HK1079774B - Aryl ureas as kinase inhibitors - Google Patents

Description

Arylureas as kinase inhibitors

Technical Field

The invention relates to an aryl urea compound and a synthetic method thereof. The compounds of the invention are useful in the treatment of the following diseases:

(i) raf-mediated diseases such as cancer,

(ii) p38 mediated diseases such as inflammation and osteoporosis, and

(iii) VEGF mediated diseases such as angiogenic diseases.

Background

Activation of the Ras signaling pathway represents a cascade of events that have a profound effect on cell proliferation, differentiation and deformation. Raf kinase, a downstream effector of Ras, is a key mediator of the transmission of these signals from cell surface receptors to the nucleus (Lowy, D.R.; Willumsen, B.M.Ann.Rev.biochem.1993, 62, 851; Bos, J.L.cancer Res.1989, 49, 4682). It has been shown that administration of a inactivated antibody to raf kinase inhibits the raf kinase signaling pathway or co-expresses either a major negative raf kinase or a major negative MEK (substrate for raf kinase), thereby inhibiting the action of active ras and thereby causing the reversion of the phenotype of the deformed cells towards normal growth (see: Daum et al, Trends biochem. Sci.1994, 19, 474-80; Fridman et al, J.biol. chem.1994, 269, 30105-8). Kolch et al (Nature 1991, 349, 426-28) and further demonstrated that inhibition of raf expression by antisense RNA blocks cell proliferation in cell membrane-associated oncogenes. Similarly, inhibition of raf kinase (by antisense oligodeoxyribonucleic acids) has been associated with inhibition of growth of various human tumor types in vitro and in vivo (Monia et al, nat. Med.1996, 2, 668-75). Thus, small molecule inhibitors of Raf kinase activity are important drugs for the treatment of Cancer (Naumann, U.S.; Eisenmann-Tappe, I.; Rapp, U.R. RecentrtResults Cancer Res.1997, 143, 237; Monia, B.P; Johnston, J.F.; Geiger, T..; Muller, M.S.; Fabbro, D.Nature Medicine 1996, 2, 668).

Inhibition of p38 has been shown to inhibit the production of cytokines (e.g., TNF α, IL-1, IL-6, IL-8) and proteolytic enzymes (e.g., MMP-1, MMP-3) in vitro and/or in vivo. 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.; Blumethal, 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.; Yound, P.R.Nature 1994, P.R.372, 739).

Clinical studies have linked TNF α production and/or signaling with a variety of diseases including rheumatoid arthritis (maini.j. royal gel. physicians London 1996, 30, 344). In addition, excessive levels of TNF α are implicated in a variety of inflammatory and/or immunomodulatory diseases, including acute rheumatic fever (Yegin et al, Lancet 1997, 349, 170), bone resorption (Pacific et al, J.Clin.Endocrinol.Metabol.1997, 82, 29), postmenopausal osteopetrosis (Pacific et al, J.bone Mineral Res.1996, 11, 1043), septicemia (Blackwell et al, Br.J.Anaesth.1996, 77, 110), gram-negative leukemia (Debets et al, prog.Clin.Biol.Res.1989, 308, 463), septic shock (Tracey et al, Nature 1987, 330, 662; Girardin et al, New England J.Im.1988, 397, endotoxin shock (Begeler et al, Science 5, Sauter et al, Sagna 1989, 1995, Nature et al, Nature Medhol.1989, Microbiol.1989, USA), systemic inflammatory response (Occidum et al, USA) 1, Nature et al, Nature J.1989, Nature J.17, Nature et al, Nature J.17, Nature et al, Nature J. 1989, Nature J. 15, Nature et al, Nature J. 15, Nature syndrome J. mounting J.19, Christenergym et al, inflammatory response shock (Bengalenic shock, J.19, J. 7, USA), endotoxin shock, J.19, Christein, J. 7, USA., USA, 47, 97) include crohn's disease (vanDeventer et al, aliment. pharmacol. therapeu.1996, 10(suppl.2), 107; vanDullemen et al, Gastroenterology 1995, 109, 129) and ulcerative colitis (Masuda et al, j.clin.lab.immunol.1995, 46, 111), ja-hertz reaction (Fekade et al, New England j.med.1996, 335, 311), asthma (amarani et al, rev.malad.respir.1996, 13, 539), adult respiratory distress syndrome (Roten et al, am.rev.respir.dis.1991, 143, 590; suter et al, am. rev. respir. dis.1992, 145, 1016), acute pulmonary fibrotic disorders (Pan et al, pathol. int.1996, 46, 91), pulmonary Sarcoidosis (Ishioka et al, sarcodosis Vasculitis Diffuse Lung dis.1996, 13, 139), allergic respiratory disorders (Casale et al, am. j. respir. cell mol. biol.1996, 15, 35), stone end deposition disorders (Gossart et al, j. immunol.1996, 156, 1540; vanhee et al, eur. respir j.1995, 8, 834), coal mining lung dust deposition disease (Borm et al, am. rev. respir. dis.1988, 138, 1589), alveolar injury (horiouchi et al, am. j. respir. cell mol.biol.1996, 14, 1044), liver failure (Gantner et al, j. pharmacol. exp.thermal.1997, 280, 53), liver disease during acute inflammation (Kim et al, j.biol. chem.1997, 272, 1402), severe alcoholic hepatitis (Bird et al, ann. lnter. med.1990, 112, 917), malaria (Grau et al, munol. rev.1989, 112, 49; taveme et al, parasitol.today 1996, 12, 290) include plasmodium falciparum malaria (Perlmann et al, infection.immunet.1997, 65, 116) and cerebral malaria (Rudin et al, am.j.pathol.1997, 150, 257), non-insulin dependent diabetes mellitus (NIDDM; stephens et al, j.biol.chem.1997, 272, 971; ofei 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), injury after heart disease (Malkiel et al, mol.med.today 1996, 2, 336), atherosclerosis (parkus et al, j.pathol.1996, 179, a46), Alzheimer's disease (fagarsan et al, Brain res.1996, 723, 231; aisen et al, Gerontology1997, 43, 143), acute encephalitis (Ichiyama et al, J.neurol.1996, 243, 457), brain injury (Cannon et al, Crit.Care Med.1992, 20, 1414; hansbrough et al, surg. clin. n. am.1987, 67, 69; marano et al, surg.gynecol.obstetr.1990, 170, 32), multiple sclerosis (m.s.; style.adv.neuroisomunol.1996, 6, 143; matusevicius et al, J.Neuroommunin.1996, 66, 115) including demyelination and oligomendacyte loss during multiple sclerosis (Brosnan et al, Brain Pathol.1996, 6, 243), advanced cancers (MucWierzgon et al, J.biol.Regula Homeostatic Agents 1996, 10, 25), lymphoid malignancies (Levy et al, Crit.Rev.Immunol.1996, 16, 31), pancreatitis (Exley et al, Gut 1992, 33, 1126) including systemic complications in acute pancreatitis (McKay et al, Br.J.Surg.1996, 83, 919), poor wound healing in infections and cancers (Buck et al, am.J.Pathol.1996, 195, 149, myelodysplasia syndrome (Raba. Milza et al, Australian. J.1996, 1988, 1987, 65, bile necrosis et al, bile necrosis (Rheol. J.1988, 1988, 1987, Australia) and bile necrosis (Rheumatoid et al, Rheol. J.358, 19817, 2, Australia J.7, 2, Edman, 2, J. 2, J. 2, J. 7, neurology1996, 46, 1633). TNF α levels are also associated with the following disorders: host-versus-graft responses (Piguet et al, Immunol. Ser.1992, 56, 409) include ischemic reperfusion injury (Colletti et al, J.Clin. invest.1989, 85, 1333) and allograft rejection including kidney (Maury et al, J.Exp. Med.1987, 166, 1132), liver (Imagawa et al, Transplantation 1990, 50, 219), heart (Boling et al, Transplantation 1992, 53, 219), and skin (Stevens et al, Transplantation. Proc.1990, 22, 1924), lung allograft rejection (Grossman et al, Immunol. Allergy Clin. N.Am.1989, 9, 153) include chronic lung allograft rejection (lithangitis; Loganinchi et al, Loceorv. 1990, J.1990, 1989, 99, and Hippo. 99, and hip replacement induced by hip surgery). TNF alpha is also associated with infectious diseases (reviewed: Beutler et al, crit. Care Me.1993, 21, 5423; Degre. biotherapy 1996, 8, 219) including tubercle bacilli (Rook et al, Med. Malad. Infect.1996, 26, 904), helicobacter pylori infection during gastric ulcer disease (Beales et al, Gastroenterology 1997, 112, 136), Chagas disease caused by Trypanosoma cruzi infection (Chandrasekar 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 (Fischer et al, J.Immunol.1990, 144, 4663), meningococcal infection (Warneal et al, Lancet. 1987, 22, 1987, 65, J.31. Scorpion virus), Haematococcus virus infection (Biophys. J.31, J.31. Scorpion, J.31, J.7. Biophys. Scorpion, J.31, J.7, J.Biophys. Biophys, J. Hematodes, J., clin.res.1986, 34, 491a), sendai virus (Goldfield et al, proc.nat 'l.acad.sci.usa 1989, 87, 1490), Theiler's encephalomyelitis virus (Sierra et al, Immunology 1993, 78, 399), and human immunodeficiency virus (HIV; pol i.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).

There are many diseases that are thought to be mediated by excessive or undesirable matrix-destroying metalloprotease (MMP) activity or due to an imbalance in the ratio of MMPs to Tissue Inhibitors of Metalloproteases (TIMPs). These include osteoarthritis (Woessner et al, J.biol. chem.1984, 259, 3633), rheumatoid Arthritis (Mullins et al, Biochim. Biophys.acta 1983, 695, 117; Wooley et al, Arthroritis Rheum.1977, 20, 1231; Gravallese et al, Arthroritis Rheum.1991, 34, 1076), septic Arthritis (Williams et al, Arthroritis Rheum.1990, 33, 533), tumor metastasis (Reich et al, Cancer Res.1988, 48, 3307; Matrisian et al, Proc.Nat. Acnead.Sci., USA 1986, 83, dy3), periodontal disease (Overall et al, J.iodordo.1987, 22, ulcer (BurnS. Acrons. Sci.Sci., USA 19881), coronary atherosclerotic plaque formation (Sterons et al, coronary artery angiosperm et al, 1989, coronary artery angiosperm et al, coronary artery angiosperm 1989, coronary artery angiosperm 62, coronary artery angiosperm 1, coronary artery angiosperm 62, coronary artery angiosperm 1, coronary artery angiosperm 1989, coronary artery angiosperm 1, coronary artery angiosperm 62, coronary artery angiosperm 1, coronary artery angiosperm 62, coronary artery angiosperm et al, coronary artery angiosperm, j. invest, dermaltol, 1982, 79, 208), degenerative cartilage loss following traumatic joint injury, MMP activity-mediated osteopenia, temporomandibular joint disease, and demyelinating diseases of the nervous system (Chantry et al, j. neurohem, 1988, 50, 688).

Because inhibition of p38 results in inhibition of TNF α formation and MMP formation, inhibition of the mitogen-activated protein (MAP) kinase p38 enzyme provides a means for treating the above-mentioned diseases including osteoporosis and inflammatory diseases such as rheumatoid arthritis and COPD (Badger, a.m.; Bradbeer, j.n.; Votta, b.; Lee, j.c.; Adams, j.l.; Griswold, d.e.j.pharm.expert.ther.1996, 279, 1453).

Angiogenesis involves the formation of new blood vessels from endothelial cell precursors or angioblasts. The first vascular structure in the embryo is formed by angiogenesis. Angiogenesis involves the development of capillaries from existing blood vessels and is the primary mechanism by which blood vessels are formed in organs such as the brain and kidneys. Although angiogenesis is limited during embryonic development, angiogenesis phenomena can also occur in adults, for example during pregnancy, the female cycle, or wound healing.

One major regulator of angiogenesis and vasculogenesis in embryonic development as well as in some angiogenesis-dependent diseases is vascular endothelial growth factor (VEGF; also known as vascular permeability factor, VPF). VEGF represents a class of mitogen isozymes that exist as homodimers due to alternative RNA splicing. The VEGF isozymes are 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). Hypoxia (Shweiki et al, Nature 1992, 359, 843) and various cytokines and growth factors such as interleukin-1, interleukin-6, epidermal growth factor and transforming growth factor all induce VEGF expression.

At present, VEGF and VEGF family members have been reported to bind to one or more of three transmembrane receptor tyrosine kinases (Mustonen et al, J.cell biol., 1995, 129, 895), VEGF receptor-1 (also known as flt-i (fms-like tyrosine kinase-1)), VEGFR-2 (also known as a receptor containing a kinase insert domain (KDR); murine analogs of KDR are known as fetal liver kinase-1 (flk-1)), and VEGFR-3 (also known as flt-4). KDR and flt-1 have been shown to have different signal transduction properties (Waltenberger et al, J.biol.chem.1994, 269, 26988); park et al, Oncogene 1995, 10, 135). Thus, KDR undergoes strong ligand-dependent tyrosine phosphorylation in intact cells, whereas flt-1 shows a weak response. In this case, binding to KDR is a critical requirement for eliciting an intact VEGF-mediated biological response profile.

In vivo, VEGF plays an important role in angiogenesis and induces angiogenesis and vascular permeabilization. Deregulated VEGF expression contributes to the development of many diseases characterized by aberrant angiogenesis and/or hyperpermeability processes. Thus, modulation of the VEGF-mediated signaling cascade may provide a useful model for controlling aberrant angiogenesis and/or hyperpermeability processes.

Angiogenesis is considered an absolute requirement for tumor growth above 1-2 mm. Oxygen and nutrients are supplied to the tumor cells below this diffusion threshold. However, each tumor is dependent on angiogenesis after it reaches a certain size in order to continue growing. Tumorigenic cells in the hypoxic region of tumors respond to stimulation of VEGF formation, which triggers activation of quiescent endothelial cells, stimulating neovascularization (Shweiki et al, proc.nat' l.acad.sci., 1995, 92, 768). In addition, VEGF production in tumor regions where there is no angiogenesis is likely to occur via the ras signaling pathway (Grugel et al, J.biol.chem., 1995, 270, 25915; Rak et al, Cancer Res.1995, 55, 4575). In situ hybridization studies have demonstrated that VEGF mRNA is strongly upregulated in a variety of Human tumors, including lung (Mattern et al, Br. J. Cancer 1996, 73, 931), thyroid (Viglietto et al, Oncogene 1995, 11, 1569), breast (Brown et al, Human Pathol.1995, 26, 86), gastrointestinal (Brown et al, Cancer Res.1993, 53, 4727; Suzuki et al, Cancer Res.1996, 56, 3004), kidney and bladder (Brown et al, am. J. Pathol.1993, 1431, 1255), ovary (Olson et al, Cancer Res.1994, 54, 1255), and cervix (Guidi et al, J. Nat' Cancer Inst.1995, 87, 12137) carcinomas, as well as angiosarcoma (Hashimoto et al, Lab. 73, Inv. 859, Natl. tumor, Natl. observer J. 1252, Nature et al, Natl. Col. J. 1992., Phr J. 1259, et al, Nature J. 1252, 1992, et al, Nature J. Onluk. Col. J. Col., 1255). Neutralizing monoclonal antibodies to KDR have been shown to be effective in blocking angiogenesis in tumours (Kim et al, Nature1993, 362, 841; Rockwell et al, mol.cell.Differ.1995, 3, 315).

For example, in extreme oxygen deficient conditions, high expression of VEGF can lead to intraocular angiogenesis, leading to high proliferation of blood vessels, and ultimately blindness. These cascade of events have been observed in a variety of retinopathies, including diabetic retinopathy, ischemic retinal vein occlusion, prematurity retinopathy (Aiello et al, New Engl. J. Med.1994, 331, 1480; Peer et al, Lab. invest.1995, 72, 638), and age-related muscle degeneration (AMD; see: Lopez et al, invest. optonalmol. Vis. Sci.1996, 37, 855).

In Rheumatoid Arthritis (RA), pannus ingrowth is likely mediated by angiogenic factors. The level of immunoreactive VEGF is high in synovial fluid of RA patients, while VEGF levels are low in other forms of arthritic patients or patients with degenerative joint disease (Koch et al, j. immunol.1994, 152, 4149). The angiogenesis inhibitor AGM-170 has been shown to inhibit joint neovascularization in a murine collagen arthritis model (Peacock et al, j.expert.med.1992, 175, 1135).

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

Because inhibition of KDR can result in inhibition of VEGF-mediated angiogenesis and osmosis, KDR inhibitors are useful in the treatment of diseases characterized by aberrant angiogenesis and/or hyperpermeability processes, including those listed above.

Disclosure of Invention

The present invention relates to compounds of formula (I):

wherein:

y is OR¹Or NHR²，

Hal is chlorine or bromine, and Hal is chlorine or bromine,

R¹is H or C₁-C₆An alkyl group, a carboxyl group,

R²is H, OH, CH₃Or CH₂OH，

Z¹And Z²Are each H or OH, wherein Z¹Or Z²Only one of which may be OH,

X¹-X⁷each independently H, OH or O (CO) C_1-4Alkyl radicals, and

n is a number of 0 or 1,

with the following conditions: at least one of the following conditions a-c is satisfied,

a)Z¹or Z²Is an OH group, and is a hydroxyl group,

b)R²is an OH group, and is a hydroxyl group,

c) n is a number of 1, and n is,

or a salt thereof, such as a pharmaceutically acceptable salt thereof, or an isolated stereoisomer thereof (hereinafter collectively referred to as the compounds of the invention). The term stereoisomer is understood to include diastereomers, enantiomers, geometric isomers and the like.

One skilled in the art will recognize that some of the compounds of formula (I) may exist in different geometric isomeric forms. In addition, some of the compounds of the present invention have 1 or more asymmetric carbon atoms and thus may exist in optically active form as well as in the form of racemates or non-racemic mixtures, as well as in the form of diastereomers and diastereomeric mixtures thereof. All such compounds, including cis-isomers, trans-isomers, diastereomeric mixtures, racemates, non-racemic mixtures of enantiomers, substantially pure and pure enantiomers, are included within the scope of the invention. Herein, substantially pure enantiomer means that more than 5% by weight of the corresponding opposite enantiomer is absent.

Optical isomers can be prepared by resolution of the racemic mixture by conventional methods, e.g., using optically active acids or bases to form diastereomeric salts. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Mixtures of diastereomers can be separated into individual diastereomers by methods known to those skilled in the art, e.g., chromatography and fractional crystallization, depending on differences in their physicochemical properties. The optically active base or acid is liberated in the separated diastereoisomeric salt. Another different method of separating optical isomers involves the use of chiral chromatography columns (e.g., chiral HPLC columns), which should be optimally selected to maximize separation of the enantiomers. Suitable chiral HPLC columns are manufactured by Diacel et al, e.g. chiralel OD and chiralel OJ. The optically active compounds of formula (I) can also be prepared by using optically active starting materials.

The invention also includes analogs of the compounds of the invention.

Preferred are compounds of the invention wherein n is 1. These compounds include in particular those in which in formula (I) n is 1 and Y is NHR²And R²Is H or CH₃Wherein n is 1 and X in formula (I)¹-X⁷Compounds of the invention which are each H, where in (I) n is 1 and Z¹And Z²Compounds of the invention of the formula (I), in which n is 1 and Z is H¹Is H and Z²Is OH or Z¹Is OH and Z²The compounds of the invention being H, wherein n in formula (I) is 1 and X¹-X⁷At least one of which is OH or O (CO) C_1-4Alkyl of the compounds of the invention, wherein n is 1 and Y is NHR²And R²Is CH₂OH wherein n is 1 and Y is NHR²And R²Compounds of the invention which are OH, and compounds of the invention wherein n is 1 and Y is OH in formula (I).

Other preferred compounds of the invention are those wherein Z is in formula (I)¹Is H and Z²Is OH or Z¹Is OH andZ²is H. These compounds include in particular, those in which Z is the radical of the formula (I)¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, and n is 0, wherein Z in formula (I)¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, n is 0, Y is NHR²And R²Is H or CH₃Wherein Z in formula (I)¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, n is 0 and X¹-X⁷The compounds of the invention are each H, wherein Z in formula (I)¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, n is 0 and X¹-X⁷At least one of which is OH or O (CO) C_1-4Alkyl compounds of the invention, wherein Z in formula (I)¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, n is 0, Y is NHR²And R is²Is CH₂OH in which Z in formula (I)¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, n is 0, Y is NHR²And R is²The compounds of the invention which are OH, and wherein Z is in the formula (I)¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, n is 0 and Y is OH.

Further preferred compounds of the invention are those wherein Y in formula (I) is NHR²And R is²Is OH. These compounds include in particular those in which Y in formula (I) is NHR²And R is²Is OH and n is 0, wherein Y in formula (I) is NHR²And R is²Is OH, n is 0 and X¹-X⁷Compounds of the invention which are each H, wherein Y in formula (I) is NHR²And R is²Is OH, n is 0 and Z¹And Z²Compounds of the invention which are each H, wherein Y in formula (I) is NHR²And R is²Is OH, n is 0, and Z¹Is H and Z²Is OH or Z¹Is OH and Z²A compound of the invention which is H, and wherein Y in formula (I) is NHR²And R is²Is OH, n is 0 and X¹-X⁷At least one of which is OH or O (CO) C_1-4Alkyl groups.

Also preferred compounds of the invention are those wherein Y in formula (I) is OH. These compounds include, in particular, the compounds of the invention in which Y is OH and n is 0 in formula (I), in which Y is OH and n is 0 and X¹-X⁷Compounds of the invention which are each H, in which in formula (I) Y is OH and n is 0 and Z¹And Z²Compounds of the invention which are each H, in which in formula (I) Y is OH and n is 0 and Z¹Is H and Z²Is OH or Z¹Is OH and Z²And (ii) a compound of the invention which is H, and wherein Y is OH and n is 0 and X in formula (I)¹-X⁷At least one of which is OH or O (CO) C_1-4Alkyl groups.

Particularly preferred compounds include:

4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide,

4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide,

4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide 1-oxide,

4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide 1-oxide,

4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-hydroxymethyl-2-pyridinecarboxamide 1-oxide,

4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-hydroxymethyl-2-pyridinecarboxamide 1-oxide, and salts, stereoisomers, and prodrugs thereof.

A preferred subgroup of compounds according to the invention comprises compounds of formula (II) or salts or stereoisomers thereof,

wherein:

y is OR¹Or NHR²，

Hal is chlorine or bromine, and Hal is chlorine or bromine,

R¹is H or C₁-C₆An alkyl group, a carboxyl group,

R²is H, OH, CH₃Or CH₂OH，

Z¹And Z²Are each H or OH, wherein Z¹Or Z²Only one of which may be OH,

X⁴-X⁷each independently H, OH or O (CO) C_1-4Alkyl radicals, and

n is a number of 0 or 1,

with the following conditions: at least one of the following conditions a-c is satisfied,

a)Z¹or Z²Is an OH group, and is a hydroxyl group,

b)R²is an OH group, and is a hydroxyl group,

c) n is 1.

These compounds include the compounds of the present invention wherein n is 1 in formula (II) wherein n is 1 and Z is¹And Z²The compounds of the invention are each H, where n in formula (II) is 1, Z¹And Z²Are H and X, respectively⁴-X⁷The compounds of the invention in which at least one is OH, wherein n in formula (II) is 1, Z¹And Z²Respectively H and Y is NHR²And R is²Is H or CH³Wherein n in formula (II) is 0, wherein n in formula (II) is 0 and Z¹Is H and Z²Is OH or Z¹Is OH and Z²The compound of the present invention is H, wherein n in the formula (II) is 0, Z¹And Z²Are respectively H, and X⁴-X⁷The compounds of the invention in which at least one is OH, wherein n in formula (II) is 0, Z¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, and X⁴-X⁷The compounds of the invention in which at least one is OH, wherein n in formula (II) is 0, Z¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, and Y is NHR²And R is²Is H or CH₃Wherein n in formula (II) is 0, Z¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, Y is NHR²And R²A compound of the invention which is OH, and wherein Y in formula (II) is NHR²、R²Is OH, n is 0 and X⁴-X⁷Compounds of the invention in which at least one is OH.

Another subset of preferred compounds of the invention includes compounds of formula (III) or a salt or isolated stereoisomer thereof:

wherein:

y is OR¹Or NHR²，

Hal is chlorine or bromine, and Hal is chlorine or bromine,

R¹is H or C₁-C₆An alkyl group, a carboxyl group,

R²is H, OH, CH₃Or CH₂OH，

Z¹And Zu is independently H or OH, wherein Z¹Or Z²Only one of which may be OH, and n is 0 or 1,

with the following conditions: at least one of the following conditions a-c is satisfied,

a)Z¹or Z²Is an OH group, and is a hydroxyl group,

b)R²is an OH group, and is a hydroxyl group,

c) n is 1.

These compounds include those in which n in formula (III) is 1 and Z¹And Z²H in which n in formula (III) is 1, Z¹And Z²H, Y are NHR respectively²And R is²Is H or CH₃Wherein n in formula (III) is 0 and Z¹Is H and Z²Is OH or Z¹Is OH and Z²The compound of the present invention is H, wherein n in the formula (III) is 0, Z¹Is H and Z²Is OH or Z¹Is OH and Z²Is H, Y is NHR²And R is²Is H or CH₃And a compound of the present invention wherein Y in the formula (III) is OH.

The invention further relates to processes and methods for preparing the novel compounds of the invention. These processes and methods include, but are not limited to, the reaction of 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] methyl)]Amino } carbonyl) amino]Phenoxy- } -N-methyl-2-pyridinecarboxamide and 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) -phenyl group)]Amino } carbonyl) amino]Piperidine epoxidation of phenoxy } -N-methyl-2-pyridinecarboxamide to their corresponding pyridine-1-oxides, the formaldehyde oxidation of any of the urea nitrogens of the compounds of the present invention to N-hydroxyurea, for X in the compounds of the present invention¹-X⁷Oxidation of an arbitrary position represented thereby replacing a hydrogen atom with a hydroxyl group, p-4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl group)]Amino } carbonyl) amino]Phenoxy } -N-methyl-2-pyridinecarboxamide and 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl group)]Amino } carbonyl) amino]The N-methyl amide of phenoxy } -N-methyl-2-pyridinecarboxamide is hydroxylated to form the corresponding hydroxymethyl amide, the N-methyl amide is hydroxylated to the hydroxamic acid, the N-methyl amide is demethylated to form the unsubstituted amide, the N-methyl amide is hydrolyzed to the carboxylic acid and mixtures thereof. In addition, the present invention relates to the pair X¹-X⁷The hydroxyl groups in the positions are esterified, for example to form acetates.

A preferred process of the present invention comprises a process for preparing 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, or a pharmaceutically acceptable salt thereof, or an isolated stereoisomer thereof, which comprises reacting 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide oxidation to the corresponding pyridine-1-oxide, and a process for preparing 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide, or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide or a pharmaceutically acceptable salt thereof, or an isolated stereoisomer, which comprises oxidizing 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide or 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide - {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide is oxidized to the corresponding pyridine-1-oxide.

The compounds prepared by these methods are included in the scope of the present invention. The invention also includes compounds obtained by transformation, including metabolic transformation, of 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] -amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide:

a) one or more of the phenyl hydrogens is substituted with a hydroxyl group,

b) hydroxylating the N-methyl amide to hydroxymethyl amide or hydroxamic acid,

c) demethylating the N-methylamide to an unsubstituted amide,

d) oxidizing one or more of the urea nitrogens by ═ NH to ═ NOH,

e) the N-methyl amide is hydrolyzed to carboxylic acid,

f) oxidation of pyridine nitrogen to pyridine-1-oxide, or

g) a combination of a-f, and a combination of a-f,

with the following conditions: performing at least one of steps b), d) and f).

Particularly preferred are compounds prepared by transformation, including metabolic transformation, of 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide:

a) hydroxylating the N-methyl amide to hydroxymethyl amide or hydroxamic acid,

b) demethylating the N-methylamide to an unsubstituted amide,

c) oxidizing one or more of the urea nitrogens by ═ NH to ═ NOH,

d) the N-methyl amide is hydrolyzed to carboxylic acid,

e) oxidation of pyridine nitrogen to pyridine-1-oxide, or

f) a combination of a-f, and a combination of a-f,

with the following conditions: performing at least one of steps a), c) and e).

It is to be understood that the term "pyridine-1-oxide" as used in the present patent includes 1-oxo-pyridine and 1-hydroxy-pyridine, and that these three terms are interchangeable with one another in the present invention. For example, chemlinnovation Software, inc^TMv.3.01 in ChemDraw where Y ═ NHCH₃、Hal＝Cl、Z¹And Z⁷The compound of formula III, H, and N-1, identified as N- [ 4-chloro-3- (trifluoromethyl) phenyl]({4- [ 1-hydroxy-2- (N-methylcarbamoyl) (4-pyridyloxy)]Phenyl } amino) formamide.

The invention further relates to pharmaceutical compositions comprising one or more compounds of the invention.

These compositions include pharmaceutical compositions comprising an effective amount of at least one compound of the invention and a physiologically acceptable carrier. Preferred pharmaceutical compositions comprise an effective amount of the following compounds: 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] -phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide, or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide or pharmaceutically acceptable salts thereof An acceptable salt, an isolated stereoisomer, or a mixture thereof, and a physiologically acceptable carrier.

Pharmaceutically acceptable salts of these compounds are also within the scope of the invention.

The salts of the invention are in particular pharmaceutically acceptable salts of the compounds of formula (I), for example organic or inorganic acid addition salts of the compounds of formula (I). Suitable inorganic acids include, but are not limited to, halogen acids (e.g., hydrochloric acid), sulfuric acid, or phosphoric acid. Suitable organic acids include, but are not limited to, carboxylic acids, phosphonic acids, sulfonic acids, sulfamic acids, specific examples of which include acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, 2-or 3-hydroxybutyric acid, gamma-aminobutyric acid (GABA), gluconic acid, glucoheptonic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, glucaric acid, galactaric acid, amino acids (such as glutamic acid, aspartic acid, N-methylglycine, acetylaminoacetic acid, N-acetylaspartamide or N-acetylcysteine), pyruvic acid, acetoacetic acid, methanesulfonic acid, 4-toluenesulfonic acid, benzenesulfonic acid, phosphoserine, and 2-or 3-glycerophosphoric acid.

The formation of prodrugs to enhance the properties of the parent compound, including solubility, absorption, biological stability and release time, is well known in the art (see: Pharmaceutical Dosage forms and Drug Delivery Systems, sixth edition, edited by Ansel et al, published by Williams & Wilkins, pages 27-29 (1995), the contents of which are incorporated herein by reference). The conventional prodrugs of the disclosed oxazolyl-phenyl-2, 4-diamino-pyrimidine compounds are primarily utilizing the primary drug biotransformation reaction and are also considered to be within the scope of the present invention. The major drug biotransformation reactions include N-dealkylation, O-dealkylation, aliphatic hydroxylation, aromatic hydroxylation, N-oxidation, S-oxidation, deamination, hydrolysis, glucuronidation, sulfation and acetylation (see: Goodman and Gilman' S The pharmacological basis of Therapeutics (ninth edition), eds: Molinoff et al, McGraw-Hill, pp.11-13 (1996), The contents of which are incorporated herein by reference).

The invention also relates to methods of treating and preventing diseases such as inflammatory and angiogenic diseases, and osteoporosis in mammals by administering the compounds of the invention or pharmaceutical compositions comprising the compounds of the invention.

These methods include methods of treating or preventing osteoporosis, inflammation, angiogenic diseases (excluding cancer) in a mammal by administering to said mammal an effective amount of a compound of the present invention. Preferred are methods of treating or preventing osteoporosis, inflammation and angiogenic diseases (excluding cancer) in a mammal by administering to the mammal an effective amount of: 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } - -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide, or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide or a pharmaceutically acceptable salt thereof An acceptable salt, an isolated stereoisomer, or a mixture thereof.

The invention also relates to methods of treating or preventing cancer and other hyperproliferative diseases by administering a compound of the invention or a pharmaceutical composition comprising one or more compounds of the invention and a cytotoxic agent.

These methods include methods of treating or preventing a hyperproliferative disorder in a mammal by administering to said mammal an effective amount of a compound of the present invention. Preferred is a method of treating or preventing a hyperproliferative disorder in a mammal by administering to the mammal an effective amount of: 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide 1-oxide, or 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide or a pharmaceutically acceptable salt thereof An acceptable salt, or an isolated stereoisomer, or a mixture thereof.

In a method of treating or preventing a hyperproliferative disorder in a mammal by administering an effective amount of a compound of the present invention, one or more additional compounds or compositions, such as an anti-cancer compound or composition, may be administered to the mammal, but they are not compounds or compositions according to the present invention, and are preferably cytotoxic compounds or compositions. A method of treating or preventing a hyperproliferative disease in a mammal comprising administering to said mammal an effective amount of 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-bromo-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide, 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide, or 4- {4- [ ({ [ 4-bromo-3- (trifluoroethyl) phenyl ] Amino } carbonyl) amino ] phenoxy } 2-pyridinecarboxamide 1-oxide or a pharmaceutically acceptable salt or isolated stereoisomer thereof or a mixture thereof and a cytotoxic compound or composition.

Optional antiproliferative agents which may be added to the compositions of the present invention include, but are not limited to, the compounds listed in the chemotherapeutic regimens for cancer in Merck Index 11 (1996), which is incorporated herein by reference, for example asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, levoasparaginase, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, doxorubicin (doxorubicin), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifene, streptozocin, tamoxifen, thioguanine, topotecan, mezzein, and, Vinblastine, vincristine, and vindesine.

Other antiproliferative agents suitable for use in The compositions of The present invention include, but are not limited to, those described in Goodmanand Gilman's The pharmaceutical basic of Therapeutics (ninth edition), editions: molinoff et al, McGraw-Hill, published at pages 1225-1287 (1996), the contents of which are incorporated herein by reference, known compounds for the treatment of neoplastic diseases, such as aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, cladribine, busulfan, diethylstilbestrol, 2' -difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine (erythrohydroxyynonylenine), ethinylestradiol, 5-fluorodeoxyuridine monophosphate, fluoroadenosine phosphate, fluorometholone, flutamide, hydroxyprogesterone heptanoate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, stavudine, N-phosphorylacetyl-L-aspartate (PALA), plicamycin, semustine, and the like, Teniposide, testosterone propionate, thiotepa, trimethylmelamine, uridine, and vinorelbine.

Other antiproliferative agents suitable for use in the compositions of the present invention include, but are not limited to, anticancer agents, such as oxaliplatin, gemcitabine, gefitinib, taxotere, BCNU, CCNU, DTIC, vidarabine, cytarabine, trastuzumab, actinomycin D, epothilone, irinotecan, raloxifene and topotecan.

Treatment of hyperproliferative diseases

Cancer and hyperproliferative diseases are defined as follows. These diseases include, but are not limited to, cancers of solid tumors such as breast, respiratory, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and their distant metastases. These diseases also include lymphomas, sarcomas and leukemias.

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 respiratory tract cancers include, but are not limited to, small cell and non-small cell lung cancers, as well as bronchomas and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to, brain stem and 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 and testicular cancer.

Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal vulvar cancer, and uterine sarcoma.

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.

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

Examples of eye 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 fibroplasic changes), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.

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

Head and neck cancers include, but are not limited to, laryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and lip and oral cavity 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, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

These diseases have been characterized in humans, but also in other mammals, similar etiologies exist, and thus can be treated with the pharmaceutical compositions of the present invention.

In general, the use of cytotoxic and/or cell proliferation inhibitors in combination with an aryl urea compound raf kinase inhibitor can achieve the following objectives: (1) better efficacy in reducing tumor growth or even eliminating tumors than when administered alone, (2) lower amounts of chemotherapeutic agents can be administered, (3) make chemotherapy more tolerable to the patient with fewer damaging drug complications than when the chemotherapeutic agent alone and certain other combination therapies are used, (4) enable the treatment of a wider range of different cancer types in mammals, particularly humans, (5) produce a higher response rate in the treated patient, (6) provide longer survival times in the treated patient than when treated with standard chemotherapeutic methods, (7) allow tumors to develop for longer periods of time, and/or (8) produce efficacy and tolerance at least as good as when administered alone as when compared to known cases where combination therapy with anti-cancer agents produces antagonism.

The invention relates to a combination comprising the following components: (a) a compound according to the invention, (b) at least one other chemotherapeutic cytotoxic or cell proliferation inhibitor; or a pharmaceutically acceptable salt of any of components (a) or (b).

The invention also relates to a pharmaceutical composition comprising the following components: (1) quantitative amounts of (a) a compound of the invention and (b) at least one other cytotoxic or cell proliferation inhibitor in amounts which, in combination, are effective for the treatment of cancer, either of components (a) or (b), if at least one salt-forming group is present, may also be in the form of a pharmaceutically acceptable salt, and (2) one or more pharmaceutically acceptable carrier molecules.

The invention also relates to a method of treating cancer by administering a compound according to the invention and at least one other chemotherapeutic agent which is a cytotoxic or cell proliferation inhibitor. The amounts of the compound according to the invention and the cytotoxic or cytostatic agent administered to the mammal should together produce an effective therapeutic effect on the hyperproliferative diseases as described above. Thus, the compounds according to the invention are effective against raf kinase mediated cancer. However, these compounds are also effective against cancers that are not mediated by raf kinase.

In a preferred embodiment, the present invention provides a method for the treatment of cancer in a mammalian, particularly human, patient, comprising administering a compound according to the invention, optionally together with cytotoxic or cell proliferation inhibitors, including but not limited to DNA topoisomerase I and II inhibitors, DNA intercalators, alkylating agents, microtubule disruptors, hormone and growth factor receptor agonists or antagonists, other kinase inhibitors and antimetabolites.

In another embodiment, the disclosed method is the administration of a chemotherapeutic agent comprising a compound of the present invention and an inhibitor of cytotoxicity and cell proliferation, either orally or by intravenous injection or infusion.

In another embodiment, a composition comprising a compound of the invention or a cytotoxic or cell proliferation inhibitor may be administered in the form of a tablet, liquid, topical gel, inhalant or sustained release composition.

In one embodiment of the invention, the compounds according to the invention may be administered to a patient suffering from cancer simultaneously with the cytotoxic or cell proliferation inhibitor in the same formulation, or more typically by different formulations, and often using different routes of administration. Administration may also be sequential in any order.

In another embodiment, the compound according to the invention is administered in tandem with a cytotoxic or cell proliferation inhibitor, wherein the compound according to the invention may be administered once or more times daily or, up to 28 consecutive days, and the cytotoxic or cell proliferation inhibitor is administered simultaneously or intermittently over the same period of time.

In another embodiment of the invention, the compounds according to the invention may be administered to a patient orally, intravenously, intramuscularly, subcutaneously or parenterally in a dose ranging between about 0.1 to 200mg/kg of total body weight.

In another embodiment, the cytotoxic or cytostatic agent may be administered to the patient at an intravenous, intramuscular, subcutaneous, or parenteral dose ranging between about 0.1mg to 200mg per kg of patient body weight.

In addition, the present invention relates to a method of inhibiting the proliferation of cancer cells comprising contacting the cancer cells with a pharmaceutical preparation or product of the invention. The invention also relates particularly to a method of treating a proliferative disease comprising contacting a patient suspected of having cancer, cells, tissues or body fluids of said patient with a pharmaceutical composition or formulation of the invention.

The invention also relates to compositions comprising the compounds according to the invention and other cytotoxic or cell proliferation inhibitors in an amount according to the invention.

The invention further relates to a kit comprising separate doses of the two chemotherapeutic agents described above in separate containers. The combination of the invention may also be formed in vivo, for example in a patient.

The term "cytotoxic agent" refers to a drug that kills or eliminates cancer cells upon administration. The term "cell proliferation inhibitor" refers to a cytoreductive effect that limits tumor proliferation after administration, rather than inducing cytotoxicity, which eliminates cancer cells from the patient's total viable cell population. Chemotherapeutic agents described herein, such as irinotecan, vinorelbine, gemcitabine, and paclitaxel, are considered cytotoxic agents. These cytotoxic and cytostatic agents have a wide range of applications as chemotherapeutic agents in the treatment of various cancer types and are well known.

These and other cytotoxic/cell proliferation inhibitors may be administered in accordance with conventional formulations and administration protocols known for administration alone.

General preparation method

The compounds of the present invention may be prepared by known chemical reactions and methods. The following general preparation methods are, however, used to assist the reader in synthesizing the compounds of the invention according to the specific examples detailed in the experimental section below.

If not specifically defined below, all of the various groups in these methods are as defined in the general description. When variable groups or substituents represented by a given symbol are used multiple times in a given structure, it is understood that each of these groups or substituents can independently vary within the definition that is expressed. It will be appreciated that the compounds of the present invention, each having optional functional groups, cannot be prepared by one of the methods listed below. Within the scope of each process, the optional substituents which are stable to the reaction conditions or, if desired, the functional groups which can participate in the reaction are present in protected form and these protecting groups can be removed at a suitable stage by methods known to the person skilled in the art.

The compounds of the present invention can be prepared according to conventional chemical methods and/or the methods described below from commercially available starting materials or starting materials that can be prepared according to conventional chemical methods. The general preparation of the compounds of the invention is given below, and the preparation of representative compounds is specifically illustrated in examples 1 and 2.

The ureas and hydroxyureas of formula (I) can be prepared by a variety of simple methods known in the art. General methods for forming these compounds can be found in: "Advanced Organic Chemistry", J.March, John Wiley and Sons, 1985, and "Comprehensive Organic transformations", R.C. Larock, VCH Publishers, 1989. The contents of these documents are incorporated herein by reference.

More specifically, the pyridine-1-oxides of the present invention (n ═ 1 in formula (I)) can be prepared from the corresponding pyridines using oxidation conditions known in the art. Some examples are as follows:

with peracids, e.g. m-chloroperbenzoic acid, in chlorinated solvents, e.g. dichloromethane, dichloroethane or chloroform (Markgraf et al Tetrahedron 1991, 47, 183)

With (Me) in the presence of catalytic amounts of acid in a chlorinated solvent, e.g. dichloromethane₃SiO)₂(Coperet et al Tetrahedron Lett.1998, 39, 761)

Use of perfluoro-cis-2-butyl-3-propyloxaziridine in several combinations of halogenated solvents (Amone et al Tetrahedron 1998, 54, 7831)

Use of hypofluoric acid-acetonitrile complex in chloroform (Dayan et al, Synthesis 1999, 1427)

The use of oxone in the presence of a base such as KOH in water (Robker et al, J.chem.Res., Synop.1993, 10, 412)

Use of magnesium monoperoxyphthalate in the presence of glacial acetic acid (Klemm et al, J.Heterococcus chem.1990, 6, 1537)

Use of hydrogen peroxide in the presence of water and acetic acid (Lin a.j., org.prep.proceed.int.1991, 23(1), 114)

Use of dimethyldioxirane in acetone (Boyd et al, J. chem. Soc., PerkinTrans.1991, 9, 2189)

The starting material for the above oxidation reaction is a diaryl urea, which contains a 2-acyl-pyridine in the side chain. Specific methods for the preparation of these urea compounds are described in the patent literature and are also applicable to the compounds of the present invention, see for example: PCT International application WO 0042012 by Riedl, B.et al entitled "O-carboxyaryl substituted diphenylureas as raf kinase inhibitors" and PCT International application WO 0041698 by Riedl, B.et al entitled "O-carboxyaryl substituted diphenylureas as p38 kinase inhibitors".

Wherein Z¹Is OH and Z²Hydroxyureas of formula (I) that are H can be prepared as follows:

substituted nitrobenzenes of formula (II) known in the art are converted to hydroxyanilines of formula (III) using a variety of conditions known to those skilled in the art, for example, sodium borohydride in the presence of a transition metal catalyst (Yanada et al, chem. Lett.1989, 951, and references cited therein), or N-methyl dihydroacridine in the presence of perchloric acid (Fukuzumi et al, J. chem. Soc., Perkin Trans. II 1991, 9, 1393, and references cited therein).

In the second step, the hydroxyaniline of formula (III) can be converted to the corresponding hydroxyurea by reaction with an isocyanate or equivalent in the same manner as urea is prepared. Examples of such reactions can be found in the prior art (Hoffman et al, j.med.chem.1964, 7, 665, and Stoffel et al, ber.dtsch.chem.ges.1972, 105, 3115).

Similarly, wherein Z¹Is H and Z²The hydroxyurea of formula (I) which is OH can be prepared according to the same method by suitably replacing the reagents.

In both cases, the preparation of arylamine fragments has been described in detail in the patent literature, see, for example, Miller s, et al, PCT international application WO 9932463 entitled "inhibition of p38 kinase using symmetrical and asymmetrical diphenyl ureas; PCT International application WO 9932436 by Miller, S et al entitled "inhibition of raf kinase Using symmetrically and asymmetrically substituted diphenylureas"; PCT international application WO 9932111 entitled "inhibition of p38 kinase activity using substituted heterocyclic ureas" to Dumas, j, et al; PCT international application WO 9932106 entitled "method of treating tumors by inhibiting raf kinase using N-heteroaryl-N' - (hetero) aryl urea" to Dumas, j, et al; PCT international application WO 9932110 entitled "inhibition of p38 kinase activity using aryl and heteroaryl substituted heterocyclic ureas," to Dumas, j, et al; PCT international application WO 9932455 entitled "inhibition of raf kinase using aryl and heteroaryl substituted heterocyclic ureas" to Dumas, j, et al; PCT International application WO 0042012 by Riedl, B. et al entitled "O-carboxyaryl substituted diphenylureas as raf kinase inhibitors"; PCT International application WO 0041698 by Riedl, B. et al entitled "O-carboxyaryl substituted diphenylureas as p38 kinase inhibitors".

Wherein Y is NHCH₂The methylolamides of formula (I) of OH can be prepared by reacting the corresponding unsubstituted amide (Y ═ NH) according to various methods known in the art₂) Hydroxylation is carried out, for example, using aqueous formaldehyde in the presence of ethanol and sodium hydroxide (Weaver et al, j.org.chem.1951, 16, 1111) or in the presence of potassium carbonate (Haworth et al, j.chem.soc.1946, 1003).

Hydroxamic acids of formula (I) wherein Y is NHOH can be prepared by amidation of the corresponding ester (Y ═ oalkyl) according to a variety of methods known in the art, for example using hydroxylamine in the presence of acetic acid and water (Boshagen, h., ber.dtsch.chem.ges.1967, 100, 954). The same compounds can be prepared from the corresponding acid (Y ═ OH) by one-pot activation with ethyl chloroformate followed by reaction with hydroxylamine in methanol (Reddy et al, Tetrahedron lett.2000, 41(33), 6285) or by activation of the acid to 1-acylimidazole followed by reaction with hydroxylamine hydrochloride (stabb et al, angelwald chem., 1962, 74, 407).

Finally, the urea compound may be further processed by using methods well known to those skilled in the art.

The invention also relates to pharmaceutical compositions comprising a compound of the invention and a physiologically acceptable carrier.

The compounds of the present invention may be administered in unit dosage forms by the oral, topical, parenteral, injection, inhalation or spray, or rectal routes. Administration by injection includes intravenous, intramuscular, subcutaneous and parenteral injection, as well as the use of infusion techniques. The one or more compounds may be associated with one or more non-toxic pharmaceutically acceptable carriers, which may also include other active ingredients, if desired.

The compositions for oral administration may be prepared according to any method known in the art to be suitable for the manufacture of pharmaceutical compositions. Such compositions may comprise one or more substances selected from the group consisting of: diluents, sweetening agents, flavoring agents, coloring agents and preservatives to provide a palatable preparation. Tablets contain the active ingredient in admixture with pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, and binding agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated according to known methods to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glycerol monostearate or glycerol distearate may be employed. These compounds can also be prepared as solid, rapid-release dosage forms.

Oral formulations may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oily medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol (heptadecene ethylene oxide), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, for example polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide or with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents are described above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present.

The compounds of the present invention may also be presented as non-aqueous liquid formulations, such as oily suspensions, which may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil or sesame oil, or in a mineral oil, for example liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may also be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical composition of the present invention may also be an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or mixtures thereof. Suitable emulsifying agents are natural gums, for example gum acacia or gum tragacanth, natural phosphatides, for example soya bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain humectants, preservatives, flavouring and colouring agents.

The compounds of the present invention may also be administered in the form of suppositories for rectal administration. These compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

For all use regimens of the compounds of the invention disclosed herein, the oral daily dosage regimen is preferably 0.01-200mg/kg body weight. The daily dose for administration by injection (including intravenous, intramuscular, subcutaneous and parenteral injection) and using infusion techniques is preferably 0.01-200mg/kg body weight. The daily dosage regimen for rectal administration is 0.01-200mg/kg body weight. The daily dosage regimen for topical administration is preferably 0.1-200mg, and is administered 1-4 times daily. The daily dosage regimen for administration by inhalation is preferably from 0.01 to 10mg/kg body weight. The dosage units used to provide these dosage regimens may be administered 1 or more times per day, or may be administered on a longer term basis, such as a weekly or monthly basis.

It will be appreciated by those skilled in the art that the particular method of administration will depend on a variety of factors that are typically considered in administering a treatment. One skilled in the art will also recognize that the specific dosage level for a particular patient will depend upon a variety of factors including the specific activity of the compound being administered, the age, body weight, health, sex, diet, time and route of administration, rate of excretion, and the like of the patient. One skilled in the art will further recognize that the optimal treatment regimen, i.e., the mode of treatment and the daily dosage and duration of the compounds of the invention, can be determined by one skilled in the art using routine therapeutic experimentation.

The compounds of the invention can be prepared from known compounds (or from starting materials which can be prepared from known compounds), for example by the general preparation methods described herein. The activity of a given compound to inhibit raf, p38, or KDR (VEGFR2) kinases can be assayed routinely, e.g., according to the methods described herein.

All applications, patents, and publications cited above and below are incorporated herein by reference in their entirety, including non-provisional application No. 09/425,228, filed 10/22/1999, and non-provisional application No. 09/458,548, filed 1/12/2001.

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. The following examples are therefore intended to illustrate the invention, but are in no way intended to be limiting.

Examples

Unless otherwise indicated, all reactions were carried out in flame-dried or oven-dried glassware under positive pressure of dry argon or dry nitrogen with magnetic stirring. Sensitive liquids and solutions were transferred via syringe or catheter and then introduced into the reaction through a rubber septum. Unless otherwise stated, the term "concentration under reduced pressure" is a Buchi rotary evaporator using about 15 mmHg. The term "under high vacuum" means a vacuum of 0.4 to 1.0mmHg, unless otherwise specified.

All temperatures are uncorrected degrees Celsius (C.). All parts and percentages are by weight unless otherwise indicated. Commercial grade reagents and solvents were used without purification.

Thin Layer Chromatography (TLC) was performed by using Whatman^®Pre-coated glass-supported silica gel 60AF-254250 μm plates. The color development of the plate is achieved by one or more of the following techniques: (a) ultraviolet irradiation, (b) exposure to iodine vapor, (c) immersion of the plate in a 10% ethanolic phosphomolybdate solution, followed by heating, (d) immersion of the plate in a cerium sulfate solution, followed by heating, and/or (e) immersion of the plate in an acidic ethanolic solution of 2, 4-dinitrophenylhydrazine, followed by heating. Column chromatography (flash chromatography) was performed by using 230-400 mesh EM Science^®Silica gel.

Melting points (mp) were determined using a Thomas-Hoover melting point apparatus or a Mettler FP66 automated melting point apparatus and were not corrected. Fourier transform infrared spectra were obtained using a Mattson 4020 Galaxyseries spectrophotometer. Proton (C)¹H) Nuclear Magnetic Resonance (NMR) Spectroscopy with a general electric GN-Omega 300(300MHz) spectrometer and Me₄Si (. delta.0.00) or residual protic solvent (CHCl)₃δ 7.26; MeOH δ 3.30; DMSO δ 2.49) as standard. Carbon (C)¹³C) NMR spectroscopy was performed using a General Electric GN-Omega 300(75MHz) spectrometer with solvent (CDCl)₃δ77.0；MeOD-d₃δ49.0；DMSO-d₆δ 39.5) as standard. Low resolution Mass Spectra (MS) and High Resolution Mass Spectra (HRMS) were obtained with Electron Impact (EI) mass spectra or fast atom impact (FAB) mass spectra. Electron impact mass spectrometry (EI-MS) was obtained using a Hewlett Packard 5989A mass spectrometer equipped with a Vacumetrics Desorption Chemical Ionization Probe for sample introduction. The ion source was maintained at 250 ℃. Electron bombardment ionization was performed at an electron energy of 70eV and a trapping current of 300. mu.A. Liquid cesium assisted ion mass spectrometry (FAB-MS), the latest version of fast atom bombardment, was obtained by using a Kratos Concept 1-H spectrometer. Chemical ionization mass spectrometry (CI-MS) was performed by using a Hewlett Packard MS-Engine (59)89A) Using methane or ammonia as reagent gas (1X 10)^-4torr-2.5×10^-4torr). Direct insertion Desorption Chemical Ionization (DCI) probes (Vaccumetrics, Inc.) were ramped up from 0-1.5amps in 10 seconds and held at 10amps until all sample traces disappeared (approximately 1-2 minutes). The spectra were scanned starting at 50-800amu, 2 seconds per scan. HPLC-electrospray mass spectrometry (HPLCES-MS) was obtained by using Hewlett-Packard 1100 HPLC, equipped with a quaternary pump, a variable wavelength detector, a C-18 column, and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned at 120-800amu using variable ion times, depending on the number of ions in the ion source. Gas chromatography-ion selective mass spectrometry (GC-MS) was obtained using a Hewlett Packard 5890 gas chromatograph equipped with a HP-1 methylsiloxane column (0.33mM coating; 25 m.times.0.2 mM) and a Hewlett Packard 5971 mass selective detector (ionization energy 70 eV). Elemental analysis was performed using Robertson Microlit Labs (Madison n.j.).

All compounds showed NMR spectra, LRMS and elemental analysis or HRMS consistent with their structures.

Example 1

Preparation of N- [ 4-chloro-3- (trifluoromethyl) phenyl ] -N' - {4- [2- (N-methylcarbamoyl) -1-oxo- (4-pyridyloxy) ] phenyl } urea

Adding N- [ 4-chloro-3- (trifluoromethyl) phenyl group under stirring]-N' - { -4- [2- (N-methylcarbamoyl) (4-pyridyloxy)]Phenyl } urea (500mg, 1.08mmol) in anhydrous CH₂Cl₂To a mixture of (2.2mL) and anhydrous THF (2.2mL) was added 3-chloroperbenzoic acid (77% pure, 1.09g, 4.86mmol, 4.5 equivalents), and the resulting mixture was heated at 40 ℃ for 33 hours. The resulting mixture was concentrated under reduced pressure and the crude product was passed through MPLC (Biotage)^®(ii) a Gradient from 20% acetone/hexane to 50% acetone/hexane). In EtOAcRecrystallizing to obtain N- [ 4-chloro-3- (trifluoromethyl) phenyl ] as white solid]-N' - {4- [2- (N-methylcarbamoyl) -1-oxo- - - (4-pyridyloxy)]Phenyl } urea (293mg, 57%): mp (uncorrected) 232-234 ℃; TLC (50% acetone/hexane) R_f0.13；¹H-NMR(DMSO-d₆)δ11.48(brs，1H)，9.19(s，1H)，8.98(s，1H)，8.38(d，J＝5.8Hz，1H)，8.10(d，J＝2.5Hz，1H)，7.64(dd，J＝8.2Hz，2.6Hz，1H)，7.61(d，J＝8.4Hz，1H)，7.57(d，J＝8.7Hz，2H)，7.54(d，J＝2.6Hz，1H)，7.28(dd，J＝5.7Hz，2.5Hz，1H)，7.18(d，J＝8.8Hz，2H)，2.86(d，J＝5.0Hz，3H)；HPLC EI-MS m/z 481((M+H)⁺)。C₂₁H₁₆ClFN₄O₄The calculated value of (a): c, 52.46%; h, 3.33%; n, 11.65%, found: c, 52.22%; h, 3.39%; n, 11.49 percent.

Example 2

Preparation of N- [ 4-chloro-3- (trifluoromethyl) phenyl ] -N' - {4- [ 2-carbamoyl-1-oxo- (4-pyridyloxy) ] phenyl } urea

Step 1: preparation of 4-chloro-2-pyridinecarboxamide

To a mixture of methyl 4-chloro-2-picolinate hydrochloride (1.0g, 4.81mmol) in concentrated aqueous ammonia (32mL) was added ammonium chloride (96.2mg, 1.8mmol, 0.37 equiv) with stirring, and the heterogeneous reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into EtOAc (500mL) and water (300 mL). The organic layer was washed with water (2 × 300mL) and saturated NaCl solution (1 × 300mL), dried (MgSO4), and then concentrated in vacuo to afford 4-chloro-2-pyridinecarboxamide as a beige solid (604.3mg, 80.3%): TLC (50% EtOAc/Hexane) R_f0.20；¹H-NMR(DMSO-d₆)δ8.61(d，J＝5.4Hz，1H)，8.20(brs，1H)，8.02(d，J＝1.8Hz，1H)，7.81(brs，1H)，7.76to7.73(m，1H)。

Step 2: preparation of 4- (4-aminophenoxy) -2-pyridinecarboxamide

To a solution of 4-aminophenol (418mg, 3.83mmol) in anhydrous DMF (7.7mL) was added potassium tert-butoxide (447mg, 3.98mmol, 1.04 eq) in one portion. The reaction mixture was stirred at room temperature for 2 hours, then a solution of 4-chloro-2-pyridinecarboxamide (600mg, 3.83mmol, 1.0 eq.) in anhydrous DMF (4mL) was added. The reaction mixture was stirred at 80 ℃ for 3 days and then poured into a mixture of EtOAc and saturated NaCl solution. The organic layer was washed sequentially with saturated NH4Cl solution, saturated NaCl solution, dried (MgSO4), and then concentrated under reduced pressure. Chromatography of the crude product on MPLC (Biotage)^®(ii) a Gradient from 100% EtOAc up to 10% MeOH/50% EtOAc/40% hexanes) to give 4-chloro-5-trifluoromethylaniline (510mg, 58%) as a brown solid.¹H-NMR(DMSO-d₆)δ8.43(d，J＝5.7Hz，1H)，8.07(brs，1H)，7.66(brs，1H)，7.31(d，J＝2.7Hz，1H)，7.07(dd，J＝5.7Hz，2.7Hz，1H)，6.85(d，J＝9.0Hz，2H)，6.62(d，J＝8.7Hz，2H)，5.17(brs，2H)；HPLC EI-MS m/z230((M+H)+.

And step 3: preparation of N- [ 4-chloro-3- (trifluoromethyl) phenyl ] -N' - {4- [ 2-carbamoyl- (4-pyridyloxy) ] phenyl } urea

A mixture of 4-chloro-5-trifluoromethylaniline (451mg, 2.31mmol, 1.1 equiv.) and 1, 1' -carbonyldiimidazole (419mg, 2.54mmol, 1.2 equiv.) in dry dichloroethane (5.5mL) was stirred under argon at 65 ℃ for 16 h. After cooling to room temperature, a solution of 4- (4-azaphenoxy) -2-pyridinecarboxamide (480mg, 2.09mmol) in anhydrous THF (4.0mL) was added. And the reaction mixture was stirred at 60 ℃ for 4 hours. The reaction mixture was poured into EtOAcThe organic layer was washed with water (2 ×) and saturated NaCl reaction (1 ×), dried (MgSO4), filtered, and evaporated in vacuo. Chromatography on MPLC (Biotage)^®(ii) a Gradient 100% EtOAc to 2% MeOH/EtOAc) to afford N- [ 4-chloro-3- (trifluoromethyl) phenyl ] as a white solid]-N' - {4- [ 2-carbamoyl- (4-pyridyloxy)]Phenyl } urea (770mg, 82%): TLC (EtOAc) R_f0.11, 100% ethyl acetate;¹H-NMR(DMSO-d₆)δ9.21(s，1H)，8.99(s，1H)，8.50(d，J＝5.6Hz，1H)，8.11(s，1H)，8.10(s，1H)，7.69(brs，1H)，7.64(dd，J＝8.2Hz，2.1Hz，1H)，7.61(s，1H)，7.59(d，J＝8.8Hz，2H)，7.39(d，J＝2.5Hz，1H)，7.15(d，J＝8.9Hz，2H)，7.14(m，1H)；MS LC-MS(MH⁺＝451)。C₂₀H₁₄ClF₃N₄O₃the calculated value of (a): c, 53.29%; h, 3.13%; n, 12.43%, found: c, 53.33%; h, 3.21%; and N, 12.60%.

And 4, step 4: preparation of N- [ 4-chloro-3- (trifluoromethyl) phenyl ] -N' - {4- - [ 2-carbamoyl-1-oxo- (4-pyridyloxy) ] phenyl } urea

According to the formula with N- [ 4-chloro-3- (trifluoromethyl) phenyl]-N' - {4- [2- (N-methylcarbamoyl) -1-oxo- (4-pyridyloxy)]Phenyl } urea in the same manner as described for N- [ 4-chloro-3- (trifluoromethyl) phenyl ]]-N' - {4- [ 2-carbamoyl- (4-pyridyloxy)]Phenyl } urea (240.0mg, 0.53mmol) N- [ 4-chloro-3- (trifluoromethyl) phenyl ] was obtained as a white solid]-N' - {4- [ 2-carbamoyl-1-oxo- (4-pyridyloxy)]Phenyl } urea (125.6mg, 51%): TLC (5% MeOH/CH)₂Cl₂)R_f0.17；¹H-NMR(DMSO-d₆)δ10.72(d，J＝4.3Hz，1H)，9.21(s，1H)，8.99(s，1H)，8.36(d，J＝7.2Hz，1H)，8.31(d，J＝4.1Hz，1H)，8.10(d，J＝2.3Hz，1H)，7.65(dd，J＝8.7Hz，2.3Hz，1H)，7.60(d，J＝8.9Hz，1H)，7.57(d，J＝9.0Hz，2H)，7.54(d，J＝3.8Hz，1H)，7.28(dd，J＝7.2Hz，3.8Hz，1H)，7.18(d，J＝9.0Hz，2H)；HPLC EI-MS m/z 467((M+H)⁺；C₂₀H₁₄ClF₃N₄O₄ 0.5H₂Calculated value of O: c, 50.49%; h, 3.18%; n, 11.78%, found: c, 50.69%; h, 2.86%; n, 11.47 percent.

Biological examples

P38 kinase in vitro assay

The in vitro inhibitory properties of these compounds were tested using p38 kinase inhibition. In vitro kinase assays were performed in 96-well microtiter plates, from which p38 activity was determined. Recombinant human p38 (0.5. mu.g/mL) and substrate (myelin basic protein, 5. mu.g/mL) in kinase buffer (25mM Hepes, 20mM MgCl)₂And 150mM NaCl) and compound. Adding 1. mu. Ci per well³³P-labeled ATP (10. mu.M) to a final volume of 100. mu.L. The reaction was carried out at 32 ℃ for 30 minutes and then stopped with 1M HCl solution. The amount of radioactivity incorporated into the substrate was determined by capturing the substrate on negatively charged glass fiber filter paper with a 1% phosphoric acid solution and then reading with a scintillation counter. Negative controls included substrate + ATP alone.

LPS-induced TNF alpha formation in mice

The in vivo inhibitory properties of selected compounds were determined using an in vivo model of murine LPS-induced TNF α formation. Ten BALB/c mice (Charles River Breeding Laboratories; Kingston, n.y.) per group were treated with vehicle or compound by the route described. After 1 hour, endotoxin (e.coli Lipopolysaccharide (LPS)100 μ g) was administered intraperitoneally (i.p.). After 90 minutes, animals were euthanized by carbon dioxide asphyxiation, and then plasma was obtained from each animal by cardiac acupuncture and placed into heparinized tubes. The sample was clarified by centrifugation at 12500 Xg for 5 minutes at 4 ℃. The supernatant was poured into a new tube, which was stored at-20 ℃ as needed. TNF α levels in serum were determined using a commercially available murine TNF ELISA kit (Genzyme).

The above two biological examples can be used to demonstrate that the compounds of the invention inhibit p38 kinase in vitro and in vivo, and thus determine their utility in the treatment of p38 mediated diseases such as inflammation and osteoporosis.

In vitro assay for raf kinase

In an in vitro kinase assay, raf was incubated with MEK in 20mM Tris-HCl, pH8.2, containing 2mM 2-mercaptoethanol and 100mM NaCl. The protein solution (20. mu.L) was mixed with water (5. mu.L) or with the compound obtained by diluting a 10mM compound DMSO stock solution with distilled water. Adding 25 μ L of [ gamma-³³P]ATP (1000-₂Thereby initiating a kinase reaction. The reaction compounds were incubated at 32 ℃ for typically 22 minutes. The reaction was collected on a phosphocellulose pad, washed free with 1% phosphoric acid solution, and then quantified for phosphorylation by liquid scintillation counting, from which analysis was performed³³Incorporation of P into proteins. For high throughput screening, 10 μ M ATP and 0.4 μ M MEK were used. In some experiments, an equal amount of Laemmli sample buffer was added, thereby stopping the kinase reaction. The sample was boiled for 3 minutes, and subjected to electrophoresis on a 7.5% Laemmli gel, thereby resolving the protein. The gel was fixed, dried and then exposed to a developing plate (Fuji). Phosphorylation was analyzed using the Fujix Bio-Imaging Analyzer System. In this experiment, the compounds of examples 1 and 2 showed > 50% inhibition at 10 micromolar, which is a significant in vitro inhibition of raf kinase.

Tumor cell proliferation assay

For in vitro growth experiments, human tumor cell lines were used in standard proliferation experiments for anchorage-dependent cell growth on plastic or anchorage-independent cell growth in soft agar, including but not limited to HCT116 and DLD-1. Human tumor cell lines were obtained from ATCC (Rockville MD.) and stored in RPMI containing 10% heat-inactivated fetal bovine serum and 200mM glutamine. Cell culture media and additives are available from Gibco/BRL (Gaithersburg,MD.) but not fetal bovine serum (JRH Biosciences, Lenexa, Kans.). In a standard proliferation assay for anchorage-dependent growth, 3X 10³Individual cells were seeded in 96-well tissue culture plates and allowed to incubate at 5% CO₂The incubators were connected overnight at 37 ℃. Compounds were titrated in a series of dilutions of medium and added to 96-well plates. Cells are grown for 5 days, with new media containing the compound typically being added at day 3. Measurement of metabolic activity, where standard XTT colorimetric assay (Boehringer Mannheim) was measured at OD490/560 with a standard ELISA plate reader, cells were collected on glass fiber pads using a cell collector, and then measured by scintillation counting³The amount of H-thymidine incorporated, and thus the proliferation effect, was monitored.

For anchorage-independent cell growth, in 24-well tissue culture plates, at 1 × 10³-3×10³Amounts cells were plated in 0.4% seaopaque agarose in RPMI complete medium, with a bottom layer of 0.64% agar contained only in RPMI complete medium. Complete medium plus serial dilutions of compound were added to each well, followed by 5% CO₂Incubate in the incubator at 37 ℃ for 10-14 days, with new media containing compounds added at 3-4 day intervals. Colony formation was monitored and total cell mass, average colony size and colony number were quantified using Image capture technology and Image analysis software (Image ProPlus, media Cybernetics).

The above two experiments determined that the compounds of formula I are effective in inhibiting raf kinase activity as well as inhibiting cancer cell growth.

KDR (VEGFR2) assay

In Sf9 insect cells, the cytoplasmic kinase domain of KDR kinase is expressed as a 6His fusion protein. The KDR kinase domain fusion protein is purified on a Ni + + chelating column. 96-well ELISA plates were coated overnight at 4 ℃ with 5. mu.g of poly (Glu 4; Tyrl) (Sigma Chemical Co., St Louis, Mo.) in 100. mu.l of HEPES buffer (20mM HEPES, pH7.5, 150mM NaCl, 0.02% thimerosal). Before use, the plates were washed with HEPES, NaCl buffer, and then blocked with 1% BSA, 0.1% Tween20 in HEPES, NaCl buffer.

Test compounds were serially diluted from 4mM up to 0.12. mu.M in 100% DMSO at a semilogarithmic dilution. These dilutions were further diluted 20-fold in water to give compound solutions in 5% DMSO. Mu.l of assay buffer (20mM HEPES, pH7.5, 100mM KCl, 10mM MgCl) was loaded onto assay plates₂，3mM MnCl₂0.05% glycerol, 0.005% Triton X-100, 1 mM-mercaptoethanol, with or without 3.3. mu.M ATP), 5. mu.l of the diluted compound was added to a final volume of 100. mu.l. The final concentration in 0.25% DMSO was between 10- μ M and 0.3 nM. The assay was started by adding 10. mu.l (30ng) of the KDR kinase domain.

The analyte was incubated with the test compound or vehicle alone for 60 minutes at room temperature with gentle agitation. Wells were washed and Phosphotyrosine (PY) probed with anti-Phosphotyrosine (PY) -mAb clone 4G10(Upstate Biotechnology, Lake plain, n.y.). The PY/anti-PY complex was detected with an anti-mouse IgG/HRP conjugate (Amersham International plc, Buckinghamshire, UK). Phosphotyrosine was quantified by incubation with 100. mu.l of 3, 3 ', 5, 5' -tetramethylbenzidine solution (Kirkegaardand Perry, TMB Microwell monocomponent peroxidase substrate). The color development was stopped by adding 100. mu.l of a 1% HCl-based stop solution (Kirkegaard and Perry, TMB one-component stop solution).

The optical density was determined spectrophotometrically at 450nm in a 96-well plate reader SpectraMax250(Molecular Devices). Background (no ATP in the assay) OD values were subtracted from all OD values and percent inhibition was calculated according to equation 1 below:

% inhibition ═ OD (vehicle control) -OD (containing compound)) × 100-

(OD (vehicle control) -OD (without ATP addition))

Determination of I by least squares analysis procedure Using Compound concentration vs. percent inhibitionC₅₀The value is obtained.

Cell mechanistic assay-inhibition of 3T3KDR phosphorylation

NIH3T3 cells expressing the full-length KDR receptor were grown in DMEM (Life Technologies, inc., Grand Island, n.y.) supplemented with 10% newborn bovine serum, low glucose, 25mM/L sodium pyruvate, pyridoxine hydrochloride, and 0.2mg/ml G418(Life Technologies, inc., Grand Island, n.y.). Cells were stored in humidified 5% CO₂Atmosphere and 37 ℃ in a collagen I coated T75 flask (Becton Dickinson Labware, Bedford, Mass.).

15000 cells were plated in collagen I coated 96-well plates in DMEM growth medium. After 6 hours, the cells were washed and the medium was replaced with DMEM without serum. After overnight culture to make cells quiescent, the medium was replaced with Dulbecco phosphate buffered saline (life technologies inc., Grand Island, n.y.) containing 0.1% bovine albumin (sigma chemical co., St Louis, Mo.). In 1% final concentration of DMSO, various concentrations (0-300nM) of test compound were added to the cells, and the cells were incubated at room temperature for 30 min. The cells were then treated with VEGF (30ng/ml) for 10 min at room temperature. After stimulation with VEGF, the buffer was removed and the cells were lysed for 30 minutes by adding 150. mu.l of extraction buffer (50mM Tris, pH7.8, supplemented with 10% glycerol, 50mM BGP, 2mM EDTA, 10mM NaF, 0.5mM NaVO4, and 0.3% TX-100) at 4 ℃.

To assess receptor phosphorylation, 100 μm of each cell lysate was added to each well of an ELISA plate pre-coated with 300ng of antibody C20(Santa Cruz biotechnology, inc., Santa Cruz, Calif.). After incubation for 60 min, the plates were washed and binding KDR to phosphotyrosine was probed using anti-phosphotyrosine mAb clone 4G10(Upstate Biotechnology, Lake plain, n.y.). The plates were washed and each well was incubated with anti-mouse IgG/HRP conjugate (Amersham International plc, Buckinghamshire, uk) for 60 minutes. The wells were washed and phosphotyrosine was quantified by adding 100. mu.l of 3, 3 ', 5, 5' -tetramethylbenzidine (Kirkegaard and Perry, TMB Microwell monocomponent peroxidase substrate) solution to each well. The color development was stopped by adding 100. mu.l of a one-pack stop solution of HCl base (Kirkegaard and Perry, TMB).

The Optical Density (OD) was determined spectrophotometrically at 450nm in a 96-well plate reader (SpectraMax 250, Molecular Devices). Background (no VEGF in the assay) OD values were subtracted from all OD values and percent inhibition was calculated according to equation 2 below:

% inhibition ═ OD (VEGF control) -OD (containing compound)) × 100-

(OD (VEGF control) -OD (without VEGF addition))

IC of some exemplary substances was determined by least squares analysis procedure using compound concentration versus percent inhibition₅₀The value is obtained.

In vivo analysis of VEGFR inhibition: matrigel^®Model of angiogenesis

Preparation of Matrigel Plugs and in vivo phases: matrigel ® (collagen BiomedicalProducts, Bedford, Mass.) is a basement membrane extract of murine tumors, consisting mainly of lamin, collagen IV and heparin sulfate proteolycan. It is in sterile liquid form at 4 ℃, but rapidly forms a solid gel at 37 ℃.

Liquid Matrigel at 4 ℃ was mixed with SK-MEL2 human tumor cells transfected with a plasmid carrying a selectable marker and containing the murine VEGF gene. Tumor cells were selectively grown in vitro and then 2X 10⁶The cells were mixed with cold liquid Matrigel at a ratio of 0.5ml each. 0.5ml was implanted subcutaneously near the midline of the abdomen using a 25 gauge needle. Test compounds in solution in ethanol/Cremaphor EL/saline (12.5%: 75%) were administered at po 1 time daily on the day of implantation at doses of 30, 100 and 300mg/kg, respectively. Mice were euthanized 12 days after implantation and Matrigel pellets were collected for analysis of hemoglobin content.

And (3) analysis of hemoglobin: the Matrigel pellets were placed in 4 volumes (W/v) of 4 ℃ lysis buffer (20mM Tris pH7.5, 1mM EGTA, 1mM EDTA, 1% Triton X-100[ EMscience, Gibbstown, N.J. ], and complete EDTA-free protease inhibitor cocktail [ Mannheim, Germany ]), and then homogenized at 4 ℃. The homogenate was incubated on ice for 30 minutes while shaking and centrifuged at 14K × g and 4 ℃ for 30 minutes. The supernatant was transferred to a refrigerated microcentrifuge tube and stored at 4 ℃ for hemoglobin analysis.

Murine hemoglobin (Sigma Chemical co., st.louis, Mo.) was suspended in autoclaved water (BioWhittaker, Inc, Walkersville, Md.) in an amount of 5 mg/ml. Standard curves ranging from 500. mu.g/ml to 30. mu.g/ml were generated in lysis buffer (supra). Duplicate standard curves and cell lysis samples were added in a quantity of 5 μ l/well on a polystyrene 96-well plate. TMB substrate was reconstituted in 50ml of acetic acid solution at room temperature using Sigma plasma hemoglobin kit (Sigma Chemical co., st. 100 microliters of substrate was added to each well, followed by 100 microliters/well of hydrogen peroxide solution at room temperature. The plates were incubated at room temperature for 10 minutes.

The optical density was determined spectrophotometrically at 600nm in a 96-well plate reader SpectraMax250 microplate spectrophotometer system (molecular devices, Sunnyvale, CA). Background lysis buffer readings were subtracted from all wells. The total sample hemoglobin content is calculated according to the following equation:

total hemoglobin (sample cell lysate volume) x (hemoglobin concentration)

The average total hemoglobin of the Matrigel samples without cells was subtracted from each of the total hemoglobin Matrigel samples with cells. Percent inhibition was calculated according to the following equation. % inhibition (mean total hemoglobin of drug-treated tumor cell lysate) × 100 ═

(mean Total hemoglobin of tumor cell lysate without drug treatment)

The three experiments described above demonstrate that the compounds of formula I are active in inhibiting VEGF receptor kinase activity and inhibiting angiogenesis.

In vivo assay for antitumor Activity

In vivo experiments of the inhibition of raf kinase mediated tumors (e.g. solid cancers) by compounds were performed as follows: at 1 × 10⁶Amount of cells CDI nu/nu mice (6-8 weeks old) were injected subcutaneously into the flank, which contained human colon adenocarcinoma cell lines. When the tumor size is between 50-100mg, a dose of 10, 30, 100, or 300mg/Kg is administered to the mice i.p., i.v., or p.o. starting at about day 10. Animals were dosed for 14 consecutive days with tumor size monitored 2 times per week with calipers. The inhibitory effect of the compounds on p38, raf and VEGFR kinases and thus on tumor growth (e.g. in vivo cancer) can be further demonstrated in vivo by Monia et al (nat. Med.1996, 2, 668-75).

The above examples can be repeated with similar success by substituting the general outline or the specific description of the reactants and/or operating conditions of the present invention for the above examples.

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

Claims (21)

1. A compound selected from the group consisting of:

4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide,

4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide 1-oxide,

2. a process for preparing a compound of claim 1, comprising:

oxy 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide,

to give 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -N-methyl-2-pyridinecarboxamide 1-oxide,

or oxidizing 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide,

to give 4- {4- [ ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } carbonyl) amino ] phenoxy } -2-pyridinecarboxamide 1-oxide.

3. The method of claim 2, wherein the nitrogen of the pyridyl ring is oxidized to the corresponding pyridine-1-oxide.

4. A pharmaceutical composition comprising at least one compound of claim 1 and a physiologically acceptable carrier.

5. The pharmaceutical composition of claim 4, further comprising an antiproliferative agent.

6. The pharmaceutical composition of claim 5, wherein the antiproliferative agent is selected from the group consisting of: asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, levo-asparaginase, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifene, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine, vindesine, aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, cladribine, busulfan, equol, 2' -difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine, ethinylestradiol, 5-fluorodeoxyuridine monophosphate, fluoroadenosine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone heptanoate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, oxaliplatin, gemcitabine, gefitinib, taxotere, BCNU, CCNU, DTIC, vidarabine, cytarabine, trastuzumab, actinomycin D, pentostatin, N-phosphorylacetyl-L-aspartic acid, plicamycin, semustine, teniposide, testosterone propionate, thiotepa, trimethylmelamine, uridine, and vinorelbine.

7. The pharmaceutical composition of claim 4, further comprising an anti-cytotoxic agent or a cell proliferation-inhibiting chemotherapeutic agent.

8. The pharmaceutical composition of claim 7, wherein the cytotoxic or cytostatic chemotherapeutic agent is selected from the group consisting of: DNA topoisomerase I and II inhibitors, DNA intercalators, alkylating agents, microtubule disruptors, hormone and growth factor receptor agonists or antagonists, other kinase inhibitors and antimetabolites.

9. Use of at least one compound as claimed in claim 1 for the manufacture of a medicament for the treatment or prevention of osteoporosis, inflammation and angiogenic diseases in mammals, but excluding cancer.

10. Use of a composition according to claim 4 in the manufacture of a medicament for the treatment or prevention of osteoporosis, inflammation and angiogenic diseases in a mammal, but said diseases do not include cancer.

11. Use of at least one compound according to claim 1 for the preparation of a medicament for the treatment or prevention of a hyperproliferative disease in a mammal.

12. Use of a pharmaceutical composition according to claim 5 in the manufacture of a medicament for the treatment or prevention of a hyperproliferative disease in a mammal.

13. Use of a pharmaceutical composition according to claim 5 in the manufacture of a medicament for the treatment of a hyperproliferative disease in a mammal, wherein said antiproliferation is in a separate pharmaceutical composition.

14. Use of a pharmaceutical composition according to claim 7 in the manufacture of a medicament for the treatment or prevention of cancer.

15. Use of a pharmaceutical composition according to claim 7 in the manufacture of a medicament for the treatment of cancer, wherein the anti-cytotoxic agent or cell proliferation-inhibiting chemotherapeutic agent is in a separate pharmaceutical composition.

16. Use of at least one compound as claimed in claim 1 in the manufacture of a medicament for the treatment or prevention of osteoporosis, inflammation and angiogenic diseases in a mammal, but excluding raf mediated cancer.

17. A kit comprising a single dose of a cytotoxic or cytostatic agent and a single dose of a compound according to claim 1.

18. A pharmaceutical composition comprising at least one compound of claim 1 and an antiproliferative agent.

19. A pharmaceutical composition comprising at least one compound of claim 1 and an anti-cytotoxic agent or a cell proliferation inhibiting chemotherapeutic agent.

20. Use of a pharmaceutical composition according to claim 18 in the manufacture of a medicament for the treatment or prevention of a hyperproliferative disease in a mammal.

21. Use of a pharmaceutical composition according to claim 19 in the manufacture of a medicament for the treatment or prevention of cancer.