HK1183231A - Synthetic metabolites of fluoro substituted omega-carboxyaryl diphenyl urea for the treatment and prevention diseases and conditions - Google Patents

Description

Anabolic products of fluoro-substituted omega-carboxyaryl diphenyl ureas for the treatment and prevention of diseases and disorders

Technical Field

The present invention is directed to novel compounds, pharmaceutical compositions comprising such compounds and the use of these compounds or compositions, alone or in combination with anti-cancer drugs, for the treatment of diseases and disorders mediated by aberrant VEGFR, PDGFR, raf, p38 and/or flt-3 kinase signaling.

Background

Activation of the ras signaling pathway is indicative of a cascade of events that have profound effects on cell proliferation, differentiation and transformation. Raf kinase, a downstream effector of ras, is thought to be a key mediator of these signals from cell surface receptors to reach the nucleus (Lowy, D.R.; Willumsen, B.M.Ann.Rev.biochem.1993,62,851; Bos, J.L.cancer Res.1989,49,4682). Inhibition of the effect of active ras by administering an inactivated antibody to raf kinase or by co-expressing dominant negative raf kinase or dominant negative MEK (a substrate for raf kinase) has been shown to result in the reversion of transformed cells to 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 (Nature1991, 349, 426-28) have further shown that blocking cell proliferation in membrane-associated oncogenes by antisense RNA can inhibit raf expression. Similarly, in vivo or in vitro inhibition of raf kinase (by antisense oligodeoxynucleotides) has also been linked to inhibition of growth of various human tumor types (Monia et al, nat. Med.1996,2,668-75).

To support more than 1-2mm³Progressive tumor growth in size, recognizing that tumor cells require a functional stroma, consisting of fibroblastsCell, smooth muscle cell, endothelial cell, extracellular matrix proteins and soluble factors (Folkman, j., semin. oncol.2002.29(6 Suppl 16), 15-8). Tumors induce the formation of stromal tissue by secreting soluble growth factors, such as PDGF and transforming growth factor-beta (TGF- β), which stimulate host cells to secrete complementing factors, such as Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), and Vascular Endothelial Growth Factor (VEGF). These stimulatory factors induce the formation of new blood vessels (or angiogenesis), which carry oxygen and nutrients into the tumor and allow it to grow and provide a metastatic pathway. It is believed that some therapies for inhibiting mesenchymal formation will inhibit the growth of epithelial tumors of various histological types (George, d.semin. oncol.2001.28(5 Suppl 17),27-33; Shaheen, r.m., et al, Cancer res.2001,61(4),1464-8; Shaheen, r.m., et al, Cancer res.1999,59(21), 5412-6). However, drugs targeting a single pathway may have limited efficacy due to the complex nature and multiple growth factors involved in the angiogenic process and tumor progression. There is a need to provide treatments for a variety of critical signaling pathways utilized by tumors to induce angiogenesis in the host stroma. These include PDGF (a potent stimulator of mesenchymal formation) (Ostman, a.and c.h.hellin, adv.cancer res.2001,80, 1-38), FGF (a chemoattractant and mitogen for fibroblasts and endothelial cells) and VEGF (a potent modulator of vascularization).

PDGF is a key regulator of mesenchyme formation, which is secreted by many tumors in a paracrine fashion and is thought to promote the growth of fibroblasts, smooth muscle cells and endothelial cells, promoting mesenchyme formation and angiogenesis. PDGF was originally identified as the v-sis oncogene product of the simian sarcoma virus (Heldin, c.h., et al, j.cell.sci.suppl.1985,3, 65-76). The growth factor consists of two peptide chains, called a or B chains, whose main amino acid sequence has 60% homology. The chains are cross-linked by disulfide bonds to form a 30kDa mature protein consisting of AA, BB or AB homo-or heterodimers. PDGF is found at high levels in platelets and is expressed by endothelial cells and vascular smooth muscle cells. In addition, PDGF production is upregulated under hypoxic conditions (Kourembanas, s., et al., Kidney int.1997,51(2), 438-43), such as those found in poorly vascularized tumor tissues. PDGF binds with high affinity to the PDGF receptor, a 1106 amino acid transmembrane tyrosine kinase receptor with a molecular weight of 124kDa (hellin, c.h., a.ostman, andl.ronnstrand, biochim.biophysis.acta 1998,1378(1), 79-113). PDGFR has been found to be a homodimeric or heterodimeric chain with 30% homology over its entire amino acid sequence and 64% homology between its kinase domains (helldin, c.h., et al. PDGFR is a member of the family of tyrosine kinase receptors with split-kinase domains, including VEGFR-2 (KDR), VEGFR-3 (flt-4), c-kit and flt-3. The PDGF receptor is expressed primarily in fibroblasts, smooth muscle cells, pericytes and to a lesser extent in neurons of the central nervous system, mesangial cells, lesch cells and schwann cells. Upon binding to the receptor, PDGF induces receptor dimerization and undergoes autophosphorylation and transphosphorylation of tyrosine residues (which increases the kinase activity of the receptor) and promotes recruitment of downstream effectors through activation of the SH2 protein binding domain. A number of signaling molecules form complexes with activated PDGFR, including PI-3 kinase, phospholipase C-. gamma., src, and GAP (GTPase activating protein for p 21-ras) (Soskic, V., et al. biochemistry 1999,38(6), 1757-64). PDGF activates the Rho signaling pathway that induces cellular movement and migration through activation of PI-3 kinase, and mitogenesis through activation of the p21-ras and MAPK signaling pathways through activation of GAP.

In adults, the primary function of PDGF is believed to be to promote and increase the rate of Wound healing and maintain vascular homeostasis (Baker, e.a. and d.j.leaper, Wound repairregen.2000,8(5),392-8, and Yu, j., a.moon, and h.r.kim, biochem. biophysis. res. commu.2001, 282(3), 697-. 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 also known to help maintain vascular homeostasis. During the development of new blood vessels, PDGF recruits pericytes and smooth muscle cells required for vascular structural integrity. PDGF is thought to play a similar role in tumor neovascularization. As part of its role in angiogenesis, PDGF controls interstitial fluid pressure, regulating vascular permeability through its regulation of the interaction between connective tissue cells and the extracellular matrix. Inhibition of PDGFR activity reduces interstitial pressure and promotes cytotoxic influx into 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).

PDGF can promote tumor growth directly through paracrine or autocrine stimulation of PDGFR receptors on stromal or tumor cells, or by expanding the receptors or activating the receptors using recombination. Overexpression of PDGF is likely to transform human melanoma cells and keratinocytes by the direct effect of PDGF on the induction of mesenchyme formation and angiogenesis-two cell types that do not express the PDGF receptor (Forsberg, K., et al. Proc. Natl. Acad. Sci. USA.1993,90(2),393-7; Skobe, M.and N.E.Fusenig, Proc. Natl. Acad. Sci. USA.1998,95(3), 1050-5). This paracrine stimulation of tumor stroma has also been observed in colon, 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, J.M., et al, Cancer Res.1994,54(20), 5455-63), where the tumor expresses PDGF but does not express a receptor. Autocrine stimulation of tumor cell growth, most of which analyzed tumors express ligand PDGF and the receptor, has been reported in glioblastomas (Fleming, t.p., et al. Cancer res.1992,52(16), 4550-3), soft tissue sarcomas (Wang, j., m.d. coltrera, and a.m.go, Cancer res.1994,54(2), 560-4), ovarian cancers (Henriksen, r., et al. Cancer res.1993,53(19), 4550-4), prostate cancers (Fudge, k., c.y. Wang, and m.e.stears, mod.pathol.1994,7 (5)), pancreatic cancers (Funa, k., et al. Cancer res.1990,50(3), 53-53) and lung cancers (antoniads, h.n., ac., nat. 549, 7 (89), nat. 89, usa), et al. 3989 (usa). A small number of such receptors have been found to be non-ligand dependent activated, but have been reported in chronic myelogenous leukemia (CMML), where a chromosomal translocation event forms a fusion protein between the Ets-like transcription factor TEL and the PDGF receptor. In addition, activating mutations in PDGFR have been found in gastrointestinal stromal tumors, where activation of c-kit is not involved (Heinrich, m.c., et al, Science 2003,9, 9).

Another major regulator of angiogenesis and vasculogenesis (vasculogenesis) in embryonic development and in some angiogenesis-dependent diseases is vascular endothelial growth factor (VEGF; also known as vascular permeability factor, VPF). VEGF represents a family of subtypes of mitogens that exist as homodimers due to alternative RNA splicing. The VEGF subtype is highly specific for vascular endothelial cells (for a review see: Farrara et al Endocr. Rev.1992,13,18; Neufield et al FASEB J.1999,13, 9).

VEGF expression is induced by hypoxia (Shweiki et al Nature 1992,359,843) and also by various cytokines and growth factors, such as interleukin-1, interleukin-6, epidermal growth factor and transforming growth factor. To date, VEGF and VEGF family members have been reported to bind to one or more of the three transmembrane receptor tyrosine kinases (Mustonenet al. J. cell biol.1995,129, 895), including VEGF receptor-1 (also known as flt-1 (fms-like tyrosine kinase-1)), VEGFR-2 (also known as a receptor containing kinase insert (KDR); mouse analogs of VEGFR-2 are 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). VEGFR-2 therefore undergoes strong ligand-dependent tyrosine phosphorylation in intact cells, whereas flt-1 shows a weaker response. Therefore, binding to VEGFR-2 is considered to be a key requirement for the induction of all VEGF-mediated biological responses.

In vivo, VEGF plays a major role in angiogenesis and induces angiogenesis and permeabilization of blood vessels. Dysregulated expression of VEGF leads to the development of a number of diseases characterized by abnormal angiogenic and/or high permeability processes. It is believed that modulation of the VEGF-mediated signaling cascade with certain agents may provide useful control of aberrant angiogenesis and/or high permeability processes. Tumorigenic cells within the hypoxic region of a tumor respond by stimulating VEGF production, which causes activation of quiescent endothelial cells to stimulate the formation of new blood vessels (Shweiki et al proc.nat' l.acad.sci.1995,92,768). Furthermore, VEGF production in tumor regions without angiogenesis can occur through the ras signaling pathway (Grugelet al.J.biol.chem.1995,270,25915; Rak et al.cancer Res.1995,55,4575). VEGF mRNA has been shown to be strongly upregulated in situ hybridization studies in a variety of human tumors including lung Cancer (Matern et al Br. J. Cancer 1996,73, 931), thyroid Cancer (Viglietto et al oncogene 1995,11, 1569), breast Cancer (Brown et al human Pathol.1995,26, 86), gastrointestinal Cancer (Brown et al Cancer Res.1993,53,4727; Suzuki et al Cancer Res.1996,56,3004), renal and bladder Cancer (Brown et al am. J. Pathol.1993,143I, 1255), ovarian Cancer (Olson et al Cancer Res.1994,54,1255) and cervical Cancer (Guidi et al J. Nat' Cancer Inst.1995,87,12137), as well as angiosarcoma (Hashimoto et al Lab. 1995, enc. 32. enc. J. et al. J. 387. enc. et al. J. et al., EP J. 3864, et al J. 1985; tumor enc. J. et al. J. 387.) and a few enc. J. Olymp. J. et al. J. 387. A tumor (tumor) 2, EP. Olymp. J. et al., EP, 1993). Neutralizing mabs to VEGFR-2 have been shown to effectively block tumor angiogenesis (Kim et al Nature 1993,362,841; Rockwell et al mol.cell.Differ.1995,3,315).

Overexpression of VEGF, for example in the case of extreme hypoxia, can lead to intraocular angiogenesis, to hyperproliferation of blood vessels and ultimately to blindness. Such a cascade of events has been observed in a number of retinopathies, including diabetic retinopathy, ischemic retinal vein occlusion, retinopathy of prematurity (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. opthalmolol. vis. sci.1996,37,855).

In Rheumatoid Arthritis (RA), pannus ingrowth may be mediated by the production of angiogenic factors. Immunoreactive VEGF levels are higher in the synovial fluid of patients with RA, while VEGF levels are lower in the synovial fluid of patients with other forms of arthritis or degenerative joint disease (Koch et al J. Immunol.1994,152, 4149). The angiogenesis inhibitor AGM-170 has been shown to prevent neovascularization of the joints in a rat collagen arthritis model (Peacock et al.J.Exper. Med.1992,175, 1135).

Increased VEGF expression has also been demonstrated in psoriatic skin and in bullous diseases associated with epidermal blister formation, such as bullous pemphigoid, erythema multiforme, and dermatitis herpetiformis (Brown et al j. invest. dermotol.1995, 104, 744).

Vascular endothelial growth factors (VEGF, VEGF-C, VEGF-D) and their receptors (VEGFR-2, VEGFR-3) are not only key regulators of tumor angiogenesis, but also of lymphangiogenesis. VEGF, VEGF-C and VEGF-D are expressed in most tumors, mainly during tumor growth and often at significantly increased levels. VEGF expression is triggered by hypoxia, cytokines, oncogenes such as ras, or by inactivation of tumor suppressor genes (McMahon, G.Oncologenist 2000,5(suppl.1),3-10; McDonald, N.Q.; Hendrickson, W.A. cell 1993,73, 421-.

The biological activity of VEGF is mediated by binding to its receptor. VEGFR-3 (also known as flt-4) is predominantly expressed in the lymphatic endothelium of normal adult tissues. The function of VEGFR-3 is required for the formation of new lymphatic vessels, but not for the maintenance of existing ones. VEGFR-3 is also upregulated in the vascular endothelium in tumors. Recently, VEGF-C and VEGF-D, ligands for VEGFR-3, have been identified as modulators of lymphangiogenesis in mammals. Lymphangiogenesis induced by tumor-associated lymphangiogenic factors promotes the growth of new blood vessels into the tumor, allowing tumor cells to access the systemic circulation. Cells that invade the lymphatic vessels can enter the blood stream via the thoracic duct. Tumor expression studies have allowed a direct comparison of the expression of VEGF-C, VEGF-D and VEGFR-3 with the patho-clinical factors directly associated with the ability of primary tumors to spread (e.g., lymph node metastasis, lymphatic infiltration, secondary metastasis, disease-free survival). In many cases, these studies show a statistical correlation between the expression of lymphopoietic factors and the metastatic capacity of primary solid tumors (Skobe, M.et al. Nature Med.2001,7(2),192-198; Stacker, S.A.et al. Nature Med.2001,7(2),186-191; Makinen, T.et al. Nature Med.2001,7(2),199-205; Maniota, S.J.et al. EMBO J.2001,20(4), (672-82; Karpanen, T.et al. cancer Res.2001,61(5), (1786-90; Kudbo, H.et al. blood 2000,96(2), 546-53).

Hypoxia appears to be an important stimulus for VEGF production in malignant cells. Activation of p38 MAP kinase is required for VEGF induction by tumor cells in response to hypoxia (Blsache, F.et al. biochem. Biophys. Res. Commun.2002,296,890-896; Shemirani, B.et al. oral Oncology 2002,38, 251-257). In addition to participating in angiogenesis by modulating VEGF secretion, p38 MAP kinase also promotes malignant cell invasion and migration of different tumor types by modulating collagenase activity and expression of the urokinase plasminogen activator (Laferriere, J.et al.J.biol.chem.2001,276, 33762-33772; Wemarsterck, 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).

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. The mitogen-activated protein (MAP) kinase p38 is involved in the IL-1 and TNF signaling pathways (Lee, J.C.; Laydon, J.T.; McDonnell, P.C.; Gallagher, T.F.; Kumar, S.; Green, D.; McNulty, D.; Blumenhal, M.J., Heys, J.R.; Landvatter, S.W.; Stricker, J.E.; McLaughlin, M.M.; Siemens, I.R.; Fisher, S.M.; Livi, G.P.; White, J.R.; Adams, J.L.; Ynd, P.R.Nature 1994,372,739).

Clinical studies have linked Tumor Necrosis Factor (TNF) production and/or signaling to a number of diseases, including rheumatoid arthritis (maini.j. royal gel. physics london 1996,30, 344). In addition, excessive levels of TNF have been implicated in a variety of inflammatory and/or immunomodulatory diseases, including acute rheumatic fever (Yeast et al Lancet 1997,349,170), bone resorption (Pacific et al J. Clin. Endocrinol. Metabol.1997,82, 29), postmenopausal osteoporosis (Pacific et al J. bone Mineral Res.1996,11,1043), sepsis (Blackwell et al Br. J. Anaeth. 1996,77,110), septic sepsis (Deschell 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), endotoxin shock (Beutler et al science 1985,229,869; Mahe. Proc. Nature. Sci. Natl. Sci. Sa. J.1988, 319, 76), inflammatory bowel disease (inflammatory bowel syndrome, USA. 15. 10. inflammatory bowel syndrome, USA. 10. inflammatory bowel syndrome (Chronic disease, USA. 10. 19813, 19847, Salmon et al, Salmon. (inflammatory bowel disease), 46,111)), Heres Hiemer's reaction (Jarisch-Herxheimer reactions) (Fekade et al.New England J.Med.1996,335, 311), asthma (Amani et al.Rev.Malad.Respir.1996,13,539), adult respiratory distress syndrome (Roten et al.Rev.Respir.Dis.1991, 143,590; Suter et al.Am.Rev.Respir.Dis.1992,145, 1016), acute pulmonary fibrosis (Pan et al.Pathol.int.1996,46, 91), pulmonary sarcoidosis (Ishiokait. Sarcois Vasculus diffiulu.1996, 13,139), allergic respiratory system disease (Casale et al.J.Respir.Biol.35, thermal Lung disease (Gondrome. 1997, 9. acute respiratory disease, 19, acute Lung disease, 14, 75, 7, 19, acute Lung disease, malaria, 9, 35, 9, acute Lung disease, 9, acute Lung disease, 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 et al.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), post-cardiac injury (Malkinea. mol. med. Today 1996,2, 336), atherosclerosis (Parums. J.Pathol.1996,179, A46), Alzheimer's disease (Fagarsan. in Res.1996, 231; ai Gerono logy et al, 43,143, encephalitis, acute encephalitis, et al.1997, acute brain trauma (acute brain trauma, 2, biomedical, 198, 22, biomedical, 198, 2,1, 198, biomedical, 2, biomedical, 198, biomedical, 14,2, 1,2, and acute brain trauma, 1,2, 1,2, 1,2, 1,2, 1,7, 2,1, 7,1, 2,16, 31), pancreatitis (alley et al gut 1992,33, 1126) (including systemic complications in acute pancreatitis (McKay et al br. j. surg.1996,83,919)), poor wound healing in infection, inflammation and cancer (Buck et al am. j. pathol.1996,149, 195), myelodysplastic syndrome (Raza et al int. j. healol. 1996,63,265), systemic lupus erythematosus (male et al arthritis rheum.1989,32,146), biliary cirrhosis (Miller et al am. j. gastroenterology.1992, 87,465), intestinal necrosis (Sun et al j. clin. invest.1988,81,1328), psoriasis (christophers. str.163j. dermotol.1996, 37, S4), radiation injury (radiation injury j. gul. dlich. J.1988, 3) and monoclonal antibody administration after surgery, 6746, in example, monoclonal antibody 6746, 1996). Levels of TNF have also been associated with host anti-graft responses (Piguet et al, immunol.ser.1992,56,409), including ischemia reperfusion injury (Colletti et al, j. clin.invest.1989,85,1333) and allograft rejection (including those in kidney (heart et al, j. exp. med.1987,166, 1132), liver (Imagawa et al, transplantation 1990,50, 219), heart (balling et al, transplantation 1992,53, 283) and skin (Stevens et al, transplantation. proc.1990,22,1924)), lung allograft rejection (Grossman et al, immunol. allergy clin.n.am.1989,9,153) (including lung chronic allograft rejection (lochiatus bronchitis; lochia. j. odoral. 1990, total joint replacement, 35) and hip replacement resulting from hip replacement, 2.35). TNF is also associated with infectious diseases (reviewed: Beutler et al, Crit. Care Med.1993,21,5423; Degre. biotherapy 1996,8, 219), including tuberculosis (Rook et al. Med. inlay. infection. 1996,26,904), helicobacter pylori infection during peptic ulcer disease (Belles et al. gastroenterology 1997,112,136), Czochralski disease caused by Trypanosoma cruzi infection (Chandraseka et al. biochem. Biophys.Res. Commun.1996,223, 365), shiga-like toxin effect caused by E.coli infection (Harel et al. J.Clin. Invest.1992,56, 40), enterotoxin A effect caused by staphylococcal infection (Fisher et al. J.Imnol.1990, 144, 4663), meningococcal infection (Wature. Lance et al. 1987; Staphylococcus infection; Osbec. J.1987, 1987. 1987; Ki. Marseki virus infection, 76, Marseki infection, Marseki virus infection (Biofel. Res. J. Res. 144, 369. Marsein. J. infection, 369. 1987, 19, Marseira. infection, Marseira. J. infection, Marseira. infection, Sw. 1987, Marseira. Marseh infection, 19, Marseira. Marseh infection (SEQ. Marseh. 1987, Marseh. Marseh infection), 87,1490), Taylor encephalomyelitis virus (Sierra et al. immunology 1993,78, 399) and human immunodeficiency virus (HIV; Poli.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).

Many diseases are thought to be mediated by excessive or unwanted activity of matrix-destructive metalloproteinases (MMPs) or by an imbalance in the ratio between MMPs and Tissue Inhibitors of Metalloproteinases (TIMPs). These include osteoarthritis (Woesner et al J. biol. chem.1984,259, 3633), rheumatoid arthritis (Mullins et al Biochim. Biophys. acta 1983,695,117; Woolley et al Arthrotis Rheum.1977,20,1231; Gravallese et al Arthrotis Rheum.1991,34,1076), septic arthritis (Williams et al Arthrotis Rheum.1990,33,533), tumor metastasis (Reich et al cancer Res.1988,48,3307; Matrisian et al Proc. Nat. Acad. Sci., USA 1986,83, 9413), periodontal disease (Overall et al J. iodotals. 1987,22, 81), corneal ulceration (Burma. Burm. Thyss. 1986,83, 9413), periodontal disease (Bursa et al J. Perolor et al J. 1987,22, 81), thrombotic lesions (Bursa et al J. Threels et al J. 1984, 1989), thrombotic lesions of the aorta (Bureau. J. 1989), thrombotic lesions of the aorta et al (intravascular lesion. 1981, Klotch. 1988), thrombotic lesion; Thromboplastic arterial thrombosis, Kleine et al (Threon. 1989, Kleinem et al (Thromb. 1986, 2. 1988), thrombotic lesions of the atherosclerotic lesion), thrombotic lesions of the arterial thrombosis of the human aortic system (thrombosis of the arterial thrombosis of the human, 62,8, III), thrombotic system, III), thrombotic system, III, Temporomandibular joint disease and demyelinating diseases of the nervous system (Chantryet al.j. neurochem.1988,50,688).

Because inhibition of p38 results in inhibition of TNF production and MMP production, it is believed that inhibition of the mitogen-activated protein (MAP) kinase p38 enzyme may provide a method of treating the diseases listed above, including osteoporosis and inflammatory disorders such as rheumatoid arthritis and Chronic Obstructive Pulmonary Disease (COPD) (Badger, A.M.; Bradbeer, J.N.; Votta, B.; Lee, J.C.; Adams, J.L.; Griswold, D.E.J.Pharm.Exper.Ther.1996,279, 1453).

Hypoxia appears to be an important stimulus for VEGF production in malignant cells. Activation of p38 MAP kinase is required for VEGF induction by tumor cells in response to hypoxia (Blachke, 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 through regulation of VEGF secretion, p38 MAP kinase also promotes malignant cell invasion and migration of different tumor types by regulating collagenase activity and expression of the 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 is also expected to affect tumor growth by interfering with the signaling cascade associated with angiogenesis and malignant cell invasion.

Certain ureas have been described as having activity as inhibitors of serine-threonine kinases and/or tyrosine kinases. In particular, the use of certain ureas as active ingredients in pharmaceutical compositions for the treatment of cancer, angiogenic diseases, inflammatory disorders has been demonstrated.

For cancer and angiogenesis, see:

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，93rdAACR 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.

for p38 mediated diseases, including inflammatory disorders, see:

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 J.B.，Book of Abstracts，Protein Kinases：Novel Target Identification andValidation 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.

omega-carboxyaryl diphenylureas are disclosed in WO00/42012 (published on 7/20.2000);

WO00/41698 (published on 7/20/2000); the following published U.S. applications:

US2002-0165394-A1, published 11/7/2002,

US2001-003447-A1, published 2001, 10 months and 25 days,

US2001-0016659-A1, published on 8/23/2001,

US2002-013774-A1, published in 2002, 9/26,

and pending U.S. applications:

09/758,547, application date 2001, 1/12,

09/889,227, application date 2001, 7/12,

09/993,647, application date 2001, 11/27,

10/042,203, App. Pup. No. 2002,1, 11, and

10/071,248, application date 2002, 2/11,

disclosure of Invention

The following anabolic products of omega-carboxy aryl diphenylureas of formula I have been found to be potent inhibitors of raf kinase, VEGFR kinase, p38 kinase and PDGFR kinase, which are all molecular targets of interest for the treatment and prevention of osteoporosis, inflammatory disorders, hyperproliferative disorders and angiogenic disorders, including cancer.

The present invention provides, for example,

(i) novel anabolic products of said compounds of formula (I), salts thereof, prodrugs thereof and metabolites thereof,

(ii) pharmaceutical compositions comprising said compounds, and

(iii) use of the anabolic products or compositions as a sole agent or in combination with cytotoxic therapy for the treatment of diseases and disorders mediated by raf, VEGFR, PDGFR, flt-3 and p 38.

The compounds of formula I, salts thereof, prodrugs thereof, and metabolites thereof described below are collectively referred to as "compounds of the invention". Formula I is as follows:

the anabolic products of the compounds of the present invention include oxidized derivatives of formula I, wherein one or more urea nitrogens are substituted with a hydroxyl group. The anabolic products of the compounds of the present invention also include analogs wherein the methylamide group of the compound of formula I is hydroxylated and then demethylated by metabolic degradation. The anabolic products of the compounds of the present invention further include oxidized derivatives in which the pyridine nitrogen atom is in the form of an N-oxide (e.g., bearing a hydroxyl substituent), forming these structures referred to in the art as 1-oxo-pyridine and 1-hydroxy-pyridine.

The plural forms of the words compounds, salts, and the like used herein are also taken to mean a single compound, salt, and the like.

In particular, the invention relates to synthetic forms of the M2, M3, M4 and M5 metabolites of said compound of formula I, the structures of which are shown below:

the relationship of the above mentioned anabolic products to the parent compound of formula I is illustrated in figure 1.

The M2 and M5 metabolites of the compounds of formula I are particularly preferred.

Metabolite M-2

Metabolite M-5

As an embodiment of the invention, the biotransformation of the compound of formula I was studied in vitro with liver microsomes and hepatocytes, and in vivo in the plasma of some species. In humans, the N-oxide (M-2) and the demethylated N-oxide (M-5) are of great significance. Both metabolites in synthetic form showed steady state systemic exposure (area under the curve [ AUC ], mg x h/L) similar to regorafenib (parent compound) in patients in phase I studies with doses of 160mg taken at 3 weeks/1 week off.

The pharmacological activity of the anabolic products of the compounds of the formula I, in particular the M-2 and M-5 metabolites, is also of interest. In vitro, biochemical and cellular kinase phosphorylation assays indicate that the anabolic products of the compounds of formula I are broad-spectrum kinase inhibitors. In preclinical models in vivo, the metabolites are effective against tumor growth and further inhibit tumor vasculature. The anabolic products also show an acute effect on Vascular Endothelial Growth Factor (VEGF) -induced hypotension in a pharmacodynamic rat model.

The term "synthetic" is art-recognized and refers to production by in vitro chemical or enzymatic synthesis.

The term "isolated" as used herein means that the referenced material is removed from its natural environment (e.g., cells or tissues) or from the body. Thus, an isolated metabolite may not contain some or all of the cellular components in which the native material is naturally present (e.g., cytoplasmic or membrane components, such as microsomes).

The term "purified" as used herein refers to a material that has been separated under conditions that reduce or eliminate the presence of an unrelated material (i.e., a contaminant), including precursor materials from which the material was obtained. For example, the purified metabolite is preferably substantially free of other metabolites or precursor compounds from which the purified metabolite is derived. The term "substantially free" as used herein may be used in the context of an analytical test for the substance in question. Preferably, the purified material that is substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, more preferably at least 99% pure. Purity can be assessed by chromatography, gel electrophoresis, HPLC, NMR or mass spectrometry, compositional analysis, bioassay, and other methods known in the art.

Salt (salt)

Pharmaceutically acceptable salts of said anabolic products of formula I are also within the scope of the present invention. The term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic or organic acid addition salts of the compounds of the present invention. See, e.g., s.m. berge, et al, "Pharmaceutical Salts," j.pharm. sci.1977,66, 1-19.

Representative salts of the compounds of the present invention include conventional non-toxic salts, such as those obtained from inorganic or organic acids by methods well known in the art. For example, such acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cinnamate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate, maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectate (pectinate), persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfonate, pivalate, fumarate, picrate, etc, Tartrate, thiocyanate, tosylate and undecanoate salts.

The salts or prodrugs of the compounds of formula I may contain one or more asymmetric centers. The asymmetric carbon atoms may be present in the (R) or (S) configuration or the (R, S) configuration. Substituents on the ring may also be present in cis or trans form. All such configurations (including enantiomers and diastereomers) are intended to be included within the scope of the present invention. Preferred isomers are those having a configuration that produces more of the desired biological activity. Isolated, pure or partially purified isomers or racemic mixtures of the compounds of the present invention are also included within the scope of the present invention. Purification of the isomers and separation of the isomeric mixtures may be accomplished by standard techniques known in the art.

The specific method utilized in the preparation of the anabolic products used in the embodiments of the present invention is described in example 4. The salt forms of the anabolic products of formula (I) are described in the examples.

Application method

The present invention provides compounds that modulate one or more signal transduction pathways involving raf, VEGFR, PDGFR, p38 and/or flt-3 kinase. Raf is an important signaling molecule involved in the regulation of many key cellular processes, including cell growth, cell survival and invasion. It is a member of the Ras/raf/MEK/ERK pathway. This pathway is present in most tumor cells. VEGFR, PDGFR and flt-3 are transmembrane receptor molecules that, when stimulated by appropriate ligands, trigger the Ras/raf/MEK/ERK cellular signaling pathway, leading to a cascade of cellular events. Each of said receptor molecules has tyrosine kinase activity.

The VEGFR receptors are stimulated by Vascular Endothelial Growth Factor (VEGF) and are important control points in the regulation of endothelial cell development and function. The PDGF-beta receptor regulates cell proliferation and survival in many cell types, including mesenchymal cells. Flt-3 is a receptor for FL ligand. It is similar in structure to c-kit and regulates the growth of pluripotent hematopoietic cells, affecting the development of T cells, B cells and dendritic cells.

Any gene or isoform (including wild-type and mutant forms) of raf, VEGFR, PDGFR, p38 and/or flt-3 can be modulated according to the invention. Raf or Raf-1 kinases are a family of serine/threonine kinases that include 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 invention. Activated b-raf mutations (e.g., the V599E mutant) have been identified in various cancers, including melanoma, and the compounds described herein can be utilized to inhibit their activity.

The term "modulate" means that the functional activity of the pathway (or a component thereof) is altered compared to the normal activity in the absence of the compound. Such effects include any quality or degree of modulation, including increase (increasing), promotion (agoning), increase (enriching), enhancement (enhancing), promotion, stimulation, reduction (degrading), blocking, inhibition, reduction (reducing), reduction (differentiating), antagonism, and the like.

The compounds of the invention may also modulate one or more of the following processes including, but not limited to, for example, cell growth (including, e.g., differentiation, cell survival and/or spread), tumor regression, endothelial cell growth (including, e.g., differentiation, cell survival and/or spread), angiogenesis (vasculogenesis), lymphangiogenesis (lymphangiogenesis), and/or hematopoiesis (e.g., T cell and B cell development, dendritic cell development, etc.).

Without wishing to be bound by any theory or mechanism of action, it has been found that the compounds of the present invention have the ability to modulate kinase activity. However, the methods of the invention are not limited to any particular mechanism or how the compounds achieve their therapeutic effect. The term "kinase activity" denotes a catalytic activity in which gamma-phosphate from Adenosine Triphosphate (ATP) is transferred to an amino acid residue (e.g., serine, threonine or tyrosine) in a protein substrate. The compounds can modulate kinase activity, e.g., inhibit kinase activity by directly competing with ATP for the ATP-binding pocket of the kinase, inhibit kinase activity by producing a conformational change in the enzyme structure that affects its activity (e.g., by interfering with the three-dimensional structure of the biological activity), and the like.

Kinase activity can generally be determined using conventional assay methods. Kinase assays generally include the kinase, substrate, buffer, and components of the detection system. Conventional kinase assays involve the reaction of a protein kinase with a peptide substrate and ATP (e.g., 32P-ATP) to produce a phosphorylated end product (e.g., a phosphoprotein when a peptide substrate is used). The resulting final product can be detected using any suitable method. When radioactive ATP is used, the radiolabeled phosphoprotein can be separated from unreacted γ -32P-ATP using affinity membrane or gel electrophoresis, and then visualized on a gel using autoradiography or detected using a scintillation counter. Non-radioactive methods may also be used. The methods may utilize antibodies that recognize phosphorylated substrates, such as antibodies against phosphotyrosine. For example, the kinase can be incubated with a substrate in the presence of ATP and a kinase buffer under conditions effective for the enzyme to phosphorylate the substrate. The reaction mixture can be separated, for example, electrophoretically, and then the phosphorylation of the substrate can be measured, for example, by Western blotting using an antibody against phosphotyrosine. The antibody may be labeled with a detectable label (e.g., an enzyme (e.g., HRP, avidin, or biotin), a chemiluminescent reagent, etc.). Other methods may utilize enzyme-linked immunosorbent assay (ELISA) formats, affinity membrane separation, fluorescence polarization assays, luminescence assays, and the like.

An alternative to the radioactive format is time-resolved fluorescence resonance energy transfer (TR-FRET). The method is performed after a standard kinase reaction, in which a substrate such as biotinylated poly (GluTyr) is phosphorylated by a protein kinase in the presence of ATP. The final product was then detected with europium chelate phosphate-specific antibodies (anti-phosphotyrosine or anti-phosphoserine/threonine) and streptavidin-APC which bound the biotinylated substrate. The two components are sterically linked together upon binding, and energy transfer from the phospho-specific antibody to the receptor (SA-APC) generates fluorescence readings in the same format.

The compounds of the invention may be used to treat and/or prevent any disease or disorder mediated by one or more cell signaling pathways involving raf, VEGFR, PDGFR, p38 and/or flt-3 kinases. The term "treatment" is used in a conventional manner, e.g., treatment or care of a subject for the purpose of striking (fighting), alleviating (alleviating), reducing (reliving), ameliorating a condition of a disease or disorder, etc. The compounds may also be described as useful for the treatment and/or prevention of diseases and/or conditions mediated by signaling molecules. The term "mediated" means, for example, that the signaling molecule is part of a pathway that is aberrant or disturbed in the disease and/or disorder.

Diseases and conditions that may be treated include any of the diseases mentioned above and below, as well as:

raf-associated diseases include, for example, cell proliferative disorders, cancer, tumors, and the like;

VEGFR-2 related diseases include, for example, cancer, tumor growth, inflammatory diseases, rheumatoid arthritis, retinopathy, psoriasis, glomerulonephritis, asthma, chronic bronchitis, atherosclerosis, transplant rejection, conditions involving angiogenesis, and the like;

VEGFR-3 related diseases include, for example, cancer, corneal diseases, inflamed corneas (e.g., Hamrah, am. J. Path.2003,163, 57-68), corneal transplants (Cursiefen et al, Cornea 2003,22, 273-81), lymphoproliferations, disorders involving lymphangiogenesis, and the like;

PDGFR- β associated diseases include, for example, diseases or disorders characterized by cell proliferation, cell matrix production, cell motility, and/or extracellular matrix production. Specific examples include, for example, tumors, malignancies, cancers, metastases, chronic myeloid leukemia, inflammation, kidney disease, diabetic nephropathy, mesangial proliferative glomerulonephritis, fibrotic disorders, atherosclerosis, restenosis, hypertension-associated arteriosclerosis, venous bypass graft arteriosclerosis, scleroderma, interstitial lung disease, synovial disorders, arthritis, leukemia, lymphoma, and the like;

flt-3 related diseases include, for example, immune related disorders, blood cell disorders, conditions involving hematopoietic cell development (e.g., T cells, B cells, dendritic cells), cancer, anemia, HIV, acquired immunodeficiency syndrome, and the like.

p 38-related diseases include inflammatory disorders, immunoregulatory disorders, and other disorders associated with aberrant cytokine production (particularly TNF- α) or aberrant MMP activity. These disorders include, but are not limited to, rheumatoid arthritis, Chronic Obstructive Pulmonary Disease (COPD), osteoporosis, crohn's disease, and psoriasis.

In addition, the compounds of the present invention may be used to treat conditions and disorders disclosed in U.S. patent No.6,316,479, such as glomerulosclerosis, interstitial nephritis, interstitial pulmonary fibrosis, atherosclerosis, wound scarring, and scleroderma.

The compounds of the present invention also have a wide range of therapeutic activities to treat or prevent a wide range of disease progression such as inflammatory disorders, coronary restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, inflammation, certain renal diseases associated with proliferation of mesangial cells and ocular diseases associated with retinal vessel proliferation, psoriasis, cirrhosis, diabetes, atherosclerosis, restenosis, vascular graft restenosis, in-stent restenosis, angiogenesis, ocular diseases, pulmonary fibrosis, bronchiolitis obliterans, glomerulonephritis, rheumatoid arthritis.

The present invention also provides methods for treating, preventing, modulating, etc., one or more of the following conditions in humans and/or other mammals: retinopathy, including diabetic retinopathy, ischemic retinal vein occlusion, retinopathy of prematurity, and age-related macular degeneration; rheumatoid arthritis, psoriasis or bullous diseases associated with subepidermal blister formation including bullous pemphigoid, erythema multiforme or dermatitis herpetiformis, rheumatic fever, bone resorption, postmenopausal osteoporosis, sepsis, gram negative sepsis, septic shock, endotoxic shock, toxic shock syndrome, systemic inflammatory response syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), Herslem's Himerle's reaction, asthma, adult respiratory distress syndrome, acute pulmonary fibrosis, pulmonary sarcoidosis, allergic respiratory disease, silicosis, pneumosilicosis, alveolar injury, liver failure, liver disease during acute inflammation, severe alcoholic hepatitis, malaria (Plasmodium falciparum and cerebral malaria), non-insulin dependent diabetes mellitus (NIDDM), congestive heart failure, post-cardiac injury, post-viral infection, post-inflammatory liver disease, chronic inflammatory conditions of the lung disease, chronic inflammatory conditions of the lungs, chronic inflammatory, Atherosclerosis, alzheimer's disease, acute encephalitis, brain injury, multiple sclerosis (demyelination and oligodendrocyte loss in multiple sclerosis), advanced cancer, lymphoid malignancies, pancreatitis, poor wound healing in infection, inflammation and cancer, myelodysplastic syndrome, systemic lupus erythematosus, biliary cirrhosis, intestinal necrosis, radiation injury, toxicity following administration of monoclonal antibodies, host versus graft reaction (ischemia reperfusion injury and allograft rejection of kidney, liver, heart and skin), lung allograft rejection (obliterative bronchitis), or complications due to total hip replacement, and infectious diseases selected from the group consisting of tuberculosis, helicobacter pylori infection during peptic ulcer disease, chagas disease due to trypanosoma cruzi infection, Shiga-like toxin action due to E.coli infection, enterotoxin A action due to staphylococcal infection, meningococcal infection and infection from Borrelia burgdorferi, Treponema pallidum infection, Cytomegalovirus infection, influenza virus infection, Teller encephalomyelitis virus infection and infection with Human Immunodeficiency Virus (HIV), papilloma, glioblastoma (blastoglioma), Kaposi's sarcoma, melanoma, Lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin's disease, Burkitt's disease, arthritis, rheumatoid arthritis, diabetic retinopathy, angiogenesis, restenosis, in-stent restenosis, restenosis in-stent, restenosis, radiation-induced by E.coli infection, and infection with B.C Vascular graft restenosis, pulmonary fibrosis, cirrhosis, atherosclerosis, glomerulonephritis, diabetic nephropathy, embolic microangiopathy syndrome (thrombomicangiopathy syndrome), transplant rejection, psoriasis, diabetes, wound healing, inflammation and neurodegenerative diseases, hyperimmune imbalance, hemangioma, myocardial angiogenesis, coronary and cerebral collateral vascularization, ischemia, corneal diseases, flushing, neovascular glaucoma, retinopathy of prematurity, macular degeneration, wound healing, helicobacter ulcer-related diseases, bone fractures, endometriosis, diabetic conditions, cat scratch fever, thyroid hyperplasia, asthma or edema after burn, trauma, chronic lung disease, stroke, polyps, synovitis, chronic and allergic inflammation, ovarian hyperstimulation syndrome, pulmonary and cerebral edema, keloids, fibrosis, lung and brain edema, Liver cirrhosis, carpal tunnel syndrome, adult respiratory distress syndrome, ascites, ocular disorders, cardiovascular disorders, Crow-fukase (poems) disease, crohn's disease, glomerulonephritis, osteoarthritis, multiple sclerosis, transplant rejection, lyme disease, sepsis, cerebral retinal vascular disease (von Hippel Lindau disease), pemphigoid, osteitis deformans, polycystic kidney disease, sarcoidosis, thyroiditis, hyperviscosity syndrome, Osler-webber-rand (Osler-Weber-Rendu disease), chronic occlusive lung disease, radiation, hypoxia, preeclampsia, menorrhagia, endometriosis, infection by herpes simplex virus, ischemic retinopathy, corneal angiogenesis, herpes zoster, human immunodeficiency virus, parapoxvirus, protozoa, toxoplasmosis, tumor-related fluid exudation, and edema.

The compounds of the invention may have more than one of the mentioned activities and may therefore target a number of signal transduction pathways. Thus, these compounds can achieve therapeutic and prophylactic effects, which are usually only obtained when combinations of different compounds are used. For example, the ability to inhibit neovascularization (e.g., associated with VEGFR-2 and VEGFR-3 function) (e.g., blood and/or lymph) and cell proliferation (e.g., associated with raf and PDGFR-beta function) using a single compound is particularly beneficial in the treatment of cancer and other cell proliferation disorders that can be promoted by neovascularization. The invention therefore relates in particular to compounds having at least anti-cell-proliferation and anti-angiogenic (i.e. angiogenesis-inhibiting) activity. Any disorder or condition that would benefit from inhibition of blood vessel growth and cell proliferation can be treated according to the present invention. The use of a single compound is also beneficial because its range of activity can be more precisely defined.

As indicated above, the present invention relates to methods of treating and/or preventing diseases and disorders associated with raf, VEGFR, PDGFR, p38 and/or flt-3; and/or modulating one or more pathways, polypeptides, genes, diseases, disorders, etc., associated with raf, VEGFR, PDGFR, p38, and/or flt-3. These methods generally involve administering an effective amount of a compound of the invention, where the effective amount is an amount useful to achieve the desired result. The compounds may be administered by an effective route in any effective form, as discussed in more detail below.

The methods comprise modulating tumor cell proliferation, including inhibiting cell proliferation. The latter means that the growth and/or differentiation of the tumor cells is reduced (reduced), decreased (degraded), decreased (diminished), slowed down, etc. The term "proliferation" includes any process associated with cell growth and division and includes differentiation and apoptosis. As discussed above, raf kinases play a key role in the activation of the cytoplasmic signaling cascade involved in cell proliferation, differentiation and apoptosis. For example, inhibition of c-raf by antisense oligonucleotides was found to block cell proliferation (see above). Any amount of inhibition is considered to be therapeutically effective.

Included in the methods of the present invention are methods of treating hyperproliferative disorders in a mammal, including humans in need thereof, using the anabolic products (including salts, prodrugs, and compositions thereof) of the compounds described above (compounds of formula I), including administering to the mammal an amount of the anabolic products, pharmaceutically acceptable salts, and compositions thereof of the compounds of the present invention effective to treat the disorder. Hyperproliferative disorders include, but are not limited to, solid tumors, such as breast, respiratory, brain, genital, digestive, urinary tract, eyeball, liver, skin, head and neck, thyroid, parathyroid, and their distant metastases. These disorders also include lymphomas, sarcomas, and leukemias.

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 and any downstream or upstream member of the signaling pathway of which it is a part. As described previously, the compounds of the present invention can be used to treat cancer without regard to the mechanism responsible for it. Cancers of any organ can be treated, including, but not limited to, for example, colon cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, skin cancer, pancreatic cancer, gastric cancer, colorectal cancer, renal cell carcinoma, hepatocellular carcinoma, melanoma, and the like.

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

Examples of cancers of the respiratory tract include, but are not limited to, small cell and non-small cell lung cancers as well as bronchial adenomas and pleural pneumococcal carcinomas.

Examples of brain cancers include, but are not limited to, brainstem and hypothalamic gliomas (hypophtalmic gliomas), cerebellar and cerebral astrocytomas, medulloblastomas, ependymomas, and neuroectodermal and pineal tumors.

Tumors of the male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine sarcomas.

Tumors of the digestive tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small intestine, and salivary gland cancers.

Urethral tumors include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, and urethral cancers.

Eyeball cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (with or without fibrolamellar variants), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.

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.

Head and neck cancer includes, but is not limited to, laryngeal, hypopharyngeal, nasopharyngeal and/or oropharyngeal cancer, lip cancer, and oral cancer.

Lymphomas include, but are not limited to, AIDS-related lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Hodgkin's disease, and lymphomas of the central nervous system.

Sarcomas include, but are not limited to sarcomas of soft tissue, osteosarcomas, malignant fibrous histiocytomas, lymphosarcomas, rhabdomyosarcomas.

Leukemias include, but are not limited to, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia.

In addition to inhibiting tumor cell proliferation, the compounds of the invention may also cause tumor regression, e.g., a reduction in tumor size, or a reduction in the range of cancer in the body.

The invention also relates to a method of modulating angiogenesis and/or lymphangiogenesis in a system comprising cells, comprising administering to the system an effective amount of a compound as described herein. The system comprising the cell may be an in vivo system, such as a tumor in a patient, an isolated organ, tissue, or cell, an in vitro system (CAM, BCE, etc.), an animal model (e.g., in vivo, subcutaneous, cancer model), a host in need of treatment (e.g., a patient with a disease having an angiogenic and/or lymphangiogenic component (e.g., cancer)), and the like.

Inappropriate and ectopic expression of angiogenesis can be harmful to living beings. Many pathological conditions are associated with growth of unrelated blood vessels. Such disorders include, for example, diabetic retinopathy, neovascular glaucoma, psoriasis, retrolental cellulose proliferation, angiofibroma, inflammation, and the like. In addition, the increased blood supply associated with cancer and tumor tissue encourages growth, resulting in rapid tumor enlargement and metastasis. In addition, the growth of new blood and lymphatic vessels in tumors provides escape routes for traitorous cells (renegade cells), encouraging metastasis and eventual spread of the cancer.

Systems that can be used to measure angiogenesis and/or lymphangiogenesis and its inhibition include, for example, neovascularization of tumor explants (e.g., U.S. Pat. No.5,192,744;6,024,688), membrane (CAM) assays of chicken chorioallantoic membranes (e.g., Taylor and Folkman, Nature1982,297,307-312; Eliceiri et al, J.Cell biol.1998,140, 1255-1263), Bovine Capillary Endothelial (BCE) cell assays (e.g., U.S. Pat. No.6,024,688; Polverini, P.J.et al, Methods enzymol.1991,198, 440-450), migration assays, and HUVEC (human umbilical vascular endothelial cell) growth inhibition assays (e.g., U.S. Pat. No.6,060,449) and the use of an ear model (e.g., Szuba et al, U.2002, 16(14), EB 1985-7).

Modulation of angiogenesis may be determined by any other method. For example, the degree of tissue vascularity is typically determined by assessing the number and density of blood vessels present in a given sample. For example, microvascular density (MVD) can be estimated by counting the number of endothelial clusters in a high power microscopic field, or by detecting specific markers of microvascular endothelium or other markers of growing or established blood vessels (e.g., CD31, also known as platelet endothelial cell adhesion molecule or PECAM). The CD31 antibody can be used in conventional immunohistochemical methods to immunostain tissue sections, for example, U.S. Pat. Nos. 6,017,949, Dellas et al, Gyn. Oncol.1997,67, 27-33; and in other documents. Other markers of angiogenesis include, for example, Vezf1 (e.g., Xiaong et al, Dev. Bio.1999,206, 123-141), angiogenin, Tie-1, and Tie-2 (e.g., Sato et al, Nature 1995,376, 70-74).

In addition, the invention relates to methods of screening patients to determine their sensitivity to the compounds of the invention. For example, the invention relates to methods of determining whether a disorder is modulated with a compound disclosed herein, comprising measuring the expression or activity of raf, VEGFR-2, VEGFR-3, PDGFR- β, p38, and/or flt-3 in a sample comprising cells or cell extracts, wherein the sample is obtained from a cell or subject having the disorder. When the results of the assay indicate that one or more of the mentioned genes (and/or polypeptides encoded thereby) differ from the normal state, this determines that the disorder or condition can be treated with a compound of the invention, i.e. when the expression or activity is increased in the disorder as compared to a normal control, then the disorder or condition can be modulated by the compound. The method may further comprise the step of comparing expression in the sample to a normal control, or to expression in a sample obtained from normal or unaffected tissue. The comparison may be done manually against an electronic form of standard (e.g., against a database), etc. The normal control may be a standard sample provided for the assay, which may be obtained from adjacent but unaffected tissue of the same patient, or it may be a predetermined value, or the like. Gene expression, protein expression (e.g., abundance in a cell), protein activity (e.g., kinase activity), and the like can be determined.

For example, the presence, amount and/or activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and/or flt-3 in a biopsy sample from a cancer patient can be determined. Increased expression or activity of one or more of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38, and/or flt-3 may indicate that the cancer may be targeted for treatment with a compound of the invention. For example, as described in the examples below, the activity of raf can be monitored by its ability to initiate a cascade that leads to ERK phosphorylation (i.e., raf/MEK/ERK) to yield phosphorylated-ERK. Increased levels of phospho-ERK in cancer samples indicate an increase in raf activity, indicating the use of the compounds of the invention for treating this cancer.

Measuring expression includes determining or detecting the amount of the polypeptide present in or shed by the cell, and measuring the mRNA that is caused to form, wherein the amount of mRNA present is believed to reflect the amount of polypeptide produced by the cell. In addition, the genes for raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38, and/or flt-3 can be analyzed to determine whether there is a genetic defect that results in aberrant expression or polypeptide activity.

Polypeptide detection can be performed by any available method, e.g., by western blotting, ELISA, dot blotting, immunoprecipitation, Radioimmunoassay (RIA), immunohistochemistry, and the like. For example, tissue sections can be prepared and labeled with specific antibodies (either indirect or direct) and viewed microscopically. The amount of polypeptide can be quantified without observation, for example, by preparing a lysate of the sample of interest and then determining the amount of polypeptide per tissue amount by ELISA or Western blotting. Antibodies and other specific binding reagents may be used. There is no limitation on how the detection is performed.

Assays that allow for quantification of a target nucleic acid (e.g., genes, mRNA, etc. of raf, VEGFR, PDGFR, p38, and/or flt-3) in a sample and/or detection of the presence/absence of a target nucleic acid in a sample can be utilized. The assay may be performed at the single cell level, or in a sample comprising a plurality of cells, wherein the assay is "averaged" in expression over the complete collection of cells and tissues present in the sample. Any suitable assay format may be used, including, but not limited to, for example, southern blot Analysis, northern blot Analysis, polymerase chain reaction ("PCR") (e.g., Saiki et al, Science 1988,241,53; U.S. Pat. No.4,683,195, 4,683,202, and 6,040,166; PCR Protocols: A Guide to Methods and Applications, Innis et al, eds., Academic Press, New York, 1990), reverse transcription polymerase chain reaction ("RT-PCR"), Anchor PCR, cDNA end rapid amplification ("E") (e.g., Schaefer in Gene Cloning and Analysis: Current Innovation, pp. 99-115, 1997), ligase chain reaction ("LCR") (EP 320308), Single-sided PCR (Ohara et al, Proc. Natl. Acad. Sci.1989,86, 5673), index chain reaction ("LCR") such as LCR. No.5,262,311, in situ hybridization Methods, 365954; WO 67567, 5,262,311; in. 5,262,311; see, Natl.Acad.Sci.,93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126, Welsh et al, Nucleic Acid Res.,20:4965-4970,1992, and U.S. Pat. No.5,487,985), as well as other RNA fingerprinting techniques, Nucleic Acid sequence-based amplification ("NASBA"), and other transcription-based amplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO 88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854, 5,424,186, 5,700,637, 5,874,219 and 6,054,270; PCTWO 92/10092; PCT WO 90/15070), Qbeta replicase (PCT/US 87/00880), strand displacement amplification ("SDA"), repair chain reactions ("RCR"), nuclease protection assays, subtractive-based methods, rapid scanning, and the like. Additional useful methods include, but are not limited to, for example, template-based amplification methods, competitive PCR (e.g., U.S. Pat. No.5,747,251), redox-based assays (e.g., U.S. Pat. No.5,871,918), Taqman-based assays (e.g., Holland et al, Proc. Natl. Acad, Sci.1991,88,7276-. Any method suitable for single cell analysis of gene or protein expression may be used, including in situ hybridization, immunocytochemistry, MACS, FACS, flow cytometry, and the like. For single cell assays, expression products can be assayed using antibody, PCR, or other types of nucleic acid amplification (e.g., Brady et al, methods mol. & cell. biol.1990,2,17-25; Eberwine et al, Proc. Natl. Acad. Sci.1992,89,3010-3014; U.S. Pat. No.5,723, 290). These and other methods can be carried out in a conventional manner, for example as described in the publications mentioned. The activity of raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and/or flt-3 can be determined in conventional manner, for example as described in the examples below, or using standard assays for kinase activity.

The invention also provides methods of evaluating the efficacy of a compound of the invention in treating a disorder, comprising one or more of the following steps, in any effective order, e.g., administering an amount of the compound, measuring the expression or activity of raf, VEGFR-2, VEGFR-3, PDGFR- β, p38 and/or flt-3 (see above), and determining the effect of the compound on the expression or activity. For example, a biopsy sample may be taken from a patient who has been treated with a compound of the invention and the presence and/or activity of the mentioned signaling molecule is determined. Similarly, as described above, a decrease in the level of phospho-ERK in cancer tissues (e.g., as compared to normal tissues or prior to treatment) indicates that the compound is effective in vivo and therapeutically. The methods can be used to determine the appropriate dosage and dosing regimen, e.g., how much compound to administer and what the frequency of administration is. By monitoring its effect on signaling molecules in the tissue, a clinician can determine an appropriate treatment regimen and whether it achieves a desired effect, e.g., modulating or inhibiting the signaling pathway.

The compounds of the invention may 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 comprising biological material. This includes one or more of the following steps, in any effective order: (i) contacting the sample comprising biological material with a compound of the invention, and (ii) determining whether the compound binds to the material. The compound may be labeled, or it may be used as a competitor for a labeled compound (e.g., labeled ATP).

The invention also provides methods for the treatment, prevention, modulation, and the like, of diseases and disorders in mammals, comprising administering a compound of the invention, in combination with another modulator of the signal transduction pathway, including but not limited to raf, VEGFR-2, VEGFR-3, PDGFR, p38, and/or flt-3. These may be present in the same composition or in separate formulations or dosage units. Administration may be by the same or different routes, and may be simultaneous or sequential.

The following publications relate to the modulation of VEGFR-3 and their descriptions of disease states mediated by VEGFR-3 and assays for determining such activity are incorporated herein.

WO95/33772 Alitalo，et.al.

WO95/33050 Charnock-Jones，et.al..

WO96/39421 Hu，et.al.

WO98/33917 Alitalo，et.al.

WO02/057299 Alitalo，et.al.

WO02/060950 Alitalo，et.al.

WO02/081520 Boesen，et.al.

The publications below relate to the modulation of VEGFR-2 and their description of the disease states mediated by VEGFR-2 and the assays for determining such activity are incorporated herein.

EP0882799 Hanai，et.al.

EP1167384 Ferraram，et，al.

EP1086705 Sato，et.al.

EP11300032 Tesar，et.al.

EP1166798 Haberey，et.al.

EP1166799 Haberey，et.al.

EP1170017 Maini，et.al.

EP1203827 Smith

WO02/083850 Rosen，et.al.

The following publications relate to the modulation of flt-3 and their description of flt-3 mediated disease states and assays for determining such activity is incorporated herein.

2002/0034517 Brasel，et.al.

2002/0107365 Lyman，et.al.

2002/0111475 Graddis，et.al.

EP0627487 Beckermann，et.al.

WO9846750 Bauer，et.al.

WO9818923 McWherter，et.al.

WO9428391 Beckermann，et al.

WO9426891 Birnbaum，et.al.

The following publications relate to the modulation of PDGF/PDGFR and their description of PDGFR- β mediated disease states and assays for determining such activity is incorporated herein.

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.

5,817,310 Ramakrishnan，et.al.

5,833,986 LaRochelle，et.al.

5,863,739 LaRochelle，et.al.

5,872,218 Wolf，et.al.

5,882,644 Chang，et.al.

5,891,652 Wolf，et.al.

5,976,534 Hart，et.al.

5,990,141 Hirth，et.al.

6,022,854 Shuman

6,043,211 Williams，et.al.

6,110,737 Escobedo，et.al.

6,207,816B1 Gold，et.al.

6,228,600B1 Matsui，et.al.

6,229,002B1 Janjic，et.al.

6,316,603B1 McTigue，et.al.

6,372,438B1 Williams，et.al.

6,403,769B1 La Rochelle，et.al.

6,440,445B1 Nowak，et.al.

6,475,782B1 Escobedo，et.al.

WO02/083849 Rosen，et.al.

WO02/083704 Rosen，et.al.

WO02/081520 Boesen，et.al.

WO02/079498 Thomas，et.al.

WO02/070008 Rockwell，et.al.

WO09959636 Sato，et.al.

WO09946364 Cao，et.al.

WO09940118 Hanai，et.al.

WO9931238 Yabana，et.al.

WO9929861 Klagsbrun，et.al.

WO9858053 Kendall，et.al.

WO9851344 Maini，et.al.

WO9833917 Alitalo，et.al.

WO9831794 Matsumoto，et.al.

WO9816551 Ferrara，et.al.

WO9813071 Kendall，et al.

WO9811223 Martiny-Baron，et.al.

WO9744453 Chen，et.al.

WO9723510 Plouet，et.al.

WO9715662 Stinchcomb，et.al.

WO9708313 Ferrara，et.al.

WO9639515 Cao，et.al.

WO9623065 Smith，et.al.

WO9606641 Fleurbaaij，et.al.

WO9524473 Cao，et.al.

WO9822316 Kyowa

WO9521868 Rockwell，et.al.

WO02/060489 Xia，et.al.

PDGFR-β

EP0869177 Matsui，et.al.

WO09010013 Matsui，et.al.

WO9737029 Matsui，et.al.

PDGFR-α

EP1000617 Lammers，et.al.

EP0869177 Matsui，et.al.

EP0811685 Escobedo，et.al.

Pharmaceutical compositions based on the compounds of the invention.

The invention also relates to pharmaceutical compositions comprising the compounds of the invention and their pharmaceutically acceptable salts. These compositions can be used to achieve a desired pharmacological effect by administration to a patient in need thereof. For the purposes of the present invention, a patient is a mammal, including a human, in need of treatment for a particular condition or disease. Accordingly, the present invention includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of the present invention or a salt thereof. The term "pharmaceutically acceptable carrier" means any carrier that is relatively non-toxic and non-injurious to a patient at concentrations consistent with effective activity of the active ingredient such that any side effects caused by the carrier do not detract from the beneficial effects of the active ingredient. A pharmaceutically effective amount of a compound is that amount which produces an effect or exerts an effect on the particular condition being treated. The compounds of the present invention may be administered in any effective conventional dosage unit form including immediate release, sustained release and timed release preparations, orally, parenterally, topically, nasally, ocularly, sublingually, rectally, vaginally, and the like, in association with a pharmaceutically acceptable carrier known in the art.

For oral administration, the compounds may be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, troches, fluxes, powders, solutions, suspensions, or emulsions and may be prepared according to methods known in the art for the manufacture of pharmaceutical compositions. The solid unit dosage form may be a capsule, which may be of the ordinary hard-or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.

In another embodiment, the compounds of the present invention may be formulated into tablets with: conventional tablet bases (e.g., lactose, sucrose, and corn starch) are combined with: binders (e.g., gum arabic, corn starch, or gelatin), disintegrants (e.g., potato starch, alginic acid, corn starch, guar gum, tragacanth gum, gum arabic) intended to aid tablet disintegration and dissolution following administration, lubricants (e.g., talc, stearic acid or magnesium stearate, calcium stearate, or zinc stearate) intended to improve the flow of tablet particles and prevent tablet material from adhering to tablet die and punch surfaces, dyes, colorants, and flavorants (e.g., peppermint flavorant, wintergreen flavorant, or cherry flavorant) intended to increase the aesthetic characteristics of the tablet and make it more acceptable to patients. Suitable excipients for use in oral liquid dosage forms include dicalcium phosphate and diluents (e.g., water and alcohols such as ethanol, benzyl alcohol and polyvinyl alcohol), with or without the addition of pharmaceutically acceptable surfactants, suspending agents or emulsifying agents. Various other materials may be present in the form of coatings or used to modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both.

The dispersed powders and granules are suitable for preparing aqueous suspensions. Which provides a mixture of the active ingredient together with dispersing or wetting agents, suspending agents and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are those already mentioned above. Additional excipients, such as those sweetening, flavoring and coloring agents described above, may also be present.

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, for example gum acacia and gum tragacanth, (2) naturally-occurring phosphatides, for example soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, (4) condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

Oil suspensions may be prepared by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspending agent may comprise a thickening agent such as beeswax, hard paraffin or cetyl alcohol. The suspension may also contain one or more preservatives (e.g., ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents such as sucrose or saccharin.

Syrups and elixirs (elixir) may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. The formulation may also contain a demulcent and a preservative (e.g., ethyl or n-propyl paraben), as well as fragrances and colorants.

The compounds of the present invention may also be administered parenterally, i.e., subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly, or intraperitoneally, in the form of an injectable formulation of the compound in a physiologically acceptable diluent, containing a pharmaceutically acceptable carrier in the form of a sterile liquid or liquid mixture (e.g., water, saline, aqueous dextrose and related sugar solutions), an alcohol (e.g., ethanol, isopropanol, or cetyl alcohol), a glycol (e.g., propylene glycol or polyethylene glycol), a glycerol ketal (e.g., 2-dimethyl-1, 1-dioxolane-4-methanol), an ether (e.g., polyethylene glycol 400), an oil, a fatty acid ester, a fatty acid glyceride, or an acetylated fatty acid glyceride, with or without the addition of a pharmaceutically acceptable surfactant (e.g., a soap or detergent), a suspending agent (e.g., pectin, a suspending, Carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose) or an emulsifying agent, as well as other pharmaceutically acceptable adjuvants.

Examples of oils which may be used in the parenteral formulations of the invention are petroleum, animal oils, vegetable oils or oils of synthetic origin, for example peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, vaseline oil and mineral oil. Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty acid alkali metal salts, fatty acid ammonium salts, and fatty acid triethanolamine salts; and suitable detergents include cationic detergents such as dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; anionic detergents such as alkyl, aryl and alkylene sulfonates, alkyl, alkylene, ether and monoglyceride sulfates and sulfosuccinates; nonionic detergents such as fatty amine oxides, fatty acid alkanolamides, poly (oxyethylene-oxypropylene), ethylene oxide copolymers or propylene oxide copolymers; and amphoteric detergents such as alkyl-beta-aminopropionates, 2-alkylimidazoline quats, and mixtures.

The parenteral compositions of the invention typically comprise from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be advantageously employed. To minimize or eliminate irritation at the injection site, the composition may comprise a nonionic surfactant having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in the formulation ranges from about 5% to about 15% by weight. The surfactant may be a single component having the above HLB or may be a mixture of two or more components having the desired HLB.

Examples of surfactants for use in parenteral formulations are polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide and a hydrophobic base formed by the condensation of propylene oxide and propylene glycol.

The pharmaceutical composition may be in the form of a sterile injectable aqueous suspension. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents, for example, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin, condensation products of an alkylene oxide with fatty acids (for example polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (for example heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (for example polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (for example polyoxyethylene sorbitan monooleate).

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, for example, water, ringer's solution, isotonic sodium chloride solution and isotonic glucose solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compositions of the present invention may also be administered in the form of suppositories for rectal administration. These compositions can be formulated 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 materials are, for example, cocoa butter and polyethylene glycols.

Another formulation for use in the method of the present invention employs a transdermal delivery device ("patch"). The transdermal patch may be used to provide continuous or discontinuous infusion of a compound of the present invention in controlled amounts. The structure of transdermal patches and their use for delivering agents are well known in the art (see, e.g., U.S. Pat. No.5,023,252, issued 6/11/1991, incorporated by reference herein). The patch may be configured for continuous, pulsed or on-demand delivery of a pharmaceutical formulation.

Controlled release formulations for parenteral administration include liposome, polymeric microsphere and polymeric gel formulations known in the art.

It may be desirable or necessary to introduce the pharmaceutical composition into a patient via a mechanical delivery device. The structure of such mechanical delivery devices and the use thereof for the delivery of pharmaceutical agents are well known in the art. Direct techniques, such as those used to administer drugs directly to the brain, typically involve placing a drug delivery catheter in the ventricular system of the patient to bypass the blood brain barrier. One such implantable delivery system for delivering agents to specific anatomical regions of the body is described in U.S. Pat. No.5,011,472 (issued 4/30 1991).

The compositions of the present invention may also contain other conventional pharmaceutically acceptable compounding ingredients, commonly referred to as carriers or diluents, as necessary or desired. Conventional procedures for preparing the compositions in suitable dosage form may be utilized. The ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: powell, M.F.et al, "Complex of Excipients for particulate Formulations" PDAjournal of Pharmaceutical Science & Technology 1998,52(5),238- "311; strickley, R.G, "partial Formulations of Small molecular therapeutics marked in the United States (1999) -Part-1," PDA journal of Pharmaceutical Science & Technology 1999,53(6), 324-349; and Nema, S.et al, "Excipients and the same Use in Injectable Products," PDA Journal of Pharmaceutical Science & Technology 1997,51(4), 166-.

Commonly used pharmaceutical ingredients suitable for formulating the compositions for their intended route of administration include:

acidulants (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 (triethanolamine), 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 CClF 3);

air displacement agents (examples include, but are not limited to, nitrogen and argon);

antifungal preservatives (examples include, but are not limited to, benzoic acid, butyl paraben, ethyl paraben, methyl paraben, propyl paraben, 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);

adhesive materials (examples include, but are not limited to, block polymers, natural and synthetic rubbers, 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);

a carrier (examples include, but are not limited to, acacia syrup, aromatic elixir, cherry syrup, cocoa syrup, orange syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection, and bacteriostatic water for injection);

chelating agents (examples include, but are not limited to, edetate disodium and ethylenediaminetetraacetic acid);

coloring agents (examples include, but are not limited to FD & C Red No.3, FD & C Red No.20, FD & C Yellow No.6, FD & C Blue No.2, D & C Green No.5, D & COAnge No.5, D & 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, gum arabic, 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);

fragrances (examples include, but are not limited to, anise oil, cinnamon oil, cocoa powder, menthol, orange oil, peppermint oil, and vanillin);

humectants (examples include, but are not limited to, glycerin, propylene glycol, sorbitol);

abrasives (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, petrolatum, hydrophilic petrolatum, white ointment, yellow ointment, and rose water ointment);

penetration enhancers (transdermal delivery) (examples include, but are not limited to, monohydric or polyhydric alcohols, mono or polyhydric alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalins, terpene ethers, 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 rinsing);

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

suppository bases (examples include, but are not limited to, cocoa butter and polyethylene glycol (mixtures));

surfactants (examples include, but are not limited to, benzalkonium chloride, nonoxynol 10, octoxynol 9 (oxytoxynol 9), tween 80, sodium lauryl sulfate, and sorbitan monopalmitate);

suspending agents (examples include, but are not limited to, agar, bentonite, carbomer, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, kaolin, methylcellulose, tragacanth and magnesium aluminum silicate);

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

tablet antiadherents (examples include, but are not limited to, magnesium stearate and talc);

tablet binders (examples include, but are not limited to, gum arabic, alginic acid, sodium carboxymethylcellulose, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, 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 phosphate, sorbitol, and starch);

tablet coatings (examples include, but are not limited to, liquid glucose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, ethylcellulose, cellulose acetate phthalate, and shellac);

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

tablet disintegrating agents (examples include, but are not limited to, alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose, polacrilin potassium, sodium alginate, sodium starch glycolate, and starch);

tablet glidants (examples include, but are not limited to, colloidal silicon dioxide, 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 polishes (examples include, but are not limited to, carnauba wax and white wax);

thickening agents (examples include, but are not limited to, beeswax, cetyl alcohol, and paraffin wax);

tonicity agents (examples include, but are not limited to, glucose and sodium chloride);

viscosity increasing agents (examples include, but are not limited to, alginic acid, bentonite, carbomer, sodium carboxymethylcellulose, methylcellulose, sodium alginate, and gum tragacanth); and

wetting agents (examples include, but are not limited to, heptadecaethyleneoxycetanol, lecithin, sorbitol monooleate, polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).

The pharmaceutical composition according to the invention is illustrated below:

sterile IV solution: sterile water for injection is used to prepare a 5mg/mL solution of the desired compound of the invention and, if necessary, to adjust the pH. The solution was diluted with sterile 5% glucose to 1-2mg/mL for administration and administered as an IV infusion over 60 minutes.

Lyophilized powder for IV administration: sterile preparations can be prepared from (i) 100-1000mg of the compound required according to the invention as lyophilisate, (ii) 32-327mg/mL of sodium citrate and (iii) 300-3000mg of dextran 40. The formulation is reconstituted with sterile saline for injection or 5% glucose to a concentration of 10 to 20mg/mL, further diluted with saline or 5% glucose to 0.2-0.4 mg/mL, and administered either as an IV bolus or by IV infusion over 15-60 minutes.

Intramuscular suspension: the following solutions or suspensions can be prepared for intramuscular injection:

50mg/mL of the desired water-insoluble compound of the invention

5mg/mL sodium carboxymethylcellulose

4mg/mL Tween 80

9mg/mL sodium chloride

9mg/mL benzyl alcohol

Hard shell capsule: a large number of unit capsules were prepared by filling each standard two-piece hard gelatin (galantine) capsule with the following ingredients: 100mg of powdered active ingredient, 150mg of lactose, 50mg of cellulose and 6mg of magnesium stearate.

Soft gelatin capsules: a mixture of the active ingredient dissolved in a digestible oil (such as soybean oil, cottonseed oil or olive oil) is prepared and injected by a positive displacement pump into molten gelatin to form a soft gelatin capsule containing 100mg of the active ingredient. The capsules are washed and dried. The active ingredient may be dissolved in a mixture of polyethylene glycol, glycerol and sorbitol to prepare a water-miscible drug mixture.

And (3) tablet preparation: large quantities of tablets are prepared by conventional procedures to give a dosage unit of 100mg of active ingredient, 0.2mg of colloidal silicon dioxide, 5mg of magnesium stearate, 275mg of microcrystalline cellulose, 11mg of starch and 98.8mg of lactose. Suitable aqueous as well as non-aqueous coatings may be used to increase palatability, improve elegance and stability or delay absorption.

Immediate release tablets/capsules: these are solid oral dosage forms prepared by conventional and novel processes. These units are taken orally and do not use water for rapid dissolution and delivery of the medicament. The active ingredient is mixed in a liquid containing ingredients such as sugar, gelatin, pectin and sweeteners. These liquids are solidified by freeze-drying and solid-state extraction techniques into solid tablets or lozenges. The pharmaceutical compound can be compressed with a viscoelastic and thermoelastic sugar and a polymer or effervescent component to prepare a porous matrix for immediate release, without the need for water.

Dosage of the pharmaceutical composition of the invention

Effective dosages of the compounds of the present invention for the treatment of each of the desired indications can be readily determined based on standard laboratory techniques known for evaluating compounds useful for the treatment of any of the above-mentioned disorders, by standard toxicity tests and by standard pharmacological assays for determining treatment of the above-mentioned conditions in mammals, and by comparing these results with the results of known agents for treating the condition. The amount of the active ingredient to be administered in the treatment of one of these conditions may vary widely according to the following considerations: the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient being treated, and the nature and extent of the condition being treated.

The total amount of the active ingredient to be administered may range from about 0.001mg/kg to about 200mg/kg body weight per day, and preferably from about 0.1mg/kg to about 50mg/kg body weight per day. The unit dose may preferably contain from about 5mg to about 4000mg of the active ingredient and may be administered one or more times per day. The daily dose for oral administration will preferably be from 0.1 to 50mg/kg of total body weight. The daily dose given by injection (including intravenous, intramuscular, subcutaneous and parenteral) and using infusion techniques will preferably be from 0.1 to 10mg/kg of total body weight. The daily rectal dosage regimen will preferably be from 0.1 to 50mg/kg of total body weight. The daily vaginal dosage regimen will preferably be from 0.1 to 50mg/kg of total body weight. The daily topical regimen will preferably be from 0.1 to 10mg/kg, administered one to four times daily. Transdermal concentrations will preferably be those required to maintain a daily dose of from 0.1 to 10 mg/kg. The daily inhalation dosage regimen will preferably be from 0.1 to 10mg/kg of total body weight. Other dosages and amounts can be selected by conventional methods.

The specific initial and sustained dosing regimen for each patient will vary depending upon the nature and severity of the condition, the activity of the particular compound employed, the age and general condition of the patient, the time of administration, the route of administration, the rate of drug excretion, the drug combination, and the like, as determined by the attending physician. The desired mode of treatment and the number of doses of a compound of the invention or a pharmaceutically acceptable salt or ester or composition thereof can be determined by one skilled in the art using routine therapeutic testing.

Combinations of the compounds and compositions of the present invention with additional active ingredients.

The compounds of the present invention may be administered as the sole pharmaceutical formulation or in combination with one or more other pharmaceutical formulations, wherein the combination does not cause unacceptable side effects. This may be particularly relevant for the treatment of hyperproliferative diseases, such as cancer. In this case, the compounds of the invention may be conjugated with known cytotoxic agents, signal transduction inhibitors or with other anti-cancer agents, as well as mixtures and conjugates thereof.

In one embodiment, the compounds of the present invention may be combined with cytotoxic anticancer agents. Examples of such agents can be found in the Merck Index 11th edition (1996). These agents include, but are not limited to, asparaginase (aspargine), bleomycin (bleomycin), carboplatin (carboplatin), carmustine (carmustine), chlorambucil (chlorambucil), cisplatin (cisclin), asparaginase (colaspase), cyclophosphamide, cytarabine (cytarabine), dacarbazine (dacrbazine), actinomycin D (dactinomycin), daunorubicin (daunorubicin), doxorubicin (doxorubin) (adriamycin), epirubicin (epirubicin), etoposide (etoposide), 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan (irinotecan), folinic acid (leucovorin), lomustine (lomustine), mechlorethamine (mellothamine), 6-mercaptopurine, sodium methicillin (mestranilide), methotrexate (prednisone), mitomycin (prednisolone (prednisone), mitomycin (mitomycin), mitomycin (mitomycin), and mitomycin (mitomycin C (mitomycin) Raloxifene (raloxifen), streptozocin (streptozocin), tamoxifen (tamoxifen), thioguanine (thioguanine), topotecan (topotecan), vinblastine (vinblastine), vincristine (vincristine), and vindesine (vindesine).

Other cytotoxic drugs suitable for use with The compounds of The present invention include, but are not limited to, those compounds that are well known for use in The treatment of neoplastic diseases in Goodman and Gilman's The pharmacological basis of Therapeutics (ninth edition, 1996, McGraw-Hill). These include, but are not limited to, aminoglutethimide (aminoglutethimide), L-asparaginase, azathioprine (azathioprine), 5-azacytidine cladribine (5-azacytidine cladribine), busulfan (busufan), diethylstilbestrol (diethyltilbenol), 2' -difluorodeoxycytidine, docetaxel (docetaxel), erythrohydroxynonyladenine (erythrohydroxyxynyladine), ethinylestradiol, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate (fludarabinepenthalt), fluoxymesterone (fluoroxymesterone), flutamide (flutamide), hydroxyprogesterone hexanoate (hydroxyprogestin acetate), idarubicin (idarubicin), interferon, hydroxyprogesterone acetate (hydroxyprogesterone acetate), megestrol (acetylketonuronate), phosphonominophen-L (phosphonoketol), megestrol (phosphonoketonurenine), medroxyprogesterone (acetylketonurenine), megestrol acetate (phosphonominol acetate (pentostatin), medetoxynol (L-acetate (L-acetate), medetostatin (phosphonominol (phosphonominophen-L (phosphono-L, doxylamine (phosphonominophen-D), medrystatin (phosphono), medryptostatin (dox), medryptophan), medryptostatin (e), medrystatin (guane), and (guanylurea), medryne (guanylurea), medrystatin (e), medryne, plicamycin (plicamycin), semustine (semustine), teniposide (teniposide), testosterone propionate (testosterone propionate), thiotepa (thiotepa), trimethylmelamine, uridine (uridine), and vinorelbine (vinorelbine).

Other cytotoxic anticancer drugs suitable for use in combination with the compounds of the present invention include newly discovered cytotoxic components such as oxaliplatin (oxaliplatin), gemcitabine (gemcitabine), capecitabine (capecitabine), epothilone (epothilone) and its natural or synthetic derivatives, temozolomide (temozolomide) (Quinn et al, j.clin. Oncology 2003,21(4), 646-.

In another embodiment, the compounds of the present invention may be combined with other signal transduction inhibitors. Of particular interest are signal transduction inhibitors (Raymond et al, Drugs 2000,60 (supply.1), 15-23; Harari et al, Oncogene 2000,19(53), 6102-. Examples of such agents include, but are not limited to, antibody therapies such as Herceptin (Herceptin) (trastuzumab), Erbitux (cetuximab), and pertuzumab (pertuzumab). Examples of such therapies also include, but are not limited to, small molecule kinase inhibitors such as ZD-1839/Iressa (Baselaga et al., Drugs 2000,60 (supply.1), 33-40), OSI-774/Tarceva (Pollack et al. J. phase. exp. Ther.1999,291(2), 739-748), CI-1033 (Bridges, curr. Med. chem.1999,6, 825-843), GW-2016 (Lackey et al.,92nd AACR Meeting, New Orleanes, 24-28.2001, act 4582), CP-724,714 (Jani et al., Proceedings of the American Society for clinical analysis 2004,23, act 3122), HKI-272 (Rabidra et al., Cancer et al., EOs. 65, EOc. for the Society for clinical analysis 2004,23, act 3122), and Cancer-80 (Cancer et al., RTC. 76, RTC. RTM. 11-5. RTM., RTC. RTM. 11, 76, RTC. RTM. As well as Experimental results in FIGS.

In another embodiment, the compounds of the invention may bind to other signal transduction inhibitors of receptor kinases targeting the family of the split-kinase domains (VEGFR, FGFR, PDGFR, flt-3, c-kit, c-fms, etc.) and their respective ligands. Examples of such agents include, but are not limited to, antibodies such as Avastin (Avastin) (bevacizumab). Examples of such drugs also include, but are not limited to, 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), SU-11248 (Demetri et al, Proceedings of the American Society for Clinical Oncol 2004,23, abstract 3001), ZD-6474 (Hennequin et al, 92 AACR Meeting, New Orleanans, 2001, 24-28, abstract 2004), AG-13736 (Herbst et al, Clin. Cancer Res.2003, 2003,16 (Mercury. 2003), Experimental for example, European 7363, European 7375, European 7395, European 7375, European 7395, European 731, European 7395, 45, abstrates 3989), CHIR-258 (Lee et al, Proceedings of the American Association of Cancer Research2004,45, abstrates 2130), MLN-518 (Shen et al, Blood 2003,102,11, abstrates 476), and AZD-2171 (Hennequin et al, Proceedings of the American Association of Cancer Research2004,45, abstrates 4539).

In another embodiment, the compounds of the invention may bind to inhibitors of the Raf/MEK/ERK transduction pathway (Avruch et al, Recent prog.Horm.Res.2001,56, 127-. These include, but are not limited to, PD-325901 (Sebolt-Leopol et al, Proceedings of the American Association of Cancer Research2004,45, abstrate 4003) and ARRY-142886 (Wallace et al, Proceedings of the American Association of Cancer Research2004,45, abstrate 3891).

In another embodiment, the compounds of the present invention may bind to inhibitors of histone deacetylase. Examples of such agents include, but are not limited to suberoylanilide hydroxamic acid (SAHA), LAQ-824 (Ottmann et al, Proceedings of the American Society for Clinical Research2004, 23, abstract 3024), LBH-589 (Becket et al, Proceedings of the American Society for Clinical Research2004, 23, abstract 3025), MS-275 (Ryan et al, Proceedings of the American Society for Clinical Research2004,45, abstract 2452), and FR-901228 (Piekarz et al, Proceedings of the American Society for Clinical Research2004, 23, abstract 3028).

In another embodiment, the compounds of the present invention may be combined with other anti-cancer agents (e.g., proteasome inhibitors, and m-TOR inhibitors). These include, but are not limited to, bortezomib (Mackay et al, Proceedings of the American society for Clinical Oncology 2004,23, prostractic 3109) and CCI-779 (Wu et al, Proceedings of the American Association of cancer research2004,45, prostractic 3849).

In general, the treatment of cancer using cytotoxic and/or cytostatic anticancer agents in combination with a compound or composition of the invention will exert the following effects:

(1) results in better efficacy in reducing tumor growth or even eliminating tumors than either drug administered alone,

(2) provides for the administration of smaller amounts of the chemotherapeutic agent administered,

(3) providing a well-tolerated chemotherapy treatment in a patient with fewer harmful pharmacological complications than observed in single agent chemotherapy and certain other combination therapies,

(4) provides a method for treating a broad spectrum of different cancer types in mammals, particularly in humans,

(5) provide a higher response rate in the treated patient,

(6) provides a longer survival time in the treated patient compared to standard chemotherapy treatment,

(7) provide longer tumor regression time, and/or

(8) Compared to known examples, where other cancer drugs in combination produce antagonism, results in at least as good efficacy and tolerability as the drugs used alone.

Aspects of the invention include, but are not limited to:

in one embodiment, the present invention provides the following aspects

Aspect 1 an anabolic product of formula (I) or a salt thereof, or a prodrug thereof, or an isolated stereoisomer thereof.

Aspect 2. a synthetic M2, M3, M4, or M5 metabolite of a compound of formula I, or a salt of said metabolite, wherein the structures of said M2, M3, M4, and M5 metabolites are:

metabolite M-2

Metabolite M-3

Metabolite M-4

Metabolite M-5

Aspect 3 pharmaceutically acceptable salts of the anabolic products according to aspect 2 which are

a) Basic salts of organic or inorganic acids, said acids being hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, trifluoromethanesulfonic, benzenesulfonic, p-toluenesulfonic (tosylate), 1-naphthalenesulfonic, 2-naphthalenesulfonic, acetic, trifluoroacetic, malic, tartaric, citric, lactic, oxalic, succinic, fumaric, maleic, benzoic, salicylic, phenylacetic or mandelic acid; or

b) Acid salts of organic or inorganic bases containing alkali metal cations, alkaline earth metal cations, ammonium cations, aliphatically substituted ammonium cations, or aromatically substituted ammonium cations.

Aspect 4. the anabolic product according to aspect 2, which is a metabolite of 4{4- [3- (4-chloro-3-trifluoromethylphenyl) -ureido ] -3-fluorophenoxy } -pyridine-2-carboxylic acid carboxamide.

Aspect 5 pharmaceutically acceptable salts of the anabolic product according to aspect 4, which are basic salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid (tosylate), 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, or mandelic acid.

Aspect 6 a compound which is the hydrochloride, benzenesulfonate or methanesulfonate salt of an anabolic product according to aspect 2.

Aspect 7. a pharmaceutical composition comprising an anabolic product according to aspect 2 and a physiologically acceptable carrier.

Aspect 8. a pharmaceutical composition comprising an anabolic product according to aspect 4 and a physiologically acceptable carrier.

Aspect 9. a pharmaceutical composition for treating a disease modulated by a protein kinase and associated with an abnormality in the protein kinase signal transduction pathway in a human or other mammal, comprising an anabolic product according to aspect 2 and a physiologically acceptable carrier.

Aspect 10. a pharmaceutical composition for the treatment of a hyperproliferative disorder comprising an anabolic product according to aspect 4 and a physiologically acceptable carrier.

Aspect 11. a pharmaceutical composition for treating cancer cell growth comprising an anabolic product according to aspect 2 and a physiologically acceptable carrier.

Aspect 12 a method for modulating tyrosine kinase signal transduction comprising administering an anabolic product according to aspect 2 to a human or other mammal.

A method for the treatment or prevention of a disease in a human or other mammal which is modulated by a tyrosine kinase and which is associated with an abnormality in the tyrosine kinase signal transduction pathway, which method comprises administering an anabolic product according to aspect 2 to the human or other mammal.

A method for treating or preventing a disorder mediated by VEGFR-2 in a human and/or other mammal, comprising administering an anabolic product according to aspect 2 to the human or other mammal.

Aspect 15 a method for the treatment or prevention of a PDGFR-mediated disorder in a human and/or other mammal, which method comprises administering an anabolic product according to aspect 2 to the human or other mammal.

A method for the treatment or prevention of a disorder mediated by raf in a human and/or other mammal, comprising administering an anabolic product according to aspect 2 to the human or other mammal.

Aspect 17 a method for the treatment or prevention of a disorder mediated by p38 in a human and/or other mammal, the method comprising administering an anabolic product according to aspect 2 to the human or other mammal.

A method for treating or preventing a VEGF-mediated disorder in a human and/or other mammal, the method comprising administering an anabolic product according to aspect 2 to the human or other mammal.

A method for the treatment or prophylaxis of hyperproliferative, inflammatory and/or angiogenic disorders in a human and/or other mammal, comprising administering an anabolic product according to aspect 2 to the human or other mammal.

A method for the treatment or prevention of a hyperproliferative disorder in a human and/or other mammal, said method comprising administering an anabolic product according to aspect 2 to the human or other mammal.

The method as in aspect 20, wherein the hyperproliferative disorder is cancer.

Aspect 22. the method as in aspect 21, wherein the method comprises administering the anabolic product according to aspect 2 to a human or other mammal in combination with one or several additional anti-cancer agents.

A method for the treatment or prevention of a disease characterized by abnormal angiogenesis or hyper-osmotic processes in a human and/or other mammal, the method comprising administering an anabolic product according to aspect 2 to the human or other mammal.

The method as in aspect 23, for treating or preventing a disease characterized by abnormal angiogenesis or hyper-osmotic processes in a human and/or other mammal, comprising administering an anabolic product according to aspect 2 to the human or other mammal simultaneously with an additional anti-angiogenic drug, in the same formulation or in a different formulation.

A method for treating or preventing one or more of the following conditions in a human and/or other mammal:

tumor growth, retinopathy, ischemic retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration; rheumatoid arthritis, psoriasis, bullous diseases associated with epidermoid blister formation, including bullous pemphigoid, erythema multiforme or dermatitis herpetiformis, rheumatoid arthritis, osteoarthritis, septic arthritis, tumor metastasis, periodontal disease, corneal ulceration, proteinuria and coronary thrombosis from atherosclerotic plaques, aneurysmal aortic disease, birth control, dystrophic epidermolysis bullosa, degenerative cartilage loss following traumatic joint injury, osteopenia mediated by MMP activity, temporomandibular joint disease or demyelinating diseases of the nervous system,

the method comprises administering an anabolic product according to aspect 2 to a human or other mammal.

A method for treating or preventing one or more of the following conditions, as well as another condition, in a human and/or other mammal: tumor growth, retinopathy, ischemic retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration; rheumatoid arthritis, psoriasis, bullous diseases associated with epidermoid blister formation including bullous pemphigoid, erythema multiforme or dermatitis herpetiformis;

the other condition is selected from:

rheumatic fever, bone resorption, postmenopausal osteoporosis, sepsis, gram-negative sepsis, septic shock, endotoxic shock, toxic shock syndrome, systemic inflammatory response syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), Helmholter's reaction, asthma, adult respiratory distress syndrome, acute pulmonary fibrosis, pulmonary sarcoidosis, allergic respiratory disease, silicosis, coal dust lung, alveolar injury, liver failure, liver disease during acute inflammation, severe alcoholic hepatitis, malaria (Plasmodium falciparum and cerebral malaria), non-insulin dependent diabetes mellitus (NIDDM), congestive heart failure, post-cardiac injury, atherosclerosis, Alzheimer's disease, acute encephalitis, brain injury, multiple sclerosis (demyelination and oligodendrocyte loss in multiple sclerosis) Advanced cancers, lymphoid malignancies, pancreatitis, poor wound healing in infection, inflammation and cancer, myelodysplastic syndrome, systemic lupus erythematosus, biliary cirrhosis, intestinal necrosis, radiation injury, toxicity following administration of monoclonal antibodies, host versus graft reactions (ischemia reperfusion injury and allograft rejection of kidney, liver, heart, and skin), lung allograft rejection (obliterative bronchitis), and complications due to total hip replacement,

the method comprises administering an anabolic product according to aspect 2 to a human or other mammal.

A method for treating or preventing one or more of the following conditions and an infectious disease in a human and/or other mammal: tumor growth, retinopathy, diabetic retinopathy, ischemic retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration; rheumatoid arthritis, psoriasis, bullous diseases associated with epidermolysis bullosa, bullous pemphigoid, erythema multiforme and dermatitis herpetiformis,

the infectious disease is selected from:

tuberculosis, helicobacter pylori infection during peptic ulcer disease, chagas disease caused by trypanosoma cruzi infection, shiga-like toxin effect caused by escherichia coli infection, enterotoxin a effect caused by staphylococcus infection, meningococcal infection and infection from borrelia burgdorferi, treponema pallidum infection, cytomegalovirus infection, influenza virus infection, taylor encephalomyelitis virus infection and Human Immunodeficiency Virus (HIV) infection;

the method comprises administering an anabolic product according to aspect 2 to a human or other mammal.

Aspect 28. the method as in aspect 22, wherein the additional anti-cancer agent is selected from asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, asparaginase, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, doxorubicin (adriamycin), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, folinic acid, lomustine, nitrogen mustard, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifene, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine, vindesine, aminoglutethimide, L-asparaginase, azathioprine, 5-azadrobine, Busulfan, diethylstilbestrol, 2' -difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine, ethinylestradiol, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartic acid (PALA), plicamycin, semustine, teniposide, testosterone propionate, thiotepa, trimethylmelamine, uridine and vinorelbine, oxaliplatin, gemcitabine, capecitabine, epothilones and natural or synthetic derivatives thereof, tositumomab, trabebedecretin and temozolomide, trastuzumab, cetuximab, bevacizumab, valacil, valvacizumab, and temozolol, Pertuzumab, ZD-1839 (Iressa), OSI-774 (Tarceva), CI-1033, GW-2016, CP-724,714, HKI-272, EKB-569, STI-571 (gleevec), PTK-787, SU-11248, ZD-6474, AG-13736, KRN-951, CP-547,632, CP-673,451, CHIR-258, MLN-518, AZD-2171, PD-325901, ARRY-142886, suberoylanilide hydroxamic acid (SAHA), LAQ-824, LBH-589, MS 275-275, FR 901228, bortezomib, and CCI-779.

Aspect 29 the method as in aspect 22, wherein the additional anti-cancer agent is a cytotoxic agent selected from the group consisting of DNA topoisomerase I and II inhibitors, DNA intercalators, alkylating agents, antimetabolites, cell cycle blockers, microtubule interferents, and Eg5 inhibitors.

The method as in aspect 22, wherein the additional anti-cancer agent is selected from the group consisting of growth factor receptor signaling inhibitors, histone deacetylase inhibitors, PKB pathway inhibitors, Raf/MEK/ERK pathway inhibitors, mTOR pathway inhibitors, and proteasome inhibitors.

A method for treating or preventing one or more of the following conditions in a human and/or other mammal:

rheumatic fever, bone resorption, postmenopausal osteoporosis, sepsis, gram-negative sepsis, septic shock, endotoxic shock, toxic shock syndrome, systemic inflammatory response syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), Helmholter's reaction, asthma, adult respiratory distress syndrome, acute pulmonary fibrosis, pulmonary sarcoidosis, allergic respiratory disease, silicosis, coal dust lung, alveolar injury, liver failure, liver disease during acute inflammation, severe alcoholic hepatitis, malaria (Plasmodium falciparum and cerebral malaria), non-insulin dependent diabetes mellitus (NIDDM), congestive heart failure, post-cardiac injury, atherosclerosis, Alzheimer's disease, acute encephalitis, brain injury, multiple sclerosis (demyelination and oligodendrocyte loss in multiple sclerosis) Advanced cancers, lymphoid malignancies, pancreatitis, poor wound healing in infection, inflammation and cancer, myelodysplastic syndrome, systemic lupus erythematosus, biliary cirrhosis, intestinal necrosis, psoriasis, radiation injury, toxicity following administration of monoclonal antibodies, host versus graft reactions (ischemia reperfusion injury and allograft rejection of kidney, liver, heart, and skin), lung allograft rejection (obliterative bronchitis), and complications due to total hip replacement,

the method comprises administering an anabolic product according to aspect 2 to a human or other mammal.

A method for treating or preventing one or more of the following conditions in a human and/or other mammal:

tuberculosis, helicobacter pylori infection during peptic ulcer disease, Chagas disease caused by Trypanosoma cruzi infection, Shiga-like toxin effect caused by Escherichia coli infection, enterotoxin A effect caused by staphylococcal infection, meningococcal infection and infection from Borrelia burgdorferi, Treponema pallidum infection, cytomegalovirus infection, influenza virus infection, Tayloencephalomyelitis virus infection and Human Immunodeficiency Virus (HIV) infection,

the method comprises administering an anabolic product according to aspect 2 to a human or other mammal.

Aspect 33. a method for treating or preventing osteoporosis, inflammation and angiogenesis disorders other than cancer in a human and/or other mammal by administering to said mammal an effective amount of an anabolic product of aspect 2.

A method for treating or preventing cancer in a human or other mammal comprising administering a single active ingredient to the human or other mammal in combination with inhibition of raf/MEK/ERK pathway mediated tumor cell proliferation and inhibition of PDGF and VEGF mediated angiogenesis, wherein the active ingredient comprises an anabolic product according to aspect 2.

The method of aspect 35. aspect 34, wherein said inhibition of tumor cell proliferation is caused by inhibition of raf kinase and said inhibition of angiogenesis is caused by dual inhibition of PDGFR- β and VEGFR-2 kinase.

Aspect 36. a method for treating or preventing cancer in a human or other mammal comprising administering a single active ingredient to the human or other mammal in combination with inhibition of raf pathway mediated tumor cell proliferation and inhibition of PDGF or VEGF mediated angiogenesis, wherein the active ingredient comprises an anabolic product according to aspect 2.

Aspect 37. a method for treating and/or preventing a disease and/or disorder in a subject in need thereof, comprising administering an effective amount of an anabolic product according to aspect 2.

The method of aspect 38. aspect 37, wherein the method comprises causing regression of a tumor in the subject or cells derived therefrom.

The method of aspect 39, aspect 37, wherein the method comprises inhibiting lymphangiogenesis.

The method of aspect 40. aspect 37, wherein the method comprises inhibiting angiogenesis.

Aspect 41 the method of aspect 37, wherein the method comprises inhibiting lymphangiogenesis and angiogenesis.

The method of aspect 42. aspect 37, wherein the method comprises stimulating the proliferation of hematopoietic progenitor cells.

The method of aspect 43. aspect 37, wherein the method comprises treating a disorder mediated by raf, VEGFR-2, VEGFR-3, PDGFR-beta, p38 and/or flt-3 in a mammalian subject.

The method of aspect 44. aspect 37, wherein the method comprises determining whether a disorder is modulated by the compound, comprising measuring expression or activity of raf, VEGFR-2, VEGFR-3, PDGFR- β, p38 and/or flt-3 in a sample comprising cells or cell extracts, which is modulated by the compound when the expression or activity in the disorder is different from a normal control, wherein the sample is obtained from a subject or cell having the disorder.

Aspect 45 the method of aspect 44, further comprising comparing expression in said sample and a normal control.

Aspects 46. aspect 37, wherein the method comprises evaluating the efficacy of the compound in treating a disorder, comprising administering the compound, measuring the expression or activity of raf, VEGFR-2, VEGFR-3, PDGFR- β, p38 and/or flt-3, and determining the effect of the compound on the expression or activity.

Aspect 47. the method of aspect 37, wherein the method comprises determining the presence of raf, VEGFR-2, VEGFR-3, PDGFR- β, p38 and/or flt-3 in a sample of biological material, contacting the sample with the compound and determining whether the compound binds to the material.

The method of aspect 48. aspect 37, wherein the method comprises treating a tumor in a subject in need thereof, comprising administering an effective amount of the compound, wherein the amount is effective to inhibit tumor cell proliferation and neovascularization.

Aspect 49 the anabolic product according to aspect 2, which is an M-2 or M-5 metabolite having the structural formula below, or a salt thereof, or a prodrug thereof or an isolated stereoisomer thereof:

metabolite M-2

Metabolite M-5

Aspect 50 a method for treating or preventing a tumor or vascular disease in a subject, comprising administering an anabolic product according to aspect 49 to the subject.

Aspect 51. a method of making an anabolic product according to aspect 2, comprising contacting the compound of formula I or a salt thereof with a liver microsome preparation and isolating the metabolite from the preparation under conditions sufficient for metabolism of the compound of formula I.

Drawings

The various features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in connection with the accompanying drawings in which like reference characters designate the same or similar parts throughout the several views, and wherein:

figure 1 shows metabolites of regorafenib from in vitro and in vivo studies.

FIG.2 shows the kinase selectivity profile of regorafenib and its N-oxide (M-2) and demethylated N-oxide (M-5) anabolic products.

Figure 3 shows that M-2 and M-5 anabolic products significantly inhibited tumor growth of human xenografts in mice.

FIG.4 shows that regorafenib and its anabolic products M-2 and M-5 inhibit acute hypotensive effects of VEGF in anesthetized rats.

FIG.5 shows that M-2 anabolic products of regorafenib affect vascular extravasation after a single oral dose of 7.5mg/kg in the rat GS9L glioblastoma model. The determination was performed using kinetic DCE-MRI analysis.

Figure 6 shows plasma concentrations of regorafenib, M-2, and M-5 anabolic products at day 1 and day 21 after administration of 160mg regorafenib co-precipitated tablets to patients with colorectal cancer (n =9, initial data). Data were from the same study as shown in table 4.

Examples

The invention is further illustrated by the following non-limiting examples.

Abbreviations used in this specification are as follows:

preparation of 4-amino-3-fluorophenol

A dry flask purged with argon was charged with 10% Pd/C (80 mg), followed by a solution of 3-fluoro-4-nitrophenol (1.2 g, 7.64 mmol) in ethyl acetate (40 mL). The mixingIn the presence of H₂Stirred under atmosphere for 4 hours. The mixture was filtered through a pad of celite and the solvent was evaporated under reduced pressure to give the desired product as a tan solid (940 mg, 7.39 mmol; yield 97%); 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 carboxamide

A solution of 4-amino-3-fluorophenol (500 mg, 3.9 mmol) in N, N-dimethylacetamide (6 mL) cooled to 0 ℃ was treated with potassium tert-butoxide (441 mg, 3.9 mmol) and the brown solution was stirred at 0 ℃ for 25 minutes. To the mixture was added a solution of 4-chloro-N-methyl-2-pyridinecarboxamide (516 mg, 3.0 mmol) in dimethylacetamide (4 mL). The reaction was heated at 100 ℃ for 16 hours. The mixture was cooled to room temperature and washed with H₂O (20 mL) was quenched and extracted with ethyl acetate (4X 40 mL). Combined organic phases with H₂O (2X 30 mL) and dried (MgSO)₄) And evaporated to yield a reddish brown oil. 1H-NMR showed the presence of residual dimethylacetamide, so the oil was dissolved in diethyl ether (50 mL) and further washed with brine (5X 30 mL). The organic layer was dried (MgSO)₄) And concentrated to give 950mg of the desired product as a red-brown solid, which was used in the next step without purification.

The preparation of 4-chloro-N-methyl-2-pyridinecarboxamide is described in Bank ston et al, org. Proc. Res. Dev.2002,6(6), 777-.

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

To a solution of 4- (4-amino-3-fluorophenoxy) pyridine-2-carboxylic acid carboxamide (177 mg, 0.68 mmol) in toluene (3 mL) was added 4-chloro-3- (trifluoromethyl) phenyl isocyanate (150 mg, 0.68 mmol). The mixture was stirred at room temperature for 72 hours. The reaction was concentrated under reduced pressure and the resulting residue was triturated with ether. The resulting solid was collected by filtration and dried under vacuum for 4 hours to obtain the title compound (155 mg, 0.32 mmol; yield 47%); 1H-NMR (DMSO-d6)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)483.06M/z = (M + 1).

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

The compound described in example 1 as the free base (2.0 g) was dissolved in anhydrous tetrahydrofuran (15 mL) and 4M HCl/dioxane (excess) was added. The solution was then concentrated under vacuum to yield 2.32 grams of an off-white solid. The crude salt was dissolved in hot ethanol (125 mL), activated carbon was added and the mixture was heated at reflux for 15 minutes. The hot suspension was filtered through a pad of celite 521 and cooled to room temperature. The flask was placed in a refrigerator overnight. The crystalline solid was collected by suction filtration, washed with ethanol, then hexane and air dried. The mother liquor was concentrated and crystallization (in the refrigerator) was carried out overnight. The second batch of solids was collected and combined with the first batch. The colorless salt was dried in a vacuum oven at 60 ℃ for two days. The yield of the hydrochloride obtained was 1.72g (79%).

Melting point: 215 ℃ C

Elemental analysis:

example 3: preparation of 4{4- [3- (4-chloro-3-trifluoromethylphenyl) -ureido ] -3-fluorophenoxy } -pyridine-2-carboxylic acid methylamide methanesulfonate

The free base form of the compound described in example 1 (2.25 g) was dissolved in ethanol (100 mL) and methanesulfonic acid stock solution (excess) was added. The solution was then concentrated in vacuo to give a yellow oil. Ethanol was added and concentrated again to give 2.41g of an off-white solid. The crude salt was dissolved in hot ethanol (about 125 mL) and then slowly cooled to crystallize. After reaching room temperature, the flask was placed in a refrigerator overnight. Collecting colorless crystal substances through suction filtration; the resulting filter cake was washed with ethanol, then hexane and air dried to give 2.05g of material, which was dried overnight at 60 ℃ in a vacuum oven.

Melting point: 231 ℃ C

Elemental analysis:

example 4: preparation of 4{4- [3- (4-chloro-3-trifluoromethylphenyl) -ureido ] -3-fluorophenoxy } -pyridine-2-carboxylic acid methylamide phenylsulfonate

The compound described in example 1 as the free base (2.25 g) was suspended in ethanol (50 mL) and benzenesulfonic acid (0.737 g) dissolved in ethanol (50 mL) was added. The mixture was heated under vigorous stirring. All solid material dissolved to give a reddish solution. The solution was cooled to room temperature and the flask was scraped. Crystals were difficult to form, some seeds were found, added to the solution and left in the refrigerator overnight. A light Grayish tan (Grayish-tan) solid formed in the flask; the material was broken up by suction filtration and collected. The solid was washed with ethanol, then hexane and air dried. The weighed product: 2.05g, yield 69%.

Melting point: 213 deg.C

Elemental analysis:

example 5: preparation of the anabolic product of the Compound of formula I

As a representative example, an anabolic product of a compound of formula I may be obtained by incubating a parent regorafenib compound with liver microsomes.

The metabolites of the present application may also be obtained by synthetic methods.

The metabolites of the present application can be purified using techniques known in the art.

Example 6: metabolic profile in animals

N-oxide (M-2) and hydroxymethyl (M-3) metabolites were identified in vitro as metabolites of regorafenib by incubation with liver enzymes of various mammalian species (human, canine, rat, mouse). Studies have shown that regorafenib and its metabolites exhibit high protein binding rates in human and animal species.

The metabolic profile is shown in figure 1 and table 1 below.

TABLE 1 [14C ] metabolism profile of regorafenib (20 μ M) incubated with different species of liver microsomes (protein concentration 0.5mg/mL, 60 min).

Example 7 pharmacological Condition

After oral administration of 10mg/kg regorafenib to mice for 5 days, exposure of the N-oxide (M-2) accounted for about 16% of the total AUC (R + M-2+ M-5), while the contribution of M-5 relative to the total AUC was about 2%. The data are shown in table 2 below. Regorafenib exposure reached about 17% of total exposure after oral administration of 10mg/kg M-2 to mice, indicating that reduction of N-oxide is a relevant metabolic pathway in vivo, while M-5 accounts for 5% of total exposure.

TABLE 2 pharmacokinetic parameters of regorafenib and its metabolites M-2 and M-5 at steady state following oral administration of regorafenib and its metabolites M-2 and M-5 to female NMRI-Foxn-1 mice.

Example 8 broad selectivity profiles for kinase panels

The broad selectivity profile of the kinase panel of regorafenib and its metabolites M-2 and M-5 was performed by Ambit Biosciences (San Diego, CA, US) using an active site competition binding assay (Fabian et al. Nat Biotechnol 2005;23: 329-336). A total of 402 kinases were analysed using a single dose of 1 μ M compound. Binding inhibition activity was expressed as a percentage of kinase still bound compared to DMSO-treated controls. The potency of the compounds is reflected by the ring size.

In a competitive binding assay, M-2 and M-5 show similar but different kinase selectivity profiles than regorafenib. The results are shown in fig. 2.

Example 9 Biochemical characterization of M-2 and M-5 metabolites

Both M-2 and M-5 characteristics show potent pharmacological activity. In biochemical kinase assays, M-2 and M-5 showed similar but different inhibition profiles than regorafenib. In cellular assays, M-2 and M-5 inhibited key targets such as Vascular Endothelial Growth Factor (VEGF) receptor 2, TIE-2, and mutant and wild-type c-KIT and B-RAF, the IC50 value for M-2 was very similar to that of regorafenib and the IC50 value for M-5 was slightly higher than that of regorafenib. The data are shown in table 3.

TABLE 3 pharmacological activity of regorafenib, M-2, and M-5 in cellular kinase phosphorylation assays.

Example 10 tumor growth inhibition by M-2 and M-5 metabolites

Human and rat tumor models were used to determine the in vivo effects of M-2 and M-5 anabolic products on tumor growth and tumor vasculature.

In these studies, mice bearing xenografts of the human breast cancer cell line MDA-MB-231 (K-RASG 13D, B-RAFG 464V) or the human colorectal cancer cell line HT-29 (B-RAFV 600E) were treated with 10mg/kg of regorafenib, M-2 or M-5 orally (daily x 27) starting on day 11 after receiving the tumor. Tumor growth inhibition is given as relative tumor area (p <0.05 vs. solvent in all cases (vehicle); n =8 mice/group). The tumor growth inhibition assay was performed using DCE-MRI.

As shown in figure 3, both orally administered anabolics exhibited potent dose-dependent Tumor Growth Inhibition (TGI) in both HT-29 colorectal and MDA-MB-231 breast cancer xenografts in preclinical mice, achieving significant 62/58% and 54/50% TGI, respectively, compared to the 10mg/kg solvent control group (figure 3).

Example 11 inhibition of VEGF-induced hypotension

The acute effect of the anabolic products on Vascular Endothelial Growth Factor (VEGF) -induced hypotension was studied using a rat pharmacodynamic model.

In these studies, intravenous bolus injection of VEGF (9. mu.g/kg) was initially used to induce transient hypotension in rats (see inset of FIG. 4). The antagonism of regorafenib and its anabolic products against VEGF-induced hypotension was subsequently investigated. For this purpose, regorafenib, M-2 or M-5 was administered at the indicated doses after 10 minutes of intravenous pretreatment with solvents (FIG. 4). The reduction (difference between pre-injection and minimum) in diastolic (blue bar) and systolic (orange bar) pressures was calculated. The results are shown in FIG. 4.

Example 12 Effect on tumor vasculature

The pharmacodynamics of regorafenib and M-2 on tumor vasculature in vivo was analyzed by DCE-MRI analysis using the macromolecular contrast agent Gadomer-17 in GS9L rat fischer rates carrying glioblastoma. In these studies, rats bearing glioblastoma in their thighs were treated with a single dose of 7.5mg/kg M-2 or regorafenib and analyzed for inhibition of Gadomer-17 exudation at 2,6 and 24 hours post-treatment.

The results are shown in fig.5, the left panel shows the results for regorafenib and the right panel shows the results for M-2 anabolic products. The Y-axis on the left shows tumor AUC values, normalized to thigh muscle. The right Y-axis shows the serum levels of regorafenib, M-2, M-4 and M-5. Thus, the contributions of regorafenib, M-2, M-4, and M-5 can be determined separately.

Example 13 in vivo pharmacological Studies

In colon cancer patients treated daily with 160mg co-precipitated tablets for 21 days, M-2 and M-5 showed a systemic exposure very similar to regorafenib. The results are shown in Table 4 and FIG.6 (B). As shown in table 4, the biotransformation of regorafenib in patients with colorectal cancer resulted in significantly elevated levels of demethylated and oxidized M-2 and M-5 anabolic products. The anabolic products were observed after one or more daily administrations with 160mg co-precipitated tablets.

Continuous daily dosing for 19 days in patients with colorectal cancer results in a 25-fold and 2-fold increase in AUC for M-5 and M-2 anabolic products, respectively. Cmax values were similarly increased 42-fold and 5-fold, respectively. These values are reflected in the comparison with the initial dose.

To further characterize the metabolites, plasma concentrations of regorafenib, M-2, and M-5 were observed daily on days 1 and 21 after administration of 160mg regorafenib co-precipitated tablets to patients with colorectal cancer (n =9, initial data). The results are shown in FIG. 6. Multiple peaks of plasma concentrations of regorafenib, M-2 and M-5 were observed, most likely due to enterohepatic circulation. The overall Cmax level of the parent compound and M2/M5 metabolite was 11mg/L on day 21. The pharmacologically relevant total plasma concentration was estimated to be about 2.5mg/L for 3 days after the last administration.

TABLE 4 pharmacokinetic parameters (geometric mean (% CV), initial data) of regorafenib, M-2, and M-5 on days 1 and 21 after administration of 160mg regorafenib co-precipitated tablet to patients with colorectal cancer

Example 14 c-raf (raf-1) Biochemical assay

The c-raf biochemical assay was performed with the c-raf enzyme activated (phosphorylated) by Lck kinase. Lck activated c-raf (Lck/c-raf) was originally prepared in Sf9 insect cells by co-infecting the cells with baculovirus expressing GST-c-raf (from amino acid 302 to amino acid 648) and Lck (full length) under the control of the polyhedrin promoter. The multiplicity of infection of the two baculoviruses used was 2.5 and the cells were harvested 48 hours post infection.

The MEK-1 protein was prepared in Sf9 insect cells by infecting the cells with a baculovirus expressing the GST-MEK-1 (full-length) fusion protein at a multiplicity of 5, and the cells were harvested 48 hours post infection. Similar purification procedures were used for GST-c-raf 302-648 and GST-MEK-1.

Transfected cells were suspended at 100mg wet cell biomass/mL in a buffer containing 10mM sodium phosphate, 140mM sodium chloride pH 7.3, 0.5% Triton X-100, and a protease inhibitor cocktail. The cells were disrupted with a Polytron homogenizer and centrifuged at 30,000g for 30 minutes. The 30,000g supernatant was applied to GSH-Sepharose. The resin was washed with a buffer containing 50mM Tris, pH 8.0, 150mM NaCl and 0.01% Triton X-100. The glutathione transferase-labeled protein was eluted with a solution containing 100mM glutathione, 50mM Tris, pH 8.0, 150mM NaCl and 0.01% Triton X-100. The purified protein was dialyzed into a buffer containing 20mM Tris, pH 7.5, 150mM NaCl and 20% glycerol.

Test compounds were serially diluted in DMSO using 3-fold diluent to stock concentrations typically ranging from 50 μ M to 20nM (final concentration in the assay is from 1 μ M to 0.4 nM). The c-Raf biochemical assay was performed as a radioactive filter mat assay in Costar 96 well polypropylene plates (Costar 3365). The plate was loaded with 75. mu.L of solution containing 50mM HEPES pH 7.5, 70mM NaCl, 80ng Lck/c-raf and 1. mu.g MEK-1. Subsequently, 2 μ L of each of the serially diluted compounds was added to the reaction, followed by addition of ATP. The reaction was initiated with 25. mu.L of ATP solution containing 5. mu. MATP and 0.3. mu. Ci [33P ] -ATP. The plates were sealed and incubated at 32 ℃ for 1 hour. The reaction was quenched by the addition of 50 μ L of 4% phosphoric acid and collected onto P30 filtermat (perkinelmer) using a Wallac Tomtec harvester. The filter cotton was washed with 1% phosphoric acid and then with deionized water. The filters were microwave dried, soaked in scintillation fluid and read in a Wallac 1205Betaplate counter (Wallac inc., atlanta, GA, usa). The results are expressed as percent inhibition.

% inhibition = [100- (Tib/Ti) ] × 100, wherein

Tib = (counts per minute with inhibitor) - (background)

Ti = (counts per minute without inhibitor) - (background)

The compounds of the invention showed potent inhibition of raf kinase in this assay.

Example 15: in vitro assay for p38 kinase

P38 (expressed in E.coli) purified and tagged with histidine was activated in vitro by MMK-6 to a high specific activity. Using a microtiter format, all reactions were performed in 100 μ L volumes, and the reactions were diluted with assay buffer (25 mM HEPES 7.4, 20mM MgCl2, 150mM NaCl) to yield 0.05 μ g/well of activated p38 and 10 μ g/well of myelin basic protein. Test compounds (5 μ Ι _ of 10% DMSO in water) were prepared and diluted into the assay buffer to include a final concentration range from 5nM to 2.5 μ Μ. The kinase assay was initiated by adding 25. mu.L of ATP mix to a final concentration of 10. mu.M cold ATP and 0.2. mu. Ci [ gamma. -33P ] ATP per well (200-400 dpm/pmol ATP). The plates were incubated at 32 ℃ for 35 minutes and the reaction was quenched with 7 μ Ι _ of 1N aqueous HCl. The resulting samples were collected on a P30 Filtermat (Wallac, Inc.) using a TomTec 1295 collector (Wallac, Inc.) and counted in a LKB 1205Betaplate liquid scintillation counter (Wallac, Inc.). Negative controls included only substrate plus ATP. SW1353 cell assay: SW1353 cells (human chondrosarcoma) were seeded (1000 cells/100 μ L DMEM 10% FCS/well) in 96-well plates and incubated overnight. After replacing the medium, cells were contacted with test compound for 1 hour at 37 ℃, at which time human IL-1 (1 ng/mL, Endogen, Woburn, WA) and recombinant human TNF α (10 ng/mL) were added. Cultures were incubated at 37 ℃ for 48 hours and the IL-6 value of the supernatant was then determined by ELISA. The compounds of the present invention showed significant inhibition of p38 kinase.

Example 16: Bio-Plex Phospho-ERK 1/2 immunoassay

A 96-well pERK immunoassay using a laser flow cytometer (Bio-Rad) platform was established to measure inhibition of basal pERK in breast cancer cell lines. MDA-MB-231 cells were plated at 50,000 cells/well in complete growth medium in 96-well microtiter plates. For the effect of test compounds on basal pERK1/2 inhibition, the next day after plating, MDA-MB-231 cells were transferred into DMEM with 0.1% BSA and incubated with test compounds diluted 1:3 in 0.1% DMSO to a final concentration of 3 μ M to 12 nM. Cells were incubated with test compounds for 2 hours, washed, and lysed in Bio-Plex whole cell lysate A. Samples were diluted with buffer B1: 1 (v/v) and transferred directly to assay plates or frozen at-80 ℃ until processing. 50 μ L of diluted MDA-MB-231 cell lysate was incubated with approximately 2000 Bio-Plex beads (conjugated with anti-ERK 1/2 antibody) of 5 microns overnight at room temperature on a shaker. The following day, biotinylated phospho-ERK 1/2 sandwich immunoassay was performed, the beads were washed 3 times during each incubation period and then 50 μ LPE-streptavidin was used as the imaging agent. The relative fluorescence units of pERK1/2 were detected by counting 25 beads with high sensitivity using a Bio-Plex flow cell (probe). IC50 was calculated using an Excel spreadsheet-based program using untreated cells as the maximum and no cells (beads only) as the background.

The compounds of the invention showed significant inhibition in this assay.

Example 17 Flk-1 (murine VEGFR-2) Biochemical assay

The assay was performed in a 96-well opaque plate (Costar 3915) in TR-FRET format. The reaction conditions were as follows: 10 μ M ATP, 25nM poly GT-biotin, 2nM europium-labeled phosphorylated-Tyr Ab, 10nM APC, 7nM Flk-1 (kinase domain), 1% DMSO, 50mM HEPES pH 7.5, 10mM MgCl₂0.1mM EDTA, 0.015% BRIJ, 0.1mg/mL BSA, 0.1% mercaptoethanol. The reaction is initiated by the addition of an enzyme. The final reaction volume in each well was 100. mu.L. Approximately 1.5-2.0 hours after reaction initiation, plates were read on a Perkinelmer Victor V multi-label counting plate at 615 and 665 nM. For each well, the signal is taken as a ratio: (665 nm/615 nm). times.10000.

The compounds of the present invention showed significant inhibitory effect on VEGFR2 kinase.

Example 18: biochemical assay of mouse PDGFR FRET

The assay was performed in a format of 96-well blackboard (Costar 3915). The following reactants were used: europium-labeled anti-phosphotyrosine antibodies pY20, Perand streptavidin-APC, poly GT-biotin, and mouse PDGFR. The reaction conditions were as follows: in assay buffer (50 mM HEPES pH 7.5, 10mM MgCl)₂0.1mM EDTA, 0.015% BRIJ 35, 0.1mg/mL BSA, 0.1% mercaptoethanol) 1nM mouse PDGFR was conjugated with 20 μ MATP, 7nM poly GT-biotin, 1nM pY20 antibody, 5nM streptavidin-APC and 1% DMSO. The reaction is initiated by the addition of an enzyme. The final reaction volume in each well was 100. mu.L. After 90 minutes, the reaction was stopped by adding 10. mu.L/well of 5. mu.M staurosporine. After the reaction had stopped for about 1 hour, plates were read on a Perkin Elmer Victor V multi-label counting plate at 615 and 665 nM. For each well, the signal is taken as a ratio: (665 nm/615 nm). times.10000. The compound of the invention shows a remarkable inhibitory effect on PDGFR kinase.

For IC50 production of PDGFR and Flk-1, compounds were added and then enzyme priming was performed. A50-fold stock solution was prepared with compounds serially diluted 1:3 in 50% DMSO/50% distilled water. Add 2. mu.L of the stock solution to the assay to give the final compound concentration in the range of 10. mu.M-4.56 nM in 1% DMSO. Data are expressed as percent inhibition: % inhibition =100- ((signal with inhibitor-background)/(signal without inhibitor-background)) × 100.

Example 19: MDA-MB231 proliferation assay

Human breast cancer cells (MDA MB-231, NCI) were grown in standard growth medium (DMEM) supplemented with 10% heat-inactivated FBS at 37 ℃ in humidified incubator at 5% CO₂(v/v). Cells were plated at 3000 cells per well in 90 μ L growth medium in 96 well dishes. To determine the T0h CTG value, 100. mu.L of CellTiter was plated 24 hours laterGlo luminescent reagent (Promega) was added to each well and incubated for 30 min at room temperature. Luminescence was recorded in a Wallac Victor II instrument. The CellTiter-Glo reagent causes cell lysis and produces a luminescent signal proportional to the amount of ATP present, which in turn is directly proportional to the number of cells present.

Test compounds were dissolved in 100% DMSO to prepare 10mM stock solutions. The stock was further diluted 1:400 in growth medium to produce a working stock of 25 μ M 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 μ L of diluted test compound was added to each culture well to give a final volume of 180 μ L. Cells with and without each test compound were incubated for 72 hours at which time ATP-dependent luminescence was measured as described previously to generate T72h values. Optionally, the IC50 value can be determined using a least squares analysis program using compound concentration and percent inhibition.

% inhibition = [1- (T72 h test-T0 h)/(T72 h control-T0 h) ] × 100, wherein

T72h test = ATP-dependent luminescence at 72 hours in the presence of test product

T72h control = ATP-dependent luminescence at 72 hours in the absence of test product

T0h = ATP-dependent luminescence at time zero

Using this assay, the compounds of the invention showed significant proliferation inhibition.

Example 20: pPDGFR-beta sandwich ELISA in AoSMC cells

100K P3-P6 Aortic SMCs were plated in SGM-2 in each well of a 12-well cluster at 1000. mu.L volume/well using standard cell culture techniques. The following day, cells were washed once with 1000 μ LD-PBS, and then plasma was starved overnight in 500 μ L of SBM (smooth muscle cell substrate medium) containing 0.1% BSA. Compounds were diluted in DMSO with a 10-fold dilution step at a dose range from 10 μ M to 1 nM. The final DMSO concentration was 0.1%. Old medium was removed by quickly inverting the tank, and then 100 μ L of each dilution was added to the corresponding cell well over 1 hour at 37 ℃. Cells were then stimulated with 10ng/mLPDGF-BB ligand at 37 ℃ for 7 min. The medium was decanted and 150. mu.L of isotonic cell lysis buffer containing protease inhibitor tablets (intact; EDTA-free) and 0.2mM sodium vanadate was added. Cells were lysed on a shaker in a cold room at 4 ℃ for 15 minutes. The lysates were placed in a microcentrifuge tube, to which 15 μ L of agarose conjugated anti-PDGFR- β antibody was added and incubated overnight at 4 ℃. The following day, the beads were washed 3 times with 50 volumes of PBS and boiled in 1 xlds sample buffer for 5 minutes. Samples were electrophoresed on a 3-8% gradient Tris-acetate gel and transferred to nitrocellulose membranes. Membranes were blocked in 1% BSA/TBS-T for 1 hour and then incubated with anti-phosphorylated-PDGFR-. beta. -b (Tyr-857) antibody (1: 1000 dilution) in blocking solution for 1 hour. After 3 washes with TBS-T, the membranes were incubated in goat anti-rabbit HRP IgG (1: 25000 dilution) for 1 hour. Three more washes were performed before adding ECL substrate. The film was exposed to Hyperfilm-ECL. Subsequently, the membrane was peeled off and re-detected with an anti-PDGFR- β antibody against total PDGFR- β (reprobed).

Table a illustrates representative results of in vitro kinase biochemical assays for p38 kinase, PDGFR kinase, and VEGFR2 kinase. All three kinase targets are involved in mesenchymal activation and endothelial cell proliferation, causing angiogenesis and providing blood supply to tumor tissue.

TABLE A

Table B illustrates representative results of two cellular assays for raf kinase activity, which are (i) inhibition of pERK in MDA-MB231 cells, a machine readout for raf kinase activity, and (ii) a proliferation assay for MDA-MB231 cells, a functional assay for raf kinase activity. In addition, table B illustrates the results of PDGFR- β phosphorylation driven by PDGFR in aortic smooth muscle cells, which is a machine readout for PDGFR kinase inhibition.

TABLE B

In summary, the compounds of the present invention provide a unique combination of inhibition of angiogenesis and tumor cell proliferation. It also has an improved inhibition profile for some key kinase targets such as raf, p38, PDGFR and VEGFR-2, all of which are molecular targets of interest for the treatment of osteoporosis, inflammatory diseases and hyperproliferative diseases, including cancer.

It is believed that one skilled in the art can, using the preceding information and information available in the art, utilize the present invention to its fullest extent. It will be apparent to those skilled in the art that variations and modifications of the present invention can be made without departing from the spirit or scope of the invention as set forth herein. All publications, applications and patents cited above and below are incorporated herein by reference.

The subject headings set forth above and below are intended to guide certain information that may be found in the application, but are not intended as the only source of information that may be found in the application with respect to such headings.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Accordingly, the foregoing preferred specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the examples, all temperatures are set forth uncorrected in degrees celsius and all parts and percentages are by weight unless otherwise indicated.

The complete disclosures of all applications, patents, and publications cited herein and of the publications cited below are incorporated by reference into this specification.

1.Wilhelm S et al.“Regorafenib(BAY 73-4506)：identification of clinically relevantmetabolites and their preclinical pharmacology.”American Society of ClinicalOncology(ASCO)Annual Meeting，2010.Abstract no.1666(enclosed).

2.Wilhelm S et al.Mol Cancer Ther 2009；8(suppl)：abs B42.

3.Eisen T et al.Eur J Cancer Suppl 2009；7：424，abs O-71053.

4.Strumberg D et al.J Clin Oncol(Meeting Abstracts)2009；27：16ls，abs 35604.

5.Fabian MA et al.Nat Biotechnol 2005；23：329-336

The foregoing examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the foregoing examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims (4)

1. M2, M3, M4 or M5 anabolic products of the compound of formula I or salts or prodrugs or stereoisomers of said anabolic products, wherein the structures of said compound of formula I and said M2, M3, M4 or M5 anabolic products are:

metabolite M-2

Metabolite M-3

Metabolite M-4

Metabolite M-5

2. The anabolic product according to claim 1, or a salt or a prodrug or a stereoisomer thereof, wherein the M2 or M5 anabolic product has the following structural formula:

metabolite M-2

Metabolite M-5

3. A method of inhibiting tumor growth in a subject comprising administering an anabolic product according to claim 1, or a pharmaceutical composition comprising the anabolic product, to the subject.

4. A method of making an anabolic product according to claim 1, comprising contacting a compound of formula I, or a salt thereof, with a liver microsome preparation and isolating the metabolite from the preparation under conditions sufficient to metabolize the compound of formula I.