WO2005019177A1 - Aminopiperidine amide derivatives as vla-1 integrin antagonists and uses thereof - Google Patents

Aminopiperidine amide derivatives as vla-1 integrin antagonists and uses thereof Download PDF

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WO2005019177A1
WO2005019177A1 PCT/US2004/026207 US2004026207W WO2005019177A1 WO 2005019177 A1 WO2005019177 A1 WO 2005019177A1 US 2004026207 W US2004026207 W US 2004026207W WO 2005019177 A1 WO2005019177 A1 WO 2005019177A1
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piperidin
acryloyl
dichloro
thiophen
benzo
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PCT/US2004/026207
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French (fr)
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Steven A. Boyd
Scott Miller
Allen Thomas
Rui Xu
Yvan Lehuerou
Indrani Gunawardana
Gan Zhang
Jason Demeese
Martin Mclaughlin
Matthew Yanik
Mark L. Lupher, Jr.
Irina C. Jacobson
Eugene Thorsett
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Icos Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D211/62—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
    • C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
    • C04B35/00—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/622—Forming processes; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/626—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B
    • C04B35/63—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B using additives specially adapted for forming the products, e.g.. binder binders
    • C04B35/632—Organic additives
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • This invention relates to compounds which are VLA-1 integrin antagonists. This invention also relates to compositions containing such compounds and methods of treatment using such compounds in treating diseases mediated, at least in part, by the VLA-1 integrin.
  • Integrins are heterodimeric cell surface proteins composed of two noncovalently linked polypeptide chains, a. and ⁇ . Integrins are the major receptor for cell adhesion to extracellular matrix and play important roles in certain cell-cell and cell-matrix adhesion events. These integrin-medi- ated adhesion events are critical for both normal and pathophysiological processes during cell activation, migration, proliferation and differentiation (for reviews see Hynes (1992) Cell 69: 11 ; Springer (1994) Cell 75:301; Hynes (2002) Cell 110: 613 ) .
  • VLA-1 very late antigen-1 is an integrin heterodimer composed of an alpha chain (CD49a, ⁇ l) and a beta chain (CD29, ⁇ l) .
  • VLA-1 is one member of a family of four ⁇ l integrin molecules that have been shown to bind to the extracellular matrix proteins, collagen and laminin.
  • the ⁇ l integrin collagen receptors include ⁇ l ⁇ l (VLA-1), ⁇ 2 ⁇ l (VLA-2), ⁇ lO ⁇ l and ⁇ ll ⁇ l. These four collagen receptors share overlapping but distinct expression profiles. They also appear to have distinct ligand preferences in vitro (Tulla et al., (2001) J. Biol . Chem. 52:48206).
  • ⁇ l ⁇ l has been shown to bind more effectively to type IV collagen than type I collagen while ⁇ 2 ⁇ l binds to type I collagen better than to type IV collagen (Dickeson et al., (1999) J. Biol . Chem. 274:32182).
  • VLA-1 is expressed on smooth muscle cells, microvascular endothelial cells, fibroblasts, osteo- blasts and chondrocytes. In addition, VLA-1 is also expressed on activated cells of the immune system including effector T cells, macrophages, and NK cells (de Fougerolles et al., (2000) J. Clin . Invest . 105: 121 ) ; however, it does not appear to be expressed on B cells or neutrophils. VLA-1 is ex- pressed on T cells in various disease states including in the joints of arthritis patients (HemLer et al., (1986) J. Clin . Invest .
  • mice- generated by homologous recombination are viable and fertile and have no overt phenotype, demonstrating that the molecule is not required for development (Gardner et al., (1996) Dev. Biol . 275:301).
  • no increased incidence of infection was noted in the mutant mice.
  • embryonic fibroblasts derived from mutant animals show a striking absence of adhesion to collagen IV, they show no deficit in adhesion to collagen I.
  • no compensatory upregulation of other collagen binding receptors could be identified, suggesting instead that VLA-1 may have redundant roles during development.
  • Inhibiting VLA-1 function using ⁇ l null mice and/or blocking anti- ⁇ l antibodies has shown efficacy either prophylactically or therapeutically or both in several animal models of inflammatory disease including 1) delayed-type hypersensitivity as a model of general inflammatory disease (de Fougerolles et al., (2000) J. Clin . Invest . 105: 121 ) ; 2) contact hypersensitivity as a model for skin allergic reactions (A.R. de Fougerolles et al., 2000, J. Clin . Invest . , 105: 121 ) ; 3) anticollagen mAb-induced arthritis as a model of rheumatoid arthritis (de Fougerolles et al., (2000) J. Clin .
  • TNBS- and DSS-induced colitis as models of inflammatory bowel disease (Fiorucci et al., (2002) Immunity 1 7: 169 ; Kriegl- stein et al., (2002) J. Clin . Invest. 110: 1113) .
  • VLA-1 has been shown to mediate adhesion to and migration across collagen matrix. Therefore, VLA-1 expression may be critical for allowing the effector cells to enter the site of inflammation.
  • mAbs against ⁇ l have also been shown to block collagen-induced cytokine release, including release of TNF- ⁇ , a key mediator in arthritis (Miyake et al . , (1994) Eur. J. Immunol . 24 : 2000) .
  • MMP matrix metalloproteinase
  • VLA-1 is an upstream regulator of multiple disease promoting factors
  • Fibrosis is a common response to chronic injury and represents a paradigm for the cycle of parenchymal wound healing in a variety of tissues (reviewed in Bataller et al. (2001) Semin . Liver Dis . 21 : 431 ; Bissell. (1998) J. Gastroenterol . 33 : 295 ) .
  • this wound healing process can result in pathologic tissue scarring, which results from the progression of several defined steps.
  • an infiltrate consisting of inflammatory cells and platelets and resident " yofibro- blasts" (identified as hepatic stellate cells in the liver and differentiated mesangial cells in the kidney) , accumulates at the site of injury.
  • the local extracellular matrix (ECM) is altered by de novo production of collagen by the myofibro- blasts.
  • ECM extracellular matrix
  • the myofibroblasts migrate and align within the wound site and proliferate.
  • the myofibroblasts contract the collagen, forming the fibrotic scar which contributes to tissue dysfunction. It is generally believed that a similar process results in scarring within tissues of the liver, kidney, lung, and skin.
  • VLA-1 is expressed on myofibroblasts in vitro and in vivo and is believed to regulate their pathologic functions.
  • Alports syndrome is a genetic disorder characterized by progressive glomeruloneph- ritis resulting in fibrosis of the kidneys and ultimately kidney failure. Alports syndrome affects approximately 1 in 5000 people and is caused by mutations in the type IV collagen genes. This condition has been mimicked in mice by knocking out the gene of the ⁇ 3 chain of type IV collagen (Alport mouse) . Double knockout mice for both type IV collagen and ⁇ l integrin have a delayed onset and slowed progression of glomerular disease (Cosgrove et al., (2000) Am . J. Pa thol . 257:1649).
  • ⁇ l mAb blocks hepatic stellate cell' adhesion to collagen and endothelin-stimulated hepatic stellate cell- mediated contraction of collagen lattices in vi tro
  • VLA-1 is the sole integrin utilized by contracting hepatic stellate cells in vivo (Racine Sampson et al., (1997) J. Biol . Chem . 272:30911).
  • blocking anti- ⁇ l antibody has shown efficacy therapeutically in two independent models of fibrotic kidney disease (Kagami et al., (2002) Lab. Invest . 82 : 1219 ; Cook et al . , (2002) Am . J. Pathol . 252:1265).
  • VLA-1 may also play a role in regulation of tumor vascularization (angiogenesis) and tumor cell metastasis in many forms of cancer.
  • VLA-1 may regulate tumor angiogenesis by two distinct mechanisms: 1) by regulating the proliferation potential of the vascular endothelial fibro- blasts (Pozzi et al., (1998) J. Cell . Biol . 142 : 581 ; Senger et al . , (2002) Am . J. Pa thol .
  • VLA-1 inhibitors there are only two descriptions for VLA-1 inhibitors in the patent literature, and both describe large molecular weight polypeptides.
  • the first is a mAb to VLA-1 (WO 02/083854-A2) and the second is a disintegrin isolated from cobra venom (WO 02/22571-A2) . Therefore, there still exists a need in the art for low molecular weight antagonists, specific inhibitors of VLA-1-dependent cell adhesion that have improved pharmacokinetic and pharmacodynamic properties such as oral bioavail- ability and significant duration of action.
  • Such compounds would prove to be useful for the treatment, prevention or suppression of various pathologies mediated by VLA-1 binding and cellular adhe- sion, migration, activation or differentiation.
  • the present invention provides aminopiper- idine amide compounds which are antagonists to the VLA-1 integrin.
  • this invention is directed to a compound of Formula I:
  • a and B together with the nitrogen atom bound thereto, form a 4-8 membered nitrogen containing heterocyclic group containing 1 to 2 nitrogen atoms, wherein said heterocyclic group may be optionally substituted with 1 to 3 additional substituents each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cyclo- alkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, hydroxy, alkoxy, thioalkyl, and halo, wherein the one or more alkyl and sub- stituted alkyl substituents, if present, may be attached to either a carbon or a nitrogen atom in said heterocyclic group, wherein the one or more hydroxy, alkoxy, alkylsulfanyl and halo substituents, if present, may not be attached to a nitrogen atom in said heterocyclic group, and wherein the one or more hydroxy, al
  • R 1 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, fluoroalkyl, and alkyl;
  • R 4 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heterocyclic, and cycloalkyl;
  • R 6 and R 7 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, amino, substituted amino, aminocar- bonyloxy, aminoacyl, aminosulfonyl, sulfonylamino, acylamino, aminoacylamino, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, aryl, substituted aryl, OR a , acyloxy, oxycarbonyl- amino, thioalkyl, thioaryl, thioalkylaryl, thioalk- ylheteroaryl, NHS0 2 NR a R a , SC(0)R a , and SC(0)NR a 2 , or R 6 and R 7 , together with the carbon atom bound thereto, form a cycloalkyl, substituted cycloalkyl, heterocyclic, or a substituted heterocyclic group; R 8 is selected from the group consisting of
  • R 9 and R 10 are independently hydrogen, halo, alkyl, substituted alkyl, amino, substituted amino, aminocarbonyloxy, aminoacyl, acylamino, aminoacylamino, aminosulfonyl, sulfonylamino, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, aryl, substituted aryl, -OR a , acyloxy, oxycarbonylamino, thioalkyl, thioaryl, thioalkylaryl,
  • ⁇ Formula I exhibiting a biological activity of at least fifty percent inhibition of VLA-1 when tested at a concentration of 50 ⁇ M.
  • compounds of Formula I exhibiting a biological activity of at least fifty percent inhi- bition VLA-1 when tested at a concentration of 40 ⁇ M.
  • compounds of Formula I exhibiting a biological activity of at least fifty percent inhibition VLA-1 when tested at a concentration of 20 ⁇ M.
  • a and B, together with the nitrogen atom bound thereto preferably form a piperidine, pyrrolidine, or azetidine ring.
  • R 1 is selected from the group consisting of aryl, substi- tuted aryl, heteroaryl, and substituted heteroaryl, and even more preferably R 1 is selected from the group consisting of substituted aryl and substituted ⁇ heteroaryl'.
  • R 1 groups are selected from the group consisting of:
  • R 2 , R 3 , R 4 , and R 6 are hydrogen.
  • R 7 is selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, substituted alkyl- ene, aryl, and heteroaryl.
  • Particularly preferred R 7 groups are selected from the group consisting of: hydrogen; fluoro; methyl; aminomethyl; phenylamidomethylene;
  • 2-thiophenylmethyleneamidomethylene cyclopropyl-1-phenyl-l-amidomethylene; phenylethyleneamidomethylene; phenyloxymethyleneamidomethylene; 4-chlorophenyloxy-dimethylmethyleneamido-ethylene; cyclopentyl-1- (4-chlorophenyl) -1- amidomethylene;
  • 2-phenylcyclopropaneamidomethylene phenylmethyleneoxymethyleneamidomethylene; methylaminoamidomethylene; morpholinylamidomethylene; phenylmethyleneaminoamidomethylene; phenylethyleneaminoamidomethylene; ' methoxyamidomethylene; phenylmethyleneoxyamidomethylene;
  • 2-thiophenemethyleneamido cyclopropyl-1-phenyl-l-amido; phenylethyleneamido; phenyloxymethylenea ido; 4-chlorophenyloxy-l, 1-dimethylmethylene- amido; phenylethyl-ene-amido; phenylmethoxyloxymethyleneamido; methylaminoamido; dimethylaminoamido; morpholine-N-amido; phenylmethyleneaminoamido; phenylethyleneaminoamido; methoxyamido; phenylmethyleneoxyamido; phenylsulfonamido;
  • R 8 groups are selected from the groups consisting of CR a R a C (0) OR a , -C0 2 R a , where R a is as defined above, and, when R 9 and R 10 form an oxo group, then R 8 is preferably hydroxy.
  • R 8 groups are selected from the group consisting of: carboxyl
  • Preferred R 9 groups are selected from the group consisting of hydrogen, alkyl, aminoacyl, acylamino, and aryl.
  • R 9 groups are selected from the group consisting of: methyl; hydrogen; phenyl; and
  • Preferred R 10 groups are selected from the group consisting of hydrogen and alkyl. Particular- ly preferred R 10 groups are selected from the group consisting of methyl and hydrogen.
  • R 7 and R 9 together with the carbon atoms bound thereto form a cycloalkylene or cycloalkenylene group such as, for example, cyclopropylene or cyclohexenylene; R 9 and
  • Aminopiperidine amide derivatives within the scope of this invention are exemplified by those set forth in Tables I, IT, and III as follows:
  • R 1 is 2,3- dichlorobenzo[b] thiophen-4-yl
  • R 9 and R 10 are H
  • R 8 is -C(0)0H
  • R 7 is propylamidomethylene; methoxymethylenea idomethylene; t-butyl-amidomethylene; methylthioethyleleneamido ethylene; t-butyl-methyleneamidomethylene; cyclopropyla idomethylene; cyclopentylamidomethylene; cyclohexylamidomethylene;
  • 2-thiophenylmethyleneamidomethylene • cyclopropyl-1-phenyl-l-amidomethylene; phenylethyleneamidomethylene; phenyloxymethyleneamidomethylene;
  • 2-phenylcyclopropaneamidomethylene phenylmethyleneoxymethyleneamidomethylene; methylaminoamidomethylene; morpholinylamidomethylene; phenylmethyleneaminoamidomethylene; phenylethyleneaminoamidomethylene; methoxyamidomethylene; phenylmethyleneoxyamidomethylene;
  • 2-thiophenemethyleneamido 0 cycloprop.yl-1-phenyl-1-amido; phenylethyleneamido; phenyloxymethyleneamido;
  • this invention provides ' pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound defined herein..
  • this inven- tion is directed to a method for. assaying a biological sample from a mammalian patient suspected of having a,, disease,, condition or disorder mediated, at least ' in ' part, by VLA-1, which method comprises obtaining a biological sample from said patient and assaying said sample for the presence of VLA-1,.
  • this invention is directed to a method for inhibiting adhesion of mammalian cells to the extracellular matrix mediated, at least in part, by VLA-1, which method comprises contacting said cells with a compound or pharmaceutical composition of this invention.
  • this invention is directed to a method for treating a disease, condition or disorder whose progression is regulated, at least in part, by VLA-1 expression or activity in a mammalian patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound or composi- tion of this invention.
  • said disease, disorder, or condition is selected from the group consisting of asthma, trachoma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes, inflamma- tory bowel disease, multiple sclerosis, rheumatoid arthritis, tissue transplantation, tumor metastasis, tumor migration, and/or tumor growth, proliferation of fibroblasts in cancer, solid tumors, meningitis, encephalitis, stroke, cerebral traumas, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia, acute leukocyte-mediated lung injury, and fibrotic diseases.
  • said disease, disorder, or condition is a fibrotic disease.
  • said fibrotic disease is selected from the group consisting of systemic sclerosis, mixed connective tissue disease, fibrodysplasia, fibrocystic disease, sarcoidosis, and myositis.
  • said fibrotic disease has a manifestation of fibrotic vascular intimal hypertrophy, and is selected from the group consisting of vasculitis, polyarteritis nodosa, and temporal arteritis.
  • said fibrotic disease has a manifestation of fibrotic hypertrophy of skin and/or muscle tissue, and is selected from the group consisting of scleroderma, eosinophilic fasciitis, discoid lesions associated with lupus or discoid lupus, and surgical adhesions.
  • said fibrotic disease has a manifestation of fibrotic hypertrophy of nerve tissue, and is selected from the group consisting of cerebrosclerosis, annular sclerosis, diffuse sclerosis, and lobar sclerosis.
  • said fibrotic disease has a manifestation of fibrotic hypertrophy, or fibrosis of lung tissue, and is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, the fibrotic element of pneumoconiosis, pulmonary sarcoidosis, fibrosing alveolitis, the fibrotic or hypertrophic element of cystic fibrosis, chronic obstructive pulmonary disease, adult respiratory distress syndrome, and emphysema.
  • said fibrotic disease has a manifestation of fibrotic hypertrophy, or fibrosis of prostate, liver, the pleura, or pancreas, and is selected from the group consisting of benign prostatic hypertrophy (BPH) , nonalcoholic steato hepatitis, and fibrosis of the liver.
  • BPH benign prostatic hypertrophy
  • nonalcoholic steato hepatitis fibrosis of the liver.
  • said fibrotic disease has a manifestation of fibrotic hypertrophy, or fibrosis of the kidney, and is selected from the group consisting of chronic renal failure, lupus nephritis, alports syndrome, glomerulonephritis, and diabetic nephritis.
  • said disease, disorder, or condition is cancer.
  • said cancer is a tumor or a neoplasm selected from the group consisting of carcinomas, adenocarcinomas, and sarcomas .
  • said can- cer is selected from the group consisting of growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, human soft tissue carcinoma, cancer metastases, squamous cell carcinoma, esophageal squamous cell carcinoma, oral carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producing tumors, nonsmall cell cancers, breast cancer, gastrointestinal cancers, urological cancers, malignancies of the female gen- ital tract, malignancies of the male genital tract, kidney cancer, brain cancer, bone cancers, skin cancers, thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer, trophoblastic neoplasms, hemangiopericytom
  • said cancer is a cell proliferative disorders and is selected from the group consisting of angiogenesis- mediated diseases, benign tumors, acoustic neuromas, neurofibromas, pyogenic granulomas, biliary tract cancer, choriocarcinoma, esophageal cancer, gastric cancer, intraepithelial neoplasms, lung cancer, and neuroblastomas .
  • a compound or composition of this inven- tion may be administered to the mammal by a suitable route, such- as orally, intravenously, parenterally, transdermally, topically, rectally, or intranasally.
  • Mammals include, for example, humans and other primates, pet or companion animals, such as dogs and cats, - laboratory animals, such .as rats, mice and- rabbits, and farm animals, such as horses, pigs, sheep, and cattle.
  • the present invention is directed to novel aminopiperidine amide derivatives.
  • alkyl refers to mono- valent alkyl groups having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, t-butyl, n- heptyl, octyl, and the like.
  • Substituted alkyl refers to an alkyl group having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryl- oxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl, thiol, thioalkyl, heteroaryl, substituted hetero- aryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino .
  • Fluoroalkyl refers to an alkyl group having from 1 to 4 carbon atoms and from 2 to 7 fluoro atoms.
  • Hydrox refers to the group -OH.
  • Alkylene refers to divalent alkylene groups having from 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methylene, n-heptyl- ene, 1, 3-octylene, and the like.
  • Substituted alkylene refers to an alkylene group having from 1 to 5 substituents selected from the group consisting of substituents defined for substituted alkyl.
  • Alkoxy refers to the group “alkyl-O—” which includes, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec- butoxy, n-pentoxy, n-hexoxy, 1, 2-dimethylbutoxy, and the like.
  • Substituted alkoxy refers to the group “substituted alkyl-O-.”
  • Acyl refers to the groups H—C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C (0) -, substituted alkenyl-C (0) -, cycloalkyl-C (0) -, substituted cycloalkyl-C (0) -, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C (0) -, substituted hetero- aryl-C(O), heterocyclic-C (0) -, and substituted heterocyclic-C (0) - .
  • Acylamino refers to the group —C(0)NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl heteroaryl, substituted heteroaryl, heterocyclic,. substituted heterocyclic, and where each R is option- ally joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic ring.
  • Acyloxy refers to the groups alkyl- C(0)0-, substituted alkyl-C (0) 0-, alkenyl-C (0) 0-, substituted alkenyl-C (0) 0-, aryl-C(0)0-, substituted aryl-C(0)0-, cycloalkyl-C (0) 0-, substituted cycloalkyl-C (0)0-, heteroaryl-C (0) 0-, substituted heteroaryl-C (0)0-, heterocyclic-C (0) 0-, and substituted heterocyclic-C (0) 0- .
  • Alkenyl refers to monovalent alkenyl groups having from 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms, and having at least 1 and preferably from 1-2 sites of alkenyl unsatura- tion.
  • Substituted alkenyl refers to alkenyl groups having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryl- oxy, cyano, halogen, hydroxyl, nitro, carboxyl, car- ⁇ -.
  • boxyl esters cycloalkyl, substituted cycloalkyl, thiol, thioalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino, provided that the hydroxyl or the thio group is not pendent to an unsaturated carbon • atom.
  • Amino refers to the group -NH 2 .
  • substituted amino refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where each R is optionally joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic ring provided that both R's are not hydrogen.
  • Aminosulfonyl refers to the group -S0 2 NR'R', wherein each R' is independently selected from the group consisting of hydrogen, alkyl, sub- stituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted cyclo- alkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where each R is optionally joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic ring.
  • Sulfonylamino refers to the group -NR'R'S0 2 , wherein each R' is as defined above.
  • aminoacyl refers to the groups -NRC(O)- alkyl, -NRC (0) substituted alkyl, -NRC (0) alkenyl, -NRC (0) substituted alkenyl, -NRC (0) cycloalkyl, -NRC- (0) substituted cycloalkyl, -NRC (0) aryl, -NRC (0) substituted aryl, -NRC (0) heteroaryl, -NRC (0) substituted heteroaryl, -NRC (O) eterocyclic, and -NRC (0) substituted heterocyclic, where R is hydrogen or alkyl.
  • Aminocarbonyloxy refers to the groups
  • -NRC(0)0-alkyl -NRC (0)0 substituted alkyl, -NRC(O)- O-Cycloalkyl, -NRC (0)0 substituted cycloalkyl, -NRC- (O)O-aryl, -NRC (0)0 substituted aryl, -NRC (0) 0-het- eroaryl, -NRC (0)0 substituted heteroaryl, -NRC (0)0- heterocyclic, and -NRC (0)0 substituted heterocyclic, where R is hydrogen or alkyl.
  • Oxycarbonylamino refers to the groups -0C(0)Q where Q is amino or substituted amino.
  • Aminocarbonylamino or “aminoacylamino” refers to the groups -QC(0)Q where each Q is independently amino or substituted amino.
  • Aryl or “Ar” refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multi- pie condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-1, 4-benzoxazin-3 (4H) - one-7-yl, and the like) provided that the point of attachment is on an aromatic carbocyclic group atom.
  • Preferred aryls include phenyl and naphthyl.
  • Substituted aryl refers to aryl groups which are substituted with from 1 to 3 substituents selected from the group consisting of hydroxy, . acyl, acylamino, acyloxy, alkyl, substituted alkyl, ⁇ alkoxy, substituted alkoxy, alkenyl, substituted
  • Aryloxy refers to the group ' -aryl-O— which includes, by way of' example, phenoxy, naph- thoxy, and the like'.
  • Substituted aryloxy refers to substituted aryl-O— groups.
  • Carboxyl refers to the group -COOH- and salts thereof.
  • Carboxyl esters refer to the group -COOR
  • R is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic.
  • Cycloalkyl refers to monovalent cyclic alkyl groups of from 3 to 8 carbon atoms having a single cyclic ring including, by way of example, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like.
  • Cycloalkylene refers to divalent cyclic alkyl groups of from 3 to 8 carbon atoms having a single cyclic ring including, by way of example, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, and the like.
  • Cycloalkenylene refers to divalent cyclic alkenyl groups of from 4 to 8 carbon atoms having a single cyclic ring and 1-2 sites of unsat- uration including, by way of example, cyclobutenyl- ene, cyclopentenylene, cyclooctenylene, and the like.
  • Halo or halogen refers to fluoro, chloro, bromo and iodo and preferably is fluoro, chloro or bromo.
  • Heteroaryl refers to an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring.
  • Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizin- yl or benzothienyl) provided that the point of attachment is to a heteroaryl group atom and further provided that the heteroaryl 'group contains at least five ring atoms.
  • Preferred heteroaryls include pyridyl, pyrrolyl, indolyl and furyl .
  • Substituted heteroaryl refers to heter- oaryl groups which are substituted with from 1 to 3 substituents selected from the group of' substituents defined for .substituted aryl.
  • Heterocycle or “heterocyclic” refers to a monovalent saturated or unsaturated, but not aro- matic, group having a single ring or multiple condensed rings, from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur or oxygen within the ring wherein, in fused ring systems, one or more the rings can be aryl or heteroaryl, provided that the heterocyclic ring has at least 4 atoms and further provided that the point of attachment is to a heterocyclic ring atom.
  • Substituted heterocyclic refers to net-' erocycle groups which are substituted with from 1 to 3 substituents selected from the group of substituents defined for substituted cycloalkyl.
  • heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydro- indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinox- aline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperi- dine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquino
  • Heterocyclene refers to divalent heterocyclic groups of from 3 to 8 carbon atoms having a single cyclic ring.
  • Heterocyclenylene refers to divalent heterocyclic groups of from 4 to .8 carbon atoms ' having a single cyclic ring and 1-2 sites of unsat- uration.
  • Thiol refers to the group —SH.
  • Thioalkyl refers to the group —S-alkyl.
  • Substituted thioalkyl refers to the group —S substituted alkyl.
  • Thioaryl refers to the group —S-aryl.
  • Thioalkylaryl refers to the group -S- alkylene-aryl, S substituted alkylene aryl / S alkylene substituted aryl or -S substituted alkylene substituted aryl.
  • Thioalkylheteroaryl refers to the group -S alkylene heteroaryl, S substituted alkylene heteroaryl, S alkylene substituted heteroaryl or -S substituted alkylene substituted heteroaryl.
  • “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound of Formula I which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkyl- ammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids,- such as hydrochloride, hydrobro- mide, tartrate, mesylate, acetate, maleate, oxalate, and the like.
  • Prodrugs are- compounds which convert (e.g., hydrolyze, metabolize) in vivo • to a compound of the invention.
  • the effectiveness of an orally administered drug is dependent upon the drug'.s efficient transport across the mucosal epi-; • thelium and .its stability in entero-hepatic circula- • tion.
  • Drugs that are effective after parenteral administration but less effective orally, or whose ' plasma half-life is' considered too short, may be chemically modified- into a prodrug form.
  • the pro- drug should have a pharmacokinetic profile that is different from that of the parent, enabling easier absorption across the mucosal epithelium-, better * salt formulation and/or solubility, and/or improved systemic stability (for an increase in plasma half- life, for example) .
  • Many chemical modifications may be suitable for the creation of the prodrugs accord- ing to the invention, including:
  • Ester or amide derivatives which may be cleaved by, for example, esterases or lipases.
  • ester derivatives the ester is derived from the carboxylic acid moiety of the drug molecule by known means.
  • amide derivatives the amide may be de- rived from the carboxylic acid moiety or the amine moiety of the drug molecule by known means.
  • a peptide may be coupled to the- drug molecule via amide bond formation with the amine or carboxylic acid moiety of the drug molecule by known means.
  • Bioactivity as used herein re- fers to an inhibition concentration when tested in at least one of the assays outlined in Example A or B.
  • substituted as used with, for example, “substituted alkyl” does not include poly- mers derived therefrom but are limited to a maximum of 3 substituents groups, e.g., Ar-Ar-Ar.
  • tautomer refers to an isomer in which migration of a hydrogen atom results in two or more structures.
  • the compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction tempera- tures, times, mole ratios of reactants, solvents, pressures) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and P. G. M. Wuts, "Protecting Groups in Organic Synthesis, Second Edition," Wiley, New York, 1991, and references cited therein.
  • the compounds of this inven- tion may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoiso- mers (and enriched mixtures) are included within the scope of this invention, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chi- ral column chromatography, chiral resolvinc? agents, and the like.
  • each R 2 H, CH 3 , CF 3
  • R 1 and R 3 are as defined for Formula I
  • Pd ( 0 ) refers to metallic palladium
  • Cinnamic acid intermediates 3 may be prepared by a Heck-type, palladium-mediated coupling (e . g . , using tetrakis- (o-tolyl phosphine ) palladium- (0), Pd 2 (dba) 3 , Pd(OAc) 2 , or the like) of halo substituted aromatic derivatives 2 with an appropriate olefinic substrate 1 (e.g., methyl acrylate, ethyl crotonate, or the like; S. J. Buchwald, Chem . Eur. J. 1999, 5, 3107-3112) .
  • the intermediate ester may require a separate hydrolysis step to allow for further elaboration.
  • R H or CH 3
  • R 1 is as defined for Formula I
  • Substituted aromatic aldehydes 5 may be prepared from aromatic carboxylic acids 4 by utilizing a reducing agent (for example, borane-THF, LiAlH 4 ) to reduce the acid to the benzyl alcohol. The resulting alcohols can then undergo partial oxidation by standard methods (Jones, Swern, Moffat) to the desired aldehydes.
  • a reducing agent for example, borane-THF, LiAlH 4
  • the aldehyde may be reacted with a Wittig, Horner-Emmons, or Wadsworth-Emmons reagent (e.g., 2- (diethoxyphos- phoryl) -propionic acid ethyl ester, or the like) in a solvent (CH 2 C1 2 , CH 2 C1CH 2 C1, or like) and a base (NaOH, NaOEt, NaH, or the like) to furnish the desired cinnamic ester.
  • a base NaOH, NaOEt, NaH, or the like
  • R 1 , R 2 , R 3 are as defined for Formula I m is as defined above
  • the cinnamic acid is activated (for example, using thionyl chloride, or oxalyl chloride, or 1- (3-dimethylamino- propyl) -3-ethylcarbodiimide and N-hydroxysuccin- imide, or the like) and reacted with 7, usually in the presence of a tertiary amine base (e.g., diiso- propylethylamine, triethylamine, N-methyl morph- oline, or the like) to provide amides 8.
  • a tertiary amine base e.g., diiso- propylethylamine, triethylamine, N-methyl morph- oline, or the like
  • the protected amino group of 8 is subsequently deprotected to the amine 9 using appropriate reagents (e.g., anhydrous HC1 to remove a Boc group) .
  • R 1 , R 2 , R 3 , R 6 , R 7 , R 9 , Rl° are as defined for
  • a ' tertiary amine base e.g., diisopropylethylamine, or the like
  • R 6 or R 7 R a regioisomeric mixture of amides 11 may be obtained, which can be isolated by conventional techniques as described below.
  • R 1 , R 2 , R 3 , R 6 , R 7 are as defined for Formula I
  • R" is as defined above m is as defined above
  • P 2 is a suitable protecting group such as t-butyl and the like
  • amines 9 may be coupled with a mono-protected diacid 12 (for example, mono- ethyl malonate, mono-ethyl succinate, or the like) to give an amide-ester intermediate 13, as shown in Scheme 5.
  • a mono-protected diacid 12 for example, mono- ethyl malonate, mono-ethyl succinate, or the like
  • Amide formation protocols are as described for Scheme 3.
  • Subsequent deprotection of the ester group of 13, using appropriate reagents for example, TFA to remove tert-butyl ester groups
  • R 6 , R 7 , R 1 , R 2 , R 3 are as defined above
  • amide formation protocols described in Scheme 3.
  • amine 16 can be coupled with a cinnamic acid 6 as described for Scheme 3 followed by deprotection of P 2 using appropriate reagents (for example, TFA to remove tert-butyl ester groups) to provide amides 11.
  • R 1 , R 2 , R 3 are as defined for Formula I
  • Scheme 7 illustrates an alternative procedure in which the order of these coupling steps may be reversed.
  • Cyclic amines bearing a protected amine group (7, e.g., 4-N-Boc-aminopiperidine, or the like) may be coupled with an acryloyl chloride 17 in the presence of an appropriate base (e.g.,
  • the acrylamide olefin 18 may be further elaborated by palladium-mediated coupling with a halo substituted aromatic derivative (using protocols described for Scheme 1) to provide cinnamides 8.
  • each P 2 is a suitable carboxy protecting group m is as defined above
  • R 1 , R 2 , R 3 , R 6 , R 7 are as defined for Formula I
  • a dialkyl malonate e.g., di- ethyl malonate, or the like
  • the resulting amines 24 can be coupled with a cinnamic acid 6 using amide formation protocols described for Scheme 3, thus providing malonamides 25.
  • Deprotection of the ester group Ra gives desired acids 26.
  • R' substitution on aryl group -as defined above
  • Scheme 9 illustrates the preparation of 5- bromobenzothiophenes, used for the preparation of the corresponding cinnamic acids.
  • a suitable phen- ylsulfanyl acetic acid 28 is prepared from a thio- phenol 27 using bromoacetic acid under standard conditions.
  • Sulfide 28 is selectively brominated, e.g., with bromine in a solvent such as dichloro- methane, glacial acetic acid, or the like, in the presence of iron or iodine as catalysts.
  • the reaction is typically performed at room temperature for 1 to 96 hours.
  • Bromide 29 is then converted to an acid halide intermediate by treatment with an in- organic acid halide (for example, thionyl chloride, phosphorous trichloride, phosphorous pentachloride, phosphrous tribromide, oxalyl chloride, or the like) in an inert solvent (for example dichloromethane or the like), at a temperature in the range of 0°C to 110 °C for about 1 to 48 hours.
  • the volatiles are then removed under reduced pressure and the residue is dissolved in an appropriate solvent, typically dichloromethane, and subjected to Friedel-Crafts cyclization by treatment with a Lewis acid such as AICI 3 or poiyphosphoric acid.
  • the reaction is generally carried out at -78 °C to 25°C.
  • the resulting intermediate ketone product is reduced without isolation by a hydride reducing reagent- (for exam- pie, NaBH or the like) to " provide alcohol 30.
  • Alcohol 30 may be used directly in the next step, or first purified by chromatography or recrystalliza- tion as appropriate.
  • the dehydration of 30 to benzothiophene 31 " is accomplished by treatment " with a Lewis or proti.c acid (for example, boron trifluor-" ide etherate.) in an appropriate solvent, (for example glacial acetic acid or the like) at a temperature ranging from ambient temperature to the reflux temperature of the solvent.
  • a Lewis or proti.c acid for example, boron trifluor-" ide etherate.
  • an appropriate solvent for example glacial acetic acid or the like
  • R 1 , R 2 , R 3 , R 6 , R 7 are as defined for Formula I
  • R 10 ' is a suitable substituent m is as defined above
  • R 1 , R 2 , R 3 are as defined for Formula I
  • Scheme 11 illustrates the preparation of succinamides in which an aminomethyl group is appended to the ⁇ -position of the succinamide chain.
  • the diester 35 can be selectively mono-depro- tected to give carboxylic acid intermediate 36 (for example, using lithium hydroxide to hydrolyze a methyl ester selectively over a tert-butyl ester) .
  • the monocarboxylic acid 36 is coupled to amines 9 using amide formation protocols described for Scheme 3.
  • the amino group of 37 can be revealed by selective deprotection, and then functionalized by reaction with activated carboxyl- and sulfonyl-containing inputs (e.g., acid chlorides, sulfonyl chlorides, carbamoyl chlorides, isocyanates, anhydrides, chloroformates, or the like) in the presence of an appropriate base (e.g., diisopropylethylamine, or the like), to provide, after ester cleavage, the amine-derivatized compounds 39.
  • activated carboxyl- and sulfonyl-containing inputs e.g., acid chlorides, sulfonyl chlorides, carbamoyl chlorides, isocyanates, anhydrides, chloroformates, or the like
  • an appropriate base e.g., diisopropylethylamine, or the like
  • the amino group of 38 may be functionalized selectively in the presence of the, carboxylic acid using the activate
  • R 6 and R 7 are as defined for Formula I
  • Het is a suitable heteroaryl or heterocyclic
  • the carboxyl group of an nitrogen-protected ⁇ -amino acid, compound 100 is reduced using conventional techniques such as the use of a reducing agent includ- ing, for example, lithium aluminum hydride to provide for the corresponding alcohol, compound 200.
  • a reducing agent includ- ing, for example, lithium aluminum hydride
  • the reaction is preferably conducted in an inert diluent such as tetrahydrofuran, diethyl ether, and the like at a temperature preferably from about -78°C to about 25°C.
  • the reaction is continued until substantial completion which typically occurs from within 0.5 to 18 hours.
  • compound 200 can be recovered by conventional methods including neutralization, extraction, precipitation, chromatography, filtration, and the like or used in the next step of the reaction without purification and/or isolation.
  • halo group e.g., chloro
  • a suitable halogenating agent include, for example, inorganic acid halides, such as thionyl chloride, phosphorous trichloride, phosphorous tribromide or phosphorous pentachloride, under conventional conditions.
  • this reaction is conducted using about 1 to 5 molar equivalents of the inorganic acid halide, either neat or in an inert solvent, such as dichloromethane or carbon tetrachloride, at temperature in the range of about 0°C to about 80°C for about 1 to about 48 hours.
  • a catalyst such as DMF, may also be used in this reaction.
  • compound 300 can be recovered by conventional methods including neutralization, extraction, precipitation, chromatography, filtration, and the like or used in the next step of the reaction without purification and/or isolation.
  • the compounds of the subject invention are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, parenteral, transdermal, topical, rectal, and intranasal. These compounds are effective as both injectable and oral compositions.
  • Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • This invention also includes pharmaceutical compositions which contain, as the active ingredient, , one or more of the compounds of the subject invention above associated with pharmaceu- 5 tically acceptable carriers. In making the compositions of this invention, the active ingredient is usually mixed with ' an excipient, . diluted by an
  • ⁇ ' excipient or enclosed within such a carrier which can be in the form of a capsule, sachet-, paper or
  • The. excipient- employed is typical- ' • ly an excipient suitable for administration to' human subjects or other mammals-.
  • the excipient serves as a diluent, it can be a solid, semi-solid, • • or liquid material, which acts as a vehicle, carrier- - •
  • the com- " ' positions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups-, aerosols (as. a solid or in a liquid medium) , ointments contain-
  • the active compound for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders .
  • the active compound 25 necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is
  • the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
  • excipients include lactose, dextrose, sucrose,- sorbitol, anni- tol, starches, ' gum acacia, calcium phosphate, al- ginates, tragacanth, gelatin, calcium silicate, microcrysta-lline cellulose, polyvinylpyrrolidone, cellulose,' sterile water,- syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as. talc, magnesium stearate, " and mineral oil; wetting agents; .emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates; sweetening agents; and flavoring agents.--
  • the compositions of . the invention can .be formulated so as to provide quick, sustained ; or delayed release of the active ingredient after ' administration to • the patient by -employing procedures known in the art. :
  • the quantity of active- component, that is- the compound according to • the subject invention, in the pharmaceutical composition and unit dosage- form - thereof may be varied or adjusted widely depending upon the particular application, the potency of the particular compound and the desired concentration.
  • the compositions are preferably formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg, usually about 5 to about 100 mg, occasionally about 10 to about 30 mg, of the active ingredient.
  • unit dosage forms re- fers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the compound of the subject "invention above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier (s).
  • the active compound is effective over a wide dosage range and is generally administered in a pharmaceutically or therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the ⁇ relevant circumstances, including the condition to be treated, the severity of the condition being treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the compounds or pharmaceutical compositions thereof will be administered by any appropriate route, such as orally, topically, transdermally, and/or par- enterally at a dosage to obtain and maintain a concentration, that is, an amount, or blood-level of active component in the animal undergoing treatment that will be therapeutically effective.
  • a dosage that is, an amount, or blood-level of active component in the animal undergoing treatment that will be therapeutically effective.
  • such therapeutically effective amount of dosage of active component i.e., an effective dosage
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre- formulation composition containing a homogeneous mixture of a compound of the present invention.
  • a pharmaceutical excipient for preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre- formulation composition containing a homogeneous mixture of a compound of the present invention.
  • these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformula- tion is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the present inven- tion may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • liquid forms in which the novel compositions of the present invention may be incorpo- rated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceu-.. tically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra.
  • the compositions are' administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized .solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine.
  • Solution, sus- pension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • Hard gelatin capsules containing the following ingredients are prepared:
  • the above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
  • a tablet ⁇ formula is prepared using the in-; gredients below:
  • the components are blended and compressed to form tablets, each weighing 240 mg.
  • a dry powder inhaler formulation is prepared containing the following components:
  • the active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
  • Tablets each containing 30 mg of active ingredient, are prepared, as follows
  • Capsules each containing 40 mg of medicament are made as follows:
  • the active ingredient, -starch ' ' and magnesium stearate are blended, passed through, a No. 20 mesh U.S. sieve, and filled into hard gelatin cap- sules in 150 mg quantities,
  • Suppositories each containing 25 mg of active ingredient are made as follows:
  • Suspensions each containing 50 mg of medicament per 5.0 mL dose are made as follows:
  • the active ingredient, sucrose and x ' anthan. gum are blended, passed through a No. 10 mesh U.S. . * ; sieve, and then mixed with a previously made solution of the microcrystalline cellulose ' and sodium carboxymethyl cellulose in water..
  • the sodium ben- : zoate, flavor, and, color are diluted with some of the water and added with stirring,, Sufficient water is then added to produce the required volume.
  • a subcutaneous formulation may be prepared as follows:
  • a topical formulation may be prepared as follows :
  • the white soft paraffin is heated until molten.
  • the liquid paraffin and emulsifying wax are incorporated and stirred until dissolved.
  • the active ingredient is added and stirring is continued until dispersed.
  • the mixture is then cooled until solid.
  • An intravenous formulation may be prepared as follows:
  • transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts .
  • the construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent 5,023,252, herein incorpo- rated by reference.
  • patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • Indirect techniques usually involve formulating the composi- tions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid soluble drugs.
  • Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood brain barrier.
  • the delivery of hydrophilic drugs may be enhanced by intra arterial infusion of hypertonic . solutions which can transiently open the blood brain barrier. 5
  • suitable formulations for use in the present invention can be found in Remington ' s Phar- : . maceutical Sciences, Mack -.Publishing Company, Philadelphia, PA, 17th ed. (1985) . •' • As noted above, the compounds described
  • the compounds may be encapsulated, introduced into the lumen of liposomes, 15 - prepared as a colloid, or other conventional techniques may be employed which provide an ..extended ' serum half life of the compounds.
  • a variety of methods are available for ' preparing liposomes, as described in, e.g., Szoka et al., U.S. Patent Nos. 20 4,235,871, 4,501,728 and 4, 837, 028, each of which is • incorporated- herein by reference.
  • the compounds and/or compositions of this invention can be employed to bind VLA-1 in biolog- 25 ical samples, for instance in mammalian patients suspected of having a disease, condition or disorder mediated, at least in part, by VLA-1. Accordingly, these compounds have utility in, for example, assaying such samples for VLA-1 mediated adhesion.
  • compounds of this invention and/or pharmaceutical compositions thereof inhibit, in vivo, adhesion of mammalian cells to the extracellular matrix medi- 5 ated, at least in part by VLA-1 and, accordingly, .- can be used in the treatment, prevention, or amelioration of diseases;, conditions, or disorders whose progression or symptoms is regulated, at least in part, by VLA-1 expression or activity.
  • Such di- 10. seases, conditions, or disorders include, but. are not limited to, inflammatory diseases, fibrotic . > diseases, and cancer.
  • diseases, 'conditions,, and .-. disorders which are expected to be treatable by the 15- compounds and/or compositions of the present invention ' include, but are- not limited to, asthma, - • ' trachoma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes (including acute juvenile onset diabetes), inflammatory bowel disease (including 20 ' ulcerative colitis -and Crohn's disease)-,- multiple sclerosis, rheumatoid arthritis, tissue transplantation, tumor metastasis, migration, and/or growth (including angiogenesis), proliferation of fibroblasts in cancer, solid tumors, meningitis, enceph- 25 alitis, stroke, and other cerebral traumas, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia, acute leukocyte-mediated lung injury such as that which occurs in adult respiratory distress syndrome, and fibrotic diseases, such 30 as fibrotic diseases of the
  • Fibrotic diseases which are expected to be treatable by the compounds and/or compositions of the present invention include systemic sclerosis, mixed connective tissue disease, fibrodysplasia, fibrocystic disease, sarcoidosis, myositis (e.g., polymyositis, primary idiopathic polymyositis, childhood polymyositis, dermatomyositis, childhood dermatomyositis, primary idiopathic dermatomyositis in adults, inclusion body myositis, polymyositis, or dermatomyositis associated with malignant tumors) .
  • myositis e.g., polymyositis, primary idiopathic polymyositis, childhood polymyositis, dermatomyositis, childhood dermatomyositis, primary idiopathic dermatomyositis in adults, inclusion body myositis, polymyo
  • Dermatomyositis can be associated with fibrosing or hypertrophic aspects, including fibrosing alveolitis and pulmonary fibrosis.
  • Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis in such diseases.
  • Amelioration includes reducing the rate of progression of a disease.
  • fibrotic diseases are diseases that have as a manifestation fibrotic vascular intimal hypertrophy.
  • diseases include vascu- litis (including coronary artery vasculitis) , poly- arteritis nodosa or temporal arteritis.
  • Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate vascular intimal hypertrophy in such diseases.
  • fibrotic diseases further include ⁇ diseases that have as a manifestation fibrotic hypertrophy of skin and/or muscle tissue. These diseases include scleroderma, eosinophilic fasciit- 5 is, discoid lesions associated with lupus or discoid ⁇ .
  • Treatment using the ..'.- compounds and/or compositions of the present invention is expected to- treat, prevent, reduce, or ameliorate such indications, -or hypertrophy or 10.- fibrosis of skin or muscle tissue.
  • Fibrotic diseases further include ' diseases that have as a -manifestation fibrotic hypertrophy of , nerve tissue. These diseases include cerebroscler- .. osis, annular sclerosis. diffuse sclerosis and ⁇ • 15' lobar sclerosis. Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent,- reduce-, ' or ameliorate such diseases, or hypertrophy, .fibrotic hypertrophy, or -.:-. fibrosis' of nerve tissue in such diseases. 20 These fibrotic diseases further include fibrotic lung diseases that have as a manifestation . fibrotic hypertrophy, or fibrosis of lung tissue.
  • pulmonary ' fibrosis or interstitial lung disease or interstitial pulmonary 25. fibrosis
  • idiopathic pulmonary fibrosis the fibrotic element of pneumoconiosis (which is associated with exposure to environmental hazards such as smoking, asbestos, cotton lint, stone dust, mine dust and other particles)
  • pulmonary sarcoidosis 30 fibrosing alveolitis
  • the fibrotic or hypertrophic element of cystic fibrosis chronic obstructive pulmonary disease, adult respiratory distress syndrome and emphysema.
  • Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis in such diseases.
  • Such fibrotic diseases further include diseases that have as a manifestation fibrotic hypertrophy, or fibrosis of prostate, liver, the pleura (e.g., pleurisy, pleural fibrosis) or pancreas.
  • diseases include benign prostatic hypertrophy (BPH) , nonalcoholic steato hepatitis and fibrosis of the liver.
  • BPH benign prostatic hypertrophy
  • Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis in such diseases.
  • fibrotic diseases further include diseases that have as a manifestation fibrotic. hypertrophy, or fibrosis of the kidney, such as chronic renal failure, lupus nephritis, alports syndrome, glomerulonephritis and diabetic nephritis.
  • Treatment using the compound and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis of the kidney.
  • Cancers which are expected to be treatable by the compounds and/or compositions of the present invention typically occur in mammals.
  • Tumors or neoplasms include growths of
  • tissue cells in which -the multiplication of the cells is uncontrolled and progressive. Some such growths are benign,- but others are termed “malignant” and can lead to death of the organism. Malignant neoplasms or “cancers” are distinguished
  • Tumors or neoplasms which are- expected to ,- be treatable by the compounds and/or compositions of' 0 the present invention include, but are hot limited to., solid tumors, i.e., carcinomas, adenocarcinomas, ' and sarcomas.
  • Carcinomas include those malignant neoplasms derived from epithelial cells, which infil- - trate (invade) the surrounding tissues and give rise 5 to metastases.
  • Adenocarcinomas are carcinomas derived from granular tissue, or from tissues which form recognizable glandular structures.
  • sarcomas are tumors whose cells are embedded in a fibrillar 0 or homogenous substance like embryonic connective tissue.
  • VLA-1 may be associated with adult and pediatric oncology in various forms of cancer, for example, growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, human soft tissue carcinoma (including Ewing's sarcoma) , cancer metastases (including lymphatic metas- tases), squamous cell carcinoma (particularly of the head and neck), esophageal squamous cell carcinoma, oral carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producing tumors, nonsmall cell cancers, breast cancer (including small cell carcinoma and ductal carcinoma) , gastrointestinal cancers (including stomach cancer, colon cancer, colorectal cancer, polyps associated with colorectal neop
  • compositions of the present invention also include angiogenesis-mediated diseases, . benign - tumors (e.g., hemangiomas)., acoustic neuromas, neurofibromas, pyogenic granulomas, biliary tract cancer, choriocarcinoma, ' esophageal cancer, gastric ⁇
  • the biological -activity of the compounds identified -above may be assayed in a variety of sys ⁇ - , terns.- For example, extracellular matrix, such as
  • ⁇ 25 smooth muscle cells, microvascular endothelial cells, fibroblasts, osteoblasts, chondrocytes, and activated cells of the immune system including effector T cells, macrophages and NK cells.
  • a number of transfected cell lines can also be used,
  • VLA-1 can also be tested for the ability to inhibit binding between VLA-1 and extracellular matrix such as collagen IV, or between VLA-1 and a labeled compound known to bind VLA-1 such as a compound and/or composition of this invention or anti- bodies to VLA-1.
  • the extracellular matrix can be soluble or immobilized on a solid surface.
  • VLA-1 may also be expressed as a recombi- nant fusion protein having acidic and basic leucine- .. zipper tails so that binding to extracellular matrix may be detected in an immunoassay.
  • the labeling systems can be in a vari- ety of forms.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • a wide variety of labels may be used.
  • the component may be labeled by any one of several methods. The most common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 14 C, or 32 P labeled compounds, and the like.
  • Nonradioactive labels include europium, as well as ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation. Appropriate in vivo models for demonstrating efficacy in treating inflammatory responses in- elude DTH (delayed type hypersensitivity) in mice, rats, guinea pigs, or primates, as well as other inflammatory or fibrotic models dependent upon VLA-1 integrin.
  • Compounds having the desired biological activity may be modified as necessary to provide desired properties such as improved pharmacological properties (e.g., in vivo stability, bio-availability) , or the ability to be detected in diagnostic applications. Stability can be assayed in a variety of ways such as by measuring the half-life of the compounds during incubation with peptidases or human plasma or serum.
  • a wide variety of labels may be linked to the compounds, which may provide, directly or indirectly, a detectable signal.
  • the compounds and/or compositions of the subject invention may be modified in a variety of ways for a variety of end purposes while still re- taining biological activity.
  • various reactive sites may be introduced for linking to particles, solid substrates, macromolecules, and the like.
  • Labeled compounds can be used in a variety of in vivo or in vitro applications.
  • a wide variety of labels may be employed, such as radionuclides (e.g., gamma-emitting radioisotopes such as tech- netium-99 or indium-Ill), fluorescers (e.g., fluorescein) , enzymes, enzyme substrates, enzyme cofac- tors, enzyme inhibitors, chemiluminescent compounds, bioluminescent compounds, and the like.
  • radionuclides e.g., gamma-emitting radioisotopes such as tech- netium-99 or indium-Ill
  • fluorescers e.g., fluorescein
  • enzymes enzyme substrates
  • enzyme cofac- tors enzyme inhibitors
  • chemiluminescent compounds chemiluminescent compounds
  • bioluminescent compounds bioluminescent compounds
  • In vitro uses include diagnostic applications such as monitoring inflammatory responses by detecting the presence of cells expressing VLA-1.
  • the compounds and/or compositions of this invention ⁇ can also be used for isolating or labeling such cells.
  • the compounds and/or compositions of the invention can be used to assay for potential inhibitors of VLA- 1/Extracellular matrix interactions.
  • radioisotopes are typically used in accordance with well known techniques.
  • the radioisotopes may be bound to the com- pound either directly or indirectly using intermediate functional groups.
  • chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules have been used to bind com- pounds to metallic ion radioisotopes.
  • the complexes can also be labeled with a paramagnetic isotope for purposes of m vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR) , both of which are well known.
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • any conventional method for visualizing diagnostic images can be used.
  • gamma- and positron-emitting radioisotopes are used for camera imaging and paramagnetic isotopes are used for MRI.
  • the compounds can be used to monitor the course of amelioration of an inflamma- tory response in an individual. By measuring the increase or decrease in cells expressing VLA-1 it is possible to determine whether a particular therapeutic regimen aimed at ameliorating the disease is effective.
  • Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. -Suitable formulations for use in the present invention are found in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).
  • compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the progression or symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on the disease condition being treated as well as by the judgment of the attending clini- cian depending upon factors such as the severity of the disease, disorder or condition, the age, weight and general condition of .the patient, and the like.
  • the compounds administered to a patient are typically in the form of pharmaceutical composi- tions described above.- These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The .resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administra- ' tion.-
  • the pH of the compound preparations typically, will be between about 3 and 11, more- preferably from about 5 to .-9, and most- ' preferably from about 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will .,- result in the formation o.f pharmaceutical salts.
  • the therapeutic dosage of the compounds and/or compositions of the present invention will vary according to, for example, the particular' use. • ' for ' which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of .the prescribing physician.
  • the dose will typically be in the range of about 100 ⁇ g to about 50 mg per kilogram body weight per day, preferably about 5 mg to about 20 mg per kilogram body weight per day.
  • the dose will typically be in the range of about 20 ⁇ g to about 500 ⁇ g per kilogram body weight, preferably about 100 ⁇ g to about 300 ⁇ g per kilogram body weight.
  • Alternative routes of administration contemplated include, but are not limited to, intranasal, transdermal, inhaled, subcutaneous and intramuscular. Effective doses can be extrapolated from dose-re- sponse curves derived from in vi tro or animal model test systems.
  • the compounds and/or compositions of the subject invention will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5Q/ED50.
  • Compounds that exhibit large therapeutic indices are preferred.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range which includes the IC 50 (the concentration of the test compound which achieves a half-maximal inhibition of activity) as determined in cell culture.
  • IC 50 the concentration of the test compound which achieves a half-maximal inhibition of activity
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • Method A Varian HPLC System-':' (Pumps: Varian ProStar Solvent Delive.ry ' System, " Model 210; Detector: Varian ProStar UV-VIS Detector, Model
  • Method B Varian HPLC System: (Pumps: Varian ProStar Solvent Delivery System,' Model 210; Detector: Rainin Dynamax Absorbance Detector, Model UV-DII; Autosampler: Varian ProStar Autosampler, Model 430) .
  • Analytical column YMC ODS-AQ,
  • Solvent A H 2 0, 0.01% HFBA, 1.0% IPA.
  • Solvent B Acetonitrile, 0.01%HFBA, 1.0% IPA.
  • Flow. Rate 2.0 mL/min.
  • Gradient Program O.OOmin 95% Solvent A, 5% Solvent B; 0.12min 95% Solvent A, 5% Solvent B; 4.00 min 5% Solvent A, 95% Solvent B; 4.18min 5% Solvent A, 95% Solvent B; 4.30min 95% Solvent A, 5% Solvent B; 5.30mih 95% Solvent A, ' 5% Solvent B.
  • Method C Varian HPLC System: (Pumps: Varian ProStar Solvent Delivery System, Model 210; Detector: Varian ProStar PDA, Model 330; Autosampler: Varian ProStar Autosampler, Model 430) .
  • Analytical column YMC ODS-AQ, .6x50mm, S3m, ' •' Waters Corporation. Detection: - 220nm and 254 ⁇ m.
  • Solvent A ,H 2 0, 0,01% HFBA, 1.0% IPA.
  • '' Solvent B Acetonitrile, 0.01%HFBA, 1.0% IPA.
  • Flow Rate 2.0 • mL/min.
  • Method D Berger SFC (Berger- Dual Pump Fluid Control Module, Model FCM-1200; Berger Thermal Control Module, Model TCM-2000; Hewlett Packard 1100 Series DAD, Model G1315A; Alcott Autosampler, Model 718AL) .
  • Method E Agilent Technologies 1100 HPLC System (Pump: QuatPump Model G1311A; Detector: DAD, Model G1315B; Column Compartment: Model G1216A; Autosampler: ALS, Model G1313A; Degasser: Model G1322A) .
  • Analytical column Chiralcel OD-RH, 150x4.6 mm. Detection: 220nm.
  • HPLC Conditions 1.0 mL/min, Isocratic, 70% (H 2 0, 0.01%TFA), 30% (ACN, 5' 0.01%TFA), 35°C.
  • TSP HPLC System (Pump: TSP ⁇ SpectraSYSTEM ® P4000; Detector: ' ⁇ TSP SpectraSYSTEM ® UV2000, 220nm and 254m; Autosampler: TSP Spectra-
  • TSP HPLC System . (Pump: TSP SpectraSYSTEM ® P4000; Detector: TSP 'SpectraSYSTEM ® UV2000, 220nm and 254m; Autosampler: TSP Spectra-
  • TSP HPLC System (Pump: TSP SpectraSYSTEM ® P4000; Detector: TSP SpectraSYSTEM ® • UV2000, 220nm and 254m; Autosampler: TSP Spectra- •• SYSTEM ® AS3000; Degasser: " TSP SpectraSYSTEM ® Model : SCMIOOO Solvent Degasser) . Detection: 220nm and
  • TSP HPLC System . (Pump: TSP Spec- ' traSYSTEM ® P2000; Detector: TSP ⁇ SpectraSYSTEM ® UV2000, 220nm and 254m; Autosampler: TSP Spectra- SYSTEM ® AS3000; Degasser: TSP SpectraSYSTEM ® Model SCMIOOO Solvent Degasser) . Detection: 220nm and 254nm. Analytical column: Zorbax Extend C18 Rapid Resolution ® , 50x4.6mm, 3.5m, 80A, Agilent Technologies.
  • Solvent A 10 mM Ammonium Acetate in H 2 0.
  • Solvent B 10 mM Ammonium Acetate in Acetonitrile, 1.0% IPA.
  • Flow Rate 2.0 mL/min.
  • Gradient Pro- gram O.OOmin 95% Solvent A, 5% Solvent B; 0.02min 95% Solvent A, 5% Solvent B; 4.00 min 5% Solvent A, • 95% Solvent B; 4.30min 5% Solvent A, 95% Solvent B; 4.50min 95% Solvent A, 5% Solvent B; 5.50min 95% 5 Solvent A, 5% Solvent B.
  • Method J Mass spectrometers: LCQTMDuo and LCQTMDeca.
  • Pump Series 1100, Quat pump model G1311A, Agilent Technologies.
  • Detector Series 1100, Column model G1216A, Agilent Technologies.
  • the aqueous filtrate was extracted with MTBE (2 L) and the crystalline solid was then combined with the basic aqueous layer. This mixture was acidified with 12 M HCl to pH 1 with stirring to produce a white solid, which was collected by filtration and washed with water. The solids were dried under high vacuum to give the indicated product as a white powder (82.7 g, 89%).
  • Example 1A A solution of Example 1A (5.43 g, 25.0 mmol) in DMF (25 L) was stirred with 4-N-Boc-amino- piperidine (7.51 g, 37.5 mmol), 1-hydroxybenzotri- azole hydrate (7.66 g, 50.0 mmol), N-methylmorph- oline (8.25 mL, 75.0 mmol), and 1- (3-dimethylamino- propyl) -3-ethylcarbodiimide hydrochloride (9.59 g, 50.0 mmol) under N for 16 h. The reaction was diluted with methylene chloride (200 mL) and water (200 mL) , and the phases were separated.
  • the organic phase was then washed with IN aqueous HCl (2x100 mL) followed by a sat. solution of NaHC0 3 (200 mL) .
  • the organic phase was dried (Na 2 S0 4 ) , filtered, and concentrated to a white solid (9.88 g, 99%) which was carried forward without purification.
  • the Boc group was removed by stirring a suspension of the white solid (9.88 g, 24.7 mmol) with methanol (20 mL) , THF (100 mL) and 4N HCL in dioxane (54 mL, 124 mmol) under N 2 for 16 h.
  • Example 2 was prepared from Example IB (54 mg, 0.18 mmol) and itaconic anhydride (17 mg, 0.15 mmol) as detailed in Example IC to provide the title compound (33 mg, 53%) as a white solid.
  • Example IB 54 mg, 0.18 mmol
  • succinic anhydride 15 mg, 0.15 mmol
  • Example IC succinic anhydride
  • the crude solid was dissolved in IN aqueous- NaOH (10 mL) and extracted with diethyl ether (3x10 mL) .
  • the aqueous ;.' phase was then acidified with 6N aqueous HCl, and extracted with methylene chloride (3x20 mL) .
  • the organic phase was dried (Na 2 S0 4 ) , filtered, and concentrated to obtain Example 3 as a white solid (22 mg, 37%).
  • Example 4A The title compound was prepared from Example 4A (1.84 mg, 3.42 mmol) according to the Boc deprotection protocol described for Example IB.
  • the product (1.27 g, 85%) was obtained as a fluffy, white powder after flash chromatographic purification (3-7% methanol in methylene chloride) .
  • Bohdan block was charged with a solution of Example 4B in chloroform (0.084 M, 1.0 mL, 0.084 mmol), and a solution of cis- 1, 2, 3, 6-tetrahydrophthalic anhydride in chloroform (0.24 M, 0.29 mL, 0.070 mmol, via Packard liquid handler) .
  • the block was shaken for 16 h at 500 RPM (Bohdan block shaker) .
  • PS-Benzaldehyde 105 mg, 0.14 mmol, via Argoscoop ® , Argonaut, Foster City, CA, USA
  • chloroform 1.0 mL
  • Example 4C was analyzed by LCMS (results in Table 1) .
  • Example 9 After synthesis according " to the procedure described for Example 4C, but omitting the purification step (digestion in toluene)', a solution of Example 9 (0.140- mmol, prepared on twice the scale) in methylene chloride (10 mL) was shaken with more of the scavenging reagent ' MP-TsOH (183 mg, 0.28 mmol)- for 2 h at 300 RPM. • The reaction was filtered rinsing the resins with methylene chloride (10x0.5 mL) . The filtrate was concentrated, and Example 9 • was analyzed by LCMS (results in Table 1) .
  • the title compound was prepared from 3- [2, 3-Dichloro-4- (2-methoxy-phenylsulfanyl) -phenyl] - acrylic acid (1.30 g, 3.66' mmol; WO 00/59880, WO 00/39081) and 3-N-Boc-aminopiperidine (880 mg, 4.39 ' mmol) according to the amide coupling protocol for Example IB.
  • the product (1.89 g, 96%) was ' obtained as a white powder and carried ' forward' to the next step without purification.
  • Example 14A The title compound was prepared from Example 14A (1.77 g, 3.29 mmol) according to the Boc deprotection protocol described for Example IB.
  • the product (1.30 g, 90%) was obtained as a fluffy, white powder after flash chromatographic purification (3-7% methanol in methylene chloride) ⁇ NMR
  • Example 14C was analyzed by LCMS (results ' in Table 1) .
  • Example 18A A solution of Example 18A (1.00 g, 3.29 mmol) in absolute EtOH (13 mL) was stirred with Pd on C (348 mg, 0.164 mmol; 10% Pd, 50% water, Degussa type) under an H 2 atmosphere for 16 h. The suspension was filtered through celite, rinsing with EtOH. The filtrate was concentrated. The resulting resi- due was triturated with diethyl ether and filtered. The resulting sticky solid was dissolved in methylene chloride and concentrated to give Example 18B as a foamy, yellow solid.
  • Pd on C 348 mg, 0.164 mmol; 10% Pd, 50% water, Degussa type
  • Example 18C A solution of Example 18C (650 mg, 1.20 mmol) in 1:1 methanol/THF (6 mL) was stirred with 2.4 M aqueous LiOH (1.0 mL, 2.4 mmol) for 2.5 h. 5 The solvent was removed in vacuo, and the residue was suspended in H 2 0 (10 mL) and extracted with ethyl acetate (10 mL) . The aqueous phase was acidified by dropwise addition of 1M aqueous HCl, and then extracted with ethyl acetate (10 mL) , dried (Na 2 S0 4 ) ,Q filtered, and concentrated to give Example 18D as an off-white solid (200 mg, 33%) .
  • Example 18B The title compound was prepared according to the procedure for Example 18B, except using the product of Example 19A in place of the product from 18A and the diethyl ether trituration was not performed.
  • MS (ESI+) m/z 233 (M+H) + .
  • the title compound was prepared from 3- [2, 3-dichloro-4- (4-fluoro-phenylsulfanyl) -phenyl-] - acrylic acid (98 mg, 0.287 mmol; synthesized in a similar manner to the acrylic acid used in Example 4A by methodology described in WO 00/59880, WO 00/39081 except that 4-fluorothiophenol was substituted for 2-methoxythiophenol) and Example 19B (100 mg, 0.431 mmol) according to amide coupling protocol described for Example IB, with the following purification step added. The product was purified by flash chromatography eluting with 3% meth- anol in methylene chloride.
  • Example 19C (87 mg, 85%) was obtained as a white foam.
  • Example 19C The title compound was prepared from Example 19C (75 mg, 0.14 mmol) according to saponifica- tion conditions described for Example 18D, except that the aqueous phase was not extracted with ethyl acetate before acidification with 1 M aqueous HCl. The resulting precipitate was filtered, washed with CH 3 CN (5 mL) , and dried under vacuum to obtain Example 19D (50 mg, 70%) as a white solid.
  • Example 20A To a solution of Example 20A (58.0- g, 245 mmol) in acetic acid (250 mL) .was- added, iron chips (3.5 g, 63 mmol). The mixture was gently heated to, dissolve Example 20A. The.- mixture was .cooled to room temperature and bromine (25. mL, 490. mmol) was added over 20 min by addition funnel. The reaction was stirred for 4 days, then more bromine (10 mL) was added in one portion -and stirring continued for 5 h. Next, a third bolus . of bromine (10 mL) was added and the mixture stirred 12 h. The mixture was then quenched by careful addition of a saturated solution of NaHS0 4 and stirred for 45 min.
  • bromine 25. mL, 490. mmol
  • Example 20B (76.7 g, . 99%) containing 90% bromination at the 4-position.
  • Example 20B To a suspension of Example 20B (50.0 g, 158 mmol) in THF (160 mL) was added 3 drops of DMF followed by oxalyl chloride (21.1 g, 166.1 mmol) over a 15 min period. The mixture was heated to
  • the solid was dried under high vacuum for 14 h to give an intermediate ketone, which was used in the next step without purification.
  • the ketone was resuspended in ethanol (500 mL, absolute) and NaBH 4 (12 g, 316 mmol) was added portionwise while cooling on an ice bath. The reaction was warmed to room temperature after addition was complete. After 2 h, 5 the reaction was transferred to a 4 L Erlenmeyer flask and quenched with 4 M aqueous HCl (caution, vigorous gas evolution) . The mixture was concentrated in vacuo, diluted with water (200 mL) , and extracted with ethyl acetate (2x500 mL) .
  • Example 20C (23. Og, 48% for 3 steps) as a red oil that solidified upon standing.
  • Example 20C 23. Og, 76.7 mmol
  • acetic acid 230 mL
  • the mixture was sonicated for 30 min.
  • BF 3 -Et 2 0 (15 mL, 115 mmol) was added dropwise over 1 min at room temperature.
  • the homogeneous mixture was diluted with water (500 mL) and a white precipitate formed.
  • the solid was collected by filtration and washed with water (200 mL) .
  • the solid was dissolved in ethyl acetate (500 L) and washed with a saturated solution of NaHC0 3 (200 mL) followed by brine (200 mL) .
  • Example 20D 9.80 g, 35.0 mmol
  • Pd 2 (dba) 3 796 mg, 0.869 mmol
  • o-(Tol)3P 809 mg, 2.66 mmol
  • the flask was purged with N 2 (3x) ,. and then charged with anhydrous DMF (70 mL) , methyl acrylate (8.98 g, 104 mmol) and triethylamine (14.5 mL, 104 mmol).
  • the reaction was purged with N 2 (2x) again.
  • the mixture was stirred under N 2 at 100 °C for 16h.
  • the reaction was cooled to room temperature and a thick precipitate formed.
  • Example 20E The suspension was partitioned between methylene chloride (300 mL) and water (100 mL) . The phases were separated, and the organic phase was washed with 0.5 N aqueous HCl (4x100 mL) . The organic phase was dried (Na 2 S0) , filtered and concentrated to obtain a beige fluffy solid (11.2 g) . The crude product was purified by partial dissolution (digestion) in hot heptanes followed by cooling to room temperature and filtration to give Example 20E (8.22g, 82%).
  • Example 20F was obtained as an off-white powder (6.60 g, 84%) after recrystallization from hot toluene.
  • Example 20F (2.00 g, 7.32 mmol) was reacted with 4-N-Boc-amino-piperidine (2.20 g, 11.0 mmol) according to the amide coupling protocol described for Example IB.
  • Example 20G was obtained as an orange-colored solid (3.34 g, 100%) which was carried forward without purification.
  • Example 20G (4.52 g, 9.93 mmol) according to the procedure described for Example IB.
  • Example 2OH was obtained as a waxy, creme-colored solid (3.10 g, 88%).
  • Example . 20J (152 mg, 53% of theoretical recovery) was obtained as a white powder. : ⁇ H NMR (DMS0-d 6 , 400 MHz)
  • Example 2OK -Benzyl-3-propionyl-oxazolidin-2-one Adapted from Levy, D.E. et al., J. Med. Chem . 1998, 41, 199-223.
  • (R) -4-benzyl-oxazolidin-2-one 30.0 g, 169.3 mmol
  • THF 300mL, 99.9% anhy- drous
  • the flask was cooled to -78°C (dry ice/acetone).
  • n-BuLi 70 mL, 169 mmol, 2.41 M in hexanes
  • Bromo-acetic acid tert-butyl ester (67.7 g, 347 mmol) was added dropwise over 20 min. Stirring was continued for, 30min at -78 °C. The mixture was warmed to 0°C by placing the flask in an ice water bath for 20 min. The mixture was quenched by pouring into a 1 L separatory funnel containing saturated NH 4 C1 (200 mL) . The mixture was extracted with EtOAc (300 mL) , the phases were separated and the organic was washed with IN aqueous HCl (100 mL) , saturated NaHC0 3 (100 ⁇ mL) , and brine (100 mL) .
  • the organic was dried (Na 2 S0) , filtered, and concentrated.
  • the crude product was purified using a Biotage Flash 65 column. The column was wetted with solvent (9:1 hexane/MTBE; 1 L) and wet-mounted with the crude (dissolved in 50 mL DCM; two columns were performed to purify all of the product) .
  • the title compound was obtained as a white, crystalline solid (29.1 g, 65%), and was pure by HPLC, LCMS and X H NMR.
  • Example 20L (29.0 g, 83.5 mmol) and a solution of 4:1 THF/water (300 mL) .
  • the mixture was cooled to 0°C (ice water bath) and 30% aqueous H 2 0 2 (17.0 g, 501 mmol) was added dropwise over 10 min while stirring.
  • An aqueous solution of LiOH (2 M, 4.73 g, 117 mmol) was added, and the reaction was stirred for 4 h in the ice bath.
  • the reaction was quenched by careful addition of Na 2 S0 3 (2.73 M, 14.7 g, 117 mmol).
  • the THF was removed in vacuo and the resulting suspension was extracted with DCM (400 mL) .
  • the aqueous phase was reserved (contains product) and the DCM phase was washed with 0.1 N aqueous NaOH (100 mL) .
  • the DCM was dried (Na 2 S0 4 ) , filtered and concentrated in vacuo to afford the chiral auxiliary (reusable) .
  • the aqueous phase was cooled in an ice bath and acidified with 2 N aqueous HCl to pH 4.
  • the cloudy solution was extracted with EtOAc (300mL) , dried (Na 2 S0 4 ) , filtered and concentrated to give the product as a colorless oil, which solidified to a white solid upon standing.
  • Example 20J 150 mg, 0.23 mmol
  • methylene chloride 5 L
  • piperidine 115 ⁇ L, 1.16 mmol
  • the solution was washed with IN aqueous HCl (2x10 mL) , dried (Na 2 S0 4 ) , filtered, and concentrated.
  • the crude product was purified by Biotage Flash 40 elut- ng with 10% methanol in methylene chloride (200 mL) to elute the 9H-fluoren-9-yl by-products, followed by 40% methanol in methylene chloride (800 mL) to elute the title compound 20N (69 mg, 63%), which was obtained as a white powder in 98% regioisomeric pur- ity.
  • the title compound can be made from Example 2OH and Example 20M as described in Example IB followed by deprotection of the tert- butyl ester with TFA according to the method described in Example 23E.
  • Example 21 N- ⁇ 1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl ⁇ - (S) -2-methyl- succinamic acid
  • Example 21 (regioisomer of Example 20N) was prepared in greater than 90% regioisomeric purity according to the same reaction protocol described for Example 20N.
  • Example 2OH 89 mg, 0.25 mmol
  • (R) -methyl succinic anhydride 29 mg, 0.25 mmol; prepared from (R) -2-methyl succinic acid according to the literature: Davies, S. G. ; Dixon, D. J. J. Chem . Soc . f Perkin Trans . 1 1998, 2635- 2643) as described in the synthesis of Example 201.
  • Example 22 105 mg, 89%) was obtained as a beige solid.
  • Example 23A has also been prepared by esterification of Boc- ⁇ -alanine with methanol (Hayashida, Osamu, et al, J. Org. Chem . 2002, 67, 8291-8298) .
  • the reaction was' stirred for 5 min, and then removed from the -78 °C bath and immediately ⁇ submerged in an ice water bath. The mixture was stirred for 10 min (note: leaving the reaction for longer periods of time results in- poor yields), and then poured into” a saturated solution of NHC1 (300 mL) . The mixture was shaken, and then diluted with ethyl acetate (700 mL) . The phases were shaken again, and then separated. The organic phase was • washed with IN aqueous HCl (2x100 mL) , a, saturated ' solution of NaHC0 3 (200 mL) , and brine (200 mL) .
  • Example 23C rac-2- (tert-Butoxycarbonylamino- methyl) -succinic acid 4-tert-butyl ester
  • the title compound was prepared from Example 23B (7.56 g, 23.8 mmol) according to the procedure described for Example 18D, except the amount of lithium hydroxide hydrate was reduced (1.50 g, 35.7 mmol) to 1.5 equivalents.
  • Example 23C (6.78 g, 94%) was obtained as a pale yellow oil, which solidified upon standing.
  • Example 23C 910 mg, 3.00 mmol
  • Example 20H 888 mg
  • Example 23D (1.03 g, 64%) was obtained as a white powder after purification using a Biotage Flash 40 chromatography system, eluting with the following gradient: 1:1 ethyl
  • Example 23D A solution of Example 23D (1.00 g, 1.56 mmol) in methylene chloride (20 mL) was stirred with trifluoroacetic acid (2.4 mL, 31 mmol) under N 2 for 16 h at room temperature. The reaction was then concentrated in vacuo . The residue was resuspended in methylene chloride (10, mL), and the solvent was concentrated again.- This process was repeated (3x) in order to remove residual trifluoroacetic acid. The crude product was triturated with diethyl ether (10 mL) , and the resulting white solid was filtered. Example 23E (890 mg, 95%) was obtained as a white powder.
  • Example 23E To a stirred suspension of Example 23E (300 mg, 0.50 mmol) and diisopropylethylamine (0.35 mL, 2.0 mmol) in methylene chloride (10 mL) at 0°C (ice bath) was added benzoyl chloride (0.24 M solu- tion in chloroform, 2.1 mL, 0.50 mmol) dropwise. After addition was complete, the reaction was stirred for 30 min at 0°C. The reaction was then quenched by addition of 1 N aqueous HCl (5 mL) . The heterogeneous mixture was extracted with 20% meth- anol in methylene chloride (30 mL) .
  • the organic phase was washed with IN aqueous HCl (3x20 mL) . Before each aqueous extraction, methanol (5 mL) had to be added to maintain two homogeneous phases. The ' organic phase was dried (Na 2 S0 4 ) , filtered, and concentrated. The crude product was purified by partial dissolution (or digestion) in hot acetonitrile (10 L) followed by cooling to room temperature. The resulting white solid.was filtered, washing with acetonitrile (3x2 mL) . A second digestion, in ethyl- acetate, was performed to raise the purity above 95%. The powder was dried (CaS0 4 dessicator) under high vacuum for 2 days.
  • Example 24 (135 mg, 46%) was obtained as a white powder.
  • Example 23E 150 mg, 0.25 mmol
  • 3-m.ethoxybenzoyl chloride (0.24 M solution in chloroform, 1.0 mL, , 0.24 mmol) according to the procedure described by Example 24.
  • Example 25 99 mg, 64.7% was obtained as a white powder after purification by partial dis-:- solution (or digestion) in hot acetonitrile (10 mL) , followed by cooling to room temperature and filtra- tion.
  • X H NMR DMSO-d 6 , 400 MHz ⁇ 12.1 (bs, IH) ,
  • Example 23E 150 mg, 0.25 mmol
  • a'nd 3-Chlorobenzoyl chloride (0.24 M solution in chloroform, 1.0 mL, 0.24 mmol) according to the procedure described- by Example 24.
  • Example 26 (93 mg, 60%) was obtained as a white powder after purification' by partial dis- solution (or digestion) in hot acetonitrile (10 mL) followed by cooling to room temperature and filtration.
  • Example 2 The title compound was prepared from Exam- pie 23E (150 mg, 0.25 mmol) and phenyl isocyanate (0.24 M solution in chloroform, 1.0 mL, 0.24 mmol) according to the procedure described by Example 24, except the amount of diisopropylethylamine (0.11 mL, 0.63 mmol) was reduced to 2.5 equivalents.
  • Example, 27 (89 mg, 59%) was obtained as a white powder after purification by partial dissolution (or digestion) in hot acetonitrile (10 mL) followed by cooling to room temperature and filtration.
  • Example 28A Bromine (0.79 mL,.- 15 mmol) was added drop- wise to a stirred solution of Example 28A (2.97 g, 15.4 mmol) in CH 2 C1 2 (50 mL) at 0°C. The resulting mixture was stirred at room temperature overnight.
  • the title compound 28D (774mg, 79%) was prepared from Example 28C by the method described in Example 20F.
  • Example 28D 100 mg, 0.380 mmol, H0Bt.H 2 0 (87 mg, 0.570 mmol), NMM (0.105 mL, 0.950 mmol) and azetidin-3-yl-carbamic acid tert- butyl ester (79 mg, 0.456 mmol), in dry DMF (2 mL) was treated with EDCI (109 mg, 0.570 mmol) at 0°C and allowed to stir at ambient temperature under N 2 atmosphere for 18 h. The reaction mixture was diluted with warm 2% methanol/EtOAc (40 mL) , washed with water (2x10 mL) and brine (1x10 mL) .
  • Example 28E 120 mg, 0.288 mmol
  • CH 2 C1 2 1 mL
  • 4N HCl in dioxane 4 mL
  • the reaction was monitored by HPLC at 1 h intervals. After 2 hours the solvent was removed under reduced pressure and dried under vacuum for 5 h to provide the HCl salt of the amine (110 mg, 108.6%) as a white solid.
  • Example 20F 100 mg, 0.366 mmol
  • azetidin-3-yl-carbamic acid tert-butyl ester 76 mg, 0.439 mmol
  • Example 23B The title compound was prepared by the procedures described in Example 23B, substituting Example 23A with phenyl-acetic acid methyl ester.
  • Example 23C The title compound was prepared by the procedures described in Example 23C, substituting Example 23B with Example 31A.
  • Example 23E The title ' compound was prepared by the procedures described in Example 23E, substituting Example 23D with Example 33E.
  • Examples 34-118 were prepared by procedures described in Examples 25 or 26.
  • these compounds can be made via solution phase method in solvents such as chloroform or tetrahydrofuran, with the following reagents: PS-DIEA, PS-DMAP, and corresponding acyl chlorides or isocyanates, by shaking in a 24-well format Bohdan mini-block at 550 rpm for 16 hrs.
  • the work- up consisted of rinsing with DMF and/or ' THF.
  • the crude solid was purified by preparative HPLC (Methods F or J) .
  • LCMS Methodhod I
  • the title compound was prepared by the procedures described in Example 119A, substituting 4-bromoisoquinoline with 3-bromothianaphthalene.
  • the crude product was purified by flash column chromatography (1:4 EtOAc/Hexanes) to give the title compound as a brown oil .
  • a soluble form of the ⁇ l ⁇ l heterodimer (sVLA-1-LZ) was generated by truncating each chain of the heterodimer at the beginning of the transmem- rane region and adding an acidic and basic leucine zipper sequence to the ⁇ l and ⁇ l chains, respective- y.
  • the ⁇ l chain (described in U.S. Patent Applica- ion Publication No. 2003/0088061) was truncated after residue P1141 of the pro-peptide and the 47 amino acid acidic leucine zipper cassette was added.
  • the ⁇ l chain (Genbank accession no. P05556) was truncated after residue D728 of the pro-peptide and the 47 amino acid basic leucine zipper cassette was added.
  • the purified protein was stored at -70°C.
  • ⁇ l-LZ construct which has the extracellular domain of ⁇ l fused to a C-terminal leucine zipper sequence.
  • the extracellular domain of ⁇ l was amplified using standard PCR methods and reagents in order to add restriction sites for the subcloning.
  • the sequences of the primers used in the PCR reaction were:
  • 3' primer ⁇ l-04: ATT ACG CGT TGG CAC TCT GCC CGG
  • the primers above were used in a PCR reaction with an ⁇ l cDNA clone (described previously, U.S. Patent Application Publication No. 2003/0088061) .
  • the resulting PCR product was sub- cloned 5' to the acidic leucine zipper sequence in the mammalian expression vector pDEF38 (described in U.S. Patent Application Publication No. 2003/0088061) .
  • the resulting plasmid was verified by sequencing.
  • the extracellular domain of ⁇ l likewise was amplified using standard PCR methods and reagents in order to add restriction sites for the subcloning.
  • sequences of the primers used in the PCR reaction were: 5' primer: ⁇ l-02: ATT CTC GAG ACC GCC ACC ATG AAT TTA CAA CCA ATT TTC TGG (SEQ. ID. 3) 3' primer: ⁇ l-03: GTT CCA TTC ACC CCG TTC TTG C (SEQ. ID. 4)
  • the 5' end of the ⁇ -1 insert was generated by PCR from a ⁇ l cDNA and subcloned 5' to the basic leucine zipper sequence in the mammalian expression.
  • vector pNEF38 (described in U.S. Patent Application Publication No. 2003/00.88061) . The; resulting plasmid was verified by sequencing.
  • VNIQKKNCHM EGKETVCINA TVCFDVKLKS KEDTIYEADL QYRVTLDSLR QISRSFFSGT QERKVQRNIT VRKSECTKHS FYMLDKHDFQ DSVRITLDFN LTDPENGPVL DDSLPNSVHE YIPFAKDCGN KEKCISDLSL HVATTEKDLL IVRSQNDKFN VSLTVKNTKD SAYNTRTIVH YSPNLVFSGI EAIQKDSCES NHNITCKVGY PFLRRGEMVT FKILFQFNTS YLMENVTIYL SATSDSEEPP ETLSDNVVNI SIPVKYEVGL QFYSSASEYH ISIAANETVP EVINSTEDIG NEINIFYLIR KSGSFPMPEL
  • Beta 1 Leucine Zipper (basic) construct protein sequence :
  • Dilution Buffer alone or in combination with, anti- ⁇ l mAb (Immunodiagnostic, #8149a, 2.5 ⁇ g/mL), 10 mM EDTA, DMSO or DMSO+2X inhibitor was added to the wells of the plate, followed by 50 ⁇ L/well Collagen IV-biotin at 2 ⁇ g/mL in Dilution buffer.
  • Collagen IV-biotin was generated by biotinylating human Collagen IV (Sigma-Aldrich, Milwaukee, WI, USA) using a biotin labeling kit (Pierce Biotechnology, Rockford, IL, USA) following the manufacturer's0 protocol.
  • the plates were incubated for 1 hour at room temperature, and washed four times with Wash Buffer (300 ⁇ L/well) .
  • the plates were then incubated with 100 ⁇ L/well of 1:1000 diluted (with H 2 0) Strepavidin-Europium (PerkinElmer, Boston, MA, USA) 5 for 30 min. at room temperature.
  • the plates were ⁇ :... then washed four times with Wash -Buffer (300 ⁇ L/- well) .
  • One hundred ⁇ L/well of Delphia- Enhancement Solution (PerkinElmer; diluted 1:1 with. dH 2 0) was added, and the plates were shaken for 5 minutes.
  • Binding was -then analyzed, by time resolve fluorescence (TRF) using a Victor Plate-reader (Perkin- -; Elmer). Results were analyzed using the equations i..,- below. The percent of inhibition was plotted versus-. • ' .. the log concentration of .inhibitor across a twelve 15;-,. point titration, and a linear regression trend-line was drawn.
  • TRF signal Collagen-IV-biotin in

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Abstract

Compounds of formula (I) that are VLA-1 integrin antagonists are disclosed. Also disclosed are compositions containing such compounds and methods of using such compounds in treating diseases mediated, at least in part, by the VLA-1 integrin.

Description

AMINOPIPERIDINE AMIDE DERIVATIVES AS VLA-1 INTEGRIN ANTAGONISTS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of provisional U.S. patent application Serial No. 60/495,757, filed August 14, 2003.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
This invention relates to compounds which are VLA-1 integrin antagonists. This invention also relates to compositions containing such compounds and methods of treatment using such compounds in treating diseases mediated, at least in part, by the VLA-1 integrin.
STATE OF THE ART
Integrins are heterodimeric cell surface proteins composed of two noncovalently linked polypeptide chains, a. and β. Integrins are the major receptor for cell adhesion to extracellular matrix and play important roles in certain cell-cell and cell-matrix adhesion events. These integrin-medi- ated adhesion events are critical for both normal and pathophysiological processes during cell activation, migration, proliferation and differentiation (for reviews see Hynes (1992) Cell 69: 11 ; Springer (1994) Cell 75:301; Hynes (2002) Cell 110: 613 ) . VLA-1 (very late antigen-1) is an integrin heterodimer composed of an alpha chain (CD49a, αl) and a beta chain (CD29, βl) . VLA-1 is one member of a family of four βl integrin molecules that have been shown to bind to the extracellular matrix proteins, collagen and laminin. The βl integrin collagen receptors include αlβl (VLA-1), α2βl (VLA-2), αlOβl and αllβl. These four collagen receptors share overlapping but distinct expression profiles. They also appear to have distinct ligand preferences in vitro (Tulla et al., (2001) J. Biol . Chem. 52:48206). For example, αlβl has been shown to bind more effectively to type IV collagen than type I collagen while α2βl binds to type I collagen better than to type IV collagen (Dickeson et al., (1999) J. Biol . Chem. 274:32182).
VLA-1 is expressed on smooth muscle cells, microvascular endothelial cells, fibroblasts, osteo- blasts and chondrocytes. In addition, VLA-1 is also expressed on activated cells of the immune system including effector T cells, macrophages, and NK cells (de Fougerolles et al., (2000) J. Clin . Invest . 105: 121 ) ; however, it does not appear to be expressed on B cells or neutrophils. VLA-1 is ex- pressed on T cells in various disease states including in the joints of arthritis patients (HemLer et al., (1986) J. Clin . Invest . 78:696), lesions of giant cell arteritis patients (Schaufelberger et al., (1993) Clin . Exp. Immunol . 92:421), arterio- sclerotic plaques, thyroid infiltrates of patients with autoimmune thyroid disease (Paolieri et al., (1992) J. Endocrinol . Invest . 25:63) and lungs of chronic bronchitis (Saetta et al., (1993) Am . Rev. Respir. Dis . 247:301) , sarcoidosis (reviewed in HemLer (1990) Annu . Rev. Immunol . 8 : 365 ) and asthma patients (Corrigan et al., (1991) Int . Arch . Allergy Appl . Immunol . 94 : 210 ) .
Despite this broad expression profile, αl null mice- generated by homologous recombination are viable and fertile and have no overt phenotype, demonstrating that the molecule is not required for development (Gardner et al., (1996) Dev. Biol . 275:301). In addition, no increased incidence of infection was noted in the mutant mice. While embryonic fibroblasts derived from mutant animals show a striking absence of adhesion to collagen IV, they show no deficit in adhesion to collagen I. Despite the absence of an overt phenotype, no compensatory upregulation of other collagen binding receptors could be identified, suggesting instead that VLA-1 may have redundant roles during development.
Together these data suggest that inhibiting VLA-1 function should be nontoxic.
Inhibiting VLA-1 function using αl null mice and/or blocking anti-αl antibodies has shown efficacy either prophylactically or therapeutically or both in several animal models of inflammatory disease including 1) delayed-type hypersensitivity as a model of general inflammatory disease (de Fougerolles et al., (2000) J. Clin . Invest . 105: 121 ) ; 2) contact hypersensitivity as a model for skin allergic reactions (A.R. de Fougerolles et al., 2000, J. Clin . Invest . , 105: 121 ) ; 3) anticollagen mAb-induced arthritis as a model of rheumatoid arthritis (de Fougerolles et al., (2000) J. Clin . Invest . 105: 121 ) ; and 4) TNBS- and DSS-induced colitis as models of inflammatory bowel disease (Fiorucci et al., (2002) Immunity 1 7: 169 ; Kriegl- stein et al., (2002) J. Clin . Invest. 110: 1113) .
The mechanism for the reduction in inflammation seen with the blocking anti-αl mAb and in αl null mice results from multiple effects. VLA-1 has been shown to mediate adhesion to and migration across collagen matrix. Therefore, VLA-1 expression may be critical for allowing the effector cells to enter the site of inflammation. mAbs against αl have also been shown to block collagen-induced cytokine release, including release of TNF-α, a key mediator in arthritis (Miyake et al . , (1994) Eur. J. Immunol . 24 : 2000) . In addition, VLA-1 also regulates matrix metalloproteinase (MMP) expression (Gardner et al . , (1996) Dev. Biol . 175: 301 ; Pozzi et al. (2000) Proc. Natl . Acad. Sci . USA 97:2202; Pozzi et al., (2002) Oncogene 21:272; Lochter et al., (1999) Mol Biol Cell 10: 211 ) . Therefore, inhibiting VLA-1 provides a way of reducing inflammation through the synergistic action of a variety of mechanisms (i.e., VLA-1 is an upstream regulator of multiple disease promoting factors) .
Fibrosis is a common response to chronic injury and represents a paradigm for the cycle of parenchymal wound healing in a variety of tissues (reviewed in Bataller et al. (2001) Semin . Liver Dis . 21 : 431 ; Bissell. (1998) J. Gastroenterol . 33 : 295 ) . When overactive, this wound healing process can result in pathologic tissue scarring, which results from the progression of several defined steps. First, an infiltrate, consisting of inflammatory cells and platelets and resident " yofibro- blasts" (identified as hepatic stellate cells in the liver and differentiated mesangial cells in the kidney) , accumulates at the site of injury. Second, the local extracellular matrix (ECM) is altered by de novo production of collagen by the myofibro- blasts. Third, the myofibroblasts migrate and align within the wound site and proliferate. Finally, the myofibroblasts contract the collagen, forming the fibrotic scar which contributes to tissue dysfunction. It is generally believed that a similar process results in scarring within tissues of the liver, kidney, lung, and skin.
VLA-1 is expressed on myofibroblasts in vitro and in vivo and is believed to regulate their pathologic functions. Alports syndrome is a genetic disorder characterized by progressive glomeruloneph- ritis resulting in fibrosis of the kidneys and ultimately kidney failure. Alports syndrome affects approximately 1 in 5000 people and is caused by mutations in the type IV collagen genes. This condition has been mimicked in mice by knocking out the gene of the α3 chain of type IV collagen (Alport mouse) . Double knockout mice for both type IV collagen and αl integrin have a delayed onset and slowed progression of glomerular disease (Cosgrove et al., (2000) Am . J. Pa thol . 257:1649). In addition, inhibition of TGF-βl with a soluble receptor construct had a synergistic effect with the inacti- vation of αl, slowing the onset and severity of glomerular disease. These results correlated with a dramatic decrease in the accumulation of myofibroblasts and macrophages in the tubular interstitium of double knockout mice (Sampson et al., (2001) J. Biol . Chem . 275:34182) . Another report studying the effects of αl expression in transfected glomerular mesangial cells showed that VLA-1 expression levels influenced the cell growth, cell size and collagen matrix remodeling ability of these cells (Kaga i et al., (2000) Kidney Int . 58:1088). In addition, αl mAb blocks hepatic stellate cell' adhesion to collagen and endothelin-stimulated hepatic stellate cell- mediated contraction of collagen lattices in vi tro, and VLA-1 is the sole integrin utilized by contracting hepatic stellate cells in vivo (Racine Sampson et al., (1997) J. Biol . Chem . 272:30911). Furthermore, blocking anti-αl antibody has shown efficacy therapeutically in two independent models of fibrotic kidney disease (Kagami et al., (2002) Lab. Invest . 82 : 1219 ; Cook et al . , (2002) Am . J. Pathol . 252:1265).
VLA-1 may also play a role in regulation of tumor vascularization (angiogenesis) and tumor cell metastasis in many forms of cancer. For example, VLA-1 may regulate tumor angiogenesis by two distinct mechanisms: 1) by regulating the proliferation potential of the vascular endothelial fibro- blasts (Pozzi et al., (1998) J. Cell . Biol . 142 : 581 ; Senger et al . , (2002) Am . J. Pa thol . 160: 195 ) , and 2) by regulating the production of matrix metallo- proteinase 9 which in turn regulates the activity of angiostatin, a potent angiogenesis inhibitor (Pozzi et al., (2000) PNAS 97:2202; Pozzi et al., (2002) Oncogene 22:272) . Furthermore, the metastatic potential of malignant melanoma cell lines correlates with the expression level of VLA-1 (Schadendorf et al., (1996) Br. J. Cancer, 74:194), and blocking VLA-1 binding inhibits tumor cell invasion across reconstituted basement membrane through inhibition of matrix metalloproteinase 3 (stromelysin-1) expression (Lochter et al., (1999) Mol . Biol . Cell, 10: 211 ) .
Currently, there are only two descriptions for VLA-1 inhibitors in the patent literature, and both describe large molecular weight polypeptides. The first is a mAb to VLA-1 (WO 02/083854-A2) and the second is a disintegrin isolated from cobra venom (WO 02/22571-A2) . Therefore, there still exists a need in the art for low molecular weight antagonists, specific inhibitors of VLA-1-dependent cell adhesion that have improved pharmacokinetic and pharmacodynamic properties such as oral bioavail- ability and significant duration of action. Such compounds would prove to be useful for the treatment, prevention or suppression of various pathologies mediated by VLA-1 binding and cellular adhe- sion, migration, activation or differentiation. SUMMARY OF THE INVENTION
The present invention provides aminopiper- idine amide compounds which are antagonists to the VLA-1 integrin. In one of its composition aspects, this invention is directed to a compound of Formula I:
Figure imgf000009_0001
wherein A and B, together with the nitrogen atom bound thereto, form a 4-8 membered nitrogen containing heterocyclic group containing 1 to 2 nitrogen atoms, wherein said heterocyclic group may be optionally substituted with 1 to 3 additional substituents each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cyclo- alkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, hydroxy, alkoxy, thioalkyl, and halo, wherein the one or more alkyl and sub- stituted alkyl substituents, if present, may be attached to either a carbon or a nitrogen atom in said heterocyclic group, wherein the one or more hydroxy, alkoxy, alkylsulfanyl and halo substituents, if present, may not be attached to a nitrogen atom in said heterocyclic group, and wherein the one or more hydroxy, alkoxy, and halo substituents, if present, may not be attached to a carbon atom which is adjacent to a nitrogen atom in said heterocyclic group, and further wherein A together with the nitrogen atom bound thereto form a 4-8 membered heterocyclic group containing two nitrogen atoms, then the two nitrogen atoms are either adjacent to each other, or are separated by at least two carbon atoms,
R1 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
R2 and R3 are independently selected from the group consisting of hydrogen, fluoroalkyl, and alkyl;
R4 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heterocyclic, and cycloalkyl;
R6 and R7 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, amino, substituted amino, aminocar- bonyloxy, aminoacyl, aminosulfonyl, sulfonylamino, acylamino, aminoacylamino, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, aryl, substituted aryl, ORa, acyloxy, oxycarbonyl- amino, thioalkyl, thioaryl, thioalkylaryl, thioalk- ylheteroaryl, NHS02NRaRa, SC(0)Ra, and SC(0)NRa 2, or R6 and R7, together with the carbon atom bound thereto, form a cycloalkyl, substituted cycloalkyl, heterocyclic, or a substituted heterocyclic group; R8 is selected from the group consisting of
CRaRaC(0)ORa, C02Ra, C(0)NRa 2, C(0)NRaORa, C(0)NHS02Ra, CRaR heteroaryl (e.g., tetrazolyl) , CRaRa substituted heteroaryl, CN, and (CRa 2)pOH, wherein p is 0, 1 or 2; R9 and R10 are independently hydrogen, halo, alkyl, substituted alkyl, amino, substituted amino, aminocarbonyloxy, aminoacyl, acylamino, aminoacylamino, aminosulfonyl, sulfonylamino, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, aryl, substituted aryl, -ORa, acyloxy, oxycarbonylamino, thioalkyl, thioaryl, thioalkylaryl, thioalkylheteroaryl, NHS02NRa Ra, SC(0)Ra, and -SC(0)NRa 2, or R7-C-C-R9 can form a cycloalkylene, cycloalkenylene, heterocyclene, or heterocyclenylene group, or R9 and R10, together with the carbon atom attached thereto, form a >C=0 (oxo) group, with the proviso that when R9 and R10 form an oxo group, R8 is not -CN, or R9 and R10, or R6 and R7, together with the carbon atom attached thereto, form a vinyl group of the formula >C=CR1:LR12 where R11 and R12 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, or R9-C-R10, together with the carbon atom attached thereto, form a group selected from the group consisting of cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic group; wherein each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substi- tuted heterocyclic, and, in the case of -NRa 2, each Ra, together with the nitrogen atom bound thereto, can form a heterocyclic or substituted heterocyclic - group or in the case of CRaRaC (0) 0Ra, the two R can. be an oxo group, with the proviso that when the two Ra are an oxo group, then • R9 and R10 are not an oxo group; and pharmaceutically acceptable salts, prodrugs, or tautomers thereof.
In a preferred embodiment are compounds of ■ Formula I exhibiting a biological activity of at least fifty percent inhibition of VLA-1 when tested at a concentration of 50 μM. In a more preferred embodiment are compounds of Formula I exhibiting a biological activity of at least fifty percent inhi- bition VLA-1 when tested at a concentration of 40 μM. In a still more preferred embodiment are compounds of Formula I exhibiting a biological activity of at least fifty percent inhibition VLA-1 when tested at a concentration of 20 μM. In a preferred embodiment of this invention, A and B, together with the nitrogen atom bound thereto, preferably form a piperidine, pyrrolidine, or azetidine ring.
In another preferred embodiment, R1 is selected from the group consisting of aryl, substi- tuted aryl, heteroaryl, and substituted heteroaryl, and even more preferably R1 is selected from the group consisting of substituted aryl and substituted ■ heteroaryl'.
Examples of particularly preferred R1 groups are selected from the group consisting of:
2, 3-dichlorophenyl;
2, 3-dichloro-4- (2-methoxyphenylthio) - phenyl; ■
2, 3-dichloro-4- (4-fluorophenylthio) phenyl; 4-methylthio-2, 3-dichlorophenyl; and
2, 3-dichlorobenzo [b] thiophen-4-yl .
In another preferred embodiment, R2, R3, R4, and R6 are hydrogen.
In yet another preferred embodiment, R7 is selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, substituted alkyl- ene, aryl, and heteroaryl. Particularly preferred R7 groups are selected from the group consisting of: hydrogen; fluoro; methyl; aminomethyl; phenylamidomethylene;
(2-methoxyphenyl) amidomethylene; (2-chlorophenyl) amidomethylene; phenyl; pyridin-3-yl; quinolin-3-yl; phenylaminocarbonylaminomethylene; propylamidomethylene; methoxymethyleneamidomethylene; t-butyl-amidomethylene; methylthioethyleneamidomethylene; t-butyl-methyleneamidomethylene; cyclopropylamidomethylene; cyclopentylamidomethylene; cyclohexylamidomethylene;
4-N-acetylpiperidinylamidomethylene; cyclopentylmethylenamidomethylene; piperidinyl-N-ethyleneamidomethylene; 2-methoxyphenylamidomethylene; i-propylamidomethylene; cyclobutylamidomethylene;
2-pyridynylamidomethylene;
3-pyridinylamidomethylene; 4-pyridinylamidomethylene;
2-thiophenylamidomethylene;
2-furanoylamidomethylene; benzo [1,3] -dioxole-5-amidomethylene;
3-methoxyphenylmethyleneamidomethylene; 4-methoxyphenylamidomethylene;
2-thiophenylmethyleneamidomethylene; cyclopropyl-1-phenyl-l-amidomethylene; phenylethyleneamidomethylene; phenyloxymethyleneamidomethylene; 4-chlorophenyloxy-dimethylmethyleneamido-ethylene; cyclopentyl-1- (4-chlorophenyl) -1- amidomethylene;
2-phenylcyclopropaneamidomethylene; phenylmethyleneoxymethyleneamidomethylene; methylaminoamidomethylene; morpholinylamidomethylene; phenylmethyleneaminoamidomethylene; phenylethyleneaminoamidomethylene; ' methoxyamidomethylene; phenylmethyleneoxyamidomethylene;
R-phenylamido; ;
S-phenylamido; methylamido; propylamido; methoxymethyleneamido; t-butyl-amido; ' thiomethylethyleneamido; t-butylmethyleneamido; cyclopropylamido; cyclopentylamido; cyclohexylamido;
4-N-acetylipiperidinylamido; cyclopentylmethylene amido; piperidynyl-N-ethylene amido; 3-methoxyphenylamido;
4-methoxyphenylamido; i-propylamido;
3-chlorophenylamido;
4-chlorophenylamido; cyclobutylamido;
2-pyridinylamido; 3-pyridinylamido;
4-pyridinylamido;
2-thiophenylamido;
2-furanoylamido; 5-isoxazolylamido;
4-biphenylamido; phenylmethylene amido;
4-chlorophenylmethylene amido;
3-methoxyphenylmethyleneamido; 4-methoxyphenylmethyleneamido;
2-thiophenemethyleneamido; cyclopropyl-1-phenyl-l-amido; phenylethyleneamido; phenyloxymethylenea ido; 4-chlorophenyloxy-l, 1-dimethylmethylene- amido; phenylethyl-ene-amido; phenylmethoxyloxymethyleneamido; methylaminoamido; dimethylaminoamido; morpholine-N-amido; phenylmethyleneaminoamido; phenylethyleneaminoamido; methoxyamido; phenylmethyleneoxyamido; phenylsulfonamido;
3-methoxyphenylamido; and
3-chlorophenylamido .
Preferred R8 groups are selected from the groups consisting of CRaRaC (0) ORa, -C02Ra, where Ra is as defined above, and, when R9 and R10 form an oxo group, then R8 is preferably hydroxy.
Particularly preferred R8 groups are selected from the group consisting of: carboxyl;
-CH(C6H4)C00H;
-CH2COOH; and when R9 and R10 form an oxo group, hydroxy.
Preferred R9 groups are selected from the group consisting of hydrogen, alkyl, aminoacyl, acylamino, and aryl.
Particularly preferred R9 groups are selected from the group consisting of: methyl; hydrogen; phenyl; and
-NHC(0)CF3.
Preferred R10 groups are selected from the group consisting of hydrogen and alkyl. Particular- ly preferred R10 groups are selected from the group consisting of methyl and hydrogen.
In other preferred embodiments, R7 and R9, together with the carbon atoms bound thereto form a cycloalkylene or cycloalkenylene group such as, for example, cyclopropylene or cyclohexenylene; R9 and
R10, together with the carbon atom bound thereto form an oxo group [>C(0)] or a vinyl group of the formula >C=CR11R12 where R11 and R12 are as defined above such as, for example, >C=CH2. Aminopiperidine amide derivatives within the scope of this invention are exemplified by those set forth in Tables I, IT, and III as follows:
Figure imgf000018_0001
Figure imgf000019_0001
Φ=p enyl
Additional compounds are those having the structure set forth in Table 1, wherein R1 is 2,3- dichlorobenzo[b] thiophen-4-yl; R9 and R10 are H; R8 is -C(0)0H; and R7 is propylamidomethylene; methoxymethylenea idomethylene; t-butyl-amidomethylene; methylthioethyleleneamido ethylene; t-butyl-methyleneamidomethylene; cyclopropyla idomethylene; cyclopentylamidomethylene; cyclohexylamidomethylene;
4-N-acetylpiperidinylamido ethylene; cyclopentylmethylenamidomethylene; piperidinyl-N-ethyleneamidomethylene;
2-methoxyphenylamidomethylene; i-propylamidomethylene; cyclobutylamidomethylene; 2-pyridynylamidomethylene;
3-pyridinylamidomethylene;
4-pyridinylamidomethylene;
2-thiophenylamidomethylene;
2-furanoylamidomethylerie; benzo [1, 3] -dioxole-5-amidomethylene;
3-methoxyphenylmethyleneamidomethylene;
4-methoxyphenylamidomethylene;
2-thiophenylmethyleneamidomethylene; • cyclopropyl-1-phenyl-l-amidomethylene; phenylethyleneamidomethylene; phenyloxymethyleneamidomethylene;
4-chlorophenyloxy-dimethylmethyleneamido- methylene; cyclopentyl-1- (4-chlorόphenyl) -1-amido- ' methylene;
2-phenylcyclopropaneamidomethylene; phenylmethyleneoxymethyleneamidomethylene; methylaminoamidomethylene; morpholinylamidomethylene; phenylmethyleneaminoamidomethylene; phenylethyleneaminoamidomethylene; methoxyamidomethylene; phenylmethyleneoxyamidomethylene;
R-phenylamido; S-phenylamido; methylamido; propylamido; methoxymethyleneamido; t-butyl-amido; thiomethylethyleneamido; 5. t-butylmethyleneamido; cyclopropylamido; cyclopentylamido; cyclohexylamido;
4-N-acetylipiper.idinylamido;0 cyclopentylmethylene amido; . piperidynyl-N-ethylene amido;
3-methoxyphenylamido;
4-methoxyphenylamido; i-propylamido; 5 3-chlorophenylamido;
4-chlorophenylamido; cyclobutylamido;
2-pyridinylamido;
3-pyridinylamido; 0 4-pyridinylamido;
2-thiophenylamido;
2-furanoylamido; •
5-isoxazolylamido;
4-biphenylamido; 5 phenylmethylene amido;
4-chlorophenylmethylene amido;
3-methoxyphenylmethyleneamido;
4-methoxyphenylmethyleneamido;
2-thiophenemethyleneamido; 0 cycloprop.yl-1-phenyl-1-amido; phenylethyleneamido; phenyloxymethyleneamido;
4-chlorophenyloxy-l, 1-dimethylmethylene- amido; phenylethyl-ene-amido; phenylmethoxyloxymethyleneamido; methylaminoamido; dimethylaminoamido; morpholine-N-amido; phenylmethyleneaminoamido; phenylethyleneaminoamido; methoxyamido; phenylmethyleneoxyamido; phenylsulfonamido;
3-methoxyphenylamido; 3-chlorophenylamido; ;- phenyl; phenylmethylene; phenylethylene; ■
2-bromo-phenylmethylene; 3-bromo-phenylmethylene;
4-bromo-phenylmethylene;
2-biphenyl-methylene;
3-biphenyl-methylene;
4-biphenyl-methylene; and methoxy.
Figure imgf000023_0001
Φ=phenyl
Figure imgf000023_0002
Specific compounds within the scope of this invention are exemplified by the following com- pounds:
(S) -N-{1- [3- (2,3-dichlorophenyl) -acryloyl] -piper- idin-4-yl}-2-methyl-succinamic acid; N-{ 1- [3- (2, 3-dichlorophenyl) -acryloyl] -piperidin-4- yl} -2-methylene-succinamic acid; N-{ 1- [3- (2, 3-dichlorophenyl) -acryloyl] -piperidin-4- yl}-succinamic acid;
Cis-6- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsul- fanyl) -phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) - cyclohex-3-enecarboxylic acid;
Cis-6- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsul- fanyl) -phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) - cyclohex-3-enecarboxylic acid;
4- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-ylcarbamoyl) -2-phenyl- butyric acid;
2- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) cyclo- propanecarboxylic acid; 4- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) -3-methyl- butyric acid;
4- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-ylcarbamoyl) -butyric acid;
N- (l-{ 3- [2 , 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-yl)-(S) -2- (2,2,2- trifluoro-acetylamino) -succinamic acid;
N- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-yl) -2-methyl-succinam- ic acid;
N- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -2-phenyl-succinam- ic acid; N- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -2, 2-dimethyl- succinamic acid;
4- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-3-ylcarbamoyl) -3-methyl- butyric acid;
N- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-3-yl) - (S) -2- (2,2,2- trifluoro-acetylamino) -succinamic acid; N- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-3-yl) -2-methyl-succinam- ic acid;
N- (l-{3- [2, 3-dichloro-4- (4-fluoro-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -malonamic acid; N- (l-{3- [2, 3-dichloro-4- (4-fluoro-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -2-fluoro-malonamic acid;
N-{ 1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- (S) -3-methyl-succinamic acid; N-{l-[3-(6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- (S) -2-methyl-succinamic acid; N-{ 1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- (R) -2-methyl-succinamic acid; rac-3-aminomethyl-N-{ 1- [3- ( 6, 7-dichloro-benzo [b] - thiophen-5-yl) -acryloyl] -piperidin-4-yl} -succinamic acid trifluoroacetic acid salt; rac-3- (benzoylamino-methyl) -N-{ 1- [3- (6, 7-dichloro- benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- succinamic acid; rac-N-{ 1- [3- ( 6, 7-dichlorobenzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl}-3- [ (3-methoxy-benzoyl- amino) -methyl] -succinamic acid; rac-3- [ (3-chlorobenzoylamino) -methyl] -N-{1- [3- (6, 7- dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin- 4-yl}-succinamic acid; rac-N-{ 1- [3- (6, 7-dichlorobenzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl}-3- [ (3-phenyl-ureido) - methyl] -succinamic acid; N-{ 1- [3- (2, 3-Dichloro-4-methylsulfanyl-phenyl) - acryloyl] -azetidin-3-yl}-3-methyl-succinamic acid; N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryloyl] -azetidin-3-yl} -3-methyl-succinamic acid; N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryl- oyl] -piperidin-4-yl}-3-phenyl-succinamic acid;
N-{ 1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}-3-pyridin-3-yl-succinamic acid; N-{ 1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -3-methyl-piperidin-4-yl}-3-methyl-succinamic acid;
3-(Butyrylamino-methyl)-Λ/-{1-[3-(6,7-dichloro-benzo[ώ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
Λ/-{1-[3-(67-Dichloro-benzo[jb]lhiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2-rrιethoxy-acetylamino)-methyl]-succinamic acid
Λ/-{1-[3-(6 -Dichloro-benzo[fo]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2,2-dimethyl-propionylamino)-methyl]- succinamic acid /-{1-t3-(6 -Dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-methylsulfanyl-propionylamino)-methyl]- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[ι ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3,3-dimethyl-butyrylamino)-methyl]-succinamic acid
3-[(Cyclopropanecarbonyl-amino)-methyl]-Λ/-{1-[3-(6,7-dichloro-benzo[£)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid 3-[(Cyclopentanecarbonyl-amino)-methyl]-Λ/-{1-[3-(6,7-dichloro-benzo[it)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
3-[(Cyclohexanecarbonyl-amino)-methyl]-W-{1-[3-(6l7-dichloro-benzo[i']thiophen-5-yl)-acryloyl]-piperidin-4-yI}-succinamic acid
3-{[(1-Acetyl-piperidine-4-carbonyl)-amino]-methyl}-Λ-{1-[3-(6,7-dichloro-benzo[ι ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
3-[(2-Cyclopentyl-acetylamino)-methyl]-Λ-{1-[3-(6,7-dichloro-benzo[ι ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[ύ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-piperidin-1-yl-propionylamino)-methyl]- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2-methoxy-benzoylamino)-methyl]-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[ιb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-methyl-butyrylamino)-methyl]-succinamic acid
3-[(Cyclobutanecarbonyl-amino)-methyl]-Λ-{1-[3-(6,7-dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic • acid
Λ/- 1-[3-(6,7-Dichloro-benzo[/)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(pyridine-2-carbonyl)-amino]-methyl}-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[j ]thiop en-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(pyridine-3-carbonyl)-amino]-methyl}-succinamic acid
/V-{1-[3-(6,7-Dichloro-benzo[ή]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(pyridine-4-carbonyl)-amino]-methyl}-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(thiophene-2-carbonyl)-amino]-methyl}- succinamic acid
W-{1-[3-(6,7-Dichloro-benzo[fc]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(furan-2-carbonyl)-amino]-methyl}-succinamic acid
3-{[(Benzot1,3]dioxole-5-carbonyl)-amino]-methyl}-/V-{1-[3-(6,7-dichloro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
Λ-{1-[3-(6,7-Dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[2-(3-methoxy-phenyl)-acetylamino]-methyl}- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[/3]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[2-(4-methoxy-phenyl)-acetylamino]-methyl}- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2-thiophen-2-yl-acetylamino)-methyl]-succinamic acid Λ/-{1-[3-(6,7-Dichloro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(1-phenyl-cyclopropanecarbonyl)-amino]-methyl}- succinamic acid
/V-{1-[3-(6,7-Dichloro-benzo[ 3]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-phenyl-propionylamino)-methyl]-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2-phenoxy-acetyIamino)-methyl]-succinamic acid
3-{[2-(4-Chloro-phenoxy)-2-methyl-propionylamino]-methyl}-Λ/-{1-[3-(6l7-dichloro-benzo[ιb]thiophen-5-yl)-acryloyl]- piperidin-4-yl}-succinamic acid
3-({[1-(4-Chloro-phenyl)-cyclopentanecarbonyl]-amino}-methyl)-Λ/-{1-[3-(6,7-dichloro-benzo[i3]thiophen-5-yl)-acryloyl]-
■ piperidin-4-yl}-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(2-phenyl-cycIopropanecarbonyl)-amino]-methyl}-
. succinamic acid
3-[(2-BenzyIoxy-acetylamino)-methyl]-Λ/-{1-[3-(6,7-dichloro-benzo[b]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic . acid
, W-{1-[3-(6,7-Dichloro-benzo[/>]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-methyl-ureido)-methyl]-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(morpholine-4-carbonyl)-amino]-methyl}- succinamic acid
3-(3-Benzyl-ureidomethyl)-Λ/-{1-[3-(6,7-dichloro-benzo[ώ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid.
Λ/-{1-[3-(6,7-Dic loro-benzo[J!)]thioρhen-5-yl)-acryloyl]-piperidin-4-yl}-3-(3-ph8nethyl-ureidomethyl)-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[6]thiophen-5-yl)-acryloyI]-piperidin-4-yl}-3-(methoxycarbonylamino-methyl)-succinamic acid'
3-S-Benzoylamino-Λ -{1-[3-(6,7-dichIoro-benzo[/)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
3-R-Benzoylamino-Λ -{1-[3-(6,7-dichloro-benzo[fe]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
3-R-Acetylamino-Λ/-{1-[3-(6,7-dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
3-R-Butyrylamino-/V-{1-[3-(6,7-dichloro-benzo[j ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[ύ]thiophen-5-yl)-acry!oyl]-piperidin-4-yl}-3-(2-methoxy-acetylamino)-succinamic acid
Λ -R-{1-[3-(6J-Dichloro-benzo[d]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-(2l2-dimethyl-propionylamino)- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzotό]t iophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3-methylsulfanyl-propionylamino)-succinamic acid Λ/-R~{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3,3-dimethyl-butyrylamino)-succinamic acid
3R-(Cyclopropanecarbonyl-amino)-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl) -acryloyl]-piperidin-4-y!}-succinamic acid
3R-(Cyclopentanecarbonyl-amino)-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
3-R-(Cyclohexanecarbonyl-amino)-Λ/-{1-[3-(6,7-dichloro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}
-succinamic acid
3R-[(1-Acetyl-piperidine-4-carbonyl)-amino]-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)-acryloyl]- piperidin-4-yl}-succinamic acid
3-R-(2-Cyclopentyl-acetylamino)-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)-acryloy!]-piperidin-4-yl}- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-y!)-acryloyl]-piperidin-4-yl}-3- (3-piperidin-1-yl-propionylamino) -succinamic acid '
Λ/R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(2-methoxy-benzoylamino)-succinamic acid
Λ/-R-{1-[3-(6J-Dichloro-benzo[ό]t iophen-5-yl)-acryloyl]-piperidin-4-yl}-3- (4-methoxy-benzoylamino)-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzotό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3-methyl-butyrylamino)-succinamic acid
3-R(2-Chloro-benzoylamino)-Λ/-{1-[3-(6,7-dic loro-benzo[D]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
3-R-(4-Chloro-benzoylamino)-Λ -{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
3R-(Cyclobutanecarbonyl-amino)-Λ/-{1-[3-(6,7-dichloro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}
-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[o]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(pyridine-2-carbonyl)-amino]- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[i ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(pyridine-3-carbonyl)-amino]- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzotD]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(pyridine-4-carbonyl)-amino]- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[/3]thiophen-5-y!)-acryloyl]-piperidin-4-yl}-3-[(thiophene-2-carbonyl)-amino]- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[j ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(furan-2-carbonyl)-amino]- succinamic acid Λ/-R-{1-[3-(6,7-Dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(isoxazole-5-carbonyl)-amino]- succinamic acid
3-R-[(Biphenyl-4-carbonyl)-amino]-Λ/-{1-[3-(6,7-dichloro-benzo[o]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[ib]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-phenylacetylamino- succinamic acid
3R-[2-(4-Chloro-phenyl)-acetylamino]-Λ/-{1-[3-(6,7-dichloro-benzo[6]thiophen-5-yl)-acryloyl]- piperidin-4-yl}- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[2-(3-methoxy-phenyl)- acetylaminoj-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[ib]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[2-(4-methoxy-phenyl)
-acetylamino]-succinamic acid
Λ-R-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidi -4-yl}-3- (2-thiophen-2-yl-acetylamino)-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3- [(1-phenyl-cyclopropanecarbonyl)-amino]-succinamic acid
Λ/R-{1-[3-(6,7-Dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin; -yl}- 3-(3-phenyl-propionylaminoj-succinamic acid
Λ/f?-{1 -[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyi]-piperidin-4-yl}- 3-(2τphenoxy-acety lamino)-succinamic. acid
3R-[2-(4-Chloro-phenoxy)-2-methyl-propionylamino]-Λ/-{1-[3-(6,7-dichloro-benzo[ό]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid 3R-{[1-(4-Chloro-phenyl)-cyclopentanecarbonyl]-amino}-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
Λ/f?-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3- (3-phenyl-acryloylamino)-succinamic acid
3-R-(2-Benzyloxy-acetylamino)-Λ/-{1-[3-(6,7-dichloro-benzo[ό]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
Λ/R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl} -3-(3-methyl-ureido)-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3- (3,3-dimethyl-ureido)-succinamic acid
Λ/R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3 -[(morpholine-4-carbonyl)-amino]-succinamic acid
3-R(3-Benzyl-ureido)-Λ/-{1-[3-(6,7-dichloro-benzo[b]thiophen-5-yl)-acryloyl] -piperidin-4-yl}-succinamic acid
Λ-R-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3-phenethyl-ureido)-succinamic acid Λ/f?-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-methoxycarbonyiamino-succinamic acid
3-R-Benzyloxycarbonylamino-Λ-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl) -acryloyl]-piperidin-4-yl}-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3 -(3-methoxy-benzoylamino)-succinamic acid
3-R-(3-Chloro-benzoylamino)-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
3-R-Benzenesulfonylamino-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)-acryioyl]- piperidin-4-yl}-succinamic acid and
N-{1- [3- (6, 7-dichloro-benzo [b] thioρhen-5-yl) -acryloyl] -piperidin-4-yl}-3-quinolin-3-yl-succinamic acid. In another aspect, this invention provides ' pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound defined herein..
In one of the method aspects, this inven- tion is directed to a method for. assaying a biological sample from a mammalian patient suspected of having a,, disease,, condition or disorder mediated, at least' in' part, by VLA-1, which method comprises obtaining a biological sample from said patient and assaying said sample for the presence of VLA-1,. In another aspect, this invention is directed to a method for inhibiting adhesion of mammalian cells to the extracellular matrix mediated, at least in part, by VLA-1, which method comprises contacting said cells with a compound or pharmaceutical composition of this invention.
In another one of its method aspects, this invention is directed to a method for treating a disease, condition or disorder whose progression is regulated, at least in part, by VLA-1 expression or activity in a mammalian patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound or composi- tion of this invention.
In a preferred embodiment, said disease, disorder, or condition is selected from the group consisting of asthma, trachoma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes, inflamma- tory bowel disease, multiple sclerosis, rheumatoid arthritis, tissue transplantation, tumor metastasis, tumor migration, and/or tumor growth, proliferation of fibroblasts in cancer, solid tumors, meningitis, encephalitis, stroke, cerebral traumas, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia, acute leukocyte-mediated lung injury, and fibrotic diseases.
In another preferred embodiment, said disease, disorder, or condition is a fibrotic disease. In still another preferred embodiment, said fibrotic disease is selected from the group consisting of systemic sclerosis, mixed connective tissue disease, fibrodysplasia, fibrocystic disease, sarcoidosis, and myositis. In yet another preferred embodiment, said fibrotic disease has a manifestation of fibrotic vascular intimal hypertrophy, and is selected from the group consisting of vasculitis, polyarteritis nodosa, and temporal arteritis. In yet another preferred embodiment, said fibrotic disease has a manifestation of fibrotic hypertrophy of skin and/or muscle tissue, and is selected from the group consisting of scleroderma, eosinophilic fasciitis, discoid lesions associated with lupus or discoid lupus, and surgical adhesions. In yet another preferred embodiment, said fibrotic disease has a manifestation of fibrotic hypertrophy of nerve tissue, and is selected from the group consisting of cerebrosclerosis, annular sclerosis, diffuse sclerosis, and lobar sclerosis. In yet another preferred embodiment, said fibrotic disease has a manifestation of fibrotic hypertrophy, or fibrosis of lung tissue, and is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, the fibrotic element of pneumoconiosis, pulmonary sarcoidosis, fibrosing alveolitis, the fibrotic or hypertrophic element of cystic fibrosis, chronic obstructive pulmonary disease, adult respiratory distress syndrome, and emphysema. In yet another preferred embodiment, said fibrotic disease has a manifestation of fibrotic hypertrophy, or fibrosis of prostate, liver, the pleura, or pancreas, and is selected from the group consisting of benign prostatic hypertrophy (BPH) , nonalcoholic steato hepatitis, and fibrosis of the liver.
In yet another preferred embodiment, said fibrotic disease has a manifestation of fibrotic hypertrophy, or fibrosis of the kidney, and is selected from the group consisting of chronic renal failure, lupus nephritis, alports syndrome, glomerulonephritis, and diabetic nephritis.
In another preferred embodiment, said disease, disorder, or condition is cancer. In a preferred embodiment, said cancer is a tumor or a neoplasm selected from the group consisting of carcinomas, adenocarcinomas, and sarcomas .
In another preferred embodiment, said can- cer is selected from the group consisting of growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, human soft tissue carcinoma, cancer metastases, squamous cell carcinoma, esophageal squamous cell carcinoma, oral carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producing tumors, nonsmall cell cancers, breast cancer, gastrointestinal cancers, urological cancers, malignancies of the female gen- ital tract, malignancies of the male genital tract, kidney cancer, brain cancer, bone cancers, skin cancers, thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer, trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma.
In still yet another preferred embodiment, said cancer is a cell proliferative disorders and is selected from the group consisting of angiogenesis- mediated diseases, benign tumors, acoustic neuromas, neurofibromas, pyogenic granulomas, biliary tract cancer, choriocarcinoma, esophageal cancer, gastric cancer, intraepithelial neoplasms, lung cancer, and neuroblastomas .
A compound or composition of this inven- tion may be administered to the mammal by a suitable route, such- as orally, intravenously, parenterally, transdermally, topically, rectally, or intranasally.
Mammals include, for example, humans and other primates, pet or companion animals, such as dogs and cats, - laboratory animals, such .as rats, mice and- rabbits, and farm animals, such as horses, pigs, sheep, and cattle.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and Overview
As discussed above, the present invention is directed to novel aminopiperidine amide derivatives.
It is to be understood that the terminal- ogy used herein is. for the purpose of describing ■ particular embodiments only and is not intended to limit the scope of the present invention. It must " be noted that as used herein and in the claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates other- wise. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: As used herein, "alkyl" refers to mono- valent alkyl groups having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, t-butyl, n- heptyl, octyl, and the like.
"Substituted alkyl" refers to an alkyl group having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryl- oxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl, thiol, thioalkyl, heteroaryl, substituted hetero- aryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino .
"Fluoroalkyl" refers to an alkyl group having from 1 to 4 carbon atoms and from 2 to 7 fluoro atoms. "Hydroxy" refers to the group -OH.
"Alkylene" refers to divalent alkylene groups having from 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methylene, n-heptyl- ene, 1, 3-octylene, and the like.
"Substituted alkylene" refers to an alkylene group having from 1 to 5 substituents selected from the group consisting of substituents defined for substituted alkyl. "Alkoxy" refers to the group "alkyl-O—" which includes, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec- butoxy, n-pentoxy, n-hexoxy, 1, 2-dimethylbutoxy, and the like.
"Substituted alkoxy" refers to the group "substituted alkyl-O-."
"Acyl" refers to the groups H—C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C (0) -, substituted alkenyl-C (0) -, cycloalkyl-C (0) -, substituted cycloalkyl-C (0) -, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C (0) -, substituted hetero- aryl-C(O), heterocyclic-C (0) -, and substituted heterocyclic-C (0) - .
"Acylamino" refers to the group —C(0)NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl heteroaryl, substituted heteroaryl, heterocyclic,. substituted heterocyclic, and where each R is option- ally joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic ring. "Acyloxy" refers to the groups alkyl- C(0)0-, substituted alkyl-C (0) 0-, alkenyl-C (0) 0-, substituted alkenyl-C (0) 0-, aryl-C(0)0-, substituted aryl-C(0)0-, cycloalkyl-C (0) 0-, substituted cycloalkyl-C (0)0-, heteroaryl-C (0) 0-, substituted heteroaryl-C (0)0-, heterocyclic-C (0) 0-, and substituted heterocyclic-C (0) 0- .
"Alkenyl" refers to monovalent alkenyl groups having from 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms, and having at least 1 and preferably from 1-2 sites of alkenyl unsatura- tion.
"Substituted alkenyl" refers to alkenyl groups having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryl- oxy, cyano, halogen, hydroxyl, nitro, carboxyl, car- ■-. boxyl esters, cycloalkyl, substituted cycloalkyl, thiol, thioalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino, provided that the hydroxyl or the thio group is not pendent to an unsaturated carbon • atom.
"Amino" refers to the group -NH2. "Substituted amino" refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where each R is optionally joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic ring provided that both R's are not hydrogen.
"Aminosulfonyl" refers to the group -S02NR'R', wherein each R' is independently selected from the group consisting of hydrogen, alkyl, sub- stituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted cyclo- alkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where each R is optionally joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic ring.
"Sulfonylamino" refers to the group -NR'R'S02, wherein each R' is as defined above.
"Aminoacyl" refers to the groups -NRC(O)- alkyl, -NRC (0) substituted alkyl, -NRC (0) alkenyl, -NRC (0) substituted alkenyl, -NRC (0) cycloalkyl, -NRC- (0) substituted cycloalkyl, -NRC (0) aryl, -NRC (0) substituted aryl, -NRC (0) heteroaryl, -NRC (0) substituted heteroaryl, -NRC (O) eterocyclic, and -NRC (0) substituted heterocyclic, where R is hydrogen or alkyl. "Aminocarbonyloxy" refers to the groups
-NRC(0)0-alkyl, -NRC (0)0 substituted alkyl, -NRC(O)- O-Cycloalkyl, -NRC (0)0 substituted cycloalkyl, -NRC- (O)O-aryl, -NRC (0)0 substituted aryl, -NRC (0) 0-het- eroaryl, -NRC (0)0 substituted heteroaryl, -NRC (0)0- heterocyclic, and -NRC (0)0 substituted heterocyclic, where R is hydrogen or alkyl.
"Oxycarbonylamino" refers to the groups -0C(0)Q where Q is amino or substituted amino.
"Aminocarbonylamino" or "aminoacylamino" refers to the groups -QC(0)Q where each Q is independently amino or substituted amino.
"Aryl" or "Ar" refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multi- pie condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-1, 4-benzoxazin-3 (4H) - one-7-yl, and the like) provided that the point of attachment is on an aromatic carbocyclic group atom. Preferred aryls include phenyl and naphthyl. 5 "Substituted aryl" refers to aryl groups which are substituted with from 1 to 3 substituents selected from the group consisting of hydroxy, . acyl, acylamino, acyloxy, alkyl, substituted alkyl, ■ alkoxy, substituted alkoxy, alkenyl, substituted
10' alkenyl, amino,. substituted amino, aminoacyl, -amino-" carbonyloxy, aminocarbonylamino, aryl, substituted aryl, aryloxy, substituted aryloxy, carboxyl, car- !■. boxyl esters, cyano, thiol, thioalkyl, substituted thioalkyl, cycloalkyl, substituted cycloalkyl, halo,
15. nitro, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.
"Aryloxy" refers to the group'-aryl-O— which includes, by way of' example, phenoxy, naph- thoxy, and the like'.
20 ' "Substituted aryloxy" refers to substituted aryl-O— groups.
"Carboxyl" refers to the group -COOH- and salts thereof.
"Carboxyl esters" refer to the group -COOR
25 where R is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic.
30 "Cycloalkyl" refers to monovalent cyclic alkyl groups of from 3 to 8 carbon atoms having a single cyclic ring including, by way of example, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like.
"Substituted cycloalkyl" refers to a cycloalkyl group, preferably of from 3 to 8 carbon atoms, having from 1 to 5 substituents selected from the same group of substituents as defined for substituted alkyl as well as oxo (=0) and thioxo (=S) groups . "Cycloalkylene" refers to divalent cyclic alkyl groups of from 3 to 8 carbon atoms having a single cyclic ring including, by way of example, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, and the like. "Cycloalkenylene" refers to divalent cyclic alkenyl groups of from 4 to 8 carbon atoms having a single cyclic ring and 1-2 sites of unsat- uration including, by way of example, cyclobutenyl- ene, cyclopentenylene, cyclooctenylene, and the like.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo and preferably is fluoro, chloro or bromo.
"Heteroaryl" refers to an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizin- yl or benzothienyl) provided that the point of attachment is to a heteroaryl group atom and further provided that the heteroaryl 'group contains at least five ring atoms. Preferred heteroaryls include pyridyl, pyrrolyl, indolyl and furyl .
"Substituted heteroaryl" refers to heter- oaryl groups which are substituted with from 1 to 3 substituents selected from the group of' substituents defined for .substituted aryl.
"Heterocycle" or "heterocyclic" refers to a monovalent saturated or unsaturated, but not aro- matic, group having a single ring or multiple condensed rings, from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur or oxygen within the ring wherein, in fused ring systems, one or more the rings can be aryl or heteroaryl, provided that the heterocyclic ring has at least 4 atoms and further provided that the point of attachment is to a heterocyclic ring atom. -
"Substituted heterocyclic" refers to net-' erocycle groups which are substituted with from 1 to 3 substituents selected from the group of substituents defined for substituted cycloalkyl.
Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydro- indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinox- aline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperi- dine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6, 7-tetrahydrobenzo [b] - thiophene, thiazole, thiazolidine, thiophene, benzo- [b] thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl) , piperidinyl, pyrrol- idine, tetrahydrofuranyl, and the like.
"Heterocyclene" refers to divalent heterocyclic groups of from 3 to 8 carbon atoms having a single cyclic ring.
"Heterocyclenylene" refers to divalent heterocyclic groups of from 4 to .8 carbon atoms' having a single cyclic ring and 1-2 sites of unsat- uration. "Thiol" refers to the group —SH.
"Thioalkyl" refers to the group —S-alkyl. "Substituted thioalkyl" refers to the group —S substituted alkyl.
"Thioaryl" refers to the group —S-aryl. "Thioalkylaryl" refers to the group -S- alkylene-aryl, S substituted alkylene aryl/ S alkylene substituted aryl or -S substituted alkylene substituted aryl.
"Thioalkylheteroaryl" refers to the group -S alkylene heteroaryl, S substituted alkylene heteroaryl, S alkylene substituted heteroaryl or -S substituted alkylene substituted heteroaryl.
"Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound of Formula I which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkyl- ammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids,- such as hydrochloride, hydrobro- mide, tartrate, mesylate, acetate, maleate, oxalate, and the like.
"Prodrugs" as used herein, are- compounds which convert (e.g., hydrolyze, metabolize) in vivo • to a compound of the invention. The effectiveness of an orally administered drug is dependent upon the drug'.s efficient transport across the mucosal epi-;• thelium and .its stability in entero-hepatic circula- • tion. Drugs that are effective after parenteral administration but less effective orally, or whose ' plasma half-life is' considered too short, may be chemically modified- into a prodrug form. The pro- drug should have a pharmacokinetic profile that is different from that of the parent, enabling easier absorption across the mucosal epithelium-, better * salt formulation and/or solubility, and/or improved systemic stability (for an increase in plasma half- life, for example) . Many chemical modifications may be suitable for the creation of the prodrugs accord- ing to the invention, including:
(1) Ester or amide derivatives which may be cleaved by, for example, esterases or lipases. For ester derivatives, the ester is derived from the carboxylic acid moiety of the drug molecule by known means. For amide derivatives, the amide may be de- rived from the carboxylic acid moiety or the amine moiety of the drug molecule by known means.
(2) Peptides that may be recognized by specific or nonspecific proteinases. A peptide may be coupled to the- drug molecule via amide bond formation with the amine or carboxylic acid moiety of the drug molecule by known means.
(3) Derivatives that accumulate at a site of action through membrane selection of a prodrug form or modified prodrug form.
(4) Any combination of 1 to 3.
It will further be appreciated by those skilled in the art that certain moieties known to those skilled in the art as "pro-moieties," for example as described in "Design of Prodrugs" by H. Bundgaard (Elsevier) 1985, may be placed on appropriate functionalities when such functionalities are present in compounds of the invention also to form a "prodrug." Further, certain compounds of the invention may act as prodrugs of other compounds of the invention. All protected derivatives, and pro- drugs, of the compounds of the invention are included within the scope of the invention.
"Biological activity" as used herein re- fers to an inhibition concentration when tested in at least one of the assays outlined in Example A or B.
The terms "substituted" as used with, for example, "substituted alkyl" does not include poly- mers derived therefrom but are limited to a maximum of 3 substituents groups, e.g., Ar-Ar-Ar. The term "tautomer" refers to an isomer in which migration of a hydrogen atom results in two or more structures.
Compound Preparation
The compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction tempera- tures, times, mole ratios of reactants, solvents, pressures) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and P. G. M. Wuts, "Protecting Groups in Organic Synthesis, Second Edition," Wiley, New York, 1991, and references cited therein.
Furthermore, the compounds of this inven- tion may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoiso- mers (and enriched mixtures) are included within the scope of this invention, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chi- ral column chromatography, chiral resolvinc? agents, and the like.
Compounds in the present invention may be better understood by the following synthetic Schemes that illustrate methods for the synthesis of compounds of the invention.
Scheme 1
Figure imgf000047_0001
3 each R2 = H, CH3, CF3
R1 and R3 are as defined for Formula I
Pd ( 0 ) refers to metallic palladium
Cinnamic acid intermediates 3 may be prepared by a Heck-type, palladium-mediated coupling (e . g . , using tetrakis- (o-tolyl phosphine ) palladium- (0), Pd2(dba)3, Pd(OAc)2, or the like) of halo substituted aromatic derivatives 2 with an appropriate olefinic substrate 1 (e.g., methyl acrylate, ethyl crotonate, or the like; S. J. Buchwald, Chem . Eur. J. 1999, 5, 3107-3112) . The intermediate ester may require a separate hydrolysis step to allow for further elaboration.-
Scheme 2
"acetate equivalent"
base, solvent
Figure imgf000048_0001
(optional hydrolysis)
Figure imgf000048_0002
R = H or CH3
R1 is as defined for Formula I
An alternative preparation of substituted cinnamic acids 6 is given in Scheme 2. Substituted aromatic aldehydes 5 may be prepared from aromatic carboxylic acids 4 by utilizing a reducing agent (for example, borane-THF, LiAlH4) to reduce the acid to the benzyl alcohol. The resulting alcohols can then undergo partial oxidation by standard methods (Jones, Swern, Moffat) to the desired aldehydes. Further elaboration is possible by reacting the aldehyde with an acetate equivalent (using malonic acid, Meldrum's acid, or the like) and a base (such as piperidine, or the like) in a solvent (e.g., pyridine or 2, 6-lutidine) to give the resulting cinnamic acids 6. In an alternate procedure (M. Mikolajczyk, Synthesis 1976, 6, 396), the aldehyde may be reacted with a Wittig, Horner-Emmons, or Wadsworth-Emmons reagent (e.g., 2- (diethoxyphos- phoryl) -propionic acid ethyl ester, or the like) in a solvent (CH2C12, CH2C1CH2C1, or like) and a base (NaOH, NaOEt, NaH, or the like) to furnish the desired cinnamic ester. The ester may undergo hydrol- ysis to the acid 6 with the use of a base (LiOH,
NaOH, or the like) in a solvent (THF-water, CHC12- water) .
Scheme 3
Figure imgf000049_0001
deprotection
Figure imgf000050_0001
Figure imgf000050_0002
wherein R1, R2, R3 are as defined for Formula I m is as defined above
Cinnamides of amino substituted cyclic amines may be constructed by coupling of a cinnamic acid 6 with an appropriately protected amino substituted cyclic amine 7 (e.g., P1=Boc, Fmoc, or the like; m=0-4), as shown in Scheme 3. The cinnamic acid is activated (for example, using thionyl chloride, or oxalyl chloride, or 1- (3-dimethylamino- propyl) -3-ethylcarbodiimide and N-hydroxysuccin- imide, or the like) and reacted with 7, usually in the presence of a tertiary amine base (e.g., diiso- propylethylamine, triethylamine, N-methyl morph- oline, or the like) to provide amides 8. The protected amino group of 8 is subsequently deprotected to the amine 9 using appropriate reagents (e.g., anhydrous HC1 to remove a Boc group) .
Scheme 4
(optional base)
Figure imgf000050_0003
10
Figure imgf000051_0001
when n=l, then R"=R9 and/or R10 when n=2, then first R"=R9 and/or R10 and second
CHR"-COOH=R8
R1, R2, R3, R6, R7, R9, Rl° are as defined for
Formula I m is as defined above
Scheme 4 illustrates the elaboration of amino cinnamides 9 -with various cyclic anhydrides 10 (for example, substituted succinic and glutaric anhydrides, or- the like) to give succinamides (1.1, n=l) and glutaramides (11, n=2) . 'The use of a ' tertiary amine base (e.g., diisopropylethylamine, or the like) is optional. In cases /where R6 or R7 R", a regioisomeric mixture of amides 11 may be obtained, which can be isolated by conventional techniques as described below.
Scheme 5
coupling
Figure imgf000051_0002
12 deprotection
Figure imgf000052_0001
13
Figure imgf000052_0002
R1, R2, R3, R6, R7 are as defined for Formula I
R" is as defined above m is as defined above
P2 is a suitable protecting group such as t-butyl and the like
Alternatively, amines 9 may be coupled with a mono-protected diacid 12 (for example, mono- ethyl malonate, mono-ethyl succinate, or the like) to give an amide-ester intermediate 13, as shown in Scheme 5. Amide formation protocols are as described for Scheme 3. Subsequent deprotection of the ester group of 13, using appropriate reagents (for example, TFA to remove tert-butyl ester groups) yields malonamides (11, n=0) , succinamides (11, n=l), or glutaramides (11, n=2) . Scheme 6
Figure imgf000053_0001
14 12
deprotection
Figure imgf000053_0002
15
Figure imgf000053_0003
i, P2, n, R", m are as defined above R6, R7, R1, R2, R3 are as defined above
In another variation for preparing the compounds of the invention, shown in Scheme 6, the mono-protected diacid 12 is first coupled with an appropriately protected amino substituted cyclic amine 14 (Pχ=Boc group, or the like; m=0-4) using amide formation protocols described in Scheme 3. After removal of the protecting group (e.g., anhydrous HCl for Boc removal, or the like) , amine 16 can be coupled with a cinnamic acid 6 as described for Scheme 3 followed by deprotection of P2 using appropriate reagents (for example, TFA to remove tert-butyl ester groups) to provide amides 11.
Scheme 7
Figure imgf000054_0001
17
Figure imgf000054_0002
Pi, m, Pd(0) are as defined above
R1, R2, R3 are as defined for Formula I
Scheme 7 illustrates an alternative procedure in which the order of these coupling steps may be reversed. Cyclic amines bearing a protected amine group (7, e.g., 4-N-Boc-aminopiperidine, or the like) may be coupled with an acryloyl chloride 17 in the presence of an appropriate base (e.g.,
DIEA, or the like) . The acrylamide olefin 18 may be further elaborated by palladium-mediated coupling with a halo substituted aromatic derivative (using protocols described for Scheme 1) to provide cinnamides 8.
Scheme 8
Figure imgf000055_0001
23
deprotect
Figure imgf000055_0002
24
coupling hydrolysis
Figure imgf000055_0003
25
Figure imgf000056_0001
26
each P2 is a suitable carboxy protecting group m is as defined above
R1, R2, R3, R6, R7 are as defined for Formula I
Some of the analogues described herein contain a malonamide moiety, which can be prepared as described in Scheme 5, or by direct displacement of the ester group of a dialkyl malonate (e.g., di- ethyl malonate, or the like) with amines 14 (e.g., Pι=benzyl, or the like) leading to malonamides 23. After deprotection (for example, using H2 and Pd/C to remove an N-benzyl group, or the like) , the resulting amines 24 can be coupled with a cinnamic acid 6 using amide formation protocols described for Scheme 3, thus providing malonamides 25. Deprotection of the ester group Ra gives desired acids 26.
Scheme 9
Figure imgf000056_0002
27
Figure imgf000057_0001
28
Figure imgf000057_0002
29
Figure imgf000057_0003
30 31
R' =substitution on aryl group -as defined above
Scheme 9 illustrates the preparation of 5- bromobenzothiophenes, used for the preparation of the corresponding cinnamic acids. A suitable phen- ylsulfanyl acetic acid 28 is prepared from a thio- phenol 27 using bromoacetic acid under standard conditions. Sulfide 28 is selectively brominated, e.g., with bromine in a solvent such as dichloro- methane, glacial acetic acid, or the like, in the presence of iron or iodine as catalysts. The reaction is typically performed at room temperature for 1 to 96 hours. Bromide 29 is then converted to an acid halide intermediate by treatment with an in- organic acid halide (for example, thionyl chloride, phosphorous trichloride, phosphorous pentachloride, phosphrous tribromide, oxalyl chloride, or the like) in an inert solvent (for example dichloromethane or the like), at a temperature in the range of 0°C to 110 °C for about 1 to 48 hours. The volatiles are then removed under reduced pressure and the residue is dissolved in an appropriate solvent, typically dichloromethane, and subjected to Friedel-Crafts cyclization by treatment with a Lewis acid such as AICI3 or poiyphosphoric acid. The reaction is generally carried out at -78 °C to 25°C. The resulting intermediate ketone product is reduced without isolation by a hydride reducing reagent- (for exam- pie, NaBH or the like) to" provide alcohol 30.
Alcohol 30 may be used directly in the next step, or first purified by chromatography or recrystalliza- tion as appropriate. The dehydration of 30 to benzothiophene 31" is accomplished by treatment" with a Lewis or proti.c acid (for example, boron trifluor-" ide etherate.) in an appropriate solvent, (for example glacial acetic acid or the like) at a temperature ranging from ambient temperature to the reflux temperature of the solvent. Upon completion of the reaction, the resulting 5-bromo benzothiophene 31 is recovered by conventional means such as neutralization, precipitation, filtration, recrystallization, and the like. Scheme 10
Figure imgf000059_0001
Figure imgf000059_0002
11 , R6/R7 R"
Figure imgf000059_0003
coupling separate isomers
deprotect
Figure imgf000059_0004
32
Figure imgf000060_0001
11, isomers separates , n, R" are .as defined above
R1, R2, R3, R6, R7 are as defined for Formula I
Some of the diamides 11 prepared according to Scheme 4 as regioisomeric mixtures (R6 or R7≠R"), were separated chromatographically as 9-fluprenyl- methyl esters 32 as shown in Scheme 10. These fluorenyl esters 32 were synthesized by coupling the carboxylic acid group of the mixture of regioisomeric compounds 11 with 9-fluorenylmethanol using an appropriate activating reagent (e.g., dicyclohexyl- carbodiimide, or the like) . After chromatographic separation, the 9-fluorenylmethyl group was removed without regioisomeric scrambling of R6 or R7 and R" using piperidine, or the like, to generate pure regioisomeric compounds 11.
Scheme 11
Figure imgf000060_0002
33 hydrolysis
Figure imgf000061_0001
35
Figure imgf000061_0002
36
coupling
Figure imgf000061_0003
37
Figure imgf000061_0004
Figure imgf000062_0001
R10' is a suitable substituent m is as defined above R1, R2, R3 are as defined for Formula I
Scheme 11 illustrates the preparation of succinamides in which an aminomethyl group is appended to the β-position of the succinamide chain. Alkylation of an ester of ,β-glycine (33, P2=:Boc group, or the like; alk=methyl, ethyl, or the like) with an -bromo acetate ester 34 (Rx=tert-butyl, or another group with orthogonal deprotection conditions to alk) gives an aminomethyl succinate diester 35. The diester 35 can be selectively mono-depro- tected to give carboxylic acid intermediate 36 (for example, using lithium hydroxide to hydrolyze a methyl ester selectively over a tert-butyl ester) . The monocarboxylic acid 36 is coupled to amines 9 using amide formation protocols described for Scheme 3. Simultaneous deprotection of P and Rx of amides 37 can be achieved using appropriate reagents (for example, TFA in cases where P2=Boc and Rx=tert-butyl, or the like) to provide amino acids 38. The amino group of 37 can be revealed by selective deprotection, and then functionalized by reaction with activated carboxyl- and sulfonyl-containing inputs (e.g., acid chlorides, sulfonyl chlorides, carbamoyl chlorides, isocyanates, anhydrides, chloroformates, or the like) in the presence of an appropriate base (e.g., diisopropylethylamine, or the like), to provide, after ester cleavage, the amine-derivatized compounds 39. Alternatively, the amino group of 38 may be functionalized selectively in the presence of the, carboxylic acid using the activated carboxyl- and sulfonyl-containing inputs described above.
The preparation of the β-heteroaryl group (Ra is heteroaryl, substituted heteroaryl in Formula I) can be accomplished by well known techniques, an example of which is provided in Scheme 12 below:
Scheme 12
Figure imgf000063_0001
100
200
Figure imgf000063_0002
R6 and R7 are as defined for Formula I
Het is a suitable heteroaryl or heterocyclic
Figure imgf000063_0003
600 Specifically, in Scheme 12 above, the carboxyl group of an nitrogen-protected α-amino acid, compound 100, is reduced using conventional techniques such as the use of a reducing agent includ- ing, for example, lithium aluminum hydride to provide for the corresponding alcohol, compound 200. The reaction is preferably conducted in an inert diluent such as tetrahydrofuran, diethyl ether, and the like at a temperature preferably from about -78°C to about 25°C. The reaction is continued until substantial completion which typically occurs from within 0.5 to 18 hours. Upon -completion of the reaction, compound 200 can be recovered by conventional methods including neutralization, extraction, precipitation, chromatography, filtration, and the like or used in the next step of the reaction without purification and/or isolation.
The alcohol, compound 200, is converted to a halo group (e.g., chloro) again by conventional methods such as contact with a suitable halogenating agent to provide for compound 300. Suitable halogenating agents include, for example, inorganic acid halides, such as thionyl chloride, phosphorous trichloride, phosphorous tribromide or phosphorous pentachloride, under conventional conditions. Generally, this reaction is conducted using about 1 to 5 molar equivalents of the inorganic acid halide, either neat or in an inert solvent, such as dichloromethane or carbon tetrachloride, at temperature in the range of about 0°C to about 80°C for about 1 to about 48 hours. A catalyst, such as DMF, may also be used in this reaction. Upon completion of the reaction, compound 300 can be recovered by conventional methods including neutralization, extraction, precipitation, chromatography, filtration, and the like or used in the next step of the reaction without purification and/or isolation.
The conversion of compound 300 into a Grignard reagent, compound 400, follows conventional techniques well documented in the literature includ- ing Gutsche et al., "Fundamentals of Organic Chemistry," Prentice Hall (1975), pp. 238 et seq. Specifically, at least an equimolar amount of metallic magnesium is added to compound 3 in a suitable inert diluent such as diethyl ether to form the Grignard reagent, compound 400. This compound, typically in a single reaction vessel, is reacted with at least an equimolar equivalent of a heteroaryl or heterocyclic halide, i.e., Het--X (compound 500) to provide for compound 600.
Pharmaceutical Formulations
When employed as pharmaceuticals, the compounds of the subject invention are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, parenteral, transdermal, topical, rectal, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. This invention also includes pharmaceutical compositions which contain, as the active ingredient,, one or more of the compounds of the subject invention above associated with pharmaceu- 5 tically acceptable carriers. In making the compositions of this invention, the active ingredient is usually mixed with' an excipient, . diluted by an
■ ' excipient or enclosed within such a carrier which can be in the form of a capsule, sachet-, paper or
10" other container.. The. excipient- employed is typical- '• ly an excipient suitable for administration to' human subjects or other mammals-. When, the excipient serves as a diluent, it can be a solid, semi-solid, •• or liquid material, which acts as a vehicle, carrier- -•
15 or medium for the active ingredient. Thus, the com-" ' positions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups-, aerosols (as. a solid or in a liquid medium) , ointments contain-
20 ing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders .
In preparing a formulation, it may be
25 necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the
30 active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose,- sorbitol, anni- tol, starches, ' gum acacia, calcium phosphate, al- ginates, tragacanth, gelatin, calcium silicate, microcrysta-lline cellulose, polyvinylpyrrolidone, cellulose,' sterile water,- syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as. talc, magnesium stearate, " and mineral oil; wetting agents; .emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates; sweetening agents; and flavoring agents.-- The compositions of . the invention can .be formulated so as to provide quick, sustained ; or delayed release of the active ingredient after ' administration to • the patient by -employing procedures known in the art. :
The quantity of active- component, that is- the compound according to • the subject invention, in the pharmaceutical composition and unit dosage- form - thereof may be varied or adjusted widely depending upon the particular application, the potency of the particular compound and the desired concentration. The compositions are preferably formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg, usually about 5 to about 100 mg, occasionally about 10 to about 30 mg, of the active ingredient. The term "unit dosage forms" re- fers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Preferably, the compound of the subject "invention above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier (s). The active compound is effective over a wide dosage range and is generally administered in a pharmaceutically or therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the< relevant circumstances, including the condition to be treated, the severity of the condition being treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
In therapeutic use for treating, or combating, inflammation in warm-blooded animals, the compounds or pharmaceutical compositions thereof will be administered by any appropriate route, such as orally, topically, transdermally, and/or par- enterally at a dosage to obtain and maintain a concentration, that is, an amount, or blood-level of active component in the animal undergoing treatment that will be therapeutically effective. Generally, such therapeutically effective amount of dosage of active component (i.e., an effective dosage) will be in the range of about 0.1 to about 100, more preferably about 1 to about 50 mg/kg of body weight/day. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre- formulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformula- tion is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
The tablets or pills of the present inven- tion may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorpo- rated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceu-.. tically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are' administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized .solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, sus- pension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
The following formulation examples illustrate representative pharmaceutical compositions of the present invention. Formulation Example 1
Hard gelatin capsules containing the following ingredients are prepared:
Figure imgf000071_0001
The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
Formulation Example 2
A tablet ■ formula is prepared using the in-; gredients below:
Figure imgf000071_0002
The components are blended and compressed to form tablets, each weighing 240 mg.
Formulation Example 3
A dry powder inhaler formulation is prepared containing the following components:
Figure imgf000071_0003
The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
Formulation Example 4
Tablets, each containing 30 mg of active ingredient, are prepared, as follows
Figure imgf000072_0001
The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules so produced are dried at 50 °C to 60 °C and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg. Formulation Example 5
Capsules, each containing 40 mg of medicament are made as follows:
Figure imgf000073_0001
The active ingredient, -starch' 'and magnesium stearate are blended, passed through, a No. 20 mesh U.S. sieve, and filled into hard gelatin cap- sules in 150 mg quantities,
Formulation Example 6
Suppositories, each containing 25 mg of active ingredient are made as follows:
Figure imgf000073_0002
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool. Formulation Example 7
Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as follows:
Figure imgf000074_0001
The active ingredient, sucrose and x'anthan. gum are blended, passed through a No. 10 mesh U.S. .*; sieve, and then mixed with a previously made solution of the microcrystalline cellulose 'and sodium carboxymethyl cellulose in water.. The sodium ben- : zoate, flavor, and, color are diluted with some of the water and added with stirring,, Sufficient water is then added to produce the required volume.
Formulation Example 8
Figure imgf000074_0002
The active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425.0 mg quantities. Formulation Example 9
A subcutaneous formulation may be prepared as follows:
Figure imgf000075_0001
Formulation Example 10
A topical formulation may be prepared as follows :
Figure imgf000075_0002
The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid.
Formulation Example 11
An intravenous formulation may be prepared as follows:
Figure imgf000075_0003
Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices ("patches") . Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts . The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent 5,023,252, herein incorpo- rated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
Frequently, it will be desirable or necessary to introduce the pharmaceutical composi- tion to the brain, either directly or indirectly. Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to bypass the blood brain barrier. One such implantable delivery system used for the transport of biological factors to specific anatomical regions of the body is described in U.S. Patent 5,011,472 which is herein incorporated by reference.
Indirect techniques, which are generally preferred, usually involve formulating the composi- tions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid soluble drugs. Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood brain barrier. Alterna- tively, the delivery of hydrophilic drugs may be enhanced by intra arterial infusion of hypertonic . solutions which can transiently open the blood brain barrier. 5 Other suitable formulations for use in the present invention can be found in Remington ' s Phar- : . maceutical Sciences, Mack -.Publishing Company, Philadelphia, PA, 17th ed. (1985) . •' • As noted above, the compounds described
10. herein are suitable, for use in a variety of drug • delivery systems described above. Additionally, in order to enhance the in vivo serum half life of the administered compound, the compounds may be encapsulated, introduced into the lumen of liposomes, 15 - prepared as a colloid, or other conventional techniques may be employed which provide an ..extended' serum half life of the compounds. A variety of methods are available for' preparing liposomes, as described in, e.g., Szoka et al., U.S. Patent Nos. 20 4,235,871, 4,501,728 and 4, 837, 028, each of which is • incorporated- herein by reference.
Utility
The compounds and/or compositions of this invention can be employed to bind VLA-1 in biolog- 25 ical samples, for instance in mammalian patients suspected of having a disease, condition or disorder mediated, at least in part, by VLA-1. Accordingly, these compounds have utility in, for example, assaying such samples for VLA-1 mediated adhesion. In addition, it is contemplated that compounds of this invention and/or pharmaceutical compositions thereof inhibit, in vivo, adhesion of mammalian cells to the extracellular matrix medi- 5 ated, at least in part by VLA-1 and, accordingly, .- can be used in the treatment, prevention, or amelioration of diseases;, conditions, or disorders whose progression or symptoms is regulated, at least in part, by VLA-1 expression or activity. Such di- 10. seases, conditions, or disorders include, but. are not limited to, inflammatory diseases, fibrotic . > diseases, and cancer.
For example, diseases, 'conditions,, and .-. disorders which are expected to be treatable by the 15- compounds and/or compositions of the present invention' include, but are- not limited to, asthma, - • ' trachoma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes (including acute juvenile onset diabetes), inflammatory bowel disease (including 20' ulcerative colitis -and Crohn's disease)-,- multiple sclerosis, rheumatoid arthritis, tissue transplantation, tumor metastasis, migration, and/or growth (including angiogenesis), proliferation of fibroblasts in cancer, solid tumors, meningitis, enceph- 25 alitis, stroke, and other cerebral traumas, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia, acute leukocyte-mediated lung injury such as that which occurs in adult respiratory distress syndrome, and fibrotic diseases, such 30 as fibrotic diseases of the lung, kidney, liver and vasculature (including idiopathic pulmonary fibro- sis, systemic sclerosis, glomerulonephritis, chronic hepatitis, chronic renal failure, and nonalcoholic steatohepatitis) .
Fibrotic diseases which are expected to be treatable by the compounds and/or compositions of the present invention include systemic sclerosis, mixed connective tissue disease, fibrodysplasia, fibrocystic disease, sarcoidosis, myositis (e.g., polymyositis, primary idiopathic polymyositis, childhood polymyositis, dermatomyositis, childhood dermatomyositis, primary idiopathic dermatomyositis in adults, inclusion body myositis, polymyositis, or dermatomyositis associated with malignant tumors) . Dermatomyositis can be associated with fibrosing or hypertrophic aspects, including fibrosing alveolitis and pulmonary fibrosis. Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis in such diseases. Amelioration includes reducing the rate of progression of a disease.
Among these fibrotic diseases are diseases that have as a manifestation fibrotic vascular intimal hypertrophy. These diseases include vascu- litis (including coronary artery vasculitis) , poly- arteritis nodosa or temporal arteritis. Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate vascular intimal hypertrophy in such diseases. These fibrotic diseases further include ■ diseases that have as a manifestation fibrotic hypertrophy of skin and/or muscle tissue. These diseases include scleroderma, eosinophilic fasciit- 5 is, discoid lesions associated with lupus or discoid ■.', lupus or surgical adhesions. Treatment using the ..'.- compounds and/or compositions of the present invention is expected to- treat, prevent, reduce, or ameliorate such indications, -or hypertrophy or 10.- fibrosis of skin or muscle tissue.
Fibrotic diseases further include ' diseases that have as a -manifestation fibrotic hypertrophy of , nerve tissue. These diseases include cerebroscler- .. osis, annular sclerosis. diffuse sclerosis and ■• 15' lobar sclerosis. Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent,- reduce-, ' or ameliorate such diseases, or hypertrophy, .fibrotic hypertrophy, or -.:-. fibrosis' of nerve tissue in such diseases. 20 These fibrotic diseases further include fibrotic lung diseases that have as a manifestation . fibrotic hypertrophy, or fibrosis of lung tissue. These diseases include pulmonary ' fibrosis (or interstitial lung disease or interstitial pulmonary 25. fibrosis), idiopathic pulmonary fibrosis, the fibrotic element of pneumoconiosis (which is associated with exposure to environmental hazards such as smoking, asbestos, cotton lint, stone dust, mine dust and other particles) , pulmonary sarcoidosis, 30 fibrosing alveolitis, the fibrotic or hypertrophic element of cystic fibrosis, chronic obstructive pulmonary disease, adult respiratory distress syndrome and emphysema. Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis in such diseases.
Such fibrotic diseases further include diseases that have as a manifestation fibrotic hypertrophy, or fibrosis of prostate, liver, the pleura (e.g., pleurisy, pleural fibrosis) or pancreas. These diseases include benign prostatic hypertrophy (BPH) , nonalcoholic steato hepatitis and fibrosis of the liver. Treatment using the compounds and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis in such diseases.
These fibrotic diseases further include diseases that have as a manifestation fibrotic. hypertrophy, or fibrosis of the kidney, such as chronic renal failure, lupus nephritis, alports syndrome, glomerulonephritis and diabetic nephritis. Treatment using the compound and/or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy, or fibrosis of the kidney.
Cancers which are expected to be treatable by the compounds and/or compositions of the present invention typically occur in mammals. Mammals in- elude, for example, humans and other primates, pet or companion animals, such as dogs and cats, labor- atory animals, such as rats, mice and rabbits, and farm animals, such as horses, pigs, sheep, and cattle.
Tumors or neoplasms include growths of
- 5 tissue cells in which -the multiplication of the cells is uncontrolled and progressive. Some such growths are benign,- but others are termed "malignant" and can lead to death of the organism. Malignant neoplasms or "cancers" are distinguished
1,0 from benign growths in that, in addition to exhibiting aggressive' cellular proliferation, they can •• ι . invade surrounding tissues and metastasize. Moreover, malignant neoplasms -are characterized in that •- ■ they show a greater loss of differentiation (greater -
15- "dedifferentiation") and - organization relative to •:. one another and to surrounding tissues-:.; This property is called "anaplasia. " •
Tumors or neoplasms which are- expected to ,- be treatable by the compounds and/or compositions of' 0 the present invention include, but are hot limited to., solid tumors, i.e., carcinomas, adenocarcinomas, ' and sarcomas. Carcinomas include those malignant neoplasms derived from epithelial cells, which infil- - trate (invade) the surrounding tissues and give rise 5 to metastases. Adenocarcinomas are carcinomas derived from granular tissue, or from tissues which form recognizable glandular structures. Another broad category of cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar 0 or homogenous substance like embryonic connective tissue. VLA-1 may be associated with adult and pediatric oncology in various forms of cancer, for example, growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, human soft tissue carcinoma (including Ewing's sarcoma) , cancer metastases (including lymphatic metas- tases), squamous cell carcinoma (particularly of the head and neck), esophageal squamous cell carcinoma, oral carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producing tumors, nonsmall cell cancers, breast cancer (including small cell carcinoma and ductal carcinoma) , gastrointestinal cancers (including stomach cancer, colon cancer, colorectal cancer, polyps associated with colorectal neoplasia, pancreatic cancer and liver cancer) , urological cancers (including bladder cancer, especially primary superficial bladder tumors, invasive transitional cell carcinoma of the bladder, and muscle-invasive bladder cancer -and prostate cancer) , malignancies of the female genital tract (including ovarian carcinoma, primary peritoneal epithelial neoplasms, cervical carcinoma, uterine endometrial cancers, vaginal cancer, cancer of the vulva, uterine cancer and solid tumors in the ovarian follicle) malignancies of the male genital tract (including testicular cancer and penile cancer) , kidney cancer (including renal cell carcinoma) , brain cancer (including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas and metastatic tumor cell invasion in central nervous system) , bone cancers (including osteomas and osteosarcomas) , skin cancers (including malignant melanoma, tumor progression of human skin keratinocytes and squamous cell cancer) , thyroid cancer, retinoblastoma, neuro- . 5 blastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms ' s tumors, gall bladder • ; cancer, trophoblastic neoplasms,, hemangiopericytoma, - and Kaposi's sarcoma. Cancers arid other cell pro-
- .:■ , liferative disorders treatable by the compounds
10 and/ox compositions of the present invention also include angiogenesis-mediated diseases, . benign - tumors (e.g., hemangiomas)., acoustic neuromas, neurofibromas, pyogenic granulomas, biliary tract cancer, choriocarcinoma, ' esophageal cancer, gastric ■
15' cancer, int aepithelial neoplasms, lung cancer, and' •• neuroblastom s . ; ,
The biological -activity of the compounds identified -above may be assayed in a variety of sys^- , terns.- For example, extracellular matrix, such as
20 collagen IV, can be immobilized on a solid surface
• • and adhesion of cells expressing -VLA-1 can be measured in the presence or absence of compound. Using such formats, large .numbers of compounds can be screened. Cells suitable for this assay include
25 smooth muscle cells, microvascular endothelial cells, fibroblasts, osteoblasts, chondrocytes, and activated cells of the immune system including effector T cells, macrophages and NK cells. A number of transfected cell lines can also be used,
30 including, for example, CHO, K562, and the like. The compounds and/or compositions of the invention can also be tested for the ability to inhibit binding between VLA-1 and extracellular matrix such as collagen IV, or between VLA-1 and a labeled compound known to bind VLA-1 such as a compound and/or composition of this invention or anti- bodies to VLA-1. -In these assays, the extracellular matrix can be soluble or immobilized on a solid surface. VLA-1 may also be expressed as a recombi- nant fusion protein having acidic and basic leucine- .. zipper tails so that binding to extracellular matrix may be detected in an immunoassay.
Many assay formats employ labeled assay components. ■ The labeling systems can be in a vari- ety of forms. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. A wide variety of labels may be used. The component may be labeled by any one of several methods. The most common method of detection is the use of autoradiography with 3H, 125I, 35S, 14C, or 32P labeled compounds, and the like. Nonradioactive labels include europium, as well as ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. The choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation. Appropriate in vivo models for demonstrating efficacy in treating inflammatory responses in- elude DTH (delayed type hypersensitivity) in mice, rats, guinea pigs, or primates, as well as other inflammatory or fibrotic models dependent upon VLA-1 integrin. Compounds having the desired biological activity may be modified as necessary to provide desired properties such as improved pharmacological properties (e.g., in vivo stability, bio-availability) , or the ability to be detected in diagnostic applications. Stability can be assayed in a variety of ways such as by measuring the half-life of the compounds during incubation with peptidases or human plasma or serum.
For diagnostic purposes, a wide variety of labels may be linked to the compounds, which may provide, directly or indirectly, a detectable signal. Thus, the compounds and/or compositions of the subject invention may be modified in a variety of ways for a variety of end purposes while still re- taining biological activity. In addition, various reactive sites may be introduced for linking to particles, solid substrates, macromolecules, and the like.
Labeled compounds can be used in a variety of in vivo or in vitro applications. A wide variety of labels may be employed, such as radionuclides (e.g., gamma-emitting radioisotopes such as tech- netium-99 or indium-Ill), fluorescers (e.g., fluorescein) , enzymes, enzyme substrates, enzyme cofac- tors, enzyme inhibitors, chemiluminescent compounds, bioluminescent compounds, and the like. Those of ordinary skill in the art will know of other suitable labels for binding to the complexes, or will be able to ascertain such using routine experimentation. The binding of these labels is achieved using standard techniques common to those of ordinary skill in the art.
In vitro uses include diagnostic applications such as monitoring inflammatory responses by detecting the presence of cells expressing VLA-1. The compounds and/or compositions of this invention ■ can also be used for isolating or labeling such cells. In addition, as mentioned above, the compounds and/or compositions of the invention can be used to assay for potential inhibitors of VLA- 1/Extracellular matrix interactions.
For in vivo diagnostic Imaging to identify, e.g., sites of inflammation,, radioisotopes are typically used in accordance with well known techniques. The radioisotopes may be bound to the com- pound either directly or indirectly using intermediate functional groups. For instance, chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules have been used to bind com- pounds to metallic ion radioisotopes.
The complexes can also be labeled with a paramagnetic isotope for purposes of m vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR) , both of which are well known. In general, any conventional method for visualizing diagnostic images can be used. Usually gamma- and positron-emitting radioisotopes are used for camera imaging and paramagnetic isotopes are used for MRI. Thus, the compounds can be used to monitor the course of amelioration of an inflamma- tory response in an individual. By measuring the increase or decrease in cells expressing VLA-1 it is possible to determine whether a particular therapeutic regimen aimed at ameliorating the disease is effective. Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. -Suitable formulations for use in the present invention are found in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).
The amount administered to the patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the progression or symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on the disease condition being treated as well as by the judgment of the attending clini- cian depending upon factors such as the severity of the disease, disorder or condition, the age, weight and general condition of .the patient, and the like.
The compounds administered to a patient are typically in the form of pharmaceutical composi- tions described above.- These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The .resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administra- ' tion.- The pH of the compound preparations typically, will be between about 3 and 11, more- preferably from about 5 to .-9, and most- 'preferably from about 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will .,- result in the formation o.f pharmaceutical salts. The therapeutic dosage of the compounds and/or compositions of the present invention will vary according to, for example, the particular' use. • ' for 'which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of .the prescribing physician. For example, for oral administration, the dose will typically be in the range of about 100 μg to about 50 mg per kilogram body weight per day, preferably about 5 mg to about 20 mg per kilogram body weight per day. In the alternative, for intravenous administration, the dose, will typically be in the range of about 20 μg to about 500 μg per kilogram body weight, preferably about 100 μg to about 300 μg per kilogram body weight. Alternative routes of administration contemplated include, but are not limited to, intranasal, transdermal, inhaled, subcutaneous and intramuscular. Effective doses can be extrapolated from dose-re- sponse curves derived from in vi tro or animal model test systems.
In general, the compounds and/or compositions of the subject invention will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5Q/ED50. Compounds that exhibit large therapeutic indices are preferred.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound and/or composition used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range which includes the IC50 (the concentration of the test compound which achieves a half-maximal inhibition of activity) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The following synthetic and biological examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees Celsius.
EXAMPLES
Unless otherwise stated all temperatures are in degrees Celsius. Also, in these examples and elsewhere, abbreviations have the following meanings:
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
The following analytical HPLC methods (or LC Methods) are referred to the Examples. The gradient profiles were straight-line gradients increasing or decreasing linearly over the time period indicated.
Method A: Varian HPLC System-':' (Pumps: Varian ProStar Solvent Delive.ry' System," Model 210; Detector: Varian ProStar UV-VIS Detector, Model
345; Autosampler: Varian ProStar Autosampler, Model 430). Analytical column: YMC ODS-AQ, 4.6x50 mm, S3 μ, Waters Corporation. Detection: 220 nm and 254ηm. Solvent A: H20, 0.1% TFA, 1.0%. IPA. Solvent B: cetonitrile, 0.05% TFA, 1.0% IPA. Flow Rate: 2.0 mL/min. Gradient Program: .O.OOmin 95% Solvent , A, 5% Solvent B; 0.05min.95% Solvent A, 5% Solvent B; 4.12 min 5% Solvent A, 95% Solvent B; 4.24min 5% Solvent A, 95% Solvent B;'4.30min 95% Solvent A, 5% Solvent B; 5.00min 95% Solvent A, 5% Solvent B.
Method B: Varian HPLC System: (Pumps: Varian ProStar Solvent Delivery System,' Model 210; Detector: Rainin Dynamax Absorbance Detector, Model UV-DII; Autosampler: Varian ProStar Autosampler, Model 430) . Analytical column: YMC ODS-AQ,
4.6x50mm, S3m, Waters Corporation. Detection: 220nm and 254nm. Solvent A: H20, 0.01% HFBA, 1.0% IPA. Solvent B: Acetonitrile, 0.01%HFBA, 1.0% IPA. Flow. Rate: 2.0 mL/min. Gradient Program: O.OOmin 95% Solvent A, 5% Solvent B; 0.12min 95% Solvent A, 5% Solvent B; 4.00 min 5% Solvent A, 95% Solvent B; 4.18min 5% Solvent A, 95% Solvent B; 4.30min 95% Solvent A, 5% Solvent B; 5.30mih 95% Solvent A,' 5% Solvent B.
Method C: Varian HPLC System: (Pumps: Varian ProStar Solvent Delivery System, Model 210; Detector: Varian ProStar PDA, Model 330; Autosampler: Varian ProStar Autosampler, Model 430) . Analytical column: YMC ODS-AQ, .6x50mm, S3m, '•' Waters Corporation. Detection: - 220nm and 254ήm. Solvent A: ,H20, 0,01% HFBA, 1.0% IPA. ''Solvent B: Acetonitrile, 0.01%HFBA, 1.0% IPA. Flow Rate: 2.0 • mL/min. Gradient - Program: O.OOmin 95% Solvent A, 5% Solvent B; 0.12min 95% Solvent A, 5% Solvent B; 4.00 rain 5% Solvent A, 95% Solvent B; 4.18min 5% Solvent A,- 95% Solvent B; 4.30min 5% 'Solvent -A, 95% Solvent B; 5.00min 95% Solvent A-,' 5% Solvent B : 5.30min 95%' .Solvent A, 5% Solvent B.
Method D: Berger SFC (Berger- Dual Pump Fluid Control Module, Model FCM-1200; Berger Thermal Control Module, Model TCM-2000; Hewlett Packard 1100 Series DAD, Model G1315A; Alcott Autosampler, Model 718AL) . Column: Chiralcel OJ-R 150x4.6 mm. Detection: 258nm. SFC Conditions: 2 mL/min, 10%MeOH ■ in C02, 35°C, 200 bar.
Method E: Agilent Technologies 1100 HPLC System (Pump: QuatPump Model G1311A; Detector: DAD, Model G1315B; Column Compartment: Model G1216A; Autosampler: ALS, Model G1313A; Degasser: Model G1322A) . Analytical column: Chiralcel OD-RH, 150x4.6 mm. Detection: 220nm. HPLC Conditions: 1.0 mL/min, Isocratic, 70% (H20, 0.01%TFA), 30% (ACN, 5' 0.01%TFA), 35°C.
Method F: Mass Spectrometer: Finnigan LCQ™Duo, APCI. TSP HPLC System: (Pump: TSP ■ SpectraSYSTEM® P4000; Detector:' ■ TSP SpectraSYSTEM® UV2000, 220nm and 254m; Autosampler: TSP Spectra-
10. SYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser)'.. Detection: .220nm and 254nm. Analytical -column: YMC ODS-AQ, - 4.6x50mm, . S3m, Waters . Corporation. Solvent C: H20, 0.01% HFBA, 1.0% IPA. .Solvent D : Acetonitrile, 0.01%
15 HFBA, 1.0% IPA. Flow Rate: 2.0 mL/miή: Gradient Program: O.OOmin 95% Solvent C,. 5% Solvent D; 0.02min 95% Solvent C, 5% Solvent D; 4.00 min 5% ' ' Solvent C, -95% -Solvent D-; '4.30mm 5% Solvent C, 95% Solvent D; ,4-.50min 95% Solvent C, 5% Solvent D;
20 5.50min 95% Solvent C, 5% Solvent D.
Method G: Mass- Spectrometer:, Finnigan LCQ™Duo, ESI. TSP HPLC System: . (Pump: TSP SpectraSYSTEM® P4000; Detector: TSP 'SpectraSYSTEM® UV2000, 220nm and 254m; Autosampler: TSP Spectra-
25 SYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser) . Detection: 220nm and 254nm. Analytical column: YMC ODS-AQ 4.6x50mm S3m, Waters Corporation. Solvent C: 10 mM Ammonium Acetate in H20. Solvent D: 10 M Ammonium Acetate
30 in Acetonitrile, 1.0% IPA. Flow Rate: 2.0 mL/min. ' Gradient Program: O.OOmin 75% Solvent C,25% Solvent D; 0.02min 75% Solvent C, 25% Solvent D; 4.00 in 5% Solvent C, 95% Solvent D; 4.30min 5% Solvent C, 95% Solvent D; 4.50min 75% Solvent C, 25% Solvent D; 5.50min 75% Solvent C, 25% Solvent D. Method H,: . Mass Spectrometer: Finnigan
■ LCQ™Duo, ESI. TSP HPLC System: (Pump: TSP SpectraSYSTEM® P4000; Detector: TSP SpectraSYSTEM® • UV2000, 220nm and 254m; Autosampler: TSP Spectra- •• SYSTEM® AS3000; Degasser: " TSP SpectraSYSTEM® Model : SCMIOOO Solvent Degasser) . Detection: 220nm and
254nm'. Analytical, column: Zorbax Extend C18 Rapid - - Resolution®,, 50x4."6mm, 3.5m, 8θA, Agilent Technol- . ogies. Solvent ,C: 10 mM Ammonium Acetate in H20. '■ ' Solvent D: 10 mM Ammonium Acetate in Acetonitrile,- ' 1.0%. IPA. Flow Rate: 2.0 mL/min. Gradient Pro- - gram: O.OOmin 95% Solvent C, 5%. Solvent D; 0.02min ,
95% Solvent C, 5% Solvent D; 4.00 min 5% Solvent C, : 95% Solvent D; 4.30min 5% Solvent C, 95% Solvent D; '. 4.50min 95% Solvent C, 5% Solvent D; 5.'50min 95% Solvent C, 5% Solvent D.
Method I: Mass Spectrometer:. Finnigan ,,- LCQ™Duo, ESI. TSP HPLC System: . (Pump: TSP Spec- ' traSYSTEM® P2000; Detector: TSP ■ SpectraSYSTEM® UV2000, 220nm and 254m; Autosampler: TSP Spectra- SYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCMIOOO Solvent Degasser) . Detection: 220nm and 254nm. Analytical column: Zorbax Extend C18 Rapid Resolution®, 50x4.6mm, 3.5m, 80A, Agilent Technologies. Solvent A: 10 mM Ammonium Acetate in H20. Solvent B: 10 mM Ammonium Acetate in Acetonitrile, 1.0% IPA. Flow Rate: 2.0 mL/min. Gradient Pro- gram: O.OOmin 95% Solvent A, 5% Solvent B; 0.02min 95% Solvent A, 5% Solvent B; 4.00 min 5% Solvent A, • 95% Solvent B; 4.30min 5% Solvent A, 95% Solvent B; 4.50min 95% Solvent A, 5% Solvent B; 5.50min 95% 5 Solvent A, 5% Solvent B.
Method J: Mass spectrometers: LCQ™Duo and LCQ™Deca. Pump: Series 1100, Quat pump model G1311A, Agilent Technologies. Detector: Series 1100, Column model G1216A, Agilent Technologies.
10 Autosampler:' Series 1100 ALS model G1313A, Agilent Technologies. Degasser: Series 1100, Degasser model G1322A, Agilent Technologies. ELSD: Sedex model 75, Sedere. Analytical column: YMC ODS-AQ 4.6x50mm S3μ, Waters Corporation. Solvent A: H20,
15. 0.01% HFBA, 1.0% IPA. Solvent B: Acetonitrile, 0.01%HFBA, 1.0% IPA. Flow Rate: 2.0 mL/min.
1 '- , Gradient Program: O.OOmin 95% Solvent A, 5% Solvent' B; 0.02min 95% Solvent A, 5% Solvent B; 4.10 min 5% '', Solvent A, 95% Solvent B;.4.30min 5% Solvent A, 95%
20 Solvent B; 4.50min 95% Solvent A, 5% Solvent B;
5.50min 95% Solvent A, 5% Solvent B. Purity based on 220 nm wavelength channel.
Figure imgf000099_0001
Example 1
(S) -N-{1- [3- (2, 3-Dichloro-phenyl) -acryloyl] -piperidin- 4-yl} -2-methyl-succinamic acid (55:45 regioisomeric mixture of 2-methyl and 3-methyl-succinamic acids)
Figure imgf000099_0002
Example 1A 3- (2, 3-Dichloro-phenyl) -acrylic acid
To a solution of 2, 3-dichlorobenzaldehyde (75 g, 429 mmol) in pyridine (220 mL) was added malonic acid (82 g, 788 mmol) and piperidine (3.5 g, 41 mmol) . The mixture was heated at 110 °C until gas evolution had ceased, and then heated for an additional 3 hours at that temperature. The mixture was concentrated in vacuo, dissolved in 3.0 M NaOH (1 L) and H20 (2 L) with heating at 90 °C to give complete dissolution, and then allowed to cool overnight. The resulting white crystalline solid was collected by filtration and washed with water. The aqueous filtrate was extracted with MTBE (2 L) and the crystalline solid was then combined with the basic aqueous layer. This mixture was acidified with 12 M HCl to pH 1 with stirring to produce a white solid, which was collected by filtration and washed with water. The solids were dried under high vacuum to give the indicated product as a white powder (82.7 g, 89%). XH NMR (acetone-d6, 400 MHz) δ 8.06 (d, J=16.0 Hz, 1H) , 7.86 (dd, J=7.8, 1.2 Hz, 1H) , 7.66 (dd, J=7.9, 1.3 Hz, 1H) , 7.44 (t, J=8.0 Hz, 1H) , 6.60 (d, J=15.6 Hz, 1H) .
Figure imgf000100_0001
Example IB
1- (4-Anιino-piperidin-l-yl) -3- (2, 3-dichlorophenyl) -propenone
A solution of Example 1A (5.43 g, 25.0 mmol) in DMF (25 L) was stirred with 4-N-Boc-amino- piperidine (7.51 g, 37.5 mmol), 1-hydroxybenzotri- azole hydrate (7.66 g, 50.0 mmol), N-methylmorph- oline (8.25 mL, 75.0 mmol), and 1- (3-dimethylamino- propyl) -3-ethylcarbodiimide hydrochloride (9.59 g, 50.0 mmol) under N for 16 h. The reaction was diluted with methylene chloride (200 mL) and water (200 mL) , and the phases were separated. The organic phase was then washed with IN aqueous HCl (2x100 mL) followed by a sat. solution of NaHC03 (200 mL) . The organic phase was dried (Na2S04) , filtered, and concentrated to a white solid (9.88 g, 99%) which was carried forward without purification. The Boc group was removed by stirring a suspension of the white solid (9.88 g, 24.7 mmol) with methanol (20 mL) , THF (100 mL) and 4N HCL in dioxane (54 mL, 124 mmol) under N2 for 16 h. The suspension was 5 diluted with diethyl ether (200 L) , and the white : solid was filtered, washing with diethyl ether (3x50 • mL) . , The solid was partitioned between methylene chloride (100 m'L) and IN aqueous NaOH (100 L) . The phases were separated, and the aqueous phase was re-0 extracted with methylene chloride (2x100 mL) . The combined organic phases were washed with brine (100 mL) , dried (Na2S0) , filtered, and concentrated to obtain the product as a waxy, white solid (7.0 g, 95%). αH NMR (CDC13, 400 MHz) δ 7.95 (d, J=15.2 Hz,5 1H), 7.48 (dd, J=7.81, 1.56 Hz, .1H) , 7.44 (dd,
J=8.20, 1.56 Hz, 1H) , 7.20 (m, 1H) , 6.85 (d, J=15.2 Hz, 1H), 4.58 (m, 1H) , 4.03 (m, 1H) , 3.19 (m, 1H , 2.97 (m, 1H), 2.87 (m, 1H) , 1.91 ( , 2H) , 1.27-1.55 (m, 4H) . LCMS (ESI + ; Method G) m/z 2.99, 301 (M+H)+;θ' Rt=2.00 min (220 nm, 100 area%) .
Figure imgf000101_0001
Example 1C
(S) -N-{1- [3- (2, 3-Dichloro-phenyl) -acryloyl ]-piperidin-4-yl }- 2-methyl-succinamic acid (55:45 regioisomeric mixture of 2-methyl and 3-methyl-succinamic acids) A solution of Example IB (54 mg, 0.18 mmol) in CHC13 (3.25 mL) was shaken (400 RPM) with (S) -methyl succinic anhydride (17 mg, 0.15 mmol; prepared from (S) -2-methyl succinic acid according to the literature: Davies, S. G.; Dixon, D. J. J. Chem . Soc , Perkin Trans . 1 1998, 2635-2643) for 16 h at room temperature. PS-Benzaldehyde (226 mg, 0.300 mmol) was added (Argoscoop®, Argonaut, Foster City, CA, USA) . The reaction was shaken (400 RPM) for an additional 6 h, and then filtered, rinsing the resin with methylene chloride (3x2 mL) . The filtrate was concentrated. A solution of IN aqueous HCl (5 mL) was added to precipitate the product. The solid was filtered, washing with water (2x2 L) . The product was dried (CaS04 dessicator) under high vacuum for 16 h. The product was obtained as a white powder (47 mg, 76%) . XH NMR (10% DMS0-d6 in CDC13, 400 MHz; 55:45 mixture of 2-methyl and 3- ethyl succinamide regioisomers, respectively) δ 7.96 (d, J=15.6 Hz, 2H) , 7.51 (m, 4H) , 7.24 (m, 2H) , 6.88 (d, J=15.6 Hz, 2H) , 4.53 (m, 2H) , 3.93-4.09 (m, 4H) , 3.29 (m, 2H) , 2.87-2.99 (m, 3H) , 2.70 (m, 2H) , 2.54 (m, 1H) , 2.38 (m, 1H) , 2.27 (m, 1H) , 1.92-2.07 (m, 4H), 1.42 (m, 4H) , 1.22 (d, J=7.03 Hz, 3H; CH3 of 2-methyl succinamide), 1.18 (d, J=6.25 Hz, 3H; CH3 of 3-methyl succinamide) . Regiochemical assignment was based on correlation of COSY, HMQC, HMBC and APT multidimensional NMR experiments. LCMS (APCI+; Method F) m/z 413, 415 (M+H)+; Rt=2.63 min (220 nm, 100 area%; regioisomers not separated) . Enantio- eric purity (>98%) was determined by chiral HPLC (Method E) : Example IC, 2-methyl succinamide: Rt=10.5 min (60 area%) , 3-methyl succinamide regioisomer: Rt=14.3 min (40 area%); chiral HPLC (Method E) analysis of a mixture of the enantiomers gave different retention times: Rt=16.2 min (46 area%), Rt=25.2 min (54 area%) . Neither of the "R" enantiomers were detected (<2%) by chiral HPLC of Example IC.
Figure imgf000103_0001
Example 2
N-{1- [3- (2, 3-Dichloro-phenyl) -acryloyl] -piperidin-4-yl}- 2-methylene-succinamic acid (78:22 regioisomeric mixture of 2-methylene and 3-methylene-succinamic acids)
Example 2 was prepared from Example IB (54 mg, 0.18 mmol) and itaconic anhydride (17 mg, 0.15 mmol) as detailed in Example IC to provide the title compound (33 mg, 53%) as a white solid. 1H NMR (DMS0-d6, 400 MHz, 78:22 mixture of regioisomers) δ 12.46 (bs, IH) , 8.01 (m, IH) , 7.96 (m, 0.2H, minor regioisomer), 7.91 (d, J=7.80 Hz, IH) , 7.79 ( , IH) , 7.68 (dd, J=7.80, 1.17 Hz, IH) , 7.35-7.45 (m, 2H) , 6.1 (d, J=1.95 Hz, IH) , 5.78 (m, 0.2H, minor regioisomer), 5.65 (d, J=1.56 Hz, IH) , 5.47 (m, 0.2 H, minor regioisomer), 4.23 (m, 2H) , 3.82 (m, IH) ,
3.18-3.30 (m, IH, overlaps with H20) , 3.08 (s, 2H) , 2.90 (m, IH) , 1.78 (m, 2H) , 1.30 ( , 2H) . Regio- chemical assignment was based on' correlation of COSY, HSQC, and HMBC multidimensional NMR experiments. LCMS (APCI+; Method F) m/z 411, 413 (M+H)+; Rt=2.63 min (220 nm, 67.4 area%) and 2.67 (220 nm, 23.9 area%) .
Figure imgf000104_0001
Example 3
N~{1- [3- (2, 3-Dichloro-phenyl) -acryloyl] -piperidin- 4-yl}-succinamic acid
The title compound was prepared from • Example IB (54 mg, 0.18 mmol) and succinic anhydride (15 mg, 0.15 mmol) as detailed in Example IC, with the following purification step added: the crude solid was dissolved in IN aqueous- NaOH (10 mL) and extracted with diethyl ether (3x10 mL) . The aqueous ;.' phase was then acidified with 6N aqueous HCl, and extracted with methylene chloride (3x20 mL) . The organic phase was dried (Na2S04) , filtered, and concentrated to obtain Example 3 as a white solid (22 mg, 37%). XH NMR (DMSO-d6, 400 MHz) δ 12.07 (bs, IH), 8.01 (dd, J=7.80, 1.17 Hz IH) , 7.87 (d, J=7.80, IH), 7.80 (d, J=15.2 Hz, IH) , 7.67 (dd, J=8.19, 1.56 Hz, IH) , 7.35-7.45 (m, 2H) , 4.23 (m, 2H) , 3.82 (m, IH) , 3.24 (m, IH) , 2.90 (m, IH) , 2.42 (m, 2H) , 2.31 (m, 2H) , 1.78 (m, 2H) , 1.28 (m, 2H) . LCMS (APCI+; Method F) m/z 399,-401 (M+H)+; Rt=2.55 min (220 nm, 100 area%) .
Various syntheses of Examples 4 through 16 are presented in Table 1 below.
Figure imgf000105_0001
Example 4
Cis-6- (l-{3- [2, 3-Dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) -cyclohex-3- enecarboxylic acid
Figure imgf000105_0002
Example 4A
(l-{'3- [2, 3-Dichloro-4- (2-methoxy-phenylsulfanyl) -phenyl] - acryloyl } -piperidin-4-yl ) -carbamic acid tert-butyl ester
The title compound was prepared from 3- [2, 3-Dichloro-4- (2-methoxy-phenylsulfanyl) -phenyl] - acrylic acid (1.30 g, 3.66 mmol; WO 00/59880, WO 00/39081) and 4-N-Boc-aminopiperidine (880 mg,
4.39 mmol) according to the amide coupling protocol for Example IB. The product (1.96 g, 100%) was obtained as a beige powder and carried forward to the next step without purification. 1H NMR (CDC13, 400 MHz) δ 7.92 (d, J=15.6 Hz, IH) , 7.47 (m, 2H) , 7.26 (m, IH, overlaps with CHC13) , 7.-02 (m, 2H) , 6.74 (d, J=15.6 Hz, IH) , 6.55 (d, J=8.98 Hz, IH) , 4.58 (m, IH) , 4.45 (m, IH) , 3.95 (m, IH) , 3.83 (s, 3H) , 3.69 (m, IH) , 3.19 (m, IH) , 2.85 (m, IH) , 2.02 (m, 2H) , 1.44 (s, 9H) , 1.33 (m, 2H) .
Figure imgf000106_0001
Example 4B
1- (4-Amino-piperidin-l-yl) -3- [2, 3-dichloro-4- (2- methoxy-phenylsulfanyl) -phenyl] -propeonone
The title compound was prepared from Example 4A (1.84 mg, 3.42 mmol) according to the Boc deprotection protocol described for Example IB. The product (1.27 g, 85%) was obtained as a fluffy, white powder after flash chromatographic purification (3-7% methanol in methylene chloride) . 1H NMR (CDCI3, 4Q0 MHz) δ 7.91 (d, J=15.6 Hz, IH) , 7.47 (m, 2H) , 7.27 (m, IH, overlaps with CHC13) , 7.02 (m, 2H) , 6.76 (d, J=14.5 Hz, IH) , 6.56 (d, J=8.2 Hz, IH) , 4.57 (m, IH) , 3.99 (m, IH) , 3.84 (s, 3H) , 3.15 ( , IH) , 2.94 (m, IH) , 2.83 (m, IH) , 1.89 (m, 2H) , 1.52 (bs, 2H, overlaps with HOD), 1.30 (m, 2H) . Analytical HPLC (Method A) : Rt=2.83 min (94.5 area%, 220 nm) .
Figure imgf000107_0001
Example 4C (Procedure A in Table 1)
Cis-6- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) -cyclohex-3- enecarboxylic acid
A 24-well format, Bohdan block was charged with a solution of Example 4B in chloroform (0.084 M, 1.0 mL, 0.084 mmol), and a solution of cis- 1, 2, 3, 6-tetrahydrophthalic anhydride in chloroform (0.24 M, 0.29 mL, 0.070 mmol, via Packard liquid handler) . The block was shaken for 16 h at 500 RPM (Bohdan block shaker) . PS-Benzaldehyde (105 mg, 0.14 mmol, via Argoscoop®, Argonaut, Foster City, CA, USA) and chloroform (1.0 mL) were added, and the block was shaken for an additional 16 h at 600 RPM. The reaction was filtered rinsing the resins with methylene chloride (10x0.5 mL) . The filtrate was concentrated, and the crude product was purified by partial dissolution (digestion) in hot toluene followed with cooling to room temperature and filtration. Example 4C was analyzed by LCMS (results in Table 1) .
Figure imgf000108_0001
Example 9 (Proceudre B in Table 1)
N- (1-{3- [2 3-Dichloro-4- (2-methoxy-phenylsulfanyl) ■ phenyl] -acryloyl}-piperidin-4-yl) - (S) -2- (2,2, 2- trifluoro-acetylamino) -succinamic acid
After synthesis according "to the procedure described for Example 4C, but omitting the purification step (digestion in toluene)', a solution of Example 9 (0.140- mmol, prepared on twice the scale) in methylene chloride (10 mL) was shaken with more of the scavenging reagent' MP-TsOH (183 mg, 0.28 mmol)- for 2 h at 300 RPM. • The reaction was filtered rinsing the resins with methylene chloride (10x0.5 mL) . The filtrate was concentrated, and Example 9 • was analyzed by LCMS (results in Table 1) .
Figure imgf000108_0002
Example 14
4- (l- { 3- [2 , 3-Dichloro-4- (2-methoxy-phenylsulfanyl) phenyl] -acryloyl } -piperidin-3-ylcarboamoyl) -3- methyl-butyric acid
Figure imgf000109_0001
Example 14A
( l- { 3- [2 , 3-Dichlbro-4- ( 2-methoxy-phenylsulfanyl ) phenyl ] -acryloyl } -piperidin-3-yI ) -carbamic acid tert-butyl ester "
The title compound was prepared from 3- [2, 3-Dichloro-4- (2-methoxy-phenylsulfanyl) -phenyl] - acrylic acid (1.30 g, 3.66' mmol; WO 00/59880, WO 00/39081) and 3-N-Boc-aminopiperidine (880 mg, 4.39' mmol) according to the amide coupling protocol for Example IB. The product (1.89 g, 96%) was' obtained as a white powder and carried' forward' to the next step without purification. 1H'NMR (CDC13, 400 MHz) δ 8.00 (br d, J=12.1 Hz, IH), -7.47 (m, 2H) , 7.30 (m, IH-)1, 7.02 (m, 2H) , 6.79 (br d, J=12.9 Hz, IH) , 6.55 (d, J=8.59 Hz, IH) , 4.60 (bs, IH) , 3.89 (m, IH) , 3.83 (s, 3H) , 3.66 (m, 3H) , 3.45 (m, IH) , 1.91 (m, IH), 1.6-1.8 ( , 3H) , 1.3-1.5 (m, 9H) .
Figure imgf000110_0001
Example 14B
1- (3-Amino-piperidin-l-yl) -3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) -phenyl] -pfopenone
The title compound was prepared from Example 14A (1.77 g, 3.29 mmol) according to the Boc deprotection protocol described for Example IB. The product (1.30 g, 90%) was obtained as a fluffy, white powder after flash chromatographic purification (3-7% methanol in methylene chloride) Η NMR
(CDC13, 400 MHz) δ 7.93 (br d, J=15.6 Hz, IH) , 7.47 (m, 2H), 7.26 (m, IH, overlaps with CHCI3) , 7.02 (m, 2H) , 6.77 (m, IH) , 6.56 (d, J=8.59 Hz, IH) , 4.21-
4.48 (m, 1 H) , 3.84 (m, 4H) , 2.60-3.17 (m, 3H) , 1.99 (m, IH) , 1.78 (m, IH) , 1.53 (m, 2H) , 1.34 (m, 2H) . Analytical HPLC (Method A): Rt=2.86 min (95.9 area%, 220 nm) .
Figure imgf000111_0001
Example 14C (Procedure C in Table 1)
4- (l-{3-[2,3-Dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-3-ylcarbamoyl) -3- methyl-butyric acid
The title compound was prepared according to the same procedure described for Example 4C, except using the product in 14B in place of the product from 4B, and the purification step (digestion in toluene) was omitted. Example 14C was analyzed by LCMS (results' in Table 1) .
There is no Example 17.
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0002
The detailed description of the observed mass is described in the designated LC Method.
Figure imgf000114_0001
Figure imgf000115_0001
Example 18
N- (l-{3- [2, 3-Dichloro-4- (4-fluoro-phenylsulfanyl) phenyl] -acryloyl}-piperidin-4-yl) -malonamic acid
H
Figure imgf000115_0002
Examp] .e 18A
N- (l-Benzyl-piperidin-4-yl) malonamic acid ethyl ester
A, stirred mixture of 4-amino-l-benzyl- piperidin'e (1.00 g, 5.25 mmol) and diethyl malonate (7.90 mL, 52.0 mmol) was heated to 100°C under N2 for 7 h. The reaction was allowed to cool "to room temperature and stirring was continued for 16 h. The crude product was purified by flash chromatography, using methylene chloride to elute the excess diethyl malonate, followed by 5% methanol in methylene chloride to elute the product. Example 18A (1.00 g, 63%) was obtained as a yellow oil which solidified upon standing. XE NMR (CDC13, 400 MHz) δ 7.29-7.32 ( , 4H) , 7.26 (m, IH) , 7.07 (bd, J=6.64 Hz, IH) , 4.20 (q, J=7.03 Hz, 2H) , 3.84 (m, IH) , 3.49 (s, 2H) , 3.28 (s, 2H), 2.78 (m, 2H) , 2.15 ( , 2H) , 1.91 (m, 2H), 1.53 (m, 2H) , 1.29 (t, J=7.03 Hz, 3H) .
Figure imgf000116_0001
Example 18B N-Piperidin-4-yl-malonamic acid ethyl ester
A solution of Example 18A (1.00 g, 3.29 mmol) in absolute EtOH (13 mL) was stirred with Pd on C (348 mg, 0.164 mmol; 10% Pd, 50% water, Degussa type) under an H2 atmosphere for 16 h. The suspension was filtered through celite, rinsing with EtOH. The filtrate was concentrated. The resulting resi- due was triturated with diethyl ether and filtered. The resulting sticky solid was dissolved in methylene chloride and concentrated to give Example 18B as a foamy, yellow solid. XH NMR (CDC13, 400 MHz) δ 7.62 (bd, J=7.42 Hz, IH) , 4.19 (q, J=7.16 Hz, 2H) , 4.06 (m, IH), 3.43 (m, 2H) , 3.29-3.34 (m, 2H) , 2.98 (m, 2H), 2.14 (m, 2H) , 1.87 (m, 2H) , 1.29 (t, J=7.22 Hz, 3H) .
Figure imgf000117_0001
Example 18C
N- (l-{3-[2,3-Dichloro-4-(4-fluoro-phenylsulfanyl) phenyl] -acryloyl }-piperidin-4-yl) -malonamic acid ethyl ester
The title compound was prepared from 3- [2, 3-dichloro-4- (4-fluoro-phenylsulfanyl) -phenyl] - acrylic acid (415 mg, 1.21 mmol; synthesized in a similar manner to the acrylic acid used in Example 4A by methodology described in WO 00/59880, WO 00/39081 except that 4-fluorothioρhenol was substituted for 2-methoxythiophenol) and Example 18B (390 mg, 1.8 mmol) according to amide coupling protocol described for Example IB. The product (650 mg, ,100%) was obtained as an off-white solid. Η NMR
(DMS0-d6, 400 MHz) δ 8.31 (bd, J=7.81 Hz, IH) , 7.90 (d, J=8.59 Hz, IH) , 7.73 (d, J=15.2 Hz, IH) , 7.66 (m, 2H), 7.41 (m, 2H) , 7.29 (d, J=15.6 Hz, IH) , 6.67 (d, J=8.59 Hz, IH), 4.24 (m, IH) , 4.12 (m, IH) , 4.06 (q, J=7.16 Hz, 2H) , 3.83 (m, IH) , 3.22 (s, 2H) , 2.96 (m, 2H) , 1.77 (m, 2H) , 1.31 (m, 2H) , 1.17 (t, J=7.03 Hz, 3H) . LCMS (APCI+; Method F) m/z 539, 541 (M+H)+; Rt=3.77 min (220 nm, 86.6 area%) .
Figure imgf000118_0001
Example 18D
N- (1-{3- [2, 3-Dichloro-4- (4-fluoro-phenylsulfanyl) phenyl] -acryloyl}-piperidin-4-yl) -malonamic acid
A solution of Example 18C (650 mg, 1.20 mmol) in 1:1 methanol/THF (6 mL) was stirred with 2.4 M aqueous LiOH (1.0 mL, 2.4 mmol) for 2.5 h. 5 The solvent was removed in vacuo, and the residue was suspended in H20 (10 mL) and extracted with ethyl acetate (10 mL) . The aqueous phase was acidified by dropwise addition of 1M aqueous HCl, and then extracted with ethyl acetate (10 mL) , dried (Na2S04) ,Q filtered, and concentrated to give Example 18D as an off-white solid (200 mg, 33%) . lH NMR (DMS0-d6, 400 MHz) δ 12.43 (bs, IH) , 8.05 (d, J=7.42 Hz, IH) , 7.87 (d, J=8.59 Hz, IH) , 7.73 (d, J=15.2 Hz, IH) , 7.65 (m, 2H) , 7.4 (m, 2H) , 7.26 (d, J=15.6 Hz, IH) , 6.665 (d, J=8.59 Hz, IH) , 4.25 (m, IH) , 4.09 (m, IH) , 3.82 (m, IH) , 3.23 (m, IH) , 3.10 (s, 2H) , 2.92 (m, IH) , 1.79 (m, 2H) , 1.28 (m, 2H) . LCMS (APCI+; Method F) m/z 511, 513 (M+H)+; Rt=3.44 min (220 nm, 95.4 area%) .
Figure imgf000119_0001
Example 19
N- (l-{3- [2, 3-Dichloro-4- (4-fluoro-phenylsulfanyl) phenyl] -acryloyl}-piperidin-4-yl) -2-fluoro- malonamic acid
Figure imgf000119_0002
Example 19A
N- (l-Benzyl-piperidin-4-yl) -2-fluoro-malonamic. acid ethyl ester
The title compound was prepared according to the procedure for Example 18A/ except using diethyl fluoromalonate in place of diethyl malonate. X NMR (CDC13, 400 MHz) δ 7.23-7.34 (m, 5H) , 6.24 (bd, J=5.86 Hz, IH) , 5.22 (d, 2JHF=49.2 Hz, IH) , 4.33 (m, 2H), 3.84 (m, IH) , 3.49 (s, 2H) , 2.82 (m, 2H) , 2.13 (m, 2H) , 1.92 (m, 2H) , 1.53 (m, 2H) , 1.33 (m, 3H) . LCMS (APCI+; Method F) m/z 323 (M+H)+; Rt=1.83 min (220 nm, 100 area%) .
Figure imgf000120_0001
Example 19B 2-Fluoro-N-piperidin-4-yl-malonamic acid ethyl ester
The title compound was prepared according to the procedure for Example 18B, except using the product of Example 19A in place of the product from 18A and the diethyl ether trituration was not performed. XH NMR (CDC13, 400 MHz) δ 6.23 ' (m, IH) , 5.23 (d, 2JHF =48.8 Hz, lH),-4.34 (m, • 2H) , 3.92 (m, IH) , 3.09 (m, 2H) , 2.69 (m, 2H) , 1.94 (m, 2H) , 1.32-1.42 (m, 5H) . MS (ESI+) m/z 233 (M+H)+.
Figure imgf000120_0002
Example 19C
N- (l-{3- [2, 3-Dichloro-4- (4-fluoro-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -2-fluoro- malonamic acid ethyl ester
The title compound was prepared from 3- [2, 3-dichloro-4- (4-fluoro-phenylsulfanyl) -phenyl-] - acrylic acid (98 mg, 0.287 mmol; synthesized in a similar manner to the acrylic acid used in Example 4A by methodology described in WO 00/59880, WO 00/39081 except that 4-fluorothiophenol was substituted for 2-methoxythiophenol) and Example 19B (100 mg, 0.431 mmol) according to amide coupling protocol described for Example IB, with the following purification step added. The product was purified by flash chromatography eluting with 3% meth- anol in methylene chloride. Example 19C (87 mg, 85%) was obtained as a white foam. 1H NMR (DMS0-d6, 400 MHz) δ 8.56 (bd, J=7.81 Hz, IH) , 7.87 (d, J=8.59 Hz, IH) , 7.73 (d, J=15.6 Hz, IH) , 7.65 (m, 2H) , 7.38 (m, 2H) , 7.27 (d, J=15.2 Hz, IH) , 6.66 (d, J=8.20 Hz, IH) , 5.45 (d, 2JHF =47.6 Hz, IH) , 4:33 (m, IH) , 4.11-4.25 (m, 3H) , 3.89 (m, IH) , 3.20 (m, IH) , 2.85 (m, IH), 1.72-1.81 (m, 2H) , 1.37 (m, 2H) , 1.20 (t, J=7.03 Hz, 3H) . LCMS (APCI+; Method F) m/z 557, 559 (M+H)+; Rt=3.88 min (220 nm, 86.1 area%) .
Figure imgf000121_0001
Example 19D
N- (l-{3- [2,3-Dichloro-4- (4-fluoro-phenylsulfanyl; phenyl] -acryloyl}-piperidin-4-yl) -2-fluoro- malonamic acid
The title compound was prepared from Example 19C (75 mg, 0.14 mmol) according to saponifica- tion conditions described for Example 18D, except that the aqueous phase was not extracted with ethyl acetate before acidification with 1 M aqueous HCl. The resulting precipitate was filtered, washed with CH3CN (5 mL) , and dried under vacuum to obtain Example 19D (50 mg, 70%) as a white solid. XH NMR (DMSO-d6, 400 MHz) δ 8.48 (d, J=7.81 Hz, IH) , 7.87 (d, J=8.59 Hz, IH) , 7.73 (d, J=15.2 Hz, IH) , 7.66 (m, 2H) , 7.40 (m, -2H) , 7.27 (d, J=15.2 Hz, IH) , 6.66 (d, J=8.98 Hz, IH) , 5.27 (d, 2JHF=48.8 Hz, IH) , 4.32 (m, IH) , 4.14 ( , IH) , 3.88 (m, IH) , 3.2-3.5 (m, IH, buried under H20 peak), 2.:85 (m, IH) , 1.75 (m, .2H), 1.36 (m, 2H) . LCMS (APCI+; Method F with an alternative gradient program: O.OOmin 75% Solvent C, 25%- Solvent D; 0.02min 75% Solvent C, 25% Solvent - D; 4.00 min 5% Solvent C, 95% Solvent D; 4.30min 5% Solvent C, 95% Solvent D; 4.50min 75% Solvent C, 25% Solvent D; 5.50min 75% Solvent C, 25% Solvent D) m/z 529, 532 (M+H)+; Rt=3.01 min (220 nm, 100 area%).
Figure imgf000122_0001
Example 20
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl}- (S) -3-methyl- succinamic acid
Figure imgf000123_0001
. . Example 20A (2, 3-Dichloro-phenylsulfanyl) -acetic acid
A 2 L, 3-neck flask, with a mechanical stir shaft, was charged with 2, 3-dichlorothiophenol (50.0 g, 279 mmol) and bromoacetic acid (40.7 g, 293 mmol) . The mixture was suspended in water (300 mL) and 3 M aqueous NaOH (300 L, 900 mmol) was added. A reflux condenser was attached and a thermocouple was placed in the remaining port. The mixture was heated to 95°C internal temperature. The mixture was refluxed 15 h and then cooled to room temperature. Once the mixture had cooled, 4 M aqueous HCl (400 mL, 1.6 mol) was added and the mixture extracted with ethyl acetate (2x1.25 L) . The combined organic layers were' dried (MgS04) , filtered and con- centrated to an off-white solid. To the solids was added hexanes (150 mL) and the mixture heated to reflux with stirring for 15 min. The mixture was cooled to room temperature and collected by filtration and washed with additional hexanes (100 L) . The solid was dried under vacuum affording Example 20A (55.6 g, 84%) as a white solid. 1H NMR (acetone d6, 400 MHz) δ 7.39-7.43 (m, 2H) , 7.31-7.37 (m, IH) , 3.96 (s, 2H) .
Figure imgf000124_0001
Example 2OB
(4-Bromo-2, 3-dichloro-phenylsulfanyl) acetic acid
To a solution of Example 20A (58.0- g, 245 mmol) in acetic acid (250 mL) .was- added, iron chips (3.5 g, 63 mmol). The mixture was gently heated to, dissolve Example 20A. The.- mixture was .cooled to room temperature and bromine (25. mL, 490. mmol) was added over 20 min by addition funnel. The reaction was stirred for 4 days, then more bromine (10 mL) was added in one portion -and stirring continued for 5 h. Next, a third bolus . of bromine (10 mL) was added and the mixture stirred 12 h. The mixture was then quenched by careful addition of a saturated solution of NaHS04 and stirred for 45 min. A white precipitate was collected by filtration and washed with water. The solid was dried, under vacuum for two days in a desiccator, followed by six hours. in a vacuum oven at 70°C, affording Example 20B (76.7 g, . 99%) containing 90% bromination at the 4-position. XH NMR (acetone d6, 400 MHz) δ 7.70 (d, J=8.98 Hz, IH) , 7.35 (d, J=8.59 Hz, IH) , 4.00 (s, 2H) .
Figure imgf000125_0001
Example 20C 5-Bromo-6, 7-dichloro-2, 3-dihydro-benzo [b] thiophen-3-ol
To a suspension of Example 20B (50.0 g, 158 mmol) in THF (160 mL) was added 3 drops of DMF followed by oxalyl chloride (21.1 g, 166.1 mmol) over a 15 min period. The mixture was heated to
50°C for 3 h. Next, the mixture was cooled to room temperature and concentrated in vacuo to obtain an intermediate acid chloride (51.3 g) as a dark yellow oil that was used in the next step without purifica- tion. A I L flask was charged with A1C13 (179 g,
1.35 mol) and methylene chloride (250 L) , and then cooled to -78 °C (dry ice/acetone). A solution of the acid chloride (prepared above) in methylene chloride (150 mL) was added dropwise over 20 min. When addition was complete the dry ice bath was removed and the reaction was allowed to warm to room temperature and stir for an additional 1 h. The mixture was quenched by slowly pouring onto ice (2 L) . The mixture was extracted with ethyl acetate (2x1 L) , and the combined organic phases were washed with brine (500 mL) . The red organic layer was dried (MgS04) , filtered and concentrated to an orange solid. The solid was dried under high vacuum for 14 h to give an intermediate ketone, which was used in the next step without purification. The ketone was resuspended in ethanol (500 mL, absolute) and NaBH4 (12 g, 316 mmol) was added portionwise while cooling on an ice bath. The reaction was warmed to room temperature after addition was complete. After 2 h, 5 the reaction was transferred to a 4 L Erlenmeyer flask and quenched with 4 M aqueous HCl (caution, vigorous gas evolution) . The mixture was concentrated in vacuo, diluted with water (200 mL) , and extracted with ethyl acetate (2x500 mL) . The 0 combined organic phases were washed with brine (250 mL) , dried (MgS0) , filtered and concentrated to give a dark brown oil (45 g) . Purification by chromatography on silica gel (Biotage Flash 75 L, 10% ethyl acetate in hexanes followed by 20% ethyl acetate in5 hexanes) afforded Example 20C (23. Og, 48% for 3 steps) as a red oil that solidified upon standing. 1H NMR (CDC13, 400 MHz) δ 7.53 (d, J=0.78 Hz, IH) , 5.44 (m, IH) , 3.66 (dd, J=12.1, 6.63 Hz, IH) , 3.35 (dd, J=12.1, 4.68 Hz, IH) , 2.23 (d, J=8.19 Hz, IH) .
Figure imgf000126_0001
Example 20D (-j 5-Bromo-6, 7-dichloro-benzo [b] thiophene
To a 1 L flask was added Example 20C (23. Og, 76.7 mmol) and acetic acid (230 mL) . The mixture was sonicated for 30 min. BF3-Et20 (15 mL, 115 mmol) was added dropwise over 1 min at room temperature. After 15 min the homogeneous mixture was diluted with water (500 mL) and a white precipitate formed. After stirring for an additional 15 min, the solid was collected by filtration and washed with water (200 mL) . The solid was dissolved in ethyl acetate (500 L) and washed with a saturated solution of NaHC03 (200 mL) followed by brine (200 mL) . The organic phase was- dried (MgS04) , filtered and concentrated. The resulting pink solid was recrystallized from boiling hexanes . (200 mL) . The crystals (14.3 g) that formed had a dark red color. The mother liquor was concentrated to give a . tan solid (6.6 g) which was combined with the first crop to yield Example 20D (20.9 g, 97%).- XH NMR (CDC13, 400 MHz) δ.7.97 (s, IH) , 7.51 (d, J=5.47 Hz,. 1H), 7.24 (d, J=5..47 Hz, IH) .
Figure imgf000127_0001
Example 20E
3- (6, 7-Dichloro-benzo [b] thiophen- 5-yl) -acrylic acid methyl ester
A 250 mL round bottom flask was charged with Example 20D (9.80 g, 35.0 mmol), Pd2(dba)3 (796 mg, 0.869 mmol), and o-(Tol)3P (809 mg, 2.66 mmol). The flask was purged with N2 (3x) ,. and then charged with anhydrous DMF (70 mL) , methyl acrylate (8.98 g, 104 mmol) and triethylamine (14.5 mL, 104 mmol). The reaction was purged with N2 (2x) again. The mixture was stirred under N2 at 100 °C for 16h. The reaction was cooled to room temperature and a thick precipitate formed. The suspension was partitioned between methylene chloride (300 mL) and water (100 mL) . The phases were separated, and the organic phase was washed with 0.5 N aqueous HCl (4x100 mL) . The organic phase was dried (Na2S0) , filtered and concentrated to obtain a beige fluffy solid (11.2 g) . The crude product was purified by partial dissolution (digestion) in hot heptanes followed by cooling to room temperature and filtration to give Example 20E (8.22g, 82%). XH NMR (CDC13, 400 MHz) δ 8.18 (d, J=16.0 Hz, IH) , 7.97 (s, IH) , 7.53 (d, J=5.47 Hz, IH) , 7.36 (d, J=5.47 Hz, IH) , 6.48 (d, J=15.6 Hz, IH) , 3.85 (s, 3H) .
Figure imgf000128_0001
Example 2OF
3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acrylic acid
The title compound was prepared from Example 20E (8.22 g, 28.6 mmol) according to the procedure described for Example 18D. Example 20F was obtained as an off-white powder (6.60 g, 84%) after recrystallization from hot toluene. 1H NMR (10% DMSO-de in CDC13, 400 MHz) δ 8.12 (d, J=16.4 Hz, IH) , 8.06 (s, IH) , 7.60 (d, J=5.47 Hz, IH) , 7.41 (d, J=5.47 Hz, IH) , 6.49 (d, J=16.0 Hz, IH) .
Figure imgf000129_0001
Example 20G
{1- [3- (6, 7-Dichloro-benzo [b] thiophen- 5-yl) -acryloyl] -piperidin-4-yl}-carbamic acid tert-butyl ester
Example 20F (2.00 g, 7.32 mmol) was reacted with 4-N-Boc-amino-piperidine (2.20 g, 11.0 mmol) according to the amide coupling protocol described for Example IB. Example 20G was obtained as an orange-colored solid (3.34 g, 100%) which was carried forward without purification. 1H NMR (CDC1 , 400 MHz) δ 8.08 (d, J=15.-6 Hz, IH) , 7.94 (s, IH) , 7.52 (d, J=5.47 Hz, IH) , 7.35 (d, J=5.47 Hz, IH) , 6.90 (d, J=15.2 Hz, IH) , 4.65 (m, IH) , 4.49 (m, IH) , 4.06 (m, IH) , 3.73 (m, IH) , 3.26 (m, IH) , 2.89 (m, IH), 2.05 (m, 2H) , 1.46 (s, 9H) , 1.40 (m, 2H) . LCMS (APCI+; Method, F) m/z 455, 457 (M+H)+; Rt=3.77 min (220 nm, 100 area%) .
Figure imgf000130_0001
Example 2OH
1- (4-Amino-piperidin-l-yl) -3- (6, 7-dichlorobenzo [b] thiophen-5-yl) -propenone
The Boc group was removed from Example 20G (4.52 g, 9.93 mmol) according to the procedure described for Example IB. Example 2OH was obtained as a waxy, creme-colored solid (3.10 g, 88%). Η NMR
(DMSO-de, 400 MHz) δ 8.55 (s, IH) , 7.98 (d, J=5.46 Hz, IH) , 7.89 (d, J=15.2 Hz, IH) , 7.56 (d, J=5.46 Hz, IH) , 7.42 (d, J=15.2 Hz, IH) , 4.26 (dd, J=40.9, 13.3 Hz, 2H) , 3.20 (t, J=ll.l Hz, IH) , 2.86 (m, 2H) , 2.15 (bs, 2H) , 1.78 (m, 2H) , 1.18 (m, 2H) . LCMS (ESI+; Method H) m/z 355, 357 (M+H)+; Rt=2.42 min (220 nm, 100 area%) .
Figure imgf000130_0002
Example 201
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen- 5-yl) -acryloyl] -piperidin-4-yl}-3- methyl-succinamic acid (1:1 regioisomeric mixture of 2-methyl and 3-methyl- succinamic acids) A solution of Example 20H (312 mg, 0.88 mmol) in methylene chloride (10 mL) was stirred with DIEA (230 μL, 1.32 mmol) and (S) -methyl succinic anhydride (100 mg, 0.88 mmol; prepared from (S)-2- methyl succinic acid according to the literature: Davies, S. G. ; Dixon, D. J. J. Chem . Soc , Perkin Trans . 1 1998, 2635-2643) under N2 for -1 h. The reaction was diluted with methylene chloride (20 mL) and methanol (5 mL) and washed with IN aqueous HCl (10 mL) . The aqueous phase was back-extracted with 10% methanol in methylene chloride (2x20 mL) . The combined organic phases were washed with brine (20 mL) , dried (Na2S04) , filtered, and concentrated to obtain a waxy solid (412 mg, 100%) which contained a 1:1 mixture of Example 2ON and its regioisomer
Example 21 as analyzed by chiral HPLC (Method D) : Example 20N: Rt=14.2 min (50.2 area%) and Example 21: Rt=19.8 min (49.8 area%) . LCMS (ESI-; Method H) m/z 467, 469 (M-H) -; Rt=2.40 min (220 nm, 100 area%; regioisomers not separated) .
Figure imgf000131_0001
Example 20J
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl] -piperidin- 4-yl}- (S) -3-methyl-succinamic acid 9H-fluoren-9- ylmethyl ester A solution of the 1:1 regioisomeric mixture of carboxylic acids from Example 201 (412 mg, 0.88 mmol) in methylene chloride (20 mL) was stirred with DMAP (21 mg, 0.18 mmol) and (9H-fluoren-9-yl) - 5 methanol (258 mg, -1.-3-2 mmol) . The reaction was cooled to 0°C under N and dicyclohexylcarbodiimide (272 mg, 1.32 mmol) was added. The reaction was stirred for 16 h at rt under N2.. The resulting suspension was filtered, rinsing the solid .with ethyl-
10: ene chloride. The filtrate was concentrated and the. regioisomers. were separated by Biotage Flash 40 using the following, solvent gradient: 3:1 EtOAc/- hexanes (500 mL) , 9:1 EtOAc/hexanes (500 mL) , and neat EtOAc (1 L) . , Mixed fractions were rechromato--
15 graphed using a similar elution 'gradient. Example . 20J (152 mg, 53% of theoretical recovery) was obtained as a white powder. : αH NMR (DMS0-d6, 400 MHz)
• ' δ 8.55 (d, J=3.12 Hz, IH) , 7.98 (d, J=5-.46 Hz, IH) , 7.90 (m, 4H) , 7.67 (d, J=7:.02 Ez; 2H) , 7.55 (m, IH) ,
20 7.42' (m, 3H) , 7.34 _ (m, 2H) , 4.32 (m, 3H) , 4.24 (m, 2H) , 3.83 (m, IH) , 3.26 (m, IH) , 2.89 (m, IH) , 2.67 (m, 2H) , 2.36 (m, IH) , 1.77 (m, 2H) , 1.29 (m, 2H) , 1.02 (d, J=4.68 Hz, 3H) .
Figure imgf000132_0001
Example 2OK -Benzyl-3-propionyl-oxazolidin-2-one Adapted from Levy, D.E. et al., J. Med. Chem . 1998, 41, 199-223. To an oven-dried 2L flask under N2 was added (R) -4-benzyl-oxazolidin-2-one (30.0 g, 169.3 mmol) and THF (300mL, 99.9% anhy- drous) via canula. The flask was cooled to -78°C (dry ice/acetone). n-BuLi (70 mL, 169 mmol, 2.41 M in hexanes) was added dropwise over a 30 min period. After the addition was complete, the mixture was stirred for 30 min and propionyl chloride (17.2 g, 186 mmol) was added dropwise. Stirring was continued for l^h at -78 °C. The reaction was allowed to warm to room temperature over 1 h and quenched by pouring into a 1 L separatory funnel containing saturated NH4C1 (300 mL) . The mixture was extracted with ethyl acetate (600 mL) . The organic phase was washed with saturated NaHC03 (200 L) and brine (200 mL) . The organic phase was dried (Na2S04) , filtered and concentrated to give Example 20K (42.2 g, 107%) as a light-colored oil. The crude product was carried forward without purification.
Figure imgf000133_0001
(S) -4- (4-Benzyl-2-oxo-oxazolidin-3-yl) - 3-methyl-4-oxo-butyric acid tert-butyl ester
To an oven-dried 1 L flask under N2 was added THF (250 mL, 99.9% anhydrous) and diisopropyl- amine (14.3 g, 141 mmol). The flask was cooled to 0°C (ice bath) and n-BuLi (56 mL, 141 mmol, 2.54 M in hexanes) was added dropwise. The flask was cooled to -78 °C (dry ice/acetone bath) . Example 20K (30.0 g, 128.6 mmol, dissolved in 40 mL anhydrous THF) was added dropwise over 30 min. The yellow solution was stirred at -78°C for 2 h. Bromo-acetic acid tert-butyl ester (67.7 g, 347 mmol) was added dropwise over 20 min. Stirring was continued for, 30min at -78 °C. The mixture was warmed to 0°C by placing the flask in an ice water bath for 20 min. The mixture was quenched by pouring into a 1 L separatory funnel containing saturated NH4C1 (200 mL) . The mixture was extracted with EtOAc (300 mL) , the phases were separated and the organic was washed with IN aqueous HCl (100 mL) , saturated NaHC03 (100 ■ mL) , and brine (100 mL) . The organic was dried (Na2S0) , filtered, and concentrated. The crude product was purified using a Biotage Flash 65 column. The column was wetted with solvent (9:1 hexane/MTBE; 1 L) and wet-mounted with the crude (dissolved in 50 mL DCM; two columns were performed to purify all of the product) . The title compound was obtained as a white, crystalline solid (29.1 g, 65%), and was pure by HPLC, LCMS and XH NMR.
Figure imgf000135_0001
Example 20M (S) -2-Methyl-succinic acid 4-tert-butyl ester
To a 1 L flask was added Example 20L (29.0 g, 83.5 mmol) and a solution of 4:1 THF/water (300 mL) . The mixture was cooled to 0°C (ice water bath) and 30% aqueous H202 (17.0 g, 501 mmol) was added dropwise over 10 min while stirring. An aqueous solution of LiOH (2 M, 4.73 g, 117 mmol) was added, and the reaction was stirred for 4 h in the ice bath. The reaction was quenched by careful addition of Na2S03 (2.73 M, 14.7 g, 117 mmol). The THF was removed in vacuo and the resulting suspension was extracted with DCM (400 mL) . The aqueous phase was reserved (contains product) and the DCM phase was washed with 0.1 N aqueous NaOH (100 mL) . The DCM was dried (Na2S04) , filtered and concentrated in vacuo to afford the chiral auxiliary (reusable) . The aqueous phase was cooled in an ice bath and acidified with 2 N aqueous HCl to pH 4. The cloudy solution was extracted with EtOAc (300mL) , dried (Na2S04) , filtered and concentrated to give the product as a colorless oil, which solidified to a white solid upon standing. To remove trace amounts of the auxiliary (which alters the optical rotation) the product was purified using a Biotage Flash 40 column, eluting with 30% EtOAc in hexanes. The title compound (13.4 g, 85%) is less polar than the auxiliary. [ ]D=-6.56° (c=0.904, CHC13) ; literature: [α]D=-7.0° (0=0.86, CHC13) ; Davies, S. J. Chem . Soc , Perkin Trans 1 : Org. and Biorg. Chem . 1998, 1 7, 2635-2644. XH NMR (CDC13, 400 MHz) δ 11.73 (bs, IH) , 2.91 (m, IH) , 2.64 (dd, J=16.4, 8.91 Hz, IH) , 2.36 (dd, J=16.4, 5.85 Hz, IH) , 1.44 (s, 9H) , 1.24 (d, J=7.4 Hz, 3H) .
Figure imgf000136_0001
Example 2ON
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5- yl) -acryloyl] -piperidin-4-yl}- (S) -3- methyl-succinamic acid
A solution of Example 20J (150 mg, 0.23 mmol) in methylene chloride (5 L) was shaken with piperidine (115 μL, 1.16 mmol) for 16 h at 300 RPM. The solution was washed with IN aqueous HCl (2x10 mL) , dried (Na2S04) , filtered, and concentrated. The crude product was purified by Biotage Flash 40 elut- ng with 10% methanol in methylene chloride (200 mL) to elute the 9H-fluoren-9-yl by-products, followed by 40% methanol in methylene chloride (800 mL) to elute the title compound 20N (69 mg, 63%), which was obtained as a white powder in 98% regioisomeric pur- ity. Alternatively the title compound can be made from Example 2OH and Example 20M as described in Example IB followed by deprotection of the tert- butyl ester with TFA according to the method described in Example 23E. XH NMR :(DMSO-d6, 400 MHz) δ 8.56 (s, IH) , 8.12 (d, J=6.63 Hz, IH) , 7.98 (d, J=5.07 Hz, IH) , 7.89 (d, J=15.2 Hz, IH)', 7.55 (d, 5.' J=5.46 Hz, IH) , 7.42 (d, J=15.2 Hz, IH) , 4.24 (dd, J=28.9, 12.9 Hz, 2H) , 3.82 (m, IH) , 3.29 (m, IH) , 2.95 ( , IH) , 2.63. (m, IH) , 2.33 (dd, J=15.6, 7.41 Hz, IH) , 2.07 (m, IH) , 1.78 (m, 2H) , 1.33 (m, 2H) , 1.00 (d, J=7.02 Hz, 3H) . Regiochemical assignment0 was based on correlation of COSY, HMBC, HSQC and
TOCSY multidimensional NMR experiments.' , LCMS (ESI-; Method H) m/z 467, -469 (M-H)-; Rt=2.40 min (220 nm, 100% area) . Enantiomeric purity (>98%) and regioisomeric purity (98%) were determined by chiral HPLC5- (Method D) : Example 20N: Rt=14.0 min (98.1 area%) and the regioisomer, Example 21: ■. Rt=19.7 min (1.9 area%) ; chiral HPLC analysis (Method D) of a mixture of the enantiomers, Example 22, gave distinguishable retention times: Rt=12.6'min (50.6 area%) , Rt=16.10 min (49.4 area%) . The "R" enantiomer was not detected (<2%) by chiral HPLC of Example 20K.
Figure imgf000137_0001
Example 21 N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl}- (S) -2-methyl- succinamic acid Example 21 (regioisomer of Example 20N) was prepared in greater than 90% regioisomeric purity according to the same reaction protocol described for Example 20N. 1H NMR (DMSO-d6) δ 8.52 (s, IH), 7.98 (d, J=7.41 Hz, IH) , 7.92 (d, J=15.2 Hz, IH) , 7.92 (d, J=5.46 Hz, IH) , 7.53 (d, J=5.07 Hz, IH) , 7.39 (d, J=15.2 Hz, IH) , 4.26 (dd, J=45.6, 12.5 Hz, 2H), 3.87 (m, IH) , 3.3 (m, IH; overlaps with water) , 2.94 (m, IH), 2.68 (m, IH) , 2.44 (dd, J=14.8, 7.02 Hz, IH) , 2.12 (dd, J=14.4, 7.41 Hz,
IH) , 1.82 (m, 2H) , 1.35 (m, 2H) , 1.06 (d, J=7.02 Hz, 3H) . Regiochemical assignment was based on correlation of COSY, HMBC, HSQC and TOCSY multidimensional NMR experiments. LCMS (APCI+; Method F) m/z 469, 471 (M+H)+; Rt=2.98 min (220 nm, 100 area%) .
Enantiomeric purity (>98%) and regioisomeric purity (91%) were determined by chiral HPLC (Method D) : Example 2ON: Rt=13.9 min (8.9 area%) and Example 21: Rt=19.2 min (91.1 area%) ; chiral HPLC analysis (Method D) of a mixture of the enantiomers, Example 22, gave distinguishable retention times: Rt=12.6 min (50.6 area%), Rt=16.1 min (49.4 area%) . The "R" enantiomer of Example 21 was not detected (<2%) by chiral HPLC.
Figure imgf000139_0001
Example 22
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5- yl) -acryloyl] -piperidin-4-yl}- (R) -2-methyl- succinamic acid (1:1 regioisomeric mixture of 2-methyl and 3-methyl-succinamic acids)
The title compound was prepared as a 1:1 regioisomeric mixture from Example 2OH (89 mg, 0.25 mmol) and (R) -methyl succinic anhydride (29 mg, 0.25 mmol; prepared from (R) -2-methyl succinic acid according to the literature: Davies, S. G. ; Dixon, D. J. J. Chem . Soc . f Perkin Trans . 1 1998, 2635- 2643) as described in the synthesis of Example 201. Example 22 (105 mg, 89%) was obtained as a beige solid. XH NMR (DMSO-d6, 400 MHz, 1:1 mixture of regioisomers) δ 12.1 (bs, 2H) , 8.56 (s, 2H) , 7.98 (d, J=5.46 Hz, 2H) , 7.82-7.92 (m, 4H) , 7.56 (d, J=5.46 Hz, 2H) , 7.43 (d, J=15.2 Hz, 2H) , 4.26 (m, 4H) , 3.84 (m, 2H) , 3.28 (m, 2H) , 2.92 (m, 2H) , 2.60- 2.74 (m, 2H) , 2.39-2.48 (m, 2H) , 2.11-2.23 (m, 2H) , 1.79 (m, 4H), 1.31 (m, 4H) , 1.06 (d, J=7.02 Hz, 3H; 2-methyl succinamic acid regioisomer), 1.02 (d, J=7.02 Hz, 3H; 3-methyl succinamic acid regioisomer) . Regiochemical assignment was based on cor- relation of COSY, HMBC, HSQC and TOCSY multidimensional NMR experiments. Enantiomeric purity (>98%) was verified by chiral HPLC (Method D) : Example 22: Rt=12.6 min (50.6 area%) , Rt=16.1 min (49.4 area%) . The enantiomers gave distinguishable retention times, Example 20N: Rt=14.2 min (50.2 area%) and Example 21: Rt=19.8 min (49.8 area%) . LCMS (APCI+; Method F) m/z 469, 471 (M+H) +; . Rt=2.99 min (220 nm, 100 area%; regioisomers not separated) .
Figure imgf000140_0001
Example 23 rac-3-Aminomethyl-N-{l- [3- (6, 7-dichlorobenzo [b] thiophen-5-yl) -acryloyl] -piperidin- 4-yl] -succinamic acid trifluoroacetic acid salt
.0 NHBoc
0
Example 23A
3-tert-Butoxycarbonylamino- propionic acid methyl ester
To a stirred solution of β-alanine methyl ester hydrochloride (14.0 g, 100 mmol) and diiso- propylethylamme (44 mL, 250 mmol) in methylene chloride (500 mL) at 0°C (ice bath) was added di- tert-butyl dicarbonate (21.8 mL, 95 mmol) dropwise. The reaction was removed from the ice bath, and allowed to warm to room temperature (gas evolution observed, vent adequately) . The' reaction was stirred for 16 h. The mixture was washed with water (200 mL) , IN aqueous HCl (2x150 mL; much gas evolution, vent adequately) , and a saturated solution of- NaHC03 (200 mL)'. The organic phase was dried
(Na2S04) , filtered, and concentrated to obtain Example 23A as a pale yellow oil (20.0 g, 98.5%). Example 23A has also been prepared by esterification of Boc-β-alanine with methanol (Hayashida, Osamu, et al, J. Org. Chem . 2002, 67, 8291-8298) .■■ XE NMR
(CDC13, 400 MHz) δ 5.06 (m, 1H), ,3.70 (s, 3H) , 3.40 (m, 2H) , 2.53 ( , 2H) , 1.44 (s, 9H) .
Figure imgf000141_0001
Example 23B rac-2- (ter-Butoxycarbony'lamino- methyl) -succinic acid 4-tert-butyl ester 1-methyl ester
To a stirred solution of diisopropylamine (16 mL, 115 mmol) in THF (500 mL, 99.9% anhydrous) at 0°C (ice bath) under N2 was added n-butyllithium (2.56 M in hexanes, 45 mL, 115 mmol) dropwise. The solution was cooled to -78 °C (dry ice/acetone), and Example 23A (10.2 g, 50.0 mmol) was added dropwise. The yellow suspension was stirred at -78 °C for 3 h. tert-Butyl bromoacetate (12.7 g, 65.0 mmol) was then added dropwise. The reaction was' stirred for 5 min, and then removed from the -78 °C bath and immediately ■ submerged in an ice water bath. The mixture was stirred for 10 min (note: leaving the reaction for longer periods of time results in- poor yields), and then poured into" a saturated solution of NHC1 (300 mL) . The mixture was shaken, and then diluted with ethyl acetate (700 mL) . The phases were shaken again, and then separated. The organic phase was • washed with IN aqueous HCl (2x100 mL) , a, saturated ' solution of NaHC03 (200 mL) , and brine (200 mL) . The organic phase was dried (Na2S04) , filtered, and concentrated. The crude product (14.0 g) was purified using a Biotage Flash 75 chromatography system, eluting with the following gradient: 10% ethyl ace- tate in hexanes followed by 15% ethyl acetate in hexanes. Example 23B was- obtained as a colorless oil (6.67 g, 42%). 1H NMR (CDC13, 400 MHz) δ 4.93 (m, IH) , 3.72 (s, 3H) , 3.38 (m, 2H) , 2.96 ( , IH) , 2.63 (dd, J=16.8, 7.80- Hz, IH) , 2.50 (dd, J=16.8, 5.85 Hz, IH) , 1.39-1.54 (m, 18H) . LCMS (ESI+;
Method H) m/z 340 (M+Na)+; Rt=3.18 min (220 nm, 100 area%) .
Figure imgf000142_0001
Example 23C rac-2- (tert-Butoxycarbonylamino- methyl) -succinic acid 4-tert-butyl ester The title compound was prepared from Example 23B (7.56 g, 23.8 mmol) according to the procedure described for Example 18D, except the amount of lithium hydroxide hydrate was reduced (1.50 g, 35.7 mmol) to 1.5 equivalents. Example 23C (6.78 g, 94%) was obtained as a pale yellow oil, which solidified upon standing. E NMR (CDC13, 400 MHz) δ 10.61 (bs, IH) , 5.04 (m, 1H),- 3.41 (m, 2H) , 2.98 (m, IH) , 2.65 (dd, J=16.8, 7.41 Hz, IH) , 2.38-2.57 (m,- IH) , 1.37-
•10. 1..54 (m, 18H) . LCMS (ESI;. Method H) m/z 302 (M-H)-; Rt=2.07 min.
Figure imgf000143_0001
Example 23D rac-3- (tert-Butoxycarbonylamino-methyl) -N-{1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl)- acryloyl] -piperidin-4-yl}-succinamic acid tert-butyl ester
The title compound was prepared from Example 23C (910 mg, 3.00 mmol) and Example 20H (888 mg,
15 2.50 mmol) according to the amide coupling procedure described for Example IB. Example 23D (1.03 g, 64%) was obtained as a white powder after purification using a Biotage Flash 40 chromatography system, eluting with the following gradient: 1:1 ethyl
20 acetate/hexanes, 3:1 ethyl acetate/hexanes, then 9:1 ethyl acetate/hexanes. XH NMR (CDC13, 400 MHz) δ 8.56 (bs, IH) , 7.98 (d, J=5.46 Hz, IH) , 7.86-7.94 (m, 2H) , 7.55 (d, J=5.07 Hz, IH) , 7.44 (d, J=15.2 Hz, IH) , 6.77 (m, IH) , 4.29 (m, 2H) , 3.85 (m, IH) , 3.28 (m, IH), 3.10 ( , IH) , 2.89-2.98 (m, 2H), 2.68 (m, IH) , 2.43 (dd, J=16.0, 9.75 Hz, IH) 2.27 (dd, J=16.0, 4.68 Hz, IH) , 1.78 (m, 2H) , 1.27-1.45 (m, 20H) . LCMS (ESI+; Method I) m/z 662,' 664 (M+Na)+; Rt=4.16 min (220 nm, 100 area%) .
Figure imgf000144_0001
Example 23E
rac-3-Aminomethyl-N-{l- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- succinamic acid trifluoroacetic acid salt
A solution of Example 23D (1.00 g, 1.56 mmol) in methylene chloride (20 mL) was stirred with trifluoroacetic acid (2.4 mL, 31 mmol) under N2 for 16 h at room temperature. The reaction was then concentrated in vacuo . The residue was resuspended in methylene chloride (10, mL), and the solvent was concentrated again.- This process was repeated (3x) in order to remove residual trifluoroacetic acid. The crude product was triturated with diethyl ether (10 mL) , and the resulting white solid was filtered. Example 23E (890 mg, 95%) was obtained as a white powder. 1E NMR (DMS0-d6, 400 MHz) δ 12.5 (bs, IH) , 8.56 (s, IH), 8.15 (d, J=7.80 Hz, IH) , 7.99 (d, J=5.07 Hz, IH) , 7.77-7.95 (m, 4H) , 7.56 ' (d, J=5.46 Hz, IH) , 7.44 (d, J=15.2 Hz., IH) , 4.25 (m, 2H) , 3.89 (m, IH) , 3.32 ( , IH) , 2.84-3.11 (m, 4H) , 2.58 (m, IH, overlaps DMSO) , 1.82- (m, 2H) ,- 1.37 (m, 2H) . LCMS (ESI-; Method I) m/z 482, 484 (M-H)-; Rt=2.64 min (220 nm, 100 area%) .
Figure imgf000145_0001
Example 24 rac-3- (Benzoylamino- ethyl) -N-{1- [3- (6, 7-dichlorobenzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- succinamic acid
To a stirred suspension of Example 23E (300 mg, 0.50 mmol) and diisopropylethylamine (0.35 mL, 2.0 mmol) in methylene chloride (10 mL) at 0°C (ice bath) was added benzoyl chloride (0.24 M solu- tion in chloroform, 2.1 mL, 0.50 mmol) dropwise. After addition was complete, the reaction was stirred for 30 min at 0°C. The reaction was then quenched by addition of 1 N aqueous HCl (5 mL) . The heterogeneous mixture was extracted with 20% meth- anol in methylene chloride (30 mL) . The organic phase was washed with IN aqueous HCl (3x20 mL) . Before each aqueous extraction, methanol (5 mL) had to be added to maintain two homogeneous phases. The ' organic phase was dried (Na2S04) , filtered, and concentrated. The crude product was purified by partial dissolution (or digestion) in hot acetonitrile (10 L) followed by cooling to room temperature. The resulting white solid.was filtered, washing with acetonitrile (3x2 mL) . A second digestion, in ethyl- acetate, was performed to raise the purity above 95%. The powder was dried (CaS04 dessicator) under high vacuum for 2 days. Example 24 (135 mg, 46%) was obtained as a white powder. 1H NMR (DMS0-d6, 400 MHz) δ 12.1 (bs, IH) , 8.55 (s, IH) , 8.43 (m, IH) , 7.93-8.00 ( , 2H) , 7.89 (d, J=15.2 Hz,- IH) , 7.80- 7.85 (m, 2H) , 7.56 (d, J=5.07 Hz, IH) , 7.37-7.53 (m, 4H) , 4.14-4.33 (m, 2H) , 3.86 (m, IH) , 3.21-3.45 (m, 3H, overlaps with water), 2.90 (m, 2H) , 2.55 (m, IH, overlaps with DMSO), 2.40 (dd, J=16.4, 5.07 Hz, IH) , 1.77 (m, 2H) , 1.31 (m, 2H) . LCMS (ESI+; Method I) m/z 588, 590 (M+H)+, 610, 612 (M+Na)+; Rt=2.80 min (220 nm, 100 area%; 254 nm, 96.9 area%) .
Figure imgf000147_0001
Example 25
rac-N-{l- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl}-3- [ (3-methoxy- benzoylamino) -methyl] -succinamic acid
The title compound was prepared from Example 23E (150 mg, 0.25 mmol) and 3-m.ethoxybenzoyl chloride (0.24 M solution in chloroform, 1.0 mL, , 0.24 mmol) according to the procedure described by Example 24. Example 25 (99 mg, 64.%) was obtained as a white powder after purification by partial dis-:- solution (or digestion) in hot acetonitrile (10 mL) , followed by cooling to room temperature and filtra- tion. XH NMR (DMSO-d6, 400 MHz δ 12.1 (bs, IH) ,
8.55 (s, IH) , 8.40 (m, IH) , 7.98 (d, J=5.46 Hz, IH) , 7.94 (d, J=7.80 Hz, IH) , 7.89 (d, J=15.2 Hz, IH) ,
7.56 (d, J=5.46 Hz, IH) , 7.30-7.46 (m, 4H) , 7.07 (m, IH) , 4.13-4.30 (m, 2H) , 3.86 (m, IH) , 3.78 (m, 3H, 3-methoxy group split into two singlets due to hindered rotation), 3.31-3.32 (m, 3H, overlaps water)', 2.89 (m, 2H) , 2.55 (m, IH, overlaps DMSO), 2.38 (dd, J=16.4 Hz, 5.07 Hz, IH)/ 1.77 (m, 2H) , 1.32 (m, 2H) . LCMS (ESI-; Method I) m/z 616, 618 (M-H)-; Rt=2.86 min (220 nm, 96.4 area%) .
Figure imgf000148_0001
Example 26
rac-3- t (3-Chloro-benzoylamino) -methyl] -N-{1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl}-succinamic acid
The title compound was prepared from '■ Example 23E (150 mg, 0.25 mmol) a'nd 3-Chlorobenzoyl chloride (0.24 M solution in chloroform, 1.0 mL, 0.24 mmol) according to the procedure described- by Example 24. Example 26 (93 mg, 60%) was obtained as a white powder after purification' by partial dis- solution (or digestion) in hot acetonitrile (10 mL) followed by cooling to room temperature and filtration. XH NMR (DMSO-d6, 400 MHz) δ 8.96 (m, IH) , 8.52 (s, IH), 8.13 (m, IH) , 7.75-8.00 (m, 4H) , 7.33-7.57 (m, 4H) , 4.12-4.29 (m, 2H) , 3.85 (m, IH) , 3.20-3.47 (m, 3H, overlaps water), 2.92 (m, 2H) , 2.39 (m, IH) , 2.26 (m, IH), 1.76 (m, 2H) , 1.31 (m, 2H)'. LCMS (ESI-; Method I) m/z 622, 624 (M-H)-; Rt=2.95 min (220 nm, 1O0 area%; 254 nm, 93.9 area%) .
Figure imgf000149_0001
Example 27
rac-N-{l- [3- (6, 7-Dichloro-benzo [b]thiophen-5-yl) ■ acryloyl] -piperidin-4-yl}-3- [ (3-plienyl-ureido) - methyl] -succinamic acid
The title compound was prepared from Exam- pie 23E (150 mg, 0.25 mmol) and phenyl isocyanate (0.24 M solution in chloroform, 1.0 mL, 0.24 mmol) according to the procedure described by Example 24, except the amount of diisopropylethylamine (0.11 mL, 0.63 mmol) was reduced to 2.5 equivalents. Example, 27 (89 mg, 59%) was obtained as a white powder after purification by partial dissolution (or digestion) in hot acetonitrile (10 mL) followed by cooling to room temperature and filtration. 1H NMR (DMSO-d6, 400 MHz) δ 12.1 (bs, IH) , 8.50-8.62 (m, 2H) , 7.96- 8.06 (m, 2H), 7.90 (d, J=15.2 Hz, IH) , 7.56 (d, J=5.46 Hz, IH), 7.35-7.47 (m, 3H) , 7.15-7.26 (m, 2H) , 6.87 (m, IH) , 6.11 (m, IH) , 4.27 (m, 2H) , 3.87 (m, IH), 3.18-3.32 (m, 3H, overlaps water), 2.94 (m, IH) , 2.77 (m, IH) , 2.45-2.56 (m, H, overlaps DMSO), 2.33 (dd, J=16.4, 5.85 Hz, IH) , 1.80 (m, 2H) , 1.36 (m, 2H) . LCMS (ESI-; Method I) m/z 601, 603 (M-H)-; Rt=2.87 min (220-nm, "100 area%).
Figure imgf000150_0001
Example 28
N-{l-[3-(2,3-Dichloro-4-methylsulfanyl- phenyl) -acryloyl] -azetidin-3-yl}-3- methyl-succinacmic acid
Figure imgf000150_0002
Example '28A l-Bromo-2, 3-dichloro-4-methylsulfanyl-benzene
Dimethyl sulfate (2.90 mL, 30.7 mmol) was added dropwise to a stirred solution of NaOH (1.28 g, 32.1 mmol) and 2, 3-dichlorobenzenethiol (5.00 g, 27.9 mmol) in water (50 mL) at 0°C. The- reaction mixture was heated at 110 °C for 2 h and allowed to cool to ambient temperature. The reaction mixture was extracted with ether and the organic layer was washed with water and brine, dried (MgS04) , and filtered. Evaporation of the solvent followed by recrystallization from hexane gave the title compound (3.89 g, 72%) as a white solid. XE NMR (CDC13, 400 MHz) δ 7.24 (dd, J=8.2 Hz, J=1.6 Hz, IH) , 7.18 (t, J=8.2 Hz, IH) , 7.04 (dd, J=8.2 Hz, J=l .6 Hz, IH) , 2.48 (s, 3H) .
Figure imgf000151_0001
Example 28B
l-Bromo-2, 3-dichloro-4-methylsulfanyl-benzene
Bromine (0.79 mL,.- 15 mmol) was added drop- wise to a stirred solution of Example 28A (2.97 g, 15.4 mmol) in CH2C12 (50 mL) at 0°C. The resulting mixture was stirred at room temperature overnight.
Additional bromine was added to the reaction mixture in 0.05 mL portions until the reaction was completed, as indicated by HPLC. The reaction mixture was diluted with CH2C12 and washed with 10% NaHSθ3. The organic layer was dried (MgS04) and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 1% ' ' EtOAc/hexane) to provide the title compound (3.68 g, 88%) as a white solid. 1H NMR (CDC13, 400 MHz) δ 7.50 (d, J=8.4 Hz, IH) , 6.91 (d, J=8.4 Hz, IH) , 2.47 (s, 3H) .
Figure imgf000152_0001
Example 28C
3- (2, 3-Dichloro-4-methylsulfanyl- phenyl) -acrylic acid methyl ester
The title compound was prepared from
Example 28B (1.60 g, 5.916 mmol) and methylacrylate
(1.6 mL, 17.75 mmol) by the procedure described in
Example 20E. XE NMR (400 MHz, DMSO-d6) δ 7.92 (d,
J=8.59 Hz, IH), 7.87 (d, J=16.01 -Hz, IH) , 7.29 (d,
J=8.59 Hz, IH) , 6.72 (d, J=16.01 Hz, IH) , 3.72 (s, '3H), 2.55 (s, 3H) .
Figure imgf000152_0002
Example 28D
3- (2, 3-Dichloro-4-methylsulfanyl- phenyl) -acrylic acid
The title compound 28D (774mg, 79%) was prepared from Example 28C by the method described in Example 20F. XE NMR (400 MHz, DMSO-d6) δ 12.63 (s, IH), 7.89 (d, J=8.59 Hz, IH) , 7.82 (d, J=16.01 Hz, IH) , 7.27 (d, J=8.59 Hz, IH) , 6.60 (d, J=15.62 Hz, IH) , 2.54 (s, 3H) . Analytical HPLC (Method C) Rt=3.165 min (220 nm, 96 area%) .
Figure imgf000153_0001
Example 28E
{1- [3- (2, 3-Dichloro-4-methylsulfanyl- phenyl) -acryloyl] -azetidin-3-yl}- carbamic acid tert-butyl ester
A solution of Example 28D (100 mg, 0.380 mmol), H0Bt.H20 (87 mg, 0.570 mmol), NMM (0.105 mL, 0.950 mmol) and azetidin-3-yl-carbamic acid tert- butyl ester (79 mg, 0.456 mmol), in dry DMF (2 mL) was treated with EDCI (109 mg, 0.570 mmol) at 0°C and allowed to stir at ambient temperature under N2 atmosphere for 18 h. The reaction mixture was diluted with warm 2% methanol/EtOAc (40 mL) , washed with water (2x10 mL) and brine (1x10 mL) . The organic phase was separated (while keeping warm, otherwise product precipitates out) , dried (Na2S04) , filtered, and evaporated to dryness. The residue obtained was crystallized from methanol to provide the desired product (121 mg, 76.26%) as a white solid. αH NMR (400 MHz, DMSO) δ 7.93 (d, J=8.59 Hz, IH) , 7.70 (d, J=15.62 Hz, IH) , 7.60 (br d, J=7.81 Hz, IH) , 7.30 (d, J=8.59 Hz, IH) , 6.78 (d, J=15.62
Hz, IH), 4.54-4.50 (m, IH) , 4.35-4.27 (m, IH) , 4.18- 4.13 (m, IH), 4.09-4.06 (m, IH) , 3.82-3.78 (m, IH) , 2.57 (s, 3H), 1.40 (s, 9H) . LCMS (APCI+; Method F) m/z 417, 419 (M+H)+; Analytical HPLC (Method C) Rt=3.487 min (220 nm, 100 area %) .
Figure imgf000154_0001
Example 28F
1- (3-Amino-azetidin-l-yl) -(2,3- dichloro-4-methylsulfanyl-phenyl] propenone HCl salt
A suspension of Example 28E (120 mg, 0.288 mmol) in CH2C12 (1 mL) was treated with 4N HCl in dioxane (4 mL) at 0°C under N2 atmosphere for 30 min and allowed to stir at ambient temperature for 2 h. The reaction was monitored by HPLC at 1 h intervals. After 2 hours the solvent was removed under reduced pressure and dried under vacuum for 5 h to provide the HCl salt of the amine (110 mg, 108.6%) as a white solid. XH NMR (DMSO, 400 MHz) δ 8.64 (br s, 2H), 7.99 (d, J=8'.59 Hz, IH) , 7.74 (d, J=15.62 Hz, IH) , 7.30 (d, J=8.59 Hz, IH) , 6.85 (d, J=15.62 Hz, IH), 4.59-4.55 (m, IH) , 4.33-4.28 (m, IH) , 4.22-4.17 (m, IH) , 4.00-3.96 (m, IH) . LCMS (APCI+; Method F) m/z 317, 318 (M+H)+. Analytical HPLC (Method C) Rt=2.066 min (220 nm, 100 area %, 254 nm 100 area
■ό ) .
Figure imgf000155_0001
Example 28G
N- { 1- [3- (2, 3-Dichloro-4-methylsulfanyl-phenyl] ■ azetidin-3-yl} -3-methyl-succinamic acid tert-butyl ester
The title compound 28G (75 mg, 44.37%) was . prepared from Example 28F and Example 20M as de- cribed in Example 28E. XE NMR (DMSO, 400 MHz) δ 8.55 (d, J=6.64 Hz, IH) , '7.94 (d, J=8.59 Hz, IH) , 7.71 (d, J=15.62 Hz, IH) ,, 7.30 (d, J=8.59 Hz, ' IH) , 6.81 (d, J=15.62 Hz, IH) , 4.61-4.53 (m, IH) , 4.50-4.43 (m, IH) , 4.19 (m, IH) , 4.09-4.01 (m, IH) , 3.83-3.78 - (m, IH) , 2.67-2.59 (m, IH) , 2.57 (s, 3H) , ' 2.21 ' (m, IH) , 1.37 (s, 9H), 1.03 (d, J=7.03 Hz, 3H) . LCMS . (APCI+; Method F) m/z 487, 489 (M+H)+. .Analytical ■ HPLC (Method C) Rt=3.201 min (220 nm, 100 area %, ' 254 nm .100 area %) .
Figure imgf000155_0002
Example 28H
N-{1- [3- (2, 3-Dichloro-4-methylsulfanyl-phenyl) - acryloyl] -azetidin-3-yl} -3-methyl-succinamic acid The title compound (51 mg, 82%) was pre- ared from Example 28G as described in Example 23E. XH NMR (DMSO, 400 MHz) δ 12.08 (bs, IH) , 8.55 (d, J=6.64 Hz, IH) , 7.92 (d, J=9.37 Hz, IH) , 7.72 (d, J=15.62 Hz, IH) , 7.30 (d, J=8.59 Hz, IH) , 6.80 (d, J=15.23 Hz, IH), 4.58-4.53 (m, IH) , 4.48-4.42 (m, IH), 4.22-4.17 (m, IH) , 4.09-4.03 (m, IH) , 3.83-3.77 (m, IH) , 2.67-2.62 (m, IH) , 2.57 (s, 3H) , 2.25-2.19 (m, IH) , 1.04 (d, J= 7.03 Hz, 3H) . LCMS (APCI+; Method F) m/z 431, 433 (M+H)+. Analytical HPLC (Method C) Rt=2.556 min (220 nm, 85 area %) .
Figure imgf000156_0001
Example 29
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) - acryloyl] -azetidin-3-yl] -3-methyl-succinamic acid
Figure imgf000156_0002
Example 29A
{1- [3- (6, 7-Dichlσro-benzo [b] thiophen-5-yl) acryloyl] -azetidin-3-yl}-carbamic acid tert-butyl ester The title compound (139 mg, 89%) was pre- ared from Example 20F (100 mg, 0.366 mmol) and azetidin-3-yl-carbamic acid tert-butyl ester (76 mg, 0.439 mmol) as described in Example 28E. Example 29A was obtained as a white powder. 1H NMR (400 MHz, DMSO) δ 8.48 (s, IH) , 7.98 (d, J=5.47 Hz, IH) , 7.84 (d, J=15.62 Hz, IH) , 7.62 (br d, J=7.03 Hz, IH) , 7.58 (d, J=5.47 Hz, IH) , 6.78 (d, J=15.62 Hz, IH) , 4.55 (t, J=8.59 Hz, 'IH), 4.38-4.30 (m, IH) , 4.20- 4.15 (m, IH) , 4.13-4.09 (m, IH) , 3,84-3.80 (m, IH) , 1.40 (s, 9H) . LCMS (APCI+; Method F) m/z 427, 429 (M+H)+; Analytical HPLC (Method C) Rt=3.473 min (220 nm, 100 area %) .
Figure imgf000157_0001
• Example 29B
1- (3-Amino-azetidin-l-yl) -3- (6, 7-dichlorobenzo [b] thiophen-5-yl) -propenone HCl salt
The title compound was prepared from
Example 29A as described in Example 28F. Η NMR
(DMSO, 400 MHz) δ 8.79 (s, 2H) , 8.54 (s, IH) , 7.99 (d, J=5.47 Hz, IH) , 7.87 (d, J=15.62 Hz, IH) , 7.58 (d, J=547 Hz, IH) , 6.94 (d, J=15.23 Hz, IH) , 4.65- 4.59 (m, IH) , 4.38-4.35 (m, IH) , 4.24-4.19 (m, IH) , 4.11-4.06 (m, IH) , 4.04-4.00 (m, IH) . LCMS (APCI+; Method F) m/z 327, 329 (M+H)+. Analytical HPLC (Method C) Rt=2.147 min (220 n , 100 area %, 254 nm 97.64 area %) .
Figure imgf000158_0001
Example 29C
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryloyl] - azetidin-3-yl}-3-methyl-succinamic acid tert-butyl ester
The title compound 29C (42 mg, 25%) was prepared from Example 29B as described in Example 28G. XH NMR (DMSO, 400 MHz) δ 8.57 (d, J=6.64 Hz, IH) , 8.49 (s, IH) , 7.98 (d, J=5.47 Hz, IH) , 7.85 (d, J=15.62 Hz, IH) , 7.57 (d, J=5.47 Hz, IH) , 6.89 (d,d, Jι=1.5.6 Hz, J2=15.62 Hz, IH) , 4.59 (m, IH) , 4.48 (m, IH) , 4.21 (m, IH) , 4.11 ( , IH) , 3.83 (m, IH) , 2.64 (m, IH) , 2.22 (m, IH) , 1.38 (s, 9H) , 1.04 (d, J=7.03 Hz, 3H) . LCMS (APCI+; Method F) m/z 497, 498 (M+H) + . Analytical HPLC (Method C) Rt=3.'376 min (220 nm, 100 area %) .
Figure imgf000158_0002
Example 29D
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryloyl]- azetidin-3-yl } -3-methyl-succinamic acid The title compound 29D was prepared from Example 29C as described in Example 28H. LCMS (APCI+; Method F) m/z 441, 443 (M+H)+. Analytical HPLC (Method C) Rt=2.370 min (220 nm, 85 area %) .
There is no. Example 30.
Figure imgf000159_0001
Example 31 -
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl} -3-phenyl-succinamic acid
Figure imgf000159_0002
Example 31A
2-Phenyl-succinic acid 4-tert-butyl ester 1-methyl ester
The title compound was prepared by the procedures described in Example 23B, substituting Example 23A with phenyl-acetic acid methyl ester. 1H NMR (CDC13, 400 MHz) δ 7.30 (m, 5H) , 4.02 (dd, J=10.0 Hz, J=5.6 Hz, IH) , 3.68 (s, 3H) , 3.10 (dd, J=16.4 Hz, J=10.0 Hz, IH) , 2.60 (dd, J=16.4 Hz, J=5.6 Hz, IH) , 1.40 (s, 9H) .
Figure imgf000160_0001
Example 3IB
2-Phenyl-succinic acid 4-tert-butyl ester
The title compound was prepared by the procedures described in Example 23C, substituting Example 23B with Example 31A. XH NMR (DMSO-d6, 400 MHz) δ 7.29 (m, 5H),'3.86' (dd, J=10.2 Hz, J=5.9'Hz,
IH) 2.90 (dd, J=16.4 Hz, J=10.2 Hz, IH) , 2.53 (dd,
J=16.4 Hz, J=5.5 Hz, IH) , 1.34 (s, 9H) . LCMS (ESI-; Method H) m/z 249 (M-H)-; Rt=2.37 min (220 nm, 100 area%) .
Figure imgf000160_0002
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryloyl] - piperidin-4-yl}-3-phenyl-succinamic acid tert-butyl ester The title compound was prepared by the procedures described in Example 23D, substituting Example 23C with Example 3IB. XE NMR (CDC13, 400 MHz) δ 8.04 (d, J=15.2 Hz, IH) , 7.91 (s, IH) , 7.52 (d, J=5.5 Hz, IH) , 7.31 (m, 7H) , 6.85 (br d) , 5.42 (d, J=7.6 Hz, IH) , 4.55 (m, IH) , 4.01 (m, IH) , 3.83 (dd, J=8.6, J=5.9 Hz, IH) , 3.24 (m, IH) , 3.17 (dd, J=16.4, J=9.0 Hz, IH) , 2.86 (m, IH) , 2.56 (dd, J=16.4, J=5.9 Hz, IH) , 1.98 (m, 2H) , 1.38 (s, 9H) , 1.24 (m, 2H) . LCMS (APCI+; Method I) m/z 587, 589 (M+H)+; Rt=4.28 min (220 rim, 100 area%) .
Figure imgf000161_0001
Example 31D
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl}-3-phenyl-succinamic acid
The title compound was prepared by the procedures described in Example 23E, substituting Example 23D with Example 31C. 1H NMR (DMSO-d6, 400 MHz) δ 12.12 (s, IH) , 8.54 (d, J=9.4 Hz, IH) , 8.06 (d, J=7.6 Hz, IH), 7.98 (s, IH) , 7.88 (d, J=14.8 Hz, IH) , 7.55 (m, IH) , 7.40 (m, IH) , 7.31 (m, 4H) , 7.22 (m, IH), 4.20 (m, 2H) , 3.87 (m, IH) , 3.80 (m, IH) , 3.24 (m, IH) , 2.93 (m, 2H) , 2.53 (m, IH) , 1.83 (m, ' IH) , 1.64 (m, IH) , 1.35 (m, IH) , 1.20 (m, IH) . LCMS (APCI+; Method I) m/z 531, 533 (M+H)+; Rt=3.29 min (220 nm, 100 area%) .
Figure imgf000162_0001
Example 32
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl}-3-pyridin-3-yl- succinamic acid
Figure imgf000162_0002
Example 32A
2-Pyridin-3-yl-succinic acid 4-tert-butyl ester 1-ethyl ester
The title compound was prepared by the procedures described in Example 23B, substituting Example 23A with pyridin-3-yl-acetic acid ethyl ester. X NMR (CDC13, 400 MHz) δ 8.57 (d, J=2.4 Hz, IH) , 8.54 (dd, J=4.8 Hz, J=l .6 Hz, IH) , 7.67 (dt, J=8.0 Hz, J=1.6 Hz, IH) , 7.29 (dd, J=8.0 Hz, J=4.8 Hz, IH) , 4.16 (m, 2H) , 4.05 (dd, J=9.2 Hz, J=6.4 Hz, IH), 3.11 (dd, J=16.4 Hz, J=9.2 Hz, IH) , 2.63 (dd, J=16.4 Hz, J=6.4 Hz, IH) , 1.40 (s, 9H) , 1.21 (t, J=7.2 Hz, 3H) . LCMS (APCI+; Method I) m/z 280 (M+H)+; Rt=3.19 min (220 nm, 100 area%) .
Figure imgf000163_0001
Example 32B
2-Pyridin-3-yl-succinic acid 4-tert-butyl ester
The title compound was prepared by the procedures described in Example 23C, substituting Example 23B with Example 32A. XH NMR (DMSO-d6, 400 MHz) δ 8.52 (d, J=2.0 Hz, IH) , 8.47 (dd, J=4.8 Hz, J=1.6 Hz, IH) , 7.72 (dt, J=7.8 Hz, J=2.0 Hz, IH) , 7.36 (dd, J=7.8 Hz, J=4.8 Hz, IH) , 3.95 (dd, J=9.4 Hz, J=6.2 Hz, IH) , 2.96 (dd, J=16.4 Hz, J=9.8 Hz, IH) , 2.62 (dd, J=16.4 Hz, J=6.2 Hz, IH) , 1.34 (s, 9H) . LCMS (APCI+; Method I) m/z 252 (M+H)+; Rt=1.80 min (220 nm, 100 area%) .
Figure imgf000164_0001
Example 32C
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryloyl] ■ piperidin-4-yl}-3-pyridin-3-yl-succinamic acid tert- butyl ester
The title compound was prepared by the procedures described in Example 23D, substituting Example 23C with Example 32B. 1H NMR (CD.C13, 400 MHz) δ 8.51 (m, 2H),.8.04 (d, J=15.6 Hz, IH) , 7.91 (s, IH) , 7.71 (dt, J=8.2 Hz, J=2.0 Hz, IH) , 7.51 (d, J=5.6 Hz, IH) , 7.33 (d, J=4.2 Hz, IH) , 7.26 (m, IH) , 6.85 (d, J=15.2 Hz, IH) , 5.91 (d, J=7.8 Hz, IH) , 4.60 (m, IH) , 4.02 (m, 2H) , 3.81 (dd, J=9.4 Hz, J=5.8 Hz, IH) , 3.23 (m, IH) , 3.15 (dd, J=16.8 Hz, J=9.4 Hz, IH) , 2.85 (m, IH) , 2.57 (dd, J=16.8 Hz, J=5.5 Hz, IH) , 1.99 (m, 2H) , 1.39 (s, 9H) , 1.30 (m, 2H) . LCMS (APCI+; Method I) m/z 588, 590 (M+H)+; Rt=3.73 min (220 nm, 100 area%) .
Figure imgf000165_0001
Example 32D
N-{l-[3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl}-3-pyridin-3-yl- succinamic acid
The title compound was prepared by the procedures described in Example 23E, substituting Example 23D with Example 32C. αH NMR (DMSO-d6, 400 MHz) δ 12.27 (s, IH) , 8.55 (m, 3H) , 8.18 (d, J=7.6 Hz, IH), 7.97 (s, IH), 7.88 (m, 2H) , 7.48 (m, 3H) , 4.21 (m, 2H), 3.97 (m, IH) , 3.80 (m, IH) , 3.26 (m, IH) , 3.00 (dd, J=16.4 Hz, J=9.0 Hz, 1H),,2.91 (m, IH) , 2.63 (dd, J=16.4 Hz, J=5.9 Hz, IH)', 1.84 (m, IH) , 1.66 (m, IH), 1.35 (m, IH) , 1.21 (m, IH) . LCMS (APCI+; Method I) m/z 532, 534 (M+H)+; Rt=2.67 min (220 nm, 100 area%) .
Figure imgf000166_0001
Example 33
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl}-3-quinolin-3- yl-succinamic acid
Figure imgf000166_0002
Example 33A Quinolin-3-yl-acetic acid tert-butyl ester
To a solution of 3-bromoquinoline (0.208 g, 1.00 mmol) in THF (5 mL) was added (1-tert- butoxy-vinyloxy) -tert-butyl-dimethyl-silane (0.472 g, 2.05 mmol), Pd(Tol3P) 2C12 (0.039 g, 0.05 mmol) and KOAc (0.196 g, 2.00 mmol). The mixture was heated at reflux for 48 h under nitrogen. After cooling to room temperature, the reaction was quenched with sat. NH4CI, diluted with Et20 and stirred for 20 min. Phases were separated. The aqueous phase was extracted with Et20. The combined organic layers were washed with water, brine, dried and concen- rated. The residue was purified by flash- chromatog- aphy (silica gel, gradient elution from 10:1 to 6:1 hexanes/EtOAc) to give the title compound (0.202 g, 83%) as a pale yellow oil. XH NMR (CDC13, 400 MHz) δ 8.84 (d, J=2.0 Hz, IH) , 8.09 (d, J=8.4 Hz, IH) , 8.06 (s, IH) , 7.80 (d, J=8.0 Hz, IH) , 7.69 (t, J=8.0 Hz, IH), 7.54 (t, J=8.0 Hz, IH) , 3.72 (s, 2H) , 1.45 (s, 9H) . LCMS (APCI+; Method I) m/z 244 (M+H)+; Rt=3.36 min (220 nm, 100 area%) .
Figure imgf000167_0001
Example 33B Quinolin-3-yl-acetic acid methyl ester
To a stirred solution of Example 33A
(0.153 g, 0.629 mmol) in MeOH (2 mL) was added hydrochloride in ether (2 M, 2 mL) . After stirring for 48 h, the reaction mixture was concentrated in vacuo. The resulting solid was suspended in IPA- ether (1:5) and stirred for 30 min. The mixture was filtered to give the title compound (0.105 g, 83%) as a white solid. XH NMR (CDC13, 400 MHz) δ 8.84 (d, J=2.0 Hz, IH) , 8.11 (d, J=8.4 Hz, IH) , 8.08 (s, IH) , 7.80 (d„ J=9.6 Hz, IH) , 7.70 (t, J=8.0 Hz, IH) , 7.55 (t, J=8.0 Hz, IH) , 3.83 (s, 2H) , 3.73 (s, 3H) . LCMS (APCI+; Method I) m/z 202 (M+H)+; Rt=2.54 min (220 nm, 100 area%) .
Figure imgf000168_0001
Example 33C
2-Quinolin-3-yl-succinic acid 4-tert-butyl ester 1-methyl ester
The title compound was prepared by the procedures described in Example 23B, substituting Example 23A with Example 33B. lE NMR (CDC13, 400 MHz) δ 8.87 (d, J=2.4 Hz, IH) , 8.10 (d, J=8.8 Hz, IH) , 8.07 (d, J=2.0 Hz, IH) , 7.80 (d, J=8.0 Hz, IH) , 7.71 (t, J=8.0 Hz, IH) , 7.56 (t, J=8.0 Hz, IH) , 4.26 (dd, J=9.6 Hz, J=6.0 Hz, IH) , 3.70 (s, 3H) , 3.24 (dd, J=16.8 Hz, J=9.6 Hz, IH) , 2.74 (dd, J=16.8 Hz, ,J=6.0 Hz, IH), 1.40 (s, 9H) . LCMS (APCI+; Method I) m/z 316 (M+H)+; Rt=3.15 min (220 nm, 100 area%) .
Figure imgf000168_0002
Example 33D
2-Quinolin-3-yl-succinic acid 4-tert-butyl ester
The title compound was prepared by the procedures described in Example 23C, substituting Example 23B with Example 33D. XE NMR (DMSO-d6, 400 MHz) δ 12.75 (s, IH) , 8.88 (d, J=2.4 Hz, IH) , 8.28 (d, J=2.0 Hz, IH) , 8.01 (d, J=8.2 Hz, IH) , 7.97 (d, J=8.2 Hz, IH) , 7.75 (t, J=8.0 Hz, IH) , 7.61 (t, J=8.0 Hz, IH) , 4.17 (dd, J=9.4 Hz, J=6.2 Hz, IH) , 3.10 (dd, J=16.4 Hz, J=9.4 Hz, IH) , 2.76 (dd, J=16.4 Hz, J=6.2 Hz, IH) , 1.34 (s, 9H) . LCMS (APCI+; Method I) m/z 302 (M+H)+; Rt=2.13 min (220 nm, 100 area%) .
Figure imgf000169_0001
Example 33E
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl}-3-quinolin-3-yl- succinamic acid tert-butyl ester
The title compound was prepared by the procedures described in Example 23D, substituting Example 23C with Example 33D. XE NMR (CDC13, 400 MHz) XE NMR (CDC13, 400 MHz) δ 8.83 (d, J=2.0 Hz, IH), 8.15 (d, J=2.0 Hz, IH) , 8.06 (d, J=8.4 Hz, IH) 8.01 (d, J=15.6 Hz, IH) , 7.89 (br s, IH) , 7.79 (d, J=8.4 Hz, IH) , 7.69 (t, J=6.8 Hz, IH) , 7.55 (d, J=6.8 Hz, IH) , 7.51 (t, J=8.4 Hz, IH) , 7.33 (br s, IH) , 6.84 (m, IH) , 5.87 (d,. J=7.6 Hz, IH) , 4.61 (m, IH) , 4.03 (m, 2H) , 4.16 (m, 2H) , 3.27 (dd, J=16.8
Hz, J=9.6 Hz, IH) , 3.20 (m, IH) , 2.86 (m, IH) , 2.68 (dd, J=16.8 Hz, J=5.2 Hz, IH) , 2.09 (m, IH) , 1.93 (m, IH) , 1.43 (m, 2H) , 1.39 (s, 9H) . LCMS (APCI+;
Method I) m/z 638 - 640 (M+H)+; Rt=4.04 min (220 nm,
100 area%) .
Figure imgf000170_0001
Example 33F
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) acryloyl] -piperidin-4-yl}-3-quinolin-3-yl- succinamic acid
The title' compound was prepared by the procedures described in Example 23E, substituting Example 23D with Example 33E. XE NMR (DMSO-d6, ■ 400 MHz) δ 8.98 (s, IH) , 8.53 (br d, IH) , 8.38 (s, IH) , ■'8.26 (d, J=7.2 Hz, IH) , 8.04 (m, 2H), 7.97 (m, IH), ' 7.87 (d, J=15.2 Hz, IH) , 7.80 (m, IH) , 7.67 (m, IH) , 7.54 (m, IH) , 7.38 (m, IH) , 4.16 (m, 3H) , 3.83 (m, IH) , 3.25 (m, IH) , 3.13 (dd, J=16.4. Hz, J=8.8 Hz, IH), 2.90 (m, IH) , 2.76 (dd, J=16.4 Hz, J=6.4 Hz, IH) , 1.86 (m, IH) , 1.65 (m, IH) , 1.37 (m, IH) , 1.18 (m, IH) . LCMS (APCI+; Method I) m/z 582, 584 (M+H)+; Rt=2.88 min (220 nm, 100 area%) .
The following examples 34-118 were prepared by procedures described in Examples 25 or 26. Alternatively, these compounds can be made via solution phase method in solvents such as chloroform or tetrahydrofuran, with the following reagents: PS-DIEA, PS-DMAP, and corresponding acyl chlorides or isocyanates, by shaking in a 24-well format Bohdan mini-block at 550 rpm for 16 hrs. The work- up consisted of rinsing with DMF and/or 'THF. The crude solid was purified by preparative HPLC (Methods F or J) . LCMS (Method I) .
TABLE II
Figure imgf000172_0001
I I -
Z.0Z9Z0/t00ZSfl/I3d LL lO/ςOOZ OΛV
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
- 176 -
Figure imgf000177_0001
- 177 -
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
- IS
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
- 189 -
Figure imgf000190_0001
191 -
Figure imgf000192_0001
- 192 -
Figure imgf000193_0001
- 193 -
Figure imgf000194_0001
- 194 -
Figure imgf000195_0001
- 195 -
Figure imgf000196_0001
- 961 -
L0Z9Z0/t00ZSΑ/lDd LLUIO/SOOZ OΛV 197 -
Figure imgf000198_0001
Figure imgf000199_0001
- 861 -
Z.OZ9ZO/tOOZSfl/X3d Z.-.Ϊ6Ϊ0/S00Z OΛV - 199 -
Figure imgf000200_0001
Figure imgf000201_0001
- 002 -
Z.0Z9Z0/t00ZSfl/X3d LLUIO/SOOZ OΛV
Figure imgf000202_0001
- 102 -
_.0Z9Z0/t00ZSfl/X3d LLUIO/SOOZ OΛV
Figure imgf000203_0001
_.0Z9Z0/t00ZSfl/X3d LLUIO/SOOZ OΛV
Figure imgf000204_0001
εo2 -
Z.0Z9Z0/t00ZSfl/X3d LLUIO/SOOZ OΛV
Figure imgf000205_0001
- 02 -
Z.OZ9ZO/tOOZSfl/X3d LLUIO/SOOZ OΛV
Figure imgf000206_0001
Figure imgf000206_0002
Example 119
N-[l-(3-Isoquinolin-4-yl-acryloyl)-piperidin-4-yl]-3-methyl-succinamic acid
Figure imgf000206_0003
Example 119A
3-Isoquinolin-4-yl-acrylic acid methyl ester
To a solution of 4-bromoisoquinoline (1 g, 4 •±..8o muimiiiol) in THF (20 mL) were added Pd(OAc)2 (432 mg, 1.9 mmol), P(o-tol)3 (2.9 g, 9.6 mmol), Νal (360 mg, 2.4 mmol), and Et3Ν (2 mL, 14.4 mmol) under a N2 atmosphere. After stirring 30 min, methyl acrylate (0.9 mL, 9.6 mmol) was added and the reaction mixture heated to 60°C overnight. The reaction mixture was filtered, concentrated, then purified by flash column chromatography (1:1 EtOAc/Hexanes) to give the title compound as an orange solid. LCMS (Method I) m/z 214 (M+H)+; Rt = 2.58 min.
Figure imgf000207_0001
Example 119B
N-[l-(3-Isoquinolin-4-yl-acryloyl)-piperidin-4-yl]-3-methyl-succinamic acid
The title compounds were prepared by procedures described in Examples 20F, 20G, 20H and 201. The final product was purified by preparative -HPLC (Method F) . LCMS (Method I) m/z 396 (M+H)+
Figure imgf000207_0002
Example 120
N- [ 1 -(3 -B enzα[δ]thiophen-3 -yl-acryloy l)-piperidin-4-yl]-3 -methyl-succinamic acid
Figure imgf000208_0001
Example 120A
3-Benzo [b] thiophen-3-yl-acrylic acid methyl ester
The title compound was prepared by the procedures described in Example 119A, substituting 4-bromoisoquinoline with 3-bromothianaphthalene. The crude product was purified by flash column chromatography (1:4 EtOAc/Hexanes) to give the title compound as a brown oil .
Figure imgf000208_0002
Example 120B
N-[l-(3-Benzo[έ]thiophen-3-yl-acryloyl)-piperidin-4-yl]-3-methyl-succinamic acid
The title compounds were prepared by pro- cedures described in Examples 20F, 20G, 20H, and
201. The final product was purified by preparative HPLC (Method F) . LCMS (Method I) m/z 401 (M+H)+ Example 121
7V-{l-[3-(6,7-Dichloro-benzo[A]thiophen-5-yl)-acryloyl]-3-methyl-piperidin-4-yI}-3-methyI-succinamic acid
Figure imgf000209_0001
Example 121A.
4-Amino-3-methyl-piperidine-l-carboxylic acid benzyl ester
Figure imgf000209_0002
To l-benzyl-3-methyl-piperidin-l-one (3.2 g,16 mmol) in benzene (100 ml) was added benzyl chloroformate (7.25 g, 42.5 mmol), and the solution refluxed for 15 hours. After concentration and purification by chromatography (dichloromethane as eluant) the product was isolated as clear oil (3.80 g, 98%) . This material was carried to the next step and diluted in methanol (100ml), then treated with ammonium acetate (11 g, 138 mmol) and stirred at room temperature for 3 hrs . The solution was cooled to 0°C for 10 min, and sodium cyanoborohydride (0.97 g) was added. The resultant suspension was stirred at 0°C for 10 min and then at room temperature for 4 hrs. The reaction was concentrated, diluted with water and brought to pH 3-4 with 1 N HCl. The aqueous phase was washed with ethyl acetate, brought to pH 10 with 1 N NaOH and extracted with dichloromethane, dried over MgS04 and concentrated to yield the final product as aclear oil (2.6 g, 69%). LCMS (Method G) m/z 249 (M+H)+
Example 12IB
7V-{l-[3-(6,7-DichIoro-benzo[6]thiophen-5-yl)-acryIoyl]-3-methyl-piperidin-4-yl}-3-methyl-succinamic acid
Figure imgf000210_0001
The title compounds were prepared by procedures leading to Example 201. The final product was purified by preparative HPLC (Method F) . LCMS (Method G) m/z 483 (M+H)+
From the foregoing description, various modifications and changes in the composition and methods will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein. The following Methods may be used to test compounds of this invention. Unless otherwise indi- ated, the reagents used in the following examples are commercially available and may be purchased from Sigma-Aldrich Company, Inc. (Milwaukee, WI, USA) or Alfa Aesar (Ward Hill, MA, USA) .
Example A
Inhibition of soluble VLA-1 binding to Collagen IV
Generation of sVLA-1-LZ
A soluble form of the αlβl heterodimer (sVLA-1-LZ) was generated by truncating each chain of the heterodimer at the beginning of the transmem- rane region and adding an acidic and basic leucine zipper sequence to the αl and βl chains, respective- y. The αl chain (described in U.S. Patent Applica- ion Publication No. 2003/0088061) was truncated after residue P1141 of the pro-peptide and the 47 amino acid acidic leucine zipper cassette was added. The βl chain (Genbank accession no. P05556) was truncated after residue D728 of the pro-peptide and the 47 amino acid basic leucine zipper cassette was added. Both constructs were then expressed in CHO DG44 cells, and the heterodimer was purified from the culture supernatant using an antibody affinity column specific for the leucine zipper sequences, followed by size-exclusion chromatography on an S200 sizing column (Pharmacia, now Pfizer Inc., New York, NY USA) using methods standard to the art. (See also, U.S. Patent Application Publication No.
2003/0088061) . The purified protein was stored at -70°C.
Briefly, an αl-LZ construct was generated which has the extracellular domain of αl fused to a C-terminal leucine zipper sequence. The extracellular domain of αl was amplified using standard PCR methods and reagents in order to add restriction sites for the subcloning. The sequences of the primers used in the PCR reaction were:
5' primer: αl-01: ATT CTC GAG ACC GCC ACC ATG GTC
CCC AGG CGT CC (SEQ. ID. 1)
3' primer: αl-04: ATT ACG CGT TGG CAC TCT GCC CGG
TAG (SEQ. ID. 2)
The primers above were used in a PCR reaction with an αl cDNA clone (described previously, U.S. Patent Application Publication No. 2003/0088061) . The resulting PCR product was sub- cloned 5' to the acidic leucine zipper sequence in the mammalian expression vector pDEF38 (described in U.S. Patent Application Publication No. 2003/0088061) . The resulting plasmid was verified by sequencing. The extracellular domain of βl likewise was amplified using standard PCR methods and reagents in order to add restriction sites for the subcloning. The sequences of the primers used in the PCR reaction were: 5' primer: βl-02: ATT CTC GAG ACC GCC ACC ATG AAT TTA CAA CCA ATT TTC TGG (SEQ. ID. 3) 3' primer: βl-03: GTT CCA TTC ACC CCG TTC TTG C (SEQ. ID. 4)
In order to generate a βl leucine zipper soluble molecule, the 5' end of the β-1 insert was generated by PCR from a βl cDNA and subcloned 5' to the basic leucine zipper sequence in the mammalian expression. vector pNEF38 (described in U.S. Patent Application Publication No. 2003/00.88061) . The; resulting plasmid was verified by sequencing.
Alpha 1 Leucine Zipper (Acidic) construct protein sequence:
MVPRRPASLE VTVACIWLLT VILGFCVSFN VDVKNSMTFS GPVEDMFGYT VQQYENEEGK WVLIGSPLVG QPKNRTGDVY KCPVGRGESL PCVKLDLPVN TSIPNVTEVK ENMTFGSTLV TNPNGGFLAC GPLYAYRCGH LHYTTGICSD VSPTFQVVNS
• IAPVQECSTQ LDIVIVLDGS NSIYPWDSVT AFLNDLLERM .
DIGPKQTQVG IVQYGENVTH EFNLNKYSST EEVLVAAKKI
VQRGGRQTMT ALGTDTARKE AFTEARGARR GVKKVMVIVT
DGESHDNHRL KKVIQDCEDE NIQRFSIAIL GSYNRGNLST " EKFVEEIKSI ASEPTEKHFF NVSDELALVT IVKTLGERIF
ALEATADQSA ASFEMEMSQT GFSAHYSQDW VMLGAVGAYD
■' WNGTWMQKA SQIIIPRNTT FNVESTKKNE' PLASYLGYTV
NSATASSGDV LYIAGQPRYN HTGQVIIYRM EDGNIKILQT
LSGEQIGSYF GSILTTTDID KDSNTDILLV GAPMYMGTEK ' EEQGKVYVYA LNQTRFEYQM SLEPIKQTCC SSRQHNSCTT
- ENKNEPCGAR FGTAIAAVKD LNLDGFNDIV IGAPLEDDHG
GAVYIYHGSG KTIRKEYAQR IPSGGDGKTL KFFGQSIHGE
MDLNGDGLTD VTIGGLGGAA LFWSRDVAVV KVTMNFEPNK
VNIQKKNCHM EGKETVCINA TVCFDVKLKS KEDTIYEADL QYRVTLDSLR QISRSFFSGT QERKVQRNIT VRKSECTKHS FYMLDKHDFQ DSVRITLDFN LTDPENGPVL DDSLPNSVHE YIPFAKDCGN KEKCISDLSL HVATTEKDLL IVRSQNDKFN VSLTVKNTKD SAYNTRTIVH YSPNLVFSGI EAIQKDSCES NHNITCKVGY PFLRRGEMVT FKILFQFNTS YLMENVTIYL SATSDSEEPP ETLSDNVVNI SIPVKYEVGL QFYSSASEYH ISIAANETVP EVINSTEDIG NEINIFYLIR KSGSFPMPEL
KLSISFPNMT SNGYPVLYPT GLSSSENANC RPHIFEDPFS INSGKKMTTS TDHLKRGTIL DCNTCKFATI TCNLTSSDIS
QVNVSLILWK PTFIKSYFSS LNLTIRGELR SENASLVLSS . SNQKRELAIQ ISKDGLPGRV PTRSSADLVP RGSTTAPSAQ
LEKELQALEK ENAQLEWELQ ALEKELAQ (SEQ. ID. 5)
Beta 1 Leucine Zipper (basic) construct protein sequence :
MNLQPIFWIG LISSVCCVFR QTDENRCLKA NAKSCGECIQ ' AGPNCGWCTN STFLQEGMPT SARCDDLEAL KKKGCPPDDI ENPRGSKDIK KNKNVTNRSK GTAEKLKPED. IHQIQPQQLV LRLRSGEPQT FTLKFKRAED YPIDLYYLMD LSYSMKDDLE NVKSLGTDLM NEMRRITSDF RIGFGSFVEK TVMPYISTTP AKLRNPCTSE QNCTTPFSYK NVLSLTNKGE VFNELVGKQR ISGNLDSPEG GFDAIMQVAV CGSLIGWRNV ■ TRLLVFSTDA ; GFHFAGDGKL GGIVLPNDGQ CHLENNMYTM. SHYYDYPSIA HLVQKLSENN IQTIFAVTEE FQPVYKELKN LIPKSAVGTL SANSSNVIQL IIDAYNSLSS EVILENGKLS EGVTISYKSY CKNGVNGTGE NGRKCSNISI GDEVQFEIST TSNKCPKKDS DSFKIRPLGF TEEVEVIL Y ICECECQSEG' IPESPKCHEG ' . NGTFECGACR CNEGRVGRHC ECSTDEVNSE DMDAYCRKEN SSE1CSNNGE CVCGQCVCRK RDNTNEIYSG KFCECDNFNC DRSNGLICGG NGVCKCRVCE CNPNYTGSAC DCSLDTSTCE ASNGQICNGR GICECGVCKC TDPKFQGQTC EMCQTCLGVC AEHKECVQCR AFNKGEKKDT CTQECSYFNI TKVESRDKLP QPVQPDPVSH CKEKDVDDCW FYFTYSVNGN NEVMVHVVEN PECPTGPDTS SADLVPRGST TAPSAQLKKK LQALKKKNAQ LKWKLQALKK KLAQ (SEQ. ID. 6) Signal Sequences are shown in italics Mature polypetides are shown in plain text Acidic and Basic Leucine Zippers are underlined and bold
Screening Assay for Measuring sVLA-1-LZ Binding to Collagen IV
5 A biochemical assay for measuring binding of sVLA-1-LZ to Collagen IV using time-resolved '.' ' fluorescence, suitable for high-throughput screening, was developed. Briefly, 96-well Immulon 4 ELISA plates were coated with sVLA-1-LZ at 2 μg/mL0 in 50mM NaHC0, pH'9.2, .and incubated overnight at 4°. The1 plates were then- washed' two times with 300 μL/well Wash Buffer (TBS;."0.1% Tween-20; 2mM MgCl2) , and blocked for 1 -hour with 200 μL/well Blocking Buffer (2% BSA; CMF-PBS," 2mM MgCl2) a room tempera-5 ture. Compounds of this invention were prepared in DMSO at 200X the desired- final assay concentration. ■ Final concentrations were1:' selected from a range between 0.01 nM-100 μM. DMSO inhibitor stocks were then diluted to 2X final ' concentration in Dilution0 Buffer (TBS;.0.01% BSA; 2mM MgCl2) . Fifty μL/well
Dilution Buffer, alone or in combination with, anti- αl mAb (Immunodiagnostic, #8149a, 2.5 μg/mL), 10 mM EDTA, DMSO or DMSO+2X inhibitor was added to the wells of the plate, followed by 50 μL/well Collagen IV-biotin at 2 μg/mL in Dilution buffer. Collagen IV-biotin was generated by biotinylating human Collagen IV (Sigma-Aldrich, Milwaukee, WI, USA) using a biotin labeling kit (Pierce Biotechnology, Rockford, IL, USA) following the manufacturer's0 protocol. The plates were incubated for 1 hour at room temperature, and washed four times with Wash Buffer (300 μL/well) . The plates were then incubated with 100 μL/well of 1:1000 diluted (with H20) Strepavidin-Europium (PerkinElmer, Boston, MA, USA) 5 for 30 min. at room temperature. The plates were ■:... then washed four times with Wash -Buffer (300 μL/- well) . One hundred μL/well of Delphia- Enhancement Solution (PerkinElmer; diluted 1:1 with. dH20) was added, and the plates were shaken for 5 minutes. 10,, Binding was -then analyzed, by time resolve fluorescence (TRF) using a Victor Plate-reader (Perkin- -; Elmer). Results were analyzed using the equations i..,- below. The percent of inhibition was plotted versus-. • '.. the log concentration of .inhibitor across a twelve 15;-,. point titration, and a linear regression trend-line was drawn.
Specific binding = TRF signal (Collagen-IV-biotin in
Dilution Buffer plus Ab, DMSO only or DMSO + inhbitor) -TRF 20. . signal (Collagen-IV-biotin with '
EDTA)
% inhibition=
Figure imgf000216_0001
As a variation in the assay, the effect of 25 serum protein in the assay was determined by substituted Collagen IV-biotin at 2μg/mL in FBS, instead of Dilution buffer, resulting in a final serum concentration of 50% in the assay. The percent inhibition in the presence and absence of 50% FBS was then compared.
Example B
Inhibition of VLA-1 dependent K562-ocl cell adhesion to Collagen IV
Generation of K562-αl cells
The αl cDNA was subcloned into the mammal- ian expression vector pMH-Neo (Hahn, W. C. et al., ■, 1993. Gene 127: 261 ) . The resulting clone αl/- pMHneo/40 was verified by sequencing. The αl/- pMHneo/40 construct was introduced into K562 cells by electroporation. The transfected cells were initially maintained in complete RPMI with 10% FBS. Two days after the transfection, the cells were spun down and resuspended in complete RPMI with 10% FBS and 0.5mg/mL G418 (Sigma-Aldrich) to select for the neomycin resistance conferred by the pMHneo plasmid. Transfectants with functional αl expression were selected by panning the cells for binding to Type IV collagen (an αl ligand) . In panning experiments, K562 transfectants were stimulated with 20ng/mL of PMA and allowed to adhere to plate-bound Type IV collagen. After a 30-60 min incubation at 37 °C, the unbound cells were washed away and the adherent cells were recovered with versene. Cellular expression of VLA-1 was verified by FACS analysis with an αl mAb. K562-αl cell adhesion assay to Collagen IV
The day prior to the assay, the K562-αl cells were split 1:2 into fresh culture media (RPMI- 1640, 10% FBS) and cultured at 37 °C in C02. Ninety- 5 six well Immulon-4 plates were coated with either
1.25 μg/mL collagen IV (Sigma) in CMF-PBS or the Dl "-"• mAb at 5μg/mL in Coating Buffer (50 mM sodium carbonate, 50 mM sodium bicarbonate, pH 9.6), and incubated overnight at 4°C. On the day of the 0 assay, the plates were washed with D-PBS and then blocked with 1% BSA/D-PBS for 1.5-2.0 hours at room temperature. Compounds of this "invention were prepared in DMSO at 66.67X the desired final assay concentration. Final concentrations were selected5 from a range between 1 nM-100 μM. Deep-well blocks (96-square well titer plates, Beckman) were prepared with 700μL RPMI/well. DMSO solutions of diluted compound were then added to the deep-well block (10.5μL diluted compound/700μL RPMI) to generate0' 1.5X solutions. After blocking, the Immulon-4 plates were aspirated and 200μL RPMI only, RPMI + 1.5X DMSO/RPMI Solution, or RPMI + 1.5X compound in DMSO/RPMI solution was added to each well. The plates were then incubated at 37 °C in 5% C02 for 10-5 30 min. K562-αl cells were counted, spun down, washed once with RPMI and resuspended in RPMI+60ng/- L PMA at a density of 1.0X106 cells/mL. One hundred μL cell suspension was then added to each well and incubated at 37 °C in 5% C02 for 30 min. Adherent0 cells were then fixed to the plate with 14% glutar- aldehyde/D-PBS for 1.5 hours. Plates were washed with water and the cells were stained with 5% crystal violet for 5 min. at room temperature. The plates were washed with water again, and the crystal violet dye was extracted from the cells with 200 μL/well 70% ethanol. ' The plates were read on a plate reader at A570nm and A410nm. Results were analyzed using the equations below. The percent inhibition was plotted versus the log concentration of inhibitor across an eight point titration, and a linear regression trend-line was drawn. As a varia-1 tion in the assay, the effect of serum protein in the assay was determined by substituting RPMI+50% FBS instead of RPMI. The percent inhibition in the presence and absence of 5.0% FBS was then compared.
Percent cells binding = / A570-A410 (binding to Collagen IV) x 100
A570-A410 (binding to Alphal antibody)
Percent Inhibition-
Figure imgf000219_0001
It is contemplated that all of this invention will exhibit at least fifty -percent inhibition in one or more of the above assays when tested- at a concentration of 50 μM.

Claims

WHAT IS CLAIMED IS:
A compound having a structural formula
Figure imgf000220_0001
wherein A and B, . together with the nitrogen atom bound thereto, form a 4-8 membered nitrogen containing heterocyclic group containing 1 to 2 nitrogen atoms, wherein said heterocyclic group may be optionally substituted with 1 to 3 additional substituents each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, hydroxy, alkoxy, thioalkyl, and halo, wherein the one or more alkyl and substituted alkyl substituents, if present, may be attached to either a carbon or a nitrogen atom in said heterocyclic group, wherein the one or more hydroxy, alkoxy, alkylsulfanyl and halo substituents, if present, may not be attached to a nitrogen atom in said heterocyclic group, and wherein the one or more hydroxy, alkoxy, and halo substituents, if present, may not be attached to a carbon atom which is adjacent to a nitrogen atom in said heterocyclic group, and further wherein A together with the nitrogen atom bound thereto form a 4-8 membered heterocyclic group containing two nitrogen atoms, then the two nitrogen atoms are either adjacent to each other, or are separated by at least two carbon atoms,
R1 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
R2 and R3 are independently selected from the group consisting of hydrogen, fluoroalkyl and alkyl;
R4 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heterocyclic, and cycloalkyl;
R5 and R7 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, amino, substituted amino, aminocar- bonyloxy, aminoacyl, aminosulfonyl, sulfonylamino, acylamino, aminoacylamino, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, aryl, substituted aryl, ORa, acyloxy, oxycarbonyl- amino, thioalkyl, thioaryl, thioalkylaryl, thioalk- ylheteroaryl, NHS02NRaRa, SC(0)Ra, and SC(0)NRa 2, or R6 and R7, together with the carbon atom bound thereto, form a cycloalkyl, substituted cyclo- alkyl, heterocyclic, or a substituted heterocyclic group;
R8 is selected from the group consisting of CRaRaC(0)0Ra, C02Ra, C(0)NRa 2, C(0)NRaORa, C(0)NHS02Ra, CRaRa heteroaryl (e. g. , - tetrazolyl) , CRaRa substituted . heteroaryl, CN, and (CRa 2)p0H, wherein p is 0, 1 or . 2;
R9 and R10 are independently hydrogen, halo, alkyl, substituted alkyl, amino,' . substituted , ,. amino, aminocarbonyloxy, aminoacyl, acylamino, '< amino'acylamino, aminosulfonyl, sulfonylamino heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, aryl, substituted aryl, -ORa, acyloxy, oxycarbonylamino, thioalkyl, thioaryl, •■ thioalkylaryl, thioalkylheteroaryl, NHS02NRaRa,
SC(0)Ra, and -SC(0)NRa 2, '• •' or R7-C-C-R9 can form a cycloa-lkylene, cycloalkenylene, heterocyclene, or heterocyclenylene group, 1 or R9 and R10, together . with the carbon atom attached thereto, form a >C=0 (oxo) group, with the proviso that when R9 and R1" form an oxo group, R8 ' is not -CN, or R9 and R10, or R6 and R7, together with the carbon atom attached thereto, form a vinyl group of the formula >C=CR R12 where R11 and R12 are independently selected' from the group consisting of hydrogen, alkyl, substituted alkyl, aryl and substi- . tuted aryl, or R9-C-R10, together with the carbon atom attached thereto, form a group selected from the group consisting of cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic group; wherein each Ra is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, and, in the case of -NRa 2, each Ra, together with the nitrogen atom bound thereto, can form a heterocyclic or substituted heterocyclic group or in the case of CRaRaC (0) ORa, the two Ra can - be an oxo group, with the proviso that when the two Ra are an oxo group, then R9 and R10 are not an oxo group; and a pharmaceutically acceptable salt, or prodrug, or tautomer thereof.
2. The compound of claim 1 wherein A and B, together with the nitrogen atom bound thereto, form a piperidine ring.
3. The compound of claim 1 wherein A and B, together with the nitrogen atom bound thereto, form a pyrrolidine ring.
4. The compound of claim 1 wherein A and B together with the nitrogen atom bound thereto form a azetidine ring.
5. The compound of claim 1 wherein R1 is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl.
6. The compound of claim 5 wherein R1 is selected from the group consisting of substituted aryl and substituted heteroaryl.
7. The compound of claim 6 wherein R1 is selected from the group consisting of:
2, 3-dichlorophenyl;
2, 3-dichloro-4- (2-methoxyphenylthio) - phenyl;
2, 3-dichloro-4- (4-fluorophenylthio) phenyl; 4-methylthio-2, 3-dichlorophenyl; and 2,3-dichlorobenzo[b] thiophen-4-yl.
8. The compound of claim 1 wherein R2, R3, R4, and R6 are hydrogen.
9. The compound of claim 1 wherein R7 is selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, substituted alkylene, aryl, and heteroaryl.
10. The compound of claim 9 wherein R7 is selected from the group consisting of: hydrogen; fluoro; methyi; - aminomethyl; phenylamidomethylene;
(2-methoxyphenyl) amidomethylene;
(2-chlorophenyl) amidomethylene; phenyl; pyridin-3-yl; quinolin-3-yl; phenylaminocarbonylaminomethylene; propylamidomethylene;' "' methoxymethyleneamidomethylene; t-butyl-amidomethylene; methylthioethyleleneamidomethylene; t-butyl-methyleneamidomethylene; cyclopropylamidomethylene; cyclopentylamidomethylene; cyclohexylamidomethylene; 4-N-acetylpiperidinylamidomethylene; cyclopentylmethylenamidomethylene; piperidinyl-N-ethyleneamidomethylene; 2-methoxyphenylamidomethylene; i-propylamidomethylene; cyclobutylamidomethylene; 2-pyridynylamidomethylene; 3-pyridinylamidomethylene; 4-pyridinylamidomethylene; 2-thiophenylamidomethylene; 2-furanoylamidomethylene; benzo [1, 3] -dioxole-5-amidomethylene;
3-methoxyphenylmethyleneamidomethylene;
4-methoxyphenylamidomethylene;
2-thiophenylmethyleneamidomethylene; cyclopropyl-1-phenyl-l-amidomethylene; phenylethyleneamidomethylene; phenyloxymethyleneamidomethylene;
4-chlorophenyloxy-dimethylmethyleneamido- methylene;' cyclopentyl-1- (4-chlorophenyl) -1-amido- methylene;
2-phenylcyclopropaneamidomethylene; phenylmethyleneoxymethyleneamidomethylene; methylaminoamidomethylene; morpholinylamidomethylene; phenylmethyleneaminoamidomethylene; phenylethyleneaminoamidomethylene; methoxyamidomethylene; • phenylmethyleneoxyamidomethylene;
R-phenylamido; • ,. - -
S-phenylamido; ■ methylamido; propylamido; methoxymethylene.amido; t-butyl-amido; thiomethylethyleneamido; t-butylmethyleneamido; • cyclopropyiamido; cyclopentylamido; cyclohexylamido;. 4-N-acetylipiperidinylamido; cyclopentylmethylene amido; piperidynyl-N-ethylene amido;
3-methoxyphenylamido;
4-methoxyphenytamido; i-propylamido;
3-chlorophenylamido;
4-chlorophenylamido; cyclobutylamido; .
.2-pyridinylamido;
3-pyridinylamido;
4-pyridinylamido;
2-thiophenylamido;
2-furanoylamido;'
5-isoxazolylamido;
4-biphenylamido;'-■ phenylmethylene .amido;- '■
4-chlorophenylmethylene amido;'
3-methoxyphenylmethyleneamido; -methoxyphenylmethyleneamido;
2-thiophenemethyleneamido; cyclopropyl-l-phenyl-1-amido; phenylethyleneamido; phenyloxymethyleneamido;
4-chlorophenyloxy-l, 1-dimethylmethylene- amido; phenylethyl-ene-amido; phenylmethoxyloxymethyleneamido; methylaminoamido; dimethylaminoamido; morpholine-N-amido; phenylmethyleneaminoamido; phenylethyleneaminoamido; methoxyamido; phenylmethyleneoxyamido; phenylsulfonamido; 3-methoxyphenylamido; and 3-chlorophenylamido .
11. The compound of claim 1 wherein R8 is selected from the group consisting of CRaRaC (0) ORa, -C02Ra, where Ra is as defined above, and, when R9 and R10 form an oxo group, then R8 is preferably hydroxy.
12. The compound of claim 11 wherein R8 is selected from the group consisting of: carboxyl; -CH(C6H4)COOH; -CH2COOH; and hydroxyl .
13. The compound of claim 1 wherein R9 is selected from the group consisting of hydrogen, alkyl, acylamino, and aryl.
14. The compound of claim 13 wherein R9 is selected from the group consisting of: methyl; hydrogen; phenyl; and . -NHC(0)CF3.
15. The compound of claim 1 wherein R10 is selected from the group consisting of hydrogen and alkyl. . ■•
16,. T-ke- compound of claim 1 wherein R7 and R9, together with the carbon atoms bound thereto, form a cycloalkylene or cycloalkenylene group.
17. The compound of claim 1 wherein R9 and R10, together with the carbon atom bound thereto, form an oxo group.
18. The compound of claim 1 wherein R9 and R10, together with, the carbon atom bound thereto, form a vinyl group of the formula >C=CR11R12 where R11 and R12 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.
19. A compound selected from. the group consisting of:
(S) -N-{1- [3- (2, 3-dichlorophenyl) -acryloyl] -piper- idin-4-yl} -2-methyl-succinamic acid;
N-{ 1- [3- (2, 3-dichlorophenyl) -acryloyl] -piperidin-4- yl } -2-methylene-succinamic acid;
N-{ 1- [3- (2, 3-dichlorophenyl) -acryloyl] -piperidin-4- yl} -succinamic acid;
Cis-6- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) -phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) - Cyclohex-3-enecarboxylic acid;
Cis-6- (l-{ 3- [2,"3-"dichloro-4- (2-methoxy-phenylsulfanyl) -phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) - Cyclohex-3-enecarboxylic acid;
4- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-ylcarbamoyl) -2-phenyl- butyric acid;
2- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-ylcarbamoyl) -cyclopro- panecarboxylic acid;
4- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-ylcarbamoyl) -3-methyl- butyric acid;
4- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-ylcarbamoyl) -butyric acid;
N- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) - (S) -2- (2, 2, 2- trifluoro-acetylamino) -succinamic acid; N- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -2-methyl-succinamic acid;
N- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -2-phenyl-suc- cinamic acid; ■ N- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-4-yl) -2, 2-dimethyl- succinamic acid;
4- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-3-ylcarbamoyl) -3-methy1-
-.-. butyric acid; . -
' N- (l-{3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-3-yl) - (S) -2- (2, 2, 2-tri- fluoro-acetylamino) -succinamic acid;
'; N- (l-{ 3- [2, 3-dichloro-4- (2-methoxy-phenylsulfanyl) - phenyl] -acryloyl }-piperidin-3-yl) -2-methyl-succinamic acid; ■ .'• N- (l-{3- [2, 3-dichloro-4- (4-fluoro-phenylsulfanyl) - phenyl] -acryloyl}-piperidin-4-yl) -malonamic acid; N- (l-{ 3- [2, 3-dichloro-4- (4-fluoro-phenylsulfanyl) - phenyl] -acryloyl } -piperidin-4-yl) -2-fluoro-malonamic acid;
N-{1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- (S) -3-methyl-succinamic acid; N-{1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- (S) -2-methyl-succinamic acid; N-{ 1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}- (R) -2-methyl-succinamic acid; rac-3-aminomethyl-N-{ 1- [3- (6, 7-dichloro-benzo [b] - thiophen-5-yl) -acryloyl] -piperidin-4-yl} -succinamic acid trifluoroacetic acid salt; rac-3- (benzoylamino-methyl) -N-{1- [3- (6, 7-dichlorobenzo [b] thiophen-5-yl)--acryloyl]-piperidin-4-yl}- succinamic acid; rac-N-{l- [3- (6, 7-dichlorobenzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl}-3- [(3-methoxy-benzoyl- amino) -methyl] -succinamic acid; rac-3- [ (3-Chlorobenzoylamino) -methyl] -N-{1- [3- (6, 7- . dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin- 4-yl}-succinamic acid; :- .' rac-N-{l- [3- (6, 7-dichlorobenzo [b] thiophen-5-yl) - acryloyl] -piperidin-4-yl} -3- [ (3-phenyl-ureido) - methyl] -succinamic acid; ..
N-{ 1- [3- (2, 3-Dichloro-4-methylsulfanyl- henyl) - acryloyl] -azetidin-3-yl}-3-methyl-succinamic acid; N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryl- ' • oyl] -azetidin-3-yl} -3-methyl-succinamic acid; •■ v,
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -acryl- : oyl] -piperidin-4-yl}-3-phenyl-succinamic acid; :„•
N-{1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) - acryloyl] -3-methyl-piperidin-4-yl } -3-methyl- succinamic acid;
N-{1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}-3-pyridin-3-yl-succinamic acid; N-{ 1- [3- (6, 7-dichloro-benzo [b] thiophen-5-yl) -acryloyl] -piperidin-4-yl}-3-quinolin-3-yl-succinamic acid; 3-(Butyrylamino-methyI)-Λ/-{1-[3-(6,7-dichloro-benzo[ή]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
Λ/^1-[3-(6,7-Dichloro-beπzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2-methoxy-acetylarτιino)-nrιet yl]-succinarnic acid Λ/-{1-[3-(6,7-Dichloro-benzo[/)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2,2-dimethyl-propionylamino)-methyl]- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[d]thiophen-5-yl)-acιyloyl]-piperidin-4-yl}-3-[(3-methylsulfanyI-propionylamino)-methyl]- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[i ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3,3-dimethyl-butyrylamino)-methyl]-succinamic acid
3-[(Cyclopropanecarbonyl-amino)-methyl]-Λ/-{1-[3-(6,7-dichloro-benzo[£)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
3-[(Cyclopentanecarbonyl-amino)-methyl]-Λ/-{1-[3-(6,7-dic loro-benzo[/)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
3-[(Cyclohexanecarbonyl-a ino)-methyl]-Λ/-{1-[3-(6,7-dichloro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succina ic acid
3-{[(1-Acetyl-piperidine-4-carbonyl)-amino]-methyl}-Λ/-{1-[3-(6,7-dichloro-benzo[ι ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
3-[(2-Cyclopentyl-acetylamino)-methy!]-Λ-{1-[3-(6,7-dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succfnamic * acid
Λ/-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-piperidin-1-yl-propionylamino)-methyl]- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[fe]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2-methoxy-benzoylamino)-methyl]-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[ι ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-methyl-butyrylamino)-methyl]-succinamic acid
3-[(Cyclobutanecarbonyl-amino)-methyl]-Λ-{1-[3-(6,7-dic loro-benzo[f)]thiophen-5-yl)-acryloyl] piperidin-4-yl}-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[d]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(pyridine-2-carbonyl)-amino]-methyl}-succinamic acid
Λ-{1-[3-(6,7-Dichloro-benzo[j ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(pyridine-3-carbonyI)-amino]-methyl}-succinamic acid
Λ-{1-[3-(6,7-Dichloro-benzo[ι ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(pyridine-4-carbonyl)-amino]-methyl}-succinamic acid
W-{1-[3-(6,7-Dichloro-benzo[ib]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(thiophene-2-carbonyl)-amino]-methyl}- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[fe]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(furan-2-carbonyl)-amino]-methyl}-succinamic acid 3-{[(Benzo[1,3]dioxole-5-carbonyl)-amino]-methyl}-Λ/-{1-[3-(6,7-dichloro-benzo[i]thiophen-5ryl)-acryloyl]-piperidin-4-yl}- succinamic acid /-{1 -[3-(6,7-DichIoro-benzo[£ι]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[2-(3-methoxy-phenyl)-acetylamino]-methy I}- succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[b]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[2-(4-methoxy-phenyl)-acetylamino]-methyl}- succinamic acid
Λ/-{1 -[3-(6,7-Dichloro-benzo[i)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(2-thiophen-2-yl-acetylamino)-methyl]-succinamic- acid
Λ/-{1-[3-(6 -Dichloro-b,enzo[/3]thiophen-5-yl)-acιyloyl]-piperidin-4-yl 3-{[(1-phenyl-cyclopropaπecarbonyl)-amino]-met yl}- succinamic acid
Λ/-{1 -[3-(6 -Dichloro-.benzo[ft]thiophen-5-yl)-acryloy]]-piperidin-4-yl}-3-[(3-phenyl-propionylamino)-methyI]-succinamic ,
: " ' "' " ' acid
Λ/-{1-[3-(6 -Dichlorθ benzo[6]thiophen-5-yl -acryloyl37piperidin-4-y!}-3-[(2-phenqxy-acetylamino)-rnethyl]-succinamic acid ,
3-{[2-(4-Chloro-phenoxy)-2-methyl-propionylamino]-methyl}- /-{1-[3-(6,7-dichloro-benzo[i ]thiophen-5-yl)-acryloyl]- , , t piperidin-4-yl}-succinamic acid
3-({[1-(4-Chloro-phenyl)-cyclopentanecarbonyl]-amino}-methyl)-Λ-{1-[3-(6,7-dichloro-benzo[ιb]thiophen-5-yl)-acryloyl]- • .. piperidin-4-yl}-succinamic acid
Λ-{1-[3-(6,7-Dichloro-benzo[J ]thiophen-5-yl)-acryloyl]-piperidin-4-yI}-3-{[(2-phenyl-cyclopropanecarbonyl)-amino]-methyl}-' ,, , . succinamic acid
3-[(2-Benzyloxy-acetylamino)-methyl]-/V-{1-[3-(6,7-dichloro-benzo[i ]thiophen-5-yl)-acryIoyl]-piperidin-4-yl}-succinamic-
Λ/-{1-[3-(6,7-Dichlorρ-benzo[ft]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(3-methyl-ureido)-methyl}-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[/3]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-{[(morpholine-4-carbonyI)-amino]-methyl}- succinamic acid
3-(3-Benzyl-ureidomethyl)-/V-{1-[3-(6,7-dichloro-benzo[ib]thiophen-5-y!)-acryIoyl]-piperidin-4-yl}-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[ft]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-(3-phenethyl-ureidomethyl)-succinamic acid
Λ/-{1-[3-(6,7-Dichloro-benzo[j ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-(methoxycarbonylamino-methyl)-succinamic acid
3-S-Benzoylamino-Λ/-{1-[3-(6,7-dichloro-benzo[ύ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
3-R-Benzoylamino-Λ/-{1-[3-(6,7-dichloro-benzo[/)]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid 3-R-Acetylamino-Λ/-{1-t3-(6>7rdichloro-benzo[ύ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid .
3-R-Butyrylamino-Λ-{1-t3-(6,7-dichloro-benzo[ύ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[6]thiophen-5-yl)-acryIoyl]-piperidin-4-yl}-3-(2-methoxy-acetylamino)-succinamic acid
Λ -R-{1-[3-(6,7-Dichloro-benzo[ύ]thiophen-5-yl)-acryloy!]-piperidin-4-yl}-3-(2,2-dimethyl-propionylamino)- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- ";
>r3-(3-methylsulfanyl-propionylamino)-succinamic acid .
Λ/-R-{1-[3-(6l7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3,3-dimethyl-butyrylamino)-succinamic acid
3R-(Cyclopropahecarbonyl-amino)-Λ-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl) -acryloyl]-piperidin-4-yl}-succinarhic acid
, ,.- 3R-(Cyclopentanecarbonyl-amino)-Λ/-{1-[3-(6,7-dic loro-benzo[D]thiophen 5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
. 3-R-(Cyclohexanecarbonyl-amino)-Λ/-{1-[3-(6,7-dichlorp-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}l
-succinamic acid
3R-[(1-Acetyl-piperidine-4-carbonyl)-amino]-Λ/-{1-[3-(6,7-dic loro-benzo[ό]thiophen-5-yl)-acryloyl]-, piperidin-4-yl}-succinamic acid
3-R-(2-Cyclopentyl-acetylamino)-Λ/-{1-[3-(6J-dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
J Λ-R-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3- ; , :
(3-piperidin-1-yl-propionylamino) -succinamic acid
Λ/R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyi]-piperidin-4-yl}- 3-(2-methoxy-benzoylamino)-succinamic acid
Λ/-R-{i-[3-(6,7-Dichloro-benzo[D]t iophen-5-yl)-acryloyl]-piperidin-4-yl}-3- (4-methoxy-benzoylamino)-succinamic acid
W-R-{1-t3-(6,7-Dichloro-benzo[ø]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3-methyl-butyrylamino)-succinamic acid
3-R(2-Chloro-benzoylamino)-Λ/-{1-[3-(6,7-dichloro-benzo[o]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
3-R-(4-Chloro-benzoylamino)-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid 3R-(Cyclobutanecarbonyl-amino)-Λ/-{1-[3-(6,7-dichloro-benzo[j ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}
-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[Λ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(pyridine-2-carbonyl)-amino]-. succinamic acid Λ/-R-{1-[3-(6,7-Dichloro-benzo[ώ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(pyridine-3-carbonyl)-amino]- succinamic acid /-R-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(pyridine-4-carbonyl)-amino]- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[jb]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(thiophene-2-carbonyl)-amino]- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[i ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[(furan-2-carbonyl)-amino]- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[d]thiophen-5-yl)-acryloyl]-pιperidin-4-yl}-3-[(isoxazole-5-carbonyl)-amino]- succinamic acid
3-R-I(Biphenyl-4-carbonyl)-amino]-Λ/-{1-[3-(6,7-dichloro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- succinamic acid
Λ-R-{1-[3-(6,7-Dichloro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-phenylacetylamino- succinamic acid
3R-[2-(4-Chloro-phenyl)-acetylamino]-Λ-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)-acryloyl]- piperidin-4-yl}- succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[2-(3-methoxy-phenyl)- acetylamino]-succinamic acid
Λ/-R-{1-[3-(6,7-Dic loro-benzo[o]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3-[2-(4-methoxy-phenyl)
-acetylaminoj-succinamic acid
Λ-R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3- (2-thiophen-2-yl-acetylamino)-succinamic acid
Λ-R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3- [(1-phenyl-cyclopropanecarbonyl)-amino]-succinamic acid
Λ/R-{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3-phenyl-propionylamino)-succinamic acid
Λ/ -{1-[3-(6,7-Dichloro-benzo[ό]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(2-phenoxy-acetylamino)-succinamic acid
3R-[2-(4-Chloro-phenoxy)-2-met yl-propionylamino]-Λ/-{1-[3-(6,7-dichloro-benzotD]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid 3R-{[1-(4-Chloro-phenyl)-cyclopentanecarbonyl]-amino}-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
Λ/f?-{1-[3-(6,7-Dichloro-benzo[D]t iophen-5-yl)-acryloyl]-piperidin-4-yl}-3- (3-phenyl-acryloylamino)-succinamic acid 3-R-(2-Benzyloxy-acetylamino)-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
ΛR-{1-[3-(6,7-Dichloro-benzo[D]thiop en-5-yl)-acryloyl]-piperidin-4-yl} -3-(3-methyl-ureido)-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[ώ]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3- (3,3-dimethyl-ureido)-succinamic acid
Λ/R-{1-[3-(6,7-Dic loro-benzo[6]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3 -[(morpholine-4-carbonyl)-amino]-succinamic acid
3-R(3-Benzyl-ureido)-Λ/-{1-[3-(6l7-dichloro-benzo[D]thiophen-5-yl)-acryloyl] • -piperidin-4-yl}-succinamic acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[D]thiophen-5-yl)-acryloyl]-piperidin-4-yl}- 3-(3-phenethyl-ureido)-succinamic acid
Λ/R-{1-[3-(6,7-Dichloro-benzo[Dithiophen-5-yl)-acryloyl]-piperidin^4-yi}- 3-met oxycarbonylamino-succinamic acid
3-R-BenzyIoxyca onylamino-Λ/-{1-[3-(6,7-dichloro-benzo[ό]thiophen-5-yl), v -acryloyl]-piperidin-4-yl}-succinamic'acid
Λ/-R-{1-[3-(6,7-Dichloro-benzo[o]thiophen-5-yl)-acryloyl]-piperidin-4-yl}-3 -(3-methoxy-benzoylamino)-succinamic acid
3-R-(3-Chloro-benzoylamino)-Λ/-{1-[3-(6,7-dichloro-benzo[ό]thiophen-5-yl)- acryloyl]-piperidin-4-yl}-succinamic acid
3-R-Beηzenesulfonyiamino-Λ/-{1-[3-(6,7-dichloro-benzo[D]thiophen-5-yl)-acryloyl]- piperidin-4-yl}-succinamic.acid and a pharmaceutically acceptable salt or prodrug thereof.
20. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of claim 1. ■ •'
21. A method of assaying a biological sample from a mammalian patient suspected of having a disease, condition or disorder mediated, at least in part, by VLA-1, which method comprises obtaining a biological sample from said patient and assaying said sample for the presence of VLA-1.
22. A method of inhibiting adhesion of mammalian cells to an extracellular matrix mediated, at least in part, by VLA-1, which method comprises contacting said cells with a compound of claim 1.
23. A method for treating a disease, condition, or disorder whose progression is regulated, at least in part, by VLA-1. expression or activity in a mammalian patient in need thereof comprising administering to said patient a therapeutically effective amount -of a compound1 of claim 1.
24. The method of claim 23 wherein said disease, condition, or disorder is selected from the
• ■ group consisting of'■ asthma, trachoma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, tissue transplantation, .tumor metastasis, tumor migration, and/or tumor growth, proliferation of fibroblasts in cancer, solid tumors, meningitis, encephalitis, stroke, cerebral traumas, nephritis, retinitis, atopic dermatitis, . psoriasis, myocardial ischemia, acute leukocyte- mediated lung injury, and fibrotic diseases.
25. The method of claim 24 wherein said disease, condition, or disorder is a fibrotic disease .
26. The method of claim 25 wherein said fibrotic disease is selected from the group consisting of systemic sclerosis, mixed connective tissue disease, fibrodysplasia, fibrocystic disease, sarcoidosis, and myositis.-
27. The method of claim 25 wherein said fibrotic disease has a manifestation of fibrotic vascular intimal hypertrophy, and is selected from the group consisting of vasculitis, polyarteritis nodosa, and temporal arteritis. ■
28. The method of claim 25 wherein said fibrotic disease has a manifestation of fibrotic hypertrophy of skin or muscle tissue, and is selected from the group consisting of scleroderma, eosinophilic fasciitis, discoid lesions associated with lupus or discoid lupus, and surgical adhesions.
29. The method,of claim 25 wherein said fibrotic disease has a manifestation of fibrotic hypertrophy of nerve tissue, and is selected from the group consisting of cerebrosclerosis, annular sclerosis, diffuse sclerosis, and lobar sclerosis.
30. The method of claim 25 wherein said fibrotic disease has a manifestation of fibrotic hypertrophy, or fibrosis of lung tissue, and is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, the fibrotic element of pneumoconiosis, pulmonary sarcoidosis, fibrosing alveolitis, the fibrotic or hypertrophic element of cystic fibrosis, chronic
' - obstructive pulmonary disease, adult respiratory distress syndrome,- and emphysema.
31. The method of claim 25 wherein said fibrotic disease has a manifestation of fibrotic -;' "'•'. hypertrophy, -or fibrosis -of prostate, liver, the pleura, or pancreas, and is selected from the group consisting of benign prostatic hypertrophy, nonalcoholic steato hepatitis, and fibrosis of the liver.
32. The method of claim 25 wherein said fibrotic disease has a manifestation of fibrotic hypertrophy, ' or fibrosis Of the kidney, and is selected from the group consisting of chronic renal
' failure, lupus nephritis, alports syndrome, glomerulonephritis, and diabetic nephritis.
33. The method of claim 23 wherein said disease, disorder, or condition is a cancer.
34. The method according to claim 33 wherein said cancer is a tumor or a neoplasm selected from the group consisting of a carcinoma, an adenocarcinoma, and a sarcoma.
35. The method of claim 33 wherein said cancer is selected from the group consisting of growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, human soft tissue carcinoma, cancer metastases, squamous cell carcinoma, esophageal squamous cell carcinoma, oral carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producing tumors, nonsmall cell cancers, breast cancer, gastrointestinal cancers, urological cancers, malignancies of the female genital tract, malignancies of the male genital tract, kidney cancer, brain cancer, bone cancers, skin cancers, thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer, trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma.
36. The method of claim 33 wherein said cancer is a cell proliferative disorder, and is selected from the group consisting of angiogenesis- mediated diseases, benign tumors, acoustic neuromas, neurofibromas, pyogenic granulomas, biliary tract cancer, choriocarcinoma, esophageal cancer, gastric cancer, intraepithelial neoplasms, lung cancer, and neuroblastomas .
37. The method of claim 23 wherein said administration is selected from the group consisting of orally, intravenously, parenterally, transdermal- ly, topically, rectally, and intranasally.
38. The method according to claim 23 wherein said mammal is selected from the group consisting of humans, primates, pet or companion animals, laboratory animals, and- farm animals.
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