Tetrahydroisoquinoline-3-carboxylic acid alkoxyguanidines as integrin antagonists

FIELD OF THE INVENTION

[0002] The present invention relates to novel tetrahydroisoquinoline-3-carboxylic acid alkoxyguanidine compounds that are antagonists of alpha V (αv) integrins, for example αvβ3 and αvβ5 integrins, their pharmaceutically acceptable salts, and pharmaceutical compositions thereof.

[0003] BACKGROUND ART

[0004] Integrins are cell surface glycoprotein receptors which bind extracellular matrix proteins and mediate cell-cell and cell-extracellular matrix interactions (generally referred to as cell adhesion events) (Hynes, R. O., Cell 69:11-25 (1992)). These receptors are composed of noncovalently associated alpha (α) and beta (β) chains which combine to give a variety of heterodimeric proteins with distinct cellular and adhesive specificities (Albeda, S. M., Lab. Invest. 68:4-14 (1993)). Recent studies have implicated integrins in the regulation of cellular adhesion, migration, invasion, proliferation, apoptosis and gene expression (Albeda, S. M., Lab. Invest. 68:4-14 (1993): Juliano, R., Cancer Met. Rev. 13:25-30 (1994); Ruoslahti, E. and Reed, J. C., Cell 77:477-478 (1994); and Ruoslahti, E. and Giancotti, F. G., Cancer Cells 1:119-126 (1989)).

[0005] One member of the integrin family which has been shown to play a significant role in a number of pathological conditions is the integrin αvβ3, or vitronectin receptor (Brooks, P. C., DN&P 10(8):456-461 (1997)). This integrin binds a variety of extracellular matrix components and other ligands, including fibrin, fibrinogen, fibronectin, vitronectin, laminin, thrombospondin, and proteolyzed or denatured collagen (Cheresh, D. A., Cancer Met. Rev. 10:3-10 (1991) and Shattil, S. J., Thromb. Haemost. 74:149-155 (1995)). The two related αv integrins, αvβ5 and αvβ1 (also vitronectin receptors), are more specific and bind vitronectin (αvβ5) or fibronectin and vitronectin (αvβ1) exclusively (Horton, M., Int. J Exp. Pathol. 71:741-759 (1990)). αvβ3 and the other integrins recognize and bind to their ligands through the tripeptide sequence Arg-Gly-Asp (“RGD”) (Cheresh, D. A., Cancer Met. Rev. 10:3-10 (1991) and Shattil, S. J., Thromb. Haemost. 74:149-155 (1995)) found within all the ligands mentioned above.

[0006] The αvβ3 integrin has been implicated in a number of pathological processes and conditions, including metastasis and tumor growth, pathological angiogenesis, and restenosis. For example, several studies have clearly implicated αvβ3 in the metastatic cascade (Cheresh, D. A., Cancer Met. Rev. 10:3-10 (1991); Nip, J. et al., J. Clin. Invest. 95:2096-2103 (1995); and Yun, Z., et al., Cancer Res. 56:3101-3111 (1996)). Vertically invasive lesions in melanomas are also commonly associated with high levels of αvβ3, whereas horizontally growing noninvasive lesions have little if any αvβ3 (Albeda, S. M., et al., Cancer Res. 50:6757-6764 (1990)). Moreover, Brooks et al. (in Cell 79:1157-1164 (1994)) have demonstrated that systemic administration of αvβ3 antagonists disrupts ongoing angiogenesis on chick chorioallantoic membrane (“CAM”), leading to the rapid regression of histologically distinct human tumors transplanted onto the CAM. These results indicate that antagonists of αvβ3 may provide a therapeutic approach for the treatment of neoplasia (solid tumor growth).

[0007] αvβ3 has also been implicated in angiogenesis, which is the development of new vessels from preexisting vessels, a process that plays a significant role in a variety of normal and pathological biological events. It has been demonstrated that αvβ3 is up-regulated in actively proliferating blood vessels undergoing angiogenesis during wound healing as well as in solid tumor growth. Also, antagonists of αvβ3 have been shown to significantly inhibit angiogenesis induced by cytokines and solid tumor fragments (Brooks, P. C. et al., Science 264:569-571 (1994); Enenstein, J. and Kramer, R. H., J. Invest. Dermatol. 103:381-386 (1994); Gladson, C. L., J. Neuropathol. Exp. Neurol 55:1143-1149 (1996); Okada, Y., et al, Amer. J. Pathol. 149:37-44 (1996); and Brooks, P. C., et al., J. Clin. Invest. 96:1815-1822 (1995)). Such αvβ3 antagonists would be useful for treating conditions that are associated with pathological angiogenesis, such as rheumatoid arthritis, diabetic retinopathy, macular degeneration, and psoriasis (Nicosia, R. F. and Madri, J. A., Amer. J. Pathol. 128:78-90 (1987); Boudreau, N. and Rabinovitch, M., Lab. Invest. 64:187-199 (1991); and Brooks, P. C., Cancer Met. Rev. 15:187-194 (1996)).

[0008] There is also evidence that αvβ3 plays a role in neointimal hyperplasia after angioplasty and restenosis. For example, peptide antagonists and monoclonal antibodies directed to both αvβ3 and the platelet receptor αIIbβ3 have been shown to inhibit neointimal hyperplasia in vivo (Choi, E. T., et al., J. Vasc. Surg. 19:125-134 (1994); and Topol, E. J., et al., Lancet 343:881-886 (1994)), and recent clinical trials with a monoclonal antibody directed to both αIIbβ3 and αvβ3 have resulted in significant reduction in restenosis, providing clinical evidence of the therapeutic utility of β3 antagonists (Topol, E. J., et al, Lancet 343:881-886 (1994)).

[0009] It has also been reported that αvβ3 is the major integrin on osteoclasts responsible for attachment to bone. Osteoclasts cause bone resorption. When bone resorbing activity exceeds bone forming activity, the result is osteoporosis, a condition which leads to an increased number of bone fractures, incapacitation and increased mortality. Antagonists of αvβ3 have been shown to be potent inhibitors of osteoclastic activity both in vitro (Sato, M., et al, J. Cell Biol. 111: 1713-1723 (1990)) and in vivo (Fisher, J. E., et al., Endocrinology 132:1411-1413 (1993)).

[0010] Lastly, White (in Current Biology 3(9):596-599 (1993)) has reported that adenovirus uses αvβ3 for entering host cells. The αvβ3 integrin appears to be required for endocytosis of the virus particle and may be required for penetration of the viral genome into the host cell cytoplasm. Thus compounds which inhibit αvβ3 could be useful as antiviral agents.

[0011] The αvβ5 integrin has been implicated in pathological processes as well. Friedlander et al have demonstrated that a monoclonal antibody for αvβ5 can inhibit VEGF-induced angiogenesis in rabbit cornea and chick chorioalloantoic membrane, indicating that the αvβ5 integrin plays a role in mediating growth factor-induced angiogenesis (Friedlander, M. C., et al, Science 270:1500-1502 (1995)). Compounds that act as αvβ5 antagonists could be used to inhibit pathological angiogenesis in tissues of the body, including ocular tissue undergoing neovascularization, inflamed tissue, solid tumors, metastases, or tissues undergoing restenosis.

[0012] Discovery of the involvement of αvβ3 and αvβ5 in such processes and pathological conditions has led to an interest in these integrins as potential therapeutic targets, as suggested in the preceding paragraphs. A number of specific antagonists of αvβ3 and αvβ5 that can block the activity of these integrins have been developed. One major group of such antagonists includes nonpeptide mimetics and organic-type compounds. For example, a number of organic nonpeptidic mimetics have been developed that appear to inhibit tumor cell adhesion to a number of αvβ3 ligands, including vitronectin, fibronectin, and fibrinogen (Greenspoon, N., et al., Biochemistry 32:1001-1008 (1993); Ku, T. W., et al., J. Amer. Chem. Soc. 115:8861-8862 (1993); Hershkoviz, R., et al., Clin. Exp. Immunol. 95:270-276 (1994); and Hardan, L., et al., Int. J. Cancer 55:1023-1028 (1993)).

[0013] Additional organic compounds developed specifically as αvβ3 or αvβ5 integrin antagonists or as compounds useful in the treatment of αv-mediated conditions have been described in several recent publications.

[0014] For example, U.S. Pat. No. 5,731,324, issued Mar. 24, 1998, discloses bicyclic compounds of formula:

[0015] wherein the bicyclic nucleus is preferably selected from the group consisting of benzopyran, isoquinoline, isoquinolone, tetrahydronaphthalene, dihydronaphthalene and tetralone. The compounds are disclosed to be useful as glycoprotein IIb/IIIa antagonists for the prevention of thrombosis.

[0016] PCT Published Application WO 97/06791, published February 1997, discloses methods for inhibition of angiogenesis in tissue using vitronectin αvβ5 antagonists.

[0017] More recently, PCT Published Application WO 97/23451, published Jul. 3, 1997, discloses tyrosine derivatives of the general formula:

[0018] wherein

[0019] X is C1-6alkylene or 1,4-piperidyl;

[0020] Y is absent, O, CONH or —-C≡C—;

[0021] R1 is H, CN, N3, NH2, H2N—C(═NH), or H2N—C(═NH)—NH, where the primary amino groups can also be provided with conventional amino protective groups;

[0022] R2 and R3 are independently H, A, A-SO2—, Ar—SO2—, camphor-10-SO2, COOA or a conventional amino protective group;

[0023] A and R4 are independently H, C1-10alkyl, or benzyl; and

[0024] Ar is phenyl or benzyl, each of which is unsubstituted or monosubstituted by CH3;

[0025] and their physiologically acceptable salts.

[0026] The disclosed compounds are described as αv-integrin inhibitors (especially αvβ3 inhibitors) useful in the treatment of tumors, osteoporoses, and osteolytic disorders and for suppressing angiogenesis.

[0027] PCT Published Application WO 98/00395, published Jan. 8, 1998, discloses novel tyrosine and phenylalanine derivatives as av integrin and GPIIb/IIIa antagonists having the general formula:

[0028] wherein

[0029] X can be, among other groups, alkyl, aryl or cycloalkyl;

[0030] Y and Z can be alkyl, O, S, NH, C(═O), CONH, NHCO, C(═S), SO2NH, NHSO2, CA═CA′ or —C≡C—;

[0031] R1 can be H2N—C(═NH) or H2N-(C═NH)—NH;

[0032] R2 is A, aryl or aralkyl;

[0033] R3 is hydrogen or A;

[0034] R4 is hydrogen, halogen, OA, NHA, NAA′, —NH-Acyl, —O-Acyl, CN, NO2, SA, SOA, SO2A, SO2Ar or SO3H; and

[0035] A and A′ can be hydrogen, alkyl or cycloalkyl.

[0036] The publication discloses the use of the compounds in pharmaceutical preparations for the treatment of thrombosis, infarction, coronary heart disease, tumors, arteriosclerosis, infection and inflammation.

[0037] A need continues to exist for non-peptide compounds that are potent and selective integrin antagonists, and which possess greater bioavailability or fewer side-effects than currently available integrin antagonists.

SUMMARY OF THE INVENTION

[0038] The present invention is directed to novel tetrahydroisoquinoline-3-carboxylic acid alkoxyguanidine compounds having Formula I (below). Also provided is a process for preparing compounds of Formula I. The novel compounds of the present invention exhibit inhibition of αvβ3 and αvβ5 integrin receptor binding. Also provided is a method of treating αvβ3 integrin- and αvβ5 integrin-mediated pathological conditions such as tumor growth, metastasis, osteoporosis, restenosis, inflammation, macular degeneration, diabetic retinopathy, and rheumatoid arthritis in a mammal in need of such treatment comprising administering to said mammal an effective amount of a compound of Formula I. Further provided is a pharmaceutical composition comprising a compound of Formula I and one or more pharmaceutically acceptable carriers or diluents.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention is directed to compounds of Formula I:

[0040] and pharmaceutically acceptable salts thereof; wherein

[0041] R1 is hydrogen, alkyl, aralkyl, R11SO2, R11OOC, R11CO or R11CH2, where R11 is (i) hydrogen, or (ii) alkyl, cycloalkyl, camphor-10-yl, alkenyl, alkynyl, heterocycle, aryl, aralkyl, or aralkenyl, any of which can be optionally substituted by one or more alkyl, alkenyl, aryl, aryloxy (further optionally substituted by nitro, halo, or cyano), aralkyl, aryldiazenyl (further optionally substituted by amino, alkylamino, or dialkylamino), alkoxy, haloalkyl, haloalkoxy, alkylcarbonylamino, alkylsulfonyl, mono- or di-alkylamino, hydroxy, carboxy, cyano, nitro, halo, or a heteroaryl which is optionally substituted with one or more alkyl, haloalkyl, or halo;

[0042] and when R1 is R11CO, then R11 can also be N-attached pyrrolidinyl, piperidinyl or morpholinyl;

[0043] R2 is hydrogen or a functionality which acts as a prodrug ( i.e., converts to the active species by an endogenous biological process such as an esterase, lipase, or other hydrolases), such as alkyl, aryl, aralkyl. dialkylaminoalkyl, 1-morpholinoalkyl, 1-piperidinylalkyl, pyridinylalkyl, alkoxy(alkoxy)alkoxyalkyl, or (alkoxycarbonyl)oxyethyl;

[0044] R3 is hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, hydroxy, alkoxy, aralkoxy, aryloxy, heteroaryloxy, or mono- or di- alkylamino;

[0045] R4, R5, and R6 are independently hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl or carboxyalkyl;

[0046] or R3 and R4 are taken together to form —(CH2)y—, where y is zero (a bond), 1 or 2, while R5 and R6 are defined as above; or R3 and R6 are taken together to form —(CH2)q—, where q is zero (a bond), or 1 to 8, while R4 and R5 are defined as above; or R4 and R5 are taken together to form —(CH2)r—, where r is 2-8, while R3 and R6 are defined as above;

[0047] R7 is hydrogen, alkyl, aralkyl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl or carboxyalkyl;

[0048] R8, R9, and R10 are independently hydrogen, alkyl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, alkoxycarbonyloxy, cyano or —COORw;

[0049] Rw is alkyl, cycloalkyl, phenyl, benzyl,

[0050] where Ra and Rb are independently hydrogen, alkyl, alkenyl or phenyl; Rc is hydrogen, alkyl, alkenyl or phenyl; Rd is hydrogen, alkyl, alkenyl or phenyl; and Re is aralkyl or alkyl;

[0051] n is from zero to 8; and m is from zero to 4, provided that n is other than zero when R3 is hydroxy, alkoxy, aralkoxy, aryloxy, heteroaryloxy, or mono- or dialkylamino.

[0052] Preferred compounds of the present invention are those of Formula I wherein:

[0053] R1 represents hydrogen, C1-6 alkyl, C6-10 ar(C1-6)alkyl, R11SO2, R11OOC, R11 CO or R11CH2, where R11 is hydrogen, C1-6 alkyl, C6-10 ar(C1-6)alkyl, C4-7 cycloalkyl(C1-4)alkyl, camphor-10-yl, or C6-10 aryl substituted by one or more C1-6 alkyl, C2-6 alkenyl, C6-10 aryl, C6-10 ar(C1-6)alkyl, C6-10 aryloxy (further optionally substituted by nitro, halo, or cyano), C6-10 aryldiazenyl (further optionally substituted by amino, C1-4 alkylamino or di-(C1-4)alkylamino), C1-6 alkoxy, halo(C1-6)alkyl, halo(C1-6)alkoxy, C1-6 alkylcarbonylamino, C1-6 alkylsulfonyl, mono- or di-(C1-6)alkylamino, hydroxy, carboxy, cyano, nitro, halo, or a heteroaryl which is optionally substituted with one or more C1-6 alkyl, halo(C1-6)alkyl, or halo;

[0054] and when R1 is R11CO, then R11 can also be N-attached pyrrolidinyl, piperidinyl or morpholinyl.

[0055] Preferred values of R1 include hydrogen, t-butylcarbonyl, butylsulfonyl, propylsulfonyl, optionally substituted benzylsulfonyl, optionally substituted phenylsulfonyl, pentylsulfonyl, 4-tolylsulfonyl, naphthylsulfonyl and camphor-10-sulfonyl.

[0056] Especially preferred compounds are those of Formula I wherein:

[0057] R1 is R11SO2 wherein R11 is hydrogen, C1-6 alkyl, C4-7 cycloalkyl, camphor-10-yl, C2-6 alkenyl, C2-6 alkynyl, thienyl, thiazolyl, benzo[b]thiophenyl, pyrazolyl, chromanyl, imidazolyl, benzo

[0058] [2,3-c]1,2,5-oxadiazole, C6-10 aryl, C6-10 ar(C1-6)alkyl, or C6-10 ar(C2-6)alkenyl, any of which can be optionally substituted by one or more C1-6 alkyl, C2-6 alkenyl, C6-10 aryl. C6-10 aryloxy (further optionally substituted by nitro, halo, or cyano), C6-10 ar(C1-6)alkyl, 4-dimethylaminophenyldiazenyl, C1-6 alkoxy, halo(C1-6)alkyl, halo(C1-6)alkoxy, C1-6 alkylcarbonylamino, C1-6 alkylsulfonyl, mono- or di-(C1-6)alkylamino, hydroxy, carboxy, cyano, nitro, halo, or pyrazolyl which is optionally substituted with one or more C1-6 alkyl, halo(C1-6)alkyl, or halo.

[0059] Suitable values of R11 include methyl, butyl, chloropropyl, phenyl, benzyl, methylphenyl, ethylphenyl, propylphenyl, butylphenyl, tert-butylphenyl, pentylphenyl, phenylphenyl, camphoryl, nitrophenyl, nitrophenylmethyl, cyanophenyl, chlorophenyl, fluorophenyl, bromophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl, acetylaminophenyl, butoxyphenyl, biphenyl, vinylphenyl, methoxyphenyl, methylsulfonylphenyl, 4-(3-chloro-2-cyanophenoxy)phenyl, 4-(1,1-dimethylpropyl)phenyl, 6-chloro-2-methylphenyl, 2-methyl-5-nitrophenyl, 2,3,4-trichlorophenyl, 4-bromo-2,5-difluorophenyl, 5-bromo-2-methoxyphenyl, 2-chloro-5-(trifluoromethyl)phenyl, 4-(2-chloro-6-nitrophenoxy, 4-bromo-2-(trifluoromethoxy)phenyl, 3-chloro-2-cyanophenyl, 3-chloro-2-methylphenyl, 2-methyl-5-nitrophenyl, 4-methyl-3-nitrophenyl, 2,5-bis(2,2,2-trifluoroethoxy)phenyl, 2-chloro-4-(trifluoromethyl)phenyl, 4-chloro-2,5-dimethylphenyl, 5-chloro-2-methoxyphenyl, 4,6-dichloro-2-methylphenyl, 4-bromo-2-methylphenyl, 4-bromo-2-ethylphenyl, 2,4,6-trimethylphenyl, 2,3,4,5,6-pentamethylphenyl, 3,5-dichloro-2-hydroxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl, 2,5-dimethylphenyl, 2-chloro-4-(trifluoromethyl)phenyl, 3,5-dichlorophenyl, 2,6-dichlorophenyl, 2,3-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2,4-dichlorophenyl, 3,4-dibromophenyl, 2,6-difluorophenyl, 3,4-difluorophenyl, 2,4,5-trichlorophenyl, 2,4,6-trichlorophenyl, 2,4,6-tris(methylethyl)phenyl, 4-bromo-2-ethylphenyl, 4-chloro-3-nitrophenyl, 2-methoxy-5-methylphenyl, 5-fluoro-2-methylphenyl, 4-methoxy-2,3,6-trimethylphenyl, 3-chloro-4-methylphenyl, 1-methylimidazol-4-yl, carboxyphenyl, naphthyl, 2,2,5,7,8-pentamethyl-chroma-6-yl, thienyl, 5-chloro-2-thienyl, 3-bromo-5-chloro-2-thienyl, 4-bromo-2,5-dichloro-3-thienyl, 4,5-dibromo-2-thienyl, 4-bromo-5-chloro-2-thienyl, 5-bromo-2-thienyl, 2,5-dichloro-3-thienyl, 2-(acetylamino)-4-methyl-1,3-thiazol-5-yl, 5-chloro-1,3-dimethylpyrazol-4-yl, 5-[1-methyl-5-(trifluoromethyl)-pyrazol-3-yl]-2-thienyl, 5-chloro-3-methylbenzo[b]thiophen-2-yl, 5-chloro-1,3-dimethylpyrazol-4-yl, 4-[4-(dimethylaminophenyl)diazenyl]phenyl, 4-[3-(amidinoaminooxy)-propoxy]-phenyl, benzo[2,3-c]1,2,5-oxadiazol-4-yl, and 2-phenylvinyl.

[0060] Preferred R2 groups include hydrogen, C1-6 alkyl and benzyl.

[0061] Preferred values of R3 include hydrogen, C1-6 alkyl, C6-10 ar(C1-6)alkyl, C6-10 aryl, C2-10 hydroxyalkyl, C2-10 aminoalkyl, C2-7 carboxyalkyl, mono(C1-4 alkyl)amino(C1-8)alkyl, and di(C1-4 alkyl)amino(C1-8)alkyl. Suitable values of R3 include methyl, ethyl, propyl, n-butyl, benzyl, phenylethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 2-aminoethyl, carboxymethyl, 2-carboxyethyl, 3-carboxypropyl, 4-carboxybutyl and 2-(dimethylamino)ethyl.

[0062] Preferred compounds are those of Formula I in which R4, R5 and R6 are independently hydrogen, C1-6 alkyl, C6-10 ar(C1-6)alkyl, C6-10 aryl, C2-10 hydroxyalkyl or C2-7 carboxyalkyl. Useful values of R4, R5, and R6 include hydrogen, methyl, ethyl, propyl, n-butyl, benzyl, phenylethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, carboxymethyl, 2-carboxyethyl, 3-carboxypropyl and 4-carboxybutyl. In the most preferred embodiments, R4, R5 and R6 are each hydrogen.

[0063] Preferred values of R7 include hydrogen or C1-6 alkyl.

[0064] Preferred values of R8, R9 and R10 in Formula I include hydrogen, hydroxy, C1-6 alkyl, C1-6 alkoxy, cyano or —CO2Rw, where Rw, in each instance, is preferably one of C1-4 alkyl, C4-7 cycloalkyl, phenyl, or benzyl. Suitable values of R8, R9 and R10 include hydrogen, methyl, ethyl, propyl, n-butyl, hydroxy, methoxy, ethoxy, cyano, —CO2CH3, —CO2CH2CH3 and —CO2CH2CH2CH3. In the most preferred embodiments, R8, R9 and R10 are each hydrogen.

[0065] Preferred values of n in Formula I include zero to 6, more preferably zero to 4, and most preferably zero, 1, or 2.

[0066] Preferred values of m include zero to 4, and most preferably zero, 1, or 2.

[0067] Useful compounds of the present invention include, without limitation:

[0068] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2,5-dimethoxyphenyl)-sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0069] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-(phenylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0070] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-(2-naphthylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0071] (3S)-7-[3-(Amidinoaminooxy)propoxy)]-2-{[2-(methylsulfonyl)-phenyl]sulfonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0072] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-(butylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0073] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2,6-dichlorophenyl)-sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0074] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2-methyl-5-nitrophenyl)sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0075] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-{[(7,7-dimethyl-2-oxobicyclo[2.2.1]heptyl)methyl]sulfonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;

[0076] or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof. Preferred salts include the HCl and TFA (trifluoroacetic acid) salts.

[0077] It is also to be understood that the present invention is considered to include stereoisomers as well as optical isomers, e.g. mixtures of enantiomers as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in selected compounds of the present series.

[0078] When any variable occurs more than one time in any constituent or in Formula I, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

[0079] The term “alkyl” as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 12 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl. Preferred alkyl groups have from 1 to 6 carbon atoms.

[0080] The term “alkenyl” is used herein to mean a straight or branched chain radical of 2-20 carbon atoms, unless the chain length is limited thereto, including, but not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Preferably, the alkenyl chain is 2 to 10 carbon atoms in length, more preferably, 2 to 8 carbon atoms in length, most preferably from 2 to 4 carbon atoms in length.

[0081] The term “alkoxy” is used herein to mean a straight or branched chain radical of 1 to 20 carbon atoms, unless the chain length is limited thereto, bonded to an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably the alkoxy chain is 1 to 10 carbon atoms in length, more preferably 1 to 8 carbon atoms in length.

[0082] The term “aryl” as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, naphthyl or tetrahydronaphthyl.

[0083] The term “aryloxy” as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, bonded to an oxygen atom. Examples include, but are not limited to, phenoxy, naphthoxy, and the like.

[0084] The term “heteroaryl” as employed herein refers to groups having 5 to 14 ring atoms; 6, 10 or 14 Λ electrons shared in a cyclic array; and containing carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfur heteroatoms (where examples of heteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinyl groups).

[0085] The term “aralkyl” or “arylalkyl” as employed herein by itself or as part of another group refers to C1-6 alkyl groups as discussed above having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.

[0086] The term “cycloalkyl” as employed herein by itself or as part of another group refers to cycloalkyl groups containing 3 to 9 carbon atoms. Typical examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl.

[0087] The term “heterocycle” as used herein, except where noted, represents a stable 5- to 7-membered mono- or bicyclic or stable 7- to 10-membered bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. Especially useful are rings containing one oxygen or sulfur, one to three nitrogen atoms, or one oxygen or sulfur combined with one or two nitrogen atoms. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic groups include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, chromanyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzo[b]thiophenyl, benzo[2,3-c]1,2,5-oxadiazolyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same as morpholinyl.

[0088] The term “halogen” or “halo” as employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine with chlorine being preferred.

[0089] The term “monoalkylamine” as employed herein by itself or as part of another group refers to an amino group which is substituted with one alkyl group having from 1 to 6 carbon atoms.

[0090] The term “dialkylamine” as employed herein by itself or as part of another group refers to an amino group which is substituted with two alkyl groups, each having from 1 to 6 carbon atoms.

[0091] The term “hydroxyalkyl” as employed herein refers to any of the above alkyl groups substituted by one or more hydroxyl moieties.

[0092] The term “carboxyalkyl” as employed herein refers to any of the above alkyl groups substituted by one or more carboxylic acid moieties.

[0093] The term “haloalkyl” as employed herein refers to any of the above alkyl groups substituted by one or more chlorine, bromine, fluorine or iodine with fluorine and chlorine being preferred, such as chloromethyl, iodomethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 2-chloroethyl.

[0094] The term “haloalkoxy” as used herein refers to any of the above haloalkyl groups bonded to an oxygen atom, such as trifluromethoxy, trichloromethoxy, and the like.

[0095] Another aspect of the present invention is a process for preparing a tetrahydroisoquinoline-3-carboxylic acid alkoxyguanidine compound of Formula I, comprising reacting a compound of Formula II:

[0096] or a salt, hydrate, solvate or prodrug thereof, wherein R1, R2, R3, R4, R5, R6, m and n are as defined above, with a deprotection reagent and a guanidinylating reagent, to form a compound of Formula III:

[0097] or a salt, hydrate, solvate or prodrug thereof, where R1, R2, R3, R4, R5, R6, R8, R9, m and n are as defined as above. Preferred deprotection reagents include hydrazine or methylamine. Preferred guanidinylating reagents include aminoiminosulfonic acid, 1H-pyrazole-1-carboxamidine hydrochloride, N,N′-bis(tert-butoxycarbonyl)-S-methylisothiourea, or N-R8, N-R9-1H-pyrazole-1-carboxamidine, where R8 and R9 are defined as above.

[0098] The compounds of the present invention may be prepared by the general procedures outlined in Schemes I, II, and III (below), where R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, Rw, n, and m are as defined above.

[0099] Scheme I outlines the synthetic steps to produce compounds of the present invention where R1 is R11CO— or R11OOC— or R11CH2—. The carboxyl group of the (3S)-1,2,3,4-tetrahydro-7-hydroxy-isoquinoline-3-carboxylic acid 1 is protected as an ester by methods well known in the art (Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis, Springer-Verlag, Berlin (1984)). The resulting amine is reacted with acyl chlorides (R11COCl) in the presence of a suitable base such as a tertiary amine to produce carboxamides 2 (R1═R11CO). Alternatively, the carboxamides 2 may be produced by the reaction of (3S)-1,2,3,4-tetrahydro-7-hydroxy-isoquinoline-3-carboxylate with carboxylic acids (R11COOH) by any of the known peptide coupling reagents, such as 1,3-dicyclohexylcarbodiimide or Castro's reagent (BOP) (Castro, B., et al., Tetrahedron Letter 1219 (1975)). Still alternatively, the (3S)-1,2,3,4-tetrahydro-7-hydroxy-isoquinoline-3-carboxylate can be converted to carboxamides 2 (R1═R11OOC) by reaction with chloroformates (R11OCOCl) in the presence of a base, such as a tertiary amine. Still alternatively, reductive amination of the secondary amine can be achieved by reaction with an aldehyde (R11CHO) under reducing conditions to give 2 (R1═R11CH2). The preferred reducing agent is tetramethylammonium triacetoxyborohydride. Alternatively, sodium triacetoxyborohydride or sodium cyanoborohydride may be used. As an alternative to reduction methods, the (3S)-1,2,3,4-tetrahydro-7-hydroxy-isoquinoline-3-carboxylate may be reacted with R11CH2L, where L is a reactive leaving group, such as a halide or sulfonate, to produce the carboxamide 2 (R1═R11CH2).

[0100] The phenolic functionality of 2 is coupled to alcohol 3, where L is a reactive leaving group, such as a halide or sulfonate, under basic conditions, such as cesium carbonate in a solvent such as acetonitrile. Alternatively, the phenolic functionality of 2 may be coupled to 3 (L═OH) using a Mitsunobu coupling procedure (Mitsunobu, O., Synthesis 1 (1981)). Preferred coupling conditions include using a trialkylphosphine or triarylphosphine, such as tri-n-butylphosphine or triphenylphosphine, in a suitable solvent, such as tetrahydrofuran, and an azodicarbonyl reagent, such as diethyl azodicarboxylate or 1,1′-(azodicarbonyl)dipiperidine.

[0101] Alcohol 4 is converted to 5 employing a Mitsunobu reaction with a N-hydroxycyclic imide derivative such as N-hydroxyphthalimide. Unveiling of the phthalimide protecting group of 5 is accomplished using standard conditions well known in the art (Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley and Sons, Inc. New York (1999)), for example using hydrazine or methylamine. An alternative method is using sodium borohydride in a mixture of an appropriate alcohol (e.g., ethanol/water) followed by acidification.

[0102] Guanidinylation of the resulting alkoxyamine to 6 is achieved using standard reagents such as aminoiminosulfonic acid (Miller, A. E. and Bischoff, J. J., Synthesis 777 (1986)), or 1H-pyrazole-1-carboxamidine hydrochloride (Bernatowicz, M. S. et al., J. Org. Chem. 57 (8), 2497 (1992)), or with substituted guanidinylating reagents such as N,N′-bis(tert-butoxycarbonyl)-S-methylisothiourea (Bergeron, R. J. and McManis, J. S., J. Org. Chem. 52:1700 (1987)) or N-R8, N-R9-1H-pyrazole-1-carboxamidine, where R8 and R9 are defined as above for Formula I. When R8 and R9 are protecting groups, for example t-butyloxycarbonyl (Boc), the compound can be optionally reacted with R10OH using standard Mitsunobu reaction condition as reviewed above to produce alkylated compounds 7. These protecting groups can be optionally removed by treatment with acid, usually trifluoroacetic acid in a suitable solvent such as dichloromethane or water, or by HCl gas dissolved in a suitable solvent, such as 1,4-dioxane to produce targeted compounds 8.

[0103] Scheme II outlines the synthetic steps to produce compounds of the present invention where R1 of Formula I is R11SO2—. Thus, compound 9, where R1 is N-benzyloxycarbonyl (Cbz) is removed by catalytic hydrogenation using a catalyst such as palladium on carbon and hydrogen to reveal the amino functionality, which is subsequently sulfonylated with sulfonyl chlorides (R11SO2Cl) or sulfoanhydrides (R11SO2)2O to produce sulfonamides 10. When R8 and R9 are protecting groups, for example t-butyloxycarbonyl (Boc), the compound can be optionally reacted with R11OH using standard Mitsunobu reaction condition as reviewed above to produce alkylated compounds 11. These protecting groups can be optionally removed by treatment with acid, usually trifluoroacetic acid in a suitable solvent such as dichloromethane or water, or by HCl gas dissolved in a suitable solvent, such as 1,4-dioxane to produce targeted compounds 12.

[0104] Further functionalization of the amidinoaminooxy group in 8 and 12 (where R1 is R11SO2) is described in Scheme III. The aminooxy nitrogen of 8 and 12 may be optionally alkylated using basic conditions such as solid sodium bicarbonate in a suitable solvent such as N,N-dimethylformamide with R7X, where X is a reactive leaving group such as a halide or sulfonate to give 13. Additionally, 13 may be reacted with pyrocarbonates such as diethyl pyrocarbonate in a suitable solvent such as acetonitrile or N,N-dimethylformamide in the presence of a tertiary amine base such as N,N-diisopropylethylamine to give carbamates of either mono- or di-substitution on the amidino nitrogens as in 14 and 15 as well as tri-carbamates with additional substitution on the aminooxy nitrogen as in 16.

[0105] Compounds of the present invention can be tested for the ability to inhibit or antagonize αvβ3 or αvβ5 cell surface receptors by assays known to those of ordinary skill in the art. Such assays are described in Example 9 herein.

[0106] The present invention relates to a method of treating αvβ3 integrin- or αvβ5 integrin-mediated conditions by selectively inhibiting or antagonizing αvβ3 and αvβ5 cell surface receptors, which method comprises administering a therapeutically effective amount of a compound selected from the class of compounds depicted by Formula I, wherein one or more compounds of Formula I is administered in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants and if desired other active ingredients.

[0107] More specifically, the present invention provides a method for inhibition of the αvβ3 cell surface receptor. Most preferably, the present invention provides a method for inhibiting bone resorption, treating osteoporosis, inhibiting humoral hypercalcemia of malignancy, treating Paget's disease, inhibiting tumor metastasis, inhibiting neoplasia (solid tumor growth), inhibiting angiogenesis including tumor angiogenesis, treating diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity and other neo-vascular eye diseases, inhibiting arthritis, psoriasis and periodontal disease, and inhibiting smooth muscle cell migration including neointimal hyperplasia and restenosis.

[0108] The present invention also provides a method for inhibition of the αvβ5 cell surface receptor. Most preferably, the present invention provides a method for inhibiting angiogenesis associated with pathological conditions such as inflammatory disorders such as immune and non-immune inflammation, chronic articular rheumatism and psoriasis, disorders associated with inappropriate or inopportune invasion of vessels such as restenosis, capillary proliferation in atherosclerotic plaques and osteoporosis, and cancer associated disorders, such as solid tumors, solid tumor metastases, angiofibromas, retrolental fibroplasia, hemangiomas, Kaposi sarcoma and similar cancers which require neovascularization to support tumor growth. The present invention also provides a method for treating eye diseases characterized by angiogenesis, such as diabetic retinopathy, age-related macular degeneration, presumed ocular histoplasmosis, retinopathy of prematurity, and neovascular glaucoma.

[0109] The compounds of the present invention are useful in treating cancer, including tumor growth, metastasis and angiogenesis. For example, compounds of the present invention can be employed to treat breast cancer and prostate cancer.

[0110] The compounds of the present invention may be administered in an effective amount within the dosage range of about 0.01 mg/kg to about 300 mg/kg, preferably between 1.0 mg/kg to 100 mg/kg body weight. Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.

[0111] The pharmaceutical compositions of the present invention can be administered to any animal that can experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.

[0112] The pharmaceutical compositions of the present invention can be administered by any means that achieve their intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, or ocular routes. Alternatively, or concurrently, administration can be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0113] In addition to the pharmacologically active compounds, the pharmaceutical preparations of the compounds can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. The pharmaceutical preparations of the present invention are manufactured in a manner that is, itself, known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

[0114] Suitable excipients are, in particular, fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders, such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents can be added, such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings, that, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.

[0115] Other pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules that may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids such as fatty oils or liquid paraffin. In addition, stabilizers may be added.

[0116] Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example water-soluble salts and alkaline solutions. Especially preferred alkaline salts are ammonium salts prepared, for example, with Tris, choline hydroxide, bis-Tris propane, N-methylglucamine, or arginine. In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400). Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

[0117] The compounds of the present invention may be administered to the eye in animals and humans as a drop, or within ointments, gels, liposomes, or biocompatible polymer discs, pellets or carried within contact lenses. The intraocular composition may also contain a physiologically compatible ophthalmic vehicle as those skilled in the art can select using conventional criteria. The vehicles may be selected from the known ophthalmic vehicles which include but are not limited to water, polyethers such as polyethylene glycol 400, polyvinyls such as polyvinyl alcohol, povidone, cellulose derivatives such as carboxymethylcellulose, methylcellulose and hydroxypropyl methylcellulose, petroleumn derivatives such as mineral oil and white petrolatum, animal fats such as lanolin, vegetable fats such as peanut oil, polymers of acrylic acid such as carboxylpolymethylene gel, polysaccharides such as dextrans and glycosaminoglycans such as sodium chloride and potassium, chloride, zinc chloride and buffer such as sodium bicarbonate or sodium lactate. High molecular weight molecules can also be used. Physiologically compatible preservatives which do not inactivate the compounds of the present invention in the composition include alcohols such as chlorobutanol, benzalknonium chloride and EDTA, or any other appropriate preservative known to those skilled in the art.

[0118] The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.

EXAMPLE 1

(3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2,5-dimethoxyphenyl)sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0119]

[0120] 1. (3S)-2-[(tert-Butyl)oxycarbonyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline carboxylic acid

[0121] To a mixture of (3S)-1,2,3,4-tetrahydro-7-hydroxy-isoquinoline-3-carboxylic acid (1.01 g, 5.23 mmol), sodium bicarbonate (0.88 g, 10.5 mmol), tetrahydrofuran (30 mL), and water (30 mL) was added di-tert-butyl dicarbonate (1.26 g, 5.78 mmol) at room temperature. The mixture was stirred overnight and concentrated. The residue was diluted with dichloromethane and water, and acidified with 10% HCl until pH 4. The white solid formed was filtered, the filtrate was separated, and the aqueous layer was extracted with dichloromethane. The organic phase was dried over Na2SO4 and concentrated to a white solid which was combined with the solid from filtration to give the title compound (1.35 g. 88.0%).

[0122]

1
H NMR (CDCl3/MeOH-d4) δ6.99 (dd, 1 H, J=5.2, 8.0 Hz), 6.67-6.57 (m, 2 H), 5.01 (dd, 0.4 H, J=3.0, 5.9 Hz), 4.72 (t, 0.6 H, J=5.3 Hz), 4.60 (m, 1 H), 4.42 (m, 1 H), 3.19-3.05 (m, 2 H), 1.52 (s, 4.5 H), 1.46 (s, 4.5 H).

[0123] 2. Methyl (3S)-2-[(tert-butyl)oxycarbonyl]-7-hydroxy,-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0124] To a solution of the product (1.35 g, 4.61 mmol) of the preceding step in methanol (10 mL) and benzene (6 mL) at 4° C. was added 2.0 M (trimethylsilyl)diazomethane in hexane (3.0 mL, 6.0 mmol). After 2 hours at 4° C., more 2.0 M (trimethylsilyl)diazomethane in hexane (0.7 mL, 1.4 mmol) was added. After additional 1.5 hours, the solution was concentrated to give the title compound as white foam (1.43 g, 100%).

[0125]

1
H NMR (CDCl3) δ6.99 (m, 1 H), 6.71-6.62 (m, 2 H), 5.11 (dd, 0.4 H, J=3.0, 5.8 Hz), 4.74 (t, 0.6 H, J=5.4 Hz), 4.63 (m, 1 H), 4.45 (m, 1 H), 3.66 (s, 1.8 H), 3.60 (s, 1.2 H), 3.15-3.07 (m, 2 H), 1.52 (s, 3.6 H), 1.46 (s, 5.4 H).

[0126] 3. Methyl (3S)-2-[(tert-butyl)oxycarbonyl]-7-(3-hydroxypropoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0127] A mixture of the product (706 mg, 2.30 mmol), as prepared from the preceding step, 3-bromo-1-propanol (390 mg, 2.81 mmol), cesium carbonate (1.12 g, 3.44 mmol), and acetonitrile (10 mL) was heated at 55° C. for 5 hours. After removal of the solvent, the residue was purified by flash chromatography to provide the title compound as a clear oil (636 mg, 75.8%).

[0128]

1
H NMR (CDCl3) δ7.04 (d, 1 H, J=8.4 Hz), 6.74-6.65 (m, 2 H), 5.12 (dd, 0.5 H, J=3.0, 6.0 Hz), 4.76 (t, 0.5 H, J=5.4 Hz), 4.67 (m, 1 H), 4.51-4.42 (m, 1 H), 4.09 (t, 2 H, J=5.9 Hz), 3.85 (m, 2 H), 3.64 (s, 1.5 H), 3.61 (s, 1.5 H), 3.21-3.05 (m, 2 H), 2.03 (t, 2 H, J=5.9 Hz), 1.52 (s, 4.5 H), 1.45 (s, 4.5 H).

[0129] 4. Methyl (3S)-2-[(tert-butyl)oxycarbonyl]-7-[3-(1,3-dioxoisoindolin-2-yloxy)propoxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0130] To a solution of the product (515 mg, 1.41 mmol), as prepared in the preceding step, triphenylphosphine (555 mg, 2.12 mmol), N-hydroxyphthalimide (345 mg, 2.12 mmol), and tetrahydrofuran (15 mL) was added diethyl azodicarboxylate (370 mg, 2.13 mmol). After stirring at room temperature overnight, the reaction solution was concentrated and flash chromatographed (SiO2) to give the title compound as a yellow oil (650 mg, 90.3%).

[0131]

1
H NMR (CDCl3) δ7.85-7.82 (m, 2 H), 7.77-7.75 (m, 2 H), 7.04 (dd, 1 H, J=4.4, 8.2 Hz), 6.77-6.74 (m, 2 H), 5.12 (dd, 0.5 H, J=2.9, 5.9 Hz), 4.76-4.74 (m, 0.5 H), 4.72-4.66 (m, 1 H), 4.51-4.45 (m, 1 H), 4.41 (t, 2 H, J=6.1 Hz), 4.22 (m, 2 H), 3.64 (s, 1.5 H), 3.62 (s, 1.5 H), 3.21-3.05 (m, 2 H), 2.24 (m, 2 H), 1.53 (s, 4.5 H), 1.45 (s, 4.5 H).

[0132] 5. Methyl (3S)-7-[3-(1,3-dioxoisoindolin-2-yloxy)propoxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0133] The product (650 mg, 1.27 mmol) of the preceding step in dichloromethane (6 mL) was treated with trifluoroacetic acid (1.5 mL) for 1 hour at room temperature and concentrated. The residue was partitioned between dichloromethane and saturated sodium bicarbonate. The organic phase was dried, concentrated, and flash chromatographed to give the title compound as a white solid (324 mg, 62.0%).

[0134]

1
H NMR (CDCl3) δ7.85-7.82 (m, 2 H), 7.77-7.74 (m, 2 H), 7.02 (d, 1 H, J=8.4 Hz), 6.76 (dd, 1 H, J=2.5, 8.4 Hz), 6.61 (d, 1 H, J=2.2 Hz), 4.41 (t, 2 H, J=6.1 Hz), 4.20 (t, 2 H, J=6.1 Hz), 4.08 (d, 2 H, J=5.5 Hz), 3.78 (s, 3 H), 3.72 (dd, 1 H, J=4.6, 10.2 H 3.02 (dd, 1 H, J=4.6, 15.9 Hz), 2.87 (dd, 1 H, J=10.2, 15.8 Hz), 2.26-2.20 (m,2 H).

[0135] 6. Methyl (3S)-2-[(2,5-dimethoxyphenyl)sulfonyl]-7-[3-(1,3-dioxoisoindolin-2-yloxy)propoxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0136] A solution of the product (62 mg, 0.15 mmol), as prepared in the preceding step, 2,5-dimethoxybenzenesulfonyl chloride (144 mg, 0.61 mmol), triethylamine (126 μL, 0.91 mmol) in dichloromethane (2 mL) was stirred at room temperature for 3 hours. The solvent was evaporated and the residue was flash chromatographed to provide the title compound as a clear oil (92 mg, 100%).

[0137]

1
H NMR (CDCl3) δ7.85-7.81 (m, 2 H), 7.78-7.75 (m, 2 H), 7.53 (d, 1 H, J=3.0 Hz), 7.03 (dd, 1 H, J=3.0, 9.0 Hz), 6.99 (d, 1 H, J=8.4 Hz), 6.85 (d, 1 H, J=9.0 Hz), 6.73 (dd, 1 H, J=2.0, 8.4 Hz), 6.58 (s, 1 H), 5.05 (dd, 1 H, J=2.6, 6.1 Hz), 4.69 (d, 1 H, J=16.0 Hz), 4.61 (d, 1 H, J=16.0 Hz), 4.39 (t, 2 H, J=6.1 Hz), 4.17 (t, 2 H, J=6.1 Hz), 3.81 (s, 3 H), 3.71 (s,3 H), 3.59 (s, 3 H), 3.13-3.00 (m, 2 H), 2.24-2.18 (m, 2 H).

[0138] 7. tert-Butyl 3-[(3-{(3S)-2-[(2,5-dimethoxyphenyl)sulfonyl]-3-(methoxycarbonyl)(7-1,2,3,4-tetrahydroisoquinolyloxy)}propoxy)-amino](2Z)-2-aza-3-[(tert-butoxy)carbonylamino]prop-2-enoate

[0139] The product (92 mg, 0.15 mmol) of the preceding step in tetrahydrofuran (1 mL) was treated with hydrazine hydrate (28 μL, ˜0.57 mmol) for 1 hour. After removal of the solvent in vacuo, the residue was purified with flash chromatography to give a clear oil. To this oil were added N,N-dimethylformamide (1 mL) and N,N-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (56 mg, 0.18 mmol). After stirring overnight at room temperature, the solvent was evaporated and the residue was flash chromatographed to yield the title compound as a clear oil (46 mg, 42%).

[0140]

1
H NMR (CDCl3) δ7.52 (d, 1 H, J=2.8 Hz), 7.02 (dd, 1 H, J=2.9, 9.0 Hz), 6.98 (d, 1 H, J=8.4 Hz), 6.85 (d, 1 H, J=8.9 Hz), 6.69 (d, 1 H, J=6.4 Hz), 6.54 (s, 1 H), 5.06-5.05 (m, 1 H), 4.70-4.59 (m, 2 H), 4.24-4.11 (m, 2 H), 3.98 (m, 2 H), 3.81 (s, 3 H), 3.71 (s, 3 H), 3.58 (s, 3 H), 3.13-3.00 (m, 2 H), 2.15-2.07 (m, 2 H), 1.49 (s, 18 H).

[0141] 8. (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2,5-dimethoxyphenyl)-sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0142] The product (46 mg, 0.064 mmol ) of the preceding step in methanol (1 mL) was treated with 1.0 M potassium hydroxide (0.25 mL, 0.25 mmol) in water for 2 hours at room temperature. The solution was concentrated in vacuo to dryness to produce a white solid. This solid was treated with trifluoroacetic acid (0.4 mL) in dichloromethane (1 mL) for 3 hours. After concentration, the residue was purified on Water's sep-pak (SiO2, 2 g) to give the title compound as a white solid (10 mg, 31%).

[0143]

1
H NMR (CDCl3/MeOH-d4) δ7.50 (m, 1 H), 7.05 (dd, 1 H, J=2.6, 9.0 Hz), 7.01 (d, 1 H, J=8.4 Hz), 6.87 (d, 1 H, J=9.0 Hz), 6.69 (d, 1 H, J=8.3 Hz), 6.54 (s, 1 H), 4.91 (m, 1 H), 4.61 (d, 1 H, J=16.0 Hz), 4.52 (d, 1 H, J=15.8 Hz), 4.07-4.03 (m, 4 H), 3.82 (s, 3H), 3.68 (s, 3 H), 3.14 (d, 1 H, J=15.0 Hz), 2.97-2.92 (m, 1 H), 2.1 (t, 2 H, J=5.8 Hz). Mass spectrum (LCMS, ESI) calcd. for C22H28N4O8S: 509 (M+H). Found: 509.

EXAMPLE 2

[0144] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-(phenylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0145] The title compound was prepared according to the synthesis described in Example 1, except that benzenesulfonyl chloride was substituted for 2,5-dimethoxybenzenesulfonyl chloride in step 6.

[0146]

1
H NMR (CDCl3/MeOH-d4) δ7.83 (d, 2 H, J=7.7 Hz), 7.58-7.55 (m, 1 H), 7.50-7.46 (m, 2 H), 6.98 (d, 1 H, J=8.4 Hz), 6.68 (d, 1 H, J=8.4 Hz), 6.57 (s, 1 H), 4.82 (d, 1 H, J=4.0 Hz), 4.59 (d, 1 H, J=15.7 Hz), 4.50 (d, 1 H, J=15.5 Hz), 4.06-4.02 (m, 4 H), 3.16 (d, 1 H, J=15.1 Hz), 2.96 (dd, 1 H, J=6.1, 15.6 Hz), 2.12-2.08 (m, 2 H). Mass spectrum (LCMS, ESI) calcd. for C20H24N4O6S: 449 (M+H). Found: 449.

EXAMPLE 3

[0147] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-(2-naphthylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0148] The title compound was prepared similarly to Example 1, except that naphthyl-2-ylsulfonyl chloride was used in step 6.

[0149]

1
H NMR (CDCl3/MeOH-d4) δ8.44 (s, 1 H), 7.98-7.87 (m, 3 H), 7.78 (d, 1 H, J=8.6 Hz), 7.65-7.58 (m, 2 H), 6.97 (d, 1 H, J=8.2 Hz), 6.67 (d, 1 H, J=8.1 Hz), 6.57 (s, 1 H), 5.00 (d, 1 H, J=4.4 Hz), 4.57 (m, 2 H), 4.03-4.01 (m, 4 H), 3.16 (d, 1 H, J=15.9 Hz), 3.02 (m, 1 H), 2.05 (t 2 H, J=5.5 Hz). Mass spectrum (LCMS, ESI) calcd. for C24H26N4O6S: 499 (M+H). Found: 499.

EXAMPLE 4

[0150] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-{[2-(methylsulfonyl)phenyl]sulfonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0151] The title compound was prepared similarly to Example 1, except that 2-methylsulfonylphenylsulfonyl chloride was used in step 6.

[0152]

1
H NMR (CDCl3/MeOH-d4) δ8.32 (m, 1 H), 7.81 (m, 1 H), 7.48 (m, 1 H), 7.02 (dd, 1 H, J=6.9 Hz), 6.70 (mn, 1 H), 6.57 (mn, 1 H), 5.30 (m, 1 H), 4.64 (d, 1 H, J=14.8 Hz), 4.48 (d, 1 H, J=14.4 Hz), 4.30-4.22 (mn, 3 H), 3.38 (mn, 3 H), 2.09 (mn, 2 H). Mass spectrum (LCMS, ESI) calcd. for C2H26N4O8S2: 527 (M+H). Found: 527.

EXAMPLE 5

[0153] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-(butylsulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0154] The title compound was prepared similarly to Example 1, except that n-butylsulfonyl chloride was used in step 6.

[0155]

1
H NMR (CDCl3/MeOH-d4) δ7.06 (d, 1 H, J=8.4 Hz), 6.74 (d, 1 H, J=8.3 Hz), 6.61 (s, 1 H), 4.83 (m, 1 H), 4.60 (s, 2 H), 4.08-4.05 (m, 4 H), 3.26 (d, 1 H, J=15.0 Hz), 3.18-3.08 (m, 3 H), 2.11 (t, 2 H, J=5.7 Hz), 1.83-1.77 (m, 2 H), 1.47-1.41 (m, 2 H), 0.94 (t, 3 H, J=7.3 Hz). Mass spectrum (LCMS, ESI) calcd. for C18H28N4O6S: 429 (M+H). Found: 429.

EXAMPLE 6

[0156] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2,6-dichlorophenyl)sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0157] 1. Methyl (3S)-7-hydroxy-2-[benzyloxycarbonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0158] To a mixture of (3S)-1,2,3,4-tetrahydro-7-hydroxy-isoquinoline-3-carboxylic acid (1.00 g, 5.18 mmol), sodium bicarbonate (0.88 g, 10.5 mmol), tetrahydrofuran (30 mL), and water (30 mL) was added benzyl chloroformate (0.84 mL, 5.88 mmol) at room temperature. The mixture was stirred overnight and concentrated to about 10 mL. The residue was acidified with 20% HCl until pH 4. The white solid formed was filtered and washed with water, and the filtrate was extracted with dichloromethane (×3). The organic layer was dried, concentrated, and combined with the white solid from filtration. To a solution of this compound (1.85 g, 5.66 mmol) in methanol (15 mL) and benzene (10 mL) at 4° C. was added 2.0 M (trimethylsilyl)diazomethane (4.2 mL, 8.4 mmol) in hexane. After 2 hours at 4° C., the solution was concentrated to give the title compound as a yellow oil (1.99 g, 100%).

[0159]

1
H NMR (CDCl3) δ7.40-7.30 (m, 5 H), 6.96 (d, 1 H, J=8.1 Hz), 6.67-6.64 (m, 1 H), 6.57 (d, 1 H, J=14.3 Hz), 5.25-5.11 (m, 3 H), 4.68 (d, 1 H, J=14.9 Hz), 4.55-4.45 (m, 1 H), 3.60 (s, 1.5 H), 3.52 (s, 1.5 H), 3.19-3.06 (m, 2 H).

[0160] 2. Phenylmethyl (3S)-7-[3-(1,3-dioxoisoindolin-2-yloxy)propoxy]-3-(methoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-2-carboxylate

[0161] A mixture of the product (990 mg, 2.90 mmol), as prepared from the preceding step, 3-bromo-1-propanol (450 mg, 3.24 mmol), cesium carbonate (1.23 g, 3.78 mmol), and acetonitrile (15 mL) was heated at 50° C. for 3 hours. After removal of the solvent under reduced pressure, the residue was filtered and washed with dichloromethane. The filtrate was concentrated to provide a yellow oil (1.13 g). To a solution of this oil (1.13 g, 2.83 mmol), triphenylphosphine (1.14 g, 4.35 mmol), N-hydroxyphthalimide (660 mg, 4.05 mmol), and tetrahydrofuran (20 mL) was added diethyl azodicarboxylate (760 mg, 4.37 mmol). After stirring at room temperature overnight, the reaction solution was concentrated and flash chromatographed (SiO2) to give the title compound as a clear oil (1.35 g, 85.5%).

[0162]

1
H NMR (CDCl3) δ7.84-7.82 (m, 2 H), 7.75 (m, 2 H), 7.43-7.34 (m, 5 H), 7.04 (d, 1 H, J=8.3 Hz), 6.77-6.66 (m, 2 H), 5.30-5.14 (m, 2.5 H), 4.94 (m, 0.5 H), 4.80-4.74 (m, 1 H), 4.60-4.51 (m, 1 H), 4.41-4.39 (m, 2 H), 4.24-4.19 (m, 3 H), 3.63 (s, 1.5 H), 3.55 (s, 1.5 H), 3.18-3.11 (m, 2 H), 2.25-2.21 (m, 2 H).

[0163] 3. tert-Butyl 3-[(3-{(3S)-3-(methoxycarbonyl)-2-[benzyloxycarbonyl](7-1,2,3,4-tetrahydroisoquinolyloxy)}propoxy)amino](2Z)-2-aza-3-[(tert-butoxy)carbonylamino]prop-2-enoate

[0164] The product (1.35 g, 2.48 mmol) of the preceding step in tetrahydrofuran (15 mL) was treated with hydrazine hydrate (0.65 mL, ˜13.4 mmol) for 1 hour. The white solid formed from the reaction was filtered and washed with diethyl ether. The filtrate was concentrated to give a white solid. To this solid were added N,N-dimethylformamide (10 mL) and NN-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (0.97 g 3.13 mmol). After stirring overnight at room temperature, the solvent was evaporated and the residue was flash chromatographed to yield the title compound as a clear oil (0.70 g, 43%).

[0165]

1
H NMR (CDCl3) δ9.06 (s, 1 H), 7.72 (s, 1 H), 7.42-7.27 (m, 5 H), 7.03 (d, 1 H, J=8.3 Hz), 6.74-6.61 (m, 2 H), 5.26-5.13 (m, 2.5 H), 4.95-4.93 (m, 0.5 H), 4.76 (d, 1 H, J=16.4 Hz), 4.59-4.50 (m, 1 H), 4.22-4.18 (m, 2 H), 4.04-4.00 (m, 2 H), 3.62 (s, 1.5 H), 3.54 (s, 1.5 H), 3.23-3.07 (m, 2 H), 2.17-2.13 (m, 2 H), 1.48 (s, 18 H).

[0166] 4. tert-Butyl 3-({3-[(3S)-3-(methoxycarbonyl)(7-1,2,3,4-tetrahydroisoquinolyloxy)]propoxy}amino)(2Z)-2-aza-3-[(tert-butoxy)carbonylamino]prop-2-enoate

[0167] A mixture of the product (0.70 g, 1.07 mmol), as prepared in the preceding step, 10% palladium on carbon (65 mg), methanol (20 mL), and chloroform (0.70 g, 5.88 mmol) was stirred under H2 balloon for 3 hours. The mixture was filtered through Celite, the filtrate was concentrated and flash chromatographed to give the title compound as a clear oil (282 mg, 50.6%).

[0168]

1
H NMR (CDCl3) δ9.06 (s, 1 H), 7.72 (s, 1 H), 7.01 (d, 1 H, J=8.4 Hz), 6.74 (dd, 1 H, J=2.5, 8.4 Hz), 6.58 (d, 1 H, J=2.3 Hz), 4.23 (t, 2 H, J=6.1 Hz), 4.18-4.07 (m, 2 H), 4.03 (t, 2 H, J=6.2 Hz), 3.81-3.76 (m, 1 H), 3.78 (s, 3 H), 3.09-3.04 (m, 1 H), 2.92 (dd, 1 H, J=10.1, 15.9 Hz), 2.18-2.12 (m, 2 H), 1.49 (s, 18 H).

[0169] 5. tert-Butyl 3-[(3-{(3S)-2-[(2,6-dichlorophenyl)sulfonyl]-3-(methoxycarbonyl)(7-1,2,3,4-tetrahydroisoquinolyloxy)}propoxy)-amino](2Z)-2-aza-3-[(tert-butoxy)carbonylamino]prop-2-enoate

[0170] A solution of the product (118 mg, 0.226 mmol), as prepared in the preceding step, 2,6-dichlorobenzenesulfonyl chloride (166 mg, 0.676 mmol), triethylamine (160 μL, 1.15 mmol), and dichloromethane (2 mL) was stirred at room temperature for 3 hours. The solvent was evaporated and the residue was flash chromatographed to provide the title compound as a clear oil (133 mg, 80.5%).

[0171]

1
H NMR (CDCl3) δ9.06 (s, 1 H), 7.71 (s. 1 H), 7.46 (d, 2 H, J=8.0 Hz), 7.34-7.30 (m, 1 H), 7.01 (d, 1 H, J =8.4 Hz), 6.73 (d. 1 H, J=8.4 Hz), 6.58 (s, 1 H), 5.19 (t, 1 H, J=4.0 Hz), 4.70 (m, 2 H), 4.24-4.19 (m, 2 H), 4.00 (t, 2 H, J=6.1 Hz), 3.58 (s, 3 H), 3.23 (m, 2 H), 2.14 (t, 2 H, J=6.1 HHz), 1.49 (s, 18 H).

[0172] 6. (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2,6-dichlorophenyl)-sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0173] The product (133 mg, 0.182 mmol ) of the preceding step in methanol (2 mL) was treated with 1.0 M potassium hydroxide (0.50 mL, 0.50 mmol) in water for 2 hours at room temperature. The solution was concentrated in vacuo to dryness to produce a white solid. This solid was treated with trifluoroacetic acid (0.5 mL) in dichloromethane (1 mL) for 3 hours. After concentration, the residue was purified on Water's sep-pak (SiO2, 2 g) to give the title compound as a white solid (84 mg, 89%).

[0174]

1
H NMR (CDCl3/MeOH-d4) δ7.53 (d, 2 H, J=8.3 Hz), 7.45-7.41 (m, 1 H), 7.06 (d, 1 H, J=8.5 Hz) 6.75 (dd, 1 H, J=2.4, 8.4 Hz), 6.62 (d, 1 H, J=2.2 Hz), 5.11 (dd, 1 H, J=2.3, 6.2 Hz), 4.76 (d, 1 H, J=15.8 Hz), 4.51 (d, 1 H, J=15.7 Hz), 4.10-4.04 (m, 4 H), 3.31-3.18 (m, 2 H), 2.17-2.11 (m, 2 H). Mass spectrum (LCMS, ESI) calcd. for C20H22Cl2N4O6S: 517 (M+H). Found: 517.

EXAMPLE 7

[0175] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-[(2-methyl-5-nitrophenyl)sulfonyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0176] The title compound was prepared similarly to Example 6, except that 2-methyl-5-nitrobenzenesulfonyl chloride was used in step 5.

[0177]

1
H NMR (CDCl3/MeOH-d4) δ8.87 (d, 1 H, J=2.4 Hz), 8.33 (dd, 1 H, J=2.4, 8.3 Hz), 7.57 (d, 1 H, J=8.4 Hz), 7.08 (d, 1 H, J=8.5 Hz), 6.76 (dd, 1 H, J=2.5, 8.4 Hz), 6.60 (d, 1 H, J=2.3 Hz), 4.92 (m, 1 H), 4.69 (d, 1 H, J=15.7 Hz), 4.09-4.04 (m, 4 H), 3.36-3.34 (m, 2 H), 3.20-3.16 (m, 1 H), 2.73 (s, 3 H), 2.16-2.10 (m, 2 H). Mass spectrum (LCMS, ESI) calcd. for C21H25N5O8S: 508 (M+H). Found: 508.

EXAMPLE 8

[0178] (3S)-7-[3-(Amidinoaminooxy)propoxy]-2-{[(7,7-dimethyl-2-oxobicyclo [2.2.1 ]heptyl)methyl]sulfonyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid trifluoroacetic acid salt

[0179] The title compound was prepared similarly to Example 6, except that (1S)-(+)-10-camphorsulfonyl chloride was used in step 5.

[0180]

1
H NMR (CDCl3/MeOH-d4) δ7.10 (d, 1 H, J=8.4 Hz), 6.78 (d, 1 H, J=8.3 Hz), 6.71 (s, 1 H), 4.89 (m, 1 H), 4.72 (d, 1 H,J=15.5 Hz), 4.10 (m, 4 H), 3.54 (d, 1 H, J=14.8 Hz), 3.35-3.16 (m, 3 H), 3.07 (d, 1 H, J=14.8 Hz), 2.43-2.38 (m, 2 H), 2.18-2.08 (m, 4 H), 1.96 (d, 1 H, J=18.6 Hz), 1.78-1.71 (m, 1 H), 1.52-1.45 (m, 1 H), 1.09 (s, 3 H), 0.87 (s, 3 H). Mass spectrum (LCMS, ESI) calcd. for C24H34N4O7S: 523 (M+H). Found: 523.

EXAMPLE 9

In Vitro Inhibition of Purified Enzymes αvβ3-vitronectin Assay

[0181] The assay was based on the method of Niiya (Niiya, K., et al., Blood 70:475-483 (1987)). All the steps were performed at room temperature. Costar 9018 flat-bottom 96-well ELISA plates were coated overnight with I 00 μL/well of 0.4 μg/mL human αvβ3 (Chemicon CC1019) in TS buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2). Plates were blocked for 2 hours with 200 μL/well of TS buffer containing 1% BSA (TSB buffer), and washed 3 times with 200 μL/well of PBST buffer. Controls or test compound were mixed with 0.5 μg/mL of human vitronectin (Chemicon CC080) that had been biotinylated in-house with sulfo-NHS-LC-LC-biotin (Pierce 21338, 20:1 molar ratio), and 100 μL/well of these solutions (in TSB buffer) were incubated for 2 hours. The plate was then washed 5 times with PBST buffer, and 100 μL/well of 0.25 μg/mL NeutrAvidin-horseradish peroxidase conjugate (Pierce 31001) in TSB buffer was incubated for 1 hour. Following a 5-fold PBST buffer wash, the plate was developed by adding 100 μL/well of 0.67 mg o-phenylenediamine dihydrochloride per mL of 0.012% H2O2, 22 mM sodium citrate, 50 mM sodium phosphate, pH 5.0 at room temperature. The reaction was stopped with 50 μuL/well of 2M H2SO4, and the absorbence at 492 nm was recorded. Percent (%) inhibition was calculated from the average of two separate determinations relative to buffer controls (no test compound added), and a four parameter fit (Marquardt, D. W., J Soc. Indust. Appl. Math. 11:431-441 (1963)) was used to estimate the half maximal inhibition concentration (IC50). IC50 values for inhibition of the αvβ3-vitronectin interaction by compounds 1, 2 and 3 of the invention are presented in Table I.

1TABLE IInhibition of theαvβ3-Vitronectin InteractionExample No.αvβ3 IC50 (nM)1 73211003 500

Fibrinogen-IIb-IIa Assay

[0182] The assay is based on the method of Dennis (Dennis, M. S., el al., Proteins 15:312-231 (1993)). Costar 9018 flat-bottom 96-well ELISA plates are coated overnight at 4° C. with 100 μL/well of 10 μL/mL human fibrinogen (Calbiochem 341578) in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM CaCl2, 0.02% NaN3 (TAC buffer), and blocked for 1 hour at 37° C. with 150 μL/well of TAC buffer containing 0.05% Tween 20 and 1% bovine serum albumin (TACTB buffer). After washing 3 times with 200 μL/well of 10 mM Na2HPO4 pH 7.5, 150 mM NaCl, 0.01% Tween 20 (PBST buffer), controls or test compound (0.027-20.0 μM) are mixed with 40 μg/mL human GPIIbIIIa (Enzyme Research Laboratories) in TACTB buffer, and 100 μL/well of these solutions are incubated for 1 hour at 37° C. The plate is then washed 5 times with PBST buffer, and 100 μL/well of a monoclonal anti-GPIIbIIIa antibody in TACTB buffer (1 μg/mL, Enzyme Reasearch Laboratories MabGP2b3a) was incubated at 37° C. for 1 hour. After washing (5 times with PBST buffer), 100 μL/well of goat anti-mouse IgG conjugated to horseradish peroxidase (Kirkegaard & Perry 14-23-06) is incubated at 37° C. for 1 hour (25 ng/mL in PBST buffer), followed by a 6-fold PBST buffer wash. The plate is developed by adding 100 μL/well of 0.67 mg o-phenylenediamine dihydrochloride per mL of 0.012% H2O2, 22 mM sodium citrate, 50 mM sodium phosphate, pH 5.0 at room temperature. The reaction is stopped with 50 μL/well of 2 M H2SO4, and the absorbence at 492 nm is recorded. IC50 values for inhibition of the fibrinogen-GPIIb-IIIa interaction is calculated as described for the αvβ3-vitronectin assay.

αvβ5-vitronectin Assay

[0183] The assay is similar to the αvβ3-vitronectin assay. Costar 9018 flat-bottom 96-well ELISA plates are coated overnight at room temperature with 100 μL/well of 1 μg/mL human αvβ5 (Chemicon CC1023) in TS buffer. Plates are blocked for 2 hours at 30° C. with 150 μL/well of TSB buffer, and washed 3 times with 200 μL/well of PBST buffer. Controls or test compound (0.027-20 μM) are mixed with 1 μg/mL of human vitronectin (Chemicon CC080) that has been biotinylated in-house with sulfo-NHS-LC-LC-biotin (Pierce 21338, 20:1 molar ratio), and 100 μL/well of these solutions (in TSB buffer) are incubated at 30° C. for 2 hours. The plate is then washed 5 times with PBST buffer, and 100 μL/well of 0.25 μg/mL NeutrAvidin- horseradish peroxidase conjugate (Pierce 31001) in TSB buffer is incubated at 30° C. for 1 hour. Following a 6-fold PBST buffer wash, the plate is developed and results are calculated as described for the fibrinogen-IIbIIIa assay.

EXAMPLE 10

Tablet Preparation

[0184] Tablets containing 25.0, 50.0, and 100.0 mg, respectively, of the compound of Example 1 (“active compound”) are prepared as illustrated below:

2TABLET FOR DOSES CONTAINING FROM25-100 MG OF THE ACTIVE COMPOUNDAmount-mgActive compound25.050.0100.00Microcrystalline cellulose37.25100.0200.0Modified food corn starch37.254.258.5Magnesium stearate0.500.751.5

[0185] All of the active compound, cellulose, and a portion of the corn starch are mixed and granulated to 10% corn starch paste. The resulting granulation is sieved, dried and blended with the remainder of the corn starch and the magnesium stearate. The resulting granulation is then compressed into tablets containing 25.0, 50.0, and 100.0 mg, respectively, of active ingredient per tablet.

EXAMPLE 11

Intravenous Solution Preparation

[0186] An intravenous dosage form of the compound of Example I (“active compound”) is prepared as follows:

3Active compound0.5-10.0mgSodium citrate5-50mgCitric acid1-15mgSodium chloride1-8mgWater for injection (USP)q.s. to 1ml

[0187] Utilizing the above quantities, the active compound is dissolved at room temperature in a previously prepared solution of sodium chloride, citric acid, and sodium citrate in Water for Injection (USP, see page 1636 of United States Pharmacopeia/National Formulary for 1995, published by United States Pharmacopeial Convention, Inc., Rockville, Md. (1994).

[0188] Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents and publications cited herein are fully incorporated by reference herein in their entirety.

Tetrahydroisoquinoline-3-carboxylic acid alkoxyguanidines as integrin antagonists

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