AU1000399A - Synthetic matrix metalloprotease inhibitors and uses thereof - Google Patents

Description

&Ref 31417801

-AUSTRAUA.

PATENTS ACT 1990 FOR A STANDARD PATENT

ORAL

Name and Address of.Applicant: Actual Inventor(s): Address for Service: invention Title: Glyconmed Incorporated 860 Atlantic Avenue Alameda California 94501 UNITED STATES OF AMERICA Daniel E. Levy, Damilan Grobeiny, Peng Cho Tang, Kevin R. Holme. Richard E. Galardy, Gregory S. Shultz. Assad Nematalla,'John H. M~usser Spruson Ferguson,.Patent Attorneys Level 33 ~t martins Tower, 31 Market Street Syieney, New South Wales, 2000. Australia Synthetic Matrix Metalloprotease Inhibito:"i and Uses Thereof The following -statemfent is a fulli description of this invention, including the best method of performing it known tc me/us:-

IA

SYNITHETIC MATRIX M TL LO ER TE ASaEN IH-1 aQB6a AND.. USE THEREC',E This app'ication is a continuation-in-part of Unied States patent application Serial No. 08/044,324 filed April.7,1993. Serial No. (~1O 944,324 is a continuation-in-part of United States Patent No. 5,268,384, issued on December 7, 1993. Unite~d States Patent No.-<.68,384 is a canti r'l ion-in-part of United States Patent No. 5,239,078 issued on August 24, 1993 and United States Patent No.

10 5,189,178 issued on February 23,1993, both of which are also continuation8-in-part of Unitea States Patent No. 5,18:3,900 issued on February 2, 1993. Serial No.

08/044,324 iz also a continuation of United States Patent No. 5,270C,326 issued on December 14, 1993, wbich in turn is a continuation of United States Patent No.

5,114,953 issued on May 19,1992.

Technical Field The invention relates to synthtetic compounds that are inhibitors of matrix metalloproteases, and certain medical applications thereof.

*There are a number of enzymes whiich. effect the breakdown of structural proteins and which are structurally related mneta!Loproteases. Th6se include human skin fibroblast collagenase, human skin fibroblast gelatinase. human neutrophii collagenase and gelatisiase. and human stromelysin. These are zinc-containing metalloprotease enzymes, as are the angiotensiri-converiny enzymes and the enkephalinaSes.

Collager'ase and related enzymes are important in mediating the symptomology of a number of diseases, including rheumatoid arthritis (Mullins, D.E., et al., 51ochim Biptys AJI (1983) 695:117-214): the metastasis of tumor cells iidBroadhurst, MAJ., et al., EP application 276436 (1987), Reich, -'at al., Canicer Res (1988) 4B73307-3312); and Various ulceratled conditions. Ulcerative conditions can result in the cornea as the result of alkali bums or as a result of infe-Aion by Pseudoman-naS rtgnDsaAca ihamoeba. He~i aim~lex and vaccirtia. viruses.

Other conditions characterized by unwanted matrix metalloprotease activity include perio&iintal disease, epidermolysis bullosa and scieritis.

In view of the involvement of collagenase in a nutaber of disease conditions, attempts have been made to prepare inhibitors to this enzyme. A number of such inhibitors are disclosed in EP applications 126,974 (published 1984) and. 159,396 (published 1935) assigned to G.D. aarle. These inhibitors are secondary amines which contain oxtc gubstituents at the 2-position in both substituents bonded to the amino nitrogen.

More closety related to the compounds of the present invention are those disclosed in U.S. prtAni-4,599,361 and 4,743,587, also assigned td~ G.D. Seare.

These compounds are hydroxylamine dipeptide derivatives which -conta in, as a part of the compound, a tyro-sine or derivatized tyrosine residue or certain analogs thereof.

Other compounds that contain sulthydryl moieties as well as residues of aromatic amino acids such as phenylalanine and tryptophan are disclosed in PCT application WO 88/068990. Some of these compounds also contain i-butyl side chains.

FPapplication 498,665, itiventors Beckett., R. P. et al describes the process for ,*reparation of hydroxamic acid derivatives and uses thereof.

application WO 92121360, inventors Sahoo, S. et al, describes substituted ~0 N.-csa.biaxyalkylpeptidyl derivat ives and applications of these compounds for treating ~-.wifvo~ diseases including osteoarthritis, rheumatoid arthritis, certain cancers and ornea ulceration.

~appication 497.192. inventors Lobb., R. et ai. presents peptide collagenase with pharmacological properties.

2E t S. Patent No. 4,681,894, inventors Murray.. W. et al, presents hydroxamic .acids and estes8 that are useful anti-inflammatory agents.

U.S. Patent No. 4,943,587, inventors Cetenko et al.. describe hydroxamate derivatives of selected nonsteroidal anti-inflamnmator acyl residues. Medical aopiications of the compounds are. als6 shown.

U.S. Patent No.. 4,918.105. inverntors Ca-riwri-zhr. et al., presents compounds with callagenase-inhibiting activity. Certain nealapplications a rG described including arthritis, ulcer-Ation, and tufrn: invas*r, 2 a: EP application 574,758, inventors Broadhurst., M.J. et al, presents hydroxamic acids derivatives as collagenase inhibitorsfor treatmentand prevention of degenerative joint disease, invasive tumors, atherosclerosis and multiple sclerosis.

PCT application WO 93/23075, inventors Lang, C. et al, describes the use of matrix metalloproteinase inhibitors for the treatment of vascular leakage syndrome and collagenase induced disease.

Inhibitors have also been disclosed for the related protease, thermolysin.

These include hydroxamic peptide derivatives described by Nishino, et at., Biochemistry (1979) 18:4340-4347: Nishino, et al.. Biochemistry (1978) 11:2846-2850. Tryptophan is also known to be therapeutic in various conditions, some of which may involve coliagenase (see, for example. JP 57/058626; U.S.

S4.698,342; 4,291,048). Also, inhibitors of bacterial collagenases have been S disclosed in U.S. 4,558,034.

It has now been found that the compounds described below have superior inhibiting activity with respect to matrix metalloproteases. The invention compounds add to the repertoire of agents available for the treatment of conditions and diseases which are characterized by unwanted activity by the class of proteins which d.stroy structural proteins and designated "matrix metalloprotease" herein.

Compounds of the invention are also useful for treating diseases that have as one component unwanted angiogenesis. Angiogenesis is defined as the growth of new blood vessels, in particular, capillaries. The ingrowth of such capillaries and ancillary blood vessels is essential for tumor growth and is thus an unwanted physiological response which encourages the spread of malignant tissue and metastases. Inhibition of angiogenesis is therefore envisioned as a component of effective treatment of malignancy. Neovascularization of the eye is a major cause of blindness. One form of this condition, proliferative diabetic retinopathy, results from diabetes; blindness can also be caused by neovascular glaucoma. Inhibition of angiogenesis is useful in teating these conditions also, PCT application WO 91/11193, published 25 January 1991 describes the isolation of a collagenase inhibitor from carti!aoe which inhibits the formation of b!ood vessels. This composition, designated cartilage-derived inhibitor (CDI), is reported to inhibit tumor-induced angiogenesis in the rabbit comeal pocket assay 3 t i L and to inhibit capillary tube formation, It is further speculated that other collagenase inhibitors such aspeptides or antibodies immunoreactive with collagenase will also have the ability to inhibit blood vessel formation.

In addition, EP application 424,193 published 24 April 1991, describes the activity of actinonin as an angiogenesis inhibitor. Actinonin is an antibiotic produced by a particular strain of Streotomyces and is a modified peptide structure.

As disclosed in the two foregoing applications, unwanted levels of angiogenesis are present not only in association with tumor growth, but also are the cause of blindness resulting from diabetic relinopathy and other ocular pathologies.

Compounds of the invention are also useful for treating a certain form of shock, hypovolemic shock and related syndromes. In general, hypovolemic shock can be described as widespread hypoperfusion of cells and tissue due to reduction in blood volume or cardiac output or redistribution of blood resulting in an inadequate effective circulating volume.

Hypovolemic shock, and models for studying this condition are described by Chaudry and Ayala in 'Immunological Aspects of Hemorrhage" Landes Co., Austin, Texas, 1992). Generally, hypovolemic shock due to reduced blood flow S associated with blood loss results in "sludging" of the blood and capillary "plugging' S by erythrocytes, platelets and neutrophils. This in turn leads to the insufficient delivery of oxygen and nutrients to cells and tissues, deficient clearance of other metabolites, and to activation of neutrophils and platelets. This oxidant stress (Hypoxia) and release of other factors from the endothelium and macrophages stimulates the arachidonic acid cascade and the production of chemoattractant and inflammation mediators, leading to further neutrophii infiltration. The activation of neutrophits, platelets, macrophages and the complement cascade leads to the release of numerous biologically active agents including cytokines. These factors stimulate expression of adhesion molecules on the surface of the endothelium.

neutrophils and leukocytes which permit binding and ultimately migration of the neutrophils and leukocytes through the extracellular matrix (ECM) and basement membrane of blood vessels and capillaries. :nis migration or extravasation is attributed to the action of a number of extracellular matrix degrading enzymes including matrix metalloproteinases. serine proteases and endoglycosidases e~ausn~ss~p l l-ra~ers~--~ 1 (i.e.heparanases), which are released by activated neutrophils, leukocytes and/or platelets. The damage to the ECM and basement membrane results in increased vascular permeability, and infiltration of organs by neutrophils and leukocytes.

An analogous series of events is associated with septic shock except, and most critically, the key mediators of the inflammatory response are unlikely to be the same as those that cause hypovolemic shock. The initial blood volume reduction in septic shock occurs as a result of blood pooling after endotoxin stimulate neutrophil activation and the release of inflammation mediating cytokines (TNF, IL-1 and IL-6, TGF- etc.).

It is important to keep in mind that hypovolemic and septic shock are distinct diseases. Hypovolemic shock is a general collapse of the circulatory system that S can be caused by many events including any trauma to the circulatory system (e.g.

gun shot wound, automobile injury, bums, stabbing, and so on). Septic shock, on the other hand, is caused by bacterial infection. Thus, as mentioned above, the causes of these diseases are highly likely to be distinct.

Ischemia/reperfusion injury (UIRI) is another instance where inflammation mediated cell and organ damage result after a reduced blood flow state (ischemia).

The vascular damage associated with hypovolemic shock, and the resulting infiltration of neutrophils and leukocytes into the various organs leads to tissue damage and ultimately multiple organ failure (MOF) and acute respiratory distress S syndrome (ARDS). The destructive agents and mediators are numerous and include cytokines, enzymes and various other inflammatory agents. MOF and ARDS can occur in severe shock and often result in death. For therapeutic agents to be effective in shock, they must protect the microvasculalure and various organs (liver, kidney, heart, spleen and gut) from failure. The importance of protecting or restoring gut function and intestinal function in hemorrhagic shock and I/R injury has been reported, and correlates with reduced septic complications, and long-term survival.

Disclosure of the Invention The methods and compositions of the invention are preferably utilized for preventing or treating certain diseases that have as their underlying cause the k t

A

activation anWVr the exnfesSicn Of unwanted matrix metalloprotease activity. -ijch diseases include skin disorders, keratoconus, restenois hypovolemic shock, wounds, ulcers, particularly of the cornea or mouth or skin, or those disea-se states that are benefitted by uncantrotied angiogenesis, reproductive disease. or conditions such as premature cervical dilation, anti fertility, benign prostatic hypertrophy, and eridometriosis. Regarding the latter, the invention is directed to a method for treating cancer, preferably by inhibiting angiogenesis which facilitates or is required for the growth and spread of cancer throughout a patients body. Further diseases that are prevented or treatable by the invention compounds include those.

mediated by unwanted lymphocyte infiltralion into tissues or organs. such as that which occurs during and is responsible for organ rejection.

Some members of the class of matrix metalloprotease inhibitors'are known in the ait; others are described and claimedin U.S. Serial No. 07/747,751, filed August 1991; 071747,752. filed 20 August 1991; 071615,798. filed 21 November 1990; U-S-Patent No. 4,996,358; EP 0497192; WO 92/09565; and EP 0498665, the disclosures of which are Incorporated herein by reference.

This application is a continuationinpar of United Stares patent applicat ion Serial No. 081044,324 filed April 7,199-3. Serial No. 08/044.324 is a continuationin-part of Un'e States PaetNo. 52834 sudo eebr7 1993. United States Patent No. 5,268.384 is a continuation-in-part of United States Patent No. 5.239,078 issued on Auaust 24, 1993 and United States Paten! No.

5.189,178 issued on February 23,1993, both of which are also continuationsinpart of United States Patent No. 5, 1 3,9OO issued on February 2, 1993- Serial No.

06/44,24 s aso cotiuation of United States Patent No. 5,270,326 issued on December 14, 1993, which in turn is a continuation of United States Patent No.

5,114,953 issuid on May 19, 1992. All patents, patent appfications and publicaions discussed or cited herein are understood to be incorporated by reference to the same extent as if each individual pubticaio or patent applicaticin was speaifically and individually set forth in its entirety.

A summary of the art-known synthetic matrix metalioprotae inhibitors is found in EP application 423,943 published 24 April 1991. This application -assembles the structures of the synthetic maktrix metailoproteases known in Lfie ant and claim-ns their use in the treatment of oe~myeiflatifl9 diseases of the nervous system- The present invention is directed to the use of these compounds, as well as those disclosed in the above-referenced U.S- appications, for the above described uses, and other uses set forth below.

Another objiect of the present inventionl is the description of improved, etflcient and cost effective methods for the synthesis of matrix metalloprotleaSe inhibitors, prodrugs of the inhibitors, derivatives and analogues thereof- Fgure Figure 1 shows light microscopic photographs of mouse skin 'exposed to PdiBu (panel or PdiBu and compound 5A (panel B) and stained with hemnaloxYlin and eosin three days latter- Figure 2 shows that the protease levels present in mastectomy fluid samples collected on days 1 to 7 after surger were an average of 0.75 0.06 pg equivalenits of collagenasefml of wound fluid.

*Figure 3 compares the protease levels present in mastectomy wound fl uids collected from closed (collected on different days post operationl), open, or chronic wounds. Note that closed wounds exhibited marginal protease activity, while open wound fluid contained an average protease level of 199 59 jirgml. and fluids collected from chronic wounds contained an average protease level of 125 Figure 4 shows the effect of three protease ihbitots on the pruieaste ac .iY chronic wound fluid. Compound 5A very effectively inhibited proteolytic degradatiort of Azocoll (approximately 96% of initial proteolytic activity) at final m-i-entratidfls of 40 1 ,iil l(100 M) or 4 ig/ml (10iiM). EDTA. a nonspecific inhib~itor of metalloProteinaSes, also effectively reduced protease activity. approximately 961%.

PMSF. a nonspecific inhibitor of serine proteases. reduced proteolytic activity approximately 65% at a concentration of 500 VpM.

Figure 5 shows the effects of the inhibitors, compound 5.PMSF and EDTA on protease activity present in open and chronic wounds. Compound 5A and EDTA were very effective inhibitors while PFASF did not significantly reduce t~iO proteolyti.activity of the wound fflu-;ds 1k I- W

I

Figure 6 shows the effects of compound 5A S1209. ULQwi. MP50. and EDTA on the proteolytic degradation of Azocoll by wound fluids.

Figure 7 shows the effects of the inhibitors compounds 5A. 21A. 3A and SlQ 2 on protease activity present in chronic wound fluid.

Figure 8 shows the effects of the inhibitor compound 5A on thioglycollate induced peritonitis.

Figure 9 shows the antimetastatic effect of compound A in a murine melanoma (B16-F10) model.

Figure 10A is a bar graph showing changes in maximal velocity of the clearance of indocyanine green (1C6) (active transport process) ii sham-operated (Sham), normal saline-treated (Saline), and 5A treated rats at 2 and4 h after the initiation of crystalloid resuscitation following hemorrhage. There were 6 animals in each group. The trauma-hemorrhage and resuscitation protocol and 5A infusion procedure is described in Example 51. Data are presented as means SE and compared by one-way ANOVA and Tukey's test "P 0.05 as compared with sham-operated group; 'P 0.05 as compared with saline-treated group.

Figure 10B is a bar graph showing changes in the efficiency (Km) of the indocyanine green (ICG) transport in sham-operated (Sham), normal saline-treated (Saline), and 5A treated rats at 2 and 4 h after the initiation of crystalloid resuscitation. See the legend to Fig. 10A for further details.

Figure 11A is a bar graph showing changes in mean arteriai pressure (MAP) in sham-operated (Sham), normal saline-treated (Saline), and 5A treated rats at 2 and 4 h after the initiation of crystalloid resuscitaticn. See the legend to Fig. 10A for further details.

Figure 11B is a bar graph showing changes in heart rate (HR) in sham-operated :(Sham), normal saline-treated (Saline), and 5A treated rats at 2 and 4 h after the initiation of crystalloid resuscitation. See the legend to Fig. 10A for further details.

Figure 12 includes graphs A, B and C showing .changes in cardiac output (CO.

stroke volume (SV. and total peripheral resistance (TPR, C) in sham-operated 30 (Sham), normal saline-treated (Saline), and 5 treated rats at 2 and 4 h after the initiation of crystalloid resuscitation. Seethe legend to Fig. 10A or further details.

Figure 13 includes graphs A. B. C and D showing changes in organ surface ,-~-iWhU---rrua~U-rUc ~)T microvascular blood flow (MEF) in the iver lKidney spl-een and small intestine in sham-operated (Sham), normal salne-treated (Saline), and -e& treated rats at 2 and 4 h after the initiation of crystalloid resuscitation- See the legend to Fig. 10A for further details- S Figure 14 Shows the aelfects of the inhibitor 5A in the anti-restenotic assay (4 da).Amnstaino Acaused a significant decrease in, number of cells per unit area -when compared to 0MC treated controls- Figure 15 shows the effects of the inhibitor in the anti-restenotic assay day). Administration of 5A caused a significant decrease in intimal crass-sectional to area when compared to WCG treated controls- Modes f arring Out the Invention The inhib~itory compounds of the invention are synthetic inhibitors of mammalian matrix metalloproteases. Matrix metalloroeases ilude %Without limitation human skin fibroblast collagenase, humnan skin fibroblast gelatinase, human neutrophil collagenase and gelatinase, and human stromelysin. These are zinc-containing metalloprotease enzymes, as are the angiotensin- converting enzymes and the enkephatinases- As used hierein, *mammalian matrix metalloprotease' means any zinc-containing enzyme found in mammalian sources that is capable of catalyzing the breakdown of collagen, gelatin or proteoglycan, under suitable assay conditions.

Appropriate assay conditions c-an be found, for example, in U.S, patent 4,743,587, which references the procedure of Cawston. et al., Anal Biochern (1979) 20,-340-345, use of a synthetic substrate is descibed by Weingarien, Et at., j3iochgm Biophyt Res Comm (1984) IJ9:1 184-1187. Any standard method fcr 25 analyzing the breakdown of these structural proteins can, of cour-se. be used- The matrix, metalloprotease enzymes referred to in the herein invention are all zinc-containing proteases which are similar in structure to, for example, human stromelysin or skin fibroblast collagenase.

The ability of candidate compounds to inhibit matrix metalloprolease activity can, of course, be tested in the assays described above- Isolated matrx metalloprotease enzymes can be used to confim the in-hibiting activity of the invention compounds, or. crude exiracts -which contain the -ianqe of enzymes capable of tissue breakdown can be used.

Specifically, assay of inhibition activity can be conducted as follows. Inhibitors may be assayed against crude or purified human skin fibroblast collagenase, or purified human gingival fibroblast collagenase using the synthetic thiol ester substrate at pH 6.5 exactly as described by Kortylewicz Galardy, J Med Chem (1990) 33:263-273. at a collagenase concentration of 1-2 nM. The candidate inhibitors are tested for their ability to inhibit crude collagenase and galatinase from human skin fibroblasts, crude collagenase and gelatinase from purulent human neutrophils in this assay. The results may be set forth in terms of Ki, the calculated dissociation constant for the inhibitor complex with enzyme. Ki values for effective inhibitors are 500 nM for purified enzyme in this assay. Por purified human skin collagenase, excellent inhibitors show Ki values of 10nM. Assays for inhibition of human stromeiysin are conducted as described by Teahan, et al., Biochemistry (1989) 20:8497-8501.

Assay of inhibition activity can be conducted using a fluorogenic substrate developed by Knight et al., FEBS Letters (1992) 6:263. This method uses a fluorometer to determine the rate of substrate hydrolysis in a 96 well plate format.

Reaction rates are determined at several substrate concentrations, and multiple inhibitor levels. The data are analyzed by Lineweaver-Burk, Dixon and Henderson plot analyses (Enzyme Kinetics (1975) Irwin Segal (John Wiley Sons, Inc., publishers), and as described by Henderson, Biochem J. (1972) 127:321). The substrate levels range from 10-301M. The inhibitor levels are chosen to give 0-90% inhibition (0-500nM). Inhibitors may be assayed against purified 72kD gelatinase, 92kD gelatinase, neutrophil collagenase, stromelysin or other proteases as appropriate. The enzymes are activated at 370C with APMA (4aminophenylmercuric acetate), and the assays are carried out at room temperature.

The synthetic compounds that are successful in these assays for mammalian matrix metalloprotease inhibition are generally small molecules containing at least one amide bond and have a variety of sidechain substituents. Examples of such compounds known in the art are given, as set forth above, in EP application 423,943. incorporated herein by reference.

-ml\ V i Other suitable inhibitors are of the formula: R 7

-NR

6 CH-CHQN HOOX L[R 4 2 13 (1) R 7

-NR

6 -OCCH- C=CHON CHGOX L J

M

R R (2 wherein each RI is independently H or alkyl (I1-8C) and R2 is H or alkyl (1 -8C) or -NHZ wherein Z is -RI I, -CORI', or -COORil where RI is an alkyl group; or wherein the proximal RI and R2 taken together are -CCH 2 wherein p is R3 is H or alkyl (1-4C); R4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene; n nis0, 1or 2;mris 0or1; and X is -OR5, -NHRs, -M or -NH(CH 2 )qM; wherein R5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-160); -Lo ~M is an amino acid residue or amide thereof or the residue of a cyclic amine or, heterocyclic amine; q is an integer of from 1-8; and R6 is H or lower alkyl (1 -40) and R7 is H. lower alkyl or an acyl group, and the -CONR3- amide bond shown is optional'iy replaced by a modified isosteric bond, such as -CH 2 NR3-, -CH 2 CHR3-, -CH=CR3-, -COCHR3-.

-CHOHOHR3-, -NR3CO-, -CF=0R3-, and the like.

Other compounds of the invention include compounds of the formulas Y-CH---CHCON -CHCOX (3) or FOO -ICO LR"IIJR IR 2 R3 R4 (4) wherein each RI is independently H or alkyl (1 -80) and R2 is H or alkyl (1 -8C) or NHZ wherein Z is 1. -0011i1 or -COORl I where RI is an alkyl (1 -e0) g roup;, or wherein the proximal R1 and R2 taken together are -(0H 2 wherein p R3 is H or alkyl (1-4C); R4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene; n nis0, 1or 2; mis 0or1; and X is -0R5, -NHR5. -M or -NH(CH2),M; wherein IRS is H or substituted or unsubstituted alkyt (1-12C), aryl (6-12C), aryl alkyl (6-16C); *M is an amino acid residue or amide thereof or the residue of a cyclic amine or heterocyclic amine; q is an integer of from 1-8; and -26 Y is selected from the group consisting of R7ONR600NRG6-, R6 2 NCONOR7-, and R600NOR7-,or -COOR12, wherein each R6 is independently H- or lower alkyl (1-40); R7 sH lower alkyl (1-4C) or an acyl group, and R12 is H, alkyl (1-60) or -CH 2 -O-acyl group, and wherein the -CQNR3- amide bond shown is optionally replaced by a modified isosteric bond, such as -CH 2 NR3-, -CH 2 CHR3-, -CH=CR3-, -COCHR3-, -CHOHCHR3-, -NR300-, -CF=CR3-, and the like.

12 Other compounds of the invention include compounds of the formula HONO--CH CHCON -CHCOX R64-R L"q J R 2 wherein each Ri is independently H or alkyl aryl alkyl (1-4C) or aryl-S.

alky! (1-4C) and R2 is H or alkyl alkenyl aryl aikyl (1-6C) or -NHZ wherein Z is -Ru -CORli or -COOR"l where R" is an alkyl group; or wherein the proximal Ri and R2 taken together are -(CH 2 wherein D n isO0, 1 cr2;, P3 is H or alkyl (1-4C); R4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene, unsubstituted or substituted aryl methylene, alkyl (1I-1 2C); X is -OR5, -NHR5, -M or -NH(CH 2 )gM; wherein R5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); M is an amino acid residue or amide thereof or the residue dt a cyclic amine or heterocyclic am-ine; is an integer of from 1-8; and Ra is H or CH 3 R9 is optionally substituted aryl, aryl alkyl (1 substituted or unsubstituted alkyl (1-120), -0-alkyl -S-alkyl -OCORO, -OCOORlO, 5-methvi-2-oxo- 1 ,3-dioxol-4-yl, -COQH, COORIO, -CONH 2 and RIO is alkyl (1 -1 A prodrug is defined as a chemically protected compound which is substantially biologically inactive, and wherein the biologically active form of the compound is released within the body of a patient preferably as a result of hydrolysis by enzymes such as esterases.

'Aikyr has its conventional meaning as a straight chain, branched chain or cyclic saturated hydrocarbyl.xesidue such as methyl, ethyl. isobutyl (i-Ru), cyclohexyl, t-butyl (t-Bul or the like. The alkyl substituents of the invention are of the number of carbons noted which may be substituted with 1 or 2 substituents.

Substituents are gener'ally those which do not interflere With the activity of the 13 compound, including hydroxyl, CBZO-, CBZNH-, amino, and the like. Aryl refers to aromatic ring systems such as phenyl, naphthyl,'pyridyl, quinolyl, indolyl, and the like; aryl alkyl refers to aryl residues linked to the position indicated through an alkyl residue. In all cases the aryl portion may be substituted or unsubstituted. 'Acyl" refers to a substituent of the formula RCO- wherein R is alkyl or arylalkyl as above-defined. The number of carbons in the acyl group is generally 1-15; however as the acyl substituent is readily hydroylsed in vivo the nature of the group is relatively unimportant. 'Cyclic amines' refer to those amines where the nitrogen is part of a heterocyclic ring, such as piperidine, 'heterocyclic amines' refer to such heterocycles which contain an additional heteroatom, such as morpholine. Bn refers to a benzyl group (CH 2 Ph), Piv refers to a pivalyi group (CO-t-Bu), D refers to a phenyl group.

In the compounds of formulas 1 and 3, preferred embodiments for R, and R2 include those wherein each Ri is H or methyl and R2 is alkyl of 3-8C, especially isobutyl, 2-methyl butyl, or isopropyl. Especially preferred is isobutyl. Preferred also are those compounds of all of formulas 1-4, wherein n=1 or m=1.

In all of formulas 1-4, preferred embodiments of R3 are H and methyl, especially

H.

R

4 is a fused or conjugated bicycle aromatic system linked through a methylene 20 group to the molecule. By 'fused or conjugated bicyclo aromatic system' is meant a two-ringed system with aromatic character which may, further, contain one or more heteroatoms such as S, N, or O. When a heteroatom such as N is included, the system as it forms a part of formulas 1-4, may contain an acyl protecting group attached to the nitrogen. Representative bicycle fused aromatic systems :25 include naphthyl, indolyl, quinolinyl, and isoquinolinyl. Representative conjugated systems include biphenyl, 4-phenylpyrimidyl, 3-phenylpyridyl and the like. In all cases, any available position of the fused or conjugated bicyclic system can be used for attachment through the methylene. The fused or conjugated aromatic system may further be substituted by 1-2 alkyl (1-4C) residues and/or hydroxy or any ring nitrogens may be acylated. Preferred acylation is acetylation.

Preferred embodiments of R4 include 1-(2-methyl naphthyl)methylene; 1-quinolyl methylene; 1-naphthyl methylene; 2-naphthyl methylene; 1-isoquinolyl methylenle; 3-isoquiriolyl methylene; 3-thionaphthenyl methylene; 3-CUmaronyl methylenle; 3.(5.methylifldolyl)metliyile; 3-(2-hydroxyifldolyl)methyfene; biphenyl methylene; and 4.phenylpyrimidyl methylene; and the substituted forms thereof.

Many of these substituents as part of an amino acid residue are described in Greenstein and Winitz. 'Chemistry of the Amino Acids' (1961) 2:2731-2741 (John Wiley Sons, NY).

A particularly preferred embodiment of R4 is 3-indolylmethylene or its N-acylated derivative-i.e., that embodiment wherein the 'C-terminal' amino acid is a tryptophan residue or a protected form thereof. A preferred configuration at the carbon to which R4 is bound is that corresponding to L-tryptophan.

Preferred embodiments of X are those of the formula NHRs wherein R5 is H, substituted or unsubstituted alkyl (1-12C) or aryl alkyl (6-120) Particularly preferred substitutions on R5 are a hydroxyl group, or a pheriylmethoxyCarbamyl (CBZN H-) residue. In addition, the compound may be extended by embodiments wherein X is an additional amino acid residue, particularly a gilycyll residue, which may also be amidated as described.

In general. the compounds that are hydroxamates are obtained by converting a carboxylic acid or ester. precursor of the formulas ROOC -CH-CHCOOH (6) or1 ROOC ~Hi-C=HCOH (7) wherein R is H or alkyl to the corresponding hydroxamates by treating these compounds or their activated forms with hydrox\ylamne under conditions which effect the conversion.

With respect to starting materials, the components forming the -NR3-CHR400X -dl~-wam~da~ssaR LOI L~.Z moiety are readily available in the case of tryptophan and its analogs as esters or amides. As set forth above, many analogous fused bicyclo aromatic amino acids are described by Greenstein and Winitz (supra). Amino acids corresponding to those wherein R4 is 1-(2-methyl naphthyl)methylene; 1-quinolyl-methylene; 1-naphthyl methylene; 1-isoquinolyl methylene; and 3-isoquinolyl methylene can be prepared from the bicyclo aromatic methylene halides using the acetamido malonic ester synthesis of amino acids, as is well understood in the art. The methylene halides themselves can be prepared from their corresponding carboxylic acids by reduction with lithium aluminum hydride and bromination of the resulting alcohol with thionyl bromide.

In general, the hydroxylamine reagent is formed in situ by mixing-the hydroxylamine hydrochloride salt with an excess of KOH in methanol and removing the precipitated potassium chloride by filtration. The filtrate is then stirred with the precursor activated carboxylic acid or ester of formula 6 or 7 for several hours at room temperature, and the mixture is then evaporated to dryness under reduced pressure. The residue is acidified, then extracted with a suitable organic solvent such as ethyl acetate, the extract washed with aqueous potassium bisulfate and salt, and then dried with a solid drying agent such as anhydrous magnesium sulfate.

The extract is then again evaporated to dryness and crystallized.

:-20 The substituted forms of the hydroxamate which include -NHOR7 are synthesized in an analogous manner but substituting H 2 NOR7, wherein R7 is lower alkyl or acyl (1-4C) for hydroxyfamine per se. The resulting O-alkyl or acyl ydroxamate can then be further alkylated, if desired, to obtain the R70NR5derivative of the carboxylic acid. Similarly, HNR6OH may be reacted with the carboxylic acid to obtain the HONRe- derivative. CH 3 NHOH and H 2

NOCH

3 are commercially available.

I

To prepare the starting materials of formulas 6 and 7, the monoesterifted carboxylic acid of the formula R-ONR'-OC- CH--CHCON- CHCOX 1 R2 R R 4 or r-I

R

7

-ONR

6 CHCON-CHCOX (9) 1 R' 1R2 R R 4 is reacted with the acid of the formula NHR3CHR'COX wherein X is other than OH under conditions wherein the condensatiorn to form the amide bond occurs. Such conditions typically comprise mixture of the two components in a nonaqueous anhydrous polar aprotic solvent in the presence of 20 base and a condensing agent such as a carbodiimide. Thus, the formation of the amide linkage can be catalyzed in the presence of standard dehydration agents such as the carbodiimides, for example dicyclohexyl carbodiimide, or N, N-carbonyl diimidazole. The product is then recovered a, a mixture of diastereomers of formula 6 or 7. This mixture is preferably used for the conversion to the hydroxamate and one of the resulting diastereomers is crystallized directly from the product mixture.

Alternatively, the diastereomers are separated by flash chromatography before conversion to the hydroxamate and recovered separately. This process is less preferred as compared to the process wherein separation of the diastereomers is reserved until the final product is obtained.

in the notation used in the examples, the isomer is defined as that which migrates faster on TLC; the isomer as that which migrates more slowly. When the form of tryptophan or other amino acid containing a fused bicycloaromatic ring system is used as the residue, and Ri is H, in general, the form is that which contains the corresponding configuration at the carbon containing the R2 substituent in the final hydroxamate product. However, in Example 2. below, where D-tryptophan is included in the composition, the 'B isomer contains vhat would correspond to an configuration at the carbon containing R2 in the compounds of 17 formula 1.

When Rs andior R7 alkyl. the correspondcing 0- or N-alkyl hydroxylamifle is reacted with th e methyl ester 4A as performed for unsubstituted hydroxylainfe in Example 1I. Alternatively, the methyl ester 4A can be saponified to its corresponding carboxylic acid and activated with oxalyl chloride or other condensing agent. The alkyl hydroxylamifle can then be reacted with the activated carboxylic acid to give the 0- or N-substituted hydroxamic acid. 0- and N-methylhydroxylamifle can be purchased from the Aldrich Chemical Company.

Other N-aikyl hydroxyarnifles can be synthesized by conversion of aliphatic aldehydes to their oximes, followed by reduction to the N-alkyl hydroxylamllie with borane-pJyridifle complex in. the presence of 6N HCI (Kawase, M. and Kikugawa, YJ, Ch in Poc Prkin Trans (1979) 1:643. Other 0-alkyl hydroxylamifles can be synthesized by the general methods given by Roberts, Derlivatives of Hydroxylamifle,' Chapter 6.4 iin Barton, et al.. eds., Comrehefsve rqn i g 's Chemistr (1979) 2:187-18S kPergamoh Press, Oxford). The two general methods employed are displacementby R70- of a leaving group from hydroxylamifle sultoniz acid or chloramne. and 0-iyation of a hydroxarnic acid with R7-X followed by hydrolysis:

.R

7 0 NH1I.S0%H (or NH2 Cl) NH-0R 7 or H,0' .77 -RCO-INOH R X

RCO-NI-OR

7

NHO

2

R,

For R7 acyl, a hydroxamfic acid of this invention can be acylated with an, acid chloride, anhydride. other acylating agent to, give the compounds of this class.

In some cases the derivatized maleic and succinic acid residues required for synthesis of the invention compounds are commercially available. If not, these can readily be prepared, in embodiments wherein R' is H or alkyl (I18C) by reaction of'a 2-oxocarboxylic ester of the formula R200COOR in a Wittig reaction with an alkyl acetate or a-tripheflyl-phosphorany1idefle alkanoaie.

The methyl acetate or. alkanoate is preferred. but any suitable ester can be employed. This reaction is conducted in a nonaqiueous, nonpolar sblvent usually at room temperature. The resultant coimpound is of the formula ROOCCR1--CR2COOR', wherein R and H'are residues of esterifying alkyl or arylalkyl alcohols.

if the compounds of formula 7 are desired, this product is condensed with ,he appropriate tryotophan or analogous derivative; if the compounds of formula 6 are desired, the intermediate is reduced using hydrogen with a suitable cataly'st. The sequence of reactions to obtain those embodiments wherein R, is H or alkyl, n is 1 and m is 0, and R2 is alkyl are shown in Reaction Scheme 1.

Schemne 1 4i3P=CHCCJOCTh 1 4- R 2

ZCOCOOR!-"

1H21

H

3 COOCHC=C-COOR* b, H 3

COOCCH

2

-COOR*

R 1.

E+Z

R+S

Vonmfla 2 Formula (1I Th~ hydrogenation ecinw- mv R! %%.hen R' is bcnzyl For those embodirrents wherein R, and Rz zke together are (CH- 2 the compounds of the i~wention are prepared anak, 4t.u.-:v to the manner set forth in Reaction Scheme 1. except that the inlerrmer -ate c6 Ine iormula ROOCCHRICHR200QH is prepared from t wcrespondn yiakn dicarboxyi tid yc~ela dic &boxyfic acid anhydride; 1 .2-cyclohexafle dicarboxyfic anhydride or 1 .2-cycloheplane dicauboxyll'c anhydride- For compounds wherein -CQNRt3- is in modiffied isosteuic form, these forms can be prepared by methods known in the art. The following references describe preparation of peptide analogs which include these alternative-linking moieties: Spatota. VegAData~ (March 1983), VoL 1, Issue 3. OPeptide Backbone Modifications' (general review); Spatola. AF.. in *Chemistry and Biochemistry of Amino Acids Peptides and Proteins.' (1983) B. Weinstein, eds., Marcel Dekker, New York, p. 267 (general review); Morley. Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, et al, tnt J Pep, Pro( Res (1979) 14-177-185

(-CH

2 NR3-, -GH 2 C1HR3-); Spatola, et at., Life Sc (1986) .:1243-12 9

(-CH

2 Hann, J Chem SocernTrs119) 307-314 (-CH-CR3-. Cis and trans); Almquist. et al_ 1L~22Ei(90 2a1 32398 K-CCH3-) Jennings- White, et at., Teitedrn Lett (1982) 21.2533 (-COCHR3-); Szetke, M_ et at.. European application EP 45665 (1982) CkET7:39405 (1982) (-CH(OH)CHR3-); Holladay, et at.. ITthedronLan (1983) 2474401-4404 (-C(OH)CHr); and Hruby, Life Sci (1982) a1*189-199 (-CH 2 Preferred compounds of formula or include: ti-ONHCQCH 2 CH(n-hexyl)-CO-L-Trp-NHMe-; *:go HONHCOCH 2 CH(fl-pefntyl)-CO-L-Trp-NHMe;

H-ONHCOCH

2 CH(i-pentyi)-CO-L-Trp-NHMe;

HONHCOCH

2 CH(ethyl)-CO-L-Trp-NHMe, HONHCQCHaCH(ethy)-CO-L-Trp-NHCH 2 CH3; HONHCOCH4 2 CH(e)CO-L-rp-NHCH 2

CH

2

OH-

.Ze 5 HONHCOCH2CH(ethy)-CO-L-TP-NHcyctoheCyl.

MeONHCOCHaCH(iBu)-CO-L-Trp-NHEt- EtONMeGOCH 2 CHiBu)-CO-L-Trp-NHEt: MeONHCOCH- 2 CH~iBu)-CO-L-Aia(2-naphthyl)-NHEt; EtONMeCOCH 2 CH(iBu-CQ-L-Ala(2-laphthl)-NHEt;

HONHCOCH

2 GH(i-Bu)CO-L-Trp-NHMe; HONHCOCH-ICH(i-Bu)CO-L-N-Merp-NHMe; HON HCOCH2CH(i-Bu)CO-L-Trp-NH(CH2)OH;

HONHCOCH

2 CH(i-BU)CQ-L-Trp-NH(S)CHMePh-,

HONHCOCH

2 CHCi-Bu)CO..L-TFp-NH(CH 2 6

NH.-CBZ

HON HCOCH 2 CH(i-Bu)CO.L-Ata(2-naphthy[) NHIe; KiNHCOH 2 CH(i-Bu)CO-L-Trp-4H(CH) 4 CH2.

HONHCOCH

2 GH(i-Bu)CO.L -Trp-piperidine;

HONHCOCH

2 CH1-Bu)co-L-Trp.NH(CH 2 )I 1

CH

3

HONHCOCH-

2 CH(i-Bu)CO-L-Trp-NHcyclohexyj

HONHCOCH

2 CN iBu)-L-Trp-OH: H-ONMeCOCH 2 CH(i-Bu)CO-L-Trp-NHMe; HONEtCOGH2CH(i-Bu)CO-L-Trp-NHMe- CH3COONHCOCH 2 CH(i-Bu)CO.L-Trp-N.HMe;

>C.OONHCOCH

2 CH(i-Bu)CO-L-Trp-NHMe-; GH3CQONMeCOCHaCH(i-Bu)CO-L-Trp-NHMe;, and 15 4OCOONEtCOCH2CH(i-Bu)CO-L.-Trp-NHMe.

The reverse hydroxamates and hydroxyureas of formulas 3 and 4 are more stable biologically than the corresponding hydroxamates per se. This has been confirmed in Carter. et al-, J Pharrmacgl EMp Thor (1991) 25:920--937; Jackson, et al., JL-Mber Ce(1988) 3-1:499,500. Young, et al-. FAEU (1991) 5~:A1273; Hahn, at al., J Phamaco Ex Ther (1-091) no-.94-i02: Trarnposcb;, KCM., et al., tkgnt.Actws (1990) 3Q--443-450; Argentieri. el al.: Kimball, E, et al-, 5th In! Con! Inflammation Research Assoc.. Whitehaven, PA.

September 23-27 1990, Abstract 100; and Hfuang, et al., j Me..Chem (1989) 2a-1836-1842. Thus, while somewhat more complicated to synthesize, these analogs offer physiological characteristics which are advantageous in the applications of these compounds to therapy.

The reverse hydroxamates and hycdroxyureas of the invem~ion are obtainable using the standard techniques of synthetic organic chemistry (see Challis. et al., 'Amides and Related Compounds' in 'Comprehensiv Organic Chemis try,' Barton. et al.. eds. (1979) 2 1036 MI5) Pergmo Prs.Oodafute described below.

With respect to starting materials, the components forming the -NR3-CHR4GOX moiety are readily available in the case of tryptophan and its analogs as esters or amides- As set forth above, many analogous fused bicyclo aromatic amino acids are described by Greenstein and Winitz (supra)- Amino acids corresponding to those wherein R4 is I -(2-methyl naphthyl)methylene; 1 -quinolyl-methylene; 1-naphthyt methyiene; 1-isoquinalyi methylene; and 3-isoquinolyl methylene can be prepared fromn the bicyclo aromatic methylene halides using the acetamido malonic ester synthesis of amino acids, as is well understood in the art. The methylene halides themselves can be prepared from their corresponding carboxylic acids by reduction with lithium aluminum hydride and bromination of the resulting alcohol with thionyl bromide- Depending on the functional group symbolized by Y, the stage of synthesis at which this moiety is brought into the compound of the invention varies- For those ermbodiments wherein Y is R70NR6CONR6- and wherein n 1 or 2.

5 the compounds are prepared by acylating an ci, P or y amino acid, respectively with methyl or ethyl chloroforrfate, condensing the resulting amino acid with a protected form of the moiety .NRaCHR400X and reacting the resulting carboethoxy 'dipeptide' with hydroxylamine or a substituted hydroxylam ine as. described by Fieser, L. et at., TReagents for Organic Synthesis* (1967) 1:479 (John Wiley Sons, New York).

This sequence of reactions is shown in Reaction Schem e 1A.

Scheme I A EtOCOCI W CH2-CHCOOH- EIOCO- CH2 CHC'OOII CDI HN CH- COX EtOCON- H CI-CO-N-CH-o

NMOR'

2 ON-CON- CH-CHCO-N- CH- COX R" R~ R2 3~ Alternatively, the m, B or -f amino acid is temporarily protected using, for example, carbobelzoxy or tertiary butytpxyc-arbol and coupliN it to the carboxy-termia-protected amino acid moiety. containing .R4. The potectiag group is then riemoved by hydrogeflclysis or acidolysiS as appropriate a-rd the deprotected a. (3 or y amino croup is reatted with an aclvated cwbn-i- acid such as carbonydiimfidazole. The resultant is then reactedwith hyJroXY~arnik1 or substituted hyd~cytam ine to obtain the desired prc'kct. Thi seqjyce of cin issmarized in Reaction Sch& 2.(rTh aua -Am. IM reppiresents an imidazole residue.) PrN-C -CCHN- CH- COX 'If 1J 2 '3 4 1-N- CH- CHCO- N- CH- COX 6 P1 '2 3

R

6 R R R~ R 4 Irn-CO-lin NH-OR 7

R

7 ON -COCN- CH- CHCO- N- CH- COX 6 1 'l6 1 R3p3 The appropriate a, B3 or y amino acids are prepared by general methods as set forth by Jones, JJ-l., et al., in 'Amino Acids,' p. 834 (Barton, et al., eds.) ('Comprehensive Organic Chemistry' (1979) Vol. 2. Pergamon Press), Such ,:2D methods include, for example, homologation by Amndt-Eisteit synthesis of the corresponding N-protected a-amino acid and more generally the addition of nitrogen nucleophiles such as phthalimide to a,13-unsaturated esters, acids or nitriles.

In a second class of hyciroxyureas, Y has the formula R6 2 NCONOR7- and n is 0.

1 or 2. These compounds are prepared from the corresponding a. B or y hydroxyamino acids of the formula R 7 0NH(CHR1)nCHR2COOH. When both R6 are H, this intermediate is converted to the desired hydroxyurea by reaction with silicon tetraisocyanate, as described by Fieser and Fieser, *Reagents for Organic Synthesis' (1968) .1:4Y9 (Jolin Wiley Sons, New York). The reaction is conducted with the hydroyt group protected or substituted by A7. The resulting hydroxyurea is then coupled to the component of the formula HNR3CHR4COX to obtain the desired 24 product. Alternatively, the amide is first formed and the N-hydroxyl dipeptide is treated with the reagent.

Alternatively, when Y is R6HNCO-NOR7, wherein R6 is alkyl, the above O-protected a, 1 or y N-hydroxyamino acid is reacted with the relevant aikylisocyanate R6NCO to produce the desired product.

When Y is of the formula R6 2

NCO-NOR

7 wherein both R6 are alkyl, the a, 1 or y N.hydroxyamino acid is reacted with an activated form of carbonic acid, for example, carbonyl-dimidazole or bis-p-nitrophenylcarbonate, and then with the diamine R6 2 NH wherein both R6 are alkyl groups. This is followed by deprotection, if desired.

Conditions for the foregoing can be found in the descriptions of analogous preparations for tripeptides as described by Nishino, et al., Bfochmistrv (1979) 18:4340-4346.

The 3-N-hydroxyamino acids used as intermediates in the foregoing synthesis can be prepared by a malonic ester synthesis in which diethyl malonate is alkylated 15 twice, one with R2-Br and then with benzylchloromethyl ether, for example, for the case wherein Ri is H. The product is saponified, decarboxylated, hydrogenated, and oxidized to give the aldehyde in a manner similar to the synthesis of a homologous aldehyde described by Kortylewicz, et al., Biochemistry (1984) 2:2083-2087. The desired 0-hydroxyamino acid is then obtained by addition of P::0 protected (or alkylated, if R7 is alkyl or acylated if R7 is acyl) hydroxylamine. The corresponding compound wherein R1 is alkyl can be prepared in an analogous manner wherein the second alkylation utilizes benzyl-O-CHRIC. The homologous ketone was described by Galardy, et al., Biochemistry (1985) A4:7607-7612.

Finally, those compounds wherein Y is of the formula R6CONOR7-, the reverse hydroxymates, can be prepared by acylation of the corresponding a, 6 or 7 N-hydroxy dipeptide. Alternatively, the N-hydroxyamino acid can be acylated, followed by condensation to form the amide bond in the compounds of the invention. The acylation method is described by, for example, Nishino, et al., Biochemistrv (1979) 1:4340-4346, cited above.

Alternatively, for those compounds wherein n=1 and RI is H, the'compounds can be prepared by condensing the ylide 1,1-dimethoxy- 5 S .s 2-(triphenylphosphoranylidefle) ethane prepared from triphenylphosphine and 1 aI .dimethoxy-2-bromoethafle With 4-methyl-2-oxopentanoic acid. The product is then hydrogenated to obtain 4,4-dimethoxy-2isobutybutr~oic acid which is coupled to the moiety R3NHCHR4COX to0 obtain 4,4-dimethoxy-2-isobutylbutanoyl-NR3CH-R 4 00X. Treatment with aqueous acid yields the aldehyde 2-isobutyl.4-oxobutanoy-NR3CHR 4 00X. The oxime is prepared by reaction with hydroxylamine and reduced to the corresponding N-substituted hydroxylamine.

Acylation of both the hydroxaminol oxygen and nitrogen followed by hydrolysis of the 0-acyl group provides the N-acyl reverse hydroxymates. (Summers, J.B.a et at., J-e-Ce (1988) 3-1.1960-1 964.) For compounds wherein -CONR3- is in modified isosteric form, tfiese forms cani be prepared by methods known in the art, as set forth above.

Preferred compounds of formulas and include: EtONHCQNMe-CH 2 CH(i~u)-CO-L-Trp-NH Et; 415 EtCONOH-CH 2 CH(iBu)-CO-L-Tr p-NHEt; a n-PrOOEt-CH 2 GH(iBu)-CO-L-Trp-NHEt; EtNHCONOMe-CH 2 CH(iBu)-CO-L-Trp-NHEt; MeNH-CONOH.CH 2 CHCiBu)-CO-L-Trp-NHEt; EtONHCONKe-CH2CH(iBu)-CO-L-Ala(2-lSphthyI)-N HEt;

ECONOH.CH-

2 0H(iBu).CO-L-Ala(2-flaphthyl)-NHEt; n.PrCONOEt-CH2CH(iBu)-CQ-L-Ala(2-naphlNHt EtNHCONOMe-CHsCH (iBu).CO- ;i(-naphthyl)-N HEt;, MeN HCONOH-Gi- 2 CHkibu -CO-L-Ala(2-naphthyl)-NHEt;

HONHCONHCH

2 CH (iBu)-OO-L-TrpNHMe: .25 HONHCONHCH 2

CH

2 CH(iBu)-CQ-L-TrpNHMe; HONHCONHCHQiBu)CO--L-TrpNHMe-,

H

2 NCQN(O.H)CH(iBu)CO-L-TrpNHMe;

HN(OH-)CH

2 CH(iBu)CO-L-TrpNHMe;

NCON(OH)CH

2

CH

2 C iu)CQ-L-TrpNHMe; CI20NO)HiuCO-~L3TpIIe 3 C0N(OH)CHaG(iBu)CO-L-TrpNHMe;an 26 "4 CH3CON(OH)CHH 2 CHCH(iBu)COL-TrpNHMe.

Administration and Use As set forth in the Background section above, a number of diseases are known to be mediated by excess or undesired matrix-destroying metallo-protease activity.

Thus the compounds of the invention can be applied to treat or prevent such diseases. These include tumor metastasis, rheumatoid arthritis, skin inflammation, ulcerations, particularly of the cornea or mouth, reaction to infection, and the like.

Also intended to come within the definition of diseases that can be treated by the invention inhibitors are wounds, preferably chronic dermal wounds, The inhibitors of the invention are, however, useful in any ulcerative skin condition, including, for example, decubitus ulcers, ulcers of the mouth, or other conditions where wound healing is slow. Thus, the compounds of the invention are useful in therapy with regard to conditions involving this unwanted activity.

._1"15 The compounds of the instant invention are particularly useful for treating or preventing psoriasis. Psoriasis is a common inflammatory skin disease of uncertain etiology, which is characterized by prominent epidermal hyperplasia, mixed inflammatory infiltrates and vascular alterations. The molecular mechanism(s) responsible for epidermal hyperplasia in psoriasis and other skin disorders remain 20 unresolved. However, various growth factors, cytokines and proto-oncogenes have been implicated in the transduction of growth-promoting signals from the extracellular environment into the epidermal keratinocyte. Current treatment of psoriasis and other hyperproliferative skin disorders includes a variety of topical .steroids, keratolytic, systemic chemotherapy and UV-light exposure. However, these available therapies are limited by toxicities as well as tachyphylaxis.

Still another condition responsive to the matrix metalloprotease inhibitors of the invention, particularly collagenase inhibitors, include restenosis fo!lowing angioplasty. The healthy arterial wall is composted of an outer adventitial layer of fibroblasts, a central medial layer of smooth muscle cells and a luminal intimal layer of endothelial cells. It has been postulated that one cause of restenosis following balloon angioplasty is the production and release of collagenase by'smooth muscle cells that causes degradation of the intima (Southgate, K. M. et al (1992) Bioche-m, 27 I I I' -I I- 1 ,T h. 288:93-99). This, in turn, facilitates migration of the smooth muscle cells into the intima where they continue to proliferate to form the fibrous plaques that are characteristic of restenosis. Thus, matrix metailoprotease inhibitors of the invention would prevent or inhibit restenosis when administered before or after angioplasty.

Yet another application of the matrix metalloprotease inhibitors of the invention is the treatment or prevention of cancer, particularly metastatic and invasive cancers. Cancer cells migrate from their primary site of origin to remote secondary sites by extravasation into the blood, and subsequent extravasation out of the blood to the target organ. Thus, it would be possible to prevent or eliminate metastasis if extravasation of cancer cells could be controlled. Since a key process in extravasation is the breakdown of the extracellular matrix by enzymes secreted by cancer cells, particularly collagenases, the collagenase inhibitors of the invention have significant applications for the treatment or prevention of cancer.

As mentioned above, the collagenase inhibitors of the invention are useful in 5 any ulcerative skin condition, including, for example, decubitus ulcers, ulcers of the mouth, or other conditions where wound healing is slow. Similar conditions susceptible to treatment by the compounds of the invention include comeal or scleral melting associated with keratomalacia, scleromalacia perforans and connective tissue diseases. An example of the latter is keratoconus which involves thinning and central protuberance of the cornea. Type IV coll genase is thought to be responsible, at least in part, for the disease.

Compounds which are synthetic inhibitors of mammalian metalloproteases are useful to inhibit angiogenesis. These compounds can therefore be formulated into S pharmaceutical compositions for use in inhibiting angiogenesis in conditions characterized by an unwanted level of such blood vessel growth.

Still another condition responsive to the matrix metalloprotease inhibitors of the invention, particularly collagenase inhibitors, include treatment of shock, including for example, hypovolemic shock and septic shock. The mechanism of many diseases such as hypovolemic shock are complex and the result of multiple causes.

Accordingly, 'treating" as used herein indicates a methodology which interferes with one or more causes or events and thereby has a beneficial impact dn the individual being treated. It is understood that to treat' hypovolemic shock includes preventing, i 28 I II~ m hm delaying or in some way reducing the onset of symptoms without perhaps actually removing the cause for shock completely. Accordingly, treatment with the present invention compositions may extend life and/or improve its quality even though the individual being treated ultimately succumbs to shock.

The invention compositions are also applicable to the prevention of such shock in patients that are at a high risk of developing the disease. For instance, certain conditions carry a high risk of developing hypovolemic shock including hemorrhage, trauma, bums, polyuria. vomiting, and diarrhea. See, Circulatory Shock (1992) 91:7. Thus, a patient hospitalized for one of these conditions may be administered the compositions of the invention to prevent the development of hypovolemic shock.

Consequently, while reference throughout the patent application is hade to methods of treating hypovolemic shock it will be understood by the skilled practitioner of this art that such terminology encompasses preventing shock as well.

Standard pharmaceutical formulation techniques are used, such as those '.15 disclosed in Reminaton's Pharmaceutical Sciences, Mack Publishing Comoany, Easton, PA. latest edition.

SFor indications to be treated systemically, it is preferred that the compounds be injected or administered orally. These conditions include tumor growth and metastasis. The compounds can be formulated for injection using excipients conventional for such purpose such as physiological saline. Hank's solution, Ringer's solution, and the like. Injection can be intravenous, intramuscular, intraperitoneal or subcutaneous. Dosage levels are of the order of 0.1 mg/kg of subject to 100 mg/kg of subject, depending, of course, on the nature of the condition, the nature of the subject, the particular embodiment of the invention compounds chosen, and the nature of the formulation and route of administration.

In addition to administration by injection, the compounds of the invention can iso be formulated into compositions for transdermal or transmucosal delivery by including agents which effect penetration of these tissues, such as bile salts, fus;dic acid derivatives, cholic acid, and the like. The compounds can also be used in liposome-based delivery systems and in formulations for topical and oral S- administration depending on the nature of the condition to be treated. Oral S- administration is especially advantageous for those compounds wherein the moiety 29 r c- -CONR3 is in a modified isosteric form or for prodrug forms. These compounds resist the hydrolytic action of the digestive tract. Oral formulations include syrups, tablets, capsules, and the like, or the compound may be administered in food or juice.

The inhibitors of the invention can oe targeted to specific locations where vascularization occurs by using targeting ligands. For example, to focus the compounds to a tumor, the inhibitor is conjugated to an antibody or fragment thereof which is immunoreactive with a tumor marker as is generally understood in the preparation of immunotoxins in general. The targeting ligand can also be a ligand suitable for a receptor which is present on the tumor. Any targeting ligand which specifically reacts with a marker for the intended target tissue can be-used. Methods for coupling the compounds to the targeting ligand are well known and are similar to those described below for coupling to carrier. The conjugates are formulated and administered as described above.

15 For localized conditions, topical administration is preferred. For example, to treat diabetes-induced retinopathy or neovascular glaucomas, direct application to the affected eye may employ a formulation as eyedrops or ointment or gel or aerosol. For this treatment, the compounds of the invention can also be formulated as gels or ointments, or can be incorporated into collagen or a hydrophilic polymer shield. The materials can also be inserted as a contact lens or reservoir or as a subconjunctival formulation.

'n all of the foregoing, of course, the compounds of the invention can be administered alone or as mixtures, and the compositions may further include additional drugs or excipients as appropriate for the indication.

Conditions that benefit from angiogenesis inhibition thus include, generally, cancer, including angiosarcoma, Kaposi's sarcoma, glioblastoma multiforme, hemangioblastoma, including von Hippel-Lindan disease and hemangiopericytoma: eye conditions, such as diabetic retinopathy and neovascular glaucoma; immune system conditions, such as rheumatoid arthritis. anglolymphoid S hyperp!asia with eosinophiiia; and skin conditions, such as cavernous herrangioma (including Kasabach-Merritt syndrome) and psoriasis. S The following examples are intended to illustrate but not to limit the invention. .t 30 8 m* *5i I'm .m dl m Ma il SQ f I Thes exaple desrib theprearation of ceftain compounds of the invetn and their activity in inhibiting mamm~fiafl metalloproteases.

In the examples below, TLC solvent systems are as follows: (A)ithyl acetate/methanol ethyl acetatemethalol ethyl acetate; (D) ethyl acetateimethanlol ethyl acetatelhexane chloroiortrt methanollacetic acid chlorofornmlethafloVacetic acid (85:10:1).

Preparation of N- D. L.

2 -,sobutylVl.3NhydroXncar p vlam2idol .roo Ioyl- typoab nk~hYI lhi A suspension of 5 g (0.033 mol) of the sodium salt of 4-methyl-2- oxopentanoi acid and 5.65 g (0.033 mol) of benzyl bromide in 10 ml -of anhydrous dimethylformamide was stirred for 4 days at room temperature. After evaporation of the solvent under reduced pressure the residue was diluted to 100 ml with hexane and washed with water (3 x 20 ml) and saturated sodium chloride and dried over anhydrous magnesium sulfate. Evaporaton of solvent gave 6.4 g (88% yield) of the benzyl ester of 4-rnethyl-2-oxopefltaloic acid (1 as a colorless oil.

A mixture of 6.4 g (0.029 mol) of (1 and 9.7 g (0.029 mol) of methyl- (tripheflylPhosphoranyhidene)acetate in 100 mL of dry methylene chloride was S stirred for 12 hr at room temperature and evaporated to dryness. The residute was extracted with hexane (3 x 50 rnL). The hexane solution was washed with sodium bicarbonate (2 x 30 rnL, water andl saturated sodium chloride and dried over anhydrous magnesium sulfate. Evaporation of the solvent gave 8,01 g (1000% ::yield) of benzyl 2 -isobutyl-3-(metioxytcarbonyl).propictiate as a mixture E and Z isomers.

A mixture of fl.01 (0.029 mol) of ()and 1 g of 10% palladium on ca rbon in *mL of methanol was hydrogenated at room temperature under 4 atmospheres of hydrogen gas for 8 hr. After removal of the catalyst by filtration the filtrate was evapoa todyesudrrduced pressure to give 4.7.g (86% yield) of 2 -isobutyb,3-(methoxycarbonyl)-propionic acid 0~ as a colorless oil.

To a-mixture of 0.65 g (4.5 mrnol) of, a? nd 0.57 g (4,5 mmol) of 'oxalyl chloride in 10 niL oDf dry methylene chloride 0.1 niL of anhydrous dimethylfcrrnamfide was 31777 added. After stirring for I hr at room temperature the solverit was evaporated unkder reduced Pressure and the residua was diluted to 5 mL with arnhydrous dimethylformamide and 1.06 g (4.1 rnrol) of the hydrochloride salt et L-tryptophan methylamide (Kortylewicz and Galardy, J Med Ch=in (1990) 2263-273) was 5 added followed by addition of 1.3 rnL (9.3 mmol) of triethylamifle at -100 C. This was stirred for 7 hr at room, temperature and evaporated to dryness at room tempnwature under reduced pitessure. Tie residue was diluted to 150 mL with ethyl acetae and washed with water (2 x 15 mL), 10% potassium bisulfate (5 x 20 mL), 10% -todiurn bicarbonate (2 x 20 rnL, saturated sodium chloride and dried over anhydrous magnesiu~m sulfate and then evaporated to give 1.6 g (83% yield) of L-P-isabutyl-3- (rnethoxycarbofy)-propartoyi]-.&tryptophal methylamnide as a.

mixture of diastereomers, 4A and 4k.

Isomers 4A and AB were separated by flash chromatography (Sqc el, ethyl acetate).

15Isomer 4A, mp=1 34-1370 C. RI(C)=0.3 7 *Isomer 457 mp=1 56-1 -r8 0 C. 0.2.

Alternatively, the mixture of AA and _4 was conveeted difecity to its tz~roxamate as described below- In this case, 5A was crystallized from the mixture 0A jA and M.

A warm mixture of 0.22 g (3.96 rnrnol) of potassium hydroxide in 1.rn.L Uf 20 methanol was added to a warm mixture of 0.184 g (2.65 minol) of the -ydrochlo ride salt of hydroxyl-amine. After cooling in ice- under an argon atmosphere tkhe potassium chloride was filtered off and 0.5 g (1.32 mimol) of (AN *-added to the filtrate. The resulting!mixture was stirred for 7 hr at room tempert'ttie and evaporated to. dryness under reduced pressure. The residue was supeded kl 100 mL of ethyl acetate and washed with 10 mL of 101% potassiujm 1 iuji-ae auae sodium chloride and dried over anhydrous magnesium S111fate ane. evaporated to dryness under reduced pressure. The residue was crystafliaed frts. ethyl 3ctate to give 0.28 g (56% yield) of pure 5 A.

Isomer 4k was converted to its corresponding hydroxamric acid 5k (72% yieldl as described !or 4A:y mp=176-1820 C. KD)7-0.46.

Isomer 0: mp=1l 57-1620 C. R 1 (D)=0.39.

32

WIN

For the case wherein toe 4/ mixture is used, the ~Acan be crystallized directly from the ressidue As described above.

In a similar inawer to that set forth above, but substituting for 4-methyb-2-oxopentanoic acid, 2-oxopentarioic acid, 3-methyi-2-oxobutyric acid.

6 2-oxohexanoic acid, 5-rriethyl-2-oxohexanoic acid, or 2-decanoic acid, the corresponding compounds of formula 1 are prepared wherein R' is H and R2 is an n-propyl, i-propyl, n-butyl, 2-niethylbuty, and n-octyl, respectively. In addition, following the procedures set forth hereinabove in Example 1, but omitting'the step of hydrogenating the intermediate obtained by the Wittig reaction, the corresponding compounds of formula 2 wherein RI is H and R2 is as set forth above are obtained- To synthesize the comnpounds containing acylated forms of the indolyl residue, the intermediate ester of formula 3 or 4 is deesterified and acylated prior to conversion to the hydroxamate. For illustration. 4A is deester iied with sodium hydroxide in ethanol and then acidified to give N-(L.2-isobutyl.3- Is carboxyp-ropanoyl)-L-tryptophan methylamide. which is treated with the anhydride of an alkyl (1-4C) carboxylic acid to obtain N-(L-2-isobutyl- 3-carboxvpropanoyl)-L-((N-acylindolyl)-tryptophan methylamide. This intermediate is then treated with oxalyl chloride followed by hydroxytamine at low temperature to *give the corresponding hydroxAmate.

Examlole 2 Prevaration of N-12-isobulyl- -(N-hydrovcarbon rlarniqIo)- prolanoyll-Q-trytoghan methylamide 7t T he mixture of the two diastereoisomers of N.{2-isobutyl-3-(methoxycarbonyi)-proparnoytj-D-tryptophan methyl amide 5A was prepared as described for 4ABf in Examptl-1 The mixture was crystallized from ethyl aceiate to give. after two recrystallizations, 0.26 g of the pure diastereomer r mp 155-1570 C.

R- (C)=O0.32. was converted into its hydroxamic acid 78 by the method described in Example Itin 50% yield (119 mg): mp 157-1590 C, RrD0..

3~3 Preparation of N-j2-iSobu!Yl-3-(N-hydOxvamidOabfnf propanoWL-N-methyl-LA-tryphafl methyfam2ide-(9A The reaction of N-methyl-L-tryptophlaflmethylamide, prepared as described in Example I for L-tiy ptpa-ehlmd.wt performed as described for 4 gave crude N-[D,L-2-isobutyl-3-(methoxyCarbofyl)-propaloyi- N-methyl-L-tryptophan methylamide 8A.B which was crystallized from ethyl acetate to give 76 mg (19% yield) of A: mp 171-1740 C. PFj(C)=OA0.

BA was converted into A by the method described in Example 1 in 45% yield (34 mg): mp 180-1 830 C, Rt(D)=-0.54.

Examrzle 4 Preparation of N-[2isobutyl-3-(N-hydroxyamidOcarboflyl)- N.[DL~isobutlK l-(m 3-hO-yabhflyl)-Paflne -L-rnfahhl)aalfe1Q was prepared as described in Example 1 from L-3-(2-naphthyl)-alan ine methylamide and 2. The crude product was chromatographed on 60 g of silica, gel in ethyl acetate-.hexane 1,1 to yield 12.mg yield) of 410A: mp 151-1580 C.

Rt(C)=O0.69.

*~21 OQA was converted into the hydroxamate I !A as in Example I in 30% yield (3 mg): mp, 179-13 10GC, Rf(DO.17- MS-FAB 400 Example Preparation of N-2-isobuty!-3-1N-h:droxyamid-,cadSI'bl- propanoyi-IZ-trytoohan 2-hvdroxvathvlamide (1 3A The hydrochloride salt of L-try ptophan-2- hydroxy-ethylamide was prepared and coupled with I as described for ihe hydrochloride salt of L-tryptophan mnelylamide in Example 1. except that was activated with 1.1 -carbonytdiimidazole fo minutes in methylerie chloride at roomn temrperature. The crude product was a mixture of 0.7 g (67% yield) of the dias'ereoisorners jgg Ri(C) 12A 0.38, RA(C) 1:2A crystallized from ethyl acetate in 35% yield (0.18 mrP 161-1630 C.

RI(C)=0.3 8 1 2A was converted into N-2iouy--N-yrxaioab~y) propenoyt].L-tryptaphafl 2-hydroxyethylamide 13A as in Examle1i3%yid(2 mp 162-1630 C. MS-FAB (rn.Iz) 419 (Mt- Preparation of N.2isobu t-l3IN'hro~yamidmrb)d)-l or qalyl1L~tYt0a _amyamide Sp to The hydrochloride salt of L-tryptophafl amylam~ide was prepared as described in Example 1 for L-tryptophafl methylamide and was reacted with that had been activated with 1,1 Sarbnydimidazole for 20 minutes in dichlorornetharie at room ternperature- The mixture of the two ditastereom~erS of N-DL2iouy3(itoyaro )ooaoiLtytpa arnylamide I 'A L (90% yield) was converted to its corresponding hydroxamic acids as described for 4A. Slow evaporatIon of the ethyl acetate solution gave 0-343 g (71 ot 15A.13: mp 160-16300C_ MS-FAB 445

H)_

Examole 7 Preparation of N-2iou13 -vrocmdc~gjyL oroan~ll-LtrV~O~ha oieridinarride f E L-tryptophafl piperidiramide was reacted with a as pe-rformed in Example 1 for p 4 1-tryptophan methylamide to give 1.14 g (89% yield) of N -(D 4 2 5 Qbutyl.34methoxycarbony1)-proPaoy.Ltryptophan piperidinamide 16A& as a foam; Rg{C) (ij) OJ4. (165) 0.67.

13A was converted into crude i 7A identically to !A iEamle 1 in 88% yield (570 Rt(b) C17A) 0.41. C178) 0.30. Crude 17&B was chromatographed On 180 g Of Splica gel in. 120A isopropaflol in ethyl acetate to give 140 mg (25% yield) of 1 a. after crystalUzatioi from ethyl acette rpI6.700 MS,;FAB 4 Preparatign of N~f~sb _rqa o n-tvoopa dodeovlamide (19A)l The reaction of L-tryptophan dodecylamide was prepared in a manner analogous to that described for L-tryptophan methylamide in Example 1- This ester was reacted with 2 as described in Example 1 to give crude -sbtl3 ehxcront-rpnlLtytpa dodecylamide I 8A. B in 930/ yield as a mixture of isomers 1 8A and 1 8B. This mixture was chrornatographed on 15G g of silica gel in ethyl acetate'-hexafle, 1:2, to yield 0.62 g of the mixture of the two isomers:

W

1 E) 18A 0.37. Rt(E) 18B 0-29.

Crystallization by slow evaporation frc- a ethyl acetate gave 0-38 g of 18A contaminated by approximately 10% of 18B by TLC and NMR analysis: mp 1335C- 18A was converted to its corresponding hydroxaomic acid as described in Example 1. except that the potassium salt of 1 9A crystallized from the alkaline eaction mrixture in 81% yield (222 The potassium salt of I!A (54 mg) was dissolved in 2 mL of boiling methanol. a few drops of water were added, and the solution was acidified to pH 6 with 0.1 N hydrochloric acid and diluted with water to give 50 mg (100% yield) of 1-9A-- mp 155-1590 C, MS-FAB (mlvz) 543 (M,

H).

16 Preparation-of N42-obutyF-f-damidocatbflvloruoaI1oyl1L-trVtopthaf (S -methvlbenazyamide (21 MF The reaction of L-tryptophan (S)-methyibenzyiarnide with 3- was performed as described in Example 1 to give, after crystallizatio from ethyl acetate, 330 mg (5 1% yield) of N-[2-isobutyt-3-(methoxycarbonyl)-propanoyll L-tryptophan (S)-mnethylbenzylamide 2 A; mp 160-1620 C, RdC)=07 was converted into hydroxamate 21 A by the identical method used in Example 1 in 38% yield (76 mg): mp 165-1660. P 4 MS-FAB (mlz) 479 Exampole ~Preartion of N-fL-2-isobuty-34N -hydroxvan1idocarbonyl)- Qfgovaovl- ~L-trytoghanf& -nyiethqxvcarbonyI-1 -amninohe v~mide 127Al ~15To prepare I-amino-6 -phenylmethoxycarbonyiamiro-hexa.-e C21), an *equimolar mixture (0.01 moo) of 1,6-dianiinohexane and benzaldehyde in 25 mL of methylene chloride was stirred for 5 hr in the presence of 1.5 g of anhydrous magnesium sulfate at room temperature. After removing the drying agent -by filtration the filtrate was evaporated to dryness under reduced pressure to give 2 9 :20(100% yield) of crude 1-ario-6-phenyla-mino-hexane 22 as a colorless oi; NMR(CDCb) 1.1 1.9(m. IOHI hexae NH 2 2.6(m. 2H, CH-1):j 3.51(m, 2H-, hexane CHz-6): 71-7.8 (in 5H. aromatic); 8_16,1s, 1H. fmine CH'. To a mixture of 2 g (0.L'1 ,Ttot of 2a and mL (101 mol) of triethyiamine ip 20 ML of dichioromnethanea. Then I 3a~ g (6.01 moll of benzybchloroformate was added dropwise at -50 C. The resultting rinixtur~ was stirred fot 0.5 hr at 00 C and for 2 h r at room temperature then, diuted to,53 rnL. with mnethylene chloride and washed with water (20 ml). 2% sodium bicarbonte, (20 ml). water and saturated sodium chloride and dried over anh drous magnecs.n suffate, Aftser evaporation of solvent under reduced pressure the residue ss is~cved in- S nizL of ethanol and 10 ml- of 2N hydrochloric acid was addad. The restiting mi,,ture was stirred fr6h tro ternperature then. evaporated to dryness under reduced pre sure. The residue was diluted to 50 mL with water and washed with ethyt ether (2 x 15S ml), The water 37 phase was evaporated under red,.ed pressure and the product 2a was purified by crystallization from a small portion of water with a yield of 42%; mp 175-1780 C.

To prepare the dipeptide analog (N-(L-2-isobutyl-3- methoxycabonyl)propanoyl-L-tryptophan for derivatization to 23: To a mixture of 1.754 g (9.32 mmol) of 2-isobutyl-3-methoxycarbonylpropionic acid 3 in 4 mL of 50% anhydrous DMF in methylene chloride 1.66 g (10.2 mmol) of N,N'-carbonyl-diimidazole (CDI) was added at room temperature. After 15 minutes of stirring at room temperature, 3.08 g (9.31 mmol) of the hydrochloride salt of L-tryptophan benzyl ester was added.

The resulting mixture was stirred overnight at room temperature, then diluted to mL with ethyl acetate and washed with 5% sodium bicarbonate (2 k 15 ml), water (2 x 15 ml), saturated sodium chloride solution and dried over magnesium sulfate.

Evaporation of the solvent under reduced pressure gave 4.32 g (100% yield) of 24, the benzyl ester of 25 as a colorless foam, which was used in the next step without further purification.

Hydrogen gas was bubbled through a mixture of 4.32 g (9.31 mmol) of 24 and S 0.5 g of 10% palladium on carbon in 15 mL of methanot for 2 hr while methanol was added to keep the volume of the reaction mixture constant. The catalyst was filtered S off and washed with a fresh portion of methanol (15 ml) and the filtrate was evaporated to dryness under reduced pressure. Evaporation of the solvent under 20 reduced pressure and drying of the residue in vacuo gave 3.08 g (88% yield) of acid AB as a mixture of two diastereoisomers, In the form of a colorless glassy solid.

This was separated to give isomers 2A and 25 by flash chromatography (silica gel: ethyl acetate; Ri(25A)=0.24, The compound 25A was converted to N-[L-2-isobutyl-3- methoxycarbonylpropanoyl]-L-tryptophan(carbonylaminohexyl)amide as follows. A mixture of 0.55 g (1.47 mmol) of 25A and 0.24 g (1,48 mmol) of CDI in 1 mL of 2% dimethylformamide in methylene chloride was stirred for 0.5 hr at room temperature and 0,42 g (1.47 mmol) of 23 was added. After stirring overnight at room temperature, the mixture was diluted to 50 mL with chloroform and washed with 2% potassium bisulfate (2 x 10 mi), water (10 mi), 5% sodium bicarbonate (2 x 10 ml), water (2 x 10 ml) and saturated sodium chloride and dried over anhydrous magnesium sulfate. Evaporation of the solvent under reduced pressure gave 0.8 g 38 of the crude ~Awhich was purified by flash chromatography (silica gel; ethyl *acetatefhexane 25:5): Yield 56%; Rt(E)=0.57.

When the product 2aA is substituted for 4A in Example 1. the idipptical process afforded the title compound A9ZA, melting at 102.10800C, in 46% yield', R,(D)=0.63.

Pr~paration of N-fL-2-IsobutyI-3-(N'-hvdr6Xyam idocarbonyl)loroganoyll-L-trvptgotign cyclohexylamide (26A)I When cyclohexylamine is substituted for 22 in Example 10, the identical process afforded the title compound 23A melting at 199-20300C, ir' 49% yield; Rf 1.

Pren~aration of N-U±Wois-2-(N 4 -hvdroxvamidocarbonvl)- S cyclohexylcarbonylj.L-typtooan methylaMide (29A.B) A mixture of 2 g (0.013 mol) of cls-1,2-cyciohexane-dicarboxylic anhydride in mL of methanol was refluxed for 5 hr, then evaporated to dryness under reduced pressure to give 2.41 g (100% %field) of cis-2-methoxy-carbonylcyclohexanecarboxylic acid. When this was substituted forj in Example 1, the identical process Afforded the title compound, melting at 140-14400C, in 36% yield; Rr(D)=0.53, 0.47.

Preparation of N-t(±)trans-2-(N'.hydroXyamidocarbonylwcvcohexylCatbonyj4 L-twytoohan methylamide (3A&B 2 'Mhen trans- 1.2-cyclohexanedicarboxylic anhydride was substituted for cis-1.2-cyclohexane-dicarboxylic anhydride In Example 12, the identical process afforded the title compound 30.B melIting At 167-1740 C, in 37% yield;, R,(D)=0.57.

39 Prepration of N1 2 is bt I- .N.h droxygmidogsarbony oonyl-titOhf a1A was prepared from 2,A in Example 10 in a similar manner to the preparation of 5A in Example 1 in 75% yield (128 mg) and isolated as a foamn from ethyl acetate: RI(F)=0.55, MS-FAB (mlz) (Ma- A small sample of 31 A recrystallized from ethyl acetate had a melting point of 116.1200

C.

110 Peaair ofN(.2.sbutyl.3-carboxYDrQ~o~aol-Ljrooh n (aminohexyl-1 1mird 1.9A') A mixture of 0.5 g (8.24 mmol) of M~ in 0.4 mL of 2N potassium hydroxide in methanol was stirred overnlight at room temperature. then evaporated to dryness under reduced pressure. The residue was diluted to 15 mL with water and acidified -T:4S to PH 2 with 1iN hydrochloric acid. The crude free acid of 26A was taken up with ethyl acetate (3 x 15 ml) and the organic phase was dried over anhydrous magnesium sulfate and evaporated to dryness to give 0.459g(92% Yield) of M~ as a colorless foam.

Hydrogen gas was bubbled through a mixture of 0.295 g (6.6 mmol) of the free acid of 2_eA in 15 mL of methanol for 2 hr, in the presence of 0.129g of palladium on carbon at room temperature. The catalyst was filtered off, washed with ethanol, (2 x 20 ml) and the filtrate was evaporated to dryness under reduced pressure to give 0.3 g (92% yield) of the title compound ZRA as a colorless foam; RI)0.08.

ExamRle 16 -The reaction of L-tryptophanyt-glycine me thyl ester with acid a.per'omda described for 2MA gave crude N-[N-(D,U-2-isobuty13mehoxycaboypoa~i--rpohnt-lcn methyl ester U in 87%yeld as a mitr fdiastereomerS 33A and 33.Isomers,=? and were separated by flash chiromatography (silica gel; ethyl acetate). IsomeraM mp =r 15-1550 C; Rj(C) =0.46.

Esters 33. were transformed to free acids 34. by saponificetion with two equivalent of methanolic potassium hydroxide, as described for 25A. Isomer 24A 6 yield 92%; mp 96-10200; RI(G) 0.31.

Isomer 20 yield 93%; rnp 99-10Q50 C; Rj(G) 0.25.

Preocralion of N-f(+)cis-2-carboXv~ycly lcarbonyll.

L-ty1popn mnethylamide To a mixture of 0.281 g (1.82 mmol) of cis- 1,2-cyclohexanedicarboxylic anhydride and 0.47 g of the hydrochloride salt of L-Trp-NHMe in 0.5 mL of dimethylformamide 0.51 mL of triethylamine was added at room temperature. After 2 hr of stirring the resulting mixture was diluted to 10 mL with water and 25 mL of 45 ethyl acetate was added. The resulting mixture was acidified to pH 2 with potassium bisulfate and the organic phase was washed with water (2 x 15 ml), *saturated sodium chloride and dried over anhydrous magnesium sulfate and evaporated to dryness. The title compound M was purified by crystallization from an ethyl, acetate-hexane mixture. Yield 48%; mp =105-1 120 C; Rf(G) =0,65, 0.6 1.

Preparation of-r(tsn I l .L-tryrotophgn mehylam do When trans- 1,2-cyclohexanedicarboxylic anhydride is substituted for cis- 1,2-cyclohexanedicarboxlic anhydride in Example 17, the identical process afforded the title compound N~ in 56% yield: mp =167-1740 C; Rf(G) =0.67, 0.61.

41 Pregaration of N-f2-lsobu-tvl-3-(N'-acetoxyamldocrbonvLl~ropnoI1I-L-IEV12ophan methvlamide (37A) -z To 97.5 mg (0.25 mmol) of 56 (Example 1) in 0.5 mi of dimethyl-formam ide was added 25.5 mng (0.25 mmol) of acetic anhydride and 37 mg (0.25 mmoi) of I ,8.diazabicyclo[5.4.Ol-undec-7-ene (DBU) at room temperature. After standing overnight, the DMF was evaporated under high vacuum and the residue taken up in a mixture of equal volumes of ethyl acetate and 2% potassium bisulfate. 'The ethyl acetate layer was washed with 2% potassium bisulfate, water, and brine, dried over magnesium sulfate, and evaporated to give a solid. The solid was dissolved in a 1:1 mixture of hot ethyl acetate: hexane, which upon standing at room temperature gave 71 mg (66% yield) of solid product ZA: mp=1 84-1 860 C; Rf(G)=-0.68.

:15 Pregaration of N4-12-Isoblutvl-3-(N-benzoxvamidocarbonfl)oprooanoyll-L-trytophan methylamide 131A) To 30.5 mg (0,25 mmol) of benzoic acid in 1 mi of tetrahydrofuran was added 40.5 mg (0.25 mmol) of cartboryl-diirnidazole. After 10 minutes, 97 mg (0.25. mmol) of compound MA from Example 1 was added in 1 ml of dimethylforrnamide. After .1:20 minutes, the reaction mixture was evaporated to, dryness under high vacuum, and dissolved in a mixture of equal volumes of ethyl acetate and water. The ethyl acetate layer was washed with 5% sodium bicarbonate, water, 2% sodium bisulf ate, water, and brine, and dried over magnesium sulfate. Evaporation of the ethyl acetate layer to a small volume gave 50 mg (41 of the title (,ompound, 3A mp=1 87,167.50CG- Fr(G)=0.54.

Example 21 Preraration of N-(2R-2-Carboxyethyl-4-metlhyloentanoyQl L-trygtonhan (S)-methylbenzyl am.d (3A) N-tsoc-L-Tryptophan (10.04 g, 32.99 mmole) was dissolved in tetrahydroturan (100 ml) and carbonyldiimidazole (5.35 g, 32.99 mmole) was -added. After stirring for I hour at room temperature. (S)-methylbenzylamlne (4.25 mi. 32:99 mmole) was added., The reaction was stirred at room temperature for 16 hours after which the *42ii solvent waS removed and the residue waE dissolved in ethyl acetate (100 MI). The resulting mixture was washed with 0.1 N HCt (3 X 60 ml), saturated aqueous sodium bicarbonate (2 x 50 and brine (2 X 20 ml). After drying over anhydrous magnesium sulfate, the solutionl Was filtered and concentrated to dryness to give Boc.L-tryptophafl (S)-l.pheflethyl amide N-tert-butyl carbamate (4D, 13.78 g, 100% yield).

The compound 4Q (2.00 9, 4.91 mmole) was dissolved in methanol (40 mL) and 6N aqueous hydrochloric acid (20 mL) was added. The reaction was stirred at room temperature for 2 hours and concentrated to dryness to provide L-tryptophafl

I

phenethyl amide hydrochloride salt (41, 1.69 g, 95% yield) as a colbrless solidthe compound 4.1 was coupled with MA (see Examples 23 and 24 for the synthesis and structure ef W) in the presence of carbonlyl diimidazole as described above to yield N-2--ehx-abnlehi4-ehletny)Ltytpa .(S)-methylbelzyI amide (42-A).

The compound 42A (10.04 g. 21.05 mmoles) was dissolved in tetrahydrofuran (200mL),6N hdrohloric acid (200 was added to the solution and the reaction was stirred at room temperature ior i1 hurs after which it was washed with, chorfrm (3 X 200 mL) Th -t-rif~ organic extracts were'wahdih0.N sodium hydro~ide (3 X 150 mQ-. Tbe hlorofoirm layer was concentrated to 150 mL .20 nd ashed again with 0.IN sodium hydroxidq (X50m).Te combined aqueous extracts were acidified to pH =2 with concentrated hydrochloric -acid and washed with ethylacetata (3 X 50 mQ-. Mhe combined ethyl acetate extracts were didovr ahyr'5ageimsulf ate, filtered and concentrated to dryness. The residue is recrysalized from ethylacetate (75 mQ- to yield -ab~iehl--~typnaoy)Ltytpa (S)-methylbelzyl amide C(9A1 After f Rtration and concentration~ of the mother liquor, a second crop of was obtained by recrystlliatiof from dichloramnethafle V75 ml-) and the combined crops of MA (3.72 g, 38%) were dried under vacuum.

it !s n-oteworthy that Compound M2 was recovered from the chloroform layer, which was dried over anhydrouJs magnesium sulfate. filtered, and concenitated.

The residue was purifted on. silica gel (50% ethylacetateftexane) to give recovefed 42A (2.49,25%).

43 methyipentaly'-LtYtnhR(3 L-Tryptophafl (1.22 g. 6.0 ramole) was dissolved in water (5.00 mL) and 6N saqueous sodium hydroxide (0.90 mL). The resulting solution was cooled to 00 C.

D.L-2.lsobutyl-3-(methoxycarbonl)propionic acid Q~ 0.94 a. 5.0 mmole) was dissolved in dichlorornethane (3.00 mL) and oxaly chloride (0.44 mL, 5.0 mmole) was added. The resulting solution was cooled to 0G C and added to the L-tryptophari solution over 1 hour. During the addition, the reaction was vigorously stirred and the pH was maintained above 9 by the periodic additioni of 100 jil portions of 6N aqueous sodium hydroxide. After the addition, the reaction was stirred at room temperature overnight and subsequently diluted with water and ethyl acetate. After acidifying the reaction to pH- 2 With 10% aqueous potassium bisulfite.

the aqueous layer was removed and the ethyl acetate layer was washed with 2% potassium bisulfite (2 x 10 ml), water (2 x 10 ml), and brine. The organic phase was dried over magnesium sulfate, filtered, and concentrated. Purification of the residue on silica gel (chloroform then chlo-roform! methanol) gave (0.28 g,29% recovered P.yield) and A,1 (1.11 g, 60% yield).

It is noteworthy that compound Aa can be converted to 4.ZA by coupling with S.

qO methylbenzylamine and the resultant product can be resolved by crystallizing from ZJethanol. 39A can be produced from compound!L2.A by hydrolysis as described above in Example 21.

Prebaration of D.-k1j2Soytl34thmoXarbo Y,1-, This method describes an improved method for the synltsis A D,--sbtl;-mloxcr~nl-rpoi acid GI-Mmleic af hy-nCPd. O*g 7.65 motes) was dissolved in hot toluene (500 mL), fittered, and alkwved Ic~ Mzuc 's producing a finely divided crystalline precipitate.ThreJingmxueashr* coated in: a refrigerator- 2-MethylproOenrd was wondensed ito nold toluene (I Qt.

The cold malec anhydride suspension was teansfs.rred, weith the aidofdiir> -44 toluene (300 rnL) to the glass liner of a Parr 2 gallon stirred autoclave. The 2-methyipropene solution was then added followed by 4-tert-butyl catechiol (2 g).

The autoclave was sealed and heated, with stirring, to 1700 C over 45 minutes arnd maintained at that temperature for an additional.9.5 hours. The reaction was then allowed to cool to 600 C over 7 hours after which. the autoclave was vented. After an additional 1.5 hours, the internal temperature had reached 4500C and the autoclave was opened. The solvent was removed and the residue was distilled under aspirator pressure through a 10 Inch Vacuum jacketed Vigreux column fitted with a partial take off head to give unreacted maleic anhydride (139.4 g, 18.6% yield) and fl-methallylsuccinic anhydride (765.3 g. 79.8% yield).

1-Methallysuccinic anhydride (539 g. 3.50 mole) w~s dissolved-in toluene (800 mL) in a 1700 mL Parr stainless steel bottle fitted with a heating jacket. 10% PdIC (3.60 g) was added and the bottle was connected to a Parr 2 L shaker fitted with two,' 4 L hydrogen tanks, After several evacuations and risfilling with hydrogen, the .11i shaker was started with the heater controlled for 6000C. The initial pressure wvas When the pressure fell to 10 lbs. the tanks were, repressurized to 60 lbs.. After two hours, the controller was reset to 7000 and the reaction was continued for another 12 hours. The heat was then turned off arnd after another, 5 hours, the shaker was stopped. The reaction vessel was evacuated and the reactior- mixi'jre was filtered and concentrated. Distillation of the residue through the Vigreux column described above (P 9-10 mrm Hg, T 1800 C) gavo isobuty-succinic anhydride (5329 9, 97.6% yield).

Isobutylsuccinic anhydride (501 q4 3.21 mole) was dissolved in anhydrou methanol (500 mL) and allwed. to stand at room temperature 5 days. The methanol was removed and the residue was mixed with pe-roleum ellher (W00 rnL and refrigerated overnight. The solids were filtered anid the filtrate *was put in a freezer. After prolonged storage, the solids were filtered and the combi~ned -solids wererecystIlized from petroleum eCher to give the Corpound 375g 59 yield).

After concentrating th* com-bzned preIum ether filltraes fromn abova. 188 9 mole) of the crude inateiat (mosty onItng of (S3.rnathoxycarboS- etylhexanoic aCid was miked wi'th Sodium hydroxide (120 g. 3 moles) and water (700 mL). The mixture was stirred on a steam bath tor 14 hurs.The ixtue was fitered and ac"idified with concentrated sulfuric acid m)ad refrigerated. The suspension of sodium sulfate and thedidwsftrd an tesldweehruhlwahed with ethyl acetate (500 mL}. The aqueous solution was washed with ethyl acetate and the combined ethyl acetate phases were concentrated and dried under vacuum to give the diacid (162.8 9, 93.6% yield). The diacid was heated with acetic anhydride (1150 ml) under a 10 inch vact un jacketed Vigraux column. Acetic acid was removed over several hours at I1I8.12000C. -VWhen the temperature began to fall, the pressure was gradually lo reduced to remove the excess acetic anhydride. Isobutylsucciflic anhydride (138 9, 88.5% yield) was collected by distillation (P 10 mm Hg, T =1440 0) and recycled back into the final step of this preparation.

Exe yIgl 24 D, -sbtl3Cehoyabnl-fpoi acid CJ. 17.55 g, 93.35 mrnole) was dissolved in diethyl ether (85 ml) and S-methyl benzytlamine (13.24 mL.

102.69 rnmole) was added. The reaction was stirred at room temperature for 24 hours and the solids were removed by filtration. Recrysiallization oi the solids from ethanol/diethylether gave the sal 44A (9.24 g. 32% mass recovery, 64% Yield) as a single diastereotner.

A4A-(1.29 g.4.18 mmole) was dissolved in saturated aqueous sodium bicaronfate ana washed, three times. with ethyl acetate, The combined ethyl actt aerwee washed with saturated aqueous sodium bicarbonate and the combined aus~ layers were acidified itaueous 6N hydrochloric acid and washted three times with ethyl acetate. The combined ethyl acetate layers were washed with aqueous saturated sodium chlofide, dried over anhydrous magnesiurn suitate, filtered and concen~trated to give 3A (G.77 g. 98% yield. [ctJE)+19.S deg.).

46 Preparation of T2lsbt~3N4dOyriOa10ll A suspension of t-BOC-tryptophan (6 9) in dichloromethane (20 ml) was mixed with carbonyl diimidazole (3.26 g) and -stirred at room temperature for 20 minutes.

4-Morpholinyt-fl-propylamine (2.88 ml) was added to this mixture and this was stirred overnight at room temperature. que-nched with water (100 mrl). and extracted with ethyl acetate (300 ml). The organic layer was washed with brine. dried over sodium sulfate, filtered and concentrated to provide the crude morpholine amide

(J

The compound was dissolved in a solvent mixture containing dichloromethane (30 ml). trifluo-roacetic acid (30 ml) and anisole (6 and stirred at room temperature for 2 hours. The solution was mixed with hexane (60 ml) followed by diethylether (120 ml), the supernatent. was decanted and the residue was evaporated to dryness to yield the trifluoroacetic acid salt MAn.

A solution of 4-(t-butoxy)-2R-isobutyucCMii acid (0.46 g) (see European Patent Application No. 92301051.8, filed on July 2, 1992V'and carbonyl diimidazole (0.32 g) in dichloromethane (3 ml) was stirred at roam t emperatu re for 20 minutes, and added to a solution of 46 (1.18 g) arnd diisopropyrlethytamine- (0.74 ml) in dimethylforamide (3 ml). This mixture was stirred overnight at room temperature and extracted with ethylacetate (2 x 50 ml) and washed with sodium bicarbonate ml). The crude was purified on a slagecoun (dichloromethane: methanol.

9 5:5) to provide the t-butyl ester (47. 1.0 g).

The compound 47 was dissolved in dichloromethane (3 ral), trifluoroacetic acid (3 ml) and anisole (1.2 ml). stirred at room temperature for 2 hours and evaporated to dryness- The residue (0.2 g) was mixed with solid sodium bicarbonate (0.1 a).

dissolved in water (2 ml) and purified on a. C 18 column by a gradient of water to acetonitnie in water to provide the sodium, salt 4.

A. solution of the free acid of AD~ (0.1 g) in teirahydrofuran (1 ml) at '120 C was mixed with N-ethylmorphoinle (0.05 ml)j and isobutyichloroformate (0.046 mnl) and stirred for 30 minutes.,.This was added to a solution of Q..benzylhydroxyarnine hydrochloride saft (0,028 M) and N-ethylmor pholine (0.05 ml) in dimethytforamicie (I 47 ml) and stirred overnight gradually increasing the. temperature from -1ao C to ro~om temperature-. The reaction mixtureD was quenched with water -(20 ml). extracted with 0 ethyl acetate (50 Ml) arnd washed wit h sodium bicarbonate solution ml), dried.

arid evaporated. The crude mixure was purified on a silica gel column (dichloromethane: methanol, 9.1) to provide the 0-berizythydroxamide t'6g.

The comnpound A~lg) was dissolved in methanol (20 ml), 5% palladium on carbon (50 mg) was added and the reaction mixture was stirred under hydrogen atmosphere (balloon pressure) for 6 hrs. The mixture was diluted with methanol ml). fitered through celite and concentrated. The residue was crystallized with methanol and ethyl acetate to provide the Compound ~Q(640 mg)-.

Prenaration of N-2lgu o l proo Ltt IAiie (52) When 4-rnorpholinyl ethylamine is substituted for 4 -morpholinyl-n-propylamine in Example 25,' the identical process afforded the sodium salt (51 and the Preoaration of Nf 2 -sobu fy ftv rbonyp,.g 0 1 4N-1,-Boc-L-tryptophan (5.07g, 16-66 mmoles) was dissolved in tetrahydrofuran mL) and carbonyldiimidazole (2.70 g. 16.66 mmole) was added to the solution.

The reaction was stirred for 1 hour at rom temperature, 4 -aminomethyipyridine (1.69 m L. 16.66 mmole) was added and the reaetioi w-.as stirred at room temperature for 3 hours. The solvent was removed arnd the residue was thoroughly washed with water and dried under vacuum to provide N-t-Boc-L-ryptophn(4.

pyridyt -methylamide -4 (6-38g,~ 97% yieid).

The cornpoundi 4 (6-38 g. 16.19 imatle) was dissolved in 2N HCI, stirred at roorn tempera for 24 hours. and concentrated to drynes s to give L-tryptophan-(4.

Sof 4 (t-butoxy)-2R-isobutylsucinie acid (Vi.9g .16 immate) i 48 tetrahydrotur-an (20 mL.) was mixed wihcarbonyldiirnidazOle (0.84 g. 5.16 mmoles); the reaction was stirred at room temperature for 1 hour and 9 1.8g 5. 16 mmnofe) was added followed by triethylamine (1.44 mL., 10.32 mmole)- The raction was stirred at room temperature for 18 hours. diluted with ethyl acetate (50 mL). washed with saturated aqueous sodium bicarbonate (3X 10 ml-) and brine (10, mL). The organic phase was dried over anhydrous magnesium sulfate, filtered. concentrated, and-the res.1due -was recrystallized from ethyl acetatelhexane to give the t-butyl ester (lASo, 4dyield'.

The compound 56 (0.12 g, 0.24 mmole) was dissolved in dichloromethane (1 mL). trifluoroacetic acid (1 mL.) was added and the reaction was stirred at room temperature for 2.5hours and then concentrated to dryness. The residue was combined w-ah saturated aqueous sodium bicarbonate (5 ml) and eitracted with ethyl acetate (3 ml.. T-he ethyl acetate was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was recrytaallized from ethyl acetate (1 ml) to yield the-Compound (22.6 mg. 21% yield), Preparaioin of N-2Iou--irn royirpny -L-tygtoohan-tbenzylamide (57) M Wen benzyamine is substituted for 4 -rnorpholinyl..n-propytamine in Example 25. the identical process afforded the Compound 57.

Preva!!ation. of N--sobub-3-h dr orboniprpp ~~-L-tryotonhan-2(ohenylethvmie(8 When 2-phenyiethylarnine is substituted for 4 -morpholinyt-n-propylamine in Example 25. the identical process afforded the CompoundQ.

49.

Prep aration of N-12-ISobut-3-HYdro ,Oabon~roafOl When 4-(2-aminoethyl)benzeflesiUlfonamide is substitued for 4-morpholinyl-npropylamine in Example 25, the identical process afforded the corresponding carboxylit acid Q and t~he Compound Q Example al Preparation of N4DL--isobuyl-3-(ydoxycarboflVj oropanovll-L-trvphan methytamide (61) N-{DL2-isabutyl-3-(hydroxycroyib)lpr0paloyTL-ryptophan methylamide (4 g) was dissolved in letrahydrofuran- The solution was cooled in an ice-bath, and 6M HCI (200 ml) was added. The resulting mixture was stirred at 20-250 C for 16-18 hours. The reaction mixture was cooled in an ice-bath and 6N NaGiH was- added until a pH of 2 was attained. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (200 ml). The organic layers were combined, dried, fitered and concentrated to dryness to yield the Compound ELI '10.5 The crude product was purified by flash chromatography us"ing methanok ethyl acetate gradient at 5% and 10% to yield pure fzi (6-8 g)- Prep~artion of N4[D.L-2-tsotuty-3-(hydroxyQarbonyi)orooanoll-L.-trvatohafl methylamide (62'l N4DL-2-isobutyl.3-(hydroyarboflyt)-propalOYl-L-tryptophafl methylamide (4 25 20 g) was dissolved in methanol (300 ml) and lithium hydroxide 14.33 9) and water (100 ml) was added to the solution at room temperature- The reaction mixture was stirred at 20-250, C for 12-16 hours. neutrahzed with 1N HCl (100 mt. pH The excess methanol was re moved in vacuo and the aqueous layer was extracted with ethyl acetate (2 x 300 ml). The combined organic layers were dried over magnesium sulfate, fikered and concentrated to dryness to yield the title compound i~a (25.7 ).The crude product was recrystallized from acetonitrile (20 l) *to yield pure 62 (6.5 g).

Precaration of N jLnZisobutvI,3-(Y-,dM 0- oydc tnviprO;2ajcvr IryptO~han rnetljlamTide(5A M N- (2RS-2-Methoxycarb cnylmethyi-4 -Mnethyipentartoy)- L-tryptophan (4)was Prepared as described ru' nExample 22- The compound 4C was coupled with niethylam the in tetrahy:ioiuran at room temperature. overnight, using car-KoryT&iidazc o yId a of mixture of 4A and 4B.

Thre mixture of aA and 4B was converted directly to its hydroxarnm acid as descnbe 1 in Exam pie I above. The compound 5A was crystallized from the mixture t0o sAand Base hydrolysis of the mother liquor containing L on neutralization to pH S, 1 foloe by arilepotii >..ctalline L-tryptophan which is recycledf!i use in the synthesis of E~xample 34 Preparation of N-rR-2-lsobutvl-34N-hVdroxyabwl~amido)- 4.penyt-b2utanoflL-typoohal-Tethytamide A solution of 4-(t-butoxy)-2R-is ,obutyl succinic acild (2-3 g, 10 mmol) in tetrahydrofuran was added to a soiuition of lithium diisepropylamide in ~O tetrahydrofur-an'(25 M moll at -45.0 C. The mixture was gradually warmed to 00 C and stirred for 3 hours- Benzyl bromide (1-5 ml) was added and the mixture was stirred overnight at ro-cm temperature. The mixture was evaporated and the residue was partitioned 1b-etween dPIut6;adium ,bicartx'ate solution and ethyl acetate. The aqueous layer was acidified with 1 N hydrochloric acid and extracted with ethyl acetate (2 x 100 ml). The comnbined organic layers were washed With water (50 ml).

brine, dried and concentrated to yie-ld the benzyi derivative g).

Asolution of idimetylforrnamide (6 ml) was mixed with carbonyl7 diiridazole (0.24 g) and stirres al. rceom temp~erature for 30 min- L-t-ypophnmethyam hydrochlorde- (2-2 -J-ws added and the mixture was stire vrih at room temperaturc-. The rnxturew s iluted, wih hyl aeAte(0 l.wse with wte (5_l.floeby sodiun bicarbcoate solution and brine, dried and concentrated-. The cno-da was mted on a sili~e methanol) to yield the t-butyl ester f? (1.8 g, The t-butyl ester fiA was converted to the Compound according to the procedure described in Example Preparation of N4-R-2-lsobutyl-3.hydroxycarbonylrooanolI -L-tryptophan-3-(N'N'-dimethyfl-n-propylamlde When 3-N'N'-dimethylamino propylamine is substituted for 4-rnorpholinyl-npropyiamine in Example 25, the identical process afforded the Compound f.

Preparation of N-rR-2-lsobuty-3-(N-benzyloxyamid' roy prpnylttga methylamide (671 A suspension of 5A (4.19 g, 12.62 rnmol), benzyl bromide (1.8 ml, 12.62,mmol) 45 and potassium carbonate (2.6 g, 18.88 mmol) in dlmethyiformamide (20 ml) was stirred overnight at, room temperature. The mixture was poured into ethyl acetate (300 ml), the organic layer was washed with water (2 x 50 ml), brine, dried and concentrated. The crude was crystallized In ethyl acetate to yield the Compound 67 (3.7 g,61%) Example 37 Preparation of N-R 2lutyl-3-(N'-benzyl.N'-hydromy-aridocarboyl.

opropanoll-L-tcyDtophan methvlamide (69l A suspension of SA (800 mg. 2.06 mmol), benzyl bromide (0.7 ml) and potassium carbonate (1.1 g) in dirneihylformamide (5 ml) was stirred overnight at room temperature. The mixture was poured into ethyl acetate (100 ml), the organic layer was washed with water (2 x 50 ml), brine, dried and concentrated. The crude was purified on a silica gel column (2:1 ethyl acetate:hexane) to yield compound 68 (363 mg. the N,O-bis-benzyl derivative of faZ.

solution of compound (50 mg) and 5% palladium-on-carbon (5mg) ill methanol (5 ml) was stirred under an hydrogen atmosphere (balloont pressure) for hours. The mixture was filtered over celite, followed by purification on a silica gel 52 column (95:5 chloroform -methanol) to yield the Compound 6 (38 mg).

Preoaration of N-.R.2-.sobutvl-3-(N'--flucroohenvlmethyl- N' .hdroxaminocarbonvyl)-ropanoyl-L-t than methvyamide (71 A suspension of 67 (200 mg, 0.42 mmol), p-fluorobenzyl bromide (64 pl, 0.51 mmol) and potassium carbonate (87 mg) in dimethylformamide (1 ml) was stirred overnight at room temperature. The mixture was poured into ethyl acetate (30 ml), the organic layer was washed with water (2 x 10 ml), brine, dried and concentrated.

The crude was purified on a silica gel column (95:5 chloroform- met anol) to yield the benzyl derivative IZ (100 mg, 41%).

A solution of Compound ZQ (100 mg) and 5% palladium-on-carbon (10 mg) in methanol (5 ml) was stirred overnight under an hydrogen atmosphere (balloon pressure). The mixture was filtered over celite, followed by purification on a silica 15 gel column (95:5 chloroform -methanol) to yield the Compound Z1 (50 mg).

S .xample 39 Preoaration of N-rR-2-1sobutyl-3-(N'-methoxvmethvl-N'-hydroxvamidocarbonyl-orooanoyl]-L-trytophan methylamide (73) 2"20 A suspension of 7. (1 g, 2.1 mmol), iodomethyl pivalate (760 mg, 3 mmol) and potassium carbonate (430 mg) in dimethylformamide (5 mi) was stirred overnight at room temperature. Additional iodomethyl pivalate (760 mg) and potassium carbonate (430 mg) were added and the mixture was stirred overnight at room temperature. The mixture was poured into ethyl acetate (200 ml), the organic layer was washed with water (2 x 50 ml), brine, dried (Na 2

SO

4 and concentrated. The crudewas purified on a silica gel column (95:5 chloroform-methanol) to yield the ester 72 (300 mg, 24%).

A solution of Compound 22 (300 mg) and 5% palladium-on-carbon (100 mg) in methanol (10 ml) was stirred overnight under an hydrogen atmosphere (balloon pressure). The mixture was filtered over celite, followed by purification on a silica gel column (95:5 chloroform-methanol) to yield the Compound Z7 (60 mg).

S. 53 l53 Preparation of N-rR-2-lsobutlI-3-(N -Diva lylm ethyl-N'-hydroxyamldocarbonvll-grooanol1-L-tryotoohan methylamide 4 A solutibn of compound Z2 (300 mg) and 5% palladium-on-carbon (100 mg) In ethyl acetate (10 ml) was stirred overnight under an hydrogen atmosphere. The mixture was filtered over celite. followed by purification on a silica gel column (95:5 chloroform-methanol) to yield the Compound 74 (100 mg).

Example 41 Pregaration of N-{R-2-lsobutyl-3-(N'-carboxylmethyhNfiydroxv! amidocarbonyfl-prooanoyil-L-trvo~toohan methylamide (-76) A suspension of 6_7(180 mg) and potassium carbonate (100 mg) in dimethylformamide (3 ml) was stirred at room temperature for30 min. Bromoacetic acid berizyl ester (72 g1) was added arnd the mixture was stirred for 4 hours. The mixture i was poured into ethyl acetate (10 ml), the organic layer was washied with water (2 x 20 ml), brine, dried and concentrated. The crude was purified on a silica gel column (1 methanol in dichloromethane) to yield the ester Za (50 mg).

A solution of compound Za. (50 mg) and 5% palladium-on-carbon (S mg) In methanol (2 ml) was stirred under an hydrogen atmosphere for 2 hours, The mixture was filtered over celite, followed by purification on a silica gel column (95:5 chloroform -methanol) to yield the Compound M~ (18 mg).

Examgle 42 Preuaraton of Doc- D-gamm a-Hyd roxa midoglutamyl-L-t cypto phan- (SLmthylbenzylamide (Z9) BocD-glutamic acid gamma benzyl ester was coupled to L-tryptophan-Smethylbenzylamide using carbonyldiidazole in a mixture of dichlomethane and dimethylfom~namide. The reaction mixture was diluted with ethyl acetate and water and the orgainc phase was washed with sodium bicarbonate, potassium hydrogen sulfate, water and brine. Evaporation of the the ethyl actate layer gave the fully protected. intermediate Z1- Deprotection of the intermediate 2_2 by hydrogenolysis with palladium-on- IAl charcoal in methanol, followed by filtration oi the catalyst and evaporation yielded the carboxylic acid 78.

Hydroxaminolysis of the acid Zj. using potassium salt of the hy~oxylamirie hydrochloride in methanol gave the Compound 79.

P1reparation of Boc-D-beta-hydroxamidoaspartyl-l- -rooptharn-(S) methvlbenzylamide (82) Boc-D-aspartic acid gamma benzyl ester was coupled to L-tryptophan-(S)methylbenzylamide using carbonyldijimidazole in a mixture of dictilomethane and dimethylformamide. The reaction mixture was diluted with ethyl acetate and water and the orgaino phase was washed with sodium bicarbonate, potaisium hydrogen sulfate, water and brine. Evaporation of the the ethyl actale layer gave the fully protected intermediate B.

Deprotection of the intermediate 5-Q by hydrogenolysis with palladium-oncharcoal in methanol, followed by filtration of the catalyst and evaporation yielded the carboxylic acid El.

Hydroxaminolysis of the acid AL using potassium salt of the hydroxylamine.

hydrochloride in methanol gave the Compound 2.

Example 44 Pregaration of N-ID2.L-2-lsobutyl-3-(N'-h,.ydroxyamidoc Ltnyl) nrooanoyll-trygtophan methylamide L-tryptophan is reacted with phosgene 1o yield the N-carboxyanhydfide 25 derivative ~.Compound BI Is rbacted with methylamino (I equivalent) followed by.

treatmenL with hydrochloric acid to yield the salt 34.

Isobutyl succinic anhydride in tetrahydrofuran is treated at room temperature With L4, in the presence of a base, to yield a mixture of carboxylic acids 85 and B6.

These are esterified with methanot under mild conditions to yield the respective ester$ A avid 7.

The ix-tire of, 4 and _Z is conerd directly to its hydroxamate -as describcai W Example 1 above. The compound 5A is crystallized from the mixture of A and I IC4'44 Alternative'y the acids Il and Rfi can be reacted with acetic anhydride or other coupling agents to yield sucoinirnide derivative 89, which on further hydroxaminolysis gives 5A in high yield.

pe4 Applying the methods set forth above, the following invention compounds are synthesized:

HONHCOCH

2 CH(n-hexyl).GO-L-Trp-NHMe; HON HCOCH 2 CH(n-pentyl)-CO-L-Trp-NHMe: HON HCOCH 2 GH(i-pentyl).CO-L-Trp-Nie;

HQNHCOCH

2 CH(ethyl)-CO.L-Trp-NHMe, HONHOOCH2CH(ethyl).COL-Trp-NH-CH 2 CH3;

HONHCOCH

2 CH(ethyl)CO-L-Trp-NHCH 2

CH

2 0H;

HONHCOCH

2 CH(ethyl).COL-Trp-NHcycohexyl.

2i~sMeONHCOCH 2 CH(iBu)-CO..L-Trp-NHEt; EtONMeCOCH 2 CH(i~u)-CO-L-Trp-NHEt; MSONHCOCH2CH(iBu)-CO-L.A&(2.flSpithyI)-NHEI; EtONMeCOCH 2 CH(ilu).CO-L-Ala(2-naphthyl)-NHEt; ON -CHzCH(iBu).CO-L-Trp-NHt ~oEtCONO)H-OH 2 CH(iBu)-CO..L-Trp-NHEt; n-PTGONOEt-CH 2 CH(iBu)-CO.L-Trp-NHEt; EtNHCONOMe.CH 2 CH(i~u)-CO-L-Trp-NHEt, MeNHCONOH-CHaCH(iBu).CO-L-Trp..NHEt; EtONHCONMe-CH 2 CHi(iBu)-CO-L-Ala(2-naphthy)-NHEt, *64.2 EtCO NOH-CH 2 CH(iBu)..CO-L.Ala(2.naphthyl).NHEt; n-PrCONOEt.CH2CH(iBu)-CO.L-Ala(2-naphthyl).NH Et-: EtNHCONOMe-OH2CH(i~u).COL-Ala(2-naphthyl).NHr.t; MeNHCONOH.CH2CH(i~u)COL-Ala(2-naphthyl).wHEt;

HONHCONHCH

2 CH(i~u)-CO.L-TrpNHMe;

HONHCONH-CH

2

CH

2 CH(iBu).COL-TrpNHMe: HONHCONHCH(iBu)CQ-L.TrpNHMe, 56 *4

H

2 NCON(OH)CH(iBU)CLTrpNH N(OH)CH2 CH(i8u)COL-Tr1NHMe; H2NCON(OH)CH 2

CH

2 CH(iBu)CC>L-TrpNHMe-,

CH

3 CON(OH)CH(iBu)CO-L..TrpNHMe; s

CH-

3 GON(OH)CH 2 CH(iBu)COL-T;.pNHMe;

CH

3 CON(H)CH2

CH

2 CH(iBu)C-TpNH HNOHGOCH 2 CH(i-Bu)CO-L-TFp-.NHMS, HONHCOCH 2 CH (i-Bu)CO-LTrpNH('CH2)3-(4morphoinyI); HONHCOCH 2 CH(i-Bu)CO-L.TrpNH(C;H2)2-(4morphoiiyI);

HOOCCH

2 OH(i-Bu)COLTrpNHCH2Ph., HQOCCHaCH(i-Bu)COLTrpNHCH2CH(-4pyridyl)- HOOCCHZCH(I-Bu)COL-TrpNH(CH2)2Ph; HOOCCH-,CH(i-Bu)CO-L-TrpNH(CH2)2- (4-benzenesultoflamide); HOOCCHU.-Bu)CH2 CO-L-TIpNHMe; HOOCCH2CHO.-Bu)CO-L-TrpNHMe; HONHCOCH(i-Bu)CH 2 00.L-T(pNHMe; BflONHCOCH 2 CH(i-Bu)CO-L.TrpNH~e; ON(Bfl)COCH 2 CH(-Bu)COLTrpNHMe; HON(p-fluoro-Bf)OCHCH(iBu)COLTrpNHMe; BflON(CH 20

CH

3

)COCH

2 CH{i.8u)CO-LTrpNHMe; HKON(CH 2 OPiv)COCHaCH(iBu)COLTrpNHMe- 2 CH(i-8u)CO.L-TrpNHMe, HONHCOCH(Bn)CHQ.-Bu)CO-L-TrpNHMe; HOCHC~-uC-LTpHC23C3 HOOC(CH 2 2 CH(NHCOO-1-8u)CO-L-TrpNCH(CH3) Ph; HONHOC(CH 2 2 CH(NHCOOtBL)CO-L-TrNCH(CH3)Ph-, .HOOCCH 2 H(NHCOtBu)COLTrpNC()h and HONHOGCH 2 CH(NHCOOtu)CO.LTrpNCH(CH3)Ph.

57 Determination of thie inhibitory activity E.1 certain of the compounds preparch.- is conducted as described above, arnd provides the results shown in Taole 1 and Table 2.

9 U. 9~ *9t*

F

o0ad If Aiiia- mamis 0." 2 7A 3 9A 4 HIA 13A 6 15A 7 17A,B 8 19A 9 21A 27A 11 28A *tS 12 29A,B ,'i4p{OCOCH 2 CH(i.Bu)CO-L-Trp-NHMe 0o -1100-Cr-CHCH(i-Bu)CO-L-Trp-NHMe 1 N4HOHCOCH 2 C;H(t-Bu)CO-D-Trp-,NHMe NH1CQCH 2 ,CH(i-Bu)CO-L-N-MeTrp-NHMe 5 00

NHOHCOCH

2 CH'ti-Bu)CO-L-Ala(2-naphthyl)NH .e

NHOHCQCH

2 CH'li-Bu)CO-L-Trp-NH(GHR) 2

OH

NHOHCOCH

2 GCH(i-Bu)CQ-L-Ttp-NH (CH 2 )4OHs

NHOHCOCH

2 CHQi-Bu)CO-L-Trp-pipendine

NHOHCOCH

2 CHQi-Bu)CO-L-Trp-NH(CH 2 11 CHa

NIIOHOOCH

2 OH~i-13u)CO-L-Trp-NH(S9)GHMePh

NHOHCOCH

2 CH(i-Bu)CO-L-Trp-NH(GH 2 6 N H-C8Z

NHOIICOCH

2 CH(i-Bu)CO-L-Trp-NHcycbohexyI cis-UNOC4? 0. L-Trp-Nl-Me 13 3OAB >10,000 300 3 13 5,10,000 *55*44

S

*5*S =aS- 0 L-Trp-Nlihfk 14 31A

CO.CH

2 CH(i-Bu)-L-Tfp-O H 200 32A HQOC-CH 2 GHi-Bu)CO-L-'Trp-NH(CH 2

)NH

2 I" HO0H >~10,000 No-~ 16 34A 348 17 35 Table (ggontiaued) Comyoundfl

HOCO-CH

2 CHi-Bu}CO-L-Trp-Gly-OH

HQCO-CH

2 CH(i-Bu)CG.L-Trp-Gly-OH 10.000 >10.000 >10,000 b *.d5 *bb& 0- L-.Trp-NHMe 18 36 >10,000 Ixan-HOCOJ- 0 L-.Trp-INIIAe camp#

SA

9A ~~kQ abl 0.26 0.22 27 0.12 298 >1000 54 -~23 32 ->1000 29 -0.3 -0.3 0.37 40 0-62 0.2 0.001 0.1 0.1 4. o 14. 34A 348 36 19331 V2 57 0.19 Table 2 (continued) 72k 9 G2L Strom N- MGEQ 57 57 37 5145 20 2.2 58 79 -10587 26 59 265 -38178 .60161-- -3 78 >10000 >10000 >10000 >1I00W0 2000 79 >10000 21098 >1000 >10000 81 >10000 >10000 >100om >10000 500 82 9262 3585 >10000 326-r- s0 Units for all the above measurements are in nmoles.

a Natural human 72k0 gelatinaseb MID0 gelatinase c =Stromelysifl d Recombinant neutrophil collagenase e =Human gingival fibrblast collagenase lnhiion of Angioenesis A crude extract (30 mglL protein) of Walker 256 carcinoma. a rat malignant tumor, was incorporated ito Hydron, a slow retease.~Potymner, in 1.5 mm diameter pellets. Pellets were implanted in the stroma of the corneas of anesthetized albino rats. A canwila was chronically implanted in the inferior vena cama through which 10 mglmL of comnpound 5A in DMSQ in water was infused continuously for six days at the rate of 0.8 rr.Li hr- Controls received only the DMSQ solution. After six days, the animals were re-anesthetized and perfused intra-arterially with India ink in order to visualize the comeal vessels. The eyes were then enucleated and fixed in formalin. Control eyes which received only the DMSQ solution show massive vessel growth toward the pellet from the limbus. The animals receiving compound IA show vessels much shorter and/or much finer h in the controls, barely filling with ink.

ExamIDAe 4 Treatment of Psoriasis The effect of the compound 5A on psoriasis was studied usintg a i0 phorbol ester'induced epidermal hyperplasia, mouse model. The 12-0-tetradecanoyphorbol-1 3-acetate (TPA) hypeplsa model was coe as it is a widely accepted model for screening antiproliferative agerdis.A single, topical application, of TPA produces a pronounced epideimqad hyperplasia and a strong inflammatory response in mice as is obsc-rved in P25 psoriasis (Argyris. T.S. (1980) Am. J. Pathol. 98: pages 63S-648) In this model system., epidermal hyperplasia Is cr evide-i! histologically at 3 to 5 days following TPA treatment. tn adiiti- r to P other, more stable, phorbol esters produce the samre effectinhVi~ phorbol-12,13 dibutyroyl (PdiBul.

The following procedures was carried out to test he Tto~ invention compounds in the above described epiderma! hypc-roias. n~ The phorbol ester, PdiBu (20 rimol in 20 ±liactn)wsapedJr t both ears of hairless mice (v)(approximately I cmZ each). The test compounds (in total volume of 20 p.1) were then applied to the right ear immediately (15 to 30 min) following PdiBu. The left car of each animal received an equivalent amount (20 p.1) of ve-hicle- Test compounds (and S vehicle) wete reapplied at 6 and 18 hours following Pdi18u. Treatment times were staggered to allow exact time intervals to be obtained. At 30 hours after FdiBu, animals were anesthetized, and ear thickness values were obtained using a microcaliper, T1he weights of punch biopsies (6mm) were then obtained- Histology was performed on selected samples taken at 10 hours.

Test compounds includedS~A (10mg/mi in ETOK), a negative control, acetchydroxamic acid (AHA 2m /mI in ETOH). and iluiocnonide (LidexQ) as a positive control (0,05% in vehicle of alcohol [35%1, diisopropyl adlpate, citric acid and propyiene glycol)- Left ear in each animal serned as vehicie-treated controls (PdiBu :ETOH).. Thus, the controls for this series include: 1) Untreated controls; 2) Pdigu plus vehicle alone (included for each mouse tested); 3) PdiBu plus AH-A. 4) PdiBu plus LidexR as a positive control.

Table 3 shows the results- 64 Effects pf Compound MA on PdiBu- ruedE EidiaHyevap Ear ThIcknessz Topical 5 (051 jirOVeqf; 20 jil of 10M91mI 200 llg/crn2) applied at each of three timepoints (o025, and 24 hours) signifficantly inhilbited Pdi~u-induced ear thickness: -71ic", ess (inch x 10-a) %LQ o Cnro I Control (untreated)a 'k3..3 ±01(10% 12 to PdiBU 30.4±2.1 22b Vehicle PdiBu-*+ 18.6± 1.6 140%

A

Skin BionsV- Weight: Topical-5-A (0.51 timof/ear applied at eachi of two timepoits: 0.25, and 24 hours) significantly inhibited Pdi~u-induced increase in-punch biopsy weight (6mm): Weight (mg) of Control n Control 9.2±0.2 12 (untreated)a PdiBU I- 22.9% 7 .8C** Vehicle Pcl8U.+ 13A,±.5 146% 7c aUntreated control values were poolee-ifrom three experiments Results represent mean E for thr-e s-xperiMeriq- 416 mr ase C: ults represent mean± S.E-Jor three expe.-ir"ntts 16 h ,dosage, was iid.

gyjAnalysis of stained skin samples revealed that topical inhibited the PdlBu-induced: I) migration of inflammatory cells both into dermis and epidermis; 2) extravasatlon of RBC's; 3) epidermal hyperplasia; and 4) resulted in more normal appearing epidermial morphology (i.e.

reduction of PdiBu-induced parakeratosis, and roeduction of irregular basaloid, spinous and granular cell shape &nd distribution).

NIiHistologic analysis were performed both on ear and flank samples with similar results; both sites received 20 nmol PdiBu; however, ear received 20 idl 6001 (200 fig) over an area of approximatefy I cm2. while flank received 50 tiI (500 jig) over an area of approximately 4 cm 2 Skin samples were prepared using standard histologic methods and stained with hematoxylinleosin.

I shows typical sections of mouse skin exposed to PdiBu (Panel A) or PdiBu and MA (Panel It Is clear that ~Acompletely prevents PdiBu-induced epidermal hyperplasia.

AIAA..Qgnlr1 Topical AHA (0.53 l.Lmol/ear applied at each of three timepoints: 0.25, 6.0, and 24 hours) did not alter the PdiBu-induced increase in ear thickness and biopsy weight: Thickness inch x 10W) ht a~ Control 13.3± 0.1 9.2±0.2 12 PdiBu 35.5 ±1.3 25.9 ±0.8 6(! Vehicle 9Pdi~u +AHA 36.8±1.3 2. 12 6 d Untreated control values were pooled from three experiments.

Results represent mean for two experiments.

66t Positive Control: LidexR (fluocinonide) significantly inhibited the PdiBu-induced increase in ear thickness and biopsy weight: Thickness Weglahtn Control 13.3 ±0.1 9.2 0.2 12 (untreated)t PdiBu+ 33.6 1.2 24.4 1.1 69 Vehicle PdiBu AHA 15.8 0.4 9.7 0.3 69 Untreated control values were pooled from three experiments.

9 Results represent mean S.E. for single experiment.

To summarize, 5A demonstrated potent anti-inflammatory activity in this standard in .YLo model for psoriasis. The extent of anti-inflammatory activity was nearly comparable to that observed with LidexR, the positive control. The reduction of PdiBu-induced ear weight was accorr-panied by 0 similar inhibition of ear thickening. Moreover, decreased inflammation and epidermal hyperplasia were evident by histologic analysis.

Acetohydroxamic acid (AHA) was used as a negative control since it is devoid of inhibitory effects on MMPs. It did not alter PdiBu-induced effects on ear thickness or weight.

Examole 48 Treatment of Chronic Dermal Wounds Experiments were done to show the presence of matrix metalloproteinase activity in certain types of wounds, and the inhibition of such protease activity with the appropriate invention inhibitor.

Fluids were collected from 3 types of human wounds termed closed spontaneously healing wounds, open slowly healing wounds, ad open chronic wounds, in the first category were fluids collected from the chest S67 wall of women following mastectomy surgery. Fluids ifom non-Infected wounds that were left open for valid surgical reasons were considered open slowly healing wounds. An occlusive dressing was placed over the wound bed, and fluids were collected after 2-6 hours of occlusion. Finaffy, fluids were also collected from chronic open wounds by covering the wounds with occlusive dressings. Wounds were considered to be chronic if they were not clinically infected and had been open and not healing for more than 4 weeks.

The various wound fluids were assayed for matrix metalloprotetnase activity using the Azocoll assay essentially as described by Chavira, et al., Analytic Biochemistry (1984), 1,U:446.

The fluids were centrifuged, and the supernatants fi ltered'using a 0.45 u sterile Gelman filter. The filtrates were stored frozen at -800 C until tested for protease activity.

The effects of four inhibitors on the protease activity present In wound fluids were determined. The inhibitors were: compounds fNHOHCOCH2CH(i-Bu)CO-tryptophan-NHMeJ, 21.A [NHOHCOCH2CH(i-Bu)CO-tryptophan-NHCH.MePh], [A HOOCCH2CH(i-B3u)OO-tryptophan-NHCHMeP'h], and 20S1209 fNHOHCOCH2CHQl-Bu)CO-tyrosine-O MeNH Mel.

These innibitors were compared to certain other inhibitors and these were UL001 [HSCHzCH(O1-ICH(0H 3 )2)CO-Phe-Ala-NH 2 I obtained from Peptides International and MP506, obtained from Elastin Products. EDTA (ethyleneciaiine tetraacetic acid) and PMSF (phenylmethylsulfonyl we~ also studied.

Sto~t solutions of the four inhibitors were all prepared at a concentration of 800 pig/ml. Due to different solubility properties of the inhibitors, different techniques were utilized. A was dissolved in an amount of warmed propylene glycol to give a final concentraticel- of then dissolved in 10mM citrate, pH 5.5, containing 0.05% methyl cellulose.

ii~was dissolved in 1% DMSO then dissolved in I0mM citrate, pH containing 0.05% methyl'cellulose. 39QA was dissolved in propylene glycol (to give a final concentration of 2.4%1 then dissolved ini 1MIM CaCl2, 50 mIM 68

I

B

Tris-C, pH 7.8. 21A was dissolved in propylene glycol methyl cellulose and 10 mM citrate, pH 8.

The protease substrate, Azocoll, was obtained from Sigma Chemical Corporation and it is a substrate for collagenase/gelatinase-like_ metalloproteinase enzymes and general proteases. Clostridium histolyticum collagenase (the crude form of the enzyme) was from Worthington B-ochemicals. General chemicals including TRIS buffer, DMSO, and CaC12 were from Sigma Chemical Corporation.

Briefly, 900 pl of the Azocoll substrate suspension in buftter (5 u.g/mi in 50 mM TRIS, pH 7.8, ImM CaCI 2 was added to 1.5 ml microcentrifuge tubes then 50 .I of inhibitor (cr buffer) and 50 pi of chronic wound fluid (or collagenase standard)' jre added to the reaction tube. The reaction tubes Swere placed at 370 C in a shaker that inverted the tubes 30 times per minute.

After 24 hours of incubation, the reaction tubes were centrifuges at 10,000 X g and the absorbance of the supematant solution was measured at 520 nm with a Milton-Roy spectrophotometer. Proteolysis of Azocoll substrate generates soluble colored fragments from the insoluble Azocoll substrate.

I Wound fluid samples were incubated alone or with the inhibitors. Controls included incubation of the AzocoI substrate with the assay buffer to measure spontaneous degradation of the substrate. A standard curve for digestion of the Azocoll substrate was generated by incubation of the Azocoll with crude bacterial collagenase. Protease levels were expressed as net pg of collagenase equivalent per ml of chronic wound fluid. In the figures, certain of the wound fluids are referred to by an individual name.

Figure 2 shows the results. Mastectomy fluid samples collected on days 1 to 7 after surgery contained low levels of protease activity with an average of 0.75 ±0.06 gg equivalents of collagenase/mi of wound fluid. In contrast, figure 3 shows that wound fluids collected from open wounds contained an average protease level of 199 59 pg/ml of wound fluid, and fluids collected from chronic wounds contained an average protease level of 125 95 pg/ml. Only one of the thirteen samples of fluids, L. Smith, from chronic or open wounds did not contain measurable levels of Azocoli hydrolysis activity. The protease levels of the remaining twelve samples of 69 S- I open and chronic wounds were all higher than the levels measured in mastectomy fluids and ranged from 2 to 585 Ig/ml wound fluid.

Thus it is clear that fluids collected from chronic and open wounds contain very high levels of Azocoll-degrading protease activity compared to fluid collected from mastectomy drains. This suggests that the in vivo environment of open or chronic wounds contains proteases that have the ability to degrade extracellular matrix proteins of wounds.

Having established the levels of protease activity in various wound fluids, the effect of three protease inhibitors were measured. As shown in Figure 4, 5A very effectively inhibited proteolytic degradation of Azocoll (approximately 96% of initial proteolytic activity) by a chronic wound fluid when added at final concentrations of 40 g/mi (100 lM) or 4 ig/ml (10 pM), Lower concentrations of 5A [4 ig/ml (1 pM) and 0.4 Igml (0.1 piM)] inhibited approximately 92% of nontreated protease levels. Addition of EDTA, which is a nonspecific inhibitor of metalloproteinases, also effectively reduced protease activity (inhibited approximately However, the effective concentrations of 5A were gM while the concentrations of EDTA were mM.

Addition of PMSF, a nonspecific inhibitor of serine proteases, reduced proteolytic activity approximately 65% when added to a concentration of 500 IIM. Lower concentrations of PMSF (500 UM and 50 pM) actually slightly increase the levels of protease activity. CW in the figure refers to wound fluid not treated with protease inhibitor.

To conclude, both 5A and EDTA effectively inhibited the protease activity of a chronic wound fluid, but 5A was much more potent that EDTA.

PMSF was not an effective inhibitor except at the highest concentration.

This indicates that much of the proteolytic activity present in the chronic wound fluid was due to metalloproteinases. The inhibition observed with high concentration of PMSF was most likely due to nonspecific inhibition of non-serine proteases which has been reported at high concentrations of PMSF (see, for example. Arch. Blochem. Biophys. 124:70).

Additional experiments were conducted to ascertain the effects ot certain inhibitors on open and chronic wounds, and the results are shown in Figure 5 5A and EDTA were very effective Inhibitors while PMSF did not 70 significantly reduce proteolytic activity of the wound fluids.

To summarize, k and EDTA consistently reduced proteolytic activity by 95% in open or chronic wound fluids with high levels of Azocoll-degrading protease activity. PMSF did not reduce the leyels of proteolytic activity. This supports the general concept that open and chronic wounds consistently have elevated levels of matrix metallo-proteinases which can effectively be inhibited A further experiment was performed to determine the effects of a series of matrix metalloprotease inhibitors on the proteolytic degradation of Azocoll -by wound fluid,.. In this experiment, SIPM,~ UI-001, and EDTA inhibited proteolytic activity of wound fluids very effectively, Fot example, these inhibitors- reduced proteolytic activity of Christ! wound fluid by 97% to 94% (from 404 jgg collagenaselmI to 9 to 18 gig cotlagenase/mI wound fluid).

However, MP0 was substantially less effective than the other inhibitors. In 16. 3 of the 4 open and chronic wounds, MPSQ4 did not reduce the levels of protease atvt.Fgr6sumizes the results.

Rased on the abo-ve results, 5A and S=2D are very effective irthitIors of Azooolt-dagrading proteases In a larger series'of wound fluids.

MP506 was a somnewhat less effective inhibitor than MA or S12 2b Finally, expetiments were conducted to ascertain the effects of M,.

and a1P09 on the protease activity present in chronic wound iluid., For these experiments, due to flhe different solubility properties of the inhibitors, differo~t techniques were utilized. 5A was dissolved in an amount of warmed propylene glycol to give a final concentration of then disscved ini 10 mM citrate. pH 5.5, containing 0.05% methyl cellulose.

20-9 was dissolved in 1% DMSQ then dissolved In 10mM citrate. pH contain~ing 0.05%/ methyl cellulose. -MA was dissolved in propyiene glycol (to give a final concentration of then dissolved in 1 mM CaC1 2 50 Mn ~0 .Tfm-Cl, pH 7.8. al A was dissolved in propylene glycol (2,401%) methyl cellulose and 10 mM citrate. pH 8 *As shown in Figure 7. the chronic wound fluid conteine~t a:-high level ot pmot~ass activity with an average of 284 ±52 Vig coltagenase 71 1 equivalents/mi of wound fluid. Addition of a at 800 tig/ml reduced the level of protease activity by 84% to 45± 1 gig collagenase equivalentsimi Of wound fluid. Lower concentrations of .5 resulted in sK- tly higher levels of protew-r. activity up to go0±6 g.g coliagenase equivalents/ml of yound fluid.

S 21A and S1209 also effectively inhibited the protease activity of the chronic wound fluid, with the highest concentration (800 gig/ml) inhibiting 94% and respectively (see Table In contrast, even the highest level of M9 only inhibited 23% of the protease activity, and 13% and 30% increases were measured at the 8 lig/mI and 0.8 tig/ml concentrations.

To summarize, all three inhibitors at the highest concentration reduced the proteolytic degradation of Azacoll. However, 3.9A- was significantly less potent that 5A, 21A, or S1209 and tower concentrat ions, of aM actually increased the level of proteolytic activity of the chronic wound fluid.

ThoWljjajteftIduced PertnisAsa The effect of 5A on preventing thioglycotlate induced infiltratiob-of neutrophils in the peritoneal cavity was measured by the following P* O procedure.

6-8 week old Balb/c mate mice were used (n =10 each for the treated and control groups), Thioglycollate suspension (5.9 gAl in deionized-water).

was boiled until the color was golden, then cooled yielding a pink color. The.

lavage soio consisted of saline supplemented with bovine serf-im albumin (100 m9!1 00 ml) and 5U heparmi.l Thioglypollate (lmVanimal) was injected intraperitoneally at time T=0O. iA (800 ji9'ml. I mllanimal) was iniectcrd i. p. at time T=0,5, 1.0, and hr. post thioglycollate injectkn, Calls were collected by lavage at T 3.0 hr.

post thioglycollate administration. The animals were euthanized by CO:! ~asphyxiation, and the abdominal skin vras cut with surgical sisr orva the pezitoneal cavity. Five mli! lvage fluid. was injected into the peritoneum. and a suspension w mede by gentle massage. Th~e fluid was removed by syringe. recovervlg.;,&- 4I 80% Mf the injected fluid. Tolal cell 12 counts in the lavage fluid werze determined by Coulter Counter.

The average number of cells in the peritoneum of mice treated wilh SA was significanty.1reduced (p=0.01 4 1) from the average number of cel ls in the peritoneum of mice injected with vehicle alone. Figure. 8 shows the.

effects of the inhibitor ~Aon thioglycollate induced peritonitis assay.

Exampole Metastasis Assay In C57 Mice This assay was performed to determine the antimeiastatic potential of j~in an in vivo experimental metastasis assay in 057 mice. Female 057 B3US mice. 4-6 weeks old (15-20 grams), from Simonsen Laboratories (Gilroy,, CA) Were. used fior this asy Tne B16-FIO murine meilanoma wvas itained from the American Type Tissue Culture Collection, and routinely wobae-pled in DMEM Ii (Dtilbeccces modified eagles medium) 10% fetal COO serum in plastic tissue culture flasks in a humidified 5% C02 irncubator &i 370 C, 1316,171 tumor cells were harvested with trypsin-EDTA (othyleriedisk in~rwact acid), washed with PBS (phosphate buffered sciline, 2x resuspended at a ::.concentration of 5 x I105 ceUs/mI, and held on ice prior to injection. Animals were challenged wVith 5x I0J cers in a volume bf 0:1'ml intravenously animalsArealmert group). Animals were randomly distributed prior to assignment to treatment groups- Azsus~nsknf0Arrl, 4% (wM v~infiter s-terilized carboxy methyl cellulosO or spsio vetkVle was injected into the peroeu(i)fCS BU mice 24 h out prier to tumor ce1l challe nge. 30 minuteU6ms noir to -challengs, at imats were- dosed with aA or suspension vehicle, and 50,000 BI6-F-;0,1vmqr -cel Jnjected iv-, in a solution of A in ophthalfmic vehicle (WQO p.g/M11 or tpht a~ic veice alone, after incubation for 30 minutes at rooMa reer,%.azure prior to Injection 1 nI/mouse). Animals were then do~seciwith A (15-0 mtp'kq/d. ip) sut nSion of Vehicle fo-r 4 additional days.

surv~vai timres we-a recorded for animals in the various treatment gosand resulta were evaluated uisin~g Chi-Squafed statislical analysis.

Result~s of the assay are shown in Fig. 9. Rhere was a significant 4 increase in teSuivtFl Of mice treated with Aversus vehicie contlrot on days 35-37 (p<c0,05).

E~ffect of 5A on Hovoeic Shock ina.

Non-Heinarini--ed Aninal Thlis aSSaY 'as Weiotmed to determine !he eliect 0l on cardiovascular funclion, hepatocellular lunction and microcircujation.

fatt wing 4rauma-ftemorrtiage anrd rssiain experiment reliates to metalfoprotease inhibitors in treating hYPOvOIT~rn Iho,-k when administered systemically (via i-v. iection.

'4 infusion or otNhXi"poropriate route) during resuscitation following hemc~rj s~d ock in a non-heparinized animal, Seet Chaudry et 438 Awspo Qjr hoc 2:1. wsrared as a suspension at 100 ;4'winRir*,u- llae Prov-Daleyrats (weighing approyirnately 300 g. 6 in each group: s-,han. saliha and A)we" lightly anesthetized with ether and various lood vwecannulated for th~e study (Wang, P. et al. Am, I. PhysioL.

(19q0) 259:AW<). Following hemnorrhiage. Ringer's lactate (3X maximum 9b. ~bieeKne -AMonie) was- given over 45 minutes followed by Ringer's lactate (2X maxkium bleedout volume) over 60 minutes. A dose of suspension (0.75 mit. 40 mng/mi) was given subcutaneously at the time of completion of th' 3X sPingers lactate. Two times Ringer's, lactate containing A 1 mg/ml)- was provided as a second poriokn of-,he fluid resuscitation (total do-se of approximately 107 mgfkg body weight), ShaM-operated animals underwent blood vessel cannulation and midlitie incision, but hernorrhagi end resuscitation was not induced. These rats did not receive A, Salinetreated underwent traumna-hemnorrhage and fluid resuscitation plus vehicle (an -eq'al voume 60 Pormal saline) treatment.

At 2 and 41N ur;,after th-e completion of R-ngers lactate i'esuScitarlon.

thie following parariiairs were (t nitored: 1. eaoel~rfnto

K

and V~ associatad wiqth indocyarline green clearan-v in (Figures I OA and 81.Wag.P. t tAm. J, Physiol. (1 990) 259:R645. Wang, p, et al J U4j.

74 310 Res. (1990) 48:464) mean arteria- Pressure (MAP) arnd heart rate (HR) (Figures I IA and cardiac function (Wang, P. et at, J. Surg. Res, (1991) 50:163), (cardiac output stroke volume (SV) and total peripheral resistance- (TPR) Figures 12A, B3 and CI; and ml icovascu lar*Wod flow (MBF) in liver, kiney, spleen and small intestine (Figures 13A, B. C -id 0, respectively)- in Figures 10-13, date. are- P-7sented for three diff-atent experimental groups; consisting of wla operated (POOt control), (2) saline treated (negative control) and Hepatoceltula r funcion was monitored using. idocyanine Gleamff-.kinetics, where the and correlate ith. the efficiency of active transport, and rate of clearance, respectivey. The maximal clearance rate NmVx, Fig. 10) at 2h after initiating resvstcitation was significapnny ihighert in the 5A group than the saline groult, an8d was about 85% of shAm level. The effect was slightly reduced, but si1gnifficant at 4h, and ttl twr -o@ta Sa~lne ticated animals. The afficiency of active transport (tW at 2h f~r the group w.as higher (1-50Y4C0) than in the sham and. satine groapc., At 4h the A group had decreased (125%) to values nearer shxt nd about threefold higher than. Ihe saline treated group. The.$ rsui sggs an improved hepabc function relative to saline _croup.

Cricfunction of the A grouw;imrvdeate t-3.safine treated negative ontrol animals, Th6. mean arter-at pre (Fig I IA) for A Arid saline treated animals wsntsgii~~ ifrnSdwr Abdut.60-70% of the sham group. Heart- rate AT13. i184 for th ree grOLSPS were about the same ind~itating that neilher c~a~r; r Iteatm-ent significantly affected, heart rate. Cardiac, itpuf fF4T 12A) hn tho 5A i6-Gup was significantly elevated relat!-ve to t" saiegru nd was about 80% of sham group. Similarly. stroke Yluze (Fig, 1213 Ie- the.6 Mgroup was elevatea irelative to saline QreD'ip 8nd wa~ h~Ia'4c hmgoj..~ (g.I2C) was lowet following, resuscitalion ixM -AA 9MuP vrsus wn-alr r shamn treatment. Tne- enhanced cardiac o4ut rv!wvfjnc in te treated group. telative to saline treated groUP. ar the~w~ lalrphd~ resistance, il1 indicate improved kJaric cloni blood!suppfy rid fed ced' resistance to blood flow.

I

The microvascular blood flow~ (IMBF) was measured at 2h and 4h after onset of resuscitation, as shown in Fig- 13A-D. All the data show group to have MBF elevated relative to the saline group. C-eneratly, the IABF recovered to within. 80-100% of sham using. 5A versus 45-65% f6T saline.

The improved microvascular blood flow for 5.A treated animals supports improved tissue perfusion for the respective organs.

The results indicate that administration of MA during ciytalloid resuscitation followed by severe hemorrhage restores or significantly improves cardiac output, hepatocetular function (Vu and Kt) and surface microvascular blood flow in the liver, kidney, spleen and small intestine at 2 and 4 hours after the completion of the fluid resuscitation. Treatment with decreases total peripheral resistance. Administration of j& has hio significant effects on mean arterial pressure, heart rate or systemic hemnatocrit as compared with saline treated group. Thus, administration of 5A as an adjunct following trauma-hemorrhage and fluid resuscitation significantly improves cardiovascular and hepatocellular function even in the absence of blood resuscitation.

Examplg 52 Anti Restenotic ACtivy of 03 4tyjL9loon catheter iniuQIr 5A admizqistration Male Sprague-Dawley rats (3-3.5 months old).were subject to balloon catheter injury of the common carotid artery (Clowes at a. Lab. Inve5L (1983 A27208-215). Immediately before surgery, rats (n 6) were injected i.p.

with A at a dose of 100 mg/kg (total volume injected: I ml). and rats (riz were injected with an. equal vofume of 4% carboxymethylcetlutose vehicle (CINC). Rats were treated for the next four days with daily injections of either 100 mg/kg 5A. or CM4G vehicle. Rats wete sacrificed at 4 days after injury.

They were injected with 25 mg/kg BrdU subcutaneously at 17. 9 and I hours before sacrifice to-label replicating vascular smooth muscle Cells. A catheter.

as inserted into the aorta, and the carotids were fixed by perfusion with 4% paraformnaldehyde at physiologic pressure. After carefully dissedting out the common carotid, the vessels were embedded in paraffin, cross-sectioned 76 slow Ri, Ali.

1_4 and sta ined -with anti-BrdU arilibody and hematoxylin. and an index of medical smooth muscle cell replication was obtained by expressing the labelled SMCs (Lindler et aL. J. Clin. I nves. (1992) RQ20442049).

After flxaticn, the common carotis were pinned out with the intimal surface facing upwards. and ceIls present on the intimal surface were stained with an anti-body to histone proteins, With this procedure. only the surface cells are stained-, the antibody does not penetrate past the internal elastic lamnina. The surface cells were counted and the number of cells per unit area was determined. Administration of 5A caused a significant decrease in number of cells per unit area when compared to CMC treated, controls (Figure 14). There was a very large decrease in cell migration into the intima at 4 days after balloon injury.

I da lloon gatee ijrySA administrafio Surgery was performed, and !A-was administered daily for 10 days as described above fort the 4 day animals (n=5 for b oth groups). The aninialls were injected with BrdU before sacrifice to label replicating cells in thermedia, and intimra- By 10 days after injury, the normal vessel forms a thikened rieointimra; smooth muscle migt~tion in to the infima and subserqent proliferation-contribute to this thickening. Cross-s-ections of the vessel were cut and stained with anti-Brdt) and hematoxylin. Intimral crosssectional areas were measured using a computer digitizing system.

Administration of J.A.caused a significant -decrease in intirnal cross-sectional area when compared to 0MG. treated controls (Figure 15), Intimal cell repkiation rate was significantly higher in the SA treated rats than in the controls. Despite the increased rate on irtimal cell replication. lesion thickness was dramatically reduced in the 5Atreated group. suggesting that smooth muscle cell migration into the intirna was inhibited.

All data are presented as mean +SEM. and the data were analyzed by student's test.,

Claims (8)

1. A method to prevent or treat disease in an animal, wherein said disease is caused by unwanted mammalian matrix metalloprotease activity, said method comprising administering to an animal suffering from said disease an effective amount, aud for an s effective timne a syndhetic mammalian mtrix metalloprotease inhibitor.

2. The method of claima 1 wh~Lsaid disease is selected from the croup consisting of skin disorders, keratoconus. restenosis. rheumatoid arthritis, wounds. cancer, angiogenesis and. shock.

3. The rmthoyd of claim 2 wherein said disease is rheumnatoid arthritis. 0 4- The method of claim 2 wherein said disease is restenosis. The method of claim 2 wherein said wound is a chronic derrmal wound.

6.The method of claim 2 wherein said wound is a. shock, including hypovolenie x shock and septic shock. The method of claim 2wherein said (,iSease is cancer. Ss The method of claim 2 whcrein sai disease is angoigenesis. The mnethed of claim I wherein said inhibitor is of the faimula- ON6- C CHCON-CHCOX R R 2 R 3 R 4 or R7LONROC j-HCON -CHCOX I I (2) Rlj K4 R 3 R 4 wherein each RE is indendentl~y H or alkyl (1-SC) and R 2 -is H or, alkyl (I or -NHZ wherein Z is. -RR t -CORtt or -COQRII -is an alkvt group, or whertin the proximal RI and R 2 tkek toether are -(CHi 2 wherein p R 3 is H or alkyl (1-4Q; R 4 .is fused or Lonjusgte unsubstituted or substituted bicycloaryl Now n isO0. 1Or2; mis0; an is -ORS,'NH5 4-or NH(CH 2 )M wherein R5 is H or sub iknea (XI Mis arn am inmacid residue or amide thereof or Ihe residue of amine, or hercF amine;- q is w-tinzeof from 1 nd R6 is H or lower alkyt (1 -40) and R1?. is ii,, wr aik~4U- an acyl group. and wherein the P71R- amide bond shown is -optonaily replaced bya aimodified isbsteric, bon-! s64et',,A from the group consisting of -CH 2 NR3-----0H 2 CHiR3-, -CH'-CR -COCHRa-, -CHOHCHR3-. -NR3.CQ-, n -GF=G-R- Thes method of claim 1 wvhereinc ak inhibitor is of lhejorrnula: y (r-KL.CCON -CI RP- R3-R Y-C CHCON CHOX .1 4~.a aaaa wherein each R! is inld :endenty H or-alWy (1-8C) and R2 is5 H~ or alkyl (14aC) or -NHZvwherein Z is -911. -CORT, or -COOR" where R'is an atV group; or wtherein the proximal P, and R2 taken together are -C2 Ris H or aiky; (I R4 is fused corconjugated unsubstitted or substitited bicvclrcarA4 Me, a~ne; nis0. 1or 2: rnis 0or 1;arid X is -ORS. 44HRs. Al 'or -NH(CI-4qM; Aierein flS s H or substItuvtedl or~ A' M is an amino acid residue or amnide thereof or the residue of a cyclic amine or heterocyclic amine; q is an Integer of from 1-8; and Y is selected from the group consisting of R70NRGCONR6-. R5 2 NCCNOR7_. R5CONOR7- and -COQR12; wherein each R6 is independently H or lower alkyl (1-4C0); R7 is H, lower alky', (1-4C) or an acyl group and R12 is H or alkyl (1 -60) or -OCH 2 O-acyl; and wherein -CONR3- amide bond shown is optionally replaced by a modified isosteric bond selected from the group consisting of -CH 2 NR3-, -CI- 2 CHR3_, -CH=CR3-, -COCHR3- 3 -CHOHCHR3-, -NR3CO-, and -CF:-CR3-,

11. The method of claim 10 wherein Y is -COQR12; and Riz is H or alkyl (I -6C) or -OCH- 2 0-acyl.

12. The method of claim 1 wherein said inhibitor is of the formula: HON OC CH-CHN- GX .1111 R~JR 9 LI R2 OO3 R4 wherein each R, is independently H or alkyl aryl alkyl (1-4C) or aryl-S-alkyl (I -4C) and R2 Is H or alkyl (I1-8C), alkenyl aryl alkyl (1 :2P6C), or -NHZ wherein Z is -R1 1 -CORI" or -COORI1 where RI' is an aikyl group, or wherein the proximal RI and R2 taken together are -(CH 2 )p- 2wherein p n is 0. 1 or 2; R3 is H or alkyl (1-4C); R4 is fused or conjugated unsubstituted or substituted bicycloaryt methylene, unsubstit~uted or substituted anjlmethylene. alkyl X is -0R5, -NHRS, -M or -NH(CH2)qM; wherein R5 is H or substituted or unsubstiluted alkyl (1-12C), aryl (6-120), aryl alkyl (6-160); M is an amino acid residue or amnide thereof or th-3 residue a cyclic amine or heterocyclic amine, q is an integer of from 1-8; and RO is H or CH 3 RS is optionally substituted aryl, aryl alkyl substituted or unsubstitued .'lkyl (1-12C). -0-alkyl -S-alkyl -QCORIO, OCC)ORi0, 5-methyl-2-oxo- 1.3-dioxol-4-yl. -COOH, -COORIC, -CONH 2 and RIO is alkyl (1 -12C)~.R3 is H or alkyl (1 -4C).

513. The method of claim 10 wherein the inhibitor ir NHOHCOCH 2 CH(i-Bu)C04...Trp.NHMe. 14. A composition for preventing or treaing disease in an animal, wherein said disease is caused by unwanted mammalian matrix metalioprotease activity, said cornoosition comprises a synthetic mammalian matrix metalloprotease Inhibitor. The composition of claim 14 wherein said Inhibitor is of the formufla: 1-N1-C-c CO- CHCOX or HO R' R2 R3 F4 (2) wherein each R I Is independently H or alkyl (1 -8C) and R2 is H or alkyl (1 -80) or -NHZ wherein Zis 1,CORI I or.-COORI Iwhere R I I;s an P6 alkyl, group; or wherein the proximal RI and R2 taken together are wherein p R3 is H or alkyl (14C); R4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene: n0 i~s0, Ior 2; m is 0or 1, and X is -0R5, -NHR-5. -M or -NH(CH, 2 wherein RS is H or substituted or unsubstituted alkyl (1-120). aryl (6-12C), aryl alkyl 16C); or MWs an amino acid residue or amide thereof or the residue of a cyclic amine or heterocyclic amine: q is an integer of from 1 and R6 is H or lower alkyl (1 -4C) and R7 is lower alkyl (1 -4C) or an acy! group, and wherein the -CQNR3- am ide bond shown is Optionally replaced by a modified isosteric bond selected from the group consisting of -CH 2 NR3-, -CH 2 CHRa., -CH=CR3-, -COCH-R3-, -CHOHCHR3-, -NR300-. and -CF=CR3-. 16. The composition of claim 14 wherein said inhibitor is of the formula: Y CH 2 -CHCON CHCOX or Y CHC-CHCON -CHOOX [Rim IlR2 34(4) wherein each R1 is independently H or alkyl (1-8C) and R2 is H or alkyl (1-8C) or-;NHZ wherein Zis-R", -CORil or -COOR"l where R s an alkyl group; -or wherein the proximal RI and R2 taken together are -C2p wherein p R3 is H or alkyl (1 -4C); R4 is fused or conjugated unsubstituted or substituted bicycloaryl 1126 methylene: n is0, 1or 2; m is 0or 1;and X is -OR5, -NHR5, -M or -NH(CH 2 )qM; wherein R5 is H or substituted or unsubstituted alkyl (1-12C). aryl (6-120), aryl alkyl (6-16C); M is an amino acid residue or amide thereof or the residue of a cyclic amine or heterocyclic amine; q is an integer of from 1-8; and Y is selected from the group consisting of R 7 ONRG00NR6- R62NCONcR7-. R6CONOR7- and -COOR12; wherein each R6 is independently H or lower alkyl R7 is H, lower alkyl (1-4C) or an acyl group and R12 is H or alkyl (1-6C) or -CCH20-acyl; and wherein -CONR3- amide bond shown is optionally replaced by a modified isosteric bond selected from the group consisting of -CH 2 NR3-, CH 2 CH-R3-, -C.H=GR3-, -COCHR3-. -CHOHCHR 3 -NR3CO-, and -CF=CR3-. 17. The composition of claim 16 wherein Y is -COOR12; and R12 is H or ,Alkyl (1 -6C) or -OCH 2 O-acyl. 18. The composition of claim 14 wherein said inhibitor is of the formula: HONO--CH- CHCON -CHCOX -1 12i 1 1 R R 2 R3 R 4 'RH Un wherein each Ri is independently H or alkyl (I1-80), aryi alkyl (174C) or aryl-S-alkyl (1-4C) and R2 is H or alkyl alkenyl aryl alkyl (I- Is 60) or -NHZ wherein Z is .R1I1 .CQR I or -COOR"I where RI' is an alkyl group; or wherein the proximal Ri and R2 taken together are -C2p wherein p n is 0, 1cor 2; *O R2 is H or alkyl (1-4C); R4 is fused or conjugated unsubstiluted or substituted bicycloaryl methylene, unsubst1ituted or substituted ayI methylene, alkyl (1-120); X is -QR5, -NHR5, -M or -NH(CH 2 wherein R5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); 2 amine M is an amino acid residue or amide thereof or the residue of a cyclic amn rheterocyclic amine; qis an integer of from 1-8; and R6 is H orCHs; R9 is optibnally substituted aryl, aryl alkyl (1 substituted or unsubstituted alkyl (1-12C), -0-alkyl -S-alkyl (44-6C), -OCORlO, OCOORlO, 5-methyl-2-oxo-1,3-dioxo-4-yl, -COQH. -COORIO, -CONH 2 and RIO is aikyl (1-120). 19. 'the composition of claim 16 wherein the inhibitor is NHOHCOCH 2 Ci4(i--Su)CQ--L-Trp-NHMe. A process -for the- synthesis of L-tryptophan hydroxarnic acid derivative which process comprises the steps of; coupling L-tryptophMr with an acid or acid anhydride to yield a L- tryptophan. derivative;, coupling said derivative with an amine to yield a L-t.yptophan amide derivative; converting said L-tryptophan amnide derivative to a L-tryptophan hydroxamic acid derivative.

1021. The process of claim 20 wherein said L-tryptophan hydroxb mic acid derivative is N-[D,L-2-isobutyl-3- (N'-hydroxy amidocarborWAi).opanoyl]- tryptcphan methylamide and which process further, 6ornprises the step of, coupling of L Ityptopi.an with D.L-2-isobutyl-3-(methoxy-carbonyl)- propionic acid to yield N-(2RS-2-methoxy-carbonyl-4-methyl-pentanoyl). L-tryptaphan (42); coupling of said L-tryptophan derivative (42) with methylamine to yield N-[D,L-2-isobutyl-3-(methoxvcarbonyl)-propanoyhL -tryptophan methylamide 2& converting said methylamide to its hydroxamic, acid'derivate and crystallizing and rcxcvering of N-[D,L2-isobutyl-3- (N'-hydroxycarbony-lamido)-propanoyl]-L-tryptophan methylam ide 22. The process of claim 20 wherein said L-tryptophan hydroxamic acid derivative is N-fjD,L-2-isobutyi-3- (N'-hydroxy- -2 amidocarbonyl).propanoylJ- tryptophan methylamide ().and which process further comprises the steps of, reacting L-tryptophan with phosgene to yield N-carboxyanhydride derivative (83); reacting said N-carboxyanhydride derivative (83) methylamine followed by treatment with hydrochloric acid to yield the salt 84; treating isobutyl succinic anhydride in tetrahydrofuran is with said salt 34, in the presence of a base, to yield a mixture of carboxcylic acids and 84 esterification of said carb;oxylic acids 5 and 2 with mathanol under mild conditions to yield the respective esters 4 and 87' converting said mixture of 4 and BZ to respective hydroxamic acidF and and crystallizing and recoverie'g of 5A from said mixture of A and 23. The processot claim 20 wherein said L-tryptophan hi-iroxamic acid derivative is N-[D,L.2-isobutyj-3- (N'-hydroxy- amidocarbony)-pr-op~noyi]-tryptophan methylamide and which process further comprises the steps of, reacting L-tryptophan with phosgene to yield N-carboxyanhydride derivative 4 reacting said N-carboxyanhydride derivative ()methylamine followed by treatment with hydrochloric acid to yield the salt 84; treating isobutyl-succinic anhydride in tetrahydrofuran is with said salt L4 in the presence of a base, to yield a mixture ct carboxylic acids 55 and reacting said acids K and K with acetic anhydride or other coupling agents to yield suc~cinimide derivative 89; ~O yeldconverting said supcinimide. derivative 29 by hydroxaminolysis to yil hy~droxamic acid recovering said hydroxarnic acid 24. A process for the synthesis of N-(2-methoxy-carbonyl-4-methyl- pentanoyl)-L-tryptophan (S)-Methylbenzy! Amide (33Al which process -omorises the steps of; coupling of L-tryptophan with an acid to yield a L-Irypop ri derivative; coupling of said L-tryptophan derivative with an amine to yield a L-try'tophan amide derivative;, and hydrolyzing said L-tryptophan amide to yt--ld N (2-ehoxycarbonyl-4- methyl-pentanovi)-L. iryptophan (S)-methvl-benzy! a m ide Q-0, A). T he process of claim 24 which process comprises the steps of, coupling of L-tryptophan with 0, L-2-isobutyt-3-(methoxy-carbony). I *4 -2 so propnIIc. -acki to Yield N (2S2mtoycroy-4mtv-etr~~) Ltryptaprian (4W; coupling of said L-tryptophafl derivalive {4,I w.ith() niethylbenzylamifle to yield N-(2-methoxy~amrofly[' 4 methyl- pentanoyl)-L-tryptophafl ISI-methyl-beflzytFaide anda -hydrolyzing said aleth ylbelzyi amide with aqueous SN hydrochloricI acid to yiela 2mtoyat-t -4mty-ei~ry) L-tryptophan (S)-methyl-b-riZyl amide (39-A. 26. A process for the synthesis of D.L 2-iobutyl- 3-(rnethoxycabOflyl)- propionic acid (which process comprises the steps of; coupling maleic anhiydride with 2-methylpropefle to yield 1-methalivi- succinic anhydride; reducing said-I3-methallyl-succinic anhydride to isobutylsuccifltc anh-ydride; treating said izo~utylstk.iflic anhydride with anhydrous methanol to yield D,L-2-isobutyl-3--(mE-, Ihoxycarbonyl)-propionic ac;d and recovering said D,L-2-isobutyl-3-(metho)Cycarboly)-proptonic acid 27. A process of claim 26 which further comprises resolving the diastereomeric mixture D,L-2-isobuy-3-(meioxycarbonyU)-propionic acid (~)into a single diastereoisorner (2A). 2B. The process of claim 27 which process comprises the steps of; coupling said diastereorneric mixture D. L-2-isobutyl-3- (methoxy- carbony)-prpiolic acid with S-methy! berizylanine to yield a diasteriorfl salt mixture; recrystallization of said diastertomeric salt mixture from ethanoldiathyl ether to yield a single diasteriomerio salt 1,44); treating saio salt (44 wvith saturated aqueous sodium bicarbonate solution followed by aqueous 6N hydrochloric acid to yield (R--sbtl3(~ehxcr- ii-rp~ri acid (aA). I 29. Th e p--dUCE Of Ihbe press of any L~ne ofcliS0 o2 3:Aopoition including a sy, hetic inantmilian nia'triy, retal;*PUOt~i~ hiirsubstalillially. as hereinLNefote- descr -ibed .vith reference t n n~o h sy, 31a mialc acid derivative. 31y.opA (S-Mthes iZ fu mid 1e, sbsftntaa hyrbforedsnbdw re,. ?ni1jVa e oe ecie ih ference to at,, one of the Example Ricar E.-4ad patn ro rer o h A hpc i -a oiatd ero 32R.O AE GU O -I.irtlesnhss fr -e