NZ507696A - Triazole, oxadiazole and thiadiazole derivatives useful as cysteine protease inhibitors - Google Patents

Abstract

A cysteine protease inhibitor or a pharmaceutically acceptable salt thereof has the formula (I) wherein: R1 is optionally substituted alkyl or alkenyl, alkylamino, dialkylamino, alkyldialkylamino, cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl or optionally substituted aryl, arylalkyl or arylalkenyl optionally comprising 1-4 heteroatoms selected from N, O and S and Y and X are independently O, S or N where the N being optionally substituted with alkyl or alkenyl, or optionally substituted aryl, arylalkyl or arylalkenyl optionally comprising 1-3 heteroatoms selected from N, O and S provided at least one of Y or X is N Z is a cysteine protease binding moiety of the formula (II); AA1, AA2, AA3, AA4 and AA5 are independently an amino acid residue or amino acid residue mimetic; a direct bond or absent; R4 and R4' are independently -C(O)R5, -C(O)NHR5, -S(O)2R5, - C(O)OR5, -CH2R5 or R5 or together R4 and R4' form a ring comprising 5-7 atoms selected from C, N, S and O and R5 is as defined in the specification.

Description

50769® WO 99/54317 PCT/US99/08501 CYSTEINE PROTEASE INHIBITORS Background of the Invention Numerous cysteine proteases have been identified in biological systems. A "protease" is 5 an enzyme which degrades proteins or peptides into smaller components. The term "cysteine protease" refers to proteases which are distinguished by the presence of a cysteine residue which plays a critical role in the catalytic process. Mammalian systems, including humans, normally degrade and process proteins via a variety of mechanisms including the actions of cysteine proteases. However, when present at elevated levels or when abnormally activated, or where 10 introduced into a biological system in the context of a viral, bacterial or parasitic infection, cysteine proteases are thought to be involved in numerous pathophysiological processes and disease states.

For example, calcium-activated neutral proteases C'calpains") comprise a family of intracellular cysteine proteases which are ubiquitously expressed in mammalian tissues. Three 15 major calpains have been identified: calpain I and II, and p94. The calpain family of cysteine proteases has been implicated in many diseases and disorders, including stroke, neurodegeneration, such as Alzheimer's disease, amyotrophy and motor neuron damage; acute central nervous system injury, muscular dystrophy, bone resorption, platelet aggregation, cataracts and inflammation. Calpain I has been implicated in excitatory amino-acid induced 20 neurotoxicity disorders including ischemia, hypoglycemia and epilepsy. The cysteine protease p94, a muscle-specific member of the' calpain family, has been identified as a gene product responsible.for limb girdle muscular dystrophy (Barrett A.J., et al. ICOP Newsletter, 1-2 (1996)).

Lysosomal cysteine proteases or cathepsins (including cathepsins B, C, H, L, S, 0 and 02/K) belong to the papain superfamily of cysteine proteases. They are widely distributed and 25 differentially expressed among tissues. Intracellularly, they serve a variety of digestive and processing functions. Extracellularly, they may be involved in tissue remodeling and in pathologies such as arthritis, inflammation, myocardial infarction, Alzheimer's disease, cancer and muscular dystrophy (Elliott E., et al., Per. In Drug Disc, and Des., 6:12-32 (1996)).

Interleukin-1 P converting enzyme ("ICE") is a member of the caspase family of cysteine proteases which catalyzes the formation of interleukin-1 P (IL-ip), as well as the formation of Printed from Mimosa 10/24/2000 13:39:28 page -3- PCT/US99/D8501 interferon-y inducing factor (IGIF) from their inactive precursors, proIL-ip and pro-IGIF, respectively. Interleukin-1 P is an immunoregulatoiy protein implicated in inflammation, diabetes, septic shock, rheumatoid arthritis and Alzheimer's disease. ICE and/or other caspases have also been linked to the apoptotic cell death of neurons which is implicated in a variety of S neurodegenerative disorders including Parkinson's disease, ischemia and amyotrophic lateral sclerosis (ALS)(Dinarello C., et al., New Eng. J. Med, 328:106-113 (1993)).

Cysteine proteases are also produced by various viral pathogens and appear to be involved in every stage of reproduction including DNA and RNA translation and synthesis, and capsid formation (Gorbalenya A., et al., Per. In Drug Disc., 6:64-86 (1996); Krausslich etal., 10 Ann. Rev. Biochem., 57:701-54 (1988)). Examples of viral pathogens include Picornaviridae, which includes the genera Enterovirus, Rhinovirus, Cardiovirus, and Aphthovirus, which cause numerous human disease syndromes, ranging from fatal paralysis, encephalitis, meningitis, hepatitis and myocarditis to the common cold (Krausslich et al., Ann. Rev. Biochem., 57:701-54 (1988)). The picomaviral 3C proteinases, which are produced by all picornaviruses, are 15 responsible for processing viral polyproteins, an essential stage in viral growth (Malcolm B., et al. Biochemistry, 34:8172-8179 (1995)).

In addition, parasitic cysteine proteinases play significant roles in host-parasite interactions and pathogenesis (Robertson C., et al., Pers. in Drug Disc, and Des., 6:99-118 (1996)). For example, most of the proteinase activity detected in trypanosomes and various 20 Leishmania species has been characterized as belonging to the cysteine protease class. Other proteases are produced by Clostridium histolyticum and malaria parasites, such as Plasmodium falciparum and Plasmodium vinckei strains, and Schistosoma.

Cancer procoagulant, CP, a cysteine proteinase from malignant cells, has emerged as a probable activator of the coagulation system in cancer (Alessio M.G., et al., Eur. J. Haematol, 25 45:78-81 (1990); Gordon S., Methods in Em., 244:568-581 (1994); Gordon S., Sem. in Thromb. andHemo., 18,4:424-433 (1992)).

Existing cysteine protease inhibitors are primarily irreversible in nature; only weakly inhibit the enzymatic activity of the targeted protease and/or are toxic. Thus, there is a need for effective inhibitors of cysteine proteases as therapeutic and as prophylactic agents for the 2 Printed from Mimosa 10/24/2000 13:39:28 page -4- treatment and/or prevention of cysteine protease mediated pathologies.

It is an object of the present invention to go some way towards overcoming this need and/or to provide the public with a useful choice.

The present invention, relates to cysteine protease inhibitors of the general formula (I): wherein Z is a cysteine protease binding moiety, Z bong a carbonyl containing group, preferably an amino carbonyl containing group, wherein die carbon of the heterocycle is attached directly to the carbonyl group of Z.

In the above formula (I), R| is alkyl or alkenyl optionally substituted with 1-3 halo or hydroxy; alkylamino, dialkylamino, alkyldialkylamino; or cycloalkyl, alkylcycloalkyl, alkeaylcydoalkyl, (CrC,2)aryl> (C3-C12)arylalkyl or (Cj-Cu)aiylalkcnyl optionally comprising 1-4 heteroatoms selected from N, O and S, and optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl,, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (CrCc)aiyl, -CKQ-Cyaiyl, arylcarboxamide, alkylthio or haloalkylthio; and X and Y are independiently O, S or N, where N is optionally substituted with alkyl or alkenyl optionally substituted with 1-3 halo atoms; (Cj-C^aiyl, arylalkyl or arylalkenyl optionally comprising 1-3 hetteroatoms selected from N, O and S, and optionally substituted with halo, cyano, nitre, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, arylcarboxamide, alkylthio or haloalkylthio; provided that ait least one of Y or X is N. It will be understood that where Y or X is substituted nitrogen, both Y and X must be nitrogen.

In one embodiment, Rt is methyl, dimethylamino, phenyl or benzyl optionally substituted with methyl, halo, methylenadioxy, methoxy, dimethoxy, trimethoxy, trifluoromethyl and dimethylamino.

Summary of fee Invention N-Y CO According to several preferred embodiments, X is 0 and Y is N; X is N and Y is O; or both X and Y are N.- Typically Z comprises 1 to 5 amino acid residues or mimetics thereof. Thus, Z may, for example, comprise a pentapqptidyl, tetrapeptidyl, tripeptidyl or dipeptidyl binding moiety. According to a preferred embodiment, Z is of the formula (II): ALA5-AA4-AA3-AAj-AA j - 84 (n) wherein AA,, AA2, AA3, AA^ and AAj are independently an amino acid residue or amino acid residue mimetic; a direct bonid or absent; and R, and R«' are independently -C(0)Rs, -C(0)NHR5, -SCO^R* -C(0)0R* CH2Rs or Rs, IS where Rj is H, alkyl, alkenyl or alkynyl optionally substituted with halo, cyano, nitre, haloalkyl, amino, aminoalkyl, dialkylamino, haloalkoxy, carboxyl, carboalkoxy or alkylcarboxamide; cycloalkyl, alkylcycloalkyl, ((CyCI2) aryl or (Cj-Ca)arylalkyl optionally comprising 1-4 heteroatoms selected from N» O and S, and optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyll, dialkylamino, haloalkoxy, carboxyl, carboalkoxy, 20 alkylcarboxamide, alkyl, alkenyl,- alkynyl or (C,-CI2)aiyl;J or together R4 and R4' form a ring comprising 5-7 atoms selected from C,N, S and O. Typical terminal R* groups include Cbz, succinic acid derivatives of the formulas ^OJCHC-CHjCHCCHjWCHjCOOH, -C(0)CH2CHjC00H, and -Cd0)CH2CH2C(0)0C(CH3)3; toluenesulfonyl, methane sulfonyl, FMOC, (0-mentbyloxy-CO- ;and acetyl.

Preferably, the amino acids are selected from arginine or an arginine mimetic, proline; aspartic and glutamic acid and the aryl and alkyl esters thereof, alanine and glycine optionally substituted at the a-carbon or a-nhrogen with alkyl, cycloalkyl or aryl; leucine, isoleudne; cysteine optionally substituted at the sulfur atom with alkyl, alkenyl or phenyl optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkoxy, INTELLECTUAL PROPERTY OFFICE 0FN2.

JUL 2003 RECEIVED haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, arylcarboxamide, alkylthio or haloalkylthio; phenylalanine, homo-phenylalanine, dehydro-phenylalanine, indoline-2-carboxylic acid; tetrahydroisoquinoline-2-carboxylic acid optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl,, dialkylamino, alkyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, 5 alkylcarboxamide, arylcarboxamide, alkylthio or haloalkylthio; tyrosine, serine or threonine optionally substituted with allkyl or aryl; tryptophan, histidine, methionine, valine, norvaline, norleucine, octahydroindole-2-carboxylic acid; asparagine, glutamine and lysine optionally substituted at the nitrogen atcom with alkyl, alkenyl, alkynyl, alkoxy alkyl, alkylthioalkyl, alkylaminoalkyl, dialkylamiruoalkyl, carboxyalkyl, alkoxycarbonyl alkyl or cycloalkyl, bicycloalkyl, cycloalkyl alkyl, bicycloalkyl alkyl or fused aryl-cycloalkyl alkyl optionally comprising 1 or more heteroaitonis selected from N, 0 and S.

R* R2' and R2" are independently H; alkyl or alkenyl optionally substituted with 1-3 halo, hydroxy, thio, alkylthio;, amino, alkylamino, dialkylamino, alkylguanidinyl, dialkylguanidinyl, guanidinyll; -RCOR', -RCOOR', -RNRH'R0 or -RC(0)NR'R" where R is alkyl or alkenyl, and R', R" and R° are independently H, alkyl, alkenyl, cycloalkyl or (Cj-CJaiyl; or cycloalkyl, alkylcycloalkyl, alkcnylcycloalkyl, alkyl-oxyaryl, alkyl-thioaryl, (C5-C12) atyl, (Cj-C)2)arylalkyl or (Cj-C|j)arylalkenyl optionally comprising 1-4 heteroatoms selected from N, O and S, and optionadly substituted with hydroxy, halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, annidine, alkylamidine, dialkylamidine, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl,, carboalkoxy, alkylcarboxamide, (CJ-Cs)aiyl> -0-(Cj-Cg)aryl, AA| is tof the formula (Ilia): (ma) wherein X' is N; and.

INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 RECEIVED arylcarboxamide, alkylthio or haloalkylthio; or R2 and R2' together with X' form a ring comprising 4-7 atoms selected from C, N, S and O, said ring optionally subsitituted with hydroxy, halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, amidine, alkylamidine, dialkyl amidine, alkyl, alkenyl, alkynyl, S alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (C^C^aryl, -0-(C3-C6)aryI, arylcarboxamide, alkylthio or haloalkylthio.

AA2 may be a residue of the formula (Illb): K- (Illb) IS or selected from a residue mimetic of formulas IV to XXIV: 6 Printed from Mimosa 10/24/2000 13:39:28 page -8- n / *3 (VI) 0 G—R/C fY i (Vin) 0 y** vV". V/i VY*tV (-°YtV (X) (XI) (Ml) *3 ff . /"V i , Y HVtV Vr-rV v (XIII) (XIV) o (XV) < y o (XVI) (XVII) ■« v ° // (xvni) pax> 6' x*-/' 4 >■ (XX) *3 NJ0 O vVrV \WV /xxII) ' nncrm ^ /wim ' Printed from Mimosa 10/24/2000 13:39:28 page -9- wherein X" is CR', or N; Rj, R'j and R"j are independently H; alkyl or alkenyl optionally substituted with 1-3 halo, hydroxy, thio, alkylthio, amino, alkylamino, dialkylamino, alkylguanidinyl, dialkylguanidinyl, guanidinyrl; -RCOR', -RCOOR' or -RC(0)NR'R" where R is alkyl or alkenyl, and R' and R" are independently H, alkyl, alkenyl, cycloalkyl or (Cj-CJaryl; or cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkyl-oxyaryl, alkyl-thioaryl, (Cj-C,]) aryl, (Cj-C|2>arylalkyl or (Cj-C,i)arylalkenyl optionally comprising 1-4 heteroatoms selected from N, O and S, and ' optionally substituted with hydroxy, halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, amidine, alkylamidine, dialkylamidine, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (Cj-C^aryl, -O-tCj-C^aryl, arylcarboxamide, alkylthio oir haloalkylthio; m is 0,1 or 2; n is 0,1 or 2; G is -C(0>, -NHC(0>, -S(0)i-, -0C(0>, -CH2- or a direct bond; R«, R7, R'<f R't are independently H, alkyl, alkenyl, halo, alkoxy, carboxyl, carboalkoxy, amino, aminoalkyl, dialkylamino; cycloalkyl, (Cj-Q) aryl or (Cj-Q) arylalkyl optionally comprising 1-3 heteroatoms:selected from N, O and S, and optionally substituted with alkyl, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, alkoxy, haloalkoxy, carboxyll, carboalkoxy, alkylcarboxamide, alkylthio, guanidine, alkylguanidine, dialkylguaniidine, amidine, alkylamidine or dialkylamidine; and U, V, W and Y' are imdependently or together N, C, C(0), N(R,) where R^ is H, alkyl, halo, alkoxy, carboalkoxy, cycloalkoxy, carboxyl, alkylthio, amino, alkylamino, dialkylamino, or aryl, fused aryl or cycloalkyl optionally comprising 1 or more heteroatoms selected from O, S and N, and optionally subshnted with halo or alkyl; N(R|q) where R,0 is H, alkyl, alkenyl or cycloalkyl, aryl, arylalkyl or- fused aryl-cycloalkyl optionally comprising 1-4 heteroatoms selected from N, O and S, amd optionally substituted with alkyl, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, alkylthio, guanidine; alkylguanidine, dialkylguanidine, amidine, alkylamidine or dialkylamidiine; or C(R|iXR12) where Rn and R|2 are independently or together 8 FCT/US99/D8501 H, alkyl, alkythio, alkythioalkyl or cycloalkyl, alkylcycloalkyl, phenyl or phenyl alkyl optionally subsituted with guanidine, carboalkoxy, hydroxy, haloalkyl, alkylthio, alkylguanidine, dialkylguanidine, amidine, alkylamidine or dialkylamidine.

In a preferred embodiment, X' and X" are C, and R'2 and R'3 are H.

Where Z is a calpain binding moiety, preferably R2 is benzyl optionally substituted with alkoxy; H2NC(=*NH,)NHCH2CH2CH2-; -R'-C(=*NH2)NH2; -R'-NHC(=+NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected from N, S or O; and R" and R° are alkyl or cycloalkyl; or CH]SCH2CH2-, 10 HOOCCCHjJiCHj-, cyclohexyl-CH2-, imidazolyl-CH2, benzyl optionally substituted with OH or -O-benzyl, (CH3)2CHCH2-, (CH3)2CH-, CH3CH2CH2- or CH3(CHj)2CHj-; and R3 is -CH2-benzyl, benzyl, (CH3)3C-, (CH3)jCCH2-, (CH^CH-, CH3(CHj)2CH2-, CHjCHjCHfCHj)- or (CH})2CHCH3-. Preferably, R} is benzyl, isoquinolinyl, quinolinyl, naphthyl or HOOCCH2C(CH2CH(CH3)2)-; or R, is Cbz wherein the phenyl is optionally substituted with 15 nitro. Additionally, R« may be toluenesulfbnyl, methanesulfonyl, FMOC or (+)-menthyl-oxy-CO-.

In another embodiment, X' and/or X" are N.

In one embodiment, AA3 is leucine, AA« and AAS are direct bonds or absent, and R5 is alkyl.

Several particular embodiments include those where Z is Ri-Leu-Leu-Leu-; RrLeu-Leu-; R,-Leu-Leu-Phe-; Rt-Leu-Abu-; R^-Val-Phe-; R^-Leu-Leu-Nle-; R,-Ala-t-BuGly-Val- R,-t-BuGIy-Val- R,-Leu-Leu-Met-; or R,-Leu-Nle-. 9 Printed from Mimosa 10/24/2000 13:39:28 page -11- Preferably, Z is Cbz-Leu-Nle-; or Cbz-Leu-Val-.

Z may also be a cysteine cathepsin binding moiety, where preferably R2 is CH3-, (CH3)2CH-, (CHJJjCHCHJ-, CH3(CHJ)2CHr, H2NC(=*NHj)NHCH2CH2CHr; -R'-C(=*NH2)NH2; -R'-NHC(=+NR")NR°; pr -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl 5 optionally substituted with one or more heteroatoms selected from N, S or O; and R" and R" are alkyl or cycloalkyl; benzyl or -CH2-benzyl optionally substituted with OH or -OR' where R' is alkyl or aryl; CHjCH(-O-benzyl)- or benzyl-S-CH2-; and R3 is H, (CHjJjCH-, (CHj)2CHCH2-, CHJCCHjJJCH,-, H2N(CHJ)3CH2-, H2N(CHJ)2CH2-, H2NC(=*NH2)NHCHiCH2CH2-; -R'-C(=+NHi)NH2; -R'-NHC(=*NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl 10 optionally substituted with one or more heteroatoms selected from N, S or O; and R" and R° are alkyl or cycloalkyl; benzyl, benzyl substituted with hydroxy and halo; or (naphthyl)-CH2-.

In one embodiment, Z is a cathepsin B binding moiety, where preferably, R2 and R3 are independently benzyl, -CH2-benzyl, H2NC(="NHj)NHCH2CH2CH2-; -R'-C(=<NH2)NHj; -R'-NHC(="NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted 15 with one or more heteroatoms selected from N, S or 0; and R" and R° are alkyl or cycloalkyl; H2N(CH2)]CH2- or H2N(CH2)jCH2-; and preferably AA3 is lie, Leu, absent or a direct bond.

According to a particular embodiment, -AAj-AA,- are selected from: -Phe-hPhe-; -Arg-hPhe-; -Arg mimetic-hPhe-; -Leu-hPhe-; and -Om-hPhe.

Z may be a cathepsin L binding moiety, where R3 is preferably benzyl or (CHj^CHCHj-; and R2 is -CH2-benzyl.

Where Z is a cathepsin S binding moiety; preferably R2 and R} are alkyl; more preferably (CHj^CH-, (CH3)2CHCHj- or CH,(CHj)jCH2-.

In another embodiment, R3 is benzyl, (CHj^CHCH^ or (CHj^CH-; and R2 is -CH2-benzyl. According to one particular embodiment, AA3, AA» and AA, are direct bonds or absent; Rs is benzyl, isoquinolinyl, quinolinyl, naphthyl or HOOCCH2C(CH2CH(CH])2)-; or R, is Cbz.

Printed from Mimosa 10/24/2000 13:39:28 page -12- Where Z is a cathepsin H binding moiety; Z is preferably Ri-hPhe-;or ■ HOhPhe-.

Z may also be a cathepsin K binding moiety; where preferably R} is benzyl, 5 (CH3)2CHCH2- or (CH3)jCH-; and preferably AA3 is Gly; and AA< is Val or D-Val.

In another embodiment, - AAj is Arg, Arg mimetic or hPhe; AA2 is Pro; AAj is Gly; and 10 AA« is Val or D-Val; or preferably Z is R,-Pro-AA|-; R^-Gly-Pro-AA,-; Rj-Val-Gly-Pro-AA,-; D-Val-Gly-Pro-AA|-; or R«-D-Val-Gly-Pro-AA,; where AA, is Apa, Arg or Arg mimetic, or hPhe.

Other embodiments include compounds where Z is R,-AA3-Leu-hPhe-; R^-AAj-Phe-hPhe-; or R,-AA3-Val-hPhe-; where AA3 is Gly, Val, D-Val, a direct bond or absent Where Z is a caspase binding moiety; preferably R2 is -RCOOR'; where preferably R is -CHj- and preferably R' is H; where preferably AA3 and AA4 are amino acid residues and AA3 is 25 a direct bond.

Where Z is an interleukin-1 P converting enzyme binding moiety; AA« may be optionally substituted tyrosine or leucine; AA3 may be valine, glutamate or an ester of glutamale; and R} may be -CH, or (CHj^CH-.

In another embodiment, R3 is -CH3 or imidazolyl-CH2-; AA3 is valine or glutamate; and 11 Printed from Mimosa 10/24/2000 13:39:28 page -13- PCT/US99/D8501 Rj is -CH,.

Z may also be RrAA3-AA«-AA3-Pro-AA,; where AA| is Asp or Asp ester; where -AAj-AAi-AAj- may be -Ala-; -Glu-; -Val-; -Tyr-Ala-; -Tyr-Glu-; -Tyr-Val-; -Leu-Ala-; -Leu-Glu-; or -Leu-Val-.

In yet a further embodiment where Z is an interleukin-1 p converting enzyme binding moiety, AA2 is of the formula (VI); wherein X" is CR'a where preferably R', is H; and R2 is -RCOOR' where R is alkyl or alkenyl, and R' is H, alkyl, alkenyl, cycloalkyl or (C3-C6) aryl. In another, AA« and AAS are direct bonds or absent, AA3 is Tyr or Tyr(0-R') or a direct 10 bond or absent; R2 is -RCOOR' where R is alkyl or alkenyl, and R' is H, alkyl, alkenyl, cycloalkyl or (C3-C6) aryl; R« is phenyl or benzyl substituted with halo; and R5 may be benzyl, isoquinolinyl, quinolinyl, naphthyl or HOOCCH2C(CH2CH(CH3)2)-.

Where Z is a YAMA binding moiety, preferably R2 is -RCOOR' where preferably R is -CHj- and AA4 is Asp or an ester thereof. In another embodiment, AA3 is optionally substituted 15 glutamine or glutamic acid or an ester thereof. In yet another embodiment R2 is (CH3)2CH- or CH3SCH2CH2-.

Where Z is a FLICE binding moiety, preferably R2 is -RCOOR', where preferably R is -CH2-; AA4 is optionally substituted lysine; and preferably AA3 is glutamic acid; and R3 is (CH3)jCH-.

Z may also be a viral or microbial cysteine protease binding moiety. In one embodiment, Z is a gingipain binding moiety. Where Z is a gingipain K binding moiety; R2 is preferably -RNR'R"R° where preferably R is (CpCOalkyl; R' is H; and preferably R" and R° are H or (C,-C3)alkyl. In one embodiment R2 is *H3N(CH2)3CH2-. Where Z is a gingipain R binding moiety, preferably R2 is H2NC(=tNH2)NHCH2CH2CH2-; -R'-C(=*NH2)NH2; -R'-NHC(=+NR")NR°; or 25 -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected from N, S or O; and R" and R° are alkyl or cycloalkyl.

According to one embodiment, AAZ is proline, where Z is R^-Leu-Pro-AA, where AA, is arginine or an arginine mimetic.

Z may also be a human coronavirus protease binding moiety, where preferably R2 is 12 Printed from Mimosa 10/24/2000 13:39:28 page -14- H2NC(=*NH2)NHCH,CHjCHj-; -R'-C(=*NH2)NH2; -R'-NHC(=*NR")NR°; or-R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected from N, S or O; and R" and R° are alkyl or cycloalkyl; and preferably R3 is (CH^CH-, (CHj)2CHCH2- or CHj^HJjCHj-; AA3 is Asp or ester thereof. Leu, Arg or Arg mimetic, or 5 direct bond; AA4 and AA5 are direct bonds or absent; and R* is alkyl.

Where Z is a hepatitis A virus 3C proteinase binding moiety, R2 is preferably -RC(0)NR'R" where R' and R" are H or -CH3; or RCOOR' where R' is CH3; and AA3 and AA4 are amino acid residues. Preferably, AA, is Leu; R3 is -CH} and AA} is Ala.

Z may also be a hepatitis A virus 3C proteinase binding moiety, where Z is 10 R,-Leu-AA3-Thr-Gln-; R4-Tip-AA3-Thr-Gln-; R«-VaI-AA3-Thr-Gln-; R^-lle-AAj-Thr-Gln-; or RrD-Leu-AAj-Thr-Gln-; where AA3 is Arg or Arg mimetic.

Where Z is an Ad2 23K protease binding moiety, Rz and R3 are preferably H; AAj is alanine; AA4 is leucine; AAj is a direct bond; and R« is absent.

Where Z is a human rhinovirus 3C protease binding moiety, preferably R2 is RCOOR' where R is -CH2-; R3 is benzyl; and AA3 is leucine, isoleucine or a direct bond. 20 In yet a further embodiment, R2 is -RC(0)NR'R" where R' and R" are H, -CH3 or - CH2CHj; or RCOOR'where R'is-CH, or-CH2CH3.

Z may also be a human picornain 2A protease; where R} is -CH(OR')CH} where R' is H, alkyl or aryl; and preferably R^isa hydrophobic side chain. Alternatively, AA, is Val or dehydro-Phe; AA2 is Pro; and AA3 is Val. Examples include compounds CE-2072, CE-2060 and 25 CE-2061, the structures of which are shown below.

In another embodiment, Z is R,-Ala-Ala-Pro-Val-; or R,-Ala-Ala-Pro-Ala-.

Additionally, Z may be a protozoan protease binding moiety, such as a Trypanosoma, 13 Printed from Mimosa 10/24/2000 13:39:28 page -15- WO 99/54317 PCT/US99/08501 Leishmania or Schistosoma protease binding moiety. The protease may be a cathepsin L- or cathepsin B-like protease. In one embodiment, R2 is H2N(CH2)3CH2-, H2NC(=*NH2)NHCH2CH2CH2-; -R'-C(=*NH2)NH2; -R'-NHC(=*NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aiyl or arylalkyl optionally substituted with one or more heteroatoms 5 selected from N, S or O; and R" and R° are alkyl or cycloalkyl; -CH2-benzyl or benzyl optionally substituted with OH; and preferably R3 is benzyl, (CH3)2CHCH2- or (CH3)2CH-; and AAj is Phe, Leu, Pro or a direct bond. In One example, R, is Boc or Sue.

Z may be also selected from -Pro-Phe-Arg-; -Phe-Arg-;-Val-Leu-Lys-; -Leu-Val-Tyr-; Suc-Leu-Tyr- or -Phe-Ala-.

Where Z is a Plasmodium protease binding moiety, preferably R2 is (CHj^CH-, -CH2- benzyl, benzyl or phenyl optionally substituted with hydroxyl; H2NC(=*NH2)NHCH2CH2CHj-; -R'-C(=*NH2)NH2; -R'-NHC(=*NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aiyl or arylalkyl optionally substituted with one or more heteroatoms selected from N, S or O; and R" and R° are alkyl or cycloalkyl; or -R'-N(R")(R°) where R' is alkyl, and R" and Ra are alkyl or 15 cycloalkyl; or alkylimidazoyl; and R3 is benzyl, (CHj^CHCHj-, (CH3)jCH-, HOCH2- or -CHjOR'.

In one embodiment, Z is R,-Phe-Arg-; R^-Phe-Carginine mimetic)-; R,-Phe-Lys-; R,-Leu-hPhe-; Rj-Val-Leu-Arg-; R^-Phe(e-Z)-Lys-; R,-Val-Leu-{Arg mimetic)-25 R,-Phe-Val-; or R4-Phe-Ser(OBzl)-.

In another embodiment, Z is R,-Phe-AA,-; or R,-Leu-AA|-; 14 Printed from Mimosa 10/24/2000 13:39:28 page -16- where AA, is optionally substituted lysine; and where R, may be moipholino. In a further embodiment, AA3, AA, and AAS are direct bonds or absent, and R« is Cbz.

The present invention also provides methods of inhibiting the enzymatic activity of one or more cysteine proteases comprising contacting a protease with an inhibitory amount of a compound described herein.

Preferably the compound is selected from [2-[5-(3-methylbenzyl)-l,3,4-oxadiazolyl]carbonyl]-2-(S)-methylpropyl]-L-phenylalanamide-(3R)-(isobutyl)succinicacid; Acetyl-L-leucyl-N-[l-[2-[5-phenyl]-l,3,4-oxadiazolyl]caibonyl]-4-(guanidino)-butyl-L-leucyl amide; Acetyl-L-leucyl-N-[l-[3-[5-methyl]-l,2,4-oxadiazolyl]carbonyl]-ethyl-L-leucylamide; Acetyl-L-leucyI-N-[l-p-[5-methyl]-l,2,4-oxadiazoIyl]carbonyl]-4-(guanidino)-butyl-L-leucyl amide; Acetyl-L-tyrosinyl-L-valyl-N-[l-[2-[(5-phenyl)-I,3,4-oxadiazolyl]carbonyl]-2-carboxy-ethyl]-L-alanine amide; Acetyl-L-Aspartyl-Valyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl] carbonyl]-2-(carboxy>-ethyl]-L-glutamyl amide; (Benzyloxycarbonyl)-L-valyI-N-[l-(2-[5-(3-methylbenzyI)-l,3,4-oxadiazDlyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide; (t-butoxysuccinyl)-L-valyl-N-[l-[3-[5-(3-trifluoromethylbenzyl)-l,2,4-oxadiazolyl]carbonyl]-2-benzylidone]-L-prolinamide; and Carboxysuccinyl-L-valyl-N-[l-[3-[5-(3-trifluoromethylbenzyl)-1,2,4-oxadia2olyl]carbonyl]-2-benzylidone]-L-prolinamide.

The present invention also provides a method of inhibiting cancer cell growth or tumor progression or tumor metastasis or invasion, by inhibiting die enzymatic activity of cysteine proteases associated with such growth or progression, such as cathepsin B or cathepsin L.

Further provided is a method of inhibiting microbial cell or viral growth or reproduction by inhibiting the en^matic activity of cysteine proteases associated with such growth or reproduction. Suitable pathogenic targets include, by example only, hepatitis A virus 3C IS Printed from Mimosa 10/24/2000 13:39:28 page -17- proteinase, hepatitis C virus endopeptidase 2, picomain 3C rhinovirus protease, encephalomyelitis virus endopeptidase 2 and picomain 2A protease.

The present invention also provides a method of treating the symptoms associated with allergic responses by inhibiting the enzymatic activity of cysteine proteases associated with 5 certain allergens, such as, for example Der p I.

The invention provides a method of treating the symptoms associated with neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis. The invention further provides a method of treating the symptoms associated with stroke.

Further provided is a method of treating the symptoms associated with inflammatory and degenerative diseases, such as arthridities, including rheumatoid arthritis or osteoarthritis, or periodontal disease.

As used herein, the term "cysteine protease binding moiety" means a chemical group capable of binding to the substrate binding site of a cysteine protease, typically defined in the 15 literature as the S,-S„ site. The term includes both peptides and peptide mimetics. Preferably, the binding moiety is selected such that when linked to the keto-heterocycle, the moiety provides the resulting compound with inhibitory activity against the target cysteine protease of less than 100 fiM (K, value); and more preferably of less than 10 |iM.

As used herein, the term "optionally substituted" means, when substituted, mono to fully 20 substituted.

As used herein, the term "independently" means that the substituents may be the same or different.

As used herein, the term "alkyl" means CrC|j, however, preferably C]-CT.

As used herein, the term "alkenyl" means CrCu, however, preferably Cj-C,. 25 As used herein, the term "alkynyl" means CpCu, however, preferably Ci-C,.

It will be understood that alkyl, alkenyl and alkynyl groups, whether substituted or unsubstituted, may be linear or branched.

As used herein, the term "aryl," unless otherwise stated, means aryl groups preferably comprising 5 to 12 carbons, and more preferably 5 to 6 carbons. Unless otherwise indicated, the 16 Printed from Mimosa 10/24/2000 13:39:28 page -18- term aryl includes mono-and bi-cyclic, as well as fused ring systems. As used herein, the term "arylalkyl" includes mono-substituted alkyl groups (e.g., benzyl), as well as di-substituted alkyl groups such as -alkyl(phenyl)2 (e.g., -CH(phenyl)j). As used herein, where the term "aiylalkyl" or "arylalkenyl" is defined by the general formula (C„-Cr)aiylalkyl or (C„.C,)arylalkenyl, x and y S refer to the number of carbons making up the aiyl group. The alkyl group is as defined above. As used here, the term "arylalkenyl" includes aryl compounds having an alkenyl chain comprising 1-3 or more double bonds. Exemplary arylalekenyl groups include =CH-CH2-aryl and -CH=CH-aryl, where aryl is preferably phenyl.

As used herein, the term "arginine mimetic" means an amino acid residue with a side 10 chain substituent of the formula -R'-C(=*NH2)NH2; -R'-NHC(=*NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected from N, S or 0; and R"and R"are alkyl or cycloalkyl.

As used herein, the term "Cbz" means benzyloxycarbonyl; and the term "Mu " means moipholino. 1S Pharmaceutical^ acceptable salts of the compounds described above are within the scope of the invention.

Brief Description of the Drawings Figure 1 shows the inhibition of the production of mature IL-lp in THP-1 cell line by 20 certain compounds of the present invention.

Figures 2 A and 2B show the inhibition of the production of mature IL-ip in whole blood by certain compounds of the present invention.

Figure 3 is a schematic representation of the synthesis of a compound according to the invention (CM-0019).

Detailed Description of the Invention The present invention provides compounds which are useful as cysteine protease inhibitors. These compounds are characterized by their relatively low molecular weight, reversible inhibition, high potency and selectivity with respect to various types of cysteine 17 Printed from Mimosa 10/24/2000 13:39:28 page -19- proteases. The compounds can be implemented to prevent, alleviate and/or otherwise treat diseases which are mediated by the effects associated with the presence of cysteine proteases. Their usage is of particluar importance as they relate to various human treatments in vivo and as well as diagnostic tools in vitro.

Peptidyl inhibitors of serine proteases comprising serine protease binding moieties and certain keto-heterocycles have been previously described (see WO 96/16080). It has been surprisingly found that compounds comprising cysteine protease binding moieties and these keto-heterocycles are highly potent and specific inhibitors of a wide variety of cysteine proteases as well. The inhibiting activity can be directed against any cysteine protease by identifying the 10 binding moiety specific for that protease. The characteristics for the Pt... P„ residues (using substrate nomenclature by Schechter and Berger (Biochem. Biophys. Res. Commun. 27:157 (1967); Biochem. Biophys. Res. Commun. 32:898 (1968)), which define the minimum recognition sequence of enzymes for small synthetic peptide substrates or inhibitors are known for many enzymes or can be determined by measuring rates of hydrolysis of various substrates. 15 Some examples are listed in Table 1.

Table 1. Cysteine proteases and exemplary recognition elements.

Cysteine Protease PI P2 Other Reference Calpain I and II large hydrophobic e.g. Nva, Phe, Abu Leu, bulky aliphatic. hPhe 18 Calpain I Arg or Aig-mimetic, Lys, Tyr, Val, NIe, Tyi<0-Bzl), Leu, Abu, Phe /-butyl-Gly, Leu, ValhPhe 38 Papain hPhe, Arg or its mimetics, Agly, Aala bulky, non-polar, Phe 22 Cathepsins Cysteine (in general) bulky hydrophobic residues like hPhe, Phe, Met, Abu, Nva, or Arg and its mimetics Val, Leu 18,21,22,25 18 Printed from Mimosa 10/24/2000 13:39:28 page -20- PCT/US99/D8501 Cathepsin B hPhe, Phe, Tyr, SeKOBzl), Thr(OBzl), Cys(SBzl), Arg or its mimetics, Gly Phe, Arg or its mimetics, Leu, Tyr, Np2, Lys, Ornithine P3-large hydrophobic aromatic, lie 42 Cathepsin S Val, Nle, hPhe, Phe Leu, Phe, Val 42,43 Cathepsin L hPhe, Lys Phe, Leu 42 Cathepsin K Arg or its mimetics, hPhe, Leu Pro, Leu, Phe, Val P3 - Leu 44 Cathepsin H Arg or its mimetics, hPhe Caspases Asp P4 - determines the specificity within the caspase family 22,40 Interleukin-1 p converting enzyme Asp P3-Val, Glu or ester thereof P4 - Tyr, Leu 22,36,41,45,48 Caspase 3 (YAMA) Asp P4-Lys 40,41 Caspase 8 (FLICE) Asp Val P3 - Glu P4-Asp 40,41 Picomain 3C Gin or its derivatives (e.g. dimethylGln, Azogln), Glu and its derivatives Phe, Gly P3-Ik P4-small hydrophobic residues 9 Human Rhinovims 3C protease Gin or its derivatives (e.g. dimethylGln, Azogln), Glu and its derivatives Phe P3 - Leu 8 Hepatitis A Vims 3C proteinase Gin or its derivatives (e.g. dimethylGln, Azogln),' Glu and its derivatives Ala, Val, Leu, Nle, Phe P3 - Arg or its mimetics, Ala P4 - hydrophobic residues 14 Human Corona Virus protease Arg or its mimetics Val, Leu, Nle P3 - Asp or its esters 1 Hepatitis C Virus endopeptidase 2 Leu Leu P3 - Arg or its mimetics 8 19 Printed from Mimosa 10/24/2000 13:39:28 page -21- Ad2 23K protease Gly Gly P3 - Ala, P4-Leu 8 Trypanosoma, Leishmaitia protease Arg or its mimetics, Lys, Tyr, Ala Phe, Leu, Val P3 - Pro .Val. Leu 23 Picomain 2A Gin or its derivatives (e.g. dimethylGln, Azogln), Glu and its derivatives, Tyr, Val, Ala Thr, Gly, Pro P3 - Ala 9,46 Gingipain K Lys, Om ,47 Gingipain R Arg or its mimetics Pro, Leu P3-Lcu Malarial hemoglobinase hPhe, Arg or its mimetics, Lys, Val, Ser(OBzl), ImNva, Tyr large hydrophobic residues, e.g. Phe, Leu P3 - Val 23a Nva=norvaline; Abu=a-aminobutyric acid; Agly=azaglycyl; Aala=azaalanyl; Np2=2-naphthylalanine; Nle=norleucine; Eps=epoxysuccinyl In addition to altering the binding moiety Z, the substituent on the heterocycle (i.e., R,) can be varied to further increase the specificity of these compounds toward the desired cysteine IS protease.

By way of example, the compound CM-00I9 comprises the binding moiety specific for papain and a substituted 1,3,4-oxadiazole: CM-0019 Printed from Mimosa 10/24/2000 13:39:28 page -22- By way of further example, the compounds CQ-0010 and CQ-0011 inhibit caspases. Compounds CQ-0002 and CQ-0008 are analogs of leupeptin, the structure of which is provided below for comparison. o .r^CH, o O 3 O CH3 CQ-0002 Compounds CQ-0004,0008,0010 and 0011 are represented below: -o N^-CHJ ^OH ch3 i CHJn N-o O ^-CHj O CQ40I0 OK CHj CQ400S NH n K NH, a,, KKy° V"~>, ° >0 ^ C<H»11 21 Printed from Mimosa 10/24/2000 13:39:28 page -23- Other specific inhibitors include compounds CE-2072, CE-2060 and CE-2061, which have shown inhibitory activity against picomain 2A protease (100% inhibition at 100 |iM): The compounds of the present invention, salts thereof, and their intermediates can be prepared or manufactured as described herein or by various methods known in the chemical art, as well as by extension and modification of methods previously described (see WO 96/16080, incorporated herein by reference). 22 Printed from Mimosa 10/24/2000 13:39:28 page -24- An alternative method has been used where suitably protected peptides are converted by the action of an activating coupling reagent such as BOP-CI or HBTU to a Weinreb amide. The Weinreb amide is then reacted with a 5-substituted 2-lithio-l,3,4-oxadiazole at appropriate temperatures ranging from -78°C to -25°C in a suitable solvent such as THF or ether to provide 5 the desired keto-oxadiazoles in a single step. Protecting groups, if present, are then removed to provide the enzyme inhibitors in an efficient and convergent manner. A number of efficient methods to synthesize 5-substituted 1,3,4-oxadiazoles are known in the art. Conveniently, these compounds can be synthesized in a single step by refluxing hydrazides of common carboxylic acids with excess ethyl orthofoimate at high temperature. The excess orthoformate is hydrolyzed 10 in the workup and the 5-substituted 1,3,4-oxadiazoles are often obtained in essentially pure form without further purification necessary. This entire method of synthesis is illustrated in general form in scheme 1 below. Instances where R2 correlates to the amino acid side chains of aspartic acid, arginine, and alanine are provided in the Examples.

Scheme 1.

OCH, 23 Printed from Mimosa 10/24/2000 13:39:28 page -25- where AA„ means AA2... AAS.

The compounds described herein are useful in inhibiting the activity of cysteine proteases, by contacting the compound with the targeted protease, either in an in vivo or an in vitro environment. As used herein, the term "contacting" means directly or indirectly causing the 5 inhibitor and the protease to come into physical association with each other. Contacting thus includes physical acts such as placing the inhibitor and protease together in a container, or administering the inhibitors to a patient. Thus, for example, administering a compound of the invention to a human patient evidencing a disease or disorder associated with abnormal and/or aberrant activity of such proteases in a method for inhibiting the enzymatic activity of such 10 proteases which are associated with disease or disorder, falls within the scope of the definition of the term "contacting." Pharmaceutically acceptable salts of the cysteine protease inhibitors also fall within the scope of the compounds as disclosed herein. The term "pharmaceutically acceptable salts" as used herein includes organic and inorganic acid addition salts such as chloride, acetate, maleate, 1S fumarate, tartrate and citrate. Examples of pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt or potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of pharmaceutically acceptable ammonium salts are ammonium salt, trishydroxymethylaminomethane and tetramethylammonium salt. Examples of pharmaceutically acceptable amino acid addition salts 20 are salts with lysine, glycine and phenylalanine.

Cysteine proteases which may be inhibited by the compounds described herein include mammalian, bacterial, parasite, viral, fungal, insect and plant cysteine proteases. Cysteine proteases include papain, actinidain, aleurain (barley), allergen (Dermatophagoides), allergen (Euroglyphus), ananain (Ananas comosus), asclepain (Asepias syriaca), bleomycin hydrolase, 25 calotropin (Calotropis), caricain, clostripain, cathepsin B, cathepsin H, cathepsin L, cathepsin S, cathepsin O, cathepsin K, cathepsin T, chymopain, cysteine aminopeptidase (Lactococcus), cysteine endopeptidases 2 and 3 (barley), cysteine endopeptidases (Brassica napus), cysteine endopeptidase (Caenorhahditis), cysteine endopeptidases 1 and 2 (Dictyostelium), cysteine endopeptidase (Entamoeba), cysteine endopeptidases 1 and 2 (Haemonchus), cysteine 24 Printed from Mimosa 10/24/2000 13:39:28 page -26- PCMJS99/08501 endopeptidase (Hemerocallis), cysteine endopeptidases 1,2 and 3 (Homarus), cysteine endopeptidase (Leishmania), cysteine endopeptidase (mungbean), cysteine endopeptidase (Ostertagia), cysteine endopeptidase (pea), cysteine endopeptidase (Plasmodium), cysteine protease tpr (Porphyromonas), cysteine endopeptidase (Tetrahymena), cysteine endopeptidase (Theileria), cysteine endopeptidase (tobacco), cysteine endopeptidase (Trypanosoma), dipeptidyl peptidase I, endopeptidase (baculovirus of Autographa), endopeptidase EP-C1 (Phaseolus vulgaris), glycyl endopeptidase, oryzain (includes a, p and y) (rice), bromelain (including stem-and fruit bromelain), ficin, thaumatopain (Thaumatococcus); gingipain R and gingipain K; calpains, including calpain (Schistosoma), calpain I, calpain II, calpain p94, calcium-binding protein PMP41, sol gene product (Drosophila); streptopain and cysteine endopeptidase (Porphyromonas)-, picomain 2A, picomain 3C, apothovirus endopeptidase, caxdiovirus endopeptidase, comovirus endopeptidase, nepovirus endopeptidase; tobacco etch virus NIa endopeptidase, hepatitis C virus endopeptidase 2, adenovirus endopeptidase; tobacco etch virus HC-proteinase; chestnut blight virus p29 endopeptidase; chestnut blight virus p48 endopeptidase; sindbis virus nsP2 endopeptidase; mouse hepatitis virus endopeptidase, avian infectious bronchitis virus endopeptidase; a-clostripain; ubiquitin carboxyl-terminal hydrolase; deubiquinating enzyme (DOA4 protein), ubiquitin-specific processing peptidase 1, ubiquitin-specific processing peptidase 2, ubiquitin-specific processing peptidase 3, tre oncogene protein (human), unp protein (mouse); hemoglobinase (Schistosoma), legumain (jack bean); interleukin converting enzyme and caspases, such as caspase 2 (ICH-1), caspase 3 (CPP32, YAMA), caspase 4 (ICErel-II), caspase 5 (ICErel-III), caspase 6 (Mch2), caspase 7 (Mch3), caspase 8 (FLICE, Mch5), caspase 9 (Mch6, ICE-LAP6), caspase 10 (Mch4); pyroglutamyl-peptidase I; microsomal ER60 protein endopeptidase; prepilin leader peptidase; PRRS arteritis virus PCP «-endopeptidase, equine arteritis virus Nsp2 endopeptidase; foot and mouth disease virus L proteinase; hepatitis A viral protease; human corona virus protease; encephalomyelitis virus endopeptidase; malarial hemoglobinase; drosophila hedgehog virus gene product; dipeptidyl peptidase I (cathepsin C); Derpl (dust mite); Y-glutamyl hydrolase; Actinide (Actinidia); yeast cysteine proteinase E, yeast proteinase D, yeast proteinase F; cancer procoagulant; and histolysis Enzyme inhibitors for cysteine proteases may be useful as potential therapeutic Printed from Mimosa 10/24/2000 13:39:28 page -27- drugs for humans or animals, as diagnostic or research tools, as antibacterial agents, herbicides, fungicides or pesticides. Potential indications for cysteine protease inhibitors described herein, used in prophylaxis, cure or therapy, include: Cardiovascular disorders-ischemia reperfusion injury from transplantation and/or vascular surgery, angiogenesis, neovascularization, acute cardiac allograft dysfunction, ischemic cardiac damage, chemotherapy-induced myocardial suppression; Inflammatory disorders - rheumatoid arthritis and other inflammatory aithritidies, inflammatory bowel disease, inflammatory peritonitis, sepsis, systemic inflammatory response syndrome, multiple organ failure; Musculoskeletal disoiders—osteoarthritis, osteoporosis, muscular dystrophy, myositis; Neurological disorders- multiple sclerosis, stroke, Alzheimer's disease, prion-associated disorders, ataxia telangiectasia, central nervous system injury; Pulmonary disorders- asthma, COPD, adult respiratory distress syndrome, Wegener's granulomatosis, emphysema; Allergic, immunologic and autoimmune disorders-house dust mite allergy, transplant rejection, graft verses host disease, Type 1 diabetes mellitus, autoimmune thyroiditis, psoriasis, antibody-mediated autoimmune diseases, lupus erythematosus, Sjogren's syndrome, autoimmune encephalomyelitis; Solid tumors, lymphomas, leukemias and other malignancies and related disorders—acute and chronic myelogenous leukemia, neuronal cancer, cancer invasion and metastasis, tumor angiogenesis, B and T cell lymphomas, acute and chronic lymphocytic leukemia, resistance to chemotherapy, cancer associated coagulopathies (including deep venous thrombosis, coronary artery disorder, pulmonary embolism, disseminated intravascular coagulation), Hodgkins disease, carcinomas of the colon, liver, lung, breast, kidney, stomach, pancreas, esophagus, oral pharynx, intestine, thyroid, prostate, bladder, brain; osteo-sarcoma, chondro-sarcoma and liposarcoma; neuroblastoma; melanoma; and carcinomas derived from amnion and/or chorion); Infectious diseases and associated syndromes-septic shock (including Gram-negative sepsis), HIVinfection and AIDS, genital herpes, zoster, chickenpox, EBV infections and encephalitis, CMV-choreoretinitis or encephalitis, cytomegalovirus infections in neonates 26 Printed from Mimosa 10/24/2000 13:39:28 page -28- (including related pneumonitis), opportunistic infections in immunocompromised individuals (including AIDS and transplant patients), dysentery, hepatitis C, hepatitis A, keratoconjuctovitis, bronchopneumonia (including pneumonia in immunocompromised individuals), gastroenteritis, malaria, rhinovirus, polio, enterovirus infections, common cold, aseptic meningitis, foot and 5 mouth disease, Klebsiella pneumonia infection, escherichia coli or staphylococcus epidermidis, leprosy bacteremia, otitis media, lambliasis, non-atopic sinusitis, fulminant hepatitis; Kidney disorders-polycystic kidney disease, glomerulonephritis; Other miscellaneous disorders—periodontal disease, alcohol hepatitis, prostate hypertrophy, trauma, cutaneous mastocytosis, radiation- and HIV-induced diarrhea, cachexia 10 (including acompanying cancer and malnutrition).

Examples of cysteine proteases and associated disease are described in Table 2.

Table 2.

Cysteine Protease Disease State References Interleukin lp converting Stroke, traumatic brain Patel, et al., FASEB, 10:587- enzyme (ICE, Caspase 1) injury, organ transplant 597 (1996);. rejection and septic shock.

Barretal., Bio/Technology, Inflammatory disorders 12:487-493 (1994); Epstein, including the arthritides, such New Eng. J. Med., 328:106- as rheumatoid arthritis and 113 (1993). osteoarthritis, inflammatory bowel disease, ulcerative colitis, pancreatitis, and inflammatory peritonitis, asthma. 27 Printed from Mimosa 10/24/2000 13:39:28 page -29- YAMA (Apopain, CPP32, Diseases in which Barret al., Bio/Technology, Caspase 3), FLICE (Mch5, disregulated apoptosis plays a 12:487-493 (1994) Caspase 8), and other role in pathology: caspases Solid tumors, B cell lymphoma, chronic lymphocytic leukemia, prostate hypertrophy, preneoplastic liver foci, resistance to chemotherapy, stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, prion-associated disorders, ataxia telangiectasia, ischemic cardiac damage, chemotherapy-induced myocardial suppression, AIDS, type I diabetes, lupus erythematosus, Sjogren's syndrome, glomerulonephritis, dysentery, inflammatory bowel disease, radiation- and HIV induced diarrhea, polycystic kidney disease, anemia or erythropoiesis.

Malarial Hemoglobinase Malaria Rockett, et al., FEB, 259:257- 259(1990) 28 Printed from Mimosa 10/24/2000 13:39:28 page -30- Derpl Asthma, house dust mite Kalsheker, et al., Biochem. allergy Biophys. Research Comm., 221:59-61 (1996) Gingipain K and Adult Periodontitis Wingrove, et al., J. Biol Gingipain R Chem., 267:18902-18907 (1992); DiScipio et al., Immun., 87:660-667 (1996) Cathepsin B, Cathepsin L, Osteoarthritis, osteoporosis, Velasco, et al., J. Biol.

Cathepsin S, Cathepsin 0 and rheumatoid arthritis, Chem., 269:27136-27142 Cathepsin K Alzheimer's disease, cancer (1994); Takeda et al., FEBS invasion and Metastasis, Letters, 359:78-80 (1995); Parkinson's disease, Elliott et al., Persp. in Drug leukemia, lymphoma, Disc, and Des., 6:12-32 hodgkin's disease, tumors, (1996) including those of the bladder, brain, lung, pancreas, prostate, stomach and thyroid Cancer Procoagulant Cancer (including carcinomas Alessio et al., Eur. J. of the liver, lung, breast, Haematology, 45:78-81 kidney, colon, kidney; osteo-, (1990); Gordon, Seminars in chondro-, and liposaicoma; Thrombosis and Hemostasis, neuroblastoma; melanoma; 18:424-433 (1992) nonlympocytic leukemia; lymphocytic leukemia) 29 Printed from Mimosa 10/24/2000 13:39:28 page -31- Calpain I and II Osteoporosis, stroke, CNS injury, Alzheimer's disease Additionally, diseases involving dysregulated Karlsson, et al., Neurobiology of Aging, 16:901-906(1995); Squier, et al., J. Cell.

Physiol., 159:229-237 apoptosis as listed for caspase above. (1994).

Calpain p94 Muscular dystrophy Calpain p94 and limb-girdle muscular dystrophy, ICOP Letters 1996.

Hepatitis C Virus Endopeptidase 2 and Hepatitis C Virus NS3 Endopeptidase Hepatitis C Grakoui, et al., Proc. Nat. Acad. Sciences, 90:10583-10587(1993) Picomain 2A and Picomain Rhinovirus, polio, enterovirus Palmenberg, J. Cell. 3C Proteases infection, common cold, aseptic meningitis, polio Biochem. 33:191-198 (1987); Cordingley, et al., J.

Virology, 5037-5045 (Dec 1989) Hepatitis A Viral Protease Hepatitis A Krausslich, et al., Annu. Rev.

Biochem., 57:701-754 (1988).

Foot and Mouth Disease Foot and mouth disease Roberts et al., Virology Virus L Protease (Cattle) 213:140-146(1995) Although the compounds described herein and/or their salts may be administered as the pure chemicals, it is preferable to present the active ingredient as a pharmaceutical composition. The invention thus further provides the use of a pharmaceutical composition comprising one or Printed from Mimosa 10/24/2000 13:39:28 page -32- more compounds and/or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and or prophylactic ingredients. The camer(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

Pharmaceutical compositions include those suitable for oral, topical or parenteral (including intramuscular, subcutaneous and intravenous) administration. The compositions may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with liquid carriers, solid matrices, semi-solid 10 carriers, finely divided solid carriers or combination thereof, and then, if necessary, shaping the product into the desired delivery system.

Pharmaceutical compositions suitable for oral administration may be presented as discrete unit dosage forms such as hard or soft gelatin capsules, capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or as granules; as a 15 solution, a suspension or as an emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art., e.g., with enteric coatings.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, 20 solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservative.

The compounds may also be formulated for parenteral administration (e.g., by injection, 25 for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small bolus infusion containers or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained 31 Printed from Mimosa 10/24/2000 13:39:28 page -33- by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the compounds may be formulated as ointments, creams or lotions, or as the active ingredient of a transdermal patch. Suitable 5 transdermal delivery systems are disclosed, for example, in Fisher et al. (U.S. Patent No. 4,788,603) or Bawas et al. (U.S. Patent No. 4,931,279,4668,504 and 4,713,224). Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, 10 dispersing agents, suspending agents, thickening agents, or coloring agents. The active ingredient can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122,4,383,529, or 4,051,842.

Compositions suitable for topical administration in the mouth include unit dosage forms such as lozenges comprising active ingredient in a flavored base, usually sucrose and acacia or 15 tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the active ingredient in a suitable liquid carrier.

When desired, the above-described compositions can be adapted to provide sustained release of the active ingredient employed, e.g., by combination thereof with certain hydrophilic 20 polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.

The pharmaceutical compositions according to the invention may also contain other adjuvants such as flavorings, coloring, antimicrobial agents, or preservatives.

The compositions may also be administered via inhalation, using a suitable delivery vehicle.

It will be further appreciated that the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. 32 Printed from Mimosa 10/24/2000 13:39:28 page -34- PCT/US99/D8501 In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg/day, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; for example, containing to 1000 mg,' conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 pM, most preferably, about 1 to 50 nM, most preferably, about 2 to about 30 |iM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

The inhibitors described herein may be also used for the detection and quantification of the activity of a cysteine protease in a pure sample, mixture or biological fluid or tissue. The activity can be measured with a protease substrate in the absence and presence of a known concentration of the inhibitor. Specific inhibitors can also be used to confirm that the observed activity is due to a particular protease.

The inhibitors described herein may also be used to identify and purify cysteine proteases. The inhibitors can be covalently linked to a solid support, such as an affinity column or beads used in batch methods, and used to purify a protease or enrich a mixture containing the protease. The inhibitor may be linked to the solid support or bead either directly or via a linker of variable length, such that linkage does not interfere with the binding properties (see, e.g., 33 Printed from Mimosa 10/24/2000 13:39:28 page -35- WO 99/54317 PCT/US99/08501 Thomberry, N., Methods in Enz., 244:615-31 (1994)) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of 5 the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

The following examples are given to illustrate the invention and are not intended to be inclusive in any manner: Examples Abbreviations used herein are defined as follows: DMF • dimethylformamide; HBTU - 2-( 1 H-benzotriazole-1 -yl)-1,1,3,3-tetramethyluronium 15 hexafluorophosphate; DIEA - diisopropylethylamine; THF - tetrahydrofuran; CH3CN - acetonitrile; EDTA-Na2 - ethylenediaminetetraacedc acid disodium salt; Mtr - 4-methoxy-2,3,6-trimethylbenzene sulfonyl; Bop-Cl • bis(2-oxo-3-oxazolidinyl)phosphinic chloride; EtOH -ethylalcohol; EtOAc • ethylacetate; LDA - lithium diisopropylamide; EDCI - l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NMM - N-methyl morpholine; 20 HOBT - 1-hydroxybenzotriazole; TFA - trifluoroacetic acid.

Example I - Synthesis of [[2-[5-(3-methylbenzyl)-l,3,4-oxadiazolyl]carbonyl]-2-(S)-methylpropyl]-L-phenylalanamide-(3R)-(isobutyl)succinic acid (CM-0019).

The intennediate (benzyloxycarbonyl)-L-valyl-N-[l-(2-[5-(3-methylbenzyl)-l,3,4-25 oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-I-proliBamide was prepared as follows: a. 3-(S)-Amino-2-(R,S)-hydraxy~4-methyl pentanoic acid.

To a solution containing 3-(S)-[(benzyloxycaibonyl)amino]-2-acetoxy-4-methylpentanenitrile (see example 1 of WO 96/16080) (15.2 g, 50.0 mmol) in 183 mL of dioxane was added 183 mL of concentrated hydrochloric acid and 7.45 mL of anisole. The 34 Printed from Mimosa 10/24/2000 13:39:28 page -36- reaction mixture was heated to reflux ovemight. The hydrolysis reaction was allowed to cool to room temperature and then concentrated in vacuo. The resulting aqueous solution was extracted with ether (2X). The aqueous phase was placed on a Dowex 50X8-100 column (H+ form, preeluted with deionized water to pH = 7). The column was eluted with 2.0 N ammonium hydroxide and the pure fractions concentrated to afford 5.53 g (75 %) of 3-(S)-amino-2-(& S)-hydroxy-4-methylpentanoic acid as a pale yellow solid. FAB MS [M+H] m/z; Calcd: 148, Found: 148. b. 2-(S)-[(Berayloxycarbonyl)amino]-2-(R,S)-hydroxy-4-methyl pentanoic acid To a solution under an atmosphere of nitrogen containing 1.0 g (6.8 mmol) of 3-(S)-amino-2-(/f,S)-hydroxy-4-methylpentanoic acid in 9.5 mL of 1 N NaOH and 10 mL of dioxane was added 1.43 g (8.4 mmol) of benzyl chlorofoimate. The pH was maintained above pH 8 with 1 N NaOH as needed. The reaction mixture was allowed to stir at room temperature ovemight The reaction was diluted with water and washed with ether. The aqueous layer was acidified with 1 N HC1 to pH = 2 and extracted with ether (2X). The combined organic layers were dried over magnesium sulfate, filtered and evaporated in vacuo to afford 1.75 g (92%) of 3-(S)-[(benzyloxycarbonyl)amiiio]-2-(iJfS)-hydroxy-4-methylpentanoic acid as a light yellow viscous oil. FAB MS [M+H] m/z; Calcd: 282, Found: 282. c. 3-(S)-[(Benzyloxylcarbonyl)amino]-2-(R,S)-acetoxy-4-methyl pentanoic acid To a solution of 3-(S)-[benzyloxycarbonyl)amino]-2-(.R,S)-hydroxy-4-methylpentanoic acid (1.70 g, 6.04 mmol) and pyridine (4.9 mL) was added acetic anhydride (5.7 mL, 6.17 g, 60.4 mmol) dropwise at room temperature. The reaction was allowed to stir ovemight and was diluted with ethyl acetate and washed with water (2X). The organic layer was dried over magnesium sulfate, filtered and evaporated in vacuo to give a thick oil. The residue was purified by column chromatography on silica gel with 15% methanol/dichloromethane to afford 1.56 g (80%) of 3-(5>[(benzyloxycarbonyl)amino]-2-(& <S)-acetoxy-4-methyl pentanoic acid as a light yellow viscous oil. FAB MS [M+H] m/z; Calcd: 324, Found: 324. d. l-[(3-Methylphenylacetyl)-2-(2-(R,S)-acetoxy)-3-(S)-[(benzyloxycarbonyl)aminoJ-4-methylpentanoyl] hydrazine.

To a solution containing 3-(S)-[(bcnzyloxycarbonyl)amino]-2-(/J,S)-acetoxy-4- Printed from Mimosa 10/24/2000 13:39:28 page -37- methylpentanoic acid (2.3 g, 7.11 mmol) in 40 mL of DMF under a nitrogen atmosphere at 0°C was added 1.31 g (9.69 mmol) of HOBT and 1.36 g (7.09 mmol) of EDCI. After stirring for 30 minutes, 1.20 g (7.31 mmol) of 3-methylphenyl acetic hydrazide [prepared analogously to the monoacid hydrazides cited by Rabins et al. {J. Org. Chem., 30:2486 (1965))] and 1.0 mL (9.10 5 mmol) of NMM were added. The reaction was allowed to warm to room temperature and stir overnight. The reaction was diluted with ethyl acetate and washed with 5% potassium hydrogen sulfate, saturated sodium bicarbonate, brine and water. The organic phase was dried over magnesium sulfate, filtered and evaporated under reduced pressure. The residue was purified by column chromotography on silica gel with 10% methanol/dichloromethane to afford 2.31 g 10 (89.0%) of the title compound as a white solid. FAB MS [M+H] m/z; Calcd: 470, Found: 470. e. ]-[2-[5-(3-MethyIbenzyl)-l, 3,4-oxodiazolyl]-l-acetoxy-2-(S)-(benzyloxycarbonyl)amino]-3-methylbutane.

A solution containing 2.31 g (4.92 mmol) of l-[(3-methylpheny!acetyl)-2-(2-(R, S)-acetoxy)-3-(S)-[(benzyloxycarbonyl)amino]-4-methylpentanoyl]hydrazine in 25 mL of pyridine 15 and 1.88 g (9.86 mmol) of toluene sulfonyl chloride was heated at reflux under a nitrogen atmosphere for 72 hours. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate and washed with water. The organic phase was dried over magnesium sulfate, filtered and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with 5% ethyl acetate/hexane to afford 1.41 g (63.5%) of the title 20 compound. FAB MS [M+H] m/z; Calcd: 452, Found: 452. f. l-[2-[5-(3-Methylbenzyl)-1.3,4-oxadiazolyl)]-2-(S)-(benzyloxycarbonyl)amino]-3-methylbutan-l-ol.

A solution containing 1.80 g (3.99 mmol) of l-[2-[5-{3-methylbenzyl)-1,3,4-oxadiazolyl)-l-l-acetoxy-2-(5Mbenzyloxycarbonyl)amino]-3-methylbutane and 0.72 g (5.21 mmol) of 25 potassium carbonate in 30 mL of methanol and 8 mL of water was allowed to stir at room temperature for 30 minutes. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate and washed with water. The organic phase was dried over magnesium sulfate, filtered and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with 60% ethyl acetate/hexane to afford 1.46 (89.3%) of the title 36 Printed from Mimosa 10/24/2000 13:39:28 page -38- compound. FAB MS [M+H] m/z; Calcd: 410, Found: 410. g. l-[2-[5-(3-Methylbenzyl)-l, 3,4-oxadiazolyl]-2-(S)-Amino-3-methylbutan-l-ol hydrochloride.

To a solution containing 1.31 g (3.20 mmol) of 1 -[2-[5-(3-methylbenzyl)-l ,3,4-5 oxadiazolyl]-2-(S)-(benzyloxycarbonyl)amino]-3-methylbutan-l-ol in 25 mL of trifluoroacetic acid under a nitrogen atmosphere at 0°C was added 0.43 mL (3.94 mmol) of thioanisole. The' reaction was allowed to warm to room temperature overnight The solvent was removed under reduced pressure and the residue dissolved in ether and cooled to -78 °C under a nitrogen atmosphere. To this solution was added 3 mL (3 mmol) of 1N hydrochloric acid in ether. The 10 resulting white solid was allowed to settle and the ether decanted. Additional ether was added and decanted (3X). The solid was dried under vacuum to afford 0.92 g (92.2%) of the title compound. FAB MS [M+H] m/z; Calcd: 276, Found: 276. (4S)-4-Benzyl-3-[4 '-(methyl)pentanoylJ-2-oxazolidinone IS a. 4-Methylvaleric acid (6.S6 g, SS.6 mmol) was dissolved in diy CH2C12(40 ml) under N2 and chilled to 4°C. Oxalyl chloride (S.4 mL, 63.5 mmol) was added; followed by 4 drops of dry DMF. Rapid CO2 evolution occured. The reaction mixture was allowed to warm to ambient temperature over 2 h; no more C02 evolution was apparent. Solvents were stripped by rotary evaporation and the acid chloride was distilled in vacuo. 'H-NMR (300 MHz, CDC13) 6 0.88-20 0.93 (m, 6H), 1.59-1.64 (m, 3H), 2.90 (t, 2H, J= 7.5 Hz). b. (S)-(-)-4-benzyl-2-oxazolidinone (8.93 g, 50.4 mmol) was dissolved in dry THF under N2 and chilled to -78°C. nButyl lithium (1.6 M in hexane, 32 mL, 50.4 mmol) was added dropwise to maintain temperature < -70°C. The mixture was stirred 25 rain at -78°C, then a solution of the acid chloride prepared above in dry THF (30 mL) was added dropwise to maintain 25 temperature <-65°C. The reaction mixture was stirred overnight and allowed to warm to 15°C. The reaction was quenched by careful addition of saturated NH4CI (70 mL). THF was removed under reduced pressure and the resultant aqueous slurry was extracted with EtOAc (100 mL). The organic layer was washed with 0.5 N NaOH, H20, brine. The organic layer was dried over MgS04, filtered and evaporated in vacuo to return 12.7 g of crude yellow oil. The crude material 37 Printed from Mimosa 10/24/2000 13:39:28 page -39- PCT/US99/D8501 was purified by silica gel chromatography (10% EtOAc/hexane) and dried in vacuo to return 9.0 g (69% yield) of pale yellow oil. C-18 HPLC RT = 16.5 min., 96% pure at 215 mn (10-100% solv. B/25 min; solvent A= 0.1% (v/v)TFA/H20; solvent B = 0.1% TFA/acetonitrile; FAB-MS m/z 276(M+H)*; 'H-NMR (300 MHz, CDC13) <50.94 (d, 6H, 7=6.3 Hz, e-[(CH3)J), 1.53-1.72 5 (m, 3H, P CH2,g CH), 2.76 (dd, 1H, 7=13.3,9.6 Hz, oxazolidinone 5-CHH), 2.90-2.97 (m, 2H), 3.29 (dd, 1H,7=13.2,3.3 Hz, oxazolidinone 5CJHH), 4.15-4.20 (m, 2H), 4.64-4.70 (m, 1H, oxazolidinone 4-CH), 7.17-7.39 (m, 5H, Ph-H). c. (4S)-4-Benzyl-3-(2'R)-2'-[[(tert-butoxycarbonyl)methyl]-4'-(methyl)pentanoyl]-2-10 oxazolidinone.

Diisopropylamine (5.05 mL, 36 mmol) was diluted with diy THF (20 mL) and chilled to -20°C under N2. n-Butyl lithium (1.6 M in hexane, 23 mL, 36 mmol) was added dropwise to maintain the temperature <-10°C. The temperature was increased to 4°C and stirred 30 minutes to generate LDA. The flask was chilled to -78°C and (4S)-4-Benzyl-3-[4'-(methyl)pentanoyl]-15 2-oxazolidinone in diy THF (15 mL) was added dropwise to maintain the temperature < -70"C. The reaction was stirred 30 minutes at -78 °C then t-butylbromoacetate (4.9 mL, 33 mmol) in dry THF was added dropwise to maintain the temperature <-65°C. The mixture was stirred and allowed to warm to -10°C overnight. After 15 hours, the reaction was quenched by careful addition of water followed by evaporation of the THF. Water (100 mL) was added to the sluny. 20 and the crude mixtrure was extracted with EtOAc (100 mL). The organic layer was washed with water and brine; then dried over MgS04, filtered and dried in vacuo to leave 13.3 g crude yellow oil. Silica gel chromatography in 15% EtOAc/hexane returned 7.84 g (61% yield) of a white solid product.

C-18 HPLC RT = 19.5 min., 99% pure at 215 nm (10-100% solv. B/25 min; solvent A= 0.1% 25 (v/v)TFA/H20; solvent B = 0.1% TFA/acetonitrile; FAB-MS m/z 390 (M+H)+, 334 (M-tBu+H)*. 'H-NMR (300 MHz, CDC13) <50.92 (d, 3H, 7- 6.0 Hz), 0.94 (d, 3H, 7=5.8 Hz), 1.28-1.40 (m, 1H, CHMe2), 1.43 (s, 9H3), 2.49 (dd, 1H, 7=16.7,4.6 Hz), 2.72 (dd, 1H,7=10.3,1.1 Hz), C02C (CH3)j), 2.76 (dd, 1H, 7=10.3,2.2 Hz, Ph-CHH), 3.35 (dd, 1H, 7=13.5,3.1 Hz), 4.15-4.18 (m, 2H), 4.21 -4.26 (m, 1H), 4.52-4.61 (m, 1H), 7.27-7.34 (m, 5H, Ph-H). 38 Printed from Mimosa 10/24/2000 13:39:28 page -40- d. (2R)-2-[(tert-Butoxycarbonyl)methylJ-4-(methyI)pentanoic acid (4S)-4-BenzyI-3-(2'R)-2'-[[(/e/7-butoxycarbonyl)methyl]-4,-(methyl)pentanoyl]-2-oxazolidinone (5.89,15.1 mmol) was dissolved in dry THF(100 mL) and water (25 mL) was added. The mixture was chilled to 4 °C under N2. H202 (7.6 mL) was added followed by dropwise addition of LiOH (0.76 g, 18.2 mmol) in H20 (20 mL) over 20 minutes. The mixture was stirred for 1 hour and allowed to warm to ambient temperature. The mixture was again chilled in an ice bath and quenched by addition of NajSOj (3.1 g) in water (20 mL). THF was removed by rotovap, the remaining aqueous layer was washed with EtOAc (4x70 mL), then acidified to approx. pH 2 with conc. HC1 after layering with fresh EtOAc. The mixture was immediately extracted with EtOAc (3x80mL). The combined EtOAc extracts were dried over MgSO<, filtered, and evaporated to return 3.29 g clear oil (95% crude yield) which showed no traces of starting material by HPLC. This material was used without further purification. 'H-NMR (300 MHz, CDC1,) <50.90 (d, 3H, 7=6.4 Hz, CH3), 0.94 (d, 3H, 7=6.5 Hz, CH3), 1.20-1.48 (m, 1H, CHMej), 1.44 (s, 9H, C(CH3)3), 1.50-1.72 (m, 2H), 2.37(dd, IH,7=16.4,2.4 Hz), 2.59 (dd, 1H, 7=16.4,2.6 Hz), 2.80-2.95 (m, IH). e. Tert-butyl(3R)-3-(isobutyl)succinyl-L-phenylalanyI methyl ester.

To a solution of tert-butyl-(3R)-3-(isobutyl)succinate (10.82 g, 47.0 mmols), in 90 ml of dry DMF was added HBTU (17.45 g, 46.0 mmols), followed by DIEA (18.43 g, 142.6 mmols). After stiiring for 10 min, L-phenylalanine methyl ester hydrochloride (10.0 g, 46.36 mmols) was added. This was allowed to stir at room temperature ovemight. The solvent was removed under reduced pressure and the residue dissolved in 200 ml ethyl acetate. This solution was washed with water, 1 M HC1 (2x), saturated NaHC03 (2x), brine, and the organics were dried with anhydrous MgS04. The mixture was filtered and the solvent removed under reduced pressure. The residue crystallized to an off-white solid upon sitting overnight, giving 13.3 g (74%) of tert-butyl(3R)-3-(isobutyl)succinyl-L-phenylalanyl methyl ester. C-18 HPLC RT = 16.9 min., 98% pure at 215 nm (10 to 100% solvent B/25 min; solvent A = 0.1% TFA/HZ0; solvent B = 0.1% TFA/acetonitrile).

FAB-mass spectrum: m/z (M+H)* = 392; Theory = 392. 39 Printed from Mimosa 10/24/2000 13:39:28 page -41- PCT/US99/D8501 'H NMR (CDClj) 5 [0.85 (d, J= 7.5 hz); 0.88 (d, J= 7.5 hz); 6H]; [1.10-1.23 (m, IH)]; [1.44 (s, 9H)]; [1.45-1.65 (m, 2H)]; [2.22-2.32 (m, IH)]; [2.50-2.66 (m, 2H)]; [3.10 (d, J= 6 Hz) 2H]; [3.69 (s,3H)]; [4.82-4.92 (m, IH)]; [6.19 (d, J= 9 hz), IH]; [7.13-7.33 (m, 5H)]. f. Tert-buty!(3R)-3-(isobutyl)succinyl-L-phenyIalanine.

A solution of tert-butyl(3R)-3-(isobutyl)succinyl-phenylalanyl methyl ester (2.0 g, 5.10 mmols) in 5 ml methanol was cooled to 4°C in an ice bath. To this solution was added 4 ml of an aqueous solution of lithium hydroxide (333 mg, 7.94 mmols.), and this solution was stined and allowed to waim to room temperature ovemight. The solution was concentrated to an oil 10 under reduced pressure. The residue was dissolved in 100 ml ethyl acetate, washed with 10% citric acid, water, and dried with anhydrous MgSO,. The mixture was filtered and the solvent was removed under reduced pressure, vacuum dried ovemight to give 1.8 g (95%)of tert-butyI(3R)-3-(isobutyl)succinyl-L-phenylalanine as a light yellow oil. C-18 HPLC RT - 14.7 min., 95% pure at 254 nm (10 to 100% solvent B/25 min; solvent A = 0.1% TFA/H20; solvent B 15 =0.1% TFA/acetonitrile).

FAB mass spectrum: M+H = 378; theory = 378.

•H NMR (CDC13) 8 [0.83 (dj = 6.0 hz); 0.85 (d, J = 6.0 hz); 6H]; [1.10-1.25 (m, IH)]; [1.44 (s, 9H)]; [1.45-1.65 (m, 2H)]; [2.24-2.35 (m, IH)]; [2.48-2.58 (m, 2H)]; [3.07-3.27 (m,2H)]; [4.79-4.93 (m, IH)]; [6.36 (d, J = 9 Hz), 1HJ; [7.20-7.41 (m, 5H)]. g. Tert-butyl(3R)-3-(isobutyl)succinyl-[[2-[5-(3-methylbenzyl)-I,3,4-oxadiazolyl]-(R,S)-hydroxymethyl]-2-(S)-methyIpropylJ-L-phenylalaninamide.

To a solution of tert-butyl(3R)-3-(isobutyl)succinyl-phenylalanine (1.8 g, 4.80 mmols) in 40 ml1 DMF was added HOBT (676 mg, 5.0 mmols). This was cooled in an ice bath to 4"C. 25 EDCI (921 mg, 4.80 mmols) was then added. After stirring for 30 minutes, a solution of [2-[5-(3-methylben2yl)-l,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-l-(R,S)-ol hydrochloride^.50 g, 4.24 mmols) in 20 ml. DMF was added dropwise, followed by N-methyl moiphoiine (0.77 g, 7.66 mmols) and the reaction allowed to stir and warm to room temperature overnight. Most of the solvent was removed under reduced pressure and the mixture was diluted with ethyl acetate. 40 Printed from Mimosa 10/24/2000 13:39:28 page -42- PCT/US99/D8501 It was then washed with saturated NaHCO,, 5% KHS04, brine, and the organics dried with anhydrous MgSO). The mixture was filtered and the solvent removed under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate:hexane 50:50 to 65:35) to give 1.30 g, 43% of tert-butyl(3R)-3-(isobutyl)succinyl-[[2-[5-(3- methylbenzyl)-l,3,4-5 oxidiazolyl]-(R.S)-hydroxymethyl]-2-(S)-methylpropyl]-L-phenylalaninamide as an off-white foamy solid. C-18 HPLC RT = 18.3,18.7 min. diastereomers, 90% pure at 215 nm (10 to 100% solvent B/25 min; solvent A = 0.1% TFA/H20; solvent B = 0.1% TFA/acetonitrile).

FAB mass spectrum: m/z (M+H)+ = 635; theory = 635. h. tert-Butyl-(3R)-3-(isobutyl)succinyl-[[2-[5-(3-melhylbenzyl)-l, 3,4-oxadiazo!yl]carbonyl]-2-(S)-methylpropyl]-L-phenylalaninamide.

To a stirred mixture of N-chlorosuccinimide (1.07g, 8.0 mmols) in 25 ml dry toluene at 4°C was added 0.84 ml (11.45 mmols) dimethyl sulfide (DMS) under a nitrogen atmosphere. A white precipitate formed after the addition of DMS. After 30 minutes, the resulting suspension 15 was cooled to -25°C using a carbon tetrachloride and dry ice bath. A solution of tert-butyl(3R)-3-(isobutyl)succinyl-[[2-[5-(3-methylbenzyl)-l,3l4-oxadiazolyl]-(R,S)-hydroxymethyl]-2-(S)-methylpropyl]-L-phenylalaninamide (1.25g, 1.97 mmols) in 30 ml dry toluene was added dropwise. The resulting mixture was stirred for 1.5 h at -25°C and 1.19 ml (8.5 mmols) of triethylamine was added. After 15 minutes, the cold bath was removed, and the reaction 20 monitored by TLC; silica gel; ethyl acetate:hexane (30:70). After 1 h, the mixture was diluted with 500 ml ethyl acetate and washed with saturated NaHCO} brine and the organics dried with anhydrous MgSO<. The mixture was filtered and the solvent removed under reduced pressure. The residue was purified by column chromatography (silica gel, methanol:chloroform, 0.5:99.5 to 2.5:97.5) to give tert-butyl(3R)-3-(isobutyl)succinyl-[[2-[5-(3-methylben2yl)-l ,3,4-25 oxadiazolyl]carbonyl]-2-(S)-methylpropy!]-L-phenylalaninamide as an off white foamy solid; 1.0 g, (80.2%). C-18 HPLC RT = 20.2,20.7 min. diastereoisomers, 90% pure at 215 nm (10 to 100% solvent B/25 min; solvent A = 0.1% TFA/H20; solvent B = 0.1% TFA/acetonitrile). FAB mass-spectrum: m/z (M+H)+ = 633; theory = 633. 41 Printed from Mimosa 10/24/2000 13:39:28 page -43- i. [[2-[5-(3-methylbenzyl)-l,3,4-oxadiazolyl]carbonyl]-2-(S)-methylpropyl]-L-phenylalaninamide-(3R)-(isobutyl)succinic acid To a solution of teit-butyl(3R)-3-(isobutyl)succinyl-[2-[5-(3-methylbenzyl)-l,3,4-oxadiazolyl]carbonyl]-2-(S)-methylpropyl]-L-phenylalaninamide (1.0 g, 1.S8 mmol) in 25 ml 5 dichloromethane (DCM) cooled to 4°C in an ice bath, was added 25 ml trifluoroacetic acid (TFA). This was stirred for 1 h. The solvent and TFA are removed under reduced pressure, followed by coevaporation with DCM (3x). The material was purified via gradient RP-HPLC CH3CN:H20 (25:75 to 100:0 in 60 minutes) to give 292 mg (32%, 0.51 mmols) of which 52 mg was pure as a white solid after lyophilization. C-18 HPLC RT = 15.8 min., 95% pure at 215 nm 10 (10 to 100% solvent B/25 min; solvent A = 0.1% TFA/H20; solvent B = 0.1% TFA/acetonitrile). FAB Mass spectrum: m/z (M+H)* = 577; theory = 577. 'H-NMR(400 MHz, CDC1,)6 0.76 (d, 3H, .7=6.8 Hz), 0.85 (d, 3H, 7=6.4 Hz), 0.87 (d, 3H, 7=6.4 Hz), 0.93 (d, 3H, 7=6.8 Hz), 1.25-1.32 (m, IH), 1.48-1.61 (m, 2H), 2.28-2.36 (m, 2H), 2.35 (s, 3H), 2.44-2.49 (m, IH), 2.61-2.69 (m, 2H), 2.95 (dd, IH, 7=13.6,8.4 Hz), 3.09 (dd, IH, 7=16.6,6.4 Hz),4.24 (s, 2H), 4.67 (dt, IH, 15 7=8.0,6.8 Hz), 5.19 (dd, IH, 7=8.4,6.0 Hz), 6.52 (br. d, IH, 7=8.4 Hz), 6.81 (br. d,lH, 7=7.6 Hz), 6.94-6.99 (m, IH), 7.10-7.19 (m, 7H). ,3C-NMR(100 MHz, CDC13) 61126,19.54,21.33, 22.13,22.71,25.75,30.84,31.82,36.74,38.16,40.54,41.29,55.16,61.55,126.1,126.8,128.6, 128.8,129.0,129.1,129.7,132.5,136.2, 139.0,160.2,167.9,171.6,175.0,175.7, 184.4.

Example II -Acetyl-L-leucyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl]carbonyl]-4-(guanidino)-butyl]-L-leucyl]amide (CQ-0002). 2-Phenyl-1,3,4-oxadiazole intermediate Benzoyl hydrazide (200 mg) freshly crystallized from chloroform was suspended in 5 mL of triethyl orthofbimate and heated at reflux under nitrogen in a 160 °C oil bath for 3 hours. The 25 mixture was cooled to room temperature, chilled in ice, and treated with 50 mL water and 10 mL 10% KHS04 solution. The mixture was stirred approximately 2 minutes then 50 mL of EtOAc was added and stirring continued for 10 minutes. The organic layer was separated and the aqueous layer was extracted three times with ethyl acetate. All ethyl acetate layers were combined and were washed with 10% sodium bicarbonate solution and saturated sodium chloride 42 Printed from Mimosa 10/24/2000 13:39:28 page -44- solution. Drying over sodium sulfate, rotary evaporation and further drying under high vacuum provided 204 mg of an analytically pure oil which crystallizes upon standing. Commercially available benzoylhydrazide (Aldrich) in this reaction may be used, but the resulting product often contains a minor impurity which can be removed following the cyclization, by flash S chromatography on silica gel eluting with 0-10% acetone in hexane.

'H-NMR-CDClj 7.49-7.62 (m, 3H), 8.12 (d, 7=6,2H), 8.49 (s, IH).

A. Acetyl-L-leucyl-L-leucyl-arginine(Mtr) (N-methyl)-(N-Methoxy)-amide\ Acetyl-Leu-Leu-OH (133 mg) and arginine (Mtr) -N-methyl-N-methoxy amide (200 mg) were dissolved in 10 mL of DMF and were treated with 243 uL of DIEA and 212 mg of HBTU. The reaction stirred at room temperature for IS hours and was worked up according to method A. Drying over NajSO*, rotary evaporation of the solvent and flash chromatography on silica gel (50% acetone in hexane) provided 270 mg of the title compound as a foam.

B. Acetyl-L-leucyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl]carbonyl]-4-[(4-methoxy-2,3,6-trimethyl-benzenesulphonyl)-guanidino]-butyl]-L-leucy! amide: 2-phenyl-1,3,4-oxadiazole (194 mg) in 2 mL of dry THF was chilled to -78s C. n-Butyllithium (1.46 mmole) was added as a 2.5 M solution in hexanes. The reaction stirred 20 minutes at -78°C and was then placed in a 0°C cooling bath. Acetyl-Leu-Leu-Arg-(Mtr)-N-(CH])-OCHj was then added in 2 mL of dry THF. The reaction was placed in a room temperature water bath and stirred 1 hour, then the solution was chilled to 0°C, and 20 mL of saturated ammonium chloride solution was added under nitrogen with rapid stirring. After the several minutes of vigourous stirring the solution was extracted with EtOAc. The ethyl acetate solution was washed with saturated sodium chloride solution, dried over sodium sulfate and concentrated to a pale brown oil by rotary evaporation.

C. Acetyl-L-leucyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl]carbonyl]-4-(guanidino)-butylJ-L-leucyl amide: One half of the crude product from step B was dissolved in a pre-formed solution of 2 mL of TFA and 100 uL thioanisole. The reaction stirred under nitrogen for 4 hours. The solvent was removed in vacuo, and the product was precipitated with dry ether. The precipitate was taken up in methanol and concentrated in vacuo, the residue was triturated with 43 Printed from Mimosa 10/24/2000 13:39:28 page -45- PCT/US99/D8501 dry ether, and dried in vacuo to provide 22 mg of the title compound as a colorless powder. Samples for biological testing were obtained by reverse phase C18 chromatography (5-80% CHjCN, 0.1% TFA, over 40 minutes). MS m/z (M+H)* 571 (CQ-0002).

Example III - Synthesis ofAcetyl-L-leucyl-N-[l-[3-[5-methyl]-l,2,4-oxadiazolyl]carbonyl]-ethyl-L-leucyl amide (CQ-0004)" A. N'-benzyloxycarbonyl-L-alanine (N-methyl-N-methoxy)amide: Cbz-L-Alanine (1.0 g) was dissolved in 10 mL dry DMF with 1.55 mL of DIEA. HBTU (1.78 g) was added and the reaction Stirred 30 minutes. Dimethyl hydroxyl amine hydrochloride (0.87 g) was added followed by 1.55 mL additional DIEA. The reaction stirred approximately 15 hours at room temperature. Work up according to general method A, drying over anhydrous sodium sulfate, rotary evaporation, and drying under high vacuum produced 0.96 g of a colorless solid.

B. N'-bertzyloxycarbonyl-L-alaninal: A solution of 6 mL of 1 M lithium aluminum hydride in THF was chilled under nitrogen to 0"C and a solution of compound A (0.68 g ) in 4 mL DMF was added dropwise. After stirring 15 minutes at 0°C the reaction was carefully quenched with 20 mL of EtOAc and 10 mL of 10% KHS04 solution. The organic layer was washed with 1 N HC1 and 10% NaHCO} solution. Drying over sodium sulfate, removal of the solvent by rotary evaporation, and drying under high vacuum provided 0.38 g of a colorless oil.

C. 2-(R,S)-3-(S)-[(benzyloxycarbonyl)amino]-2-hydroxy-butanenitrile: Compound B (1.2 g) triethylamine (0.532 mL) and acetone cyanohydrin (1.56 mL) were dissovled in lOmL of dry CH2C12 and stirred at room temperature for approximately 15 hours. The solvent was removed in vacuo and the residue was taken up in Et20 and washed with saturated sodium chloride solution. Drying over anhydrous sodium sulfate, rotary evaporation, and pumping under high vacuum provided 1.3 g of the cyanohydrin.

D. 2-(R,S)-3-(S)-[(berayloxycarbonyl)ammo]-2-acetoxy-butanenltrile\ Compound C (1.3 g) was dissolved in 2 mL of dry pyridine and was treated with 3.17 mL of acetic anhydride. The reaction stirred at room temperature for 3 hours and was then diluted with ethyl acetate and washed with water. Drying of sodium sulfate, rotary evaporation, and pumping under high vacuum provided 1.34 g of the title compound as an oil. 44 Printed from Mimosa 10/24/2000 13:39:28 page -46- £. l-(R,S)-2-(S)-J-[(N-hydroxy)carboximideamido]-l-acetoxy-2-[(benzyloxycarbonyl aminoj-propane: Compound D (1.34 g) was dissolved in 21 mL of EtOH and 4.2 mL of water and treated with hydroxylamine hydrochloride (0.422 g) and sodium acetate (0.991 g) and heated at 40 C for 3 hours. The solvent was removed in vacuo and the residue was suspended in EtOAc 5 and washed with water. Drying over sodium sulfate and evaporation of the solvent provided 1.1 g of crude material which was used without further purification.

F. I-(R,S)-2-(S)-I-[3-[5-(methyl)-I,2,4-oxadiazolyl]-l-acetoxy-2(benzyloxycarbonyl)-aminoJJ-propane: Compound E (0.45 g) was suspended in 5 mL toluene and treated with 185 uL of acetic anhydride. The reaction was refluxed for approximately 15 hours, after which the solvent was removed by rotary evaporation and purified by flash chromatography on silica gel eluting with 1:1 hexane:EtOAc to provide 0.36 g of title compound.

G. l-(R,S)-2-(S)-l-[3-[5-(methyl)J-1.2,4-oxadiazoIyI]-2-[(benzyloxycarbonyI)-aminoJ-propan-J-ol: Acetate F (180 mg) was dissolved in 3 mL of MeOH and treated with a solution of 90 mg of K2C03 in 1 mL of water. After approximately 20 minutes the reaction mixture was diluted with EtOAc and washed with water. Drying over MgS04> rotary evaporation and drying under high vacuum provided 0.160 g of the title compound.

H. l-(R,S)-2-(S)-l-[3-[5-(methyl)-l,2,4-oxadiazolyl]-2-amino]-propan-l-ol TFA salt: Compound G was taken in 2 mL of trifluoroacetic acid and chilled to 0°C. Thioanisole (100 uL) was added and the reaction was allowed to warm to room temperature and stirred approximately 15 additional hours. The solvent was removed in vacuo and traces of remaining TFA were removed by rotary evaporation from dichloromethane and methanol. The crude product was partially purified by elution through a pre-packed CI 8 mini-column (Waters Sep-Pak) with acetonitrile in water. Lyophilization of appropriate fractions provided the title compound 0.14 g, which was used without further purification.

I. l-(R,S)-2-(S)-(Bemylaxycarbonyl)-L-leucyl-N-[l-[(3-[5-(methyl)-l,2,4-axadiazolyl]- hydroxymethyl]-ethyl-]-L-leucine amide: Compound H (0.14 g) and Acetyl-Leu-Leu-OH were dissolved in DMF (3 mL) and were treated with DIEA (90 uL) and HBTU (234 mg). The reaction was allowed to stir approximately 15 hours at room temperature. The reaction mixture was diluted with EtOAc and washed with water. The water wash was extracted with 45 Printed from Mimosa 10/24/2000 13:39:28 page -47- dichloromethane. All organic layers were combined and concentrated in vacuo. The residue was purified by preparative CI8 reverse phase chromatography (5-60% CH3CN, 0.1% TFA) to provide 0.110 g of the title compound upon lyophilization. 7. Acetyl-L-leucyl-N-[l-[3-[5-methyl-l,2,4-oxadiazolyl]carbonyl]-ethyl]-L-leucylamide: 5 N-chlorosuccinimide (7S.4 mg was suspended in dry toluene and chilled to 0°C. Dimethyl sulfide (60 uL) was added and the suspension stirred 30 minutes at 0°C and was then chilled to -25 °C. Compound I ( 60 mg) was added in 2 mL dichloromethane and the reaction stirred 2.5 hours at -25 °C. Triethyl amine (84 uL) was added and the reaction wanned to room temperature. After stirring 1 hour the reaction mixture was diluted with EtOAc and was washed 10 with water. Drying over anhydrous sodium sulfate and removal of the solvent by rotary evaporation provided 60 mg of crude product. Flash chromatography on silica gel provided 30 mg of the title compound as a colorless solid. MS 424 (M+H). 1 H-NMR <50.89 -0.94 (m, 12 H), 1.50 (d, 7=9.6,3H), 1.53-1.69 (m, 6H), 2.01 (s, 3H), 2.70 (s, 3H), 4.50-4.52 (m, 2H), 5.34-5.39 (m, IH) 6.23 (d, 7=11, IH), 6.81 (d, 7=10.9, IH), 7.07 (d, 15 7=8.8,1 H). 13C-NMR <512.4,17.6,22.1,22.2,22.8 (2 carbons), 23.1,24.7,24.8,40.8,41.1, 52.7,164.1,170.3,171.3,172.3,178.3, 190.

Example IV - A cetyl-L-leucyl-N-[l-[3-[5-methyl-l, 2,4-oxadiazolylJcarbonyl]-4-(guanidino)-butylJ-L-leucyl amide (CQ-0007) 20 A. N"-t-butoxycarbonyl-L-Arg(Mtr)-(N-methyl-N-methoxy)amide: Boc-L-Arg(Mtr)-OH .00 g (10.3 mmole) was suspended in dry DMF (10 mL),followed by N,0 dimethyl hydroxylamine hydrochloride (1.25 g) and DIEA (5.4 mL). HBTU (4.28 g) was added and the reaction stirred approximately 15 hours at room temperature. The reaction was worked up according to general method A, and the EtOAc solution was dried over NajSO* and concentrated 25 to 5.23 g of a colorless foam.

B. N'-t-butoxycarbonyl-L-(Mtr)-argininal: Compound A (2.00 g) was dissolved in 20 mL of diy THF and chilled to 0CC. To this solution was added 4.72 mL of a 1 M solution of LiAlH, in THF, dropwise over 30 minutes at 0°C. The reaction was quenched at 0°C by the slow addition of 50 mL EtOAc, followed by 15 mL of 10% KHS04 solution. The mixture was 46 Printed from Mimosa 10/24/2000 13:39:28 page -48- PCMJS99/D8501 partitioned between 100 mL EtOAc and 50 mL 1 N HC1 solution. The organic layer was washed with 1 N HC1 solution and saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and concentrated by rotary evaporation. Drying under high vacuum provided 1.74 g of a white solid.

C. 2-(R,S)-3-(S)-[(t-butoxycarbonyl)amino]-6-[(4-methoxy-2,3,6-lrimethyl- benzenesulphonyl)-guanidinoJ-2-hydroxy-hexanenitrile: Compound B (1.70 g) was dissolved in 25 mL of methanol and was treated with 0.941 g of potassium cyanide. The reaction was allowed to stir at room temperature for approximately 15 hours. The reaction mixture was then partitioned between 150 mL EtOAc and 25 mL 1N HC1. The organic layer was washed with 1 N 10 HC1 and dried over anhydrous sodium sulfate solution. Rotary evaporation and further drying under high vacuum provided 1.62 g of the title compound.

D. 2-(R,S)-3-(S)~[(t-butoxycarbonyl)amino]-6-[(4-methoxy-2,3,6- trimethyl-benzenesulphonyl)-guanidino]-2-acetoxy-hexanenitrile: Compound C (1.62 g) was dissolved in 10 mL of dry pyridine and treated dropwise with 0.62 mL of acetic anhydride. The reaction was 15 allowed to stir at room temperature for 3 hours. The solution was diluted with 100 mL EtOAc and washed three times with equal volumes of 1 N HC1 after drying over anhydrous sodium sulfate the solution was concentrated by rotary evaporation to an oil, and purified by flash chromatography on silica gel (50-75% EtOAc in hexanes, step gradient) to provide 0.79 g of the title compound and 0.64 g of mixed fractions containing traces of compound B. 20 E. l-(R,S)-2-(S)-l-[(N-hydroxy)carboximideamido]-l-acetoxy-2-[(t-butoxycarbonyl)- ammo]-5-[(4-methoxy-2,3,6-trimethyl-benzenesulphonyl)-guamdino]-pentane: Compound D (0.79 g) was dissolved in 45 mL of EtOAc and 3.9 mL of water and was treated with 0.174 g of sodium acetate and 0.129 g of hydroxylamine hydrochloride. In an analogous fashion the mixed fractions containing compound D (0.64 g) were dissolved in 36.5 mL ethanol and 3.2 mL of 25 water and were treated with 0.141 g of sodium acetate and 0.105 g of hydroxylamine hydrochloride. The reactions were heated at 45°C for 4 hours with stirring. HPLC analysis showed veiy similar profiles for both reactions. The reactions were diluted with EtOAc, washed with water and saturated sodium chloride solution and were then dried over anhydrous sodium sulfate and concentrated by rotary evaporation. The combined products were purified by flash 47 Printed from Mimosa 10/24/2000 13:39:28 page -49- chromatography (1-4 % MeOH in EtOAc, step gradient) to provide 0.77 g of the title compound.

F. l-(R,S)-2-(S)-l-[3-[5-(methyl)-l,2,4-oxadiazolyl]-2-amino-5-[(4-methoxy-2,3,6-trimethyl-benzenesulphonyl)-guanidino]-pentan-I-ol trifluoroacetate salt: Compound E (0.74 g) was dissolved in 6.5 mL of dry chloroform and treated with 0.27 mL of triethyl amine and 0.153 mL of acetic anhydride and allowed to stir 4 hours at room temperature. The reaction was diluted with 50 mL toluene and refluxed for approximately 15 hours in a 120°C oil bath. The volatile solvents were removed by rotary evaporation and the residue was worked up according to method A. Drying over sodium sulfate, concentration by rotary evaporation, and flash chromatography on silica gel eluting with EtOAc provided 0.34 g of a colorless oil. A portion of 10 this material 0.17 g was dissolved in 4 mL of MeOH and chilled to 0°C. To this solution was added 90 uL of a 4 N solution of K2C03. The reaction stirred two hours and was then partitioned between 40 mL of EtOAc and 5 mL water. The organic layer was washed with saturated sodium chloride solution and dried over sodium sulfate. The ethyl acetate was removed by rotary evaporation and traces of ethyl acetate were removed by rotary evaporation from 15 dichloromethane. The resulting residue was diluted in 1.33 mL of dichloromethane and chilled to 0°C. Trifluoroacetic acid (0.57 mL) was added and the reaction stirred 1.5 hour at 0°C. The solvent was rapidly removed in vacuo and the product was dissolved in dichloromethane and concentrated to dryness by rotary evaporation.

G. l-(R,S)-2-(S)-L-leucyl-N-[l-[(3-[5-(methyl)-1.2,4-oxadiazolyl]-hydroxymethyl]-4-[(4-20 methoxy-2,3,6-trimethyl-benzenesulphonyl)-guanidino)J-butylJ-L-leucine amide: Compound F (146 mg) and Acetyl-Leu-Leu-OH (82 mg) were dissolved in 5 mL of dry DMF and treated with 200 uL of DIEA, followed by 30 mg of HBTU. After 5 minutes an additional 100 uL of DIEA was added and the reaction stirred approximately 15 hours at room temperature. The reaction was diluted with EtOAc and washed with saturated NaHC03 solution and saturated sodium 25 chloride solution. After removal of the solvent by rotary evaporation the product was purified by preparative C18 reverse phase chromatography (5-60% CH3CN, 0.1% TFA) to provide 122 mg of the title compound.

H. Acetyl-L-leucyl-N-[l-[3-[(5-metkyl)-l,2,4-oxadiazolylJcarbonyl1-4-1(4-methoxy-2,3,6-trimethyl-benzenesulphonyl)-guanidino]-butyl]-L-leucylamide: N-chlorosuccinimide (45 48 Printed from Mimosa 10/24/2000 13:39:28 page -50- PCT/US99/Q8501 mg) and dimethyl sulfide (61 uL) in 2.5 mL of toluene were chilled to 0°C with stirring. Stirred at 0°C for 30 minutes. The mixture was then chilled to — -25°C in a diy ice/carbon tetrachloride bath, then compound G (100 mg) was added by dropwise addition in a mixture of 2.5 mL of dichloromethane and 1.5 mL of toluene. The reaction stirred at -25°C for 3 hours then 100 uL of 5 triethyl amine was added. After 5 minutes the cooling bath was removed, and the reaction stirred I hour. The reaction mixture was diluted with EtOAc, and washed with saturated sodium bicarbonate solution and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate solution and concentrated to an oil.

I. Acetyl-L-leucyl-N-[l-[3-[(5-methyl)-l,2,4-oxadiazolyl]carbonyl]-4-(guanidino)-butyl-10 L-leucyl amide: Compound H was taken up in 1.75 mL of TFA and chilled to 0°C. Thioanisole (90 uL) was added and the reaction stirred 1 hour at 0°C, and 4 hours at room temperature. The volatile solvents were removed by rotaiy evaporation, and residual TFA was removed by adding dichloromethane and concentrating to dryness on the rotovap. Reversed phase C18 preparative chromatography provided the title compound. FAB MS m/z (M+H)* 509 (CQ-0008).

Example V - Synthesis of Acetyl-L-tyrosinyl-L-valyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl]carbonyI]-2-carboxy-ethyl]-L-alanine amide (CQ-0010) A. N'-Benzyloxycarbonyl-L-Aspartyl(O-t-butyl) N-methyl-N-methoxy amide: Cbz-L-Aspartic Acid (O-t-butyl) (1.0 g, 2.93 mmole), N.O-dimethyl hydroxyl amine hydrochloride 20 (0.357 g, 3.66 mmole, were suspended in 15 mL of DMF and treated with 1.53 mL (8.79 mmoles) of DIEA under N2 atmosphere. HBTU (1.22 g, 3.22 mmoles) was added and the reaction stirred approximately 15 hours at room temperature. The reaction mixture was worked up according to general extractive work up method A. Drying over NajS04, rotaiy evaporation of the solvent and further drying under high vacuum provided 1.1 g as a colorless glassy solid. 25 B. L-Aspartyl-(O-t-butyl) N-methyl-N-methoxy amide: Compound A (1 g) was dissolved in 20 mL methanol containing 5% (v/v) formic acid. The solution was deoxygenated with nitrogen bubbling then treated with approximately 200 mg of palladium black. The reaction stirred under nitrogen for 3 hours, and was then filtered through celite. The celite was washed well with methanol and the filtrates were combined and concentrated by rotaiy evaporation. 49 Printed from Mimosa 10/24/2000 13:39:28 page -51- Residual methanol and formic acid were chased off by the addition and rotary evaporation of CH2C1] and 50:50 CH2Cl2:hexane. Drying under high vacuum provided 790 mg of an oil.

C. Acetyl-L-tyrosinyl-L-Valyl-L-Alcmyl-L-Aspartyl-(0-t-butyl) N-methyl-N-methoxy amide'. Compound B (150 mg) and Acetyl-Tyr-Val-Ala-OH (230 mg, prepared using 5 conventional peptide synthesis) were combined and suspended in 10 mL of DMF. DIEA (305 uL) was added followed by HBTU. Product from normal extractive work up, and the 1 N HC1 washes were combined after evaporation and purified by preparative HPLC chromatography (5 -60% CHjCN 0.1 % TFA, over 30 minutes) to provide a lyophilized fraction of 85 mg of 94% pure material, which was carried on to the anion coupling reaction. 10 D. Acetyl-L-tyrosinyl-L-vaIyI-N-[J-[2-[(5-pheny!)'l,3,4-oxadiazolyl]carbonyl]-2- (carboxy-t-butyl)-ethylJ-L-alanine amide: 2-Phenyl-l,3,4,-oxadizaole (169 mg, 1.16 mmole) was dissolved in 2 mL diy THF, and chilled to -78 C. n-Butyl lithium (510 uL, 2.5 M solution in hexane) was added via syninge, after 20 minutes compound C (88 mg, 0.145 mmole) was added via syninge in 3 mL dry THF and the reaction was allowed to warm to room temperature. 15 After 15 minutes 20 mL of saturated NH4C1 solution was carefully added under nitrogen, and the solution was rapidly stirred for several minutes. The resulting solution was extracted with EtOAc, dried over Na2S04 and concentrated. The resulting product was dissolved in H20/ CHjCN and concentrated by freeze-drying. Reverse phase preparative HPLC chromatography (5-60% CHjCN, 0.1% TFA, 30 minute gradient) provided 25.8 mg of a colorless powder upon 20 lyophilization.

K Acetyl-L-tyrosinyl-L-valyl-N-[I•[2-[(5-phenyl)-l,3,4-oxadiazolylJcarbonylJ-2-carboxy-ethyl]-L-alanine amide: Compound D (25 mg) was treated with 2 mL of TFA and stirred at room temperature for 2 hours. Hie TFA was removed on the rotovap, and remaining entrained solvent was removed by adding CH2C12 and CH3CN and evaporating. The crude 25 product was purified by reverse phase HPLC chromatography (5- 60% CHjCN, 0.1% TFA, 30 minute gradient). Lyophilization of appropriate fractions provided 15.7 mg of a colorless lyophilate. MaldiMS M+Na 659 observed. MS FAB (M+H)* 637. 1 H-NMR: <5 0.77 (m, 6H), 1.1-1.2 (m, 3H), 1.74 (s, 3H) 1.9 (m, IH), 2.57 - 2.8 (m, 2H), 2.75-3.34 (m, 2H) 4.14 (m, IH) 4.3 (m, IH), 4.44 (m, IH) 5.3 (m, IH), 6.61 (m, 2H), 7.01 (m, 2H), 50 Printed from Mimosa 10/24/2000 13:39:28 page -52- 7.70 (m, 3H) 7.73 (m, 2H) 7.74 - 8.00 (m, 2H) 8.1 (m, 2H) 8.78 (m, IH), 9.13 (bs, IH) 12.65 (bs, IH). "C-NMR <5 17.8,17.9,19.0,22.3,30.6,34.9,36.3,47.5,52.7,54.0, 57.0,114.6,122.3, 127.2,129.5,129.9,132.9,155.6,159.6, 164.9,169.0,170.2,171.1,171.3,172.2,172.3,183.8.

Example VI - Acetyl-L-Aspartyl-Valyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl] carbonylJ-2-(carboxy)-ethyl]-L'-glutamyl amide (CQ-0011) A. Acetyl-L-Aspartyl(Ot-Bu)-L-Valyl-L-GIutamyl (O-t-Bu)-L-Aspartyl-(O-t-butyl) N-methyl-N-methoxy amide: Acetyl-Asp(0-t-Bu)-Val-Glu-(0-t-Bu)-OH (0.302 g, 0.586 mmoles, prepared by conventional peptide synthesis) and H-Asp-(0-t-Bu)-N-(CH3)-0CH3 (0.150 g, 0.645 mmole, prepared as in example VIII) were combined in 5 mL of DMF and DIEA (305 uL) was added. HBTU (277 mg) was added. After 2 hours an additional 200 uL of DIEA was added and the reaction was allowed to stir approximately 15 hours at room temperature. The reaction was worked up according to method A, dried over NajS04 and concentrated to an oil. Preparative reverse phase chromatography (C18,5 - 60% CH3CN, 0.1% TFA, 30 minute gradient), and 15 lyophilization of appropriate fractions provided 0.231 g of a colorless lyophilate.

B. Acetyl-L-Aspartyl(0-t-Bu)-ValyI-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazotyl]carboTiyl]-2-(carboxy-0-t'butyl)-ethyl]-L-glutamyI(0-t -Bu) amide: 2-Phenyl-l,3,4,-oxadiazole (161 mg, 1.1 mmole) was dissolved in 2 mL dry THF, and chilled to -78 °C. n-Butyl lithium (485 uL, 2.5 M solution in hexane) was added via syringe, after 20 minutes compound A (100 mg, 0.138 mmole) was added via syringe in 3 mL dry THF and the reaction was allowed to warm to room temperature. After 60 minutes 20 mL of saturated NH«C1 solution was carefully added under nitrogen, and the solution was rapidly stined for several minutes. The resulting solution was extracted with EtOAc, dried over NajSO, and concentrated. The resulting product was dissolved in H20/ CH3CN and concentrated by freeze-drying. Reverse phase preparative HPLC 25 chromatography (5-60% CH3CN, 0.1 % TFA, 30 minute gradient) provided 34 mg of a colorless powder upon lyophilization.

C. Acetyl-L-Aspartyl-Valyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl] earbonyl]-2-(carboxy)-ethyl]-L-glutamyl amide: Compound B (25 mg) was treated with 2 mL mL of TFA and stirred at room temperature for 5.75 hours. The TFA was removed on the rotovap, and 51 Printed from Mimosa 10/24/2000 13:39:28 page -53- remaining entrained solvent was removed by adding CH2C12 and CH2CN and evaporating The crude product was purified by reverse phase HPLC chromatography (5-60% CHjCN, 0.1% TFA, 30 minute gradient). Lyophilization of appropriate fractions provided 15.1 mg of a colorless lyophilate. MaldiMS M+Na 669 observed. MS FAB (M+H)" 647.

'H-NMR 8 0.73 (m, 6H), 1.75 -1.95 (m, 2H), 1.82 (s, 3H), 1.88 (m, 2H), 2.40 - 2.70 (m, 2H) 2.75 -3.05 (m, 2H), 4.14 (m, IH), 4.30 (m, IH), 4.58 (m, IH), 5.33 (m, IH) 7.55 (m, IH) 7.61-7.73 (m, 3H), 8.02 (m, IH), 8.10 (m, 2H), 8.25 (m, IH) 8.72 - 8.82 (m, IH), 12.3 (bs, 3H). "C-NMR 317.5,18.9,22.3,27.2,29.8,30.6,34.8,35.5,49.3,51.2,52.7,57.0,122.3,127.2,129.5, 132.9,159.6,164.9,169.4,170.4,170.5,171.2, 171.3,173.7,183.8.

Example VII • General extractive work up Method A The reaction mixture was diluted with 5-10 volumes of EtOAc and washed three times each with equivalent volumes of 1 N HC1 solution, then saturated NaHC03 solution, and finally with saturated NaCl solution.

Example VIII - Inhibitory Activity Against Cathepsin B and L, Papain and Gingipain The enzyme cathepsin B (E.C. 3.422.01) was obtained from Calbiochem (San Diego, CA); cathepsin L (E.C. 3.4.22.15) from Athens Research and Technology Inc. (Athens,GA); and papain (E.C. 3.4.22.02) from Sigma (St Louis, MO).

Cbz-Phe-Arg-NHMec (-NHMec: 7-(4-methyl)coumaiylamide) was obtained from BachemCalifornia, be. (Torrance, CA). All other reagents were obtained from Sigma.

The enzymes used in enzyme assays with methylcoumarylamides were activated as described elsewhere (Barret, et al., Methods Enzymol. 80:535-561 (1981); Brdnune, et al., Biochem. J., 264:475-481 (1989). Cathepsin L was assayed in 0.34 M sodium acetate buffer, pH 25 5.5, containing 0.1% (v/v) Brij 35,2.5 mM dithiothreitol (DTT) and 5 mM Naj-EDTA.

Cathepsin B was assayed under the same conditions, except that the buffer was adjusted to pH 6. Papain was assayed in 50 mM sodium phosphate buffer, pH 6.8, containing 0.2 M sodium chloride, 2 mM DTT, 1 mM Naj-EDTA and 0.025 % Brij 35 (v/v).

Initial velocities of enzymatic reactions were measured spectrofluorometrically (X„ - 370 52 Printed from Mimosa 10/24/2000 13:39:28 page -54- nm, Xjn, 460 nm) with a Quanta Master QM1 (Photon Technologies International, South Brunswick, NJ). Stock solutions of the enzymes were diluted into the buffer, equilibrated at room temperature, and preincubated without or with increasing concentrations of inhibitors. The reactions were started by addition of substrate. A total of 4 to 8 inhibitor concentrations were 5 used to determine IC30 values. In all cases the substrate concentrations were much smaller than the K„ value, and the IC*, values measured approximated the K, directly (Cheng, et al., Biochemical Pharmacology, 22:3099-3108 (1973)).

Gingipain assay - All assays were carried out in a 96 well microtiter plate reader and cleavage of BAPNA (Na-benzoyl-DL-arginine-p-nitroanilide hydrochloride) was detected at 405 10 nm.

All assays were performed as follows: 180 |il assay buffer (50 mM Tris, 5 nM CaCl2 and 10 nM cysteine, at pH 7.6) was mixed with 10 (il gingipain R (RGP). The mixture was incubated for 5 min. at room temperature to reduce and activate RGP. 10 |il of each inhibitor were added at various concentrations. These mixtures were incubated for 10 min. at room 15 temperature to allow the inhibitors to complex with RGP. 50 fil of 10 mM BAPNA substrate was added. A two minute assay was performed with a final volume of250 (il, and a final BAPNA concentration of 2 mM. 2 mM BAPNA was sufficient excess of substrate such that substrate depletion did not occur within a 10 minute assay time.. For this reason, two minute assays were performed 20 whereby the in mOD/min change in absorbance at 405 nm was used as the initial velocity reading. In order to titrate RGP against leupeptin and to determine % activity, these velocity readings were transformed on a percent scale where the 100% control contained no inhibitor. The initial velocity values were also entered into Giaphpad Prism regression program along with the various inhibitor concentrations to obtain the ICj0 values. All data represents the minimum 25 of duplicates, and at times triplicate sets.

Assay results are presented in Table 3. As shown, CM-0019B is an inhibitor of papain and cathepsin L and more selective against cathepsin B than is leupeptin. Compound CQ-0002, which shares the same recognition sequence (Leu-Leu-Arg) with the broad spectrum inhibitor leupeptin, is nearly as potent as leupeptin versus cathepsin B, but surpisingly has a much higher 53 Printed from Mimosa 10/24/2000 13:39:28 page -55- PCT/US99/D8501 degree of specificity. In addition, compound CQ-0002 inhibits gingipain R with a potency equivalent to that of leupeptin. Compounds CQ-0004 and CQ-0008 are also potent and selective cathepsin L inhibitors.

Table 3.

K, [nM] Values for Cysteine Protease Inhibitors COMPOUND PAPAIN CATHEPSIN.B* CATHEPSIN L* GINGIPAIN R1 Leupeptin 1.0 ±0.06 6.1 ± 1.2 0.62 ±0.10 .8 (IC50) CM-0019B" 85 3,000 -100 CQ-0002 1,200±280 324 ±46 6.0 ±0.98 28 (IC50) CQ-0004 25600 ±5340 27200 ±1900 61 ±14 CQ-0008 8590 ±1860 1240±182 7.13 ±0.32 ' Human enzyme. 6 "B" denotes remake of larger quantities of corresponding CQ number.

Example IX - Inhibitory Activity Against Caspases Assay for ICE inhibition To examine the ability of the caspase family inhibitors, CQ-0010 and CQ-0011, to inhibit human IL-lp production, two different assays were employed. In the first, the human monocytic 20 cell line, THP-1, was stimulated with E. coli lipopolysaccharide (LPS serotype 0127-88; Sigma Chemical Co., St Louis, MO) in the presence and absence of the inhibitors. This cell line synthesizes and secretes IL-lp and TNFa as well as other cytokines upon LPS stimulation. The second assay used freshly-isolated human whole blood similarly stimulated with LPS. THP-1 assay. Two x 10* THP-1 cells were added to 24 well plates in 1 ml RPMI 25 supplemented with 1% FCS, glutamine and 5 x 10"3 M mercaptoethanol. Two-fold serial dilutions of the inhibitors, CQ-0010, CQ-0011 and the commmercially available Ac-YVAD-CHO (Biomol Research Laboratories Inc., Plymouth Meeting, PA), were pre incubated with the cells for 15 min at 37°C. LPS was then added to a final concentration of lug/ml and the plates 54 Printed from Mimosa 10/24/2000 13:39:28 page -56- PCT/US99/D8501 incubated for 4 hr at 37°C. All incubations were earned out in a humidified inciter with 5% C02 in air.

Supematants were harvested after 4 hr and assayed by ELISA for the presence of TNFa and IL-ip using commercially available kits (PerSeptive Biosystems, Framingham, MA and 5 R&D Systems, Minneapolis, MN, respectively).

Human whole blood assay: Heparinized whole blood (19.7 U heparin per ml) from healthy volunteers was collected and dispensed into 12 x 75 mm polystyrene tubes (0.25 ml per tube). The inhibitors, CQ-0010, CQ-0011 and Ac-YVAD-CHO were dissolved in DMSO, then diluted and added to the tubes in 0.25 ml and preincubated with the blood for 15 min at 37°C.

LPS was then added to a final concentration of 10 or 100 ug/ml.

The tubes were loosely-capped and incubated in a water bath for 4 hr at 37°C after which they were immersed briefly in an ice-water bath. Supematants were harvested by centrifiigation and stored at -70DC. The presence of TNFa and IL-lp was detected by commercially-available ELISA kits. ] 5 Assay for other caspase and granzyme B inhibition Inhibition constants were measured photometrically for YAMA (caspase 3), Lap3 (caspase 7), FLICE (caspase 8), Mch2 (caspase 6) and granzyme B. The buffer used for all enzymes consisted of 50 mM Hepes, 100 mM sodium chloride, 10% (v/v) sucrose, 0.1% (v/v) CHAPS and 10 mM dithiothreitol (DTT). In the case of granzyme B, only 1 mM DTT was used.

Enzymes were incubated at 37°C for 10 minutes in 100 uL well plates and synthetic substrate and inhibitor were added simultaneously. Final substrate concentration was 20 |iM in all cases. The synthetic substrate Ac-DEVD-pNA was used for all caspases and Succ-AAPD-pNA was used for granzyme B. The appearance of product was monitored over 10 minutes at 410 nm using a Spectromax 340 and IC30 curves were calculated from the initial slopes at varying inhibitor concentrations and inhibition constants were calculated.

The results are shown in Table 4. 55 Printed from Mimosa 10/24/2000 13:39:28 page -57- b IC50 ((iM) values of reduction of EL-ip release from THP-1 cell line. c IC50 (|iM) values of reduction of IL-ip release in whole blood assay.

The results indicate that CQ-0010 is an extremely potent and specific inhibitor of IL-ip production, capable of almost completely inhibiting the production of this cytokine at 5 jiM (Figure 1) while having no dose-dependent effect on levels of TNFa produced (results not shown). The ICn of CQ-0010 was estimated from these dose curves to be 0.3 fiM. CQ-0011 also inhibited ILip production but with approximately 10-fold less potency (Figure 1; Table 4).

In the whole blood assay, CQ-0010 was again equipotent to Ac-YVAD-CHO with an IC50 of 0.3-0.5 (iM (Figures 2a and b).

It should be noted that CQ-0010 was equipotent to the aldehyde equivalent (Ac-YVAD-CHO) in inhibiting ICE, but showed improved inhibition against FLICE with a Kj of s 20 nM. 56 Table 4.

Inhibition of Caspases - Comparison with Ac-YVAD-CHO Caspases Ac-YVAD-CHO Ac-YVAD-het Ac-DVED-het (CQ-0010) (CQ-0011) YAMA (CPP32, 3.3 *0.1 Caspase 3) Mch2 (Caspase 6) 100 33 6.7 Lap3 Not Active Not Active *0.03 (slow) (Mch3, Caspase 7) FLICE 3.7 *0.02 $0.03 (slow) (Mch5, Caspase 8) ICE 0.3" 0.3b 3b (Caspase 1) 0.3 - 0.5e 0.3 - 0.5e Granzyme Not Active Not Active Not Active 1 Values given are Kj in jiM, unless otherwise indicated.

Printed from Mimosa 10/24/2000 13:39:28 page -58- PCT/US99/D8501 Compound CQ-0011 is a potent inhibitor of Lap3 and FLICE. The compounds are selective and potent caspase inhibitors as shown by their inactivity with respect to granzyme B. 57 Printed from Mimosa 10/24/2000 13:39:28 page -59- The following references are incorporated herein: 1.

Appleyard G., et al., J. Virol., 66:363-366 (1985). 2.

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Barrett A., et al. ICOP Newsletter, 1-2 (Dec. 1996). 4.

Dinaiello C., et al., New Eng. J. of Med., 328:106-113 (1993).

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Gordon S., Meth. in Era., 244:568-581 (1994).

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Robertson C., et al., Pers. in Drug Disc, and Des., 6:99-118 (1996). 23a.

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. Storer, A., Pers. in Drug Disc. andDes., 6:33-46 (1996). 26.

SquierM., etal., J. of Cell. Phys., 159:229-237(1994). 58 Printed from Mimosa 10/24/2000 13:39:28 page -60- 27.

Takeda A., et al., FEBS, 359:78-80 (1995). 28.

Tchoupe J., et al., BBA, 1076:149-151 (1991). 29.

Yoshida K., et al., Jap. Cir. J., 59:40 (1995).

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Claims (1)

1. WO 99/54317 PCT/US99/08501 We claim: 1. A cysteine protease inhibitor of formula (I): 5 N—Y zXQkR JC , 10 wherein Z is a cysteine protease binding moiety; R, is alkyl or alkenyl optionally substituted with 1-3 halo or hydroxy; alkylamino, dialkylamino, alkyldialkylanuino; or cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, (Cj-C12) aryl, (Cj-C12)arylalkyl or (Cj-C12)sarylalkenyl optionally comprising 1-4 heteroatoms selected from N, O and S, and optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkenyl,, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, IS alkylcarboxamide, (Cj-C^)aryl, -O-tCj-C^aryl, arylcarboxamide, alkylthio or haloalkylthio; and Y and X are independently O, S or N, said N being optionally substituted with alkyl or alkenyl optionally substituted with 1-3 halo atoms; (Cj-Cg) aryl, arylalkyl or arylalkenyl optionally comprising 1-3 heteroatoms selected fiom N, O and S, and optionally substituted with halo, cyano, nitro, haloalkyl,, amino, aminoalkyl, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, 20 haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, arylcarboxamide, alkylthio or haloalkylthio; wherein at least one of Y or X is N; and 25 wherein Z is of the formula (II): AA5-AA4-AA3-AA2-AA1 • (II) 60 INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 RECEIVED wherein AAt> AA2, AAj, AA« and AA5 are independently an amino acid residue or amino-add residue mimetic; a direct bond or absent; and R, and R,1 are independently -CCOJR,, -C(0)NHRj, -55(0)^, -C(0)0R„ ->CH2RS or Rs, where Rj is H, alkyl, alkenyl or alkynyl optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, haloalkoxy, carboxyl, carboalkoxy or alkylcarboxamide; " cycloalkyl, alkylcycloalkyl, CCj-C12) aiyl or (Cj-CI2)aiylalkyl optionally comprising 1-4 heteroatoms selected fiom N.', O and S, and optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, alkyl, alkenyl, alkynyl or (Cj-Ci^l; or together R4 and R4' form a ring comprising 5-7 atoms selected fiom C, N, S and O; and wherein AA] is of the formula (Ilia): R2, R2' and R2" are inidependently H; alkyl or alkenyl optionally substituted with 1-3 halo, hydroxy, thio, alkylthio/, amino, alkylamino, dialkylamino, alkylguanidinyl, dialkylguanidinyl, guanidinyll; -RCOR', -RCOOR', -RNR'R'R" or -RQOJNR'R" where R is alkyl or alkenyl and R', R" and R° are independently H, alkyl, alkenyl, cycloalkyl or (Cj-C4)aryl; or cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkyl-oxyaryl, alkyl-thioaiyl, (Cs-Cl2) aryl, (Cj-C12)arylalkyl or (Cr C12)aiylalkenyl optionally comprising 1-4 heteroatoms selected fiom N, O and S, and optionally substituted with hydroxy, hallo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, amidine, alkylamidine, dialkylamidine, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (Cj-C6)aryl, -0-(Cj-Cs)aryl, arylcarboxamide, alkylthio or haloalkylthio; or Rj and R2' together with X' form a ring comprising 4-7 atoms selected fiom C, N, S and O, said ring optionally substituted with hydroxy, halo, cyano, nitro, haloalkyl, aminoalkyl, dialkylamino, amiidiiie, alkylamidine, dialkyl amidine, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, (Cj-C^aryl, -O^Cj-QJaryl, arylcarboxamide, alkylthio or haloalkylthio; (ni«) wherein X' N;and. or a pharmaceutically acceptable salt thereof. INTELLECTUAL PROPERTY OFFICE OF N.Z. 61 10 JUL 2003 RFP.FIVFD 15 2. An inhibitor of claim. 1 wherein Z comprises a pentapeptidyl binding moiety. 3. An inhibitor of claim: 1 wherein Z comprises a tetrapeptidyl binding moiety. 4. An inhibitor of claim 1 wherein Z comprises a tripeptidyl binding moiety. An inhibitor of claimi 1 wherein Z comprises a dipeptidyl binding moiety. 20 6. An inhibitor of claim 1 wherein the amino acids are selected from arginine or an arginine mimetic, proline; aspartic and glutamic acid and the aiyl and alkyl esters thereof; alanine and leucine, isoleutine; cysteine optionally substituted at die sulliar atom with alkyl, alkenyl or 25 phenyl optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, arylcarboxamide, alkylthio or haloalkylthio; phenylalanine, homo-phenylalanine, dehydro-phenylalamne, indoline-2-cauboxylic acid; tetrahydroisoquinoline-2-caiboxylic acid optionally substituted with halo, cyano. nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkoxy, haloalkoxy, carboxyl, carboallkoxy, alkylcarboxamide, arylcarboxamide, alkylthio or haloalkylthio; tyrosine, serine: or threonine optionally substituted with alkyl or aiyl; tryptophan, histidine, methionine, valine, norvaline, norleucine, octahydroindole-2-carboxyIic acid; asparagine, glutamine and Iysane optionally substituted at die nitrogen atom with alkyl, alkenyl, 5 alkynyl, alkoxy alkyl, alkyhhitoalkyl, alkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, alkoxycarbonyl alkyl or cycloalkyl, bicycloalkyl, cycloalkyl alkyl, bicycloalkyl alkyl or fused aryl-cycloalkyl alkyl optionally comprising 1 or more heteroatoms selected from N, O and S. 7. An inhibitor of claim 1 wherein AA2 is of die formula (mb): glycine optionally substituted at the a-carbon or a-nitxogen with alkyl, cycloalkyl or aiyl; 10 (Mb) or selected from a compound >of formulas IV to XXIV: 62 INTELLECTUAL PROPERTY OFFICE i OF NZ 1 0 JUL 2003 RFPFIV/Pn WO 99/54317 PCT/US99/D8501 H (IV) G—r6 (VII) ° t (X) can) (XUI (xvn) yp. *«. \_ VW? & o (XVI) f a _• txvni) (XK)' o- W~ (XX) ^ .V" VP K> Xh O . vrji vVrV vjr/ (xxo° / (xxn) * (xxm) Rj pcxivj 3 INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 RECEIVED wherein X" is CR'j or N; R}, R'j and R"s are independently H; alkyl or alkenyl optionally substituted with 1-3 halo, hydroxy, thio, alkylthio„ amino, alkylamino, dialkylamino, alkylguanidinyl, dialkylguanidinyl, guanidinyli; -RCOR', -RCOOR' or -RC(0)NR'R" where R is alkyl or alkenyl, and R' and R" are independently H, alkyl, alkenyl, cycloalkyl or (Cj-CgJaryl; or cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkyl-oxyaiyl, alkyl-thioaryl, (CrCl2) aiyl, (Cs-C,2)arylalkyl or (C5-C12)arylalkenyl optionally comprising 1-4 heteroatoms selected fiom N, O and S, and optionally substituted with hydroxy, halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, amidine, alkylamidine, dialkylamidine, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboallkoxy, alkylcarboxamide, (Cs-C6)aryl, -0-(Cj-Cg)aryl, arylcarboxamide, alkylthio or haloalkylthio; m is 0,1 or 2; n is 0,1 or 2; Gis-C(0)-,-NHC(0J)-,-S(O)j-,-OC(O)-, -CH2- or a direct bond; R*, R7, R'4i R't are independently H, alkyl, alkenyl, halo, alkoxy, carboxyl, carboalkoxy, amino, aminoalkyl, dialkylaimino; cycloalkyl, (Cj-Cg) aryl or (Cj-Cg) arylalkyl optionally comprising 1-3 heteroatoms selected fiom N, O and S, and optionally substituted with alkyl, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, alkoxy, haloalkoxy, carboxyl,, carboalkoxy, alkylcarboxamide, alkylthio, guanidine, alkylguanidine, dialkylguanidine, amidine, alkylamidine or dialkylamidine; and U, V, W and Y' are independently or together N, C, C(0), N(R,) where R, is H, alkyl, halo, alkoxy, carboalkoxy, cycloalkoxy, carboxyl, alkylthio, amino, alkylamino, dialkyiamino, or aryl, fused aryl or cycloalkyl optionally comprising 1 or more heteroatoms selected fiom O, S and N, and optionally subsisted with halo or alkyl; N(R10) where R10 is H, alkyl, alkenyl or cycloalkyl, aryl, arylalkyl or jfused aryl-cycloalkyl optionally comprising 1-4 heteroatoms selected fiom N, 0 and S, and optionally substituted with alkyl, alkenyl, alkynyl, halo, cyano, nitro, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, alkoxy, haloalkoxy, carboxyl, ■ carboalkoxy, alkylcarboxamiide, alkylthio, guanidine, alkylguanidine, dialkylguanidine, amidine, alkylamidine or dialkylamidime; or C(Rt i)(RI2) where R,, and Ru are independently or together 64 INTELLECTUAL PROPERTY OFFICE OF HI. 1 0 JUL 2003 RECEIVED WO 99/54317 PCT/US99/D8501 H, alkyl, alkythio, alkythioalkyl or cycloalkyl, alkylcycloalkyl, phenyl or phenyl alkyl optionally subsituted with guanidine, carboalkoxy, hydroxy, haloalkyl, alkylthio, alkylguanidine, dialkylguanidine, amidine, alkylamidine or dialkylamidine. 5 8. An inhibitor of claim 7 wherein AA„ AA4 and AA, are a direct bond or absent; or an amino acid selected from arginine or an arginine mimetic, proline; aspartic and glutamic acid and the aryl and alkyl esters thereof; alanine or glycine optionally substituted at the oc-carbon or a-nitrogen with alkyl, cycloalkyl or aryl; leucine, isoleucine; cysteine optionally substituted at the sulfur atom with alkyl, alkenyll or phenyl optionally substituted with halo, cyano, nitro, haloalkyl, 10 amino, aminoalkyl, dialkylamiino, alkyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, arylcarboxamide, alkylthio or haloalkylthio; phenylalanine, homo-phenylalanine, dehydro-phenylalanine, indoline-2-carboxylic acid; tetrahydroisioquinoline-2-carboxylic acid optionally substituted with halo, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamide, 15 arylcarboxamide, alkylthio or haloalkylthio; tyrosine, serine or threonine optionally substituted with alkyl or aryl; tryptophan,, histidine, methionine, valine, norvaline, norleucine, octahydroindole-2-carboxylic acid; asparagine, glutamine and lysine optionally substituted at the nitrogen atom with alkyl, alkenyl, alkynyl, alkoxyalkyl, alkylthioalkyl, alkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, alkoxycarbonyl alkyl or cycloalkyl, bicycloalkyl, cycloalkyl 20 alkyl, bicycloalkyl alkyl or fused aryl-cycloalkyl alkyl optionally comprising 1 or more heteroatoms selected fiom N, 0 and S. 9. An inhibitor of claim 1 wherein X' is N. 25 10. An inhibitor of claim 7 wherein X" is N. 11. An inhibitor of claim 1 wherein X' is CR'2, and R'2 is H. 12. An inhibitor of claim 7' wherein X" is CR'j, and R'j is H. 65 INTELLECTUAL PROPERTY I OFFICE OF N.Z 1 12 AUG 2003 RECEIVED > WO 99/54317 PCT/US99/08501 13. An inhibitor of claim 8 wherein Z is a calpain binding moiety. 14. An inhibitor of claim 13 wherein Rj is CH3SCH2CH2-, HOOCCCH^CHj-, cyclohexyl-CH2-, imidazolyI-CH2, (CH3)2CHCH2-, (CHjfcCH-, CH3CH2CH2- or CHrfCH^CHrf benzyl 5 optionally substituted with alkoxy, OH or -O-benzyl; H3NC(=*'*NHj)NHCH2CH2CH2-; -R'- C(=+NH2)NH2; -R'-NHC(=*NR")NR°; or -R'-NR'R0 where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected fiom N, S or O alkyl; and R" and R° are alkyl or cycloalkyl. 10 15. An inhibitor of claim 14 wherein Rj is-CHj-benzyl, benzyl, (CHj),C-,(CH3)}CCH2-, (CHOjCH-, CH3(CH2)jCH2-j iCH,CH2CH(CH,)- or (CH^CHCHj-. 16. An inhibitor of claim 15 wherein R, is benzyl, isoquinolinyl, quinolinyl, naphthyl or HOOCCH2CCCH2CH(CH3)2)-. 15 17. An inhibitor of claim 15 wherein R, is Cbz wherein the phenyl is optionally substituted with nitro. 18. An inhibitor of claim 15 wherein R< is tohienesulfonyl, methanesulfonyl, FMOC or (+)-20 menthyloxy-CO-. 19. An inhibitor of claim 14 wherein AA3 is leucine, AA» and AA, are direct bonds or absent, and Rj is alkyl. 25 20. An inhibitor of claim .6 wherein Z is Ri-Leu-Leu-Leu-; R,-Leu-Lea-; R^-Leu-Leu-Phe-; RrLeu-Abi*-;. 66 INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 RECEIVED WO 99/54317 PCT/US99/08S01 R«-Val-Phe-; Rj-Leu-Leu-NUe-; Ri-Ala-t-BuGl;y-Val-; R,-t-BuGly-Val-; 5 R^-Leu-Leu-Met-; or R^Leu-Nle-. 21. An inhibitor of claim 6 wherein Z is Cbz-Leu-Nle-; or Cbz-Leu-Val-. 10 22. An inhibitor of claim .8 wherein Z is a cysteine cathepsin binding moiety. 23. An inhibitor of claim 22 wherein Rj is CH3-, (CHj)iCH-, (CHj^CHCHj-, CHjCCH^CHj-, CH3CH(-0-lbenzyI)- or benzyl-S-CH2-; benzyl or -CH2-benzyl optionally substituted with OH or -OR' where R' is alkyl or aryl; H2NC(=+NH2)NHCH2CH2CH2-; -R'- 15 C(=,NH2)NH2; -R'-NHC(=rNlR")NR0; or -R'-NR'R" where R' is cycloalkyl, aryl or arylalkyl optionally substituted with ome or more heteroatoms selected fiomN, S or O alkyl; and R* and R° are alkyl or cycioalkyL 24. An inhibitor of claim 23 wherein R, is H, (CHjfcCH-, (CHj^CHCHj-, CH^CH^CHr, 20 benzyl optionally substituted with hydroxy and halo; (naphthyl)-CH2-; H^CCHj^CHz-, . HjN(GH2)2CHj-> H2NC(=+NHI2)NHCH2CH2CH2-; -R'-C(=+NH2)NH2; -R'-NHC(=4NR*)NR°; or -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected fiom N„ S or O alkyl; and R" and R° are alkyl or cycloalkyl. 25 25. An inhibitor ofclaim 22- whereinZ is a cathepsin B binding moiety. 26. An inhibitor of claim 25 wherein R2 and R3 are independently benzyl, -CHj-benzyl, HjNfCHjJjCHj-, H^CH^CH,-, HiNC(='NHJ)NHCHaCH1CHr; -R'-C(=+NH2)NH2; -R'-NHC(=*NR")NR°; or -R'-NIR"R° where R' is cycloalkyl, aryl or arylalkyl optionally 67 IIULLUUMJF^tRTY0FF/cTl 10 JUL 2003 RECEIVED WO 99/54317 PCT/US99/D8501 substituted with one or more hwteroatoms selected fiom N, S or O alkyl; and R" and R° are alkyl or cycloalkyl 27. An inhibitor of claim 25 or 26 wherein AAj is lie or Leu. 5 28. An inhibitor of claim 26 wherein -AA2-AA|- are -Phe-hPhe-; -Arg-hPhe-; -Arg mimetic-hPhe-; 10 -Lys-hPhe-; at -Orn-hPhe. 29. . An inhibitor ofclaim;22 wherein Z is a cathepsin L,0,K, or H binding moiety. IS 30. An inhibitor of claim .29 wherein Z is a cathepsin L binding moiety. 31. An inhibitor of d§im30 wherein R3 is benzyl or (CH^CHCH?-. 32. An inhibitor of claim 30 or 31 whereiaRj is -CHj-benzyl. 20 33 An inhibitor of claim 22 wherein Z is a cathepsin S binding moiety. 34. An inhibitor of claim 33 wherein R2 and Rj are alkyl. < 25 35. An inhibitor of claim 34 wherein R2 and R3 are independently (CH^CH-, (CHj^CHCHj-or CHj^CHj^CHj-. 36. An inhibitor of claim 33 wherein R3 is benzyl, (CH^CHCHj- or (CH^CH-. 68 INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 receivfo WO 99/54317 PCT/US99/08501 37. An inhibitor of claim 33 or 36 wherein R2 is -CH2-benzyL 38. An inhibitor of claim 37 wherein AA3, AA4 and AA3 are direct bonds or absent. 5 39. An inhibitor of claim 38 wherein R* is benzyl, isoquinolinyl, quinolinyl, naphthyl or HOOCCH2C(CH2CH(CHj)I)-. 40. An inhibitor of claim 39 wherein R, is Cbz. 10 41. An inhibitor of claim 29 wherein Z is a cathepsin H binding moiety. 42. An inhibitor of claim 41whereinZis R^-hPhe-; or HCMiPhe-. 15 43. An inhibitor of claim. 29 wherein Z is a cathepsin K binding moiety. 44. An inhibitor of claimi 43 wherein R, is benzyl, (CH3)jCHCHj- or (CH^CH-. 20 45. An inhibitor of claimi43 or 44 wherein AAj Is Gly; and AA* Is Val or D-Val. 46. An inhibitor of ciaimt 6 wherein Z is a cathepsin K binding moiety; and AA, is Arg, Arg mimetic or hPhe; AAjisPro; 25 AA3isGly;aind AA* is Val oir D-Val. 47 An inhibitor of claim: 6 wherein Z is a cathepsin Kbinding moiety; and is R,-Pro-AA,-; 69 INTELLECTUAL PROPERTY OFFICE OF N.Z. 10 JUL 2003 RECEIVFn WO 99/54317 PCT/US99/D8501 R,-GIy-Pro-AAr; Ri-Val-Gly-Pro-AA,; D-Val-Gly-Pro-AA,-; or R,-D-Val-Gly-Pro-AA^ where 5 AA, is Apa,Aig or Aug mimetic, or hPhe. An inhibitor of claim 22 wherein Z is Rt-AAj-Leu-hlPhe-; R^-AAj-Phc-hFhe-; or R,-AA3-Val-hPhe-; where AA, is Gly, Vail, D-Val, a direct bond or absent 49. An inhibitor of claim 22 wherein Z is Mu-Val-hPhe-. IS so. An inhibitor of claim 8 wherein Z is a caspase binding moiety. 51. An inhibitor of claim 50 wherein Rg is-RCOOR'. 52. An inhibitor of claim 51 wherein R is -CHr and R' is H. 20 53. An inhibitor of claim 51 wherein AA} and AA« are amino acid residues and AA, is a direct bond. 54. An inhibitor of claim 53 wherein Z is an interleukin-1 p converting enzyme binding 25 moiety. 55. An inhibitor of claim 54 wherein AA* is optionally substituted tyrosine or leucine. 56. An inhibitor of claim 55 wherein AA3 is valine, glutamate or an ester thereof. 48. 10 70 INTELLECTUAL PROPERTY OFFICE OF N.Z. I 0 JUL 2003 RECEIVFri WO 99/54317 PCT/US99/D8501 57. An inhibitor of claimt 56 wherein R3 is -CH3 or (CH^CH-. 58. An inhibitor of claim 54 wherein R3 is -CH3 or imidazolyl-CH2-; AA3 is valine or glutamate; and Rj is 5 -CHj. 59. An inhibitor of claim 6- wherein Z is an inteiieukin-lp converting enzyme binding moiety, and is R,-AArAA(-AAj-Pro-AA,; where 10 AA, is Asp or Asp ester. 60. An inhibitor of claiun 59 wherein -AAj-AA4-AA3- is -Ala-; -Glu-; 15 -Val-; -Tyr-Ala-; -Tyr-Glu-; -Tyr-Val-; -Leu-Ala-; 20 -Leu-Glu-; oir -Leu-Val-. 61. An inhibitor of claim 8' wherein Z is an interleukin-1 P converting enzyme binding moiety and AAg is of the formula (VI); 25 wherein X" is CR'j; R2 is -RCOOR' vdietre R is alkyl or alkenyl, and R' is H, alkyl, alkenyl, cycloalkyl or (Cj-CsJaryl. 62. An inhibitor of clainn 61 wherein R3 and R'j are H. WO 99/54317 PCT/US99/08S01 63. An inhibitor of claim 61 wherein AA3, AA4 and AAj are direct bonds or absent, and R2 is -RCOOH where R is-CH,-. 64. An inhibitor of claim 63 wherein Rg is phenyl or benzyl substituted with halo. 5 65. An inhibitor of claim 64 wherein Rj is benzyl, isoquinolinyl, quinolinyl, naphthyl or HOOCCH2C(CH2CH(CH3)j>. 66. An inhibitor of claim 51. wherein Z is a YAMA binding moiety, where R is -CH2- and 10 AA4 is Asp or an ester thereof.. 67. . An inhibitor of claim 66 wherein AAj is optionally substituted glutamine, or glutamic acid or an ester thereof. 15 68. An inhibitor of claim 67 wherein R2 is (CHj^CH- or CH3SCH2CH2-. 69. An inhibitor of claim 51 wherein Z is a FLICE binding moiety, where Rg is -CH2- and AA| is optimally substituted lysine. 20 70. Ah Inhibitor of claim 69 wherein AAj is glutamic acid. 71. An inhibitor of claim 70 wherein R3 is (CHj^CH-. 72. An inhibitor of claim 8 wherein Z is a viral or microbial cysteine protease binding 25 moiety. 73. An inhibitor of claim 72 - wherein Z is a gingipain binding moiety. 74. An inhibitor of chum 73 wherein Z is a gingipain K binding moiety. WO 99/54317 PCT/US99/08501 75. An inhibitor of claim 74i wherein R2 is RNR'R'R0 where R' is H; R" and R° are H or alkyl. 76. An inhibitor of claim 73 wherein Z is a gingipain R binding moiety. 5 77. An inhibitor of claimi 76 wherein R2 is HjNCC^NHjJNHCHjCHjCHj-; -R'-C(=,kNHj)NH2; -R'-NHC(=*NR")NR°; or -R'-NR"R0 where R' is cycloalkyl, aryl or arylalkyl optionally substituted with oine or more heteroatoms selected from N, S or 0 alkyl; and R" and R° are alkyl or cycloalkyl. 10 An inhibitor of claimi 6 wherein Z is a gingipain binding moiety, and AA2 is proline. 78. 79. An inhibitor of claimi 78 wherein Z is R.rLeu-Pro-AA,-, where AA, is arginine or an arginine mimetic. 80. An inhibitor of claimi 72 wherein Z is a human coronavirus protease binding moiety, and Rgis H2NC(=+NHJNHCHjCHjCH2-; -R'-C^UHjJNHj; -R'-NHC(=+NR")NR0; or -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected fiom NT, S or O alkyl; and R" and Rs are alkyl or cycloalkyl. 81- An inhibitor of claim 80 wherein R3 is (CHjJjCH-, (CH^CHCHj- or CH3(CH2)2CH2-; AA3 is Asp or an ester thereof; Leu, Arg or Arg mimetic, or direct bond; AAi and AA, are direct bonds or absent; and Rj is alkyl. 82. An inhibitor of claim 72 wherein Z is a hepatitis A virus 3C proteinase binding moiety, and 73 INTaLECTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 RECEIVED WO 99/54317 PCT/US99/08501 R2 is -RC(0)NRrR" where R' and R" are H or -CH3; or RCOOR' where R' is CH3; and AAj and AA« are amino acid residues. 83. An inhibitor of claim: 82 wherein AA« is Leu. 5 84 An inhibitor of claim 83 wherein R3 is -CH, and AA3 is Ala. 85. An inhibitor of claim 6 wherein Z is a hepatitis A virus 3C proteinase Winding moiety, and is 10 R,-Leu-AA3-TIhr-Gln-; R,-Trp-AAj-Tliir-Gln-; R,-VaI-AArTlhr-Gln-; R^-Ile-AAj-Thir-Gln-; or R^D-Leu-AA^-Thr-Gln-; IS where AA3 is Aig or Arg mimetic. 86. An inhibitor of claim 12 wherein Z is an Ad2 23K protease binding moiety, and R2 and R3are H; AA3 is alanine; 20 AAi is leucine; AA® is a direct bond; and R, is absent. g7. An inhibitor of claim 72 ■wherein Z is a human rhinovirus 3C protease binding moiety, 25 and R{ is RCOOR' where ;R is -CH2-; R3 is benzyl; and AA3 is leucine or a direct bond. ?4 J 'NTELLtUIlJAL(HROPERTY OFFICE 10 JUL 2003 RECEIVED WO 99/54317 PCT/US99/08501 88. An inhibitor of claim 72 wherein Z is a human rhinovirus 3C protease binding moiety, andRj is -RC(0)NR'R" where R' and R" are H or -CH3; or RCOOR' where R' is -CH, or -CH2CH,; or X' isNandR2is-CHj. 5 89. An inhibitor of claim 72 wherein Z is human picomain 2A protease. 90. An inhibitor of claim 79 wherein R, is -CH(OR')CH3 where R' is H, alkyl or aiyl. 10 91. An inhibitor of claim 90 wherein R2 is a hydrophobic side chain. 92. An inhibitor of claim ® wherein Z is a human picomain 2A protease binding moiety, and is R^Ala-Ala-Pno-Val-; or 15 R^Ala-Ala-Pno-Ala-. 93. An inhibitor of claim 8 wherein Z is a protozoan protease binding moiety. 94. An inhibitor of claim. 93 wherein Z is a Trypanosoma, Schistosoma or Leishmama 20 protease binding moiety. 95. An inhibitor of claim. 94 wherein Rj is benzyl optionally subsituted with OH; HjNCX-^NHjJNHCHjCHjCHi-; -R'-C^NH^NHj; -R'-NHC(=*NR")NR°; or -R'-NR"R° where R' is cycloalkyl, atyl ffir arylalkyl optionally substituted with one or more heteroatoms 25 selected fiom N,S or Oalkyll; and R" and R# are alkyl or cycloalkyl. 96. An inhibitor of claimi 95 wherein Rj is benzyl, (CHj^CHCHj- or (CHj^CH-; and AA3 is Phe, Leu, Pro or a direct bomd. 75 INTELLECTUAL PROPERTY OFFICE OF N.Z. 10 JUL 2003 RECEIVED WO 99/54317 PCT/US99/08501 97. An inhibitor of claim'96 wherein R« is Boc or Sue. 98. An inhibitor of claim. 93 wherein Z is a Plasmodium protease binding moiety. 5 99. An inhibitor of claim 98 wherein Rj is (CHj^CH-; -CH2-benzyl, benzyl or phenyl . optionally substituted with hydroxyl;-alkylimidazoyl; HjNC^NHONHGHjCHjCHj-; -R'-CC^NHJNHj; -R'-NHC(=+NR")NR°; or -R'-NR"R° where R' is cycloalkyl, aryl or arylalkyl optionally substituted with one or more heteroatoms selected fiomN, S or O alkyl; and R* and R° are alkyl or cycloalkyl. 10 100. An inhibitor of claim 99 wherein R, is benzyl, (CH3)jCHCHj-, (CH^CH-, HOCHr or -CH2OR'. 101- An inhibitor of claim 98 whereinZis 15 R,-Phe-Arg-; Ri-Phe-(argininie mimetic)-; R^-Val-LeiKarginine mimetic)-; R^-Phe-Lys-; R«-Leu-hPhe-; 20 R«-Val-Leu-Arjg-; R«-Phe(e-Z)-Lyrs-; R,-Phe-Val-; oar R,-Phe-Ser(OBtzl)-. 25 102. An inhibitor of claim 98 whereinZis R,-Phe-AA,-; or Rt-Leu-AA,-; wherein AA| is optionally substituted lysine. 76 INTELLECTUAL PROPERTY OFFICE OF N.Z. 10 JUL 2003 RECEIVED WO 99/54317 PCT/US99/D8501 103. An inhibitor of claim 102 wherein R* is moipholino. 104. An inhibitor of claim 102 wherein is Cbz. 5 105. A use of a compound of claim 1 in the manufacture of a medicament for inhibiting the enzymatic activity of one or more cysteine proteases in a patient in need thereof. 106. A use of a compound of claim 8 in the manufacture of a medicament for inhibiting the enzymatic activity of one or more cysteine proteases in a patient in need thereof. 10 107. A use of a compound of claim 13 in the manufacture of a medicament for inhibiting the enzymatic activity of one or more cysteine proteases in a patient in need thereof. 108. A use of a compound of claim 22 in the manufacture of a medicament for inhibiting the j 5 enzymatic activity of one or more cysteine proteases in a patient in need thereof. 109. A use of a compound of claim 50 in the manufacture of a medicament for inhibiting the enzymatic activity of one or more cysteine proteases in a patient in need thereof. 2q 110. The use of claim 109 wherein the caspase is human interleukin-1 (5 converting enzyme. 111. A use of a compound of claim 72 in the manufacture of a medicament for inhibiting the enzymatic activity of a viral or microbial cysteine protease in a patient in need thereof. The use of claim 111 wherein the cysteine protease is human coronavirus. The use of claim 111 wherein the microbial cysteine protease is gingipain. 112. 25 113. 77 ! 'MicutUTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 RECEIVED WO 99/54317 PCT/US99/08501 114. A use of a compound of claim 93 'in the manufacture of a medicament for inhibiting the enzymatic activity of a protozoan protease in a patient in need thereof. 115. The use of claim 114 wherein the protozoan protease is a Trypanosoma, Schistosoma or Leishmania protease. 116. The use of claim 114 wherein the protozoan protease is a Plasmodium protease. 117. A use of a compound of claim 1 in the manufacture of a medicament for inhibiting the enzymatic activity of cancer procoagulant in a patient in need thereof. 118. A use of a compound of claim 1 in the manufacture of a medicament for inhibiting the enzymatic activity of cysteine proteases associated with apoptosis in pathological states in a patient in need thereof. 119. A use of a compound of claim 1 or claim 2 in the manufacture of a medicament for inhibiting cancer cell growth or tumor progression or tumor metastasis or invasion, by inhibiting the enzymatic activity of cysteine proteases associated with such growth or progession, in a patient in need thereof. 120. The use of claim 119j wherein said protease is cathepsin B or cathepsin L. i 121. A use of a compound of claim 1 or claim 2 in the manufacture of a medicament for inhibiting microbial cell or viral growth or reporudction by inhibiting the enzymatic activity of cysteine proteases associated with such growth or reproduction, in a patient in need thereof. 122. The use of claim 121 wherein the cysteine protease is hepatitis A virus 3G proteinase. 123. The use of claim 121 '* wberein cysteine protease is hepatitis C virus endopeptidase WO 99/54317 PCT/US99/08501 2. wherein the cysteine protease is picomain 3C ihinovirus wherein the cysteine protease is foot and mouth disease virus L 126. The use of claim 121 wherein the cysteine protease is encephalomyelitis virus endopeptidase 2. 127. The use of claim 121 wherein the cysteine protease is picomain 2A protease 128. A use of a compound of claim 1 or claim 2 in the manufacture of a medicament for treating the symptoms associated with allergic response by inhibiting the enzymatic activity of cysteine proteases associated with such response, in a patient in need thereof. 129. The use of claim 128 wherein the protease is Der p I. 130. A use of a compound of claim 1 or claim 2 in the manufacture of a medicament for treating the symptoms associated with a neurodegenerative disorder by inhibiting the eiuymatic activity of cysteine proteases associated with such disease, in a patient in need thereof. 131. The use of claim 130 wherein the neurodegenerative disorder is Alzheimer's disease, Parkinson's disease or multiple sclerosis. 132. The use of claim 131 wherein said disorder is a result of isdiemic-reperfiisioii injury. 133. The use of claim 132 wherein the ischemic-reperfusion injury Is stroke. 124. The use of claim 121 protease. 125. The use of claim 121 proteinase. 79 INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 0 JUL 2003 RECEIVED WO 99/54317 PCT/US99/08501 134. The use of claim 132 wherein the ischemic-reperfusion injury is myocardial infarction, transplantation, vascular injury or cardiovascular collapse or shock. 135. A use of a compound of claim 1 or claim 2 in the manufacture of a medicament for 5 treating the symptoms associated with inflammatory and degenerative diseases by inhibiting the enzymatic activity of cysteine proteases associated with such diseases, in a patient in need thereof. 136. The use of claim 135 wherein . the inflammatory disease is an arthridity. JO '37. The use of claim 136 wherein the arthridity is rheumatoid arthiritis or osteoarthritis. 138. The use of claim 135 wherein the inflammatory disease is periodontal disease. 139. The use of claim 105 wherein the compound is [2-[5-(3-mcthyIbenzyl)-1,3,4- 15 oxadiazolyl]carbonyl]-2-(S)-nnethylpropyl]-L-phenylalanainide-{3R)-(isobutyl)succinic acid. 140. The use of claim 105 wherein the compound is: Acetyl-L-leucyl-N-[l-[2-[5-phenyl]-13,4-oxadiazolyl]caibonyl]-4-(guanidino)-butyl-L- leucyl amide; 20 Acetyl-L-lcucyl-N-[ 1 -[3-[5-methyl]-1,2,4-oxadiazolyl}carbonyl]-ethyl-L-leucyl amide; Acetyl-L-leucyl-N-[l-[3-[5-methyI]-l,2,4-oxadiazolyl]carbonyl]-4-(gu«nidino)-butyl-L-leucyl amide; Acetyl-L-tyrosinyl-L-^valyl-N-[l-[2-[(5-phenyl)-l,3,4-oxadiazolyl]carbonyl]-2-caiboxy-ethyl]-L-alanine amide; or 25 Acetyl-L-Aspartyl-Vadyl-N-[l-[2-[(5-phenyI)-1.3,4-oxadiazolyl] carbonyl]-2-{carboxy)- ethyl]-L-glutamyl amide. 141. The use of claim 105 wherein the compound is (t-butoxysucciinyl)-L-valyl-N-[ 1 -[3-[5-(3-trif]uoromethyIbeiizyl)-1,2,4- 80 0FF'^ 10 JUL 2003 RECEIVED oxadiazolyl]carbonyl]-2-benz^lidone]-L-prolinamide; or carboxysuccin;yl-L-valyI-N-[l-[3-[5-(3-trifluororaethylben2yl)-1 ,2,4-oxadiazolyl]carbonyl]-2-benzyIidone]-L-prolinamide. 5 142. The use of claim 101 wherein the compound is (benzyloxycarbonyl)-L-valyl-N-[l -(2-[5-(3-methylbenzyl>-1,3,4-oxiadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide. 143. a method of detecting or quantifying the activity of a cysteine protease in a pure sample, mixture or a biological fluid or tissue, comprising contacting said protease with a compound of claim lor 2. 144. A method of purifying a cysteine protease in a sample, comprising contacting said >, protease with a compound of'claim 1 or 2. 145. A cysteine protease inhibitor of Formula (I) as claimed in claim 1 substantially as herein described or exemplified and with or without reference to the accompanying drawings. 146. A use as claimed in any one of claims 105-109, 111,114, 117-119, 121,130 and 135 substantially as herein described or exemplified and with or without reference to the 20 accompanying drawings. 147. A method as claimed in claim 143 or 144 substantially as herein described or exemplified and with or without reference to the accompanying drawings. 25 81 INTELLECTUAL PROPERTY OFFICE OF N.Z. 10 JUL 2003 RECEIVED