# Patent

## Title
Compounds and compositions as c-kit and PDGFR kinase inhibitors

## Abstract

The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with abnormal or deregulated kinase activity, particularly diseases or disorders that involve abnormal activation of c-kit, PDGFR and PDGFR kinases.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 U.S. national phase application of international application number PCT/US2008/062568 filed 2 May 2008, which application claims priority to U.S. provisional patent application No. 60/916,051, filed 4 May 2007. The full disclosure of these applications is incorporated herein by reference in its entirety and for all purposes.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with abnormal or deregulated kinase activity, particularly diseases or disorders that involve abnormal activation of c-kit, PDGFRα and PDGFRβ kinases.
2. Background
The protein kinases represent a large family of proteins, which play a central role in the regulation of a wide variety of cellular processes and maintaining control over cellular function. A partial, non-limiting, list of these kinases include: receptor tyrosine kinases such as platelet-derived growth factor receptor kinase (PDGF-R), the nerve growth factor receptor, trkB, and the fibroblast growth factor receptor, FGFR3, B-RAF; non-receptor tyrosine kinases such Abl and the fusion kinase BCR-Abl, Lck, Bmx and c-src; and serine/threonine kinases such as c-RAF, sgk, MAP kinases (e.g., MKK4, MKK6, etc.) and SAPK2α and SAPK2β. Aberrant kinase activity has been observed in many disease states including benign and malignant proliferative disorders as well as diseases resulting from inappropriate activation of the immune and nervous systems.
The novel compounds of this invention inhibit the activity of one or more protein kinases and are, therefore, expected to be useful in the treatment of kinase-associated diseases.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compounds of Formula I:




L is selected from —NC(O)—, —NC(O)N— and —C(O)N—;
R1, R2a and R2b are each independently selected from hydrogen, hydroxy, C3-8heterocycloalkyl, C1-4alkyl, C1-4alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4alkyl, —NR10R11, —OX1R8; wherein X1 is selected from a bond and C1-4alkylene; R8 is C3-12cycloalkyl; or R1 and R2a or R1 and R2b together with the carbon atoms to which R1 and R2a or R2b are attached form phenyl; R10 and R11 are independently selected from hydrogen, C1-4alkyl, C1-4alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4alkyl, C3-8heterocycloalkyl, C1-10heteroaryl; or R10 and R11 together with the nitrogen to which R10 and R11 are both attached form C3-8heterocycloalkyl or C1-10heteroaryl;
R3, R4, R5, R6 and R7 are independently selected from hydrogen, halo, cyano, C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, C3-8heterocycloalkyl, —OX2R9, —S(O)0-2R9 and —NR12R13; wherein X2 is selected from a bond and C1-4alkylene; and each R9 is independently selected from hydrogen, C1-6alkyl, C1-6alkoxy, C3-12cycloalkyl, C3-8heterocycloalkyl and —NR12R13; wherein said cycloalkyl or heterocycloalkyl of R9 is optionally substituted with 1 to 3 radicals independently selected from C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, halo-substituted-C1-6alkyl and —NR12R13; wherein R12 and R13 are independently selected from hydrogen and C1-6alkyl; or R4 and R5 together with the carbon atoms to which R4 and R5 are attached form C3-8heteroaryl;
with the proviso that at least one of R3, R4, R5, R6 or R7 has a sulfur directly linked to the phenyl ring of Formula I to which R3, R4, R5, R6 and R7 are attached; and the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof; and the pharmaceutically acceptable salts and solvates (e.g. hydrates) of such compounds.
In a second aspect, the present invention provides a pharmaceutical composition which contains a compound of Formula I or a N-oxide derivative, individual isomers and mixture of isomers thereof; or a pharmaceutically acceptable salt thereof, in admixture with one or more suitable excipients.
In a third aspect, the present invention provides a method of treating a disease in an animal in which inhibition of kinase activity, particularly c-kit, PDGFRα and/or PDGFRβ activity, can prevent, inhibit or ameliorate the pathology and/or symptomology of the diseases, which method comprises administering to the animal a therapeutically effective amount of a compound of Formula I or a N-oxide derivative, individual isomers and mixture of isomers thereof, or a pharmaceutically acceptable salt thereof.
In a fourth aspect, the present invention provides the use of a compound of Formula I in the manufacture of a medicament for treating a disease in an animal in which kinase activity, particularly c-kit, PDGFRα and/or PDGFRβ activity, contributes to the pathology and/or symptomology of the disease.
In a fifth aspect, the present invention provides a process for preparing compounds of Formula I and the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof, and the pharmaceutically acceptable salts thereof.


DETAILED DESCRIPTION OF THE INVENTION
Definitions
“Alkyl” as a group and as a structural element of other groups, for example halo-substituted-alkyl and alkoxy, can be either straight-chained or branched. C1-4-alkoxy includes, methoxy, ethoxy, and the like. Halo-substituted alkyl includes trifluoromethyl, pentafluoroethyl, and the like.
“Aryl” means a monocyclic or fused bicyclic aromatic ring assembly containing six to ten ring carbon atoms. For example, C5-10aryl as used in this application, includes but is not limited to phenyl or naphthyl, preferably phenyl. “Arylene” means a divalent radical derived from an aryl group.
“Heteroaryl” is a 5 to 15 member, unsaturated ring system containing 1 to 3 heteroatoms independently selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)2—, wherein R is hydrogen, C1-4alkyl or a nitrogen protecting group. For example, C1-10heteroaryl (“C1-10” meaning between one and ten carbon atoms are present in the ring system), as used in this application includes, but is not limited to, pyrazolyl, pyridinyl, indolyl, thiazolyl, 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl, furanyl, benzo[b]furanyl, pyrrolyl, 1H-indazolyl, imidazo[1,2-a]pyridin-3-yl, oxazolyl, benzo[d]thiazol-6-yl, 1H-benzo[d][1,2,3]triazol-5-yl, quinolinyl, 1H-indolyl, 3,4-dihydro-2H-pyrano[2,3-b]pyridinyl and 2,3-dihydrofuro[2,3-b]pyridinyl, 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl, etc.
“Cycloalkyl” means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing the number of ring atoms indicated. For example, C3-10cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
“Heterocycloalkyl” means a 3 to 8 member, saturated or partially unsaturated ring system containing 1 to 3 heteroatoms independently selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)2—, wherein R is hydrogen, C1-4alkyl or a nitrogen protecting group. For example, C3-8heterocycloalkyl as used in this application to describe compounds of the invention includes, but is not limited to, morpholino, pyrrolidinyl, azepanyl, piperidinyl, isoquinolinyl, tetrahydrofuranyl, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinylone, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.
“Halogen” (or halo) preferably represents chloro or fluoro, but may also be bromo or iodo.
“Kinase Panel” is a list of kinases comprising Abl(human), Abl(T315I), JAK2, JAK3, ALK, JNK1α1, ALK4, KDR, Aurora-A, Lck, Blk, MAPK1, Bmx, MAPKAP-K2, BRK, MEK1, CaMKII(rat), Met, CDK1/cyclinB, p70S6K, CHK2, PAK2, CK1, PDGFRα, CK2, PDK1, c-kit, Pim-2, c-RAF, PKA(h), CSK, PKBα, cSrc, PKCα, DYRK2, Plk3, EGFR, ROCK-I, Fes, Ron, FGFR3, Ros, Flt3, SAPK2α, Fms, SGK, Fyn, SIK, GSK3β, Syk, IGF-1R, Tie-2, IKKβ, TrKB, IR, WNK3, IRAK4, ZAP-70, ITK, AMPK(rat), LIMK1, Rsk2, Axl, LKB1, SAPK2β, BrSK2, Lyn (h), SAPK3, BTK, MAPKAP-K3, SAPK4, CaMKIV, MARK1, Snk, CDK2/cyclinA, MINK, SRPK1, CDK3/cyclinE, MKK4(m), TAK1, CDK5/p25, MKK6(h), TBK1, CDK6/cyclinD3, MLCK, TrkA, CDK7/cyclinH/MAT1, MRCKβ, TSSK1, CHK1, MSK1, Yes, CK1d, MST2, ZIPK, c-Kit (D816V), MuSK, DAPK2, NEK2, DDR2, NEK6, DMPK, PAK-4, DRAK1, PAR-1Bα, EphA1, PDGFRβ, EphA2, Pim-1, EphA5, PKBβ, EphB2, PKCβI, EphB4, PKCδ, FGFR1, PKCη, FGFR2, PKCθ, FGFR4, PKD2, Fgr, PKG1β, Flt1, PRK2, Hck, PYK2, HIPK2, Ret, IKKα, RIPK2, IRR, ROCK-II(human), JNK2α2, Rse, JNK3, Rsk1(h), PI3 Kγ, PI3 Kδ and PI3-Kβ. Compounds of the invention are screened against the kinase panel (wild type and/or mutation thereof) and inhibit the activity of at least one of said panel members.
“Treat”, “treating” and “treatment” refer to a method of alleviating or abating a disease and/or its attendant symptoms.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The c-kit gene encodes a receptor tyrosine kinase and the ligand for the c-kit receptor is called the stem cell factor (SCF), which is the principal growth factor for mast cell survival. The activity of the c-kit receptor protein tyrosine kinase is regulated in normal cells, and the normal functional activity of the c-kit gene product is essential for maintenance of normal hematopoeisis, melanogenesis, genetogenesis, and growth and differentiation of mast cells. Mutations that cause constitutive activation of c-kit kinase activity in the absence of SCF binding are implicated in various diseases ranging from mastocyclosis to malignant human cancers.
In one embodiment, with reference to compounds of Formula I, L is selected from —NC(O)— and —C(O)N—; R1, R2a and R2b are independently selected from hydrogen, hydroxy, C3-8heterocycloalkyl, C1-4alkyl, C1-4alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4alkyl, —NR10R11, —OX1R8; wherein X1 is selected from a bond and C1-4alkylene; R8 is C3-12cycloalkyl; or R1 and R2 together with the carbon atoms to which R1 and R2a are attached form phenyl; R10 and R11 are independently selected from hydrogen, C1-4alkyl, C1-4alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4alkyl, C3-8heterocycloalkyl, C1-10heteroaryl; or R10 and R1l together with the nitrogen to which R10 and R11 are both attached form C3-8heterocycloalkyl or C1-10heteroaryl; R3 and R7 are independently selected from hydrogen and halo; R4 and R6 are independently selected from hydrogen, C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, —S(O)0-2R9; and R5 is selected from halo, C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, C3-8heterocycloalkyl, —OX2R9 and —S(O)0-2R9; wherein X2 is selected from a bond and C1-4alkylene; and each R9 is independently selected from hydrogen, C1-6alkyl, C3-12cycloalkyl and C3-8heterocycloalkyl; wherein said cycloalkyl or heterocycloalkyl of R9 is optionally substituted with 1 to 3 radicals independently selected from C1-6alkyl and halo-substituted-C1-6alkyl; or R4 and R5 together with the carbon atoms to which R4 and R5 are attached form C3-8heteroaryl, with the proviso that at least one of R3, R4, R5, R6 or R7 has a sulfur directly linked to the phenyl ring.
In another embodiment, R1 is selected from hydrogen, hydroxy, pyrrolidinyl, morpholino, methoxy, difluoro-methoxy, 2-fluoro-ethoxy and methyl; or R1 and R2a together with the carbon atoms to which R1 and R2a are attached form phenyl (that is, the pyridyl ring of the Markush structure is fused to a phenyl ring formed from R1 and R2a thereby creating an isoquinolinyl ring group).
In another embodiment, R4 and R6 are independently selected from hydrogen, methyl-sulfonyl, methyl, 2-fluoro-ethoxy, methyl-piperazinyl-sulfonyl, propoxy, isobutoxy, 2,2,2-trifluoroethoxy, 2,3-difluoro-2-(fluoromethyl)propoxy, butoxy and cyclopropyl-methoxy; R5 is selected from hydrogen, halo, methyl-sulfonyl, methyl, methoxy, ethoxy and morpholino; or R4 and R5 together with the carbon atoms to which R4 and R5 are attached form thiazolyl.
In another embodiment are compounds selected from: N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 4-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzo[d]thiazole-6-carboxamide; 2-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 4-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; 4-ethoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(5-morpholinopyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(5-(pyrrolidin-1-yl)pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)-4-morpholinobenzamide; N-(3-(4-(5-methoxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(3-(4-(5-(2-fluoroethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(3-(4-(5-(cyclopropylmethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(5-methylpyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-(2-fluoroethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-propoxybenzamide; 3-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-butoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-(cyclopropylmethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(3-(4-(isoquinolin-4-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3-(methylsulfonyl)benzamide; 4-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(3-(4-(5-(difluoromethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(4-methylpiperazin-1-ylsulfonyl)benzamide; N-(3-(4-(5-hydroxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; 3-isobutoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-(2,2,2-trifluoroethoxy)benzamide; and 3-(2,3-difluoro-2-(fluoromethyl)propoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide.
In one embodiment, the invention provides methods for treating a disease or condition modulated by the c-kit and PDGFRα/β kinase receptors, comprising administering compounds of Formula I, or pharmaceutically acceptable salts or pharmaceutical compositions thereof.
Examples of c-kit and/or PDGFRα/β mediated disease or conditions which may be mediated using the compounds and compositions of the invention include but are not limited to a neoplastic disorder, an allergy disorder, an inflammatory disorder, an autoimmune disorder, a graft-versus-host disease, a Plasmodium related disease, a mast cell associated disease, a metabolic syndrome, a CNS related disorder, a neurodegenerative disorder, a pain condition, a substance abuse disorder, a prion disease, a cancer, a heart disease, a fibrotic disease, idiopathic arterial hypertension (IPAH), or primary pulmonary hypertension (PPH).
Examples of a mast cell associated disease which may be treated using compounds and compositions of the invention include but are not limited to acne and Propionibacterium acnes, Fibrodysplasia ossificans progressiva (FOP), inflammation and tissue destruction induced by exposure to chemical or biological weapons (such as anthrax and sulfur-mustard), Cystic fibrosis; renal disease, inflammatory muscle disorders, HIV, type II diabetes, cerebral ischemia, mastocytosis, drug dependence and withdrawal symptoms, CNS disorders, preventing and minimizing hair loss, bacterial infections, interstitial cystitis, inflammatory bowel syndrome (IBS), inflammatory bowel diseases (IBD), tumor angiogenesis, autoimmune diseases, inflammatory diseases, Multiple Sclerosis (MS), allergic disorders (including asthma), and bone loss.
Examples of neoplastic disorders which may be treated using the compounds and compositions of the invention include but are not limited to mastocytosis, gastrointestinal stromal tumor, small cell lung cancer, non-small cell lung cancer, acute myelocytic leukemia, acute lymphocytic leukemia, myelodyplastic syndrome, chronic myelogenous leukemia, colorectal carcinoma, gastric carcinoma, testicular cancer, glioblastoma or astrocytoma.
Examples of allergy disorders which may be treated using the compounds and compositions of the invention include but are not limited to asthma, allergic rhinitis, allergic sinusitis, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis, insect bite skin inflammation, or blood sucking parasite infestation.
Examples of inflammatory disorders which may be treated using the compounds and compositions of the invention include but are not limited to rheumatoid arthritis, conjunctivitis, rheumatoid spondylitis, osteoarthritis or gouty arthritis.
Examples of autoimmune disorders which may be treated using the compounds and compositions of the invention include but are not limited to multiple sclerosis, psoriasis, intestine inflammatory disease, ulcerative colitis, Crohn's disease, rheumatoid arthritis, polyarthritis, local or systemic scleroderma, systemic lupus erythematosus, discoid lupus erythematosis, cutaneous lupus, dermatomyositis, polymyositis, Sjogren's syndrome, nodular panarteritis, autoimmune enteropathy or proliferative glomerulonephritis.
Examples of graft-versus-host diseases which may be treated using the compounds and compositions of the invention include but are not limited to organ transplantation graft rejection, such as kidney transplantation, pancreas transplantation, liver transplantation, heart transplantation, lung transplantation, or bone marrow transplantation.
Examples of metabolic syndrome which may be treated using the compounds and compositions of the invention include but are not limited to type I diabetes, type II diabetes, or obesity.
Examples of CNS related disorders which may be treated using the compounds and compositions of the invention include but are not limited to depression, dysthymic disorder, cyclothymic disorder, anorexia, bulimia, premenstrual syndrome, post-menopause syndrome, mental slowing, loss of concentration, pessimistic worry, agitation, self-deprecation and decreased libido, an anxiety disorder, a psychiatric disorder or schizophrenia.
Examples of depression conditions which may be treated using the compounds and compositions of the invention include but are not limited to bipolar depression, severe or melancholic depression, atypical depression, refractory depression, or seasonal depression. Examples of anxiety disorders which may be treated using the compounds and compositions of the invention include but are not limited to anxiety associated with hyperventilation and cardiac arrhythmias, phobic disorders, obsessive-compulsive disorder, posttraumatic stress disorder, acute stress disorder, and generalized anxiety disorder. Examples of psychiatric disorders which may be treated using the compounds and compositions of the invention include but are not limited to panic attacks, including psychosis, delusional disorders, conversion disorders, phobias, mania, delirium, dissociative episodes including dissociative amnesia, dissociative fugue and dissociative suicidal behavior, self-neglect, violent or aggressive behavior, trauma, borderline personality, and acute psychosis such as schizophrenia, including paranoid schizophrenia, disorganized schizophrenia, catatonic schizophrenia, and undifferentiated schizophrenia.
Examples of neurodegenerative disorder which may be treated using the compounds and compositions of the invention include but are not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, the prion diseases, Motor Neuron Disease (MND), or Amyotrophic Lateral Sclerosis (ALS).
Examples of pain conditions which may be treated using the compounds and compositions of the invention include but are not limited to acute pain, postoperative pain, chronic pain, nociceptive pain, cancer pain, neuropathic pain or psychogenic pain syndrome.
Examples of substance use disorders which may be treated using the compounds and compositions of the invention include but are not limited to drug addiction, drug abuse, drug habituation, drug dependence, withdrawal syndrome or overdose.
Examples of cancers which may be treated using the compounds and compositions of the invention include but are not limited to melanoma, colon cancer, gastrointestinal stromal tumor (GIST), small cell lung cancer, or other solid tumors.
Examples of fibrotic diseases which may be treated using the compounds and compositions of the invention include but are not limited to hepatitis C(HCV), liver fibrosis, nonalcoholic steatohepatitis (NASH), scleroderma, cirrhosis in liver, pulmonary fibrosis, or bone marrow fibrosis.
In another embodiment, the invention provides methods for treating a disease or condition modulated by the c-kit and/or PDGFRα/β kinase receptor, comprising administering compounds of Formula I, or pharmaceutically acceptable salts or pharmaceutical compositions thereof
Pharmacology and Utility
Compounds of the invention modulate the activity of kinases and, as such, are useful for treating diseases or disorders in which kinases, contribute to the pathology and/or symptomology of the disease. Examples of kinases that are inhibited by the compounds and compositions described herein and against which the methods described herein are useful include, but are not limited to c-kit, PDGFRα, PDGFRβ, Lyn, MAPK14 (p38delta), PDGFRα, PDGFRβ, ARG, BCR-Abl, BRK, EphB, Fms, Fyn, KDR, LCK, b-Raf, c-Raf, SAPK2, Src, Tie2 and TrkB kinase.
Mast cells (MC) are tissue elements derived from a particular subset of hematopoietic stem cells that produce a large variety of mediators most of which having strong pro-inflammatory activities. Since MCs are distributed in almost all the body sites, hypersecretion of mediators by activated elements can lead to multiple organ failures. Mast cells are, therefore, central players involved in many diseases. The present invention relates to a method for treating mast cell associated diseases comprising administering a compound capable of depleting mast cells or a compound inhibiting mast cell degranulation, to a human in need of such treatment. Such compounds can be chosen from c-kit inhibitors and more particularly non-toxic, selective and potent c-kit inhibitors. Preferably, said inhibitors are unable to promote death of IL-3 dependent cells cultured in presence of IL-3.
Mast cell associated diseases include, but are not limited to: acne and Propionibacterium acnes (acne encompasses all forms of chronic inflammation of the skin including those induced by Propionibacterium acnes); an extremely rare and disabling genetic disorder of connective tissue known as Fibrodysplasia ossificans progressiva (FOP); the detrimental effects of inflammation and tissue destruction induced by exposure to chemical or biological weapons (such as anthrax, sulfur-mustard, etc.); Cystic fibrosis (a lung, digestive and reproductive systems genetic disease); renal disease such as Acute nephritic syndrome, glomerulonephritis, renal amyloidosis, renal interstitial fibrosis (the final common pathway leading to end-stage renal disease in various nephropathies); inflammatory muscle disorders including myositis and muscular dystrophy; HIV (for example, depleting HIV infected mast cells can be a new route for treating HIV infection and related diseases); treating type II diabetes, obesity and related disorders (mast cells regulate a number of the processes that contribute to the development of atherosclerosis, including hyperglycemia, hypercholesterolemia, hypertension, endothelial dysfunction, insulin resistance, and vascular remodeling; cerebral ischemia; mastocytosis (a very heterogeneous group of disorders characterized by an abnormal accumulation of mast cells in different tissues, mainly in the skin and the bone marrow, but also in spleen, liver, lymph nodes, and the gastrointestinal tract); drug dependence and withdrawal symptoms (particularly drug addiction, drug abuse, drug habituation, drug dependence, withdrawal syndrome and overdose); CNS disorders (particularly depression, schizophrenia, anxiety, migraine, memory loss, pain and neurodegenerative diseases); promoting hair growth (including preventing and minimizing hair loss); bacterial infections (particularly infections caused by FimH expressing bacteria); interstitial cystitis (a chronic inflammation of the bladder wall resulting in tissue damage, especially at the interstices between the cells in the lining of the bladder); Inflammatory bowel diseases (generally applied to four diseases of the bowel, namely Crohn's disease, ulcerative colitis, indeterminate colitis, and infectious colitis); tumor angiogenesis; autoimmune diseases (particularly multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis and polyarthritis, scleroderma, lupus erythematosus, dermatomyositis, pemphigus, polymyositis, vasculitis and graft-versus host diseases); inflammatory diseases such as rheumatoid arthritis (RA); Multiple Sclerosis (MS); allergic disorders (particularly asthma, allergic rhinitis, allergic sinusitis, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, allergic contact dermatitis, erythema nodosum, erythema multiforme, cutaneous necrotizing venulitis and insect bite skin inflammation, bronchial asthma); nasal polyposis; inflammatory bowel disease (IBD) and inflammatory bowel syndrome (IBS); and bone loss.
PDGF (Platelet-derived Growth Factor) is a very commonly occurring growth factor, which plays an important role both in normal growth and also in pathological cell proliferation, such as is seen in carcinogenesis and in diseases of the smooth-muscle cells of blood vessels, for example in atherosclerosis and thrombosis. Compounds of the invention can inhibit PDGF receptor (PDGFR) activity and are, therefore, suitable for the treatment of: tumor diseases, such as gliomas, sarcomas, prostate tumors, and tumors of the colon, breast, and ovary; hypereosinophilia; fibrosis such as lung fibrosis, liver fibrosis and scleroderma; pulmonary hypertension; and cardiovascular diseases.
The Ras-Raf-MEK-ERK signaling pathway mediates cellular response to growth signals. Ras is mutated to an oncogenic form in  ̃15% of human cancer. The Raf family belongs to the serine/threonine protein kinase and it includes three members, A-Raf, B-Raf and c-Raf (or Raf-1). The focus on Raf being a drug target has centered on the relationship of Raf as a downstream effector of Ras. However, recent data suggests that B-Raf may have a prominent role in the formation of certain tumors with no requirement for an activated Ras allele (Nature 417, 949-954 (1 Jul. 2002). In particular, B-Raf mutations have been detected in a large percentage of malignant melanomas.
Existing medical treatments for melanoma are limited in their effectiveness, especially for late stage melanomas. The compounds of the present invention also inhibit cellular processes involving b-Raf kinase, providing a new therapeutic opportunity for treatment of human cancers, especially for melanoma.
The compounds of the present invention also inhibit cellular processes involving c-Raf kinase. c-Raf is activated by the ras oncogene, which is mutated in a wide number of human cancers. Therefore inhibition of the kinase activity of c-Raf may provide a way to prevent ras mediated tumor growth [Campbell, S. L., Oncogene, 17, 1395 (1998)].
Compounds of the present invention, can be used to treat non-malignant proliferative disorders, such as atherosclerosis, thrombosis, psoriasis, scleroderma and fibrosis, as well as for the protection of stem cells, for example to combat the hemotoxic effect of chemotherapeutic agents, such as 5-fluoruracil, and in asthma.
Compounds of the present invention show useful effects in the treatment of disorders arising as a result of transplantation, for example, allogenic transplantation, especially tissue rejection, such as especially obliterative bronchiolitis (OB), i.e. a chronic rejection of allogenic lung transplants. In contrast to patients without OB, those with OB often show an elevated PDGF concentration in bronchoalveolar lavage fluids.
Compounds of the present invention are also effective in diseases associated with vascular smooth-muscle cell migration and proliferation (where PDGF and PDGF-R often also play a role), such as restenosis and atherosclerosis. These effects and the consequences thereof for the proliferation or migration of vascular smooth-muscle cells in vitro and in vivo can be demonstrated by administration of the compounds of the present invention, and also by investigating its effect on the thickening of the vascular intima following mechanical injury in vivo.
The trk family of neurotrophin receptors (trkA, trkB, trkC) promotes the survival, growth and differentiation of the neuronal and non-neuronal tissues. The TrkB protein is expressed in neuroendocrine-type cells in the small intestine and colon, in the alpha cells of the pancreas, in the monocytes and macrophages of the lymph nodes and of the spleen, and in the granular layers of the epidermis (Shibayama and Koizumi, 1996). Expression of the TrkB protein has been associated with an unfavorable progression of Wilms tumors and of neuroblastomas. TkrB is, moreover, expressed in cancerous prostate cells but not in normal cells. The signaling pathway downstream of the trk receptors involves the cascade of MAPK activation through the Shc, activated Ras, ERK-1 and ERK-2 genes, and the PLC-gamrnal transduction pathway (Sugimoto et al., 2001).
The kinase, c-Src transmits oncogenic signals of many receptors. For example, over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of c-src, which is characteristic for the malignant cell but absent from the normal cell. On the other hand, mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.
The Tec family kinase, Bmx, a non-receptor protein-tyrosine kinase, controls the proliferation of mammary epithelial cancer cells.
Fibroblast growth factor receptor 3 was shown to exert a negative regulatory effect on bone growth and an inhibition of chondrocyte proliferation. Thanatophoric dysplasia is caused by different mutations in fibroblast growth factor receptor 3, and one mutation, TDII FGFR3, has a constitutive tyrosine kinase activity which activates the transcription factor Stat1, leading to expression of a cell-cycle inhibitor, growth arrest and abnormal bone development (Su et al., Nature, 1997, 386, 288-292). FGFR3 is also often expressed in multiple myeloma-type cancers. Inhibitors of FGFR3 activity are useful in the treatment of T-cell mediated inflammatory or autoimmune diseases including but not limited to rheumatoid arthritis (RA), collagen II arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease, sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerative colitis), celiac disease and myasthenia gravis.
The activity of serum and glucocorticoid-regulated kinase (SGK), is correlated to perturbed ion-channel activities, in particular, those of sodium and/or potassium channels and compounds of the invention can be useful for treating hypertension.
Lin et al (1997) J. Clin. Invest. 100, 8: 2072-2078 and P. Lin (1998) PNAS 95, 8829-8834, have shown an inhibition of tumor growth and vascularization and also a decrease in lung metastases during adenoviral infections or during injections of the extracellular domain of Tie-2 (Tek) in breast tumor and melanoma xenograft models. Tie2 inhibitors can be used in situations where neovascularization takes place inappropriately (i.e. in diabetic retinopathy, chronic inflammation, psoriasis, Kaposi's sarcoma, chronic neovascularization due to macular degeneration, rheumatoid arthritis, infantile haemangioma and cancers).
Lck plays a role in T-cell signaling. Mice that lack the Lck gene have a poor ability to develop thymocytes. The function of Lck as a positive activator of T-cell signaling suggests that Lck inhibitors may be useful for treating autoimmune disease such as rheumatoid arthritis.
JNKs, along with other MAPKs, have been implicated in having a role in mediating cellular response to cancer, thrombin-induced platelet aggregation, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis and heart disease. The therapeutic targets related to activation of the JNK pathway include chronic myelogenous leukemia (CML), rheumatoid arthritis, asthma, osteoarthritis, ischemia, cancer and neurodegenerative diseases. As a result of the importance of JNK activation associated with liver disease or episodes of hepatic ischemia, compounds of the invention may also be useful to treat various hepatic disorders. A role for JNK in cardiovascular disease such as myocardial infarction or congestive heart failure has also been reported as it has been shown JNK mediates hypertrophic responses to various forms of cardiac stress. It has been demonstrated that the JNK cascade also plays a role in T-cell activation, including activation of the IL-2 promoter. Thus, inhibitors of JNK may have therapeutic value in altering pathologic immune responses. A role for JNK activation in various cancers has also been established, suggesting the potential use of JNK inhibitors in cancer. For example, constitutively activated JNK is associated with HTLV-1 mediated tumorigenesis [Oncogene 13:135-42 (1996)]. JNK may play a role in Kaposi's sarcoma (KS). Other proliferative effects of other cytokines implicated in KS proliferation, such as vascular endothelial growth factor (VEGF), IL-6 and TNFα, may also be mediated by JNK. In addition, regulation of the c-jun gene in p210 BCR-ABL transformed cells corresponds with activity of JNK, suggesting a role for JNK inhibitors in the treatment for chronic myelogenous leukemia (CML) [Blood 92:2450-60 (1998)].
Certain abnormal proliferative conditions are believed to be associated with raf expression and are, therefore, believed to be responsive to inhibition of raf expression. Abnormally high levels of expression of the raf protein are also implicated in transformation and abnormal cell proliferation. These abnormal proliferative conditions are also believed to be responsive to inhibition of raf expression. For example, expression of the c-raf protein is believed to play a role in abnormal cell proliferation since it has been reported that 60% of all lung carcinoma cell lines express unusually high levels of c-raf mRNA and protein. Further examples of abnormal proliferative conditions are hyper-proliferative disorders such as cancers, tumors, hyperplasia, pulmonary fibrosis, angiogenesis, psoriasis, atherosclerosis and smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. The cellular signaling pathway of which raf is a part has also been implicated in inflammatory disorders characterized by T-cell proliferation (T-cell activation and growth), such as tissue graft rejection, endotoxin shock, and glomerular nephritis, for example.
The stress activated protein kinases (SAPKs) are a family of protein kinases that represent the penultimate step in signal transduction pathways that result in activation of the c-jun transcription factor and expression of genes regulated by c-jun. In particular, c-jun is involved in the transcription of genes that encode proteins involved in the repair of DNA that is damaged due to genotoxic insults. Therefore, agents that inhibit SAPK activity in a cell prevent DNA repair and sensitize the cell to agents that induce DNA damage or inhibit DNA synthesis and induce apoptosis of a cell or that inhibit cell proliferation.
Mitogen-activated protein kinases (MAPKs) are members of conserved signal transduction pathways that activate transcription factors, translation factors and other target molecules in response to a variety of extracellular signals. MAPKs are activated by phosphorylation at a dual phosphorylation motif having the sequence Thr-X-Tyr by mitogen-activated protein kinase kinases (MKKs). In higher eukaryotes, the physiological role of MAPK signaling has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways (particularly via MKK4 and MKK6) could lead to the development of treatments and preventive therapies for human diseases associated with MAPK signaling, such as inflammatory diseases, autoimmune diseases and cancer.
The family of human ribosomal S6 protein kinases consists of at least 8 members (RSK1, RSK2, RSK3, RSK4, MSK1, MSK2, p70S6K and p70S6 Kb). Ribosomal protein S6 protein kinases play important pleotropic functions, among them is a key role in the regulation of mRNA translation during protein biosynthesis (Eur. J. Biochem 2000 November; 267 (21): 6321-30, Exp Cell Res. Nov. 25, 1999; 253 (1):100-9, Mol Cell Endocrinol. May 25, 1999; 151 (1-2):65-77). The phosphorylation of the S6 ribosomal protein by p70S6 has also been implicated in the regulation of cell motility (Immunol. Cell Biol. 2000 August; 78 (4):447-51) and cell growth (Prog. Nucleic Acid Res. Mol. Biol., 2000; 65:101-27), and hence, may be important in tumor metastasis, the immune response and tissue repair as well as other disease conditions.
The SAPK's (also called “jun N-terminal kinases” or “JNK's”) are a family of protein kinases that represent the penultimate step in signal transduction pathways that result in activation of the c-jun transcription factor and expression of genes regulated by c-jun. In particular, c-jun is involved in the transcription of genes that encode proteins involved in the repair of DNA that is damaged due to genotoxic insults. Agents that inhibit SAPK activity in a cell prevent DNA repair and sensitize the cell to those cancer therapeutic modalities that act by inducing DNA damage.
BTK plays a role in autoimmune and/or inflammatory disease such as systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple vasculitides, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, and asthma. Because of BTK's role in B-cell activation, inhibitors of BTK are useful as inhibitors of B-cell mediated pathogenic activity, such as autoantibody production, and are useful for the treatment of B-cell lymphoma and leukemia.
CHK2 is a member of the checkpoint kinase family of serine/threonine protein kinases and is involved in a mechanism used for surveillance of DNA damage, such as damage caused by environmental mutagens and endogenous reactive oxygen species. As a result, it is implicated as a tumor suppressor and target for cancer therapy.
CSK influences the metastatic potential of cancer cells, particularly colon cancer.
Fes is a non-receptor protein tyrosine kinase that has been implicated in a variety of cytokine signal transduction pathways, as well as differentiation of myeloid cells. Fes is also a key component of the granulocyte differentiation machinery.
Flt3 receptor tyrosine kinase activity is implicated in leukemias and myelodysplastic syndrome. In approximately 25% of AML the leukemia cells express a constitutively active form of auto-phosphorylated (p) FLT3 tyrosine kinase on the cell surface. The activity of p-FLT3 confers growth and survival advantage on the leukemic cells. Patients with acute leukemia, whose leukemia cells express p-FLT3 kinase activity, have a poor overall clinical outcome. Inhibition of p-FLT3 kinase activity induces apoptosis (programmed cell death) of the leukemic cells.
Inhibitors of IKKα and IKKβ (1 & 2) are therapeutics for diseases which include rheumatoid arthritis, transplant rejection, inflammatory bowel disease, osteoarthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, psoriasis, multiple sclerosis, stroke, systemic lupus erythematosus, Alzheimer's disease, brain ischemia, traumatic brain injury, Parkinson's disease, amyotrophic lateral sclerosis, subarachnoid hemorrhage or other diseases or disorders associated with excessive production of inflammatory mediators in the brain and central nervous system.
Met is associated with most types of the major human cancers and expression is often correlated with poor prognosis and metastasis. Inhibitors of Met are therapeutics for diseases which include cancers such as lung cancer, NSCLC (non small cell lung cancer), bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), pediatric malignancy, neoplasms of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumors, brain stem glioma or pituitary adenomas), cancers of the blood such as acute myeloid leukemia, chronic myeloid leukemia, etc, Barrett's esophagus (pre-malignant syndrome) neoplastic cutaneous disease, psoriasis, mycoses fungoides and benign prostatic hypertrophy, diabetes related diseases such as diabetic retinopathy, retinal ischemia and retinal neovascularization, hepatic cirrhosis, cardiovascular disease such as atherosclerosis, immunological disease such as autoimmune disease and renal disease. Preferably, the disease is cancer such as acute myeloid leukemia and colorectal cancer.
The Nima-related kinase 2 (Nek2) is a cell cycle-regulated protein kinase with maximal activity at the onset of mitosis that localizes to the centrosome. Functional studies have implicated Nek2 in regulation of centrosome separation and spindle formation. Nek2 protein is elevated 2- to 5-fold in cell lines derived from a range of human tumors including those of cervical, ovarian, prostate, and particularly breast.
p70S6K-mediated diseases or conditions include, but are not limited to, proliferative disorders, such as cancer and tuberous sclerosis.
In accordance with the foregoing, the present invention further provides a method for preventing or treating any of the diseases or disorders described above in a subject in need of such treatment, which method comprises administering to said subject a therapeutically effective amount (See, “Administration and Pharmaceutical Compositions”, infra) of a compound of Formula I or a pharmaceutically acceptable salt thereof. For any of the above uses, the required dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired.
Administration and Pharmaceutical Compositions
In general, compounds of the invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per body weight. An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g. in divided doses up to four times a day or in retard form. Suitable unit dosage forms for oral administration comprise from ca. 1 to 50 mg active ingredient.
Compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal, inhaled or suppository form. Pharmaceutical compositions comprising a compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
Compounds of the invention can be administered in therapeutically effective amounts in combination with one or more therapeutic agents (pharmaceutical combinations). For example, synergistic effects can occur with other asthma therapies, for example, steroids and leukotriene antagonists.
For example, synergistic effects can occur with other immunomodulatory or anti-inflammatory substances, for example when used in combination with cyclosporin, rapamycin, or ascomycin, or immunosuppressant analogues thereof, for example cyclosporin A (CsA), cyclosporin G, FK-506, rapamycin, or comparable compounds, corticosteroids, cyclophosphamide, azathioprine, methotrexate, brequinar, leflunomide, mizoribine, mycophenolic acid, mycophenolate mofetil, broncho dilators, 15-deoxyspergualin, immunosuppressant antibodies, especially monoclonal antibodies for leukocyte receptors, for example MHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands, or other immunomodulatory compounds, such as CTLA41g. Where the compounds of the invention are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.
The invention also provides for a pharmaceutical combinations, e.g. a kit, comprising a) a first agent which is a compound of the invention as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. The kit can comprise instructions for its administration.
The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g. a compound of Formula I and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g. a compound of Formula I and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the 2 compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of 3 or more active ingredients.
Processes for Making Compounds of the Invention
The present invention also includes processes for the preparation of compounds of the invention. In the reactions described, it can be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups can be used in accordance with standard practice, for example, see T. W. Greene and P. G. M. Wuts in “Protective Groups in Organic Chemistry”, John Wiley and Sons, 1991.
Compounds of Formula I, wherein L is —NHC(O)—, can be prepared by proceeding as in the following Reaction Schemes I:




wherein R1, R2a, R2b, R3, R4, R5, R6 and R7 are as described in the Summary of the Invention. A compound of Formula I can be prepared by reacting of a compound of formula 2 with a compound of formula 3 in the presence of a suitable solvent (for example, DMF, and the like), a suitable coupling agent (for example, HATU, and the like) and a suitable base (for example, DIEA, and the like). The reaction is carried out in a temperature range of about 0° C. to about 60° C. and can take up to 24 hours to complete.
Compounds of Formula I, wherein L is —C(O)NH—, can be prepared by proceeding as in the following Reaction Schemes II:




wherein R1, R2, R3, R4, R5, R6 and R7 are as described in the Summary of the Invention. A compound of Formula I can be prepared by reacting of a compound of formula 4 with a compound of formula 5 in the presence of a suitable solvent (for example, DMF, and the like), a suitable coupling agent (for example, HATU, and the like) and a suitable base (for example, DIEA, and the like). The reaction is carried out in a temperature range of about 0° C. to about 60° C. and can take up to 24 hours to complete.
Detailed examples of the synthesis of compounds of formula I can be found in the Examples, infra.
Additional Processes for Making Compounds of the Invention
A compound of the invention can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. Alternatively, the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.
The free acid or free base forms of the compounds of the invention can be prepared from the corresponding base addition salt or acid addition salt from, respectively. For example a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).
Compounds of the invention in unoxidized form can be prepared from N-oxides of compounds of the invention by treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like) in a suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80° C.
Prodrug derivatives of the compounds of the invention can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs can be prepared by reacting a non-derivatized compound of the invention with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the like).
Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, “Protecting Groups in Organic Chemistry”, 3rd edition, John Wiley and Sons, Inc., 1999.
Compounds of the present invention can be conveniently prepared, or formed during the process of the invention, as solvates (e.g., hydrates). Hydrates of compounds of the present invention can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol.
Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. While resolution of enantiomers can be carried out using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, or preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley And Sons, Inc., 1981.
In summary, the compounds of Formula I can be made by a process, which involves:
(a) those of reaction schemes I and II, and
(b) optionally converting a compound of the invention into a pharmaceutically acceptable salt;
(c) optionally converting a salt form of a compound of the invention to a non-salt form;
(d) optionally converting an unoxidized form of a compound of the invention into a pharmaceutically acceptable N-oxide;
(e) optionally converting an N-oxide form of a compound of the invention to its unoxidized form;
(f) optionally resolving an individual isomer of a compound of the invention from a mixture of isomers;
(g) optionally converting a non-derivatized compound of the invention into a pharmaceutically acceptable prodrug derivative; and
(h) optionally converting a prodrug derivative of a compound of the invention to its non-derivatized form.
Insofar as the production of the starting materials is not particularly described, the compounds are known or can be prepared analogously to methods known in the art or as disclosed in the Examples hereinafter.
One of skill in the art will appreciate that the above transformations are only representative of methods for preparation of the compounds of the present invention, and that other well known methods can similarly be used.
EXAMPLES
The present invention is further exemplified, but not limited, by the following examples that illustrate the preparation of compounds of Formula I according to the invention.
Experimental Preparation of Intermediates
Synthesis of 6-methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine 5




To 2-amino-4-nitro toluene 1 (0.033 mol) in n-butanol (29 mL) is added nitric acid (2.1 g, 65% in water) followed by cyanamide solution in water (2 mL, 0.047 mmol). The resulting mixture is heated at reflux for 25 h. After cooling to 0° C., filtration and washing with 1:1=ethanol:diethyl ether (30 mL), 2-methyl-5-nitrophenyl guanidine nitrate 2 is obtained. To 2-methyl-5-nitrophenyl guanidine 2 (0.0074 mol) in isopropanol (15 ME) is added 3 (0.0074 mol) and sodium hydroxide flakes (0.008 mol). The resulting mixture is heated at reflux for 12 h. After cooling to 0° C., the product is collected by filtration and washed with isopropanol (6 mL) and methanol (3 mL) to afford 4. 1H NMR (400 MHz, d6-DMSO) δ 9.31 (s, 1H), 9.24 (s, 1H), 8.78 (m, 1H), 8.70 (m, 1H), 8.61 (m, 1H), 8.47 (m, 1H), 7.88 (m, 1H), 7.55 (m, 3H), 2.39 (s, 3H). MS (m/z) (M+1)+: 278.2.
Reactant 3 can be obtained by the following procedures. A mixture of 3-acetylpyridine (2.47 mol) and N,N-dimethylformamide dimethylacetal (240 mL) is heated at reflux for 16 h. The solvent is removed in vacuo and hexanes (100 mL) is added to the residue to crystallize a solid. The solid is recrystallized from methylene chloride-hexanes to give 3-dimethylamino-1-(3-pyridyl)-2-propen-1-one 3.
1H NMR (400 MHz, d-chloroform) δ 9.08 (d, J=2.4 Hz, 1H), 8.66 (m, 1H), 8.20 (m, 1H), 7.87 (m, 1H), 7.37 (m, 1H), 5.68 (d, J=16.4 Hz, 1H), 3.18 (s, 3H), 2.97 (s, 3H).
A reactor is charged with concentrated hydrochloric acid (17 mL) followed by stannous chloride dehydrate (0.03 mol). The mixture is stirred for 10 min and then cooled to 0-5° C. A solution of compound 4 (5.6 mmol) in ethyl acetate (3 mL) is then slowly added (during 3-4 minutes) while maintaining the temperature at 0-5° C. The reaction mixture is brought to rt and stirred for 1.5 h. To this is added water (50 mL) followed by a slow addition of 50% sodium hydroxide solution (40 mL). The resulting mixture is extracted with chloroform (2×25 mL). The organic layer is washed with water thoroughly and evaporated. The residue is dissolved in ethyl acetate (2 mL), cooled to 0-10° C. and maintained at this temperature for 1 h. The resulting precipitate is collected by filtration and washed with ethyl acetate (1 mL) to provide 1.0 g of 5. 1H NMR (400 MHz, d-chloroform) δ 9.26 (d, J=2.0 Hz, 1H), 8.71 (m, 1H), 8.48 (d, J=6.8 Hz, 1H), 8.34 (m, 1H), 7.59 (d, J=4.0 Hz, 1H), 7.41 (m, 1H), 7.12 (m, 1H), 7.04 (m, 1H), 6.42 (m, 1H), 3.50 (bs, 2H), 2.24 (s, 3H).
Synthesis of 4-(5-bromopyridin-3-yl)-N-(2-methyl-5-nitrophenyl)pyrimidin-2-amine 8




1-(5-Bromo-pyridin-3-yl)-3-dimethylamino-propenone 7 is prepared from 1-(5-bromo-pyridin-3-yl)-ethanone with a similar protocol used for the synthesis of 3. LC/MS (m/z) (M+1)+: 386.1, 388.1.
Condensation with N-(2-methyl-5-nitro-phenyl)-guanidine nitrate with a protocol similar to the one used for the preparation of 4 affords [4-(5-bromo-pyridin-3-yl)-pyrimidin-2-yl]-(2-methyl-5-nitro-phenyl)-amine 8. 1H NMR (400 MHz, d-CDCl3) δ 9.33 (d, J=2.4 Hz, 1H), 9.09 (d, J=1.6 Hz, 1H), 8.74 (d, J=2.4 Hz, 1H), 8.61 (t, J=2.0 Hz, 1H), 8.53 (d, J=4.8 Hz, 1H), 8.01 (s, 1H), 7.82 (dd, J=8.4, 2.4 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.22 (d, J=4.8 Hz, 1H), 7.10 (s, 1H), 2.41 (s, 3H). LC/MS (m/z) (M+1)+: 255.0, 257.0.
Synthesis of 6-methyl-N1-(4-(5-(pyrrolidin-1-yl)pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine 10




(2-Meth yl-5-nitro-phenyl)-[4-(5-pyrrolidin-1-yl-pyridin-3-yl)-pyrimidin-2-yl]-amine 9 is prepared by mixing 8 (0.40 g, 1.04 mmol), pyrrolidine (3.12 mmol), K3PO4 (0.44 g, 2.08 mmol), CuI (38.5 mg, 0.10 mmol) and L-proline amino acid (48.1 mg, 0.21 mmol) in DMSO (8 ml) and heating at 160° C. in a microwave oven for 20 min. The cooled mixture is partitioned between water and ethyl acetate. The organic layer is separated, and the aqueous layer is extracted with ethyl acetate. The combined organic layers are washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue is dissolved in DMSO (4 mL) and purified with HPLC to afford pure product 9. 1H NMR (400 MHz, d-CDCl3) δ 9.23 (d, J=2.4 Hz, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.58 (s, 1H), 8.10 (s, 1H), 8.04 (s, 1H), 7.90 (dd, J=8.4, 2.4 Hz, 1H), 7.44 (s, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.30 (d, J=5.2 Hz, 1H), 3.43-3.48 (m, 4H), 2.49 (s, 3H), 2.12-2.16 (m, 4H). LC/MS (m/z) (M+1)+: 377.2.
4-Methyl-N-3-[4-(5-pyrrolidin-1-yl-pyridin-3-yl)-pyrimidin-2-yl]-benzene-1,3-diamine 10 is prepared by hydrogenation of 9 in EtOH in the presence of 10% Pd/C and 1 atm hydrogen at rt (30%). Solvent is removed after filtration through Celite and the product is used without further purification. LC/MS (m/z) (M+1)+: 347.2.
By substituting the pyrrolidine with other amines (e.g. morpholine) different intermediates similar to 9 and 10 can be synthesized.
Synthesis of N1-(4-(5-methoxypyridin-3-yl)pyrimidin-2-yl)-6-methylbenzene-1,3-diamine




A mixture of 3-bromo-5-methoxypyridine (3.0 g, 16 mmol) and Pd(PPh3)4 is degassed several times and purged with N2. Tributyl(1-ethoxyvinyl)tin (7.04 mL, 20.8 mmol) and toluene (15 mL) are added to the mixture. The resulting reaction mixture is heated at 150° C. for 30 minutes in a microwave oven. After the reaction is complete, the reaction mixture is filtered through a pad of Celite, washed with MeOH and concentrated to give a residue. To the above residue is added HCl (1N, 25 mL) and THF (25 mL) and the mixture is stirred at room temperature for 2 h. The solvent is removed in vacuo and the residue is dissolved in EtOAc. The organic layer is washed with Na2CO3 solution, dried with Na2SO4, filtered and concentrated to afford a residue which is purified by silica gel column chromatography (EtOAc:hexanes=1:1) to afford 11 as a light yellow solid. 1H NMR (400 MHz, d6-DMSO) δ 8.75 (d, J=1.6 Hz, 1H), 8.52 (d, J=3.2 Hz, 1H), 7.74 (dd, J=1.6, 3.2 Hz, 1H), 3.91 (s, 3H), 2.64 (s, 3H). MS (m/z) (M+1)+: 152.1.
3-(Dimethylamino)-1-(5-methoxypyridin-3-yl)prop-2-en-1-one 12 is prepared from 11 by reacting with diethoxy-N,N-dimethylmethanamine using a similar procedure for the preparation of 3. 1H NMR (400 MHz, d6-DMSO) δ 8.69 (d, J=2.0 Hz, 1H), 8.37 (d, J=2.8 Hz, 1H), 7.76 (d, J=12.0 Hz, 1H), 7.70 (dd, J=1.6, 2.8 Hz, 1H), 5.86 (d, J=12.0 Hz, 1H), 3.89 (s, 3H), 3.17 (s, 3H), 2.95 (s, 3H). MS (m/z) (M+1)+: 207.1
Condensation of 12 with N-(2-methyl-5-nitro-phenyl)-guanidine nitrate 2 using a similar procedure for the preparation of 4 affords 4-(5-methoxypyridin-3-yl)-N-(2-methyl-5-nitrophenyl)pyrimidin-2-amine 13. 1H NMR (400 MHz, d6-DMSO) δ 9.23 (s, 1H), 8.93 (d, J=1.2 Hz, 1H), 8.80 (d, J=2.0 Hz, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.44 (d, J=2.8 Hz, 1H), 8.00 (s, 1H), 7.91 (dd, J=2.0, 8.4 Hz, 1H), 7.62 (d, J=5.2 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 3.90 (s, 3H), 2.44 (s, 3H). MS (m/z) (M+1)+: 338.1
N3-[4-(5-Methoxy-pyridin-3-yl)-pyrimidin-2-yl]-4-methyl-benzene-1,3-diamine 14 is prepared by hydrogenation of 13 using a similar protocol for the preparation of 10. 1H NMR (400 MHz, d6-DMSO) δ 8.90 (d, J=1.6 Hz, 1H), 8.71 (s, 1H), 8.51 (d, J=5.2 Hz, 1H), 8.45 (d, J=2.8 Hz, 1H), 8.01 (dd, J=2.0, 3.2 Hz, 1H), 7.43 (d, J=5.2 Hz, 1H), 6.91 (d, J=8.0 Hz, 1H), 6.87 (d, J=2.4 Hz, 1H), 6.38 (dd, J=2.4, 8.0 Hz, 1H), 4.87 (s, 2H), 3.95 (s, 3H), 2.12 (s, 3H). MS (m/z) (M+1)+: 308.1
Synthesis of N1-(4-(5-(2-fluoroethoxy)pyridin-3-yl)pyrimidin-2-yl)-6-methylbenzene-1,3-diamine 17




Compound 13 (220 mg, 0.65 mmol) is dissolved in DCM and treated with BBr3 (3.25 mmol) at −78° C. overnight. The mixture is diluted with DCM and washed with 1 N aqueous NaOH. The resultant aqueous phase is acidified with excess 1N HCl and extracted with DCM. Concentration gives 15 which is used without further purification. LC/MS (m/z) (M+1)+: 324.2.
Compound 15 (87.0 mg, 0.27 mmol) is heated with CsHCO3 (103.6 mg, 0.54 mmol) and 1-bromo-2-fluoro-ethane (102.0 mg, 0.80 mmol) in acetonitrile (2.0 mL) at reflux overnight. The mixture is purified by preparative LC/MS to afford 16. LC/MS (m/z) (M+1)+: 370.1.
Compound 16 (50.0 mg, 0.14 mmol) is heated with SnCl2.H2O (77.2 mg, 0.41 mmol) in EtOH (2.0 mL) at reflux for 1 h. The mixture is dissolved in NaOH (1N, 50 mL) and extracted with DCM. The organic layer is separated, dried over Na2SO4, filtered and concentrated to give a crude product which is used without further purification. LC/MS (m/z) (M+1)+: 340.1.
By substituting 1-bromo-2-fluoro-ethane with other alkyl halides (e.g. bromomethylcyclopropane) different intermediates similar to 16 and 17 can be synthesized.
Synthesis of N-(2-methyl-5-nitrophenyl)-4-(5-methylpyridin-3-yl)pyrimidin-2-amine 21




5-Methyl-nicotinonitrile (2.08 g, 17.6 mmol) is heated at reflux in dry THF (20 mL) with MgMeBr (3 M solution in Et2O, 10 mL, 30 mmol) for 2 h. After cooling down, aq. Na2CO3 is added slowly to quench the reaction. Extractions with DCM affords the crude mixture which is purified by silica gel chromatography to yield 1-(5-methyl-pyridin-3-yl)-ethanone 18 (0.7 g, 30%). LC/MS (m/z) (M+1)+: 136.1.
1-(5-Methyl-pyridin-3-yl)-ethanone 19 is prepared using a similar procedure for the preparation of 3 by reacting 18 with diethoxy-N,N-dimethylmethanamine. 1H NMR (400 MHz, d6-DMSO) δ 8.87 (d, J=1.2 Hz, 1H), 8.49 (d, J=1.2 Hz, 1H), 8.02-8.04 (m, 1H), 7.75 (d, J=12.0 Hz, 1H), 5.85 (d, J=12.0 Hz, 1H), 3.16 (s, 3H), 2.94 (s, 3H), 2.35 (s, 3H). LC/MS (m/z) (M+1)+: 191.2.
To 2-methyl-5-nitrophenyl guanidine 2 (2.0 mmol) in n-butanol (15 mL) is added 19 (2.0 mmol) and sodium hydroxide flakes (2.0 mmol). The resulting mixture is heated at 180° C. for 40 min in a microwave oven. After cooling to 0° C., the product is collected by filtration and washed with ether (20 mL) and methanol (10 mL) to afford 20. 1H NMR (400 MHz, d6-DMSO) δ 9.19 (s, 1H), 9.13 (d, J=1.6 Hz, 1H), 8.91 (d, J=2.0 Hz, 1H), 8.63 (d, J=5.2 Hz, 1H), 8.57 (d, J=1.2 Hz, 1H), 8.37 (s, 1H), 7.90 (dd, J=8.0, 2.4 Hz, 1H), 7.59 (d, J=5.2 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 2.45 (s, 3H), 2.41 (s, 3H). LC/MS (m/z) (M+1)+: 322.2.
Compound 21 is prepared by hydrogenation of 20 in MeOH in the presence of 10% Pd/C and hydrogen balloon at rt. Solvent is removed after filtration through Celite and the product is used without further purification (95%). LC/MS (m/z) (M+1)+: 292.1.
Synthesis of 3-hydroxy-4-methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide 23




6-Methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine 5 (578 mg, 2.09 mmol), 4-iodo-3-hydroxy-benzoic acid (587 mg, 2.22 mmol) and HATU (960 mg, 2.52 mmol) are dissolved in dry DMF (5 mL) at rt. Diisopropylethylamine (0.732 mL, 4.2 mmol) is added dropwise to the solution. After 30 min, the mixture is added slowly to saturated aqueous NaHCO3. The solid is filtered, washed with water and dried under vacuum overnight to afford the product 22 as a tanish solid (630 mg, 60%). LC/MS (M+1): 524.1.
Compound 22 (580 mg, 1.11 mmol), CuI (42 mg, 0.272 mmol), L-proline (61 mg, 0.444 mmol), 1 N NaOH (0.5 mL) and NaSO2Me (267 mg, 2.22 mmol) are suspended in DMSO (3 mL) and heated at 180° C. in a microwave oven for 25 min. The mixture is diluted with concentrate aqueous ammonia and washed by EtOAc. 1 N aq. HCl is added to aqueous phase until pH=7 and the solid precipitate is collected by filtration to afford product 23 (80%). LC/MS (M+1): 476.1.
Synthesis of Final Compounds
Type A
N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3 (methylsulfonyl)benzamide A1




6-Methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine 5 (5 mmol), 3-(methylsulfonyl)benzoic acid (6 mmol) and HATU (6 mmol) are dissolved in dry DMF (5 mL) at rt. Diisopropylethylamine (6 mmol) is added dropwise to the solution. After 30 min, the mixture is added slowly to saturated aqueous NaHCO3. The solid is filtered, washed with water and dried under vacuum overnight to afford the product A1 as a light yellow solid. 1H NMR (400 MHz, d6-DMSO) δ 10.5 (s, 1H), 9.33 (s, 1H), 9.04 (s, 1H), 8.75 (d, J=1.7 Hz, 1H), 8.63 (d, J=7.5 Hz, 1H), 8.55 (d, J=5.0 Hz, 1H), 8.47 (s, 1H), 8.29 (d, J=8 Hz, 1H), 8.13 (d, J=8.6 Hz, 1H), 8.11 (s, H), 7.83 (t, J=8 Hz, 1H), 7.66 (dd, J=7.3, 5.3 Hz, 1H), 7.47 (m, 2H), 7.25 (d, J=8.4 Hz, 1H), 3.29 (s, 3H), 2.55 (s, 6H, 2Me-HOAc), 2.24 (s, 3H). LC/MS (m/z) (M+1)+: 460.2.
Similar procedures are used to prepare compounds A2-A5 using LC/MS for purification.
N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide A2
1H NMR (400 MHz, DMSO) δ 10.52 (s, 1H), 9.26 (s, 1H), 8.90 (s, 1H), 8.69 (s, 1H), 8.61 (d, J=8.0 Hz, 1H), 8.50 (d, J=4.8 Hz, 1H), 8.08-8.14 (m, 3H), 8.00-8.07 (m, 2H), 7.65 (m, 1H), 7.40 (m, 1H), 7.23 (m, 1H), 3.22 (s, 3H), 2.21 (s, 3H). LC/MS (m/z) (M+1)+: 460.2.
4-Chloro-3-methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide A3
1H NMR (400 MHz, d6-DMSO) δ 10.6 (s, 1H), 9.3 (s, 1H), 9.04 (s, 1H), 8.72 (d, J=3.8 Hz, 1H), 8.55 (m, 3H), 8.3 (dd, J=8.3, 2.1 Hz, 1H), 8.08 (d, J=1.5 Hz, 1H), 7.94 (d, J=8.3 Hz, 1H), 7.6 (dd, J=7.9, 4.8 Hz, 1H), 7.48 (m, 2H), 7.24 (d, J=8.3 Hz, 1H), 3.44 (s, 3H), 2.24 (s, 3H). LC/MS (m/z) (M+1)+: 494.1.
N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)benzo[d]thiazole-6-carboxamide A4
1H NMR (400 MHz, DMSO) δ 10.20 (s, 1H), 9.31 (s, 1H), 9.02 (s, 1H), 8.74 (d, J=3.2 Hz, 1H), 8.58 (m, 1H), 8.53 (d, J=4.8 Hz, 1H), 8.06 (s, 1H), 7.62 (m, 1H), 7.45 (d, J=5.2 Hz, 1H), 7.41 (d, J=7.2 Hz, 1H), 7.15-7.22 (m, 3H), 7.09-7.13 (m, 1H), 2.22 (s, 3H). LC/MS (m/z) (M+1)+: 439.2.
N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-2-chloro-4-(methylsulfonyl)benzamide A5
1H NMR (400 MHz, d6-DMSO) δ 10.65 (s, 1H), 9.29 (s, 1H), 9.03 (s, 1H), 8.72 (d, J=4.4 Hz, 1H), 8.55 (m, 2H), 8.06 (s, 1H), 8.00 (d, J=9.2, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.60 (m, 1H), 7.45 (d, J=4.8 Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 7.24 (d, J=8.4 Hz, 1H), 3.34 (s, 3H), 2.24 (s, 3H). LC/MS (m/z) (M+1)+: 494.1.
4-Methanesulfonyl-3-methyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide A6




Intermediate 6 is prepared similar to A1. Compound 6 (0.1 mmol), CuI (0.075 mmol), L-proline (0.1 mmol), 1 N NaOH (0.1 mL) and NaSO2Me (0.15 mmol) are suspended in DMSO (0.5 mL) and heated at 180° C. in a microwave oven for 5 min. The mixture is purified by preparative LC/MS to afford A6. 1H NMR (400 MHz, DMSO) δ 10.45 (s, 1H), 9.24 (s, 1H), 8.89 (s, 1H), 8.67 (d, J=2.0 Hz, 1H), 8.49 (m, 2H), 8.09 (s, 1H), 8.01 (d, J=8.4, 1H), 7.90-7.93 (m, 2H), 7.56 (m, 1H), 7.39 (d, J=5.6 Hz, 2H), 7.23 (d, J=8.4 Hz, 1H), 3.32 (s, 3H), 2.70 (s, 3H), 2.21 (s, 3H). LC/MS (m/z) (M+1)+: 474.2.
Similar procedures are used to prepare A7, A8
N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-methyl-3-(methylsulfonyl)benzamide A7
1H NMR (400 MHz, DMSO) δ 10.46 (s, 1H), 9.23 (s, 1H), 8.87 (d, J=7.2 Hz, 1H), 8.67 (m, 1H), 8.50 (m, 2H), 8.05 (d, J=12.8 Hz, 1H), 7.58 (m, 2H), 7.39 (m, 2H), 7.29 (m, 1H), 7.22 (m, 2H), 3.23 (s, 3H), 2.69 (s, 3H), 2.21 (s, 3H). LC/MS (m/z) (M+1)+: 474.2.
N-(3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-ethoxy-3-(methylsulfonyl)benzamide A8
1H NMR (400 MHz, DMSO) δ 10.37 (s, 1H), 9.23 (s, 1H), 8.89 (s, 1H), 8.66 (d, J=3.6 Hz, 1H), 8.48 (m, 2H), 8.37 (d, J=2.0 Hz, 1H), 8.25 (d, J=8.8 Hz, 1H), 8.05 (s, 1H), 7.55 (m, 1H), 7.39 (m, 3H), 7.22 (d, J=8.0 Hz, 1H), 4.30 (m, 2H), 3.27 (s, 3H), 2.20 (s, 3H), 1.41 (t, J=6.4 Hz, 3H). LC/MS (m/z) (M+1)+: 504.2.
Type B
4-Methanesulfonyl-N-{4-methyl-3-[4-(5-pyrrolidin-1-yl-pyridin-3-yl)-pyrimidin-2-ylamino]-phenyl}-benzamide B1




Final compound B1 is prepared from 10 with a procedure similar to the one used for the preparation of A1. 1H NMR (400 MHz, d6-DMSO) δ 10.5 (s, 1H), 9.12 (s, 1H), 8.6 (s, 1H), 8.59 (s, 1H), 8.18 (m, 3H), 8.09 (m, 3H), 7.89 (s, 1H), 7.54 (d, J=5.1 Hz, 1H), 7.5 (dd, J=8.2, 2.1 Hz, 1H), 7.23 (d, J=8.5 Hz, 1H), 3.3 (m, 4H), 3.29 (s, 3H), 2.24 (s, 3H), 1.86 (m, 4H). LC/MS (m/z) (M+1)+: 530.1.
Type C
3-Methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-morpholin-4-yl-benzamide C1




A3 (20 mg, 0.04 mmol) and morpholine (0.07 mL, 0.8 mmol) are heated at 130° C. in a microwave oven for 30 min. Purification by preparative LC/MS affords C1. LC/MS (m/z) (M+1)+: 545.3.
Type D
4-Methanesulfonyl-N-{3-[4-(5-methoxy-pyridin-3-yl)-pyrimidin-2-ylamino]-4-methyl-phenyl}-benzamide D1




Final compound D1 is obtained from 14 using a similar procedure to the preparation of A1. 1H NMR (400 MHz, d4-MeOH) δ 9.29 (s, 1H), 8.82 (m, 1H), 8.7 (d, J=2.2 Hz, 1H), 8.6 (d, J=5.6 Hz, 1H), 8.36 (s, 1H), 8.17 (m, 2H), 8.11 (m, 2H), 7.66 (d, J=5.6 Hz, 1H), 7.35 (m, 2H), 4.09 (s, 3H), 3.19 (s, 3H), 2.35 (s, 3H). LC/MS (m/z) (M+1)+: 490.2.
N-(3-{4-[5-(2-Fluoro-ethoxy)-pyridin-3-yl]-pyrimidin-2-ylamino}-4-methyl-phenyl)-4-methanesulfonyl-benzamide D2




Final compound D2 is prepared from 17 with a similar procedure used for the preparation of A1. 1H NMR (400 MHz, d6-DMSO) δ 10.5 (s, 1H), 9.04 (s, 1H), 8.93 (d, J=1.6 Hz, 1H), 8.54 (d, J=5.2 Hz, 1H), 8.46 (d, J=2.8 Hz, 1H), 8.16 (m, 3H), 8.07 (m, 3H), 7.5 (m, 2H), 7.24 (d, J=8.5 Hz, 1H), 4.7 (d, J=47.8 Hz, 2H), 4.37 (d, J=30 Hz, 2H), 3.29 (s, 3H), 2.24 (s, 3H). LC/MS (m/z) (M+1)+: 522.2.
Type E
4-Methanesulfonyl-N-{4-methyl-3-[4-(5-methyl-pyridin-3-yl)-pyrimidin-2-ylamino]-phenyl}-benzamide E1




Final compound E1 is prepared from 21 with a similar procedure used for the preparation of A1. LC/MS (m/z) (M+1)+: 474.2.
Type F
3-(2-Fluoro-ethoxy)-4-methanesulfonyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide F1




Compound 23 (0.04 mmol), CsHCO3 (0.1 mmol) and 1-bromo-2-fluoro-ethane (0.04 mmol) are heated in dry acetonitrile (0.5 mL) at 150° C. in a microwave oven for 10 min. Purification by preparative LC/MS affords final compound F1. 1H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 9.31 (s, 1H), 9.01 (s, 1H), 8.73 (s, 1H), 8.55 (m, 2H), 8.10 (s, 1H), 7.95 (m, 1H), 7.76 (s, 1H), 7.71 (m, 1H), 7.60 (m, 1H), 7.46 (m, 2H), 7.25 (d, J=8.0 Hz, 1H), 4.92 (m, 1H), 4.80 (m, 1H), 4.62 (m, 1H), 4.55 (m, 1H), 3.32 (s, 3H), 2.25 (s, 3H). LC/MS (m/z) (M+1)+: 522.2.
Similar procedures are used to prepare compounds F2-F8
Type G
N-(3-(4-(isoquinolin-4-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3-(methylsulfonyl)benzamide G1




Final compound G1 is prepared from 27 with a similar procedure used for the preparation of A1. LC/MS (m/z) (M+1)+: 510.2.
By repeating the procedures described in the above examples (intermediates and final compounds), using appropriate starting materials, the following compounds of Formula I, as identified in Table 1, are obtained.









TABLE 1





Example #
Structure
MS [M + 1]+







A1




460.2


 


A2




460.1


 


A3




494.1


 


A4




440.2


 


A5




494.2


 


A6




474.2


 


A7




474.2


 


A8




504.2


 


A9




490.1


 


A10




544.2


 


B1




530.1


 


B2




546.2


 


C1




545.3


 


D1




490.2


 


D2




522.2


 


D3




530.2


 


D4




526.2


 


D5




476.1


 


E1




474.2


 


F1




522.2


 


F2




518.2


 


F3




490.2


 


F4




532.2


 


F5




530.2


 


F6




532.2


 


F7




558.1


 


F8




586.2


 


G1




510.2









Assays
Compounds of the present invention are assayed to measure their capacity to selectively inhibit the proliferation of wild type Ba/F3 cells and Ba/F3 cells transformed with Tel c-kit kinase and Tel PDGFR fused tyrosine kinases. In addition, compounds of the invention selectively inhibit SCF dependent proliferation in Mo7e cells. Further, compounds are assayed to measure their capacity to inhibit Abl, ARG, BCR-Abl, BRK, EphB, Fms, Fyn, KDR, c-Kit, LCK, PDGF-R, b-Raf, c-Raf, SAPK2, Src, Tie2 and TrkB kinases.
Proliferation Assay: BaF3 Library—Bright Glo Readout Protocol
Compounds are tested for their ability to inhibit the proliferation of wt Ba/F3 cells and Ba/F3 cells transformed with Tel fused tyrosine kinases. Untransformed Ba/F3 cells are maintained in media containing recombinant IL3. Cells are plated into 384 well TC plates at 5,000 cells in 50 ul media per well and test compound at 0.06 nM to 10 μM is added. The cells are then incubated for 48 hours at 37° C., 5% CO2. After incubating the cells, 25 μL of BRIGHT GLO® (Promega) is added to each well following manufacturer's instructions and the plates are read using Analyst GT—Luminescence mode—50000 integration time in RLU. IC50 values, the concentration of compound required for 50% inhibition, are determined from a dose response curve.
Mo7e Assay
The compounds described herein are tested for inhibition of SCF dependent proliferation using Mo7e cells which endogenously express c-kit in a 96 well format. Briefly, two-fold serially diluted test compounds (Cmax=10 μM) are evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability is measured by using a MTT colorimetric assay from Promega.
c-kit HTRF Protocol
An aliquot (5 μL) of a 2× concentration of c-kit enzyme mix 25 ng c-kit (5 ng/μL) and 2 μM of Biotin-EEEPQYEEIPIYLELLP-NH2 peptide in kinase buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 0.01% BSA, 0.1% Brij35, 1 mM DTT, 5% glycerol, 0.05 mM Na3VO4) is added to each well of a 384 proxiplate (Packard). Each well of the last row of the proxiplate has 5 μL of c-kit enzyme mix without c-kit to ascertain the background level. Compounds of the invention are added to each well and the plates are incubated for 30 minutes at room temperature. 2×ATP (40 μM) in kinase buffer (5 μL) is added to each well and the plate is incubated at room temperature form 3 hours. Detection mix (50% KF, 40% kinase buffer, 10% EDTA, 1:100 diluted Mab PT66-K (cat#61T66KLB) and 1:100 diluted Streptavidin-XL (cat#611SAXLB)0 (10 μL) is added to each well and the plates are further incubated for 1 to 2 hours at room temperature. The HTRF signal is then read on a detector.
Human TG-HA-VSMC Proliferation Assay
Human TG-HA-VSMC cells (ATCC) are grown in DMEM supplemented with 10% FBS to 80-90% confluence prior to resuspending in DMEM supplemented with 1% FBS and 30 ng/mL recombinant human PDGF-BB at 6e4 cells/mL. Cells are then aliquoted into 384 well plates at 50 uL/well, incubated for 20 h at 37° C., then treated with 0.5 uL of 100× compounds for 48 h at 37° C. After the treatment, 25 uL of CellTiter-Glo is added to each well for 15 min, then the plates are read on the CLIPR (Molecular Devices).
PDGFRα & β Lance Assay Protocol
An aliquot (2.5 μL) of a 2× concentration of PDGFRβ peptide and ATP mix (4 μM biotin-βA-βA-βA-AEEEEYVFLEAKKK peptide, 20 μM ATP in assay buffer (20 mM Hepes, 54 mM MgCl2, 0.01% BSA, 0.05% Tween-20, 1 mM DTT, 10% glycerol, 50 μM Na3VO4)) is added to each well of a 384 proxiplate (Packard). The plates are centrifuged and compounds of the invention (50 nL) are added to each well via a pintool dispenser. To each well is added (2.5 μL) of a 2× concentration of enzyme mix (PDGFRα at 4.5 ng/μL (cat# PV4117) or PDGFRβ at 1.5 ng/μL (cat# PV3591) in assay buffer) or assay buffer alone without PDGFRα/β enzyme. The plates are incubated for 1.5 hours at room temperature. Detection mix (5 μL; 50% 1M KF, 40% kinase buffer, 10% EDTA, 1:100 diluted Mab PT66-K (cat#61T66KLB) and 1:100 diluted Streptavidin-XL (cat#611SAXLB) is added to each well and the proxiplate is incubated for 1 hour at room temperature before reading the HTRF signal on a detector.
Ba/F3 FL FLT3 Proliferation Assay
The murine cell line used is the Ba/F3 murine pro-B cell line that over expresses full length FLT3 construct. These cells are maintained in RPMI 1640/10% fetal bovine serum (RPMI/FBS) supplemented with penicillin 50 μg/mL, streptomycin 50 μg/mL and L-glutamine 200 mM with the addition of murine recombinant IL3. Ba/F3 full length FLT3 cells undergo IL3 starvation for 16 hours and then plated into 384 well TC plates at 5,000 cells in 25 uL media per well and test compound at 0.06 nM to 10 μM is added. After the compound addition FLT3 ligand or IL3 for cytotoxicity control are added in 25 ul media per well at the appropriate concentrations. The cells are then incubated for 48 hours at 37° C., 5% CO2. After incubating the cells, 25 μL of BRIGHT GLO® (Promega) is added to each well following manufacturer's instructions and the plates are read using Analyst GT—Luminescence mode—50000 integration time in RLU.
Inhibition of Cellular BCR-Abl Dependent Proliferation (High Throughput Method)
The murine cell line used is the 32D hemopoietic progenitor cell line transformed with BCR-Abl cDNA (32D-p210). These cells are maintained in RPMI/10% fetal calf serum (RPMI/FCS) supplemented with penicillin 50 μg/mL, streptomycin 50 μg/mL and L-glutamine 200 mM. Untransformed 32D cells are similarly maintained with the addition of 15% of WEHI conditioned medium as a source of IL3.
50 μL of a 32D or 32D-p210 cells suspension are plated in Greiner 384 well microplates (black) at a density of 5000 cells per well. 50 nL of test compound (1 mM in DMSO stock solution) is added to each well (STI571 is included as a positive control). The cells are incubated for 72 hours at 37° C., 5% CO2. 10 μL of a 60% Alamar Blue solution (Tek diagnostics) is added to each well and the cells are incubated for an additional 24 hours. The fluorescence intensity (Excitation at 530 nm, Emission at 580 nm) is quantified using the AcquestTM system (Molecular Devices).
Inhibition of Cellular BCR-Abl Dependent Proliferation
32D-p210 cells are plated into 96 well TC plates at a density of 15,000 cells per well. 50 μL of two fold serial dilutions of the test compound (Cmax is 40 μM) are added to each well (STI571 is included as a positive control). After incubating the cells for 48 hours at 37° C., 5% CO2, 15 μL of MTT (Promega) is added to each well and the cells are incubated for an additional 5 hours. The optical density at 570 nm is quantified spectrophotometrically and IC50 values, the concentration of compound required for 50% inhibition, determined from a dose response curve.
Effect on Cell Cycle Distribution
32D and 32D-p210 cells are plated into 6 well TC plates at 2.5×106 cells per well in 5 mL of medium and test compound at 1 or 10 μM is added (STI571 is included as a control). The cells are then incubated for 24 or 48 hours at 37° C., 5% CO2. 2 mL of cell suspension is washed with PBS, fixed in 70% EtOH for 1 hour and treated with PBS/EDTA/RNase A for 30 minutes. Propidium iodide (Cf=10 μg/ml) is added and the fluorescence intensity is quantified by flow cytometry on the FACScaliburTM system (BD Biosciences). Test compounds of the present invention demonstrate an apoptotic effect on the 32D-p210 cells but do not induce apoptosis in the 32D parental cells.
Effect on Cellular BCR-Abl Autophosphorylation
BCR-Abl autophosphorylation is quantified with capture Elisa using a c-abl specific capture antibody and an antiphosphotyrosine antibody. 32D-p210 cells are plated in 96 well TC plates at 2×105 cells per well in 50 μL of medium. 50 μL of two fold serial dilutions of test compounds (Cmax is 10 μM) are added to each well (STI571 is included as a positive control). The cells are incubated for 90 minutes at 37° C., 5% CO2. The cells are then treated for 1 hour on ice with 150 μL of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA and 1% NP-40) containing protease and phosphatase inhibitors. 50 μL of cell lysate is added to 96 well optiplates previously coated with anti-abl specific antibody and blocked. The plates are incubated for 4 hours at 4° C. After washing with TBS-Tween 20 buffer, 50 μL of alkaline-phosphatase conjugated anti-phosphotyrosine antibody is added and the plate is further incubated overnight at 4° C. After washing with TBS-Tween 20 buffer, 90 μL of a luminescent substrate are added and the luminescence is quantified using the AcquestTM system (Molecular Devices). Test compounds of the invention that inhibit the proliferation of the BCR-Abl expressing cells, inhibit the cellular BCR-Abl autophosphorylation in a dose-dependent manner.
Effect on Proliferation of Cells Expressing Mutant Forms of Bcr-Abl
Compounds of the invention are tested for their antiproliferative effect on Ba/F3 cells expressing either wild type or the mutant forms of BCR-Abl (G250E, E255V, T315I, F317L, M351T) that confers resistance or diminished sensitivity to STI571. The antiproliferative effect of these compounds on the mutant-BCR-Abl expressing cells and on the non transformed cells were tested at 10, 3.3, 1.1 and 0.37 μM as described above (in media lacking IL3). The IC50 values of the compounds lacking toxicity on the untransformed cells were determined from the dose response curves obtained as describe above.
FGFR3 (Enzymatic Assay)
Kinase activity assay with purified FGFR3 (Upstate) is carried out in a final volume of 10 μL containing 0.25 μg/mL of enzyme in kinase buffer (30 mM Tris-HCl pH7.5, 15 mM MgCl2, 4.5 mM MnCl2, 15 μM Na3VO4 and 50 μg/mL BSA), and substrates (5 μg/mL biotin-poly-EY(Glu, Tyr) (CIS-US, Inc.) and 3 μM ATP). Two solutions are made: the first solution of 5 μL contains the FGFR3 enzyme in kinase buffer was first dispensed into 384-format ProxiPlate® (Perkin-Elmer) followed by adding 50 nL of compounds dissolved in DMSO, then 5 μL of second solution contains the substrate (poly-EY) and ATP in kinase buffer was added to each wells. The reactions are incubated at room temperature for one hour, stopped by adding 10 μL of HTRF detection mixture, which contains 30 mM Tris-HCl pH7.5, 0.5 M KF, 50 mM ETDA, 0.2 mg/mL BSA, 15 μg/mL streptavidin-XL665 (CIS-US, Inc.) and 150 ng/mL cryptate conjugated anti-phosphotyrosine antibody (CIS-US, Inc.). After one hour of room temperature incubation to allow for streptavidin-biotin interaction, time resolved florescent signals are read on Analyst GT (Molecular Devices Corp.). IC50 values are calculated by linear regression analysis of the percentage inhibition of each compound at 12 concentrations (1:3 dilution from 50 nM to 0.28 nM). In this assay, compounds of the invention have an IC50 in the range of 10 nM to 2 μM.
FGFR3 (Cellular Assay)
Compounds of the invention are tested for their ability to inhibit transformed Ba/F3-TEL-FGFR3 cells proliferation, which is depended on FGFR3 cellular kinase activity. Ba/F3-TEL-FGFR3 are cultured up to 800,000 cells/mL in suspension, with RPMI 1640 supplemented with 10% fetal bovine serum as the culture medium. Cells are dispensed into 384-well format plate at 5000 cell/well in 50 μL culture medium. Compounds of the invention are dissolved and diluted in dimethylsufoxide (DMSO). Twelve points 1:3 serial dilutions are made into DMSO to create concentrations gradient ranging typically from 10 mM to 0.05 μM. Cells are added with 50 nL of diluted compounds and incubated for 48 hours in cell culture incubator. AlamarBlue® (TREK Diagnostic Systems), which can be used to monitor the reducing environment created by proliferating cells, are added to cells at final concentration of 10%. After an additional four hours of incubation in a 37° C. cell culture incubator, fluorescence signals from reduced AlamarBlue® (Excitation at 530 nm, Emission at 580 nm) are quantified on Analyst GT (Molecular Devices Corp.). IC50 values are calculated by linear regression analysis of the percentage inhibition of each compound at 12 concentrations.
b-Raf—Enzymatic Assay
Compounds of the invention are tested for their ability to inhibit the activity of b-Raf. The assay is carried out in 384-well MaxiSorp plates (NUNC) with black walls and clear bottom. The substrate, IκBα is diluted in DPBS (1:750) and 15 μL is added to each well. The plates are incubated at 4° C. overnight and washed 3 times with TBST (25 mM Tris, pH 8.0, 150 mM NaCl and 0.05% Tween-20) using the EMBLA plate washer. Plates are blocked by Superblock (15 μL/well) for 3 hours at room temperature, washed 3 times with TBST and pat-dried. Assay buffer containing 20 μM ATP (10 μL) is added to each well followed by 100 nL or 500 nL of compound. B-Raf is diluted in the assay buffer (1 μL into 25 μL) and 10 μL of diluted b-Raf is added to each well (0.4 μg/well). The plates are incubated at room temperature for 2.5 hours. The kinase reaction is stopped by washing the plates 6 times with TBST. Phosph-IκBα (Ser32/36) antibody is diluted in Superblock (1:10,000) and 15 μL is added to each well. The plates are incubated at 4° C. overnight and washed 6 times with TBST. AP-conjugated goat-anti-mouse IgG is diluted in Superblock (1:1,500) and 15 μL is added to each well. Plates are incubated at room temperature for 1 hour and washed 6 times with TBST. 15 μL of fluorescent Attophos AP substrate (Promega) is added to each well and plates are incubated at room temperature for 15 minutes. Plates are read on Acquest or Analyst GT using a Fluorescence Intensity Program (Excitation 455 nm, Emission 580 nm).
b-Raf—Cellular Assay
Compounds of the invention are tested in A375 cells for their ability to inhibit phosphorylation of MEK. A375 cell line (ATCC) is derived from a human melanoma patient and it has a V599E mutation on the B-Raf gene. The levels of phosphorylated MEK are elevated due to the mutation of B-Raf. Sub-confluent to confluent A375 cells are incubated with compounds for 2 hours at 37° C. in serum free medium. Cells are then washed once with cold PBS and lysed with the lysis buffer containing 1% Triton X100. After centrifugation, the supernatants are subjected to SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes are then subjected to western blotting with anti-phospho-MEK antibody (ser217/221) (Cell Signaling). The amount of phosphorylated MEK is monitored by the density of phospho-MEK bands on the nitrocellulose membranes.
Upstate KinaseProfilerTM—Radio-Enzymatic Filter Binding Assay
Compounds of the invention are assessed for their ability to inhibit individual members of the kinase panel. The compounds are tested in duplicates at a final concentration of 10 μM following this generic protocol. Note that the kinase buffer composition and the substrates vary for the different kinases included in the “Upstate KinaseProfilerTM” panel. Kinase buffer (2.5 μL, 10×-containing MnCl2 when required), active kinase (0.001-0.01 Units; 2.5 μL), specific or Poly(Glu-4-Tyr) peptide (5-500 μM or 0.01 mg/ml) in kinase buffer and kinase buffer (50 μM; 5 μL) are mixed in an eppendorf on ice. A Mg/ATP mix (10 μL; 67.5 (or 33.75) mM MgCl2, 450 (or 225) μM ATP and 1 μCi/μl [γ-32P]-ATP (3000 Ci/mmol)) is added and the reaction is incubated at about 30° C. for about 10 minutes. The reaction mixture is spotted (20 μL) onto a 2 cm×2 cm P81 (phosphocellulose, for positively charged peptide substrates) or Whatman No. 1 (for Poly (Glu-4-Tyr) peptide substrate) paper square. The assay squares are washed 4 times, for 5 minutes each, with 0.75% phosphoric acid and washed once with acetone for 5 minutes. The assay squares are transferred to a scintillation vial, 5 ml scintillation cocktail are added and 32P incorporation (cpm) to the peptide substrate is quantified with a Beckman scintillation counter. Percentage inhibition is calculated for each reaction.
Compounds of Formula I, in free form or in pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, for example, as indicated by the in vitro tests described in this application.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

## Claims

1. A compound of Formula I:



in which
L is selected from —NHC(O)—, —NHC(O)N— and —C(O)NH—;
R1, R2a and R2b are each independently selected from hydrogen, hydroxy, C3-8heterocycloalkyl, C1-4-alkyl, C1-4-alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4alkyl, —NR10R11, —OX1R8; wherein X1 is selected from a bond and C1-4alkylene; R8 is C3-12cycloalkyl; or R1 and R2a or R1 and R2b together with the carbon atoms to which R1 and R2a or R2b are attached form phenyl; R10 and R11 are independently selected from hydrogen, C1-4-alkyl, C1-4-alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4-alkyl, C3-8heterocycloalkyl, C1-10heteroaryl; or R10 and R11 together with the nitrogen to which R10 and R11 are both attached form C3-8heterocycloalkyl or C1-10heteroaryl;
R3, R4, R5, R6 and R7 are independently selected from hydrogen, halo, cyano, C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, C3-8heterocycloalkyl, —OX2R9, —S(O)0-2R9 and —NR12R13; wherein X2 is selected from a bond and C1-4alkylene; and each R9 is independently selected from hydrogen, C1-6alkyl, C1-6alkoxy, C3-12cycloalkyl, and C3-8heterocycloalkyl; wherein said cycloalkyl or heterocycloalkyl of R9 is optionally substituted with 1 to 3 radicals independently selected from C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, halo-substituted-C1-6alkyl and —NR12R13; wherein R12 and R13 are independently selected from hydrogen and C1-6alkyl; or R4 and R5 together with the carbon atoms to which R4 and R5 are attached form C3-8heteroaryl;

with the proviso that at least one of R3, R4, R5, R6 or R7 has a sulfur directly linked to the phenyl ring;
and the pharmaceutically acceptable salts thereof.

2. The compound of claim 1 in which:
L is selected from —NHC(O)— and —C(O)NH—;
R1, R2a and R2b are independently selected from hydrogen, hydroxy, C3-8heterocycloalkyl, C1-4alkyl, C1-4alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4alkyl, —NR10R11, —OX1R8; wherein X1 is selected from a bond and C1-4alkylene; R8 is C3-12cycloalkyl; or R1 and R2 together with the carbon atoms to which R1 and R2a are attached form phenyl; R10 and R11 are independently selected from hydrogen, C1-4alkyl, C1-4alkoxy, halo-substituted-C1-4alkoxy, halo-substituted-C1-4alkyl, C3-8heterocycloalkyl, C1-10heteroaryl; or R10 and R11 together with the nitrogen to which R10 and R11 are both attached form C3-8heterocycloalkyl or C1-10heteroaryl;
R3 and R7 are independently selected from hydrogen and halo;
R4 and R6 are independently selected from hydrogen, C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, —S(O)0-2R6; and
R5 is selected from halo, C1-6alkyl, C1-6alkoxy, halo-substituted-C1-6alkoxy, C3-8heterocycloalkyl, —OX2R9 and —S(O)0-2R9; wherein X2 is selected from a bond and C1-4alkylene; and each R9 is independently selected from hydrogen, C1-6alkyl, C3-12cycloalkyl and C3-8heterocycloalkyl; wherein said cycloalkyl or heterocycloalkyl of R9 is optionally substituted with 1 to 3 radicals independently selected from C1-6alkyl and halo-substituted-C1-6alkyl; or R4 and R5 together with the carbon atoms to which R4 and R5 are attached form C3-8heteroaryl, with the proviso that at least one of R3, R4, R5, R6 or R7 has a sulfur directly linked to the phenyl ring.

3. The compound of claim 2 in which R1 is selected from hydrogen, hydroxy, pyrrolidinyl, morpholino, methoxy, difluoro-methoxy, 2-fluoro-ethoxy and methyl; or R1 and R2a together with the carbon atoms to which R1 and R2a are attached form phenyl.

4. The compound of claim 3 in which R4 and R6 are independently selected from hydrogen, methyl-sulfonyl, methyl, 2-fluoro-ethoxy, methyl-piperazinyl-sulfonyl, propoxy, isobutoxy, 2,2,2-trifluoroethoxy, 2,3-difluoro-2-(fluoromethyl)propoxy, butoxy and cyclopropyl-methoxy; R5 is selected from hydrogen, halo, methyl-sulfonyl, methyl, methoxy, ethoxy and morpholino; or R4 and R5 together with the carbon atoms to which R4 and R5 are attached form thiazolyl.

5. The compound of claim 1 selected from: N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 4-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzo[d]thiazole-6-carboxamide; 2-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 4-methyl-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; 4-ethoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(5-morpholinopyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(5-(pyrrolidin-1-yl)pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)-4-morpholinobenzamide; N-(3-(4-(5-methoxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(3-(4-(5-(2-fluoroethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(3-(4-(5-(cyclopropylmethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(5-methylpyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-(2-fluoroethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-propoxybenzamide; 3-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-butoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; 3-(cyclopropylmethoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(3-(4-(isoquinolin-4-yl)pyrimidin-2-ylamino)-4-methylphenyl)-3-(methylsulfonyl)benzamide; 4-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(methylsulfonyl)benzamide; N-(3-(4-(5-(difluoromethoxy)pyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-3-(4-methylpiperazin-1-ylsulfonyl)benzamide; N-(3-(4-(5-hydroxypyridin-3-yl)pyrimidin-2-ylamino)-4-methylphenyl)-4-(methylsulfonyl)benzamide; 3-isobutoxy-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide; N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)-3-(2,2,2-trifluoroethoxy)benzamide; and 3-(2,3-difluoro-2-(fluoromethyl)propoxy)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-(methylsulfonyl)benzamide.

6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 in combination with a pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable excipient is suitable for parenteral administration.

8. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable excipient is suitable for oral administration.