N-CARBOXYALKYL DERIVATIVES AS ANTIDEGENERATIVE

ACTIVE AGENTS

BACKGROUND OF THE INVENTION

This application is directed to novel N-carboxyalkyl derivatives which are useful as inhibitors of matrix metalloendoproteinase and are useful in the treatment of matrix metalloendoproteinase-mediated diseases.

The disability observed in osteoarthritis (OA) and rheumatoid arthritis (RA) is largely due to the loss of articular cartilage. No therapeutic agent in the prior art is known to prevent the attrition of articular cartilage in these diseases.

"Disease modifying antirheumatic drugs" (DMARD's), i.e., agents capable of preventing or slowing the ultimate loss of joint function in OA and RA are widely sought. Generic nonsteroidal antiinflammatory drugs (NSAIDs) may be combined with such agents to provide some relief from pain and swelling.

Stromelysin (aka. proteoglycanase, matrix metalloproteinase-3 (MMP-3), procollagenase activator, "transin"), coUagenase (aka. interstitial coUagenase, matrix metalloproteinase-1 (MMP-1)), and gelatinase (aka. type IV coUagenase, matrix metalloproteinase-2 (MMP-2), 72kDa-gelatinase or type V coUagenase, matrix metalloρroteinase-9, (MMP-9), 92kDa-gelatinase) are metalloendoproteinases secreted by fibroblasts and chondrocytes, and are capable of degrading the major connective tissue components of articular cartilage or basement membranes. Elevated levels of both enzymes have been detected in joints of arthritic humans and animals: K.A. Hasty, R.A. Reife, A.H. Kang, J.M. Stuart, "The role of stromelysin in the cartilage destruction that accompanies inflammatory arthritis", Arthr. Rheum.. 33, 388-97 (1990); S.M. Krane, E.P. Amento, M.B. Goldring, S.R. Goldring, and M.L. Stephenson, "Modulation of matrix synthesis and degradation in joint inflammation", The Control of Tissue Damage. A.B. Glauert (ed.), Elsevier Sci. Publ..

Amsterdam, 1988, Ch. 14, pp 179-95; A. Blanckaert, B. Mazieres, Y. Eeckhout, G. Vaes, "Direct extraction and assay of coUagenase from human osteoarthritic cartilage", Clin. Chim. Acta. 185 73-80 (1989). Each enzyme is secreted from these cells as an inactive proenzyme which is subsequently activated. There is evidence that stromelysin may be the in vivo activator for coUagenase, implying a cascade for degradative enzyme activity: A. Ho, H. Nagase, "Evidence that human rheumatoid synovial matrix metalloproteinase 3 is an endogenous activator of procollagenase", Arch Biochem Biophys.. 267, 211-16 (1988); G. Murphy, M.I. Crockett, P.E. Stephens, BJ. Smith, A.J.P. Docherty, "Stromelysin is an activator of procollagenase", Biochem. J.. 248, 265-8 (1987); Y. Ogata, J.J. Engh. Id, H. Nagase, "Matrix metalloprotease-3 (stromelysin) activates the precusor for human matrix metalloproteinase-9," J. Biol. Chem. 267, 3581-84 (1992). Inhibiting stromelysin could limit the activation of coUagenase and gelatinase as well as prevent the degradation of proteoglycan.

That stromelysin inhibition may be effective in preventing articular cartilage degradation has been demonstrated in vitro by measuring the effect of matrix metalloendoproteinase inhibitors on proteoglycan release from rabbit cartilage explants: C.B. Caputo, L.A. Sygowski, S.P. Patton, DJ. Wolanin, A. Shaw, R.A. Roberts, G. DiPasquale, J. Orthopaedic Res.. 6, 103-8 (1988).

There is an extensive Uterature on the involvement of these metalloproteinases in arthritis, but there is very little to guide one in developing a specific inhibitor for each enzyme.

In preliminary studies of rabbit proteoglycanase with substrates and inhibitors, Uttle was found to indicate the enzyme's requirements for hydrolysis or inhibition beyond a preference for hydrophobic residues at the Pi' position: A. Shaw, R.A. Roberts, D.J. Wolanin, "Small substrates and inhibitors of the metalloproteoglycanase of rabbit articular chondrocytes", Adv. Inflam. Res.. 12, 67-79 (1988). More extensive studies with a series of substrates and inhibitors revealed that stromelysin wiU tolerate nearly every amino acid residue around the scissile bond: S.J. Netzel-Arnett, G.B. Fields, H. Nagase, K. Suzuki,

W.G.I. Moore, H. Brikedal-Hansen, H.E. Van Wart, "Comparative sequence Specificities of human fibroblast and Neutrophil matrix metalloproeinases and inhibitors" ~ Matrix supplement No. 1: H. Brikedal-Hansen, Z. Werk, H.E. Van Wart, Eds.; Gustov Fisher Verlag:New York 1992; K.T. Chapman, I.E. Kopka, P.L. Durette, C. K. Esser, T.J. Lanza, M. Izquierdo-Martin, L. Niedzwiecki, B. Chang, R.K. Harrison, D.W. Kuo, T.-Y. Lin, R.L. Stein, W.K. Hagmann, J. Med. Chem. 36, 4293-4301 (1993).

Human rheumatoid synovial coUagenase has been shown to share ~ 50% homology with human stromelysin: S.E. Whitham, G. Murphy, P. Angel, H.J. Rahmsdorf, B.J. Smith, A. Lyons, T.J.R. Harris, J.J. Reynolds, P. Herrlich, AJ.P. Docherty, "Comparison of human stromelysin and coUagenase by cloning and sequence analysis", Biochem. J.. 240, 913-6 (1986). Many coUagenase inhibitors have been designed around the cleavage site of the α-chain sequence of Type II collagen: W.H. Johnson, N.A. Roberts, N. Brokakoti, "CoUagenase inhibitors: their design and potential therapeutic use", J. Enzvme Inhib.. 2,1-22 (1987); M.A. Schwartz and H.E. Van Wart, "Synthetic Inhibitors of Bacterial and Mammalian Interstitial Collagenases", In Prog. Med. Chem. vol. 29; G.P. Ellis and D.K. Luscombe, Eds.; Elsevier Sc. Publ.: 1992; Ch 8, pp 271-334.

Gelatinase (MR ~ 72,000) has been isolated from rheumatoid fϊbroblasts: Y. Okada, T. Morodomi, J.J. Engh d, K. Suzuki, A. Yasui, I. Nakanishi, G. Salvesen, H. Nagase, "Matrix metalloproteinase 2 from human rheumatoid synovial fϊbroblasts", Eur. J.. Biochem.. 194, 721-30 (1990). The synthesis of the proenzyme is not coordinately regulated with the other two metalloproteinases and its activation may also be different. The role of gelatinase in the tissue destruction of articular cartilage appears different from the other two enzymes and, therefore, its inhibition may provide additional protection from degradation. A higher molecular weight gelatinase (MR ~ 92,000; aka. type-V coUagenase, matrix metalloproteinase-9, MMP-9) is also secreted by fϊbroblasts and monocytes and may be involved in cartilage degradation: M. Mohtai, R.L. Smith, D.J. Schurman, Y. Tsuji, F.M.

Torti, N.I. Hutchinson, W.G. Stetler-Stevenson, G.I. Goldberg "Expression of 92-kD Type IV Collagenase/Gelatinase (Gelatinase B) in Osteoarthritic Cartilage and Its Induction in Normal Articular Cartilage by Interleukin 1", J. Clin. Invest. 92, 179-185 (1993).

The significant proportion of homology between human fibroblast coUagenase, stromelysin, and gelatinase leads to the possibility that a compound that inhibits one enzyme may to some degree inhibit all of them.

Compounds that inhibit coUagenase, which possess structural portions akin to those of the instant invention include those encompassed by U.S. 4,511,504, U.S. 4,568,666, EPO 520,573A1, PCT WO94/00119A, and EPO 126974 Al.

Compounds of related structure that are claimed to inhibit stromelysin (proteoglycanase) are encompassed by U.S. 4,771,037, and PCT WO92/21360A,

Stromelysin and coUagenase inhibitors are believed to have utility in preventing articular cartilage damage associated with septic arthritis. Bacterial infections of the joints can elicit an inflammatory response that may then be perpetuated beyond what is needed for removal of the infective agent resulting in permanent damage to structural components. Bacterial agents have been used in animal models to elicit an arthritic response with the appearance of proteolytic activities. See J.P. Case, J. Sano, R. Lafyatis, E.F. Remmers, G.K. Kumkumian, R.L. Wilder, "Transin/stromelysin expression in the synovium of rats with experimental erosive arhtritis arthritis", J. Clin Invest.. 84, 1731-40 (1989); R.J. Williams, R.L. Smith, D.J. Schurman, "Septic Arthritis: Staphylococcal induction of chondrocyte proteolytic activity", Arthr. Rheum.. 33, 533-41 (1990).

Inhibitors of stromelysin, coUagenase, and gelatinase are believed to be useful to control tumor metastasis, optionally in combination with current chemotherapy and/or radiation. See L.M. Matrisian, G.T. Bowden, P. Krieg, G. Furstenberger, J.P. Briand, P. Leroy, R. Breathnach, "The mRNA coding for the secreted protease transin is expressed more abundantly in malignant than in benign

tumors", Proc. Natl. Acad. Sci.. USA. 83, 9413-7 (1986); S.M. Wilhelm, I.E. Collier, A. Kronberger, A.Z. Eisen, B.L. Manner, G.A. Grant, E.A. Bauer, G. I. Goldberg, "Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells", Ibid.. 84, 6725-29 (1987); Z. Werb et al-, "Signal transduction through the fibronectin receptor induces coUagenase and stromelysin gene expression", J. Cell Biol.. 109, 872- 889 (1989); L.A. Liotta, CN. Rao, S.H. Barsky, "Tumor invasion and the extracellular matrix", Lab. Invest.. 49, 636-649 (1983); R. Reich, B. Stratford, K. Klein, G. R. Martin, R. A. Mueller, G. C. Fuller, "Inhibitors of coUagenase IV and cell adhesion reduce the invasive activity of malignant tumor cells", in Metastasis: Ciba Foundation Symposium: Wiley, Chichester, 1988, pp. 193-210.

Secreted proteinases such as stromelysin, coUagenase, and gelatinase play an important role in processes involved in the movement of cells during metastasic tumor invasion. Indeed, there is also evidence that the matrix metalloproteinases are overexpressed in certain metastatic tumor cell lines. In this context, the enzyme functions to penetrate underlying basement membranes and allow the tumor cell to escape from the site of primary tumor formation and enter circulation. After adhering to blood vessel walls, the tumor ceUs use these same metalloendoproteinases to pierce underlying basement membranes and penetrate other tissues, thereby leading to tumor metastasis. Inhibition of this process would prevent metastasis and improve the efficacy of current treatments with chemotherapeutics and/or radiation.

These inhibitors should also be useful for controUing periodontal diseases, such as gingivitis. Both coUagenase and stromelysin activities have been isolated from fibroblasts isolated from inflammed gingiva: V.J. Uitto, R. Applegren, P.J. Robinson, "CoUagenase and neutral metalloproteinase activity in extracts of inflammed human gingiva", J. Periodontal Res.. 16, 417-424(1981). Enzyme levels have been correlated to the severity of gum disease: CM. Overall, O.W. Wiebkin, J.C. Thonard, "Demonstration of tissue

collagenase activity in vivo and its relationship to inflammation severity in human gingiva", J. Periodontal Res.. 22, 81-88 (1987).

Proteolytic processes have also been observed in the ulceration of the cornea following alkali burns: S.I. Brown, CA. Weller, H.E. Wasserman, "Collagenolytic activity of alkali-burned corneas", Arch. Opthalmol.. 81, 370-373 (1969). Mercapto-containing peptides do inhibit the coUagenase isolated from alkali-burned rabbit cornea: F.R. Burns, M.S. Stack, R.D. Gray, C.A. Paterson, Invest. Opthalmol.. 30, 1569-1575 (1989). Treatment of alkali-burned eyes or eyes exhibiting corneal ulceration as a result of infection with inhibitors of these metalloendoproteinases in combination with sodium citrate or sodium ascorbate and/or antimicrobials may be effective in preventing developing corneal degradation.

Stromelysin has been implicated in the degradation of structural components of the glomerular basement membrane (GBM) of the kidney, the major function of which is to restrict passage of plasma proteins into the urine; W.H. Baricos, G. Murphy, Y. Zhou, H.H. Nguyen, S.V. Shah, "Degradation of glomerular basement membrane by purified mammalian metalloproteinases", Biochem. J.. 254, 609-612 (1988). Proteinuria, a result of glomerular disease, is excess protein in the urine caused by increased permeability of the GBM to plasma proteins. The underlying causes of this increased GBM permeability are unknown, but proteinases including stromelysin may play an important role in glomerular diseases. Inhibition of this enzyme may alleviate the proteinura associated with kidney malfunction.

Inhibition of stromelysin activity may prevent the rupturing of atherosclerotic plaques leading to coronary thrombosis. The tearing or rupture of atherosclerotic plaques is the most common event initiating coronary thrombosis. Destabilization and degradation of the connective tissue matrix surrounding these plaques by proteolytic enzymes or cytokines released by infiltrating inflammatory cells has been proposed as a cause of plaque Assuring. Such tearing of these plaques can cause an acute thrombolytic event as blood rapidly flows out of the blood vessel. High levels of stromelysin RNA message have been

found to be localized to individual cells in atherosclerotic plaques removed from heart transplant patients at the time of surgery: A.M. Henney, P.R. Wakeley, M.J. Davies, K. Foster, R. Hembry, G. Murphy, S. Humphries, "Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization", Proc. Nat'l. Acad. Sci. USA. 88, 8154-8158 (1991). Inhibition of stromelysin by these compounds may aid in preventing or delaying the degradation of the connective tissue matrix that stabilizes the atherosclerotic plaques, thereby preventing events leading to acute coronary thrombosis. It is also believed that inhibitors of matrix metalloproteinases would have utility in treating degenerative aortic disease associated with thinning of the medial aortic wall. Aneurysms are often associated with atherosclerosis in this tissue. Increased levels of the degradative activities of the matrix metalloproteinases have been identified in patients with aortic aneurysms and aortic stenosis: N. Vine, J.T. Powell, "MetaUoproteinases in degenerative aortic diseases", Clin. Sci.. 81, 233-9 (1991). Inhibition of these enzymes may aid in preventing or delaying the degradation of aortic tissue, thus preventing events leading to acute and often times fatal aortic aneurysms.

It is also believed that specific inhibitors of stromelysin and coUagenase should be useful as birth control agents. There is evidence that expression of metaUoendoproteinases, including stromelysin and coUagenase, is observed in unfertilized eggs and zygotes and at further cleavage stages and increased at the blastocyst stage of fetal development and with endoderm differentiation: C.A. Brenner, R.R. Adler, D.A. Rappolee, R.A. Pedersen, Z. Werb, "Genes for extracellular matrix- degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development", Genes & Develop.. 3, 848-59 (1989). By analogy to tumor invasion, a blastocyst may express metalloproteinases in order to penetrate the extracellular matrix of the uterine wall during implantation. Inhibition of stromelysin and coUagenase during these early developmental processes should presumably prevent normal embryonic development and/or implantation in the uterus. Such intervention would constitute a novel

method of birth control. In addition there is evidence that coUagenase is important in ovulation processes. In this example, a covering of collagen over the apical region of the folUcle must be penetrated in order for the ovum to escape. CoUagenase has been detected during this process and an inhibitor has been shown to be effective in preventing ovulation: J.F. Woessner, N. Morioka, C Zhu, T. Mukaida, T. Butler, W.J. LeMaire "Connective tissue breakdown in ovulation", Steroids. 54, 491-499 (1989). There may also be a role for stromelysin activity during ovulation: C.K.L. Too, G.D. Bryant-Greenwood, F.C Greenwood, "Relaxin increases the release of plasminogen activator, coUagenase, and proteo-glycanase from rat granulosa cells in vitro", Endocrin.. 115, 1043-1050 (1984).

Collagenolytic and stromelysin activity have also been observed in dystrophobic epidermolysis bullosa: A. Kronberger, K.J. Valle, A.Z. Eisen, E.A. Bauer, J. Invest. Dermatol.. 79 208-211 (1982); D. Sawamura, T. Sugawara, I. Hashimoto, L. Bruckmer-Tuderman, D. Fujimoto, Y. Okada, N. Utsumi, H. Shikata, Biochem. Biophvs. Res. Commun.. 174, 1003-8 (1991). Inhibition of metalloendoproteinases should limit the rapid destruction of connective components of the skin.

In addition to extracellular matrix comprising structural components, stromelysin can degrade other in vivo substrates including the inhibitors o -proteinase inhibitor and may therefore influence the activities of other proteinases such as elastase: P. G. Winyard, Z. Zhang, K. Chidwick, D. R. Blake, R. W. Carrell, G. Murphy, "Proteolytic inactivation of human αi-antitrypsin by human stromelysin", FEBS Letts.. 279, 1, 91-94 (1991). Inhibition of the matrix metalloendoproteinases may potentiate the antiproteinase activity of these endogenous inhibitors.

SUMMARY OF THE INVENTION

The invention encompasses novel N-carboxyalkyl derivatives of Formula I which are useful as inhibitors of matrix metalloendoproteinase and are useful in the treatment of matrix

metalloendoproteinase mediated diseases including degenerative diseases (such as defined above) and certain cancers.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention encompasses a compound of Formula I

or a pharmaceutically acceptable salt thereof wherein:

Rl is hydrogen, substituted Cl-6alkyl, or substituted C2-6 alkenyl wherein the substituent is selected from the group consisting of:

(a) hydrogen,

(b) carboxy, (c) aminocarbonyl,

(d) Cl-6alkoxy,

(e) Cl-6alkylthio,

(f) Cl-6alkylsulfonyl,

(g) Cl-6alkylcarbonyl, (h) Cl-6a_kylcarbonyl,

(i) aryl wherein the aryl is selected from the group consisting of:

(1) phenyl,

(2) naphthyl,

(3) pyridyl,

(4) f ryl,

(5) pyrryl,

(6) thienyl,

(7) isothiazolyl,

(8) imidazolyl,

(9) benzimidazolyl,

(10) tetrazolyl,

(11) pyrazinyl,

(12) pyrimidyl,

(13) quinolyl,

(14) isoquinolyl,

(15) benzofuryl,

(16) isobenzofuryl,

(17) benzothienyl,

(18) pyrazolyl,

(19) indolyl,

(20) isoindolyl,

(21) purinyl,

(22) carbazolyl,

(23) isoxazolyl,

(24) thiazolyl,

(25) oxazolyl,

(26) benzthiazolyl, and

(27) benzoxazolyl, optionally mono and di-substituted with substituents independently selected from Ci-6alkyl, Ci-6alkyl-oxy, halo, hydroxy, amino, Cμ

6alkylamino, aminoCi-6alkyl, carboxyl, carboxylCl-6alkyl, and Cl-

6alkylcarbonyl;

(j) aryloxy wherein aryl is defined in item (i) immediately above, (k) aroyl wherein aryl is defined in item (i) immediately above, (1) amino or mono- or di-substituted amino wherein the substituents are independently selected from Ci-6 alkyl and aryl as defined (i) immediate above, (m) arylthio wherein aryl is defined in item (i) immediately above, (n) arylsulfinyl wherein aryl is defined in item (i) immediately above, (o) arylsulfonyl wherein aryl is defined in item (i) immediately above,

(P) o

— N-C-0-R _b Ra

wherein Ra and Rb are each independently hydrogen, Cl-6 alkyl, optionally substituted aryl wherein aryl and its substituents are as defined in (i) above, or wherein Ra and Rb are joined together with the nitrogen and oxygen atoms to which they are attached to form a saturated or unsaturated cyclic-urethane, or a saturated or unsaturated benzofused cyclic- urethane, wherein the urethane ring contains 5, 6, 7, or 8 atoms, said ring containing two heteroatoms N and O such as;

wherein t is 1, 2 or 3, or

(q) O

— N I -C-N I -R _b ° a Re

wherein Ra Rb. and Re are each independently hydrogen, Cl-6 alkyl, optionally substituted aryl wherein aryl and its substituents areas defined in (i) above, or wherein Ra and Rb are joined together with the nitrogen atoms to which they are attached to form a saturated or unsaturated cyclic-urea or saturated or unsaturated benzofused cyclic-urea, said urea ring containing 5, 6, 7, or 8 atoms and two heteroatoms which are nitrogens such as;

wherein t is 1, 2 or 3, or

(r)

wherein R _a and Rb are each independently hydrogen, Ci-6 alkyl, optionally substituted aryl wherein aryl and its substituents areas defined in (i) above, or wherein Ra and Rb are joined together with the nitrogen and sulfur atoms to which they are attached, to form a saturated or unsaturated cyclic-sulfonamide or saturated or unsaturated benzofused cyclic- sulfonamide ring, said sulfonamide ring containing 5, 6, 7, or 8 atoms and two heteroatoms which are N and S such as ;

wherein t is 1, 2 or 3, or

(s)

O ll

-C-N-R _b R _α

wherein R _a and Rb are each independently hydrogen, Cl-6 alkyl, optionally substituted aryl wherein aryl and its substituents areas defined in (i) above, or wherein Ra and Rb are joined together with the nitrogen atom to which they are attached, to form a saturated or unsaturated heterocycle or staurated or unsaturated benzofused heterocycle ring, said ring containing 5, 6, 7, or 8 atoms and a heteroatom which is nitrogen such as;

wherein t is 1, 2 or 3, or

(t)

O

wherein Ra and Rb are each independently hydrogen, Cl-6 alkyl, optionally substituted aryl wherein aryl and its substituents areas defined in (i) above, or wherein Ra and Rb are joined together with the nitrogen atom to which they are attached, to form a saturated or unsaturated heterocycle or saturated or unsaturated benzofused heterocycle ring, said ring containing 5, 6, 7, or 8 atoms including a heteroatom which is nitrogen such as,

wherein t is 1, 2 or 3, or

R2 is biaryl wherein the aryl group is selected from the group consisting of: (1) phenyl,

(2) naphthyl,

(3) pyridyl,

(4) pyrryl,

(5) furyl, (6) thienyl,

(7) isothiazolyl,

(8) imidazolyl,

(9) benzimidazolyl,

(10) tetrazolyl, (11) pyrazinyl,

(12) pyrimidyl,

(13) quinolyl,

(14) isoquinolyl,

(15) benzofuryl, (16) isobenzofuryl,

(17) benzothienyl,

(18) pyrazolyl,

(19) indolyl,

(20) isoindolyl,

(21) purinyl,

(22) carboxazolyl,

(23) isoxazolyl,

(24) thiazolyl,

(25) oxazolyl, (26) benzthiazolyl, and

(27) benzoxazolyl, optionally mono or di-substituted with substitutents independently selected from Cl-6alkyl, Cl-6alkyloxy, hydroxyCl-6alkyl, Cμ 6alkoxyCi-6alkyl, Ci-6alkylthio, Ci-6alkylsulfinyl, Ci-6alkylsulfonyl, halo, haloalkyl, hydroxy, amino, Cl-6alkylamino, aminoCl-6alkyl, aminosulfonyl, aminocarbonyl, ureido, sulfonylureido, Ci- 6alkoxycarbonyl, carboxyl, carboxylCl-6alkyl, and Cl-6alkylcarbonyl;

R3 ΪS

(a) H,

(b) Z, where Z is a pharmaceutically acceptable counterion,

(c) Cl-lOalkyl,

(d) aryl or aryl Cl-.3alkyl, wherein the aryl group is selected from the group consisting of

(1) phenyl, and

(2) substituted phenyl, wherein the substitutent is carboxy, carboxyC 1-3 alkyl, aminocarbonyl, C 1 -6alkylaminocarbonyl;

R4 is X-R5 wherein X is a single bond or an amino acid of formula II

wherein Rd and Re are individually selected from: (a) hydrogen,

(b) Ci-6alkyl,

(c) mercapto Cl-6alkyl,

(d) hydroxy Cl-6alkyl,

(e) carboxy Cl-6alkyl, (f) amino substituted C2-6alkyl

(g) aminocarbonyl Ci-6alkyl, (h) mono- or di-Cl-6alkyl amino C2-6alkyl, (i) guanidino C2-6alkyl,

(j) substituted phenyl Ci-6alkyl, wherein the substitutent is hydrogen,hydroxy, carboxy, Cl-4 alkyl, or Cl_4alkyloxy,

(k) substituted indolyl Ci-6alkyl, wherein the substitutent is hydrogen, hydroxy, carboxy, Cl-4 alkyl, or Cl_4alkyloxy, (1) substituted imidazolyl C2-6alkyl wherein the substitutent is hydrogen, hydroxy, carboxy, Cl-4 alkyl, or Cl-4alkyloxy, (m) substituted pyridyl Cl-6alkyl wherein the substitutent is hydrogen, hydroxy, carboxy, Cl-4 alkyl, or Cl_4alkyloxy, (n) substituted pyridylamino Cl_6alkyl wherein the substitutent is hydrogen, hydroxy, carboxy, Cl-4 alkyl, or

Ci-4alkyloxy,

(o) substituted pyrimidinyl Cl-6alkyl wherein the substitutent is hydrogen, hydroxy, carboxy, Cl-4 alkyl, or Ci-4alkyloxy,

R5 is

wherein Rf and Rg are each individually selected from the group consisting of:

(a) H,

(b) substituted Cl-lθalkyl wherein the substituents are independently selected from hydrogen, Cl-6alkyloxy,

hydroxy, halo, amino, Cl-6alkylamino, carboxyl, and C\.

6alkylcarbonyl;

(C) Aryl or arylCi-6alkyl, wherein the aryl group is selected from the group consisting of

(1) phenyl,

(2) naphthyl,

(3) pyridyl,

(4) pyrryl,

(5) furyl,

(6) thienyl,

(7) isothiazolyl,

(8) imidazolyl,

(9) benzimidazolyl,

(io: i tetrazolyl,

(n: ) pyrazinyl,

(12; ) pyrimidyl,

(i3: ) quinolyl,

(i4: ) isoquinolyl,

(is: ) benzofuryl,

(i6: ) isobenzofuryl,

(i7 ) benzothienyl,

(is; i pyrazolyl,

(i9; ) indolyl,

(2o; ) isoindolyl,

(2i; ) purinyl,

(22; ) carbazolyl,

(23; > isoxazolyl,

(24; ) benzthiazolyl,

(25; I benzoxazolyl,

(26; > thiazolyl, and

(27; ) oxazolyl. optionally mono or di-substituted with substitutents independently selected from C; l_6alkyl, Cl-6alkyloxy, hydroxyCi-6alkyl, Cμ

6alkoxyCi-6alk l, Ci-6alkylthio, Ci-6alkylsulfinyl, Ci-6alkylsulfonyl,

halo, haloalkyl, hydroxy, amino, Ci-6alkylamino, aminoCi-6alkyl, aminosulfonyl, aminocarbonyl, ureido, sulfonylureido, Cμ 6alkoxycarbonyl, carboxyl, carboxylCl-6alkyl, and Cμ 6alkylcarbonyl;or

(d) Rf and Rg are joined together with the nitrogen atom to which they _^ are attached, to form a heterocycle ring, wherein the heterocycle is selected from the group consisting of

(1) morpholine,

(2) thiomorpholine,

(3) . thiomorpholine sulfone,

(4) pyrrolidine,

(5) piperazine,

(6) piperidine,

(7) 3-ketopiperazine, and

(8) 2-ketopiperazine; optionally mono or di-substituted with substitutents independently selected from Cμ6alkyl, Cl-6alkyloxy, halo, hydroxy, amino, Cμ 6alkylamino, carboxyl, carboxylCl-6alkyl, and Cl-6alkylcarbonyl.

For purposes of this specification, and as appreciated by those of skill in the art, the unsaturated rings such as those described in definitions Rl (p), (q), (r), (s), and (t) are intended to include rings wherein a double bond is present at one, two or more of the available positions.

For purposes of this specification biaryl such as in definition R2 is intended to include identical aryl groups, such as biphenyls, and heterogenous biaryls, such as furyl-phenyl, and substituted biaryls, such as p-phenyl-naphthyl or phenyl-(3-methyl- naphthyl).

Pharmaceutically acceptable counter-ions include those from salts of inorganic bases such as aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium. These counter-ions also include those from salts derived from

pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N_- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmoφholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

One genus of this embodiment concerns compounds wherein:

n = 2-3; and

R is hydrogen or mono- or di-substituted Cμβalkyl wherein the substituents are independently selected from the group consisting of:

(a) hydrogen,

(b) Ci-6alkoxy,

(c) aryl group selected from the group consisting of:

(1) phenyl,

(2) naphthyl,

(3) pyridyl,

(4) pyrryl,

(5) furyl,

(6) thienyl,

(7) isothiazolyl,

(8) imidazolyl,

(9) benzimidazolyl,

(10) tetrazolyl,

(11) pyrazinyl,

(12) pyrimidyl,

(13) quinolyl,

(14) isoquinolyl,

(15) benzofuryl,

(16) isobenzofuryl,

(17) benzothienyl,

(18) pyrazolyl,

(19) indolyl,

(20) isoindolyl,

(21) purinyl,

(22) carboxazolyl,

(23) isoxazolyl,

(24) thiazolyl,

(25) oxazolyl,

(26) benzthiazolyl, and

(27) benzoxazolyl, optionally mono- or di-substituted with substitutents independently selected from Cμ6alkyl, Cμβalkyloxy, chloro, fluoro, bromo, hydroxy, amino, Cμ6alkylamino, aminoCμόalkyl, carboxyl, carboxylCl-6alk :yl, or Cμ^alkylcarbonyl,

(d)

0 I I

— N-C-0-R _b

R _a

wherein Ra and Rb are each independently hydrogen, Cl-6 alkyl, optionally substituted aryl wherein aryl and its optional substituents are as defined in (c) above, or wherein Ra and Rb are joined together with the nitrogen and oxygen atoms to which they are attached to form a saturated or unsaturated cyclic-urethane, or a saturated or unsaturated benzofused cyclic- urethane, wherein the urethane ring contains 5, 6, 7, or 8 atoms, said ring containing two heteroatoms N and O;

(e)

— N I -C-N I -R _b ^D a e

wherein R _a Rb _. and Re are each independently hydrogen, Cμ6 alkyl, optionally substituted aryl wherein aryl and its optional substituents areas defined in (c) above, or wherein Ra and Rb are joined together with the nitrogen atoms to which they are attached to form a saturated or unsaturated cyclic-urea or saturated or unsaturated benzofused cyclic-urea, said urea ring containing 5, 6, 7, or 8 atoms and two heteroatoms which are nitrogens;

( )

wherein Ra and Rb are each independently hydrogen, Cμ6 alkyl, optionally substituted aryl wherein aryl and its optional substituents areas defined in (c) above, or wherein Ra and Rb are joined together with the nitrogen and sulfur atoms to which they are attached, to form a saturated or unsaturated cycUc-sulfonamide or saturated or unsaturated benzofused cyclic- sulfonamide ring, said sulfonamide ring containing 5, 6, 7, or 8 atoms and two heteroatoms which are N and S;

(g)

O II

-C-N-R _b

wherein R _a and Rb are each independently hydrogen, Cμ6 alkyl, optionally substituted aryl wherein aryl and its optional substituents areas defined in (c) above, or wherein Ra and Rb are joined together with the nitrogen atom to which they are attached, to form a saturated or unsaturated heterocycle or staurated or unsaturated benzofused heterocycle ring, said ring containing 5, 6, 7, or 8 atoms and a heteroatom which is nitrogen.

Within this sub-class are the compounds wherein

R2 is biaryl wherein the aryl group is selected from the group consisting of:

(1) phenyl,

(2) naphthyl,

(3) thienyl,

(4) imidazolyl,

(5) pyrimidyl,

(6) benzofuryl,

(7) pyridyl,

(8) benzothienyl, optionally mono or di-substituted with substitutents independently selected from Cμόalkyl, Cl-6alkyloxy, hydroxyCl_6alkyl, Cμ 6alkoxyCl-6alkyl, Cμ6alkylthio, Cμ6alkylsulfmyl, Cμ6alkylsulfonyl, halo, haloalkyl, hydroxy, amino, Cμ6alkylamino, aminoCl-6alkyl, aminosulfonyl, aminocarbonyl, ureido, sulfonylureido, Cμ 6alkoxycarbonyl, carboxyl, carboxylCμόalkyl, and Cμόalkylcarbonyl.

R4 is X-R5 wherein X is a single bond or an amino acid of formula II

II

wherein Rd and Re are individually selected from:

(a) hydrogen,

(b) Cl-4alkyl, (c) mercapto Cμ3alkyl,

(d) hydroxy Cμ4alkyl,

(e) carboxy Cμ4alkyl,

(f) amino substituted C2-4alkyl

(g) aminocarbonyl Cμ4alkyl, (h) mono- or di-Cμ4alkyl amino C2-4alkyl,

(i) guanidino C2-4alkyl, (j) substituted phenyl Cl-4alkyl, wherein the substitutent is hydrogen,hydroxy, carboxy, Cl-3 alkyl, or Cμ3alkyloxy, (k) substituted indolyl Cμ3alkyl, wherein the substitutent is hydrogen, hydroxy, carboxy, Cl-3alkyl, or Cμ3alkyloxy, (1) substituted imidazolyl C2-4alkyl wherein the substitutent is hydrogen, hydroxy, carboxy, Cl-3 alkyl, or Cμ3alkyloxy,

R5 is

wherein Rf and Rg are each individually selected from the group consisting of:

(a) H,

(b) substituted Cl-6alkyl wherein the substituents are independently selected from hydrogen, Cμ4alkyloxy, hydroxy, chloro, fluoro, bromo, amino, Cμ4alkylamino, carboxyl, and Cμ4alkylcarbonyl;

(c) aryl or arylCl-6alkyl, wherein the aryl group is selected from the group consisting of

(1) phenyl,

(2) naphthyl,

(3) pyridyl,

(4) thienyl,

(5) tetrazolyl,

(6) pyrazinyl,

(7) pyrimidyl,

(8) benzofuryl,

(9) benzothienyl,

(10) pyrazolyl,

(11) indolyl, optionally mono or di-substituted with substitutents independently selected from Cl-4alkyl, Cμ4alkyloxy, hydroxyd-4alkyl, Cμ 4alkoxyCl-4alkyl, fluoro, chloro, bromo, hydroxy, amino, Cμ 4alkylamino, aminoCμ4alkyl, carboxyl, carboxylCμ4alkyl, and Cμ 4alkylcarbonyl.

A smaller group within this group are the compounds wherein:

R3 is

(a) H,

(b) Z, where Z is a pharmaceutically acceptable counterion,

(c) Cl-4alkyl,

(d) aryl or aryl Cl-3alkyl, wherein the aryl group is selected from the group consisting of

(1) phenyl, and

(2) substituted phenyl, wherein the substitutent is carboxy, carboxyC 1-3 alkyl, aminocarbonyl.

R2 is arylCμ4alkyl, or biarylCμ4alkyl wherein the aryl group is selected from the group consisting of phenyl, thienyl, pyridyl, or naphthyl.

Exemplifying the present invention is the following compounds: N-[l(R)-Carboxyethyl]-α-(S)-2-(4-fluorobiρhenyl)-glycyl- (S)-2-(tert-butyl)glycine, N-Phenyl Amide.

This invention also concerns pharmaceutical composition and methods of treatment of stromelysin-mediated or implicated disorders or diseases (as described above) in a patient (which shall be defined to include man and/or mammalian animals raised in the dairy, meat, or fur industries or as pets) in need of such treatment comprising administration of the stromelysin inhibitors of Formula I as the active constituents.

Similarly, this invention also concerns pharmaceutical compositions and methods of treatment of coUagenase mediated or implicated disorders or diseases (as described above) in a patient in need of such treatment comprising administration of the coUagenase inhibitors of Formula (I) as the active constituents.

Similarly, this invention also concerns pharmaceutical compositions and methods of treatment of gelatinase-mediated or implicated disorders or diseases (as described above) in a patient in need of such treatment comprising administration of the gelatinase inhibitors of Formula (I) as the active constituents.

Moreover the invention also encompasses compositions, treatment, and method for co-administration of a compound of Formula I with a PMN elastase inhibitor such as those described in EP 0 337 549 which published on October 18, 1989, which is hereby incorporated by reference.

As may be appreciated by those of skill in the metalloendoproteinase art, it may be important to treat a patient with a matrix metalloendoproteinase mediated disease with an inhibitor that is specific for one or more matrix metalloendoproteinase (such as stromelysin, or coUagenase or gelatinase). For example, it may be advantageous to treat a pateint with a compound that is a potent

inhibitor of stromelysin but is a weak inhibitor of coUagenase, or vice versa.

Compounds of the instant invention are conveniently prepared using the procedures described generally below and more explicitly described in the Example section thereafter.

SCHEME1

SCHEME 1 (CONT'D.)

The inhibitors described in Scheme 1 can be prepared as follows: A biaryl derivative is acylated with succinic anhydride in the presence of a Lewis acid. The resulting biaryl ketone is reduced by catalytic hydrogenation to afford a biarylalkyl carboxylic acid. An This arylalkyl carboxylic acid is first converted to its corresponding acid chloride using oxalyl chloride and this is used to acylate lithio-4- benzyl-2-oxazolidinone. The derived imide is reacted with strong base, in this case potassium hexamethyl disilazide, and subsequently treated with triisopropylbenzenesulfonylazide (trisyl azide) to from the α-azido imide. The chiral auxiliary is then remove with lithium hydroperoxide and the resulting acid azide coupled to an amine. The azide is subsequently reduced to the amine by catalytic hydrogenation. The amine is reacted with benzyl lactate O-triflate in the presence of N,N- diisopropylethylamine to form the benzyl ester of an N-carboxyalkyl derivative. Removal of the benzyl ester by catalytic hydrogenation affords the desired inhibitors.

Compounds of the present invention have inhibitory activities with respect to metalloendoproteinases such as stromelysin, coUagenase and gelatinase. The capacity to inhibit the hydrolysis of peptidyl substrates by these matrix metalloproteinases may be demonstrated in assays in which the extent of enzymatic cleavage of a peptidyl substrate is determined with varying concentrations of inhibitor by methodology as detailed in the following literature reference: K.T. Chapman, I.E. Kopka, P.L. Durette, C. K. Esser, T.J. Lanza, M. Izquierdo-Martin, L. Niedzwiecki, B. Chang, R.K. Harrison, D.W. Kuo, T.-Y. Lin, R.L. Stein, W.K. Hagmann, J. Med. Chem. 36, 4293- 4301 (1993).

This invention also relates to a method of treatment for patients (including man and/or mammalian animals raised in the dairy, meat, or fur industries or as pets) suffering from disorders or diseases which can be attributed to stromelysin as previously described, and more specifically, a method of treatment involving the administration of the matrix metalloendoproteinase inhibitors of Formula (I) as the active constituents.

Accordingly, the compounds of Formula (I) can be used among other things in the treatment of osteoarthritis and rheumatoid arthritis, and in diseases and indications resulting from the over- expression of these matrix metaUoendoproteinases such as found in certain metastatic tumor cell lines.

For the treatment of rheumatoid arthritis, osteoarthritis, and in diseases and indications resulting from the over-expression of matrix metalloendoproteinases such as found in certain metastatic tumor cell lines or other diseases mediated by the matrix metalloendo¬ proteinases, the compounds of Formula (I) may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In addition to the treatment of warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, etc., the compounds of the invention are effective in the treatment of humans.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or

acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Patents 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropy. methyl- cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth, naturally-occurring phosphatides, for example, soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also

be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of Formula (I) may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non- irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jelUes, solutions or suspensions, etc., containing the compounds of Formula (I) are employed. (For purposes of this application, topical application shall include mouth washes and gargles.)

Dosage levels of the order of from about 0.05 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 2.5 mg to about 7 gms. per patient per day). For example, inflammation may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day (about 0.5 mg to about 3.5 gms per patient per day).

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.

- 35 -

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

The following Examples are intended to illustrate the preparation of compounds of Formula I, and as such are not intended to limit the invention as set forth in the claims appended, thereto.

EXAMPLE 1

N-[l(R)-Carboxyethyl]-α-(S)-2-(4-fluorobiphenyl)-glycyl( S)-2-(tert- butvDglvcine. N-Phenyl Amide

Step 1: 4-(4-Fluorobiphenyl)-4-oxo-butanoic acid

A 500-mL three-neck round-bottom flask equipped with a mechanical stirrer, thermometer, and reflux condenser was charged with methylene chloride (125 mL). 4-Fluorobiphenyl (20.0 g, 0.116 mol) was added followed by succinic anhydride (11.6 g, 0.116 mol). The mixture was cooled in an ice-bath and aluminum chloride (30.9 g, 0.232 mol) was added in six portions at 15-minute intervals. The ice- bath was applied as needed to keep the temperature near 15°C. After the last addition, the mixture was stirred for 90 min at room temperature and then for an additional 90 min at gentle reflux (37- 39°C). The cooled reaction mixture was poured into a stirred mixture of ice (300 g) and concentrated hydrochloric acid (40 mL). Additional water (500 mL) was added and the resulting solid filtered, washed with water, and dried by suction. Recrystallization from hot acetic acid afforded pure title compound after drying in vacuo at 50°C; yield 19.6 g (62%); 400 MHz iH NMR (CDCI3): δ 2.83 (t, 2H), 3.33 (t, 2H), 7.11- 8.07 (m, 8H).

Step 2: 4-(4-Fluorobiphenyl)-butanoic acid

A mixture of 4-(4-fluorobiphenyl)-3-oxo-butanoic acid (19.6 g, 72.0 mmol) in glacial acetic acid (185 mL) and methanol (75 mL) was stirred at 60°C in the presence of 20% palladium hydroxide- on-carbon (1.2 g) under 40 p.s.i. of hydrogen for several hours until thin layer chromatography (20% ethyl acetate in hexane containing 1% acetic acid) indicated complete reduction. The catalyst was removed by filtration through a pad of Celite, and the filtrate was evaporated under

diminished pressure. After several co-evaporations with toluene, the title compound was obtained a white crystalline solid that was dried in vacuo; yield 18.3 g (98%); 400 MHz iH NMR (CDCI3): δ 1.98 (quintet, 2H), 2.40 (t, 2H), 2.69 (t, 2H), 7.08-7.52 (m, 8H).

Step 3: 3-(4-(4-Fluorobiphenyl)-butanoyl)-(S)-4-phenylmethyl-2- oxazolidinone

4-(4-Fluorobiphenyl)-butanoic acid (18.3 g, 70.8 mmol) was taken up in methylene chloride (220 mL). N,N-Dimethylform- amide (362 μL) was added, and the solution was cooled in an ice bath. Oxalyl chloride (6.85 mL, 78.5 mmol) was added with stirring over 10 min., and the solution was stirred at ice temperature for 30 min and then at room temperature for 2 h. The reaction mixture was evaporated under diminished pressure and dried in vacuo for one hour. To a solution of (S)-4-phenylmethyl-2-oxazolidinone (11.4 g, 64.3 mmol) in dry THF (185 mL) cooled to -78°C was added n-butyl lithium (1.6 M in hexanes) (43.4 mL, 69.4 mmol) dropwise with stirring while maintaining the temperature below -60°C. After 15 min., a solution of the acid chloride in THF (30 mL) was added, and the mixture was stirred for 30 min at -78°C. The cooling bath was removed, and stirring was continued for one hour. After quenching with saturated aqueous ammonium chloride (55 mL), ethyl acetate (175 mL) and water (55 mL) were added, and the organic layer separated. The organic layer was washed with 2 N hydrochloric acid, saturated sodium hydrogencarbonate solution, saturated brine solution, dried (sodium sulfate), and evaporated. Crystallization from diethyl ether afforded the desired product. The filtrate was evaporated and subjected to flash silica gel chromatography eluting with 15% ethyl acetate in hexane. A

total of 22.5 grams of the title compound was obtained; yield 76%; 400 MHz IH NMR (CDC13): δ 2.05 (m, 2H), 2.70-2.78 (m, 3H), 2.98 (m, 2H), 3.28 (dd, IH), 4.15 (m, 2H), 4.64 (m, IH), 7.07-7.52 (m, 13H).

Step 4: 3-[(S)-2-Azido-4-(4-fluorobiphenyl)-butanoyl]-(S)-4- phenylmethyl-2-oxazolidinone

Potassium hexamethyldisUazide (KHMDS) (0.5 M solution in toluene) (6.85 mL, 3.43 mmol) was added to a solution of 3-(4-(4- fluorobiphenyl)-butanoyl)-(S)-4-phenylmethyl-2-oxazolidinone (1.30 g, 3.11 mmol) in dry THF (13 mL) dropwise with stirring at -78°C under a nitrogen atmosphere. The reaction mixture was stirred for 30 min at -78°C, and a solution of trisyl azide (1.20 g, 3.88 mmol) in THF (5 mL) was added dropwise with stirring. The mixture was stirred at -78 °C for 10 min. and then quenched with glacial acetic acid (0.82 mL, 14.3 mmol). The cooling bath was removed, and the mixture was stirred overnight at room temperature. It was then concentrated and partitioned between ethyl acetate and water. The organic layer was washed with saturated brine solution, dried (sodium sulfate), and evaporated. The product was purified by flash siUca gel chromatography eluting with 10% ethyl acetate in hexane; yield 1.1 g (77%): 400 MHz IH NMR (CDCI3): δ 2.12 (m, IH), 2.20 (m, IH), 3.30 (dd, IH), 4.16 (m, 2H), 4.57 (m, IH), 5.00 (dd, IH).

Step 5: (SV2-Azido-4-(4-fluorobiphenyl)-butanoic acid

To a solution of 3-[(S)-2-azido-4-(4-fluorobiphenyl)- butanoyl]-(S)-4-phenylmethyl-2-oxazolidinone (1.1 g, 2.40 mmol) in THF (37 mL) and water (11.6 mL) were added 30% hydrogen peroxide (1.34 mL) and lithium hydroxide monohydrate (112 mg, 2.67 mmol). The reaction mixture was stirred overnight at room temperature. The mixture was cooled in an ice bath, and a solution of sodium sulfite (1.86 g) in water (15 mL) followed by saturated sodium hydrogencarbonate solution (15 mL). After removal of the THF by evaporation under diminished pressure, the residue was partitioned between ethyl acetate (75 mL) and 2 N hydrochloric acid (25 mL). The organic layer was washed with saturated brine solution, dried (sodium sulfate), and evaporated. The product was purified by flash silica gel ehromatography (packed as a slurry in 20% ethyl acetate in hexane) eluting initially with 20% ethyl acetate in hexane followed by 25% ethyl acetate in hexane containing 1% acetic acid. The title compound was obtained as a white crystalline solid; yield 510 mg (71%); 400 MHz IH NMR (CDC13): δ 2.10 (m, IH), 2.20 (m, IH), 2.40 (m, 2H), 4.90 (dd, IH), 7.07-7.50 (m, 8H).

Step 6: (S)-2-Azido-4-(4-fluorobiphenyl)-butanoyl-(S)-2-(tert- hutylV lvcine. N-phenyl amide

To a solution of (S)-2-azido-4-(4-fluorobiphenyl)-butanoic acid (506 mg, 1.69 mmol) in methylene chloride (10 mL) were added N-hydroxybenzotriazole (342 mg, 2.53 mmol) and (S)-2-(tert-butyl)- gly cine N-phenyl amide (366 mg, 1.77 mmol). The reaction mixture was cooled in an ice-bath, and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (389 mg, 2.03 mmol) was added. The reaction mixture was stirred overnight at room temperature, diluted with methylene chloride, washed with water, 2 N hydrochloric acid, saturated sodium hydrogencarbonate solution, saturated brine solution, dried (sodium sulfate), and evaporated. The product was purified by flash siUca gel ehromatography eluting with 20% ethyl acetate in hexane; yield 610 mg (74%); 400 MHz IH NMR (CDCI3): δ 1.10 (s, 9H), 2.19 (m, IH), 2.28 (m, IH), 2.78 (m, 2H), 4.00 (dd, IH), 4.46 (d, IH), 7.08-7.51 (m, 13H), 7.89 (s, IH).

Step 7: (S)-2-Amino-4-(4-fluorobiρhenyl)-butanoyl-(S)-2-(tert- butvP-glvcine. N-phenyl amide

A solution of (S)-2-azido-4-(4-fluorobiphenyl)-butanoyl- (S)-2-(tert-butyl)-glycine N-phenyl amide (308 mg, 0.632 mmol) in methanol (10 mL) was stirred in the presence of 10% palladium-on- charcoal (55 mg) under an atmosphere of hydrogen for 5 hours. The catalyst was removed by filtration through a pad of Celite, and the filtrate was evaporated. The product was purified by flash silica gel ehromatography eluting with 2% methanol in methylene chloride; yield 220 mg (75.5%).

Step 8: N-[l (R)-(Benzyloxycarbonyl)ethyl]-α-(S)-2-(4-fluoro- hiphenylVglvcyl-(SV2-(tert-butyl glycine. N-phenyl amide

To a solution of benzyl (S)-lactate (86 mg, 0.477 mmol) in dry methylene chloride (3.5 mL) cooled to 0°C was added trifluoro- methanesulfonic anhydride (86 μL, 0.511 mmol) with stirring under an inert atmosphere. After 5 min. at 0°C, 2,6-lutidine (62.4 μL, 0.536 mmol) was added in one portion. After stirring for 10 min. at 0°C, N,N-diisopropylethylamine (92 μL, 0.528 mmol) was added followed immediately by a solution of (S)-2-amino-4-(4-fluorobiphenyl)- butanoyl-(S)-2-(tert-butyl)-glycine N-phenyl amide (220 mg, 0.477 mmol) in methylene chloride (2 mL). The cooling bath was removed, and the mixture was stirred overnight at room temperature. The mixture was diluted with methylene chloride which was successively washed with water, saturated sodium hydrogencarbonate solution, saturated brine solution, dried (sodium sulfate), and evaporated. Purification was achieved by means of flash sUica gel ehromatography eluting with 20% ethyl acetate in hexane. The product was obtained as a white crystalline solid; yield 70.4 mg (24%); mass spectrum: m/z 624 (M + 1); 400 MHz IH NMR (CD3OD): δ 1.08 (s, 9H), 1.31 (d, 3H), 1.92 (m, IH), 2.02 (m, IH), 2.73 (m, 2H), 3.48 (q, IH), 4.40 (s, IH), 5.12 (m, 2H), 7.07-7.58 (m, 18H).

Step 9: N-[l (R)-(Carboxyethyl]-α-(S)-2-(4-fluorobiphenyl)-glycyl-

(SV2-(tert-butyl)glycine. N-phenyl amide

A solution of N-[l(R)-(benzyloxycarbonyl)ethyl]- -(S)-2- (4-fluorobiρhenyl)-glycyl-(S)-2-(tert-butyl)glycine, N-phenyl amide (70 mg, 0.112 mmol) in methanol (4 mL) was stirred in the presence of 20% palladium hydroxide-on-carbon (20 mg) under an atmosphere of hydrogen for 2 hours. The catalyst was removed by filtration through an Anotop 25 disposable syringe filter (0.2 μm). The filtrate was evaporated to give the title compound as an amorphous solid; yield 58.2 mg (97%); mass spectrum: m/z 534 (M + 1); 400 MHz IH NMR

(CD3OD): 1.12 (s, 9H), 1.51 (d, 3H), 2.18 (m, 2H), 2.70 (m, 2H), 3.62 (q, IH), 4.18 (dd, IH), 4.52 (s, IH), 7.08-7.57 (m, 13H).

EXAMPLE 2

ENZYME INHIBITON ASSAYS

Inhibition of Human Fibroblast Stromelysin.

Activation: Human recombinant stromelysin was purchased from Celltech (Slough, U.K.) as a proenzyme of 55kD in a buffer consisting of 20 mM Tris, 10 mM CaCl2, 0.05% Brij-35, and 0.2% NaN3, pH=7.5. Briefly, to 1.0 mL of a 2.2 μM solution of prostromelysin was added 20 μL of a 1.0 μM solution of trypsin in assay buffer (20 mM HEPES, lOmM CaCl2, 0.05% Brij-35, pH=7.5, [trypsin]fi _n al = 20 nM. The solution was incubated at 37°C for 30 minutes. The reaction was quenched by addition of a 50 fold molar excess of soybean trypsin inhibitor bound to agarose (Sigma), and the solution centrifuged to remove the trypsi inhibitor complex.

Ki Determinations: Stock solutions of inhibitors were prepared by dissolving the compounds in DMSO. The inhibitors were further diluted in assay buffer to eight different concentrations encompassing the approximate Ki, and covering a 200 fold range. To 50 μL of each of the inhibitor solutions was added 25 μL of an 8 nM solution of trypsin-activated stromelysin, [DMSO] = 1.8%. The solution was allowed to incubate for 4 h to reach equilibrium. To this solution was added 60 μL of a 12.8 μM solution of the substrate Arg-Pro-Lys-Pro- Leu-Ala-Phe-Trp-NH2, and the reaction was allowed to proceed for 18 h. In the reaction [S] = 5.7 μM and [E] = 1.5 nM. The reaction was quenched by addition of 50 μL of 0.15M phosphoric acid, and 100 μL of the reaction mixture was injected onto the HPLC. Since the reactions were run under first order conditions, ([S]«K _. Km = 0.5 mM), the pseudo first order rate constant, k ₀ bs. was determined for each of the inhibitor concentrations from the peak area corresponding to unreacted substrate in the inhibited sample, and the peak area for substrate at time = 0: areajnhib In = -ko _bs t area _t = o Values of Ki were determined from the ratio of the rate constants for inhibited and control sample (no inhibitor) plotted as a function of the inhibitor concentration, and fit to the following equation:

inhib *

^control 1 + [I] Ki

Inhibition of Human Fibroblast CoUagenase

Activation. Human fibroblast coUagenase was purchased from Celltech (Slough, U.K.). The material was received as a proenzyme of 54 kD at a concentration of 1.2 μM in a buffer consisting of 20 mM Tris, 5 mM CaCl2, 0.15 M NaCI, and 0.01% NaN3. The material was activated with trypsin using the same procedure as for stromelysin, with the addition that the activation buffer contained 40 nM prostromelysin.

5 Ki Determinations. Stock solutions of inhibitors were prepared by dissolving the material in DMSO The inhibitors were further diluted in assay buffer to eight different concentrations encompassing the approximate Ki and covering a 200 fold range. Final DMSO concentration was 2.8%. To 50 uL of the inhibitor solutions was added 0 25 uL of a 108 nM solution of trypsin activated coUagenase. The solution was allowed to incubated for 4 h to reach equilibrium. To this solution was added 60 uL of a 56 μM solution of the substrate DNP- Pro-Leu-Gly-Leu-Trp-Ala-dArg-NH2, and the reaction was allowed to proceed for 18 h. In the incubation mixture [S] = 25 μM, [E]= 20 nM. 5 The reaction was quenched by addition of 50 uL of 0.15 M phosphoric acid. The reaction mixture was injected onto the HPLC. The calculation of Ki was the same as for stromelysin.

o Inhibition of Human Gelatinase A.

Activation: Human 72kD gelatinase was purchased from Celltech (Slough, U.K.) as a proenzyme at a concentration of 1.5 μM in a buffer consisting of 20 mM Tris, 5mM CaCl2 150 mM NaCI, 0.01% brij, 0.02% NaN3, pH = 7.5 . The proenzyme was activated by incubation of

500 μL proenzyme with 50 μL of a 11 mM solution aminophenyl mercuric acetate in NaOH (pH=l l) at 25°C for 120 min.

Ki Determinations. Determination of Ki values for gelatinase A were identical to that of coUagenase and stromelysin with the exception that incubation of the enzyme-inhibitor mixture with the substrate was performed for only 2 h. [S] = 25 μM, [E]= 20 nM.

Employing the assays described herein, the compound described in Example 1 inhibited the matrix metalloproteinases as follows: stromelysin Ki = 22 nM; coUagenase Ki = 3.1 μM; gelatinase-A Ki = 37 nM.

EXAMPLE 3

IN VIVO EVALUATION OF INHIBITORS

Recombinant human stromelysin (rhSLN) was purchased from Celltech, Ltd, UK, and purified in our laboratories. It was standardized in vitro by activating with trypsin and titrations of its proteolytic activity for After time and dose studies, 100 μg of activated rhSLN dissolved in 0.5 ml of stromelysin buffer (25 mM Tris-HCl, 0.7 M NaCI, 10 mM CaCl2, 0.05% Brij, 0.02% NaN3, pH 7.5 ) was injected intraarticularly into the synovial cavity of a stifle joint of 8 - 10 wk female New Zealand White rabbits (Hazelton Farms, Denver, PA) that had been anesthetized with a mixture (3:2, v/v) of ketamine HC1 (100 mg/kg; Ketaset®, Aveco Co, Inc, Ft Dodge IN) and xylazine (20 mg/kg; Rompum®, Mobay Corp, Shawnee KA). As a control, the contralateral stifle joint was injected with 0.5 ml of stromelysin buffer. 1 hr after the intraarticular injection of activated rhSLN, the animals were euthanized with an overdose of sodium pentobarbital and each joint was lavaged twice with 0.5 ml of PBS. The total mass of proteoglycans lavaged from the stifle joint of the rhSLN-injected animal minus the mass of proteoglycans lavaged from the contralateral control

joint (net activated rhSLN-induced increase in proteoglycans in synovial fluid) is a quantitative assessment of the in vivo potency of activated rhSLN.

Investigational compounds were dissolved in 100% DMSO and then diluted to 2% DMSO, 2% Cremaphore® (polyoxyethylene glycerol triricinoleate, BASF Corp, Parsippany NJ), 96% 50 mM phosphate- buffered saline, pH 7.0. The compounds were either injected intravenously or per orally at various times prior to the intraarticular injection of activated rhSLN. The capacity of the investigational compounds to reduce the net amount of proteoglycans in the synovial cavity is a quantitative measurement of their potency and is expressed as % inhibition or ED50-