USOO7662824B2

(12) United States Patent (10) Patent No.: US 7,662,824 B2 Leonard et al. (45) Date of Patent: Feb. 16, 2010

(54) ACYLEHYDRAZONES AS KINASE JP 11302173 11, 1999 MODULATORS WO 990 1434 1, 1999 WO 99.15500 4f1999 (75) Inventors: Kristi Leonard, West Chester, PA (US); WO 9951579 10, 1999 Tianbao Lu, Churchville, PA (US); WO 200006566 2, 2000 Robert W. Tuman, Chalfont, PA (US); W 38: 28 Dana L. Johnson, Upper Black Eddy, WO 2OOOO71493 11, 2000 PA (US); Anna C. Maroney, Media, PA WO 2001023386 4/2001 (US); Jan L. Sechler, Doylestown, PA WO 2002OOO661 1, 2002 (US); Richard W. Connors, WO 2002028823 4/2002 Harleysville, PA (US); Richard S. WO 2002040507 5, 2002 Alexander, Newark, DE (US); Maxwell WO 2002O83630 10, 2002 D. Cummings, Wayne, PA (US); Robert WO 2002O85888 10, 2002 A. Galemmo, Paoli, PA (US); Thomas WO 2002O96361 12/2002 P. Markotan, Morgantown, PA (US) WO 2002O96426 12/2002 s s WO 2003O37328 5, 2003 (73) Assignee: Janssen Pharmaceutica NV, Beerse W S.O.R. 3. (BE) WO 2004.0561.78 T 2004 (*) Notice: Subject to any disclaimer, the term of this WO 2005.005378 1, 2005 patent is extended or adjusted under 35 U.S.C. 154(b) by 90 days. OTHER PUBLICATIONS Wikipedia, definition of “Medicine”.* (21) Appl. No.: 11/377,077 STN search result STN-1 1377.077A-clear art. Fahmy etal. Egyptian Journal of Chemistry, (2003), 46(2),313-327.* (22) Filed: Mar 16, 2006 Bieche, et al., Int. J. Cancer 82 (1999), pp. 908-910. O O Camp et al., Cancer 86 (1999), pp. 2259-2265. (65) Prior Publication Data DiRenzo et al., J. Endocrinol. Invest 18 (1995), pp. 134-139. US 2007/006661.0 A1 Mar. 22, 2007 DiRenzo et al., Oncogene 19 (2000), pp. 1547-1555. DiRenzo et al., Int. J. Cancer 58(1994), pp. 658-662. Related U.S. Application Data (Continued) (60)60) Provisional"S"E. application No. 60/689,917, filedE. on Jun. Primary Examiner Yong Chu filed on Mar. 18, 2005. (57) ABSTRACT

(51) A6Int. IK Cl. 3/496 (2006.01) The invention is directed- 0 to acylhydrazones compounds of A6 IK 3/404 (2006.01) Formula I: CO7D 405/4 (2006.01) CO7D 405/2 (2006.01) CO7D 407/4 (2006.01) O (52) U.S. Cl...... 514/254.09: 514/414: 514/418; 514/422:514/323: 514/469: 514/416: 544/373; 544/144; 546/201: 548/454: 548/483: 548/518; RI -NH 548/525; 548/490; 549/462 (58) Field of Classification Search ...... 514/.414, 514/418, 352.2, 323,422, 254.09, 469: 544/144, O 544/373; 546/201: 548/454, 483,518, 525; 5497462 R2 N See application file for complete search history. where R', R and A are as defined herein, the use of such (56) References Cited compounds as protein tyrosine kinase modulators, particu FOREIGN PATENT DOCUMENTS larly inhibitors ofc-Met, and the use of such compounds tO reduce or inhibit kinase activity of c-Met in a cellor a subject, CA 2352.194 4/2001 and modulate c-Met expression in a cell or subject, and the DE 19829229 1, 1999 use of such compounds for preventing or treating in a subject DE 1994.6289 3, 2001 a cell proliferative disorder and/or disorders related to c-Met. DE 101 17823 10, 2002 The present invention is further directed to pharmaceutical EP 672659694.808 12/20011, 1999 compositions comprising the compounds. of the present EP 1193248 4/2002 invention and to methods for treating conditions such as can EP 1229034 4/2005 cers and other cell proliferative disorders. JP 11007O93 1, 1999 JP 1101.5090 1, 1999 12 Claims, 2 Drawing Sheets US 7,662.824 B2 Page 2

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U.S. Patent Feb. 16, 2010 Sheet 2 of 2 US 7,662.824 B2

C. c. v. C W (6) tibleMudstun O %, US 7,662,824 B2 1. 2 ACYLHYDRAZONES AS KINASE none derivatives as vasopressin V2 receptor antagonists); MODULATORS WO 9739748 (acrylic acids as modulators of molecules with phosphotyrosine recognition units); WO 973 1910 (heterocy CROSS REFERENCE TO RELATED clic fibrinogen receptor antagonists); WO9729073 (substi APPLICATIONS tuted amide derivatives as protein-farnesyltransferase inhibi tors); U.S. Pat. No. 5,550,003 (silver halide photographic This application claims priority to U.S. Provisional Appli photosensitive material); EP 694808 (Process of forming cation for Patent No. 60/663,282, filed Mar. 18, 2005, and Super high-contrast negative images and silver halide photo U.S. Provisional Application for Patent No. 60/689,917 filed graphic material and developer being used therefore); and EP Jun. 13, 2005, the entire disclosure of which are hereby incor 10 672659 (L-Arginine aldehyde peptide derivatives useful as porated in their entirely. antithrombotic agents). Protein kinases are enzymatic components of the signal FIELD OF THE INVENTION transduction pathways which catalyze the transfer of the ter minal phosphate from ATP to the hydroxy group of tyrosine, The invention relates to novel compounds that function as 15 serine and/or threonine residues of proteins. Thus, com protein tyrosine kinase modulators. More particularly, the pounds which inhibit protein kinase functions are valuable invention relates to novel compounds that function as inhibi tools for assessing the physiological consequences of protein tors of c-Met. kinase activation. The overexpression or inappropriate expression of normal or mutant protein kinases in mammals BACKGROUND OF THE INVENTION has been a topic of extensive study and has been demonstrated to play a significant role in the development of many diseases, The present invention relates to acylhydrazones as inhibi including diabetes, angiogenesis, psoriasis, restenosis, ocular tors of tyrosine kinases, including c-Met. Acylhydrazones diseases, Schizophrenia, rheumatoid arthritis, atherosclero known in the art, including those with therapeutic properties, sis, cardiovascular disease and cancer. The cardiotonic ben include: CHEMCATS Accession No. 2004:37.93350 25 efits of kinase inhibition has also been studied. In sum, inhibi Ambinter Stock Screening Collection. (1 Jan. 2004); WO tors of protein kinases have particular utility in the treatment 2003037328 (oxindole hydrazide modulators of protein of human and animal disease. tyrosine phosphatases); WO 2005.005378 (indolinone The hepatocyte growth factor (HGF) (also known as scatter hydrazides as C-Met inhibitors); WO 2004.0561.78 (pesticidal factor) receptor, c-Met, is a receptor tyrosine kinase which chloropyridinamino derivatives); US 2004067996 (hydanto 30 regulates cell proliferation, morphogenesis, and motility. The ins as inhibitors of matrix metalloproteinases and/or TNF-C. c-Met gene is translated into a 170 kD protein which is pro converting enzyme (TACE)); WO 2003097649(1-oxa-diben cessed into a cell surface receptor composed of a 140 kD B ZoaZulenes as inhibitors of tumor necrosis factor production transmembrane subunit and 50 kD glycosylated extracellular and intermediates for the preparation thereof); WO C. Subunit. 2003037328 (oxindole hydrazide modulators of protein 35 Mutations inc-Met, over-expression of c-Met and/or HGF/ tyrosine phosphatases (PTPs)): WO 2002096426 (spiro SF, expression of c-Met and HGF/SF by the same cell, and fused hydantoin derivatives as inhibitors of matrix metallo overexpression and/or aberrant c-Met signaling is presentina proteinases); DE 101 17823 and WO 2002083630 (ethanedia variety of human solid tumors and is believed to participate in mides as inhibitors of blood coagulation factor Xa for the angiogenesis, tumor development, invasion, and metastasis. treatment of thromboembolic illnesses); WO 2002040507 40 Cell lines with uncontrolled c-Met activation, for example, (novel compounds for use in radioimmunoscintigraphy and/ are both highly invasive and metastatic. A notable difference or radioimmunotherapy of cancers); EP 1193248 and WO between normal and transformed cells expressing c-Met 2002028823 (arylmalonamides and -malonamic esters with receptor is that phosphorylation of the tyrosine kinase domain as Factor VIIa inhibitors with antithrombotic activity); WO in tumor cells is often independent of the presence of ligand. 2002000661 (pyrrolo2,3-dipyrimidines as immunosuppres 45 C-Met mutations/alterations have been identified in a num sive agents); DE 1994.6289 and WO 2001023386 (2-phenyl ber of human diseases, including tumors and cancers—for 5,6-dihydro-imidazo[4,5,1-jk]1,4)benzodiazepin-7(4H)- instance, hereditary and sporadic human papillary renal car ones as poly(ADP ribose) polymerase inhibitors); WO cinomas, breast cancer, colorectal cancer, gastric carcinoma, 2000071511 (benzamidines as inhibitors of factor Xa); WO glioma, ovarian cancer, hepatocellular carcinoma, head and 2000071493 (benzamidines and arylamidines as inhibitors of 50 neck squamous cell carcinomas, testicular carcinoma, basal factor Xa); WO 2000071509 (benzamidines and aryla cell carcinoma, liver carcinoma, sarcoma, malignant pleural midines as inhibitors of factor Xa); U.S. Pat. No. 6,075,044 mesothelioma, melanoma, multiple myeloma, osteosarcoma, (Isobenzofurandione and hydrazonodihydroindolone deriva pancreatic cancer, prostate cancer, synovial sarcoma, thyroid tives as protozoacides, acting as inhibitors of purine salvage carcinoma, non-Small cell lung cancer (NSCLC) and Small phosphoribosyltransferases); JP 11302173 (Benzamide 55 cell lung cancer, transitional cell carcinoma of urinary blad derivatives as histone deacetylase inhibitors for treating der, testicular carcinoma, basal cell carcinoma, liver carci tumors and other diseases); WO9951579 (III(alkoximino) noma—and leukemias, lymphomas, and myelomas—for alkoxymethylphenylaloximinoacetates and analogs as instance, acute lymphocytic leukemia (ALL), acute myeloid agrochemical fungicides); JP 11015090 (silver halide photo leukemia (AML), acute promyelocytic leukemia (APL), graphic material for printing platemaking); JP 11007093 60 chronic lymphocytic leukemia (CLL), chronic myeloid leu (photographic emulsion containing high-sensitive oxalylhy kemia (CML), chronic neutrophilic leukemia (CNL), acute drazine derivative for graphic arts); WO990 1434 (imidazole undifferentiated leukemia (AUL), anaplastic large-cell lym containing quinoline and benzazepine derivatives as inhibi phoma (ALCL), prolymphocytic leukemia (PML), juvenile tors of farnesyl protein transferase); DE 19829229 and U.S. myelomonocyctic leukemia (JMML), adult T-cell ALL AML Pat. No. 6,235,787 (hydroxycarbamoylalkylcarboxylic acid 65 with trilineage myelodysplasia (AML/TMDS), mixed lin hydrazides as inhibitors of tumor necrosis factor and trans eage leukemia (MLL), myelodysplastic syndromes (MDSs), forming growth factor release); WO 9825901 (spiro indoli myeloproliferative disorders (MPD), multiple myeloma, US 7,662,824 B2 3 4 (MM), myeloid sarcoma, non-Hodgkin’s lymphoma and pressed in Some cases of human leukemia and lymphoma. Hodgkin’s disease (also called Hodgkin’s lymphoma). Leuk. Res. 18 (1994), pp. 7-16: E. Tolnay, C. Kuhnen, T. See Maulik G. Shrikhande A, Kijima T. Ma PC, Morrison Wiethege et al., Hepatocyte growth factor/scatter factor and PT, Salgia R., Role of the hepatocyte growth factor receptor, its receptor c-Met are over-expressed and associated with an c-Met, in oncogenesis and potential for therapeutic inhibi increased microVessel density in malignant pleural mesothe tion. Cytokine Growth Factor Rev. 2002 February; 13(1):41 lioma. J. Cancer Res. Clin. Oncol. 124 (1998), pp. 291-296; 59, and cites therein: Bieche, M. H. Champeme and R. J. Klominek, B. Baskin, Z. Liu et al., Hepatocyte growth Lidereau, Infrequent mutations of the MET gene in sporadic factor/scatter factor stimulates chemotaxis and growth of breast tumours (letter). Int. J. Cancer 82 (1999), pp. 908-910; malignant mesothelioma cells through c-met receptor. Int. J. R. L. Camp, E. B. Rimm and D. L. Rimm, Met expression is 10 Cancer 76 (1998), pp. 240-249: Thirkettle, P. Harvey, P. S. associated with poor outcome in patients with axillary lymph Hasleton et al., Immunoreactivity for cadherins, HGF/SF. node negative breast carcinoma. Cancer 86 (1999), pp. 2259 met, and erbB-2 in pleural malignant mesotheliomas. Histo 2265; L. Nakopoulou, H. Gakiopoulou, A. Keramopoulos et pathology 36 (2000), pp. 522-528; P. G. Natali, M. R. Nicotra, al., c-met tyrosine kinase receptor expression is associated M. F. Di Renzo et al., Expression of the c-Met/HGF receptor with abnormal beta-catenin expression and favourable prog 15 in human melanocytic neoplasms: demonstration of the rela nostic factors in invasive breast carcinoma. Histopathology tionship to malignant melanoma tumour progression. Br. J. 36 (2000), pp. 313-325; C. Liu, M. Park and M. S. Tsao, Cancer. 68 (1993), pp. 746-750; O. Hjertner, M. L. Torgersen, Over-expression of c-met proto-oncogene but not epidermal C. Seidel et al., Hepatocyte growth factor (HGF) induces growth factor receptor orc-erbB-2 in primary human colorec interleukin-11 secretion from osteoblasts: a possible role for tal carcinomas. Oncogene. 7 (1992), pp. 181-185; K. Umeki, HGF in myeloma-associated osteolytic bone disease. Blood G. Shiota and H. Kawasaki, Clinical significance of c-met 94 (1999), pp. 3883-3888; C. Liu and M. S. Tsao, Invitro and oncogene alterations in human colorectal cancer. Oncology in vivo expressions of transforming growth factor-alpha and 56 (1999), pp. 314-321: H. Kuniyasu, W.Yasui, Y. Kitadai et tyrosine kinase receptors in human non-Small-cell lung car al., Frequent amplification of the c-met gene in Scirrhous type cinomas. Am. J. Pathol. 142 (1993), pp. 1155-1162: M. Oliv stomach cancer. Biochem. Biophys. Res. Commun. 189 25 ero, M. Rizzo, R. Madeddu et al., Over-expression and acti (1992), pp. 227-232; H. Kuniyasu, W. Yasui, H. Yokozaki et Vation of hepatocyte growth factor/scatter factor in human al., Aberrant expression of c-met mRNA in human gastric non-small-cell lung carcinomas. Br. J. Cancer 74 (1996), pp. carcinomas. Int. J. Cancer 55 (1993), pp. 72-75; W. S. Park, 1862-1868; E. Ichimura, A. Maeshima, T. Nakajima et al., R. R. Oh, Y. S. Kim et al., Absence of mutations in the kinase Expression of c-met/HGF receptor in human non-small cell domain of the Met gene and frequent expression of Met and 30 lung carcinomas in vitro and in vivo and its prognostic sig HGF/SF protein in primary gastric carcinomas. Apmis 108 nificance. Jpn. J. Cancer Res. 87 (1996), pp. 1063-1069; (2000), pp. 195-200; J. H. Lee, S.U. Han, H. Cho et al., A Takanami, F.Tanana, T. Hashizume et al., Hepatocyte growth novel germline juxtamembrane Met mutation in human gas factor and c-Met/hepatocyte growth factor receptor in pulmo tric cancer. Oncogene 19 (2000), pp. 4947-4953: T. nary adenocarcinomas: an evaluation of their expression as Moriyama, H. Kataoka, H. Tsubouchi et al., Concomitant 35 prognostic markers. Oncology 53 (1996), pp. 392–397: J. M. expression of hepatocyte growth factor (HGF), HGF activator Siegfried, L. A. Weissfeld, J. D. Luketich et al., The clinical and c-met genes in human glioma cells in vitro. FEBS Lett. significance of hepatocyte growth factor for non-Small cell 372 (1995), pp. 78-82;Y. W. Moon, R. J. Weil, S. D. Packet lung cancer. Ann Thorac. Surg. 66 (1998), pp. 1915-1918; M. al., Missense mutation of the MET gene detected in human Tokunou, T. Niki, K. Eguchi et al., c-MET expression in glioma. Mod. Pathol. 13 (2000), pp. 973-977; M. Di Renzo, 40 myofibroblasts: role in autocrine activation and prognostic M. Olivero, T. Martone et al., Somatic mutations of the met significance in lung adenocarcinoma. Am J. Pathol. 158 oncogene are selected during metastatic spread of human (2001), pp. 1451-1463: R. Ferracini, M. F. Di Renzo, K. HNSC carcinomas. Oncogene 19 (2000), pp. 1547-1555; K. Scotlandi et al., The Met/HGF receptor is over-expressed in Suzuki, N. Hayashi, Y. Yamada et al., Expression of the c-met human osteosarcomas and is activated by either a paracrine or proto-oncogene in human hepatocellular carcinoma. Hepa 45 an autocrine circuit. Oncogene 10 (1995), pp. 739-749; M. F. tology 20 (1994), pp. 1231-1236: W. S. Park, S. M. Dong, S. Di Renzo, M. Olivero, D. Katsaros et al., Over-expression of Y. Kim et al., Somatic mutations in the kinase domain of the the Met/HGF receptor in ovarian cancer. Int. J. Cancer Met/hepatocyte growth factor receptor gene in childhood 58(1994), pp. 658-662; H. M. Sowter, A. N. Corps and S. K. hepatocellular carcinomas. Cancer Res. 59 (1999), pp. 307 Smith, Hepatocyte growth factor (HGF) in ovarian epithelial 310; L. Schmidt, K. Junker, G. Weirich et al., Two North 50 tumour fluids stimulates the migration of ovarian carcinoma American families with hereditary papillary renal carcinoma cells. Int. J. Cancer 83 (1999), pp. 476-480; M. Ebert, M. and identical novel mutations in the MET proto-oncogene. Yokoyama, H. Friess et al., Co-expression of the c-met proto Cancer Res. 58 (1998), pp. 1719-1722; J. Fischer, G. oncogene and hepatocyte growth factor in human pancreatic Palmedo, R. von Knobloch et al., Duplication and over-ex cancer. Cancer Res. 54 (1994), pp. 5775-5778; L. L. Pisters, pression of the mutant allele of the MET proto-oncogene in 55 P. Troncoso, H. E. Zhau et al., c-met proto-oncogene expres multiple hereditary papillary renal cell tumours. Oncogene. sion in benign and malignant human prostate tissues. J. Urol. 17 (1998), pp. 733-739; Z. Zhuang, W. S. Park, S. Packet al., 154 (1995), pp. 293–298: P. A. Humphrey, X. Zhu, R. Zarne Trisomy 7-harbouring non-random duplication of the mutant garet al., Hepatocyte growth factor and its receptor (c-MET) MET allele in hereditary papillary renal carcinomas. Nat in prostatic carcinoma. Am J. Pathol. 147 (1995), pp. 386 Genet 20 (1998), pp. 66-69; M. Olivero, G. Valente, A. 60 396; K. Rygaard, T. Nakamura, M. Spang-Thomsen et al., Bardelli et al., Novel mutation in the ATP-binding site of the Expression of the proto-oncogenes c-met and c-kit and their MET oncogene tyrosine kinase in a HPRCC family. Int. J. ligands, hepatocyte growth factor/scatter factor and stem cell Cancer 82 (1999), pp. 640-643; L. Schmidt, K. Junker, N. factor, in SCLC cell lines and xenografts. Br. Cancer 67 Nakaigawa et al., Novel mutations of the MET proto-onco (1993), pp. 37–46; Y. Oda, A. Sakamoto, T. Saito et al., gene in papillary renal carcinomas. Oncogene 18 (1999), pp. 65 Expression of hepatocyte growth factor (HGF)/scatter factor 2343-2350; M. Jucker, A. Gunther, G. Gradlet al., The Met/ and its receptor c-MET correlates with poor prognosis in hepatocyte growth factor receptor (HGFR) gene is over-ex synovial sarcoma. Hum. Pathol. 31 (2000), pp. 185-192; M.F. US 7,662,824 B2 5 6 Di Renzo, M. Olivero, G. Serini et al., Over-expression of the pression in node-positive breast cancer identifies patients c-MET/HGF receptor in human thyroid carcinomas derived with poor clinical outcome independent of Her2/neu. Int. J. from the follicular epithelium. J. Endocrinol. Invest 18 Cancer 2005; 113: 678-682. (1995), pp. 134-139; K. Gohji, M. Nomi, Y. Niitani et al., Independent prognostic value of serum hepatocyte growth 5 SUMMARY OF THE INVENTION factor in bladder cancer. J. Clin. Oncol. 18 (2000), pp. 2963 2971. The present invention provides novel acylhydrazones (the compounds of Formula I) as protein tyrosine kinase modula Because of the role of aberrant HGF/SF-Met signaling in tors, particularly inhibitors of c-Met, and the use of such the pathogenesis of various human cancers, inhibitors of 10 compounds to reduce or inhibit kinase activity of c-Met in a c-Met receptor tyrosine kinase have broad applications in the cell or a subject, and modulate c-Met expression in a cell or treatment of cancers in which Met activity contributes to the Subject, and the use of Such compounds for preventing or invasive/metastatic phenotype, including those in which treating in a subject a cell proliferative disorder and/or disor c-Met is not overexpressed or otherwise altered. Inhibitors of ders related to c-Met. c-Met also inhibit angiogenesis and therefore are believed to 15 Illustrative of the invention is a pharmaceutical composi have utility in the treatment of diseases associated with the tion comprising a compound of Formula I and a pharmaceu formation of new vasculature, such as rheumatoid, arthritis, tically acceptable carrier. Another illustration of the present retinopathy. See, Michieli P. MaZZone M. Basilico C, Cavassa invention is a pharmaceutical composition prepared by mix S, Sottile A, Naldini L. Comoglio PM. Targeting the tumor ing any of the compounds of Formula I and a pharmaceuti and its microenvironment by a dual-function decoy Met cally acceptable carrier. receptor. Cancer Cell. 2004 July; 6(1):61-73. Other features and advantages of the invention will be Over-expression of c-Met is also believed to be a poten apparent from the following detailed description of the inven tially useful predictor for the prognosis of certain diseases, tion and from the claims. Such as, for example, breast cancer, non-Small cell lung car cinoma, pancreatic endocrine neoplasms, prostate cancer, 25 DESCRIPTION OF THE FIGURES esophageal adenocarcinoma, colorectal cancer, salivary gland carcinoma, diffuse large B-cell lymphoma and FIG. 1 shows the time course of the inhibitory effects of endometrial carcinoma. compounds of the present invention on the growth of U87MG See Herrera LJ, El-Hefnawy T. Queiroz de Oliveira PE, tumor Xenografts in nude mice. Raja S. Finkelstein S. Gooding W. Luketich JD, Godfrey TE, 30 FIG. 2 shows shows the effects of oral administration of Hughes S.J., The HGF Receptor c-Met Is Overexpressed in compounds of the present invention on the final weight of Esophageal Adenocarcinoma. Neoplasia. 2005 January: U87MG tumors xenografts in nude mice. 7(1): 75-84; Zeng Z, Weiser MR, D'Alessio M. Grace A, Shia J. Paty P B., Immunoblot analysis of c-Met expression in DETAILED DESCRIPTION OF THE INVENTION human colorectal cancer: overexpression is associated with 35 advanced stage cancer. Clin Exp Metastasis. 2004; 21 (5): 409-17; Hey, Peng Z. Pan X, Wang H, OuyangY. Expression Definitions and correlation of c-Met and estrogen receptor in endometrial As used herein, the following terms are intended to have carcinomas Sichuan Da Xue Xue Bao Yi Xue Ban. 2003 the following meanings (additional definitions are provided January: 34(1):78-9, 88 (English Abstract Only); Tsukinoki 40 where needed throughout the Specification): K, Yasuda M, Mori Y., Asano S. Naito H, Otay, Osamura RY, The term “alkenyl, whether used alone or as part of a WatanabeY. Hepatocyte growth factor and c-Met immunore Substituent group, for example, "Calkenyl(aryl), refers to activity are associated with metastasis in high grade salivary a partially unsaturated branched or straight chain monovalent gland carcinoma. Oncol Rep. 2004 November; 12(5):1017 hydrocarbon radical having at least one carbon-carbon 21; Kawano R, Ohshima K. Karube K, Yamaguchi T, Kohno 45 double bond, whereby the double bond is derived by the S, Suzumiya J. Kikuchi M., Tamura K. Prognostic signifi removal of one hydrogen atom from each of two adjacent cance of hepatocyte growth factor and c-MET expression in carbon atoms of a parent alkyl molecule and the radical is patients with diffuse large B-cell lymphoma. Br. J Haematol. derived by the removal of one hydrogen atom from a single 2004 November; 127(3):305-7: Lengyel E, Prechtel D, Resau carbonatom. Atoms may be oriented about the double bond in J. H. Gauger K. Welk A. Lindemann K. Salanti G. Richter T. 50 either the cis (Z) or trans (E) conformation. Typical alkenyl Knudsen B, Vande Woude G. F. Harbeck N. C-Met overex radicals include, but are not limited to, ethenyl, propenyl, pression in node-positive breast cancer identifies patients allyl(2-propenyl), butenyl and the like. Examples include with poor clinical outcome independent of Her2/neu. Int J Cisalkenyl or Calkenyl groups. Cancer. 2005 Feb. 10; 113(4):678-82; Hansel D. E. Rahman The term “C” (where a and b are integers referring to a A, House M. Ashfaq R. Berg K, Yeo C J, Maitra A. Met 55 designated number of carbon atoms) refers to an alkyl, alk proto-oncogene and insulin-like growth factor binding pro enyl, alkynyl, alkoxy or cycloalkyl radical or to the alkyl tein 3 overexpression correlates with metastatic ability in portion of a radical in which alkyl appears as the prefix root well-differentiated pancreatic endocrine neoplasms. Clin containing from a to b carbon atoms inclusive. For example, Cancer Res. 2004 Sep. 15: 10(18 Pt 1):6152-8: Knudsen BS, C. denotes a radical containing 1, 2, 3 or 4 carbon atoms. Edlund M. Prostate cancer and the met hepatocyte growth 60 The term “alkyl, whether used alone or as part of a sub factor receptor. Adv. Cancer Res. 2004:91:31-67; D Masuya, stituent group, refers to a saturated branched or straight chain C Huang, D Liu, T Nakashima, et al., The tumour-stromal monovalent hydrocarbon radical, wherein the radical is interaction between intratumoral c-Met and stromal hepato derived by the removal of one hydrogen atom from a single cyte growth factor associated with tumour growth and prog carbon atom. Unless specifically indicated (e.g. by the use of nosis in non-Small-cell lung cancer patients. British Journal 65 a limiting term Such as "terminal carbon atom'). Substituent of Cancer. 2004: 90:1552-1562; Ernst Lengyel, Dieter Pre variables may be placed on any carbon chain atom. Typical chtel, James H. Resau, Katja Gauger, et al. C-Met overex alkyl radicals include, but are not limited to, methyl, ethyl, US 7,662,824 B2 7 8 propyl, isopropyl and the like. Examples include Cisalkyl, and the other is a heterocyclyl ring. Typical benzo-fused Calkyl and Calkyl groups. heterocyclyl radicals include 1,3-benzodioxolyl (also known The term “alkylamino” refers to a radical formed by the as 1.3-methylenedioxyphenyl), 2,3-dihydro-1,4-benzodioxi removal of one hydrogen atom from the nitrogen of an alky nyl (also known as 1,4-ethylenedioxyphenyl), benzo-dihy lamine, such as butylamine, and the term “dialkylamino” dro-furyl, benzo-tetrahydro-pyranyl, benzo-dihydro-thienyl refers to a radical formed by the removal of one hydrogen and the like. atom from the nitrogen of a secondary amine, Such as dibu The term "carboxyalkyl refers to an alkylated carboxy tylamine. In both cases it is expected that the point of attach group Such as tert-butoxycarbonyl, in which the point of ment to the rest of the molecule is the nitrogen atom. attachment to the rest of the molecule is the carbonyl group. The term “alkynyl, whether used alone or as part of a 10 The term “cyclic heterodionyl refers to a heterocyclic Substituent group, refers to a partially unsaturated branched compound bearing two carbonyl Substituents. Examples or straight chain monovalent hydrocarbon radical having at include thiazolidine diones, oxazolidine diones and pyrroli least one carbon-carbon triple bond, whereby the triple bond dine diones. is derived by the removal of two hydrogenatoms from each of The term “cycloalkenyl refers to a partially unsaturated two adjacent carbon atoms of a parent alkyl molecule and the 15 cycloalkyl radical derived by the removal of one hydrogen radical is derived by the removal of one hydrogen atom from atom from a hydrocarbon ring system that contains at least a single carbon atom. Typical alkynyl radicals include ethy one carbon-carbon double bond. Examples include cyclohex nyl, propynyl, butynyl and the like. Examples include enyl, cyclopentenyl and 12.5,6-cyclooctadienyl. C2-salkynyl or C2-alkynyl groups. The term “cycloalkyl refers to a saturated or partially The term “alkoxy' refers to a saturated or partially unsat unsaturated monocyclic or bicyclic hydrocarbon ring radical urated branched or straight chain monovalent hydrocarbon derived by the removal of one hydrogen atom from a single alcohol radical derived by the removal of the hydrogen atom ring carbon atom. Typical cycloalkyl radicals include cyclo from the hydroxide oxygen Substituent on a parent alkane, propyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, alkene or alkyne. Where specific levels of saturation are cyclohexenyl, cycloheptyl and cyclooctyl. Additional intended, the nomenclature “alkoxy”, “alkenyloxy” and 25 examples include Cscycloalkyl, Cs scycloalkyl, C-12cy “alkynyloxy” are used consistent with the definitions of alkyl, cloalkyl, Cocycloalkyl, decahydronaphthalenyl, and 2.3.4. alkenyl and alkynyl. Examples include Csalkoxy or 5,6,7-hexahydro-1H-indenyl. Calkoxy groups. The term “fused ring system” refers to a bicyclic molecule The term “alkoxyether refers to a saturated branched or in which two adjacent atoms are present in each of the two straight chain monovalent hydrocarbon alcohol radical 30 derived by the removal of the hydrogen atom from the cyclic moieties. Heteroatoms may optionally be present. hydroxide oxygen substituent on a hydroxyether. Examples Examples include benzothiazole, 1,3-benzodioxole and include 1-hydroxyl-2-methoxy-ethane or 1-(2-hydroxyl decahydronaphthalene. ethoxy)-2-methoxy-ethane groups. The term "hetero' used as a prefix for a ring system refers The term “aralkyl refers to a C- alkyl group containing 35 to the replacement of at least one ring carbon atom with one an aryl substituent. Examples include benzyl, phenylethyl or or more atoms independently selected from N. S., O or P. 2-naphthylmethyl. It is intended that the point of attachment Examples include rings wherein 1, 2, 3 or 4 ring members are to the rest of the molecule be the alkyl group. a nitrogen atom; or, 0, 1, 2 or 3 ring members are nitrogen The term “aromatic' refers to a cyclic hydrocarbon ring atoms and 1 member is an oxygen or Sulfur atom. system having an unsaturated, conjugated at electron system. 40 The term "heteroaralkyl refers to a C- alkyl group con The term “aryl” refers to an aromatic cyclic hydrocarbon taining a heteroaryl Substituent. Examples include furylm ring radical derived by the removal of one hydrogen atom ethyl and pyridylpropyl. It is intended that the point of attach from a single carbon atom of the ring system. Typical aryl ment to the rest of the molecule be the alkyl group. radicals include phenyl, naphthalenyl, fluorenyl, indenyl, The term "heteroaryl refers to a radical derived by the aZulenyl, anthracenyl and the like. 45 removal of one hydrogen atom from a ring carbon atom of a The term “arylamino” refers to an amino group. Such as heteroaromatic ring system. Typical heteroaryl radicals ammonia, Substituted with an aryl group. Such as phenyl. It is include furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imida expected that the point of attachment to the rest of the mol Zolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triaz ecule is through the nitrogen atom. olyl, thiadiazolyl pyridinyl, pyridazinyl, pyrimidinyl, pyrazi The term “benzo-fused cycloalkyl refers to a bicyclic 50 nyl, indolizinyl, indolyl, isoindolyl, benzobfuryl, benzob. fused ring system radical wherein one of the rings is benzene thienyl, indazolyl, benzimidazolyl, benzthiazolyl, purinyl, and the other is a cycloalkyl or cycloalkenyl ring. Typical 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, benzo-fused cycloalkyl radicals include indanyl, 1.2.3,4-tet phthalzinyl, quinazolinyl, quinoxalinyl, 1.8-naphthyridinyl, rahydro-naphthalenyl, 6,7,8,9,-tetrahydro-5H-benzocyclo pteridinyl and the like. heptenyl, 5,6,7,8,9,10-hexahydro-benzocyclooctenyl and the 55 The term "heteroaryl-fused cycloalkyl refers to a bicyclic like. A benzo-fused cycloalkyl ring system is a Subset of the fused ring system radical wherein one of the rings is aryl group. cycloalkyl and the other is heteroaryl. Typical heteroaryl The term “benzo-fused heteroaryl refers to a bicyclic fused cycloalkyl radicals include 5,6,7,8-tetrahydro-4H-cy fused ring system radical wherein one of the rings is benzene clohepta(b)thienyl, 5,6,7-trihydro-4H-cyclohexa(b)thienyl, and the other is a heteroaryl ring. Typical benzo-fused het 60 5,6-dihydro-4H-cyclopenta(b)thienyl and the like. eroaryl radicals include indolyl, indolinyl, isoindolyl, benzo The term "heterocyclyl refers to a saturated or partially blfuryl, benzobthienyl, indazolyl, benzthiazolyl, quinoli unsaturated monocyclic ring radical derived by the removal nyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, and of one hydrogen atom from a single carbon or nitrogen ring the like. A benzo-fused heteroaryl ring is a subset of the atom. Typical heterocyclyl radicals include 2H-pyrrole, heteroaryl group. 65 2-pyrrolinyl or 3-pyrrolinyl), pyrrolidinyl, 1,3-dioxolanyl, The term “benzo-fused heterocyclyl refers to a bicyclic 2-imidazolinyl (also referred to as 4,5-dihydro-1H-imida fused ring system radical wherein one of the rings is benzene Zolyl), imidazolidinyl, 2-pyrazolinyl, pyrazolidinyl, tetra US 7,662,824 B2 10 Zolyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl. thiomorpholinyl, piperazinyl, azepanyl, hexahydro-1,4-diaz -continued epinyl and the like. DMF dimethylformamide The term "squaryl refers to a cyclobutyl 12 dione radical. DMSO dimethylsulfoxide The term "substituted refers to a core molecule on which DIEA diisopropylethylamine one or more hydrogenatoms have been replaced with one or EDTA ethylenediaminetetraaceticacid more functional radical moieties. Substitution is not limited EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to a core molecule, but may also occur on a substituent radi EtOAC ethyl acetate cal, whereby the Substituent radical becomes a linking group. HOBT 1-hydroxybenzotriazole hydrate The term “independently selected refers to one or more 10 HBTU O-benzotriazol-1-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate Substituents selected from a group of Substituents, wherein -Pr(OH isopropyl alcohol the substituents may be the same or different. LC/MS (ESI) Liquid chromatography/mass spectrum The substituent nomenclature used in the disclosure of the (electrospray ionization) present invention was derived by first indicating the atom MeOH Methyl alcohol having the point of attachment, followed by the linking group 15 NMM N-methylmorpholine NMR nuclear magnetic resonance atoms toward the terminal chain atom from left to right, PS polystyrene Substantially as in: RT room temperature NaHMDS Sodium hexamethyldisilaZane TEA triethylamine TFA trifluoroacetic acid or by first indicating the terminal chain atom, followed by the THF tetrahydrofuran linking group atoms toward the atom having the point of TLC thin layer chromatography attachment, Substantially as in: Ph(C1)alkylamido (C1)alkyl Formula I 25 either of which refers to a radical of the Formula: The present invention comprises compounds of Formula I:

O Formula I

C1-C5alkyl us N -C 1-C5alkyl

Lines drawn into ring systems from Substituents indicate that the bond may be attached to any of the suitable ring 35 atOmS. When any variable (e.g. R) occurs more than one time in any embodiment of Formula I, each definition is intended to be independent. The terms “comprising”, “including, and "containing are 40 used herein in their open, non-limited sense. pharmaceutically acceptable salts, N-oxides, and stere Nomenclature ochemical isomers thereof, wherein: Except where indicated, compound names were derived using nomenclature rules well known to those skilled in the 45 A is a benzo-fused heterocyclic ring or a benzo-fused het art, by either standard IUPAC nomenclature references, such eroaryl ring, wherein the point of attachment of said ring is as Nomenclature of Organic Chemistry, Sections A, B, C, D, through the benzo moiety, said ring is optionally substituted E, F and H. (Pergamon Press, Oxford, 1979, Copyright 1979 with —NH, —CH, —CF —NO. —CN, Cl, F, Br, or IUPAC) and A Guide to IUPAC Nomenclature of Organic carbonyl: Compounds (Recommendations 1993), (Blackwell Scientific 50 R" is hydrogen or halogen; Publications, 1993, Copyright 1993 IUPAC), or commer cially available Software packages such as Autonom (brand of R’ is selected from the group consisting of Formula II, For nomenclature software provided in the ChemDraw Ultra(R) mula III or Formula IV: office suite marketed by CambridgeSoft.com) and ACD/In dex NameTM (brand of commercial nomenclature software 55 marketed by Advanced Chemistry Development, Inc., Tor Formula II onto, Ontario). Abbreviations As used herein, the following abbreviations are intended to have the following meanings (additional abbreviations are 60 Formula III provided where needed throughout the Specification): R3 N-1 Boc tert-butoxycarbonyl 65 R X DCM dichloromethane O US 7,662,824 B2 11 12

-continued -continued six Formula IV B 11- 5 N N O 2N, S

wherein n is 2, 3 or 4: 10 B is a nitrogen containing heterocyclic ring, preferably selected from holy NH, R.S.C \ RSC \ 15 N CO or 2 cyN S N s HN N s 2O

R4 R4 OR /\\ s s/\\ s X. O, \ / O1 \ / 25 N N

R4 R4 30 2

O R4 R4 35 2 s X=0. N N R is hydrogen or alkyl; H R" is H, alkyl, hydroxyalkyl, hydroxyl, or—C(O)alkyl: and n S R is OH, NH-alkyl, N-alkyl, or NH. 40 N As used hereafter, the terms “compound of Formula I and l X “compounds of Formula I are meant to include also the 2 Y: N pharmaceutically acceptable salts, N-oxides and stere ochemical isomers thereof. H EMBODIMENTS OF FORMULAI X-Cl X=0. Preferred embodiments of the invention are compounds of N O Formula I wherein one or more of the following limitations are present: 50 S R" is hydrogen, C1 or Br; X-NH. O, A is selected from: N

55 H N

N N ho s s X- HX=0. 4. S 60

H US 7,662,824 B2 13 14 R" is hydrogen, C1 or Br; -continued A is

\ NH2, M O N N OR Y; and M O 10

sé' R’ is Formula III wherein: so NH2, and n is 2 or 3: 15 R is hydrogen; and \ R is hydroxyl, methylamino, ethylamino, propylamino, M isopropylamino, dimethylamino, diethylamino, dipro O pylamino, methylethylamino, or methylpropylamino. Other preferred embodiments of the invention are com pounds of Formula I wherein one or more of the following N. and limitations are present: N H 25 R" is hydrogen, C1 or Br; A is R’ is Formula II wherein: n is 2 or 3: B is morpholinyl, piperidinyl, piperazinyl, or pyrrolidinyl: 30 R is hydrogen; and OR N R is hydrogen, hydroxyl, hydroxymethyl, methyl, ethyl, propyl or isopropyl. Another preferred embodiment of the invention are com 35 pounds of Formula I wherein one or more of the following R’ is Formula IV wherein: limitations are present: B is morpholinyl, piperidinyl, piperazinyl, or pyrrolidinyl: R" is hydrogen, C1 or Br; and 40 R" is hydrogen, hydroxyl, hydroxymethyl, methyl, ethyl, A is propyl or isopropyl. Other preferred embodiments of the invention are com pounds of Formula I wherein one or more of the following 45 limitations are present: R" is hydrogen, C1 or Br; A is O R N ; and

OR N ; and 55

R’ is Formula II wherein: n is 2 or 3: R’ is Formula III wherein: B is morpholinyl, piperidinyl, piperazinyl, or pyrrolidinyl: 60 n is 2 or 3; and R is hydrogen; and R is hydrogen; and R" is hydrogen, hydroxyl, hydroxymethyl, methyl, ethyl, R is hydroxyl, dimethylamino, diethylamino, or dipropy propyl or isopropyl. lamino. Other preferred embodiments of the invention are com 65 Other embodiments of the invention are compounds of pounds of Formula I wherein one or more of the following Formula I wherein one or more of the following limitations limitations are present: are present: US 7,662,824 B2 15 16 R" is hydrogen, C1 or Br; prodrug derivatives are described in, for example, "Design of A is Prodrugs’, ed. H. Bundgaard, Elsevier, 1985. Stereochemically Isomeric Forms One skilled in the art will recognize that the compounds of Formula I may have one or more asymmetric carbon atoms in their structure. It is intended that the present invention include within its scope single enantiomer forms of the compounds, OR \,. and racemic mixtures, and mixtures of enantiomers in which an enantiomeric excess is present. 10 The term “single enantiomeras used herein defines all the possible homochiral forms which the compounds of Formula I and their N-oxides, addition salts, quaternary amines or R’ is Formula IV wherein: physiologically functional derivatives may possess. B is piperazine; and Stereochemically pure isomeric forms may be obtained by R" is hydrogen, methyl, ethyl, propyl or isopropyl. the application of art known principles. Diastereoisomers 15 may be separated by physical separation methods such as Pharmaceutically Acceptably Salts fractional crystallization and chromatographic techniques, The compounds of the present invention may also be and enantiomers may be separated from each other by the present in the form of pharmaceutically acceptable salts. selective crystallization of the diastereomeric salts with opti For use in medicines, the salts of the compounds of this cally active acids or bases or by chiral chromatography. Pure invention refer to non-toxic pharmaceutically acceptable Stereoisomers may also be prepared synthetically from appro salts.” FDA approved pharmaceutically acceptable salt forms priate stereochemically pure starting materials, or by using (Ref. International J. Pharm. 1986, 33, 201-217, J. Pharm. Stereoselective reactions. Sci., 1977, January, 66(1), p1) include pharmaceutically The term "isomer' refers to compounds that have the same acceptable acidicfanionic or basic/cationic salts. composition and molecular weight but differ in physical and/ Pharmaceutically acceptable acidic/anionic salts include, 25 or chemical properties. Such Substances have the same num and are not limited to acetate, benzenesulfonate, benzoate, ber and kind of atoms but differ in structure. The structural bicarbonate, bitartrate, bromide, calcium edetate, camsylate, difference may be in constitution (geometric isomers) or in an carbonate, chloride, citrate, dihydrochloride, edetate, edisy ability to rotate the plane of polarized light (enantiomers). late, estolate, esylate, fumarate, glyceptate, gluconate, The term “stereoisomer refers to isomers of identical con glutamate, glycolylarsanilate, hexylresorcinate, hydrabam 30 stitution that differin the arrangement of their atoms in space. ine, hydrobromide, hydrochloride, hydroxynaphthoate, Enantiomers and diastereomers are stereoisomers wherein an iodide, isethionate, lactate, lactobionate, malate, maleate, asymmetrically substituted carbon atom acts as a chiral cen mandelate, mesylate, methylbromide, methylnitrate, methyl ter. Sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, The term "chiral” refers to the structural characteristic of a phosphate/diphosphate, polygalacturonate, Salicylate, Stear 35 molecule that makes it impossible to Superimpose it on its ate, Subacetate. Succinate, Sulfate, tannate, tartrate, teoclate, mirror image. tosylate and triethiodide. Organic or inorganic acids also The term “enantiomer refers to one of a pair of molecular include, and are not limited to, hydriodic, perchloric, Sulfuric, species that are mirror images of each other and are not phosphoric, propionic, glycolic, methanesulfonic, hydroxy Superimposable. ethanesulfonic, oxalic, 2-naphthalenesulfonic, p-toluene The term "diastereomer' refers to stereoisomers that are Sulfonic, cyclohexanesulfamic, saccharinic or trifluoroacetic 40 not mirror images. acid. The symbols “R” and “S” represent the configuration of Pharmaceutically acceptable basic/cationic salts include, Substituents around a chiral carbon atom(s). and are not limited to aluminum, 2-amino-2-hydroxymethyl The term “racemate' or “racemic mixture” refers to a com propane-1,3-diol (also known as tris(hydroxymethyl)ami position composed of equimolar quantities of two enantio nomethane, tromethane or “TRIS), ammonia, benzathine, 45 meric species, wherein the composition is devoid of optical t-butylamine, calcium, calcium gluconate, calcium hydrox activity. ide, chloroprocaine, choline, choline bicarbonate, choline The term "homochiral' refers to a state of enantiomeric chloride, cyclohexylamine, diethanolamine, ethylenedi purity amine, lithium, LiOMe, L-lysine, magnesium, meglumine, The term “optical activity” refers to the degree to which a NH, NH-OH, N-methyl-D-glucamine, piperidine, potas 50 homochiral molecule or nonracemic mixture of chiral mol sium, potassium-t-butoxide, potassium hydroxide (aqueous), ecules rotates a plane of polarized light. procaine, quinine, Sodium, Sodium carbonate, sodium-2-eth The term “geometric isomer refers to isomers that differin ylhexanoate (SEH), sodium hydroxide, triethanolamine the orientation of Substituent atoms in relationship to a car (TEA) or zinc. bon-carbon double bond, to a cycloalkyl ring or to a bridged 55 bicyclic system. Substituent atoms (other than H) on each Prodrugs side of a carbon-carbon double bond may be in an E or Z The present invention includes within its scope prodrugs of configuration. In the “E” (opposite sided) configuration, the the compounds of the invention. In general. Such prodrugs Substituents are on opposite sides in relationship to the car will be functional derivatives of the compounds which are bon-carbon double bond; in the “Z” (same sided) configura readily convertible in vivo into an active compound. Thus, in tion, the Substituents are oriented on the same side in rela the methods of treatment of the present invention, the term 60 tionship to the carbon-carbon double bond. Substituent atoms “administering shall encompass the means for treating, ame (other than H) attached to a carbocyclic ring may be in a cis or liorating or preventing a syndrome, disorder or disease trans configuration. In the “cis' configuration, the Substitu described herein with a compound specifically disclosed or a ents are on the same side in relationship to the plane of the compound, or prodrug thereof, which would obviously be ring; in the “trans' configuration, the Substituents are on included within the scope of the invention albeit not specifi 65 opposite sides in relationship to the plane of the ring. Com cally disclosed for certain of the instant compounds. Conven pounds having a mixture of “cis' and “trans' species are tional procedures for the selection and preparation of suitable designated “cis/trans'. US 7,662,824 B2 17 18 It is to be understood that the various substituent stereoi General Reaction Scheme Somers, geometric isomers and mixtures thereofused to pre Compounds of Formula I can be prepared by methods pare compounds of the present invention are either commer known to those who are skilled in the art. The following cially available, can be prepared synthetically from reaction Schemes are only meant to represent examples of the commercially available starting materials or can be prepared invention and are in no way meant to be a limit of the inven as isomeric mixtures and then obtained as resolved isomers tion. using techniques well-known to those of ordinary skill in the Aryl and heteroaryl acetic acids can be accessed by meth art. ods known in the art (Journal of Medicinal Chemistry, 1986, The isomeric descriptors “R” “S” “E” “Z” “cis,” and 29 (11), 2326-2329; Bioorganic and Medicinal Chemistry “trans' are used as described herein for indicating atom con 10 Letters, 2004, 14(14),3799-3802; EP 1229034A1 20020807; figuration(s) relative to a core molecule and are intended to be Tetrahedron Letters, 2003, 44 (35), 6745-6747: Synthetic used as defined in the literature (IUPAC Recommendations Communications, 1997, 27 (22), 3839-3846). Several for Fundamental Stereochemistry (Section E), Pure Appl. examples of aryl acetic acid synthesis are illustrated in Chem., 1976, 45:13-30). Scheme 1. Benzofused heterocyclic compound (1-1), (Jour The compounds of the present invention may be prepared nal of Medicinal Chemistry, 1996, 29(11), 2362-2369; Jour as individual isomers by either isomer-specific synthesis or 15 nal of Medicinal Chemistry, 1997, 40(7), 1049-1062), is resolved from an isomeric mixture. Conventional resolution treated with N-bromosuccinimide in carbon tetrachloride to techniques include forming the free base of each isomer of an give compound 1-2. Nitrophenylacetic acid (1-3), (Bioor isomeric pair using an optically active salt (followed by frac ganic and Medicinal Chemistry Letters, 1998, 8 (1), 17-22; tional crystallization and regeneration of the free base), form Organic Letters, 2002, 4 (16), 2675-2678; WO 00/06566, ing an ester oramide of each of the isomers of an isomeric pair Helvitica Chemica Acta, 1976, 59 (3), 855-866) is reduced (followed by chromatographic separation and removal of the with conditions such as hydrogenation in the presence of chiral auxiliary) or resolving an isomeric mixture of either a palladium on activated carbon in a solvent such as methanol starting material or a final product using preparative TLC to give compound 1-4, which is then treated with triethyl (thin layer chromatography) or a chiral HPLC column. orthoformate in toluene to give 1-5. Compound 1-6 can be 25 treated with an appropriate amine to give Compound 1-7. Polymorphs Compound 1-8 can be treated with hydroxylamine hydro Furthermore, compounds of the present invention may chloride in ethanol, followed by a Mitsunobu reaction to give have one or more polymorph or amorphous crystalline forms Compound 1-9. This can be treated with sodium hydroxide in and as such are intended to be included in the scope of the methanol to give Compound 1-10. invention. In addition, some of the compounds may form Solvates with water (i.e., hydrates) or common organic Sol 30 vents, and Such are also intended to be encompassed within Scheme 1 the scope of this invention. HO N-Oxides NBS Ho The compounds of Formula I may be converted to the 35 O CC14 corresponding N-oxide forms following art-known proce X dures for converting a trivalent nitrogen into its N-oxide form. 1-1 Said N-oxidation reaction may generally be carried out by reacting the starting material of Formula I with an appropriate HO N organic or inorganic peroxide. Appropriate inorganic peroX 40 ides comprise, for example, hydrogen peroxide, alkali metal O X or earth alkaline metal peroxides, e.g. sodium peroxide, potassiumperoxide; appropriate organic peroxides may com 1-2 prise peroxy acids such as, for example, benzenecarboper N NO oXoic acid or halo Substituted benzenecarboperoxoic acid, e.g. 3-chlorobenzenecarboperoxoic acid, peroxoalkanoic 45 HO H2, Pd/C acids, e.g. peroxoacetic acid, alkylhydroperoxides, e.g. thutyl - 2 X methanol hydro-peroxide. Suitable solvents are, for example, water, O lower alcohols, e.g. ethanol and the like, hydrocarbons, e.g. 1-3 toluene, ketones, e.g. 2-butanone, halogenated hydrocarbons, N NH2 e.g. dichloromethane, and mixtures of Such solvents. 50 Tautomeric Forms HO - OC2 X (EtO)3CHtoluene Some of the compounds of Formula I may also exist in their O tautomeric forms. Such forms although not explicitly indi 1-4 cated in the present application are intended to be included 55 N N within the scope of the present invention. HO y Preparation of Compounds of the Present Invention During any of the processes for preparation of the com 1 Null pounds of the present invention, it may be necessary and/or O desirable to protect sensitive or reactive groups on any of the 60 1-5 molecules concerned. This may be achieved by means of HO 2 conventional protecting groups, such as those described in HNXH Protecting Groups, P. Kocienski. Thieme Medical Publishers, --- 2000; and T. W. Greene & P. G. M. Wuts, Protective Groups O in Organic Synthesis, 3' ed. Wiley Interscience, 1999. The 65 protecting groups may be removed at a convenient Subse 1-6 quent stage using methods known in the art. US 7,662,824 B2 20

-continued -continued NH2 HO N 5 O N X 1-7 O 10 O 1. HNOHHCl, 1. H ethanol -- 2. PPh3, DEAD, O THF OH 15 1-8 O 1. NaOH N Her O M methanol O 2O 1-9 HO O,

O N O 25 X-O, 1-10 wherein X is NH, O or S. 30 The following compounds can be synthesized by methods known in the art: S Cl, sNOss O, X foX-4.N4.N1S1SN NSONNNN X 35 HO rO

40 where A is selected from: X O, 45 N X s/ YN4Ns/ NN

O O, O O, and N to H 55 so29 N boyS See, for example, Journal of Medicinal Chemistry, 1997, 40 (7), 1049-1058, and references therein; and WO 2002O85888. Synthesis of aryland heteroaryl acetyl chlorides and aryl and heteroaryl acetic acid hydrazides can also be accessed by s NH, 65 methods known in the art (see, Bulletin de la Societe holyO Chimique de France, 1964. 2, 245-247; and Helvitica Chemica Acta, 1928, 11, 609-656). Compound 2-1 is treated US 7,662,824 B2 21 22 with oxalyl chloride in DCM to give Compound 2-2, which is treated with anhydrous hydrazine in DCM to give hydrazide 2-3. Alternatively, Compound 2-1 can be treated with acetic anhydride, followed by hydrazine in water to give Compound 2-3. Acetic acid methyl ester 2-4 can be treated with aqueous 5 hydrazine in ethanol to give Compound 2-3.

Scheme 2 1. Ac2O 2. HNNH2, H2O

HO C oxalyl chloride --- -- DCM DCM O O 2-1 2-2

Synthesis of 4-chloro-1H-indole-6-carboxylic acid or 4-fluoro-1H-indole-6-carboxylic acid can be accessed by methods known in the art (see, Chemical and Pharmaceutical 35 Bulletin 1981, 29(11),3145; and Organic Syntheses, 13,525, 1933). Compound 3-1 can be treated with hydrochloric acid, Sodium nitrite and copper chloride to give Compound 3-2, which can be treated with sodium hydroxide in methanol to give compound 3-3. Alternatively, Compound 3-1 can be 40 treated with hydrochloric acid, sodium nitrite and tetrafluo roboric acid in water and ethanol to give Compound 3-4, which can be treated with sodium hydroxide in methanol to give Compound 3-5.

NH2 N C O O NaOH, methanol 1. C. HCl, NaNO2, CuC - -- O X. HO O X. N

3-1 3-2 3-3 HCl, NaNO ethanol US 7,662,824 B2 23 24

-continued F

N F -O N NaOH, methanol N

HO O OX. O NH \ O 3-4 3-5

15 Synthesis of 3,3-dibromo-1,3-dihydro-indol-2-ones can be wherein Z is a nucleophile selected from NH, NH(alkyl). accomplished by methods known in the art (see, Tetrahedron N(alkyl), and Letters, 1987, 28 (35), 4027-30). Triethylamine and HBTU are added to a solution of Compound 4-1 in DMF, followed by addition of an amine to give Compound 4-2; a cyclic amine is 2O pictured, for illustrative purposes only; the amine might also sy bean alkylamine or a Substituted alkylamine. Compound 4-2 B NH. is treated with pyridinium tribromide in aqueous t-butanol to 1.Y--- give Compound 4-3. Alternatively, triethylamine and HBTU are added to a solution of Compound 4-1 in DMF, followed by as addition of anhydroxyl substituted alkyl amine to give Com pound 4-4. Methanesulfonyl chloride is added at o C. to a Synthesis substituted 1H-indole-2,3-diones can be accom solution of Compound 4-4 in THF and triethylamine, fol- plished by methods known in the art (see, Tetrahedron Let lowed by the addition of an amine Z-Hat room temperature to 1984, 25 (29), 3099-31 OO). C d 5-1 (also C give Compound 4-5. This can be further reacted with pyri- so ters, ( ). r ). ompoun -1 (also Com dinium tribromide in aqueous t-butanol to give Compound pound 4-3) is dissolved in a solution of methanol and water 4-3. and heated to reflux for several hours to give compound 5-2.

Scheme 4 R1 RI RI N HBTU, TEA, N pyridinium B DMF , tribromide HO N ', -N N aq. tBuOH O H B NH R4 H 1's--- 2 N O R R H 4-1 4-2 4-3

R3 HBTU, TEA, HO Y1 NH DMF

R R1 i N 1. CHSOCl, TEA,THF N E.idini 2. Z-H a.d. tBuOH HO N1 N N Z N1 N N C O O 4-4 4-5 US 7,662,824 B2 25 26

-continued Scheme 5 R1 N-NH. R Br Br 5 W

MeOH O O HO R2 N R2 N 6-2 5-1 10 RI O also Compound 4-3 Synthesis of 1H-Indole-2,3-diones can be accessed by O is methods known in the art (see, Journal of Organic Chemistry, R2 N 1956, 21, 171-173; and Tetrahedron Letters, 1998, 39 (42), 7679-7682). Triethylamine and HBTU can be added to a 5-2 solution of Compound 7-1 (see, Synthesis, 1998, 11, 1599 1603; Journal of Agricultural and Food Chemistry, 1969, 17, also Compound 4-3 20 83-85; and WO 2004.050619) in DMF, followed by addition Synthesis of 3-hydrazono-1,3-dihydro-indol-2-ones can ofanamine to give Compound 7-2 (a cyclic amine is pictured, be accessed by methods known in the art (see, European for illustrative purposes only; the amine might also be analkyl Journal of Medicinal Chemistry 1983, 18 (3), 261-267). A amine or a substituted alkyl amine). Compound 7-2 can be solution of Compound 6-1 (also Compound 4-3) in THF is treated with trichloroacetaldehyde, hydroxylamine hydro treated with aqueous hydrazine at room temperature to give chloride and sodium sulfate in water, followed by treatment compound 6-2. with sulfuric acid to give Compound 7-3. Alternatively, tri ethylamine and HBTU can be added to a solution of Com Scheme 6 pound 7-1 in DMF, followed by addition of an hydroxyl RI 30 substituted alkyl amine to give Compound 7-4. Methane Br sulfonyl chloride can be added at 0° C. to a solution of Br Compound 7-4 in THF and triethylamine, followed by the O it re- addition of an amine Z-H at room temperature to give Com pound 7-5. This can be further reacted with trichloroacetal R2 N 35 dehyde, hydroxylamine hydrochloride and sodium sulfate in 6-1 water, followed by treatment with sulfuric acid to give Com pound 7-3.

Scheme 7 R1 RI RI HBTU, TEA, 1. CC13CHO O DMF , HNOHHCI HO '', -N Naso, to NH2 B NH R4 - NH2 2. H.SO, O O 41's--- O R2 N 7-1 7-2 7-3

R3 HO h HBTTEA, Y1

RI RI 1. CCICHO HNOHHCI Na2SO4, H2O 1. CHSOCl, TEA, THF 2. H2SO4 HO N 2. Z-H N Y1 NH2 N1 NH2 O O 7-4 7-5 US 7,662,824 B2 27 28 wherein Z is a nucleophile selected from NH, NH(alkyl). An alternative method of accessing compounds of Formula N(alkyl), and I is illustrated in Scheme 9. Compound 9-1 is heated to reflux in a solution of acetic acid hydrazide 2-3, p-toluenesulfonic acid and ethanol to give Compound 9-2, an example of For mula I.

Compounds of Formula I can be accessed by methods known in the art (see, Bioorganic and Medicinal Chemistry 10 Scheme 9 Letters 2002, 12 (24)3601-3604.). One such example is illus- O trated in Scheme 8. Triethylamine and HBTU are added at room temperature to a solution of acetic acid 2-1 in DMF, followed by addition of Compound 8-1 to give Compound RI HN-NH 8-2, an example of Formula I. Alternatively, Compound 8-1 15 O can be added to a solution of triethylamine and THF, followed by addition of acetyl chloride 2-2 to give Compound 8-2. O Compound 2-3 Compounds of Formula I in which the A ring is a benzod p-TsOH isoxazol-3-ylamine can be synthesized in the following man- R2 N citing ner: Compound 8-1 is treated with triethylamine, Compound 20 9-1 1-6 and HBTU in DMF to give Compound 8-3. Further reac tion with acetohydroxamic acid and potassium carbonate in DMF gives Compound 8-2.

Scheme 8 O

R1 N-NH O HBTU, TEA, DMF W CN Compound 1-6 O HON ls N R2 N F H DMF 8-3 K2CO3

O O

RI HO R1 N-NH N-NH W Compound 2-1 W O HBTU O TEA, DMF R2 N R2 N 8-1 8-2

O DIEA,THF C

Compound 2-2 US 7,662,824 B2 29 30 Representative Compounds -continued O

N-NH Representative compounds of the present invention syn thesized by the aforementioned methods are presented below. O Examples of the synthesis of specific compounds are pre N sented thereafter. Preferred compounds are numbers 2, 5, 6, 7, H 10 8, 9, 10, 11, 12, and 14; more preferred compounds are 9-2 numbers 2, 5, 8, 9, and 11; most preferred compounds are numbers 5, 8, and 11.

Example Number Structure

H Br N-N W OH O O H O N nu1\-N N O

B r N1 N W N O O O N N H O

N-N-- H N O O

H Br N-N W S^ O O N-N-N-Nnu-1N1 N O O

H Br N-N W O O US 7,662,824 B2 31 32

-continued

Example Number Structure

Br

N

Br

N

Br

10 Br HO

11 Br

12

HO

13 Br US 7,662,824 B2 34

-continued

Example Number Structure

14 Br N1 N W O O H O -Na-1a-N N O

15 B W O O H O HO N-1N1 N N H O

16 H Br Y N

N H O

17 B r N1 N N W O y N O N N- N O H O

18 H Br 1 N

N

19 Br N-NH

N US 7,662,824 B2 35 36 EXAMPLE1 c. (S)-4-Bromo-1H-indole-6-carboxylic acid 3-(2- hydroxymethyl-pyrrolidin-1-yl)-propylamide (S)-4-Bromo-3-(2-2,3-dihydro-benzofuran-5-yl acetyl)-hydrazono-2-oxo-2,3-dihydro-1H-indole-6- carboxylic acid 3-(2-hydroxymethyl-pyrrolidin-1- 5 yl)-propyl-amide OH Br

H OH Br N-NH 10 N-1-N-N N N O O O n-1N1 N(O) O 15 A solution methanesulfonyl chloride (0.13 mL, 1.68 mmol) in THF (1 mL) was added to a solution of 4-bromo 1H-indole-6-carboxylic acid (3-hydroxy-propyl)-amide a. 4-Bromo-1H-indole-6-carboxylic acid (0.50 g, 1.68 mmol) and triethylamine (0.25 mL, 1.84 mmol) intetrahydrofuran (5 mL) at 0°C. and slowly warmed to room temperature over 30 minutes. (S)-(+)-2-pyrrolidinemethanol (1.0 g, 9.88 mmol) was added and stirred at room temperature Br overnight. The reaction was concentrated in vacuo and puri fied by column chromatography (5% methanol in dichlo 25 romethane) to yield 0.50 g (78%) of (S)-4-bromo-1H-indole 6-carboxylic acid 3-(2-hydroxymethyl-pyrrolidin-1-yl)- HO N propyl-amide. Mass spectrum (LCMS, ESI pos.): Calcd for CHBrNO. 379.09. Found 380.0; (M+H). 30 d. (S)-4-Bromo-3-hydrazono-2-oxo-2,3-dihydro-1H A solution of sodium hydroxide (14.08 g., 352.1 mmol) in indole-6-carboxylic acid 3-(2-hydroxymethyl-pyrro water (100 mL) was added a to a solution of 4-bromo-1H lidin-1-yl)-propyl-amide indole-6-carboxylic acid methyl ester in methanol (14.91 mL, 58.7 mmol) at room temperature and stirred for 2.5 hours. The 35 reaction mixture was concentrated in vacuo, 1N HCl was added, and the product was extracted with ethyl acetate to give 13.0 g of 4-bromo-1H-indole-6-carboxylic acid, which was used in the next step with no further purification. "H NMR (400 MHz, CDOD) & 8.13 (t, J=1.0 Hz, 1H), 7.87 (d. 40 OH J=1.3 Hz, 1H), 7.53 (t, J=3.4 Hz, 1H), 8.13 (d. J=4.2 Hz, 1H). N-NH. b. 4-Bromo-1H-indole-6-carboxylic acid (3-hydroxy-propyl)-amide 45 N

Br

50

HO N-1N1 N H Pyridinium tribromide (1.67 g. 5.2 mmol) was added in O several shots at room temperature to a solution of (S)-4- 55 bromo-1H-indole-6-carboxylic acid 3-(2-hydroxymethyl pyrrolidin-1-yl)-propyl-amide (0.50 g, 1.30 mmol) dis 3-Aminopropanol (1.36 g. 18.1 mmol) was added to a solution of 4-bromo-1H-indole-6-carboxylic acid (4.35 g, solved in t-butanol: water (6:1) 7 mL. The reaction was 18.1 mmol), triethylamine (4.60 mL, 45.3 mmol), and O-ben concentrated in vacuo and taken up in THF and water. Hydra Zotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluoro 60 zine hydrate (1 mL) was added to the reaction and the bipha phosphate (HBTU) (6.87 g., 18.1 mmol) in DMF (40 mL) and sic mixture was stirred at room temperature for 30 minutes. stirred at room temperature overnight. The reaction mixture Ethyl acetate was added and the product was extracted to give was concentrated in vacuo and purified by column chroma 0.223 gof (S)-4-bromo-3-hydrazono-2-oxo-2,3-dihydro-1H tography (10% methanol in dichloromethane) to yield 3.0 g indole-6-carboxylic acid3-(2-hydroxymethyl-pyrrolidin-1- (56%) of 4-bromo-1H-indole-6-carboxylic acid (3-hydroxy 65 yl)-propyl-amide, which was used in the next step with no propyl)-amide. Mass spectrum (LCMS, ESI pos.): Calcd for further purification. Mass spectrum (LCMS, ESI pos.): Calcd CHBrNO. 296.02. Found 296.9; (M+H). for CHBrNO. 423.09. Found 424.1; (M+H).

US 7,662,824 B2 47 48 b) Benzofuran-5-yl-acetic acid (4-bromo-6-(4-me b) Benzooxazol-5-yl-acetic acid (4-bromo-6-(4-me thyl-piperazine-1-carbonyl)-2-oxo-1,2-dihydro-in thyl-piperazine-1-carbonyl)-2-oxo-1,2-dihydro-in dol-3-ylidenel-hydrazide hydrochloride dol-3-ylidenel-hydrazide

B - N Br -N N 10 O O s-,N -SO2 s-,N- -N SCC) N O 15 The title compound was synthesized using the same pro The title compound was synthesized using the same pro cedure as in Example 2. "H NMR (400 MHz, DMSO) & 12.73 cedure as in Example 2. "H NMR (400 MHz, CDOD) 87.72 (brs, 1H), 11.58 (bris 1H), 8.71 (s, 1H), 7.80 (s, 1H), 7.71 (d. (s, 1H), 7.66 (s, 1H), 7.44 (d. J=7.6 Hz, 1H), 7.42 (s, 1H), 7.35 J=8.3 Hz, 1H), 7.43 (d. J=8.4 Hz, 1H), 7.29 (s, 1H), 6.90 (s, (d. J–7.8 Hz, 1H), 7.02 (s, 1H), 6.79 (s, 1H), 4.32 (brs, 2H), 1H), 4.30 (brs, 1H), 3.60 (m, 4H), 3.40 (m, 4H), 3.10 (s.3H). 3.55 (m, 4H), 3.22 (m, 4H), 2.95 (S. 3H). Mass spectrum Mass spectrum (LCMS, ESI pos.): Calcd for CH BrNO: (LCMS, ESI pos.): Calcd for CHBrNO: 523.1. Found: 524.1. Found: 525.0; (M+H). 524.1 (M+H). EXAMPLE 1.8 EXAMPLE 17 25 Benzothiophen-5-yl-acetic acid (4-bromo-6-(4-me thyl-piperazine-1-carbonyl)-2-oxo-1,2-dihydro-in Benzooxazol-5-yl-acetic acid (4-bromo-6-(4-methyl dol-3-ylidenel-hydrazide hydrochloride piperazine-1-carbonyl)-2-oxo-1,2-dihydro-indol-3- ylidenel-hydrazide 30 B - N NN Br -N Yory 35 s-,N----> - O SCC O

O 40 The title compound was synthesized using the same pro cedure as in Example 2 (benzothiophen-5-yl-acetic acid was prepared according to Meyer, M.D., Hancock, A. A., et al., J. a) BenZooxazol-5-yl-acetic acid Med. Chem. 1997, 40 (7), 1049-1062). "H NMR (400 MHz, DMSO) & 12.75 (s, 1H), 11.76 (brs, 1H), 11.24 (brs, 1H), 45 7.96 (m, 1H), 7.89 (s, 1H), 7.76 (d. J=5.5 Hz, 1H), 7.42 (d. J=5.4 Hz, 1H), 7.39 (m, 2H), 7.00 (s, 1H), 6.79 (s, 1H), 4.30 (brs, 2H), 3.51 (m, 4H), 3.09 (m, 4H), 2.75 (s, 3H). Mass spectrum (LCMS, ESI pos.): Calcd for CHBrNOS: HO N 50 540.2: found: 540.1 (M+H). EXAMPLE19 (3-Amino-benzodisoxazol-5-yl)-acetic acid 4-bromo-6-(4-methyl-piperazine-1-carbonyl)-2- 55 oxo-1,2-dihydro-indol-3-ylidenel-hydrazide The mixture of (3-amino-4-hydroxy-phenyl)-acetic acid (250 mg, 1.5 mmol) and triethyl orthoformate (30 mg, 2.0 mmol) in toluene was refluxed for 3 h, cooled to room tem O perature and concentrated. The resulting Solid was collected, 60 N Br O Y. washed with 1:3 ethyl acetate:hexane and dried to give the title compound (220 mg, 83%). "H NMR (400 MHz, DMSO) QN /NH MNH2 & 12.4 (bs, 1H), 8.70 (s, 1H), 7.68 (d. J=6.7 Hz, 1H), 7.66 (s, 1H), 7.31 (d. J=6.8 Hz, 1H), 3.33 (s. 2H). Mass spectrum 65 O N ro (LCMS, ESI pos.): Calcd for CH-NO: 177.2. Found: 178.2 (M+H). US 7,662,824 B2 49 50 a) (3-Cyano-4-fluoro-phenyl)-acetic acid Biological Activity In Vitro Assays The following representative in vitro assays were per NC formed in determining the biological activities of compounds CO2H within the scope of the invention. They are given to illustrate the invention in a non-limiting fashion. EXAMPLEA The title compound was prepared as a yellow solid from 10 Cloning, Expression, and Purification of 3-bromo-4-fluorophenylacetic acid using the method of deS Recombinant c-Met Protein olms et al. (J. Med. Chem. 2003, 46 (14), 2980). H NMR (400 MHz, DMSO-d) & 12.6(bs, 1H), 7.80 (dd, J=6.3 Hz, 2.4 This example describes the cloning, expression, and puri HZ, 1H), 7.66 (m, 1H), 7.46 (t, J–9 HZ, 1H), 3.66 (s. 2H). fication of the cytoplasmic domain of c-Met which has the 15 c-Met receptor tyrosine kinase activity. The cytoplasmic b) (3-Cyano-4-fluoro-phenyl)-acetic acid (4-bromo domain has 435 amino acids and shows high homology with 6-(4-methyl-piperazine-1-carbonyl)-2-oxo-1,2-dihy the SRC family of tyrosine kinases (Park et al., 1987, Proc dro-indol-3-ylidenel-hydrazide Natl AcadSci USA. 84(18):6379-83). A clNA for the cytoplasmic domain of Met receptor, containing the tyrosine kinase domain, was amplified by PCR. Oligonucleotides were custom synthesized by Gibco F BRL (Carlsbad, Calif.). Forward oligonucleotide metkinF2 is identical to nucleotides 3068-3097 of the nucleotide r) - JuC. 25 sequence listed in NM 000245, except that nucleotides between 3073 and 3078 have been altered to create a BamHI site for cloning purposes. Reverse oligonucleotide S.C. N CN metkinR2a is identical to nucleotides 4378-4348 of the O N O complementary sequence of that listed in NM 000245 30 except that nucleotides between 4372-4367 have been altered The title compound was prepared as an orange Solid, from to create a XhoI site (underlined) for cloning purposes. The the product of the preceding step, using the same procedure as oligonucleotides were used as PCR primers to amplify Met in Example 2. 'H NMR (400 MHz, DMSO) & 12.73 (bs, 1H), receptor cytoplasmic domain cDNA from Quick Clone pla 11.60 (bs 1H), 7.93 (dd, J=6.3 Hz, 2.0 Hz, 1H), 7.78 (m, 1H), cental clNA (Clontech: Palo Alto, Calif.). Amplification was 7.39 (t, J–7.5 Hz, 1H), 7.29 (s, 1H), 6.91 (s, 1H), 4.26 (bs, 35 performed using Taq DNA polymerase (Gibco-BRL: Carls 2H), 3.61 (bm, 4H), 2.42 (bm, 4H), 2.27 (s.3H). Mass spec bad, Calif.), 1.25 mM each dNTP, 200 nM each oligo, in a 50 trum (LCMS, ESI pos.): Calcd for CHNOBrF: 527.4. ul volume. The thermocycle profile was 30 cycles of each Found: 527.0/529.0 (M+H). containing 94°C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute, on a Perkin Elmer 9600 thermocycler. c) (3-Amino-benzodisoxazol-5-yl)-acetic acid 40 The amplified cDNA for the cytoplasmic domain of Met 4-bromo-6-(4-methyl-piperazine-1-carbonyl)-2- receptor was cloned onto an expression vector. The PCR oxo-1,2-dihydro-indol-3-ylidenel-hydrazide product was digested with BamHI (New England Biolabs; Beverly, Mass.) and XhoI (New England Biolabs). A digested 1.3 kb product was isolated and purified from a 1% agarose 45 gel using Gene Clean (Qbiogene; Irvine, Calif.). Vector O pFastEacFITa (Gibco-BRL) was digested with BamHI and N Br O Y. XhoI (New England Biolabs) and the 4.7 kb linear fragment was purified from a 1% agarose gel using Gene Clean QN /NH M NH2 (Biol O1). The 1.3 kb Met cDNA fragment was ligated to 50 pFastEacFITa vector at 4°C. for 16 hours with T4DNA ligase (New England Biolabs) in a final volume of 10 ul. Cloning the O N ro Met cytoplasmic domain cDNA clone into the BamHI site of pFastEacFITaplaced the cDNA in-frame with the His-6 tag of The product of the preceding step (0.057 g., 0.108 mmol), the vector to allow for expression of an N-terminal His acetohydroxamic acid (0.042 g, 0.559 mmol), and powdered 55 tagged protein. Half the ligation mix (5 uL) was used to potassium carbonate (0.119 g, 0.863 mmol) were dissolved in transform 50 ul DH5O. competent E. coli cells (Gibco-BRL). 5 mL of anhydrous DMF under argon and heated to 50° C. The transformation mix was plated onto LB agarose plates under rubber septum for 24 h. The crude was purified twice by containing 100 ug/ml amplicillin and incubated for 16 hours at preparative TLC on silica gel (first with 15% MeOH/DCM, 37° C. Colonies were picked from these plates and grown in then with 20% MeOH/DCM, saturated with ammonia), then 60 LB broth containing 100 ug/ml ampicillin for 16 hours. Plas by HPLC (10-90% MeCN in water with 0.1% TFA over 30 mid DNA was isolated using Qiagen plasmid DNA purifica min, on a semi-prep C8 Betasil column), giving the title tion reagents (Qiagen; Valencia, Calif.) and clones screened compound as a yellow solid. "H NMR (400 MHz, CDC1/ by digest with BamHI/XhoI. Three clones which had the CDOD) & 7.70 (s, 1H), 7.60 (d. J=8.6 Hz, 1H), 7.35 (m, 2H), appropriate size fragment released from the digest were Sub 6.99 (s, 1H), 4.35 (s. 2H), 3.92 (bm, 4H), 3.30 (bm, 4H), 2.91 65 mitted to ACGT, Inc for DNA sequence analysis. (s, 3H). Mass spectrum (LCMS, ESI pos.): Calcd for One clone, pFastEach Tnetkin-15, contained no muta C.H.N.O.Br. 540.4. Found: 539.9/541.9 (M+H). tions in the cloned c-Met cytoplasmic domain was used to US 7,662,824 B2 51 52 generate a recombinant baculovirus for expression. Recom A major advantage of this format is that it allowed for the binant baculovirus was generated using the Gibco BRL Bac development of an autophosphorylation assay using Ni-che To-Bac system following the protocol specified by the manu late plates which bind the hexa-his tag on the recombinant facturer. Briefly, DH1 OBac cells were transformed with Met kinase. Autophosphorylation assay allows to use a pFastBacFITmetkin-15, clones were selected, viral DNA iso 5 known substrate, Met kinase itself, for the phosphorylation. lated, and screened by PCR for Met cDNA insert. Sf9 insect The DELFIA Met autophosphorylation assay is very sensi cells were transfected with the recombinant baculovirus tive with a signal to noise ratio in excess of 50:1. DNA. Media containing POviral stock was collected and used The assay procedure for screening is as follows. The puri for 2 subsequent rounds of viral amplification. fied His6 tagged cytoplasmic domain of c-Met was diluted to Multiple concentrations of amplified viral stock were used 10 a concentration of 500 ng/ml in enzyme dilution buffer (50 to infect Sf9 cells. Cells were harvested 24, 48, and 72 hours mM Tris-HCl, pH8.0, 0.1% BSA) and dispensed to assay post transfection. Infected Sf9 cells were lysed in 50 mM plates at a volume of 50 ul per well. Black opaque HisGrab Tris-HCl pH 8.0, 150 mM NaCl, 150 mMimidazole, 1.0 mM Nickel coated 96 well plates (Pierce, Rockford, Ill.) were PMSF, 0.5% NP40, 3.5ug/ml leupeptin, 3.5ug/ml aprotinin selected to run the assay. Next, 2.5ul of compound in 40% and total protein concentration determined in a BCA assay 15 DMSO was added to test wells, 2.5 ul of 40% DMSO only (Pierce; Rockford, Ill.). Cell lysates were separated on a was added to the negative control wells. The autophosphory 4-15% SDS-PAGE then transferred to nitrocellulose mem lation reaction was initiated upon the addition of 50 ul of brane for immunoblot analysis. Nitocellulose blots were reaction buffer, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl, 0.1 probed with an anti-His6 antibody to confirm expression of mM NaVO, 1 mM DTT, 1 uM ATP Plates were incubated the His-tagged met kinase protein. Optimal viral concentra at room temperature for 1 hour followed by 2 washes with 200 tion to Sf9 cell ratio was determined by examination of ul/well of PBS. Europium conjugated anti-phosphotyrosine lysates collected from different infection conditions. Maxi antibody, Eu-PY20 from Perkin Elmer was diluted to 50 mal protein recovery occurred 48 hours post infection. ng/ml in Delfia AB buffer (Perkin Elmer, Boston, Mass.), A Small-scale expression/purification of the His-tagged added to the 96 well assay plates at a volume of 1001 ul/well, cytoplasmic domain of Met receptor was performed. Sf9 25 and incubated at room temperature for 2 hours. Assay plates insect cells transfected with the recombinant baculovirus that were then washed 4 times each with 200 ul/well of Delfia expresses the His-tagged cytoplasmic domain of Met recep wash buffer (Perkin Elmer). After the final wash 150 ul of tor were lysed in buffer containing 50 mM Tris-HCl pH 8.0, Delfia Enhancement solution (Perkin Elmer) was added to 150 mM. NaCl, 150 mM imidazole, 1.0 mM PMSF, 0.5% each well of the assay plate and incubated at room tempera NP40, 3.5 ug/ml leupeptin, 3.5 lug/ml aprotinin. The lysate 30 ture for 1 hour. Plates were read on an LJLAnalyst instrument was incubated with 5 ml of a 50% solution of Ni-agarose (Molecular Devices; Sunnyvale, Calif.) with filter settings of beads (Qiagen) in PBS for 2 hours rotating at 4°C. to capture 360 excitation, 620 emission, and 410 dichroic. ICs values the His-tagged protein. The lysate containing His-tagged pro were calculated using Graphpad Prism software (Graphpad tein bound to Ni-agarose beads was loaded onto a 10 ml Software: San Diego, Calif.). column. Ni-agarose beads were allowed to pack and Super 35 natant allowed to flow through. The packed column was then EXAMPLE C washed with 60 ml of wash buffer (same as lysis buffer).5 ml of elution buffer (50 mM Tris-HCl pH 8, 150 mM. NaCl, 150 Fluorescence Polarization Assay on the Kinase mMimidazole, 1.0 mM PMSF) was added to the column and Activity of c-Met 10 fractions (0.5 ml volume each) were collected. Small 40 aliquots of each fraction were separated by 4-15% SDS A fluorescence polarization assay (FP) was developed for PAGE and either transferred to nitrocellulose for immunoblot screening of compounds that decreases the kinase activity of analysis or processed for Coomassie stain (Bio-Safe Safe c-Met. Coomassie, Bio-Rad). The major protein band on the Coo A protein (6H-TEV-cMet) comprising c-Met kinase massie stain gel has the appropriate size for His6-MetKin (52 45 domain was cloned, recombinantly expressed and purified kD), corresponding to the His-tagged protein detected by following a procedure similar to that as described in Example immunoblot. Protein concentration as estimated from the A. The protein comprises a His tag consisting of six histidines at its N-terminal, a TEV protease cleavage site, and the c-Met Coomassie stain gel was approximately 2 mg/ml. kinase domain. The c-Metkinase domain consists amino acid Recombinant viral stock was transferred to the contract lab, Pan Vera (Madison, Wis.) for large-scale expression and 50 1046-1390 of the c-Met protein as listed in GenBank protein purification of His6-MetKin in quantities sufficient for High accession number P08581. Throughput Screening. A 60 L scale up and 4 step purification For the c-Met kinase assay, the 6H-TEV-cMet was used as scheme yielded 98.4 mg of protein that is more than 95% the enzyme, and a poly GlyTyr peptide (Sigma, P-0275) was pure. used as the Substrate. The kinase reaction was performed in a 55 volume of 10 ul in HE microplates (LJL Biosystems). Assay conditions were as follows: 100 mM HEPES pH 7.5, 0.01% EXAMPLEB Tween-20, 1 mM DTT, 5 mM MgCl, 10 uM ATP, 10 g/ml poly GlyTyr. The kinase reaction was initiated upon addition Delfia Autophosphorylation Kinase Assay on the of 10 nM 6H-TEV-cMet. After a 12 minute incubation at c-Met 60 room temperature the reaction was terminated with 1.2 of 50 mM EDTA A DELFIA time resolved fluorescence assay was devel Phosphorylation of the peptide substrate was detected with oped for Screening of compounds that decreases the auto PanVera Tyrosine Kinase Assay Kit, Green reagents (P2837). phosphorylation thus the kinase activity of c-Met. Five minutes after the addition of EDTA, 10ul of antibody/ The DELFIA assay is non-radioactive. The autophophory 65 tracer mix (Pan Vera reagents, 1:1:3 dilution of antibody: lation of the c-Met is measured by an anti-phosphotyrosine Tracer: FP dilution buffer, Cat. ii. P2837) was added to each antibody coupled to an Europium tag. well. Plates were allowed to incubate for 30 minutes at room US 7,662,824 B2 53 54 temperature and fluorescence polarization was measured incubate for 60 minutes. Plates were read on an LJL Analyst with an LJL Analyst instrument (Molecular Devices; Sunny instrument (Molecular Devices; Sunnyvale, Calif.) with filter vale, Calif.) with excitation=485 nm filter and emission=530 settings of 360 excitation, 620 emission, and 410 dichroic. nm filter. ICs values were calculated from triplicate sets of ICso values were calculated using Graphpad Prism Software data using Graphpad Prism software (Graphpad Software: 5 (Graphpad Software; San Diego, Calif.). San Diego, Calif.). Aqueous Solubility Assay EXAMPLED Compounds of the present invention were tested for aque ous solubility in order to obtain optimal solubility without adversely affecting the potency of the compound. Aqueous A Cell Based ELISA Assay for c-Met 10 Phosphorylation solubility was determined in the following manner: Com pounds were taken from a 10 mM DMSO (dimethyl sulfox A cell based ELISA assay was developed to evaluate the ide) stock solution and diluted in water, PBS (phosphate ability of compounds to inhibit HGF stimulated c-Met phos buffered saline), aqueous HCl at pH2 and DMSO. Water, PBS phorylation in cells. 15 and aqueous HCl at pH2 having a final DMSO content of S114 cells were seeded to a 96 well tissue culture treated 0.4%, with the final DMSO concentration reaching 40 uM. dish at a concentration of 5x10" per well. After a 16-20 hour Samples were placed in the incubator and shaken for 15 incubation, culture medium was removed and replaced with minutes. Aqueous solubility was then determined by LC/MS serum free medium supplemented with 0.5% BSA. Test com under the following conditions: pound was then added and incubated with the cells for 60 Mobile Phase A: 0.05% CFCOH+5.0% CHCN in HO minutes, followed by the addition of 1 ul HGF at 2.5 g/ul for Mobile Phase B: 0.05% CFCOH in CHCN 15 minutes. Cells were then lysed with the addition of 25ul of Gradient: 5-95% B in 2.5 minutes ice cold 3x RIPA buffer (50 mM Tris HCl, pH 7.5, 1% Triton, Run Time: 3.1 min 1% IGEPAL, 0.25% deoxycholic acid, 150 mM. NaCl, 1 mM Flow Rate: 0.4 ml/min sodium orthovanidate, 1 mM sodium fluoride, and 1 tablet 25 Injection Volume: 15-30 uL (as needed) protease cocktail inhibitor (Boheringer Mannheim, cat. Column: Princeton Chromatography, PrincetonSPHER #1697498). Cell lysates were then transferred to NUNC Max C30 200A 5u, 50x2.0 mm with 2.0x23 mm guard car isorp plates coated with anti-c-Met receptor antibody AF276 tridges (R&D Systems). Lysates were incubated with the antibody Solubility results were calculated under the assumption coated plates for 1 hour at room temperature. Plates were 30 that compound solubility in DMSO is 100% at 40 uM. washed with Delfia wash buffer (Perkin Elmer, Boston, Mass.) and 100 ul of 0.25 ug/ml europium conjugated PT66 In Vitro Biological Data anti-phosphotyrosine antibody (Perkin Elmer, Boston, The activity of representative compounds of the present Mass.). Following another 1 hour incubation at room tem invention is presented in the chart below. All activities are in perature the plates were washed three times with Delfia wash 35 uM and data is accepted as valid if the 95% confidence inter buffer (Perkin Elmer). After the final wash, 150 ml of Delfia vals calculated by Graphpad prism are within 2 fold of the enhancer solution (Perkin Elmer) was added and allowed to ICso

c-Met FP c-Met Delfia Solubility Example Enz ICSO Autophos in H2O Number Structure M IC50 M (uM)

1 H O.OO6 Br N1 N W OH O O H O NS1a-N N H O

2 O.O10 43.85 B r N1

O N N H US 7,662,824 B2 55 56

-continued

c-Met FP c-Met Delfia Solubility Example EZICSO Autophos in H2O Number Structure IC50 M (uM)

38.06

37.9

O NN-1- N H

H 38.76 Br N-N W O O O N H

O.OOS O.OOS 43.95 Br

O

42.74 Br W O O O N N H

41.08 Br

N

O.OOS Br US 7,662,824 B2 57 58

ontinued

c-Met FP c-Met Delfia Solubility Example EZICSO Autophos in H2O Number Structure IC50 M (uM)

10 Br HO

11 O.OOOS Br

12

HO NS-1a-N O

13 H HO Br N-N W O O H O O N H O

14 Br W N O O O N H

15 B r N1 W O O O N H

16 Br N1 N W O O O N H US 7,662,824 B2 59 60

-continued c-Met FP c-Met Delfia Solubility Example EZICSO Autophos in H2O Number Structure M IC50 M (uM) 17 H O.656 Br W 1N Ny NN ~ O O O lu N H O

18 4.70 Br N1 N

1 O S N N N C:-H O O

19 B NH2 O.158 r N-NH

Sr. W O \M N- N N O O H O

In Vivo Assays 35. In pilot growth studies, the following conditions were iden The following representative in vivo assay was performed tified as permitting U87MG cell growth in nude mice as in determining the biological activities of compounds within Subcutaneous solid tumor Xenografts: Immediately prior to the scope of the invention. They are given to illustrate the injection, cells were washed in PBS and counted, suspended into a sing R fth ds ofth at a concentration of 1x107 cells/mL, and loaded into 1 cc inventionean1-lumor was evaluated eITIcacy i. OT Vivo a SubSe using OThe a nude COmpoundS R U87MG OThe 40 Syringes equipped with 25 gauge needles. Female athymic human tumor Xenograft regression model nude mice weighing no less than 20-21 grams were inocu Female athymic nude mice (CD-1, nu/nu, 9-10 weeks old) lated Subcutaneously in the left inguinal region of the thigh were obtained from Charles River Laboratories (Wilmington, with 1x10° tumor cells in a delivery volume of 0.1 mL. Mass.) and were maintained according to NIH standards. All 45 Tumors were allowed to grow to a pre-determined size prior mice were group housed (5 mice? cage) under clean-room to initiation of dosing. Approximately 2 weeks after tumor conditions in sterile micro-isolator cages on a 12-hour light/ cellinoculation, mice bearing Subcutaneous tumors were ran dark cycle in a room maintained at 21-22 C. and 40-50% domly assigned to treatment groups such that all treatment humidity. Mice were fed irradiated standard rodent diet and groups had similar starting mean tumor Volumes of ~100 water ad libitum. All animals were housed in a Laboratory 50 mm. Mice were dosed IP (intra-peritoneal) with vehicle NNESSES (control group) or compound at various doses either twice Laboratory Animal Care ( LAC). All procedures involv- daily (b.i.d.) or once-daily (q.d.). Dosing was continued for in Nis We COi. th com R with the NIH 11 consecutive days, depending on the kinetics of tumor ,de for the Care and Use of Laplan Animals and all 55 growth and size of tumors in vehicle-treated control mice. If protocols were approved by an Internal Animal Care and Use tumors in the control mice reached ~10% of body weight Committee (IACUC) (~2.0 grams), the study was to be terminated. Compounds of The human glioblastoma U-87MG cell line was obtained the present invention were prepared fresh daily as a clear from the American Type Culture Collection (ATCC Number: solution ((a) 1, 1 5 and 3 mg/mL) in 20%f0 HPBCD and admin HTB-14) and propagated in minimum essential (Eagle) 60 istered IP as described above. During the study, tumor growth medium containing 2 mML-glutamine, 1.5 g/L Sodium bicar- was measured three times-a-week (M, W, F) using electronic bonate, 0.1 mM non-essential amino acids, 1.0 mM sodium Vernier calipers. Tumor Volume (mm) was calculated using pyruvate, and 10% FBS (fetal bovine serum). U87MG cells the formula (LXW) /2, where L-length (mm) and W width express both c-Met and its ligand HGF, therefore strong anti- (shortest distance in mm) of the tumor. Body weight was tumor activity against U87MG tumor growth in the nude 65 measured three times-a-week and a loss of body weight >10% mouse tumor Xenograft model is anticipated to be a desirable was used as an indication of lack of compound tolerability. quality of the invention. Unacceptable toxicity was defined as body weight loss >20% US 7,662,824 B2 61 62 during the study. Mice were closely examined daily at each disorders include pre-existing conditions related to c-Met dose for overt clinical signs of adverse, drug-related side expression (or over expression) and/or c-Met mutation. effects. In one example, the invention provides methods for pre On the day of study termination, a final tumor Volume and venting in a subject a cell proliferative disorder or a disorder final body weight were obtained on each animal. Mice were related to c-Met, comprising administering to the Subject a euthanized using 100% CO and tumors were immediately prophylactically effective amount of a pharmaceutical com excised intact and weighed, with final tumor wet weight position comprising the compound of Formula I and a phar (grams) serving as a primary efficacy endpoint. maceutically acceptable carrier. Administration of said pro The time course of the inhibitory effects of compounds of phylactic agent can occur prior to the manifestation of the present invention on the growth of U87MG tumors is 10 symptoms characteristic of the cell proliferative disorder or illustrated in FIG. 1. Values represent the mean (tsem) of 15 disorder related to c-Met, such that a disease or disorder is mice per treatment group. Percent inhibition (%. 1) of tumor prevented or, alternatively, delayed in its progression. growth was calculated versus tumor growth in the vehicle treated Control group on the last study day. Statistical signifi In another example, the invention pertains to methods of cance versus Control was determined by Analysis of Variance 15 treating in a subject a cell proliferative disorder or a disorder (ANOVA) followed by Dunnett's t-test: * p-0.05. The 30 related to c-Met comprising administering to the Subject a mpk (mg compound per kg animal weight) dosing group was therapeutically effective amount of a pharmaceutical compo terminated on day 3 due to treatment related deaths of 50% of sition comprising the compound of Formula I and a pharma the mice in the group. Dosing of the 15 mpk group was ceutically acceptable carrier. Administration of said thera reduced from b.i.d. administration to q.d. administration on peutic agent can occur concurrently with the manifestation of day 4 due to overt toxicity. The 10 mpkgroup continued to be symptoms characteristic of the disorder. Such that said thera dosed b.i.d throughout the course of the study except for days peutic agent serves as a therapy to compensate for the cell 6 and 7 which were administered q.d. proliferative disorder or disorders related to c-Met. A similar reduction of final tumor weight was noted at In another example, the invention pertains to methods of study termination. (See FIG. 2). Values represent the mean 25 modulating in a Subject a cell proliferative disorder or a dis (tsem) of 15 mice per treatment group. Percent Inhibition order related to c-Met, such that modulation of the level of was calculated versus the mean tumor weight in the vehicle c-Met expression or of c-Met activity may act to ameliorate treated control group. Statistical significance versus Control the cell proliferative disorder or a disorder related to c-Met, was not reached for either treatment group as determined by comprising administering to the Subject a therapeutically ANOVA followed by Dunnett's t-test analysis. 30 effective amount of a pharmaceutical composition compris ing the compound of Formula I and a pharmaceutically Methods of Treatment/Prevention acceptable carrier. In another aspect of this invention, compounds of the The term “prophylactically effective amount” refers to an invention can be used to inhibit tyrosine kinase activity or amount of an active compound or pharmaceutical agent that expression, including c-Metactivity, reduce kinase activity or 35 expression, including c-Met activity, and modulate expres inhibits or delays in a Subject the onset of a disorder as being sion of c-Met in a cell or a subject, or to treat disorders related sought by a researcher, Veterinarian, medical doctor or other to c-Met kinase activity or expression in a subject. Inhibition clinician. of c-Met activity is believed to indirectly modulate c-Met The term “therapeutically effective amount’ as used herein, refers to an amount of active compound or pharma expression. 40 In one embodiment to this aspect, the present invention ceutical agent that elicits the biological or medicinal response provides a method for reducing or inhibiting the kinase activ in a Subject that is being sought by a researcher, Veterinarian, ity of c-Met, and modulate expression of c-Met in a cell medical doctor or other clinician, which includes alleviation comprising the step of contacting the cell with a compound of of the symptoms of the disease or disorder being treated. Formula I. The present invention also provides a method for 45 Methods are known in the art for determining therapeuti reducing or inhibiting the kinase activity of c-Met, and modu cally and prophylactically effective doses for the instant phar late expression of c-Met in a subject comprising the step of maceutical composition. administering a compound of Formula I to the Subject. The As used herein, the term “composition' is intended to present invention further provides a method of inhibiting cell encompass a product comprising the specified ingredients in proliferation in a cell comprising the step of contacting the 50 the specified amounts, as well as any product which results, cell with a compound of Formula I. directly or indirectly, from combinations of the specified The kinase activity or expression of c-Met in a cell or a ingredients in the specified amounts. subject can be determined by procedures well known in the As used herein, the terms “disorders related to c-Met, or art, such as the c-Met kinase assay described herein. Inhibi “disorders related to c-Met receptor tyrosine kinase' shall tion of c-Met kinase activity in cells can also be measured by 55 include diseases associated with or implicating c-Met activ determining the level of c-Met phosphorylation using an ity, for example, the overactivity of c-Met, and conditions that ELISA assay format such as the one described here. accompany with these diseases. The term “overactivity of The term “subject’ as used herein, refers to an animal, c-Met' refers to either 1) c-Met expression in cells which preferably a mammal, most preferably a human, who has normally do not express c-Met; 2) c-Met expression by cells been the object of treatment, observation or experiment. 60 which normally do not express c-Met; 3) increased c-Met The term “contacting as used herein, refers to the addition expression leading to unwanted cell proliferation; or 4) muta of compound to cells such that compound is taken up by the tions leading to constitutive activation of c-Met. Examples of cell. “disorders related to c-Met' include disorders resulting from In other embodiments to this aspect, the present invention over stimulation of c-Met due to abnormally high amount of provides both prophylactic and therapeutic methods for treat 65 c-Met or mutations in c-Met, or disorders resulting from ing a Subject at risk of (or Susceptible to) developing a cell abnormally high amount of c-Met activity due to abnormally proliferative disorder or a disorder related to c-Met. Such high amount of c-Met or mutations in c-Met. US 7,662,824 B2 63 64 It is known that overactivity of c-Met has been implicated toxins/podophyllbtoxins (e.g. etoposide, teniposide); in the pathogenesis of a number of diseases, such as cell aromatase inhibitors (e.g., anastroZole, letrozole, exemes proliferative disorders, neoplastic disorders and cancers. tane); anti-estrogen compounds (e.g., tamoxifen, fulves The term “cell proliferative disorders' refers to unwanted trant), antifolates (e.g., premetrexed disodium); hypomethy cell proliferation of one or more subset of cells in a multicel lating agents (e.g., azacitidine); biologics (e.g., gemtuzamab, lular organism resulting inharm (i.e., discomfort or decreased cetuximab, rituximab, pertuzumab, trastuzumab, bevaci life expectancy) to the multicellular organisms. Cell prolif Zumab, erlotinib); antibiotics/anthracylines (e.g. idarubicin, erative disorders can occur in different types of animals and actinomycin D. bleomycin, daunorubicin, doxorubicin, mito humans. Cell proliferative disorders include neoplastic disor mycin C, dactinomycin, carminomycin, daunomycin); anti ders (as used herein, a “neoplastic disorder” refers to a tumor 10 metabolites (e.g., clofarabine, aminopterin, cytosine arabino resulting from abnormal or uncontrolled cellular growth) and side, methotrexate); tubulin-binding agents (e.g. other cell proliferative disorders. combretastatin, colchicine, nocodazole); topoisomerase Examples of cell proliferative disorders related to c-Met, inhibitors (e.g., camptothecin). Further useful agents include include tumors and cancers—for instance, hereditary and Verapamil, a calcium antagonist found to be useful in combi sporadic human papillary renal carcinomas, breast cancer, 15 nation with antineoplastic agents to establish chemosensitiv colorectal cancer, gastric carcinoma, glioma, ovarian cancer, ity in tumor cells resistant to accepted chemotherapeutic hepatocellular carcinoma, head and neck squamous cell car agents and to potentiate the efficacy of Such compounds in cinomas, testicular carcinoma, basal cell carcinoma, liver drug-sensitive malignancies. See Simpson WG, The calcium carcinoma, sarcoma, malignant pleural mesothelima, mela channel blocker Verapamil and cancer chemotherapy. Cell noma, multiple myeloma, osteosarcoma, pancreatic cancer, Calcium. 1985 December; 6(6):449-67. Additionally, yet to prostate cancer, synovial sarcoma, thyroid carcinoma, non emerge chemotherapeutic agents are contemplated as being Small cell lung cancer (NSCLC) and Small cell lung cancer, useful in combination with the compound of the present transitional cell carcinoma of urinary bladder, testicular car invention. cinoma, basal cell carcinoma, liver carcinoma—including In another embodiment of the present invention, the com leukemias, lymphomas, and myelomas—for instance, acute 25 pound of the present invention may be administered in com lymphocytic leukemia (ALL), acute myeloid leukemia bination with radiation therapy. As used herein, “radiation (AML), acute promyelocytic leukemia (APL), chronic lym therapy refers to a therapy comprising exposing the Subject phocytic leukemia (CLL), chronic myeloid leukemia (CML), in need thereof to radiation. Such therapy is known to those chronic neutrophilic leukemia (CNL), acute undifferentiated skilled in the art. The appropriate scheme of radiation therapy leukemia (AUL), anaplastic large-cell lymphoma (ALCL), 30 will be similar to those already employed in clinical therapies prolymphocytic leukemia (PML), juvenile myelomonocyctic wherein the radiation therapy is used alone or in combination leukemia (JMML), adult T-cell ALL, AML with trilineage with other chemotherapeutics. myelodysplasia (AML/TMDS), mixed lineage leukemia In another embodiment of the present invention, the com (MLL), myelodysplastic syndromes (MDSs), myeloprolif pound of the present invention may be administered in com erative disorders (MPD), multiple myeloma, (MM), myeloid 35 bination with a gene therapy. As used herein, “gene therapy' sarcoma, non-Hodgkin’s lymphoma and Hodgkin’s disease refers to a therapy targeting on particular genes involved in (also called Hodgkin’s lymphoma)—and diseases associated tumor development. Possible gene therapy strategies include with the formation of new vasculature. Such as rheumatoid, the restoration of defective cancer-inhibitory genes, cell arthritis, retinopathy. transduction or transfection with antisense DNA correspond Other cell proliferative disorders in which overactivity of 40 ing to genes coding for growth factors and their receptors, c-Met has been implicated in their pathogenesis include can RNA-based strategies such as ribozymes, RNA decoys, anti cers in which c-Met activity contributes to the invasive/meta sense messenger RNAs and small interfering RNA (siRNA) static phenotype, including cancers in which c-Met is not molecules and the so-called 'Suicide genes. overexpressed or otherwise altered. In other embodiments of this invention, the compound of In a further embodiment to this aspect, the invention 45 the present invention may be administered in combination encompasses a combination therapy for treating or inhibiting with an immunotherapy. As used herein, “immunotherapy' the onset of a cell proliferative disorder or a disorder related refers to a therapy targeting particular protein involved in to c-Met in a Subject. The combination therapy comprises tumor development via antibodies specific to such protein. administering to the Subject a therapeutically or prophylacti For example, monoclonal antibodies against Vascular endot cally effective amount of a compound of Formula I, and one 50 helial growth factor have been used in treating cancers. or more other anti-cell proliferation therapy including che Where a second pharmaceutical is used in addition to a motherapy, radiation therapy, gene therapy and immuno compound of the present invention, the two pharmaceuticals therapy. may be administered simultaneously (e.g. in separate or uni In an embodiment of the present invention, the compound tary compositions) sequentially in either order, at approxi of the present invention may be administered in combination 55 mately the same time, or on separate dosing schedules. In the with chemotherapy. As used herein, chemotherapy refers to a latter case, the two compounds will be administered within a therapy involving a chemotherapeutic agent. A variety of period and in an amount and manner that is sufficient to chemotherapeutic agents may be used in the combined treat ensure that an advantageous or synergistic effect is achieved. ment methods disclosed herein. Chemotherapeutic agents It will be appreciated that the preferred method and order of contemplated as exemplary, include, but are not limited to: 60 administration and the respective dosage amounts and platinum compounds (e.g., cisplatin, carboplatin, oxalipl regimes for each component of the combination will depend atin); taxane compounds (e.g., paclitaxcel, docetaxol); cam on the particular chemotherapeutic agent being administered potothecin compounds (irinotecan, topotecan); Vinca alka in conjunction with the compound of the present invention, loids (e.g., Vincristine, vinblastine, Vinorelbine); anti-tumor their route of administration, the particular tumor being nucleoside derivatives (e.g., 5-fluorouracil, leucovorin, gem 65 treated and the particular host being treated. citabine, capecitabine); alkylating agents (e.g., cyclophos As will be understood by those of ordinary skill in the art, phamide, carmustine, lomustine, thiotepa); epipodophyllo the appropriate doses of chemotherapeutic agents will be US 7,662,824 B2 65 66 generally similar to or less than those already employed in daily depending on the particular agent and the condition clinical therapies wherein the chemotherapeutics are admin being treated. Tamoxifen is advantageously administered istered alone or in combination with other chemotherapeu orally in a dosage of 5 to 50 mg, preferably 10 to 20 mg twice tics. a day, continuing the therapy for Sufficient time to achieve and The optimum method and order of administration and the maintain a therapeutic effect. Toremifene is advantageously dosage amounts and regime can be readily determined by administered orally in a dosage of about 60 mg once a day, those skilled in the art using conventional methods and in continuing the therapy for Sufficient time to achieve and view of the information set out herein. maintain a therapeutic effect. AnastroZole is advantageously By way of example only, platinum compounds are advan administered orally in a dosage of about 1 mg once a day. tageously administered in a dosage of 1 to 500 mg per square 10 Droloxifene is advantageously administered orally in a dos meter (mg/m) of body surface area, for example 50 to 400 age of about 20-100 mg once a day. Raloxifene is advanta mg/m, particularly for cisplatin in a dosage of about 75 geously administered orally in a dosage of about 60 mg once mg/m and for carboplatin in about 300 mg/m per course of a day. Exemestane is advantageously administered orally in a treatment. Cisplatin is not absorbed orally and must therefore dosage of about 25 mg once a day. be delivered via injection intravenously, Subcutaneously, 15 By way of example only, biologics may be advantageously intratumorally or intraperitoneally. administered in a dosage of about 1 to 5 mg per square meter By way of example only, taxane compounds are advanta (mg/m) of body surface area, or as known in the art, if geously administered in a dosage of 50 to 400 mg per square different. For example, trastuzumab is advantageously meter (mg/m) of body surface area, for example 75 to 250 administered in a dosage of 1 to 5 mg/m particularly 2 to 4 mg/m, particularly for paclitaxel in a dosage of about 175 to mg/m per course of treatment. 250 mg/m and for docetaxel in about 75 to 150 mg/m per Dosages may be administered, for example once, twice or course of treatment. more per course of treatment, which may be repeated for By way of example only, camptothecin compounds are example every 7, 14, 21 or 28 days. advantageously administered in a dosage of 0.1 to 400 mg per The compounds of the present invention can be adminis square meter (mg/m) of body surface area, for example 1 to 25 tered to a Subject systemically, for example, intravenously, 300 mg/m, particularly for irinotecan in a dosage of about orally, Subcutaneously, intramuscular, intradermal, or 100 to 350 mg/m and for topotecanin about 1 to 2 mg/m per parenterally. The compounds of the present invention can also course of treatment. be administered to a subject locally. Non-limiting examples By way of example only, Vinca alkaloids may be advanta of local delivery systems include the use of intraluminal geously administered in a dosage of 2 to 30 mg per square 30 medical devices that include intravascular drug delivery cath meter (mg/m) of body surface area, particularly for vinblas eters, wires, pharmacological stents and endoluminal paving. tine in a dosage of about 3 to 12 mg/m, for vincristine in a The compounds of the present invention can further be dosage of about 1 to 2 mg/m, and for vinorelbine in dosage administered to a Subject in combination with a targeting of about 10 to 30 mg/m per course of treatment. agent to achieve high local concentration of the compound at By way of example only, anti-tumor nucleoside derivatives 35 the target site. In addition, the compounds of the present may be advantageously administered in a dosage of 200 to invention may be Formulated for fast-release or slow-release 2500 mg per square meter (mg/m) of body surface area, for with the objective of maintaining the drugs or agents in con example 700 to 1500 mg/m. 5-fluorouracil (5-FU) is com tact with target tissues for a period ranging from hours to monly used via intravenous administration with doses rang weeks. ing from 200 to 500 mg/m (preferably from 3 to 15 mg/kg/ 40 day). Gemcitabine is advantageously administered in a The present invention also provides a pharmaceutical com dosage of about 800 to 1200 mg/m and capecitabine is position comprising a compound of Formula I in association advantageously administered in about 1000 to 2500 mg/m with a pharmaceutically acceptable carrier. The pharmaceu per course of treatment. tical composition may contain between about 0.1 mg and By way of example only, alkylating agents may be advan 45 1000 mg, preferably about 100 to 500 mg. of the compound, tageously administered in a dosage of 100 to 500 mg per and may be constituted into any form suitable for the mode of square meter (mg/m) of body surface area, for example 120 administration selected. to 200 mg/m, particularly for cyclophosphamide in a dosage The phrases “pharmaceutically acceptable” refer to of about 100 to 500 mg/m, for chlorambucil in a dosage of molecular entities and compositions that do not produce an about 0.1 to 0.2 mg/kg of body weight, for carmustine in a 50 adverse, allergic or other untoward reaction when adminis dosage of about 150 to 200 mg/m, and for lomustine in a tered to an animal, or a human, as appropriate. Veterinary uses dosage of about 100 to 150 mg/m per course of treatment. are equally included within the invention and “pharmaceuti By way of example only, podophyllotoxin derivatives may cally acceptable Formulations include Formulations for be advantageously administered in a dosage of 30 to 300 mg both clinical and/or veterinary use. per square meter (mg/m2) of body Surface area, for example 55 Carriers include necessary and inert pharmaceutical 50 to 250 mg/m, particularly for etoposide in a dosage of excipients, including, but not limited to, binders, Suspending about 35 to 100 mg/m and for teniposide in about 50 to 250 agents, lubricants, flavorants, Sweeteners, preservatives, mg/m per course of treatment. dyes, and coatings. Compositions Suitable for oral adminis By way of example only, anthracycline derivatives may be tration include Solid forms, such as pills, tablets, caplets, advantageously administered in a dosage of 10 to 75 mg per 60 capsules (each including immediate release, timed release square meter (mg/m) of body surface area, for example 15 to and Sustained release Formulations), granules, and powders, 60 mg/m, particularly for doxorubicin in a dosage of about and liquid forms, such as Solutions, syrups, elixirs, emulsions, 40 to 75 mg/m, for daunorubicin in a dosage of about 25 to and Suspensions. Forms useful for parenteral administration 45 mg/m, and for idarubicin in a dosage of about 10 to 15 include sterile solutions, emulsions and Suspensions. mg/m per course of treatment. 65 The pharmaceutical composition of the present invention By way of example only, anti-estrogen compounds may be also includes a pharmaceutical composition for slow release advantageously administered in a dosage of about 1 to 100 mg of a compound of the present invention. The composition US 7,662,824 B2 67 68 includes a slow release carrier (typically, a polymeric carrier) sprays, drops, ampoules, auto-injector devices or Supposito and a compound of the present invention. ries; for oral parenteral, intranasal. Sublingual or rectal Slow release biodegradable carriers are well known in the administration, or for administration by inhalation or insuf art. These are materials that may form particles that capture flation. Alternatively, the composition may be presented in a therein an active compound(s) and slowly degrade/dissolve form Suitable for once-weekly or once-monthly administra under a suitable environment (e.g., aqueous, acidic, basic, tion; for example, an insoluble salt of the active compound, etc) and thereby degrade/dissolve in body fluids and release Such as the decanoate salt, may be adapted to provide a depot the active compound(s) therein. The particles are preferably preparation for intramuscular injection. For preparing Solid nanoparticles (i.e., in the range of about 1 to 500 nm in compositions such as tablets, the principal active ingredientis diameter, preferably about 50-200 nm in diameter, and most 10 mixed with a pharmaceutical carrier, e.g. conventional tablet preferably about 100 nm in diameter). ing ingredients such as corn starch, lactose, Sucrose, Sorbitol, The present invention also provides methods to prepare the talc, Stearic acid, magnesium Stearate, dicalcium phosphate pharmaceutical compositions of this invention. The com or gums, and other pharmaceutical diluents, e.g. water, to pound of Formula I, as the active ingredient, is intimately form a solid preFormulation composition containing a homo admixed with a pharmaceutical carrier according to conven 15 geneous mixture of a compound of the present invention, or a tional pharmaceutical compounding techniques, which car pharmaceutically acceptable salt thereof. When referring to rier may take a wide variety of forms depending on the form these preFormulation compositions as homogeneous, it is of preparation desired for administration, e.g., oral or meant that the active ingredient is dispersed evenly through parenteral Such as intramuscular. In preparing the composi out the composition so that the composition may be readily tions in oral dosage form, any of the usual pharmaceutical Subdivided into equally effective dosage forms such as tab media may be employed. Thus, for liquid oral preparations, lets, pills and capsules. This solid preFormulation composi Such as for example, Suspensions, elixirs and solutions, Suit tion is then subdivided into unit dosage forms of the type able carriers and additives include water, glycols, oils, alco described above containing from 0.1 to about 500 mg of the hols, flavoring agents, preservatives, coloring agents and the active ingredient of the present invention. The tablets or pills like; for Solid oral preparations such as, for example, pow 25 of the novel composition can be coated or otherwise com ders, capsules, caplets, gelcaps and tablets, Suitable carriers pounded to provide a dosage form affording the advantage of and additives include starches, Sugars, diluents, granulating prolonged action. For example, the tablet or pill can comprise agents, lubricants, binders, disintegrating agents and the like. an inner dosage and an outer dosage component, the latter Because of their ease in administration, tablets and capsules being in the form of an envelope over the former. The two represent the most advantageous oral dosage unit form, in 30 components can be separated by an enteric layer which serves which case solid pharmaceutical carriers are obviously to resist disintegration in the stomach and permits the inner employed. If desired, tablets may be sugar coated or enteric component to pass intact into the duodenum or to be delayed coated by standard techniques. For parenterals, the carrier in release. A variety of material can be used for such enteric will usually comprise sterile water, though other ingredients, layers or coatings, such materials including a number of for example, for purposes such as aiding Solubility or for 35 polymeric acids with Such materials as shellac, acetyl alcohol preservation, may be included. Injectable Suspensions may and cellulose acetate. also be prepared, in which case appropriate liquid carriers, The liquid forms in which the compound of Formula I may Suspending agents and the like may be employed. In prepa be incorporated for administration orally or by injection ration for slow release, a slow release carrier, typically a include, aqueous solutions, Suitably flavored syrups, aqueous polymeric carrier, and a compound of the present invention 40 or oil suspensions, and flavored emulsions with edible oils are first dissolved or dispersed in an organic solvent. The Such as cottonseed oil, Sesame oil, coconut oil or peanut oil, as obtained organic solution is then added into an aqueous solu well as elixirs and similar pharmaceutical vehicles. Suitable tion to obtain an oil-in-water-type emulsion. Preferably, the dispersing or Suspending agents for aqueous suspensions, aqueous solution includes Surface-active agent(s). Subse include synthetic and natural gums such as tragacanth, acacia, quently, the organic Solvent is evaporated from the oil-in 45 alginate, dextran, Sodium carboxymethylcellulose, methyl water-type emulsion to obtain a colloidal Suspension of par cellulose, polyvinyl-pyrrolidone or gelatin. The liquid forms ticles containing the slow release carrier and the compound of in Suitably flavored Suspending or dispersing agents may also the present invention. include the synthetic and natural gums, for example, traga The pharmaceutical compositions herein will contain, per canth, acacia, methyl-cellulose and the like. For parenteral dosage unit, e.g., tablet, capsule, powder, injection, teaspoon 50 administration, sterile Suspensions and solutions are desired. ful and the like, an amount of the active ingredient necessary Isotonic preparations which generally contain Suitable pre to deliver an effective dose as described above. The pharma servatives are employed when intravenous administration is ceutical compositions herein will contain, per unit dosage desired. unit, e.g., tablet, capsule, powder, injection, Suppository, tea Advantageously, compounds of Formula I may be admin spoonful and the like, from about 0.01 mg to 200 mg/kg of 55 istered in a single daily dose, or the total daily dosage may be body weight per day. Preferably, the range is from about 0.03 administered in divided doses of two, three or four times to about 100 mg/kg of body weight per day, most preferably, daily. Furthermore, compounds for the present invention can from about 0.05 to about 10 mg/kg of body weight per day. be administered in intranasal form via topical use of suitable The compounds may be administered on a regimen of 1 to 5 intranasal vehicles, or via transdermal skin patches well times per day. The dosages, however, may be varied depend 60 known to those of ordinary skill in that art. To be administered ing upon the requirement of the patients, the severity of the in the form of a transdermal delivery system, the dosage condition being treated and the compound being employed. administration will, of course, be continuous rather than The use of either daily administration or post-periodic dosing intermittent throughout the dosage regimen. may be employed. For instance, for oral administration in the form of a tablet Preferably these compositions are in unit dosage forms 65 or capsule, the active drug component can be combined with Such as tablets, pills, capsules, powders, granules, sterile an oral, non-toxic pharmaceutically acceptable inert carrier parenteral Solutions or Suspensions, metered aerosol or liquid Such as ethanol, glycerol, water and the like. Moreover, when US 7,662,824 B2 69 70 desired or necessary, Suitable binders; lubricants, disintegrat Commonly, stents are inserted into the lumen in a non ing agents and coloring agents can also be incorporated into expanded form and are then expanded autonomously, or with the mixture. Suitable binders include, without limitation, the aid of a second device in situ. A typical method of expan starch, gelatin, natural Sugars such as glucose or beta-lactose, sion occurs through the use of a catheter-mounted angio corn Sweeteners, natural and synthetic gums such as acacia, plasty balloon which is inflated within the stenosed vessel or tragacanth or sodium oleate, Sodium Stearate, magnesium body passageway in order to shear and disrupt the obstruc Stearate, Sodium benzoate, sodium acetate, Sodium chloride tions associated with the wall components of the vessel and to and the like. Disintegrators include, without limitation, obtain an enlarged lumen. Self-expanding stents as described starch, methyl cellulose, agar, bentonite, Xanthan gum and the in U.S. Pat. No. 6,776,796 (Falotico et al.) may also be uti like. 10 lized. The combination of a stent with drugs, agents or com The daily dosage of the products of the present invention pounds which prevent inflammation and proliferation, may may be varied over a wide range from 1 to 5000 mg per adult provide the most efficacious treatment for post-angioplasty human per day. For oral administration, the compositions are restenosis. preferably provided in the form of tablets containing, 0.01, Compounds of the invention can be incorporated into or 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 15 affixed to the stent in a number of ways and in utilizing any 200, 250 and 500 milligrams of the active ingredient for the number of biocompatible materials. In one exemplary symptomatic adjustment of the dosage to the patient to be embodiment, the compound is directly incorporated into a treated. An effective amount of the drug is ordinarily supplied polymeric matrix. Such as the polymer polypyrrole, and Sub at a dosage level of from about 0.01 mg/kg to about 200 sequently coated onto the outer surface of the stent. The mg/kg of body weight per day. Particularly, the range is from compound elutes from the matrix by diffusion through the about 0.03 to about 15 mg/kg of body weight per day, and polymer. Stents and methods for coating drugs on Stents are more particularly, from about 0.05 to about 10 mg/kg of body discussed in detail in the art. In another exemplary embodi weight per day. The compound of the present invention may ment, the stent is first coated with as a base layer comprising be administered on a regimen up to four or more times per a solution of the compound, ethylene-co-vinylacetate, and day, preferably of 1 to 2 times per day. 25 polybutylmethacrylate. Then, the stent is further coated with Optimal dosages to be administered may be readily deter an outer layer comprising only polybutylmethacrylate. The mined by those skilled in the art, and will vary with the outlayer acts as a diffusion barrier to prevent the compound particular compound used, the mode of administration, the from eluting too quickly and entering the Surrounding tissues. strength of the preparation, the mode of administration, and The thickness of the outer layer or topcoat determines the rate the advancement of the disease condition. In addition, factors 30 at which the compound elutes from the matrix. Stents and associated with the particular patient being treated, including methods for coating are discussed in detail in WIPO publica patient age, weight, diet and time of administration, will tion WO9632907, U.S. Publication No. 2002/0016625 and result in the need to adjust dosages. references disclosed therein. The compounds of the present invention can also be admin The solution of the compound of the invention and the istered in the form of liposome delivery systems. Such as 35 biocompatible materials/polymers may be incorporated into Small unilamellar vesicles, large unilamellar vesicles, and or onto a stent in a number of ways. For example, the Solution multilamellar vesicles. Liposomes can beformed from a vari may be sprayed onto the stent or the stent may be dipped into ety of lipids, including but not limited to amphipathic lipids the solution. In a preferred embodiment, the solution is Such as phosphatidylcholines, sphingomyelins, phosphati sprayed onto the stent and then allowed to dry. In another dylethanolamines, phophatidylcholines, cardiolipins, phos 40 exemplary embodiment, the solution may be electrically phatidylserines, phosphatidylglycerols, phosphatidic acids, charged to one polarity and the stent electrically changed to phosphatidylinositols, diacyl trimethylammonium propanes, the opposite polarity. In this manner, the solution and stent diacyl dimethylammonium propanes, and Stearylamine, neu will be attracted to one another. In using this type of spraying tral lipids such as triglycerides, and combinations thereof. process, waste may be reduced and more control over the They may either contain cholesterol or may be cholesterol 45 thickness of the coat may be achieved. Compound is prefer free. ably only affixed to the outer surface of the stent which makes The compounds of the present invention can also be admin contact with one tissue. However, for some compounds, the istered locally. Any delivery device. Such as intravascular entire stent may be coated. The combination of the dose of drug delivery catheters, wires, pharmacological stents and compound applied to the Stent and the polymer coating that endoluminal paving, may be utilized. The delivery system for 50 controls the release of the drug is important in the effective Such a device may comprise a local infusion catheter that ness of the drug. The compound preferably remains on the delivers the compound at a rate controlled by the administer. stent for at least three days up to approximately six months The present invention provides a drug delivery device com and more, preferably between seven and thirty days. prising an intraluminal medical device, preferably a stent, and Any number of non-erodible biocompatible polymers may a therapeutic dosage of a compound of the invention. 55 be utilized in conjunction with the compounds of the inven The term “stent” refers to any device capable of being tion. It is important to note that different polymers may be delivered by a catheter. A stent is routinely used to prevent utilized for different stents. For example, the above-described vascular closure due to physical anomalies Such as unwanted ethylene-co-vinylacetate and polybutylmethacrylate matrix inward growth of vascular tissue due to Surgical trauma. It works well with stainless steel stents. Other polymers may be often has a tubular, expanding lattice-type structure appropri 60 utilized more effectively with stents formed from other mate ate to be left inside the lumen of a duct to relieve an obstruc rials, including materials that exhibit Superelastic properties tion. The stent has a lumen wall-contacting Surface and a Such as alloys of nickel and titanium. lumen-exposed surface. The lumen-wall contacting Surface is Restensosis is responsible for a significant morbidity and the outside surface of the tube and the lumen-exposed surface mortality following coronary angioplasty. Restenosis occurs is the inner surface of the tube. The stent can be polymeric, 65 through a combination of four processes including elastic metallic or polymeric and metallic, and it can optionally be recoil, thrombus formation, intima hyperplasia and extracel biodegradable. lular matrix remodeling. Several growth factors have been US 7,662,824 B2 71 72 recently identified to play a part in these processes leading to tumor cell, tumor vasculature or tumor stroma, is preferably a restenosis. See Schiele T Met. al., 2004, “Vascular resteno Surface-expressed, Surface-accessible or Surface-localized sis—striving for therapy.” Expert Opin Pharmacother. 5(11): component. The antibody targeting agents also include anti 2221-32. Vascular smooth muscle cells (VSMC) express bodies or antigen-binding fragments thereof, that bind to an c-Met receptor. Exposure to hepatocyte growth factor, the intracellular component that is released from a necrotic tumor ligand for c-Met, stimulates these cells to exhibit a migratory cell. Preferably such antibodies are monoclonal antibodies, or phenotype. See Taher et. al., Hepatocyte growth factor trig antigen-binding fragments thereof, that bind to insoluble gers signaling cascades mediating vascular Smooth muscle intracellular antigenCs) present in cells that may be induced to cell migration. Biochem Biophy's Res Commun. (2002) 298 be permeable, or in cell ghosts of Substantially all neoplastic (1):80-6: Morishita R, Aoki M. Yo Y. Ogihara T. Hepatocyte 10 and normal cells, but are not present or accessible on the growth factor as cardiovascular hormone: role of HGF in the exterior of normal living cells of a mammal. In the present pathogenesis of cardiovascular disease. Endocr J. (2002) invention, the targetable or accessible component might be June; 49(3):273-84. Since VSMC migration from the media the c-Met receptor as it is accessible and expressed on or near to the intima of arteries plays a role in the development of the target tissues. atherosclerosis and restenosis, antagonists of c-Met kinase 15 As used herein, the term “antibody' is intended to refer activity are believed to present a viable therapeutic strategy in broadly to any immunologic binding agent such as IgG, IgM, the treatment of these diseases. IgA, IgE, F(ab')2, a univalent fragment such as Fab', Fab, Accordingly, the present invention provides a method for Dab, as well as engineered antibodies Such as recombinant the treatment of disorders related to c-Met, including resteno antibodies, humanized antibodies, bispecific antibodies, and sis, intimal hyperplasia or inflammation, in blood vessel the like. The antibody can be either the polyclonal or the walls, comprising the controlled delivery, by release from an monoclonal, although the monoclonal is preferred. There is a intraluminal medical device, Such as a stent, of a compound of very broad array of antibodies known in the art that have the invention in therapeutically effective amounts. immunological specificity for the cell Surface of virtually any Methods for introducing a stent into a lumen of a body are Solid tumor type (see a Summary Table on monoclonal anti well known and the compound-coated Stents of this invention 25 are preferably introduced using a catheter. As will be appre bodies for solid tumors in U.S. Pat. No. 5,855.866 to Thorpe ciated by those of ordinary skill in the art, methods will vary etal). Methods are known to those skilled in the art to produce slightly based on the location of stent implantation. For coro and isolate antibodies against tumor (U.S. Pat. No. 5,855.866 nary Stent implantation, the balloon catheterbearing the stent to Thorpe et al., and U.S. Pat. No. 6,34.2219 to Thorpe et al.). is inserted into the coronary artery and the stent is positioned 30 Techniques for conjugating therapeutic moiety to antibod at the desired site. The balloon is inflated, expanding the stent. ies are well known, see, e.g., Amon et al., “Monoclonal Anti As the stent expands, the stent contacts the lumen wall. Once bodies For Immunotargeting Of Drugs In Cancer Therapy”, the stent is positioned, the balloon is deflated and removed. in Monoclonal Antibodies And Cancer Therapy, Reisfeld et The stent remains in place with the lumen-contacting Surface al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et bearing the compound directly contacting the lumen wall 35 al., “Antibodies For Drug Delivery', in Controlled Drug Surface. Stent implantation may be accompanied by antico Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53. (Mar agulation therapy as needed. cel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cyto Optimum conditions for delivery of the compounds for use toxic Agents. In Cancer Therapy: A Review', in Monoclonal in the stent of the invention may vary with the different local Antibodies 84: Biological And Clinical Applications, delivery systems used, as well as the properties and concen 40 Pinchera et al. (eds.), pp. 475-506 (1985). Similar techniques can also be applied to attach compounds of the invention to trations of the compounds used. Conditions that may be opti non-antibody targeting agents. Those skilled in the art will mized include, for example, the concentrations of the com know, or be able to determine, methods of forming conjugates pounds, the delivery volume, the delivery rate, the depth of with non-antibody targeting agents, such as Small molecules, penetration of the vessel wall, the proximal inflation pressure, oligopeptides, polysaccharides, or other polyanionic com the amount and size of perforations and the fit of the drug 45 delivery catheter balloon. Conditions may be optimized for pounds. inhibition of smooth muscle cell proliferation at the site of Although any linking moiety that is reasonably stable in injury Such that significant arterial blockage due to restenosis blood, can be used to link the compounds of the present does not occur, as measured, for example, by the proliferative invention to the targeting agent, biologically-releasable ability of the Smooth muscle cells, or by changes in the 50 bonds and/or selectively cleavable spacers or linkers are pre vascular resistance or lumen diameter. Optimum conditions ferred. “Biologically-releasable bonds' and “selectively can be determined based on data from animal model studies cleavable spacers or linkers' still have reasonable stability in using routine computational methods. the circulation, but are releasable, cleavable or hydrolyzable Another alternative method for administering compounds only or preferentially under certain conditions, i.e., within a of this invention may be by conjugating the compound to a 55 certain environment, or in contact with a particular agent. targeting agent which directs the conjugate to its intended site Such bonds include, for example, disulfide and trisulfide of action, i.e., to vascular endothelial cells, or to tumor cells. bonds and acid-labile bonds, as described in U.S. Pat. Nos. Both antibody and non-antibody targeting agents may be 5,474,765 and 5,762.918 and enzyme-sensitive bonds, used. Because of the specific interaction between the target including peptide bonds, esters, amides, phosphodiesters and ing agent and its corresponding binding partner, a compound 60 glycosides as described in U.S. Pat. Nos. 5,474,765 and of the present invention can be administered with high local 5,762.918. Such selective-release design features facilitate concentrations at or near a target site and thus treats the Sustained release of the compounds from the conjugates at the disorder at the target site more effectively. intended target-site. The antibody targeting agents include antibodies or anti The present invention provides a pharmaceutical compo gen-binding fragments thereof, that bind to a targetable or 65 sition comprising an effective amount of a compound of the accessible component of a tumor cell, tumor vasculature, or present invention conjugated to a targeting agent and a phar tumor stroma. The “targetable or accessible component of a maceutically acceptable carrier. US 7,662,824 B2 73 74 The present invention further provides a method of treating of a disorder related to c-Met, particularly a tumor, compris -continued ing administering to a subject a therapeutically effective amount of a compound of Formula I conjugated to a targeting agent. When proteins such as antibodies or growth factors, or and polysaccharides are used as targeting agents, they are prefer ably administered in the form of injectable compositions. The injectable antibody solution will be administered into a vein, artery or into the spinal fluid over the course of from 2 minutes 10 to about 45 minutes, preferably from 10 to 20 minutes. In certain cases, intradermal and intracavitary administration are advantageous for tumors restricted to areas close to par ticular regions of the skin and/or to particular body cavities. In 15 the A ring being optionally substituted with NH, addition, intrathecal administrations may be used for tumors —CH-CF, NO,-CN, C1, F, or Br; located in the brain. R" hydrogen or halogen; Therapeutically effective dose of the compound of the present invention conjugated to a targeting agent depends on R°is selected from the group consisting of Formula II, the individual, the disease type, the disease state, the method Formula III or Formula IV; of administration and other clinical variables. The effective dosages are readily determinable using data from an animal Formula II model. Experimental animals bearing Solid tumors are fre R3 quently used to optimize appropriate therapeutic doses prior to translating to a clinical environment. Such models are : B N l known to be very reliable in predicting effective anti-cancer 25 R11s, --- N-1 strategies. For example, mice bearing Solid tumors, are O widely used in pre-clinical testing to determine working Formula III ranges of therapeutic agents that give beneficial anti-tumor R3 effects with minimal toxicity. R N While the foregoing specification teaches the principles of 30 Ya1 the present invention, with examples provided for the purpose O of illustration, it will be understood that the practice of the Formula IV invention encompasses all of the usual variations, adaptations and/or modifications as come within the scope of the follow 35 - B N ing claims and their equivalents. R4 --- O We claim: 1. A compound of Formula I: wherein n is 2, 3 or 4: 40 B is a nitrogen containing heterocyclic ring; R is hydrogen or alkyl; Formula I R" is H. alkyl, hydroxyalkyl, hydroxyl, or—C(O)alkyl: and 45 R is OH, NH-alkyl, N-alkyl, or NH. 2. A compound of claim 1, wherein: R" is hydrogen, C1 or Br; A is selected from:

50

R2 N 55 pharmaceutically acceptable salts, N-oxides, and stere ochemical isomers thereof, wherein: A is selected from: 60

65 US 7,662,824 B2 75 76 and A is R2 is Formula II wherein: R’ is Formula IV wherein: n is 20r3; B is morpholinyl, piperidinyl, piperazinyl, or pyrrolidi B is morpholinyl, piperidinyl, piperazinyl, or pyrrolidi- nyl; and s s s nyl: 5 s R is hydrogen; and R" is hydrogen, hydroxyl, hydroxymethyl, methyl, R" is hydrogen, hydroxyl, hydroxymethyl, methyl, ethyl, propyl or isopropyl. ethyl, propyl or isopropyl. 6. A compound of claim 1, wherein: 3. A compound of claim 2, wherein: R" is hydrogen, C1 or Br; A is 10

O O O O

4. A compound of claim 1, wherein: A is R" is hydrogen, C1 or Br; 20 R is Formula III wherein: A is n is 20r 3: R is hydrogen; and R is hydroxyl, dimethylamino, diethylamino, or dipro 25 pylamino.

OR N ... and 7. A compound of claim 1, wherein s R" is hydrogen, C1 or Br; O O

30 R’ is Formula III wherein n is 20r 3: R is hydrogen; and OR N ... and R is hydroxyl, methylamino, ethylamino, propylamino, s isopropylamino, dimethylamino, diethylamino, dipro- 35 O O pylamino, methylethylamino, or methylpropylamino. 5. A compound of claim 1, wherein: A is R" is hydrogen, C1 or Br; 2 Rf is Formula IV wherein: 40 B is piperazine; and R" is hydrogen, methyl, ethyl, propyl or isopropyl. 8. A pharmaceutical composition comprising a compound OR N and of claim 1 and a pharmaceutically acceptable carrier. O O 9. A combination of a chemotherapeutic agent and a com 45 pound as claimed in claim 1. 10. A compound selected from the group consisting of:

B r N1 Sr. O O N- N O

B r N1

O O N O H O US 7,662,824 B2 77 78

-continued Br N1 W O O O N H

Br N1 W O O N-nu N O

Br

H N1-S-N N O

Br

1. n-1N1 N O

H HO W O s O H R N O

Br

N

B r N1 W ; and O O N US 7,662,824 B2 79 80

-continued

B r N1 W O O N O 1. nu1N1 N H O

11. A compound selected from the group consisting of:

Br Y

N

H Br N

N N-1N N

H

W O s O N

B r N1 W ; and O O US 7,662,824 B2 81 82

-continued

B r N1 w W ...Yo O O N O nu1N1 N H O

12. A compound selected from the group consisting of:

H

N W s

r H O O O N nu-1N1 N N H O

B r N1 W ; and O O O

N1a- NH O

B r N1 w / ..Yo O O N O nu-1N1 N H O