Fluoro substituted omega-carboxyaryl diphenyl urea for the treatment and prevention of diseases and conditions

The invention relates to new compounds, pharmaceutical compositions containing such compounds and the use of these compounds or compositions for the treatment of diseases and conditions mediated by abnormal signaling of VEGFR, PDGFR, raf, p38, and / or flt-3 kinases, alone or in combination with agents anti-cancer.

Background of the invention [0002] Activation of the ras signal processing pathway is a cascade of events that have a profound effect on cell proliferation, differentiation and transformation. Raf kinase, a distal effector of ras, is considered the main mediator of these signals from cell surface receptors to the nucleus (Lowy, DR; Willumsen, BM Ann. Rev. Biochem. 1993, 62, 851; Bos, JL Cancer Res. 1989, 49 , 4682). Inhibition of the effect of active ras by inhibition of the raf kinase signaling pathway as a result of administration of inactivating raf kinase antibodies or by co-expression of a dominant raf negative kinase or a dominant negative MEK, a raf kinase substrate, has been shown to lead to the transformation of transformed cells into a normal growth phenotype (see Daum et al. Trends Biochem. Sci. 1994, 19, 474-80; Fridman et al. J. Biol. Chem. 1994, 269, 30105-8. Kolch et al. (Nature 1991, 349, 426-28) further indicated that inhibition of raf expression by antisense RNA blocks cell proliferation in membrane-associated oncogenes. Similarly, raf kinase inhibition (by antisense oligodeoxynucleotides) has been correlated in vitro and in vivo with inhibition of the growth of many types of human tumors (Monia et al., Nat. Med. 1996, 2, 668-75).

[0003] It has been found to sustain tumor growth advances over a size of 1-2 mm³, tumor cells require a functional stroma, a supportive structure composed of fibroblast, smooth muscle cells, endothelial cells, intercellular matrix proteins and soluble factors (Folkman, J., Semin. Oncol. 2002. 29 (6 Suppl 16), 15-8). Tumors induce stromal tissue formation by the secretion of soluble growth factors such as PDGF and transforming growth factor-beta (TGFbeta), which in turn stimulate the secretion of complement cells such as fibroblast growth factor (FGF), epidermal growth factor (EGF) by the host cells and vascular endothelial growth factor (VEGF). These stimulants induce the formation of new blood vessels, or angiogenesis, which supply oxygen and nutrients to the tumor and allow it to grow, which opens the way to metastasis. It is believed that certain therapies aimed at inhibiting stromal formation will inhibit the growth of epithelial tumors from a wide variety of histological types. (George, D. Semin. Oncol. 2001. 28 (5 Suppl 17), 27-33; Shaheen, RM, et al., Cancer Res. 2001, 61 (4), 14648; Shaheen, RM, et al. Cancer Res. 1999, 59 (21), 5412-6). However, due to the complex nature and multiplicity of growth factors involved in angiogenesis and tumor progression, a single pathway agent may have limited effectiveness. It is desirable to provide therapy for several key signaling pathways used by tumors to induce angiogenesis in the host stroma. These include PDGF, a strong stimulator of stroma formation (Ostman, A. and CH Heldin, Adv. Cancer Res. 2001, 80, 1-38), FGF, a chemoattractant and mitogen for fibroblasts and endothelial cells, and VEGF, a strong vascularization regulator.

[0004] PDGF is a key regulator of stromal formation that is secreted by many tumors in a para-secretory manner and is believed to promote the growth of fibroblasts, smooth muscle cells and endothelium, promoting stroma formation and angiogenesis. PDGF was initially identified as a product of the simian sarcoma virus v-sis oncogene (Heldin, CH, et al., J. Cell. Sci. Suppl. 1985, 3, 65-76). The growth factor is formed of two peptide chains, referred to as A or B chains, which have 60% homology in the primary amino acid sequence. The chains are cross-linked with disulfide bridges to produce a mature 30 kDa protein composed of AA, BB or AB homo- or heterodimers. PDGF is found in large amounts in platelets, and is expressed by endothelial and vascular smooth muscle cells. In addition, PDGF production is upregulated under low oxygen conditions, such as those found in poorly vascularized tumor tissue (Kourembanas, S., et al., Kidney Int. 1997, 51 (2), 438-43). PDGF binds with high affinity to the PDGF receptor, composed of 1106 amino acids, 124 kDa transmembrane tyrosine kinase receptor (Heldin, CH, A. Ostman, and L. Ronnstrand, Biochim. Biophys. Acta 1998, 1378 (1), 79-113 ). PDGFR is found as homo- or heterodimeric chains that have 30% homology together in their amino acid sequence and 64% homology between their kinase domains (Heldin, CH, et al. Embo J 1988, 7 (5), 1387-93) . PDGFR is a member of the family of tyrosine kinase receptors with separated kinase domains, which include VEGFR-2 (KDR), VEGFR-3 (flt-4), c-kit, and flt-3. The PDGF receptor is expressed mainly on fibroblasts, smooth muscle cells and pericytes, and to a lesser extent on neurons, kidney mesangium, Leydig and Schwann cells of the central nervous system. After binding to the PDGF receptor, it induces receptor dimerization and undergoes auto- and trans-phosphorylation of tyrosine residues, which increases the kinase activity of the receptors and promotes the recruitment of further effectors by activating SH2 protein binding domains. Many signal molecules form complexes with activated PDGFR, including PI-3-kinase, C-gamma phospholipase, src and GAP (GTPase activating protein for p21-ras) (Soskic, V., et al. Biochemistry 1999, 38 (6) , 1757-64). By activating PI-3-kinase, PDGF activates the Rho signaling pathway inducing cell motility and migration, and by activating GAP, it induces mitogenesis by activating p21-ras and the MAPK signaling pathway.

[0005] In adults, PDGF's main function is believed to be to facilitate and increase the rate of wound healing and to maintain blood vessel homeostasis (Baker, EA and DJ Leaper, Wound Repair Regen. 2000, 8 (5), 392-8, and Yu, J., A. Moon, and HR Kim, Biochem. Biophys. Res. Commun. 2001, 282 (3), 697-700). PDGF is found in high concentrations in platelets and is a potent chemoattractant for fibroblasts, smooth muscle cells, neutrophils and macrophages. In addition to its role in wound healing, PDGF is known to help maintain vascular homeostasis. During the development of new blood vessels, PDGF recruits pericytes and smooth muscle cells that are necessary for structural vessel integrity. PDGF is thought to play a similar role during tumor neovascularization. As part of its role in angiogenesis, PDGF controls the pressure of interstitial fluid, regulating vascular permeability by regulating interactions between connective tissue cells and the intercellular matrix. Inhibition of PDGFR activity may lower interstitial pressure and facilitate the supply of cytotoxic substances to tumors, improving the anti-tumor efficacy of these agents (Pietras, K., et al. Cancer Res. 2002, 62 (19), 5476-84; Pietras, K., et al. Cancer Res. 2001, 61 (7), 2929-34).

[0006] PDGF may promote tumor growth by paracrine or autocrine stimulation of PDGFR receptors on stromal cells or directly on tumor cells, or by receptor amplification or receptor activation by recombination. Overexpressed PDGF can transform human melanoma cells and keratinocytes (Forsberg, K., et al. Proc. Natl. Acad. Sci. US A. 1993, 90 (2), 393-7; Skobe, M. and NE Fusenig, Proc. Natl. Acad. Sci. US A. 1998, 95 (3), 1050-5), two types of cells that do not express PDGF receptors, probably due to the direct effect of PDGF on stroma formation and induction of angiogenesis. This paracrine stimulation of the tumor stroma is also observed in colorectal, lung, breast and prostate cancers (Bhardwaj, B., et al. Clin. Cancer Res. 1996, 2 (4), 773-82; Nakanishi, K., et al. Mod. Pathol. 1997, 10 (4), 341-7; Sundberg, C., et al. Am. J. Pathol. 1997, 151 (2), 479-92; Lindmark, G., et al. Lab. Invest. 1993, 69 (6), 682-9; Vignaud, JM, et al, Cancer Res. 1994, 54 (20), 5455-63) where tumors express

PDGF, but not the receptor. Autocrine stimulation of tumor cell growth has been described, where a large proportion of the analyzed tumors express PDGF ligand and receptor in gliomas (Fleming, TP, et al. Cancer Res. 1992, 52 (16), 4550-3), soft tissue sarcomas (Wang, J ., MD Coltrera, and AM Gown, Cancer Res. 1994, 54 (2), 560-4) and ovarian cancers (Henriksen, R., et al. Cancer Res. 1993, 53 (19), 4550-4), prostate (Fudge, K., CY Wang, and ME Steams, Mod. Pathol. 1994, 7 (5), 549-54), pancreas (Funa, K., et al. Cancer Res. 1990, 50 (3), 748-53) and lungs (Antoniades, HN, et al., Proc. Natl Acad. Sci. USA 1992, 89 (9), 3942-6). Ligand-independent receptor activation is less pronounced and has been found in chronic myelomonocytic leukemia (CMML), where the chromosomal translocation event forms a fusion protein between the Ets-like TEL transcription factor and the PDGF receptor. In addition, activating mutations in PDGFR have been found in gastrointestinal stromal tumors in which c-kit activation is not involved (Heinrich, MC, et al., Science 2003, 9, 9).

[0007] Another significant regulator of angiogenesis and angiogenesis in immune development and certain angiogenesis-dependent diseases is vascular endothelial growth factor (VEGF; also called vascular permeability factor, VPF). VEGF is a family of mitogen isoforms found in homodimeric forms as a result of alternative RNA splicing. VEGF isoforms are highly specific to vascular endothelial cells (for review see Farrara et al. Endocr. Rev. 1992, 13, 18; Neufield et al. FASEB J. 1999, 13, 9).

[0008] VEGF expression is induced by hypoxia (Shweiki et al. Nature 1992, 359, 843) as well as by many cytokines and growth factors such as interleukin-1, interleukin-6, epidermal growth factor and transforming growth factor. VEGF and members of the VEGF family have been reported to bind one or more of three transmembrane receptor tyrosine kinases (Mustonen et al. J. Cell Biol. 1995, 129, 895), VEGF-1 receptor (also known as flt-1 (fms-like tyrosine kinase-1)), VEGFR-2 (also known as kinase insertion domain receptor (KDR); mouse VEGFR2 analogue is known as fetal liver kinase-1 (flk-1)), and VEGFR-3 (also known as flt-4). VEGFR-2 and flt-1 have been shown to have different signal transduction properties (Waltenberger et al. J. Biol. Chem. 1994, 269, 26988); Park et al. Oncogene 1995, 10, 135). Thus, VEGFR-2 undergoes strong ligand-dependent tyrosine phosphorylation in intact cells, while flt-1 shows a weak response. Thus, it is believed that binding to VEGFR-2 is a critical requirement for the induction of full spectrum VEGF-mediated biological responses.

[0009] In vivo, VEGF plays a central role in angiogenesis and induces angiogenesis and increasing the permeability of blood vessels. Deregulated VEGF expression contributes to the development of many diseases that are characterized by abnormal angiogenesis and / or hyperpermeability processes. It is believed that regulation of VEGF-mediated transduction of the signal cascade by certain agents can provide useful control over abnormal angiogenesis and / or hyperpermeability processes. Oncogenic cells in hypoxic tumor regions react by stimulating VEGF production, which activates inactive endothelial cells to stimulate the formation of new blood vessels. (Shweiki et al. Proc. Nat'l. Acad. Sci. 1995, 92, 768). In addition, VEGF production in tumor regions where there is no angiogenesis may follow the signal processing ras pathway (Grugel et al. J. Biol. Chem. 1995, 270, 25915; Rak et al. Cancer Res. 1995, 55, 4575) . In situ hybridization studies have shown that VEGF mRNA is highly upregulated in a number of human cancers, including lung cancer (Mattem et al. Br. J. Cancer 1996, 73, 931), thyroid (Viglietto et al. Oncogene 1995, 11, 1569), nipple (Brown et al. Human Pathol. 1995, 26, 86), gastrointestinal tract (Brown et al. Cancer Res. 1993, 53, 4727; Suzuki et al. Cancer Res. 1996, 56, 3004), kidneys and bladder (Brown et al. Am. J. Pathol 1993, 1431, 1255), ovaries (Olson et al. Cancer Res. 1994, 54, 1255), and cervix (Guidi et al. J. Nat'l Cancer Inst. 1995, 87, 12137), as well as angiosarcoma (Hashimoto et al. Lab. Invest. 1995, 73, 859) and several intracranial tumors (Plate et al. Nature 1992, 359, 845; Phillips et al. Int. J. Oncol. 1993, 2, 913; Berkman et al. J. Clin. Invest., 1993, 91, 153). Neutralization of anti-VEGFR-2 monoclonal antibodies has been shown to be effective in blocking tumor angiogenesis (Kim et al. Nature 1993, 362, 841; Rockwell et al. Mol. Cell. Differ. 1995, 3, 315).

[0010] Overexpression of VEGF, e.g. under extreme hypoxia, can lead to intraocular angiogenesis, which causes excessive blood vessel growth, eventually leading to blindness. Such a cascade of events has been observed in many retinopathies, including diabetic retinopathy, ischemic retinal vascular occlusion, and extrashedular fibroplasia (Aiello et al. New Engl. J. Med. 1994, 331, 1480; Peer et al. Lab. Invest. 1995, 72, 638), and age-related macular degeneration (AMD; see, Lopez et al. Invest. Opththalmol. Vis. Sci. 1996, 37, 855).

[0011] In rheumatoid arthritis (RA), the internal growth of vascular scales can be mediated by the production of angiogenic factors. Immunoreactive VEGF levels are high in the synovial fluid of RA patients, while VEGF levels were low in the synovial fluid of patients with other forms of arthritis, including degenerative joint disease (Koch et al. J. Immunol. 1994, 152, 4149). The AGM-170 angiogenesis inhibitor has been shown to prevent arthritis in a rat collagen arthritis model (Peacock et al. J. Exper. Med. 1992, 175, 1135).

[0012] Increased expression of VEGF in psoriatic skin as well as bullous disorders associated with the formation of subcutaneous blisters, such as bullous pemphigoid, erythema multiforme and herpetic dermatitis (Brown et al. J. Invest. Dermatol. 1995, 104, 744) have also been shown .

[0013] Vascular endothelial growth factor (VEGF, VEGF-C, VEGF-D) and their receptors (VEGFR-2, VEGFR-3) are not the only key regulators of tumor angiogenesis, but also lymphangiogenesis. VEGF, VEGF-C and VEGF-D are expressed in most tumors, mainly during periods of tumor growth, often in significantly increased amounts. VEGF expression is stimulated by hypoxia, cytokines, oncogenes such as ras, or by inactivation of tumor suppression genes (McMahon, G. Oncologist 2000, 5 (Suppl. 1), 3-10; McDonald, NQ; Hendrickson, WA Cell 1993, 73, 421-424) [0014] The biological activity of VEGF is mediated by binding to their receptors. VEGFR-3 (also called flt-4) is mainly expressed on lymphatic endothelium in normal adult tissues. VEGFR-3 functions are needed to create a new lymphatic vessel, but not to support existing lymphatic vessels. VEGFR-3 is also upregulated on endothelial blood vessels in tumors. VEGF-C and VEGF-D, ligands for VEGFR-3, have recently been identified as regulators of lymphangiogenesis in mammals. Lymphangiogenic factors induced by tumor-associated lymphangiogenesis may promote the growth of new vessels to the tumor, providing tumor cells with access to systemic circulation. Cells that attack the lymphatic vessels may find their way into the bloodstream through the thoracic duct. Tumor expression studies have allowed direct comparison of VEGF-C, VEGF-D and VEGFR-3 expression with clinopathological factors that are directly related to the ability of primary tumors to spread (e.g. lymph node involvement, lymphatic invasion, secondary metastasis and disease-free survival) ). In many cases, these studies show a statistical correlation between lymphangiogenic factor expression and the ability of the primary solid tumor to metastasize (Skobe, M. et al. Nature Med. 2001, 7 (2), 192-198; Stacker, SA et al. Nature Med. 2001, 7 (2), 186-191; Makinen, T. et al. Nature Med. 2001, 7 (2), 199-205; Mandriota, SJ et al. EMBO J. 2001, 20 (4), 672-82; Karpanen, T. et al Cancer Res. 2001, 61 (5), 178690; Kubo, H. et al Blood 2000, 96 (2), 546-53).

[0015] Hypoxia appears to be an important stimulus for VEGF production in malignant cells. Activation of MAP p38 kinase is necessary for induction of VEGF by tumor cells in response to hypoxia (Blaschke, F. et al. Biochem. Biophys. Res. Commun. 2002, 296, 890-896; Shemirani, B. et al. Oral Oncology 2002 , 38, 251-257). In addition to its involvement in angiogenesis by regulating VEGF secretion, MAP kinase p38 promotes invasion of malignant cells and migration of various types of tumors by regulating collagenase activity and expression of urokinase plasminogen activator (Laferriere, J. et al. J. Biol. Chem. 2001, 276, 33762 -33772; Westermarck, J. et al Cancer Res. 2000, 60, 7156-7162; Huang, S. et al J. Biol. Chem. 2000, 275, 12266-12272; Simon, C. et al Exp Cell Res. 2001, 271, 344-355).

[0016] Inhibition of mitogen activated protein kinase (MAPK) p38 has been shown to inhibit cytokine production (e.g. TNF, IL-1, IL-6, IL-8) and proteolytic enzyme production (e.g. MMP-1, MMP-3) in vitro and / or in vivo. Mitogen activated protein (MAP) p38 kinase is involved in IL-1 and TNF signaling pathways (Lee, JC; Laydon, JT; McDonnell, PC; Gallagher, TF; Kumar, S .; Green, D .; McNulty, D .; Blumenthal, MJ; Heys, JR; Landvatter, SW; Stricker, JE; McLaughlin, M. M .; Siemens, IR; Fisher, SM; Livi, GP; White, JR; Adams, JL; Yound, PR Nature 1994, 372, 739).

[0017] Clinical studies have linked the production and / or signaling of tumor necrosis factor (TNF) to many diseases, including rheumatoid arthritis (Maini. J. Royal Coll. Physicians London 1996, 30, 344). In addition, excessive TNF levels have been associated with many inflammatory and / or immunomodulatory diseases, including acute joint rheumatism (Yegin et al. Lancet 1997, 349, 170), bone resorption (Pacifici et al. J. Clin. Endocrinol. Metabol. 1997, 82, 29), postmenopausal osteoporosis (Pacifici et al. J. Bone Mineral Res. 1996, 11, 1043), sepsis (Blackwell et al. Br. J. Anaesth. 1996, 77, 110), gram-negative sepsis (Debets et al. Prog. Clin. Biol. Res. 1989, 308, 463), septic shock (Tracey et al. Nature 1987, 330, 662; Girardin et al. New England J. Med. 1988, 319, 397) , endotoxic shock (Beutler et al. Science 1985, 229, 869; Ashkenasi et al. Proc. Nat'l. Acad. Sci. US 1991, 88, 10535), toxic shock syndrome, (Saha et al. J. Immunol. 1996, 157, 3869; Lina et al. FEMS Immunol. Med. Microbiol. 1996, 13, 81), systemic inflammatory response syndrome ( Anon. Crit. Care Med. 1992, 20, 864), inflammatory bowel diseases (Stokkers et al. J Inflamm. 1995-6, 47, 97) including Crohn's disease (van Deventer et al. Aliment. Pharmacol. Therapeu. 1996 , 10 (Suppl. 2), 107; van Dullemen et al. Gastroenterology 1995, 109, 129) and ulcerative colitis (Masuda et al. J. Clin. Lab. Immunol. 1995, 46, 111), Jarisch-Herxheimer reactions (Fekade et al. New England J. Med. 1996, 335, 311), asthma (Amrani et al. Rev. Malad. Respir.

1996, 13, 539), adult respiratory distress syndrome (Roten et al. Am. Rev. Respir. Dis. 1991, 143, 590; Suter et al. Am. Rev. Respir. Dis. 1992, 145, 1016), acute fibrous lung diseases (Pan et al. Pathol. Int. 1996, 46, 91), pulmonary sarcoidosis (Ishioka et al. Sarcoidosis Vasculitis Diffuse Lung Dis. 1996, 13, 139), allergic respiratory diseases (Casale et al. Am J. Respir. Cell Mol. Biol. 1996, 15, 35), silicosis (Gossart et al. J. Immunol. 1996, 156, 1540; Vanhee et al. Eur. Respir. J. 1995, 8, 834), pulmonary sclerosis of coal miners (Borm et al. Am. Rev. Respir. Dis. 1988, 138, 1589), alveolar damage (Horinouchi et al. Am. J. Respir. Cell Mol. Biol 1996, 14, 1044), liver failure (Gantner et al. J. Pharmacol. Exp. Therap. 1997, 280, 53), liver disease during acute inflammation (Kim et al. J. Biol. Chem. 1997, 272, 1402), acute alcoholic hepatitis (Bird et al. Ann. Intern. Med. 1990, 112, 917), malaria (Grau et al. Immunol. Rev. 1989, 112, 49; Taverne et al. Parasitol. Today 1996, 12, 290) including malaria Plasmodium falciparum (Perlmann et al. Infect. Immunit. 1997, 65, 116) and cerebral malaria (Rudin et al. Am. J. Pathol. 1997, 150, 257), non-insulin dependent diabetes mellitus (NIDDM; Stephens et al. J. Biol. Chem. 1997, 272, 971; Ofei and in Diabetes 1996, 45, 881), congestive heart failure (Doyama et al. Int. J. Cardiol. 1996, 54, 217; McMurray et al. Br. Heart J. 1991, 66, 356), damage after heart disease (Malkiel et al. Mol. Med. Today 1996, 2, 336), atherosclerosis (Parums et al. J. Pathol. 1996, 179, A46), Alzheimer's disease (Fagarasan et al. Brain Res. 1996, 723, 231; Aisen et al. Gerontology 1997, 43, 143), acute encephalitis (Ichiyama et al. J. Neurol. 1996, 243, 457), brain trauma (Cannon et al. Crit. Care Med. 1992, 20, 1414; Hansbrough et al. Surg. Clin. N. Am. 1987, 67, 69; Marano et al. Surg. Gynecol. Obstetr. 1990, 170, 32), multiple sclerosis (MS; Coile. Adv. Neuroimmunol. 1996, 6, 143; Matusevicius et al. J. Neuroimmunol. 1996, 66, 115) including demyelination and loss of oligodendrocytes in multiple sclerosis (Brosnan and in Brain Pathol. 1996, 6, 243), advanced cancer (MucWierzgon et al. J. Biol. Regulators Homeostatic Agents 1996, 10, 25), lymphatic cancers (Levy et al. Crit. Rev. Immunol. 1996, 16, 31), pancreatitis (Exley et al. Gut 1992, 33, 1126) including systemic complications in acute pancreatitis (McKay et al. Br. J. Surg. 1996, 83, 919), impaired wound healing in infection, inflammation and cancer (Buck et al. Am. J Pathol. 1996, 149, 195), myelodysplasia syndromes (Raza et al. Int. J. Hematol. 1996, 63, 265), systemic lupus erythematosus (Maury et al. Arthritis Rheum. 1989, 32, 146), biliary cirrhosis liver (Miller et al. Am. J. Gasteroenterolog. 1992, 87, 465), intestinal necrosis (Sun et al. J. Clin. Invest. 1988, 81, 1328), psoriasis (Christophers. AU. J. Dermatol. 1996, 37, S4), radiation damage (Redlich et al. J. Immunol. 1996, 157, 1705), and toxicity after administration of monoclonal antibodies, such as OKT3 (Brod et al. Neurology 1996, 46, 1633). TNF levels have also been associated with graft versus host reactions (Piguet et al. Immunol. Ser. 1992, 56, 409) including reperfusion ischemic injury (Colletti et al. J. Clin. Invest. 1989, 85, 1333) and allograft rejection, including kidney (Maury et al. J. Exp. Med. 1987, 166, 1132), liver (Imagawa et al. Transplantation 1990, 50, 219), heart (Bolling et al. Transplantation 1992, 53, 283), and skin (Stevens et al. Transplant. Proc. 1990, 22, 1924), pulmonary allograft rejection (Grossman et al. Immunol. Allergy Clin. N. Am. 1989, 9, 153) , including chronic pulmonary allograft rejection (bronchitis obliterans; LoCicero et al. J. Thorac. Cardiovasc. Surg. 1990, 99, 1059), as well as complications associated with hip replacement (Cirino et al. Life Sci. 1996, 59, 86). TNF has also been associated with infectious diseases (reviewed by Beutler et al. Crit. Care Med. 1993, 21, 5423; Degre. Biotherapy 1996, 8, 219), including tuberculosis (Rook et al. Malad. Infect. 1996 , 26, 904), Helicobacter pylori infection during peptic ulcer disease (Beales et al. Gastroenterology 1997, 112, 136), Chaga disease due to infection by Trypanosoma cruzi (Chandrasekar et al. Biochem. Biophys. Res. Commun. 1996, 223, 365), the effect of Shiga-like toxin as a result of E. coli infection (Harel et al. J. Clin. Invest. 1992, 56, 40), the effect of enterotoxin A as a result of staph infection (Fischer et al. J. Immunol. 1990, 144, 4663), meningococcal infection (Waage et al. Lancet 1987, 355; Ossege et al. J. Neurolog. Sci. 1996, 144, 1), and Borrelia burgdorferi infections (Brandt et al. Infect. Immunol 1990, 58, 983), Treponema pallidum (Chamberlin et al. Infect. Immunol. 1989, 57, 2872), cytomegalovirus (CMV; Geist et al. Am. J. Respir. Cell Mol. Biol. 1997, 16, 31), influenza virus (Beutler et al. Clin. Res. 1986, 34, 491a) , Sendai virus (Goldfield et al. Proc. Nat'l. Acad. Sci. USA 1989, 87, 1490), Theler's encephalomyelitis virus (Sierra et al. Immunology 1993, 78, 399), and human immunodeficiency virus (HIV; Pol. Proc. Nat'l. Acad. Sci. USA 1990, 87, 782; Vyakaram et al. AIDS 1990, 4, 21; Badley et al. J. Exp. Med. 1997, 185, 55).

[0018] Many diseases are considered to be mediated by excess or undesirable matrix metalloprotease (MMP) activity or by an imbalance in the ratio of MMP to tissue metalloprotease inhibitors (TIMPs). These include osteoarthritis (Woessner et al. J. Biol. Chem. 1984, 259, 3633), rheumatoid arthritis (Mullins et al. Biochim. Biophys. Acta 1983, 695, 117; Woolley et al. Arthritis Rheum. 1977, 20, 1231; Gravallese et al. Arthritis Rheum. 1991, 34, 1076), septic arthritis (Williams et al. Arthritis Rheum. 1990, 33, 533), tumor metastasis (Reich et al. Cancer Res. 1988, 48, 3307; Matrisian et al. Proc. Nat'l Acad. Sci., USA 1986, 83, 9413), periodontal disease (Overall et al. J. Periodontal Res. 1987, 22, 81), corneal ulcer (Bums et al. Invest. Opthalmol. Vis. Sci. 1989 , 30, 1569), proteinuria (Baricos et al. Biochem. J. 1988, 254, 609), coronary thrombosis after detachment of the atherosclerotic plaque (Henney et al. Proc. Nat'l. Acad. Sci., USA 1991, 88, 8154), aortic aneurysm (Vine et al. Clin. Sci. 1991, 81, 233), pregnancy prevention (Woessner et al. Steroids 1989, 54, 491), dystrophobic bullous epidermal separation ( Kronberger et al. J. Invest. Dermatol. 1982, 79, 208), degenerative loss of cartilage after traumatic joint damage, osteopene mediated by MMP activity, temporomandibular joint disease and demyelinating diseases of the nervous system (Chantry et al. J. Neurochem. 1988, 50, 688).

[0019] Since inhibition of p38 leads to inhibition of TNF production and MMP production, it is believed that inhibition of the enzyme, mitogen activated protein (MAP) p38 kinase, may be an approach to the therapy of the above-mentioned diseases, including osteoporosis and inflammatory disorders such as rheumatoid arthritis and COPD (Badger, AM; Bradbeer, JN; Votta, B .; Lee, JC; Adams, JL; Griswold, DEJ Pharm. Exper. Ther. 1996, 279, 1453).

[0020] Hypoxia appears to be an important stimulus for VEGF production in malignant cells. Activation of p38 kinase is necessary for the induction of VEGF by tumor cells in response to hypoxia (Blaschke, F. et al. Biochem. Biophys. Res. Commun. 2002, 296, 890-896; Shemirani, B. et al. Oral Oncology 2002, 38, 251-257). In addition to involvement in angiogenesis through regulation of VEGF secretion, p38 kinase promotes invasion of malignant cells and migration of various types of tumors by regulating collagenase activity and expression of urokinase plasminogen activator (Laferriere, J. et al. J. Biol. Chem. 2001, 276, 33762-33772; Westermarck, J. et al Cancer Res. 2000, 60, 7156-7162; Huang, S. et al J. Biol. Chem. 2000, 275, 12266-12272; Simon, C. et al. Exp. Cell Res 2001, 271, 344-355). Thus, inhibition of p38 kinase should also affect tumor growth by interfering with signaling cascades associated with angiogenesis and invasion of malignant cells.

[0021] Certain ureas have been described as having activity as serine-threonine kinase and / or tyrosine kinase inhibitors. In particular, the usefulness of certain ureas as active ingredients in pharmaceutical compositions has been demonstrated for the treatment of cancer, angiogenesis disorders, and inflammatory disorders.

[0022] Cancer and angiogenesis are described in:

Smith et al., Bioorg. Med. Chem. Lett. 2001, 11, 2775-2778.

Lowinger et al., Clin. Cancer Res. 2000, 6 (suppl.), 335.

Lyons et al., Endocr.-Relat. Cancer 2001, 8, 219-225.

Riedl et al., Book of Abstracts, 92nd AACR Meeting, New Orleans, LA, USA, abstract 4956.

Khire et al., Book of Abstracts, 93rd AACR Meeting, San Francisco, CA, USA, abstract 4211. Lowinger et al., Curr. Pharm. Design 2002, 8, 99-110.

Carter et al., Book of Abstracts, 92ndAACR Meeting, New Orleans, LA, USA, abstract 4954. Vincent et al., Book of Abstracts, 38th ASCO Meeting, Orlando, FL, USA, abstract 1900. Hilger et al., Book of Abstracts, 38th ASCO Meeting, Orlando, FL, USA, abstract 1916. Moore et al., Book of Abstracts, 38th ASCO Meeting, Orlando, FL, USA, abstract 1816. Strumberg et al., Book of Abstracts, 38th ASCO Meeting , Orlando, FL, USA, abstract 121. [0023] P38-mediated diseases, including inflammatory disorders, are described in:

Redman et al., Bioorg. Med. Chem. Lett. 2001, 11, 9-12.

Dumas et al., Bioorg. Med. Chem. Lett. 2000, 10, 2047-2050.

Dumas et al., Bioorg. Med. Chem. Lett. 2000, 10, 2051-2054.

Ranges et al., Book of Abstracts, 220th ACS National Meeting, Washington, DC, USA, MEDI 149.

Dumas et al., Bioorg. Med. Chem. Lett. 2002, 12, 1559-1562.

Regan et al., J. Med. Chem. 2002, 45, 2994-3008.

Pargellis et al., Nature Struct. Biol. 2002, 9 (4), 268-272.

Madwed JB, Book of Abstracts, Protein Kinases: Novel Target Identification and Validation for Therapeutic Development, San Diego, CA, USA, March 2002.

Pargellis C. et al., Curr. Opin. Invest., Drugs 2003, 4, 566-571.

Branger J. et al., J. Immunol. 2002, 168, 4070-4077.

Branger J. et al., Blood 2003, 101, 4446-4448.

[0024] Omega-carboxyaryldiphenylureas are disclosed in WO00 / 42012, published: July 20, 2000,

WO00 / 41698, published: July 20, 2000, the following published US applications:

US2002-0165394-A1, published on November 7, 2002,

US2001-003447-A1, published on October 25, 2001,

US2001-0016659-A1, published on August 23, 2001,

US2002-013774-A1, published on September 26, 2002, and US applications pending at the same time:

09 / 758.547, filed on January 12, 2001,

09 / 889,227, filed on July 12, 2001,

09 / 993,647, filed on November 27, 2001,

10 / 042,203, filed on January 11, 2002 and

10 / 071,248, filed on February 11, 2002.

Description of the invention [0025] It has now been discovered that an omega-carboxyaryldiphenyl urea of ​​formula I below, containing a 2-fluoro-4- (2- (N-methylcarbamoyl) -4-pyridyloxy) phenylene group, bonded to urea, is a strong raf kinase inhibitor, VEGFR kinases, p38 kinases and PDGFR kinases, which are all interesting molecular targets for the treatment and prevention of osteoporosis, inflammatory disorders, hyperproliferative disorders, and angiogenesis disorders, including cancer.

[0026] The present invention relates to e.g.

(i) a new compound of formula (I), its salts, prodrugs and metabolites, (ii) pharmaceutical compositions containing such a compound, and (iii) the use of that compound or composition for the treatment of diseases and conditions mediated by raf, VEGFR, PDGFR, flt -3 and p38 as the only agent or in combination with cytotoxic therapies.

[0027] The compound of the following formula I, its salts, prodrugs and metabolites, are collectively referred to as "compounds of the invention". Formula I is as follows:

<img file="PL1663978T3_D0001.tif" />

[0028] The metabolites of the compound of the present invention include oxidized derivatives of formula I in which one or more urea nitrogen atoms are substituted with a hydroxyl group. The metabolites of the compound of the present invention also include analogs in which the methylamide group of the compound of formula I is hydroxylated and then methylated by metabolic degradation. The metabolites of the compound of the present invention further include oxidized derivatives in which the pyridine nitrogen atom is in the form of an N-oxide (e.g., it contains a hydroxyl substituent), which leads to structures referred to in the art as 1-oxopyridine and 1-hydroxypyridine.

[0029] Where the plural, words, compounds, salts, and the like are used, it should be understood that it also means a single compound, salt or the like.

[0030] The use of pharmaceutically acceptable salts of the compounds of formula I is also within the scope of this invention. The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. See, e.g., SM Berge, et al. "Pharmaceutical

Salts, "J. Pharm. Sci. 1977, 66, 1-19.

[0031] Representative salts of the compound of the present invention include ordinary non-toxic salts, e.g. inorganic or organic acid salts prepared by well-known methods. For example, such acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphors, camphorsulfonate, cinnamate, cyclopentanopropionate, digluconate, dodecyl sulfate, ethane sulfate, phosphonate heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate, malate, mandelate, methanesulfonate, Naphthalene sulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfonate, tartrate, thiocyanate, tosylate and undecanoate.

[0032] Salts or prodrugs of compounds of formula I may contain one or more asymmetric centers. Asymmetric carbon atoms can exist in the (R) or (S) configuration or (R, S) configuration. Substituents on the ring may also be in cis or trans form. All such configurations (including enantiomers and diastereomers) are within the scope of the present invention. Preferred isomers are isomers with configurations that give more desirable biological activity. Isolated, pure or partially purified isomers or racemic mixtures of the compounds of the invention are also within the scope of the present invention. Purification of such isomers and separation of isomeric mixtures can be carried out by standard techniques known in the art.

[0033] The specific method used in the preparation of the compound used in this embodiment of the invention is described in example 1. The salts of the compound of formula (I) are described in examples 2, 3 and 4.

Methods of use [0034] The present invention provides compounds that can modulate one or more signal processing pathways, including raf, VEGFR, PDGFR, p38 and / or flt-3 kinases. Raf is an important signaling molecule involved in the regulation of several key cellular processes, including cell growth, cell survival and invasion. It is part of the Ras / raf / MEK / ERK trail. This pathway occurs in most cancer cells. VEGFR, PDGFR and flt-3 are transmembrane receptor molecules that, stimulated by the appropriate ligand, activate the Ras / raf / MEk / ERK cell signaling pathway, leading to a cascade of cellular events. Each of these receptor molecules has tyrosine kinase activity.

[0035] VEGFR receptors are stimulated by vascular endothelial growth factor (VEGF) and are important checkpoints in regulating the development and function of endothelial cells. The PDGF-beta receptor regulates cell growth and survival in many cell types, including mesenchymal cells. Flt-3 is a FL ligand receptor. It is structurally similar to c-kit and modulates the growth of pluripotent hematopoietic cells, affecting the development of T cells, B cells and dendritic cells.

[0036] Any raf or VEGFR, PDGFR, p38 and / or flt-3 gene or isoform may be modulated in accordance with the present invention, including wild type and mutants. Raf or raf-1 kinase is a serine / threonine kinase family that contains at least three family members, a-raf, b-raf and c-raf or raf-1. See, e.g., Dhillon and Kolch, Arch. Biochem. Biophys. 2002, 404, 3-9. C-raf and b-raf are preferred targets for the compounds of the present invention. Activation of b-raf mutation (e.g. the V599E mutant) has been identified in a variety of cancers, including melanoma, and the compounds described herein can be used to inhibit their activity.

[0037] The term "modulate" means that the functional activity of the pathway (or an element thereof) is changed compared with its normal activity in the absence of the compound. This effect includes any type or degree of modulation, including increasing, agonizing, increasing, strengthening, facilitating, stimulating, reducing, blocking, inhibiting, weakening, lowering, antagonizing, etc.

[0038] The compounds of the present invention may also modulate one or more of the following processes, including but not limited to e.g. cell growth (including, e.g., differentiation, cell survival and / or growth), growth of cancer cells (including, e.g., differentiation, survival cells, and / or growth), tumor regression, endothelial cell growth (including e.g. differentiation, cell survival, and / or growth), angiogenesis (blood vessel growth), lymphangiogenesis (lymphatic vessel growth), and / or hematopoiesis (e.g. T and B cell development, dendritic cell development, etc.).

[0039] Without being limited to any theory or mechanism of action, it has been found that the compounds of the present invention have the ability to modulate kinase activity. The methods of the present invention are, however, not limited to any particular mechanism or method for the compounds to achieve their therapeutic effect. The term "kinase activity" means catalytic activity in which adenosine triphosphate (ATP) gammaphosphate is transferred to an amino acid residue (e.g. serine, threonine or tyrosine) in the protein substrate. The compound may modulate kinase activity, e.g., by inhibiting it by directly competing with ATP for an ATP-binding kinase pocket, by causing a conformational change in the structure of the enzyme affecting its activity (e.g., by destroying a biologically active three-dimensional structure), etc.

[0040] Kinase activity can be determined routinely using conventional testing methods. Kinase assays typically include a kinase enzyme, substrates, buffers, and detection system components. A typical kinase assay utilizes a protein kinase reaction with a peptide substrate and ATP, such as³²P-ATP to form a phosphorylated end product (for example phosphoprotein when a peptide substrate is used). The resulting end product can be detected using any suitable method. When radioactive ATP is used, the radioactively labeled phosphoprotein can be separated from unreacted gamma-³²P-ATP using an affinity membrane or gel electrophoresis followed by gel visualization using autoradiography or scintillation counter detection. Non-radioactive methods can also be used. The methods may use an antibody that recognizes a phosphorylated substrate, e.g., an anti-phosphotyrosine antibody. For example, the kinase enzyme may be incubated with the substrate in the presence of ATP and kinase buffer under conditions that are effective for the enzyme phosphorylating the substrate. The reaction mixture can be separated, e.g., electrophoretic, and then the phosphorylation of the substrate can be measured, e.g., by Western hybridization using an anti-phosphotyrosine antibody. The antibody may be labeled with a detectable label, e.g. an enzyme such as HRP, avidin or biotin, chemiluminescent reagents, etc. Other methods may use ELISA methods, membrane separation with affinity, fluorescence polarization tests, luminescence tests, etc. [0041] An alternative to the radioactive method is time-resolved resonance fluorescent energy transfer (TR-FRET). This method uses a standard kinase reaction in which a substrate, e.g. biotinylated poly (GluTyr), is phosphorylated by a protein kinase in the presence of ATP. The final product can then be detected with a phosphospecific antibody of europium chelate (anti-phosphotyrosine or phosphoserine / threonine) and streptavidin-APC, which binds the biotinylated substrate. The two components are spatially converging upon binding, and the energy transfer from the phosphospecific antibody to the acceptor (SA-APC) gives a fluorescent reading in a homogeneous format.

[0042] The compounds of the present invention can be used for the treatment and / or prevention of a disease or condition mediated by one or more cell signal processing pathways involving raf kinases, VEGFR, PDGFR, p38 and / or flt-3. The term "treatment" is used conventionally, e.g., as therapeutic treatment or patient care to combat, alleviate, weaken, relieve, improve, etc. a condition, disease or disorder. The compounds can also be described as used for the treatment and / or prevention of diseases and / or conditions mediated by signal molecules. Ter16 min "mediated" indicates, for example, that the signal molecule is part of a pathway that is aberrant or disturbed in the disease and / or condition.

[0043] Diseases and conditions that can be treated include any of the above and below, as well as:

Raf-related diseases include, e.g., cell proliferative disorders, cancer, cancer, etc .;

VEGFR-2-related diseases include e.g. cancer, tumor growth, inflammatory disease, rheumatoid arthritis, retinopathy, psoriasis, glomerulonephritis, asthma, chronic bronchitis, atherosclerosis, transplant rejection, conditions associated with angiogenesis, etc .;

VEGFR-3 related diseases include e.g. cancer, keratosis, keratitis (e.g. Hamrah, Am. J. Path. 2003, 163, 57-68), corneal transplant (Cursiefen et al., Cornea 2003, 22, 273 -81), lymphatic hyperplasia, conditions associated with lymphangiogenesis, etc .;

PDGFR-beta-related diseases include, e.g., diseases or conditions characterized by cell proliferation, cell matrix production, cell movement, and / or intercellular matrix production. Specific examples include e.g. tumors, malignancies, cancer, metastases, chronic myelogenous leukemia, inflammation, kidney disease, diabetic nephropathy, mesangial hyperplasia of glomerulonephritis, fibrotic conditions, atherosclerosis, recurrence of the narrowing, hypertension associated with hypertension, sclerosis-associated graft , interstitial lung diseases, synovial disorders, arthritis, leukemia, lymphomas, etc .;

Flt-3 related diseases include, e.g., immune related disorders, blood cell disorders, conditions associated with the development of hematopoietic cells (e.g. T cells, B cells, dendritic cells), cancer, anemia, HIV, acquired immunodeficiency syndrome, etc.

P38-related diseases include inflammatory disorders, immunomodulatory disorders, and other disorders associated with abnormal cytokine production, particularly TNF-alpha, or abnormal MMP activity. These disorders include, but are not limited to, rheumatoid arthritis, COPD, osteoporosis, Crohn's disease, and psoriasis.

[0044] In addition, the compounds of the present invention can be used to treat the conditions and diseases disclosed in US Patent No. 6,316,479, e.g. glomerulosclerosis, interstitial nephritis, interstitial pulmonary fibrosis, atherosclerosis, scarring wounds and scleroderma.

[0045] The compounds of the present invention also have broad therapeutic activity for the treatment or prevention of the progression of many diseases, such as inflammation, recurrence of coronary artery stenosis, tumor associated angiogenesis, atherosclerosis, autoimmune diseases, inflammation, certain kidney diseases associated with cell growth. glomerular or mesangial, and eye diseases associated with retinal vascular hyperplasia, psoriasis, liver cirrhosis, diabetes, atherosclerosis, recurrence of narrowing, recurrence of stenosis in vessel transplantation, stenotic stenosis, angiogenesis, eye diseases, pulmonary fibrosis, bronchiolitis obliterans, glomerulonephritis, rheumatoid arthritis.

[0046] The present invention also provides the treatment, prevention, modulation etc. of one or more of the following conditions in humans and / or other mammals: retinopathy, including diabetic retinopathy, ischemic retinal vein occlusion, extracellular fibroplasia and age-related macular degeneration ; rheumatoid arthritis, psoriasis, or a bullous disorder associated with subepidermal blister formation, including pemphigoid, erythema multiforme, or herpetic dermatitis, rheumatic fever, bone resorption, postmenopausal osteoporosis, sepsis, Gram-negative septicemia, septic shock, endoscopic shock toxic shock syndrome, systemic inflammatory response syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), Jarisch-Herxheimer reaction, asthma, adult respiratory distress syndrome, acute fibrotic lung disease, pulmonary sarcoidosis, allergic respiratory disease, silicosis, pollinosis of coal miners, alveolar damage, liver failure, liver disease during acute inflammation, acute alcoholic hepatitis, malaria ( malaria (Plasmodium falciparum and cerebral malaria), non-insulin dependent diabetes mellitus (NIDDM), congestive heart failure, damage after heart disease, atherosclerosis, Alzheimer's disease, acute encephalitis, brain injury, multiple sclerosis (demyelination and loss of oligodendrocytes in multiple sclerosis), advanced cancer, lymphatic cancers, pancreatitis, impaired wound healing in infection, inflammation and cancer, syndromes myelodysplasia, systemic lupus erythematosus, biliary cirrhosis, intestinal necrosis, radiation damage / toxicity following the administration of monoclonal antibodies, graft versus host reaction (reperfusion ischemic injury and renal, liver, heart and skin allograft rejection), pulmonary allograft rejection (bronchitis obliterans), or complications associated with complete hip replacement, selected infectious disease from tuberculosis, Helicobacter pylori infection during peptic ulcer disease, Chaga disease from Trypanosoma cruzi infection, Shiga-like toxin action in E infection. coli, the effect of enterotoxin A in staphylococcal infection, meningococcal infection, and Borrelia burgdorferi infection, Treponema pallidum, cytomegalovirus, influenza virus, encephalomyelitis virus and human immunodeficiency virus (HIV), papilloma, glioma, sarcoma lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease, Burkitt's disease, arthritis, rheumatoid arthritis, diabetic retinopathy, angiogenesis, recurrence of stenosis, recurrence of stenosis in the stent, recurrence of stenosis in vascular transplantation, pulmonary fibrosis, pulmonary fibrosis arteries, glomerulonephritis, diabetic nephropathy, thrombotic microangiopathy syndromes, transplant rejection, psoriasis, diabetes, wound healing, inflammation, and neurodegenerative diseases, hyperimmune disorders, hemangioma, myocardial angiogenesis, collateral coronary and cerebral vasculature, ischemia, corneal disease, rubeosis, gonorrhea with neovascularization, extracellular fibroplasia, and macular degeneration, wound healing Helicobacter, fractures, endometriosis, diabetic condition, cat scratch disease, thyroid hyperplasia, asthma or swelling after burns, injury, chronic lung disease, stroke, polyps, cysts, synovitis, chronic and allergic inflammation, ovarian hyperstimulation syndrome, pulmonary and brain edema, keloids, fibrosis, cirrhosis, carpal tunnel syndrome, adult respiratory distress syndrome, water ascites, eye condition, cardiovascular status, CrowFukase disease (POEMS), Crohn's disease, glomerulonephritis, osteoarthritis, multiple sclerosis, transplant rejection, Lyme disease, sepsis, retinal hemangioma, pemphigoid, Paget's disease, cystic kidney disease, sarcoidosis, pancreatitis, hyperviscosity syndrome, Osler-Weber-Rendu disease, chronic obstructive pulmonary disease, irradiation, hypoxia, preeclampsia, abundant menstruation, endometriosis, herpes virus, ischemic retinopathy, corneal angiogenesis, herpes zoster, human immunodeficiency virus, Newcastle disease virus, protozoa, toxoplasmosis, and tumor-related exudates and edema.

[0047] The compounds of the present invention may exhibit more than one of said activities, and thus may reach many signal processing pathways.

Thus, these compounds may have therapeutic and prophylactic effects that are usually only achieved using combinations of different compounds. For example, the ability to inhibit new vessel formation (e.g. associated with VEGFR-2 and VEGFR-3) (e.g. blood and / or lymph) and cell proliferation (e.g. associated with raf and PDGFR-beta) using a single compound is particularly beneficial in the treatment of cancer and other disorders related to cell proliferation facilitated by neovascularization. Accordingly, the invention relates in particular to compounds that have at least anti-cell proliferative and anti-angiogenesis (i.e. inhibit angiogenesis) activity. Any disorder or condition that benefits from inhibiting angiogenesis and cell growth can be treated in accordance with the invention. The use of a single compound is also advantageous because the extent of its activity can be more accurately determined. [0048] As indicated above, the present invention relates to methods of treating and / or preventing diseases and conditions; and / or modulating one or more pathways, polypeptides, genes, diseases, conditions etc. associated with raf, VEGFR, PDGFR, p38 and / or flt-3. These methods generally involve administering effective amounts of the compounds of the present invention, wherein the effective amount is the amount of the compound that is useful in achieving the desired result. The compounds can be administered in any effective form by any effective route, as discussed in more detail below.

[0049] The methods include modulating tumor cell growth, including inhibiting cell growth. That is, the growth and / or differentiation of tumor cells is weakened, lowered, reduced, slowed down, etc. The term "hyperplasia" includes any process that relates to cell growth and division, and includes differentiation and apoptosis. as discussed above, raf kinases play a key role in activating the cytoplasmic signaling cascade involved in cell proliferation, differentiation and apoptosis. For example, studies have revealed that inhibition of c-raf by antisense oligonucleotides can block cell proliferation (see above). Any degree of inhibition is considered therapeutic.

[0050] The methods of the present invention include a method of using the compound described above (compound of Formula I), including salts, prodrugs, metabolites (oxidized derivatives) and compositions thereof, for the treatment of hyperproliferative disorders in mammals, comprising administering to a mammal, including a person in need thereof, the compound of the present invention, a pharmaceutically acceptable salt, a prodrug, a metabolite (oxidized derivative) and its composition in an amount which is effective in treating the disorder. Hyperplastic disorders include, but are not limited to, solid tumors such as breast, respiratory, brain, reproductive organs, gastrointestinal tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and distant metastases. These disorders also include lymphomas, sarcomas and leukemia.

[0051] Any tumor or cancer can be treated, including, but not limited to, cancers having one or more mutations in raf, ras and / or flt-3, as well as any earlier or later element of the signaling pathways of which they are part. As discussed earlier, cancer can be treated with a compound of the present invention regardless of the mechanism that is responsible for it. Cancers of any organ can be treated, including, but not limited to, e.g. cancer of the colon, pancreas, breast, prostate, bone, liver, kidney, lung, testicles, skin, pancreas, stomach, colon, kidney cells, hepatocellular carcinoma, melanoma, etc.

[0052] Examples of breast cancer include, but are not limited to, invasive ductal cancer, invasive lobular cancer, in situ ductal cancer, and lobular in situ cancer.

[0053] Examples of respiratory cancers include, but are not limited to, small cell and non-small cell lung cancer, as well as bronchial adenoma and lung and pleural blastoma. [0054] Examples of brain cancers include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebral astrocytoma, neural tubercle, ependymoma, as well as neuroectodermal cancer and pineal gland.

[0055] Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.

[0056] Tumors of the gastrointestinal tract include, but are not limited to, cancers of the anus, colon, large intestine, esophagus, gallbladder, stomach, pancreas, rectum, small intestine, and salivary glands.

[0057] Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, pelvis, ureter and urethral cancers.

[0058] Eye cancers include, but are not limited to, eye melanoma and retinoblastoma.

[0059] Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinomas with or without a fibrolamellar variant), bile duct cancer (intrahepatic bile duct cancer), and mixed bile and hepatocellular carcinoma.

[0060] Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

[0061] Head and neck cancers include, but are not limited to, larynx, throat, nasopharynx, and / or oropharyngeal, and lips and mouth cancer.

[0062] Lymphomas include, but are not limited to, ADDS associated lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma.

[0063] Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. [0064] Leukemias include, but are not limited to, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and haired cell leukemia.

[0065] In addition to inhibiting tumor cell growth, the compounds of the present invention may also cause tumor regression, e.g., a decrease in tumor size, or the extent of cancer in the body.

[0066] The present invention also relates to methods for modulating angiogenesis and / or lymphangiogenesis in a system comprising cells, comprising administering to the system an effective amount of a compound described herein. The cell-containing system may be an in vivo system, such as a patient's tumor, in isolated organs, tissues or cells, an in vitro test system (CAM, BCE, etc.), animal models (e.g., in vivo subcutaneous cancer models), hosts in need of treatment (eg. hosts suffering from diseases having an angiogenic and / or lymphangiogenic component, such as cancer) etc.

[0067] Inadequate and ectopic expression of angiogenesis may be harmful to the body. Several pathological conditions are associated with the growth of external blood vessels. These include, e.g., diabetic retinopathy, neovascular glaucoma, psoriasis, extra-lenticular fibrous hyperplasia, hemangioma, inflammation, etc. In addition, the increased blood supply associated with cancerous and cancerous tissue increases growth, leading to rapid tumor enlargement and metastasis. In addition, the growth of new blood and lymphatic vessels in the tumor provides an escape route for the changed cells, promoting metastasis and further spread of the cancer.

[0068] Useful systems for measuring angiogenesis and / or lymphangiogenesis and their inhibition include, e.g., neovascularization of tumor explants (e.g., US Patent Nos. 5,192,744; 6,024,688), Chicken Chorionic Allantoic (CAM) test (e.g. Taylor and Folkman, Nature 1982, 297, 307-312; Eliceiri et al., J. Cell Biol. 1998, 140, 12551263), endothelial cell assay of bovine capillary (BCE) (e.g. US Patent No. 6024688; Polverini, PJ et al., Methods Enzymol. 1991, 198, 440-450), migration assays and attempt to inhibit the growth of HUVEC (human umbilical cord endothelial cells) (e.g., US Pat. No. 6,060,449), and the use of the rabbit ear model (e.g. Szuba et al., FASEB J. 2002 , 16 (14), 1985-7).

[0069] Modulation of angiogenesis can be determined by any other method. For example, the degree of tissue vascularization is typically determined by assessing the number and density of vessels present in a given sample. For example, microvessel density (MVD) can be estimated by counting endothelial clusters in a highly magnifying microscope field or by detecting a microvascular endothelium specific marker or other markers for growing or established blood vessels, such as CD31 (also known as endothelial-cell adhesion molecule or PECAM) ). The CD31 antibody can be used in conventional immunohistological methods for immunostaining tissue sections as described in, e.g., US Patent No. 6,017,949; Dellas et al., Gyn. Oncol. 1997, 67, 27-33; and other. Other markers of angiogenesis include; e.g. Vezfl (e.g. Xiang et al., Dev. Bio. 1999, 206, 123-141), angiopoietin, Tie-1 and Tie-2 (e.g. Sato et al., Nature 1995, 376, 70-74) .

[0070] Furthermore, the present invention relates to methods of screening patients for determining their sensitivity to compounds of the present invention. For example, the invention relates to methods for determining whether a condition can be modulated by a compound disclosed in the invention, comprising measuring the expression or activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and / or flt-3 in a sample containing cells or extract a cell where a sample was obtained from a cell or a patient in which the condition is present. When the results of the determination indicate that one or more of said genes (and / or polypeptides that they encode) is different from the normal state, this identifies the condition as being treatable with a compound of the present invention, i.e. the disorder or condition can be modulated by a compound when expression or activity is increased in this condition compared to normal control. The method may further comprise the step of comparing the expression in the sample with normal control, or the expression in the sample obtained from normal or intact tissue. The comparison can be made manually, relative to the template, in electronic form (eg in a database), etc. The normal control can be a standard sample, which was attached to the sample; it may come from adjacent but intact tissue from this same patient; or it can be an early value etc. Gene expression, protein expression (e.g. cell abundance), protein activity (e.g. kinase activity) etc. can be determined.

[0071] For example, a biopsy from a cancer patient may be assessed for the presence, amount and / or activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and / or flt-3. Increased expression or activity of one or more thereof may indicate that the cancer may be the target of therapy with a compound of the present invention. For example, as described in the examples below, raf activity can be monitored by its ability to initiate a cascade leading to ERK phosphorylation (i.e., raf / MEK / ERK), resulting in phospho-ERK. Increased phospho-ERK levels in cancer samples show that its raf activity is increased, suggesting the use of the compounds of the present invention for its treatment.

[0072] Measurement of expression includes determining or detecting the amount of polypeptide present in or disseminated by the cell, as well as measuring associated mRNA, wherein the amount of mRNA present is to reflect the amount of polypeptide produced by the cell. In addition, raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and / or Flt-3 genes can be analyzed to determine if there is a gene defect responsible for abnormal expression or activity of the polypeptide.

[0073] The detection of the polypeptide can be carried out by any available method, e.g. by Western hybridization, ELISA, dot blot, immuno-precipitation, RIA, immunohistochemistry etc. For example, tissue sections can be prepared and labeled with a specific antibody (directly or indirectly), with microscopic visualization. The amount of polypeptide can be evaluated without visualization, e.g. by preparing the lysate of a given sample and then determining by ELISA or Western method the amount of polypeptide relative to the amount of tissue. Antibodies and other specific binding agents can be used. There is no limit to how you perform detection.

[0074] Assays that allow quantifying and / or the presence / absence of the target nucleic acid (eg, genes, mRNA etc., for raf, VEGFR, PDGFR, p38 and / or flt-3) in the sample can be used. Tests can be carried out at the level of a single cell or in a multi-cell sample, so that the test "averages" the expression over the entire set of cells and tissue present in the sample. Any suitable type of sample may be used, including, but not limited to, e.g. Southern hybridization analysis, Northern hybridization analysis, polymerase chain reaction ("PCR") (e.g., Saiki et al., Science 1988, 241, 53; US Patent Nos. 4683195, 4683202 and 6040166; PCR Protocols: A Guide to Methods and Applications , red, Innis et al., Academic Press, New York, 1990), polymerase chain reaction with reverse transcriptase ("RT-PCR"), anchored PCR, rapid amplification of cDNA ends ("RACE") (e.g., Schaefer at Gene Cloning and Analysis: Current Innovations, p. 99-115, 1997), ligase chain reaction ("LCR") (EP 320 308), unilateral PCR (Ohara et al., Proc. Natl. Acad. Sci. 1989, 86, 5673-5677), indexing methods (e.g. US Patent No. 5508169), in situ hybridization, differential imaging (e.g., Liang et al., Nucl. Acid. Res. 1993, 21, 3269 3275; US Patent Nos. 5262311, 5599672 and 5965409; WO97 / 18454; Prashar and Weissman, Proc. Natl. Acad. Sci., 93: 659-663, and US Patent Nos. 6010850 and 5712126; Welsh et al., Nucleic Acid Res., 20: 4965-4970, 1992, and US Patent No. 5,487985) and other RNA fingerprinting techniques, nucleic acid sequence amplification ("NASBA") and other transcription-based amplification systems ( e.g. US Patent Nos. 5409818 and 5554527; WO 88/10315), polynucleotide systems (e.g. U.S. Patent Nos. 5,143,854, 5,424,186; 5700637, 5874219, and 6054270; PCT WO 92/10092; PCT WO 90/15070), Qbeta replicase (PCT / US87 / 00880), thread displacement amplification ("SDA"), chain repair reaction ("RCR"), nuclease protection tests, subtraction-based methods, Rapid-Scan, etc. Additional useful methods include, but are not limited to, e.g. matrix-based amplification methods, competitive PCR (e.g., U.S. Patent No. 5,747,251), redox-based assays (e.g. U.S. Patent No. 5,871,918), Taqman-based trials (e.g., Holland et al., Proc. Natl. Acad, Sci. 1991, 88, 7276-7280; U.S. Patent Nos. 5210015 and 5994063), real-time monitoring on fluorescence (e.g., US Patent No. 5,928,907), molecular energy transfer markers (e.g., US Patent Nos. 5,348,853, 5532129, 5565322, 6030787, and 6117635; Tyagi and Kramer, Nature Biotech., 14: 303-309, 1996) . Any method suitable for analyzing expression of a single cell gene or protein may be used, including in situ hybridization, immunocytochemistry, MA15 CS, FACS, flow cytometry, etc. For a single cell assay, expression products can be measured using antibodies, PCR, or other types of amplification. nucleic acid (e.g., Brady et al., Methods Mol. &amp; Cell. Biol. 1990, 2, 17-25; Eberwine et al., Proc. Natl. Acad. Sci. 1992, 89, 3010-3014; US Patent No. 5723290). These and other methods can be used conventionally, e.g. as described in said publications.

[0075] Raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and / or flt-3 activity can be assessed routinely, e.g. as described in the examples below, or using standard assays for kinase activity.

[0076] The present invention also provides methods for assessing the efficacy of a compound of the present invention in treating a disorder, comprising one or more of the following steps in any effective order, e.g., administering a certain amount of the compound, measuring the expression or activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and / or flt-3 (see above), determining the effect of the compound on expression or activity. For example, biopsy samples can be taken from patients who have been treated with a compound of the present invention and then tested for the presence and / or activity of said signal molecules. Similarly, as mentioned above, decreases in phospho-ERK levels in cancer tissue (e.g., compared to normal tissue or before therapy) indicate that the compound shows in vivo efficacy and therapeutic effect. The method can be used to determine appropriate doses and dosage regimens, e.g. how much compound to give and how often to give it. By monitoring its effect on signaling molecules in tissue, the clinician can determine the appropriate therapy protocol and determine the achievement of the desired effect, e.g., modulation or inhibition of the signal processing pathway.

[0077] The compounds of the present invention can also be used as markers to determine the presence and amount of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and / or flt-3 in a sample containing biological material. This includes one or more of the following steps in any effective order: (i) contacting the sample containing the biological material of the compound of the present invention, and (ii) determining whether the compound binds to the material. The compound may be labeled, or it may be used as a competitor with labeled compound, such as labeled ATP.

[0078] The invention also provides methods of treating, preventing, modulating etc., diseases and conditions in mammals, comprising administering a compound of the present invention with another modulator of a signal processing pathway containing, among others, raf, VEGFR, PDGFR, p38 and / or flt- 3. They may be present in the same composition or in separate preparations or dosage units. Administration can occur by the same or different routes, and may be simultaneous or consecutive.

[0079] The following publications relate to VEGFR-3 modulation and are included herein for the description of disease states mediated by VEGFR-3 and assays to determine such activity.

WO95 / 33772 Alitalo, et al. WO95 / 33050Charnock-Jones, and In ..

WO96 / 39421Hu, et al.

WO98 / 33917Alitalo, et al.

WO02 / 057299 Alitalo, et al.

WO02 / 06095 Alitalo, et al.

WO02 / 081520Boesen, et al.

[0080] The following publications relate to VEGFR-2 modulation and are included herein for the description of disease states mediated by VEGFR-2 and assays to determine such activity.

EP0882799 Hanai, et al.

EP1167384 Ferraram, et al.

EP 1086705 Sato, et al.

EP11300032 Tesar, et al.

EP1166798 Haberey, et al.

EP1166799 Haberey, et al.

EP1170017 Maini, et al.

EP1203827 Smith

WO02 / 083850Rosen, et al.

[0081] The following publications relate to flt-3 modulation and are included herein for the description of disease states mediated by flt-3 and assays to determine such activity.

2002/0034517 Brasel, et al.

2002/0107365 Lyman, et al.

2002 / 0111475Graddis, et al.

EP0627487 Beckermann, et al.

WO9846750 Bauer, et al.

WO9818923 McWherter, et al.

WO9428391 Beckermann, et al.

WO9426891 Bimbaum, et al.

[0082] The following patents and publications relate to PDGF / PDGFR modulation and are incorporated herein for the description of disease states mediated by PDGFR-beta and assays to determine such activity.

5,094,941 Hart, et al.

5,371,205 Kelly, et al.

5,418,135 Pang 5,444,151 Vassbotn, et al.

5,468,468 LaRochelle, et al.

5,567,584 Sledziewski, et al.

5,618,678 Kelly, et al.

5,620,687 Hart, et al.

5,648,076 Ross, et al.

5,668,264 Janjic, et al.

5,686,572 Wolf, et al.

<td>5,817,310 Ramakrishnan, et al.</td>

<td>5,833,986 LaRochelle, et al.</td>

<td>5,863,739 LaRochelle, et al.</td>

<td>5,872,218 Wolf, et al.</td>

<td>5,882,644, Chang, et al.</td>

<td>5,891,652 Wolf, et al.</td>

<td>5,976,534 Hart, et al.</td>

<td>5,990,141 Hirth, et al.</td>

<td>6,022,854 Shuman</td>

<td>6,043,211 Williams, et al.</td>

<td>6,110,737 Escobedo, et al.</td>

<td>6,207,816B1 Gold, et al.</td>

<td>6,228,600B1 Matsui, et al.</td>

<td>6,229,002B1 Janjic, et al.</td>

<td>6,316,603B1 McTigue, et al.</td>

<td>6,372,438B1 Williams, et al.</td>

<td>6,403,769B1 La Rochelle, et al.</td>

<td>6,440,445B1 Nowak, et al.</td>

<td>6,475,782B1 Escobedo, et al.</td>

<td>WO02 / 083849 Rosen, et al.</td>

<td>WO02 / 083704 Rosen, et al.</td>

<td>WO02 / 081520 Boesen, et al.</td>

<td>WO02 / 079498 Thomas, et al.</td>

<td>WO02 / 070008 Rockwell, et al.</td>

<td>WO09959636 Sat, et al.</td>

<td>WO09946364 Cao, et al.</td>

<td>WO09940118 Hanai, et al.</td>

<td>WO9931238 Yabana, et al.</td>

<td>WO9929861 Klagsbrun, et al.</td>

<td>WO9858053 Kendall, et al.</td>

<td>WO9851344 Maini, et al.</td>

<td>WO9833917 Alitalo, et al.</td>

<td>WO9831794 Matsumoto, et al.</td>

<td>WO9816551 Ferrara, et al.</td>

<td>WO9813071 Kendall, et al.</td>

<td>W09811223 Martiny-Baron, et al.</td>

<td>WO9744453 Chen, et al.</td>

<td>WO9723510 Plouet, et al.</td>

<td>WO9715662 Stinchcomb, et al.</td>

<td>WO9708313 Ferrara, et al.</td>

<td>WO963951 Cao, and In.</td>

<td>WO9623065 Smith, et al.</td>

<td>WO9606641 Fleurbaaij, et al.</td>

<td>WO9524473 Cao, et al.</td>

<td>WO9822316 Kyowa</td>

<td>WO9521868 Rockwell, et al.</td>

<td>WO02 / 060489Xia, et al.</td>

PDGFR-beta [0083]

EP0869177 Matsui, et al. WO09010013 Matsui, et al. WO9737029 Matsui, et al.

PDGFR-alpha [0084]

EP 1000617 Lammers, et al. EP0869177 Matsui, et al. EP0811685 Escobedo, et al.

Pharmaceutical compositions based on compounds of the present invention [0085] This invention also relates to pharmaceutical compositions comprising a compound of the present invention and pharmaceutically acceptable salts thereof. The compositions can be used to achieve the desired pharmacological effect by administration to a patient in need thereof. The patient, for the purposes of this invention, is a mammal, including a human, in need of treatment for a particular condition or disease. Thus, the present invention includes pharmaceutical compositions composed of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound, or a salt thereof, of the present invention. The term "pharmaceutically acceptable carrier" is intended to mean any carrier that is relatively non-toxic and harmless to the patient at concentrations consistent with the active activity of the active ingredient, so that no side effects attributed to the carrier would impair the beneficial effects of the active ingredient. A pharmaceutically effective amount of a compound is an amount that results in or affects the particular condition being treated. The compound of the present invention can be administered with well-known pharmaceutically acceptable carriers using any effective conventional unit dosage form, including immediate, slow and temporary release, oral, parenteral, topical, nasal, ophthalmological, ophthalmic, sublingual, rectal, vaginal, and the like.

[0086] For oral administration, the compound may be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, feet, powders, solutions, suspensions or emulsions that can be prepared in accordance with those known in the art. methods of making pharmaceutical compositions. Solid unit dosage forms can be in the form of a capsule, which can be a regular hard or soft gelatin capsule containing, e.g. surfactants, glidants and inert fillers such as lactose, sucrose, calcium phosphate and corn starch.

[0087] In another embodiment, the compounds of the present invention may be tableted with conventional tablet media such as lactose, sucrose and corn starch, in combination with binders such as gum arabic, corn starch or gelatin, disintegrants to aid disintegration and reconstitution of the tablet after administration, such as potato starch, alginic acid, corn starch, and guar gum, tragacanth, acacia, lubricants, which are intended to support the flow of tablet granules and prevent adhesion of the tablet material to the surface of the mold and tableting punch, such as talc, stearic acid or magnesium, calcium or zinc stearate, dyes, coloring agents and flavors such as peppermint, oil wintergreen or cherry essence, which are to improve the aesthetic value of tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include dicalcium phosphate and diluents such as water and alcohols, e.g., ethanol, benzyl alcohol and polyethylene alcohols, without or with the addition of a pharmaceutically acceptable surfactant, suspending or emulsifying agent. Various other materials may be present, such as coating or otherwise modifying the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac and / or sugar.

[0088] Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are for example represented by the agents mentioned above. Additional excipients, e.g. the sweetening, flavoring and coloring agents described above may also be present.

[0089] The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifying agents may be (1) naturally occurring gums such as acacia and tragacanth, (2) naturally occurring phospholipids such as soy and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, e.g. sorbitan monooleate, (4) condensation products of these partial esters with ethylene oxide, e.g. polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

[0090] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as, for example, peanut oil, olive oil, sesame oil or coke oil, or in a mineral oil, such as liquid paraffin. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Suspensions may also contain one or more preservatives, e.g. p30 ethyl or n-propyl hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweeteners such as sucrose or saccharin.

[0091] Syrups and elixirs may be formulated with sweetening agents such as, for example, glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain a soothing agent and a preservative, such as methyl and propyl paraben, as well as flavoring and coloring agents.

[0092] The compounds of the present invention may also be administered parenterally, i.e. subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly or intraperitoneally, as injected doses of the compound in a physiologically acceptable diluent with a pharmaceutical carrier which may be a sterile liquid or mixture of liquids such as water , saline, dextrose and related sugars, alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as polyethylene glycol 400, oil, fatty acid, fatty acid ester or fatty acid glyceride, or acetylated fatty acid glyceride, without or with the addition of a pharmaceutically acceptable surfactant such as soap or detergent, a suspending agent such as pectin, carbomers, methyl cellulose, hydroxypropyl methyl cellulose or carboxymethyl cellulose, or an emulsifying agent, and other pharmaceutical adjuvants.

[0093] Examples of oils that can be used in parenteral formulations of the invention are those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petroleum jelly and mineral oil. Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable fatty acid esters are e.g. ethyl oleate and isopropyl myristate. Suitable soaps include alkali metal, ammonium and triethanolamine fatty acid salts and suitable detergents include cationic detergents, e.g. dimethyl dialkyl ammonium halides, alkyl pyridinium halides and alkyl amine acetates; anionic detergents, e.g., alkyl, aryl and olefin sulfonates and sulfosuccinates, alkyl, olefin, ether and monoglyceride sulfates; non-ionic detergents, e.g. fatty amine oxides, fatty acid alkanolamides and copolymers of poly (oxyethylene-oxypropylene) or ethylene oxide or propylene oxide; and amphoteric detergents, e.g., alkyl-beta-aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, as well as mixtures.

[0094] The parenteral compositions of the present invention will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers can also be used advantageously. To reduce or eliminate irritation at the injection site, such compositions may contain a non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) from about 12 to about 17. The amount of surfactant in such a preparation ranges from about 5% to about 15% by weight. The surfactant can be one component and have the above HLB or it can be a mixture of two or more components with the desired HLB.

[0095] An example of surfactants used in parenteral formulations is a class of polyethylene sorbitan fatty acid esters, e.g. sorbitan monooleate and high molecular weight ethylene oxide adducts with a hydrophobic base, formed by condensation of propylene oxide with propylene glycol.

[0096] The pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents, e.g. sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, tragacanth and acacia; dispersing or wetting agents, which may be naturally occurring phospholipids, such as lecithin, condensation products of alkylene oxide with fatty acid, e.g. polyoxyethylene stearate, condensation products of ethylene oxide with long-chain aliphatic alcohol, e.g. heptadekaethyleneoxycetanol, condensation products of ethylene oxide with a partial ester derived from fatty acid and hexitol, such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide with a partial ester derived from fatty acid and hexitol anhydride, e.g. polyoxyethylene sorbitan monooleate

[0097] The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. Diluents and solvents that can be used are e.g. water, Ringer's solution, isotonic sodium chloride solutions and isotonic glucose solutions. In addition, sterile vegetable oils are conventionally used as solvents or suspending agents. For this purpose, any mild vegetable oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the injection preparations.

[0098] The composition of the invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such material is, for example, cocoa butter and polyethylene glycol.

[0099] Another formulation used in the methods of the present invention uses transdermal delivery devices ("patches"). Such transdermal patches may be used to achieve continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known (see, e.g., US Patent No. 5,033,252, issued June 11, 1991, incorporated herein by reference). Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[0100] Controlled release formulations for parenteral administration include known liposome formulations, with polymer microspheres and a polymer gel.

[0101] It may be desirable or necessary to administer the pharmaceutical composition to a patient with a mechanical administration device. The construction and use of mechanical delivery devices for the delivery of pharmaceuticals is well known. Direct techniques, e.g., for delivering the drug directly to the brain, typically include placing the drug delivery catheter into the patient's ventricular system to bypass the blood-brain barrier. One such implanted delivery system, used to transport agents to specific anatomical areas of the body, is described in U.S. Patent No. 5,011,472, issued April 30, 1991.

[0102] The compositions of the invention may also contain other conventional pharmaceutically acceptable formulating ingredients, generally referred to as carriers or diluents, if desired or necessary. Conventional procedures for preparing such compositions in appropriate dosage forms can be used. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, MF et al., "Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science &amp; Technology 1998, 52 (5), 238311; Strickley, RG "Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999) -Part-1" PDA Journal of Pharmaceutical Science &amp; Technology 1999, 53 (6), 324-349; and Nema, S. et al., "Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science &amp; Technology 1997, 51 (4), 166-171.

[0103] Commonly used pharmaceutical ingredients that can be used as suitable to formulate compositions for the intended route of administration include:

· Acidifying agents (examples include, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid);

Alkalizing agents (examples include but are not limited to ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine);

· Adsorbents (examples include but are not limited to powdered cellulose and activated carbon);

· Aerosol propellants (examples include but are not limited to carbon dioxide, CCl2F2, F2ClC-CClF2 and CClF3) · air displacement agents (examples include but are not limited to nitrogen and argon);

Antifungal preservatives (examples include but are not limited to benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate);

Antimicrobial preservatives (examples include but are not limited to benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal);

Antioxidants (examples include but are not limited to ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite);

· Binding materials (examples include but are not limited to block polymers, natural and synthetic rubber, polyacrylates, polyurethanes, silicones, polysiloxanes and styrene-butadiene copolymers);

· Buffering agents (examples include but are not limited to potassium metaphosphate, dipotassium phosphate, sodium acetate, anhydrous sodium citrate and sodium citrate dihydrate) · carrier agents (examples include, but are not limited to, arabic gum syrup, aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, syrup orange, syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride for injection and bacteriostatic water for injection) chelating agents (examples include but are not limited to disodium edetate and ethylenediaminetetraacetic acid) dyes (examples include but are not limited to FD&amp;C Red No. 3, FD&amp;C Red No. 20, FD&amp;C Yellow No. 6, FD&amp;C Blue No. 2, D&amp;C Green No. 5, D&amp;C Orange No. 5, D&amp;C Red No. 8, caramel and red iron oxide);

· Clarifying agents (examples include but are not limited to bentonite);

· Emulsifying agents (examples include, but are not limited to, acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyoxyethylene 50 monostearate);

· Encapsulating agents (examples include but are not limited to gelatin and cellulose acetate phthalate) flavors (examples include but are not limited to anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin);

· Moisturizers (examples include but are not limited to glycerol, propylene glycol and sorbitol);

· Wet milling agents (examples include, but are not limited to, mineral oil and glycerin);

· Oils (examples include, but are not limited to, peanut oil, mineral oil, olive oil, peanut oil, sesame oil and vegetable oil);

Ointment bases (examples include but are not limited to lanolin, hydrophilic ointment, polyethylene glycol ointment, petroleum jelly, hydrophilic petroleum jelly, wax ointment, yellow ointment and rose water ointment);

· Penetration enhancers (transdermal administration) (examples include, but are not limited to, monohydric or polyhydric alcohols, mono- or poly-valent alcohols, saturated or unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, ethereal oils, phosphate derivatives, phosphate derivatives) , terpenes, amides, ethers, ketones and ureas) plasticizers (examples include but are not limited to diethyl phthalate and glycerol);

· Solvents (examples include, but are not limited to, ethanol, corn oil, cottonseed oil, glycerol, isopropanol, mineral oil, oleic acid, peanut oil, purified water, water for injection, sterile water for injection and sterile water for irrigation);

· Stiffeners (examples include, but are not limited to, cetyl alcohol, cetyl ester wax, microcrystalline wax, paraffin, stearyl alcohol, white wax and yellow wax);

Suppository bases (examples include but are not limited to cocoa butter and polyethylene glycols (mixtures));

· Surfactants (examples include but are not limited to benzalkonium chloride, nonoxynol 10, oxoxoxol 9, polysorbate 80, sodium lauryl sulfate and sorbitan mono palmitate);

· Suspending agents (examples include, but are not limited to, agar, bentonite, carbomers, sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, kaolin, methyl cellulose, tragacanth and bentonitant);

Sweeteners (examples include but are not limited to aspartame, dextrose, glycerol, mannitol, propylene glycol, sodium saccharin, sorbitol and sucrose);

Anti-sticking agents for tablets (examples include but are not limited to magnesium stearate and talc);

Tablet binding agents (examples include but are not limited to gum arabic, alginic acid, sodium carboxymethyl cellulose, compressible sugar, ethyl cellulose, gelatin, liquid glucose, methyl cellulose and pregelatinized starch);

Tablet and capsule diluents (examples include but are not limited to dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sodium carbonate, sodium phosphate, sorbitol and starch);

Tablet coating agents (examples include but are not limited to liquid glucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate phthalate and shellac);

Tablet direct compression excipients (examples include but are not limited to dibasic calcium phosphate);

Tablet disintegrants (examples include, but are not limited to, alginic acid, calcium carboxymethyl cellulose, microcrystalline cellulose, potassium polacrylin, sodium alginate, sodium starch glycolate and starch);

Tablet glidants (examples include but are not limited to colloidal silica, corn starch and talc);

Tablet lubricants (examples include but are not limited to calcium stearate, magnesium stearate, mineral oil, stearic acid and zinc stearate);

Tablet / capsule opacifiers (examples include but are not limited to titanium dioxide);

Tablet polish agents (examples include but are not limited to carnauba wax and white wax);

· Thickeners (examples include but are not limited to beeswax, cetyl alcohol and paraffin);

· Tonicity agents (examples include but are not limited to dextrose and sodium chloride);

· Viscosity enhancers (examples include, but are not limited to, alginic acid, bentonite, carbomers, sodium carboxymethyl cellulose, methyl cellulose, sodium alginate and tragacanth); and wetting agents (examples include, but are not limited to, heptadekaethyleneoxycetanol, lecithin, sorbitan monooleate, polyoxyethylene sorbitan monooleate, and polyoxyethylene stearate).

[0104] The pharmaceutical compositions of the present invention can be illustrated as follows:

Sterile intravenous solution: A 5 mg / ml solution of the desired compound of the present invention is prepared using sterile water for injection, and the pH is adjusted as needed.

The solution is diluted for administration to 1-2 mg / ml with sterile 5% dextrose and administered as an intravenous infusion over 60 minutes.

Lyophilized powder for intravenous administration: A sterile preparation may be prepared from (i)

100 - 1000 mg of the desired compound of the present invention as lyophilized powder, (ii) 32-327 mg / ml sodium citrate, and (iii) 300 - 3000 mg Dextran 40. The preparation is reconstituted with sterile saline or 5% dextrose for injection to a concentration of 10 to 20 mg / ml, followed by dilution with saline or 5% dextrose to 0.2-0.4 mg / ml, and administered as an intravenous injection or intravenous infusion over 15-60 minutes.

Intramuscular suspension: The following solution or suspension for intramuscular injection may be prepared:

mg / ml of the desired water-insoluble compound of the present invention mg / ml sodium carboxymethyl cellulose 4 mg / ml Tween 80 9 mg / ml sodium chloride 9 mg / ml benzyl alcohol

Hard capsules: Many unit capsules are made into standard two-part hard gelatin capsules, 100 mg powdered active ingredient, 150 mg lactose, 50 mg cellulose and 6 mg magnesium stearate.

Soft gelatin capsules: A mixture of the active ingredient is prepared in an edible oil, such as soybean oil, cottonseed oil or olive oil, and injected with a positive displacement pump into molten gelatin to form soft gelatin capsules containing 100 mg of active ingredient. The capsules are washed and dried. The active ingredient can be dissolved in a mixture of polyethylene glycol, glycerin and sorbitol to give a water-miscible medicine.

Tablets: A large number of tablets are prepared by known methods, such that the dosage unit contains 100 mg of active ingredient, 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch and 98.8 mg of lactose. Appropriate aqueous and non-aqueous coatings can be applied to improve acceptability, improve appearance and durability, or delay absorption.

Instant-release tablets / capsules: These are solid oral dosage forms made by conventional and new processes. These units are taken orally without water for immediate reconstitution and drug delivery. The active ingredient is mixed with a liquid containing an ingredient such as sugar, gelatin, pectin and sweeteners. These liquids are solidified into solid tablets or caplets by freeze drying and solid state extraction. Drug compounds can be compressed with viscoelastic and thermoelastic sugars and polymers or effervescent ingredients to form porous matrices for immediate release without the need for water.

Dosage of the pharmaceutical compositions of the present invention [0105] Based on standard laboratory techniques to evaluate compounds useful for the treatment of any of the aforementioned disorders, using standard toxicity tests and standard pharmacological attempts to determine the therapy of the conditions described above in mammals, and by comparing these results with the results for known drugs that are used to treat these conditions, effective dosages of the compounds of the present invention can be easily determined for the treatment of any desired indication. The amount of active ingredient administered in the treatment of one of these conditions may vary widely taking into account factors such as the particular compound and unit dose employed, the mode of administration, the duration of therapy, the age and sex of the patient being treated, and the nature and severity of the condition being treated.

[0106] The total amount of active ingredient administered may range from about 0.001 mg / kg to about 200 mg / kg, and preferably from about 0.1 mg / kg to about 50 mg / kg body weight per day. The unit dose may preferably contain from about 5 mg to about 4000 mg of active ingredient, and may be administered one or more times daily. The daily dosage for oral administration will preferably be from 0.1 to 50 mg / kg of total body weight. The daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injection, and the use of infusion techniques will preferably be from 0.1 to 10 mg / kg of total body weight. The daily rectal dosage regimen will preferably be from 0.1 to 50 mg / kg of total body weight. The daily vaginal dosage regimen will preferably be from 0.1 to 50 mg / kg of total body weight. The daily topical dosage regimen will preferably be from 0.1 to 10 mg / kg when administered one to four times a day. The concentration for transdermal administration will preferably be such as to maintain a daily dose of 0.1 to 10 mg / kg. The daily dosage regimen by inhalation will preferably be from 0.1 to 10 mg / kg of total body weight. Other dosages and amounts may be chosen routinely.

[0107] The specific initial and follow-up dosage regimen for each patient will vary depending on the nature and severity of the condition as determined by the attending diagnostician, the activity of the particular compound employed, the age and general condition of the patient, administration time, route of administration, rate of drug excretion, combination of drugs and the like. The desired treatment regimen and the number of doses of the compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be determined by those skilled in the art using conventional therapy tests.

Combining the compounds and compositions of the present invention with additional active ingredients [0108] The compounds of the present invention may be administered as separate pharmaceutical agents or in combination with one or more other pharmaceutical agents, if such combination does not cause unacceptable harmful side effects. This can be especially important when treating hyperproliferative diseases such as cancer. In this case, the compound of the present invention can be combined with known cytotoxic agents, signal transduction inhibitors or other anti-cancer agents, as well as with mixtures and combinations thereof.

[0109] In one embodiment, the compounds of the present invention can be combined with cytotoxic anti-cancer agents. Examples of such measures can be found in the 11th Merck Index (1996). Such agents include, without limitation, asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, adriamycinyl, etirubamycin, etirubinyl, Irinotecan, leucovorin, lomustine, mechloretamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifene, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine and vindesine. [0110] Other cytotoxic drugs suitable for use with the compounds of the invention include, but are not limited to, recognized compounds used in cancer therapy according to The Pharmacological Basis of Therapeutics by Goodman and Gilman (9th edition, 1996, McGraw-Hill). These agents include, without limitation, aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, cladribine, busulfan, diethylstilbestrol, 2 ', 2'-difluoro-rodeoxycytidine, docetaxel, erythrohydroxynonyl adenine, ethinylestrodeurine, 5-fluoridine, 5-fluoridine , fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA), plamamycin, semustine, teniposide, testosterone propionate, thiotepa, trimethylmelamine, uridine and vinorelbine.

[0111] Other cytotoxic anti-cancer agents suitable for use in combination with the compounds of the invention also include newly discovered cytotoxic active agents such as oxaliplatin, gemcitabine, capecitabine, epothilone and its natural or synthetic derivatives, temozolomide (Quinn et al., J. Clin. Oncology 2003, 21 (4), 646-651), tositumomab (Bexxar), trabedectin (Vidal et al., Proceedings of the American Society for Clinical Oncology 2004, 23, abstract 3181), and inhibitors of spindle protein kinesin Eg5 (Wood and in., Pharmacol Curr. Opin. 2001, 1, 370-377).

[0112] In another embodiment, the compounds of the present invention may be combined with other signal transduction inhibitors. Particularly interesting are signal transduction inhibitors that target the EGFR family such as EGFR, HER-2 and HER-4 (Raymond et al., Drugs 2000, 60 (Suppl. 1), 15-23; Harari et al., Oncogene 2000, 19 (53), 6102-6114), and their respective ligands. Examples of such agents include, without limitation, antibody therapies such as Herceptin (trastuzumab), Erbitux (cetuximab) and pertuzumab. Examples of such therapies also include, without limitation, small molecule kinase inhibitors such as ZD-1839 / Iressa (Baselga et al., Drugs 2000, 60 (Suppl. 1), 33-40), OSI-774 / Tarceva (Pollack et al. J. Pharm. Exp. Ther. 1999, 291 (2), 739-748), CI-1033 (Bridges, Curr. Med. Chem. Chem. 1999, 6, 825-843), GW-2016 (Lackey et al., 92nd AACR Meeting, New Orleans, March 24-28, 2001, abstract 4582), CP-724,714 (Jani et al., Proceedings of the American Society for Clinical Oncology 2004, 23, abstract 3122), HKI-272 (Rabindran et al., Cancer Res. 2004, 64, 3958-3965), and EKB-569 (Greenberger et al., 11th NCI-EORTCAACR Symposium on New Drugs in Cancer Therapy, Amsterdam, November 7-10, 2000, abstract 388).

[0113] In another embodiment, the compounds of the present invention may be combined with other signal transduction inhibitors directed to receptor kinases from families of split kinase domains (VEGFR, FGFR, PDGFR, flt-3, c-kit, c-fms, and the like) , and their respective ligands. These agents include, without limitation, antibodies such as Avastin (bevacizumab). These agents also include, without limitation, small molecule inhibitors such as STI-571 / Gleevec (Zvelebil, Curr. Opin. Oncol., Endocr. Metab. Invest.,

Drugs 2000, 2 (1), 74-82), PTK-787 (Wood et al., Cancer Res. 2000, 60 (8), 2178-2189), SU41

11248 (Demetri et al., Proceedings of the American Society for Clinical Oncology 2004, 23, abstract 3001), ZD-6474 (Hennequin et al., 92nd AACR Meeting, New Orleans, March 2428, 2001, abstract 3152), AG-13736 (Herbst et al., Clin. Cancer Res. 2003, 9, 16 (suppl 1), abstract C253), KRN-951 (Taguchi et al., 95th AACR Meeting, Orlando, FL, 2004, abstract 2575), CP- 547,632 (Beebe et al., Cancer Res. 2003, 63, 7301-7309), CP-673,451 (Roberts et al., Proceedings of the American Association of Cancer Research 2004, 45, abstract 3989), CHIR-258 (Lee et al., Proceedings of the American Association of Cancer Research 2004, 45, abstract 2130), MLN-518 (Shen et al., Blood 2003, 102, 11, abstract 476), and AZD-2171 (Hennequin et al., Proceedings of the American Association of Cancer Research 2004, 45 , abstract 4539).

[0114] In another embodiment, the compounds of the present invention can be combined with inhibitors of the Raf / MEK / ERK transduction pathway (Avruch et al., Recent Prog. Horm. Res. 2001, 56, 127-155), or the PKB pathway (act) (Lawlor et al., J. Cell Sci. 2001, 114, 2903-2910). These include, without limitation, PD-325901 (Sebolt-Leopold et al., Proceedings of the American Association of Cancer Research 2004, 45, abstract 4003), and ARRY-142886 (Wallace et al., Proceedings of the American Association of Cancer Research 2004, 45, abstract 3891).

[0115] In another embodiment, the compounds of the present invention may be combined with histone deacetylase inhibitors. Examples of such agents include, without limitation, hydroxamic acid suberoilanilide (SAHA), LAQ-824 (Ottmann et al., Proceedings of the American Society for Clinical Oncology 2004, 23, abstract 3024), LBH-589 (Beck et al., Proceedings of the American Society for Clinical Oncology 2004, 23, abstract 3025), MS275 (Ryan et al., Proceedings of the American Association of Cancer Research 2004, 45, abstract 2452), and FR-901228 (Piekarz et al., Proceedings of the American Society for Clinical Oncology 2004, 23, abstract 3028).

[0116] In another embodiment, the compounds of the present invention may be combined with other anti-cancer agents such as proteasome inhibitors and m-TOR inhibitors. These include, without limitation, bortezomib (Mackay et al., Proceedings of the American Society for Clinical Oncology 2004, 23, Abstract 3109), and CCI-779 (Wu et al., Proceedings of the

American Association of Cancer Research 2004, 45, abstract 3849).

[0117] Generally, the use of a cytotoxic and / or cytostatic anti-cancer agent in combination with a compound or composition of the present invention for the treatment of cancer is for:

(1) achieving better efficacy in attenuating tumor growth or even eradicating the tumor compared to administering each agent individually, (2) ensuring administration of smaller amounts of chemotherapeutics used, (3) providing chemotherapy well tolerated by a patient with fewer harmful pharmacological complications than in the case of single agent chemotherapy and some other combination therapies, (4) providing treatment for a wider range of different types of cancers in mammals, especially humans, (5) ensuring a higher response rate among treated patients, (6) ensuring higher survival among treated patients compared to standard chemotherapies, (7) providing longer time for progression cancer, and / or (8) obtaining results of efficacy and tolerability at least as good as results for agents used individually, compared to known cases, in which other combinations of anti-cancer agents give antagonistic effects.

Examples [0118] The abbreviations used in this description are as follows:

HPLC high pressure liquid chromatography

MS mass spectrometry

ES electrospray ionization

DMSO dimethyl sulfoxide

MP melting point

NMR nuclear resonance spectroscopy

TLC thin layer chromatography rt room temperature

Preparation of 4-amino-3-fluorophenol [0119]

<img file="PL1663978T3_D0002.tif" />

[0120] To a dry argon purged flask, 10% Pd / C (80 mg) was added, followed by

3-fluoro-4-nitrophenol (1.2 g, 7.64 mmol) as a solution in ethyl acetate (40 mL). The mixture was stirred under H atmosphere₂ for 4 hours. The mixture was filtered through a celite pad and the solvent was evaporated under reduced pressure to give the desired product as a brown solid (940 mg, 7.39 mmol; 97% yield);¹H-NMR (DMSO-d6) 4.38 (s, 2H), 6.29-6.35 (m, 1H), 6.41 (dd, J = 2.5, 12.7, 1H), 6 , 52-6.62 (m, 1H), 8.76 (s, 1H).

Preparation of 4- (4-amino-3-fluorophenoxy) pyridine-2-carboxylic acid methylamide [0121]

<img file="PL1663978T3_D0003.tif" />

[0122] A solution of 4-amino-3-fluorophenol (500 mg, 3.9 mmol) in N, N-dimethylacetamide (6 mL) cooled to 0 ° C was treated with potassium t-butoxide (441 mg, 3.9 mmol) and the brown solution was allowed to stir at 0 ° C for 25 minutes. To the mixture, 4-chloro-N-methyl-2-pyridinecarboxamide (516 mg, 3.0 mmol) was added as a solution in dimethylacetamide (4 mL). The reaction mixture was heated at 100 ° C for 16 hours. The mixture was cooled to room temperature, quenched with H₂O (20 ml) and extracted with ethyl acetate (4 x 40 ml). The combined organic phases were washed with H₂O (2 x 30 mL), dried (MgSO₄) and evaporated, and a red-brown oil was obtained. ¹H-NMR indicated the presence of residual dimethylacetamide, so the oil was dissolved in diethyl ether (50 mL) and then washed with brine (5 x 30 mL). The organic layer was dried (MgSO₄) and concentrated to give 950 mg of the desired product as a red-brown solid which was used in the next step without purification.

[0123] The method for producing 4-chloro-N-methyl-2-pyridinecarboxamide is described by Bankston et al., Org. Proc. Res. Dev. 2002, 6 (6), 777-781.

Example 1: Preparation of 4- {4- [3- (4-chloro-3-trifluoromethylphenyl) ureido] -3-fluorophenoxy} pyridine-2-carboxylic acid methylamide [0124]

<img file="PL1663978T3_D0004.tif" />

To a solution of 4- (4-amino-3-fluorophenoxy) pyridine-2-carboxylic acid methylamide (177 mg, 0.68 mmol) in toluene (3 mL) was added 4-chloro-344 (trifluoromethyl) phenyl isocyanate (150 mg , 0.68 mmol). The mixture was stirred at room temperature for 72 hours. The reaction mixture was concentrated under reduced pressure and the residue was triturated with diethyl ether. The resulting solid was collected by filtration and dried under reduced pressure for 4 hours to give the title compound (155 mg, 0.32 mmol; 47% yield); ¹H-NMR (DMSOd6) 2.78 (d, J = 4.9, 3H), 7.03-7.08 (m, 1H), 7.16 (dd, J = 2.6, 5.6, 1H), 7.32 (dd, J = 2.7, 11.6, 1H), 7.39 (d, J = 2.5, 1H), 7.60 (s, 2H), 8.07- 8.18 (m, 2H), 8.50 (d, J = 5.7, 1H), 8.72 (s, 1H), 8.74-8.80 (m, 1H), 9.50 ( s, 1H); MS (HPLC / ES) 483.06 m / z = (M + 1).

Example 2: Preparation of 4 {4- [3- (4-chloro-3-trifluoromethylphenyl) ureido] -3-fluorophenoxy} pyridine-2-carboxylic acid methylamide hydrochloride [0126] The compound of Example 1 as the free base (2.0 g) dissolved in anhydrous tetrahydrofuran (15 ml) and 4M HCl / dioxane (excess) added. The solution was then concentrated under reduced pressure to obtain 2.32 g of an off-white solid. The crude salt was dissolved in hot ethanol (125 mL), activated charcoal was added and the mixture was heated at reflux for 15 minutes. The hot suspension was filtered through a celite pad 521 and allowed to cool to room temperature. The flask was placed in the freezer overnight. The crystalline solid was collected by suction filtration, washed with ethanol followed by hexane and air dried. The mother liquor was concentrated and crystallized (in the freezer) overnight. The second crop of solid was collected and combined with the first. The colorless salt was dried in a vacuum dryer at 60 ° C for two days. 1.72 g (79%) of the hydrochloride were obtained. Melting point: 215 ° C

Elemental analysis:

<td></td><td>calculated</td><td>It was found</td>

<td>C</td><td> 48,57</td><td> 48,68</td>

<td>H</td><td> 3,11</td><td> 2,76</td>

<td>N</td><td> 10,79</td><td> 10,60</td>

<td>cl</td><td> 13,65</td><td> 13,63</td>

<td>F</td><td> 14,63</td><td> 14,88</td>

Example 3: Preparation of 4- {4- [3- (4-chloro-3-trifluoromethylphenyl) ureido] -3-fluorophenoxy} pyridine-2-carboxylic acid methylamide mesylate [0127] The compound of example 1 as the free base (2.25 g) dissolved in ethanol (100 ml) and methanesulfonic acid stock solution (excess) added. The solution was then concentrated under reduced pressure to give a yellow oil. Ethanol was added and concentration repeated to give 2.41 g of an off-white solid. The crude salt was dissolved in hot ethanol (~ 125 mL) and then slowly cooled to crystallize. After reaching room temperature, the flask was placed in the freezer overnight. The colorless crystalline material was collected by suction filtration; the filter cake was washed with ethanol and then with hexane and air dried to give 2.05 g of substance which was dried in a vacuum oven at 60 ° C overnight. Melting point: 231 ° C

Elemental analysis:

<td></td><td>calculated</td><td>It was found</td>

<td>C</td><td> 45,64</td><td> 45,34</td>

<td>H</td><td> 3,31</td><td> 3,08</td>

<td>N</td><td> 9,68</td><td> 9,44</td>

<td>cl</td><td> 6,12</td><td> 6,08</td>

<td>F</td><td> 13,13</td><td> 13,42</td>

<td>S</td><td> 5,54</td><td> 5,59</td>

Example 4: Preparation of 4 {4- [3- (4-chloro-3-trifluoromethylphenyl) ureido] -3-fluorophenoxy} pyridine-2-carboxylic acid methylamide phenyl sulfonate [0128] The compound of Example 1 as the free base (2.25 g ) was suspended in ethanol (50 ml) and benzenesulfonic acid (0.737 g) in ethanol (50 ml) was added. The mixture was heated with vigorous stirring. All solid dissolved and a reddish solution was obtained. The solution was allowed to cool to room temperature and the flask was scratched. Crystal formation was difficult to achieve, some seeding material was found, added to the solution and placed in the freezer overnight. A grayish brown solid formed in the flask; which was broken up and collected by suction filtration.

The solid was washed with ethanol followed by hexane and air dried. Weighted product: 2.05 g, 69% yield. Melting point: 213 ° C

Elemental analysis:

<td></td><td>calculated</td><td>It was found</td>

<td>C</td><td> 50,59</td><td> 50,24</td>

<td>H</td><td> 3,30</td><td> 3,50</td>

<td>N</td><td> 8,74</td><td> 8,54</td>

<td>F</td><td> 11,86</td><td> 11,79</td>

<td>cl</td><td> 5,53</td><td> 5,63</td>

<td>S</td><td> 5,00</td><td> 5,16</td>

Example 5: c-raf biochemical assay (raf-1) [0129] The c-raf biochemical assay was performed with the c-raf enzyme that was activated (phosphorylated) by Lck kinase. Activated Lck c-raf (Lck / c-raf) was produced in Sf9 insect cells by cell co-infection with baculoviruses expressing, under the control of the polyhedrin promoter, GST-c-raf (from amino acid 302 to amino acid 648) and Lck (full length). Both baculoviruses were used at 2.5 times the infection and the cells were harvested 48 hours after infection.

[0130] MEK-1 protein was produced in Sf9 insect cells by infection of the cells with baculovirus expressing the GST-MEK-1 fusion protein (full length) at an infection rate of 5 and harvesting cells 48 hours after infection. A similar purification procedure was used for GST-c-raf 302-648 and GST-MEK-1.

Transfected cells were suspended at 100 mg wet cell biomass per ml in buffer containing 10 mM sodium phosphate, 140 mM sodium chloride pH 7.3, 0.5% Triton X-100 and a protease inhibitor cocktail. Cells were broken in a homogenizer

Polytron and centrifuged at 30,000g for 30 minutes. The 30000g supernatant was given on GSHSepharose. The resin was washed with buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100. GST-labeled proteins were eluted with a solution containing 100 mM glutathione, 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100. The purified proteins were dialyzed into a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl and 20% glycerol.

[0131] Test compounds were serially diluted in DMSO using three-fold dilutions to base concentrations typically ranging from 50 μΜ to 20 nM (final test concentrations range from 1 μM to 0.4 nM). The biochemical c-Raf assay was performed as an assay on a radioactive filtermat in 96-well Costar polypropylene plates (Costar 3365). 75 μΐ of a solution containing 50 mM HEPES pH 7.5, 70 mM NaCl, 80 ng Lck / c-raf and 1 μg MEK-1 were placed on the plates. 2 μl of serially diluted individual compounds were then added to the reaction mixture before the addition of ATP. The reaction was started with 25 μl ATP solution containing 5 μΜ ATP and 0.3 LiCi [33P] -ATP. Plates were sealed and incubated at 32 ° C for 1 hour. The reaction was stopped by adding 50 μl 4% phosphoric acid and the product was collected on P30 filtermat mats (PerkinElmer) using a Wallac Tomtec Harvester. The filtermat filters were washed first with 1% phosphoric acid and then with deionized H₂A. Filters were dried in a microwave, soaked in scintillation fluid and read on a Wallac 1205 Betaplate Counter (Wallac Inc., Atlanta, GA, USA). Results are expressed as percent inhibition.

% braking = [100 - (T_b / T_and)] X100 where

T_b = (number of counts per minute with inhibitor) - (background)

T_and = (counts per minute without inhibitor) - (background) [0132] The compound of the present invention shows strong raf kinase inhibition in this assay.

Example 6: In vitro p38 kinase assay [0133] Purified and labeled His p38 α2 (expressed in E. Coli) was activated in vitro with MMK-6 for high specific activity. Using the microplate, all reactions were carried out in a 100 μL volume with reagents diluted to give 0.05 μg / well of activated p38 α2 and 10 μg / well of primary myelin protein in assay buffer (25 mM HEPES 7.4, 20 mM MgCl₂, 150 mM NaCl). Test compounds (5 μl of a 10% solution of DMSO in water) were prepared and diluted for testing to cover a range of final concentrations from 5 nM to 2.5 μΜ. The kinase assay was started by adding 25 μl ATP cocktail to a final concentration of 10 μΜ cold ATP and 0.2 LiCi [gam25 ma-³³P] ATP per well (200-400 dpm / pmol ATP). The plate was incubated at 32 ° C for 35 min. and the reaction was stopped with 7 μl of a 1 N aqueous HCl solution. Samples were collected on a P30 Filtermat (Wallac, Inc.) using TomTec 1295 Harvester (Wallac, Inc.), and counted in a LKB 1205 Betaplate Liquid Scintillation Counter (Wallac, Inc.). Negative controls included substrate plus only ATP. SW1353 cellular assay: cells

SW1353 (human chondrosarcoma) was seeded (1000 cells / 100 μl DMEM 10%

FCS / well) into 96-well plates and incubated overnight. After media exchange, the cells were exposed to the test compounds for 1 hour at 37 ° C, and then human IL-1 (1 ng / ml, Endogen, Woburn, WA) and recombinant human TNFalpha (10 ng / ml) were added. Cultures were incubated for 48 hours at temperature

37 ° C, then the IL-6 values ​​in the supernatant were determined by ELISA. The compound of the present invention shows significant inhibition of p38 kinase.

Example 7: Bio-Plex Fosfo-ERK immun immunoassay.

[0134] A 96-well immunoassay from pERK using a laser flow cytometry platform (Bio-Rad) was set to measure inhibition of basal pERK in a breast cancer cell line. MDA-MB-231 cells were plated at 50,000 cells per well in 96-well microtiter plates in full growth medium. To assess the effect of test compounds on basic inhibition of pERK1 / 2, the next day after plating, MDA-MB-231 cells were transferred to DMEM with 0.1% BSA and incubated with test compounds diluted 1: 3 to a final concentration of 3 μΜ to 12 nM in 0.1% DMSO. Cells were incubated with test compounds for 2 hours, washed and lysed in lysis buffer of whole Bio-Plex A cells. Samples were diluted with B 1: 1 buffer (by volume) and directly transferred to the test plate or frozen at -80 ° C until testing. 50 μΐ of diluted MDA-MB-231 cell lysates were incubated with approximately 2000 5 μm Bio-Plex beads conjugated with anti-ERK1 / 2 antibody overnight on a shaker at room temperature. The next day, a sandwich sandwich assay with biotinylated phospho-ERK1 / 2 was performed, the beads were washed 3 times during each incubation and then 50 μΐ PE-strepavidin was used as developer. Relative fluorescence units pERK1 / 2 were detected by counting 25 balls in a Bio-Plex flow cell (probe) at high sensitivity. IC50 was calculated assuming untreated cells as maximum and no cells (beads only) as a background using an Excel-based program. The compound of the present invention shows significant inhibition in this assay.

Example 8: Flk-1 biochemical assay (mouse VEGFR-2) [0135] This assay was performed in 96 well opaque plates (Costar 3915) in TR-FRET format. The reaction conditions were: 10 μΜ ATP, 25 nM poly GT-biotin, 2 nM Eu-labeled phospho-Tyr Ab, 10 nM APC, 7 nM Flk-1 (kinase domain), 1% DMSO, 50 mM HEPES pH 7.5 , 10 mM MgCl₂, 0.1 mM EDTA, 0.015% BRIJ, 0.1 mg / ml BSA, 0.1% mercaptoethanol). The reaction was initiated by adding the enzyme. The final reaction volume in each well was 100 gl. Plates were read at 615 and 665 nM on a Perkin Elmer Victor V Multilabel meter approximately 1.5-2.0 hours after the start of the reaction. The signal was calculated as the ratio: (665 nm / 615 nm) * 10,000 for each one hundred 49 days. The compound of the present invention shows significant inhibition of VEGFR2 kinase.

Example 9: FRP biochemical assay of mouse PDGFR [0136] This assay was performed in a 96 well black plate (Costar 3915). The following reagents were used: Europium labeled anti-phosphotyrosine pY20 antibody (Perand streptavidin-APC; poly GT-biotin z, and mouse PDGFR. The reaction conditions are: 1 nM mouse PDGFR combined with 20 μ z ATP, 7 nM poly GT-biotin, 1 nM pY20 antibody, 5 nM streptavidin-APC, and 1% DMSO in assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 0.015% BRIJ 35, 0.1 mg / ml BSA, 0.1% mercaptoethanol). The reaction was initiated by adding the enzyme. The final reaction volume in each well was 100 gl. After 90 minutes, the reaction was stopped by adding 10 μl / well of 5 μΜ of staurosporin. Plates were read at 615 and 665 nm on a Perkin Elmer VictorV Multilabel counter 1 hour after stopping the reaction. The signal was calculated as the ratio: (665 nm / 615 nm) * 10,000 for each well. The compound of the present invention exhibits significant inhibition of PDGFR kinase.

[0137] To obtain IC₅₀ for PDGFR and Flk-1, compounds were added before enzyme initiation. A 50-fold base plate was prepared with compounds serially diluted 1: 3 in a 50% DMSO / 50% dH2O solution. The addition of 2 g stock sample solution gave final concentrations of 10 μΜ - 4.56 nM in 1% DMSO. Data are expressed as percentage inhibition:% inhibition = 100 - ((Signal with inhibitor - background) / (Signal without inhibitor - background)) * 100 Example 10: MDA-MB231 proliferation assay [0138] Human breast cancer cells (MDA MB-231 , NCI) were grown in standard growth medium (DMEM) supplemented with 10% heat inactivated FBS at 37 ° C in 5% CO₂ (by volume) in a humidified incubator. Cells were plated at a density of 3000 cells per well in 90 g growth medium in a 96 well culture plate. To determine the value of T_0h CTG, 24 hours after plate placement, 100 g of CellTiter-Glo Luminescent Reagent (Promega) was added to each well and incubated at room temperature for 30 minutes. Luminescence was recorded with a Wallac Victor II device. Reagent

CellTiter-Glo causes cell lysis and the formation of a luminescent signal proportional to the amount of ATP present, which in turn is directly proportional to the number of cells present.

[0139] Test compounds were dissolved in 100% DMSO to obtain 10 mM stock solutions. They were then diluted 1: 400 in growth medium to give 25 μΜ working stock solutions of the test compound in 0.25% DMSO. Test compounds were serially diluted in growth medium containing 0.25% DMSO to maintain a constant DMSO concentration for all wells. 60 μΐ of diluted test compound was added to each culture well to give a final volume of 180 g. Cells with and without individual test compounds were incubated for 72 hours, and then ATP-dependent luminescence was measured as described previously to obtain T values_72h. Optionally, IC values₅₀ can be determined by a least squares analysis program using compound concentrations as a function of percentage inhibition.

% ΙκΐΙΙΙΟ ^ ΙΠΚΙ = ^{[1 - -} ) ^{/ (T}72hko_ntrole ^{- T.}0h ^{)] x 100} where

T_{72h test} = ATP-dependent luminescence after 72 hours in the presence of test compound

T_{72h ctrl} = ATP-dependent luminescence after 72 hours in the absence of test compound

T_0h = ATP-dependent zero time luminescence [0140] The compound of the present invention shows significant inhibition of proliferation in this assay.

Example 11: pPDGFR-beta ELISA sandwich assay in AoSMC cells [0141] 100K P3-P6 aortic SMC was plated in each well of a 12-well set in 1000 g / well SGM-2 using standard cell culture techniques. The next day, the cells were washed once with 1000 gL D-PBS, then cultured in serum with 500 lI. SBM (basic medium of smooth muscle cells) with

0.1% BSA overnight. Compounds were diluted in a dose range from (10 μΜ to 1 nM in 10-fold dilution steps in DMSO. Final DMSO concentration 0.1%). Old medium was removed by rapid inversion on the sink, then 100 g of each dilution was added to the appropriate cell well for 1 hour at 37 ° C. Cells were then stimulated with 10 ng / ml PDGF-BB ligand for 7 minutes at 37 ° C. The medium was decanted and 150 g of isotonic lysis buffer with a protease inhibitor tablet (complete; no EDTA) and 0.2 mM sodium vanadate were added. Cells were lysed for 15 minutes at 4 ° C on a shaker in a cool room.

Lysates were placed in Eppendorf tubes to which 15 g of agarose conjugated anti-PDGFR-beta antibody was added and incubated at 4 ° C overnight.

The next day, the beads were washed in 50 volumes of PBS three times and boiled in 1x LDS sample buffer for 5 minutes. Samples were analyzed on gels with a 3-8% Tris acetate gradient and transferred to nitrocellulose. Membranes were blocked in 1% BSA / TBS-T for 1 hour before incubation with anti-phospho-PDGFR-b antibody (Tyr-857) in blocking buffer (1: 1000 dilution) for 1 hour. After three washes with TBS-T, membranes were incubated in goat anti-rabbit HRP IgG (1: 25,000 dilution) for 1 hour. Washing was carried out three more times before the ECL substrate was added. The membranes were exposed to Hyperfilm-ECL. Membranes were then removed and re-probed with PDGFR-beta antibody for total PDGFR-beta.

Table 1 illustrates the results of in vitro biochemical kinase assays for p38 kinase, PDGFR kinase and VEGFR2 kinase. All three target kinases are involved in stromal activation and endothelial cell proliferation, leading to angiogenesis and blood supply to cancerous tissue.

Table 1

<td></td><td>mPDGFR IC50, nM</td><td>mVEGFR2 IC50, nM</td><td>p38 IC50, nM</td>

<td>Example 1</td><td> 83</td><td> 5,5</td><td> 24</td>

[0142] Table 2 illustrates the results of two cellular assays for raf kinase activity, which are (i) pERK inhibition in MDA-MB231 cells, mechanistic reflection of raf kinase activity, and (ii) MDA-MB231 cell proliferation assay, functional raf kinase assay . In addition, Table 2 illustrates the results of PDGFR-stimulated PDGFR-beta phosphorylation in aortic smooth muscle cells, which is a mechanistic reflection of PDGFR kinase inhibition.

Table 2 [0143]

<td></td><td>pERK in cells (MDAMB-231) IC50, nM</td><td>Growth (MDA-MB231) IC50, nM</td><td>pPDGFR (AoSMC) IC50, nM</td>

<td>Example 1</td><td> 22</td><td> 600</td><td> 43,6</td>

[0144] Generally, the compounds of the present invention provide a unique combination of angiogenesis inhibition and tumor cell growth. They also show an improved inhibition profile against several key target kinases such as raf, p38, PDGFR and VEGFR-2, all of which are of interest as molecular targets in the treatment of osteoporosis, inflammatory diseases and hyperplasia, including cancer.