitalopram (LEXAPRO, CIPRALEX), clomipramine (ANAFRANIL), duloxetine (CYMBALTA), femoxetine (MALEXIL), fenfluramine (PONDIMIN), norfenfluramine, fluoxetine (PROZAC), fluvoxamine (LUVOX), indalpine, milnacipran (IXEL), paroxetine (PAXIL, SEROXAT), sertraline (ZOLOFT, LUSTRAL), trazodone (DESYREL, MOLIPAXIN), venlafaxine (EFFEXOR), zimelidine (NORMUD, ZELMID), bicifadine, desvenlafaxine (PRISTIQ), brasofensine, vilazodone, cariprazine and tesofensine;
(xviii) Glycine transporter-1 inhibitors such as paliflutine, ORG-25935, and ORG26041; and mGluR modulators such as AFQ-059 and amantidine;
(xix) AMPA-type glutamate receptor modulators such as perampanel, mibampator, selurampanel, GSK-729327, and N-{(3S,4S)-4-[4-(5-cyanothiophen-2-yl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide;
(xx) P450 inhibitors, such as ritonavir;
(xxi) tau therapy targets, such as davunetide;
and the like.
The present invention further comprises kits that are suitable for use in performing the methods of treatment described above. In one embodiment, the kit contains a first dosage form comprising one or more of the compounds of the present invention and a container for the dosage, in quantities sufficient to carry out the methods of the present invention.
In another embodiment, the kit of the present invention comprises one or more compounds of the invention.
In one embodiment, the compound of the present invention is:
[(2S,4R)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile;
[(2R,4S)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2;
8-chloro-1-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline;
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(1-methyl-1H-1,2,3-triazol-4-yl) methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
[cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 1;
[cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 2;
8-(difluoromethyl)-2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyl tetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-(difluoromethyl)-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-methylpyrazin-2-yl)methanol, DIAST 1;
{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-methylpyrazin-2-yl)methanol, DIAST 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methoxy-1H-pyrazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methoxy-1H-pyrazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-fluoro-2-[(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
2-cyclopentyl-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
[cis-4-(8-chloro-2-methyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 1;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1;
2-[(5-methylpyrazin-2-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-2H-tetrazol-2-yl)methyl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
2-[(3-methyl-1,2-oxazol-5-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
2-[(5-methyl-1,3-oxazol-2-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo [4,5-c]quinoline-8-carbonitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-{[5-(trifluoromethyl)pyrazin-2-yl]methyl}-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline;
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-yl)methyl]-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-2H-tetrazol-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2-oxazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2-oxazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 1;
2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1-[(3R)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-[cis-2-(difluoromethyl)tetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,2-oxazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
8-chloro-1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,2,3-thiadiazol-4-ylmethyl)-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-fluoro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-imidazo[4,5-c]quinoline;
2-(1,3-benzoxazol-2-ylmethyl)-1-[cis-3-fluorocyclopentyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1H-1,2,4-triazol-1-ylmethyl)-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-2-[(5-methylpyrazin-2-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline;
1-[cis-3-fluorocyclopentyl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-(1,3-thiazol-4-ylmethyl)-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
2-[(5-methyl-1,3,4-oxadiazol-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(2,2-difluoropropyl)-2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline;
8-fluoro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-{[5-(trifluoromethyl)pyrazin-2-yl]methyl}-1H-imidazo[4,5-c]quinoline;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,3,4-thiadiazol-2-yl) methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-ylmethyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-[cis-3-fluorocyclopentyl]-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline;
3-{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}-2-methylpropanenitrile, DIAST 2;
8-fluoro-1-[cis-3-fluorocyclopentyl]-2-(1,2,3-thiadiazol-4-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
3-{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}propanenitrile;
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(5-methyl-2H-tetrazol-2-yl)methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-tetrazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(1-methyl-1H-1,2,4-triazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline;
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-tetrazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,3,4-thiadiazol-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2;
8-(difluoromethyl)-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline;
8-(difluoromethyl)-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1;
8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-yl)methyl]-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolone, ENT 1; or
[5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl]methanol or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of the present invention is:
8-chloro-2-{[5-(2H3)methylpyrazin-2-yl]methyl}-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-2-{[5-(2H3)methylpyrazin-2-yl](2H2)methyl}-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-2-{[5-(2H2)methylpyrazin-2-yl]methyl}-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
8-chloro-2-{[5-(2H1)methylpyrazin-2-yl]methyl}-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline;
[5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl](2H2)methanol; or
[5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl](2H1)methanol,
or a pharmaceutically acceptable salt thereof.
In another embodiment of the present invention, the compound of Formula I has
R1 is ethyl or
R2 is
and
R3 is chloro, cyano, difluoromethyl, or trifluoromethyl,
or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of Formula I has
R1 is
R2 is
and
R3 is chloro or cyano,
or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of the present invention of Formula I has
R1 is
R2 is
and
R3 is chloro,
or a pharmaceutically acceptable salt thereof.
In yet another embodiment, the compound of the present invention of Formula I has
R1 is
R2 is
and
R3 is chloro,
or a pharmaceutically acceptable salt thereof.
In another embodiment, the present invention directed at a method of treating a disease or disorder selected from the group consisting of Crohn's disease, Parkinson's disease, Lewy body dementia, frontotemporal dementia, corticobasal dementia, progressive supranuclear palsy, leprosy, Alzheimer's disease, tauopathy disease and Alpha-synucleinopathy in a patient, the method comprising administering to a patient in need of treatment thereof a therapeutically effective amount of a compound of Formula (I) or pharmaceutically acceptable salt thereof.
In yet another embodiment of the present invention, the treatment of a disease or disorder is selected from the group consisting of Crohn's disease, Parkinson's disease, Lewy body dementia, frontotemporal dementia, corticobasal dementia, progressive supranuclear palsy, leprosy, Alzheimer's disease, tauopathy disease and Alpha-synucleinopathy.
In another embodiment, the treatment of a disease or disorder is selected from the group consisting of Lewy body dementia, frontotemporal dementia, corticobasal dementia, progressive supranuclear palsy, leprosy, inflammatory bowel disease, inflammatory bowel syndrome, Alzheimer's disease, tauopathy diseases, Alpha-synucleinopathy, Parkinson's disease, Parkinson's disease with dementia, Parkinson's disease at risk syndrome, Lewy body variant of Alzheimer's disease, combined Parkinson's disease and Alzheimer's disease, multiple system atrophy, striatonigral degeneration, olivopontocerebellar atrophy, Shy-Drager syndrome, ulcerative colitis, juvenile parkinsonism, Steele-Richardson-Olszewski disease, Lytico-Bodig or parkinsonism-dementia-ALS complex of Guam, cortical basal ganglionic degeneration, progressive pallidal atrophy, Parkinsonism-dementia complex, pallidopyramidal disease, hereditary juvenile dystonia-parkinsonism, autosomal dominant Lewy body disease, Huntington disease, Wilson disease, hereditary ceruloplasmin deficiency, Hallervorden-Spatz disease, olivopontocerebellar and spinocerebellar degenerations, Machado-Joseph disease, familial amyotrophy-dementia-parkinsonism, disinhibitiondementia-parkinsonism-amyotrophycomplex, Gerstmann-Strausler-Scheinker disease, familial progressive subcortical gliosis, Lubag (x-linked dystonia parkinsonism), familial basal ganglia calcification, mitochondrial cytopathies with striatal necrosis, ceroid lipofuscinosis, familial Parkinsonism with peripheral neuropathy, Parkinsonism-pyramidal syndrome, neuroacanthocytosis and hereditary hemochromatosis.
In yet another embodiment of the present invention, the treatment of a disease or disorder is selected from a neurological disorder, most preferably Parkinson's disease, (but also other neurological disorders such as migraine; epilepsy; Alzheimer's disease; Niemann-Pick type C; brain injury; stroke; cerebrovascular disease; cognitive disorder; sleep disorder) or a psychiatric disorder (such as anxiety; factitious disorder; impulse control disorder; mood disorder; psychomotor disorder; psychotic disorder; drug dependence; eating disorder; and pediatric psychiatric disorder) in a mammal, preferably a human, comprising administering to said mammal a therapeutically effective amount of a compound of Formula I or pharmaceutically acceptable salt thereof. In addition, the compounds of Formula I and pharmaceutically acceptable salts thereof may also be employed in methods of treating other disorders associated with LRRK2 such as Crohn's disease, leprosy and certain cancers, such as kidney, breast, lung, prostate, lung and blood cancer.
The text revision of the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) (2000, American Psychiatric Association, Washington D.C.) provides a diagnostic tool for identifying many of the disorders described herein. The skilled artisan will recognize that there are alternative nomenclatures, nosologies, and classification systems for disorders described herein, including those as described in the DMS-IV-TR, and that terminology and classification systems evolve with medical scientific progress.
General Synthetic Schemes
The compounds of Formula I may be prepared by the methods described below, together with synthetic methods known in the art of organic chemistry, or modifications and transformations that are familiar to those of ordinary skill in the art. The starting materials used herein are commercially available or may be prepared by routine methods known in the art [such as those methods disclosed in standard reference books such as the Compendium of Organic Synthetic Methods, Vol. 1-XIII (published by Wiley-Interscience)]. Preferred methods include, but are not limited to, those described below.
During any of the following synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by means of conventional protecting groups, such as those described in T. W. Greene, Protective Groups in Organic Chemistry, John Wiley & Sons, 1981; T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Chemistry, John Wiley & Sons, 1991; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Chemistry, John Wiley & Sons, 1999, which are hereby incorporated by reference.
Compounds of Formula I, or their pharmaceutically acceptable salts, can be prepared according to the Reaction Schemes discussed herein below. Unless otherwise indicated, the substituents in the Schemes are defined as above. Isolation and purification of the products is accomplished by standard procedures, which are known to a chemist of ordinary skill.
One skilled in the art will recognize that in many cases, the compounds in Reaction Schemes 1 through 9 may be generated as a mixture of diastereomers and/or enantiomers; these may be separated at various stages of the synthetic schemes using conventional techniques or a combination of such techniques, such as, but not limited to, crystallization, normal-phase chromatography, reversed phase chromatography and chiral chromatography, to afford the single enantiomers of the invention.
It will be understood by one skilled in the art that the various symbols, superscripts and subscripts used in the schemes, methods and examples are used for convenience of representation and/or to reflect the order in which they are introduced in the schemes, and are not intended to necessarily correspond to the symbols, superscripts or subscripts in the appended claims. The schemes are representative of methods useful in synthesizing the compounds of the present invention. They are not to constrain the scope of the invention in any way.
The reactions for preparing compounds of the invention can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.
Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).
Compounds of Formula I and intermediates thereof may be prepared according to the following reaction schemes and accompanying discussion. Unless otherwise indicated, R1, R2 and R3 in the reaction schemes and discussions that follow are as defined as the same as hereinabove. In general the compounds of this invention may be made by processes which include processes analogous to those known in the chemical arts, particularly in light of the description contained herein. Certain processes for the manufacture of the compounds of this invention and intermediates thereof are provided as further features of the invention and are illustrated by the following reaction schemes. Other processes may be described in the experimental section. The schemes and examples provided herein (including the corresponding description) are for illustration only, and not intended to limit the scope of the present invention.
Reaction Scheme 1 depicts the preparation of compounds of Formula (I). Referring to Scheme 1, compounds 1.1 and 1.2 are either commercially available or can be made by methods described herein or other methods well known to those skilled in the art. In the compound of formula 1.1 the group designated LG represents an appropriate leaving group such as a halide (e.g., chloro or bromo) or triflate which is suitable to undergo nucleophilic displacement when reacted with the amine of formula 1.2. In the amine compound of formula 1.2, the group designated PG represents an appropriate amine protecting group such as an acid-labile protecting group selected from 2,4-dimethoxybenzyl (DMB), 4-methoxybenzyl (PMB) and t-butoxycarbonyl (Boc). The compounds of formulae 1.1 and 1.2 can be reacted, for example, in the presence of an appropriate base such as N,N-diisopropylethylamine (Hunig's base) or triethylamine in a suitable solvent such as acetonitrile or N,N-dimethylformamide (DMF) to afford the compound of formula 1.3. The reaction is typically carried out at an elevated temperature, such as 50 to 100° C. for a period of 1 to 48 hours. Removal of the protecting group, such as an acid-labile protecting group (PG) from the compound of formula 1.3 can typically be accomplished by treatment of 1.3 with an appropriate acid such as acetic acid, trifluoroacetic acid or hydrochloric acid to provide the compound of formula 1.4. Also, it is to be understood that in certain instances the compound of formula 1.1 can be reacted with an unprotected amine of formula R2—NH2 to arrive directly to a compound of formula 1.4. Reduction of the nitro group in the compound of formula 1.4 using conditions congruent with the functionality present affords the compound of formula 1.5. For example, the nitro group in the compound of formula 1.4 can be reduced to the corresponding amine of formula 1.5 by treatment of 1.4 with zinc dust and ammonium hydroxide in methanol or alternatively by hydrogenation of 1.4 using an appropriate catalyst such as platinum(IV) oxide in an appropriate solvent such as methanol, acetonitrile or a mixture thereof. Coupling the diamine compound 1.5 with the carboxylic acid of formula 1.6 then provides the desired compound of Formula I, also denoted as 1.7. The coupling reaction with the diamine of formula 1.5 and the carboxylic acid of formula 1.6 can be carried out in an appropriate solvent such as N,N-dimethylformamide or N-propylacetate in the presence of an appropriate base such as N,N-diisopropylethylamine and a coupling reagent such as 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphirane 2,4,6-trioxide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). The coupling reaction is often heated between 60° C. and 110° C.
Reaction Scheme 2 depicts the preparation of compounds of formula 1.7′, which is a compound of Formula I in which R2 is the chiral 2-methyltetrahydropyran-4-yl moiety as shown. Using a published procedure, Prins reaction of the compound 2.1 with the compound 2.2 generates the pyran 2.3. Chiral resolution to produce the separated enantiomers, using an enzyme-based method, affords the compound of formula 2.5 after hydrolysis of the resolved ester 2.4. Oxidation of 2.5 provides ketone 2.6, which is reacted with the compound of formula 2.7 using reductive amination chemistry to provide the protected amine of formula 2.8. The protected amine of formula 2.8 can be reacted with the compound of formula 1.1 in a manner analogous to that previously described in Scheme 1 to provide the compound of formula 1.3′. The compounds of formulae 1.4′, 1.5′ and 1.7′ can then be prepared in a manner analogous to the methods described in Scheme 1 for the compounds of formulae 1.4, 1.5 and 1.7, respectively.
Reaction Scheme 3 depicts the preparation of compounds of formula 3.13, which is a compound of Formula I in which R2 is the chiral 2-cyanomethyltetrahydropyran-4-yl moiety as shown. Using a published procedure, Prins reaction of the compound 3.1 with but-3-en-1-ol generated the pyran 3.2. Oxidation of 3.2 gave ketone 3.3 which was reacted with dimethoxybenzylamine using reductive amination chemistry to provide the protected amine of formula 3.4. The protected amine of formula 3.4 can be reacted with the compound of formula 1.1 in a manner analogous to that previously described in Scheme 1 to provide the compound of formula 3.5. Removal of the protecting group under acidic conditions afforded 3.6. The nitro group of 3.6 is reduced by catalytic hydrogenation or by treatment with a metal such as zinc or iron to afford the diamine 3.7. Acylation of 3.7 with acid 3.8 under a variety of coupling conditions known to those skilled in the art affords 3.9. The amide 3.9 can be dehydrated under thermal conditions to afford 3.10. Deprotection of 3.10 with a Lewis acid such as BCl3, TMSI, AlCl3 or through palladium-catalyzed hydrogenolysis afford the alcohol 3.11. The alcohol 3.11 can be converted to an activated leaving group such as, but not limited to, a sulfonate such as the mesylate 3.12. The compounds of formulae 3.13 can then be prepared by nucleophilic displacement of the mesylate with cyanide anion.
Reaction Scheme 4 depicts the preparation of compounds of formula 4.8, which is a compound of Formula I in which R2 is the chiral 2-methyltetrahydropyran-4-yl moiety and R3 is cyano as shown. The reaction begins from known acid 4.1, which is reacted with N-hydroxy-2-nitroethenamine prepared in situ to afford 4.2. The nitroamine 4.2 was treated with an agent that activated the carboxylic acid followed by condensation to afford quinolone 4.3. The phenol of 4.3 can be converted to the activated chloride 4.4 with phosphorous oxychloride or thionyl chloride. Chloride 4.4 can undergo nucleophilic displacement with an appropriate amine such as 2.8 to afford 4.5. 4.5 can be deprotected to provide 4.6 which, in turn, is reduced to provide diamine 4.7. Compounds of formula 4.8 can be made from 4.7 by condensation with an appropriate acid R1CO2H in a manner similar to that previously described.
Reaction Scheme 5 depicts the preparation of compounds of the formula 5.6, which is a compound of Formula I in which R2 is the chiral 2-methyltetrahydropyran-4-yl moiety and R3 is the difluoromethyl group as shown. Compound 5.1 is treated with 2,2-difluoro-1-phenylethan-1-one and a suitable palladium complex such as cataCXium A Pd G2 and base such as tri-potassium phosphate n-hydrate in an inert solvent such as toluene to afford compound 5.2. The benzoyl group of 5.2 can be removed with a base such as sodium hydroxide or potassium hydroxide in water or other similar conditions. Alternatively the benzoyl is removed in alcohol solvent with sodium methoxide. The protecting group of 5.3 (such as a DMB group) can be removed as previously described and the nitro group of 5.4 can be reduced to provide the diamine 5.5. Compounds of formula 5.6 can be made from 5.5 in a manner similar to that previously described by condensation of 5.5 with an appropriate acid R1CO2H.
Reaction Scheme 6 depicts the preparation of compounds of the formula 6.9, which is a compound of Formula I in which R2 is the chiral 4,4-difluoro-1-methylpyrrolidin-3-yl moiety and R3 is cyano as shown. This amine is available through a procedure described in US Published Patent Application 20150141402. This series of compounds may be prepared as in the examples above, through formation of the chloride 6.3 through reaction of 6.2 with phosphorous oxychloride or thionyl chloride in a suitable inert solvent. The chloride was treated with amine 6.4 in the presence of a suitable base such as Hunig's base (N,N-diisopropylethylamine) or triethylamine to afford 6.5. The protecting group is removed by treatment of 6.5 with an acid such as trifluoroacetic acid or hydrochloric acid. The secondary amine 6.6 can be methylated through a standard reductive amination using formaldehyde and a reducing agent such as sodium triacetoxyborohydride or sodium cyanoborohydride. The nitro group of compound 6.7 can be reduced through hydrogenation over a platinum catalyst or alternatively the nitro group can be reduced with a suitable metal such as iron or zinc. The claimed compounds 6.9 can be made from 6.8 through condensations with a suitable acid R1CO2H under the conditions described previously.
Reaction Scheme 7 depicts the preparation of compounds of the formula 7.5, which is a compound of Formula I in which R2 is the chiral 3,3-difluorotetrahydro-2H-pyran-4-amine moiety as shown. The chloride 7.1 is treated with amine 7.2 in the presence of a suitable base such as Hunig's base or triethylamine to afford 7.3. The nitro group of compound 7.3 can be reduced through hydrogenation over a platinum catalyst or alternatively the nitro group can be reduced with a suitable metal such as iron or zinc. The compounds 7.5 can then be made from 7.4 through condensation with a suitable acid R1CO2H under the conditions described previously.
Reaction Scheme 8 depicts the preparation of compounds of the formula 8.5, which is a compound of Formula I in which R2 is the chiral (R)-1-methylpyrrolidin-3-amine moiety and R3 is cyano as shown. The chloride was treated with chiral amine 8.2 in the presence of a suitable base such as Hunig's base or triethylamine to afford 8.3. The nitro group of compound 8.3 can be reduced through hydrogenation over a platinum catalyst or alternatively the nitro group can be reduced with a suitable metal such as iron or zinc. The compounds 8.5 can be made from 8.4 through condensation with a suitable acid R1CO2H under the conditions described previously.
Reaction Scheme 9 depicts the preparation of compounds of the formula 9.8, which is a compound of Formula I in which R2 is the chiral 2-methyltetrahydropyran-4-yl moiety and R3 is trifluoromethyl as shown. The chloride 9.3 was treated with amine 2.8 in the presence of a suitable base such as Hunig's base or triethylamine to afford 9.5. Removal of the protecting group under acidic conditions affords 9.6. The nitro group of compound 9.6 can be reduced through hydrogenation over a platinum catalyst or alternatively the nitro group can be reduced with a suitable metal such as iron or zinc. The claimed compounds 9.8 can be made from 9.7 through condensation with a suitable acid R1CO2H under the conditions described previously.
The methods generically described in Schemes 1 through 9 are not to be construed in a limiting manner. It is to be understood by one skilled in the art that variation in the order of certain reaction steps and conditions may be employed to provide compounds of Formula I. The selection of which approach is best to utilize can be made by one skilled in the art of organic synthesis. More specific examples of the methods used to prepare compounds of Formula I are provided below in the Examples, and likewise these methods are also not to be construed by one skilled in the art in a limiting manner.
Experimental Procedures
The following illustrate the synthesis of various compounds of the present invention. Additional compounds within the scope of this invention may be prepared using the methods illustrated in these Examples, either alone or in combination with techniques generally known in the art.
Experiments were generally carried out under inert atmosphere (nitrogen or argon), particularly in cases where oxygen- or moisture-sensitive reagents or intermediates were employed. Commercial solvents and reagents were generally used without further purification. Anhydrous solvents were employed where appropriate, generally AcroSeal® products from Acros Organics, Aldrich Sure/Sear™ from SigmaAldrich, or DriSolv® products from EMD Chemicals. In other cases, commercial solvents were passed through columns packed with 4 Å molecular sieves, until the following QC standards for water were attained: a) <100 ppm for dichloromethane, toluene, N,N-dimethylformamide and tetrahydrofuran; b) <180 ppm for methanol, ethanol, 1,4-dioxane and diisopropylamine. For very sensitive reactions, solvents were further treated with metallic sodium, calcium hydride or molecular sieves, and distilled just prior to use. Products were generally dried under vacuum before being carried on to further reactions or submitted for biological testing. Mass spectrometry data is reported from either liquid chromatography-mass spectrometry (LCMS), atmospheric pressure chemical ionization (APCI) or gas chromatography-mass spectrometry (GCMS) instrumentation. Chemical shifts for nuclear magnetic resonance (NMR) data are expressed in parts per million (ppm, δ) referenced to residual peaks from the deuterated solvents employed. In some examples, chiral separations were carried out to separate enantiomers or diastereomers of certain compounds of the invention (in some examples, the separated enantiomers are designated as ENT 1 and ENT 2, according to their order of elution, and the separated diastereomers are designated as DIAST 1 and DIAST 2, according to their order of elution). In some examples, the optical rotation of an enantiomer was measured using a polarimeter. According to its observed rotation data (or its specific rotation data), an enantiomer with a clockwise rotation was designated as the (+)-enantiomer and an enantiomer with a counter-clockwise rotation was designated as the (−)-enantiomer. Racemic compounds are indicated by the presence of (+/−) adjacent to the structure; in these cases, indicated stereochemistry represents the relative (rather than absolute) configuration of the compound's substituents.
Reactions proceeding through detectable intermediates were generally followed by LCMS, and allowed to proceed to full conversion prior to addition of subsequent reagents. For syntheses referencing procedures in other Examples or Methods, reaction conditions (reaction time and temperature) may vary. In general, reactions were followed by thin-layer chromatography or mass spectrometry, and subjected to work-up when appropriate. Purifications may vary between experiments: in general, solvents and the solvent ratios used for eluents/gradients were chosen to provide appropriate Rfs or retention times. All starting materials in these Preparations and Examples are either commercially available or can be prepared by methods known in the art or as described herein.
Reactions were performed in air or, when oxygen- or moisture-sensitive reagents or intermediates were employed, under an inert atmosphere (nitrogen or argon). When appropriate, reaction apparatuses were dried under dynamic vacuum using a heat gun, and anhydrous solvents (Sure-Seal™ products from Aldrich Chemical Company, Milwaukee, Wis. or DriSolv™ products from EMD Chemicals, Gibbstown, N.J.) were employed. Commercial solvents and reagents were used without further purification. When indicated, reactions were heated by microwave irradiation using Biotage Initiator or Personal Chemistry Emrys Optimizer microwaves or the like. Reaction progress was monitored using thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS) and high performance liquid chromatography (HPLC), analyses. TLC was performed on pre-coated silica gel plates with a fluorescence indicator (254 nm excitation wavelength) and visualized under UV light and/or with I2, KMnO¬4, CoCl2, phosphomolybdic acid, and/or ceric ammonium molybdate stains. LCMS data were acquired on an Agilent 1100 Series instrument with a Leap Technologies autosampler, Gemini C18 columns, MeCN/water gradients, and either TFA, formic acid, or ammonium hydroxide modifiers or similar equipment. The column eluent was analyzed using Waters ZQ mass spectrometer scanning in both positive and negative ion modes from 100 to 1200 Da. Other similar instruments were also used. HPLC data were acquired on an Agilent 1100 Series instrument using Gemini or XBridge C18 columns, MeCN/water gradients, and either TFA or ammonium hydroxide modifiers and comparable equip-ment. Purifications were performed by medium performance liquid chromatography (MPLC) using Isco CombiFlash Companion, AnaLogix IntelliFlash 280, Biotage SP1, or Biotage Isolera One instruments and pre-packed Isco RediSep or Biotage Snap silica cartridges and the like. Chiral purifications were performed by chiral supercritical fluid chromatography (SFC) using Berger or Thar instruments and similar instruments; Chi-ralPAK-AD, -AS, -IC, Chiralcel-OD, or -OJ columns; and CO2 mixtures with MeOH, EtOH, iPrOH, or MeCN, alone or modified using TFA or iPrNH2. UV detection was used to trigger fraction collection.
Mass spectrometry data are reported from LCMS analyses. Mass spectrometry (MS) was performed via atmospheric pressure chemical ionization (APCI), electrospray Ionization (ESI), electron impact ionization (EI) or electron scatter (ES) ionization sources. Proton nuclear magnetic spectroscopy (1H NMR) chemical shifts are given in parts per million downfield from tetramethylsilane and were recorded on on 300, 400, 500, or 600 MHz Varian spectrometers. Chemical shifts are expressed in parts per million (ppm, 6) referenced to the deuterated solvent residual peaks. The peak shapes are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br s, broad singlet; app, apparent. Analytical SFC data were acquired on a Berger analytical instrument as described above. Optical rotation data were acquired on a PerkinElmer model 343 polarimeter using a 1 dm cell. Silica gel chromatography was performed primarily using a medium pressure Biotage or ISCO systems using columns pre-packaged by various commercial vendors including Biotage and ISCO.
Unless otherwise noted, chemical reactions were performed at room temperature (about 23 degrees Celsius).
The compounds and intermediates described below were named using the naming convention provided with ACD/ChemSketch 2012, File Version C10H41, Build 69045 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). The naming convention provided with ACD/ChemSketch 2012 is well known by those skilled in the art and it is believed that the naming convention provided with ACD/ChemSketch 2012 generally comports with the IUPAC (International Union for Pure and Applied Chemistry) recommendations on Nomenclature of Organic Chemistry and the CAS Index rules.
In the experimental sections that follow the following abbreviations may be used. ACN is acetonitrile; Ac2O is acetic anhydride; br is broad; ° C. is degrees Celsius; CDCl3 is deutero chloroform; CD3OD is deutero methanol; CH3NO2 is nitromethane; d is doublet; DCM is dichloromethane; DEA is diethylamine; DIAST is diastereomer; DIEA is N,N-diisopropylethylamine; DMB is dimethoxybenzyl; DMSO is dimethyl sulfoxide, EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ENT is enantiomer; EtOAc is ethyl acetate; EtOH is ethanol; ES is electrospray; FA is formic acid; g is gram; h is hour; HCl is hydrochloric acid; H2 is hydrogen; H2O is water; HPLC is high performance liquid chromatography; Hz is hertz; K2CO3 is potassium carbonate; L is liter; LC is liquid chromatography; LCMS is liquid chromatography mass spectrometry; m is multiplet; M is molar; MeOH is methanol; MgSO4 is magnesium sulfate; MHz is megahertz; min is minute; mL is milliliter, mM is millimole; μL is microliter; μM is micromole; MS is mass spectrometry; MsCl is methane sulfonyl chloride; MTBE is methyl tert-butyl ether; NADPH is nicotinamide adenine dinucleotide phosphate; N2 is nitrogen; NEt3 is triethylamine; NaHCO3 is sodium bicarbonate; Na2SO4 is sodium sulfate; NH4Cl is ammonium chloride; NH4HCO3 is ammonium hydrogen carbonate; NH4OH is ammonium hydroxide; NMR is nuclear magnetic resonance, PE is petroleum ether; PSI is pounds per square inch; Pt/C is platinum on carbon; RT is retention time or room temperature depending on context; s is singlet; SFC is super critical fluid chromatography; t is triplet; TFA is trifluoroacetic acid; THF is tetrahydrofuran; TLC is thin-layer chromatography; and T3P is propyl phosphonic anhydride.
Preparation P1
(2R,4R)—N-(2,4-Dimethoxybenzyl)-2-methyltetrahydro-2H-pyran-4-amine (P1)
Step 1. Synthesis of cis-2-methyltetrahydro-2H-pyran-4-ol (C1)
But-3-en-1-ol (39.0 mL, 453 mmol) and acetaldehyde (25.5 mL, 454 mmol) were combined in aqueous sulfuric acid (20% w/w, 565 g) and stirred at 80° C. for 5 days. The reaction mixture was cooled to room temperature and extracted with diethyl ether, and then with dichloromethane; the combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 25% ethyl acetate in heptane) afforded the product as a colorless oil. Yield: 11.2 g, 96.4 mmol, 21%. 1H NMR (400 MHz, CDCl3) δ 3.99 (ddd, J=11.8, 4.9, 1.7 Hz, 1H), 3.71-3.80 (m, 1H), 3.35-3.46 (m, 2H), 1.82-1.98 (m, 3H), 1.48 (dddd, J=12.5, 12.4, 11.1, 4.9 Hz, 1H), 1.21 (d, J=6.2 Hz, 3H), 1.14-1.24 (m, 1H).
Step 2. Synthesis of (2R,4R)-2-methyltetrahydro-2H-pyran-4-yl butanoate (C2)
Ethenyl butanoate (78.6 mL, 620 mmol) and Novozyme 435 (immobilized Candida antarctica lipase B, 25 g) were added to a solution of C1 (150 g, 1.29 mol) in tetrahydrofuran (1.3 L). The reaction mixture was stirred at room temperature for 2 hours, whereupon it was filtered through a pad of diatomaceous earth, which was then rinsed twice with dichloromethane. The combined filtrates were concentrated in vacuo and purified via silica gel chromatography (Gradient: 0% to 10% ethyl acetate in heptane), providing the product as an oil. Yield: 51.5 g, 276 mmol, 45%. The absolute configurations of C2 and subsequent intermediates were confirmed via an X-ray structural determination carried out on C32 (see Preparation P10). 1H NMR (400 MHz, CDCl3) δ 4.82-4.92 (m, 1H), 3.99 (ddd, J=11.9, 4.9, 1.7 Hz, 1H), 3.42-3.52 (m, 2H), 2.25 (t, J=7.4 Hz, 2H), 1.92-2.00 (m, 1H), 1.84-1.91 (m, 1H), 1.52-1.69 (m, 3H), 1.28 (ddd, J=12, 11, 11 Hz, 1H), 1.20 (d, J=6.2 Hz, 3H), 0.94 (t, J=7.4 Hz, 3H).
Step 3. Synthesis of (2R,4R)-2-methyltetrahydro-2H-pyran-4-ol (C3)
A solution of C2 (51.5 g, 276 mmol) in methanol and tetrahydrofuran (1:1, 700 mL) was treated with a solution of lithium hydroxide (19.9 g, 831 mmol) in water (120 mL), and the reaction mixture was stirred overnight at room temperature. After removal of the organic solvents via concentration under reduced pressure, the aqueous residue was extracted 4 times with dichloromethane; the combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo to afford the product as a colorless oil. Yield: 27.3 g, 235 mmol, 85%. 1H NMR (400 MHz, CDCl3) δ 3.99 (ddd, J=11.8, 4.8, 1.7 Hz, 1H), 3.71-3.80 (m, 1H), 3.35-3.47 (m, 2H), 1.82-1.98 (m, 3H), 1.48 (dddd, J=12.5, 12.4, 11.1, 4.8 Hz, 1H), 1.21 (d, J=6.2 Hz, 3H), 1.14-1.24 (m, 1H).
Step 4. Synthesis of (2R)-2-methyltetrahydro-4H-pyran-4-one (C4)
A solution of C3 (27.3 g, 235 mmol) in acetone (980 mL) was cooled in an ice bath and treated drop-wise with Jones reagent (2.5 M, 103 mL, 258 mmol). The reaction mixture was stirred for 10 minutes at 0° C., then warmed to room temperature, stirred for a further 30 minutes, and cooled to 0° C. 2-Propanol (18 mL, 240 mmol) was added, and stirring was continued for 30 minutes. After the mixture had been concentrated in vacuo, the residue was partitioned between water and dichloromethane; the aqueous layer was extracted 3 times with dichloromethane, and the combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to provide the product as a light yellow oil. Yield: 23 g, 200 mmol, 85%. 1H NMR (400 MHz, CDCl3) δ 4.25 (ddd, J=11.5, 7.4, 1.3 Hz, 1H), 3.70 (dqd, J=12.2, 6.1, 2.7 Hz, 1H), 3.64 (ddd, J=12.2, 11.6, 2.8 Hz, 1H), 2.55 (dddd, J=14.6, 12.4, 7.4, 1.0 Hz, 1H), 2.37 (ddd, J=14.4, 2.3, 2.3 Hz, 1H), 2.21-2.31 (m, 2H), 1.29 (d, J=6.2 Hz, 3H).
Step 5. Synthesis of (2R,4R)—N-(2,4-dimethoxybenzyl)-2-methyltetrahydro-2H-pyran-4-amine (P1)
1-(2,4-Dimethoxyphenyl)methanamine (20.3 mL, 135 mmol) was added to a solution of C4 (10.3 g, 90.2 mmol) in methanol (200 mL), and the reaction mixture was stirred for 1 hour at room temperature. It was then cooled to −78° C.; lithium borohydride solution (2 M in tetrahydrofuran, 45.1 mL, 90.2 mmol) was added drop-wise, and stirring was continued at −78° C. for 2 hours. After slowly warming to room temperature overnight, the reaction mixture was quenched via careful addition of saturated aqueous sodium bicarbonate solution. Ethyl acetate (250 mL) and sufficient water to solubilize the precipitate were added, and the aqueous layer was extracted with ethyl acetate; the combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 5% methanol in dichloromethane) provided the product as a colorless oil (10.4 g). Similar purification of mixed fractions afforded additional product (3.7 g). Combined yield: 14.1 g, 53.1 mmol, 59%. 1H NMR (400 MHz, CDCl3) δ 7.13 (d, J=8.0 Hz, 1H), 6.42-6.47 (m, 2H), 3.99 (ddd, J=11.6, 4.6, 1.5 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.76 (s, 2H), 3.36-3.45 (m, 2H), 2.63-2.73 (m, 1H), 1.85-1.92 (m, 1H), 1.78-1.85 (m, 1H), 1.38 (dddd, J=13, 12, 11, 4.7 Hz, 1H), 1.20 (d, J=6.2 Hz, 3H), 1.10 (ddd, J=11, 11, 11 Hz, 1H).
Preparation P2
cis-2-[(Benzyloxy)methyl]-N-(2,4-dimethoxybenzyl)tetrahydro-2H-pyran-4-amine (P2)
Step 1. Synthesis of 2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-ol (C5)
A solution of (benzyloxy)acetaldehyde (25.0 g, 166 mmol) and but-3-en-1-ol (12.0 g, 166 mmol) in dichloromethane (550 mL) was added in a drop-wise manner to a 0° C. solution of trifluoroacetic acid (57 g, 500 mmol) in dichloromethane (500 mL). The reaction mixture was stirred at room temperature (20° C.) for 18 hours, whereupon it was concentrated in vacuo. After the residue had been dissolved in methanol (450 mL), it was treated with potassium carbonate (80 g, 580 mmol), and the reaction mixture was stirred for 5 hours at 20° C. A reaction mixture from a similar reaction employing (benzyloxy)acetaldehyde (20.0 g, 133 mmol) was added, and the combined mixtures were filtered. The filtrate was concentrated under reduced pressure, and partitioned between water (500 mL) and ethyl acetate (200 mL). The aqueous layer was then extracted with ethyl acetate (2×150 mL), and the combined organic layers were concentrated in vacuo. Silica gel chromatography (Gradient: 20% to 25% ethyl acetate in petroleum ether) provided the product as a yellow oil. From examination of the 1H NMR spectrum this material was assumed to be a mixture of the cis and trans isomers. Combined yield: 42.9 g, 193 mmol, 64%. 1H NMR (400 MHz, CDCl3) δ 7.39-7.26 (m, 5H), 4.64-4.53 (m, 2H), [4.29-4.25 (m), 4.11-3.76 (m), and 3.59-3.40 (m), total 6H], [1.96-1.83 (m), 1.71-1.48 (m), and 1.36-1.24 (m), total 4H, assumed; partially obscured by water peak].
Step 2. Synthesis of 2-[(benzyloxy)methyl]tetrahydro-4H-pyran-4-one (C6)
Pyridinium chlorochromate (48 g, 220 mmol) was added to a solution of C5 (22.9 g, 103 mmol) in dichloromethane (350 mL), and the reaction mixture was stirred at room temperature (20° C.) for 18 hours. It was then combined with a similar reaction carried out using C5 (20 g, 90 mmol), and the mixture was filtered, then concentrated in vacuo. The residue was purified via chromatography on silica gel (Eluent: 20% ethyl acetate in petroleum ether), affording the product as a colorless oil. Combined yield: 36.2 g, 164 mmol, 85%. 1H NMR (400 MHz, CDCl3) δ 7.40-7.27 (m, 5H), 4.65-4.58 (m, 2H), 4.36 (ddd, J=11.5, 7.5, 1.5 Hz, 1H), 3.85 (dddd, J=11, 5, 4, 3 Hz, 1H), 3.72 (ddd, J=12.3, 11.5, 2.8 Hz, 1H), 3.58 (dd, half of ABX pattern, J=10.5, 4.0 Hz, 1H), 3.55 (dd, half of ABX pattern, J=10.3, 5.3 Hz, 1H), 2.63 (dddd, J=15, 12, 7.5, 1 Hz, 1H), 2.56-2.47 (m, 1H), 2.40-2.32 (m, 2H).
Step 3. Synthesis of cis-2-[(benzyloxy)methyl]-N-(2,4-dimethoxybenzyl)tetrahydro-2H-pyran-4-amine (P2)
1-(2,4-Dimethoxyphenyl)methanamine (23 g, 140 mmol) was added to a solution of C6 (20 g, 91 mmol) in methanol (275 mL). The reaction mixture was stirred at room temperature (20° C.) for 24 hours, whereupon it was cooled to −78° C. and treated in a drop-wise manner with lithium borohydride (2 M solution in tetrahydrofuran; 46.0 mL 92.0 mmol). The reaction mixture was allowed to slowly warm to room temperature, and was then stirred at room temperature overnight. This was combined with a similar reaction mixture that employed C6 (16.18 g, 73.5 mmol) and concentrated in vacuo. The residue was mixed with saturated aqueous sodium bicarbonate solution (300 mL) and water (200 mL), and extracted with ethyl acetate (4×200 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified via chromatography on silica gel (Gradient: 0% to 9% methanol in dichloromethane) to provide the product as a light yellow oil. Combined yield: 52.0 g, 140 mmol, 85%. LCMS m/z 371.9 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 7.38-7.25 (m, 5H), 7.12 (d, J=8.0 Hz, 1H), 6.46 (d, half of AB quartet, J=2.5 Hz, 1H), 6.43 (dd, half of ABX pattern, J=8.0, 2.5 Hz, 1H), 4.58 (AB quartet, JAB=12.0 Hz, ΔνAB=23.2 Hz, 2H), 4.07 (ddd, J=11.5, 4.5, 1.5 Hz, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.75 (s, 2H), 3.59-3.39 (m, 4H), 2.75-2.65 (m, 1H), 1.91-1.80 (m, 2H), 1.48-1.35 (m, 1H), 1.23-1.12 (m, 1H).
Preparation P3
N4-{cis-2-[(Benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}-6-chloroquinoline-3,4-diamine (P3)
Step 1. Synthesis of 4,6-dichloro-3-nitroquinoline (C7)
N,N-Dimethylformamide (3.1 mL, 40 mmol) and thionyl chloride (97%, 6.9 mL, 93 mmol) were added to a suspension of 6-chloro-3-nitroquinolin-4-ol (15.38 g, 68.48 mmol) in dichloromethane (140 mL), and the reaction mixture was heated at reflux. After 5 hours, it was cooled to room temperature, diluted with additional dichloromethane (25 mL), and poured into saturated aqueous sodium bicarbonate solution (250 mL). The aqueous layer was extracted with dichloromethane (100 mL), then passed through a plug of diatomaceous earth, which was subsequently rinsed with dichloromethane (50 mL). The combined organic layers and organic filtrate were dried over magnesium sulfate, filtered, and concentrated in vacuo to afford the product as a pale tan solid. Yield: 16.8 g, quantitative. 1H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H), 8.42 (d, J=2.2 Hz, 1H), 8.17 (d, J=8.9 Hz, 1H), 7.89 (dd, J=9.0, 2.2 Hz, 1H).
Step 2. Synthesis of N-{cis-2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}-6-chloro-N-(2,4-dimethoxybenzyl)-3-nitroquinolin-4-amine (C8)
Compound C7 (17.2 g, 70.8 mmol) was slowly added to a solution of P2 (20.8 g, 56.0 mmol) and N,N-diisopropylethylamine (21.7 g, 168 mmol) in acetonitrile (300 mL). The reaction mixture was stirred for 16 hours at room temperature (25° C.), at which time LCMS analysis indicated conversion to the product: LCMS m/z 578.0 (chlorine isotope pattern observed) [M+H]+. The reaction mixture was concentrated to half its original volume, diluted with water (400 mL), and extracted with ethyl acetate (2×300 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified via silica gel chromatography (Gradient: 0% to 25% ethyl acetate in petroleum ether) to provide the product as a yellow solid. Yield: 26.1 g, 45.2 mmol, 81% yield. 1H NMR (400 MHz, CDCl3) δ 9.02 (s, 1H), 8.22 (d, J=2.5 Hz, 1H), 7.98 (d, J=9.0 Hz, 1H), 7.69 (dd, J=9.0, 2.5 Hz, 1H), 7.36-7.25 (m, 5H), 6.82 (br d, J=8.5 Hz, 1H), 6.22-6.18 (m, 2H), 4.57 (AB quartet, JAB=12.3 Hz, ΔνAB=9.1 Hz, 2H), 4.40-4.27 (m, 2H), 4.15-4.07 (m, 1H), 3.83-3.73 (m, 1H), 3.69 (s, 3H), 3.59-3.40 (m, 4H), 3.54 (s, 3H), 2.00-1.91 (m, 3H), 1.78-1.66 (m, 1H).
Step 3. Synthesis of N-{cis-2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}-6-chloro-3-nitroquinolin-4-amine (C9)
Trifluoroacetic acid (11.8 g, 103 mmol) was slowly added drop-wise to a 20° C. solution of C8 (6.00 g, 10.4 mmol) in dichloromethane (50 mL). The reaction mixture was stirred for 1 hour, whereupon LCMS analysis indicated conversion to the product: LCMS m/z 427.9 (chlorine isotope pattern observed) [M+H]+. It was then combined with the reaction mixture from a similar transformation of C8 (1.95 g, 3.37 mmol) and concentrated in vacuo. The residue was diluted with saturated aqueous sodium bicarbonate solution (200 mL) and extracted with ethyl acetate (4×100 mL); the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure, providing the product as a yellow solid (6.40 g) that contained some ethyl acetate by 1H NMR analysis. Combined yield, corrected for solvent: 5.69 g, 13.3 mmol, 96%. 1H NMR (400 MHz, CDCl3) δ 9.36 (s, 1H), 9.07 (br d, J=9.0 Hz, 1H), 8.10 (d, J=2.0 Hz, 1H), 7.97 (d, J=9.0 Hz, 1H), 7.73 (dd, J=9.0, 2.0 Hz, 1H), 7.38-7.26 (m, 5H), 4.59 (AB quartet, JAB=12.0 Hz, ΔνAB=7.2 Hz, 2H), 4.34-4.22 (m, 1H), 4.18 (ddd, J=12.0, 4.5, 1.5 Hz, 1H), 3.69-3.62 (m, 1H), 3.62-3.52 (m, 2H), 3.49 (dd, component of ABC pattern, J=10.3, 4.3 Hz, 1H), 2.21-2.12 (m, 2H), 1.88-1.76 (m, 1H), 1.66-1.55 (m, 1H).
Step 4. Synthesis of N4-{cis-2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}-6-chloroquinoline-3,4-diamine (P3)
Platinum on carbon (5%; 1.37 g) was added in one portion to a 20° C. solution of C9 (6.0 g, 14 mmol) in tetrahydrofuran (200 mL). The reaction mixture was purged with argon, then saturated with hydrogen and stirred under 50 psi of hydrogen for 3 hours at 20° C. Filtration and concentration of the filtrate in vacuo provided the product as a brown solid. Yield: 5.75 g, 14.4 mmol, quantitative. LCMS m/z 397.8 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.90 (d, J=9.0 Hz, 1H), 7.73 (d, J=2.0 Hz, 1H), 7.39 (dd, J=9.0, 2.0 Hz, 1H), 7.36-7.24 (m, 5H), 4.56 (AB quartet, JAB=12.3 Hz, ΔνAB=9.9 Hz, 2H), 4.09 (ddd, J=12, 4.5, 1 Hz, 1H), 3.90 (br s, 2H), 3.57-3.40 (m, 5H), 3.39-3.31 (br m, 1H), 1.91-1.82 (m, 2H), 1.66-1.53 (m, 1H), 1.43-1.33 (m, 1H).
Preparation P4
3-Amino-4-[(4,4-difluoro-1-methylpyrrolidin-3-yl)amino]quinoline-6-carbonitrile (P4)
Step 1. Synthesis of 4-hydroxy-3-nitroquinoline-6-carbonitrile (C10)
This reaction was run in two identical batches. A mixture of 6-bromo-3-nitroquinolin-4-ol (25.0 g, 92.9 mmol), potassium hexacyanoferrate(II) trihydrate (13.7 g, 32.4 mmol), 1,1′-bis(diphenylphosphino)ferrocene (5.15 g, 9.29 mmol), sodium carbonate (11.8 g, 111 mmol), and palladium(II) acetate (1.04 g, 4.63 mmol) in N,N-dimethylformamide (350 mL) was heated at 140° C. for 16 hours. The reaction mixture was cooled to room temperature, and the two batches were combined and filtered through diatomaceous earth. The filter cake was slowly rinsed with N,N-dimethylformamide (200 mL) and tert-butyl methyl ether (3.0 L) while the filtrate was stirred. A dark solid precipitated from the filtrate during the stirring, and the resulting mixture was stirred at 20° C. for 15 minutes, and then filtered. This second filtrate was concentrated in vacuo to a volume of approximately 40 mL; the residue was diluted with tert-butyl methyl ether (˜200 mL), and the resulting yellow precipitate was collected by filtration, and then triturated with ethyl acetate (˜200 mL). The product was obtained as a deep yellow solid. Combined yield: 20 g, 93 mmol, 50%. LCMS m/z 216.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.51 (d, J=2.0 Hz, 1H), 7.83 (dd, J=8.5, 1.5 Hz, 1H), 7.69 (d, J=8.5 Hz, 1H).
Step 2. Synthesis of 4-chloro-3-nitroquinoline-6-carbonitrile (C11)
To a 15° C. solution of C10 (5.00 g, 23.2 mmol) in N,N-dimethylformamide (30 mL) was added phosphorus oxychloride (9.85 g, 64.2 mmol), and the reaction mixture was stirred at 15° C. for 1.5 hours. It was then poured into ice water (100 mL) and the resulting suspension was filtered. The collected solids were dissolved in tetrahydrofuran (100 mL) and filtered through a pad of silica gel. Concentration of the filtrate in vacuo afforded the product as a white solid. Yield: 3.10 g, 13.3 mmol, yield 57%. 1H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 8.83 (d, J=1.8 Hz, 1H), 8.35 (d, J=8.8 Hz, 1H), 8.10 (dd, J=8.8, 1.8 Hz, 1H).
Step 3. Synthesis of tert-butyl 4-[(6-cyano-3-nitroquinolin-4-yl)amino]-3,3-difluoro pyrrolidine-1-carboxylate (C12)
tert-Butyl 4-amino-3,3-difluoropyrrolidine-1-carboxylate (prepared using the method described by D. C. Behenna et al., in U.S. Patent Application 2015 0141402 A1, May 21, 2015; 2.30 g, 10.3 mmol) was dissolved in acetonitrile (20 mL). N,N-Diisopropylethylamine (2.01 g, 15.5 mmol) and C11 (3.04 g, 13.0 mmol) were added to this solution, and the reaction mixture was stirred for 14 hours at 20° C. After removal of volatiles in vacuo, purification via silica gel chromatography (Gradient: 9% to 17% tetrahydrofuran in petroleum ether) provided the product as a pale yellow solid. Yield: 3.20 g, 7.63 mmol, 74%. 1H NMR (400 MHz, CDCl3) δ 9.52 (s, 1H), 9.21-9.04 (br m, 1H), 8.48 (br s, 1H), 8.20 (d, J=8.8 Hz, 1H), 8.00 (dd, J=8.6, 1.5 Hz, 1H), 4.88-4.74 (m, 1H), 4.23 (br dd, J=9.7, 8.8 Hz, 1H), 4.05-3.89 (br m, 1H), 3.89-3.75 (m, 1H), 3.60 (ddd, J=11.4, 8.4, 1.3 Hz, 1H), 1.51 (s, 9H).
Step 4. Synthesis of 4-[(4,4-difluoropyrrolidin-3-yl)amino]-3-nitroquinoline-6-carbonitrile (C13)
Trifluoroacetic acid (1 mL) was added to a 15° C. solution of C12 (1.10 g, 2.62 mmol) in dichloromethane (2 mL). After the reaction mixture had been stirred for 1 hour at 15° C., at which time LCMS analysis indicated conversion to the product: LCMS m/z 320.1 [M+H]+, it was concentrated in vacuo and neutralized via addition of aqueous sodium bicarbonate solution (60 mL). The resulting mixture was extracted with ethyl acetate (3×50 mL), and the combined organic layers were concentrated under reduced pressure to afford the product as a pale yellow solid. Yield: 810 mg, 2.54 mmol, 97%. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 9.00 (s, 1H), 8.68-8.57 (br m, 1H), 8.13 (br AB quartet, JAB=8.5 Hz, ΔνAB=48.4 Hz, 2H), 4.61-4.43 (m, 1H), 3.58 (dd, J=12.0, 7.5 Hz, 1H), 3.41-3.28 (m, 1H), 3.26-3.12 (m, 1H), 3.12 (dd, J=11.8, 7.3 Hz, 1H).
Step 5. Synthesis of 4-[(4,4-difluoro-1-methylpyrrolidin-3-yl)amino]-3-nitroquinoline-6-carbonitrile (C14)
Sodium triacetoxyborohydride (2.15 g, 10.1 mmol) was added to a 0° C. mixture of C13 (810 mg, 2.54 mmol) in acetonitrile (5 mL). An aqueous solution of formaldehyde (37%, 824 mg, 10.2 mmol) was added to the 0° C. reaction mixture over 20 minutes, and stirring was then continued at room temperature for 1 hour, at which time LCMS analysis indicated conversion to the product: LCMS m/z 334.1 [M+H]+. After solvents had been removed via concentration in vacuo, the residue was basified to pH 8 by addition of aqueous sodium bicarbonate solution, filtered, and concentrated under reduced pressure, providing the product as a red solid. Yield: 780 mg, 2.34 mmol, 92%. 1H NMR (400 MHz, CDCl3), characteristic peaks: δ 9.59 (br d, J=8.8 Hz, 1H), 9.48 (s, 1H), 8.55 (br s, 1H), 8.14 (d, J=8.4 Hz, 1H), 7.96 (dd, J=8.8, 1.3 Hz, 1H), 3.29-3.03 (m, 3H), 2.86 (ddd, J=9.9, 5.1, 2.0 Hz, 1H), 2.47 (s, 3H).
Step 6. Synthesis of 3-amino-4-[(4,4-difluoro-1-methylpyrrolidin-3-yl)amino]quinoline-6-carbonitrile (P4)
Palladium on carbon (10%; 1 g) was added to a solution of C14 (3.00 g, 9.00 mmol) in methanol (30 mL), and the reaction mixture was hydrogenated under a balloon of hydrogen for 2 hours at 25° C. It was then filtered through diatomaceous earth, concentrated in vacuo, and purified via silica gel chromatography (Gradient: 17% to 33% tetrahydrofuran in petroleum ether), providing the product as a pale yellow solid. Yield: 1.30 g, 4.29 mmol, 48%. 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 8.24 (d, J=1.8 Hz, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.60 (dd, J=8.8, 1.8 Hz, 1H), 4.32-4.19 (m, 1H), 4.09-3.96 (m, 3H), 3.18-2.97 (m, 3H), 2.64 (ddd, J=9.7, 6.6, 1.8 Hz, 1H), 2.41 (s, 3H).
Preparation P5
6-Chloro-N4-(3,3-difluorotetrahydro-2H-pyran-4-yl)quinoline-3,4-diamine (P5)
Step 1. Synthesis of tert-butyl (trans-3-hydroxytetrahydro-2H-pyran-4-yl)carbamate (C15)
A solution of trans-4-azidotetrahydro-2H-pyran-3-ol (see M. Chini et al., Tetrahedron 1994, 50, 1261-1274) (14.8 g, 103 mmol) and di-tert-butyl dicarbonate (23.0 g, 105 mmol) in ethyl acetate (345 mL) was added to palladium on carbon (10%, 1.5 g) and the reaction mixture was stirred under 50 psi of hydrogen at 20° C. to 25° C. for 22 hours. It was then filtered through diatomaceous earth and the filter pad was rinsed with ethyl acetate and methanol. The combined filtrates were concentrated in vacuo and the residue was triturated once with a mixture of dichloromethane (10 mL) and [9:1 petroleum ether/tetrahydrofuran] (60 mL), affording the product as a white solid. Yield: 15.8 g. 72.7 mmol, 71%. 1H NMR (400 MHz, CDCl3) δ 4.71-4.62 (br s, 1H), 4.01 (dd, J=11, 4 Hz, 1H), 3.98-3.87 (m, 2H), 3.57-3.42 (m, 2H), 3.40 (ddd, J=12, 12, 2 Hz, 1H), 3.13 (dd, J=11.0, 9.5 Hz, 1H), 1.96-1.88 (m, 1H), 1.59-1.47 (m, 1H, assumed; partially obscured by water peak), 1.47 (s, 9H).
Step 2. Synthesis of tert-butyl (3-oxotetrahydro-2H-pyran-4-yl)carbamate (C16)
A solution of C15 (35.1 g, 162 mmol) in dichloromethane (540 mL) was treated with [1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one] (Dess-Martin periodinane; 81.6 g, 192 mmol) and stirred at 25° C. for 18 hours. The reaction mixture was treated with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium thiosulfate solution (250 mL); after stirring for 30 minutes, the layers were separated and the aqueous layer was extracted twice with dichloromethane (200 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 10% to 30% ethyl acetate in petroleum ether) afforded the product as a yellow oil (27.95 g) that contained some aromatic material derived from the oxidizing reagent. This material was taken directly to the following step. 1H NMR (400 MHz, CDCl3), product peaks only: δ 5.49-5.38 (br s, 1H), 4.55-4.42 (m, 1H), 4.08 (AB quartet, JAB=14.8 Hz, ΔνAB=40.3 Hz, 2H), 4.07-3.99 (m, 1H), 3.89 (ddd, J=12.0, 11.5, 3.0 Hz, 1H), 2.75-2.63 (m, 1H), 1.96-1.81 (m, 1H), 1.44 (s, 9H).
Step 3. Synthesis of tert-butyl (3,3-difluorotetrahydro-2H-pyran-4-yl)carbamate (C17)
A solution of C16 (from the previous step; 27.95 g) in dichloromethane (124 mL) was slowly added to a 0° C. suspension of difluoro-4-morpholinylsulfonium tetrafluoroborate (XtalFluor-M®; 39.5 g, 163 mmol) and triethylamine trihydrofluoride (28.6 g, 177 mmol) in dichloromethane (384 mL), and the reaction mixture was allowed to slowly warm to 25° C. After three days, the reaction mixture was treated with saturated aqueous sodium bicarbonate solution (500 mL) and extracted with dichloromethane (500 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Eluent: 10% ethyl acetate in petroleum ether) provided the product as a yellow solid. Yield: 8.93 g, 37.6 mmol, 23% over two steps. 1H NMR (400 MHz, CDCl3) δ 4.91-4.75 (br m, 1H), 4.18-3.94 (m, 3H), 3.55-3.43 (m, 1H), 3.46 (dd, J=30.4, 12.8 Hz, 1H), 2.07-1.97 (m, 1H), 1.86-1.71 (m, 1H), 1.47 (s, 9H).
Step 4. Synthesis of 3,3-difluorotetrahydro-2H-pyran-4-amine, hydrochloride salt (C18)
A solution of hydrogen chloride in methanol (4 M, 16.8 mL, 67.2 mmol) was added to a 10° C. solution of C17 (3.18 g, 13.4 mmol) in methanol (35 mL). After the reaction mixture had stirred at 10° C. for 1 hour, it was concentrated in vacuo to afford the product as a white solid. Yield: 2.32 g, 13.4 mmol, quantitative. 1H NMR (400 MHz, DMSO-d6) δ 9.03-8.89 (br s, 3H), 4.06-3.57 (m, 4H, assumed; partially obscured by water peak), 3.57-3.47 (m, 1H), 2.20-2.08 (m, 1H), 1.88-1.72 (m, 1H).
Step 5. Synthesis of 6-chloro-N-(3,3-difluorotetrahydro-2H-pyran-4-yl)-3-nitroquinolin-4-amine (C19)
N,N-Diisopropylethylamine (9.2 mL, 52.8 mmol) was added to a 10° C. solution of C7 (3.2 g, 13.2 mmol) and C18 (2.32 g, 13.4 mmol) in acetonitrile (40 mL) and the reaction mixture was stirred at 10° C. for 16 hours. It was then combined with two additional reactions carried out using C7 (1.2 g, 4.9 mmol and 90 mg, 0.37 mmol) and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 20% ethyl acetate in petroleum ether) provided the product as a yellow solid. Combined yield: 4.5 g, 13 mmol, 70%. LCMS m/z 344.0 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.40 (s, 1H), 8.60 (br d, J=10.1 Hz, 1H), 8.05 (d, J=1.8 Hz, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.77 (dd, J=9.2, 2.2 Hz, 1H), 4.40-4.26 (m, 1H), 4.17-4.02 (m, 2H), 3.59 (br ddd, J=12, 12, 1 Hz, 1H), 3.48 (dd, J=29.0, 12.8 Hz, 1H), 2.40-2.32 (m, 1H), 2.28-2.16 (m, 1H).
Step 6. Synthesis of 6-chloro-N4-(3,3-difluorotetrahydro-2H-pyran-4-yl)quinoline-3,4-diamine (P5)
A mixture of C19 (4.40 g, 12.8 mmol) and platinum on carbon (5%; 250 mg) in tetrahydrofuran (50 mL) was degassed with nitrogen at 20° C., and then subjected to hydrogenation at 50 psi and 20° C. for 2 hours. The reaction mixture was filtered, and the filter cake was washed with tetrahydrofuran (3×10 mL). The combined filtrates were concentrated in vacuo, combined with the crude product from a similar reaction carried out using C19 (100 mg, 0.29 mmol), and purified via silica gel chromatography (Gradient: 0% to 20% methanol in dichloromethane) to provide the product as a yellow solid. Combined yield: 3.9 g, 12.4 mmol, 95%. LCMS m/z 314.1 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.90 (d, J=9.3 Hz, 1H), 7.78 (d, J=2.0 Hz, 1H), 7.41 (dd, J=9.0, 2.2 Hz, 1H), 4.10-4.01 (m, 2H), 3.99-3.93 (br s, 2H), 3.85-3.69 (m, 2H), 3.51-3.42 (m, 1H), 3.44 (dd, J=31.3, 12.7 Hz, 1H), 2.10-1.95 (m, 2H).
Preparation P6
6-Chloro-N4-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]quinoline-3,4-diamine (P6)
Step 1: Synthesis of tert-butyl [(3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl]carbamate (C20)
A solution of (3R,4S)-4-aminotetrahydro-2H-pyran-3-ol (see M. A. Brodney et al., in PCT International Pat. Appl. No. WO 2016009297 A1, published Jan. 21, 2016) (383 mg, 3.27 mmol) and di-tert-butyl dicarbonate (714 g, 3.27 mmol) in dichloromethane (33 mL) was treated with triethylamine (0.46 mL, 3.3 mmol) and the reaction mixture was stirred at room temperature overnight. Concentration in vacuo afforded the product as an off-white solid. Yield: 707 mg, 3.25 mmol, 99%. 1H NMR (400 MHz, CDCl3) δ 4.69-4.56 (br s, 1H), 4.02 (br dd, J=11.3, 4.7 Hz, 1H), 3.96-3.86 (m, 2H), 3.58-3.44 (m, 2H), 3.40 (ddd, J=12.1, 11.7, 2.3 Hz, 1H), 3.13 (dd, J=11.3, 9.4 Hz, 1H), 1.96-1.87 (m, 1H), 1.58-1.48 (m, 1H, assumed; partially obscured by water peak), 1.47 (s, 9H).
Step 2. Synthesis of tert-butyl [(4S)-3-oxotetrahydro-2H-pyran-4-yl]carbamate (C21)
A solution of C20 (707 mg, 3.25 mmol) in dichloromethane (40 mL) was treated with [1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one] (95%; 1.74 g, 3.90 mmol) and stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (50 mL) and saturated aqueous sodium thiosulfate solution (50 mL) and stirred for 30 minutes. The aqueous layer was extracted twice with dichloromethane, and the combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 20% to 80% ethyl acetate in heptane) provided the product as a white solid. Yield: 546 mg, 2.54 mmol, 78%. GCMS m/z 215.1 [M+]. 1H NMR (400 MHz, CDCl3) δ 5.49-5.36 (br s, 1H), 4.49-4.36 (m, 1H), 4.05 (AB quartet, JAB=14.6 Hz, ΔνAB=38.1 Hz, 2H), 4.04-3.96 (m, 1H), 3.85 (ddd, J=12.1, 11.3, 3.1 Hz, 1H), 2.70-2.59 (m, 1H), 1.92-1.78 (m, 1H), 1.41 (s, 9H).
Step 3: Synthesis of tert-butyl [(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]carbamate (C22)
A solution of C21 (540 mg, 2.51 mmol) in dichloromethane (5 mL) was slowly added to a 0° C. suspension of difluoro-4-morpholinylsulfonium tetrafluoroborate (1.22 g, 5.02 mmol) and triethylamine trihydrofluoride (0.9 mL, 5.5 mmol) in dichloromethane (10 mL). The ice bath was removed and the reaction mixture was stirred at room temperature overnight, then at 40° C. for 90 minutes. After cooling to room temperature, the reaction mixture was carefully treated with saturated aqueous sodium bicarbonate solution {Caution: gas evolution}. The aqueous layer was extracted twice with dichloromethane, and the combined organic layers were washed with water, dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 15% to 45% ethyl acetate in heptane) provided the product as a yellow solid, which was used in the following step. By 1H NMR analysis, this material was somewhat impure. GCMS m/z 138.1 {[M-(2-methylprop-1-ene and carbon dioxide)]+H}+. 1H NMR (400 MHz, CDCl3), product peaks only: δ 4.93-4.81 (m, 1H), 4.16-3.93 (m, 3H), 3.51-3.43 (m, 1H), 3.45 (dd, J=30.4, 12.9 Hz, 1H), 2.05-1.96 (m, 1H), 1.83-1.71 (m, 1H), 1.45 (s, 9H).
Step 4: Synthesis of (4S)-3,3-difluorotetrahydro-2H-pyran-4-amine, hydrochloride salt (C23)
Concentrated hydrochloric acid (2 mL) was added to a solution of C22 (from the previous step; ≤2.51 mmol) in ethanol (10 mL), and the reaction mixture was stirred at room temperature overnight. Removal of solvents in vacuo provided the product as a brown solid. Yield: 155 mg, 0.893 mmol, 36% over two steps. GCMS m/z 137.1 [M+]. 1H NMR (400 MHz, CD3OD) δ 4.09-3.86 (m, 3H), 3.65 (dd, J=31.2, 12.9 Hz, 1H), 3.65-3.56 (m, 1H), 2.23-2.14 (m, 1H), 2.03-1.90 (m, 1H).
Step 5: Synthesis of 6-chloro-N-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]-3-nitroquinolin-4-amine (C24)
N,N-Diisopropylethylamine (0.41 mL, 2.4 mmol) was added to a mixture of C7 (190 mg, 0.782 mmol) and C23 (136 mg, 0.783 mmol) in acetonitrile (3 mL), and the reaction mixture was stirred at 60° C. overnight. After cooling to room temperature, the reaction mixture was concentrated in vacuo and partitioned between water and ethyl acetate. A small amount of saturated aqueous sodium bicarbonate solution was added to adjust the aqueous layer to pH 9, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo, and purified via silica gel chromatography (Gradient: 5% to 35% ethyl acetate in heptane), affording the product as a bright yellow solid. Yield: 164 mg, 0.477 mmol, 61%. LCMS m/z 344.4 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 8.61 (br d, J=10.2 Hz, 1H), 8.04 (d, J=8.6 Hz, 1H), 8.04 (d, J=2.3 Hz, 1H), 7.77 (dd, J=9.0, 2.3 Hz, 1H), 4.40-4.26 (m, 1H), 4.17-4.02 (m, 2H), 3.59 (br ddd, J=12, 12, 1.5 Hz, 1H), 3.48 (dd, J=29.1, 12.7 Hz, 1H), 2.40-2.32 (m, 1H), 2.29-2.16 (m, 1H).
Step 6: Synthesis of 6-chloro-N4-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]quinoline-3,4-diamine (P6)
Zinc powder (97.5%, 312 mg, 4.65 mmol) was added to a slurry of C24 (160 mg, 0.466 mmol) in methanol (3 mL) and concentrated ammonium hydroxide solution (3 mL). The reaction mixture was stirred at room temperature for 2 hours, whereupon it was filtered through diatomaceous earth. The filter pad was rinsed with dichloromethane and methanol, and the combined filtrates were concentrated in vacuo. The residue was diluted with half-saturated aqueous sodium chloride solution and extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered, concentrated under reduced pressure, and purified via chromatography on silica gel (Gradient: 0% to 100% ethyl acetate in heptane) to provide the product as a pale tan oil. Yield: 78 mg, 0.249 mmol, 54%. LCMS m/z 314.4 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.89 (d, J=9.0 Hz, 1H), 7.77 (d, J=2.3 Hz, 1H), 7.39 (dd, J=8.8, 2.1 Hz, 1H), 4.08-3.89 (m, 3H), 3.84-3.68 (m, 2H), 3.49-3.40 (m, 1H), 3.42 (dd, J=31.4, 12.7 Hz, 1H), 2.07-1.94 (m, 2H).
Preparation P7
N4-[(2R,4R)-2-Methyltetrahydro-2H-pyran-4-yl]-6-(trifluoromethyl)quinoline-3,4-diamine
Step 1. Synthesis of 3-nitro-6-(trifluoromethyl)quinolin-4-ol (C25)
A solution of 6-(trifluoromethyl)quinolin-4-ol (2.00 g, 9.38 mmol) in concentrated nitric acid (10 mL) was stirred for 14 hours at 50° C., whereupon it was poured into water (50 mL). The resulting solid was isolated via filtration, providing the product as a pale yellow solid. Yield: 1.80 g, 6.97 mmol, 74%. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 8.46 (s, 1H), 8.11 (d, J=9.0 Hz, 1H), 7.92 (d, J=8.5 Hz, 1H).
Step 2. Synthesis of 4-chloro-3-nitro-6-(trifluoromethyl)quinoline (C26)
Phosphorus oxychloride (3.25 mL, 34.9 mmol) was added to a 15° C. solution of compound C25 (3.00 g, 11.6 mmol) in N,N-dimethylformamide (10 mL), and the reaction mixture was stirred for 2 hours at 15° C. It was then poured into water (80 mL). Collection of the precipitate via filtration provided the product as a solid (2.40 g). This material was impure by 1H NMR analysis, and was taken directly into the following step. 1H NMR (400 MHz, DMSO-d6), product peaks only: δ 9.22 (s, 1H), 8.40 (br s, 1H), 8.03 (br d, J=8.5 Hz, 1H), 7.92-7.97 (m, 1H).
Step 3. Synthesis of N-(2,4-dimethoxybenzyl)-N-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-3-nitro-6-(trifluoromethyl)quinolin-4-amine (C27)
N,N-Diisopropylethylamine (3.36 g, 26.0 mmol) and P1 (2.43 g, 9.16 mmol) were slowly added to a 15° C. solution of C26 (from the previous step; 2.40 g, 8.68 mmol) in acetonitrile (30 mL), and the reaction mixture was stirred for 30 minutes at 80° C. Water (100 mL) was added, and the resulting mixture was extracted with ethyl acetate (3×100 mL). The combined organic layers were concentrated in vacuo, and the residue was purified via silica gel chromatography (Gradient: 9% to 25% ethyl acetate in petroleum ether) to provide the product as a yellow solid. Yield: 3.40 g, 6.73 mmol, 58% over 2 steps. 1H NMR (400 MHz, CDCl3) δ 9.11 (s, 1H), 8.60 (br s, 1H), 8.15 (d, J=9.0 Hz, 1H), 7.92 (dd, J=8.8, 1.8 Hz, 1H), 6.84 (d, J=8.0 Hz, 1H), 6.22 (dd, J=8.3, 2.3 Hz, 1H), 6.16 (d, J=2.0 Hz, 1H), 4.33-4.44 (m, 2H), 4.02-4.10 (m, 1H), 3.77-3.87 (m, 1H), 3.68 (s, 3H), 3.50 (s, 3H), 3.36-3.46 (m, 2H), 1.95-2.10 (m, 3H), 1.67-1.78 (m, 1H), 1.23 (d, J=6.0 Hz, 3H).
Step 4. Synthesis of N-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-3-nitro-6-(trifluoromethyl)quinolin-4-amine (C28)
Trifluoroacetic acid (7.67 g, 67.3 mmol) was added to a 15° C. solution of compound C27 (3.40 g, 6.73 mmol) in dichloromethane (30 mL), and the reaction mixture was stirred for 30 minutes at 15° C. Solvents were removed in vacuo, and the residue was diluted with water (100 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layers were concentrated in vacuo to afford the product (2.50 g) as a pale yellow solid, a portion of which was used directly in the following step. LCMS m/z 355.8 [M+H]+.
Step 5. Synthesis of N4-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-6-(trifluoromethyl) quinoline-3,4-diamine (P7)
Iron powder (314 mg, 5.62 mmol) and ammonium chloride (301 mg, 5.63 mmol) were added to a solution of C28 (from the previous step, 200 mg, ≤0.54 mmol) in ethanol (5 mL) and water (1 mL), and the reaction mixture was stirred for 1 hour at 80° C. It was then filtered through diatomaceous earth, and the filtrate was concentrated in vacuo. Silica gel chromatography (Gradient: 9% to 33% ethyl acetate in petroleum ether) afforded the product as a pale grey solid. Yield: 140 mg, 0.430 mmol, 80% over 2 steps. LCMS m/z 325.9 [M+H]+.
Preparation P8
3-Amino-4-{[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]amino}quinoline-6-carbonitrile (P8)
Step 1. Synthesis of 4-{(2,4-dimethoxybenzyl)[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]amino}-3-nitroquinoline-6-carbonitrile (C29)
To a solution of C11 (8.81 g, 37.7 mmol) in acetonitrile (80 mL) was added P1 (11.0 g, 41.5 mmol), followed by N,N-diisopropylethylamine (5.85 g, 45.3 mmol). The reaction mixture was stirred for 2 hours at room temperature, whereupon it was concentrated in vacuo and purified via silica gel chromatography (Eluent: 4:1 petroleum ether/ethyl acetate), affording the product as a viscous orange oil that slowly solidified. Yield: 15.0 g, 32.4 mmol, 86%. LCMS m/z 313.0 [M-(2,4-dimethoxybenzyl)+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.55 (br dd, J=1.3, 1 Hz, 1H), 8.15 (d, J=1.0 Hz, 2H), 6.88 (d, J=8.0 Hz, 1H), 6.24-6.30 (m, 2H), 4.33 (br AB quartet, JAB=14.5 Hz, ΔνAB=12 Hz, 2H), 3.76-3.92 (m, 2H), 3.62 (s, 3H), 3.42 (s, 3H), 3.3-3.4 (m, 2H, assumed; largely obscured by water peak), 1.83-2.00 (m, 2H), 1.70-1.83 (m, 1H), 1.42-1.54 (m, 1H), 1.09 (d, J=6.0 Hz, 3H).
Step 2. Synthesis of 4-{[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]amino}-3-nitro quinoline-6-carbonitrile (C30)
A mixture of C29 (15.0 g, 32.4 mmol) and trifluoroacetic acid (18.5 g, 162 mmol) in dichloromethane (150 mL) was stirred at room temperature for 30 minutes, whereupon it was concentrated to a volume of 20 mL and treated with saturated aqueous sodium bicarbonate solution (200 mL). The aqueous layer was extracted with dichloromethane (3×150 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to provide the product as a yellow solid. Yield: 5.68 g, 18.2 mmol, 56%. LCMS m/z 313.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.06-9.09 (m, 2H), 8.30 (br d, J=9.0 Hz, 1H), 8.14 (dd, half of ABX pattern, J=8.7, 1.6 Hz, 1H), 8.01 (d, half of AB quartet, J=8.8 Hz, 1H), 3.87-3.93 (m, 1H), 3.69-3.82 (m, 1H), 3.3-3.5 (m, 2H, assumed; largely obscured by water peak), 1.87-2.03 (m, 2H), 1.60-1.72 (m, 1H), 1.36-1.47 (m, 1H), 1.11 (d, J=6.0 Hz, 3H).
Step 3. Synthesis of 3-amino-4-{[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]amino}quinoline-6-carbonitrile (P8)
Ethanol (60 mL) and water (15 mL) were added to a mixture of C30 (5.68 g, 18.2 mmol), iron (10.2 g, 183 mmol), and ammonium chloride (9.73 g, 182 mmol). The reaction mixture was heated to 80° C. for 1 hour, whereupon it was diluted with ethanol (100 mL) and filtered. The filtrate was concentrated in vacuo, and the resulting solid was partitioned between saturated aqueous sodium bicarbonate solution (100 mL) and dichloromethane (300 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford the product as a brown solid. Yield: 4.73 g, 16.8 mmol, 92%. LCMS m/z 282.9 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 8.55 (d, J=1.2 Hz, 1H), 8.51 (s, 1H), 7.90 (d, J=8.8 Hz, 1H), 7.60 (dd, J=8.5, 1.8 Hz, 1H), 3.92-4.00 (m, 1H), 3.58-3.69 (m, 1H), 3.39-3.50 (m, 2H), 1.78-1.94 (m, 2H), 1.56-1.69 (m, 1H), 1.29-1.40 (m, 1H), 1.17 (d, J=6.0 Hz, 3H).
Preparation P9
3-Amino-4-{[(3R)-1-methylpyrrolidin-3-yl]amino}quinoline-6-carbonitrile (P9)
Step 1. Synthesis of 4-{[(3R)-1-methylpyrrolidin-3-yl]amino}-3-nitroquinoline-6-carbonitrile (C31)
N,N-Diisopropylethylamine (251 mg, 1.94 mmol) was added to a 20° C. solution of C11 (210 mg, 0.899 mmol) and (3R)-1-methylpyrrolidin-3-amine (77.9 mg, 0.778 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 20° C. for 2 hours, whereupon it was concentrated in vacuo. Purification of the residue via silica gel chromatography (Gradient: 0% to 1% methanol in dichloromethane) afforded the product as a yellow solid. Yield: 210 mg, 0.706 mmol, 91%. LCMS m/z 297.9 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 10.04-10.15 (br m, 1H), 9.45 (s, 1H), 8.55 (d, J=1.5 Hz, 1H), 8.07 (d, half of AB quartet, J=8.5 Hz, 1H), 7.92 (dd, half of ABX pattern, J=8.5, 1.8 Hz, 1H), 4.65-4.74 (m, 1H), 3.02-3.10 (m, 1H), 2.84-2.90 (m, 1H), 2.80 (dd, half of ABX pattern, J=9.9, 5.6 Hz, 1H), 2.61-2.71 (m, 1H) 2.46 (s, 3H), 2.41-2.50 (m, 1H), 2.06-2.16 (m, 1H).
Step 2. Synthesis of 3-amino-4-{[(3R)-1-methylpyrrolidin-3-yl]amino}quinoline-6-carbonitrile (P9)
To a solution of C31 (100 mg, 0.336 mmol) in a mixture of ethanol (1 mL) and water (0.25 mL) were added ammonium chloride (36 mg, 0.673 mmol) and iron powder (75.1 mg, 1.34 mmol), and the reaction mixture was stirred at 80° C. for 1 hour. It was then filtered, and the filter cake was washed with methanol (30 mL). The organic layer from the combined filtrates was concentrated in vacuo and purified via silica gel chromatography (Gradient: 0% to 15% methanol in dichloromethane), affording the product as a yellow solid. Yield: 112 mg, assumed quantitative. 1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 8.65-8.71 (br s, 1H), 8.58 (s, 1H), 7.89 (d, J=8.5 Hz, 1H), 7.62 (dd, J=8.5, 2.0 Hz, 1H), 5.56-5.70 (br s, 1H), 5.43 (d, J=10.5 Hz, 1H), 4.32-4.46 (br m, 1H), 2.81 (s, 3H), 1.84-1.95 (m, 1H).
Preparation P10
6-Chloro-N4-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]quinoline-3,4-diamine (P10)
Step 1. Synthesis of 6-chloro-N-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-3-nitroquinolin-4-amine (C32)
Compound C7 (12.2 g, 50.2 mmol) was added to a solution of P1 (13.3 g, 50.1 mmol) and N,N-diisopropylethylamine (13.1 mL, 75.2 mmol) in acetonitrile (250 mL), and the reaction mixture was heated to 55° C. overnight. After concentration in vacuo, the residue was partitioned between aqueous sodium bicarbonate solution (100 mL) and dichloromethane (150 mL). The aqueous layer was extracted with dichloromethane (2×50 mL) and the combined organic layers were treated with trifluoroacetic acid (25 mL). {Caution: exotherm!}. After 20 minutes, saturated aqueous sodium carbonate solution (150 mL) was added portion-wise, and the mixture was allowed to stir for 10 minutes. The aqueous layer was extracted twice with dichloromethane, and the combined organic layers were concentrated in vacuo, providing a reddish solid (17.3 g); this was triturated with diethyl ether (230 mL) to afford a yellow solid (14.0 g). A portion of this solid (10 g) was subjected to purification via supercritical fluid chromatography (Column: Lux Amylose-2, 5 μm; Mobile phase: 65:35 carbon dioxide/methanol), providing the product as a crystalline solid. The indicated absolute configuration was determined via single crystal X-ray structural determination on this material: see below. Yield: 7.1 g, 22 mmol, 62% (yield corrected for material omitted from purification). 1H NMR (400 MHz, CDCl3) δ 9.36 (s, 1H), 9.11 (br d, J=9 Hz, 1H), 8.12 (d, J=2.0 Hz, 1H), 7.98 (d, J=8.9 Hz, 1H), 7.73 (dd, J=8.9, 2.2 Hz, 1H), 4.21-4.33 (m, 1H), 4.08-4.15 (m, 1H), 3.50-3.60 (m, 2H), 2.11-2.22 (m, 2H), 1.77 (dddd, J=12, 12, 12, 5 Hz, 1H), 1.49 (ddd, J=12, 12, 11 Hz, 1H), 1.28 (d, J=6.2 Hz, 3H).
Single-Crystal X-Ray Structural Determination of C32
Single Crystal X-Ray Analysis
Data collection was performed on a Bruker APEX diffractometer at room temperature. Data collection consisted of omega and phi scans.
The structure was solved by direct methods using SHELX software suite in the space group P212121. The structure was subsequently refined by the full-matrix least squares method. All non-hydrogen atoms were found and refined using anisotropic displacement parameters.
The hydrogen atom located on nitrogen was found from the Fourier difference map and refined with distances restrained. The remaining hydrogen atoms were placed in calculated positions and were allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms.
Analysis of the absolute structure using likelihood methods (Hooft, 2008) was performed using PLATON (Spek, 2003). The results indicate that the absolute structure has been correctly assigned. The method calculates that the probability that the structure is correct is 100.0. The Hooft parameter is reported as 0.017 with an esd of 0.09.
The final R-index was 4.8%. A final difference Fourier revealed no missing or misplaced electron density.
Pertinent crystal, data collection and refinement information is summarized in Table A. Atomic coordinates, bond lengths, bond angles, and displacement parameters are listed in Tables B-E.
SOFTWARE AND REFERENCES
SHELXTL, Version 5.1, Bruker AXS, 1997.
PLATON, A. L. Spek, J. Appl. Cryst. 2003, 36, 7-13.
MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M. Towler, and J. van de Streek, J. Appl. Cryst. 2006, 39, 453-457.
OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H. Puschmann, J. Appl. Cryst. 2009, 42, 339-341.
R. W. W. Hooft, L. H. Strayer, and A. L. Spek, J. Appl. Cryst. 2008, 41, 96-103.
H. D. Flack, Acta Cryst. 1983, A39, 867-881.
TABLE A
Crystal data and structure refinement for C32.
Empirical formula
C15H16ClN3O3
Formula weight
321.76
Temperature
273(2) K
Wavelength
1.54178 Å
Crystal system
Orthorhombic
Space group
P212121
Unit cell dimensions
a = 6.7882(13) Å
α = 90°
b = 10.0703(19) Å
β = 90°
c = 21.883(4) Å
γ = 90°
Volume
1495.9(5) Å3
Z
4
Density (calculated)
1.429 Mg/m3
Absorption coefficient
2.415 mm−1
F(000)
672
Crystal size
0.22 × 0.16 × 0.10 mm3
Theta range for data collection
4.04 to 70.57°
Index ranges
−8 <= h <= 7, −12 <= k <=
12, −26 <= l <= 24
Reflections collected
12473
Independent reflections
2784 [Rint = 0.1613]
Completeness to
97.3%
theta = 70.57°
Absorption correction
Empirical
Max. and min. transmission
0.7943 and 0.6187
Refinement method
Full-matrix least-squares
on F2
Data/restraints/parameters
2784/1/204
Goodness-of-fit on F2
1.130
Final R indices [l > 2σ(l)]
R1 = 0.0481, wR2 = 0.1164
R indices (all data)
R1 = 0.0514, wR2 = 0.1254
Absolute structure parameter
−0.02(2)
Extinction coefficient
0.0061(8)
Largest diff. peak and hole
0.236 and −0.393 e · Å−3
TABLE B
Atomic coordinates (×104) and equivalent isotropic displacement
parameters (Å2 × 103) for C32. U(eq) is defined as one-third
of the trace of the orthogonalized Uij tensor.
x
y
z
U(eq)
C(1)1294(3)
−465
(2)
8392
(1)
41(1)
C(2)2045(4)
−1731
(2)
8096
(1)
47(1)
C(3)5002(4)
−692
(3)
7811
(1)
59(1)
C(4)4408(4)
620
(3)
8086
(1)
50(1)
C(5)2992(3)
394
(2)
8615
(1)
37(1)
C(6)2190(3)
2218
(2)
9392
(1)
33(1)
C(7)2088(3)
3612
(2)
9478
(1)
36(1)
C(8)2116(3)
4182
(2)
10060
(1)
41(1)
C(9)2196(3)
2165
(2)
10525
(1)
36(1)
C(10)2142(3)
1467
(2)
9960
(1)
33(1)
C(11)1948(3)
75
(2)
9985
(1)
39(1)
C(12)1914(4)
−574
(2)
10537
(1)
47(1)
C(13)2053(4)
111
(2)
11090
(1)
49(1)
C(14)2179(3)
1449
(2)
11077
(1)
46(1)
C(15)394(5)
−2575
(3)
7835
(1)
72(1)
Cl(1)1654(2)
−2285
(1)
10550
(1)
79(1)
N(1)2317(3)
1690
(2)
8834
(1)
44(1)
N(2)2029(3)
4530
(2)
8976
(1)
46(1)
N(3)2205(3)
3529
(2)
10573
(1)
44(1)
O(1)3340(3)
−1422
(2)
7603
(1)
56(1)
O(2)1960(3)
4131
(2)
8443
(1)
59(1)
O(3)2016(4)
5719
(2)
9091
(1)
78(1)
TABLE C
Bond lengths [Å] and angles [°] for C32.
C(1)—C(2)
1.518
(3)
C(1)—C(5)
1.521
(3)
C(2)—O(1)
1.425
(3)
C(2)—C(15)
1.517
(3)
C(3)—O(1)
1.421
(3)
C(3)—C(4)
1.507
(4)
C(4)—C(5)
1.522
(3)
C(5)—N(1)
1.464
(3)
C(6)—N(1)
1.336
(2)
C(6)—C(7)
1.418
(3)
C(6)—C(10)
1.456
(3)
C(7)—C(8)
1.396
(3)
C(7)—N(2)
1.436
(3)
C(8)—N(3)
1.304
(3)
C(9)—N(3)
1.378
(3)
C(9)—C(14)
1.406
(3)
C(9)—C(10)
1.422
(3)
C(10)—C(11)
1.409
(3)
C(11 )—C(12)
1.374
(3)
C(12)—C(13)
1.395
(3)
C(12)—Cl(1)
1.733
(2)
C(13)—C(14)
1.351
(3)
N(2)—O(3)
1.223
(2)
N(2)—O(2)
1.236
(3)
C(2)—C(1)—C(5)
111.09
(18)
O(1)—C(2)—C(15)
107.09
(19)
O(1)—C(2)—C(1)
110.31
(17)
C(15)—C(2)—C(1)
112.5
(2)
O(1)—C(3)—C(4)
111.7
(2)
C(3)—C(4)—C(5)
109.98
(19)
N(1)—C(5)—C(1)
112.00
(18)
N(1)—C(5)—C(4)
108.27
(17)
C(1)—C(5)—C(4)
108.68
(15)
N(1 )—C(6)—C(7)
121.25
(17)
N(1)—C(6)—C(10)
125.16
(17)
C(7)—C(6)—C(10)
113.60
(16)
C(8)—C(7)—C(6)
121.78
(18)
C(8)—C(7)—N(2)
115.67
(17)
C(6)—C(7)—N(2)
122.51
(18)
N(3)—C(8)—C(7)
125.41
(18)
N(3)—C(9)—C(14)
116.46
(18)
N(3)—C(9)—C(10)
123.97
(19)
C(14)—C(9)—C(10)
119.54
(17)
C(11)—C(10)—C(9)
117.44
(18)
C(11)—C(10)—C(6)
123.46
(17)
C(9)—C(10)—C(6)
119.03
(16)
C(12)—C(11)—C(10)
120.51
(18)
C(11)—C(12)—C(13)
121.77
(19)
C(11)—C(12)—Cl(1)
119.23
(16)
C(13)—C(12)—Cl(1)
119.00
(17)
C(14)—C(13)—C(12)
118.66
(19)
C(13)—C(14)—C(9)
121.96
(19)
C(6)—N(1)—C(5)
132.47
(17)
O(3)—N(2)—O(2)
120.82
(18)
O(3)—N(2)—C(7)
118.24
(18)
O(2)—N(2)—C(7)
120.93
(17)
C(8)—N(3)—C(9)
115.92
(17)
C(3)—O(1)—C(2)
111.14
(16)
Symmetry transformations used to generate equivalent atoms.
TABLE D
Anisotropic displacement parameters (Å2 × 103) for C32.
The anisotropic displacement factor exponent takes the
form: −2π2[h2 a*2U11 + . . . + 2 h k a* b* U12].
U11
U22
U33
U23
U13
U12
C(1)
48
(1)
44(1)
31(1)
0
(1)
−2
(1)
−4
(1)
C(2)
70
(2)
38(1)
33(1)
0
(1)
−9
(1)
−3
(1)
C(3)
62
(2)
71(2)
45(1)
−12
(1)
15
(1)
1
(1)
C(4)
61
(1)
54(1)
36(1)
−7
(1)
12
(1)
−13
(1)
C(5)
50
(1)
38(1)
24(1)
−5
(1)
1
(1)
−2
(1)
C(6)
33
(1)
38(1)
30(1)
−4
(1)
2
(1)
0
(1)
C(7)
36
(1)
36(1)
38(1)
0
(1)
4
(1)
−1
(1)
C(8)
43
(1)
35(1)
44(1)
−9
(1)
3
(1)
−1
(1)
C(9)
34
(1)
44(1)
31(1)
−8
(1)
2
(1)
6
(1)
C(10)
30
(1)
41(1)
28(1)
−4
(1)
4
(1)
2
(1)
C(11)
49
(1)
40(1)
28(1)
−4
(1)
3
(1)
2
(1)
C(12)
60
(1)
43(1)
39(1)
2
(1)
6
(1)
8
(1)
C(13)
60
(1)
57(1)
29(1)
6
(1)
3
(1)
15
(1)
C(14)
53
(1)
58(1)
26(1)
−7
(1)
2
(1)
11
(1)
C(15)
97
(2)
53(2)
65(2)
−7
(1)
−25
(2)
−21
(2)
Cl(1)
138
(1)
40(1)
60(1)
9
(1)
18
(1)
5
(1)
N(1)
67
(1)
36(1)
29(1)
−3
(1)
0
(1)
3
(1)
N(2)
49
(1)
40(1)
47(1)
5
(1)
2
(1)
−1
(1)
N(3)
50
(1)
44(1)
37(1)
−12
(1)
0
(1)
2
(1)
O(1)
82
(1)
56(1)
32(1)
−14
(1)
6
(1)
−2
(1)
O(2)
87
(1)
53(1)
38(1)
8
(1)
8
(1)
3
(1)
O(3)
127
(2)
35(1)
73(1)
5
(1)
−4
(1)
−4
(1)
TABLE E
Hydrogen coordinates (×104) and isotropic displacement
parameters (Å2 × 103)for C32.
x
y
z
U(eq)
H(1A)
451
−690
8735
49
H(1B)
515
31
8099
49
H(2A)
2765
−2251
8401
57
H(3A)
5887
−535
7470
71
H(3B)
5704
−1210
8114
71
H(4A)
3779
1166
7777
60
H(4B)
5569
1085
8231
60
H(5)
3684
−67
8945
45
H(8)
2068
5104
10083
49
H(11)
1842
−409
9624
47
H(13)
2060
−345
11459
59
H(14)
2257
1911
11444
55
H(15A)
−305
−2077
7531
108
H(15B)
−495
−2820
8157
108
H(15C)
938
−3361
7654
108
H(111)
2170(50)
2330(30)
8481(13)
95
Step 2. Synthesis of 6-chloro-N4-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]quinoline-3,4-diamine (P10)
Zinc dust (97.5%, 12.3 g, 183 mmol) was added in one portion to a suspension of C32 (7.40 g, 23.0 mmol) in methanol (100 mL) and concentrated ammonium hydroxide (100 mL). After 1 hour, the reaction mixture was filtered through diatomaceous earth; the filter pad was rinsed with dichloromethane (70 mL). The combined filtrates were diluted with water, and the aqueous layer was extracted with dichloromethane (2×60 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified via silica gel chromatography (Gradient: 40% to 100% ethyl acetate in heptane) to provide the product. Yield: 3.68 g, 12.6 mmol, 55%. 1H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.91 (d, J=8.9 Hz, 1H), 7.74 (d, J=2.2 Hz, 1H), 7.40 (dd, J=8.9, 2.2 Hz, 1H), 4.02 (br dd, J=12, 5 Hz, 1H), 3.88 (br s, 2H), 3.29-3.56 (m, 4H), 1.82-1.96 (m, 2H), 1.56 (dddd, J=12, 12, 12, 5 Hz, 1H), 1.21-1.31 (m, 1H), 1.21 (d, J=6.2 Hz, 3H).
Preparation P11
6-(Difluoromethyl)-N4-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]quinoline-3,4-diamine (P11)
Step 1. Synthesis of 6-chloro-N-(2,4-dimethoxybenzyl)-N-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-3-nitroquinolin-4-amine (C33)
To a solution of P1 (3.90 g, 14.7 mmol) and N,N-diisopropylethylamine (8.53 mL, 49.0 mmol) in acetonitrile (74 mL) was added C7 (4.00 g, 16.5 mmol), and the reaction mixture was heated at 50° C. for 16 hours. It was then concentrated in vacuo, and the residue was partitioned between ethyl acetate (100 mL) and saturated aqueous sodium bicarbonate solution (100 mL), whereupon the aqueous layer was extracted with ethyl acetate (2×150 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution (150 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Silica gel chromatography (Gradient: 0% to 80% ethyl acetate in heptane) afforded the product as an orange solid. Yield: 6.00 g, 12.7 mmol, 86%. LCMS m/z 472.5 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.02 (s, 1H), 8.24 (d, J=2.4 Hz, 1H), 7.98 (d, J=9.0 Hz, 1H), 7.69 (dd, J=9.0, 2.4 Hz, 1H), 6.83 (d, J=8.2 Hz, 1H), 6.24-6.18 (m, 2H), 4.34 (br s, 2H), 4.08-4.00 (m, 1H), 3.82-3.70 (m, 1H), 3.69 (s, 3H), 3.55 (s, 3H), 3.49-3.38 (m, 2H), 2.02-1.85 (m, 3H), 1.66-1.52 (m, 1H, assumed; partially obscured by water peak), 1.21 (d, J=6.3 Hz, 3H).
Step 2. Synthesis of 2-(4-{(2,4-dimethoxybenzyl)[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]amino}-3-nitroquinolin-6-yl)-2,2-difluoro-1-phenylethanone (C34)
A pressure tube (250 mL) was charged with chloro[(di(1-adamantyl)-N-butylphosphine)-2-(2-aminobiphenyl)]palladium(11) (cataCXium® A Pd G2; 85.0 mg, 0.127 mmol), C33 (3.00 g, 6.36 mmol), and potassium phosphate tribasic monohydrate (5.86 g, 25.4 mmol). The vial was then evacuated and charged with nitrogen. This evacuation cycle was repeated twice, whereupon a solution of 2,2-difluoro-1-phenylethanone (1.68 mL, 12.7 mmol) in toluene (37 mL) was added, and the reaction mixture was heated at 110° C. for 24 hours. After cooling to room temperature, the reaction mixture was partitioned between saturated aqueous ammonium chloride solution (250 mL) and ethyl acetate (250 mL). The organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and purified via chromatography on silica gel (Gradient: 0% to 100% ethyl acetate in heptane), providing the product as an orange solid. Yield: 2.07 g, 3.50 mmol, 55%. LCMS m/z 592.3 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 8.49 (s, 1H), 8.17 (d, half of AB quartet, J=8.6 Hz, 1H), 8.08-7.99 (m, 3H), 7.65 (dd, J=8, 7 Hz, 1H), 7.50 (dd, J=8, 7 Hz, 2H), 6.83 (d, J=8.2 Hz, 1H), 6.20 (br d, J=8.6 Hz, 1H), 6.15 (s, 1H), 4.35 (br s, 2H), 4.04-3.96 (m, 1H), 3.81-3.70 (m, 1H), 3.69 (s, 3H), 3.47 (s, 3H), 3.30-3.18 (m, 2H), 2.07-1.87 (m, 3H), 1.76-1.64 (m, 1H), 1.21 (d, J=6.3 Hz, 3H).
Step 3. Synthesis of 6-(difluoromethyl)-N-(2,4-dimethoxybenzyl)-N-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-3-nitroquinolin-4-amine (C35)
Potassium hydroxide (1.97 g, 35.1 mmol) and water (1.22 mL, 67.7 mmol) were added to a solution of C34 (2.0 g, 3.38 mmol) in toluene (20 mL), and the resulting biphasic reaction mixture was vigorously stirred at 100° C. for 11 hours. An aliquot of the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution and ethyl acetate; LCMS analysis of the organic layer indicated the presence of both starting material and product. The reaction mixture was cooled to room temperature and partitioned between saturated aqueous sodium bicarbonate solution (125 mL) and ethyl acetate (150 mL). The organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and subjected to silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane), which failed to separate C35 from C34. The isolated mixture was resubjected to the reaction conditions for 24 hours, then worked up in the same manner; the crude residue (once again a mixture of C35 and C34) was again subjected to the original reaction conditions, this time for 48 hours. The reaction mixture was cooled to room temperature, and partitioned between saturated aqueous sodium bicarbonate solution (125 mL) and ethyl acetate (150 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford an oily, orange residue (1.85 g) that contained both C35 and C34 by LCMS analysis. This material was used directly in step 4. LCMS m/z 488.5 [M+H]+.
Improved conversion of C34 to 6-(difluoromethyl)-N-(2,4-dimethoxybenzyl)-N-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-3-nitroquinolin-4-amine (C35).
Potassium hydroxide (267 mg, 4.76 mmol) was added to a solution of C34 (470 mg, 0.794 mmol) in toluene (4.7 mL) and water (0.28 mL, 16 mmol). The reaction mixture was heated to 100° C. for 24 hours, whereupon it was cooled to room temperature and partitioned between water (150 mL) and dichloromethane (150 mL). The aqueous layer was extracted with dichloromethane (2×100 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 80% ethyl acetate in heptane) afforded the product as an orange solid. Yield: 337 mg, 0.691 mmol, 87%. 1H NMR (400 MHz, CDCl3) δ 9.10 (s, 1H), 8.45 (br s, 1H), 8.14 (d, J=8.6 Hz, 1H), 7.83 (br dd, J=8.6, 1 Hz, 1H), 6.86 (d, J=8.2 Hz, 1H), 6.81 (t, JHF=56.3 Hz, 1H), 6.23 (dd, half of ABX pattern, J=8.2, 2.4 Hz, 1H), 6.17 (d, half of AB quartet, J=2.4 Hz, 1H), 4.38 (br AB quartet, JAB=14 Hz, ΔνAB=8 Hz, 2H), 4.08-4.02 (m, 1H), 3.88-3.78 (m, 1H), 3.69 (s, 3H), 3.49 (s, 3H), 3.46-3.36 (m, 2H), 2.07-1.94 (m, 3H), 1.73-1.62 (m, 1H), 1.22 (d, J=6.3 Hz, 3H).
Step 4. Synthesis of 6-(difluoromethyl)-N-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-3-nitroquinolin-4-amine (C36)
A solution of C35 and C34 (from step 3; 1.85 g, 3.38 mmol) in dichloromethane (25 mL) was cooled to 0° C. and treated with trifluoroacetic acid (1.16 mL, 15.1 mmol). The reaction mixture was allowed to warm to room temperature, and was stirred at room temperature for 20 minutes, whereupon it was cooled to 0° C., diluted with dichloromethane (20 mL) and basified to pH 8 via addition of saturated aqueous sodium bicarbonate solution (100 mL). The aqueous layer was extracted with dichloromethane (2×50 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution (150 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified via chromatography on silica gel (Gradient: 0% to 100% ethyl acetate in heptane) followed by trituration with diethyl ether (50 mL), providing the product as a yellow solid. Yield over two steps: 0.70 g, 2.1 mmol, 62%. LCMS m/z 338.3 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.44 (s, 1H), 9.34 (br d, J=7.8 Hz, 1H), 8.37 (s, 1H), 8.11 (d, J=8.6 Hz, 1H), 7.86 (d, J=8.6 Hz, 1H), 6.84 (t, JHF=56.5 Hz, 1H), 4.39-4.26 (m, 1H), 4.18-4.08 (m, 1H), 3.61-3.48 (m, 2H), 2.27-2.12 (m, 2H), 1.89-1.74 (m, 1H), 1.58-1.46 (m, 1H), 1.28 (d, J=6.3 Hz, 3H).
Step 5. Synthesis of 6-(difluoromethyl)-N4-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]quinoline-3,4-diamine (P11)
A Parr reactor was charged with a solution of C36 (0.70 g, 2.1 mmol) in tetrahydrofuran (150 mL), followed by platinum on carbon (5%; 600 mg). The mixture was purged three times with nitrogen, backfilling with hydrogen, whereupon it was hydrogenated for 2 hours at 30 psi. The reaction mixture was then diluted with tetrahydrofuran (50 mL), and filtered through a pad of diatomaceous earth. The filter pad was washed with tetrahydrofuran (3×50 mL), and the combined filtrates were concentrated in vacuo, dissolved in dichloromethane (15 mL), and filtered through an Acrodisc® filter. The filtrate was concentrated under reduced pressure to afford the product as a dark brown solid. The product was somewhat impure, as judged by 1H NMR analysis. Yield: 547 mg. 1.78 mmol, 85%. LCMS m/z 308.4 [M+H]+. 1H NMR (400 MHz, CDCl3), characteristic peaks: δ 8.56 (s, 1H), 8.06 (d, J=8.6 Hz, 1H), 7.93 (br s, 1H), 7.57 (br d, J=8.6 Hz, 1H), 6.83 (t, JHF=56.3 Hz, 1H), 4.03 (ddd, J=11.7, 4.7, 1.6 Hz, 1H), 3.87 (br s, 2H), 3.61-3.49 (m, 1H), 3.49-3.39 (m, 2H), 1.98-1.90 (m, 1H), 1.90-1.82 (m, 1H), 1.63-1.51 (m, 1H), 1.21 (d, J=6.3 Hz, 3H).
Preparation P12
N4-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-6-fluoroquinoline-3,4-diamine (P12)
Step 1. Synthesis of tert-butyl 3,3-difluoro-4-[(6-fluoro-3-nitroquinolin-4-yl)amino]pyrrolidine-1-carboxylate (C37)
To a 15° C. solution of 4-chloro-6-fluoro-3-nitroquinoline (10.0 g, 44.1 mmol) in acetonitrile (50 mL) was added N,N-diisopropylethylamine (6.84 g, 52.9 mmol), followed by addition of tert-butyl 4-amino-3,3-difluoropyrrolidine-1-carboxylate (prepared using the method described by D. C. Behenna et al., in U.S. Patent Application 2015 0141402 A1, May 21, 2015; 9.81 g, 44.1 mmol). The reaction mixture was stirred at 20° C. for 48 hours, whereupon it was concentrated in vacuo and purified via chromatography on silica gel (Gradient: 9% to 17% tetrahydrofuran in petroleum ether) to afford the product as a pale yellow solid. Yield: 16.8 g, 40.7 mmol, 92%. 1H NMR (400 MHz, CDCl3) δ 9.39 (5, 1H), 8.87-8.69 (br m, 1H), 8.13 (dd, J=9.5, 5.5 Hz, 1H), 7.79-7.70 (br d, J=8 Hz, 1H), 7.63 (ddd, J=9.0, 7.5, 2.5 Hz, 1H), 4.87-4.71 (br m, 1H), 4.31-4.09 (br m, 1H), 4.04-3.84 (br m, 1H), 3.84-3.69 (m, 1H), 3.63-3.51 (br m, 1H), 1.50 (s, 9H).
Step 2. Synthesis of N-(4,4-difluoropyrrolidin-3-yl)-6-fluoro-3-nitroquinolin-4-amine (C38)
Trifluoroacetic acid (50 mL) was added to a 15° C. solution of C37 (16.8 g, 40.7 mmol) in dichloromethane (100 mL), and the reaction mixture was stirred for 3 hours at 15° C. LCMS analysis at this point indicated product formation (LCMS m/z 313.1 [M+H]+), and the reaction mixture was concentrated in vacuo. The residue was taken up in aqueous sodium bicarbonate solution (200 mL) and extracted with ethyl acetate (3×150 mL). Concentration of the combined organic layers under reduced pressure afforded the product as a pale yellow solid. Yield: 12.5 g, 40.0 mmol, 98%. 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.30 (br d, J=9.2 Hz, 1H), 8.26 (dd, J=10.6, 2.6 Hz, 1H), 8.04 (dd, J=9.0, 5.9 Hz, 1H), 7.85-7.78 (m, 1H), 4.53-4.39 (m, 1H), 3.58 (dd, J=11.9, 7.5 Hz, 1H), 3.39-3.25 (m, 1H), 3.24-3.09 (m, 1H), 3.08 (dd, J=11.9, 7.5 Hz, 1H).
Step 3. Synthesis of N-(4,4-difluoro-1-methylpyrrolidin-3-yl)-6-fluoro-3-nitro quinolin-4-amine (C39)
Sodium triacetoxyborohydride (33.9 g, 160 mmol) was added to a 0° C. mixture of C38 (12.5 g, 40.0 mmol) in acetonitrile (150 mL). An aqueous solution of formaldehyde (37%; 13.0 g, 160 mmol) was slowly added over 20 minutes, and the reaction mixture was stirred at room temperature for 1 hour; LCMS analysis at this point indicated that the reaction was complete (LCMS m/z 327.1 [M+H]+). After the reaction mixture had been concentrated to dryness, the residue was basified to pH 8 by addition of aqueous sodium bicarbonate solution. The resulting solid was collected via filtration to provide the product as a red solid. Yield: 11.8 g, 36.2 mmol, 90%. 1H NMR (400 MHz, CDCl3), characteristic peaks: δ 9.35 (s, 1H), 9.22 (br d, J=9.2 Hz, 1H), 8.07 (dd, J=9.0, 5.5 Hz, 1H), 7.83 (dd, J=10.1, 2.6 Hz, 1H), 7.58 (ddd, J=9.2, 7.5, 2.6 Hz, 1H), 3.27 (dd, J=9.7, 6.2 Hz, 1H), 3.15-3.05 (m, 2H), 2.79 (ddd, J=9.9, 5.9, 2.0 Hz, 1H), 2.45 (s, 3H).
Step 4. Synthesis of N4-(4,4-difluoro-1-methylpyrrolidin-3-yl)-6-fluoroquinoline-3,4-diamine (P12)
Palladium on carbon (10%, 3.85 g) was added to a solution of C39 (11.8 g, 36.2 mmol) in methanol (100 mL), and the resulting mixture was hydrogenated (30 psi) at 25° C. for 1 hour. This reaction mixture was combined with a similar reaction mixture employing C39 (3.60 g, 11.0 mmol) and filtered through diatomaceous earth. The filtrate was concentrated in vacuo and purified using chromatography on silica gel (Gradient: 9% to 17% tetrahydrofuran in petroleum ether). The product was obtained as a pale yellow solid. Combined yield: 8.40 g, 28.3 mmol, 60%. LCMS m/z 297.1 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 7.95 (dd, J=9.0, 5.5 Hz, 1H), 7.43 (dd, J=10.6, 2.6 Hz, 1H), 7.23 (ddd, J=9.0, 8.1, 2.6 Hz, 1H), 4.26-4.12 (m, 1H), 4.05-3.89 (br s, 2H), 3.79 (br d, J=11.0 Hz, 1H), 3.23-2.93 (m, 3H), 2.63-2.55 (m, 1H), 2.38 (s, 3H).
Preparation P13
6-Chloro-N4-(4,4-difluoro-1-methylpyrrolidin-3-yl)quinoline-3,4-diamine (P13)
Step 1. Synthesis of tert-butyl 4-[(6-chloro-3-nitroquinolin-4-yl)amino]-3,3-difluoropyrrolidine-1-carboxylate (C40)
To a solution of C7 (13.1 g, 53.9 mmol) in acetonitrile (60 mL) was added N,N-diisopropylethylamine (11.3 mL, 64.9 mmol), followed by addition of a solution of tertbutyl 4-amino-3,3-difluoropyrrolidine-1-carboxylate (prepared using the method described by D. C. Behenna et al., in U.S. Patent Application 2015 0141402 A1, May 21, 2015; 12.0 g, 54.0 mmol) in acetonitrile (5 mL). After the reaction mixture had been stirred at 20° C. for 32 hours, it was diluted with water (100 mL). The resulting solid was collected by filtration and purified via chromatography on silica gel (Gradient: 0% to 25% tetrahydrofuran in petroleum ether), affording the product as a yellow solid. Yield: 12.0 g, 28.0 mmol, 52%. LCMS m/z 428.7 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.41 (s, 1H), 8.91-8.78 (br m, 1H), 8.08 (br s, 1H), 8.06 (d, J=9.0 Hz, 1H), 7.79 (dd, J=9.0, 2.0 Hz, 1H), 4.86-4.72 (br m, 1H), 4.30-4.12 (br m, 1H), 4.03-3.86 (br m, 1H), 3.86-3.71 (m, 1H), 3.64-3.52 (br m, 1H), 1.51 (s, 9H).
Step 2. Synthesis of 6-chloro-N-(4,4-difluoropyrrolidin-3-yl)-3-nitroquinolin-4-amine (C41)
Trifluoroacetic acid (60 mL) was added to a solution of C40 (11.9 g, 27.8 mmol) in dichloromethane (100 mL), and the reaction mixture was stirred at 20° C. for 12 hours. Solvents were then removed via concentration in vacuo, and the residue was carefully basified by addition of aqueous sodium bicarbonate solution (500 mL). The resulting mixture was extracted with 2-methyltetrahydrofuran (2×200 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide the product as a yellow solid (10.9 g), which was used in the following step. LCMS m/z 328.5 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.57 (s, 1H), 8.42-8.29 (br s, 1H), 7.94 (br AB quartet, JAB=8 Hz, ΔνAB=26 Hz, 2H), 4.45-4.30 (br m, 1H), 3.57-3.46 (br m, 1H), 3.33-3.22 (m, 1H, assumed; partially obscured by water peak), 3.21-2.98 (m, 3H).
Step 3. Synthesis of 6-chloro-N-(4,4-difluoro-1-methylpyrrolidin-3-yl)-3-nitroquinolin-4-amine (C42)
Sodium triacetoxyborohydride (26.8 g, 126 mmol) was added to a 0° C. solution of C41 (from the previous step; 10.4 g, 26.5 mmol) in acetonitrile (110 mL). An aqueous solution of formaldehyde (37%; 10.3 g, 127 mmol) was added over 20 minutes, and the reaction mixture was stirred at room temperature for 1 hour. It was then combined with a similar reaction mixture derived from C41 (from the previous step; 500 mg, 1.27 mmol) and concentrated in vacuo. The residue was basified to pH 8 by addition of aqueous sodium bicarbonate solution, and the resulting solid was collection via filtration to afford the product as a red solid. Combined yield: 8.60 g, 25.1 mmol, 90% over two steps. LCMS m/z 342.6 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.38 (s, 1H), 9.30 (br d, J=9.2 Hz, 1H), 8.18 (d, J=2.2 Hz, 1H), 8.01 (d, J=8.8 Hz, 1H), 7.75 (dd, J=9.2, 2.2 Hz, 1H), 4.83-4.71 (m, 1H), 3.27 (ddd, J=10.1, 6.2, 0.9 Hz, 1H), 3.16-3.07 (m, 2H), 2.81 (ddd, J=9.9, 5.7, 2.0 Hz, 1H), 2.46 (s, 3H).
Step 4. Synthesis of 6-chloro-N4-(4,4-difluoro-1-methylpyrrolidin-3-yl)quinoline-3,4-diamine (P13)
Platinum(IV) oxide (5.0 g, 22 mmol) was added to a solution of C42 (8.50 g, 24.8 mmol) in methanol (100 mL), and the resulting mixture was hydrogenated at 25° C. for 4 hours, using a balloon of hydrogen. The reaction mixture was combined with a similar reaction mixture employing C42 (100 mg, 0.292 mmol), filtered through diatomaceous earth, and concentrated in vacuo. Chromatography on silica gel (Gradient: 17% to 100% tetrahydrofuran in petroleum ether) provided the product as a brown oil that solidified upon standing overnight. Combined yield: 5.02 g, 16.1 mmol, 64%. LCMS m/z 312.9 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.90 (d, J=9.0 Hz, 1H), 7.81 (d, J=2.0 Hz, 1H), 7.41 (dd, J=8.8, 2.3 Hz, 1H), 4.29-4.16 (m, 1H), 3.95 (br s, 2H), 3.86 (br d, J=11.0 Hz, 1H), 3.19-2.96 (m, 3H), 2.61 (ddd, J=9, 7, 2 Hz, 1H), 2.41 (s, 3H).
Examples 1 and 2
[(2S,4R)-4-(8-Chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile (1) and [(2R,4S)-4-(8-Chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile (2)
Step 1. Synthesis of 1-{cis-2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}-8-chloro-2-ethyl-1H-imidazo[4,5-c]quinoline (C43)
A solution of P3 (800 mg, 2.01 mmol) in propanoic acid (10 mL) and 1,1,1-triethoxypropane (10 mL) was stirred at 110° C. for 2.5 hours, whereupon it was combined with a similar reaction carried out using P3 (100 mg, 0.251 mmol), and poured into water. The resulting mixture was neutralized with solid potassium carbonate and extracted with ethyl acetate (2×100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification via silica gel chromatography (Gradient: 0% to 2% methanol in dichloromethane) provided the product as a yellow solid. Yield: 875 mg, 2.01 mmol, 89%. LCMS m/z 436.1 [M+H]+.
Step 2. Synthesis of [cis-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]methanol (C44)
A 0° C. solution of C43 (875 mg, 2.01 mmol) in dichloromethane (17 mL) was treated with boron trichloride (1 M solution; 6.02 mL, 6.02 mmol) and the reaction mixture was stirred at 20° C. for 2 hours, whereupon it was poured into aqueous sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (2×50 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified via silica gel chromatography (Gradient: 0% to 2.8% methanol in dichloromethane) to afford the product as an off-white, foamy solid. Yield: 490 mg, 1.42 mmol, 71%. LCMS m/z 346.0 [M+H]+.
Step 3. Synthesis of [cis-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]methyl methanesulfonate (C45)
To a 0° C. solution of C44 (490 mg, 1.42 mmol) in dichloromethane (10 mL) were added triethylamine (430 mg, 4.25 mmol) and methanesulfonyl chloride (195 mg, 1.70 mmol). The reaction mixture was stirred at 20° C. for 1 hour, whereupon it was poured into water (50 mL) and extracted with ethyl acetate (2×50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to provide the product as a yellow, foamy solid (640 mg), which was taken directly to the following step. LCMS m/z 423.8 (chlorine isotope pattern observed) [M+H]+.
Step 4. Synthesis of [(2S,4R)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile (1) and [(2R,4S)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile (2)
To a solution of C45 (from the previous step; 1.42 mmol) in dimethyl sulfoxide (15 mL) was added tetraethylammonium cyanide (708 mg, 4.53 mmol). The reaction mixture was heated at 80° C. for 16 hours, whereupon it was cooled, poured into water, and extracted with ethyl acetate (2×100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 2.8% methanol in dichloromethane) afforded a racemic mixture of 1 and 2 as an off-white, foamy solid. Yield of racemic product: 349 mg, 0.984 mmol, 69% over two steps.
This material was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)]. The first-eluting enantiomer was designated as 1, and the second-eluting enantiomer as 2; both were obtained as solids. The indicated absolute configurations for 1 and 2 were assigned on the basis of an X-ray structural determination carried out on 2 (see below).
For 1, Yield: 118 mg, 0.333 mmol, 34% for the separation. LCMS m/z 354.7 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.12 (s, 1H), 8.83-8.63 (v br m, 1H), 8.18 (d, J=9.0 Hz, 1H), 7.72 (dd, J=9.0, 2.0 Hz, 1H), 5.37-5.13 (v br m, 1H), 4.45-4.31 (m, 1H), 4.06-3.97 (m, 1H), 3.88 (ddd, J=12.0, 12.0, 2.5 Hz, 1H), 3.21 (q, J=7.5 Hz, 2H), 2.94-2.44 (br m, 2H), 2.88 (dd, half of ABX pattern, J=17.1, 4.5 Hz, 1H), 2.78 (br dd, half of ABX pattern, J=17.1, 6.5 Hz, 1H), 2.31-2.14 (br m, 1H), 2.14-1.97 (br m, 1H), 1.52 (t, J=7.3 Hz, 3H).
For 2, Yield: 88.8 mg, 0.250 mmol, 25% yield for the separation. LCMS m/z 354.7 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.11 (s, 1H), 8.82-8.59 (v br m, 1H), 8.17 (d, J=9.0 Hz, 1H), 7.71 (dd, J=9.0, 2.0 Hz, 1H), 5.39-5.12 (v br m, 1H), 4.44-4.31 (m, 1H), 4.06-3.96 (m, 1H), 3.88 (ddd, J=12, 12, 3 Hz, 1H), 3.20 (q, J=7.5 Hz, 2H), 2.88-2.69 (br m, 1H), 2.88 (dd, half of ABX pattern, J=17.1, 4.0 Hz, 1H), 2.78 (br dd, half of ABX pattern, J=17.1, 6.5 Hz, 1H), 2.67-2.46 (br m, 1H), 2.29-2.14 (br m, 1H), 2.14-1.97 (br m, 1H), 1.52 (t, J=7.3 Hz, 3H).
A sample of 2 was crystallized from 2-methyltetrahydrofuran/hexanes via vapor diffusion and used to determine the absolute configuration via X-ray crystallography:
Single-crystal X-ray structural determination of 2
Single Crystal X-Ray Analysis
Data collection was performed on a Bruker APEX diffractometer at room temperature. Data collection consisted of omega and phi scans. Resolution was limited by diffraction of the crystal to approximately 0.9 angstroms.
The structure was solved by direct methods using SHELX software suite in the monoclinic space group P21. The structure was subsequently refined by the full-matrix least squares method. All non-hydrogen atoms were found and refined using anisotropic displacement parameters.
The hydrogen atoms were placed in calculated positions and were allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms.
Analysis of the absolute structure using likelihood methods (Hooft, 2008) was performed using PLATON (Spek). The results indicate that the absolute structure has been correctly assigned. The method calculates that the probability that the structure is correct is 100.0. The Hooft parameter is reported as 0.045 with an Esd of 0.002.
The final R-index was 5.1%. A final difference Fourier revealed no missing or misplaced electron density.
Pertinent crystal, data collection and refinement information is summarized in Table F. Atomic coordinates, bond lengths, bond angles, and displacement parameters are listed in Tables G, H, and J.
SOFTWARE AND REFERENCES
SHELXTL, Version 5.1, Bruker A X S, 1997.
PLATON, A. L. Spek, J. Appl. Cryst. 2003, 36, 7-13.
MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M. Towler, and J. van de Streek, J. Appl. Cryst. 2006, 39, 453-457.
OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H. Puschmann, J. Appl. Cryst. 2009, 42, 339-341.
R. W. W. Hooft, L. H. Strayer, and A. L. Spek, J. Appl. Cryst. 2008, 41, 96-103.
H. D. Flack, Acta Cryst. 1983, A39, 867-881.
TABLE F
Crystal data and structure refinement for 2.
Empirical formula
C19H19ClN4O
Formula weight
354.84
Temperature
296(2) K
Wavelength
1.54178 Å
Crystal system
Monoclinic
Space group
P21
Unit cell dimensions
a = 9.3184(7) Å
α = 90°
b = 6.9545(5) Å
β = 94.437(3)°
c = 13.5545(9) Å
γ = 90°
Volume
875.76(11) Å3
Z
2
Density (calculated)
1.346 Mg/m3
Absorption coefficient
2.045 mm−1
F(000)
372
Crystal size
0.120 × 0.120 × 0.060 mm3
Theta range for data
17.720 to 69.948°
collection
Index ranges
−11 <= h <= 11, −8 <= k <=
8, −16 <= l <= 16
Reflections collected
5772
Independent reflections
2717 [Rint = 0.0396]
Completeness to
94.9%
theta = 70.57°
Absorption correction
Empirical
Refinement method
Full-matrix least-squares
on F2
Data/restraints/
2717/1/228
parameters
Goodness-of-fit on F2
1.054
Final R indices
R1 = 0.0508, wR2 = 0.1118
[l > 2σ(l)]
R indices (all data)
R1 = 0.0659, wR2 = 0.1203
Absolute structure
0.04(2)
parameter
Extinction coefficient
0.000(5)
Largest diff. peak
0.220 and −0.238 e · Å−3
and hole
TABLE G
Atomic coordinates (×104) and equivalent isotropic displacement
parameters (Å2 × 103) for 2. U(eq) is defined as one-third
of the trace of the orthogonalized Uij tensor.
x
y
z
U(eq)
Cl(1)5544(1)
6289
(3)
6228
(1)
78(1)
O(1)5128(3)
5926
(5)
1381
(2)
49(1)
N(1)11747(3)
6220
(7)
5643
(2)
50(1)
N(2)11630(3)
6286
(7)
2918
(2)
47(1)
N(3)9236(3)
6080
(6)
2976
(2)
40(1)
N(4)2604(6)
2224
(10)
539
(4)
95(2)
C(1)7761(4)
6231
(8)
5086
(2)
44(1)
C(2)7371(4)
6246
(9)
6030
(3)
49(1)
C(3)8389(5)
6178
(9)
6850
(3)
53(1)
C(4)9805(4)
6144
(9)
6676
(3)
50(1)
C(5)10282(4)
6180
(8)
5716
(2)
41(1)
C(6)9224(3)
6166
(7)
4891
(2)
38(1)
C(7)9817(4)
6160
(8)
3950
(2)
38(1)
C(8)11288(3)
6242
(8)
3887
(2)
41(1)
C(9)12213(4)
6262
(9)
4759
(3)
50(1)
C(10)10387(4)
6206
(9)
2390
(3)
44(1)
C(11)10260(4)
6227
(12)
1285
(3)
61(1)
C(12)11494(7)
7201
(11)
842
(4)
83(2)
C(13)7694(4)
5811
(7)
2668
(3)
42(1)
C(14)6917(5)
7699
(7)
2409
(4)
51(1)
C(15)5324(5)
7311
(9)
2160
(4)
62(1)
C(16)5752(5)
4147
(7)
1661
(3)
46(1)
C(17)7371(5)
4335
(7)
1852
(4)
51(1)
C(18)5357(5)
2758
(9)
813
(4)
62(1)
C(19)3808(6)
2452
(9)
659
(4)
66(1)
TABLE H
Bond lengths [Å] and angles [°] for 2.
Cl(1)—C(2)
1.744
(4)
O(1)—C(16)
1.406
(6)
O(1)—C(15)
1.431
(6)
N(1)—C(9)
1.306
(5)
N(1)—C(5)
1.377
(5)
N(2)—C(10)
1.315
(4)
N(2)—C(8)
1.376
(5)
N(3)—C(10)
1.386
(4)
N(3)—C(7)
1.389
(4)
N(3)—C(13)
1.477
(5)
N(4)—C(19)
1.132
(7)
C(1)—C(2)
1.357
(5)
C(1)—C(6)
1.410
(5)
C(1)—H(1)
0.9300
C(2)—C(3)
1.405
(5)
C(3)—C(4)
1.359
(6)
C(3)—H(3)
0.9300
C(4)—C(5)
1.407
(5)
C(4)—H(4)
0.9300
C(5)—C(6)
1.432
(5)
C(6)—C(7)
1.428
(5)
C(7)—C(8)
1.381
(5)
C(8)—C(9)
1.408
(5)
C(9)—H(9)
0.9300
C(10)—C(11)
1.493
(5)
C(11)—C(12)
1.499
(8)
C(11)—H(11A)
0.9700
C(11)—H(11B)
0.9700
C(12)—H(12A)
0.9600
C(12)—H(12B)
0.9600
C(12)—H(12C)
0.9600
C(13)—C(17)
1.522
(6)
C(13)—C(14)
1.527
(6)
C(13)—H(13)
0.9800
C(14)—C(15)
1.520
(7)
C(14)—H(14A)
0.9700
C(14)—H(14B)
0.9700
C(15)—H(15A)
0.9700
C(15)—H(15B)
0.9700
C(16)—C(17)
1.516
(6)
C(16)—C(18)
1.524
(6)
C(16)—H(16)
0.9800
C(17)—H(17A)
0.9700
C(17)—H(17B)
0.9700
C(18)—C(19)
1.458
(8)
C(18)—H(18A)
0.9700
C(18)—H(18B)
0.9700
C(16)—O(1)—C(15)
111.5
(3)
C(9)—N(1)—C(5)
117.9
(3)
C(10)—N(2)—C(8)
105.0
(3)
C(10)—N(3)—C(7)
106.3
(3)
C(10)—N(3)—C(13)
128.7
(3)
C(7)—N(3)—C(13)
125.0
(3)
C(2)—C(1)—C(6)
120.7
(3)
C(2)—C(1)—H(1)
119.6
C(6)—C(1)—H(1)
119.6
C(1)—C(2)—C(3)
122.1
(4)
C(1)—C(2)—Cl(1)
118.8
(3)
C(3)—C(2)—Cl(1)
119.1
(3)
C(4)—C(3)—C(2)
117.9
(3)
C(4)—C(3)—H(3)
121.0
C(2)—C(3)—H(3)
121.0
C(3)—C(4)—C(5)
122.7
(3)
C(3)—C(4)—H(4)
118.6
C(5)—C(4)—H(4)
118.6
N(1)—C(5)—C(4)
116.9
(3)
N(1)—C(5)—C(6)
124.8
(3)
C(4)—C(5)—C(6)
118.3
(3)
C(1)—C(6)—C(7)
127.9
(3)
C(1)—C(6)—C(5)
118.0
(3)
C(7)—C(6)—C(5)
114.0
(3)
C(8)—C(7)—N(3)
105.0
(3)
C(8)—C(7)—C(6)
120.6
(3)
N(3)—C(7)—C(6)
134.4
(3)
C(7)—C(8)—N(2)
111.4
(3)
C(7)—C(8)—C(9)
119.7
(3)
N(2)—C(8)—C(9)
129.0
(3)
N(1)—C(9)—C(8)
122.9
(3)
N(1)—C(9)—H(9)
118.5
C(8)—C(9)—H(9)
118.5
N(2)—C(10)—N(3)
112.2
(3)
N(2)—C(10)—C(11)
122.9
(3)
N(3)—C(10)—C(11)
124.8
(3)
C(10)—C(11)—C(12)
113.7
(4)
C(10)—C(11)—H(11A)
108.8
C(12)—C(11)—H(11A)
108.8
C(10)—C(11)—H(11B)
108.8
C(12)—C(11)—H(11B)
108.8
H(11A)—C(11)—H(11B)
107.7
C(11)—C(12)—H(12A)
109.5
C(11)—C(12)—H(12B)
109.5
H(12A)—C(12)—H(12B)
109.5
C(11)—C(12)—H(12C)
109.5
H(12A)—C(12)—H(12C)
109.5
H(12B)—C(12)—H(12C)
109.5
N(3)—C(13)—C(17)
115.1
(3)
N(3)—C(13)—C(14)
112.9
(4)
C(17)—C(13)—C(14)
110.4
(3)
N(3)—C(13)—H(13)
105.8
C(17)—C(13)—H(13)
105.8
C(14)—C(13)—H(13)
105.8
C(15)—C(14)—C(13)
109.6
(4)
C(15)—C(14)—H(14A)
109.7
C(13)—C(14)—H(14A)
109.7
C(15)—C(14)—H(14B)
109.7
C(13)—C(14)—H(14B)
109.7
H(14A)—C(14)—H(14B)
108.2
O(1)—C(15)—C(14)
110.5
(4)
O(1)—C(15)—H(15A)
109.6
C(14)—C(15)—H(15A)
109.6
O(1)—C(15)—H(15B)
109.6
C(14)—C(15)—H(15B)
109.6
H(15A)—C(15)—H(15B)
108.1
O(1)—C(16)—C(17)
110.8
(4)
O(1)—C(16)—C(18)
106.5
(3)
C(17)—C(16)—C(18)
111.3
(4)
O(1)—C(16)—H(16)
109.4
C(17)—C(16)—H(16)
109.4
C(18)—C(16)—H(16)
109.4
C(16)—C(17)—C(13)
108.7
(4)
C(16)—C(17)—H(17A)
109.9
C(13)—C(17)—H(17A)
109.9
C(16)—C(17)—H(17B)
109.9
C(13)—C(17)—H(17B)
109.9
H(17A)—C(17)—H(17B)
108.3
C(19)—C(18)—C(16)
112.3
(4)
C(19)—C(18)—H(18A)
109.1
C(16)—C(18)—H(18A)
109.1
C(19)—C(18)—H(18B)
109.1
C(16)—C(18)—H(18B)
109.1
H(18A)—C(18)—H(18B)
107.9
N(4)—C(19)—C(18)
179.6
(7)
TABLE J
Anisotropic displacement parameters (Å2 × 103) for 2.
The anisotropic displacement factor exponent takes the form: −2π2[h2
a*2U11 + . . . + 2 h k a* b* U12].
U11
U22
U33
U23
U13
U12
Cl(1)60(1)
124
(1)
52
(1)
−11
(1)
13
(1)
3
(1)
O(1)39(1)
58
(2)
49
(1)
−5
(2)
−7
(1)
2
(2)
N(1)50(2)
49
(2)
48
(2)
4
(2)
−16
(1)
−6
(2)
N(2)36(2)
54
(2)
50
(2)
3
(2)
−2
(1)
3
(2)
N(3)36(2)
52
(2)
33
(1)
3
(2)
−4
(1)
−5
(2)
N(4)55(3)
132
(5)
96
(4)
−37
(3)
0
(2)
−28
(3)
O(1)46(2)
48
(2)
38
(2)
−2
(2)
−6
(1)
−1
(3)
C(2)54(2)
51
(3)
42
(2)
−8
(3)
2
(2)
−1
(3)
C(3)72(3)
52
(3)
35
(2)
−3
(3)
0
(2)
−7
(3)
C(4)65(2)
46
(2)
37
(2)
0
(2)
−15
(2)
−8
(3)
C(5)50(2)
31
(2)
41
(2)
2
(2)
−12
(1)
−5
(2)
C(6)44(2)
31
(2)
36
(2)
−1
(2)
−7
(1)
−2
(2)
C(7)43(2)
32
(2)
37
(2)
0
(2)
−9
(1)
0
(2)
C(8)35(2)
39
(2)
46
(2)
5
(2)
−6
(1)
1
(2)
C(9)42(2)
51
(3)
54
(2)
−1
(3)
−12
(2)
−2
(3)
C(10)
36
(2)
52
(2)
45
(2)
2
(2)
0
(1)
1
(3)
C(11)
43
(2)
94
(4)
46
(2)
0
(3)
2
(2)
3
(4)
C(12)
72
(4)
119
(6)
59
(3)
21
(3)
13
(2)
−10
(3)
C(13)
37
(2)
57
(3)
32
(2)
2
(2)
−3
(1)
−6
(2)
C(14)
42
(2)
51
(3)
58
(3)
−10
(2)
−5
(2)
2
(2)
C(15)
39
(2)
68
(4)
79
(3)
−26
(3)
−8
(2)
6
(2)
C(16)
50
(2)
51
(3)
37
(2)
3
(2)
−4
(2)
−12
(2)
C(17)
49
(3)
44
(3)
58
(3)
−6
(2)
−16
(2)
5
(2)
C(18)
55
(3)
61
(3)
68
(3)
−14
(3)
−11
(2)
−4
(2)
C(19)
63
(3)
78
(4)
55
(3)
−18
(3)
−3
(2)
−13
(3)
Examples 3 and 4
1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1 (3) and 1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2 (4)
Step 1. Synthesis of 4-bromo-5-methyl-1H-1,2,3-triazole (C46)
N-Bromosuccinimide (5.89 g, 33.1 mmol) was added to a solution of 4-methyl-1H-1,2,3-triazole (2.50 g, 30.1 mmol) in chloroform (30 mL), and the reaction mixture was stirred for 16 hours at room temperature (15° C.). It was then diluted with dichloromethane (100 mL), washed with water (2×100 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to provide the product as a white solid (4.9 g), which was used directly in the next step.
Step 2. Synthesis of tert-butyl (4-bromo-5-methyl-2H-1,2,3-triazol-2-yl)acetate (C47)
tert-Butyl bromoacetate (8.8 g, 45 mmol) was added in one portion to a mixture of C46 (from the previous step, 4.9 g, 30.1 mmol) and cesium carbonate (17.6 g, 54.0 mmol) in N,N-dimethylformamide (80 mL). The reaction mixture was stirred at room temperature (20° C.) for 16 hours, whereupon it was diluted with water (100 mL) and extracted with ethyl acetate (2×80 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (2×100 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 15%, ethyl acetate in petroleum ether) provided the product as a colorless oil. Yield: 4.00 g, 14.5 mmol, 48% over 2 steps.
Step 3. Synthesis of tert-butyl (4-methyl-2H-1,2,3-triazol-2-yl)acetate (C48), methyl (4-methyl-2H-1,2,3-triazol-2-yl)acetate (C49), and (4-methyl-2H-1,2,3-triazol-2-yl)acetic Acid (C50)
A mixture of C47 (3.50 g, 12.7 mmol) and palladium on carbon (10%, 500 mg) in methanol (35 mL) was stirred under hydrogen (40 psi) for 4 hours at room temperature (17° C.). The reaction mixture was filtered, and the filtrate was concentrated in vacuo, providing a yellow oil (3.00 g). On the basis of 1H NMR, the product was assigned as a mixture of C48 (tert-butyl ester), C49 (methyl ester), and C50 (carboxylic acid); this material was taken directly to the following step for ester hydrolysis. 1H NMR peaks (400 MHz, CD3OD) δ [7.50 (s) and 7.49 (s), total 1H], [5.23 (s), 5.17 (s), and 5.10 (s), total 2H], 3.75 (s, from methyl ester), 2.30 (s, 3H), 1.46 (s, from tert-butyl ester).
Step 4. Synthesis of (4-methyl-2H-1,2,3-triazol-2-yl)acetic Acid (C50)
A mixture of C48, C49, and C50 (from the previous step, 3.00 g, mmol) in trifluoroacetic acid (3 mL) was stirred for 2 hours at room temperature (17° C.). After removal of solvent in vacuo, the residue was dissolved in tetrahydrofuran (10 mL) and treated with aqueous sodium hydroxide solution (2 M, 10 mL). The reaction mixture was stirred for 1 hour at room temperature (17° C.), concentrated in vacuo, and partitioned between water (50 mL) and dichloromethane (20 mL). The aqueous layer was extracted with dichloromethane (2×20 mL), and then acidified with 1 M aqueous hydrochloric acid to a pH of 1. This acidic aqueous layer was extracted with ethyl acetate (3×40 mL), and the combined ethyl acetate layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide the product as a yellow solid. Yield: 1.9 g, 13 mmol, 100% over 2 steps. 1H NMR (400 MHz, CDCl3) δ 7.46 (s, 1H), 5.25 (s, 2H), 2.34 (s, 3H).
Step 5. Synthesis of N-{6-cyano-4-[(4,4-difluoro-1-methylpyrrolidin-3-yl)amino]quinolin-3-yl}-2-(4-methyl-2H-1,2,3-triazol-2-yl)acetamide (C51)
This experiment was carried out in two identical batches. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (139 mg, 0.725 mmol) was added to a solution of P4 (100 mg, 0.330 mmol) and C50 (55.8 mg, 0.395 mmol) in pyridine (1.0 mL). After the reaction mixture had been stirred at 25° C. for 1 hour, at which time LCMS analysis indicated conversion to the product: LCMS m/z 427.2 [M+H]+, the two batches were combined, diluted with water (50 mL), and extracted with ethyl acetate (3×50 mL). The combined organic layers were concentrated in vacuo and purified via silica gel chromatography (Gradient: 17% to 50% ethyl acetate in petroleum ether) to provide the product as a white solid. Yield: 210 mg, 0.492 mmol, 75%. 1H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 8.29 (d, J=1.5 Hz, 1H), 8.11 (d, J=8.8 Hz, 1H), 8.08 (br s, 1H), 7.80 (dd, J=8.8, 1.5 Hz, 1H), 7.56 (s, 1H), 5.34 (s, 2H), 4.77 (br d, J=10.8 Hz, 1H), 4.30-4.17 (m, 1H), 3.10 (dd, J=9.8, 6.4 Hz, 1H), 3.07-2.95 (m, 2H), 2.68 (ddd, J=9.8, 5.9, 2.0 Hz, 1H), 2.42 (s, 3H), 2.40 (s, 3H).
Step 6. Synthesis of 1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1 (3) and 1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2 (4)
2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 0.92 mL, 1.5 mmol) was added to a 15° C. solution of C51 (210 mg, 0.492 mmol) in N,N-dimethylformamide (1 mL) and propyl acetate (4 mL). The reaction mixture was stirred for 14 hours at 110° C., whereupon it was cooled and treated with aqueous sodium bicarbonate solution (60 mL). The resulting mixture was extracted with ethyl acetate (3×60 mL), and the combined organic layers were concentrated in vacuo to provide a racemic mixture of 3 and 4 as a white solid. Yield of racemic product: 180 mg, 0.441 mmol, 90%.
This material was separated into its component enantiomers via supercritical fluid chromatography [Column: Regis Technologies, (S,S)-Whelk-0® 1, 10 μm; Mobile phase: 55:45 carbon dioxide/(2-propanol containing 0.1% ammonium hydroxide)]. The first-eluting product was designated as 3, and was obtained as a solid. Yield: 76.0 mg, 0.186 mmol, 42% for the separation. LCMS m/z 409.0 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 10.2-9.4 (v br s, 1H), 9.44 (s, 1H), 8.33 (d, J=8.8 Hz, 1H), 7.86 (dd, J=8.6, 1.7 Hz, 1H), 7.43 (s, 1H), 6.41-6.09 (m, 2H), 5.96 (d, J=15.6 Hz, 1H), 3.75-3.57 (br m, 1H), 3.70 (dd, J=11.7, 11.7 Hz, 1H), 3.17-3.03 (m, 1H), 3.15 (dd, J=11.2, 11.2 Hz, 1H), 2.65 (br s, 3H), 2.32 (s, 3H).
The second-eluting product, also isolated as a solid, was designated as 4. Yield: 68.6 mg, 0.168 mmol, 38% for the separation. LCMS m/z 409.1 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 10.1-9.5 (v br s, 1H), 9.44 (s, 1H), 8.33 (d, J=8.8 Hz, 1H), 7.86 (dd, J=8.8, 1.5 Hz, 1H), 7.43 (s, 1H), 6.36-6.10 (m, 2H), 5.96 (d, J=15.6 Hz, 1H), 3.75-3.57 (br m, 1H), 3.70 (dd, J=11.7, 11.2 Hz, 1H), 3.17-3.03 (m, 1H), 3.15 (dd, J=11.7, 11.2 Hz, 1H), 2.65 (br s, 3H), 2.32 (s, 3H).
Example 5
8-Chloro-1-[(4S)-3,3-difluorotetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline (5)
A 0° C. solution of P6 (75 mg, 0.24 mmol), (5-methyl-1,2-oxazol-3-yl)acetic acid (57.4 mg, 0.407 mmol), and N,N-diisopropylethylamine (0.11 mL, 0.63 mmol) in tetrahydrofuran (4 mL) was treated drop-wise with 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 0.28 mL, 0.47 mmol), and the reaction mixture was allowed to warm to room temperature overnight. The resulting solution was concentrated in vacuo, and the residue was dissolved in toluene (5 mL) and stirred at 110° C. for 72 hours, whereupon it was cooled to room temperature and partitioned between saturated aqueous sodium chloride solution and ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated. Silica gel chromatography (Gradient: 30% to 100% ethyl acetate in heptane) afforded the product as a pale tan foam. From analysis of the 1H NMR, this material was presumed to exist as a mixture of rotamers. Yield: 79 mg, 0.189 mmol, 79%. LCMS m/z 419.5 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CDCl3) δ [9.27 (s) and 9.27 (s), total 1H], [8.52 (br s) and 8.11 (br s), total 1H], [8.22 (d, J=9.0 Hz) and 8.19 (d, J=9.0 Hz), total 1H], 7.66-7.57 (m, 1H), [6.11 (s) and 6.05 (s), total 1H], 5.69-5.43 (m, 1H), [4.59 (AB quartet, JAB=16.8 Hz, ΔνAB=19.5 Hz) and 4.50 (AB quartet, JAB=15.8 Hz, ΔνAB=11.8 Hz), total 2H], 4.43-4.27 (m, 2H), 3.92-3.63 (m, 2H), [3.30-3.17 (m) and 3.17-3.04 (m), total 1H], [2.40 (s) and 2.38 (s), total 3H], [2.23-2.14 (m) and 1.95-1.85 (m), total 1H].
Example 6
2-[(6-Methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c] quinoline-8-carbonitrile, formate salt (6)
Step 1. Synthesis of lithium (6-methylpyrimidin-4-yl)acetate (C52)
n-Butyllithium (2.5 M in hexanes; 5.00 mL, 12.5 mmol) was slowly added dropwise to a −78° C. solution of 4,6-dimethylpyrimidine (1.08 g, 9.99 mmol) in tetrahydrofuran (20 mL). After the reaction mixture had been stirred for 20 minutes at −78° C., solid carbon dioxide (dry ice, 5.0 g) was added, and the reaction mixture was warmed to room temperature (15° C.) and stirred for 1 hour. Water (3.0 mL) was then added, and the resulting mixture was concentrated in vacuo to provide the product as a white solid. Yield: 1.53 g, 9.68 mmol, 97%. 1H NMR (400 MHz, D20) δ 8.78 (s, 1H), 7.28 (s, 1H), [3.60 (s) and 3.59 (br s), total 2H], 2.43 (s, 3H).
Step 2. Synthesis of 2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, formate salt (6)
This synthesis was carried out in library format. A mixture of P9 (100 μmol), C52 (130 μmol), and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 100 μL, 170 μmol) was treated with N,N-diisopropylethylamine (300 μmol) and 1,4-dioxane (1 mL), and the reaction vial was capped and shaken at 110° C. for 16 hours. After solvents had been removed using a Speedvac® concentrator, the residue was purified via reversed-phase HPLC (Column: Agela Durashell C18, 5 μm; Mobile phase A: 0.225% formic acid in water; Mobile phase B: acetonitrile; Gradient: 0% to 31% B) to afford the product. Yield: 1.5 mg, 3.5 μmol, 4%. LCMS m/z 384 [M+H]+. Retention time: 2.38 minutes (Conditions for analytical HPLC. Column: Waters XBridge C18, 2.1×50 mm, 5 μm; Mobile phase A: 0.05% ammonium hydroxide in water; Mobile phase B: acetonitrile; Gradient: 5% B for 0.5 minutes; 5% to 100% B over 2.9 minutes; 100% B for 0.8 minutes; Flow rate: 0.8 mL/minute).
Examples 7 and 8
8-Chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1 (7) and 8-Chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2 (8)
Step 1. Synthesis of N-{6-chloro-4-[(3,3-difluorotetrahydro-2H-pyran-4-yl)amino]quinolin-3-yl}-2-(5-methylpyrazin-2-yl)acetamide (C53)
1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (183 mg, 0.955 mmol) was added to a solution of P5 (150 mg, 0.478 mmol) and (5-methylpyrazin-2-yl)acetic acid (94.6 mg, 0.622 mmol) in pyridine (0.80 mL). The reaction mixture was stirred at 25° C. for 4 hours, whereupon it was combined with a similar reaction carried out using P5 (10.0 mg, 31.9 μmol), diluted with water (2 mL), and extracted with ethyl acetate (3×3 mL). The combined organic layers were concentrated in vacuo to afford the product as a brown oil, which was used directly in the following step. Combined yield: 214 mg, 0.478 mmol, 94%. LCMS m/z 448.2 [M+H]+.
Step 2. Synthesis of 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1 (7) and 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2 (8)
2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 608 mg, 0.955 mmol) was added to a 110° C. solution of C53 (214 mg, 0.478 mmol) in propyl acetate (1 mL), and the reaction mixture was stirred at 110° C. for 48 hours. It was then concentrated in vacuo and purified via silica gel chromatography (Gradient: 0% to 3% methanol in dichloromethane) to provide a racemic mixture of 7 and 8 as a yellow oil. Yield of racemic product: 150 mg, 0.349 mmol, 73%.
The enantiomers were separated using supercritical fluid chromatography ([Column: Chiral Technologies ChiralCel OD, 5 μm; Mobile phase: 7:3 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]; each enantiomer was then individually subjected to reversed-phase HPLC purification (Column: Agela Durashell, 5 μm; Mobile phase A: 0.05% ammonium hydroxide in water; Mobile phase B: acetonitrile; Gradient: 32% to 52% B). The first-eluting enantiomer was designated as 7, and the second-eluting enantiomer as 8. Both 7 and 8 were obtained as solids, and from analysis of the 1H NMR spectra, both were presumed to exist as a mixture of rotamers.
For 7, yield: 21.3 mg, 49.6 μmol, 14% for the separation. LCMS m/z 429.8 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CD3OD) δ [9.10 (s) and 9.06 (s), total 1H], 8.72-8.42 (m, 3H), [8.17 (d, J=8.8 Hz) and 8.15 (d, J=8.8 Hz), total 1H], 7.76-7.68 (m, 1H), [6.11-5.96 (m) and 5.96-5.80 (m), total 1H], 4.9-4.66 (m, 2H, assumed; partially obscured by water peak), 4.39-4.17 (m, 2H), 4.08-3.77 (m, 2H), [3.35-3.21 (m) and 3.17-3.04 (m), total 1H, assumed; partially obscured by solvent peak], [2.57 (s) and 2.54 (s), total 3H], [2.42-2.33 (m) and 2.32-2.21 (m), total 1H].
For 8, yield: 32.6 mg, 75.8 μmol, 22% for the separation. LCMS m/z 429.7 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CD3OD) δ [9.10 (s) and 9.06 (s), total 1H], 8.71-8.43 (m, 3H), [8.17 (d, J=8.8 Hz) and 8.15 (d, J=9 Hz), total 1H], 7.76-7.69 (m, 1H), [6.10-5.96 (m) and 5.96-5.81 (m), total 1H], 4.9-4.67 (m, 2H, assumed; partially obscured by water peak), 4.39-4.17 (m, 2H), 4.08-3.77 (m, 2H), [3.35-3.21 (m) and 3.17-3.04 (m), total 1H, assumed; partially obscured by solvent peak], [2.57 (s) and 2.54 (s), total 3H], [2.42-2.33 (m) and 2.32-2.22 (m), total 1H].
Example 9
1-[(2R,4R)-2-Methyltetrahydro-2H-pyran-4-yl]-2-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline (9)
Step 1. Synthesis of (1-methyl-1H-1,2,3-triazol-4-yl)methanol (C54)
Lithium aluminum hydride (685 mg, 18.0 mmol) was added to a 0° C. suspension of ethyl 1-methyl-1H-1,2,3-triazole-4-carboxylate (1.40 g, 9.02 mmol) in tetrahydrofuran (20 mL) and the reaction mixture was stirred at 0° C. for 1 hour. Water was then added drop-wise at 0° C. until no further gas evolution was observed, whereupon sodium sulfate was added, and the mixture was stirred for 10 minutes. The mixture was then filtered, and the filtrate was concentrated in vacuo, affording the product as a yellow oil. Yield: 700 mg, 6.19 mmol, 69%. 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 5.15 (t, J=5.5 Hz, 1H), 4.49 (d, J=5.5 Hz, 2H), 4.01 (s, 3H).
Step 2. Synthesis of (1-methyl-1H-1,2,3-triazol-4-yl)methyl methanesulfonate (C55)
Methanesulfonyl chloride (851 mg, 7.43 mmol) was added to a 0° C. solution of C54 (700 mg, 6.19 mmol) and triethylamine (1.00 g, 9.88 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at 0° C. for 2 hours, whereupon water (100 mL) was added, and the mixture was extracted with dichloromethane (2×100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to provide the product as a yellow oil, which was used directly in the next step. Yield: 800 mg, 4.18 mmol, 68%.
Step 3. Synthesis of (1-methyl-1H-1,2,3-triazol-4-yl)acetonitrile (C56)
To a solution of C55 (800 mg, 4.18 mmol) in acetonitrile (20 mL) was added potassium cyanide (1.50 g, 23.0 mmol). The reaction mixture was stirred at 60° C. overnight, whereupon it was treated with water (150 mL) and extracted with dichloromethane (3×100 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (80 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to afford the product as a brown solid. Yield: 200 mg, 1.64 mmol, 39%. 1H NMR (400 MHz, CDCl3) δ 7.61 (s, 1H), 4.13 (s, 3H), 3.89 (br s, 2H).
Step 4. Synthesis of (1-methyl-1H-1,2,3-triazol-4-yl)acetic acid (C57)
A solution of C56 (200 mg, 1.64 mmol) in concentrated hydrochloric acid (4 mL) was stirred at 60° C. for 2 hours. After the reaction mixture had cooled to room temperature, it was diluted with water (10 mL) and washed with tert-butyl methyl ether (2×20 mL). The aqueous layer was then concentrated to dryness, providing the product as a brown solid. Yield: 200 mg, 1.42 mmol, 87%. LCMS m/z 142.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 1H), 4.01 (s, 3H), 3.66 (s, 2H).
Step 5. Synthesis of N-[4-{[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]amino}-6-(trifluoromethyl)quinolin-3-yl]-2-(1-methyl-1H-1,2,3-triazol-4-yl)acetamide (C58)
1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (118 mg, 0.615 mmol) was added in one portion to a solution of P7 (100 mg, 0.307 mmol) and C57 (52.1 mg, 0.369 mmol) in pyridine (0.8 mL), and the reaction mixture was stirred at 25° C. for 16 hours. It was then poured into water (50 mL) and extracted with ethyl acetate (3×30 mL); the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to afford the product as a red oil (160 mg), which was used directly in the following step. LCMS m/z 449.2 [M+H]+.
Step 6. Synthesis of 1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-8-(trifluoromethyl)-1H-imidazo[4,5-c]quinoline (9)
2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (1.6 M solution in ethyl acetate; 0.669 mL, 1.07 mmol) was added to a solution of C58 (from the previous step; ≤0.307 mmol) in N,N-dimethylformamide (1 mL) and propyl acetate (4 mL). The reaction mixture was stirred at 110° C. for 16 hours, whereupon it was poured into water (40 mL) and extracted with ethyl acetate (2×30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 1.5% methanol in dichloromethane), followed by reversed-phase HPLC (Column: Agela Durashell C18, 5 μm; Mobile phase A: 0.05% ammonium hydroxide in water; Mobile phase B: acetonitrile; Gradient: 5% to 95% B) afforded the product as a solid. Yield: 29.5 mg, 68.5 μmol, 22% over two steps. LCMS m/z 431.1 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.35 (s, 1H), 9.13-8.89 (br s, 1H), 8.38 (d, J=9.0 Hz, 1H), 7.86 (br d, J=8.5 Hz, 1H), 7.64-7.54 (br s, 1H), 5.53-5.38 (m, 1H), 4.62 (s, 2H), 4.29 (dd, J=12.0, 5.0 Hz, 1H), 4.07 (s, 3H), 3.83-3.68 (m, 2H), 2.77-2.57 (m, 1H), 2.50-2.31 (m, 1H), 2.0-1.59 (m, 2H, assumed; partially obscured by water peak), 1.32 (d, J=6.5 Hz, 3H).
Examples 10 and 11
[cis-4-(8-Chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 1 (10) and [cis-4-(8-Chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 2 (11)
Step 1. Synthesis of N-[4-({cis-2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}amino)-6-chloroquinolin-3-yl]cyclobutanecarboxamide (C59)
1-[3-(Dimethylamino)propy]-3-ethylcarbodiim ide hydrochloride (771 mg, 4.02 mmol) was added to a solution of P3 (800 mg, 2.01 mmol) and cyclobutanecarboxylic acid (221 mg, 2.21 mmol) in pyridine (20 mL). The reaction mixture was stirred at 25° C. for 40 hours, whereupon it was concentrated in vacuo and partitioned between water (80 mL) and ethyl acetate (80 mL). The aqueous layer was extracted with ethyl acetate (80 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide the product as a foamy, orange solid (1.01 g), which was used directly in the following step. LCMS m/z 479.9 (chlorine isotope pattern observed) [M+H]+.
Step 2. Synthesis of 1-{cis-2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}-8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinoline (C60)
A solution of C59 (from the previous step; 2.01 mmol) in acetic acid (20 mL) was stirred at 110° C. for 16 hours. This was combined with a similar reaction carried out using C59 (154 mg, 0.321 mmol) and concentrated in vacuo. The residue was mixed with half-saturated aqueous sodium bicarbonate solution (100 mL) and extracted with ethyl acetate (100 mL); the organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford the product as a yellow solid. Combined yield: 1.07 g, 2.32 mmol, quantitative over two steps. LCMS m/z 462.0 (chlorine isotope pattern observed) [M+H]+.
Step 3. Synthesis of [cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]methanol (C61)
Boron trichloride (1 M solution; 6.95 mL, 6.95 mmol) was added in portions to a 10° C. solution of C60 (1.07 g, 2.32 mmol) in dichloromethane (30 mL). The reaction mixture was stirred at 25° C. for 1 hour, whereupon it was poured into saturated aqueous sodium bicarbonate solution (80 mL) and extracted with dichloromethane (2×50 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and purified using silica gel chromatography (Gradient: 0% to 2% methanol in dichloromethane) to provide the product as an off-white solid. Yield: 643 mg, 1.73 mmol, 75%. LCMS m/z 371.9 (chlorine isotope pattern observed) [M+H]+.
Step 4. Synthesis of [cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]methyl methanesulfonate (C62)
Triethylamine (525 mg, 5.19 mmol) and methanesulfonyl chloride (0.160 mL, 2.07 mmol) were added to a solution of C61 (643 mg, 1.73 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at 25° C. for 1 hour, whereupon it was poured into water (50 mL) and extracted with ethyl acetate (2×50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to provide the product as a foamy, light yellow solid. Yield: 750 mg, 1.67 mmol, 96%. LCMS m/z 449.8 (chlorine isotope pattern observed) [M+H]+.
Step 5. Synthesis of [cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 1 (10) and [cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 2 (11)
Tetraethylammonium cyanide (781 mg, 5.00 mmol) was added to a solution of C62 (750 mg, 1.67 mmol) in dimethyl sulfoxide (15 mL), and the reaction mixture was heated at 80° C. for 16 hours. It was then diluted with tert-butyl methyl ether (100 mL), and washed sequentially with water (2×50 mL) and saturated aqueous sodium chloride solution (50 mL). The combined aqueous layers were extracted with tert-butyl methyl ether (50 mL), whereupon the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 2% methanol in dichloromethane) afforded a racemic mixture of 10 and 11 as a light yellow, foamy solid. Yield of racemic product: 613 mg, 1.61 mmol, 96%.
A portion of this material (300 mg, 0.788 mmol) was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AS, 5 μm; Mobile phase: 3:2 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first-eluting enantiomer was designated as 10, and was obtained as a solid. Yield: 91.1 mg, 0.239 mmol, 30% for the separation. LCMS m/z 381.0 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.14 (s, 1H), 8.72-8.55 (br s, 1H), 8.17 (d, J=8.5 Hz, 1H), 7.71 (dd, J=9.0, 2.0 Hz, 1H), 5.23-4.97 (v br m, 1H), 4.36 (dd, J=11.8, 5.3 Hz, 1H), 4.18-4.08 (m, 1H), 4.03-3.95 (m, 1H), 3.86 (ddd, J=12.0, 12.0, 2.5 Hz, 1H), 2.88 (dd, half of ABX pattern, J=17.1, 4.0 Hz, 1H), 2.77 (dd, half of ABX pattern, J=17.1, 6.5 Hz, 1H), 2.73-2.42 (m, 6H), 2.33-1.93 (m, 4H).
The second-eluting enantiomer, also isolated as a solid, was designated as 11. Yield: 93.9 mg, 0.247 mmol, 31% for the separation. LCMS m/z 381.0 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.14 (s, 1H), 8.72-8.54 (br s, 1H), 8.17 (d, J=9 Hz, 1H), 7.71 (d, J=9 Hz, 1H), 5.25-4.96 (v br m, 1H), 4.36 (dd, J=12, 5 Hz, 1H), 4.19-4.07 (m, 1H), 4.03-3.95 (m, 1H), 3.86 (br dd, J=12, 12 Hz, 1H), 2.88 (dd, half of ABX pattern, J=17.1, 4.0 Hz, 1H), 2.77 (dd, half of ABX pattern, J=17.1, 6.0 Hz, 1H), 2.73-2.42 (m, 6H), 2.33-1.92 (m, 4H).
Example 12
8-(Difluoromethyl)-2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline (12)
Step 1. Synthesis of ethyl (4-methoxy-1H-pyrazol-1-yl)acetate (C63)
Ethyl bromoacetate (5.46 g, 32.7 mmol) was added in one portion to a mixture of 4-methoxy-1H-pyrazole, hydrochloride salt (4.00 g, 29.7 mmol) and potassium carbonate (8.62 g, 62.4 mmol) in N,N-dimethylformamide (40 mL) at room temperature (30° C.). The reaction mixture was stirred at room temperature (30° C.) for 16 hours, whereupon it was diluted with water (200 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (2×150 mL), dried over sodium sulfate, filtered and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 30% ethyl acetate in petroleum ether) afforded the product as a colorless oil. Yield: 4.45 g, 24.2 mmol, 81%. 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J=0.8 Hz, 1H), 7.15 (d, J=0.8 Hz, 1H), 4.80 (s, 2H), 4.24 (q, J=7.2 Hz, 2H), 3.76 (s, 3H), 1.29 (t, J=7.2 Hz, 3H).
Step 2. Synthesis of (4-methoxy-1H-pyrazol-1-yl)acetic acid (C64)
Aqueous sodium hydroxide solution (2 M; 24.2 mL, 48.4 mmol) was added in one portion to a solution of C63 (4.45 g, 24.2 mmol) in tetrahydrofuran (30 mL) at room temperature (29° C.), and the reaction mixture was stirred at room temperature (29° C.) for 3 hours. It was then concentrated under reduced pressure, diluted with water (50 mL), and extracted with dichloromethane (2×30 mL). The organic layers were discarded, and the aqueous layer was acidified to pH 1 with 1 M hydrochloric acid, and extracted with ethyl acetate (4×50 mL). After the combined ethyl acetate layers had been dried over sodium sulfate, they were filtered and concentrated in vacuo, providing the product as a white solid. Yield: 2.80 g, 17.9 mmol, 74%. 1H NMR (400 MHz, DMSO-d6) δ 7.44 (s, 1H), 7.21 (s, 1H), 4.80 (s, 2H), 3.65 (s, 3H).
Step 3. Synthesis of 8-(difluoromethyl)-2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline (12)
To a solution of P11 (50 mg, 0.16 mmol) in toluene (1.5 mL) were added C64 (26.7 mg, 0.171 mmol) and N,N-diisopropylethylamine (31.2 μL, 0.179 mmol), followed by 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 0.107 mL, 0.180 mmol). The reaction mixture was heated at 60° C. for 90 minutes, and then at 100° C. for 4 hours, whereupon it was partitioned between ethyl acetate (10 mL) and saturated aqueous sodium bicarbonate solution (10 mL). The organic layer was dried over sodium sulfate, filtered, concentrated in vacuo, and purified via silica gel chromatography (Gradient: 0% to 15% methanol in dichloromethane), providing the product as an off-white solid. Yield: 51 mg, 0.12 mmol, 75%. LCMS m/z 428.5 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.28 (s, 1H), 8.98-8.81 (br s, 1H), 8.34 (d, J=8.6 Hz, 1H), 7.92 (d, J=9.0 Hz, 1H), 7.48 (s, 1H), 7.30 (s, 1H), 7.08 (t, JHF=56.0 Hz, 1H), 5.82 (s, 2H), 5.44-5.29 (br m, 1H), 4.23 (dd, J=11.7, 5.1 Hz, 1H), 3.81-3.66 (m, 2H), 3.71 (s, 3H), 2.76-2.55 (br m, 1H), 2.47-2.24 (br m, 1H), 1.90-1.56 (br m, 2H), 1.28 (d, J=6.3 Hz, 3H).
Example 13
8-(Difluoromethyl)-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyl tetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline (13)
Reaction of P11 (50 mg, 0.16 mmol) with (5-methylpyrazin-2-yl)acetic acid was carried out using the method described for synthesis of 12 from P11 in Example 12. In this case, silica gel chromatography was carried out twice (Gradient: 0% to 15% methanol in dichloromethane), affording the product as a light orange solid. Yield: 39 mg, 92 μmol, 58%. LCMS m/z 424.5 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.19 (s, 1H), 9.03-8.87 (br s, 1H), 8.64 (s, 1H), 8.48 (s, 1H), 8.33 (d, J=8.6 Hz, 1H), 7.90 (d, J=8.6 Hz, 1H), 7.08 (t, JHF=56.0 Hz, 1H), 5.51-5.31 (br m, 1H), 4.80 (s, 2H), 4.26 (dd, J=12.1, 5.1 Hz, 1H), 3.84-3.66 (m, 2H), 2.84-2.65 (br m, 1H), 2.55 (s, 3H), 2.52-2.35 (br m, 1H), 2.13-1.84 (br m, 2H), 1.31 (d, J=5.9 Hz, 3H).
Examples 14 and 15
{8-Chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-methylpyrazin-2-yl)methanol, DIAST 1 (14) and {8-Chloro-1-[(2R,4R)-2-methyl tetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-methylpyrazin-2-yl) methanol, DIAST 2 (15)
Step 1. Synthesis of 8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline (C65)
Formic acid (310 mL) was added to a mixture of iron powder (34.7 g, 621 mmol), ammonium chloride (33.2 g, 621 mmol), and C32 (20 g, 62.2 mmol) in 2-propanol (310 mL) at room temperature (14° C.). The reaction mixture was heated at 80° C. for 16 hours, whereupon it was diluted with ethanol (300 mL), and filtered. The collected solids were washed with 2-propanol (200 mL) and dichloromethane (100 mL), and the combined filtrates were concentrated in vacuo, then co-evaporated with ethanol (200 mL). The residue was diluted with dichloromethane (300 mL), basified via addition of saturated aqueous sodium bicarbonate solution (500 mL), and then filtered through diatomaceous earth; the filter pad was washed with dichloromethane (300 mL). The aqueous layer of the combined filtrates was extracted with dichloromethane (4×100 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Silica gel chromatography (Gradient: 0% to 5% methanol in dichloromethane) afforded a solid, which was washed with a mixture of petroleum ether and ethyl acetate (3:1, 100 m L) and with petroleum ether (50 m L) to provide the product as a beige solid. Yield: 10.05 g, 33.3 mmol, 54%. LCMS m/z 301.8 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.35 (s, 1H), 8.25 (d, J=9.0 Hz, 1H), 8.19 (s, 1H), 8.09 (d, J=2.3 Hz, 1H), 7.66 (dd, J=8.8, 2.3 Hz, 1H), 5.02 (tt, J=12.0, 3.8 Hz, 1H), 4.30 (ddd, J=11.9, 4.6, 1.6 Hz, 1H), 3.77-3.89 (m, 2H), 2.33-2.46 (m, 2H), 2.09-2.22 (m, 1H), 1.83-1.95 (m, 1H), 1.38 (d, J=6.3 Hz, 3H).
Step 2. Synthesis of {8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-methylpyrazin-2-yl)methanol, DIAST 1 (14) and {8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-methylpyrazin-2-yl)methanol, DIAST 2 (15)
A vial was charged with C65 (100 mg, 0.33 mmol), and the vial was evacuated and flushed with nitrogen; this procedure was repeated twice, tetrahydrofuran (1.6 mL) was added, and the solution was cooled to −78° C. 2,2,6,6-Tetramethylpiperidinylmagnesium chloride, lithium chloride complex (1 M solution in tetrahydrofuran and toluene; 0.497 mL, 0.497 mmol) was added, and the reaction mixture was allowed to stir for 1 hour at −78° C. In a separate vial, 5-methylpyrazine-2-carbaldehyde (80.9 mg, 0.662 mmol) was dissolved in tetrahydrofuran (1.6 mL), and the resulting solution was cooled in a dry ice/acetone bath for 10 minutes. This solution was then added to the reaction mixture, which was subsequently allowed to stir while slowly warming to 15° C. After 1 hour, it was combined with two similar reaction mixtures derived from C65 (50 mg, 0.17 mmol; 100 mg, 0.33 mmol), and the resulting mixture was diluted with water (20 mL) and extracted with ethyl acetate (3×15 mL). The combined organic layers were concentrated in vacuo and subjected to reversed-phase HPLC (Column: Phenomenex Synergi Max-RP, 10 μm; Mobile phase A: 0.1% trifluoroacetic acid in water; Mobile phase B: acetonitrile; Gradient: 15% to 45% B), affording a diastereomeric mixture of 14 and 15 as a viscous, brick-red oil. Combined yield of diastereomeric mixture: 180 mg, 0.425 mmol, 51%.
This material was separated into its component diastereomers via supercritical fluid chromatography [Column: Regis Technologies, (S,S)-Whelk-0® 1, 10 μm; Mobile phase: 3:2 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first-eluting diastereomer, obtained as a light yellow solid, was designated as 14. Yield: 58.6 mg, 0.138 mmol, 32% for the separation. LCMS m/z 423.9 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.12 (br s, 1H), 8.92 (s, 1H), 8.83-8.74 (br s, 1H), 8.48 (s, 1H), 8.18 (d, J=9.0 Hz, 1H), 7.74 (dd, J=9.0, 2.0 Hz, 1H), 6.51 (s, 1H), 5.58-5.46 (m, 1H), 4.29 (dd, J=11.8, 5.3 Hz, 1H), 3.80-3.66 (br m, 1H), 3.66-3.52 (br m, 1H), 2.79-2.66 (m, 1H), 2.60 (s, 3H), 2.42-2.27 (br m, 1H), 2.13-2.00 (br m, 1H), 1.77-1.63 (br m, 1H), 1.28 (br d, J=5.5 Hz, 3H).
The second-eluting diastereomer, also isolated as a light yellow solid, was designated as 15. Yield: 56.8 mg, 0.134 mmol, 32% for the separation. LCMS m/z 423.9 (chlorine isotope pattern observed) [M+H]+. 1H NMR (400 MHz, CD3OD) δ 9.12 (br s, 1H), 8.92 (s, 1H), 8.82-8.74 (br s, 1H), 8.47 (br s, 1H), 8.18 (d, J=8.5 Hz, 1H), 7.74 (dd, J=9.0, 2.0 Hz, 1H), 6.50 (s, 1H), 5.57-5.46 (m, 1H), 4.21 (dd, J=11.8, 4.8 Hz, 1H), 3.82-3.70 (br m, 1H), 3.62-3.47 (br m, 1H), 2.71-2.57 (br m, 1H), 2.59 (s, 3H), 2.48-2.35 (m, 1H), 2.24-2.13 (br m, 1H), 1.63-1.50 (br m, 1H), 1.34 (d, J=6.0 Hz, 3H).
Examples 16 and 17
1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1 (16) and 1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2 (17)
This reaction was carried out in library format. N,N-Diisopropylethylamine (52 μL, 30 μmol) was added to a mixture of 1H-1,2,4-triazol-1-ylacetic acid (100 μmol) and P12 (29.6 mg, 100 μmol) in a 3:2 mixture of ethyl acetate and toluene (0.5 mL). 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 0.19 mL, 0.32 mmol) was added, and the reaction vial was shaken and heated at 70° C. for 10 hours, then at 110° C. for 3 hours. It was then partitioned between half-saturated aqueous sodium bicarbonate solution (1.5 mL) and ethyl acetate (2.4 mL) and subjected to vortexing. The organic layer was eluted through a solid phase extraction cartridge (6 mL) charged with sodium sulfate (˜1 g); this extraction procedure was repeated twice, and the combined eluents were concentrated in vacuo. Purification via reversed-phase HPLC (Column: Waters Sunfire C18, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water; Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 5% B for 1.0 minute, followed by 5.0% to 75% B over 7.5 minutes, followed by 75% to 100% B) provided a racemic mixture of the two products. Separation into the component enantiomers was carried out using supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 85:15 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting enantiomer was designated as 16. Yield: 4.9 mg, 13 μmol, 13%. LCMS m/z 388.5 [M+H]+. Retention time: 2.91 minutes [Analytical conditions, Column: Chiral Technologies Chiralpak AD-H, 4.6×100 mm, 5 μm; Mobile phase: 80:20 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute].
The second-eluting enantiomer was designated as 17. Yield: 2.0 mg, 5.2 μmol, 5%. LCMS m/z 388.3 [M+H]+. Retention time: 3.31 minutes, using the same analytical conditions.
Examples 18 and 19
1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1 (18) and 1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2 (19)
(4-Methyl-1H-1,2,3-triazol-1-yl)acetic acid and P12 were used to generate a racemic mixture of 18 and 19, using the method described in Examples 16 and 17. Separation into the component enantiomers was carried out using supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 3:2 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting enantiomer was designated as 18. Yield: 4.0 mg, 10 μmol, 10%. LCMS m/z 402.8 [M+H]+. Retention time: 1.68 minutes [Analytical conditions, Column: Chiral Technologies Chiralpak AD-H, 4.6×100 mm, 5 μm; Mobile phase: 3:2 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute].
The second-eluting enantiomer was designated as 19. Yield: 3.7 mg, 9.2 μmol, 9%. LCMS m/z 402.6 [M+H]+. Retention time: 4.1 minutes, using the same analytical conditions.
Examples 20 and 21
1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1 (20) and 1-(4,4-Difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2 (21)
(5-Methylpyrazin-2-yl)acetic acid and P12 were used to generate a racemic mixture of 20 and 21, using the method described in Examples 16 and 17. Separation into the component enantiomers was carried out using supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 85:15 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting enantiomer was designated as 20. Yield: 2.0 mg, 4.8 μmol, 5%. LCMS m/z 413.9 [M+H]+. Retention time: 2.66 minutes [Analytical conditions, Column: Chiral Technologies Chiralpak AD-H, 4.6×100 mm, 5 μm; Mobile phase: 80:20 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 200 bar; Flow rate: 1.5 mL/minute].
The second-eluting enantiomer was designated as 21. Yield: 1.8 mg, 4.4 μmol, 4%. LCMS m/z 413.9 [M+H]+. Retention time: 3.3 minutes, using the same analytical conditions.
Examples 22 and 23
8-Chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 1 (22) and 8-Chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2 (23)
This reaction was carried out in library format. N,N-Diisopropylethylamine (52 μL, 300 μmol) was added to a mixture of [4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]acetic acid (this may be synthesized according to the method described by M. D. Andrews et al., PCT International Application WO 2014053967 A1, Apr. 10, 2014; 100 μmol) and P13 (31.2 mg, 100 μmol) in a 3:2 mixture of ethyl acetate and toluene (0.5 mL). 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 0.19 mL, 0.32 mmol) was then added, and the reaction vial was shaken and heated at 70° C. for 10 hours, then at 110° C. for 3 hours. The reaction mixture was then partitioned between half-saturated aqueous sodium bicarbonate solution (1.5 mL) and ethyl acetate (2.4 mL) and subjected to vortexing. The organic layer was eluted through a solid phase extraction cartridge (6 mL) charged with sodium sulfate (˜1 g); this extraction procedure was repeated twice, and the combined eluents were concentrated in vacuo. Purification via reversed-phase HPLC (Column: Waters Sunfire C18, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water; Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile; Gradient: 5% B for 1.0 minute, followed by 5.0% to 75% B over 7.5 minutes, followed by 75% to 100% B) provided a racemic mixture of the two products. Separation into the component enantiomers was carried out using supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 3:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting enantiomer was designated as 22. Yield: 4.9 mg, 11 μmol, 11%. LCMS m/z 447.9 [M+H]+. Retention time: 2.4 minutes [Analytical conditions, Column: Chiral Technologies Chiralpak AD-H, 4.6×100 mm, 5 μm; Mobile phase: 3:2 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 m L/minute].
The second-eluting enantiomer was designated as 23. Yield: 4.8 mg, 11 μmol, 11%. LCMS m/z 448.2 (chlorine isotope pattern observed) [M+H]+. Retention time: 2.95 minutes, using the same analytical conditions.
Examples 24 and 25
8-Chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1 (24) and 8-Chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2 (25)
1H-1,2,4-Triazol-1-ylacetic acid and P13 were used to generate a racemic mixture of 24 and 25, using the method described in Examples 22 and 23. Separation into the component enantiomers was carried out using supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 85:15 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. In this case, the enantiomers were not fully separated, but the samples described are enriched in the indicated enantiomer. The first-eluting enantiomer was designated as 24. Yield: 2.3 mg, 5.7 μmol, 6%. LCMS m/z 404.5 (chlorine isotope pattern observed) [M+H]+. Retention time: 3.7 minutes [Analytical conditions, Column: Chiral Technologies Chiralpak AD-H, 4.6×100 mm, 5 μm; Mobile phase: 75:25 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute].
The second-eluting enantiomer was designated as 25. Yield: 1.0 mg, 2.5 μmol, 2%. LCMS m/z 403.9 [M+H]+. Retention time: 3.9 minutes, using the same analytical conditions.
Examples 26 and 27
8-Chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methoxy-1H-pyrazol-1-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 1 (26) and 8-Chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2 (27)
This reaction was carried out in library format. N,N-Diisopropylethylamine (52 μL, 300 μmol) was added to a mixture of C64 (100 μmol) and P5 (31.2 mg, 99 μmol) in a 3:2 mixture of ethyl acetate and toluene (0.5 mL). 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 0.19 mL, 0.32 mmol) was then added, and the reaction vial was shaken and heated at 70° C. for 2 hours, then at 110° C. for 6 hours. The reaction mixture was then partitioned between half-saturated aqueous sodium bicarbonate solution (1.5 mL) and ethyl acetate (2.4 mL) and subjected to vortexing. The organic layer was eluted through a solid phase extraction cartridge (6 mL) charged with sodium sulfate (˜1 g); this extraction procedure was repeated twice, and the combined eluents were concentrated in vacuo. Purification via reversed-phase HPLC (Column: Waters XBridge C18, 5 μm; Mobile phase A: 0.03% ammonium hydroxide in water; Mobile phase B: 0.03% ammonium hydroxide in acetonitrile; Gradient: 5% to 100% B) provided a racemic mixture of the two products. Separation into the component enantiomers was carried out using supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OJ-H, 5 μm; Mobile phase: 92:8 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting enantiomer was designated as 26. Yield: 1.8 mg, 4.1 μmol, 4%. LCMS m/z 434.5 (chlorine isotope pattern observed) [M+H]+. Retention time: 1.98 minutes [Analytical conditions, Chiral Technologies Chiralcel OJ-H, 4.6×100 mm, 5 μm; Mobile phase: 90:10 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute].
The second-eluting enantiomer was designated as 27. Yield: 1.8 mg, 4.1 μmol, 4%. LCMS m/z 435.5 [M+H]+. Retention time: 2.25 minutes, using the same analytical conditions.
TABLE 1
Method of preparation, structure, and physicochemical data for Examples 28-55.
1H NMR (400 MHz,
CDCl3) δ; Mass
Method of
spectrum, observed ion
Preparation;
m/z [M + H]+ or HPLC
Non-
retention time; Mass
commercial
spectrum m/z [M + H]+
Example
starting
(unless otherwise
Number
materials
Structure
indicated)
28
Example 61
2.33 minutes2; 437
29
P73
9.39 (s, 1H), 9.09-8.91 (br s, 1H), 8.60 (s, 1H), 8.44-8.36 (m, 2H), 7.88 (dd, J = 8.8, 1.3 Hz, 1H), 5.39-5.23 (m, 1H), 4.68 (s, 2H), 4.30 (dd, J = 12.1, 5.1 Hz, 1H), 3.77-3.62 (m, 2H), 2.82-2.61 (br m, 1H), 2.57 (s, 3H), 2.54- 2.36 (br m, 1H), 1.97-1.6 (br m, 2H, assumed; partially obscured by water peak), 1.33 (d, J = 6.2 Hz, 3H); 442.0
30
Examples 10 and 114; P8
2.80 minutes2; 361
31
Examples 1 and 25; P3
1H NMR (400 MHz, CD3OD) δ 9.08 (s, 1H), 8.8-8.4 (v br s, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 5.6- 5.1 (v br s, 1H), 4.42- 4.30 (m, 1H), 4.06-3.96 (m, 1H), 3.88 (br dd, J = 12, 12 Hz, 1H), 2.92- 2.83 (m, 1H), 2.86 (s, 3H), 2.78 (dd, half of ABX pattern, J = 17.1, 6.5 Hz, 1H), 2.75-2.37 (v br m, 2H), 2.33-2.19 (br m, 1H), 2.17-2.05 (br m, 1H); 340.9 (chlorine isotope pattern
observed)
32
Examples 3 and 46; P4
10.0-9.45 (v br s, 1H), 9.40 (s, 1H), 8.33 (d, J = 8.8 Hz, 1H), 7.85 (dd, J = 8.8, 1.8 Hz, 1H), 5.95- 5.78 (m, 1H), 5.02-4.78 (br m, 1H), 4.57 (d, J = 16.3 Hz, 1H), 3.70- 3.61 (m, 1H), 3.69 (dd, J = 11.9, 11.4 Hz, 1H), 3.23 (dd, J = 11.4, 11.4 Hz, 1H), 3.08 (ddd, J = 23.8, 11.0, 7.0 Hz,
1H), 2.64 (br s, 3H), 2.60
(s, 3H); 409.8
33
Examples 7 and 8; P9
10.33-10.20 (br s, 1H), 9.38 (s, 1H), 8.58 (br s, 1H), 8.39 (br s, 1H), 8.31 (d, J = 8.8 Hz, 1H), 7.82 (dd, J = 8.6, 1.7 Hz, 1H), 5.77-5.65 (m, 1H), 4.72 (s, 2H), 3.43 (dd, J = 8.8, 6.8 Hz, 1H), 3.37 (dd, J = 11.2, 4.4 Hz, 1H), 2.80 (dd, J = 10.8, 10.8 Hz, 1H), 2.62-2.53 (m, 1H), 2.57 (s, 6H), 2.52-
2.40 (m, 1H), 2.25-2.15
(m, 1H); 384.2
34
Example 67; P9
2.50 minutes8; 374
35
Example 6; P9
1.98 minutes2; 373
36
Example 6; P9, C64
2.56 minutes8; 388
37
Examples 3 and 4; P9
10.15-9.86 (v br s, 1H), 9.40 (s, 1H), 8.34 (d, J = 8.5 Hz, 1H), 7.86 (dd, J = 9.0, 1.5 Hz, 1H), 5.92- 5.77 (br m, 1H), 5.04 (AB quartet, downfield doublet is broadened, JAB = 16.6 Hz, ΔνAB = 22.7 Hz, 2H), 3.47 (dd, J = 8.5, 8.5 Hz, 1H), 3.40 (dd, J = 11.0, 5.0 Hz, 1H),
3.14-2.96 (br m, 1H),
2.89-2.73 (m, 1H), 2.77
(s, 3H), 2.64 (br s, 3H),
2.55-2.31 (m, 2H); 390.0
38
Examples 3 and 49; P9
10.33-10.19 (br s, 1H), 9.40 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.83 (dd, J = 8.8, 1.3 Hz, 1H), 6.71 (br s, 1H), 5.66-5.55 (m, 1H), 4.69 (AB quartet, JAB = 16.7 Hz, ΔνAB = 12.2 Hz, 2H), 3.43 (dd, J = 8.8, 7.5 Hz, 1H), 3.38 (dd, J = 11.2, 4.2 Hz, 1H), 2.83 (dd, J = 11.0, 10.6 Hz, 1H), 2.62-2.54 (m,
1H), 2.57 (s, 3H), 2.51-
2.39 (m, 1H), 2.31-2.21
(m, 1H), 2.29 (br s, 3H);
373.0
39
Example 6; P8
2.94 minutes2; 453
40
Examples 3 and 410; C7, C52
9.66-9.52 (br s, 1H), 9.26 (s, 1H), 9.04 (br s, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.61 (dd, J = 9.0, 2.5 Hz, 1H), 7.21 (br s, 1H), 5.71-5.59 (m, 1H), 4.67 (AB quartet, downfield doublet is broadened, JAB = 15.8 Hz, ΔνAB =11.1 Hz, 2H), 3.39-3.30 (m, 2H), 2.76 (dd, J = 10.5,
10.5 Hz, 1H), 2.58 (ddd,
half of ABXY pattern,
J = 11.0, 9.0, 5.5 Hz, 1H),
2.54-2.41 (m, 1H), 2.51
(s, 3H), 2.50 (s, 3H),
2.26-2.14 (br m, 1H);
393.0 (chlorine isotope
pattern observed)
41
P7, C643
9.43 (s, 1H), 9.07-8.91 (br s, 1H), 8.42 (d, J = 8.8 Hz, 1H), 7.91 (dd, J = 8.6, 1.5 Hz, 1H), 7.29 (s, 1H), 7.14 (s, 1H), 5.72 (s, 2H), 5.45-5.33 (m, 1H), 4.27 (dd, J = 12.1, 5.1 Hz, 1H), 3.77-3.62 (m, 2H), 3.67 (s, 3H), 2.71-2.54 (br m, 1H), 2.45-2.28 (br m, 1H), 1.73-1.42 (br m, 2H, assumed; partially obscured by water
peak), 1.31 (d, J = 6.2 Hz,
3H); 446.1
42
Examples 3 and 410
1H NMR (400 MHz, CD3OD) δ 9.89-9.76 (br s, 1H), 9.08 (s, 1H), 8.13 (d, J = 9.2 Hz, 1H), 7.70 (dd, J = 8.8, 2.2 Hz, 1H), 5.79-5.67 (m, 1H), 4.80 (s, 2H), 3.43-3.33 (m, 2H), 2.93 (dd, J = 11.0, 10.6 Hz, 1H), 2.66-2.55 (m, 1H), 2.59 (s, 3H), 2.54-2.43 (m, 2H), 2.52 (s, 3H); 383.0 (chlorine
isotope pattern
observed)
43
Example 12; P13
3.00 minutes11; 404.2 (chlorine isotope pattern observed)
44
Example 1212; P13
3.08 minutes11; 448.3 (chlorine isotope pattern observed)
45
Example 12; P13
3.13 minutes11; 419.3 (chlorine isotope pattern observed)
46
Examples 3 and 413; P5
From analysis of the 1H NMR, this Example was presumed to exist as a mixture of rotamers; [9.31 (s) and 9.30 (s), total 1H], [8.53 (br s) and 8.13 (br s), total 1H], [8.27 (d, J = 9.0 Hz) and 8.20 (d, J = 9.0 Hz), total 1H], 7.72-7.64 (m, 1H), [7.68 (s) and 7.56 (s), total 1H], [6.28 (d,
J = 15.6 Hz) and 6.13 (d,
J = 15.6 Hz), total 1H],
[6.04-5.89 (m) and 5.72-
5.59 (m), total 1H], [5.84
(d, J = 15.6 Hz) and 5.82
(d, J = 15.6 Hz), total 1H],
4.47-4.32 (m, 2H), 3.94-
3.70 (m, 2H), 3.31-3.16
(m, 1H), [2.36 (s) and
2.33 (s), total 3H], [2.15-
2.07 (m) and 1.84-1.75
(m), total 1H]; 419.0
(chlorine isotope pattern
observed)
47
Examples 22 and 2314; P13
2.51 minutes15; 446.5
48
Examples 22 and 2316; P13
2.55 minutes17; 420.2
49
Examples 22 and 237,18; P13
1.45 minutes19; 421.2
50
Examples 22 and 2320; P13
1.9 minutes17; 429.6
51
Examples 22 and 2321; P13
1.65 minutes17; 420.1
52
Examples 22 and 2321; P13
1.91 minutes17; 419.5
53
Examples 16 and 1712,22; P12
4.8 minutes19; 432.5
54
Examples 26 and 2723; P5
3.31 minutes24; 405.6
55
Examples 26 and 2712,25; P5
2.43 minutes26; 449.5
1. The requisite 6-fluoro-N4-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]quinoline-3,4-diamine was synthesized from 6-fluoro-3-nitroquinolin-4-ol using the general method described in Preparation P7 for synthesis of P7 from C25, except that the final reduction was carried out via hydrogenation over platinum on carbon, rather than treatment with iron powder and ammonium chloride.
2. Conditions for analytical HPLC. Column: Waters XBridge C18, 2.1×50 mm, 5 μm; Mobile phase A: 0.0375% trifluoroacetic acid in water; Mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; Gradient: 1% to 5% B over 0.6 minutes; 5% to 100% B over 3.4 minutes; Flow rate: 0.8 mL/minute.
3. In this case, the amide formation and ring closure were carried out in separate steps: condensation of the appropriate amine and carboxylic acid was effected with 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide and either triethylamine or N,N-diisopropylethylamine. The intermediate amide was cyclized via heating with 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide and N,N-diisopropylethylamine in N,N-dimethylformamide.
4. Amide formation between P8 and cyclopentanecarboxylic acid was effected using dimethyl carbonate and N,N-diisopropylethylamine, affording N-(6-cyano-4-{[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]amino}quinolin-3-yl)acetamide. This material was converted to Example 30 using the method described for synthesis of C60 from C59 in Examples 10 and 11.
5. The racemate of Example 31 was separated into its component enantiomers via supercritical fluid chromatography [(Column: Chiral Technologies Chiralpak AD, 5 μm; Mobile phase: 3:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first-eluting compound was Example 31. The enantiomer of Example 31, [cis-4-(8-chloro-2-methyl-1H-imidazo[4,5-c]quinolin-1-yl)tetrahydro-2H-pyran-2-yl]acetonitrile, ENT 2, was the second-eluting enantiomer, LCMS m/z 341.0 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 1660 nM.
6. The racemate of Example 32 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)]. The first-eluting compound was Example 32. The enantiomer of Example 32, 1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline-8-carbonitrile, ENT 2, was the second-eluting enantiomer, LCMS m/z 409.8 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 473 nM.
7. Reaction of 5-methyl-1H-tetrazole with methyl bromoacetate in the presence of triethylamine afforded methyl (5-methyl-2H-tetrazol-2-yl)acetate, which was hydrolyzed with lithium hydroxide to provide the requisite (5-methyl-2H-tetrazol-2-yl)acetic acid.
8. Conditions for analytical HPLC. Column: Waters XBridge C18, 2.1×50 mm, 5 μm; Mobile phase A: 0.05% ammonium hydroxide in water; Mobile phase B: acetonitrile; Gradient: 5% B for 0.5 minutes; 5% to 100% B over 2.9 minutes; 100% B for 0.8 minutes; Flow rate: 0.8 mL/minute.
9. Methyl (5-methyl-1,3-oxazol-2-yl)acetate was synthesized using the procedure described by A. S. K. Hashmi et al., Organic Letters 2004, 6, 4391-4394. Ester hydrolysis was carried out using hydrochloric acid, to provide the requisite (5-methyl-1,3-oxazol-2-yl)acetic acid.
10. The requisite 6-chloro-N4-[(3R)-1-methylpyrrolidin-3-yl]quinoline-3,4-diamine was synthesized from C7, using the method described in Preparation P9. The reduction of the nitro group in this case was carried out via hydrogenation over platinum(IV) oxide.
11. Conditions for analytical HPLC. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water (v/v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); Gradient: 5.0% B for 1 minute, then linear from 5.0% to 95% B over 3.0 minutes, then 95% B for 1 minute. Flow rate: 2 mL/minute.
12. The requisite [4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]acetic acid may be synthesized according to the method described by M. D. Andrews et al., PCT International Application WO 2014053967 A1, Apr. 10, 2014.
13. The racemate of Example 46 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies ChiralCel OD, 5 μm; Mobile phase: 7:3 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first-eluting compound was Example 46. The enantiomer of Example 46, 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 419.1 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 21.4 nM; LRRK2, G2019S mutant 1050, 16.1 nM.
14. The racemate of Example 47 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 65:35 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The second-eluting compound was Example 47. The enantiomer of Example 47, 8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-yl)methyl]-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-1H-imidazo[4,5-c]quinoline, ENT 1, was the first-eluting enantiomer, LCMS m/z 444.3 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 97.3 nM.
15. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak ADH, 4.6×100 mm, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 200 bar; Flow rate: 1.5 mL/minute.
16. The racemate of Example 48 (Example 82) was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 4:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The second-eluting compound was Example 48. The enantiomer of Example 48, 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1, was the first-eluting enantiomer, LCMS m/z 420.1 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 145 nM.
17. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak ADH, 4.6×100 mm, 5 μm; Mobile phase: 3:2 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 120 bar; Flow rate: 1.5 mL/minute.
18. The racemate of Example 49 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 3:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting compound was Example 49. The enantiomer of Example 49, 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-2H-tetrazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 421.1 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 46.2 nM; LRRK2.
19. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak ADH, 4.6×100 mm, 5 μm; Mobile phase: 1:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 120 bar; Flow rate: 1.5 mL/minute.
20. The racemate of Example 50 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 3:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting compound was Example 50. The enantiomer of Example 50, 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 429.2 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 181 nM.
21. The racemate of Examples 51 and 52 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 3:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting compound was Example 51, and the second-eluting enantiomer was Example 52.
22. The racemate of Example 53 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 55:45 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The second-eluting compound was Example 53. The enantiomer of Example 53, 1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 1, was the first-eluting enantiomer, LCMS m/z 432.7 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 229 nM.
23. The racemate of Example 54 was separated into its component enantiomers via supercritical fluid chromatography [Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 4:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The second-eluting compound was Example 54. The enantiomer of Example 54, 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1 (Example 93), was the first-eluting enantiomer, LCMS m/z 405.3 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 11.0 nM; LRRK2.
24. Conditions for analytical HPLC. Column: Phenomenex Lux Amylose-1, 4.6×100 mm, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute.
25. The racemate of Example 55 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies ChiralCel OD-H, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. The first-eluting compound was Example 55. The enantiomer of Example 55, 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 449.3 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 15.3 nM.
26. Conditions for analytical HPLC. Column: Chiral Technologies ChiralCel ODH, 4.6×100 mm, 5 μm; Mobile phase: 3:2 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 120 bar; Flow rate: 1.5 mL/minute.
TABLE 2
Structure and mass spectral data for Examples 56-94.
1H NMR (400 MHz, CDCl3) δ and
Mass spectrum m/z [M + H]+; or
HPLC or SFC retention time and
Example
Mass spectrum m/z [M + H]+ (unless
Number
Structure
otherwise indicated)
56
9.52 (br s, 1H), 9.39 (s, 1H), 8.35 (d, J = 8.8 Hz, 1H), 7.87 (br d, J = 8.4 Hz, 1H), 5.72-5.59 (m, 1H), 4.71 (AB quartet, JAB = 16.5 Hz, ΔνAB = 12.1 Hz, 2H), 3.70-3.61 (m, 2H), 3.56-3.42 (m, 1H), 3.31-3.15 (m, 2H), 3.02-2.92 (m, 1H), 2.69- 2.55 (m, 1H), 2.59 (s, 3H), 2.50- 2.38 (m, 1H); 442.11
57
2.61 minutes2; 403
58
2.48 minutes2; 399
59
9.29 (s, 1H), 8.61-8.47 (br m, 1H), 8.23 (d, J = 8.8 Hz, 1H), 7.65 (dd, J = 9.0, 1.5 Hz, 1H), 6.00 (br s, 1H), 5.86 (td, J = 55.5, 3.1 Hz, 1H), 5.32- 5.18 (br m, 1H), 4.52 (s, 2H), 4.42 (dd, J = 11.9, 5.3 Hz, 1H), 3.95-3.82 (m, 1H), 3.77 (br dd, J = 11.9, 11.0 Hz, 1H), 2.89-2.58 (br m, 2H), 2.39 (s, 3H), 1.91-1.68 (br m, 2H); 433.0 (chlorine isotope pattern observed)3,4
60
From analysis of 1H and 2- dimensional NMR data, this Example was presumed to exist as a mixture of rotamers. 1H NMR (600 MHz, CDCl3), characteristic peaks: δ 9.29 (s, 1H), [8.54 (br s) and 8.13 (br s), total 1H], [8.26 (d, J = 8.8 Hz) and 8.22 (d, J = 9.4 Hz), total 1H], 7.67-7.62 (m, 1H), [6.12 (s) and 6.07 (s), total 1H], 5.67-5.48 (m, 1H), [4.61 (AB quartet, JAB = 16.7 Hz, ΔνAB = 30.6 Hz) and 4.52 (AB
quartet, JAB = 15.8 Hz, ΔνAB = 14.8 Hz),
total 2H], 4.42-4.30 (m, 2H),
3.90-3.67 (m, 2H), [3.28-3.19 (m) and
3.17-3.08 (m), total 1H], [2.41
(s) and 2.39 (s), total 3H]; 419.3
(chlorine isotope pattern observed)5
61
2.44 minutes2; 390
62
From analysis of the 1H NMR, this Example was presumed to exist as a mixture of rotamers. 1H NMR (400 MHz, CD3OD) δ 9.85-9.71 (br m, 1H), [9.14 (s) and 9.13 (s), total 1H], 8.17-8.11 (m, 1H), 7.86 (s, 1H), 7.75-7.69 (m, 1H), 6.27 (AB quartet, JAB = 15.6 Hz, ΔνAB = 16.4 Hz, 2H), 5.89-5.78 (m, 1H), 3.41- 3.3 (m, 3H, assumed; partially obscured by solvent peak), 2.86
(dd, J = 11.0, 10.6 Hz, 1H), 2.64-
2.55 (m, 1H), 2.52 (s, 3H), 2.47-
2.28 (m, 1H), 2.33 (s, 3H); 382.2
(chlorine isotope pattern observed)
63
2.73 minutes2; 4346
64
2.30 minutes2; 383
65
2.80 minutes2; 412
66
9.41 (s, 1H), 9.10-8.94 (br m, 1H), 8.43 (d, J = 9.0 Hz, 1H), 8.32 (s, 1H), 8.01 (s, 1H), 7.93 (dd, J = 8.8, 1.8 Hz, 1H), 5.88 (s, 2H), 5.48-5.36 (m, 1H), 4.37-4.30 (m, 1H), 3.82-3.69 (m, 2H), 2.80-2.62 (br m, 1H), 2.53- 2.35 (br m, 1H), 1.95-1.59 (br m, 2H, assumed; partially obscured by water peak), 1.35 (d, J = 6.0 Hz, 3H); 417.1
67
9.64-9.49 (br m, 1H), 9.25 (s, 1H), 8.58-8.55 (m, 1H), 8.39 (br s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.60 (dd, J = 9.0, 2.0 Hz, 1H), 5.73-5.62 (m, 1H), 4.78-4.67 (m, 2H), 3.40-3.32 (m, 2H), 2.78 (dd, J = 11.0, 10.6 Hz, 1H), 2.64-2.42 (m, 2H), 2.56 (s, 3H), 2.52 (s, 3H), 2.27-2.17 (m, 1H); 393.1 (chlorine isotope pattern observed)
68
From analysis of the 1H NMR, this Example was presumed to exist as a mixture of rotamers. 9.41 (s, 1H), [8.95 (d, J = 1.2 Hz) and 8.94 (d, J = 1.6 Hz), total 1H], 8.35 (d, J = 8.6 Hz, 1H), 7.85 (dd, J = 8.6, 2.0 Hz, 1H), 6.01-5.99 (m, 1H), [5.54-5.49 (m) and 5.44-5.32 (m), total 2H], 4.53 (s, 2H), 2.76-2.46 (m, 4H), 2.40 (br s, 3H), 2.15-1.92 (m, 2H); 376.4
69
2.70 minutes2; 433
70
9.40 (s, 1H), 9.16-8.87 (br m, 1H), 8.42 (d, J = 8.8 Hz, 1H), 7.91 (br d, J = 8.8 Hz, 1H), 5.31-5.10 (br m, 1H), 4.75 (s, 2H), 4.33 (dd, J = 12.1, 5.1 Hz, 1H), 3.82-3.67 (m, 2H), 2.86-2.33 (br m, 2H), 2.54 (s, 3H), 2.07-1.78 (br m, 2H), 1.35 (d, J = 6.2 Hz, 3H); 432.1
71
2.42 minutes2; 397
72
2.36 minutes7; 392
73
2.77 minutes2; 446
74
9.41 (s, 1H), 9.11-8.88 (br m, 1H), 8.42 (d, J = 8.8 Hz, 1H), 7.90 (dd, J = 8.8, 1.3 Hz, 1H), 5.40-5.29 (m, 1H), 4.96 (s, 2H), 4.31 (dd, J = 11.9, 4.8 Hz, 1H), 3.81-3.69 (m, 2H), 2.82-2.62 (br m, 1H), 2.77 (s, 3H), 2.51-2.34 (br m, 1H), 1.99-1.7 (m, 2H, assumed; largely obscured by water peak), 1.34 (d, J = 6.2 Hz, 3H); 448.0
75
From analysis of the 1H NMR, this Example was presumed to exist as a mixture of rotamers. 1H NMR (400 MHz, CD3OD) δ [9.13 (s) and 9.09 (s), total 1H], [8.71-8.67 (m) and 8.49-8.45 (m), total 1H], [8.18 (d, J = 8.8 Hz) and 8.17 (d, J = 8.8 Hz), total 1H], 7.78-7.70 (m, 1H), [6.11- 5.97 (m) and 5.81-5.65 (m), total 1H], 4.78-4.58 (m, 2H), 4.40-4.20 (m, 2H), 4.09-3.79 (m, 2H), [3.36- 3.22 (m) and 3.11-2.98 (m), total
1H, assumed; partially obscured by
solvent peak], [2.62 (s) and 2.59
(s), total 3H], [2.48-2.39 (m) and
2.38-2.30 (m), total 1H]; 420.1
(chlorine isotope pattern observed)8
76
1H NMR (400 MHz, CD3OD) δ 9.26 (s, 1H), 9.21-9.07 (br m, 1H), 8.94 (br s, 1H), 8.39 (d, J = 8.8 Hz, 1H), 7.99 (dd, J = 8.8, 1.5 Hz, 1H), 7.53 (s, 1H), 5.43-5.28 (br m, 1H), 4.77 (s, 2H), 4.25 (dd, J = 12.0, 5.1 Hz, 1H), 3.79-3.66 (m, 2H), 2.78-2.61 (br m, 1H), 2.54 (s, 3H), 2.48-2.32 (br m, 1H), 2.14-1.88 (br m, 2H), 1.29 (d, J = 6.4 Hz, 3H); 442.2
77
2.31 minutes7; 396
78
Characteristic 1H NMR peaks: δ 9.27 (s, 1H), 8.24 (d, J = 9.0 Hz, 1H), 7.66 (dd, J = 8.8, 2.3 Hz, 1H), 4.42- 4.32 (br m, 1H), 3.84-3.59 (m, 3H), 3.52-3.40 (m, 1H), 3.29-3.21 (m, 1H), 2.25-2.04 (br m, 2H), 1.64-1.6 (m, 3H, assumed; partially obscured by water peak), 1.40 (d, J = 6.0 Hz, 3H); 369.0 (chlorine isotope pattern observed)9,10
79
9.24 (s, 1H), 8.58 (s, 1H), 8.27 (dd, J = 9.0, 6.0 Hz, 1H), 8.17-8.11 (m, 1H), 7.45 (ddd, J = 9.3, 7.8, 2.8 Hz, 1H), 5.62-5.51 (m, 1H), [5.52-5.46 (m) and 5.38-5.33 (m), JHF = 54 Hz, total 1H], 5.06 (s, 2H), 2.81-2.67 (m, 2H), 2.66-2.60 (m, 1H), 2.56- 2.42 (m, 1H), 2.16-1.93 (m, 2H); 372.06,11
80
2.40 minutes2; 355
81
2.87 minutes7; 43212
82
3.08 minutes13; 418.3 (chlorine isotope pattern observed)
83
3.07 minutes13; 405.2 (chlorine isotope pattern observed)
84
2.93 minutes13; 418.3 (chlorine isotope pattern observed)
85
3.03 minutes14; 458.515
86
1.92 minutes16; 405.617
87
1.78 minutes18; 420.219
88
1.44 minutes20; 420.521
89
3.14 minutes22; 435.2 (chlorine isotope pattern observed)23
90
3.04 minutes13; 413.4
91
3.19 minutes13; 413.4
92
3.35 minutes24; 436.5 (chlorine isotope pattern observed)25
93
2.44 minutes26; 405.3 (chlorine isotope pattern observed)27
94
3.53 minutes24; 445.5 (chlorine isotope pattern observed)28
1. tert-Butyl (3R)-3-aminopyrrolidine-1-carboxylate and C11 were used to synthesize tert-butyl (3R)-3-[(3-amino-6-cyanoquinolin-4-yl)amino]pyrrolidine-1-carboxylate, according to the method described for synthesis of P9 in Preparation P9. This material was converted to tert-butyl (3R)-3-{8-cyano-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinolin-1-yl}pyrrolidine-1-carboxylate using the method described for synthesis of 3 and 4 in Examples 3 and 4. Removal of the protecting group with trifluoroacetic acid was followed by alkylation with 2,2,2-trifluoroethyl trifluoromethanesulfonate and N,N-diisopropylethylamine, providing Example 56.
2. Conditions for analytical HPLC. Column: Waters XBridge C18, 2.1×50 mm, 5 μm; Mobile phase A: 0.0375% trifluoroacetic acid in water; Mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; Gradient: 1% to 5% B over 0.6 minutes; 5% to 100% B over 3.4 minutes; Flow rate: 0.8 mL/minute.
3. Reaction of P3 with (5-methyl-1,2-oxazol-3-yl)acetic acid, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide, and N,N-diisopropylethylamine afforded 1-{cis-2-[(benzyloxy)methyl]tetrahydro-2H-pyran-4-yl}-8-chloro-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline, which was debenzylated with boron trichloride and oxidized using Dess-Martin periodinane [1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one]. The resulting cis-4-{8-chloro-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinolin-1-yl}tetrahydro-2H-pyran-2-carbaldehyde was converted to racemic 8-chloro-1-[cis-2-(difluoromethyl)tetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline via treatment with (diethylamino)sulfur trifluoride.
4. Example 59 was isolated from the corresponding racemic mixture via supercritical fluid chromatography (Column: Chiral Technologies Chiralpak AD, 5 μm; Mobile phase: 7:3 carbon dioxide/methanol). Example 59 was the first-eluting enantiomer. The enantiomer of Example 59, 8-chloro-1-[cis-2-(difluoromethyl)tetrahydro-2H-pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 433.0 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 631 nM.
5. Example 60 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OJ-H, 5 μm; Mobile phase: 85:15 carbon dioxide/(ethanol containing 0.2% ammonium hydroxide)]. Example 60 was the first-eluting enantiomer. The enantiomer of Example 60, 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 419.3 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 42.2 nM.
6. Reaction of 1,2,3-thiadiazol-4-ylmethanol with methanesulfonyl chloride and triethylamine, followed by displacement using potassium cyanide and hydrolysis in concentrated hydrochloric acid, provided the requisite 1,2,3-thiadiazol-4-ylacetic acid.
7. Conditions for analytical HPLC. Column: Waters XBridge C18, 2.1×50 mm, 5 μm; Mobile phase A: 0.0375% trifluoroacetic acid in water; Mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; Gradient: 10% to 100% B over 4.0 minutes; Flow rate: 0.8 mL/minute.
8. Example 75 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IC, 10 μm; Mobile phase: 55:45 carbon dioxide/(2-propanol containing 0.1% ammonium hydroxide)]. Example 75 was the first-eluting enantiomer. The enantiomer of Example 75, 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 420.0 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, not determined.
9. Treatment of ethyl 3-oxobutanoate with lithium trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, and N,N-diisopropylethylamine provided ethyl 3-{[(trifluoromethyl)sulfonyl]oxy}but-2-enoate. This was reacted with zinc cyanide in the presence of tetrakis(triphenylphosphine)palladium(0) to afford ethyl 3-cyanobut-2-enoate, which was subjected to hydrogenation over palladium on carbon, followed by hydrolysis with sodium hydroxide, to yield the requisite 3-cyanobutanoic acid.
10. Example 78 was isolated from the corresponding diastereomeric mixture via supercritical fluid chromatography [Column: Phenomenex Lux Cellulose-2, 10 μm; Mobile phase: 3:2 carbon dioxide/(2-propanol containing 0.1% ammonium hydroxide)]. Example 78 was the second-eluting diastereomer. The diastereomer of Example 78, 3-{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}-2-methylpropanenitrile, DIAST 1, was the first-eluting diastereomer, LCMS m/z 369.0 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 37.2 nM.
11. Example 79 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AY, 10 μm; Mobile phase: 3:2 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. Example 79 was the second-eluting enantiomer. The enantiomer of Example 79, 8-fluoro-1-[cis-3-fluorocyclopentyl]-2-(1,2,3-thiadiazol-4-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1, was the first-eluting enantiomer, LCMS m/z 372.0 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 7.54 nM.
12. Reaction of 5-methyl-1H-tetrazole with methyl bromoacetate in the presence of triethylamine afforded methyl (5-methyl-2H-tetrazol-2-yl)acetate, which was hydrolyzed with lithium hydroxide to provide the requisite (5-methyl-2H-tetrazol-2-yl)acetic acid.
13. Conditions for analytical HPLC. Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: 0.05% trifluoroacetic acid in water (v/v); Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); Gradient: 5.0% B for 1 minute, then linear from 5.0% to 95% B over 3.0 minutes, then 95% B for 1 minute. Flow rate: 2 mL/minute.
14. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak ADH, 4.6×100 mm, 5 μm; Mobile phase: 75:25 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 200 bar; Flow rate: 1.5 mL/minute.
15. Example 85 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 4:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 85 was the first-eluting enantiomer. The enantiomer of Example 85, 1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-2-[(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 458.3 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 55.9 nM.
16. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak ADH, 4.6×100 mm, 5 μm; Mobile phase: 70:30 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute.
17. Example 83 was separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 4:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 86 was the second-eluting enantiomer. The enantiomer of Example 86, 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-(1H-tetrazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 1, was the first-eluting enantiomer, LCMS m/z 407.1 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 271 nM.
18. Conditions for analytical HPLC. Column: Chiral Technologies Chiralcel ODH, 4.6×100 mm, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute.
19. Example 87 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies ChiralCel OD-H, 5 μm; Mobile phase: 4:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 87 was the first-eluting enantiomer. The enantiomer of Example 87, 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 420.2 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 37.8 nM.
20. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak ADH, 4.6×100 mm, 5 μm; Mobile phase: 60:40 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 120 bar; Flow rate: 1.5 mL/minute.
21. Example 88 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 3:2 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 88 was the first-eluting enantiomer. The enantiomer of Example 88, 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 420.5 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 261 nM.
22. Conditions for analytical HPLC. Column: Chiral Technologies Chiralpak ADH, 4.6×100 mm, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 200 bar; Flow rate: 1.5 mL/minute.
23. Example 89 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 85:15 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 89 was the second-eluting enantiomer. The enantiomer of Example 89, 8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline, ENT 1, was the first-eluting enantiomer, LCMS m/z 434.8 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, not determined.
24. Conditions for analytical HPLC. Column: Chiral Technologies Chiralcel OJ-H, 4.6×100 mm, 5 μm; Mobile phase: 9:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute.
25. Example 92 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OJ-H, 5 μm; Mobile phase: 95:5 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 92 was the first-eluting enantiomer. The enantiomer of Example 92, 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-[(5-methyl-1,3,4-thiadiazol-2-yl) methyl]-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 436.5 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 33.7 nM.
26. Conditions for analytical HPLC. Column: Phenomenex Lux Amylose-1, 4.6×100 mm, 5 μm; Mobile phase: 7:3 carbon dioxide/(methanol containing 0.2% ammonium hydroxide); Back pressure: 150 bar; Flow rate: 1.5 mL/minute.
27. Example 93 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 4:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 93 was the first-eluting enantiomer. The enantiomer of Example 93, 8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 405.6 [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 10.3 nM.
28. Example 94 was isolated from the corresponding racemic mixture via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak AD-H, 5 μm; Mobile phase: 3:1 carbon dioxide/(methanol containing 0.2% ammonium hydroxide)]. Example 94 was the first-eluting enantiomer. The enantiomer of Example 94, 8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-yl)methyl]-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinoline, ENT 2, was the second-eluting enantiomer, LCMS m/z 445.3 (chlorine isotope pattern observed) [M+H]+, and exhibited the following biological data: LRRK2, WT IC50, 9.35 nM.
Example 95
[5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl]methanol (95)
Step 1. Synthesis of benzyl 2-(5-methylpyrazin-2-yl)acetate
A suspension containing 2-(5-methylpyrazin-2-yl)acetic acid (1.00 g, 6.57 mmol) and benzyl alcohol (853 mg, 7.89 mmol, 0.820 mL) in tetrahydrofuran (26.3 mL) was treated with N,N-diisopropylethylamine (1.72 mL, 9.86 mmol) and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in N,N-dimethylformamide; 4.69 mL, 7.89 mmol). The solids slowly dissolved as the reaction mixture was stirred at room temperature for 20 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution, and then extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 80% ethyl acetate in heptane) afforded the product as a yellow oil. Yield: 1.2 g, 76%. LCMS m/z 243.4 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J=1.5 Hz, 1H), 8.43 (d, J=1.5 Hz, 1H), 7.43-7.30 (m, 5H), 5.20 (s, 2H), 3.91 (s, 2H), 2.58 (s, 3H).
Step 2. Synthesis of benzyl (5-methyl-4-oxidopyrazin-2-yl)acetate
A solution of benzyl 2-(5-methylpyrazin-2-yl)acetate (1.22 g, 5.03 mmol) in dichloromethane (50 mL) was placed under house vacuum and the reaction flask was refilled with nitrogen; this procedure was carried out three times. The solution was cooled to 0° C. and m-chloroperbenzoic acid (mCPBA; 886 mg, 5.13 mmol) was added in one portion, while keeping the solution temperature at 0° C. The reaction mixture was allowed to slowly warm to room temperature and was stirred for 20 hours, whereupon it was quenched with saturated aqueous sodium bicarbonate solution. The aqueous layer was extracted with dichloromethane, and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified via silica gel chromatography (Gradient: 0% to 80% ethyl acetate in heptane) to afford the product as a colorless oil, which became a white solid upon standing. Two-dimensional NMR NOE studies indicated that this material was the desired regioisomer. Yield: 616 mg, 47%. LCMS m/z 259.2 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.20 (s, 1H), 7.44-7.31 (m, 5H), 5.20 (s, 2H), 3.84 (s, 2H), 2.47 (s, 3H).
The regioisomeric N-oxide was also isolated (200 mg, 15%), as well as some starting material (205 mg, 17%).
Step 3. Synthesis of benzyl {5-[(acetyloxy)methyl]pyrazin-2-yl}acetate
A solution of benzyl (5-methyl-4-oxidopyrazin-2-yl)acetate (591 mg, 2.29 mmol) in acetic anhydride (9.15 mL) was heated to 70° C. for 1 hour, and then at 100° C. for 24 hours. The reaction mixture was then cooled to room temperature, and the acetic anhydride and acetic acid were removed under vacuum on a rotary evaporator. The residue was dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate solution. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 70% ethyl acetate in heptane) provided the product as a yellow oil. Yield: 392 mg, 57%. LCMS m/z 301.2 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.62 (d, J=1.5 Hz, 1H), 8.59 (d, J=1.5 Hz, 1H), 7.44-7.31 (m, 5H), 5.27 (s, 2H), 5.20 (s, 2H), 3.96 (s, 2H), 2.19 (s, 3H).
Step 4. Synthesis of {5-[(acetyloxy)methyl]pyrazin-2-yl}acetic acid
A mixture of benzyl {5-[(acetyloxy)methyl]pyrazin-2-yl}acetate (390 mg, 1.30 mmol) and palladium on carbon (150 mg, 10% Pd basis) in ethyl acetate (13.0 mL) was placed in a Hastelloy reactor and the atmosphere was purged three times with nitrogen, and then purged three times with hydrogen. The reaction mixture was stirred at room temperature under 30 psi hydrogen for 2 hours, whereupon it was filtered. The filter cake was washed with ethyl acetate, and the combined filtrates were concentrated in vacuo to provide the product as a yellow oil. Yield: 186 mg, 68% mass recovery. Spectral data and thin-layer chromatographic analysis indicated that the product was contaminated with the product of hydrogenolysis of the acetoxy group (˜3:4 methyl to acetoxymethyl by NMR). This mixture was carried to the next step without further purification.
Step 5. Synthesis of [5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl]methyl acetate
A mixture of P10 (246 mg, 0.843 mmol) and {5-[(acetyloxy)methyl]pyrazin-2-yl}acetic acid (186 mg, 0.885 mmol, as a mixture from the previous step) in toluene (17.7 mL) was treated with N,N-diisopropylethylamine (176 μL, 1.01 mmol) and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; (1.51 mL, 2.53 mmol). The reaction mixture was heated to 70° C. for 1 hour, and then at 110° C. for 4 hours. The reaction mixture was allowed to cool to ambient temperature and was then quenched by addition of saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) afforded two products. The desired product was obtained as a light brown oil. Yield: 206 mg, 50%. LCMS m/z 466.2 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.28 (s, 1H), 8.73 (s, 1H), 8.67 (s, 1H), 8.59 (s, 1H), 8.24 (d, J=9.0 Hz, 1H), 7.66 (dd, J=8.9, 2.1 Hz, 1H), 5.30 (br s, 1H), 5.27 (s, 2H), 4.73 (s, 2H), 4.34 (dd, J=12.0, 5.1 Hz, 1H), 3.73 (br s, 2H), 2.75 (br s, 1H), 2.48 (br s, 1H), 2.17 (s, 3H), 1.85 (br s, 1H), 1.74 (br s, 1H), 1.38 (d, J=6.1 Hz, 3H). Also obtained was a light yellow solid identified as the desacetoxy product 8-chloro-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline (95A). Yield: 131 mg, 36%.
Step 6. Synthesis of [5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl]methanol (95)
To a solution of [5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}methyl)pyrazin-2-yl]methyl acetate (206 mg, 0.442 mmol) in methanol (10 mL) was added potassium carbonate (61.1 mg, 0.442 mmol). The resulting white suspension was stirred at room temperature for 30 minutes, whereupon it was diluted with water and extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 20% methanol in dichloromethane) afforded a light yellow foam (151 mg). This material was recrystallized from diethyl ether and heptane to provide the product as a light yellow solid. Yield: 130 mg, 69%. LCMS m/z 424.2 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 9.29 (s, 1H), 8.72-8.67 (m, 2H), 8.59 (s, 1H), 8.24 (d, J=8.9 Hz, 1H), 7.66 (dd, J=8.9, 2.1 Hz, 1H), 5.31 (br s, 1H), 4.87 (d, J=5.4 Hz, 2H), 4.73 (s, 2H), 4.34 (dd, J=12.1, 5.2 Hz, 1H), 3.74 (br s, 2H), 2.91 (br s, 1H), 2.76 (br s, 1H), 2.48 (br s, 1H), 1.88 (br s, 1H), 1.75 (br s, 1H), 1.38 (d, J=6.1 Hz, 3H).
Example 96
8-chloro-2-{[5-(2H3)methylpyrazin-2-yl]methyl}-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline (96); [5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(2H3)methyl)pyrazine-2-yl]methanol (96B)
To 1.2 g of 8-chloro-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinoline (95A) (yellow solid) was added 5.7 g of deuterated acetic acid (CD3CO2D) in a first container. The mixture was stirred at 120° C. for 20 hours and then concentrated. Proton NMR suggested >90% D/H exchange on the pyrazine methyl group.
In a second container, to 3.0 g of 8-chloro-2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]quinolone was added 50 mL deuterated acetic acid. The mixture was stirred at 120° C. for 24 hours and then concentrated.
The concentrated residues from the first and second containers were combined and dissolved in 75 mL of deuterated acetic acid. This solution was stirred at 120° C. for 24 hours and then concentrated. The residue was dissolved in 50 mL of deuterated acetic acid and stirred at 120° C. for 24 hours and then concentrated. The residue was dissolved in 120 mL ethyl acetate and washed with 60 mL saturated aqueous sodium carbonate solution. The organic layer was dried over magnesium sulfate and concentrated to furnish 4.4 g of a dark solid.
Part of this sample, 2.4 g, was dissolved in 100 mL acetic acid and stirred at room temperature for 24 hours and then concentrated. The residue was dissolved in 100 mL of acetic acid and stirred at room temperature for 24 hours and the concentrated. The residue was dissolved in 150 mL ethyl acetate, washed with 80 mL of 3:1 brine/ammonium hydroxide. Organic layer was dried over magnesium sulfate, concentrated to furnish 2.4 g of a dark color, which was purified using a step gradient method (20% B hold from 0 to 1.5 minutes, 20% to 70% B from 1.5 to 10 minutes, and finally 70 to 100% from 10 to 12 minutes; with mobile phase A being 0.05% formic acid in water and mobile phase B 0.05% formic acid in acetonitrile) on a Phenomenex Gemini NX C18 150 mm×21.2 mm 5 um column at a flow rate of 27 mL/min. The collected fractions were lyophilized to furnish off-white fluffy solid samples with a combined weight of 2.12 g.
Analytical data: [M+H]+ observed 411.178 (predicted 411.178); HPLC retention time 4.12 min on a C18 100 mm×3.0 mm 2.6 um column with 5% B from 0 to 1.5 min, 5 to 100% B from 1.5 to 4.0 min and hold at 100% from 4.0 to 5.4 min (A 0.1% formic acid in water, B 0.1% formic acid in acetonitrile); 1H NMR (600 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.66 (s, 2H), 8.46 (d, J=1.5 Hz, 1H), 8.19 (d, J=8.9 Hz, 1H), 7.74 (dd, J=8.9, 2.2 Hz, 1H), 5.27 (m, 1H), 4.77 (s, 2H), 4.16 (m, 1H), 3.69 (m, 1H), 3.60 (m, 1H), 2.47 (m, 1H), 2.21 (m, 1H), 2.09-1.92 (m, 1H), 1.85 (m, 1H), 1.22 (d, J=6.1 Hz, 3H).
Biological Assays
LRRK2 Assay
LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat #PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: The compound dose response was prepared by diluting compound to a top concentration of 0.3 mM in 100% DMSO and serial diluted by half-log in DMSO to give an 11 point curve, 100× final assay concentration. Using Echo acoustic dispensing, 60 nL of compound was transferred to a low volume Corning 384-well assay plate. 3 μL of a working solution of substrate (200 nM LRRKtide, 2 mM ATP) prepared in assay buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to the 60 nL compound assay plate. The kinase reaction was started with 3 μL of a working solution of LRRK2 enzyme at a concentration of 4 μg/m L. The final reaction concentrations were 100 nM LRRKtide, 1 mM ATP, 2 μg/mL LRRK2 enzyme and a compound dose response with a top dose of 3 μM. The reaction was allowed to progress at room temperature for 30 minutes and then stopped with the addition of 6 μL of detection buffer (20 mM Tris pH 7.6, 0.01% NP-40, 6 mM EDTA with 2 nM terbium labeled antiphospho-ERM). After an incubation of 1 hour at room temperature, the plate was read on an Envision with an excitation wavelength of 340 nm and a reading emission at both 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data. Inhibition of mutant G2019S LRRK2 (Invitrogen cat #PV4881) was measured in the exact same method. All final concentrations of substrate ATP and enzyme were the same.
TABLE 3
IUPAC name and biological data for Examples 1-96
LRRK2,
LRRK2,
WT
G2019S
IC50 (nM);
IC50 (nM);
(Number
(Number
of
of
Example
determinat
determinat
Number
IUPAC Name
ions)
ions)
1
[(2S,4R)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]
10.2
(2)
8.87
(1)
quinolin-1-yl)tetrahydro-2H-pyran-2-yl]
acetonitrile
2
[(2R,4S)-4-(8-chloro-2-ethyl-1H-imidazo[4,5-c]
1530
(2)
N.D.a
quinolin-1-yl)tetrahydro-2H-pyran-2-yl]
acetonitrile
3
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-
31.8
(5)
N.D.
methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo
[4,5-c]quinoline-8-carbonitrile, ENT 1
4
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(4-
17.6
(5)
3.31
(1)
methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-imidazo
[4,5-c]quinoline-8-carbonitrile, ENT 2
5
8-chloro-1-[(4S)-3,3-difluorotetrahydro-2H-
14.6
(2)
9.41
(1)
pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-
1H-imidazo[4,5-c]quinoline
6
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(3R)-1-
6.39
(2)
11.3
(1)
methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]
quinoline-8-carbonitrile, formate salt
7
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
47.9
(2)
39.7
(1)
yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo
[4,5-c]quinoline, ENT 1
8
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
11.8
(2)
11.6
(1)
yl)-2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo
[4,5-c]quinoline, ENT 2
9
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
11.8
(2)
5.55
(1)
[(1-methyl-1H-1,2,3-triazol-4-yl)methyl]-8-
(trifluoromethyl)-1H-imidazo[4,5-c]quinoline
10
[cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]
1790
(2)
N.D.
quinolin-1-yl)tetrahydro-2H-pyran-2-yl]
acetonitrile, ENT 1
11
[cis-4-(8-chloro-2-cyclobutyl-1H-imidazo[4,5-c]
18.0
(2)
5.06
(1)
quinolin-1-yl)tetrahydro-2H-pyran-2-yl]
acetonitrile, ENT 2
12
8-(difluoromethyl)-2-[(4-methoxy-1H-pyrazol-1-
8.77
(2)
4.38
(1)
yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-
pyran-4-yl]-1H-imidazo[4,5-c]quinoline
13
8-(difluoromethyl)-2-[(5-methylpyrazin-2-yl)
9.72
(3)
7.77
(2)
methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-
pyran-4-yl]-1H-imidazo[4,5-c]quinoline
14
{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-
14.2
(3)
12.8
(3)
pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-
methylpyrazin-2-yl)methanol, DIAST 1
15
{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-
16.3
(3)
18.4
(3)
pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}(5-
methylpyrazin-2-yl)methanol, DIAST 2
16
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
731
(2)
N.D.
2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]
quinoline, ENT 1
17
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
6.49
(2)
7.57
(1)
2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]
quinoline, ENT 2
18
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
263
(2)
N.D.
2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
19
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
5.43
(2)
8.12
(1)
2-[(4-methyl-1H-1,2,3-triazol-1-yl) methyl]-1H-
imidazo[4,5-c]quinoline, ENT 2
20
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
4.12
(2)
3.31
(1)
2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-
c]quinoline, ENT 1
21
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
235
(2)
N.D.
2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-
c]quinoline, ENT 2
22
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
138
(3)
N.D.
2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]
methyl}-1H-imidazo[4,5-c]quinoline, ENT 1
23
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
2.35
(2)
1.35
(1)
2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]
methyl}-1H-imidazo[4,5-c]quinoline, ENT 2
24
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
2.77
(3)
1.19
(1)
2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]
quinoline, ENT 1
25
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
318
(2)
N.D.
2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]
quinoline, ENT 2
26
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-
5.90
(1)
N.D.
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
27
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-yl)-
29.1
(3)
20.3
(1)
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 2
28
8-fluoro-2-[(2-methylimidazo[2,1-b][1,3,4]
7.18
(2)
4.59
(1)
thiadiazol-6-yl)methyl]-1-[(2R,4R)-2-methyl
tetrahydro-2H-pyran-4-yl]-1H-imidazo[4,5-c]
quinoline, formate salt
29
2-[(5-methylpyrazin-2-yl)methyl]-1-[(2R,4R)-2-
6.47
(6)
4.13
(5)
methyltetrahydro-2H-pyran-4-yl]-8-
(trifluoromethyl)-1H-imidazo[4,5-c]quinoline
30
2-cyclopentyl-1-[(2R,4R)-2-methyltetrahydro-2H-
20.6
(2)
22.0
(2)
pyran-4-yl]-1H-imidazo[4,5-c]quinoline-8-
carbonitrile, formate salt
31
[cis-4-(8-chloro-2-methyl-1H-imidazo[4,5-c]
7.86
(2)
8.67
(1)
quinolin-1-yl)tetrahydro-2H-pyran-2-yl]
acetonitrile, ENT 1
32
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-2-[(5-
4.59
(2)
5.98
(1)
methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-
imidazo[4,5-c]quinoline-8-carbonitrile, ENT 1
33
2-[(5-methylpyrazin-2-yl)methyl]-1-[(3R)-1-
8.35b
(2)
6.82b
(1)
methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]
quinoline-8-carbonitrile
34
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-2H-
5.53
(2)
5.04
(1)
tetrazol-2-yl)methyl]-1H-imidazo[4,5-c]quinoline-
8-carbonitrile, formate salt
35
2-[(3-methyl-1,2-oxazol-5-yl)methyl]-1-[(3R)-1-
7.57
(2)
6.17
(1)
methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]
quinoline-8-carbonitrile, formate salt
36
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-[(3R)-1-
5.00
(2)
4.63
(1)
methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]
quinoline-8-carbonitrile, formate salt
37
1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(5-methyl-
5.26
(2)
12.6
(1)
1,3,4-thiadiazol-2-yl)methyl]-1H-imidazo[4,5-
c]quinoline-8-carbonitrile
38
2-[(5-methyl-1,3-oxazol-2-yl)methyl]-1-[(3R)-1-
7.49
(2)
12.9
(1)
methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]
quinoline-8-carbonitrile
39
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
17.4
(4)
9.30
(3)
{[5-(trifluoromethyl)pyrazin-2-yl]methyl}-1H-
imidazo[4,5-c]quinoline-8-carbonitrile, formate
salt
40
8-chloro-2-[(6-methylpyrimidin-4-yl)methyl]-1-
9.73
(2)
13.9
(1)
[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo[4,5-c]
quinoline
41
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-
11.1
(2)
3.87
(1)
[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-
(trifluoromethyl)-1H-imidazo[4,5-c]quinoline
42
8-chloro-2-[(5-methyl-1,2,4-oxadiazol-3-yl)
12.9
(2)
26.0
(1)
methyl]-1-[(3R)-1-methylpyrrolidin-3-yl]-1H-
imidazo[4,5-c]quinoline
43
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
5.87
(2)
8.53
(1)
2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo[4,5-c]
quinoline, trifluoroacetate salt
44
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
6.02
(2)
4.53
(1)
2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-yl]
methyl}-1H-imidazo[4,5-c]quinoline,
trifluoroacetate salt
45
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
8.13
(2)
5.82
(1)
2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-
imidazo[4,5-c]quinoline, trifluoroacetate salt
46
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
9.31
(2)
7.66
(1)
yl)-2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
47
8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-
2.80
(2)
1.42
(1)
yl)methyl]-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
1H-imidazo[4,5-c]quinoline, ENT 2
48
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
3.27
(2)
0.938
(1)
2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 2
49
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
4.48
(2)
1.34
(1)
2-[(5-methyl-2H-tetrazol-2-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
50
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
5.06
(2)
1.11
(1)
2-[(5-methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-
c]quinoline, ENT 1
51
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
5.78
(2)
3.49
(1)
2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-imidazo
[4,5-c]quinoline, ENT 1
52
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
15.1
(2)
12.9
(1)
2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 2
53
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
7.51
(2)
7.49
(1)
2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-
yl]methyl}-1H-imidazo[4,5-c] quinoline, ENT 2
54
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
10.3
(1)
N.D.
yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-
imidazo[4,5-c]quinoline, ENT 2
55
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
11.3
(3)
6.49
(1)
yl)-2-{[4-(methoxymethyl)-1H-1,2,3-triazol-1-
yl]methyl}-1H-imidazo[4,5-c]quinoline, ENT 1
56
2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1-
6.46
(2)
6.48
(1)
[(3R)-1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl]-1H-
imidazo[4,5-c]quinoline-8-carbonitrile
57
2-[(4-methoxy-1H-pyrazol-1-yl)methyl]-1-
7.43
(2)
5.40
(1)
[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-
imidazo[4,5-c]quinoline-8-carbonitrile, formate
salt
58
8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-
5.35
(2)
3.00
(1)
pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-
imidazo[4,5-c]quinoline, formate salt
59
8-chloro-1-[cis-2-(difluoromethyl)tetrahydro-2H-
6.60
(2)
8.16
(1)
pyran-4-yl]-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-
1H-imidazo[4,5-c]quinoline, ENT 1
60
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
11.4
(2)
16.3
(1)
yl)-2-[(5-methyl-1,2-oxazol-3-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
61
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
5.79
(2)
4.17
(1)
(1,3-thiazol-2-ylmethyl)-1H-imidazo[4,5-
c]quinoline-8-carbonitrile, formate salt
62
8-chloro-1-[(3R)-1-methylpyrrolidin-3-yl]-2-[(4-
5.78
(3)
6.50
(1)
methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-
imidazo[4,5-c]quinoline
63
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
6.96
(2)
3.04
(1)
(1,2,3-thiadiazol-4-ylmethyl)-8-(trifluoromethyl)-
1H-imidazo[4,5-c]quinoline, formate salt
64
8-fluoro-1-[(2R,4R)-2-methyltetrahydro-2H-
9.80
(2)
6.87
(1)
pyran-4-yl]-2-(1,3-thiazol-2-ylmethyl)-1H-
imidazo[4,5-c]quinoline, formate salt
65
2-(1,3-benzoxazol-2-ylmethyl)-1-[cis-3-
8.56
(2)
6.33
(1)
fluorocyclopentyl]-1H-imidazo[4,5-
c]quinoline-8-carbonitrile
66
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
8.32
(2)
8.01
(1)
(1H-1,2,4-triazol-1-ylmethyl)-8-(trifluoromethyl)-
1H-imidazo[4,5-c]quinoline
67
8-chloro-2-[(5-methylpyrazin-2-yl)methyl]-1-
8.03
(3)
8.55
(1)
[(3R)-1-methylpyrrolidin-3-yl]-1H-imidazo [4,5-
c]quinoline
68
1-[cis-3-fluorocyclopentyl]-2-[(5-methyl-1,2-
8.32b
(2)
12.3b
(2)
oxazol-3-yl)methyl]-1H-imidazo[4,5-c] quinoline-
8-carbonitrile
69
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
7.39
(2)
3.33
(1)
(1,3-thiazol-4-ylmethyl)-8-(trifluoromethyl)-1H-
imidazo[4,5-c]quinoline, formate salt
70
2-[(5-methyl-1,3,4-oxadiazol-2-yl)methyl]-1-
13.7
(2)
7.59
(1)
[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-8-
(trifluoromethyl)-1H-imidazo[4,5-c] quinoline
71
8-chloro-1-(2,2-dimethyltetrahydro-2H-pyran-4-
13.7
(2)
11.9
(2)
yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-imidazo
[4,5-c]quinoline, formate salt
72
8-chloro-1-(2,2-difluoropropyl)-2-[(4-methoxy-
12.1
(2)
7.17
(1)
1H-pyrazol-1-yl)methyl]-1H-imidazo[4,5-c]
quinoline, formate salt
73
8-fluoro-1-[(2R,4R)-2-methyltetrahydro-2H-
16.9
(2)
13.0
(1)
pyran-4-yl]-2-{[5-(trifluoromethyl)pyrazin-2-
yl]methyl}-1H-imidazo[4,5-c]quinoline, formate
salt
74
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
26.1
(2)
13.5
(1)
[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-8-
(trifluoromethyl)-1H-imidazo[4,5-c] quinoline
75
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
25.7
(2)
26.5
(1)
yl)-2-[(5-methyl-1,2,4-oxadiazol-3-yl) methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
76
2-[(6-methylpyrimidin-4-yl)methyl]-1-[(2R,4R)-2-
17.3
(2)
12.0
(1)
methyltetrahydro-2H-pyran-4-yl]-8-
(trifluoromethyl)-1H-imidazo[4,5-c]quinoline
77
8-chloro-1-[cis-3-fluorocyclopentyl]-2-[(5-
13.0
(2)
11.8
(1)
methylpyrazin-2-yl)methyl]-1H-imidazo[4,5-c]
quinoline, formate salt
78
3-{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-
25.8
(2)
13.2
(1)
pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}-2-
methylpropanenitrile, DIAST 2
79
8-fluoro-1-[cis-3-fluorocyclopentyl]-2-(1,2,3-
9.73
(3)
9.75
(1)
thiadiazol-4-ylmethyl)-1H-imidazo[4,5-c]
quinoline, ENT 2
80
3-{8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-
9.40
(2)
11.3
(2)
pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-
yl}propanenitrile, formate salt
81
1-[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-2-
16.9
(2)
7.50
(1)
[(5-methyl-2H-tetrazol-2-yl)methyl]-8-
(trifluoromethyl)-1H-imidazo[4,5-c]quinoline,
formate salt
82
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
6.33
(2)
4.64
(1)
2-[(4-methyl-1H-1,2,3-triazol-1-yl)methyl]-1H-
imidazo[4,5-c]quinoline, trifluoroacetate salt
83
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
7.91
(2)
11.6
(1)
2-(1H-tetrazol-1-ylmethyl)-1H-imidazo[4,5-
c]quinoline, trifluoroacetate salt
84
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
10.2
(2)
18.2
(1)
2-[(1-methyl-1H-1,2,4-triazol-3-yl)methyl]-1H-
imidazo[4,5-c]quinoline, trifluoroacetate salt
85
1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-8-fluoro-
2.62
(2)
3.31
(1)
2-[(2-methylimidazo[2,1-b][1,3,4] thiadiazol-6-
yl)methyl]-1H-imidazo[4,5-c] quinoline, ENT 1
86
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
6.66
(2)
4.40
(1)
2-(1H-tetrazol-1-ylmethyl)-1H-imidazo [4,5-
c]quinoline, ENT 2
87
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
7.04
(2)
4.51
(1)
2-[(4-methyl-2H-1,2,3-triazol-2-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
88
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
9.25
(2)
8.16
(1)
2-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
89
8-chloro-1-(4,4-difluoro-1-methylpyrrolidin-3-yl)-
13.9
(2)
8.03
(1)
2-[(5-methyl-1,3,4-thiadiazol-2-yl)methyl]-1H-
imidazo[4,5-c]quinoline, ENT 2
90
8-(difluoromethyl)-1-[(2R,4R)-2-methyltetrahydro-
14.7
(2)
7.32
(1)
2H-pyran-4-yl]-2-[(4-methyl-1H-1,2,3-
triazol-1-yl)methyl]-1H-imidazo[4,5-c]quinoline,
trifluoroacetate salt
91
8-(difluoromethyl)-2-[(5-methyl-1,2-oxazol-3-
18.0
(4)
8.40
(3)
yl)methyl]-1-[(2R,4R)-2-methyltetrahydro-2H-
pyran-4-yl]-1H-imidazo[4,5-c]quinoline,
trifluoroacetate salt
92
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
10.9
(3)
6.91
(1)
yl)-2-[(5-methyl-1,3,4-thiadiazol-2-yl) methyl]-1H-
imidazo[4,5-c]quinoline, ENT 1
93
8-chloro-1-(3,3-difluorotetrahydro-2H-pyran-4-
11.9
(3)
8.70
(1)
yl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1H-
imidazo[4,5-c]quinoline, ENT 1
94
8-chloro-2-[(4-cyclopropyl-1H-1,2,3-triazol-1-
6.35
(3)
6.48
(1)
yl)methyl]-1-(3,3-difluorotetrahydro-2H-pyran-4-
yl)-1H-imidazo[4,5-c]quinoline, ENT-1
95
[5-({8-chloro-1-[(2R,4R)-2-methyltetrahydro-2H-
4.60
(13)
3.50
(4)
pyran-4-yl]-1H-imidazo[4,5-c]quinolin-2-yl}
methyl)pyrazin-2-yl]methanol
95A
8-chloro-2-[(5-methylpyrazin-2-yl)methyl]-1-
7.32
(6)
6.06
(5)
[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-
imidazo[4,5-c]quinoline
96
8-chloro-2-{[5-(2H3)methylpyrazin-2-yl]methyl}-1-
3.21
(1)
2.16
(1)
[(2R,4R)-2-methyltetrahydro-2H-pyran-4-yl]-1H-
imidazo[4,5-c]quinoline
aNot determined
bIn this case, the biological data was obtained using the formate salt of the Example.
Intrinsic Clearance (CLint) in Human Liver Microsomes
Incubations (in duplicate) contained either 95A or 96, or both 95A and 96 at final concentrations of 1 μM, human liver microsomes (BD Biosciences Bedford, Mass., 0.25 μM CYP protein equivalent to 0.801 mg/mL protein concentration), NADPH (1.3 mM), MgCl2 (3.3 mM) and potassium phosphate buffer (100 mM, pH 7.4). The final reaction volume (500 μL) contained 0.003% DMSO, 0.5% acetonitrile. The incubations were conducted at 37° C. and aliquots (50 μL) were removed at 0, 5, 10, 15, 20, 30, 45 and 60 minutes and quenched by addition to cold acetonitrile containing mass spectrometry (MS) internal standard (200 μL). Quenched incubations were vortex for 1 minute followed by centrifugation at 3000 rpm for 5 minutes at room temperature (Allegra X-12R, Beckman Coulter, Fullerton, Calif.). The supernatant (150 μL) was then removed and added to a 96-deep well injection plate containing 150 μL of water with 0.1% formic acid (v/v), the plates were capped and vortex for 1 minute and subsequently analyzed using LC-MS/MS as described below. Control incubations were prepared similar without adding the NADPH cofactor to monitor for any non-CYP/FMO metabolism. Discrete standard curves (0.5-2000 nM) were prepared, processed, and analyzed as described above.
The amount of substrate (95A or 96) and metabolite (95 or 96B) were measured and the results are shown in Tables 4a and Table 4b. Example 96 has a decreased intrinsic clearance (increased half-life=T1/2) in comparison to its corresponding undeuterated form (95A) which can be beneficial (e.g., decreased dosage) while maintaining beneficial properties. In addition, Example 96 has a lower rate of metabolite (96B) formation in comparison to the undeuterated metabolite (95) formed from Example 95A. In the combined substrates (competition) experiment, Example 96 shows a decreased intrinsic clearance (increased T1/2) in comparison to its corresponding undeuterated form (95A), and has a lower rate of metabolite formation in comparison to the undeuterated form (95A).
TABLE 4a
CLint in the human liver microsome
assay using individual substrates
Rate of
CLint
metabolite
Example
(μL/min/mg)
T½ (min)
(nmol/min/mg)
95A
15.98
54.4
8.54
96
6.68
90.4
1.10
TABLE 4b
CLint in the human liver microsome assay
using combined substrates - competition
Rate of
CLint
metabolite
Example
(μL/min/mg)
T½ (min)
(nmol/min/mg)
95A and 96
13.98
65.9
7.39
(for 95A)
95A and 96
6.86
126.6
1.65
(for 96)
Intrinsic Clearance (CLint) in Cynomolgus Monkey Liver Microsomes
Incubations (in duplicate) contained either 95A or 96, or both 95A and 96 at final concentrations of 1 μM, pooled cynomolgus monkey liver microsomes (Xenotech, LLC, Lenexa, Kans., 0.25 μM CYP protein equivalent to 0.21 mg/mL protein concentration), NADPH (1.3 mM), magnesium chloride (3.3 mM) and potassium phosphate buffer (100 mM, pH 7.4). The final reaction volume (500 μL) contained 0.003% DMSO, 0.5% acetonitrile. The incubations were conducted at 37° C. and aliquots (50 μL) were removed at 0, 5, 10, 15, 20, 30, 45 and 60 minutes and quenched by addition to cold acetonitrile containing mass spectrometry (MS) internal standard (200 μL). Quenched incubations were vortexed for 1 minute followed by centrifugation at 3000 rpm for 5 minutes at room temperature (Allegra X-12R, Beckman Coulter, Fullerton, Calif.). The supernatant (150 μL) was then removed and added to a 96-deep well injection plate containing 150 μL of water with 0.1% formic acid (v/v). The plates were capped and vortexed for 1 minute and subsequently analyzed using LC-MS/MS as described below. Control incubations were prepared similar without adding the NADPH cofactor to monitor for any non-CYP/FMO metabolism. Discrete standard curves (0.5-2000 nM) were prepared, processed, and analyzed as described above.
The amount of substrate (95A or 96) and metabolite (95 or 96B) were measured and the results are shown in Tables 5a and Table 5b. Example 96 has a decreased intrinsic clearance (increased half-life=T1/2) in comparison to its corresponding undeuterated form (95A) which can be beneficial (e.g., decreased dosage) while maintaining beneficial properties. In addition, Example 96 has a lower rate of metabolite (96B) formation in comparison to the undeuterated form (95A). In the combined substrates (competition) experiment, Example 96 showed a similar trend when compared to the individual substrate incubations. Example 96 showed a decreased intrinsic clearance (increased half-life=T1/2) in comparison to its corresponding undeuterated form (95A), and has a lower rate of metabolite formation in comparison to the undeuterated form (95A).
TABLE 5a
CLint in the monkeyliver microsome
assay using individual substrates
Rate of
CLint
metabolite
Example
(μL/min/mg)
T½ (min)
(nmol/min/mg)
95A
215.1
15.3
109.05
96
150.7
21.9
22.83
TABLE 5b
CLint in the monkeyliver microsome assay
using combined substrates - competition
Rate of
CLint
metabolite
Example
(μL/min/mg)
T½ (min)
(nmol/min/mg)
95A and 96
192.8
17.2
97.19
(for 95A)
95A and 96
110.4
29.9
25.19
(for 96)
Human Liver Microsomes Enzyme Kinetics
Incubations (in triplicate) contained 95A or 96 (1-1000 μM, final concentrations), pooled human liver microsomes (BD Biosciences, Bedford, Mass., 0.25 protein concentration, NADPH (1.3 mM), magnesium chrolide (5 mM) and potassium phosphate buffer (100 mM, pH 7.4). The final reaction volume (100 μL) contained 1 acetonitrile. The incubations were conducted at 37° C. At time points of 15 minutes for 95A, or 30 min for 96, 50 μL of incubate was quenched by the addition of 200 μL of cold acetonitrile containing 0.1% formic acid (v/v) and mass spectrometry (MS) internal standard. Quenched samples were vortexed for 1 minute followed by centrifugation at 3000 rpm for 5 minutes at room temperature (Allegra X-12R, Beckman Coulter, Fullerton, Calif.). The supernatant (150 μL) was placed into a clean injection sample block and dried down under nitrogen gas, then reconstituted with 150 μL of water containing 0.1% formic acid (v/v). The plates were capped and vortexed for 1 minute and subsequently analyzed using LC-MS/MS as described below. Formation of metabolites 95 (from substrate 95A) and 96B (from substrate 96) was quantitated using a standard curve (0.5-5000 nM) generated using a synthetic standard of substrate 95. Standard curve samples were prepared, processed, and analyzed as described above.
The kinetics of metabolite 95 or 96B formation determined in human liver microsomes are shown in Table 6. Again in this example, Example 96 has a decreased intrinsic clearance in comparison to its corresponding undeuterated form (95A) which can be beneficial (e.g., decreased dosage) while maintaining beneficial properties.
TABLE 6
Kinetic parameters for formation of Metabolites
95 or 96B from Substrates 95A or 96, respectively,
in the human liver microsomes
Vmax
CLint
Example
Km (μM)
(nmol/min/mg)
(μL/min/mg)
95A
15.23
0.229
15.06
96
64.93
0.0554
0.85
LC-MS/MS Analyses for Data Reported in Tables 4a, 4b, 5a, 5b and 6
Disappearance of substrates 95A and 96 and formation of metabolites 95 or 96B were determined using an LC-MS/MS system which is comprised of an AB Sciex 6500 triple quadrupole mass spectrometer equipped with an electrospray source (AB Sciex, Framingham, Mass.) and Agilent Technologies Infinity 1290 (Santa Clara, Calif.). A binary gradient was employed with a flow rate of 0.500 mL/min, using 0.1% formic acid in water as the aqueous mobile phase (solvent A) and 0.1% formic acid in acetonitrile (solvent B) as the organic phase. The LC gradient profile begins at 5% solvent B which was ramped to 98% B over 2 minutes and then held for 0.20 minutes and returned to initial conditions (5% B) over 0.5 minutes, for a total run time of 3.00 minutes. The analytical column used was a Phenomenex Kinetex 2.6 μm, 2.1×50 mm (Phenomenex, Torrance, Calif.), with an injection volume of 10 μL. The mass spectrometer was run under positive mode with the source temperature set to 500° C., ionization voltage set to 4.5 kV. The following MS/MS transitions were utilized: for substrate 95A (408→310), substrate 96 (411→313), metabolite 95 (424→326), and metabolite 96B (426→328). Analytes were quantified using Analyst software, version 1.6.2 or earlier (AB Sciex, Framingham, Mass.).
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application for all purposes.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
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16492558
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pfizer 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 27th, 2022 09:01AM
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Apr 27th, 2022 09:01AM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Apr 12th, 2022 12:00AM
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Oct 4th, 2018 12:00AM
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https://www.uspto.gov?id=US11299500-20220412
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Crystalline form of lorlatinib free base hydrate
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This invention relates to a crystalline form of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclo-tetradecine-3-carbonitrile (lorlatinib) free base hydrate (Form 24). This invention also relates to pharmaceutical compositions comprising Form 24, and to methods of using Form 24 and such compositions in the treatment of abnormal cell growth, such as cancer, in a mammal.
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11299500
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1. A crystalline form of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile (lorlatinib) free base hydrate, having a powder X-ray diffraction (PXRD) pattern comprising peaks at 2θ values of: 8.8, 9.7, 17.6 and 18.8 °2θ±0.2 °2θ.
2. The crystalline form of claim 1, having a PXRD pattern further comprising a peak at the 2θ value of: 10.9 °20±0.2 °2θ.
3. The crystalline form of claim 1, having a 13C solid state NMR spectrum comprising resonance (ppm) values of: 40.2, 41.2 and 136.2 ppm±0.2 ppm.
4. The crystalline form of claim 1, having a 19F solid state NMR spectrum comprising a resonance (ppm) value of: −104.0 ppm±0.2 ppm.
5. The crystalline form of lorlatinib free base hydrate of claim 1, for use as a medicament for treating cancer in a mammal, wherein the cancer is selected from the group consisting of ALK-positive non-small cell lung cancer (NSCLC) mediated by anaplastic lymphoma kinase (ALK) or c-ros oncogene 1 receptor tyrosine kinase (ROS1), ALK-positive anaplastic large cell lymphoma (ALCL), and ALK-positive neuroblastoma.
6. The crystalline form of lorlatinib free base hydrate of claim 1, for use in the treatment of cancer in a mammal, wherein the cancer is selected from the group consisting of ALK-positive non-small cell lung cancer (NSCLC) mediated by anaplastic lymphoma kinase (ALK) or c-ros oncogene 1 receptor tyrosine kinase (ROS1), ALK-positive anaplastic large cell lymphoma (ALCL), and ALK-positive neuroblastoma.
7. A pharmaceutical composition comprising the crystalline form of lorlatinib free base hydrate of claim 1, and a pharmaceutically acceptable carrier or excipient.
8. A method of treating cancer in a mammal comprising administering to the mammal a therapeutically effective amount of the crystalline form of lorlatinib free base hydrate of claim 1, wherein the cancer is selected from the group consisting of ALK-positive non-small cell lung cancer (NSCLC) mediated by anaplastic lymphoma kinase (ALK) or c-ros oncogene 1 receptor tyrosine kinase (ROS1), ALK-positive anaplastic large cell lymphoma (ALCL), and ALK-positive neuroblastoma.
9. A crystalline form of lorlatinib free base hydrate, having a 13C solid state NMR spectrum comprising two or more resonance (ppm) values of: 40.2, 41.2 and 136.2 ppm±0.2 ppm.
10. The crystalline form of claim 9, having a 13C solid state NMR spectrum further comprising the resonance (ppm) value of: 128.1 ppm±0.2 ppm.
11. The crystalline form of claim 9, having a 13C solid state NMR spectrum further comprising the resonance (ppm) value of: 145.3 ppm±0.2 ppm.
12. The crystalline form of claim 9, having a 19F solid state NMR spectrum comprising a resonance (ppm) value of: −104.0 ppm±0.2 ppm.
13. A crystalline form of lorlatinib free base hydrate, having a 19F solid state NMR spectrum comprising a resonance (ppm) value of: −104.0 ppm±0.2 ppm.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This Application is the national stage filing under 35 U.S.C. 371 of Patent Cooperation Treaty Patent Application No. PCT/IB2018/057735, filed Oct. 4, 2018, which claims the benefit of priority from U.S. Provisional Application No. 62/570,326 filed Oct. 10, 2017, and U.S. Provisional Application No. 62/727,734 filed Sep. 6, 2018, the contents of each of which are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to a new crystalline form of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiaza-cyclotetradecine-3-carbonitrile (lorlatinib) free base hydrate (Form 24), to pharmaceutical compositions comprising Form 24, and to methods of using Form 24 and such compositions in the treatment of abnormal cell growth, such as cancer, in mammals.
Description of the Related Art
The compound (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetra-hydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), represented by the formula (I):
has been assigned the International Nonproprietary Name (INN) lorlatinib, as described in WHO Drug Information, Vol. 29, No. 4, page 541 (2015). Lorlatinib is a potent, macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) receptor tyrosine kinase. Lorlatinib demonstrates nanomolar potency against both wild type and resistance mutant forms of ALK and ROS1, including ALK G1202R and ROS1 G2032R. It was designed to penetrate the blood-brain barrier and has demonstrated antitumor activity in ALK-positive NSCLC xenograft models and intracranial ALK-positive tumor models.
Preparation of the free base of lorlatinib as an amorphous solid is disclosed in International Patent Publication No. WO 2013/132376 and U.S. Pat. No. 8,680,111. Preparation of an acetic acid solvate and two hydrated forms of lorlatinib free base is disclosed in International Patent Publication No. WO 2014/207606. Preparation of an anhydrous crystalline free base (Form 7) of lorlatinib is disclosed in International Patent Publication No. 2017/021823. Preparation of a crystalline maleate salt of lorlatinib is disclosed in International Patent Publication No. WO 2017/175091. The contents of each of the foregoing documents are incorporated herein by reference in their entirety.
Chromosomal rearrangements of ALK and ROS1 define distinct molecular subtypes of non-small cell lung cancer (NSCLC), accounting for an estimated 2-7% and 1-3% of NSCLC cases, respectively. (Soda et al., Identification of the transforming EML4-ALK fusion gene in non-small cell lung cancer, Nature 2007; 448:561-566; Rikova et al., Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer, Cell 2007; 131:1190-1203; Bergethon et al., ROS1 rearrangements define a unique molecular class of lung cancers, J Clin Oncol. 2012; 30 (8): 863-70).
The ALK/ROS1/c-MET inhibitor crizotinib is the standard first-line therapy for patients with advanced or metastatic ALK- or ROS1-positive NSCLC. As observed clinically for other tyrosine kinase inhibitors, mutations in ALK and ROS1 that confer acquired resistance have been observed for crizotinib as well as second-generation ALK inhibitors, such as ceritinib, alectinib, and brigatinib (Gainor et al. Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALK-Rearranged Lung Cancer, Cancer Discov. 2016; 6 (10): 1118-33; Awad et al., Acquired Resistance to Crizotinib from a Mutation in CD74-ROS1, N Engl J Med 2013; 368:2395-2401). Thus, there remains a need to identify compounds having novel activity profiles against wild-type and mutant forms of ALK and ROS1.
The present invention provides a novel crystalline form of lorlatinib free base, hydrate Form 24, having desirable properties relative to anhydrous lorlatinib free base (Form 7) and lorlatinib acetic acid solvate (Form 3), such as improved physical stability in aqueous-based formulations (e.g., topical formulations), and improved properties relative to lorlatinib hydrate (Form 1), which had unfavorable properties such as high hygroscopicity and multiple thermal transitions.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the invention provides a novel crystalline form of lorlatinib free base hydrate (Form 24). Form 24 is characterized by one or more of the following methods: (1) powder X-ray diffraction (PXRD) (2θ); (2) 13C solid state NMR spectroscopy (ppm); or (3) 19F solid state NMR spectroscopy (ppm).
In a first aspect, the invention provides lorlatinib free base hydrate (Form 24), which is characterized by having:
(1) a powder X-ray diffraction (PXRD) pattern (2θ) comprising: (a) one, two, three, four, five, or more than five peaks selected from the group consisting of the peaks in Table 1 in °2θ±0.2 °2θ; (b) one, two, three, four or five peaks selected from the group consisting of the characteristic peaks in Table 1 in °2θ±0.2 °2θ; or (c) peaks at 2θ values essentially the same as shown in FIG. 1; or
(2) a 13C solid state NMR spectrum (ppm) comprising: (a) one, two, three, four, five, or more than five resonance (ppm) values selected from the group consisting of the values in Table 2 in ppm±0.2 ppm; (b) one, two, three, four or five resonance (ppm) values selected from the group consisting of the characteristic values in Table 2 in ppm±0.2 ppm; or (c) resonance (ppm) values essentially the same as shown in FIG. 2; or
(3) a 19F solid state NMR spectrum (ppm) comprising: (a) one, two or three resonance (ppm) values selected from the group consisting of the values in Table 3 in ppm±0.2 ppm; or (b) resonance (ppm) values essentially the same as shown in FIG. 3;
or a combination of any two or three of the foregoing embodiments (1)(a)-(c), (2)(a)-(c) or (3)(a)-(b), provided they are not inconsistent with each other.
In another aspect, the invention further provides a pharmaceutical composition comprising lorlatinib free base hydrate (Form 24), according to any of the aspects or embodiments described herein, and a pharmaceutically acceptable carrier or excipient.
The crystalline form of lorlatinib free base hydrate (Form 24), according to any of the aspects or embodiments described herein, may be used for the treatment of abnormal cell growth, such as cancer, in a mammal.
In one aspect, the invention provides a method of treating abnormal cell growth in a mammal, including a human, comprising administering to the mammal a therapeutically effective amount of the crystalline form of lorlatinib free base hydrate (Form 24).
In another aspect, the invention provides the crystalline form of lorlatinib free base hydrate (Form 24) for use as a medicament, particularly for use in the treatment of abnormal cell growth in a mammal, including a human.
In another aspect, the invention provides a method of treating abnormal cell growth in a mammal, comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising the crystalline form of lorlatinib free base hydrate (Form 24), according to any of the aspects or embodiments described herein.
In another aspect, the invention provides use of the crystalline form of lorlatinib free base hydrate (Form 24), or a pharmaceutical composition comprising such Form 24, according to any of the aspects or embodiments described herein, in a method of treating abnormal cell growth in a mammal.
In yet another aspect, the invention provides use of the crystalline form of lorlatinib free base hydrate (Form 24), according to any of the aspects or embodiments described herein, in the manufacture of a medicament for the treatment of abnormal cell growth in a mammal.
In another aspect, the invention provides the crystalline form of lorlatinib free base hydrate (Form 24) according to any of the aspects or embodiments described herein, or a pharmaceutical composition comprising such Form 24, for use as a medicament.
In another aspect, the invention provides the crystalline form of lorlatinib free base hydrate (Form 24) according to any of the aspects or embodiments described herein, or a pharmaceutical composition comprising such Form 24, for use in the treatment of abnormal cell growth in a mammal.
In frequent embodiments of each of the aspects described herein, the abnormal cell growth is cancer. In some such embodiments, the abnormal cell growth is cancer mediated by ALK or ROS1. In some embodiments, the abnormal cell growth is cancer mediated by ALK. In other embodiments, the abnormal cell growth is cancer mediated by ROS1. In further embodiments, the abnormal cell growth is cancer mediated by at least one genetically altered tyrosine kinase, such as a genetically altered ALK or a genetically altered ROS1 kinase. In some such embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), neuroblastoma, anaplastic large cell lymphoma (ALCL) and gastric cancer. In specific embodiments, the cancer is non-small cell lung cancer (NSCLC). In particular embodiments, the cancer is NSCLC mediated by ALK or ROS1, and more particularly, NSCLC mediated by a genetically altered ALK or a genetically altered ROS1.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1. PXRD pattern of lorlatinib free base hydrate (Form 24).
FIG. 2. 13C Carbon CPMAS spectrum of lorlatinib free base hydrate (Form 24). Peaks marked by hash marks are spinning sidebands.
FIG. 3. 19Fluorine MAS spectrum of lorlatinib free base hydrate (Form 24). Peaks marked by hash marks are spinning sidebands.
FIG. 4. PXRD pattern of uncoated prototype tablet of lorlatinib free base hydrate (Form 24).
FIG. 5. 13Carbon CPMAS spectrum of uncoated prototype tablet of lorlatinib free base hydrate (Form 24).
FIG. 6. Fluorine MAS spectrum of uncoated prototype tablet of lorlatinib free base hydrate (Form 24). Peaks marked by hash marks are spinning sidebands.
DETAILED DESCRIPTION OF THE INVENTION
The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art.
As used herein, the singular form “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “a” substituent includes one or more substituents.
The term “about” means having a value falling within an accepted standard of error of the mean, when considered by one of ordinary skill in the art.
As used herein, the term “essentially the same” means that variability typical for a particular method is taken into account. For example, with reference to X-ray diffraction peak positions, the term “essentially the same” means that typical variability in peak position and intensity are taken into account. One skilled in the art will appreciate that the peak positions (2θ) will show some variability, typically as much as ±0.2° 2θ. Further, one skilled in the art will appreciate that relative peak intensities will show inter-apparatus variability as well as variability due to degree of crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art, and should be taken as qualitative measures only. Similarly, 13C and 19F solid state NMR spectrum (ppm) show variability, typically as much as ±0.2 ppm.
The term “crystalline” as used herein, means having a regularly repeating arrangement of molecules or external face planes. Crystalline forms may differ with respect to thermodynamic stability, physical parameters, x-ray structure and preparation processes.
The term “amorphous” refers to a disordered solid state.
The term “solvate” as used herein, means having, in a lattice, a stoichiometric or non-stoichiometric amount of a solvent such as water, acetic add, methanol, etc., or mixtures thereof, bound by non-covalent intermolecular forces.
The term “hydrate” may be used specifically to describe a solvate comprising water.
The term “channel hydrate” may be used to describe stoichiometric or non-stoichiometric hydrates having water filled one dimensional channels or two dimensional planes in the crystal structure. The amount of water in the crystal lattice of a non-stoichiometric hydrate may vary with the partial pressure of water in the surrounding atmosphere and with temperature.
The invention described herein may be suitably practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms.
In one aspect, the invention provides lorlatinib free base hydrate (Form 24). As disclosed herein, Form 24 is a crystalline form of lorlatinib free base hydrate having improved physical stability in aqueous-based formulations, for example topical formulations. The methods described herein provide lorlatinib free base hydrate (Form 24) which is substantially pure and substantially free of alternative forms, including the anhydrous free base (Form 7) and the solvated forms (Forms 1, 2 and 3) disclosed previously. In preferred embodiments of each of the aspects described herein, lorlatinib free base hydrate (Form 24) is substantially pure.
As used herein, the term “substantially pure” means that the crystalline form contains at least 90%, preferably at least 95%, more preferably at least 97%, and most preferably at least 99% by weight of the indicated crystalline form (e.g., Form 24). Alternatively, it will be understood that “substantially pure” means that the crystalline form contains less than 10%, preferably less than 5%, more preferably less than 3%, and most preferably less than 1% by weight of impurities, including other polymorphic, solvated or amorphous forms.
As described herein, Form 24 was characterized by PXRD, and 13C and 19F solid state NMR spectroscopy. Such crystalline forms may be further characterized by additional techniques, such as Raman spectroscopy, Fourier-Transform InfraRed Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA) or Differential Thermal Analysis (DTA). Dynamic vapor sorption (DVS) methods may be used to explore the sorption/desorption behavior of Form 24 under various conditions of temperature and relative humidity (RH).
In some embodiments of each of the aspects of the invention, lorlatinib free base hydrate (Form 24) is characterized by its powder X-ray diffraction (PXRD) pattern. In other embodiments of each of the aspects of the invention, lorlatinib free base hydrate (Form 24) is characterized by its 13C solid state NMR spectrum. In still other embodiments of each of the aspects of the invention, lorlatinib free base hydrate (Form 24) is characterized by its 19F solid state NMR spectrum. Lorlatinib free base hydrate (Form 24) may be uniquely identified relative to lorlatinib solid forms described previously (e.g., Forms 1, 2, 3 and 7) by the characteristic peaks identified herein, using each of these techniques alone or in combination.
In further embodiments, lorlatinib free base hydrate (Form 24) is characterized by a combination of two or three of these methods. Exemplary combinations including two or more of the following are provided herein: powder X-ray diffraction (PXRD) pattern (2θ); 13C solid state NMR spectrum (ppm); or 19F solid state NMR spectrum (ppm). It will be understood that other combinations of techniques may be used to uniquely characterize lorlatinib free base hydrate (Form 24) disclosed herein.
In one embodiment, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising one or more peaks at 2θ values selected from the group consisting of: 8.8, 9.7, 10.9, 17.6 and 18.8 °2θ±0.2 °2θ. In another embodiment, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising two or more peaks at 2θ values selected from the group consisting of: 8.8, 9.7, 10.9, 17.6 and 18.8 °2θ±0.2 °2θ. In another embodiment, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising three or more peaks at 2θ values selected from the group consisting of: 8.8, 9.7, 10.9, 17.6 and 18.8 °2θ±0.2 °2θ.
In another embodiment, Form 24 has a PXRD pattern comprising peaks at 2θ values of: 8.8, 17.6 and 18.8 °2θ±0.2 °2θ. In some such embodiments, Form 24 has a PXRD pattern further comprising a peak at the 2θ value of: 9.7 °2θ±0.2 °2θ. In other such embodiments, Form 24 has a PXRD pattern further comprising a peak at the 2θ value of: 10.9° 2θ±0.2 °2θ.
In another embodiment, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising a peak at a 2θ value of: 8.8° 2θ±0.2 °2θ. In another embodiment, Form 24 has a PXRD pattern comprising a peak at a 2θ value of: 9.7 °2θ±0.2 °2θ. In another embodiment, Form 24 has a PXRD pattern comprising a peak at a 2θ value of: 10.9° 2θ±0.2 °2θ. In another embodiment, Form 24 has a PXRD pattern comprising a peak at a 2θ value of: 17.6° 2θ±0.2 °2θ. In another embodiment, Form 24 has a PXRD pattern comprising a peak at a 2θ value of: 18.8 °2θ±0.2 °2θ.
In another embodiment, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising peaks at 2θ values of: 8.8, 9.7, 17.6 and 18.8 °2θ±0.2 °2θ. In another embodiment, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising peaks at 2θ values of: 8.8, 10.9, 17.6 and 18.8 °2θ±0.2 °2θ. In yet another embodiment, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising peaks at 2θ values of: 8.8, 9.7, 10.9, 17.6 and 18.8 °2θ±0.2 °2θ. In some such embodiments, the PXRD pattern further comprises one or more additional peaks at 2θ values selected from the group consisting of the peaks in Table 1.
In specific embodiments, lorlatinib free base hydrate (Form 24) has a PXRD pattern comprising: (a) one, two, three, four, five, or more than five peaks selected from the group consisting of the peaks in Table 1 in °2θ±0.2 °2θ; (b) one, two, three, four or five peaks selected from the group consisting of the characteristic peaks in Table 1 in °2θ±0.2 °2θ; or (c) peaks at 2θ values essentially the same as shown in FIG. 1.
In one embodiment, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising one or more resonance (ppm) values selected from the group consisting of: 40.2, 41.2, 128.1, 136.2 and 145.3 ppm±0.2 ppm. In another embodiment, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising two or more resonance (ppm) values selected from the group consisting of: 40.2, 41.2, 128.1, 136.2 and 145.3 ppm±0.2 ppm. In another embodiment, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising three or more resonance (ppm) values selected from the group consisting of: 40.2, 41.2, 128.1, 136.2 and 145.3 ppm±0.2 ppm.
In some embodiments, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising the resonance (ppm) values of: 40.2, 41.2 and 136.2 ppm±0.2 ppm. In some such embodiments, Form 24 has a 13C solid state NMR spectrum further comprising the resonance (ppm) value of: 128.1 ppm±0.2 ppm. In other such embodiments, Form 24 has a 13C solid state NMR spectrum further comprising the resonance (ppm) value of: 145.3 ppm±0.2 ppm. In still other such embodiments, Form 24 has a 13C solid state NMR spectrum further comprising the resonance (ppm) values of: 128.1 and 145.3 ppm±0.2 ppm.
In another embodiment, Form 24 has a 13C solid state NMR spectrum comprising the resonance (ppm) values of: 40.2, 41.2, 128.1 and 136.2 ppm±0.2 ppm. In another embodiment, Form 24 has a 13C solid state NMR spectrum comprising the resonance (ppm) values of: 40.2, 41.2, 136.2 and 145.3 ppm±0.2 ppm. In another embodiment, Form 24 has a 13C solid state NMR spectrum comprising the resonance (ppm) values of: 40.2, 41.2, 128.1, 136.2 and 145.3 ppm±0.2 ppm.
In specific embodiments, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum (ppm) comprising: (a) one, two, three, four, five, or more than five resonance (ppm) values selected from the group consisting of the values in Table 2 in ppm±0.2 ppm; (b) one, two, three, four or five resonance (ppm) values selected from the group consisting of the characteristic values in Table 2 in ppm±0.2 ppm; or (c) resonance (ppm) values essentially the same as shown in FIG. 2.
In one embodiment, lorlatinib free base hydrate (Form 24) has a 19F solid state NMR spectrum comprising a resonance (ppm) value of: −104.0 ppm±0.2 ppm.
In another embodiment, Form 24 has a 19F solid state NMR spectrum (ppm) comprising: (a) one, two or three resonance (ppm) values selected from the group consisting of the values in Table 3 in ppm±0.2 ppm; or (b) resonance (ppm) values essentially the same as shown in FIG. 3.
In further embodiments, lorlatinib free base hydrate (Form 24) is characterized by a combination of two or three of the embodiments described above that are not inconsistent with each other.
Exemplary embodiments that may be used to uniquely characterize lorlatinib free base hydrate (Form 24) are provided below.
In one embodiment, lorlatinib free base hydrate (Form 24) has a powder X-ray diffraction pattern comprising peaks at 2θ values of: 8.8, 17.6 and 18.8 °2θ±0.2 °2θ.
In another embodiment, lorlatinib free base hydrate (Form 24) has a powder X-ray diffraction pattern comprising peaks at 2θ values of: 8.8, 9.7, 17.6 and 18.8 °2θ±0.2 °2θ.
In another embodiment, lorlatinib free base hydrate (Form 24) has a powder X-ray diffraction pattern comprising peaks at 2θ value of: 8.8, 10.9, 17.6 and 18.8 °2θ±0.2 °2θ.
In another embodiment, lorlatinib free base hydrate (Form 24) has a powder X-ray diffraction pattern comprising peaks at 2θ value of: 8.8, 9.7, 10.9, 17.6 and 18.8 °2θ±0.2 °2θ.
In still another embodiment, lorlatinib free base hydrate (Form 24) has a 19F solid state NMR spectrum comprising the resonance (ppm) value of: −104.0 ppm±0.2 ppm.
In another embodiment, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising resonance (ppm) values of: 40.2, 41.2 and 136.2 ppm±0.2 ppm.
In another embodiment, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising resonance (ppm) values of: 40.2, 41.2, 128.1 and 136.2 ppm±0.2 ppm.
In yet embodiment, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising resonance (ppm) values of: 40.2, 41.2, 136.2 and 145.3 ppm±0.2 ppm.
In still another embodiment, lorlatinib free base hydrate (Form 24) has a 13C solid state NMR spectrum comprising resonance (ppm) values of: 40.2, 41.2, 128.1, 136.2 and 145.3 ppm±0.2 ppm.
In a further embodiment, lorlatinib free base hydrate (Form 24) has: (a) a powder X-ray diffraction pattern comprising peaks at 2θ values of: 8.8, 17.6 and 18.8 °2θ±0.2 °2θ; and (b) a 13C solid state NMR spectrum comprising resonance (ppm) values of: 40.2, 41.2 and 136.2 ppm±0.2 ppm.
In a further embodiment, lorlatinib free base hydrate (Form 24) has: (a) a powder X-ray diffraction pattern comprising peaks at 2θ values of: 8.8, 17.6 and 18.8 °2θ±0.2 °2θ; and (b) a 19F solid state NMR spectrum comprising the resonance (ppm) value of: −104.0 ppm±0.2 ppm.
In a further embodiment, lorlatinib free base hydrate (Form 24) has: (a) a 13C solid state NMR spectrum comprising resonance (ppm) values of: 40.2, 41.2 and 136.2 ppm±0.2 ppm; and (b) a 19F solid state NMR spectrum comprising the resonance (ppm) value of: −104.0 ppm±0.2 ppm.
In another aspect, the invention provides a pharmaceutical composition comprising lorlatinib free base hydrate (Form 24) characterized according to any of the embodiments described herein, and a pharmaceutically acceptable carrier or excipient.
In another aspect, the invention provides method of treating abnormal cell growth in a mammal, preferably a human, comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition of the invention.
The term “therapeutically effective amount” as used herein refers to that amount of a compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, and/or (4) relieving to some extent (or, preferably, eliminating) one or more signs or symptoms associated with the cancer.
As used herein, “mammal” refers to a human or animal subject. In certain preferred embodiments, the mammal is a human.
The term “treating”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term “treatment”, as used herein, unless otherwise indicated, refers to the act of treating as “treating” is defined immediately above. The term “treating” also includes adjuvant and neo-adjuvant treatment of a subject.
“Abnormal cell growth”, as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). Abnormal cell growth may be benign (not cancerous), or malignant (cancerous). In frequent embodiments of the methods provided herein, the abnormal cell growth is cancer.
As used herein “cancer” refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth. The term “cancer” includes but is not limited to a primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of different type from latter one.
In some embodiments, the abnormal cell growth is cancer mediated by an anaplastic lymphoma kinase (ALK). In some such embodiments, the ALK is a genetically altered ALK. In other embodiments, the abnormal cell growth is cancer mediated by ROS1 kinase. In some such embodiments, the ROS1 kinase is a genetically altered ROS1 kinase. In frequent embodiments, the abnormal cell growth is cancer, in particular NSCLC. In some such embodiments, the NSCLC is mediated by ALK or ROS1. In specific embodiments, the cancer is NSCLC is mediated by genetically altered ALK or genetically altered ROS1.
The moisture sorption/desorption behavior for lorlatinib free base hydrate (Form 24) revealed that this structure gradually sorbs/desorbs different levels of water as a result of a change of relative humidity. Such a moisture profile is typical of a channel (variable) hydrate. Analysis indicates that both sorption and desorption processes for all humidity levels did not reach equilibrium easily, as the structure sorbs and desorbs water very slowly and gradually. The observed behavior was found to be reproducible for different batches of Form 24. The water levels changes between a dry and wet state of Form 24 were examined as well. The change of mass for Form 24 between these two states was below 1%. Although the structure gradually increased in weight over time when exposed to high relative humidity, the stoichiometric water levels for mono or hemi-hydrate forms were not achieved. Form 24 may in some instances be referred as a non-stoichiometric hydrate or a non-stoichiometric channel hydrate.
Pharmaceutical compositions of the present invention may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.
Exemplary parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. The pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus for oral administration, tablets containing various excipients, such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Preferred materials include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be apparent, to those skilled in this art. For examples, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).
EXAMPLES
The examples and preparations provided below further illustrate and exemplify particular aspects and embodiments of the invention. It is to be understood that the scope of the present invention is not limited by the scope of the following examples.
General Method 1. Powder X-ray Diffraction (PXRD)
The PXRD data in FIGS. 1 and 4 were collected according to the following general protocol.
Instrument Method
PXRD patterns were collected on a Bruker-AXS Ltd. D4 powder X-ray diffractometer fitted with an automatic sample changer, a theta-theta goniometer, automatic beam divergence slit, and a PSD Vantec-1 detector. The X-ray tube voltage and amperage were set to 40 kV and 40 mA respectively. The diffractometer was aligned and a calibration check performed using a corundum reference material on the day of data collection. Data was collected at the Cu K-alpha wavelength using a step size of 0.018 degrees and scan time and 11.3 hours scanning from 2.0 to 65.0 degrees 2-theta. The sample powders were prepared by placing the powder in low background cavity holder. The sample powder was pressed by a glass slide to ensure that a proper sample height was achieved. The prototype 25 mg uncoated tablet (lot 00708989-0037-3) sample was cleaved to unsure appropriate height and also smooth and even tablet surface. It was mounted on to a set specimen holder, secured with blue tack and covered with Kapton film. Data were collected using Bruker DIFFRAC software and analysis was performed by DIFFRAC EVA software (Version 3.1).
Peak Picking Method
The PXRD patterns collected were imported into Bruker DIFFRAC EVA software. The peak selection carried out manually was carefully checked to ensure that all peaks had been captured and all peak positions had been accurately assigned. The peak list for peaks with 2-theta lower than 25 2-theta degrees was extracted from the Bruker software into Microsoft Excel and peak intensities were normalized relative to highest intensity peak equaling 100%. A typical error of ±0.2° 2-theta in peak positions applies to this data. The minor error associated with this measurement can occur as a result of a variety of factors including: (a) sample preparation (e.g., sample height), (b) instrument, (c) calibration, (d) operator (including those errors present when determining the peak locations), and (e) the nature of the material (e.g. preferred orientation and transparency errors). Therefore peaks are considered to have a typical associated error of ±0.2° 2-theta. When two peaks, in the list, are considered to overlap (±0.2° 2-theta) the less intense peak has been removed from the listing. Peaks existing as shoulders, on a higher intensity adjacent peak, have also been removed from the peak list. While the shoulders may be >0.2° 2-theta from the position of the adjacent peak, they are not considered as discernible from the adjacent peak. In order to obtain the absolute peak positions, the powder pattern should be aligned against a reference. This could either be the simulated powder pattern from the crystal structure of the same form solved at room temperature, or an internal standard e.g. silica.
The PXRD profile for the active pharmaceutical ingredient (API) is provided in FIG. 1 and selected peak positions are provided in Table 1. The PXRD profile for a 25 mg uncoated prototype tablet is provided in FIG. 4.
General Method 2. Solid State NMR (ssNMR) Spectroscopy
The carbon CPMAS and fluorine MAS ssNMR data in FIGS. 2, 3, 5 and 6 were collected according to the following general protocol.
Instrument Method
Solid state NMR (ssNMR) analysis on Form 24 was conducted at ambient temperature on a Bruker-BioSpin CPMAS probe positioned into a Bruker-BioSpin Avance III 500 MHz (1H frequency) NMR spectrometer. The packed rotor was oriented at the magic angle and spun at 15.00 kHz. The carbon ssNMR spectrum was collected using a proton decoupled cross-polarization magic angle spinning experiment. A phase modulated proton decoupling field of 80-90 kHz was applied during spectral acquisition. The cross-polarization contact time was set to 2 ms and the recycle delay to 5 seconds. The number of scans was adjusted to obtain an adequate signal to noise ratio. The carbon spectrum was referenced using an external standard of crystalline adamantane, setting its upfield resonance to 29.5 ppm (as determined from neat TMS). The fluorine ssNMR spectrum was collected using a proton decoupled direct polarization magic angle spinning experiment. A phase modulated proton decoupling field of 80-90 kHz was applied during spectral acquisition. The recycle delay was set to 50 seconds. The number of scans was adjusted to obtain an adequate signal to noise ratio. The direct polarization fluorine spectrum was referenced using an external standard of 50/50 volume/volume of trifluoroacetic acid and water, setting its resonance to −76.54 ppm.
Peak Picking Method
Automatic peak picking was performed using Bruker-BioSpin TopSpin version 3.2 software. Generally, a threshold value of 5% relative intensity was used to preliminary select peaks. The output of the automated peak picking was visually checked to ensure validity and adjustments were manually made if necessary.
Although specific 13C and 19F solid state NMR peak values are reported herein, a range for these peak values exists due to differences in instruments, samples, and sample preparation. This is common practice in the art of solid state NMR because of the variation inherent in peak values. A typical variability for a 13C and 19F chemical shift x-axis value is on the order of plus or minus 0.2 ppm for a crystalline solid. The solid state NMR peak heights reported herein are relative intensities. The solid state NMR intensities can vary depending on the actual setup of the experimental parameters and the thermal history of the sample.
The 13C solid state NMR spectrum of Form 24 API is provided in FIG. 2 and selected peak positions are provided in Table 2. 19F solid state NMR spectrum of Form 24 API is provided in FIG. 3 and selected peak positions are provided in Table 3. The 13C solid state NMR and 19F solid state NMR spectra for a 25 mg uncoated prototype tablet are provided in FIGS. 5 and 6.
Example 1
Preparation of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetra-decine-3-carbonitrile (lorlatinib) Free Base Hydrate (Form 24)
A sample of anhydrous lorlatinib free base (Form 7) (1.52, 3.74 mmol) was added to a 15 mL vial. 4.80 mL of isopropyl acetate and 0.20 mL of water was added to give a water activity of approximately 0.35 and the mixture was stirred. The mixture was left to slurry for 24 hours on a roller mixer before the sample was isolated using vacuum filtration briefly to yield a damp product. A sample of the isolated product was analyzed using PXRD and was confirmed to be pure isopropyl acetate solvate of lorlatinib. The experiment gave a yield of 1.49777 g of damp isopropyl acetate solvate, which was not rigorously dried before taking into the next step. Four samples of the isopropyl acetate solvate, (306.88, 298.74, 298.31 and 303.50 mg, respectively) were added to four separate 2 mL vials. 700 μL of toluene was added to each of the vials along with magnetic stirrer bars and the vials were capped. The mixtures were heated to 80° C. and left to slurry for 72 hours before the slurry was isolated by centrifuge filtration. The samples were transferred to 0.22 μm nylon centrifuge filters and centrifuged at 13,200 rpm at 20° C. for 10 minutes. The isolated products were analyzed using PXRD and confirmed to be pure lorlatinib free base hydrate (Form 24).
Characterization of Lorlatinib Free Base Hydrate (Form 24)
PXRD Data
FIG. 1 shows PXRD data for lorlatinib free base hydrate (Form 24), collected according to General Method 1. A list of PXRD peaks at diffraction angles 2-Theta ° (° 2θ)±0.2 °2θ and their relative intensities is provided in Table 1. Characteristic PXRD peaks distinguishing Form 24 are indicated by an asterisk (*).
TABLE 1
PXRD Peak List for Form 24 (2-Theta °)
Angle
Intensity
°2θ ± 0.2 °2θ
%
8.8*
28.1
9.7*
100.0
10.9*
10.6
11.9
9.6
12.3
11.1
13.1
12.1
15.7
18.4
16.0
21.0
17.0
27.0
17.6*
32.7
18.8*
20.7
19.5
37.2
20.7
22.6
21.0
23.0
21.7
37.3
22.3
56.1
22.9
26.5
23.4
33.5
ssNMR data
FIG. 2 shows the carbon CPMAS spectrum of lorlatinib free base hydrate (Form 24), which was collected according to General Method 2. Chemical shifts are expressed in parts per million (ppm) and are referenced to external sample of solid phase adamantane at 29.5 ppm. A list of ssNMR 13C chemical shifts (ppm) for Form 24 is provided in Table 2 in ppm±0.2 ppm. Characteristic 13C ssNMR chemical shifts (ppm)distinguishing Form 24 are indicated by an asterisk (*).
TABLE 2
13C ssNMR peak list for Form 24 (ppm)
13C Chemical Shifts
Relative
[ppm ± 0.2 ppm]
Intensity
23.8
50
24.2
56
25.0
48
29.5
37
30.9
41
31.6
41
38.2
38
40.2*
41
41.2*
36
47.3
49
47.8
43
71.3
44
72.3
79
109.6
9
110.8
7
112.7
19
113.4
42
115.0
100
115.8
59
116.5
51
117.4
60
118.5
49
126.1
23
126.7
33
127.5
27
128.1*
51
129.8
30
132.1
35
132.5
38
133.0
34
136.2*
53
137.0
32
138.5
30
139.2
33
139.9
29
142.2
34
142.8
36
143.8
47
144.4
38
145.3*
29
149.9
23
150.2
23
162.5
17
163.3
20
164.3
12
165.3
10
168.2
23
169.2
24
169.7
26
FIG. 3 shows the fluorine MAS (ssNMR) spectrum of lorlatinib free base hydrate (Form 24), collected according to General Method 2. Chemical shifts are expressed in parts per million (ppm) referenced to an external sample of trifluoroacetic acid (50% V/V in H2O) at −76.54 ppm.
The 19F ssNMR chemical shift (ppm) for Form 24 is provided in Table 3 in ppm±0.2 ppm. The characteristic ssNMR 19F chemical shifts (ppm) distinguishing Form 24 are indicated by an asterisk (*).
TABLE 3
19F ssNMR Peak list for Form 24 (ppm)
19F Chemical Shifts
Relative
[ppm ± 0.2 ppm]
Intensity
−104.0*
85
−107.9
100
−108.2
89
Example 2
Representative Drug Product Formulations of Lorlatinib Free Base hydrate (Form 24)
Prototype tablets comprising Form 24 of lorlatinib free base hydrate may be prepared using conventional excipients commonly used in tableted formulations.
Tablets typically contain from 1-30% of API on a w/w basis. Microcrystalline cellulose (e.g., 35-60% w/w), dibasic calcium phosphate anhydrous (DCP) (e.g., 10-35% w/w) may be used as tablet fillers. Other fillers, e.g., lactose monohydrate, may also be used in place of DCP. Sodium starch glycolate (e.g., 2-5% w/w) may be used as a disintegrant. Magnesium stearate (0.5-1.5% w/w) may be used as a lubricant.
Tablets may be manufactured using a dry granulation process prior to compression. In this process the crystalline material is blended with some proportion of the excipients falling within the typical ranges outlined above and the blend is dry granulated using a roller compactor. The granule may be milled as part of this process. The granules are blended with remainder of any of the excipients (e.g., magnesium stearate) prior to compression.
A representative tablet formulation of Form 24 is provided in Table 4.
TABLE 4
Example composition of prototype 25 mg uncoated DCP tablet,
comprising 10% w/w of lorlatinib free base hydrate (Form 24)
Component
Role
Mass %
lorlatinb free base hydrate
Active ingredient
10.00
Form 24
Cellulose Avicel PH 102
Filler
57.33
Dibasic Calcium Phosphate
Filler
28.67
DiCAFOS A12
Sodium Starch Glycolate
Disintegrant
3.00
(Explotab)
Magnesium Stearate
Lubricant
1.00
FIG. 4 shows the PXRD patterns of the prototype 25 mg uncoated tablet comprising lorlatinib free base hydrate (Form 24). FIGS. 5 and 6 show the 13C and 19F ssNMR spectra for the prototype 25 mg uncoated tablets, respectively.
Example 3
Water Sorption/Desorption Study for Lorlatinib Free Base Hydrate (Form 24)
A DVS method was employed to study the sorption/desorption behavior of Form 24.
Method: The method involved a standard sorption run from 0% RH to 90% RH, employing humidity rise at steps of 10% RH at 20° C., followed by a de-sorption run down to 0% RH and a second sorption/desorption cycle from 0% RH to 90% RH in 10% step change of RH at 20° C.
Results
Under the standard method, the material gradually increased the mass uptake of water at each humidity step from 0 to 90% RH. Specific mass increases due to water sorption at each relative humidity are provided in Table 5.
TABLE 5
Percent water absorbed in Form 24
Targeted relative
% Water
% Water
humidity (% RH)
First Cycle
Second Cycle
0
0.00
0.00
10
0.06
0.06
20
0.13
0.13
30
0.19
0.20
40
0.27
0.27
50
0.34
0.35
60
0.43
0.44
70
0.52
0.54
80
0.64
0.65
90
0.78
0.78
A significant mass increase was observed at 90% RH compared to 0% RH. For both sorption cycles, the mass uptake at each humidity level exhibits an initial increase and then slowly continues to gradually increase with time. This may indicate that the rate of sorption and de-sorption is limited primarily by the rate of diffusion of water molecules through the pores of the crystal. Similar behavior was observed in the desorption cycles (i.e., a slight initial drop followed up by a gradual continuous drop). The de-sorption isotherms displayed very little hysteresis with respect to sorption isotherm, another feature typical of channel hydrates.
Since the increase of mass (sorption) and decrease of mass (de-sorption) gradually continued to change at each humidity level, it was hypothesized that equilibrium at each humidity had not been reached.
For reference, stoichiometrically, a monohydrate corresponds to 4.2% change in mass and a 1:3 water: API stoichiometry corresponds to 1.4% change in mass. The measured change in mass for Form 24 was lower than the theoretically calculated stoichiometry, and therefore this structure can be considered a non-stoichiometric hydrate.
Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention.
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16650505
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pfizer 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 12th, 2022 12:12PM
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Apr 12th, 2022 12:12PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Jun 19th, 2012 12:00AM
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Aug 4th, 2004 12:00AM
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https://www.uspto.gov?id=US08201590-20120619
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Method and apparatus for filling a container
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A method and apparatus for filling with powder (4) a container (8) having an open end, including positioning an outlet of a hopper (2) containing powder above the open end of the container (8), mechanically agitating the hopper so as to cause powder (4) to be transferred from the hopper to the container and mechanically agitating the container, wherein the steps of mechanically agitating are conducted by at least a predetermined amount sufficient to ensure that the container is filled with powder at a predetermined density.
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8201590
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1. An apparatus for filling with powder a container having an open end, the apparatus including: a support for the container; a hopper having an outlet and being selectively moveable relative to the support to position the outlet above the open end of a supported container; a clamp for clamping the hopper and the container together; a dispenser for mechanically tapping the hopper and the container so as to cause powder to be transferred from the hopper to the container, the dispenser being arranged to tap the clamped hopper and container; and a controller for operating the dispenser by at least a predetermined amount sufficient to ensure that powder in the container reaches a predetermined density.
2. A method of filling with powder a container having an open end, the method including: positioning an outlet of a hopper containing powder above the open end of the container; mechanically agitating the hopper so as to cause powder to be transferred from the hopper to the container; and mechanically agitating the container; wherein the hopper and the container are clamped together and the steps of mechanically agitating include tapping the clamped hopper and container by at least a predetermined amount sufficient to ensure that the container is filled with powder at a predetermined density.
3. A method according to claim 2 further including: using the volume of the container to define a predetermined volume for the powder.
4. A method according to claim 3 further including: filling the entire volume of the container with powder, the volume of the container equaling the predetermined volume.
5. A method according to claim 4 further including: for at least some of the step of mechanically agitating the hopper, spacing the outlet of the hopper away from the open end of the container so as to overfill the container; and after the steps of mechanically agitating, removing excess powder from the open end of the container.
6. A method according to claim 4 further including; positioning the outlet of the hopper across the open end of the container such that the container is filled level with the open end.
7. A method according to claim 3 further including: positioning the outlet of the hopper at a predetermined level within the container so as to define, with the container, the predetermined volume, the predetermined volume being smaller than the volume of the container.
8. A method according to claim 2 further including: providing the outlet of the hopper with one of an orifice, mesh, screen and grid to separate the powder in the hopper from the container.
9. A method according to claim 8 further including: providing the orifice, mesh, screen or grid with a hole-size small enough that bulk density powder will not flow through under gravity, but large enough to allow powder to fall through during the step of mechanically agitating.
10. A method according to claim 8 or 9 further including: providing the orifice, mesh, screen or grid with a hole-size of approximately 0.5 mm.
11. A method according to claim 2 wherein the steps of mechanically agitating include lifting the hopper and container by 1 to 10 mm, then letting the hopper and container fall under gravity to a substantially fixed position.
12. A method according to claim 2 wherein the step of mechanically agitating provides an acceleration of approximately 1000 G to powder in the hopper and container.
13. A method according to claim 2 wherein the steps of mechanically agitating include tapping the clamped hopper and container between 50 and 500 times.
14. A method according to claim 2 further including: tapping vibrating the hopper and/or container at a frequency between 100 Hz and 1 kHz.
15. A method according to claim 2, 11 or 12 further including: providing a powder-tight seal between the hopper and container during at least part of the step of mechanically agitating the hopper.
16. A method according to claim 2 including: adjusting the amount of mechanical agitation of the container so as to vary the density of the powder in the container, thereby compensating for batch-to-batch variations in the powder.
17. A method according to claim 2 wherein at least the step of mechanically agitating the container provides impulses to the powder in a direction from the open end of the container into the container.
18. A method of simultaneously filling with powder a plurality of containers having respective open ends, the method including: providing a hopper having a plurality of outlets positioning the plurality of outlets above corresponding open ends of the containers; and simultaneously conducting the method of claim 2, 11 or 12 for each container.
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18
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The present invention relates to a method of filling with powder a container having an open end, a method of simultaneously filling a plurality of such containers and an apparatus for carrying out such methods.
When factory packing unit doses of drug into individual containers, there is a requirement to achieve protection of the drug from the atmosphere. The fill weight (drug mass) must be accurate, aiming for better than 5% RSD (relative standard deviation).
Cohesive powders are difficult to push into a small container, because they stick to the walls and to themselves, causing inhomogeneous filling. If high force is used to overcome this, then the powders compact into a solid mass. This is especially disadvantageous for DPI (dry powder inhalation) applications where the powder must be sucked out of the container by the patients inhaled air stream.
Methods are known for filling. Dosators tubes can be used. The tube is pushed into a powder bed, lifted out with powder stuck in the tube and moved to the container. The powder is then pushed out of tube into the container. It is also known to push a container into a powder bed upside down, such that powder sticks in the container, and then wipe off the excess. It is also known to tap powder into the container, weigh the container and stop tapping when the container holds the right amount. Finally, it is known to suck powder into a transfer tube of known volume, transfer the tube to the container and blow out the powder into the container.
Generally these methods have difficulty filling a small container so that it is full to the brim with no powder deposited on the surface surrounding the container, and the density in the pocket is higher than bulk density.
Wo 97/05018 describes a method and apparatus for filling cavities, in particular for filling cavities with a powder which is in a free-flowing agglomerated form and is made to flow from a hopper by subjecting the hopper to vibration. It indicates that it is possible to accurately switch the flow of the powder on and off using vibrations. The cavities may be formed in a disc with a circular configuration. The disc can be placed on a turntable and subjected to vibrations. This document explains that the effect of the vibrations is to cause the cavities at the periphery of the dose ring to fill uniformly with powder as they pass underneath the hopper outlet. The vibrations also to cause excess powder in the cavities and on the upper face of the dose ring to move along the face to the next cavity or to fall of the edge of the dose ring. This document also teaches the possibility of locking the dose holder (in which the cavities are formed) and the hopper into engagement, such that the powder flows directly into each cavity and the upper face of the dose holder between the cavities remains clean of powder.
Thus, WO 97/05018 teaches a system which uses vibrations to ensure that each cavity is properly filled. The vibrations ensure that powder flows from the hopper to the cavity and then ensures that the powder continues to flow within the cavity, such that it does not leave spaces or air pockets to the side or in the middle of the cavity and, in this sense, achieves a uniform density. However, in WO 97/05018, the actual density of the resulting powder in the cavity is not considered. WO 97/05018 proposes one system where vibrations are provided until the cavity is completely full and another system which, on the basis that the flow rate of powder into the cavity is substantially constant provided that the amplitude and frequency of vibration remains constant, determines the fill weight by carefully timing the duration of vibrator operation. There is no consideration of the fact that, for a particular volume, for instance the total volume of the cavity, the density of the powder can vary, such that fill weight will vary. This is different to merely ensuring that the volume is full of powder and has no air pockets or spaces.
It is an object of the present invention to overcome or at least reduce the shortcomings of previous methods and devices.
The present invention is based on the realisation that a predetermined mechanical agitation of a container containing powder will result in that powder settling over time to a stable predetermined and reproducable density. The mechanical agitation should cause vertical acceleration to the particles of powder and is preferably produced by tapping.
According to the present invention there is provided a method of filling with powder a container having an open end, the method including positioning an outlet of a hopper containing powder above the open end of the container, mechanically agitating the hopper so as to cause powder to be transferred from the hopper to the container, and mechanically agitating the container wherein the steps of mechanically agitating are conducted by at least a predetermined amount sufficient to ensure that the container is filled with powder at a predetermined density.
By mechanically agitating the hopper, powder will be transferred from the hopper to the container. By then mechanically agitating the container, the powder will settle in the container and be brought to the reproducible condition known as “tap density”. The powder will be brought to the tap density after a predetermined amount of agitation to the container. Further agitation will not increase the density by any significant amount. Hence, in this way, it is not necessary to monitor the amount of powder in the container. The amount of agitation provided to the container can be measured, for instance, by the time for which the container is agitated, the number of taps given to the container or the frequency or magnitude of vibration. Where the container is of a known volume and filling is carried out to a predetermined level, for instance, determined by the outlet of the hopper, a known mass of powder can be provided on the basis of a predetermined density. Additionally, it is possible to end the tapping at a point before tap density has been achieved. During the last portion of tapping to reach tap density, the container will be fully filled with powder with the density increasing slowly with each tap.
Additionally, in this range, typically above 90% of tap density, the behavior of the powder is very reproducible. Thus, by altering the number of taps used, it is possible to fully fill the container and control the density of the powder within it in a reproducible way over the range from 90% to 100% of tap density. This enables small alterations to the fill weight to be achieved. This is useful to allow for batch-to-batch variations in the powder.
Preferably the method includes using the volume of the container to define a predetermined volume for the powder.
In this way, a predetermined mass can be achieved by virtue of the predetermined volume.
Preferably the method further includes filling the entire volume of the container with powder, the volume of the container equalling the predetermined volume.
In this way, the volume of the container can be used to determine the mass of powder.
Preferably the method includes, for at least some of the step of mechanically agitating the hopper, spacing the outlet of the hopper away from the open end of the container so as to overfill the container and, after the steps of mechanically agitating, removing excess powder from the open end of the container.
In particular, it is preferred that the hopper fills the container and powder is caused to settle in the container before the hopper is moved away from the open end of the container. By further agitating the hopper when spaced from the open end of the container, it is ensured that the container is completely filled with powder. This overcomes the possibility that, when the hopper is moved away from the open end of the container, it takes with it some powder from the top of the container.
Preferably the method further includes positioning the outlet of the hopper across the open end of the container such that the container is filled level with the open end.
In this way, the outlet of the hopper defines the predetermined volume of powder as being the volume of the container up to a position level with the open end.
Alternatively, the method further includes positioning the outlet of the hopper at a predetermined level within the container so as to define, with the container, the predetermined volume, the predetermined volume being smaller than the volume of the container.
In this way, the container can still be used to defme a predetermined volume. However, since the outlet of the hopper extends to a position within the container, the top surface of the predetermined volume of powder in the container is below the level of the open end. In this way, there is a reduced likelihood of any powder being deposited on the container around its open end. Furthermore, the predetermined volume can easily be adjusted by adjusting the extent to which the outlet of the hopper protrudes into the container.
Preferably the method further includes providing the outlet of the hopper with one of an orifice, mesh, screen and grid to separate the powder in the hopper from the container.
This provides an effective way of maintaining powder in the hopper until the mechanical agitation is provided to the hopper.
Preferably the method further includes providing the orifice, mesh, screen or grid with a hole-size small enough that bulk density powder will not flow through under gravity, but large enough to allow powder to fall through during the step of mechanically agitating.
In this way, the hopper can be moved to and from the container without dropping any significant quantities of powder.
Preferably the method further includes providing the orifice, mesh, screen or grid with a hole-size of approximately 0.5 mm.
Other hole-sizes may be more appropriate depending on the properties of the powder.
Preferably one or both of the steps of mechanically agitating includes tapping the hopper and/or container.
Hence, the hopper and/or container can be tapped to provide the mechanical agitation for transferring the powder and/or settling the powder.
The tapping, unlike mere general non-specific vibration, does not merely cause the powder particles to move around and, hence, flow more freely, but actually provides positive impulses to the powder, in particular to move in a direction determined by the tapping direction. Hence, preferably, the tapping is in a direction from the open end of the container into the container so as to provide impulses to the powder particles in that direction. Usually, where filling occurs by means of gravity, the open end of the container is orientated to face upwards, such that the tapping is provided in a vertical downward direction.
Preferably the steps of mechanically agitating include lifting the hopper and container by 1 to 10 mm, then letting the hopper and container fall under gravity to a substantially fixed position.
This tapping of the hopper and container causes transfer of powder from the hopper to the container and suitable settling of the powder in the container.
Preferably the step of mechanically agitating provides an acceleration of approximately 1000 G to powder in the hopper and container.
This acceleration to the powder can be provided as described above or with any suitable movement of the hopper and/or container. It is appropriate for settling the powder to the required density.
Preferably the steps of mechanically agitating include tapping the hopper and/or the container between 50 and 500 times.
Depending on the nature of the powder and the size of the predetermined volume, this will provide sufficient mechanical agitation to ensure that the container is filled with powder and the powder settles to the required density. There is thus no need to weigh the container.
Preferably the steps of mechanically agitating include vibrating the hopper and/or container.
This is an alternative way of causing transfer of powder and/or settling of the powder. It may be used in conjunction with tapping as described above.
To achieve the required mechanical agitation, it is insufficient to provide general non-specific vibration to the container. General vibration merely causes powder particles to move relative to one another and over one another and, hence, to cause improved flow of powder. While this is useful in causing powder to transfer from the hopper to the container and to cause the powder to completely fill the container, the resulting density of the powder remains insufficiently well-defined.
In order to provide the mechanical agitation required to produce the settling of the powder to the reproducible condition of tap density, it is necessary to arrange the vibrations in the required so as to give the powder particles the impulses as described above for tapping. Indeed, the profile of the vibration movement should also be arranged to move the powder particles in a manner similar to as if they were subjected to tapping. In this sense, vibrations considered as suitable for the mechanical agitation could be considered as a series of consecutive taps, rather than more general non-specific “vibrations” as would normally be understood by the skilled person.
In view of the above, it will be appreciated that tapping is particularly advantageous.
Preferably the method further includes vibrating the hopper and/or container at a frequency between 100 Hz and 1 kHz.
For most general powders, this provides suitable mechanical agitation for transferring and settling the powder.
Preferably the method further includes providing a powder-tight seal between the hopper and container during at least part of the step of mechanically agitating the hopper.
In this way, when the mechanical agitation of the hopper releases powder from the hopper, that powder transfers correctly to the container and does not spill on to surfaces around the container.
Preferably the present invention further includes mechanically connecting the hopper to the container such that mechanical agitation of one of the hopper and container causes mechanical agitation of the other of the hopper and container such that the steps of mechanically agitating the hopper and container are conducted simultaneously by mechanically agitating the hopper and container together.
In this way, it is only necessary to provide the mechanical agitation to the hopper and container as a single unit. For example, the hopper and container can be dropped together as a single unit so as to provide appropriate tapping. Furthermore, vibrations applied to one or other of the hopper and container will vibrate both of the hopper and container.
According to the present invention, there is also provided a method of simultaneously filling with powder a plurality of containers having respective open ends, the method including:
providing a hopper having a plurality of outlets positioning the plurality of outlets above corresponding open ends of the containers; and
simultaneously conducting the method defined above for each container.
In this way, a plurality of containers can be filled together. In particular, since the process of mechanical agitation ensures that each of the containers is filled with the same density, it is not necessary to monitor each of the containers separately, for instance by weighing. Hence, it is also possible for the plurality of containers to be provided together in a single carrier.
Following, the methods defined above it is then possible to seal a lidding sheet to the container to seal the powder in place.
According to the present invention, there is also provided an apparatus for filling with powder a container having an open end, the apparatus including:
a support for the container;
a hopper having an outlet and being selectively moveable relative to the support to position the outlet above the open end of a supported container,
a dispenser for mechanically agitating the hopper and container so as to cause powder to be transferred from the hopper to the container; and
a controller for operating the dispenser by at least a predetermined amount sufficient to ensure that powder in the container reaches a predetermined density.
In particular, the apparatus may be arranged to carry out any of the methods described above, for instance simultaneously filling a plurality of containers, optionally forming part of a single carrier.
The invention will be more clearly understood from the following description, given by way of example only, with reference to the accompanying drawings, in which:
FIGS. 1(a) and (b) illustrate an embodiment of the present invention;
FIG. 2 illustrates separation of a hopper from a container according to the present invention;
FIG. 3 illustrates an alternative method according to the present invention;
FIGS. 4(a) and (b) illustrate an alternative method and hopper according to the present invention;
FIG. 5 illustrates one example of the present invention applied to a plurality of containers;
FIG. 6(a) to (e) illustrate alternative arrangements for the outlet of the hopper according to the present invention;
FIG. 7 illustrates schematically an arrangement for providing taps to a container and hopper according to the present invention; and
FIG. 8 illustrates the position, velocity and acceleration profiles against time.
There is a requirement to fill a container with a predetermined mass of a powdered drug or drug and excipient formulation
Where the volume of the container can be accurately controlled, then the mass of powder
that would fill the container can also be accurately controlled if the powder in the container has a uniform and reproducibly density.
Factory filled unit dose DPI's need to be accurately filled at high speed. Many DPI's have arrays of containers on a plane surface. It is advantageous for achieving rapid filling of a number of the containers in parallel rather than sequentially.
It is useful to be able to trim the dose mass by a small amount (˜±5%), without a major equipment change, to enable the filling system to account for small variation in drug concentration on the formulation.
The present application describes a means of using tapping or vibration both to transfer powder from a supply hopper into the container and simultaneously to distribute the powder throughout the container with a uniform and reproducible density.
The supply hopper is fitted with an orifice at the bottom that abuts the opening of the container. The supply hopper and container can be clamped together and both items are then tapped in a way that causes powder to pass through a mesh in the outlet of the hopper into the container under the action of gravity. Tapping or vibration fills the container with powder from the hopper and also settles the powder in the container so that it approaches the reproducible condition known as “tap density”. At this point, the hopper and container are separated. The orifice size and shape is chosen so that powder will not fall through them unless tapped and hence the surface of the powder in the container is defined by the position of the mesh during filling.
The method can be used to fill a plurality of containers from a single hopper provided with the necessary number of orifices. Even though some containers will fill before others, providing that sufficient taps are used to ensure all the containers are full, then the density in each will be substantially the same.
The level of fill can be set to be beneath the opening of the container by the use of a hopper with an orifice plate that protrudes through the opening face to a set level within the container.
The method also has the advantage that it fills a container with powder at a high density without compacting the powder in a way that causes cohesive powders to stick together in the pocket.
FIG. 1 (a) shows a cross section and FIG. 1 (b) a top view of a basic arrangement for implementation of the concept
Powder 1 is placed in the hopper 2. Hopper 2 has an opening in the bottom 7 whose area is appropriate for the opening of the container 8. The open area of the hopper 7 is covered by a thin plate with holes in it that form an orifice 3. The hopper 2 and the container 8 are clamped together and then tapped. Tapping or vibration is in the form of short pulses of high acceleration. They can take many forms and be applied in various directions depending upon the geometry and powder properties. For the basic example, a tapping or vibration mode is assumed that lifts both hopper and container up to a distance between 1 mm and 10 mm's and then lets them fall under gravity to impact a hard flat surface. This may be achieved by using a cam as shown in FIG. 7 and results in the powder undergoing a rapid deceleration from a downward velocity. The inertia of the powder over the openings in the mesh causes it to fall into the container. On each tap, a discrete mass of powder 4 falls into the container. The nature of the powder is such that the mass transferred on each tap is not very consistent. Hence, an accurate mass cannot be achieved just by tapping or vibration a preset number of times. Tapping or vibration continues past the point that the container is full, i.e. where the powder is touching the underside of the mesh 3. Further tapping or vibration densifies the powder in the container and if tapping or vibration continues a long time the powder will achieve what is known as tap density.
Tap density is a very reproducible property of a powder. Tap density is typically 20% to 100% higher than the bulk density (lightly poured into a container).
It is not necessary to tap by an amount to reach full tap density provided that the condition which is achieved has the necessary repeatability to achieve the filling accuracy required. Typically between 50 and 500 taps have been found to be suitable. Where required, the number of taps may be used to adjust the fill weight of the container to accommodate batch-to-batch variation of the powder.
After tapping or vibration has finished then the hopper 2 and the full container 9 are separated as shown in FIG. 2 without causing any vibration of the hopper which would be likely to cause powder to fall out of the hopper onto the surfaces surrounding the container. The result is a container full to the brim with powder at a controlled and uniform density. Thus, an accurate fill mass is achieved.
FIG. 3 shows a variation that might be preferred where the powder is extremely cohesive and would stick to the underside of the mesh 10. If the amount stuck varies, then this would adversely affect the accuracy. Hence, for this example, after separation, the hopper is tapped with the container stationary. This deposits powder above the surface 11, ensuring the container is completely full. The excess can then be removed by a doctor blade 12 leaving the container brim full.
FIG. 4 shows another embodiment developed to fill a container to an accurate level and with a reproducible density. In this case the level of fill is below the brim. Here, the mesh plate protrudes downwards in a way that it fully fills the open area of the container at a preset distance below the opening 15.
Filling below the brim makes it easier to seal the container without spilling any powder or having any of the powder get on the sealing surface around the rim of the container.
Filling is as described previously. However, the container 9 is only filled to the height at which the mesh plate was located, not to the brim. FIG. 4b shows the hopper and container after the filling. It can be seen that the container is filled to a height below the top of the container and that a=b where b is the depth by which the mesh plate protrudes under the hopper. Obviously, the fill depth can be set by the design of the hopper and mesh plate. Small adjustments to the fill height can also be made by shimming the position of the container with respect to the hopper.
FIG. 5 shows another arrangement where the hopper has multiple mesh plates in its base positioned so that several containers can be fitted to the hopper at the same time, each being supplied through its own mesh plate. FIG. 5 shows a single hopper 16 with 3 mesh plates 17a, 17b, 17c and three containers 18a, 18b, 18c.
Filling takes place as before. The FIG. 5 shows the system mid way through the tapping or vibration sequence. As shown, the container 18c is almost full whereas container 18b is only half full. However, as tapping or vibration continues both containers will be completely filled and the additional tapping or vibration will settle the powder in the container to close to top density. There is no limit to the number of containers that can be filled simultaneously. This enables a rapid filling rate to be achieved. For example, a system that fills 30 containers in parallel using 100 taps at a tap rate of ten per second has an average fill rate of 3 containers/second.
FIG. 6 shows cross sections of various types of mesh plate. FIG. 6(a) shows an orifice plate that might be manufactured by milling holes in a sheet of material. For example the plate might have a thickness (t) of 0.5 mm and holes of 0.5 mm diameter (d) drilled in it in a rectangular or hexagonal array of 1 mm pitch (p). Such an orifice plate might be suitable for dispensing powder with particles in the range 0.005 mm to 0.01 mm.
However, it has been noted that such a geometry can cause some variation where the powder separates as the mesh is lifted clear of the powder in the container. Specifically the powder is seen sometimes to separate at the bottom of the hole 20 leaving a plane surface and sometimes at the top of the hole 21 leaving behind a pillar of powder on the surface of the powder in the container.
This uncertainly of the separation point can lead to a significant variation in the fill weight.
FIG. 6(b) shows one way to overcome this where the thickness of the mesh plate is made much thinner than the hole diameter. For typical pharmaceutical powders this means an orifice plate thickness in the region of 0.05 mm to 0.1 mm. Whilst such mesh plates are often used and can be readily manufactured by etching or laser machining they are somewhat fragile for a production environment and may vibrate excessively on larger containers where high tapping or vibration forces are being used. FIG. 6(c) shows a version with tapered holes with the larger dimension d1, on the hopper side. Such an arrangement causes the powder always to break at the smaller opening d2 at the container side of the plate. The angle of the taper will have an optimum value for any specific powder where too shallow an angle does not force the break off always to be at the bottom and too steep an angle compresses the powder passing through the hole potentially leading to blockages.
FIG. 6(d) shows a version with tapered holes with the larger dimension d2 on the container side. In this case the powder will separate-at the hopper side of the plate. However, the large taper angle allows the powder within the hole to drop into the container as the orifice plate is lifted away ensuring that the point of separation is accurately controlled.
These tapered orifices allow a robust and stiff orifice plate to be used whilst maintaining accurate control of the separation location. The selection between positive or negative tapers is governed by the properties of the powder, particularly its cohesiveness.
FIG. 6(e) shows an orifice plate with a slot hole instead of an array of circular holes. The retention of powder over a slot is primarily governed by the width of the slot (w) By making the length (1) of the slot much greater than the width a large open area, rapid filling can be achieved along with good powder retention during separation
FIG. 7 shows one means of creating the tapping or vibration. The container and hopper are rigidly connected to the follower of a cam 20. The cam profile 21 causes the cam follower to be raised up and then allowed to free fall under gravity and to be rapidly stopped as it impacts the lower cam surface 22. FIG. 8 shows the position, velocity and acceleration profiles plotted against time. The cam profile 21 is designed to lift the hopper with a low acceleration and then to let it all downwards under gravity so as not to cause the powder in the hopper to become air born and then to stop the downward motion of the powder in the hopper and in the container within a very short space of time by impaction with a solid surface. The impact causes a very high peak of acceleration. If the hopper is allowed to fall 3 mm and stops on impact over a distance 3 microns then the peak deceleration would be 1000 g (or ≈ 10,000 m/s2). Powder immediately over a hole in the mesh is unsupported and a portion of it is pushed through the hole into the container. The remaining powder rapidly comes to rest after the impact, typically in less than 0.01 seconds. This repeated tapping or vibration at up to 100 taps per second can be made without changing the behavior compared to a slower rate of tapping or vibration.
It has been noted that some powders fill more uniformly and quickly where a vibration is used rather than discrete taps. Vibration is characterized as being a cyclic motion where the cycle time is short enough that the powder is still in motion at the start of the next cycle. Typically vibration in the frequency range 100 Hz to 1 KHz would be suitable. Vibration can be used either vertically or horizontally.
Combinations of tapping or vibration and vibration can also be advantageous either sequentially or simultaneously. This is especially applicable to cohesive powders where a high tapping or vibration force promotes transfer form the hopper through the mesh and the vibration assists settling and distribution of the powder in the container without compaction.
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10581668
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pfizer, limited
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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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141/73
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Nov 5th, 2013 12:00AM
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Jul 5th, 2012 12:00AM
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https://www.uspto.gov?id=US08575336-20131105
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Indazoles
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The present invention relates to compounds of formula (I):
to pharmaceutically acceptable salts therefore and to pharmaceutically acceptable solvates of said compounds and salts, wherein the substituents are defined herein; to compositions containing such compounds; and to the uses of such compounds in the treatment of various diseases, particularly asthma and COPD.
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8575336
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1. A compound of formula (I):
or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein:
R1 is halo;
R2 is C1-C6 alkyl optionally substituted by one or more fluorine atoms;
X is a bond, —CO—, —SO2— or —CH2—;
R3 is Aryl1, Het1 or Het2, each of which is optionally substituted by 1 substituent —Y—R4 and/or 1-4 substituents each independently selected from R5;
n is 1 or 2;
Aryl1 is phenyl or naphthyl;
Het1 is (i) a 6-membered aromatic heterocycle containing 1-3 N atoms or (ii) a 5-membered aromatic heterocycle containing either (a) 1-4 N atoms or (b) 1 O or S atom and 0-3 N atoms;
Het2 is (i) a 10-membered bicyclic aromatic heterocycle containing 1-4 N atoms or (ii) a 9-membered bicyclic aromatic heterocycle containing either (a) 1-4 N atoms or (b) 1 O or S atom and 0-3 N atoms or (iii) an 8-membered bicyclic aromatic heterocycle containing (a) 1-4 N atoms or (b) 1 O or S atom and 1-3 N atoms or (c) 2 O or S atoms and 0-2 N atoms;
Y is a bond or —O—;
R4 is Aryl2 or Het3;
R5 is C1-C6 alkyl, C3-C8 cycloalkyl, halo, —CN, —OR6, —NR7R8, —SR6, —SOR9, —SO2R9, —COR6, —OCOR6, —COOR6, —NR6COR6, —CONR7R8, —NR6SO2R9, —SO2NR7R8, —NR6CONR7R8, —NR6COOR9 or —NR6SO2NR7R8;
R6 is H, C1-C6 alkyl or C3-C8 cycloalkyl, said C1-C6 alkyl being optionally substituted by halo or C3-C8 cycloalkyl;
R7 and R8 are (a) each independently H, C1-C6 alkyl or C3-C8 cycloalkyl, said C1-C6 alkyl being optionally substituted by —NR10R11, wherein R1 and R11 are C1-C6 alkyl or taken together with the nitrogen atom to which they are attached form a 4-, 5- or 6-membered saturated heterocyclic ring containing 1-2 nitrogen atoms or 1 nitrogen and 1 oxygen atom, said heterocyclic ring being optionally substituted by one or more C1-C6 alkyl or C3-C8 cycloalkyl groups; or, (b) are taken together with the nitrogen atom to which they are attached to form a 4-, 5- or 6-membered saturated heterocyclic ring containing 1-2 nitrogen atoms or 1 nitrogen and 1 oxygen atom, said heterocyclic ring being optionally substituted by one or more C1-C6 alkyl or C3-C8 cycloalkyl groups;
R9 is C1-C6 alkyl or C3-C8 cycloalkyl;
Aryl2 is phenyl or naphthyl, said phenyl and naphthyl being optionally substituted with 1-5 substituents selected from C1-C6 alkyl, C3-C8 cycloalkyl, halo, —CN, —OR6, —NR7R8, —SR6, —SOR9, —SO2R9, —COR6, —OCOR6, —COOR6, —NR6COR6, —CONR7R8, —NR6SO2R9, —SO2NR7R8, —NR6CONR7R8, —NR6COOR9 and —NR6SO2NR7R8; and
Het3 is a 3 to 8-membered saturated or partially unsaturated monocyclic heterocycle, containing 1 or 2 heteroatoms selected from O and N, said heterocycle being optionally substituted by 1-5 substituents selected from C1-C6 alkyl, C3-C8 cycloalkyl, halo, oxo, —OR6, —NR7R8, —SR6, —SOR9, —SO2R9, —COR6, —OCOR6, —COOR6, —NR6COR6, —CONR7R8, —NR6SO2R9, —SO2NR7R8, —NR6CONR7R8, —NR6COOR9 and —NR6SO2NR7R8.
2. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein R1 is fluoro.
3. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein R2 is —CH2CH3 or —CH2CF3.
4. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein n is 1.
5. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein n is 2.
6. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein X is a bond.
7. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein X is —CO—.
8. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein X is —SO2—.
9. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein X is —CH2—.
10. A compound claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, wherein R3 is phenyl, thiazolyl, quinolinyl, pyrimidinyl, [1,8]naphthyridinyl or pyridyl, each of which is optionally substituted by 1 substituent selected from piperidininyl, (fluorophenyl)oxy, phenyloxy and morpholinyl and 1-2 substituents each independently selected from fluoro, chloro, cyano, methoxy and hydroxyl.
11. A compound of claim 1, which is:
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(4-fluoro-phenyl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-isothiazol-3-yl-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-isothiazol-3-yl-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-piperidin-1-yl-pyrazin-2-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(6-phenoxy-pyridin-3-yl)-methanone;
5-Ethyl-2-fluoro-4-{3-[5-(6-morpholin-4-yl-pyridine-3-sulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(6-phenoxy-pyridine-3-sulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
(5-Chloro-pyridin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-methanone;
2-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carbonyl}-isonicotinonitrile;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-(4-fluoro-phenyl)methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-isothiazol-3-yl-methanone;
5-Ethyl-2-fluoro-4-{3-[5-(4-fluoro-benzenesulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-fluoro-phenoxy)-pyrazin-2-yl]-methanone;
4-[3-(6-Benzyl-1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-5-ethyl-2-fluoro-phenol;
(5-Chloro-pyridin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
5-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carbonyl}-pyridine-2-carbonitrile;
5-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carbonyl}-pyridine-2-carbonitrile;
5-Ethyl-2-fluoro-4-[3-(5-quinolin-6-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(4-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(3-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
4-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-ylmethyl}-pyridine-2-carbonitrile;
5-Ethyl-2-fluoro-4-{3-[5-(3-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-[3-(5-quinolin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(6-phenoxy-pyridin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5′-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
3-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-ylmethyl}-pyridine-2-carbonitrile;
5-Ethyl-2-fluoro-4-{3-[5-(4-fluoro-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-[3-(5-[1,8]naphthyridin-2-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol;
(2-{6-[5-Fluoro-4-hydroxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl)-(5-piperidin-1-yl-pyrazin-2-yl)-methanone;
(2-{6-[5-Fluoro-4-hydroxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl)-(4-fluoro-phenyl)-methanone; or
4-[3-(5-Benzyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-2-fluoro-5-(2,2,2-trifluoro-ethyl)-phenol;
or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.
12. A compound of claim 1, which is:
{5-[(2-Dimethylamino-ethyl)-methyl-amino]-pyrazin-2-yl}-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-pyrrolidin-1-yl-ethylamino)-pyrazin-2-yl]-methanone;
[5-(2-Dimethylamino-ethylamino)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
[5-(4-Dimethylamino-piperidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-{5-[ethyl-(2-hydroxy-ethyl)-amino]-pyrazin-2-yl}-methanone;
[5-((R)-3-Dimethylamino-pyrrolidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
[5-((S)-3-Dimethylamino-pyrrolidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-etrahydroimidazo[4,5c]pyridin-5-yl}-[5-(2-piperidin-1-yl-ethylamino)-pyrazin-2-yl]-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-piperazin-1-yl-ethylamino)-pyrazin-2-yl]-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-(4-methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-morpholin-4-yl-pyrazin-2-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-idazo[4,5c]pyridin-5-yl}-[5-(4-methyl-piperidin-1-yl)-pyrazin-2-yl]-methanone;
(5-Cyclopentylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-morpholin-4-yl-ethylamino)-pyrazin-2-yl]-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(4-isopropyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-pyrrolidin-1-yl-pyrazin-2-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-[5-(ethyl-methyl-amino)-pyrazin-2-yl]-methanon;
(5-Cyclohexylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
(5-Dimethylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
(5-Azetidin-1-yl-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
2-Fluoro-4-{3-[5-(4-fluoro-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5′-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(6-phenoxy-pyridin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(4-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)phenol;
2-Fluoro-4-{3-[5-(4-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(3-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(3-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-[3-(5-quinolin-6-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-[3-(5-quinolin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-[3-(5-[1,8]naphthyridin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol;
((3R,5S)-3,5-Dimethyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-((S)-3-methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
((2S,5R)-2,5-Dimethyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-(3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.
13. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, and a pharmaceutically acceptable excipient.
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This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/512,144, filed Jul. 27, 2011, which is incorporated herein by reference in its entirety for all purposes.
FIELD OF THE INVENTION
The present invention relates to indazoles, pharmaceutical compositions comprising such compounds and their use as medicaments. More particularly, the present invention provides 6-phenyl-1H-indazole derivatives which are Janus Kinase (JAK) inhibitors and useful for the treatment of allergic and respiratory conditions, particularly chronic obstructive pulmonary disease.
BACKGROUND OF THE INVENTION
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the US and is characterized by airflow obstruction that is not fully reversible with bronchodilators. The airflow limitation is usually progressive and is associated with an abnormal inflammatory response of the lungs to noxious particles or gases, primarily cigarette smoke. Symptoms are typically breathing-related (e.g. chronic cough, exertional dyspnea, expectoration and wheeze). Patients experience periods of stable disease interspersed with inflammatory exacerbations resulting in acute decline in lung function and often hospitalization.
Current treatment guidelines recommend bronchodilators as the mainstay of COPD drug treatment. However, anti-inflammatory inhaled corticosteroids (ICS) and bronchodilator/inhaled corticosteroid combination products, are extensively used. Whilst inhaled corticosteroids do provide some benefits with respect to short term lung function improvements and exacerbation frequency, they do not address the corticosteroid-refractory inflammation which is characteristic of this disease and thought to play a key role in disease progression. There is a clear medical need for anti-inflammatory therapies in COPD that will address the chronic inflammatory component of the disease and ultimately provide symptomatic relief, a reduction in exacerbation frequency and an amelioration of exacerbation severity.
The Janus kinase (JAK) family of receptor associated tyrosine kinases, JAK 1, JAK 2, JAK 3 and tyrosine kinase 2 (TYK2), are involved in signal transduction associated with a variety of inflammatory cytokines. JAK kinases can function as either hetero or homo-dimers, phosphorylating STAT transcription factors which regulate inflammatory gene transcription. Oral JAK 1/JAK 3 inhibitors such as CP-690550 have shown impressive anti-inflammatory activity in inflammatory diseases such as rheumatoid arthritis and psoriasis.
Many JAK dependent cytokines are thought to play key roles in the pathology of COPD which involves the interplay of multiple inflammatory cells such as T lymphocytes, neutrophils, macrophages and lung epithelium. For example the JAK 1/JAK 3 heterodimer plays a key role in T lymphocyte survival and activation whereas JAK 2 is thought to be critical for regulation of neutrophil activation and apoptosis. JAK 1 and JAK 2 play an important role in IL-13 mediated inflammatory signaling in macrophages, which is thought to link acute inflammatory events to chronic progressive disease. Importantly JAK 1, JAK 2 and TYK 2 also play an important role in signaling mediated by IFNγ, a cytokine associated with the chronic inflammation observed in COPD, which modulates the activity of T cells, epithelium and macrophages whilst not being modulated by corticosteroids.
Macrophage phagocytosis of bacteria is impaired in the lungs of COPD patients, potentially in part due to high local IFNγ levels. In vitro studies with isolated patient cells have shown that JAK inhibitors increase phagocytotic rate in the presence of IFNγ. Consequently, as well as exerting a direct anti-inflammatory effect, JAK inhibitors may also increase the ability of the lung to maintain a sterile environment.
JAK inhibitors are therefore likely to have utility in the treatment of a range of inflammatory diseases, including lung diseases such as COPD, asthma and pulmonary vascular disease. Compounds which have a broad inhibitory activity across the range of Janus kinases, in particular, are likely to have a potent anti-inflammatory effect. However, such a selectivity profile can also lead to undesirable side-effects in systemically circulating compounds, particularly anemia and neutropenia associated with JAK 2 inhibition. For the treatment of lung diseases, it is therefore particularly favourable to provide JAK inhibitors which can be administered by inhalation and which inhibit Janus kinases locally in the lung without having a significant systemic exposure.
There is thus a need to provide new JAK inhibitors that are potent, selective inhibitors of Janus kinases with appropriate metabolic stability and pharmacokinetic properties, particularly compounds which can be administered by inhalation and are active in lung tissue whilst having poor systemic penetration or high systemic lability.
SUMMARY OF THE INVENTION
The invention therefore provides, as embodiment E1, a compound of formula (I):
or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein:
R1 is halo;
R2 is C1-C6 alkyl optionally substituted by one or more fluorine atoms;
X is a bond, —CO—, —SO2— or —CH2—;
R3 is Aryl1, Het1 or Het2, each of which is optionally substituted by 1 substituent —Y—R4 and/or 1-4 substituents each independently selected from R5;
n is 1 or 2;
Aryl1 is phenyl or naphthyl;
Het1 is (i) a 6-membered aromatic heterocycle containing 1-3 N atoms or (ii) a 5-membered aromatic heterocycle containing either (a) 1-4 N atoms or (b) 1 O or S atom and 0-3 N atoms;
Het2 is (i) a 10-membered bicyclic aromatic heterocycle containing 1-4 N atoms or (ii) a 9-membered bicyclic aromatic heterocycle containing either (a) 1-4 N atoms or (b) 1 O or S atom and 0-3 N atoms or (iii) an 8-membered bicyclic aromatic heterocycle containing (a) 1-4 N atoms or (b) 1 O or S atom and 1-3 N atoms or (c) 2 O or S atoms and 0-2 N atoms;
Y is a bond or —O—;
R4 is Aryl2 or Het3;
R5 is C1-C6 alkyl, C3-C8 cycloalkyl, halo, —CN, —OR6, —NR7R8, —SR6, —SOR9, —SO2R9, —COR6, —OCOR6, —COOR6, —NR6COR6, —CONR7R8, —NR6SO2R9, —SO2NR7R8, —NR6CONR7R8, —NR6COOR9 or —NR6SO2NR7R8;
R6 is H, C1-C6 alkyl or C3-C8 cycloalkyl, said C1-C6 alkyl;
R7 and R8 are each independently H, C1-C6 alkyl or C3-C8 cycloalkyl or are taken together with the nitrogen atom to which they are attached to form a 4-, 5- or 6-membered saturated heterocyclic ring containing 1-2 nitrogen atoms or 1 nitrogen and 1 oxygen atom, said heterocyclic ring being optionally substituted by one or more C1-C6 alkyl or C3-C8 cycloalkyl groups;
R9 is C1-C6 alkyl or C3-C8 cycloalkyl;
Aryl2 is phenyl or naphthyl, said phenyl and naphthyl being optionally substituted with 1-5 substituents selected from C1-C6 alkyl, C3-C8 cycloalkyl, halo, —CN, —OR6, —NR7R8, —SR6, —SOR9, —SO2R9, —COR6, —OCOR6, —COOR6, —NR6COR6, —CONR7R8, —NR6SO2R9, —SO2NR7R8, —NR6CONR7R8, —NR6COOR9 and —NR6SO2NR7R8; and
Het3 is a 3 to 8-membered saturated or partially unsaturated monocyclic heterocycle, containing 1 or 2 heteroatoms selected from O and N, said heterocycle being optionally substituted by 1-5 substituents selected from C1-C6 alkyl, C3-C8 cycloalkyl, halo, oxo, —OR6, —NR7R8, —SR6, —SOR9, —SO2R9, —COR6, —OCOR6, —COOR6, —NR6COR6, —CONR7R8, —NR6SO2R9, —SO2NR7R8, —NR6CONR7R8, —NR6COOR9 and —NR6SO2NR7R8.
The invention also provides, as embodiment E2, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R2, n, X and R3 are as defined in embodiment E1 and R1 is fluoro.
The invention also provides, as embodiment E3, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, n, X and R3 are as defined in embodiment E1 and R2 is —CH2CH3 or —CH2CF3.
The invention also provides, as embodiment E4, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, X and R3 are as defined in embodiment E1 and n is 1.
The invention also provides, as embodiment E5, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, X and R3 are as defined in embodiment E1 and n is 2.
The invention also provides, as embodiment E6, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is defined in any one of embodiments E1, E4 or E5, R3 is as defined in embodiment E1 and X is a bond.
The invention also provides, as embodiment E7, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is defined in any one of embodiments E1, E4 or E5, R3 is as defined in embodiment E1 and X is —CO—.
The invention also provides, as embodiment E8, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is defined in any one of embodiments E1, E4 or E5, R3 is as defined in embodiment E1 and X is —CH2—.
The invention also provides, as embodiment E9, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is defined in any one of embodiments E1, E4 or E5, R3 is as defined in embodiment E1 and X is —SO2—.
The invention also provides, as embodiment E10, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is as defined in any one of embodiments E1, E4 or E5, X is as defined in any one of embodiments E1, E6, E7, E8 or E9 and R3 is phenyl, thiazolyl, quinolinyl, pyrimidinyl, [1,8]naphthyridinyl or pyridyl, each of which is optionally substituted by 1 substituent —Y—R4 and 1-4 substituents each independently selected from R5.
The invention also provides, as embodiment E11, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is as defined in any one of embodiments E1, E4 or E5, X is as defined in any one of embodiments E1, E6, E7, E8 or E9 and R3 is phenyl, thiazolyl, quinolinyl, pyrimidinyl, [1,8]naphthyridinyl or pyridyl, each of which is optionally substituted by 1 substituent selected from piperidininyl, (fluorophenyl)oxy, phenyloxy and morpholinyl and 1-2 substituents each independently selected from fluoro, chloro, cyano, methoxy and hydroxy.
The invention also provides, as embodiment E12, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is as defined in any one of embodiments E1, E4 or E5, X is as defined in any one of embodiments E1, E6, E7, E8 or E9 and R3 is fluorophenyl, methoxyphenyl, thiazolyl, hydroxyphenyl, phenyl, quinolinyl, [1,8]naphthyridinyl, (piperidinyl)pyridyl, (piperidinyl)pyrimidinyl, ((fluorophenyl)oxy)pyrimidinyl, (phenyloxy)pyridyl, (morpholinyl)pyridyl, chloropyridyl or cyanopyridyl.
The invention also provides, as embodiment E13, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein R1 is as defined in either of embodiments E1 or E2, R2 is as defined in either of embodiments E1 or E3, n is defined in any one of embodiments E1, E4 or E5, and —X—R3 is (fluorophenyl)carbonyl, (thiazolyl)carbonyl, benzyl, ((piperidinyl)pyrimidinyl)carbonyl, ((phenoxy)pyridyl)carbonyl, ((morpholinyl)pyridyl)sulphonyl, ((phenoxy)pyridyl)sulphonyl, (chloropyridyl)carbonyl, (cyanopyridyl)carbonyl, (fluorophenyl)carbonyl, (thiazolyl)carbonyl, (fluorophenyl)sulphonyl, ((fluorophenoxy)pyrimidinyl)carbonyl, (quinolinyl)methyl, (hydroxyphenyl)methyl, (cyanopyridyl)methyl, (methoxyphenyl)methyl, ((phenoxy)pyridyl)methyl, ((piperidinyl)pyridyl)methyl, ((cyanopyridyl)methyl, (fluorophenyl)methyl or ([1,8]naphthyridinyl)methyl.
The invention also provides, as embodiment E14, a compound of formula:
or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or pharmaceutically acceptable salt, wherein X is as defined in any one of embodiments E1, E6, E7, E8 or E9 and R3 is as defined in any one of embodiments E1, E10, E11 or E12 or —X—R3 is as defined in embodiment E13.
Particularly preferred compounds of formula (I) include:
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(4-fluoro-phenyl)methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-isothiazol-3-yl-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-isothiazol-3-yl-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-piperidin-1-yl-pyrazin-2-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(6-phenoxy-pyridin-3-yl)-methanone;
5-Ethyl-2-fluoro-4-{3-[5-(6-morpholin-4-yl-pyridine-3-sulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(6-phenoxy-pyridine-3-sulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
(5-Chloro-pyridin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-methanone;
2-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carbonyl}-isonicotinonitrile;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-(4-fluoro-phenyl)methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-isothiazol-3-yl-methanone;
5-Ethyl-2-fluoro-4-{3-[5-(4-fluoro-benzenesulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-fluoro-phenoxy)-pyrazin-2-yl]-methanone;
4-[3-(6-Benzyl-1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-5-ethyl-2-fluoro-phenol;
(5-Chloro-pyridin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
5-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carbonyl}-pyridine-2-carbonitrile;
5-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carbonyl}-pyridine-2-carbonitrile;
5-Ethyl-2-fluoro-4-[3-(5-quinolin-6-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(4-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(3-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
4-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-ylmethyl}-pyridine-2-carbonitrile;
5-Ethyl-2-fluoro-4-{3-[5-(3-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-[3-(5-quinolin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(6-phenoxy-pyridin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-{3-[5-(3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5′-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
3-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-ylmethyl}-pyridine-2-carbonitrile;
5-Ethyl-2-fluoro-4-{3-[5-(4-fluoro-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol;
5-Ethyl-2-fluoro-4-[3-(5-[1,8]naphthyridin-2-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol;
(2-{6-[5-Fluoro-4-hydroxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl)-(5-piperidin-1-yl-pyrazin-2-yl)-methanone;
(2-{6-[5-Fluoro-4-hydroxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl)-(4-fluoro-phenyl)-methanone;
4-[3-(5-Benzyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-2-fluoro-5-(2,2,2-trifluoro-ethyl)-phenol;
or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.
Other preferred compounds of formula (I) include:
{5-[(2-Dimethylamino-ethyl)methyl-amino]-pyrazin-2-yl}-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-pyrrolidin-1-yl-ethylamino)-pyrazin-2-yl]-methanone;
[5-(2-Dimethylamino-ethylamino)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
[5-(4-Dimethylamino-piperidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-{5-[ethyl-(2-hydroxy-ethyl)-amino]-pyrazin-2-yl}-methanone;
[5-((R)-3-Dimethylamino-pyrrolidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
[5-((S)-3-Dimethylamino-pyrrolidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-etrahydroimidazo[4,5c]pyridin-5-yl}-[5-(2-piperidin-1-yl-ethylamino)-pyrazin-2-yl]-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-piperazin-1-yl-ethylamino)-pyrazin-2-yl]-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-(4-methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-morpholin-4-yl-pyrazin-2-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-idazo[4,5c]pyridin-5-yl}-[5-(4-methyl-piperidin-1-yl)-pyrazin-2-yl]-methanone;
(5-Cyclopentylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-morpholin-4-yl-ethylamino)-pyrazin-2-yl]-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(4-isopropyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-pyrrolidin-1-yl-pyrazin-2-yl)-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-[5-(ethyl-methyl-amino)-pyrazin-2-yl]-methanon; (5-Cyclohexylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
(5-Dimethylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
(5-Azetidin-1-yl-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
2-Fluoro-4-{3-[5-(4-fluoro-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5′-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(6-phenoxy-pyridin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(4-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)phenol;
2-Fluoro-4-{3-[5-(4-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)phenol;
2-Fluoro-4-{3-[5-(3-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-{3-[5-(3-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-[3-(5-quinolin-6-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-[3-(5-quinolin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol;
2-Fluoro-4-[3-(5-[1,8]naphthyridin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol;
((3R,5S)-3,5-Dimethyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-((S)-3-methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
((2S,5R)-2,5-Dimethyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone;
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-(3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone;
or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.
Most preferred is {2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-piperidin-1-yl-pyrazin-2-yl)-methanone or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.
The present invention also provides: a method of treating a disease for which a JAK inhibitor is indicated, in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt; the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for the manufacture of a medicament for treating a disease or condition for which a JAK inhibitor is indicated; a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use as a medicament; a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use in the treatment of a disease or condition for which a JAK inhibitor is indicated; a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, and a pharmaceutically acceptable excipient; a pharmaceutical composition for the treatment of a disease or condition for which a JAK inhibitor is indicated, comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.
The disease or condition for which a JAK inhibitor is indicated is preferably an allergic or respiratory condition such as allergic rhinitis, nasal congestion, rhinorrhea, perennial rhinitis, nasal inflammation, asthma of all types, chronic obstructive pulmonary disease (COPD), chronic or acute bronchoconstriction, chronic bronchitis, small airways obstruction, emphysema, chronic eosinophilic pneumonia, adult respiratory distress syndrome, exacerbation of airways hyper-reactivity consequent to other drug therapy, pulmonary vascular disease (including pulmonary arterial hypertension), acute lung injury, bronchiectasis, sinusitis, allergic conjunctivitis, idiopathic pulmonary fibrosis or atopic dermatitis, particularly asthma or chronic obstructive pulmonary disease, most particularly chronic obstructive pulmonary disease.
Other diseases and conditions of interest are inflammation (including neuroinflammation), arthritis (including rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematous arthritis, osteoarthritis and gouty arthritis), pain, fever, pulmonary sarcoisosis, silicosis, cardiovascular disease (including atherosclerosis, myocardial infarction, thrombosis, congestive heart failure and cardiac reperfusion injury), cardiomyopathy, stroke, ischaemia, reperfusion injury, brain edema, brain trauma, neurodegeneration, liver disease, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), nephritis, retinitis, retinopathy, macular degeneration, glaucoma, diabetes (including type 1 and type 2 diabetes), diabetic neurorpathy, viral and bacterial infection, myalgia, endotoxic shock, toxic shock syndrome, autoimmune disease, osteoporosis, multiple sclerosis, endometriosis, menstrual cramps, vaginitis, candidiasis, cancer, fibrosis, obesity, muscular dystrophy, polymyositis, Alzheimer's disease, skin flushing, eczema, psoriasis, atopic dermatitis and sunburn.
Types of asthma include atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, intrinsic asthma caused by pathophysiologic disturbances, extrinsic asthma caused by environmental factors, essential asthma of unknown or inapparent cause, bronchitic asthma, emphysematous asthma, exercise-induced asthma, allergen induced asthma, cold air induced asthma, occupational asthma, infective asthma caused by bacterial, fungal, protozoal, or viral infection, non-allergic asthma, incipient asthma, wheezy infant syndrome and bronchiolytis.
The treatment of asthma includes palliative treatment for the symptoms and conditions of asthma such as wheezing, coughing, shortness of breath, tightness in the chest, shallow or fast breathing, nasal flaring (nostril size increases with breathing), retractions (neck area and between or below the ribs moves inward with breathing), cyanosis (gray or bluish tint to skin, beginning around the mouth), runny or stuffy nose, and headache.
The present invention also provides any of the uses, methods or compositions as defined above wherein the compound of formula (I), or pharmaceutically acceptable salt thereof, or pharmaceutically acceptable solvate of said compound or salt, is used in combination with another pharmacologically active compound, particularly one of the functionally-defined classes or specific compounds listed below. Generally, the compounds of the combination will be administered together as a formulation in association with one or more pharmaceutically acceptable excipients.
Suitable agents for use in combination therapy with a compound of formula (I), or pharmaceutically acceptable salt thereof, or pharmaceutically acceptable solvate of said compound or salt, particularly in the treatment of respiratory disease, include:
a 5-lipoxygenase activating protein (FLAP) antagonist;
a leukotriene antagonist (LTRA) such as an antagonist of LTB4, LTC4, LTD4, LTE4, CysLT1 or CysLT2, e.g. montelukast or zafirlukast;
a histamine receptor antagonist, such as a histamine type 1 receptor antagonist or a histamine type 2 receptor antagonist, e.g. loratidine, fexofenadine, desloratidine, levocetirizine, methapyrilene or cetirizine;
an α1-adrenoceptor agonist or an α2-adrenoceptor agonist, e.g. phenylephrine, methoxamine, oxymetazoline or methylnorephrine;
a muscarinic M3 receptor antagonist, e.g. tiotropium or ipratropium;
a dual muscarinic M3 receptor antagononist/β2 agonist;
a PDE inhibitor, such as a PDE3 inhibitor, a PDE4 inhibitor or a PDE5 inhibitor, e.g. theophylline, sildenafil, vardenafil, tadalafil, ibudilast, cilomilast or roflumilast;
sodium cromoglycate or sodium nedocromil;
a cyclooxygenase (COX) inhibitor, such as a non-selective inhibitor (e.g. aspirin or ibuprofen) or a selective inhibitor (e.g. celecoxib or valdecoxib);
a glucocorticosteroid, e.g. fluticasone, mometasone, dexamethasone, prednisolone, budesonide, ciclesonide or beclamethasone;
an anti-inflammatory monoclonal antibody, e.g. infliximab, adalimumab, tanezumab, ranibizumab, bevacizumab or mepolizumab;
a β2 agonist, e.g. salmeterol, albuterol, salbutamol, fenoterol or formoterol, particularly a long-acting β2 agonist;
an intigrin antagonist, e.g. natalizumab;
an adhesion molecule inhibitor, such as a VLA-4 antagonist;
a kinin B1 or B2 receptor antagonist;
an immunosuppressive agent, such as an inhibitor of the IgE pathway (e.g. omalizumab) or cyclosporine;
a matrix metalloprotease (MMP) inhibitor, such as an inhibitor of MMP-9 or MMP-12;
a tachykinin NK1, NK2 or NK3 receptor antagonist;
a protease inhibitor, such as an inhibitor of elastase, chymase or catheopsin G;
an adenosine A2a receptor agonist;
an adenosine A2b receptor antagonist;
a urokinase inhibitor;
a dopamine receptor agonist (e.g. ropinirole), particularly a dopamine D2 receptor agonist (e.g. bromocriptine);
a modulator of the NFκB pathway, such as an IKK inhibitor;
a further modulator of a cytokine signalling pathway such as an inhibitor of JAK kinase, syk kinase, p38 kinase, SPHK-1 kinase, Rho kinase, EGF-R or MK-2;
a mucolytic, mucokinetic or anti-tussive agent
an antibiotic;
an antiviral agent;
a vaccine;
a chemokine;
an epithelial sodium channel (ENaC) blocker or Epithelial sodium channel (ENaC) inhibitor;
a nucleotide receptor agonist, such as a P2Y2 agonist;
a thromboxane inhibitor;
niacin;
a 5-lipoxygenase (5-LO) inhibitor, e.g. Zileuton;
an adhesion factor, such as VLAM, ICAM or ELAM;
a CRTH2 receptor (DP2) antagonist;
a prostaglandin D2 receptor (DP1) antagonist;
a haematopoietic prostaglandin D2 synthase (HPGDS) inhibitor;
interferon-β;
a soluble human TNF receptor, e.g. Etanercept;
a HDAC inhibitor;
a phosphoinositotide 3-kinase gamma (PI3Kγ) inhibitor;
a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor;
a CXCR-1 or a CXCR-2 receptor antagonist;
an IRAK-4 inhibitor; and
a TLR-4 or TLR-9 inhibitor;
including the pharmaceutically acceptable salts of the specifically named compounds and the pharmaceutically acceptable solvates of said specifically named compounds and salts.
Besides being useful for human treatment, compounds of formula (I) are also useful for veterinary treatment of companion animals, exotic animals and farm animals.
When used in the present application, the following abbreviations have the meanings set out below:
AcOH is acetic acid;
APCI (in relation to mass spectrometry) is atmospheric pressure chemical ionization;
BOP is (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate;
Calc is calculated;
CDCl3 is deuterochloroform;
CO2Et is ethyl carboxylate;
DCC is N,N′-dicyclohexylcarbodiimide;
DCM is dichloromethane;
DEA is diethylamine;
DIAD is diisopropyl azodicarboxylate;
DIEA is N,N-diisopropylethylamine;
DIPEA is N,N-diisopropylethylamine;
DMA is N,N-dimethylacetamide;
DMF is dimethylformamide;
DMSO-d6 is fully deuterated dimethyl sulphoxide;
EDC/EDCI is N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
ES (in relation to mass spectrometry) is electrospray;
Et is ethyl;
EtOAc is ethyl acetate
Ex is Example;
h is hour(s);
HATU is N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate;
HBTU is N,N,N′N-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate;
HCl is hydrochloric acid;
1H NMR or 1H NMR is proton nuclear magnetic resonance;
HOAt is 1-hydroxy-7-azabenzotriazole;
HOBt is 1-hydroxybenzotriazole;
HPLC is high performance liquid chromatography;
H2SO4 is sulphuric acid;
IPA is isopropyl alcohol;
iPr is isopropyl;
K2CO3 is potassium carbonate;
KMnO4 is potassium permanganate;
KOH is potassium hydroxide;
KOAc is potassium acetate;
LCMS is liquid chromatography mass spectrometry;
LRMS is low resolution mass spectrometry;
NMM is 4-methylmorpholine;
Me is methyl;
MeCN is acetonitrile;
MeOD-d4 is fully deuterated methanol;
MgSO4 is magnesium sulphate;
2-MeTHF is 2-methyltetrahydrofuran;
min is minute(s);
MS is mass spectroscopy;
NaCl is sodium chloride;
NaH is sodium hydride;
NBS is N-bromosuccinimide;
NIS is N-iodosuccinimide;
NMP is N-methylpyrrolidine;
Obs is observed;
Pd(OAc)2 is palladium(II)acetate;
RT is retention time;
SEM-Cl is (2-chloromethoxy-ethyl)-trimethyl-silane;
SPhos is 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl;
STAB is sodium (tri-acetoxy) borohydride;
TBTU is O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate;
TEA is triethylamine;
TFA is trifluoroacetic acid;
THF is tetrahydrofuran;
tBME is 2-mMethoxy-2-methyl-propane;
p-TsOH is para-toluene sulfonic acid.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those of ordinary skill in the art.
The phrase “therapeutically effective” is intended to qualify the amount of compound or pharmaceutical composition, or the combined amount of active ingredients in the case of combination therapy. This amount or combined amount will achieve the goal of treating the relevant condition.
The term “treatment,” as used herein to describe the present invention and unless otherwise qualified, means administration of the compound, pharmaceutical composition or combination to effect preventative, palliative, supportive, restorative or curative treatment. The term treatment encompasses any objective or subjective improvement in a subject with respect to a relevant condition or disease.
The term “preventive treatment,” as used herein to describe the present invention, means that the compound, pharmaceutical composition or combination is administered to a subject to inhibit or stop the relevant condition from occurring in a subject, particularly in a subject or member of a population that is significantly predisposed to the relevant condition.
The term “palliative treatment,” as used herein to describe the present invention, means that the compound, pharmaceutical composition or combination is administered to a subject to remedy signs and/or symptoms of a condition, without necessarily modifying the progression of, or underlying etiology of, the relevant condition.
The term “supportive treatment,” as used herein to describe the present invention, means that the compound, pharmaceutical composition or combination is administered to a subject as a part of a regimen of therapy, but that such therapy is not limited to administration of the compound, pharmaceutical composition or combination. Unless otherwise expressly stated, supportive treatment may embrace preventive, palliative, restorative or curative treatment, particularly when the compounds or pharmaceutical compositions are combined with another component of supportive therapy.
The term “restorative treatment,” as used herein to describe the present invention, means that the compound, pharmaceutical composition or combination is administered to a subject to modify the underlying progression or etiology of a condition. Non-limiting examples include an increase in forced expiratory volume in one second (FEV 1) for lung disorders, decreased rate of a decline in lung function over time, inhibition of progressive nerve destruction, reduction of biomarkers associated and correlated with diseases or disorders, a reduction in relapses, improvement in quality of life, reduced time spent in hospital during an acute exacerbation event and the like.
The term “curative treatment,” as used herein to describe the present invention, means that compound, pharmaceutical composition or combination is administered to a subject for the purpose of bringing the disease or disorder into complete remission, or that the disease or disorder is undetectable after such treatment.
The term “selective”, when used to describe a functionally-defined receptor ligand or enzyme inhibitor means selective for the defined receptor or enzyme subtype as compared with other receptor or enzyme subtypes in the same family. For instance, a selective PDE5 inhibitor is a compound which inhibits the PDE5 enzyme subtype more potently than any other PDE enzyme subtype. Such selectivity is preferably at least 2 fold (as measured using conventional binding assays), more preferably at least 10 fold, most preferably at least 100 fold.
The term “alkyl”, alone or in combination, means an acyclic, saturated hydrocarbon group of the formula CnH2n+1 which may be linear or branched. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl and hexyl. Unless otherwise specified, an alkyl group comprises from 1 to 6 carbon atoms.
The carbon atom content of alkyl and various other hydrocarbon-containing moieties is indicated by a prefix designating a lower and upper number of carbon atoms in the moiety, that is, the prefix Ci-Cj indicates a moiety of the integer “i” to the integer “j” carbon atoms, inclusive. Thus, for example, C1-C6alkyl refers to alkyl of one to six carbon atoms, inclusive.
The term “hydroxy,” as used herein, means an OH radical.
Het3 is a saturated or partially saturated (i.e. non aromatic) heterocycle and may be attached via a ring nitrogen atom (when the heterocycle is attached to a carbon atom) or a ring carbon atom (in all cases). Equally, when substituted, the substituent may be located on a ring nitrogen atom (if the substituent is joined through a carbon atom) or a ring carbon atom (in all cases). Specific examples include oxiranyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, piperazinyl, azepanyl, oxepanyl, oxazepanyl and diazepinyl.
Het3 may be fully saturated or partially unsaturated, i.e. may have one or more degrees of unsaturation but may not be fully aromatic.
Het1 is an aromatic heterocycle and may be attached via a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (when the heterocycle is attached to a carbon atom). Equally, when substituted, the substituent may be located on a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (if the substituent is joined through a carbon atom). Specific examples include thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl.
Het2 is an aromatic heterocycle and may be attached via a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (when the heterocycle is attached to a carbon atom). Equally, when substituted, the substituent may be located on a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (if the substituent is joined through a carbon atom). Het2 is aromatic and is therefore necessarily a fused bicycle. Specific examples include imidazo[2,1-b][1,3]thiazolyl, benzofuranyl, benzothienyl, indolyl, benzimidazolyl, indazolyl, benzotriazolyl, pyrrolo[2,3-b]pyridyl, pyrrolo[2,3-c]pyridyl, pyrrolo[3,2-c]pyridyl, pyrrolo[3,2-b]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrazolo[4,3-d]pyridyl, pyrazolo[4,3-c]pyridyl, pyrazolo[3,4-c]pyridyl, pyrazolo[3,4-b]pyridyl, isoindolyl, indazolyl, purinyl, indolizinyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, pyrazolo[1,5-a]pyridyl, pyrrolo[1,2-b]pyridazinyl, imidazo[1,2-c]pyrimidinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, 1,6-naphthyridinyl, 1,7-naphthyridinyl, 1,8-naphthyridinyl, 1,5-naphthyridinyl, 2,6-naphthyridinyl, 2,7-naphthyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl, pyrido[2,3-d]pyrazinyl, pyrido[3,4-b]pyrazinyl, pyrimido[5,4-d]pyrimidinyl, pyrazino[2,3-b]pyrazinyl and pyrimido[4,5-d]pyrimidine.
The term “cycloalkyl” means a means a monocyclic, saturated hydrocarbon group of the formula CnH2n−1. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Unless otherwise specified, a cycloalkyl group comprises from 3 to 8 carbon atoms.
The term “oxo” means a doubly bonded oxygen.
The term “alkoxy” means a radical comprising an alkyl radical that is bonded to an oxygen atom, such as a methoxy radical. Examples of such radicals include methoxy, ethoxy, propoxy, isopropoxy, butoxy and tert-butoxy.
The term “halo” means, fluoro, chloro, bromo or iodo.
As used herein, the terms “co-administration”, “co-administered” and “in combination with”, referring to a combination of a compound of formula (I) and one or more other therapeutic agents, includes the following:
simultaneous administration of such a combination of a compound of formula (I) and a further therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient,
substantially simultaneous administration of such a combination of a compound of formula (I) and a further therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient,
sequential administration of such a combination of a compound of formula (I) and a further therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and
sequential administration of such a combination of a compound of formula (I) and a further therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner.
The term ‘excipient’ is used herein to describe any ingredient other than a compound of formula (I). The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. The term “excipient” encompasses diluent, carrier or adjuvant.
One way of carrying out the invention is to administer a compound of formula (I) in the form of a prodrug. Thus, certain derivatives of a compound of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into a compound of formula (I) having the desired activity, for example by hydrolytic cleavage, particularly hydrolytic cleavage promoted by an esterase or peptidase enzyme. Such derivatives are referred to as ‘prodrugs’. Further information on the use of prodrugs may be found in ‘Pro-drugs as Novel Delivery Systems’, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and ‘Bioreversible Carriers in Drug Design’, Pergamon Press, 1987 (Ed. E. B. Roche, American Pharmaceutical Association). Reference can also be made to Nature Reviews/Drug Discovery, 2008, 7, 355 and Current Opinion in Drug Discovery and Development, 2007, 10, 550.
Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as ‘pro-moieties’ as described, for example, in ‘Design of Prodrugs’ by H. Bundgaard (Elsevier, 1985).
Thus, a prodrug in accordance with the invention is (a) an ester or amide derivative of a carboxylic acid in a compound of formula (I); (b) an ester, carbonate, carbamate, phosphate or ether derivative of a hydroxyl group in a compound of formula (I); (c) an amide, imine, carbamate or amine derivative of an amino group in a compound form formula (I); (d) a thioester, thiocarbonate, thiocarbamate or sulphide derivatives of a thiol group in a compound of formula (I); or (e) an oxime or imine derivative of a carbonyl group in a compound of formula (I).
Some specific examples of prodrugs in accordance with the invention include:
(i) where the compound of formula (I) contains a carboxylic acid functionality (—COOH), an ester thereof, such as a compound wherein the hydrogen of the carboxylic acid functionality of the compound of formula (I) is replaced by C1-C8 alkyl (e.g. ethyl) or (C1-C8 alkyl)C(═O)OCH2— (e.g. tBuC(═O)OCH2—);
(ii) where the compound of formula (I) contains an alcohol functionality (—OH), an ester thereof, such as a compound wherein the hydrogen of the alcohol functionality of the compound of formula (I) is replaced by —CO(C1-C8 alkyl) (e.g. methylcarbonyl) or the alcohol is esterified with an amino acid;
(iii) where the compound of formula (I) contains an alcohol functionality (—OH), an ether thereof, such as a compound wherein the hydrogen of the alcohol functionality of the compound of formula (I) is replaced by (C1-C8 alkyl)C(═O)OCH2— or —CH2OP(═O)(OH)2;
(iv) where the compound of formula (I) contains an alcohol functionality (—OH), a phosphate thereof, such as a compound wherein the hydrogen of the alcohol functionality of the compound of formula (I) is replaced by —P(═O)(OH)2 or —P(═O)(ONa)2 or —P(═O)(O−)2Ca2+;
(v) where the compound of formula (I) contains a primary or secondary amino functionality (—NH2 or —NHR where R≠H), an amide thereof, for example, a compound wherein, as the case may be, one or both hydrogens of the amino functionality of the compound of formula (I) is/are replaced by (C1-C10)alkanoyl, —COCH2NH2 or the amino group is derivatised with an amino acid;
(vi) where the compound of formula (I) contains a primary or secondary amino functionality (—NH2 or —NHR where R≠H), an amine thereof, for example, a compound wherein, as the case may be, one or both hydrogens of the amino functionality of the compound of formula (I) is/are replaced by —CH2OP(═O)(OH)2.
Certain compounds of formula (I) may themselves act as prodrugs of other compounds of formula (I). It is also possible for two compounds of formula (I) to be joined together in the form of a prodrug. In certain circumstances, a prodrug of a compound of formula (I) may be created by internally linking two functional groups in a compound of formula (I), for instance by forming a lactone.
References below to compounds of formula (I) are taken to include the compounds themselves and prodrugs thereof. The invention includes such compounds of formula (I) as well as pharmaceutically acceptable salts of such compounds and pharmaceutically acceptable solvates of said compounds and salts.
Pharmaceutically acceptable salts of the compounds of formula (I) include acid addition and base salts.
Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, naphatlene-1,5-disulfonic acid and xinofoate salts.
Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).
Pharmaceutically acceptable salts of compounds of formula (I) may be prepared by one or more of three methods:
(i) by reacting the compound of formula (I) with the desired acid or base;
(ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of formula (I) or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or
(iii) by converting one salt of the compound of formula (I) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
The compounds of formula (I), and pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. The term ‘solvate’ is used herein to describe a molecular complex comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ may be employed when said solvent is water.
A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates—see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995). Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.
When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.
Also included within the scope of the invention are multi-component complexes (other than salts and solvates) wherein the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallisation, by recrystallisation from solvents, or by physically grinding the components together—see Chem Commun, 17, 1889-1896, by O. Almarsson and M. J. Zaworotko (2004). For a general review of multi-component complexes, see J Pharm Sci, 64 (8), 1269-1288, by Haleblian (August 1975).
The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term ‘amorphous’ refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterised by a change of state, typically second order (‘glass transition’). The term ‘crystalline’ refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterised by a phase change, typically first order (‘melting point’).
The compounds of formula (I) may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described as ‘thermotropic’ and that resulting from the addition of a second component, such as water or another solvent, is described as ‘lyotropic’. Compounds that have the potential to form lyotropic mesophases are described as ‘amphiphilic’ and consist of molecules which possess an ionic (such as —COO−Na+, —COO−K+, or —SO3−Na+) or non-ionic (such as —N−N+(CH3)3) polar head group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and A. Stuart, 4th Edition (Edward Arnold, 1970).
Hereinafter all references to compounds of formula (I) include references to pharmaceutically acceptable salts, solvates, multi-component complexes and liquid crystals thereof and to solvates, multi-component complexes and liquid crystals of pharmaceutically acceptable salts thereof.
The compounds of formula (I) may exhibit polymorphism and/or one or more kinds of isomerism (e.g. optical, geometric or tautomeric isomerism). The compounds of formula (I) may also be isotopically labelled. Such variation is implicit to the compounds of formula (I) defined as they are by reference to their structural features and therefore within the scope of the invention.
Compounds of formula (I) containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound of formula (I) contains an alkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers are possible. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism (‘tautomerism’) can occur. This can take the form of proton tautomerism in compounds of formula (I) containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.
The pharmaceutically acceptable salts of compounds of formula (I) may also contain a counterion which is optically active (e.g. d-lactate or l-lysine) or racemic (e.g. dl-tartrate or dl-arginine).
Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person. Chiral compounds of formula (I) (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture. Chiral chromatography using sub- and supercritical fluids may be employed. Methods for chiral chromatography useful in some embodiments of the present invention are known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography with Packed Columns), pp. 223-249 and references cited therein). In some relevant examples herein, columns were obtained from Chiral Technologies, Inc, West Chester, Pa., USA, a subsidiary of Daicel® Chemical Industries, Ltd., Tokyo, Japan.
When any racemate crystallises, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer. While both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art—see, for example, Stereochemistry of Organic Compounds by E. L. Eliel and S. H. Wilen (Wiley, 1994).
The present invention includes all pharmaceutically acceptable isotopically-labelled compounds of formula (I) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Isotopically-labelled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed. In particular, hydrogen atoms may be replaced by deuterium atoms since such deuterated compounds are sometimes more resistant to metabolism.
Also included within the scope of the invention are active metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug, often by oxidatation or dealkylation. Some examples of metabolites in accordance with the invention include
(i) where the compound of formula (I) contains a methyl group, an hydroxymethyl derivative thereof (—CH3→—CH2OH):
(ii) where the compound of formula (I) contains an alkoxy group, an hydroxy derivative thereof (—OR→—OH);
(iii) where the compound of formula (I) contains a tertiary amino group, a secondary amino derivative thereof (—NRR′→—NHR or —NHR′);
(iv) where the compound of formula (I) contains a secondary amino group, a primary derivative thereof (—NHR→—NH2);
(v) where the compound of formula (I) contains a phenyl moiety, a phenol derivative thereof (-Ph→-PhOH); and
(vi) where the compound of formula (I) contains an amide group, a carboxylic acid derivative thereof (—CONH2→COOH).
For administration to human patients, the total daily dose of a compound of formula (I) is typically in the range of 0.01 mg to 500 mg depending, of course, on the mode of administration. In another embodiment of the present invention, the total daily dose of a compound of formula (I) is typically in the range of 0.1 mg to 300 mg. In yet another embodiment of the present invention, the total daily dose of a compound of formula (I) is typically in the range of 1 mg to 30 mg. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. These dosages are based on an average human subject having a weight of about 65 kg to 70 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.
In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a prefilled capsule, blister or pocket or by a system that utilises a gravimetrically fed dosing chamber. Units in accordance with the invention are typically arranged to administer a metered dose or “puff” containing from 1 to 5000 μg of drug. The overall daily dose will typically be in the range 1 μg to 20 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
A compound of formula (I) can be administered per se, or in the form of a pharmaceutical composition, which, as active constituent contains an efficacious dose of at least one compound of the invention, in addition to customary pharmaceutically innocuous excipients and/or additives.
Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
Compounds of formula (I) may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays and liquid formulations.
Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
Compounds of formula (I) may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986, by Liang and Chen (2001).
For tablet dosage forms, depending on dose, the drug may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 weight % to 25 weight %. In one embodiment of the present invention, the disintegrant will comprise from 5 weight % to 20 weight % of the dosage form. Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate. Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet. Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %. In one embodiment of the present invention, lubricants comprise from 0.5 weight % to 3 weight % of the tablet. Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.
Exemplary tablets contain up to about 80% drug, from about 10 weight % to about 90 weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant.
Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. Formulations of tablets are discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).
Consumable oral films for human or veterinary use are typically pliable water-soluble or water-swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive and typically comprise a compound of formula (I), a film-forming polymer, a binder, a solvent, a humectant, a plasticiser, a stabiliser or emulsifier, a viscosity-modifying agent and a solvent. Some components of the formulation may perform more than one function. The film-forming polymer may be selected from natural polysaccharides, proteins, or synthetic hydrocolloids and is typically present in the range 0.01 to 99 weight %, more typically in the range 30 to 80 weight %. Other possible ingredients include anti-oxidants, colorants, flavourings and flavour enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, surfactants and taste-masking agents. Films in accordance with the invention are typically prepared by evaporative drying of thin aqueous films coated onto a peelable backing support or paper. This may be done in a drying oven or tunnel, typically a combined coater dryer, or by freeze-drying or vacuuming.
Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release includes delayed, sustained, pulsed, controlled, targeted and programmed release. Suitable modified release formulations for the purposes of the invention are described in U.S. Pat. No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in Pharmaceutical Technology On-line, 25(2), 1-14, by Verma et al (2001). The use of chewing gum to achieve controlled release is described in WO-A-00/35298.
Compounds of formula (I) may also be administered directly into the blood stream, into muscle, or into an internal organ. Such parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous administration. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
Compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally.
The compounds of formula (I) can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler, as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane, or as nasal drops. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin. Delivery by inhalation is the preferred route of administration for the compounds of the present invention.
The pressurised container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound of formula (I) comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the compound, a propellant as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.
Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from 1 μg to 20 mg of the compound of the invention per actuation and the actuation volume may vary from 1 μl to 100 μl. A typical formulation may comprise a compound of formula (I), propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for intranasal administration. Formulations for intranasal administration may be formulated to be immediate and/or modified release using, for example, PGLA. Modified release includes delayed, sustained, pulsed, controlled, targeted and programmed release.
Compounds of formula (I) may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline.
Compounds of formula (I) may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste, bioavailability and/or stability when using any of the aforementioned modes of administration. Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in international patent publications WO-A-91/11172, WO-A-94/02518 and WO-A-98/55148.
Inasmuch as it may desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound of formula (I), may conveniently be combined in the form of a kit suitable for coadministration of the compositions. Thus, a kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of formula (I), and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like. Such a kit is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically comprises directions for administration and may be provided with a so-called memory aid.
All the compounds of formula (I) can be made by the specific and general experimental procedures described below in combination with the common general knowledge of one skilled in the art (see, for example, Comprehensive Organic Chemistry, Ed. Barton and Ollis, Elsevier; Comprehensive Organic Transformations: A Guide to Functional Group Preparations, Larock, John Wiley and Sons). In the general methods that follow, R1, R2, R3, X and n have the meanings given in embodiment E1 described above unless otherwise stated.
Compounds of formula (I) can be made by reacting a compound of formula:
with a compound of formula:
R3—X-LG1 (III)
in which LG1 is a suitable leaving group such as a halogen atom. The reaction will typically be carried out in a suitable inert solvent in the presence of a base such as diisopropylethylamine. When X is —SO2—, for example, a sulphonyl chloride (LG1=Cl) may be used. In a typical procedure, a solution of the compound of formula (II) in DMF is treated with one equivalent of the sulphonyl chloride and 1 equivalent of diisopropylethylamine and stirred at room temperature. When X is —CH2-, an alkyl bromide (LG1=Br) may be used. In a typical procedure, a solution of the compound of formula (II) in DMF is treated with 1.1 equivalents of the alkyl bromide and 1.1 equivalents of diisopropylethylamine and stirred at 50° C. When X is —CO—, an acid chloride (LG1=Cl) may be used. In a typical procedure, a solution of the compound of formula (II) in DMF is treated with 1.1 equivalents of the acid chloride and 1.1 equivalents of diisopropylethylamine and stirred at room temperature.
Where X is a carbonyl group, the leaving group LG1 may be created in situ from the corresponding carboxylic acid of formula
R3—CO2H (IV)
by using a condensation reagent such as HATU. In a typical procedure, a solution of the compound of formula (II) in DMF is treated with 1.1 equivalents of HATU and 1.1 equivalents of diisopropylethylamine and stirred at room temperature for 30 minutes. An equivalent of the acid of formula (IV) is then added. For a general review on amide bond formation, see Chem. Soc. Rev., 2009, 38(2), 606-631
Where X is —CH2—, an aldehyde of formula:
R3—CHO (V)
may alternatively be condensed with a compound of formula (II) under reducing conditions in order to provide the desired compound of formula (I). In a typical procedure, a solution of the compound of formula (II) in DMF is treated with the 1.5 equivalents of the aldehyde of formula (V), 2 equivalents of diisopropylethylamine and 1.5 equivalents of acetic acid and stirred at room temperature for one hour. Sodium triacetoxyborohydride (1.5 equivalents) is then added and stirring continued at room temperature.
Compounds of formula (II) can be assembled by successive aryl-heteroaryl and heteroaryl-heteroaryl organometallic coupling reactions. One example of a possible reaction sequence is shown in Scheme 1 (PG=protecting group, LG=leaving group, M=metal species; where multiple protecting groups are shown, they may be the same or different). Free NH groups will generally need to be protected during these reactions. Suitable protecting groups, their introduction and their removal are all part of the common general knowledge of the skilled person—see, for instance, ‘Protective Groups in Organic Chemistry’ by Wuts and Greene (Wiley-Blackwell).
Suitable reaction conditions for the various steps necessary to prepare and react together the compounds in Scheme 1 may be found in the specific Preparations listed below. For a general review on organometallic cross-coupling chemistry, see ‘Handbook of Organopalladium Chemistry for Organic Synthesis’ (Volume 1) edited by Ei-ichi Negishi (John Wiley & Sons).
Compounds of formula (I) can also be prepared by treating a compound of formula:
with an acid (e.g. concentrated hydrochloric acid). PG1 is an acid-labile protecting group and C═Y is a carbonyl group or an acid-labile, protected form of a carbonyl group (e.g. a ketal). The reaction will usually be performed in a suitable inert solvent with heating.
Compounds of formula (X) can be made from precursors of formula:
Compounds of formula (XI) can be assembled using the aryl-heteroaryl bond forming reactions discussed above.
The skilled person will appreciate that many compounds of formula (I) may be interconverted by functional group manipulation.
The starting materials necessary for carrying out the methods described above are in many cases commercially available and may otherwise be described in the literature or in the Preparations below or may be made using analogous procedures to those described in the literature or in the Preparations below.
Supplementing the general methods presented above, the following experimental details illustrate specifically how certain compounds of formula (I) may be prepared. All Examples are compounds of formula (I). Preparations are intermediates useful in the synthesis of compounds of formula (I).
The following HPLC methods have been used in the characterization of the Examples below:
Method A
HPLC
conditions
Analytical (QC)
Preparative
Column
Gemini-NX 3 μm C18
Gemini-NX 5 μm C18
110A
21.2 × 100 mm
Temperature
Ambient
Ambient
Detection
UV 225 nm-ELSD-MS
UV 225 nm-ELSD-
MS
Injection volume
5 μL
1000 μL
Flow rate
1.5 mL/min
18 mL/min
Mobile phase
A: H2O + 0.1%
A: H2O + 0.1% DEA
ammonium acetate
B: MeCN + 0.1% DEA
B: MeCN + 0.1%
ammonium acetate
Time
(min)
% B
Time (min)
% B
Gradient
0
5
0-1.0
5
0-3.0
5-95
1.0-7.0
5-98
3.0-4.0
95
7.0-9.0
98
4.0-4.1
95-5
9.0-9.10
98-5
4.1-5.0
5
9.10-10
5
Method B
HPLC
conditions
Analytical (QC)
Preparative
Column
Gemini-NX3 μm C18
Gemini-NX 5 μm C18
110A
21.1 × 100 mm
Temperature
Ambient
Ambient
Detection
UV 225 nm-ELSD-MS
UV 225 nm-ELSD-
MS
Injection volume
5 μL
1000 μL
Flow rate
1.5 mL/min
18 mL/min
Mobile phase
A: H2O + 0.1% formic
A: H2O + 0.1% formic
B: MeCN + 0.1% formic
B: MeCN + 0.1% formic
acid
acid
Time
(min)
% B
Time (min)
% B
Gradient
0
5
initial
20
0-3.0
5-95
1
20
3.0-4.0
95
5.4
70
4.0-4.1
95-5
6.33
98
4.1-5.0
5
6.4
20
7
20
Method C
HPLC
conditions
Preparative
Column
Phenomenex Luna C18
5 μm-100 Å
21.2 × 150 mm
Temperature
Ambient
Detection
UV 254 nm-ELSD-
MS
Injection volume
1000 μL
Flow rate
18 mL/min
Mobile phase
A: H2O + 0.05% formic
acid
B: MeCN + 0.05%
formic acid
Time (min)
% B
Gradient
0-2.5
5
2.5-17.5
5-95
17.5-22.5
95
22.5-22.6
95-5
22.6-23.0
5
Method D
HPLC
conditions
Preparative
Column
Zorbax SB C18 5 μm-100 Å
21.2 × 150 mm
Temperature
Ambient
Detection
UV 254 nm-ELSD-
MS
Injection volume
1000 μL
Flow rate
20 mL/min
Mobile phase
A: H2O + 0.05%
NH4OAc
B: MeCN + 0.05%
NH4OAc
Time (min)
% B
Gradient
0-2.5
5
2.5-17.5
5-95
17.5-22.5
95
22.5-22.6
95-5
22.6-23.0
5
Method E
HPLC
conditions
Preparative
Column
Luna Phenyl-Hexyl
5 μm-100 Å
21.2 × 150 mm
Temperature
Ambient
Detection
UV 254 nm-ELSD-
MS
Injection volume
1000 μL
Flow rate
20 mL/min
Mobile phase
A: H2O + 0.05%
NH4OAc
B: MeCN + 0.05%
NH4OAc
Time (min)
% B
Gradient
0-2.5
5
2.5-17.5
5-95
17.5-22.5
95
22.5-22.6
95-5
22.6-23.0
5
Method F
HPLC
conditions
Preparative
Column
Xterra RP18
19-250 mm
Temperature
Ambient
Detection
UV 254 nm-ELSD-
MS
Injection volume
1000 μL
Flow rate
16 mL/min
Mobile phase
A: H2O + 0.05%
NH4OAc
B: MeCN + 0.05%
NH4OAc
Time (min)
% B
Gradient
0-2.5
5
2.5-17.5
5-95
17.5-22.5
95
22.5-22.6
95-5
22.6-25.0
5
Method G
HPLC
conditions
Preparative
Column
Sunfire C18 30 × 100 mm
5 u
Temperature
Ambient
Detection
UV 254 nm-ELSD-
MS
Injection volume
1000 μL
Flow rate
16 mL/min
Mobile phase
A: H2O + 0.05%
NH4OAc
B: MeCN + 0.05%
NH4OAc
Time (min)
% B
Gradient
0-2.5
5
2.5-17.5
5-95
17.5-22.5
95
22.5-22.6
95-5
22.6-25.0
5
Example 1
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(4-fluoro-phenyl)-methanone
To a solution of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) in DMF (1 mL), was added HATU (32 mg, 84 μmol), and DIPEA (56 μL, 320 μmol). The reaction mixture was stirred at room temperature for 30 minutes. 4-Fluoro-benzoic acid (11.2 mg, 80 μmol) was added to the reaction mixture and stirring was continued for 18 hours. Saturated aqueous sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture. The resulting solid was collected by filtration, washing with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by HPLC Method B to afford 7.7 mg of the title compound.
LCMS (Method A): RT 2.52 min (100% area), ES m/z 500.182 [M+H]+.
Example 2
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-isothiazol-3-yl-methanone
The title compound was prepared from 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) and isothiazole-3-carboxylic acid (11 mg, 80 μmol) using the same method as described in Example 1. The crude material was purified by HPLC Method A to afford 7.7 mg of the title compound.
LCMS (Method A) RT 2.39 min (100% area), ES m/z 489.143 [M+H]+.
Example 3
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-isothiazol-3-yl-methanone
To a solution of N-(1-benzyl-4,4-diethoxy-piperidin-3-yl)-6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboxamidine (Preparation 9, 6.06 g, 9.41 mmol) in ethanol (34 mL) was added concentrated hydrochloric acid (12M, 15.8 mL, 189 mmol). The reaction mixture was heated at 65° C. for 18 hours. The reaction mixture was concentrated in vacuo and recharged with fresh ethanol (34 mL) and concentrated hydrochloric acid (12M, 15.8 mL, 189 mmol). The reaction mixture was heated at 65° C. for a further 4 hours. Water (20 mL) was added to the reaction mixture at 65° C. and then the reaction was allowed to cool slowly to room temperature. The solvents were removed in vacuo and the residue was partitioned between 2-MeTHF (200 mL) and saturated sodium hydrogen carbonate aqueous solution (100 mL). The organic layer was washed with further saturated sodium hydrogen carbonate aqueous solution (100 mL). The combined aqueous layers were re-extracted with 2-MeTHF (250 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo to yield a brown foam. The crude material was dissolved in MeCN (150 mL) and ethanol (30 mL) and heated at 50° C. for 2 days. The product crystallised from this solution and was collected by filtration and dried in vacuo to give the title compound as a crystalline white solid (3.53 g) in an 80% yield.
1H NMR (400 MHz, CD3OD) δ ppm 1.04 (t, 3H), 2.52 (q, 2H), 2.81 (t, 2H), 2.91 (t, 2H), 3.62 (s, 2H), 3.80 (s, 2H), 6.87 (d, 1H), 6.92 (d, 1H), 7.11 (d, 1H), 7.26-7.43 (m, 6H), 8.22 (d, 1H).
LCMS: m/z 468 [M+H]+, 466 [M−H]−.
Example 4
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-d]pyridin-5-yl}-(5-piperidin-1-yl-pyrazin-2-yl)-methanone
To a solution of 5-piperidin-1-yl-pyrazine-2-carboxylic acid (Preparation 43, 10.7 g, 51.8 mmol) in DMF (200 mL) was added DIPEA (24.6 mL, 141 mmol) and HATU (21.5 g, 56.5 mmol) and the resulting mixture was stirred at room temperature for 10 minutes before being added dropwise to a suspension of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol dihydrochloric acid salt (Preparation 11, 19.5 g, 47.1 mmol) in DMF (200 mL) over 30 minutes, using a further 75 mL DMF to wash the vessel. The reaction mixture was then stirred at room temperature for 18 hours. A further portion of 5-piperidin-1-yl-pyrazine-2-carboxylic acid (1.07 g, 5.18 mmol) in DMF (40 mL) was treated with DIPEA (2.46 mL, 14.1 mmol) and activated with HATU (2.15 g, 5.65 mmol) and the resulting mixture was stirred at room temperature for 10 minutes before being added to the original reaction mixture which was then stirred for a further 4 hours at room temperature. The reaction mixture was poured onto water (1.2 L) and the pH was adjusted to 7 with sodium hydroxide solution. The resulting suspension was stirred at room temperature for 30 minutes. The precipitate was collected by filtration, washed with water (400 mL) and then dried under vacuum. The crude material was dissolved in ethanol (113 mL) and treated with a 1M aqueous solution of sodium hydroxide. The reaction mixture was stirred at room temperature for 18 hours. The precipitate was collected by filtration, washed with a cold solution of 1:3 1M sodium hydroxide:ethanol (100 mL) and dried under vacuum to give the sodium salt of the title compound, 16.14 g. This material was dissolved in water (100 mL) and treated with a 10% aqueous solution of citric acid (10 mL) to adjust the pH to 4. A few drops of 1M sodium hydroxide solution were added to bring the pH to 7. The resulting suspension was stirred at room temperature for 1 hour and the solid was collected by filtration, washed with water and then dried under vacuum to give the title compound as a white solid (13.864 g) in an 89% yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 0.94 (t, 3H), 1.54-1.60 (m, 2H), 1.60-1.67 (m, 2H), 2.38 (q, 2H), 2.71-2.83 (m, 2H), 3.64-3.71 (m, 4H), 3.85-3.98 (m, 4H), 4.63-4.78 (m, 2H), 6.66 (d, 1H), 6.73 (d, 1H), 7.00-7.08 (m, 1H), 7.16-7.24 (m, 1H), 816-8.25 (m, 1H), 8.29 (s, 1H), 8.37 (s, 1H).
LCMS: m/z 567 [M+H]+, 565 [M−H]−.
Example 5
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(6-phenoxy-pyridin-3-yl)-methanone
The title compound was prepared from 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) and 6-phenoxy-nicotinic acid (17 mg, 80 μmol) using the method of Example 1. The crude material was purified by HPLC Method A to afford 3.1 mg of the title compound.
LCMS (Method A): RT 2.67 min (100% area), ES m/z 575.213 [M+H]+.
Example 6
5-Ethyl-2-fluoro-4-{3-[5-(6-morpholin-4-yl-pyridine-3-sulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-O]pyridin-2-yl]-1H-indazol-6-yl}-phenol
To a solution of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) in DMF (1 mL), was added 6-morpholin-4-yl-pyridine-3-sulfonyl chloride (21 mg, 80 μmol) and DIPEA (56 μL, 320 μmol). The reaction mixture was stirred at room temperature for 18 hours. Saturated aqueous sodium hydrogen carbonate solution (5 mL) was added and the resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by HPLC Method A to afford 18.7 mg of the title compound.
LCMS (Method A): RT 2.58 min (100% area), ES m/z 602.206 [M−H]−.
Example 7
5-Ethyl-2-fluoro-4-{3-[5-(6-phenoxy-pyridine-3-sulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol
The title compound was prepared from 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) and 6-phenoxy-pyridine-3-sulfonyl chloride (22 mg, 80 μmol) using the method of Example 6. The crude material was purified by HPLC Method A to afford 8.4 mg of the title compound.
LCMS (Method A): RT 2.82 min (100% area), ES m/z 611.18 [M+H]+.
Example 8
(5-Chloro-pyridin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-methanone
To a solution of 5-chloro-pyridine-2-carboxylic acid (13.2 mg, 85 μmol) in DMF (1 mL) was added HATU (32 mg, 85 μmol) and the resulting reaction mixture was stirred at room temperature for 30 minutes. 5-Ethyl-2-fluoro-4-[3-(1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 32, 50 mg, 80 μmol) and DIPEA (56 μL, 320 μmol) were added and stirring was continued at room temperature for 18 hours. Saturated aqueous sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by HPLC Method A to afford 20.5 mg of the title compound.
LCMS (Method A): RT 2.76 min (100% area), ES m/z 531.163 [M−H]−.
Example 9
2-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carbonyl}-isonicotinonitrile
The title compound was prepared from 4-cyano-pyridine-2-carboxylic acid (12.5 mg, 85 μmol) and 5-ethyl-2-fluoro-4-[3-(1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 32, 50 mg, 80 μmol) using the method of Example 8. The crude material was purified by HPLC Method B to afford 6.5 mg of the title compound.
LCMS (Method A): RT 2.66 min (100% area), ES m/z 522.198 [M+H]+.
Example 10
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-(4-fluoro-phenyl)-methanone
The title compound was prepared from 4-fluoro-benzoic acid (12.5 mg, 85 μmol) and 5-ethyl-2-fluoro-4-[3-(1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 32, 50 mg, 80 μmol) using the method of Example 8. The crude material was purified by HPLC Method B to afford 14.5 mg of the title compound.
LCMS (Method A): RT 2.76 min (100% area), ES m/z 514.198 [M+H]+.
Example 11
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepin-6-yl}-isothiazol-3-yl-methanone
The title compound was prepared from isothiazole-3-carboxylic acid (11 mg, 85 μmol) and 5-ethyl-2-fluoro-4-[3-(1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 32, 50 mg, 80 μmol) using the method of Example 8. The crude material was purified by HPLC Method B to afford 10.3 mg of the title compound.
LCMS (Method A): RT 2.54 min (100% area), ES m/z 503.159 [M+H]+.
Example 12
5-Ethyl-2-fluoro-4-{3-[5-(4-fluoro-benzenesulfonyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol
To a solution of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) in DMF (1 mL), was added 4-fluoro-benzenesulfonyl chloride (16 mg, 80 μmol), and DIPEA (56 μL, 320 μmol). The reaction mixture was stirred at room temperature for 4 hours. Saturated aqueous sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by reverse phase chromatography (Method C) to afford 7 mg of the title compound.
1H NMR (400 MHz, CD3OD) δ ppm 1.02 (t, 3H), 2.52 (q, 2H), 3.78-3.81 (m, 2H), 4.37-4.29 (m, 2H), 6.88-6.96 (m, 2H), 7.15 (d, 1H), 7.32-7.36 (m, 2H), 7.40 (s, 1H), 7.89-7.95 (m, 2H), 8.21 (d, 1H).
LCMS: m/z 536 [M+H]+.
Example 13
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-fluoro-phenoxy)-pyrazin-2-yl]-methanone
To a solution of 5-(2-fluoro-phenoxy)-pyrazine-2-carboxylic acid (Preparation 45, 19 mg, 80 μmol) in DMF (1 mL) was added HBTU (32 mg, 85 μmol) and the resulting reaction mixture was stirred at room temperature for 30 minutes. 5-Ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) and DIPEA (56 μL, 320 μmol) were added and stirring was continued at room temperature for 18 hours. Saturated aqueous sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by HPLC Method B to afford 8.3 mg of the title compound.
LCMS (Method A): RT 2.97 min (100% area), ES m/z 594.199 [M−H]−.
Example 14
4-[3-(6-Benzyl-1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-5-ethyl-2-fluoro-phenol
To a solution of 5-ethyl-2-fluoro-4-[3-(1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 32, 50 mg, 80 μmol) in DMF (1 mL), was added benzyl bromide (14.4 mg, 10 μL, 85 μmol), and DIPEA (56 μL, 320 μmol). The reaction mixture was heated at 50° C. for 18 hours. The reaction mixture was then cooled to room temperature and partitioned between EtOAc (50 mL) and water (50 mL). The organic layer was washed with brine (50 mL), dried over sodium sulfate and concentrated in vacuo to furnish a brown oil. The crude material was purified by reverse phase chromatography (Method C) to afford 4 mg of the title compound.
1H NMR (400 MHz, CD3OD) δ ppm 1.02 (t, 3H), 2.55 (q, 2H), 3.08-3.11 (m, 4H), 3.34-3.38 (m, 4H), 4.23 (s, 2H), 6.88-6.94 (m, 2H), 7.18 (d, 1H), 7.41-7.4 (m, 3H), 7.52-7.55 (m, 2H), 8.20 (d, 1H), 8.39 (br s, 1H).
LCMS: m/z 482 [M+H]+, 480 [M−H]−.
Example 15
(5-Chloro-pyridin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
To a solution of 5-chloro-pyridine-2-carboxylic acid (12 mg, 80 μmol) in DMF (1 mL) was added HBTU (32 mg, 85 μmol) and the resulting reaction mixture was stirred at room temperature for 30 minutes. 5-Ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) and DIPEA (56 μL, 320 μmol) were added and stirring was continued at room temperature for 18 hours. Saturated aqueous sodium hydrogen carbonate solution (5 mL) was added to the reaction mixture. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by reverse phase chromatography (Method C) to afford 3.3 mg of the title compound.
1H NMR (400 MHz, CD3OD) δ ppm 1.02 (t, 3H), 2.52 (q, 2H), 2.88-2.96 (m, 2H), 3.79-3.82 (m, 2H), 4.15-4.19 (m, 2H), 6.82-6.96 (m, 2H), 7.08-7.18 (m, 1H), 7.39-7.41 (m, 1H), 7.69-7.71 (m, 1H), 7.99-8.01 (m, 1H), 8.19-8.21 (d, 1H), 8.38-8.42 (m, 1H).
LCMS: m/z 517 [M+H]+, 515 [M−H]−.
Example 16
5-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carbonyl}-pyridine-2-carbonitrile
To a solution of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 80 μmol) in DMF (1 mL), was added 6-cyano-nicotinoyl chloride (Preparation 46, 17 mg, 96 μmol), and DIPEA (56 μL, 320 μmol). The reaction mixture was stirred at room temperature for 72 hours and then saturated aqueous sodium hydrogen carbonate solution (5 mL) was added. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by reverse phase chromatography (Method C) to afford 6.9 mg of the title compound.
1H NMR (400 MHz, CD3OD) δ ppm 1.02 (t, 3H), 2.52 (q, 2H), 2.85-2.98 (m, 2H), 3.75-3.79 (m, 2H), 4.15-4.19 (m, 2H), 6.82-6.96 (m, 2H), 7.08-7.18 (m, 1H), 7.38-7.40 (m, 1H), 8.00 (d, 1H), 8.15-8.20 (m, 2H), 8.22 (d, 1H).
LCMS: m/z 508 [M+H]+, 506 [M−H]−.
Example 17
5-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carbonyl}-pyridine-2-carbonitrile
To a solution of 5-ethyl-2-fluoro-4-[3-(1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 32, 28 mg, 44 μmol) in DMF (1 mL), was added 6-cyano-nicotinoyl chloride (Preparation 46, 20.9 mg, 141 μmol), and DIPEA (31 μL, 176 μmol). The reaction mixture was stirred at room temperature for 18 hours and then saturated aqueous sodium hydrogen carbonate solution (5 mL) was added. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by HPLC Method A to afford 10.3 mg of the title compound.
LCMS (Method A): RT 2.69 min (100% area), ES m/z 522.198 [M−H]−.
Example 18
5-Ethyl-2-fluoro-4-[3-(5-quinolin-6-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol
To a solution of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) in DMF (1 mL), was added quinoline-6-carbaldehyde (31 mg, 198 μmol), DIPEA (34 mg, 46 μL, 264 μmol) and AcOH (11.8 mg, 11 μL, 198 μmol). The reaction mixture was stirred at room temperature for 1 hour. STAB (42 mg, 198 μmol) was added and stirring was continued for 18 hours. The reaction mixture was partitioned between EtOAc (10 mL) and saturated aqueous sodium hydrogen carbonate solution (10 ml). The organic layer was washed with further saturated aqueous sodium hydrogen carbonate solution (2×10 mL), dried over magnesium sulfate and concentrated in vacuo to furnish a brown oil. The crude material was purified by HPLC Method A to afford 20.6 mg of the title compound.
LCMS (Method A): RT 2.59 min (100% area), ES m/z 519.223 [M−H]−.
Example 19
5-Ethyl-2-fluoro-4-{3-[5-(4-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol
The title compound was prepared from 4-hydroxy-benzaldehyde (24.2 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 18. The crude material was purified by HPLC Method A to afford 31.4 mg of the title compound.
LCMS (Method A): RT 2.32 min (100% area), ES m/z 482.207 [M−H]−.
Example 20
5-Ethyl-2-fluoro-4-{3-[5-(3-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-d]pyridin-2-yl]-1H-indazol-6-yl}-phenol
The title compound was prepared from 3-hydroxy-benzaldehyde (24.2 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 18. The crude material was purified by HPLC Method A to afford 39.6 mg of the title compound.
LCMS (Method A): RT 2.39 min (100% area), ES m/z 484.207 [M+H]+.
Example 21
4-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-d]pyridin-5-ylmethyl}-pyridine-2-carbonitrile
The title compound was prepared from 4-formyl-pyridine-2-carbonitrile (26 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 18. The crude material was purified by HPLC Method B to afford 16 mg of the title compound.
LCMS (Method B): RT 1.75 min (100% area), ES m/z 494.203 [M+H]+.
Example 22
5-Ethyl-2-fluoro-4-{3-[5-(3-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol formic acid salt
To a solution of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) in DMF (1 mL), was added 3-methoxy-benzaldehyde (27 mg, 198 μmol), DIPEA (34 mg, 46 μL, 264 μmol) and AcOH (11.8 mg, 11 μL, 198 μmol). The reaction mixture was stirred at room temperature for 1 hour. STAB (42 mg, 198 μmol) was added and stirring was continued for 18 hours. Saturated aqueous sodium hydrogen carbonate solution (5 ml) was added to the reaction mixture. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by HPLC Method B to afford 30.1 mg of the title compound.
LCMS (Method A): RT 2.64 min (100% area), ES m/z 496.223 [M−H]−.
Example 23
5-Ethyl-2-fluoro-4-[3-(5-quinolin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol
The title compound was prepared from quinoline-3-carbaldehyde (31 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 22. The crude material was purified by HPLC Method B to afford 14.3 mg of the title compound.
LCMS (Method A): RT 2.59 min (100% area), ES m/z 519.223 [M+H]+.
Example 24
5-Ethyl-2-fluoro-4-{3-[5-(6-phenoxy-pyridin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol
The title compound was prepared from 6-phenoxy-pyridine-3-carbaldehyde (39 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 22. The crude material was purified by HPLC Method A to afford 24.7 mg of the title compound.
LCMS (Method B): RT 2.53 min (100% area), ES m/z 561.234 [M+H]+.
Example 25
5-Ethyl-2-fluoro-4-{3-[5-(3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5′-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol
The title compound was prepared from 3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5′-carbaldehyde (38 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 22. The crude material was purified by HPLC Method A to afford 22.1 mg of the title compound.
LCMS (Method B): RT 2.22 min (100% area), ES m/z 552.281 [M+H]+.
Example 26
3-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-ylmethyl}-pyridine-2-carbonitrile
The title compound was prepared from 3-formyl-pyridine-2-carbonitrile (26 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 22. The crude material was purified by HPLC Method A to afford 14.9 mg of the title compound.
LCMS (Method A): RT 1.46 min (100% area), ES m/z 494.203 [M+H]+.
Example 27
5-Ethyl-2-fluoro-4-{3-[5-(4-fluoro-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-phenol
The title compound was prepared from 4-fluoro-benzaldehyde (25 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 22. The crude material was purified by HPLC Method A to afford 3.4 mg of the title compound.
LCMS (Method A): RT 2.77 min (100% area), ES m/z 486.203 [M+H]+.
Example 28
5-Ethyl-2-fluoro-4-[3-(5-[1,8]naphthyridin-2-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol
The title compound was prepared from [1,8]naphthyridine-2-carbaldehyde (31 mg, 198 μmol) and 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt (Preparation 25, 50 mg, 132 μmol) using the method of Example 22. The crude material was purified by HPLC Method B to afford 22.5 mg of the title compound.
LCMS (Method B): RT 2.28 min (100% area), ES m/z 520.218 [M+H]+.
Example 29
(2-{6-[5-Fluoro-4-hydroxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl)-(5-piperidin-1-yl-pyrazin-2-yl)-methanone diethylamine salt
The title compound was prepared from 5-piperidin-1-yl-pyrazine-2-carboxylic acid (Preparation 44, 35 mg, 168 μmol) and 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (Preparation 39, 100 mg, 168 μmol) using the method of Example 8. The crude material was purified by HPLC Method A to afford 29.0 mg of the title compound as the diethylamine salt.
LCMS (Method B): RT 2.66 min (100% area), ES m/z 621.227 [M+H]+.
Example 30
(2-{6-[5-Fluoro-4-hydroxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl)-(4-fluoro-phenyl)-methanone diethylamine salt
The title compound was prepared from 4-fluoro-benzoic acid (24 mg, 168 μmol) and 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (Preparation 39, 100 mg, 168 μmol) using the method of Example 8. The crude material was purified by HPLC Method A to afford 33.3 mg of the title compound.
LCMS (Method B): RT 2.62 min (100% area), ES m/z 554.154 [M+H]+.
Example 31
4-[3-(5-Benzyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-O]pyridin-2-yl)-1H-indazol-6-yl]-2-fluoro-5-(2,2,2-trifluoro-ethyl)-phenol diethylamine salt
To a solution of 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (Preparation 39, 100 mg, 168 μmol) in DMF (1 mL), was added benzyl bromide (28.8 mg, 20 μL, 168 μmol), and DIPEA (120 μL, 672 μmol). The reaction mixture was heated at 80° C. for 3 hours. The reaction mixture was then cooled to room temperature and saturated aqueous sodium hydrogen carbonate solution (5 mL) was added. The resulting solid was collected by filtration and washed with further saturated aqueous sodium hydrogen carbonate solution. The crude material was purified by HPLC Method A to afford 10.6 mg of the title compound.
LCMS (Method A): RT 2.67 min (100% area), ES m/z 522.184 [M+H]+.
Example 32
{5-[(2-Dimethylamino-ethyl)-methyl-amino]-pyrazin-2-yl}-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
To a solution of (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 0.154 mmol) in DMSO (1 mL) were added DIPEA (0.08 mL, 0.463 mmol) and N,N,N-trimethylethylendiamine (31.56 mg, 0.308 mmol) and the mixture stirred at room temperature for 18 hours. The crude reaction mass was purified by prep-HPLC Method C to afford the title compound as an off white solid (25 mg, 28%).
1H NMR (400 MHz, DMSO) δ (ppm): 1.01 (t, 3H), 2.18 (s, 6H), 2.44 (m, 4H), 2.78 (bs, 2H), 3.13 (s, 3H), 3.70 (t, 3H), 3.94 (bs, 2H), 4.66-4.76 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 8.12 (s, 1H), 8.31 (d, 1H), 8.38 (d, 1H), 12.51 (s, 1H), 13.21 (s, 1H);
LCMS: Rt=2.59 min; m/z 584[M+H]+
Example 33
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-pyrrolidin-1-yl-ethylamino)-pyrazin-2-yl]-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (50 mg, 96 μmol) and (2-(pyrrolidin-1-yl)ethanamine, 50 mg, 132 μmol) using the method of Example 32. The crude material was purified by HPLC Method E to afford (30 mg, 52%) of the title compound.
1H NMR (400 MHz, DMSO) δ (ppm): 0.98-1.01 (t, 3H), 1.13 (s, 3H), 1.22 (bs, 2H), 1.68 (bs, 3H), 1.90 (s, 1H), 2.11 (s, 1H), 2.60 (t, 2H), 2.78 (bs, 3H), 3.42 (m, 2H), 3.91 (bs, 2H), 4.64-4.68 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.08 (d, 1H), 7.36 (s, 1H), 7.60 (bs, 1H), 7.95 (s, 1H), 8.29-8.33 (m, 2H), 9.80 (bs, 1H), 12.52 (s, 1H), 13.22 (s, 1H);
LCMS: Rt=5.71 min; m/z 596.4[M+H]+.
Example 34
[5-(2-Dimethylamino-ethylamino)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (100 mg, 193 μmol) and N,N-dimethylethylendiamine, (34 mg, 386 μmol) using the method of Example 32. The crude material was purified by HPLC Method E to afford (50 mg, 46%) of the title compound as white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.03 (t, 3H), 2.18 (s, 6H), 2.40-2.45 (m, 4H), 2.77 (bs, 2H), 2.93-2.98 (m, H), 3.16 (s, 3H), 3.38-3.44 (m, 2H), 3.78 (m, 2H), 4.66 (m, 2H), 6.90 (d, 1H), 6.99 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 7.53 (bs, 1H), 7.96 (s, 1H), 8.29 (s, 2H):
LCMS: Rt=2.53 min; m/z 570[M+H]+.
Example 35
[5-(4-Dimethylamino-piperidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and 4-N,N-dimethylaminopiperidine, (40 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method C to afford (60 mg, 64%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.01 (t, 3H), 1.39-1.41 (m, 2H), 1.83-1.86 (m, 2H), 2.18 (s, 6H), 2.32-2.38 (m, 2H), 2.78 (bs, 2H), 2.95-3.01 (t, 2H), 3.90 (s, 2H), 3.92-3.94 (m, 2H), 4.42-4.45 (m, 2H), 4.67-4.75 (m, 2H), 6.87 (d, 1H), 6.92 (d, 1H), 7.03 (bs, 1H), 7.37 (s, 1H), 8.33-8.39 (m, 3H), 12.50 (bs, 1H), 13.22 (bs, 1H);
LCMS: Rt=5.46 min; m/z 610.4[M+H]+
Example 36
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-{5-[ethyl-(2-hydroxy-ethyl)-amino]-pyrazin-2-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (120 mg, 231 μmol) and N-ethylaminoethanol, (41 mg, 463 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (60 mg, 64%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.99-1.03 (t, 3H), 1.13-1.16 (t, 3H), 2.32-2.41 (m, 2H), 2.78 (bs, 2H), 2.94-2.97 (m, 1H), 3.61 (bs, 5H), 3.94 (m, 2H), 4.65-4.86 (m, 3H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (bs, 1H), 7.36 (s, 1H), 8.14 (s, 1H), 8.31-8.38 (m, 2H), 9.82 (bs, 1H), 12.51 (bs, 1H), 13.20 (bs, 1H);
LCMS: Rt=2.69 min; m/z 571.4[M+H]+.
Example 37
[5-((R)-3-Dimethylamino-pyrrolidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and (R)-3-dimethylaminopyrrolidine, (35 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method C to afford (35 mg, 38%) of the title compound as white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.03 (t, 3H), 1.75 (m, 2H), 2.21 (s, 6H), 2.79-2.81 (m, 3H), 3.20 (m, 2H), 3.41-3.47 (m, 2H), 3.71 (m, 1H), 3.77-3.81 (m, 1H), 3.92 (bs, 2H), 4.66-4.74 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 7.98 (s, 1H), 8.31 (d, 1H), 8.39 (m, 1H), 12.50 (s, 1H), 13.21 (s, 1H);
LCMS: Rt=2.61 min; m/z 596.4[M+H]+.
Example 38
[5-((S)-3-Dimethylamino-pyrrolidin-1-yl)-pyrazin-2-yl]-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and (S)-3-dimethylaminopyrrolidine, (35 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method C to afford (38 mg, 40%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.03 (t, 3H), 1.75-1.83 (m, 2H), 2.21 (s, 6H), 2.79-2.81 (m, 3H), 3.18-3.23 (m, 2H), 3.43-3.45 (m, 2H), 3.69-3.77 (m, 1H), 3.79-3.81 (m, 1H), 3.90 (bs, 2H), 4.66 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 7.98 (s, 1H), 8.32 (m, 1H), 8.39 (m, 1H), 12.51 (s, 1H), 13.22 (s, 1H);
LCMS: Rt=2.61 min; m/z 596.4[M+H]+.
Example 39
{2-[6-[2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-etrahydroimidazo[4,5c]pyridin-5-yl}-5-(2-piperidin-1-yl)ethylamino)-pyrazin-2-yl]-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (120 mg, 231 μmol) and (2-(piperidine-1-yl)ethanamine, (59 mg, 463 μmol) using the method of Example 32. The crude material was purified by HPLC Method G to afford (38 mg, 27%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.09 (t, 3H), 1.39 (Brs, 2H), 1.39-1.50 (m, 3H), 2.32 (m, 3H), 2.43-2.54 (m, 4H), 2.77 (Brs, 2H), 3.33-3.42 (m, 2H), 3.90 (Brs, 2H), 4.68 (m, 2H), 6.90-6.92 (d, 1H), 7.00-7.03 (d, 1H), 7.09 (m, 1H), 7.37 (s, 1H), 7.48 (s, 1H) 7.94 (s, 1H), 8.29-8.32 (s, 2H),
LCMS: Rt=2.64 min; m/z 610.2 [M+H]+.
Example 40
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-piperazin-1-yl-ethylamino)-pyrazin-2-yl]-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and (2-(piperazinyl-1-yl)ethanamine, (40 mg, 309 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (26 mg, 28%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.97-1.00 (t, 3H), 1.20 (s, 2H), 2.40-2.43 (m, 2H), 2.50 (s, 2H), 2.76 (Brs, 2H), 3.51-3.54 (d, 2H), 3.64 (Brs, 4H), 3.87-3.93 (d, 2H), 4.41-4.42 (m, 1H), 4.64-4.68 (d, 2H) 6.87-6.89 (d, 1H), 6.97-7.08 (m, 2H), 7.34 (s, 1H), 8.29 (s, 2H), 8.37 (s, 1H), 9.79 (s, 1H), 12.48 (s, 1H), 13.17 (s, 1H);
LCMS: Rt=2.59 min; m/z 612.4 [M+H]+.
Example 41
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-(4-methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and 1-methyl-piperazine, (31 mg, 309 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (21 mg, 23%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.90-1.03 (m, 6H), 2.22 (s, 3H), 2.40-2.49 (m, 4H), 2.78 (Brs, 2H), 3.67 (s, 3H), 3.88 (m, 2H), 4.66 (m, 2H), 6.90-6.92 (d, 1H), 7.00-7.03 (d, 2H), 7.37 (s, 1H) 8.32 (s, 3H), 9.82 (s, 1H), 12.50 (s, 1H), 13.20 (s, 1H);
LCMS: Rt=2.63 min; m/z 582.6 [M+H]+.
Example 42
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-morpholin-4-yl-pyrazin-2-yl)-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (100 mg, 154 μmol) and morpholine, (34 mg, 386 μmol) using the method of Example 32. The crude material was purified by HPLC Method F to afford (42 mg, 38%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.99-1.03 (t, 3H), 2.78 (Brs, 2H), 3.65-3.72 (q, 8H), 3.88 (d, 2H), 4.67-4.77 (m, 2H), 6.90 (d, 1H), 2H) 7.00-7.11 (m, 2H), 7.37 (s, 1H), 8.26 (s, 2H), 8.42 (s, 1H), 12.50 (s, 1H), 13.20 (s, 1H);
LCMS: Rt=3.23 min; m/z 569.4 [M+H]+.
Example 43
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-idazo[4,5c]pyridin-5-yl}-[5-(4-methyl-piperidin-1-yl)-pyrazin-2-yl]-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (100 mg, 154 μmol) and 4-methyl-piperidine, (38 mg, 386 μmol) using the method of Example 32. The crude material was purified by HPLC Method F to afford (34 mg, 30%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.92-0.93 (d, 3H), 0.99-1.03 (t, 3H), 1.10-1.16 (m, 2H), 1.69-1.75 (m, 3H), 1.85 (s, 1H), 2.78 (Brs, 2H), 2.91-2.98 (t, 2H), 3.93 (Brs, 2H), 4.42-4.45 (d, 2H), 4.66-4.75 (m, 2H) 6.90-6.92 (d, 1H), 7.00-7.03 (d, 2H), 7.37 (s, 1H), 8.31-8.39 (m, 3H), 12.51 (s, 1H), 13.21 (s, 1H);
LCMS: Rt=3.11 min; m/z 581.4 [M+H]+.
Example 44
(5-Cyclopentylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (90 mg, 173 μmol) and cylopenylamine, (30 mg, 347 μmol) using the method of Example 32. The crude material was purified by HPLC Method G to afford (28 mg, 28%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.03 (t, 3H), 1.45-1.51 (m, 2H), 1.56-1.57 (m, 2H), 1.69 (m, 2H), 1.93-1.95 (m, 2H), 2.53 (m, 1H), 2.78 (Brs, 2H), 3.16 (s, 1H), 3.91 (Brs, 2H), 4.14-4.19 (m 1H), 4.64 (m, 2H), 6.90-6.92 (d, 1H), 7.00-7.03 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 7.62 (s, 1H) 7.88 (s, 1H), 8.29 (s, 2H),
LCMS: Rt=2.98 min; m/z 567.6 [M+H]+.
Example 45
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-[5-(2-morpholin-4-yl-ethylamino)-pyrazin-2-yl]-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (100 mg, 193 μmol) and 2-morpholin-4-yl-ethylamine, (30 mg, 347 μmol) using the method of Example 32. The crude material was purified by HPLC Method F to afford (28 mg, 28%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.99-1.03 (t, 3H), 2.49-2.57 (m, 2H), 2.77 (Brs, 2H), 3.16 (s, 2H), 3.32 (s, 3H), 3.56-3.58 (m, 2H), 2H) 3.90 (m, 2H) 4.69 (m, 2H) 6.90-6.92 (d, 1H), 7.00-7.03 (d, 1H), 7.09 (m, 1H), 7.37 (s, 1H), 7.53 (Brs, 1H), 7.95 (s, 1H), 8.29 (s, 2H);
LCMS: Rt=5.89 min; m/z 612.4 [M+H]+.
Example 46
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(4-isopropyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and 1-isopropyl-piperazine, (40 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (18 mg, 19%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.95-1.03 (m, 9H), 2.53 (m, 4H), 2.68-2.78 (m, 3H), 3.65 (s, 4H), 3.89-3.95 (m, 2H), 4.70 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 8.31 (m, 2H), 8.39 (d, 1H), 9.82 (bs, 1H), 12.51 (s, 1H), 13.20 (s, 1H);
LCMS: Rt=6.52 min; m/z 610.4[M+H]+.
Example 47
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-(5-pyrrolidin-1-yl-pyrazin-2-yl)-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and pyrrolidine (22 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (18 mg, 19%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.01 (t, 3H), 1.97 (bs, 4H), 2.78 (bs, 2H), 3.50 (s, 4H), 3.93 (bs, 2H), 4.65-4.76 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.08 (d, 1H), 7.36 (s, 1H), 7.96 (s, 1H), 8.31 (d, 1H), 8.39 (s, 1H), 9.86 (bs, 1H), 12.52 (s, 1H), 13.22 (s, 1H);
LCMS: Rt=2.85 min; m/z 553.4[M+H]+.
Example 48
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-[5-(ethyl-methyl-amino)-pyrazin-2-yl]-methanon
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and ethyl-methylamine (18 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method E to afford (38 mg, 46%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.01 (t, 3H), 1.12 (t, 3H), 2.78 (m, 2H), 3.11 (s, 3H), 3.65 (m, 2H), 3.93 (m, 2H), 4.76 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 8.13 (s, 1H), 8.31 (m, 1H), 8.39 (s, 1H), 9.84 (bs, 1H), 12.51 (s, 1H), 13.22 (s, 1H);
LCMS: Rt=2.85 min; m/z 541.6[M+H]+.
Example 49
(5-Cyclohexylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (126 mg, 243 μmol) and cyclohexylamine (48 mg, 487 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (29 mg, 20%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 0.99-1.04 (m, 3H), 1.18-1.35 (m, 6H), 1.62-1.84 (m, 6H), 1.90-1.93 (m, 1H), 2.78 (m, 2H), 3.32 (m, 1H), 3.76-3.91 (m, 2H), 4.66-4.72 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 7.51 (bs, 1H), 7.88 (s, 1H), 8.23 (s, 1H), 12.50 (bs, 1H), 13.22 (bs, 1H);
LCMS: Rt=3.06 min; m/z 581.6[M+H]+.
Example 50
(5-Dimethylamino-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-,4,6,7-tetrahydro-imidazo[4,5-O]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and dimethylamine HCl (25 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (17 mg, 21%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.01 (t, 3H), 2.79 (m, 2H), 3.15 (s, 6H), 3.93 (m, 2H), 4.70 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.01 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 8.15 (s, 1H), 8.31 (m, 1H), 8.40 (s, 1H), 9.84 (bs, 1H), 12.51 (s, 1H), 13.20 (s, 1H);
LCMS: Rt=2.81 min; m/z 527.4[M+H]+.
Example 51
(5-Azetidin-1-yl-pyrazin-2-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-O]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (80 mg, 154 μmol) and azetdine HCl (29 mg, 308 μmol) using the method of Example 32. The crude material was purified by HPLC Method D to afford (19 mg, 23%) of the title compound as off-white solid.
1H NMR (400 MHz, DMSO) δ (ppm): 1.03 (t, 3H), 2.43-2.49 (m, 2H), 2.78 (bs, 2H), 3.37 (m, 2H), 3.87-3.95 (m, 2H), 4.13 (t, 4H), 4.65-4.70 (m, 2H), 6.90 (d, 1H), 7.00 (d, 1H), 7.09 (d, 1H), 7.37 (s, 1H), 7.83 (s, 1H), 8.33-8.37 (m, 2H), 9.84 (bs, 1H), 12.52 (s, 1H), 13.21 (s, 1H);
LCMS: Rt=2.78 min; m/z 539.4[M+H]+.
Example 52
2-Fluoro-4-{3-[5-(4-fluoro-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol
To a solution of 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (Preparation 39, 100 mg, 0.21 mmol) and KOAc (22.75 mg, 0.23 mmol) in MeOH (1 mL), 4-fluorobenzaldehyde (57.54 mg, 0.46 mmol) was added and the mixture stirred at room temperature for 1 hr followed by portionwise addition of sodium triacetoxy borohydride (162.12 mg, 0.76 mmol) over 2 hrs. The mixture was thereafter stirred at room temperature for 18 hrs. The reaction mixture was concentrated and the residue partitioned between saturated sodium bicarbonate solution & ethyl acetate. The organic phase was dried over sodium sulphate, evaporated in vacuo, purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as a light yellow solid in 30.35% yield, 35 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.66 (m, 2H), 2.76-2.80 (m, 2H), 3.44-3.56 (m, 4H), 3.71 (s, 2H), 7.07-7.10 (m, 2H), 7.13-7.19 (m, 2H), 7.36 (s, 1H), 7.41-7.43 (m, 2H), 8.29-8.36 (m, 1H), 10.13 (s, 1H), 12.21-12.34 (m, 1H), 13.19 (s, 1H);
LCMS: Rt=3.17 min; m/z 540.4 [M+H]+
Example 53
2-Fluoro-4-{3-[5-(3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5′-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and 3,4,5,6-tetrahydro-2H-[1,2′]bipyridinyl-5-carbaldehyde (88.2 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 16.99% yield, 22 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 1.53 (m, 6H), 2.64 (m, 2H), 2.73-2.77 (m, 2H), 3.41-3.57 (m, 10H), 6.78 (d, 1H), 7.02-7.10 (m, 2H), 7.14-7.17 (m, 1H), 7.36 (s, 1H), 7.48 (d, 1H), 8.03 (s, 1H), 8.30-8.35 (m, 1H), 10.15 (s, 1H), 12.22-12.34 (m, 1H), 13.20 (s, 1H);
LCMS: Rt=3.17 min; m/z 606.2 [M+H]+
Example 54
2-Fluoro-4-{3-[5-(6-phenoxy-pyridin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and 6-phenoxy-pyridine-3-carbaldehyde (92.3 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 23.6% yield, 31 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.67 (m, 2H), 2.77 (m, 2H), 3.45-3.56 (m, 4H), 3.70 (s, 2H), 6.78 (d, 1H), 7.00-7.22 (m, 6H), 7.36 (s, 1H), 7.39-7.43 (t, 2H), 7.84 (d, 1H), 8.11 (s, 1H), 8.30-8.35 (m, 1H), 10.15 (s, 1H), 12.24-12.36 (m, 1H), 13.21 (s, 1H);
LCMS: Rt=3.20 min; m/z 615.4 [M+H]+
Example 55
2-Fluoro-4-{3-[5-(4-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and 4-methoxybenzaldehyde (63.1 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 22.1% yield, 26 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.65 (m, 2H), 2.73-2.79 (m, 2H), 3.37-3.47 (m, 2H), 3.50-3.56 (m, 2H), 3.64 (s, 2H), 3.75 (s, 3H), 6.90-6.92 (m, 2H), 7.02-7.10 (m, 2H), 7.13-7.16 (m, 1H), 7.27-7.29 (m, 2H), 7.36 (s, 1H), 8.29-8.36 (m, 1H), 10.13 (s, 1H), 12.20-12.35 (m, 1H), 13.19 (s, 1H);
LCMS: Rt=3.15 min; m/z 552.2 [M+H]+
Example 56
2-Fluoro-4-{3-[5-(4-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and 4-hydroxybenzaldehyde (56.6 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 24.4% yield, 28 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.66 (m, 2H), 2.73-2.78 (m, 2H), 3.40-3.43 (m, 2H), 3.47-3.56 (m, 2H), 3.59 (s, 2H), 6.72-6.74 (d, 2H), 7.02-7.10 (m, 2H), 7.14-7.18 (m, 3H), 7.36 (s, 1H), 8.29-8.36 (m, 1H), 9.27 (s, 1H), 10.13 (s, 1H), 12.20-12.32 (m, 1H), 13.19 (s, 1H);
LCMS: Rt=2.86 min; m/z 538.2 [M+H]+
Example 57
2-Fluoro-4-{3-[5-(3-methoxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and 3-methoxybenzaldehyde (63.1 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 21.2% yield, 25 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.66 (m, 2H), 2.76-2.80 (m, 2H), 3.45-3.56 (m, 4H), 3.69 (s, 2H), 3.75 (s, 3H), 6.83-6.85 (d, 1H), 6.94 (m, 2H), 7.02-7.10 (m, 2H), 7.14 (d, 1H), 7.24-7.28 (t, 1H), 7.36 (s, 1H), 8.29-8.36 (m, 1H), 10.15 (s, 1H), 12.23-12.35 (m, 1H), 13.20 (s, 1H);
LCMS: Rt=3.14 min; m/z 552 [M+H]+
Example 58
2-Fluoro-4-{3-[5-(3-hydroxy-benzyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-d]pyridin-2-yl]-1H-indazol-6-yl}-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and 3-hydroxybenzaldehyde (56.6 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 26.1% yield, 30 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.66 (m, 2H), 2.76-2.81 (m, 2H), 3.43-3.53 (m, 4H), 3.63 (s, 2H), 6.64-6.66 (d, 1H), 6.76-6.81 (m, 2H), 7.02-7.16 (m, 4H), 7.36 (s, 1H), 8.30-8.32 (m, 1H), 9.28 (d, 1H), 10.13 (s, 1H), 12.21-12.33 (m, 1H), 13.19 (s, 1H);
LCMS: Rt=2.90 min; m/z 538.2 [M+H]+
Example 59
2-Fluoro-4-[3-(5-quinolin-6-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and quinoline-6-carbaldehyde (72.9 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 22.9% yield, 28 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.67 (m, 2H), 2.83.2.89 (m, 2H), 3.49-3.55 (m, 4H), 3.93 (s, 2H), 7.01-7.17 (m, 3H), 7.36 (s, 1H), 7.51 (m, 1H), 7.80 (d, 1H), 7.95 (s, 1H), 7.99 (d, 1H), 8.29-8.38 (m, 2H), 8.87 (d, 1H), 10.13 (s, 1H), 12.21-12.33 (m, 1H), 13.19 (s, 1H);
LCMS: Rt=2.90 min; m/z 573.6 [M+H]+
Example 60
2-Fluoro-4-[3-(5-quinolin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and quinoline-3-carbaldehyde (72.9 mg, 0.46 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 27.8% yield, 34 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.67-2.70 (m, 2H), 2.83.2.90 (m, 2H), 3.47-3.58 (m, 4H), 3.95 (s, 2H), 7.01-7.17 (m, 3H), 7.36 (s, 1H), 7.59 (t, 1H), 7.74 (t, 1H), 8.02 (t, 2H), 8.29-8.38 (m, 2H), 8.93 (d, 1H), 10.14 (s, 1H), 12.23-12.36 (m, 1H), 13.20 (s, 1H);
LCMS: Rt=2.97 min; m/z 573.6 [M+H]+
Example 61
2-Fluoro-4-[3-(5-[1,8]naphthyridin-3-ylmethyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol
The title compound was prepared from 2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol (100 mg, 0.21 mmol) and [1,8]naphthyridine-3-carbaldehyde (146.1 mg, 0.92 mmol) using the method of Example 51. The crude material was purified initially over silica and finally by Prep TLC (Mobile Phase: 10% MeOH-DCM) to afford the title compound as an off white solid in 9.0% yield, 22 mg.
1H NMR (400 MHz, DMSO) δ (ppm): 2.72 (m, 2H), 2.89.2.92 (m, 2H), 3.50-3.54 (m, 2H), 3.60-3.63 (m, 2H), 4.08 (s, 2H), 7.02-7.17 (m, 3H), 7.36 (s, 1H), 7.61-7.64 (m, 1H), 7.82-7.85 (m, 1H), 8.29-8.38 (m, 2H), 8.44-8.47 (m, 2H), 9.06 (m, 1H), 10.15 (s, 1H), 12.26-12.39 (m, 1H), 13.21 (s, 1H);
LCMS: Rt=2.86 min; m/z 574.2 [M+H]+
Example 62
((3R,5S)-3,5-Dimethyl-3,4,5,6-tetrahydro-2H-[1,2]bipyrazinyl-5′-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
To a stirring solution of (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (150 mg, 0.154 mmol) in DMSO (1.5 mL) were added DIPEA (0.143 mL, 0.87 mmol) and (2R,6S)-2,6-Dimethyl-piperazine-1-carboxylic acid tert-butyl ester (124 mg, 0.58 mmol) and the mixture stirred at room temperature for 18 hours. The crude reaction mass was purified by prep-HPLC Method F to afford (3R,5S)-5′-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carbonyl}-3,5-dimethyl-2,3,5,6-tetrahydro-[1,2′]bipyrazinyl-4-carboxyl is acid tert-butyl ester as an off white solid (80 mg, 40%).
LCMS: Rt=3.18 min; m/z 696.6 [M+H]+.
To a stirring solution of (3R,5S)-5′-{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carbonyl}-3,5-dimethyl-2,3,5,6-tetrahydro-[1,2′]bipyrazinyl-4-carboxylic acid tert-butyl ester (80 mg, 0.114 mmol) in dioxane (3 mL), 10% dioxane-HCl (2 mL) was added and the mixture stirred at RT for 18 hours. The reaction mass was evaporated in vacuo and the resulting solid triturated with ether to afford the title compound (HCl salt) as an off white solid (62 mg 91%).
1H NMR (400 MHz, DMSO): 1.06 (t, 3H), 1.22-1.34 (s, 6H), 2.94 (Brs, 2H), 3.01-3.07 (t, 2H), 3.32 (Brs, 2H), 3.97 (m, 2H), 4.61-4.64 (d, 2H), 4.84 (s, 2H), 6.94 (d, 1H), 7.03 (d, 1H), 7.32 (s, 1H), 7.58 (s, 1H), 8.41-8.48 (m, 3H), 9.27 (m, 1H), 9.64 (m, 1H), 9.79 (s, 1H), 14.33 (s, 1H);
LCMS: Rt=2.65 min; m/z 596.4 [M+H]+.
Example 63
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-((S)-3-methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (200 mg, 386 μmol) and (S)-2-methyl-piperazine-1-carboxylic acid tert-butyl ester (155 mg, 773 μmol) using the method from Example 61. After purification by HPLC Method E and deprotection using HCl/dioxan, the title compound (58 mg, 25% yield over two steps) was obtained as off-white solid (HCl-salt).
1H NMR (400 MHz, DMSO): 0.82 (t, 3H), 1.23-1.31 (s, 3H), 2.93 (Brs, 2H), 3.07-3.18 (m, 3H), 3.97 (Brs, 2H), 4.50-4.53 (d, 2H), 4.83 (s, 2H), 6.93-6.95 (d, 1H), 7.03-7.06 (d, 1H), 7.28-7.30 (m, 1H), 7.57 (s, 1H), 8.37 (m, 1H), 8.44 (s, 1H), 8.49 (s, 1H), 9.24 (s, 1H), 9.40 (m, 1H), 9.94 (s, 1H), 14.25 (s, 1H);
LCMS: Rt=2.61 min; m/z 582.4 [M+H]+.
Example 64
((2S,5R)-2,5-Dimethyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-{2-[6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridin-5-yl}-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (100 mg, 193 μmol) and (2S,5R)-2,5-Dimethyl-piperazine-1-carboxylic acid tert-butyl ester (84 mg, 386 μmol) using the method from Example 61. After purification by HPLC Method E and deprotection using HCl/dioxan, the title compound (43 mg, 37% yield over two steps) was obtained as off-white solid (HCl-salt).
1H NMR (400 MHz, DMSO): 0.82 (t, 3H), 1.33-1.34 (m, 6H), 2.95 (Brs, 3H), 3.11-3.14 (d, 1H), 3.37 (s, 1H), 3.51-3.62 (m, 5H), 3.83 (Brs, 2H), 4.01 (m, 1H), 4.84 (m, 3H), 6.94-6.96 (d, 1H), 7.03-7.06 (d, 1H), 7.31 (Brs, 1H), 7.58 (s, 1H), 8.36 (s, 2H), 8.49 (s, 1H), 9.33 (m, 1H), 9.53 (s, 1H), 14.33 (s, 1H);
LCMS: Rt=2.55 min; m/z 596.2 [M+H]+.
Example 65
{2-[6-(2-Ethyl-5-fluoro-4-hydroxy-phenyl)-1H-indazol-3-yl]-1,4,6,7-tetrahydro-imidazo[4,5-]pyridin-5-yl}-(3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-5′-yl)-methanone
The title compound was prepared from (5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (200 mg, 386 μmol) and piperazine-1-carboxylic acid tert-butyl ester (144 mg, 773 μmol) using the method from Example 61. After purification by HPLC Method E and deprotection using HCl/dioxan, the title compound (64 mg, 29% yield over two steps) was obtained as off-white solid (HCl-salt).
1H NMR (400 MHz, DMSO): 1.00-1.03 (t, 3H), 2.94 (Brs, 2H), 3.22 (s, 4H), 3.93-3.95 (m, 6H), 4.84 (s, 2H), 6.94-6.96 (d, 1H), 7.03-7.06 (d, 1H), 7.30-7.33 (m, 1H), 7.58 (s, 1H), 8.42 (s, 2H), 8.50 (s, 1H), 9.29 (Brs, 2H), 9.95 (s, 1H), 14.30 (s, 1H);
LCMS: Rt=2.48 min; m/z 568.2 [M+H]+.
Preparation 1
6-Bromo-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbaldehyde
To a solution of 6-bromo-1H-indazole-3-carbaldehyde (13.97 g, 61.9 mmol) in DCM (150 mL) was added p-TsOH (2.36 g, 12.4 mmol) and the mixture was cooled to 0° C. 3,4-Dihydro-2H-pyran (8.47 mL, 92.8 mmol) was added dropwise to the solution and the reaction was stirred at room temperature overnight. The reaction mixture was diluted with DCM (200 mL) and washed with a solution of saturated aqueous sodium hydrogen carbonate (500 mL). The aqueous layer was re-extracted with DCM (500 mL) and the combined organic layers were washed with brine (2×1 L), dried over MgSO4 and concentrated in vacuo to yield a black oil. The crude material was refluxed in cyclohexane (20 mL) and filtered while hot. The filtrate was concentrated in vacuo and the residue was stirred in heptane for 48 hours. The resulting solid was collected by filtration to give the title compound (13.87 g) in a 73% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.71-1.80 (m, 3H), 2.10-2.20 (m, 2H), 2.49-2.57 (m, 1H), 3.76-3.82 (m, 1H), 3.98-4.03 (m, 1H), 5.78 (dd, 1H), 7.46 (dd, 1H), 7.87 (d, 1H), 8.16 (d, 1H), 10.22 (s, 1H).
Preparation 2
6-Bromo-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbonitrile
To a solution of 6-bromo-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbaldehyde (Preparation 1, 60 g, 194 mmol) in MeCN (1.5 L) was added triethylamine (68.5 mL, 485 mmol) and hydroxylamine hydrochloride (20 g, 291 mmol). The reaction was heated at 60° C. for 3 hours. The reaction was cooled to 0° C., further triethylamine (220 mL, 1.55 mol) was added and TFAA (109 mL, 776 mmol) was added dropwise. The reaction was allowed to warm to room temperature and stirred for 2 hours. Water (2 L) was added to the reaction mixture and the resulting solid was collected by filtration. The solid was dissolved in DCM (1 L) and the resulting solution was washed with water (2×500 mL). The organic layer was dried over MgSO4 and concentrated in vacuo to give the title compound as a white solid (58.59 g) in a 99% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.71-1.80 (m, 3H), 2.08-2.18 (m, 2H), 2.43-2.50 (m, 1H), 3.73-3.79 (m, 1H), 3.92-3.96 (m, 1H), 5.77 (dd, 1H), 7.47 (dd, 1H), 7.69 (d, 1H), 7.93 (d, 1H).
Preparation 3
4-Bromo-5-ethyl-2-fluoro-phenol
To a solution of 5-ethyl-2-fluoro-phenol (WO-2007/002313, 76.36 g, 545 mmol) in MeCN (2.5 L) was added copper (II) bromide (361.5 g, 1.619 mol). The resulting suspension was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was suspended in EtOAc (3 L) and filtered through a pad of Arbocel®. The filtrate was washed with water (2 L) and brine (2 L), dried over MgSO4 and concentrated in vacuo to furnish the title compound (119 g) in 100% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.19 (t, 3H), 2.86 (q, 2H), 5.20 (s, 1H), 6.90 (d, 1H), 7.25 (d, 1H).
Preparation 4
[2-(4-Bromo-5-ethyl-2-fluoro-phenoxymethoxy)-ethyl]-trimethyl-silane
To a solution of 4-bromo-5-ethyl-2-fluoro-phenol (Preparation 3, 80 g, 365 mmol) in DCM (1 L) was added DIPEA (70 mL, 401 mmol) and SEM-Cl (71 mL, 401 mmol). The resulting solution was stirred at room temperature for 18 hours. The reaction mixture was washed with water (1 L), dried over MgSO4 and concentrated in vacuo to yield the crude product. This material was purified by silica gel chromatography eluting with 30% DCM in heptane to give the title compound as a pale yellow oil (109.8 g, 86%).
1H NMR (400 MHz, CDCl3) δ ppm 0.00 (s, 9H), 0.92-0.97 (m, 2H), 1.19 (t, 3H), 2.67 (q, 2H), 3.77-3.81 (m, 2H), 5.22 (s, 2H), 7.08 (d, 1H), 7.25 (d, 1H).
Preparation 5
2-[2-Ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane
To a solution of [2-(4-bromo-5-ethyl-2-fluoro-phenoxymethoxy)-ethyl]-trimethyl-silane (Preparation 4, 105 g, 300.6 mmol) in dioxane (1 L) was added bis(pinacolato)diboron (76.4 g, 300.6 mmol) and KOAc (88.5 g, 902 mmol). The resulting suspension was degassed with nitrogen, Pd(dppf)Cl2 (24.54 g, 30.1 mmol) was added and the reaction was heated at reflux for 18 hours. The reaction was cooled to room temperature and the solvent was removed in vacuo. The resulting black solid was suspended in EtOAc (2 L) and filtered through Arbocel®, washing with further EtOAc. The filtrate was washed with water (1.5 L) and brine (1.5 L), dried over MgSO4 and concentrated in vacuo to yield the title compound as a black oil (155.5 g, 130%) that was used crude in the next step.
1H NMR (400 MHz, CDCl3) δ ppm 0.00 (s, 9H), 0.93-0.97 (m, 2H), 1.17 (t, 3H), 1.32 (s, 12H), 2.86 (q, 2H), 3.73-3.82 (m, 2H), 5.25 (s, 2H), 7.01 (d, 1H), 7.47 (d, 1H).
Preparation 6
6-[2-Ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbonitrile
To a solution of 6-bromo-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbonitrile (Preparation 2, 28.5 g, 93 mmol) and 2-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (Preparation 5, 73.8 g, 112 mmol) in dioxane (500 mL) was added a solution of potassium phosphate (59.2 g, 279 mmol) in water (120 mL). The mixture was degassed with nitrogen and then tetrakis(triphenylphosphine) palladium(0) (10.8 g, 9.3 mmol) was added. The reaction mixture was heated at 110° C. for 18 hours. The reaction mixture was concentrated in vacuo and the residue was redissolved in EtOAc (1 L) and filtered through Arbocel®, washing with EtOAc (2×500 mL). The combined organic phases were concentrated in vacuo to give a brown oil. The residue was purified by column chromatography on silica gel eluting with 10% EtOAc in heptane to give the title compound as a viscous oil (37.4 g) in an 81% yield.
1H NMR (400 MHz, CDCl3) δ ppm 0.00 (s, 9H), 0.97 (t, 2H), 1.06 (t, 3H), 1.66-1.77 (m, 3H), 2.05-2.17 (m, 2H), 2.44-2.48 (m, 3H), 3.67-3.73 (m, 1H), 3.82 (t, 2H), 3.90-3.94 (m, 1H), 5.28 (s, 2H), 5.77 (dd, 1H), 6.95 (d, 1H), 7.13 (d, 1H), 7.25 (d, 1H), 7.58 (s, 1H), 7.80 (d, 1H).
Preparation 7
6-[2-Ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboximidic acid methyl ester
To a solution of 6-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbonitrile (Preparation 6, 37.42 g, 75.6 mmol) in methanol (700 mL) was added sodium methoxide (12.21 g, 226.8 mmol) and the reaction mixture was then stirred at room temperature for 18 hours. The solvent was removed in vacuo and the residue was partitioned between EtOAc (1 L) and water (500 mL). The organic layer was washed with water (500 mL), dried over MgSO4 and concentrated in vacuo to give the title compound as a gum (37.26 g) in a 94% yield.
1H NMR (400 MHz, CDCl3) δ ppm 0.00 (s, 9H), 0.97 (t, 2H), 1.06 (t, 3H), 1.61-1.76 (m, 3H), 2.03-2.15 (m, 2H), 2.47-2.56 (m, 3H), 3.67-3.72 (m, 1H), 3.82 (t, 2H), 3.97-4.01 (m, 1H), 4.03 (s, 3H), 5.28 (s, 2H), 5.71 (dd, 1H), 6.97 (d, 1H), 7.12 (d, 1H), 7.15 (d, 1H), 7.46 (s, 1H), 8.03 (d, 1H), 8.45 (s, 1H).
LCMS: m/z 528 M+H+.
Preparation 8
N-(1-Benzyl-4,4-diethoxy-piperidin-3-yl)-6-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboxamidine
To a solution of 6-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboximidic acid methyl ester (Preparation 7, 17.15 g, 32.49 mmol) in ethanol (100 mL) was added a solution of 1-benzyl-4,4-diethoxy-piperidin-3-ylamine (Tetrahedron, 1995, 51, 13447-13454; 9.51 g, 34.2 mmol) in ethanol (70 mL). Acetic acid (3.56 mL, 62.1 mmol) was added and the reaction mixture was heated at 50° C. for 18 hours. The reaction mixture was concentrated in vacuo and the residue was partitioned between EtOAc (400 mL) and saturated sodium hydrogen carbonate aqueous solution (300 mL). The organic layer was washed with further saturated sodium hydrogen carbonate aqueous solution (300 mL). The combined aqueous layers were re-extracted with EtOAc (400 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel eluting with DCM:methanol:ammonia (80:20:2) to give the title compound (11.31 g) in a 45% yield.
1H NMR (400 MHz, CDCl3) δ ppm 0.00 (s, 9H), 0.95-0.99 (m, 2H), 1.04-1.07 (m, 3H), 1.11-1.19 (m, 6H), 1.58-1.82 (m, 4H), 1.84-1.93 (m, 2H), 2.06-2.31 (m, 3H), 2.46-2.55 (m, 2H), 2.55-2.71 (m, 3H), 2.71-2.85 (m, 1H), 3.47-3.75 (m, 7H), 3.80-3.84 (m, 2H), 3.95-4.05 (m, 1H), 5.27 (s, 2H), 5.68-5.76 (m, 1H), 6.98-7.01 (m, 1H), 7.11-7.24 (m, 5H), 7.29-7.36 (m, 2H), 7.44-7.49 (m, 1H), 8.11-8.28 (m, 1H).
LCMS: m/z 774 M+H+.
Preparation 9
N-(1-Benzyl-4,4-diethoxy-piperidin-3-yl)-6-(2-ethyl-5-fluoro-4-hydroxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboxamidine
To a solution of N-(1-benzyl-4,4-diethoxy-piperidin-3-yl)-6-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboxamidine (Preparation 8, 7.28 g, 9.4 mmol) in ethanol (32 mL) was added concentrated hydrochloric acid (12M, 3.92 mL, 47 mmol) and the resulting solution was allowed to stir at room temperature for 18 hours. The reaction mixture was cooled to 0° C. and neutralised by dropwise addition of a saturated aqueous solution of sodium hydrogen carbonate (150 mL). The mixture was extracted with EtOAc (2×200 mL). The combined organic layers were washed with further saturated aqueous sodium hydrogen carbonate solution (100 mL), dried over MgSO4 and concentrated in vacuo to give the title compound as a foam (6.06 g).
1H NMR (400 MHz, CDCl3) δ ppm 1.01-1.05 (m, 3H), 1.11-1.19 (m, 6H), 1.58-1.82 (m, 3H), 1.84-1.93 (m, 2H), 2.06-2.22 (m, 2H), 2.23-2.35 (m, 1H), 2.43-2.49 (m, 2H), 2.55-2.71 (m, 3H), 2.79-2.89 (m, 1H), 3.44-3.68 (m, 7H), 3.95-4.05 (m, 1H), 4.17-4.26 (m, 1H), 5.68-5.76 (m, 1H), 6.87-6.95 (m, 2H), 7.11-7.24 (m, 4H), 7.29-7.36 (m, 2H), 7.44-7.49 (m, 1H), 8.13-8.24 (m, 1H).
LCMS: m/z 644 M+H+.
Preparation 10
4,4-Diethoxy-3-{[6-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboximidoyl]-amino}-piperidine-1-carboxylic acid tert-butyl ester
To a solution of 6-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboximidic acid methyl ester (Preparation 7, 31.4 g, 59.5 mmol) in ethanol (140 mL) was added a solution of 3-amino-4,4-diethoxy-piperidine-1-carboxylic acid tert-butyl ester (US-2004/0229862, 18.02 g, 62.48 mmol) in ethanol (100 mL). Acetic acid (6.81 mL, 119 mmol) was added and the reaction mixture was heated at 50° C. for 18 hours. The reaction mixture was concentrated in vacuo and azeotroped with toluene (100 mL) to give the title compound as a foam (54.7 g).
1H NMR (400 MHz, CDCl3) δ ppm 0.03 (s, 9H), 0.96-1.00 (m, 2H), 1.05-1.08 (m, 3H), 1.17-1.31 (m, 6H), 1.46 (s, 9H), 1.69-1.75 (m, 2H), 1.93-2.19 (m, 4H), 2.52-2.58 (m, 2H), 3.47-3.58 (m, 2H), 3.59-3.70 (m, 4H), 3.70-3.75 (m, 4H), 3.84-3.88 (m, 2H), 3.92-4.07 (m, 3H), 5.32 (s, 2H), 5.94-5.99 (m, 1H), 7.01 (d, 1H), 7.21 (d, 1H), 7.27 (d, 1H), 7.69 (s, 1H), 8.10 (s, 1H).
LCMS: m/z 784 M+H+.
Preparation 11
5-Ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol dihydrochloric acid salt
To a solution of 4,4-diethoxy-3-{[6-[2-ethyl-5-fluoro-4-(2-trimethylsilanyl-ethoxymethoxy)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboximidoyl]-amino}-piperidine-1-carboxylic acid tert-butyl ester (Preparation 10, 46.65 g, 55.27 mmol) in ethanol (200 mL) was added concentrated hydrochloric acid (12M, 100 mL, 1.2 mol) and the resulting solution was stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo and azeotroped with toluene (100 mL) and DCM (2×100 mL). The resulting gum was dried under vacuum for 3 hours. The crude material was triturated in MeCN (300 mL) and the resulting solid was collected by filtration. The solid was dissolved in ethanol (250 mL) and treated with concentrated hydrochloric acid (12M, 77.2 mL, 927 mmol). The resulting solution was heated at 40° C. for 18 hours, then at 50° C. for 2 hours. The solvents were removed in vacuo and the resulting gum was triturated in MeCN (200 mL). The solid which formed was collected by filtration, washed with further MeCN (200 mL) and dried under vacuum to give the title compound as a beige solid (23.5 g, 88% yield, dihydrochloride salt).
1H NMR (400 MHz, CD3OD) δ ppm 1.05 (t, 3H), 2.53 (q, 2H), 3.22 (t, 2H), 3.73 (t, 2H), 4.55 (s, 2H), 6.90-6.96 (m, 2H), 7.35 (d, 1H), 7.58 (s, 1H), 8.21 (d, 1H). LCMS: m/z 378 M+H+.
Preparation 12
1-Bromo-2-ethyl-5-fluoro-4-methoxy-benzene
To a solution of 4-ethyl-1-fluoro-2-methoxy-benzene (WO-2010/090537, 12.2 g, 79.1 mmol) in MeCN (150 mL) was added a solution of NBS (14.4 g, 80.7 mmol) in MeCN (50 mL). The resulting solution was stirred at room temperature for 18 hours. The solvent was removed in vacuo and the residue was diluted with diethyl ether (150 mL). Precipitated solid was removed by filtration and the filtrate was washed with sodium sulfite aqueous solution (100 mL) and brine (100 mL), dried over MgSO4 and concentrated in vacuo to give the title compound as a yellow oil (18 g) in a 97% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.21 (t, 3H), 2.69 (q, 2H), 3.87 (s, 3H), 6.82 (d, 1H), 7.24 (d, 1H).
Preparation 13
2-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane
To a solution of 1-bromo-2-ethyl-5-fluoro-4-methoxy-benzene (Preparation 12, 18.0 g, 77.2 mmol) in dioxane (100 mL) were added SPhos (4.12 g, 10.0 mmol), 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (14.8 g, 116 mmol) and triethylamine (10.7 mL, 77.2 mmol). The reaction mixture was degassed with nitrogen prior to the addition of dichlorobis(acetonitrile)palladium (II) (801 mg, 3.09 mmol). The reaction mixture was then heated at 110° C. for 18 hours, cooled to room temperature and filtered through a pad of Celite, washing with EtOAc. The solvent was removed in vacuo and the residue was redissolved in EtOAc (30 mL) and washed with water (30 mL). The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude material was triturated with methanol and the resulting solid was collected by filtration to give the title compound as a beige solid (13.8 g) in a 64% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.18 (t, 3H), 1.32 (s, 12H), 2.87 (q, 2H), 3.89 (s, 3H), 6.76 (d, 1H), 7.46 (d, 1H).
LCMS: m/z 281 M+H+.
Preparation 14
6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazole
To a solution of 6-bromo-1-(tetrahydro-pyran-2-yl)-1H-indazole (WO-2010/027500, 2.25 g, 8.0 mmol) and 2-(2-ethyl-5-fluoro-4-methoxy-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (Preparation 13, 2.24 g, 8.0 mmol) in dioxane (32 mL) was added potassium phosphate (5.1 g, 24 mmol) as a solution in water (8 mL). The reaction mixture was degassed with nitrogen and treated with tetrakis(triphenylphosphine) palladium(0) (1.85 g, 1.6 mmol). The reaction mixture was heated at 110° C. for 18 hours, cooled to room temperature and filtered through a pad of Arbocel®, washing with EtOAc (2×100 mL). The filtrate was washed with water (100 mL), dried over MgSO4 and concentrated in vacuo. The crude material was purified by column chromatography on silica gel eluting with 10% EtOAc in heptane to give the title compound as a white solid (2.024 g) in a 71% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.12 (t, 3H), 1.62-1.81 (m, 3H), 2.07-2.17 (m, 2H), 2.54-2.63 (m, 3H), 3.70-3.76 (m, 1H), 3.95 (s, 3H), 4.01-4.07 (m, 1H), 5.71 (dd, 1H), 6.90 (d, 1H), 7.01 (d, 1H), 7.09 (dd, 1H), 7.46 (s, 1H), 7.71 (d, 1H), 8.05 (s, 1H).
Preparation 15
6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-1H-indazole
To a solution of 6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazole (Preparation 14, 1.8 g, 5.07 mmol) in methanol (100 mL) was added concentrated hydrochloric acid (12M) and the resulting solution was heated at 60° C. overnight, cooled to room temperature and concentrated in vacuo. The residue was redissolved in EtOAc (50 mL) and washed with saturated sodium hydrogen carbonate aqueous solution (50 mL). The organic layer was dried over MgSO4 and concentrated in vacuo to yield the title product (1.465 g) in 95% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.09 (t, 3H), 2.56 (q, 2H), 3.95 (s, 3H), 6.90 (d, 1H), 6.98 (d, 1H), 7.10 (dd, 1H), 7.38 (s, 1H), 7.76 (d, 1H), 8.14 (br s, 1H), 10.69 (br s, 1H)
LCMS: m/z 271 M+H+.
Preparation 16
6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-3-iodo-1H-indazole
To a solution of 6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1H-indazole (Preparation 15, 1.46 g, 5.4 mmol) in DMF (20 mL) was added KOH (1.14 g, 20.3 mmol) and the mixture was stirred for 5 minutes. A solution of iodine (2.75 g, 10.8 mmol) in DMF (5 mL) was slowly added and the reaction was stirred at room temperature for 30 minutes. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (100 mL). The organic layer was washed with water (2×100 mL) and saturated sodium metabisulfite aqueous solution (100 mL), dried over MgSO4 and concentrated in vacuo to furnish the title compound (1.94 g) in a 91% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.08 (t, 3H), 2.55 (q, 2H), 3.95 (s, 3H), 6.90 (d, 1H), 6.97 (d, 1H), 7.16 (dd, 1H), 7.37 (s, 1H), 7.52 (d, 1H), 10.64 (br s, 1H).
LCMS: m/z 397 M+H+.
Preparation 17
6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-3-iodo-1-(tetrahydro-pyran-2-yl)-1H-indazole
To a solution of 6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-3-iodo-1H-indazole (Preparation 16, 1.94 g, 4.9 mmol) in DCM (10 mL) was added p-TsOH (187 mg, 982 μmol) and the mixture was cooled to 0° C. 3,4-Dihydro-2H-pyran (660 μL, 7.4 mmol) was added dropwise to the solution and the reaction was stirred at room temperature overnight. The reaction mixture was diluted with DCM (5 mL) and washed with saturated sodium hydrogen carbonate aqueous solution (20 mL). The organic layer was dried over MgSO4 and concentrated in vacuo to yield a black oil. The residue was purified by column chromatography (Biotage SNAP 10 g) eluting with a gradient of 20% EtOAc in heptane to give the title compound as a colourless oil (2.09 g) in an 89% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.11 (t, 3H), 1.60-1.64 (m, 1H), 1.72-1.76 (m, 2H), 2.02-2.10 (m, 2H), 2.50-2.58 (m, 3H), 3.69-3.73 (m, 1H), 3.95 (s, 3H), 4.01-4.05 (m, 1H), 5.68 (dd, 1H), 6.90 (d, 1H), 7.10 (d, 1H), 7.15 (dd, 1H), 7.42-7.46 (m, 2H).
LCMS: m/z 481 M+H+.
Preparation 18
6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-3-trimethylstannanyl-1H-indazole
To a solution of 6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-3-iodo-1-(tetrahydro-pyran-2-yl)-1H-indazole (Preparation 17, 2.09 g, 4.35 mmol) in toluene (24 mL) was added 1,1,1,2,2,2-hexamethyl-distannane (1 mL, 4.79 mmol) followed by tetrakis(triphenylphosphine) palladium(0) (100 mg, 87 μmol). The reaction mixture was degassed with nitrogen and heated at 100° C. for 18 hours. The reaction was then cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with 10% EtOAc in heptane to give the title compound as a colourless oil (1.47 g) in a 65% yield.
1H NMR (400 MHz, CDCl3) δ ppm 0.47 (t, 9H), 1.12 (t, 3H), 1.60-1.64 (m, 1H), 1.73-1.78 (m, 2H), 2.05-2.18 (m, 2H), 2.57 (q, 2H), 2.59-2.67 (m, 1H), 3.70-3.76 (m, 1H), 3.95 (s, 3H), 4.06-4.09 (m, 1H), 5.74 (dd, 1H), 6.91 (d, 1H), 7.00-7.06 (m, 2H), 7.48 (s, 1H), 7.68 (s, 1H).
LCMS: m/z 519 M+H+.
Preparation 19
6,7-Dihydro-4H-imidazo[4,5-c]pyridine-1,5-dicarboxylic acid di-tert-butyl ester
To a solution of 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine (21.3 g, 109 mmol) in methanol (250 mL) was added DIPEA (47.3 mL, 272 mmol) and a solution of di-tert-butyl dicarbonate (59.3 g, 272 mmol) in methanol (130 mL). The reaction mixture was stirred at room temperature for 18 hours and concentrated in vacuo to yield an oil. The residue was redissolved in EtOAc (500 mL) and the resulting solution was washed with water (500 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with 30% EtOAc in DCM to yield the title compound as a white solid (26.85 g) in 76% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.48 (s, 9H), 1.60 (s, 9H), 2.61-2.92 (m, 2H), 3.62-3.72 (m, 2H), 4.59-4.65 (m, 2H), 7.98 (s, 1H).
Preparation 20
1,4,6,7-Tetrahydro-imidazo[4,5-d]pyridine-5-carboxylic acid tert-butyl ester
To a solution of 6,7-dihydro-4H-imidazo[4,5-c]pyridine-1,5-dicarboxylic acid di-tert-butyl ester (Preparation 19, 26.8 g, 82.9 mmol) in methanol (210 mL) was added 1M sodium hydroxide aqueous solution (170 mL, 170 mmol). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with 10% citric acid aqueous solution (250 mL), basified to pH8 and extracted with DCM (2×500 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo to give the title compound as a brown foam (18.5 g) in a 97% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.47 (s, 9H), 2.64-2.72 (m, 2H), 3.68-3.75 (m, 2H), 4.43-4.53 (m, 2H), 7.52 (s, 1H).
Preparation 21
2-Iodo-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester
To a solution of 1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (Preparation 20, 18.g, 80.62 mmol) in THF (300 mL) was added NIS (27.2 g, 121 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with EtOAc (800 mL), washed with sodium thiosulfate aqueous solution (3×700 mL) and brine (500 mL), dried over MgSO4 and concentrated in vacuo to give the title compound as a yellow solid (18.25 g) in a 64.8% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 2.66-2.74 (m, 2H), 3.64-3.75 (m, 2H), 4.42-4.54 (m, 2H).
Preparation 22
2-Iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester
To a solution of 2-iodo-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (Preparation 21, 17.25 g, 49.4 mmol) in THF (250 mL) was added NaH (60% in paraffin oil, 2.08 g, 51.9 mmol) and the resulting solution was stirred at room temperature for 1.5 hours. The reaction mixture was cooled to 0° C. and SEM-Cl (9.18 mL, 51.9 mmol) was added dropwise. The reaction was stirred at room temperature for 18 hours, cooled to 0° C. and quenched carefully with water (500 mL). The resulting solution was extracted with EtOAc (2×500 mL) and the combined organics layers were dried over MgSO4, filtered through a pad of silica and concentrated in vacuo to give the title compound (23.4 g) in a 99% yield.
1H NMR (400 MHz, CDCl3) δ ppm 0.00 (s, 9H), 0.90-0.95 (m, 2H), 1.47 (s, 9H), 2.64-2.76 (m, 2H), 3.54 (t, 2H), 3.63-3.75 (m, 2H), 4.43-4.57 (m, 2H), 5.15 (s, 2H).
Preparation 23
2-[6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazol-3-yl]-1-(2-trimethylsilanyl-ethoxymethyl)-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester
To a solution of 6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-3-trimethylstannanyl-1H-indazole (Preparation 18, 735 mg, 1.53 mmol) in toluene (6 mL) was added 2-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (Preparation 22, 805 mg, 1.68 mmol), copper (1) iodide (60 mg, 310 μmol) and tetrakis(triphenylphosphine) palladium(0) (173 mg, 150 μmol). The reaction mixture was degassed with nitrogen, heated at 100° C. for 18 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with 20% EtOAc in toluene to give the title compound as a foam (801 mg) in a 74% yield.
1H NMR (400 MHz, CDCl3) δ ppm −0.12 (s, 9H), 0.81-0.90 (m, 2H), 1.10 (t, 3H), 1.50 (s, 9H), 1.67-1.83 (m, 3H), 2.12-2.20 (m, 2H), 2.57 (q, 2H), 2.58-2.60 (m, 1H), 2.81-2.84 (m, 2H), 3.49-3.56 (m, 2H), 3.72-3.82 (m, 3H), 3.95 (s, 3H), 4.01-4.04 (m, 1H), 4.60-4.63 (m, 2H), 5.74-5.76 (m, 1H), 5.83-5.86 (m, 1H), 5.98-6.00 (m, 1H), 6.90 (d, 1H), 7.03 (d, 1H), 7.19 (dd, 1H), 7.46 (s, 1H), 8.42 (d, 1H).
LCMS: m/z 706 M+H+.
Preparation 24
2-[6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-1H-indazol-3-yl]-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine trihydrochloride salt
To a solution of 2-[6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazol-3-yl]-1-(2-trimethylsilanyl-ethoxymethyl)-1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (Preparation 23, 801 mg, 1.13 mmol) in methanol (20 mL) was added concentrated hydrochloric acid (12M, 8 mL) and the resulting solution was heated at 60° C. for 18 hours. The reaction mixture was cooled to room temperature and concentrated in vacuo to furnish the title compound (739 mg).
1H NMR (400 MHz, CD3OD) δ ppm 1.04 (t, 3H), 2.55 (q, 2H), 3.25-3.26 (m, 2H), 3.62-3.65 (m, 2H), 3.89 (s, 3H), 4.48-4.52 (m, 2H), 6.94 (d, 1H), 7.02 (d, 1H), 7.30-7.32 (m, 1H), 7.56 (s, 1H), 8.27-8.29 (m, 1H).
LCMS: m/z 392 M+H+.
Preparation 25
5-Ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt
A 1M solution of boron tribromide in DCM (4.54 mL, 4.54 mmol) was added dropwise to a solution of 2-[6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1H-indazol-3-yl]-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine (Preparation 24, 739 mg, 1.13 mmol) in DCM (10 mL) at 0° C. The resulting solution was stirred at room temperature for 18 hours. Further boron tribromide (4.54 mL, 4.54 mmol) was added dropwise and the reaction was allowed to stir at room temperature for 5 hours. Precipitated solid was collected by filtration, washed with tBME, then triturated with EtOAc to yield the title compound as the trihydrobromide salt (665 mg) in a 94% yield.
1H NMR (400 MHz, CD3OD) δ ppm 1.05 (t, 3H), 2.52 (q, 2H), 3.25-3.26 (m, 2H), 3.75-3.76 (m, 2H), 4.60-4.61 (m, 2H), 6.89-6.96 (m, 2H), 7.32-7.41 (m, 1H), 7.58-7.59 (m, 1H), 8.22-8.23 (m, 1H).
LCMS: m/z 378 M+H+.
Preparation 26
4,5,7,8-Tetrahydro-imidazo[4,5-d]azepine-1,6-dicarboxylic acid di-tert-butyl ester
To a solution of 1,4,5,6,7,8-Hexahydro-imidazo[4,5-d]azepine (WO-2000/063208; 5.0 g, 23.5 mmol) in methanol (60 mL) was added DIPEA (5.6 mL, 59.6 mmol) and a solution of BOC-anhydride (13.07 g, 59.6 mmol) in methanol (30 mL). The reaction mixture was stirred at room temperature for 18 hours and concentrated in vacuo to yield an oil. The residue was redissolved in DCM (250 mL) and the resulting solution was washed with water (100 mL) and saturated aqueous ammonium chloride solution (100 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with 50% EtOAc in heptane to yield the title compound as a brown oil (6.87 g) in 85% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.40 (s, 9H), 1.53 (s, 9H), 2.84-2.87 (m, 2H), 3.10-3.12 (m, 2H), 3.50-3.62 (m, 4H), 7.87 (s, 1H).
Preparation 27
4,5,7,8-Tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester
To a solution of 4,5,7,8-tetrahydro-imidazo[4,5-d]azepine-1,6-dicarboxylic acid di-tert-butyl ester (Preparation 26, 6.87 g, 20.37 mmol) in methanol (60 mL) was added an aqueous 1M solution of sodium hydroxide (40.7 mL, 40.7 mmol). The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was partitioned between DCM (100 mL) and water (100 mL). The organic layer was dried over MgSO4 and concentrated in vacuo to give the title compound as a brown foam (4.8 g) in a 99% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 2.78-2.83 (m, 4H), 3.56-3.62 (m, 4H), 7.40 (s, 1H).
Preparation 28
2-Iodo-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester
To a solution of 4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester (Preparation 27, 4.8 g, 20.23 mmol) in THF (60 mL) was added NIS (4.78 g, 21.24 mmol). The reaction was stirred at room temperature for 18 hours and the solvent was then removed in vacuo. The residue was dissolved in EtOAc (200 mL) and the resulting solution was washed with sodium thiosulfate solution (150 mL). The aqueous layer was re-extracted with EtOAc (150 mL) and the combined organic layers were washed with brine (150 mL), dried over MgSO4 and concentrated in vacuo to give the title compound as a tan solid (6.14 g) in an 84% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9H), 2.79-2.87 (m, 4H), 3.53-3.59 (m, 4H).
LCMS: m/z 364 M+H+.
Preparation 29
2-Iodo-1-(2-trimethylsilanyl-ethoxymethyl)-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester
To a solution of 2-iodo-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester (Preparation 28, 2.2 g, 6.06 mmol) in THF (35 mL) was added NaH (60% in paraffin oil, 254 mg, 6.36 mmol) and the resulting solution was stirred at room temperature for 1.5 hours. The reaction mixture was cooled to 0° C. and SEM-Cl (1.13 mL, 6.36 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 18 hours and then cooled to 0° C. and quenched carefully with water (100 mL). The resulting solution was extracted with EtOAc (2×100 mL) and the combined organic layers were dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (Biotage SNAP 100 g) eluting with 40% EtOAc in DCM to give the title compound (2.34 g) in a 78% yield.
1H NMR (400 MHz, CDCl3) δ ppm −0.01 (s, 9H), 0.89-0.93 (m, 2H), 1.46 (s, 9H), 2.82-2.92 (m, 4H), 3.54-3.66 (m, 6H), 5.16 (s, 2H).
LCMS: m/z 494 M+H+.
Preparation 30
2-[6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazol-3-yl]-1-(2-trimethylsilanyl-ethoxymethyl)-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester
To a solution of 6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-3-trimethylstannanyl-1H-indazole (Preparation 18, 735 mg, 1.53 mmol) in toluene (6 mL) was added 2-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester (Preparation 29, 829 mg, 1.68 mmol), copper (I) iodide (60 mg, 310 μmol) and tetrakis(triphenylphosphine) palladium(0) (173 mg, 150 μmol). The reaction mixture was degassed with nitrogen, heated at 100° C. for 18 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with 20% EtOAc in toluene to give the title compound as a foam (633 mg) in a 57% yield.
1H NMR (400 MHz, CDCl3) δ ppm −0.13 (s, 9H), 0.80-0.84 (m, 2H), 1.09 (t, 3H), 1.50 (s, 9H), 1.65-1.84 (m, 3H), 2.10-2.18 (m, 2H), 2.56 (q, 2H), 2.58-2.64 (m, 1H), 2.91-3.08 (m, 4H), 3.50-3.54 (m, 2H), 3.63-3.76 (m, 5H), 3.95 (s, 3H), 3.99-4.04 (m, 1H), 5.74 (dd, 1H), 5.83-5.88 (m, 1H), 5.97-6.03 (m, 1H), 6.90 (d, 1H), 7.03 (d, 1H), 7.18 (dd, 1H), 7.45 (s, 1H), 8.40-8.43 (m, 1H).
LCMS: m/z 720 M+H+.
Preparation 31
2-[6-(2-Ethyl-5-fluoro-4-methoxy-phenyl)-1H-indazol-3-yl]-1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepine trihydrochloride salt
To a solution of 2-[6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1-(tetrahydro-pyran-2-yl)-1H-indazol-3-yl]-1-(2-trimethylsilanyl-ethoxymethyl)-4,5,7,8-tetrahydro-1H-imidazo[4,5-d]azepine-6-carboxylic acid tert-butyl ester (Preparation 30, 633 mg, 879 μmol) in methanol (20 mL) was added concentrated hydrochloric acid (12M, 8 mL) and the resulting solution was heated at 60° C. for 18 hours. A further amount of concentrated hydrochloric acid (12M, 4 mL) was added and the reaction mixture was heated at 60° C. for a further 18 hours. The reaction mixture was cooled to room temperature and concentrated in vacuo to furnish the title compound (410 mg).
1H NMR (400 MHz, CD3OD) δ ppm 1.07 (t, 3H), 2.60 (q, 2H), 3.35-3.37 (m, 4H), 3.62-3.63 (m, 4H), 3.93 (s, 3H), 6.99 (d, 1H), 7.07 (d, 1H), 7.35 (d, 1H), 7.59 (s, 1H), 8.24 (m, 1H).
LCMS: m/z 406 M+H+.
Preparation 32
5-Ethyl-2-fluoro-4-[3-(1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepin-2-yl)-1H-indazol-6-yl]-phenol trihydrobromide salt
A 1M solution of boron tribromide in DCM (3.18 mL, 3.18 mmol) was added dropwise to a solution of 2-[6-(2-ethyl-5-fluoro-4-methoxy-phenyl)-1H-indazol-3-yl]-1,4,5,6,7,8-hexahydro-imidazo[4,5-d]azepine (Preparation 31, 410 mg, 796 μmol) in DCM (10 mL) at 0° C. The resulting solution was stirred at room temperature for 18 hours. The precipitated solid was collected by filtration, washed with tBME, then triturated with EtOAc to yield the title compound (380 mg) in a 75% yield.
1H NMR (400 MHz, CD3OD) ppm 1.05 (t, 3H), 2.52 (q, 2H), 3.36-3.39 (m, 4H), 3.63-3.66 (m, 4H), 6.89-6.96 (m, 2H), 7.34 (d, 1H), 7.57 (s, 1H), 8.22 (d, 1H).
LCMS: m/z 392 M+H+.
Preparation 33
(2-Bromo-4-fluoro-5-methoxy-phenyl)-methanol
To a solution of (4-fluoro-3-methoxy-phenyl)-methanol (10.0 g, 64.04 mmol) in MeCN (160 mL) was added a solution of NBS (11.4 g, 64.04 mmol) in MeCN (50 mL) and the resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo and the residue was suspended in diethyl ether (200 mL). Solid material was removed by filtration and washed with further diethyl ether. The filtrate was washed with water (200 mL) and brine (100 mL), dried over MgSO4 and concentrated in vacuo to give the title compound as a white solid (14.4 g) in a 96% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.94 (t, 1H), 3.90 (s, 3H), 4.70 (d, 2H), 7.14 (d, 1H), 7.27 (d, 1H).
Preparation 34
1-Bromo-2-bromomethyl-5-fluoro-4-methoxy-benzene
Phosphorus tribromide (11.56 mL, 122.5 mmol) was added to a solution of (2-bromo-4-fluoro-5-methoxy-phenyl)-methanol (Preparation 33, 14.4 g, 61.26 mmol) in DCM (235 mL) at 0° C. The reaction was allowed to warm to room temperature and stirred at that temperature for 18 hours. The reaction mixture was cooled to 0° C. and quenched by slow addition of saturated sodium hydrogen carbonate aqueous solution until effervescence had ceased. The layers were separated and the aqueous layer was extracted with DCM (2×100 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo to give the title compound as a white solid (17.48 g) in a 96% yield.
1H NMR (400 MHz, CDCl3) δ ppm 3.89 (s, 3H), 4.55 (s, 2H), 7.04 (d, 1H), 7.29 (d, 1H).
Preparation 35
1-Bromo-5-fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-benzene
To a solution of 1-bromo-2-bromomethyl-5-fluoro-4-methoxy-benzene (Preparation 34, 10.84 g, 36.4 mmol) in DMF (80 mL) was added copper (I) iodide (1.746 g, 9.09 mmol) and the solution was degassed with nitrogen. To this solution was added difluoro-fluorosulfonyl-acetic acid methyl ester (11.57 mL, 90.9 mmol) and the resulting reaction mixture was heated at 120° C. for 4 hours. The reaction mixture was cooled to 0° C., diluted with EtOAc (60 mL) and stirred for 10 minutes at 0° C. A solution of ammonium hydroxide (60 mL) was added dropwise and the mixture was stirred as it warmed from 0° C. to room temperature over 20 minutes. Ethyl acetate (200 mL) and water (100 mL) were added and the layers were separated. The aqueous layer was further extracted with EtOAc (2×100 mL). The combined organic layers were washed with water (100 mL) and brine (100 mL), dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by column chromatography on silica gel eluting with 20% EtOAc in heptane to give the title compound as a yellow solid (7.356 g) in a 70% yield.
1H NMR (400 MHz, CDCl3) δ ppm 3.56 (q, 2H), 3.89 (s, 3H), 6.94 (d, 1H), 7.32 (d, 1H).
LCMS: m/z 288 M+H+.
Preparation 36
2-[5-Fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-4,4,5,5-tetramethyl-1,3,2]dioxaborolane
To a solution of 1-bromo-5-fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-benzene (Preparation 35, 7.07 g, 26.82 mmol) in dioxane (100 mL) was added 4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl] (8.17 g, 32.18 mmol) and KOAc (7.9 g, 80.46 mmol). The mixture was degassed with nitrogen prior to the addition of [1,1-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) (2.3 g, 2.68 mmol). The reaction mixture was stirred at 110° C. for 18 hours, then cooled to room temperature and concentrated in vacuo. The residue was dissolved in methanol and filtered through Arbocel®, washing with methanol. The filtrate was concentrated in vacuo and then partitioned between EtOAc (200 mL) and water (200 mL). The aqueous layer was extracted with further EtOAc (2×100 mL). The combined organic layers were washed with water (200 mL) and brine (150 mL), dried over Na2SO4 and concentrated in vacuo to give the title compound as an oil (8.96 g) in a 100% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.31 (s, 12H), 3.78 (q, 2H), 3.95 (s, 3H), 6.85 (d, 1H), 7.53 (d, 1H).
Preparation 37
6-[5-Fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbonitrile
To a solution of 6-bromo-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbonitrile (Preparation 2, 3.99 g, 13.03 mmol) and 2-[5-fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (Preparation 36, 7.46 g, 15.63 mmol) in dioxane (60 mL) was added a solution of potassium phosphate (18.8 g, 39.09 mmol) in water (12 mL). The mixture was degassed with nitrogen, treated with tetrakis(triphenylphosphine) palladium(0) (3.01 g, 2.6 mmol) and heated at 110° C. for 18 hours. The reaction mixture was concentrated in vacuo and the residue was redissolved in EtOAc (500 mL) and filtered through Arbocel®, washing with EtOAc (2×500 mL). The combined organic phases were washed with water (300 mL), dried over MgSO4 and concentrated in vacuo to give a brown oil. The residue was purified by column chromatography on silica gel, eluting with 25% EtOAc in heptanes, to give the title compound (1.737 g) in a 31% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.68-1.80 (m, 3H), 2.11-2.17 (m, 2H), 2.45-2.53 (m, 1H), 3.27-3.41 (m, 3H), 3.74 (m, 1H), 3.96 (s, 3H), 5.80 (dd, 1H), 7.06 (m, 2H), 7.24 (m, 1H), 7.84 (s, 1H), 7.87 (d, 1H).
Preparation 38
6-[5-Fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboximidic acid methyl ester
To a solution of 6-[5-fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carbonitrile (Preparation 37, 1.737 g, 4.00 mmol) in methanol (40 mL) was added sodium methoxide (648 mg, 12.0 mmol) and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was partitioned between EtOAc (50 mL) and water (50 mL) and the aqueous layer was extracted with further EtOAc (2×50 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo to give the title compound as an oily solid (1.64 g) in an 88% yield.
1H NMR (400 MHz, CDCl3) δ ppm 1.63-1.78 (m, 3H), 2.07-2.18 (m, 2H), 2.53-2.61 (m, 1H), 3.28-3.42 (m, 3H), 3.96 (s, 3H), 4.00-4.04 (m, 1H), 4.07 (s, 3H), 5.74 (dd, 1H), 7.05 (d, 1H), 7.09 (d, 1H), 7.14 (dd, 1H), 7.52 (s, 1H), 8.10 (d, 1H).
Preparation 38
2-{6-[5-Fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine trihydrochloride salt
To a solution of 6-[5-fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1-(tetrahydro-pyran-2-yl)-1H-indazole-3-carboximidic acid methyl ester (Preparation 37, 1.64 g, 3.78 mmol) in ethanol (5 mL) was added a solution of 3-amino-4,4-diethoxy-piperidine-1-carboxylic acid tert-butyl ester (US-2004/0229862, 1.15 g, 3.97 mmol) in ethanol (7.5 mL). Acetic acid (430 μL, 7.56 mmol) was added and the reaction mixture was heated at 50° C. for 18 hours and then concentrated in vacuo to give a brown oil. The oil was dissolved in ethanol (15 mL) and the resulting solution was treated with concentrated hydrochloric acid (12M, 4.75 mL, 56.7 mmol) and then heated at 80° C. for 18 hours. The solvent was removed in vacuo to yield the title compound (2.03 g) in a 97% yield.
1H NMR (400 MHz, CD3OD) δ ppm 3.20-3.26 (m, 2H), 3.40-3.54 (m, 2H), 3.66-3.75 (m, 2H), 3.80 (s, 3H), 4.51-4.55 (m, 2H), 7.15 (d, 1H), 7.24 (d, 1H), 7.33 (d, 1H), 7.62 (s, 1H), 8.27 (d, 1H).
LCMS: m/z 446 M+H+.
Preparation 39
2-Fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-5-(2,2,2-trifluoro-ethyl)-phenol dihydrobromide salt
Boron tribromide (750 μL, 7.83 mmol) was added driowuse to a solution of 2-{6-[5-fluoro-4-methoxy-2-(2,2,2-trifluoro-ethyl)-phenyl]-1H-indazol-3-yl}-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine trihydrochloride salt (Preparation 38, 2.03 g, 3.66 mmol) in DCM (25 mL) at 0° C. The resulting solution was stirred at room temperature for 18 hours. Further boron tribromide (2 mL, 20.7 mmol) was added dropwise and the reaction mixture was allowed to stir at room temperature for 18 hours. The precipitated solid was collected by filtration, washed with DCM and triturated with EtOAc to yield the title compound as the dihydrobromide salt (1.56 g) in a 67% yield.
1H NMR (400 MHz, CD3OD) δ ppm 3.24 (dd, 2H), 3.41 (q, 2H), 3.75 (dd, 2H), 4.58 (s, 2H), 7.05-7.09 (m, 2H), 7.34 (d, 1H), 7.60 (s, 1H), 8.26 (d, 1H).
LCMS: m/z 432 M+H+.
Preparation 40
5-Hydroxy-pyrazine-2-carboxylic acid methyl ester
Thionyl chloride (152 mL, 2.08 mol) was added dropwise at −20° C. to methanol (5 L). After the addition was completed, the mixture was stirred at this temperature for 30 minutes. Then 5-hydroxy-pyrazine-2-carboxylic acid (100 g, 714 mmol) was added, and the mixture was heated at reflux for 2 hours. The reaction mixture was concentrated in vacuo, and the residue was recrystallised from methanol (400 mL) to give 71 g (464 mmol) of the title compound in 65% yield.
Preparation 41
5-Chloro-pyrazine-2-carboxylic acid methyl ester
A mixture of 5-hydroxy-pyrazine-2-carboxylic acid methyl ester (Preparation 40, 50 g, 324 mmol) and POCl3 (500 mL, 5.36 mol) was heated under reflux for 1.5 hours and then poured onto ice. The resulting mixture was extracted with ether (4×500 mL). The organic layers were concentrated in vacuo, and the residue was recrystallised from toluene to give the title compound (30.8 g) in a 55% yield.
Preparation 42
5-Piperidin-1-yl-pyrazine-2-carboxylic acid methyl ester
To a solution of 5-chloro-pyrazine-2-carboxylic acid methyl ester (Preparation 41, 85 g, 492 mmol) in DMF (365 mL) was added DIPEA (129 mL, 738 mmol) and piperidine (58.4 mL, 591 mmol) and the resulting solution was stirred at room temperature for 18 hours. The reaction mixture was poured onto water (4 L) and the resulting precipitate was collected by filtration to give the title compound as a white solid (85.12 g) in a 78% yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 1.50-1.56 (m, 4H), 1.60-1.62 (m, 2H), 3.68-3.73 (m, 4H), 3.80 (s, 3H), 8.35 (s, 1H), 8.60 (s, 1H).
Preparation 43
5-Piperidin-1-yl-pyrazine-2-carboxylic acid
5-Piperidin-1-yl-pyrazine-2-carboxylic acid methyl ester (Preparation 42, 85.1 g, 384 mmol) was added to a solution of sodium hydroxide (61.5 g, 1.53 mol) in water (760 mL). The mixture was stirred mechanically for 1 hour at room temperature. THF (300 mL) was added and stirring was continued for 3 hours. The volatile solvents were removed in vacuo and the remaining aqueous solution was adjusted to pH 4. The mixture was cooled on ice to induce precipitation of the product. The resulting solid was collected by filtration and dried in vacuo to give the title compound as a white solid (56 g) in a 70% yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 1.43-1.61 (m, 6H), 3.60-3.70 (m, 4H), 8.28 (s, 1H), 8.58 (s, 1H), 12.59 (br s, 1H).
LCMS: m/z 208 [M+H]+.
Preparation 44
5-(2-Fluoro-phenoxy)-pyrazine-2-carboxylic acid methyl ester
2-Fluorophenol (21.6 g, 233 mmol) was dissolved in DMF (250 mL) under a calcium chloride drying tube. The solution was cooled to 0° C., and then 60% NaH in paraffin oil (9.3 g, 233 mmol) was added in small portions. After the main portion of NaH had dissolved, 5-chloro-pyrazine-2-carboxylic acid methyl ester (Preparation 41, 40.2 g, 233 mmol) was added. The mixture was refluxed for 1 hour and then poured into water (1 L). The aqueous mixture was extracted with ether (3×300 mL), and the combined organic layers were washed with 2% sodium hydroxide aqueous solution (400 mL) and filtered through a layer of silica gel (40/60 μm). The filtrate was concentrated in vacuo to yield the title compound which was used without further purification.
Preparation 45
5-(2-Fluoro-phenoxy)-pyrazine-2-carboxylic acid
5-(2-Fluoro-phenoxy)-pyrazine-2-carboxylic acid methyl ester (Preparation 44, 57.9 g, 233 mmol) was added to a solution of KOH (15 g, 267 mmol) in 78% ethanol (330 mL). The solution was stirred at room temperature for 18 hours, and the formed precipitate was collected by filtration. The resulting solid was dissolved in water (200 mL), and the solution was acidified with aqueous hydrochloric acid. The formed precipitate was collected by filtration, dried and recrystallised from 41% ethanol (265 mL) to give the title compound (28.3 g, 120.8 mmol) in 51.8% yield.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.29-7.48 (m, 4H), 8.75 (s, 2H), 13.51 (s, 1H).
LCMS: m/z 235.1 [M+H]+.
Preparation 46
6-Cyano-nicotinoyl chloride
6-Cyano-nicotinic acid (120 mg, 810 μmol) was suspended in toluene (1 mL) and thionyl chloride (119 μL, 1.62 mmol) was added dropwise followed by one drop of DMF. The reaction mixture was refluxed for 2.5 hours and then allowed to cool to room temperature for 18 hours. The solvents were removed in vacuo and the residue was azeotroped with toluene to furnish the title compound as a brown oil (134 mg) which was used in further experiments without purification.
LCMS: m/z 167.02 M+H+.
The activity of the compounds of formula (I) may be assessed in the following assays.
Preparation 47
(5-chloropyrazin-2-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone
To a solution of 5-ethyl-2-fluoro-4-[3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl]-phenol preparation 25 (500 mg, 1.326 mmol) and 7 (209.54 mg, 1.326 mmol) in dry DMF (10 ml), DIPEA (0.65 ml, 3.978 mmol) and T3P (2.38 ml, 3.978 mmol) were added and the reaction mixture was stirred at room temperature for 1 h. TLC & LCMS showed product formation. The reaction mixture was evaporated in vacuo, ice water was added to form solid precipitate which was washed with water, saturated sodium bi carbonate and pentane to afford a brown solid (460 mg, 67.08%).
1H NMR (400 MHz, DMSO) δ (ppm): 1.04 (t, 3H), 2.66-2.81 (m, 2H), 3.72 (m, 1H), 4.04 (t, 1H), 4.57 (s, 1H), 4.74 (s, 1H), 6.89-6.92 (m, 1H), 6.98-7.13 (m, 2H), 7.36 (d, 1H), 8.31 (d, 1H), 8.75 (d, 1H), 8.86-8.87 (m, 1H), 9.82 (s, 1H), 12.55 (s, 1H), 13.21 (s, 1H);
LCMS: Rt=2.89 min; m/z 518.4 [M+H]+.
JAK3 Isolated Enzyme High ATP Caliper Endpoint Assay
4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using PF-00956980-00 at a top concentration of 4 mM is also prepared. High percentage effect (HPE) is defined by 500 μM PF-00956980-00 and 0% effect (ZPE) is defined by 100% DMSO. Greiner 384 well plates are prepared containing 400 nl of serially diluted compound, standard and HPE/ZPE. Final top assay concentration is 80 μM as the assay dilution factor is fifty.
JAK3 enzyme (Invitrogen) stock solution is made up at 4.1 μM in sterile water. JAK3 enzyme stock is diluted to 2 nM in assay buffer (10 mM HEPES free acid pH 7.5, 10 mM HEPES free base pH 7.5, 10 mM MgCL2, 0.0005% Tween-20, 0.01% BSA) containing 2 mM DTT (all supplied by Sigma). ATP is made up at 10 mM stock in sterile water and diluted to 800 μM in assay buffer. Peptide (American peptide company) is made up at 30 mM in 100% DMSO and diluted to 3 μM in assay buffer. Stop buffer comprises 140 mM HEPES, 22.5 mM EDTA (Sigma) and 0.15% coating reagent (Caliper Life Sciences).
Assays are performed in Greiner polypropylene 384 well plates. Following compound preparation within the plate 10 μl of enzyme in assay buffer containing DTT is added using a Multidrop Micro. Final assay concentration of enzyme is 1 nM. Compound and enzyme are pre-incubated for 60 minutes at room temperature using low evaporation lids before addition of 10 μl ATP/peptide mixture in assay buffer using a Multidrop Micro. Final assay concentrations are 400 μM ATP and 1.5 μM peptide. Plates are foil sealed and incubated for a further 60 minutes at room temperature. Stop solution is added to the plates (20 μl/well) using a Multidrop Micro and plates are loaded onto the Caliper EZReader II. Data is generated by the shift in mobility of non-phosphorylated peptide substrates and phosphorylated products by electrophoresis within a chip and detected via LED induced fluorescence. Data is analysed using LabChip EZReader software which calculates the relative heights of the substrate and product peaks and reports product/product plus substrate peak ratio. Test compound data are subsequently expressed as percentage inhibition defined by HPE and ZPE values for each plate. Percentage inhibition in the presence of test compound is plotted against compound concentration on a log scale to determine an IC50 from the resultant sigmoid curve.
JAK1 Isolated Enzyme High ATP Caliper Endpoint Assay
4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using PF-00956980 (commercially available from Sigma Aldrich) at a top concentration of 4 mM is also prepared. High percentage effect (HPE) is defined by 500 μM PF-00956980 and 0% effect (ZPE) is defined by 100% DMSO. Greiner 384 well plates are prepared containing 400 nl of serially diluted compound, standard and HPE/ZPE. Final top assay concentration is 80 μM as the assay dilution factor is fifty.
JAK1 enzyme (Invitrogen) stock solution is made up at 5.2 μM in sterile water. JAK1 enzyme stock is diluted to 20 nM in assay buffer (10 mM HEPES free acid pH 7.5, 10 mM HEPES free base pH 7.5, 10 mM MgCL2, 0.0005% Tween-20, 0.01% BSA) containing 2 mM DTT (all supplied by Sigma) with the addition of one protease tablet per 25 mls buffer (Roche). ATP is made up at 10 mM stock in sterile water and diluted to 5 mM in assay buffer. Peptide H236 (Caliper Life Sciences) is made up at 1.5 mM in 100% DMSO and diluted to 3 μM in assay buffer. Stop buffer comprises 140 mM HEPES, 22.5 mM EDTA (Sigma) and 0.15% coating reagent (Caliper Life Sciences).
Assays are performed in Greiner polypropylene 384 well plates. Following compound preparation within the plate 10 μl of enzyme in assay buffer containing DTT is added using a Multidrop Micro. Final assay concentration of enzyme is 10 nM. Compound and enzyme are pre-incubated for 30 minutes at room temperature using low evaporation lids before addition of 10 μl ATP/peptide mixture in assay buffer using a Multidrop Micro. Final assay concentrations are 2.5 mM ATP and 1.5 μM peptide. Plates are foil sealed and incubated for a further 120 minutes at room temperature. Stop solution is added to the plates (20 μl/well) using a Multidrop Micro and plates are loaded onto the Caliper EZReader II. Data is generated by the shift in mobility of non-phosphorylated peptide substrates and phosphorylated products by electrophoresis within a chip and detected via LED induced fluorescence. Data is analysed using LabChip EZReader software which calculates the relative heights of the substrate and product peaks and reports product/product plus substrate peak ratio. Test compound data are subsequently expressed as percentage inhibition defined by HPE and ZPE values for each plate. Percentage inhibition in the presence of test compound is plotted against compound concentration on a log scale to determine an IC50 from the resultant sigmoid curve.
The following Table shows the available IC50 data for Examples 1-31 in the JAK-1 and JAK-3 isolated enzyme high ATP Caliper endpoint assays described above.
Example
JAK-3 IC50
JAK-1 IC50
number
(nM)
(nM)
1
<0.6
<0.7
2
0.5
46
3
1.0
3.7
4
0.5
5.4
5
No data
No data
6
1.3
8.4
7
1.5
2.5
8
1.4
2.9
9
1.9
18.9
10
1.3
4.5
11
1.0
2.5
12
1.6
1.2
13
3.6
1.5
14
1.4
0.8
15
1.0
No data
16
1.9
0.7
17
1.4
3.4
18
1.4
4.2
19
2.2
4.3
20
5.6
2.6
21
1.4
5.9
22
2.6
3.0
23
1.2
1.7
24
2.4
6.2
25
1.4
4.0
26
4.1
4.0
27
No data
No data
28
10.9
13.7
29
42
1.1
30
6.7
7.7
31
186
66
As a comparator compound, Example 24(c) of WO-2001/002369 was tested. It gave an IC50 of 119 nM in the JAK-3 assay and an IC50 of 120 nM in the JAK-1 assay.
JAK1/3 Whole Cell Reporter Gene Assay
4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using PF-00956980 at a top concentration of 10 mM is also prepared. High percentage effect (HPE) is defined by 10 mM PF-00956980 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted by addition of 39 μl assay media (Optimem with 100 uM NEAA, 10 uM sodium pyruvate and 100 U penicillin/100 ug streptomycin (Invitrogen)) using a Multidrop Combi. This dilutes test compounds to a top concentration of 100 μM. Final top assay concentration is 10 μM as the assay dilution factor is ten. Final DMSO concentration is 0.25%.
CD40 ligand is a member of the TNF superfamily and activates B cells. CD40 (Invitrogen) is prepared at 0.1 mg/ml in PBS minus Ca2+, minus Mg2+. The concentration of CD40 required for activation is predetermined by CD40 titration with the cell line. Interleukin-4 (1 L-4, Invitrogen) is used as the co-activator and functions by binding to the IL-4 receptor complex leading to recruitment and activation of JAK1 and JAK3 tyrosine kinases. IL-4 is prepared at 1 mg/ml in sterile water to generate a stock solution. This is further diluted to 100 ng/ml in assay media. Inhibition of the STATE-beta-lactamase reporter response is measured in the presence of IL-4 at an approximate EC50 concentration.
Beta lactamase dye reagent comprises three components and is made up by adding 1 part CCF4 dye, 5 parts solution B and 77 parts Live Blazer-substrate mixture.
Assays are performed in Greiner 384 well black polypropylene clear bottomed plates. The Invitrogen Cellsensor STAT6-bla-RA-1 cell line is thawed, counted and resuspended at 1.88×106 cells/ml. Cells are stimulated with CD40 ligand by addition of 5.56 μl of 0.1 mg/ml stock per 1 ml of cell suspension. Cells are plated out at 60000 cells/well, 32 μl/well and incubated at 37° C., 5% CO2. After 18 hours 4 μl test compound is added to the plate using a Platemate Plus. Plates are incubated at 37° C., 5% CO2 for 60 minutes using low evaporation lids before addition of 4 μl IL-4 at a concentration of 100 ng/ml. Plates are incubated at 37° C., 5% CO2 for a further five hours before addition of 10 μl beta lactamase dye. After reagent addition plates are incubated at 37° C., 5% CO2 for 18 hours. Beta lactamase fluorescence signal is read at 460 nm (blue) and 530 nm (green) and a ratio calculated using an Envision. Test compound data are expressed as percentage inhibition defined by HPE and ZPE values for each plate. Percentage inhibition in the presence of test compound is plotted against compound concentration on a log scale to determine an IC50 from the resultant sigmoid curve.
Example 4 gave an IC50 of 140 nM in this assay.
JAK1 and JAK2 PathHunter Assay
4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using PF-00956980 at a top concentration of 10 mM is also prepared. High percentage effect (HPE) is defined by 10 mM PF-00956980 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted by addition of 65 μl compound diluent (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Multidrop Combi. This dilutes test compounds to a top concentration of 60 μM. Final top assay concentration is 10 μM as the assay dilution factor is six. Final DMSO concentration is 0.25%.
Prolactin (Peprotech) is used for the agonist challenge. Prolactin is prepared at 40 μM in compound diluent to generate a stock solution and further diluted to 6 μM in compound diluent. A standard curve is prepared in compound diluent. Prolactin is also diluted to a concentration of 15 nM (2.5 nM fac). Antagonism of the JAK1 or JAK2 prolactin response is measured in the presence of prolactin at an approximate EC80 concentration for JAK1 and approximately EC100 for JAK2.
PathHunter detection reagent comprises three components and is made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer.
Assays are performed in Greiner white 384 well plates. The PathHunter U2OS cell line expressing the cytosolic tyrosine kinase JAK1 or JAK2 and the membrane bound cytokine receptor prolactin is plated out using OptiMEM (Invitrogen) at 5000 cells/well, 20 μl/well and incubated at 37° C., 5% CO2. After 18 hours 5 μl test compound is added to the plate using a Platemate Plus. Plates are incubated at 37° C., 5% CO2 for 60 minutes before addition of 5 μl prolactin at a concentration of 15 nM. Plates are incubated at room temperature for a further 180 minutes before addition of 10 μl detection reagent. After reagent addition plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision. Test compound data are expressed as percentage inhibition defined by HPE and ZPE values for each plate. Percentage inhibition in the presence of test compound is plotted against compound concentration on a log scale to determine an IC50 from the resultant sigmoid curve.
Example 4 gave a JAK-1 IC50 of 75 nM and a JAK-2 IC50 of 176 nM in this assay.
Functional Assessment of JAK Inhibitory Potency Using hrIL-2 and haCD3 Stimulated IFNγ Production in Human Isolated PBMC.
Isolation of Human Peripheral Blood Mononuclear Cells (PBMC)
Peripheral venous blood from healthy volunteers of either sex was collected into 50 ml centrifuge tubes (Corning) containing 1 ml of 5 mg/ml heparin (Sigma H3400) in distilled water. The heparininsed blood was diluted using an equal volume of sterile Dulbeccos phosphate buffered saline (PBS: Invitrogen 14190) before decanting into 50 ml Leucosep tubes (Sigma A0561). The Leucosep tubes were centrifuged at 400 g for 30 min at room temperature and the buffycoat at the Ficoll:Plamsa interface collected into clean centrifuge tubes and the volume made up to 50 ml using PBS and centrifuging at 200 g for 10 min at room temperature. The supernatent was discarded and the pellet resuspended in assay media (Dulbeccos Modified Essential Medium (DMEM: Invitrogen 11971025) containing 5% Foetal Bovine Serum, 100 U/ml penicilin/100 μg/ml streptomycin (Sigma P4458) at 2×106 lymphocytes per ml for IFNγ experiments and at 1×106 lymphocytes per ml for the pYSTAT5 experiments.
hrIL2/haCD3 Stimulated IFNγ Production
180 μl of PBMC cell suspension was added to each well of a sterile 96 well, flat bottomed plate (Corning-Costar 3598). After a 1 h incubation at 37° C., 10 μl of test compound dilution (final assay concentration range of 0.3 nM to 1 μM in half log increments) or vehicle (2% DMSO in Hanks Balanced Salt Solution (Sigma H8264)) was added to the appropriate well and the pates incubated at 37° C., in 95% O2/5% CO2 for 1 h. 10 μl of 200 ng/ml IL-2 (R&D systems 202-IL):20 μg/ml aCD3 (BD Biosciences 555329) (final assay concentrations of 10 ng/ml and 1 μg/ml respectively) in assay buffer was added and the plates incubated at 37° C., in 95% O2/5% CO2 for 18 h. Plates were removed from the incubator and centrifuged at 200 g for 5 min at room temperature. 100 μl supernatent was collected, diluted 1:4 and IFNγ content determined using a commercially available IFNγ ELISA kit (Invitrogen CHC1233) as per the manufacturer's instructions. Absorbances were measured using a Spectramax 190/250 plate reader (Molecular Devices). The IFNγ concentration of test wells was expressed as % of the IFNγ concentration produced in wells exposed to IL-2/aCD3 in the absence of test compound, and IC50 values determined using a 4 parameter curve fit.
Example 4 gave an IC50 of 70 nM in this assay.
hrIL2/haCD3 Stimulated pYSTAT5 in PBMC Lymphocytes
90 μl of PBMC cell suspension was added to each well of a 96 well plate (Corning-Costar 3598) along with 10 μl of test compound dilution giving a final assay concentration range of 0.03 nM to 1 μM in half log increments. The plates were incuated at 37° C., in 95% O2/5% CO2 for 1 h before 10 μl rhIL2 (3 μg/ml final assay concentration) was added to appropriate wells and the plates incubated at the stated conditions for a further 15 min. 25 μl of 20% formaldehyde (Tousimis) was added to all wells and the plates left at room temperature for 10 min prior to centrifugation at 400 g for 4 min at room temperature. 200 μl PBS was added to each well and centrifugation repeated as just described. The supernatent was removed and 50 μl of a 1:50 dilution of Mouse anti-human CD3 (BD Biosciences 555329) in 0.1% BSA (Sigma A7906)/PBS added to each well (excluding control wells) and the plates incubated at room temperature in the dark for 30 min. 150 μl of 0.1% BSA/PBS was added to each well and the plate centrifuged at 400 g for 4 min before the supernatent was discarded and 100 μl ice-cold Phosflow Perm Buffer III (BD Biosciences 612599) added. The plates were briefly vortexed and incubated on ice in the dark for 30 min before 100 μl of 0.1% BSA/PBS was added and the plates centrifuged again as just described. 20 μl of a 1:20 dilution of AF647 anti-phospho-STAT5 antibody (in PBS) was added to the wells (excluding controls) and incubated in the dark at room temperature for 30 min before adding 180 μl of 0.1% BSA/PBS and centrifuging as already described. Once the supernatent was discarded the cells were resuspended in 100 μl of 2% formaldehyde and the plates stored at 4° C. overnight. Plates were read the next day on a FACS Canto (Becton Dickinson). Lymphocytes were gated on PE immunofluorescence and the AF647 signal used as a measure of pYSTAT5 expression. IC50 values were generated in Excel using a four parameter curve fit.
Example 4 gave an IC50 of 45 nM in this assay.
For determination of compound duration of action (DoA) cells were incubated with compound at an approximate IC80 concentration for 1 h before being washed by cenrifugation and resuspension in assay media without compound. At set intervals after wash cells were stimulated with IL-2:aCD3 for 15 min and the plates processed as described above. 100% inhibition was defined as reduction of pYSTAT5 levels down to basal. DoA was calculated as the time taken for the inhibition to reverse by 50% (T50%).
Example 4 gave a DoA of >8.8 hours in this assay.
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13542153
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pfizer limited
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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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540/578
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Sep 22nd, 2009 12:00AM
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Jan 4th, 2007 12:00AM
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https://www.uspto.gov?id=US07592362-20090922
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Substituted imidazoles
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This invention relates to a range of alpha substituted 2-benzyl substituted imidazole compounds and pharmaceutically acceptable salts and solvates thereof, to compositions comprising such compounds, processes for their synthesis and their use as parasiticides.
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7592362
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1. Compound
2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole;
or a pharmaceutically acceptable salt thereof, which has the formula
2. A pharmaceutical, veterinary or agricultural composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a suitable excipient or carrier.
3. The compound of claim 1 wherein the compound is a free base.
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3
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This invention relates to imidazoles having parasiticidal properties. The compounds of interest are substituted imidazoles and, more particularly, the invention relates to alpha substituted 2-benzyl imidazoles.
There is a need for improved antiparasitic agents for use with mammals, including humans and animals, and in particular there is a need for improved insecticides and acaricides. Furthermore there is a need for improved topical products with convenient administration and which contain one or more of such antiparasitic agents which can be used to effectively treat ectoparasites, such as insects and acarids, and particularly aracids such as mites and ticks. Such products would be particularly useful for the treatment of companion animals, such as cats, dogs and horses, and livestock, such as cattle. There is equally a need for agents to control parasitic infestations in animal hosts other than mammals, including insects such as bees, which are susceptible to parasites such as varroa mites.
The compounds currently available for insecticidal and acaricidal treatment of companion animals and livestock do not always demonstrate good activity, good speed of action, or a long duration of action. Most treatments contain hazardous chemicals that can have serious consequences when either used too often or when used in excess of recommended quantities. Many products have toxic side effects and some are lethal to cats when accidentally ingested. They are not always suitable for use as a topical or spot-on formulation and some topical and spot-on formulations are disadvantaged by common side effects in animals and owners. Persons applying these insecticidal and acaricidal agents are advised to limit their exposure to the chemicals by wearing gloves and avoiding inhalation of the chemical vapours. Pet collars and tags have been utilised to overcome some problems, but these are susceptible to chewing and therefore are disadvantageous since the compound may be accidentally orally ingested. Thus, treatments currently achieve varying degrees of success depending on a variety of factors including toxicity and the method of administration. In some cases toxicity may be attributed to their non-selective activity at various receptors. In addition it has recently been shown that some current agents are becoming ineffective as the parasites develop resistance.
The present invention overcomes one or more of the various disadvantages of, or improves upon, the properties of existing compounds. In particular the present invention develops some new alpha substituted 2-benzyl imidazoles which demonstrate such properties.
Heterocyclic derivatives have been disclosed in the prior art as having insecticidal and acaricidal activity against agricultural pests, for example International patent application publication no. WO 03/092374.
Generic disclosures also exist in the prior art of heterocyclic derivatives which optionally encompass alpha substituted 2-benzyl imidazoles. For example, international patent application publication no. WO 2005/007188 describes a generic structure, which optionally encompasses alpha substituted 2-benzyl imidazoles for the inhibition of the hatching of an ectoparasite egg; international patent application publication no. WO 2004/103959 describes a generic structure which optionally encompasses alpha substituted 2-benzyl imidazoles for use as antibacterial agents; international patent applications publication nos WO 01/00586 and WO 99/28300 both describe a generic structure which optionally encompasses alpha substituted 2-benzyl imidazoles and discloses their adrenergic activity; and U.S. Pat. No. 6,103,733 describes a generic structure which optionally encompasses alpha substituted 2-benzyl imidazoles for increasing blood serum and HDL cholesterol levels. However, none of this prior art exemplifies any alpha substituted 2-benzyl imidazoles, nor does the prior art indicate that such compounds would be useful against a spectrum of parasites relevant to companion animals and livestock or against the range of ectoparasite lifecycle stages.
Thus, it is an aim of the present invention to overcome one or more of the various disadvantages of, or improve on the properties of, known compounds. In particular it is an aim of the invention to develop some new alpha substituted 2-benzyl substituted imidazoles. It is a further aim that such new compounds have the same or improved activity when compared to the prior art compounds against parasites. It is another aim of the present invention to develop compounds which have a similar or decreased toxicity profile when compared to the prior art compounds. It is yet another aim to develop compounds which demonstrate selectivity for the octopaminergic receptor, a known invertebrate neurotransmitter, over the ubiquitous animal adrenergic receptor. Furthermore, it is an aim of the invention to reduce the exposure of both humans and animals to the treatment by developing compounds which can be dosed as a low volume spot-on or topical application. The compounds of the present invention have especially good ability to control arthropods as shown by the results of tests demonstrating their potency and efficacy. In particular, the compounds of the present invention are active against ticks and they are able to prevent ticks from attaching to, and feeding from, the host animal. It is yet another aim of the present invention to provide compounds which have good speed of action when compared to those of the prior art and hence an improved efficacy against the transmission of tick borne diseases.
It is also desirable that the compounds of the present invention should have one or more of the same or improved duration of action, an improved pharmacokinetic profile, improved safety, improved persistence, improved solubility or other improved physicochemical and formulation properties such as good spreading after topical application compared to those of the prior art.
Thus, according to the present invention, there is provided a compound of formula (I):
wherein:
R1, R2, R3, R4, R5 are independently selected from the group consisting of hydrogen, cyano, nitro, halo, hydroxy, C1-4 alkyl optionally substituted by one or more hydroxy groups, C3-6 cycloalkyl optionally substituted by one or more C1-4 alkyl or halo groups, C1-4 alkoxy, C1-4 haloalkyl, C1-4 haloalkoxy, phenyl, amino, NRxRy, and S(O)nR10;
R6 is selected from the group consisting of hydrogen, —C0-2alkyleneR7, —C1-2alkyleneOR7, —C0-2alkyleneC(O)R7, —C1-2alkyleneOC(O)R7, —C1-2alkyleneOC(O)OR7, —C0-2alkyleneC(O)OR7, —C1-2alkyleneN(H)C(O)R7, —C1-2alkyleneN(R7)C(O)R7, —C0-2alkyleneC(O)NHR7, —C0-2alkyleneC(O)NR15R16, —C1-2alkyleneNHC(O)NR15R16, —C1-2alkyleneNR7C(O)NR15R16, —C1-2alkyleneOC(O)NHR7, —C1-2alkyleneOC(O)NR15R16, —C0-2alkyleneCH═N(R7), —C1-2alkyleneP(═O)(NR15R16)(NR15R16), —C0-2alkyleneSi(R7)3, and —C0-2alkyleneS(O)nR10;
where the C0-2alkylene or C1-2alkylene of R6 may, where chemically possible, optionally be substituted by one or more substituents selected from the group consisting of C1-6 alkyl, C3-6 cycloalkyl, C1-4 alkylene(C3-6 cycloalkyl), C0-6 alkylenephenyl, which C0-2alkylene or C1-2alkylene substituent may in turn be optionally further substituted, where chemically possible, by one or more substituents selected from the group consisting of hydrogen, cyano, nitro, halo, formyl, oxo, hydroxy, C(O)OH, C1-4 alkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, C1-4 alkyleneC1-4 alkyoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, C1-4 haloalkoxy, amino, C1-4 alkylamino, C1-4 dialkylamino, and S(O)nR10;
where each R7, R15 and R16, where chemically possible, is independently selected from the group consisting of hydrogen, C1-8 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C1-4 alkylene(C3-6 cycloalkyl), C1-4 alkyleneC1-4 alkoxy, C1-6 haloalkyl, C0-6 alkylenephenyl, C0-6 alkylenenaphthyl, C0-6 alkylene(tetrahydronaphthyl), and C0-2 alkylene(Het), where Het is selected from oxetanyl, tetrahydropyranyl, piperidinyl, morpholinyl, furyl, pyridyl, benzofuranyl, benzothiazolyl, indolyl, 2,3-benzodioxolyl, 2,3-dihydro-1,4-benzodioxinyl, indolyl and 1,5-naphthyridinyl;
or R15 and R16 together with the nitrogen to which they are attached may form a three to seven-membered saturated or unsaturated heterocyclic ring optionally containing one or more further N, O or S atoms or SO2 groups;
where each of the above R7, R15 or R16 groups may independently include one or more optional substituents where chemically possible selected from hydrogen, cyano, nitro, halo, formyl, oxo, hydroxy, C(O)OH, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, C1-4 alkyleneC1-4 alkyoxy, C1-4 alkoxyC1-4 alkoxy, C1-4 alkanoyl, —C(O)OC1-4 alkyl, C1-4 haloalkyl, C3-6 halocycloalkyl, C1-4 haloalkoxy, C1-4 haloalkanoyl, —C(O)OC1-4 haloalkyl, phenyl, 4-halophenyl, 4-alkoxyphenyl, 2-cyanophenyl, phenoxy, 4-halophenoxy, benzyloxy, 4-halobenzyloxy, benzoyl, pyrazolyl, triazolyl, 2-halo-4-pyrimidinyl, 2-phenylethyl, amino, C1-4 alkylamino, C1-4 dialkylamino, C(O)N(C1-4 alkyl)2, N(C1-4 alkylene)C(O)(C1-4 alkyl) and S(O)nR10;
R8 and R9 are independently selected from the group consisting of hydrogen, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, C1-4 haloalkoxy and C0-4 alkylenephenyl but with the proviso that R8 and R9 are not both hydrogen;
where each of R8 and R9 may independently include one or more optional substituents where chemically possible selected from hydrogen, cyano, halo, hydroxy, C1-4 alkyl, C3-6 cycloalkyl, C1-4 alkoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, C1-4 haloalkoxy, and S(O)nR10;
or R8 and R9 together with the carbon to which they are attached may form a three to six membered carbocyclic, saturated ring, which ring is optionally substituted with one or more substituents selected from the group consisting of halo, C1-2 alkyl, C1-2 alkoxy, C1-2 haloalkyl, C1-2 haloalkoxy;
R11 and R12 are independently selected from the group consisting of hydrogen, halo, cyano, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, and C1-4 haloalkoxy;
where Rx and Ry are independently selected from hydrogen, C1-4 alkyl, C1-4 haloalkyl, and S(O)nR10;
each n is independently 0, 1 or 2;
and each R10 is independently hydrogen, hydroxy, C1-4 alkyl, C1-4 haloalkyl, 4-halophenyl, amino, C1-6 alkyl amino and di C1-6 alkyl amino;
or a pharmaceutically acceptable salt or a prodrug thereof.
In particular, there is provided a compound of formula (I):
wherein:
R1, R2, R3, R4, R5 are independently selected from the group consisting of hydrogen, cyano, nitro, halo, hydroxy, C1-4 alkyl, C3-6 cycloalkyl, C1-4 alkoxy, C1-4 haloalkyl, C1-4 haloalkoxy, amino, NRxRy, and S(O)nR1;
R6 is selected from the group consisting of hydrogen, —C0-2alkyleneR7, —C1-2alkyleneOR7, —C0-2alkyleneC(O)R7, —C1-2alkyleneOC(O)R7, —C1-2alkyleneOC(O)OR7, —C0-2alkyleneC(O)OR7, —C1-2alkyleneN(H)C(O)R7, —C1-2alkyleneN(R7)C(O)R7, —C0-2alkyleneC(O)NHR7, —C0-2alkyleneC(O)NR15R16, —C1-2alkyleneNHC(O)NR15R16, —C1-2alkyleneNR7C(O)NR15R16, —C1-2alkyleneOC(O)NHR7, —C1-2alkyleneOC(O)NR15R16, —C0-2alkyleneCH═N(R7), —C1-2alkyleneP(═O)(NR15R16)(NR15R16), —C0-2alkyleneSi(R7)3, and —C0-2alkyleneS(O)nR10;
where the C0-2alkylene or C1-2alkylene of R6 may, where chemically possible, optionally be substituted by one or more substituents selected from the group consisting of C1-6 alkyl, C3-6 cycloalkyl, C1-4 alkylene(C3-6 cycloalkyl), C0-6 alkylenephenyl, which C0-2alkylene or C1-2alkylene substituent may in turn be optionally further substituted, where chemically possible, by one or more substituents selected from the group consisting of hydrogen, cyano, nitro, halo, formyl, oxo, hydroxy, C(O)OH, C1-4 alkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, C1-4 alkyleneC1-4 alkyoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, C1-4 haloalkoxy, amino, C1-4 alkylamino, C1-4 dialkylamino, and S(O)nR10;
where each R7, R15 and R16, where chemically possible, is independently selected from the group consisting of hydrogen, C1-8 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C1-4 alkylene(C3-6 cycloalkyl), C1-4 alkyleneC1-4 alkoxy, C1-6 haloalkyl, C0-6 alkylenephenyl;
or R15 and R15 together with the nitrogen to which they are attached may form a three to seven-membered saturated or unsaturated heterocyclic ring optionally containing one or more further N, O or S atoms;
where each of the above R7, R15 or R16 groups may independently include one or more optional substituents where chemically possible selected from hydrogen, cyano, nitro, halo, formyl, oxo, hydroxy, C(O)OH, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, C1-4 alkyleneC1-4 alkyoxy, C1-4 alkanoyl, —C(O)OC1-4 alkyl, C1-4 haloalkyl, C3-6 halocycloalkyl, C1-4 haloalkoxy, C1-4 haloalkanoyl, —C(O)OC1-4 haloalkyl, phenyl, 4-halophenyl, 4-alkoxyphenyl, amino, C1-4 alkylamino, C1-4 dialkylamino, C(O)N(C1-4 alkyl)2, N(C1-4 alkylene)C(O)(C1-4 alkyl) and S(O)nR10;
R8 and R9 are independently selected from the group consisting of hydrogen, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, C1-4 haloalkoxy and C0-4 alkylenephenyl but with the proviso that R8 and R9 are not both hydrogen;
where each of R8 and R9 may independently include one or more optional substituents where chemically possible selected from hydrogen, cyano, halo, hydroxy, C1-4 alkyl, C3-6 cycloalkyl, C1-4 alkoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, C1-4 haloalkoxy, and S(O)nR10;
or R8 and R9 together with the carbon to which they are attached may form a three to six membered carbocyclic, saturated ring, which ring is optionally substituted with one or more substituents selected from the group consisting of halo, C1-2 alkyl, C1-2 alkoxy, C1-2 haloalkyl, C1-2 haloalkoxy;
R11 and R12 are independently selected from the group consisting of hydrogen, halo, cyano, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, and C1-4 haloalkoxy;
where Rx and Ry are independently selected from hydrogen, C1-4 alkyl, C1-4 haloalkyl, and S(O)nR10;
each n is independently 0, 1 or 2;
and each R10 is independently hydrogen, hydroxy, C1-4 alkyl, C1-4 haloalkyl, amino, C1-6 alkyl amino and di C1-6 alkyl amino;
or a pharmaceutically acceptable salt or a prodrug thereof.
In the definition of R1, R2, R3, R4 and R5, “C1-4 alkyl optionally substituted by one or more hydroxy groups” means an alkyl group with between one and four carbon atoms, which may be unsubstituted or may be substituted at any available position with a hydroxy group. For reasons of chemical stability, it is preferred that no carbon atom should be substituted with more than one hydroxy group. Accordingly, alkyl groups with up to four hydroxy substituents are foreseen. Preferred are alkyl groups with no more than two hydroxy substituents. Examples include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl and 2,3-dihydroxypropyl.
In the definition of R1, R2, R3, R4 and R5, “C3-6 cycloalkyl optionally substituted by one or more C1-4 alkyl or halo groups” means a cycloalkyl group with between three and six carbon atoms in the ring, which may be unsubstituted or may be substituted at any available position with an alkyl group of between one and four carbon atoms or a halogen atom. In the case of alkyl substituents, it is preferred that not more than four such substituents be present, and more preferred that not more than two such substituents be present. Examples include 1-methylcyclopropyl, 2,5-dimethylcyclopentyl and 4-tert-butylcyclohexyl. In the case of halo substituents, any degree of substitution up to complete substitution is foreseen. In the case of cyclohexyl therefore, up to eleven halo substituents may be present. While each halo group may be independently selected, it may be preferred to have all halo substituents the same. Preferably the halo is chloro or fluoro. Geminal disubstitution at any methylene position may be preferred ver monosubstitution. Examples include 2,2-dichlorocyclopropyl and perfluorocyclohexyl. Substitution with both alkyl and halo groups is also foreseen. An example is 2,2-difluoro-1-methylcyclobutyl.
Preferably, each of R1, R2, R3, R4, R5 are independently selected from hydrogen, halo eg chloro or fluoro, C1-4 alkyl eg methyl or ethyl, C3-4 cycloalkyl eg cyclopropyl, C1-4 alkoxy eg methoxy or ethoxy, C1-4 haloalkyl eg trifluoromethyl, trifluoroethyl, C1-4 haloalkoxy eg trifluoromethoxy or trifluoroethoxy, and S(O)nR10 where n is 0 and R10 is preferably selected from C1-4 alkyl such as methyl or ethyl or C1-4 haloalkyl such as trifluoromethyl or trifluoroethyl to form for example trifluoromethylthio or trifluoroethylthio. More preferably each of R1, R2, R3, R4, R5 are independently selected from hydrogen, halo eg chloro, C1-4 alkyl eg methyl or ethyl, C1-4 alkoxy eg methoxy or ethoxy, and C1-4 haloalkyl eg trifluoromethyl, trifluoroethyl. Most preferably each of R1, R2, R3, R4, R5 are independently selected from hydrogen, and C1-4 alkyl eg methyl or ethyl.
Most preferably two of R1, R2, R3, R4, and R5 are independently selected from C1-4 alkyl eg methyl or ethyl, preferably methyl, and three of R1, R2, R3, R4, and R5 are H. Even more preferably R1 and R2 are selected from C1-4 alkyl eg methyl or ethyl, preferably methyl, and R3, R4 and R5 are H.
Further suitable compounds include those where at least one of R1, R2, R3, R4, and R5 is independently selected from C1-4 haloalkyl eg trifluoromethyl, trifluoroethyl, preferably trifluoromethyl, with the others of R1, R2, R3, R4, and R5 being H. Preferably R2 is C1-4 haloalkyl eg trifluoromethyl, trifluoroethyl preferably trifluoroethyl, with the others of R1, R3, R4, and R5 being H.
Other suitable compounds include those where at least one of R1, R2, R3, R4, and R5 is independently selected from C1-4 alkoxy eg methoxy or ethoxy preferably methoxy, with the others of R1, R2, R3, R4, and R5 being H. Preferably R2 and R3 are selected from C1-4 alkoxy eg methoxy or ethoxy preferably methoxy, and R1, R4 and R5 are H.
Other suitable compounds include those where at least one of R1, R2, R3, R4, and R5 is independently selected from halo eg chloro or fluoro, with the others of R1, R2, R3, R4, and R5 being H.
Other suitable compounds include those where at least one of R1, R2, R3, R4, and R5 is independently selected from halo eg chloro or fluoro, and another one of R1, R2, R3, R4, and R5 is independently selected from C1-4 alkyl eg methyl or ethyl, with the others of R1, R2, R3, R4, and R5 being H.
Most preferred compounds are those where R1 and R2 are methyl and R3, R4, and R5 are hydrogen.
Preferably R6 is selected from the group consisting of hydrogen; —C0-2alkyleneR7; —C1-2alkyleneOR7; —C1-2alkyleneOC(O)R7; —C1-2alkyleneOC(O)OR7; —C0-2alkyleneC(O)OR7; —C1-2alkyleneOC(O)NHR7; —C1-2alkyleneOC(O)NR15R16; and —C0-2alkyleneS(O)nR10. More preferably R6 is selected from the group consisting of hydrogen; —C0-2alkyleneR7; —C1-2alkyleneOR7; —C1-2alkyleneOC(O)R7; —C1-2alkyleneOC(O)OR7; and —C0-2alkyleneC(O)OR7. Even more preferably R5 is selected from the group consisting of hydrogen; —C0-2alkyleneR7; —C1-2alkyleneOC(O)R7 and —C0-2alkyleneC(O)OR7. Most preferably R6 is hydrogen.
Preferably R7, R15 and R16 are, where chemically possible, independently selected from the group consisting of hydrogen; C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl; C3-8 cycloalkyl for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; C1-4 alkylene(C3-6 cycloalkyl) for example cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl; C1-6 haloalkyl for example fluoromethyl, trifluoromethyl, chloromethyl, fluoroethyl, chloroethyl, trifluoroethyl and trifluoropropyl; and C0-6 alkylphenyl for example phenyl, phenylmethyl and phenylethyl. More preferably R7, R15 and R16 are, where chemically possible, independently selected from the group consisting of hydrogen; C1-6 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl, n-pentyl, n-hexyl; C1-4 alkylene(C3-6 cycloalkyl) for example cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl. Even more preferably R7, R15 and R16 are, where chemically possible, independently selected from the group consisting of hydrogen and C1-4 alkyl for example methyl, ethyl, propyl, isopropyl, n-butyl and tert-butyl.
Further suitable compounds include those where R7, R15 and R16 are, where chemically possible, optionally substituted with one or more substituents selected from the group consisting of halo for example fluoro or chloro, C1-4 alkyl for example methyl or ethyl preferably methyl, C3-6 cycloalkyl, for example cyclopropyl, cyclobutyl or cyclopentyl preferably cyclopentyl, C1-4 alkoxy for example methoxy or ethoxy, C1-4 haloalkyl for example fluoromethyl, chloromethyl, trifluoromethyl, fluoroethyl, chloroethyl or trifluoroethyl, preferably trifluoroethyl or trifluoromethyl, and S(O)nR10 for example methylsulphonyl or dimethyl amido sulphonyl. Examples of R7, R15 and R16 groups which have then been so substituted include for example branched alkyl groups such as 2-methylbutyl, 3-methylbutyl, substituted sulphonyl groups such as methylsulphonylmethyl, methylsulphonylethyl, dimethylamidosulphonylmethyl and dimethylamidosulphonylethyl and substituted phenyl groups such as 4-chlorophenyl, 4-nitrophenyl, 4-fluorophenyl, 4-methoxyphenyl, 2,4-dichlorophenyl, 4-chlorophenylmethyl, 4-nitrophenylmethyl, 4-fluorophenylmethyl, 4-methoxyphenyl methyl, 2,4-dichlorophenylmethyl, 4-chlorophenylethyl, 4-nitro phenyl ethyl, 4-fluorophenylethyl, 4-methoxyphenylethyl, and 2,4-dichlorophenylethyl.
Suitably when R15 and R16 together with the nitrogen to which they are attached form a three to seven-membered saturated or unsaturated heterocyclic ring optionally containing one or more further N, O or S atoms it is preferred that the ring is a five or six membered ring, is saturated and comprises one further heteroatom selected from N, O or S. Suitable examples of such rings include pyrrolidinyl, pyrazolidinyl, imidazolinyl, thiazolidinyl, isoxazolidinyl, piperidinyl, piperazinyl, morpholinyl, or thiomorpholinyl. Preferred rings include pyrrolidinyl, thiazolidinyl, morpholinyl, or thiomorpholinyl. Such rings may optionally be further substituted with one or more groups, preferably selected from the group consisting of oxo, C(O)OH, halo for example fluoro or chloro, and C1-4 alkyl for example methyl or ethyl preferably methyl. For example any heterocyclic sulphur atoms may be optionally substituted with one or more oxo groups to form for example 1,1-dioxothiazolidinyl or 1,1-dioxothiomorpholinyl substitutents.
Suitable compounds include those where, when the R6 group comprises a one carbon alkylene moiety, that said alkylene moiety is optionally substituted with one or two substituents. Further suitable compounds also include those where, when the R6 group comprises a two carbon alkylene moiety, that said alkylene moiety is optionally substituted with one, two, three or four substituents which may be independently orientated on either the alpha or beta carbon positions with respect to the imidazole nitrogen to which the R6 substitutent is bound.
Suitably when the C0-2alkylene or C1-2alkylene of R6 is substituted with one or more substitutents it is preferred that such substituents are independently selected from the group consisting of hydrogen; C1-4 alkyl for example methyl or ethyl; C3-6 cycloalkyl for example cyclopropyl; C1-4 alkyleneC3-6 cycloalkyl for example cyclopropylmethyl or cyclopropylethyl; C1-4 alkoxy for example methoxy or ethoxy; C1-4 alkyleneC1-4 alkyoxy for example methoxy methyl, methoxy ethyl, ethoxy methyl or ethoxy ethyl; C1-4 haloalkyl for example fluoromethyl, trifluromethyl, fluoroethyl or 1,1,1-trifluoroethyl; phenyl, benzyl and 4-trifluoromethylbenzyl. More preferably such substituents are independently chosen from the group consisting of hydrogen; C1-4 alkyl for example methyl or ethyl; C3-6 cycloalkyl for example cyclopropyl; C1-4 alkyleneC3-6 cycloalkyl for example cyclopropylmethyl or cyclopropylethyl; C1-4 haloalkyl for example fluoromethyl, trifluromethyl, fluoroethyl or 1,1,1-trifluoroethyl; and phenyl.
Suitable compounds include those where R6 is selected from the group consisting of —C0-2alkyleneR7, preferably where R6 is CH2R7, and where R7 is selected from the group consisting of C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl; C3-8 cycloalkyl for example cyclopropyl, cyclobutyl, cyclopentyl; C1-6 haloalkyl for example trifluoromethyl, and trifluoroethyl; and C0-6 alkylenephenyl for example phenyl which is optionally substituted to form for example 4-methoxy phenyl, 4-trifluoromethylphenyl. Further suitable compounds also include those where R6 is selected from the group consisting of —C0-2alkyleneR7, preferably where R6 does not comprise an additional alkylene moiety (ie is CoalkyleneR7)7, and where R7 is selected from the group consisting of C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl, preferably methyl and ethyl; C3-8 cycloalkyl for example cyclopropyl, cyclobutyl, cyclopentyl, preferably cyclopropyl; C1-6 haloalkyl for example trifluoromethyl, and trifluoroethyl; and C0-6 alkylenephenyl for example phenyl which is optionally substituted to form for example 4-methoxy phenyl, 4-trifluoromethylphenyl.
A further group of suitable compounds include those where R6 is selected from the group consisting of —C1-2alkyleneOR7, preferably where R6 is CH2OR7, and where R7 is selected from the group consisting of C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl. Examples of such so substituted R6 groups include methoxymethyl, ethoxymethyl, methoxyethyl, ethoxyethyl, propoxymethyl, propoxyethyl, isopropoxyethyl, butoxymethyl, sec-butoxyoxymethyl, isobutoxymethyl, tert-butoxymethyl, butoxyethyl, sec-butoxyoxyethyl, isobutoxyethyl, tert-butoxyethyl, pentyloxymethyl, pentyloxyethyl, hexyloxymethyl, hexyloxyethyl.
A still further group of suitable compounds include those where R6 is selected from the group consisting of —C1-2alkyleneOC(O)R7, preferably where R6 is CH2OC(O)R7, and where R7 is C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl, which R7 in turn may be optionally further substituted. Examples of such so substituted R6 groups include acetyloxymethyl, acetyloxyethyl, propionyloxymethyl, propionyloxyethyl, butyryloxymethyl, butyryloxyethyl, isobutyryloxymethyl, isobutyryloxyethyl, pentanoyloxymethyl, pentanoyloxyethyl, 2-methylbutyryloxymethyl, 2-methylbutyryloxyethyl, 3-methylbutyryloxymethyl, 3-methylbutyrylcarbonyloxy)ethyl, 2,2-dimethylpropionyloxymethyl, 2,2-dimethylpropionyloxyethyl hexanoyloxymethyl, hexanoyloxyethyl, heptanoyloxymethyl, heptanoyloxyethyl. Further suitable examples of compounds where R6 is selected from the group consisting of —C1-2alkyleneOC(O)R7, preferably where R6 is CH2OC(O)R7, also include those where R7 is C1-4 alkylene(C3-6 cycloalkyl) for example cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl, and cyclcohexylethyl. Examples of such so substituted R6 groups include cyclopropylacetyloxymethyl, cyclopropylpropionyloxymethyl, cyclobutylacetyloxymethyl, cyclobutylpropionyloxymethyl, cyclopentylacetyloxymethyl, cyclopentylpropionyloxymethyl, cyclopentylbutyryloxymethyl, cyclohexylacetyloxymethyl, and cyclcohexylpropionyloxymethyl, cyclopropylacetyloxyethyl, cyclopropylpropionyloxyethyl, cyclobutylacetyloxyethyl, cyclobutylpropionyloxyethyl, cyclopentylacetyloxyethyl, cyclopentylpropionyloxyethyl, cyclopentylbutyryloxyethyl, cyclohexylacetyloxyethyl, and cyclcohexylpropionyloxyethyl. Preferably R6 is 3-cyclopentylpropionyloxymethyl. It is preferred that in such compounds R7 is preferably C1-8 alkyl, more preferably ethyl or tert-butyl, and most preferably tert-butyl.
A yet further group of suitable compounds include those where R6 is selected from the group consisting of —C1-2alkyleneOC(O)OR7, preferably where R6 is CH2OC(O)OR7, and where R7 is C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl, which may in turn be optionally further substituted. Examples of such so substituted R6 groups include methoxycarbonyloxymethyl, methoxycarbonyloxyethyl, ethoxycarbonyloxymethyl, ethoxycarbonyloxyethyl, propoxycarbonyloxymethyl, propoxycarbonyloxyethyl, isopropoxycarbonyloxymethyl, isopropoxycarbonyloxyethyl, butoxycarbonyloxymethyl, butoxycarbonyloxyethyl, isobutoxycarbonyloxymethyl, isobutoxycarbonyloxyethyl, pentyloxycarbonyloxymethyl, pentyloxycarbonyloxyethyl, 2-methylbutoxycarbonyloxymethyl, 2-methylbutoxycarbonyloxyethyl, 3-methylbutoxycarbonyloxymethyl, 3-methylbutoxycarbonyloxyethyl, 2,2-dimethylpropoxycarbonyloxymethyl, 2,2-dimethylpropoxycarbonyloxyethyl, hexyloxycarbonyloxymethyl, hexyloxycarbonyloxyethyl. Further suitable examples of compounds where R6 is selected from the group consisting of —C1-2alkyleneOC(O)OR7, preferably where R6 is CH2OC(O)OR7, also include those where R7 is selected from the group consisting of C3-6 cycloalkyl for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; C1-4 alkylene(C3-6 cycloalkyl) for example cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl; C1-6 haloalkyl for example trifluoromethyl, and 2,2,2-trifluoroethyl; and C0-6 alkylphenyl for example phenyl which is optionally further substituted to form for example 4-methoxyphenyl, 4-trifluoromethylphenyl-4-methoxybenzyl. Examples of such so substituted R6 groups include cyclopropyloxycarbonyloxymethyl, cyclobutyloxycarbonyloxymethyl, cyclopentyloxycarbonyloxymethyl or cyclohexyloxycarbonyloxymethyl cyclopropyloxycarbonyloxyethyl, cyclobutyloxycarbonyloxyethyl, cyclopentyloxycarbonyloxyethyl or cyclohexyloxycarbonyloxyethyl; C1-4 alkylene(C3-6 cycloalkyl) for example cyclopropylmethyloxycarbonyloxymethyl, cyclopropylethyloxycarbonyloxymethyl, cyclobutylmethyloxycarbonyloxymethyl, cyclobutylethyloxycarbonyloxymethyl, cyclopentylmethyloxycarbonyloxymethyl, cyclopentylethyloxycarbonyloxymethyl, cyclohexylmethyloxycarbonyloxymethyl, cyclohexylethyloxycarbonyloxymethyl, cyclopropylmethyloxycarbonyloxyethyl, cyclopropylethyloxycarbonyloxyethyl, cyclobutylmethyloxycarbonyloxyethyl, cyclobutylethyloxycarbonyloxyethyl, cyclopentylmethyloxycarbonyloxyethyl, cyclopentylethyloxycarbonyloxyethyl, cyclohexylmethyloxycarbonyloxyethyl, cyclohexylethyloxycarbonyloxyethyl; C1-6 haloalkyl for example trifluoromethyloxycarbonyloxymethyl, and 2,2,2-trifluoroethyloxycarbonyloxymethyl, trifluoromethyloxycarbonyloxyethyl, and 2,2,2-trifluoroethyloxycarbonyloxyethyl; and C0-6 alkylphenyl for example phenyloxycarbonyloxymethyl which is optionally further substituted to form for example 4-methoxyphenyloxycarbonyloxymethyl, 4-trifluoromethylphenyloxycarbonyloxymethyl, 4-methoxybenzyloxycarbonyloxymethyl.
A still yet further group of suitable compounds include those where R6 is selected from the group consisting of —C0-2alkyleneC(O)OR7, preferably where R6 is C(O)OR7, and where R7 is C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl, which may in turn be optionally further substituted. Examples of such so substituted R6 groups include methoxycarbonyl, methoxycarbonylmethyl, methoxycarbonylethyl, ethoxycarbonyl, ethoxycarbonylmethyl, ethoxycarbonylethyl, propoxycarbonyl, propoxycarbonylmethyl, propoxycarbonylethyl, isopropoxycarbonyl, isopropoxycarbonylmethyl, isopropoxycarbonylethyl, butoxycarbonyl, butoxycarbonylmethyl, butoxycarbonylethyl, isobutoxycarbonyl, isobutoxycarbonylmethyl, isobutoxycarbonylethyl, pentyloxycarbonyl, pentyloxycarbonylmethyl, pentyloxycarbonylethyl, 2-methylbutoxycarbonyl, 2-methylbutoxycarbonylmethyl, 2-methylbutoxycarbonylethyl, 3-methylbutoxycarbonyl, 3-methylbutoxycarbonylmethyl, 3-methylbutoxycarbonylethyl, 2,2-dimethylpropoxycarbonyl, 2,2-dimethylpropoxycarbonylmethyl, 2,2-dimethylpropoxycarbonylethyl, hexyloxycarbonyl, hexyloxycarbonylmethyl, hexyloxycarbonylethyl. Further suitable examples of compounds include those where R6 is selected from the group consisting of —C0-2alkyleneC(O)OR7, preferably where R6 is C(O)OR7, also include those where R7 is selected from the group consisting of C0-6 alkylphenyl for example phenyl which in turn is optionally substituted to form for example 4-methoxy phenyl, 4-trifluoromethyl phenyl. Examples of such so substituted R6 groups include phenyloxycarbonyl, phenyloxycarbonylmethyl, phenyloxycarbonylethyl.
An even further group of suitable compounds include those where R6 is selected from the group consisting of —C1-2alkyleneOC(O)NHR7, preferably where R6 is CH2OC(O)NHR7, and where R7 is selected from the group consisting of C1-8 alkyl for example methyl, ethyl, n-propyl, isopropyl, butyl, tert-butyl; C3-6 cycloalkyl for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; C1-6 haloalkyl for example trifluoromethyl, and trifluoroethyl; and C0-6 alkylphenyl for example phenyl, phenylmethyl or phenylethyl which C0-6 alkylphenyl is optionally substituted to form for example 4-methoxyphenyl, 4-trifluoromethylphenyl, 2,4-dichlorophenyl, 4-methoxyphenylmethyl, 4-trifluoromethylphenylmethyl, 2,4-dichlorophenylmethyl, 4-methoxyphenylethyl, 4-trifluoromethylphenylethyl, or 2,4-dichlorophenylethyl.
Preferred are those compounds where R6 is selected from the group consisting of hydrogen, —C0-2alkyleneR7 and —C1-2alkyleneOC(O)R7 and where R7 is selected from the group consisting of C1-8 alkyl. Even more preferred compounds are those where R6 is hydrogen.
Preferably, each R8 and R9 are independently selected from the group consisting of hydrogen; C1-4 alkyl eg methyl or ethyl, preferably methyl; C1-4 haloalkyl for example trifluoromethyl, trichloromethyl, trichloroethyl or trifluoroethyl, preferably trifluoromethyl; C1-4 alkoxy for example methoxy or ethoxy, preferably methoxy; and C0-4 alkylenephenyl for example phenyl, phenylmethyl or phenylethyl, but with the proviso that R8 and R9 are not both hydrogen. More preferably each R8 and R9 are independently selected from the group consisting of hydrogen and C1-4 alkyl eg methyl or ethyl, preferably methyl but again with the proviso that R8 and R9 are not both hydrogen. Most preferably R8 is methyl and R9 is hydrogen.
Suitably when either one or more of R8 or R9 are phenyl, the phenyl group is optionally substituted with one or more substitutents selected from the group consisting of fluoro, chloro, methoxy or trifluoromethyl.
Suitably when R8 and R9 together with the carbon to which they are attached may form a three to six membered carbocyclic, saturated ring it is preferred that the ring is a three membered ring.
Preferably each of R11 and R12 are independently selected from the group consisting of hydrogen, C1-2 alkyl eg methyl or ethyl, preferably methyl, and C1-2 alkoxy for example methoxy or ethoxy, preferably methoxy. More preferably at least one of R11 and R12 is hydrogen. Most preferably both of R11 and R12 are hydrogen.
A further group of suitable compounds of the present invention are those of formula (LV) where: each of R1, R2, R3, R4, R5 are independently selected from hydrogen and C1-4 alkyl eg methyl or ethyl, preferably methyl;
each R8 and R9 are independently selected from the group consisting of hydrogen and C1-4 alkyl eg methyl or ethyl, preferably methyl; and
each R11 and R12 are hydrogen or a pharmaceutically acceptable salt or a prodrug thereof. Preferably, in compounds of formula (LV): R1, R2 and R8 are selected from C1-4 alkyl eg methyl or ethyl, preferably methyl, R3, R4, R5 and R9 are H.
It will be understood that throughout the application all references to formula (I) apply equally to compounds of the formula (LV).
Furthermore, it will be understood that all the suitable groups and preferences applied to R1-R12, Ra, Rb and n for formula (I) apply equally to compounds of the formula (LV).
A further group of preferred compounds are the compounds of formula (XXXX)
wherein R1 to R5 are selected from hydrogen, halo, C1-4 alkyl, C1-4 haloalkyl and CN, and R8 is C1-3 alkyl. Preferably, at least two of R1 to R5 are hydrogen, and more preferably at least three of R1 to R5 are hydrogen. Preferably, the groups from R1 to R5 that are not hydrogen are selected from chloro, fluoro, methyl, ethyl, difluoromethyl and trifluoromethyl, and more preferably from fluoro, chloro and methyl. Preferably R8 is methyl or ethyl, and more preferably R8 is methyl.
A further group of preferred compounds are the compounds of formula (XXXXI)
wherein R1 to R5 are selected from hydrogen, halo, C1-4 alkyl, C1-4 haloalkyl and CN, R7 is phenyl optionally substituted by one or more groups selected from cyano, nitro, halo, formyl, hydroxy, C(O)OH, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, C1-4 haloalkoxy, pyrazolyl, triazolyl, amino, C1-4 alkylamino, and C1-4 dialkylamino, and R8 is C1-3 alkyl. Preferably, at least two of R1 to R5 are hydrogen, and more preferably at least three of R1 to R5 are hydrogen. Preferably, the groups from R1 to R5 that are not hydrogen are selected from chloro, fluoro, methyl, ethyl, difluoromethyl and trifluoromethyl, and more preferably from fluoro, chloro and methyl. Preferably R7 is phenyl optionally substituted by one or two groups selected from cyano, chloro, fluoro, hydroxy, C1-3 alkyl, C1-3 alkoxy and C1-2 haloalkyl. Preferably R8 is methyl or ethyl, and more preferably R8 is methyl.
A further group of preferred compounds are the compounds of formula (XXXXII)
wherein R1 to R5 are selected from hydrogen, halo, C1-4 alkyl, C1-4 haloalkyl and CN, R7 is selected from C1-3alkylenephenyl optionally substituted by on the phenyl ring by one or more groups selected from cyano, halo, hydroxy, C(O)OH, C1-4 alkyl, C3-6 cycloalkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, and C1-4 haloalkoxy, C1-8 alkyl optionally substituted by one or two C1-4 alkoxy groups, C3-6 cycloalkyl, C1-3alkyleneC3-6cycloalkyl, and C1-6 haloalkyl, and R3 is C1-3 alkyl. Preferably, at least two of R1 to R5 are hydrogen, and more preferably at least three of R1 to R5 are hydrogen. Preferably, the groups from R1 to R5 that are not hydrogen are selected from chloro, fluoro, methyl, ethyl, difluoromethyl and trifluoromethyl, and more preferably from fluoro, chloro and methyl. Preferably R7 is C1-8alkyl or C1-6haloalkyl. Preferably R8 is methyl or ethyl, and more preferably R8 is methyl.
A further group of preferred compounds are the compounds of formula (XXXXIII)
wherein R1 to R5 are selected from hydrogen, halo, C1-4 alkyl, C1-4 haloalkyl and CN, R7 is selected from C1-3alkylenephenyl optionally substituted by on the phenyl ring by one or more groups selected from cyano, halo, hydroxy, C(O)OH, C1-4 alkyl, C3-6 cycloalkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, and C1-4 haloalkoxy, C1-8 alkyl optionally substituted by one or two C1-4 alkoxy groups, C3-6 cycloalkyl, C1-3alkyleneC3-6cycloalkyl, and C1-6 haloalkyl, and R8 is C1-3 alkyl.
Preferably, at least two of R1 to R5 are hydrogen, and more preferably at least three of R1 to R5 are hydrogen. Preferably, the groups from R1 to R5 that are not hydrogen are selected from chloro, fluoro, methyl, ethyl, difluoromethyl and trifluoromethyl, and more preferably from fluoro, chloro and methyl. Preferably R7 is C1-8alkyl or C1-6haloalkyl. Preferably R8 is methyl or ethyl, and more preferably R8 is methyl.
A further group of preferred compounds are the compounds of formula (XXXXIV)
wherein R1 to R5 are selected from hydrogen, halo, C1-4 alkyl, C1-4 haloalkyl and CN, R7 is selected from C1-3alkylenephenyl optionally substituted by on the phenyl ring by one or more groups selected from cyano, halo, hydroxy, C(O)OH, C1-4 alkyl, C3-6 cycloalkyl, C1-4 alkyleneC3-6 cycloalkyl, C1-4 alkoxy, —C(O)OC1-4 alkyl, C1-4 haloalkyl, and C1-4 haloalkoxy, C1-8 alkyl optionally substituted by one or two C1-4 alkoxy groups, C3-6 cycloalkyl, C1-3alkyleneC3-6cycloalkyl, and C1-6 haloalkyl, and R8 is C1-3 alkyl. Preferably, at least two of R1 to R5 are hydrogen, and more preferably at least three of R1 to R5 are hydrogen. Preferably, the groups from R1 to R5 that are not hydrogen are selected from chloro, fluoro, methyl, ethyl, difluoromethyl and trifluoromethyl, and more preferably from fluoro, chloro and methyl. Preferably R7 is C1-8alkyl or C1-6haloalkyl, and more preferably R7 is isobutyl. Preferably R8 is methyl or ethyl, and more preferably R8 is methyl.
Preferred individual compounds of the invention are selected from the compounds of the Examples described herein.
More preferred individual compounds of the invention are selected from:
2-[(2,3-dimethylphenyl)(methoxy)methyl]-1H-imidazole;
2-[1-(2,5-dimethylphenyl)ethyl]-1H-imidazole;
2-[1-(2,4-dimethylphenyl)ethyl]-1H-imidazole;
2-[1-(3,4-dimethylphenyl)ethyl]-1H-imidazole;
2-{1-[2-(trifluoromethyl)phenyl]ethyl}-1H-imidazole;
(2,3-dimethylphenyl)(1H-imidazol-2-yl)methanol;
2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl propionate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-methylbutanoate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl butyrate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-cyclopentylpropanoate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl heptanoate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pentanoate
2-[1-(4-chloro-3-methylphenyl)ethyl]-1H-imidazole
2-[1-(3,5-dimethylphenyl)ethyl]-1H-imidazole
1-benzyl-2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 4-methoxybenzyl carbonate
1-(cyclopropylmethyl)-2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-methyl-1H-imidazole
cyclopropylmethyl {2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl carbonate
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-methylbutyl carbonate
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl isopropyl carbonate
cyclobutyl {2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl carbonate
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 2,2,2-trifluoroethyl carbonate
2-[1-(2,3-dimethylphenyl)ethyl]-1-ethyl-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-(4-methoxybenzyl)-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-(methoxymethyl)-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-[4-(trifluoromethyl)benzyl]-1H-imidazole
4-fluorophenyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
isobutyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
isopropyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
2-[1-(3-methylphenyl)ethyl]-1H-imidazole
or a pharmaceutically acceptable salt or prodrug thereof.
More preferred individual compounds of the present invention are selected from:
2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole;
2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole;
2-[(1R)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate;
{2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methylpivalate;
{2-[(1R)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methylpivalate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl propionate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-methylbutanoate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl butyrate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-cyclopentylpropanoate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl heptanoate;
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pentanoate
2-{1-[2-(trifluoromethyl)phenyl]ethyl}-1H-imidazole;
2-[1-(2,5-dimethylphenyl)ethyl]-1H-imidazole
2-[1-(4-chloro-3-methylphenyl)ethyl]-1H-imidazole
2-[1-(3,5-dimethylphenyl)ethyl]-1H-imidazole
1-benzyl-2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 4-methoxybenzyl carbonate
1-(cyclopropylmethyl)-2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-methyl-1H-imidazole
cyclopropylmethyl {2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl carbonate
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-methylbutyl carbonate
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl isopropyl carbonate
cyclobutyl {2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl carbonate
{2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 2,2,2-trifluoroethyl carbonate
2-[1-(2,3-dimethylphenyl)ethyl]-1-ethyl-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-(4-methoxybenzyl)-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-(methoxymethyl)-1H-imidazole
2-[1-(2,3-dimethylphenyl)ethyl]-1-[4-(trifluoromethyl)benzyl]-1H-imidazole
4-fluorophenyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
isobutyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
isopropyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
2-[1-(3-methylphenyl)ethyl]-1H-imidazole
or a pharmaceutically acceptable salt or prodrug thereof.
Even more preferred compounds of the present invention are 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole, and {2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate, or a pharmaceutically acceptable salt or prodrug thereof.
The most preferred compound of the present invention is 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole, or a pharmaceutically acceptable salt or prodrug thereof.
Included within the scope of the present invention are all stereoisomers such as enantiomers and diasteromers, all geometric isomers and tautomeric forms of the compounds of formula (I), including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counterion is optically active, for example, D-lactate or L-lysine, or racemic, for example, DL-tartrate or DL-arginine.
It is to be understood that compounds of formula (I) may contain one or more asymmetric carbon atoms, thus compounds of the invention can exist as two or more stereoisomers. In particular it will be understood that when R8 and R9 are different substitutents a stereocentre exists at the carbon atom to which they are attached—the benzylic carbon. Suitable compounds for use in this invention include those where the absolute stereochemistry at the benzylic carbon has the “S configuration”. Further suitable compounds for use in this invention include those where the absolute stereochemistry at the benzylic carbon has the “R configuration”. Such stereoisomers can be resolved and identified by one skilled in the art using known techniques.
The present invention includes the individual stereoisomers of the compounds of formula (I) together with mixtures thereof. Preferred compounds of formula (I) include those of formula (IA) and formula (IB) which possess the stereochemistry shown below.
It will be understood that throughout the application all references to formula (I) apply equally to compounds of the formulae (IA) and (IB).
Furthermore, it will be understood that all the suitable groups and preferences applied to R1-R12, Ra, Rb and n for formula (I) apply equally to compounds of the formulae (IA) and (IB).
In one particular embodiment of the invention preferred compounds are those of the formula (IA).
In one particular embodiment of the invention preferred compounds are those of the formula (IB).
Preferred compounds of the present invention include 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole, 2-[(1R)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole, {2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methylpivalate, {2-[(1R)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methylpivalate or a pharmaceutically acceptable salt or prodrug thereof.
Even more preferred compounds of the present invention are 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole, 2-[(1R)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole, or a pharmaceutically acceptable salt or prodrug thereof with the formulae shown below.
Most preferred is 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole.
Geometric isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor, stereoselective synthesis from a prochiral precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, fractional crystallization or chiral high pressure liquid chromatography (HPLC). Reference is made herein to “Enantiomers, Racemates and Resolutions” J. Jacques and A. Collet, published by Wiley, NY, 1981; and “Handbook of Chiral Chemicals” chapter 8, Eds D. Ager and M. Dekker, ISBN:0-8247-1058-4.
Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, an acid or base such as tartaric acid or 1-phenylethylamine. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% isopropanol, typically from 2 to 20%, and from 0 to 5% of an alkylamine, typically 0.1% diethylamine. Concentration of the eluant affords the enriched mixture.
Stereoisomeric conglomerates may be separated by conventional techniques known to those skilled in the art—see, for example, “Stereochemistry of Organic Compounds” by E L Eliel (Wiley, New York, 1994).
In the compounds according to formula (I) the term ‘halo’ means a group selected from fluoro, chloro, bromo or iodo.
Alkyl, alkylene, alkenyl, alkynyl and alkoxy groups, containing the requisite number of carbon atoms, can be unbranched or branched. Examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl. Examples of alkoxy include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy and t-butoxy. Examples of alkylene include —CH2—, —CH(CH3)— and —C2H4—. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
For the avoidance of doubt, it will be understood that throughout the application all references to pharmaceutically acceptable compounds includes references to veterinarily acceptable compounds or agriculturally acceptable compounds. Furthermore it will be understood that throughout the application all references to pharmaceutical activity includes references to veterinary activity or agricultural activity.
Pharmaceutically acceptable salts of the compounds of formula (I) include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids, which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
The pharmaceutically, veterinarily and agriculturally acceptable acid addition salts of certain of the compounds of formula (I) may also be prepared in a conventional manner. For example, a solution of a free base may be treated with the appropriate acid, either neat or in a suitable solvent, and the resulting salt isolated either by filtration or by evaporation under reduced pressure of the reaction solvent. For a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
The compounds of the invention may exist in both unsolvated and solvated forms. The term ‘solvate’ is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D2O, d6-acetone, d6-DMSO.
Hereinafter and throughout the application all references to compounds of formula (I) include references to salts, solvates and complexes thereof and to solvates and complexes of salts thereof.
As stated, the invention includes all polymorphs of the compounds of formula (I) as hereinbefore defined.
Included within the scope of the invention are complexes such as clathrates, drug-host inclusion complexes wherein, in contrast to the aforementioned solvates, the drug and host are present in stoichiometric or non-stoichiometric amounts. Also included are complexes of the drug containing two or more organic and/or inorganic components which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionised, partially ionised, or non-ionised. For a review of such complexes, see J Pharm Sci, 64 (8), 1269-1288 by Haleblian (August 1975).
The present invention includes all pharmaceutically acceptable isotopically-labelled compounds of formula (I) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H and 3H, carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123I and 125I, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, and sulphur, such as 35S.
Within the scope of the invention are so-called ‘prodrugs’ of the compounds of formula (I). Thus certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body of an animal, be converted by the host or parasite into compounds of formula (I) having the desired activity, for example, by hydrolytic or enzymatic cleavage.
Such derivatives are referred to as ‘prodrugs’. It will be appreciated that certain compounds of formula (I) may themselves act as pro-drugs of other compounds of formula (I). Further information on the use of prodrugs may be found in ‘Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and ‘Bioreversible Carriers in Drug Design’, Pergamon Press, 1987 (ed. E B Roche, American Pharmaceutical Association).
Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as ‘pro-moieties’ as described, for example, in “Design of Prodrugs” by H Bundgaard (Elsevier, 1985).
Some examples of prodrugs in accordance with the invention include:
(i) where the compound of formula (I) contains a carboxylic acid functionality (—COOH), an ester thereof, for example, replacement of the hydrogen with (C1-C8)alkyl;
(ii) where the compound of formula (I) contains an alcohol functionality (—OH), an ether thereof, for example, replacement of the hydrogen with (C1-C6)alkanoyloxymethyl; and
(iii) where the compound of formula (I) contains a primary or secondary amino functionality (—NH2 or —NHR where R≠H), an amide thereof, for example, replacement of one or both hydrogens with (C1-C10)alkanoyl.
Prodrugs in accordance with the invention can, for example, be produced by reacting compounds of formula (I) wherein R6 is H with certain moieties known to those skilled in the art as ‘pro-drug moieties’ as described, for example, in “Design of Prodrugs” by H Bundgaard (Elsevier, 1985); “Design and application of prodrugs,” Textbook of Drug Design and Discovery, (3 Edition), 2002, 410-458, (Taylor and Francis Ltd., London); and references therein. Examples of substituents include: alkyl amines, aryl amines, amides, ureas, carbamates, carbonates, imines, enamines, imides, sulfenamides, and sulfonamides. The hydrocarbon portion of these groups contain C1-6 alkyl, phenyl, heteroaryl such as pyridyl, C2-6 alkenyl, and C3-8 cycloalkyl; wherein each of the above groups may include one or more optional substituents where chemically possible independently selected from: halo; hydroxy; C1-6 alkyl, C1-6 haloalkyl and C1-6 alkoxy.
Further examples of replacement groups in accordance with the foregoing example and examples of other prodrug types may be found in the aforementioned references.
A prodrug that is administered to a test animal and metabolized by the host according to the invention can be readily identified by sampling a body fluid for a compound of formula (I).
Finally, certain compounds of formula (I) may themselves act as prodrugs of other compounds of formula (I).
In a further aspect, the present invention provides processes for the preparation of a compound of formula (I), or a pharmaceutically, veterinarily or agriculturally acceptable salt thereof, or a pharmaceutically, veterinarily or agriculturally acceptable solvate (including hydrate) of either entity, as illustrated below.
The following processes are illustrative of the general synthetic procedures which may be adopted in order to obtain the compounds of the invention.
It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods, for example as described in “Protective Groups in Organic Synthesis” by T W Greene and P G M Wuts, John Wiley & Sons Inc (1999), and references therein. Thus, when one or more of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 R12, R15 and R16 contain reactive functional groups then additional protection may be provided according to standard procedures during the synthesis of compounds of formula (I). In the processes described below, for all synthetic precursors used in the synthesis of compounds of formula (I), the definitions of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 R11 R12, R15 and R16, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 R12, R15 and R16, are as defined for formula (I), are intended to optionally include suitably protected variants, P1, P2, P3, P4, P5, P6, P7, P8, P9, P10 P11 P12, P15 and P16. Such suitable protecting groups for these functionalities are described in the references listed below and the use of these protecting groups where needed is specifically intended to fall within the scope of the processes described in the present invention for producing compounds of formula (I) and its precursors. When suitable protecting groups are used, then these will need to be removed to yield compounds of formula (I). Deprotection can be effected according to standard procedures including those described in the references listed below. When R6 is a protecting group it is preferred that it is chosen from benzyl, p-methoxybenzyl, diethoxymethyl, allyl and trityl.
Compounds of formula (I) may be obtained from other compounds of formula (I) by standard procedures such as electrophilic or nucleophilic substitution, organometallic catalysed cross coupling reactions and functional group interconversions known to those skilled in the art. For example, compounds of formula (I) in which one or more of R1, R2, R3, R4 and R5 are CO2Rc wherein Rc=alkyl, may be transformed into compounds of formula (I) in which one or more of R1, R2, R3, R4 and R5 are CO2Rd wherein Rd=NH2 upon treatment with ammonium hydroxide at 85° C. for 2 h. Similarly compounds of formula (I) wherein one or more of R1, R2, R3, R4 and R5 are CO2R wherein R═NH2 upon treatment with a dehydrating agent such as thionyl chloride at low temperatures in an anhydrous solvent such as N,N-dimethylformamide produce the corresponding nitrile compound.
Compounds of formula (I), wherein R8 is C1-C4 alkyl, R9, R11 and R12 are hydrogen and R6 is hydrogen or alkyl and R1, R2, R3, R4 and R5 are as defined previously
may be synthesised from compounds of formula (II) using standard hydrogenation procedures. For example, compounds of formula (II) wherein Ra is hydrogen, and Rb is hydrogen or alkyl may be reduced to compounds of formula (I) in a suitable protic solvent such as methanol or propan-2-ol under a hydrogen atmosphere at temperatures up to 60° C. and elevated pressure up to 300 psi in the presence of 10% palladium on carbon or Freiborg activated 10% palladium on carbon for up to 72 h.
Compounds of formula (I) in which one or more of R1, R2, R3, R4 and R5 are optionally halo, and the remainder of R1, R2, R3, R4 and R5 are as previously defined, may be accessed from compounds of formula (II) in which one or more of R1, R2, R3, R4 and R5 are optionally halo by hydrogenation procedures. Thus, compounds of formula (II) may be reduced to give compounds of formula (I) under a hydrogen atmosphere at temperatures up to 60° C. and elevated pressure up to 200 psi in the presence of 10% palladium on carbon and a chelating agent such as zinc bromide in a standard protic solvent such as methanol or propan-2-ol.
Alternatively, compounds of formula (I) may be obtained from compounds of formula (II) by transfer hydrogenation conditions. For example, ammonium formate or formic acid or ammonium formate in the presence of formic acid may be used to generate an in situ source of hydrogen which when in the presence of a hydrogenation catalyst such as 10% palladium on carbon in an alcoholic solvent such as propan-2-ol, for 2-3 hours at temperatures up to 80° C. can be used to effect the transformation of compounds of formula (II) to compounds of formula (I). Optionally reactions using formic acid as the hydrogen source may be performed without alcoholic solvents.
Stereoselective hydrogenations may be performed to yield a preferred stereoisomer using chiral catalysts, in accordance with standard organic chemistry textbooks or literature precedent. For example there are many known homogeneous and heterogeneous catalytic methods using transition metals such as palladium, rhodium and ruthenium. One particularly preferred catalyst is bis(norbornadiene)rhodium(I) tetrafluoroborate. Enantiopure ligands that have been utilised to effect enantioselective hydrogenations have been referenced in the literature and illustrative examples of homochiral ligands include phospholanes such as Duphos and its analogues, ferrocenyl ligands such as Josiphos, 1-[(R)-2-diphenylphosphino)ferrocenyl]ethyldi-tert-butylphosphine, biphenyl ligands such as (+/−)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthalene (BINAP) and miscellaneous ligands such as Prophos, Diamp, Bicp, Monophos. References providing details of enantioselective hydrogenations include Y. Yamanori, T. Imamoto, Reviews on Heteroatom Chemistry, 1999, 20, 227; T. Clark, C. Landis, Tetrahedron: Asymmetry, 2004, 15, 14, 2123; H. Blaser, Topics in Catalysis, 2002, 19, 1, 3; H. Blaser et al, Synthetic Methods of organometallic and inorganic chemistry, 2002, 10, 78; Pure and Applied Chemistry, 1999, 71, 8, 1531; Pure and Applied Chemistry, 1998, 70, 8, 1477; U. Berens et al, Speciality Chemicals, 2000, 20, 6, 210; M. T. Reetz, Pure and Applied Chemistry, 1999, 71, 8, 1503; D. J. Bayston et al, Speciality Chemicals, 1998, 18, 5, 224; C. Saluzzo and M. Lemaire, Advanced Synthesis and Catalysis, 2002, 344, 9, 915; H. Kumobayashi, Synlett, 2001, (Spec Issue) 1055.
Thus, enantiomerically enriched compounds of formula (I) may be obtained from achiral compounds of formula (II) by stereoselective hydrogenation. For example, compounds of formula (II) wherein Ra is hydrogen, and Rb is hydrogen or alkyl may be reduced to compounds of formula (I) in a suitable protic solvent such as methanol under a hydrogen atmosphere at ambient temperatures and elevated pressure up to 60 psi in the presence of a rhodium catalyst such as bis(norbornadiene)rhodium(I) tetrafluoroborate and chiral ligand such as 1-[(R)-2-diphenylphosphino)ferrocenyl]ethyldi-tert-butylphosphine to give optically enriched compounds of formula (I).
Chiral resolution can be utilised to enhance the enantiomeric purity of compounds of formula (I). For example, an acid salt can be enantioselectively formed upon addition of an enantiomerically pure chiral acid such as di-p-toluoyl-L-tartaric acid in a suitable protic solvent such as methanol. Using this process one enantiomer preferentially forms a crystalline salt which can be removed by filtration whereas the other enantiomer remains in the mother liquor. Upon separately basifying the salt and mother liquor with a suitable base such as sodium hydroxide (1N), the enantiomers are resolved to give separated optically enriched compounds of formula (I).
Alternatively, racemic compounds of formula (I) may be resolved using chiral HPLC procedures, known to those skilled in the art, to give enantiomerically pure compounds of formula (I).
Compounds of formula (II) wherein R6 is a protecting group such as benzyl or substituted benzyl e.g. p-methoxybenzyl, may be deprotected and reduced under hydrogenation conditions to give compounds of formula (I) wherein R6 is hydrogen.
Imidazole ring formation can also be utilised to access compounds of formula (I), other synthetic methods are precedented in textbooks and the literature. One illustrative example is from desirably substituted phenylacetonitrile reactants, for example a compound such as 2-(2,3-dimethylphenyl)propanenitrile may be reacted with an appropriately substituted ethylenediamine for example, the p-toluenesulfonic acid salt of ethylenediamine at elevated temperatures ranging from 140°-180° C. to form the compound of formula (I) wherein R1, R2 and R8 are methyl and R3, R4, R5, R6 and R9, R11 and R12 are hydrogen.
Another example of imidazole ring formation is from the reaction of suitably 2-substituted 2-aryl-1,1-dibromoethenes and an appropriately substituted ethylenediamine at room temperature to give the intermediate 2-substituted 2-arylmethylimidazoline. Standard oxidation procedures such as Swern oxidation can transform the intermediate 2-substituted 2-arylmethylimidazoline into compounds of formula (I).
Compounds of formula (II) may be prepared by Wittig chemistry by the reaction of a compound of formula (X) with the appropriate alkylphosphonium salt-derived phosphorus ylid. For example treatment of a methyltriphenylphosphonium halide with a strong base in a suitable solvent, followed by the addition of (X), will produce a compound of formula (II) wherein both Ra and Rb are hydrogen. Preferably the base reagent is a solution of n-butyllithium in hexane, the solvent is ether or tetrahydrofuran and the reaction is conducted at from about room temperature to about 35° C.
Compounds of formula (II) may undergo functional group interconversion into other compounds of formula (II). For example, wherein one or more of R1, R2, R3, R4 and R5 are bromo or iodo, and R6 is protected with a suitable protecting group such as benzyl, palladium catalysed coupling reactions such as Stille, Heck and Suzuki coupling reactions may be effected. For example, treatment of such organohalide compounds of formula (II) with a suitable boronic acid such as an alkyl or aryl boronic acid, in an inert solvent such as toluene, in the presence of a suitable base such as potassium phosphate, a suitable phosphine ligand such as tricyclohexylphosphine and palladium acetate under an inert atmosphere at elevated temperatures up to 120° C. for up to 18 h provides the corresponding alkylated or arylated compound of formula (II). Similarly, compounds of formula (II) wherein one or more of R1, R2, R3, R4 and R5 are bromo or iodo, and R6 is protected with a suitable protecting group such as benzyl, may undergo transmetallation reaction with a palladium catalyst such as [1,1-bis(diphenylphosphino)ferrocene]palladium (II) chloride followed by cross coupling with a suitable boronic anhydride such as trialkylboroxine under an inert atmosphere, in the presence of a mild base such as sodium carbonate and a suitable inert solvent such as dioxane and water at elevated temperatures up to 120° C. Alternatively, compounds of formula (II) wherein one or more of R1, R2, R3, R4 and R5 are bromo or iodo, and R6 is protected with a suitable protecting group such as benzyl, may undergo nucleophilic substitution reactions. For example, nitrile compounds may be formed upon treatment of such a halo compound of formula (II) in a polar solvent such as N,N-dimethylacetamide with a cyanide source such as copper cyanide at temperatures up to 150° C. for 3 days to give the corresponding compound of formula (II) wherein one or more of R1, R2, R3, R4 and R5 are cyano, and R6 is protected with a suitable protecting group such as benzyl. Nitrile compounds of formula (II) may also be formed from the corresponding halo compound of formula (II) upon treatment with a cyanide source such as sodium cyanide in the presence of a suitable transmetallating agent such as nickel bromide in a polar solvent such as N-methylpyrrolidinone and heating in a 150 W microwave at up to 150° C. for 5 min. Nitrile compounds of formula (II) may also be formed from the corresponding halo compound of formula (II) from the reaction of a suitable cyanide source such as potassium hexacyanoferrate, a transmetallating agent such as copper iodide, a salt such as potassium iodide, and a coordinating agent such as dimethylethylenediamine in a polar solvent such as N-methylpyrrolidinone under an inert atmosphere at elevated temperatures up to 140° C. for up to 60 hours.
Compounds of formula (II) may be prepared from compounds of formula (III) by standard dehydration conditions, optionally R6 may be a suitable protecting group e.g. benzyl, or substituted benzyl.
Thus, dehydration may be effected under acidic conditions. For example compounds of formula (III) may be treated with an inorganic acid such as hydrochloric acid (4-6N) or concentrated sulphuric acid, for up to 72 h, optionally in an organic miscible solvent such as acetonitrile, optionally at elevated temperatures up to 60° C. Alternatively, dehydration may result from heating compounds of formula (III) at reflux with an organic acid such as trifluoroacetic acid or p-toluenesulphonic acid in an aprotic solvent such as toluene. Otherwise, compounds of formula (III) may be dehydrated using Eaton's reagent, typically stirring at room temperature for several hours neat or in a polar solvent such as methanol. Dehydration may also be effected by treating a compound of formula (III) with thionyl chloride in a polar solvent such as acetonitrile.
Compounds of formula (III) may be used to directly access compounds of formula (I) upon treatment with Pearlman's catalyst in a suitable protic solvent such as methanol under a hydrogen atmosphere, in cases wherein R6 is a benzylic protecting group a deprotected compound of formula (I) will be obtained wherein R6 is hydrogen. Alternatively, compounds of formula (III) wherein R6 is a protecting group such as benzyl may be deprotected, dehydrated and reduced simultaneously by hydrogenation under acidic conditions. For example, upon treatment of compounds of formula (III) with a hydrogen source such as ammonium formate in the presence of an acid such as formic acid and 10% palladium on carbon, optionally for up to 72 h, compounds of formula (I) wherein R6 is hydrogen are obtained.
Compounds of formula (II) may be obtained by dehydrohalogenation procedures, known to the skilled man, from compounds of formula (III) for example by standard chlorination followed by dehydrochlorination procedures.
Alternatively, compounds of formula (II) can be obtained by transition metal catalysed cross-coupling reactions by utilizing methods known in the literature. For these reactions, it may be necessary to protect the basic imidazole, optionally R6 may include a suitable protecting group such as diethoxymethyl. Thus, suitably protected organozincates such as compounds of formula (V), wherein X is halo for example chloro or bromo, can be coupled with suitably substituted styrenes such as compounds of formula (IV) wherein Y is a group suitable for transmetallation such as OTf, Cl, Br or I in the presence of a palladium catalyst such as Pd(PPh3)3.
Standard deprotection of compounds of formula (II) wherein R6 is a suitable protecting group provides compounds of formula (II) in which R6 is hydrogen. For example, when R6 is diethoxymethyl treatment with an organic acid such as trifluoroacetic acid or an inorganic acid such as hydrochloric acid provides compound (II) wherein R6 is hydrogen. Similarly, deprotection of compounds of formula (II) wherein R6 is a benzyl moiety protecting group may easily be effected by hydrogenation.
Compounds of formula (III) wherein R6 is a protecting group can be formed by 1,2-addition of a suitably protected organometallic compound (VI) to the corresponding ketone (VII) where chemically feasible for example wherein R1, R2, R3, R4 and R5 may be chosen independently from alkyl, chloro, and Ra and Rb may be chosen from alkyl.
For example, compound (VI) may be reacted with ketone (VII) in an aprotic solvent such as tetrahydrofuran at temperatures typically ranging from −80 to 0° C. to give compounds of formula (III), which can be readily deprotected to give a compound of formula (III) wherein R6 is H if desired.
Alternative organometallic chemistry may be utilized to yield a compound of formula (III), wherein R6 is a suitable protecting group such as benzyl, when an organometallic compound of formula (VIII), wherein X may be a halo e.g. chloro or bromo, is added to a ketone of formula (IX) wherein R6 is a protecting group.
Similarly, compounds of formula (III) wherein R6 is optionally a suitable protecting group such as benzyl may also be accessed by organometallic addition to a protected ketone (X), suitable organometallic reagents include Grignard reagents and organolithium reagents. For example, a Grignard reagent such as methylmagnesium chloride may be added to a solution of compound (X) in an anhydrous, aprotic solvent such as tetrahydrofuran, toluene or diethyl ether at −10°-0° C. for up to 4 h to provide compounds of formula (III) wherein Ra and Rb are H.
Compounds of formula (III) wherein R6 is a protecting group such as benzyl may be deprotected using standard hydrogenation conditions such as 10% palladium on carbon in a protic solvent at elevated pressure and temperature to give deprotected compounds of formula (III) wherein R6 is hydrogen. Deprotecting compounds of formula (III) in a stepwise manner, before dehydration to produce compounds of formula (II), allows compounds of formula (II) to be stereoselectively reduced to give compounds of formula (I) if desired.
Compounds of formula (IV), (V), (VI), (VII), (VIII), and (IX) may readily be accessed by utilisation of literature methods or simple modifications thereof as would be routinely employed by a skilled man. For example, compounds of formula (V) can be prepared by stirring a 1-protected imidazole with n-butyllithium at reduced temperature, typically −60 to −20° C. followed by the addition of zinc chloride and allowing to warm to room temperature.
For example, compound (VI) may be obtained in situ by treatment of a protected imidazole reactant, with an organolithium reagent such as n-butylithium in an aprotic solvent such as tetrahydrofuran at reduced temperatures typically ranging from −80 to 0° C. Suitable protecting groups include diethoxymethyl.
For example, compounds of formula (IX) may be synthesised by acylating a suitably substituted imidazole using acid chlorides. Thus, heating for several hours a suitable acid chloride with a 1-protected imidazole in the presence of a mild base such as triethylamine provides compounds of formula (IX).
Compounds of formula (VII) may be accessed in a number of ways. Some methods utilise simple precursors as detailed below.
Compounds of formula (VII) may be prepared by the addition of a chelating agent such as Fe(acac)3 and a Grignard reagent such as methylmagnesium bromide to a suitably substituted acid chloride (XI) at reduced temperatures, typically −20° C. in a suitable aprotic solvent. Acid chlorides (XI) may be prepared by the reaction of the corresponding benzoic acid (XII) with thionyl chloride or oxalyl chloride, at elevated temperatures, typically 100° C. for several hours.
Compounds of formula (VII) may also be prepared by reaction of an acid anhydride such as acetic anhydride, with a phenyl Grignard reactant (XIII) in an aprotic solvent. Alternatively, amides, or acid chlorides may be used in place of the acid anhydride. Compounds of formula (XIII) may be formed in situ by reacting a suitable bromobenzene derivative with magnesium turnings in an anhydrous, aprotic solvent such as tetrahydrofuran.
Similarly, compounds of formula (VII) may be prepared by reacting a Grignard reactant e.g. methylmagnesium bromide with an amide e.g. a suitably substituted benzoylmorpholine (XIV) at reflux in a suitable solvent such as tetrahydrofuran.
Compounds of formula (VII) may also be obtained from reaction of a suitable benzoic acid (XII) with an organolithium reactant, for example methyllithium, at reduced temperatures in an anhydrous aprotic solvent such as tetrahydrofuran.
Compounds of formula (VII) may be obtained by Friedel Crafts acylation of suitably functionalized phenyl moieties. For example, a functionalized phenyl reactant can be treated with a Lewis acid such as aluminium chloride, in the presence of a suitable acylating agent such as acetyl chloride, in an aprotic solvent such as dichloromethane at room temperature for up to 18 h to give the desired compounds of formula (VII).
Alternatively compounds of formula (VII) may by obtained in a two step procedure from a suitably substituted halobenzene, preferably bromo or iodo benzene. For example a bromobenzene compound may be transmetallated with an organometallic reagent such as n-butyllithium in an anhydrous, apolar solvent such as tetrahydrofuran at low temperatures down to −80° C. followed by electrophilic quenching with an aldehyde to give the corresponding secondary alcohol which may be oxidized under standard conditions, for example using Dess Martin periodinane, to give compounds of formula (VII) wherein Ra is selected from H, C1-4alkyl, or C0-4alkylenephenyl and Rb=C1-4alkyl, or C0-4alkylenephenyl.
Compounds of formula (VII) may also be formed from the corresponding aryliodide and boronic acids using palladium chemistry in a carbon monoxide atmosphere. Thus, heating aryliodides with carbon monoxide, methylboronic acid and palladium tetrakis triphenylphosphine provides compounds of formula (VII) wherein Ra and Rb are H.
Compounds of formula (VII) may undergo standard chemical reactions and functional group interconversion reactions known to the skilled man to give other compounds of formula (VII). Thus, compounds of formula (VII) may be chlorinated using standard reagents such as Selectafluor™ and sodium chloride. Also, suitably substituted halo compounds of formula (VII) may undergo standard palladium catalysed cross coupling reactions such as Suzuki, Stille, Heck reactions to give a variety of standard products. For example, bromo or iodo compounds of formula (VII) may undergo alkylation and arylation reactions via Suzuki coupling reactions upon treatment with an organoborane e.g. triethyl borane in the presence of [1,1-bis(diphenylphosphino)ferocene]palladium (II) chloride, and potassium carbonate in an aprotic solvent such as N,N-dimethylformamide to give alkyl or aryl substituted compounds of formula (VII).
Compounds of formula (X) may be obtained from the reaction of acid chlorides of formula (XI) and imidazoles of formula (XV) wherein R6 is a suitable protecting group in a suitable aprotic solvent such as toluene or acetonitrile in the presence of a mild base such as triethylamine at temperatures ranging from −10°-130° C.
Suitably functionalised acid chlorides of formula (XI) may be synthesized from the corresponding acid upon treatment with thionyl chloride at 80° C. for ˜1 hour. Alternatively, acid chlorides may be synthesized from carboxylic acids upon treatment with oxalyl chloride in an aprotic solvent such as toluene at room temperature for up to 4 hours. Suitably functionalized acids may be obtained by utilising standard literature procedures available to the skilled man, thus substituents may be introduced via electrophilic or nucleophilic substitution or cross coupling reactions or via functional group interconversion.
Compounds of formula (X) can also be synthesized by oxidation of compounds of formula (XVI) by suitable oxidising agents, wherein R6 is hydrogen or a suitable protecting group.
One such oxidation may include Dess Martin oxidation conditions. For example, a compound of formula (X), may be prepared by stirring the corresponding compound of formula (XVI) at room temperature with Dess-Martin Periodinane in a suitable polar solvent such as dichloromethane.
Compounds of formula (XVI) may be formed by the 1,2-addition of a suitably protected organometallic compound to a suitable aldehyde. Thus reaction of an organolithium compound of formula (VI) and a corresponding aldehyde of formula (XVII), in an anhydrous, aprotic solvent such as tetrahydrofuran at temperatures ranging from −80-0° C. provides compounds of formula (XVI).
It is to be understood that precursors to compounds of formula (I) and compounds of formula (I) themselves may undergo functional group interconversion in order to deliver alternative compounds of formula (I). For example compounds of formula (I) wherein R6 is hydrogen may be reacted with alkylating agents of the formula L-C0-2alkyleneR7, L-C1-2alkyleneOR7, L-C1-2alkyleneC(O)R7, L-C1-2alkyleneOC(O)R7, L-C1-2alkyleneOC(O)OR7, L-C1-2alkyleneC(O)OR7, L-C1-2alkyleneN(H)C(O)R7, L-C1-2alkyleneN(R7)C(O)R7, L-C1-2alkyleneC(O)NHR7, L-C1-2alkyleneNHC(O)NR15R16, L-C1-2alkyleneNR7C(O)NR15R16, L-C1-2alkyleneC(O)NR15R16, L-C1-2alkyleneOC(O)NHR7, L-C1-2alkyleneOC(O)NR15R16, to provide compounds wherein R6 is —C0-2alkyleneR7, —C1-2alkyleneOR7, —C1-2alkyleneC(O)R7, —C1-2alkyleneOC(O)R7, —C1-2alkyleneOC(O)OR7—C1-2alkyleneC(O)OR7, —C1-2alkyleneN(H)C(O)R7, —C1-2alkyleneN(R7)C(O)R7, —C1-2alkyleneC(O)NHR7, —C1-2alkyleneNHC(O)NR15R16, —C1-2alkyleneNR7C(O)NR15R16, —C1-2alkyleneC(O)NR15R16, —C1-2alkyleneOC(O)NHR7, —C1-2alkyleneOC(O)NR15R16. L is a suitable leaving group such as Cl, Br, I, or a sulfonate such as trifluoromethanesulfonate. For example compounds of formula (I) wherein R6 is hydrogen may be reacted with alkylating agents in the presence of a mild base such as cesium carbonate, potassium carbonate, triethylamine, or diisopropylethylamine, in an aprotic solvent such as acetone, 1-methyl-2-pyrrolidinone, dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide optionally in the presence of a salt such as sodium iodide. Generally the alkylation reaction will proceed for up to 72 h at room temperature, optionally the reaction may be heated to reflux or may be microwaved at 200 W for up to 1 h.
Alkylating agents of the form Cl—CH2OC(O)R7 may be produced from the reaction of the acid chloride ClC(O)R7 with paraformaldehyde in the presence of a Lewis acid such as zinc chloride at temperatures up to 80° C. for 2-3 hours. Under alkylating conditions such reagents give compounds of formula (I) wherein R6 is CH2OC(O)R7.
Alkylating agents of the form L-CH2OC(O)OR7 may be produced from the reaction of the alcohol HOR7 with chloromethyl chloroformate in an aprotic solvent such as dichloromethane at temperatures ranging from 0° C. to room temperature. Under alkylating conditions such reagents give compounds of formula (I) wherein R6 is CH2OC(O)OR7.
Alkylating agents of the form L-CH2OC(O)NHR7, may be produced from the reaction of the amine R7NH2 with chloromethyl chloroformate in an aprotic solvent such as dichloromethane at temperatures ranging from −10° C. to room temperature. Under alkylating conditions such reagents give compounds of formula (I) wherein R6 is CH2OC(O)NHR7.
Alkylating agents of the form L-CH2OC(O)NR15R16, may be produced from the reaction of the amine R15R16NH with chloromethyl chloroformate in an aprotic solvent such as dichloromethane optionally in the presence of a mild base such as diisopropylethylamine at temperatures ranging from −0° C. to room temperature. Under alkylating conditions such reagents give compounds of formula (I) wherein R6 is CH2OC(O)NR15R16.
Compounds of formula (I) wherein R6 is hydrogen may be reacted with acylating agents of the formula ClC(O)R7, O[OC(O)R7]2, ClC(O)OR7, ClC(O)NHR7, ClC(O)NR15R16, to provide compounds wherein R6 is —C(O)R7, —OC(O)R7, —C(O)OR7, —C(O)NHR7, —C(O)NR15R16. For example compounds of formula (I) wherein R6 is hydrogen may be reacted with acylating agents in the presence of a mild base such as triethylamine, or pyridine in an aprotic solvent such as dichloromethane, tetrahdyrofuran or acetonitrile at temperatures ranging from room temperature to 100° C. for between 1 and 36 h.
It is possible to form the acylating agent ClC(O)OR7 in situ. Thus a compound of formula (I) wherein R6 is hydrogen, may be reacted with phosgene or diphosgene in an anhydrous solvent such as dichloromethane or acetonitrile in the presence of a mild base such as pyridine in the presence of an alcohol R7OH at ambient temperature to give the compound of formula (I) wherein R6 is C(O)OR7.
Compounds of formula (I) wherein R6 is hydrogen may be reacted with phosphorylating agents of the formula Cl—P(═O)[N(R7)2(R7)2] to give compounds of formula (I) wherein R6 is P(═O)[N(R7)2(R7)2]. For example reaction with a corresponding bis(dialkylamino)phosphoryl chloride e.g. bis(dimethylamino)phosphoryl chloride in an aprotic solvent such as dichloromethane.
Compounds of formula (I) wherein R6 is hydrogen may be reacted with silating agents of the formula Cl—Si(R7) to give compounds of formula (I) wherein R6 is Si(R7)3 For example reaction with a corresponding alkylsilane or arylsilane e.g. chlorotrimethylsilane in an aprotic solvent such as dichloromethane or tetrahydrofuran.
Compounds of formula (I) wherein R6 is hydrogen may be reacted with sulphonating agents of the formula Cl—S(═O)2R10 to give compounds of formula (I) wherein R6 is S(═O)2R10. For example reaction with a corresponding sulphonyl chloride e.g. methanesulphonyl chloride in an aprotic solvent such as dichloromethane, optionally with a weak base such as triethylamine.
Compounds of formula (I) wherein R6 is hydrogen may be reacted with cyanogen bromide in an aprotic solvent such as dichloromethane, optionally with a weak base such as diisopropylethylamine to give compounds of formula (I) wherein R6 is CN.
Compounds of formula (III) may be alkylated to give compounds of formula (I) wherein R9 is C1-C4 alkoxy. Thus, treatment of compounds of formula (III) with a strong base such as sodium hydride in an aprotic solvent such as tetrahydrofuran followed by addition of an alkylating agent will provide compounds of formula (I) wherein R9 is C1-C4 alkoxy.
Compounds of formula (II) may be cyclopropanated to give compounds of formula (I) wherein R8 and R9 together form a cyclopropyl ring. Compounds of formula (II) may be reacted with a carbenoid species, CRdRe. For example, when Rd═Re═F, a reactive species such as trimethylsilyl difluoro(fluorosulfonyl)acetate (TFDA) may be reacted with a compound of formula (II), with an optional apolar solvent at elevated temperature in the presence of sodium fluoride to yield a product of formula (I) after deprotection, wherein the cyclopropyl ring is substituted with fluoro.
Other specific methods include treatment of chloroform with base, preferably under phase transfer catalysis conditions, thermolysis of a suitable organometallic precursor such as an aryl trifluoromethyl, trichloromethyl, or phenyl(trifluoromethyl) mercury derivative or treatment with a diazoalkane in the presence of a transition metal catalyst and treatment with a diazoalkane in the absence of a transition metal catalyst followed by thermolysis of the intermediate pyrazoline, or generation from a sulphur ylid.
Moreover, persons skilled in the art will be aware of variations of, and alternatives to, the processes described which allow the compounds defined by formula (I) to be obtained.
It will also be appreciated by persons skilled in the art that, within certain of the processes described, the order of the synthetic steps employed may be varied and will depend inter alia on factors such as the nature of other functional groups present in a particular substrate, the availability of key intermediates, and the protecting group strategy (if any) to be adopted. Clearly, such factors will also influence the choice of reagent for use in the said synthetic steps.
The skilled person will appreciate that the compounds of the invention could be made by methods other than those herein described, by adaptation of the methods herein described and/or adaptation of methods known in the art, for example the art described herein, or using standard textbooks such as “Comprehensive Organic Transformations—A Guide to Functional Group Transformations”, R C Larock, Wiley-VCH (1999 or later editions).
It is to be understood that the synthetic transformation methods mentioned herein are exemplary only and they may be carried out in various different sequences in order that the desired compounds can be efficiently assembled. The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound.
Further examples of replacement groups in accordance with the foregoing examples and examples of other prodrug types may be found in the aforementioned references.
The present invention also relates to intermediates of formula (LX) below:
where:
R1-R12, Ra, Rb, and n are all as defined for formula (I) above or a pharmaceutical salt or a prodrug thereof. With reference to formula (LX), suitably R1 and R2 are selected from C1-4 alkyl and R3, R4 and R5 are hydrogen.
The present invention also relates to intermediates of formula (LXV) below:
where:
R1-R12, Ra, Rb, and n are all as defined for formula (I) above and where Pg is a chemical protecting group or a pharmaceutical salt or a prodrug thereof. With reference to formula (LXV), suitably R1 and R2 are selected from C1-4 alkyl and R3, R4 and R5 are hydrogen.
The present invention also relates to intermediates of formula (LXX) below:
where:
R1-R12, Ra, Rb, and n are all as defined for formula (I) above and where Pg is a chemical protecting group or a pharmaceutical salt or a prodrug thereof. With reference to formula (LXX), suitably R1 and R2 are selected from C1-4 alkyl and R3, R4 and R5 are hydrogen.
It will be understood that throughout the application all references to formula (I) apply equally to compounds of the formulas (LX), (LXV) and (LXX) above.
Furthermore, it will be understood that all the suitable groups and preferences applied to R1-R12, Ra, Rb, and n for formula (I) apply equally to compounds of the formulas (LX), (LXV) and (LXX) above.
Finally, certain compounds of formula (I) may themselves act as intermediates in the preparation of other compounds of formula (I).
One of ordinary skill in the art would understand that Pg in the formulas (LX), (LXV) and (LXX) above can represent a wide range of possible protecting group and the specific group required will depend on the final compounds to be made and can be readily selected by one of ordinary skill. Preferred protecting groups include benzyl, para-methoxybenzyl, allyl, trityl, or 1,1-diethoxymethyl, preferably benzyl.
This invention also relates to a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, together with a pharmaceutically acceptable diluent or carrier, which may be adapted for oral, parenteral or topical administration.
Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in ‘Remington's Pharmaceutical Sciences’, 19th Edition (Mack Publishing Company, 1995).
Compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, or spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.
The methods by which the compounds may be administered include oral administration by capsule, bolus, tablet, powders, lozenges, chews, multi and nanoparticulates, gels, solid solution, films, sprays, or liquid formulation. Liquid forms include suspensions, solutions, syrups, drenches and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet. Oral drenches are commonly prepared by dissolving or suspending the active ingredient in a suitable medium.
Compounds of the present invention may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). The compounds may be administered alone or in a formulation appropriate to the specific use envisaged, the particular species of host mammal being treated and the parasite involved. Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term “excipient” is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
Thus compositions useful for oral administration may be prepared by mixing the active ingredient with a suitable finely divided diluent and/or disintegrating agent and/or binder, and/or lubricant etc. Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.
For oral dosage forms, depending on dose, the drug may make up from 1 wt % to 80 wt % of the dosage form, more typically from 5 wt % to 60 wt % of the dosage form. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 wt % to 25 wt %, preferably from 5 wt % to 20 wt % of the dosage form.
Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Examples of diluents include lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
Oral formulations may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 wt % to 5 wt % of the tablet, and glidants may comprise from 0.2 wt % to 1 wt % of the tablet.
Lubricants include magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 wt % to 10 wt %, preferably from 0.5 wt % to 3 wt % of the tablet.
Exemplary tablets contain up to about 80% drug, from about 10 wt % to about 90 wt % binder, from about 0 wt % to about 85 wt % diluent, from about 2 wt % to about 10 wt % disintegrant, and from about 0.25 wt % to about 10 wt % lubricant.
The formulation of tablets is discussed in “Pharmaceutical Dosage Forms: Tablets, Vol. 1”, by H. Lieberman and L. Lachman, Marcel Dekker, N.Y., 1980 (ISBN 0-8247-6918-X).
The compounds may be administered topically to the skin or mucosa, that is dermally or transdermally. This is a preferred method of administration and as such it is desirable to develop active compounds, which are particularly suited to such formulations. Typical formulations for this purpose include pour-on, spot-on, dip, spray, mousse, shampoo, powder formulation, gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated—see, for example, J Pharm Sci, 88 (10), 955-958 by Finnin and Morgan (October 1999). Pour-on or spot-on formulations may be prepared by dissolving the active ingredient in an acceptable liquid carrier vehicle such as butyl digol, liquid paraffin or a non-volatile ester, optionally with the addition of a volatile component such as propan-2-ol. Alternatively, pour-on, spot-on or spray formulations can be prepared by encapsulation, to leave a residue of active agent on the surface of the animal, this effect may ensure that the compounds of formula (I) have increased persistence of action and are more durable, for example they may be more waterfast.
Agents may be added to the formulations of the present invention to improve the persistence of such formulations on the surface of the animal to which they are applied, for example to improve their persistence on the coat of the animal. It is particularly preferred to include such agents in a formulation which is to be applied as a pour-on or spot-on formulation. Examples of such agents acrylic copolymers and in particular fluorinated acrylic copolymers. A particular suitable reagent is Foraperle™ (Redline Products Inc, Texas, USA).
Certain topical formulations may include unpalatable additives to minimize accidental oral exposure.
Injectable formulations may be prepared in the form of a sterile solution, which may contain other substances, for example enough salts or glucose to make the solution isotonic with blood. Acceptable liquid carriers include vegetable oils such as sesame oil, glycerides such as triacetin, esters such as benzyl benzoate, isopropyl myristate and fatty acid derivatives of propylene glycol, as well as organic solvents such as pyrrolidin-2-one and glycerol formal. The formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.01 to 10% by weight of the active ingredient.
Alternatively, the compounds can be administered parenterally, or by injection directly into the blood stream, muscle or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as powdered a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. The solubility of compounds of formula (I) used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
Such formulations are prepared in a conventional manner in accordance with standard medicinal or veterinary practice.
These formulations will vary with regard to the weight of active compound contained therein, depending on the species of host animal to be treated, the severity and type of infection and the body weight of the host. For parenteral, topical and oral administration, typical dose ranges of the active ingredient are 0.01 to 100 mg per kg of body weight of the animal. Preferably the range is 0.1 to 10 mg per kg.
Formulations may be immediate and/or modified controlled release. Controlled release formulations include modified release formulations including delayed-, sustained-, pulsed-, controlled, targeted, or programmed release. Suitable modified release formulations for the purposes of the invention are described in U.S. Pat. No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in Verma et al, Pharmaceutical Technology On-line, 25(2), 1-14 (2001). The use of chewing gum to achieve controlled release is described in WO 00/35298. Alternatively, compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and PGLA microspheres.
As an alternative the compounds may be administered to a non-human animal with the feedstuff and for this purpose a concentrated feed additive or premix may be prepared for mixing with the normal animal feed.
All the aforementioned aqueous dispersions or emulsions or spraying mixtures can be applied, for example, to crops by any suitable means, chiefly by spraying, at rates which are generally of the order of about 100 to about 1,200 liters of spraying mixture per hectare, but may be higher or lower (eg. low or ultra-low volume) depending upon the need or application technique. The compounds or compositions according to the invention are conveniently applied to vegetation and in particular to roots or leaves having pests to be eliminated. Another method of application of the compounds or compositions according to the invention is by chemigation, that is to say, the addition of a formulation containing the active ingredient to irrigation water. This irrigation may be sprinkler irrigation for foliar pesticides or it can be ground irrigation or underground irrigation for soil or for systemic pesticides.
The concentrated suspensions, which can for example be applied by spraying, are prepared so as to produce a stable fluid product which does not settle (fine grinding) and usually contain from about 10 to about 75% by weight of active ingredient, from about 0.5 to about 30% of surface-active agents, from about 0.1 to about 10% of thixotropic agents, from about 0 to about 30% of suitable additives, such as anti-foaming agents, corrosion inhibitors, stabilizers, penetrating agents, adhesives and, as the carrier, water or an organic liquid in which the active ingredient is poorly soluble or insoluble. Some organic solids or inorganic salts may be dissolved in the carrier to help prevent settling or as antifreezes for water.
The wettable powers (or powder for spraying) are usually prepared so that they contain from about 10 to about 80% by weight of active ingredient, from about 20 to about 90% of a solid carrier, from about 0 to about 5% of a wetting agent, from about 3 to about 10% of a dispersing agent and, when necessary, from about 0 to about 80% of one or more stabilizers and/or other additives, such as penetrating agents, adhesives, anti-caking agents, colorants, or the like. To obtain these wettable powders, the active ingredient(s) is(are) thoroughly mixed in a suitable blender with additional substances which may be impregnated on the porous filler and is(are) ground using a mill or other suitable grinder. This produces wettable powders, the wettability and the suspendability of which are advantageous. They may be suspended in water to give any desired concentration and this suspension can be employed very advantageously in particular for application to plant foliage.
The “water dispersible granules (WG)” (granules which are readily dispersible in water) have compositions which are substantially close to that of the wettable powders. They may be prepared by granulation of formulations described for the wettable powders, either by a wet route (contacting finely divided active ingredient with the inert filler and a little water, e.g. 1 to 20% by weight, or with an aqueous solution of a dispersing agent or binder, followed by drying and screening), or by a dry route (compacting followed by grinding and screening).
Depending on the method of application or the nature of the composition or use thereof, the rates and concentrations of the formulated compositions may vary according. Generally speaking, the compositions for application to control arthropod, plant nematode, helminth or protozoan pests usually contain from about 0.00001% to about 95%, more particularly from about 0.0005% to about 50% by weight of one or more compounds of formula (I), or pesticidally acceptable salts thereof, or of total active ingredients (that is to say the compound of formula (I), or a pesticidally acceptable salt thereof, together with: other substances toxic to arthropods or plant nematodes, anthelmintics, anticoccidials, synergists, trace elements or stabilizers). The actual compositions employed and their rate of application will be selected to achieve the desired effect(s) by the farmer, livestock producer, medical or veterinary practitioner, pest control operator or other person skilled in the art.
The compounds of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.
Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.
Compounds of the invention can also be mixed with one or more biologically active compounds or agents including insecticides, acaricides, anthelmintics, fungicides, nematocides, antiprotozoals, bactericides, growth regulators, entomopathogenic bacteria, viruses or fungi to form a multi-component pesticide giving an even broader spectrum of pharmaceutical, veterinary or agricultural utility. Thus, the present invention also pertains to a composition comprising a biologically effective amount of compounds of the invention and an effective amount of at least one additional biologically active compound or agent and can further comprise one or more of surfactant, a solid diluent or a liquid diluent. Specific further active compounds include those described in International Patent Application No WO0 2005/090313, at pages 39 to 44.
It be may desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound in accordance with the invention, may conveniently be combined in the form of a kit suitable for coadministration of the compositions.
Thus the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of formula (I) in accordance with the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.
The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically comprises directions for administration and may be provided with a so-called memory aid.
The compounds of the invention, i.e. those of formula (I), possess parasiticidal activity in humans, animals, insects and plants. They are particularly useful in the treatment of ectoparasites.
This invention also relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing, for use as a medicament.
A further aspect of this invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, for the manufacture of a medicament for the treatment of a parasitic infestation.
In one embodiment this invention is useful for the manufacture of a medicament for the treatment of a parasitic infestation in humans.
In one embodiment this invention is useful for the manufacture of a medicament for the treatment of a parasitic infestation in animals.
In one embodiment this invention is useful for the manufacture of a medicament for the treatment of a parasitic infestation in insects.
In one embodiment this invention is useful for the manufacture of a medicament for the treatment of a parasitic infestation in plants.
An even further aspect of this invention relates to a method of treating a parasitic infestation in a mammal which comprises treating said mammal with an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing.
A yet further aspect of this invention relates to a method of preventing a parasitic infestation in a mammal which comprises treating said mammal with an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing.
In a still further embodiment this invention also relates to a method of controlling disease transmission in a mammal which comprises treating said mammal with an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing.
According to another aspect of the present invention, there is provided a method for the control of arthropod, plant nematode or helminth pests at a locus which comprises the treatment of the locus (e.g. by application or administration) with an effective amount of a compound of general formula (I), or a pesticidally acceptable salt thereof.
For the avoidance of doubt, references herein to “treatment” as used herein includes references to curative, palliative and prophylactic treatment, references to “control” (of parasites and/or pests etc.) include kill, repel, expel, incapacitate, deter, eliminate, alleviate, minimise, eradicate.
The compounds of the invention have utility in the control of arthropod pests. They may, in particular, be used in the fields of veterinary medicine, livestock husbandry and the maintenance of public health: against arthropods which are parasitic internally or externally upon vertebrates, particularly warm-blooded vertebrates, including man and domestic animals such as dogs, cats, cattle, sheep, goats, equines, swine, poultry and fish for example Acarina, including ticks (e.g. Ixodes spp., Boophilus spp. e.g. Boophilus microplus, Amblyomma spp., Hyalomma spp., Rhipicephalus spp. e.g. Rhipicephalus appendiculatus, Haemaphysalis spp., Dermacentor spp., Omithodorus spp. (e.g. Omithodorus moubata), mites (e.g. Damalinia spp., Dermanyssus gallinae, Sarcoptes spp. e.g. Sarcoptes scabiei, Psoroptes spp., Chorioptes spp., Demodex spp., Eutrombicula spp.), specific further arthropod pests include those described in International Patent Application No WO 2005/090313; Diptera (e.g. Aedes spp., Anopheles spp., Muscidae spp. e.g. Stomoxys calcitrans and Haematobia irritans, Hypoderma spp., Gastrophilus spp., Simulium spp.); Hemiptera (e.g. Triatoma spp.); Phthiraptera (e.g. Damalinia spp., Linognathus spp.); Siphonaptera (e.g. Ctenocephalides spp.); Dictyoptera (e.g. Periplaneta spp., Blatella spp.) and Hymenoptera (e.g. Monomorium pharaonis). The compounds of the present invention also have utility in the field of control of plant pests, soil inhabiting pests and other environmental pests.
The present invention is particularly useful in the control of arthropod pests in mammals, in particular humans and animals. Preferably this invention is useful in the control of arthropod pests in animals which includes livestock such as cattle, sheep, goats, equines, swine and companion animals such as dogs and cats.
The compounds of the invention are of particular value in the control of arthropods which are injurious to, or spread or act as vectors of diseases in, man and domestic animals, for example those hereinbefore mentioned, and more especially in the control of ticks, mites, lice, fleas, midges and biting, nuisance and myiasis flies. They are particularly useful in controlling arthropods which are present inside domestic host animals or which feed in or on the skin or suck the blood of the animal, for which purpose they may be administered orally, parenterally, percutaneously or topically.
The compounds of the invention are of value for the treatment and control of the various lifecycle stages of parasites including egg, nymph, larvae, juvenile and adult stages.
According to another aspect of the present invention, there is provided a method for the control of arthropod pests of insects which comprises treatment of the insect with an effective amount of a compound of general formula (I), or a pesticidally acceptable salt thereof. Compounds of the present invention may also be used for the treatment of infections caused by mites, and in particular varoaa mites. In particular compounds of the present invention may also be used for the treatment of varoaa mite infection in bees.
According to another aspect of the present invention, there is provided a method for the control of arthropod pests of plants which comprises treatment of the plant with an effective amount of a compound of general formula (I), or a pesticidally acceptable salt thereof. The compounds of the invention also have utility in the control of arthropod pests of plants. The active compound is generally applied to the locus at which the arthropod infestation is to be controlled at a rate of about 0.005 kg to about 25 kg of active compound per hectare (ha) of locus treated, preferably 0.02 to 2 kg/ha. Under ideal conditions, depending on the pest to be controlled, the lower rate may offer adequate protection. On the other hand, adverse weather conditions and other factors may require that the active ingredient be used in higher proportions. For foliar application, a rate of 0.01 to 1 kg/ha may be used. Preferably, the locus is the plant surface, or the soil around the plant to be treated.
According to another aspect of the present invention, there is provided a method for the protection of timber which comprises treatment of the timber with an effective amount of a compound of general formula (I), or a pesticidally acceptable salt thereof. Compounds of the present invention are also valuable in the protection of timber (standing, felled, converted, stored or structural) from attack by sawflies or beetles or termites. They have applications in the protection of stored products such as grains, fruits, nuts, spices and tobacco, whether whole, milled or compounded into products, from moth, beetle and mite attack. Also protected are stored animal products such as skins, hair, wool and feathers in natural or converted form (e.g. as carpets or textiles) from moth and beetle attack; also stored meat and fish from beetle, mite and fly attack. Solid or liquid compositions for application topically to timber, stored products or household goods usually contain from about 0.00005% to about 90%, more particularly from about 0.001% to about 10%, by weight of one or more compounds of formula (I) or pesticidally acceptable salts thereof.
The liquid compositions of this invention may, in addition to normal agricultural use applications be used for example to treat substrates or sites infested or liable to infestation by arthropods (or other pests controlled by compounds of this invention) including premises, outdoor or indoor storage or processing areas, containers or equipment or standing or running water.
The present invention also relates to a method of cleaning animals in good health comprising the application to the animal of compound of formula (I) or a veterinarily acceptable salt. The purpose of such cleaning is to reduce or eliminate the infestation of humans with parasites carried by the animal and to improve the environment in which humans inhabit.
The biological activity of the compounds was tested using one or more of the test methods outlined below.
In Vitro Tick Assay
Application of octopamine agonists to acarids for example, ticks, causes distinct behavioural changes compared to untreated control ticks. Treated ticks become agitated and move constantly, this prevents ticks attaching and feeding on a host animal to which the compound has been applied. Normal behaviour of ticks is to go into stasis when all other external stimuli are removed. Agitation and movement can be measured in vitro in the laboratory to predict efficacy and potency in vivo.
The assay was run using unfed Rhipicephalus sanguineus (brown dog tick) and precoated glass vials with an inner surface area of 34.5 cm2. Each compound was tested in duplicate.
Compound (345 μg) was dissolved in isopropyl alcohol (500 μl) and delivered to each vial. The vials were placed on a tilting roller in a fume hood for 2 hours to allow the isopropyl alcohol to evaporate giving a compound concentration for each vial of 10 μg/cm2. Five R. sanguineus (male and female) were added to each coated vial and the vial sealed with a firm wad of cotton wool. Vials were then kept, undisturbed, on the bench at room temperature. Observation and recordings of activity were taken at 24, 48 and 72 hours after addition of ticks to the vials. The ED100 value was determined as the lowest dose at which all five ticks were seen moving around inside the vial.
Octopamine Activity
One skilled in the art could determine agonist activity of compounds against insect octopamine receptors expressed in CHO cells by adapting the methods described in B. Maqueira, H. Chatwin, P. D. Evans, J. Neurochemistry, 2005, 94, 2, 547. Compound activity can be measured as an increase in cAMP by various methods known to a skilled person and can be recorded as % Vmax (Vmax=maximal octopamine response) and EC50.
Adrenergic Activity
Methods from literature procedures were simply adapted, as could be readily performed by one skilled in the art, in order to determine α2 adrenergic activity of the compounds. Suitable procedures include those described in J J. Meana, F. Barturen, J. A. Garcia-Sevilla, Journal of Neurochemistry, 1989, 1210; and D. J. Loftus, J. M. Stolk, D. C. U'Pritchard, Life Sciences, 1984, 35, 610.
EXAMPLES
The following Examples illustrate the preparation of compounds of the formula (I).
In the following Examples, structures are depicted as follows:
Unless specified otherwise, the wedge and dashed bonds indicate absolute stereochemistry as drawn at this chiral centre, a wiggly bond indicates that the absolute stereochemistry is unknown but the compound is a single stereoisomer at this chiral centre. Straight bonds emanating from a chiral centre indicate that the stereoisomers are not resolved and a mixture of stereoisomers is present.
When the source of a simple precursor is unspecified these compounds may be obtained from commercial suppliers or according to literature procedures. The following is a list of commercial suppliers for such compounds:
Sigma-Aldrich, P O Box 14508, St. Louis, Mo., 63178, USA
Lancaster Synthesis Ltd., Newgate, White Lund, Morecambe, Lancashire, LA3 3BN, UK
Maybridge, Trevillett, Tintagel, Cornwall, PL34 0HW, UK
Fluorochem Ltd., Wesley Street, Old Glossop, Derbyshire, SK13 7RY, UK
ASDI Inc, 601 Interchange Blvd., Newark, Del., 19711, USA
Alfa Aesar, 26 Parkridge Road, Ward Hill, Mass., 01835, USA
Bionet Research Ltd., Highfield Industrial Estate, Camelford, Cornwall, PL32 9QZ, UK
Acros Organics, Janssens Pharmaceuticalaan 3A, Geel, 2440, Belgium
Apin Chemicals Ltd., 3D Milton Park, Abingdon, Oxfordshire, OX14 4RU, UK
Pfaltz & Bauer, Inc., 172 East Aurora Street, Waterbury, Conn. 06708, USA
Trans World Chemicals, Inc., 14674 Southlawn Lane, Rockville, Md. 20850, USA
Peakdale Molecular Ltd., Peakdale Science Park, Sheffield Road, Chapel-en-le-Frith, High Peak, SK23 0PG, UK
TCI America, 9211 N. Harborgate Street, Portland, Oreg. 97203, USA
Fluka Chemie GmbH, Industriestrasse 25, P.O. Box 260, CH-9471 Buchs, Switzerland
JRD Fluorochemicals Ltd., Unit 11, Mole Business Park, Leatherhead, Surrey, KT22 7BA, UK
Instruments Used
In the following experimental details, nuclear magnetic resonance spectral data were obtained using Varian Inova 300, Varian Inova 400, Varian Mercury 400, Varian Unityplus 400, Bruker AC 300 MHz, Bruker AM 250 MHz or Varian T60 MHz spectrometers, the observed chemical shifts being consistent with the proposed structures. N.m.r chemical shifts are quoted in p.p.m downfield from tetramethylsilane. Mass spectral data were obtained on a Finnigan ThermoQuest Aqa, a Waters micromass ZQ, Bruker APEX II FT-MS or a Hewlett Packard GCMS System Model 5971 spectrometer. The calculated and observed ions quoted refer to the isotopic composition of lowest mass. HPLC means high performance liquid chromatography. Analytical HPLC data was collected on a HP1100 Series HPLC system. Preparative HPLC data was collected using a Gilson Preparative HPCL system.
CHN microanalysis data were collected using Exeter Analytical CE 440 instruments by Warwick Analytical Service, (University of Warwick Science Park, Barclays Venture Centre, Sir William Lyons Road, Coventry, CV4 7EZ).
Optical rotation data was collected using a Perkin Elmer Polarimeter 341 by Warwick analytical Service, (University of Warwick Science Park, Barclays Venture Centre, Sir William Lyons Road, Coventry, CV4 7EZ).
Example 1
2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazole
A solution of the compound of Preparation 194 (11.0 g, 38.1 mmol) and palladium(II) hydroxide (1.10 g, 7.83 mmol) in methanol (100 ml) was heated to 60° C. at a pressure of 300 psi under a hydrogen atmosphere for 18 h. The reaction mixture was then filtered and concentrated in vacuo and the residue was re-crystallised from hot acetonitrile (50 ml) to give the title compound (3.27 g).
Experimental MH+ 201.3; expected 201.1 1H-NMR (CD3OD): 1.50-1.55 (3H), 2.15-2.20 (3H), 2.20-2.25 (3H), 4.40-4.50 (1H), 6.80-6.85 (1H), 6.90-6.92 (2H), 6.95-7.00 (2H) Rhip. Funct. ED100 mg/cm2=0.1
Alternative Synthesis
A solution of the compound of Preparation 1 (72 mg, 0.36 mmol) in methanol (5 ml) was hydrogenated at 100 psi and 60° C. using palladium (10 wt % on carbon, 10 mg), overnight. The mixture was filtered and the filtrate concentrated in vacuo. The residue was dissolved in methanol (1 ml) and diethylamine (2-3 drops, 1 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [30:70 to 98:2]. The appropriate fractions were concentrated in vacuo to give the title compound (26 mg).
Experimental MH+ 201.2; expected 201.1 1H-NMR (d6-DMSO): 1.65-1.72 (3H), 2.13-2.18 (3H), 2.24-2.31 (3H), 4.43-4.52 (1H), 6.89-6.92 (2H), 7.00-7.03 (1H), 7.03-7.11 (2H)
Alternative Synthesis
To a mixture of the compound of Preparation 1 (1.0 g, 3.26 mmol) and ammonium formate (1.0 g, 15.9 mmol) in formic acid (20 ml) was added palladium (10% wt % on carbon, 1.0 g). The reaction mixture was heated at 100° C. for 72 h, filtered and concentrated in vacuo. The residue was triturated with methanol:ethyl acetate [1:9] to give the title compound (200 mg).
Experimental MH+ 201.3; expected 201.1 1H-NMR (CD3OD): 1.65-1.70 (3H), 2.20-2.25 (3H), 2.25-2.30 (3H), 4.80-4.90 (1H), 6.80-6.85 (1H), 7.00-7.10 (2H), 7.35-7.40 (2H)
Alternative Synthesis
A mixture of the crude compound of Preparation 13 (500 mg, 2.3 mmol) and palladium (10 wt % on carbon, 223 mg) in formic acid (10 ml) was heated at reflux for 36 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give the crude title compound
Experimental MH+ 201.3; expected 201.1
Example 2
2-{1-[2-Methyl-3-(trifluoromethyl)phenyl]ethyl}-1H-imidazole
A mixture of the compound of Preparation 148 (2.0 g, 5.8 mmol) and palladium (10 wt % on carbon, 500 mg) in methanol (25 ml) was heated at 60° C. under a hydrogen atmosphere (150 psi) for 24 h. The mixture was filtered through Arbocel® and the filtrate was concentrated in vacuo.
The residue was purified by flash chromatography (silica), eluting with methanol. The appropriate fractions were combined and concentrated to give the title compound (11 mg).
1H-NMR (CD3OD): 1.58-1.62 (3H), 2.40-2.43 (3H), 4.56-4.62 (1H), 6.90-6.94 (2H), 7.21-7.29 (2H), 7.47-7.51 (1H) Experimental MH+ 255.3; expected 255.1 Rhip. Funct. ED100 mg/cm2=>1
Example 3
2-[1-(1H-Imidazol-2-yl)ethyl]-6-methylbenzonitrile
To a solution of the compound of Preparation 167 (50 mg, 0.17 mmol) in 2-propanol (2 ml) was added ammonium formate (105 mg, 1.67 mmol) and palladium (10 wt % on carbon, 36 mg). The reaction mixture was heated at 80° C., under nitrogen, for 2 h and then cooled. The mixture was filtered through Arbocel®, washing through with 2-propanol, and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile:water (9:1, 4 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [30:70 (20 min) to 95:5 (21 min)]. The appropriate fractions were combined and concentrated to give the title compound (8 mg).
Experimental MH+ 212.1; expected 212.1 1H-NMR (d6-Acetone): 1.64-1.66 (3H), 2.50-2.51 (3H), 4.59-4.61 (1H), 6.90-7.05 (2H), 7.19-7.21 (1H), 7.23-7.25 (1H), 7.42-7.45 (1H) Rhip. Funct. ED100 mg/cm2=0.3
Similarly Prepared were:
Ex.
From
MH+ Found/
Rhip. Funct.
No.
Ar
Name
Prep.
Expected
ED100 mg/cm2
4
2-[1-(3-Ethylphenyl)ethyl]-1H- imidazole
146
201.3 201.1
>1
5
2-[1-(3-Cyclopropylphenyl)- ethyl]-1H-imidazole
138
213.2 213.1
>10
6
2-(1-Biphenyl-3-ylethyl)-1H- imidazole
137
249.4 249.1
<=10
7
2-[1-(2-Fluoro-3-methylphenyl)- ethyl]-1H-imidazole
143
205.2 205.1
0.1, 0.3, <=0.03
8
2-{1-[2-Methyl-5- (trifluoromethyl)phenyl]-ethyl}- 1H-imidazole
144
255.3 255.1
>1
9
2-[1-(3-Ethyl-2-methylphenyl)- ethyl]-1H-imidazole
176
215.4 215.2
1
10
3-[1-(1H-Imidazol-2-yl)ethyl]-5- methylbenzonitrile
169
212.3 212.1
<=10
11
3-[1-(1H-Imidazol-2-yl)ethyl]-2- methylbenzonitrile
168
212.2 212.1
<=10
12
2-{1-[2-(Difluoromethyl)-3- methylphenyl]ethyl}-1H- imidazole
181
237.2 237.1
>1
Example 4
1H-NMR (CDCl3): 1.14-1.21 (3H), 1.62-1.70 (3H), 2.53-2.62 (2H), 4.15-4.22 (1H), 6.80-6.85 (2H), 6.97-7.02 (2H), 7.02-7.07 (1H), 7.15-7.21 (1H)
Example 5
1H-NMR (d6-DMSO): 0.58-0.62 (2H), 0.83-0.87 (2H), 1.50-1.54 (3H), 1.80-1.84 (1H), 4.03-4.05 (1H), 6.80-6.88 (3H), 6.95-6.98 (3H), 7.10-7.14 (1H)
Example 6
1H-NMR (CD3OD): 1.68-1.74 (3H), 4.25-4.34 (1H), 6.95-6.97 (2H), 7.19-7.21 (1H), 7.27-7.45 (6H), 7.55-7.59 (2H)
Example 7
1H-NMR (d6-DMSO): 1.42-1.50 (3H), 2.15-2.20 (3H), 4.37-4.41 (1H), 6.71-6.75 (1H), 6.89-6.98 (3H), 7.03-7.06 (1H)
Example 8
1H-NMR (CD3OD): 1.61-1.65 (3H), 2.39-2.42 (3H), 4.51-4.58 (1H), 6.94-6.98 (2H), 7.32-7.36 (2H), 7.37-7.41 (1H)
Example 9
1H-NMR (d6-Acetone): 1.11-1.19 (3H), 1.55-1.59 (3H), 2.26-2.28 (3H), 2.60-2.68 (2H), 4.45-4.52 (1H), 6.89-6.93 (2H), 6.97-7.01 (3H)
Example 10
1H-NMR (CDCl3): 1.65-1.71 (3H), 2.35-2.38 (3H), 4.19-4.24 (1H), 6.98-7.00 (2H), 7.28-7.34 (3H)
Example 11
1H-NMR (d6-Acetone): 1.60-1.63 (3H), 2.58-2.59 (3H), 4.55-4.50 (1H), 6.90-6.95 (2H), 7.29-7.33 (1H), 7.50-7.60 (2H)
Example 12
1H-NMR (CDCl3): 1.70-1.75 (3H), 2.32-2.34 (3H), 4.54-4.60 (1H), 6.91-6.93 (2H), 7.21-7.25 (3H)
Example 13
1-Benzyl-2-{1-[3-(difluoromethyl)phenyl]ethyl}-1H-imidazole
To a solution of the compound of Preparation 142 (100 mg, 0.32 mmol) in 2-propanol (4 ml) was added ammonium formate (406 mg, 6.44 mmol) and palladium (10 wt % on carbon, 137 mg). The reaction mixture was heated at 80° C., under nitrogen, for 18 h and then cooled. The mixture was filtered through Arbocel®, washing through with 2-propanol, and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile:water (9:1, 4 ml) and purified by automated preparative liquid chromatography (Gilson system, 100 mm×30 mm LUNA C18(2) 5 μm column, 40 ml/min) using an acetonitrile:water gradient [40:60 (20 min) to 95:5 (25 min)]. The appropriate fractions were combined and concentrated to give the title compound (8 mg).
Experimental MH+ 313.4; expected 313.2 1H-NMR (d6-Acetone): 1.58-1.61 (3H), 4.25-4.31 (1H), 4.99-5.03 (1H), 5.10-5.14 (1H), 6.80-6.82 (1H), 6.94-6.98 (3H), 7.00-7.02 (1H), 7.20-7.25 (3H), 7.38-7.41 (4H) Rhip. Funct. ED100 mg/cm2=>1
Similarly Prepared Was:
Ex.
From
MH+ Found/
Rhip. Funct.
No.
Ar
Name
Prep.
Expected
ED100 mg/cm2
14
1-Benzyl-2-{1-[2-(difluoromethyl)- 3-methylphenyl]ethyl}-1H- imidazole
181
327.2 327.2
>1
Example 14
1H-NMR (CD3OD): 1.59-1.62 (3H), 2.30-2.31 (3H), 4.54-4.59 (1H), 6.80-6.83 (3H), 6.88-6.90 (1H), 7.00-7.02 (2H), 7.15-7.17 (1H), 7.18-7.20 (2H), 7.26-7.27 (1H)
Example 15
2-[1-(2-Methyl-3-propylphenyl)ethyl]-1H-imidazole
To a solution of the compound of Preparation 136 (720 mg, 2.3 mmol) in 2-propanol (20 ml) was added ammonium formate (1.0 g, 20 mmol) and palladium (10 wt % on carbon, 300 mg). The reaction mixture was heated at 80° C., under nitrogen, for 72 h and then cooled. The mixture was filtered through Arbocel®, washing through with 2-propanol, and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile (2 ml) and diethylamine (2-3 drops) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm LUNA C18(2) AX 5 μm column, 40 ml/min) using an acetonitrile:water gradient [40:60 (15 min) to 95:5 (15.5 min)]. The appropriate fractions were combined and concentrated to give the title compound (74 mg).
Experimental MH+ 229.3; expected 229.2 1H-NMR (d6-Acetone): 0.95-1.00 (3H), 1.51-1.60 (5H), 2.13-2.15 (3H), 2.58-2.61 (2H), 4.47-4.52 (1H), 6.85-6.90 (2H), 6.96-7.00 (3H) Rhip. Funct. ED100 mg/cm2<=10
Example 16
2-{1-[2-(Trifluoromethyl)phenyl]ethyl}-1H-imidazole
A mixture of the compound of Preparation 51 (212 mg, 0.88 mmol) and palladium (10 wt % on carbon, 500 mg) in methanol (10 ml) was heated at 60° C. under a hydrogen atmosphere (150 psi) for 60 h. The mixture was filtered through Arbocel® and the filtrate was concentrated in vacuo.
The residue was dissolved in methanol (2 ml) diethylamine (2-3 drops) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm LUNA C18(2) 10 μm column, 40 ml min) using an acetonitrile:water gradient [35:65 to 95:5]. The appropriate fractions were concentrated in vacuo to give the title compound (58 mg).
Experimental MH+ 241.3; expected 241.1 1H-NMR (CD3OD): 1.60-1.66 (3H), 4.53-4.61 (1H), 6.88-6.95 (2H), 7.31-7.39 (2H), 7.48-7.53 (1H), 7.62-7.68 (1H) Rhip. Funct. ED100 mg/cm2=3
Similarly Prepared were:
Ex.
From
MH+ Found/
Rhip. Funct.
No.
Ar
Name
Prep.
Expected
ED100 mg/cm2
17
2-[1-(2,5-Dimethyl- phenyl)ethyl]-1H- imidazole
49
201.4 201.1
>10
18
2-[1-(2,6-Dimethyl- phenyl)ethyl]-1H- imidazole
66
201.3 201.1
>1
19
2-[1-(3,5-Dimethyl- phenyl)ethyl]-1H- imidazole
50
201.4 201.1
0.3, 1
20
2-[1-(3-Methylphenyl)- ethyl]-1H-imidazole
48
187.3 187.1
0.1
21
2-(1-Phenylethyl)-1H- imidazole
57
173.2 173.1
1
22
2-[1-(4-Methylphenyl)- ethyl]-1H-imidazole
58
187.3 187.1
>1
23
2-(1-Mesitylethyl)-1H- imidazole
59
215.4 215.2
>1
24
2-{1-[3-(Trifluoro- methyl)- phenyl]ethyl}- 1H-imidazole
60
241.3 241.1
>1
25
2-{1-[4-(Trifluoro- methyl)- phenyl]ethyl}-1H- imidazole
61
241.3 241.1
>1
26
2-[1-(3-Methoxy-2- methylphenyl)ethyl]-1H- imidazole
62
217.3 217.1
>1
27
2-[1-(2-Ethyl-3-methyl- phenyl)ethyl]-1H- imidazole
63
215.3 215.2
<=10
28
2-{1-[3-(Trifluorometh- oxy)phenyl]ethyl}-1H- imidazole
65
257.1 257.1
<=10
29
2-[1-(2,6-Difluoro-3- methylphenyl)ethyl]-1H- imidazole
73
223.2 223.1
>1
30
2-[1-(3,5-Difluoro- phenyl)ethyl]-1H- imidazole
10
209.2 209.1
>1
31
2-{1-[2-Fluoro-3- (trifluoromethyl)phenyl] ethyl}-1H-imidazole
12
259.1 259.1
>1
Example 17
1H-NMR (d6-Acetone): 1.60-1.70 (3H), 2.08-2.15 (3H), 2.21-2.30 (3H), 4.40-4.50 (1H), 6.81-6.92 (2H), 6.93-6.99 (1H), 7.00-7.08 (2H)
Example 18
1H-NMR (CDCl3): 1.68-1.72 (3H), 2.04-2.12 (6H), 4.49-4.55 (1H), 6.86-6.91 (2H), 6.95-6.98 (2H), 7.00-7.05 (1H)
Example 19
1H-NMR (CD3OD): 1.55-1.60 (3H), 2.19-2.21 (6H), 4.05-4.15 (1H), 6.75-7.80 (3H), 6.85-6.90 (2H)
Example 20
1H-NMR (CDCl3): 1.67-1.71 (3H), 2.28-2.30 (3H), 4.12-4.18 (1H), 6.90-6.93 (2H), 7.00-7.06 (2H), 7.17-7.23 (2H)
Example 21
1H-NMR (CDCl3): 1.67-1.72 (3H), 4.14-4.21 (1H), 6.89-6.94 (2H), 7.18-7.25 (3H), 7.26-7.33 (2H)
Example 22
1H-NMR (CDCl3): 1.67-1.70 (3H), 2.29-2.31 (3H), 4.12-4.18 (1H), 6.89-6.92 (2H), 7.10-7.12 (4H)
Example 23
1H-NMR (CD3OD): 1.55-1.65 (3H), 2.00-2.10 (3H), 2.14-2.17 (3H), 2.18-2.20 (3H), 4.40-4.50 (1H), 6.80-6.90 (1H), 6.90-6.95 (3H)
Example 24
1H-NMR (CDCl3): 1.69-1.73 (3H), 4.23-4.30 (1H), 6.92-6.97 (2H), 7.31-7.35 (2H), 7.52-7.56 (2H)
Example 25
1H-NMR (CDCl3): 1.69-1.74 (3H), 4.22-4.30 (1H), 6.92-6.97 (2H), 7.31-7.35 (2H), 7.52-7.56 (2H)
Example 26
1H-NMR (CDCl3): 1.64-1.68 (3H), 2.09-2.13 (3H), 3.77-3.81 (3H), 4.38-4.45 (1H), 6.72-6.77 (2H), 6.87-6.89 (2H), 7.09-7.15 (1H)
Example 27
1H-NMR (CD3OD): 1.03-1.09 (3H), 1.58-1.63 (3H), 2.29-2.31 (3H), 2.65-2.75 (2H), 4.42-4.48 (1H), 6.82-6.85 (2H), 6.92-7.00 (3H)
Example 28
1H-NMR (CD3OD): 1.60-1.65 (3H), 4.20-4.26 (1H), 6.90-6.93 (2H), 7.03-7.06 (2H), 7.18-7.20 (1H), 7.33-7.37 (1H)
Example 29
1H-NMR (CDCl3): 1.68-1.72 (3H), 2.17-2.20 (3H), 4.60-4.65 (1H), 6.70-6.75 (1H), 6.90-6.93 (2H), 6.95-7.00 (1H)
Example 32
2-[1-(2,3,5-Trimethylphenyl)ethyl]-1H-imidazole
A mixture of the compound of Preparation 64 (150 mg, 0.52 mmol) and palladium (10 wt % on carbon, 15 mg) in 2-propanol (5 ml) was heated at 60° C. under a hydrogen atmosphere (200 psi) for 18 h. The mixture was filtered through Arbocel® and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile (1.22 ml) and diethylamine (2-3 drops) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm LUNA C18(2) 5 μm column, 40 ml/min) using an acetonitrile:water gradient [32:68 (20 min) to 95:5 (21 min)]. The appropriate fractions were combined and concentrated to give the title compound (30 mg).
Experimental MH+ 215.4; expected 215.2 1H-NMR (CD3OD): 1.57-1.60 (3H), 2.15-2.19 (6H), 2.20-2.22 (3H), 4.35-4.39 (1H), 6.80-6.82 (1H), 6.87-6.90 (3H) Rhip. Funct. ED100 mg/cm2=>1
Example 33
2-[1-(2,3-Dimethylphenyl)propyl]-1H-imidazole
A mixture of the compound of Preparation 47 (255 mg, 1.2 mmol) and palladium (10 wt % on carbon, 50 mg) in 2-propanol (50 ml) was heated at 40° C. under a hydrogen atmosphere (200 psi) for 18 h. The mixture was filtered through Arbocel® and the filtrate was concentrated in vacuo. The residue was re-crystallised from warm diethyl ether (5 ml) and the solid was triturated with further diethyl ether (5 ml) to give the title compound (175 mg).
Experimental MH+ 215.3; expected 215.2 1H-NMR (CDCl3): 0.87-0.95 (3H), 1.90-2.03 (1H), 2.11-2.16 (3H), 2.23-2.27 (3H), 2.28-2.38 (1H), 4.19-4.25 (1H), 6.85-6.90 (2H), 7.01-7.07 (3H) Rhip. Funct. ED100 mg/cm2=1
Example 34
2-[1-(2-Chloro-3-methylphenyl)ethyl]-1H-imidazole
A mixture of the compound of Preparation 67 (1.51 g, 6.8 mmol) and palladium hydroxide (20 wt % Pd on carbon, 500 mg) in 2-propanol (100 ml) was heated at 50° C. under a hydrogen atmosphere (200 psi) for 18 h. The mixture was filtered through Arbocel® and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile (1 ml) and diethylamine (2-3 drops) and purified by automated preparative liquid chromatography (Gilson system, 100 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [35:65 (15 min) to 95:5 (15.5 min)]. The appropriate fractions were combined and concentrated to give the title compound (21 mg).
Experimental MH+ 221.3; expected 221.1 1H-NMR (d6-DMSO): 1.49-1.53 (3H), 2.34-2.37 (3H), 4.58-4.62 (1H), 6.79-6.81 (1H), 6.95-7.00 (2H), 7.10-7.13 (1H), 7.18-7.20 (1H) Rhip. Funct. ED100 mg/cm2=0.1
Similarly Prepared were:
Ex.
From
MH+ Found/
Rhip. Funct.
No.
Ar
Name
Prep.
Expected
ED100 mg/cm2
35
2-[1-(2,4-Dichloro- phenyl)ethyl]-1H- imidazole
56
241.2 241.0
>1
36
2-[1-(4-Chloro-3-methyl- phenyl)ethyl]-1H- imidazole
74
221.3 221.1
>10
37
2-[1-(2,3-Dichloro- phenyl)ethyl]-1H- imidazole
52
241.2 241.0
>1
38
2-[1-(3,4-Dichloro- phenyl)ethyl]-1H- imidazole
53
241.2 241.0
3
39
2-[1-(3-Chlorophenyl)- ethyl]-1H-imidazole
54
201.3 201.3
>1
40
2-[1-(2-chloro-4- Methoxyphenyl)ethyl]- 1H-imidazole
75
237.3 237.1
>1
41
2-[1-(3-Chloro-2- methoxyphenyl)ethyl]- 1H-imidazole
77
237.3 237.1
>1
42
2-[1-(3-Chloro-4- methoxyphenyl)ethyl]- 1H-imidazole
76
237.3 237.1
>1
43
2-[1-(2,5-Dichloro- phenyl)ethyl]-1H- imidazole
55
241.2 241.0
>1
44
2-[1-(3-Chloro-4-methyl- phenyl)ethyl]-1H- imidazole
68
221.3 221.1
>1
45
2-[1-(3-Chloro-2-methyl- phenyl)ethyl]-1H- imidazole
69
221.3 221.1
>1
46
2-[1-(2-Chloro-5- methoxyphenyl)ethyl]- 1H-imidazole
70
237.2 237.1
<=10
47
2-[1-(2-Chloro-5-methyl- phenyl)ethyl]-1H- imidazole
71
221.3 221.1
—
Example 36
1H-NMR (CD3OD): 1.53-1.62 (3H), 2.20-2.28 (3H), 4.10-4.20 (1H), 6.83-6.92 (2H), 6.92-6.99 (1H), 7.06-7.11 (1H), 7.14-7.21 (1H)
Example 38
1H-NMR (CD3OD): 1.60-1.65 (3H), 4.20-4.30 (1H), 6.90-7.00 (2H), 7.10-7.15 (1H), 7.36-7.40 (1H), 7.40-7.44 (1H)
Example 39
1H-NMR (CD3OD): 1.60-1.63 (3H), 4.20-4.24 (1H), 6.95-6.97 (2H), 7.14-7.16 (1H), 7.19-7.27 (3H)
Example 40
1H-NMR (d6-Acetone): 1.55-1.65 (3H), 3.75-3.81 (3H), 5.18-5.25 (1H), 6.80-6.85 (1H), 6.95-6.98 (1H), 7.10-7.20 (2H), 7.35-7.40 (1H)
Example 43
1H-NMR (CD3OD): 1.58-1.61 (3H), 4.60-4.64 (1H), 6.95-6.97 (2H), 7.07-7.08 (1H), 7.18-7.20 (1H), 7.37-7.39 (1H)
Example 44
1H-NMR (CD3OD): 1.58-1.61 (3H), 2.24-2.26 (3H), 4.15-4.20 (1H), 6.89-6.91 (2H), 7.00-7.02 (1H), 7.17-7.19 (2H)
Example 46
1H-NMR (CD3OD): 1.57-1.60 (3H), 3.62-3.63 (3H), 4.60-4.65 (1H), 6.60-6.61 (1H), 6.72-6.75 (1H), 6.91-6.93 (2H), 7.21-7.24 (1H)
Example 48
2-[1-(2,3-Difluorophenyl)ethyl]-1H-imidazole
To a solution of the compound of Preparation 2 (320 mg, 1.55 mmol) in 2-propanol (20 ml) was added ammonium formate (1.47 g, 23.3 mmol) and palladium (10 wt % on carbon, 495 mg). The reaction mixture was heated at 80° C. for 18 h and then cooled. The mixture was filtered through Arbocel®, washing through with 2-propanol (10 ml), and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile (2 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [30:70 (20 min) to 95:5 (21 min)]. The appropriate fractions were combined and concentrated to give the title compound (10 mg).
Experimental MH+ 209.4; expected 209.1 1H-NMR (CD3OD): 1.62-1.65 (3H), 4.54-4.61 (1H), 6.90-6.96 (2H), 7.02-7.25 (3H) Rhip. Funct. ED100 mg/cm2=3
Similarly Prepared were:
Rhip. Funct.
Ex.
From
MH+ Found/
ED100
No.
Ar
Name
Prep.
Expected
mg/cm2
49
2-[1-(5-Methoxy-2,4- dimethylphenyl)ethyl]- 1H-imidazole
11
231.2 231.1
<=10
50
2-[1-(4-Fluoro-3-methyl- phenyl)ethyl]-1H- imidazole
4
205.2 205.1
>1
51
2-[1-(2,6-Difluoro- phenyl)ethyl]-1H- imidazole
5
209.2 209.1
>1
52
2-[1-(3-Fluoro-2-methyl- phenyl)ethyl]-1H- imidazole
6
205.3 205.1
>1
53
2-[1-(3-Fluorophenyl)- ethyl]-1H-imidazole
7
191.3 191.1
>1
54
2-{1-[2-Chloro-3- (trifluoromethyl)phenyl]- ethyl}-1H-imidazole
8
275.1 275.1
>1
55
2-[1-(3-Fluoro-5-methyl- phenyl)ethyl]-1H- imidazole
9
205.3 205.1
0.3
56
2-{1-[3-Methyl-2- (trifluoromethyl)phenyl]- ethyl}-1H-imidazole
72
255.3 255.1
>1
Example 49
1H-NMR (d6-DMSO): 1.45-1.49 (3H), 2.00-2.02 (3H), 2.19-2.21 (3H), 3.60-3.61 (3H), 4.23-4.27 (1H), 6.70-6.72 (1H), 6.77-6.79 (1H), 6.83-6.85 (1H), 6.92-6.95 (1H)
Example 50
1H-NMR (CDCl3): 1.62-1.67 (3H), 2.15-2.20 (3H), 4.19-4.24 (1H), 6.82-6.87 (3H), 6.97-7.01 (2H)
Example 51
1H-NMR (CDCl3): 1.75-1.80 (3H), 4.68-4.73 (1H), 6.82-6.87 (2H), 6.94-6.96 (2H), 7.17-7.21 (1H)
Example 52
1H-NMR (CDCl3): 1.63-1.66 (3H), 2.18-2.20 (3H), 4.39-4.44 (1H), 6.90-7.00 (4H), 7.10-7.15 (1H)
Example 53
1H-NMR (CDCl3): 1.70-1.74 (3H), 4.18-4.23 (1H), 6.90-7.00 (4H), 7.00-7.02 (1H), 7.17-7.20 (1H)
Example 54
1H-NMR (CDCl3): 1.69-1.74 (3H), 4.79-4.85 (1H), 6.94-6.98 (2H), 7.26-7.31 (1H), 7.41-7.44 (1H), 7.55-7.58 (1H)
Example 55
1H-NMR (d6-Acetone): 1.60-1.64 (3H), 2.25-2.27 (3H), 4.19-4.24 (1H), 6.75-6.82 (2H), 6.85-6.98 (3H)
Example 56
1H-NMR (CD3OD): 1.60-1.64 (3H), 2.50-2.54 (3H), 4.63-4.67 (1H), 6.90-6.93 (2H), 7.07-7.10 (1H), 7.18-7.20 (1H), 7.30-7.34 (1H)
Example 57
2-[1-(2-Fluoro-5-methylphenyl)ethyl]-1H-imidazole
A mixture of the compound of Preparation 3 (74 mg, 0.31 mmol), palladium (10 wt % on carbon, 140 mg) and ammonium formate (394 mg, 6.4 mmol) in 2-propanol (20 ml) was heated at 80° C. for 24 h. The mixture was filtered through Arbocel® and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile:methanol (1 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [50:50 (20 min) to 98:2 (20.5 min)]. The appropriate fractions were combined and concentrated to give the title compound (49 mg).
Experimental MH+ 205.1; expected 205.1 1H-NMR (d6-Acetone): 1.60-1.63 (3H), 2.20-2.22 (3H), 4.43-4.47 (1H), 6.84-6.86 (1H), 6.90-7.00 (2H), 7.00-7.07 (2H) Rhip. Funct. ED100 mg/cm2=>1
Example 58
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazole
The compound of Example 1 (750 mg, 3.75 mmol) was dissolved in ethanol (4 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 50×50 mm ID Chiralcel OD, 20 μm column, 50 ml/min) using ethanol:hexane [10:90] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (370 mg).
Retention time=5.79 min Chiralcel OD-H, 250×4.6 mm ID, 5 μm column, ethanol:hexane [10:90], 1 ml/min Experimental MH+ 201.3; expected 201.1 1H-NMR (CD3OD): 1.56-1.60 (3H), 2.18-2.20 (3H), 2.22-2.24 (3H), 4.45-4.50 (1H), 6.80-6.86 (3H), 6.95-6.99 (2H) Optical rotation, (25° C., methanol, 5.035 mg/ml, path length 100 mm): 365 nm=+266.93, 546 nm=+88.43, 589 nm=+73.58 Rhip. Funct. ED100 mg/cm2=0.1
Alternative Synthesis
To a solution of the compound of Preparation 1 (600 g, 3 mol) in methanol (6.0 l) was added bis(norbornadiene)rhodium (I) tetrafluoroborate (1.50 g) and S(+)-1-[(R)-2-diphenyl phosphinoferrocenyl]ethylditert.butylphosphine (2.61 g) and the reaction mixture was heated at 25° C., under a hydrogen atmosphere (45-60 psi), for 10 h. The reaction was monitored by HPLC, (upon completion:starting material <0.1%, optical purity 93-94%). To the mixture was added charcoal (60 g) and the solution was stirred for 30 min. The mixture was filtered through Hyflo Super Cel®, washing through with methanol (2×300 ml). To the filtrate was added di-p-toluoyl-L-tartaric acid (1.2 kg, 3.08 mol). The reaction mixture was stirred at room temperature for 1 h and the solid material formed was collected by filtration. To the solid salt was added dichloromethane (6.0 l) and aqueous sodium hydroxide solution (1N, 6.0 l) and the reaction mixture was stirred for 30 min. The organic layer was separated and was washed with aqueous sodium hydroxide solution (1N, 2×3.0 L). The organic layer was extracted with hydrochloric acid (1N, 3×2.0 L). The combined acidic aqueous layer was adjusted to pH 10 by addition of aqueous sodium hydroxide solution (1N) and the resulting precipitate was collected by filtration and dried in vacuo, at 50° C., to give the title compound (optical purity 98.58%). The process of di-p-toluoyl-L-tartaric acid salt formation and generation of free base was repeated once more to give the title compound (0.359 kg, optical purity 99.66%) after second resolution.
Example 59
2-[(1R)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazole
The compound of Example 1 (750 mg, 3.75 mmol) was dissolved in ethanol (4 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 50×50 mm ID Chiralcel OD, 20 μm column, 50 ml/min) using ethanol:hexane [10:90] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (370 mg).
Retention time=7.84 min Chiralcel OD-H, 250×4.6 mm ID, 5 μm column, ethanol:hexane [10:90], 1 ml/min Experimental MH+ 201.3; expected 201.1 1H-NMR (CD3OD): 1.56-1.60 (3H), 2.18-2.20 (3H), 2.22-2.24 (3H), 4.43-4.48 (1H), 6.80-6.86 (3H), 6.95-6.99 (2H) Optical rotation, (25° C., methanol, 5.24 mg/ml, path length 100 mm): 365 nm=−262.79, 546 nm=−86.26, 589 nm=−72.23 Rhip. Funct. ED100 mg/cm2=>10
Example 60
2-[(1R*)-1-(3-Methylphenyl)ethyl]-1H-imidazole
The compound of Example 20 (40 mg, 0.22 mmol) was dissolved in ethanol (1 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 250×20 mm ID Chiralpak AD-H, 5 μm column, 15 ml/min) using ethanol:hexane [5:95] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (16 mg).
Retention time=7.93 min Chiralpak AD-H, 250×4.6 mm ID, 5 μm column, ethanol:hexane [10:90], 1 ml/min Experimental MH+ 187.2; expected 187.1 1H-NMR (CD3OD): 1.58-1.62 (3H), 2.23-2.27 (3H), 4.12-4.18 (1H), 6.87-6.89 (2H), 6.94-7.01 (3H), 7.09-7.14 (1H) Rhip. Funct. ED100 mg/cm2=0.3
Example 61
2-[(1R*)-1-(3-Methylphenyl)ethyl]-1H-imidazole
The compound of Example 20 (40 mg, 0.22 mmol) was dissolved in ethanol (1 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 250×20 mm ID Chiralpak AD-H, 5 μm column, 15 ml/min) using ethanol:hexane [5:95] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (15 mg).
Retention time=6.06 min Chiralpak AD-H, 250×4.6 mm ID, 5 μm column, ethanol:hexane [10:90], 1 ml/min Experimental MH+ 187.2; expected 187.1 1H-NMR (CD3OD): 1.58-1.62 (3H), 2.23-2.27 (3H), 4.12-4.18 (1H), 6.87-6.90 (2H), 6.94-7.00 (3H), 7.09-7.14 (1H) Rhip. Funct. ED100 mg/cm2=0.1
Example 62
1-Benzyl-2-[(1R*)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
The compound of Example 76 (540 mg, 1.8 mmol) was dissolved in ethanol (2 ml) and hexane (2 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 50×50 mm ID Chiralcel OD, 20 μm column, 40 ml/min) using ethanol:hexane [5:95] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (240 mg).
Retention time=5.82 min Chiralcel OD-H, 250×4.6 mm ID, 5 μm column, ethanol:hexane [10:90], 1 ml/min Experimental MH+ 291.3; expected 291.1 1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.19-2.26 (6H), 4.31-4.36 (1H), 4.68-4.72 (1H), 4.90-4.94 (1H), 6.70-6.72 (1H), 6.90-7.00 (6H), 7.20-7.25 (3H) Rhip. Funct. ED100 mg/cm2=0.3
Example 63
1-Benzyl-2-[1-(1R*)-(2,3-dimethylphenyl)ethyl]-1H-imidazole
The compound of Example 76 (540 mg, 1.8 mmol) was dissolved in ethanol (2 ml) and hexane (2 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 50×50 mm ID Chiralcel OD, 20 μm column, 40 ml/min) using ethanol:hexane [5:95] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (260 mg).
Retention time=8.80 min Chiralcel OD-H, 250×4.6 mm ID, 5 μm column, ethanol:hexane [10:90], 1 ml/min Experimental MH+ 291.3; expected 291.1 Rhip. Funct. ED100 mg/cm2=>3
Example 64
2-[(1R*)-1-(2-Fluoro-3-methylphenyl)ethyl]-1H-imidazole
The compound of Example 7 (18 mg, 0.09 mmol) was dissolved in ethanol:hexane (1:1, 2 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 250×20 mm ID Chiralcel OD-H, 5 μm column, 15 ml/min) using ethanol:hexane [5:95] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (6 mg). Retention time=6.15 min Chiralcel OD-H, 250×4.6 mm ID, 5 μm column, ethanol:hexane [10:90], 1 ml/min
Rhip. Funct. ED100 mg/cm2=0.3
Example 65
2-[(1R*)-1-(2-Fluoro-3-methylphenyl)ethyl]-1H-imidazole
The compound of Example 7 (18 mg, 0.09 mmol) was dissolved in ethanol:hexane (1:1, 2 ml) and the enantiomers were separated by automated preparative liquid chromatography (Gilson system, 250×20 mm ID Chiralcel OD-H, 5 μm column, 15 ml/min) using ethanol:hexane [5:95] as the mobile phase. The appropriate fractions were combined and concentrated to give the title compound (7 mg).
Retention time=6.90 min Chiralcel OD-H, 250×4.6 mm ID 5 μm column, ethanol:hexane [10:90], 1 ml/min Rhip. Funct. ED100 mg/cm2=1
Example 66
{2-[(1R*)1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
To a suspension of the compound of Example 1 (120 mg, 0.6 mmol) and potassium carbonate (246 mg, 1.8 mmol) in dimethylformamide (4 ml) was added chloromethyl pivalate (215 μl, 1.5 mmol) and the reaction mixture stirred at room temperature overnight. Water (10 ml) was added and the mixture then extracted with ethyl acetate (2×10 ml). The organic layers were combined, washed with water (10 ml) and brine (10 ml), dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in acetonitrile (2 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×21.2 mm LUNA C18(2) 5 μm column) using an acetonitrile:water gradient [50:50 to 95:5]. The appropriate fractions were concentrated in vacuo to give the title compound (136 mg).
Experimental MH+ 315.4; expected 315.2 1H-NMR (CD3OD): 0.96-0.99 (9H), 1.58-1.61 (3H), 2.28-2.30 (3H), 2.35-2.38 (3H), 4.62-4.72 (1H), 5.53-5.66 (2H), 6.60-6.64 (1H), 6.90-7.00 (3H), 7.18-7.19 (1H) Rhip. Funct. ED100 mg/cm2=0.03
Example 67
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl propionate
To a suspension of the compound of Example 1 (120 mg, 0.6 mmol) and caesium carbonate (731 mg, 1.8 mmol) in acetone (4 ml), under nitrogen, was added chloromethyl propionate (Eur. J. Pharm. Sci: 24; 5; 2005; 433-440, 183 mg, 1.5 mmol) and the reaction mixture was stirred at room temperature for 18 h. The mixture was filtered and the filtrate was concentrated in vacuo.
The residue was dissolved in acetonitrile (1.5 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×21.2 mm LUNA C18(2) 5 mm column) using an acetonitrile:water gradient [50:50 to 95:5]. The appropriate fractions were concentrated to give the title compound (137 mg).
Experimental MH+ 287.4; expected 287.2 1H-NMR (CD3OD): 0.84-0.11 (3H), 1.53-1.59 (3H), 1.82-2.02 (2H), 2.22-2.30 (3H), 2.31-2.38 (3H), 4.60-4.68 (1H), 5.41-5.48 (1H), 5.64-5.69 (1H), 6.50-6.56 (1H), 6.83-6.99 (3H), 7.13-7.16 (1H) Rhip. Funct. ED100 mg/cm2=0.01
Similarly Prepared from Example 1 were:
MH+
Rhip. Funct.
Ex.
Found/
ED100
No.
R6
Precursor
From
Expected
mg/cm2
68
Chloromethyl 3-methyl- butanoate
Ref. 1
315.5 315.2
0.03
69
Chloromethyl heptanoate
Prep. 117
343.5 343.2
0.03
70
Chloromethyl butyrate
—
301.5 301.2
0.01
71
Chloromethyl 3- cyclopentylpropanoate
Prep. 116
355.6 355.3
<=0.03
72
Chloromethyl pentanoate
Ref. 2
315.4 315.2
0.03
73
Chloromethyl 3,3- dimethylbutanoate
Prep. 118
329.4 329.2
0.01
74
Chloromethyl 2-methyl- propanoate
Ref. 3
301.6 301.3
<0.03 <=0.1
Ref. 1: Acta Chem. Scand. Ser. B; EN; 36; 7; 1982; 467-474.
Ref. 2: J. Am. Chem. Soc.; 43; 1921; 665
Ref. 3: EP-79782, Example 6
Example 68
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-methyl butanoate
1H-NMR (CD3OD): 0.75-0.80 (6H), 1.54-1.58 (3H), 1.70-1.80 (1H), 1.80-1.84 (2H), 2.23-2.30 (3H), 2.31-2.34 (3H), 4.60-4.68 (1H), 5.48-5.55 (1H), 5.61-5.68 (1H), 6.51-6.58 (1H), 6.83-6.95 (2H), 6.95-6.99 (1H), 7.12-7.18 (1H)
Example 69
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl heptanoate
1H-NMR (CD3OD): 0.82-0.89 (3H), 1.08-1.3 (6H), 1.3-1.4 (2H), 1.52-1.59 (3H), 1.88-1.98 (2H), 2.24-2.30 (3H), 2.30-2.35 (3H), 4.60-4.69 (1H), 5.42-5.51 (1H), 5.62-5.71 (1H), 6.50-6.56 (1H), 6.86-6.99 (3H), 7.12-7.16 (1H)
Example 70
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl butyrate
1H-NMR (CD3OD): 0.78-0.83 (3H), 1.35-1.47 (2H), 1.55-1.61 (3H), 1.90-1.98 (2H), 2.28-2.32 (3H), 2.33-2.37 (3H), 4.61-4.70 (1H), 5.48-5.55 (1H), 5.64-5.72 (1H), 6.53-6.59 (1H), 6.90-6.96 (2H), 6.97-7.01 (1H), 7.16-7.20 (1H)
Example 71
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-cyclopentylpropanoate
1H-NMR (CD3OD): 0.92-1.06 (2H), 1.35-1.45 (2H), 1.47-1.55 (2H), 1.56-1.63 (6H), 1.63-1.73 (2H), 1.91-2.00 (2H), 2.28-2.33 (3H), 2.33-2.38 (3H), 4.62-4.71 (1H), 5.48-5.55 (1H), 5.68-5.73 (1H), 6.52-6.59 (1H), 6.88-7.01 (3H), 7.18-7.19 (1H)
Example 72
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pentanoate
1H-NMR (CD3OD): 0.82-0.86 (3H), 1.13-1.24 (2H), 1.31-1.40 (2H), 1.56-1.61 (3H), 1.87-2.00 (2H), 2.26-2.31 (3H), 2.32-2.36 (2H), 4.61-4.69 (1H), 5.46-5.52 (1H), 5.66-5.72 (1H), 6.52-6.58 (1H), 6.88-7.00 (3H), 7.15-7.17 (1H)
Example 73
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3,3-dimethylbutanoate
1H-NMR (d6-Acetone): 0.89-0.92 (9H), 1.58-1.60 (3H), 1.96-1.97 (2H), 2.29-2.31 (3H), 2.38-2.40 (3H), 4.60-4.64 (1H), 5.50-5.54 (1H), 5.70-5.74 (1H), 6.67-6.69 (1H), 6.90-6.96 (2H), 6.99-7.01 (1H), 7.12-7.14 (1H)
Example 74
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 2-methyl propanoate
1H-NMR (d6-Acetone): 0.91-0.94 (6H), 1.58-1.60 (3H), 2.20-2.24 (1H), 2.24-2.26 (3H), 2.36-2.38 (3H), 4.60-4.64 (1H), 5.66-5.70 (1H), 5.68-5.70 (1H), 6.90-6.95 (2H), 6.98-7.00 (1H), 7.12-7.14 (1H)
Similarly Prepared from Example 58 was:
MH+
Rhip. Funct.
Ex.
Found/
ED100
No.
R6
Precursor
From
Expected
mg/cm2
75
Chloromethyl propionate
Ref. 4
287.2 287.2
<=0.03, <=0.01, 0.3
Ref. 4: J. Am. Chem. Soc.; 43; 1921; 660
Example 75
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl propionate
1H-NMR (d6-Acetone): 0.89-0.95 (3H), 1.57-1.60 (3H), 2.03-2.06 (2H), 2.27-2.29 (3H), 2.32-2.35 (3H), 4.60-4.65 (1H), 5.44-5.50 (1H), 5.71-5.76 (1H), 6.62-6.64 (1H), 6.90-7.00 (3H), 7.14-7.16 (1H)
Example 76
1-Benzyl-2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
To a suspension of the compound of Example 1 (100 mg, 0.50 mmol) and caesium carbonate (407 mg, 1.25 mmol) in acetone (4 ml) was added benzyl bromide (171 mg, 1.00 mmol). The reaction mixture was stirred at room temperature, under nitrogen, for 18 h and then concentrated in vacuo. The residue was partitioned between water (10 ml) and ethyl acetate (10 ml) and the two layers were separated. The aqueous layer was extracted with ethyl acetate (10 ml) and the combined organic phases were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol:water (9:1, 2 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [60:40 to 95:5]. The appropriate fractions were concentrated in vacuo to give the title compound (100 mg).
Experimental MH+ 291.0; expected 291.2 1H-NMR (d6-Acetone): 1.45-1.55 (3H), 2.15-2.20 (3H), 2.20-2.24 (3H), 4.26-4.35 (1H), 4.65-4.70 (1H), 4.85-4.93 (1H), 6.66-6.70 (1H), 6.82-7.00 (6H), 7.17-7.28 (3H) Rhip. Funct. ED100 mg/cm2=0.01
Similarly Prepared from Example 1 were:
MH+
Rhip. Funct.
Ex.
Precursor
Found/
ED100
No.
R6
(all commercially available)
Expected
mg/cm2
77
1-(Bromomethyl)-3-methoxybenzene
321.4 321.2
>1
78
(1-Bromoethyl)benzene
305.4 305.2
>1
Example 77
2-[1-(2,3-Dimethylphenyl)ethyl]-1-(3-methoxybenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.50-1.56 (3H), 2.19-2.28 (6H), 3.66-3.72 (3H), 4.30-4.39 (1H), 4.66-4.71 (1H), 4.86-4.94 (1H), 6.40-6.44 (1H), 6.48-6.52 (1H), 6.71-6.80 (2H), 6.90-6.99 (3H), 7.00-7.02 (1H), 7.14-7.20 (1H)
Example 78
2-[1-(2,3-Dimethylphenyl)ethyl]-1-(1-phenylethyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.72-1.79 (3H), 1.80-1.90 (3H), 2.23-2.31 (6H), 5.05-5.11 (1H), 5.48-5.58 (1H), 6.64-6.70 (1H), 6.70-6.80 (2H), 6.88-6.95 (1H), 6.95-7.00 (1H), 7.04-7.20 (4H), 7.66-7.70 (1H)
Example 79
1-[4-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)phenyl]-1H-1,2,4-triazole
To a mixture of the compound of Example 58 (90 mg, 0.45 mmol) and caesium carbonate (244 mg, 0.75 mmol) in 1-methyl-2-pyrrolidinone (1 ml) was added 1-[4-(bromomethyl)phenyl]-1H-1,2,4-triazole (83 μl, 0.5 mmol). The reaction mixture was stirred at room temperature for 18 h and then concentrated in vacuo. The residue was dissolved in 1-methyl-2-pyrrolidinone (0.8 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×22.4 mm LUNA C18(2) 5 μm column, 20 ml/min) using an acetonitrile:water gradient [15:85 (3 min) to 98:2 (11 min)]. The appropriate fractions were combined and concentrated to give the title compound (57 mg).
Experimental MH+ 358.5; expected 358.2 1H-NMR (CDCl3): 1.61-1.66 (3H), 2.13-2.21 (6H), 4.20-4.26 (1H), 4.60-4.80 (2H), 6.63-6.66 (1H), 6.80-6.82 (1H), 6.87-6.98 (4H), 7.11-7.13 (1H), 7.47-7.51 (2H), 8.04-8.06 (1H), 8.43-8.45 (1H) Rhip. Funct. ED100 mg/cm2<=10
Example 80
1-[3-(Benzyloxy)benzyl]-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
To a mixture of the compound of Example 58 (90 mg, 0.45 mmol) and caesium carbonate (244 mg, 0.75 mmol) in 1-methyl-2-pyrrolidinone (1 ml) was added 1-(benzyloxy)-3-(bromomethyl)benzene (139 mg, 0.50 mmol). The reaction mixture was stirred at room temperature for 48 h and then filtered through a Whatman PTFE filter tube (5 μm).
The filtrate was purified by automated preparative liquid chromatography (Gilson system, 150 mm×22.4 mm LUNA C18(2) 5 μm column, 20 ml/min) using an acetonitrile:water gradient [50:50 (15 min) to 98:2 (20 min)]. The appropriate fractions were combined and concentrated to give the title compound (31 mg).
Experimental MH+ 397.5; expected 397.2 1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.19-2.25 (6H), 4.33-4.38 (1H), 4.65-4.70 (1H), 4.90-4.95 (1H), 4.98-5.00 (2H), 6.46-6.51 (2H), 6.74-6.76 (1H), 6.84-6.98 (4H), 7.00-7.01 (1H), 7.15-7.20 (1H), 7.31-7.42 (5H) Rhip. Funct. ED100 mg/cm2<=10
Similarly Prepared from Example 58 were:
MH+
Rhip. Funct.
Ex.
Found/
ED100
No.
R6
Precursor
Expected
mg/cm2
81
1-(Bromomethyl)-4-(methyl- sulfonyl)benzene
369.4 369.1
1
82
[4-(Bromomethyl)phenyl] (phenyl)methanone
395.4 395.2
<=10
83
Methyl 4-(bromomethyl)- benzoate
349.4 349.2
<=10
84
4-(Bromomethyl)pyridine
292.4 292.2
<=10
85
3-(Bromomethyl)benzonitrile
316.4 316.2
>0.01
86
2-(Bromomethyl)benzonitrile
316.4 316.2
>0.01
87
3-(Bromomethyl)-4-fluoro- benzonitrile
334.4 334.2
>0.01
88
1-(Bromomethyl)-3,5-dimethoxy- benzene
351.5 351.2
>0.01
89
1-(Bromomethyl)-4-methoxy- benzene
321.4 321.2
>0.01
90
1-(Bromomethyl)-3-methoxy- benzene
321.4 321.2
>0.01
91
Methyl 4-(bromomethyl)-3- methoxybenzoate
379.5 379.2
>0.01
92
1-[4-(Bromomethyl)phenyl]-1H- pyrazole
357.5 357.2
>0.01
93
1-(Bromomethyl)-4-fluoro- benzene
309.4 309.2
<=10
94
4-(Bromomethyl)-1,2-difluoro- benzene
327.2 327.2
>0.01
95
1-(Bromomethyl)-2-fluoro- benzene
309.3 309.2
>0.01
96
1-(Bromomethyl)-3- (difluoromethoxy)benzene
357.2 357.2
<=10
97
1-(Bromomethyl)-2,3-difluoro- benzene
327.2 327.2
>0.01
98
1-(Bromomethyl)-3-fluoro- benzene
309.3 309.2
>0.01
99
1-(Bromomethyl)-2,4-difluoro- benzene
327.4 327.2
>0.01
100
1-(Bromomethyl)-3,5-difluoro- benzene
327.4 327.2
>0.01
101
2-(Bromomethyl)-1,3-difluoro- benzene
327.4 327.2
>0.01
102
1-(Bromomethyl)-2-chloro-4- fluorobenzene
343.4 343.1
>0.01
103
2-(Bromomethyl)-1,4-difluoro- benzene
327.4 327.2
>0.01
104
1-(Bromomethyl)-4-chloro-2- fluorobenzene
343.4 343.1
>0.01
105
2-(Bromomethyl)-1,3,4-trifluoro- benzene
345.4 345.2
>0.01
106
1-(Bromomethyl)-2,4,5-trifluoro- benzene
345.5 345.2
>0.01
107
1-(Bromomethyl)-4-methyl- benzene
305.5 305.2
<=10
108
2-(Bromomethyl)-1,3,5-trifluoro- benzene
345.4 345.2
>0.01
109
1-(Bromomethyl)-2-methyl- benzene
305.4 305.2
<=10
110
1-(Bromomethyl)-4-fluoro-2- (trifluoromethyl)benzene
377.5 377.2
>0.01, >0.03
111
1-(Bromomethyl)-2-fluoro-3- methylbenzene
323.2 323.2
>0.01, >0.03
112
1-(Bromomethyl)-3- (trifluoromethyl)benzene
359.2 359.2
<=10
113
1-(Bromomethyl)-4-chloro- benzene
325.1 325.1
<=10
114
1-(Bromomethyl)-4- (trifluoromethyl)benzene
359.2 359.2
<=10
115
1-(Bromomethyl)-3-chloro- benzene
325.1 325.1
>0.01
116
1-(Bromomethyl)-2- (trifluoromethyl)benzene
359.2 359.2
>0.01
117
4-(Bromomethyl)-2-fluoro-1- (trifluoromethyl)benzene
377.2 377.2
<=10
118
2-(Bromomethyl)-4-fluoro-1- (trifluoromethyl)benzene
377.2 377.2
<=10
119
1-(Bromomethyl)-3-chloro-2- fluorobenzene
343.2 343.1
<=10
120
1-(Bromomethyl)-3,5-dimethyl- benzene
319.4 319.2
<=10
121
1-(Bromomethyl)-2-ethyl- benzene
No mass ion
<=10
122
1-(Bromomethyl)-2- (trifluoromethoxy)benzene
375.4 375.2
<=10
123
1-(Bromomethyl)-3- (trifluoromethoxy)benzene
375.4 375.2
>0.1
124
4′-(Bromomethyl)biphenyl-2- carbonitrile
392.5 392.2
>0.1
125
1-(Bromomethyl)-4-iodobenzene
417.3 417.1
>0.1
126
1-(Bromomethyl)-4- [(trifluoromethyl)thio]benzene
391.4 391.1
<=10
127
1-(Bromomethyl)-3-(4-fluoro- phenoxy)benzene
401.4 401.2
>0.1
128
1-(Bromomethyl)-4-tert-butyl- benzene
347.5 347.2
<=10
129
2-(Bromomethyl)-1,4-dichloro- benzene
359.4 359.1
<=10
130
2-(Bromomethyl)-1-chloro-4- (trifluoromethyl)benzene
393.4 393.1
1
131
2-(Bromomethyl)-4-chloro-1- methylbenzene
339.4 339.2
1
132
2-(Bromomethyl)naphthalene
341.4 341.2
<=10
133
4-(Bromomethyl)-1,2-dichloro- benzene
359.4 359.1
<=10
134
2-(Bromomethyl)-1,3-dichloro- benzene
359.4 359.1
>0.1
135
4-(Bromomethyl)biphenyl
367.4 367.2
>0.1
Example 81
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[4-(methylsulfonyl)benzyl]-1H-imidazole
1H-NMR (CDCl3): 1.60-1.64 (3H), 2.09-2.11 (3H), 2.18-2.20 (3H), 2.97-3.00 (3H), 4.19-4.23 (1H), 4.70-4.76 (1H), 4.79-4.85 (1H), 6.61-6.65 (1H), 6.80-6.81 (1H), 6.84-6.95 (4H), 7.14-7.17 (1H), 7.72-7.78 (2H)
Example 82
[4-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)phenyl](phenyl)methanone
1H-NMR (CDCl3): 1.64-1.70 (3H), 2.17-2.19 (3H), 2.22-2.24 (3H), 4.23-4.29 (1H), 4.70-4.77 (1H), 4.80-4.86 (1H), 6.80-6.84 (1H), 6.85-6.92 (3H), 6.94-7.00 (2H), 7.18-7.20 (1H), 7.45-7.52 (2H), 7.59-7.62 (1H), 7.68-7.72 (2H), 7.75-7.79 (2H)
Example 83
Methyl 4-({2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)benzoate
1H-NMR (d6-Acetone): 1.53-1.61 (3H), 2.15-2.22 (6H), 3.83-3.85 (3H), 4.28-4.35 (1H), 4.80-4.86 (1H), 5.02-5.10 (1H), 6.70-6.74 (1H), 6.86-6.98 (5H), 7.05-7.07 (1H), 7.80-7.84 (2H)
Example 84
4-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)pyridine
1H-NMR (CDCl3): 1.61-1.64 (3H), 2.10-2.12 (3H), 2.21-2.23 (3H), 4.12-4.20 (1H), 4.60-4.66 (1H), 4.75-4.80 (1H), 6.65-6.71 (3H), 6.82-6.83 (1H), 6.89-6.96 (2H), 7.12-7.13 (1H), 8.40-8.44 (2H)
Example 85
3-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)benzonitrile
1H-NMR (d6-Acetone): 1.55-1.58 (3H), 2.17-2.20 (6H), 4.38-4.41 (1H), 4.93-4.98 (1H), 5.05-5.09 (1H), 6.63-6.65 (1H), 6.80-6.87 (2H), 6.97-7.00 (2H), 7.12-7.13 (1H), 7.18-7.20 (1H), 7.37-7.40 (1H), 7.52-7.54 (1H)
Example 86
2-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)benzonitrile
1H-NMR (d6-Acetone): 1.55-1.58 (3H), 2.16-2.20 (6H), 4.29-4.33 (1H), 4.93-4.98 (1H), 5.10-5.14 (1H), 6.66-6.68 (1H), 6.80-6.86 (2H), 6.95-7.00 (3H), 7.10-7.11 (1H), 7.57-7.59 (2H)
Example 87
3-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)-4-fluorobenzonitrile
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.16-2.18 (3H), 2.20-2.22 (3H), 4.42-4.46 (1H), 5.01-5.05 (1H), 5.06-5.11 (1H), 6.60-6.68 (2H), 6.80-6.82 (2H), 7.00-7.01 (1H), 7.15-7.16 (1H), 7.21-7.25 (1H), 7.60-7.62 (1H)
Example 88
1-(3,5-Dimethoxybenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.20-2.25 (6H), 3.62-3.66 (6H), 4.33-4.38 (1H), 4.60-4.64 (1H), 4.81-4.85 (1H), 6.02-6.05 (2H), 6.36-6.38 (1H), 6.76-6.79 (1H), 6.90-6.98 (3H), 7.00-7.01 (1H)
Example 89
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(4-methoxybenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.20-2.25 (6H), 3.76-3.78 (3H), 4.36-4.40 (1H), 4.60-4.64 (1H), 4.80-4.84 (1H), 6.68-6.70 (1H), 6.79-6.85 (4H), 6.90-7.00 (4H)
Example 90
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(3-methoxybenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.20-2.25 (6H), 3.65-3.67 (3H), 4.36-4.40 (1H), 4.62-4.66 (1H), 4.90-4.94 (1H), 6.40-6.42 (1H), 6.48-6.50 (1H), 6.72-6.74 (1H), 6.79-6.81 (1H), 6.95-7.00 (3H), 7.00-7.01 (1H), 7.17-7.20 (1H)
Example 91
Methyl 4-({2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)-3-methoxybenzoate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.16-2.18 (3H), 2.20-2.22 (3H), 3.84-3.90 (6H), 4.32-4.36 (1H), 4.81-4.86 (2H), 6.46-6.48 (1H), 6.71-6.73 (1H), 6.85-6.90 (2H), 6.97-6.98 (1H), 7.01-7.02 (1H), 7.39-7.41 (1H), 7.50-7.51 (1H)
Example 92
1-[4-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)phenyl]-1H-pyrazole
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.20-2.24 (6H), 4.36-4.40 (1H), 4.78-4.82 (1H), 4.96-5.00 (1H), 6.44-6.46 (1H), 6.70-6.72 (1H), 6.90-7.00 (5H), 7.04-7.06 (1H), 7.63-7.69 (3H), 8.21-8.23 (1H)
Example 93
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(4-fluorobenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.19-2.24 (6H), 4.32-4.36 (1H), 4.71-4.75 (1H), 4.90-4.94 (1H), 6.65-6.67 (1H), 6.89-6.95 (3H), 6.95-7.00 (4H), 7.00-7.01 (1H)
Example 94
1-(3,4-Difluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.55-1.58 (3H), 2.20-2.23 (6H), 4.36-4.40 (1H), 4.80-4.84 (1H), 4.97-5.01 (1H), 6.62-6.70 (3H), 6.82-6.90 (2H), 6.96-6.98 (1H), 7.04-7.05 (1H), 7.06-7.10 (1H)
Example 95
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2-fluorobenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.59 (3H), 2.20-2.24 (6H), 4.38-4.42 (1H), 4.80-4.90 (2H), 6.60-6.63 (1H), 6.64-6.66 (1H), 6.88-6.95 (3H), 7.00-7.03 (2H), 7.06-7.09 (1H), 7.25-7.28 (1H)
Example 96
1-[3-(Difluoromethoxy)benzyl]-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.19-2.23 (6H), 4.32-4.38 (1H), 4.79-4.83 (1H), 4.97-5.01 (1H), 6.61-6.63 (1H), 6.70-6.79 (2H), 6.81-6.82 (1H), 6.88-6.99 (2H), 7.00-7.05 (2H), 7.25-7.30 (1H)
Example 97
1-(2,3-Difluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.59 (3H), 2.20-2.24 (6H), 4.39-4.43 (1H), 4.89-4.93 (1H), 5.00-5.04 (1H), 6.39-6.41 (1H), 6.61-6.63 (1H), 6.81-6.90 (2H), 6.95-7.00 (2H), 7.06-7.07 (1H), 7.14-7.19 (1H)
Example 98
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(3-fluorobenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.19-2.23 (6H), 4.32-4.36 (1H), 4.76-4.80 (1H), 4.96-5.00 (1H), 6.53-6.55 (1H), 6.70-6.75 (2H), 6.89-6.99 (4H), 7.02-7.04 (1H), 7.22-7.25 (1H)
Example 99
1-(2,4-Difluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.59 (3H), 2.20-2.24 (6H), 4.39-4.43 (1H), 4.81-4.91 (2H), 6.60-6.65 (2H), 6.77-6.80 (1H), 6.83-6.86 (1H), 6.89-6.99 (3H), 7.01-7.03 (1H)
Example 100
1-(3,5-Difluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.59 (3H), 2.19-2.23 (6H), 4.36-4.40 (1H), 4.81-4.85 (1H), 5.00-5.04 (1H), 6.40-6.44 (2H), 6.69-6.71 (1H), 6.78-6.82 (1H), 6.83-6.90 (2H), 6.98-6.99 (1H), 7.09-7.10 (1H)
Example 101
1-(2,6-Difluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.26-2.28 (3H), 2.37-2.39 (3H), 4.60-4.64 (1H), 4.72-4.77 (1H), 5.05-5.10 (1H), 6.57-6.59 (1H), 6.85-6.84 (3H), 6.97-7.05 (3H), 7.39-7.43 (1H)
Example 102
1-(2-Chloro-4-fluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.59 (3H), 2.16-2.20 (6H), 4.32-4.37 (1H), 4.81-4.85 (1H), 4.96-5.00 (1H), 6.29-6.32 (1H), 6.69-6.71 (1H), 6.82-6.88 (3H), 6.99-7.03 (2H), 7.20-7.22 (1H)
Example 103
1-(2,5-Difluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.19-2.24 (6H), 4.40-4.44 (1H), 4.87-4.91 (1H), 4.96-5.00 (1H), 6.16-6.20 (1H), 6.67-6.70 (1H), 6.82-6.88 (2H), 6.96-7.00 (2H), 7.06-7.09 (2H)
Example 104
1-(4-Chloro-2-fluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.59 (3H), 2.19-2.23 (6H), 4.39-4.44 (1H), 4.82-4.86 (1H), 4.96-5.00 (1H), 6.49-6.53 (1H), 6.65-6.68 (1H), 6.80-6.90 (2H), 6.96-6.99 (2H), 7.07-7.08 (1H), 7.16-7.19 (1H)
Example 105
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2,3,6-trifluorobenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.21-2.23 (3H), 2.36-2.38 (3H), 4.57-4.61 (1H), 4.85-4.89 (1H), 4.97-5.01 (1H), 6.47-6.50 (1H), 6.81-6.89 (2H), 6.90-7.01 (3H), 7.23-7.28 (1H)
Example 106
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2,4,5-trifluorobenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.18-2.20 (3H), 2.21-2.23 (3H), 4.40-4.45 (1H), 4.90-4.94 (1H), 4.95-5.01 (1H), 6.27-6.32 (1H), 6.61-6.63 (1H), 6.80-6.88 (2H), 6.99-7.00 (1H), 7.10-7.11 (1H), 7.11-7.15 (1H)
Example 107
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(4-methyl benzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.55 (3H), 2.20-2.22 (3H), 2.22-2.26 (6H), 4.30-4.36 (1H), 4.61-4.65 (1H), 4.80-4.85 (1H), 6.70-6.72 (1H), 6.78-6.81 (2H), 6.90-6.99 (4H), 7.03-7.06 (2H)
Example 108
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2,4,6-trifluorobenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.21-2.23 (3H), 2.30-2.32 (3H), 4.57-4.61 (1H), 4.76-4.80 (1H), 4.84-4.88 (1H), 6.47-6.49 (1H), 6.81-6.92 (5H), 6.97-6.99 (1H)
Example 109
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2-methyl benzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.55-1.58 (3H), 2.02-2.04 (3H), 2.10-2.12 (3H), 2.21-2.23 (3H), 4.21-4.36 (1H), 4.65-4.69 (1H), 4.78-4.82 (1H), 6.47-6.49 (1H), 6.75-6.77 (1H), 6.80-6.82 (1H), 6.95-7.00 (3H), 7.04-7.07 (1H), 7.18-7.20 (2H)
Example 110
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[4-fluoro-2-(trifluoromethyl)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.07-2.09 (3H), 2.15-2.17 (3H), 4.28-4.32 (1H), 5.00-5.04 (1H), 5.16-5.20 (1H), 6.38-6.41 (1H), 6.78-6.80 (1H), 6.85-6.88 (2H), 7.00-7.04 (2H), 7.16-7.19 (1H), 7.41-7.43 (1H)
Example 111
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2-fluoro-3-methylbenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.59 (3H), 2.20-2.25 (9H), 4.38-4.42 (1H), 4.80-4.86 (2H), 6.44-6.48 (1H), 6.64-6.66 (1H), 6.87-6.97 (4H), 7.00-7.01 (1H), 7.10-7.14 (1H)
Example 112
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[3-(trifluoromethyl)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.52-1.56 (3H), 2.18-2.21 (6H), 4.36-4.40 (1H), 4.90-4.95 (1H), 5.08-5.12 (1H), 6.67-6.70 (1H), 6.81-6.90 (2H), 6.98-6.99 (1H), 7.10-7.16 (3H), 7.40-7.44 (1H), 7.50-7.52 (1H)
Example 113
1-(4-Chlorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.18-2.22 (6H), 4.31-4.38 (1H), 4.86-4.91 (1H), 4.95-4.99 (1H), 6.67-6.70 (1H), 6.81-6.97 (5H), 7.03-7.04 (1H), 7.20-7.23 (1H)
Example 114
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[4-(trifluoromethyl)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.52-1.56 (3H), 2.17-2.20 (6H), 4.31-4.39 (1H), 4.90-4.96 (1H), 5.07-5.12 (1H), 6.67-6.70 (1H), 6.83-6.89 (2H), 6.99-7.04 (3H), 7.09-7.10 (1H), 7.49-7.53 (2H)
Example 115
1-(3-Chlorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.58 (3H), 2.00-2.05 (6H), 4.34-4.39 (1H), 4.78-4.82 (1H), 4.98-5.02 (1H), 6.69-6.71 (1H), 6.79-6.83 (2H), 6.87-6.97 (3H), 7.04-7.05 (1H), 7.19-7.23 (2H)
Example 116
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[2-(trifluoromethyl)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.58 (3H), 2.01-2.03 (3H), 2.16-2.18 (3H), 4.22-4.28 (1H), 4.99-5.03 (1H), 5.15-5.20 (1H), 6.40-6.42 (1H), 6.78-6.80 (1H), 6.89-6.92 (2H), 7.01-7.04 (2H), 7.40-7.44 (2H), 7.70-7.72 (1H)
Example 117
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[3-fluoro-4-(trifluoromethyl)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.54-1.58 (3H), 2.10-2.17 (6H), 4.38-4.42 (1H), 5.00-5.05 (1H), 5.12-5.18 (1H), 6.60-6.70 (2H), 6.80-6.85 (3H), 7.00-7.01 (1H), 7.16-7.17 (1H), 7.45-7.52 (1H)
Example 118
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[5-fluoro-2-(trifluoromethyl)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.09-2.15 (6H), 4.31-4.38 (1H), 5.02-5.07 (1H), 5.10-5.15 (1H), 5.95-5.98 (1H), 6.78-6.80 (1H), 6.81-6.88 (2H), 7.04-7.05 (1H), 7.10-7.16 (2H), 7.72-7.76 (1H)
Example 119
1-(3-Chloro-2-fluorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.55-1.58 (3H), 2.20-2.23 (6H), 4.39-4.44 (1H), 4.90-4.95 (1H), 5.00-5.05 (1H), 6.50-6.54 (1H), 6.61-6.63 (1H), 6.80-6.90 (2H), 6.95-7.00 (2H), 7.09-7.10 (1H), 7.30-7.35 (1H)
Example 120
1-(3,5-Dimethylbenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.17-2.19 (6H), 2.21-2.25 (6H), 4.35-4.40 (1H), 4.60-4.65 (1H), 4.80-4.85 (1H), 6.42-6.45 (2H), 6.71-6.73 (1H), 6.81-6.82 (1H), 6.90-7.00 (4H)
Example 121
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2-ethylbenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 0.98-1.02 (3H), 1.56-1.59 (3H), 2.16-2.17 (3H), 2.21-2.22 (3H), 2.30-2.38 (2H), 4.25-4.34 (1H), 4.71-4.77 (1H), 4.80-4.85 (1H), 6.57-6.59 (1H), 6.73-6.80 (2H), 6.94-7.00 (3H), 7.05-7.09 (1H), 7.20-7.23 (2H)
Example 122
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[2-(trifluoromethoxy)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.55-1.59 (3H), 2.16-2.22 (6H), 4.31-4.38 (1H), 4.82-4.87 (1H), 4.99-5.02 (1H), 6.58-6.60 (1H), 6.67-6.70 (1H), 6.84-6.91 (2H), 6.97-6.99 (1H), 7.01-7.02 (1H), 7.16-7.20 (1H), 7.30-7.40 (2H)
Example 123
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[3-(trifluoromethoxy)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.59 (3H), 2.19-2.22 (6H), 4.36-4.40 (1H), 4.80-4.85 (1H), 5.00-5.05 (1H), 6.70-6.72 (1H), 6.78-6.80 (1H), 6.82-6.94 (3H), 6.99-7.00 (1H), 7.06-7.08 (1H), 7.16-7.19 (1H), 7.35-7.38 (1H)
Example 124
4′-({2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl)biphenyl-2-carbonitrile
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.20-2.21 (6H), 4.38-4.42 (1H), 4.81-4.85 (1H), 5.00-5.05 (1H), 6.76-6.78 (1H), 6.90-7.02 (5H), 7.07-7.08 (1H), 7.42-7.45 (2H), 7.58-7.61 (2H), 7.78-7.80 (1H), 7.82-7.84 (1H)
Example 125
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(4-iodobenzyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 1.97-1.99 (3H), 2.00-2.02 (3H), 4.30-4.35 (1H), 4.75-4.80 (1H), 4.96-5.00 (1H), 6.61-6.64 (2H), 6.68-6.70 (1H), 6.86-6.95 (3H), 7.01-7.02 (1H), 7.58-7.60 (2H)
Example 126
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-{4-[(trifluoromethyl)thio]benzyl}-1H-imidazole
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.17-2.20 (6H), 4.31-4.38 (1H), 4.83-4.88 (1H), 5.03-5.08 (1H), 6.67-6.69 (1H), 6.82-6.88 (2H), 6.95-7.00 (3H), 7.07-7.08 (1H), 7.50-7.54 (2H)
Example 127
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-[3-(4-fluorophenoxy)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.19-2.23 (6H), 4.33-4.38 (1H), 4.67-4.71 (1H), 4.89-4.93 (1H), 6.50-6.52 (1H), 6.64-6.68 (2H), 6.80-6.82 (1H), 6.86-7.00 (6H), 7.14-7.19 (2H), 7.22-7.25 (1H)
Example 128
1-(4-tert-Butylbenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.13-1.15 (9H), 1.51-1.53 (3H), 2.20-2.26 (6H), 4.36-4.40 (1H), 4.66-4.70 (1H), 4.82-4.87 (1H), 6.70-6.73 (1H), 6.80-6.82 (2H), 6.90-7.00 (4H), 7.25-7.28 (2H)
Example 129
1-(2,5-Dichlorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.55-1.59 (3H), 2.11-2.20 (6H), 4.37-4.41 (1H), 4.89-4.93 (1H), 5.02-5.06 (1H), 6.19-6.21 (1H), 6.76-6.79 (1H), 6.80-6.82 (1H), 6.82-6.84 (1H), 7.00-7.01 (1H), 7.09-7.10 (1H), 7.19-7.22 (1H), 7.37-7.39 (1H)
Example 130
1-[2-Chloro-5-(trifluoromethyl)benzyl]-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.60 (3H), 2.10-2.12 (3H), 2.18-2.20 (3H), 4.39-4.44 (1H), 4.99-5.05 (1H), 5.12-5.18 (1H), 6.57-6.59 (1H), 6.71-6.80 (3H), 7.01-7.03 (1H), 7.10-7.12 (1H), 7.50-7.53 (1H), 7.58-7.60 (1H)
Example 131
1-(5-Chloro-2-methylbenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.56-1.60 (3H), 2.02-2.04 (3H), 2.10-2.12 (3H), 2.20-2.21 (3H), 4.24-4.28 (1H), 4.71-4.78 (1H), 4.88-4.93 (1H), 6.32-6.34 (1H), 6.78-6.80 (1H), 6.90-7.00 (4H), 7.13-7.17 (1H)
Example 132
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(2-naphthylmethyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.17-2.21 (6H), 4.38-4.41 (1H), 4.87-4.91 (1H), 5.10-5.14 (1H), 6.74-6.78 (1H), 6.90-6.93 (2H), 6.98-6.99 (1H), 7.01-7.08 (2H), 7.28-7.29 (1H), 7.43-7.46 (2H), 7.70-7.80 (2H), 7.81-7.84 (1H)
Example 133
1-(3,4-Dichlorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.19-2.22 (6H), 4.35-4.40 (1H), 4.85-4.90 (1H), 5.00-5.06 (1H), 4.98-5.00 (2H), 6.63-6.66 (1H), 6.78-6.80 (1H), 6.81-6.87 (3H), 6.99-7.00 (1H), 7.10-7.11 (1H), 7.30-7.33 (1H)
Example 134
1-(2,6-Dichlorobenzyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.60-1.63 (3H), 2.30-2.32 (3H), 2.40-2.42 (3H), 4.66-4.72 (2H), 5.05-5.09 (1H), 6.41-6.43 (1H), 6.67-6.70 (1H), 6.81-6.82 (1H), 6.96-7.01 (2H), 7.40-7.46 (3H)
Example 135
1-(Biphenyl-4-ylmethyl)-2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.54-1.56 (3H), 2.20-2.22 (6H), 4.36-4.40 (1H), 4.80-4.84 (1H), 4.97-5.00 (1H), 6.71-6.73 (1H), 6.93-6.99 (4H), 7.06-7.07 (1H), 7.36-7.37 (1H), 7.42-7.46 (3H), 7.55-7.58 (2H), 7.60-7.63 (2H)
Example 136
Cyclopropylmethyl {2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl carbonate
To a mixture of the compound of Example 1 (100 mg, 0.50 mmol) and caesium carbonate (407 mg, 1.25 mmol) in acetone (5 ml) and under nitrogen was added Preparation 119 (205 mg, 1.25 mmol). The reaction mixture was stirred at room temperature for 60 h and then concentrated in vacuo. To the residue was added water (10 ml) and ethyl acetate (20 ml) and the two layers were separated. The organic phase was then dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in acetonitrile (0.8 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [55:45 (20 min) to 98:2 (20.1 min)]. The appropriate fractions were combined and concentrated to give the title compound (74 mg).
Experimental MH+ 329.4; expected 329.2 1H-NMR (d6-Acetone): 0.20-0.30 (2H), 0.50-0.60 (2H), 1.05-1.15 (1H), 1.55-1.60 (3H), 2.30-2.40 (6H), 3.80-3.95 (2H), 4.60-4.70 (1H), 5.40-5.45 (1H), 5.70-5.80 (1H), 6.65-6.70 (1H), 6.90-6.95 (2H), 6.95-7.00 (1H), 7.17-7.20 (1H) Rhip. Funct. ED100 mg/cm2=0.01
Similarly Prepared from Example 1 were:
Rhip.
MH+
Funct.
Ex.
From
Found/
ED100
No.
R6
Precursor
Prep.
Expected
mg/cm2
137
Chloromethyl 4- methoxybenzyl carbonate
120
395.3, 395.2
0.1
138
Chloromethyl 2,2,2-trifluoroethyl carbonate
124
357.3, 357.1
0.03
139
Chloromethyl 3- methylbutyl carbonate
121
345.4, 345.2
<=0.03
140
Chloromethyl isopropyl carbonate
122
317.3, 317.2
0.01
141
Chloromethyl cyclobutyl carbonate
123
329.4, 329.2
0.03
Example 137
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 4-methoxybenzyl carbonate
1H-NMR (d6-Acetone): 1.50-1.60 (3H), 2.25-2.35 (6H), 3.80-3.85 (3H), 4.60-4.65 (1H), 4.95-5.10 (2H), 5.40-5.50 (1H), 5.70-5.80 (1H), 6.60-6.65 (1H), 6.90-7.00 (5H), 7.15-7.18 (1H), 7.25-7.35 (2H)
Example 138
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 2,2,2-trifluoroethyl carbonate
1H-NMR (d6-Acetone): 1.50-1.60 (3H), 2.20-2.30 (6H), 4.50-4.70 (3H), 5.50-5.55 (1H), 5.80-5.85 (1H), 6.60-6.64 (1H), 6.85-7.00 (3H), 7.14-7.18 (1H)
Example 139
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl 3-methylbutyl carbonate
1H-NMR (d6-Acetone): 0.80-0.90 (6H), 1.40-1.50 (2H), 1.55-1.60 (3H), 1.60-1.65 (1H), 2.20-2.30 (6H), 4.00-4.10 (2H), 4.60-4.65 (1H), 5.40-5.45 (1H), 5.70-5.75 (1H), 6.60-6.65 (1H), 6.85-6.90 (2H), 6.95-7.00 (1H), 7.10-7.15 (1H)
Example 140
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl isopropyl carbonate
1H-NMR (d6-Acetone): 1.10-1.20 (6H), 1.50-1.60 (3H), 2.20-2.30 (6H), 4.60-4.65 (1H), 4.70-4.75 (1H), 5.38-5.42 (1H), 5.65-5.70 (1H), 6.60-6.64 (1H), 6.85-6.90 (2H), 6.95-7.00 (1H), 7.10-7.14 (1H)
Example 141
Cyclobutyl {2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl carbonate
1H-NMR (d6-Acetone): 1.50-1.60 (3H), 1.60-1.75 (2H), 1.90-2.00 (2H), 2.20-2.24 (2H), 2.25-2.30 (6H), 4.55-4.60 (1H), 4.70-4.80 (1H), 5.38-5.41 (1H), 5.65-5.70 (1H), 6.60-6.64 (1H), 6.85-6.90 (2H), 6.95-7.00 (1H), 7.10-7.13 (1H)
Example 142
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl (2,4-dichlorobenzyl)carbamate
To a mixture of the compound of Example 1 (100 mg, 0.5 mmol) and caesium carbonate (163 mg, 0.5 mmol) in anhydrous acetone (2 ml) was added dropwise the compound of Preparation 125 (134 mg, 0.5 mmol) in anhydrous acetone (1 ml). The reaction mixture was stirred at room temperature for 4 days and then ethyl acetate (5 ml) and water (5 ml) was added. The two layers were separated and the aqueous layer was extracted with ethyl acetate (2×5 ml). The combined organic layers were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol (1.5 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [55:45 (20 min) to 95:5 (21 min)]. The appropriate fractions were combined and concentrated to give the title compound (106 mg).
Experimental MH+ 432.3; expected 432.1 1H-NMR (d6-Acetone): 1.48-1.55 (3H), 2.23-2.27 (3H), 2.29-2.33 (3H), 4.22-4.30 (2H), 4.60-4.65 (1H), 5.38-5.41 (1H), 5.59-5.62 (1H), 6.60-6.63 (1H), 6.81-6.95 (3H), 7.08-7.10 (1H), 7.20-7.26 (1H), 7.41-7.50 (1H) Rhip. Funct. ED100 mg/cm2=1
Example 143
1-{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}ethyl methyl[2-(methylsulfonyl)ethyl]carbamate
To a mixture of the compound of Example 1 (100 mg, 0.5 mmol) and caesium carbonate (163 mg, 0.5 mmol) in anhydrous acetone (2 ml) was added dropwise Preparation 133 (230 mg, 0.5 mmol) in anhydrous acetone (1 ml). The reaction mixture was stirred at room temperature for 14 days and then dichloromethane (5 ml) and water (5 ml) was added. The two layers were separated and the aqueous layer was extracted with dichloromethane (2×5 ml). The combined organic layers were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol (1.5 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×21.4 mm LUNA C18(2) 5 μm column, 20 ml/min) using an acetonitrile:water gradient [20:80 (3 min) to 98:2 (16 min)]. The appropriate fractions were combined and concentrated to give the title compound (7 mg).
Experimental MH+ 408.4; expected 408.2 Rhip. Funct. ED100 mg/cm2=0.3
Similarly Prepared from Example 1 were:
Rhip.
MH+
Funct.
Ex.
From
Found/
ED100
No.
R6
Precursor
Prep.
Expected
mg/cm2
144
1-Chloroethyl morpholine-4- carboxylate
134
358.5 358.2
<=10
145
Chloromethyl thiomorpholine-4- carboxylate 1,1-dioxide
126
392.4 392.2
<=10
146
1-(Chloromethyl) 2-methyl (2S)- pyrrolidine-1,2-dicarboxylate
127
386.4 386.2
>1
147
Chloromethyl cyclohexylcarbamate
128
356.4 356.2
<=10
148
Chloromethyl [2-(2,4-dichloro- phenyl)ethyl]carbamate
129
446.3 446.1
>1
149
Chloromethyl cyclohexyl(methyl)- carbamate
130
370.5 370.2
<=10
150
Chloromethyl benzyl(methyl)- carbamate
131
378.5 378.2
<=10
151
Chloromethyl methyl(2-phenyl- ethyl)carbamate
132
392.5 392.2
<=10
152
1-(2-Chloroethyl) 2-methyl (2S)- pyrrolidine-1,2-dicarboxylate
135
400.4 400.2
>1
Example 144
1-{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}ethyl morpholine-4-carboxylate
1H-NMR (CD3OD): 1.06-1.09 (3H), 1.58-1.60 (3H), 2.30-2.31 (3H), 2.40-2.41 (2H), 3.39-3.46 (4H), 3.50-3.43 (4H), 4.88-4.92 (1H), 6.17-6.20 (1H), 6.38-6.40 (1H), 6.89-6.92 (1H), 6.99-7.01 (1H), 7.23-7.24 (1H)
Example 145
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl thiomorpholine-4-carboxylate 1,1-dioxide
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.25-2.27 (3H), 2.37-2.39 (3H), 2.75-2.85 (2H), 2.90-3.00 (2H), 3.35-3.42 (2H), 3.76-3.80 (2H), 4.61-4.65 (1H), 5.68-5.69 (2H), 6.62-6.64 (1H), 6.89-6.95 (2H), 6.98-7.01 (1H), 7.09-7.10 (1H)
Example 146
1-({2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl) 2-methyl (2S)-pyrrolidine-1,2-dicarboxylate
1H-NMR (d6-Acetone): 1.50-1.55 (3H), 1.78-1.89 (3H), 2.10-2.20 (1H), 2.22-2.27 (3H), 2.34-2.39 (3H), 3.58-3.63 (3H), 4.59-4.64 (1H), 6.59-6.61 (1H), 6.82-6.90 (3H), 7.01-7.06 (1H)
Example 147
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl cyclohexylcarbamate
1H-NMR (d6-Acetone): 1.02-1.20 (3H), 1.20-1.30 (2H), 1.51-1.54 (3H), 1.54-1.57 (1H), 1.61-1.68 (2H), 1.75-1.82 (2H), 2.22-2.24 (3H), 2.30-2.33 (3H), 3.22-3.30 (1H), 4.60-4.64 (1H), 5.31-5.34 (1H), 5.54-5.57 (1H), 6.60-6.62 (1H), 6.82-6.83 (1H), 6.83-6.87 (1H), 6.95-6.98 (1H), 7.03-7.04 (1H)
Example 148
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl [2-(2,4-dichlorophenyl)ethyl]carbamate
1H-NMR (d6-Acetone): 1.50-1.56 (3H), 2.22-2.24 (3H), 2.30-2.32 (3H), 2.85-2.90 (2H), 3.30-3.35 (2H), 4.60-4.64 (1H), 5.30-5.33 (1H), 5.55-5.58 (1H), 6.60-6.62 (1H), 6.82-6.90 (2H), 6.96-6.98 (1H), 7.03-7.04 (2H), 7.19-7.24 (2H), 7.40-7.41 (1H)
Example 149
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl cyclohexyl(methyl)carbamate
1H-NMR (d6-Acetone): 1.00-1.10 (1H), 1.20-1.40 (4H), 1.41-1.60 (6H), 1.70-1.80 (2H), 2.10-2.16 (3H), 2.30-2.42 (6H), 4.61-4.70 (1H), 5.40-5.60 (2H), 6.62-6.65 (1H), 6.81-6.82 (1H), 6.83-6.98 (2H), 7.10-7.15 (1H)
Example 150
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl benzyl(methyl)carbamate
1H-NMR (d6-Acetone): 1.41-1.59 (3H), 2.18-2.26 (3H), 2.30-2.40 (3H), 2.70-2.80 (3H), 4.00-4.05 (1H), 4.25-4.38 (1H), 4.60-4.73 (1H), 5.59-5.69 (2H), 6.60-6.70 (1H), 6.80-7.00 (3H), 7.01-7.06 (1H), 7.10-7.19 (2H), 7.20-7.33 (3H)
Example 151
{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl methyl(2-phenylethyl)carbamate
1H-NMR (d6-Acetone): 1.33-1.41 (3H), 1.50-1.56 (3H), 2.17-2.20 (1H), 2.20-2.22 (3H), 2.22-2.26 (2H), 2.36-2.39 (3H), 2.45-2.55 (1H), 4.62-4.70 (1H), 5.60-5.70 (2H), 6.67-6.70 (1H), 6.82-6.92 (3H), 7.10-7.18 (2H), 7.18-7.24 (2H), 7.24-7.27 (2H)
Example 152
1-(1-{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}ethyl) 2-methyl (2S)-pyrrolidine-1,2-dicarboxylate
1H-NMR (CD3OD): 1.10-1.13 (3H), 1.56-1.60 (3H), 1.85-1.90 (3H), 2.20-2.24 (1H), 2.30-2.32 (3H), 2.39-2.41 (3H), 3.40-3.50 (2H), 3.60-3.63 (1H), 3.70-3.75 (2H), 4.29-4.33 (1H), 4.79-4.83 (1H), 6.10-6.13 (1H), 6.37-6.40 (1H), 6.90-9.93 (1H), 7.00-7.03 (1H), 7.20-7.23 (1H)
Example 153
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-methyl-1H-imidazole
To a mixture of the compound of Example 58 (100 mg, 0.5 mmol) and caesium carbonate (407 mg, 1.25 mmol) in acetone (4 ml) was added iodomethane (78 μl, 1.25 mmol). The reaction mixture was stirred at room temperature, under nitrogen, for 4 h and then concentrated in vacuo. to the residue was added water (10 ml) and the solution was extracted with ethyl acetate (2×10 ml). The combined extracts were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol (1 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×21.4 mm LUNA C18(2) 5 μm column, 20 ml/min) using an acetonitrile:water gradient [20:80 (3 min) to 98:2 (16 min)]. The appropriate fractions were combined and concentrated to give the title compound (40 mg).
Experimental MH+ 215.3; expected 215.1 1H-NMR (d6-Acetone): 1.45-1.50 (3H), 2.20-2.30 (6H), 3.10-3.15 (3H), 4.35-4.45 (1H), 6.55-6.60 (1H), 6.80-6.85 (1H), 6.85-6.90 (2H), 6.90-6.95 (1H) Rhip. Funct. ED100 mg/cm2=0.3
Similarly Prepared from Example 1 was:
MH+
Rhip. Funct.
Ex.
Found/
ED100
No.
R6
Precursor
Expected
mg/cm2
154
(Bromomethyl)cyclopropane
255.2; 255.2
1
Example 154
1-(Cyclopropylmethyl)-2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole
1H-NMR (d6-Acetone): 0.00-0.05 (1H), 0.10-0.20 (1H), 0.30-0.45 (2H), 0.80-0.90 (1H), 1.50-1.60 (3H), 2.20-2.30 (6H), 3.30-3.50 (2H), 4.40-4.50 (1H), 6.60-6.65 (1H), 6.80-6.90 (2H), 6.90-6.95 (1H), 7.05-7.10 (1H)
Similarly Prepared from Example 58 was:
MH+
Ex.
Found/
Rhip. Funct.
No.
R6
Precursor
Expected
ED100 mg/cm2
155
Bromoethane
229.2; 229.2
1
Example 155
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-ethyl-1H-imidazole
1H-NMR (d6-Acetone): 0.90-1.00 (3H), 1.50-1.60 (3H), 2.20-2.35 (6H), 3.50-3.70 (2H), 4.40-4.50 (1H), 6.60-6.63 (1H), 6.80-6.90 (2H), 6.90-7.00 (2H)
Example 156
{2-[1-(2,5-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
To a mixture of the compound of Example 17 (58 mg, 0.29 mmol) and caesium carbonate (236 mg, 0.72 mmol) in acetone (5 ml) was added chloromethyl pivalate (87 mg, 0.58 mmol). The reaction mixture was stirred at room temperature and under nitrogen for 18 h and then concentrated in vacuo. To the residue was added diethyl ether and the solution was passed through a silica plug (10 g), eluting with diethyl ether. The appropriate fractions were combined and concentrated to give the title compound (78 mg).
1H-NMR (CDCl3): 1.01-1.05 (9H), 1.61-1.65 (3H), 2.13-2.16 (3H), 2.35-2.37 (3H), 4.38-4.44 (1H), 5.34-5.38 (1H), 5.47-5.51 (1H), 6.65-6.67 (1H), 6.85-6.89 (1H), 6.96-7.02 (3H) Experimental MH+ 315.4; expected 315.2 Rhip. Funct. ED100 mg/cm2<=10
Similarly Prepared by Alkylation with Chloromethyl Pivalate were:
Rhip.
MH+
Funct.
Ex.
From
Found/
ED100
No.
AR
Ex.
Expected
mg/cm2
157
26
331.3 331.2
<=10
158
16
355.3 355.2
0.3, 1
159
24
355.3 355.2
<=10
160
20
301.2 301.2
0.1
161
18
315.2 315.2
>1
162
39
321.1 321.1
0.3
163
37
355.2 355.1
<=10
164
27
329.4 329.2
<=1, 1
165
43
355.1 355.1
>1
166
54
389.2 389.1
0.3
167
21
287.3 287.2
1, 0.3
Example 157
{2-[1-(3-Methoxy-2-methylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
1H-NMR (CDCl3): 1.01-1.05 (9H), 1.61-1.65 (3H), 2.23-2.25 (3H), 3.79-3.81 (3H), 4.45-4.55 (1H), 5.33-5.36 (1H), 5.42-5.46 (1H), 6.40-6.43 (1H), 6.65-6.68 (1H), 6.96-7.01 (3H)
Example 158
(2-{1-[2-(Trifluoromethyl)phenyl]ethyl}-1H-imidazol-1-yl)methyl pivalate
1H-NMR (CDCl3): 0.93-0.96 (9H), 1.69-1.73 (3H), 4.61-4.68 (1H), 5.51-5.60 (2H), 6.98-7.01 (2H), 7.24-7.31 (2H), 7.37-7.42 (1H), 7.61-7.64 (1H)
Example 159
(2-{1-[3-(Trifluoromethyl)phenyl]ethyl}-1H-imidazol-1-yl)methyl pivalate
1H-NMR (CDCl3): 0.96-0.99 (9H), 1.68-1.72 (3H), 4.30-4.36 (1H), 5.52-5.56 (1H), 5.65-5.69 (1H), 6.98-7.01 (2H), 7.36-7.38 (2H), 7.42-7.45 (2H)
Example 160
{2-[1-(3-Methylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
1H-NMR (CDCl3): 1.02-1.05 (9H), 1.68-1.71 (3H), 2.25-2.27 (3H), 4.19-4.25 (1H), 5.51-5.62 (2H), 6.93-7.01 (4H), 7.11-7.16 (1H)
Example 161
{2-[1-(2,6-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
1H-NMR (CDCl3): 1.03-1.06 (9H), 1.72-1.76 (3H), 2.00-2.10 (6H), 4.54-4.60 (1H), 5.13-5.17 (1H), 5.32-5.36 (1H), 6.92-7.02 (5H)
Example 162
{2-[1-(3-Chlorophenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
1H-NMR (CDCl3): 1.00-1.03 (9H), 1.66-1.70 (3H), 4.20-4.27 (1H), 5.51-5.66 (2H), 6.97-7.01 (2H), 7.04-7.08 (1H), 7.12-7.20 (3H)
Example 164
{2-[1-(2-Ethyl-3-methylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
1H-NMR (CD3OD): 0.98-1.00 (9H), 1.20-1.24 (3H), 1.60-1.63 (3H), 2.37-2.38 (3H), 2.80-2.86 (2H), 4.60-4.65 (1H), 5.53-5.56 (1H), 5.66-5.70 (1H), 6.61-6.63 (1H), 6.90-6.95 (2H), 6.99-7.01 (1H), 7.18-7.19 (1H)
Example 166
(2-{1-[2-Chloro-3-(trifluoromethyl)phenyl]ethyl}-1H-imidazol-1-yl)methyl pivalate
1H-NMR (CDCl3): 0.96-0.99 (9H), 1.69-1.72 (3H), 4.94-5.01 (1H), 5.60-5.64 (1H), 5.72-5.76 (1H), 7.03-7.05 (2H), 7.26-7.30 (1H), 7.40-7.43 (1H), 7.55-7.59 (1H)
Example 167
[2-(1-Phenylethyl)-1H-imidazol-1-yl]methyl pivalate
1H-NMR (CDCl3): 1.01-1.04 (9H), 1.68-1.72 (3H), 4.22-4.28 (1H), 5.51-5.61 (2H), 6.96-7.00 (2H), 7.13-7.19 (3H), 7.22-7.27 (2H)
Example 168
3-[1-(1H-Imidazol-2-yl)ethyl]benzonitrile
To a solution of the compound of Preparation 196 (264 mg, 1.23 mmol) in anhydrous N,N-dimethyl formamide (3 ml), at −15° C., was added dropwise thionyl chloride (0.20 ml, 2.7 mmol). The reaction mixture was allowed to warm to 0° C. over 4 h and then poured into an ice:water mixture. The mixture was extracted with ethyl acetate and the extracts were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in warm methanol (1 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm Sunfire C18 10 μm column, 120 ml/min) using an acetonitrile:water gradient [20:80 (2 min) to 95:5 (18.5 min)]. The appropriate fractions were combined and concentrated to give the title compound (7 mg).
Experimental MH+ 198.1; expected 198.1 1H-NMR (CDCl3): 1.68-1.72 (3H), 4.22-4.26 (1H), 6.97-7.00 (2H), 7.40-7.43 (1H), 7.50-7.55 (3H) Rhip. Funct. ED100 mg/cm2=>1
Example 169
1-Benzyl-2-[1-(3-methyl phenyl)ethyl]-1H-imidazole
To a mixture of the compound of Example 20 (500 mg, 2.68 mmol) and caesium carbonate (2.19 g, 6.71 mmol) in acetone (20 ml) was added benzyl bromide (0.64 ml, 5.37 mmol). The reaction mixture was stirred at room temperature for 18 h, filtered through Celite® and then concentrated in vacuo.
The residue was dissolved in acetonitrile (2 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [50:50 (20 min) to 95:5 (21 min)]. The appropriate fractions were combined and concentrated to give the title compound (272 mg).
Experimental MH+ 277.4; expected 277.2 1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.11-2.12 (3H), 4.08-4.13 (1H), 4.90-4.95 (1H), 5.01-5.06 (1H), 6.90-6.91 (1H), 6.91-7.00 (6H), 7.10-7.14 (1H), 7.21-7.25 (3H) Rhip. Funct. ED100 mg/cm2=>1
Example 170
1-Methyl-2-[1-(3-methylphenyl)ethyl]-1H-imidazole
To a mixture of the compound of Example 20 (500 mg, 2.68 mmol) and caesium carbonate (2.19 g, 6.71 mmol) in acetone (20 ml) was added methyl iodide (0.33 ml, 5.37 mmol). The reaction mixture was stirred at room temperature for 2 h, filtered through Celite® and then concentrated in vacuo.
The residue was dissolved in acetonitrile (2 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [50:50 (20 min) to 95:5 (21 min)]. The appropriate fractions were combined and concentrated to give the title compound (230 mg).
Experimental MH+ 201.4; expected 201.1 1H-NMR (d6-Acetone): 1.59-1.62 (3H), 2.12-2.14 (3H), 4.17-4.21 (1H), 6.80-6.81 (1H), 6.89-6.90 (1H), 6.96-7.02 (3H), 7.14-7.17 (1H) Rhip. Funct. ED100 mg/cm2=>1
Example 171
1-{2-[1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}ethyl morpholine-4-carboxylate
To a mixture of the compound of Example 1 (100 mg, 0.5 mmol) and caesium carbonate (163 mg, 0.5 mmol) in anhydrous acetone (2 ml) was added Preparation 134 (96 mg, 0.50 mmol) in anhydrous acetone (1 ml). The reaction mixture was stirred at room temperature for 14 days and then diluted with water (5 ml) and ethyl acetate (5 ml). The two layers were separated and the aqueous phase was extracted with ethyl acetate (2×5 ml). The combined organic phases were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in acetonitrile (1.5 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×21.4 mm LUNA C18(2) 5 μm column, 20 ml/min) using an acetonitrile:water gradient [20:80 (3 min) to 98:2 (16 min)]. The appropriate fractions were combined and concentrated to give the title compound (6 mg).
Retention time 6.13 min (Gilson system, 150 mm×4.6 mm LUNA C18(2) 5 μm column, 15 ml/min) using a 0.1% trifluoroacetic acid:acetonitrile gradient [95:5 (5 min) to 2:98 (9 min)]. Experimental MH+ 358.5; expected 358.2 1H-NMR (CD3OD): 1.55-1.60 (3H), 1.62-1.65 (3H), 2.27-2.30 (3H), 2.30-2.33 (3H), 2.90-3.00 (2H), 3.39-3.49 (2H), 4.61-4.65 (1H), 6.50-6.56 (2H), 6.85-6.89 (1H), 6.99-7.03 (1H), 7.17-7.18 (1H) Rhip. Funct. ED100 mg/cm2=1
Example 172
{2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1H-imidazol-1-yl}methyl pivalate
To a mixture of the compound of Example 58 (500 mg, 2.5 mmol) and caesium carbonate (1.79 g, 5.5 mmol) in anhydrous acetone (10 ml) was added chloromethyl pivalate (0.43 ml, 3.0 mmol). The reaction mixture was stirred at room temperature for 18 h. To the mixture was added dichloromethane (10 ml) and water (10 ml) and the two layers were separated. The aqueous phase was extracted with dichloromethane (2×10 ml) and the combined organic phases were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in acetonitrile (2 ml) and purified by automated preparative liquid chromatography (Gilson system, 250 mm×50 mm LUNA C18(2) 10 μm column, 120 ml/min) using an acetonitrile:water gradient [55:45 (20 min) to 95:5 (20.5 min)]. The appropriate fractions were combined and concentrated to give the title compound (410 mg).
Experimental MH+ 315.2; expected 315.2 1H-NMR (d6-Acetone): 0.97-1.00 (9H), 1.57-1.60 (3H), 2.28-2.30 (3H), 2.37-2.39 (3H), 4.60-4.65 (1H), 5.56-5.60 (1H), 5.65-5.69 (1H), 6.75-6.78 (1H), 6.90-7.00 (3H), 7.12-7.13 (1H)
Example 173
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-(3,3,3-trifluoropropyl)-1H-imidazole
To a mixture of the preparation of Example 58 (50 mg, 0.25 mmol) and caesium carbonate (203 mg, 0.62 mmol) in acetonitrile (2.5 ml) was added 1,1,1-trifluoro-3-iodopropane (73 μl, 0.62 mmol). The reaction mixture was heated at 100° C. in a microwave (200 W) for 45 min and then concentrated in vacuo. To the residue was added water (10 ml) and the mixture was extracted with ethyl acetate (2×10 ml). The combined extracts were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in acetonitrile (1 ml) and diethylamine (2-3 drops) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [50:50 (20 min) to 98:2 (20.1 min)]. The appropriate fractions were combined and concentrated to give the title compound (20 mg).
Experimental MH+ 297.3; expected 297.2 1H-NMR (d6-Acetone): 1.55-1.60 (3H), 1.79-1.90 (1H), 2.23-2.25 (3H), 2.43-2.62 (4H), 3.81-3.89 (2H), 4.46-4.53 (1H), 6.58-6.61 (1H), 6.85-6.92 (2H), 6.96-6.99 (1H), 7.06-7.08 (1H) Rhip. Funct. ED100 mg/cm2<=10
Similarly Prepared from Example 58 was:
MH+
Ex.
Found/
Rhip. Funct.
No.
R6
Precursor
Expected
ED100 mg/cm2
174
2- Bromopropane
243.3 243.2
>1
Example 174
2-[(1S)-1-(2,3-Dimethylphenyl)ethyl]-1-isopropyl-1H-imidazole
1H-NMR (d6-Acetone): 0.80-0.83 (3H), 1.29-1.32 (3H), 1.57-1.59 (3H), 2.25-2.27 (3H), 2.30-2.32 (3H), 4.00-4.06 (1H), 4.41-4.45 (1H), 6.58-6.60 (1H), 6.85-6.88 (2H), 6.95-6.97 (1H), 7.06-7.07 (1H)
Example 175
2-[1-(2,3-Dimethylphenyl)ethyl]-1-(4-methoxybenzyl)-1H-imidazole
To a solution of the compound of Example 1 (100 mg, 0.5 mmol) and N,N-diisopropylethylamine (77 mg, 0.6 mmol) in dichloromethane, under nitrogen, was added 4-methoxybenzyl bromide (151 mg, 0.75 mmol). The reaction mixture was stirred at room temperature for 90 min and water (10 ml) was added. The layers were separated, and the aqueous layer washed with dichloromethane (15 ml). The combined organics were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol:water (9:1, 3 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×4.6 mm LUNA C18(2) 10 μm column, 20 ml/min) using an acetonitrile:water gradient [60:40 to 95:5]. The appropriate fractions were concentrated in vacuo to give the title compound (10 mg).
Experimental MH+ 321.5; expected 321.2 1H-NMR (d6-Acetone): 1.48-1.53 (3H), 2.16-2.21 (3H), 2.21-2.24 (3H), 3.70-3.75 (3H), 4.31-4.36 (1H), 4.58-4.64 (1H), 4.74-4.81 (1H), 6.62-6.69 (1H), 6.72-6.82 (4H), 6.85-6.98 (4H) Rhip. Funct. ED100 mg/cm2=0.3
Similarly Prepared from Example 1 were:
MH+
Rhip. Funct.
Ex.
Found/
ED100
No.
R6
Precursor
Expected
mg/cm2
176
1-(Bromomethyl)-4- (trifluoromethyl)benzene
359.3 359.2
>1, 1
177
Bromo(methoxy)methane
245.4 245.2
0.1
Example 176
2-[1-(2,3-Dimethylphenyl)ethyl]-1-[4-(trifluoromethyl)benzyl]-1H-imidazole
1H-NMR (d6-Acetone): 1.51-1.58 (3H), 2.12-2.21 (6H), 4.32-4.40 (1H), 4.91-4.99 (1H), 5.08-5.16 (1H), 6.68-6.78 (1H), 6.84-6.92 (2H), 6.96-7.06 (3H), 7.07-7.12 (1H), 7.48-7.56 (2H)
Example 177
2-[1-(2,3-Dimethylphenyl)ethyl]-1-(methoxymethyl)-1H-imidazole
1H-NMR (d6-Acetone): 1.54-1.60 (3H), 2.28-2.32 (3H), 2.33-2.37 (3H), 3.04-3.09 (3H), 4.54-4.61 (1H), 4.82-4.90 (1H), 4.92-4.99 (1H), 6.72-6.78 (1H), 6.88-7.00 (3H), 7.08-7.12 (1H)
Example 178
2-[1-(2,3-Dimethylphenyl)ethyl]-1-(2-methoxybenzyl)-1H-imidazole
1-(Bromomethyl)-2-methoxybenzene (J. Indian Chem. Soc.; 28, 1951, 277; 150 mg, 0.75 mmol) was added to a suspension of the compound of Example 1 (100 mg, 0.5 mmol) and caesium carbonate (406 mg, 1.2 mmol) in acetone (4 ml), under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 18 h. The mixture was filtered and the filtrate was concentrated in vacuo. To the residue was added water (10 ml) and ethyl acetate (10 ml) and the two layers were separated. The aqueous layer was washed with a further portion of ethyl acetate (10 ml) and the combined organic layers were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol:water (9:1, 2 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [20:80 to 98:2]. The appropriate fractions were concentrated in vacuo to give the title compound (24 mg).
Experimental MH+ 321.5; expected 321.2 1H-NMR (d6-Acetone): 1.48-1.53 (3H), 2.14-2.19 (3H), 2.20-2.26 (3H), 3.78-3.82 (3H), 4.32-4.40 (1H), 4.64-4.78 (2H), 6.49-6.54 (1H), 6.68-6.72 (1H), 6.73-6.80 (1H), 6.86-6.98 (5H), 7.19-7.24 (1H) Rhip. Funct. ED100 mg/cm2<=10
Example 179
1-Benzyl-2-[1-(2-fluoro-3-methylphenyl)ethyl]-1H-imidazole
To a solution of the compound of Example 7 (69 mg, 0.34 mmol) and triethylamine (57 μl, 0.41 mmol) in anhydrous tetrahydrofuran (3 ml), under nitrogen, was added benzyl bromide (81 μl, 0.68 mmol). The reaction mixture was stirred at room temperature for 11 days and then concentrated in vacuo. To the residue was added saturated aqueous sodium hydrogen carbonate solution (10 ml) and the mixture was extracted with ethyl acetate (2×10 ml). The combined extracts were dried (MgSO4), filtered through silica and the filtrate was concentrated in vacuo to give the title compound (66 mg).
Experimental MH+ 295.2; expected 295.2 1H-NMR (d6-Acetone): 1.56-1.60 (3H), 2.19-2.22 (3H), 4.43-4.50 (1H), 4.95-5.00 (1H), 5.04-5.10 (1H), 6.90-6.99 (5H), 7.00-7.04 (2H), 7.10-7.17 (3H) Rhip. Funct. ED100 mg/cm2=1
Example 180
4-Fluorophenyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
To a solution of the compound of Example 1 (100 mg, 0.5 mmol) in anhydrous tetrahydrofuran (2 ml) was added triethylamine (0.08 ml, 0.6 mmol), followed by 4-fluorophenyl chloroformate (0.26 ml, 2.0 mmol). The reaction mixture was then stirred at room temperature, under nitrogen, for 1 h. To the mixture was added ethyl acetate (10 ml) and water (10 ml) and the two layers were separated. The aqueous layer was washed with ethyl acetate (10 ml) and the combined organic phases were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in acetonitrile (1 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 10 μm column, 40 ml/min) using an acetonitrile:water gradient [55:45 (20 min) to 98:2 (20.1 min)]. The appropriate fractions were concentrated in vacuo to give the title compound (60 mg).
Experimental MH+ 339.3; expected 339.1 1H-NMR (d6-Acetone): 1.50-1.60 (3H), 2.15-2.25 (6H), 4.40-4.50 (1H), 6.80-6.85 (1H), 6.90-7.00 (3H), 7.00-7.05 (1H), 7.15-7.25 (2H), 7.35-7.40 (1H) Rhip. Funct. ED100 mg/cm2=0.03
Example 181
Benzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
To a solution of the compound of Example 58 (7.50 g, 37.4 mmol) and triethylamine (5.74 ml, 41.2 mmol) in dichloromethane (100 ml), cooled in an ice bath, was added dropwise benzyl chloroformate (21.4 ml, 150 mmol). The reaction mixture was allowed to warm to room temperature and stirred under nitrogen for 3 h. The mixture was cooled, before addition of aqueous sodium hydrogen carbonate solution and dichloromethane and the two layers were separated. The aqueous phase was extracted with dichloromethane and the combined organic phases were dried (MgSO4) and concentrated in vacuo. To the residue was added cyclohexane and the solution was filtered and concentrated in vacuo.
The residue was purified by flash chromatography (silica) with gradient elution, ethyl acetate:cyclohexane [10:90 to 100:0]. The appropriate fractions were combined and concentrated to give the title compound (9.96 g).
Experimental MH+ 335.2; expected 335.2 1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.22-2.29 (6H), 5.05-5.10 (1H), 5.23-5.25 (2H), 6.50-6.52 (1H), 6.83-6.87 (1H), 6.97-7.00 (2H), 7.28-7.31 (2H), 7.35-7.38 (3H), 7.50-7.51 (1H) Rhip. Funct. ED100 mg/cm2=>0.03
Similarly Prepared from Example 1 was:
Ex.
MH+ Found/
Rhip. Funct.
No.
R6
Precursor
Expected
ED100 mg/cm2
182
Isopropyl chlorocarbonate
287.4 287.2
0.03
Example 182
Isopropyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.10-1.20 (6H), 1.45-1.55 (3H), 2.20-2.35 (6H), 4.90-5.00 (1H), 5.00-5.10 (1H), 6.40-6.45 (1H), 6.80-6.85 (1H), 6.90-7.00 (2H), 7.40-7.45 (1H)
Example 183
Isobutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
To a solution of the compound of Example 58 (1.00 g, 5.0 mmol) and triethylamine (0.77 ml, 5.5 mmol) in anhydrous dichloromethane (10 ml) was added dropwise isobutyl chloroformate (2.60 ml, 20 mmol). The reaction mixture was stirred at room temperature for 18 h and then concentrated in vacuo. The residue was partitioned between water (10 ml) and ethyl acetate (10 ml) and the two layers were separated. The aqueous layer was extracted with ethyl acetate (10 ml) and the combined organic layers were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in acetonitrile:water (9:1, 4 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×30 mm LUNA C18(2) 5 μm column, 40 ml/min) using an acetonitrile:water gradient [65:35 to 95:5]. The appropriate fractions were combined and concentrated to give the title compound (150 mg).
Experimental MH+ 301.4; expected 301.2 1H-NMR (CDCl3): 0.89-0.95 (6H), 1.60-1.63 (3H), 1.91-2.00 (1H), 2.28-2.39 (6H), 3.95-4.04 (2H), 5.05-5.11 (1H), 6.62-6.65 1H), 6.90-7.01 (3H), 7.40-7.41 (1H) Rhip. Funct. ED100 mg/cm2=0.03
Similarly Prepared by Acylation with Isobutyl Chlorocarbonate were:
Ex.
From
MH+ Found/
Rhip. Funct.
No.
Ar
Ex.
Expected
ED100 mg/cm2
184
52
305.4 305.2
0.3
185
29
323.6 323.2
1
186
1
301.4 301.2
0.01
Example 184
Isobutyl 2-[1-(3-fluoro-2-methylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (CDCl3): 0.83-0.95 (6H), 1.60-1.64 (3H), 1.91-1.99 (1H), 2.35-2.38 (3H), 3.97-4.02 (2H), 5.00-5.03 (1H), 6.59-6.61 (1H), 6.80-6.85 (1H), 6.98-7.03 (2H), 7.39-7.40 (1H)
Example 185
Isobutyl 2-[1-(2,6-difluoro-3-methylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (CDCl3): 0.85-0.96 (6H), 1.74-1.78 (3H), 1.95-2.00 (1H), 2.17-2.20 (3H), 3.98-4.04 (2H), 4.99-5.03 (1H), 6.65-6.71 (1H), 6.90-6.97 (2H), 7.39-7.41 (1H)
Example 186
Isobutyl 2-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.80-0.90 (6H), 1.40-1.50 (3H), 1.80-1.90 (1H), 2.20-2.30 (6H), 3.90-4.00 (2H), 5.00-5.10 (1H), 6.45-6.50 (1H), 6.80-6.85 (1H), 6.90-6.95 (2H), 7.45-7.50 (1H)
Example 187
2-[1-(2,3-Dimethylphenyl)ethyl]-N,N-dimethyl-1H-imidazole-1-sulfonamide
To a solution of the compound of Example 1 (100 mg, 0.5 mmol) and triethylamine (77 μl, 0.55 mmol) in anhydrous tetrahydrofuran (4 ml), was added dimethylsulphamoyl chloride (59 μl, 0.55 mmol). The reaction mixture was stirred at 60° C., under nitrogen, for 36 h and then concentrated in vacuo. The residue was partitioned between water (10 ml) and ethyl acetate (10 ml) and the two layers were separated. The aqueous layer was extracted with ethyl acetate (10 ml) and the combined organic layers were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol (1.5 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm LUNA C18(2) 10 μm column, 120 ml/min) using an acetonitrile:water gradient [20:80 to 95:5]. The appropriate fractions were combined and concentrated to give the title compound (49 mg).
Experimental MH+ 308.2; expected 308.1 1H-NMR (d6-Acetone): 1.50-1.56 (3H), 2.22-2.26 (3H), 2.31-2.36 (3H), 2.50-2.57 (6H), 4.95-5.01 (1H), 6.70-6.74 (1H), 6.85-6.95 (2H), 7.00-7.04 (1H), 7.40-7.41 (1H) Rhip. Funct. ED100 mg/cm2<=10
Example 188
2-Ethoxy-1-(ethoxymethyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
To a mixture of Example 58 (500 mg, 2.5 mmol) and pyridine (0.44 ml, 5.5 mmol) in anhydrous dichloromethane (5 ml), at 0° C. and under nitrogen, was added phosgene (20% in toluene, 1.44 ml, 2.75 mmol). The mixture was stirred at 0° C. for 20 min, before addition of 1,3-diethoxypropan-2-ol (407 mg, 2.75 mmol). The reaction mixture was stirred at room temperature for 30 min and then poured into ice water (10 ml). The mixture was adjusted to pH 7 by addition of solid sodium hydrogen carbonate and the two layers were separated. The aqueous phase was extracted with dichloromethane (2×10 ml) and the combined extracts were dried (MgSO4) and concentrated in vacuo.
The residue was dissolved in methanol (1.5 ml) and purified by automated preparative liquid chromatography (Gilson system, 150 mm×50 mm Sunfire C18 10 μm column, 120 ml/min) using an acetonitrile:water gradient [60:40 (20 min) to 98:2 (20.5 min)]. The appropriate fractions were combined and concentrated to give the title compound (435 mg).
Experimental MH+ 375.2; expected 375.2 1H-NMR (d6-Acetone): 0.97-1.02 (3H), 1.05-1.10 (3H), 1.55-1.59 (3H), 2.25-2.28 (3H), 2.32-2.35 (3H), 3.32-3.50 (5H), 3.52-3.58 (3H), 5.05-5.12 (1H), 6.54-6.57 (1H), 6.84-6.89 (1H), 6.96-6.99 (2H), 7.49-7.50 (1H) Rhip. Funct. ED100 mg/cm2=0.03
Example 189
Cyclopropylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
To a mixture of the compound of Example 58 (100 mg, 0.5 mmol) and pyridine (90 μl, 1.1 mmol) in anhydrous acetonitrile (1 ml), at 0° C. and under nitrogen, was added diphosgene (33 μl, 0.28 mmol). The mixture was stirred at 0° C. for 30 min, before addition of cyclopropylmethanol (43 μl, 0.55 mmol). The reaction mixture was stirred at room temperature for 1 h and then filtered.
The filtrate was purified by automated preparative liquid chromatography (Gilson system, 150 mm×22.4 mm LUNA C18(2) 5 μm column, 20 ml/min) using an acetonitrile:water gradient [15:85 (3 min) to 98:2 (16 min)]. The appropriate fractions were combined and concentrated to give the title compound (30 mg).
Experimental MH+ 299.4; expected 299.2 1H-NMR (CDCl3): 0.20-0.30 (2H), 0.50-0.60 (2H), 1.02-1.10 (1H), 1.59-1.65 (3H), 2.30-2.29 (6H), 3.96-4.05 (2H), 5.03-5.10 (1H), 6.62-6.65 (1H), 6.91-7.00 (3H), 7.40-7.41 (1H) Rhip. Funct. ED100 mg/cm2=0.03
Similarly Prepared from Example 58 were:
Ex.
MH+ Found/
Rhip. Funct.
No.
R6
Precursor
Expected
ED100 mg/cm2
190
2-Isopropoxyethanol
331.8 331.2
<=10
191
3-(3-Propoxypropoxy)- propan-1-ol
403.5 403.3
<=10
192
2-Ethoxyethanol
317.2 317.2
0.03
193
Cyclobutanol
300.0 299.2
<=10
194
2-Cyclohexylethanol
355.2 355.2
0.03
195
Tetrahydro-2H-pyran- 4-ylmethanol
343.9 343.2
<=10
196
3-(3-Methoxypropoxy)- propan-1-ol
375.2 375.2
0.03
197
(2-Methylcyclopropyl)- methanol
270.0 269.2 decarboxylates
<=10
198
3-[3-(3-Butoxy- propoxy)-propoxy]- propan-1-ol
476.1 475.3
>0.03
199
2,2,2-Trifluoroethanol
327.9 327.1
<=10
200
Cyclobutylmethanol
313.2 313.2
0.03
201
(1-Methylcyclopropyl)- methanol
313.9 313.2
202
2-Cyclopropylethanol
313.8 313.2
<=10
203
1,3-Dimethoxypropan- 2-ol
348.0 347.2
>0.03
204
(3-Methyloxetan-3- yl)methanol
329.9 329.2
>0.03
205
5-Methylhexan-1-ol
300.0 299.2 decarboxyl
>0.03
206
3-(4-Fluorophenoxy)- propan-1-ol
397.2 397.2
0.03
207
2,2,3,3,3-Pentafluoro- propan-1-ol
377.8 377.1
<=10
208
2-(Methylthio)ethanol
319.8 319.1
<=10
209
Ethanol
273.7 273.2
>0.03
210
3-Cyclohexylpropan-1- ol
369.9 369.3
<=10
211
3-Methylbutan-1-ol
315.7 315.2
>0.03
212
2-Isopropylcyclo- hexanol
369.9 369.3
<=10
213
2-Methoxyethanol
303.2 303.2
<=0.03, 0.1
214
Tetrahydro-2H-pyran- 4-ol
370.8 (MeCN adduct) 370.2
<=0.03
215
3-Cyclopentylpropan- 1-ol
355.9 355.2
>0.03
216
1-Methylpiperidin-4-ol
342.9 342.2
<=10
217
4,4,4-Trifluorobutan-1- ol
355.2 355.2
0.03
218
Cyclopentanol
313.8 313.2
<=10
219
(1-Methylcyclohexyl)- methanol
—
>0.03
220
Cyclopentylmethanol
328.1 327.2
<=10
221
4-Methylpentan-1-ol
329.8 329.2
>0.03
222
(1-Propylcyclobutyl)- methanol
355.9 355.2
>0.03
223
2-[(4-Chlorophenyl)- thio]ethanol
415.9 415.1
>0.03
224
(2S)-2-Methylbutan-1- ol
316.0 315.2
>0.03
225
3-(Methylthio)propan- 1-ol
333.8 333.2
>0.03
226
Cyclohexylmethanol
341.2 341.2
0.03
227
3-Ethoxypropan-1-ol
332.0 331.2
>0.03
228
2-Methylcyclohexanol
—
>0.03
229
2-(2,6-Dimethyl- morpholin-4-yl)ethanol
387.1 386.2
<=10
230
Pentan-1-ol
315.8 315.2
<=10
231
trans-4-Methyl- cyclohexanol
298.1 297.2 decarboxylates
<=10
232
2-Propylpentan-1-ol
—
>0.03
233
2-Ethylbutan-1-ol
—
<=10
234
2,2-Dimethylpropan-1- ol
315.8 315.2
<=10
235
Bicyclo[2.2.1]hept-2-yl- methanol
354.0 353.2
>0.03
236
3,3-Dimethylbutan-1-ol
330.1 329.2
>0.03
237
4-Isopropylcyclo- hexanol
369.3 369.3
<=0.03< =0.01
238
3-(Ethylthio)propan-1- ol
347.9 347.2
>0.03
239
Propan-1-ol
287.7 287.2
>0.03
240
3-Methoxy-3- methylbutan-1-ol
346.0 345.2
>0.03
241
3-(Dimethylamino)-2,2- dimethylpropan-1-ol
359.0 358.2
<=10
242
4-Methoxybutan-1-ol
332.1 331.2
>0.03
243
2,2,4-Trimethylpentan- 1-ol
357.3 357.3
0.03
244
Butan-1-ol
301.8 301.2
>0.03
245
3-Fluoro-3-methyl- butan-1-ol
333.8 333.2
>0.03
246
2-Isobutoxyethanol
346.0 345.2
0.1
247
4-Cyclohexylbutan-1-ol
383.3 383.3
0.1
248
4-Methylpent-3-en-1-ol
327.9 327.2
<=10
249
Pentan-3-ol
315.9 315.2
>0.03
250
(2S)-Pentan-2-ol
—
>0.03
251
(3S)-3,7-Dimethyl- octan-1-ol
386.1 385.3
>0.03
252
2-Propoxyethanol
331.8 331.2
>0.03
253
2,3-Dimethylpentan-1- ol
343.2 343.2
0.1
254
2,2-Dimethylbutan-1-ol
—
>0.03
255
Methyl 3-hydroxy-2,2- dimethylpropanoate
360.0 359.2
<=10
Example 190
2-Isopropoxyethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.00-1.09 (6H), 1.51-1.55 (3H), 2.25-2.27 (3H), 2.36-2.38 (3H), 3.50-3.63 (3H), 4.32-4.36 (2H), 5.08-5.12 (1H), 6.55-6.58 (1H), 6.82-6.87 (1H), 6.95-6.98 (2H), 7.45-7.47 (1H)
Example 191
3-(3-Propoxypropoxy)propyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.81-0.92 (3H), 0.97-1.13 (6H), 1.42-1.53 (2H), 1.57-1.60 (3H), 2.25-2.40 (6H), 3.21-3.40 (4H), 3.50-3.61 (3H), 5.00-5.15 (2H), 6.50-6.58 (1H), 6.83-6.91 (1H), 6.95-6.98 (2H), 7.47-7.50 (1H)
Example 192
2-Ethoxyethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.02-1.09 (3H), 1.52-1.57 (3H), 2.24-2.26 (3H), 2.37-2.39 (3H), 3.40-3.48 (2H), 3.59-3.66 (2H), 4.30-4.37 (2H), 5.05-5.11 (1H), 6.55-6.58 (1H), 6.84-6.90 (1H), 6.95-6.99 (2H), 7.48-7.49 (1H)
Example 193
Cyclobutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 1.60-1.65 (1H), 1.70-1.75 (1H), 1.98-2.08 (2H), 2.22-2.30 (5H), 2.36-2.39 (3H), 4.98-5.06 (2H), 6.48-6.50 (1H), 6.84-6.87 (1H), 6.95-6.98 (2H), 7.50-7.52 (1H)
Example 194
2-Cyclohexylethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.81-0.97 (2H), 1.10-1.30 (4H), 1.42-1.55 (5H), 1.59-1.70 (5H), 2.27-2.29 (3H), 2.36-2.38 (3H), 4.20-4.30 (2H), 5.04-5.09 (1H), 6.50-6.53 (1H), 6.85-6.89 (1H), 6.95-6.99 (2H), 7.46-7.47 (1H)
CHN Analysis Predicted: % C=74.54; % H=8.53; % N=7.90. Observed: % C=74.31; % H=8.50; % N=7.95.
Example 195
Tetrahydro-2H-pyran-4-ylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.17-1.24 (2H), 1.40-1.48 (2H), 1.54-1.57 (3H), 1.80-1.90 (1H), 2.27-2.29 (3H), 2.37-2.39 (3H), 3.20-3.30 (2H), 3.79-3.84 (2H), 4.01-4.15 (2H), 5.02-5.09 (1H), 6.51-6.54 (1H), 6.83-6.86 (1H), 6.95-6.98 (2H), 7.50-7.51 (1H)
Example 196
3-(3-Methoxypropoxy)propyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.95-1.04 (4H), 1.18-1.21 (3H), 1.51-1.56 (3H), 2.27-2.29 (3H), 2.37-2.39 (3H), 3.20-3.23 (2H), 3.24-3.40 (3H), 3.40-3.59 (2H), 5.04-5.10 (2H), 6.49-6.59 (1H), 6.82-6.88 (1H), 6.96-6.99 (2H), 7.46-7.48 (1H)
Example 197
(2-Methylcyclopropyl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.21-0.26 (1H), 0.40-0.48 (1H), 0.62-0.81 (2H), 0.92-0.97 (3H), 1.53-1.56 (3H), 2.26-2.28 (3H), 2.37-2.39 (3H), 4.00-4.12 (2H), 5.05-5.10 (1H), 6.56-6.59 (1H), 6.86-6.89 (1H), 6.95-6.98 (2H), 7.50-7.51 (1H)
Example 198
3-[3-(3-Butoxypropoxy)propoxy]propyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.15-1.20 (3H), 1.30-1.40 (2H), 1.41-1.50 (2H), 1.53-1.56 (3H), 2.25-2.29 (3H), 2.33-2.37 (3H), 3.20-3.25 (1H), 3.30-3.41 (4H), 3.47-3.61 (5H), 5.00-5.05 (1H), 5.10-5.17 (2H), 6.50-6.54 (1H), 6.82-6.86 (1H), 6.95-6.98 (2H), 7.50-7.53 (1H)
Example 199
2,2,2-Trifluoroethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.26-2.28 (3H), 2.36-2.38 (3H), 4.80-4.91 (2H), 5.05-5.10 (1H), 6.56-6.59 (1H), 6.86-6.88 (1H), 6.96-6.98 (1H), 7.00-7.01 (1H), 7.50-7.51 (1H)
Example 200
Cyclobutylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.55 (3H), 1.70-1.80 (2H), 1.80-1.90 (2H), 1.90-2.02 (2H), 2.25-2.27 (3H), 2.35-2.37 (3H), 4.12-4.21 (2H), 5.03-5.09 (1H), 6.53-6.57 (1H), 6.85-6.89 (1H), 6.95-6.98 (2H), 7.46-7.47 (1H)
Example 201
(1-Methylcyclopropyl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.30-0.40 (2H), 0.45-0.55 (2H), 1.02-1.05 (3H), 1.53-1.56 (3H), 2.23-2.25 (3H), 2.34-2.36 (3H), 4.00-4.07 (2H), 5.10-5.15 (1H), 6.57-6.59 (1H), 6.84-6.86 (1H), 6.95-6.98 (2H), 7.51-7.53 (1H)
Example 202
2-Cyclopropylethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.00-0.05 (2H), 0.35-0.40 (2H), 0.60-0.68 (1H), 1.45-1.56 (5H), 2.24-2.26 (3H), 2.35-2.38 (3H), 4.20-4.35 (2H), 5.05-5.13 (1H), 6.51-6.53 (1H), 6.84-6.88 (1H), 6.92-6.98 (2H), 7.49-7.50 (1H)
Example 203
2-Methoxy-1-(methoxymethyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.29-2.31 (3H), 2.36-2.38 (3H), 3.29-3.35 (6H), 3.41-3.51 (4H), 5.05-5.12 (2H), 6.53-6.55 (1H), 6.85-6.87 (1H), 6.95-6.98 (2H), 7.49-7.51 (1H)
Example 204
(3-Methyloxetan-3-yl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.25-1.26 (3H), 1.55-1.58 (3H), 2.26-2.28 (3H), 2.36-2.38 (3H), 4.23-4.29 (3H), 4.39-4.44 (3H), 5.05-5.10 (1H), 6.58-6.60 (1H), 6.84-6.87 (1H), 6.95-6.98 (2H), 7.50-7.52 (1H)
Example 205
5-Methylhexyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.80-0.84 (6H), 1.15-1.20 (2H), 1.25-1.30 (2H), 1.49-1.61 (5H), 2.26-2.28 (3H), 2.37-2.39 (3H), 4.19-4.25 (2H), 5.05-5.10 (1H), 6.56-6.58 (1H), 6.84-6.86 (1H), 6.95-6.98 (2H), 7.48-7.50 (1H)
Example 206
3-(4-Fluorophenoxy)propyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.05-2.10 (2H), 2.24-2.26 (3H), 2.34-2.37 (3H), 3.97-4.01 (2H), 4.36-4.40 (1H), 4.01-4.05 (1H), 5.02-5.07 (1H), 6.50-6.53 (1H), 6.82-6.90 (3H), 6.91-6.96 (2H), 7.00-7.05 (2H), 7.51-7.52 (1H)
CHN Analysis
Predicted: % C=69.68; % H=6.36; % N=7.07.
Observed: % C=69.70; % H=6.37; % N=7.07.
Example 207
2,2,3,3,3-Pentafluoropropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.57 (3H), 2.25-2.27 (3H), 2.35-2.38 (3H), 4.85-4.99 (2H), 5.03-5.09 (1H), 6.55-6.58 (1H), 6.85-6.99 (2H), 7.00-7.01 (1H), 7.45-7.46 (1H)
Example 208
2-(Methylthio)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.02-2.05 (3H), 2.24-2.26 (3H), 2.35-2.38 (3H), 2.61-2.72 (2H), 4.30-4.41 (2H), 5.03-5.10 (1H), 6.52-6.56 (1H), 6.85-6.91 (1H), 6.95-6.99 (2H), 7.50-7.51 (1H)
Example 209
Ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.17-1.21 (3H), 1.51-1.53 (3H), 2.26-2.28 (3H), 2.36-2.38 (3H), 4.19-4.26 (2H), 5.03-5.08 (1H), 6.51-6.53 (1H), 6.85-6.88 (1H), 6.96-6.99 (2H), 7.47-7.49 (1H)
Example 210
3-Cyclohexylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.79-0.89 (2H), 1.10-1.25 (6H), 1.51-1.54 (3H), 1.58-1.70 (7H), 2.25-2.27 (3H), 2.35-2.38 (3H), 4.12-4.25 (2H), 5.02-5.09 (1H), 6.50-6.53 (1H), 6.84-6.89 (1H), 6.95-6.98 (2H), 7.49-7.54 (1H)
Example 211
3-Methylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.81-0.87 (6H), 1.43-1.60 (6H), 2.27-2.29 (3H), 2.36-2.38 (3H), 4.19-4.27 (2H), 5.03-5.08 (1H), 6.51-6.53 (1H), 6.86-6.88 (1H), 6.96-6.99 (2H), 7.47-7.49 (1H)
Example 212
2-Isopropylcyclohexyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.50-0.54 (3H), 0.70-0.80 (3H), 1.00-1.20 (3H), 1.20-1.45 (3H), 1.50-1.60 (3H), 1.61-1.72 (3H), 1.80-1.89 (1H), 2.24-2.28 (3H), 2.35-2.38 (3H), 4.60-4.65 (1H), 4.70-4.76 (1H), 5.00-5.08 (1H), 6.35-6.45 (1H), 6.82-6.90 (1H), 6.95-6.98 (2H), 7.49-7.54 (1H)
Example 213
2-Methoxyethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.25-2.27 (3H), 2.31-2.33 (3H), 3.22-3.23 (3H), 3.45-3.60 (2H), 4.30-4.40 (2H), 5.04-5.11 (1H), 6.56-6.58 (1H), 6.84-6.86 (1H), 6.95-6.99 (2H), 7.44-7.45 (1H)
Example 214
Tetrahydro-2H-pyran-4-yl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.55 (3H), 1.90-1.96 (1H), 2.09-2.11 (1H), 2.24-2.27 (3H), 2.32-2.34 (3H), 3.70-3.83 (4H), 5.02-5.09 (1H), 5.34-5.37 (1H), 6.50-6.53 (1H), 6.83-6.86 (1H), 6.96-6.99 (2H), 7.44-7.45 (1H)
Example 215
3-Cyclopentylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.99-1.05 (2H), 1.22-1.28 (2H), 1.48-1.62 (9H), 1.69-1.74 (3H), 2.26-2.28 (3H), 2.36-2.38 (3H), 4.15-4.22 (2H), 5.03-5.08 (1H), 6.51-6.53 (1H), 6.85-6.87 (1H), 6.95-6.98 (2H), 7.47-7.49 (1H)
Example 216
1-Methylpiperidin-4-yl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 1.60-1.71 (3H), 1.79-1.88 (2H), 2.10-2.21 (4H), 2.27-2.29 (3H), 2.36-2.38 (3H), 2.50-2.60 (2H), 4.70-4.80 (1H), 5.03-5.10 (1H), 6.52-6.54 (1H), 6.82-6.88 (1H), 6.95-6.98 (2H), 7.49-7.50 (1H)
Example 217
4,4,4-Trifluorobutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.55 (3H), 1.89-1.96 (3H), 2.10-2.21 (4H), 2.37-2.39 (3H), 4.21-4.26 (1H), 4.35-4.40 (1H), 5.02-5.09 (1H), 6.50-6.53 (1H), 6.83-6.86 (1H), 6.96-6.99 (2H), 7.47-7.48 (1H)
Example 218
Cyclopentyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 1.55-1.75 (6H), 1.80-1.90 (2H), 2.25-2.27 (3H), 2.35-2.37 (3H), 5.05-5.11 (1H), 5.18-5.20 (1H), 6.52-6.54 (1H), 6.83-6.98 (3H), 7.44-7.45 (1H)
Example 219
(1-Methylcyclohexyl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.92-0.93 (3H), 1.20-1.40 (5H), 1.40-1.49 (5H), 1.53-1.56 (3H), 2.26-2.28 (3H), 2.36-2.38 (3H), 3.96-4.02 (2H), 5.10-5.15 (1H), 6.56-6.59 (1H), 6.85-6.87 (1H), 6.95-6.98 (2H), 7.48-7.50 (1H)
Example 220
Cyclopentylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.17-1.25 (2H), 1.45-1.70 (11H), 2.15-2.21 (1H), 2.25-2.26 (3H), 2.35-2.37 (3H), 4.04-4.19 (2H), 5.04-5.10 (1H), 6.52-6.55 (1H), 6.83-6.88 (1H), 6.95-6.98 (2H), 7.47-7.48 (1H)
Example 221
4-Methylpentyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.80-0.85 (6H), 1.12-1.20 (2H), 1.50-1.56 (4H), 1.58-1.61 (2H), 2.27-2.29 (3H), 2.35-2.37 (3H), 4.14-4.19 (1H), 4.20-4.24 (1H), 5.04-5.08 (1H), 6.52-6.55 (1H), 6.85-6.87 (1H), 6.93-6.97 (2H), 7.47-7.49 (1H)
Example 222
(1-Propylcyclobutyl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.81-0.85 (3H), 1.20-1.26 (2H), 1.42-1.46 (2H), 1.52-1.56 (3H), 1.75-1.86 (6H), 2.26-2.28 (3H), 2.35-2.37 (3H), 4.15-4.22 (2H), 5.09-5.13 (1H), 6.58-6.60 (1H), 6.85-6.87 (1H), 6.95-6.98 (2H), 7.43-7.45 (1H)
Example 223
2-[(4-Chlorophenyl)thio]ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.54-1.56 (3H), 2.25-2.27 (3H), 2.35-2.37 (3H), 3.20-3.25 (2H), 4.36-4.40 (2H), 5.00-5.05 (1H), 6.51-6.53 (1H), 6.85-6.87 (1H), 6.91-6.95 (2H), 7.30-7.40 (5H)
Example 224
(2S)-2-Methylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.90-0.95 (3H), 1.10-1.20 (1H), 1.32-1.41 (1H), 1.54-1.56 (3H), 1.65-1.71 (1H), 2.25-2.27 (3H), 2.34-2.36 (3H), 4.03-4.05 (2H), 5.04-5.08 (1H), 6.52-6.54 (1H), 6.83-6.86 (1H), 6.96-7.00 (2H), 7.48-7.50 (1H)
Example 225
3-(Methylthio)propyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.54 (3H), 1.85-1.92 (2H), 2.05-2.07 (3H), 2.27-2.29 (3H), 2.36-2.38 (3H), 2.42-2.46 (2H), 4.22-4.30 (2H), 4.23-4.29 (1H), 4.32-4.38 (1H), 6.51-6.53 (1H), 6.83-6.86 (1H), 6.96-6.99 (2H), 7.51-7.53 (1H)
Example 226
Cyclohexylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.82-0.99 (2H), 1.05-1.23 (3H), 1.50-1.55 (3H), 1.56-1.70 (6H), 2.25-2.27 (3H), 2.37-2.39 (3H), 3.98-4.08 (2H), 5.02-5.09 (1H), 6.49-6.52 (1H), 6.83-6.86 (1H), 6.96-6.99 (2H), 7.48-7.49 (1H)
CHN Analysis
Predicted: % C=74.08; % H=8.29; % N=8.23.
Observed: % C=74.09; % H=8.27; % N=8.27.
Example 227
3-Ethoxypropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.02-1.06 (3H), 1.54-1.57 (3H), 1.80-1.86 (2H), 2.26-2.28 (3H), 2.36-2.38 (3H), 3.35-3.40 (4H), 4.21-4.30 (2H), 5.03-5.05 (1H), 6.52-6.54 (1H), 6.83-6.86 (1H), 6.96-6.99 (2H), 7.50-7.52 (1H)
Example 228
2-Methylcyclohexyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.70-0.78 (3H), 1.00-1.10 (2H), 1.20-1.30 (2H), 1.40-1.45 (1H), 1.55-1.58 (3H), 1.60-1.64 (1H), 1.65-1.75 (2H), 1.80-1.85 (1H), 2.26-2.28 (3H), 2.36-2.38 (3H), 4.30-4.36 (1H), 5.00-5.05 (1H), 6.40-6.44 (1H), 6.82-6.86 (1H), 6.96-6.99 (2H), 7.50-7.52 (1H)
Example 229
2-(2,6-Dimethylmorpholin-4-yl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.98-1.03 (3H), 1.53-1.56 (3H), 1.59-1.65 (1H), 2.21-2.24 (1H), 2.27-2.28 (3H), 2.38-2.39 (3H), 2.42-2.58 (2H), 2.60-2.63 (1H), 2.65-2.67 (1H), 3.20-3.29 (1H), 3.35-3.41 (1H), 4.30-4.35 (2H), 5.04-5.09 (1H), 6.54-6.56 (1H), 6.82-6.87 (1H), 6.95-7.00 (2H), 7.49-7.50 (1H)
Example 230
Pentyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.81-0.86 (3H), 1.20-1.33 (4H), 1.53-1.56 (3H), 1.59-1.62 (2H), 2.26-2.27 (3H), 2.35-2.36 (3H), 4.17-4.25 (2H), 5.04-5.10 (1H), 6.52-6.54 (1H), 6.83-6.87 (1H), 6.95-6.97 (2H), 7.49-7.50 (1H)
Example 231
trans-4-Methylcyclohexyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.82-0.90 (3H), 1.00-1.10 (1H), 1.20-1.45 (4H), 1.52-1.55 (3H), 1.65-1.70 (2H), 1.85-1.95 (2H), 2.24-2.30 (6H), 4.60-4.66 (1H), 5.02-5.10 (1H), 6.52-6.55 (1H), 6.82-6.98 (3H), 7.46-7.49 (1H)
Example 232
2-Propylpentyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.90-0.97 (6H), 1.20-1.38 (8H), 1.54-1.58 (3H), 1.65-1.72 (1H), 2.27-2.29 (3H), 2.36-2.38 (3H), 4.10-4.19 (2H), 5.10-5.15 (1H), 6.55-6.58 (1H), 6.85-6.88 (1H), 6.96-6.99 (2H), 7.44-7.46 (1H)
Example 233
2-Ethylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.90-0.96 (6H), 1.24-1.36 (4H), 1.54-1.58 (4H), 2.27-2.29 (3H), 2.36-2.38 (3H), 4.10-4.15 (1H), 4.16-4.20 (1H), 5.07-5.11 (1H), 5.57-5.59 (1H), 6.83-6.87 (1H), 6.95-6.98 (2H), 7.46-7.48 (1H)
Example 234
2,2-Dimethylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.95-0.98 (9H), 1.54-1.57 (3H), 2.26-2.28 (3H), 2.32-2.34 (3H), 3.90-3.94 (1H), 3.96-4.00 (1H), 5.09-5.13 (1H), 6.57-6.59 (1H), 6.84-6.88 (1H), 6.94-6.98 (2H), 7.55-7.57 (1H)
Example 235
Bicyclo[2.2.1]hept-2-ylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.62-0.70 (1H), 1.10-1.16 (2H), 1.25-1.35 (3H), 1.40-1.48 (2H), 1.52-1.57 (3H), 1.61-1.66 (1H), 2.12-2.19 (2H), 2.28-2.30 (3H), 2.37-2.39 (3H), 4.19-4.27 (2H), 5.03-5.08 (1H), 6.51-6.53 (1H), 6.84-6.88 (1H), 6.96-6.99 (2H), 7.46-7.48 (1H)
Example 236
3,3-Dimethylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.89-0.92 (3H), 1.48-1.57 (5H), 2.28-2.30 (3H), 2.37-2.39 (3H), 4.20-4.25 (1H), 4.30-4.35 (1H), 5.02-5.08 (1H), 6.51-6.53 (1H), 6.84-6.87 (1H), 6.96-6.99 (2H), 7.45-7.47 (1H)
Example 237
4-Isopropylcyclohexyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.81-0.86 (6H), 1.03-1.20 (2H), 1.20-1.29 (2H), 1.38-1.48 (2H), 1.54-1.57 (3H), 1.75-1.81 (2H), 1.90-2.00 (2H), 2.27-2.29 (3H), 2.37-2.39 (3H), 4.60-4.67 (1H), 5.02-5.08 (1H), 6.49-6.52 (1H), 6.84-6.87 (1H), 6.96-6.99 (2H), 7.46-7.48 (1H)
CHN Analysis
Predicted: % C=74.96; % H=8.75; % N=7.60.
Observed: % C=74.98; % H=8.78; % N=7.58.
Example 238
3-(Ethylthio)propyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.15-1.20 (3H), 1.55-1.58 (3H), 1.83-1.89 (2H), 2.28-2.30 (3H), 2.36-2.38 (3H), 2.42-2.50 (4H), 4.25-4.30 (1H), 4.33-4.38 (1H), 5.04-5.08 (1H), 6.53-6.55 (1H), 6.85-6.88 (1H), 6.96-6.99 (2H), 7.50-7.52 (1H)
Example 239
Propyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.82-0.86 (3H), 1.55-1.58 (3H), 1.60-1.66 (2H), 2.27-2.29 (3H), 2.36-2.38 (3H), 4.10-4.20 (2H), 5.04-5.10 (1H), 6.53-6.55 (1H), 6.85-6.88 (1H), 6.96-6.99 (2H), 7.49-7.51 (1H)
Example 240
3-Methoxy-3-methylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.10-1.12 (6H), 1.53-1.55 (3H), 1.70-1.80 (2H), 2.26-2.28 (3H), 2.37-2.38 (3H), 3.09-3.10 (3H), 4.21-4.31 (2H), 5.03-5.07 (1H), 6.51-6.53 (1H), 6.85-6.88 (1H), 6.97-6.99 (2H), 7.46-7.47 (1H)
Example 241
3-(Dimethylamino)-2,2-dimethylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.83-0.90 (6H), 1.52-1.55 (3H), 2.15-2.20 (6H), 2.23-2.24 (3H), 2.35-2.36 (3H), 4.00-4.08 (2H), 5.10-5.16 (1H), 6.52-6.55 (1H), 6.83-6.87 (1H), 6.95-6.99 (2H), 7.49-7.50 (1H)
Example 242
4-Methoxybutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.47-1.55 (5H), 1.61-1.66 (2H), 2.28-2.30 (3H), 2.37-2.39 (3H), 3.21-3.22 (3H), 3.30-3.34 (2H), 4.20-4.26 (2H), 5.04-5.08 (1H), 6.52-6.54 (1H), 6.83-6.86 (1H), 6.97-7.00 (2H), 7.48-7.49 (1H)
Example 243
2,2,4-Trimethylpentyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.82-0.90 (6H), 0.95-0.97 (6H), 1.20-1.22 (2H), 1.53-1.57 (3H), 1.61-1.71 (1H), 2.25-2.27 (3H), 2.35-2.37 (3H), 3.95-4.01 (2H), 5.10-5.15 (1H), 6.55-6.58 (1H), 6.84-6.87 (1H), 6.95-7.00 (2H), 7.49-7.50 (1H)
CHN Analysis
Predicted: % C=74.12; % H=9.05; % N=7.86.
Observed: % C=74.22; % H=9.05; % N=7.91.
Example 244
Butyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.82-0.88 (3H), 1.21-1.28 (2H), 1.54-1.60 (5H), 2.28-2.30 (3H), 2.37-2.39 (3H), 4.17-4.25 (2H), 5.03-5.07 (1H), 6.52-6.54 (1H), 6.82-6.85 (1H), 6.97-7.00 (2H), 7.47-7.49 (1H)
Example 245
3-Fluoro-3-methylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.30-1.33 (3H), 1.37-1.39 (3H), 1.53-1.56 (3H), 1.85-1.95 (2H), 2.29-2.31 (3H), 2.36-2.38 (3H), 4.30-4.34 (1H), 4.39-5.02 (1H), 5.03-5.07 (1H), 6.52-6.54 (1H), 6.85-6.88 (1H), 6.96-6.99 (2H), 7.45-7.46 (1H)
Example 246
2-Isobutoxyethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.80-0.85 (6H), 1.53-1.57 (3H), 1.65-1.79 (1H), 2.26-2.28 (3H), 2.35-2.37 (3H), 3.15-3.20 (2H), 3.57-3.64 (1H), 4.31-4.40 (2H), 5.06-5.15 (1H), 6.57-6.60 (1H), 6.84-6.87 (1H), 6.95-6.99 (2H), 7.47-7.48 (1H)
Example 247
4-Cyclohexylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.80-0.90 (2H), 1.10-1.25 (8H), 1.50-1.70 (10H), 2.27-2.29 (3H), 2.37-2.39 (3H), 4.15-4.26 (2H), 5.02-5.10 (1H), 6.50-6.52 (1H), 6.84-6.87 (1H), 6.96-7.00 (2H), 7.47-7.48 (1H)
Example 248
4-Methylpent-3-en-1-yl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.60 (6H), 1.62-1.64 (3H), 2.28-2.30 (3H), 2.25-2.35 (2H), 2.37-2.39 (3H), 4.11-4.21 (2H), 5.02-5.10 (2H), 6.53-6.57 (1H), 6.84-6.89 (1H), 6.96-6.99 (2H), 7.42-7.43 (1H)
Example 249
1-Ethylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.72-0.78 (3H), 1.44-1.53 (7H), 2.25-2.28 (3H), 2.32-2.35 (3H), 4.74-4.79 (1H), 5.01-5.06 (1H), 6.42-6.44 (1H), 6.82-6.86 (1H), 6.96-6.99 (2H), 7.50-7.52 (1H)
Example 250
(1S)-1-Methylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.89-0.93 (3H), 1.10-1.22 (5H), 1.41-1.52 (5H), 2.25-2.28 (3H), 2.36-2.39 (3H), 4.89-4.95 (1H), 5.01-5.06 (1H), 6.41-6.46 (1H), 6.85-6.90 (1H), 6.96-6.99 (2H), 7.47-7.49 (1H)
Example 251
(3S)-3,7-Dimethyloctyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.91-0.96 (3H), 1.10-1.16 (3H), 1.20-1.30 (3H), 1.38-1.43 (1H), 1.45-1.55 (5H), 1.60-1.65 (1H), 2.29-2.31 (3H), 2.36-2.38 (3H), 4.20-4.30 (2H), 5.05-5.10 (1H), 6.51-6.53 (1H), 6.83-6.85 (1H), 6.96-6.98 (2H), 7.46-7.48 (1H)
Example 252
2-Propoxyethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.90-0.95 (3H), 1.42-1.52 (5H), 2.27-2.29 (3H), 2.36-2.38 (3H), 3.31-3.37 (2H), 3.59-3.64 (2H), 4.30-4.36 (2H), 5.07-5.12 (1H), 6.57-6.59 (1H), 6.83-6.85 (1H), 6.95-6.98 (2H), 7.46-7.48 (1H)
Example 253
2,3-Dimethylpentyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.75-0.88 (6H), 1.10-1.21 (1H), 1.30-1.43 (2H), 1.52-1.55 (3H), 1.79-1.88 (1H), 2.25-2.27 (3H), 2.34-2.36 (3H), 4.00-4.25 (2H), 5.02-5.11 (1H), 6.50-6.52 (1H), 6.84-6.87 (1H), 6.96-6.99 (2H), 7.44-7.46 (1H)
Example 254
2,2-Dimethylbutyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.79-0.83 (3H), 0.90-0.92 (6H), 1.25-1.33 (2H), 1.52-1.55 (3H), 2.24-2.26 (3H), 2.33-2.35 (3H), 3.90-3.95 (1H), 4.00-4.04 (1H), 5.10-5.15 (1H), 6.57-6.59 (1H), 6.85-6.88 (1H), 6.94-6.97 (2H), 7.49-7.50 (1H)
Example 255
3-Methoxy-2,2-dimethyl-3-oxopropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.19-1.24 (6H), 1.51-1.54 (3H), 2.23-2.26 (3H), 2.31-2.34 (3H), 3.59-3.60 (3H), 4.20-4.30 (2H), 5.05-5.11 (1H), 6.57-6.60 (1H), 6.82-6.86 (1H), 6.95-6.99 (2H), 7.19-7.20 (1H)
Example 256
4-Butoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
To a mixture of the compound of Example 58 (200 mg, 1.0 mmol) and pyridine (177 μl, 2.2 mmol) in anhydrous acetonitrile (3 ml), at 0° C. and under nitrogen, was added diphosgene (132 μl, 1.1 mmol). The mixture was allowed to warm to room temperature and stirred for 10 min, before addition to (4-butoxyphenyl)methanol (198 mg, 1.1 mmol) via syringe. The reaction mixture was stirred at room temperature for 30 min and filtered.
The filtrate was purified by automated preparative liquid chromatography (Gilson system, 150 mm×22.4 mm LUNA C18(2) 10 μm column, 24 ml/min) using an acetonitrile:water gradient [15:85 (3 min) to 98:2 (16 min)]. The appropriate fractions were combined and concentrated to give the title compound (6 mg).
Experimental MH+ 364.0; expected 363.2 (compound de-carboxylates) 1H-NMR (d6-Acetone): 0.92-0.98 (3H), 1.43-1.55 (5H), 1.69-1.78 (2H), 2.24-2.30 (6H), 3.97-4.01 (2H), 5.04-5.09 (1H), 5.17-5.18 (2H), 6.48-6.50 (1H), 6.83-6.88 (3H), 6.95-6.98 (2H), 7.10-7.13 (2H), 7.44-7.45 (1H) Rhip. Funct. ED100 mg/cm2=0.03
Similarly Prepared from Example 58 were:
Ex.
MH+ Found/
Rhip. Funct.
No.
R6
Precursor
Expected
ED100 mg/cm2
257
Biphenyl-4-ylmethanol
412.1 411.2
<=10
258
2,2-Diphenylethanol
426.0 425.0
<=10
259
(3,5-Difluorophenyl)methanol
371.2 371.2
0.1
260
(4-Chlorophenyl)methanol
369.8 369.1
>0.03
261
{4-[(4-Fluorobenzyl)oxy]- phenyl}methanol
460.0 459.2
<=10
262
(2,4,5-Trimethylphenyl)- methanol
334.0 333.2 decarboxylates
>0.03
263
(2,4-Dimethylphenyl)- methanol
363.9 363.2
>0.03
264
1-Naphthylmethanol
342.1 341.2 decarboxylates
0.03
265
Mesitylmethanol
334.0 333.2 decarboxylates
<=10
266
[4-(1H-1,2,4-Triazol-1-yl)- phenyl]methanol
—
>0.03
267
(4-tert-Butylphenyl)methanol
348.1 347.2 decarboxylates
<=10
268
(2-Fluorophenyl)methanol
310.0 309.2 decarboxylates
0.01
269
[4-(Benzyloxy)phenyl]- methanol
—
<=10
270
(Pentafluorophenyl)- methanol
423.0 423.1
<=10
271
Biphenyl-2-ylmethanol
—
<=10
272
(3-Phenoxyphenyl)methanol
384.0 383.2 decarboxylates
<=10
273
(2,3,5-Trifluorophenyl)- methanol
389.9 389.1
>0.03
274
(2-Chloro-4-fluorophenyl)- methanol
—
>0.03
275
(4-Fluoro-3-methoxyphenyl)- methanol
383.9 383.2
>0.03
276
(2,6-Dichlorophenyl)- methanol
403.9 403.1
>0.03
277
2-(4-tert-Butylphenyl)ethanol
406.0 405.3
>0.03
278
(2R)-2-Phenylpropan-1-ol
363.9 363.2
279
2-Mesitylethanol
392.0 391.2
>0.03
280
2-(4-Chlorophenyl)ethanol
383.9 383.2
0.03
281
2-(4-Isopropyl-2-methyl- phenyl)ethanol
406.0 405.3
<=10
282
2-(4-Methylphenyl)ethanol
364.0 363.2
<=10
283
(1S)-1-Phenylpropan-1-ol
363.9 363.2
<=10
284
2-(2,5-Dimethylphenyl)- ethanol
377.2 377.2
0.1
285
3-Phenylpropan-1-ol
363.9 363.2
>0.03
286
(2S)-2-Phenylpropan-1-ol
363.2 363.2
0.03
287
2-(3-Methylphenyl)ethanol
363.9 363.2
>0.03
288
2-Phenylethanol
349.9 349.2
<=10
289
2-(2-Methylphenyl)ethanol
363.2 363.2
0.03
290
2-[2-(2-Chloropyrimidin-4- yl)phenyl]ethanol
—
<=10
291
(2,3,4-Trifluorophenyl)- methanol
389.1 389.1
0.03
292
[2-(2-Phenylethyl)phenyl]- methanol
396.1 395.2 decarboxylates
>0.03
293
(5-Fluoro-2-methylphenyl)- methanol
324.1 323.2 decarboxylates
>0.03
294
(Pentamethylphenyl)- methanol
—
>0.03
295
[4-(Benzyloxy)-3-methoxy- phenyl]methanol
428.1 427.2 decarboxylates
>0.03
296
(2-Chlorophenyl)methanol
326.0 325.1 decarboxylates
>0.03
297
(2-Methoxy-5-methylphenyl)- methanol
336.1 335.2 decarboxylates
<=10
298
(3-Fluorophenyl)methanol
310.0 309.2 decarboxylates
>0.03
299
(4-Ethoxyphenyl)methanol
336.0 335.2 decarboxylates
<=10
300
(2,4-Difluorophenyl)methanol
—
>0.03
301
(2,4-Dimethoxy-3-methyl- phenyl)methanol
366.0 365.2 decarboxylates
<=10
302
(2-Fluoro-5-methoxyphenyl)- methanol
339.9 339.2 decarboxylates
>0.03
303
(4-Fluoro-2-methoxyphenyl)- methanol
340.0 339.2 decarboxylates
>0.03
304
(4-Chloro-2-fluorophenyl)- methanol
—
<=10
305
(2,5-Dimethoxyphenyl)- methanol
—
<=10
306
(3-Ethoxyphenyl)methanol
335.9 335.2 decarboxylates
>0.03
307
(2,5-Dichlorophenyl)- methanol
359.9 359.1 decarboxylates
>0.03
308
(2,6-Difluorophenyl)methanol
328.0 327.2 decarboxylates
>0.03
309
(3,5-Dichlorophenyl)- methanol
359.9 359.1 decarboxylates
>0.03
310
(5-Chloro-2-methoxyphenyl)- methanol
355.9 355.1 decarboxylates
>0.03
311
(3,4-Dimethylphenyl)- methanol
319.9 319.2 decarboxylates
>0.03
312
(4-Bromophenyl)methanol
369.9 369.1 decarboxylates
<=10
313
[4-(Cyclopentyloxy)-3- methoxyphenyl]methanol
406.1 405.2 decarboxylates
<=10
314
(2,3,5,6-Tetrafluorophenyl)- methanol
408.0 407.1 decarboxylates
>0.03
315
(3-Methoxy-4-methylphenyl)- methanol
380.0 379.2
<=10
316
(4-Methylphenyl)methanol
305.8 305.2 decarboxylates
>0.03
317
4-(Hydroxymethyl)- benzonitrile
361.0 360.2
<=10
318
(2-Ethoxyphenyl)methanol
380.1 379.2
<=10
319
(2-Fluoro-5-methylphenyl)- methanol
324.0 323.2 decarboxylates
<=10
320
(2,5-Difluoro-4-methyl- phenyl)methanol
385.2 385.2
<=0.03
321
(2,3,6-Trifluorophenyl)- methanol
389.9 389.1
>0.03
322
(2-Methylbiphenyl-3-yl)- methanol
426.0 425.2
<=10
323
(2-Methoxyphenyl)methanol
322.0 321.2 decarboxylates
<=0.03
324
(4-Bromo-2-fluorophenyl)- methanol
431.9 431.1
>0.03
325
(2,3-Dimethoxyphenyl)- methanol
396.0 395.2
>0.03
326
(2,3-Dichlorophenyl)- methanol
403.9 403.1
>0.03
327
(4-Butylphenyl)methanol
392.1 391.2
<=10
328
(3-Methoxyphenyl)methanol
365.9 365.2
<=10
329
(3,4-Dichlorophenyl)- methanol
403.9 403.1
>0.03
330
(3,4-Diethoxyphenyl)- methanol
380.1 379.2 decarboxylates
<=10
331
(3-Methylphenyl)methanol
350.1 349.2
>0.03
332
(4-Isopropylphenyl)methanol
378.0 377.2
>0.03
333
(3-Chlorophenyl)methanol
369.9 369.1
>0.03
334
(3,4-Difluorophenyl)methanol
327.8 327.2 decarboxylates
>0.03
335
(2-Chloro-3,4-dimethoxy- phenyl)methanol
385.9 485.2 decarboxylates
>0.03
336
(2-Methylphenyl)methanol
349.9 349.1
>0.03
337
(2-Chloro-6-fluorophenyl)- methanol
387.1 387.1
<=0.03
338
(4-Methoxyphenyl)methanol
321.9 321.2 decarboxylates
<=10
339
(2,3,5,6-Tetramethylphenyl)- methanol
391.1 391.2
<=10
340
(3,4,5-Trifluorophenyl)- methanol
345.1 345.1 decarboxylates
>0.03
341
(2,5-Difluorophenyl)methanol
327.1 327.2 decarboxylates
>0.03
342
(3,5-Dimethylphenyl)- methanol
319.1 319.2 decarboxylates
>0.03
343
[4-(1H-Pyrazol-1-yl)phenyl]- methanol
—
<=10
344
(3-Chloro-4-methylphenyl)- methanol
340.0 339.2 decarboxylates
>0.03
345
(4-Ethoxy-3-methoxyphenyl)- methanol
365.1 365.2 decarboxylates
>0.03
346
3-(Hydroxymethyl)- benzonitrile
360.1 360.2
<=10
347
(2-Methoxy-4-methylphenyl)- methanol
335.2 335.2 decarboxylates
>0.03
348
(4-Fluorophenyl)methanol
309.1 309.2 decarboxylates
<=0.03
Example 257
Biphenyl-4-ylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.23-2.30 (6H), 5.09-5.13 (1H), 5.29-5.32 (2H), 6.50-6.52 (1H), 6.84-6.86 (1H), 6.96-6.99 (2H), 7.35-7.39 (3H), 7.42-7.46 (2H), 7.57-7.58 (1H), 7.60-7.68 (4H)
Example 258
2,2-Diphenylethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.48-1.56 (3H), 2.25-2.35 (6H), 4.40-4.47 (1H), 4.79-4.87 (2H), 5.00-5.05 (1H), 6.55-6.58 (1H), 6.82-6.88 (2H), 6.95-6.99 (1H), 7.19-7.21 (1H), 7.21-7.24 (2H), 7.29-7.39 (8H)
Example 259
3,5-Difluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.56 (3H), 2.22-2.24 (3H), 2.28-2.30 (3H), 5.02-5.10 (1H), 5.22-5.25 (1H), 5.32-5.37 (1H), 6.48-6.50 (1H), 6.84-7.00 (6H), 7.59-7.60 (1H)
Example 260
4-Chlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.22-2.28 (6H), 5.02-5.07 (1H), 5.21-5.28 (2H), 6.48-6.51 (1H), 6.84-6.87 (1H), 6.97-6.99 (2H), 7.25-7.28 (2H), 7.36-7.39 (2H), 7.50-7.51 (1H)
Example 261
4-[(4-Fluorobenzyl)oxy]benzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.22-2.30 (6H), 5.04-5.13 (3H), 5.19-5.20 (2H), 6.51-6.53 (1H), 6.82-6.86 (1H), 6.95-7.00 (4H), 7.11-7.20 (2H), 7.22-7.25 (2H), 7.45-7.46 (1H), 7.50-7.56 (2H)
Example 262
2,4,5-Trimethylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.15-2.17 (3H), 2.19-2.23 (6H), 2.23-2.25 (3H), 5.02-5.06 (1H), 5.17-5.20 (1H), 5.21-5.24 (1H), 6.48-6.51 (1H), 6.84-6.86 (1H), 6.96-6.99 (3H), 7.00-7.01 (1H), 7.46-7.48 (1H)
Example 263
2,4-dimethylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
Example 264
1-Naphthylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.46-1.51 (3H), 2.19-2.26 (6H), 5.04-5.10 (1H), 5.75-5.79 (2H), 6.52-6.55 (1H), 6.83-6.87 (1H), 6.90-6.96 (2H), 7.42-7.50 (3H), 7.53-7.58 (2H), 7.95-7.99 (2H), 8.00-8.03 (1H)
Example 265
Mesitylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
Example 266
4-(1H-1,2,4-Triazol-1-yl)benzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.22-2.24 (3H), 2.30-2.32 (3H), 5.03-5.08 (1H), 5.31-5.39 (2H), 6.49-6.52 (1H), 6.84-6.86 (1H), 6.97-7.00 (2H), 7.41-7.44 (2H), 7.56-7.58 (1H), 7.80-7.83 (2H), 8.13-8.14 (1H), 9.02-9.04 (1H)
Example 267
4-tert-Butylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.28-1.33 (9H), 1.52-1.55 (3H), 2.22-2.28 (6H), 5.02-5.07 (1H), 5.20-5.22 (2H), 6.51-6.53 (1H), 6.84-6.86 (1H), 6.95-6.98 (2H), 7.20-7.23 (2H), 7.38-7.41 (2H), 7.50-7.51 (1H)
Example 268
2-Fluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.22-2.28 (6H), 5.02-5.07 (1H), 5.28-5.31 (1H), 5.37-5.40 (1H), 6.51-6.53 (1H), 6.83-6.85 (1H), 6.95-6.98 (2H), 7.16-7.20 (2H), 7.30-7.33 (1H), 7.40-7.43 (1H), 7.46-7.47 (1H)
Example 269
4-(Benzyloxy)benzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.24-2.29 (6H), 5.02-5.07 (1H), 5.11-5.13 (2H), 5.18-5.20 (2H), 6.48-6.50 (1H), 6.81-6.84 (1H), 6.94-7.00 (4H), 7.20-7.24 (2H), 7.33-7.41 (3H), 7.42-7.45 (3H)
Example 270
Pentafluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.25-2.27 (3H), 2.29-2.31 (3H), 4.99-5.02 (1H), 5.38-5.41 (1H), 5.42-5.45 (1H), 6.40-6.42 (1H), 6.80-6.83 (1H), 6.90-6.92 (1H), 6.97-6.98 (1H), 7.46-7.47 (1H)
Example 271
Biphenyl-2-ylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.15-2.17 (3H), 2.19-2.21 (3H), 4.98-5.02 (1H), 5.19-5.21 (2H), 6.48-6.50 (1H), 6.85-6.95 (3H), 7.20-7.24 (2H), 7.30-7.38 (5H), 7.39-7.41 (2H), 7.41-7.44 (1H)
Example 272
3-Phenoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.54-1.57 (3H), 2.23-2.25 (3H), 2.28-2.30 (3H), 5.02-5.10 (1H), 5.20-5.27 (2H), 6.51-6.54 (1H), 6.83-6.85 (1H), 6.91-7.04 (7H), 7.13-7.17 (1H), 7.30-7.41 (3H), 7.49-7.50 (1H)
Example 273
2,3,5-Trifluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.22-2.25 (3H), 2.29-2.32 (3H), 5.00-5.05 (1H), 5.30-5.33 (1H), 5.40-5.43 (1H), 6.48-6.50 (1H), 6.82-6.85 (1H), 6.93-6.98 (3H), 7.25-7.29 (1H), 7.57-7.59 (1H)
Example 274
2-Chloro-4-fluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.21-2.27 (6H), 5.02-5.06 (1H), 5.30-5.34 (1H), 5.36-5.40 (1H), 6.49-6.51 (1H), 6.84-6.86 (1H), 6.95-6.98 (2H), 7.09-7.12 (1H), 7.32-7.34 (1H), 7.39-7.41 (1H), 7.49-7.51 (1H)
Example 275
4-Fluoro-3-methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.01-2.03 (3H), 2.21-2.26 (6H), 3.80-3.81 (3H), 5.05-5.10 (1H), 5.20-5.23 (2H), 6.47-6.49 (1H), 6.84-6.87 (2H), 6.95-6.98 (2H), 7.09-7.16 (2H), 7.49-7.51 (1H)
Example 276
2,6-Dichlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.20-2.26 (6H), 5.00-5.05 (1H), 5.70-5.72 (1H), 5.77-5.79 (1H), 6.47-6.49 (1H), 6.81-6.83 (1H), 6.95-6.98 (2H), 7.42-7.43 (1H), 7.49-7.52 (3H)
Example 277
2-(4-tert-Butylphenyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.23-1.27 (9H), 1.50-1.53 (3H), 2.23-2.30 (6H), 2.85-2.93 (2H), 4.38-4.42 (2H), 5.00-5.05 (1H), 6.54-6.56 (1H), 6.85-6.88 (1H), 6.91-6.95 (2H), 7.16-7.19 (2H), 7.35-7.38 (2H), 7.42-7.43 (1H)
Example 278
(2R)-2-Phenylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.22-1.26 (3H), 1.50-1.53 (3H), 2.25-2.27 (3H), 2.30-2.32 (3H), 3.12-3.18 (1H), 4.24-4.30 (2H), 5.00-5.06 (1H), 6.52-6.54 (1H), 6.85-6.90 (2H), 6.90-6.94 (1H), 7.20-7.25 (3H), 7.29-7.32 (2H), 7.37-7.39 (1H)
Example 279
2-Mesitylethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.55 (3H), 2.20-2.21 (3H), 2.24-2.26 (6H), 2.27-2.29 (3H), 2.36-2.38 (3H), 4.20-4.25 (2H), 5.01-5.06 (1H), 6.51-6.53 (1H), 6.80-6.81 (2H), 6.85-6.87 (1H), 6.95-6.99 (2H), 7.46-7.48 (1H)
Example 280
2-(4-Chlorophenyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.24-2.26 (3H), 2.26-2.28 (3H), 2.90-2.96 (2H), 4.40-4.50 (2H), 5.01-5.06 (1H), 6.52-6.54 (1H), 6.83-6.96 (3H), 7.20-7.23 (2H), 7.25-7.27 (2H), 7.39-7.40 (1H)
Example 281
2-(4-Isopropyl-2-methylphenyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.19-1.26 (9H), 1.50-1.55 (3H), 2.25-2.29 (6H), 2.85-2.92 (1H), 3.04-3.15 (1H), 4.22-4.31 (2H), 5.00-5.10 (1H), 6.52-6.55 (1H), 6.82-6.96 (3H), 7.15-7.21 (4H), 7.38-7.39 (1H)
Example 282
2-(4-Methylphenyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.25-2.28 (6H), 2.30-2.31 (3H), 2.84-2.89 (2H), 4.35-4.42 (2H), 5.01-5.09 (1H), 6.53-6.55 (1H), 6.82-6.87 (1H), 6.95-6.98 (2H), 7.08-7.09 (4H), 7.40-7.41 (1H)
Example 283
1-Phenylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.89-0.93 (3H), 1.54-1.57 (3H), 1.79-1.84 (1H), 1.90-2.00 (1H), 2.30-2.36 (6H), 5.00-5.05 (1H), 5.60-5.64 (1H), 6.47-6.49 (1H), 6.89-6.93 (1H), 6.98-7.02 (2H), 7.19-7.21 (2H), 7.29-7.33 (3H), 7.60-7.61 (1H)
Example 284
2-(2,5-Dimethylphenyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.56 (3H), 2.20-2.25 (6H), 2.29-2.36 (6H), 2.81-2.94 (2H), 4.31-4.41 (2H), 5.00-5.05 (1H), 6.50-6.52 (1H), 6.84-6.98 (5H), 7.00-7.02 (1H), 7.42-7.43 (1H)
Example 285
3-Phenylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 1.90-1.98 (2H), 2.23-2.25 (3H), 2.36-2.38 (3H), 2.60-2.64 (2H), 4.20-4.26 (2H), 5.04-5.08 (1H), 6.51-6.53 (1H), 6.85-6.88 (1H), 6.96-6.98 (2H), 7.16-7.19 (3H), 7.22-7.25 (2H), 7.46-7.48 (1H)
Example 286
2-Phenylpropyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.20-1.24 (3H), 1.51-1.55 (3H), 2.25-2.27 (3H), 2.32-2.34 (3H), 3.05-3.09 (1H), 4.25-4.39 (2H), 5.02-5.10 (1H), 6.51-6.53 (1H), 6.81-6.95 (3H), 7.19-7.22 (1H), 7.27-7.30 (4H), 7.37-7.38 (1H)
CHN Analysis
Predicted: % C=76.21; % H=7.23; % N=7.73.
Observed: % C=76.07; % H=7.24; % N=7.63.
Example 287
2-(3-Methylphenyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.22-2.25 (6H), 2.32-2.35 (3H), 4.38-4.42 (2H), 5.01-5.05 (1H), 6.55-6.57 (1H), 6.85-6.88 (1H), 6.92-7.00 (3H), 7.00-7.05 (2H), 7.14-7.18 (1H), 7.40-7.41 (1H)
Example 288
2-Phenylethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.26-2.28 (3H), 2.31-2.33 (3H), 2.90-2.96 (2H), 4.39-4.44 (2H), 5.02-5.06 (1H), 6.52-6.55 (1H), 6.85-6.95 (3H), 7.20-7.30 (5H), 7.39-7.40 (1H)
Example 289
2-(2-Methylphenyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.27-2.30 (6H), 2.33-2.35 (3H), 2.85-2.99 (2H), 4.35-4.42 (2H), 5.02-5.09 (1H), 6.50-6.52 (1H), 6.84-6.87 (1H), 6.96-6.99 (2H), 7.10-7.19 (4H), 7.41-7.42 (1H)
CHN Analysis
Predicted: % C=76.21; % H=7.23; % N=7.73.
Observed: % C=76.22; % H=7.22; % N=7.66.
Example 290
2-[2-(2-Chloropyrimidin-4-yl)phenyl]ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.21-2.29 (6H), 3.10-3.20 (1H), 3.20-3.28 (1H), 4.40-4.50 (2H), 4.97-5.03 (1H), 6.50-6.52 (1H), 6.83-6.91 (3H), 7.18-7.29 (4H), 7.35-7.38 (1H), 7.40-7.42 (1H), 8.75-8.77 (1H)
Example 291
2,3,4-Trifluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.22-2.30 (6H), 5.01-5.07 (1H), 5.25-5.30 (1H), 5.39-5.41 (1H), 6.42-6.44 (1H), 6.81-6.85 (1H), 6.92-6.97 (2H), 7.16-7.20 (2H), 7.44-7.46 (1H)
CHN Analysis
Predicted: % C=64.94; % H=4.93; % N=7.21.
Observed: % C=64.90; % H=4.93; % N=7.21.
Example 292
2-(2-Phenylethyl)benzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.21-2.25 (6H), 2.80-2.85 (2H), 2.90-2.95 (2H), 5.01-5.06 (1H), 5.20-5.24 (1H), 5.31-5.35 (1H), 6.49-6.52 (1H), 6.82-6.85 (1H), 6.94-6.97 (2H), 7.12-7.20 (4H), 7.20-7.26 (3H), 7.30-7.34 (2H), 7.46-7.47 (1H)
Example 293
5-Fluoro-2-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.20-2.29 (9H), 5.01-5.06 (1H), 5.20-5.25 (1H), 5.31-5.35 (1H), 6.48-6.51 (1H), 6.82-6.85 (1H), 6.92-7.00 (2H), 7.00-7.05 (2H), 7.20-7.24 (1H), 7.56-7.58 (1H)
Example 294
Pentamethylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.45-1.49 (3H), 2.00-2.02 (3H), 2.11-2.13 (6H), 2.18-2.22 (9H), 2.22-2.23 (3H), 4.98-5.02 (1H), 5.31-5.35 (1H), 5.40-5.43 (1H), 6.40-6.43 (1H), 6.81-6.84 (1H), 6.90-6.94 (2H), 7.43-7.45 (1H)
Example 295
4-(Benzyloxy)-3-methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.21-2.27 (6H), 3.78-3.80 (3H), 5.04-5.11 (3H), 5.18-5.20 (2H), 6.50-6.53 (1H), 6.80-6.90 (2H), 6.90-7.00 (4H), 7.30-7.40 (3H), 7.43-7.46 (2H)
Example 296
2-Chlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.20-2.26 (6H), 5.04-5.09 (1H), 5.30-5.40 (2H), 6.50-6.53 (1H), 6.83-6.85 (1H), 6.96-7.00 (2H), 7.30-7.33 (2H), 7.39-7.41 (1H), 7.43-7.45 (1H), 7.54-7.55 (1H)
Example 297
2-Methoxy-5-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.20-2.30 (9H), 3.78-3.80 (3H), 5.04-5.12 (1H), 5.20-5.27 (2H), 6.52-6.54 (1H), 6.85-6.90 (2H), 6.95-7.00 (2H), 7.00-7.02 (1H), 7.15-7.18 (1H), 7.42-7.44 (1H)
Example 298
3-Fluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.22-2.28 (6H), 5.02-5.07 (1H), 5.12-5.19 (2H), 6.49-6.51 (1H), 6.82-6.84 (1H), 6.96-7.00 (2H), 7.02-7.10 (3H), 7.36-7.40 (1H), 7.57-7.58 (1H)
Example 299
4-Ethoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.37-1.40 (3H), 1.50-1.53 (3H), 2.23-2.28 (6H), 4.00-4.05 (2H), 5.03-5.07 (1H), 5.17-5.20 (2H), 6.49-6.51 (1H), 6.82-6.90 (3H), 6.95-6.99 (2H), 7.10-7.14 (2H), 7.44-7.46 (1H)
Example 300
2,4-Difluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.22-2.27 (6H), 5.01-5.05 (1H), 5.21-5.24 (1H), 5.35-5.39 (1H), 6.43-6.45 (1H), 6.91-6.93 (1H), 6.95-7.00 (2H), 7.00-7.09 (2H), 7.37-7.40 (1H), 7.44-7.46 (1H)
Example 301
2,4-Dimethoxy-3-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.57-1.60 (3H), 2.09-2.11 (3H), 2.22-2.27 (6H), 3.71-3.73 (3H), 3.79-3.81 (3H), 4.45-4.54 (3H), 6.69-6.72 (1H), 6.89-6.91 (2H), 6.95-7.00 (3H), 7.10-7.12 (1H)
Example 302
2-Fluoro-5-methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.22-2.27 (6H), 3.79-3.81 (3H), 5.02-5.07 (1H), 5.21-5.24 (1H), 5.26-5.29 (1H), 6.51-6.54 (1H), 6.83-6.86 (1H), 6.90-6.96 (4H), 7.03-7.06 (1H), 7.49-7.51 (1H
Example 303
4-Fluoro-2-methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.22-2.26 (6H), 3.80-3.81 (3H), 5.02-5.07 (1H), 5.19-5.22 (1H), 5.23-5.26 (1H), 6.49-6.52 (1H), 6.62-6.66 (1H), 6.80-6.90 (2H), 6.95-6.98 (2H), 7.21-7.24 (1H), 7.42-7.44 (1H)
Example 304
4-Chloro-2-fluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.21-2.27 (6H), 5.01-5.05 (1H), 5.21-5.25 (1H), 5.35-5.39 (1H), 6.47-6.50 (1H), 6.82-6.86 (1H), 6.92-6.96 (2H), 7.20-7.22 (1H), 7.25-7.35 (2H), 7.47-7.49 (1H)
Example 305
2,5-Dimethoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.21-2.25 (6H), 3.72-3.76 (6H), 5.05-5.09 (1H), 5.20-5.24 (2H), 6.54-6.57 (1H), 6.82-6.93 (6H), 7.47-7.49 (1H)
Example 306
3-Ethoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.35-1.39 (3H), 1.52-1.55 (3H), 2.22-2.27 (6H), 4.00-4.04 (2H), 5.03-5.07 (1H), 5.20-5.24 (2H), 6.51-6.53 (1H), 6.80-6.89 (4H), 6.95-6.98 (2H), 7.20-7.24 (1H), 7.50-7.52 (1H)
Example 307
2,5-Dichlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.21-2.27 (6H), 5.01-5.06 (1H), 5.30-5.34 (1H), 5.39-5.43 (1H), 6.50-6.53 (1H), 6.84-6.87 (1H), 6.92-6.97 (2H), 7.40-7.47 (3H), 7.48-7.50 (1H)
Example 308
2,6-Difluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.21-2.27 (6H), 5.01-5.06 (1H), 5.32-5.36 (1H), 5.40-5.44 (1H), 6.50-6.53 (1H), 6.82-6.85 (1H), 6.90-6.95 (2H), 7.42-7.44 (1H), 7.50-7.55 (1H)
Example 309
3,5-Dichlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.22-2.24 (3H), 2.27-2.30 (3H), 5.01-5.06 (1H), 5.20-5.24 (1H), 5.31-5.35 (1H), 6.50-6.53 (1H), 6.85-6.88 (1H), 6.95-6.99 (2H), 7.29-7.31 (1H), 7.42-7.44 (1H), 7.57-7.59 (1H)
Example 310
5-Chloro-2-methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.21-2.27 (6H), 3.79-3.81 (3H), 5.03-5.09 (1H), 5.20-5.23 (1H), 5.27-5.30 (1H), 6.51-6.53 (1H), 6.84-6.86 (1H), 6.90-6.94 (2H), 7.00-7.03 (1H), 7.21-7.23 (1H), 7.35-7.38 (1H), 7.50-7.51 (1H)
Example 311
3,4-Dimethylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.20-2.25 (6H), 2.26-2.30 (6H), 5.05-5.10 (1H), 5.17-5.21 (2H), 6.50-6.53 (1H), 6.83-6.85 (1H), 6.95-6.97 (2H), 6.98-7.03 (2H), 7.05-7.08 (1H), 7.44-7.46 (1H)
Example 312
4-Bromobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.22-2.29 (6H), 5.01-5.06 (1H), 5.20-5.29 (2H), 6.43-6.45 (1H), 6.83-6.86 (1H), 6.95-6.99 (2H), 7.09-7.11 (2H), 7.49-7.53 (3H)
Example 313
4-(Cyclopentyloxy)-3-methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 1.58-1.62 (2H), 1.70-1.81 (4H), 1.82-1.89 (2H), 2.22-2.28 (6H), 3.75-3.77 (3H), 4.80-4.83 (1H), 5.06-5.10 (1H), 5.17-5.19 (2H), 6.50-6.53 (1H), 6.80-6.90 (3H), 6.93-6.98 (3H), 7.47-7.48 (1H)
Example 314
2,3,5,6-Tetrafluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.22-2.28 (6H), 5.00-5.05 (1H), 5.39-5.43 (1H), 5.45-5.48 (1H), 6.42-6.45 (1H), 6.80-6.83 (1H), 6.89-6.91 (1H), 6.95-6.97 (1H), 7.47-7.48 (1H), 7.59-7.63 (1H)
Example 315
3-Methoxy-4-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.07-2.09 (3H), 2.22-2.28 (6H), 3.78-3.80 (3H), 5.04-5.09 (1H), 5.20-5.22 (2H), 6.50-6.53 (1H), 6.78-6.80 (1H), 6.82-6.88 (2H), 6.95-6.98 (2H), 7.18-7.20 (1H), 7.50-7.51 (1H)
Example 316
4-Methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.55 (3H), 2.22-2.30 (9H), 5.02-5.06 (1H), 5.20-5.23 (2H), 6.50-6.53 (1H), 6.83-6.86 (1H), 6.95-6.98 (2H), 7.15-7.19 (3H), 7.45-7.46 (1H)
Example 317
4-Cyanobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.21-2.28 (6H), 5.02-5.08 (1H), 5.35-5.42 (2H), 6.49-6.52 (1H), 6.88-6.92 (1H), 6.97-7.00 (2H), 7.40-7.43 (2H), 7.58-7.59 (1H), 7.70-7.73 (2H)
Example 318
2-Ethoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.20-1.26 (3H), 1.50-1.53 (3H), 2.20-2.24 (6H), 3.97-4.03 (2H), 5.05-5.13 (1H), 5.21-5.24 (1H), 5.33-5.36 (1H), 6.53-6.55 (1H), 6.82-6.89 (2H), 6.92-6.98 (3H), 7.19-7.21 (1H), 7.30-7.33 (1H), 7.46-7.47 (1H)
Example 319
2-Fluoro-5-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.22-2.31 (9H), 5.02-5.10 (1H), 5.20-5.23 (1H), 5.31-5.34 (1H), 6.50-6.52 (1H), 6.82-6.86 (1H), 6.94-6.98 (2H), 7.00-7.04 (1H), 7.10-7.13 (1H), 7.20-7.24 (1H), 7.45-7.46 (1H)
Example 320
2,5-Difluoro-4-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.22-2.31 (9H), 5.01-5.06 (1H), 5.20-5.24 (1H), 5.30-5.33 (1H), 6.46-6.48 (1H), 6.82-6.99 (3H), 7.00-7.10 (2H), 7.49-7.53 (1H)
Example 321
2,3,6-Trifluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.21-2.28 (6H), 5.01-5.06 (1H), 5.35-5.39 (1H), 5.41-5.45 (1H), 6.44-6.46 (1H), 6.81-6.85 (1H), 6.93-6.98 (2H), 7.05-7.08 (1H), 7.40-7.45 (2H)
Example 322
(2-Methyl biphenyl-3-yl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.17-2.18 (3H), 2.21-2.26 (6H), 5.05-5.11 (1H), 5.36-5.41 (2H), 6.50-6.52 (1H), 6.85-6.88 (1H), 6.93-6.98 (2H), 7.20-7.32 (5H), 7.38-7.40 (1H), 7.41-7.45 (2H), 7.56-7.57 (1H)
Example 323
2-Methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.22-2.29 (6H), 3.79-3.80 (3H), 5.03-5.11 (1H), 5.20-5.30 (2H), 6.51-6.53 (1H), 6.82-6.99 (4H), 7.00-7.02 (1H), 7.18-7.20 (1H), 7.33-7.36 (1H), 7.45-7.46 (1H)
Example 324
4-Bromo-2-fluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.55 (3H), 2.21-2.27 (6H), 5.01-5.06 (1H), 5.22-5.26 (1H), 5.32-5.36 (1H), 6.45-6.47 (1H), 6.82-6.85 (1H), 6.95-6.98 (2H), 7.20-7.23 (1H), 7.35-7.38 (1H), 7.40-7.43 (1H), 7.51-7.53 (1H)
Example 325
2,3-Dimethoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.22-2.27 (6H), 3.69-3.70 (3H), 3.83-3.84 (3H), 5.04-5.09 (1H), 5.24-5.27 (2H), 6.51-6.53 (1H), 6.79-6.81 (1H), 6.84-6.87 (1H), 6.95-6.98 (2H), 7.00-7.06 (2H), 7.45-7.46 (1H)
Example 326
2,3-Dichlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.54-1.57 (3H), 2.21-2.26 (6H), 5.02-5.08 (1H), 5.35-5.40 (1H), 5.41-5.45 (1H), 6.47-6.49 (1H), 6.84-6.87 (1H), 6.97-7.00 (2H), 7.21-7.23 (1H), 7.30-7.33 (1H), 7.57-7.58 (1H), 7.58-7.59 (1H)
Example 327
4-Butylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 0.88-0.94 (3H), 1.30-1.40 (2H), 1.50-1.61 (5H), 2.23-2.26 (6H), 2.80-2.83 (2H), 5.02-5.10 (1H), 5.20-5.21 (2H), 6.50-6.52 (1H), 6.83-6.86 (1H), 6.95-6.99 (2H), 7.17-7.20 (4H), 7.47-7.48 (1H)
Example 328
3-Methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.22-2.27 (6H), 3.88-4.00 (3H), 5.04-5.09 (1H), 5.21-5.23 (2H), 6.52-6.54 (1H), 6.81-6.90 (4H), 6.95-6.99 (2H), 7.21-7.25 (1H), 7.46-7.47 (1H)
Example 329
3,4-Dichlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.23-2.28 (6H), 5.02-5.08 (1H), 5.20-5.24 (1H), 5.27-5.31 (1H), 6.44-6.46 (1H), 6.84-6.87 (1H), 6.96-6.99 (2H), 7.20-7.22 (1H), 7.50-7.56 (3H)
Example 330
3,4-Diethoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.31-1.39 (6H), 1.50-1.55 (3H), 2.22-2.28 (6H), 3.96-4.07 (4H), 5.03-5.09 (1H), 5.17-5.18 (2H), 6.52-6.54 (1H), 6.80-6.90 (3H), 6.95-7.00 (3H), 7.45-7.46 (1H)
Example 331
3-Methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.23-2.30 (9H), 5.04-5.09 (1H), 5.20-5.24 (2H), 6.49-6.51 (1H), 6.84-6.87 (1H), 6.95-6.98 (2H), 7.05-7.10 (2H), 7.15-7.18 (1H), 7.20-7.23 (1H), 7.48-7.49 (1H)
Example 332
4-Isopropylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.20-1.24 (6H), 1.52-1.55 (3H), 2.23-2.28 (6H), 5.03-5.08 (1H), 5.20-5.22 (2H), 6.49-6.51 (1H), 6.83-6.86 (1H), 6.96-6.99 (2H), 7.20-7.24 (4H), 7.50-7.51 (1H)
Example 333
3-Chlorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.22-2.24 (3H), 2.27-2.29 (3H), 5.02-5.07 (1H), 5.21-5.30 (2H), 6.49-6.51 (1H), 6.85-6.88 (1H), 6.95-6.99 (2H), 7.20-7.22 (1H), 7.35-7.39 (3H), 7.57-7.58 (1H)
Example 334
3,4-Difluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.22-2.24 (3H), 2.28-2.30 (3H), 5.02-5.07 (1H), 5.20-5.24 (1H), 5.25-5.29 (1H), 6.44-6.46 (1H), 6.83-6.86 (1H), 6.95-6.99 (2H), 7.09-7.12 (1H), 7.21-7.28 (2H), 7.56-7.57 (1H)
Example 335
2-Chloro-3,4-dimethoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.22-2.26 (6H), 3.80-3.81 (3H), 3.90-3.92 (3H), 5.02-5.06 (1H), 5.21-5.29 (2H), 6.49-6.51 (1H), 6.85-6.89 (1H), 6.94-7.00 (3H), 7.09-7.12 (1H), 7.44-7.46 (1H)
Example 336
2-Methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.20-2.29 (9H), 5.02-5.07 (1H), 5.21-5.25 (1H), 5.29-5.34 (1H), 6.48-6.51 (1H), 6.84-6.89 (2H), 6.90-6.96 (2H), 7.16-7.25 (3H), 7.50-7.51 (1H)
Example 337
2-Chloro-6-fluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.21-2.25 (6H), 5.01-5.06 (1H), 5.39-5.67 (2H), 6.49-6.52 (1H), 6.82-6.87 (1H), 6.90-6.95 (2H), 7.19-7.24 (1H), 7.35-7.38 (1H), 7.43-7.44 (1H), 7.50-7.56 (1H)
Example 338
4-Methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.24-2.29 (6H), 3.80-3.81 (3H), 5.02-5.10 (1H), 5.19-5.20 (2H), 6.50-6.53 (1H), 6.83-6.90 (3H), 6.95-6.99 (2H), 7.20-7.23 (2H), 7.43-7.44 (1H)
Example 339
2,3,5,6-Tetramethylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.44-1.47 (3H), 2.02-2.06 (3H), 2.09-2.12 (6H), 2.19-2.23 (9H), 4.98-5.03 (1H), 5.33-5.35 (1H), 5.30-5.03 (1H), 6.40-6.42 (1H), 6.80-6.84 (1H), 6.90-6.94 (2H), 7.00-7.01 (1H), 7.43-7.44 (1H)
Example 340
3,4,5-Trifluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.54 (3H), 2.22-2.24 (3H), 2.29-2.31 (3H), 5.02-5.08 (1H), 5.20-5.24 (1H), 5.30-5.34 (1H), 6.45-6.47 (1H), 6.83-6.86 (1H), 6.93-6.97 (2H), 7.10-7.16 (2H), 7.57-7.58 (1H)
Example 341
2,5-Difluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.22-2.28 (6H), 5.02-5.09 (1H), 5.23-5.26 (1H), 5.36-5.40 (1H), 6.50-6.52 (1H), 6.82-6.86 (1H), 6.92-6.96 (2H), 7.05-7.10 (1H), 7.19-7.23 (2H), 7.51-7.52 (1H)
Example 342
3,5-Dimethylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.22-2.31 (12H), 5.05-5.15 (1H), 5.18-5.20 (2H), 6.52-6.54 (1H), 6.87-6.90 (3H), 6.95-6.98 (3H), 7.50-7.51 (1H)
Example 343
4-(1H-Pyrazol-1-yl)benzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.22-2.28 (6H), 5.05-5.14 (1H), 5.25-5.31 (2H), 6.43-6.45 (2H), 6.83-6.86 (1H), 6.96-6.99 (2H), 7.39-7.42 (2H), 7.53-7.54 (1H), 7.69-7.70 (1H), 7.80-7.83 (2H), 8.35-8.36 (1H)
Example 344
3-Chloro-4-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.21-2.33 (9H), 5.02-5.10 (1H), 5.20-5.27 (2H), 6.47-6.49 (1H), 6.83-6.86 (1H), 6.94-6.97 (2H), 7.10-7.21 (2H), 7.33-7.36 (1H), 7.51-7.53 (1H)
Example 345
4-Ethoxy-3-methoxybenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
Example 346
3-Cyanobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.22-2.28 (6H), 5.02-5.09 (1H), 5.28-5.31 (1H), 5.38-5.41 (1H), 6.43-6.45 (1H), 6.84-6.86 (1H), 6.95-6.98 (2H), 7.55-7.60 (3H), 7.61-7.63 (1H), 7.85-7.88 (1H)
Example 347
2-Methoxy-4-methylbenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.53 (3H), 2.21-2.25 (6H), 2.32-2.34 (3H), 3.78-3.79 (3H), 5.06-5.11 (1H), 5.19-5.25 (2H), 6.50-6.53 (1H), 6.72-6.74 (1H), 6.81-6.90 (2H), 6.93-6.97 (2H), 7.05-6.08 (1H), 7.42-7.43 (1H)
Example 348
4-Fluorobenzyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.23-2.27 (6H), 5.01-5.06 (1H), 5.21-5.29 (2H), 6.47-6.49 (1H), 6.82-6.85 (1H), 6.95-6.98 (2H), 7.05-7.11 (2H), 7.32-7.38 (2H), 7.48-7.50 (1H)
Example 349
(7-Methoxy-1,3-benzodioxol-5-yl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
To a mixture of the compound of Example 1 (180 mg, 0.90 mmol) and pyridine (146 μl, 1.80 mmol) in anhydrous acetonitrile (3 ml), at 0° C. and under nitrogen, was added diphosgene (54 μl, 89 mg, 0.45 mmol). The mixture was allowed to warm to room temperature and stirred for 10 min, before addition of (7-methoxy-1,3-benzodioxol-5-yl)methanol (137 mg, 0.75 mmol) in acetonitrile (1 ml), via syringe. The reaction mixture was stirred at room temperature for 30 min and then filtered.
The filtrate was purified by automated preparative liquid chromatography (Gilson system, 150 mm×22.4 mm LUNA C18(2)5 μm column, 20 ml/min) using an acetonitrile:water gradient [15:85 to 98:2]. The appropriate fractions were combined and concentrated to give the title compound (20 mg).
Experimental MH+ 365.9 (minus 44); expected 409.2 or 365.2 1H-NMR (d6-Acetone): 1.50-1.54 (3H), 2.24-2.32 (6H), 3.80-3.81 (3H), 5.05-5.12 (1H), 5.15-5.18 (2H), 6.00-6.01 (2H), 6.50-6.53 (2H), 6.63-6.64 (1H), 6.82-6.86 (1H), 6.95-6.97 (2H), 7.49-7.50 (1H) Rhip. Funct. ED100 mg/cm2=0.03
Similarly Prepared from Example 58 were:
Ex.
MH+ Found/
Rhip. Funct.
No.
R6
Precursor
Expected
ED100 mg/cm2
350
2-Naphthylmethanol
385.9 385.2
<=10
351
(4-Phenyl-2-furyl)- methanol
—
>0.03
352
(6-Phenoxypyridin-3-yl)- methanol
428.9 428.2
<=10
353
5-(6-Fluoro-1H-indol-1- yl)pentan-1-ol
449.0 448.2
>0.03
354
2-(6-Methoxy-1,5- naphthyridin-4-yl)- ethanol
432.0 431.2
<=10
355
2-(2-Naphthyl)ethanol
399.9 399.2
<=10
356
1-Benzofuran-2- ylmethanol
331.8 331.2 decarboxylates
0.1
357
2,3-Dihydro-1,4- benzodioxin-6-yl- methanol
350.0 349.2 decarboxylates
>0.03
358
(2-Phenyl-1,3- benzothiazol-5-yl)- methanol
469.0 468.2
<=10
359
(3-Ethyl-5,5,8,8- tetramethyl-5,6,7,8- tetrahydronaphthalen-2- yl)methanol
430.1 429.3 decarboxylates
<=10
Example 350
2-Naphthylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.53-1.56 (3H), 2.20-2.22 (3H), 2.26-2.28 (3H), 5.05-5.12 (1H), 5.40-5.45 (2H), 6.51-6.53 (1H), 6.85-6.96 (3H), 7.18-7.21 (1H), 7.51-7.56 (3H), 7.80-7.81 (1H), 7.83-7.95 (3H)
Example 351
(4-Phenyl-2-furyl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.48-1.56 (3H), 2.20-2.30 (6H), 4.42-4.51 (1H), 4.80-4.86 (2H), 6.49-6.53 (2H), 6.85-7.01 (10H) h
Example 352
(6-Phenoxypyridin-3-yl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.23-2.29 (6H), 5.01-5.10 (1H), 5.20-5.30 (2H), 6.44-6.47 (1H), 6.84-6.88 (1H), 6.90-6.98 (3H), 7.12-7.15 (2H), 7.20-7.24 (1H), 7.40-7.46 (2H), 7.50-7.51 (1H), 7.65-7.67 (1H), 8.10-8.12 (1H)
Example 353
5-(6-Fluoro-1H-indol-1-yl)pentyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.22-1.28 (2H), 1.52-1.55 (3H), 1.60-1.66 (2H), 1.80-1.85 (2H), 2.24-2.26 (3H), 2.35-2.37 (3H), 4.16-4.26 (4H), 5.01-5.06 (1H), 6.40-6.42 (1H), 6.51-6.53 (1H), 6.80-6.90 (2H), 6.96-6.99 (2H), 7.19-7.21 (1H), 7.22-7.23 (1H), 7.40-7.41 (1H), 7.49-7.52 (1H)
Example 354
2-(6-Methoxy-1,5-naphthyridin-4-yl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.43-1.46 (3H), 2.21-2.26 (6H), 3.42-3.60 (2H), 4.00-4.01 (3H), 4.61-4.75 (2H), 4.92-4.97 (1H), 6.51-6.53 (1H), 6.82-6.95 (3H), 7.08-7.10 (1H), 7.29-7.30 (1H), 7.42-7.44 (1H), 8.20-8.23 (1H), 8.61-8.63 (1H)
Example 355
2-(2-Naphthyl)ethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.55 (3H), 2.24-2.30 (6H), 3.10-3.15 (2H), 4.45-4.60 (2H), 5.00-5.07 (1H), 6.52-6.55 (1H), 6.82-6.95 (3H), 7.39-7.49 (4H), 7.71-7.72 (1H), 7.80-7.87 (3H)
Example 356
1-Benzofuran-2-ylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.52-1.56 (3H), 2.21-2.23 (3H), 2.30-2.32 (3H), 5.03-5.10 (1H), 5.39-5.42 (2H), 6.45-6.47 (1H), 6.81-6.98 (4H), 7.21-7.24 (1H), 7.30-7.34 (1H), 7.45-7.51 (2H), 7.62-7.64 (1H)
Example 357
2,3-Dihydro-1,4-benzodioxin-6-ylmethyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.24-2.29 (6H), 4.21-4.24 (4H), 5.03-5.11 (3H), 6.50-6.53 (1H), 6.78-6.80 (2H), 6.82-6.88 (2H), 6.96-6.99 (2H), 7.46-7.48 (1H)
Example 358
(2-Phenyl-1,3-benzothiazol-5-yl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.51-1.54 (3H), 2.21-2.22 (3H), 2.27-2.28 (3H), 5.02-5.09 (1H), 5.20-5.28 (2H), 6.50-6.52 (1H), 6.82-6.98 (3H), 7.36-7.39 (1H), 7.58-7.61 (4H), 8.00-8.05 (2H), 8.14-8.18 (2H)
Example 359
(3-Ethyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl 2-[(1S)-1-(2,3-dimethylphenyl)ethyl]-1H-imidazole-1-carboxylate
1H-NMR (d6-Acetone): 1.09-1.15 (3H), 1.20-1.30 (12H), 1.50-1.54 (3H), 1.64-1.66 (4H), 2.20-2.29 (6H), 2.50-2.60 (2H), 5.04-5.10 (1H), 5.22-5.24 (2H), 6.52-6.55 (1H), 6.81-6.84 (1H), 6.92-6.95 (2H), 7.20-7.21 (1H), 7.37-7.38 (1H), 7.44-7.45 (1H)
Preparations
Preparation 1
2-[1-(2,3-Dimethylphenyl)vinyl]-1H-imidazole
The compound of Preparation 13 (80 mg, 0.37 mmol) was stirred at 50° C. in thionyl chloride (2 ml) for 1 h. The reaction was quenched into iced water (5 ml) and then basified with dilute aqueous sodium hydroxide solution. The aqueous phase was then extracted with dichloromethane (2×10 ml). The combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (72 mg).
Alternative Synthesis
A solution of thionyl chloride (37 ml, 498 mmol) in acetonitrile (200 ml) was added to the compound of Preparation 13 (48.90 g, 226 mmol). The resulting solution was stirred at room temperature for 2 h, then poured into ice/water (600 ml), during which time the internal temperature was maintained at <25° C. The reaction mixture was then neutralised by the addition of aqueous sodium hydroxide solution (4N) while maintaining the temperature at <35° C. The mixture was adjusted to pH 6, and the suspension obtained was filtered at room temperature. The light beige crystalline solid obtained was washed with water (100 ml) and dried in vacuo at 60° C. to give the title compound (30.6 g).
Experimental MH+ 199.2; expected 199.1
Alternative Synthesis
To a solution of the compound of Preparation 195 (1.0 kg, 3.25 mol) in 2-propanol (10 L) was added palladium (10 wt. % on carbon, 100.0 g) and the reaction mixture was heated at 60° C. under a hydrogen atmosphere (45-60 psi) for 24 h. The mixture was cooled and filtered through Hyflo Super Cel®, washing through with 2-propanol (2×250 ml). The filtrate was concentrated in vacuo and diluted with acetonitrile (1300 ml) and stirred to get a solution. To this solution, was then added dropwise sulphuric acid (conc., 1.2 L). The reaction mixture was stirred at 55° C. for 18 h. The mixture was cooled to −5° C., quenched with water (12.5 l), and adjusted to pH 10 by addition of aqueous sodium hydroxide solution (50%). The resulting solid was collected by filtration, reslurried with water (15.0 L) filtered, washed with water (2.5 L) and dried in vacuo at 50° C. to give the title compound (0.413 kg, purity by HPLC 99.80%).
Preparation 2
2-[1-(2,3-Difluorophenyl)vinyl]-1H-imidazole
A solution of the compound of Preparation 14 (240 mg, 1.1 mmol) and thionyl chloride (1.56 ml, 21.4 mmol) in acetonitrile (5 ml) was heated at 70° C. for 10 h and then stirred at room temperature for 18 h. The mixture was concentrated in vacuo and to the residue was added toluene. This solution was concentrated in vacuo and the process was repeated. The residue was then partitioned between ethyl acetate (50 ml) and saturated aqueous sodium hydrogen carbonate solution (30 ml). The two layers were separated and the aqueous layer was extracted with ethyl acetate (2×40 ml). The combined organic phases were dried (MgSO4) and stirred with activated charcoal, before being filtered and concentrated in vacuo to give the title compound (325 mg).
Experimental MH+ 207.1; expected 207.1
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
3
2-[1-(2-Chloro-6-fluoro-3-
237.1
88
1-(2-Chloro-6-fluoro-3-methylphenyl)-
methylphenyl)vinyl]-1H-imidazole
237.1
1-(1H-imidazol-2-yl)ethanol
4
2-[1-(4-Fluoro-3-methylphenyl)
203.1
15
1-(4-Fluoro-3-methylphenyl)-1-(1H-
vinyl]-1H-imidazole
203.1
imidazol-2-yl)ethanol
5
2-[1-(2,6-Difluorophenyl)vinyl]-1H-
207.3
16
1-(2,6-Difluorophenyl)-1-(1H-
imidazole
207.1
imidazol-2-yl)ethanol
6
2-[1-(3-Fluoro-2-methylphenyl)
203.3
17
1-(3-Fluoro-2-methylphenyl)-1-(1H-
vinyl]-1H-imidazole
203.1
imidazol-2-yl)ethanol
7
2-[1-(3-Fluorophenyl)vinyl]-1H-
189.3
18
1-(3-Fluorophenyl)-1-(1H-imidazol-
imidazole
189.1
2-yl)ethanol
8
2-{1-[2-Chloro-3-(trifluoromethyl)-
273.1
21
1-[2-Chloro-3-(trifluoromethyl)-
phenyl]vinyl}-1H-imidazole
273.0
phenyl]-1-(1H-imidazol-2-yl)ethanol
9
2-[1-(3-Fluoro-5-methylphenyl)-
—
19
1-(3-Fluoro-5-methylphenyl)-1-(1H-
vinyl]-1H-imidazole
imidazol-2-yl)ethanol
10
2-[1-(3,5-Difluorophenyl)vinyl]-1H-
—
22
1-(3,5-Difluorophenyl)-1-(1H-
imidazole
imidazol-2-yl)ethanol
11
2-[1-(5-Methoxy-2,4-
229.3
20
1-(1H-Imidazol-2-yl)-1-(5-methoxy-
dimethylphenyl)vinyl]-1H-
229.1
2,4-dimethylphenyl)ethanol
imidazole
12
2-{1-[2-Fluoro-3-(trifluoromethyl)-
257.4
23
1-[2-Fluoro-3-(trifluoromethyl)-
phenyl]vinyl}-1H-imidazole
257.1
phenyl]-1-(1H-imidazol-2-yl)ethanol
Preparation 13
1-(2,3-Dimethylphenyl)-1-(1H-imidazol-2-yl)ethanol
1-(Diethoxymethyl)imidazole (76.0 g, 446 mmol) and N,N,N,N-tetramethylethylene diamine (67.6 mL, 446 mmol) were dissolved in 2-methyltetrahydrofuran (400 ml) and cooled to −40° C., under nitrogen. n-Butyl lithium (2.5M in hexane, 180 ml, 446 mmol) was added slowly maintaining the reaction temperature at <−25° C. throughout. The reaction mixture was stirred for an hour and allowed to warm to 0° C., after which 2,3-dimethylacetophenone (44.00 g, 297.00 mmol) was added whilst maintaining the reaction temperature at <15° C. throughout. The reaction was stirred at room temperature overnight and then quenched with aqueous hydrochloric acid (2N, 1 l). The mixture was extracted with ethyl acetate (500 ml) and to the aqueous layer was added sodium carbonate. The aqueous layer was further extracted with ethyl acetate (800 ml) and the combined extracts were washed with water (500 ml), dried (MgSO4) and concentrated in vacuo to give the title compound (48.9 g).
Experimental MH+ 217.2; expected 217.1
Alternative Synthesis
To a solution of methylmagnesium bromide (0.63 ml, 0.88 mmol) was added a stirred solution of the compound of Preparation 24 (80 mg, 0.4 mmol) in anhydrous tetrahydrofuran at 0° C. The reaction mixture was stirred for 30 min, quenched with saturated ammonium chloride solution, basified with saturated sodium hydrogen carbonate solution and extracted with dichloromethane (2×3 ml). The organic layers were dried (MgSO4) and concentrated in vacuo to give the title compound (85 mg)
Experimental MH+ 217.2; expected 217.1
Preparation 14
1-(2,3-Difluorophenyl)-1-(1H-imidazol-2-yl)ethanol
To a solution of the compound of Preparation 25 (450 mg, 2.2 mmol) in tetrahydrofuran (5 ml), at 0° C., was added methylmagnesium bromide (3M in diethyl ether, 2.16 ml, 6.5 mmol) and the reaction mixture was stirred at room temperature for 1 h. To the mixture was added hydrochloric acid (0.1M, 15 ml) and the mixture basified by addition of saturated aqueous sodium hydrogen carbonate solution. The mixture was extracted with ethyl acetate (3×20 ml) and the combined organics were dried (MgSO4) and concentrated in vacuo to give the title compound (240 mg)
Experimental MH+ 225.1; expected 225.1
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
15
1-(4-Fluoro-3-methylphenyl)-1-(1H-
221.1
26
(4-Fluoro-3-methylphenyl)(1H-
imidazol-2-yl)ethanol
221.1
imidazol-2-yl)methanone
16
1-(2,6-Difluorophenyl)-1-(1H-
—
27
(2,6-Difluorophenyl)(1H-
imidazol-2-yl)ethanol
imidazol-2-yl)methanone
17
1-(3-Fluoro-2-methylphenyl)-1-(1H-
—
28
(3-Fluoro-2-methylphenyl)(1H-
imidazol-2-yl)ethanol
imidazol-2-yl)methanone
18
1-(3-Fluorophenyl)-1-(1H-imidazol-
—
29
(3-Fluorophenyl)(1H-imidazol-2-
2-yl)ethanol
yl)methanone
19
1-(3-Fluoro-5-methylphenyl)-1-(1H-
—
31
(3-Fluoro-5-methylphenyl)(1H-
imidazol-2-yl)ethanol
imidazol-2-yl)methanone
20
1-(1H-Imidazol-2-yl)-1-(5-methoxy-
247.4
34
1H-Imidazol-2-yl(5-methoxy-
2,4-dimethylphenyl)ethanol
247.1
2,4-dimethylphenyl)methanone
Preparation 16
1H-NMR (CD3OD): 2.00-2.05 (3H), 6.84-6.95 (4H), 7.26-7.34 (1H)
Preparation 18
1H-NMR (CD3OD): 1.89-1.92 (3H), 6.89-6.96 (3H), 7.18-7.23 (2H), 7.25-7.31 (1H)
Preparation 19
1H-NMR (CDCl3): 1.20-1.25 (3H), 2.21-2.27 (3H), 6.62-6.66 (1H), 6.80-7.00 (3H), 7.41-7.49 (1H)
Preparation 21
1-[2-Chloro-3-(trifluoromethyl)phenyl]-1-(1H-imidazol-2-yl)ethanol
To a solution of the compound of Preparation 30 (1.1 g, 4.0 mmol) in tetrahydrofuran (10 ml), at −78° C., was added dropwise methyllithium (1.6M in diethyl ether, 3 ml, 4.8 mmol). After stirring for 2 h, cold hydrochloric acid (0.1M) was added and the mixture was adjusted to pH 7 by addition of potassium carbonate. The mixture was extracted with ethyl acetate and the combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (600 mg).
Similarly Prepared were:
Prep.
MH+ Found/
From
No
Name
Expected
Prep.
From
22
1-(3,5-Difluorophenyl)-1-(1H-
225.4
32
(3,5-Difluorophenyl)(1H-imidazol-
imidazol-2-yl)ethanol
225.1
2-yl)methanone
23
1-[2-Fluoro-3-(trifluoromethyl)-
275.5
33
[2-Fluoro-3-(trifluoromethyl)-
phenyl]-1-(1H-imidazol-2-
275.1
phenyl](1H-imidazol-2-yl)-
yl)ethanol
methanone
Preparation 24
(2,3-Dimethylphenyl)(1H-imidazol-2-yl)methanone
To the compound of Preparation 201 (200 mg, 1.0 mmol) in dichloromethane (10 ml) was added Dess Martin Periodinane (15% in dichloromethane, 3 ml) and the reaction mixture was stirred at room temperature for 30 min. The mixture filtered through silica, eluting with diethyl ether and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography (silica), with gradient elution, diethyl ether:dichloromethane [0:1 to 1:1]. The appropriate fractions were combined and concentrated to give the title compound (100 mg).
Experimental MH+ 201.2; expected 201.1
Preparation 25
(2,3-difluorophenyl)(1H-imidazol-2-yl)methanone
To a solution of the compound of Preparation 37 (350 mg, 1.67 mmol) in dichloromethane (20 ml) was added Dess-Martin Periodinane (780 mg, 1.80 mmol) and the reaction mixture was stirred at room temperature for 1 h. The mixture was filtered through silica, washing through with dichloromethane and ethyl acetate and the filtrate was concentrated in vacuo. To the residue as added ethyl acetate (100 ml) and the solution was washed with aqueous sodium metabisulphite solution (10%, 40 ml). The aqueous phase was extracted with ethyl acetate (100 ml) and the combined organic phases were dried (MgSO4) and concentrated in vacuo to give the title compound (450 mg)
Experimental MH+ 209.1; expected 209.1
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
26
(4-Fluoro-3-methylphenyl)(1H-
205.3
38
(4-Fluoro-3-methylphenyl)(1H-
imidazol-2-yl)methanone
205.1
imidazol-2-yl)methanol
27
(2,6-Difluorophenyl)(1H-imidazol-2-
209.1
39
(2,6-Difluorophenyl)(1H-imidazol-2-
yl)methanone
209.1
yl)methanol
28
(3-Fluoro-2-methylphenyl)(1H-
205.3
40
(3-Fluoro-2-methylphenyl)(1H-
imidazol-2-yl)methanone
205.1
imidazol-2-yl)methanol
29
(3-Fluorophenyl)(1H-imidazol-2-
191.1
41
(3-Fluorophenyl)(1H-imidazol-2-
yl)methanone
191.1
yl)methanol
30
[2-Chloro-3-(trifluoromethyl)-
No data
42
[2-Chloro-3-(trifluoromethyl)-
phenyl](1H-imidazol-2-yl)methanone
phenyl](1H-imidazol-2-yl)methanol
31
(3-Fluoro-5-methylphenyl)(1H-
205.3
43
(3-Fluoro-5-methylphenyl)(1H-
imidazol-2-yl)methanone
205.1
imidazol-2-yl)methanol
32
(3,5-Difluorophenyl)(1H-imidazol-2-
209.3
44
(3,5-Difluorophenyl)(1H-imidazol-2-
yl)methanone
209.1
yl)methanol
33
[2-Fluoro-3-(trifluoromethyl)phenyl]-
259.4
46
[2-Fluoro-3-(trifluoromethyl)phenyl]-
(1H-imidazol-2-yl)methanone
259.1
(1H-imidazol-2-yl)methanol
Preparation 34
1H-Imidazol-2-yl(5-methoxy-2,4-dimethylphenyl)methanone
To a solution of the compound of Preparation 45 (433 mg, 1.8 mmol) in ethyl acetate (10 ml) was added manganese (IV) oxide (810 mg, 9.3 mmol) and the reaction mixture was stirred at room temperature for 3 h. The mixture was filtered through Arbocel®, washing through with ethyl acetate, and the filtrate was concentrated in vacuo to give the title compound (440 mg).
Experimental MH+ 231.3; expected 231.1
Preparation 35
1-(2,3-Dimethylphenyl)propan-1-one
A mixture of the compound of Preparation 192 (1.0 g, 6.1 mmol) and Dess-Martin Periodinane (2.58 g, 6.1 mmol) in dichloromethane (20 ml) was stirred at room temperature for 1 h. The mixture was then purified by column chromatography (silica), eluting with dichloromethane:cyclohexane [1:1]. The appropriate fractions were combined and concentrated to give the title compound (0.95 g).
1H-NMR (CDCl3): 1.11-1.19 (3H), 2.23-2.29 (6H), 2.79-2.87 (2H), 7.07-7.12 (1H), 7.17-7.27 (3H)
Preparation 36
(1-Benzyl-1H-imidazol-2-yl)(2,3-dimethylphenyl)methanone
A solution of 2,3-dimethylbenzoic acid (100 g, 666 mmol) in thionyl chloride (350 ml) was heated at 80° C. for 1 h, before cooling to room temperature and concentrating in vacuo. To the residue was added toluene (100 ml) and the solution was again concentrated in vacuo. The intermediate acid chloride was added to a mixture of 1-benzylimidazole (100 g, 632 mmol) and triethylamine (100 ml) in acetonitrile (1 l) and the reaction mixture was heated at reflux for 18 h. The reaction mixture was concentrated in vacuo and to the residue was added diethyl ether (500 ml) and ethyl acetate (50 ml). This solution was washed with water (500 ml) and saturated aqueous sodium hydrogen carbonate solution (500 ml), filtered through silica gel (100 g) and concentrated in vacuo to give the title compound (182 g).
Experimental MH+ 291.4; expected 291.1
Alternative Synthesis
To a solution of 2,3-dimethylbenzoic acid (2.0 kg, 13.2 mol) in toluene (20 L) was added N,N-dimethylformamide (20 ml), followed by oxalyl chloride (2.0 kg, 15.6 mol) at room temperature. The reaction mixture was stirred at room temperature for 4 h and monitored by thin layer chromatography. If necessary, excess oxalyl chloride (25 g) was added until no starting material was observed. Excess toluene and oxalyl chloride were removed by distillation under vacuum at temperatures below 70° C. To the residue was added toluene (150 ml) and the mixture was again concentrated in vacuo to give 2,3-dimethylbenzoyl chloride (2.0 kg).
To a solution of 1-benzyl-1H-imidazole (1.69 Kg, 10.56 mol) in dichloromethane (14.0 L), at −7° C., was added triethylamine (1.61 kg, 10.56 mol). A solution of 2,3-dimethylbenzoyl chloride (2.0 kg, 11.99 mol) in dichloromethane (6.0 L) was then added dropwise and the reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by Thin layer Chromatography. After completion of the reaction, the reaction mixture was diluted with water (5.0 L) and the mixture was stirred for a further 15 min. The two layers were separated and the organic phase was concentrated in vacuo. To the residue was added toluene (8.0 L) and the solution was cooled to −5° C., before addition of hydrochloric acid (5N, 8.0 L). The two layers were separated and the aqueous layer was adjusted to pH 9-12, by addition of aqueous sodium hydroxide solution (50%), and extracted with toluene (4.0 L and then 8.0 L). The combined organic phases were concentrated in vacuo to give the title compound (2.8 kg).
Preparation 37
(2,3-Difluorophenyl)(1H-imidazol-2-yl)methanol
To a solution of 1-(diethoxymethyl)-1H-imidazole (1.65 ml, 10.1 mmol) in tetrahydrofuran (15 ml), at −60° C. and under nitrogen, was added n-butyllithium (2.5 M in hexanes, 4.03 ml, 10.1 mmol). The reaction mixture was stirred at −60° C. for 1 h, before addition of 2,3-difluorobenzaldehyde (1.00 ml, 9.2 mmol), and then allowed to warm to room temperature over 18 h. The mixture was concentrated in vacuo and to the residue was added ethyl acetate (50 ml) and hydrochloric acid (3M, 50 ml). The two layers were separated and the aqueous phase was basified with aqueous sodium hydroxide solution (20%) and extracted with ethyl acetate (3×100 ml). The combined organic phases were dried (MgSO4) and concentrated in vacuo and the residue was re-crystallised from 2-propanol to give the title compound (1.25 g)
Experimental MH+ 211.1; expected 211.1
Similarly Prepared were:
Prep.
MH+ Found/
No
Name
Expected
From
From
38
(4-Fluoro-3-methylphenyl)(1H-
207.2
—
4-Fluoro-3-methyl-
imidazol-2-yl)methanol
207.1
benzaldehyde
39
(2,6-Difluorophenyl)(1H-imidazol-2-
211.1
—
2,6-Difluoro-benzaldehyde
yl)methanol
211.1
40
(3-Fluoro-2-methylphenyl)(1H-
207.3
—
3-Fluoro-2-methyl-
imidazol-2-yl)methanol
207.1
benzaldehyde
41
(3-Fluorophenyl)(1H-imidazol-2-
193.1
—
3-Fluoro-benzaldehyde
yl)methanol
193.1
42
[2-Chloro-3-(trifluoromethyl)-
—
—
2-Chloro-3-(trifluoromethyl)-
phenyl](1H-imidazol-2-yl)methanol
benzaldehyde
43
(3-Fluoro-5-methylphenyl)(1H-
207.3
—
3-Fluoro-5-methyl-
imidazol-2-yl)methanol
207.1
benzaldehyde
44
(3,5-Difluorophenyl)(1H-imidazol-2-
211.3
—
3,5-Difluoro-benzaldehyde
yl)methanol
211.1
45
1H-Imidazol-2-yl(5-methoxy-2,4-
233.3
Prep.
5-Methoxy-2,4-dimethyl-
dimethylphenyl)methanol
233.1
190
benzaldehyde
46
[2-Fluoro-3-(trifluoromethyl)-
261.4
—
2-Fluoro-3-(trifluoromethyl)-
phenyl](1H-imidazol-2-yl)methanol
261.1
benzaldehyde
Preparation 47
2-[1-(2,3-Dimethylphenyl)prop-1-en-1-yl]-1H-imidazole
A solution of the compound of Preparation 83 (350 mg, 1.52 mmol) in hydrochloric acid (2N, 50 ml) was heated at reflux for 18 h. The reaction mixture was concentrated in vacuo and the residue was partitioned between dichloromethane (20 ml) and aqueous sodium hydrogen carbonate solution (20 ml). The two layers were separated and the aqueous phase was extracted with dichloromethane (2×20 ml). The combined organic phases were dried (MgSO4) and concentrated in vacuo to give the title compound (255 mg).
Experimental MH+ 213.2; expected 213.1
Preparation 48
2-[1-(3-Methylphenyl)vinyl]-1H-imidazole
A solution of the compound of Preparation 78 (850 mg, 4.2 mmol) in hydrochloric acid (6N, 20 ml) was heated at reflux for 18 h. The reaction mixture was concentrated in vacuo and the residue was partitioned between dichloromethane (20 ml) and water (10 ml). The mixture was adjusted to pH 7 by addition of saturated aqueous sodium hydrogen carbonate solution and the two layers were separated. The organic phase was dried (MgSO4) and concentrated in vacuo to give the title compound (800 mg).
Experimental MH+ 185.3; expected 185.1
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
49
2-[1-(2,5-Dimethylphenyl)vinyl]-
217.0
80
1-(2,5-Dimethylphenyl)-1-(1H-
1H-imidazole
217.3
imidazol-2-yl)ethanol
50
2-[1-(3,5-Dimethylphenyl)vinyl]-
199.3
82
1-(3,5-Dimethylphenyl)-1-(1H-
1H-imidazole
199.1
imidazol-2-yl)ethanol
51
2-{1-[2-
239.4
79
1-(1H-imidazol-2-yl)-1-[2-
(Trifluoromethyl)phenyl]vinyl}-
239.1
(trifluoromethyl)phenyl]ethanol
1H-imidazole
52
2-[1-(2,3-Dichlorophenyl)vinyl]-
239.2
84
1-(2,3-Dichlorophenyl)-1-(1H-
1H-imidazole
239.0
imidazol-2-yl)ethanol
53
2-[1-(3,4-Dichlorophenyl)vinyl]-
239.2
85
1-(3,4-Dichlorophenyl)-1-(1H-
1H-imidazole
239.0
imidazol-2-yl)ethanol
54
2-[1-(3-Chlorophenyl)vinyl]-1H-
205.1
86
1-(3-Chlorophenyl)-1-(1H-imidazol-2-
imidazole
205.3
yl)ethanol
55
2-[1-(2,5-Dichlorophenyl)vinyl]-
239.2
87
1-(2,5-Dichlorophenyl)-1-(1H-
1H-imidazole
239.0
imidazol-2-yl)ethanol
56
2-[1-(2,4-Dichlorophenyl)vinyl]-
239.2
98
1-(2,4-Dichlorophenyl)-1-(1H-
1H-imidazole
239.0
imidazol-2-yl)ethanol
57
2-(1-Phenylvinyl)-1H-imidazole
171.2
99
1-(1H-Imidazol-2-yl)-1-phenylethanol
171.1
58
2-[1-(4-Methylphenyl)vinyl]-1H-
185.3
100
1-(1H-Imidazol-2-yl)-1-(4-methylphenyl)
imidazole
185.1
ethanol
59
2-(1-Mesitylvinyl)-1H-imidazole
213.4
101
1-(1H-Imidazol-2-yl)-1-mesitylethanol
213.1
60
2-{1-[3-
239.3
102
1-(1H-Imidazol-2-yl)-1-[3-
(Trifluoromethyl)phenyl]vinyl}-
239.1
(trifluoromethyl)phenyl]-ethanol
1H-imidazole
61
2-{1-[4-
239.3
103
1-(1H-imidazol-2-yl)-1-[4-
(Trifluoromethyl)phenyl]vinyl}-
239.1
(trifluoromethyl)phenyl]-ethanol
1H-imidazole
62
2-[1-(3-Methoxy-2-
215.3
104
1-(1H-Imidazol-2-yl)-1-(3-methoxy-2-
methylphenyl)vinyl]-1H-
215.1
methylphenyl)ethanol
imidazole
63
2-[1-(2-Ethyl-3-methylphenyl)-
213.3
105
1-(2-Ethyl-3-methylphenyl)-1-(1H-
vinyl]-1H-imidazole
213.1
imidazol-2-yl)ethanol
64
2-[1-(2-Bromo-3,5,6-trimethylphenyl)
291.3
106
1-(2-Bromo-3,5,6-trimethylphenyl)-1-
vinyl]-1H-imidazole
291.0
(1H-imidazol-2-yl)ethanol
65
2-{1-[3-
255.1
107
1-(1H-imidazol-2-yl)-1-[3-
(Trifluoromethoxy)phenyl]vinyl}-
255.1
(trifluoromethoxy)phenyl]ethanol
1H-imidazole
66
2-[1-(2,6-Dimethylphenyl)vinyl]-
199.3
109
1-(2,6-Dimethylphenyl)-1-(1H-
1H-imidazole
199.1
imidazol-2-yl)ethanol
Preparation 67
2-[1-(2-Chloro-3-methylphenyl)vinyl]-1H-imidazole
A solution of the compound of Preparation 97 (1.22 g, 5.2 mmol) in Eaton's Reagent (15 ml) was stirred at room temperature for 18 h. To the mixture was added ethyl acetate and saturated aqueous sodium hydrogen carbonate solution and the two layers were separated. The organic phase was washed with brine, dried (MgSO4) and concentrated in vacuo to give the title compound (1.00 g).
Experimental MH+ 219.3; expected 219.1
Similarly Prepared were:
Prep.
MH+ Found/
From
No
Name
Expected
Prep.
From
68
2-[1-(3-Chloro-4-methylphenyl)-
219.3
93
1-(3-Chloro-4-methylphenyl)-1-
vinyl]-1H-imidazole
219.1
(1H-imidazol-2-yl)ethanol
69
2-[1-(3-Chloro-2-methylphenyl)
219.3
94
1-(3-Chloro-2-methylphenyl)-1-
vinyl]-1H-imidazole
219.1
(1H-imidazol-2-yl)ethanol
70
2-[1-(2-Chloro-5-methoxyphenyl)
235.3
95
1-(2-Chloro-5-methoxyphenyl)-1-
vinyl]-1H-imidazole
235.1
(1H-imidazol-2-yl)ethanol
71
2-[1-(2-Chloro-5-methylphenyl)
219.3
96
1-(2-Chloro-5-methylphenyl)-1-
vinyl]-1H-imidazole
219.1
(1H-imidazol-2-yl)ethanol
72
2-{1-[3-Methyl-2-(trifluoromethyl)-
253.3
89
1-(1H-Imidazol-2-yl)-1-[3-methyl-
phenyl]vinyl}-1H-imidazole
253.1
2-(trifluoromethyl)phenyl]ethanol
73
2-[1-(2,6-Difluoro-3-methylphenyl)
221.3
108
1-(2,6-Difluoro-3-methylphenyl)-1-
vinyl]-1H-imidazole
221.1
(1H-imidazol-2-yl)ethanol
74
2-[1-(4-Chloro-3-methylphenyl)-
219.1
81
1-(4-Chloro-3-methylphenyl)-1-
vinyl]-1H-imidazole
219.3
(1H-imidazol-2-yl)ethanol
Preparation 75
2-[1-(2-Chloro-4-methoxyphenyl)vinyl]-1H-imidazole
A solution of the compound of Preparation 90 (703 mg, 2.7 mmol) in trifluoroacetic acid (15 ml) was heated at 50° C. for 18 h. The reaction mixture was concentrated in vacuo and the residue was neutralised by addition of aqueous sodium hydrogen carbonate solution. The mixture was extracted with ethyl acetate and the combined extracts were concentrated in vacuo to give the title compound (469 mg).
Experimental MH+ 235.3; expected 235.1
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
76**
2-[1-(3-Chloro-4-methoxyphenyl)
235.3
92
1-(3-Chloro-4-methoxyphenyl)-1-(1H-
vinyl]-1H-imidazole
235.1
imidazol-2-yl)ethanol
77**
2-[1-(3-Chloro-2-methoxyphenyl)
235.3
91
1-(3-Chloro-2-methoxyphenyl)-1-(1H-
vinyl]-1H-imidazole
235.1
imidazol-2-yl)ethanol
**The reaction to yield Preparation 76 gave some of Preparation 77 since Preparation 92 contained some Preparation 91 and vice versa.
Preparation 78
1-(1H-Imidazol-2-yl)-1-(3-methyl phenyl)ethanol
To a solution of 1-(diethoxymethyl)-1H-imidazole (935 mg, 5.5 mmol) in anhydrous tetrahydrofuran (6 ml), at −78° C., was added n-butyllithium (2.5M in hexanes, 2.2 ml, 5.5 mmol). The mixture was allowed to warm to 0° C. and then added to a solution of 1-(3-methylphenyl)ethanone (670 mg, 5.0 mmol) in anhydrous tetrahydrofuran (5 ml), also at 0° C. The reaction mixture was stirred at 0° C. for 30 min and then at room temperature for 1 h. The mixture was poured into cold hydrochloric acid (4N, 10 ml) and stirred for 20 min. The mixture was adjusted to pH 7 by addition of sodium hydrogen carbonate and then extracted with dichloromethane. The combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (850 mg).
Experimental MH+ 203.3; expected 203.1
Similarly Prepared were:
Prep.
MH+ Found/
No
Name
Expected
From
From
79
1-(1H-Imidazol-2-yl)-1-[2-
257.3
—
1-[2-(Trifluoromethyl)phenyl]-
(trifluoromethyl)phenyl]ethanol
257.1
ethanone
80
1-(2,5-Dimethylphenyl)-1-(1H-
217.3
—
1-(2,5-Dimethylphenyl)-
imidazol-2-yl)ethanol
217.1
ethanone
81
1-(4-Chloro-3-methylphenyl)-1-
237.3
—
1-(4-Chloro-3-methylphenyl)-
(1H-imidazol-2-yl)ethanol
237.1
ethanone
82
1-(3,5-Dimethylphenyl)-1-(1H-
217.3
Prep.
1-(3,5-Dimethylphenyl)-
imidazol-2-yl)ethanol
217.1
113
ethanone
83
1-(2,3-Dimethylphenyl)-1-(1H-
231.1
Prep. 35
1-(2,3-Dimethylphenyl)propan-
imidazol-2-yl)propan-1-ol
231.1
1-one
84
1-(2,3-Dichlorophenyl)-1-(1H-
257.2
—
1-(2,3-Dichlorophenyl)ethanone
imidazol-2-yl)ethanol
257.0
85
1-(3,4-Dichlorophenyl)-1-(1H-
257.3
—
1-(3,4-Dichlorophenyl)ethanone
imidazol-2-yl)ethanol
257.0
86
1-(3-Chlorophenyl)-1-(1H-
3223.3
—
1-(3-Chlorophenyl)ethanone
imidazol-2-yl)ethanol
223.1
87
1-(2,5-Dichlorophenyl)-1-(1H-
257.2
—
1-(2,5-Dichlorophenyl)ethanone
imidazol-2-yl)ethanol
257.0
88
1-(2-Chloro-6-fluoro-3-
255.2
—
1-(2-Chloro-6-fluoro-3-methylphenyl)
methylphenyl)-1-(1H-imidazol-
255.1
ethanone
2-yl)ethanol
89
1-(1H-Imidazol-2-yl)-1[3-
271.4
Prep.
1-[3-Methyl-2-(trifluoromethyl)-
methyl-2-(trifluoromethyl)-
271.1
170
phenyl]-ethanone
phenyl]ethanol
90
1-(2-Chloro-4-methoxyphenyl)-
253.3
J. Org.
1-(2-Chloro-4-methoxyphenyl)-
1-(1H-imidazol-2-yl)ethanol
253.1
Chem.,
ethanone
2002, 67,
23, 8043
91*
1-(3-Chloro-2-methoxyphenyl)-
235.3
Prep.
1-(3-Chloro-2-methoxyphenyl)-
1-(1H-imidazol-2-yl)ethanol
235.1
173
ethanone
92*
1-(3-Chloro-4-methoxyphenyl)-
235.3
Prep.
1-(3-Chloro-4-methoxyphenyl)
1-(1H-imidazol-2-yl)ethanol
235.1
174
ethanone
93
1-(3-Chloro-4-methylphenyl)-1-
237.3
Prep.
1-(3-Chloro-4-methylphenyl)
(1H-imidazol-2-yl)ethanol
237.1
114
ethanone
94
1-(3-Chloro-2-methylphenyl)-1-
237.3
Prep.
1-(3-Chloro-2-methylphenyl)-
(1H-imidazol-2-yl)ethanol
237.1
111
ethanone
95
1-(2-Chloro-5-methoxyphenyl)-
253.2
Prep.
1-(2-Chloro-5-methoxyphenyl)
1-(1H-imidazol-2-yl)ethanol
253.1
175
ethanone
96
1-(2-Chloro-5-methylphenyl)-1-
237.3
Prep.
1-(2-Chloro-5-methylphenyl)-
(1H-imidazol-2-yl)ethanol
237.1
112
ethanone
97
1-(2-Chloro-3-methylphenyl)-1-
237.3
Prep.
1-(2-Chloro-3-methylphenyl)-
(1H-imidazol-2-yl)ethanol
237.1
110
ethanone
98
1-(2,4-Dichlorophenyl)-1-(1H-
257.2
—
1-(2,4-Dichlorophenyl)ethanone
imidazol-2-yl)ethanol
257.0
99
1-(1H-Imidazol-2-yl)-1-phenylethanol
189.3
—
1-Phenylethanone
189.1
100
1-(1H-Imidazol-2-yl)-1-(4-
203.3
—
1-(4-Methylphenyl)ethanone
methylphenyl)ethanol
203.1
101
1-(1H-Imidazol-2-yl)-1-
231.4
—
1-Mesitylethanone
mesitylethanol
231.1
102
1-(1H-Imidazol-2-yl)-1-[3-
257.3
—
1-[3-(Trifluoromethyl)phenyl]-
(trifluoromethyl)phenyl]ethanol
257.1
ethanone
103
1-(1H-Imidazol-2-yl)-1-[4-
257.3
—
1-[4-(Trifluoromethyl)phenyl]-
(trifluoromethyl)phenyl]ethanol
257.1
ethanone
104
1-(1H-Imidazol-2-yl)-1-(3-
233.3
Prep.
1-(3-Methoxy-2-methylphenyl)
methoxy-2-methylphenyl)-
233.1
115
ethanone
ethanol
105
1-(2-Ethyl-3-methylphenyl)-1-
231.3
Prep.
1-(2-Ethyl-3-methylphenyl)-
(1H-imidazol-2-yl)ethanol
231.1
178
ethanone
106
1-(2-Bromo-3,5,6-trimethylphenyl)-
—
Prep.
1-(2-Bromo-3,5,6-trimethylphenyl)
1-(1H-imidazol-2-
180
ethanone
yl)ethanol
107
1-(1H-Imidazol-2-yl)-1-[3-
—
—
1-[3-(Trifluoromethoxy)-
(trifluoromethoxy)phenyl]ethanol
phenyl]ethanone
108
1-(2,6-Difluoro-3-methylphenyl)-
239.2
—
1-(2,6-Difluoro-3-methylphenyl)
1-(1H-imidazol-2-
239.1
ethanone
yl)ethanol
109
1-(2,6-Dimethylphenyl)-1-(1H-
216.4
—
1-(2,6-Dimethylphenyl)-
imidazol-2-yl)ethanol
216.1
ethanone
*The reaction to yield Preparation 91 gave some of Preparation 92 since Preparation 173 contained some Preparation 174 and vice versa.
Preparation 107
1H-NMR (CD3OD): 1.89-1.94 (3H), 6.93-6.97 (2H), 7.08-7.13 (1H), 7.33-7.41 (3H)
Preparation 110
1-(2-Chloro-3-methylphenyl)ethanone
To a solution of 2-chloro-3-methylbenzoic acid (1.71 g, 10.0 mmol) in anhydrous tetrahydrofuran (10 ml), at 0° C. and under nitrogen, was added methyllithium (1.6M in diethyl ether, 13.1 ml, 21.0 mmol), via syringe. The reaction mixture was stirred at 0° C. for 30 min and then allowed to warm to room temperature over 1 h. To the reaction mixture was added cold hydrochloric acid (1M, 100 ml) and dichloromethane (110 ml). The mixture was adjusted to pH 7 by addition of saturated aqueous sodium hydrogen carbonate solution and the two layers were separated. The aqueous layer was extracted with further dichloromethane and the combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (1.19 g).
1H-NMR (CDCl3): 2.37-2.39 (3H), 2.57-2.60 (3H), 7.15-7.20 (1H), 7.23-7.31 (2H)
Similarly Prepared were:
Prep. No
Name
1H-NMR (CDCl3)
From
111
1-(3-Chloro-2-methylphenyl)-
2.44-2.46(3H), 2.52-2.54(3H),
3-Chloro-2-methylbenzoic
ethanone
7.13-7.18(1H), 7.40-7.45(1H)
acid
112
1-(2-Chloro-5-methylphenyl)-
1.67-1.69(3H), 2.29-2.30(3H),
2-Chloro-5-
ethanone
7.13-7.18(1H), 7.22-7.25(1H),
methylbenzoic acid
7.30-7.32(1H)
113
1-(3,5-Dimethylphenyl)-
2.32-2.35(6H), 2.53-2.55(3H),
3,5-Dimethylbenzoic
ethanone
7.16-7.18(1H), 7.52-7.54(2H)
acid
114
1-(3-chloro-4-Methylphenyl)-
2.38-2.41(3H), 2.53-2.55(3H),
3-Chloro-4-
ethanone
7.27-7.30(1H), 7.69-7.72(1H),
methylbenzoic acid
7.88-7.90(1H)
Preparation 115
1-(3-Methoxy-2-methylphenyl)ethanone
A solution of 2-methyl-3-methoxybenzoic acid (10.0 g, 60.2 mmol) in thionyl chloride (50 ml) was heated at reflux for 1 h and then cooled and concentrated in vacuo. To the residue was added tetrahydrofuran (100 ml) and iron (III) acetylacetonate (638 mg, 1.8 mmol) and the solution was cooled to −20° C., before addition of methylmagnesium bromide (3M in diethyl ether, 22.1 ml, 66.2 mmol). After stirring for 15 min, the mixture was poured into saturated aqueous ammonium chloride solution and extracted with dichloromethane. The combined extracts were washed with saturated aqueous sodium hydrogen carbonate solution, dried (MgSO4) and concentrated in vacuo.
The residue was purified by flash chromatography (silica), eluting with pentane:dichloromethane [1:1]. The appropriate fractions were combined and concentrated to give the title compound (7.60 g).
1H-NMR (CDCl3): 2.27-2.29 (3H), 2.50-2.53 (3H), 3.79-3.83 (3H), 6.90-6.94 (1H), 7.10-7.14 (1H), 7.16-7.21 (1H)
Preparation 116
Chloromethyl 3-cyclopentylpropanoate
Cyclopentylpropionyl chloride (2.0 g, 12.4 mmol) was added to a mixture of paraformaldehyde (377 mg, 13.0 mmol) and zinc chloride at room temperature under nitrogen. The reaction mixture was heated to 75° C. for 3 hours, cooled and the mixture distilled (90°-100° C.) to give the title compound (1.10 g)
1H-NMR (CDCl3): 1.00-1.10 (2H), 1.45-1.75 (9H), 2.35-2.40 (2H), 5.70-5.75 (2H)
Similarly Prepared were:
Prep.
No
Name
1H-NMR (CDCl3):
From
117
Chloromethyl
0.90-0.95(3H),
Heptanoyl chloride
heptanoate
1.20-1.40(6H),
1.60-1.70(2H),
2.30-2.40(2H,
5.70-5.75(2H)
118
Chloromethyl 3,3-
—
3,3-Dimethylbutanoyl
dimethylbutanoate
chloride
Preparation 119
Chloromethyl cyclopropylmethyl carbonate
To a solution of cyclopropylmethanol (0.39 ml, 5.0 mmol) and pyridine (0.40 ml, 5.0 mmol) in dichloromethane (4 ml), at 0° C. and under nitrogen, was added dropwise chloromethyl chlorocarbonate (0.40 ml, 4.5 mmol). The reaction mixture was stirred at 0° C. for 30 min and then at room temperature for 2 h. To the mixture was added diethyl ether (15 ml) and the solid material was collected by filtration and washed with diethyl ether (10 ml). The combined organic phases were dried (MgSO4) and concentrated in vacuo to give the title compound (725 mg) which was used directly.
Similarly Prepared were:
MH+
Prep.
Found/
No
Name
Expected
From
120
Chloromethyl 4-
345.4
(4-Methoxy-
methoxybenzyl carbonate
345.4
phenyl)methanol
121
Chloromethyl 3-methylbutyl
—
3-Methylbutan-1-ol
carbonate
122
Chloromethyl isopropyl
—
Propan-2-ol
carbonate
123
Chloromethyl cyclobutyl
—
Cyclobutanol
carbonate
124
Chloromethyl 2,2,2-
—
2,2,2-Trifluoroethanol
trifluoroethyl carbonate
Preparation 125
Chloromethyl (2,4-Dichlorobenzyl)carbamate
To a solution of the compound of 1-(2,4-dichlorophenyl)methanamine (0.15 ml, 1.1 mmol) in anhydrous dichloromethane (2 ml), at −10° C. and under nitrogen, was added dropwise 3-chloropropanoyl chloride (0.12 ml, 1.1 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 18 h. To the mixture was added dichloromethane (5 ml) and water (5 ml) and the two layers were separated. The aqueous layer was extracted with dichloromethane (10 ml) and the combined organic layers were dried (MgSO4) and concentrated in vacuo to give the title compound (285 mg).
1H-NMR (CDCl3): 4.35-4.39 (2H), 5.76-5.79 (2H), 7.11-7.15 (1H), 7.37-7.44 (2H)
Similarly Prepared were:
Prep. No
Name
1H-NMR (CDCl3):
From
126
Chloromethyl
2.99-3.14(4H), 3.98-4.08(4H),
Thiomorpholine 1,1-dioxide
thiomorpholine-4-
5.78-5.81(2H)
carboxylate 1,1-dioxide
127
1-(Chloromethyl) 2-methyl
1.90-2.00(3H), 2.20-2.30(2H),
Methyl L-prolinate
(2S)-pyrrolidine-1,2-
3.45-3.55(2H), 3.76-3.79(3H),
hydrochloride
dicarboxylate
4.35-4.42(2H), 5.70-5.73(2H)
128
Chloromethyl cyclohexyl-
1.04-1.16(4H), 1.23-1.35(3H),
Cyclohexanamine
carbamate
1.83-1.93(3H), 3.42-3.52(1H),
5.66-5.69(2H)
129
Chloromethyl[2-(2,4-
2.93-3.00(2H), 3.45-3.51(2H),
2-(2,4-Dichlorophenyl)-
dichlorophenyl)ethyl]-
5.72-5.75(2H), 7.13-7.22(2H),
ethanamine
carbamate
7.36-7.41(1H)
130
Chloromethyl cyclohexyl-
1.00-1.13(1H), 1.26-1.48(4H),
N-Methylcyclohexan-amine
(methyl)-carbamate
1.62-1.86(5H), 2.76-2.88(3H),
3.77-4.04(1H), 5.76-5.84(2H)
131
Chloromethyl benzyl-
2.86-2.94(3H), 4.48-4.54(2H),
N-Methyl-1-phenyl-
(methyl)-carbamate
5.83-5.85(2H), 7.19-7.39(5H)
methanamine
132
Chloromethyl methyl(2-
1.50-1.67(5H), 2.61-2.71(2H),
N-Methyl-2-
phenylethyl)carbamate
5.75-5.90(2H), 7.23-7.40(5H)
phenylethanamine
Preparation 133
1-Chloroethyl [2-(methylsulfonyl)ethyl]carbamate
To a solution of 2-(methylsulfonyl)ethanamine (176 mg, 1.1 mmol) and N,N-diisopropylethylamine (0.38 ml, 2.2 mmol) in anhydrous dichloromethane (2 ml), at 0° C., was added dropwise 3-chloropropanoyl chloride (0.12 ml, 1.1 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 62 h. To the mixture was added water (5 ml) and the two layers were separated. The aqueous layer was extracted with dichloromethane (2×5 ml) and the combined organic layers were dried (MgSO4) and concentrated in vacuo to give the title compound (260 mg).
Similarly Prepared were:
Prep. No
Name
From
134
1-Chloroethyl morpholine-4-
Morpholine
carboxylate
135
1-(1-Chloroethyl) 2-methyl (2S)-
2-methyl (2S)-pyrrolidine-
pyrrolidine-1,2-dicarboxylate
2-carboxylate
Preparation 136
1-Benzyl-2-[1-(3-cyclopropyl-2-methylphenyl)vinyl]-1H-imidazole
To a solution of the compound of Preparation 140 (1.04 g, 3.0 mmol) in toluene (30 ml) was added potassium phosphate (1.88 g, 8.9 mmol) and cyclopropyl boronic acid (304 mg, 3.5 mmol). The mixture was de-gassed and tricyclohexylphosphine (83 mg, 0.3 mmol) was added. The mixture was de-gassed again, before addition of palladium (II) acetate (33 mg). The reaction mixture was then heated at reflux for 18 h. The mixture was poured into ethyl acetate and water and the two layers were separated. The organic phase was washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was filtered through silica, eluting with ethyl acetate:cyclohexane [1:1] and the filtrate was concentrated in vacuo to give the title compound (720 mg).
1H-NMR (d6-DMSO): 0.44-0.47 (2H), 0.80-0.83 (2H), 1.20-1.25 (1H), 1.70-1.80 (2H), 1.89-1.91 (3H) 5.22-5.24 (1H), 5.61-5.63 (1H), 6.80-6.84 (2H), 6.84-6.86 (1H), 6.86-6.89 (2H), 7.00-7.02 (1H), 7.17-7.23 (3H), 7.40-7.45 (1H)
Similarly Prepared were:
MH+
Prep.
Found/
No
Name
Expected
From Prep.
From
137
1-Benzyl-2-(1-biphenyl-3-yl-
337.2
147 and phenyl
1-Benzyl-2-[1-(3-bromophenyl)
vinyl)-1H-imidazole
337.2
boronic acid
vinyl]-1H-imidazole
138
1-Benzyl-2-[1-(3-cyclopropyl-
301.3
147
1-Benzyl-2-[1-(3-bromophenyl)
phenyl)vinyl]-1H-imidazole
301.2
vinyl]-1H-imidazole
Preparation 139
1-Benzyl-2-[1-(2-bromo-3-methylphenyl)vinyl]-1H-imidazole
To a suspension of the compound of Preparation 149 (3.1 g, 8.3 mmol) in acetonitrile (30 ml) was added thionyl chloride (12.2 ml, 167 mmol) and the reaction mixture was heated at 60° C., under nitrogen, for 11 h. The mixture was concentrated in vacuo and to the residue was added acetonitrile. This solution was concentrated in vacuo and the process was repeated. To the final residue was added 2-propanol (40 ml) and activated charcoal and the mixture was heated at 60° C. for 1 h. The mixture was concentrated in vacuo to give the title compound (3.1 g).
Experimental MH+ 353.3; expected 353.1
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
140
1-Benzyl-2-[1-(3-bromo-2-
353.0
151
1-(1-Benzyl-1H-imidazol-2-yl)-1-(3-
methylphenyl)vinyl]-1H-imidazole
353.1
bromo-2-methylphenyl)ethanol
141
1-Benzyl-2-{1-[3-bromo-2-
389.4
152
1-(1-Benzyl-1H-imidazol-2-yl)-1-[3-
(difluoromethyl)phenyl]vinyl}-1H-
389.1
bromo-2-(difluoromethyl)-
imidazole
phenyl]ethanol
142
1-Benzyl-2-{1-[3-(difluoromethyl)-
311.2
153
1-(1-Benzyl-1H-imidazol-2-yl)-1-[3-
phenyl]vinyl}-1H-imidazole
311.1
(difluoromethyl)phenyl]ethanol
143
1-Benzyl-2-[1-(2-fluoro-3-
293.3
154
1-(1-Benzyl-1H-imidazol-2-yl)-1-(2-
methylphenyl)vinyl]-1H-imidazole
293.1
fluoro-3-methylphenyl)ethanol
144
1-Benzyl-2-{1-[2-methyl-5-
—
155
1-(1-Benzyl-1H-imidazol-2-yl)-1-[2-
(trifluoromethyl)phenyl]vinyl}-1H-
methyl-5-(trifluoromethyl)phenyl]-
imidazole
ethanol
145
1-Benzyl-2-[1-(3-bromo-5-
—
156
1-(1-Benzyl-1H-imidazol-2-yl)-1-(3-
methylphenyl)vinyl]-1H-imidazole
bromo-5-methylphenyl)ethanol
146
1-Benzyl-2-[1-(3-ethylphenyl)-
289.2
177
1-(1-Benzyl-1H-imidazol-2-yl)-1-(3-
vinyl]-1H-imidazole
289.2
ethylphenyl)ethanol
147
1-Benzyl-2-[1-(3-bromophenyl)
339.0
150
1-(1-Benzyl-1H-imidazol-2-yl)-1-(3-
vinyl]-1H-imidazole
339.0
bromophenyl)ethanol
Preparation 145
1H-NMR (CDCl3): 2.20-2.25 (3H), 4.80-4.84 (2H), 5.66-5.70 (1H), 5.81-5.84 (1H), 6.88-6.95 (4H), 7.10-7.14 (1H), 7.21-7.29 (5H)
Preparation 148
1-Benzyl-2-{1-[2-methyl-3-(trifluoromethyl)phenyl]vinyl}-1H-imidazole
A solution of the compound of Preparation 157 (4.90 g, 13.6 mmol) in Eaton's Reagent (50 ml) was stirred at room temperature for 40 h. The mixture was poured into ice/water (200 ml) and adjusted to pH 7 by addition of saturated aqueous sodium hydrogen carbonate solution. The mixture was extracted with ethyl acetate (2×100 ml) and the combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (3.1 g).
Experimental MH+ 343.3; expected 343.1
Preparation 149
1-(1-Benzyl-1H-imidazol-2-yl)-1-(2-bromo-3-methylphenyl)ethanol
To a solution of the compound of Preparation 158 (3.38 g, 9.5 mmol) in tetrahydrofuran (30 ml), at 0° C. and under nitrogen, was added dropwise methylmagnesium bromide (3M, 6.34 ml, 19 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 18 h. To the mixture was added hydrochloric acid (0.1M, 25 ml), and the solution was basified by addition of saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted with ethyl acetate (4×30 ml). The combined extracts were washed with brine (20 ml), dried (MgSO4) and concentrated in vacuo to give the title compound (3.10 g).
Experimental MH+ 371.3; expected 371.1
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
150
1-(1-Benzyl-1H-imidazol-2-yl)-1-
—
159
(1-Benzyl-1H-imidazol-2-yl)(3-
(3-bromophenyl)ethanol
bromophenyl)methanone
151
1-(1-Benzyl-1H-imidazol-2-yl)-1-
371.2
160
(1-Benzyl-1H-imidazol-2-yl)(3-
(3-bromo-2-methylphenyl)ethanol
371.1
bromo-2-methylphenyl)methanone
152
1-(1-Benzyl-1H-imidazol-2-yl)-1-
407.4
161
(1-Benzyl-1H-imidazol-2-yl)[3-
[3-bromo-2-(difluoromethyl)-
407.1
bromo-2-(difluoromethyl)phenyl]-
phenyl]ethanol
methanone
153
1-(1-Benzyl-1H-imidazol-2-yl)-1-
329.3
162
(1-Benzyl-1H-imidazol-2-yl)[3-
[3-(difluoromethyl)phenyl]ethanol
329.1
(difluoromethyl)phenyl]-methanone
154
1-(1-Benzyl-1H-imidazol-2-yl)-1-
311.5
163
(1-Benzyl-1H-imidazol-2-yl)(2-
(2-fluoro-3-methylphenyl)ethanol
311.2
fluoro-3-methylphenyl)methanone
155
1-(1-Benzyl-1H-imidazol-2-yl)-1-
361.0
164
(1-Benzyl-1H-imidazol-2-yl)[2-
[2-methyl-5-(trifluoromethyl)-
361.2
methyl-5-(trifluoromethyl)phenyl]-
phenyl]ethanol
methanone
156
1-(1-Benzyl-1H-imidazol-2-yl)-1-
—
165
(1-Benzyl-1H-imidazol-2-yl)(3-
(3-bromo-5-methylphenyl)ethanol
bromo-5-methylphenyl)methanone
157
1-(1-Benzyl-1H-imidazol-2-yl)-1-
361.3
166
(1-Benzyl-1H-imidazol-2-yl)[2-
[2-methyl-3-(trifluoromethyl)-
361.2
methyl-3-(trifluoromethyl)phenyl]-
phenyl]ethanol
methanone
Preparation 156
1H-NMR (CDCl3): 2.07-2.15 (3H), 2.42-2.48 (3H), 4.93-5.00 (2H), 6.75-6.93 (5H), 7.05-7.16 (5H)
Preparation 158
(1-Benzyl-1H-imidazol-2-yl)(2-bromo-3-methylphenyl)methanone
A solution of 2-bromo-3-methylbenzoic acid (2.0 g, 9.3 mmol) in thionyl chloride (4.75 ml, 65.1 mmol) was heated at 65° C., under nitrogen, for 3 h. The mixture was concentrated in vacuo and to the residue was added acetonitrile (25 ml). This solution was concentrated in vacuo and the process was repeated. To the final residue was added acetonitrile (25 ml), 1-benzylimidazole (1.62 g, 10.2 mmol) and triethylamine (1.44 ml, 10.2 mmol) and the reaction mixture was heated at 60° C., under nitrogen, for 18 h. The mixture was concentrated in vacuo and to the residue was added ethyl acetate (80 ml). The solution was washed with water (40 ml) and saturated aqueous sodium hydrogen carbonate solution (40 ml), dried (MgSO4) and concentrated in vacuo to give the title compound (3.38 g).
Similarly Prepared were:
MH+
Prep.
Found/
From
No
Name
Expected
Prep.
From
159
(1-Benzyl-1H-imidazol-2-yl)(3-
341.1
—
3-Bromobenzoic acid
bromophenyl)methanone
341.0
160
(1-Benzyl-1H-imidazol-2-yl)(3-bromo-
355.1
—
3-Bromo-2-methylbenzoic acid
2-methylphenyl)-methanone
355.0
161
(1-Benzyl-1H-imidazol-2-yl)[3-bromo-
391.2
182
3-Bromo-2-(difluoromethyl)-
2-(difluoromethyl)-phenyl]methanone
391.0
benzoic acid
162
(1-Benzyl-1H-imidazol-2-yl)[3-
—
187
3-(Difluoromethyl)benzoic acid
(difluoromethyl)phenyl]-methanone
163
(1-Benzyl-1H-imidazol-2-yl)(2-fluoro-
295.3
—
2-Fluoro-3-methylbenzoic acid
3-methylphenyl)methanone
295.1
164
(1-Benzyl-1H-imidazol-2-yl)[2-methyl-
345.3
—
2-Methyl-5-(trifluoromethyl)-
5-(trifluoromethyl)phenyl]-methanone
345.1
benzoic acid
165
(1-Benzyl-1H-imidazol-2-yl)(3-bromo-
—
188
3-Bromo-5-methylbenzoic acid
5-methylphenyl)methanone
166
(1-Benzyl-1H-imidazol-2-yl)[2-methyl-
345.2
—
2-Methyl-3-(trifluoromethyl)-
3-(trifluoromethyl)-phenyl]methanone
345.1
benzoic acid
Preparation 162
1H-NMR (CDCl3): 5.69-5.71 (2H), 6.81-6.85 (1H), 7.17-7.20 (2H), 7.21-7.24 (2H), 7.30-7.40 (3H), 7.71-7.73 (1H), 8.39-8.42 (2H)
Preparation 165
1H-NMR (CDCl3): 2.35-2.38 (3H), 5.61-5.65 (2H), 7.15-7.37 (8H), 7.46-7.50 (1H), 7.92-7.97 (1H)
Preparation 167
2-[1-(1-benzyl-1H-imidazol-2-yl)vinyl]-6-methylbenzonitrile
A mixture of the compound of Preparation 139 (150 mg, 0.43 mmol), potassium hexacyanoferrate(II) (dried in vacuo at 85° C., 36 mg, 0.08 mmol), copper(I) iodide (8 mg), potassium iodide (7 mg), 1-methyl-2-pyrrolidinone (2 ml) and dimethylethylenediamine (49 μl) was placed in a pressure tube and degassed with nitrogen (×3). The tube was sealed and heated at 140° C. for 100 h. To the mixture was added ethyl acetate (10 ml) and water (10 ml) and the two layers were separated. The organic phase was dried (MgSO4) and concentrated in vacuo to give the title compound (160 mg)
Experimental MH+ 300.3; expected 300.2
Preparation 168
3-[1-(1-Benzyl-1H-imidazol-2-yl)vinyl]-2-methylbenzonitrile
To a solution of the compound of Preparation 140 (100 mg, 0.28 mmol) in 1-methyl-2-pyrrolidinone (3 ml) was added sodium cyanide (28 mg, 0.57 mmol) and nickel (II) bromide (62 mg, 0.28 mmol). The reaction mixture was sealed and heated in a microwave (150 W) at 150° C. for 5 min. To the mixture was added water (10 ml) and the solution was extracted with diethyl ether (4×10 ml). The combined extracts were washed with water (10 ml) and brine (10 ml), dried (MgSO4) and concentrated in vacuo. To the residue was added 2-propanol (15 ml) and activated charcoal and the solution was heated at 60° C. for 1 h. The mixture was then filtered through Arbocel® and the filtrate was concentrated in vacuo to give the title compound (40 mg).
Experimental MH+ 300.4; expected 300.2
Preparation 169
3-[1-(1-Benzyl-1H-imidazol-2-yl)vinyl]-5-methylbenzonitrile
To a solution of the compound of Preparation 145 (1.1 g, 3.1 mmol) in N,N-dimethylacetamide (30 ml) was added copper (I) cyanide (641 mg, 7.1 mmol) and the reaction mixture was heated at 150° C. for 3 days. The reaction mixture was poured into ethyl acetate and the mixture was washed with water and brine. The aqueous phase was filtered and the solid material was collected by filtration and dissolved in ethyl acetate, water and N,N,N′,N′-tetramethylethylenediamine. The two layers were separated and the organic phase was washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was filtered through charcoal and silica, eluting with ethyl acetate and the filtrate was concentrated in vacuo to give the title compound (210 mg).
1H-NMR (CDCl3): 2.24-2.28 (3H), 4.80-4.92 (2H), 5.56-5.60 (1H), 5.78-5.82 (1H), 6.88-6.97 (3H), 7.10-7.14 (1H), 7.19-7.33 (6H)
Preparation 170
1-[3-Methyl-2-(trifluoromethyl)phenyl]ethanone
To a solution of the compound of Preparation 171 (427 mg, 2.1 mmol) in dichloromethane (20 ml) was added Dess-Martin periodinane (25%, 3.83 ml, 2.3 mmol) and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was filtered through silica, eluting with dichloromethane, followed by diethyl ether. The filtrate was washed with saturated aqueous sodium hydrogen carbonate solution, dried (MgSO4) and concentrated in vacuo. To the residue was added dichloromethane and the solution was filtered through silica. The filtrate was dried (MgSO4) and concentrated in vacuo. The residue was dissolved in tetrahydrofuran and re-concentrated to give the title compound (332 mg).
1H-NMR (CDCl3): 1.80-1.85 (3H), 3.75-3.79 (3H), 7.04-7.07 (1H), 7.31-7.33 (1H), 7.40-7.43 (1H)
Preparation 171
1-[3-Methyl-2-(trifluoromethyl)phenyl]ethanol
To a solution of the compound of Preparation 172 (500 mg, 2.1 mmol) in tetrahydrofuran (22 ml), at −78° C., was added n-butyllithium (2.5M in hexanes, 0.92 ml, 2.3 mmol). After stirring for 45 min, acetaldehyde (0.14 ml, 2.5 mmol) was added and the reaction mixture was allowed to warm to room temperature over 18 h. To the mixture was added saturated aqueous ammonium chloride solution and the mixture was extracted with ethyl acetate. The combined extracts were washed with brine, dried (MgSO4) and concentrated in vacuo to give the title compound (600 mg).
1H-NMR (CDCl3): 1.42-1.45 (3H), 2.49-2.53 (3H), 5.38-5.42 (1H), 7.16-7.19 (1H), 7.40-7.43 (1H), 7.65-7.68 (1H)
Preparation 172
1-Bromo-3-methyl-2-(trifluoromethyl)benzene
A mixture of 2-bromo-6-methylbenzoic acid (10.0 g, 47.0 mmol) and sulphur tetrafluoride (5.02 g, 46.5 mmol) was heated in hydrofluoric acid (930 mg, 46.5 mmol) at 110° C. To the reaction mixture was added ethyl acetate and water and the two layers were separated. The organic phase was dried (MgSO4) and concentrated in vacuo. The residue was distilled under reduced pressure (bp 30-33° C. at 1 mmHg) to give the title compound (1.83 g).
Preparation 173
1-(3-Chloro-2-methoxyphenyl)ethanone
To the compound of Preparation 193 (697 mg, 4.1 mmol) in acetone (30 ml) was added potassium carbonate (1.13 g, 8.2 mmol), followed by methyl iodide (2.0 ml, 4.66 g, 32.8 mmol). The reaction mixture was heated at 40° C. for 18 h, cooled and concentrated in vacuo. The residue was partitioned between ethyl acetate and water and the organic phase was separated, washed with brine, dried (MgSO4) and concentrated in vacuo to give the title compound (450 mg) as a mixture of regioisomers.
1H-NMR (CDCl3): 2.58-2.62 (3H), 3.92-3.95 (3H), 7.47-7.51 (1H), 7.82-7.86 (1H), 7.94-7.97 (1H)
Similarly Prepared was:
Prep.
From
No
Name
Prep.
From
174***
1-(3-chloro-4-
193
1-(3-chloro-4-
methoxyphenyl)-ethanone
hydroxyphenyl)ethanone
***The reaction to yield Preparation 174 gave some of Preparation 173 since Preparation 193 contained a mixture of 1-(3-chloro-2-hydroxyphenyl)ethanone and 1-(3-chloro-4-hydroxyphenyl)ethanone
Preparation 174
1H-NMR (CDCl3): 2.58-2.62 (3H), 3.92-3.95 (3H), 7.47-7.51 (1H), 7.82-7.86 (1H), 7.94-7.97 (1H)
Preparation 175
1-(2-Chloro-5-methoxyphenyl)ethanone
To a solution of SELECTFLUOR™ (5.0 g, 14.1 mmol) and sodium chloride (825 mg, 14.1 mmol) in acetonitrile (200 ml), under nitrogen, was added 1-(3-methoxyphenyl)ethanone (1.94 ml, 14.1 mmol) and the reaction mixture was stirred at room temperature for 5 days. To the mixture was added distilled water (200 ml) and the solution was extracted with dichloromethane (2×100 ml). The combined extracts were dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography (silica) with gradient elution, ethyl acetate:cyclohexane [5:95 to 10:90]. The appropriate fractions were combined and concentrated to give the title compound (1.12 g).
1H-NMR (CDCl3): 2.59-2.65 (3H), 3.76-3.80 (3H), 6.88-6.91 (1H), 7.01-7.04 (1H), 7.25-7.28 (1H)
Preparation 176
1-Benzyl-2-[1-(3-ethyl-2-methylphenyl)vinyl]-1H-imidazole
To a solution of the compound of Preparation 140 (207 mg, 0.3 mmol) in N,N-dimethylformamide (28 ml) was added potassium carbonate (1.17 g, 8.5 mmol), [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride (550 mg) and triethylborane (1M, 6.79 ml, 6.8 mmol). The reaction mixture was heated at reflux for 60 h, cooled and concentrated in vacuo. To the residue was added ethyl acetate and water and the two layers were separated. The organic phase was washed with water and brine, dried (MgSO4) and concentrated in vacuo to give the title compound (110 mg).
Experimental MH+ 303.2; expected 303.2
Preparation 177
1-(1-Benzyl-1H-imidazol-2-yl)-1-(3-ethylphenyl)ethanol
To a solution of the compound of Preparation 150 (500 mg, 1.4 mmol) in N,N-dimethylformamide (14 ml) was added potassium carbonate (193 mg, 1.4 mmol) and triethylborane (1M, 3.36 ml, 3.4 mmol). The mixture was de-oxygenated and [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride (114 mg) was added. The reaction mixture was heated at 50° C. for 18 h, cooled and concentrated in vacuo. To the residue was added ethyl acetate and water and the two layers were separated. The organic phase was washed with water and brine, dried (MgSO4) and concentrated in vacuo to give the title compound (500 mg).
Experimental MH+ 307.3; expected 307.2
Preparation 178
1-(2-Ethyl-3-methylphenyl)ethanone
To a solution of the compound of Preparation 179 (367 mg, 1.7 mmol) in anhydrous N,N-dimethylformamide (10 ml), under nitrogen, was added potassium carbonate (4.52 g, 32.7 mmol), followed by [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride (141 mg) and triethylborane (1M in tetrahydrofuran, 4.13 ml, 4.13 mmol). The reaction mixture was heated at 50° C. for 18 h, filtered and concentrated in vacuo. The residue was purified by flash chromatography (Biotage™ 40M cartridge), eluting with ethyl acetate:pentane [5:95]. The appropriate fractions were combined and concentrated to give the title compound (160 mg).
1H-NMR (CDCl3): 1.15-1.20 (3H), 2.35-2.38 (3H), 2.55-2.58 (3H), 2.74-2.81 (2H), 7.12-7.17 (1H), 7.23-7.27 (1H), 7.36-7.40 (1H)
Preparation 179
1-(2-Bromo-3-methylphenyl)ethanone
To a solution of 1-(2-amino-3-methylphenyl)ethanone (Helv. Chim. Acta; EN; 62, 1979, 271-303,) (850 mg, 5.7 mmol) in hydrobromic acid (9 ml, 5.7 mmol) and water (6 ml), at 0° C., was added aqueous sodium nitrite solution (503 mg, 7.3 mmol) and the mixture was stirred for 15 min. This mixture was added to copper (I) bromide (899 mg, 6.3 mmol) in hydrobromic acid (9 ml, 5.7 mmol) at 60° C. and the reaction mixture was heated at 95° C. for a further 30 min. After cooling, the mixture was poured into an ice/water slurry and extracted with ethyl acetate. The combined extracts were dried (MgSO4) and concentrated in vacuo. To the residue was added ethyl acetate:cyclohexane [1:4] and the solution was filtered through silica. The filtrate was concentrated in vacuo to give the title compound (1.06 g).
Preparation 180
1-(2-Bromo-3,5,6-trimethylphenyl)ethanone
To a mixture of 1-bromo-2,4,5-trimethylbenzene (5.0 g, 25.0 mmol) and acetyl chloride (2.45 ml, 34.5 mmol) in dichloromethane (50 ml) was added aluminium chloride (4.42 g, 33.1 mmol) in dichloromethane (50 ml) and the reaction mixture was stirred at room temperature for 18 h. The mixture was poured into water and the two layers were separated. The organic phase was washed with saturated aqueous sodium hydrogen carbonate solution, dried (MgSO4) and filtered through silica. The filtrate was concentrated in vacuo to give the title compound (5.50 g).
1H-NMR (CDCl3): 2.13-2.17 (3H), 2.22-2.27 (3H), 2.28-2.34 (3H), 2.48-2.54 (3H), 6.89-6.92 (1H)
Preparation 181
1-Benzyl-2-{1-[2-(difluoromethyl)-3-methylphenyl]vinyl}-1H-imidazole
To a solution of the compound of Preparation 141 (140 mg, 0.36 mmol) in 1,4-dioxane:water (9:1, 10 ml) was added trimethylboroxine (50 μl, 0.36 mmol) and sodium carbonate (114 mg, 1.08 mmol). The mixture was degassed, before addition of [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride (30 mg). The reaction mixture was heated at 100° C. for 18 h, cooled and concentrated in vacuo. To the residue was added ethyl acetate and the solution was washed with water, dried (MgSO4) and filtered through silica. The filtrate was concentrated in vacuo to give the title compound (110 mg).
Experimental MH+ 325.3; expected 325.2
Preparation 182
3-Bromo-2-(difluoromethyl)benzoic acid
To a solution of the compound of Preparation 183 (4.05 g, 11.9 mmol) in tetrahydrofuran (120 ml) was added aqueous sodium hydroxide solution (1M, 24.30 ml, 24.3 mmol) and the reaction mixture was stirred at room temperature for 18 h. The mixture was partitioned between diethyl ether and water and the two layers were separated. The aqueous layer was acidified with hydrochloric acid (2M) and extracted with ethyl acetate. The combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (3.06 g).
Experimental MH+ 251.1; expected 251.0
Preparation 183
Benzyl 3-bromo-2-(difluoromethyl)benzoate
To a solution of the compound of Preparation 185 (4.1 g, 12.9 mmol) in dichloromethane (130 ml) was added (diethylamino)sulphur trifluoride (5.06 ml, 38.6 mmol) and the reaction mixture was stirred at room temperature for 18 h. To the mixture was added additional dichloromethane and saturated aqueous sodium hydrogen carbonate solution and the two layers were separated. The organic phase was concentrated in vacuo and the residue was filtered through silica, eluting with dichloromethane. The filtrate was concentrated in vacuo to give the title compound (4.05 g).
1H-NMR (CDCl3): 5.23-5.25 (1H), 5.33-5.35 (2H), 7.30-7.41 (4H), 7.59-7.64 (2H), 7.85-7.90 (1H)
Similarly Prepared was:
Prep.
No.
Name
1H-NMR (CDCl3)
From
184
Methyl 3-
3.85-3.88(3H), 6.47-6.76(1H),
Methyl 3-
(difluoromethyl)-
7.44-7.50(1H), 7.61-7.66(1H),
formyl-
benzoate
8.05-8.13(2H)
benzoate
Preparation 185
Benzyl 3-bromo-2-formylbenzoate
To a solution of the compound of Preparation 186 (4.77 g, 14.9 mmol) in ethyl acetate (150 ml) was added manganese (IV) oxide (12.95 g, 148.9 mmol) and the reaction mixture was stirred at room temperature for 1 h. The mixture was filtered and the filtrate was concentrated in vacuo to give the title compound (4.11 g).
Experimental MH+ 319.1; expected 319.0
Preparation 186
Benzyl 3-bromo-2-(hydroxymethyl)benzoate
A solution of 4-bromo-2-benzofuran-1(3H)-one (4.02 g, 18.9 mmol) in aqueous sodium hydroxide solution (1M, 18.9 ml) was heated at 100° C. for 1 h. The solution was concentrated in vacuo and the residue was dissolved in toluene and re-concentrated. To a solution of the residue in N,N-dimethylformamide (20 ml) was added benzyl bromide (2.26 ml, 18.90 mmol) and the reaction mixture was stirred at room temperature for 14 days. The mixture was poured into water and the resulting precipitate was collected by filtration, washed with water and pentane and dried to give the title compound (4.77 g).
Experimental MH+ 321.1; expected 321.0
Preparation 187
3-(Difluoromethyl)benzoic acid
To a solution of the compound of Preparation 184 (188 mg, 1.0 mmol) in tetrahydrofuran (5 ml) was added lithium hydroxide monohydrate (85 mg, 2.0 mmol). The reaction mixture was stirred at room temperature for 18 h and then acidified by addition of hydrochloric acid (1M). To the mixture was added water (5 ml) and brine (5 ml) and the solution was extracted with ethyl acetate (3×10 ml). The combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (290 mg).
1H-NMR (CDCl3): 6.50-6.80 (1H), 7.50-7.57 (1H), 7.69-7.74 (1H), 8.14-8.21 (2H)
Preparation 188
3-Bromo-5-methylbenzoic acid
To a solution of the compound of Preparation 189 (10.0 g, 43.5 mmol) in acetic acid (45 ml) was added hydrochloric acid (12M, 14.1 ml) and the reaction mixture was heated at 70° C. for 1 h. The solution was cooled to 0° C. and aqueous sodium nitrite solution (5M, 3.0 g, 43.5 mmol) was added. After 1 h, the mixture was cooled to −15° C. and aqueous hypophosphorous acid (50%, 23 ml, 170 mmol) was added dropwise. The reaction mixture was allowed to warm to 10° C. over 2 h and filtered. The solid material was washed with water and cyclohexane and the product was dried to give the title compound (8.6 g).
1H-NMR (CDCl3): 2.32-2.36 (3H), 7.64-7.66 (1H), 7.71-7.74 (1H), 7.79-7.82 (1H)
Preparation 189
2-Amino-3-bromo-5-methylbenzoic acid
To a solution of 2-amino-5-methylbenzoic acid (25.0 g, 170 mmol) in acetic acid (250 ml) was added bromine (10 ml, 195 mmol) and the reaction mixture was stirred at room temperature for 2 h. The mixture was filtered and the solid material was washed with water and cyclohexane and dried to give the title compound (33.4 g).
1H-NMR (CDCl3): 2.08-2.13 (3H), 7.42-7.45 (1H), 7.53-7.56 (1H)
Preparation 190
5-Methoxy-2,4-dimethylbenzaldehyde
To a solution of the compound of Preparation 191 (4.12 g, 14 mmol) in 1,4-dioxane (30 ml) was added aqueous sodium carbonate solution (15M, 2.80 ml, 42 mmol). After purging with nitrogen, trimethylboroxine (1.95 ml, 14 mmol) was added, followed by [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.03 g). The reaction mixture was heated at 100° C. for 18 h, cooled and filtered through Arbocel®. The filtrate was concentrated in vacuo and to the residue was added dichloromethane. The solution was filtered through silica and the filtrate was concentrated in vacuo to give the title compound (988 mg).
1H-NMR (CDCl3): 2.19-2.23 (3H), 2.53-2.57 (3H), 3.80-3.85 (3H), 7.21-7.24 (2H), 10.23-10.25 (1H)
Preparation 191
2,4-Dibromo-5-methoxybenzaldehyde
To a solution of 3-methoxybenzaldehyde (5.00 g, 4.47 ml, 36.7 mmol) in methanol (245 ml) was added aqueous sodium bromide solution (5M, 36.7 ml, 184 mmol) and aqueous OXONE® solution (45.00 g, 73.4 mmol). The reaction mixture was stirred at room temperature for 18 h, before addition of aqueous sodium thiosulphate solution (1M, 200 ml) and ethyl acetate (400 ml). The two layers were separated and the organic phase was washed with water and brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by flash chromatography (Biotage) eluting with ethyl acetate:cyclohexane [10:90]. The appropriate fractions were combined and concentrated to give the title compound (4.12 g).
1H-NMR (CDCl3): 3.89-3.93 (3H), 7.37-7.39 (1H), 7.79-7.82 (1H), 10.20-10.23 (1H)
Preparation 192
1-(2,3-Dimethylphenyl)propan-1-ol
To a solution of 2,3-dimethylbenzaldehyde (1.0 g, 7.5 mmol) in anhydrous tetrahydrofuran (50 ml), at −78° C. and under nitrogen, was added ethyllithium (0.5M in benzene:cyclohexane 9:1, 14.9 ml, 7.5 mmol), dropwise via syringe. The reaction mixture was stirred at −78° C. for 30 min and then poured into ice cold hydrochloric acid (2N, 20 ml). The mixture was extracted with ethyl acetate (2×50 ml) and the combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (1.22 g).
1H-NMR (CDCl3): 0.93-0.99 (3H), 1.68-1.76 (2H), 2.18-2.22 (3H), 2.25-2.28 (3H), 4.87-4.92 (1H), 7.03-7.13 (2H), 7.28-7.38 (1H)
Preparation 193
1-(3-Chloro-2-hydroxyphenyl)ethanone and 1-(3-chloro-4-hydroxyphenyl)ethanone
A solution of 2-chlorophenyl acetate (1.98 g, 11.6 mmol) in 1,2-dichlorobenzene (10 ml) was added dropwise to a solution of aluminium chloride (1.90 g, 13.9 mmol) in 1,2-dichlorobenzene (10 ml). The reaction mixture was heated at 100° C. for 24 h and then cooled, before addition of dichloromethane (10 ml). The mixture was poured into hydrochloric acid (10%, 12 ml), at 0° C., and the two layers were separated. The aqueous phase was extracted with dichloromethane and the combined organic phases were washed with water, dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography (silica), eluting with cyclohexane. The appropriate fractions were combined and concentrated to give the title compound as a 1:1 mixture of regioisomers (1.0 g).
Preparation 194
1-Benzyl-2-[1-(2,3-dimethylphenyl)vinyl]-1H-imidazole
To a suspension of the compound of Preparation 195 (500 mg, 1.63 mmol) in acetonitrile (10 ml) was added thionyl chloride (0.2 mmol, 2.74 mmol) and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo and the residue was triturated with ethyl acetate to give the title compound (450 mg).
Experimental MH+ 289.3; expected 289.2
Alternative Synthesis
A solution of the compound of Preparation 195 (82.00 g, 267.6 mmol) in Eaton's Reagent (380 ml) was stirred at room temperature for 18 h. The reaction mixture was poured onto ice and the solution was washed with diethyl ether and adjusted to pH 7 by addition sodium carbonate. The aqueous layer was extracted with ethyl acetate and the combined organic extracts were concentrated in vacuo to give the title compound (79.0 g).
Experimental MH+ 289.4; expected 289.2
Preparation 195
1-(1-Benzyl-1H-imidazol-2-yl)-1-(2,3-dimethylphenyl)ethanol
To a solution of the compound of Preparation 36 (182 g, 626.8 mmol) in tetrahydrofuran (1 l), at 0° C., was added methylmagnesium chloride (3M in tetrahydrofuran, 271 ml, 814 mmol). The reaction mixture was stirred at room temperature for 18 h and then poured into hydrochloric acid (2M, 500 ml). To the mixture was added diethyl ether (500 ml) and saturated aqueous sodium chloride solution (100 ml) and the two layers were separated. To the aqueous layer was added ethyl acetate (500 ml) and sodium carbonate (50 g) and the organic layer was separated. The resulting solid material was collected by filtration and triturated with diethyl ether (300 ml) to give the title compound (82 g).
Experimental MH+ 307.4; expected 307.2
Alternative Synthesis
To a solution of methylmagnesium chloride (3M in tetrahydrofuran, 5.0 L, 15.2 mol), under nitrogen, was added a solution of the compound of Preparation 36 (2.8 kg, 9.6 mol) in toluene (6.0 L), at −10° C. The reaction mixture was stirred at −10° C. for 4 h and then quenched by the dropwise addition of aqueous ammonium chloride solution (20%, 14.0 L). The resulting solid was collected by filtration and then was slurried with water (2×10 L) and filtered. The residue obtained is further slurried in acetonitrile (14 L) and filtered. The solid material collected by filtration was washed with acetonitrile (2×4 L) and dried in vacuo at 50° C. to give the title compound (2.63 kg, 99.75% pure by HPLC).
Preparation 196
3-[1-(1H-Imidazol-2-yl)ethyl]benzamide
A solution of the compound of Preparation 197 (311 mg, 1.35 mmol) in ammonium hydroxide (28% in water, 15 ml) was heated at 85° C. for 2 h. The mixture was then cooled and concentrated in vacuo to give the title compound (364 mg).
Experimental MH+ 216.2; expected 216.1
Preparation 197
Methyl 3-[1-(1H-imidazol-2-yl)ethyl]benzoate
To a solution of the compound of Preparation 198 (477 mg, 1.5 mmol) in 2-propanol (10 ml) was added ammonium formate (945 mg, 15.0 mmol) and palladium (10 wt. % on carbon, 168 mg) and the reaction mixture was heated at 80° C. for 18 h. The mixture was filtered through Arbocel® and the filtrate was concentrated in vacuo to give the title compound (270 mg).
Experimental MH+ 231.4; expected 231.1
Preparation 198
Methyl 3-[1-(1-benzyl-1H-imidazol-2-yl)vinyl]benzoate
A mixture of the compound of Preparation 199 (2.55 g, 7.3 mmol) and thionyl chloride (2.12 ml, 29.1 mmol) in acetonitrile (20 ml) was stirred at room temperature for 18 h. The mixture was concentrated in vacuo and the residue was partitioned between ethyl acetate and aqueous sodium hydrogen carbonate solution. The two layers were separated and the organic phase was filtered through silica and charcoal and concentrated in vacuo. The residue was purified by column chromatography (silica), with gradient elution, ethyl acetate:cyclohexane [1:1 to 4:1 to 1:0]. The appropriate fractions were combined and concentrated to give the title compound (2.32 g).
Experimental MH+ 319.3; expected 319.1
Preparation 199
Methyl 3-[1-(1-benzyl-1H-imidazol-2-yl)-1-hydroxyethyl]benzoate
To a solution of the compound of Preparation 200 (3.82 g, 11.9 mmol) in tetrahydrofuran (25 ml), at 0° C., was added methylmagnesium chloride (3M in tetrahydrofuran, 5.17 ml, 15.5 mmol) and the reaction mixture was stirred at room temperature for 18 h. The mixture was poured into a mixture of ice, hydrochloric acid (2M) and diethyl ether and the two layers were separated. The aqueous layer was adjusted to pH 7 by addition of solid sodium hydrogen carbonate and then extracted with ethyl acetate. The combined extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (2.55 g).
Experimental MH+ 337.4; expected 337.1
Preparation 200
Methyl 3-[(1-benzyl-1H-imidazol-2-yl)carbonyl]benzoate
A solution of 3-(methoxycarbonyl)benzoic acid (2.53 g, 14.0 mmol) in thionyl chloride (7.17 ml, 98.30 mmol) was heated at reflux for 1 h. The mixture was cooled and concentrated in vacuo and to the residue was added toluene. This solution was concentrated in vacuo and to the residue was added anhydrous acetonitrile (24 ml), 1-benzyl-1H-imidazole (2.22 g, 14.0 mmol) and triethylamine (2.35 ml, 16.9 mmol). The reaction mixture was heated at reflux for 18 h and then cooled and concentrated in vacuo. To the residue was added ethyl acetate and the solution was washed with water and saturated aqueous sodium hydrogen carbonate solution. The organic phase was filtered through silica, eluting with ethyl acetate, and the filtrate was concentrated in vacuo to give the title compound (3.82 g).
Experimental MH+ 321.3; expected 321.1
Preparation 201
(2,3-Dimethylphenyl)(1H-imidazol-2-yl)methanol
To a solution of 1-(diethoxymethyl)-1H-imidazole (698 mg, 4.10 mmol) in anhydrous tetrahydrofuran (7 ml), at −78° C., was added n-butyllithium (2.5 M in hexane, 1.64 ml, 4.1 mmol). The reaction mixture was stirred at −78° C. for 1 h, before addition of a solution of 2,3-dimethylbenzaldehyde (500 mg, 3.73 mmol) in tetrahydrofuran (3 ml). The reaction mixture was stirred at −78° C. for 2 h, warmed to room temperature and quenched with ice cold hydrochloric acid (4M, 20 ml). The reaction mixture was concentrated in vacuo and to the residue was added water (20 ml). The solution was extracted with diethyl ether (2×20 ml) and the aqueous layer was basified by addition of solid sodium hydrogen carbonate. This solution was extracted with ethyl acetate (3×20 ml) and the combined organic phases were dried (MgSO4) and concentrated in vacuo to give the title compound (543 mg)
Experimental MH+ 203.1; expected 203.1
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11619735
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pfizer limited
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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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514/396
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Jul 8th, 2014 12:00AM
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Jul 8th, 2011 12:00AM
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https://www.uspto.gov?id=US08772293-20140708
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Chemical compounds
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The invention relates to sulfonamide derivatives, to their use in medicine, to compositions containing them, to processes for their preparation and to intermediates used in such processes.
More particularly the invention relates to new sulfonamide Nav1.7 inhibitors of formula (I):
or pharmaceutically acceptable salts thereof, wherein Z1, Ra, Rb, R1, R2, R3, R4 and R5 are as defined in the description.
Nav 1.7 inhibitors are potentially useful in the treatment of a wide range of disorders, particularly pain.
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8772293
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1. A compound of formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, wherein said heteroaryl is optionally substituted on a ring carbon atom by F or CI; or
R1 is a ‘C-linked’ 5-membered heteroaryl comprising one or two nitrogen atoms and one sulphur atom, wherein said heteroaryl is optionally substituted on a ring carbon atom by F or CI;
R2, R3 and R4 are independently H, F, CI or —OCH3;
R5 is CN, F, CI or R6;
Ra is phenyl optionally substituted by one to three substituents that are independently F, CI, CN, H2N(C1-C4)alkylene-, (C1-C4)alkylNH(C1-C4)alkylene-, (C3-C8)cycloalkyl or R6; or
Ra is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, wherein said heteroaryl is optionally substituted by R7 or R8, or both R7 and R8;
Rb is H, F, CI, CN or R6;
R6 is (C1-C4)alkyl or (C1-C4)alkyloxy, each optionally substituted by one to eight F;
Z1 is phenyl optionally substituted by one to three substituents that are independently F, CI or R6; or
Z1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, wherein said heteroaryl is optionally substituted by R7 or R8, or both R7 and R8;
R7 is attached to a Z1 ring carbon and is F, CI, NR9R10, R6, (C3-C8)cycloalkyl or Het';
R8 is attached to a Z1 ring nitrogen and is (C1-C4)alkyl or (C3-C8)cycloalkyl, each optionally substituted by, one to three F; or
R8 is attached to a Z1 ring nitrogen and is a ‘C-linked’ Het1;
Het1 ‘is a 3- to 8-membered saturated monoheterocycloalkyl comprising one or two ring members selected from —N(R11)— and —O—, wherein said monoheterocycloalkyl is optionally substituted on a ring carbon atom by one to three substituents that are independently F, (C1-C6)alkyl, (C1-C4)alkyloxy(Co-C4)alkylene or (C3-C8)cycloalky; and .
R9, R10 and R11 are independently H, (C1-C6)alkyl or (C3-C8)cycloalkyl; or, when Het1 is ‘N-linked’, R11 is absent from that nitrogen atom.
2. The compound according to claim 1 of the following formula:
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, wherein said heteroaryl is optionally substituted on a ring carbon atom by F or Cl; or
R1 is a ‘C-linked’ 5-membered heteroaryl comprising one or two nitrogen atoms and one sulphur atom, wherein said heteroaryl is optionally substituted on a ring carbon atom by F or CI;
R2, R3 and R4 are independently H, F, CI or —OCH3;
R5 is CN, F, CI or R6;
Ra is phenyl optionally substituted by one to three substituents that are independently from F, CI or R6;
Rb is H, F, CI or R6;
R6 is (C1-C4)alkyl or (C1-C4)alkyloxy, each optionally substituted by one to three F;
Z1 is phenyl optionally substituted by one to three substituents that are independently F, CI or R6; or
Z1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, wherein said heteroaryl is optionally substituted by R7 or R8, or both R7 and R8;
R7 is attached to a Z1 ring carbon and is F, CI, NR9R10, R6, (C3-C8)cycloalkyl or Het1;
R8 is attached to a Z1 ring nitrogen and is (C1-C4)alkyl, (C3-C8)cycloalkyl or ‘C-linked’ Het1;
Het1 is a 3- to 8-membered saturated monoheterocycloalkyl comprising one or two ring members selected from —N(R11)— and —O—, wherein said monoheterocycloalkyl is optionally substituted on a ring carbon atom by one to three substituents that are independently F, (C1-C6)alkyl, (C1-C4)alkyloxy(Co-C4)alkylene or (C3-C8)cycloalky; and
R9, R10 and R11 are independently H, (C1-C6)alkyl or (C3-C8)cycloalkyl; provided that when Het1 is ‘N-linked’, R11 is absent from that nitrogen atom.
3. The compound according to claim 2 wherein R1 is a ‘C-linked’ heteroaryl selected from thiazolyl, thiadiazolyl, pyridazinyl or pyrimidinyl, wherein said heteroaryl is optionally substituted on a ring carbon atom by F or Cl.
4. The compound according to claim 2 wherein R1 is a ‘C-linked’ heteroaryl selected from thiazolyl or thiadiazolyl, wherein said heteroaryl is optionally substituted on a ring carbon atom by F.
5. The compound according to claim 4 wherein R2, R3 and R4 are independently H or F.
6. The compound according to claim 5 wherein R5 is CN, F or Cl.
7. The compound according to claim 6 wherein Ra is phenyl, optionally substituted by R6.
8. The compound according claim 7 wherein Rb is H.
9. The compound according to claim 8 wherein Z1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, wherein said heteroaryl is optionally substituted by R7 or R8, or both R7 and R8.
10. The compound according claim 8 wherein Z1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, wherein said heteroaryl being is optionally substituted by R8.
11. The compound according to claim 10 wherein said ‘C-linked’ 5- or 6-membered heteroaryl is pyrazolyl or pyridazinyl, wherein said heteroaryl is optionally substituted by R8.
12. The compound according to claim 10 wherein said ‘C-linked’ 5- or 6-membered heteroaryl is pyridazinyl or pyrazolyl, wherein said pyrazolyl is optionally substituted by methyl or a ‘C-linked’ 3- to 4-membered saturated monoheterocycloalkyl comprising one —N((C1-C2)alkyl)- ring member.
13. A pharmaceutical composition comprising a compound according to claim 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
14. The pharmaceutical composition according to claim 13 further comprising one or more additional therapeutic agents.
15. The compound according to claim 1 that is
3-Cyano-4-{[3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide;
5-Chloro-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-pyrimidin-2-ylbenzenesulfonamide;
3-Chloro-N-pyridazin-3-yl-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}benzenesulfonamide;
5-Chloro-2-fluoro-4-{[3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-Chloro-2-fluoro-4-{[3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-Chloro-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
3-Cyano-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide;
3-Fluoro-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-1,3-thiazol-2-ylbenzenesulfonamide;
3-Chloro-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
3-Cyano-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-Chloro-2-fluoro-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
3-Cyano-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide;
5-Chloro-2-fluoro-4-({3-[1-(1-methylazetidin-3-yl)-1H-pyrazol-5-yl]-2′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-Chloro-2-fluoro-4-({3-[1-(1-methylazetidin-3-yl)-1H-pyrazol-5-yl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide; -4-ylloxy)-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
3-Cyano-N-(5-fluoro-1,3-thiazol-2-yl)-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonamide;
3-Cyano-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide;
3-Cyano-4-{[3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide;
3-Cyano-4-{[3′-methoxy-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-(1,3-thiazol-2-yl)benzenesulfonamide;
3-Cyano-4-{[2′-methoxy-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-(1,3-thiazol-2-yl)benzenesulfonamide;
3-Cyano-N-(5-fluoropyridin-2-yl)-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonamide;
4-{[3′-(Aminomethyl)-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-3-cyano-N-(1,3-thiazol-2-yl)benzenesulfonamide;
5-Chloro-2-fluoro-N-(5-fluoropyridin-2-yl)-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonamide;
3-Cyano-4-({2′-[(methylamino)methyl]-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl}oxy)-N-(1,3-thiazol-2-yl)benzenesulfonamide;
5-Chloro-4-{[2-chloro-4′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
4-{[3-(3-Amino-1H-pyrazol-4-yl)-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-4-{[2-chloro-3′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-4-{[2-chloro-2′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-4-{[2-chloro-5-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-4-{[4′-chloro-3-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-2-fluoro-4-{2-(pyridazin-4-yl)-4-[6-(trifluoromethyl)pyrid in-3-yl]phenoxy}-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-2-fluoro-4-{2-(pyridazin-4-yl)-4-[6-(trifluoromethyl)pyrid in-2-yl]phenoxy}-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
4-{[3-(5-Amino-1H-pyrazol-4-yl)-3′-cyanobiphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-2-fluoro-4-{2-(pyridazin-4-yl)-4-[2-(trifluoromethyl) pyridin-4-yl]phenoxy}-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-2-fluoro-4-({3-[2-(piperazin-1-yl)pyridin-4-yl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide;
5-Chloro-2-fluoro-4-({3-[2-(piperazin-1-yl)pyridin-4-yl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-(pyrimidin-4-yl)benzenesulfonamide;
5-Chloro-4-[(6-chloro-3′-fluoro-4-pyridazin-4-ylbiphenyl-3-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-Chloro-4-[(6-chloro-4′-fluoro-4-pyridazin-4-ylbiphenyl-3-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-Chloro-4-[(6-chloro-2′-fluoro-4-pyridazin-4-ylbiphenyl-3-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-chloro-4-[(3′-cyano-3-pyridazin-4-ylbiphenyl-4-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide;
5-chloro-2-fluoro-4-{[3-(2-piperazin-1-ylpyridin-4-yl)-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-pyrimidin-2-ylbenzenesulfonamide;
or a ;pharmaceutically acceptable salt thereof.
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15
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This application claims benefit of U.S. Provisional Application No. 61/362,927, filed Jul. 9, 2010; and U.S. Provisional Application No. 61/492,525, filed Jun. 2, 2011; each application is hereby incorporated by reference in its entirety for any purpose.
The invention relates to sulfonamide derivatives, to their use in medicine, to compositions containing them, to processes for their preparation and to intermediates used in such processes.
Voltage-gated sodium channels are found in all excitable cells including myocytes of muscle and neurons of the central and peripheral nervous system. In neuronal cells, sodium channels are primarily responsible for generating the rapid upstroke of the action potential. In this manner sodium channels are essential to the initiation and propagation of electrical signals in the nervous system. Proper and appropriate function of sodium channels is therefore necessary for normal function of the neuron. Consequently, aberrant sodium channel function is thought to underlie a variety of medical disorders (see Hubner C A, Jentsch T J, Hum. Mol. Genet., 11(20): 2435-45 (2002) for a general review of inherited ion channel disorders) including epilepsy (Yogeeswari et al., Curr. Drug Targets, 5(7): 589-602 (2004)), arrhythmia (Noble D., Proc. Natl. Acad. Sci. USA, 99(9): 5755-6 (2002)) myotonia (Cannon, S C, Kidney Int. 57(3): 772-9 (2000)), and pain (Wood, J N et al., J. Neurobiol., 61(1): 55-71 (2004)).
There are currently at least nine known members of the family of voltage-gated sodium channel (VGSC) alpha subunits. Names for this family include SCNx, SCNAx, and Navx.x. The VGSC family has been phylogenetically divided into two subfamilies Nav1.x (all but SCN6A) and Nav2.x (SCN6A). The Nav1.x subfamily can be functionally subdivided into two groups, those which are sensitive to blocking by tetrodotoxin (TTX-sensitive or TTX-s) and those which are resistant to blocking by tetrodotoxin (TTX-resistant or TTX-r).
The Nav1.7 (PN1, SCN9A) VGSC is sensitive to blocking by tetrodotoxin and is preferentially expressed in peripheral sympathetic and sensory neurons. The SCN9A gene has been cloned from a number of species, including human, rat, and rabbit and shows ˜90% amino acid identity between the human and rat genes (Toledo-Aral et al., Proc. Natl. Acad. Sci. USA, 94(4): 1527-1532 (1997)).
An increasing body of evidence suggests that Nav1.7 may play a key role in various pain states, including acute, inflammatory and/or neuropathic pain. Deletion of the SCN9A gene in nociceptive neurons of mice led to a reduction in mechanical and thermal pain thresholds and reduction or abolition of inflammatory pain responses (Nassar et al., Proc Natl Acad Sci USA, 101(34): 12706-11 (2004)). In humans, Nav1.7 protein has been shown to accumulate in neuromas, particularly painful neuromas (Kretschmer et al., Acta. Neurochir. (Wien), 144(8): 803-10 (2002)). Gain of function mutations of Nav1.7, both familial and sporadic, have been linked to primary erythermalgia, a disease characterized by burning pain and inflammation of the extremities (Yang et al., J. Med. Genet., 41(3): 171-4 (2004), and paroxysmal extreme pain disorder (Waxman, S G Neurology. 7; 69(6): 505-7 (2007)). Congruent with this observation is the report that the non-selective sodium channel blockers lidocaine and mexiletine can provide symptomatic relief in cases of familial erythermalgia (Legroux-Crepel et al., Ann. Dermatol Venereol., 130: 429-433) and carbamazepine is effective in reducing the number and severity of attacks in PEPD (Fertleman et al, Neuron.; 52(5):767-74 (2006). Further evidence of the role of Nav1.7 in pain is found in the phenotype of loss of function mutations of the SCN9A gene. Cox and colleagues (Nature, 444(7121):894-8 (2006)) were the first to report an association between loss-of-function mutations of SNC9A and congenital indifference to pain (CIP), a rare autosomal recessive disorder characterized by a complete indifference or insensitivity to painful stimuli. Subsequent studies have revealed a number of different mutations that result in a loss of function of the SCN9A gene and the CIP phenotype (Goldberg et al, Clin Genet.; 71(4): 311-9 (2007), Ahmad et al, Hum Mol. Genet. 1; 16(17): 2114-21 (2007)).
Nav 1.7 inhibitors are therefore potentially useful in the treatment of a wide range of disorders, particularly pain, including: acute pain; chronic pain; neuropathic pain; inflammatory pain; visceral pain; nociceptive pain including post-surgical pain; and mixed pain types involving the viscera, gastrointestinal tract, cranial structures, musculoskeletal system, spine, urogenital system, cardiovascular system and CNS, including cancer pain, back and orofacial pain.
Certain inhibitors of voltage gated sodium channels useful in the treatment of pain are known. Thus WO-A-2005/013914 discloses heteroarylamino sulfonylphenyl derivatives, WO-A-2008/118758 aryl sulphonamides and WO-A-2009/012242 N-thiazolyl benzenesulfonamides.
There is, however, an ongoing need to provide new Nav1.7 inhibitors that are good drug candidates.
Preferably compounds are selective Nav1.7 channel inhibitors. That is, preferred compounds show an affinity for the Nav1.7 channel over other Nav channels. In particular, they should show an affinity for the Nav1.7 channel which is greater than their affinity for Nav1.5 channels. Advantageously, compounds should show little or no affinity for the Nav1.5 channel.
Selectivity for the Nav1.7 channel over Nav1.5 may potentially lead to one or more improvements in side-effect profile. Without wishing to be bound by theory, such selectivity is thought to reduce any cardiovascular side effects which may be associated with affinity for the Nav1.5 channel. Preferably compounds demonstrate a selectivity of 10-fold, more preferably 30-fold, most preferably 100-fold, for the Nav 1.7 channel when compared to their selectivity for the Nav1.5 channel whilst maintaining good potency for the Nav1.7 channel.
Furthermore, preferred compounds should have one or more of the following properties: be well absorbed from the gastrointestinal tract; be metabolically stable; have a good metabolic profile, in particular with respect to the toxicity or allergenicity of any metabolites formed; or possess favourable pharmacokinetic properties whilst still retaining their activity profile as Nav1.7 channel inhibitors. They should be non-toxic and demonstrate few side-effects. Ideal drug candidates should exist in a physical form that is stable, non-hygroscopic and easily formulated.
We have now found new sulphonamide Nav1.7 inhibitors.
According to a first aspect of the invention there is provided a compound of formula (I)
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising (a) one or two nitrogen atoms or, when 5-membered, (b) one or two nitrogen atoms and one sulphur atom, said heteroaryl being optionally substituted on a ring carbon atom by F or Cl;
R2, R3 and R4 are independently H, F, Cl or —OCH3;
R5 is CN, F, Cl or R6;
Ra is (a) phenyl optionally substituted by one to three substituents independently selected from F, Cl, CN, H2N(C1-C4)alkylene-, (C1-C4)alkylNH(C1-C4)alkylene-(C3-C8)cycloalkyl or R6; or (b) a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, said heteroaryl being optionally substituted by R7 or R8, or both R7 and R8;
Rb is H, F, Cl, CN or R6;
R6 is (C1-C4)alkyl or (C1-C4)alkyloxy, each optionally substituted by, valency permitting, one to eight F;
Z1 is (a) phenyl, optionally substituted by one to three substituents independently selected from F, Cl or R6; or (b) a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, said heteroaryl being optionally substituted by R7 or R8, or both R7 and R8;
R7 is attached to a Z1 ring carbon and is selected from F, Cl, NR9R10, R6, (C3-C8)cycloalkyl or Het1;
R8 is attached to a Z1 ring nitrogen and is selected from (a) (C1-C4)alkyl or (C3-C8)cycloalkyl, each optionally substituted by, valency permitting, one to three F; or (b) ‘C-linked’ Het1;
Het1 is a 3- to 8-membered saturated monoheterocycloalkyl comprising one or two ring members selected from —NR11— and —O—, said monoheterocycloalkyl being optionally substituted on a ring carbon atom by one to three substituents independently selected from F, (C1-C6)alkyl, (C1-C4)alkyloxy(C0-C4)alkylene and (C3-C8)cycloalkyl; and
R9, R10 and R11 are independently selected from H, (C1-C6)alkyl or (C3-C8)cycloalkyl; or, when Het1 is ‘N-linked’, R11 is absent from that nitrogen atom.
Described below are a number of embodiments (E) of this first aspect of the invention, where for convenience E1 is identical thereto.
E1 A compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof.
E2 A compound according to E1 of the following formula
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising (a) one or two nitrogen atoms or, when 5-membered, (b) one or two nitrogen atoms and one sulphur atom, said heteroaryl being optionally substituted on a ring carbon atom by F or Cl;
R2, R3 and R4 are independently H, F, Cl or —OCH3;
R5 is CN, F, Cl or R6;
Ra is phenyl optionally substituted by one to three substituents independently selected from F, Cl or R6;
Rb is H, F, Cl or R6;
R6 is (C1-C4)alkyl or (C1-C4)alkyloxy, each optionally sustituted by one to three F;
Z1 is (a) phenyl, optionally substituted by one to three substituents independently selected from F, Cl or R6; or (b) a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, said heteroaryl being optionally substituted by R7 or R8, or both R7 and R8;
R7 is attached to a Z1 ring carbon and is selected from F, Cl, NR9R10, R6, (C3-C8)cycloalkyl or Het1;
R8 is attached to a Z1 ring nitrogen and is from (C1-C4)alkyl, (C3-C8)cycloalkyl or ‘C-linked’ Het1;
Het1 is a 3- to 8-membered saturated monoheterocycloalkyl comprising one or two ring members selected from —NR11— and —O—, said monoheterocycloalkyl being optionally substituted on a ring carbon atom by one to three substituents independently selected from F, (C1-C6)alkyl, (C1-C4)alkyloxy(C0-C4)alkylene and (C3-C8)cycloalkyl; and
R9, R19 and R11 are independently selected from H, (C1-C6)alkyl or (C3-C8)cycloalkyl; or, when Het1 is ‘N-linked’, R11 is absent from that nitrogen atom.
E3 A compound according to E1 or E2 wherein R1 is a ‘C-linked’ heteroaryl selected from thiazolyl, thiadiazolyl, pyridazinyl or pyrimidinyl, said heteroaryl being optionally substituted on a ring carbon atom by F or Cl.
E4 A compound according to any of E1 to E3 wherein R1 is a ‘C-linked’ heteroaryl selected from thiazolyl or thiadiazolyl, said heteroaryl being optionally substituted by on a ring carbon atom F.
E5 A compound according to any of E1 to E4 wherein R1 is ‘C-linked’ thiadiazolyl, such as ‘C-linked’ 1,3,4-thiadiazolyl.
E6 A compound according to any of E1 to E5 wherein R2, R3 and R4 are independently H or F.
E7 A compound according to any of E1 to E6 wherein R2, is H or F; and R3 and R4 are both H.
E8 A compound according to any of E1 to E7 wherein R5 is CN, F or Cl.
E9 A compound according to any of E1 to E8 wherein R5 is CN or Cl.
E10 A compound according to any of E1 to E9 wherein Ra is phenyl, optionally substituted by R6.
E11 A compound according to any of E1 to E10 wherein Ra is phenyl optionally substituted by Cl or CF3.
E12 A compound according to any of E1 to E11 wherein Rb is H.
E13 A compound according to any of E1 to E12 wherein Z1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, said heteroaryl being optionally substituted by R7 or R8, or both R7 and R8.
E14 A compound according to any of E1 to E13 wherein Z1 is a ‘C-linked’ heteroaryl selected from pyrazolyl and pyridazinyl, said heteroaryl being optionally substituted by R7 or R8, or both R7 and R8.
E15 A compound according to any of E1 to E13 wherein Z1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, said heteroaryl being optionally substituted by R8.
E16 A compound according to any of E1 to E13 or E15 wherein Z1 is a ‘C-linked’ 5- or 6-membered heteroaryl comprising one or two nitrogen atoms, said heteroaryl being optionally substituted by methyl or a ‘C-linked’ 3- to 6-membered saturated monoheterocycloalkyl comprising one or two ring members selected from NH or —N(C1-C4)alkyl.
E17 A compound according to any of E1 to E14 wherein Z1 is a ‘C-linked’ heteroaryl selected from pyrazolyl and pyridazinyl, said heteroaryl being optionally substituted by R8.
E18 A compound according to any of E1 to E14 or E17 wherein Z1 is a ‘C-linked’ heteroaryl selected from pyrazolyl and pyridazinyl, said heteroaryl being optionally substituted by methyl or a ‘C-linked’ 3- to 6-membered saturated monoheterocycloalkyl comprising one or two ring members selected from NH or —N(C1-C4)alkyl.
E19 A compound according to any of E1 to E14 or E17 to E18 wherein Z1 is a ‘C-linked’ pyridazinyl or ‘C-linked’ pyrazolyl, said pyrazolyl being optionally substituted by methyl or a ‘C-linked’ 3- to 4-membered saturated monoheterocycloalkyl comprising one —N(C1-C2)alkyl ring member.
E20 A compound according to E1 which is the compound of any one of:
Examples 1 to 20;
4-{[3′-(aminomethyl)-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-3-cyano-N-(1,3-thiazol-2-yl)benzenesulfonamide;
Example 22;
3-cyano-4-({2′-[(methylamino)methyl]-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl}oxy)-N-(1,3-thiazol-2-yl)benzenesulfonamide;
Examples 24 to 33;
5-Chloro-2-fluoro-4-({3-[2-(piperazin-1-yl)pyridin-4-yl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide; or
Examples 35 to 40;
or a pharmaceutically acceptable salt thereof.
Alkyl, alkylene, and alkoxy groups, containing the requisite number of carbon atoms, can be unbranched or branched. Examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl and t-butyl. Examples of alkoxy include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy and t-butoxy. Examples of alkylene include methylene, 1,1-ethylene, 1,2-ethylene, 1,1-propylene, 1,2-propylene, 1,3-propylene and 2,2-propylene.
Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
Halo means fluoro, chloro, bromo or iodo.
The term ‘C-linked’ used in the definitions of formula (I) means that the group in question is joined via a ring carbon. The term ‘N-linked’ used in the definitions of formula (I) means that the group in question is joined via a ring nitrogen.
Specific examples of 5- or 6-membered heteroaryl used in the definitions of formula (I) include pyrrolyl, pyrazolyl, imidazoyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl. Except as expressly defined above, when such heteroaryls are substituted, the substituent may be located on a ring carbon (in all cases) or a ring nitrogen with appropriate valency (if the substituent is joined through a carbon atom).
Specific examples of Het1 include oxiranyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, azepanyl, oxepanyl, oxazepanyl and diazepinyl.
Hereinafter, all references to compounds of the invention include compounds of formula (I) or pharmaceutically acceptable salts, solvates, or multi-component complexes thereof, or pharmaceutically acceptable solvates or multi-component complexes of pharmaceutically acceptable salts of compounds of formula (I), as discussed in more detail below.
Preferred compounds of the invention are compounds of formula (I) or pharmaceutically acceptable salts thereof.
Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.
Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
The skilled person will appreciate that the aforementioned salts include ones wherein the counterion is optically active, for example d-lactate or l-lysine, or racemic, for example dl-tartrate or dl-arginine.
For a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
Pharmaceutically acceptable salts of compounds of formula (I) may be prepared by one or more of three methods:
(i) by reacting the compound of formula (I) with the desired acid or base;
(ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of formula (I) using the desired acid or base; or
(iii) by converting one salt of the compound of formula (I) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
The compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in both unsolvated and solvated forms. The term ‘solvate’ is used herein to describe a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D2O, d6-acetone and d6-DMSO.
A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates—see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995), incorporated herein by reference. Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.
When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.
The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term ‘amorphous’ refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterised by a change of state, typically second order (glass transition'). The term ‘crystalline’ refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterised by a phase change, typically first order (‘melting point’).
Also included within the scope of the invention are multi-component complexes (other than salts and solvates) of compounds of formula (I) or pharmaceutically acceptable salts thereof wherein the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallisation, by recrystallisation from solvents, or by physically grinding the components together—see Chem Commun, 17, 1889-1896, by O. Almarsson and M. J. Zaworotko (2004), incorporated herein by reference. For a general review of multi-component complexes, see J Pharm Sci, 64 (8), 1269-1288, by Haleblian (August 1975), incorporated herein by reference.
The compounds of the invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described as ‘thermotropic’ and that resulting from the addition of a second component, such as water or another solvent, is described as ‘lyotropic’. Compounds that have the potential to form lyotropic mesophases are described as ‘amphiphilic’ and consist of molecules which possess an ionic (such as —COO−Na+, —COO−K+, or —SO3−Na+) or non-ionic (such as —N−N+(CH3)3) polar head group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and A. Stuart, 4th Edition (Edward Arnold, 1970), incorporated herein by reference.
The compounds of the invention may be administered as prodrugs. Thus certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as ‘prodrugs’. Further information on the use of prodrugs may be found in ‘Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and ‘Bioreversible Carriers in Drug Design’, Pergamon Press, 1987 (ed. E B Roche, American Pharmaceutical Association).
Prodrugs can, for example, be produced by replacing appropriate functionalities present in a compound of formula (I) with certain moieties known to those skilled in the art as ‘pro-moieties’ as described, for example, in “Design of Prodrugs” by H Bundgaard (Elsevier, 1985).
Examples of prodrugs include phosphate prodrugs, such as dihydrogen or dialkyl (e.g. di-tert-butyl) phosphate prodrugs. Further examples of replacement groups in accordance with the foregoing examples and examples of other prodrug types may be found in the aforementioned references.
Also included within the scope of the invention are metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites in accordance with the invention include, where the compound of formula (I) contains a phenyl (Ph) moiety, a phenol derivative thereof (-Ph>-PhOH);
Compounds of the invention containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Included within the scope of the invention are all stereoisomers of the compounds of the invention and mixtures of one or more thereof.
Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.
Mixtures of stereoisomers may be separated by conventional techniques known to those skilled in the art; see, for example, “Stereochemistry of Organic Compounds” by E. L. Eliel and S. H. Wilen (Wiley, New York, 1994.
The scope of the invention includes all crystal forms of the compounds of the invention, including racemates and racemic mixtures (conglomerates) thereof. Stereoisomeric conglomerates may also be separated by the conventional techniques described herein just above.
The scope of the invention includes all pharmaceutically acceptable isotopically-labelled compounds of the invention wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.
Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H and 3H, carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123I and 125I, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, and sulphur, such as 35S.
Certain isotopically-labelled compounds of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e. 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
Also within the scope of the invention are intermediate compounds as hereinafter defined, all salts, solvates and complexes thereof and all solvates and complexes of salts thereof as defined hereinbefore for compounds of formula (I). The invention includes all polymorphs of the aforementioned species and crystal habits thereof.
When preparing a compound of formula (I) in accordance with the invention, a person skilled in the art may routinely select the form of intermediate which provides the best combination of features for this purpose. Such features include the melting point, solubility, processability and yield of the intermediate form and the resulting ease with which the product may be purified on isolation.
The compounds of the invention may be prepared by any method known in the art for the preparation of compounds of analogous structure. In particular, the compounds of the invention can be prepared by the procedures described by reference to the Schemes that follow, or by the specific methods described in the Examples, or by similar processes to either.
The skilled person will appreciate that the experimental conditions set forth in the schemes that follow are illustrative of suitable conditions for effecting the transformations shown, and that it may be necessary or desirable to vary the precise conditions employed for the preparation of compounds of formula (I). It will be further appreciated that it may be necessary or desirable to carry out the transformations in a different order from that described in the schemes, or to modify one or more of the transformations, to provide the desired compound of the invention.
In addition, the skilled person will appreciate that it may be necessary or desirable at any stage in the synthesis of compounds of the invention to protect one or more sensitive groups, so as to prevent undesirable side reactions. In particular, it may be necessary or desirable to protect amino groups. The protecting groups used in the preparation of the compounds of the invention may be used in conventional manner. See, for example, those described in ‘Greene's Protective Groups in Organic Synthesis’ by Theodora W Greene and Peter G M Wuts, fourth edition, (John Wiley and Sons, 2006), in particular chapter 7 (“Protection for the Amino Group”), incorporated herein by reference, which also describes methods for the removal of such groups.
In the following general methods, R1, R2, R3, R4, R5, Ra, Rb and Z1 are as previously defined for a derivative of the formula (I) unless otherwise stated. Pg1 is a suitable amino protecting group, such as dimethoxybenzyl, methoxymethyl or ethoxyethyl. Pg2 is H or is a suitable hydroxy protecting group, such as methoxy or benzyl. Lg is a suitable leaving group, such as halo (e.g. Br) or a sulphonate ester (e.g mesylate). M is an optionally substituted/ligated metal or boron group suitable for cross coupling reactions, such as trialkylstannane, dihydroxyborane, dialkoxyborane or halozinc.
Where ratios of solvents are given, the ratios are by volume.
According to a first process, compounds of formula (I) may be prepared by the process illustrated in Scheme 1.
Compounds of formula (I) can be prepared from compounds of formula (II) according to reaction step (v) by deprotection methods under acidic conditions. Suitable acids include HCl, formic acid or trifluoroacetic acid. Preferred methods comprise HCl in 1,4-dioxane at room temperature.
Compounds of formula (II) can be prepared from compounds of formula (IV) according to reaction step (iv) by nucleophilic aromatic substitution reaction with a phenol of formula (III) under basic reaction conditions. Suitable conditions include potassium carbonate in DMF or DMSO, sodium hydride in NMP or DMF, sodium hydroxide or potassium hydroxide in 1,4-dioxane and water or DMSO or potassium tert-butoxide in THF at from room temperature to 150° C. Preferred conditions comprise 2 equivalents of potassium carbonate in DMSO at room temperature.
Compounds of formula (III) are either commercially available or can be prepared according to Schemes 5 and 6.
Compounds of formula (IV) can be prepared from compounds of formula (VI) according to reaction step (iii) by displacement of a sulfonyl chloride with compounds of formula (VII) under basic reaction conditions. Typical conditions comprise lithium hexamethyldisilazane in THF at −78° C.
Alternatively compounds of formula (IV) can be prepared from compounds of formula (V) according to reaction step (ii) by introduction of protecting group Pg1, such as dimethoxybenzyl or methoxymethyl, under basic reaction conditions or Mitsunobu conditions. Preferred conditions comprise N,N-diisopropylethylamine in dichloromethane at room temperature.
Compounds of formula (V) can be prepared from compounds of formula (VI) according to reaction step (i) by displacement of a sulfonyl chloride under basic reaction conditions with compounds of formula (IX), for example lithium hexamethyldisilazane, diazabicyclo(2.2.2)octane, triethylamine, NaOH or pyridine. Preferred conditions comprise NaOH in 1,4-dioxane and water at room temperature.
Compounds of formula (VII) can be prepared from compounds of formula (VIII) according to reaction step (vi) by Curtius rearrangement through generation of an acyl azide using diphenylphosphoryl azide.
Alternatively compounds of formula (VII) may be prepared from compounds of formula (IX) according to reaction step (vii) through the processes outlined for reaction step (ii) or by reductive amination with an aldehyde. Typical reaction conditions comprise dimethoxybenzaldehyde in toluene at 110° C. followed by reduction with sodium borohydride.
Alternatively compounds of formula (VII) may be prepared from compounds of formula (X) according to reaction step (viii) by nucleophilic aromatic substitution reaction on compounds of formula (XI). Typical reaction conditions comprise triethylamine in ethanol under microwave irradiation at 120° C. for 15 minutes.
According to a second process, compounds of formula (I) may be prepared by the process illustrated in Scheme 2.
Compounds of formula (I) can be prepared from compounds of formula (V) by nucleophilic aromatic substitution reaction with compounds of formula (III) according to process step (iv), under conditions described above for Scheme 1 step (iv). Preferred conditions comprise potassium carbonate in dimethylformamide at 80-100° C.
According to a third process, compounds of formula (I) may be prepared by the process illustrated in Scheme 3.
Compounds of formula (I) can be prepared from compounds of formula (XV) by reaction according to process step (i) by displacement of a sulfonyl chloride with compounds of formula (IX) under basic reaction conditions, such as those described above for Scheme 1 step (i).
Alternatively compounds of formula (I) can be prepared from compounds of formula (XV) by reaction according to process step (iii) by displacement of a sulfonyl chloride under basic reaction conditions with compounds of formula (VII) to yield compounds of formula (XVa), followed by a deprotection according to step (v) under conditions described above for Scheme 1 step (v).
Compounds of formula (XV) can be prepared from compounds of formula (XIV) according to process step (x) by a Sandmeyer reaction. Typical conditions comprise sodium nitrite in HCl, acetic acid and water, followed by sulfur dioxide in acetic acid with copper chloride at 0° C.
Compounds of formula (XIV) can be prepared from compounds of formula (XIII) by a reduction reaction according to process step (ix), for example hydrogenation, a suitable metal reduction or use of sodium dithionite. Preferred conditions comprise calcium chloride in the presence of iron in ethanol/water.
Compounds of formula (XIII) can be prepared from compounds of formula (XII) by nucleophilic aromatic substitution reaction with compounds of formula (III) according to process step (iv), as described above for Scheme 1 step (iv). Preferred conditions comprise potassium carbonate in dimethylformamide at 0° C.
According to a fourth process, compounds of formula (I) may be prepared by the process illustrated in Scheme 4.
Compounds of formula (I) may be prepared from compounds of formulae (XVI) and (XVII) according to process step (iv) followed by process step (v), under conditions described in Scheme 1 steps (iv) and (v).
Compounds of formula (XVII) can be prepared from compounds of formula (IV) according to process step (xi) by nucleophilic aromatic substitution reaction under basic conditions. Preferred conditions comprise potassium tert-butoxide in THF followed by a suitable acid deprotection such as HCl in dioxane, or trimethylsilylethanol and potassium carbonate in DMSO at room temperature.
According to a fifth process, compounds of formula (III) may be prepared by the process illustrated in Scheme 5.
Compounds of formula (III) can be prepared by cross-coupling compounds of formula (XVIII) with compounds formula (XXII) according to process step (xii), followed, as appropriate, by deprotection of any protecting group present according to process step (xiv).
Cross-coupling is conveniently effected in the presence of a suitable catalyst system (e.g. palladium or nickel) and base. Typical Suzuki coupling conditions comprise 1.2-3 equivalents of boronic acid, and 0.01-0.25 equivalents of palladium catalyst with phosphine base ligands in an organic solvent at a temperature of from 50° C. to 100° C. Preferred Suzuki conditions comprise bis(tri-tert-butylphosphine) palladium (0) and potassium carbonate in 1,4-dioxane at 100° C. Alternatively, Stille coupling conditions may be employed. Preferred Stille conditions comprise a trialkylstannane and caesium fluoride in dimethylformamide at 45° C.
Deprotection according to process step (xiv) may be effected, as required, under conventional conditions. Where Pg2 is benzyl, deprotection is conveniently effected by hydrogenation over palladium on carbon.
Compounds of formula (XVIII) can be prepared from compounds of formula (XIX) according to process step (xiii) by an electrophilic halogenation reaction. Preferred conditions comprise N-iodosuccinimide in acetic acid at 0° C.
Compounds of formula (XIX) can be prepared from compounds of formula (XX) according to process step (xii) by cross-coupling reaction with compounds for formula (XXI) under conditions described above in step (xii).
According to a sixth process, compounds of formula (III) may be prepared by the process illustrated in Scheme 6.
Compounds of formula (III) can be prepared from compounds of formulae (XXIII) and (XXIV) according to process step (xii) under conditions described for Scheme 5 step (xii) followed, as required, by deprotection under conventional conditions according to process step (xiv). Where Pg2 is benzyl, deprotection is conveniently effected by hydrogenation over palladium on carbon.
According to a seventh process, compounds of formula (I) wherein Z1 is a C-linked 5-membered heteroaryl comprising two nitrogen atoms optionally substituted by R8 may be prepared by the process illustrated in Scheme 7.
Compounds of formula (I) can be prepared from compounds of formula (XXXI) according to process step (v) by a suitable deprotection under conditions described in Scheme 1 step (v).
Compounds of formula (XXXI) can be prepared from compounds of formula (XXVIII) according to process step (xvi) by cyclisation with compounds of formula (XXX) or hydrazine. Preferred conditions comprise heating to 70° C. in ethanol for 3 hours.
Compounds of formula (XXX) can be prepared from compounds of formula (XXIX) according to process step (xvii) by bimolecular nucleophilic substitution displacement of a mesylate of formula (XXIX) with hydrazine. Preferred conditions comprise heating the mesylate of formula (XXIX) in neat hydrazine at 95° C. for 18 hours.
Compounds of formula (XXVIII) can be prepared from compounds of formulae (IV) and (XXVII) according to reaction step (iv) under conditions described in Scheme 1 step (iv).
Compounds of formula (XXVII) can be prepared from compounds of formula (XXVI) according to reaction step (xv) by reaction with N,N-dimethylformamide dimethylacetal. Preferred conditions comprise N,N-dimethylformamide dimethylacetal in iso-propyl alcohol at 45° C.
Compounds of formula (XXVI) can be prepared from compounds of formulae (XXI) and (XXV) according to process step (xii) under conditions described for Scheme 5 step (xii). Preferred conditions comprise palladium tetrakis triphenyl phosphine and potassium carbonate in 1,4-dioxane and water at 60° C.
Compounds of formulae (III), (VI), (VIII), (IX), (X), (XI), (XII), (XVI), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV) and (XXIX) are either commercially available, known from the literature, easily prepared by methods well known to those skilled in the art, or can be made according to preparations described herein.
All new processes for preparing compounds of formula (I), and corresponding new intermediates employed in such processes, form further aspects of the present invention.
Compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products or may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.
They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term ‘excipient’ is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
In another aspect the invention provides a pharmaceutical composition comprising a compound of the invention together with one or more pharmaceutically acceptable excipients.
Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in “Remington's Pharmaceutical Sciences”, 19th Edition (Mack Publishing Company, 1995).
Suitable modes of administration include oral, parenteral, topical, inhaled/intranasal, rectal/intravaginal, and ocular/aural administration.
Formulations suitable for the aforementioned modes of administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays, liquid formulations and buccal/mucoadhesive patches.
Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
The compounds of the invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986, by Liang and Chen (2001).
For tablet dosage forms, depending on dose, the drug may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmel lose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.
Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.
Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet. Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.
Exemplary tablets contain up to about 80% drug, from about 10 weight % to about 90 weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant. Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of tablets is discussed in “Pharmaceutical Dosage Forms: Tablets”, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).
Suitable modified release formulations for the purposes of the invention are described in U.S. Pat. No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in “Pharmaceutical Technology On-line”, 25(2), 1-14, by Verma et al (2001). The use of chewing gum to achieve controlled release is described in WO 00/35298.
The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
The solubility of compounds of formula (I) used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microspheres.
The compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated—see, for example, J Pharm Sci, 88 (10), 955-958, by Finnin and Morgan (October 1999).
Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.
The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
The pressurised container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.
Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from 1 μg to 20 mg of the compound of the invention per actuation and the actuation volume may vary from 1 μl to 100 μl. A typical formulation may comprise a compound of formula (I), propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.
In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or “puff” containing from 1 μg to 100 mg of the compound of formula (I). The overall daily dose will typically be in the range 1 μg to 200 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, microbicide, vaginal ring or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.
The compounds of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.
The compounds of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.
Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.
For administration to human patients, the total daily dose of the compounds of the invention is typically in the range 1 mg to 10 g, such as 10 mg to 1 g, for example 25 mg to 500 mg depending, of course, on the mode of administration and efficacy. For example, oral administration may require a total daily dose of from 50 mg to 100 mg. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. These dosages are based on an average human subject having a weight of about 60 kg to 70 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.
As noted above, the compounds of the invention are useful because they exhibit pharmacological activity in animals, i.e., Nav1.7 channel inhibition. More particularly, the compounds of the invention are of use in the treatment of disorders for which a Nav1.7 inhibitor is indicated. Preferably the animal is a mammal, more preferably a human.
In a further aspect of the invention there is provided a compound of the invention for use as a medicament.
In a further aspect of the invention there is provided a compound of the invention for the treatment of a disorder for which a Nav1.7 inhibitor is indicated.
In a further aspect of the invention there is provided use of a compound of the invention for the preparation of a medicament for the treatment of a disorder for which a Nav1.7 inhibitor is indicated.
In a further aspect of the invention there is provided a method of treating a disorder in an animal (preferably a mammal, more preferably a human) for which a Nav1.7 inhibitor is indicated, comprising administering to said animal a therapeutically effective amount of a compound of the invention.
Disorders for which a Nav1.7 inhibitor is indicated include pain, particularly neuropathic, nociceptive and inflammatory pain.
Physiological pain is an important protective mechanism designed to warn of danger from potentially injurious stimuli from the external environment. The system operates through a specific set of primary sensory neurones and is activated by noxious stimuli via peripheral transducing mechanisms (see Millan, 1999, Prog. Neurobiol., 57, 1-164 for a review). These sensory fibres are known as nociceptors and are characteristically small diameter axons with slow conduction velocities. Nociceptors encode the intensity, duration and quality of noxious stimulus and by virtue of their topographically organised projection to the spinal cord, the location of the stimulus. The nociceptors are found on nociceptive nerve fibres of which there are two main types, A-delta fibres (myelinated) and C fibres (non-myelinated). The activity generated by nociceptor input is transferred, after complex processing in the dorsal horn, either directly, or via brain stem relay nuclei, to the ventrobasal thalamus and then on to the cortex, where the sensation of pain is generated.
Pain may generally be classified as acute or chronic. Acute pain begins suddenly and is short-lived (usually twelve weeks or less). It is usually associated with a specific cause such as a specific injury and is often sharp and severe. It is the kind of pain that can occur after specific injuries resulting from surgery, dental work, a strain or a sprain. Acute pain does not generally result in any persistent psychological response. In contrast, chronic pain is long-term pain, typically persisting for more than three months and leading to significant psychological and emotional problems. Common examples of chronic pain are neuropathic pain (e.g. painful diabetic neuropathy, postherpetic neuralgia), carpal tunnel syndrome, back pain, headache, cancer pain, arthritic pain and chronic post-surgical pain.
When a substantial injury occurs to body tissue, via disease or trauma, the characteristics of nociceptor activation are altered and there is sensitisation in the periphery, locally around the injury and centrally where the nociceptors terminate. These effects lead to a hightened sensation of pain. In acute pain these mechanisms can be useful, in promoting protective behaviours which may better enable repair processes to take place. The normal expectation would be that sensitivity returns to normal once the injury has healed. However, in many chronic pain states, the hypersensitivity far outlasts the healing process and is often due to nervous system injury. This injury often leads to abnormalities in sensory nerve fibres associated with maladaptation and aberrant activity (Woolf & Salter, 2000, Science, 288, 1765-1768).
Clinical pain is present when discomfort and abnormal sensitivity feature among the patient's symptoms. Patients tend to be quite heterogeneous and may present with various pain symptoms. Such symptoms include: 1) spontaneous pain which may be dull, burning, or stabbing; 2) exaggerated pain responses to noxious stimuli (hyperalgesia); and 3) pain produced by normally innocuous stimuli (allodynia—Meyer et al., 1994, Textbook of Pain, 13-44). Although patients suffering from various forms of acute and chronic pain may have similar symptoms, the underlying mechanisms may be different and may, therefore, require different treatment strategies. Pain can also therefore be divided into a number of different subtypes according to differing pathophysiology, including nociceptive, inflammatory and neuropathic pain.
Nociceptive pain is induced by tissue injury or by intense stimuli with the potential to cause injury. Pain afferents are activated by transduction of stimuli by nociceptors at the site of injury and activate neurons in the spinal cord at the level of their termination. This is then relayed up the spinal tracts to the brain where pain is perceived (Meyer et al., 1994, Textbook of Pain, 13-44). The activation of nociceptors activates two types of afferent nerve fibres. Myelinated A-delta fibres transmit rapidly and are responsible for sharp and stabbing pain sensations, whilst unmyelinated C fibres transmit at a slower rate and convey a dull or aching pain. Moderate to severe acute nociceptive pain is a prominent feature of pain from central nervous system trauma, strains/sprains, burns, myocardial infarction and acute pancreatitis, post-operative pain (pain following any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and back pain. Cancer pain may be chronic pain such as tumour related pain (e.g. bone pain, headache, facial pain or visceral pain) or pain associated with cancer therapy (e.g. postchemotherapy syndrome, chronic postsurgical pain syndrome or post radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormonal therapy or radiotherapy. Back pain may be due to herniated or ruptured intervertabral discs or abnormalities of the lumber facet joints, sacroiliac joints, paraspinal muscles or the posterior longitudinal ligament. Back pain may resolve naturally but in some patients, where it lasts over 12 weeks, it becomes a chronic condition which can be particularly debilitating.
Neuropathic pain is currently defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. Nerve damage can be caused by trauma and disease and thus the term ‘neuropathic pain’ encompasses many disorders with diverse aetiologies. These include, but are not limited to, peripheral neuropathy, diabetic neuropathy, post herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain and pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy and vitamin deficiency. Neuropathic pain is pathological as it has no protective role. It is often present well after the original cause has dissipated, commonly lasting for years, significantly decreasing a patient's quality of life (Woolf and Mannion, 1999, Lancet, 353, 1959-1964). The symptoms of neuropathic pain are difficult to treat, as they are often heterogeneous even between patients with the same disease (Woolf & Decosterd, 1999, Pain Supp., 6, S141-S147; Woolf and Mannion, 1999, Lancet, 353, 1959-1964). They include spontaneous pain, which can be continuous, and paroxysmal or abnormal evoked pain, such as hyperalgesia (increased sensitivity to a noxious stimulus) and allodynia (sensitivity to a normally innocuous stimulus).
The inflammatory process is a complex series of biochemical and cellular events, activated in response to tissue injury or the presence of foreign substances, which results in swelling and pain (Levine and Taiwo, 1994, Textbook of Pain, 45-56). Arthritic pain is the most common inflammatory pain. Rheumatoid disease is one of the commonest chronic inflammatory conditions in developed countries and rheumatoid arthritis is a common cause of disability. The exact aetiology of rheumatoid arthritis is unknown, but current hypotheses suggest that both genetic and microbiological factors may be important (Grennan & Jayson, 1994, Textbook of Pain, 397-407). It has been estimated that almost 16 million Americans have symptomatic osteoarthritis (OA) or degenerative joint disease, most of whom are over 60 years of age, and this is expected to increase to 40 million as the age of the population increases, making this a public health problem of enormous magnitude (Houge & Mersfelder, 2002, Ann Pharmacother., 36, 679-686; McCarthy et al., 1994, Textbook of Pain, 387-395). Most patients with osteoarthritis seek medical attention because of the associated pain. Arthritis has a significant impact on psychosocial and physical function and is known to be the leading cause of disability in later life. Ankylosing spondylitis is also a rheumatic disease that causes arthritis of the spine and sacroiliac joints. It varies from intermittent episodes of back pain that occur throughout life to a severe chronic disease that attacks the spine, peripheral joints and other body organs.
Another type of inflammatory pain is visceral pain which includes pain associated with inflammatory bowel disease (IBD). Visceral pain is pain associated with the viscera, which encompass the organs of the abdominal cavity. These organs include the sex organs, spleen and part of the digestive system. Pain associated with the viscera can be divided into digestive visceral pain and non-digestive visceral pain. Commonly encountered gastrointestinal (GI) disorders that cause pain include functional bowel disorder (FBD) and inflammatory bowel disease (IBD). These GI disorders include a wide range of disease states that are currently only moderately controlled, including, in respect of FBD, gastro-esophageal reflux, dyspepsia, irritable bowel syndrome (IBS) and functional abdominal pain syndrome (FAPS), and, in respect of IBD, Crohn's disease, ileitis and ulcerative colitis, all of which regularly produce visceral pain. Other types of visceral pain include the pain associated with dysmenorrhea, cystitis and pancreatitis and pelvic pain.
It should be noted that some types of pain have multiple aetiologies and thus can be classified in more than one area, e.g. back pain and cancer pain have both nociceptive and neuropathic components.
Other types of pain include:
pain resulting from musculo-skeletal disorders, including myalgia, fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism, dystrophinopathy, glycogenolysis, polymyositis and pyomyositis;
heart and vascular pain, including pain caused by angina, myocardical infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma and skeletal muscle ischemia;
head pain, such as migraine (including migraine with aura and migraine without aura), cluster headache, tension-type headache mixed headache and headache associated with vascular disorders;
erythermalgia; and
orofacial pain, including dental pain, otic pain, burning mouth syndrome and temporomandibular myofascial pain.
A Nav1.7 inhibitor may be usefully combined with another pharmacologically active compound, or with two or more other pharmacologically active compounds, particularly in the treatment of pain. Such combinations offer the possibility of significant advantages, including patient compliance, ease of dosing and synergistic activity.
In the combinations that follow the compound of the invention may be administered simultaneously, sequentially or separately in combination with the other therapeutic agent or agents.
A Nav1.7 inhibitor of formula (I), or a pharmaceutically acceptable salt thereof, as defined above, may be administered in combination with one or more agents selected from:
an alternative Nav1.7 channel modulator, such as another compound of the present invention or a compound disclosed in WO 2009/012242;
an alternative sodium channel modulator, such as a Nav1.3 modulator (e.g. as disclosed in WO2008/118758); or a Nav1.8 modulator (e.g. as disclosed in WO 2008/135826, more particularly N-[6-Amino-5-(2-chloro-5-methoxyphenyl)pyridin-2-yl]-1-methyl-1H-pyrazole-5-carboxamide);
an inhibitor of nerve growth factor signaling, such as: an agent that binds to NGF and inhibits NGF biological activity and/or downstream pathway(s) mediated by NGF signaling (e.g. tanezumab), a TrkA antagonist or a p75 antagonist;
a compound which increases the levels of endocannabinoid, such as a compound with fatty acid amid hydrolase inhibitory (FAAH) activity, in particular those disclosed in WO 2008/047229 (e.g. N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridine-2-yl]oxy}benzylidene)piperidene124-1-carboxamide);
an opioid analgesic, e.g. morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine;
a nonsteroidal antiinflammatory drug (NSAID), e.g. aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin or zomepirac;
a barbiturate sedative, e.g. amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, theamylal or thiopental;
a benzodiazepine having a sedative action, e.g. chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam;
an H1 antagonist having a sedative action, e.g. diphenhydramine, pyrilamine, promethazine, chlorpheniramine or chlorcyclizine;
a sedative such as glutethimide, meprobamate, methaqualone or dichloralphenazone;
a skeletal muscle relaxant, e.g. baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine;
an NMDA receptor antagonist, e.g. dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine, EN-3231 (MorphiDex®, a combination formulation of morphine and dextromethorphan), topiramate, neramexane or perzinfotel including an NR2B antagonist, e.g. ifenprodil, traxoprodil or (−)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone;
an alpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, or 4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl) quinazoline;
a tricyclic antidepressant, e.g. desipramine, imipramine, amitriptyline or nortriptyline;
an anticonvulsant, e.g. carbamazepine, lamotrigine, topiratmate or valproate;
a tachykinin (NK) antagonist, particularly an NK-3, NK-2 or NK-1 antagonist, e.g. (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione (TAK-637), 5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine (2S,3S);
a muscarinic antagonist, e.g oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin, solifenacin, temiverine and ipratropium;
a COX-2 selective inhibitor, e.g. celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib;
a coal-tar analgesic, in particular paracetamol;
a neuroleptic such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, sertindole, aripiprazole, sonepiprazole, blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox, asenapine, lurasidone, amisulpride, balaperidone, palindore, eplivanserin, osanetant, rimonabant, meclinertant, Miraxion® or sarizotan;
a vanilloid receptor agonist (e.g. resinferatoxin) or antagonist (e.g. capsazepine);
a beta-adrenergic such as propranolol;
a local anaesthetic such as mexiletine;
a corticosteroid such as dexamethasone;
a 5-HT receptor agonist or antagonist, particularly a 5-HT1B/1D agonist such as eletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan;
a 5-HT2A receptor antagonist such as R(+)-alpha-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL-100907);
a 5-HT3 antagonist, such as ondansetron
a cholinergic (nicotinic) analgesic, such as ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403), (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594) or nicotine;
Tramadol®;
a PDEV inhibitor, such as 5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil), (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]-pyrido[3,4-b]indole-1,4-dione (IC-351 or tadalafil), 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (vardenafil), 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide, 3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide;
an alpha-2-delta ligand such as gabapentin, pregabalin, 3-methylgabapentin, (1α,3α,5α)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, (3S,5R)-3-aminomethyl-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-octanoic acid, (2S,4S)-4-(3-chlorophenoxy)proline, (2S,4S)-4-(3-fluorobenzyl)-proline, [(1R,5R,6S)-6-(aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, 3-(1-aminomethyl-cyclohexylmethyl)-4H-[1,2,4]oxadiazol-5-one, C-[1-(1H-tetrazol-5-ylmethyl)-cycloheptyl]-methylamine, (3S,4S)-(1-aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, (3S,5R)-3-aminomethyl-5-methyl-octanoic acid, (3S,5R)-3-amino-5-methyl-nonanoic acid, (3S,5R)-3-amino-5-methyl-octanoic acid, (3R,4R,5R)-3-amino-4,5-dimethyl-heptanoic acid and (3R,4R,5R)-3-amino-4,5-dimethyl-octanoic acid;
metabotropic glutamate subtype 1 receptor (mGluR1) antagonist;
a serotonin reuptake inhibitor such as sertraline, sertraline metabolite demethylsertraline, fluoxetine, norfluoxetine (fluoxetine desmethyl metabolite), fluvoxamine, paroxetine, citalopram, citalopram metabolite desmethylcitalopram, escitalopram, d,l-fenfluramine, femoxetine, ifoxetine, cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine and trazodone;
a noradrenaline (norepinephrine) reuptake inhibitor, such as maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, buproprion metabolite hydroxybuproprion, nomifensine and viloxazine (Vivalan®), especially a selective noradrenaline reuptake inhibitor such as reboxetine, in particular (S,S)-reboxetine;
a dual serotonin-noradrenaline reuptake inhibitor, such as venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine, clomipramine, clomipramine metabolite desmethylclomipramine, duloxetine, milnacipran and imipramine;
an inducible nitric oxide synthase (iNOS) inhibitor such as S-[2-[(1-iminoethyl)amino]ethyl]-homocysteine, S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine, S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine, (2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile; 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile, (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3 pyridinecarbonitrile, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile, N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine, or guanidinoethyldisulfide;
an acetylcholinesterase inhibitor such as donepezil;
a prostaglandin E2 subtype 4 (EP4) antagonist such as N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamide or 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoic acid;
a microsomal prostaglandin E synthase type 1 (mPGES-1) inhibitor;
a leukotriene B4 antagonist; such as 1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic acid (CP-105696), 5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valeric acid (ONO-4057) or DPC-11870, and
a 5-lipoxygenase inhibitor, such as zileuton, 6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxy-methyl]-1-methyl-2-quinolone (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),1,4-benzoquinone (CV-6504).
There is also included within the scope the present invention combinations of a compound of the invention together with one or more additional therapeutic agents which slow down the rate of metabolism of the compound of the invention, thereby leading to increased exposure in patients. Increasing the exposure in such a manner is known as boosting. This has the benefit of increasing the efficacy of the compound of the invention or reducing the dose required to achieve the same efficacy as an unboosted dose. The metabolism of the compounds of the invention includes oxidative processes carried out by P450 (CYP450) enzymes, particularly CYP 3A4 and conjugation by UDP glucuronosyl transferase and sulphating enzymes. Thus, among the agents that may be used to increase the exposure of a patient to a compound of the present invention are those that can act as inhibitors of at least one isoform of the cytochrome P450 (CYP450) enzymes. The isoforms of CYP450 that may be beneficially inhibited include, but are not limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19 and CYP3A4. Suitable agents that may be used to inhibit CYP 3A4 include ritonavir, saquinavir, ketoconazole, N-(3,4-difluorobenzyl)-N-methyl-2-{[(4-methoxypyridin-3-yl)amino]sulfonyl}benzamide and N-(1-(2-(5-(4-fluorobenzyl)-3-(pyridin-4-yl)-1H-pyrazol-1-yl)acetyl)piperidin-4-yl)methanesulfonamide.
It is within the scope of the invention that two or more pharmaceutical compositions, at least one of which contains a compound of the invention, may conveniently be combined in the form of a kit suitable for coadministration of the compositions. Thus the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like. The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically comprises directions for administration and may be provided with a so-called memory aid.
In another aspect the invention provides a pharmaceutical product (such as in the form of a kit) comprising a compound of the invention together with one or more additional therapeutically active agents as a combined preparation for simultaneous, separate or sequential use in the treatment of a disorder for which a Nav1.7 inhibitor is indicated.
It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.
In the non-limiting Examples and Preparations that are set out later in the description, and in the aforementioned Schemes, the following the abbreviations, definitions and analytical procedures may be referred to:
AcOH is acetic acid,
Cs2CO3 is caesium carbonate;
Cu(acac)2 is copper (II) acetylacetonate;
CuI is copper (I) iodide;
Cu(OAc)2 is copper (II) acetate;
DAD is diode array detector;
DCM is dichloromethane; methylene chloride;
DIPEA is N-ethyldiisopropylamine, N,N-diisopropylethylamine;
DMAP is 4-dimethylaminopyridine;
DMF is N,N-dimethylformamide;
DMSO is dimethyl sulphoxide;
EDTA is ethylenediaminetetraacetic acid;
ELSD is evaporative light scattering detection;
Et2O is diethyl ether;
EtOAc is ethyl acetate;
EtOH is ethanol;
HCl is hydrochloric acid;
IPA is isopropanol;
Ir2(OMe)2COD2 is bis(1,5-cyclooctadiene)di-μ-methoxydiiridium (I);
K2CO3 is potassium carbonate;
KHSO4 is potassium hydrogen sulphate;
KOAc is potassium acetate;
KOH is potassium hydroxide;
K3PO4 is potassium phosphate tribasic;
LCMS is liquid chromatography mass spectrometry (Rt=retention time)
LiOH is lithium hydroxide;
MeOH is methanol;
MgSO4 is magnesium sulphate;
NaH is sodium hydride;
NaHCO3 is sodium hydrogencarbonate;
Na2CO3 is sodium carbonate;
NaHSO3 is sodium bisulphate;
NaHSO4 is sodium hydrogensulphate;
NaOH is sodium hydroxide;
Na2SO4 is sodium sulphate;
NH4Cl is ammonium chloride;
NMP is N-Methyl-2-pyrrolidone;
Pd/C is palladium on carbon;
Pd(PPh3)4 is palladium tetrakis;
Pd(dppf)2Cl2 is [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane;
THF is tetrahydrofuran;
THP is tetrahydropyran;
TLC is thin layer chromatography; and
WSCDI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
1H Nuclear magnetic resonance (NMR) spectra were in all cases consistent with the proposed structures. Characteristic chemical shifts (δ) are given in parts-per-million downfield from tetramethylsilane using conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The following abbreviations have been used for common solvents: CDCl3, deuterochloroform; d6-DMSO, deuterodimethylsulphoxide; and CD3OD, deuteromethanol.
Mass spectra, MS (m/z), were recorded using either electrospray ionisation (ESI) or atmospheric pressure chemical ionisation (APCI). When relevant, and unless stated otherwise, the m/z data provided are for isotopes 19F, 35Cl and 79Br.
Automated Preparative High Performance Liquid Chromatography (Auto-HPLC)
Certain compounds of the Examples and Preparations were purified using Automated Preparative High Performance Liquid Chromatography (HPLC). Reversed-phase HPLC conditions were either on Fraction Lynx systems or on a Trilution system.
In the case of the Fractionlynx system, Samples were submitted dissolved in 1 mL of DMSO. Depending on the nature of the compounds and the results of a pre-analysis, the purification was performed under either acidic (‘A-HPLC’), or basic (‘B-HPLC’) conditions at ambient temperature. A-HPLC was carried out on a Sunfire Prep C18 OBD column (19×100 mm, 5 μm). B-HPLC was carried out on an Xterra Prep MS C18 (19×100 mm, 5 μm), both from Waters. A flow rate of 18 mL/min was used with mobile phase A: water+0.1% modifier (v/v) and B: acetonitrile+0.1% modifier (v/v). For acidic runs the modifier was formic acid, for basic run the modifier was diethylamine. A Waters 2525 binary LC pump supplied a mobile phase with a composition of 5% B for 1 min then ran from 5% to 98% B over 6 min followed by a 2 min hold at 98% B.
Detection was achieved using a Waters 2487 dual wavelength absorbance detector set at 225 nm followed in series by a Polymer Labs PL-ELS 2100 detector and a Waters ZQ 2000 4 way MUX mass spectrometer in parallel. The PL 2100 ELSD was set at 30° C. with 1.6 L/min supply of Nitrogen. The Waters ZQ MS was tuned with the following parameters:
ES+ Cone voltage: 30 v Capillary: 3.20 kv
ES− Cone voltage: −30 v Capillary: −3.00 kv
Desolvation gas: 600 L/hr
Source Temp: 120° C.
Scan range 150-900 Da
The fraction collection was triggered by both MS and ELSD.
Quality control (QC) analysis was performed using a LCMS method. Acidic runs were carried out on a Sunfire C18 (4.6×50 mm, 5 μm), basic runs were carried out on a Xterra C18 (4.6×50 mm, 5 μm), both from Waters. A flow rate of 1.5 mL/min was used with mobile phase A: water+0.1% modifier (v/v) and B: acetonitrile+0.1% modifier (v/v). For acidic runs the modifier was formic acid, for basic run the modifier was ammonia. A Waters 1525 binary LC pump ran a gradient elution from 5% to 95% B over 3 min followed by a 1 min hold at 95% B. Detection was achieved using a Waters MUX UV 2488 detector set at 225 nm followed in series by a Polymer Labs PL-ELS 2100 detector and a Waters ZQ 2000 4 way MUX mass spectrometer in parallel. The PL 2100 ELSD was set at 30° C. with 1.6 L/min supply of Nitrogen. The Waters ZQ MS was tuned with the following parameters:
ES+ Cone voltage: 25 v Capillary: 3.30 kv
ES− Cone voltage: −30 v Capillary: −2.50 kv
Desolvation gas: 800 L/hr
Source Temp: 150° C.
Scan range 160-900 Da
Where the reversed-phase Trilution system was used (T-HPLC) the conditions were as follows:
Mobile phase A: 0.1% formic acid in water
Mobile phase B: 0.1% formic acid in acetonitrile
Column: Phenomenex C18 Luna 21.5 mm×15 cm with 5 micron particule size
Gradient: 95-5% A over 15 min, 15 min hold, 15 ml/min flow rate
UV: 200 nm-400 nm
Temperature: Room temperature
Liquid Chromatography Mass Spectrometry
Unless carried out by Auto-HPLC (under conditions of A-HPLC or B-HPLC) as just decriberd, LCMS conditions were run according to one of the conditions given below (where ratios of solvents are given, the ratios are by volume):
Acidic 2 Minute LCMS
Mobile phase A: 0.1% formic acid in water
Mobile phase B: 0.1% formic acid in 70% methanol: 30% isopropanol
Column: C18 phase Phenomenex 20×4.0 mm with 3 micron particle size
Gradient: 98-10% A over 1.5 min, 0.3 min hold, 0.2 re-equilbration, 2 ml/min flow rate
UV: 210 nm-450 nm DAD
Temperature: 75° C.
Or
Mobile phase A: 0.1% formic acid in water
Mobile phase B: 0.1% formic acid in acetonitrile
Column: C18 phase Phenomenex 20×4.0 mm with 3 micron particle size
Gradient: 70-2% A over 1.5 min, 0.3 min hold, 0.2 re-equilbration, 1.8 ml/min flow rate
UV: 210 nm-450 nm DAD
Temperature: 75° C.
Acidic 4.5 Minute LCMS
Mobile phase A: 0.05% formic acid in water
Mobile phase B: acetonitrile
Column: Phenomenex Gemini C18 45×45 mm with 5 micron particle size
Gradient: 80-50% A over 0.5 min, 50-2% A over 3 min, 1 min hold, 0.2 min re-equilibration, 2.0 ml/min flow rate
UV: 220 nm-254 nm DAD
Temperature: 40° C.
Acidic 8 Minute LCMS
Mobile phase A: 0.05% formic acid in water
Mobile phase B: acetonitrile
Column: Phenomenex Gemini C18 45×45 mm with 5 micron particle size
Gradient: 80-50% A over 0.5 min, 50-2% A over 3 min, 4.5 min hold, 0.2 min re-equilibration, 2.0 ml/min flow rate
UV: 220 nm-254 nm DAD
Temperature: 40° C.
Acidic 6 Minute LCMS
Mobile phase A: 0.1% formic acid in water
Mobile phase B: 0.1% formic acid in acetonitrile
Column: C18 phase Waters Sunfire 50×4.6 mm with 5 micron particle size
Gradient: 95-5% A over 3 min, 1 min hold, 2 min re-equilibration, 1.5 ml/min flow rate
UV: 210 nm-450 nm DAD
Temperature: 50° C.
Basic 6 Minute LCMS
Mobile phase A: 0.1% ammonium hydroxide in water
Mobile phase B: 0.1% ammonium hydroxide in acetonitrile
Column: C18 phase Fortis 50×4.6 mm with 5 micron particle size
Gradient: 95-5% A over 3 min, 1 min hold, 2 min re-equilibration, 1 ml/min flow rate
UV: 210 nm-450 nm DAD
Temperature: 50° C.
Acidic 30 Minute LCMS
Mobile phase A: 0.1% formic acid in water
Mobile phase B: 0.1% formic acid in acetonitrile
Column: Phenomenex C18 phase Gemini 150×4.6 mm with 5 micron particle size
Gradient: 98-2% A over 18 min, 2 min hold, 1 ml/min flow rate
UV: 210 nm-450 nm DAD
Temperature: 50° C.
Basic 30 Minute LCMS
Mobile phase A: 10 mM ammonium acetate in water
Mobile phase B: 10 mM ammonium acetate in methanol
Column: Phenomenex Phenyl Hexyl 150×4.6 mm with 5 micron particle size
Gradient: 98-2% A over 18 min, 2 min hold, 1 ml/min flow rate
UV: 210 nm-450 nm DAD
Temperature: 50° C.
EXAMPLE 1
3-Cyano-4-{[3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
3-Cyano-N-(2,4-dimethoxybenzyl)-4-{[3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide (Preparation 20, 386 mg, 0.52 mmol) was dissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (13 mL) and stirred at room temperature for 18 hours. The reaction was concentrated in vacuo and purified using silica gel column chromatography (1% acetic acid in dichloromethane to 10% methanol and 1% acetic acid in dichloromethane gradient elution) followed by a second purification using silica gel column chromatography (0%-15% methanol in dichloromethane gradient elution) to afford the title compound (76 mg, 25%).
1HNMR (CD3OD): δ 7.05 (m, 1H), 7.39 (m, 1H), 7.67 (m, 2H), 7.91-8.05 (m, 7H), 8.20 (m, 1H), 9.20 (m, 1H), 9.50 (m, 1H)
LCMS Rt=5.14 minutes MS m/z 581 [MH]+
EXAMPLE 2
5-Chloro-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-pyrimidin-2-ylbenzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-pyrimidin-2-ylbenzenesulfonamide (Preparation 48) 356 mg, 0.47 mmol) was dissolved in 1,4-dioxane (1.5 mL) and a 4M solution of hydrogen chloride in 1,4-dioxane (2.4 mL) added. The mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo and the resulting residue purified by reverse phase preparative HPLC (Trilution method) to afford the title compound as a white solid (120 mg, 42%).
1HNMR (CD3OD): δ 6.85 (m, 1H), 6.95 (m, 1H), 7.30 (m, 1H), 7.80 (m, 2H), 7.90 (m, 3H), 8.00 (m, 2H), 8.10 (m, 1H), 8.40 (m, 2H), 9.20 (m, 1H), 9.50 (m, 1H)
LCMS Rt=3.12 minutes MS m/z 602 [MH]+
EXAMPLE 3
3-Chloro-N-pyridazin-3-yl-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}benzenesulfonamide
A mixture of 3-Chloro-N-(methoxymethyl)-N-pyridazin-3-yl-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}benzenesulfonamide and 3-chloro-N-[(3E)-2-(methoxymethyl)pyridazin-3(2H)-ylidene]-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}benzenesulfonamide (Preparation 49, 85 mg, 0.13 mmol) were dissolved in dichloromethane (1 mL) and a 4M solution of hydrogen chloride in 1,4-dioxane (0.34 mL) added. The mixture was stirred at room temperature for 15 minutes. The reaction mixture was concentrated in vacuo and the resulting residue purified by reverse phase preparative HPLC (Trilution method) to afford the title compound as a white solid (26 mg, 33%).
1HNMR (CD3OD): δ 7.10 (m, 1H), 7.20 (m, 1H), 7.60 (m, 1H), 7.80 (m, 3H), 7.90 (m, 4H), 8.05 (m, 3H), 8.30 (m, 1H), 9.20 (m, 1H), 9.55 (m, 1H).
LCMS Rt=3.20 minutes MS m/z 584 [MH]+, 582 [MH]−
EXAMPLE 4
5-Chloro-2-fluoro-4-{[3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 23, 339 mg, 0.45 mmol) was dissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (10 mL). The mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and the resulting residue purified by reverse phase preparative HPLC (Trilution method) to afford the title compound as a white solid (75 mg, 27%).
1HNMR (CD3OD): δ 6.91 (d, 1H), 7.22 (d, 1H), 7.48-7.57 (m, 2H), 7.58 (t, 1H), 7.62 (t, 1H), 7.71 (t, 1H), 7.82 (d, 1H), 7.92-7.97 (m, 1H), 8.00 (d, 1H), 8.58 (s, 1H), 9.20 (d, 1H), 9.43 (s, 1H)
LCMS Rt=3.26 minutes MS m/z 608.1 [MH]+, 606.1 [MH]−
EXAMPLE 5
5-Chloro-2-fluoro-4-O-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 22, 266 mg, 0.35 mmol) was dissolved a in 4M solution of hydrogen chloride in 1,4-dioxane (10 mL) and stirred at room temperature for 3 hours. A precipitate formed which was collected by filtration and triturated with acetonitrile to afford a solid. The filtrate and solid were combined and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (0%-20% methanol in dichloromethane gradient elution) to afford the title compound as a solid (67 mg, 31%).
1HNMR (CD3OD): δ 6.95 (m, 1H), 7.40 (m, 1H), 7.70 (m, 2H), 7.90 (m, 1H), 7.95-8.05 (m, 5H), 8.60 (s, 1H), 9.25 (m, 1H), 9.55 (m, 1H)
LCMS Rt=3.43 minutes MS m/z 608 [MH]+, 606 [MH]−
EXAMPLE 6
5-Chloro-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 24, 213 mg, 0.28 mmol) was dissolved a in 4M solution of hydrogen chloride in 1,4-dioxane (7 mL) and stirred at room temperature for 3 hours. A precipitate formed which was collected by filtration and purified by reverse phase preparative HPLC (Trilution method) to afford the title compound as a solid (44 mg, 26%).
1HNMR (CD3OD): δ 6.95 (m, 1H), 7.30 (m, 1H), 7.50-7.70 (m, 2H), 7.80 (m, 1H), 7.90-8.05 (m, 5H), 8.60 (s, 1H), 9.25 (m, 1H), 9.55 (m, 1H)
LCMS Rt=3.44 minutes MS m/z 608 [MH]+, 606 [MH]−
EXAMPLE 7
3-Cyano-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
3-Cyano-N-(2,4-dimethoxybenzyl)-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide (Preparation 26, 210 mg, 0.29 mmol) was dissolved a in 4M solution of hydrogen chloride in 1,4-dioxane (7 mL) and stirred at room temperature for 5 hours. A precipitate formed which was collected by filtration and purified by trituration with dichloromethane followed by silica gel column chromatography (0%-15% methanol in dichloromethane gradient elution) to afford the title compound as a solid (77 mg, yield).
1HNMR (d6-DMSO): δ 7.11 (d, 1H), 7.47 (d, 1H), 7.82 (d, 2H), 7.84-7.88 (m, 3H), 8.00-8.10 (m, 3H), 8.18 (d, 2H), 8.24 (s, 1H), 9.23 (d, 1H), 9.50 (s, 1H).
LCMS Rt=4.89 minutes MS m/z 581 [MH]+
EXAMPLE 8
3-Fluoro-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-1,3-thiazol-2-ylbenzenesulfonamide
A solution of 3-fluoro-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonyl chloride (Preparation 31, 350 mg, 0.79 mmol) and 2-amino thiazole (158 mg, 1.58 mmol) in pyridine (2 mL) was stirred at room temperature for 12 hours. The reaction mixture was concentrated in vacuo and the residue acidified to pH 4-5 with a 1M aqueous solution of hydrogen chloride. The mixture was extraction with ethyl acetate (3×10 mL). The organic layer was separated and washed sequentially with water (3×5 mL) and brine (1×5 mL), then dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (100-200 mesh silica gel, 35% ethyl acetate in hexane) followed by trituration with pentane to afford the title compound as an off white solid (75 mg, 19%).
1HNMR (d6-DMSO): δ 3.76 (s, 3H), 6.31 (s, 1H), 6.87 (d, 1H), 7.14 (t, 1H), 7.25 (d, 1H), 7.28 (d, 1H), 7.38-7.40 (m, 2H), 7.48 (t, 2H), 7.56 (d, 1H), 7.65 (d, 1H), 7.72-7.76 (m, 3H), 12.82 (s, 1H).
EXAMPLE 9
3-Chloro-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
3-Pyridazin-4-ylbiphenyl-4-ol (Preparation 4, 40 mg, 0.16 mmol) and 3-chloro-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 17, 72 mg, 0.16 mmol) were dissolved in dimethylsulfoxide (2 mL). Potassium carbonate (67 mg, 0.5 mmol) was added and the reaction stirred at room temperature for 16 hours. The crude material was partitioned between ethyl acetate (20 mL) and water (20 mL), the organic layer separated, concentrated in vacuo, dissolved in trifluoroacetic acid (1 mL) and the solution stirred for 16 hours at room temperature. The reaction was then concentrated in vacuo and purified by silica gel column chromatography (ISCO™, 12 g silica, 50-100% ethyl acetate in heptane gradient elution). The appropriate fractions were combined and concentrated in vacuo to afford the title compound as a white solid (41 mg, 49%).
1HNMR (CD3OD): δ 7.18 (d, 1H), 7.21 (m, 2H), 7.39 (m, 1H), 7.45 (m, 2H) 7.69 (d, 1H) 7.78 (d, 1H), 7.84 (m, 2H) 7.95(m, 1H) 7.99 (m, 1H), 8.78(s, 1H), 9.24 (m, 1H) 9.51 (m, 1H).
LCMS Rt=1.66 minutes MS m/z 522 [MH]+
EXAMPLE 10
3-Cyano-4-[(3-Pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
3-Pyridazin-4-ylbiphenyl-4-ol (Preparation 4, 50 mg, 0.2 mmol) and 3-cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 15, 87.3 mg, 0.2 mmol) were dissolved in dimethylsulfoxide (2 mL). Potassium carbonate (83 mg, 0.6 mmol) was added and the reaction stirred at room temperature for 16 hours. The crude material was partitioned between ethyl acetate (20 mL) and water (20 mL), the organic layer separated, concentrated in vacuo, dissolved in trifluoroacetic acid (1 mL) and the solution stirred for 16 hours at room temperature. The reaction was concentrated in vacuo then purified by reverse phase column chromatography (ISCO™, 12 g, C18, 20:1 water:acetonitrile to 1:4 water:acetonitrile). The appropriate fractions were combined and concentrated in vacuo to afford the title compound as a white solid (25 mg, 24%).
1HNMR (CD3OD): δ 7.04 (d, 1H), 7.41 (m, 2H), 7.49 (m, 2H) 7.73 (m, 2H) 7.90 (m, 1H) 7.97 (m, 3H), 8.17 (d, 1H) 8.54 (s, 1H), 9.19 (m, 1H) 9.45 (m, 1H)
LCMS Rt=1.61 minutes MS m/z 513 [MH]+
EXAMPLE 11
5-Chloro-2-fluoro-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 25, 124 mg, 0.18 mmol) was dissolved in trifluoroacetic acid (1 mL) and the solution stirred for 16 hours at room temperature. The reaction was concentrated in vacuo then purified by reverse phase column chromatography (ISCO™, 12 g, C18, 20:1 water:acetonitrile to 1:4 water:acetonitrile). The appropriate fractions were combined and concentrated in vacuo to afford the title compound as a white solid (61 mg, 63%).
1HNMR (CD3OD): δ 7.20 (d, 1H) 7.28 (d, 1H), 7.39 (m, 1H), 7.48 (m, 2H) 7.78 (m, 2H) 7.84 (m, 1H) 7.90 (d, 1H), 7.94 (m, 1H) 7.98 (d, 1H) 8.78 (s, 1H), 9.27 (m, 1H) 9.50 (m, 1H).
LCMS Rt=1.71 minutes MS m/z 540 [MH]+
EXAMPLE 12
3-Cyano-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
3-Pyridazin-4-ylbiphenyl-4-ol (Preparation 4, 50 mg, 0.2 mmol) and 3-cyano-4-fluoro-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide (Preparation 47, 50 mg, 0.2 mmol) were dissolved in dimethylsulfoxide (2 mL). Potassium carbonate (83 mg, 0.6 mmol) was added and the reaction heated to 90° C. for 16 hours. The crude material was then purified by reverse phase column chromatography (ISCO™, 4 g, C18, 20:1 water:acetonitrile to 3:2 water acetonitrile). The appropriate fractions were combined and concentrated in vacuo to afford the title compound as an off white solid (25 mg, 24%).
1HNMR (CD3OD): δ 6.98 (d, 1H) 7.33 (d, 1H), 7.37 (m, 1H), 7.46 (m, 2H) 7.70 (m, 2H) 7.84 (m, 1H) 7.93 (m, 3H), 8.00 (m, 1H) 8.16 (d, 1H), 9.17 (m, 1H) 9.45 (m, 1H)
LCMS Rt=1.23 minutes MS m/z 550 [MK]+
EXAMPLE 13
5-Chloro-2-fluoro-4-({3-[1-(1-methylazetidin-3-yl)-1H-pyrazol-5-yl]-2′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
To a suspension of 4-{[3-(1-azetidin-3-yl-1H-pyrazol-5-yl)-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 46, 42.9 mg, 0.0659 mmol) in methanol (0.10 mL), dichloromethane (1.72 mL) and acetic acid (0.10 mL) was added formaldehyde (37% w/w, 16.7 μL, 0.224 mmol). The reaction was then stirred under nitrogen at room temperature for 45 minutes. Sodium triacetoxyborohydride (42.6 mg, 0.201 mmol) was added to the reaction which was stirred for 18 hours at room temperature. The reaction was diluted with dichloromethane (20 mL) and washed with water (3×2 mL). The combined aqueous phases were extracted with dichloromethane (3×5 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a white solid (58.0 mg). The solid was purified by B-HPLC to afford the title compound.
LCMS Rt=2.54 minutes (basic QC method) MS m/z 665 [MH]+, 663 [MH]−
EXAMPLE 14
5-Chloro-2-fluoro-4-({3-[1-(1-methylazetidin-3-yl)-1H-pyrazol-5-yl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
To a suspension of 4-{[3-(1-azetidin-3-yl-1H-pyrazol-5-yl)-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 41, 46.3 mg, 0.0711 mmol) in methanol (0.11 mL), dichloromethane (1.86 mL) and acetic acid (0.11 mL) was added formaldehyde (37% w/w, 19.6 μL, 0.263 mmol). The reaction was then stirred under nitrogen at room temperature for 45 minutes. Sodium triacetoxyborohydride (45.9 mg, 0.217 mmol) was added to the reaction which was stirred for 18 hours at room temperature. The reaction was diluted with dichloromethane (20 mL) and washed with water (3×2 mL). The combined aqueous phases were extracted with dichloromethane (3×5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a clear gum (58.0 mg). The clear gum was purified by B-HPLC to afford the title compound.
LCMS Rt=2.77 minutes (acidic QC method) MS m/z 665 [MH]+, 663 [MH]−
EXAMPLE 15
3-Cyano-N-(5-fluoro-1,3-thiazol-2-yl)-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonamide
To a stirred solution of 3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-ol (Preparation 28, 188 mg, 0.75 mmol) and potassium carbonate (173 mg, 1.25 mmol) in N,N-dimethylformamide (2.5 mL) was added 3-cyano-4-fluoro-N-(5-fluoro-1,3-thiazol-2-yl)benzenesulfonamide (Preparation 34, 151 mg, 0.5 mmol) and the reaction mixture was stirred at 80° C. After stirring for 16 hours, the reaction mixture was cooled to room temperature. Saturated aqueous ammonium chloride (10 mL) was added to the reaction mixture and the mixture was extracted with dichloromethane (3×10 mL). The collected organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain a crude residue. The residue was purified by silica gel column chromatography (40% ethyl acetate in dichloromethane elution) to afford the title compound as a white solid (127 mg, 48%).
1HNMR (d6-DMSO): δ 3.78 (s, 3H), 6.26 (d, 1H), 6.96 (d, 1H), 7.35-7.44 (m, 3H), 7.47-7.55 (m, 3H), 7.76-7.81 (m, 2H), 7.84 (d, 1H), 7.90-7.95 (m, 2H), 8.14 (d, 1H)
LCMS Rt=3.24 minutes MS m/z 532 [MH]+
EXAMPLE 16
3-Cyano-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
To a stirred solution of 3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-ol (Preparation 28, 44.1 mg, 0.176 mmol) and potassium carbonate (30.4 mg, 0.22 mmol) in N,N-dimethylformamide (1 mL) was added 3-cyano-4-fluoro-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide (Preparation 47, 50.0 mg, 0.176 mmol) and the reaction mixture was stirred at 100° C. After stirring for 24 hours, the reaction mixture was cooled to room temperature. A 1M aqueous solution of hydrogen chloride (10 mL) was added to the reaction mixture and the mixture was extracted with dichloromethane (3×10 mL). The combined organic layer was concentrated in vacuo to obtain the title compound (90 mg, 99%).
LCMS Rt=3.22 minutes MS m/z 515 [MH]+
EXAMPLE 17
3-Cyano-4-{[3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
3-Cyano-N-(2,4-dimethoxybenzyl)-4-{[3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide (Preparation 21, 400 mg, 0.55 mmol) was dissolved in a 4M solution of HCl in 1,4-dioxane (9 mL). The mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was purified by silica gel column chromatography (0%-15% methanol in dichloromethane gradient elution), followed by trituration in tert-butylmethyl ether. The residue was purified further by a silica plug column (0%-20% methanol in dichloromethane) to afford the title compound (73 mg, 23%) as a white solid.
1HNMR (400 MHz, CD3OD): δ 7.01 (d, 1H), 7.34 (d, 1H), 7.49 (d, 1H), 7.52-7.60 (m, 2H), 7.62-7.70 (m, 2H), 7.79 (d, 1H), 7.83-7.95 (m, 2H), 8.01 (d, 1H), 8.17 (s, 1H), 9.18 (d, 1H), 9.42 (s, 1H)
EXAMPLE 18
3-Cyano-4-{[3′-methoxy-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-(1,3-thiazol-2-yl)benzenesulfonamide
To a solution of 4-[4-bromo-2-(2-methyl-2H-pyrazol-3-yl)-phenoxy]-3-cyano-N-(2,4-dimethoxy-benzyl)-N-thiazol-2-yl-benzenesulfonamide (Preparation 88, 98.5 mg, 0.148 mmol), 3-methoxyphenylboronic acid (48 mg, 0.32 mmol), and potassium carbonate (62.5 mg, 0.452 mmol) in toluene (3 mL) was added tetrakistriphenylphosphinepalladium (0) (22.5 mg, 0.0195 mmol) and the mixture was sparged two times with argon. The reaction mixture was heated at reflux for 4.5 hours. After cooling to room temperature the reaction mixture was partitioned between ethyl acetate and water. The organic layer was separated and washed with saturated sodium chloride solution, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by automated flash column chromatography using a 0-100% EtOAc/hexanes gradient to yield a clear oil. This oil was dissolved in methylene chloride (5 mL) and treated with trifluoroacetic acid (1 mL, 10 mmol). After stirring for 1 hour, the reaction mixture was concentrated in vacuo and purified by automated flash column chromatography (0%-5% methanol in dichloromethane gradient elution) to yield the title compound (49 mg, 61%) as a white solid.
1HNMR (400 MHz, d6-DMSO): δ 3.70 (s, 3H), 3.86 (s, 3H), 6.28 (m, 1H), 6.90 (m, 2H), 6.96 (m, 1H), 7.36 (m, 2H), 7.38 (m, 2H), 7.43 (m, 1H), 7.54 (m, 1H), 7.89 (m, 1H), 7.94 (m, 2H), 8.14 (m, 1H), 12.80 (s, 1H).
LCMS Rt=1.70 minutes; MS m/z 544 [MH]+
EXAMPLE 19
3-Cyano-4-{[2′-methoxy-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-N-(1,3-thiazol-2-yl)benzenesulfonamide
To a solution of 4-[4-bromo-2-(2-methyl-2H-pyrazol-3-yl)-phenoxy]-3-cyano-N-(2,4-dimethoxy-benzyl)-N-thiazol-2-yl-benzenesulfonamide (Preparation 88, 98.5 mg, 0.148 mmol), 2-methoxyphenylboronic acid (48 mg, 0.32 mmol), and potassium carbonate (62.5 mg, 0.452 mmol) in toluene (3 mL) was added tetrakis(triphenylphosphine)palladium(0) (22.5 mg, 0.0195 mmol) and the mixture was sparged two times with argon. The reaction mixture was then heated at reflux for 4.5 hours and then allowed to cool to room temperature. The reaction mixture was diluted with ethyl acetate and washed with water and saturated sodium chloride solution. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by automated flash column chromatography using a 0-100% EtOAc/hexanes gradient to give a clear oil. This oil was dissolved in methylene chloride (5 mL) and treated with trifluoroacetic acid (1 mL, 10 mmol). After stirring for 1 hour, the reaction mixture was concentrated in vacuo and purified by automated flash column chromatography (0%-5% methanol in dichloromethane gradient elution) to yield the title compound (50 mg, 61%) as a white solid.
1HNMR (400 MHz, d6-DMSO): δ 3.81 (s, 3H), 3.86 (s, 3H), 6.29 (m, 1H), 6.92 (m, 1H), 6.97 (m, 1H), 7.09 (m, 1H), 7.19 (m, 1H), 7.34 (m, 2H), 7.48 (m, 3H), 7.70 (m, 1H), 7.77 (m, 1H), 7.96 (m, 1H), 8.15 (m, 1H), 12.80 (s, 1H).
LCMS Rt=1.63 minutes; MS m/z 544 [MH]+
EXAMPLE 20
3-Cyano-N-(5-fluoropyridin-2-yl)-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonamide
In a pressure sealed vial, 3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-ol (Preparation 107, mg, 0.19 mmol), 3-cyano-4-fluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (WO2010079443, 50 mg, 0.17 mmol) and potassium carbonate (70 mg, 0.51 mmol) were stirred at 90° C. in dimethyl sulfoxide for 18 hours. The mixture was cooled down to room temperature and treated with 2M hydrochloric acid (5 mL). The mixture was stirred for 1 hour and the resulting precipitate was filtered and purified by preparative HPLC to afford the title compound (17 mg, 18%) as a white solid.
LCMS Rt=3.73 minutes, MS m/z 526 [MH]+
EXAMPLE 21
4-{[3′-(Aminomethyl)-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}-3-cyano-N-(1,3-thiazol-2-yl)benzenesulfonamide, hydrochloride salt
tert-Butyl-{[4′-{2-cyano-4-[(1,3-thiazol-2-ylamino)sulfonyl]phenoxy}-3′(1-methyl-1H-pyrazol-5-yl)biphenyl-3-yl]methyl}carbamate (Preparation 77, 380 mg, 0.59 mmol) was dissolved in dichloromethane (20 mL), 4M HCl in 1,4-dioxane (4 mL) was added and the reaction was stirred for 18 hours at room temperature. The reaction mixture was concentrated in vacuo, slurried in cold diethyl ether (20 ml) then filtered to afford the title compound (342 mg, 99%) as a yellow solid as the hydrochloride salt.
1HNMR (400 MHz, d6-DMSO): δ 3.76 (s, 3H), 4.09 (m, 2H), 6.23 (d, 1H), 6.87 (d, 1H), 6.94 (d, 1H), 7.28 (d, 1H), 7.34 (d, 1H), 7.50 (m, 3H), 7.78 (m, 1H), 7.87 (d, 1H), 7.94 (m, 3H), 8.10 (d, 1H), 8.42 (br s, 3H).
LCMS Rt=1.02 minutes MS m/z 543 [MH]+
EXAMPLE 22
5-Chloro-2-fluoro-N-(5-fluoropyridin-2-yl)-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide (Preparation 105, 23 mg, 0.04 mmol), 3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-ol (Preparation 107, 9 mg, 0.04 mmol) and potassium carbonate (15 mg, 0.11 mmol) in dimethyl sulfoxide (1 mL) were stirred at room temperature for 2 hours. The mixture was treated with aqueous 2M HCl (3 mL). The resulting mixture was extracted with dichloromethane (3 mL). The dichloromethane layer was dried through a phase separating cartridge followed by treatment with trifluoroacetic acid (500 μL). The mixture was stirred for 2 hours and allowed to stand at room temperature for 18 hours. The mixture was then treated with a saturated solution of ammonium chloride (5 mL). The dichloromethane layer was separated, dried through a phase separating cartridge and evaporated in vacuo. The residue was purified by preparative HPLC to afford the title compound (14 mg, 45%).
LCMS Rt=2.55 minutes, MS m/z 551 [M-H]−.
EXAMPLE 23
3-Cyano-4-({2′-[(methylamino)methyl]-3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl}oxy)-N-(1,3-thiazol-2-yl)benzenesulfonamide, trifluoroacetate salt
tert-Butyl-{[4′-{2-cyano-4-[(1,3-thiazol-2-ylamino)sulfonyl]phenoxy}-3′(1-methyl-1H-pyrazol-5-yl)biphenyl-2-yl]methyl}methylcarbamate (Preparation 65, 64 mg, 0.01 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.2 mL) was added and the reaction was stirred for 18 hours at room temperature. The reaction mixture was concentrated in vacuo and purified by reverse phase preparative HPLC to afford the title compound (22 mg, 37%) as a white solid as the trifluoroacetate salt.
LCMS Rt=2.29 minutes MS m/z 557 [MH]+, 555 [M-H]−
EXAMPLE 24
5-Chloro-4-{[2-chloro-4′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-4-(2-chloro-4′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 70, 220 mg, 0.30 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (1 mL) was added. The reaction was stirred at room temperature for 3 hours. Methanol (5 mL) was added to quench the reaction and the suspension was stirred vigorously for 1 hour. The resulting precipitate was filtered through Celite™ and washed with methanol and the filtrate concentrated in vacuo. The residue was suspended in hot methanol (5 mL) and the remaining solids filtered off. The filtrate was concentrated in vacuo and the residue triturated with ethyl acetate and filtered to give the title compound (77 mg, 43%) as a white solid.
1HNMR (400 MHz, d6-DMSO): δ 7.33 (m, 3H), 7.52 (s, 1H), 7.58 (dd, 2H), 7.78 (s, 1H), 7.92 (d, 2H), 8.80 (s, 1H), 9.26 (d, 1H), 9.46 (s, 1H).
LCMS Rt=3.34 minutes MS m/z 592 [M35ClH]+.
EXAMPLE 25
4-{[3-(3-Amino-1H-pyrazol-4-yl)-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-(3-(3-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)biphenyl-4-yloxy)-N-(1,3,4-thiadiazol-2-yl)benzene sulfonamide (Preparation 86, 0.15 g, 0.17 mmol) was dissolved in acetonitrile (2 mL). Potassium carbonate (117 mg, 0.85 mmol), sodium dithionite (0.15 g, 0.85 mmol) and water (1 mL) were added and the reaction was heated at 40° C. for 3 hours. After cooling to room temperature, the reaction was partitioned between ethyl acetate (50 mL) and water (30 mL). The organic layer was dried over magnesium sulphate, filtered and concentrated in vacuo. The crude residue was dissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (2.5 mL). The reaction was stirred at room temperature for 18 hours, concentrated in vacuo and the residue was purified by reverse phase HPLC using acetonitrile/water (5/95 to 95/5 with 0.05% formic acid as eluent to give the title compound (5.2 mg, 7%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 3.30 (s, 2H), 6.55 (d, 1H), 7.22 (d, 1H), 7.62 (m, 4H), 7.90 (m, 4H), 8.55 (s, 1H).
LCMS Rt=2.75 minutes, MS m/z 611 [MH]+
EXAMPLE 26
5-Chloro-4-{[2-chloro-3′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-4-(2-chloro-3′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 66, 165 mg, 0.22 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (1 mL) was added. The reaction was stirred at room temperature for 3 hours. Methanol (5 mL) was added to quench the reaction and the suspension stirred vigorously for 18 hours. The mixture was diluted with dichloromethane (5 mL) and the resulting precipitate was filtered through Celite™ and washed with dichloromethane (2×5 mL) and the filtrate concentrated in vacuo. The residue was dissolved in dichloromethane and methanol and passed through a short silica plug eluting with dichloromethane/methanol (98:2). The material obtained was further purified by silica gel column chromatography using (dichloromethane/methanol/acetic acid 97:3:0.5) to give the title compound (17 mg, 13%) as a white solid.
1HNMR (400 MHz, d6-DMSO): δ 6.28 (t, 1H), 7.40 (m, 3H), 7.53 (m, 2H), 7.82 (s, 1H), 7.94 (m, 2H), 8.82 (s, 1H), 9.28 (d, 1H), 9.52 (s, 1H).
LCMS Rt=3.36 minutes MS m/z 592 [MH]+
EXAMPLE 27
5-Chloro-4-{[2-chloro-2′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
2-Chloro-2′-fluoro-5-(pyridazin-4-yl)biphenyl-4-ol (Preparation 74, 100 mg, 0.4 mmol) was dissolved in DMSO (2 mL) and potassium carbonate (92 mg, 0.66 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-l)benzenesulfonamide (Preparation 16, 154 mg, 0.33 mmol). The reaction was stirred at room temperature for 18 hours and then partitioned between ethyl acetate (50 mL) and water (40 mL). The ethyl acetate was separated, dried over anhydrous MgSO4 filtered, and evaporated to give 5-chloro-4-(2-chloro-2′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (220 mg) which was used without further purification in the next stage.
LCMS Rt=3.70 minutes, MS m/z 742 [MH]+.
The crude 5-chloro-4-(2-chloro-2′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (220 mg) was dissolved in a 4M solution of HCl in 1,4-dioxane (10 mL) and stirred at room temperature for 3 hours. The resulting precipitate was collected and purified by reverse phase chromatography using acetonitrile:water:0.05% formic acid followed by chromatography on silica gel eluting with dichloromethane/methanol 9:1 to give the title compound (8.5 mg, 3.5%) as a white solid.
LCMS Rt=2.94 minutes, MS m/z 592 [MH]+.
EXAMPLE 28
5-Chloro-4-{[2-chloro-5-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-4-(2-chloro-5-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 53, 231 mg, 0.29 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (1 mL) added. The resulting solution was stirred at room temperature for 18 hours. Methanol (2 mL) was added and the reaction was stirred for 10 minutes. The resulting mixture was evaporated and azeotroped with methanol (2×10 mL). The residue was partitioned between ethyl acetate (50 mL) and water (20 mL). The ethyl acetate was separated and dried over MgSO4 and evaporated. The residue was chromatographed on silica eluting with dichloromethane:methanol:acetic acid 100:0:0 to 95:5:0.5 in 1% stages of methanol. The column product was stirred in dichloromethane (10 mL) for 20 minutes, the solid filtered off, and stirred in dichloromethane (5 mL) at reflux for 10 minutes. The solid was filtered to give the title compound (45 mg, 25%) as a white solid.
1HNMR (400 MHz, d-6DMSO): δ 7.38 (d, 1H), 7.56 (s, 1H), 7.74 (m, 1H), 7.80 (m, 1H), 7.84-7.94 (m, 6H), 8.80 (s, 1H), 9.27 (d, 1H), 9.50 (s, 1H).
LCMS (5.0 min) Rt=3.52 minutes, MS m/z 642 [MH]+.
EXAMPLE 29
5-Chloro-4-{[4′-chloro-3-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-4-(4′-chloro-3-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 57, 135 mg, 0.17 mmol) was dissolved in a 4M solution of HCl in dioxane (5 mL), methanol (5 mL) added and the resulting mixture was stirred at 50° C. for 6 hours. The reaction mixture was evaporated and the residue partitioned between ethyl acetate (50 mL) and water (50 mL). The ethyl acetate was separated, dried over MgSO4, filtered and evaporated. The residue was chromatographed on silica eluting with a gradient of dichloromethane:methanol:acetic acid 100:0:0 to 95:4:0.4 to give the title compound (75 mg, 68%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 7.23 (d, 1H), 7.27 (d, 1H), 7.84, (d 1H), 7.94 (m, 3H), 8.10 (m, 1H), 8.13 (s, 1H), 8.20 (s, 1H), 8.80 (s, 1H), 9.31 (d, 1H), 9.55 (s, 1H).
LCMS (5.0 min) Rt=3.56 minutes, MS m/z 642 [MH]+.
EXAMPLE 30
5-Chloro-2-fluoro-4-{2-(pyridazin-4-yl)-4-[6-(trifluoromethyl)pyridin-3-yl]phenoxy}-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-(2-(pyridazin-4-yl)-4-(6-(trifluoromethyl)pyridin-3-yl)phenoxy)-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 58, 250 mg, 0.329 mmol) was dissolved in a 4M solution of HCl in 1,4-dioxane (0.9 mL, 3.29 mmol). The reaction mixture was stirred at room temperature for 18 hours and then concentrated in vacuo. The resulting residue was purified by reverse phase chromatography on the ISCO system using acetonitrile:water0.1% formic acid to afford the title compound (160 mg, 80%) as a white solid.
1HNMR (400 MHz, d-6DMSO): δ 7.30 (dd, 2H), 7.80 (d, 1H), 7.89-8.02 (m, 3H), 8.20 (d, 1H), 8.50 (d, 1H), 8.75 (s, 1H), 9.20 (d, 1H), 9.31 (d, 1H), 9.56 (s, 1H).
19F NMR (400 MHz, d-6DMSO): δ −66.2, −106.7
LCMS Rt=3.13 minutes, MS m/z 609 [MH]+.
EXAMPLE 31
5-Chloro-2-fluoro-4-{2-(pyridazin-4-yl)-4-[6-(trifluoromethyl)pyridin-2-yl]phenoxy}-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-(2-pyridazin-4-yl)-6-(trifluoromethyl)pyridine-2-yl)phenoxy-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 82, 50 mg, 0.06 mmol) was dissolved in 4M solution of HCl in 1,4-dioxane (2 mL). The reaction was stirred at room temperature for 5 hours, concentrated in vacuo and the residue was purified by reverse phase HPLC using acetonitrile:water:0.5% formic acid to give the title compound (9.8 mg, 25%) as a white solid.
1HNMR (400 MHz, d6-DMSO): δ 7.35 (d, 1H), 7.38 (d, 1H), 7.90 (m, 3H), 8.24 (m, 1H), 8.30 (m, 1H), 8.40 (s, 1H), 8.45 (d, 1H), 8.80 (s, 1H), 9.36 (d, 1H), 9.58 (s, 1H).
LCMS Rt=3.27 minutes, MS m/z 609 [MH]+.
EXAMPLE 32
4-{[3-(5-Amino-1H-pyrazol-4-yl)-3′-cyanobiphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-4-(3′-cyano-3-(3-nitro-1H-pyrazol-4-yl)biphenyl-4-yloxy)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 62, 51 mg, 0.0853 mmol) was dissolved in acetonitrile (1 mL) and heated at 50° C. Potassium carbonate (58.8 mg, 0.426 mmol) followed by sodium dithionite (59.4 mg, 0.341 mmol) and water (1 mL) were added. The reaction mixture was heated at 50° C. for 2 hours, cooled to room temperature and partitioned between EtOAc (10 mL) and water (5 mL). The aqueous phase was separated and extracted with EtOAc (2×3 mL) and the combined organic phases were washed with a saturated solution of brine (3 mL), dried over MgSO4 and concentrated in vacuo. The crude residue was purified by reverse phase HPLC.
LCMS Rt=2.44 minutes, MS m/z 568 [MH]+.
EXAMPLE 33
5-Chloro-2-fluoro-4-{2-(pyridazin-4-yl)-4-[2-(trifluoromethyl)pyridin-4-yl]phenoxy}-N-1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-(2-pyridazin-4-yl)-4-(2-trifluoromethyl)-pyridine-4-yl)phenoxy)-N-1,3,4-thidiazol-2-yl)benzenesulfonamide (Preparation 78, 0.2 g, 0.26 mmol) was dissolved in a mixture of a 4M solution of HCl in 1,4-dioxane (4 mL) and methanol (3 mL). The reaction was stirred at room temperature for 5 hours, concentrated in vacuo and residue was purified by reverse phase HPLC using acetonitrile:water:0.5% formic acid to give the title compound (26 mg, 16%) as a white solid.
1HNMR (400 MHz, CD3OD): δ 7.02 (d, 1H), 7.14 (d, 1H), 8.02 (m, 4H), 8.10 (d, 2H), 8.60 (s, 1H), 8.80 (d, 1H), 9.12 (d, 1H), 9.57 (s, 1H).
LCMS Rt=3.10 minutes, MS m/z 609 [MH]+.
EXAMPLE 34
5-Chloro-2-fluoro-4-({3-[2-(piperazin-1-yl)pyridin-4-yl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, hydrochloride
tert-Butyl 4-(4-(4-(2-chloro-4-(N-(2,4-dimethoxybenzyl)-N-(1,3,4-thiadiazol-2-yl)sulfamoyl)-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 113, 340 mg, 0.361 mmol) was dissolved in methanol (1 mL) and a 4M solution of hydrogen chloride in 1,4-dioxane (3 mL) was added. The reaction mixture was stirred at room temperature for 3 hours and then concentrated in vacuo. The resulting residue was purified by reverse phase chromatography using the ISCO™ system and acetonitrile/water 5/95-95/5 with 0.1% formic acid as eluent to afford the title compound (90 mg, 34%) as a white solid.
1HNMR (400 MHz, d-6DMSO): δ 3.08 (m, 4H), 3.69 (m, 4H), 6.74 (d, 1H), 6.91 (d, 1H), 7.00 (s, 1H), 7.32 (d, 1H), 7.69 (d, 1H), 7.80-7.95 (m, 4H), 7.98 (d, 2H), 8.11 (d, 1H), 8.56 (s, 1H), 9.06 (br s, 1H).
19F NMR (376 MHz, DMSO-d6): δ −107.4, −60.9.
LCMS Rt=2.53 minutes. MS m/z 691 [MH]+.
EXAMPLE 35
5-Chloro-2-fluoro-4-({3-[2-(piperazin-1-yl)pyridin-4-yl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-(pyrimidin-4-yl)benzenesulfonamide hydrochloride salt
A 4M solution of hydrogen chloride in 1,4-dioxane (10 mL) was added to a solution of tert-butyl 4-(4-(4-(2-chloro-4-(N-(2,4-dimethoxybenzyl)-N-(pyrimidin-4-yl)sulfamoyl)-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 109, 310 mg, 0.332 mmol) in methanol (2 mL). The reaction mixture was stirred at room temperature for 3 hours and then concentrated in vacuo. The residue was purified by reverse phase chromatography (acetonitrile/water with 0.1% formic acid) to afford the title compound (196 mg, 82%) as a white solid.
1HNMR (400 MHz, d-6DMSO): δ 3.08 (br s, 4H), 3.72 (br s, 4H), 6.61 (d, 1H), 6.70 (d, 1H), 6.94 (d, 1H), 7.04 (s, 1H), 7.30 (d, 1H), 7.78-7.87 (m, 5H), 7.95-7.99 (m, 3H), 8.12 (d, 1H), 8.26 (s, 1H).
19FNMR (376 MHz, d6-DMSO): δ −108.02 (F), −60.91 (CF3).
LCMS Rt=2.95 minutes, m/z 685 [MH]+.
EXAMPLE 36
5-Chloro-4-[(6-chloro-3′-fluoro-4-pyridazin-4-ylbiphenyl-3-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-4-(6-chloro-3′-fluoro-4-(pyridazin-4-yl)biphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 97, 60 mg, 0.08 mmol) was dissolved in a 4M solution of HCl in dioxane (5 mL). The reaction was stirred for 18 hours at room temperature and then evaporated in vacuo. The residue was dissolved in ethyl acetate (5 mL) washed with water (2×5 mL), dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography eluting with dichloromethane:methanol:acetic acid (97:2.7:0.3) to give 30 mg of the title compound. The compound was further purified by preparative HPLC to give the title compound (3.6 mg, 7.5%) as a solid.
1H NMR (400 MHz, CDCl3): δ 7.21-7.36 (m, 4H), 7.40 (s, 1H), 7.49-7.55 (m, 1H), 7.83 (d, 1H), 7.87-7.89 (m, 1H), 8.02 (s, 1H), 8.75 (s, 1H), 9.28-9.29 (m, 1H), 9.44-9.45 (m, 1H).
19F NMR (400 MHz, CDCl3): δ −107, −113.
LCMS (4.5 min acidic run) Rt=3.34 minutes, m/z 592 [MH]+.
EXAMPLE 37
5-Chloro-4-[(6-chloro-4′-fluoro-4-pyridazin-4-ylbiphenyl-3-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-4-(6-chloro-4′-fluoro-4-(pyridazin-4-yl)biphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 101, 100 mg, 0.13 mmol) was dissolved in a 4M solution of HCl in dioxane (10 mL). The reaction mixture was stirred for 18 hours at room temperature. Methanol (50 mL) was added to the reaction mixture and the suspension was concentrated in vacuo. The crude residue was purified by reverse phase semi preparative HPLC (solvent A: 0.05% formic acid in acetonitrile, solvent B: 0.05% formic acid in water; flow rate: 12.5 ml/min; gradient: 0 min 10% A, 2.5 min 10% A, 32.5 min 95% A, 37.5 min 95% A then return to initial conditions) to afford the title compound (46 mg, 60%) as a solid.
1H NMR (400 MHz, CDCl3): δ 6.94 (m, 1H), 7.22 (m, 3H), 7.52 (m, 2H), 7.90 (s, 1H), 7.95 (m, 2H), 8.55 (s, 1H), 9.23 (m, 1H), 9.47 (m, 1H).
19F NMR (400 MHz, CD3OD+CD3CN drops): δ −107.7, −115.3
LCMS Rt=3.31 minutes MS m/z 592 [MH]+.
EXAMPLE 38
5-Chloro-4-[(6-chloro-2′-fluoro-4-pyridazin-4-ylbiphenyl-3-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
Hydrogen chloride in dioxane (4M, 1.5 mL, 6.00 mmol) was added to a solution of 5-chloro-4-(6-chloro-2′-fluoro-4-(pyridazin-4-yl)biphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 92, 220 mg, 0.27 mmol) in methanol (1.5 mL) and the reaction stirred at room temperature for 18 hours. The mixture was evaporated to dryness and dissolved in dimethylsulfoxide (4.0 mL) and methanol (2.0 mL). The resulting precipitate was filtered, washed with methanol (2.0 mL) and the filtrate purified by preparative HPLC using a Phenomenex Luna C18 5 u 110 A 21.2×150 mm using acetonitrile:water as eluent to give the title compound (92 mg, 56%) as a beige coloured solid.
1H-NMR (400 MHz, CDCl3): δ 7.26 (d, 1H), 7.32 (m, 1H), 7.34 (m, 1H), 7.40 (m, 1H), 7.44 (m, 1H), 7.52 (m, 1H), 7.87 (d, 1H), 7.93 (dd, 1H), 8.02 (s, 1H), 8.78 (s, 1H), 9.31 (dd, 1H), 9.49 (t, 1H).
19F-NMR (400 MHz, CDCl3): δ −106.66, −114.13
LCMS (4.5 min) Rt=3.26 minutes MS m/z 592 [MH]+.
EXAMPLE 39
5-chloro-4-[(3′-cyano-3-pyridazin-4-ylbiphenyl-4-yl)oxy]-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-4-(3′-cyano-3-(pyridazin-4-yl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 118, 550 mg, 0.77 mmol) was dissolved in methanol (2 mL) and a 4M solution of hydrogen chloride in 1,4-dioxane (10 mL) was added. The reaction mixture was stirred at room temperature for 18 hours and then concentrated in vacuo. The residue was co-evaporated with methanol and then purified by reverse phase chromatography (acetonitrile/water both with 0.1% formic acid) to give the title compound (303 mg, 70%) as a white solid.
LCMS Rt=2.62 minutes, MS m/z 565 [MH]+.
1HNMR (400 MHz, d-6DMSO): δ 7.31-7.25 (m, 2H), 7.68 (t, 1H), 7.86 (d, 1H), 7.94-7.91 (m, 2H), 7.99-7.97 (m, 1H), 8.16-8.13 (m, 2H), 8.35 (s, 1H), 8.80 (s, 1H), 9.30 (d, 1H), 9.55 (s, 1H).
19FNMR (376 MHz, d-6DMSO): δ −106.67 (s, 1 F)
EXAMPLE 40
5-chloro-2-fluoro-4-{[3-(2-piperazin-1-ylpyridin-4-yl)-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-pyrimidin-2-ylbenzenesulfonamide
tert-Butyl 4-(4-(4-(2-chloro-4-(N-(2,4-dimethoxybenzyl)-N-(pyrimidin-2-yl)sulfamoyl)-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 122, 360 mg, 0.385 mmol) was dissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (5 mL). The reaction mixture was stirred at room temperature for 20 hours and then concentrated in vacuo. The residue was purified by reverse phase chromatography (acetonitrile/water both with 0.1% formic acid) to give the title compound (30 mg, 11%) as a white solid.
1HNMR (400 MHz, d6-acetone): δ 3.12 (br s, 4H), 3.72 (br s, 4H), 6.25 (d, 1H), 6.60 (s, 1H), 6.74-6.72 (m, 1H), 7.17-7.13 (m, 1H), 7.61-7.56 (m, 5H), 8.02-7.99 (m, 2H), 8.34-8.32 (m, 3H), 8.65 (br s, 2H)
19FNMR (376 MHz, acetone-d6): δ −108.65 (F), −62.52 (CF3)
LCMS Rt=2.36 minutes, MS m/z 685 [MH]+.
The following Examples may be prepared by the methods described in the aforementioned Schemes, foregoing Examples and the corresponding Preparations, or by processes similar to either:
5-chloro-2-fluoro-4-{[3-(2-piperazin-1-ylpyridin-4-yl)-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3-thiazol-4-ylbenzenesulfonamide;
4-{[3-(5-amino-1H-pyrazol-4-yl)-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide; and
5-chloro-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3-thiazol-4-ylbenzenesulfonamide.
PREPARATION 1
3′-(trifluoromethyl)biphenyl-4-ol
An aqueous solution of sodium hydrogen carbonate (6.9 g in 18 mL water, 82 mmol) was added to a stirred solution of 3-trifluoromethylbenzeneboronic acid (7.77 g, 41 mmol) and 4-iodophenol (6.0 g, 30 mmol) in 1,4-dioxane (90 mL). The reaction mixture was degassed, then tetrakis(triphenylphosphine) palladium (0) (1.58 g, 1.36 mmol) was added and the reaction mixture heated at 100° C. for 18 hours. The mixture was diluted with a 2M aqueous solution of HCl and extracted with ethyl acetate (50 mL). The organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (5%-40% ethyl acetate in heptane gradient elution) to afford the title compound (2.17 g, 30%) as an oil.
1HNMR (d6-DMSO): δ 4.95 (br s, 1H), 6.95 (m, 2H), 7.45-7.60 (m, 4H), 7.70 (m, 1H), 7.80 (m, 1H).
PREPARATION 2
3-Iodo-3′-(trifluoromethyl)biphenyl-4-ol
To a solution of 3′-(trifluoromethyl)biphenyl-4-ol (Preparation 1, 2.17 g, 9.11 mmol) in acetic acid (20 mL) at 0° C. was added N-iodosuccinimide (2.05 g, 9.11 mmol). The reaction was allowed to warm to room temperature and stirred for 48 hours before the addition of water (20 mL). The reaction mixture was extracted with dichloromethane (2×20 mL) and the combined extracts were washed with saturated aqueous sodium thiosulfate solution, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (2%-20% ethyl acetate in heptane gradient elution) to afford the title compound (1.30 g, 39%).
LCMS Rt=3.54 minutes MS m/z 363 [M-H]
PREPARATION 3
3-Pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-ol
To a solution of 4-(tributylstannyl)pyridazine (1.71 g, 4.64 mmol) and 3-iodo-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 2, 1.30 g, 3.57 mmol) in N,N-dimethylformamide (20 mL) was added caesium fluoride (1.10 g, 7.14 mmol). The mixture was degassed before the addition of tetrakis(triphenylphosphine) palladium (0) (412 mg, 0.357 mmol), then heated to 45° C. for 4 hours. The reaction was concentrated in vacuo and purified by silica gel column chromatography (0%-20% methanol in dichloromethane gradient elution). The residue was triturated with acetonitrile and filtered to afford the title compound (420 mg, 37%) as a solid.
1HNMR (d6-DMSO): δ 7.23 (m, 1H), 7.60-7.75 (m, 3H), 7.83 (m, 1H), 7.96-8.10 (m, 3H), 9.23 (s, 1H), 10.48 (m, 1H)
LCMS Rt=2.93 minutes MS m/z 317 [MH]+
PREPARATION 4
3-Pyridazin-4-ylbiphenyl-4-ol
3-Iodobiphenyl-4-ol (1 g, 3.4 mmol) was mixed with 4-(tributylstannyl)pyridazine (1.25 g 3.4 mmol), caesium fluoride (1.03 g 6.8 mmol), tetrakis(triphenylphosphine) palladium (0) (195 mg, 0.17 mmol) and copper iodide (128 mg 0.68 mmol) in acetonitrile (10 mL). The reaction was degassed 3 times before being placed under nitrogen and heated to 45° C. for 16 hours. The reaction was diluted with acetonitrile (20 mL), washed with heptane (2×20 mL) then absorbed onto silica and purified by silica gel column chromatography (ISCO™, 40 g, 50%-100% ethyl acetate in heptane gradient elution) to afford the title compound (270 mg, 32%) as a pale yellow solid.
1HNMR (CDCl3): δ 7.11 (d, 1H), 7.31 (m, 1H), 7.42 (m, 2H) 7.63 (m, 1H) 7.68 (m, 2H) 7.78 (m, 1H), 7.98 (m, 1H) 9.23 (m, 1H), 9.57 (m, 1H) 10.38 (br s, 1H)
LCMS Rt=1.52 minutes MS m/z 249 [MH]+
PREPARATION 5
4-Bromopyridazine hydrobromide
3-Bromofuran (15 g, 102 mmol) and potassium acetate (27.6 g, 281 mmol) were suspended in acetic acid (90 mL). Bromine (5.26 mL, 102 mmol) in acetic acid (45 mL) was added dropwise. The reaction mixture was then stirred for one hour and then concentrated in vacuo and azeotropically dried with toluene (×3). The residue was dissolved in ethanol (150 mL) and hydrazine hydrate (15 mL, 309 mmol) was added dropwise to the solution, which was then stirred at room temperature for two hours. The reaction was diluted with tert-butylmethyl ether (300 mL) and a solution of saturated aqueous brine (200 mL). The aqueous layer was separated and extracted with further tert-butylmethyl ether and then with ethyl acetate (×2). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was dissolved in 1,4-dioxane (500 mL) and hydrobromic acid in acetic acid (15 mL) was added dropwise. A brown solid formed. The reaction mixture was concentrated in vacuo and the resulting solid triturated with acetone and filtered to yield the title compound (11 g, 46%) as a brown solid.
1HNMR (d6-DMSO): δ 8.11 (m, 1H), 9.11 (d, 1H), 9.49 (s, 1H)
LCMS Rt=0.75 minutes MS m/z 159 [MH]+
PREPARATION 6
4-(5-Chloro-2-methoxyphenyl)pyridazine
To an argon purged flask containing toluene (187 mL), ethanol (20.6 mL) and a 2M aqueous solution of sodium carbonate (132.3 mL) was added 4-bromopyridazine hydrobromide (Preparation 5, 15 g, 64 mmol), 5-chloro-2-methoxybenzeneboronic acid (13.4 g, 72 mmol) and tetrakis(triphenylphosphine) palladium (0) (3.2 g, 2.8 mmol). The flask was purged with argon again, then the reaction mixture heated to 110° C. for 4 hours. The mixture was filtered through Celite™ and the filtrate concentrated in vacuo. The residue was partitioned between ethyl acetate and water. The organic layer was washed with water and brine, then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude residue was dissolved in ethyl acetate and extracted with a 2M aqueous solution of hydrogen chloride (×3). The aqueous layer was basified with sodium hydrogen carbonate and extracted with ethyl acetate. The organic layer was then washed with water and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford the title compound (8 g, 58%).
1HNMR (CDCl3): δ 3.84 (s, 3H), 6.96 (d, 1H), 7.34 (s, 1H), 7.38-7.41 (dd, 1H), 7.61 (d, 1H), 9.20-9.21 (d, 1H), 9.37 (s, 1H).
LCMS Rt=2.89 minutes MS m/z 221 [MH]+
PREPARATION 7
4-Chloro-2-pyridazin-4-ylphenol
To a stirred solution of 4-(5-chloro-2-methoxyphenyl)pyridazine (Preparation 6, 22 g, 100 mmol) in dichloromethane (200 mL) at 0° C. was added drop wise a solution of boron tribromide (48 mL, 499 mmol) in dichloromethane (200 mL). The reaction mixture was stirred at room temperature for 18 hours. The reaction was quenched by pouring onto crushed ice and basifying the mixture to pH 8 with sodium hydrogen carbonate. The mixture was extracted with dichloromethane. The aqueous layer was extracted further with ethyl acetate. The organics were combined and dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (0%-4% methanol in dichloromethane gradient elution) to afford the title compound (16.5 g, 80%)
1HNMR (d6-DMSO): δ 6.99 (d, 1H), 7.31 (dd, 1H), 7.53 (d, 1H), 7.86 (m, 1H), 9.20 (d, 1H), 9.41 (s, 1H), 10.45 (s, 1H).
LCMS Rt=2.81 minutes Ms m/z 207 [MH]+
PREPARATION 8
3-Pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-ol
To a solution of 4-chloro-2-pyridazin-4-ylphenol (Preparation 7, 1.5 g, 7.26 mmol) and 4-trifluoromethylbenzene boronic acid (3.45 g, 18.1 mmol) in 1,4-dioxane (20 mL) was added a solution of potassium carbonate (2.0 g, 14.5 mmol) in water (4 mL). The reaction mixture was degassed, then bis(tri-t-butylphosphine) palladium (0) (371 mg, 0.726 mmol) added and the reaction mixture heated to 100° C. for 18 hours. The mixture was diluted with a 2M aqueous solution of hydrogen chloride and brine, then extracted with ethyl acetate. The organic layer was separated and dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (0%-100% ethyl acetate in dichloromethane gradient elution) to afford the title compound as a solid (940 mg, 41%).
1HNMR (d6-DMSO): δ 7.13 (d, 1H), 7.68-7.77 (m, 3H), 7.83-8.02 (m, 4H), 9.23 (s, 1H), 9.58 (s, 1H), 10.52 (br s, 1H).
LCMS Rt=2.36 minutes MS m/z 317 [MH]+
PREPARATION 9
3-Pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-ol
To a solution of 4-chloro-2-pyridizin-4-ylphenol (Preparation 7, 1.5 g, 7.26 mmol) and 2-trifluoromethylbenzene boronic acid (3.45 g, 18.1 mmol) in 1,4-dioxane (20 mL) was added a solution of potassium carbonate (2.0 g, 14.5 mmol) in water (4 mL). The reaction mixture was degassed, then bis(tri-t-butylphosphine) palladium (0) (371 mg, 0.726 mmol) added and the reaction mixture heated to 100° C. for 18 hours. Further 2-trifluoromethylbenzene boronic acid (2.76 g, 14.5 mmol), potassium carbonate (2.0 g, 14.5 mmol) and bis(tri-t-butylphosphine)palladium (0) (371 mg, 0.726 mmol) were added and the mixture heated for a further 24 hours at 115° C. The reaction still did not reach completion, therefore tetrakistriphenylphosphine palladium (0) (200 mg, 0.173 mmol) was added. As no further progression of the reaction was observed the mixture was diluted with a 2M aqueous solution of hydrogen chloride and extracted with ethyl acetate. The organic layer was separated and dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (0%-100% ethyl acetate in dichloromethane gradient elution) to afford the title compound as a solid (466 mg, 20%).
1HNMR (CDCl3): δ 7.25 (d, 1H), 7.31-7.38 (m, 3H), 7.41-48 (m, 1H), 7.52-7.60 (m, 1H), 7.75 (d, 1H), 7.90-7.95 (m, 1H), 9.10-9.18 (m, 1H), 9.68 (s, 1H)
LCMS Rt=2.37 minutes MS m/z 317 [MH]+
PREPARATION 10
3-Chloro-4-fluoro-N-(pyridazin-3-yl)benzenesulfonamide
To a solution of pyridazin-3-amine (5.0 g, 52.63 mmol) in anhydrous acetonitrile (250 mL) was added 3-chloro-4-fluorobenzenesulfonyl chloride (12.05 g, 52.63 mmol) followed by 1,4-diazabicyclo[2,2,2]octane (5.9 g, 52.63 mmol). The reaction was stirred at room temperature for 18 hours. A solid was observed which was collected by filtration and washed with acetonitrile. The filtrate was concentrated in vacuo and the resulting residue purified by silica gel column chromatography (0%-10% methanol in chloroform gradient elution) to afford the title compound (5.3 g, 35%)
1HNMR (d6-DMSO): δ 7.58 (m, 1H), 7.74 (m, 1H), 7.85 (m, 1H), 7.93 (m, 1H), 8.02 (m, 1H), 8.35 (br m, 1H), 14.62 (br, s 1H).
PREPARATION 11
3-Chloro-4-fluoro-N-(methoxymethyl)-N-(pyridazin-3-yl)benzenesulfonamide and 3-chloro-4-fluoro-N-[(3E)-2-(methoxymethyl)pyridazin-3(2H)-ylidene]benzenesulfonamide
To 3-chloro-4-fluoro-N-(pyridazin-3-yl)benzenesulfonamide (Preparation 10, 850 mg, 3.0 mmol) in dichloromethane (20 mL) at 0° C. was added N,N-diisopropylethylamine (0.77 mL, 4.4 mmol) and chloromethyl methyl ether (0.25 mL, 3.2 mmol). The reaction mixture was stirred at room temperature for 3 hours. The mixture was diluted with ethyl acetate, and washed sequentially with a 1N aqueous solution of sodium hydroxide, water and brine. The organics were dried over anhydrous anhydrous sodium sulfate, filtered and concentrated in vacuo to afford the title compounds as a brown foam (910 mg, 91%). The product was isolated as a mixture of regioisomers that were used without separation in the next step.
LCMS Rt=1.26 minutes and 1.52 minutes MS m/z 332 [MH]+
PREPARATION 12
N-(2,4-dimethoxybenzyl)pyrimidin-2-amine
A mixture of 2-chloropyrimidine (1.37 g, 12 mmol), 2,4-dimethoxybenzylamine (2.61 g, 15.6 mmol) and triethylamine (2.51 mL, 18 mmol) in ethanol (8 mL) was heated in a Biotage Initiator™ microwave at 120° C. for 15 minutes. The reaction mixture was diluted with water and extracted with dichloromethane (×3). The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20-50% ethyl acetate in heptane gradient elution) to afford the title compound as a white solid (2.14 g, 72%).
1HNMR (CD3OD): δ 3.76 (s, 3H), 3.83 (s, 3H), 4.47 (s, 2H), 6.42 (m, 1H), 6.52 (m, 1H), 6.58 (m, 1H), 7.14 (m, 1H), 8.24 (m, 2H)
PREPARATION 13
5-Chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-pyrimidin-2-yl-benzenesulfonamide
A solution of (2,4-dimethoxybenzyl)-pyrimidin-2-yl-amine (Preparation 12, 736 mg, 3 mmol) in anhydrous tetrahydrofuran (20 mL) was cooled to −78° C. before the addition of a 1M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (3.30 mL, 3.30 mmol). The reaction was allowed to warm to 0° C. for 30 minutes before cooling again to −78° C. The resulting solution was added to a solution of 3-chloro-4,6-difluorobenzenesulfonyl chloride (890 mg, 3.6 mmol) in tetrahydrofuran (10 mL) at −78° C. After 30 minutes at this temperature the reaction was warmed to room temperature and stirred for 24 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride solution and extracted into ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (50-100% dichloromethane in heptane gradient elution) to afford the title compound as a white solid (260 mg, 19%).
1HNMR (d6-DMSO): δ 3.73 (s, 3H), 3.75 (s, 3H), 5.27 (s, 2H), 6.47 (m, 1H), 6.57 (m, 1H), 7.01 (m, 1H), 7.18 (m, 1H), 7.82 (m, 1H), 8.10 (m, 1H), 8.57 (m, 2H).
LCMS Rt=1.77 minutes MS m/z 456 [MH]+
PREPARATION 14
N-(2,4-Dimethoxybenzyl)-1,3,4-thiadiazol-2-amine
2,4-Dimethoxybenzaldehyde (771 g, 4.64 mol) was added to a suspension of 2-amino-1,3,4-thiadiazole (391.2 g, 3.87 mol) in xylene (5.87 L) and heated to reflux for 18 hours. Dean-Stark apparatus was used to remove the water. The reaction mixture was cooled to 5° C. and diluted with 2-methyltetrahydrofuran (2.93 L). Sodium tetrahydroborate (73.17 g, 1.93 mol) was added as a single portion. Methanol (782.8 mL) was then added slowly over 30 minutes, maintaining the temperature below 15° C. After a further 30 minutes, water (1 L) was added followed by saturated aqueous sodium bicarbonate solution (1 L) and the mixture stirred at ambient temperature for 18 hours. The biphasic mixture was diluted with 2-methyltetrahydrofuran and heated to 43° C. to aid dissolution. The layers were separated and the organic layer washed with water (3 L) before concentrating in vacuo. The resulting solid was slurried in heptanes (2.5 L), homogenised, filtered, washed with tert-butylmethyl ether and dried to afford the title compound (715 g).
1HNMR (d6-DMSO): δ 3.75 (s, 3H), 3.80 (s, 3H), 4.37 (d, 2H), 6.49 (m, 1H), 6.58 (s, 1H), 7.19 (d, 1H), 7.97 (m, 1H), 8.59 (s, 1H).
LCMS Rt=1.36 minutes MS m/z 252 [MNa]+
PREPARATION 15
3-Cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
N-(2,4-Dimethoxybenzyl)-1,3,4-thiadiazol-2-amine (Preparation 14, 5.72 g, 22.8 mmol) was dissolved in 2-methyltetrahydrofuran (100 mL) and the suspension cooled to −50° C. A 1M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (34.1 mL, 34.1 mmol) was added slowly over 15 minutes. This suspension was stirred at −50° C. for 5 minutes, warmed to 10° C. then cooled again to −78° C. A solution of 3-cyano-4-fluorobenzene-1-sulfonyl chloride (10 g, 45.5 mmol) in tetrahydrofuran (20 mL) was then added drop wise. The pale orange solution was allowed to warmed to 20° C. for 18 hours. The reaction was quenched with an aqueous solution of saturated ammonium chloride (50 mL) and stirred vigorously for 5 minutes. Ethyl acetate (100 mL) was added and the layers separated. The organic layer was washed with water (100 mL) and concentrated in vacuo to give an orange gum. The gum was dissolved in ethyl acetate and eluted through a silica plug before being purified by silica gel column chromatography (ISCO™, 50% ethyl acetate in heptane) to afford the title compound as a pale yellow oil (2.96 g).
1HNMR (CDCl3): δ 3.59 (s, 3H), 3.78 (s, 3H), 5.14 (s, 2H), 6.24 (s, 1H), 6.35 (m, 1H), 7.14 (m, 1H), 7.25 (m, 1H), 7.85 (m, 1H), 8.04 (m, 1H), 8.88 (s, 1H).
LCMS Rt=3.21 minutes MS m/z 435 [MH]+
PREPARATION 16
5-Chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
N-(2,4-Dimethoxybenzyl)-1,3,4-thiadiazol-2-amine (Preparation 14, 203.4 g, 0.809 mol) was dissolved in 2-methyltetrahydrofuran (1.63 L) and the yellow suspension cooled to between −38° C. and −45° C. A 1M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (890 mL, 0.890 mol) was added slowly over 15 minutes keeping the temperature between −38° C. and −45° C. to give an orange suspension. This orange suspension was stirred at −38° C. to −45° C. for 45 minutes and then a solution of 5-chloro-2,4-difluorobenzenesulfonyl chloride, (200 g, 0.809 mol) in 2-methyltetrahydrofuran (407 mL) added slowly over 20 minutes keeping the temperature between −38° C. and −45° C. The mixture was warmed to 15° C. over 1 hour. The reaction was quenched with a solution of ammonium chloride (203.4 g, 3.80 mol) in water (1.02 L) and stirred vigorously for 5 minutes. The layers were separated and the organic layer washed with water (813.6 mL) and concentrated in vacuo to give an orange solid which was triturated with isopropyl acetate (1.22 L) to afford the title compound as a yellow-orange solid (218.6 g).
1HNMR (CDCl3): δ 3.71 (s, 3H), 3.78 (s, 3H), 5.35 (m, 2H), 6.26 (m, 1H), 6.38 (m, 1H), 6.99 (m, 1H), 7.27 (m, 1H), 7.83 (m, 1H), 8.87 (m, 1H).
LCMS Rt=1.76 minutes MS m/z 484 [MNa]+
PREPARATION 17
3-Chloro-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
The title compound was prepared according to the procedure used in Preparation 16, using 3-chloro-4-fluorobenzene-1-sulfonyl chloride (0.91 g) to obtain the title compound as a white solid (1.3 g).
LCMS Rt=1.70 minutes MS m/z 466 [MNa]+
PREPARATION 18
N-(2,4-dimethoxybenzyl)-1,2,4-thiadiazol-5-amine
A mixture of 5-amino-1,2,4-thiadiazole (1 g, 9.89 mmol) and 2,4-dimethoxybenzaldehyde (1.81 g, 10.9 mmol) in toluene (30 mL) was refluxed under Dean-Stark conditions for 2 hours. The reaction mixture was evaporated and the residue taken up in methanol (25 mL), sodium borohydride (600 mg, 15.9 mmol) was added carefully in small portions (vigorous effervescence after each addition), and the reaction was left to stir for 18 hours at ambient temperature. A 2M aqueous solution of hydrogen chloride (1 mL) was added followed by a 2M aqueous solution of sodium hydroxide (10 mL). The bulk of the methanol was evaporated, water (20 mL) was added and the mixture extracted with ethyl acetate (2×30 mL). The combined organic phase was washed with brine (20 mL), dried, and concentrated in vacuo. The residue was purified by silica gel column chromatography (ISCO™ column 120 g; 25-60% ethyl acetate in heptane gradient elution) to furnish a semi-solid residue that was re-evaporated from heptane. tert-Butylmethyl ether (2-3 mL) was added, followed by heptane (2-3 mL). The resulting solid was collected by filtration, washed with heptane and dried to afford the title compound (1.22 g).
1HNMR (d6-DMSO): δ 3.73 (s, 3H), 3.78 (s, 3H), 4.36 (d, 2H), 6.47 (dd, 1H), 6.56 (d, 1H), 7.15 (d, 1H), 7.88 (s, 1H), 8.65 (br. s, 1H)
PREPARATION 19
3-Cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
N-(2,4-Dimethoxybenzyl)-1,2,4-thiadiazol-5-amine (Preparation 18, 42.8 g, 170 mmol) was dissolved in anhydrous tetrahydrofuran (600 mL) and stirred under a nitrogen atmosphere at −78° C. A 1M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (238 mL, 238 mmol) was added drop wise over 30 minutes maintaining the temperature between −65° C. and −70° C. The reaction mixture was left at −78° C. for 5 minutes, then allowed to warm to −10° C. over 1.5 hours. Upon reaching −10° C., the brown reaction mixture was cooled to −78° C. again, and a solution of 3-cyano-4-fluorobenzene sulfonyl chloride (48.6 g, 221 mmol) in tetrahydrofuran (200 mL) was added drop wise over 30 minutes maintaining the temperature between −65° C. and −70° C. The brown solution was allowed to warm gradually to ambient temperature and stirred for 18 hours. The reaction mixture was diluted with ethyl acetate, washed with a saturated ammonium chloride solution, and extracted with further ethyl acetate. The combined organics were dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to afford a brown residue. The residue was purified by silica gel column chromatography (10%-30% ethyl acetate in heptane gradient elution) to afford the title compound as a white solid (52.3 g, 71%).
1HNMR (CDCl3): δ 3.60 (s, 3H), 3.79 (s, 3H), 5.32 (s, 2H), 6.22 (s, 1H), 6.32-6.48 (m, 1H), 7.05-7.09 (m, 1H), 7.18-7.24 (m, 1H), 7.70-7.73 (m, 1H), 7.92-7.99 (m, 1H), 8.22 (s, 1H).
LCMS Rt=3.47 minutes
PREPARATION 20
3-Cyano-N-(2,4-dimethoxybenzyl)-4-{[3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
To a solution of 3-cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide (Preparation 19, 206 mg, 0.474 mmol) and 3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 3, 150 mg, 0.474 mmol) in dimethylsulfoxide (5 mL) was added potassium carbonate (196 mg, 1.42 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with a 1M aqueous solution of sodium hydroxide whereupon a fine precipitate formed. The mixture was extracted with ethyl acetate. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to afford the title compound as an oil (380 mg, 111%, contains residual dimethylsulfoxide). The material was used without purification in the next step.
1HNMR (CDCl3): δ 3.70-3.80 (m, 6H), 5.25 (m, 2H), 6.30 (m, 2H), 6.65 (d, 1H), 7.0 (m, 2H), 7.55-7.90 (m, 9H), 8.15 (s, 1H), 9.25 (m, 1H), 9.35 (m, 1H).
PREPARATION 21
3-Cyano-N-(2,4-dimethoxybenzyl)-4-{[3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
To a solution of 3-cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide (Preparation 19, 206 mg, 0.474 mmol) and 3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-ol (Preparation 9, 150 mg, 0.474 mmol) in dimethylsulfoxide (5 mL) was added potassium carbonate (196 mg, 1.42 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with a 1M aqueous solution of sodium hydroxide whereupon a precipitate formed. The precipitate was collected by filtration and washed with water to afford the title compound as a solid (400 mg, 115%, contains residual dimethylsulfoxide). The material was used without purification in the next step.
1HNMR (CDCl3): δ 3.42 (s, 3H), 3.72 (s, 3H), 5.22 (s, 2H), 6.03 (s, 1H), 6.23 (d, 1H), 6.62 (d, 1H), 7.00 (d, 1H), 7.10 (d, 1H), 7.31 (d, 1H), 7.42-7.53 (m, 3H), 7.55 (t, 1H), 7.61 (s, 1H), 7.63-7.78 (m, 3H), 8.10 (s, 1H), 9.27 (d, 1H), 9.30 (s, 1H).
LCMS Rt=3.97 minutes MS m/z 731 [MH]+
PREPARATION 22
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
To a solution of 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 16, 219 mg, 0.474 mmol) and 3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 3, 150 mg, 0.474 mmol) in dimethylsulfoxide (5 mL) was added potassium carbonate (196 mg, 1.42 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with a 1M aqueous solution of sodium hydroxide whereupon a precipitate formed. The solid was collected by filtration, washed with water and freeze dried to afford the title compound as a solid (266 mg, 74%).
1HNMR (CD3OD): δ 3.58 (s, 3H), 3.70 (s, 3H), 5.21 (s, 2H), 6.17 (s, 1H), 6.37 (dd, 1H), 6.83 (d, 1H), 7.17 (d, 1H), 7.28 (d, 1H), 7.64-7.71 (m, 3H), 7.91 (d, 1H), 7.97-8.02 (m, 4H), 9.08 (s, 1H), 9.23 (d, 1H), 9.50 (s, 1H).
LCMS Rt=3.55 minutes MS m/z 758 [MH]+
PREPARATION 23
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-Pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
Prepared according to Preparation 22 using 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 16, 291 mg, 0.63 mmol) and 3-pyridazin-4-yl-2′-(trifluoromethyl)biphenyl-4-ol (Preparation 9, 200 mg, 0.63 mmol) to afford the title compound as a solid (339 mg, 71%).
1HNMR (CDCl3): δ 3.57 (s, 3H), 3.64 (s, 3H), 5.21 (s, 2H), 6.17 (s, 1H), 6.25 (d, 1H), 6.42 (d, 1H), 7.03 (d, 1H), 7.17 (d, 1H), 7.28-7.31 (m, 2H), 7.41-7.50 (m, 3H), 7.55 (t, 1H), 7.60-7.63 (m, 1H), 7.72 (t, 1H), 8.78 (s, 1H), 9.17 (d, 1H), 9.38 (s, 1H).
LCMS Rt=4.13 minutes MS m/z 758 [MH]+
PREPARATION 24
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-Pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
Prepared according to Preparation 22 using 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 16, 175 mg, 0.379 mmol) and 3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-ol (Preparation 8, 120 mg, 0.379 mmol) to afford the title compound as a solid (213 mg, 74%).
1HNMR (CD3OD): δ 3.59 (s, 3H), 3.73 (s, 3H), 5.25 (s, 2H), 6.18 (s, 1H), 6.37 (d, 1H), 6.84 (d, 1H), 7.17 (d, 1H), 7.30 (d, 1H), 7.66-8.01 (m, 8H), 9.10 (s, 1H), 9.23 (d, 1H), 9.50 (s, 1H).
LCMS Rt=3.79 minutes MS m/z 758 [MH]+
PREPARATION 25
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-[(3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
3-Pyridazin-4-ylbiphenyl-4-ol (Preparation 4, 50 mg, 0.2 mmol) and 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 16, 93 mg, 0.2 mmol) were dissolved in dimethylsulfoxide (2 mL). Potassium carbonate (83 mg, 0.6 mmol) was added and the reaction stirred at room temperature for 16 hours. The crude material was partitioned between ethyl acetate (20 mL) and water (20 mL), the organic layer separated, concentrated in vacuo and purified by silica gel column chromatography (ISCO™, 12 g silica, 0-100% ethyl acetate in heptane gradient elution). The appropriate fractions were combined and concentrated in vacuo to afford the title compound as a gum (100 mg, 72%).
1HNMR (CDCl3): δ 3.66 (s, 3H) 3.73 (s, 3H) 5.28 (s, 2H) 6.24 (m, 1H), 6.35 (m, 1H), 6.51 (d, 1H) 7.18 (d, 1H) 7.22 (d, 1H) 7.45 (m, 4H), 7.60 (m, 2H) 7.78 (m, 3H) 8.81 (s, 1H) 9.23 (m, 1H), 9.45 (m, 1H)
LCMS Rt=1.82 minutes MS m/z 690 [MH]+
PREPARATION 26
3-Cyano-N-(2,4-dimethoxybenzyl)-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide
Prepared according to Preparation 22 using 3-cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,2,4-thiadiazol-5-ylbenzenesulfonamide (Preparation 19, 165 mg, 0.379 mmol) and 3-Pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-ol (Preparation 8, 120 mg, 0.379 mmol) to afford the title compound as a solid (219 mg, 79%).
1HNMR (d6-DMSO): δ 3.57 (s, 3H), 3.70 (s, 3H), 5.19 (s, 2H), 6.37 (s, 1H), 6.41 (d, 1H), 6.99 (d, 1H), 7.09 (d, 1H), 7.53 (d, 1H), 7.84 (m, 2H), 7.87-7.91 (m, 1H), 7.97-8.08 (m, 4H), 8.18 (d, 2H), 8.40 (s, 1H), 9.25 (d, 1H), 9.44 (s, 1H).
LCMS Rt=3.76 minutes MS m/z 731 [MH]+
PREPARATION 27
5-[4-(Benzyloxy)biphenyl-3-yl]-1-methyl-1H-pyrazole
Solution A: A stirred mixture of 4-(benzyloxy)-3-bromobiphenyl (7.5 g, 22.1 mmol, J. Med. Chem. 1988, 31, 1437-1445) and (1-methyl-1H-pyrazol-5-yl)boronic acid (2.8 g, 22.1 mmol) in 1,4-dioxane (59 mL) was purged with argon for 20 minutes. Tris(dibenzylideneacetone)dipalladium (0) (810 mg, 0.88 mmol) and tricyclohexyl phosphine (495 mg, 1.8 mmol) were added.
Solution B: In a separate flask dipotassium phosphate (9.4 g, 44.2 mmol) was dissolved in water (29 mL) and was also purged with argon for 20 minutes.
Solution B was added to solution A and the resulting mixture was heated at 100° C. for 18 hours. After cooling to room temperature, the mixture was filtered through a pad of silica gel, and washed with ethyl acetate. The filtrate was concentrated in vacuo, diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (20% Et2O in hexane) to afford the title compound (5.0 g, 67%).
1HNMR (d6-DMSO): δ 3.67 (s, 3H), 5.21 (s, 2H), 6.35 (s, 1H), 7.31-7.46 (m, 10H), 7.55 (s, 1H), 7.67 (d, 2H), 7.73-7.76 (m, 1H)
PREPARATION 28
3-(1-Methyl-1H-pyrazol-5-yl)biphenyl-4-ol
To a stirred solution of 5-[4-(benzyloxy)biphenyl-3-yl]-1-methyl-1H-pyrazole (Preparation 27, 3.0 g, 8.8 mmol) in methanol (26 mL) was added palladium on carbon (300 mg). The mixture was stirred under hydrogen gas for 16 hours. The reaction mixture was filtered through Celite™, and washed with tetrahydrofuran. The resulting filtrate was concentrated in vacuo. The residue was dissolved in ethyl acetate (26 mL) and degassed with argon prior to addition of palladium on carbon (300 mg). The reaction mixture was stirred under hydrogen gas for 6 hours. The reaction mixture was filtered through Celite™ and the filtrate concentrated in vacuo to give a solid. The solid was triturated with hexane to afford the title compound as a white solid (1.7 g, 77%).
1HNMR (d6-DMSO): δ 3.71 (s, 3H), 6.30 (d, 1H), 7.06 (d, 1H), 7.29 (t, 1H), 7.39-7.44 (m, 4H), 7.57-7.62 (m, 3H), 10.13 (br s, 1H)
LCMS Rt=3.23 minutes MS m/z 251 [MH]+
PREPARATION 29
5-[4-(2-Fluoro-4-nitrophenoxy)biphenyl-3-yl]-1-methyl-1H-pyrazole
To a stirred solution of 3-(1-Methyl-1H-pyrazol-5-yl)biphenyl-4-ol (Preparation 28, 600 mg, 2.39 mmol) in N,N-dimethylformamide (6 mL) at 0° C. was added potassium carbonate (332 mg, 2.39 mmol). The mixture was stirred for 30 minutes at 0° C. 3,4-Difluoronitrobenzene (318 mg, 1.99 mmol) was added drop wise to the reaction mixture and allowed to stir at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (20 mL). The organic layer was washed sequentially with water (3×10 mL) and brine (1×10 ml), then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford the title compound (870 mg, quantitative). This material was used without purification in the next step.
1HNMR (d6-DMSO): δ 3.78 (s, 3H), 6.32 (d, 1H), 7.13 (t, 1H), 7.38-7.42 (m, 3H), 7.49 (t, 2H), 7.76 (d, 2H), 7.82 (d, 1H), 7.88 (dd, 1H), 8.01 (d, 1H), 8.27 (dd, 1H).
PREPARATION 30
3-Fluoro-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}aniline
5-[4-(2-Fluoro-4-nitrophenoxy)biphenyl-3-yl]-1-methyl-1H-pyrazole (Preparation 29, 870 mg, 2.33 mmol) was dissolved in ethanol (8 mL) and water (2 mL). Iron powder (624 mg, 11.17 mmol) and CaCl2 (248 mg, 2.33 mmol) were then added and the reaction mixture was refluxed for 3 hours. After filtration through Celite™, the filtrate was concentrated in vacuo. The residue was partitioned between dichloromethane and water. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (100-200 mesh silica gel, 15% ethyl acetate in hexane) to afford the title compound (700 mg, 84%).
1HNMR (d6-DMSO): δ 3.79 (s, 3H), 5.40 (br s, 1H), 6.40 (dd, 1H), 6.43 (d, 1H), 6.50 (dd, 1H), 6.76 (d, 1H), 6.94 (t, 1H), 7.34 (t, 1H), 7.44 (t, 2H), 7.49 (d, 1H), 7.61-7.68 (m, 4H).
PREPARATION 31
3-Fluoro-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}benzenesulfonyl chloride
Solution A: To a stirred suspension of 3-fluoro-4-{[3-(1-methyl-1H-pyrazol-5-yl)biphenyl-4-yl]oxy}aniline (Preparation 30, 700 mg, 1.94 mmol) in a mixture of concentrated hydrogen chloride (1.75 mL) and acetic acid (1.75 mL) at 0° C. was added a solution of sodium nitrite (148 mg, 2.14 mmol) in water (0.87 mL) and the mixture stirred at 0° C. for 30 minutes.
Solution B: In another flask, acetic acid (3.5 mL) was saturated with sulfur dioxide at 0° C. followed by the addition of copper (II) chloride dihydrate (133 mg, 0.779 mmol) portion wise.
Solution A was added drop wise to solution B at 0° C. and stirred at room temperature for 14 hours. The reaction mixture was then diluted with water (10 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layer was neutralized with saturated aqueous sodium hydrogen carbonate solution. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (100-200 mesh silica gel, 10% ethyl acetate in hexane) to afford the title compound (350 mg, 41%).
1HNMR (d6-DMSO): δ 3.80 (s, 3H), 6.41 (s, 1H), 7.02 (d, 1H), 7.09 (t, 1H), 7.35-7.48 (m, 6H), 7.70-7.76 (m, 4H)
PREPARATION 32
3-Cyano-4-fluoro-N-1,3-thiazol-2-ylbenzenesulfonamide
3-Cyano-4-fluorobenzenesulfonyl chloride (10 g, 45.53 mmol) was added portion wise to a solution of 2-aminothiazole (5 g, 50.13 mmol) in dichloromethane (50 mL) and pyridine (18.4 mL, 228 mmol) at 0° C. The reaction mixture was allowed to warm to room temperature. After 1 hour a precipitate was observed. The mixture was stirred for 18 hours at room temperature. The mixture was sonicated for 2.5 hours until the solid had dissolved, then left to stir at room temperature for 18 hours. The reaction mixture was then concentrated in vacuo and azeotropically dried with toluene (2×100 mL). The residue was diluted carefully with a 1M aqueous solution of hydrogen chloride and stirred for 1 hour at room temperature whereupon a precipitate formed. The brown solid was collected by filtration and triturated with dichloromethane to afford the title compound as a brown solid (7.8 g, 60%).
1HNMR (d6-DMSO): δ 6.90 (m, 1H), 7.30 (m, 1H), 7.65 (t, 1H), 8.15 (m, 1H), 8.30 (m, 1H), 12.90 (br s, 1H).
LCMS Rt=2.18 minutes MS m/z 284 [MH]+, 282 [MH]−
PREPARATION 33
3-Cyano-4-fluoro-N-[(4S,5R)-5-fluoro-4-hydroxy-4,5-dihydro-1,3-thiazol-2-yl]benzenesulfonamide
3-Cyano-4-fluoro-N-1,3-thiazol-2-ylbenzenesulfonamide (Preparation 32, 1.99 g, 7.02 mmol) and 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (3.12 g, 8.81 mmol) were dissolved in acetonitrile (25 mL) and water (1 mL) and heated to 45° C. under an atmosphere of nitrogen for 24 hours. A precipitate was observed which was collected by filtration to afford the title compound as a white solid which was used without purification in the next step (1.32 g, 59%).
1HNMR (d6-DMSO): δ 5.42 (m, 1H), 6.25-6.40 (d, 1H), 7.00 (br m, 1H), 7.75 (m, 1H), 8.15 (m, 1H), 8.30 (m, 1H), 10.50 (s, 1H).
LCMS Rt=1.16 minutes MS m/z 320 [MH]+, 318 [MH]−
PREPARATION 34
3-Cyano-4-fluoro-N-(5-fluoro-1,3-thiazol-2-yl)benzenesulfonamide
To a suspension of 3-cyano-4-fluoro-N-[(4S,5R)-5-fluoro-4-hydroxy-4,5-dihydro-1,3-thiazol-2-yl]benzenesulfonamide (Preparation 33, 1.42 g, 4.45 mmol) in dichloromethane (150 mL) was added triethylamine (6.20 mL, 44.5 mmol) and acetic anhydride (1.30 mL, 13.8 mL). The reaction mixture was stirred at room temperature under an atmosphere of nitrogen for 18 hours. The mixture was washed with a 2M aqueous solution of hydrogen chloride. The organics were separated, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting residue was triturated with dichloromethane to afford the title compound as a pale yellow solid (825 mg, 62%).
1HNMR (d6-DMSO): δ 7.40 (s, 1H), 7.70 (t, 1H), 8.15 (m, 1H), 8.30 (m, 1H)
LCMS Rt=1.22 minutes MS m/z 302 [MH]+, 300 [MH]−
PREPARATION 35
tert-Butyl 3-[(methylsulfonyl)oxy]azetidine-1-carboxylate
A mixture of tert-butyl 3-hydroxyazetidine-1-carboxylate (4.98 g, 28.7 mmol) and triethylamine (4.82 mL, 62.3 mmol) in tetrahydrofuran (75 mL) was cooled to 0° C. using an ice bath. Methanesulfonyl chloride (2.46 mL, 31.8 mmol) in tetrahydrofuran (12.5 mL) was added slowly to the reaction. Once the addition was complete, the ice bath was removed and the reaction was stirred at room temperature for 4 hours. Water (100 mL) was added to the reaction, and the mixture extracted with ethyl acetate (2×150 mL). The combined organic phase was washed with brine (2×100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford the title compound as a pale yellow oil (7.11 g, 98%).
1HNMR (CDCl3): δ 1.45 (s, 9H), 3.07 (s, 3H), 4.08-4.12 (m, 2H), 4.26-4.30 (m, 2H), 5.18-5.23 (m, 1H).
LCMS Rt=2.53 minutes MS m/z 151.98 [M−Boc+H]+.
PREPARATION 36
tert-Butyl 3-hydrazinoazetidine-1-carboxylate
A suspension of tert-butyl 3-[(methylsulfonyl)oxy]azetidine-1-carboxylate (Preparation 35, 7.11 g, 28.3 mmol) in neat hydrazine monohydrate (13.7 mL, 283 mmol) was heated to 95° C. for 18 hours. The reaction was cooled to room temperature, then water (100 mL) was added and the mixture extracted with dichloromethane (5×100 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a the title compound as a clear oil (4.71 g, 89%). The compound was used without further purification in the next step.
1HNMR (CDCl3): δ 1.44 (s, 9H), 3.31 (br.s, 3H), 3.73-3.79 (m, 3H), 4.01-4.08 (m, 2H).
PREPARATION 37
1-[4-Hydroxy-4′-(trifluoromethyl)biphenyl-3-yl]ethanone
A mixture of 5-bromo-2-hydroxy acetophenone (1.00 g, 4.65 mmol), 4-(trifluoromethyl)phenylboronic acid (1.32 g, 6.97 mmol), potassium carbonate (1.30 g, 9.38 mmol), tetrakis(triphenylphosphine)palladium (0) (538 mg, 0.465 mmol) in 1,4-dioxane (30.0 mL) and water (18.0 mL) was heated to 60° C. for 18 hours under nitrogen. The reaction was allowed to cool to room temperature and concentrated in vacuo to afford a dark brown oil which was dissolved in ethyl acetate (50 mL) and filtered through Arbocel™. The Arbocel™ was washed with ethyl acetate (100 mL). The combined organics were washed sequentially with a 0.5M aqueous solution of hydrogen chloride (2×50 mL) and brine (2×100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a brown oil (2.12 g). The oil was then purified by silica gel column chromatography (5%-10% ethyl acetate in heptane gradient elution) to afford the title compound as a yellow solid (795 mg, 61%).
1HNMR (CDCl3): δ 2.72 (s, 3H), 7.11 (d, 1H), 7.65 (d, 2H), 7.71-7.75 (m, 3H), 7.94 (d, 1H), 12.34 (s, 1H).
LCMS Rt=3.63 minutes MS m/z 279.45 [MH]−.
PREPARATION 38
(2E)-3-(Dimethylamino)-1-[4-hydroxy-4′-(trifluoromethyl)biphenyl-3-yl]prop-2-en-1-one
N,N-Dimethylformamide dimethyl acetal (0.76 mL, 5.701 mmol) was added to a solution of 1-[4-hydroxy-4′-(trifluoromethyl)biphenyl-3-yl]ethanone (Preparation 37, 795 mg, 2.84 mmol) in isopropyl alcohol (4.7 mL). The reaction mixture was heated for 18 hours at 45° C. under an atmosphere of nitrogen. After 1 hour, crystallization was observed therefore the stirring was stopped. After 18 hours at 45° C., a yellow precipitate had formed. The reaction mixture was allowed to cool, the yellow precipitate was collected by filtration and washed with cold isopropyl alcohol to afford the title compound as fine yellow needle crystals (669 mg, 70%).
1HNMR (d6-DMSO): δ 3.06 (s, 3H), 3.23 (s, 3H), 6.15 (d, 1H), 6.95 (d, 1H), 7.75 (dd, 1H), 7.78 (d, 2H), 7.91 (d, 2H), 7.97 (d, 1H), 8.21 (d, 1H)
LCMS Rt=3.57 minutes MS m/z 336.44 [MH]+
PREPARATION 39
5-Chloro-N-(2,4-dimethoxybenzyl)-4-({3-[(2E)-3-(dimethylamino)prop-2-enoyl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
To a suspension of (2E)-3-(dimethylamino)-1-[4-hydroxy-4′-(trifluoromethyl)biphenyl-3-yl]prop-2-en-1-one (Preparation 38, 657 mg, 1.96 mmol) and 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 16, 879 mg, 1.90 mmol) in dimethylsulfoxide (8.0 mL) was added potassium carbonate (656 mg, 4.75 mmol). The reaction mixture was stirred for 18 hours at room temperature under an atmosphere of nitrogen. The reaction was poured into a saturated solution of aqueous ammonium chloride (20 mL) and extracted with dichloromethane (3×40 mL). The combined organic phase was washed with brine (3×40 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a thick yellow-brown oil (1.56 g). The oil was purified by silica gel column chromatography (25%-75% ethyl acetate in heptane gradient elution) to afford the title compound as an off white foam (504 mg, 34%).
1HNMR (d6-DMSO): δ 2.78 (br.s, 3H), 3.06 (br.s, 3H), 3.68 (s, 3H), 3.73 (s, 3H), 5.13 (s, 2H), 5.37 (br.s, 1H), 6.45 (dd, 1H), 6.48 (d, 1H), 6.90 (br.d, 1H), 7.09 (d, 1H), 7.35 (d, 1H), 7.52 (br.s, 1H), 7.83-7.92 (m, 5H), 7.97 (d, 2H), 9.30 (s, 1H).
19F NMR (d6-DMSO): δ −60.78 (s).
LCMS Rt=4.31 minutes MS m/z 777.18 [MH]+, 779.20 [MH]+.
PREPARATION 40
tert-Butyl 3-{5-[4-(2-chloro-4-{[(2,4-dimethoxybenzyl)(1,3,4-thiadiazol-2-yl)amino]sulfonyl}-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl]-1H-pyrazol-1-yl}azetidine-1-carboxylate
5-Chloro-N-(2,4-dimethoxybenzyl)-4-({3-[(2E)-3-(dimethylamino)prop-2-enoyl]-4′-(trifluoromethyl)biphenyl-4-yl}oxy)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 39, 504 mg, 0.648 mmol) in ethanol (10 mL) was slowly added a solution of tert-butyl 3-hydrazinoazetidine-1-carboxylate (Preparation 36, 536 mg, 2.86 mmol) in ethanol (10 mL) and acetic acid (0.23 mL) at 0° C. under nitrogen. The reaction was heated to 70° C. for 3 hours and then cooled to room temperature. The reaction mixture was neutralised to pH 7 with saturated aqueous sodium hydrogen carbonate solution (2 mL) and concentrated in vacuo to afford a yellow oil. The oil was partitioned between water (100 mL) and ethyl acetate (100 mL). The aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine (2×100 mL) dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a yellow oil (1.64 g). The oil was partially purified by silica gel column chromatography (0%-10% methanol in dichloromethane) to afford the title compound as a brown oil (419 mg) of 39% purity via LCMS. The compound was used without further purification in the next step.
LCMS Rt=4.08 minutes MS m/z 651.13 [M−Boc−DMB+H]+, 653.14 [M−Boc−DMB+H]+, 801.25 [M−Boc+H]+, 803.24 [M−Boc+H]+, 923.35 [MNa]+, 925.33 [MNa]+.
PREPARATION 41
4-{[3-(1-Azetidin-3-yl-1H-pyrazol-5-yl)-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
Trifluoroacetic acid (0.50 mL, 6.53 mmol) was added to a solution of tert-butyl 3-{5-[4-(2-chloro-4-{[(2,4-dimethoxybenzyl)(1,3,4-thiadiazol-2-yl)amino]sulfonyl}-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl]-1H-pyrazol-1-yl}azetidine-1-carboxylate (Preparation 40, 419 mg, 0.465 mmol) in dichloromethane (20 mL). The mixture was then heated to 40° C. for 18 hours under an atmosphere of nitrogen. The reaction was then cooled to room temperature and concentrated in vacuo to afford a brown residue (385.4 mg). The residue was purified by preparative HPLC (Trilution method) to afford the title compound as a white solid (46.3 mg, 11% over 2 steps).
1HNMR (d6-DMSO): δ 4.26 (d, 4H), 5.27 (t, 1H), 6.49 (s, 1H), 7.08 (d, 1H), 7.19 (d, 1H), 7.73 (d, 1H), 7.77 (d, 1H), 7.80-7.83 (m, 3H), 7.89 (dd, 1H), 7.94, (d, 2H), 8.58 (s, 1H)
LCMS Rt=2.44 minutes MS m/z 651.05 [MH]+, 653.03 [MH]+.
PREPARATION 42
1-[4-Hydroxy-2′-(trifluoromethyl)biphenyl-3-yl]ethanone
A mixture of 5-bromo-2-hydroxy acetophenone (3.00 g, 13.9 mmol), 2-(trifluoromethyl)benzeneboronic acid (3.97 g, 20.9 mmol), potassium carbonate (3.86 g, 27.9 mmol) and tetrakistriphenylphosphinepalladium (0) (1.61 g, 1.39 mmol) in 1,4-dioxane (90 mL) and water (18.0 mL) was heated to 50° C. over 2 days under an atmosphere of nitrogen. The reaction was allowed to cool to room temperature and poured into a 1M aqueous solution of hydrogen chloride (50 mL). The aqueous layer was then extracted with ethyl acetate (3×50 mL). The combined organics were washed with water (50 mL), dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to afford a brown oil. The oil was purified by silica gel column chromatography (10% ethyl acetate in heptane) to afford the title compound as a colourless oil (3.70 g, 95%).
1HNMR (CDCl3): δ 2.62 (s, 3H), 7.02 (d, 1H), 7.35 (d, 1H), 7.45 (dd, 1H), 7.49 (t, 1H), 7.59 (t, 1H), 7.72 (d, 1H), 7.77 (d, 1H)
LCMS Rt=3.67 minutes MS m/z 279 [M−H]−.
PREPARATION 43
(2E)-3-(Dimethylamino)-1-[4-hydroxy-2′-(trifluoromethyl)biphenyl-3-yl]prop-2-en-1-
N,N-Dimethylformamide dimethyl acetal (0.95 mL, 7.13 mmol) was added to a solution of 1-[4-hydroxy-2′-(trifluoromethyl)biphenyl-3-yl]ethanone (Preparation 42, 1.02 g, 3.65 mmol) in isopropyl alcohol (6.0 mL). The reaction mixture was heated for 18 hours at 45° C. under an atmosphere of nitrogen. After 1 hour, crystallization was observed therefore the stirring was stopped. After 18 hours at 45° C. a yellow precipitate had formed. The reaction mixture was allowed to cool, the yellow precipitate was filtrated and washed with cold isopropyl alcohol to afford the title compound as a yellow solid (840 mg, 69%).
1H NMR (d6-DMSO): δ 2.95 (s, 3H), 3.20 (s, 3H), 5.96 (d, 1H), 6.87 (d, 1H), 7.29 (dd, 1H), 7.44 (d, 1H), 7.59 (t, 1H), 7.71 (t, 1H), 7.82 (d, 1H), 7.86 (d, 1H), 7.93 (d, 1H)
LCMS Rt=3.41 minutes MS m/z 336.43 [MH]+.
PREPARATION 44
5-Chloro-N-(2,4-dimethoxybenzyl)-4-({3-[(2E)-3-(dimethylamino)prop-2-enoyl]-2′-(trifluoromethyl)biphenyl-4-yl}oxy)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
To a suspension of (2E)-3-(Dimethylamino)-1-[4-hydroxy-2′-(trifluoromethyl)biphenyl-3-yl]prop-2-en-1-one (Preparation 43, 809 mg, 2.41 mmol) and 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 16, 1.10 g, 2.39 mmol) in dimethylsulfoxide (10.0 mL) was added potassium carbonate (859 mg, 6.22 mmol). The reaction mixture was stirred for 18 hours at room temperature under an atmosphere of nitrogen. The reaction was poured into a saturated solution of aqueous ammonium chloride (20 mL) and extracted with dichloromethane (3×40 mL). The combined organic phase was washed with brine (3×40 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a yellow solid (1.96 g). The solid was purified by silica gel column chromatography (40%-75% ethyl acetate in heptane gradient elution) to afford the title compound as a clear glass (563 mg, 30%)
1HNMR (d6-DMSO): δ 2.77 (br.s, 3H), 3.06 (br.s, 3H), 3.68 (s, 3H), 3.73 (s, 3H), 5.13 (s, 2H), 5.35 (br.s, 1H), 6.45 (dd, 1H), 6.47 (d, 1H), 6.77 (br.d, 1H), 7.09 (d, 1H), 7.28 (d, 1H), 7.46-7.53 (m, 4H), 7.63-7.68 (m, 1H), 7.75-7.79 (m, 1H), 7.85-7.89 (m, 2H), 9.31 (s, 1H).
19F NMR (d6-DMSO): δ −55.31 (s).
LCMS Rt=3.79 minutes MS m/z 777.10 [MH]+, 779.09 [MH]+.
PREPARATION 45
tert-Butyl 3-{5-[4-(2-chloro-4-{[(2,4-dimethoxybenzyl)(1,3,4-thiadiazol-2-yl)amino]sulfonyl}-5-fluorophenoxy)-2′-(trifluoromethyl)biphenyl-3-yl]-1H-pyrazol-1-yl}azetidine-1-carboxylate
(5-Chloro-N-(2,4-dimethoxybenzyl)-4-({3-[(2E)-3-(dimethylamino)prop-2-enoyl]-2′-(trifluoromethyl)biphenyl-4-yl}oxy)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (Preparation 44, 563 mg, 0.724 mmol) in ethanol (10 mL) was slowly added a solution of tert-butyl 3-hydrazinoazetidine-1-carboxylate (Preparation 36, 603 mg, 3.22 mmol) in ethanol (10 mL) and acetic acid (0.25 mL) at 0° C. under nitrogen. The reaction was heated to 70° C. for 3 hours and then cooled to room temperature. The reaction mixture was neutralised to pH7 with saturated aqueous sodium hydrogen carbonate solution (2.0 mL) and concentrated in vacuo to afford a yellow oil. The oil was partitioned between water (100 mL) and ethyl acetate (100 mL). The aqueous layer was then extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine (2×100 mL) dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a yellow oil (1.74 g). The oil was partially purified by silica gel column chromatography (0%-10% methanol in dichloromethane gradient elution) to afford the title compound as a solid (228 mg) of 52% purity via LCMS. The compound was used without further purification in the next step.
LCMS Rt=4.06 min MS m/z 651.15 [M−Boc−DMB+H]+, 653.16 [M−Boc−DMB+H]+, 801.25 [M−Boc+H]+, 803.27 [M−Boc+H]+, 923.37 [MNa]+, 925.36 [MNa]+.
PREPARATION 46
4-{[3-(1-Azetidin-3-yl-1H-pyrazol-5-yl)-2′-(trifluoromethyl)biphenyl-4-yl]oxy}-5-chloro-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
Trifluoroacetic acid (0.55 mL, 7.18 mmol) was added to a solution of tert-butyl 3-{5-[4-(2-chloro-4-{[(2,4-dimethoxybenzyl)(1,3,4-thiadiazol-2-yl)amino]sulfonyl}-5-fluorophenoxy)-2′-(trifluoromethyl)biphenyl-3-yl]-1H-pyrazol-1-yl}azetidine-1-carboxylate (Preparation 45, 228 mg, 0.253 mmol) in dichloromethane (20 mL). The mixture was then heated to 40° C. for 18 hours under an atmosphere of nitrogen. The reaction was then cooled to room temperature and concentrated in vacuo to afford a clear oil (236.5 mg). The oil was then purified by preparative HPLC (Trilution method) to afford the title compound as a white solid (42.9 mg, 9% over 2 steps).
1HNMR (d6-DMSO): δ 4.27 (d, 4H), 5.24 (t, 1H), 6.45 (d, 1H), 7.09 (d, 1H), 7.13 (d, 1H), 7.40 (d, 1H), 7.47 (dd, 1H), 7.52 (br.d, 1H), 7.65 (br.t, 1H), 7.72-7.76 (m, 2H), 7.79, (d, 1H), 7.86 (d, 1H), 8.57 (s, 1H)
LCMS Rt=2.30 minutes MS m/z 651.05/653.06 [MH]+
PREPARATION 47
3-Cyano-4-fluoro-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide
Sodium hydroxide (5.08 g, 0.127 mmol) was dissolved in water (60 mL) and 1,4-dioxane (300 mL). 1,2,4-thiadiazol-5-amine (10 g, 100 mmol) was added and the reaction stirred for 5 minutes. 3-Cyano-4-fluorobenzene-1-sulfonyl chloride (8.25 g, 37.6 mmol) was added and the reaction was allowed to stir for 3 hours at 20° C. After this time, the reaction was poured into a 1M aqueous solution of hydrogen chloride (150 mL). This solution was extracted with ethyl acetate (3×50 mL). The combined organics were dried over sodium sulfate, filtered and concentrated to give the title compound as a brown solid.
1HNMR (d6-DMSO): δ 7.71 (m, 1H), 8.19 (m, 1H), 8.39 (m, 1H), 8.54 (s, 1H)
LCMS Rt=1.22 minutes MS m/z 283 [MH]+
PREPARATION 48
5-Chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-pyrimidin-2-ylbenzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-pyrimidin-2-yl-benzenesulfonamide (Preparation 13, 216 mg, 0.47 mmol), 3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-ol (Preparation 8, 150 mg, 0.47 mmol) and potassium carbonate (196 mg, 1.42 mmol) were stirred in dimethylsulfoxide (3 mL) at room temperature for 18 hours. A 1M aqueous solution of sodium hydroxide was added to the reaction mixture whereupon a precipitate was observed. The precipitate was collected by filtration and dissolved in ethyl acetate. The ethyl acetate was dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to afford the title compound as a solid (357 mg, 100%).
1HNMR (CDCl3): δ 3.80 (m, 6H), 5.40 (s, 2H), 6.40 (m, 2H), 6.55 (m, 1H), 6.30 (m, 1H), 7.15 (m, 2H), 7.70 (m, 7H), 8.15 (m, 1H), 8.40 (m, 2H), 9.22 (m, 1H), 9.45 (m, 1H).
LCMS Rt=4.43 minutes MS m/z 752 [MH]+
PREPARATION 49
3-Chloro-N-(methoxymethyl)-N-pyridazin-3-yl-4-{[3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}benzenesulfonamide and 3-chloro-N-[(3E)-2-(methoxymethyl)pyridazin-3(2H)-ylidene]-4-{[3-Pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-yl]oxy}benzenesulfonamide
A mixture of regioisomers 3-chloro-4-fluoro-N-(methoxymethyl)-N-(pyridazin-3-yl)benzenesulfonamide and 3-chloro-4-fluoro-N-(methoxymethyl-2H-pyridazin-3-ylidene)benzenesulfonamide (Preparation 11, 157 mg, 0.47 mmol), 3-pyridazin-4-yl-4′-(trifluoromethyl)biphenyl-4-ol (Preparation 8, 150 mg, 0.47 mmol) and potassium carbonate (196 mg, 1.42 mmol) were stirred in dimethylsulfoxide (3 mL) at room temperature for 18 hours. The reaction mixture was then heated at 100° C. for 18 hours. Ethyl acetate (15 mL) was added the mixture was extracted with water (3×5 mL). The organic phase was separated, dried and concentrated in vacuo to afford the title compound (85 mg, 46%). This material was used directly in the next step.
PREPARATION 50
2-Chloro-3′-(trifluoromethyl)biphenyl-4-ol
4-Bromo-3-chlorophenol (518 mg, 2.5 mmol), 3-(trifluoromethyl)phenylboronic acid (617 mg, 3.25 mmol), potassium fluoride (435 mg, 7.5 mmol) palladium acetate (28 mg, 0.125 mmol) and S_phos (102 mg, 0.25 mmol) were stirred in dioxane (10 mL) at 80° C. for 4 hours. The reaction mixture was partitioned between ethyl acetate (100 mL) and water (50 mL). The EtOAc was dried over MgSO4 and evaporated. The residue was resubjected to the reaction conditions and was redissolved in dioxane (10 mL). 3-(trifluoromethyl)phenylboronic acid (380 mg, 2 mmol), potassium fluoride (348 mg, 6 mmol) palladium acetate (14 mg, 0.0625 mmol) and S_phos (51 mg, 0.125 mmol) were added and the reaction stirred at 80° C. for a further 4 hours. The reaction mixture was worked up as before and the crude product was chromatographed on silica eluting with a gradient of heptane:ethyl acetate 100:0 to 75:25. Fractions containing product were evaporated. The resulting material was chromatographed on silica eluting with a gradient of cyclohexane:triethylamine:isopropyl alcohol 95:5:0 to 95:5:10 to give the title compound (310 mg, 1.14 mmol, 45%) as a colourless gum.
1HNMR (400 MHz, CDCl3): δ 6.75 (m, 1H), 6.94 (s, 1H), 7.15 (d, 1H), 7.49 (m, 1H), 7.56 (m, 2H), 7.60 (s, 1H).
LCMS (5.0 min) Rt=3.41 minutes, MS m/z 271 [M−H]−
PREPARATION 51
2-Chloro-5-iodo-3′-(trifluoromethyl)biphenyl-4-ol
2-Chloro-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 50, 310 mg, 1.14 mmol) was dissolved in acetic acid (2 mL), and cooled to 0° C. N-iodosuccinimide (256 mg, 1.14 mmol) was added followed by concentrated sulphuric acid (0.067 μL). The reaction was stirred at room temperature for 18 hours. A second portion of N-iodosuccinimide (25 mg, 0.11 mmol) was added and the reaction stirred at room temperature for a further 1 hour. The reaction mixture was partitioned between ethyl acetate (70 mL) and water (50 mL). The ethyl acetate was separated, dried over MgSO4, filtered and evaporated. The residue was chromatographed on silica eluting with a gradient of heptane:ethyl acetate 100:0 to 80:20 to give the title compound (230 mg, 0.58 mmol, 51%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 5.38 (s, 1H), 7.15 (s, 1H), 7.58 (m, 2H), 7.64 (m, 3H). MS m/z 397 [M−H]−
PREPARATION 52
2-Chloro-5-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-ol
2-Chloro-5-iodo-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 51, 230 mg, 0.57 mmol) was dissolved in acetonitrile (3 mL), 4-(tributylstannyl)pyridazine (273 mg, 0.75 mmol), caesium fluoride (173 mg, 1.14 mmol), copper iodide (22 mg, 0.114 mmol) and tetrakis(triphenylphosphine)palladium (0) (70 mg, 0.06 mmol) were added. The reaction was stirred at 45° C. for 1.5 hours and then after cooling to room temperature was partitioned between ethyl acetate (100 mL) and water (50 mL) containing 0.880 ammonia (1 mL). The mixture was stirred for 20 minutes. The ethyl acetate phase was separated and washed with an aqueous solution of potassium fluoride (1.5 g in 20 mL), 1M hydrochloric acid (20 mL) and aqueous ammonia (1 mL 0.880 in 50 mL water). The ethyl acetate was dried over MgSO4, filtered and evaporated. The residue was chromatographed on silica eluting with a gradient of dichloromethane:methanol 100:0 to 95:5 to give the title compound (144 mg, 0.40 mmol, 71%) as a white solid.
1HNMR (400 MHz, d6-DMSO): δ 7.20 (s, 1H), 7.30-7.80 (m, 5H), 7.98 (d, 1H), 9.22 (d, 1H), 9.57 (s, 1H), 10.96 (s, 1H).
LCMS (5.0 min) Rt=3.06 minutes, MS m/z 351 [MH]+
PREPARATION 53
5-chloro-4-{[2-chloro-5-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-(2,4-dimethoxybenzyl)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
2-Chloro-5-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 52, 140 mg, 0.4 mmol) was dissolved in DMSO (1 mL) and potassium carbonate (110 mg, 0.8 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 203 mg, 0.44 mmol). The reaction was stirred at room temperature for 18 hours and then partitioned between ethyl acetate (50 mL) and water (40 mL). The ethyl acetate was separated and dried over MgSO4, filtered and evaporated. The residue was chromatographed on silica eluting with a gradient of heptane:ethyl acetate 80:20 to 20:80 to give the title compound (231 mg, 0.30 mmol, 73%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 3.70 (s, 3H), 3.75 (s, 3H), 5.30 (s, 2H), 6.24 (s, 1H), 6.35 (d, 1H), 6.63 (d, 1H), 7.30 (m, 2H), 7.34, (d, 1H), 7.50-7.75 (m, 5H), 7.82 (d, 1H), 8.83 (s, 1H), 9.28 (d, 1H), 9.42 (s, 1H).
LCMS (5.0 min) Rt=3.85 minutes, MS m/z 792 M[H]+
PREPARATION 54
4′-Chloro-3′-(trifluoromethyl)biphenyl-4-ol
4-Chloro-3-(trifluoromethyl)phenylboronic acid (448 mg, 2 mmol) and 4-iodophenol (440 mg, 2 mmol) were dissolved in dioxane (10 mL). Caesium carbonate (1.95 g, 6 mmol), water (2 mL) and tetrakis(triphenylphosphine)palladium (0) (231 mg, 0.2 mmol) were added and the reaction stirred at 80° C. for 1 hour. The reaction was quenched with 2M HCl (5 mL) and partitioned between ethyl acetate (50 mL) and water (50 mL). The ethyl acetate was separated, dried over MgSO4 and evaporated. The crude product was chromatographed on silica eluting with a gradient of heptane:ethyl acetate 100:0 to 80:20 to give the title compound (150 mg, 0.55 mmol, 27%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 4.82 (br-s, 1H, OH), 6.92 (d, 2H), 7.44 (d, 2H), 7.51 (d, 1H), 7.58 (d, 1H), 7.80 (s, 1H).
LCMS (5.0 min) Rt=3.42 minutes, MS m/z 271 [M−H]−
PREPARATION 55
4′-Chloro-3-iodo-3′-(trifluoromethyl)biphenyl-4-ol
4′-Chloro-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 54, 150 mg, 0.55 mmol) was dissolved in acetic acid (5 mL) and concentrated sulphuric acid (0.032 μL) was added followed by N-iodosuccinimide (124 mg, 0.55 mmol). The reaction was stirred at room temperature for 18 hours and then partitioned between ethyl acetate (50 mL) and water (20 mL). The ethyl acetate was separated, dried over MgSO4, filtered and evaporated. The crude product was chromatographed on silica eluting with a gradient of heptane:ethyl acetate 100:0 to 85:15 to give the title compound (167 mg, 0.42 mmol, 76%) as a gum.
1HNMR (400 MHz, CDCl3): δ 5.40 (br-s, 1H, OH), 7.06 (d, 1H), 7.44 (d, 1H), 7.55 (m, 1H), 7.58 (m, 1H), 7.79 (s, 1H), 7.84 (s, 1H).
LCMS (5.0 min) Rt=3.65 minutes, MS m/z 397 [M−H]−
PREPARATION 56
4′-Chloro-3-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-ol
4′-Chloro-3-iodo-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 55, 167 mg, 0.42 mmol) was dissolved in acetonitrile (1 mL) and 4-(tributylstannyl)pyridazine (200 mg, 0.55 mmol), caesium fluoride (127 mg, 0.84 mmol), copper iodide (16 mg, 0.084 mmol) and tetrakis(triphenylphosphine)palladium (0) (46 mg, 0.04 mmol) were added. The reaction was stirred at 45° C. for 40 minutes and then partitioned between ethyl acetate (50 mL) and water (10 mL) containing 0.880 ammonia (1 mL). The mixture was stirred for 20 minutes. The ethyl acetate was separated, dried over MgSO4, filtered and evaporated. The residue was chromatographed on silica eluting with a gradient of dichloromethane:methanol: 0.880NH3 100:0:0 to 92:8:0.8 to give the title compound (95 mg, 0.27 mmol, 64%) as a white solid.
1HNMR (400 MHz, DMSO-d6): δ 7.15 (d, 1H), 7.75-7.78 (m, 2H), 7.90 (s, 1H), 8.00 (m, 2H), 8.08 (s, 1H), 9.24 (d, 1H) 9.58 (s, 1H), 10.58 (br-s, 1H, OH).
LCMS (5.0 min) Rt=3.08 minutes, MS m/z 349 [M−H]−
PREPARATION 57
5-chloro-4-{[4′-chloro-3-pyridazin-4-yl-3′-(trifluoromethyl)biphenyl-4-yl]oxy}-N-(2,4-dimethoxybenzyl)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
4′-Chloro-3-(pyridazin-4-yl)-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 56, 88 mg, 0.25 mmol) was dissolved in DMSO (1 mL), potassium carbonate (69 mg, 0.5 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 127 mg, 0.27 mmol). The reaction was stirred at room temperature for 3 hours and then partitioned between ethyl acetate (50 mL) and water (50 mL). The organic phase was separated and washed with water (2×50 mL), dried over MgSO4, filtered and evaporated. The crude product was chromatographed on silica eluting with a gradient of heptane:ethyl acetate 100:0 to 20:80 to give the title compound (135 mg, 0.17 mmol, 69%) as a white solid.
1HNMR (400 MHz, DMSO-d6): δ 3.62 (s, 3H), 3.68 (s, 3H), 5.14 (s, 2H), 6.40 (m, 2H), 7.06 (d, 1H), 7.28 (d, 1H), 7.34 (d, 1H), 7.84 (m, 2H), 7.95 (m, 2H), 8.13 (m, 1H), 8.16 (s, 1H), 8.22 (s, 1H), 9.30 (m, 2H), 9.54 (s, 1H).
LCMS (5.0 min) Rt=3.88 minutes, MS m/z 792 [MH]+
PREPARATION 58
5-chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{2-pyridazin-4-yl-4-[6-(trifluoromethyl)pyridin-3-yl]phenoxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
2-(Pyridazin-4-yl)-4-(6-(trifluoromethyl)pyridin-3-yl)phenol (Preparation 59, 360 mg, 1.134 mmol) was dissolved in DMSO (6 mL) and potassium carbonate (313 mg, 2.27 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 524 mg, 1.134 mmol). The reaction was stirred at room temperature for 18 hours and then partitioned between ethyl acetate and water. The organic layer was separated, washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified on silica gel by Biotage (20% to 100% EtOAc in heptane over 20 CV) to give the title compound (501 mg, 58%) as yellow foam.
1HNMR (400 MHz, CDCl3): δ 3.65 (s, 3H), 3.70 (s, 3H), 5.30 (s, 2H), 6.20 (s, 1H), 6.30 (d, 1H), 6.65 (d, 1H), 7.15 (d, 1H), 7.20 (m, 1H), 7.70-7.90 (m, 5H), 8.10 (d, 1H), 8.80 (s, 1H), 8.95 (s, 1H), 9.30 (d, 1H), 9.45 (s, 1H).
19F NMR (400 MHz, CDCl3): δ −68.0, −104.0.
MS No mass ion seen
PREPARATION 59
2-(Pyridazin-4-yl)-4-(6-(trifluoromethyl)pyridin-3-yl)phenol
2-Iodo-4-(6-(trifluoromethyl)pyridin-3-yl)phenol (Preparation 60, 840 mg, 2.30 mmol) was dissolved in acetonitrile (20 mL) and 4-(tributylstannyl)pyridazine (1.10 g, 2.99 mmol), caesium fluoride (698 mg, 4.60 mmol), copper iodide (87 mg, 0.46 mmol) and tetrakis(triphenylphosphine)palladium (0) (266 mg, 0.23 mmol) were added. The reaction mixture was stirred at 80° C. for 18 hours and then partitioned between ethyl acetate and water containing 0.88 ammonia. The resulting mixture was stirred for 15 minutes and then filtered through a pad of celite. The aqueous phase was separated and extracted with ethyl acetate (2×20 mL) and the combined organic phases were washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified on silica gel by Biotage (10% to 100% EtOAc in heptane over 25 CV) to give a mixture of the title compound and triphenylphosphine oxide. The residue was triturated with dichloromethane to give the title compound (360 mg, 50%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 4.9 (s, 1H), 7.05 (d, 1H), 7.75 (d, 1H), 7.90 (m, 2H), 8.05 (d, 1H), 8.30 (d, 1H), 9.00 (s, 1H), 9.2 (s, 1H), 9.6 (s, 1H).
19F NMR (400 MHz, CDCl3): δ −69.0
LCMS Rt=2.24 minutes, MS m/z 318 [MH]+.
PREPARATION 60
2-iodo-4-[6-(trifluoromethyl)pyridin-3-yl]phenol
4-(6-(Trifluoromethyl)pyridin-3-yl)phenol (Preparation 61, 1.24 g, 5.18 mmol) was dissolved in a mixture of acetic acid (10 mL), dichloromethane (10 mL) and CH3CN (10 mL) at room temperature. Concentrated sulphuric acid (0.5 mL) was then added followed by N-iodosuccinimide (1.052 g, 4.67 mmol). The reaction was stirred at room temperature for 18 hours. A further aliquot of N-iodosuccinimide (116 mg, 0.518 mmol) was added and the reaction mixture was stirred for one hour and concentrated in vacuo. The crude oil was purified on silica gel by Biotage™ (5% to 60% of EtOAc in heptane over 20 CV) and fractions containing product were evaporated to give an inseparable mixture of product/starting material 7:3 (840 mg). This was used directly in the next stage without further purification.
1HNMR (400 MHz, CDCl3): δ 6.90 (d, 1H), 7.40 (d, 1H), 7.60 (s, 1H), 7.80 (d, 1H), 7.85 (m, 1H), 8.80 (s, 1H).
19F NMR (400 MHz, CDCl3): δ −68.0
LCMS Rt=2.69 minutes, MS m/z 363 [M−H]
PREPARATION 61
4-(6-(Trifluoromethyl)pyridin-3-yl)phenol
5-Bromo-2-(trifluoromethyl)pyridine (1.18 g, 5.51 mmol), 4-hydroxyphenylboronic acid (775 mg, 5.51 mmol) and sodium carbonate (2.34 g, 22.0 mmol) were combined and dissolved in a mixture of dioxane/water (30 mL/6 mL). The reaction mixture was degassed and then tetrakistriphenylphosphinepalladium (0) (322 mg, 0.275 mmol) was added and the reaction mixture was heated at 70° C. for 18 hours. The cooled reaction mixture was partitioned between ethyl acetate and water. The organic layer was separated and washed with brine, dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified on silica gel by Biotage™ (7% to 60% EtOAc in heptane over 20 CV) to give the title compound (1.24 g, 95%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 5.30 (s, 1H), 6.95 (d, 2H), 7.50 (m, 2H), 7.85 (d, 1H), 7.95 (d, 1H), 8.85 (s, 1H).
LCMS Rt=2.71 minutes MS m/z 240 [MH]+
PREPARATION 62
5-Chloro-4-{[3′-cyano-3-(3-nitro-1H-pyrazol-4-yl)biphenyl-4-yl]oxy}-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
5-Chloro-4-(3′-cyano-3-(3-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 63, 447 mg, 0.537 mmol) was dissolved in a 4M solution of HCl in 1,4-dioxane (2.7 mL, 10.74 mmol). The reaction mixture was stirred at room temperature for 18 hours and then concentrated in vacuo. The residue was purified by reverse phase chromatography on the ISCO™ system to afford the title compound (204 mg, 64%) as a white solid.
1HNMR (400 MHz, d-6DMSO): δ 6.80 (d, 1H), 7.25 (d, 1H), 7.65 (t, 1H), 7.80 (m, 3H), 7.95 (s, 1H), 8.05 (dd, 1H), 8.20 (d, 1H), 8.25 (d, 1H), 8.80 (s, 1H).
19F NMR (400 MHz, d-6DMSO): δ −107.0
LCMS Rt=3.13 minutes, MS m/z 596 [M−H]
PREPARATION 63
5-chloro-4-({3′-cyano-3-[3-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl]biphenyl-4-yl}oxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
4′-Hydroxy-3′-(3-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)biphenyl-3-carbonitrile (Preparation 64, 250 mg, 0.64 mmol) was dissolved in DMSO (3.5 mL) and potassium carbonate (177 mg, 1.28 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 310 mg, 0.67 mmol). The reaction was stirred at room temperature for 1 hour and then partitioned between ethyl acetate and water. The aqueous phase was separated and extracted with EtOAc (3×5 mL) and the combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified on silica gel by Biotage™ (10% to 80% EtOAc in heptane over 12 CV) to give the title compound (451 mg, 85%) as a light yellow foam.
1HNMR (400 MHz, CDCl3): δ 1.60 (m, 3H), 2.00 (m, 2H), 2.20 (m, 1H), 3.60 (s, 3H), 3.75 (s, 3H), 3.80 (m, 1H), 4.05 (m, 1H), 5.20 (s, 2H), 5.40 (d, 1H), 6.2 (br s, 1H), 6.30 (d, 1H), 6.55 (d, 1H), 7.05 (d, 1H), 7.20 (d, 1H), 7.55-7.80 (m, 7H), 7.90 (s, 1H), 8.8 (s, 1H).
19F NMR (400 MHz, CDCl3): δ −104.0
LCMS Rt=3.25 minutes, MS no mass ion detected.
PREPARATION 64
4′-Hydroxy-3′-(3-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)biphenyl-3-carbonitrile
4-Chloro-2-(3-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)phenol (500 mg, 1.544 mmol), 3-cyanophenylboronic acid (453 mg, 3.09 mmol), di-mu-chlorobis[5-chloro-2-[(4-chlorophenyl)(hydroxyimino)methyl]phenyl]palladium (II) dimer (63 mg, 0.0772 mmol), tri-tert-butylphosphonium tetrafluoroborate (45 mg, 0.154 mmol), potassium carbonate (426 mg, 3.09 mmol) and tetrabutyl ammonium hydroxyde solution (1M in MeOH, 0.31 mL, 0.31 mmol) were combined in a microwave vial. DMF (7.5 mL) was added and the vial was sealed. The mixture was heated at 130° C. for 1 hour under microwave irradiation and then partitioned between ethyl acetate (25 mL) and water (10 mL). The organic layer was separated and washed with brine (10 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified on silica gel by Biotage™ (10% to 80% EtOAc in heptane over 20 CV) to give the title compound (254 mg, 41%) as brown oil
1HNMR (400 MHz, CDCl3): δ 1.70 (m, 3H), 2.00 (m, 2H), 2.20 (m, 1H), 3.85 (m, 1H), 4.05 (m, 1H), 5.30 (br s, 1H), 5.50 (d, 1H), 7.00 (d, 1H), 7.40 (s, 1H), 7.50 (m, 2H), 7.60 (d, 1H), 7.75 (d, 1H), 7.8 (m, 2H).
LCMS Rt=2.79 minutes, MS m/z 389 [M−H]
PREPARATION 65
tert-Butyl {[4′-{2-cyano-4-[(1,3-thiazol-2-ylamino)sulfonyl]phenoxy}-3′-(1-methyl-1H-pyrazol-5-yl)biphenyl-2-yl]methyl}methylcarbamate
4-[4-Bromo-2-(1-methyl-1H-pyrazol-5-yl)phenoxy]-3-cyano-N-1,3-thiazol-2-ylbenzene sulfonamide (Patent WO 2010079443, 500 mg, 0.97 mmol) was dissolved in dimethylformamide (3 mL) and added to a 5 mL microwave vial under nitrogen. Bis(pinacolato) diboron (327 mg, 1.33 mmol), (1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium (II) (71 mg, 0.1 mmol) and potassium acetate (475 mg, 4.84 mmol) were added. The reaction vessel was sealed and then heated to 100° C. for 1 hour in the microwave. The cooled reaction mixture was partitioned between ethyl acetate (20 mL) and water (20 mL). The organic layer was separated, filtered and then concentrated in vacuo. The residue was dissolved in 1,4-dioxane (3 mL) and added to a 5 mL microwave vial under nitrogen. Tert-butyl (2-iodobenzyl)methylcarbamate (European Journal of Organic Chemistry, 2010, 19, 3704-3710) (504 mg, 1.45 mmol), bis(triphenylphosphine) palladium (II) dichloride (68 mg, 0.1 mmol), potassium carbonate (334 mg, 2.42 mmol) and water (0.5 mL) were added and the reaction vessel sealed and then heated to 125° C. for 30 minutes in the microwave. The crude material was partitioned between ethyl acetate (20 mL) and 0.2M aqueous solution of HCl (20 mL). The organic layer was separated, filtered and then concentrated in vacuo and purified by silica gel column chromatography (ISCO™, 12 g silica, 99:1 DCM:formic acid to 90:10:1 DCM:MeOH:formic acid gradient to afford the title compound (630 mg, 100%) as a brown oil.
1HNMR (CDCl3): δ 1.42 (s, 9H), 2.72 (s, 3H), 3.90 (s, 3H), 3.96 (s 2H), 4.45 (br s, 1H) 6.25 (d, 1H) 6.59 (d, 1H), 6.81 (d, 1H) 7.07 (d, 1H) 7.40 (m, 8H), 7.94 (dd, 1H), 8.12 (d, 1H).
LCMS Rt=1.69 minutes MS m/z 657 [MH]+.
PREPARATION 66
5-Chloro-4-(2-chloro-3′-fluoro-5-(pyridazin-4-yl)biphenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
2-Chloro-3′-fluoro-5-(pyridazin-4-yl)biphenyl-4-ol (Preparation 67, 98 mg, 0.33 mmol) was dissolved in dimethylsulfoxide (2 mL) and 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 181 mg, 0.39 mmol) and potassium carbonate (135 mg, 0.98 mmol) were added. The reaction was stirred at room temperature for 18 hours. Water (10 mL) and ethyl acetate (15 mL) were added and the two layers were separated. The aqueous phase was extracted with ethyl acetate (10 mL). The combined organic extracts were washed with brine (10 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20% heptane in ethyl acetate) to give the title compound (165 mg, 67%) as an off-white solid.
1HNMR (400 MHz, CDCl3): δ 3.64 (s, 3H), 3.68 (s, 3H), 5.24 (s, 2H), 6.20 (s, 1H), 6.30 (d, 1H), 6.56 (d, 1H), 7.15 (m, 5H), 7.38 (dd, 1H), 7.48 (s, 1H), 7.60 (dd, 1H), 7.78 (d, 1H), 8.76 (s, 1H), 9.18 (d, 1H), 9.36 (s, 1H).
LCMS Rt=3.62 minutes MS m/z 742 [MH]+
PREPARATION 67
2-Chloro-3′-fluoro-5-(pyridazin-4-yl)biphenyl-4-ol
2-Chloro-3′-fluoro-5-iodobiphenyl-4-ol (Preparation 68, 480 mg, 1.38 mmol) and 4-(tributylstannyl)pyridazine (610 mg, 1.65 mmol) were dissolved in degassed acetonitrile (7 mL). Caesium fluoride (418 mg, 2.75 mmol) was added and the mixture further degassed. Tetrakis(triphenylphosphine) palladium (0) (159 mg, 0.14 mmol) and copper (I) iodide (79 mg, 0.41 mmol) were added and the reaction heated at 50° C. for 2 hours. The reaction mixture was cooled and filtered through celite, washing with ethyl acetate. The organic solution was washed with water and brine, dried over anhydrous MgSO4, filtered and evaporated. The residue was purified by silica gel column chromatography (20% heptane in ethyl acetate) followed by trituration with dichloromethane to give the title compound (98 mg, 24%) as a white solid.
1HNMR (400 MHz, d6-DMSO): δ 7.14 (s, 1H), 7.20 (dd, 1H), 7.34 (m, 2H), 7.26 (dd, 1H), 7.56 (s, 1H), 7.94 (d, 1H), 9.22 (d, 1H), 9.56, (s, 1H), 10.86 (s, 1H).
PREPARATION 68
2-Chloro-3′-fluoro-5-iodobiphenyl-4-ol
2-Chloro-3′-fluorobiphenyl-4-ol (Preparation 69, 500 mg, 2.25 mmol) was dissolved in dichloromethane (5 mL) and acetic acid (5 mL). Concentrated sulfuric acid (0.05 mL) was added followed by N-iodosuccinimide (480 mg, 2.13 mmol) and the reaction stirred at room temperature for 18 hours. A further portion of N-iodosuccinimide (50 mg, 0.22 mmol) was added and stirring continued at room temperature for 3 hours. Water and dichloromethane were added and the two layers separated. The organic layer was washed twice with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20% ethyl acetate in heptane) to afford the title compound (496 mg, 63%) as a yellow oil that solidified on standing.
1HNMR (400 MHz, CDCl3): δ 5.40 (s, 1H), 7.10 (m, 4H), 7.36 (dd, 1H), 7.62 (s, 1H).
LCMS Rt=2.67 minutes MS m/z 347 [M−H]−
PREPARATION 69
2-Chloro-3′-fluorobiphenyl-4-ol
3-Fluorobenzeneboronic acid (405 mg, 2.89 mmol) and 4-bromo-3-chlorophenol (500 mg, 2.41 mmol) were dissolved in dioxane (15 mL) and water (3 mL) and the solution degassed. Tetrakis(triphenylphosphine) palladium (0) (278 mg, 0.24 mmol) was added followed by caesium carbonate (2.36 g, 7.23 mmol) and the reaction was stirred at 80° C. under nitrogen for 18 hours. The reaction mixture was cooled to room temperature and partitioned between ethyl acetate and water. The aqueous layer was separated and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20% ethyl acetate in heptane) to afford the title compound (509 mg, 95%) as a colourless oil.
1HNMR (400 MHz, CDCl3): δ 6.80 (d, 1H), 7.00 (s, 1H), 7.06 (dd, 1H), 7.12 (dd, 1H), 7.18 (m, 2H), 7.36 (dd, 1H).
LCMS Rt=3.02 minutes MS m/z 221 [M−H]
PREPARATION 70
5-Chloro-4-(2-chloro-4′-fluoro-5-(pyridazin-4-yl)bi phenyl-4-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
2-Chloro-4′-fluoro-5-(pyridazin-4-yl)biphenyl-4-ol (Preparation 71, 148 mg, 0.49 mmol) was dissolved in dimethylsulfoxide (3 mL) and 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 273 mg, 0.59 mmol) and potassium carbonate (204 mg, 1.48 mmol) were added. The reaction was stirred at 20° C. for 18 hours and then partitioned between water and ethyl acetate. The aqueous phase was separated and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20% heptane in ethyl acetate) to give the title compound (224 mg, 62%) as an off-white solid.
1HNMR (400 MHz, CDCl3): δ 3.68 (s, 3H), 3.76 (s, 3H), 5.32 (s, 2H), 6.26 (s, 1H), 6.36 (d, 1H), 6.60 (d, 1H), 7.18 (d, 2H), 7.22 (s, 1H), 7.26 (d, 1H), 7.46 (dd, 2H), 7.54 (s, 1H), 7.68 (d, 1H), 7.84 (d, 1H), 8.82 (s, 1H), 9.24 (d, 1H), 9.42 (s, 1H).
LCMS Rt=3.76 minutes MS m/z 742 [MH]+.
PREPARATION 71
2-Chloro-4′-fluoro-5-(pyridazin-4-yl)biphenyl-4-ol
2-Chloro-4′-fluoro-5-iodobiphenyl-4-ol (Preparation 72, 553 mg, 1.59 mmol) and 4-(tributylstannyl)pyridazine (703 mg, 1.90 mmol) were dissolved in degassed acetonitrile (8 mL). Caesium fluoride (482 mg, 3.17 mmol) was added and the mixture further degassed. Tetrakistriphenylphosphinepalladium (0) (183 mg, 0.16 mmol) and copper (I) iodide (91 mg, 0.48 mmol) were added and the reaction heated at 50° C. for 2 hours. The reaction mixture was cooled and filtered through celite, washing with ethyl acetate. The organic solution was washed with water and brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by trituration with hot ethyl acetate to give the title compound (151 mg, 32%) as a beige solid.
1HNMR (400 MHz, d6-DMSO): δ 7.18 (s, 1H), 7.26 (dd, 2H), 7.72 (dd, 2H), 7.76 (s, 1H), 7.96 (d, 1H), 9.24 (d, 1H), 9.56, (s, 1H), 10.82 (s, 1H).
LCMS Rt=2.62 minutes MS m/z 301 [MH]+.
PREPARATION 72
2-Chloro-4′-fluoro-5-iodobiphenyl-4-ol
2-Chloro-4′-fluorobiphenyl-4-ol (Preparation 73, 503 mg, 2.26 mmol) was dissolved in dichloromethane (5 mL) and acetic acid (5 mL). Concentrated sulfuric acid (0.05 mL) was added followed by N-iodosuccinimide (508 mg, 2.26 mmol) and the reaction stirred at room temperature for 2 hours before partitioning it between water and dichloromethane. The organic layer was separated and washed twice with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20% ethyl acetate in heptane) to afford the title compound (553 mg, 70%) as an orange oil.
1HNMR (400 MHz, CDCl3): δ 5.36 (s, 1H), 7.10 (dd, 2H), 7.14 (s, 1H), 7.36 (dd, 2H), 7.62 (s, 1H).
LCMS Rt=2.64 minutes MS m/z 347 [M−H]
PREPARATION 73
2-Chloro-4′-fluorobiphenyl-4-ol
4-Fluorobenzeneboronic acid (405 mg, 2.89 mmol) and 4-bromo-3-chlorophenol (500 mg, 2.41 mmol) were dissolved in dioxane (15 mL) and water (3 mL) under nitrogen. The solution was degassed before tetrakis(triphenylphosphine) palladium (0) (278 mg, 0.24 mmol) was added followed by caesium carbonate (2.36 g, 7.23 mmol) and the reaction was stirred at 80° C. for 18 hours. The cooled reaction mixture was partitioned between ethyl acetate and water. The aqueous layer was separated and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (20% ethyl acetate in heptane) to afford the title compound (503 mg, 94%) as a tan oil which solidified on standing.
1HNMR (400 MHz, CDCl3): δ 6.78 (d, 1H), 6.98 (s, 1H), 7.08 (dd, 2H), 7.18 (d, 1H), 7.38 (dd, 2H).
LCMS Rt=2.93 minutes MS m/z 221 [M−H]
PREPARATION 74
2-Chloro-2′-fluoro-5-(pyridazin-4-yl)biphenyl-4-ol
2-Chloro-2′-fluoro-5-iodobiphenyl-4-ol (Preparation 75, 610 mg, 1.76 mmol) was dissolved in acetonitrile (3 mL) and 4-(tributylstannyl)pyridazine (843 mg, 2.28 mmol), caesium fluoride (533 mg, 3.51 mmol), copper iodide (67 mg, 0.35 mmol) and tetrakis(triphenylphosphine)palladium (0) (204 mg, 0.176 mmol) were added. The reaction was stirred at 80° C. for 18 hours and then the cooled reaction mixture was partitioned between ethyl acetate (100 mL) and water. The organic layer was separated and dried over MgSO4, filtered and evaporated. The residue was chromatographed on silica gel eluting with heptane:ethyl acetate 1:1 to 100% ethyl acetate. Fractions containing product were evaporated and then triturated with dichloromethane to give the title compound (210 mg, 40%) as a yellow solid
1HNMR (400 MHz, CD3OD): δ 7.26-7.15 (m, 3H), 7.45-7.33 (m, 2H), 7.48 (s, 1H), 8.00-7.98 (m, 1H), 9.17 (d, 1H), 9.51 (s, 1H).
19FNMR (400 MHz, CD3OD): δ −115.98
LCMS Rt=2.79 minutes, MS m/z 301 [MH]+
PREPARATION 75
2-Chloro-2′-fluoro-5-iodobiphenyl-4-ol
2-Chloro-2′-fluorobiphenyl-4-ol (Preparation 76, 430 mg, 1.93 mmol) was dissolved in DCM, and cooled to 0° C. Acetic acid (5 mL), N-iodosuccinimide (434 mg, 1.93 mmol) were added followed by concentrated sulphuric acid (0.2 mL). The reaction mixture was stirred at room temperature for 2 hours. The solvent was evaporated and the residue was purified by flash chromatography on silica gel eluting with heptane:ethyl acetate 7:3 to give the title compound (620 mg, 92%) as oil.
1HNMR (400 MHz, CDCl3): δ 5.37 (s, 1H), 7.29-7.11 (m, 4H), 7.40-7.35 (m, 1H) and 7.62 (s, 1H).
19FNMR (400 MHz, CDCl3): δ −114.01
LCMS Rt=3.40 minutes, MS m/z 347 [M−H]−
PREPARATION 76
2-Chloro-2′-fluorobiphenyl-4-ol
2-Fluorophenylboronic acid (0.405 g, 2.89 mmol) and 4-bromo-3-chlorophenol (0.500 g, 2.41 mmol) were dissolved in dioxane (10 mL). A solution of caesium carbonate (2.35 g, 7.21 mmol) in water (2 mL) was added and the reaction mixture was degassed. Tetrakis(triphenylphosphine)palladium (0) (0.280 g, 0.242 mmol) was added and reaction was further degassed before heating the reaction at 100° C. for 18 hours. The cooled reaction mixture was filtered through a pad of celite. The filtrate was diluted with EtOAc and washed with water and dried over MgSO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography on silica gel eluting with EtOAc/Heptane 2:3 to give the title compound (0.445 g, 69%) as dark solid
1HNMR (400 MHz, CDCl3): δ 4.87 (s, 1H), 6.82-6.79 (m, 1H), 7.00 (s, 1H), 7.39-7.11 (m, 5H).
19FNMR (400 MHz, CDCl3): δ −114.28
LCMS Rt=3.09 minutes, MS m/z 221 [M−H]−
PREPARATION 77
tert-Butyl {[4′-{2-cyano-4-[(1,3-thiazol-2-ylamino)sulfonyl]phenoxy}-3′-(1-methyl-1H-pyrazol-5-yl)biphenyl-3-yl]methyl}carbamate
In a 5 mL microwave vial 4-[4-bromo-2-(1-methyl-1H-pyrazol-5-yl)phenoxy]-3-cyano-N-1,3-thiazol-2-ylbenzene sulfonamide (Patent WO 2010079443, 500 mg, 0.97 mmol) was dissolved in 1,4-dioxane (3 mL) under nitrogen. (3-{[(Tert-butoxycarbonyl)amino]methyl}phenyl)boronic acid (362 mg, 1.44 mmol), bis(triphenylphosphine) palladium (II) dichloride (68 mg, 0.1 mmol), sodium carbonate (204 mg, 1.92 mmol) and water (0.5 mL) were added and the reaction vessel sealed and heated to 120° C. for 45 minutes in the microwave. The reaction mixture was partitioned between ethyl acetate (20 mL) and 0.2M aqueous HCl (20 mL). The organic layer was separated, filtered then concentrated in vacuo. The residue was purified by silica gel column chromatography (ISCO™, 12 g silica, 99:1 DCM:Acetic acid to 90:10:1 DCM:MeOH:Acetic acid gradient) to afford the title compound (380 mg, 61%) as a pale orange solid.
1HNMR (400 MHz, CDCl3): δ 1.46 (s, 9H), 3.88 (s, 3H), 5.29 (s, 2H), 6.24 (d, 1H), 6.59 (d, 1H), 6.73 (d, 1H), 7.08 (d, 1H), 7.27 (d, 1H), 7.32 (d, 1H), 7.38 (d, 1H), 7.45 (m, 1H) 7.50 (m, 2H) 7.64 (d, 1H) 7.72 (dd, 1H), 7.90 (dd, 1H), 8.09 (d, 1H), 9.40-10.20 (br s, 2H).
LCMS Rt=1.57 minutes MS m/z 643 [MH]+.
PREPARATION 78
5-chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{2-pyridazin-4-yl-4-[2-(trifluoromethyl)pyridin-4-yl]phenoxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
2-(Pyridazin-4-yl)-4-(2-(trifluoromethyl)pyridine-4-yl)phenol (Preparation 79, 0.105 mg, 0.33 mmol) was dissolved in DMSO (3 mL) and potassium carbonate (91 mg, 0.66 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzensulfonamide (Preparation 16, 153 mg, 0.33 mmol). The mixture was stirred at room temperature for 3 hours and then partitioned between ethyl acetate (40 mL) and 1M aqueous sodium hydroxide solution (10 mL). The organic layer was separated and dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate) to give the title compound (207 mg, 81%) as a beige solid.
1HNMR (400 MHz, CDCl3): δ 3.58 (s, 3H), 3.60 (s, 3H), 5.12 (s, 2H), 6.20 (d, 1H), 6.25 (d, 1H), 6.55 (d, 1H), 7.05 (d, 1H), 7.12 (m, 1H), 7.62 (m, 2H), 7.78 (m, 2H), 7.80 (d, 1H), 7.82 (s, 1H), 8.80 (m, 2H), 9.12 (d, 1H), 9.40 (s, 1H).
LCMS Rt=3.55 minutes, MS m/z 759 [MH]+.
PREPARATION 79
2-(Pyridazin-4-yl)-4-(2-(trifluoromethyl)pyridine-4-yl)phenol
2-Iodo-4-(2-trifluoromethyl)pyridine-4-ylphenol (Preparation 80, 0.65 g, 1.8 mmol) was dissolved in acetonitrile (5 mL). 4-(tributylstannyl)pyridazine (0.90 g, 2.38 mmol) and caesium fluoride (0.53 g, 3.4 mmol) were added and the mixture was degassed for 10 minutes. Copper iodide (67 mg, 0.36 mmol) and tetrakistriphenylphosphinepalladium (0) (0.20 g, 0.18 mmol) were added and the mixture was heated at 50° C. for 18 hours. The cooled reaction mixture was concentrated in vacuo and the residue was purified by silica gel column chromatography eluting with a gradient of ethyl acetate:heptane 1:1 to 100% ethyl acetate to afford the title compound (102 mg, 19%).
1HNMR (400 MHz, CD3OD): δ 7.08 (d, 1H), 7.82 (d, 1H), 8.05 (m, 2H), 8.18 (m, 2H), 8.70 (d, 1H), 9.20 (d, 1H), 9.60 (s, 1H).
LCMS Rt=2.37 minutes, MS m/z 316 [M−H]−
PREPARATION 80
2-Iodo-4-(2-trifluoromethyl)pyridine-4-ylphenol
4-(2-Trifluoromethyl)pyridine-4-yl)phenol (Preparation 81, 3.60 g, 15 mmol) was dissolved in dichloromethane (200 mL) and acetic acid (60 mL). Concentrated sulphuric acid (2 mL) followed by N-iodosuccinimide (3.21 g, 14.2 mmol) were added. The reaction mixture was stirred for 18 hours at room temperature. The mixture was concentrated in vacuo and the residue was partitioned between ethyl acetate (200 mL) and water (100 mL). The organic layer was separated and concentrated in vacuo and the residue was purified by silica gel column chromatography (gradient 6% to 40% ethyl acetate in heptane) to afford a mixture (1.64 g) of the title compound and starting material which was used without further purification.
1HNMR (400 MHz, CDCl3): δ 5.15 (br s, 1H), 7.10 (d, 1H), 7.58 (m, 1H), 7.64 (m, 1H), 7.80 (s, 1H), 7.95 (s, 1H), 8.85 (m, 1H).
PREPARATION 81
4-(2-Trifluoromethyl)pyridine-4-ylphenol
4-Bromo-2-trifluoromethylpyridine (4 g, 17 mmol), 4-hydroxybenzene boronic acid (2.45 g, 17 mmol) and sodium carbonate (5.6 g, 52 mmol) were combined and dissolved in a mixture of dioxane/water (58 mL, 6:1). The reaction mixture was degassed and then tetrakistriphenylphosphinepalladium (0) (0.98 g, 0.85 mmol) was added and the reaction mixture was heated at 70° C. for 18 hours. The cooled reaction mixture was partitioned between ethyl acetate (100 mL) and water (50 mL). The organic layer was separated and dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate:heptane 1:2) to provide the title compound (3.63 g, 84%) as a yellow solid.
1HNMR (400 MHz, CDCl3): δ 5.18 (s, 1H), 6.95 (d, 2H), 7.57 (m, 2H), 7.60 (d, 1H), 7.82 (s, 1H), 8.78 (d, 1H)
PREPARATION 82
5-chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-{2-pyridazin-4-yl-4-[6-(trifluoromethyl)pyridin-2-yl]phenoxy}-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
2-(Pyridazin-4-yl)-4-(6-trifluoromethyl)pyridine-2-yl)phenol (Preparation 83, 75 mg, 0.23 mmol) was dissolved in DMSO (2 mL) and potassium carbonate (65 mg, 0.46 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 109 mg, 0.23 mmol). The mixture was stirred at room temperature for 3 hours and then partitioned between ethyl acetate (40 mL) and 1M aqueous sodium hydroxide solution (10 mL). The organic layer was separated and dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography (1% methanol in dichloromethane) to give the title compound (52 mg, 29%) as a beige solid.
1HNMR (400 MHz, CDCl3): δ 3.60 (s, 3H), 3.65 (s, 3H), 5.25 (s, 2H), 6.08 (s, 1H), 6.25 (d, 1H), 6.42 (d, 1H), 7.05 (d, 1H), 7.10 (m, 1H), 7.45 (m, 1H), 7.78 (d, 1H), 7.90 (m, 2H), 8.08 (d, 1H), 8.12 (s, 1H), 8.88 (s, 2H), 9.10 (d, 1H), 9.20 (s, 1H).
LCMS Rt=3.10 minutes, MS m/z 759 [MH]+.
PREPARATION 83
2-(Pyridazin-4-yl)-4-(6-trifluoromethyl)pyridine-2-ylphenol
2-Iodo-4-(6-trifluoromethyl)pyridin-2-ylphenol (Preparation 84, 0.25 g, 0.68 mmol) was dissolved in acetonitrile (5 mL), then 4-(tributylstannyl)pyridazine (0.30 g, 0.82 mmol) and caesium fluoride (0.20 g, 1.36 mmol) were added and the mixture was degassed. Copper iodide (67 mg, 0.36 mmol) and tetrakis(triphenylphosphine)palladium (0) (80 mg, 0.068 mmol) were added and the mixture was heated at 70° C. for 18 hours. The cooled reaction mixture was concentrated in vacuo and the residue was purified by silica gel column chromatography eluting with a gradient of ethyl acetate:heptane (1:1 to 100:0) to afford the title compound (100 mg, 46%).
1HNMR (400 MHz, CD3OD): δ 7.02 (d, 1H), 7.50 (d, 1H), 7.95 (m, 2H), 8.02 (m, 2H), 8.18 (m, 1H), 9.10 (br s, 1H), 9.50 (br s, 1H).
LCMS Rt=2.73 minutes, MS m/z 318 [MH]+
PREPARATION 84
2-Iodo-4-(6-trifluoromethyl)pyridin-2-ylphenol
4-(6-Trifluoromethyl)pyridine-2-ylphenol (Preparation 85, 2.85 g, 12 mmol) was dissolved in dichloromethane (230 mL) and acetic acid (55 mL). Concentrated sulphuric acid (2 mL) was added followed by N-iodosuccinimide (2.41 g, 10.8 mmol). The reaction mixture was stirred for 18 hours at room temperature. The reaction mixture was concentrated in vacuo and the residue was partitioned between ethyl acetate (200 mL) and water (100 mL). The organic layer was separated, concentrated in vacuo and the residue was purified by silica gel column chromatography (gradient 6% to 40% ethyl acetate in heptane) to afford the title compound (2.16 g, 50%) as brown solid.
1HNMR (400 MHz, CDCl3): δ 5.25 (br s, 1H), 7.05 (d, 1H), 7.58 (d, 1H), 7.82 (m, 1H), 7.90 (m, 1H), 7.95 (m, 1H), 8.40 (s, 1H).
LCMS Rt=3.55 minutes, MS m/z 364 [M−H]
PREPARATION 85
4-(6-Trifluoromethyl)pyridine-2-yl)phenol
2-Bromo-6-trifluoromethylpyridine (3.5 g, 15.4 mmol), 4-hydroxybenzene boronic acid (2.12 g, 15.4 mmol) and sodium carbonate (4.2 g, 46 mmol) were dissolved in a 9:1 mixture of dioxane/water (120 mL) The reaction mixture was degassed and tetrakis(triphenylphosphine)palladium (0) (0.40 g, 0.35 mmol) was added. The reaction mixture was stirred at 80° C. for 18 hours. The cooled reaction mixture was partitioned between ethyl acetate (50 mL) and water (30 mL). The organic layer was separated and dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (ethyl acetate:heptane 1:2) to provide the title compound (2.95 g, 79%) as a yellow solid.
1HNMR (400 MHz, CDCl3): δ 4.85 (br s, 1H), 6.85 (d, 2H), 7.45 (d, 1H), 7.78 (m, 1H), 7.80 (m, 1H), 7.98 (d, 2H).
LCMS Rt=2.98 minutes, MS m/z 240 [MH]+.
PREPARATION 86
5-chloro-N-(2,4-dimethoxybenzyl)-2-fluoro-4-({3-[3-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl]-3′-(trifluoromethyl)biphenyl-4-yl}oxy)-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
3-(3-Nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)-3-(trifluoromethyl)biphenyl-4-ol (Preparation 87, 0.105 g, 0.24 mmol) was dissolved in DMSO (3 mL). 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzene sulfonamide (Preparation 16, 0.11 g, 0.27 mmol) and potassium carbonate (35 mg, 0.25 mmol) were added and the mixture was stirred at room temperature for 18 hours. The reaction mixture was partitioned between ethyl acetate (40 mL) and water (10 mL). The organic layer was separated and dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel chromatography eluting with a gradient of ethyl acetate:heptane 1:10 to 100% ethyl acetate to provide the title compound (0.15 g, 35%) as a white solid.
1HNMR (400 MHz, CDCl3): δ 1.82 (m, 3H), 1.95 (m, 2H), 2.10 (m, 1H), 3.62 (s, 3H), 3.68 (m, 1H), 3.70 (s, 3H), 3.95. (m, 1H), 4.20 (s, 2H), 5.40 (m, 1H), 6.18 (s, 1H), 6.30 (d, 1H), 6.48 (d, 1H), 7.05 (d, 1H), 7.20 (m, 2H), 7.55 (m, 1H), 7.60 (m, 2H), 7.70 (m, 2H), 7.76 (s, 2H), 8.78 (s, 1H).
LCMS Rt=3.50 minutes, MS m/z 875 [MH]+.
PREPARATION 87
3-(3-Nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)-3′-(trifluoromethyl)biphenyl-4-ol
A mixture of 3-iodo-3′-(trifluoromethyl)biphenyl-4-ol (Preparation 2, 0.5 g, 1.3 mmol), 3-nitro-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.42 g, 1.3 mmol), potassium fluoride (0.39 g, 0.65 mmol) in tetrahydrofuran (10 mL) was degassed. Bis-(tri-t-Butylphosphino)palladium (0) (35 mg, 0.068 mmol) was added and the reaction heated at 65° C. for 4 hours. After cooling the solvent was removed in vacuo and the residue was purified by silica gel chromatography (ethyl acetate:heptane 1:10) to provide the title compound (0.34 g, 61%) as a colourless oil.
1HNMR (400 MHz, CDCl3): δ 1.80 (m, 3H), 2.05 (m, 2H), 2.10 (m, 1H), 3.75 (m, 1H), 4.05 (m, 1H), 5.22 (m, 1H), 5.50. (br s, 1H), 6.90 (d, 1H), 7.45 (m, 4H), 7.62 (m, 1H), 7.70 (s, 1H), 7.78 (m, 1H).
19FNMR (400 MHz, CDCl3): δ −62
LCMS Rt=3.50 minutes, MS m/z 432 [M−H]−
PREPARATION 88
4-[4-bromo-2-(1-methyl-1H-pyrazol-5-yl)phenoxy]-3-cyano-N-(2,4-dimethoxybenzyl)-N-1,3-thiazol-2-ylbenzenesulfonamide
To a slurry of sodium hydride (54 mg, 1.4 mmol, 60% in mineral oil) in DMF (1 mL) was added 4-bromo-2-(1-methyl-1H-pyrazol-5-yl)phenol (Preparation 89, 210 mg, 0.83 mmol) as a solution in DMF (3 mL). After stirring for 30 minutes, 3-cyano-N-(2,4-dimethoxy-benzyl)-4-fluoro-N-thiazol-2-yl-benzenesulfonamide (Preparation 90, 415 mg, 0.957 mmol) was added. After 3 hours the reaction mixture was diluted with ethyl acetate (10 mL) and washed with water (5 mL) and brine (5 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. Purification by automated flash column chromatography using a 0-100% ethyl acetate/dichloromethane gradient provided the title compound (482 mg, 87%) as a yellow foam.
1HNMR (400 MHz, d6-DMSO): δ 3.66 (s, 3H), 3.82 (s, 3H), 3.90 (s, 3H), 5.02 (m, 2H), 6.20 (m, 1H), 6.34 (m, 1H), 6.41 (m, 1H), 6.63 (m, 1H), 7.15 (m, 3H), 7.45 (m, 2H), 7.65 (m, 1H), 7.71 (m, 1H), 7.85 (m, 1H), 7.91 (m, 1H).
LCMS Rt=1.83 minutes; MS m/z 666 [MH]+.
PREPARATION 89
4-Bromo-2-(1-methyl-1H-pyrazol-5-yl)phenol
To a suspension of 6-bromochromone (1.58 g, 0.0070 mol) in ethanol (30 mL) was added methylhydrazine (0.41 mL, 0.0077 mol) and boron trifluoride etherate (1.15 mL, 0.0091 mol). The reaction was heated to reflux for 22 hours. After cooling, the reaction was concentrated in vacuo and the residue purified by automated flash column chromatography using a 0-100% ethyl acetate/hexanes gradient. This provided the title compound (0.79 g, 44%) as a light yellow solid.
1HNMR (400 MHz, d6-DMSO): δ 3.70 (s, 3H), 6.30 (d, 1H), 6.96 (d, 1H), 7.36 (d, 1H), 7.47 (m, 2H), 10.28 (br s, 1H).
LCMS Rt=1.58 minutes MS m/z 253 [MH]+.
PREPARATION 90
3-Cyano-N-(2,4-dimethoxybenzyl)-4-fluoro-N-1,3-thiazol-2-ylbenzenesulfonamide
N-(2,4-Dimethoxybenzyl)-1,3-thiazol-2-yl-amine (Preparation 91, 8.010 g, 0.032 mol) was dissolved in tetrahydrofuran (100 mL) and the solution was cooled to −78° C. Lithium hexamethyldisilazide in tetrahydrofuran (35.2 mL, 1M) was added dropwise to the reaction mixture. The cooling bath was removed and the reaction mixture was allowed to stir for 30 minutes to attain room temperature before re-cooling to −78° C. and a solution of 3-cyano-4-fluorobenzenesulfonyl chloride (7.028 g, 0.032 mol) in tetrahydrofuran (80 mL) was added dropwise to the reaction mixture. The reaction was allowed to stir 30 minutes at −78° C. before pouring it into saturated aqueous ammonium chloride (50 mL). The aqueous phase was separated and extracted with ethyl acetate (3×30 mL). The combined organic phases were washed twice with 10% aqueous citric acid solution (30 mL), water (30 mL), brine (20 mL), dried over MgSO4, filtered and evaporated. The residue was purified by column chromatography (120 g silica gel column, hexanes/ethyl acetate gradient elution 100/0 to 0/100). Product fractions were combined and evaporated. The residue was triturated with 10% tert-butyl methyl ether in hexanes and the resulting off-white solid collected by filtration and rinsed with hexanes and vacuum dried to provide the title compound (3.58 g).
1H NMR (400 MHz, d6-DMSO) δ 3.64 (s, 3H), 3.72 (s, 3H), 4.99 (s, 2H), 6.44 (dd, 1H), 6.48 (d, 1H), 7.05 (d, 1H), 7.50 (dd, 2H), 7.77 (t, 1H), 8.20 (m, 1H), 8.41 (dd, 1H).
LCMS Rt=1.66 minutes MS m/z 456 [MNa]+.
PREPARATION 91
N-(2,4-Dimethoxybenzyl)-1,3-thiazol-2-amine
2,4-Dimethoxybenzaldehyde (25 g, 150 mmol), 2-aminothiazole (15.1 g, 150 mmol) and piperidine (150 mg, 1.76 mmol) were combined in dichloroethane (500 ml) and the mixture was heated to reflux over 4 Å molecular sieves for 18 hours. The sieves were removed by filtration and the reaction mixture diluted with methanol (300 ml). Sodium borohydride (25 g, 662 mmol) was added in portions and the reaction mixture heated to reflux for 2 hours. The cooled reaction mixture was quenched with water (50 mL) and the organic solvents evaporated in vacuo. The aqueous residue was extracted with ethyl acetate (2×100 mL) and the combined organic solutions extracted with 2M HCl (2×50 mL). The acidic solution was basified with solid potassium carbonate and re-extracted with ethyl acetate (2×50 mL). The organic layer was dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by column chromatography eluting with 9:1 dichloromethane:methanol to yield the title compound (24 g, 96 mmol, 64%).
1HNMR (300 MHz, CDCl3): δ 3.80 (s, 3H), 3.83 (s, 3H), 4.38 (s, 2H), 5.1 (br s, 1H), 6.45 (m, 3H), 7.09 (d, 1H), 7.21 (d, 1H).
PREPARATION 92
5-Chloro-4-(6-chloro-2′-fluoro-4-(pyridazin-4-yl)biphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
Tetrakistriphenylphosphinepalladium (0) (44 mg, 0.038 mmol) and copper (I) iodide (29 mg, 0.152 mmol) were added to a degassed mixture of 5-chloro-4-(6-chloro-2′-fluoro-4-iodobiphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 93, 600 mg, 0.759 mmol), 4-(tributylstannyl)pyridazine (364 mg, 0.987 mmol), caesium fluoride (230 mg, 1.52 mmol), and acetonitrile (5.0 mL). The reaction was heated at 45° C. for 18 hours and then the cooled reaction mixture was diluted with ethyl acetate (30 mL) and filtered through Arbocel. The filtrate was then washed with water (5 mL), brine (5 mL), dried over MgSO4, filtered and evaporated. The residue was purified by preparative HPLC using acetonitrile/water as eluent (15/85 to 95/5, Phenomenex Luna C18 5 u 110 A 21.2×150 mm) to give the title compound (220 mg, 39%) as a brown foam.
1H-NMR (400 MHz, CDCl3): δ 3.63 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 5.26 (s, 2H, NCH2), 6.16 (d, 1H, Ar), 6.29 (dd, 1H, Ar), 6.54 (d, 1H, Ar), 7.10 (s, 1H, Ar), 7.21 (m, 2H, Ar), 7.23 (m, 1H, Ar), 7.34 (m, 1H, Ar), 7.44 (m, 1H, Ar), 7.70 (m, 2H, Ar), 7.77 (d, 1H, Ar), 8.80 (s, 1H, Ar), 9.28 (dd, 1H, Ar), 9.45 (dd, 1H, Ar).
19F-NMR (400 MHz, CDCl3): δ −104.01, −114.04
LCMS (4.5 min) Rt=3.07 minutes, MS no mass ion seen.
PREPARATION 93
5-Chloro-4-(6-chloro-2′-fluoro-4-iodobiphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
6-Chloro-2′-fluoro-4-iodobiphenyl-3-ol (Preparation 94, 651 mg, 1.75 mmol) was added to a mixture of 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 671 mg, 1.85 mmol) and potassium carbonate (967 mg, 7.00 mmol) in dimethylsulfoxide (17.5 mL) and the mixture stirred at room temperature for 18 hours. The reaction was quenched by addition of 0.75 N aq. sodium hydroxide (30.0 mL) and ethyl acetate (30 mL). The aqueous layer was separated and extracted with ethyl acetate (3×10 mL). The combined organic phases were washed with brine (10 mL), dried over MgSO4, filtered and evaporated. The residue was purified on silica, eluting with ethyl acetate:heptanes (3:7) to give the title compound as a mixture of regioisomers (930 mg, 67%). Further purification by preparative HPLC using acetonitrile/water as eluent (5/95-95/5, Phenomenex Luna C18 5 u 110 A 21.2×150 mm) gave the title compound (600 mg, 43%) as a white solid.
1H-NMR (400 MHz, CDCl3): δ 3.68 (s, 3H), 3.71 (s, 3H), 5.33 (s, 2H), 6.24 (d, 1H), 6.32 (dd, 1H), 6.39 (d, 1H), 7.02 (s, 1H), 7.16 (m, 1H), 7.24 (m, 1H), 7.26 (m, 1H), 7.29 (m, 1H), 7.42 (m, 1H), 7.83 (d, 1H), 8.03 (s, 1H), 8.81 (s, 1H).
19F-NMR (400 MHz, CDCl3): δ −104.48, −113.97
LCMS (4.5 min) Rt=4.20 minutes, MS no mass ion seen.
PREPARATION 94
6-Chloro-2′-fluoro-4-iodobiphenyl-3-ol
Boron tribromide (251 mg, 2.61 mmol) was added to a solution of 2-chloro-2′-fluoro-4-iodo-5-methoxybiphenyl (Preparation 95, 671 mg, 1.85 mmol) in dichloromethane (4.7 mL) at −20° C. and the mixture allowed to warm slowly to room temperature for 18 hours. The reaction was quenched by addition of water (10.0 mL), before being diluted with dichloromethane (30 mL). The organic phase was separated and washed with water (3×5.0 mL), brine (5.0 mL), dried over MgSO4, filtered and evaporated to give a purple oil. Column chromatography purification on silica, eluting with 1:4 ethyl acetate:heptanes gave the title compound (610 mg, 94%) as a colourless oil.
1H-NMR (400 MHz, CDCl3): δ 6.97 (s, 1H, Ar), 7.15 (t, 1H, Ar), 7.22 (m, 1H, Ar), 7.28 (m, 1H, Ar), 7.39 (m, 1H, Ar), 7.76 (s, 1H, Ar),
19F-NMR (400 MHz, CDCl3): δ −114.03
LCMS (4.5 min) Rt=3.44 minutes, MS m/z 347 [M−H]−
PREPARATION 95
2-Chloro-2′-fluoro-4-iodo-5-methoxybiphenyl
N-Iodosuccinimide (683 mg, 3.04 mmol) was added to a solution of 2-chloro-2′-fluoro-5-methoxybiphenyl (Preparation 96, 749 mg, 3.16 mmol) in concentrated sulfuric acid (0.09 mL), acetic acid (4.7 mL) and dichloromethane (4.7 mL). The resulting mixture was stirred at room temperature for 18 hours and then partitioned between dichloromethane (30 mL) and water (5 mL). The organic phase was separated and washed with water (2×5 mL), brine (5 mL), dried over MgSO4, filtered and evaporated to give a red oil. Column chromatography purification on silica, eluting with dichloromethane:heptanes (1:9) gave the title compound (671 mg, 58%) as a colourless oil.
1H-NMR (400 MHz, CDCl3): δ 3.86 (s, 3H, OMe), 6.75 (s, 1H, Ar), 7.16 (t, 1H, Ar), 7.22 (m, 1H, Ar), 7.32 (m, 1H, Ar), 7.41 (m, 1H, Ar), 7.88 (s, 1H, Ar),
19F-NMR (400 MHz, CDCl3): δ −114.02
LCMS (4.5 min) Rt=3.88 minutes, No mass ion seen
PREPARATION 96
2-Chloro-2′-fluoro-5-methoxybiphenyl
Tetrakistriphenylphosphinepalladium (0) (229 mg, 0.20 mmol) was added to a degassed mixture of 2-fluorophenylboronic acid (556 mg, 3.97 mmol), 2-bromo-1-chloro-4-methoxybenzene (0.49 mL, 3.57 mmol), caesium carbonate (3.87 g, 11.9 mmol), water (5.0 mL) and dioxane (26.0 mL). The reaction was heated at 80° C. for 18 hours, cooled to room temperature and then partitioned between ethyl acetate (30.0 mL) and sat. aq. ammonium chloride (10.0 mL). The aqueous phase was separated and extracted with ethyl acetate (2×10 mL). The combined organic phases were washed with brine (5.0 mL), dried over MgSO4, filtered and evaporated to give a pale yellow oil. Column chromatography purification on silica, eluting with heptanes gave the title compound (749 mg, 88%) as a colourless oil.
1HNMR (400 MHz, CDCl3): δ 3.81 (s, 3H, OMe), 6.87-6.89 (m, 2H, Ar), 7.15 (m, 1H, Ar), 7.21 (m, 1H, Ar), 7.31-7.41 (m, 3H, Ar).
LCMS (4.5 min) Rt=3.00 minutes, No mass ion seen.
PREPARATION 97
5-Chloro-4-(6-chloro-3′-fluoro-4-(pyridazin-4-yl)biphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
5-Chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 136 mg, 0.29 mmol) was added to a solution of 6-chloro-3′-fluoro-4-(pyridazin-4-yl)biphenyl-3-ol (Preparation 98, 133 mg, 0.29 mmol) and potassium carbonate (183 mg, 0.88 mmol) in dimethylsulfoxide (5 mL). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with sodium hydroxide (1M, 5 mL) and extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was dissolved in dimethylsulfoxide:acetonitrile (2.5 mL: 1.5 mL) and then purified on the reverse phase HPLC eluting with acetonitrile:water (from 5:95 to 95:5, 30 minutes gradient then 5 minutes isocratic) to give the title compound (60 mg, 18%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 3.64 (s, 3H), 3.71 (s, 3H), 5.23 (s, 2H), 6.20 (d, 1H), 6.31 (dd, 1H), 6.52 (d, 1H), 7.08 (s, 1H), 7.13-7.23 (m, 4H), 7.43-7.48 (m, 1H), 7.69-7.71 (m, 2H), 7.80 (d, 1H), 8.80 (s, 1H), 9.28-9.30 (m, 1H), 9.44-9.45 (m, 1H).
19F NMR (400 MHz, CDCl3): δ −104, −112.
LCMS (4.5 min acidic run) Rt=3.16 minutes, MS m/z 742 [MH]+.
PREPARATION 98
6-Chloro-3′-fluoro-4-(pyridazin-4-yl)biphenyl-3-ol
Caesium fluoride (219 mg, 1.44 mmol) was added to a solution of 6-chloro-3′-fluoro-4-iodobiphenyl-3-ol (Preparation 99, 251 mg, 0.72 mmol) and 4-(tributylstannyl)pyridazine (345 mg, 0.93 mmol) in acetonitrile (5 mL). The reaction mixture was degassed and copper iodide (28 mg, 0.15 mmol) and tetrakistriphenylphosphinepalladium (0) (83 mg, 0.07 mmol) were added. The reaction mixture was stirred at 80° C. for 3 hours. The cooled reaction mixture was diluted with ethyl acetate (20 mL) and quenched with a solution of ammonia (10%, 10 mL) and stirred for a further 10 minutes. The organic layer was separated and washed with brine (1×10 mL), dried over MgSO4, filtered and concentrated in vacuo. The residue was purified using silica gel chromatography (Biotage) eluting with heptane:ethyl acetate (93:7 to 0:100) to give the title compound (185 mg, 62%) as a yellow solid.
1H NMR (400 MHz, d6-DMSO): δ 7.01 (s, 1H), 7.26-7.30 (m, 3H), 7.58-7.60 (m, 1H), 7.72 (s, 1H), 7.94-7.96 (m, 1H), 9.25-9.27 (m, 1H), 9.52-9.53 (m, 1H).
LCMS (4.5 min acidic run) Rt=2.88 minutes, MS m/z 301 [MH]+.
PREPARATION 99
6-Chloro-3′-fluoro-4-iodobiphenyl-3-ol
N-Iodosuccinimide (261 mg, 1.16 mmol) was added to a mixture of 6-chloro-3′-fluorobiphenyl-3-ol (Preparation 100, 270 mg, 1.21 mmol) and concentrated sulphuric acid (24 μL, 0.43 mmol) in acetic acid (3 mL) and dichloromethane (3 mL). The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with dichloromethane (10 mL) and washed with sodium metabisulfite (0.5M, 10 mL), dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography eluting with heptane:dichloromethane (70:30) to give the title compound (193 mg, 46%) as a pale yellow solid.
1H NMR (400 MHz, CDCl3): δ 5.28 (br-s, 1H), 6.99-7.12 (m, 3H), 7.29-7.35 (m, 1H), 7.68 (s, 1H).
19F NMR (400 MHz, CDCl3): δ −113.
LCMS (4.5 min acidic run) Rt=3.51 minutes, MS m/z 347 [M−H]
PREPARATION 100
6-Chloro-3′-fluorobiphenyl-3-ol
Caesium carbonate (1.15 g, 3.53 mmol) was added to a solution of 4-chloro-3-iodophenol (300 mg, 2.14 mmol) and 3-fluorophenylboronic acid (330 mg, 1.30 mmol) in dioxane:water (22.5 mL:4.5 mL). The reaction mixture was degassed and tetrakistriphenylphosphine palladium (0) (69 mg, 0.06 mmol) was added. The reaction mixture was stirred at 70° C. for 5 hours. The cooled reaction mixture was concentrated in vacuo and the aqueous residue was extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified on the biotage eluting with heptane:ethyl acetate (from 98:2 to 80:20) to give the title compound (270 mg, 100%) as a yellow solid.
1H NMR (400 MHz, CDCl3): δ 6.00 (br-s, 1H), 6.76-6.82 (m, 2H), 7.04-7.11 (m, 1H), 7.12-7.17 (m, 1H), 7.18-7.21 (m, 1H), 7.32 (d, 1H), 7.36-7.42 (m, 1H).
19F NMR (400 MHz, CDCl3): δ −114.
LCMS (4.5 min acidic run) Rt=3.16 minutes, MS m/z 221 [M−H]−
PREPARATION 101
5-Chloro-4-(6-chloro-4′-fluoro-4-(pyridazin-4-yl)biphenyl-3-yloxy)-N-(2,4-dimethoxybenzyl)-2-fluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide
6-Chloro-4′-fluoro-4-(pyridazin-4-yl)biphenyl-3-ol (Preparation 102, 150 mg, 0.50 mmol), 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 345 mg, 0.75 mmol), and potassium carbonate (207 mg, 1.50 mmol) were suspended in dimethyl sulfoxide (2 mL). The reaction mixture was stirred for 18 hours at room temperature. Water (50 mL) was added and the suspension was extracted with ethyl acetate (2×50 mL) and dichloromethane (3×50 mL). The organic layers were combined, dried over MgSO4, filtered and evaporated. The residue was purified by semi preparative reverse phase HPLC (solvent A: 0.05% formic acid in acetonitrile; solvent B: 0.05% formic acid in water; flow rate: 15 mL/min; gradient 0 min 5% A, 2.5 min 5% A, 22.5 min 95% A, 32.5 min 95% A then return to initial conditions) to afford the title compound (100 mg, 27%) as a glass.
1H NMR (400 MHz, CDCl3): δ 3.64 (s, 3H), 3.71 (s, 3H), 5.25 (s, 2H), 6.20 (m, 1H), 6.31 (m, 1H), 6.52 (m, 1H), 7.07 (s, 1H), 7.18 (m, 3H), 7.44 (m, 2H), 7.70 (s, 1H), 7.74 (m, 1H), 7.80 (m, 1H), 8.81 (s, 1H), 9.29 (m, 1H), 9.46 (m, 1H).
19F NMR (400 MHz, CDCl3): δ −104.0, −112.2
LCMS Rt=3.46 min MS m/z 742 [MH]+.
PREPARATION 102
6-Chloro-4′-fluoro-4-(pyridazin-4-yl)biphenyl-3-ol
A suspension of 6-chloro-4′-fluoro-4-iodobiphenyl-3-ol (Preparation 103, 300 mg, 0.86 mmol), 4-(tributylstannyl)-pyridazine (413 mg, 1.12 mmol), caesium fluoride (261 mg, 1.72 mmol), and copper (I) iodide (33 mg, 0.17 mmol) in acetonitrile (5 mL) was degassed for 20 minutes under nitrogen. Tetrakistriphenylphosphinepalladium (0) (100 mg, 0.09 mmol) was added and the reaction mixture was heated for 18 hours at 45° C. under nitrogen. The cooled reaction mixture was filtered through Arbocel and the Arbocel pad was washed with ethyl acetate (100 mL). The organic layer was washed with brine (2×15 mL), dried over MgSO4, filtered and evaporated. The residue was purified by silica gel chromatography eluting with 20% heptane in ethyl acetate to afford the title compound (150 mg, 58%) as an oil.
1H NMR (400 MHz, CDCl3): δ 6.98 (s, 1H), 7.20 (m, 2H), 7.48 (m, 2H), 7.67 (s, 1H), 8.04 (m, 1H), 9.20 (m, 1H), 9.56 (m, 1H).
19F NMR (400 MHz, CDCl3): δ −116.2
LCMS Rt=2.85 min MS m/z 299 [M−H]
PREPARATION 103
6-Chloro-4′-fluoro-4-iodobiphenyl-3-ol
To a solution of 6-chloro-4′-fluorobiphenyl-3-ol (Preparation 104, 280 mg, 1.26 mmol) in acetic acid (2.5 mL), dichloromethane (2.5 mL) and concentrated sulfuric acid (25 μL) was added N-iodosuccinimide (272 mg, 1.21 mmol) at room temperature. The reaction mixture was stirred for 18 hours at room temperature. Dichloromethane (60 mL) was added and the organic layer was washed with brine (2×20 mL), dried over MgSO4, filtered and evaporated. The residue was purified by silica gel chromatography eluting 33% dichloromethane in heptane to afford the title compound (306 mg, 70%) as a colourless oil.
1H NMR (400 MHz, CDCl3): δ 5.27 (s, 1H), 6.96 (s, 1H), 7.12 (m, 2H), 7.38 (m, 2H), 7.75 (s, 1H).
19F NMR (400 MHz, CDCl3): δ −113.8
LCMS Rt=3.52 min MS m/z 347 [M−H]
PREPARATION 104
6-Chloro-4′-fluorobiphenyl-3-ol
A solution of 4-fluorophenylboronic acid (500 mg, 3.57 mmol), 4-chloro-3-iodophenol (455 mg, 1.79 mmol) and caesium carbonate (1.75 g) in dioxane (10 mL) and water (5 mL) was degassed 1 hour with nitrogen. Tetrakis(triphenylphosphine)palladium (0) (104 mg, 0.09 mmol) was added and the reaction mixture was heated for 18 hours at 75° C. The cooled reaction mixture was concentrated in vacuo and the residual aqueous layer was extracted with ethyl acetate (3×50 mL). The combined organic layer were dried over MgSO4, filtered and evaporated. The residue was purified by silica gel chromatography eluting with 20% ethyl acetate in heptane to afford the title compound (280 mg, 70%) as a yellow oil.
1H NMR (400 MHz, CDCl3): δ 4.85 (s, 1H), 6.79 (m, 2H), 7.11 (m, 2H), 7.31 (m, 1H), 7.40 (m, 2H).
19F NMR (400 MHz, CDCl3): δ −114.0
LCMS Rt=2.96 min MS m/z 221 [M−H]
PREPARATION 105
5-Chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(5-fluoropyridin-2-yl)benzenesulfonamide
5-Chloro-2,4-difluorobenzenesulfonyl chloride (200 mg, 0.81 mmol), N-(2,4-dimethoxybenzyl)-5-fluoropyridin-2-amine (Preparation 106, 255 mg, 0.97 mmol) and pyridine (196 μL, 2.43 mmol) in dichloromethane (3 mL) were stirred at room temperature for 36 hours. The mixture was evaporated in vacuo and the residue was purified by silica gel column chromatography (5 g Varian bond-elut cartridge, heptane/ethyl acetate 100/0 to 70/30) to afford the title compound (193 mg) as a gum.
1H NMR (400 MHz, CDCl3) δ ppm 3.68 (s, 3 H), 3.76 (s, 3 H), 4.99 (s, 2 H), 6.31-6.37 (m, 2H), 6.96-7.05 (m, 1 H), 7.16 (d, 1 H), 7.29-7.36 (m, 2 H), 7.89 (dd, 1 H), 8.15-8.18 (m, 1 H).
LCMS Rt=1.74 minutes, MS no mass ion seen.
PREPARATION 106
N-(2,4-Dimethoxybenzyl)-5-fluoropyridin-2-amine
5-Fluoropyridin-2-amine (500 mg, 4.46 mmol) and 2,4-dimethoxybenzaldehyde (674 mg, 4.06 mmol) were stirred in dichloromethane (10 mL) at room temperature for 30 minutes. Sodium triacetoxyborohydride (1.3 g, 6.08 mmol) was added portion wise. The mixture was then stirred at room temperature for 18 hours before treatment with 1M aqueous sodium hydroxide solution (10 mL). The aqueous layer was separated and extracted with dichloromethane (10 mL). The combined organic layers were dried through a phase separating cartridge and evaporated to afford the title compound (1.2 g) as a brown solid.
1H NMR (400 MHz, CDCl3) δ 3.80 (s, 3 H), 3.84 (s, 3 H), 4.38 (d, 2 H), 4.84 (br. s., 1 H), 6.35 (dd, 1 H), 6.43 (dd, 2.34 Hz, 1 H), 6.48 (d, 1 H), 7.12-7.22 (m, 2 H), 7.96 (d, 1 H).
LCMS Rt=2.07 minutes, MS m/z 263 [MH]+.
PREPARATION 107
3-(1-Methyl-1H-pyrazol-5-yl)biphenyl-4-ol
To a stirred suspension of 5-[4-(benzyloxy)biphenyl-3-yl]-1-methyl-1H-pyrazole (Preparation 108, 3 g, 8.81 mmol) in methanol (26 mL) was added palladium on carbon (300 mg). The mixture was stirred at room temperature under a hydrogen atmosphere for 16 hours before filtering through a Celite™ pad. The pad was washed with tetrahydrofuran and the filtrate was evaporated in vacuo. The residue was dissolved in ethyl acetate (26 mL) and the solution degassed with argon. Palladium on carbon (300 mg) was added and the mixture was stirred at room temperature under a hydrogen atmosphere for 6 hours. The catalyst was filtered off through a Celite pad and the filtrate was evaporated in vacuo. Trituration of the residue with n-hexane afforded the title compound (1.95 g) as a white solid.
1H NMR (400 MHz, d-6DMSO) δ 3.89 (s, 3H), 6.30 (d, 1H), 7.06 (d, 1H), 7.29 (t, 1H), 7.38-7.46 (m, 4H), 7.56-7.64 (m, 3H), 10.14 (br. s, 1H).
LCMS Rt=3.29 minutes, MS m/z 251 [MH]+.
PREPARATION 108
5-[4-(Benzyloxy)biphenyl-3-yl]-1-methyl-1H-pyrazole
A solution of benzyl 3-bromobiphenyl-4-yl ether (500 mg, 1.47 mmol) and (1-methyl-1H-pyrazol-5-yl)boronic acid (185 mg, 1.47 mmol) in dioxane (4 mL) was degassed with argon for 30 minutes. Tris(dibenzylideneacetone)dipalladium (0) (54 mg, 0.06 mmol) and tricyclohexylphosphine (33 mg, 0.12 mmol) were added to the mixture under an argon atmosphere. A degassed solution of tripotassium phosphate (626 mg, 2.95 mmol) in water (2 mL) was added and the mixture was stirred at reflux for 16 hours under an argon atmosphere. The cooled reaction mixture was filtered through a pad of Celite and the filtrate evaporated in vacuo. The residue was dissolved in ethyl acetate (25 mL) and the solution was washed with water (2×10 mL), brine (10 mL), dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography (10% ethyl acetate in hexane) to afforded the title compound (320 mg).
1H NMR (400 MHz, d-6DMSO) δ 3.67 (s, 3H), 5.21 (s, 2H), 6.35 (d, 1H), 7.27-7.45 (m, 10H), 7.56 (d, 1H), 7.65-7.69 (m, 2H), 7.72-7.77 (m, 1H).
LCMS Rt=2.21 minutes, MS m/z 341 [MH]+.
PREPARATION 109
tert-butyl 4-{4-[4-(2-chloro-4-{[(2,4-dimethoxybenzyl)(pyrimidin-4-yl)amino]sulfonyl}-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl]pyridin-2-yl}piperazine-1-carboxylate
To a solution of tert-butyl 4-(4-(4-hydroxy-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 114, 200 mg, 0.401 mmol) in dimethyl sulfoxide (5 mL) was added potassium carbonate (111 mg, 0.802 mmol) followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(pyrimidin-4-yl)benzenesulfonamide (Preparation 110, 182 mg, 0.401 mmol). The reaction mixture was stirred at room temperature for 2 hours and then partitioned between ethyl acetate (20 mL) and water (10 mL). The organic layer was separated, dried over anhydrous MgSO4, filtered and evaporated. The residue was purified by flash chromatography on silica gel eluting with 30% heptane in ethyl acetate to give the title compound (320 mg, 85%) as yellow foam.
1HNMR (400 MHz, CDCl3): δ 1.49 (s, 9H), 3.55 (br s, 8H), 3.75 (s, 3H), 3.77 (s, 3H), 5.18 (s, 2H), 6.42-6.36 (m, 3H), 6.75 (d, 1H), 6.82 (s, 1H), 7.23-7.16 (m, 3H), 7.75-7.66 (m, 6H), 8.02 (d, 1H), 8.16 (d, 1H), 8.46 (d, 1H), 8.79 (s, 1H)
19FNMR (376 MHz, CDCl3): δ −106.76 (F), −62.55 (CF3)
LCMS Rt=4.49 minutes, m/z 935 [MH]+.
PREPARATION 110
5-Chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(pyrimidin-4-yl)benzenesulfonamide
N-(2,4-Dimethoxybenzyl)pyrimidin-4-amine (Preparation 111, 1.80 g, 7.35 mmol), 5-chloro-2,4-difluorobenzene-1-sulfonyl chloride (1.81 g, 7.35 mmol) and 1,4-diazabicyclo[2.2.2]octane (0.82 g, 7.35 mmol) in acetonitrile (50 mL) were stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo and the residue was partitioned between dichloromethane (30 mL) and water (15 mL). The organic layer was separated and dried over anhydrous MgSO4, filtered and evaporated. The residue was purified by flash chromatography on silica gel eluting with 10% dichloromethane in ethyl acetate to give the title compound (1.47 g, 44%) as an orange solid.
1HNMR (400 MHz, CDCl3): δ 3.77 (s, 3H), 3.78 (m, 3H), 5.23 (s, 2H), 6.43-6.41 (m, 2H), 6.98 (t, 1H), 7.16-7.14 (dd, 1H), 7.20 (d, 1H), 8.12 (t, 1H), 8.49 (d, 1H), 8.79 (s, 1H).
19FNMR (376 MHz, CDCl3) δ −105.97 (F), −100.64 (F).
LCMS Rt=3.51 minutes, no mass ion seen.
PREPARATION 111
N-(2,4-dimethoxybenzyl)pyrimidin-4-amine
6-Chloro-N-(2,4-dimethoxybenzyl)pyrimidin-4-amine (Preparation 112, 3.46 g, 12.39 mmol) was dissolved in ethanol (140 mL). The solution was degassed and then 10% palladium on carbon (0.98 g) was added followed by ammonium formate (4.55 g, 72.15 mmol) and the reaction was heated at 80° C. for 2 hours. The reaction was cooled to room temperature, filtered through pad of Celite™ and the filtrate was concentrated in vacuo. The residue was partitioned between dichloromethane (30 mL) and water (15 mL). The organic layer was separated, dried over anhydrous MgSO4, filtered and evaporated to give the title compound (2.94 g, 97%) as viscous oil.
1HNMR (400 MHz, CDCl3): δ 3.79 (s, 3H), 3.81 (m, 3H), 4.43 (br s, 2H), 5.55 (br s, 1H), 6.32 (d, 1H), 6.45-6.41 (m, 2H), 7.15 (d, 1H), 8.12 (d, 1H), 8.51 (s, 1H).
LCMS Rt=1.50 minutes, m/z 246 [MH]+.
PREPARATION 112
6-Chloro-N-(2,4-dimethoxybenzyl)pyrimidin-4-amine
N,N-Diisopropylethylamine (8.10 mL, 46.50 mmol) and 2,4-dimethoxybenzylamine (2.52 mL, 16.78 mmol) were added to a solution of 4,6-dichloropyrimidine (2.50 g, 16.78 mmol) in butanol (80 mL) and reaction mixture was heated at 100° C. for 2 hours. The reaction mixture was cooled to room temperature and washed with water (30 mL). The aqueous layer was extracted with ethyl acetate (30 mL) and the combined organic layers were dried over anhydrous MgSO4, filtered and evaporated. The residue was triturated in heptane to give the title compound (4.00 g, 85%) as a solid.
1HNMR (400 MHz, CDCl3): δ 3.80 (s, 3H), 3.83 (m, 3H), 4.40 (br s, 2H), 6.47-6.36 (m, 3H), 7.16 (d, 1H), 8.31 (s, 1H).
LCMS Rt=2.87 minutes, m/z 278 [M−H]−.
PREPARATION 113
tert-Butyl 4-{4-[4-(2-chloro-4-{[(2,4-dimethoxybenzyl)(1,3,4-thiadiazol-2-yl)amino]sulfonyl}-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl]pyridin-2-yl}piperazine-1-carboxylate
tert-Butyl 4-(4-(4-hydroxy-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 114, 200 mg, 0.400 mmol) was dissolved in dimethyl sulfoxide (3 mL) and potassium carbonate (110 mg, 0.800 mmol) was added followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 185 mg, 0.400 mmol). The reaction was stirred at room temperature for 18 hours and then partitioned between ethyl acetate (10 mL) and water (5 mL). The organic layer was separated and washed with brine (5 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified on silica gel by Biotage™ (7% to 60% ethyl acetate in heptane over 20 CV) to give the title product (340 mg, 90%) as a yellow foam.
1HNMR (400 MHz, CDCl3): δ 1.40 (s, 9H), 3.40 (m, 8H), 3.60 (s, 3H), 3.70 (s, 3H), 5.20 (s, 2H), 6.20 (s, 1H), 6.30 (m, 2H), 6.75 (d, 1H), 6.80 (s, 1H), 7.20 (m, 2H), 7.50-7.90 (m, 7H), 8.20 (d, 1H), 8.80 (s, 1H).
19F NMR (376 MHz, CDCl3): δ −105.0, −63.0
LCMS Rt=3.26 minutes, MS m/z 941 [MH]+.
PREPARATION 114
tert-Butyl 4-(4-(4-hydroxy-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate
tert-Butyl 4-(4-(4-(benzyloxy)-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 115, 1.30 g, 2.20 mmol) was dissolved in ethanol (20 mL) at room temperature and palladium hydroxide on activated charcoal (130 mg) was added. The reaction mixture was heated at 60° C. under a hydrogen atmosphere (50 psi) for 18 hours. The mixture was then filtered through a pad of Celite™, rinsed with ethanol and concentrated in vacuo. The residue was purified on silica gel by Biotage (5% to 60% ethyl acetate in heptane over 20 CV). Further purification by reverse phase using acetonitrile/water (5/95-95/5) with 0.1% formic acid as eluent gave the title compound (650 mg, 59%) as a white powder.
1HNMR (400 MHz, CDCl3): δ 1.49 (s, 9H), 3.59 (m, 8H), 6.75 (s, 1H), 6.79 (d, 1H), 7.08 (d, 1H), 7.48 (s, 1H), 7.53 (d, 1H), 7.60 (m, 4H), 8.30 (d, 1H).
19FNMR (376 MHz, CDCl3): δ −62.41
LCMS Rt=2.93 minutes, MS m/z 500 [MH]+.
PREPARATION 115
tert-Butyl 4-(4-(4-(benzyloxy)-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate
tert-Butyl 4-(4-(2-(benzyloxy)-5-chlorophenyl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 116, 1.10 g, 2.29 mmol), 4-(trifluoromethyl)phenylboronic acid (866 mg, 4.58 mmol), di-μ-chlorobis[5-chloro-2-[(4-chlorophenyl)(hydroxyimino)methyl]phenyl]palladium (II) dimer (93 mg, 0.114 mmol), tri-tert-butylphosphonium tetrafluoroborate (66 mg, 0.228 mmol), potassium carbonate (635 mg, 4.60 mmol) and tetrabutyl ammonium hydroxide (1M in methanol, 0.46 mL, 0.46 mmol) were combined in a microwave vial. Dimethylformamide (12 mL) was added and the vial was sealed. The mixture was heated at 130° C. for 2 hours in a microwave and then partitioned between ethyl acetate (15 mL) and water (5 mL). The organic layer was separated, washed with brine (5 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was dissolved in dimethylformamide (12 mL) and 4-(trifluoromethyl)phenylboronic acid (866 mg, 4.58 mmol), di-mu-chlorobis[5-chloro-2-[(4-chlorophenyl)(hydroxyimino)methyl]phenyl]palladium (II) dimer (93 mg, 0.114 mmol), tri-tert-butylphosphonium tetrafluoroborate (66 mg, 0.228 mmol), potassium carbonate (635 mg, 4.60 mmol) and tetrabutyl ammonium hydroxyde (1M in methanol, 0.46 mL, 0.46 mmol) were added. The mixture was heated at 130° C. for 1 hour in microwave and then partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated in vacuo. The oil residue was purified on silica gel by Biotage (5% to 80% ethyl acetate in heptane over 20 CV) to give the title compound as a white solid (655 mg, 48%).
1HNMR (400 MHz, CDCl3): δ 1.40 (s, 9H), 3.50 (m, 8H), 5.10 (s, 2H), 6.85 (m, 2H), 7.15 (d, 1H), 7.30 (m, 4H), 7.60 (m, 2H), 7.65-7.80 (m, 5H), 8.20 (d, 1H).
LCMS Rt=3.05 minutes, MS m/z 590 [MH]+.
PREPARATION 116
tert-Butyl 4-(4-(2-(benzyloxy)-5-chlorophenyl)pyridin-2-yl)piperazine-1-carboxylate
tert-Butyl 4-(4-(5-chloro-2-hydroxyphenyl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 117, 1.85 g, 4.755 mmol) was dissolved in dimethylformamide (10 mL) at room temperature under a nitrogen atmosphere. Potassium carbonate (1.31 g, 9.51 mmol) was added and the mixture was stirred for 10 minutes. Benzyl bromide (0.622 mL, 5.23 mmol) was added dropwise and the reaction mixture was heated at 60° C. for 18 hours and then partitioned between ethyl acetate (40 mL) and water (20 mL). The organic layer was separated and washed with brine (20 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo to give the title compound (2.20 g, 96%) as a light yellow solid.
1H NMR (400 MHz, CDCl3): δ 1.45 (s, 9H), 3.40 (m, 4H), 3.50 (m, 4H), 5.05 (s, 2H), 680 (m, 2H), 6.95 (d, 1H), 7.20-7.40 (m, 7H), 8.20 (d, 1H).
LCMS Rt=3.56 minutes MS m/z 480 [MH]+.
PREPARATION 117
tert-Butyl 4-(4-(5-chloro-2-hydroxyphenyl)pyridin-2-yl)piperazine-1-carboxylate
tert-Butyl 4-(4-bromopyridin-2-yl)piperazine-1-carboxylate (1.00 g, 2.66 mmol), 5-chloro-2-hydroxyphenylboronic acid (458 mg, 2.66 mmol) and sodium carbonate (1.13 g, 10.64 mmol) were combined and dissolved in a mixture of dioxane/water (14 mL/4 mL). The reaction mixture was degassed for 20 min with nitrogen and then tetrakistriphenylphosphinepalladium (0) (153 mg, 0.133 mmol) was added. The reaction mixture was heated at 70° C. for 18 hours and then partitioned between ethyl acetate (20 mL) and water (10 mL). The organic layer was separated, washed with brine (10 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified on silica gel by Biotage™ (10% to 60% ethyl acetate in heptane over 20 CV) to give the title compound (700 mg, 66%) as a white solid.
1H NMR (400 MHz, CD3OD): δ 1.40 (s, 9H), 3.50 (s, 8H), 6.80-6.90 (m, 2H), 6.95 (s, 1H), 7.15 (d, 1H), 7.30 (s, 1H), 8.05 (d, 1H).
LCMS Rt=2.48 minutes MS m/z 388 [M−H]−.
PREPARATION 118
5-Chloro-4-[(3′-cyano-3-pyridazin-4-ylbiphenyl-4-yl)oxy]-N-(2,4-dimethoxybenzyl)-2-fluoro-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide
4′-Hydroxy-3′-(pyridazin-4-yl)biphenyl-3-carbonitrile (Preparation 119, 330 mg, 1.21 mmol) and potassium carbonate (334 mg, 2.42 mmol) were dissolved in dimethylsulfoxide (7 mL). Then 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (Preparation 16, 558 mg, 1.21 mmol) was added and the reaction was stirred at room temperature for 2 hours. Water (15 mL) and ethyl acetate (25 mL) were added and the two layers were separated. The organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 10% dichloromethane in ethyl acetate to give the title compound (561 mg, 65%).
LCMS Rt=3.57 minutes, MS m/z 715 [MH]+.
1HNMR (400 MHz, CDCl3): δ 3.69 (s, 3H), 3.75 (s, 3H), 5.30 (s, 2H), 6.26 (s, 1H), 6.36 (d, 1H), 6.55 (d, 1H), 7.18 (d, 1H), 7.26 (d, 1H), 7.63 (t, 1H), 7.74-7.70 (m, 4H), 7.85 (d, 2H), 7.89 (s, 1H), 8.82 (s, 1H), 9.28 (d, 1H), 9.47 (s, 1H).
19FNMR (376 MHz, CDCl3): δ −104.24 (s, 1 F)
PREPARATION 119
4′-hydroxy-3′-pyridazin-4-ylbiphenyl-3-carbonitrile
A mixture of 4′-hydroxy-3′-iodobiphenyl-3-carbonitrile (Preparation 120, 715 mg, 2.23 mmol), 4-(tributylstannyl)pyridazine (904 mg, 2.45 mmol) and cesium fluoride (677 mg, 4.46 mmol) in N,N-dimethylformamide (5 mL) was degassed under nitrogen. Then tetrakistriphenylphosphinepalladium (0) (258 mg, 0.22 mmol) and copper (I) iodide (85 mg, 0.45 mmol) were added, the reaction mixture was further degassed and then heated at 60° C. for 4 hours. The cooled reaction mixture was quenched with 10% ammonia (0.88M) in water (10 mL), diluted with ethyl acetate (20 mL) and then the mixture was stirred for 20 minutes. The resulting mixture was further diluted with ethyl acetate (10 mL) and the layers were separated. The organic layer was dried over anhydrous MgSO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 10% dichloromethane in ethyl acetate to give the title compound (355 mg, 55%).
LCMS Rt=2.54 minutes, MS m/z 274 [MH]+.
1HNMR (400 MHz, d-6DMSO): δ 7.11 (d, 1H), 7.61 (t, 1H), 7.75-7.70 (m, 2H), 7.89 (d, 1H), 8.05-8.00 (m, 2H), 8.24 (d, 1H), 9.25 (d, 1H), 9.59 (s, 1H), 10.51 (s, 1H).
PREPARATION 120
4′-Hydroxy-3′-iodobiphenyl-3-carbonitrile
4′-Hydroxybiphenyl-3-carbonitrile (Preparation 121, 570 mg, 2.92 mmol) was dissolved in dichloromethane (10 mL) and acetic acid (10 mL). Then concentrated sulfuric acid (0.30 mL) and N-iodosuccinimide (657 mg, 2.92 mmol) were added at 0° C. (ice-bath cooling) and the reaction was allowed to warm to room temperature over 2 hours. The mixture was partitioned between ethyl acetate (30 mL) and water (15 mL). The organic layer was separated and dried over anhydrous MgSO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 50% ethyl acetate in heptane to give the title compound (720 mg, 77%).
LCMS Rt=3.16 minutes, MS m/z 320 [M−H]−.
1HNMR (400 MHz, CDCl3): δ 5.41 (s, 1H), 7.08 (d, 1H), 7.46-7.44 (m, 1H), 7.54-7.50 (m, 1H), 7.62-7.59 (m, 1H), 7.74-7.71 (m, 1H), 7.78 (s, 1H), 7.86 (d, 1H).
PREPARATION 121
4′-Hydroxybiphenyl-3-carbonitrile
3-Cyanophenylboronic acid (1.18 g, 8.03 mmol), 4-bromophenol (1.16 g, 6.69 mmol) and sodium carbonate (2.12 g, 20.07 mmol) were dissolved in dioxane (20 mL) and water (8 mL) and the reaction mixture was degassed under nitrogen. Tetrakistriphenylphosphinepalladium (0) (0.77 g, 0.67 mmol) was added and the reaction was stirred at 110° C. for 2 hours. The mixture was cooled to room temperature, filtered through pad of Arbocel™ and the filtrate was concentrated in vacuo. The residue was partitioned between ethyl acetate (30 mL) and water (15 mL). The organic layer was dried over anhydrous magnesium sulphate, filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 40% ethyl acetate in heptane to give the title compound as a pale yellow solid (0.58 g, 44%).
LCMS Rt=2.81 minutes, m/z mass ion not detected
1HNMR (400 MHz, CDCl3): δ 4.97 (s, 1H), 6.95 (d, 2H), 7.46 (d, 2H), 7.59-7.49 (m, 2H), 7.75 (d, 1H), 7.81 (s, 1H).
PREPARATION 122
tert-butyl 4-{4-[4-(2-chloro-4-{[(2,4-dimethoxybenzyl)(pyrimidin-2-yl)amino]sulfonyl}-5-fluorophenoxy)-4′-(trifluoromethyl)biphenyl-3-yl]pyridin-2-yl}piperazine-1-carboxylate
tert-Butyl 4-(4-(4-hydroxy-4′-(trifluoromethyl)biphenyl-3-yl)pyridin-2-yl)piperazine-1-carboxylate (Preparation 114, 240 mg, 0.480 mmol) was dissolved in dimethyl sulfoxide (3 mL) and then potassium carbonate (133 mg, 0.962 mmol) followed by 5-chloro-N-(2,4-dimethoxybenzyl)-2,4-difluoro-N-(pyrimidin-2-yl)benzenesulfonamide (Preparation 13, 219 mg, 0.480 mmol) were added. The reaction mixture was stirred at room temperature for 20 hours. The reaction was then partitioned between ethyl acetate (15 mL) and 2M HCl (5 mL). The organic layer was separated and dried over anhydrous MgSO4, filtered and evaporated. The residue was purified by flash chromatography on silica gel (gradient: 5-60% ethyl acetate in heptane) to give the title compound (380 mg, 84%) as a yellow foam.
1HNMR (400 MHz, CDCl3): δ 1.49 (s, 9H), 3.55 (br s, 8H), 3.76 (s, 6H), 5.38 (s, 2H), 6.44-6.36 (m, 3H), 6.75 (dd, 1H), 6.84 (s, 1H), 6.90 (t, 1H), 7.22-7.18 (m, 2H), 7.75-7.65 (m, 6H), 8.13-8.10 (m, 2H), 8.40 (d, 2H)
19FNMR (376 MHz, CDCl3): δ −107.11 (F), −62.50 (CF3)
LCMS Rt=4.41 minutes, m/z 935 [MH]+.
The ability of the compounds of formula (I) to block the Nav1.7 (or SCN9A) channel were measured using the assay described below.
Cell Line Construction and Maintenance
Human Embryonic Kidney (HEK) cells were transfected with an hSCN9A construct using lipofectamine reagent (Invitrogen), using standard techniques. Cells stably expressing the hSCN9A constructs were identified by their resistance to G-418 (400 μg/ml). Clones were screened for expression using the whole-cell voltage-clamp technique.
Cell Culture
HEK cells stably transfected with hSCN9A were maintained in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and 400 pg/ml G-418 in an incubator at 37° C. with a humidified atmosphere of 10% CO2. For HTS, cells were harvested from flasks by trypsinization and replated in an appropriate multi-well plate (typically 96 or 384 wells/plate) such that confluence would be achieved within 24 hours of plating. For electrophysiological studies, cells were removed from the culture flask by brief trypsinization and re-plated at low density onto glass cover slips. Cells were typically used for electrophysiological experiments within 24 to 72 hours after plating.
Electrophysiological Recording
Cover slips containing HEK cells expressing hSCN9A were placed in a bath on the stage of an inverted microscope and perfused (approximately 1 ml/minutes) with extracellular solution of the following composition: 138 mM NaCl, 2 mM CaCl2, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4, with NaOH. Pipettes were filled with an intracellular solution of the following composition: 135 mM CsF, 5 mM CsCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.3 with NaOH, and had a resistance of 1 to 2 megaohms. The osmolarity of the extracellular and intracellular solutions was 300 mOsm/kg and 295 mOsm/kg, respectively. All recordings were made at room temperature (22-24° C.) using AXOPATCH 200B amplifiers and PCLAMP software (Axon Instruments, Burlingame, Calif.).
hSCN9A currents in HEK cells were measured using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Uncompensated series resistance was typically 2 to 5 mega ohms and >85% series resistance compensation was routinely achieved. As a result, voltage errors were negligible and no correction was applied. Current records were acquired at 20 to 50 KHz and filtered at 5 to 10 KHz.
HEK cells stably transfected with hSCN9A were viewed under Hoffman contrast optics and placed in front of an array of flow pipes emitting either control or compound-containing extracellular solutions. All compounds were dissolved in dimethyl sulfoxide to make 10 mM stock solutions, which were then diluted into extracellular solution to attain the final concentrations desired. The final concentration of dimethyl sulfoxide (<0.3% dimethyl sulfoxide) was found to have no significant effect on hSCN9A sodium currents. The voltage-dependence of inactivation was determined by applying a series of depolarizing prepulses (8 sec long in 10 mV increments) from a negative holding potential. The voltage was then immediately stepped to 0 mV to assess the magnitude of the sodium current. Currents elicited at 0 mV were plotted as a function of prepulse potential to allow estimation of the voltage at which 50% of the channels were inactivated (midpoint of inactivation or V1/2). Compounds were tested for their ability to inhibit hSCN9A sodium channels by activating the channel with a 20 msec voltage step to 0 mV following an 8 second conditioning prepulse to the empirically determined V1/2. Compound effect (% inhibition) was determined by difference in current amplitude before and after application of test compounds. For ease of comparison, “estimated IC-50” (EIC50) values were calculated from single point electrophysiology data by the following equation, (tested concentration, uM)×(100-% inhibition/% inhibition). Inhibition values <20% and >80% were excluded from the calculation.
Electrophysiological assays were conducted with PatchXpress 7000 hardware and associated software (Molecular Devices Corp). All assay buffers and solutions were identical to those used in conventional whole-cell voltage clamp experiments described above. hSCN9A cells were grown as above to 50%-80% confluency and harvested by trypsinization. Trypsinized cells were washed and resuspended in extracellular buffer at a concentration of 1×106 cells/ml. The onboard liquid handling facility of the PatchXpress was used for dispensing cells and application of test compounds. Determination of the voltage midpoint of inactivation was as described for conventional whole-cell recordings. Cells were then voltage-clamped to the empirically determined V1/2 and current was activated by a 20 msec voltage step to 0 mV.
Electrophysiological assays may also be conducted using the lonworks Quattro automated electrophysiological platform (Molecular Devices Corp). Intracellular and extracellular solutions were as described above with the following changes, 100 μg/ml amphotericin was added to the intracellular solution to perforate the membrane and allow electrical access to the cells. hSCN9A cells were grown and harvested as for PatchXpress and cells were resuspended in extracellular solution at a concentration of 3-4×106 cells/ml. The onboard liquid handling facility of the Ionworks Quattro was used for dispensing cells and application of test compounds. A voltage protocol was then applied that comprised of a voltage step to fully inactivate the sodium channels, followed by a brief hyperpolarized recovery period to allow partial recovery from inactivation for unblocked sodium channels, followed by a test depolarized voltage step to assess magnitude of inhibition by test compound. Compound effect was determined based on current amplitude difference between the pre-compound addition and post-compound addition scans.
Compounds of the Examples were tested in the assay described above using the PatchXpress platform and found to have the Nav1.7 EIC50 (uM) values specified in the table below.
Ex
EIC50
1
0.0018
2
0.0081
3
0.031
4
0.0029
5
0.0013
6
0.0029
7
0.0012
8
0.032
9
0.0116
10
0.0530
11
0.0077
12
0.0019
13
0.0022
14
0.0011
15
0.0060
16
0.0015
17
0.0027
18
0.018
19
0.011
20
0.24
21
0.10
22
0.033
23
0.0051
24
0.0017
25
0.0008
26
0.0023
27
0.0009
28
0.0009
29
0.0008
30
0.023
31
0.016
32
0.0053
33
0.016
34
0.0005
35
0.0022
36
0.011
37
0.012
38
0.018
39
0.0077
40
0.001
The ability of compounds of formula (I) to block the Nav1.5 (or SCN5A) channel can also be measured using an assay analogous to that described above but replacing the SCN9A gene with the SCN5A gene. All other conditions remain the same including the same cell line and conditions for cell growth. The estimated IC50s are determined at the half inactivation for Nav1.5. These results can be compared to the EIC50 value at the Nav1.7 channel to determine the selectivity of a given compound for Nav1.7 vs Nav1.5.
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13178534
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pfizer limited
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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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514/247
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Sep 10th, 2013 12:00AM
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Jan 20th, 2011 12:00AM
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https://www.uspto.gov?id=US08528549-20130910
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Device for dispensing a plurality of unitary doses of dry powder, and inhaler comprising such device
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A device for dispensing a plurality of unitary doses of dry powder includes: first and second supports, an indexing mechanism adapted to move the first and second supports, the indexing mechanism and the supports being configured so that in a first dispensing state of the device, the first and second supports are respectively in engagement with and disengaged from the indexing mechanism, in a second dispensing state of the device, the second and first supports are respectively in engagement with and disengaged from the indexing mechanism, and a changeover mechanism for causing the device to pass from the first dispensing state to the second dispensing state, to lock the second support while the device is in the first dispensing state, and to lock the first support while the device is in the second dispensing state.
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8528549
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1. A device for dispensing a plurality of unitary doses of dry powder, comprising:
a casing provided with a mouthpiece for inhalation by a user,
first and second supports respectively for two carriers each having a plurality of housings for respective unitary doses adapted to be connected to the mouthpiece for inhalation of the dose, the first and second supports being moveably mounted within the casing for sequentially connecting the housings to the mouthpiece,
an indexing mechanism adapted to engage and move the first and second supports, the indexing mechanism and the supports being configured so that
in a first dispensing state of the device, the first support is in engagement with the indexing mechanism so as to be moveable with respect to the casing, and the second support is disengaged from the indexing mechanism so as to be stationary with respect to the casing,
in a subsequent second dispensing state of the device, the second support is in engagement with the indexing mechanism so as to be moveable with respect to the casing, and the first support is disengaged from the indexing mechanism so as to be stationary with respect to the casing,
changeover means for causing the device to pass from the first dispensing state to the second dispensing state,
wherein the changeover means are adapted to lock the second support in its stationary position while the device is in the first dispensing state, and to lock the first support in its stationary position while the device is in the second dispensing state.
2. The device according to claim 1, wherein the changeover means are adapted to release the second support from its stationary position and to place the second support into engagement with the indexing mechanism while disengaging the first support from the indexing mechanism.
3. The device according to claim 1, wherein the changeover means comprise a changeover mechanism which is arranged between the first and second supports and is moveably mounted within the casing.
4. The device according to claim 3, wherein the changeover means comprise first and second engaging portions provided on the first and second supports respectively, the changeover mechanism comprises first and second engaging sections and a locking arrangement arranged to cooperate with the first and second supports so that
the second engaging section engages the second engaging portion, and the locking arrangement prevents the changeover mechanism from moving with respect to the casing while the device is in the first dispensing state,
the first and second engaging sections engage respectively the first and second engaging portions, and the locking arrangement allows the changeover mechanism to move with respect to the casing while the first support is disengaging from the indexing mechanism,
the first engaging section engages the first engaging portion, and the locking arrangement prevents the changeover mechanism from moving with respect to the casing while the device is in the second dispensing state.
5. The device according to claim 4, wherein the first and second supports comprise respectively first and second contact surfaces, and the locking arrangement comprises first and second abutting sections arranged on the changeover mechanism to cooperate respectively with the first and second contact surfaces so that
the first abutting section abuts the first contact surface while the device is in the first dispensing state,
the first and second abutting sections do not abut the first and second contact surfaces while the first support is disengaging from the indexing mechanism,
the second abutting section abuts the second contact surface while the device is in the second dispensing state.
6. The device according to claim 5, wherein the first and second supports further comprise first and second notches arranged respectively in correspondence with the first and second engaging portions, the first and second notches being adapted to remove the abutments of the first and second abutting sections on the first and second contact surfaces while the first support is disengaging from the indexing mechanism.
7. The device according to claim 3, wherein the changeover mechanism is integrally made.
8. The device according to claim 7, wherein the first and second supports are superposed and the changeover mechanism is rotatably mounted within the casing, the changeover mechanism having first and second sides cooperating respectively with the first and second supports.
9. The device according to claim 7, wherein each of the first and second engaging sections is made of gear teeth adapted to mesh with gear teeth of each of the first and second engaging portions, and each of the first and second abutting sections comprises surfaces angularly offset with respect to the respective first and second engaging sections.
10. The device according to claim 9, wherein the respective surfaces of the first and second abutting sections are at least partly formed on the gear teeth of the first and second engaging sections.
11. The device according to claim 1, wherein the first and second supports are of circular configuration, the first and second supports being rotatably mounted within the casing about a central axis.
12. An inhaler comprising a device for dispensing a plurality of unitary doses of dry powder according to claim 1, and two carriers each having a plurality of housings for respective unitary doses, the two carriers being associated respectively with the first and second supports.
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12
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This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/296,629, filed on Jan. 20, 2010, the disclosure of which is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
The invention relates to a device for dispensing a plurality of unitary doses of dry powder, and to an inhaler comprising such device.
BACKGROUND
In particular, the invention relates to a device for dispensing a plurality of unitary doses of dry powder, comprising:
a casing provided with a mouthpiece for inhalation by a user,
first and second supports respectively for two carriers each having a plurality of housings for respective unitary doses adapted to be connected to the mouthpiece for inhalation of the dose, the first and second supports being moveably mounted within the casing for sequentially connecting the housings to the mouthpiece,
an indexing mechanism adapted to engage and move the first and second supports, the indexing mechanism and the supports being configured so that
in a first dispensing state of the device, the first support is in engagement with the indexing mechanism so as to be moveable with respect to the casing, and the second support is disengaged from the indexing mechanism so as to be stationary with respect to the casing,
in a subsequent second dispensing state of the device, the second support is in engagement with the indexing mechanism so as to be moveable with respect to the casing, and the first support is disengaged from the indexing mechanism so as to be stationary with respect to the casing,
changeover means adapted to cause the device to pass from the first dispensing state to the second dispensing state.
Such a device is known from WO-A-2005/002654.
The device disclosed in the aforementioned document comprises conduits between the housings and the mouthpiece, each conduit defining a flow path for an airstream carrying the unitary dose through inhalation by the user. The device provides for one sole conduit for each unitary dose of dry powder. In use, a user actuates the device to inhale a unitary dose of medicine in the form of dry powder through one of the conduits. Upon a subsequent actuation of the device, a new unitary dose can be inhaled through a new conduit.
Furthermore, such a device allows the dispensing of a large number of unitary doses since the unitary doses of two carriers may be dispensed successively.
With the known device, it may happen that the device jams and becomes out of order when the relative positioning of the components of the device is changed in an unexpected manner such that appropriate relative movements of the components can no longer be obtained. It is especially the case after the device has been dropped causing an offset of at least one of the components.
SUMMARY
The invention aims to solve the above mentioned problem.
To this end, according to a first aspect, the invention provides for a device of the aforementioned type wherein the changeover means are adapted to lock the second support in its stationary position while the device is in the first dispensing state, and to lock the first support in its stationary position while the device is in the second dispensing state.
Hence, the changeover means positively locks and secures the stationary support while the device is in the first and second dispensing states so as to prevent any movement of the stationary support with respect to the casing. This makes it possible to reduce the risk of an unexpected change in the relative positioning of the components. The appropriate relative movements of the components can be preserved, thereby reducing the risk of jamming.
In particular, the changeover means may be adapted to release the second support from its stationary position and to place the second support into engagement with the indexing mechanism while disengaging the first support from the indexing mechanism.
Preferably, the changeover means comprise a changeover mechanism which is arranged between the first and second supports and is moveably mounted within the casing.
In an embodiment, the changeover means may comprise first and second engaging portions provided on the first and second supports respectively, the changeover mechanism may comprise first and second engaging sections and a locking arrangement arranged to cooperate with the first and second supports so that
the second engaging section engages the second engaging portion and the locking arrangement prevents the changeover mechanism from moving with respect to the casing while the device is in the first dispensing state,
the first and second engaging sections engage respectively the first and second engaging portions, and the locking arrangement allows the changeover mechanism to move with respect to the casing while the first support is disengaging from the indexing mechanism,
the first engaging section engages the first engaging portion, and the locking arrangement prevents the changeover mechanism from moving with respect to the casing while the device is in the second dispensing state.
With such embodiment, the switching of the locking arrangement may be achieved in an accurate manner so that the second support is released at a desired moment to enable the device to pass from the first dispensing state to the second dispensing state.
The first and second supports may comprise respectively first and second contact surfaces, and the locking arrangement may comprise first and second abutting sections arranged on the changeover mechanism to cooperate respectively with the first and second contact surfaces so that
the first abutting section abuts the first contact surface while the device is in the first dispensing state,
the first and second abutting sections do not abut the first and second contact surfaces while the first support is disengaging from the indexing mechanism,
the second abutting section abuts the second contact surface while the device is in the second dispensing state.
The first and second supports may further comprise first and second notches arranged respectively on the first and second supports in correspondence with the first and second engaging portions, the first and second notches being adapted to remove the abutments of the first and second abutting sections on the first and second contact surfaces while the first support is disengaging from the indexing mechanism.
Preferably, the changeover mechanism is integrally made, for example as a one-piece moulded component. This results in a more compact device that is easy to manufacture.
In this example, the first and second supports may be superposed and the changeover mechanism may be rotatably mounted within the casing, the changeover mechanism having first and second sides cooperating respectively with the first and second supports.
Besides, each of the first and second engaging sections may be made of gear teeth adapted to mesh with gear teeth of each of the first and second engaging portions, and each of the first and second abutting sections may comprise surfaces angularly offset with respect to the respective first and second engaging sections.
In particular, the respective surfaces of the first and second abutting sections may be at least partly formed on the gear teeth of the first and second engaging sections.
The first and second supports may be of circular configuration, the first and second supports being rotatably mounted within the casing about a central axis.
According to a second aspect, the invention concerns an inhaler comprising a device for dispensing a plurality of unitary doses of dry powder as defined above, and two carriers each having a plurality of housings for respective unitary doses, the two carriers being associated respectively with the first and second supports.
BRIEF DESCRIPTION OF THE DRAWINGS
Other objects and advantages of the invention will emerge from the following disclosure made in reference to the enclosed drawings in which:
FIG. 1 is a side view of an inhaler comprising a device for dispensing a plurality of unitary doses of dry powder according to an embodiment of the invention,
FIG. 2 is an exploded view in perspective of the inhaler of FIG. 1,
FIG. 3 is an exploded view of top faces of a support and a carrier of the inhaler of FIG. 1, illustrating top surfaces of the carrier and of an anvil plate and an airway plate forming the support,
FIG. 4 is an enlarged view of the detail referenced IV on FIG. 3, illustrating a portion of the top surface of the anvil plate,
FIG. 5 is an exploded view of bottom faces of the support and the carrier of the inhaler of FIG. 1, illustrating bottom surfaces of the carrier, of the anvil plate and of the airway plate,
FIG. 6 is an enlarged view of the detail referenced VI on FIG. 5, illustrating a portion of the bottom surface of the airway plate,
FIGS. 7 and 8 are views in perspective of the support partially cut and the carrier of the inhaler of FIG. 1, illustrating the assembly of the carrier and the support and two steps of a dispensing process of a unitary dose of dry powder carried by the carrier,
FIG. 9 is a view in section along line referenced IX-IX on FIG. 2 of the support and the carrier of the inhaler of FIG. 1, illustrating barrier-forming elements at an interface between the anvil plate and the airway plate,
FIGS. 10, 11 and 12 are enlarged views of alternative embodiments of the barrier-forming elements at the interface between the anvil plate and the airway plate,
FIG. 13 is an exploded view of an actuating mechanism of the inhaler of FIG. 1,
FIG. 14 is a perspective view of a changeover component of the inhaler of FIG. 1, illustrating a set of operational features on a first side of the changeover component,
FIG. 15 is a top view of the first side of the changeover component of FIG. 14,
FIG. 16 is a perspective view of the changeover component of the inhaler of FIG. 1, illustrating a set of operational features on a second side of the changeover component,
FIG. 17 is a bottom view of the second side of the changeover component of FIG. 16,
FIG. 18 is a perspective view of the arrangement of the changeover component with respect to the first and second supports for the respective first and second carriers in the inhaler of FIG. 1,
FIGS. 19a and 19b are respectively top and bottom views of the arrangement of FIG. 18 when a last unitary dose of the first carrier is dispensed, illustrating respectively the first side of the changeover component that prevents rotation of the changeover component, and the second side of the changeover component that engages the second support,
FIGS. 20a and 20b are respectively top and bottom views of the arrangement of FIG. 18 after the last unitary dose of the first carrier has been dispensed, illustrating the first support which moves the second support through the changeover component,
FIGS. 21a and 21b are respectively top and bottom views of the arrangement of FIG. 18 when a first unitary dose of the second carrier is dispensed, illustrating respectively the second side of the changeover component that prevents rotation of the changeover component, and the first side of the changeover component that engages the first support.
DETAILED DESCRIPTION
On the Figures, same references refer to similar or analogous elements.
FIG. 1 illustrates an inhaler 1 from which a user may inhale successively unitary doses 2 of medicament in the form of dry powder.
The inhaler 1 of the illustrated embodiment includes a device 3 for dispensing the unitary doses 2 and two carriers 15, visible in particular on FIGS. 3 and 5, which carry the unitary doses 2 and which are mounted in the device 3.
On FIG. 1, the device 3 comprises a casing 5 presenting a contour with a hump-shaped part 5a and a constant-radius shaped part 5b.
The casing 5 is provided with a mouthpiece 6, formed integrally with the casing 5 or as a separate component, arranged substantially at a first end of the constant-radius shaped part 5b.
The constant-radius shaped part 5b is provided with a slot 14, partly visible on FIG. 2, extending from the mouthpiece 6 to a second end opposite to the first end. A priming lever 4 extends out of the casing 5 through the slot 14. As it will be apparent from the following description, the priming lever 4 is mounted so as to rotate around the constant-radius shaped part 5b, about a central axis A, along a stroke delimited by the slot 14. The user may actuate the priming lever 4 to prime the device 3 so that one of the unitary doses 2 may be inhaled through the mouthpiece 6.
The device 3 includes a window 7 in one side of the casing 5. The window 7 allows the user to view a counter display 8 which provides the user with an indication of how many unitary doses 2 have been dispensed and/or how many unitary doses 2 remain unused.
An L-shaped mouthpiece cover 10 may be mounted on the casing 5. The mouthpiece cover 10 comprises long 11 and short 12 hollow parts substantially perpendicular to each other. An end of the long part 11 is rotatably mounted on the casing 5 in the vicinity of the second end of the constant-radius shaped part 5b so that the long 11 and short 12 parts may selectively cover or expose, as illustrated on FIG. 1, the slot 14, the priming lever 4 and the mouthpiece 6. An actuation rib 13, the purpose of which will be explained later, extends centrally in the long part 11.
As can be seen on FIG. 2, the casing 5 is made of two halves assembled to each other to define a housing. The casing 5 comprises a central shaft 64 extending within the housing along the central axis A and on which the following components of the device 3 are mounted:
first 25a and second 25b supports which each receive one respective of the carriers 15,
an actuating mechanism 60 arranged between the first 25a and second 25b supports and comprising the priming lever 4,
a changeover mechanism 90, and
a counter mechanism 140.
With reference to FIGS. 3, 4, 5 and 6, one of the carriers 15 and the first support 25a are described, by way of example. This description can be transposed to the other carrier 15 and to the second support 25b, these being identical or at least similar to the described carrier 15 and first support 25a.
As can be seen on FIGS. 3 and 5, the carrier 15, similar to that disclosed in WO-A-2005/002654, is formed from a disc-shaped plate 16 having an axis and a central opening 17. The plate 16 is provided with a plurality of through-holes 18 extending between top and bottom surfaces of the plate 16 and defining housings for the respective unitary doses of dry powder. In the illustrated embodiment, thirty through-holes 18 are arranged at equally spaced locations, according to a circumferential array. The through-holes 18 are hence adjacent to each other in a circumferential direction and extend in radial directions with respect to the axis of the plate 16.
One location of the plate 16 is deprived of a through-hole, so that a full portion 19 is formed between two adjacent through-holes 18. An indent 20 is formed at the periphery of the plate 16 in correspondence with this full portion 19.
Each through-hole 18 may receive a cup-shaped insert 21, visible in particular on FIGS. 7 and 8, opening in the top surface of the plate 16. Each insert 21 is adapted to contain one of the unitary doses 2 of dry powder. To protect the dry powder, especially from humidity and contaminants, and to retain the inserts 21 and the dry powder in the through-holes 18, appropriate top 22 and bottom 23 lidding sheets may be secured to the top and bottom surfaces of the plate 16.
The first support 25a is of circular configuration with respect to an axis and has first and second members, consisting respectively of an anvil plate 26 and an airway plate 27.
As can be seen on FIG. 3 which shows a top surface of the anvil plate 26, the anvil plate 26 comprises a disc-shaped part 28 pierced with a central opening 32. The disc-shaped part 28 is provided with successive through-holes 29 adapted to be placed in correspondence respectively with the through-holes 18 of the carrier 15. As for the carrier 15, the though-holes 29 are adjacent to each other in a circumferential direction and extend in radial directions with respect to the axis of the first support 25a. The disc-shaped part 28 is provided with radial walls 30 each extending in a radial direction and each arranged between two adjacent through-holes 29 so as to separate them.
On FIG. 4, it can be seen that each radial wall 30 of the disc-shaped part 28 of the anvil plate 26 has a rib 31 protruding on the top surface of the anvil plate 26. Each rib 31 has a rectangular cross-section and a radial dimension which corresponds substantially to that of the adjacent through-holes 29.
The anvil plate 26 also has a securing element for attachment of the top surface of the anvil plate 26 to the airway plate 27. In the illustrated embodiment, a recessed portion 39 surrounding the central opening 32 is formed on the disc-shaped part 28 to cooperate with a securing element of the airway plate 27.
As can be seen on FIG. 5 which shows a bottom surface of the anvil plate 26, the anvil plate 26 is generally concave with a concavity formed on its bottom surface. For example, the anvil plate 26 is provided with an annular lateral wall 33 adapted to surround the external periphery of the carrier 15 so as to accommodate the carrier 15 in the concavity of the anvil plate 26. In particular, the lateral wall 33 extends perpendicularly to an outer edge of the disc-shaped part 28.
Internally, the lateral wall 33 is provided with a coupling portion formed, for example, of gear teeth 34 protruding toward the axis of the first support 25a, and with a decoupling portion formed, for example, of a smooth part 35 arranged locally and deprived of gear tooth. The lateral wall 33 also has a protrusion 36 extending toward the axis and adapted to be received in the indent 20 of the carrier 15.
Externally, the lateral wall 33 is provided with an engaging portion formed, for example, of gear teeth 37 arranged locally and protruding opposite to the axis.
The appropriate relative arrangement of the coupling and decoupling portions, of the protrusion 36 and of the engaging portion will become apparent from the following description of the device 3.
The anvil plate 26 also has a securing element for attachment of the carrier 15 to the bottom surface of the anvil plate 26. In the illustrated embodiment, the bottom surface comprises a mounting skirt 38 extending perpendicularly from the disc-shaped part 28 and adapted to be fitted in the central opening 17 of the carrier 15.
Regarding the airway plate 27, as can be seen on FIG. 3 which shows a top surface of the airway plate 27, it comprises a disc-shaped part 40 pierced with a central opening 41. The disc-shaped part 40 is provided with successive pairs of through-holes 42, 44 adjacent to each other in a circumferential direction. The through-holes 42, 44 of each pair of through-holes extend in a radial direction with respect to the axis of the first support 25a and are adapted to be placed in correspondence with one of the through-holes 29 of the anvil plate 26.
As can be seen on FIG. 5 which shows a bottom surface of the airway plate 27, the disc-shaped part 40 is provided with successive channels 43 and with radial walls 45 arranged so that the channels 43 and the radial walls 45 of the airway plate 27 may face respectively the through-holes 29 and the radial walls 30 of the anvil plate 26. The channels 43 are adjacent to each other in a circumferential direction. Each channel 43 extends in the radial direction between one pair of the through-holes 42, 44 so as to form an inlet 42, close to the axis of the first support 25a, and an outlet 44, at a distance from the axis of the first support 25a, for the channel 43. Each radial wall 45 extending in a radial direction is arranged between two adjacent channels 43 so as to separate them.
The airway plate 27 is generally concave with a concavity formed on its bottom surface adapted to accommodate the anvil plate 26, such that the anvil plate 26 is interposed between the airway plate 27 and the carrier 15. For example, the airway plate 27 is provided with an annular lateral wall 47 adapted to surround the lateral wall 33 of the anvil plate 26. In particular, the lateral wall 47 extends perpendicularly to an outer edge of the disc-shaped part 40. The lateral wall 47 presents an outer smooth contact surface 48 and a notch 49 extending locally from a free edge of the lateral wall 47.
In the illustrated embodiment, the securing element for attachment of the airway plate 27 to the top surface of the anvil plate 26 comprises a mounting flange 50 surrounding the central opening 41 and adapted to be fitted in the recessed portion 39 of the anvil plate 26.
On FIG. 6, it can be seen that each radial wall 45 of the disc-shaped part 40 of the airway plate 27 has a groove 46 formed in the bottom surface of the airway plate 27. Each groove 46 of rectangular cross-section is adapted to receive the rib 31 protruding on the corresponding radial wall 30 of the anvil plate 26.
FIG. 7 illustrates the above disclosed anvil plate 26 and airway plate 27 assembled to form the first support 25a in which one of the carrier 15 is received.
From the above, the anvil plate 26 and the airway plate 27 are secured to one another with the bottom surface of the airway plate 27 in contact with the top surface of the anvil plate 26, and the mounting flange 50 of the airway plate 27 fitted in the recessed portion 39 of the anvil plate 26. The lateral wall 47 of the airway plate 27 surrounds the lateral wall 33 of the anvil plate 26. As apparent from FIG. 2, the gear teeth 37 of the engaging portion of the anvil plate 26 extend in the notch 49 of the airway plate 27.
The channels 43 of the airway plate 27 are in communication respectively with the through-holes 29 of the anvil plate 26. In particular, the inlet 42 of each channel 43 communicates with one side of its corresponding through-hole 29 whilst the outlet 44 communicates with the opposite side of the corresponding through-hole 29.
The through-holes 29 of the anvil plate 26 and the channels 43 of the airway plate 27 form respectively first and second conduit portions which together define a plurality of conduits adapted to be connected respectively to the housings of the carrier 15. The conduits are adjacent to each other in the circumferential direction and extend in radial directions with respect to the support 25. The radial walls 30 of the anvil plate 26 and the radial walls 45 of the airway plate 27 form respectively first and second separation portions interposed between the conduits.
The carrier 15 is mounted within the first support 25a with the top lidding sheet 22 in contact with the bottom surface of the anvil plate 26, and the central opening 17 of the carrier 15 fitted on the mounting skirt 38 of the anvil plate 26. The lateral wall 33 of the anvil plate 26 surrounds the periphery of the carrier 15 with the protrusion 36 of the anvil plate 26 placed in the indent 20 of the carrier 15, thereby providing an appropriate positioning of the through-holes 18 and the full portion 19 of the carrier 15 with respect to the conduits of the first support 25a. In this regard, it will be appreciated that each housing of the carrier 15 has its own conduit formed in the first support 25a, the conduit being adapted to define a flow path for an airstream carrying the unitary dose through inhalation by a user.
In relation to FIGS. 7 and 8, a dispensing process of one of the unitary doses 2 of dry powder contained in one insert 21 is disclosed.
On FIG. 7, the insert 21 is in a storage position in which it is fully contained in the through-hole 18 of the carrier 15 and flush with the top surface of the carrier 15. The insert 21 faces the conduit of the first support 25a.
As shown on FIG. 8, by pushing the insert 21 from the side of the bottom lidding sheet 23, it is possible to move the insert 21 outwardly to a discharge position, in which the insert 21 protrudes from the top surface of the carrier 15 and extends in the through-hole 29 of the anvil plate 26. The insert 26 used to outwardly burst through the top lidding sheet 22 is still held securely in place. In this respect, the anvil plate 26 can be used to improve the predictability of the rupture of the top lidding sheet 22.
In the discharge position, the insert 21 within the conduit faces the inlet 42 of the channel 43. In this way, when the user inhales through the mouthpiece 6 of the device 3, an airstream, illustrated by an arrow on FIG. 8, may be drawn through the airway plate 27 such that it passes through the inlet 42 down into the insert 21, back up into the channel 43 and then out of the outlet 44. The unitary dose of dry powder in the insert 21 is thus picked up by the airstream, removed from the insert 21 and carried out of the first support 25a.
Suitable dimensions and shape of the conduits to ensure the dry powder is picked up, and where needed deaggregated, may resume that disclosed in WO-A-2005/002654. Besides, as in WO-A-2005/002654, a second flow path which bypasses the insert 21 may be provided to increase the overall cross sectional area available through which to inhale, and to control the overall flow resistance of the device so that it is comfortable for the user to inhale through. This second flow path may be formed by walls of the casing 5.
As can be seen on FIG. 9, in the first support 25a, when the anvil plate 26 and the airway plate 27 are assembled, the ribs 31 on the radial walls 30 of the anvil plate 26 are placed within the grooves 46 of the radial walls 45 of the airway plate 27.
Therefore, even if small gaps exist at the interface between the radial walls 30, 45 delimiting respectively the through-holes 29 of the anvil plate 26 and the channels 43 of the airway plates 27, for example because these radial walls 30, 45 are not closely tightened, the arrangement of ribs 31 and grooves 46 provides a circuitous path between two adjacent conduits.
In the case where the first and second unitary doses 2 of dry powder are placed in communication respectively with adjacent first and second conduits, the airstream created in the second conduit through inhalation by the user to pick up the second unitary dose 2 will draw the dry powder of the second unitary dose 2 without drawing that of the first unitary dose since the arrangement of rib 31 and groove 46 between corresponding radial walls 30, 45 of the anvil plate 26 and of the airway plate 27 inhibits dry powder of the first unitary dose 2 from passing from the first conduit to the adjacent second conduit.
This situation may arise when the user actuates the device, thereby moving the insert 21 containing the first unitary dose 2 in the discharge position, and is distracted before inhaling the first unitary dose 2. Subsequently, the user actuates the device once again, forgetting that he actuated it previously, thereby moving the insert 21 containing the second unitary dose 2 into the discharge position.
The rib 31 and the groove 46 of two facing radial walls of the anvil plate 26 and the airway plate 27 form barriers to the dry powder of the adjacent conduits, limiting thereby cross-dosing, i.e. the amount of dry powder inhaled when the previous unitary dose has been missed or untaken.
For example, in the device disclosed in WO-2005/002654 deprived of barrier-forming elements such as the above described rib and groove arrangement, it has been found that the cross-dosing could reach 150% or more of the nominal unitary dose, that is an excess of 50% or more of dry powder of the previous unitary dose may be inhaled when the subsequent unitary dose is inhaled.
The use of barrier-forming elements aims to reduce cross-dosing to 135% or less. In particular, a cross-dosing of less than 115% can be obtained with the barrier-forming elements.
Of course, the barrier-forming elements are not limited to the above described rib and groove arrangement. For example, the ribs 31 could be arranged on the airway plate 27 and the grooves 46 could be arranged on the airway plate 26.
Besides, in the above described embodiment, the barrier-forming elements form a baffle between the corresponding radial walls 30, 45 of the anvil plate 26 and the airway plate 27 providing a circuitous path between two adjacent conduits. Therefore, the barrier-forming elements may comprise any angled or curved interface between the corresponding walls 30, 45 of the anvil plate 26 and the airway plate 27.
In particular, the barrier-forming elements may comprise more than one rib 31 and one groove 46.
For example, as illustrated on FIG. 10, each radial wall 30 of the anvil plate 26 is provided with one radial rib 131 and one radial groove 146 adapted to cooperate respectively with one radial groove 146 and one radial rib 131 of the corresponding radial wall 45 of the airway plate 27. Besides, on FIG. 10, each rib 131 includes a first pair of opposed surfaces inclined with respect to one another, and each groove 146 includes a second pair of opposed surfaces inclined with respect to one another and complementary to the first pair of opposed surfaces of the corresponding rib 131.
FIG. 11 illustrates an alternative embodiment of the barrier-forming elements in which an additive layer 52 is interposed between the corresponding radial walls 30, 45 of the anvil plate 26 and the airway plate 27. Any appropriate gasket or adhesive in any appropriate pattern, such as continuous layers, discrete points or other, could be used as additive layer.
Besides, as shown on FIG. 12, in another alternative embodiment, the barrier-forming elements may comprise a welded connection between the corresponding radial walls 30, 45 of the anvil plate 26 and the airway plate 27. In this embodiment, the radial walls of the anvil plate 26 and the airway plate 27, or the anvil plate 26 and the airway plate 27 themselves, are made of thermoplastic material and are configured to permit the corresponding radial walls 30, 45 to be joined by an ultrasonic welding process. For example, a tipped protrusion or energy director 53 is arranged on the rib 231 and abuts the bottom surface of the groove 246. The relative movement of the anvil plate 26 and the airway plate 27 caused by ultrasonic vibrations will cause the thermoplastic material to melt and the radial walls 30, 45 to be welded.
The barrier-forming elements may implement one of the above disclosed embodiments or may combine several of them.
As can be seen on FIG. 2, within the casing 5, the first 25a and second 25b supports with their respective carriers 15 are superposed and arranged coaxially to the central axis A, the bottom surfaces of the carriers 15 facing each other. The first 25a and second 25b supports are rotatably mounted within the casing 5 about the central axis A so as to bring successively each conduit in communication with the mouthpiece 6, thus sequentially connecting the housings to the mouthpiece.
The actuating mechanism 60, illustrated in detail on FIG. 13, is arranged between the bottom surfaces of the carriers 15. The actuating mechanism 60 is adapted to expose one of the unitary doses 2 of dry powder such that it may be carried with the airstream out of the mouthpiece 6 each time the priming lever 4 is actuated.
In particular, the actuating mechanism 60 comprises a dispensing mechanism adapted to expose each unitary dose 2 to the corresponding conduit, and an indexing mechanism adapted to place each conduit in communication with the mouthpiece 6.
The actuating mechanism 60 comprises a disc-shaped chassis 61 which supports the dispensing mechanism and the indexing mechanism. The chassis is fixed to the casing 5 and comprises a hollow pivot shaft 65 fitted on the shaft 64 of the casing 5. At a location, the chassis comprises guide members 71 extending axially and defining a radial aperture between them.
The actuating mechanism 60 further comprises a priming member 62 bearing the priming lever 4 and rotatable about the central axis A so as to operate the dispensing mechanism and the indexing mechanism when the priming lever 4 is actuated.
An example of a suitable priming member 62 is disclosed in WO-A-2005/002654. The priming member 62 is formed of a disc-shaped plate moulded in plastic and having a central pivot opening 66 by which it is rotatably supported on the pivot shaft 65 of the chassis 61.
In the illustrated embodiment, the dispensing mechanism is adapted to move each insert 21 of each carrier 15 from its storage position to its discharge position. Again, an example of a suitable dispensing mechanism, implementing prodgers 69 mounted on the priming member 62, and cam surfaces 68, 75 arranged on the priming member 62 and adapted to move the prodgers 69 axially, is disclosed in WO-A-2005/002654.
In particular, the dispensing mechanism includes an elongate cam member 67 formed on the priming member 62 and separated from the remaining part of the priming member 62 by elongate openings 70 through which the abutment members 71 of the chassis 61 extend. The cam member 67 extends in a circumferential direction and presents a profile adapted to provide a limited amount of flexibility. The central cam surface 68 is provided on each of two opposite sides of the cam member 67. Besides, the lateral cam surfaces 75 extend on either side of the priming member 62, in circumferential directions along the elongate openings 70, opposite the cam member 67.
The prodgers 69 are identical to each other and clip together with the cam member 67 between them. Each prodger 69 has arms 73 extending perpendicularly to a central part arranged to cooperate with the central cam surface 68 of the cam member 67. The arms 73 extend through the elongate openings 70 of the priming member 62, and have features 72 arranged at their ends to contact the lateral cam surfaces 75 of the priming member 62.
The elongate openings 70 of the priming member 62 and the guide members 71 on the chassis 61 are arranged to hold the prodgers 69 rotationally but to allow them to move in an axial direction of the device 3, towards and away from the carriers 15 by means of the central 68 and lateral 75 cam surfaces that positively guide the prodgers 69.
As explained in WO-A-2005/002654, the actuating mechanism 60 arranges for one of the prodger 69 to be in alignment with one of the insert 21 of the corresponding carrier 15 while the other prodger 69 faces the full portion 19 of the other carrier 15. In this way, the dispensing mechanism only dispenses one unitary dose 2 of one of the carrier 15 at a time.
Operation of the dispensing mechanism is now described.
Movement of the priming lever 4 in the slot 14 of the casing 5 along its stroke from a first position close to the mouthpiece 6 to a second position at a distance from the mouthpiece 6 primes the device 3 to expose the unitary dose 2 of dry powder to the corresponding conduit.
At an initial step, when the user moves the mouthpiece cover 10 to expose the mouthpiece 6, the priming lever 4 is in its first position and both prodgers 69 are in a retracted position at one end of the cam member 67 opposite the central cam surfaces 68.
When the user moves the priming lever 4 to its second position, the priming member 62 is rotated relative to the chassis 61. The cam surfaces 68 of the cam member 67 engage the prodgers 69, respectively. The cam surface 68 that engages the prodger 69 in alignment with one of the insert 21 presses out this prodger 69 so that this prodger 69 is moved outwardly towards its corresponding carrier 15, penetrates the through-hole 18 of the carrier 15 and pushes the insert 21 in the discharge position. Meanwhile, the cam surface 68 that engages the prodger 69 in alignment with the full portion 19 deforms thanks to its flexibility.
After the user has inhaled the unitary dose 2, the mouthpiece cover 10 may be rotated back by the user. The actuation rib 13 of the mouthpiece cover 10 may engage the priming lever 4 to move it back to its first position. The lateral cam surfaces 75 of the priming member 62 retract the prodgers 69.
The indexing mechanism will now be described.
In the illustrated embodiment, the indexing mechanism is adapted to move the first 25a and second 25b supports in successive active positions in each of which one of the conduits is connected to the mouthpiece 6 so that the corresponding unitary dose 2 may be carried by the airstream through the mouthpiece 6. An example of a suitable indexing mechanism implementing an intermittent motion mechanism is disclosed in WO-A-2005/002654.
In particular, the indexing mechanism comprises a Geneva wheel 76 rotatably mounted within the casing 5 about an axis parallel to the central axis A. The Geneva wheel 76 includes a peg wheel 77 adapted to cooperate with the priming member 62 so that the Geneva wheel rotates through an angle of 120.degree. each time the priming lever 4 is actuated. The Geneva wheel 76 also includes two gears 78 coaxial with the peg wheel 77 and adapted to cooperate respectively with the coupling portions of the first 25a and second 25b supports.
The peg wheel 77 has three long pegs 79 and three short pegs 80 arranged alternately at intervals of 60.degree. around its edge.
The indexing mechanism further comprises a driving member 81 formed on an outer edge of the priming member 62. The driving member 81 is arranged so that:
when the priming lever 4 is moved from its first position to its second position so that, as explained above, the dispensing mechanism pushes the insert 21 in the discharge position, the driving member 81 does not rotate the Geneva wheel 76,
when the priming lever 4 is moved back from its second position to its first position, the driving member 81 rotates the Geneva wheel 76.
In particular, the driving member 81 is placed, in the circumferential direction, next to a portion of the priming member 62 comprising the dispensing mechanism.
The driving member 81 is provided with a leading portion 82, a ratchet pawl 83 which slopes downward toward the leading portion 82, and a slot 84 with a trailing edge 85 arranged in sequence.
The operation of the indexing mechanism will now be described in relation to one cycle defined by the movement of the priming lever 4 as it is actuated by the user. The terms “first”, “second” and “third” related to the long 79 and short 80 pegs in the following description are used in relation to one cycle. It should be understood that the “first”, “second” and “third” pegs would change in a subsequent cycle.
As indicated above, when the priming lever 4 is moved from its first position to its second position, the driving member 81 does not rotate the Geneva wheel 76. In particular, the peg wheel 77 and the driving member 81 are arranged so that the outer edge of the priming member 62 passes over the first of the short pegs 80 and slides against the first and second of the long pegs 79 adjacent on either side of the first short peg 80, the ratchet pawl 83 deforming when passing over the second short peg 80. The peg wheel 76 is therefore prevented from rotating.
When the priming lever 4 returns from its second position to its first position, the leading portion 82 passes over the first short peg 80 and the outer edge of the priming member 62 slides against the first and second long pegs 79, thereby preventing the peg wheel 77 from rotating. Then the ratchet pawl 83 engages with the first short peg 80 so that the peg wheel 77 is driven around, the second long peg 79 entering the slot 84. As the ratchet pawl 83 disengages the first short peg 80, the trailing edge 85 of the slot 84 engages the second long peg 79 and continues to drive the peg wheel 77 around. As the trailing edge 85 of the slot 84 disengages the second long peg 79, the outer edge of the priming member 62 passes over the second of the short pegs 80 adjacent to the second long peg 79 and abuts against the second and third of the long pegs 79.
The indexing mechanism causes one of each carrier 15 to be incremented by one unitary dose 2 each time the priming lever 4 is actuated.
The gear teeth 34 of the coupling portion of each airway plate 27 may be in engagement with the corresponding gear 78 of the Geneva wheel 76 so as to be moved with respect to the casing 5 successively in the active positions. The numbers of gear teeth 34 on the airway plates 34 and gears 78 are arranged so that the motion of an angle of 120.degree. of the Geneva wheel 76 increments the support 25 exactly one conduit pitch.
Therefore, the indexing mechanism rotates successively each support 25 to the next position in which one of the conduits is in communication with the mouthpiece 6 and the prodger 69 is aligned with a new insert 21. The above described operation of dispensing the unitary dose can then be repeated.
To avoid having both first 25a and second 25b supports driven simultaneously, the indexing mechanism is caused initially to drive the first support 25a and, when this has had all of its unitary doses 2 dispensed, to then drive the second support 25b.
The first 25a and second 25b supports are configured, in particular through the appropriate relative arrangement of the coupling and decoupling portions, of the protrusions 36 and of the engaging portions of the first 25a and second 25b supports, so that the device 3 presents:
a first dispensing state, in which the first support 25a is in engagement with the Geneva wheel 76 of the indexing mechanism so as to be moved with respect to the casing 5 in each active position, and the second support 25b is disengaged from the Geneva wheel 76 of the indexing mechanism so as to be stationary with respect to the casing 5,
a subsequent second dispensing state, in which the second support 25b is in engagement with the Geneva wheel 76 of the indexing mechanism so as to be moveable with respect to the casing 5 in each active position, and the first support 25a is disengaged from the Geneva wheel 76 of the indexing mechanism so as to be stationary with respect to the casing 5.
In this respect, it is arranged that the decoupling portion of the airway plate 27 of one of the first 25a and second 25b supports faces the corresponding gear 78 of the Geneva wheel 76, while the gear teeth 34 of the coupling portion of the airway plate 27 of the other of the first 25a and second 25b supports engage the corresponding gear 78 of the Geneva wheel 76. As a result, with its decoupling portion, the airway plate 27 may be disengaged from the gear 78 of the Geneva wheel 76 so that rotation of the Geneva wheel 76 does not rotate the support 25.
Besides, the decoupling portion and the protrusion 36 of each of the first 25a and second 25b supports are arranged so that when the decoupling portion faces the gear 78 of the Geneva wheel 76, the prodger 69 faces the full portion 19 of the carrier 15 and no unitary dose of this carrier 15 can be dispensed. Thus, as the indexing mechanism drives the first support 25a, in the first dispensing state of the device 3, the second support 25b remains stationary with respect to the casing 5, in an inactive position in which there is no connection between any unitary dose of the carrier 15 of this second support 25b and the mouthpiece 6. And subsequently, as the indexing mechanism drives the second support 25b, in the second dispensing state of the device 3, the first support 25a remains stationary with respect to the casing 5, in an inactive position in which there is no connection between any unitary dose of the carrier 15 of this first support 25a and the mouthpiece 6.
The embodiment described above is arranged to dispense the dry powder from each insert 21 of one carrier 15 and then subsequently the dry powder from each insert 21 of the other carrier 15.
FIGS. 14, 15, 16 and 17 illustrate the changeover mechanism 90 provided to cause the device 3 to pass from the first dispensing state to the second dispensing state.
In the illustrated embodiment, the changeover mechanism 90 is formed of an integral changeover component 91, made in one piece, for example by moulding, having first 92 and second 93 sides that extend along an axis in opposite directions from a plate 94.
On FIGS. 14 and 15, the first side 92 of the changeover component 91 comprises an axle 96 and a first engaging section 95 formed, in the illustrated embodiment, of first 95a, second 95b and third 95c gear teeth arranged in sequence along an arc.
The first gear tooth 95a extends from the axle 96 in a radial direction to a free end that presents an end surface 97 substantially perpendicular to the radial direction of the first gear tooth 95a. The first gear tooth 95a has thus a limited length in the radial direction with respect to that of the second 95b and third 95c gear teeth. Besides, the first gear tooth 95a has a height along the axis of about one half of that of the second 95b and third 95c gear teeth.
The second gear tooth 95b extends from the axle 96 in a radial direction to a free end presenting a lower engaging profile 98, close to the plate 94, of a height substantially similar to that of the first gear tooth 95a, and an upper profile 99. The upper profile 99 comprises two end surfaces angled with respect to each other, one first 100 substantially parallel to the end surface 97 of the first gear tooth 95a and offset toward the axle 96 with respect to this end surface 97, the other second 101 substantially perpendicular to the radial direction of the second gear tooth 95b.
In a similar manner, the third gear tooth 95c extends from the axle 96 in a radial direction to a free end presenting a lower engaging profile 102, close to the plate 94, of a height substantially similar to that of the first gear tooth 95a, and an upper profile 103 presenting an end surface 104 substantially perpendicular to the radial direction of the third gear tooth 95c.
The first side 92 of the changeover component 91 further comprises a tab 106 extending substantially perpendicularly to the first gear tooth 95a and tangentially to the axle 96, in a direction opposite to the engaging section 95. The tab 106 presents an abutting surface 105 substantially in alignment with the first end surface 100 of the second gear tooth 95b.
On FIGS. 16 and 17, the second side 93 of the changeover component 91 comprises an axle 110 and a second engaging section 115 formed, in the illustrated embodiment, of first 115a, second 115b and third 115c gear teeth arranged in sequence along an arc. As can be seen on FIGS. 14 and 16, the first 115a, second 115b and third 115c gear teeth of the second side 93 are substantially axially aligned respectively with the first 95a, second 95b and third 95c gear teeth of the first side 92.
The first gear tooth 115a extends from the axle 110 in a radial direction to a free end that presents an engaging profile 116.
The second gear tooth 115b extends from the axle 110 in a radial direction to a free end presenting a lower engaging profile 117, close to the plate 94, and an upper profile 118. The upper profile 118 has an end surface 120 substantially perpendicular to a radial direction along which the third gear tooth 115c extends.
The third gear tooth 115c extends from the axle 110 to a free end presenting a lower profile 121, close to the plate 94, and an upper profile 122. The lower profile 121 presents an end surface 123 substantially perpendicular to the radial direction of the third gear tooth 115c. The upper profile 122 also presents an end surface 125 substantially perpendicular to the radial direction of the third gear tooth 115c, the end surface 125 of the upper profile 122 being offset toward the axle 110 with respect to that of the lower profile 121 and being in alignment with the end surface 120 of the upper profile 118 of the second gear teeth 115b.
The second side 93 of the changeover component 91 further comprises a tab 129 extending from the axle 110 next to the third gear tooth 115c in a radial direction. The tab 129 presents an abutting surface 130 substantially in alignment with the end surfaces 120, 125 of the upper profiles 118, 122 of the second 115b and third 115c gear teeth.
As can be seen on FIG. 18, the changeover component 91 is arranged between the first 25a and second 25b supports and rotatably mounted within the casing 5 with its axis parallel to the central axis A. For example, a casing 135, visible on FIG. 13, may be formed in one piece with the chassis 61 of the actuating mechanism to rotatably support the changeover component 91.
The first 92 and second 93 sides of the changeover component 91 cooperate respectively with the first 25a and second 25b supports.
In particular, the gear teeth 95a, 95b, and 95c of the first side 92 of the changeover component 91 are adapted to mesh with the gear teeth 37 of the engaging portion of the first support 25a. And the gear teeth 115a, 115b, and 115c of the second side 93 of the changeover component 91 are adapted to mesh with the gear teeth 37 of the engaging portion of the second support 25b.
The engaging portion of the first support 25a is arranged so as to engage the engaging section of the first side 92 of the changeover component 91 after the last unitary dose 2 of the first support 25a has been dispensed and while the first support 25a is disengaging from the indexing mechanism, i.e. the indexing mechanism moves the first support 25a so as to disengage its coupling portion and to face its decoupling portion. The engaging portion of the first support 25a remains engaged with the engaging section of the first side 92 of the changeover component 91 in the second dispensing state of the device. And the engaging portion of the second support 25b is arranged so as to engage the engaging section of the second side 93 of the changeover component 91 after the last unitary dose 2 of the first support 25a has been dispensed and while the first support 25a is disengaging from the indexing mechanism. The engaging portion of the second support 25 is engaged with the engaging section of the second side 93 of the changeover component 91 in the first dispensing state of the device.
The changeover component 91 is therefore adapted to place the gear teeth 34 of the coupling portion of the second support 25b into engagement with the corresponding gear 78 of the indexing mechanism while the other gear 78 of the indexing mechanism moves the first support 25a so as to face its decoupling portion, thereby disengaging the first support 25a from the indexing mechanism.
Furthermore, the first end surface 100 borne by the second gear tooth 95b and the abutting surface 105 of the tab 106 of the first side 92 of the changeover component 91 form a first abutting section adapted to cooperate with the contact surface 48 of the first support 25a. And the end surfaces 120, 125 borne by the second 115b and third 115c gear teeth and the abutting surface 130 of the tab 129 of the second side 92 of the changeover component 91 form a second abutting section adapted to cooperate with the contact surface 48 of the second support 25b.
The abutting sections of the first and second sides are arranged at opposite location with respect to the corresponding first 95 and second 115 engaging sections, whereas first 95 and second 115 engaging sections are arranged at a same location. Reasons of such arrangement will become apparent from the following description of the operation of the changeover component 91.
The description of this operation is now made in relation to FIGS. 19a, 19b, 20a, 20b, 21a and 21b.
On FIG. 19a, in the first dispensing state of the device, whilst the first support 25a is in engagement with the indexing mechanism, the gear 78 of the Geneva wheel 76 meshing with the coupling portion of the first support 25a, the first support 25a is rotated, as shown by an arrow, successively in the active positions so that the unitary doses of the carrier 15 mounted in the first support 25a may be dispensed. Meanwhile, the second support 25b is locked in the inactive position.
Actually, until the first support 25a has reached last active position, the changeover component 91 is prevented from rotating because the first end surface 100 of the second gear tooth 95b and the abutting surface 105 of the tab 106 of the first side 92 abut the contact surface 48 of the first support 25a, shown in chain dotted line. Thanks to the limited length and height of the first tooth 95a and to the limited length of the upper profile of second tooth 95b, these first 95a and second 95b teeth do not interfere with the first support 25a.
On FIG. 19b, at this same step, the first 115a and second 115b gear teeth of the second side 93 of the changeover component 91 meshes with gear teeth 37 of the engaging portion of the second support 25 through the notch 49. Since the changeover component 91 cannot rotate, the second support 25b is also prevented from rotating.
On FIG. 20a, after the last active position has been indexed on the first support 25a and the last unitary dose 2 has been dispensed, the indexing mechanism moves the first support 25a so that the first support 25a disengages the indexing mechanism, the decoupling portion 35 being brought in correspondence with the gear 78 of the Geneva wheel 76. The first support 25a is driven to the inactive position. At the same time, thanks to the appropriate positioning of the decoupling portion 35 and the engaging portion, the notch 49 of the first support 25a faces the first end surface 100 of the second gear tooth 95b of the first side 92 of the changeover component 91, thereby removing the rotational constraint on the changeover component 91 which can rotate.
At this step, the gear teeth 37 of the engaging portion of the first support 25a that protrude into the notch 49 mesh with the engaging profile of the first gear tooth 95a arranged in the path of the engaging portion of the first support 25a. While the first support 25a keeps on rotating to the inactive position by means of the indexing mechanism, the gear teeth 37 of the engaging portion of the first support 25a which mesh with the engaging section 95 of the first side 92 of the changeover component 91 rotate the changeover component 91 as shown by an arrow.
On FIG. 20b, since the changeover component 91 is now free to rotate, the gear teeth 115a, 115b and 115c of the second side 93 of the changeover component 91 that mesh with gear teeth 37 of the engaging portion of the second support 25b rotate this second support 25b, as shown by an arrow, to space apart its decoupling portion and to place its coupling portion in engagement with the corresponding gear 78 of the indexing mechanism. Thereby, the second support 25b is spaced apart from its inactive position and can be driven to a first active position by the indexing mechanism.
On FIG. 21b, at the completion of the move illustrated on FIG. 20b, the device is in the second dispensing state. The gear teeth 115a, 115b and 115c of the second side 93 of the changeover component 91 run out of engagement with the gear teeth 37 of the engaging portion of the second support 25b so that subsequent rotational movement of the second support 25b is independent of the changeover component 91.
On the next index and all subsequent indexes of the second support 25, the changeover component 91 is prevented from rotating because the end surfaces 120, 125 of the second 115b and third 115c gear teeth and the abutting surface 130 of the tab 129 of the second side 93 of the changeover component 91 abut the contact surface 48 of the second support 25b, shown in chain dotted line.
On FIG. 21a, at this step, the third gear teeth 95c of the first side 92 of the changeover component 91 remains in mesh with the gear teeth 37 of the engaging portion of the first support 25a, thereby preventing this first support 25a from rotating.
Therefore, in the illustrated embodiment, the first and second abutting sections provide the changeover component 91 with a locking arrangement that:
in the first dispensing state of the device 3, prevents the changeover component 91 from rotating with respect to the casing 5 so that the changeover component 91 locks the second support 25b while the first support 25a is driven by the indexing mechanism successively between its first and last active positions to dispense the unitary doses 2 of its carrier 15,
while the first support 25a is disengaging from the indexing mechanism, allows the changeover component 91 to rotate with respect to the casing 5 so that the changeover component 91 releases the second support 25b and places the second support 25b into engagement with the indexing mechanism,
in the second dispensing state of the device, prevents the changeover component 91 from rotating with respect to the casing 5 so that the changeover component 91 locks the first support 25a while the second support 25b is driven by the indexing mechanism successively between its first and last active positions to dispense the unitary doses 2 of its carrier 15.
The invention is not limited to the above disclosed changeover mechanism 90. Any other suitable changeover mechanism 90 that allows for a reliable locking of the unused support 25 and for a releasing at a determined moment, when the first support 25 is in a determined position, to allow the driven support 25 to be changed, could be provided.
Besides, the invention is not limited to a device as above disclosed. For example, the device could comprise more than two supports 25 for more than two carriers. The supports could be of different types and otherwise moveable with respect to the casing. Many aspects of the present invention are applicable to devices with appropriate supports for housing a wide variety of different carriers. In particular, many of the features of the embodiment described below can be used with carriers having a blister pack construction or with carriers having various arrays of housing.
The indexing of the device, in addition to moving the next insert 21 into alignment with the prodgers, actuates the counter mechanism 140 that provides a visual indication to the user of how many unitary doses 2 have been dispensed and/or how may unitary doses 2 remain unused
An example of a suitable counter mechanism 140, implementing a unit and tens counter driven by a driving gear meshing with one of the gears 78 of the Geneva wheel 76 of the indexing mechanism, is disclosed in WO-A-2005/002654. The driving gear and the unit and tens counters are adapted to index a tens display of the counter display 8 of one number as a unit display of the counter display 8 is indexed from 9 to 0.
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13010195
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pfizer limited
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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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128/203.15
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Jul 14th, 2015 12:00AM
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Oct 15th, 2012 12:00AM
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https://www.uspto.gov?id=US09079878-20150714
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(4-phenylimidazol-2-yl) ethylamine derivatives useful as sodium channel modulators
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The present invention is directed to imidazole derivatives, to their use in medicine, to compositions containing them, to processes for their preparation and to intermediates used in such processes. More particularly the invention relates to a new imidazole NaV1.8 modulators of formula (I):
or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4 and R5 are as defined in the description. NaV1.8 modulators are useful in the treatment of a wide range of disorders, particularly pain.
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9079878
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1. A compound according to formula (I)
or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein:
R1 and R2, together with the carbon to which they are attached, form a 4- to 7-membered ring, wherein:
one member of said ring is O; and
the remaining members of said ring are CR6R7, which may be the same or different at each occurrence;
R3 is selected from the group consisting of H, (C1-C3)alkyl, cyclopropyl, cyclopropyl-CH2—, —CH2OH, —CH2OCH3, (C1-C3)fluoroalkyl, —OH, —OCH3, F, —NH2, NHCH3, —N(CH3)2 and —NHC(O)CH3;
R4 is selected from the group consisting of —CF3, —OCF3, —OCHF2, Cl and —SF5;
R5 is selected from the group consisting of H and —CH3; and
R6 and R7 are independently selected from the group consisting of H, CH3—, —OH, —OCH3, F, —NH2, —NHCH3 and —N(CH3)2.
2. The compound of formula (I) or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, according to claim 1, wherein:
R1 and R2, together with the carbon to which they are attached, form a 4- to 7-membered ring of formula
wherein m is 1, 2 or 3 and n is 1 or 2.
3. The compound of formula (I) or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, according to claim 2, wherein m is 1 and n is 1.
4. The compound of formula (I) or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, according to claim 1, wherein:
R3 is selected from the group consisting of H and (C1-C3)alkyl wherein said C1-C3)alkyl is selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
5. The compound of formula (I) or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, according to claim 1, wherein:
R5 is H.
6. The compound of formula (I) according to claim 1, wherein the compound is selected from:
3-({4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-3-amine,
3-{[4-(4-Chloro-3-methylphenyl)-1H-imidazol-2-yl]methyl}oxetan-3-amine,
3-({4-[4-(Difluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-amine,
3-({4-[4-(Pentafluoro-λ6-sulfanyl)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-amine,
4-({4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-4-amine,
3-({4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-amine,
3-(1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-amine,
3-[(1S)-1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine,
3-[(1R)-1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine,
3-(1-{4-[4-(Trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-amine,
3-[(1S)-1-{4-[4-(Trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine,
3-[(1R)-1-{4-[4-(Trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine,
3-(1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}propyl)oxetan-3-amine,
or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.
7. A pharmaceutical composition comprising a compound of formula (I) or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, as defined in claim 1, and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of claim 7 wherein the composition is adapted for topical administration.
9. The pharmaceutical composition of claim 7 wherein the composition is adapted for ocular administration.
10. The pharmaceutical composition of claim 7 which further comprises one or more additional therapeutic agents.
11. A method of treating pain, comprising administering a therapeutically effective amount of a compound of formula (I) or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, as defined in claim 1, to a subject in need of such treatment.
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11
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This application is a national stage application under 35 U.S.C. 371 of PCT/IB2012/055610, filed on Oct. 15, 2012, which claims the benefit of U.S. Provisional Patent Application No. 61/551,628, filed on Oct. 26, 2011, the disclosure of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to imidazole derivatives. More particularly, this invention relates to derivatives of (4-phenylimidazol-2-yl)ethylamine, to their use in medicine, to compositions containing them, to processes for their preparation and to intermediates used in such processes.
BACKGROUND
The imidazole derivatives of the present invention are sodium channel modulators. In particular they are modulators of the NaV1.8 sodium channel. Preferred imidazole derivatives of the invention show an affinity for the NaV1.8 channel which is greater than their affinity for other sodium channels such as the NaV1.5 sodium channel and the tetrodotoxin-sensitive sodium channels (TTX-S). The imidazole derivatives of the invention have a number of therapeutic applications and potential therapeutic applications. In particular they are useful in the treatment of pain.
Voltage-gated sodium channels are found in all excitable cells including myocytes of muscle and neurons of the central and peripheral nervous system. In neuronal cells, sodium channels are primarily responsible for generating the rapid upstroke of the action potential. In this manner sodium channels are essential to the initiation and propagation of electrical signals in the nervous system. Proper and appropriate function of sodium channels is therefore necessary for normal function of the neuron. Consequently, aberrant sodium channel function is thought to underlie a variety of medical disorders (see Hubner C. A., Jentsch T. J., Hum. Mol. Genet., 11(20): 2435-45 (2002) for a general review of inherited ion channel disorders) including epilepsy (Yogeeswari et al., Curr. Drug Targets, 5(7): 589-602 (2004)), arrhythmia (Noble D., Proc. Natl. Acad. Sci. USA, 99(9): 5755-6 (2002)) myotonia (Cannon, S. C., Kidney Int. 57(3): 772-9 (2000)), and pain (Wood, J. N. et al., J. Neurobiol., 61(1): 55-71 (2004)).
There are currently at least nine known members of the family of voltage-gated sodium channel (VGSC) alpha subunits. Names for this family include SCNx, SCNAx, and NaVx.x. The VGSC family has been phylogenetically divided into two subfamilies NaV1.x (all but SCN6A) and NaV2.x (SCN6A). The NaV1.x subfamily can be functionally subdivided into two groups, those which are sensitive to blocking by tetrodotoxin (TTX-sensitive or TTX-S) and those which are resistant to blocking by tetrodotoxin (TTX-resistant or TTX-R).
The NaV1.8 channel is a voltage-gated sodium channel which is expressed in nociceptors, the sensory neurones responsible for transducing painful stimuli. The rat channel and the human channel were cloned in 1996 and 1998 respectively (Nature 1996; 379: 257-262; Pain 1998 (November); 78(2):107-114). The NaV1.8 channel was previously known as SNS (sensory neurone specific) and PN3 (peripheral nerve type 3). The NaV1.8 channel is atypical in that it shows resistance to the blocking effects of the puffer fish toxin tetrodotoxin and it is believed to underlie the slow-voltage-gated and tetrodotoxin-resistant (TTX-R) sodium currents recorded from dorsal root ganglion neurones. The closest molecular relative to the NaV1.8 channel is the NaV1.5 channel, which is the cardiac sodium channel, with which it shares approximately 60% homology. The NaV1.8 channel is expressed most highly in the ‘small cells’ of the dorsal root ganglia (DRG). These are thought to be the C- and A-delta cells which are the putative polymodal nociceptors, or pain sensors. Under normal conditions, the NaV1.8 channel is not expressed anywhere other than subpopulations of DRG neurones. The NaV1.8 channels are thought to contribute to the process of DRG sensitisation and also to hyperexcitability due to nerve injury. Inhibitory modulation of the NaV1.8 channels is aimed at reducing the excitability of nociceptors, by preventing them from contributing to the excitatory process.
Studies have shown that NaV1.8 knock-out leads to a blunted pain phenotype, mostly to inflammatory challenges (A. N. Akopian et al., Nat. Neurosci. 1999; 2; 541-548) and that NaV1.8 knockdown reduces pain behaviours, in this case neuropathic pain (J. Lai et al., Pain, 2002 (January); 95(1-2): 143-152). Coward et al. and Yiangou et al., have shown that NaV1.8 appears to be expressed in pain conditions (Pain. 2000 (March); 85(1-2): 41-50 and FEBS Lett. 2000 (Feb. 11); 467(2-3): 249-252).
The NaV1.8 channel has also been shown to be expressed in structures relating to the back and tooth pulp and there is evidence for a role in causalgia, inflammatory bowel conditions and multiple sclerosis (Bucknill et al., Spine. 2002 (Jan. 15); 27(2):135-140: Shembalker et al., Eur J Pain. 2001; 5(3): 319-323: Laird et al., J Neurosci. 2002 (Oct. 1); 22(19): 8352-8356: Black et al., Neuroreport. 1999 (Apr. 6); 10(5): 913-918 and Proc. Natl. Acad. Sci. USA 2000: 97: 11598-11602).
Examples of modulators of the NaV1.8 sodium channel are disclosed in WO2008/135826 and WO2008/135830. There is, however, an ongoing need to provide new NaV1.8 sodium channel inhibitors that are good drug candidates. These drug candidates should have one or more of the following properties: be well absorbed from the gastrointestinal tract; be metabolically stable; have a good metabolic profile, in particular with respect to the toxicity or allergenicity of any metabolites formed; or possess favourable pharmacokinetic properties whilst still retaining their activity profile as NaV1.8 channel inhibitors. They should be non-toxic and demonstrate few side-effects. Ideal drug candidates should exist in a physical form that is stable, non-hygroscopic and easily formulated.
SUMMARY OF THE INVENTION
According to a first aspect of the invention there is provided a compound of formula (I)
or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein:
R1 and R2, together with the carbon to which they are attached, form a 4- to 7-membered ring, wherein:
one member of said ring is O: and
the remaining members of said ring are CR6R7, which may be the same or different at each occurrence;
R3 is selected from the group consisting of H, (C1-C3)alkyl, cyclopropyl, cyclopropyl-CH2—, —CH2OH, —CH2OCH3, (C1-C3)fluoroalkyl, —OH, —OCH3, F, —NH2, NHCH3, —N(CH3)2 and —NHC(O)CH3;
R4 is selected from the group consisting of —CF3, —OCF3, —OCHF2, Cl and —SF5;
R5 is selected from the group consisting of H and —CH3;
R6 and R7 are independently selected from the group consisting of H, —CH3, —OH, —OCH3, F, —NH2, NHCH3 and —N(CH3)2.
Described below are a number of embodiments (E) of this first aspect of the invention, where for convenience E1 is identical thereto.
E1 A compound of formula (I) as defined above, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or said tautomer.
E2 A compound according to E1 wherein R1 and R2, together with the carbon to which they are attached, form a 4- to 7-membered ring of formula
wherein m is 1, 2 or 3 and n is 1 or 2. Such a compound is represented by formula (Ia).
E3 A compound according to E2 wherein m is 1 and n is 1. Such a compound is represented by formula (Ib).
E4 A compound according to any of E1 to E3 wherein R3 is selected from the group consisting of H, methyl, ethyl, n-propyl and isopropyl.
E5 A compound according to any of E1 to E4 wherein R5 is H.
In a further aspect of the invention there is provided a compound according to formula (I) as described above for use as a medicament.
In a further aspect of the invention there is provided a compound according to formula (I) as described above for use in the treatment of pain.
In a further aspect of the invention there is provided a compound according to formula (I) as described above for use in the manufacture of a medicament for the treatment of pain.
In a further aspect of the invention there is provided a pharmaceutical composition comprising a compound according to formula (I) as described above and one or more pharmaceutically acceptable carriers.
In one embodiment, the pharmaceutical composition is adapted for topical administration.
In another embodiment, the pharmaceutical composition is adapted for intra-ocular administration.
In a further aspect of the invention there is provided a method for the treatment of a condition for which a NaV1.8 modulator is indicated comprising the administration to a subject of a therapeutically effective amount of a compound according to formula (I) as described above.
In a further aspect of the invention there is provided a method for the treatment of pain in a subject in need of such treatment comprising the administration to said subject of a therapeutically effective amount of a compound according to formula (I) as described above.
DETAILED DESCRIPTION OF THE INVENTION
Alkyl groups, containing the requisite number of carbon atoms, can be unbranched or branched. (C1-C3)Alkyl includes methyl, ethyl, 1-propyl and 2-propyl.
Fluoroalkyl includes monofluoroalkyl, polyfluoroalkyl and perfluoroalkyl. Examples of (C1-C3)fluoroalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, heptafluoro-n-propyl and 1,1,1,3,3,3-hexafluoro-2-propyl.
The compounds of formula (I) can exist in tautomeric forms. Specifically, the 2,4-disubstituted imidazole can exist as the (1H)-tautomer or the (3H)-tautomer. It will be understood that a 2,4-disubstituted-(3H)-imidazole may also be described as a 2,5-disubstituted-(1H)-imidazole.
The compounds of formula (I) may exist in substantially pure (1H)-tautomeric form, substantially pure (3H)-tautomeric form, or as a mixture of tautomeric forms. All such tautomers and mixtures of tautomers are included within the scope of the present invention. References herein to specific compounds should be understood to refer to the compound and/or its tautomer.
Certain compounds of formula (I) include one or more stereogenic centers and so may exist as optical isomers, such as enantiomers and disastereomers. All such isomers and mixtures thereof are included within the scope of the present invention.
Hereinafter, all references to compounds of the invention include compounds of formula (I) or pharmaceutically acceptable salts, solvates, or multi-component complexes thereof, or pharmaceutically acceptable solvates or multi-component complexes of pharmaceutically acceptable salts of compounds of formula (I), as discussed in more detail below.
Preferred compounds of the invention are compounds of formula (I) or pharmaceutically acceptable salts thereof.
Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.
Hemisalts of acids and bases may also be formed, for example, hemisulphate salts.
The skilled person will appreciate that the aforementioned salts include ones wherein the counterion is optically active, for example d-lactate or l-lysine, or racemic, for example dl-tartrate or dl-arginine.
For a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
Pharmaceutically acceptable salts of compounds of formula (I) may be prepared by one or more of three methods:
(i) by reacting the compound of formula (I) with the desired acid or base;
(ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of formula (I) using the desired acid or base; or
(iii) by converting one salt of the compound of formula (I) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
The compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in both unsolvated and solvated forms. The term ‘solvate’ is used herein to describe a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D2O, d6-acetone and d6-DMSO.
A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates—see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995), incorporated herein by reference. Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.
When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.
The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term ‘amorphous’ refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterised by a change of state, typically second order (‘glass transition’). The term ‘crystalline’ refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterised by a phase change, typically first order (‘melting point’).
Also included within the scope of the invention are multi-component complexes (other than salts and solvates) of compounds of formula (I) or pharmaceutically acceptable salts thereof wherein the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallisation, by recrystallisation from solvents, or by physically grinding the components together—see Chem Commun, 17, 1889-1896, by 0. Almarsson and M. J. Zaworotko (2004), incorporated herein by reference. For a general review of multi-component complexes, see J Pharm Sci, 64 (8), 1269-1288, by Haleblian (August 1975), incorporated herein by reference.
The compounds of the invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described as ‘thermotropic’ and that resulting from the addition of a second component, such as water or another solvent, is described as ‘lyotropic’. Compounds that have the potential to form lyotropic mesophases are described as ‘amphiphilic’ and consist of molecules which possess an ionic (such as —COO−Na+, —COO−K+, or —SO3−Na+) or non-ionic (such as —N−N+(CH3)3) polar head group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and A. Stuart, 4th Edition (Edward Arnold, 1970), incorporated herein by reference.
The compounds of the invention may be administered as prodrugs. Thus certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as ‘prodrugs’. Further information on the use of prodrugs may be found in ‘Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and ‘Bioreversible Carriers in Drug Design’, Pergamon Press, 1987 (ed. E B Roche, American Pharmaceutical Association).
Prodrugs can, for example, be produced by replacing appropriate functionalities present in a compound of formula (I) with certain moieties known to those skilled in the art as ‘pro-moieties’ as described, for example, in “Design of Prodrugs” by H Bundgaard (Elsevier, 1985).
Examples of prodrugs include phosphate prodrugs, such as dihydrogen or dialkyl (e.g. di-tert-butyl) phosphate prodrugs. Further examples of replacement groups in accordance with the foregoing examples and examples of other prodrug types may be found in the aforementioned references.
Also included within the scope of the invention are metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites in accordance with the invention include, where the compound of formula (I) contains a phenyl (Ph) moiety, a phenol derivative thereof (-Ph>-PhOH);
Compounds of the invention containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Included within the scope of the invention are all stereoisomers of the compounds of the invention and mixtures of one or more thereof.
Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.
Mixtures of stereoisomers may be separated by conventional techniques known to those skilled in the art; see, for example, “Stereochemistry of Organic Compounds” by E. L. Eliel and S. H. Wilen (Wiley, New York, 1994.
The scope of the invention includes all crystal forms of the compounds of the invention, including racemates and racemic mixtures (conglomerates) thereof. Stereoisomeric conglomerates may also be separated by the conventional techniques described herein just above.
The scope of the invention includes all pharmaceutically acceptable isotopically-labelled compounds of the invention wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.
Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H and 3H, carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123I and 125I, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, and sulphur, such as 35S.
Certain isotopically-labelled compounds of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e. 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
Also within the scope of the invention are intermediate compounds as hereinafter defined, all salts, solvates and complexes thereof and all solvates and complexes of salts thereof as defined hereinbefore for compounds of formula (I). The invention includes all polymorphs of the aforementioned species and crystal habits thereof.
When preparing a compound of formula (I) in accordance with the invention, a person skilled in the art may routinely select the form of intermediate which provides the best combination of features for this purpose. Such features include the melting point, solubility, processability and yield of the intermediate form and the resulting ease with which the product may be purified on isolation.
The compounds of the invention may be prepared by any method known in the art for the preparation of compounds of analogous structure. In particular, the compounds of the invention can be prepared by the procedures described by reference to the Schemes that follow, or by the specific methods described in the Examples, or by similar processes to either.
The skilled person will appreciate that the experimental conditions set forth in the schemes that follow are illustrative of suitable conditions for effecting the transformations shown, and that it may be necessary or desirable to vary the precise conditions employed for the preparation of compounds of formula (I). It will be further appreciated that it may be necessary or desirable to carry out the transformations in a different order from that described in the schemes, or to modify one or more of the transformations, to provide the desired compound of the invention.
In addition, the skilled person will appreciate that it may be necessary or desirable at any stage in the synthesis of compounds of the invention to protect one or more sensitive groups, so as to prevent undesirable side reactions. In particular, it may be necessary or desirable to protect amino or carboxylic acid groups. The protecting groups used in the preparation of the compounds of the invention may be used in conventional manner. See, for example, those described in ‘Greene's Protective Groups in Organic Synthesis’ by Theodora W Greene and Peter G M Wuts, third edition, (John Wiley and Sons, 1999), in particular chapters 7 (“Protection for the Amino Group”) and 5 (“Protection for the Carboxyl Group”), incorporated herein by reference, which also describes methods for the removal of such groups.
All of the imidazole derivatives of the formula (I) can be prepared by the procedures described in the general methods presented below or by routine modifications thereof. The present invention also encompasses any one or more of these processes for preparing the imidazole derivatives of formula (I), in addition to any novel intermediates used therein.
In the following general methods, Ar represents
and R1, R2, R3, R4 and R5 are as previously defined for an imidazole derivative of the formula (I) unless otherwise stated. In order to improve the legibility the schemes show structures wherein R6 and R7 are both H. Compounds wherein R6 and/or R7 are other than H may be prepared using analogous methods.
According to a first process, compounds of formula (I) may be prepared from compounds of formula (IV), as illustrated by Scheme 1.
X is a suitable leaving group, typically Br.
Y is a suitable amine protecting group, typically tert-butoxycarbonyl, benzyloxycarbonyl or alkylsulfinyl
Compounds of formula (II) are either commercially available or may be prepared according to the methods set out in Schemes 2 (for compounds wherein Y is tert-butoxycarbonyl or benzyloxycarbonyl) or 3 (for compounds wherein Y is alkylsulfinyl).
Compounds of formula (V) are either commercially available or may be prepared according to the methods set out in Scheme 4.
Compounds of formula (III) may be prepared from compounds of formula (II) according to process step (i), by alkylation with a compound of formula (V) in the presence of base in a suitable solvent. Typical conditions comprise combining an acid of formula (II) and an α-halo-ketone of formula (V) with an excess of base in a suitable solvent at a temperature between room temperature and 50° C. Preferred conditions comprise using 1.05 equivalents of α-bromo-ketone of formula (V) and 1.5 equivalents of caesium carbonate in acetonitrile at room temperature, or 1 equivalent of α-bromo-ketone of formula (V) and 1.5 equivalents of triethylamine in acetone at 50° C., or 1 equivalent of α-bromo-ketone of formula (V) and 1.5 equivalents of triethylamine in ethyl acetate at room temperature.
Compounds of formula (IV) can be prepared from compounds of formula (III) by process step (ii), a cyclisation reaction, in the presence of a suitable ammonium salt, typically ammonium acetate. Typical conditions comprise an excess of ammonium salt in a suitable organic solvent at a temperature between 100° C. and 130° C. Preferred conditions comprise 10 equivalents of ammonium acetate in anhydrous toluene at 100° C.-130° C.
Compounds of formula (I) can be prepared from compounds of formula (IV) by process step (iii), a deprotection reaction under hydrogenolysis or acidic conditions. Typical conditions are dependent on the nature of the protecting group. Where the protecting group is a tert-butoxycarbonyl group, conditions are acid mediated. Preferred conditions are an excess of HCl in 1,4-dioxane at room temperature. Where the protecting group is a benzyloxycarbonyl group, conditions are either acid mediated, typically using HBr in acetic acid at room temperature or by hydrogenolysis over a suitable hydrogenation catalyst, typically Pd/C or Pd(OH)2/C.
According to a second process, compounds of formula (VI) (i.e. compounds of formula (II) wherein Y is tert-butyloxycarbonyl or benzyloxycarbonyl, R3 is hydrogen and R1 and R2 together with the carbon atom to which they are attached form a 4- to 7-membered ring of formula
where m is 1, 2 or 3 and n is 1 or 2) may be prepared by the process illustrated by Scheme 2.
Ra is a suitable alkyl protecting group, typically methyl or ethyl.
Y is tert-butyloxycarbonyl or benzyloxycarbonyl.
m is 1, 2 or 3, and n is 1 or 2.
Compounds of formula (VII) are commercially available or can be prepared using published methods.
Compounds of formula (VIII) can be prepared from compounds of formula (VII) by a Wittig-type reaction according to process step (iv), with a ketone of formula (VII) and either a phosphonate ester in the presence of a strong base or a phosphorane in a suitable solvent. In the case of the phosphonate ester, typical conditions comprise the phosphonate ester in the presence of a strong base in anhydrous THF at 0° C. Preferred conditions comprise triethyl phosphonoacetate with 1.1 equivalents of sodium hydride in anhydrous THF at 0° C. In the case of the phosphorane, preferred conditions comprise 1.01 equivalents of (carbethoxymethylene)triphenylphosphorane in dichloromethane at 0° C.
Compounds of formula (IX) can be prepared from compounds of formula (VIII) by process step (v), a conjugate addition reaction with a Michael acceptor of formula (VIII) and ammonia. Preferred conditions comprise an excess of ammonia in an alcoholic solvent at a temperature between 100° C. and 150° C. in a sealed vessel.
Compounds of formula (X) can be prepared from compounds of formula (IX) by process step (vi), a protection reaction of an amino ester of formula (IX). Typical conditions are dependent on the nature of the amine protecting group. Where the protecting group is a benzyloxycarbonyl group, typical conditions comprise benzylchloroformate in the presence of a base in a suitable solvent. Preferred conditions comprise 1.2 equivalents of benzylchloroformate and 3 equivalents of N,N-diisopropylethylamine in acetonitrile at room temperature, or 1.3 equivalents of benzylchloroformate and an aqueous solution of sodium carbonate in tert-butylmethyl ether at 5-20° C.
Compounds of formula (VI) can be prepared from compounds of formula (X) by process step (vii), a hydrolysis reaction of a protected amino ester of formula (X). Typical conditions comprise a base in a suitable solvent at a temperature between room temperature and 75° C. Preferred conditions comprise an aqueous solution of sodium hydroxide in methanol at 75° C. or an aqueous solution of sodium hydroxide in tert-butylmethylether at room temperature.
According to a third process, compounds of formula (XI) (i.e. compounds of formula (II) wherein Y is alkylsulfinyl and R1 and R2 together with the carbon atom to which they are attached form a 4- to 7-membered ring of formula
where m is 1, 2 or 3 and n is 1 or 2) may be prepared by the process illustrated by Scheme 3.
Ra is a suitable alkyl protecting group, typically methyl or ethyl.
Q is a suitable alkyl protecting group, typically tert-butyl.
m is 1, 2 or 3, and n is 1 or 2.
Compounds of formula (XIII) are commercially available.
Compounds of formula (XII) can be prepared from compounds of formula (VII) by an imine formation reaction according to process step (viii), with a ketone of formula (VII) and a sulfinamide of formula (XIII) in the presence of base in a suitable solvent. Preferred conditions comprise 1.0 equivalent of an alkyl sulfinamide (XIII) and 1.0 equivalent of caesium carbonate in dichloromethane at room temperature.
Compounds of formula (XV) can be prepared from compounds of formula (XII) by process step (ix), addition of a lithium enolate of formula (XIV) to a sulfinime of formula (XII). The lithium enolate is formed in situ from the appropriate ester in the presence of a lithium base in a suitable solvent at −78° C. Preferred conditions comprise of 2.1 equivalents of the appropriate ester and 2 equivalents of lithium diisopropylamine in anhydrous THF at −78° C., followed by addition of the sulfinimine of formula (XII).
Compounds of formula (XI) can be prepared from compounds of formula (XV) by process step (x), a hydrolysis reaction of the protected amino ester of formula (XIII). Typical conditions comprise a base in a suitable solvent at room temperature. Preferred conditions comprise an aqueous solution of sodium hydroxide in methanol.
According to a fourth process, compounds of formula (V) may be prepared using the methods illustrated in Scheme 4.
Compounds of formula (V) can be prepared from compounds of formula (XVIII) according to process step (xiii), a halogenation reaction. Preferred bromination (whereby X is Br) reaction conditions comprise a brominating agent, such as trimethylphenylammonium tribromide, in a suitable solvent at 0° C.
If non-commercial, compounds of formula (XVIII) can be prepared from compounds of formula (XVII) according to process step (xii), displacement of a Weinreb amide. Preferred conditions comprise methyl lithium in a suitable solvent at 0° C.
Compounds of formula (XVII) can be prepared from compounds of formula (XVI) according to process step (xi), an amide bond formation. Preferred conditions comprise O,N-dimethylhydroxylamine hydrochloride and suitable base, such as triethylamine in a suitable solvent at room temperature.
Referring to the general methods above, it will be readily understood to the skilled person that where protecting groups are present, these will be generally interchangeable with other protecting groups of a similar nature, e.g. where an amine is described as being protected with a tert-butoxycarbonyl group, this may be readily interchanged with any suitable amine protecting group. Suitable protecting groups are described in ‘Protective Groups in Organic Synthesis’ by T. Greene and P. Wuts (3rd edition, 1999, John Wiley and Sons).
The present invention also relates to novel intermediate compounds as defined above, all salts, solvates and complexes thereof and all solvates and complexes of salts thereof as defined hereinbefore for imidazole derivatives of formula (I). The invention includes all polymorphs of the aforementioned species and crystal habits thereof.
When preparing imidazole derivatives of formula (I) or amino acids of formula (VI) in accordance with the invention, it is open to a person skilled in the art to routinely select the best order of steps with which to synthesise the intermediates, and to choose the form of the intermediate compounds which provides the best combination of features for this purpose. Such features include the melting point, solubility, processability and yield of the intermediate form and the resulting ease with which the product may be purified on isolation.
Compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products or may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.
They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term ‘excipient’ is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
In another aspect the invention provides a pharmaceutical composition comprising a compound of the invention together with one or more pharmaceutically acceptable excipients.
Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in “Remington's Pharmaceutical Sciences”, 19th Edition (Mack Publishing Company, 1995).
Suitable modes of administration include oral, parenteral, topical, inhaled/intranasal, rectal/intravaginal, and ocular/aural administration.
Formulations suitable for the aforementioned modes of administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays, liquid formulations and buccal/mucoadhesive patches.
Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
The compounds of the invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986, by Liang and Chen (2001).
For tablet dosage forms, depending on dose, the drug may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.
Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.
Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet. Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.
Exemplary tablets contain up to about 80% drug, from about 10 weight % to about 90 weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant. Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of tablets is discussed in “Pharmaceutical Dosage Forms: Tablets”, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).
Suitable modified release formulations for the purposes of the invention are described in U.S. Pat. No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in “Pharmaceutical Technology On-line”, 25(2), 1-14, by Verma et al (2001). The use of chewing gum to achieve controlled release is described in WO 00/35298.
The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, subcutaneous and trans-tympanic. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
The solubility of compounds of formula (I) used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microspheres.
The compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated—see, for example, J Pharm Sci, 88 (10), 955-958, by Finnin and Morgan (October 1999).
Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.
The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
The pressurised container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.
Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from 1 μg to 20 mg of the compound of the invention per actuation and the actuation volume may vary from 1 μl to 100 μl. A typical formulation may comprise a compound of formula (I), propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.
In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or “puff” containing from 1 μg to 100 mg of the compound of formula (I). The overall daily dose will typically be in the range 1 μg to 200 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, microbicide, vaginal ring or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.
The compounds of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.
The compounds of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.
Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.
For administration to human patients, the total daily dose of the compounds of the invention is typically in the range 1 mg to 10 g, such as 10 mg to 1 g, for example 25 mg to 500 mg depending, of course, on the mode of administration and efficacy. For example, oral administration may require a total daily dose of from 50 mg to 100 mg. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. These dosages are based on an average human subject having a weight of about 60 kg to 70 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.
As noted above, the compounds of the invention are useful because they exhibit pharmacological activity in animals, i.e., NaV1.8 channel modulation. More particularly, the compounds of the invention are of use in the treatment of disorders for which a NaV1.8 modulator is indicated. Preferably the animal is a mammal, more preferably a human.
In a further aspect of the invention there is provided a compound of the invention for use as a medicament.
In a further aspect of the invention there is provided a compound of the invention for the treatment of a disorder for which a NaV1.8 modulator is indicated.
In a further aspect of the invention there is provided use of a compound of the invention for the preparation of a medicament for the treatment of a disorder for which a NaV1.8 modulator is indicated.
In a further aspect of the invention there is provided a method of treating a disorder in an animal (preferably a mammal, more preferably a human) for which a NaV1.8 modulator is indicated, comprising administering to said animal a therapeutically effective amount of a compound of the invention.
Disorders for which a NaV1.8 modulator is indicated include pain, particularly neuropathic, nociceptive and inflammatory pain.
Physiological pain is an important protective mechanism designed to warn of danger from potentially injurious stimuli from the external environment. The system operates through a specific set of primary sensory neurones and is activated by noxious stimuli via peripheral transducing mechanisms (see Millan, 1999, Prog. Neurobiol., 57, 1-164 for a review). These sensory fibres are known as nociceptors and are characteristically small diameter axons with slow conduction velocities. Nociceptors encode the intensity, duration and quality of noxious stimulus and by virtue of their topographically organised projection to the spinal cord, the location of the stimulus. The nociceptors are found on nociceptive nerve fibres of which there are two main types, A-delta fibres (myelinated) and C fibres (non-myelinated). The activity generated by nociceptor input is transferred, after complex processing in the dorsal horn, either directly, or via brain stem relay nuclei, to the ventrobasal thalamus and then on to the cortex, where the sensation of pain is generated.
Pain may generally be classified as acute or chronic. Acute pain begins suddenly and is short-lived (usually twelve weeks or less). It is usually associated with a specific cause such as a specific injury and is often sharp and severe. It is the kind of pain that can occur after specific injuries resulting from surgery, dental work, a strain or a sprain. Acute pain does not generally result in any persistent psychological response. In contrast, chronic pain is long-term pain, typically persisting for more than three months and leading to significant psychological and emotional problems. Common examples of chronic pain are neuropathic pain (e.g. painful diabetic neuropathy, postherpetic neuralgia), carpal tunnel syndrome, back pain, headache, cancer pain, arthritic pain and chronic post-surgical pain.
When a substantial injury occurs to body tissue, via disease or trauma, the characteristics of nociceptor activation are altered and there is sensitisation in the periphery, locally around the injury and centrally where the nociceptors terminate. These effects lead to a heightened sensation of pain. In acute pain these mechanisms can be useful, in promoting protective behaviours which may better enable repair processes to take place. The normal expectation would be that sensitivity returns to normal once the injury has healed. However, in many chronic pain states, the hypersensitivity far outlasts the healing process and is often due to nervous system injury. This injury often leads to abnormalities in sensory nerve fibres associated with maladaptation and aberrant activity (Woolf & Salter, 2000, Science, 288, 1765-1768).
Clinical pain is present when discomfort and abnormal sensitivity feature among the patient's symptoms. Patients tend to be quite heterogeneous and may present with various pain symptoms. Such symptoms include: 1) spontaneous pain which may be dull, burning, or stabbing; 2) exaggerated pain responses to noxious stimuli (hyperalgesia); and 3) pain produced by normally innocuous stimuli (allodynia—Meyer et al., 1994, Textbook of Pain, 13-44). Although patients suffering from various forms of acute and chronic pain may have similar symptoms, the underlying mechanisms may be different and may, therefore, require different treatment strategies. Pain can also therefore be divided into a number of different subtypes according to differing pathophysiology, including nociceptive, inflammatory and neuropathic pain.
Nociceptive pain is induced by tissue injury or by intense stimuli with the potential to cause injury. Pain afferents are activated by transduction of stimuli by nociceptors at the site of injury and activate neurons in the spinal cord at the level of their termination. This is then relayed up the spinal tracts to the brain where pain is perceived (Meyer et al., 1994, Textbook of Pain, 13-44). The activation of nociceptors activates two types of afferent nerve fibres. Myelinated A-delta fibres transmit rapidly and are responsible for sharp and stabbing pain sensations, whilst unmyelinated C fibres transmit at a slower rate and convey a dull or aching pain. Moderate to severe acute nociceptive pain is a prominent feature of pain from central nervous system trauma, strains/sprains, burns, myocardial infarction and acute pancreatitis, post-operative pain (pain following any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and back pain. Cancer pain may be chronic pain such as tumour related pain (e.g. bone pain, headache, facial pain or visceral pain) or pain associated with cancer therapy (e.g. postchemotherapy syndrome, chronic postsurgical pain syndrome or post radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormonal therapy or radiotherapy. Back pain may be due to herniated or ruptured intervertabral discs or abnormalities of the lumber facet joints, sacroiliac joints, paraspinal muscles or the posterior longitudinal ligament. Back pain may resolve naturally but in some patients, where it lasts over 12 weeks, it becomes a chronic condition which can be particularly debilitating.
Neuropathic pain is currently defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. Nerve damage can be caused by trauma and disease and thus the term ‘neuropathic pain’ encompasses many disorders with diverse aetiologies. These include, but are not limited to, peripheral neuropathy, diabetic neuropathy, post herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain and pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy and vitamin deficiency. Neuropathic pain is pathological as it has no protective role. It is often present well after the original cause has dissipated, commonly lasting for years, significantly decreasing a patient's quality of life (Woolf and Mannion, 1999, Lancet, 353, 1959-1964). The symptoms of neuropathic pain are difficult to treat, as they are often heterogeneous even between patients with the same disease (Woolf & Decosterd, 1999, Pain Supp., 6, S141-S147; Woolf and Mannion, 1999, Lancet, 353, 1959-1964). They include spontaneous pain, which can be continuous, and paroxysmal or abnormal evoked pain, such as hyperalgesia (increased sensitivity to a noxious stimulus) and allodynia (sensitivity to a normally innocuous stimulus).
The inflammatory process is a complex series of biochemical and cellular events, activated in response to tissue injury or the presence of foreign substances, which results in swelling and pain (Levine and Taiwo, 1994, Textbook of Pain, 45-56). Arthritic pain is the most common inflammatory pain. Rheumatoid disease is one of the commonest chronic inflammatory conditions in developed countries and rheumatoid arthritis is a common cause of disability. The exact aetiology of rheumatoid arthritis is unknown, but current hypotheses suggest that both genetic and microbiological factors may be important (Grennan & Jayson, 1994, Textbook of Pain, 397-407). It has been estimated that almost 16 million Americans have symptomatic osteoarthritis (OA) or degenerative joint disease, most of whom are over 60 years of age, and this is expected to increase to 40 million as the age of the population increases, making this a public health problem of enormous magnitude (Houge & Mersfelder, 2002, Ann Pharmacother., 36, 679-686; McCarthy et al., 1994, Textbook of Pain, 387-395). Most patients with osteoarthritis seek medical attention because of the associated pain. Arthritis has a significant impact on psychosocial and physical function and is known to be the leading cause of disability in later life. Ankylosing spondylitis is also a rheumatic disease that causes arthritis of the spine and sacroiliac joints. It varies from intermittent episodes of back pain that occur throughout life to a severe chronic disease that attacks the spine, peripheral joints and other body organs.
Another type of inflammatory pain is visceral pain which includes pain associated with inflammatory bowel disease (IBD). Visceral pain is pain associated with the viscera, which encompass the organs of the abdominal cavity. These organs include the sex organs, spleen and part of the digestive system. Pain associated with the viscera can be divided into digestive visceral pain and non-digestive visceral pain. Commonly encountered gastrointestinal (GI) disorders that cause pain include functional bowel disorder (FBD) and inflammatory bowel disease (IBD). These GI disorders include a wide range of disease states that are currently only moderately controlled, including, in respect of FBD, gastro-esophageal reflux, dyspepsia, irritable bowel syndrome (IBS) and functional abdominal pain syndrome (FAPS), and, in respect of IBD, Crohn's disease, ileitis and ulcerative colitis, all of which regularly produce visceral pain. Other types of visceral pain include the pain associated with dysmenorrhea, cystitis and pancreatitis and pelvic pain.
It should be noted that some types of pain have multiple aetiologies and thus can be classified in more than one area, e.g. back pain and cancer pain have both nociceptive and neuropathic components.
Other types of pain include:
pain resulting from musculo-skeletal disorders, including myalgia, fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism, dystrophinopathy, glycogenolysis, polymyositis and pyomyositis;
heart and vascular pain, including pain caused by angina, myocardical infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma and skeletal muscle ischemia;
head pain, such as migraine (including migraine with aura and migraine without aura), cluster headache, tension-type headache mixed headache and headache associated with vascular disorders;
erythermalgia; and
orofacial pain, including dental pain, otic pain, burning mouth syndrome and temporomandibular myofascial pain.
A NaV1.8 modulator may be usefully combined with another pharmacologically active compound, or with two or more other pharmacologically active compounds, particularly in the treatment of pain. Such combinations offer the possibility of significant advantages, including patient compliance, ease of dosing and synergistic activity.
In the combinations that follow the compound of the invention may be administered simultaneously, sequentially or separately in combination with the other therapeutic agent or agents.
A NaV1.8 modulator of formula (I), or a pharmaceutically acceptable salt thereof, as defined above, may be administered in combination with one or more agents selected from:
an alternative NaV1.8 modulator (e.g. as disclosed in WO 2008/135826, more particularly N-[6-Amino-5-(2-chloro-5-methoxyphenyl)pyridin-2-yl]-1-methyl-1H-pyrazole-5-carboxamide);
an alternative sodium channel modulator, such as a NaV1.3 modulator (e.g. as disclosed in WO2008/118758); or a NaV1.7 channel modulator e.g. as disclosed in WO 2009/012242);
an inhibitor of nerve growth factor signaling, such as: an agent that binds to NGF and inhibits NGF biological activity and/or downstream pathway(s) mediated by NGF signaling (e.g. tanezumab), a TrkA antagonist or a p75 antagonist;
a compound which increases the levels of endocannabinoid, such as a compound with fatty acid amid hydrolase inhibitory (FAAH) activity, in particular those disclosed in WO 2008/047229 (e.g. N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridine-2-yl]oxy}benzylidene)piperidene-1-carboxamide);
an opioid analgesic, e.g. morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine;
a nonsteroidal antiinflammatory drug (NSAID), e.g. aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin or zomepirac;
a barbiturate sedative, e.g. amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobarbital, secobarbital, talbutal, theamylal or thiopental;
a benzodiazepine having a sedative action, e.g. chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam;
an H1 antagonist having a sedative action, e.g. diphenhydramine, pyrilamine, promethazine, chlorpheniramine or chlorcyclizine;
a sedative such as glutethimide, meprobamate, methaqualone or dichloralphenazone;
a skeletal muscle relaxant, e.g. baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine;
an NMDA receptor antagonist, e.g. dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine, EN-3231 (MorphiDex®, a combination formulation of morphine and dextromethorphan), topiramate, neramexane or perzinfotel including an NR2B antagonist, e.g. ifenprodil, traxoprodil or (−)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone;
an alpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, or 4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl) quinazoline;
a tricyclic antidepressant, e.g. desipramine, imipramine, amitriptyline or nortriptyline;
an anticonvulsant, e.g. carbamazepine, lamotrigine, topiratmate or valproate;
a tachykinin (NK) antagonist, particularly an NK-3, NK-2 or NK-1 antagonist, e.g. (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione (TAK-637), 5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine (2S,3S);
a muscarinic antagonist, e.g. oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin, solifenacin, temiverine and ipratropium;
a COX-2 selective inhibitor, e.g. celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib;
a coal-tar analgesic, in particular paracetamol;
a neuroleptic such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, sertindole, aripiprazole, sonepiprazole, blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox, asenapine, lurasidone, amisulpride, balaperidone, palindore, eplivanserin, osanetant, rimonabant, meclinertant, Miraxion® or sarizotan;
a vanilloid receptor agonist (e.g. resinferatoxin) or antagonist (e.g. capsazepine);
a beta-adrenergic such as propranolol;
a local anaesthetic such as mexiletine;
a corticosteroid such as dexamethasone;
a 5-HT receptor agonist or antagonist, particularly a 5-HT1B/1D agonist such as eletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan;
a 5-HT2A receptor antagonist such as R(+)-alpha-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL-100907);
a 5-HT3 antagonist, such as ondansetron
a cholinergic (nicotinic) analgesic, such as ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403), (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594) or nicotine;
Tramadol®;
a PDEV inhibitor, such as 5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil), (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]-pyrido[3,4-b]indole-1,4-dione (IC-351 or tadalafil), 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (vardenafil), 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide, 3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide;
an alpha-2-delta ligand such as gabapentin, pregabalin, 3-methylgabapentin, (1α,3α,5α)(3-amino-methyl-bicyclo[3.2.0]hept-3-yl)-acetic acid, (3S,5R)-3-aminomethyl-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-heptanoic acid, (3S,5R)-3-amino-5-methyl-octanoic acid, (2S,4S)-4-(3-chlorophenoxy)proline, (2S,4S)-4-(3-fluorobenzyl)-proline, [(1R,5R,6S)-6-(aminomethyl)bicyclo[3.2.0]hept-6-yl]acetic acid, 3-(1-aminomethyl-cyclohexylmethyl)-4H-[1,2,4]oxadiazol-5-one, C-[1-(1H-tetrazol-5-ylmethyl)-cycloheptyl]-methylamine, (3S,4S)-(1-aminomethyl-3,4-dimethyl-cyclopentyl)-acetic acid, (3S,5R)-3-aminomethyl-5-methyl-octanoic acid, (3S,5R)-3-amino-5-methyl-nonanoic acid, (3S,5R)-3-amino-5-methyl-octanoic acid, (3R,4R,5R)-3-amino-4,5-dimethyl-heptanoic acid and (3R,4R,5R)-3-amino-4,5-dimethyl-octanoic acid;
metabotropic glutamate subtype 1 receptor (mGluR1) antagonist;
a serotonin reuptake inhibitor such as sertraline, sertraline metabolite demethylsertraline, fluoxetine, norfluoxetine (fluoxetine desmethyl metabolite), fluvoxamine, paroxetine, citalopram, citalopram metabolite desmethylcitalopram, escitalopram, d,l-fenfluramine, femoxetine, ifoxetine, cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine and trazodone;
a noradrenaline (norepinephrine) reuptake inhibitor, such as maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, buproprion metabolite hydroxybuproprion, nomifensine and viloxazine (Vivalan®), especially a selective noradrenaline reuptake inhibitor such as reboxetine, in particular (S,S)-reboxetine;
a dual serotonin-noradrenaline reuptake inhibitor, such as venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine, clomipramine, clomipramine metabolite desmethylclomipramine, duloxetine, milnacipran and imipramine;
an inducible nitric oxide synthase (iNOS) inhibitor such as S-[2-[(1-iminoethyl)-amino]ethyl]-L-homocysteine, S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine, S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine, (2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile; 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile, (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3 pyridinecarbonitrile, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile, N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine, or guanidinoethyldisulfide;
an acetylcholinesterase inhibitor such as donepezil;
a prostaglandin E2 subtype 4 (EP4) antagonist such as N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamide or 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoic acid;
a microsomal prostaglandin E synthase type 1 (mPGES-1) inhibitor;
a leukotriene B4 antagonist; such as 1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic acid (CP-105696), 5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valeric acid (ONO-4057) or DPC-11870,
a 5-lipoxygenase inhibitor, such as zileuton, 6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxy-methyl]-1-methyl-2-quinolone (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),1,4-benzoquinone (CV-6504).
There is also included within the scope the present invention combinations of a compound of the invention together with one or more additional therapeutic agents which slow down the rate of metabolism of the compound of the invention, thereby leading to increased exposure in patients. Increasing the exposure in such a manner is known as boosting. This has the benefit of increasing the efficacy of the compound of the invention or reducing the dose required to achieve the same efficacy as an unboosted dose. The metabolism of the compounds of the invention includes oxidative processes carried out by P450 (CYP450) enzymes, particularly CYP 3A4 and conjugation by UDP glucuronosyl transferase and sulphating enzymes. Thus, among the agents that may be used to increase the exposure of a patient to a compound of the present invention are those that can act as inhibitors of at least one isoform of the cytochrome P450 (CYP450) enzymes. The isoforms of CYP450 that may be beneficially inhibited include, but are not limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19 and CYP3A4. Suitable agents that may be used to inhibit CYP3A4 include ritonavir, saquinavir, ketoconazole, N-(3,4-difluorobenzyl)-N-methyl-2-{[(4-methoxypyridin-3-yl)amino]sulfonyl}benzamide and N-(1-(2-(5-(4-fluorobenzyl)-3-(pyridin-4-yl)-1H-pyrazol-1-yl)acetyl)piperidin-4-yl)methanesulfonamide.
It is within the scope of the invention that two or more pharmaceutical compositions, at least one of which contains a compound of the invention, may conveniently be combined in the form of a kit suitable for coadministration of the compositions. Thus the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like. The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically comprises directions for administration and may be provided with a so-called memory aid.
In another aspect the invention provides a pharmaceutical product (such as in the form of a kit) comprising a compound of the invention together with one or more additional therapeutically active agents as a combined preparation for simultaneous, separate or sequential use in the treatment of a disorder for which a NaV1.8 modulator is indicated.
It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.
In the non-limiting Examples and Preparations that are set out later in the description, and in the aforementioned Schemes, the following the abbreviations, definitions and analytical procedures may be referred to:
AcOH is acetic acid,
Cs2CO3 is caesium carbonate;
Cu(acac)2 is copper (II) acetylacetonate;
CuI is copper (I) iodide;
Cu(OAc)2 is copper (II) acetate;
DAD is diode array detector;
DCM is dichloromethane; methylene chloride;
DIPEA is N-ethyldiisopropylamine, N,N-diisopropylethylamine;
DMAP is 4-dimethylaminopyridine;
DMF is N,N-dimethylformamide;
DMSO is dimethyl sulphoxide;
EDCl is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
EDTA is ethylenediaminetetraacetic acid;
ELSD is evaporative light scattering detection;
Et2O is diethyl ether;
EtOAc is ethyl acetate;
EtOH is ethanol;
HCl is hydrochloric acid;
IPA is isopropanol;
Ir2(OMe)2COD2 is bis(1,5-cyclooctadiene)di-μ-methoxydiiridium (I);
K2CO3 is potassium carbonate;
KHSO4 is potassium hydrogen sulphate;
KOAc is potassium acetate;
KOH is potassium hydroxide;
K3PO4 is potassium phosphate tribasic;
LCMS is liquid chromatography mass spectrometry (Rt=retention time)
LiOH is lithium hydroxide;
MeOH is methanol;
MgSO4 is magnesium sulphate;
NaH is sodium hydride;
NaHCO3 is sodium hydrogencarbonate;
Na2CO3 is sodium carbonate;
NaHSO3 is sodium bisulphite;
NaHSO4 is sodium hydrogensulphate;
NaOH is sodium hydroxide;
Na2SO4 is sodium sulphate;
NH4Cl is ammonium chloride;
NMP is N-Methyl-2-pyrrolidone;
Pd/C is palladium on carbon;
Pd(PPh3)4 is palladium tetrakis(triphenylphosphine);
Pd(dppf)2Cl2 is [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane;
THF is tetrahydrofuran;
THP is tetrahydropyran;
TLC is thin layer chromatography; and
WSCDI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
The invention is illustrated by the following representative Examples.
1H Nuclear magnetic resonance (NMR) spectra were in all cases consistent with the proposed structures. Characteristic chemical shifts (δ) are given in parts-per-million downfield from tetramethylsilane using conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The mass spectra (MS) were recorded using either electrospray ionisation (ESI) or atmospheric pressure chemical ionisation (APCI). The following abbreviations have been used for common solvents: CDCl3, deuterochloroform; DMSO-d6, deuterodimethylsulphoxide; CD3OD, deuteromethanol; THF, tetrahydrofuran. LCMS indicates liquid chromatography mass spectrometry (Rt=retention time). Where ratios of solvents are given, the ratios are by volume.
Certain compounds of the Examples and Preparations were purified using Automated Preparative High Performance Liquid Chromatography (HPLC). Reversed-phase HPLC conditions were on FractionLynx systems. Samples were submitted dissolved in 1 mL of DMSO. Depending on the nature of the compounds and the results of a pre-analysis, the purification was performed under either acidic conditions (‘A-HPLC’) or basic conditions (‘B-HPLC’) at ambient temperature. Acidic runs were carried out on a Sunfire Prep C18 OBD column (19×100 mm, 5 μm), basic runs were carried out on an Xterra Prep MS C18 (19×100 mm, 5 μm), both from Waters. A flow rate of 18 mL/min was used with mobile phase A: water+0.1% modifier (v/v) and B: acetonitrile+0.1% modifier (v/v). For acidic runs the modifier was formic acid, for basic run the modifier was diethylamine. A Waters 2525 binary LC pump supplied a mobile phase with a composition of 5% B for 1 min then ran from 5% to 98% B over 6 min followed by a 2 min hold at 98% B.
Detection was achieved using a Waters 2487 dual wavelength absorbance detector set at 225 nm followed in series by a Polymer Labs PL-ELS 2100 detector and a Waters ZQ 2000 4 way MUX mass spectrometer in parallel. The PL 2100 ELSD was set at 30° C. with 1.6 L/min supply of Nitrogen. The Waters ZQ MS was tuned with the following parameters:
ES+ Cone voltage: 30 v Capillary: 3.20 kv
ES− Cone voltage: −30 v Capillary: −3.00 kv
Desolvation gas: 600 L/hr
Source Temp: 120° C.
Scan range 150-900 Da
The fraction collection was triggered by both MS and ELSD.
Quality control (QC) analysis was performed using a LCMS method. Acidic runs were carried out on a Sunfire C18 (4.6×50 mm, 5 μm), basic runs were carried out on a Xterra C18 (4.6×50 mm, 5 μm), both from Waters. A flow rate of 1.5 mL/min was used with mobile phase A: water+0.1% modifier (v/v) and B: acetonitrile+0.1% modifier (v/v). For acidic runs the modifier was formic acid, for basic run the modifier was ammonia. A Waters 1525 binary LC pump ran a gradient elution from 5% to 95% B over 3 min followed by a 1 min hold at 95% B. Detection was achieved using a Waters MUX UV 2488 detector set at 225 nm followed in series by a Polymer Labs PL-ELS 2100 detector and a Waters ZQ 2000 4 way MUX mass spectrometer in parallel. The PL 2100 ELSD was set at 30° C. with 1.6 L/min supply of Nitrogen. The Waters ZQ MS was tuned with the following parameters:
ES+ Cone voltage: 25 v Capillary: 3.30 kv
ES− Cone voltage: −30 v Capillary: −2.50 kv
Desolvation gas: 800 L/hr
Source Temp: 150° C.
Scan range 160-900 Da
Unless carried out by Auto-HPLC (under conditions of A-HPLC or B-HPLC as just described, LCMS conditions were run according to one of the conditions given below (where ratios of solvents are given, the ratios are by volume):
6 Minute LC-MS Gradient and Instrument Conditions
Acid run: A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile Column: C18 phase Phenomenex Gemini 50×4.6 mm with 5 micron particle size. Gradient: 95-5% A over 3 min, 1 min hold, 1 ml/min. UV: 210 nm 450 nm DAD. Temperature: 50° C.
2 Minute LC-MS Gradient and Instrument Conditions
Acid run: A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile Column: C18 phase Fortis Pace 20×2.1 mm with 3 micron particle size. Gradient: 70-2% A over 1.8 min, 0.2 min hold, 1.8 ml/min. UV: 210 nm 450 nm DAD. Temperature: 75° C.
C18 30 Minute Method LC-MS Gradient and Instrument Conditions
A: 0.1% formic acid in H2O B: 0.1% formic acid in MeCN Column: Phenomenex C18 phase Gemini 150×4.6 mm with 5 micron particle size Gradient: 98-2% A over 18 min, 2 min hold, 1 ml/min. UV: 210 nm 450 nm DAD. Temperature: 50° C.
Phenyl Hexyl 30 Minute Method LC-MS Gradient and Instrument Conditions
A: 10 mM ammonium acetate in H2O B: 10 mM ammonium acetate in methanol Column: Phenomenex Phenyl Hexyl 150×4.6 mm with 5 micron particle size Gradient: 98-2% A over 18 min, 2 min hold, 1 ml/min. UV: 210 nm 450 nm DAD. Temperature: 50° C.
Unless otherwise noted, HPLC analysis conditions were run according to the conditions given below:
Ultra Acid Method HPLC Gradient and Instrument Conditions
HPLC analysis was performed using the ultra acid method. Zorbax SB-C18 (3.0×50 mm, 1.8 μm), supplied by Crawford scientific at a column temperature of 50° C. A flow rate of 1.2 mL/min was used with mobile phase A: water+0.05% TFA (v/v) and B: acetonitrile. An Agilent 1100 LC pump ran a gradient elution from 5% to 100% B over 3.5 min followed by a 1 min hold at 100% B.
Example 1
3-({4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-3-amine
Method A
To benzyl [3-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-3-yl]carbamate (Preparation 1, 0.120 g, 0.253 mmol) in acetic acid (1 mL) was added a solution of HBr in acetic acid (48%, 2 mL) and the reaction left to stir at room temperature for 1.5 hours before concentrating in vacuo. The residue was azeotroped with cyclohexane to yield an orange solid. The solid was purified by Isolute™ SCX ion exchange column eluting with methanol followed by 7M ammonia in methanol to afford a yellow oil. The oil was further purified by preparative HPLC conditions (B-HPLC) to afford the title compound.
LCMS (acidic QC method) Rt=2.16 min MS m/z 342 [MH]+
Example 2
3-{[4-(4-Chloro-3-methylphenyl)-1H-imidazol-2-yl]methyl}oxetan-3-amine
Method B
Benzyl (3-{[4-(4-chloro-3-methylphenyl)-1H-imidazol-2-yl]methyl}oxetan-3-yl)carbamate (Preparation 3, 0.150 g, 0.364 mmol) was dissolved in methanol (5 mL) and hydrogenated at 50° C. through a 20% Pd(OH)2 on carbon CATCART™ (30 mm) supplied by Thales Nanotechnology Inc® using the Thales Nanotechnology Inc® HC2 hydrogenater at a flow rate of 1 mL/min and a pressure of 1 Bar. The reaction was concentrated in vacuo. The residue was purified by preparative HPLC conditions (A-HPLC) to afford the title compound.
LCMS (acidic QC method) Rt=1.99 min MS m/z 278 [MH]+
The following examples 3 to 6 were prepared by methods analogous to Methods A and B as described for Examples 1 and 2 above. Unless otherwise noted, preparation details are as described for the method referred to.
Example 3
3-({4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydrofuran-3-amine
Prepared by Method A using benzyl [3-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydrofuran-3-yl]carbamate (Preparation 2, 0.132 g, 0.286 mmol) but without the need for initial purification via Isolute™ SCX ion exchange column to afford the title compound.
LCMS (acidic QC method) Rt=2.23 min MS m/z 328 [MH]+
Example 4
3-({4-[4-(Trifluoromethyl)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-amine
Prepared by Method B using benzyl [3-({4-[4-(trifluoromethyl)phenyl]-1H-imidazol-2-yl}-methyl)oxetan-3-yl]carbamate (Preparation 5, 0.135 g, 0.310 mmol) to afford the title compound.
LCMS (acidic QC method) Rt=2.15 min MS m/z 298 [MH]+
Example 5
3-({4-[4-(Difluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-amine
Prepared by Method B using benzyl [3-({4-[4-(difluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-yl]carbamate (Preparation 6, 0.133 g, 0.310 mmol). Purified by preparative HPLC conditions (B-HPLC) to afford the title compound.
LCMS (acidic QC method) Rt=2.30 min MS m/z 296 [MH]+
Example 6
3-({4-[4-(Pentafluoro-λ6-sulfanyl)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-amine
Prepared by Method B using benzyl [3-({4-[4-(pentafluoro-λ6-sulfanyl)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-yl]carbamate (Preparation 7, 0.100 g, 0.204 mmol). Purified by preparative HPLC conditions (B-HPLC) to afford the title compound.
LCMS (acidic QC method) Rt=2.31 min MS m/z 356 [MH]+
Example 7
4-({4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-4-amine
To tert-butyl [4-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-4-yl]carbamate (Preparation 4, 0.166 g, 0.376 mmol) was added 4M hydrogen chloride in 1,4-dioxane (3 mL) and the reaction left to stir at room temperature for 18 hours before concentrating in vacuo. The residue was purified by preparative HPLC conditions (A-HPLC) to afford the title compound.
LCMS (acidic QC method) Rt=1.98 min MS m/z 342 [MH]+
Example 8
3-({4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-amine
Benzyl [3-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-yl]-carbamate (Preparation 11, 311 g, 695 mmol) was dissolved in methanol (3.2 L). 5% Palladium on carbon E105 R/W (EVONIK) (22 g, 7 wt %) was added and the reaction hydrogenated at 40° C., 100 psi for 18 hours. Hydrogen uptake was monitored and showed the reaction to be complete after 4 hours. The mixture was cooled to room temperature and filtered over Arbocel©. The filter cake was washed with methanol (2×1 L) and the filtrate concentrated in vacuo to afford a solid. The solid was dissolved in ethyl acetate (1 L) and filtered through a carbon tablet to remove traces of palladium. The solution was warmed to 50° C. and heptane (1 L) added. The solution was cooled slowly whereupon at 40° C. crystallisation was observed. The mixture was stirred at room temperature for 72 hours. The solid was collected by filtration and washed with ethyl acetate: heptane (1:1, 250 mL). The solid was dried in vacuo at 40° C. for 18 hours to afford the title compound as a crystalline solid.
HPLC (ultra acid method) Rt=1.996 min.
Example 9
3-(1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-amine
To a solution of 2-methyl-N-[3-(1-{4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}-ethyl)oxetan-3-yl]propane-2-sulfinamide (Preparation 9, 0.320 g, 0.74 mmol) in methanol (4 mL) at 0° C. was added 4M hydrogen chloride in 1,4-dioxane (4 mL) and the reaction left to stir for 2 hours. Solid sodium hydrogen carbonate was added to the reaction, followed by a saturated aqueous solution of sodium hydrogen carbonate. The mixture was extracted with dichloromethane. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.243 g, 91% yield).
LCMS (2 min) Rt=0.96 min MS m/z 328 [MH]+
Examples 10 & 11
3-[(1S)-1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine and
3-[(1R)-1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine
Racemic 3-(1-{4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-amine (Example 9, 0.243 g, 0.743 mmol) was dissolved in ethanol (1 mL). Enantiomers were separated by chiral preparative HPLC under basic conditions at ambient temperature on a Chiralpak AD-H column (250*, 20 mm i.d) supplied by Daicel Chemical Industries. A flow rate of 18 mL/min was used with mobile phase A: heptane and B: IPA+0.1% diethylamine (v/v). Two Agilent 1200 prep pumps supplied a mobile phase with a composition of 20% B. Run time was 10 minutes per 0.1 mL injection volume. Detection was achieved using an Agilent 1200 multiple wavelength UV absorbance detector set at 220 nm.
Enantiomer 1: Rt=5.89 min. >99.5% ee (58 mg, 24%)
Enantiomer 2: Rt=8.42 min. >99.5% ee (89 mg, 37%)
Enantiomer 1: 1HNMR (CDCl3): δ 1.35 (d, 3H), 3.21 (s, 2H), 3.64 (q, 1H), 4.39 (d, 1H), 4.43 (d, 1H), 4.52 (d, 1H), 4.66 (d, 1H), 7.13-7.22 (m, 3H), 7.75 (br s, 2H).
Enantiomer 2: 1HNMR (CDCl3): δ 1.35 (d, 3H), 3.21 (s, 2H), 3.64 (q, 1H), 4.39 (d, 1H), 4.43 (d, 1H), 4.52 (d, 1H), 4.66 (d, 1H), 7.13-7.22 (m, 3H), 7.75 (br s, 2H).
Example 12
3-(1-{4-[4-(Trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-amine
Benzyl [3-(1-{4-[4-(trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-yl]carbamate (Preparation 8, 0.95 g, 2.13 mmol) was dissolved in methanol (20 mL) and hydrogenated at room temperature and 100 psi. The reaction mixture was then filtered over Arbocel© and the resulting filtrate concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound as a solid (0.42 g, 63%).
LCMS (2 min) Rt=0.75 min. MS m/z 312 [MH+], 310 [MH]−
Examples 13 & 14
3-[(1S)-1-{4-[4-(Trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine and
3-[(1R)-1-{4-[4-(Trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl]oxetan-3-amine
Racemic 3-(1-{4-[4-(trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-amine (Example 12, 0.410 g, 1.32 mmol) was dissolved in ethanol (8.2 mL). Enantiomers were separated by chiral preparative HPLC under basic conditions at ambient temperature on a Chiralpak AD-H column (250*, 21.2 mm i.d) supplied by Daicel Chemical Industries. A flow rate of 18 mL/min was used with a mobile phase of: 70% heptane+30% IPA+0.3% diethylamine (v/v) supplied by an Agilent 1200 prep pump. An injection volume of 1 mL was used per run.
Detection was achieved using an Agilent 1200 multiple wavelength UV absorbance detector set at 220 nm and 254 nm.
Enantiomer 1: Rt=4.85 min. >99.5% ee (144 mg, 35%)
Enantiomer 2: Rt=5.89 min. >97.6% ee (142 mg, 35%)
Enantiomer 1: 1HNMR (d6-DMSO): δ 1.25 (m, 3H), 3.35 (m, 1H), 4.23 (m, 1H), 4.30 (m, 1H), 4.43 (m, 2H), 7.63 (m, 3H), 7.90 (m, 2H).
Enantiomer 2: 1HNMR (d6-DMSO): δ 1.25 (m, 3H), 3.35 (m, 1H), 4.23 (m, 1H), 4.30 (m, 1H), 4.43 (m, 2H), 7.63 (m, 3H), 7.90 (m, 2H).
Example 15
3-(1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}propyl)oxetan-3-amine
To a solution of 2-methyl-N-[3-(1-{4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}-propyl)oxetan-3-yl]propane-2-sulfinamide (Preparation 10, 0.450 g, 1.01 mmol) in methanol (5 mL) at 0° C. was added 4M hydrogen chloride in 1,4-dioxane (1 mL) and the reaction left to stir for 4 hours. Solid sodium hydrogen carbonate was added to the reaction, followed by a saturated aqueous solution of sodium hydrogen carbonate. The mixture was extracted with dichloromethane. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.154 g, 45% yield).
1HNMR (CDCl3): δ 1.0 (t, 3H), 1.65 (m, 2H), 3.4 (m, 1H), 4.25 (d, 1H), 4.45 (m, 2H), 4.75 (d, 1H), 7.2-7.3 (m, 4H), 7.75 (d, 2H).
LCMS (2 min) Rt=1.57 min MS m/z 342 [MH]+, 340 [MH]−
Examples 16 & 17
3-((1S)-1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}propyl)oxetan-3-amine and
3-((1R)-1-{4-[4-(Trifluoromethoxy)phenyl]-1H-imidazol-2-yl}propyl)oxetan-3-amine
Racemic 3-(1-{4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}propyl)oxetan-3-amine (Example 15, 0.145 g, 1.01 mmol) was dissolved in a mixture of 70% heptane and 30% IPA (3 mL). Enantiomers were separated by chiral preparative HPLC under basic conditions at ambient temperature on a Chiralpak AD-H column (250*, 20 mm i.d) supplied by Daicel Chemical Industries. A flow rate of 1 mL/min was used with a mobile phase of: 90% heptane+10% IPA+0.1% diethylamine (v/v) delivered by a Waters 515 HPLC prep pump over a 20 minute run time. Detection was achieved using an Agilent 119 UV absorbance detector (UV), followed in series by a Polymer Labs PL-ELS 2100 detector (ELSD) and a Waters ZQ micromass mass spectrometer (MS).
Enantiomer 1: Rt=7.95 min. MS m/z 342 [MH]+
Enantiomer 2: Rt=10.39 min. MS m/z 342 [MH]+
QC analysis was performed under basic conditions at ambient temperature on a Chiralpak AD-H column (250*, 10 mm i.d) supplied by Daicel Chemical Industries. A flow rate of 1 mL/min was used with a mobile phase of: 80% heptane+20% IPA+0.2% diethylamine (v/v) over a 10 minute run time. Detection was achieved using an Agilent 100 detector (DAD), followed in series by a Polymer Labs PL-ELS 2100 detector (ELSD) and a Waters ZQ micromass mass spectrometer (MS).
Enantiomer 1: Rt=4.58 min. MS m/z 342 [MH]+ >99/5% ee
Enantiomer 2: Rt=5.26 min. MS m/z 342 [MH]+ >99/5% ee
Preparation 1
Benzyl [3-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-3-yl]carbamate
Method C
Ammonium acetate (1.58 g, 20.5 mmol) was suspended in anhydrous toluene (10 mL) and heated to 100° C. until fully solubilised. A solution of 2-oxo-2-[4-(trifluoromethoxy)-phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}tetrahydro-2H-pyran-3-yl)acetate (Preparation 20, 1.11 g, 2.046 mmol) in anhydrous toluene (10 mL) was added to the reaction. The temperature was increased to 120° C. and the reaction refluxed for 2.5 hours. Once cooled, the reaction was partitioned between dichloromethane (3×5 mL) and water (5 mL). The organic layer was separated by phase separation cartridge and concentrated in vacuo to give an oil. The oil was purified by silica gel column chromatography (0-50% ethyl acetate in heptane gradient elution) to afford the title compound as a yellow oil (0.48 g, 49% yield).
1HNMR (CDCl3): δ 1.45 (m, 2H), 1.7 (m, 1H), 2.2 (m, 1H), 2.85 (d, 1H), 3.35 (m, 3H), 3.75 (m, 1H), 3.85 (m, 1H), 4.95 (d, 2H), 5.05 (d, 1H), 6.85 (br s, 1H), 7.1 (d, 2H), 7.3 (m, 5H), 7.6 (br m, 2H).
LCMS (2 min) Rt=1.30 min MS m/z 476 [MH]+, 474 [MH]−
Preparation 2
Benzyl [3-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydrofuran-3-yl]carbamate
Method D
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}-tetrahydrofuran-3-yl)acetate (Preparation 13, 0.634 g, 1.317 mmol), ammonium acetate (1.9 g, 25 mmol) and molecular sieves (3 Å) were suspended in anhydrous toluene (5 mL) and heated to 110° C. for 18 hours. Once cooled the reaction was partitioned between dichloromethane (3×5 mL) and water (5 mL) The organic layer was separated by phase separation cartridge and concentrated in vacuo to give an oil. The reaction had not gone to completion by 2 min LCMS analysis, therefore the oil, ammonium acetate (1.5 g, 19 mmol) and molecular sieves (3 Å) were placed in a microwave vial with anhydrous toluene (5 mL) and heated at 150° C. for 1 hour in a Biotage Initiator™ microwave. Once cooled the reaction was partitioned between dichloromethane (3×5 mL) and water (5 mL) The organic layer was separated by phase separation cartridge and concentrated in vacuo to give an oil. The oil was purified by silica gel column chromatography (0-100% ethyl acetate in heptane gradient elution) to afford the title compound as a yellow oil (0.132 g, 22% yield).
LCMS (2 min) Rt=1.30 min MS m/z 462 [MH]+, 460 [MH]−
The following Preparations 3 to 8 were prepared by methods analogous to Methods C and D as described for Preparations 1 and 2 above. Unless otherwise noted, preparation details are as described for the method referred to.
Preparation 3
Benzyl (3-{[4-(4-chloro-3-methylphenyl)-1H-imidazol-2-yl]methyl}oxetan-3-yl)carbamate
Prepared by Method C using 2-(4-chloro-3-methylphenyl)-2-oxoethyl (3-{[(benzyloxy)-carbonyl]amino}oxetan-3-yl)acetate (Preparation 12, 0.488 g, 1.13 mmol). The mixture was partitioned between ethyl acetate and water. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.297 g, 64% yield).
LCMS (2 min) Rt=1.25 min MS m/z 412 [MH]+
Preparation 4
tert-Butyl [4-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)tetrahydro-2H-pyran-4-yl]carbamate
Prepared by Method D using 2-oxo-2-[4-(trifluoromethoxy)phenyl]ethyl {4-[(tert-butoxycarbonyl)amino]tetrahydro-2H-pyran-4-yl}acetate (Preparation 19, 0.485 g, 1.051 mmol). The residue was purified by silica gel column chromatography (0-100% ethyl acetate+3% triethylamine (v/v) in heptane gradient elution) to afford the title compound as a yellow oil (0.166 g, 36% yield).
LCMS (2 min) Rt=1.25 min MS m/z 442 [MH]+, 440 [MH]−
Preparation 5
Benzyl [3-({4-[4-(trifluoromethyl)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-yl]carbamate
Prepared by Method C using 2-oxo-2-[4-(trifluoromethyl)phenyl]ethyl (3-{[(benzyloxy)-carbonyl]amino}oxetan-3-yl)acetate (Preparation 14, 0.510 g, 1.13 mmol). The mixture was partitioned between ethyl acetate and water. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.273 g, 56% yield).
LCMS (2 min) Rt=1.32 min MS m/z 432 [MH]+
Preparation 6
Benzyl [3-({4-[4-(difluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-yl]carbamate
Prepared by Method C using 2-[4-(difluoromethoxy)phenyl]-2-oxoethyl (3-{[(benzyloxy)-carbonyl]amino}oxetan-3-yl)acetate (Preparation 15, 0.508 g, 1.13 mmol). The mixture was partitioned between ethyl acetate and water. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.269 g, 56% yield).
LCMS (2 min) Rt=1.13 min MS m/z 430 [MH]+
Preparation 7
Benzyl [3-({4-[4-(pentafluoro-λ6-sulfanyl)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-yl]carbamate
Prepared by Method C using 2-oxo-2-[4-(pentafluoro-λ6-sulfanyl)phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetate (Preparation 18, 0.9 g, 1.77 mmol). The reaction was refluxed for 18 hours. The mixture was partitioned between ethyl acetate and water. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.227 g, 26% yield).
1HNMR (CDCl3): δ 3.50 (s, 2H), 4.70 (d, 2H), 4.85 (d, 2H), 5.10 (s, 2H), 5.80 (br s, 1H), 7.3-7.45 (m, 8H), 7.65-7.8 (m, 3H).
LCMS (2 min) Rt=1.44 min MS m/z 490 [MH]+, 512 [MNa]+, 488 [MH]−
Preparation 8
Benzyl [3-(1-{4-[4-(trifluoromethyl)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-yl]carbamate
Prepared by Method C using 2-oxo-2-[4-(trifluoromethyl)phenyl]ethyl 2-(3-{[(benzyloxy)-carbonyl]amino}oxetan-3-yl)propanoate (Preparation 22, 2.15 g, 4.62 mmol). The reaction was refluxed for 12 hours. The mixture was partitioned between ethyl acetate and water. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.983 g, 48% yield).
LCMS (2 min) Rt=0.97 min. MS m/z 446 [MH]+, 444 [MH]−
Preparation 9
2-Methyl-N-[3-(1-{4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}ethyl)oxetan-3-yl]propane-2-sulfinamide
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}-propanoate (Preparation 16, 1.4 g, 3.10 mmol) and ammonium acetate (2.44 g, 31.0 mmol) were refluxed in toluene (40 mL) at 130° C. for 18 hours. Once cooled, water was added and the mixture extracted with ethyl acetate. The organic layer was dried over MgSO4 then concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (0.323 g, 24% yield).
LCMS (2 min) Rt=1.40 min MS m/z 432 [MH]+, 430 [MH]−
Preparation 10
2-Methyl-N-[3-(1-{4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}propyl)oxetan-3-yl]propane-2-sulfinamide
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}-butanoate (Preparation 17, 3.4 g, 7.3 mmol) and ammonium acetate (5.74 g, 73.0 mmol) were refluxed in toluene (40 mL) at 130° C. for 18 hours. Once cooled, water was added and the mixture extracted with ethyl acetate. The organic layer was dried over MgSO4 then concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound (2.527 g, 78% yield).
LCMS (2 min) Rt=1.52 min MS m/z 446 [MH]+, 444 [MH]−
LCMS (6 min acidic) Rt=2.30 min MS m/z 446 [MH]+, 444 [MH]−
Preparation 11
Benzyl [3-({4-[4-(trifluoromethoxy)phenyl]-1H-imidazol-2-yl}methyl)oxetan-3-yl]carbamate
Ammonium acetate (1.22 Kg, 15 mol) was stirred in toluene (12 L) and heated to 100° C. for 30 minutes until the solid had melted. A solution of 2-oxo-2-[4-(trifluoromethoxy)-phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetate (Preparation 21, 700 g, 1.5 mol) in toluene (2 L) was added rapidly and the temperature increased to 130° C. and heated at vigorous reflux for 4 hours. The reaction was cooled to room temperature, water (4 L) added and the mixture stirred for 10 minutes before leaving to stand for 2 hours. The organic layer was separated and concentrated in vacuo to afford a thick orange oil. Dichloromethane (5 L) was added and the solution gently agitated by turning slowly on the rotary for 72 hours. A white precipitate was then observed. The solution volume was reduced in vacuo to 1 L and the mixture filtered through Arbocel©. The gelatinous solid was washed with dichloromethane (2 L) and the filtrate concentrated in vacuo to afford a dark orange mobile oil. The oil was purified by silica gel column chromatography eluting with tert-butyl methyl ether to afford the title compound as a light orange oil (311 g, 46% yield).
HPLC (ultra acid method) Rt=2.532 min.
Preparation 12
2-(4-Chloro-3-methylphenyl)-2-oxoethyl (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetate
Method E
(3-{[(Benzyloxy)carbonyl]amino}oxetan-3-yl)acetic acid (Preparation 25, 0.3 g, 1.13 mmol), 2-bromo-1-(4-chloro-3-methylphenyl)ethanone (0.294 g, 1.19 mmol) and cesium carbonate (0.553 g, 1.70 mmol) were stirred in acetonitrile (10 mL) at room temperature for 2 hours. The reaction was concentrated in vacuo and partitioned between ethyl acetate and water. The organic layer was dried over MgSO4 and concentrated in vacuo to afford the title compound which was used without purification in the next step.
LCMS (2 min) Rt=1.70 min MS m/z 432 [MH]+, 454 [MNa]+, 430 [MH]−
Preparation 13
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}-tetrahydrofuran-3-yl)acetate
Method F
(3-{[(Benzyloxy)carbonyl]amino}tetrahydrofuran-3-yl)acetic acid (Preparation 24, 0.311 g, 1.11 mmol) and triethylamine (0.233 mL, 1.67 mmol) were stirred in acetone (4 mL). A solution of 2-bromo-1-[4-(trifluoromethoxy)phenyl]ethanone (0.315 g, 1.11 mmol) in acetone (4 mL) was added and the reaction heated to 50° C. for 1 hour. Rapid formation of a white precipitate was observed. The reaction was partitioned between dichloromethane and water. The organic layer was separated by phase separation cartridge and concentrated in vacuo to afford the title compound as an oil which was used without purification in the next step (0.634 g, 118% yield).
LCMS (2 min) Rt=1.73 min MS m/z 482 [MH]+, 504 [MNa]+, 480 [MH]−
The following Preparations 14 to 20 were prepared by methods analogous to Methods E and F as described for Preparations 12 and 13 above. Unless otherwise noted, preparation details are as described for the method referred to.
Preparation 14
2-Oxo-2-[4-(trifluoromethyl)phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetate
Prepared by Method E using (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetic acid (Preparation 25, 0.3 g, 1.13 mmol) and 2-bromo-1-[4-(trifluoromethyl)phenyl]ethanone (0.317 g, 1.19 mmol) to afford the title compound.
LCMS (2 min) Rt=1.68 min MS m/z 452 [MH]+, 474 [MNa]+, 450 [MH]−
Preparation 15
2-[4-(Difluoromethoxy)phenyl]-2-oxoethyl (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetate
Prepared by Method E using (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetic acid (Preparation 25, 0.3 g, 1.13 mmol) and 2-bromo-1-[4-(difluoromethoxy)phenyl]ethanone (0.315 g, 1.19 mmol) to afford the title compound.
LCMS (2 min) Rt=1.63 min MS m/z 472 [MNa]+, 448 [MH]−
Preparation 16
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}propanoate
Prepared by Method E using 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}propanoic acid (Preparation 26, 1.18 g, 4.733 mmol) and 2-bromo-1-[4-(trifluoromethoxy)phenyl]-ethanone (1.47 g, 5.21 mmol). The residue was purified by silica gel column chromatography to afford the title compound (1.413 g, 66% yield).
LCMS (2 min) Rt=1.65 min MS m/z 452 [MH]+, 474 [MNa]+, 450 [MH]−
Preparation 17
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}butanoate
Prepared by Method E using 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}butanoic acid (Preparation 27, 2.658 g, 10.1 mmol) and 2-bromo-1-[4-(trifluoromethoxy)phenyl]-ethanone (3.14 g, 11.1 mmol). The reaction was stirred at room temperature for 3 hours. The residue was purified by silica gel column chromatography to afford the title compound (3.435 g, 73% yield).
LCMS (2 min) Rt=1.68 min MS m/z 466 [MH]+, 464 [MH]−
Preparation 18
2-Oxo-2-[4-(pentafluoro-λ6-sulfanyl)phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetate
Prepared by Method E using (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetic acid (Preparation 25, 0.647 g, 2.44 mmol) and 2-bromo-1-[4-(pentafluoro-λ6-sulfanyl)phenyl]-ethanone (Preparation 40, 0.793 g, 2.44 mmol) to afford the title compound.
LCMS (2 min) Rt=1.74 min MS m/z 510 [MH]+, 532 [MNa]+
Preparation 19
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl {4-[(tert-butoxycarbonyl)amino]tetrahydro-2H-pyran-4-yl}acetate
Prepared by Method F using {4-[(tert-butoxycarbonyl)amino]tetrahydro-2H-pyran-4-yl}acetic acid (0.259 g, 1.00 mmol) and 2-bromo-1-[4-(trifluoromethoxy)phenyl]-ethanone (0.283 g, 1.00 mmol). The reaction was stirred at 50° C. for 50 minutes. The residue was isolated as a crude oil that crystallised to afford the title compound as a solid (0.485 g, 105% yield).
LCMS (2 min) Rt=1.73 min MS m/z 484 [MNa]+, 460 [MH]−
Preparation 20
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}tetrahydro-2H-pyran-3-yl)acetate
Prepared by Method F using (3-{[(benzyloxy)carbonyl]amino}tetrahydro-2H-pyran-3-yl)-acetic acid (Preparation 23, 0.6 g, 2.05 mmol) and 2-bromo-1-[4-(trifluoromethoxy)-phenyl]ethanone (0.579 g, 0.205 mmol). The reaction was stirred at 50° C. for 1.5 hours.
LCMS (2 min) Rt=1.75 min MS m/z 496, [MH]+, 518 [MNa]+, 494 [MH]−
Preparation 21
2-Oxo-2-[4-(trifluoromethoxy)phenyl]ethyl (3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)acetate
(3-{[(Benzyloxy)carbonyl]amino}oxetan-3-yl)acetic acid (Preparation 25, 1.011 Kg, 3.812 mol) was stirred in ethyl acetate (8 L). 2-Bromo-1-[4-(trifluoromethoxy)phenyl]-ethanone (1.08 Kg, 3.81 mol) was added, followed by triethylamine (585 mL, 4.19 mol). The reaction was initially fully solubilised, but a precipitate was then observed. The reaction was washed with water (2×4 L), then concentrated in vacuo to afford the title compound as a mobile orange oil (1.903 Kg, 107%, contains residual ethyl acetate).
HPLC (ultra acid method) Rt=3.290 min.
Preparation 22
2-Oxo-2-[4-(trifluoromethyl)phenyl]ethyl 2-(3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)propanoate
2-(3-{[(Benzyloxy)carbonyl]amino}oxetan-3-yl)propanoic acid (Preparation 28, 1.5 g, 5.37 mmol) and triethylamine (1.12 mL, 8.06 mmol) were stirred in ethyl acetate (50 mL). 2-Bromo-1-[4-(trifluoromethyl)phenyl]ethanone (1.51 g, 5.64 mmol) was added and the reaction was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was separated, dried over anhydrous magnesium sulphate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound as an oil (2.19 g, 88%).
1HNMR (CDCl3): δ 1.43 (m, 3H), 3.40 (m, 1H), 4.70 (m, 2H), 4.80 (m, 1H), 4.90 (m, 1H), 5.10 (m, 2H), 5.35 (m, 2H), 6.05 (br s, 1H), 7.35 (m, 5H), 7.80 (m, 2H), 8.00 (m, 2H).
Preparation 23
(3-{[(Benzyloxy)carbonyl]amino}tetrahydro-2H-pyran-3-yl)acetic acid
Method G
Ethyl (3-aminotetrahydro-2H-pyran-3-yl)acetate (Preparation 32, 1.33 g, 7.109 mmol), benzyl chloroformate (1.53 g, 8.53 mmol) and N,N-diisopropylethylamine (3.72 mL, 21.3 mmol) were stirred in anhydrous acetonitrile (30 mL) for 18 hours at room temperature. The reaction was concentrated in vacuo then partitioned between ethyl acetate and water. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with heptane:ethyl acetate:methanol (100:0:0-0:90:10). The oil isolated was then dissolved in methanol (10 mL) and a 1M aqueous solution of sodium hydroxide (10 mL) and heated to 75° C. for 18 hours. The methanol was removed in vacuo and the mixture partitioned between dichloromethane (10 mL) and water. The aqueous layer was acidified with 2M aqueous hydrogen chloride and extracted with dichloromethane (4×10 mL). The organic layer was dried over MgSO4 to afford the title compound as a oil (0.6 g, 29% yield over 2 steps).
1HNMR (CDCl3): δ 1.5-1.6 (m, 1H), 1.6-1.7 (m, 1H), 1.7-1.8 (m, 1H), 2.3 (m, 1H), 2.7 (br m, 1H), 3.0 (br m, 1H), 3.5-3.6 (m, 2H), 3.8 (m, 1H), 3.9 (d, 1H), 5.1 (s, 2H), 7.35-7.40 (m, 5H).
LCMS (2 min) Rt=1.34 min MS m/z 292 [MH]−, 316 [MNa]+
The following Preparation 24 was prepared by a method analogous to Method G as described for Preparation 23 above. Unless otherwise noted, preparation details are as described for the method referred to.
Preparation 24
(3-{[(Benzyloxy)carbonyl]amino}tetrahydrofuran-3-yl)acetic acid
Prepared by Method G using ethyl (3-aminotetrahydrofuran-3-yl)acetate (Preparation 31, 1.43 g, 8.25 mmol) to afford the title compound as an oil (0.311 g, 14% yield over 2 steps).
LCMS (2 min) Rt=1.26 min MS m/z 278 [MH]−, 302 [MNa]+
Preparation 25
(3-{[(Benzyloxy)carbonyl]amino}oxetan-3-yl)acetic acid
tert-Butyl methyl ether (2.5 L) and an aqueous solution of sodium carbonate (750 g in 2.2 L water, 7.07 mol) were stirred. Ethyl (3-aminooxetan-3-yl)acetate (Preparation 30, 875 g, 5.5 mol) was added to the reaction followed by further tert-butyl methyl ether (2.5 L). The reaction was cooled to 5° C. and benzyl chloroformate (1.21 Kg, 7.09 mol) added in a controlled manner such as to maintain the temperature below 20° C. A precipitate was observed so further water (5 L) and tert-butyl methyl ether (1.5 L) were added to solubilise the reaction mixture. The biphasic mixture was separated. The organic layer was basified with 2M aqueous solution of sodium hydroxide (3.5 L) and stirred vigorously for 18 hours. The aqueous layer was separated and the remaining organic layer washed with water (1.5 L). The aqueous layers were combined and cooled to 15° C. Isopropyl acetate (5 L) was added followed by controlled addition of a 6M aqueous solution of hydrogen chloride (1.2 L), maintaining the temperature below 17° C. The reaction was stirred for 30 minutes. Solid crystallised out in the reactor so was dissolved in a mixture of ethyl acetate and methanol (˜20 L). The solution was stirred at room temperature for 18 hours. The reaction was concentrated in vacuo to afford solid material. Ethyl acetate (5 L) was added and concentrated in vacuo. Further ethyl acetate (5 L) was added and the slurry heated to reflux to give an orange solution. The solution was cooled to 50° C. and heptane (2.5 L) added. A thick slurry was observed that was stirred at room temperature for 18 hours. The solid was filtered and dried on a sinter for 3 hours before drying in vacuo at 40° C. for 18 hours to afford the title compound as a white crystalline solid (1.07 Kg, 73% yield).
1HNMR (CDCl3): δ 3.1 (m, 2H), 4.6 (m, 2H), 4.7 (m, 2H), 5.1 (m, 2H), 7.2-7.4 (m, 5H).
Preparation 26
2-{3-[(tert-Butylsulfinyl)amino]oxetan-3-yl}propanoic acid
Methyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}propanoate (Preparation 33, 1.25 g, 4.746 mmol) was stirred in methanol (15 mL) and a 1M aqueous solution of sodium hydroxide (15 mL) for 3 hours at room temperature. The reaction was concentrated in vacuo and partitioned between diethyl ether and water. The pH of the aqueous layer was adjusted to pH3 with potassium hydrogen sulphate and extracted with dichloromethane. The organic layer was dried over MgSO4 and concentrated in vacuo to afford the title compound that was used without purification in the next step.
Preparation 27
2-{3-[(tert-Butylsulfinyl)amino]oxetan-3-yl}butanoic acid
Methyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}butanoate (Preparation 35, 2.89 g, 10.42 mmol) was stirred in methanol (30 mL) and a 1M aqueous solution of sodium hydroxide (30 mL) for 18 hours at room temperature. The reaction was concentrated in vacuo and partitioned between diethyl ether and water. The pH of the aqueous layer was adjusted to pH3 with potassium hydrogen sulphate and extracted with dichloromethane. The organic layer was dried over MgSO4 and concentrated in vacuo to afford the title compound that was used without purification in the next step.
Preparation 28
2-(3-{[(Benzyloxy)carbonyl]amino}oxetan-3-yl)propanoic acid
Ethyl 2-(3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)propanoate (Preparation 29, 43 g, 140 mmol) was stirred in methanol (200 mL) and a 1M aqueous solution of sodium hydroxide (200 mL) for 18 hours at room temperature. The reaction was concentrated in vacuo and partitioned between diethyl ether and water. The pH of the aqueous layer was adjusted to pH3 with potassium hydrogen sulphate and extracted with dichloromethane. The organic layer was dried over MgSO4 and concentrated in vacuo to afford the title compound that was used without purification in the next step.
Preparation 29
Ethyl 2-(3-{[(benzyloxy)carbonyl]amino}oxetan-3-yl)propanoate
To a solution of ethyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}propanoate (Preparation 34, 40 g, 140 mmol) in methanol (400 mL) at 0° C. was added a 4M solution of hydrogen chloride in 1,4-dioxane (72 mL). After 2 hours, a 4M aqueous solution of sodium hydroxide (400 mL) was added drop-wise until pH 7 was achieved whilst maintaining the temperature at 0° C. The methanol was removed in vacuo. The resulting solution was stirred with tetrahydrofuran (150 mL) and a 1M aqueous solution of sodium hydrogen carbonate (180 mL) at 0° C. Benzyl chloroformate (33.7 g, 187 mmol) was added and the reaction mixture stirred at room temperature for 18 hours. The organics were removed in vacuo and the resulting solution extracted with dichloromethane. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography to afford the title compound.
Preparation 30
Ethyl (3-aminooxetan-3-yl)acetate
Ethyl oxetan-3-ylideneacetate (Preparation 38, 781 g, 5.49 mol) was dissolved in 2M ammonia in ethanol (8.24 L) and heated to 100° C. in a bomb for 5 hours. The reaction was concentrated in vacuo to afford the title compound as a mobile oil (750 g, 100% yield).
1HNMR (CDCl3): δ 1.25 (t, 3H), 2.0 (br s, 2H), 2.85 (s, 2H), 4.2 (q, 2H), 4.5 (d, 2H), 4.55 (d, 2H).
Preparation 31
Ethyl (3-aminotetrahydrofuran-3-yl)acetate
Ethyl (2Z)-dihydrofuran-3(2H)-ylideneacetate (Preparation 36, 1.29 g, 8.25 mmol) was stirred in 1,4-dioxane (7 mL) in a microwave vial. A solution of 7M ammonia in methanol (5 mL) was added and the reaction heated for 4 hours at 150° C. in a Biotage Initiator™ microwave. The reaction was concentrated in vacuo, but later deemed not to have reached completion. A solution of 7M ammonia in methanol (7 mL) was added and the reaction heated again for 3 hours at 150° C. in the microwave. A further portion of 7M ammonia in methanol (3 mL) was added and the reaction heated for a further 2 hours. The reaction was concentrated in vacuo to afford the title compound along with the methyl ester where trans-esterification had occurred. The material was used without further purification in the next step.
Preparation 32
Ethyl (3-aminotetrahydro-2H-pyran-3-yl)acetate
Ethyl (2Z)-dihydro-2H-pyran-3(4H)-ylideneacetate (Preparation 37, 1.21 g, 7.11 mmol) was stirred in 1,4-dioxane (7 mL) in a microwave vial. A solution of 7M ammonia in methanol (5 mL) was added and the reaction heated for 3 hours at 150° C. in a Biotage Initiator™ microwave. A further solution of 7M ammonia in methanol (2 mL) was added and the reaction heated again for 2 hours at 150° C. in the microwave. The reaction was concentrated in vacuo and the residue dissolved in a further portion of 7M ammonia in methanol (10 mL) and heated for a further 5 hours at 150° C. in the microwave. The reaction was concentrated in vacuo to afford the title compound along with the methyl ester where trans-esterification had occurred. The material was used without further purification in the next step.
Preparation 33
Methyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}propanoate
Methyl propionate (2.71 g, 30.8 mmol) was dissolved in anhydrous THF (90 mL) and cooled to −78° C. under nitrogen. LDA (2M solution in THF, 15 mL, 30 mmol) was added drop-wise. After 1 hour at −78° C., a solution of 2-methyl-N-oxetan-3-ylidenepropane-2-sulfinamide (Preparation 39, 1.35 g, 7.703 mmol) in anhydrous THF (10 mL) was added. The reaction was gradually warmed to room temperature and stirred for 18 hours. The reaction was quenched with a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound as an oil (1.276 g, 63% yield).
1HNMR (CDCl3): δ 1.25 (s, 9H), 1.3-1.4 (m, 3H), 3.25 (m, 1H), 3.7 (m, 3H), 4.2 (br s, 0.4H), 4.45 (br s, 0.6H), 4.55 (m, 1.1H), 4.6 (m, 0.9H), 4.75 (d, 0.6H), 4.85-5.0 (m, 1.4H).
Preparation 34
Ethyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}propanoate
N,N-diisopropylamine (78 g, 770 mmol) was dissolved in anhydrous THF (200 mL) and cooled to −78° C. under nitrogen. Butyl lithium (2.5M solution in hexane, 297 mL, 743 mmol) was added drop-wise. The reaction was removed from the cooling bath for 30 minutes, then re-cooled to −78° C. A solution of ethyl propionate (72.8 g, 713 mmol) in anhydrous THF (200 mL) was added drop-wise and the reaction allowed to stir at room temperature for 1 hour. The reaction was cooled to −78° C. once again and a solution of 2-methyl-N-oxetan-3-ylidenepropane-2-sulfinamide (Preparation 39, 50 g, 285 mmol) in anhydrous THF (200 mL) was added drop-wise. The reaction was stirred at between −40° C. and −60° C. for 4 hours before being quenched with a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated in vacuo. Purification by silica gel column chromatography (ethyl acetate elution) was not successful. The title compound was obtained as a yellow oil (40 g, 51% yield) and used without further purification.
Preparation 35
Methyl 2-{3-[(tert-butylsulfinyl)amino]oxetan-3-yl}butanoate
Methyl butyrate (5.67 g, 55.5 mmol) was dissolved in anhydrous THF (100 mL) and cooled to −78° C. under nitrogen. LDA (2M solution in THF 27.1 mL, 54.2 mmol) was added drop-wise. After 1 hour at −78° C., a solution of 2-methyl-N-oxetan-3-ylidene-propane-2-sulfinamide (Preparation 39, 2.43 g, 13.88 mmol) in anhydrous THF (10 mL) was added. The reaction was gradually warmed to room temperature and stirred for 18 hours. The reaction was quenched with a saturated aqueous solution of ammonium chloride and extracted with ethyl acetate. The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound as an oil (2.89 g, 75% yield).
1HNMR (CDCl3): δ 1.00 (m, 3H), 1.25 (s, 9H), 1.6-2.0 (m, 2H), 3.0 (m, 1H), 3.7 (m, 3H), 4.3 (br s, 0.5H), 4.50 (m, 1H), 4.55 (br s, 0.5H), 4.6 (m, 0.5H), 4.65 (m, 1H), 4.9 (m, 0.5H), 4.95 (m, 1H).
Preparation 36
Ethyl (2Z)-dihydrofuran-3(2H)-ylideneacetate
Method H
Sodium hydride (60% dispersion in oil, 0.65 g, 16.3 mmol) was cooled to 0° C. under nitrogen before adding anhydrous THF (20 mL). Triethyl phosphonoacetate (3 mL, 15.1 mmol) was added slowly over 40 minutes to control gas evolution. A solution of 3-oxo-tetrahydrofuran (1 g, 11.62 mmol) in anhydrous THF (2 mL) was added and the reaction gradually warmed to room temperature and stirred for 18 hours. The reaction was concentrated in vacuo and the residue partitioned between ethyl acetate (3×50 mL) and water (30 mL). The organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-50% ethyl acetate in heptane gradient elution) to afford the title compound as an oil (1.29 g, 71% yield).
1HNMR (CDCl3): δ 1.3 (m, 3H), 2.7 (m, 1H), 3.05 (m, 0.7H), 3.2 (m, 0.3H), 3.9 (t, 1H), 3.95 (t, 0.7H), 4.15 (m, 2H), 4.4 (m, 0.7H), 4.6-4.7 (m, 0.6H), 4.75 (m, 1H), 5.7-5.85 (m, 1H).
LCMS (2 min) Rt=1.23 min MS m/z 157 [MH]+
The following Preparation 37 was prepared by methods analogous to Method H as described for Preparation 36 above. Unless otherwise noted, preparation details are as described for the method referred to.
Preparation 37
Ethyl (2Z)-dihydro-2H-pyran-3(4H)-ylideneacetate
Prepared by Method H using dihydropyran-3-one (1 g, 9.99 mmol) to afford the title compound as an oil (1.214 g, 71% yield).
1HNMR (CDCl3): δ 1.25 (m, 3H), 1.8 (m, 2H), 2.2 (m, 1H), 3.0 (m, 1H), 3.75 (m, 2H), 4.0 (s, 1H), 4.1 (m, 2H), 4.7 (s, 1H), 5.65 (m, 1H).
LCMS (2 min) Rt=1.33 min MS m/z 171 [MH]+
Dihydropyran-3-one can be prepared using the literature procedure Tet., 2004, 60, 46, 10411.
Preparation 38
Ethyl oxetan-3-ylideneacetate
To a solution of (carbethoxymethylene)triphenylphosphorane (1.95 Kg, 5.61 mol) in dichloromethane (4 L) at 0° C. was added over 1 hour, a solution of 3-oxetanone (400 g, 5.55 mol) in dichloromethane (2 L) maintaining the temperature below 10° C. The reaction was warmed gradually to room temperature and stirred for 1.5 hours. The reaction was warmed to 30° C. and dichloromethane (˜4 L) removed in vacuo. Heptane (5 L) was added and the mixture distilled under vacuum for a further 1 hour. Further heptane (2.5 L) was added, the temperature increased to 50° C. and the reaction continued to be distilled under vacuum for a further 2 hours. The mixture was cooled to 0° C. and aged for 1 hour at atmospheric pressure. The solid was collected by filtration and washed with heptane (2×2.5 L). The pale yellow filtrate was concentrated in vacuo to afford the title compound as a pale yellow mobile liquid (757 g, 96% yield).
1HNMR (CDCl3): δ 1.25 (t, 3H), 4.2 (q, 2H), 5.3 (m, 2H), 5.5 (m, 2H), 5.65 (m, 1H).
Preparation 39
2-Methyl-N-oxetan-3-ylidenepropane-2-sulfinamide
3-Oxetanone (3 g, 41.63 mmol), tert-butyl sulfinamide (5.55 g, 45.8 mmol) and titanium (IV) ethoxide (13.5 mL, 62.4 mmol) were stirred in THF (200 mL) at 40° C. for 72 hours. The mixture was cooled to room temperature and poured into a rapidly stirred aqueous solution of saturated sodium chloride (200 mL). The resulting suspension was filtered through Celite© and the filter cake washed with ethyl acetate. The organic layer was separated and washed with brine, then dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the title compound as an oil (1.37 g, 19% yield).
1HNMR (CDCl3): δ 1.3 (s, 9H), 5.4-5.5 (m, 2H), 5.65 (m, 1H), 5.8 (m, 1H).
Preparation 40
2-Bromo-1-[4-(pentafluoro-λ6-sulfanyl)phenyl]ethanone
To a solution of 1-[4-(pentafluoro-λ6-sulfanyl)phenyl]ethanone (Preparation 41, 0.6 g, 2.44 mmol) in THF (20 mL) at 0° C. was added trimethylphenylammonium tribromide (0.962 g, 2.56 mmol). After stirring for 2 hours at 0° C. the reaction was quenched with a saturated aqueous solution of sodium hydrogen carbonate. The reaction was extracted with ethyl acetate and dried over MgSO4 before concentrating in vacuo to afford the title compound that was used without further purification.
Preparation 41
1-[4-(Pentafluoro-λ6-sulfanyl)phenyl]ethanone
To a solution of N-methoxy-N-methyl-4-(pentafluoro-λ6-sulfanyl)benzamide (Preparation 42, 3.0 g, 10.3 mmol) in THF (100 mL) at 0° C. was added drop-wise methyl lithium (1.5M solution, 10.3 mL, 15.5 mmol). The reaction was stirred at 0° C. for 2 hours, then quenched with a saturated aqueous solution of ammonium acetate. The reaction was extracted with ethyl acetate and dried over MgSO4 before concentrating in vacuo to afford the title compound that was used without further purification.
1HNMR (CDCl3): δ 2.65 (s, 3H), 7.9 (d, 2H), 8.05 (d, 2H).
Preparation 42
N-Methoxy-N-methyl-4-(pentafluoro-λ6-sulfanyl)benzamide
4-(Pentafluoro-λ6-sulfanyl)benzoyl chloride (1.00 g, 3.751 mmol), O,N-dimethyl-hydroxylamine hydrochloride (0.402 g, 4.13 mmol), and triethylamine (0.835 g, 8.25 mmol) were stirred in dichloromethane for 2 hours at room temperature. The reaction was concentrated in vacuo and diethyl ether added. The solid was collected by filtration and purified by silica gel column chromatography to afford the title compound as a solid (0.557 g, 51% yield).
LCMS (2 min) Rt=1.56 min MS m/z 292 [MH]+
1HNMR (CDCl3): δ 3.4 (s, 3H), 3.55 (s, 3H), 7.8 (m, 4H).
Assay Method
The ability of the imidazole derivatives of the formula (I) to inhibit the NaV1.8 channel may be measured using the assay described below.
HEK cells stably transfected with hNav1.8, purchased from Millipore (Millipore Corp., Billerica, Mass. 01821), were maintained according to manufacturer's instructions. For electrophysiological studies, cells were removed from the culture flask by brief trypsinization and re-plated at low density onto glass cover slips. Cells were typically used for electrophysiological experiments within 24 to 72 h after plating.
Electrophysiological Recording
Cover slips containing HEK cells expressing hNav1.8 were placed in a bath on the stage of an inverted microscope and perfused (approximately 1 mL/min) with extracellular solution of the following composition: 138 mM NaCl, 2 mM CaCl2, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4, with NaOH. Pipettes were filled with an intracellular solution of the following composition: 135 mM CsF, 5 mM CsCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.3 with NaOH, and had a resistance of 1 to 2 megaohms. The osmolarity of the extracellular and intracellular solutions was 300 mOsm/kg and 295 mOsm/kg, respectively. All recordings were made at room temperature (22-24° C.) using AXOPATCH 200B amplifiers and PCLAMP software (Axon Instruments, Burlingame, Calif.).
hNav1.8 currents in HEK cells were measured using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Uncompensated series resistance was typically 2 to 5 mega ohms and >85% series resistance compensation was routinely achieved. As a result, voltage errors were negligible and no correction was applied. Current records were acquired at 20 to 50 KHz and filtered at 5 to 10 KHz.
HEK cells stably transfected with hNav1.8 were viewed under Hoffman contrast optics and placed in front of an array of flow pipes emitting either control or compound-containing extracellular solutions. All compounds were dissolved in dimethyl sulfoxide to make 10 mM stock solutions, which were then diluted into extracellular solution to attain the final concentrations desired. The final concentration of dimethyl sulfoxide (≦0.3% dimethyl sulfoxide) was found to have no significant effect on hNav1.8 sodium currents.
The voltage-dependence of inactivation was determined by applying a series of depolarizing prepulses (8 sec long in 10 mV increments) from a negative holding potential. The voltage was then immediately stepped to 0 mV to assess the magnitude of the sodium current. Currents elicited at 0 mV were plotted as a function of prepulse potential to allow estimation of the voltage at which 50% of the channels were inactivated (midpoint of inactivation or V1/2). Compounds were tested for their ability to inhibit hNav1.8 sodium channels by activating the channel with a 20 msec voltage step to 0 mV following an 8 second conditioning prepulse to the empirically determined V1/2. Compound effect (% inhibition) was determined by difference in current amplitude before and after application of test compounds. For ease of comparison, “estimated IC-50” values were calculated from single point electrophysiology data by the following equation, (tested concentration, uM)×(100−% inhibition/% inhibition). Inhibition values <20% and >80% were excluded from the calculation.
In some cases electrophysiological assays were conducted with PatchXpress 7000 hardware and associated software (Molecular Devices Corp). All assay buffers and solutions were identical to those used in conventional whole-cell voltage clamp experiments described above. hNav1.8 cells were grown as above to 50%-80% confluency and harvested by trypsinization. Trypsinized cells were washed and resuspended in extracellular buffer at a concentration of 1×106 cells/mL. The onboard liquid handling facility of the PatchXpress was used for dispensing cells and application of test compounds. Determination of the voltage midpoint of inactivation was as described for conventional whole-cell recordings. Cells were then voltage-clamped to the empirically determined V1/2 and current was activated by a 20 msec voltage step to 0 mV.
Estimated IC50 values for the compounds of formula I exemplified above are as follows.
Example No.
NaV1.8 EIC50 (μM)
1
0.19
2
0.36
3
0.26
4
0.36
5
0.48
6
0.92
7
0.40
8
0.057
9
0.026
10
0.0033
11
0.009
12
0.0097
13
0.0078
14
0.051
15
0.011
16
0.032
17
0.075
Where replicate experiments were conducted resulting in multiple sets of data for a test compound, the data presented represent the average value from all replicate experiments.
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14351893
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pfizer limited
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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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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:pfe
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Pfizer
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Sep 14th, 2021 12:00AM
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Nov 7th, 2018 12:00AM
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https://www.uspto.gov?id=US11116917-20210914
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Dispensing device
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A device for dispensing individual doses of powder from respective pockets of a disc-shaped carrier by outwardly rupturing a lidding foil by means of pressure on an opposite side surface, the device providing individual respective deaggregation flow paths for each pocket, split airstreams allowing improved entrainment of powder, a cam mechanism for outwardly rupturing the pockets, an indexing mechanism linked to the cam mechanism and a dose counter.
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11116917
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1. A device for dispensing powdered medicaments, comprising at least one carrier bearing a plurality of respective pockets, each pocket holding a dose of a powdered medicament; a mouthpiece through which to inhale an airstream carrying the dose of the powdered medicament from an opened pocket of the plurality of respective pockets; an indexing mechanism for indexing the at least one carrier between the respective pockets, from a storage position to a discharge position; a first counter ring having a first display surface displaying numbers on the first display surface, the first counter ring being rotatable about a counter axis; and a second counter ring having a second display surface indicating tens counts, the second counter ring being rotatable about the counter axis; the first counter ring being driven with the indexing mechanism, the second counter ring being positioned within the first counter ring, wherein the first display surface and the second display surface are planar and perpendicular to the counter axis.
2. The device according to claim 1, wherein the at least one carrier comprises a first carrier and a second carrier, and the indexing mechanism is configured to index each of the first carrier and the second carrier between respective pockets thereof.
3. The device according to claim 2, wherein the first carrier and the second carrier are indexed such that respective doses of powdered medicament are dispensed from each of the first and second carriers simultaneously.
4. The device according to claim 3, wherein, when the device is indexed, one pocket from the first carrier and one pocket from the second carrier are opened together.
5. The device according to claim 2, wherein the first carrier and the second carrier are indexed such that respective doses of powered medicament are dispensed from each of the first and second carriers sequentially, one at a time in an alternating fashion.
6. The device according to claim 2, wherein the first carrier and the second carrier are arranged side by side; the device comprises a housing and a priming lever extending out of the housing; the priming lever is configured to rotate about a central axis of the device; and the priming lever extends from between the first carrier and the second carrier.
7. The device according to claim 2, wherein the plurality of respective pockets of the first carrier carries a different powered medicament than the plurality of respective pockets of the second carrier.
8. The device according to claim 7, wherein the powdered medicament carried by the first carrier and the powdered medicament carried by the second carrier comprise two different medicaments that are more effective for treating asthma and/or chronic obstructive pulmonary disease together than singularly.
9. The device according to claim 1 wherein, when the device is indexed, only one of the plurality of respective pockets of the at least one carrier is opened each time the device is indexed.
10. The device according to claim 1 wherein, when the device is indexed, two of the plurality of respective pockets of the at least one carrier are opened together.
11. The device according to claim 1, wherein the device comprises a housing and a mouthpiece cover, and the mouthpiece cover is formed as a separate component from the housing, the mouthpiece cover being configured to be rotated relative to the housing to expose the mouthpiece.
12. The device according to claim 1, wherein the device comprises a housing and a priming lever extending out of the housing, and the priming lever is configured to rotate about a central axis of the device.
13. The device according to claim 12, wherein the priming lever has a first position and a second position such that a movement from the first position to the second position is arranged to prime the device, and priming the device comprises exposing at least one dose of powdered medicament carried by the at least one carrier.
14. The device according to claim 13, wherein the priming lever is adjacent to the mouthpiece at the first position.
15. The device according to claim 12, wherein a movement of the priming lever operates the indexing mechanism to index the device such that at least one unused and unopened pocket of powdered medicament is moved into a position for dispensing and is opened.
16. The device according to claim 15, wherein the device comprises a mouthpiece cover, wherein rotation of the mouthpiece cover from an open position to a closed position returns the priming lever to the first position and covers the mouthpiece.
17. The device according to claim 15, wherein when a pocket is opened but the dose of powdered medicament held therein is not inhaled, a further movement of the priming lever indexes the open pocket to a position where the powder released therefrom is held within the device.
18. The device according to claim 15, wherein each of the plurality of respective pockets is sealed by a foil, and a rupturing of the foil during indexing of the device imparts a resistance on the priming lever and thus provides tactile feedback to the user.
19. The device according to claim 18, wherein when the priming lever is rotated, the force required to continue rotating the priming lever is increased due to resistance of the foil until such time as the foil is ruptured, whereupon the force required to rotate the priming lever is reduced.
20. The device according to claim 1, wherein the at least one carrier comprises at least one disc, the at least one disc bearing a plurality of respective pockets.
21. The device according to claim 1, wherein the at least one carrier comprises at least one blister-pack array, the at least one blister-pack array bearing a plurality of respective pockets.
22. The device according to claim 1, wherein each of the plurality of respective pockets is sealed by a lidding sheet.
23. The device according to claim 22, wherein each respective dose of the powdered medicament is exposed by a peeling of the lidding sheet.
24. The device according to claim 22, wherein the lidding sheet is ruptured as a consequence of moving a pocket from a respective storage position to a respective discharge position.
25. The device according to claim 1, wherein the powdered medicament comprises at least one of a long acting beta-agonist, a steroid, or any combination thereof.
26. The device according to claim 1, wherein the powdered medicament comprises at least one of formoterol, salmeterol, fluticasone, budesonide, monetasone, or any combination thereof.
27. The device according to claim 1, further comprising an intermittent-motion mechanism for driving the second counter ring from the first counter ring.
28. The device according to claim 27, wherein the second counter ring rotates between consecutive tens counts when the first counter ring rotates between two predetermined consecutive unit counts.
29. The device according to claim 27, wherein the intermittent-motion mechanism is a Geneva mechanism.
30. The device according to claim 27, further comprising a counter gear engaged with a first gear of the first counter ring, wherein the intermittent-motion mechanism comprises a second gear, and wherein rotation of the counter gear is configured to rotate the first counter ring, with the first counter ring configured to rotate the second counter ring.
31. The device according to claim 30, wherein the first counter ring comprises a peg configured to rotate the second gear, and wherein the peg is positioned on an inner diameter of the first counter ring.
32. The device according to claim 1, wherein the first counter ring includes a pin for engaging a Geneva wheel rotatable about an axis offset from the counter axis and the second counter ring includes features engageable by the Geneva wheel.
33. The device according to claim 1, wherein the first counter ring and the second counter ring rotate about an axis parallel to the axis of the dose carriers.
34. The device according to claim 1, further comprising walls defining at least one first flow path for deaggregating the powdered medicament from an open pocket in the discharge position and at least a second flow path which bypasses the open pocket.
35. The device according to claim 34, wherein the walls defining the first flow path are configured to force the airstream to change direction such that the dose of powder in the airstream is deaggregated.
36. The device according to claim 35, wherein the walls defining the first flow path have rounded comers configured to limit a pressure drop such that the pressure drop does not exceed 4 kPa at 60 I/min.
37. The device according to claim 34, wherein each of the at least one first flow path comprises a section of relatively reduced cross-sectional area oriented so as to be directed towards an open pocket in the discharge position and configured to direct a relatively high velocity airstream into the pocket.
38. The device according to claim 37, wherein the section of relatively reduced cross-sectional area of each of the at least one first flow path is between 50% and 60% of the cross-sectional area of the smallest part of the second flow path.
39. The device according to claim 37, wherein the section of relatively reduced cross-sectional area of each of the at least one first flow path is between 2.0 mm2 and 10.0 mm2.
40. The device according to claim 1, wherein the second display surface comprises an indicia indicating that the device is nearing the end of its functional life, and the indicia is displayed when a predetermined number of doses remain within the device.
41. The device according to claim 40, wherein the indicia comprises a symbol, a color, a light, or any combination thereof.
42. The device according to claim 40, wherein the indicia is not a number “0”.
43. The device according to claim 1, wherein the indexing mechanism comprises a wheel and a plurality of gears, and wherein, upon rotation of the wheel and the plurality of gears, the at least one carrier is indexed between the respective pockets.
44. The device according to claim 43, further comprising a counter gear engaged with a first gear of the first counter ring, wherein, upon rotation of a first gear of the plurality of gears of the indexing mechanism, the counter gear rotates to drive the first counter ring.
45. The device according to claim 43, wherein the indexing mechanism comprises a ratchet.
46. A device for dispensing powdered medicaments, comprising
a first carrier bearing a plurality of respective pockets and a second carrier bearing a plurality of respective pockets, each pocket of the first and second carriers holding a dose of a powdered medicament;
a mouthpiece through which to inhale an airstream carrying the dose of the powdered medicament from an opened pocket of the plurality of respective pockets;
an indexing mechanism for indexing the at least one carrier between the respective pockets, from a storage position to a discharge position, the indexing mechanism being configured to index each of the first carrier and the second carrier between respective pockets thereof;
a first counter ring having a first display surface displaying numbers on the first display surface, the first counter ring being rotatable about a counter axis;
a second counter ring having a second display surface indicating tens counts, the second counter ring being rotatable about the counter axis; the first counter ring being driven with the indexing mechanism, the second counter ring being positioned within the first counter ring, the first display surface and the second display surface being planar and perpendicular to the counter axis;
a housing enclosing the at least one carrier; and
a priming lever extending out of the housing, the priming lever being configured to rotate about an axis of the device, the priming lever having a first position and a second position such that a movement from the first position to the second position is arranged to prime the device,
wherein priming the device comprises exposing at least one dose of powdered medicament carried by the at least one carrier; and a movement of the priming lever operates the indexing mechanism to index the device such that two unused and unopened pockets of powdered medicament, one from the first carrier and one from the second carrier, are moved into a position for dispensing and are opened.
47. The device according to claim 46, wherein the indexing mechanism comprises a wheel and a plurality of gears, and wherein, upon rotation of the wheel and the plurality of gears, the first carrier and the second carrier are indexed between the respective pockets.
48. The device according to claim 47, further comprising a counter gear engaged with a first gear of the first counter ring, wherein, upon rotation of a first gear of the plurality of gears of the indexing mechanism, the counter gear rotates to drive the first counter ring.
49. The device according to claim 47, wherein the indexing mechanism comprises a ratchet.
50. A device for dispensing powdered medicaments, comprising
at least one carrier bearing a plurality of respective pockets, each pocket holding a dose of a powdered medicament;
a mouthpiece through which to inhale an airstream carrying the dose of the powdered medicament from an opened pocket of the plurality of respective pockets;
an indexing mechanism for indexing the at least one carrier between the respective pockets, from a storage position to a discharge position;
a first counter ring having a first display surface displaying numbers on the first display surface, the first counter ring being rotatable about a counter axis; and
a second counter ring having a second display surface indicating tens counts, the second counter ring being rotatable about the counter axis; the first counter ring being driven with the indexing mechanism, the second counter ring being positioned within the first counter ring, the first display surface and the second display surface being planar and perpendicular to the counter axis;
a housing enclosing the at least one carrier;
a mouthpiece cover formed as a separate component from the housing, the mouthpiece cover being configured to be rotated relative to the housing to expose the mouthpiece; and
a priming lever extending out of the housing, the priming lever being configured to rotate about an axis of the device, the priming lever having a first position and a second position such that a movement from the first position to the second position is arranged to prime the device, wherein priming the device comprises exposing at least one dose of powdered medicament carried by the at least one carrier; and a movement of the priming lever operates the indexing mechanism to index the device such that at least one unused and unopened pocket of powdered medicament is moved into a position for dispensing and is opened.
51. The device according to claim 50, wherein the indexing mechanism comprises a wheel and a plurality of gears, and wherein, upon rotation of the wheel and the plurality of gears, the at least one carrier is indexed between the respective pockets.
52. The device according to claim 51, further comprising a counter gear engaged with a first gear of the first counter ring, wherein, upon rotation of a first gear of the plurality of gears of the indexing mechanism, the counter gear rotates to drive the first counter ring.
53. The device according to claim 51, wherein the indexing mechanism comprises a ratchet.
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53
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This application is a continuation of application Ser. No. 14/461,891, filed Aug. 18, 2014 which is a continuation of application Ser. No. 13/560,314, filed Jul. 27, 2012, now U.S. Pat. No. 8,851,070, issued Oct. 7, 2014, which is a continuation of application Ser. No. 12/795,206, filed Jun. 7, 2010, now U.S. Pat. No. 8,256,416, issued Sep. 4, 2012, which is a continuation of application Ser. No. 10/565,064, filed Jul. 7, 2006, now U.S. Pat. No. 8,181,645, issued May 22, 2012, which claims the benefit of International Patent Application No. PCT/GB2004/002748, filed Jun. 25, 2004, which claims the benefit of Great Britain Patent Application No. 0315509.0, filed Jul. 2, 2003, all of which are incorporated by reference herein in their entireties.
TECHNICAL FIELD
The present invention relates to a dispensing device, in particular for dispensing individual doses of powder from respective pockets of a carrier.
A wide variety of devices are known for dispensing doses of medicament in the form of powder for inhalation. Devices are known which contain a store of powdered medicament from which individual doses are metered as required. Devices are also known which include carriers having a plurality of pockets containing respective doses of powder. These carriers are typically in the form of blister-packs. All of these devices face problems of providing reliable, repeatable and accurate inhaled amounts of powder.
There are problems in ensuring that all of a dispensed dose of powder is entrained into the airstream for inhalation. Furthermore, some of the powder which is originally provided for inhalation may adhere to surfaces within the device. This will reduce the inhaled dose. However, more importantly, after a number of uses, previously adhered powder may become dislodged, thereby resulting in an unwanted and undesirable increase in the inhaled dose. There are other problems in providing repeatable and consistent release of powder into the inhalation airstream as desired.
In attempting to reduce these problems, previous devices suffer problems of increased size, complexity and/or cost.
U.S. Pat. No. 4,811,731 describes an inhaler having a support for a blister pack having an annular array of blisters. The support faces a tray having upstanding walls defining a flow path to a mouthpiece. In use, the support and blister pack is consecutively indexed such that powder from respective blisters is dispensed via the flow path defined by the tray and upstanding walls. Because the same tray and upstanding walls are used for all of the blisters, there is the problem that powder can become adhered to the tray and upstanding walls and then dislodged subsequently.
It is an object of the present invention to overcome or at least reduce these problems.
SUMMARY
According to the present invention, there is provided a device for dispensing individual doses of powder from respective pockets of a carrier, the device including a support for a carrier having a plurality of pockets containing respective doses of powder and a mouthpiece through which to inhale an airstream carrying a dose of powder, the device further including walls for defining individual respective first flow paths downstream of each respective pocket of a supported carrier wherein each individual respective first flow path is defined entirely by respective walls unique to that individual respective first flow path, is for connecting the corresponding respective pocket to the mouthpiece and is for deaggregating powder in the airstream.
In this way, each pocket of powder is provided with its own first flow path such that any powder which does adhere to the walls of that first flow path will not affect subsequent dispensing inhalations through the device. There are no walls in common between respective first flow paths such that powder adhering to walls of a first flow path will not affect subsequent doses. In particular, subsequent inhalations will draw airstreams through the first flow paths of the respective pockets being dispensed such that, even if previously adhered powder is dislodged in the first flow paths of previously dispensed pockets, this powder will not be part of the inhalation airstream and, hence, will not be inhaled by the user.
According to the present invention, there is also provided a device for dispensing individual doses of powder from respective pockets of a carrier, the device including: a support for a carrier having a plurality of pockets containing respective doses of powder, and a mouthpiece through which to inhale an airstream carrying a dose of powder, the device further including: walls for defining individual respective first flow paths downstream of each respective pocket of a supported carrier for connecting the corresponding respective pockets to the mouthpiece and deaggregating powder in the airstream; an arrangement for moving individually each pocket from a respective storage position to a respective discharge position, wherein each pocket, in the respective discharge position, forms an integral part of the individual respective first flow path.
Preferably, the device is for use with a carrier having pockets provided with a lidding sheet, the device allowing the lidding sheet to be ruptured as a consequence of moving a pocket from a respective storage position to a respective discharge position.
Preferably, the device further includes walls defining a second flow path connecting with the mouthpiece and bypassing the pockets.
This allows an increase in flow volume through the device and a reduction in the resistance to flow, such that a user may more easily inhale through the device. This is most important where the size of the pockets is not sufficient to allow the flow volume to reach that necessary to carry medicament into the lung. Furthermore, it also becomes possible to entrain powder in the airstream over a small, but sustained, period of time, rather than substantially all at once. The powder may be entrained during a mid-portion of inhalation, thereby improving the transfer of powder to the user. The device should preferably provide similar performance over flow rates ranging from 28.31/min to 601/min and should preferably have a pressure drop not exceeding 4 kPa at 601/min.
Preferably, with the device configured to dispense a dose of powder from one of the pockets of the supported carrier, the respective flow path connects with the second flow path downstream of the bypass and at an angle such that substantially no powder impacts with the walls defining the second flow path. Some impact could be allowed, but then preferably substantially no deposition occurs. Some powder could be allowed to be deposited on the walls defining the second flow path, but then preferably, with repeated use of the device and the second flow path, no more than 25% or preferably no more than 15% of a dose remains deposited on the walls defining the second flow path. It will be appreciated that the airstream through the second flow path also acts to scour or scavenge powder deposited on the walls defining the second flow path.
In other words, it is thus possible for a part of the second flow path to be used consecutively for all of the pockets of the carrier. However, since the respective first flow paths provide the required deaggregation, the second flow path can be arranged to provide a minimum amount of turbulence and to avoid substantially any powder adhering to its walls. By providing an appropriate angle at which the first flow paths meet the second flow path, powder can substantially be prevented from impacting the walls of the second flow path when it joins the second flow path from the respective first flow path.
Preferably, where the respective first flow path connects with the second flow path, the angle is less than 45 degrees, more preferably less than 30 degrees.
This ensures that substantially no powder adheres to the walls defining the second flow path.
Preferably, the support for the carrier and the walls defining the first flow path are moveable with a supported carrier so as to selectively connect respective first flow paths with the second flow path and, hence, selectively dispense doses of powder from respective pockets of the supported carrier.
In this way, the device can be provided with a single mouthpiece and dispensing mechanism so as to minimise cost and complexity and yet still provide each pocket of the carrier with its own respective first flow path in which deaggregation and any adherence of powder occurs.
In a preferred embodiment, the carriers are disc-shaped with a circumferential array of pockets. In this embodiment, the pockets and their respective first flow paths are indexed by rotation relative to the second flow path and mouthpiece so as to dispense consecutively doses of powder for inhalation through the mouthpiece.
Preferably, the walls defining the first flow paths include, upstream of the pockets, respective portions of relatively reduced cross-sectional area orientated so as to be directed towards respective pockets and direct a relatively high velocity airstream into the respective pockets.
Indeed, according to the present invention, there is also provided a device for dispensing a dose of powder from a pocket of a carrier, the device including a support for a carrier having a pocket containing a dose of powder and a mouthpiece through which to inhale an airstream carrying a dose of powder, the device further including walls defining first and second flow paths connecting with the mouthpiece, the first flow path connecting the pocket of the supported carrier to the mouthpiece and the second flow path bypassing the pocket, wherein the walls defining the first flow path include, upstream of the pocket, a portion of relatively reduced cross-sectional area orientated so as to be directed towards the pocket and direct a relatively high velocity airstream into the pocket.
In this way, the high velocity airstream can erode the powder in the pocket so as to progressively entrain it into the airstream, rather than merely attempt to flush the powder from the pocket. This results in the powder being entrained into the airstream over a sustained period of time. The time is preferably within the range of 0.01 s to 1.0 s and more preferably in the range of 0.2 s to 0.5 s. This provides improved inhalation characteristics.
Furthermore, by virtue of the second flow path in conjunction with the first flow path, resistance to airflow can be reduced and volume of airflow increased. The portions of relatively reduced cross-sectional area produce a small high velocity stream suitable for eroding the powder. By providing these in conjunction with the second flow path, the user is still able to inhale relatively easily through the device, despite the restriction of the respective reduced cross-sectional area portions.
As will be appreciated, this arrangement has similar advantages when used with a carrier having only a single pocket and, hence, only a single first flow path.
Preferably, each portion has a cross-sectional area between 50% and 66% of the cross-sectional area of the smallest part of the second flow path. Indeed, preferably, the cross-sectional areas of the non-reduced cross-section parts of each flow path are provided between 110% and 150% of the minimum values in their own path order to maintain the high air velocities required to keep the powder entrained without contributing significantly to pressure drop.
This allows a suitably high velocity airstream to be directed into a pocket without unduly increasing the overall resistance to inhalation and allowing a sufficiently high overall volume of airflow.
In the preferred embodiment, there is another second flow path for the other side of the device and its corresponding carrier. In use, a patient inhales through both second flow paths whilst drawing powder from the first flow path in use. Each of the second flow paths is expected to carry approximately 40%> of the total inhaled air for an average use.
Actual requirements will vary depending upon the nature of the powder and the intended user. For an easily dispensed powder, the portion forming the inlet to the pocket can be small and, for a child or patient with COPD (Chronic Obstructive Pulmonary Disease), the total pressure drop should be low. In this case, an inlet portion could be provided with a cross-sectional area of 2 mm2 and a bypass second flow path with a minimum cross-sectional area of 8 mm2, resulting in a ratio of 25%. On the other hand, with sticky powder for a healthy adult, the inlet portion could be provided with a cross-sectional area of 4 mm2 together with a bypass second flow path having a minimum cross-sectional area of 6 mm2, resulting in a ratio of 66%. Of course, intermediate values are also possible and a preferred arrangement has an inlet portion of approximately 3 mm2 with a second flow path minimum cross-sectional area of 6 mm2, resulting in a ratio of 50%.
Referring to FIG. 21(a) of the accompanying drawings, it will be noted that it is important for the cross sectional areas A1, A2 and A3 to be between 120% and 200% of the smallest cross sectional area B for the bypass flow. Similarly the cross sectional areas C1, C2 and C3 should be between 120% and 200%) of the smallest cross-sectional area of the portions D for the flow path through a pocket. The combined flow path cross sectional area E should then be greater than A3 plus C3 such that air velocity in the pocket is not reduced.
For this arrangement, the pressures at A3, C3, and E can all be the same and equal to or less than that in the mouth of the patient. The whole pressure drop due to inhalation then occurs across both B and D. For cohesive formulations, it is advantageous to have the maximum air velocity through the pocket portion. This may be achieved by minimizing the pressure at C3 during inhalation. If the mouthpiece is shaped to cause the air to expand with laminar flow by the use of a small divergence angle, typically less than 10 degrees, then it is possible to cause the pressure at C3 to be below the pressure in the mouth, thus increasing the air velocity through the pocket portion.
The ratio of B to D sets the ratio of air flowing through the bypass and the pocket.
The sum of the areas B and D sets the overall flow resistance and is preferably set to give 3 kPa to 4 kPa at 60 J/m.
For the preferred embodiment, storing individual doses of powder of approximately 20 mg in pockets having volumes of approximately 30 mm3, each portion preferably has a cross-sectional area of between 2.0 mm2 and 10.0 mm2, more preferably between 2.0 mm2 and 5.0 mm2.
The reduced cross-section is selected to be between 50%> and 90% of the area which, for the normal range of inhalation rates and volumes, provides a suitable high velocity airstream into the pocket.
FIG. 21(b) of the accompanying drawings illustrates the preferred cross sections for a particular embodiment. In particular, the minimum cross sectional area B for the bypass flow is approximately 5.0 mm2, the minimum cross sectional area D for the pocket flow is approximately 3.8 mm2 and the combined flow path cross sectional area E is approximately 12.0 mm2.
According to the present invention, there is also provided a device for dispensing individual doses of powder from respective pockets of a pair of carriers, the device including a support for two disc-shaped carriers, each disc-shaped carrier having at least one substantially planar first side surface having an annular array of cavities in which respective pockets are formed and a respective first lidding sheet sealed to the first side surface for enclosing the cavities wherein the support is for rotatably supporting the carriers about a substantially common axis, a mouthpiece through which to inhale an airstream carrying powder from the carriers, a dispensing mechanism for releasing into the airstream the powder of a respective pocket of a supported carrier and an indexing mechanism for rotating the carrier relative to the dispensing mechanism so as to enable powder to be released from different pockets.
This arrangement provides an extremely compact and efficient way of holding and dispensing a plurality of pockets of powder. A single dispensing mechanism can be provided for both carriers and the carriers may easily be moved so as to selectively bring each of their pockets in line with the dispensing mechanism and an airstream for carrying powder to the mouthpiece.
The arrangement can be used in conjunction with the features described above and is particularly effective in this regard.
Preferably, between consecutive dispensing of powder from one of said carriers, the indexing mechanism is operable to rotate both of said carriers relative to the dispensing mechanism.
In other words, where powder is dispensed from a first disc, powder will also be dispensed from the second disc before more powder is dispensed from the first disc. Hence, similarly, preferably, between consecutive dispensing of powder from the other of said carriers, the indexing mechanism is operable to rotate both of said carriers relative to the dispensing mechanism.
Preferably, the dispensing mechanism is operable to release powder from a pocket of each carrier for a single inhalation of both respective powders simultaneously.
In other words, powder is dispensed from the pocket of a first disc and also powder is dispensed from a pocket of a second disc. The user may then inhale the powder dispensed from both discs simultaneously. This allows two different medicaments to be administered simultaneously without the medicaments coming into contact until immediately before or indeed during the inhalation process.
Preferably, the mechanism is operable to dispense medicament from a pocket of each disc simultaneously.
Hence, having rotated the discs to an appropriate position with an unopened pocket of each disc available, the mechanism then opens both pockets together in one operation.
Alternatively, the dispensing mechanism may be operable to release powder from a pocket of one of the carriers for inhalation then to release powder from a pocket of the other of the carriers for inhalation.
In this way, a user may administer two different pharmaceuticals immediately one after the other or may use the inhalation device as part of a course of treatment whereby different medicaments are administered alternately after predetermined periods of time. By way of example, a steroid compound could be dispensed from one disk and a long acting beta-agonist from the other disk for the treatment of e.g. asthma or chronic obstructive pulmonary disease: Examples of long acting beta-agonists include formoterol and salmeterol and examples of steroids include fluticasone propionate, budesonide and monetasone furoate.
Indeed, the inhalation device could include three or more discs such that more complicated courses of pharmaceuticals could be administered. Indeed, the mechanism could be arranged so as to dispense a predetermined number of doses from one disc before administering a dose from the other disc.
In certain embodiments, the mechanism may be operable to release powder from a pocket of one carrier and from a pocket of the other carrier consecutively.
Such a system could be used when the powder of the two pockets is inhaled together or consecutively.
A device may be provided with two of said disc shaped carriers respectively containing powder of different medicaments.
This allows, as mentioned above, different medicaments to be dispensed from the same device.
Preferably, between consecutive dispensing of powder, the indexing mechanism is operable to rotate one of said carriers in turn between consecutive dispensing positions before rotating the other of said carriers.
In other words, the indexing mechanism is arranged to move one of the carriers between consecutive pockets whilst the other carrier remains where it is.
The dispensing mechanism and the indexing mechanism may together be operable to dispense powder from all of the pockets from one of the said carriers before dispensing powder from pockets of the other of said carriers.
This allows the use of an indexing mechanism which always moves one or other of the carriers onto its next position.
According to the present invention, there is also provided a device for dispensing individual doses of powder from respective pockets of carrier, the device including a first support for a first carrier having first and second side surfaces opposite each other, an array of cavities in which respective pockets are formed and a first lidding sheet sealed to the first side surface, a first prodger member moveable towards and away from the second side surface of a supported first carrier between a retracted and an extended position and a cam member adjacent to and moveable generally parallel with the second side surface of a supported carrier between a rest position and a primed position, wherein the cam member has a first cam surface for engaging with the first prodger member such that movement of the cam member from the rest position to the primed position moves the prodger member from the retracted position to the extended position so as to press upon the second side surface of a supported first carrier and outwardly rupture the first lidding sheet of the supported first carrier.
In this way, a compact and effective mechanism is provided for opening individual pockets of a carrier. The cam member may be provided with a relatively large amount of movement, but, since this is generally parallel to the plane of the carrier, this need not take up excessive space. At the same time, converting this large amount of movement to only the small amount of movement required for the prodger member, the user is given a large mechanical advantage such that dispensing of the powder from a pocket is relatively easy and well controlled.
Preferably, the device further includes a second support for a second carrier having first and second side surfaces opposite each other, an array of cavities in which respective pockets are formed and a first lidding sheet sealed to the first side surface, the first and second carriers being supported with respective second side surfaces facing each other, a second prodger member moveable towards and away from the second side surface of a supported second carrier between a retracted and an extended position, wherein the cam member has a second cam surface for engaging with the second prodger member such that movement of the cam member from the rest position to the primed position moves the prodger member from the retracted position to the extended position so as to press upon the second side surface of a supported second carrier and outwardly rupture the first lidding sheet of the supported second carrier.
In this way, the same advantages are achieved for a second carrier. Furthermore, these advantages are achieved using only a single cam member for dispensing from both of the two carriers. Hence, the device is very efficient in its use of space.
The device may be arranged as described above so as to achieve the same advantages. Thus, preferably, the device further includes an indexing mechanism for moving the first and second supports relative to the first and second prodger members so as to selectively align pockets of the carrier with respective prodger members.
In this way, the carriers are efficiently moved and located with respect to the dispensing mechanism and the mouthpiece.
Preferably, the indexing mechanism is arranged such that, with one of the first and second prodger members aligned with a respective pocket, the other of the first and second prodger members is aligned between respective pockets, whereby movement of the cam member from the rest position to the primed position causes only one of the first and second prodger members to outwardly rupture the first lidding sheet of the corresponding one of the first and second carriers.
In this way, although the cam member is moved in the same way for each use, the indexing mechanism positions the carriers such that the pocket of one carrier is dispensed for a particular use of the cam member. Nevertheless, the same cam member is still able to open pockets from either of the carriers. Again, this is a highly efficient use of the mechanism and also of space within the device.
Preferably, the cam member is moveable in a direction towards and away from the second side surfaces of the supported first and second carriers such that, when the other of the first and second prodger members is aligned between respective pockets, movement of the cam member from the rest position to the primed position and the resulting engagement of the other of the first and second prodger members with the corresponding cam surface causes the other of the first and second prodger members to abut the corresponding second side surface and the cam member to be moved towards the corresponding one of the first and second carriers.
Thus, for each use of the cam member, each cam surface pushes against a corresponding cam member. However, since one-prodger member will abut a second side surface between pockets and, therefore, will not itself move, the corresponding cam surface would actually cause the cam member to move away from that second side surface.
In this way, in effect, both cam surfaces contribute to movement of a prodger member to open a pocket, such that each cam surface need have only a relatively small slope.
Preferably the cam member is provided on a priming member moveable as part of the indexing mechanism.
In this way, it is not necessary for a user to operate two separate actuators. Actuating the device to move the cam member in one direction will prime the device so as to dispense a dose of powder for inhalation and then the movement of the cam member back to its rest position will index at least one of the carriers ready for another pocket of powder to be dispensed.
Preferably, the indexing mechanism is arranged such that, after the first and second carriers have been indexed past all of their respective pockets, the first and second prodger members are both aligned between pockets of respective carriers and, hence, provide resistance to movement of the cam member.
This provides a feature of “lock-out” whereby once all of the pockets of the carriers have been used and the device is effectively empty, the user is provided with a physical feedback. In particular, it becomes difficult for the user to move the priming lever of the device, since the cam member is unable to move the prodger members.
Preferably, the cam member includes an elongate flexible member having first and second cam surfaces on opposite respective sides.
In this way, when it is required for the cam member or a portion of it to move from side to side, it is sufficient for only the elongate flexible member to move.
Preferably, the first cam surface and/or the second cam surface includes at least one groove into which any stray powder from previously dispensed pockets may move.
In this way, stray powder will not interfere with the interface between the cam surfaces and the prodger members such that operation will not be impeded.
Preferably, the device is for use with carriers having the cavities formed from respective through holes between the first and second side surfaces, having second lidding sheets sealed to the second side surfaces and having respective cup shaped inserts in each cavity orientated with open portions facing the first lidding sheets, wherein the first prodger member and/or second prodger member is arranged to penetrate an aligned through hole through a second lidding sheet so as to push the corresponding second insert outwardly through the first lidding sheet.
This is a particularly effective way of dispensing doses of powder and the mechanism for moving the prodgers is particularly effective in pushing the inserts as required.
Preferably, at least one of the cam surfaces is resiliently deformable, the cam member being dimensioned so as to move the prodger members beyond the extended position such that, once a prodger member reaches its respective extended position, further movement of the cam member causes the at least one of the cam surfaces to resiliently deform.
In this way, the device can itself compensate for variations in tolerances and it is not necessary for the cam member to move the prodger members by exactly the distance required.
Preferably, each support includes a peripheral array of gear teeth and the indexing mechanism is engageable with the gear teeth so as selectively to move the supports and carriers.
This provides an advantageous way of moving and controlling the positions of the carriers.
Preferably, the indexing mechanism includes a priming member mounted for rotation about a central axis and a Geneva wheel rotatably mounted on an axis offset from the central axis for interaction with the priming member and gear teeth of the supports such that rotation of the priming member from a first position to a second position causes rotation of at least one of the supports by a predetermined angle and rotation of the priming member back from the second position to the first position causes no rotation of at least one of the supports.
Indeed, according to the present invention, there is also provided a device for dispensing individual doses of powder from respective pockets of a carrier, the device including a chassis, a first support mounted on the chassis for rotation about a central axis and for supporting a first carrier having cavities with respective pockets formed therein and arranged in a circular array centred on the central axis, the first support including an array of gear teeth centred on the central axis, a priming member mounted on the chassis for rotation about the central axis and an intermittent-motion mechanism mounted on the chassis for interaction with the priming member and gear teeth of the first support such that rotation of the priming member from a first position to a second position causes rotation of the first support by a predetermined angle and rotation of the priming member back from the second position to the first position causes no rotation of the first support.
This allows a user to move the priming member through a relatively large and imprecise range of movements whilst ensuring that the support and carrier is moved by a predetermined amount.
Preferably, the intermittent-motion mechanism is a Geneva wheel rotatably mounted on the chassis on an axis offset from the central axis.
The device may further include a second support mounted on the chassis for rotation about the central axis and for supporting a second carrier having cavities with respective pockets formed therein and arranged in a circular array centred on the central axis, the second support including an array of gear teeth centred on the central axis wherein the Geneva wheel may interact with the gear teeth of the second support such that rotation of the priming member from the first position to the second position causes rotation of the second support by a predetermined angle and rotation of the priming member back from the second position to the first position causes no rotation of the second support.
In this way, the priming member may be used to rotate both the first and second supports and their associated carriers.
Preferably, the respective arrays of gear teeth of the first and second supports are incomplete circular arrays leaving respective spaces such that, with a space positioned between the Geneva wheel, rotation of the priming member will not rotate the respective supports.
In this way, it is possible for the priming member to rotate selectively one or other of the supports.
The indexing mechanism may be actuated by a lever pivoted about the disc axis being moved through an angle between 30° and 180° and preferably the indexing movement remains constant provided that the lever moves through a minimum angle.
The mechanism preferably locates to a radial accuracy sufficient to ensure that the prodger member accurately locates on the pocket. Preferably, the force required to index the motion is between 1 N and 20 N.
Preferably, the indexing mechanism holds the carriers in place so that they will not move when subjected to shocks such as experienced when carried in the pocket or dropped onto a hard surface. It can be designed to index precisely between whatever number of pockets are on a disc.
The indexing mechanism preferably causes the selected disc carrier to increment through a fixed angle to a deferred location so that the airway of the pocket that will be opened next is aligned to the airway leading to the mouthpiece.
It is preferable that the priming lever used for indexing is not rigidly linked to the position of the disc, as this would cause any small movement of the lever to disturb the alignment of the airways. Hence it is preferable that the correct motion of the disc occurs as the lever moves through the central part of its travel and that its start and end positions are not critical to accurate operation.
Although various mechanisms could be used to achieve this type of motion, the preferred approach is to use a Geneva mechanism to allow the lost motion aspect of the indexing. A combination of gears with the Geneva mechanism can ensure that for every operation of the priming lever the carrier disc indexes a predetermined angle. For example, a carrier disc that has 31 positions would require an indexing angle of 11.61 degrees.
Preferably, the device further includes a changeover component located between the first and second supports, the first support having a first feature engaging with the changeover component and the second support having a second feature for engaging with the changeover component, wherein with the space of the second support adjacent the Geneva wheel, consecutive rotations of the priming member cause only the first support to rotate until the first feature engages the changeover component and then to move the changeover component so as to engage with the second feature and rotate the second support to a position with the space of the second support not adjacent the Geneva wheel, the space of the first support then being adjacent the Geneva wheel and consecutive rotations of the priming member causing only the second support to rotate.
In this way, it is possible to continuously operate the priming member and yet achieve automatic changeover between indexing of the first support and then the second support.
Preferably, the changeover component is arranged such that, when the priming member rotates the second support back around to the position with the space of the second support adjacent the Geneva wheel, the second feature does not engage with the changeover component and consecutive rotations of the priming member cause no rotation of either support.
In this way, the device is automatically prevented from indexing to previously used pockets of the carriers.
At this point in the operation, as described above, the first and second prodger members are preferably both aligned between pockets of respective carriers and, hence, provide resistance to movement of the cam member. This provides the feature of “lock-out”.
According to the present invention, there is also provided a device for dispensing individual doses of powder from respective pockets of a carrier, the device including first and second supports rotatable about a central axis and for supporting respective first and second carriers having cavities with respective pockets formed therein and arranged in respective first and second circular arrays centred on the central axis, a changeover component located between the first and second supports, the first support having a first feature for engaging with the changeover component and a second component having a second feature for engaging with the changeover component and an indexing mechanism arranged to rotate each of the first and second supports, wherein the indexing mechanism is arranged to rotate the first support until the first feature engages the changeover component such that the first support then moves the changeover component, the changeover component being arranged to then engage the second feature so as to rotate the second support to a position from which the indexing mechanism is arranged to rotate the second support.
In this way, a single indexing mechanism may be provided to rotate the first and second supports in sequence with changeover being achieved automatically by means of the changeover component.
Preferably, the changeover component rotates to the second support from a position at which the indexing mechanism does not rotate the second support and, when the first support moves the changeover component, the first support moves to a position which the indexing mechanism does not rotate the first support.
When the second support is rotated back around to the position at which the indexing mechanism does not rotate the second support, consecutive operations of the indexing mechanism preferably cause no rotation of either support.
In this way, the device is automatically prevented from indexing carriers to pockets which have already been used.
The changeover mechanism allows the same indexing mechanism to initially index a first carrier disc and then, at a predetermined location, index both carrier discs together for one increment and then subsequently cause the indexing mechanism to only index the second carrier disc.
The changeover action can be initiated solely by the angular position of the first carrier disc requiring no other input from the user and providing insignificant difference in the tactile feedback.
Preferably, the changeover component is supported freely between and by the first and second components.
Preferably, the device further includes a dose counter having a first counter ring having an indication of unit counts on a first display surface, the first counter ring being rotatable about a counter axis, a second counter ring having an indication of tens counts on a second display surface, the second counter ring being rotatable about the counter axis and a Geneva mechanism for driving the second counter ring from the first counter ring and rotating the second counter ring between consecutive tens counts when the first counter ring rotates between two predetermined unit counts.
In this way, the user is provided with an indication of the doses used or the doses remaining.
By providing two counter rings respectively for units and tens, relatively large display figures may be provided, while still allowing a large number of counts, for instance 40, 60 or 80. The Geneva mechanism provides a particularly effective way of allowing the tens counter ring to be incremented as required.
Preferably, the first counter ring is driven with rotation of the first support.
Hence, the count of unit doses dispensed is incremented/decremented automatically with each indexing of the device. The first counter ring may include gear teeth around its outer periphery and an intermediate gear may be provided to drive it from the indexing mechanism. Where, as described above, the indexing mechanism includes a Geneva wheel, the intermediate gear can be driven directly from the Geneva wheel.
Preferably, the counter axis is coaxial with the first support.
Hence, the first and second counter rings may rotate about the same axis as the carriers and their supports. This allows a particularly compact arrangement.
According to the present invention, there may also be provided a device for dispensing individual doses of powder from respective pockets of a carrier, the device including an indexing mechanism for indexing the carrier between respective pockets, a first counter ring having an indication of unit counts on a first display surface, the first counter ring being rotatable about a counter axis, a second counter ring having an indication of tens counts on a second display surface, the second counter ring being rotatable about the counter axis and an intermittent-motion mechanism for driving the second counter ring from the first counter ring and 5. rotating the second counter ring between consecutive tens counts where the first counter ring rotates between two predetermined consecutive unit counts, the first counter ring being driven with the indexing mechanism.
Preferably, the intermittent-motion mechanism is a Geneva mechanism.
Hence, as described above, this allows a large number of counts to be provided with relatively large display numerals.
Preferably, the first and second counter rings are positioned one within the other, with the first and second display surfaces adjacent each other.
The display surfaces may thus be generally planar (and perpendicular to the counter axis).
Preferably, the second counter ring may be positioned within the first counter ring, the first counter ring may include a pin for engaging a Geneva wheel rotatable about an axis offset from the counter axis and the second counter ring may include features engageable by the Geneva wheel.
In this way, during a complete revolution of the first counter ring, at a predetermined position of that revolution, a pin may engage the Geneva wheel so as to rotate it and, hence, rotate the second counter wheel by one increment. This arrangement allows a particularly compact design.
According to the present invention, there is also provided a device for dispensing individual doses of powder from respective pockets of a carrier, the device including: an indexing mechanism for indexing the carrier between respective pockets; a first counter ring having an indication of unit counts on a first display surface, the first counter ring being rotatable about a counter axis; a second counter ring having an indication of tens counts on a second display surface, the second counter ring being rotatable about the counter axis; and a mechanism for rotating the second counter ring between consecutive tens counts when the first counter ring rotates between two predetermined consecutive unit counts, the first counter ring being driven with the indexing mechanism; wherein the first and second counter rings are positioned one with the other, with the first and second display surfaces adjacent each other.
It will be appreciated that devices according to the present invention can be provided with or without carriers.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be more clearly understood from the following description, given by way of example only, with reference to the accompanying drawings in which:
FIGS. 1(a) to (c) illustrate operation of an assembled device according to the present invention;
FIGS. 2(a) and 2(b) illustrate a carrier for use with the present invention without and with its lidding sheets;
FIGS. 3(a) and (b) illustrate movement of an insert from the carrier of FIGS. 2(a) to (c);
FIGS. 4(a) and (b) illustrate a preferred arrangement for carriers within the device without and with supports of the device;
FIGS. 5(a) and (b) illustrate airway plates and anvil plates of the device in conjunction with corresponding carriers;
FIG. 6 illustrates an insert of a carrier pushed into its corresponding anvil plate;
FIGS. 7(a) and (b) illustrate movement of an insert of a carrier plate into a corresponding anvil plate;
FIG. 8 illustrates the housing of the preferred embodiment;
FIGS. 9(a) and 9(b) illustrate airflow paths through the preferred embodiment;
FIG. 10 illustrates the chassis and cam member assembly of the preferred embodiment;
FIG. 11 illustrates schematically operation of the dispensing mechanism of the preferred embodiment;
FIG. 12 illustrates schematically the preferred profile for the cam member;
FIG. 13 illustrates sub-assemblies of the preferred embodiment;
FIGS. 14(a) to (f) illustrate the Geneva mechanism of the indexing mechanism of an embodiment of the present invention;
FIGS. 15(a) to (e) illustrate the changeover mechanism of an embodiment of the present invention;
FIGS. 16(a) to (h) illustrate the dispensing mechanism of an embodiment of the present invention;
FIGS. 17(a) and (b) illustrate cross-sections through the components of FIGS. 16(a) to (h);
FIG. 18 illustrates pockets being opened in a device embodying the present invention;
FIGS. 19(a) to (d) illustrate the Geneva mechanism of a counter in an embodiment of the present invention;
FIGS. 20(a) to (e) illustrate operation of the counter of FIGS. 19(a) to (d); and
FIGS. 21(a) and (b) illustrate preferred cross sectional areas at various locations in an embodiment of the present invention.
DETAILED DESCRIPTION OF THE DRAWINGS
A preferred embodiment of the present invention is an inhalation device from which a user may inhale consecutive doses of medicament in the form of dry powder. The preferred embodiment is illustrated in FIGS. 1(a) to (c).
The device includes a housing 2 on which a mouthpiece cover 4 is rotatably supported.
In order to use the device, the mouthpiece cover 4 is rotated away from the housing 2. As illustrated in FIG. 1(b) this exposes a mouthpiece 6. The mouthpiece 6 may be formed integrally with the housing 2, but, as will be described below, it can also be formed as a separate component for mounting with the housing 2. This allows the material properties, for instance, colour, of the mouthpiece 6 and housing 2 to be varied easily according to the requirements of the device.
As illustrated in FIG. 1(b), a priming lever 8 extends out of the housing 2 at a position adjacent the mouthpiece 6. The priming lever 8 is mounted so as to rotate about a central axis within the device (to be discussed further below). In this way, it is moveable by the user around a periphery of the housing 2 to a position as illustrated in FIG. 1(c). Movement of the priming lever 8 from the first position illustrated in FIG. 1(b) to the second position illustrated in FIG. 1(c) is arranged to prime the device, in particular, to expose a dose of powder such that it may be carried with an airstream out of the mouthpiece 6.
It should be noted that locating the first position of the priming lever 8 adjacent the mouthpiece 6 is highly advantageous, since it discourages a user from attempting to inhale from the mouthpiece 6 before moving the priming lever 8 away from the mouthpiece 6 to the second position of FIG. 1(c). In other words, the user is encouraged to prime the device before attempting to inhale through it. Nevertheless, it should be noted that a small space is preferably provided between the mouthpiece 6 and priming lever 8 so as to allow the user to operate the priming lever 8 with his or her finger without touching the mouthpiece 6.
After use of the device, the mouthpiece cover 4 may be rotated back to its stowed position illustrated in FIG. 1(a). In this respect, an inner surface of the mouthpiece cover 4 is provided with a return actuator for engaging with the priming lever 8. In particular, when the mouthpiece cover 4 is moved from its open position of FIGS. 1(b) and (c) to its closed position of FIG. 1(a), the return actuator engages with the priming lever 8 and moves it back from its second position illustrated in FIG. 1(c) to its first position illustrated in FIG. 1(b). As will be described further below, in the preferred embodiment, this movement of the priming lever 8 operates an indexing mechanism for moving a still unused and unopened pocket of powder into line with a dispensing mechanism such that, with subsequent priming of the device, the powder of that pocket is dispensed for inhalation. By operating the indexing mechanism during the return movement of the priming lever 8 immediately after priming and release of a pocket of powder, if the released powder is not inhaled, it is indexed to a position where it can safely be held within the device.
As illustrated in FIGS. 1(a) to (c), the preferred embodiment also includes a window 10 in one side of the housing 2. The window 10 is provided so as to allow a user to view a counter display within the device. A counter mechanism indexes the counter display upon each use of the device so as to provide the user with an indication of how may doses have been dispensed and/or how may doses remain unused.
Many aspects of the present invention are applicable to devices housing a wide variety of different dose carriers. In particular, many of the features of the embodiment described below can be used with carriers having a traditional blister-pack construction, with carriers having various arrays of pockets and, in some arrangements, with some carriers having a single respective pocket. Nevertheless, the present invention is particularly advantageous when used with carriers of the form illustrated in FIGS. 2(a) and (b).
As illustrated in FIG. 2(a), each carrier 12 is formed from a disc-shaped base 14 having a substantially planar first side surface 16 opposite and parallel with a substantially planar second side surface 18. A plurality of through holes 20 are formed between the first and second side surfaces 16, 18 so as to form spaces for housing doses of powder. The base 14 is formed with an appreciable thickness so as to provide the through holes 20 with sufficient space to house the required doses of powder. The through holes 20 are arranged as a circumferential array and, in the preferred embodiment, 30 through holes are provided in the array.
As illustrated in FIG. 2(b), the first and second side surfaces 16, 18 of the base 14 are sealed with respective first and second lidding sheets 22, 24. In this way, the carrier 12 provides a plurality of pockets housing individual respective doses of powder.
As illustrated by the cross-sections of FIGS. 3(a) and (b), the pockets preferably include a respective insert 26 within each through hole 20. The inserts 26 are generally cup-shaped with their open ends facing the first lidding sheet 22. Each contain a respective dose of powder 28.
By pushing on the closed end of the insert 26 from the side of the second lidding sheet 24, it is possible to push the insert 26 outwardly from the base 14 of the carrier 12 through the first lidding sheet 22. This is illustrated in FIG. 3(b), but, for clarity, without either lidding sheet. As illustrated, with the insert 26 extending out of the base 14, it may be more convenient to provide an airflow (such as indicated by arrows) to remove powder from the pocket.
Within the housing 2 of the inhalation device, in a preferred embodiment, two of the carriers 12 are arranged coaxially side by side as illustrated in FIG. 4(a). Each carrier 12 is provided with a support 30 as illustrated in FIG. 4(b). In the illustrated embodiment, each support 30 is positioned adjacent an outwardly facing surface of its respective carrier 12. In particular, the first side surface 16 of each carrier 12 faces a respective support 30 such that a dispensing mechanism may be provided between the two carriers 12 so as to press respective inserts 26 outwardly towards the respective supports 30. The preferred arrangement for this will be described further below.
As illustrated, the priming lever 8 is positioned such that it extends between the carriers 12 and is rotatable about the common axis of the carriers 12 so as to operate a dispensing mechanism and an indexing mechanism.
In the preferred embodiment, each support 30 is made up of two components, namely an anvil plate 32 and an airway plate 34. These are illustrated in FIGS. 5(a) and (b) in conjunction with associated carriers 12.
Each anvil plate 32 has a planar surface 6 which-, in use, abuts against the first side surface 16 of the associated carrier 12 as covered by the first lidding sheet 22. Each anvil plate 32 also includes a plurality of guide through holes 38 corresponding to the through holes 20 of the associated carrier 12.
In this way, as illustrated schematically in FIG. 6, an insert 26 can be pushed out of its through hole 20 and into a corresponding guide through hole 38 of the anvil plate 32. The insert 26 is thus used to outwardly burst through the first lidding sheet 22, but is still held securely in place. Although not of a particular concern here, the anvil plate 32 also supports the first lidding sheet 22 around the through hole 20 and can be used to improve the predictability of the nature of the lidding sheet rupture.
As illustrated by the cross-section of FIG. 7(a), the anvil plate 32 includes a second surface 40 which abuts an inner surface of the associated airway plate 34. The airway plate 34 includes a pair of through holes corresponding to each guide through hole 38 of the corresponding anvil plate 32. In particular, each pair includes an inlet hole 42 and an outlet hole 44.
As illustrated in FIG. 7(a), relative to the surface 40 of the anvil plate 32 abutting the inner surface of the airway plate 34, a recessed channel 46 extends radially inwardly from the outlet 44 so as to communicate with the guide through-hole 38 of the anvil plate 32. Hence, for each guide through hole 38 of the anvil plate 32, the airway plate 34 provides, communicating with it, a corresponding inlet 42 and outlet 44 with its associated recessed channel 46. In particular, each inlet 42 communicates with one side of its associated guide through hole 38 whilst the corresponding outlet 44 communicates with the opposite side of the associated guide through hole 38.
As illustrated in FIG. 7(b), when an insert 26 is pushed outwardly of the through hole 20 of the base 14 into the guide through hole 38 of the anvil plate 32, it is positioned with the open portion of its cup-shape facing the inlet 42 (at one end of the cup-shape) and the recessed channel 46 (at the opposite end of the cup-shape). In this way, as illustrated, an airflow may be drawn through the airway plate 34 such that it passes down into the pocket formed in the insert 26, back up into the recessed channel 46 and then out of the outlet 44. Powder in the insert 26 is thus picked up by the airstream, removed from the insert 26 and carried out of the airway plate 34. A flow path is thus formed into and out of a pocket which may then connect the pocket to the mouthpiece 6 of the device.
As illustrated in FIG. 8, the housing 2 may be formed from a pair of casing halves 2a and 2b. As illustrated in FIGS. 9(a) and (b), an inner wall 50 of the casing halves 2a and 2b cooperates with the airway plate 34 so as to form a second flow path to the mouthpiece 6 which bypasses the pocket(s). Alternatively, an additional component may be provided, to define the second flow path.
As illustrated in FIG. 9(b), for each pocket formed by an insert 26, the corresponding inlet 42 of the airway plate 34 is positioned adjacent a periphery of the pocket. The corresponding outlet 44 is provided on an opposite side of the pocket such that the airstream between the inlet 42 and outlet 44 crosses the pocket and, hence, picks up any powder from the pocket.
As illustrated, the inlet 42 is formed as a portion which is directed down into the insert 26 forming the pocket.
In this way, when a user inhales through the device and creates an airstream through it, the airstream drawn through the inlet 42 will be directed down into any powder in the insert 26 so as to dislodge it and move it into the airstream so as to be carried out of the outlet 44. In the illustrated embodiment, the recessed channel 46, which connects the volume of the pocket to the outlet 44, is positioned adjacent the inlet 42. In this way, the airstream from the inlet 42 is deflected from the base of the insert 26 (and any powder there) so as to travel back towards the recessed channel 46. Powder carried in the airstream up into the recessed channel 46 is subjected to a relatively sharp change in direction. As a result of this, powder in the airstream tends to be deaggregated. Furthermore, the powder will tend to hit the surfaces of the recessed channel 46 also contributing to deaggregation.
As is clear from FIG. 9(b), the shape of the airway path is chosen to force large aggregates of powder to impact the walls as the airflow is forced to change direction, thereby deaggregating large clumps of powder. The shape is also designed to ensure that airflow over any surface within the airway is maintained at a high value to avoid excessive powder adhering to the surface. Thus corners are rounded and the cross section at each position along the tube is designed to maintain air velocity without generating excessive pressure drop.
As illustrated in FIG. 9(a), in this embodiment, the airflow through the pocket has its minimum area at the inlet to the pocket defined by the dimension “a” whereas the airflow that bypasses the pocket has its minimum cross section just before the airflow join and so is defined by the dimension A.
The air velocity is highest where the cross sectional area is smallest so this arrangement provides high velocity air to extract the powder from the pocket and uses the high velocity of the bypass air joining the powder contained in the pocket airflow to assist de-aggregation and to protect the walls from powder deposition.
The airflow velocity through the pocket is controlled mainly by the suction pressure created as the user inhales, whereas the volume flow rate is a factor of both velocity and area.
A sufficiently high air velocity should be generated to ensure that the powder is entrained in the airflow. However, if the velocity—and flow volume are too high then there is the possibility that the whole of the mass of powder in the pocket is pushed through the airway as an agglomerated clump. If this happens, the clump may not accelerate to a sufficient velocity for its impact with the walls in the airway to break it up and provide de-aggregation. It is preferred that the powder is removed gradually from the pocket by the airflow. To achieve this, a small gap 46a is provided between the surface of the powder in the pocket and the airway roof formed from the division in the airway plate 34 between the inlet 42 and recessed channel 46. This, combined with a dimension for “a” that limits the flow volume through the pocket, ensures that the powder is eroded from the pocket rather than pushed out.
To enable this, the inlet hole diameter “a” is chosen to be between 0.5 mm and 2.0 mm for pockets—of around 2.0 mm width (in a circumferential direction) and of around 7.3 mm length (in a radial direction). The value chosen depends on the properties of the powder.
In this way, the powder can be removed from the pocket over a time period ranging from between 0.1 s to 1.0 s. This is within the period of the high flow rate of the inhalation cycle and provides good de-aggregation of the powder.
It should be appreciated that, in other embodiments, it is possible for parts of the flow path through the pocket, other than the inlet hole, for instance downstream of the powder, to form the minimum cross-sectional area of that flow path. Similar considerations will still apply for the diameter “a” of the inlet hole.
The arrangement of the inlet hole 42 and channel 46 is particularly advantageous in conjunction with deep narrow pockets of powder. At a particular flow rate, for instance 10 ltr/min, the surface of the powder will be eroded by a certain depth. Increasing the flow rate to, for instance 20 ltr/min, will result in the powder being eroded by a further depth. Since inhalation by users results in flow rates which increase progressively to a maximum, powder is eroded depth by depth and the pocket is emptied gradually over an appropriate period.
Although the volume and strength of inhalation will vary between users, it is important that the device should not provide too much in the way of resistance to inhalation. In this respect, it would be extremely difficult to inhale through an inlet 42 having a desired cross sectional area. Indeed, where possible, it would result in a flow velocity which was far too high and which would entrain of all of the powder from the insert 26 far too quickly. In practice, it is found that approximately only 20% of inhaled air can be used directly for picking up and deaggregating the powder.
As illustrated in FIG. 9(b), a second flow path is formed between an inner wall 50 of the housing 2 and the outside of the airway plate 34. The second flow path bypasses the pocket and increases the overall cross sectional area available through which to inhale. By changing the values of the dimensions a and A, it is possible to change the rates of airflow between the pocket and bypass and to control the overall flow resistance of the device so that it is comfortable for the user to inhale through. A typical flow resistance for the device would be between 2 kPa and 5 Kpa for a flow volume of 601/min. Higher flow resistances are chosen for powders which are harder to deaggregate, whereas lower flow resistances are preferred for devices used by children. The recessed channel 46 and outlet 44 generally have larger cross sectional areas than the inlet 42. It is envisaged that the minimum cross sectional area for the pocket path would be 3.5 mm2 to 4.0 mm2 and for the bypass 5.0 mm2 to 6.0 mm2.
In this way, it is relatively easy to inhale through the device, since a large proportion of the airflow will be through the second flow path. Nevertheless, some of the flow will occur through the first flow path so as to entrain and deaggregate the powder as described above.
In the preferred embodiment, there is another second flow path for the other side of the device and its corresponding carrier. In use, a patient inhales through both second flow paths whilst drawing powder from the first flow path in use. Each of the second flow paths is expected to carry approximately 40% of the total inhaled air for an average use.
Actual requirements will vary depending upon the nature of the powder and the intended user. For an easily dispensed powder, the portion forming the inlet to the pocket can be small and, for a child or patient with COPD (Chronic Obstructive Pulmonary Disease), the total pressure drop should be low. In this case, an inlet portion could be provided with a cross-sectional area of 2 mm2 and a bypass second flow path with a minimum cross-sectional area of 8 mm2, resulting in a ratio of 25%). On the other hand, with sticky powder for a healthy adult, the inlet portion could be provided with a cross-sectional area of 4 mm2 together with a bypass second flow path having a minimum cross-sectional area of 6 mm2, resulting in a ratio of 66%. Of course, intermediate values are also possible and a preferred arrangement has an inlet portion of approximately 3 mm2 with a second flow path minimum cross-sectional area of 6 mm2, resulting in a ratio of 50%.
As illustrated in FIG. 9(a), the walls of the outlet 44 are orientated so as to direct the flow of air and powder into the second flow path at an angle θ relative to the flow in the second flow path. By ensuring that the angle θ is less than 45°, it is possible to substantially reduce the amount of powder which might impact with or stick to the wall 50 opposite the outlet 44. Preferably the angle θ is no greater than 45°, more preferably no greater than 30°. In this way, substantially no powder will adhere to the wall 50 forming the second flow path to the mouthpiece 6. Preferably, with repeated use of the device, no more than 25%, preferably no more than 15% of a dose remains deposited on the wall 50. In this respect, it will be appreciated that the flow from the bypass past wall 50 will act to scour or scavenge powder from the wall 50.
As mentioned above with reference to FIG. 4(b), the anvil plate 32 and airway plate 34 together form a support for a corresponding carrier 12. By means of the priming lever 8 and the indexing mechanism to be described below, a support 30 and its corresponding carrier 12 is moved to consecutive-positions to dispense powder from consecutive pockets. In this regard, it will be appreciated that each pocket has its own first flow path as formed in the airway plate 34. From the description above, it will be appreciated that turbulent flow in removing powder from the pocket and deaggregation of powder occurs within the first flow path. Thus, should any powder adhere to walls within the airway plate 34, this powder is not available for inhalation when subsequent pockets of powder are dispensed.
The device is preferably arranged such that an inlet passage that provides the air for the flow through the pocket and through the bypass is arranged so that it feeds the air only to the pocket positioned for dispensing, such as illustrated in FIGS. 9(a) and (b). The indexing of the carrier 12, anvil plate 32 and airway plate 34 after use repositions the inlet 42 and outlet 44 for a used pocket outside of the airflow for the pocket currently in use.
This arrangement ensures that, even if none of the powder from a pocket is removed after it has been opened, once it has been indexed on, then the powder will be permanently retained within the device such that it will not be inhaled along with a subsequent dose.
The supports 30 and associated carriers 12 may be rotatably mounted within the housing 2 by means of a chassis sub-assembly 58 as illustrated in FIG. 10. The chassis sub-assembly 58 is positioned between the second side surfaces 18 of the carriers 12. It extends axially along the axis of the carriers 12 and is fixed to one or both of the two halves 2a, 2b of the housing 2.
As illustrated in FIG. 10, the priming lever 8 forms part of (or could be attached to) a priming member 60. The priming member 60 has a central pivot opening 62 by which it is rotatably supported on a pivot shaft 64 of a chassis 66.
As illustrated in FIG. 13, the priming member 60 and chassis 66 are together positioned between the two carriers 12 and associated supports 30. Furthermore, the chassis 66 is mounted to the housing 2 so as to be rotatably fixed. In the illustrated embodiment, the pivot shaft 64 may itself be located on a shaft 68 provided on the inside of one or both halves 2a, 2b of the housing 2. Also, a radial extension 70 (shown in FIG. 10) may be provided on the chassis 66 to interact with an inner portion of the housing 2 so as to rotationally fix the chassis 66.
The carriers 12 and associated supports 30 may be rotationally mounted on the chassis 66.
The priming member 60 includes an elongate cam member 72 which extends in a circumferential direction and has a cam surface 74 on each of two opposites sides.
Each cam surface 74 interacts with a respective member 76 which will be described as a prodger.
Operation of the priming member 60, cam member 72, cam surfaces 74 and prodgers 76 will be described with reference to the schematic illustration of FIG. 11.
When the priming lever 8 is moved from its first position to its second position, the priming member 60 is rotated relative to the chassis 66, the carriers 12 and their supports 30 such that, in the schematic illustration FIG. 11, the cam member 72 moves upwardly.
As can be seen in FIG. 10, the priming member 60 includes elongate openings either side of the cam member 72 through which arms 80 of the prodgers 76 can extend. The chassis 66 holds the prodger 76 rotationally but allows them to move in an axial direction of the device, in other words towards and away from the carriers 12 on either side. Indeed, as illustrated, an aperture 82 exists in the chassis 66 allowing one of the prodgers 76 to extend through the chassis 66 towards a corresponding carrier 12.
As illustrated in FIG. 11, the cam surface 74 on either side of the cam member 72 is such that, as the priming member 60 rotates and the cam member 72 moves upwardly as illustrated in FIG. 11, the prodgers 76 are moved outwardly towards their respective carriers 12.
In FIG. 11, the right hand prodger 76 is illustrated in alignment with a pocket in its corresponding carrier 12. Thus, when the priming member 60 rotates and the cam member 72 moves upwardly in FIG. 11, the right hand prodger 76 will be moved outwardly towards its corresponding carrier 12, will penetrate the through hole 20 and push the insert 26a out of the first side surface 16. In this respect, FIG. 11 illustrates one insert 26b which has already been pushed out by the prodger 76.
An indexing mechanism, to be described below, rotates the right hand carrier 12 and its corresponding support 30 to the next position in which the prodger 76 is aligned with a new, unopened pocket. The operation of opening a pocket can then be repeated.
It will be appreciated from FIG. 11 that carriers 12 on either side of the priming member 60 could have respective pockets aligned with the prodgers 76 such that operation of the cam member 72 simultaneously opens pockets of the respective carriers 12. However, in the illustrated embodiment, the indexing mechanism arranges for one of the prodgers 76 to be aligned with a pocket whilst the other of the prodgers 76 is at a position between pockets. In this way, the dispensing mechanism formed from the cam member 72 and prodgers 76 only opens one pocket at a time.
Referring to FIG. 2(a), it will be seen that the preferred carrier 12 has an array of through holes 20 which includes a space 82 in which a through hole 20 is not formed.
Using carriers of this type, it is possible to position one carrier 12 with the blank portion 82 opposite a prodger 76 whilst consecutively indexing the other carrier 12 around each of its through holes 20 and the pockets they form until all have been emptied. The indexing mechanism can then rotate the empty carrier to a position in which its blank portion 82 is opposite the prodger 76 and rotate the other carrier 12 around all of the positions in which the corresponding prodger 76 aligns with the through holes 20. In this way, the same dispensing mechanism is used for dispensing powder from both carriers and using the same operation.
Although it is the intention that substantially all of the powder dispensed from the individual pockets will be removed from the device by way of inhalation, it is possible that some powder will remain within the device. Indeed, where different types of carrier are used or the device has a different application, it might be that more powder does remain within the device.
As illustrated in FIG. 10, the cam surfaces 74 are provided with one or more grooves or channels 84. Any excess powder can thus fall into the grooves 84 such that contact and movement between the cam surface 74 and the prodger 76 is not impeded.
It will be appreciated that, with the arrangement where one or other of the prodgers 76 abuts a portion 82 of a carrier 12 where there is no pocket, in order for the priming member 60 to rotate and the cam member 72 to move a prodger 76 towards the other carrier 12, it will be necessary for the cam member 72 to move away from the portion 82. In some embodiments, it might be possible to allow the entire priming member 60 to move axially or for the carriers 12 to move axially. However, in the preferred embodiment, the cam member 72 has itself a limited amount of flexibility. As illustrated, the cam member 72 is provided as an elongate member which is attached to the rest of the priming member 60 at each end with an elongate opening either side of it. This will allow sufficient flexibility for the cam member 72 to move towards and away from the carriers 12.
Considering the overall embodiment as described with reference to FIG. 1(a) to (c), it will be appreciated that it is highly desirable to ensure that the user moves the priming lever 8 through its entire length of travel so as to fully dispense a dose of powder. In particular, considering FIG. 11, it would be undesirable for a user to partly operate the priming lever 8 and priming member 60 such that a prodger 76 pushes an insert 26 far enough to partly rupture a lidding sheet on the first side surface 16, but without fully extending the insert 26 to the position illustrated in FIGS. 6 and 7(b).
As the motion of the inserts 26 is restricted by the foils 22, 24 sealing both surfaces of the carrier plate 12, a high force is required to cause the inserts 26 to start to move. This force increases to the point at which the foils 22, 24 rupture after which the force decreases substantially. Thus, the user feels a resistance to the motion of the priming lever 8 for the early part of its travel. At some point along its travel, the resistance suddenly reduces, as the foil 22, 24 rupture. The user cannot reduce the applied force instantaneously so that the priming lever 8 is rapidly pushed to the end of its available stroke. This tactile feedback encourages the user to fully open the pockets.
If the cam member 72 driving the prodgers 76 was solid as shown in FIG. 11, then the inserts 26 would be forced to the positions shown. However, in the preferred embodiment where the parts are moulded in plastic, it is impossible to control the dimensions of all parts with absolute accuracy. Thus, where the distance moved by the insert 26 is smaller than the space allowed for it, there would be a gap above the pocket but where the distance moved is greater than the distance allowed for it, the anvil plates 32 would be pushed apart from the carriers 12 by the force. This force would be transmitted to the casework causing it to deform if sufficiently high force was applied to the priming lever 8.
To avoid this potential problem, the cam member 72 is made to a form that varies its force versus distance profile along its length.
An example of a suitable form is shown in FIG. 12. The preferred embodiment includes two such members arranged back to back. The solid wedge shape profile at the right hand side as illustrated in FIG. 12 has the same profile as shown in FIG. 11. This form rigidly transmits the force applied by the priming lever 8 to the insert 26. The length of this profile is chosen so that, for all devices, the prodgers 76 will be moved sufficiently far to break the foils 22, 24 by this profile. Once the foils 22, 24 are broken, much less force is required, but the distance that the insert 26 must move may vary from device to device. Thus, for the last part of its travel, the cam member 72 cross-section is designed to provide compliance in its movement. This ensures that the cam member 72 provides sufficient force for the insert 26 to be pushed to the end of its available travel in the anvil plate 32. However, after the insert 26 is stopped at the end of its travel, the force that the cam member 72 applies to the prodger 76 is limited to that generated as it deforms. This can be much less than the force that would be applied if the prodger 76 were rigidly connected to the priming member 60.
In this way, reliable opening of the pocket is achieved using components that can be manufactured using conventional materials and moulding processes.
The action of cam member 72 and prodger 76 is further illustrated in FIGS. 16(a) to 16(h). These Figures show the cam member 72 and prodger 76 at sequential positions as the priming lever 8 and priming member 60 are moved to open a pocket. The Figures are grouped in pairs, each group giving two views of the same position.
FIGS. 16(a) and 16(b) show the prodger 76a in its fully retracted position at one end of the cam member 72. The prodgers 76a and 76b are identical components that clip together with the cam member 72 between them. Each prodger 76 has features 86 at the ends of their arms 80 that locate with additional cam surfaces 88 formed on the priming member 60 either side of the elongate openings though which the arms 80 extend.
Where a prodger member 76 has penetrated past the first surface of a carrier disc in order to push the pocket through the second surface, then it is necessary to retract the prodger member 76 before the carrier disc can be indexed to its next position.
A spring could be used to achieve this if it were positioned to press the prodger member 76 against its base surface. However, it is preferable to have an active method for retracting the prodger member 76 that acts as the cam member 72 is returned to the original position. However, where the action of returning the cam member 72 to its original position is also used to index the carrier disc, it is important to ensure that the retraction of the prodger members 76 is completed before the carrier disc is indexed.
A preferred method of achieving this is by the use of the further cam surfaces 88 located in the non-moving housing in which the cam member and carrier discs are located.
FIG. 17(a) shows a schematic cross section through the prodgers 76a and 76b also in their retracted position.
The location of prodger 76a is constrained by the surface 90 of the cam 88 and the cam surface 74 of the cam member 72. The cams 88 and cam member 72 are designed so that their thickness CI and C2 change along the direction of the primary member 60 motion. FIG. 17(b) shows the prodgers 76a and 76b in their open position where it can be seen that C2 has increased and CI decreased compared to their values at the closed position.
The cam member 72 has a rectangular cross section C2 at one end that gradually increases in area. At the point that it starts to become a compliant wedge, rather than a rigid one, the wedge splits into a central part that pushes up 74 and two side parts that push down 74a.
This arrangement provides a positive force to both open and close the prodgers 76a and 76b.
FIGS. 16(c) to 16(h) show how the concept illustrated in FIGS. 17(a) and (b) might be implemented.
FIGS. 16(c) and (d) show the prodgers 76a and 76b where the cam member 72 has completed approximately one third of its full travel. The cam member 72 over this section is of uniform thickness such that the prodgers are fully retracted. This allows the movement of the rotary priming member 60 on the return stroke over this section to drive the indexing mechanism (as will be described below).
FIGS. 16(e) and (f) show the prodgers 76a and 76b where the cam member 72 has completed two thirds of its travel. The cam member 72 along this section includes the circumferential grooves 84 mentioned above. The raised parts of the cam member 72 are sufficient to rigidly couple the force applied to the priming lever 8 to the prodgers 76a and 76b and the grooves 84 are provided solely to increase the tolerance of the mechanism to stray powder that may have collected on the cam surface 74.
FIGS. 16(g) and (h) show the prodger where the cam member 72 has completed its travel. In this section, the cam member 72 is not solid but split into a central section and two side sections arranged so that the central section presses up against one prodger whilst the two outer sections press down against the other prodger.
If the prodgers 76a and 76b reach the end of their travel before the cam member 72 reaches the end of its travel, then the thinned section of the cam member 72 at this point will be deflected, thereby limiting the force applied to the prodgers 76a and 76b over the remaining travel of the cam member 72.
In the preferred embodiment, the indexing of the two carrier assemblies (FIGS. 5(a) and (b)) is accomplished by an indexing mechanism that causes a carrier 12 to be incremented by one pocket each time the priming lever 8 is actuated and a changeover mechanism that causes the indexing mechanism initially to drive the first carrier 12 but, when the last pocket of that carrier 12 has been used, for that carrier 12 to remain stationery whilst the second carrier 12 is incremented when the indexing mechanism is actuated.
The preferred indexing mechanism illustrated in FIGS. 14(a) to (f) uses a 3 peg Geneva 100 that rotates exactly 120° each time the indexing mechanism is actuated. The Geneva peg wheel 100 has two gears co-axial with the peg-wheel arranged so that the gears can engage with teeth 35 on the airway plates 34.
To avoid having both airway plates 34 driven simultaneously, it is arranged that, at one location around the airway plate 34, the gear teeth 35 are missing. As a result, at this location, rotation of the Geneva peg wheel 100 does not rotate the airway plate 34. Thus, the indexing mechanism drives the first carrier 12 via the Geneva 100 and its gears until it reaches the end of the gear teeth 35 for that carrier 12. The next indexing moves the first carrier 12 to its non-driven position, i.e. where the gear teeth 35 are missing, and engages a changeover mechanism which rotates the second carrier 12 until its gears 35 are engaged with the gears on the Geneva peg wheel 100.
A preferred embodiment of the indexing mechanism is illustrated in FIGS. 14(a) to 14(f). In these, it can be seen that the peg wheel 100 is located with its axis parallel t0 the axis of the dose carriers 12 and rotary priming member 60.
The rotary priming member 60 incorporates many of the functional elements described previously within a single moulded component. It includes the priming lever 8, the cam member 72 and the prodger closing cams 83, as well as being the driving member for the indexing Geneva 100.
The Figures start with the priming member 60 at the end of its travel where a pocket has been opened and shows what happens as the priming lever 8 is returned to its start position by the closing of the mouthpiece cover 4.
The peg wheel 100 has six pegs 102a-102c, 103a-103c arranged at 60° intervals around its edge. Three of these pegs 102a, 102b, 102c are longer than the other three 103 a, 103b, 103 c and are shown with black ends for clarity. As the rotary priming member 60 moves from its position in FIG. 14(a) to that in FIG. 14(b), the leading part 101 of a driving member 104 formed by the priming member 60 passes over the short peg 103 a with its periphery touching the edges of the longer pegs 102a and 102c preventing the peg wheel 100 from rotating. At the position shown in FIG. 14(b), a ratchet 105, which slopes downward and forward from the driving member 104, engages with the peg 103 a. As the priming member 60 and the driving member 104 continue to move from the position of FIG. 14(b) to that of FIG. 14(c), the peg wheel 100 is driven around. To permit the peg wheel 100 to rotate, a slot 106 is cut into the driving member 104 of the priming member 60 into which the long peg 102c can enter. At the position of FIG. 14(c), the ratchet 105 is starting to disengage with the peg 103 a but the trailing edge 107 of the slot 106 now engages with the long peg 102c and continues to drive the peg wheel 100 around through to the position shown in FIG. 14(d). At the position of FIG. 14(d), the edge 108 of the driving member 104 passes over the short peg 103 c. The peg wheel 100 then continues to rotate to the position of FIG. 14(e) to complete the forward motion of the peg wheel 100. The slot 109 is provided to accommodate the long peg 102b. At this position, the dose carrier 12 has been driven so that the next pocket to be opened is beyond its desired location and the mouthpiece cover 4 that has been driving the rotary priming member 60 is fully closed.
When the priming lever 8 is pushed in the reverse direction by the user to open a pocket, the initial part of the travel, over which the prodgers 76a and 76b are not moved, takes the rotary priming member 60 plate back from the position shown in FIG. 14(e) to that shown in FIG. 14(b). The angled face 110 in the slot 109 on the rotary priming member 60 pushes on the long peg 102b causing the peg wheel 100 to rotate backwards until the two long pegs 102b and 102c are both dis-engaged from the driving edges and pressing against the outer periphery 108 of the rotary priming member 60.
This accurately defines the rotary position of the peg wheel 100, ensuring that the prodgers 76a and 76b accurately line up with the pockets. The short peg 103c, that is within the outer periphery of the rotary priming member 60, is short enough to allow the ratchet 105 to return over the top of it. Thus, after the initial movement, the peg wheel 100 is held stationary throughout the remainder of the stroke opening a pocket. Thus, each indexing operation causes the peg wheel 100 to rotate 120°. The gears above and below the plane of the peg wheel 100 are shown in FIG. 14(f) which for clarity is viewed from the opposite side from FIG. 14(a) to 14(e). FIG. 14(f) shows the gears 35 on one of the airway plates 34 engaged with the gear on the peg wheel 100. The number of gear teeth on the airway plates 34 and peg wheel 100 are arranged so that the 120° motion of the peg wheel increments the dose carrier plate exactly one pocket pitch.
The arrangement described here is advantageous in achieving precise intermittent motion control of two disks within very tight space allocation and with a minimal number of components.
As described previously, for the device to operate with two disk carrier plates, a changeover mechanism is preferably provided to cause the indexing mechanism initially to drive a first disk and, when this has had all of its pockets opened, to then drive a second disk. Such a changeover mechanism will be described with reference to FIG. 15(a) to FIG. 15(e). These Figures show the device viewed edge on with the two airway plates 34 arranged horizontally.
FIG. 15(a) shows the device in its position before a first pocket is opened.
In FIG. 1(b), airway plate 34a has been indexed by one position to the right. The two features 123 on the periphery of the airway plate 34a can be seen to have shifted.
FIG. 15(c) shows the position after the last pocket of the lower carrier 12 of airway plate 34a has been opened. The rotation has brought the features 123 right around the device to the position shown. The next indexing operation causes the lower airway plate 34a to move as before.
However, the leading feature 123 pushes on a changeover component 124 which pushes on the feature 122 on the upper airway plate 34b causing both plates 34a and 34b and carriers 12 to move together. When the upper airway plate 34b was in its original position, the prodger 76b was aligned to the missing pocket part 82 providing a hard surface against which that prodger 76b could push whilst the other prodger 76a pushed against a pocket of the lower airway plate 34a. In addition, at this location, the missing teeth on the gear 35 of the upper airway plate 34b aligned with the gear on the Geneva peg wheel 100 and, hence, rotation j of the peg wheel 100 did not index the upper airway plate 34b. However, the indexing operation performed by the changeover component 124 on the upper airway plate 34b moves the gear of the upper airway plate 34b to engage with the gear of the peg wheel 100 and aligns the first pocket of the upper carrier 12 with the prodgers 76. Simultaneously, indexing by the priming member 60 causes the lower airway plate 34a to continue to move to a position which the gear teeth 35 on the lower airway plate 34a disengage from the gear on the peg wheel 100. The priming member 60 and peg wheel 100 move the lower airway plate 34a to a position in which the missing teeth on the gear 35 of the lower airway plate 34a are aligned with the gear on the Geneva peg wheel 100 and the missing pocket segment 82 of the lower dose carrier 12 is aligned with the prodgers 76.
The clip 125 provides an interlock that prevents any frictional coupling from causing the upper airway plate 34b to move before the lower airway plate 34a has arrived at the correct location.
Thus, changeover from the indexing of one disc to the other is achieved automatically and with minimal number of components and in a very small space.
The indexing of the device, in addition to moving the next pocket into alignment with the prodgers 76, preferably actuates a dose counter that provides a visual indication to the user of the number of doses remaining. The operation of the dose indicator will be described with reference to FIGS. 19 and 20.
It is preferable that the device, when dispensing medicament, indicates to the user the number of doses remaining in the device.
It is preferable that such indication is easily readable and, as such, very small numbers indicating the remaining doses would be a disadvantage. Within the size constraints of a pocket portable device that contains 60 doses providing such a display is challenging.
The simplest arrangement of marking the carrier discs with numbers visible through windows in the casework requires, where two carrier discs are used, the user to view different windows and, in addition, the space available around the carrier disc means that the size of the numbers would be small.
A preferred method is to employ a display with separate units and tens indication, driven such that the tens display index one number as the unit display index from 9 to 0. This allows larger numbers to be used within the same casework. The two discs may be provided concentrically one within the other and preferably co axially with the axis of the device, for instance on the shaft 68 illustrated in FIG. 13. The displayed units and tens are visible through the window 10 illustrated in FIG. 1(a).
In a preferred embodiment, the display counts down to zero, but the tens disc is not provided with a “0”. Instead, it is provided with an indicator, for instance a symbol, colour light etc to indicate to the user that the device is nearing the end of its functional life.
The preferred embodiment uses another Geneva and gear arrangement that is driven from the movement of the carrier discs. It is preferable that a single counter is increment initially by the motion of the first carrier disc and subsequently by the motion of the second carrier disc such that the fact that the device contains two carrier discs is not apparent to the user.
FIG. 20(a) shows a view of the dose counter display. The counter consists of two concentric rings 130, 131 with numbers formed in the rings facing toward the outer casing 2 of the device. The outer ring is the units counter 130 and the inner ring is the tens counter 131. The window 10 is provided in the outer casework 2 is arranged to permit the user to see only one digit of the units counter 130 and the adjacent digit of the tens counter 131. In FIG. 20(a), the counter indicates that there are 21 doses left. The operation of the counter requires the units counter 130 to index by 36° every time the indexing mechanism is actuated and for the tens counter to index by 36° only as the units counter moves from displaying 9 to 0. It can be seen that the units digits are evenly distributed around the ring whereas, for the example shown in FIG. 20(a) which has 60 doses, there are only the digits 1 to 6 on the tens counter 131.
The counter is driven by a gear 133 which itself is driven by one of the gears on the indexing Geneva peg wheel 100. In the preferred embodiment described above, the indexing Geneva 100 turns through 120° for each indexing operation and the gear on it has six teeth. The gear 133 has fifteen teeth and engages with the twenty teeth 134 of the units counter illustrated in FIG. 19(a). Thus, the 120° rotation of the indexing Geneva 100 drives the units counter 130 through 36°. FIG. 19(a) shows the units counter ring 130 viewed from behind the face on which the numbers are formed.
A counter Geneva wheel 135 is shown located inside the units counter ring 130 for mounting on a fixed post which is part of chassis 66.
An actuated peg 136 for the counter Geneva mechanism is located on the inner diameter of the units counter wheel 130. This peg 136 engages with one of the three indentations 137 in the Geneva wheel 135 causing the Geneva wheel 135 to rotate by 120° as the peg 136 passes by the wheel 135 during its 36° rotation between displaying the digits 9 and 0.
It should be noted that in this Geneva mechanism, the peg 136 is on the outer larger diameter component 130 and this drives the slotted smaller wheel 135 whereas, for the indexing Geneva 100, the slots are on the larger wheel and they drive the pegs on the smaller wheel. However, both are examples of a Geneva type mechanism providing intermittent rotation with accurate location between the rotations.
The Geneva wheel includes cam faces 138 which contact against the inner wall 139 of the units counter 130 preventing the Geneva 135 rotating between indexing. To permit the Geneva 135 to rotate as it is pushed by the peg 136, there is a gap 139a in the inner wall 139 adjacent to the peg 136.
The Geneva has a 3 tooth gear on its underside engaging with pegs on the tens counter ring to drive it.
FIG. 19(a) through to FIG. 19(e) show the positions of the Geneva wheel 135, the drive gear 133 and the units counter 130 at four stages during the 36° rotation of the units counter.
FIG. 20(a) to FIG. 20(c) show the motion of the two counter wheels as they index from 21 doses to 20 doses remaining, when only the outer units counter 130 moves. FIG. 20(c) to FIG. 20(e) show the corresponding situation from 20 to 19 doses remaining where both counters index.
After the last dose has been used, the remaining doses display will read 0 indicating that the device is empty to the user.
However, if the user does not look at the display, they may actuate the device again when desiring further doses.
It is preferable that the device provides some positive feedback to the user, as it is being actuated, that it is empty.
This feedback can be in the form that the priming lever 8 cannot be moved to its operating position with the level of force normally used. This tactile feedback provides a lockout feature.
A preferred method of achieving this with the two disc device is to arrange that after the last dose has been used, the second disc indexes such that it has no pocket under the prodger. At this point, the two prodger members 76 both face surfaces of the discs without pockets. Thus as the priming lever 8 is moved, neither prodger member 76 can move onto a disc and the resulting force on the prodger members 76 is transmitted back through the drive mechanism to the priming lever 8 and hence to the user.
Whilst the user may be able to apply sufficient force to move the priming lever 8 through to its home position, this will only be possible by forcing the discs to separate against the constraint of the casework. The force required to do this can be made sufficiently greater that the normal actuation force as to be obvious to the user.
From the description, it can be seen that this mechanism provides a clear visual indication of the number of doses remaining with a minimal number of components.
The preferred embodiment described above is arranged consecutively to dispense the powder from each pocket of one carrier and then subsequently the powder from each pocket of the other carrier. However, it should be appreciated that it is also possible for a device to dispense powder from pockets alternately from one carrier and then the other carrier. Alternatively, pockets of both disks may be dispensed simultaneously.
By dispensing powder from both carriers, either one after the other or simultaneously, it is possible for the user to inhale the powder from both carriers simultaneously. This arrangement is particularly advantageous when used with disks containing different medicament. In particular, it is preferred to provide disks containing a combination of medicaments that are more effective together than singularly. By way of example, a steroid compound could be dispensed from one disk and a long acting beta agonist (LABA) from the other disk for the treatment of, for example, asthma or chronic obstructive pulmonary disease. Examples of long acting beta agonists include formoterol and salmeterol and examples of steroids include fluticasone propionate. budesonide and monetasone furoate.
It is also possible to adapt the mechanism so to as to arrange for selective dispensing from one or both carriers. Where both disks are provided with the same medicament, this may be used to vary the dispensed dosage.
Although a device has been described with reference to a particular type of carrier, in particular having through holes and sealed with lidding sheets on either side, it is also possible to use other carriers, such as more conventional blister packs. These could include inserts similar to those described above. However, alternatively, powder in the pockets themselves could outwardly burst the lidding sheet. Also certain aspects of the device are applicable with other opening arrangements such as peeling or cutting of the lidding sheet.
Finally, it should be appreciated that the device can be provided with carriers pre-installed or, alternatively, ready for use with appropriate carriers.
As illustrated in FIG. 13, the preferred arrangement described above can be provided conveniently as three assemblies for use with carriers 12. In particular, a first cover sub-assembly A receives one carrier 12 and a second cover sub-assembly B receives another carrier 12. The two cover sub-assemblies A and B are then secured to one another with a chassis sub-assembly C therebetween.
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16183364
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pfizer limited
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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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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Pfizer
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Health Care
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Pharmaceuticals & Biotechnology
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