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WO2005019200A2 - Aryl piperidine derivatives as vla-1 integrin antagonists and uses thereof - Google Patents

Aryl piperidine derivatives as vla-1 integrin antagonists and uses thereof Download PDF

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Publication number
WO2005019200A2
WO2005019200A2 PCT/US2004/026206 US2004026206W WO2005019200A2 WO 2005019200 A2 WO2005019200 A2 WO 2005019200A2 US 2004026206 W US2004026206 W US 2004026206W WO 2005019200 A2 WO2005019200 A2 WO 2005019200A2
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amino
carbonyl
phenyl
propionic acid
piperidine
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PCT/US2004/026206
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French (fr)
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WO2005019200A3 (en
Inventor
Steven A. Boyd
Jason Demeese
Indrani Gunawardana
Irina C. Jacobson
Yvan Lehuerou
Mark L. Lupher, Jr.
Martin Mclaughlin
Scott Miller
Allen Thomas
Eugene Thorsett
Rui Xu
Matthew Yanik
Gan Zhang
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Icos Corp
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Icos Corp
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07D401/04—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 directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C07D401/14—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 three or more hetero rings
    • 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
    • C07D405/14—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 three or more hetero rings
    • 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
    • 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
    • C07D417/04—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 directly linked by a ring-member-to-ring-member bond
    • 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
    • C07D417/14—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 three or more hetero rings

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, ⁇ and ⁇ . Integrins are the major receptor for cell adhesion to the extracellular matrix and play important roles in certain cell-cell and cell-matrix adhesion events. These integrin- mediated adhesion events are critical for both normal and pathophysiological processes during cell activation, migration, proliferation, and differen- tiation (for reviews see Hynes (1992) Cell 69: 11 ; Springer (1994) Cell 76: 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 . 51:48206).
  • ⁇ 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 expressed on T cells in various disease states including in the joints of arthritis patients (He Ler et al. (1986) J. Clin . Invest . 78:696), lesions of giant cell arteritis patients (Schaufelberger et al. (1993) Clin .
  • 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
  • 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. Abs against ⁇ l have also been shown to block collagen-induced cyto- kine release, including release of TNF- ⁇ , 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.
  • MMP matrix metalloproteinase
  • VLA-1 is an upstream regulator of multiple disease promoting factors
  • VLA-1 is expressed on myofibroblasts in vi tro and in vivo and is believed to regulate their pathologic functions.
  • Alport syndrome is a genetic disorder characterized by progressive glomeruloneph- ritis resulting in fibrosis of the kidneys and ultimately kidney failure. Alport 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. Pathol . 157:1649).
  • ⁇ 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 fi- brotic kidney disease (Kagami et al. (2002) Lab .
  • 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 fibroblasts (Pozzi et al. (1998) J. Cell . Biol . 142:587; Senger et al. (2002) Am . J. Pathol . 160: 195) , and 2) by regulating the production of matrix metallopro- teinase 9 which in turn regulates the activity of angiostatin, a potent angiogenesis inhibitor (Pozzi et al. (2000) PNAS 97: 2202 ; Pozzi et al .
  • 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 path- ologies mediated by VLA-1 binding and cellular adhesion, migration, activation or differentiation.
  • the present invention provides aryl piper- idine compounds that are antagonists to the VLA-1 integrin.
  • this invention is directed to a compound of Formula I:
  • Ar is selected from the group con- sisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
  • A together with the nitrogen atom bound thereto, forms 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, sub- stituted 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 and alkoxy substituents, if present, may be attached to a carbon atom in said heterocyclic group, wherein the one or more thioalkyl and the one or more halo substituents, if present,
  • W is selected from the group consisting of -C(X)-NH and -NH-C (X) -, ⁇ wherein X is oxygen or sul- fur,
  • Y is selected, from the group consisting of a bond, alkylene, and substituted alkylene;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminocarbonyloxy, oxycarbonyl- a ino / aminoacyl, acylamino, aminocarbonyla ino, aminosulfonylamino, aminosulfonyl, sulfonylamino, heterocyclic, substituted heterocyclic, aryl, sub- stituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, carboxyl, and carboxyl esters; or R 3 -C-R 4 forms a cycloalkyl, substituted cycloalkyl, heterocyclic, or substituted heterocyclic group; R 5 is selected from the group consisting of
  • R a is selected from the group consisting of hydrogen, alkyl, aryl, substituted aryl, heterocyclic (e.g., tetrazolyl) , and substi- tuted heterocyclic; with the proviso that when Y is a bond and W is C(0)NH, then R 3 and/or R 4 are not amino, substituted amino, hydroxy, or alkoxy; and pharmaceutically acceptable salts, prodrugs, and any and all tautomers thereof.
  • a and the nitrogen atom pendent thereto form a piperidine ring.
  • W is -C(0)-NH- or -NH-C(O)-.
  • Y is a bond or methylene
  • R 4 is hydrogen
  • R 5 is -C0 2 H.
  • R 3 is alkyl or substituted alkyl.
  • Particularly preferred Y groups include a bond, methylene, and substituted methylene.
  • R 3 groups include methyl, N-benzylimidazol-4-ylmethylene, and phenyl - acetamidomethylene.
  • Ar is selected from the group consisting of:
  • this invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of Formula I as de- fined herein.
  • ⁇ this invention 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 mam- malian 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 therapeu- tically effective amount of a compound or co posi- 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 di- . • sease, 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, sarcoido ' sis, 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 selec- ted from the group consisting of chronic renal fail- ure, lupus nephritis, alport syndrome, glomerulo- nephritis, and diabetic nephritis.
  • the invention is directed to a method for ameliorating cancers 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 composition of this invention.
  • 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, uro- logical 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, hemangioperi
  • 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 .
  • a compound or composition of this inven- tion may be administered to the mammal by any suitable route, such as orally, intravenously, par- enterally, 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 aryl piperidine derivatives.
  • alkyl refers to mono- valent alkyl groups having from 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms, even ' more preferably 1 to 4 carbon atoms. This term is exemplified by groups such as methyl, t-butyl, ⁇ n-
  • 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,
  • Hydroa ⁇ kyl refers to an alkyl group having from 1 to 3 hydroxy substituents provided that there is no more than one hydroxy group per
  • Haldroxy 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 ex-
  • substituted alkylene refers to an alkylene group as defined above substituted with from 1 to 3 substituents as described for substituted alkyl.
  • Alkoxy refers to the group “alkyl-0-" 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
  • Acyl refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C (0) -, alkenyl-C (0) -, substituted alkenyl-C (0) -, cycloalkyl-C (O) -, substi- tuted cycloalkyl-C (0) -, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C (0) -, substituted hetero- ⁇ aryl-C(O), heterocyclic-C (0) -, and substituted het- erocyclic-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 optionally 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 hetero- aryl-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 site, and preferably from 1-2 sites, of alkenyl unsaturation.
  • 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 aryloxy, cyano, halogen, hydroxy, nitro, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl, thiol, thioalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino, provided that the hydroxy or the
  • Amino refers to the group -NH 2 .
  • substituted amino refers to the group -NRR where each R is independently 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 groups are not hydrogen.
  • 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 (0) heterocyclic, 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(0)- O-cycloalkyl, -NRC.(O) 0-substituted cycloalkyl, - -NRC- (O)O-aryl, -NRC (0) 0-substituted- aryl, -NRC (0) O-het- eroaryl, -NRC (0) O-substituted heteroaryl, -NRC (0)0- heterocyclic, and -NRC (0) 0-substituted- heterocyclic, where R is hydrogen or alkyl.
  • aminosulfonyl refers to the group ⁇ S0 2 Q where Q is amino- or substituted amino, as defined . herein.
  • Aminosulfonylamino refers to the group QS0 2 Q where each Q is independently amino or substituted amino, as defined herein.
  • Oxycarbonylamino refers to the group -0C(0)Q where Q is amino or substituted amino, as defined herein.
  • Aminocarbonylamino refers to the group -QC(0)Q where each Q is independently amino or substituted amino, as defined herein.
  • 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 multiple 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-7yl, 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 alkenyl, amino, substituted amino, aminoacyl, amino- carbonyloxy, aminocarbonyiamino, aryl, substituted aryl, aryloxy, substituted aryloxy, carboxyl, carboxyl esters, cyano, thiol, thioalkyl, substituted thioalkyl, cycloalkyl, substituted cycloalkyl, halo, 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.
  • Substituted aryloxy refers to substituted aryl-O- groups.
  • Carboxyl refers to the group -COOH, and salts thereof.
  • Carboxyl esters refer to the group -COOR where R is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, carboxyl-substituted heteroaryl, heterocyclic, and 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.
  • 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, nitro- gen and sulfur within the ring.
  • Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indoli- zinyl or benzothienyl) provided that the point of attachment is to a heteroaryl group atom and further provided that the heteroaryl group contains five ring atoms.
  • Preferred heteroaryls include pyridyl, pyrrolyl, indolyl, and furyl.
  • Substituted heteroaryl refers to heteroaryl 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 onovalent saturated or unsaturated, but not aromatic, 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 pro- vided that the point of attachment is to a heterocyclic ring atom.
  • Substituted heterocyclic refers to heterocycle groups which are substituted with from 1 to 3 substituents selected from the group of substit- uents 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
  • Thioalkyl refers to the group -S-alkyl.
  • Substituted thioalkyl refers to the group -S-substituted alkyl.
  • Sulfonylamino refers. to the group QS0 2 where Q is amino or substituted amino, as defined herein.
  • Tautomers refers to chemical compounds that exist as a mixture of two interconvertible iso- -10- mers in equilibrium.
  • “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 15 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- 20 mide, tartrate, mesylate, acetate, maleate, oxalate, and the like.
  • Prodrugs are compounds which convert (e.g., hydrolyze, metabolize, etc.) in vivo to a compound of the invention.
  • the effective- 25 ness of an orally administered drug is dependent upon the drug's efficient- transport across the muco- sal epithelium and its stability in entero-hepatic circulation.
  • Drugs that are effective after par- enteral administration but less effective orally, or 30 whose plasma half-life is considered too short, may be chemically modified into a prodrug form.
  • a pro- drug is a drug which has been chemically modified and may be biologically inactive at its site of action, but which may be degraded or modified by one or more enzymatic or other in vivo processes to the parent bioactive form.
  • the prodrug 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 prodrugs according to the invention, including:
  • Ester or amide derivatives which may • ' be cleaved by, for example, esterases or lipases.
  • ester-' is derived from the. carboxylic acid moiety of- the drug molecule by known means.
  • the amide may be derived from the carboxylic acid moiety or the a ine 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.
  • substituted as used with, for example, “substituted alkyl” does not include polymers derived therefrom but are limited to a maximum of 3 substituents groups, e.g., alkyl-Ar-Ar-Ar.
  • 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 pre- ferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) 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 Greene and Wuts, (1991) Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, and references cited therein.
  • the compounds of this invention 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.
  • stereoisomers and enriched mixtures are included within the scope of this invention, unless otherwise indicated.
  • Pure stereoisomers may be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art.
  • racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving 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.
  • This reaction is run at a temperature of about 25°C to about 90 °C for a time sufficient for the reaction to go to completion, typically about 1 to about 12 hours.
  • the product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • the reaction also can be performed in the reverse direction by switching the halide and transition metal of the aryl groups.
  • Scheme 2 for illustrative purposes only, describes how a halopyridine such as compound 105, can be synthesized by treating a pyridine biaryl compound useful in the preparation of compounds of formula I, for example compound 104, with an oxidizing agent (e.g., m-CPBA, hydrogen peroxide, etc.), in a suitable solvent (e.g., DCM, DCE, CC1 4 ) .
  • an oxidizing agent e.g., m-CPBA, hydrogen peroxide, etc.
  • a suitable solvent e.g., DCM, DCE, CC1 4
  • the N-oxide intermediate is then reacted with a phosphorus oxyhalide (e.g., P0C13, POBr 3 ) to furnish the halopyridine.
  • the halopyridine of this reaction may be optionally substituted with substituents suitable for use in the subsequent reactions.
  • Step 1 of the reaction shown in Scheme 2 is run at a temperature of about -10 °C to about 30°C for a time sufficient for the reaction to fo to completion, typically about 1 to about 18 hours.
  • Step 2 of the reaction shown in Scheme 2 is run at a temperature of about 25 °C to about 90 °C for a time sufficient for the reaction to go to completion, typically about 1 to about 18 hours.
  • the product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • R 3 ' is either a carboxylic acid with a suitable protecting group (OCOPg, where Pg may be alkyl, or more specifically, t-butyl) or an amine with a suitable protecting group (NHPg*, where Pg* may be boc, cbz, or frnoc) , n is 0 to 4, and the halopyridine may be optionally substituted with substituents suitable for use in the subsequent reaction.
  • OCOPg a carboxylic acid with a suitable protecting group
  • Pg may be alkyl, or more specifically, t-butyl
  • NHSg* a suitable protecting group
  • n is 0 to 4
  • the halopyridine may be optionally substituted with substituents suitable for use in the subsequent reaction.
  • Scheme 3 illustrates a method for converting a halopyridine, such as compound 105, [e.g., 2- (4-methoxyphenyl)pyri- dine] , to a 2-aminopyridine, compound 106, through a melt reaction using various amines (e.g., ethyl or methyl isonipecotate or 4- ( t-butyloxycarbonyl- amino) piperidine or methyl azetidine-3-carboxylate) .
  • This reaction is typically run neat, preferably using an excess of amine, at a suitable temperature, from about 25°C to about 90°C in an inert atmosphere in a closed tube.
  • the reaction is run for a time sufficient for the reaction to go to completion, typically about 1 to 50 hours.
  • the product can be recovered using standard techniques such as filtra- tion, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • the reaction is run for a time suffi- cient for the reaction to go to completion, typically about' 1 to about 20 hours.
  • the product can be recovered using standard techniques such as filtra- tion, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification. 5
  • the nitro group of compound 108 may be reduced to form the aniline, (compound not shown) , using a, suitable reducing conditions (e.g., zinc in acetic acid or FeS0 4 (H 2 0) 7 in THF) followed by formation of a diazo salt (NaN0 2 , sulfuric acid, or
  • the resulting diazonium salt - may be reduced . (H 3 P ⁇ 4 or HMPA in ethanol or sodium stannite, see Ruchardt et al. (1977) Chem . Ber. 210:2494) to the hydrido product compound 109. This reaction is run at a temperature of about 0° to about 90 °C for a
  • the product can be recovered using standard .techniques such, as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may
  • Biaryl halides such as compound 111, (2, 3-methyl-3 ' -iodo-biphenyl, etc.) can also be synthesized from biaryl nitro compounds, such as
  • compound 107a (e.g., 2, 3-dichloro-3 ' -nitro-biphen- yl, and the like) . Reduction of the nitro group of compound 107a leads to an aniline derivative, compound 110. This intermediate can then be converted to an aryl halide, such as compound 111, by first
  • halopyridines such as compound 105
  • aryl halides such as compound 111, (e.g., 3 bromobiphenyl, 2, 3-dichloro-3 ' - iodo-biphenyl, 2-chloro-4- (m-tolyl) -pyridine)
  • aryl halides such as compound 111, can be converted to various substituted aminoaryls, such compounds 106, 109> 109a, (and, as illustrated in Scheme 5 below, compound 112) by a palladium mediated coupling (WO 00/39081; Old et al.
  • Ar 2 , R 3' , n, X, and Y' are defined herein above.
  • the coupling can be performed as described in Schemes 3 and 4 to give compound 112.
  • Compound 112 may be halogenated by using techniques described herein and/or by methods well known in the art, to give compound 112a, where Y' is a boronic acid, boronic ester, or an organo stannane.
  • compound 112a can be coupled to Ar 2 -Y' when Y' is a boronic acid, boronic ester, or an organo stannane via copper mediated Chan-Lam coupling (see Lam et al. (2002) Tetrahedron Lett . 43:3091; Chan et al .
  • a second palladium coupling (as indicated in Scheme 1) can provide compound 113 from compound 112a.
  • compound 112 may be transmetallated to give compound 114, (Miyaura et al . (1995) J. Org. Chem . 60: 1508- 7510) , which can then be coupled as in Scheme 1 to other aromatic halides to give compound 113.
  • the above reactions are run under conditions described herein or using techniques well known in the art.
  • Substituted benzothiophenes were synthesized according to Scheme 6.
  • One method entails the alkylation of bromoacetic acid compound 116 with substituted thiophenols compound 115 (3-methyl thio- henol, 2,3-dichloro thiophenol, etc.) in aqueous solvent and in the presence of a base (NaOH, Cs 2 C0 3 , and the like) to afford compound 117.
  • Bromination e.g., using iron filings and
  • ketone compound 119 by treatment with a Lewis acid such as A1C1 3 or polyphos- phoric acid.
  • a Lewis acid such as A1C1 3 or polyphos- phoric acid.
  • the reaction is generally carried out at -78°C to 25°C.
  • Reduction of the ketone is afforded by a reducing agent (NaBH 4 , LiAlH , or DIBAL-H, or the like) , in a solvent such as ethanol, at a tempera- ture of about -10 °C to about 10 °C for a period of time sufficient for completion of the reaction.
  • a reducing agent NaBH 4 , LiAlH , or DIBAL-H, or the like
  • a solvent such as ethanol
  • the intermediate alcohol is eliminated to benzothiophene compound 120 using techniques well known in the art.
  • the product can be recovered using standard tech- niques such as filtration, chromatography, distilla- tion, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • the bromide can be converted to the corresponding iodide compound 121 (Buchwald et al. (2002) J. Am . Chem . Soc. 224:14844-14845) .
  • the product can be recovered using standard techniques such as filtra- tion, chromatography, distillation, extraction, and • the like. Alternatively, the product may be used in the next step without further ' isolation and/or purification.
  • a process for preparing substituted heteroaryl derivatives is illustrated in Scheme 7, following a Hantzsch type synthesis (Beugelmans et al. (1996) J. Org. Chem . 61 : 118 ) , wherein Ar 1 , R 3 ', and n are defined herein above, and Q* is sulfur or oxygen.
  • the starting aryl ketone compound 122 is chlorinated by reaction with oxalyl chloride in toluene yielding compound 123. This reaction is run at a temperature of about 0°C to about 90 °C for a time sufficient for the reaction to go to completion, typically about 1 to 24 hours.
  • the product can be recovered using standard techniques such as filtration, chromatography, distillation, extrac- tion, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • Ureas such as compound 124
  • an aprotic solvent e.g., DCM, toluene
  • the product can be recovered using standard techniques such as fil- tration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • the product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • Scheme 8 illustrates the synthesis of sub- stituted arylethers, aryl. thioethers and amino derivatives, such as compound 128, wherein X is as defined herein, Ar i.s aryl,. substituted aryl, het- roaryl, or substituted ' heteroaryl, R 2* is alkyl, substituted alkyl, aryl, substituted aryl, ..hetercryl,- and substituted heteroaryl, and Y* is NH, 0 or S.
  • amino acid derivatives such as compound 131 are coupled to the carboxylic acids, com- pound 130, in an inert solvent (DCM, THF, DMF or the like) using any of a wide variety of well known coupling agents.
  • Suitable coupling agents include, but are not limited to, 1- (3-dimethylaminopropyl) -3- ethylcarbodimide hydrochloride (EDC) , dicyclohexyl- carbodiimide (DCC) , N, N' -carbonyldiimidazole (CDl), benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) , and diethylcyanophos- phonate (DECP) .
  • well known coupling promoters for example 1-hydroxybenzotriazole
  • the acid may be coupled utilizing solid support resins such as 1% cross linked polystyrene tetrafluoro phenol (PS-TFP) , polystyrene dicyclohexyl carbodiimide (PS- DCC) or polystyrene 1-hydroxybenzotriazole (PS-HOBT) by the use of one of the listed coupling reagents in the presence of a base (4-dimethylamino pyridine, triethyl amine, etc.) and solvent (DMF, methylene chloride.)
  • PS-TFP cross linked polystyrene tetrafluoro phenol
  • PS- DCC polystyrene dicyclohexyl carbodiimide
  • PS-HOBT polystyrene 1-hydroxybenzotriazole
  • the coupled amide compound 132 is then subjected to basic hydrolysis conditions listed above to afford the final acid compound 133.
  • the product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
  • Scheme 10 depicts the preparation of- re- versed amide analogs, wherein Ar, R 3 , R 4 , n, Pg and Pg* are as defined herein- above.
  • the protecting group Pg* on the protected amine, compound 134, (also compound 112 or compound 113 from Scheme 5 wherein R 3 ' is an amine protected with a- suitable protecting group such as Boc, Cbz, Fmoc, trifluoro- ⁇ acetamide, or the like) , is removed using conditions suitable for the protecting group, to afford the amine compound 135.
  • Amine compound 135 is then coupled using conditions listed above to an acid, compound 136, to provide for the amide compound 137.
  • the ester of the resulting amide compound 137 is then hydrolyzed using conditions listed above to yield the acid, compound 138.
  • the carboxyl group of compound 100 is reduced using conventional techniques such as the use of a conventional reducing agent including, for example, lithium aluminum hydride to provide for the corre- sponding alcohol, compound 200.
  • a conventional reducing agent including, 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, chro- matography, filtration, and the like or used in the next step of the reaction without purification and/or isolation.
  • halo group e.g., chloro
  • Suitable halogenating agents include, for example, inorganic acid halides, such as thionyl chloride, phosphorous tri- chloride, phosphorous tribromide or phosphorous pentachloride, under conventional conditions.
  • this reaction is conducted using about 1 to 5 molar equivalents of the inorganic acid halide or oxalyl chloride, 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 re- covered by conventional methods including neutrali- zation, extraction, precipitation, chromatography, filtration, and the like or used in the next step of the reaction without purification and/or isolation.
  • This compound 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.
  • a heteroaryl or heterocyclic halide i.e., Het-X (compound 500)
  • Het-X compound 500
  • - Transition-metal catalysts may be used to facilitate this transformation, for example using copper (I) (e.g., copper (I) iodide, copper (I) - bromide dimethylsulfide complex) or palladium complexes .
  • 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.
  • compositions which contain, as the active ingredient, one or more of the compounds of the subject invention above associated with pharmaceutically acceptable carriers.
  • 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 other con- tainer.
  • the excipient employed is typically 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, or: medium for the active - ingredient .
  • compositions 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 containing, ' 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 particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
  • suitable excipients include lactose, dextrose, sucrose, sorbitol, manni- tol, starches, gum acacia, calcium phosphate, algi- nates, tragacanth, gelatin, calcium silicate, micro- crystalline cellulose, polyvinylpyrrolidone, cellu- lose, sterile water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubri- eating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as ethyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • lubri- eating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying and suspending agents such as ethyl- 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.
  • 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 refers 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.
  • 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.
  • preformulation compositions 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 preformu- lation 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 pro-- vide a dosage form affording the advantage of prolonged action.
  • the-.tablet or pill can comprise an inner dosage and an outer dosage com- ponent, 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 mate- • . rials 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 incorporated 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, suspension, 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.
  • Hard gelatin capsules containing the fol- lowing 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 ingredients below:
  • the components are blended and compressed to form tablets, each weighing 240 mg.
  • a dry powder inhaler formulation is pre- pared containing the following components:
  • Tablets each containing 30 mg of active ingredient, are prepared as follows
  • the active ingredient, starch and cellulose are passed through a No. 20 mesh U'. S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrroli- done 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, pre- viously 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.
  • 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 capsules in 150 mg quantities.
  • Suppositories each containing 25 mg of active ingredient are made as follows:
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the satur- ated 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.
  • Suspensions each containing 50 mg of medicament per 5.0 mL dose are made as follows:
  • the active ingredient, sucrose and xanthan 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 benzoate, 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:
  • Another preferred formulation employed in 5 the methods of the " present invention employs txans- ⁇ dermal delivery devices ("patches") .
  • Such trans- dermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
  • transdermal patches for the delivery, of pharmaceutical agents is well known in the art. See ' , e.g., U.S. Patent 5,023,252, herein incorporated by reference.. Such patches may be constructed for continuous, pulsatile, . or on demand ' delivery, of
  • Indirect techniques usually involve formulating the compositions 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 trans- portation 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.
  • Other suitable formulations for use in the present invention can be found in Remington ' s Pharmaceutical Sciences, (1985), Seventeenth Edition, Mack Publishing Company, Philadelphia, PA.
  • the compounds described 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, 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. 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- 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 mediate ⁇ , 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.
  • diseases, condi- tions, or disorders include, but are not limited to, inflammatory diseases, fibrotic diseases, and cancer.
  • diseases, conditions, and disorders which are expected to be trea able by the 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 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- alitis, stroke, and other cerebral traumas, nephritis, retinitis, atopic dermatitis, psoriasis, myo- cardial ischemia, acute leukocyte-mediated lung injury such as that which occurs - in adult respiratory distress syndrome, and fibrotic diseases, such as fibrotic diseases of the lung, kidney, liver, and 5 vasculature (including idiopathic
  • 10.- 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,
  • Dermatomyositis can be associated with fibrosing or
  • hypertrophic aspects including fibrosing alveolitis and pulmonary fibrosis.
  • Treatment using the compounds or compositions of .the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy
  • Amelioration includes reducing the rate of progression of a disease.
  • fibrotic diseases that have as a manifestation fibrotic vascular intimal hypertrophy. These diseases include vascu-
  • fibrotic diseases include diseases that have as a ' manifestation fibrotic hypertrophy of skin -and/or muscle tissue. These diseases include scleroderma, eosinophilic fasci- itis, discoid lesions associated with lupus or,, discoid lupus, or surgical- adhesions. Treatment • • .
  • using the compounds or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such indications, or hypertrophy or-, 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 lobar sclerosis.
  • Treatment using the compounds 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.
  • fibrotic diseases further include fibrotic lung diseases that have as a manifestation fibrotic hypertrophy or fibrosis of lung tissue.
  • diseases include pulmonary fibrosis (or interstitial lung disease or interstitial pulmonary 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, fibrosing alveolitis, the fibrotic or hypertrophic element of cystic fibrosis, chronic obstructive pulmonary disease, adult respiratory distress syndrome, and emphysema.
  • Treatment using the compounds 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, alport syndrome, glomerulonephritis, and diabetic nephritis.
  • Treatment using the compound 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 or compositions of the present invention typically occur in mammals. 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.
  • 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 from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they can invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation . (greater "de- differentiation”) 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 or compositions of the present invention include, but are not limited to, solid tumors, i.e., carcinomas, adenocarcinomas, and sarcomas.
  • Carcinomas include those malignant neoplasms derived from epithelial cells which infiltrate (invade) the surrounding tissues and give rise 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 or homogenous substance like embryonic connective 5 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,
  • non-Hodgkin' s lymphoma 15- lymphoma, non-Hodgkin' s lymphoma,- cancer of the adrenal cortex, ACTH-producing tumors, nonsmail cell cancers, breast cancer '(including small cell carcinoma and ductal carcinoma) , gastrointestinal cancers (including stomach cancer, colon cancer, colo-
  • rectal 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,
  • 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 oste- omas and osteosarcomas) , skin cancers (including malignant melanoma, tumor progression of human skin keratinocytes and squamous cell cancer) , thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer, trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma.
  • Cancers and other cell proliferative disorders treatable by the compounds or 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 cancer, intraepithelial neoplasms, lung cancer, and neuroblastomas .
  • the biological activity of the compounds identified above may be assayed in a variety of systems.
  • extracellular matrix such as collagen IV
  • extracellular matrix such as collagen IV
  • adhesion of cells expressing VLA-1 can be measured in the presence or absence of compound.
  • large numbers of compounds can be screened.
  • Cells suitable for this assay include 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, including, for example, CHO, K562, and the like.
  • the compounds and 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 antibodies to VLA-1.
  • the extracellular matrix can be soluble or immobilized on a solid surface.
  • VLA-1 may also be expressed as a recombinant fusion protein having acidic and basic leucine-zipper tails so that binding to extracellular matrix -may be de- . tected 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 autoradi- ography 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.
  • 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, bioavailabil- ity) ,- 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 sig- ⁇ nal .
  • the compounds and compositions of the subject invention may be modified in a variety of ways for a variety of end purposes while still retaining 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 vi tro 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
  • the compounds and compositions of. this invention can 15 also be used for isolating. or labeling such cells.
  • the compounds and compositions of the .invention can be used to assay for potential inhibitors of VLA-1/Extraeellular matrix interactions. 20 - For in vivo diagnostic - imaging to identi-
  • radioisotopes • are typically used in accordance with well-known techniques.
  • the radioisotopes may be bound to the compound either directly or indirectly using inter- 25 mediate functional groups.
  • chelating ⁇ agents such as diethylenetriaminepentacetic acid (DTPA) , ethylenediaminetetraacetic acid (EDTA) , and similar molecules have been used to bind compounds to metallic ion radioisotopes.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the complexes can also- be labeled with a paramagnetic isotope for purposes of in vivo diag- nosis, as in magnetic resonance imaging (MRI) pr 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 inflammatory response in an ' individual. - By measuring the
  • the amount administered to the patient will vary depending- upon what is.-.being administered, the purpose of the administration, such as prophyl- - axis or therapy, the state of the patient, the manner of administration, and the like. In thera-
  • 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
  • Amounts effective for this use will depend on the disease condition being treated, as well as by the judgment of the attending clinician 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 compositions 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 lyophil- ized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • 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.
  • 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 con- dition 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 typ- ically 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-response curves ⁇ derived from in vitro or animal model test systems.
  • the compounds and/or composi- tions of the subject invention will be administered • in a therapeutically effective amount by any Of the v..
  • Toxici-ty 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 5 0 (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 LD 5 o/ED 50 .
  • 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 5 0 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 IC50 (the concentration of the test compound which achieves a half-maximal inhibition of activity) as determined in cell culture.
  • IC50 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: arian ProStar Solvent Delivery 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, 4.6 x 50 mm, S3 ⁇ , Waters Corporation. ' Detection: 220nm and 254nm.
  • Solvent A H 2 0, 0.01% HFBA, .1.0% IPA. .
  • Solvent B Acetonitrile, 0.01%HFBA, 1.0% IPA.
  • 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, 4.6 x 50 mm, S3 ⁇ , Waters Corporation. Detection: 220nm and 254nm.
  • 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 E Agilent Technologies 1100.HPLC ,' ⁇ ' System (Pump: QuatPump Model G1311A; Detector:. DAD, Model G1315B; Column Compartment: Model
  • TSP HPLC System (Pump: TSP Spec- ' traSYSTEM® P4000; Detector: TSP SpectraSYSTEM® . .. - UV2000, 220nm and 254 ; Autosampler: TSP Spectra- SYSTEM® AS3000 Degasser: ⁇ TSP SpectraSYSTEM® Model SCMIOOO Solvent Degasser) .
  • Detection -220nm and 254nm.
  • Analytical column YMC ODS-AQ, 4.6 x 50 mm, S3 ⁇ , Waters Corporation.
  • Solvent C H 2 0, 0.01% HFBA, 1.0% IPA.
  • Solvent D Acetonitrile, 0.01% HFBA, 1.0% IPA.
  • Flow Rate 2.0 mL/min. Gradient
  • TSP HPLC System (Pump: TSP SpectraSYSTEM® P4000; Detector: TSP SpectraSYSTEM® UV2000, 220nm and 254 m; Autosampler: TSP Spectra- SYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser) . Detection: 220nm and 254nm. Analytical column: YMC ODS-AQ 4.6 x 50 mm S3 ⁇ , Waters Corporation. Solvent C: 10 mM Ammonium Acetate in H ? 0. Solvent D: 10 mM Ammonium Acetate in Acetonitrile, 1.0% IPA. Flow Rate: 2.0 mL/min.
  • TSP HPLC System (Pump: TSP SpectraSYSTEM® P4000; Detector: TSP SpectraSYSTEM® UV2000, 220nm and 254 m; Autosampler: TSP SpectraSYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser) . Detection: 220nm and 254nm. Analytical column: Zorbax Extend C18 Rapid Resolution®, 50 x 4.6 mm, 3.5 ⁇ , 80A, Agilent Tech- nologies. Solvent C: 10 mM Ammonium Acetate in H 2 0.
  • Solvent D 10 mM Ammonium Acetate in Acetonitrile, 1.0% IPA. Flow Rate: 2.0 mL/min. Gradient Program: 0.00 min 95% Solvent C, 5% Solvent D; 0.02 min 95% Solvent C, 5% Solvent D; 4.00 min 5% Solvent C, 95% Solvent D; 4.30 min 5% Solvent C, 95% Solvent D; 4.50 min 95% Solvent C, 5% Solvent D;. 5.50 min 95% Solvent C, 5% Solvent D.
  • TSP HPLC System (Pump: . TSP Spec- 5 traSYSTEM® P2000; Detector: TSP SpectraSYSTEM® . UV2000, 220nm and 254 m; Autosampler: TSP SpectraSYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser) . Detection: - 220nm and 254nm. Analytical column: Zorbax Extend C18 Rapid-0 Resolution®, .50 x 4.6 mm, 3.5 ⁇ , 80A, Agilent Technologies.
  • Solvent B 0.02 min 95% Solvent A, 5% Solvent B; 4.10 min 5% Solvent A, 95% Solvent B; 4.30 min 5% Solvent A, 95% Solvent B; 4.50 min 95% Solvent A, 5% Solvent B; 5.50 min 95% Solvent A, 5% Solvent B. Purity based on 220 nm wavelength channel.
  • Example 1A (1.76 g, 80.1%) as a white solid.
  • Example IB (1.60 g, 99%) as a white solid.
  • IC 1.00 g, 3.87 mmol
  • ethyl isonipecotate 6.08 g, 38.68 mmol
  • the tube was placed under nitrogen, sealed and heated with stirring to 145°C for 24 h.
  • the mixture was cooled to ambient temperature.
  • the mixture was purified by flash chromatography on silica gel eluting with 10% 7N methanolic ammonia/- DCM to provide the title compound (0.890g, 3.87 mmol, 61%).
  • Example 2 was prepared, according to the same reaction protocol as. described for, Example 1 substituting. L-I ⁇ -benzyl histidine methyl ester di- hydrochloride for L-alanine, methyl ester HCl. The procedure provided the title . compound (0.036 mmol,. 8%) as a white solid.
  • Example 3 was prepared according to the same reaction protocol as described for Example 1 substituting 2-tributylstannanyl-pyridine for 4-tri- butylstannanyl-pyridine. The procedure resulted in the. title compound (0.0 ' 80 g, 0.190 mmol, 41%) as a ' white solid-.
  • Example 4 was prepared according to the same reaction protocol as described for Example 1 substituting 4A for 1A. The procedure resulted in the title compound (0.020 g, 0.046 mmol, 26%) as a white solid.
  • Example 5B (as a 3;2 mixture of regioiso- mers) was prepared according to the same reaction protocol as described for Example 1 substituting 5A for IB. The procedure resulted in the title compound
  • Example 6 was prepared according . to the same reaction protocol as described for Example 1 substituting 3-tributylstannanyl-pyridine for 4-tri- butylstannanylpyridine . The procedure 'resulted in the title compound (0.103-g, 0.246 mmol, 61%) as a white powder.
  • Example 7C was prepared according to the , . same reaction protocol as described for Example 1 substituting 7B for ID. The procedure resulted in
  • Example 8 was prepared according to the same reaction protocol as described for Example 7 substituting L- ⁇ -benzyl histidine methyl ester di- hydrochloride for L-alanine methyl ester HCl. The procedure resulted in the title compound (0.058 g, 0.286 mmol, 32.1%) as a white solid.
  • Example " 9 was prepared' according to the same reaction protocol as • described for 'Example 7 substituting L-2-amino-3-benzoylamino-propionic acid methyl ester HCl for L-aianine methyl ester HCl. - The procedure resulted in the title compound (0.062 ' g, 0.285 mmol, 41%) as a white solid.
  • Example 10 was prepared according to the same reaction protocol as described for Example 1 substituting 7A for IE. The procedure resulted in the title compound (0.019 g, 0.351 mmol, 15.2%) as a white solid.
  • Example 11 was prepared according to the same reaction protocol as described for Example 10 substituting L-2-amino-3-benzoylamino-propionic acid methyl ester HCl for L-alanine methyl ester HCl. The procedure resulted in the title compound as a white solid (0.110 g, 0.350 mmol, 66.0%).
  • Example 12 was prepared according to the same reaction protocol as described for Example 10 substituting L-I ⁇ -benzyl histidine methyl ester di- hydrochloride for L-alanine methyl ester HCl. The procedure resulted in the title compound (0.101 g, 0.351- mmol, 57.0%) as a white solid.
  • Example 14 was prepared according to'the • " same reaction protocol as described for Example 13 substituting L-Z ⁇ -benzyl histidine methyl ' ester ' di- hydrochloride for L-alanine methyl ester HCl. The procedure resulted in the title compound (0.030 g, 0.48 mmol, 14.0%) as a white solid.
  • LCMS (ESI-Method G) m/z 432.0 (M-2H) " , Rt 2.02 min (220 : nm, 100.0 area! .
  • Example 15B was prepared according to the same reaction protocol as described for Example 7B substituting 15A for 7A. The procedure resulted in the title compound (0.160 g, 42.0! as an off-white solid.
  • Example 15C was prepared according to the same reaction protocol as described for Example 1 substituting 15E for IE and 15G for L-alanine methyl ester to provide the title compound (0.145 g, 95! as an off-white solid.
  • Bromoacetic acid tert-butyl ester (67.7 g, 347 mmol) was added dropwise over 20 min and the mixture was stirred for 30 min at -78 ⁇ C .
  • the mixture was warmed to 0°C by placing the flask in a water-ice bath for 20 min.
  • the mixture was quenched by pouring into 1 L separatory funnel containing 200 mL of sat. aq NH 4 C1.
  • the mixture was diluted with EtOAc (300 mL) , the phases were separated and the organic phase was washed sequentially with 1 N HCl (2X100 mL) , sat.
  • Example 15F (29.0 g, 83.5 mmol) and THF/water (300 mL, 4:,1 mixture). The mixture was cooled to 0°C in an ice bath and 30! v H 2 0 2 (17.0 g, 501 mmol, in water) was added over 10
  • Example 16 was prepared as in Example IG substituting 15C for IE. The procedure provided the title compounds as an off-white solid.
  • 1 H NMR (DMSO- d 6 r 400 MHz) ⁇ 12.09 (s, IH) , 8.36 (d, J 2 .
  • Example 17D (0.350 g, 0.925 mmol) was processed as in Example IE to provide the title compound (0.292 g, 90.1!) as a cream color powder.
  • Example 17E (0.150 g, 0.428 mmol) was processed as in Example IF to provide the title compound (0.1600 g, 85.8! as a cream color powder.
  • Example 17F (0.230 g, 0.368 mmol) was processed as in Example IG to provide the title compound (0.104 g, 67.2%) as a cream color powder.
  • Example 18 was prepared as in Example 17
  • Example 19D (O.lOOg, 0.280 mmol) was pro-5 Stepd as in Example IF to provide the title compound (0.100 g, 81.0%) as a light yellow colored powder.
  • Example 19E . (0-.230 g, 0..368 mmol) for Example ID. 'The title compound (0.104 g, 67.2%) was obtained as a pale yellow powder.
  • Example . 19D (0.120 g, 0.336 mmol) was processed as in Example 19 ⁇ • substituting ⁇ L-i ⁇ J 1 -benzyl histidine methyl ester dihydrochloride for L-alanine methyl ester, to provide the title compound (0.100 g,- 50.9%) as a light yellow colored powder..- X H NMR
  • Example 21A A solution of Example 21A (298 mg, 1.205 mmol) in THF:MeOH (1:1, 6 mL) was treated with a solution of LiOH'H 2 0 (76 mg, 1.807 mmol) in water (2 mL) at ambient temperature for 18 h. The solvents were removed under reduced pressure. The residue dissolved in water (5 mL) and washed with EtOAc (2X5 mL) to remove organic impurities. Then the aqueous layer was made acidic with 10% aq HCl. The product formed was soluble in aqueous acidic medium and water was removed by evaporating with CH 3 CN to provide the lithium salt of the title compound (159 mg, 50%).
  • LCMS APCI+; Method D) m/z 220, (M+H) + .
  • Example 21B A solution of Example 21B (159 mg, 0.607 mmol), HOBt.H 2 0 (139 mg, 0.9098 mmol) , L-N x -benzyl histidine methyl ester dihydrochloride (242 mg, 0.728 mmol), and triethylamine (0.296 mL, 2.123 mmol), in dry DMF (3'mL) was treated with EDC (174 mg, 0.9098 mmol) at ambient temperature under N 2 atmosphere for 18 h. The reaction mixture was diluted' with EtOAc (10 mL) , washed with water (2x5 mL) , dried (MgS0 4 ) , filtered, and evaporated to dry- ' ness.
  • Example 22A (190 mg, 0.727 mmol) was hydrolyzed with LiOH * H 2 0 (37 mg, 0.872 mmol) as described in Example 2IB to provide the title compound (210 mg, 87! as an off-white color powder.
  • LCMS (APCI+; Method D) m/z 234, (M+H) + .
  • Example 22B 150 mg, 0.643 mmol. as described in Example 21C.
  • LCMS m/z 476, (M+H) + .
  • Example 23A (340 mg, 1.235 mmol) was hydrolyzed with LiOH.H 2 0 (78 mg, 1.852 mmol) as described in Example 21B to provide the title compound (300 mg, 84! as a white powder.
  • LCMS APCI+; Method D) m/z 248, (M+H) + .
  • Example 23D (88 mg, 39%) was obtained as a white powder.
  • Example 24A (270 mg, 0.896 mmol) was hydrolyzed with LiOH.H 2 0 (57 mg, 1.344 mmol) as described in Example 21B to provide the title compound (165 mg, 58! as a white powder.
  • X R NMR 400 MHz, DMSO-de) ⁇ 7.48-7.39 ( , 3H) , 7.18 (s, IH) , 3.73-3.65 (m, 2H), 3.06-2.94 (m, 2H) , 2.46 (m, IH) , 1.97-1.93 (m, 2H) , 1.77-1.71 (m, 2H) .
  • LCMS (APCI ; Method D) m/z 274, (M+H) + .
  • Example 24D 3-(l-Benzyl ⁇ lH-imidazol-4-yl)-2- ⁇ [1-O- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - ' ⁇ ⁇ amino ⁇ -propionic acid
  • Example 24C 150 mg, 0.292 mmol
  • Example 21D The title compound (73 mg, 50%) was obtained as a white powder.
  • Example 2.5A (259 mg, 0.937 mmol) was- hydrolyzed with LiOH'H 2 0 (59 g, 1.406 mmol) as described in Example 21B to provide the title compound (151 mg, 55! as a white powder.
  • LCMS (APCI+; Method D) m/z 249, (M+H) + .
  • Example 25C 120 mg, 0.245 mmol
  • the title compound 50 mg, 43! was obtained as a white powder.
  • Example 26A (331 mg, 1.017 mmol) was - treated with LiOH*H 2 0 (64 mg, 1.526 mmol) as described in Example 2IB to provide the title compound (180 mg, 59! as a white powder.
  • LCMS (APCI+; Method D) m/z 316, (M+H) + .
  • Example 26C The title compound was prepared from Example 26C (130 mg, 0.234 mmol) as. described in Example 21D.
  • Example 26D (70 mg, 55!) was obtained as a white powder.
  • X H NMR 400 MHz, DMSO-d 6 ) ⁇ 8.03 (d, J-8.19 Hz, IH), 7.68 (s, IH) , 7.34-7.31 (m, 2H) ,
  • Example 27A (280 mg, 0.877 mmol) was treated with LiOH'H 2 0 (55 mg, 1.315 mmol) as described in Example 21B to, provide the title compound (177.mg, 61! as a -white powder.
  • LCMS (APCI+; Method D) m/z 292, (M+H) + , (92 area %) . ⁇
  • Example 27C The title , compound was prepared from Example 27C (218 mg, 409 mmol) as described in Example 21D.
  • Example 27D (173 mg, 82%) was obtained as a white powder.
  • Example 28A (380 mg, 1.029 mmol) was hydrolyzed with LiOH"H 2 0 (86 mg, 2.058 mmol) as described in Example 21B to provide the title compound (347 mg, 98.82! as a white powder.
  • X H NMR 400 MHz, DMSO-de) ⁇ 12.31 (s, IH) , 7.46 (s, 2H) , 7.27 (s, IH) , 3.89-3.85 (m, 2H) , 2.98-2.91 (m, 2H) , 2.47-2.44 (m, IH) , 1.94-1.89 (m, 2H) , 1.66-1.56 (m, 2H) .
  • LCMS (APCI+; Method D) m/z 342, (M+H) + .
  • Example 28C 160 mg, 0.275 mmol
  • Example 28D 117 mg, 75%) was obtained as a white powder.
  • Example 29A was used for- the preparation of Example 29B directly.
  • Example' 29A (280 mg, 0.938 mmol) was treated with LiOH'H 2 0 (59 mg, 1.408 mmol) as described in Example 21B to provide the title compound ' (164 mg, 56%) as a yellow color solid.
  • Example 29C 50 mg, 0.098 mmol
  • Example 21D The crude product obtained was purified by C- 18 Reverse phase HPLC using a gradient of 5-95! CH 3 CN/H 2 0 containing 1% TFA to provide the title compound (62 mg, 92! as a bright yellow powder.
  • Example 30A (318 mg, 1.052 mmol) was treated with LiOH.H 2 0 (88 mg, 2.105 mmol) as described in Example 21B to provide the title compound (276 mg, 96%) as a white powder.
  • Example 30C The title compound was prepared from Example 30C (130 mg, 0.2522 mmol) as described in, Example 21D.
  • Example.30D (93 mg, 73.54!). was obtained as a white powder.
  • Example 31A 120 mg, 0.397 mmol was hy- drolyzed with LiOH.H 2 0 (20 mg, 0.477 mmol) as described in Example 2IB to provide the title compound (100 mg, 96! as a white powder.
  • ⁇ H NMR 400 MHz, DMSO-de) ⁇ 12.30 (br s, IH) , 7.30-7.28 (m, 2H) , 7.14- 7.12 ( , IH), 3.24-3.19 (m, 2H) , 2.74-2.68 (m, 2H) , 2.44-2.56 (m, IH) , 1.95-1.91 (m, 2H) , 1.77-1.68 (m, 2H) .
  • LCMS (APCI+; Method D) m/z 274, 276, (M+H) + .
  • Example 31C The title compound was prepared from Example 31C (130 mg, 0.2522 mmol) as ' described in Example 21D.
  • Example 31D (78 mg, 62! was obtained as a white powder.
  • Example' " 32A (45 mg, 126 mmol) ' was hydrolyzed with LiOH"H 2 0 (6 mg, 0.151 mmol) as described in Example 2IB to provide the. title compound (30 mg, 72!).
  • LCMS (APCI+; Method D) m/z 330, 332, (M+H) 4 .
  • Example 32B The title compound (31, mg, 64%) was prepared from Example 32B (28 mg, 0.085 mmol) as described in Example 21C.
  • LCMS APCI+; Method D) m/z, 571, 573 (M+H) ⁇ .
  • Example 33A (260 mg, 0.704 mmol) was hydrolyzed with LiOH"H 2 0 (59 mg, 1.41 mmol.) as described in Example 21B to provide the title compound (225 mg, 95!).
  • X E NMR 400 MHz, DMSO-d 6 ) ⁇ 12.28 (s, IH) , 6.92 (d, J L.95 Hz, 2H) , 6.82-6.80 (m, IH) , 3.74-3.69 (m, 2H) , 2.87-2.82 (m, 2H) , 2.47-2.42 (m, IH) , 1.88-1.84 (m, 2H) , 1.61-1.52 (m, 2H) .
  • LCMS (APCI+; Method D) m/z 274, 276, (M+H) + .
  • Example 33B The title compound (156 mg, 83!) was prepared from Example 33B (100 mg, 0.365 mmol) as de- scribed in Example 21C.
  • Example 33D (112 mg, 69%) was obtained as a white powder.
  • Example 34A (500 mg, 1.803 mmol) was hydrolyzed with LiOH*H 2 0 (91 mg, 2.164 mmol) as described in Example 2IB to provide the title compound (438 mg, 97%) as a white powder.
  • X E NMR 400 MHz,
  • Example 34B (100 mg, 0.401 mmcl) as described in Example 21C.
  • Example 34D (45 mg, 41%) was obtained as a white powder.
  • Example 35A was prepared by the procedure_ .de.S-cribed- in Example.21A, - substituting -l-b-romo-3- methyl-benzene with l-bromo-4-tert-butyl-benzene (250 mg, 1.173 mmol). The title compound (194 mg,
  • Example 35A (190 mg, 0.657 mmol) was hydrolyzed with LiOH.H 2 0 (33 mg, 0.7878 mmol) as described in Example 21B to provide the title compound (127 mg, 74!).
  • LCMS (APCI+; Method D) m/z 262, (M+H) + .
  • Example 35C (175 mg, 0.3482 mmol) as described in Example 21D.
  • Example 35D (55 mg, 32!) was obtained as a white powder.

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Abstract

Aryl piperidine 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-I integrin.

Description

ARYL PIPERIDINE DERIVATIVES AS VLA-1 INTEGRIN ANTAGONISTS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. provisional patent application Serial No. 60/495,740, .filed August 14, 2003.
FIELD OF THE INVENTION
BACKGROUND 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, α and β. Integrins are the major receptor for cell adhesion to the extracellular matrix and play important roles in certain cell-cell and cell-matrix adhesion events. These integrin- mediated adhesion events are critical for both normal and pathophysiological processes during cell activation, migration, proliferation, and differen- tiation (for reviews see Hynes (1992) Cell 69: 11 ; Springer (1994) Cell 76: 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 llβl. 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 . 51:48206). 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 expressed on T cells in various disease states including in the joints of arthritis patients (He Ler et al. (1986) J. Clin . Invest . 78:696), lesions of giant cell arteritis patients (Schaufelberger et al. (1993) Clin . Exp. Immunol . 92:421), arterioscler- otic plaques, thyroid infiltrates of patients with autoimmune thyroid disease (Paolieri et al. (1992) J. Endocrinol . Invest . 15: 63 ) , and lungs of chronic bronchitis (Saetta et al. (1993) Am . Rev. Respir. Dis . 147: 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 . 175: 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 (de Fougerolles et al. (2000) J. Clin. Invest. 105:121) ; 3) anti-collagen 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 17:769; Krieglstein 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. Abs against αl have also been shown to block collagen-induced cyto- kine 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 . 21:437; Bissell. (1998) J. Gastroenterol . 33:295) . When overactive, this wound healing pro- cess 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 "myofibro- 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 vi tro and in vivo and is believed to regulate their pathologic functions. Alport syndrome is a genetic disorder characterized by progressive glomeruloneph- ritis resulting in fibrosis of the kidneys and ultimately kidney failure. Alport 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. Pathol . 157:1649). In addition, inhibition of TGF-βl with a soluble receptor construct had a synergistic effect with the inactivation 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 (Kagami 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 fi- brotic kidney disease (Kagami et al. (2002) Lab .
Invest . 82 : 1219 ; Cook et al . (2002) Am . J. Pa thol . 161 : 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 fibroblasts (Pozzi et al. (1998) J. Cell . Biol . 142:587; Senger et al. (2002) Am . J. Pathol . 160: 195) , and 2) by regulating the production of matrix metallopro- teinase 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 21 : 212 ) . Furthermore, the metastatic po~ ' tential 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 ; olecular 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 path- ologies mediated by VLA-1 binding and cellular adhesion, migration, activation or differentiation. SUMMARY OF THE INVENTION
The present invention provides aryl piper- idine compounds that 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
(I)
wherein Ar is selected from the group con- sisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
A, together with the nitrogen atom bound thereto, forms 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, sub- stituted 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 and alkoxy substituents, if present, may be attached to a carbon atom in said heterocyclic group, wherein the one or more thioalkyl and the one or more halo substituents, if present, may not be attached to a nitrogen atom in said heterocyclic group, and wherein the one or more halo, thioalkyl, and the one or more hydroxy substituents, if present, may not be attached to a carbon atom which is adjacent to a nitrogen atom in said heterocyclic group;
W is selected from the group consisting of -C(X)-NH and -NH-C (X) -, ■ wherein X is oxygen or sul- fur,
Y is selected, from the group consisting of a bond, alkylene, and substituted alkylene;
R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminocarbonyloxy, oxycarbonyl- a ino/ aminoacyl, acylamino, aminocarbonyla ino, aminosulfonylamino, aminosulfonyl, sulfonylamino, heterocyclic, substituted heterocyclic, aryl, sub- stituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, carboxyl, and carboxyl esters; or R3-C-R4 forms a cycloalkyl, substituted cycloalkyl, heterocyclic, or substituted heterocyclic group; R5 is selected from the group consisting of
CRaRaC02Ra, C0C02Ra, carboxyl, carboxyl esters, -CONRaORa, -CONRaS02Ra, heterocyclic, cyano, and hy- droxyalkyl; wherein Ra is selected from the group consisting of hydrogen, alkyl, aryl, substituted aryl, heterocyclic (e.g., tetrazolyl) , and substi- tuted heterocyclic; with the proviso that when Y is a bond and W is C(0)NH, then R3 and/or R4 are not amino, substituted amino, hydroxy, or alkoxy; and pharmaceutically acceptable salts, prodrugs, and any and all tautomers thereof.
In a preferred embodiment of this invention, A and the nitrogen atom pendent thereto form a piperidine ring.
In another preferred embodiment, W is -C(0)-NH- or -NH-C(O)-.
In still another preferred embodiment, Y is a bond or methylene, R4 is hydrogen, and R5 is -C02H.
In yet another preferred embodiment, R3 is alkyl or substituted alkyl.
Particularly preferred Y groups include a bond, methylene, and substituted methylene.
Particularly preferred R3 groups include methyl, N-benzylimidazol-4-ylmethylene, and phenyl - acetamidomethylene.
In still another preferred embodiment, Ar is selected from the group consisting of:
5- (2, 3-dichlorophenyl) pyridin-3-yl;
4- (2, 3-dichlorophenyl) pyridin-2-yl; 5- (2, 3-dichlorophenyl) pyridin-2-yl; 3-methyl-5- (2, 3-dichlorophenyl) pyrdin-2- yi;
3-methyl-4- (2, 3-dichlorophenyl) pyridin-2- yl; 5-methyl-4- (2, 3-dichlorophenyl) pyridin-2- yl;
5- (2, 3-dichlorophenyl) pyridin-2-yl;
6-methyl-3- (2, 3-dichlorophenyl) phenyl;
4- (2, 3-dichlorophenyl) thiazol-2-yl; 5-bromo-pyridin-3-yl; pyrdin-3-yl;
3- (2, 3-dichlorophenyl) phenyl; .,
6-methyl-3- (2, 3-dichlorophenyl) phenyl;
3-methylphenyl; ' . 3-ethylphenyl; ■■
3-isopropylphenyl;
3-trifluoromethylphenyl; .
3-dimethylaminophenyl;
3- (4-fluorophenoxy) phenyl; 3-fluoro-5-trifluoromethylphenyl;
3, 5-trifluoromethylphenyl;
2-methylquinolin-6-yl;
3, 4-dichlorophenyl;
2 , 3-dichlorophenyl; 6, 7-dichlorobenzo [b] thiophen-5-yl;
3, 5-dichlorophenyl; benzo [1,3] dioxol-6-yl;
4-t-butylphenyl;
3-chloro-6-nitrophenyl; 5-bromo-4-fluoro-2-nitrophenyl;
5- (N-morpholino) -2-nitro-phenyl; 3- (trifluoro ethylthio) phenyl;
3- (trifluoromethoxy) phenyl;
3- (trifluoromethoxy) henyl; quinolin-3-yl quinolin-3-yl; isoquinolin-3-yl; isoquinolin-3-yl;
(4-fluorophenyloxy)benz-3-yl;
3- (4-fluorophenyloxy) ethylphenyl; quinolin-3-yl; and ' phenyl .
Aryl piperidine- amide derivatives within the scope of this invention are exemplified by those set forth in Tables I and' II as follows:
Figure imgf000013_0001
* unless otherwise indicated, the stereochemistry at this site corresponds to L-amino acids
Ex. No. Ar
4- (2, 3-dichlorophenyl) pyridin-2-yl methyl
N-benzylimidazol-4-
4- (2, 3-dichlorophenyl)pyridin-2-yl ylmethylene
5- (2, 3-dichlorophenyl) yridin-2-yl methyl
3-methyl-5-(2,3- dichlorophenyl) yrdin-2-yl methyl
Figure imgf000014_0001
Figure imgf000015_0001
* unless otherwise indicated, the stereochemistry at this site corresponds to L-amino acids
Ex . No . Ar
N-benzylimidazol-4-
27 3-fluoro-5-trifluoromethylphenyl ylmethylene
N-benzylimidazol-4-
28 3, 5-trifluoromethylphenyl ylmethylene
N-benzylimidazol-4-
29 2-methylquinolin-6-yl • ylmethylene
N-benzylimid.azol-4-
30 3, 4-dichlorophenyl ylmethylene
N-benzylimidazol- 4'
31 2, 3-dichlorophenyl ylmethylene
N-benzylimidazol-4-
32 6, 7-dichlorobenzo [b] thiophen-5-yl yl ethylene
N-benzylimidazol-4-
33 3 , 5-dichlorophenyl ylmethylene
N-benzylimidazol-4-
34 benzo [i, 3] dioxol-6-yl ylmethylene
N-benzylimidazol-4-
35 4-t-butylphenyl ylmethylene
N-benzylimidazol-4-
36 3-chloro-6-nitrophenyl ylmethylene
N-benzylimidazol-4-
37 5-bromo-4-fluoro-2-nitrophenyl ylmethyϊene
N-benzylimidazol-4-
38 5- (N-morpholino) -2-nitro-phenyl ylmethylene
N-benzylimidazol-4-
39 3- (trifluoromethylthio) phenyl ylmethylene
N-benzylimidazol-4-
40 3- (trifluoromethoxy) phenyl ylmethylene phenylacetamidomethy
41 3- (trifluoromethoxy) phenyl lene
N-benzylimidazol-4-
42 quinolin-3-yl ylmethylene phenylacetamidomethy
43 quinolin-3-yl lene
N-benzylimidazol-4-
44 isoquinolin-3-yl ylmethylene phenylacetamidomethy
45 isoquinolin-3-yl lene
Figure imgf000016_0001
* unless otherwise indicated, the stereochemistry at this site corresponds to L-amino acids
E . No. Ar
46 3- (4-fluorophenyloxymethyl) phenyl-.. N-benzylimidazol-4- ylmethylene -
47 N-benzylimidazol-4-
3- (4-fluorophenyloxy) ethylphenyl ylmethylene
49 3- ( 6r 7-dichlorobenzo [b] thiophen-5~ N-benzylimidazol-4- yl) phenyl ylmethylene
4-isoxazolyl (3, 5-dimethyl) -pyridin-3- N-benzylimidazole-4- yi l-methylene
TABLE II
Figure imgf000017_0001
* both R and S configurations are contemplated, though the illustrated stersoisomer is generally preferred
Ex. No. Ar
48 quinolin-3-yl methyl hydrogen
15 5- (2, 3-dichlorophenyl)pyridin-3-yl methyl hydrogen
16a 5- (2, 3-dichlorophenyl) pyridin-3-yl methyl hydrogen
16b 5- (2, 3-dichlorophenyl) pyridin-3-yl hydrogen methyl
3- ( 6, 7-dichlorobenz [b] thiophen-5- methyl hydrogen yl) phenyl-pyridin-3-yl
5-chloro-3-dimethyl-2- benzo [b] thiophen-pyridin-3yl methyl hydrogen
Specific compounds within the scope of this invention are exemplified by the following:
2-{ [4 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino }-propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [4 ' - (2, 3-dichlorophenyl) -3,4,5, 6-tetrahydro-2H- [1,2' ] bipyridinyl-4- carbonyl] -amino}-propionic acid;
2-{ [6' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino}-propionic acid; 2- { [6 ' - (2, 3-Dichloro-phenyl) -3 ' -methyl-3, 4,5,6- tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino}- propionic acid;
2-{ [ ' - (2, 3-Dichloro-phenyl) -3 ' -methyl-3, 4, 5, 6- tetrahydro-2H- [1,2 ' ]bipyridinyl-4-carbonyl] -amino}- propionic acid/2-{ [4 '- (2, 3-Dichloro-phenyl) -5 ' - methyl-3, 4, 5, 6-tetrahydro-2H- [1,2' ] bipyridinyl-4- carbonyl] -amino} -propionic acid;
2- { [5 ' - (2, 3-Dichloro-phenyl) -3, 4 , 5, 6-tetrahydro-2H- [1, 2 ' ]bipyridinyl-4-carbonyl] -amino} -propionic acid; 2- { [5 ' - (2 , 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4-carbonyl] -amino}-propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [5 ' - (2, 3-dichlorophenyl) -3,4,5, 6-tetrahydro-2H- [1, 3' ] bipyridinyl-4- carbonyl] -amino} -propionic acid;
3-Benzoylamino-2-{ [5 ' - (2, 3-dichloro-phenyl) -3,4,5,6- tetrahydro-2H- [1,3' ]bipyridinyl-4-carbonyl] -amino}- propionic acid; . . .
2- [ (5 ' -Bromo-3, 4 , 5, 6-tetrahydro-2H- [1, 3 ' ] bipyridin- yl-4-carbonyl) -amino] -propionic acid; *'
3-Benzoylamino-2- [ (5 ' -bromo-3, 4,5, 6-tetrahydro~2H- [1,3' ]bipyridinyl-4-carbonyl) -amino] -propionic acid; 3-(l-Benzyl-lH-imidazol-4-yl)-2-[ (5 ' -bromo-3, 4, 5, 6- tetrahydro-2H- [1,3' ] bipyridinyl-4-carbonyl) -amino] - propionic acid;
2-[ (3,4,5, 6-Tetrahydro-2H- [1,3' ] bipyridinyl-4- carbonyl) -amino] -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- [(3,4,5, 6-tetra- hydro-2H- [1,3' ] bipyridinyl-4-carbonyl) -amino] - propionic acid;
N- [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,3'] bipyridinyl-4-yl] -3-methyl-succinamic acid; N- [5 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,3'] bipyridinyl-4-yl] -3-methyl-succinamic/N- [5 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1, 3 ' ] - bipyridinyl-4-yl] -2-methyl-succinamic acid; 2, 3-Dichloro-3 ' -2 [ (piperidine-4-carbonyl) -amino] - propionic acid;
2, 3-Dichloro-4 ' -methyl-3 ' -2 [ (piperidine-4-carbonyl) - amino] -propionic acid; 2- ( { 1- [4- (2, 3-^Dichloro-phenyl) -thiazol-2-yl] -piper- idine-4-carbonyl} -amino) -propionic acid;
3-(l-Benzyl-lH-imidazol-4-yl) -2- ( {1- [4- (2, 3-di- chloro-phenyl) -thiazol-2-yl] -piperidine-4-carbonyl}- amino) -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (1-m-tolyl-piperi- dine-4-carbonyl) -amino] -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-ethyl-phen- yl) -piperidine-4-carbonyl] -amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-isopropyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid; •
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-trifluoro- methyl-phenyl) -piperidine-4-carbonyl] -amino}- ■• propionic acid; ' 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-dimethyl- amino-phenyl) -piperidine-4-carbonyl] -amino}- ' propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (4-fluoro- phenoxy) -phenyl] -piperidine-4-carbonyl} -amino) - propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-fluoro-5- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 5-bis- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (2-methyl- quinolin-6-yl) -piperidine-4-carbonyl] -amino}- propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 4-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (2, 3-dichlorophenyl) -piperidine-4-carbonyl] -amino}-propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (6, 7-dichlorobenzo [b] thiophen-5-yl) -piperidine-4-carbonyl] - amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-.{ [1- (3, 5-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid; '
2- [ (1-Benzo [1,3] dioxol-5-yl-piperidine-4-carbonyl) - amino] -3- (l-benzyl-lH-imidazol-4-yl) -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (4-tert-butyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-chloro-2- nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-bromo-4- fluoro-2-nitro-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-morpholin-4- yl-2-nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid; 3- ( l-Benzyl-lH-imidazol-4-yl) -2- { [1- ( 3-trif luoro- methylsulfanyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid;
3-(l-Benzyl-lH-imidazol-4-yl)-2-{ [1- (3-trifluoro- methoxy-phenyl) -piperidine-4-carbonyl] -amino} - propionic acid;
3-Benzoylamino-2-{ [1- (3-trifluoromethoxy-phenyl) - piperidine-4-carbonyl] -amino } -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-quinolin-3-yl- piperidine-4-carbonyl) -amino] -propionic acid;
3-Benzoylamino-2- [ (l-quinolin-3-yl-piperidine-4- carbonyl) -amino] -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-isoquinolin-4- yl-piperidine-4-carbonyl) -amino] -propionic acid; 3-Benzoyl amino-2- [ (l-isoquinolin-4-yl-pipeπdine-4- carbonyl) -amino] -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (4-fluoro- phenoxymethyl) -phenyl] -piperidine-4-carbonyl}- amino) -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-{ 3- [1- (4-fluoro- phenoxy) -ethyl] -phenyl } -piperidine-4-carbonyl) - amino] -propionic acid;
3-{ [5 ' - (l-Methyl-lH-indol-5-yl) -3,4,5, 6-tetrahydro-
2H- [1,3' ]bipyridinyl-4-carbonyl] -amino} -butyric acid;
3-Methyl-N- (l-quinolin-3-yl-piperidin-4-yl) - succinamic acid; and
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (6, 7-di- chloro-benzo [b] thiophen-5-yl) -phenyl] -piperidine-4- carbonyl}-amino) -propionic acid. In a preferred embodiment are compounds of Formula I having an IC50 of less, than or about 50 μM using the method described in Example A or Example B herein. In a more preferred embodiment are com- pounds of- Formula I having an IC50 of less than or about 40 μM using the method described in Example A or Example B herein. In a still- more preferred embodiment are compounds .of Formula I having an IC50 of less than or about 20 μM using the method described in Example A or -Example B hereini.
In another aspect, this invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of Formula I as de- fined herein.
In one of the method aspects, ■ this invention 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 mam- malian 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 therapeu- tically effective amount of a compound or co posi- 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 di- .• sease, 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, sarcoido'sis, 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 selec- ted from the group consisting of chronic renal fail- ure, lupus nephritis, alport syndrome, glomerulo- nephritis, and diabetic nephritis.
In another preferred embodiment, the invention is directed to a method for ameliorating cancers 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 composition of this invention. 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, uro- logical 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. In still yet another preferred embodiment, 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 .
A compound or composition of this inven- tion may be administered to the mammal by any suitable route, such as orally, intravenously, par- enterally, 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 aryl piperidine derivatives.
It is to be understood that the terminology 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: 5 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, even' more preferably 1 to 4 carbon atoms. This term is exemplified by groups such as methyl, t-butyl, ■ n-
•10. 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,
15 aminόacyl, aminocarbonylamino, aminocarbonylox , aryl, substituted aryl, aryloxy, substituted aryl-' - oxy, cyano, halogen, hydroxy, nitro, carboxyl, carboxyl esters, cycloalkyl/ substituted cycloalkyl, thiol, thioalkyl, heteroaryl, substituted hetero-
20 aryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino.
"Hydroxyaϊkyl" refers to an alkyl group having from 1 to 3 hydroxy substituents provided that there is no more than one hydroxy group per
25 carbon atom.
"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 ex-
30 emplified by groups such as methylene, n-heptylene, 1, 3-octylene, and the like. "Substituted alkylene" refers to an alkylene group as defined above substituted with from 1 to 3 substituents as described for substituted alkyl. "Alkoxy" refers to the group "alkyl-0-" 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 (0) -, alkenyl-C (0) -, substituted alkenyl-C (0) -, cycloalkyl-C (O) -, substi- tuted cycloalkyl-C (0) -, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C (0) -, substituted hetero- ■ aryl-C(O), heterocyclic-C (0) -, and substituted het- erocyclic-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 optionally 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 hetero- aryl-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 site, and preferably from 1-2 sites, of alkenyl unsaturation. "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 aryloxy, cyano, halogen, hydroxy, nitro, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl, thiol, thioalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and oxycarbonylamino, provided that the hydroxy 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 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 groups are not hydrogen.
"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 (0) heterocyclic, 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(0)- O-cycloalkyl, -NRC.(O) 0-substituted cycloalkyl, - -NRC- (O)O-aryl, -NRC (0) 0-substituted- aryl, -NRC (0) O-het- eroaryl, -NRC (0) O-substituted heteroaryl, -NRC (0)0- heterocyclic, and -NRC (0) 0-substituted- heterocyclic, where R is hydrogen or alkyl.
"Aminosulfonyl" refers to the group ~S02Q where Q is amino- or substituted amino, as defined . herein.
"Aminosulfonylamino" refers to the group QS02Q where each Q is independently amino or substituted amino, as defined herein.
"Oxycarbonylamino" refers to the group -0C(0)Q where Q is amino or substituted amino, as defined herein.
"Aminocarbonylamino" refers to the group -QC(0)Q where each Q is independently amino or substituted amino, as defined herein. "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 multiple 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-7yl, 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 alkenyl, amino, substituted amino, aminoacyl, amino- carbonyloxy, aminocarbonyiamino, aryl, substituted aryl, aryloxy, substituted aryloxy, carboxyl, carboxyl esters, cyano, thiol, thioalkyl, substituted thioalkyl, cycloalkyl, substituted cycloalkyl, halo, 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.
"Substituted aryloxy" refers to substituted aryl-O- groups. "Carboxyl" refers to the group -COOH, and salts thereof.
"Carboxyl esters" refer to the group -COOR where R is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, carboxyl-substituted heteroaryl, heterocyclic, and 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.
"Substituted cycloalkyl" refers to a cycloalkyl, 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.
"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, nitro- gen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indoli- zinyl or benzothienyl) provided that the point of attachment is to a heteroaryl group atom and further provided that the heteroaryl group contains five ring atoms. Preferred heteroaryls include pyridyl, pyrrolyl, indolyl, and furyl.
"Substituted heteroaryl" refers to heteroaryl 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 onovalent saturated or unsaturated, but not aromatic, 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 pro- vided that the point of attachment is to a heterocyclic ring atom.
"Substituted heterocyclic" refers to heterocycle groups which are substituted with from 1 to 3 substituents selected from the group of substit- uents 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 re- ferred to as thiamorpholmyl) , piperidinyl, pyrroli- dine, tetrahydrofuranyl, and the like. "Thiol" refers to the group -SH.
"Thioalkyl" refers to the group -S-alkyl.
"Substituted thioalkyl" refers to the group -S-substituted alkyl. 5 "Sulfonylamino" refers. to the group QS02 where Q is amino or substituted amino, as defined herein.
"Tautomers" refers to chemical compounds that exist as a mixture of two interconvertible iso- -10- mers in equilibrium.
"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 15 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- 20 mide, tartrate, mesylate, acetate, maleate, oxalate, and the like.
"Prodrugs" as used herein, are compounds which convert (e.g., hydrolyze, metabolize, etc.) in vivo to a compound of the invention. The effective- 25 ness of an orally administered drug is dependent upon the drug's efficient- transport across the muco- sal epithelium and its stability in entero-hepatic circulation. Drugs that are effective after par- enteral administration but less effective orally, or 30 whose plasma half-life is considered too short, may be chemically modified into a prodrug form. A pro- drug is a drug which has been chemically modified and may be biologically inactive at its site of action, but which may be degraded or modified by one or more enzymatic or other in vivo processes to the parent bioactive form. The prodrug 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 prodrugs according 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 derived from the carboxylic acid moiety or the a ine 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 Bundgaard, (1985) Design of Prodrugs (Elsevier) , may be placed on appropriate functionalities when such functionalities are pres- ent 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 in- eluded within the scope of the invention.
The terms "substituted" as used with, for example, "substituted alkyl" does not include polymers derived therefrom but are limited to a maximum of 3 substituents groups, e.g., alkyl-Ar-Ar-Ar.
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 pre- ferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) 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 Greene and Wuts, (1991) Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, and references cited therein. Furthermore, the compounds of this invention 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 stereoisomers (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, chiral column chromatography, chiral resolving 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
Ar1— X + Y' ^r2 Palladium coupling > Ar1— Ar2 solvent 101 102 103
Scheme -1 above, wherein Ar1, Ar2 can be aryl, substituted aryl, heteroaryl, or substituted aryl, describes the synthesis of a typical substituted biaryl, compound 103, via palladium coupling. Such biaryls are useful starting materials for the preparation of compounds of formula I above where Ar is Arx-Ar2. The coupling (Suzuki et al . (1995) Chem . Rev. 95:457; Org. React . (1997) 50:1) can be performed using compound 101, wherein X is a halogen, (e.g., 3-chloroiodo-benzene or 2-chloropyridine or the like) and compound 102, wherein Y' is a boronic acid, boronic ester, or an organo stannane of an aryl, substituted aryl, heteroaryl, and substituted heteroaryl (e.g., 3-nitrophenylboronic acid or 2- tributylstannyl pyridine) in the presence of a base (e.g., cesium carbonate or tripotassium phosphate) in a nonprotic solvent (e.g., dioxane, DME, toluene) or protic solvent (e.g., DMF, acetone/water), together with a palladium catalyst (e.g., Pd(PPh3)4, Pd2(dba)3, Pd(OAc)2). This reaction is run at a temperature of about 25°C to about 90 °C for a time sufficient for the reaction to go to completion, typically about 1 to about 12 hours. The product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification. The reaction also can be performed in the reverse direction by switching the halide and transition metal of the aryl groups.
Scheme 2
Figure imgf000039_0001
104 105
Scheme 2, for illustrative purposes only, describes how a halopyridine such as compound 105, can be synthesized by treating a pyridine biaryl compound useful in the preparation of compounds of formula I, for example compound 104, with an oxidizing agent (e.g., m-CPBA, hydrogen peroxide, etc.), in a suitable solvent (e.g., DCM, DCE, CC14) . The N-oxide intermediate is then reacted with a phosphorus oxyhalide (e.g., P0C13, POBr3) to furnish the halopyridine. The halopyridine of this reaction may be optionally substituted with substituents suitable for use in the subsequent reactions. Step 1 of the reaction shown in Scheme 2 is run at a temperature of about -10 °C to about 30°C for a time sufficient for the reaction to fo to completion, typically about 1 to about 18 hours. Step 2 of the reaction shown in Scheme 2 is run at a temperature of about 25 °C to about 90 °C for a time sufficient for the reaction to go to completion, typically about 1 to about 18 hours. The product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
Scheme 3
Figure imgf000040_0001
105 106
In scheme 3 above, Ar1 is defined above, R3' is either a carboxylic acid with a suitable protecting group (OCOPg, where Pg may be alkyl, or more specifically, t-butyl) or an amine with a suitable protecting group (NHPg*, where Pg* may be boc, cbz, or frnoc) , n is 0 to 4, and the halopyridine may be optionally substituted with substituents suitable for use in the subsequent reaction. Scheme 3 illustrates a method for converting a halopyridine, such as compound 105, [e.g., 2- (4-methoxyphenyl)pyri- dine] , to a 2-aminopyridine, compound 106, through a melt reaction using various amines (e.g., ethyl or methyl isonipecotate or 4- ( t-butyloxycarbonyl- amino) piperidine or methyl azetidine-3-carboxylate) . This reaction is typically run neat, preferably using an excess of amine, at a suitable temperature, from about 25°C to about 90°C in an inert atmosphere in a closed tube. The reaction is run for a time sufficient for the reaction to go to completion, typically about 1 to 50 hours. The product can be recovered using standard techniques such as filtra- tion, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
Scheme 4
Figure imgf000041_0001
107 108 109
Halide
Figure imgf000041_0003
formation
Figure imgf000041_0002
107a 110 111 109a
In Scheme 4 above, Ar1, R3', n, and X are defined above arid the phenyl group is further op- tionally substituted with a group suitable for use in subsequent reactions. As shown, substituted nitrohaloaryl analogs, such as compound 107, can be reacted with various amines (ethyl or methyl iso- nipecotate 'or 4- ( -butyloxycarbonyl-amino) piperi- dine or methyl azetidine-3-carboxylate, etc.) to form the nitro aniline compound 108. This reaction is typically run neat, preferably using an excess of amine, at a suitable temperature, from about 25°C to about 90 °C. The reaction is run for a time suffi- cient for the reaction to go to completion, typically about' 1 to about 20 hours. The product can be recovered using standard techniques such as filtra- tion, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification. 5 The nitro group of compound 108 may be reduced to form the aniline, (compound not shown) , using a, suitable reducing conditions (e.g., zinc in acetic acid or FeS04 (H20)7 in THF) followed by formation of a diazo salt (NaN02, sulfuric acid, or
10 HN02) . The resulting diazonium salt -may be reduced . (H3PΘ4 or HMPA in ethanol or sodium stannite, see Ruchardt et al. (1977) Chem . Ber. 210:2494) to the hydrido product compound 109. This reaction is run at a temperature of about 0° to about 90 °C for a
1.5 time sufficient for the reaction to go to completion, typically about 1 to 24 hours. The product can be recovered using standard .techniques such, as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may
20 be used in the next step without further isolation •• and/or purification.
Biaryl halides, such as compound 111, (2, 3-methyl-3 ' -iodo-biphenyl, etc.) can also be synthesized from biaryl nitro compounds, such as
25 compound 107a, (e.g., 2, 3-dichloro-3 ' -nitro-biphen- yl, and the like) . Reduction of the nitro group of compound 107a leads to an aniline derivative, compound 110. This intermediate can then be converted to an aryl halide, such as compound 111, by first
30 converting the aniline to a diazo salt by the method described above, followed by displacement with a halide such as Nal or CuBr2 (J. Chem . Soc. , Perkin Trans . 1 (1987) 645) . Furthermore, halopyridines, such as compound 105, or aryl halides such as compound 111, (e.g., 3 bromobiphenyl, 2, 3-dichloro-3 ' - iodo-biphenyl, 2-chloro-4- (m-tolyl) -pyridine) ) , can be converted to various substituted aminoaryls, such compounds 106, 109> 109a, (and, as illustrated in Scheme 5 below, compound 112) by a palladium mediated coupling (WO 00/39081; Old et al. - (1998) J. Am . Chem . Soc. 120 (91) : 9122; Prim et al. (2002) Tetrahedron 58:2041). The product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, -and the like. Alternatively, the product may be used .in the next step without further isolation and/or purification.
Scheme 5
Ar1-X 101
Figure imgf000043_0001
coupling
114 113
Alternatively the coupling partners can be reversed, as shown in Scheme 5 above, wherein Ar1,
Ar2, R3', n, X, and Y' are defined herein above. The coupling can be performed as described in Schemes 3 and 4 to give compound 112. Compound 112 may be halogenated by using techniques described herein and/or by methods well known in the art, to give compound 112a, where Y' is a boronic acid, boronic ester, or an organo stannane. Furthermore, compound 112a can be coupled to Ar2-Y' when Y' is a boronic acid, boronic ester, or an organo stannane via copper mediated Chan-Lam coupling (see Lam et al. (2002) Tetrahedron Lett . 43:3091; Chan et al . (2003) Tetrahedron Lett . 44 : 3863 ) . A second palladium coupling (as indicated in Scheme 1) can provide compound 113 from compound 112a. Alternatively, compound 112 may be transmetallated to give compound 114, (Miyaura et al . (1995) J. Org. Chem . 60: 1508- 7510) , which can then be coupled as in Scheme 1 to other aromatic halides to give compound 113. The above reactions are run under conditions described herein or using techniques well known in the art.
Scheme 6
Figure imgf000044_0001
Substituted benzothiophenes were synthesized according to Scheme 6. One method entails the alkylation of bromoacetic acid compound 116 with substituted thiophenols compound 115 (3-methyl thio- henol, 2,3-dichloro thiophenol, etc.) in aqueous solvent and in the presence of a base (NaOH, Cs2C03, and the like) to afford compound 117. Bromination (e.g., using iron filings and
Br2, or CuBr2) in acidic solvent (e.g., acetic acid) then affords compound 118. The reaction is typically performed at room temperature for 1 to 96 hours. The carboxylic acid is activated, for example by conversion to the corresponding acid chloride (thionyl chloride, oxalyl chloride, or the like, with catalytic DMF in a halogenated solvent, e.g., DCM, DCE) 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 to afford ketone compound 119 by treatment with a Lewis acid such as A1C13 or polyphos- phoric acid. The reaction is generally carried out at -78°C to 25°C.
Reduction of the ketone is afforded by a reducing agent (NaBH4, LiAlH , or DIBAL-H, or the like) , in a solvent such as ethanol, at a tempera- ture of about -10 °C to about 10 °C for a period of time sufficient for completion of the reaction. The intermediate alcohol is eliminated to benzothiophene compound 120 using techniques well known in the art. The product can be recovered using standard tech- niques such as filtration, chromatography, distilla- tion, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
In order to increase the' reactivity of the aryl halide towards palladium coupling reactions, the bromide can be converted to the corresponding iodide compound 121 (Buchwald et al. (2002) J. Am . Chem . Soc. 224:14844-14845) . The product can be recovered using standard techniques such as filtra- tion, chromatography, distillation, extraction, and • the like. Alternatively, the product may be used in the next step without further 'isolation and/or purification.
Scheme 7
Figure imgf000047_0001
122 123
H
Figure imgf000047_0002
Figure imgf000047_0003
125
Figure imgf000047_0004
126
A process for preparing substituted heteroaryl derivatives is illustrated in Scheme 7, following a Hantzsch type synthesis (Beugelmans et al. (1996) J. Org. Chem . 61 : 118 ) , wherein Ar1, R3', and n are defined herein above, and Q* is sulfur or oxygen. The starting aryl ketone compound 122 is chlorinated by reaction with oxalyl chloride in toluene yielding compound 123. This reaction is run at a temperature of about 0°C to about 90 °C for a time sufficient for the reaction to go to completion, typically about 1 to 24 hours. The product can be recovered using standard techniques such as filtration, chromatography, distillation, extrac- tion, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
Ureas, such as compound 124, can be syn- thesized by reaction of an amine (e.g., ethyl or methyl isonipecotate or 4- ( -butyloxy-carbonyl- amino)piperidine or methyl azetidine-3-carboxylate, etc.) with (trimethylsilyl) isocyanate or trimethyl- silylisothiocyanate in an aprotic solvent (e.g., DCM, toluene) . This reaction is run at a temperature of about 0°C to about 90 °C for a time sufficient for the reaction to go to completion, typically about 1 to 24 hours. The product can be recovered using standard techniques such as fil- tration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification. The product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
Combination of compounds 123 and 124 in the presence of base (LiHMDS, NaH, or the like) results in heteroaryl compounds such as .compound 125. When they are combined in the absence of base, heteroaryl compounds analogs such as compound 126 are formed. In either case the reactions are run at a temperature of about 0°C to about 90°C for a time sufficient for the reaction to go to completion, typically about 1 to 24 hours. The products can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively,- the product may be used in the next step without further isolation and/or purification.
Scheme 8
OH-Ar— X *" R — Y — Al — X
127 128
Scheme 8 illustrates the synthesis of sub- stituted arylethers, aryl. thioethers and amino derivatives, such as compound 128, wherein X is as defined herein, Ar i.s aryl,. substituted aryl, het- roaryl, or substituted' heteroaryl, R2* is alkyl, substituted alkyl, aryl, substituted aryl, ..hetercryl,- and substituted heteroaryl, and Y* is NH, 0 or S.
The Mitsunobu procedure can be utilized- on compounds such as compound 127, which are prepared from the corresponding optional .substituted Ar-X (Mitsunobu et al. (1967) Bull . Chem . Soc . Jpn . 40: 2380 ; Camp et al. (1989) J. Org. Chem. 54 : 3045 and 3049) to furnish substituted aryl ethers such as compound 128. Scheme 9
Figure imgf000050_0001
131
Figure imgf000050_0002
132 133
As illustrated in Scheme 9., (wherein Ar, R3,. R4, and n are as herein defined above, and Pg is a suitable protecting group for a carboxylic acid such as alkyl (e.g., methyl)), hydrolysis of the ester of compound 129 (e..g., compound 113 from Scheme 5), using LiOH or NaOH, or the like, in:-- an aqueous solvent (water-THF, etc.) .afforded the carboxylic acid compound 130.. This reaction- is run at a temperature of about 0°C to about 30°C for a time sufficient for the reaction to go to completion, typically about 1 to 12 hours. The product can be recovered using standard techniques such as filtra- tion, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification.
To prepare coupled amide compounds such as compound 132, amino acid derivatives such as compound 131 are coupled to the carboxylic acids, com- pound 130, in an inert solvent (DCM, THF, DMF or the like) using any of a wide variety of well known coupling agents. Suitable coupling agents include, but are not limited to, 1- (3-dimethylaminopropyl) -3- ethylcarbodimide hydrochloride (EDC) , dicyclohexyl- carbodiimide (DCC) , N, N' -carbonyldiimidazole (CDl), benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) , and diethylcyanophos- phonate (DECP) . Additionally, well known coupling promoters (for example 1-hydroxybenzotriazole
(HOBt) , N-hydroxysuccinimide , and the like) may be used to optimize the yield. Furthermore, the acid may be coupled utilizing solid support resins such as 1% cross linked polystyrene tetrafluoro phenol (PS-TFP) , polystyrene dicyclohexyl carbodiimide (PS- DCC) or polystyrene 1-hydroxybenzotriazole (PS-HOBT) by the use of one of the listed coupling reagents in the presence of a base (4-dimethylamino pyridine, triethyl amine, etc.) and solvent (DMF, methylene chloride.) The resin bound acid can then be reacted with a range of amines to furnish the corresponding amide. The coupled amide compound 132 is then subjected to basic hydrolysis conditions listed above to afford the final acid compound 133. The product can be recovered using standard techniques such as filtration, chromatography, distillation, extraction, and the like. Alternatively, the product may be used in the next step without further isolation and/or purification. Scheme 10
deprotection
Figure imgf000052_0002
Figure imgf000052_0001
134 135 amide couple
hydrolysis
Figure imgf000052_0003
Figure imgf000052_0004
137 138
Scheme 10 depicts the preparation of- re- versed amide analogs, wherein Ar, R3, R4, n, Pg and Pg* are as defined herein- above. The protecting group Pg* on the protected amine, compound 134, (also compound 112 or compound 113 from Scheme 5 wherein R3' is an amine protected with a- suitable protecting group such as Boc, Cbz, Fmoc, trifluoro- ■ acetamide, or the like) , is removed using conditions suitable for the protecting group, to afford the amine compound 135. Amine compound 135 is then coupled using conditions listed above to an acid, compound 136, to provide for the amide compound 137. The ester of the resulting amide compound 137 is then hydrolyzed using conditions listed above to yield the acid, compound 138.
Conversion of a methylene carboxylic acid to the corresponding ethylene heteroaryl derivative can be accomplished by well known techniques an example of which is provided in Scheme 11 below: Scheme 11
Figure imgf000053_0001
600
Specifically, in Scheme 11 above, (wherein P , R4, Y, W, A, Ar and X are as defined herein above, and HET is heteroaryl or substituted heteroaryl) , the carboxyl group of compound 100 is reduced using conventional techniques such as the use of a conventional reducing agent including, for example, lithium aluminum hydride to provide for the corre- sponding 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, chro- matography, 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 tri- chloride, 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 or oxalyl chloride, 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 re- covered by conventional methods including neutrali- zation, 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 including Gutsche et al. (1975) Fundamentals of Organic Chemistry, Prentice Hall, p. 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.- Transition-metal catalysts may be used to facilitate this transformation, for example using copper (I) (e.g., copper (I) iodide, copper (I) - bromide dimethylsulfide complex) or palladium complexes .
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 pharmaceutically 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 other con- tainer. The excipient employed is typically 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, or: medium for the active - ingredient . Thus, the compositions 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 containing, ' 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 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 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, manni- tol, starches, gum acacia, calcium phosphate, algi- nates, tragacanth, gelatin, calcium silicate, micro- crystalline cellulose, polyvinylpyrrolidone, cellu- lose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubri- eating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as ethyl- 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" refers 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 prefer- ably 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 preformu- lation 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- ' Λ •. tion .may be • coated or otherwise .compounded to pro-- vide a dosage form affording the advantage of prolonged action. For example, the-.tablet or pill, can comprise an inner dosage and an outer dosage com- ponent, 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 mate- • . rials 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 incorporated 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, suspension, 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 fol- lowing ingredients are prepared:
Figure imgf000060_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 ingredients below:
Figure imgf000061_0001
The components are blended and compressed to form tablets, each weighing 240 mg.
Formulation Example 3
A dry powder inhaler formulation is pre- pared containing the following components:
Figure imgf000061_0002
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 imgf000062_0001
The active ingredient, starch and cellulose are passed through a No. 20 mesh U'. S. sieve and mixed thoroughly. The solution of polyvinylpyrroli- done 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, pre- viously 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 imgf000063_0001
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 150 mg quantities.
Formulation Example 6
Suppositories, each containing 25 mg of active ingredient are made as follows:
Figure imgf000063_0002
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the satur- ated 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 imgf000064_0001
The active ingredient, sucrose and xanthan 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 benzoate, 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 imgf000064_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 imgf000065_0001
Formulation Example 10
A topical formulation may be prepared as follows:
Figure imgf000065_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 imgf000066_0001
Another preferred formulation employed in 5 the methods of the "present invention employs txans- ■ dermal delivery devices ("patches") . Such trans- dermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construc-
10. tion 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 incorporated by reference.. Such patches may be constructed for continuous, pulsatile, . or on demand' delivery, of
15. pharmaceutical agents.- ' • ,' \
Frequently, it will be- desirable or necessary to introduce the pharmaceutical composition to the brain, either directly or indirectly. Direct techniques usually ' involve placement of a drug de-
20 livery catheter into the host's ventricular system to bypass the blood-brain barrier. One such im- plantable 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
25 is herein incorporated by reference.
Indirect techniques, which are generally preferred, usually involve formulating the compositions 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 trans- portation across the blood-brain barrier. Alternatively, the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier. Other suitable formulations for use in the present invention can be found in Remington ' s Pharmaceutical Sciences, (1985), Seventeenth Edition, Mack Publishing Company, Philadelphia, PA.
As noted above, the compounds described 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, 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. 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- 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 mediateα, 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 diseases, condi- tions, 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 trea able by the 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 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- alitis, stroke, and other cerebral traumas, nephritis, retinitis, atopic dermatitis, psoriasis, myo- cardial ischemia, acute leukocyte-mediated lung injury such as that which occurs - in adult respiratory distress syndrome, and fibrotic diseases, such as fibrotic diseases of the lung, kidney, liver, and 5 vasculature (including idiopathic pulmonary fibro- • sis, systemic sclerosis, glomerulonephritis, chronic hepatitis, chronic renal failure, and nonalcoholic steatohepatitis) . - -
Fibrotic diseases which are expected to be
10.- 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,
15 - 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
20 hypertrophic aspects, including fibrosing alveolitis and pulmonary fibrosis. Treatment using the compounds or compositions of .the present invention is expected to treat, prevent, reduce, or ameliorate such diseases, or hypertrophy, fibrotic hypertrophy
25 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-
30 litis (including coronary artery vasculitis) , poly- arteritis nodosa, or temporal arteritis. Treatment using the compounds 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 fasci- itis, discoid lesions associated with lupus or,, discoid lupus, or surgical- adhesions. Treatment • • . using the compounds or compositions of the present invention is expected to treat, prevent, reduce, or ameliorate such indications, or hypertrophy or-, 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 lobar sclerosis. Treatment using the compounds 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.
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 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, fibrosing alveolitis, the fibrotic or hypertrophic element of cystic fibrosis, chronic obstructive pulmonary disease, adult respiratory distress syndrome, and emphysema. Treatment using the compounds 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, alport syndrome, glomerulonephritis, and diabetic nephritis. Treatment using the compound 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 or compositions of the present invention typically occur in mammals. 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.
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 from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they can invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation . (greater "de- differentiation") 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 or compositions of the present invention include, but are not limited to, solid tumors, i.e., carcinomas, adenocarcinomas, and sarcomas. Carcinomas include those malignant neoplasms derived from epithelial cells which infiltrate (invade) the surrounding tissues and give rise 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 or homogenous substance like embryonic connective 5 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,
1.0 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
15- lymphoma, non-Hodgkin' s lymphoma,- cancer of the adrenal cortex, ACTH-producing tumors, nonsmail cell cancers, breast cancer '(including small cell carcinoma and ductal carcinoma) , gastrointestinal cancers (including stomach cancer, colon cancer, colo-
20 rectal 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,
25 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
30 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 oste- omas and osteosarcomas) , skin cancers (including malignant melanoma, tumor progression of human skin keratinocytes and squamous cell cancer) , thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer, trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma. Cancers and other cell proliferative disorders treatable by the compounds or 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 cancer, intraepithelial neoplasms, lung cancer, and neuroblastomas .
The biological activity of the compounds identified above may be assayed in a variety of systems. For example, extracellular matrix, such as 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 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, including, for example, CHO, K562, and the like. The compounds and 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 antibodies 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 recombinant fusion protein having acidic and basic leucine-zipper tails so that binding to extracellular matrix -may be de- . tected 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 autoradi- ography 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, bioavailabil- ity) ,- 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 sig- ■ nal . Thus, the compounds and compositions of the subject invention may be modified in a variety of ways for a variety of end purposes while still retaining 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 vi tro 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 5 ordinary skill in- the art will know of other suit- • ' able labels for binding to the complexes, or will be able to ascertain such using routine experimenta- - tion. The binding of these labels is achieved using standard techniques common to those of ordinary 10. - skill in the art. :
In vi tro uses include diagnostic applica- -•"tions such as monitoring, inflammatory responses by : detecting the presence of -cells expressing VLA-1. .- The compounds and compositions of. this invention can 15 also be used for isolating. or labeling such cells. In addition, as mentioned 'above, the compounds and compositions of the .invention can be used to assay for potential inhibitors of VLA-1/Extraeellular matrix interactions. 20 - For in vivo diagnostic - imaging to identi-
- . fy,. e.g., sites of inflammation, radioisotopes • are typically used in accordance with well-known techniques. The radioisotopes may be bound to the compound either directly or indirectly using inter- 25 mediate functional groups. For instance, chelating ■ agents such as diethylenetriaminepentacetic acid (DTPA) , ethylenediaminetetraacetic acid (EDTA) , and similar molecules have been used to bind compounds to metallic ion radioisotopes. 30 The complexes can also- be labeled with a paramagnetic isotope for purposes of in vivo diag- nosis, as in magnetic resonance imaging (MRI) pr electron spin resonance (ESR) , both of which are well known. In general, any conventional method for visualizing diagnostic images can- be used. Usually 5. 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 inflammatory response in an' individual. - By measuring the
10 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. r -.
- ■ Pharmaceutical compositions of the inven-
15' tion 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, (1985) -Seventeenth Edition, Mack .Publishing Company, Philadelphia, PA.
20 The amount administered to the patient will vary depending- upon what is.-.being administered, the purpose of the administration, such as prophyl- - axis or therapy, the state of the patient, the manner of administration, and the like. In thera-
25 peutic 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
30 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 clinician 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 compositions 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 lyophil- ized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. 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 of 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 con- dition 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 typ- ically 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-response curves ■ derived from in vitro or animal model test systems. In general, the compounds and/or composi- tions of the subject invention will be administered • in a therapeutically effective amount by any Of the v.. accepted modes of administration,.for agents that ■• serve similar utilities., Toxici-ty 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 LD5o/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 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 stared, 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 imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
The following analytical HPLC methods (or LC Methods) are referred co in the Examples. The gradient profiles were straight-line gradients increasing or decreasing linearly over the time period indicated.
Method A: Varian HPLC System: Pumps: arian ProStar Solvent Delivery System, Model 210; Detector: Varian ProStar UV-VIS Detector, Model
345; Autosampler: Varian ProStar Autosampler, Model 430). Analytical column: YMC ODS-AQ, 4.6 x 50 mm, S3 microns, Waters Corporation. Detection: 220nm and 254nm. Solvent A: H20, 0.1% TFA, 1.0% IPA. Solvent B: Acetonitrile, 0.05% TFA, 1.0% IPA. Flow Rate: 2.0 mL/min. Gradient Program: 0.00 min 95% Solvent A, 5% Solvent B; 0.05 min 95% Solvent A, 5% Solvent B; 4.12 min 5% Solvent A, 95% Solvent B; 4.24 min 5% Solvent A, 95% Solvent B; 4.30 min 95% Solvent A, 5% Solvent B; 5.00 min 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, 4.6 x 50 mm, S3μ, 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: 0.00 min 95% Solvent A, 5% Solvent B;, Q'..12 min- 95% Solvent A, 5% Solvent B; 4.00 min 5% Solvent A, 95% Solvent B; 4.18 min 5% Solvent A, 95% Solvent B; 4.30 min 95% -. Solvent A, 5% Solvent B; 5.30 min 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, 4.6 x 50 mm, S3μ, 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: 0.00 min 95% Solvent A, 5% Solvent B; 0.12 min 95% Solvent A, 5% Solvent B; 4.00 min 5% Solvent A, 95% Solvent B; 4.18 min 5% Solvent A, 95% Solvent B; 4.30 min 5% Solvent A, 95% Solvent B; .5.00 min 95% Solvent A, 5% Solvent B 5.30 min 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 150 X 4.6 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; Degass;er: Model -G1322A) . Analytical column: Chiralcel OD-RH, 150 X 4.6 mm. Detection: 220nm. HPLC Conditions: 1.0 mL/min, Isocratic, 70% (H20, 0.01%TEA), 30% (ACN, 0.01%TFA), 35°C. . '
;■ , Method F: ';■ Mass Spectrometer: , Finnigan
LCQTMDuo, APCI. TSP HPLC System: (Pump: TSP Spec- ' traSYSTEM® P4000; Detector: TSP SpectraSYSTEM® . .. - UV2000, 220nm and 254 ; Autosampler: TSP Spectra- SYSTEM® AS3000 Degasser: ■■ TSP SpectraSYSTEM® Model SCMIOOO Solvent Degasser) .• Detection: -220nm and 254nm. Analytical column: YMC ODS-AQ, 4.6 x 50 mm, S3μ, Waters Corporation. Solvent C: H20, 0.01% HFBA, 1.0% IPA. Solvent D: Acetonitrile, 0.01% HFBA, 1.0% IPA. Flow Rate: 2.0 mL/min. Gradient
Program: 0.00 min 95% Solvent C, 5% Solvent D; 0.02 min 95% Solvent C, 5% Solvent D; 4.00 min 5% Solvent C, 95% Solvent D; 4.30 min 5% Solvent C, 95% Solvent D; 4.50 min 95% Solvent C, 5% Solvent D; 5.50 min 95% Solvent C, 5% Solvent D. Method G: Mass Spectrometer: Finnigan LCQTMDuo, ESI. TSP HPLC System: (Pump: TSP SpectraSYSTEM® P4000; Detector: TSP SpectraSYSTEM® UV2000, 220nm and 254 m; Autosampler: TSP Spectra- SYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser) . Detection: 220nm and 254nm. Analytical column: YMC ODS-AQ 4.6 x 50 mm S3μ, Waters Corporation. Solvent C: 10 mM Ammonium Acetate in H?0. Solvent D: 10 mM Ammonium Acetate in Acetonitrile, 1.0% IPA. Flow Rate: 2.0 mL/min. Gradient Program: 0.00 min 75% Solvent C,25% Solvent D; 0.02 min 75% Solvent C, 25% Solvent D; 4.00 min 5% Solvent C, 95% Solvent D; 4.30 min 5% Solvent C, 95% Solvent D; 4.50 min 75% Solvent C, 25% Solvent D; 5.50 min 75% Solvent C, 25% Solvent D.
Method H: Mass Spectrometer: Finnigan LCQTMDuo, ESI. TSP HPLC System: (Pump: TSP SpectraSYSTEM® P4000; Detector: TSP SpectraSYSTEM® UV2000, 220nm and 254 m; Autosampler: TSP SpectraSYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser) . Detection: 220nm and 254nm. Analytical column: Zorbax Extend C18 Rapid Resolution®, 50 x 4.6 mm, 3.5μ, 80A, Agilent Tech- nologies. 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 Program: 0.00 min 95% Solvent C, 5% Solvent D; 0.02 min 95% Solvent C, 5% Solvent D; 4.00 min 5% Solvent C, 95% Solvent D; 4.30 min 5% Solvent C, 95% Solvent D; 4.50 min 95% Solvent C, 5% Solvent D;. 5.50 min 95% Solvent C, 5% Solvent D.
Method I: ' Mass Spectrometer: Finnigan LCQTMDuo, ESI. TSP HPLC System: (Pump: . TSP Spec- 5 traSYSTEM® P2000; Detector: TSP SpectraSYSTEM® . UV2000, 220nm and 254 m; Autosampler: TSP SpectraSYSTEM® AS3000; Degasser: TSP SpectraSYSTEM® Model SCM1000 Solvent Degasser) . Detection: - 220nm and 254nm. Analytical column: Zorbax Extend C18 Rapid-0 Resolution®, .50 x 4.6 mm, 3.5μ, 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 Program: 0.00 min 95% Solvent A, 5% Solvent B; 0.02 5- min 95% Solvent A, 5% Solvent B; 4.00 min 5% Solvent A, 95% Solvent B; 4.30 min 5% Solvent A, 95% Solvent B; 4.50 min 95% Solvent A, 5% Solvent B; 5.50 n • • < 95% Solvent A, 5% Solvent B.
Method 'J:. Mass spectrometers:" LCQTMDuo 0- • and LCQTMDeca. Pump: Series 1100, Quat pump model '- G1311A, Agilent Technologies. Detector: Series • 1100, Column .model G1216A, Agilent Technologies. Autosampler: Series 1100, ALS model G1313A, Agilent Technologies. Degasser: Series 1100, Degasser 5 model G1322A, Agilent Technologies. ELSD: Sedex model 75, Sedere. Analytical column: YMC ODS-AQ 4.6 x 50 mm S3μ, Waters Corporation. Solvent A: H20, 0.01% HFBA, 1.0% IPA. Solvent B: Acetonitrile, 0.01%HFBA, 1.0% IPA. Flow Rate: 2.0 mL/min. 0 Gradient Program: 0.00 min 95% Solvent A, 5%
Solvent B; 0.02 min 95% Solvent A, 5% Solvent B; 4.10 min 5% Solvent A, 95% Solvent B; 4.30 min 5% Solvent A, 95% Solvent B; 4.50 min 95% Solvent A, 5% Solvent B; 5.50 min 95% Solvent A, 5% Solvent B. Purity based on 220 nm wavelength channel.
Figure imgf000089_0001
Example 1
2-{ [4 '- (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino} -propionic acid
Figure imgf000089_0002
Example 1A
4- (2, 3-Dichloro-phenyl) -pyridine
Following the procedure of (Fujita et al . (1995) Tetrahedron Lett . 36 (29) : 5247-5250) , a stirred solution of 4-tributylstannanyl-pyridine (5.0 g, 13.6 mmol) in 60 mL of anhydrous toluene, lithium chloride (8.24 g, 194 mmol), 1, 2-dichloro-3- iodo-benzene (2.65 mL, 9.71 mmol) was made. The mixture was then degassed several times by bubbling nitrogen through the solution via syringe and then trans-dichlorobis (triphenylphosphine) palladium(II) was added. The mixture was heated under nitrogen atmosphere at reflux for 96 hours. The reaction mixture was then allowed to cool to ambient temperature and partitioned between toluene and brine. The organic layer was separated and saturated KF was added and the mixture was stirred for 24 h. The organic layer was filtered, and condensed in vacuo to provide Example 1A (1.76 g, 80.1%) as a white solid. 1H NMR (CDC13, 400 MHz) 5 8.7 (dd, =1.56, -. 4.69 Hz, 2H) , 7.53 (dd, J=1. 56, 7.81 Hz, IH) , 7.34 ' (s, L.56, 4.29 Hz, 2H) , 7.29 (t, J=8 Hz, IH) , 7.21 (s, J=1.56, 7.42 Hz, IH)
Figure imgf000090_0001
Example IB
4- (2, 3-Dichloro-phenyl) -pyridine M-oxide
A flask was charged with 1A (1.50 g, 6.69 mmol) and 15 mL DCM and placed in an ice bath and cooled to 0°C. Next, mCPBA (1.62 g, 9.37 mmol) was added and the mixture stirred for 16 h, warming to ambient temperature. After 16 h a second bolus of mCPBA (1.62 g, 9.37 mmol) was added at ambient tem- . perature and the mixture stirred for 2 h. The mix- ■ ture was partitioned between DCM and saturated sodium metabisulfite (2X25 mL) , followed by extraction of the organic layer with saturated sodium bi- ' carbonate/brine. The organic layer was dried over sodium sulfate, filtered,' and concentrated in vacuo . The crude product was purified via silica gel flash chromatography, eluting with 5% 7 N methanolic ammonia/DCM, . to provide Example IB (1.60 g, 99%) as a white solid. 2H -NMR (CDC13, 400 MHz) δ 8.29 (d,
J=7.4 Hz, 2H) , 7.56 (dd, J=1.6, 8.2 Hz, IH) , 7.4 (d, • , J=l Λ Hz, 2H) , 7.32 (t, J=7.8 Hz, 7H) , 7.24 (dd, J=2.0, 7.8 Hz, IH) . •
Figure imgf000091_0001
Example IC
2-Chloro-4- (2, 3-dichloro-phenyl) -pyridine
To a flask under nitrogen was added IB (1.90 g, 7.91 mmol) and phosphorus oxychloride (22.3 g, 185.3 mmol). A reflux condenser was placed on . the flask and the mixture. was heated to.90 °C with stirring for 18h. The mixture was allowed to cool to ambient temperature and concentrated under reduced pressure. The remaining slurry was poured over ice chips and neutralized with saturated sodium bicarbonate. The aqueous phase obtained was extracted with CH2C12 (3X50 L) . The combined organic layers were dried (Na2S04) and concentrated in vacuo to provide the title compound (1.10 g, 7.9 mmol, 54% yield) as an orange solid. lH NMR (CDC13, 400 MHz) δ 8.48 (d, J=5.06 Hz, IH) , 7'.56 (dd, J=1.17, 7.41 Hz, IH), 7.39 (s, IH) , 7.31 (t, J=8.19 Hz, 3H) , 7.3 (dd, J=1.56, 5.07 Hz, IH) , 7.22 (dd, J=1.17, 7.02 Hz, IH) . HPLC (Method A) Rt=3.06 min (92.5 area%, 220 nm) .
Figure imgf000092_0001
Example ID
4 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1, 2 ' ]bipyridinyl-4-carboxylic 'acid methyl ester
To a 25 mL sealable tube was added IC (1.00 g, 3.87 mmol) and ethyl isonipecotate (6.08 g, 38.68 mmol). The tube was placed under nitrogen, sealed and heated with stirring to 145°C for 24 h. The mixture was cooled to ambient temperature. The mixture was purified by flash chromatography on silica gel eluting with 10% 7N methanolic ammonia/- DCM to provide the title compound (0.890g, 3.87 mmol, 61%). XH NMR (CDC13, 400 MHz) δ 8.23 (d, J=5.08 Hz, IH) , 7.49 (m, IH) , 7.23 (m, 2H) 6.65 (s, IH) , 6.62 (m, IH) , 4.26 (m, IH) , 4.15 (q, J=6.9 Hz, 2H) , 3.00 (m, 2H) , 2.55 (m, IH) , 2.01 (m, 2H) , 1.8 (m, 2H) , 1.26 (t, J=7.22 Hz, 3H) .
Figure imgf000093_0001
Example IE
4 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4-carboxylic acid
To a stirred solution of ID (0.090 g,
0.246 mmol) in THF (2 mL) was added LiOH monohydrate (0.0062 g, 0.259 mmol) dissolved in 1 mL water. The mixture was stirred for 24 h, concentrated under vacuum and acidified by adding 2.0 eq of HCl (0.1 N) lowering the pH to 3.5. The solution was concentrated by in vacuo to provide the title compound IE (0.085g, 98%) as a light brown solid with salts present. XH NMR (CDClj, 400 MHz) δ 8.59 (s, IH) , 8.25 (d, J=5.08 Hz, IH) , 7.5 (m, IH) , 7.26 (t, J=7.61 Hz, IH) , 7.21 (m, HI), 6.67 (s, IH) , 6.65 (d, J=5.08 Hz, IH) , 4.24 (m, 2H) , 3.05 (m, 2H) , 2.62 (m, IH), 2.05 (m, 2H) , 1.83 (m, 2H) . HPLC (Method A) Rt=2.10 min (87.5 area%, 220 nm) . LCMS (ESI+ Method F) m/z 351 (M+), 279.2 (M-2C1) ; Rt=2.65 min (220 nm, 90 area%) .
Figure imgf000094_0001
Example IF
2-{ [4 '-(2-, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,2' Jbipyridinyl- -carbonyl] -amino} -propionic - acid methyl ester
To a stirred solution of IE in DMF (6 mL) was added L-alanine HCl (0.0507 g, 0.363 mmol), H0Bt.H20 (0.0490 g, 0.363 mmol), NMM (0.04 mL, 0.363 mmol), and diisopropyl ethylamine (0.0367 g, 0.363, mmol). After stirring for 5 min, EDCI (0.0564 g, 0.363 mmol) was added and the mixture was stirred for 24 h. Next, 20 mL water was added and the mixture extracted with EtOAc (3X15 L) , The combined organic phases were dried over sodium sulfate, , filtered, and concentrated to obtain the product (0.242 mmol, 61%) as an off-white , solid. XE NMR . ' . (CDC13, 400 MHz) δ 8.23 (d, J=5.08 Hz, IH) , 7.48 (m, IH) , 7.26 (t, <J=7.61 Hz, IH) , 7.2 (m, IH) , 6.66 (s, IH), 6.63 (m, IH) , .6.14 (d, J=7.03 Hz, IH) , 4.61 (m, IH) , 4.37 (m, 2H) , 3.76 (s, 3H) , 2.93 (m, 2H) , 2.4
(m, IH) , 1.94 (m, 2H) , 1.79 (m, 2H) , 1.41 (d, J=7.03 Hz, 3H) .
Figure imgf000095_0001
Example IG •
2-{ [4 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1, 2 ' ]bipyridinyl-4-carbonyl] -amino} -propionic acid
To a stirred solution of IF (0.065 g,
0.1-49 mmol) in THF (5 mL) was added LiOH monohydrate (0.019 g, 0,447 mmol, dissolved in 2 mL water) and stirred for 14 h. The crude product was concentrated in vacuo and acidified tό'pH 2 by dropping in, 0.1N HCl until a white precipitate formed. The solid -was partitioned between methylene" chloride (20 mL) and 0.1' N aqueous HCl (10 mL) . The phases -were separated, and the aqueous phase' was re-extracted' with methylene chloride (2x10 mL) . The combined ' organic phases were dried over sodium sulfate, filtered, and concentrated to provide the 'title compound (0.045 g, 0.1-49 mmol, 72%) as a white solid. XH NMR (DMSO- g, 400 MHz" δ 12.37 (s, IH) , 8.14 (d, - J=5.08 Hz, IH) , 8.08 (d, J=7.42 Hz, IH) , 7.68 (m, IH), 7.42 (t, J=7.81 Hz, IH) , 7.36 (m, IH) , 6.8 (s, IH) , 6.62 (m, IH) , 4.32 (m, 2H) , 4.15 (m, IH) , 2.83 (m, 2H) , 2.43 (m, 2H) , 1.69 (m, 2H) , 1.52 (m, 2H) , 1.22 (d, J=7.42 Hz, 3H) . HPLC (Method B) Rt=2.22 min (90.5area%, 220 nm. ) LCMS (ESI+ Method F) m/z 422.1 (M+) , Rt=2.19 min (220 nm, 90.5 area%) .
Figure imgf000096_0001
Example 2
3-(l-Benzyl-lH-imidazol-4-yl) -2-{ [4 '- (2, 3-dichlorophenyl) -3, 4, 5, 6-tetrahydro-2H- [1, 2 ' ]bipyridinyl-4- ' carbonyl] -amino} -propionic acid
, Example 2, was prepared, according to the same reaction protocol as. described for, Example 1 substituting. L-I^-benzyl histidine methyl ester di- hydrochloride for L-alanine, methyl ester HCl. The procedure provided the title . compound (0.036 mmol,. 8%) as a white solid. XH NMR (DMS0-d, 400 MHz) δ 8.91 (s, IH) , 8.09 (d, J-5.85 Hz, IH) , 7.64 (m, IH) , 7.43' (m, 9H) , 7.06 (s, IH) , 6.84 (m, IH) , 5.37 (q, J-14.85, 2.34 Hz, 2H) , 4.73 (m, IH) , 4.23 (m, 2H) , 3.29 ( , IH) , 3.08 (m, 2H), ■ 2 .5 , (m, IH), 1.81 (m, ..IH), .1.61 (m, 2H) , 1.49 -(-m,- IH) . HPLC (Method C)
Rt=2.11 min (100.0 area%, 220 nm) . LCMS (ESI-Method E) m/z 576.1 (M-2H)", Rt=2.64 min (220 nm, 95.-5 area%) .
Figure imgf000097_0001
Example 3
2-{ [6'- (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,2'] bipyridinyl-4-carbonyl] -amino} -propionic. acid
' Example 3 was prepared according to the same reaction protocol as described for Example 1 substituting 2-tributylstannanyl-pyridine for 4-tri- butylstannanyl-pyridine. The procedure resulted in the. title compound (0.0'80 g, 0.190 mmol, 41%) as a ' white solid-. XH NMR (DMS0-d6, 400 MHz) δ 12.44 (s, IH), 8.08 (d, J=7.42 Hz, H) , 7.66 (m, IH) , 7.6 (m, IH) , 7.43 ( , 2H) , 6.86 (d, J=8."59 Hz, -IH) , 6.8 (d, ■ J-7..42 Hz, IH) , 4.32 ( , 2H),"4.15 (m, IH), 2.83' (m, 2H),' 2.44 ( , IH) , 1.71 (m, 2H), 1.53 (m, 2H) , 1.23 - (d, J=7.42 Hz, 3H) . HPLC (Method' B) Rt=2.78 min "' (96.1 area%, 220 nm) . LCMS (APCI+ Method D) m/z 422.1 (M+) , Rt=2.81 min (220 nm, -98.0 area%) .-
Figure imgf000098_0001
Example 4
2-{ [6'- (2, 3-Dichloro-phenyl) -3 '-methyl-3, 4,5,6- tetrahydro-2H- [1,2'] bipyridinyl-4-carbonyl] -amino} - propionic acid
Example 4 was prepared according to the same reaction protocol as described for Example 1 substituting 4A for 1A. The procedure resulted in the title compound (0.020 g, 0.046 mmol, 26%) as a white solid. XH NMR (DMS0-d6, 400 MHz) δ 12.39 (s,
IH) , 8.08 (d, J=7.42 Hz, IH) , 7.66 (m, IH) , 7.58 (d, J=7.42 Hz, IH) , 7.5 (m, IH) , 7.42 (t, J=7.81 Hz, IH) , 7.14 (d, J=7.42 Hz, IH) , 4.18 ( , IH) , 3.5 (m, 2H) , 2.69 (m, 2H) , 2.31 (m, IH) , 2.30 (s, 3H) , 1.74 (m, 3H) , 1.23 (m, 5H) . HPLC (Method B) : Λt=3.11 min (89.0 area%, 220 nm) . LCMS (APCI+ Method D) m/z 436.1 (M+) , Rt=3.13 min (220 nm, 93.4 area%) .
Figure imgf000098_0002
Example 4A
2- (2, 3-Dichloro-phenyl) -5-methyl-pyridine
To a stirred solution of 2-bromo-5-methyl- pyridine (1.00 g, 5.81 mmol) and 2,3-dichloro phen- ylboronic acid (1.33 g, 6.98 mmol) in DME (5.8 L) was added potassium carbonate (1.21 g, 8.7 mmol). The mixture was degassed by bubbling nitrogen with a syringe for 5 min through the mixture, followed by addition of Pd(PPh3)4 (0.672 g, 0.58 mmol). A reflux condenser was attached to the flask and the mixture -• heated to 90 °C for 48 h. The mixture was cooled to ambient temperature and partitioned betwee ethyl acetate and brine. The organic phase was washed with brine (3X20 mL) and dried over sodium sulfate, . filtered, and concentrated in vacuo. The resulting- oil was purified by flash chromatography on silica '' gel eluting' with 1:1 ethyl acetate: hexane to provide the title compound' (0.380 g, 5'.81 mmol, 27% yield) as a light yellow oil whi.ch solidified upon standing- ■" to an off with solid.
Figure imgf000099_0001
(CDC13, 400 MHz) δ 8.54 ' (m, IH) , 7.58 (m, IH) , 1.5- (s, 2H) , 7.44 (dd, J=1.56, 7.42 Hz, IH) , 7.43 (m, IH) , 7.27 (t, J=7.81 Hz, IH) , 2.40 (s, 3H) .
Figure imgf000100_0001
Example 5
2-{ [4 ' - (2, 3-Dichloro-phenyl) -3 ' -methyl-3, 4, 5, 6- tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino}- propionic acid/2-{ [4 ' -'(2, 3-Dichloro-phenyl) -5 ' - methyl-3, 4, 5, 6-tetrahydro-2H- [1,2'] bipyridinyl-4- carbonyl] -amino} -propionic acid
Figure imgf000100_0002
Example 5A
4- (2, 3-Dichloro-phenyl) -3-methyl-pyridin-n-oxide
To a stirred solution of 4-bromo-3-methyl- pyri.dine N-oxide (0.985 g, 5.2 mmol) (prepared from 4-bromo-3-methyl-pyridine in the same manner as in Example IB) and 2,3-dichloro phenylboronic acid (1.0 g, 5.2 mmol) in DME (5.8 mL) was added potassium carbonate (0.869 g, 6.3 mmol). The mixture was degassed by bubbling nitrogen with a syringe for 5 min through the mixture, followed by addition of Pd- (PPh3) (0.606 g, 0.52 mmol). A reflux condenser was attached to the flask and the mixture heated to 90 °C for 48 h. The mixture was cooled to ambient temperature and partitioned between ethyl acetate and brine. The organic layer was washed with brine (3X20 mL) and dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting oil was purified by flash chromatography on silica gel eluting with 4% 7 N methanolic ammonia/DCM to provide the title compound (0.483 g, 5.24 mmol, 36% yield) as a white solid. XR NMR (CDC13, 400 MHz) δ-8.18 (s, IH) , 8.14 (m, IH) , 7.57 (m, IH) , 7.33 (t, J=8 Hz, IH) , 7.15 (m, IH) , 7.08 (d, J=6.64 Hz, IH) , 2.10 (s, 3H) .
Figure imgf000101_0001
Example 5B
2-{ [4 '-(2, 3-Dichloro-phenyl) -3' -methyl-3, 4,5, 6- tetrahydro-2H- [1,2' ] bipyridinyl-4-carbonyl] -amino}- propionic acid/2-{ [4 ' - (2, 3-Dichloro-phenyl) -5 ' - methyl-3, 4, 5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4- carbonyl] -amino} -propionic acid
Example 5B (as a 3;2 mixture of regioiso- mers) was prepared according to the same reaction protocol as described for Example 1 substituting 5A for IB. The procedure resulted in the title compound
(0.060 g, 0.222 mmol, 62%) as a white solid. Η NMR
(DMS0-d6, 400 MHz) δ 12.38 (s, 1.5H), 8.15 (d, J=5.47 Hz, IH) , 8.08 (d, J=7.42 Hz, IH) , 8.01 (s, 1.5H), 7.69 (m, 1.5H), 7.44 (m, 1.5H), 7.27 (m, 1.5H), 6.79 (d, J=5.08 Hz, IH), 4.19 (m, 4.5H), 3.38 (m, 3. OH), 2.74 (m, 1.5H), 1.92 (s, 3. OH), 1.84 (s, 1.5H), 1.71 (m, 5. OH), 1.52 (m, 1.0H), 1.23 (m, 4.5H). HPLC (Method A) : Rt=2.24 min (40.1area%, 220 nm) , 2.38 min (58.0 area%, 220 nm) . LCMS (APCI+ Method D) m/z
436.1 (M+) , Rt=2.27 min (220 nm, 41.5 area%) , Rt=2.41 min (220 nm, 58.5 area%) .
Figure imgf000102_0001
Example 6 '■
2-{ [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino} -propionic acid
Example 6 was prepared according .to the same reaction protocol as described for Example 1 substituting 3-tributylstannanyl-pyridine for 4-tri- butylstannanylpyridine . The procedure 'resulted in the title compound (0.103-g, 0.246 mmol, 61%) as a white powder. 1H NMR (DMSO-d6, 400 MHz) δ 12.4 (s, IH) , 8.14 (d, J=2.73 Hz, IH) , 8.11 (d, J=7.42 Hz, IH) , 7.61 (m, 2H), 7.4 (m, 2H)', 6.9 (d, J=8.98 Hz, IH) , 4.36 (m, 2H) , 4.17 (m, IH) , 2.88 (m, 2H) , 2.44 (m, IH) , 1.73 (m, 2H) , 1.53 (m, 2H) , 1.24 (d, J=7.42 Hz, 3H) . HPLC (Method B) , Rt=2.54 min (95.0 area%, 254 nm) . LCMS (APCI+ Method D) m/z 422.1 (M+) , Rt=2.28 min (220 nm, 95.5 area%) .
Figure imgf000103_0001
2- { [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4-carbonyl] -amino} -propionic acid
Figure imgf000103_0002
Example 7A.
5 ' -Bromo-3, 4,5, 6-tetrahydro-2H- [1,3'] bipyridinyl-4- carboxylic acid ethyl ester
To a 250 mL sealable tube was "added 3,5- dibromo pyridine (5.00 g, 21.1 mmol), ethyl iso- nepicotate (16.0 g, 106 mmol) and cesium carbonate (7.22 g, 22.2 mmol). The-■ tube was vented with N2 and ' sealed. The mixture was heated to 150°C for 72 h then cooled to ambient temperature. The crude prod- uct was dissolved in toluene and filtered. The or- ' ganic layer was concentrated in vacuo and the residue was purified by flash chromatography on silica gel eluting with 1:1 EtOAc/Hexane to provide the title compound (3.95g, 60%) as a light yellow oil. XH NMR (CDC13, .400 MHz) δ 8.21 (d, J=2 . 13 Hz, IH) ,
8.1 (d, J=1.95 Hz, IH) , 7.3 (t, J=2.34 Hz, IH) , 4.18 (q, J=7.03, 14.43Hz, 2H) , 3.66 (m, 2H) , 2.89 (m, 2H) , 2 . 48 (m, IH) , 2 . 04 (m, 2H) , 1 . 87 (m, 2H) , 1 . 28 (t, J=7 . 21 Hz , 3H) .
Figure imgf000104_0001
5 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1, 3 ' ]bipyridinyl-4-carboxylic acid ethyl ester
To a stirred solution of 7A (0.800 g, 2.55 mmol) in 10 mL toluene was added 2, 3-dichlorophenyl boronic acid (0.975 g, 5.11 mmol) and cesium carbonate (1.33 g, 5.62 mmol). The mixture was degassed with nitrogen for 10 min, then Pd(PPh3)4 (0.148 g, 0.128 mmol) was added. The mixture was heated to 85°C for 18 h then cooled to ambient temperature. The crude product was partitioned between toluene and brine. The organic layer was washed (2 X 30 mL) with brine and concentrated in vacuo . The resulting oil was purified by flash chromatography on silica gel eluting with 2:1 EtOAc/Hexane to provide the title compound (0.295 g, 30.5%) as a light oil which solidified upon standing to a white solid. XH NMR (CDC13 400 MHz) δ 8.33 (d, J=2 . 13 Hz, IH) , 8.1 (d, J=1.56 Hz, IH) , 7.51 (dd, J=1.95, 7.80 Hz, IH) , 7.27 (t, J=3.9 Hz, IH) , 7.23 (m, 2H) , 4.2 (q, J=7.02, 14.04 Hz, 2H) , 3.71 (m, 2H) , 2.91 (m, 2H) , 2 . 49 (m, IH) , 2 . 05 (m, 2H) , 1 . 9 (m, 2H) , 1 . 28 (t, =7 . 41 Hz , 3H) .
Figure imgf000105_0001
.5 2-{-[5 '- (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- • [1,3'] bipyridinyl-4-carbonyl] -amino} -propionic acid .
Example 7C was prepared according to the , . same reaction protocol as described for Example 1 substituting 7B for ID. The procedure resulted in
10 the title compound (0.037 g, 0.286 mmol, 30.7%). as a' white solid. 1H NMR (DMSO-d6, 400 MHz) δ 12.46 (s, IH) , 8.35 (s, IH), 8.15 (d, J=7.41 Hz, . IH) , 7.98 (s, IH) , 7.71 (d, J=6.63 Hz, IH) , 7.44 (m, 82H) , 7.36
• '■ (s-, - IH) , 4.19 (m, IH) , 3.86 (m, 2H) , 2.79 (m, 2H) , .
15 2.39 (m, 2H) , 1.74 (m, 4H) , 1.26' (d, J=7.02 Hz, 3H) . '■ HPLC (Method C) Rt=2.19 min (100.0 area-%, 220 nm. ) LCMS (ESI+ Method G) m/z 422.2 (M+) , Rt=2.46 min (220 nm, 93.6 area% . )
Figure imgf000106_0001
Example 8
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [5 ' - (2, 3-dichlorophenyl) -3,4,5, 6-tetrahydro-2H- [1,3' ] bipyridinyl-4- carbonyl] -amino} -propionic acid
Example 8 was prepared according to the same reaction protocol as described for Example 7 substituting L-^-benzyl histidine methyl ester di- hydrochloride for L-alanine methyl ester HCl. The procedure resulted in the title compound (0.058 g, 0.286 mmol, 32.1%) as a white solid. 2H NMR (DMSO- d6, 400 MHz) δ 8.34 (m, IH) , 8.07 (d, J=7.8 Hz, IH) , 7.99 (s, IH) , 7.72 (m, 2H) , 7.34 ( , 8H) , 6.89 (s, IH), 5.13 (s, 2H) , 4.42 (m, IH) , 3.78 (m, 2H) , 2.89 (m, 4H) , 2.31 (m, IH) , 1.57 (m, 3H) , 1.23 (m, IH) HPLC (Method C) Rt=2.11 min (93.2 area%, 254 nm) . LCMS (ESI+ Method G) m/z 578.3 (M+) , Rt=2.77 min (220 nm, 100.0 area% . )
Figure imgf000107_0001
Example 9 •'•
3-Benzoylamino-2-{ [5 ' - (2, 3-dichloro-phenyl) -3,4,5,6- tetrahydro-2H- [1,3'] bipy idinyl-4-carbonyl] -amino }- propionic acid '■
Example" 9 was prepared' according to the same reaction protocol as • described for 'Example 7 substituting L-2-amino-3-benzoylamino-propionic acid methyl ester HCl for L-aianine methyl ester HCl. - The procedure resulted in the title compound (0.062 ' g, 0.285 mmol, 41%) as a white solid. ltt NMR CDMSO-" d6, 400 MHz) δ 12.68 (s, IH) , 8.87 (m, IH) , 8.55 (d, ' J=7.02 Hz, IH) , 8.47 (d,' -J=2.34 ' Hz, IH) , 8.22 (s, IH) , 7.98 (s, -IH) , 7.89 (d, J=7.02 Hz, 2H) , 7.8 (m, ' IH) ,' 7.5 (m, 5H), 4.38' (m, IH) , 3.99 (m,' 2H) , 3.64 (m, 2H) , 2.97 (m, 2H) , 2.55 (m, IH) , 1.8 (m, 2H) , 1.61 (m, 2H) .. HPLC (Method C) Rt=2.20 min (96.5 area%, 220 nm) . LCMS (ESI-Method G) m/z 539.0 (M- 2H)~, Rt=2.68 min (220 nm, 100.0 area%.)
Figure imgf000108_0001
2- [ (5 ' -Bromo-3, 4,5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4-carbonyl) -amino] -propionic acid
Example 10 was prepared according to the same reaction protocol as described for Example 1 substituting 7A for IE. The procedure resulted in the title compound (0.019 g, 0.351 mmol, 15.2%) as a white solid. XH NMR (DMSO-d6, 400 MHz) δ 12.46 (s, IH) , 8.29 (d, J=2.73 Hz, IH) , 8.14 (d, .7=7.41 Hz,
IH) , 8 (d, J=1.95 Hz, IH) , 7.52 (t, =2.14 Hz, IH) , 4.18 (m, IH) , 3.84 (m, 2H) , 2.79 (m, 2H) , 2.39 (m, IH) , 1.74 (m, 2H) , 1.62 (m, 2H) , 1.26 (d, J=7.41 Hz, 3H) . Analytical. HPLC (Method C) Rt=1.69 (100 area!, 220 nm) . LCMS (ESI-Method G) m/z 353.9 (M-2H) " Rt=2.77 min (220 nm, 100.0 area!.)
Figure imgf000109_0001
Example 11
3-Benzoylamino-2- [ (5 ' -bromo-3, 4, 5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4-carbonyl) -amino] -propionic acid
Example 11 was prepared according to the same reaction protocol as described for Example 10 substituting L-2-amino-3-benzoylamino-propionic acid methyl ester HCl for L-alanine methyl ester HCl. The procedure resulted in the title compound as a white solid (0.110 g, 0.350 mmol, 66.0%). XH NMR (DMSO-de, 400 MHz) δ 12.7 (s, IH) , 8.53 (t, J=6.04 Hz, IH) , 8.28 (d, J=2.34 Hz, IH) , 8.16 (d, J=8.19 Hz, IH) , 7.99 (d, J=1.95 Hz, IH) , 7.8 (d, J=7.8 Hz, 2H), 7.51 (m, 4H) , 4.47 (m, IH) , 3.81 (m, 2H) , 3.61 (m, 2H) , 2.81 (m, 2H) , 2.4 (m, IH) , 1.77 (m, 2H) , 1.58 (m, 2H) . Analytical HPLC (Method C) Rt=2.01 min (99.4 area!, 220 nm) . LCMS (ESI-Method G) m/z 473.0 (M-2H)", Rt=2.28 min (220 nm, 100.0 area!) .
Figure imgf000110_0001
Example 12 ■
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (5 ' -bromo-3, 4, 5, 6- tetrahydro-2H- [1,3' ] bipyridinyl-4-carbonyl) -amino] - propionic acid
Example 12 was prepared according to the same reaction protocol as described for Example 10 substituting L-I^-benzyl histidine methyl ester di- hydrochloride for L-alanine methyl ester HCl. The procedure resulted in the title compound (0.101 g, 0.351- mmol, 57.0%) as a white solid. XH NMR (DMSO- d6, 400 MHz) δ 8.27 (d, =2.34 Hz, IH) , 8.06 (d, J=7.8 Hz, IH) , 8 (d, J=1.56 Hz, IH) , 7.67 (s, IH) , 7.5 (t, J=2.14 Hz, IH) , 7.33 (m, 3H) , 7.22 (m, 2H) , 6.87 (s, IH) , 5.13 (s, 2H) , 4.39 (m, IH) , 3.78 ( , 2H) , 2.87 (m, IH) , 2.77 (m, 3H) , 2.32 (m, IH), 1.68 (m, IH) , 1.56 (m, 3H) . Analytical HPLC (Method C) Rt=2.01 min (96.0area%, 220 nm) . LCMS (ESI-Method G) m/z 548.5 (M+Na)+, Rt=3.01 min (220 nm, 100.0 area!) .
Figure imgf000111_0001
Example 13
2-[ (3,4,5,6-Tetrahydro-2H-[l,3' ] bipyridinyl-4- carbonyl) -amino] -propionic acid
Figure imgf000111_0002
Example 13A
3,4,5, 6-Tetrahydro~2H- [1,3'] bipyridinyl-4-carboxylic acid ethyl ester
To a flask was added 7A (1.00' g, 3.19 " mmol), dioxaήe (13' mL) , triethyl amine '(0.97 g, 9.58 • • mmol) and pinacol borane (0.613 g., 4.79"mmol). The mixture was degassed with N2 for 15 min and Pd2Cl2- (dppf) dcm (0.132 g, 0.16 mmol) was added. The flask was heated to 90°C for 12 h and cooled to ambient temperature. The crude product was concen- rated in vacuo and purified by flash chromatography on silica gel eluting with 15% 7'N methanolic ammonia/CM to provide the title compound (400 mg, 45%) 1H NMR (DMSO-d6, 400 MHz) δ 8.31 (d, J=2.73 Hz, IH) , 8.09 (m, IH) , 7.18 (m, 2H) , 4.16 (dd, J=l . 02 , 14.04 Hz, 2H) , 3.67 (m, 2H) , 2.85 (m, 2H) , 2.46 (m, - Ill -
IH) , 2.05 (m, 2H) , 1.89 (m, 2H) , 1.28 (t, J=7.21 Hz, 3H) .
Figure imgf000112_0001
Example 13B
2-[ (3,4,5,6-Tetrahydro-2H-[l,3']bipyridinyl-4- carbonyl ) -amino] -propionic acid
To a flask was added 13A (0.240 g, 1.02 mmol) 5 mL THF and LiOH monohydrate (0.13O g, 3.07 mmol) dissolved in 3 mL water. The mixture was stirred for 18 h and acidified to pH 1 v/ith 0.1 N HCl. All solvents were removed in vacuo and the acid obtained was treated with L-alanine methyl ester HCl as described in Example IF. The resulting methyl ester was hydrolyzed as described in Example IG. The crude product obtained was purified by C-18 reverse phase HPLC eluting with a gradient of 5-95% CH3CN containing 0.1% TFA to provide the title compound (0.006g, 0.49 mmol, 5.0%) as a white solid. (XH NMR (DMS0-d6, 400 MHz) δ 8.42 (d, J=2.73 Hz, IH) , 8.23 (d, J=7.41 Hz, IH) , 8.1 (d, J=5.07 Hz, IH) ,
7.94 (m, IH), 7.69 (m, IH) , 4.18 (m, IH) , 3.95 (m, 2H) , 2.93 (m, 2H) , 2.47 (m, IH) , 1.78 ( , 2H) , 1.62 (m, 2H) , 1.26 (d, J=7.41 Hz, 3H) . Analytical HPLC (Method C) Rt=1.37 min (52.3 area!, 220 nm) , 1.40 min (47.0 area!, 220 nm) . LCMS (ESI-Method G) m/z 276.1 (M-1H)_, Rt=1.32 min (220 nm, 100.0 area!.) •
Figure imgf000113_0001
Example 14
' 3-(l-Benzyl-lH-imidazol-4-yl)-2-['(3,4,5,6- tetrahydro-2H- [1,3'] bipyridinyl-4-carbonyl) -amino] - propionic acid
Example 14 was prepared according to'the • " same reaction protocol as described for Example 13 substituting L-Z^-benzyl histidine methyl ' ester' di- hydrochloride for L-alanine methyl ester HCl. The procedure resulted in the title compound (0.030 g, 0.48 mmol, 14.0%) as a white solid. Analytical HPLC (Method C) Rt=1.65 min (90.4 area!, 220 nm) . LCMS (ESI-Method G) m/z 432.0 (M-2H)", Rt=2.02 min (220 : nm, 100.0 area!) .
Figure imgf000114_0001
Example 15
N- [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,3'] bipyridinyl-4-yl] -3-methyl-succinamic acid
Figure imgf000114_0002
Example 15A
5 ' -Bromo-3, 4,5, 6-tetrahydro-2H- [1,3' ] bipyridinyl-4- ylamine
To a flask was added 3,5-dibromo pyridine (2.00 g, 8.44 mmol), 4-amino piperidine (0.888 g, 8.86 mmol), and cesium carbonate (2.89 g, 8.86 mmol) . The mixture was heated under N2 to 155 °C for 24 h. The mixture was cooled to rt and diluted with toluene (30 mL) , filtered, and concentrated in vacuo and the crude product obtained was purified by column chromatography on silica gel eluting with 20% 7 N methanolic ammonia/DCM to provide the title compound (0.440 g, 20.5%) as a yellow oil. LCMS (ESI+Method G) m/z 322.2 (M+) , Rt=2.57 min (220 nm, 100.0 area!) .
Figure imgf000115_0001
5'- (2, 3-Dichloro-phenyl) -3, 4, 5-, 6-tetrahydro-2H- [1, 3 ' ]bipyridinyl-4-ylamine
Example 15B was prepared according to the same reaction protocol as described for Example 7B substituting 15A for 7A. The procedure resulted in the title compound (0.160 g, 42.0!) as an off-white solid. LCMS (ESI+ Method G) m/z 256.3 (M+) , Rt=1.72 min (220 nm, 100.0 area!).
Figure imgf000115_0002
Example 15C
N- [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4-yl] -3-methyl-succinamic acid tert-butyl ester
Example 15C was prepared according to the same reaction protocol as described for Example 1 substituting 15E for IE and 15G for L-alanine methyl ester to provide the title compound (0.145 g, 95!) as an off-white solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.33 (d, J=3.12 Hz, IH) , 8.1 (d, J=1.56 Hz, IH) , 7.51 (m, IH) , 7.27 (t, J=l .8 Hz, IH) , 7.24 (d, L.95 Hz, IH) , 7.23 (m, IH) , 5.79 (m, IH) , 3.97 (m, IH) , 3.7 (m, 2H) , 2.98 ( , 2H) , 2.65 (m, 2H) , 2.32 (m, 2H), 2.05 (m, 2H) , 1.57 (m, 2H) , 1.45 (s, 9H) , 1.17 (d, =6.63 Hz, 3H. )
Figure imgf000116_0001
Example 15D
N- [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1, 3 ' ]bipyridinyl-4-yl] -3-methyl-succinamic acid
To a stirred solution of 15C (0.065 g, 0.132 mmol) in 2 mL DCM was added 0.5 L TFA. The. mixture, was stirred for 24 h and concentrated in vacuo. The residual oil was partitioned between EtOAc and brine. The organic layer was separated and dried over sodium sulfate, filtered, and concen- rated in vacuo to provide the title compound (0.035 mg, 60.0!) as an off-white solid. 1H NMR (DMS0-d6, 400 MHz) δ 12.2 (s, IH) , 8.4 (s, IH) , 8-.06 (s, • IH) , 7.81 (m, 2H), 7.5 (m, 3H) , 3.81 (m, 9H) , 2.96 (m, 2H) , 2.62 (m, IH) , 2.46 (m, IH) , 2.22 (m, IH) , 1.81 (m, 2H) , 1.48 (m, 2H) , 1.02 (d, J=6.63 Hz, 3H) HPLC (Method C) Rt=2.08 min (94.6 area!, 220 nm) . LCMS (ESI-Method G) m/z 448.7 (M-2H) + Rt=3.77 min (220 nm, 100.0 area!) .
Figure imgf000117_0001
Example 15E
4-Benzyl-3-acetyl-oxazolidm-2-one
The procedure was adapted from Levy et al. (1998) J. Med. Chem . 42:199-223. 4-Benzyl-3-pro- pionyl-oxazolidin-2-one was prepared as follows. To an oven dried 2 L flask under N2 was added (R) -4- benzyl-oxazolidin-2-one (30.0 g, 169.3 mmol) and THF (300 L by canula, dry, degassed with N2 for 30 min) . The flask was cooled in a dry ice/acetone bath to
-78°C (internal probe temperature). Next was added n-BuLi (10.84 g, 169.3 mmol, 2.41 M in hexanes) dropwise over a 30 min period to maintain reaction temperature below -68 °C. After the addition was complete the mixture was stirred for 30 min and propionyl chloride (17.2 g, 186 mmol) was added over 15 min and the mixture stirred for 1 h. The reacion was allowed to warm to rt over 1 h and quenched by pouring into a 1 L separatory funnel containing 300 mL of saturated aqueous NH4C1. The mixture was par- itioned with EtOAc (600 mL) , separated and the organic layer was washed sequentially with 200 mL saturated aqueous NaHC03 and 200 mL brine. The organic layer was dried over Na2S0, filtered, and concen- trated in vacuo to give the title compound (42.18 g, 107!) as a light colored oil.
Figure imgf000118_0001
Example 15F
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 anhydrous THF (250 mL) and diisopropylamine (14.3g, 141 mmol). The flask was cooled to 0°C in an ice bath, and n-BuLi (9.06 mL, 2.54 M, 141 mmol) was added over 10 min and the flask was cooled to -78 °C in a dry ice/acetone bath. To the resulting LDA solution was added a solution of 15E (30.0 g, 128.6 mmol) in 40 mL dry THF over 30 min. The yellow solution was stirred at -78°C for 2 h. Bromoacetic acid tert-butyl ester (67.7 g, 347 mmol) was added dropwise over 20 min and the mixture was stirred for 30 min at -78 όC . The mixture was warmed to 0°C by placing the flask in a water-ice bath for 20 min. The mixture was quenched by pouring into 1 L separatory funnel containing 200 mL of sat. aq NH4C1. The mixture was diluted with EtOAc (300 mL) , the phases were separated and the organic phase was washed sequentially with 1 N HCl (2X100 mL) , sat. aq NaHC03 (2X100 mL) , and brine (2X10& mL) . The organic layer was dried over Na2S04, concentrated and puriied on a Biotage 65 column (9:1 hexane: MTBE) . The title compound was obtained as a white crystalline solid (29.1 g, 65!) .
Figure imgf000119_0001
Example 15G
5 2-Methyl-succinic acid 4-tert-butyl ester
To a 1 L flask was added Example 15F (29.0 g, 83.5 mmol) and THF/water (300 mL, 4:,1 mixture). The mixture was cooled to 0°C in an ice bath and 30! v H202 (17.0 g, 501 mmol, in water) was added over 10
10 min with stirring. Nex was added LiOH (2 M in water, 4.73 g> 117. mmol) and the reaction was stirred for 4 h in an ice- bath. :The reaction was quenched by careful dropwise addition of Na2S03- (2.73 M) over 1 h, resulting in slight gas evolution. The
1.5' THF was removed in vacuo and the- resulting suspenion, was extracted with DCM (400 L) . ■ The aqueous phase was reserved (contained desired product) and the DCM was washed with 0.1 N NaOH (100 mL) . The organic phase was dried, filtered, and concentrated in vacuo
20 to afford the chiral auxiliary (reusable) . The com- ined aqueous phase was cooled in an ice bath and acidified with 2 N HCl to pH 4. The cloudy solution was extracted with EtOAc (300 mL) , the EtOAc layer was dried with Na2S04, filtered, and concentrated to
25 give the crude product as a colorless oil, which solidified to a white solid upon standing. The product was purified by a Biotage 40 column, eluting with 30! EtOAc/hexanes, to provide the title compound (13.4 g, 85!): [α]D=-6.56° (-7.0 ° published) (c 0.86, CHC13, 9 mg/mL), contains minuscule chiral auxiliary which alters the rotation to the + direction. (Lit: Davies et al . (1998) J. Chem . Soc , Perkin Trans . 1 17:2635-2644).
Figure imgf000120_0001
Example 16
N- [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,3' ]b pyπdinyl-4-yl] -3-methy L-succinamic/ - [5 ' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4-yl] -2-methyl-succinamic acid
Example 16 was prepared as in Example IG substituting 15C for IE. The procedure provided the title compounds as an off-white solid. 1H NMR (DMSO- d6 r 400 MHz) δ 12.09 (s, IH) , 8.36 (d, J=2 . 13 Hz, IH) , 8 (d, J=1.95 Hz, IH) , 7.86 (m, IH) , 7.71 (m, IH) , 7.45 (m, 2H) , 3.78 (m, 3H) , 2.92 (m, 2H) , 2.69 (m, IH) , 2.43 (m, IH) , 2.15 (m, IH) , 1.8 (m, 2H) , 1.48 (m, 2H) , 1.05 (d, J=7.02 Hz, 3H) , 1.02 (d, J=7.02 Hz, 3H) . Analytical HPLC (Method C) Rt=2.08 min (92.3 area!, 220 nm) . LCMS (ESI-Method G) m/z 448.7 (M-2H)" Rt=3.77 min (220 nm, 100.0 area!).
Figure imgf000121_0001
Example 17
2, 3-Dichloro-3 ' -2 [ (piperidine-4-carbonyl) -amino] propionic acid
Figure imgf000121_0002
Example 17A
2 , 3-Dichloro-3 ' -nitro-biphenyl
A solution of 2, 3-dichloro-iodobenzene (6.00 g, 22.0 mmol) in 60 mL toluene and 20 mL eth- anol was treated with 3-nitro-phenylboronic acid
(4.22 g, 25.3 mmol) and Na2C03 (2 N in H20, 30.0 mL) . Then mixture was purged with N2 for 5 min and Pd- (PPh3) (1.02 g, 0.879 mmol) was added. The mixture was heated to 90 °C for 24 h and cooled to ambient temperature and concentrated in vacuo . The residual oil was partitioned with Et20 (80 mL) and washed with dilute brine solution (3x50 mL) , dried (Na2S04) , filtered, and concentrated under reduced pressure. The resulting white solid was recrystallized from hexane and to give the title compound (2.30 g, 39!) as a fluffy white solid. H NMR (CDC13, 400 MHz) δ 8.29 ( , 2H) , 7.76 (m, IH) , 7.63 (t, J=l .61 Hz, IH) , 7.55' (m, IH) , 7.31 (t, J=7.81 Hz, IH) , 7.26 (m, IH.)
Figure imgf000122_0001
Example 17B
2, 3-Dichloro-3 ' -amino-biphenyl
To a solution of 17A (2.30 g, 8.58 mmol) in acetic acid (100 mL) was added zinc dust (5.61 g, 85.8 mmol). The mixture was stirred for 2 h, diluted with DCM (40 mL) , and filtered through Celite. The solution was partitioned with NaHCOj and extracted with DCM (3 X 25 mL) , dried (Na2S04) , filtered, and concentrated under reduced pressure to provide the title compound (1.80 g, 88%) as a light yellow colored oil. 1H NMR (CDC13, 400 MHz) δ 7.44 (m, IH) , 7.22 (m, 2H) , 6.78 (m, IH) , 6.71 (m, 2H)., 3.73 (s, 2H) .
Figure imgf000123_0001
Example 17C
2, 3-Dichloro-3 ' -iodo-biphenyl
A suspension of 17B (4 g, 21.44 mmol) in 3N H2S04 (0.15 M) at 0°C was treated with a solution of NaN02 (0.869 g, 12.60 mmol) in water (2 mL) for 1 ■■'-'■' h. Then KI (2.09 g. 12.60 mmol) and urea (151 mg, ' 2.52 mmol) in water (2 mL)- were added, stirred at - 0°C for 1 h and allowed to warm to ambient tempera- ..- '• ture over 2 h. The reaction mixture was' partitioned " with EtOAc. (50 mL) and washed with 20! NaHS03 (3x20 '•• mL) , then saturated NaHC03 solution (3x20 mL) . The "' organic layer was separated,' dried ,(Na2S0) , filtered, and concentrated under reduced pressure to-' provide the title compound (1.02 .g, 70!) as a light red oil. XH NMR (CDC13, 400 MHz) δ 7.74 (m, 2H) , 7.48 (m, IH) , 7.39 (m, IH) , 7_.25 (t, J=7.81 Hz, IH) , 7.19 .(m, 2H. )
Figure imgf000124_0001
Example 17D ■'
2, 3-Dichloro-3 ' -piperidine-4-carboxylic acid ethyl ester
To a solution of 17C (0.850 g, 2.44 mmol) in 5 mL toluene was added Cs2C03 (1.19 g, 3.65 mmol) and ethyl isonipecotate (0.460 g, 2.92 mmol). The mixture was purged with N2 for 5.min and racemic- BINAP, (0.228 g, 0.365 mmol) and Pd2 (dba),3 (0.112 g, '■ 0.122 mmol) were added and the mixture heated to 100°C for 18 h. After cooling to ambient temperature, the mixture was partitioned between dilute brine (30 mL) and toluene (50 mL) . The organic layer was washed (3X20 L) with dilute brine and concentrated in vacuo. The residual oil was puri- : fied by flash chromatography on silica gel eluting with 1:20 EtOAc/hexane to give the title compound (0.330 g, 36!) as a. light colored oil which solidified upon standing. 1H NMR (CDC13, 400 MHz) δ 7.41 (m, IH), 7.29 (m, IH) , 7.21 (m, 2H) , 6.95 (m, 2H) , 6.84 (m, IH) , 4.14 (dd, J=7.41, 14.45 Hz, 2H) , 3.66 (m, 2H) , 2.82 (m, 2H) , 2.43 (m, IH) , 2.01 (m, 2H) , 1.88 (m, 2H) , 1.26 (t, J=7.41, 3H.)
Figure imgf000125_0001
Example 17E
2, 3-Dichloro-3' -piperidine-4-carboxylic acid
Example 17D (0.350 g, 0.925 mmol) was processed as in Example IE to provide the title compound (0.292 g, 90.1!) as a cream color powder. αH NMR (CDC13, 400 MHz) δ 11.36 (s, IH) , 7.45- (m, ' IH) , 7.32 (t, J=7.81 Hz, IH) , 7.23 (m, 2H) , 7.00 (m, 2H), 6.91 ( , 1H), 3.69 (m, 2H) , 2.88 ( , 2H) , 2.52- (m, IH) , 2.08 (m, 2H) , 1.94 (m, 2H) .
Figure imgf000125_0002
Example 17F
2, 3-Dichloro-3 ' -2 [ (piperidine-4-carbon'yl) -amino] - propionic acid methyl ester
Example 17E (0.150 g, 0.428 mmol) was processed as in Example IF to provide the title compound (0.1600 g, 85.8!) as a cream color powder. λH NMR (CDCI3, 400 MHz) δ 7.44 (m, IH) , 7.31 (t, J=7.81 Hz, IH) , 7.23 (m, 2H) , 6.96 (m, 2H) , 6.85 (m, IH) , 6.27 (d, <J=7.42 Hz, IH) , 4.61 ( , IH) , 3.78 (m, 5H) , 2.78 (m, 2H) , 2.31 (m, IH) , 1.95 (m, 4H) , 1.42 (d, J=7.03 Hz, 3H) .
Figure imgf000126_0001
Example 17G
■5 2, 3-Dichloro-3 ' -2 [ (piperidine-4-carbonyl) -amino] - propionic acid
Example 17F (0.230 g, 0.368 mmol) was processed as in Example IG to provide the title compound (0.104 g, 67.2%) as a cream color powder.
1.0 XH NMR (CDC13, 400 MHz) δ 12.45 (s, 1H),'8.18 (m,
IK), 7,.71 (m, IH) , 7.48 (t, J=l .8 Hz, IH) , 7.44 (m, IH), 7.44 (m, IH) , 7.35 (t> J=7.21 Hz, IH) , 7.07 (m, IH) , 6.98 (m, 2H) , 6.85 (d, J=l .02 Hz, IH) , 4.25 (m, IH) , 3.82 (m, 2H) , 2.78 (m,- 2H) , .2.41 (m, IH) ,. 1.82
15 (m, 4H) , 1.34 (d, J=7.41 Hz, 3H) . Analytical HPLC (Method A) Rt=2.73 min (100.0 area!, 220 nm) . LCMS " (APCI+" Method D) m/z 421.1 (M*")", "Rt=2.77 min" "(220 nm, 100.0 area!) .
Figure imgf000127_0001
Example 18
2, 3-Dichloro-4 ' -methyl-3 ' -2 [ (piperidine-4-carbonyl) - amino] -propionic acid
Example 18 was prepared as in Example 17
- by substituting 4~methyl-3-nitro-phenylboronic acid for 3-nitro-phenylboronic acid. The title compound (0.090 g, 48.6!) was obtained as a cream 'color powder. 1H NMR (CDC13, 400' MHz) δ 12.4 (s, IH) , 8.08 (d, J=7.42 Hz, IH) , 7.62 (m, 1H), '7.4 (t, J=7.81 Hz, ' IH) , 7.35 (m, IH) , 7.22 '(d J=7.42 Hz, 54H) , 6.98 ( , 2H) , 4.18 (m, ' IH) , 3.11 (m, 2H) , 2.6 (m, 2H) , 2.26 (m, 3H) , 1.76 (m, 4H) ,' 1.24 (d, J=7.42 Hz, 3H) . '■' HPLC (Method A) Rt=2..65 min (93.0 area!, 254 nm) . LCMS (APCI+ Method D) m/z 435.1 (M+) , Rt=3.36 min (254 nm, 90.6 area!) .
Figure imgf000128_0001
Example 19
2- ( { 1- [4- (2 , 3-Dichloro-phenyl) -thiazol-2-yl] - piperidine-4-carbonyl } -amino) -propionic acid
Figure imgf000128_0002
.Example 19A
l-Thiocarbamoyl-piperidine-4-carboxylic acid ethyl
■ ester
To a flask was added tri ethylsilyl-thio- isocyanate (5.01 g, 38.2 mmol), 60 mL dry DCM, eth- ylisonipecotate (2.00 g, 12.72 mmol) and 4-dimethyl- amino-pyridine (0.077 g, 0.636 mmol). The mixture was stirred at 40 °C for 72 h and cooled to ambient temperature. DCM (40 mL) was added and the mixture washed with 0.3 N HCl (3x30 mL) . The organic layer was separated, dried (Na2S04) , filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel eluting with 15! 7 N methanolic ammonia/DCM to pro- vide the title compound (1.30 g, 47.2!) as a light yellow solid.
Figure imgf000129_0001
Example 19B
■ 2-Chloro-l- (2, 3-dichloro-phenyl) -ethanone
To a mixture of 2-3-dichloro-acetophenone 5 in carbon tetrachloride (9 mL) was added sulfuryl chloride (3.93 g, 29.05 mmol). The mixture was heated to 45°C for 48 h. The mixture was cooled to ambient temperature and a second bolus of sulfuryl chloride (3.93 g, 29.05 mmol) was added. The mix-
10. ture was stirred at.45°C for 72 h. The mixture was cooled to ambient temperature and concentrated under reduced vacuum to provide the title compound. The light orange solid (5.80 g, 95!) was carried on without further purification. Analytical HPLC
15 (Method A) Rt=3.07 min (7 area!, 220 nm, starting material); Rt=3.35 min (68 area!, 220 nm, Example 19B)'.
Figure imgf000130_0001
Example 19C
1- [4- (2, 3-Dichloro-phenyl) -thiazol-2-yl] -piperidine- 4-carboxylic acid ethyl ester
To a solution of 19A (1.66 g, 7.68 mmol) in 10 mL THF at 0°C was added LiHMDS (1.41 g, 8.44 mmol) . The mixture was stirred for 10 min and 19B (2.40 g, 10.74 mmol) was added. The mixture was allowed to warm to ambient temperature over 3 h. The mixture was concentrated in vacuo and purified by flash chromatography on silica gel eluting with 1:4 EtOAc:hexane to provide the title compound (0.300 g, 0%; as a light yellow solid. Note: only pure fraction collected off column. 1H NMR (CDC13, 400 MHz) δ 7.79 (dd, J=1.56, 7.80, IH) , 7.39 (dd,
J=1.56, 7.80, IH) , 7.21 (t, J=l . 8 Hz, IH) , 7.06 (s, IH) , 4.17 (q, J=7.02, 14.43 Hz, 2H) , 4 (m, 2H) , 3.14 - (m, 2H) , 2.53 (m, IH) , 2.03 (m, 2H) , 1.87 (m, 2H) , 1.27 (t, J=7.21 Hz, 3H. )
Figure imgf000131_0001
Example 19D
. ..1- [4- (2, 3-Dichloro-phenyl) --thiazol-2-yl] -piperidine-
4-carboxylic acid
5.... Example 19D (0.30 g, 0.778 mmol) was pro-
' • cessed as in Example IE to provide the title compound (0.250 g, 90.0%) as a light yellow .colored.- ..powder.
Figure imgf000131_0002
0 ■: - Example 19E . ;
2- ( { 1- [4- (2, 3-Dichloro-phenyl) -thiazol-2-yl] - piperidine-4-carbόnyl} -amino) -propionic acid methyl ester
Example 19D (O.lOOg, 0.280 mmol) was pro-5 cessed as in Example IF to provide the title compound (0.100 g, 81.0%) as a light yellow colored powder.
Figure imgf000132_0001
Example 19F
2- ({ 1- [4- ('2, 3-Dichloro-phenyl) -thiazol-2-yl]- piperidine-4-carbonyl} -amino) -propionic acid.
'• ■ Example 19F (0.104 g, 67.2%) was prepared
' as described in Example IG substituting . Example 19E .: (0-.230 g, 0..368 mmol) for Example ID. 'The title compound (0.104 g, 67.2%) was obtained as a pale yellow powder. XH NMR (DMSO-d6, 400 MHz) δ 7.79 (dd, J=1.56, 7.80 Hz, IH) , 7.4 (dd, J=1.56, 7.80 Hz, IH) , ■ ' 7.22 .(-t, J=7.8 Hz, IH) , 7.06 (s, IH) , 4.18 (q,
J=7.02, 14.40 Hz, 2H) , 4.00. (m, 2H) , 3.14 (m, 2H) , 2.53 (m, IH), 2.03 (m, 2H) , 3..87 (m, 2H) , 1.27 (t, ' J=7.21 Hz, 3H) . Analytical HPLC (Method A) Rt=2.953 min .(96 area!, 220 nm) . LCMS (APCI+ Method D) m/z 428.1 (M+) , Rt=3.13.min (220 nm, 100.0 area!.)
Figure imgf000133_0001
Example 20
3- (l-Benzyl-lH-.imidazol-4-yl).-2- ( {1- [4- (2,3- dichloro-phenyl) -thiazol-2-yll -piperidine-4- carbonyl} -amino) -propionic acid
Example.19D (0.120 g, 0.336 mmol) was processed as in Example 19■• substituting ■L-i\J1-benzyl histidine methyl ester dihydrochloride for L-alanine methyl ester, to provide the title compound (0.100 g,- 50.9%) as a light yellow colored powder..- XH NMR
(DMSO-d6-, 4,00 MHz) δ 8.05 (d, J=7.81 Hz, IH) , 7.76" . (dd, J=1.56, 8.20 Hz, IH) , 7.67 (m, IH) , 7,59 ("dd, , J=1.56, 8.20, Hz, IH) , 7.37, ,(t, J=7-.81 Hz, IH) , ,7.3 (m, 4H) , 7.19 (d, J=6.64 Hz, 2H) , 6.84 (s, IH) , 5.1 (s, 2H) , 4.37 (m, IH) , 3.82 (m, 2H) , 3.01 (m, 2H) , 2.88 (-s, IH)-., 2.76 (s, IH)., 2.34 - {-s,- -1H-) , 1.68 -(s, - IH) , 1.54 (m, 2H) , 1.44 (s, IH) . Analytical HPLC (Method A) Rt=2.64 min (93.0 area!, 220 nm) . LCMS (APCI+ Method D) m/z 584.1 (M+) , Rt=2.72 min (220 nm, 100.0 area!) .
Figure imgf000134_0001
Example 21 '
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (1-m-tolyl- piperidine-4-carbonyl) -amino] -propionic acid
Figure imgf000134_0002
■ - Example 21A -
l-m-Tolyl-piperidine-4-carboxylic acid ethyl ester
A sealed tube charged with the ' 3-bromptol- uene (159 mg, 1.325 mmol), Pd2(dba)3 (30 mg, 0.0331 mmol, 2.5 mol %) , (2 ' -dicyclohexylphosphanyl-biphen- - yl-2-yl) -dimethyl-amine (Stre or Aldrich) (39 mg, 0.0994 mmol, 7.5 mol %) , ethylisonipecotate (0.245 mL, 1.59 mmol), and powdered dried K3P04 (393 mg, 1.855 mmol) in dry DME (1 mL) was purged under N2 (3X) and heated at 90°C for 5 h under N2 atmosphere. The reaction mixture was diluted with EtOAc (10 mL) and washed with water (2x4 mL) . The organic layer was dried (Na2S04) , filtered, and evaporated to dry- ness. The crude product was purified by flash chromatography on silica gel (Biotage Flash 40 s+) eluting with 10% acetone/hexane to provide the title compound (300 mg, 91%) as a straw color liquid. LCMS (APCI+; Method D) m/z 248, (M+H)+.
Figure imgf000135_0001
Example 2IB
1-m-Tolyl-piperidine- -carboxylic acid
A solution of Example 21A (298 mg, 1.205 mmol) in THF:MeOH (1:1, 6 mL) was treated with a solution of LiOH'H20 (76 mg, 1.807 mmol) in water (2 mL) at ambient temperature for 18 h. The solvents were removed under reduced pressure. The residue dissolved in water (5 mL) and washed with EtOAc (2X5 mL) to remove organic impurities. Then the aqueous layer was made acidic with 10% aq HCl. The product formed was soluble in aqueous acidic medium and water was removed by evaporating with CH3CN to provide the lithium salt of the title compound (159 mg, 50%). LCMS (APCI+; Method D) m/z 220, (M+H)+.
Figure imgf000136_0001
Example 21C
3-(l-Benzyl-lH-imidazol-4-yl)-2-[ (1-m-tolyl- piperidine-4-carbonyl) -amino] -propionic acid methyl ester
A solution of Example 21B (159 mg, 0.607 mmol), HOBt.H20 (139 mg, 0.9098 mmol) , L-Nx-benzyl histidine methyl ester dihydrochloride (242 mg, 0.728 mmol), and triethylamine (0.296 mL, 2.123 mmol), in dry DMF (3'mL) was treated with EDC (174 mg, 0.9098 mmol) at ambient temperature under N2 atmosphere for 18 h. The reaction mixture was diluted' with EtOAc (10 mL) , washed with water (2x5 mL) , dried (MgS04) , filtered, and evaporated to dry- ' ness. The residue obtained was purified by flash chromatography on "silica gel" (Alitech SEP""pack," 2O g) eluting with .2% MeOH/CH2Cl2 followed by 7% MeOH/- CH2C12 to provide the title compound (130 mg, 47% yield) as white solid. LCMS (APCI+; Method D) m/z 461, (M+H)+. Analytical HPLC (Method C) Rt=1.532 min (254 nm, 100 area!) .
Figure imgf000137_0001
Example 2ID
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (1-m-tolyl- piperidine-4-ca'rbonyl) -amino] -propionic acid
A solution of ester Example 21C (130 mg,
0.2823 mmol) in THF:MeOH (1:1 mixture, 5 mL) was ''■'treated with a solution of LiOH'H20 (14 mg, 0.3389 mmol) in water (1 mL)' at ambient temperature for 5 h. The solvents were removed, residue dissolved in . water (3 mL) and made acidic (pH 3-4) with formic . .acid. The product formed was soluble in- water at this stage and the water was removed under reduced pressure. The residue was crystallized with a small amount of water "(1-2 mL) . The solid formed was collected by filtration and dried under vacuum (oil pump) to provide the title ' compound (60 mg, 48%) as . a white powder. XH NMR (400 MHz, DMS0-d6) δ 8.02 (d, ' J=8.19 Hz, IH) , 7.67 (s, IH) , 7.36-7.29 (m, 3H) , 7.23-7.21 (m, 2H) , 7.07 (t, J=7.80 Hz, IH) , 6.87 (s, IH) , 6.73 (s, IH) , 6.71-6.69 (m, IH) , 6.57 (d,
.7=8.19 Hz, IH) , 5.13 (s, 2H) , 4.41-4.36 (m, IH) , 3.66-3.58 (m, IH) , 2.91-2.86 (m, IH) , 2.80-2.74 (m, IH), 2.64-2.54 (m, 2H) , 2.68-2.19 (m, IH) , 2.24 (s, 3H) , 1.70-1.65 (m, IH) , 1.62-1.46 (m, 3H) . LCMS (APCI+; Method D) m/z 501, 503, (M+H)+. Analytical HPLC (Method C) Rt=1.763 min. (220 nm, 100 area!) .
Figure imgf000138_0001
Example 22
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-ethyl- phenyl) -piperidine-4-carbonyl] -amino}-propionic acid
Figure imgf000138_0002
• Example 22A
1- (3-Ethyl-phenyl) -piperidine-4-carboxylic acid ethyl ester
The title compound (275 mg, 79.4!) was prepared by the method described in Example 21A, substituting l-bromo-3-methyl-benzene with 1-bromo- 3-ethyl-benzene (245 mg, 1.325 mmol). XE NMR (400 MHz, DMS0-d6) δ 7.10 (t, J=7.80 Hz, IH) , 6.77-6.72 (m, 2H), 6.61 (d, J=7.41 Hz, IH) , 4.08 (q, J=7.02 Hz, 2H) , 3.64-3.59 (m, 2H) , 2.76-2.69 ( , 2H) , 2.54- 2.51 (m, 2H) , 2.51-2.49 ( , 2H) , 2.51-2.49 (m, 2H) , 2.48-2.44 (m, IH) , 1.91-1.87 ( , 2H) , 1.69 ( 1.59 (m, 2H), 1.19 (t, J=l .02 Hz, 3H) , 1.15 (t, J=7.41 - Hz, 3H) . LCMS (APCI+; Method D) m/z 262, (M+H)+.
Figure imgf000139_0001
Example 22B
1- (3-Ethyl-phenyl) -piperidine-4-carboxylic acid
Example 22A (190 mg, 0.727 mmol) was hydrolyzed with LiOH*H20 (37 mg, 0.872 mmol) as described in Example 2IB to provide the title compound (210 mg, 87!) as an off-white color powder. LCMS (APCI+; Method D) m/z 234, (M+H)+.
Figure imgf000139_0002
Example 22C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-ethyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid methyl ester
The title compound was prepared from Example 22B (150 mg, 0.643 mmol). as described in Example 21C. LCMS (APCI+; Method D) m/z 476, (M+H)+.
Figure imgf000140_0001
Example 22D
3- (l-Benzyl-lH-imidazol-4-yl) -2- { [1- (3-ethyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid
■5 The title -compound can.be prepared accord-
: -ing to the method described in Example. 2-lD.
Figure imgf000141_0001
Example 23
3- ( l-Benzyl-lH-imidazol-4-yl ) -2- { [1- ( 3-isopropyl- phenyl ) -piperidine-4-carbonyl] -amino } -propionic acid
Figure imgf000141_0002
Example 23A
1- (3-Isopropyl-phenyl) -piperidine-4-carboxylιc acid ethyl ester
The title compound (150 mg, 40!) was pre- pared by the procedure described in Example 21A, substituting l-bromo-3-methyl-benzene with 1-bromo- 3-isopropyl-benzene (269 mg, 1.325 mmol). LCMS (APCI+; Method D) m/z 276, (M+H)+.
Figure imgf000142_0001
Example 23B
l-(3-Isopropyl-phenyl) -piperidine-4-carboxylic acid
Example 23A (340 mg, 1.235 mmol) was hydrolyzed with LiOH.H20 (78 mg, 1.852 mmol) as described in Example 21B to provide the title compound (300 mg, 84!) as a white powder. LCMS (APCI+; Method D) m/z 248, (M+H)+.
Figure imgf000142_0002
Example 23C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-isopropyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid methyl ester
The title compound (220 mg, 74! yield) was prepared from Example 23B (150 mg, 0.6065 mmol) as described in Example 21C. LCMS (APCI+; Method D) m/z 489, (M+H)+. Analytical HPLC (Method C) Rt=1.545 min (254 nm 97 area!) .
Figure imgf000143_0001
Example 23D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-isopropyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic' acid
The .title compound was prepared from Exam- • pie 23C (220 mg, 0.450 mmol) as described in Example 21D. Example 23D (88 mg, 39%) was obtained as a white powder. XH NMR (400 MHz, DMSO-d6) δ 8.01 (d, J=7.80 Hz, IH) , 7.67 (s, IH) , 7.37-7.29 (m, 3H) , 7.29-7.21 (m, 2H) , 7.12 (t, J=7.80 Hz, IH) , 6.87 (s, IH) , 6.77 (s, IH) , 6.71 (dd, Ji=8'.19 Hz, J2Λ . 6 Hz, IH) , 6.64 (d, J=7.41 Hz, IH) , 5.13 (s, '2H) , 4.41- ■ 4.35 (m, IH) , 3.68-3.57 (m, IH) , 22.91-2.87 (m, IH) , 2.83-2.74 (m, 2H) , 22.64-2.54 (m, 2H) , 2.27-2.20 (m, IH), 1.71-1.65 (m, IH) , 1.61-1. 5' (m, 3H)., 1.18 (d, J=7.02 Hz, 6H) . LCMS (APCI+; Method D) m/z 275(M+H)+. Analytical HPLC (Method C) Rt=1.975 min (220 nm, 95 area %) .
Figure imgf000144_0001
Example 24
3- (l-Benzyl-lH-imidazol-4-yl) -2- { [1- ( 3- ■ trif luoromethyl-phenyl ) - iperidine- --carbonyl] amino } -propionic acid
Figure imgf000144_0002
Example 24A ■ -
1- (3-Trifluoromethyl-phenyl) -piperidine-4-carboxylic acid ethyl ester
The title compound (165 mg, 58!) was prepared by the procedure described in Example 21A, substituting l-bromo-3-methyl-benzene with 1-bromo- 3-trifluoromethyl-benzene (303.76 mg, 1.325 mmol). LCMS (APCI+; Method D) m/z 302, (M+H)+.
Figure imgf000145_0001
Example 24B
1- (3-Trif luoromethyl-phenyl) -piperidine-4-carboxylic acid
Example 24A (270 mg, 0.896 mmol) was hydrolyzed with LiOH.H20 (57 mg, 1.344 mmol) as described in Example 21B to provide the title compound (165 mg, 58!) as a white powder. XR NMR (400 MHz, DMSO-de) δ 7.48-7.39 ( , 3H) , 7.18 (s, IH) , 3.73-3.65 (m, 2H), 3.06-2.94 (m, 2H) , 2.46 (m, IH) , 1.97-1.93 (m, 2H) , 1.77-1.71 (m, 2H) . LCMS (APCI ; Method D) m/z 274, (M+H)+.
Figure imgf000145_0002
Example 24C
3- (l-Benzyl-lH-imidazol-4-yl) -2- { [1- (3- trifluoromethyl-phenyl) -piperidine-4-carbonyl] amino} -propionic acid methyl ester
The title compound (150 mg, 48! yield) was prepared from Example 24B (165 mg, 0.6065 mmol) as described in Example 21C. LCMS (APCI+; Method D) m/z 515, (M+H)+. Analytical HPLC (Method C) Rt=1.452 min (220 nm, 98 area!) .
Figure imgf000146_0001
Example 24D ,. , 3-(l-Benzyl~lH-imidazol-4-yl)-2-{ [1-O- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - ' ■ ■ amino} -propionic acid
The title .compound was prepared from, Example 24C (150 mg, 0.292 mmol) as described in Example 21D. The title compound (73 mg, 50%) was obtained as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (d, J=7.80 Hz, IH) , 7.68 (s, IH) , 7.41 (t, J=7.80 Hz, IH), 7.37-7.27 ( , 3H) , 7.23-7.19 (m, 3H) , 7.13 (s, IH) , 7.03 (d, J=7.41 Hz, IH) , 6.87 (s, IH) , 5.13 (s, 2H) , 4.42-4.37 (m, IH) , 3.79-3.70 (m, 2H) , 2.92- 2.87 ( , IH) , 2.81-2.67 ( , 3H) , 2.34-2.27 (m, IH) , 1.73-1.68 (m, IH) , 1.61-1.43 (m, 3H) . LCMS (APCI+; Method D) m/z 502, (M+H)+. Analytical HPLC (Method C) Rt=2.378 min (220 nm, 100 area !) .
Figure imgf000147_0001
Example 25
3- (l-Benzyl-lH-imidazol-4-yl) -2- { [1- (3- dimethylamino-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid
Figure imgf000147_0002
Example 25A
1- (3-Dimethylamino-phenyl) -piperidine-4-carboxylic acid ethyl ester
The title compound was prepared by the procedure described in Example 21A, substituting 1- bromo-3-methyl-benzene with (3-bromo-phenyl) dimeth- ylamine (270 mg, 1.325 mmol). LCMS (APCI+; Method D) m/z 277, (M+H)+.
Figure imgf000148_0001
Example 25B
1- (3-Dimethylamino-phenyl) -piperidine-4-carboxylic acid
Example 2.5A (259 mg, 0.937 mmol) was- hydrolyzed with LiOH'H20 (59 g, 1.406 mmol) as described in Example 21B to provide the title compound (151 mg, 55!) as a white powder. LCMS (APCI+; Method D) m/z 249, (M+H)+.
Figure imgf000148_0002
Example 25C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- dimethylamino-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid methyl ester
The title compound (120 mg, 40.4 yield) was prepared from Example 25B (150 mg, 0.607 mmol) as described in Example 21C. LCMS (APCI+; Method D) m/z 490, (M+H)+. Analytical HPLC (Method C) Rt=1.562 min (220 nm, 96 area!) . - 148 -
Figure imgf000149_0001
Example 25D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- dimethylamino-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid
The title compound was, prepared from Example 25C (120 mg, 0.245 mmol) as described in Example 21D. The title compound (50 mg, 43!) was obtained as a white powder. XH NMR (400 MHz, DMSO-d6) δ 8.32 (s, IH) , 7.69 (d, =7.80 Hz, IH) , 7.58 (s, IH) ,
7.30-7.2 (m, 2H) , 7.16 (d, J=6.63 Hz, IH) , 6.94 (t, J=7.80 Hz, IH) , 6.77 (s, IH) , 6.20-6.12 ( , 2H) , 5.05 (s, 2H) , 4.23 (m, IH) , 3.58-3.49 (m, 2H) , 2.88- 2.83 (m, IH), 2.80 (s, 6H) , 2.73-2.61 (m, IH) , 2.56- 2.46 (m, 2H) , 2.18-2.13 ( , IH) , 1.67-1.57 (m, IH) , 1.54-1.39 (m, 3H) . LCMS (APCI+; Method D) m/z 476, (M+H)+. Analytical HPLC (Method C) Rt=1.715 min (220 nm, 100 area!) .
Figure imgf000150_0001
Example 26
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( {1- [3- (4-fluoro- phenoxy) -phenyl] -piperidine-4-carbonyl } -amino) - propionic acid
Figure imgf000150_0002
Example 26A
1- [3- (4-Fluoro-phenoxy) -phenyl] -piperidine-4- carboxylic acid ethyl ester
The title compound was prepared by the procedure described in Example 21A, substituting 1- bromo-3-methyl-benzene with l-bromo-3- (4-fluorophenyloxy) benzene (360 mg, 1.325 mmol). LCMS (APCI+; Method D) m/z 344, (M+H)+.
Figure imgf000151_0001
Example26B
1- [3- (4-Fluoro-phenoxy) -phenyl] -piperidine-4- carboxylic acid-
Example 26A (331 mg, 1.017 mmol) was - treated with LiOH*H20 (64 mg, 1.526 mmol) as described in Example 2IB to provide the title compound (180 mg, 59!) as a white powder. LCMS (APCI+; Method D) m/z 316, (M+H)+.
Figure imgf000151_0002
Example 26C
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( {1- [3- (4-fluoro- phenoxy) -phenyl] -piperidine-4-carbonyl} -amino) - propionic acid methyl ester
The title compound (130 mg, 40! yield) was prepared from Example 26B (191 mg, 0.607 mmol) as described in Example 21C. LCMS (APCI+; Method D) m/z 557, (M+H)+. Analytical HPLC (Method C) Rt=1.57 min (220 nm, 100 area!) .
Figure imgf000152_0001
Example 26D ■ -
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (4-fluoro- phenoxy) -phenyl] -piperidine-4-carbonyl } -amino) - propionic acid '
The title compound was prepared from Example 26C (130 mg, 0.234 mmol) as. described in Example 21D. Example 26D (70 mg, 55!) was obtained as a white powder. XH NMR (400 MHz, DMSO-d6) δ 8.03 (d, J-8.19 Hz, IH), 7.68 (s, IH) , 7.34-7.31 (m, 2H) ,
7.27-7.15 (m, 6H) , 7.06-7.02 (m, 2H) , 6.86 (s, IH) ,
- 6.69 (dd, J2=8.19 Hz, J2=1.95 Hz, IH) , 6.54 (m, IH) ,
6.29 (dd, Jι=7.41 Hz, 2=1.56 Hz, IH) , 5.12 (s, 2H) ,
4.41-4.36 (m, IH) , ' 3.67-3.57 (m, 2H) , 2.91-2.86 (m, IH) , 2.80-2.74 (m, IH) , 2.68-2.57 (m, 2H) , 2.28-2.22 (m, IH) , 1.68-1.63 (m, IH) , 1.58-1.45 (m, 3H) . LCMS (APCI+; Method D) m/z 543, (M+H)+. Analytical HPLC (Method C) Rt=2.417 min. (220 nm, 100 area!).
Figure imgf000153_0001
Example 27
3- ( l-Benzyl-lH-imidazol-4-yl) -2- { [1- ( 3-f luoro-5- trif luoromethyl-phenyl) -piperidine-4-carbonyl] - amino } -propionic acid
Figure imgf000153_0002
■Example 27A ■ '
(3-Fluoro-5-trifluoromethyl-phenyl) -piperidine-4- carboxylic acid ethyl ester
. The. title, compound- was prepared by the procedure described in Example 21A, substituting 1- bromo-3-methyl-benzene with l-bromo-3-fluoro-5-tri- fluoromethyl-benzene (328 mg, 1.325 mmol).
Figure imgf000154_0001
1- (3-Fluoro~-5-trifluoromethyl-phenyl) -piperidine-4- carboxylic acid
Example 27A (280 mg, 0.877 mmol) was treated with LiOH'H20 (55 mg, 1.315 mmol) as described in Example 21B to, provide the title compound (177.mg, 61!) as a -white powder. LCMS (APCI+; Method D) m/z 292, (M+H)+, (92 area %) . ■
Figure imgf000154_0002
Example 27C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-fluoro-5- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid methyl ester
The title compound (218 mg, 68%) was prepared from Example 27B (177 mg, 0.607 mmol) as described in Example 21C. LCMS (APCI+; Method D) m/z 533, (M+H)+.
Figure imgf000155_0001
Example 27D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-fluoro-5- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid
The title, compound was prepared from Example 27C (218 mg, 409 mmol) as described in Example 21D. Example 27D (173 mg, 82%) was obtained as a white powder. 1H NMR (400 MHz, DMS0-d6) δ 8.07 (d, J=7.80 Hz, IH) , 7.67 (s, IH) , 7.36-7.33 (m, 2H) ,
7.30-7.28 (m, IH) , 7.23-7.21 (m, 2H) , 7.05-7.00 ( , 2H), 6.87-6.84 (m, 2H) , 5.13 (s, 2H) , 4.42-4.36 (m, IH), 3.85-3.75 (m, 2H) , 2.91-2.87 (m, IH) , 2.83-2.74 (m, 3H), 2.36-2.30 ( , IH) , 1.71-1.66 ( , 1H0, 1.58- 1.40 (m, 3H) . LCMS (APCI+; Method D) m/z 519,
(M+H)+. Analytical HPLC (Method C) Rt=2.50 min (220 nm, 96.3 area!) .
Figure imgf000156_0001
Example 28
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 5-bis- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - amino}-propionic acid
Figure imgf000156_0002
Example 28A
1- (3, 5-Bis-trifluoromethyl-phenyl) -piρeridine-4- carboxylic acid ethyl ester
The title compound (386 mg, 79!) was prepared by the procedure described in Example 21A, substituting l-bromo-3-methyl-benzene with 1-bromo- 3, 5-bis-trifluoromethyl-benzene (388 mg, 1.325 mmol). XR NMR (400 MHz, DMS0-d6) δ 7.46 (s, 2H) , 7.27 (s, IH) , 4.08 (q, J=7.02 Hz, 2H) , 3.90-3.85 (m, 2H) , 2.98-2.92 ( , 2H) , 2.61-2.54 (m, IH) , 1.94-1.90 (m, 2H) , 1 . 67-1. 57 (m, 2H) , 1 . 19 (t, J=7 . 02 Hz, 3H) LCMS (APCI+; Method D) m/z 370 , (M+H) + .
Figure imgf000157_0001
Example 28B
1- (3, 5-Bis-trifluoromethyl-phenyl) -piperidine-4- carboxylic acid
Example 28A (380 mg, 1.029 mmol) was hydrolyzed with LiOH"H20 (86 mg, 2.058 mmol) as described in Example 21B to provide the title compound (347 mg, 98.82!) as a white powder. XH NMR (400 MHz, DMSO-de) δ 12.31 (s, IH) , 7.46 (s, 2H) , 7.27 (s, IH) , 3.89-3.85 (m, 2H) , 2.98-2.91 (m, 2H) , 2.47-2.44 (m, IH) , 1.94-1.89 (m, 2H) , 1.66-1.56 (m, 2H) . LCMS (APCI+; Method D) m/z 342, (M+H)+.
Figure imgf000158_0001
Example 28C
3-(l-Benzyl-lH-imidazol-4-yl)-2-{ [1- (3,5-bis- trif luoromethyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid methyl ester
The title compound (165 mg, 74! yield) was prepared from Example 28B (130 mg, 0.383 mmol) as described in Example 21C. XH NMR (400 MHz, DMSO-d6)
(d, J=7.41, IH) , 7.67 (s,.. IH) 44 2H.) , 7.36-7.33 (m, 2H) , 7.28-7.20 (m, 2H) , 6.88 (s, IH) , 5.13 (s, 2H) , 4.47-4.42 (m, IH) , 3.93-3.85 (m, 2H) , 3.55 (s, 3H) , 2.89-2.78 (m., .4H) , 2.39-2.33 (m, IH) , 1.75-1.70 (m, IH) , 1.64-1.59 (IH.) , 1.58-1.43 (m, 2H)-. LCMS (APCI+; Method D) m/z 583, (M+H)+.
Figure imgf000159_0001
Example 28D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 5-bis- t ifluoromethyl-phenyl) ~piperidine-4-carbonyl] - amino} -propionic acid
The title compound was prepared from Example 28C (160 mg, 0.275 mmol) as described in Example 21D. Example 28D (117 mg, 75%) was obtained as a white powder. aH NMR (400 MHz, DMSO-d6) δ 8.09 (d, J=7.80 Hz, IH), 7.67 (s, IH) , 7.44 (s, 2H) , 7.36-
7.33 (m, 2H), 7.29-7.27 (m, 2H) , 7.23-7.21 (m, 2H) , 6.87 (s, IH), 5.13 (s, 2H) , 4.42-4.37 ( , IH) , 3.93- 3.83 (m, 2H), 2.92-2.85 (m, 2H) , 2.81-2.74 (m, 2H) , 2.37-2.32 ( , IH) , 1.74-1.69 (m, IH) , 1.61-1.56 (m, IH) , 1.53-1.44 (m, 2H) . LCMS (APCI+; Method D) m/z
569, (M+H)+. Analytical HPLC (Method C) Rt=2.704 min (220 nm, 100 area!) .
Figure imgf000160_0001
Example 29
3- ( l-Benzyl-lH-imidazol-4-yl) -2- { [ 1- ( 2 -methyl - quinolin-6-yl) -piperidine-4-carbonyl] -amino } - , . propionic acid
Figure imgf000160_0002
Example 29A
1- (2-Methyl-quinolin-6-yl) -piperidine-4-carboxylic acid ethyl ester
The title compound was . prepared by the procedure described in Example 21A, substituting 1- bromo-3-methyl-benzene with 6-brpmo-2-methyl-quin- oline. Example 29A was used for- the preparation of Example 29B directly.
Figure imgf000161_0001
Example 29B
1- (2-Methyl-quinolin-6-yl) -piperidine-4-carboxylic acid
' Example' 29A (280 mg, 0.938 mmol) was treated with LiOH'H20 (59 mg, 1.408 mmol) as described in Example 21B to provide the title compound' (164 mg, 56%) as a yellow color solid. 1H NMR (400 MHz, DMSO-de) δ 8.73 (d, J=8.58 Hz, IH) , 8.30 (d, =9.36 Hz, IH) , 7.98 (d, d, ^=22.73 Hz, J2=9.36 Hz, IH) , 7.78 (d, J=8.56 Hz, IH) , 7.53 (d, =2.73 Hz, IH), 3.94-3.88 (m, 2H) , 3.07-3.01 (m, 2H) , 2.90 (s, 3H), 2.58-2.53 (m, IH) , 1.99-1.95 ( , 2H) , 1.73-1.63 '' (m, 2H) . LCMS (APCI+; Method D)' m/z 271, (M+H)+.
Figure imgf000162_0001
Example 29C
3- ( l-Benzyl-lH-imidazol-4-yl) -2- [ [1- (2-methyl- quinolin-6-yl) -piperidine-4-carbonyl] -amino } - propionic acid methyl ester
The title compound (50. mg, 16%) was prepared from Example 29B (163 mg, 0.607 mmol) as described in Example 21C. LCMS (APCI+; Method D) m/z 512, (M+H)+. Analytical HPLC (Method C) Rt=1.565 min (220 nm, 89- area!) .
Figure imgf000162_0002
Example 29D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (2-methyl- quinolin-6-yl) -piperidine-4-carbonyl] -amino}- propionic acid
The title compound was prepared from Example 29C (50 mg, 0.098 mmol) as described in Example 21D. The crude product obtained was purified by C- 18 Reverse phase HPLC using a gradient of 5-95! CH3CN/H20 containing 1% TFA to provide the title compound (62 mg, 92!) as a bright yellow powder. XH NMR (400 MHz, DMSO-d6) δ 9.19 (s, IH) , 8.69 (d, J=8.58 Hz, IH) , 8.27 (d, J=8.58 Hz, IH) , 7.99 (d, J=9.36 Hz, IH) , 7.90 (d, d, J2=2.24 Hz, J2=9.36 Hz, IH) , 7.77 (d, J=8.58 Hz, IH) , 7.466 (m, IH) , 7.43- 7.37 (m, 6H) , 5.39 (s, 2H) , 4.58-4.52 ( , IH) , 3.95- 3.86 ( , 2H) , 3.17-3.12 (m, IH) , 2.97-2.84 (m, 3H) , 2.83 (s, 3H) , 2.36-2.27 ( , IH) , 1.74-1.68 (m, IH) , 1.60-1.52 (m, 2H) , 1.45-1.40 (m, IH) . LCMS (APCI+; Method D) m/z 498, (M+H)+, Rt=1.78 min.
Figure imgf000164_0001
Example 30
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 4-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid
Figure imgf000164_0002
Example 30A
1- (3, 4-Dichloro-phenyl) -piperidine-4-carboxylic acid ethyl ester
The title compound (322 mg, 80.5!) was prepared by the procedure described in Example 21A, -substituting 1—bromo-3-methyl-benzene with -4-bromo- 1,2-dichloro-benzene (299 mg, 1,325 mmol). 1H NMR (400 MHz, DMSO-de) δ 7.37 (d, J= .97 Hz, IH) , 7.12 (d, J=3.12 Hz, IH) , 6.93 (d, d,
Figure imgf000164_0003
. 13 Hz, IH) , 4.07 (q, J=7.02 Hz, 2H) , 3.70-3.65 (m, 2H) , 2.85-2.79 (m, 2H) , 2.56-2.52 (m, IH) , 1.89-1.85 (m, 2H) , 1.64-1.58 (m, 2H) , 1.19 (t, J=7.41 Hz, 3H) . LCMS (APCI+; Method D) m/z 302, 304, (M+H)+.
Figure imgf000165_0001
Example 30B
1- (3, 4-Dichloro-phenyl) -piperidine-4-carboxylic acid
Example 30A (318 mg, 1.052 mmol) was treated with LiOH.H20 (88 mg, 2.105 mmol) as described in Example 21B to provide the title compound (276 mg, 96%) as a white powder. XE NMR (400 MHz, DMSO-de) δ 12.27 (s, IH) , 7.37 (d, J=8.97 Hz, IH) , 7.12 (d, J=3.12 Hz, IH) , 6.93 (d, d, ι=31.2 Hz, J2=8.97 Hz, IH) , 3.69-3.64 (m, 2H) , 2.84-2.78 (m,
2H) , 2.45-2.38 ( , IH) , 1.89-1.85 (m, 2H) , 1.64-1.54 (m, 2H) . LCMS (APCI+; Method D) m/z 274, 276, (M+H)+.
Figure imgf000166_0001
Example 30C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 4-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid methyl ester
The title compound (130 mg, 69! yield) was prepared from Example 30B (100 mg, 0.365 mmol) as described in Example 21C. 1H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J=7.41 Hz, IH) , 7.66 (s, IH) , 7.38-7.30 (m, 4H) , 7.22-7.19 (m, 2H) , 7.10 (d, J=2.73 Hz, IH) , 6.91 (d, d, Jι=2.73 Hz, J2=8.97 Hz, IH) , 6.87 (s, IH) , 5.13 (s, 2H) , 4.47-4.41 (m, IH) , 3.73-3.65 (m, 2H) , 3.55 (s, 3H), 2.89-2.79 (m, 2H) , 2.75-2.66 (m, 2H), 2.34-2.26 (m, IH) , 1.69-1.65 (m, IH) , 1.59-1.44 (m, 3H) . LCMS (APCI+; Method D) m/z 515, 517, (M+H)+.
Figure imgf000167_0001
Example 30D.
3-(l-Benzyl-lH-imidazol-4-yl)-2-{ [1- (3, 4-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid
The title compound was prepared from Example 30C (130 mg, 0.2522 mmol) as described in, Example 21D. Example.30D (93 mg, 73.54!). was obtained as a white powder. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (br s, IH), 8.14 (d, J=8.19 Hz, IH) , 7.40-7.35 (m, 5H) , 7.29-7.28 (m, 2H) , 7.13 (s, IH) , 7.09 (d,
J=2.73 Hz, IH) , 6.91 (d, d, Jx=3.12 Hz, J2=8.97 Hz, IH) , 5.25 (s, 2H), 4.49-4.43 (m, IH) , 3.73-3.63 (m, 2H) , 3.03-2.98 (m, IH) , 2.88-2.82 (m, IH) , 2.73-2.63 (m, 2H) , 2.29-2.28 (m, IH) , 1.67-1.61 (m, IH) , 1.55- 1.46 (m, 2H) , 1.43-1.38 (m, IH) '. ' LCMS' (APCI+;
Method D) m/z 501, 503, (M+H)+. Analytical HPLC (Method C) Rt=2.465 min (220 nm, 100 area!).
Figure imgf000168_0001
Example 31
3- ( l-Benzyl-lH-imidazol-4-yl) -2- { [1- (2 , 3-dichlorophenyl ) -piperidine-4-carbonyl] -amino } -propionic acid
Figure imgf000168_0002
Example 31A
1- (2, 3-Dichloro-phenyl) -piperidine-4-carboxylic acid. ethyl ester
The title compound was prepared by the procedure described in Example 21A, substituting 1- ■ bromo-3-methyl-benzene with l-bromo-2, 3-dichloro- benzene (200 mg, 0.885 mmol). The title compound (124 mg, 46!) was obtained as a yellow color liquid. XE NMR (400 MHz, DMSO-d6) δ 7.30-7.29 (m, 2H) , 7.14- 7.11 (m, IH) , 4.09 (t, J=7.02 Hz, 2H) , 3.25-3.20 (m, 2H) , 2.75-2.69 (m, 2H) , 2.48 (m, IH) , 1.96-1.92 (m, 2H), 1.79-1.69 (m, 2H) , 1.20 (t, J=7.02 Hz, 3H) . LCMS (APCI+; Method D) m/z 302, 304, (M+H)+.
Figure imgf000169_0001
Example 3IB
1- (2, 3-Dichloro-phenyl) -piperidine-4-carboxylic acid
Example 31A (120 mg, 0.397 mmol) was hy- drolyzed with LiOH.H20 (20 mg, 0.477 mmol) as described in Example 2IB to provide the title compound (100 mg, 96!) as a white powder. αH NMR (400 MHz, DMSO-de) δ 12.30 (br s, IH) , 7.30-7.28 (m, 2H) , 7.14- 7.12 ( , IH), 3.24-3.19 (m, 2H) , 2.74-2.68 (m, 2H) , 2.44-2.56 (m, IH) , 1.95-1.91 (m, 2H) , 1.77-1.68 (m, 2H) . LCMS (APCI+; Method D) m/z 274, 276, (M+H)+.
Figure imgf000169_0002
Example 31C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (2, 3-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid methyl ester
The title compound (135 mg, 72! yield) was prepared from Example 31B (100 mg, 0.365 mmol) as described in Example 21C. 1H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J=7.80 Hz, IH) , 7.68 (s, IH) , 7.35-7.27 (m, 5H) , 7.21-7.19 (m, 2H) , 7.14-7.12 (m, IH) , 6.89 (s, IH) , 5.13 (s, 2H) , 4.49-4.44 (m, IH) , 3.56 (s,
3H), 3.26-3.20 ( , 2H) , 2.86-2.77 (m, 2H) , 2.66-2.57 (m, 2H) , 2.31-2.23 (m, IH) , 1.73-1.67 (m, 2H) , 1.64-
1.59 (m, 2H) . LCMS (APCI+; Method D) m/z 515, 517, (M+H)+.
Figure imgf000170_0001
Example 31
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (2, 3-dichlorophenyl) -piperidine-4-carbonyl] -amino} -propionic acid
The title compound was prepared from Example 31C (130 mg, 0.2522 mmol) as ' described in Example 21D. Example 31D (78 mg, 62!) was obtained as a white powder. λE NMR (400 MHz, DMSO-d6) δ 8.07 (d, J=8.19 Hz, IH), 7.68 (s, IH) , 7.35-7,27 (m, 5H) , 7.22-7.20 (m, 2H) , 7.14-7.12 (m, IH) , 6.87 (s, IH) , 5.13 (s, 2H) , 4.44-4.38 ( , IH) , 3,26-3.19 (m, 2H) , 2.92-2.87 (m, IH) , 2.82-2.76 (m, IH) , 2.66-2.56 (m, 2H), 2.28-2.22 (m, IH) , 1.73-1.69 (m, 2H) , 1.62-1.57 (m, 2H) . LCMS (APCI+; Method D) m/z 510, 503, (M+H)+. Analytical HPLC (Method C) Rt=2.431 min (220 nm, 100 area!) .
Figure imgf000171_0001
Example 32
3- (l-Benzyl-lH-imidazol-4-yl) -2- { [ 1- ( 6, 7-dichlorobenzo [b] hiophen-5-yl) -piperidine-4-carbonyl] - amino } -propionic acid
Figure imgf000171_0002
Example 32A
1- (6, 7-Dichioro-benzo [b] thiophen-5-yl) -piperj dine-4- carboxylic acid ethyl ester
The title compound was prepared by the procedure described in Example 21A, substituting 1- bromo-3-methyl-benzene with 5-bromo-6, 7-dichlorobenzo [b] thiophene (Example 49D, 100 mg, 0.355 mmol). The title compound (46 mg, 36!) was obtained as a yellow color liquid. LCMS (APCI+; Method D) m/z 358, (M+H)+.
Figure imgf000172_0001
Example 32B
1- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -piperidine-4- carboxylic acid ethyl ester
Example' "32A (45 mg, 126 mmol)' was hydrolyzed with LiOH"H20 (6 mg, 0.151 mmol) as described in Example 2IB to provide the. title compound (30 mg, 72!). XH NMR (400 MHz, DMSO-d6) δ 7.88 (d, J=5.46 Hz, IH) , 7.67 (s, IH) , 7.50 (d, J=5.46 Hz, IH) , 3.26-3.23 (m, 2H) , 2.78-2.72 (m, 2H) , 2.45-2.38 (m, IH) , i.99-1.93 ( , 2H) , 1.82-1.73 (m, 2H) . LCMS (APCI+; Method D) m/z 330, 332, (M+H)4.
Figure imgf000173_0001
Example 32C
3-(l-Benzyl-lH-imidazol-4-yl)-2-{ [1- (6, 7-dichlorobenzo [b] thiophen-5-yl) -piperidine-4-carbonyl] - amino} -propionic acid methyl ester
The title compound (31, mg, 64%) was prepared from Example 32B (28 mg, 0.085 mmol) as described in Example 21C. LCMS (APCI+; Method D) m/z, 571, 573 (M+H)→.
Figure imgf000173_0002
Example 32D
3-(l-Benzyl-lH-imidazol-4-yl)-2-{ [1- ( 6, 7-dichlorobenzo [b] thiophen-5-yl) -piperidine-4-carbonyl] - amino} -propionic acid
The title compound (8 mg, 26.45%) was prepared from Example 32C (31 mg, 0.0542 mmol) as de- scribed in Example 2ID. XE NMR (400 MHz, DMSO-d6) δ 8.09 (d, J=7.80 Hz, IH) , 7.88 (d, J=5.46 Hz, IH) , 7.68 (d, J=8.58 Hz, IH) , 7.67 (s, IH) , 7.50 (d, J=5.46 Hz, IH) , 7.35-7.26 (m, 3H) , 7.22-7.20 (m, 2H), 6.89 (s, IH) , 5.13 (.s, 2H) , 4.46-4.40 (m, IH) , 3.29-3.23 (m, 2H) , 2.94-2.89 (m, IH) , 2.83-2.77 (m, IH), 2.71-2.59 ( , 2H) , 2.33-2.25 (m, IH) , 1.77-1.72 (m, 2H) , 1.68-1.62 (m, 2H) . LCMS (APCI+; Method D) m/z 557, 559, (M+H)+. Analytical HPLC (Method C) Rt=557, 559 min (220 nm, ' 100 area!) .
Figure imgf000175_0001
Example 33
3- ( l-Benzyl-lH-imidazol-4-yl) -2- { [ 1- ( 3 , 5-dichloro- phenyl) -piperidine-4 -carbonyl] -amino } -propionic acid
Figure imgf000175_0002
Example 33A
1- (3, 5-Dichloro-phenyl) -piperidine-4-carboxylic acid ethyl ester
The title compound (263 mg, 65.75!) was prepared by the procedure described in Example 21A, substituting l-bromo-3-methyl-benzene with 1-bromo- 3,5-dichloro-benzene (299 mg, 1.325 mmol). XE NMR (400 MHz, DMSO-de) δ 6.93 (d, J=1.95 Hz, 2H) , 6.82 (t, J=1.95 Hz, IH) , 4.07 (q, J=7.02 Hz, 2H) , 3.75- 3.70 (m, 2H) , 2.89-2.83 (m, 2H) , 2.58-2.52 (m, IH) , 1.89-1.84 (m, 2H) , 1.63-1.53 (m, 2H) , 1.19 (t, J=7.02 Hz, 3H) . LCMS (APC +; Method D) m/z 302, 304, (M+H)+.
Figure imgf000176_0001
Example 33B
1- (3, 5-Dichloro-phenyl) -piperidine-4-carboxylic acid
Example 33A (260 mg, 0.704 mmol) was hydrolyzed with LiOH"H20 (59 mg, 1.41 mmol.) as described in Example 21B to provide the title compound (225 mg, 95!). XE NMR (400 MHz, DMSO-d6) δ 12.28 (s, IH) , 6.92 (d, J L.95 Hz, 2H) , 6.82-6.80 (m, IH) , 3.74-3.69 (m, 2H) , 2.87-2.82 (m, 2H) , 2.47-2.42 (m, IH) , 1.88-1.84 (m, 2H) , 1.61-1.52 (m, 2H) . LCMS (APCI+; Method D) m/z 274, 276, (M+H)+.
Figure imgf000176_0002
Example 33C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 5-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid methyl ester
The title compound (156 mg, 83!) was prepared from Example 33B (100 mg, 0.365 mmol) as de- scribed in Example 21C. 1H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J=7.41 Hz, IH) , 7.66 (d, J=1.56 Hz, IH) , 7.37-7.27 (m, 3H) , 7.22-7.19 (m, 2H) , 6.91 (d, J=1.56 Hz, 2H) , 6.87 (d, J=1.17 Hz, IH) , 6.82 (t, J=1.56 Hz, IH), 5.13 (s, 2H) , 4.47-4.41 (m, IH) ,
3.78-3.70 (m, 2H) , 3.55 (s, 3H) , 2.87-2.76 (m, 3H) , 2.73-2.69 (m, IH) , 2.35-2.29 (m, IH) , 1.68-1.63 (m, 2H)., 1.58-1.41 (m, 3H) ."'. LCMS (APCI+; Method D) m/z 515, 517, (M÷H)+.
Figure imgf000177_0001
Example 33D
3-(l-Benzyl-lH-imidazol-4-yl)-2-{ [1- (3, 5-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid
The title compound was prepared from Exam- pie 33C (160 mg, 0.3104 mmol) as described in Example 21D. Example 33D (112 mg, 69%) was obtained as a white powder. lE NMR (400 MHz, DMSO-d6) δ 8.05 (d, J=7.80 Hz, IH) , 7.67 (s, IH) , 7.37-7.29 (m, 3H) , 7.22-7.20 (m, 2H) , 6.91 (s, 2H) , 6.86 (s, IH) , 6.82 (s, IH) , 5.13 (s, 2H) , 4.42-4.36 (m, IH) , 3.78-3.68 (m, 2H) , 2.91-2.86 (m, IH) , 2.86-2.72 (m, 3H) , 2.34- 2.28 (m, IH) , 1,68-1.63 (m, IH) , 1.56-1.40 (m, 3H) . . LCMS (APCI+; Method D) m/z 501, ' 503, (M+H)+. - 17,7 -
Analytical HPLC (Method C) Rt=2.553 min (220 nm, 95 area!) .
Figure imgf000178_0001
;. Example 34
2- [ (1-Benzo [1,3] dioxol-5-yl-piperidine-4-carbonyl) - amino] -3- (l-benzyl-lH-imidazol-4-yl) -propionic acid
Figure imgf000178_0002
Example 34A
1-Benzo [1,3] dioxol-5-yl-piperidine-4-carboxylic acid ' ethyl ester
The title compound was prepared by the procedure described in Example 21A, substituting 1- bromo-3-methyl-benzene with 5-bromo-benzo [l,3]diox- ole (500 mg, 2.487 mmol). The title compound (516 mg, 75%) was obtained as a yellow color solid.
XH NMR (400 MHz, DMSO-d6) δ 6.74 (d, J=8.58 Hz, IH) , 6.66 (d, J=2.34 Hz, IH) , 6.33 (d, d, J2=2.34Hz, J2=8.58 Hz, IH), 5.90 (s, 2H) , 4.07 (q, J=7.21 Hz, 2H) , 3.45-3.40 (m, 2H) , 2.67-2.61 (m, 2H) , 2.46-2.39 (m, IH) , 1.90-1.85 (m, 2H) , 1.70-1.61 (m, 2H) , 1.19 (t, J=7.21 Hz, 3H) . LCMS (APCI- ; Method D) m/z 278.2 , (M+H)+.
Figure imgf000179_0001
Example 34B
1-Benzo [1, 3] dioxol-5-yl-piperidine-4-carboxylic acid
Example 34A (500 mg, 1.803 mmol) was hydrolyzed with LiOH*H20 (91 mg, 2.164 mmol) as described in Example 2IB to provide the title compound (438 mg, 97%) as a white powder. XE NMR (400 MHz,
DMS0-d6) δ 12.86 (br s, IH) , 7.51-7.28 (m, 2H) , 7.05 (m, IH) , 6.11 (s, 2H) , 3.49 (m, 4H) , 2.67-2.59 (m, IH) , 2.23-2.04 (m, 4H) . LCMS (APCI+; Method D) m/z 250, (M+H)+.
Figure imgf000180_0001
Example 34C
2- [ (1-Benzo [1, 3] dioxol-5-yl-piperidine-4-carbonyl) - amino] -3- (l-benzyl-lH-imidazol-4-yl) -propionic acid . methyl ester
The title compound (80 mg, 41%) was prepared from Example 34B (100 mg, 0.401 mmcl) as described in Example 21C. X NMR (400 MHz, DMSO-de) δ 8.25 (d, J=7.80 Hz, IH) , 7.67 (d, J=1.56 Hz, IH) , 7.37-7.27 (m, 3H), 7.22-7.19 (m, '2H) , 6.87 (d, L.17 Hz, IH) , 6.75 (d, J-8.58 Hz, IH) , 6.65 (d, J=2.34 Hz, IH) , 6.33 (d, d, Jι=2.34 Hz, J2=8.58 Hz, IH) , 5.90 (s, 2H) , 5.12 (s, 2H) , 4.457-4.42 (m, IH) , 3.55 (s, 3H) , 3.45-3.40 (m, 2H) , 2.89-2.76 (m, 2H) , 2.57-2.40 (m, 2H) , 2.23-2.17 (m, IH) , 1.70-1.62 (m, ■ IH), 1.60-1.50 (m, 3H) . LCMS (APCI+; Method D) m/z 491, (M+H)+.
Figure imgf000181_0001
Example 34 D
2- [ (1-Benzo [1, 3] dipxol-5-yl-piperidine-4-carbonyl) - amino] -3- (l-benzyl-lH-imidazol-4-yl) -propionic acid
The title compound was prepared from Example 34C (80 mg, 163 mmol) as described in Example 29D. Example 34D (45 mg, 41%) was obtained as a white powder. ^Η NMR (400 MHz, DMSO-d5) δ 9.17 (s, IH) , 8.26 (m, IH) , 7.46 (s, IH) , 7.42-7.37 (m, 5H) , 6.85 (m, 2H) , 6.55 (m, IH) , 5.97 (s, 2H) , 5.3.8,(ε,
2H) , 4.59-4.52 (m, IH) , 3.51-3.39 (m, 211), 3.16-3.12 (m, IH) , 2.97-2.90 (m, IH) , 2.80-2.66 (m, 1,H) , 2.26- 2.14 (m, 2H) , 1.72-1.62 (m, 2H) , 1.56-1.48 (m, 2H) . LCMS (APCI+; Method D) m/z 477, (M+H)+. Analytical HPLC (Method C) Rt=1.682 min (220 nm, 100 area!).
Figure imgf000182_0001
Example 35
3- ( l-Benzyl-lH-imidazol-4-yl ) -2- { [1- ( 4-tert -butyl - phenyl) -piperidine-4 -carbonyl] -amino } -propionic acid
Figure imgf000182_0002
Example 35A
1- (4-tert-Butyl -phenyl ) -piperidlne-4-carboxylic acid, ethyl ester .
Example 35A was prepared by the procedure_ .de.S-cribed- in Example.21A, - substituting -l-b-romo-3- methyl-benzene with l-bromo-4-tert-butyl-benzene (250 mg, 1.173 mmol). The title compound (194 mg,
57!) was obtained as a yellow color solid. LH NMR
(400 MHz, DMSO-de) δ 7.20 (d, J=8.97 Hz, 2H) , 6.86 (d, J=8.97 Hz, 2H), 4.08 (q, J=7.02 Hz, 2H) , 3.59- 3.54 (m, 2H), 2.73-2.67 (m, 2H) , 2.47-2.41 (m, IH) , 1.91-1.86 (m, 2H) , 1.69-1.59 (m, 2H) , 1.23 (s, 9H) , 1.19 (t, J=l .02 Hz, 3H) . LCMS (APCI+; Method D) m/z 290, (M+H)+.
Figure imgf000183_0001
Example 35B
1- (4-tert-Butyl -phenyl) -piperidine-4-carboxylic acid
Example 35A (190 mg, 0.657 mmol) was hydrolyzed with LiOH.H20 (33 mg, 0.7878 mmol) as described in Example 21B to provide the title compound (127 mg, 74!). XH NMR (400 MHz, DMSO-d6) δ 12.23 (s, IH) , 7.21 (d, J=8.58 Hz, IH) , 6.85 (d, J=8.58 Hz, IH) , 3.58-3.54 (m, 2H) , 2.72-2.65 (m, 2H) , 2.39-2.32 (m, IH) , 1.90-1.86 (m, 2H) , 1.68-1.58 ( , 2H) , 1.23 (s, 9H) . LCMS (APCI+; Method D) m/z 262, (M+H)+.
Figure imgf000183_0002
Example 35C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (4-tert-butyl- phenyl) -piperidine-4-carbonyl] -amino}-propionic acid methyl ester
The title compound (185 mg, 77!) was prepared from Example 35B (125 mg, 0.4783 mmol) as de- scribed in Example 21C. 2H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J=7.80 Hz, IH), 7.66 (d, J=1.56 Hz, IH) , 7.36-7.29 (m, 3H) , 7.23-7.20 (m, 4H) , 6.87-6.83 (m, 3H), 5.12 (s, 2H) , 4.47-4.41 (m, IH) , 3.64-3.57 (m, 2H) , 3.55 (s, 3H) , 2.89-2.73 (m, 2H) , 2.62-2.52 (m, 2H), 2.28-2.20 (m, IH) , 1.70-1.64 (m, IH) , 1.62-1.49 (m, 3H) . LCMS (APCI+; Method D) m/z 503, (M+H)+.
Figure imgf000184_0001
Example 35D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (4-tert-butyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid
The title compound was prepared from Example 35C (175 mg, 0.3482 mmol) as described in Example 21D. Example 35D (55 mg, 32!) was obtained as a white powder. XE NMR (400 MHz, DMSO-d6) δ 8.03 (d, J=7.80 Hz, IH) , 7.68 (d, J=1.17 Hz, IH) , 7.36-7.30 (m, 3H) , 7.22-7.20 (m, 4H) , 6.87-6.83 (m, 3H) , 5.13 (s, 2H) , 4.42-4.36 (m, IH) , 3.63-3.54 (m, 2H) , 2.91- 2.86 (m, IH), 2.80-2.74 (m, IH) , 2.61-2.52 (m, 2H) , 2.26-2.19 (m, IH) , 1.71-1.62 (m, IH) , 1.59-1.47 (m, 3H), 1.24 (s, 9H) . LCMS (APCI+; Method D) m/z 489, (M+H)+. Analytical HPLC (Method C) Rt=2.068 min (220 nm, 100 area!) .
Figure imgf000185_0001
Example 36
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-chloro-2- nitro-phenyl) -piperidine-4-ca.rbonyl] -amino}- propionic acid
Figure imgf000185_0002
Example 36A
1- (5-Chloro-2-hitro-phenyl) -piperidine-4-carboxylic acid ethyl ester
A solution of 4-chlbro" 2-fluoronitrόbeh- zene (5.00 g, 28.48 mmol) in dry DMF (30 mL) containing CsF (4.33 g, 28.48 mmol) was treated with ethyl isonipecotate (4.82 mL, 31.33 mmol) under N2 atmosphere at 0°C for 1 h and then at ambient tem- perature for 4 h. The reaction mixture was diluted with EtOAc (150 mL)' and washed with water (2x50 mL) . The organic layer was separated, dried (MgS04) , filtered and evaporated to dryness to provide the title compound (9.31 g, 105! crude yield) as a yellow color liquid. XH NMR (400 MHz, DMSO-d6) δ 7.86 (d, J=8.58 Hz, IH) , 7.32 (d, J=2.34 Hz, IH) , 7.11 (d, d, JE=2.34 Hz, J2=8.58 Hz, IH) , 4.09 (q, J=7.02 Hz, 2H) , 3.22-3.17 (m, 2H) , 2.93-2.87 (m, 2H) , 2.54-2.52 (m, IH), 1.91-1.87 (m, -2H) , 1.71-1.61 (m, 2H) , 1.19 (t, ' J=7.02 Hz, 3H) . LCMS (APCI+; Method D) m/z 313, (M+H)+. Analytical HPLC (Method E) Rt=3.656 min (220 nm, 100 area!) .
Figure imgf000186_0001
.;
Example 36B
1- (5-Chloro-2-nitro-phenyl) -piperidine-4-carboxylic acid
A solution of ester Example 36A (150 mg, 0.4796 mmol) in THF:MeOH (1:1, 5'- mL) was treated with a solution of LiOH"H20 (30 mg, 0.719 mmol) in water ' ("2~~mL)~"~at 'ambient" temperature ~for~l-"h". The solvents were removed, residue dissolved in water (5 mL) and adjusted to pH 3.5 with formic acid. The bright yellow precipitate, formed was filtered, washed with water (3x5 mL) , and dried under vacuum (oil pump) for .2 h to provide the title compound (140 mg, 102! yield) as yellow color powder. XE NMR (400 MHz, DMSO-de) δ 12.33 (s, IH) , 7.86 (d, J=8.58 Hz, IH) , 7.32 (d, J=2.34 Hz, IH) , 7.10 (d, d, Jι=2.34 Hz, J2=8.58 Hz, IH) , 3.21-3.17 (m, 2H) , 2.92-2.86 (m, 2H) , 2 . 45-2 . 37 (m, IH) , 1 . 91-1 . 85 (m, 2H) , 1 . 69-1 . 59 '(m, 2H) . LCMS (APCI + ; Method D) m/z 285 , 287 , . (M+H) + .
Figure imgf000187_0001
Example 36G
3-(l-Benzyl-lH-imidazol-4-yl)-2-{ [1- (5-chloro-2- nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid methyl ester
A solution of acid Example 36B (130 mg, 0.457 mmol), HOBt . H20 (105 mg, 0.685 mmol), L-N1- benzyl histidine methyl ester dihydrochloride (182 mg, 0.548 mmol), and triethylamme (0.223 mL, 1.598 mmol), in dry DMF. (3 mL) was treated with EDC (131 mg, 0.685 mmol) at -ambient temperature under. N2- atmosphere for 18 h. The reaction mixture was diluted with EtOAc , (10 mL) , washed with water (2x5 mL) , dried (MgS0) , filtered, and evaporated to dryness. The residue obtained was. purified by flash chromatography on silica- gel (Biotage 40+S) .eluting- with 5! methanol and CH2C12 to provide the title compound (238 mg, 99%) as a bright yellow powder. 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J=7.80 Hz, IH) , 7.86 (d, J=7.58 Hz, IH) , 7.66 (d, J=1.56 Hz, IH) , 7.35-7.27 (m, 4H) ,' 7.22-7.20 (m, 2H) , 7.09 (d, d, J2=2.24 Hz, J2=8.58 Hz, IH) , 6.86 (s, IH) , 5.12 (s, 2H) , 4.48-4.42 (m, IH) , 3.5β(s, 3H) , 3.22-3.12 (m, 2H) , 2.88-2.75 (m, 4H) , 2.31-2.55 (m, IH) , 1.69-1.61 ( , 2H) , 1.58-1.50 (m, 2H) . LCMS (APCI+; Method D) m/z 526, (M+H) + .
Figure imgf000188_0001
Example 36D
3-(l-Benzyl-lH-lmidazol-4-yl)-2-{ [l-(5-chloro-2- nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid
; Absolution of Example 36C (210 mg, 0.3993 mmol) in THF:MeOH (1:1,-5 mL) was- treated with a solution of LiOH'H20 (20 mg, 0.4791 _mmol).-in water (1 mL) at ambient temperature for 6 h. The solvents were removed, the residue dissolved in water (6 mL) and washed with EtOAc (2x3 mL) to remove a minor impurity. The aqueous layer was made acidic (pH 4) with formic acid. The precipitate formed was . collected by filtration; The solid was washed with water (2x5 mL) and dried under vacuum (oil pump) to provide the title compound (195 mg, 94.87%) as a yellow color solid. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (d, J=7.80 Hz, IH), 7.86 (d, J=8.97 Hz, IH) , 7.67 (d, J=1.17 Hz, IH) , 7.35-7.26 (m, 4H) , 7.22-7.20 (m, 2H) , 7.09 (d, d, J2=2.34 Hz, J2=8.97 Hz, IH) , 6.86 (d, J L.17 Hz, IH) , 5.12 (s, 2H) , 4.42-4.37' (m, IH) , 3.22-3.11 ( , 2H) , 2.92-2.85 (m, IH) , 2.82-2.74 (m, 3H) , 2.32-2.22 ( , IH) , 1.709-1.58 (m, 2H) , 1.54- 1.48 (m, 2H) . LCMS (APCI+; Method D) m/z 512, 514, (M+H)+. Analytical HPLC (Method C) Rt=2.330 min (220 nm, 99.44%, area!) .
Figure imgf000190_0001
Exa ple 37
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-bromo-4- fluoro-2-nitro-phenyl) -piperidine-4-carbonyl] - amino}-propionic acid
Figure imgf000190_0002
Example 37A
1- (5-Bromo-4-fluoro-2-nitro-phen-yl) -piperidine-4- carboxylic acid ethyl ester
■ _A_sqlution of 4-bromo 2, 5-difluoronitro- benzene (5.00 g, 21.01 mmol) in dry DMF (30 mL) containing CsF (3.19 g, 21.01 mmol) was treated with ethyl iso ipecotate (3.56 mL, 23.11 mmol) under N2 atmosphere at ambient temperature for 18 h. The bright orange color suspension was diluted with
EtOAc (150 L) and washed with water (3x50 mL) . The organic layer was separated, dried (using MgS0) , filtered, and evaporated to dryness to provide the crude title compound (8.02 g, 101.74 !) as a bright red liquid. XE NMR (400 MHz, DMSO-d6) δ 8.01 (d, =8.19 Hz, IH) , 7.68 (d, J=6.24 Hz, IH)-, 4.09 (q, J=7.02 Hz, 2H) , 3.15-3.10 (m, 2H) , 2.89-2.83 (m, 2H), 2.48-2.44 (m, IH) , 1.91-1.87 (m, 2H) , 1.70-1.60 (m, 2H) , 1.19 (t, J-7-.02 Hz, 3H) . LCMS (APCI+; Method D) m/z 375; 377, (M+H)+.
Figure imgf000191_0001
Example 37B
1- (5-Bromo-4-fluoro-2-nitro-phenyl) -piperidine-4- carboxylic acid
The title compound was prepared from Example 37A (150 mg, 0.4 mmol) as described in Example 36B. Example 37B (137 mg, 99!) was obtained as a bright orange powder. 1H NMR (400 MHz, DMSO-d6) δ 12.31 (s, IH) , 8.02 (d, J=8.19 Hz, IH) , 7.68 (d, J=_6.24 Hz,.lH), 3.14^-3.09- (m, 2-H) , 2.88-2.83 (m, 2H) , 2.42-2.34 (m, IH) , 1.91-1.86 (m, 2H) , 1.68-1.58 (m, 2H) . LCMS (APCI+; Method D) m/z 347, (M+H)+.
Figure imgf000192_0001
Example 37C
3- (l-Benzyl-lH-imidazol-4-yl)--2-{ [1- (5-bromo-4- . fluoro-2-nitro-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid methyl ester
Example 37B (135 mg, 0.389 mmol) was treated with L-N1-benzyl histidine methyl ester dihydrochloride (155 mg, 0.-467 mmol) as described in Example 36C to provide the title compound (215 mg, 94!) as a bright yellow liquid. - XH. NMP (400 MHz,
DMSO-de) δ 8.20 (d, J=7.80 Hz, IH) , 8..12 (d, .7=8.19 Hz, IH), 7.68-7.66 ( , 2H) , 7.35-7.27 (m, 3H) , 7.21- 7.19 (m, 2H) , 6.87 (d, J=1.17 Hz, IH) , 5.13 (,s, 2H) ,-.'■ 4.48-4.42 (m, IH) „ 3.56 (s, 3H) , 3.15-3.04 ( , 2H) , 2.86-2.75 (m, 4H) ,.2.89-2 21..(m, IH) , 1.68-1.6-1 (m, 2H), 1.57-1.48 (m, 2H) . LCMS (APCI+; Method- D) m/z 588, 589, (M+H)+.
Figure imgf000193_0001
Example 37D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-bromo-4- fluoro-2-nitro-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid
The title compound was 'prepared from Example 37C (210 mg, 0.399 mmol) as described in Example 36D. Example 37D (195 mg, 94.87%) was obtained as a yellow color solid. 1H NMR (400 MHz, DMSO-de) δ 8.07 (d, J=8.'19 Hz, IH) , 8.01 (d, J=8.58 Hz, IH), 7.66 (m, 2H) , 7.35-7.26 (m, 3H) , 7.22-7.20 (m, 2H) , 6.86 (s, IH) , 5.12 (s, 2H) , 4.42-4.37 (m, IH) , 3.15-3.04 ( ,.2H) , 2.92-2.87 (m, IH) , 2.83-2.74' (m, 2H), 2.27-2.19 (m, IH) , 1.69-1.61 ( , IH) , 1.58- 1.47 (m, 2H) . LCMS (APCI+; Method D) m/z 512, 514, (M+H)+ Analytical HPLC (Method C) Rt=2.425 min (220 nm, 99.44 area!) .
Figure imgf000194_0001
Example 38 '
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-morpholin-4- yl-2-nitro-phenyl) -pip.eridine-4-carbonyl] -amino} - propionic acid
Figure imgf000194_0002
Example 38A
1- (5-Morpholin-4-yl-2-nitro-phenyl) -piperidine-4- carboxylic acid ethyl ester
'•'■ A sealed, tube containing a solution of
Example 36A (500 mg, 1.599 mmol) and morpholine (2 mL, 23 mmol) was heated at 100°C for 15 h. The reaction mixture was directly purified by silica gel flash chromatography (Biotage 40+M) eluting with 10! acetone/hexane to provide the title compound (400 mg, 69! ) as a yellow powder. XH NMR (400 MHz, DMSO- de) δ 7.92 (d, J=9.36 Hz, IH) , 6.61 (d, d, J2=2.24 Hz, J2=9.36 Hz, IH) , 6.41 (d, J=2 . 13 Hz, IH) , 4.09 (q, J=7.02 Hz, 2H) , 3.70-3.70 (m, 4H) , 3.36-3.34 (m, 4H), 3.23-3.17 (m, 2H) , 2.86-2.79 (m, 2H) , 2.48 (m,
IH), 1.92-1.87 (m, 2H) , 1.78-1.68 (m, 2H) , 1.20 (t,
J= 7.02 Hz, 3H) . LCMS (APCI+; Method D) m/z 364, (M+H) .
Figure imgf000195_0001
Example 38B
1- (5-Morpholin-4-yl-2-nitro-phenyl) -piperidine-4- carboxylic acid
A solution of Example 38A(150 mg, 0.4128 mmol) in THF:MeOH (1:1, 5 mL) was' treated with a solution of LiOH.H20 (21 mg, 0.4953 mmol) in water (1 mL) at ambient temperature for 5 h. The solvents were removed, residue dissolved in water (6 mL) and washed with EtOAc (2x3 mL) to remove a minor impur- ity. The aqueous layer was made acidic (pH 4) with formic acid. The precipitate formed was filtered, washed with water (2x5 mL) , and dried under vacuum (oil pump) to provide the title compound (115 mg, 83.08!) as a yellow color solid. lE NMR (400 MHz, DMSO-de) δ 12.30 (s, IH) , 7.91 (d, J=9.36 Hz, IH) , 6.60- (d, d, J2=2.13 Hz, J2=9.36 Hz, IH) , 6.41 (d, J=2.73 Hz, IH) , 3.72-3.69 (m, 4H) , 3.36-3.33 (m, 4H) , 3.22-3.18 (m, 2H) , 2.85-2.88 (m, 2H) , 2.42- 2.36 (m, IH) , 1.92-1.87 (m, 2H) , 1.77-1.68 (m, 2H) . LCMS (APCI+; Method D) m/z 336, (M+H)+. Analytical HPLC (Method C) Rt=2.356 min (220 nm, 100 area!).
Figure imgf000196_0001
Example 38C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-morpholin-4- yl-2-nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid methyl ester
The title compound was prepared according to the procedure described in Example 36C substitut- ing Example 36B with Example 38B (110 mg, 0.328 mmol). Example 38C (130 mg, 67.64!) was obtained as a bright yellow powder. 1H NMR (400 MHz, DMSO-d5) δ 8.19 (d, J=7.41 Hz, IH) , 7.91 (d, J=9.75 Hz, IH) , 7.66 (d, J=1.17 Hz, IH) , 7.35-7.31 (m, 2H) , 7.28- 7.25 (m, IH) , 7.21-7.19 (m, 2H) , 6.87 (d, J=1.17 Hz, IH) , 6.50 (d, d, i=2.73 Hz, J2=9.36 Hz, IH) , 6.40 (d, J=2.73 Hz, IH), 5.12 (s, 2H) , 4.48-4.43 (m, IH) , 3.73-3.70 (m, 4H) , 3.56 (s, 3H) , 3.36-3.33 (m, 4H) , 3.26-3.14 (m, 2H) , 2.86-2.75 (m, 4H) , 2.33-2.24 (m, IH) , 1.73-1.65 (m, 2H) , 1.61-1.57 (m, 2H) . LCMS
(APCI+; Method D) m/z 577, (M+H)+. Analytical HPLC (Method C) Rt=2.185 min (220 nm, 98.42 area!).
Figure imgf000197_0001
Example 38D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-morpholin-4- yl-2-nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid
The title compound was prepared from Example 38C (126 mg, 0.219 mol) as described in Example 36D. Example 38D (110 mg, 89.48!) was obtained as a bright yellow powder. XH NMR (400 MHz, DMSO-d6) δ 8.07 (d, J=7.80 Hz, IH) , 7.91 (d, J=9.36 Hz, IH) ,
7.68 (s, IH), 7.34-7.31 (m, 2H) , 7.27-7.25 (m, IH) , 7.22-7.20 (m, 2H), 6.87 (s, IH) , 6.60 (d, d, Ji=2.24 Hz, J2=9.36 Hz, IH) , 6.39 (d, J=2-.24 Hz, 1H),' 5.13 (s, 2H), 4.43-4.38 (m, IH) , 3.73-3.70 (m, 4H) , 3.36- 3.34 (m, 4H) , 3.26-3.13 (m, 2H) , 2.91-2.87 (m, IH) , 2.82-2.69 (m, 3H) , 2.30-2.22 (m, IH) 1.71-1.66 (m, 2H) , 1.62-1.53 (m, 2H) . LCMS (APCI+; Method D) m/z 563, (M+H)+. Analytical HPLC (Method C) Rt=2.105 min (220 nm, 100 area!) .
Figure imgf000198_0001
Example 39
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- trifluoromethylsulfanyl-phenyl) -piperidine-4- carbonyl] -amino} -propionic acid
Figure imgf000198_0002
Example 39A
1- (3-Trifluoromethylsulfanyl-phenyl) -piperidine-4- carboxylic acid ethyl ester
The title compound (355 mg, 79.2!) was prepared by the procedure described in Example 21A, substituting l-bromo-3-methyl-benzene with 1-bromo- 3-trifluoromethyl-sulfanyl-benzene (341 mg, 1.325 mmol). XH NMR (400 MHz, DMSO-de) δ 7.36-7.32 (m, IH) , 7.19-7.15 (m, 2H) , 7.04 (d, J=7.41 Hz, IH) , 4.08 (q, J=l . 02 Hz, 2H) , 3.72-3.67 (m, 2H) , 2.87- 2.79 (m, 2H), 2.56-2.51 (m, IH) , 1.93-1.88 (m, 2H) , 1.68-1.58 (m, 2H) , 1.19 (t, J=7.02 Hz, 3H) . LCMS (APCI+) : (M+H)+=563. Analytical HPLC purity=100!, retention time 2.105 min. LCMS (APCI+; Method D) m/z 334, (M+H)+. Analytical HPLC (Method C) Rt=3.9f min (220 nm, 98.57 area!;
Figure imgf000199_0001
Example 39B
1- (3-Trifluoromethylsulfanyl-phenyl) -piperidine-4- carboxylic acid
Example 39A (350 mg, 0.1.050 mmol) was hydrolyzed with Li0H.H2O (53 mg, 1.26 mmol) as described in Example 2IB to provide the title compound (210 mg, 65.5!) as a cream color solid. LCMS (APCI+; Method D) m/z 306, (M+H)+.
Figure imgf000199_0002
Example 39C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- trifluoromethylsulfanyl-phenyl) -piperidine-4- carbonyl] -amino} -propionic acid methyl ester
The title compound (228 mg, 76.6!) was prepared from Example 39B (197 mg, 0.645 mmol) as described in Example 21C. Example 39C was obtained as a cream color solid. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J=7.41 Hz, IH) , 7.66 (s, IH) , 7.37-7.28 (m, 2H), 7.22-7.20 ( , 2H) , 7.16-7.13 (m, 2H) , 7.05-7.03 ( , IH), 6.87 (s, IH) , 5.13 (s, 2H) , 4.47-4.42 (m, IH) , 3.76-3.67 (m, 2H) , 3.55 (s, 3H) , 2.89-2.79 (m, 2H) , 2.76-2.66 (m, 2H) , 2.34-2.28 (m, IH) , 1.73-1.66 (m, IH) , 1.61-1.43 (m, 3H) . LCMS (APCI+; Method D) m/z 547, (M+H)+. Analytical HPLC (Method C) Rt=2.639 min (220 nm, 93.83 area!) .
Figure imgf000200_0001
Example 39D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- trifluoromethylsulfanyl-phenyl) -piperidine-4- carbonyl] -amino} -propionic acid"
The title compound was prepared from Example 39C (282 mg, 0.516 mmol) as described in Example 21D. Example 39D (258 mg, 91.31%) was obtained as a white powder. XE NMR (400 MHz, DMSO-d6) δ 8.03 (d, J=7.81 Hz, IH), 7.67 (s, IH) , 7.36-7.29 (m, 4H) , 7.23-7.21 (m, 2H) , 7.16-7.13 (m, 2H) , 7.04 (d,
J=7.42 Hz, IH), 6.86 (s, IH) , 5.13 (s, 2H) , 4.42- 4.37 (m, IH) , 3.76-3.65 (m, 2H) , 2.91-2.87 (m, IH) , 2.81-2.65 (m, 3H) , 2.33-2.26 (m, IH) , 1.72-1.67 (m, IH) , 1.61-1.44 (m, 3H) . LCMS (APCI+; Method D) m/z 533, (M+H)+. Analytical HPLC (Method C) Rt=2.525 min (220 nm, 97.24 area%) .
Figure imgf000201_0001
Example 40
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- trifluoromethoxy-phenyl) -piperidine-4-carbonyl] amino}-propionic acid
Figure imgf000201_0002
Example 40A
1- (3-Trifluoromethoxy-phenyl) -piperidine-4- carboxylic acid ethyl ester
The title compound (331 mg, 73.78%) was prepared by the procedure described in Example 21A, substituting l-bromo-3-methyl-benzene with 1-bromo- 3-trifluoromethoxy-benzene (0.197 mL, 1.325 mmol). XH NMR (400 MHz, DMSO-d6) δ 7.29 (t, J=8.19 Hz, IH) , 6.94 (d, d, Jι=3.12 Hz, J=8.58 Hz, IH) , 6.83 (s, IH) , 6.68 (d, J=8.19 Hz, IH) , 4.08 (q, J=7.02 Hz, ' 2H) , 3.72-3.67 (m, 2H) , 2.86-2.79 (m, 2H) , 2.58-2.53 (m, IH) , 1.91-1.87 (m, 2H) , 1.678-1.56 (m, 2H) , 1.19 (t, J=7.02 Hz, 3H) . LCMS (APCI+; Method D) m/z 318, (M+H)+. Analytical HPLC (Method C) Rt=3.792 min (220 nm, 97.24 area%) .
Figure imgf000202_0001
Example 4OB
1- (3-Trifluoromethoxy-phenyl) -piperidine-4- carboxylic acid
Example 40A (325 mg, 1.024 mmol) was hydrolyzed with LiOH.H20 (52 mg, 1.229 mmol) as de- scribed in Example 2IB to provide the title compound (289 mg, 96.62%) as an off-white solid. XE NMR (400 MHz, DMS0-d6) δ 7.28 (t, J=8.19 Hz, IH) , 6.95 (d, d, z=1.95 Hz, J2=8 . 19 Hz, IH) , 6.23 (m, IH) , 6.67 (m, IH), 3.71-3.66 (m, 2H) , 2.84-2.77 (m, 2H) , 2.44-2.37 (m, IH) , 1.91-1.85 (m, 2H) , 1.65-1.55 (m, 2H) . LCMS (APCI+; Method D) m/z 290, (M+H)+.
Figure imgf000203_0001
Example 40C
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- trifluoromethoxy-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid methyl ester
The title compound (120 mg, 82%) was prepared from Example 40B (80 mg, 0.277 mmol) as described in Example 21C. αH NMR (400 MHz, DMSO-d6) δ 8.16 (d, J=7.80 Hz, IH) , 7.66 (s, IH) , 7.37-7.27 (m, 4H) , 7.21 (d, J=8.19 Hz, 2H) , 6.93 (dd, i=8.19 Hz, ■ J2=2.24 Hz, IH) , 6.87 (s, IH) , 6.81 (s, IH) , 6.67 (d, J=8.19 Hz, IH) , 5.13 (s,, 2H) , 4.47-4.42 (m, IH) , 3.75-3.64 (m, 2H) , .3.55 (s, 3H) , 2.89-2.78 (m, 2H) , 2.75-2.66 (m, 2H) , 2.35-2.26 (m, IH) , 1.78-1.66 (m, IH) , 1.60-1.45 (m, 3H) . LCMS (APCI+; Method D) m/z " ■ 531, (M+H)+. Analytical HPLC (Method C) Rt=2.525 min (220 nm, 100 area%) .
Figure imgf000204_0001
Example 40D
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3- trifluoromethoxy-phenyl) -piperidine-4-carbonyl] - amino}-propionic acid
The title compound was prepared from Example 40C (107 mg, 0.202 mmol) as described in Example 21D. Example 40D (65 mg, 62.4%) was obtained as a white powder. XE NMR (400 MHz, DMSO-d6) δ 8.04 (d, J=7.81 Hz, IH) , 7.67 (s, IH) , 7.37-7.27 (m, 4H) ,
7.22 (d, "=8.20 Hz, IH) , 6.94 ( , IH) , 6.87 (s, IH) , 6.80 (s, IH) , 6.66 (d, J=8 .20 Hz, IH) , 5.13 (s, 2H) , 4.42-4.37 (m, IH) , 3.75-3.65 (m, 2H) , 2.91-2.86 (m, IH) , 2.81-2.65 (m, 3H) , 2.33-2.56 (m, IH) , 1.72-1.66 (m, IH) , 1.58-1.44 (m, 3H) . LCMS (APCI+; Method D) m/z 517, (M+H)+. Analytical HPLC (Method C) Rt=2.44 min (220 nm, 100 area%) .
Figure imgf000205_0001
Example 41
3-Benzoylamino-2-{ [1- (3-trifluoromethoxy-phenyl) piperidine-4-carbonyl] -amino}-propionic acid
Figure imgf000205_0002
Example 41A
3-Benzoylamino-2-{ [1- (3-trifluoromethoxy-phenyl) - piperidine-4-carbonyl] -amino}-propionic acid methyl ester
The title compound (130 mg, 86%) was prepared from Example 40B (80 mg, 277 mmol) and methyl- 2-amino-3-benzoylamino-propionate HCl salt (202 mg, 0.553 mmol) as described in Example 21C. Example 41A was obtained as a cream color solid. 1H NMR (400 MHz, DMSO-d6) δ 8.55 (t, J=5.85 Hz, IH) , 8.27 (d, J=7.80 Hz, IH), 7.86 (d, J=8.19 Hz, IH) , 7.81- 7.78 (m, 2H) , 7.54-7.45 (m, 5H) , 7.28 (t, J=8.19 Hz, IH) , 6.93 (d, d,
Figure imgf000205_0003
Hz, IH) , 6.81 (s, IH) , 6.66 (d, J=8.17 Hz, IH) , 4.4 (q, J=7.02 Hz, IH), 3.80-3.70 (m, 2H) , 3.64-3.59 (m, 5H) , 2.79-2.71 (m, 2H) , 2.44-2.37 (m, IH) , 1.78-11.73 (m, 2H) , 1.65-1.55 (m, 2H) . LCMS (APCI+; Method D) m/z 494, (M+H)+. Analytical HPLC (Method C) Rt=3.072 min (220 nm, 90.5 area%) .
Figure imgf000206_0001
Example 4IB
3-Benzoylamino-2-{ [1- (3-trifluoromethoxy-phenyl) - piperidine-4-carbonyl] -amino} -propionic acid
The title compound was prepared from Example 41A (125 mg, 0.253 mmol) as described in Example 2ID. Example 4IB (101 mg, 83%) was obtained as a white powder. XH NMR (400 MHz, DMSO-d6) δ 12.68 (br s, IH) , 8.51 (d, J=5.86 Hz, IH) , 8.13 (d, J=7.81 Hz, IH), 7.80-7.78 (m, .2H) , 7.53-7.52 (m, IH) , 7.48-7.45 (m, 2H) , 7.27 (t, "=8.20 Hz, IH) , 6.93 (d, d, ι=8.59 Hz, J2=2.34 Hz, IH) , 6.81 (s, IH) , 6.66 (d, J=7.81 Hz, IH) , 4.48-4.43 (m, IH) , 3.77-3.71 (m, 2H) , 3.62- 3.58 (m, 2H) , 2.79-2.70 (m, 2H) , 2.41-2.36 (m, IH) , 1.78-1.40 (m, 2H) , 1.65-1.55 (m, 2H) . LCMS (APCI+; Method D) m/z 480, (M+H)+. Analytical HPLC (Method C) Rt=2.811 min (220 nm, 94.96 area!).
Figure imgf000207_0001
Example 42
3- ( l-Benzyl-lH-imidazol-4-yl) -2- [ ( l-quinolin-3-yl- piperidine-4-carbonyl) -amino] -propionic acid
Figure imgf000207_0002
Example 42A
l-Quinolin-3-yl-piperidine-4-carboxylic acid ethyl ester
The title compound (325 mg, 86.25%) was i0 prepared by the procedure described in Example 21A, substituting l^-bromo-3^-methyl-benzene with 3-bromo- quinoline (276 mg, 1.325 mmol). 1H NMR (400 MHz, DMSO-de) δ 8.86 (d, J=3.12 Hz, IH) , 7.86 (d, d, i=3.51Hz, J2=6.24 Hz, IH) , 7.78 (d, d, ^=3.51 Hz, 15 J2=5.85 Hz, IH) , 7.55 (d, J=2.73 Hz, IH) , 7.49-7.45 (m, 2H), 4.10 (q, J=7.02 Hz, 2H) , 3.84-3.79 (m, 2H) , 2.94-2.88 (m, 2H) , 2.61-2.53 (m, IH) , 2.01-1.95 (m, 2H), 1.79-1.69 (m, 2H) , 1.20 (t, J=7.02Hz, 3H) . LCMS (APCI+; Method D) m/z 285, (M+H) +. Analytical 20 HPLC (Method C) Rt=2.247 min (220 nm, 97.83 area%) .
Figure imgf000208_0001
l-Quinolin-3-yl-piperidine-4-carboxylic acid
Example 42A (320 mg, 1.125 mmol) was hy- drolyzed with LiOH.H20 (57 mg, 1.35 mmol) as described in Example 2IB to provide the title compound (285 mg, 98.3%). XE NMR (400 MHz, DMSO-d6) δ 12.30 (s, IH) , 8.56 (d, J=2.73 Hz, IH) , 7.86-7.84 (m, IH) , 7.79-7.77 (m, IH) , 7.55 (d, J=2.73 Hz, IH) , 7.49- 7.45 (m, 2H), 3.83-3.79 ( , 2H) , 2.93-2.87 (m, 2H) , 2.48-2.43 (m, IH) , 1.98-1.93 (m, 2H) , 1.77-1.67 (m, 2H) . LCMS (APCI+; Method D) m/z 257, (M+H)+. Analytical HPLC (Method C) Rt=99.48 min (220 nm, 99.48 area!) .
Figure imgf000209_0001
Example 42C
3- ( l-Benzyl-lH-imidazol-4-yl ) -2- [ ( l-quinolin-3-yl- piperidine-4-carbonyl) -amino] -propionic acid methyl ester
The title compound (100 mg, 64.39!) was prepared from Example 42B (80 mg, 0.312 mmol) as described in Example 21C. Example 42C was obtained as a cream color powder. XE NMR (400 MHz, DMSO-d6) δ 8.85 (d, J=3.12 Hz, IH) , 8.21 (d, J-=7.80 Hz, IH) ,
7.87-7.84 (m, IH) , 7.80-7.72 (m, IH) , 7.67 (s, IH) , 7.53 (d, J=2.73 Hz, IH) , 7.49 (s, IH) , 7.48-7.46 (m, 2H) , 7.38-7.27 (m, 3H) , 7.23-7.20 (m, 2H) , 6.89 (s, 2H), 5.13 (s, 2H) , 4.49-4.43 (m, IH) , 3.87-3.81 (m, 2H), 3.56 (s, 3H) , 2.89-2.74 (m, 4H) , 2.39-2.31 (m, IH) , 1.79-1.74 (m, IH) , 1.69-1.58 (m, 2H) . LCMS (APCI+; Method D) m/z 498, (M+H)+. Analytical HPLC (Method C) Rt=1.863 min (220 nm, 100 area!).
Figure imgf000210_0001
Example 42D
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-quinolin-3-yl- • piperidine-4-carbonyl) -amino] -propionic acid
The title compound was prepared from Example 42C (95 mg, 0.191 mmol) as described in Example 21D. Example 42D (54 mg, 58.5!) was obtained as a cream color solid. XE NMR (400 MHz, DMSO-d6) δ 8.85 (d, J=2.73 Hz, IH) , 8.05 (d, J=7.80 Hz,'lH), 7.87- 7.84 (m, IH), 7.80-7.77 (m, IH) , 7,67 (s, IH) , 7.53 (d, J=2.73 Hz, IH) , 7.48-7.46 (m, 2H) , 7.36-7.29 (m, 3H) , 7.23-7.20 (m, 2H) , 6.88 (s, IH) , 5.13 (s, 2H) , 4.44-4.38 (m, IH) , 3.87-3.79 (m, 2H) , 3.36 (m, IH) , 2.93-2.88 (m, 2H) , 2.82-2.74 (m, 3H) , 2.37-2.31 (m, IH) ,' 1.80-1.71 (m, IH) , 1.68-1.57 (m, 4H) . LCMS
(APCI+; Method D) m/z 484, (M+H)+. Analytical HPLC (Method C) Rt=1.758 min (220 nm, 100 area!).
Figure imgf000211_0001
Example 43
3-Benzoylamino-2- [ (l-quinolin-3-yl-piperidine-4- carbonyl) -amino] -propionic acid
Figure imgf000211_0002
Example 43A
3-Benzoylamino-2- [ (l-quinolin-3-yl-piperidine-4- carbonyl) -amino] -propionic acid methyl ester
Example 43A was prepared from Example 42B (80 mg, 312 mmol) and methyl-2-amino-3-benzoylamino- propionate HCl salt (231 mg, 0.624 mmol) as described in Example 21C. The title compound (110 mg, 76.5!) was obtained as a cream color solid. XE NMR (400 MHz, DMSO-de) δ 8.85 (d, J=2. 13 Hz, IH) , 8.57 (d, J=5.85 Hz, IH) , 8.30 (d, J=8.00 Hz, IH) , 7.86- 7.56 (m, 4H) , 7.56-7.52 (m, 2H) , 7.50-7.45 (m, 4H) , 4.50 (q, J=6.63 Hz, IH) , 3.93-3.85 (m, 2H) , 3.65 (m, IH) , 3.62 (s, 3H) , 3.60 (m, IH) , 2.88-2.79 (m, 2H) , 2.48-2.39 (m, IH) , 1.89-1.81 (m, 2H) , 1.77-1.69 (m, 2H) . LCMS (APCI+; Method D) m/z 461, (M+H)+. Analytical HPLC (Method C) Rt=1.99 min (220 nm, 96.78 area!) .
Figure imgf000212_0001
Example 43B
3-Benzoylamino-2- [ (l-quinolin-3-yl-piperidine-4- carbonyl) -amino] -propionic acid
The title compound was prepared from Example 43A (105 mg, 0.228 mmol) as described in Example 21D. Example 43B (76 mg, 75!) was obtained as a cream color powder. XH NMR (400 MHz, DMSO-d6) δ 12.68 (s, IH) , 8.84 (d, J=2.73 Hz, IH) , 8.53 (t, J=5.85, IH) , 8.16 (d, J=7.80 Hz, IH) , 7.86-7.56 (m, 4H), 7.53-7.51 (m, 2H) , 7.48-4.46 (m, 4H) , 4.50-4.44 (m, IH), 3.91-3.84 (m, IH) , 3.63-3.60 (m, 2H) , 2.87- 2.79 (m, 2H) , 2.45-2.40 (m, IH) , 1.86-1.81 (m, 2H) , 1.76-1.67 (m, 2H) . LCMS (APCI+; Method D) m/z 447, (M+H)+. Analytical HPLC (Method C) Rt=1.831 min (220 nm, 100 area!) .
Figure imgf000213_0001
Example 44
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-isoquinolin-4- yl-piperidine-4-carbonyl) -amino] -propionic acid
Figure imgf000213_0002
Example 44A
l-Isoquinolin-4-yl-piperidine-4-carboxylic acid ethyl ester
Example 44A was prepared by the procedure described in Example 21A, substituting' l-bromo-3- methyl-benzene with 4-bromo-isoquinoline (276 mg, 1.325 mmol). The title compound (230 mg, 59.4!) was obtained as a yellow color solid. 1H NMR (400 MHz, DMSO-de) δ 8.99 (s, IH) , 8.16 (s, IH) , 8.10-8.04 (m, 2H), 7.81-7.77 (m, IH) , 7.69-7.65 (m, IH) , 4.13 (q, J=7.02 Hz, 2H) , 3.38-3.34 (m, 2H) , 2.91-2.85 (m, 2H) , 2.61-2.53 (m, IH) , 2.05-2.01 (m, 2H) , 1.96-1.87 (m, 2H) , 1.23 (t, J=7.02 Hz, 3H) . LCMS (APCI+; Method D) m/z 285, (M+H)+. Analytical HPLC (Method C) Rt=2.041 min (220 nm, 97.29 area!).
Figure imgf000214_0001
l-Isoquinolin-4-yl-piperidine-4-carboxylic acid
Example 44A (220 mg, 0.774 mmol) was hy- drolyzed with LiOH.H20 (39 mg, 0.928 mmol) as described in Example 2IB to provide the title compound (189 mg, 98.44!) as a cream color solid. 1H NMR (400 MHz, DMSO-de) δ 12.35 (s, IH) , 8.99 (s, IH) , 8.16 (s, IH) , 8.09 (d, J=8.18 Hz, IH) , 8.05 (d, J=7.41 Hz, IH), 7.81-7.77 (m, IH) , 7.69-7.65 (m, IH) , 3.39-3.36 (m, 2H), 2.91-2.84 (m, 2H) , 2.49-2.43 (m, IH) , 2.04- 2.00 ( , 2H) , 1.94-7.85 (m, 2H) . LCMS (APCI+; Method D) m/z 257, (M+H)+. Analytical HPLC (Method C) Rt=1.620 min (220 nm, 98.44 area!).
Figure imgf000215_0001
Example 44C
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-isoquinolin-4- yl-piperidine-4-carbonyl) -amino] -propionic acid methyl ester
Example 44C was prepared from Example 44B (80 mg, 0.312 mmol) as described in Example 21C. The title compound (75 mg, 48!) was obtained as a cream color solid. XE NMR (400 MHz, DMSO-d6) δ 8.99 (s, IH) , 8.23 (d, J=7.4lΗz, IH) , 8.16 (s, IH) ,
8.094 (d, J=l . l 98 Hz, IH) , 8.023 (d, J=8.58 Hz, IH) , 7.82-7.78 (m, IH) , 7.70-7.66 (m, 2H) , 7.35-7.30 (m, 2H) , 7.26-7.20 (m, 3H) , 6.90 (s, IH) , 5.14 (s, 2H) , 4.53-4.47 (m, IH) , 3.58 (s, 3H) , 2.93-2.74 (m, 4H) , 2.39-2.31 (m, IH) , 1.91-1.76 (m, 3H) , 1.72-1.67 (m, IH) . LCMS (APCI+; Method D) m/z 498, (M+H)+. Analytical HPLC (Method C) Rt=1.863 min (220 nm, 100 area!) .
Figure imgf000216_0001
Example 44D
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-isoquinolin-4- yl-piperidine-4-carbonyl) -amino] -propionic acid
The title compound was prepared from Example 44C (70 mg, 0.141 mmol) as described in Example 21D. Example 44D (50 mg, 73.5!) was obtained as a cream color solid. 1H NMR (400 MHz, DMSO-d6) δ 1H NMR (400 MHz, DMSO) δ 8.99 (s, IH) , 8.40 (s, IH) , 8.144 (s, IH) , 8.09(d, J=8.19 Hz, IH) , 8.02 (d,
J=8.58 Hz, IH) , 7.79 (t, J=l .02 Hz, IH) , 7.69-7.65 (m, 2H) , 7.33-7.30 (m, 2H) , 7.24-7.20 (m, 3H) , 6.84 (s, IH) , 5.12 (s, 2H) , 4.25-4.21 (m, IH) , 3.38-3.29 (m, 2H) , 2.97-2.92 (m, IH) , 2.82-2.71 (m, 3H) , 2.36- 2.28 (m, IH) , 1.87-1.73 (m, 3H) , 1.67-1.62 (m, IH) . LCMS (APCI+; Method D) m/z 484, (M+H)+. Analytical HPLC (Method C) Rt=1.82 min (220 nm, 100 area!).
Figure imgf000217_0001
Example 45
3-Benzoylamino-2- [ ( l-isoquinolin-4-yl-piperidine-4- carbonyl) -amino] -propionic acid
Figure imgf000217_0002
Example 45A
3-Benzoylamino-2- [ (l-isoquinolin-4-yl-piperidine-4- carbonyl) -amino] -propionic acid methyl ester
Example 45A was prepared from Example 44B (80 mg, 312 mmol) and methyl-2-amino-3-benzoylamino- propionate HCl salt (231 mg, 0.624 mmol) as described in Example 21C. The title compound (120 mg, 83.5!) was obtained as a yellow color solid. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, IH) , 8.58 (t, J=5.85 Hz, IH) , 8.32 (d, J=8.00 Hz, IH) , 8.16 (s, IH) , 8.08 (d, J=7.80 Hz, IH) , 8.02 (d, J=8.19 Hz, IH) , 7.83- 7.77 (m, 3H) , 7.69-7.65 (m, IH) , 7.56-7.46 (m, 3H) , 4.54 (q, J=6.63, IH) , 3.65 (m, IH) , 3.63 (s, 3H) , 3.62 (m, IH) , 3.42-3.36 ( , IH) , 2.67-2.79 (m, 2H) , 2.46-2.40 (m, IH) , 1.93-1.86 (m, 3H) , 1.25 (m, IH) , 0.86 ( , IH) . LCMS (APCI+; Method D) m/z 461, (M+H)+. Analytical HPLC (Method C) Rt=1.877 min (220 nm, 100 area!) .
Figure imgf000218_0001
Example 45B
3-Benzoylamino-2- [ (l-isoquinolin-4-yl-piperidine-4- carbonyl) -amino] -propionic acid
The title compound was prepared from Example 45A (115 mg, 0.250 mmol) as described in Example 21D. Example 45B (80 mg, 71.75!) was obtained as a cream color powder. XE NMR (400 MHz, DMSO-d6) δ 12.70 (s, IH) , 8.99 (s, IH) , 8.54 (t, J=5.85 Hz,
IH) , 8.18 (d, J=7.80 Hz, IH) , 8.16 (s, IH) , 8.08 (d, J=8.19 Hz, IH) , 8.02 (d, J=8.19 Hz, IH) , 7.84-7.80 (m, 2H) , 7,89-7.76 (m, IH) , 7,69-7.65 (m, IH) , 7.54- 7.50 (m, IH) , 7.49-7.46 (m, 2H) , 4.54-4.49 (m, IH) , 3.65-3.62 (m, 2H) , 3.41-3.34 (m, 2H) , 2.86-2.76 (m, 2H) , 2.45-2.40 (m, IH) , 1.92-1.87 (m, 4H) . LCMS (APCI+; Method D) m/z 447, (M+H)+. Analytical HPLC (Method C) Rt=1.739 min (220 nm, 100 area!).
Figure imgf000219_0001
Example 46
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (4-f luoro- phenoxymethyl ) -phenyl] -piperidine-4-carbonyl } - amino) -propionic acid
Figure imgf000219_0002
Example 46A
3- (4-Fluoro-phenoxy) benzyl bromide
A solution of 3-bromobenzylalcohol (161 mL, 1.337 mmol), Ph3P (427 mg, 1.626 mmol), and Et3N (0.413 mL, 2.962 mmol) in anhydrous THF (5 mL) was treated with 4-fluoro phenol (180 mg, 1.604 mmol) and DEAD (0.253 mL, 1.604 mmol) and stirred under N2 atmosphere at ambient temperature for 18 h. The re- action mixture was diluted with EtOAc (25 mL) and washed with water (lx 10 mL) , IN NaOH (2x10 mL) , and brine (2x10 mL) . The organic layer was separated, dried (Na2S0) , filtered, and evaporated to dryness to provide a colorless oil. This was crystallized upon standing at room temperature for 1 h. The product formed was triturated with hexane and the filtrate collected was evaporated to dryness. The residue obtained was purified by flash chromatography on silica gel (Biotage 40+ S) eluting with 5! EtOAc/hexane to provide the title compound (210 mg, 53.67%) as a colorless oil. XH NMR (400 MHz, DMSO- d6) δ 7.65 (m, IH) , 7.55 (m, IH) , 7.46-7.44 (m, IH) , 7.38-7.34 (m, IH) , 7.18-7.11 (m, 2H) , 7.04-7.01 (m, 2H) , 5.09 (s, 2H) .
Figure imgf000220_0001
Example 46B
1- [3- (4-Fluoro-phenoxymethyl) -phenyl] -piperidine-4- carboxylic acid ethyl ester
Example 46B was prepared by the procedure ' described in Example 21A, substituting l-bromd-3- - methyl-benzene with Example 46A (205 mg, 0.729 mmol) . The title compound (230 mg, 103%) was obtained as a colorless oil. LCMS (APCI+; Method D) m/z 358, (M+H)+. Analytical HPLC (Method C) Rt=3.114 min (220 nm, 92 area%) .
Figure imgf000221_0001
Example 46C
1- [3- (4-Fluoro-phenoxymethyl) -phenyl] -piperidine-4- carboxylic acid
Example 46B (60 mg, 0.168 mmol) was hydrolyzed with LiOH.H20 (8.5 mg, 0.201 mmol) as described in Example 2IB to provide the title compound (54 mg, 97.67%) as a white powder. E NMR (400 MHz, DMSO-de) δ 12.22 (s, IH) , 7.20 (t, ,7=7.81 Hz, IH) , 7.13 -7.09 (m, 2H) , 7.02-6.99 ( , 3H) , 6.88 (d, d,
J2=8.20 Hz, J2=2.34 Hz, IH) , 6.81 (d, J=7.42 Hz, IH) , 5.00 (s, 2H) , 3.66-3.61 (m, 2H) , 2.79-2.72 (m,- 2H) , 2.43-2.36 ( , IH) , 1.91-1.86 (m, 2H) , 1.67-1.57 (m, 2H) . LCMS (APCI+; Method D) m/z 330, (M+H)+.
Figure imgf000222_0001
Example 46D
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( {1- [3- (4-fluoro- phenoxymethyl) -phenyl] -piperidine-4-carbonyl}- '5 amino) -propionic acid methyl ester
Example 46D (70 mg, 75%) was prepared from Example 46C (50 mg, 0.152 mmol) as described in Example 21C. XE NMR (400 MHz, DMSO-d6) δ 8.14 (d, J=7.81 Hz, IH) , 7.66 (s, IH) , 7.36-7.28 (m, 3H) , 0 7.22-7.19 (m, 3H) , 7.14-7.09 (m,.2H), 7.03-6.99 (m, 3H) , 6.89-6.87 (m, 2H) , 6.82 (d, J=7.42 Hz, IH) , 5.12 (s, 2H), 5.00 (s, 2H) ., 4.48-4.42 (m, IH) , 3.69- 3.63 (m, 2H) , 3.55 (s, 3H) , 2.85-2.76 (m, 2H) , 2.69- 22.59 (m, 2H) , 2.30-2.25 (m, IH) , 1.71-1.64 (m, IH) , 5 1.59-1.48 (m, 3H) . LCMS (APCI+; Method D) m/z 571,
(M+H)+. Analytical HPLC (Method C) Rt=2.401 min (220 nm, 92.78 area%) .
Figure imgf000223_0001
Example 46E
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (4-fluoro- phenoxymethyl) -phenyl] -piperidine-4-carbonyl}- amino) -propionic acid
The title compound was prepared from Example 46D (65 mg, 0.114 mmol) as described in Example 21D. Example 46E (45 mg, 71%) was obtained as a white powder. XE NMR (400 MHz, DMSO-de) . δ 8.02 (d, J=7.81 Hz, IH), 7.67 (s, IH) , 7.36-7.2-8 (m, 3H) , 7.24-7.19 (m, 3H) , 7.14-7.09 (m, 2H) , 7.03-6.98 (m, 3H) , 6.89-6.86 (m, 2H), 6.82-6.81 (m, IH) , 5.12 (s, IH) , 5.01 (s, 2H) , 4.42-4.37 (irt, IH) , 3.70-3.59 (m, 2H) , 2.91-2.86 (m, IH) , 2.81-2.75 (m, >1H), 2.68-2.58 (m, 2H) , 2.29-2.23 (m, IH) , 1.73-1.65 (m, 1H),- 1.62- 1.47 (m, 3H) . LCMS (APCI+) : (M+H)+=557. LCMS (APCI+; Method D) m/z 557, (M+H)+. Analytical HPLC (Method C) Rt=2.319 min (220 nm, 92.94 area%) .
Figure imgf000224_0001
Example 47
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-{ 3- [1- (4-fluoro- phenoxy) -ethyl] -phenyl }-piperidine-4-carbonyl) - amino] -propionic acid
Figure imgf000224_0002
Example 47A
3- (4-Fluoro-phenoxy) α-methylbenzyl bromide
Example 47A was prepared by the procedure described in Example 46A, substituting 3-bromobenzyl alcohol with 1- (3-bromo-phenyl) -ethanol (250 mg, 1.243 mmol). The title compound (230 mg, 50%) was obtained as a colorless liquid. 1H NMR (400 MHz, DMSO-de) δ.7.59 (m, IH) , 7.47-7.44 ( , IH) , 7.42-7.39 ( , IH) , 7.33-7.29 (m, IH) , 7.07-7.03 (m, 2H) , 6.93- 6.89 (m, 2H), 5.50 (q, J=6.23 Hz, IH) , 1.52 (d, J=6.24 Hz, 3H) . LCMS (APCI+; Method D) m/z 295, (M+H)+.
Figure imgf000225_0001
Example 47B
l-{3- [1- (4-Fluoro-phenoxy) -ethyl] -phenyl}- piperidine-4-carboxylic acid ethyl ester
Example 47B was prepared by the procedure described in Example 21A, substituting l-bromo-3- methyl-benzene with Example 47A (225 mg, 0.762 mmol) . The title compound (140 mg, 58%) was obtained as a colorless oil. LCMS (APCI+; Method D) m/z 372, (M+H)+. Analytical HPLC (Method C) Rt=3.375 min (220 nm, 98 area%) .
Figure imgf000225_0002
Example 47C
l-{3- [1- (4-Fluoro-phenoxy) -ethyl] -phenyl }- piperidine-4-carboxylic acid
Example 47B (135 mg, 0.363 mmol) was hydrolyzed with LiOH.H20 (18 mg, 0.436 mmol) as described in Example 2IB to provide the title compound (80 mg, 49%). E NMR (400 MHz, DMSO-d6) δ 7.14 (t, J=7.80 Hz, IH) , 7.04 -6.99 (m, 2H) , 6.96 (s, IH) ,
6.91-6.87 (m, 2H) , 6.816.75 (m, 2H) , 5.35 (q, J=6.25 Hz, IH), 3.61-3.58 (m, 2H) , 2.75-2.67 (m, 2H) , 2.36- 2.29 (m, IH) , 1.89 (m, 2H) , 1.66-1.57 (m, 2H) , 1.51 (d, J=6.64 Hz, 3H) . LCMS (APCI+; Method D) m/z 344 (M+H)+.
Figure imgf000226_0001
Example 47D
3-(l-Benzyl-lH-imidazol-4-yl)-2-[ (l-{ 3- [1- (4-fluoro- phenoxy) -ethyl] -phenyl }-piperidine-4-carbonyl) - amino] -propionic acid methyl ester
Example 47D was prepared from Example 47C
(70 mg, 0.204 mmol) as described in Example 21C. The title compound (70 mg, 51%) was obtained as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J=7.42 Hz, IH) , 7.66 (s, IH) , 7.36-7.32 (m, 2H) , 7.30-7.28 (m, IH) , 7.22-7.20 (m, 2H) , 7.17-1.12 (m, IH) , 7.04-7.00 (m, 2H) , 6.95 (m, IH) , 6.91-6.87 (m, 3H) , 6.80-6.76 (m, 2H) , 5.34 (q, J=6.25 Hz, IH) , 5.12 (s, 2H), 4.47-4.42 (m, IH) , 3.65-3.52 (m, 2H) , 3.55 (s, 3H), 2.85-2.76 (m, 2H) , 2.63-2.55 (m, 2H) , 2.29-2.23 (m, IH) , 1.71-1.66 (m, IH) , 1.61-1.54 (m, 3H) , 1.52 (d, =6.25 Hz, 3H) . LCMS (APCI+ Method D) m/z 585, (M+H)+. Analytical HPLC (Method C) Rt=2.467 min (220 nm, 86.94 area%) .
Figure imgf000227_0001
Example 47E
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-{3- [1- (4-fluoro- phenoxy) -ethyl] -phenyl}-piperidine-4-carbonyl) - amino] -propionic acid
The title compound was prepared from Example 47D (65 mg, 0.1.11 mmol) as described in Example 21D. XH NMR (400 MHz, DMSO-d6) δ 8.01 (d, J=7.42 Hz, IH) , 7.67 (s, IH), 7.36-7.21 (m, IH) , 7.17-7.13 (m, 2H), 7.056-7.00 (m, 3H) , 6.94-6.86 (m, 3H) , 6.80-
6.76 (m, 2H) , 5.35 (q, J=6.23 Hz, IH) , 5.12 (s, 2H) , 4.43-4.46 (m, IH) , 3.67-3.55 (m, 2H) , 2.91-2.87 (m, IH), 2.81-2.75 (m, IH) , 2.65-2.54 (m, 2H) , 2.27-2.19 (m, IH) , 1.92-1.86 (m, IH)', 1.71-1.56 (m, 3H) , 1.52 (d, J=6.23 Hz, 3H) . LCMS (APCI+; Method D) m/z 571, (M+H)+. Analytical HPLC (Method C) Rt=2.372 min (220 nm, 83 area%) .
Figure imgf000228_0001
Example 48
3-Methyl-N- (l-quinolin-3-yl-piperidin-4-yl] succinamic acid
Figure imgf000228_0002
Example 48A
(l-Quinolin-3-yl-piperidin-4-yl) -carbamic acid tert- butyl ester
The title compound was prepared by the procedure described in Example 21A, substituting 1- bromo-3-methyl-benzene with 3-bromo-quinoline (416 mg, 2.00 mmol) and ethyl isonipecotate with piper- idin-4-yl-carbamic acid tert-butyl ester (481 mg, 2.4 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.85 (d, J=2.73 Hz, IH), 7.86-7.83 (m, IH) , 7.80-7.76 (m, IH), 7.53 (d, J=2.34 Hz, IH) , 7.48-7.45 (m, 2H) , 6.89 (br d, J=7.81 Hz, IH) , 3.83 (m, 2H) , 3.50-3.42 (m, IH) , 2.91-2.84 (m, 2H) , 1.88-1.84 (m, 2H) , 1.59- 1.50 (m, 2H) , 1.40 (s, 9H) . LCMS (APCI+; Method D) m/z 328, (M+H)+, major peak 228 [ (M-tBuC02) +H] +. Analytical HPLC (Method C) Rt=2.312 min (220 nm, 92.7 area%) .
Figure imgf000229_0001
Example 48B
l-Quinolin-3-yl-piperidin-4-ylamine . HCl
A suspension of Example 48A (350 mg, 1.07 mmol) in CH2C12 was treated with 4N HCl in dioxane (6 mL) at 0°C under N atmosphere for 30 min and allowed to stir at ambient temperature. After 2 hours the solvent was removed under reduced pressure and the residue obtained was dried under vacuum for 5 h to provide the title compound (325 mg, 95%) as a yellow solid. XE NMR (400 MHz, DMS0-d6) δ 9.23 (d, J=2.73 Hz, IH) , 8.42 (m, IH) , 8.36 (m, 2H) , 8.25 (d, J=9.76 Hz, IH) , 8.07 (d, J=9.76 Hz, IH) , 7.80-7.76 (m, 2H) , 4.09 (d, J=13.28 Hz, 2H) , 3.35-3.27 (m, IH) , 3.08 (t, J=11.71 Hz, 2H), 2.09 (m, 2H) , 1.78-1.68 (m, 2H) . LCMS (APCI+; Method D) m/z 228, (M+H)+. Analytical HPLC (Method C) Rt=1.493 min (220 nm, 94.03 area%) .
Figure imgf000230_0001
Example 48C
'3-Methyl-N- (l-quinolin-3-yl-piperidin-4-yl) - succinamic acid tert-butyl ester
, The title compound was prepared from Example 48B (150 mg, 0.500 mmol) and Example 15G (103 mg, 0.550 mmol) as described in Example 21C. Example 48C (121 mg, 61%) was obtained as a yellow solid. XE NMR (400' MHz, DMS0-d6) δ 8.86 (d, J=2.73 Hz, IH), 7.86-7.82 (m, 2H) , 7.78-7.76 (m, IH) , 7.55 (d, J=3.12 Hz, IH) , 7.48-7.45 (m, 2H) , 3.86-3.79 (m, 2H) , 3.77-3.71 (m, IH) , 2.97-2.90 (m, 2H) , 2.64-2.59 (m, IH) , 2.45 (dd, ^=15.62 Hz,' J2=8.59 Hz, IH) , 2.18 (d, d, Jι=16.00 Hz, J2=6.25 Hz, IH) , 1.87 (m, 2H) , 1.60-1.50 (m, 2H) , 1.38 (s, 9H) , 1.02 (d, J=l .03 Hz, 3H) . LCMS (APCI+; Method D) m/z 398, (M+H)+. Analytical HPLC (Method C) Rt=2.208 min (220 nm, 100 area%) .
Figure imgf000231_0001
Example 48D
3-Methyl-N- (l-quinolin-3-yl-piperidin-4-yl) - succinamic acid. HCl salt .
A solution of Example 48C (110 mg, 0.268 mmol) in CH2C12 (2 mL) was- treated with TFA (0.320 ■ mL, 4.151 mmol) under N2 atmosphere at ambient temperature for 3 h. The reaction was monitored by TLC and by HPLC at 1 h intervals. After 3 h solvents were removed and the residue obtained was evaporated with CHC12 (3X5 mL) , followed by ether (2 mL) and dried under vacuum to provide the1 title compound . (100 mg, 96.72%) as a bright yellow powder. XE NMR - (400 MHz, DMSO-de) δ 9.05 (d, J=2.73 Hz, IH) , 8.02 ' (s, IH) , 7.98-7.92 (m, 2H) , 7.85 (d, <J=7.42 Hz,- IH) , 7.65-7.62 (m, 2Η) , 3.93-3.85 (m, 2H) , 3.82-3.75 (m, IH) , 3.08-3.01 (m, 2H) , 2.65-2.60 (m, IH) , 2.51-2.47 ( , IH), 2.23-2.17 (d, d, ι=6.25 Hz, " 2=16.14 Hz, IH) , 1.89-1.85 (m, 2H) , 1.60-1.51 (m, 2H) , 1.04 (d, =6.64 Hz, 3H) . LCMS (APCI+; Method D) m/z 342,
(M+H)+. Analytical HPLC (Method C) Rt=1.660 min (220 nm, 98.7 area%) .
Figure imgf000232_0001
Example 4.9
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (6, 7- dichloro-benzo [b] thiophen-5-yl) -phenyl] -piperidine-
4-carbonyl } -amino) -propionic acid
Figure imgf000232_0002
Example 49A
(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 mL, 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 was ex- tracted with ethyl acetate (2x1.25 L) . The combined organic layers were dried (MgS04) , filtered, and concentrated to an off-white solid. To the solids were added hexanes (150 L) and the mixture heated to reflux with stirring for 15 min. The mixture was cooled to room temperature and the solid formed was collected by filtration and washed with additional hexanes (100 mL) . The solid was dried under vacuum to provide the title compound (55.6 g, 84%) as a white solid. XE NMR (acetone-d&, 400 MHz) δ 7.39- 7.43 (m, 2H) , 7.31-7.37 (m, IH) , 3.96 (s, 2H) .
Figure imgf000233_0001
Example 49B
(4-Bromo-2, 3-dichloro-phenylsulfanyl) -Acetic acid
To a solution of Example 49A (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 49A. 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 h, 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 NaHS03 and stirred for 45 min. A white precipi- tate formed 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 to provide the title compound
(76.7 g, 99%) containing 90% bro ination at the 4- position.. XE NMR (acetone-d6, 400 MHz) δ 7.70 (d, i =8.98 Hz, IH) , 7-.35 (-d, J=8.59 Hz, IH) , 4.00 (s, 2H) .
Figure imgf000234_0001
5-Bromo-6, 7-dichloro-2, 3-dihydro-benzo [b] thiophen- 3-ol
To a suspension of Example 49B (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 tern-: perature 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 purification. A I L flask was charged with A1C13 (179 g, 1.35 mol) and methylene chloride (250 mL) , 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 completed, the dry ice bath was removed and the reaction was allowed to warm to room temperature and stirred 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 L, 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, the reaction mixture 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 combined organic phases were washed with brine (250 L) , dried (MgS04), filtered, and concentrated to give a dark brown oil (45 g) . This was purified by chromatography on silica gel (Biotage Flash 75 L) eluting with 10% ethyl acetate in hexanes followed by 20% ethyl acetate in hexanes to provide the title compound (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, =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 imgf000236_0001
5-Bromo-6, 7-dichloro-benzo [b] Thiophene
To a IL flask was added Example 49C (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 precip- itate 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 mL) and washed with a saturated solution of NaHC03 (200 mL) followed by brine (200 mL) . The organic phase was dried (MgS0) , filtered, and concentrated. The resulting pink solid was recrystallized from boiling hexanes (200 mL) . The crystals (14.3 g) 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 provide the title compound (20.9 g, 97%). XE NMR (CDC13, 400 MHz) δ 7.97 (s, IH) , 7.51 (d, J=5.47 Hz, IH) , 7.24 (d, J=5.47 Hz, IH) .
Figure imgf000237_0001
5-Iodo-6, 7-dichloro-benzo [b] Thiophene
To a 250 mL flask was added 49D (5.00g, ' 17.7 mmol), copper iodide (0.170 g, 0.887 mmol,) toluene (13.5 mL, ) diglyme (3.5 mL, ) and 1,3-di-
- aminopropane- (0.22 mL, 2.66 mmol). The mixture was . heated under- N2 at 130°C for 10 days. The mixture was cooled to rt, diluted with toluene (50 mL) and washed with NH4C1/ brine (3x100 L) . The organic layer was concentrated to dryness to provide the title compound (5.10 g, 89%) as a 3:1 mixture of
- aryl iodine and aryl bromide as an orange solid. XE NMR (CDC13, 400 MHz) δ 8.24 (s, IH) , 7.51 (d, <J=5.47 Hz, IH), 7.25 (d, J=5.86 Hz, IH.)
Figure imgf000237_0002
Example 49F
5- (3-Bromo-phenyl) -6, 7-dichloro-benzo [b] thiophene
A solution of 49D (1.00 g, 3.55 mmol) in 14 mL DME was treated with 3-bromo-phenylboronic acid (0.855 g, 4.26 mmol) -and K2C03 (0.735 g, 0.532 mmol) . The mixture was purged with N2 for 15 min and Pd(PPh3)4 (0.123 g, 0.106 mmol) was added. The mixture was heated to 85 °C for 72 h and cooled to ambient temperature, and concentrated in vacuo. The residual oil was partitioned with EtOAc (50 mL) and washed with dilute brine solution (3x25 mL) , dried (Na2S04) , filtered, and concentrated under reduced pressure. The resulting white solid was purified by flash chromatography on silica gel eluting with 100% hexanes to provide the title compound (0.428 g, 33.8%) as an off-white solid. XE NMR (CDC13, 400 MHz) δ 7.65 (s, IH) , 7.59 (t, J=1.76 Hz, IH) , 7.53 (m, 2H) , 7.38 ( , IH) , 7.33 (m, 2H) .
Figure imgf000238_0001
Example 49G
1- [3- (6, 7-Dichloro-benzo [b] thiophen-5-yl) -phenyl] - piperidine-4-carboxylic acid ethyl ester
To a solution of 49F (0.072 g, 0.20 mmol) in 0.4 mL DME was added K2P03 (0.076 g, 0.362 mmol) and ethyl isonipecotate (0.047 g, 0.302 mmol). The mixture was purged with N2 for 5 min and (2'-dicyclo- hexylphosphanyl-biphenyl-2-yl) -dimethyl-amine (0.0063 g, 0.0161 mmol) and Pd2(dba)3 (0.0074 g, 0.008 mmol) was added and the mixture heated to 90 °C for 18 h. After cooling to ambient tempera- ture, the mixture was partitioned between dilute brine (10 mL) and EtOAc (10 mL) . The organic layer was washed (3X5 L) with dilute brine and concentrated in vacuo . The residual oil was purified by flash chromatography on silica gel eluting with EtOAc/Hexane 1:2 to give the title compound (0.034 g, 39!) as a light colored oil which solidified upon standing. XE NMR (CDC13, 400 MHz) δ 7.68 (s, IH) , 7.51 (d, J=5.85 Hz, 1H),7.32 (m, 2H) , 6.98 (m, 2H) , 6.91 (s, IH) , 4.17 (q, J=7.41, 14.2 Hz, 2H) , 3.7 (m, 2H), 2..84 (m, 2H), 2.44 (m, IH) , 2.04 (m, 2H) , 1.9 ■ (m, 2H) , 1.27 (t, J=7.02 Hz, 3H) .
Figure imgf000239_0001
Example 49H
1- [3- ( 6, 7-Dichloro-benzo [b] thiophen-5-yl) -phenyl] - piperidine-4-carboxylic acid
''. Example 49G (0.034 g, 0.078 mmol) was processed as in Example 17E substituting 49F for 17D to provide the title compound (0.028 g, 90%) as an off-white powder.
Figure imgf000240_0001
Example 491
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( {1- [3- (6, 7- dichloro-benzo [b] thiophen-5-yl) -phenyl] -piperidine- 4-carbonyl} -amino) -propionic acid
Example 49H (0.034 g, 0.078 mmol) was processed as in Example 17G substituting 49H for 17E to provide the title compound (0.0075 g, 45.8%) as an off-white powder. HPLC (Method C) Rt=2.83 min (85.0 area%, 220 nm) . LCMS (APCI+ Method D) m/z 633.2 (M+) , Rt=3.41 min (254 nm, 83.1 area% . )
The following Methods may be used to test compounds of this invention. Unless otherwise indicated, 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 trans- membrane region and adding an acidic and basic leu- cine zipper sequence to the αl and βl chains, respectively. The αl chain (described in U.S. Patent Application Publication No. US2003/0088061) 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. US2003/0088061) . 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. US2003/0088061) . The resulting PCR product was subcloned 5' to the acidic leucine zipper sequence in the mammalian expression vector pDEF38 (described in U.S. Patent Application Publication No. US2003/0088061) . The resulting plasmid was verified by sequencing. The extracellular domain of βl was 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. US2003/0088061) . The resulting plasmid was verified by sequencing.
Alpha 1 Leucine Zipper (Acidic) construct protein sequence:
MVPRRPAS E VTVACIWLLT VILGFCVSFN VDVKNSMTFS GPVEDMFGYT
VQQYENEEGK WVLIGSPLVG QPK RTGDVY KCPVGRGESL PCVKLDLPVN TSIPNVTEVK ENMTFGSTLV TNPNGGFLAC GPLYAYRCGH LHYTTGICSD VSPTFQWNS lAPVQECSTQ LDIVIVLDGS NSIYPWDSVT AFLNDLLERM DIGPKQTQVG IVQYGENVTH EFNLNKYSST EEVLVAAKKI VQRGGRQTMT ALGTDTARKE AFTEARGARR GVKKVMVIVT DGESHDNHRL KKVIQDCEDE NIQRFSIAIL GSYNRGNLST EKFVEEIKSI ASEPTEKHFF NVSDELALVT IVKTLGERIF ALEATADQSA ASFEMEMSQT GFSAHYSQDW VMLGAVGAYD WNGTVVMQKA 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 ETLSDNWNI 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 ISSVCCVFA QTDENRCLKA NAKSCGECIQ AGPNCG CTN 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 1IDAYNSLSS EVILENGKLS EGVTISYKSY CKNGVNGTGE NGRKCSNISI
GDEVQFEISI TSNKCPKKDS DSFKIRPLGF TEEVEVILQY ICECECQSEG IPESPKCHEG NGTFECGACR CNEGRVGRHC ECSTDEVNSE DMDAYCRKEN SSEICSNNGE 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 i talics
Mature polypeptides 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:
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/mL in 50mM NaHC03, pH 9.2, and incubated overnight at 4°C. The plates then were 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) at room tempera- ■ ture. Compounds of this invention were prepared in DMSO at 200X the desired final assay concentration. Final concentrations were selected from a range between 0.01 nM-100 μM. DMSO inhibitor stocks were then diluted to 2X final concentration in Dilution 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's protocol. The plates were incubated for 1 hour at room tempera- ture, 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) 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. Binding was then analyzed by time resolve fluorescence (TRF) using a Victor Plate-reader (PerkinElmer) . Results were analyzed using the equations below. The percent of inhibition was plotted versus the log concentration of inhibitor across a twelve point titra- tion, and a linear regression trend-line was drawn. Specific binding = TRF signal (Collagen-IV-biotin in Dilution Buffer plus Ab, DMSO only or DMSO + inhibitor) - TRF signal (Collagen-IV-biotin with EDTA)
1 - /specific binding of Collagen-IV-biotin with inhibitor in DMSO ' xlOO i specific binding of Collagen-IV-biotin with DMSO only
As a variation in the assay, the effect of serum protein in the assay was determined by substi- tuted 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-αl cell adhesion to Collagen IV
Generation of K562-αl cells
The αl cDNA was subcloned into the mammal- ian expression vector pMH-neo (Hahn et al. (1993)
Gene 127:267). 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 resus- pended in complete RPMI with 10! FBS and 0.5mg/mL G418 (Sigma-Aldrich) to select for the neomycin resistance conferred by the pMHneo plasmid. Trans- fectants with functional αl expression were selected by panning the cells for binding to Type IV collagen (an αl ligand) . In panning experiments, K562 trans- fectants 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- six well Immulon-4 plates were coated with either 1.25 μg/mL collagen IV (Sigma) in CMF-PBS or the αl mAb at 5 μg/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 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 selected 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/7OOμL RPMI) to generate 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- 30 min. K562-αl cells were counted, spun down, washed once with RPMI and resuspended in RPMI+60ng/- mL 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. Adherent 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 A57onm and A4ιonm- 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 variation 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 50! FBS was then compared. Percent cells binding = / A570-A410(binding to Collagen IV) x lOO
A570-A410(binding to Alphal antibody)
Figure imgf000249_0001
Percent Inhibition H i - / % cells binding with inhibitor xlOO % cells binding with DMSO
Figure imgf000249_0002
All of the compounds of this invention that were tested exhibited 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 for- mula:
Figure imgf000250_0001
wherein Ar is selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
A, together with the nitrogen atom bound thereto, forms 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 and alkoxy substituents, if present, may be attached to a carbon atom in said heterocyclic group, wherein the one or more thioalkyl and the one or more halo substituents, if present, may not be attached to a nitrogen atom in said heterocyclic group, and wherein the one or more halo, thioalkyl, and the one or more hydroxy substituents, if present, may not be attached to a carbon atom which is adjacent to a nitrogen atom in said heterocyclic group;
W is selected from the group consisting of -C(X)-NH- and -NH-C(X)-, wherein X is oxygen or sulfur,
Y is selected from the group consisting of a bond, alkylene, and substituted alkylene;
R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminocarbonyloxy, oxycarbonyl- amino, aminoacyl, acylamino, aminocarbonylamino, aminosulfonylamino, a inosulfonyl, sulfonylamino, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, carboxyl, and carboxyl■ esters; "or""R3-C-R4 "fofms" a cycloalkyl, substituted cycloalkyl, heterocyclic, or substituted heterocyclic group;
R5 is selected from the group consisting of CRaRaC02Ra> C0C02Ra, carboxyl, carboxyl esters, -C0- NRaORa, -C0NRaS02Ra, heterocyclic, cyano, and hydroxy- alkyl; wherein Ra is selected from the group consisting of hydrogen, alkyl, aryl, substituted aryl, heterocyclic, and substituted heterocyclic; with the proviso that when Y is a bond and W is C(0)NH, then R3 and/or R4 are not amino, substituted amino, hydroxy, or alkoxy; and a pharmaceutically acceptable salt, a prodrug, or a tautomer- thereof .
2. The compound of claim 1 wherein A together with the nitrogen atom bound thereto form a piperidine ring.
3. The compound of claim 1 wherein W is -C(0)-NH-.
4. The compound of claim 1 wherein W is -NH-C(O)-.
5. The compound of claim 1 wherein Y is a bond.
6. The compound of claim 1 wherein Y is methylene.
7. The compound of claim 1 wherein R4 is hydrogen.
8. The compound of claim 1 wherein R5 is -C02H.
9. The compound of claim 1 wherein R3 is alkyl or substituted alkyl.
10. The compound of claim 9 wherein R3 is selected from the group consisting of: methyl;
N-benzylimidazol-4-ylmethylene' and phenylacetamidomethylene .
11. The compound of claim 1 wherein Ar is selected from the group consisting of:
5- (2, 3-dichlorophenyl) pyridin-3-yl; 4- (2, 3-dichlorophenyl) pyridin-2-yl; 5- (2, 3-dichlorophenyl) pyridin-2-yl; 3-methyl-5- (2, 3-dichlorophenyl) pyrdin-2- yi;
3-methyl-4- (2, 3-dichlorophenyl) pyridin-2- yi;
5-methyl-4- (2, 3-dichlorophenyl) pyridin-2- yi;
5- (2, 3-dichlorophenyl) pyridin-2-yl;
6-methyl-3- (2, 3-dichlorophenyl) phenyl;
4- (2, 3-dichlorophenyl) thiazol-2-yl;
5-bromo-pyridin-3-yl ; pyrdin-3-yl;
3- (2, 3-dichlorophenyl) phenyl;
6-methyl-3- (2, 3-dichlorophenyl) phenyl;
3-methylphenyl;
3-ethylphenyl ;
3-isϊdpfbpylphe'riyl";
3-trifluoromethylphenyl;
3-dimethylaminophenyl ;
3- ( 4-fluorophenoxy) phenyl ;
3-fluoro-5-trifluoromethylphenyl;
3, 5-trifluoromethylphenyl;
2-methylquinolin-6-yl;
3, 4-dichlorophenyl;
2, 3-dichlorophenyl;
6, 7-dichlorobenzo [b] thiophen-5-yl; 3, 5-dichlorophenyl; benzo [1 , 3] dioxol-6-yl;
4-t-butylphenyl;
3-chloro-6-nitrophenyl;
5-bromo-4-fluoro-2-nitrophenyl;
5- (N-morpholino) -2-nitrophenyl;
3- (trifluoromethylthio) phenyl;
3- (trifluoromethoxy) phenyl;
3- (trifluoromethoxy) phenyl; quinolin-3-yl; quinolin-3-yl; isoquinolin-3-yl; isoquinolin-3-yl;
(4-fluorophenyloxy) benz-3-yl;
3- (4-fluorophenyloxy) ethylphenyl; quinolin-3-yl; and phenyl.
12. A compound selected from the group consisting of:
2- { [4 ' - (2 , 3-Dichloro-phenyl) -3, 4 , 5, 6-tetrahydro-2H- [1,2' ] bipyridinyl—4-carbonyl] -amino}-propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [4 ' - (2, 3-dichlorophenyl) -3, 4,5, 6-tetrahydro-2H- [1,2'] bipyridinyl-4- carbonyl] -amino}-propionic acid;
2-{ [6" - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1,2' ] ipyridinyl- -carbonyl] -amino} -propionic acid; 2-{ [6 ' - (2, 3-Dichloro-phenyl) -3 ' -methyl-3, 4,5,6- tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino}- propionic acid;
2-{ [4 ' - (2, 3-Dichloro-phenyl) -3 ' -methyl-3, 4, 5, 6- tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino}- propionic acid/2-{ [4 ' - (2, 3-Dichloro-phenyl) -5 ' - methyl-3, 4, 5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4- carbonyl] -amino}-propionic acid;
2-{ [5 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,2' ]bipyridinyl-4-carbonyl] -amino}-propionic acid; 2-{ [5' - (2, 3-Dichloro-phenyl) -3,4,5, 6-tetrahydro-2H- [1, 3 '-]bipyridinyl-4-Carbonyl] -aminό} -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [5 ' - (2, 3-dichlorophenyl) -3, 4, 5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4- carbonyl] -amino} -propionic acid;
3-Benzoylamino-2-{ [5 '- (2, 3-dichloro-phenyl) -3,4,5,6- tetrahydro-2H- [1, 3 ' ]bipyridinyl-4-carbonyl] -amino}- propionic acid;
2- [ (5 ' -Bromo-3, 4,5, 6-tetrahydro-2H- [1,3'] bipyridin- yl-4-carbonyl) -amino] -propionic acid; 3-Benzoylamino-2- [ (5 ' -bromo-3, 4, 5, 6-tetrahydro-2H- [1,3'] bipyridinyl-4-carbonyl) -amino] -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (5 ' -bromo-3, 4,5,6- tetrahydro-2H- [1, 3 ' ]bipyridinyl-4-carbonyl) -amino] - propionic acid; •
2-[ (3,4,5, 6-Tetrahydro-2H-[l,3' ]bipyridinyl-4-car- bonyl) -amino] -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (3,4,5, 6-tetra- hydro-2H- [1,3'] bipyridinyl-4-carbonyl) -amino] - propionic acid;
N- [5 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1, 3 ' ]bipyridinyl-4-yl] -3-methyl-succinamic acid; N- [5 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,3' ]bipyridinyl-4-yl] -3-methyl-succinamic/N- [5 ' - (2, 3-Dichloro-phenyl) -3, 4, 5, 6-tetrahydro-2H- [1,3']- bipyridinyl-4-yl] -2-methyl-succinamic acid; 2, 3-Dichloro-3 ' -2 [ (piperidine-4-carbonyl) -amino] - propionic acid;
2, 3-Dichloro-4 ' -methyl-3 ' -2 [ (piperidine-4-carbonyl) - amino] -propionic acid;
2- ( { 1- [4- (2, 3-Dichloro-phenyl) -thiazol-2-yl] -piper- idine-4-carbonyl} -amino) -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [4- (2, 3-di- chloro-phenyl) -thiazol-2-yl] -piperidine-4-carbonyl}- amino) -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (1-m-tolyl-piper- idine-4-carbonyl) -amino] -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-ethyl-phen- yl) -piperidine-4-carbonyl] -amino} -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-isopropyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-trifluoro- methyl-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-dimethyl- amino-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (4-fluoro- phenoxy) -phenyl] -piperidine-4-carbonyl} -amino) - propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-fluoro-5- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 5-bis- trifluoromethyl-phenyl) -piperidine-4-carbonyl] - amino}-propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (2-methyl- quinolin-6-yl) -piperidine-4-carbonyl] -amino} - propionic acid;
3- (l-Benzyl-lH-i idazol-4-yl) -2-{ [1- (3, 4-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (2, 3-dichlorophenyl) -piperidine-4-carbonyl] -amino}-propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (6, 7-dichlorobenzo [b] thiophen-5-yl) -piperidine-4-carbonyl] - amino} -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3, 5-dichloro- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid;
2- [ (1-Benzo [1, 3] dioxol-5-yl-piperidine-4-carbonyl) - amino] -3- (l-benzyl-lH-imidazol-4-yl) -propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (4-tert-butyl- phenyl) -piperidine-4-carbonyl] -amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-chloro-2- nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-bromo-4- fluoro-2-nitro-phenyl) -piperidine-4-carbonyl] - amino}-propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (5-morpholin-4- yl-2-nitro-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-trifluoro- methylsulfanyl-phenyl) -piperidine-4-carbonyl] - amino} -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2-{ [1- (3-trifluoromethoxy-phenyl) -piperidine-4-carbonyl] -amino}- propionic acid;
3-Benzoylamino-2-{ [1- (3-trifluoromethoxy-phenyl) - piperidine-4-carbonyl] -amino}-propionic acid; 3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-quinolin-3-yl- piperidine-4-carbonyl) -amino] -propionic acid; 3-Benzoylamino-2- [ (l-quinolin-3-yl-piperidine-4- carbonyl) -amino] -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-isoquinolin-4- yl-piperidine-4-carbonyl) -amino] -propionic acid; 3-Benzoylamino-2- [ (l-isoquinolin-4-yl-piperidine-4- carbonyl) -amino] -propionic acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( {1- [3- (4-fluoro- phenoxymethyl) -phenyl] -piperidine-4-carbonyl } - amino) -propionic • acid;
3- (l-Benzyl-lH-imidazol-4-yl) -2- [ (l-{3- [1- (4-fluoro- phenoxy) -ethyl] -phenyl }-piperidine-4-carbonyl) - amino] -propionic acid;
3-{ [5 ' - (l-Methyl-lH-indol-5-yl) -3, 4, 5, 6-tetrahydro- 2H- [1, 3 ' ]bipyridinyl-4-carbonyl] -amino} -butyric acid;
3-Methyl-N- (l-quinoliή-3-yl-piperidin-4-yl) -suc- cinamic acid; and
3- (l-Benzyl-lH-imidazol-4-yl) -2- ( { 1- [3- (6, 7-di- chloro-benzo [b] thiophen-5-yl) -phenyl] -piperidine-4- carbonyl} -amino) -propionic. acid.
13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of claim 1.
14. 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, said method comprising obtaining a biological sample from said patient and assaying said sample for the presence of VLA-1.
15. A method of inhibiting adhesion of mammalian cells to the extracellular matrix mediated, at least in part, by VLA-1, said method comprising contacting said cells with a compound of claim 1.
16. A method of treating a disease, disorder, or condition, 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 of claim 1.
17. The method of claim 16 wherein said disease, disorder, or condition 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.
18. The method of claim 17 wherein said disease, disorder, or condition is a fibrotic disease.
19. The method of claim 18 wherein said fibrotic disease is selected from the group consisting of systemic sclerosis, mixed connective tissue disease, fibrodysplasia, fibrocystic disease, sarcoidosis, and myositis.
20. The method according to claim .18 wherein said fibrotic disease has a manifestation of fibrotic vascular intimal hypertrophy, and is selected from the group consisting of vasculitis, poly- arteritis nodosa, and temporal arteritis.
21. The method according to claim 18
• wherein said fibrotic disease has a manifestation of fibrotic hypertrophy of skin and/or muscle tissue, and is selected from the group consisting of sclero- derma, eosinophilic fasciitis, discoid lesions associated with lupus or discoid lupus, and surgical adhesions.
22. The method according to claim 18 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 .
23. The method according to claim 18 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.
24. The method according to claim 18 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 (BPH) , nonalcoholic steato hepatitis, and fibrosis of the liver.
25. The method according to claim 18 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, alport syndrome, glomerulonephritis, and diabetic nephritis.
26. The method according to claim 16 wherein said disease, disorder, or condition is cancer.
27. The method according to claim 26 wherein said cancer is a tumor or a neoplasm selected from the group consisting of carcinomas, adenocarcinomas, and sarcomas.
28. The method according to claim 26 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-produc- ing 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, retino- blastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms ' s tumors, gall bladder "cancer, trophoblasti-c neoplasms, hemangiopericytoma, and Kaposi's sarcoma.
29. The method according to claim 26 wherein said cancer is a cell proliferative disorders, and is selected from the group consisting of angiogenesis-mediated diseases, benign tumors, acoustic neuromas, neurofibromas, pyogenic granul- omas, biliary tract cancer, choriocarcinoma, esoph- ageal cancer, gastric cancer, intraepithelial neoplasms, lung cancer, and neuroblastomas .
30. The method according to claim 26 wherein said administration is selected from the group consisting of orally, intravenously,, par- enterally, transdermally, topically, rectally, and intranasally.
31. The method according to claim 26 wherein said mammal is selected, from the group consisting of humans, primates, pet or companion animals, laboratory animals, and farm animals.
32. The compound of claim 1 wherein the compound exhibits a.biological...a.ct.±vity..θ-f-.-at.,least; . fifty percent inhibition when tested at a concentration of 50 μM.
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