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    • 专利摘要:
    A compound of formula (I) which is a betronicotene receptor antagonist and useful for the treatment of osteoporosis is disclosed: (I) Wherein Y is CR'R 'or NR'C (O); R 1 is C 0-6 alkyl, -Het, -C 0-6 alkyl -Ar, H, -C 1-6 alkyl, -CN or S (O) R k and g; R 2 is a residue of the formula: W is (CHR g ) a -U-CHR g ) b -; U is absent or CO, CR g 2, C ( = CR g 2), S (O) k, O, NR g, CR g OR g, CR g (OR k) CR g 2, CR g 2 CR g (OR k), C ( O) CR g 2, CR g 2 C (O), CONR i, NR i CO, OC (O), C (O) O, C (S) O, OC (S) , C (S) NR g, NR g C (S), S (O) 2 NR g, NR g S (O) 2 N = N, NR g NR g, NR g CR g 2, CR g 2 NR g , CR g 2 O, OCR g 2 , C? C, CR g = CR g , Ar or Het; G is NRe, S or O; R g is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl or Ar-C 0-6 alkyl; R k is R g , -C (O) R g , or -C (O) OR f ; R i is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl, Ar-C 0-6 alkyl, or halogen, CN, NR g 2, OR g, SR g, CO 2 R g, and CON (R g) 1 gae selected from 2 to three groups substituted by C 1-6 alkyl; R f is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl; R e is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl-C 0-6 alkyl, or (CH 2 ) k CO 2 R g ego; R b and R c are independently hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl, Het-C 0-6 alkyl, or C 3-6 cycloalkyl, -C 0-6 alkyl, halogen, CF 3 , oR f, S (O) k R f, COR f, NO 2, N (R f) 2, CO (NR f) 2, CH 2 N (R f) selected from the second or, or R b and R c is halogen, CF 3, C 1-4 alkyl, OR f, S (O) k R f, COR f, CO 2 R f, OH, NO 2, N (R f) 2, CO (NR f bonded to each other ) 2 , and CH 2 N (R f ) 2 , or a 5 to 6 membered aromatic or nonaromatic carbocyclic or heterocyclic ring optionally substituted with up to three substituents selected from CH 2 N (R f ) 2 ; Or methylenedioxy; Q 1 , Q 2 , Q 3 and Q 4 are independently nitrogen or CR y , with the proviso that no more than one of Q 1 , Q 2 , Q 3 and Q 4 is nitrogen; R 'is hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl or C 3-6 cycloalkyl -C 0-6 alkyl; R "is R ', -C (O) R ' or -C (O) OR ', and; R y is hydrogen, halo, -OR g, -SR g, -CN , -NR g R k, -NO 2 , CF 3, CF 3 S ( O) r -, -CO 2 R g, -COR g or -CONR g 2, or halo, -OR g, -SR g, -CN , -NR g R ", -NO 2, -CF 3, R'S ( O) r -, -CO 2 R g, -COR g or -CONR g 2 by an optionally substituted C 1-6 alkyl; a is 0, 1 or 2; b is 0, 1 or 2; k is 0, 1 or 2; r is 0, 1 or 2; s is 0, 1 or 2; u is 0 or 1; v is 0 or 1;
    公开号:KR20010086355A
    申请号:KR1020017001583
    申请日:1999-08-03
    公开日:2001-09-10
    发明作者:피터 제이. 맨리；윌리엄 에이치. 밀러
    申请人:스튜어트 알. 수터, 스티븐 베네티아너, 피터 존 기딩스；스미스클라인 비참 코포레이션；
    IPC主号:
    专利说明:

    [0001] VITRONECTIN RECEPTOR ANTAGONISTS [0002]
    [2] Integins are a progenitor of cell adhesion receptors, which are dendritic proteins that are expressed on various cells. These cell surface adhesion receptors include gpIIb / IIIa (fibrinogen receptor) and [alpha] v [ beta] 3 (vitronectin receptor). The fibrinogen receptor gpIIb / IIIa is expressed on the platelet surface and mediates platelet aggregation and hematopoietic clot formation at the bleeding site of the wound. Phillips et al., Blood , 1988 , 71 , 831. The vitronectin receptor V 3 is expressed on a number of cells, including endothelial cells, smooth muscle cells, osteoclasts, and tumor cells and thus has a variety of functions. The α v β 3 receptor expressed on the osteoclast membrane mediates the attachment of osteoclasts to the bone matrix, which is a key step in the bone resorption process. Ross et al . , J. Biol. Chem. 1987 , 262 , 7703. Osteoporosis is a disease characterized by excess bone resorption. The α v β 3 receptor expressed on human aortic smooth muscle cells mediates the migration of these cells into the neointima, which can cause recurrent stenosis after coronary angioplasty. Brown, etc., Cardiovscular Res., 1994, 28 , 1815. In addition, disclosed in such as Brooks, Cell, 1994, 79, 1157 ] is α v β 3 antagonist is able to promote tumor regression by inducing apoptosis of the vascular vessel . Therefore, agents that block bitronectin receptors are useful for treating diseases such as osteoporosis, restenosis, and cancer.
    [3] Vitronectin receptor is now known to refer to three different integrins represented by the α v β 1, α v β 3 and α v β 5 receptors. Horton et al. , Int. J. exp. Pathol ., 1990 , 71, 741. [alpha] v [ beta] 3 is a very potent antioxidant , including fibrin, fibrinogen, laminin, thrombospondin, bitonectin, von willebrand's factor, osteopontin and bone- It binds with various ligands. α v β 5 binds to bitronectin. The vitronectin receptor α v β 5 has been implicated in cell attachment of various cell types including microvascular endothelial cells (Davis et al . , J. Cell. Biol. , 1993 , 51 , 206) The role was confirmed. Brooks et al., Science , 1994 , 264, 569. This integrin is expressed on blood vessels of human wounded granular tissue, but not normal skin.
    [4] The vitronectin receptor is known to bind to a bone matrix protein containing a tri-peptide Arg-Gly-Asp (or RGD) motif. Hence, Horton et al . , Exp. Cell Res . 1991 , 195 , 368) discloses that RGD-containing peptides and anti-vitronectin receptor antibody (23C6) inhibit dentin reabsorption and cell diffusion by osteoclasts. Also, as described in Sato et al., J. Cell Biol. , 1990 , 11, 1713], a zodiac peptide containing the RGD sequence, is a potent inhibitor of bone resorption in tissue culture and inhibits osteoclast attachment to bone.
    [5] Certain compounds have been found to be potent inhibitors of the V 3 and V 5 receptors. In particular, such compounds have been found to be a more potent inhibitor of the bitronectin receptor than the fibrinogen receptor.
    [6] SUMMARY OF THE INVENTION
    [7] The present invention encompasses compounds of formula (I) as described hereinafter, which have pharmacological activity against the inhibition of the vitronectin receptor and are useful for the treatment of cardiovascular diseases, such as inflammation, cancer and arteriosclerosis and restenosis, and osteoporosis It is useful for the treatment of diseases in which bone resorption is a factor.
    [8] The present invention also relates to pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutical carrier.
    [9] The invention also relates to a method of treating a disease mediated by a vitronectin receptor. In particular aspects, the compounds of the present invention are useful for the treatment of diseases in which bone resorption is a factor, such as arteriosclerosis, restenosis, inflammation, cancer and osteoporosis.
    [1] The present invention relates to pharmaceutical active compounds which inhibit the vitronectin receptor and are useful in the treatment of diseases in which bone resorption is a factor, such as cardiovascular diseases such as inflammation, cancer and arteriosclerosis and restenosis, and osteoporosis.
    [10] The present invention includes novel compounds that are more potent inhibitors of the bitronectin receptor than the fibrinogen receptor. The present invention includes a compound of formula (I) or a pharmaceutically acceptable salt thereof:
    [11]
    [12] In this formula,
    [13] Y is CR ' R ' or NR ' C (O);
    [14] R 1 is C 0-6 alkyl, -Het, -C 0-6 alkyl -Ar, H, -C 1-6 alkyl, -CN or S (O) R k and g;
    [15] R 2 is a residue of the formula:
    [16]
    [17] W is (CHR g ) a -U- (CHR g ) b -
    [18] U is absent or CO, CR g 2, C ( = CR g 2), S (O) k, O, NR g, CR g OR g, CR g (OR k) CR g 2, CR g 2 CR g (OR k), C ( O) CR g 2, CR g 2 C (O), CONR i, NR i CO, OC (O), C (O) O, C (S) O, OC (S) , C (S) NR g, NR g C (S), S (O) 2 NR g, NR g S (O) 2 N = N, NR g NR g, NR g CR g 2, CR g 2 NR g , CR g 2 O, OCR g 2 , C C, CR g = CR g , Ar or Het;
    [19] G is NRe, S or O;
    [20] R g is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl or Ar-C 0-6 alkyl;
    [21] R k is R g , -C (O) R g , or -C (O) OR f ;
    [22] R i is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl, Ar-C 0-6 alkyl, or halogen, CN, NR g 2, OR g, SR g, CO 2 R g, and CON (R g) 1 gae selected from 2 to three groups substituted by C 1-6 alkyl;
    [23] R f is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl;
    [24] R e is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl-C 0-6 alkyl, or (CH 2 ) k CO 2 R g ego;
    [25] R b and R c are independently hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl, Het-C 0-6 alkyl, or C 3-6 cycloalkyl, -C 0-6 alkyl, halogen, CF 3 , oR f, S (O) k R f, COR f, NO 2, N (R f) 2, CO (NR f) 2, CH 2 N (R f) selected from the second or, or R b and R c is halogen, CF 3, C 1-4 alkyl, OR f, S (O) k R f, COR f, CO 2 R f, OH, NO 2, N (R f) 2, CO (NR f bonded to each other ) 2 , and CH 2 N (R f ) 2 , or a 5 to 6 membered aromatic or nonaromatic carbocyclic or heterocyclic ring optionally substituted with up to three substituents selected from CH 2 N (R f ) 2 ; Or methylenedioxy;
    [26] Q 1 , Q 2 , Q 3 and Q 4 are independently nitrogen or CR y , with the proviso that no more than one of Q 1 , Q 2 , Q 3 and Q 4 is nitrogen;
    [27] R 'is hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl or C 3-6 cycloalkyl -C 0-6 alkyl;
    [28] R "is R ', -C (O) R' or -C (O) OR ';
    [29] R y is hydrogen, halo, -OR g, -SR g, -CN , -NR g R k, -NO 2, CF 3, CF 3 S (O) r -, -CO 2 R g, -COR g or -CONR g 2, or halo, -OR g, -SR g, -CN , -NR g r ", -NO 2, -CF 3, r'S (O) r -, -CO 2 r g, -COR g or by -CONR g 2, optionally substituted C 1-6 alkyl;
    [30] a is 0, 1 or 2;
    [31] b is 0, 1 or 2;
    [32] k is 0, 1 or 2;
    [33] r is 0, 1 or 2;
    [34] s is 0, 1 or 2;
    [35] u is 0 or 1;
    [36] v is 0 or 1;
    [37] Also included in the present invention are pharmaceutically acceptable addition salts and complexes of the compounds of the present invention. Where the compounds of the present invention are capable of having one or more subtitle centers, unless otherwise specified, the present invention includes each unique non-racemic compound that can be synthesized and isolated by conventional methods. When the compound has a carbon-carbon double bond, both the cis (Z) and trans (E) isomers are included in the scope of the present invention. The compound , Each tautomeric form, whether in equilibrium or in a form appropriately substituted with R ', can be in the form of a tautomeric form, such as a keto-enol tautomer, And the like.
    [38] The compounds of formula (I) inhibit the binding of vitronectin and other RGD-containing peptides to the vitronectin receptor. Inhibition of the vitronectin receptor on osteoclasts inhibits osteoclast bone resorption and is useful for the treatment of diseases related to bone resorption, such as osteoporosis and osteoarthritis.
    [39] In another aspect, the invention is directed to a method for stimulating bone formation comprising administering a compound that causes increased osteocalcin release. Increased bone formation has obvious advantages for diseases that lack mineralized bone mass and require bone remodeling, such as fracture treatment and fracture prevention. Diseases or metabolic abnormalities leading to bone structure loss will also be improved with this treatment. For example, hyperparathyroidism, Paget's disease, hypercalcemia of malignant tumors, osteolytic lesions caused by bone metastasis, bone loss due to immobilization or sex hormone deficiency, Betset disease, osteomalacia, osteoporosis, The symptoms can be improved by administering the compounds of the present invention.
    [40] In addition, because the compounds of the present invention inhibit the vitronectin receptors on a number of different types of cells, these compounds are useful for the treatment of cardiovascular diseases, such as rheumatoid arthritis and psoriasis, inflammatory diseases, and arteriosclerosis and restenosis something to do. The compounds of formula (I) of the present invention can be used in the treatment of thromboembolic diseases, asthma, allergies, adult respiratory distress syndrome, graft versus host disease, tissue graft rejection, septic shock, eczema, contact dermatitis, And may be useful for the treatment and prevention of other diseases including diseases and the like. The compounds of the present invention may also be useful in the treatment of wounds.
    [41] The compounds of the present invention are also useful for treatment, including prevention of angiogenic diseases. As used herein, angiogenic diseases include conditions involving abnormal angiogenesis. When new blood vessel growth is the cause of the disease or contributes to disease-related pathology, inhibition of angiogenesis will reduce blood supply to the tissue and thereby contribute to tissue mass reduction based on blood supply requirements. Examples include tumor growth, an ongoing requirement for angiogenesis to allow tumors to grow and solid tumors to metastasize. Therefore, the compounds of the present invention prevent tumor metastasis and tumor growth by inhibiting tumor tissue angiogenesis.
    [42] Therefore, according to the method of the present invention, inhibition of angiogenesis using the compounds of the present invention can ameliorate the symptoms of the disease and, in some cases, treat the disease.
    [43] Another therapeutic target disease of the compounds of the present invention is ocular disease characterized by angiogenesis. Such ocular diseases include corneal angiogenesis diseases such as corneal neovascular disease, herpetic keratitis, platelet keratitis, pterygium associated with contact lens use and angiogenesis parasites. Additional ocular diseases also include age-related macular degeneration, presumed anterior hystoplasmosis, retinopathy of prematurity, and angiogenic glaucoma.
    [44] The present invention further provides a method of inhibiting tumor growth comprising administering an antitumor agent, such as topotecan and cisplatin, stepwise or in a physical combination, with a compound of formula (I).
    [45] Regarding the anger (I):
    [46] Suitably, R < 2 > is a residue of the formula:
    [47]
    [48] Wherein Q 1 , Q 2 and Q 3 are each CR y , Q 4 is CR y or nitrogen and u is 0, preferably each R 'is hydrogen and R "is hydrogen or C 1 and -6 alkyl, W is - (CH 2) 1-4 -, and, Q 4 is CR y and R y are hydrogen.
    [49] Optionally, R < 2 > is a residue of the formula:
    [50]
    [51] Wherein R, Q 1, Q 2, and Q 3 are each CH and u is 0, and preferably, each R 'is hydrogen, R "is hydrogen or C 1-6 alkyl, W is - (CH 2 ) 1-4 - and v is zero.
    [52] Optionally, R < 2 > is a residue of the formula:
    [53]
    [54] Wherein G is NH and R b and R c are each hydrogen, and preferably W is - (CH 2 ) 1-4 -.
    [55] Optionally, R < 2 > is a residue of the formula:
    [56]
    [57] Wherein, G is NH and R b and R c is halogen, CF 3, C 1-4 alkyl, OR f, S (O) k R f, COR f, CO 2 R f, OH, NO 2 combine with each other , N (R f) 2, CO (NR f) 2, and CH 2 N (R f) a by a substituent of up to three selected from 2, optionally substituted, 5- to 6-membered aromatic or non-aromatic carbocyclic or heterocyclic Form a cyclic ring; Or methylenedioxy. Preferably, R b and R c combine with each other to form a six-membered aromatic carbocyclic or heterocyclic ring and W is - (CH 2 ) 1-4 -.
    [58] Optionally, R < 2 > is a residue of the formula:
    [59]
    [60] Wherein R 'is hydrogen or C 1-6 alkyl, R g is hydrogen or C 1-6 alkyl and s is 0, 1 or 2, preferably W is - (CH 2) 1-4 - is.
    [61] With respect to formula (I), suitably R < 1 > is phenyl, benzyl, pyridyl, imidazole, oxazolyl or thiazolyl. Preferably, R < 1 > is phenyl. Suitably, Y is NHC (O).
    [62] Representative examples of the novel compounds of the present invention are as follows:
    [63] Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (2-amino- ;
    [64] Ethyl] amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (Compound ;
    [65] Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (2-amino-3- ; And
    [66] Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (hereinafter referred to as ";
    [67] Or a pharmaceutically acceptable salt thereof.
    [68] Where the compounds of the present invention are capable of possessing one or more subtitle centers, unless otherwise specified, the present invention includes each unique non-racemic compound that can be synthesized and isolated by conventional techniques. According to the present invention, the (S) form of the compound of formula (I) is preferred.
    [69] When the compound has an unsaturated carbon-carbon double bond, both the cis (Z) and trans (E) isomers are within the scope of the present invention. In some cases, the meaning of one substituent is not related to its meaning, or in other cases it is not related to another meaning.
    [70] The drug precursors of the compounds of the present invention are also encompassed by the present invention. The drug precursor is considered to be a covalently bonded carrier which releases the drug substance of formula (I) in vivo. Therefore, another aspect of the invention relates to novel drug precursors, which are also the compounds of formula (II) or pharmaceutically acceptable salts thereof, which are intermediates for the preparation of compounds of formula (I)
    [71]
    [72] In this formula,
    [73] Y is CR ' R ' or NR ' C (O);
    [74] R 1 is C 0-6 alkyl, -Het, -C 0-6 alkyl -Ar, hydrogen, -C 1-6 alkyl, -CN or S (O) R k and g;
    [75] R 2 is a residue of the formula:
    [76]
    [77] W is (CHR g ) a -U- (CHR g ) b -
    [78] U is absent or CO, CR g 2, C ( = CR g 2), S (O) k, O, NR g, CR g OR g, CR g (OR k) CR g 2, CR g 2 CR g (OR k), C ( O) CR g 2, CR g 2 C (O), CONR i, NR i CO, OC (O), C (O) O, C (S) O, OC (S) , C (S) NR g, NR g C (S), S (O) 2 NR g, NR g S (O) 2 N = N, NR g NR g, NR g CR g 2, CR g 2 NR g , CR g 2 O, OCR g 2 , C C, CR g = CR g , Ar or Het;
    [79] G is NRe, S or O;
    [80] R g is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl or Ar-C 0-6 alkyl;
    [81] R k is R g , -C (O) R g , or -C (O) OR f ;
    [82] R i is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl, Ar-C 0-6 alkyl, or halogen, CN, NR g 2, OR g, SR g, CO 2 R g, and CON (R g) 1 gae selected from 2 to three groups substituted by C 1-6 alkyl;
    [83] R f is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl;
    [84] R e is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl-C 0-6 alkyl, or (CH 2 ) k CO 2 R g ego;
    [85] R b and R c are independently hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl, Het-C 0-6 alkyl, or C 3-6 cycloalkyl, -C 0-6 alkyl, halogen, CF 3 , oR f, S (O) k R f, COR f, NO 2, N (R f) 2, CO (NR f) 2, CH 2 N (R f) selected from the second or, or R b and R c is halogen, CF 3, C 1-4 alkyl, OR f, S (O) k R f, COR f, CO 2 R f, OH, NO 2, N (R f) 2, CO (NR f bonded to each other ) 2 , and CH 2 N (R f ) 2 , or a 5 to 6 membered aromatic or nonaromatic carbocyclic or heterocyclic ring optionally substituted with up to three substituents selected from CH 2 N (R f ) 2 ; Or methylenedioxy;
    [86] Q 1 , Q 2 , Q 3 and Q 4 are independently nitrogen or CR y , with the proviso that no more than one of Q 1 , Q 2 , Q 3 and Q 4 is nitrogen;
    [87] R 'is hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl or C 3-6 cycloalkyl -C 0-6 alkyl;
    [88] R "is R ', -C (O) R' or -C (O) OR ';
    [89] R y is hydrogen, halo, -OR g, -SR g, -CN , -NR g R k, -NO 2, CF 3, CF 3 S (O) r -, -CO 2 R g, -COR g or -CONR g 2, or halo, -OR g, -SR g, -CN , -NR g R ", -NO 2, -CF 3, R'S (O) r -, -CO 2 R g, -COR g or by -CONR g 2, optionally substituted C 1-6 alkyl;
    [90] a is 0, 1 or 2;
    [91] b is 0, 1 or 2;
    [92] k is 0, 1 or 2;
    [93] r is 0, 1 or 2;
    [94] s is 0, 1 or 2;
    [95] u is 0 or 1;
    [96] v is 0 or 1;
    [97] Abbreviations and codes commonly used in the field of peptides and chemistry are used herein to describe the compounds of the present invention. In general, amino acid abbreviations are described in literature Eur. J. Biolchem. , 158, 9 (1984)]. The IUPAC-IUB joint commission on biochemical nomenclature follows.
    [98] C 1-4 alkyl as applied herein means an optionally substituted alkyl group of one to four carbon atoms and includes methyl, ethyl, n-propyl, isopropyl and t-butyl. C 1-6 alkyl further includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. C 0-4 alkyl and C 0-6 alkyl indicate that no further alkyl groups need be present (e.g., indicating the presence of a covalent bond).
    [99] C 1-4 alkyl or C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 oxoalkyl may optionally be replaced by an R x group, And can be used by conventional synthetic techniques. Suitable R x groups of C 1-4 alkyl, OR ', SR', C 1-4 alkylsulfonyl, C 1-4 alkyl sulfonic poksil, -CN, N (R ') 2, CH 2 N (R') 2, -NO 2, -CF 3, -CO 2 R ', -CON (R') 2, -COR ', -NR'C (O) R', F, Cl, Br, I, or CF 3 S (O) r -, wherein r is 0, 1 or 2.
    [100] Halogen or halo means F, Cl, Br and I.
    [101] As used herein, Ar, or aryl is phenyl or naphthyl, or alkyl, especially C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkoxy, CF 3 , NH 2 , OH, Means phenyl or naphthyl substituted by one to three substituents, such as those defined above for Br or I.
    [102] Het, or heterocycle represents an optionally substituted 5 or 6 membered monocyclic ring, or a 9 or 10 membered bicyclic ring containing 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, And is available by conventional chemical techniques. Examples of this heterocycle include benzofuryl, benzimidazole, benzopyran, benzothiophene, furan, imidazole, indolin, morpholine, piperidine, piperazine, pyrrole, pyrrolidine, tetrahydropyridine, pyridine, Thiazole, oxazole, thiophene, quinoline, isoquinoline, and tetra- and perhydro-quinoline and isoquinoline. Possible combinations of up to three substituents on the Het ring, which are available and stable by chemical synthesis, such as those defined above for alkyl, are within the scope of the present invention.
    [103] C 3-7 cycloalkyl means an optionally substituted carbocyclic system of 3 to 7 carbon atoms, which may contain up to two unsaturated carbon-carbon bonds. Typical C3-7 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and cycloheptyl. Combinations of up to three substituents, such as those defined above for alkyl on cycloalkyl rings that are available and stable by conventional chemical synthesis, are within the scope of the present invention.
    [104] When R b and R c are bonded to each other to form a 5 or 6 membered aromatic or nonaromatic carbocyclic or heterocyclic ring fused to the ring to which R b and R c are bonded, Lt; / RTI > is a 5 or 6 membered heterocycle selected from those listed above for cycloalkyl, cycloalkyl, cycloalkyl, cycloalkyl, cycloalkyl or cyclohexyl. Preferably, R b and R c are independently selected from the group consisting of CH 2, nitrogen, or CR x (provided that no more than two of D 1 - D 4 are nitrogen) - D 1 = D 2 = D 3 = D 4. Most preferably, when R b and R c are bonded to each other, they form a -CH = CH-CH = CH- group.
    [105] Certain radicals are used herein as abbreviations. t-Bu means tertiary butyl radical, Boc means t-butyloxycarbonyl radical, Fmoc means fluorenylmethoxycarbonyl radical, Bn means benzyl radical, Me means methyl , Et means ethyl, Ac means acetyl, Alk means C 1-4 alkyl, Nph means 1- or 2-naphthyl, and cHex means cyclohexyl. Tet means 5-tetrazolyl.
    [106] Certain reagents are used herein as abbreviation. DCC means dicyclohexylcarbodiimide, DMAP means dimethylaminopyridine, DIEA means diisopropylamine, EDC means 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride , HOBt means 1-hydroxybenzotriazole, THF means tetrahydrofuran, DIEA means diisopropylethylamine, DEAD means diethyl azodicarboxylate, and PPh 3 means triphenylphosphine, DIAD means diisopropyl azodicarboxylate, DME means dimethoxyethane, DMF means dimethylformamide, NBS means N-bromosuccinimide Pd / C means a Pd catalyst, PPA means polyphosphoric acid, DPPA means diphenylphosphoryl azide, BOP means benzotriazol-1-yloxy-tris (dimethylamino ) Phosphonium hexafluorophosphate Refers to the agent and, HF refers to hydrofluoric acid, TEA refers to triethylamine, TFA refers to trifluoroacetic acid, PCC refers to and pyrid titanium chloro chromate.
    [107] The compounds of formula (I) are generally prepared by reacting a compound of formula (III): EMI5.1 with a compound of formula (IV) wherein the reactive functional group is protected, then removing the protecting group and optionally forming a pharmaceutically acceptable salt do:
    [108]
    [109]
    [110] Wherein R 1 and R 2 are as defined in formula (I).
    [111] Compounds of formula (I) are prepared by the general methods described in Scheme I-II.
    [112]
    [113] (I-1; J. Org. Chem. , 1959 , 24, 1290) of 3-phenylglutaric acid is converted to the active form of the acid using, for example, EDC and HOBt, or SOCl 2 And this active form is reacted with a suitable 2-hydroxyethylamine, such as serine methyl ester, in a suitable solvent such as DMF, CH 2 Cl 2 , or CH 3 CN to give the hydroxyethylamide I-2. Additional bases such as triethylamine (Et 3 N), diisopropylethylamine ((i-Pr) 2 NEt) or pyridine may be used, depending on whether acid neutralization is desired or not. Many additional methods for converting carboxylic acids to amides are known and described in Compendium of Organic Synthetic Methods, Vol. I-VI, Published by Springer-Verlag), or Bodansky, " The Practice of Peptide Synthesis, " Published by Springer-Verlag. Compound I-2 is converted to an oxazole derivative via a conventional two-step process. First, Compound I-2 is reacted under dehydrating conditions, preferably in Berger's reagent ( Org. Synth. Coll. Vol. VI 1988 , 788) described by Meyers ( J. Org. Chem. , 1996 , 61 , 8207) To give the oxazoline derivative I-3. Oxazole derivative I-4 is then obtained by another oxidation in the general method developed by Barish (J. Org. CHem. 1993, 58, 4494). Other methods for the preparation of oxazoles from oxazolines are known, some of which have been described in recent reviews by Meyer ( Teterahedron , 1994 , 58 , 4494). The methyl ester of I-4 is cleaved to the corresponding carboxylic acid (I-5) using nucleophilic conditions, such as lithium iodide in refluxing pyridine. Other methods for the nucleophilic cleavage of methyl esters can be found in conventional reference books, such as, for example, " Protective Groups in Organic Synthesis, " published by Wiley-Interscience. Since simultaneous cleavage of the ethyl ester is expected under conventional saponification conditions, nucleophilic conditions may be preferred in this case. Compound I-5 is converted to the active form of the acid using 1,1'-carbonyl diimidazole and the activated form is reacted with a suitable amine such as 2- [(3-amino-1-propyl) amino] Pyridine dihydrochloride to obtain the amide derivative I-6. As discussed above, various other methods can be used to convert acid I-5 to amide I-6. The ethyl ester of I-6 is hydrolyzed with an aqueous base, for example LiOH in aqueous THF or aqueous methanol or NaOH in ethanol, and the intermediate carboxylate salt is acidified with a suitable acid, such as TFA or HCl Carboxylic acid I-7 is obtained. Alternatively, the intermediate carboxylate salt may be isolated or, if desired, or a salt of the free carboxylic acid may be prepared by methods well known to those skilled in the art.
    [114]
    [115] The carboxylic acid of the compound II-1 is selectively reduced to the corresponding alcohol (II-2) by the two-step method in the presence of the ethyl ester. The first step involves forming a mixed acid anhydride using an alkyl chloroformate, such as ethyl chloroformate, in the presence of a trialkylamine base, generally N-methylmorpholine (NMM). The reaction is typically carried out in a polar, aprotic solvent such as THF. The mixed acid anhydrides are generally not separated but rather reacted in situ with a suitable reducing agent, preferably sodium borohydride (NaBH 4 ). When NaBH 4 is used as a reducing agent, co-solvents such as methanol and ethanol are required to dissolve NaBH 4 . Alcohol II-2 is converted to the bromide II-3 by reaction with carbon tetrabromide (CBr 4 ) in the presence of triphenylphosphine (PPh 3 ) according to the general method of Downey ( Chem. Ind ., 1966 , 900) . Many additional methods are available for the conversion of alcohols to the corresponding halides and appear in conventional reference books such as " Compendium of Organic Synthetic Methods, " Published by Wiley-Interscience. Bromide II-3 is reacted with a trialkyl- or triarylphosphine, for example triphenylphosphine, in a suitable solvent such as acetonitrile to give bithig reagent II-4. When reacting a suitable base with a suitable aldehyde such as 4- [N- (tert-butoxycarbonyl) -N- (pyridin-2-yl) amino] butanal, II- To obtain olefin II-5. Here, the conversion of II-4 to II-5 is optimally established according to the general method described by Evans ( J. Am. Chem. Soc ., 1992 , 114 , 9434). The tert-butoxycarbonyl protecting group II-5 is removed under acidic conditions such as 4N HCl in 1,4-dioxane or TFA in CH 2 Cl 2 to give II-6. Conditions for the removal of the tert-butoxycarbonyl group are well known to those skilled in the art, and several useful methods are described in conventional reference books such as Green, " Protective Groups in Organic Synthesis ". Both the olefin and the N-oxide residue of II-6 are simultaneously reduced using hydrogenation conditions to obtain II-7. In general, the reaction is mediated by a palladium catalyst, preferably palladium metal on activated carbon, and occurs in an inert solvent, such as methanol, ethanol, or 2-propanol. Cyclohexane, 1,4-cyclohexadiene, formic acid, and formate salts such as potassium formate or ammonium formate are commonly used as hydrogen transfer reagents in this type of reaction. The ethyl ester of II-7 is hydrolyzed as described in Scheme I to give the carboxylic acid II-8.
    [116] Amide coupling agents used herein refer to reagents that can be used to form peptide bonds. Typical coupling methods use carbodiimides, activated acid anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPQ, BOP reagent, HOBt, N-butoxy succinimide and oxalyl chloride are typical.
    [117] Coupling methods for forming peptide bonds are generally well known in the art. The Practice of Peptide Synthesis, Spring-Verlag, Berlin, 1984, Ali et al., J. Med. Chem ., 29, 984, 1986 and J. Med. Chem. , 30, 2291, 1987) are generally described in the art and incorporated herein by reference.
    [118] In general, an amine or an aniline can be converted to N, N'-dicyclohexylcarbodiimide (DCC) and optionally N, N'-dicyclohexylcarbodiimide (DCC) in the presence of a catalyst such as 1-hydroxybenzotriazole (HOBt) and dimethylaminopyridine Is coupled with a suitable carboxylic acid substrate using the appropriate carbodiimide coupling agent. Other methods are also suitable, such as the formation of the active ester, acid anhydride or acid halide of the free carboxyl of a suitably protected amino acid substrate, and the subsequent reaction of the appropriately protected amine with the free amine in the presence of a base, optionally in the presence of a base . For example, the protected Boc-amino acid or Cbz-amidinobenzoic acid can be reacted with an iso-propanediol in the presence of a base such as N-methylmorpholine, DMAP or trialkylamine in an anhydrous solvent, such as methylene chloride or tetrahydrofuran Reacted with butyl chloroformate to form the " activated acid anhydride " which is then reacted with the free amine of another protected amino acid or aniline.
    [119] Useful intermediates of compounds of formula (I) wherein R < 2 > is benzimidazole are described in Nester et al . , J. Med. Chem., 1984 , 27 , 320). Representative methods for preparing benzimidazole compounds useful as intermediates in the present invention are also conventional in the art and can be found, for example, in EP-A 0 381 033.
    [120] The acid addition salts of the compounds are prepared in conventional manner in suitable solvents from parent acids and excess acids such as hydrochloric acid, hydrobromic acid, fluoric acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid or methanesulfonic acid . Certain compounds form acceptable internal salts or amphoteric ions. Cationic salts are prepared by reacting the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing suitable cations; Or by treatment with a suitable organic amine. Cations such as Li + , Na + , K + , Ca ++ , Mg ++ and NH 4 + are specific examples of cations present in pharmaceutically acceptable salts.
    [121] The present invention also provides a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier. Thus, the compounds of formula (I) may be used in the manufacture of medicaments. The pharmaceutical compositions of the compounds of formula (I) prepared as hereinbefore described may be formulated as a solution and lyophilized for parenteral administration. The powder may be reconstituted by adding a suitable diluent or other pharmaceutically acceptable carrier before use. The medicament may be a buffering, isotonic, aqueous solution. An example of a suitable diluent is a normal isotonic salt solution, a conventional 5% dextrose in water or a buffered sodium or ammonium acetate solution. These formulations are particularly suitable for parenteral administration, but may also be contained in a Haller or nebulizer, which is used for oral administration or as a medication that is being measured for inhalation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
    [122] Alternatively, these compounds may be encapsulated, tableted, or prepared as emulsions or syrups for occasional administration. A pharmaceutically acceptable solid or liquid carrier may be added to improve or stabilize the composition, or to facilitate the preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also contain, alone or in combination with a wax, a sustained release material such as glyceryl monostearate or glyceryl distearate. The amount of solid carrier will however preferably be from about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations can be used for purification, when necessary, by milling, mixing, granulating, and pressing; Or hard gelatine capsules, as described herein, in accordance with conventional techniques in the pharmaceutical art. When liquid carriers are used, the formulations may be in the form of syrups, elixirs, emulsions or aqueous or nonaqueous suspensions. Such liquid preparations can be administered directly orally or they can be filled into soft gelatin capsules.
    [123] For rectal administration, the compounds of the present invention may also be mixed with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycol and molded into suppositories.
    [124] The compounds described herein are antagonists of the vitronectin receptor and are useful for treating a ligand or a cell-derived disease in which the underlying pathology interacts with the vitronectin receptor. For example, these compounds are useful in the treatment of diseases where loss of bone matrix causes disease. Therefore, the compounds of the present invention are useful for the treatment of osteoporosis, hyperparathyroidism, Paget's disease, hypercalcemia of malignant tumors, osteolytic lesions caused by bone metastasis, immobilization or bone loss due to sex hormone deficiency. The compounds of the present invention are also believed to have utility as antineoplastic agents, anti-angiogenic agents, anti-inflammatory agents and anti-convulsants and are useful in the treatment of arteriosclerosis and restenosis.
    [125] The compound is administered orally or parenterally to the patient in such a manner that the concentration of the drug is sufficient to inhibit bone resorption. The pharmaceutical composition containing the compound is administered in an oral dosage of about 0.1 to about 50 mg / kg in a manner appropriate to the condition of the patient. Preferably, the oral dosage will be from about 0.5 to about 20 mg / kg. For acute treatment, parenteral administration is preferred. Although intramuscular bolus injection is also useful, intravenous infusion of peptides in 5% dextrose in water or normal saline, or similar agents with suitable excipients, is most effective. Generally, the parenteral dosage is about 0.01 to about 100 mg / kg; Preferably 0.1 to 20 mg / kg. The compounds are administered one to four times a day to achieve a total daily dosage of about 0.4 to about 400 mg / kg / day. The precise amount and method of administration of the compound is readily determined by those skilled in the art by comparing the blood concentration of the formulation to the concentration required to have a therapeutic effect.
    [126] The present invention further relates to the use of compounds of formula (I) and other inhibitors of bone resorption, such as bisphosphonates (i.e., alendronate), hormone replacement therapy, anti-estrogens, or calcitonin, in a phased or physical combination A method for inhibiting bone loss comprising administering a therapeutically effective amount of a compound of the present invention. In addition, the present invention provides a method of treating with an anabolic agent, such as a compound of the present invention and a bone morphogen protein, isoflavone, useful for preventing bone loss and / or increasing bone mass.
    [127] In addition, the present invention provides a method for inhibiting tumor growth comprising administering a compound of formula (I) and an anti-tumor agent in a stepwise or physical combination. Camptothecin compounds such as topotecan, irinotecan and 9-aminocamptothecin, and platinum-bound complexes such as cisplatin, ornaplastatin and tetrapelatin are well known chemotherapeutic agents. Compounds of camptothecin derivatives are disclosed in U.S. Patent Nos. 5,004,758, 4,604,463, 4,473,692, 4,545,880, 4,342,776, 4,513,138, 4,399,276, European Patent Application Nos. 0 418 099 and 0 088 642, Et al . , J. Med. Chem. 1986 , 29 , 2358, Wani et al . , J. Med. Chem. , 1980 , 23, 554, Wani et al . , J. Med. Chem. . 1987 , 30 , 1774, and Nita et al . , Proc. 14th International Congr. Chemotherapy. , 1985 , Anticancer Section 1 , 28], the entire disclosures of each of which are incorporated herein by reference. Platinum binding complexes, cisplatin, are commercially available from Bristol Myers-Scrub Corporation as Platinol, Can be purchased under the trade name. Useful formulations of cisplatin are described in U.S. Patent Nos. 5,562,925 and 4,310,51, the entire disclosures of which are incorporated herein by reference.
    [128] In a method of inhibiting growth of a hair growth comprising administering a compound of formula (I) and an antineoplastic agent in a stepwise or physical combination, a platinum binding compound, such as cisplatin, may be administered using slow intravenous infusion . A preferred carrier is a dextrose / saline solution containing mannitol. Dosage adjustment of the platinum binding compound can be based on about 1 and about 500 mg per square meter (mg / m 2 ) of the body surface of the treatment process. The injection of the platinum-binding compound can be carried out once or twice a week, and the main unit treatment can be repeated several times. In the course of parenteral, commonly used therapies, about 1.0 to about 2.0 mg / m < 2 > of body surface (m < 2 >) per day for about 5 consecutive days is used as the camptothecin compound. Preferably, the treatment regimen is repeated at least once every 7 to 28 days.
    [129] The pharmaceutical composition may be formulated by putting both the compound of formula (I) and the anti-tumor agent in the same container, but a formulation in a different container is preferred. When two agents are provided in solution form, they may be included in the injection / injection system for simultaneous administration or in a tandem rearrangement.
    [130] For the usual administration of a compound of formula (I) and an antineoplastic agent at the same or different times, an effective amount of a compound of formula (I) for parenteral administration, as described above, and a parenteral A single kit is prepared, including a single container, individual bottle, bag, vial or other container, such as a box, carton or other container, each having an effective amount of an anti-tumor agent for administration. Such kits may include, for example, both optionally plug freeze dried, separate containers or a pharmaceutical formulation in the same container, and a container of solution for reconstitution. What has changed is that it contains a solution for reconstitution and a lyophilized plug in two compartments of a single vessel, which can be mixed with each other before use. In this form, the antineoplastic agent and the compounds of the present invention may be individually packaged in two containers, or may be lyophilized together as a powder and presented in a single container.
    [131] When the two agents are provided in solution form, they may be contained in an injection / injection system or in tandem for simultaneous administration. For example, the compound of formula (I) may be in an intravenously injectable form, or it may be in an infusion bag connected via a tube to the antineoplastic agent of the second infusion bag. Using such a system, the patient is first subjected to a bolus-type injection or infusion of a compound of formula (I) followed by an antitumor agent injection.
    [132] Compounds can be tested with one of several biological assays to determine the concentration of compound required to have a given pharmacological effect.
    [133] Bitronectin binding inhibition
    [134] α v β 3 in the solid phase of [3 H] -SK & F- 107260 combination: buffer T (2 mM CaCl 2 and 1% octyl glucoside content) of the human placenta or human platelet α v β 3 (0.1-0.3 mg / mL) was diluted with Buffer T containing 1 mM CaCl 2 , 1 mM MnCl 2 , 1 mM MgCl 2 (buffer A) and 0.05% NaN 3 and then incubated in 96-well ELISA plates (New York, 0.1 mL / well was added immediately. 0.1 to 0.2 [mu] g of [alpha] v [ beta] 3 was added per well. Plates were incubated overnight at 4 ° C. During the experiment, the wells were washed once with Buffer A and incubated with 0.1 mL of 3.5% bovine serum albumin in the same buffer for 1 hour at room temperature. After incubation, the wells were completely inhaled and washed twice with 0.2 mL Buffer A.
    [135] The compound was dissolved in 100% DMSO to obtain a 2 mM stock solution. The compound was dissolved in binding buffer (15 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , 1 mM MgCl 2 ) And diluted to a compound concentration of 100 [mu] M. Unlabeled antagonist of various concentrations (0.001-100 μM) was added to the wells in triplicate, followed by addition of 5.0 nM [ 3 H] -SK & F-107260 (65-86 Ci / mmol).
    [136] Plates were incubated at room temperature for 1 hour. After incubation, the wells were completely inhaled and washed once with 0.2 mL of ice-cold buffer A in well-to-well form. The receptor was dissolved in 0.1 mL of 1% SDS and the bound [ 3 H] -SK & F-107260 was determined by liquid scintillation counting with the addition of 3 mL Readyseaf in a Beckman LS liquid scintillation counter with 40% . The specific binding of [ 3 H] -SK & F-107260 was determined in the presence of 2 μM SK & F-107260 and was constant at less than 1% of the total radioligand flux. The IC 50 (concentration of antagonist to inhibit [ 3 H] -SK & F-107260 binding by 50%) was determined by nonlinear, least squares curve-fitting method, which was changed from the LUNDON-2 program. The K i (dissociation constant of antagonist) was calculated by the following equation: K i = IC 50 / (1 + L / K d ), where L and K d are the concentration and dissociation of [ 3 H] -SK & F- It was constant.
    [137] The compounds of the present invention inhibit vitronectin binding to SK & F 107260 in a concentration range of about 2.0-0.2 micromoles.
    [138] The compounds of the present invention are also tested for in vitro and in vivo bone resorption by conventional analytical methods for assessing bone formation inhibition, such as the pit formation assay disclosed in EP 528 587, Cells and Cells, Materials, 1991 , Sup. 1, 69-74. ≪ / RTI >
    [139] Vascular smooth muscle movement assay
    [140] Rat or human arterial smooth muscle cells were used. Cell migration was observed in a Transwell cell culture chamber using a polycarbonate membrane (Costa) with a pore size of 8 um. The lower surface of the filter was coated with bitronectin. In DMEM supplemented with 0.2% bovine serum albumin with the cells 2.5 - 5.0 x 10 was suspended in 6 cells / mL of temperature, were pretreated with test compound at various concentrations for 20 minutes at 20 o C. Only the solvent was used as control. 0.2 mL of the cell suspension was placed in the upper compartment of the chamber. The lower compartment contained 0.6 mL of DMEM supplemented with 0.2% bovine serum albumin. From 95% atmosphere of air / 5% CO2 for 24 hours and incubated at 37 o C. After incubation, non-migrating cells on the upper surface of the filter were gently scraped off. The filter was then placed in methanol and stained with 10% DM dye. Transfer was measured by counting the number of cells that a) migrated to the bottom surface of the filter, or b) extracting cells stained with 10% acetic acid and then observing the uptake at 600 nM.
    [141] Parathyroid-removed mouse model
    [142] Each experimental group consisted of 5-6 adult male Sprague-Dawley rats (250-400 g body weight). Remove the parathyroid glands 7 days before using the rats (by Benoit, Taconic palms). All rats received an alternate dose of thyroxine every 3 days. After receiving the rats, whole blood was collected by intravenous injection into a heparinized test tube and the circulating ionized calcium concentration was measured immediately. A rat with an ionized calcium concentration (measured by a Ciba-Corning Model 634 calcium pH analyzer) of less than 1.2 mM / L is used. Each rat is equipped with an intraluminal vein and arterial catheter for test substance delivery and blood collection, respectively. Then the rats are fed a calcium-free diet and a distilled water diet. Basal calcium concentration was measured and each rat was injected intravenously with an intravenous catheter using an external syringe pump to determine the presence of a control agent or human parathyroid hormone 1-34 peptide (hPTH1-34, dose, 1.25 ug / kg / h / 0.1% bovine serum albumin, Bachem, Ca) or hPTH 1-34 and a mixture of test substances. The calcium response of each rat is specified at intervals of two hours during the 6-8 hour infusion period.
    [143] Human osteoclast reabsorption and adhesion analysis
    [144] Pit resorption and attachment assays have been developed and standardized using normal human osteoclasts derived from osteoclastoma tissue. Assay Method 1 was developed for the measurement of osteoclast pit capacity by laser confocal microscopy. Assay 2 was developed as a screen with a higher throughput ratio that the collagen fragments (released during reabsorption) are measured by competitive ELISA.
    [145] Method 1 (using a laser confocal microscope)
    [146] The fraction of cell suspension derived from human osteoclasts is removed from the liquid nitrogen storage chamber, quickly warmed to 37 ° C and washed once in RPMI-1640 medium by centrifugation (1000 rpm, 5 min at 4 ° C).
    [147] Air in the medium is inhaled and replaced with murine anti-HLA-DR antibody and then diluted 1: 3 in RPMI-1640 medium. The suspension is incubated on ice bath for 30 minutes and mixed frequently.
    [148] Cells are washed twice with cold RPMI-1640, centrifuged (1000 rpm, 5 min at 4 ° C) and then transferred to a sterile 15 ml centrifuge tube. The number of mononuclear cells is counted in an improved Newburgh coefficient chamber.
    [149] Sufficient magnetic beads (5 / mononuclear cells) coated with goat anti-mouse IgG (New York State, Great Neck, Dianal) are removed from their stock bottles and clean media (which rinses the toxic azide preservative) . This medium is removed by fixing the beads on the magnet and replaced with a clean medium.
    [150] The beads are mixed with the cells and the suspension is incubated on ice bath for 30 minutes.
    [151] The bead-coated cells are fixed on the magnet and the remaining cells (osteoclast-rich fraction) are subjected to a sterile 50 ml centrifuge tube.
    [152] A clean medium is added to the bead-coated cells to remove the trapped osteoclasts. This cleaning procedure is repeated 10 times. Discard bead-coded cells.
    [153] Surviving osteoclasts are counted to display living cells in a counting chamber using fluorescent diethate. Use a large-diameter, disposable plastic Pasteur pipette to add the sample to the chamber.
    [154] The osteoclasts are pelleted by centrifugation and the density is adjusted to the appropriate number in the EMEM medium supplemented with 10% fetal bovine serum and 1.7 g / liter of sodium bicarbonate (the number of osteoclasts varies between tumors).
    [155] 3 ml of cell suspension (per treatment compound) is applied to a 15 ml centrifuge tube. Cells are pelleted by centrifugation.
    [156] To each tube, 3 ml of the appropriate treatment compound is added (diluted to 50 uM in EMEM medium). A suitable vehicle control, a positive control (anti-bitonectin receptor mouse mononuclear antibody [87MEM1] diluted at 100 ug / ml) and a heterogeneous control (IgG 2a diluted at 100 ug / ml) are also included in the test tube. Samples are incubated at 37 ° C for 30 minutes.
    [157] 0.5 ml aliquots of cells are inoculated into a 48-well plate with sterile dentin slices and cultured at 37 ° C for 2 hours. Each process consists of quadruple.
    [158] Slices were washed 6 times in warm PBS (10 ml / well in 6-well plate) and then placed in a clean medium containing the treatment compound and control sample. Samples are incubated at 37 ° C for 48 hours.
    [159] Tartrate-resistant acid phosphatase (TRAP) method (selective staining of osteoclast-like cells)
    [160] Bone slices containing attached osteoclasts are washed with phosphate buffered saline and fixed in 2% gluteraldehyde (in 0.2 M sodium chocodelate) for 5 minutes.
    [161] They are then washed in water and incubated in TRAP buffer for 4 min at 37 ° C and incubated with 0.25 M (pH 7.4) containing 10 mM sodium tartrate (0.5 mg / ml naphthol AS-BI phosphate dissolved in N, N-dimethylformamide) Citrate buffer (pH 4.5).
    [162] After washing in cold water, the slices are soaked in cold acetate buffer (0.1 M, pH 6.2) containing 1 mg / ml fast red garnet and incubated at 4 ° C for 4 minutes.
    [163] The excess buffer is inhaled and the slice is washed with water and air dried.
    [164] TRAP positive osteoclasts (red brick / purple settling) are counted by bright field microscopy and then removed from the dentin surface by ultrasonography.
    [165] The pit capacity is measured using a Nikon / Lasertech ILM21W confocal microscope.
    [166] Assay 2 (using ELISA readings)
    [167] Human osteoclasts are enriched and prepared for screening compounds as described in the first 9 steps of Assay Method 1. For the sake of clarity, these steps are repeated below.
    [168] The fraction of cell suspension derived from human osteoclasts is removed from the liquid nitrogen storage chamber, quickly warmed to 37 ° C and washed once in RPMI-1640 medium by centrifugation (1000 rpm, 5 min at 4 ° C).
    [169] Air in the medium is inhaled and replaced with murine anti-HLA-DR antibody and then diluted 1: 3 in RPMI-1640 medium. The suspension is incubated on ice bath for 30 minutes and mixed frequently.
    [170] Cells are washed twice with cold RPMI-1640, centrifuged (1000 rpm, 5 min at 4 ° C) and then transferred to a sterile 15 ml centrifuge tube. The number of mononuclear cells is counted in an improved Newburgh coefficient chamber.
    [171] Sufficient magnetic beads (5 / mononuclear cells) coated with goat anti-mouse IgG (New York State, Great Neck, Dianal) are removed from their stock bottles and clean media (which rinses the toxic azide preservative) . This medium is removed by fixing the beads on the magnet and replaced with a clean medium.
    [172] The beads are mixed with the cells and the suspension is incubated on ice bath for 30 minutes.
    [173] The bead-coated cells are fixed on the magnet and the remaining cells (osteoclast-rich fraction) are subjected to a sterile 50 ml centrifuge tube.
    [174] A clean medium is added to the bead-coated cells to remove the trapped osteoclasts. This cleaning procedure is repeated 10 times. Discard bead-coded cells.
    [175] Surviving osteoclasts are counted to display living cells in a counting chamber using fluorescent diethate. Use a large-diameter, disposable plastic Pasteur pipette to add the sample to the chamber.
    [176] The osteoclasts are pelleted by centrifugation and the density is adjusted to the appropriate number in the EMEM medium supplemented with 10% fetal bovine serum and 1.7 g / liter of sodium bicarbonate (the number of osteoclasts varies between tumors).
    [177] In contrast to the method described above in Assay 1, the compounds are screened at 4 doses as summarized below to obtain IC 50 :
    [178] The osteoclast preparation is pre-incubated with the test compound (4 doses) or the control at 37 ° C for 30 minutes.
    [179] These are then inoculated into the wells of a 48-well tissue culture plate onto small surface bone slices and incubated at 37 ° C for a further 2 hours.
    [180] The bone slices are washed with 6 changes of warm phosphate buffered saline (PBS) to remove non-adherent cells and then returned to the wells of a 48 well plate containing the clean compound or control.
    [181] Tissue culture plates are then incubated for 48 hours at 37 ° C.
    [182] The supernatant of each well is inhaled into a separate test tube and screened in a competitive ELISA that detects c-telopeptides of type I collagen released during the resorption process. It is commercially available containing a rabbit antibody that specifically reacts with the 8-amino acid sequence (Glu-Lys-Ala-His-Asp-Bly-Gly-Arg) present in the carboxy-terminal telopeptide of the a1- Available ELISA (Denmark, Ostertometer). Results are expressed as percent resorption inhibition compared to vehicle control.
    [183] Human osteoclast adhesion assay
    [184] Human osteoclasts are enriched and prepared for screening compounds as described in the first 9 steps of Assay Method 1. For the sake of clarity, these steps are repeated below.
    [185] The fraction of cell suspension derived from human osteoclasts is removed from the liquid nitrogen storage chamber, quickly warmed to 37 ° C and washed once in RPMI-1640 medium by centrifugation (1000 rpm, 5 min at 4 ° C).
    [186] Air in the medium is inhaled and replaced with murine anti-HLA-DR antibody and then diluted 1: 3 in RPMI-1640 medium. The suspension is incubated on ice bath for 30 minutes and mixed frequently.
    [187] Cells are washed twice with cold RPMI-1640, centrifuged (1000 rpm, 5 min at 4 ° C) and then transferred to a sterile 15 ml centrifuge tube. The number of mononuclear cells is counted in an improved Newburgh coefficient chamber.
    [188] Sufficient magnetic beads (5 / mononuclear cells) coated with goat anti-mouse IgG (New York State, Great Neck, Dianal) are removed from their stock bottles and clean media (which rinses the toxic azide preservative) . This medium is removed by fixing the beads on the magnet and replaced with a clean medium.
    [189] The beads are mixed with the cells and the suspension is incubated on ice bath for 30 minutes.
    [190] The bead-coated cells are fixed on the magnet and the remaining cells (osteoclast-rich fraction) are subjected to a sterile 50 ml centrifuge tube.
    [191] A clean medium is added to the bead-coated cells to remove the trapped osteoclasts. This cleaning procedure is repeated 10 times. Discard bead-coded cells.
    [192] Surviving osteoclasts are counted to display living cells in a counting chamber using fluorescent diethate. Use a large-diameter, disposable plastic Pasteur pipette to add the sample to the chamber.
    [193] The osteoclasts are pelleted by centrifugation and the density is adjusted to the appropriate number in the EMEM medium supplemented with 10% fetal bovine serum and 1.7 g / liter of sodium bicarbonate (the number of osteoclasts varies between tumors).
    [194] Osteoclastoma-induced osteoclasts are pre-incubated with compound (4 doses) or control for 30 minutes at 37 ° C.
    [195] Cells are then inoculated onto osteopontin-coated slides (human or rat osteopontin, 2.5 ug / ml) and incubated for 2 hours at 37 ° C.
    [196] Non-adherent cells are removed by washing the slides intensely in phosphate buffered saline and the remaining cells on the slides are fixed in acetone.
    [197] The osteoclasts are stained for tartrate resistant acid phosphatase (TRAP), an optional marker for these phenotypic cells (see steps 15-17) and counted by light microscopy techniques. The results are expressed as percent adhesion inhibition compared with vehicle control.
    [198] Cell adhesion assay
    [199] Cell and cell culture
    [200] Human embryonic kidney cells (HEK293 cells) were obtained from ATCC (Cat. No. CRL 1573). Cells were cultured in Earl's minimal essential medium (EMEM) containing Earl's salts, 10% fetal bovine serum, 1% glutamine and 1% penicillin-streptomycin.
    [201] Constructs and Transfections
    [202] A 3.2 kb EcoRI-LpnI fragment of the v subunit and a 2.4 kb XbaI-XhoI fragment of the 3 subunit were ligated to the EcoRI-pvc region of the pCDN vector containing a CMV promoter and a G418 selectable marker by blunt-end binding (Aya et al., 1994) EcoRI cloning site. For stable expression, 80 x 10 6 HEK 293 cells were electrotransformed to the α v + β 3 construct (20 μg DNA of each subunit) using Gene Plus (Hensley et al., 1994) 5 x 10 5 cells / plate). Forty-eight hours later, the culture media were supplemented with 450 [mu] g / mL geneticin (G418 sulfate, GIBCO-BRL, Vetes, MD). Cells were maintained in selective medium until they were large enough for colony analysis.
    [203] Immunocytochemical analysis of transfected cells
    [204] To determine if the HEK 293 transfectant expressed the vitronectin receptor, cells were fixed on glass microscope slides by centrifugation, fixed in acetone for 2 minutes at room temperature and air dried. Specific reactivity with specific mononuclear antibodies against 23C6, V 3 complexes was examined using conventional hepatic immunofluorescence.
    [205] Cell attachment study
    [206] Corning 96-well ELISA plates were precoated overnight at 4 ° C with 0.1 mL human bitronectin (0.2 μg / mL in RPMI medium). During the experiment, the plates were washed once with RPMI medium and blocked with 3.5% BSA in RPMI medium for 1 hour at room temperature. Transfected cells were resuspended in RPMI medium and supplemented with 20 mM Hepes (pH 7.4) and 0.1% BSA at a density of 0.5 x 106 cells / mL. 0.1 mL of cell suspension was added to each well and incubated at 37 ° C for 1 hour in the presence or absence of various α v β 3 antagonists. After incubation, 0.025 mL of 10% formaldehyde solution (pH 7.4) was added and the cells were left at room temperature for 10 minutes. Plates were washed three times with 0.2 mL RPMI solution and adherent cells were stained with 0.1 mL of 0.5% toluidine blue for 20 minutes at room temperature.
    [207] Excess dye was removed by intensive washing with deionized water. Toluidine blue incorporated into the cells was eluted by adding 0.1 mL of 50% ethanol containing 50 mM HCl. Cell attachment was quantified at 600 nm optical density on a microtiter plate reader (Stirling, Va., Titan ™ Multiscan MC).
    [208] Solid phase alpha vbeta 5Bond analysis
    [209] The vitronectin receptor α v β 5 was purified from human placenta. The receptor preparation was diluted with buffer A (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM CaCl 2 , 1 mM MnCl 2 , 1 mM MgCl 2 ) and immediately added to a 96-well ELISA plate with 0.1 ml of wells . 0.1 to 0.2 [mu] g of [alpha] v [ beta] 3 was added to each well. Plates were incubated overnight at 4 ° C. During the experiment, the wells were washed once with Buffer A and incubated with 0.1 ml of 3.5% bovine serum albumin in the same buffer for 1 hour at room temperature. After incubation, the wells were inhaled completely and washed twice with 0.2 ml buffer A.
    [210] [ 3 H] -SK & F-107260 In a competitive assay, various concentrations of unlabeled antagonist (0.001-100 μM) were added to the wells and 5.0 nM of [ 3 H] -SK & F-107260 was added. Plates were incubated at room temperature for 1 hour. After incubation, the wells were inhaled completely and washed once with 0.2 ml of ice-cold Buffer A in the form of a well-to-well. The receptor was dissolved in 0.1 mL of 1% SDS and bound [ 3 H] -SK & F-107260 was eluted by liquid scintillation counting with the addition of 3 mL Readyseaf in a Beckman LS 6800 liquid scintillation counter with 40% . The specific binding of [ 3 H] -SK & F-107260 was determined in the presence of 2 μM SK & F-107260 and was constant at less than 1% of the total radioligand flux. The IC 50 (concentration of antagonist to inhibit [ 3 H] -SK & F-107260 binding by 50%) was determined by nonlinear, least squares curve-fitting method, which was changed from the LUNDON-2 program. The K i (dissociation constant of antagonist) was calculated by the following equation: K i = IC 50 / (1 + L / K d ), where L and K d are the concentration and dissociation of [ 3 H] -SK & F- It was constant.
    [211] Inhibition of RGD-mediated GPIIb-IIIa binding
    [212] Purification of GPIIb-IIIa
    [213] Ten units of old, washed human platelets (obtained from the red cross) were gently treated at 4 ° C for 2 hours in 3% octylglucoside, 20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2 mM CaCl 2 And dissolved by stirring. The lysate was centrifuged at 100,000 g for 1 hour. The resulting supernatant was applied to 5 ml lentil lectin Sepharose 4B column (EY Labs) preliminarily equilibrated with buffer A (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl 2 , 1% octylglucoside) . After incubation for 2 hours, the column was washed with 50 mL of cold Buffer A. Lectin retention GPIIb-IIIa was eluted with buffer A containing 10% dextrose. All procedures were performed at 4 ° C. GPIIb-IIIa obtained was greater than 95% as shown by SDS polyacrylamide gel electrophoresis.
    [214] Incorporation of GPIIb-IIIa in liposomes
    [215] A mixture of phosphatidylserine (70%) and phosphatidylcholine (30%) (Avanti polar fat) was dried on a glass test tube under nitrogen vapor. Purified GPIIb-IIIa was diluted to a final concentration of 0.5 mg / mL and mixed with phospholipids so that the ratio of protein to phospholipid was 1: 3 (w: w). The mixture was resuspended and sonicated for 5 minutes on a bass sonicator. The mixture was then dialyzed overnight using 12,000-14,000 molecular weight cleavage dialysis tubing connected to 1000 mM more than 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl 2 (2 changes). GPIIb-IIIa containing liposomes were subjected to centrifugation at 12,000 g for 15 minutes and resuspended with dialysis buffer to a final protein concentration of approximately 1 mg / mL. Liposomes were stored at -70 ° C until needed.
    [216] Competitive binding to GPIIb-IIIa
    [217] Binding to the fibrinogen receptor was analyzed by indirect competitive binding method using [ 3 H] -SK & F-107260 as RGD-type ligand. Binding assays were performed in 96-well filtration plate assemblies (Millipore Corporation, Bedford, Mass.) Using 0.22 um nucleophilic dual pore membranes. The wells were precoated with 0.2 mL of 10 [mu] g / mL polylysine (Sigma Chemical Co., St. Louis, Mo.) for 1 hour at room temperature to block non-specific binding. Various concentrations of unmarked benzazepine were added to the wells in quarts. [ 3 H] -SK & F-107260 was applied to each well to a final concentration of 4.5 nM and 1 μg of the purified platelet GPIIb-IIIa containing liposome was added. The mixture was incubated at room temperature for 1 hour. The GPIIb-IIIa binding [ 3 H] -SK & F-107260 was removed from the unbound by filtration using a Millipore filtration manifold and washed with ice-cold buffer (2 times, 0.2 mL each). The bound radioactivity remaining on the filter was counted in a 1.5 mL Redisolve (Beckman Liquid Scintillation Counter, Model LS 6800) with 40% efficacy on a filter (Fuller, Beckman Instruments, Inc., Calif.). Non-specific binding was determined in the presence of 2 [mu] M of the unlabelled SK & K-107260, which was consistently less than 0.14% total radioactivity added to the sample. All data is the average of four measurements.
    [218] The competitive binding data was analyzed by a nonlinear least squares fitting method. This method provides the IC 50 of the antagonist (the concentration of antagonist that inhibits the specific binding of [ 3 H] -SK & F-107260 by 50% at equilibrium). IC 50 is related to the equilibrium dissociation constant (K i ) of the antagonist based on the Cheng ' s off-off equation: K i = IC 50 / (1 + L / K d ) H] -SK & F-107260 (4.5 nM), and K d is the dissociation constant of [ 3 H] -SK & F-107260 of 4.5 nM as determined by the Scatchard assay.
    [219] Preferred compounds of the present invention have affinity of the vitronectin receptor for fibrinogen receptors greater than 10: 1. Most preferred compounds have an activity ratio of greater than 100: 1.
    [220] The efficacy of the compound of formula (I) alone or in combination with an anti-tumor agent can be determined using several implantable mouse tumor models. See U.S. Patent Nos. 5,004,758 and 5,633,016 for these models.
    [221] The following examples are provided to illustrate the present invention but not to limit the scope of the present invention. Many other embodiments will be readily apparent to those skilled in the art.
    [222] General method
    [223] Hydrogen nuclear magnetic resonance ( 1 H NMR) spectra were measured at 300 MHz and chemical shifts were reported from internal standard trimethylsilane (TMSO to ppm down (ppm)). The NMR data abbreviations are: s = Singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet, app = shows the NMR coupling constant measured in. CDCl 3 double hydrogen chloroform, DMSO-d 6 is heksayi deuterium dimethylsulfoxide, CD 3 OD is teteurayi deuterium methanol. the mass spectra ekrek Trojan spray (ES) ionization techniques The elemental analysis was performed by Quantitative Technologies Inc. (Hite House, NJ) The melting point was measured with the Thomas-Hoover melting point instrument and not calibrated. All temperatures are reported in degrees Celsius . Analchic silica gel GF and Merck silica gel 60 F-254 thin film plates were used for thin layer chromatography. Flash chromatography was performed on this Mercki gel gel 60 (230-400 mesh) ODS refers to an octadecylsilyl-derivative silica gel chromatographic support. YMC ODS-AQ Is an ODS chromatographic support which is manufactured by YMC Corporation LTd. (Japan, Kyoto). PRP-1 Is a polymer (styrene-divinylbenzene) chromatographic support, which is a registered trademark of Hamilton Corporation (Reno, Nev.). C-18 megabond el route Is a solid phase extraction column containing octadecyl-bonded silica sorbent and is a registered trademark of Barian Products (Sunnyvale, Calif.). Cell light Is a filtration aid compound consisting of pickled diatomaceous silica and is a registered trademark of Mannich Corporation (Denver, CO).
    [224] Production Example 1
    [225] Preparation of 2 - [(2-amino-1-ethyl) amino] pyridine dihydrochloric acid
    [226] a) 2 - [[2- (tert-Butoxycarbonyl) amino-1-ethyl] amino] -1- oxopyridine
    [227] To a solution of N-Boc-ethylenediamine (5.83 g, 36.39 mmol) and 2-chloropyridine-N-oxide hydrochloric acid (7.25 g, 43.67 mmol), NaHCO3 (15.29 g, 182 mmol) The mixture was heated to reflux. After 47 h, the dark brown mixture was cooled, diluted with CH 2 Cl 2 (100 mL) and suction filtered. The filtrate was concentrated and the residue was re-concentrated from toluene. The title compound (8.23 g, 89%) was obtained as a yellow solid by silica gel chromatography (10% MeOH / CH 2 Cl 2 ): 1 H NMR (250 MHz, CDCl 3 ) 隆 8.16 (dd, J = (M, 1H), 7.05-7.30 (m, 2H), 6.68 (br d, J = 8.6 Hz, 1H), 6.50-6.65 , ≪ / RTI > 4H), 1.44 (s, 9H); MS (ES) m / e 254 (M + H) < + & gt ; .
    [228] b) 2 - [[2- (tert-Butoxycarbonyl) amino-1-ethyl] amino]
    [229] (7.00 g, 27.64 mmol), 10% pd / C (5.88 g, 5.53 mmol), cyclohexene (28 mL, 276.4 mmol), and isopropanol (110 mL) was heated to reflux. After 17 hours, the reaction solution was diluted with celite Lt; / RTI > and the filtrate was concentrated. The yellow residue from toluene was chromatographed on jaenong celebration bran, after silica gel (5% MeOH / CHCl 3) . 1 H NMR (400 MHz, CDCl 3 ) 8.05-8.12 (m, 1H), 7.37-7.46 (m, 1H), 6.53-6.61 (m, 1H), 6.41 (d, J = 8.3 Hz, 1H), 5.12 (br s, 1H), 4.86 (br s, 1H), 3.26-3.51 (m, 4H), 1.44 MS (ES) m / e 238 (M + H) < + & gt ; .
    [230] c) 2 - [(2-Amino-1-ethyl) amino] pyridine dicarboxylic acid
    [231] 4 N HCl / dioxane (54 mL) in an argon stream for 2 to 0 o C in anhydrous CH 2 Cl 2 (54 mL) - [[2- (tert- butoxycarbonyl) amino-1-ethyl] amino] Pyridine (5.09 g, 21.45 mmol) in tetrahydrofuran (10 mL) and then the mixture was allowed to warm to room temperature. After 2 hours, the mixture was cooled to 0 ° C and suction filtered. The solid was washed vigorously with anhydrous ether and dried under high vacuum at 40 ° C to give the title compound (4.27 g, 95%) as a white, slightly hygroscopic solid: 1 H NMR (400 MHz, CD 3 OD) 6.27 (m, 1H), 7.92-7.98 (m, 1H), 7.19 (d, J = 9.1 Hz, 1H), 6.98-7.04 (t, J = 6.2 Hz, 2H, partially obscured by residual solvent signal); MS (ES) m / e 138 (M + H) < + & gt ; .
    [232] Production Example 2
    [233] 2 - [(3-amino-1-propyl) amino] pyridine dicarboxylic acid
    [234] a) 2 - [[[3- (tert-Butoxycarbonyl) amino-1-propyl] amino] -1- oxopyridine
    [235] The title compound (4.90 g, 92%) was prepared following the procedure of Preparation Example 1 (a), but replacing N-Boc-ethylenediamine with N-Boc-propylenediamine (3.33 g, 20 mmol) : MS (ES) m / e 268 (M + H) < + & gt ; .
    [236] b) 2 - [[[3- (tert-Butoxycarbonyl) amino-1-propyl] amino]
    [237] Amino] -1-oxopyridine is reacted with 2 - [[2- (tert-butoxycarbonyl) amino-1-propyl] amino] The title compound (3.46 g, 75%) was prepared following the method of Preparation 1 (b), except that the title compound was obtained as a white solid (4.90 g, 18.3 mmol) (M + H) < + & gt ; .
    [238] c) 2 - [(3-Amino-1-propyl) amino] pyridine dicarboxylic acid
    [239] Amino] pyridine (prepared by reacting 2 - [[2- (tert-butoxycarbonyl) amino- The title compound (2.88 g, 93%) was prepared following the method of Preparation 1 (c), except that the title compound was obtained as a white solid, MS (ES) m / e 152 (M + H) + .
    [240] Production Example 3
    [241] Preparation of 2 - [(4-amino-1-butyl) amino] pyridine
    [242] a) 2 - [[4- (tert-Butoxycarbonyl) amino-1-butyl] amino-1-oxopyridine
    [243] The title compound (0.42 g, 50%) was prepared according to the method of Preparation 1 (a), but substituting N-Boc-ethylenediamine with N-Boc-butylenediamine (0.57 mL, 2.98 mmol) MS (ES) m / e 282 (M + H) < + & gt ; .
    [244] b) 2 - [[4- (tert-Butoxycarbonyl) amino-1-butyl] amino] pyridine
    [245] Amino] -1-oxopyridine is reacted with 2 - [[4- (tert-butoxycarbonyl) amino-1-butyl] amino] -1-oxopyridine (0.88 g, 3.13 mmol), the crude title compound was prepared. This material was used without purification.
    [246] c) 2 - [(4-Amino-1-butyl) amino] pyridine
    [247] Amino] pyridine (3.13 mmol) was treated with TFA (10 mL) in CH 2 Cl 2 (10 mL). After 2 h, the mixture was concentrated under reduced pressure. The residue was taken in 1.0 N NaOH (20 mL) and extracted with CHCl 3 (4 x 50 mL) . After drying the mixed organic extracts sprocket with MgSO 4, filtered and concentrated under reduced pressure to give the title compound (122 mg, 24%) as hwangsek five days: MS (ES) m / e 166 (M + H) +.
    [248] Production Example 4
    [249] Preparation of 4- [N- (tert-butoxycarbonyl) -N- (pyridin-2-yl) amino] butanal
    [250] a) 2 - [(3-Hydroxy-1-butyl) amino] pyridine-N-
    [251] 4-amino-1-butanal (2.0 mL, 21.7 mmol), 2- chloropyridine--N- oxide hydrochloride (3.00 g, 18.0 mmol), NaHCO 3 (7.56 g, 90.0 mmol), and tert- amyl alcohol (22 mL) was heated to reflux. After 24 hours, the mixture was cooled and filtered, then the filtrate was concentrated under reduced pressure. Obtained was purified by silica gel chromatography (10% MeOH / CHCl 3) to give the title compound (2.08 g, 635) as a yellow oil: MS (ES) m / e 183 (M + H) +.
    [252] b) 2- [N- (3-Hydroxy-1-butyl) -N- (tert- butoxycarbonyl) amino] pyridine-
    [253] To a suspension of 2 - [(3-hydroxy-1-butyl) amino] pyridine-N-oxide (895 mg, 4.91 mmol) in tert-butanol (5 mL) was added di-tert- butyldimarbonate mmol). After 30 hours, the mixture was concentrated under reduced pressure. The residue was purified by chromatography (10% MeOH / CHCl 3) on silica gel to give the title compound (1.34 g, 97%) as a yellow solid: MS (ES) m / e 283 (M + H) +.
    [254] c) 4- [N- (tert-Butoxycarbonyl) -N- (pyridin-2-yl) amino]
    [255] To a solution of DMSO (0.31 mL, 3.54 mmol) in CH 2 Cl 2 (5 mL) at -78 ° C was added dropwise oxalyl chloride (0.13 mL, 1.77 mmol). After 10 minutes, CH 2 Cl 2 (5 mL ) of 2- [N- (3- hydroxy-1-butyl) -N- (tert- butoxycarbonyl) amino] -N- pyridin-oxide (500 mg, 1.77 mmol) was added dropwise. After 30 min, Et 3 N (0.81 mL, 5.84 mmol) was added and the mixture was gradually warmed to room temperature. After 20 minutes, the mixture was diluted with CH 2 Cl 2 (20 mL) and washed successively with 10 mL each of water, 10% HCl, water, and brine. The organic layer was dried over MgSO 4 , filtered and concentrated under reduced pressure to give the title compound (455 mg, 92%) as a colorless oil. This was used without purification: MS (ES) m / e 281 (M + H) < + & gt ; .
    [256] Production Example 5
    [257] Preparation of ethyl (±) -4- (4-carboxy-1,3-oxazol-2-yl) -3-phenylbutanoate
    [258] a) (±) -N- (4-ethoxycarbonyl-3-phenylbutanoyl) serine methyl ester
    [259] To a solution of ethyl (±) -4-carboxy-3-phenylbutanoate (1.00 g, 4.23 mmol) in anhydrous DMF (20 mL) was added HOBt (686 mg, 5.08 mmol), Et 3 N (1.77 mL, 12.69 mmol ), And EDC (974 mg, 5.08 mmol). After 30 minutes, L-serine methyl ester hydrochloric acid (790 mg, 5.08 mmol) was added. After 48 h, the mixture was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 (50 mL) and washed sequentially with 20 mL each of water, saturated NaHCO 3 , and water. The organic layer was dried with MgSO 4, filtered, and concentrated under reduced pressure. The residue was chromatographed on silica gel (ethyl acetate) to give the title compound (961 mg, 67%) as a yellow oil: MS (ES) m / e 338 (M +
    [260] b) Ethyl (±) -4- (4-methoxycarbonyl-1,3-oxazolin-2-yl) -3-phenylbutanoate
    [261] A solution of (±) -N- (4-ethoxycarbonyl-3-phenylbutanoyl) serine methyl ester (723 mg, 2.14 mmol) and Bergers reagent (613 mg, 2.57 mmol) in anhydrous THF (10 mL) And heated under reflux in a nitrogen stream. After 2 hours, the mixture was cooled to room temperature and concentrated under reduced pressure. The residue was chromatographed on silica gel (50% EtOAc / hexanes) to give the title compound (420 mg, 61%) as a colorless oil: MS (ES) m / e 320 (M + H) + .
    [262] c) Ethyl (±) -4- (4-methoxycarbonyl-1,3-oxazol-2-yl) -3-phenylbutanoate
    [263] Degassed (3x vacuum / N 2) a CH 2 Cl 2 (6 mL) of CuBr 2 To a suspension of (1.17 g, 5.26 mmol) of hexamethylenetetramine (737 mg, 5.26 mmol) and DBU (0.78 mL, 5.26 mmol ). After 5 minutes, CH 2 Cl 2 (2 mL ) of ethyl (± -4- (4- methoxycarbonyl-1, 3-oxazoline-2-yl) -3-phenyl-butanoate (420 mg, 1.32 The residue was chromatographed on silica gel (50% EtOAc / Hexane) to give the title compound (267 mg, 64%) as a colorless oil. %) As a white solid: MS (ES) m / e 318 (M + H) < + & gt ; .
    [264] d) Ethyl (±) -4- (4-carboxy-1,3-oxazol-2-yl) -3-phenylbutanoate
    [265] To a solution of ethyl (±) -4- (4-methoxycarbonyl-1,3-oxazol-2-yl) -3-phenylbutanoate (267 mg, 0.84 mmol) and LiI (338 mg, 2.52 mmol) in dichloromethane (5 mL) was heated to reflux. After 18 hours, the mixture was cooled to room temperature, poured into 10% HCl (150 mL), and extracted with CH 2 Cl 2 (3 x 50 mL). The combined organic extracts were dried over MgSO 4 , filtered and concentrated to give the title compound (205 mg, 80%) as a white solid: MS (ES) m / e 304 (M + H) <+> .
    [266] Production Example 6
    [267] Preparation of [2- (3-ethoxycarbonyl-2-phenylpropyl) -1,3-oxazol-4-yl] methyltriphenylphosphonium bromide
    [268] a) Ethyl (±) -4- (4-hydroxymethyl-1,3-oxazol-2-yl) -3-phenylbutanoate
    [269] To a solution of ethyl (±) -4- (4-carboxy-1,3-oxazol-2-yl) -3-phenylbutanoate (500 mg, 1.65 mmol) in anhydrous THF (8 mL) (0.22 mL, 1.99 mmol) and ethyl chloroformate (0.19 mL, 1.99 mmol) at 0 ° C. After 10 minutes, the addition of NaBH 4 (249 mg, 6.59 mmol ) at once, followed by subsequent dropwise addition of methanol (8 mL). After 30 min, the mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 20 mL). Dry the combined organic layer with MgSO 4, filtered, and concentrated under reduced pressure. The residue was chromatographed on silica gel (80% EtOAc / hexanes) to give the title compound (371 mg, 78%) as a yellow oil: Ms (ES) m / e 290 (M + H) + .
    [270] b) Ethyl (±) -4- (4-bromomethyl-1,3-oxazol-2-yl) -3-phenylbutanoate
    [271] In anhydrous THF (20 mL), PPh the ethyl (±) -4- (4- hydroxymethyl-1,3-oxazol-2-yl) -3-phenyl-butanoate (1.3 g, 4.49 mmol) solution of the 3 a (1.41 g, 5.39 mmol) and CBr 4 was added at 0 o C. After 20 minutes, the mixture was allowed to warm to room temperature. After 2 h, the mixture was concentrated under reduced pressure. The residue was chromatographed on silica gel (20% EtOAc / hexanes) to give the title compound (1.38 g, 87%) as a colorless oil: MS (ES) m / e 352 (M + H) + .
    [272] c) Preparation of [2- (3-ethoxycarbonyl-2-phenylpropyl) -1,3-oxazol-4-yl] methyltrifluoromide
    [273] Anhydrous CH 3 CN (10 mL) of ethyl (±) -4- (4- Bromo-ethyl-1,3-oxazol-2-yl) -3-phenyl probe Gaetano benzoate (1.0 g, 2.84 mmol) and PPh 3 (745 mg, 2.84 mmol) was heated under a nitrogen stream. After 3 hours, the mixture was cooled to room temperature and concentrated under reduced pressure. To give the title compound (1.66 g, 95%) was treated with Et 2 O (10 mL) and the residue as a white powder: MS (ES) m / e 535 (M + H) +.
    [274] Example 1
    [275] Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (2-amino- Manufacturing
    [276] a) Ethyl (±) -3-phenyl-4- [4 - [[[3- (pyridin- ] Butanoate
    [277] To a solution of ethyl (±) -4- (4-carboxy-1,3-oxazol-2-yl) -3-phenylbutanoate (100 mg, 0.33 mmol) in CH 2 Cl 2 (2 mL) 1-Carbonyldiimidazole (80 mg, 0.49 mmol) was added. After 2 h, Et 3 N (0.14 mL, 0.98 mmol) and 2 - [(3-amino-1-propyl) amino] pyridine dihydrochloride (110 mg, 0.49 mmol) were added. After 4 hours, the mixture was concentrated under reduced pressure. The residue was chromatographed on silica gel (75% EtOAc / hexanes) to give the title compound (131 mg, 91%) as a yellow oil: MS (ES) m / e 437 (M + H) + .
    [278] b) (+) - 3-phenyl-4- [4 - [[[3- (pyridin- Butanoic acid
    [279] THF / H 2 O) (1 : 1, 10 mL) of ethyl (±) -3- phenyl-4- [4 - [[[3- (pyridin-2-yl) amino-1-propyl] amino] carbonyl -1,3-oxazol-2-yl] butanoate (131 mg, 0.30 mmol) in dichloromethane (5 mL) was added 1 N LiOH (0.36 mL, 0.36 mmol). After 18 hours, the mixture was concentrated under reduced pressure. The residue was dissolved in water (20 mL) and acidified to pH 6 using 10% HCl. The resulting cloudy solution was extracted with CH 2 Cl 2 (3 x 75 mL). The organic extracts were dried with MgSO 4, filtered, and concentrated under reduced pressure. The residue was treated with Et 2 O and collected by filtration to give the title compound (63 mg, 51%) as a white solid: MS (ES) m / e 409 (M + H) <+> . Elemental analysis: calculated: C 22 H 24 N 4 O 4 0.75H 2 O, 1.5HCl: C, 55.44; H, 5.71; N, 11.75. Found: C, 55.49; H, 5.54; N, 11.63.
    [280] Example 2
    [281] Ethyl] amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (Compound Manufacturing
    [282] ethyl) amino] carbonyl] -1, 3-oxazol-2-yl (2-pyridyl) ] Butanoate
    [283] Except substituting 2 - [(2-amino-1-ethyl) amino] pyridine dihydrochloride (129 mg, 0.74 mmol) for 2 - [(3-amino- According to the method of Example 1 (a), the title compound (157 mg, 76%) was prepared: MS (ES) m / e 423 (M + H) &lt; + & gt ; .
    [284] b) Preparation of (±) -3-phenyl-4- [4 - [[[2- (pyridin- Butanoic acid
    [285] THF / H 2 O) (1 : 1, 4 mL) of ethyl (±) -3- phenyl-4- [4 - [[[2- (pyridin-2-yl) amino-1-ethyl] amino] carbonyl 1-yl] butanoate (157 mg, 0.37 mmol) in dichloromethane (5 mL) was added 1 N LiOH (0.56 mL, 0.56 mmol). After 24 h, the mixture was washed with Et 2 O (2 x 2 mL) and the pH of the aqueous layer was acidified to pH 6 using 10% HCl. The solution was C-18 Mega Bond El root ± column (H2O 50 mL after 10% CH 3 CN / H 2 O) 50 mL). The fractions containing the product were collected and lyophilized to give the title compound (48 mg, 33%) as a white powder: MS (ES) m / e 395 (M + H) &lt; + & gt ; . Elemental analysis: calculated: C 21 H 22 N 4 O 4 0.75H 2 O, 1.5HCl: C, 59.80; H, 5.44; N, 13.28. Found: C, 59.89; H, 5.50; N, 12.98.
    [286] Example 3
    [287] Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (2-amino-3- Manufacturing
    [288] amino] carbonyl] -1,3-oxazol-2-yl} - (2-methylpiperazin-1- ] Butanoate
    [289] The procedure of Example 1 was followed except that 2 - [(3-amino-1-propyl) amino] pyridine dihydrochloride was replaced with 2 - [(4- amino- 1 -butyl) amino] pyridine (122 mg, 0.74 mmol) (ES) m / e 451 (M + H) <+> , the title compound (197 mg, 89%
    [290] b) Preparation of (±) 3-phenyl-4- [4 - [[[4- (pyridin- Noshan
    [291] Phenyl) -4- [4 - [[[4- (pyridin-2-yl) amino- 1 -butyl] amino] carbaldehyde in THF / H 2 O (1: 1-yl] butanoate (197 mg, 0.44 mmol) in dichloromethane (5 mL) was added 1 N LiOH (0.66 mL, 0.66 mmol). After 24 h, the mixture was washed with Et 2 O (2 x 2 mL) and the pH of the aqueous layer was acidified to pH 6 using 10% HCl. The solution was treated with C-18 Megabund elutor + column (50 mL of H 2 O followed by 20% CH 3 CN / H 2 O). The fractions containing the product were collected and lyophilized to give the title compound (154 mg, 83%) as a white powder: MS (ES) m / e 423 (M + H) &lt; + & gt ; . Elemental analysis: calculated: C 23 H 26 N 4 O 4 0.75H 2 O: C, 63.36; H, 6.36; N, 12.85. Found: C, 63.55; H, 6.27; N, 12.50.
    [292] Example 4
    [293] Preparation of (±) -3-phenyl-4- [4- [5- (pyridin-2-yl) amino-1-phenyl] -1,3-oxazol-2-yl] butanoic acid
    [294] a) Ethyl (±) -3-phenyl-4- [4- (5- [N- (1-oxopyridin- 2- yl) -N- (tert- butoxycarbonyl) amino] -1,3-oxazol-2-yl] butanoate
    [295] To a solution of 4- [N- (tert-butoxycarbonyl) -N- (pyridin-2-yl) amino] butanal (140 mg, 0.5 mmol) in anhydrous DMF (5 mL) the ethoxycarbonyl-2-phenylpropyl) -1, 3-oxazol-4-yl] methyl triphenylphosphonium bromide (369 mg, 0.6 mmol) and KHMDS (1.2 mL, 0.6 mmol) was added dropwise at 0 o C . After 3.5 h, the mixture was diluted with water (20 mL) and extracted with CH 2 Cl 2 (3 x 20 mL). The combined organic extracts were dried with MgSO 4, filtered, and concentrated under reduced pressure. Chromatography on silica gel and the residue (1: 1 EtOAc / CHCl 3 % MeOH in 3) to obtain the title compound (167 mg, 62%, E / Z isomer mixture) as a yellow oil: MS (ES) m / e 536 ( M + H) &lt; + & gt ; .
    [296] b) Preparation of ethyl (±) -3-phenyl-4- [4- [5- (pyridin- 2- yl) amino- 1 -phenyl] -1,3- oxazol- 2-yl] butanoate
    [297] Amino] -1-pentyl] -1 &lt; RTI ID = 0.0 &gt; , 3-oxazol-2-yl] butanoate (167 mg, 0.31 mmol) was dissolved in 4 N HCl in dioxane (3 mL). After 1.5 h, the mixture was concentrated under reduced pressure. The residue was taken up in saturated NaHCO 3 (20 mL) and extracted with CH 2 Cl 2 (2 x 20 mL). The combined organic extracts were dried with MgSO 4, filtered, and concentrated.
    [298] The residue was mixed with 10% Pd / C (33 mg) in isopropanol (5 mL) and cyclohexane (0.63 mL, 6.2 mmol) and the mixture was heated under a stream of nitrogen. After 18 h, the mixture was cooled to room temperature, The mixture was filtered through a pad, and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (1: from 1 EtOAc / CHCl 3 5% MeOH ) to give the title compound (57% from a 75 mg, a): MS ( ES) m / e 422 (M + H) +.
    [299] c) (±) -3-Phenyl-4- [4- [5- (pyridin-
    [300] THF / H 2 O) (1 : 1, 2 mL) of ethyl (±) -3- phenyl-4- [4- [5- (pyridin-2-yl) amino-1-pentyl] -1,3- Oxazol-2-yl] butanoate (75 mg, 0.18 mmol) in DMF (5 mL) was added 1 N LiOH (0.27 mL, 0.27 mmol). After 18 h, the pH was adjusted to pH 6 using 10% HCl, after which the solution was concentrated under reduced pressure to remove the THF. The solution was diluted with C-18 Megabond El route Was column (20% CH 3 CN / H 2 O containing 0.1% TFA after the H 2 O 50 mL) 200 mL ). The fractions containing the product were collected and lyophilized to give the title compound (43 mg, 61%) as a yellow gum: MS (ES) m / e 394 (M + H) &lt; + & gt ; . Elemental analysis: Calculated: C 23 H 27 N 3 O 3 0.098 CF 3 CO 2 H: C, 68.85; H, 6.75; N, 10.38. Found: C, 69.23; H, 7.00; N, 9.98.
    [301] Example 5
    [302] Parenteral dosage unit composition
    [303] A formulation containing 20 mg of the compound of Example 1 as a sterile dry powder is prepared as follows: Dissolve 20 mg of the compound in 15 mL of distilled water. The solution is filtered under sterile conditions in a 25 mL composite-dose ampoule and lyophilized. Powders are reconstituted by the addition of 20 mL of 5% dextrose in water for intravenous and intramuscular injection. Dosage is determined by injection volume. Thereafter, the metered dose of the determined dose unit is diluted by addition to another dose of D5W for injection, or the metered dose is administered as a bottle or bag for IV drip infusion or other injection-infusion system, It can be added to other mechanisms.
    [304] Example 6
    [305] Oral dosage unit composition
    [306] Orally administered capsules are prepared by mixing and milling 50 mg of the compound of Example 1 with 75 mg of lactose and 5 mg of magnesium stearate. The resulting powder is screened and filled into hard gelatin capsules.
    [307] Example 7
    [308] Oral dosage unit composition
    [309] Tablets for oral administration are prepared by mixing 20 mg of glycerol per day, 150 mg of calcium sulfate dihydrate and 50 mg of the compound of Example 1 with 10% gelatin solution and granulating. The wet granules are screened, dried, mixed with 10 mg of starch, 5 mg of talc and 3 mg of stearic acid, and then compressed into tablets.
    [310] The above description fully discloses the process and use of the present invention. However, the present invention is not limited to the specific embodiments described above, but includes all variations within the scope of the following claims. References to publications, patents, and other publications cited herein are incorporated by reference in their entirety, including those of skill in the art.
    权利要求:
    Claims (48)
    [1" claim-type="Currently amended] A compound of formula (I): &lt; EMI ID =
    (I)

    In this formula,
    Y is CR ' R ' or NR ' C (O);
    R 1 is C 0-6 alkyl, -Het, -C 0-6 alkyl -Ar, hydrogen, -C 1-6 alkyl, -CN or S (O) R k and g;
    R 2 is a residue of the formula:

    W is (CHR g ) a -U- (CHR g ) b -
    U is absent or CO, CR g 2, C ( = CR g 2), S (O) k, O, NR g, CR g OR g, CR g (OR k) CR g 2, CR g 2 CR g (OR k), C ( O) CR g 2, CR g 2 C (O), CONR i, NR i CO, OC (O), C (O) O, C (S) O, OC (S) , C (S) NR g, NR g C (S), S (O) 2 NR g, NR g S (O) 2 N = N, NR g NR g, NR g CR g 2, CR g 2 NR g , CR g 2 O, OCR g 2 , C C, CR g = CR g , Ar or Het;
    G is NRe, S or O;
    R g is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl or Ar-C 0-6 alkyl;
    R k is R g , -C (O) R g , or -C (O) OR f ;
    R i is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl, Ar-C 0-6 alkyl, or halogen, CN, NR g 2, OR g, SR g, CO 2 R g, and CON (R g) 1 gae selected from 2 to three groups substituted by C 1-6 alkyl;
    R f is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl;
    R e is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl-C 0-6 alkyl, or (CH 2 ) k CO 2 R g ego;
    R b and R c are independently hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl, Het-C 0-6 alkyl, or C 3-6 cycloalkyl, -C 0-6 alkyl, halogen, CF 3 , oR f, S (O) k R f, COR f, NO 2, N (R f) 2, CO (NR f) 2, CH 2 N (R f) selected from the second or, or R b and R c is halogen, CF 3, C 1-4 alkyl, OR f, S (O) k R f, COR f, CO 2 R f, OH, NO 2, N (R f) 2, CO (NR f bonded to each other ) 2 , and CH 2 N (R f ) 2 , or a 5 to 6 membered aromatic or nonaromatic carbocyclic or heterocyclic ring optionally substituted with up to three substituents selected from CH 2 N (R f ) 2 ; Or methylenedioxy;
    Q 1 , Q 2 , Q 3 and Q 4 are independently nitrogen or CR y , with the proviso that no more than one of Q 1 , Q 2 , Q 3 and Q 4 is nitrogen;
    R 'is hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl or C 3-6 cycloalkyl -C 0-6 alkyl;
    R "is R ', -C (O) R' or -C (O) OR ';
    R y is hydrogen, halo, -OR g, -SR g, -CN , -NR g R k, -NO 2, CF 3, CF 3 S (O) r -, -CO 2 R g, -COR g or -CONR g 2, or halo, -OR g, -SR g, -CN , -NR g R ", -NO 2, -CF 3, R'S (O) r -, -CO 2 R g, -COR g or by -CONR g 2, optionally substituted C 1-6 alkyl;
    a is 0, 1 or 2;
    b is 0, 1 or 2;
    k is 0, 1 or 2;
    r is 0, 1 or 2;
    s is 0, 1 or 2;
    u is 0 or 1;
    v is 0 or 1;
    [2" claim-type="Currently amended] A compound according to claim 1, wherein R 2 is a residue of the formula:

    Wherein Q 1 , Q 2 , and Q 3 are each CR y , Q 4 is CR y or nitrogen, and u is zero.
    [3" claim-type="Currently amended] The method of claim 2, wherein each R 'is hydrogen, R "is hydrogen or C 1-6 alkyl, W is (CH 2) 1-4 -, and an, Q 4 is CR y, and R y is a hydrogen compound .
    [4" claim-type="Currently amended] A compound according to claim 1, wherein R 2 is a residue of the formula:

    Wherein Q 1 , Q 2 , and Q 3 are each CH and u is zero.
    [5" claim-type="Currently amended] The compound of claim 4, wherein each R 'is hydrogen, R "is hydrogen or C 1-6 alkyl, v is 0, and W is - (CH 2 ) 1-4 -.
    [6" claim-type="Currently amended] A compound according to claim 1, wherein R 2 is a residue of the formula:

    Wherein G is NH and R &lt; b & gt ; and R &lt; c &gt; are each hydrogen.
    [7" claim-type="Currently amended] The method of claim 6, wherein, W is - (CH 2) 1-4 - the compound.
    [8" claim-type="Currently amended] A compound according to claim 1, wherein R 2 is a residue of the formula:

    Wherein, G is NH and R b and R c is halogen, CF 3, C 1-4 alkyl, OR f, S (O) k R f, COR f, CO 2 R f, OH, NO 2 combine with each other , N (R f) 2, CO (NR f) 2, and CH 2 N (R f) a by a substituent of up to three selected from 2, optionally substituted, 5- to 6-membered aromatic or non-aromatic carbocyclic or heterocyclic Form a cyclic ring; Or methylenedioxy.
    [9" claim-type="Currently amended] The compound according to claim 8, wherein R b and R c are bonded to each other to form a 6-membered aromatic carbocyclic ring.
    [10" claim-type="Currently amended] 10. The method of claim 9, wherein, W is - (CH 2) 1-4 - the compound.
    [11" claim-type="Currently amended] The compound according to claim 8, wherein R b and R c are bonded to each other to form a 6-membered aromatic heterocyclic ring.
    [12" claim-type="Currently amended] W is a method according to claim 11 wherein - (CH 2) 1-4 - the compound.
    [13" claim-type="Currently amended] A compound according to claim 1, wherein R 2 is a residue of the formula:

    Wherein each R 'is hydrogen, R "is hydrogen or C 1-6 alkyl, R g is hydrogen or C 1-6 alkyl, and s is 0, 1 or 2.
    [14" claim-type="Currently amended] 14. The method of claim 13, wherein, W is - (CH 2) 1-4 - the compound.
    [15" claim-type="Currently amended] The compound according to claim 1, wherein R &lt; 1 &gt; is phenyl, benzyl, pyridyl, imidazole, oxazolyl or thiazolyl.
    [16" claim-type="Currently amended] 16. The compound of claim 15, wherein R &lt; 1 &gt; is phenyl.
    [17" claim-type="Currently amended] 2. The compound according to claim 1, wherein Y is NHC (O).
    [18" claim-type="Currently amended] 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof,
    Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (2-amino- ;
    Ethyl] amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (Compound ;
    Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (2-amino-3- ; And
    Amino] carbonyl] -1,3-oxazol-2-yl] butanoic acid (hereinafter referred to as &quot; .
    [19" claim-type="Currently amended] A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.
    [20" claim-type="Currently amended] A pharmaceutical composition comprising the compound of claim 1, an anti-tumor agent, and a pharmaceutically acceptable carrier.
    [21" claim-type="Currently amended] 21. The pharmaceutical composition according to claim 20, wherein the antitumor agent is topotecan.
    [22" claim-type="Currently amended] 21. The pharmaceutical composition according to claim 20, wherein the antitumor agent is cisplatin.
    [23" claim-type="Currently amended] A pharmaceutical composition comprising a compound of claim 1, a bone resorption inhibitor and a pharmaceutically acceptable carrier.
    [24" claim-type="Currently amended] A method for treating a disease requiring antagonism of an V 3 receptor comprising administering a compound of claim 1 to a patient.
    [25" claim-type="Currently amended] A method for treating a disease requiring antagonism of an V 5 receptor comprising administering a compound of claim 1 to a patient.
    [26" claim-type="Currently amended] A method of treating osteoporosis comprising administering to a patient a compound of claim 1.
    [27" claim-type="Currently amended] A method of inhibiting angiogenesis comprising administering to a patient a compound of claim 1.
    [28" claim-type="Currently amended] A method of inhibiting tumor growth or tumor metastasis comprising administering to a patient a compound of claim 1.
    [29" claim-type="Currently amended] A method of treating arteriosclerosis or restenosis, comprising administering to a patient a compound of claim 1.
    [30" claim-type="Currently amended] A method of treating inflammation comprising administering to a patient a compound of claim 1.
    [31" claim-type="Currently amended] A method of inhibiting tumor growth comprising administering the compound of claim 1 and an antineoplastic agent in a stepwise or physical combination.
    [32" claim-type="Currently amended] 32. The method of claim 31, wherein the anti-tumor agent is topotecan.
    [33" claim-type="Currently amended] 32. The method of claim 31, wherein the anti-tumor agent is cisplatin.
    [34" claim-type="Currently amended] A method of treating osteoporosis or inhibiting bone loss comprising administering the compound of claim 1 and a bone resorption inhibitor in a stepwise or physical combination.
    [35" claim-type="Currently amended] A compound of formula (II): &lt; EMI ID =
    &Lt;

    In this formula,
    Y is CR ' R ' or NR ' C (O);
    R 1 is C 0-6 alkyl, -Het, -C 0-6 alkyl -Ar, hydrogen, -C 1-6 alkyl, -CN or S (O) R k and g;
    R 2 is a residue of the formula:

    W is (CHR g ) a -U- (CHR g ) b -
    U is absent or CO, CR g 2, C ( = CR g 2), S (O) k, O, NR g, CR g OR g, CR g (OR k) CR g 2, CR g 2 CR g (OR k), C ( O) CR g 2, CR g 2 C (O), CONR i, NR i CO, OC (O), C (O) O, C (S) O, OC (S) , C (S) NR g, NR g C (S), S (O) 2 NR g, NR g S (O) 2 N = N, NR g NR g, NR g CR g 2, CR g 2 NR g , CR g 2 O, OCR g 2 , C C, CR g = CR g , Ar or Het;
    G is NRe, S or O;
    R g is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl or Ar-C 0-6 alkyl;
    R k is R g , -C (O) R g , or -C (O) OR f ;
    R i is hydrogen, C 1-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl, -C 0-6 alkyl, Ar-C 0-6 alkyl, or halogen, CN, NR g 2, OR g, SR g, CO 2 R g, and CON (R g) 1 gae selected from 2 to three groups substituted by C 1-6 alkyl;
    R f is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl;
    R e is hydrogen, C 1-6 alkyl or Ar-C 0-6 alkyl, Het-C 0-6 alkyl, C 3-7 cycloalkyl-C 0-6 alkyl, or (CH 2 ) k CO 2 R g ego;
    R b and R c are independently hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl, Het-C 0-6 alkyl, or C 3-6 cycloalkyl, -C 0-6 alkyl, halogen, CF 3 , oR f, S (O) k R f, COR f, NO 2, N (R f) 2, CO (NR f) 2, CH 2 N (R f) selected from the second or, or R b and R c is halogen, CF 3, C 1-4 alkyl, OR f, S (O) k R f, COR f, CO 2 R f, OH, NO 2, N (R f) 2, CO (NR f bonded to each other ) 2 , and CH 2 N (R f ) 2 , or a 5 to 6 membered aromatic or nonaromatic carbocyclic or heterocyclic ring optionally substituted with up to three substituents selected from CH 2 N (R f ) 2 ; Or methylenedioxy;
    Q 1 , Q 2 , Q 3 and Q 4 are independently nitrogen or CR y , with the proviso that no more than one of Q 1 , Q 2 , Q 3 and Q 4 is nitrogen;
    R 'is hydrogen, C 1-6 alkyl, Ar-C 0-6 alkyl or C 3-6 cycloalkyl -C 0-6 alkyl;
    R "is R ', -C (O) R' or -C (O) OR ';
    R y is hydrogen, halo, -OR g, -SR g, -CN , -NR g R k, -NO 2, CF 3, CF 3 S (O) r -, -CO 2 R g, -COR g or -CONR g 2, or halo, -OR g, -SR g, -CN , -NR g R ", -NO 2, -CF 3, R'S (O) r -, -CO 2 R g, -COR g or by -CONR g 2, optionally substituted C 1-6 alkyl;
    a is 0, 1 or 2;
    b is 0, 1 or 2;
    k is 0, 1 or 2;
    r is 0, 1 or 2;
    s is 0, 1 or 2;
    u is 0 or 1;
    v is 0 or 1;
    [36" claim-type="Currently amended] 8. A process for the preparation of a compound of formula (I) as defined in claim 1, which comprises reacting a compound of formula (III): wherein R 1 is a protected compound of formula (IV) A process for preparing a compound of formula (I)
    (III)

    (IV)

    Wherein R 1 and R 2 are as defined in formula (I).
    [37" claim-type="Currently amended] 19. A compound according to any one of claims 1 to 18 for use as a medicament.
    [38" claim-type="Currently amended] The use of a compound of formula (I) as defined in claim 1 in the manufacture of a medicament for the treatment of diseases requiring antagonism of the v 3 receptor.
    [39" claim-type="Currently amended] The use of a compound of formula (I) as defined in claim 1 in the manufacture of a medicament for the treatment of diseases requiring antagonism of the v 5 receptor.
    [40" claim-type="Currently amended] Use of a compound of formula (I) as defined in claim 1 in the manufacture of a medicament for the treatment of osteoporosis.
    [41" claim-type="Currently amended] Use of a compound of formula (I) as defined in claim 1 in the manufacture of a medicament for inhibiting angiogenesis.
    [42" claim-type="Currently amended] Use of a compound of formula (I) as defined in claim 1 in the manufacture of a medicament for inhibiting tumor growth or tumor metastasis.
    [43" claim-type="Currently amended] Use of a compound of formula (I) as defined in claim 1 in the manufacture of a medicament for the treatment of arteriosclerosis or restenosis.
    [44" claim-type="Currently amended] Use of a compound of formula (I) as defined in claim 1 in the manufacture of a medicament for the treatment of inflammation.
    [45" claim-type="Currently amended] The use of a compound of formula (I) and an antineoplastic agent as defined in claim 1 for physically combined or stepwise administration in the manufacture of a medicament for inhibiting tumor growth.
    [46" claim-type="Currently amended] 46. The use of claim 45 wherein the antineoplastic agent is topotecan.
    [47" claim-type="Currently amended] 46. The use of claim 45 wherein the antineoplastic agent is cisplatin.
    [48" claim-type="Currently amended] The use of a compound of formula (I) and a bone resorption inhibitor as defined in claim 1 for physically combined or stepwise administration in the manufacture of a medicament for the treatment of osteoporosis.
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    同族专利:
    公开号 | 公开日
    AU5336299A|2000-02-28|
    TR200100363T2|2001-06-21|
    HU0103090A3|2002-12-28|
    PL346157A1|2002-01-28|
    WO2000007544A2|2000-02-17|
    CA2339666A1|2000-02-17|
    JP2002522360A|2002-07-23|
    CZ2001447A3|2001-08-15|
    NO20010620L|2001-02-06|
    CN1322133A|2001-11-14|
    EP1102587A4|2003-03-12|
    US6458814B1|2002-10-01|
    ZA200100935B|2002-05-14|
    NO20010620D0|2001-02-06|
    CO5170532A1|2002-06-27|
    AR020121A1|2002-04-10|
    EP1102587A2|2001-05-30|
    BR9912638A|2001-05-02|
    WO2000007544A3|2000-05-18|
    IL141163D0|2002-02-10|
    HU0103090A2|2002-01-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1998-08-07|Priority to US9570398P
    1998-08-07|Priority to US60/095,703
    1999-08-03|Application filed by 스튜어트 알. 수터, 스티븐 베네티아너, 피터 존 기딩스, 스미스클라인 비참 코포레이션
    1999-08-03|Priority to PCT/US1999/017665
    2001-09-10|Publication of KR20010086355A
    优先权:
    申请号 | 申请日 | 专利标题
    US9570398P| true| 1998-08-07|1998-08-07|
    US60/095,703|1998-08-07|
    PCT/US1999/017665|WO2000007544A2|1998-08-07|1999-08-03|Vitronectin receptor antagonists|
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