COVALENT BTK INHIBITORS AND USES THEREOF

Abstract

The present invention features compounds having BTK inhibitory activity. The compounds of the invention, alone or in combination with other pharmaceutically active agents, can be used for treating or preventing various medical conditions, such as cardiovascular diseases, respiratory diseases, inflammation, and diabetes.

Claims

1. A compound having the structure of Formula I:
ABCFormula I wherein A is ##STR00052## B has the structure: ##STR00053## wherein B is conjugated to A at the nitrogen and to C at the carbonyl; the dotted line is an optional double bond; m is O or 1; n is 0, 1, or 2; o is 0 or 1; p is 1 or 2; R.sup.1 is hydrogen or combines with R.sup.3 or R.sup.4 to form a 5- to 6-membered carbocyclic ring; R.sup.2 is hydrogen or C.sub.1-C.sub.6 alkyl; X.sup.1 is S or CR.sup.3R.sup.4, wherein R.sup.3 and R.sup.4 are each, independently, hydrogen or combine with R.sup.1 to form a 5- to 6-membered carbocyclic ring, wherein if a double bond is present, then R.sup.4 is absent; and X.sup.2 is CR.sup.5R.sup.6, wherein R.sup.5 and R.sup.6 are each, independently, hydrogen or C.sub.1-C.sub.6 alkyl, wherein if a double bond is present, R.sup.6 is absent; R.sup.14 is hydrogen or C.sub.1-C.sub.6 alkyl; each R.sup.15 and R.sup.16 is, independently, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl; or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl; and C has the structure: ##STR00054## wherein p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, 4, or 5; R.sup.7 is hydrogen or amido; each R.sup.8 and R.sup.11 is, independently hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl, cyano, or halo; and R.sup.9, R.sup.10, R.sup.12, and R.sup.13 are each, independently, hydrogen or C.sub.1-C.sub.6 alkyl, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein B has the structure of Formula IIa: ##STR00055## wherein B is conjugated to A at the nitrogen and to C at the carbonyl; the dotted line is an optional double bond; m is 0 or 1; n is 0, 1, or 2; R.sup.1 is hydrogen or combines with R.sup.3 or R.sup.4 to form a 5- to 6-membered carbocyclic ring; X.sup.1 is S or CR.sup.3R.sup.4, wherein R.sup.3 and R.sup.4 are each, independently, hydrogen or combine with R.sup.1 to form a 5- to 6-membered carbocyclic ring, wherein if a double bond is present then R.sup.4 is absent; and X.sup.2 is CR.sup.5R.sup.6, wherein R.sup.5 and R.sup.6 are each, independently, hydrogen or C.sub.1-C.sub.6 alkyl, wherein if a double bond is present, R.sup.6 is absent.

3. The compound of claim 1 or 2, wherein m is 1.

4. The compound of claim 3, wherein R.sup.1 is hydrogen.

5. The compound of claim 3 or 4, wherein X.sup.1 is CR.sup.3R.sup.4.

6. The compound of claim 5, wherein R.sup.3 and R.sup.4 are both hydrogen.

7. The compound of any one of claims 3 to 6, wherein X.sup.2 is CR.sup.5R.sup.6.

8. The compound of claim 7, wherein R.sup.5 and R.sup.6 are both hydrogen.

9. The compound of any one of claims 3 to 8, wherein n is 1.

10. The compound of any one of claims 3 to 9, wherein B has the structure: ##STR00056##

11. The compound of claim 10, wherein B has the structure: ##STR00057##

12. The compound of claim 2, wherein m is 0.

13. The compound of claim 12, wherein n is 2.

14. The compound of claim 13, wherein R.sup.1 is hydrogen.

15. The compound of claim 13 or 14, wherein X.sup.1 is CR.sup.3R.sup.4.

16. The compound of claim 15, wherein R.sup.3 and R.sup.4 are both hydrogen.

17. The compound of any one of claims 13 to 16, wherein X.sup.2 is CR.sup.5R.sup.6.

18. The compound of claim 17, wherein R.sup.5 and R.sup.6 are both hydrogen.

19. The compound of any one of claims 13 to 18, wherein B has the structure: ##STR00058##

20. The compound of claim 12, wherein n is 0.

21. The compound of claim 20, wherein R.sup.1 is hydrogen.

22. The compound of claim 21, wherein X.sup.1 is S.

23. The compound of claim 21 or 22, wherein X.sup.2 is CR.sup.5R.sup.6.

24. The compound of claim 23, wherein R.sup.5 and R.sup.6 are both methyl.

25. The compound of any one of claims 20 to 24, wherein B has the structure: ##STR00059##

26. The compound of claim 25, wherein B has the structure: ##STR00060##

27. The compound of claim 21, wherein B has the structure: ##STR00061##

28. The compound of claim 27, wherein R.sup.3 and R.sup.5 are both hydrogen.

29. The compound of claim 21, wherein X.sup.2 is CR.sup.5R.sup.6.

30. The compound of claim 29, wherein R.sup.5 and R.sup.6 are both hydrogen.

31. The compound of claim 29 or 30, wherein X.sup.1 is CR.sup.3R.sup.4.

32. The compound of claim 31, wherein R.sup.4 is hydrogen.

33. The compound of anyone of claims 29 to 32, wherein R.sup.1 and R.sup.3 combine to form a 5- or 6-membered carbocyclic ring.

34. The compound of any one of claims 29 to 33, wherein B has the structure: ##STR00062##

35. The compound of claim 34, wherein B has the structure: ##STR00063##

36. The compound of claim 1, wherein B has the structure of Formula IIb: ##STR00064## wherein B is conjugated to A at the nitrogen and to C at the carbonyl; o is 0 or 1; and R.sup.2 is hydrogen or C.sub.1-C.sub.6 alkyl.

37. The compound of claim 36, wherein o is 0.

38. The compound of claim 36 or 37, wherein R.sup.2 is hydrogen.

39. The compound of claim 1 wherein B has the structure of Formula IIc: ##STR00065## wherein B is conjugated to A at the nitrogen and to C at the carbonyl; p is 1 or 2; R.sup.14 is hydrogen or C.sub.1-C.sub.6 alkyl; and each R.sup.15 and R.sup.16 is, independently, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl; or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl.

40. The compound of claim 39, wherein R.sup.14 is hydrogen.

41. The compound of claim 39 or 40, wherein p is 1.

42. The compound of any one of claims 39 to 41, wherein R.sup.15 is hydrogen.

43. The compound of any one of claims 39 to 42, wherein R.sup.16 is optionally substituted C.sub.6-C.sub.10 aryl.

44. The compound of claim 43, wherein optionally substituted C.sub.6-C.sub.10 aryl is phenyl.

45. The compound of any one of claims 39 to 42, wherein R.sup.16 is optionally substituted C.sub.1-C.sub.6 heteroalkyl.

46. The compound of claim 45, wherein optionally substituted C.sub.1-C.sub.6 heteroalkyl is ##STR00066##

47. The compound of claim 39, wherein B has the structure: ##STR00067##

48. The compound of claim 39, wherein B has the structure: ##STR00068##

49. The compound of claim 39 or 40, wherein p is 2.

50. The compound of claim 49, wherein R.sup.15 is hydrogen.

51. The compound of claim 49 or 50, wherein R.sup.16 is optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl.

52. The compound of claim 51, wherein optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl is 2-fluoro-benzyl.

53. The compound of any one of claims 49 to 52, wherein B has the structure: ##STR00069##

54. The compound of claim 53, wherein B has the structure: ##STR00070##

55. The compound of any one of claims 1 to 54, wherein C has the structure of Formula IIIa: ##STR00071## wherein p is 0, 1, 2, 3, or 4; R.sup.7 is hydrogen or amido; each R.sup.8 is, independently hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl, cyano, or halo; and R.sup.9 is hydrogen or C.sub.1-C.sub.6 alkyl.

56. The compound of claim 55, wherein p is 0.

57. The compound of claim 55 or 56, wherein R.sup.9 is hydrogen.

58. The compound of any one of claims 55 to 57, wherein R.sup.7 is amido.

59. The compound of any one of claims 55 to 58, wherein C has the structure: ##STR00072##

60. The compound of claim 59, wherein C has the structure: ##STR00073##

61. The compound of any one of claims 1 to 54, wherein C has the structure: ##STR00074## wherein q is 0, 1, 2, 3, 4, or 5; each R.sup.11 is, independently hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl, cyano, or halo; and R.sup.12 and R.sup.13 are each, independently, hydrogen or C.sub.1-C.sub.6 alkyl.

62. The compound of claim 61, wherein R.sup.10 is hydrogen.

63. The compound of claim 60 or 61, wherein R.sup.12 is hydrogen.

64. The compound of any one of claims 60 to 63, wherein R.sup.13 is C.sub.1-C.sub.6 alkyl.

65. The compound of claim 64, wherein C.sub.1-C.sub.6 alkyl is methyl.

66. The compound of any one of claims 60 to 65, wherein q is 1.

67. The compound of any one of claims 60 to 66, wherein C has the structure: ##STR00075##

68. The compound of claim 67, wherein C has the structure: ##STR00076##

69. The compound of any one of claims 61 to 68, wherein R.sup.11 is cyano.

70. The compound of any one of claims 1 to 69, wherein A is ##STR00077##

71. The compound of any one of claims 1 to 69, wherein A is ##STR00078##

72. The compound of any one of claims 1 to 69, wherein A is ##STR00079##

73. A compound having the structure of any one of compounds 1-11 in Table 1.

74. A pharmaceutical composition comprising a compound of any one of claims 1 to 73 and a pharmaceutically acceptable excipient.

75. A method of inhibiting Bruton's tyrosine kinase, said method comprising contacting a cell with a compound of any one of claims 1 to 73.

76. A method of treating a B-cell associated disease or a mast cell associated disease in a subject in need thereof, said method comprising administering an effective amount of a compound of any one of claims 1 to 73 or a pharmaceutically acceptable salt thereof, or a composition of claim 74 to said subject.

77. The method of claim 76, wherein said B-cell associated disease or said mast cell associated disease is cancer, an inflammatory disorder, or an autoimmune disorder.

78. A method of treating cancer in a subject in need thereof, said method comprising administering an effective amount of a compound of any one of claims 1 to 73 or a pharmaceutically acceptable salt thereof, or a composition of claim 74 to said subject.

79. The method of claim 78, wherein said cancer is leukemia, lymphoma, myeloma, or a pancreatic neoplasm.

80. The method of claim 78 or 79, wherein said cancer is non-Hodgkin lymphoma, B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, pancreatic insulinoma, pancreatic glucagonoma, or pancreatic gastrinoma.

81. A method of treating an inflammatory or autoimmune disorder in a subject in need thereof, said method comprising administering an effective amount of a compound of any one of claims 1 to 73 or a pharmaceutically acceptable salt thereof, or a composition of claim 74 to said subject.

82. The method of claim 81, wherein said inflammatory or autoimmune disorder is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, idiopathic thrombocytopenic purpura, glomerulonephritis, autoimmune-mediated hemolytic anemia, immune complex mediated vasculitis, or psoriasis.

Description

DETAILED DESCRIPTION OF THE INVENTION

Compounds

[0127] The invention features novel compounds of Formula I:

ABCFormula I

[0128] wherein A is

##STR00029##

[0129] B has the structure:

##STR00030##

[0130] wherein B is conjugated to A at the nitrogen and to C at the carbonyl;

[0131] the dotted line is an optional double bond;

[0132] m is 0 or 1;

[0133] n is 0, 1, or 2;

[0134] o is 0 or 1;

[0135] p is 1 or 2;

[0136] R.sup.1 is hydrogen or combines with R.sup.3 or R.sup.4 to form a 5- to 6-membered carbocyclic ring;

[0137] R.sup.2 is hydrogen or C.sub.1-C.sub.6 alkyl;

[0138] X.sup.1 is S or CR.sup.3R.sup.4, wherein R.sup.3 and R.sup.4 are each, independently, hydrogen or combine with R.sup.1 to form a 5- to 6-membered carbocyclic ring, wherein if a double bond is present, then R.sup.4 is absent; and

[0139] X.sup.2 is CR.sup.5R.sup.6, wherein R.sup.5 and R.sup.6 are each, independently, hydrogen or C.sub.1-C.sub.6 alkyl, wherein if a double bond is present, R.sup.6 is absent;

[0140] R.sup.14 is hydrogen or C.sub.1-C.sub.6 alkyl;

[0141] each R.sup.15 and R.sup.16 is, independently, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.6-C.sub.10 aryl; or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl; and

[0142] C has the structure:

##STR00031##

[0143] wherein p is 0, 1, 2, 3, or 4;

[0144] q is 0, 1, 2, 3, 4, or 5;

[0145] R.sup.7 is hydrogen or amido;

[0146] each R.sup.8 and R.sup.11 is, independently hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl, cyano, or halo; and

[0147] R.sup.9, R.sup.10, R.sup.12, and R.sup.13 are each, independently, hydrogen or C.sub.1-C.sub.6 alkyl.

[0148] In some embodiments, the compounds have kinase (e.g., BTK) inhibitory activity. In some embodiments, the compounds may be useful in pharmaceutical and diagnostic compositions containing them and medical uses. Exemplary compounds of the invention are shown in Table 1, including stereoisomers (e.g., diastereomers or enantiomers), or pharmaceutically acceptable salts thereof.

TABLE-US-00001 TABLE 1 Exemplary Compounds of the Invention Compound # Structure 1 [00032] 2 [00033] 3 [00034] 4 [00035] 5 [00036] 6 [00037] 7 [00038] 8 [00039] 9 [00040] 10 [00041] 11 [00042]

[0149] Exemplary methods for synthesizing compounds of the invention are described herein.

Pharmaceutical Uses

[0150] The compounds described herein are useful in the methods of the invention and, while not bound by theory, are believed to exert their desirable effects through their ability to inhibit the activity of BTK. The compounds of the invention having useful BTK inhibiting activity, may be useful to treat, prevent, or reduce the risk of, diseases or conditions that are ameliorated by a reduction in BTK activity, such as a B-cell related disorder or a mast cell related disorder (e.g., any disorder described herein).

[0151] Cancer

[0152] BTK is a key regulator in B-cell development, differentiation, and signaling, as well as in mast cell activation. Accordingly, activation of BTK has been implicated in the pathology of numerous proliferative disorders, including B-cell, mast cell, and other non-B-cell associated cancers.

[0153] Exemplary proliferative disorders (e.g., cancers) include leukemia, including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), and B-cell prolymphocytic leukemia (B-PLL); lymphomas, including Hodgkin and non-Hodgkin lymphoma, such as B-cell lymphomas (e.g., diffuse large B-cell lymphoma (e.g., mediastinal (thymic) large B-cell lymphoma and intravascular large B-cell lymphoma), follicular lymphoma, small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (e.g., relapsed or refractory), marginal zone B-cell lymphomas (e.g., extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma), Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis); myelomas, including multiple myeloma (plasma cell myeloma), plasmacytoma, localized myeloma, and extramedullary myeloma; and other cancers, such as pancreatic neoplasms, including pancreatic exocrine tumors (e.g., ductal adenocarcinoma, signet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas, and undifferentiated carcinomas with osteoclast-like giant cells), pancreatic cystic neoplasms (e.g., mucinous cystadenoma, serous cystadenoma, and mucinous ductal ectasia), pancreatic neuroendocrine tumors (e.g., insulinoma, glucagonoma, gastrinoma (Zollinger-Ellison syndrome), VIPoma, and somatostatinoma), papillary cystic neoplasms of the pancreas, lymphoma of the pancreas, and acinar cell tumors of the pancreas; malignant glioma; and papillary thyroid cancer.

[0154] Inflammatory Disorders (Including Autoimmune Disorders)

[0155] Inhibition of BTK has been shown to mitigate inflammation and/or suppress the production of inflammatory cytokines. Accordingly, the compounds of the invention can be used to treat or prophylactically treat inflammatory disorders, including autoimmune disorders.

[0156] Exemplary inflammatory or autoimmune disorders include rheumatoid arthritis, systemic lupus erythematosus (and associated glomerulonephritis), multiple sclerosis, and asthma. Further exemplary disorders include acute disseminated encephalomyelitis, Addison's disease, allergy, alopecia universalis, Alzheimer's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, appendicitis, atherosclerosis, autoimmune arthritis (e.g., rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Still's disease, juvenile arthritis, and mixed and undifferentiated connective tissue diseases), autoimmune hemolytic and thrombocytopenic states (e.g., autoimmune-mediated hemolytic anemia, e.g., warm autoimmune hemolytic anemia, cold autoimmune hemolytic anemia, cold agglutinin disease, and paroxysmal cold hemoglobinuria), autoimmune hepatitis, Behet's disease, blepharitis, bronchiolitis, bronchitis, bursitis, celiac disease, cervicitis, cholangitis, cholecystitis, chronic fatigue, chronic idiopathic thrombocytopenic purpura (ITP), colitis, conjunctivitis, Crohn's disease, cystitis, dacryoadenitis, dermatitis (including contact dermatitis), dermatomyositis, diabetes, dysautonomia, eczema, encephalitis, endocarditis, endometriosis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibromyalgia (fibrositis), gastritis, gastroenteritis, gingivitis, glomerulonephritis, Goodpasture's syndrome (and associated glomerulonephritis and pulmonary hemorrhage), Graves' disease, Guillain-Barr syndrome, Hashimoto's thyroiditis, hepatitis, hidradenitis suppurativa, hyperacute rejection of transplanted organs, idiopathic thrombocytopenic purpura (ITP), inflammatory bowel disease (including Crohn's disease and ulcerative colitis), inflammatory pelvic disease, interstitial cystitis, irritable bowel syndrome, juvenile arthritis, juvenile idiopathic arthritis, laryngitis, mastitis, meningitis, multiple vasculitides, myasthenia gravis, myelitis myocarditis, myocarditis, myositis, nephritis, neuromyotonia, oophoritis, opsoclonus-myoclonus syndrome, optic neuritis, orchitis, Ord's thyroiditis, osteitis, osteoarthritis, osteomyelitis, otitis, pancreatitis, Parkinson's disease, parotitis, pericarditis, peritonitis, pharyngitis, phlebitis, pleuritis, pneumonia, pneumonitis, primary biliary cirrhosis, proctitis, prostatitis, psoriasis (including psoriatic lesions in the skin), psoriatic arthritis, pyelonephritis, Reiter's syndrome, rheumatoid arthritis, rhinitis (including allergic rhinitis), rosacea, salpingitis, scleroderma, septic shock, sinusitis, Sjgren's syndrome, skin sunburn, skin sunburn, Still's disease, stomatitis, synovitis, Takayasu's arteritis, temporal arteritis, tendonitis, tissue graft rejection, tonsillitis, urethritis, urticaria, uveitis, uvitis, vaginitis, vasculitis (including antineutrophil cytoplasmic antibodies-associated vasculitis and immune complex mediated vasculitis), vulvitis, vulvodynia, warm autoimmune hemolytic anemia, and Wegener's granulomatosis.

Combination Formulations and Uses Thereof

[0157] The compounds of the invention can be combined with one or more therapeutic agents. In particular, the therapeutic agent can be one that treats or prophylactically treats any disorder described herein, such as a B-cell related disorder, cancer, or an inflammatory or autoimmune disorder.

Combination Formulations

[0158] In addition to the formulations described herein, one or more compounds of the invention can be used in combination with other therapeutic agents. For example, one or more compounds of the invention can be combined with another therapeutic agent. Exemplary therapeutic agent useful for this purpose include, without limitation, those described in U.S. Pat. Nos. 8,008,309; 7,943,618; 7,884,108; 7,868,018; 7,825,118; 7,642,255; 7,501,410; 7,405,295; 6,753,348; and 6,303,652. In particular embodiments, the compound of the invention is used in combination with an anti-cancer agent or an anti-inflammatory agent (e.g., a nonsteroidal anti-inflammatory drug, acetaminophen, a gold complex, a corticosteroid, or an immunosuppressant).

[0159] Non-limiting, exemplary anti-cancer agents include fludarabine, cyclophosphamide, methotrexate, rituximab, bendamustine, ofatumumab, dasatinib, U0126 ((2Z,3Z)-2,3-bis[amino-(2-aminophenyl)sulfanylmethylidene]butanedinitrile), PD98059 (2-(2-amino-3-methoxyphenyl)chromen-4-one), PD184352 (2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide), PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide), ARRY-142886 (6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), SB 239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol), SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one), BAY 43-9006 (sorafenib or 4-[4-[[4-chloro-3(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide), wortmannin, or LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one or a hydrochloride salt thereof). Additional non-limiting, exemplary classes of anti-cancer agents include other kinase inhibitors (e.g., a BTK inhibitor, e.g., PCI-32765 (1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one), LOB 03-0110 ((3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol), ()-terreic acid ((1R,6S)-3-hydroxy-4-methyl-7-oxabicyclo[4.1.0]hept-3-ene-2,5-dione), LFM-A13 (2-cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide), staurosporine, and dasatinib), topoisomerase I inhibitors (e.g., camptothecin and topotecan), topoisomerase II inhibitors (e.g., daunomycin and etoposide), alkylating agents (e.g., cyclophosphamide, melphalan, and carmustine (BCNU)), and anti-tubulin agents (e.g., taxol and vinblastine).

[0160] Non-limiting, exemplary anti-inflammatory agents include a nonsteroidal anti-inflammatory drug (an NSAID, e.g., non-specific and COX-2 specific cyclooxgenase enzyme inhibitors), acetaminophen, a gold complex, a corticosteroid, and an immunosuppressant. Non-limiting examples of NSAIDs include acemetacin, aspirin, celecoxib, deracoxib, diclofenac, diflunisal, ethenzamide, etodolac, etofenamate, etoricoxib, fenoprofen, flufenamic acid, flurbiprofen, hydroxychloroquine, ibuprofen, indomethacin, isoxicam, kebuzone, ketoprofen, ketorolac, lonazolac, lornoxicam, lumiracoxib, meclofenamic acid, mefenamic acid, meloxicam, metamizol, misoprostol, mofebutazone, naproxen, nabumetone, niflumic acid, piroxicam, oxaprozinpiroxicam, oxyphenbutazone, parecoxib, phenidone, phenylbutazone, piroxicam, propacetamol, propyphenazone, rofecoxib, salicylamide, salsalate, sulfasalazine, sulindac, suprofen, tiaprofenic acid, tenoxicam, tolmetin, valdecoxib, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide, 2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methylbutoxy)-5-[4-(methylsulfonyl)phenyl]-3(2H)-pyridazinone, and 2-(3,5-difluorophenyl)-3-[4-(methylsulfonyl)phenyl]-2-cyclopenten-1-one. Non-limiting examples of gold complexes include aurothioglucose, auranofin disodium aurothiomalate, sodium aurothiomalate, and sodium aurothiosulfate. Non-limiting examples of corticosteroids include cortisone, dexamethasone, methylprednisolone, prednisolone, prednisolone sodium phosphate, and prednisone. Non-limiting examples of immunosuppressants include alkylation agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine, methotrexate, leflunomide, and mycophenolate mofetil), antibodies or antibody fragments or derivatives (e.g., an anti-C5 monoclonal antibody, such as eculizumab or pexelizumab; and a TNF antagonist, such as entanercept or infliximab, or fragments or derivatives of any of these), and macrolides (e.g., cyclosporine and tacrolimus).

[0161] Combination Therapies

[0162] A compound of the invention can be used alone or in combination with other agents that have BTK-inhibiting activity, or in combination with other types of treatment (which may or may not inhibit BTK) to treat, prevent, and/or reduce the risk of cancer, an inflammatory disorder, or other disorders that benefit from BTK inhibition. In combination treatments, the dosages of one or more of the therapeutic compounds may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6, 2005). In this case, dosages of the compounds when combined should provide a therapeutic effect.

Pharmaceutical Compositions

[0163] The compounds of the invention are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention in admixture with a suitable diluent, carrier, or excipient.

[0164] The compounds of the invention may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the scope of the invention. In accordance with the methods of the invention, the described compounds or salts, solvates, or prodrugs thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0165] A compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, a compound of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.

[0166] A compound of the invention may also be administered parenterally. Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20th ed.) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19), published in 1999.

[0167] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe.

[0168] Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.

[0169] The compounds of the invention may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.

Dosages

[0170] The dosage of the compounds of the invention, and/or compositions comprising a compound of the invention, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds of the invention are administered to a human at a daily dosage of, for example, between 0.05 mg and 3000 mg (measured as the solid form). Dose ranges include, for example, between 10-1000 mg (e.g., 50-800 mg). In some embodiments, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg of the compound is administered. Preferred dose ranges include, for example, between 0.05-15 mg/kg or between 0.5-15 mg/kg.

[0171] Alternatively, the dosage amount can be calculated using the body weight of the patient. For example, the dose of a compound, or pharmaceutical composition thereof, administered to a patient may range from 0.1-50 mg/kg (e.g., 0.25-25 mg/kg). In exemplary, non-limiting embodiments, the dose may range from 0.5-5.0 mg/kg (e.g., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/kg) or from 5.0-20 mg/kg (e.g., 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg).

Diagnostic and Screening Assays

[0172] In addition to the above-mentioned therapeutic uses, a compound of the invention can also be used in diagnostic assays, screening assays, and as a research tool.

[0173] In diagnostic assays, a compound of the invention may be useful in identifying or detecting BTK activity.

[0174] In screening assays, a compound of the invention may be used to identify other compounds that inhibit BTK, for example, as first generation drugs. As research tools, the compounds of the invention may be used in enzyme assays and assays to study the extent of BTK activity. Such information may be useful, for example, for diagnosing or monitoring disease states or progression. In such assays, a compound of the invention may also be radiolabeled.

BTK In Vitro Inhibition Assays

[0175] The compounds of the present invention have been found to exhibit BTK inhibition. Compounds may be examined for their efficacy in inhibiting kinase activity by a person skilled in the art, for example, by using the methods described in Example 1 and the other examples provided herein or by methods known in the literature (e.g., Mast Cells: Methods and Protocols (eds. G. Krishnaswamy and D. S. Chi), Methods in Molecular Biology, Series 315, Humana Press, pp. 175-192, 2006).

[0176] Inhibitory activity can be determined by any useful method. For example, inhibition can be determined by the effect of a test compound on BTK autophosphorylation. Btk and varying concentrations of the test compound can be included in a [-.sup.32P]ATP-containing kinase buffer. Autophosphorylation can be analyzed by SDS/PAGE followed by electroblotting and autoradiography, where phosphorylated protein bands can be quantified by densitometry. These assays can be conducted without or with an exogenous substrate (e.g., glutathione S-transferase (GST)-IG).

[0177] In another example, inhibitory activity can be determined by the effect of a test compound on BTK binding. For example, BTK can bind to protein kinase C (PKC) in vivo, where PKC in turn phosphorylates BTK. Accordingly, an exemplary assay to assess BTK-PKC binding includes incubating PKC or cell lysates having PKC (e.g., lysates from human mast cell lines) with glutathione S-transferase (GST)-Btk beads in the absence or presence of the test compound. Then, the extent of Btk-bound PKC can be detected by any useful manner, such as by SDS/PAGE followed by immunoblotting with anti-PKC (MC5) and/or anti-BTK antibodies.

[0178] Further examples include use of cellular assays, such as by determining the effect of a test compound on cellular activation. For example, stimulated lymphoid, myeloid, or mast cells (e.g., cells stimulated with a signaling molecule, such as erythropoietin or an antigen, such as IgE) can be incubated with a test compound, and the activation of particular compounds or proteins can be measured. Exemplary compounds and proteins include histamine, leukotriene, cytokines, PKC, Janus tyrosine kinase 2 (Jak2), erythropoietin receptor (EpoR), Stat5, protein kinase B (PKB), and/or mitogen activating protein kinase (Erk1/2). In another example, as activated Btk can be phosphorylated at tyrosine 223 (Y223) and/or tyrosine 551 (Y551), cellular assays can be conducted by staining P-Y223 or P-Y551-positive cells in a population of cells (e.g., by phosphorylation-specific immunochemical staining followed by FACS analysis).

[0179] As BTK is a tyrosine kinase, additional useful assays include any tyrosine kinase assay. In particular, commercially available assays include kinase assays that detect formation of ADP, e.g., with luminescent detection, such as in an ADP-Glo Kinase Assay (Promega Corp., Madison, Wisc.). Dose response curves can be obtained by incubating BTK with a substrate (e.g., ATP or a binding partner, such as PKC) and increasing (e.g., logarithmically increasing) the concentration of a test compound. In addition, a detectable agent (e.g., a luminescent probe, such as a luciferase/luciferin reaction that measures ATP) can be used to correlate kinase activity (e.g., ATP-to-ADP conversion) with the concentration of the test compound. These data can be used to construct a dose response curve, where IC.sub.50 is the concentration of the test compound that provides about 50% inhibition.

[0180] The following non-limiting examples are illustrative of the present invention.

EXAMPLES

Example 1

Synthesis of Compound 1

[0181] ##STR00043##

Experimental Procedure:

[0182] ##STR00044##

[0183] Synthesis of tert-butyl (R)-3-(methylcarbamoyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate: To a solution of (R)-2-(tert-butoxycarbonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid (200 mg, 0.63 mmol) and (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (420 mg, 0.94 mmol) in DMF (3 mL), was added methylamine hydrochloride (84 mg, 1.26 mmol) and diisopropylethylamine (0.39 mL, 1.90 mmol). The mixture was stirred at room temperature overnight, diluted with DCM (5 mL), washed with water (3 mL3), dried over sodium sulfate and concentrated. Product was purified using silica gel column chromatography with 0-50% THF in DCM to give the title compound as a white solid (175 mg).

##STR00045##

Synthesis of tert-butyl (R)-3-(2-((R)-3-(methylcarbamoyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2-oxoethyl)piperidine-1-carboxylate: To a solution of tert-butyl (R)-3-(methylcarbamoyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate (175 mg from step 1) in DCM (5 mL) was added 4.0 M HCl in dioxane (3.0 mL). The mixture was stirred at room temperature for 2 hrs before diethylether (10 mL) was added. Solid was collected to give the deprotected compound (150 mg) as an HCl salt. To a solution of (R)-2-(1-(tert-butoxycarbonyl)piperidin-3-yl)acetic acid (109 mg, 0.45 mmol) and BOP (199 mg, 0.45 mmol) in DMF (3 mL) was added the product above (100 mg, 0. 38 mmol) and diisopropylethylamine (0.26 mL, 1.52 mmol). The mixture was stirred at room temperature overnight, diluted with DCM (5 mL), washed with water (3 mL3), dried over sodium sulfate and concentrated. Product was purified using silica gel column chromatography with 0-50% THF in DCM to give the title compound as a brown oil. It was used in the next step without further purification.

##STR00046##

Synthesis of (R)-2-(2-((R)-1-acryloylpiperidin-3-yl)acetyl)-N-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-1]indole-3-carboxamide (1): To a solution of tert-butyl (R)-3-(2-((R)-3-(methylcarbamoyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2-oxoethyl)piperidine-1-carboxylate (from step 3) in DCM (5 mL) was added 4.0M HCl in dioxane (1.5 mL). The mixture was stirred at RT for 2 hrs before diethylether (10 mL) was added. Solid was collected to give deprotected compound as an HCl salt. To a solution of the deprotected intermediate (50 mg, 0.13 mmol) in DMF (2 mL) was added acrylic acid (0.13 mmol) and HBTU (66 mg, 0.17 mmol) followed by the addition of TEA (0.083 mL, 0.60 mmol). The mixture was stirred at RT overnight, diluted with DCM (5 mL), washed with water (3 mL3), dried over sodium sulfate and concentrated. Product was purified by silica gel column chromatography with 0-10% methanol in DCM to give 1 as a yellow solid (17 mg). LC-MS (M+H).sup.+=409.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) 10.78 (d, 1H), 7.88-7.77 (m, 1H), 7.40 (d, J=7.5 Hz, 1H), 7.31-7.27 (m, 1H), 7.06-6.90 (m, 2H), 6.85-6.72 (m, 1H), 5.50-5.70 (m, 2H), 5.20-4.50 (m, 2H), 4.24-3.97 (m, 2H), 3.63-3.59 (m, 2H), 3.10-3.05 (m, 5H), 1.86-1.84 (m, 2H), 1.63 (bs, 1H), 1.12-1.20 (m, 5H).

Example 2

Synthesis of Compound 2

[0184] ##STR00047##

Experimental Procedure

[0185] ##STR00048##

Synthesis of tert-butyl (S)-(3-(4-cyanophenyl)-1-(methylamino)-1-oxopropan-2-yl)carbamate: To a solution of (S)-2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propanoic acid (200 mg, 0.69 mmol) and (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (457 mg, 1.03 mmol) in DMF (2 mL) was added methylamine hydrochloride (92 mg, 1.38 mmol) and diisopropylethylamine (0.36 mL, 2.07 mmol). The mixture was stirred at room temperature overnight, diluted with DCM (5 mL), washed with water (3 mL3), dried over sodium sulfate and concentrated. Product was purified using silica gel column chromatography with 0-50% THF in DCM to give the title compound as a white solid (160 mg).

##STR00049##

Synthesis of tert-butyl (2S,3aS,7aS)-2-(((S)-3-(4-cyanophenyl)-1-(methylamino)-1-oxopropan-2-yl)carbamoyl)octahydro-1H-indole-1-carboxylate: To a solution of tert-butyl (S)-(3-(4-cyanophenyl)-1-(methylamino)-1-oxopropan-2H)carbamate (160 mg from step 1) in DCM (5 mL) was added 4.0M HCl in dioxane (3.0 mL). The mixture was stirred at room temperature for 2 hrs before diethylether (10 mL) was added. Solid was collected to give the product (150 mg) as an HCl salt. To a solution of Boc-L-octahydroindole-2-carboxylic acid (134 mg, 0.5 mmol) and BOP (221 mg, 0.5 mmol) in DMF (3 mL) was added the product above (100 mg, 0. 42 mmol) and diisopropylethylamine (0.29 mL, 1.68 mmol). The mixture was stirred at room temperature overnight, diluted with DCM (5 mL), washed with water (3 mL3), dried over sodium sulfate and concentrated. Product was purified using silica gel column chromatography with 0-50% THF in DCM to give the title compound as a white solid (180 mg).

##STR00050##

Synthesis of (2S,3aS,7aS)-1-acryloyl-N-((S)-3-(4-cyanophenyl)-1-(methylamino)-1-oxopropan-2-yl)octahydro-1H-indole-2-carboxamide (2): To a solution of tert-butyl (2S,3aS,7aS)-2-(((S)-3-(4-cyanophenyl)-1-(methylamino)-1-oxopropan-2-yl)carbamoyl)octahydro-1H-indole-1-carboxylate (180 mg) in DCM (5 mL), was added 4.0M HCl in dioxane (1.5 mL). The mixture was stirred at RT for 2 hrs before diethylether (10 mL) was added. Solid was collected to give product as an HCl salt. To a solution of the product (30 mg, 0.077 mmol) in DMF (2 mL) was added a solution of acrylic acid (0.12 mmol) in DMF (100 uL) and HBTU (35 mg, 0.092 mmol), followed by the addition of TEA (0.043 mL, 0.31 mmol). The mixture was stirred at room temperature overnight, diluted with DCM (5 mL), washed with water (3 mL3), dried over sodium sulfate and concentrated. Product was purified using silica gel column chromatography with 0-10% methanol in DCM to give 2 as a white solid (10 mg) after collecting only the pure fractions from the column. LC-MS (M+H).sup.+=409.
.sup.1H NMR (300 MHz, CDCl.sub.3) 7.53 (d, J=8.1 Hz, 2H), 7.30 (d, 2H), 6.88 (d, J=8.1 Hz, 1H), 6.74 (bs, 1H), 6.37-6.39 (m, 2H), 5.78-5.82 (m, 1H), 4.74-4.77 (m, 1H), 4.47 (t, 1H), 3.82-3.85 (m, 1H), 3.44-3.13 (m, 2H), 2.82 (d, J=4.8 Hz, 3H), 2.35-2.01 (m, 3H), 1.81-1.10 (m, 8H).

Example 3

Synthesis of Compound 11

[0186] ##STR00051##

Example 4

Determination of BTK inhibition Activity

[0187] The compounds were assayed for BTK inhibition activity using the Invitrogen LanthaScreen Kinase Binding Assay. In short, the compounds were tested for their ability to displace a tracer (in this case Invitrogen Kinase Tracer 236) from the active site of BTK. The BTK protein used in the assay was labeled with europium (Eu), and so displacement was conveniently detected as a loss of Eu-to-tracer FRET (fluorescence resonance energy transfer) signal using a plate reader equipped to measure TR-FRET (time resolved FRET). This displacement assay is commonly used to characterize kinase inhibitors and it is predictive of kinase inhibitory activity.

[0188] Several of the compounds were also tested directly for kinase inhibitory activity using the Invitrogen Omnia assay. The Omnia assay is a real time kinetic assay that uses a phosphate-induced fluorophore to detect transfer of phosphate from ATP to a peptide. Inhibition of kinase activity in this assay reduces the rate of fluorescence increase. Compounds tested in both assays demonstrated similar IC.sub.50 values. More details and experimental protocols for both assays can be found at invitrogen.com.

[0189] Determination of IC.sub.50 Values

[0190] Various compounds of the invention (i.e., compounds of formula (I) or (Ia)) were assayed for BTK inhibition activity, as described above, and possessed IC.sub.50 values less than 1.0 M. In some embodiments, the compounds possessed IC.sub.50 values less than 0.9 M, less than 0.8 M, less than 0.5 M, less than 0.3 M, less than 0.2 M, less than 0.1 M, less than 0.09 M, less than 0.08 M, less than 0.05 M, less than 0.04 M, less than 0.03 M, less than 0.025 M, less than 0.015 M, less than 0.01 M, less than 0.005 M, less than 0.002 M, less than 0.0015 M, or less than 0.001 M. In some embodiments, the compounds possessed IC.sub.50 values from 0.0001 M to 0.9 M (e.g., from 0.0001 M to 0.8 M, from 0.0001 M to 0.5 M, from 0.0001 M to 0.3 M, from 0.0001 M to 0.2 M, from 0.0001 M to 0.1 M, from 0.0001 M to 0.09 M, from 0.0001 M to 0.08 M, from 0.0001 M to 0.05 M, from 0.0001 M to 0.04 M, from 0.0001 M to 0.03 M, from 0.0001 M to 0.025 M, from 0.0001 M to 0.015 M, from 0.0001 M to 0.01 M, from 0.0001 M to 0.005 M, 0.0002 M to 0.9 M, from 0.0002 M to 0.8 M, from 0.0002 M to 0.5 M, from 0.0002 M to 0.3 M, from 0.0002 M to 0.2 M, from 0.0002 M to 0.1 M, from 0.0002 M to 0.09 M, from 0.0002 M to 0.08 M, from 0.0002 M to 0.05 M, from 0.0002 M to 0.04 M, from 0.0002 M to 0.03 M, from 0.0002 M to 0.025 M, from 0.0002 M to 0.015 M, from 0.0002 M to 0.01 M, from 0.0002 M to 0.005 M, 0.0005 M to 0.9 M, from 0.0005 M to 0.8 M, from 0.0005 M to 0.5 M, from 0.0005 M to 0.3 M, from 0.0005 M to 0.2 M, from 0.0005 M to 0.1 M, from 0.0005 M to 0.09 M, from 0.0005 M to 0.08 M, from 0.0005 M to 0.05 M, from 0.0005 M to 0.04 M, from 0.0005 M to 0.03 M, from 0.0005 M to 0.025 M, from 0.0005 M to 0.015 M, from 0.0005 M to 0.01 M, from 0.0005 M to 0.005 M, from 0.0005 M to 0.002 M, from 0.0005 M to 0.0015 M, or from 0.0005 M to 0.001 M).

Example 5

Determination of Compound Off-Rate

[0191] Biotinylated BTK (20 nM final concentration) was premixed with Europium labeled streptavidin (10 nM final concentration) and the complex was incubated for 18 hours at room temperature with compounds at various concentrations in assay buffer (25 mM HEPES, 10 mM MgCl.sub.2, 0.5 mg/ml bovine serum albumin, 1% DMSO) in a final volume of 100 L. Following this incubation, 2 L of the BTK/compound mixture was diluted 10-fold into 18 L of buffer containing 30 nM Kinase Tracer 236 (ThermoFisher), an Alexa Fluor conjugated non-covalent compound that binds to multiple kinase active sites including BTK. The final concentration of compound after dilution was at 2-30 fold below its IC.sub.50 as determined by a competition binding assay also using Tracer 236. The TR-FRET signal generated by the proximity of the Eu-streptavidin to Tracer 236 when both bind simultaneously to BTK was read in a Tecan Infinite M1000 Pro plate reader (excitation 620 nm, emission 665 nm) at regular intervals over 6 hours. An increase in TR-FRET signal over time should be closely related to the off-rate of the compound after dilution. Controls without BTK and with BTK only, not including compound, were also included, setting the low and high ranges of the assay respectively. Control assays with non-covalent BTK inhibitors resulted in a steady increase in TR-FRET signal over time indicating that these compounds could be displaced by the tracer. A covalent BTK inhibitor, ibrutinib, completely prevented the increase in TR-FRET signal indicating that, consistent with its covalent mechanism of action, ibrutinib could not be displaced by the tracer.

Other Embodiments

[0192] While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0193] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

[0194] Other embodiments are in the claims.