US006030946A United States Patent (19) 11 Patent Number: 6,030,946 Klaus et al. (45) Date of Patent: *Feb. 29, 2000

54 REVERSIBLE CYSTEINE 56) References Cited INHIBITORS U.S. PATENT DOCUMENTS 75 Inventors: Jeffrey L. Klaus, Redwood City; David 3,859,337 1/1975 Herz et al...... 260/500.5 Rasnick, San Francisco; James T. 4.381,305 4/1983 Casagrande et al...... 424/263 Palmer, San Ramon; Elaine Yee-Lin 5,607.937 3/1997 Stuerzebecher et al...... 514/255 Kuo, San Francisco, all of Calif. FOREIGN PATENT DOCUMENTS 73 Assignee: Axys Pharmaceuticals, Inc., South San 63-275575 11/1988 Japan. Francisco, Calif. 92/08709 5/1992 WIPO. s 92/16549 10/1992 WIPO. * Notice: This patent issued on a continued pros- 94/18185 8/1994 WIPO. ecution application filed under 37 CFR OTHER PUBLICATIONS 1.53(d), and is Subject to the twenty year Hara, K., et al. "Inhibition of Calcium-Activated Neutral patent term provisions of 35 U.S.C. Protease of Monkey Cardiac Muscle by Epoxysuccinic Acid 154(a)(2). Derivatives,” Biomedical Research, 4(1):121-124 (1983). Westmarket al., “Photoregulation of Activity. Pho 21 Appl. No.: 08/657,103 tochromic, Transition-State-Analogue Inhibitors of Cys teine and Serine ,” J. Am. Chem. Soc., 22 Filed: Jun. 3, 1996 115:3416–3419 (1993). O O Turk et al., “The Cystatins: Protein Inhibitors of Cysteine Related U.S. Application Data Proteinases.” FEBS, 285(2):213–219 (1991). Giordano et al., “Synthesis and Properties of D-glucosamine 63 Cont6"Eart tion-in-part of applicationlication No. 08/474.99308/474,993, Jun. 7,7 N-peptidyl Derivativess as Substrate Analog Inhibitors of 7 Papainapain and B.B, Eur: J. Med. Chen.Chem. 26:753:76226:753:

3. ------sse '' (1991). 70.50. o; Primary Examiner Bennett Celsa s s s s s ttorney, Agent, Or Firm ehr HOhbach eSt rttOn 4359463.EE 435/94 65: 435/212. 544/121. Attorney,Herbert LLP.; Agent, David or Firm J. Brezner; Flehr RobinHobbach M. TestSilva Albritton & • Y-1s • as 56451 (57) ABSTRACT 58 Field of Search ...... 514/12, 16–19, inhibitors are provided. 514/37; 530/328-331; 435/4, 23, 24, 94.63, 99.65, 212; 544/121; 564/511 21 Claims, 11 Drawing Sheets U.S. Patent Feb. 29, 2000 Sheet 1 of 11 6,030.946

Mu-Phe-retro-(D,L)-PheSO2Ph 50.OOOO O O 50.OOOO -> N -N- NH NH O 25.OOOO Cruzain 50.0000 FIG. 1A

Mu-Phe-retro-(D,L)-LeuSO2Ph Cathepsin B 5O.OOOO O O Cathepsin L. 17.0000 -> N -NH NH so Cathepsin S 13.0000 bu O O Cruzain 5.7OOO FIG.1B

OH Mu-Tyr-retro-(D,L)-LeuSO2Ph Cathepsin B 50.OOOO O O Cathepsin L 43.0000 ~ NH NH to Cathepsin S 13.0000 bu O O Cruzain. 26.0000 FIG.1C

Mu-Phe-retro(D,L) HphSO2Ph Cathepsin B 6000.0000 O O Cathepsin L 5.2000 rNinh NH is C Cathepsin S 25.0000 O Cruzain 9.OOOO U.S. Patent Feb. 29, 2000 Sheet 2 of 11 6,030.946

OH Mu-Tyr-retro(D,L)-HphSO2Ph Cathepsin B 26.OOOO ? Cathepsin L 15.OOOO 1N1 NH NH NH- S O Cathepsin S 33.0000 ou O O Cruzain 6.2000

OH Mu-Tyr-retro(D,L)NleSO2Ph Cathepsin B 6000.0000 ( Cathepsin L 4.0000 1N NH NH NH1S O Cathepsin S 14.0000 bu O O Cruzain. 13.0000

O Mu-Np2-retro(D,L)NleSO2Ph O Cathepsin B 5O.OOOO l Cathepsin L OOOO 1N-NHN NH so Cathepsin S 0.9400 ou O O Cruzain O.3000 N-- FIG.1G O Boc-Np2-retro(D,L)NleSO2Ph O Cathepsin B 15.OOOO O Cathepsin L O3OOO ^o-NHl NH NH 1. O Cathepsin S 0.0890 O O Cruzain 0.028O U.S. Patent Feb. 29, 2000 Sheet 3 of 11 6,030,946

Piv-Np2-retro(D,L)NIeSO2Ph

Cathepsin B O.OOOO Cathepsin L 0.2000 Cathepsin S 0.2300 Cruzain 0.4200

Mu-Np2-retro(D,L)HphSO2Ph

Cathepsin B 50.OOOO Cathepsin L. 1.8000 N H N H O Cathepsin S 2.2000 C O Cruzain 1.OOOO FIG.1.J. O Boc-Np2-retro(D,L)HphSO2Ph O Cathepsin B 5.3000 X Cathepsin L 11 OOO O1 NH1N NH so Cathepsin S 2.0000 O O Cruzain O.3000 FIG.1 K Mu-Phe-pip-retro(D,L) LeuSO2Ph O Cathepsin B 50.0000 O N1S I Cathepsin L 13.0000 -> N. l NH Nu 6 Cathepsin S 51.0000 ou O Cruzain 75.OOOO U.S. Patent Feb. 29, 2000 Sheet 4 of 11 6,030,946

Mu-Phe-pip-retro(D,L)HphSO2Ph Cathepsin B 10.0000 Ol NuN1i EO Cathepsin L O. 5600 1N1 NH Cathepsin S 15000 ou O Cruzain O.21 OO

O Z-Np2-retro(D,L)NleSO22Np O Cathepsin B 6.OOOO Cathepsin L 0.0300 Or O1 NH1NNH *OO Cathepsin S 0.4300 O O Cruzain O.O290 NY ------FIG.1N 9 Bzac-Np2-retro(D,L)NleSO22Np Cathepsin B 2.OOOO O O Cathepsin L O. 1600 l NH Y

Or NH O NH OEOIO CathepsinCruzain S 0.07300.5800 FIG.1 O

BzISO2-Np2-retro(D,L)NleSO22Np O Cathepsin B 1.4000 (OU Cathepsin O. 1300

O N.YNHO *OOO CathepsinCruzain S 0.2500O. 1100 FIG.1P U.S. Patent Feb. 29, 2000 Sheet S of 11 6,030,946

(OO) O Mu-Np2-pip-retro(D,L)NeSO22Np Cathepsin B 10.0000 O N1iO OO Cathepsin L 0.62OO --- NH Nu Cathepsin S 0.93OO O O Cruzain 0.3700 FIG.1 O (OO) MeOSuc-Np2-pip-retro(DL)NleSO22Np -S Cathepsin B O.OOOO lCOOO Cathepsin L O.2OOO --~'n'- Cathepsinarepsin S O.33OO O O Cruzain O.O560 FIG. 1R CO) O Z-Np2-pip-retro(D,L)NleSO22Np O N1i Cathepsin B 27.0000 N O OO Cathepsin L O.O1 O on- O ~ Cathepsin S O. 15OO Cruzain O. 11 OO FIG. 1S (O) Bzac-Np2-pip-retro(D,L)NleSO22Np O N1i Cathepsin B 4.2OOO 1s-N-N O OO Cathepsin L 0.2300 Or O Cathepsin S 0.1400 Cruzain O.32OO U.S. Patent Feb. 29, 2000 Sheet 6 of 11 6,030.946

BzISO2-Np2-pip-retro(D,L)NleSO22Np

Cathepsin B 5. OOO Cathepsin L 0.2400 Cathepsin S O. 1400 Cruzain O.2200 FIG. 1U

PhSO2-Np2-pip-retro(D,L)NleSO22Np

I Cathepsin B 16.OOOO N Cathepsinatnepsin L. O.0240 Nu. 6 Cathepsin S 0.4500 Cruzain O.O52O FIG.1 V (O) tBac-Np2-pip-retro(D,L)NIeSO22Np (O) f Cathepsin B 67.OOOO O N Cathepsin L O.0410 *th Nu. 6 Cathepsin S 1.3000 Cruzain O. 1400 O

Mu-Phe-pip-GlySO2Ph O Cathepsin B 2000.0000 O -> N b O Cathepsin L. 21.0000 NN- Nu Cathepsin S 67.OOOO Crer Cruzain 122.0000 FIG.1X U.S. Patent Feb. 29, 2000 Sheet 7 of 11 6,030.946 CO) Z-Np2-pip-GlySO22Np Cathepsin B 7.7OOO

on-O Nu(CEOO O CathepsinCathepsin SL O.0.0210 1900 Or O Cruzain 0.0340 FIG.1 Y (O) Phac-Np2-pip-GlySO22Np (O) Cathepsin B 8.0000 ( r OO Cathepsin L 0.0600 NH Nu 6 Cathepsin S 0.4800 O Cruzain O.O350 FIG. 12 (OO) t-BuAC-Np2-pip-GlySO22Np Cathepsin B 10.0000 O N OO Cathepsin L. O.1300 X-- N ~ O Cathepsin S 8000 O Cruzain O.2600 FIG.1AA (O) Boc-Np2-pip-GlySO22NP (O) Cathepsin B 1O.OOOO 1SN 1. Cathepsin L O. 1200 N ~ Cathepsin S 14000 O Cruzain O.2600 FIG.1BB U.S. Patent Feb. 29, 2000 Sheet 8 of 11 6,030.946

Z-Np2-pip-GlySO2Bz Cathepsin B 21.0000 Cathepsin L 0.6800 Cathepsin S 0.2200 Cruzain O.62OO

(O) Phac-Np2-pip-GlySO2Bz. (O) uO Cathepsin B 40.0000 (OU Ni Cathepsin L O. 17OO NH Nsu- O Cathepsin S O.3300 O Cruzain O.32OO FIG.1DD U.S. Patent Feb. 29, 2000 Sheet 9 of 11 6,030.946

O O H O. NH E.AirH2

O R 2 O R 2 R2

O O H 4. Rafi HC* - S - o, -- 9 s N-Nil, O R2 O R2

AA 1 H R 2 O 6. PG Nsu-N 1SN H O O

O O 1 HCI" N - N HC * O R R

O R O R

AA O 5 PG N / H H O U.S. Patent Feb. 29, 2000 Sheet 10 of 11 6,030,946

O H Q R5 O N OH H O

C O R2 -1. He o N-1 O R2 O H 2. N so~.

R2 Rs. O

R RO 2 &2 S 5. N.) /NH (HCL)2 7 N)-/ N1TR O 6. R5 Rs

2 O -Ho-8. (HCL) HN) /N1TYE 6 '9 R COMPOUND 2 U.S. Patent Feb. 29, 2000 Sheet 11 of 11 6,030.946

H R O o H 6 O O O R -e-1. o N1 O R O H 2. Ro-N R

R O O R HBr” 6 O ) { 3. H2N N N1 4 HNX-( N R O Re

R (HCl)2)- 5 HN N

6. R 6 COMPOUND 1

O OR1

Re

H O R1 H OR O N )- 10. N ) PG1 N NH*HCL PG1 N N1TYE AA u-( AA \-( '9 R Re N 6 FIG.5 6,030.946 1 2 REVERSIBLE CYSTEINE PROTEASE inhibitor and target enzyme. It is a further object to provide INHIBITORS these novel cysteine protease inhibitors for use in a variety of therapeutic applications. This is a continuation-in-part of application Ser. No. 08/474,993, filed Jun. 7, 1995, abandoned. 5 In accordance with the foregoing objects, one embodi ment of the present invention provides reversible cysteine FIELD OF THE INVENTION protease inhibitors comprising two N-Substituents linked via The invention relates to novel reversible protease inhibi an ethylenediamine or a Substituted ethylenediamine, tors. The inhibitors are Selective for cysteine proteases. wherein the dissociation constant for inhibition, K, of a protease with the inhibitor, is no greater than about 100 uM, BACKGROUND OF THE INVENTION and wherein Said N-Substituents are Selected from the group Cysteine or thiol proteases contain a cysteine residue at consisting of acyl, acyl peptidyl, alkyloxycarbonyl, alky the responsible for . Since cysteine loxycarbonyl peptidyl, Sulfonyl, Sulfonyl peptidyl, peptidyl, proteases have been implicated in a number of diseases, 15 Sulfamoyl, Sulfamoyl peptidyl, Sulfinyl, Sulfinyl peptidyl, including arthritis, muscular dystrophy, inflammation, tumor carbamoyl, and carbamoyl peptidyl. invasion, glomerulonephritis, malaria, and other parasite borne infections, methods for selectively and irreversibly Also provided are reversible cysteine protease inhibitor inactivating them provide opportunities for new drug can having the formula comprising: didates. See, for example, Esser, R. E. et al., Arthritis & Rheumatism (1994) 37, 236; Meijers, M. H. M. et al., R1 R2 Agents Actions (1993), 39 (Special Conference Issue), C219; Machleidt, W. et al, Fibrinolysis (1992), 6 Suppl. 4,125; Sloane, B. F. et al., Biomed. Biochim. Acta (1991), -x 50,549; Duffy, M.J., Clin. Exp. Metastasis (1992), 10, 145; 25 R R4 Rosenthal, P. J., Wollish, W. S., Palmer, J. T., Rasnick, D., J. Clin. Investigations (1991), 88, 1467; Baricos, W. H. etal, Arch. Biochem. Biophys. (1991), 288, 468; Thornberry, N. A. et al., Nature (1992), 356, 768. In this aspect, A and X are N-substituents selected from Low molecular weight inhibitors of cysteine proteases the group consisting of acyl, acyl peptidyl, have been described by Rich, Proteinase Inhibitors (Chapter alkyloxycarbonyl, alkyloxycarbonyl peptidyl, Sulfonyl, Sul 4, “Inhibitors of Cysteine Proteinases”), Elsevier Science fonyl peptidyl, peptidyl, Sulfamoyl, Sulfamoyl peptidyl, Publishers (1986). Such inhibitors include peptide Sulfinyl, Sulfinyl peptidy, carbamoyl, and carbamoyl pepti aldehydes, which form hemithioacetals with the cysteine of dyl. R is either hydrogen or an amino acid side chain, and the protease active Site. See, for instance, Cheng, H., Keitz, 35 R is either hydrogen or an amino acid Side chain. However, P., and Jones, J. B., J. Org. Chem. (1994), 59, 7671. The either both R and R2 are hydrogen, or one of R or R2 is an disadvantage of aldehydes is their in Vivo and chemical amino acid Side chain and the other one of R and R is instabilities. hydrogen. Ramd R are hydrogen, or are bonded together Methods for selectively and irreversibly inhibiting cys to form ethylene or substituted ethylene. Additionally, the teine proteases have relied upon alkylation by peptide 40 dissociation constant for inhibition, K, of a protease with C-fluoromethyl ketones (Rasnick, D., Anal. Biochem. the inhibitor, is no greater than about 100 uM. (1985), 149, 416), diazomethyl-ketones (Kirschke, H., Shaw, E. Biochem. Biphys. Res. Commun. (1981), 101, Also provided are reversible cysteine protease inhibitors 454), acyloxymethyl ketones (Krantz, A. et al., having the formula comprising: Biochemistry, (1991), 30, 4678; Krantz, A. et al., U.S. Pat. 45 No. 5,055,451, issued Oct. 8, 1991), and ketosulfonium salts R1 R2 (Walker, B., Shaw, E., Fed. Proc. Fed. Am. Soc. Exp. Biol., (1985), 44, 1433). Other families of cysteine protease inhibitors include AN NEX epoxySuccinyl peptides, including E-64 and its analogs 50 (Hanada, K. et al., Agric. Biol. Chem (1978), 42, 523; Sumiya, S. etal., Chem. Pharm. Bull. (1992), 40, 299 Gour-Salin, B. J. et al., J. Med. Chem., (1993), 36, 720), Further provided are reversible cysteine protease inhibi C.-dicarbonyl compounds, reviewed by Mehdi, S., Bioor tors having the formula comprising: ganic Chemistry, (1993), 21, 249, and N-peptidyl-O-acyl 55 hydroxamates (Bromme, D., Neumann, U., Kirschke, H., Demuth, H- U., Biochim. Biophys. Acta, (1993), 1202, 271. An additional summary of methods for reversibly and irre versibly inhibiting cysteine proteases has recently been compiled; See Shaw, E., Advances in Enzymology and 60 Related Areas of Molecular Biology (1990), 63, 271. SUMMARY OF THE INVENTION It is an object of the present invention to provide novel 65 cysteine protease inhibitors that function reversibly, result Additionally provided are reversible cysteine protease ing in tight binding (low dissociation constants) between inhibitors having the formula comprising: 6,030.946 4 ing to a patient a therapeutically effective dose of cysteine protease inhibitor of the invention, and pharmaceutical com positions comprising the cysteine protease inhibitors of the A O invention. Additionally, the invention provides methods of detecting re-ever -- a cysteine protease in a Sample comprising assaying the R R4 O Sample for protease activity using a protease Substrate. The Sample is then assayed for protease activity in the presence In this aspect, n is from 1 to 10, PG is a protecting group, of a known concentration of a cysteine protease inhibitor of and AA is an amino acid. R is either hydrogen or an amino the invention, and determining the amount of cysteine acid Side chain, and R is either hydrogen or an amino acid protease. Side chain. However, either both R and R2 are hydrogen, or one of R or R is an amino acid side chain and the other one BRIEF DESCRIPTION OF THE DRAWINGS of R and R is hydrogen. R and R are hydrogen, or are bonded together to form ethylene or substituted ethylene. 15 FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, -SO-Ro is a Sulfonyl moiety. Additionally, the dissociation 1M, 1N, 10, 1P, 1 O, 1R, 1S, 1T, 1U, 1V, 1W, 1X, 1Y, 17, constant for inhibition, K, of a protease with the inhibitor, 1AA, 1B, 1CC, and 1DD depict the structures of some of the is no greater than about 100 uM. cysteine protease inhibitors of the invention. The Structures, abbreviated names, and kinetic data are shown, as described Further provided are reversible cysteine protease inhibitor more fully in the Examples. The kinetic data are in micro having a formula comprising: molar units. FIG. 2 depicts a Schematic Synthesis for cysteine protease inhibitors with R. Substituent groups on ethylenediamine )–( moieties. 1. Carbonyldiimidazole, NH/THF; 2. HCl/ 25 dioxane, CHCl/ether; 3. RSOCl, triethyl amine (TEA), R-it-wr. i THF; 4. LAH or suitable reducing agent, THF, reflux; 5. O R3 R4 O HCl/dioxane, CHCl/ether; 6. PG-M-OH, NMM, IBCF, THF at -10° C. In this aspect, n is from 1 to 10 and AA is an amino acid. FIG.3 depicts a Schematic Synthesis for cysteine protease R is either hydrogen or an amino acid Side chain, and R is inhibitors with R. Substituent groups on ethylenediamine either hydrogen or an amino acid Side chain. However, either moieties. 1. LiAlH (LAH) or suitable reducing agent, THF, both R and R are hydrogen, or one of R or R is an amino reflux; 2. HCl/dioxane, CHCl/ether; 3. RSOCl, TEA, acid Side chain and the other one of R and R2 is hydrogen. THF, -10° C.; 4. H/Pd/C or suitable catalyst, HCl/dioxane, R and R are hydrogen, or are bonded together to form EtOH; 5. PG-AA-OH, 4-methyl morpholine (NMM); ethylene or substituted ethylene. -SOR is a sulfonyl 35 isobutyl chloroformate (IBCF), THF, -10° C. moiety. Additionally, the dissociation constant for inhibition, FIG. 4 depicts a Schematic Synthesis for cysteine protease K, of a protease with the inhibitor, is no greater than about inhibitors with R- and Rs Substituent groups on piperazine 100 uM. moieties. 1. Activation of acid: pyr, TC, THF, -10° C.; 2. An additional aspect of the invention provides reversible bis(trimethylsilyl)acetamide (BSA), TEA, THF; 3. HBr/ cysteine protease inhibitors having a formula comprising: 40 AcOH; 4. TEA, EtOH; 5. LAH or suitable reducing agent, THF, reflux; 6. HCl/dioxane, CHCl/ether; 7. RSOCl, TEA, THF; 8. H/Pd/C or suitable catalyst, EtOH; 9. pyr, R1 R2 TC, THF, -10° C.; 10. compound 2, BSA, TEA, THF. FIG. 5 depicts a Schematic Synthesis for cysteine protease )-( 45 inhibitors with R and R. Substituent groups on piperazine R- c-ever i moieties. Step 1 is the activation of the acid: pyridine (pyr), R R4 O thionyl chloride (TC), THF, at -10° C.; 2. bis(trimethylsilyl) acetamide (BSA), TEA, THF; 3. HBr/AcOH; 4. TEA, In this aspect, n is from 1 to 10, PG is a protecting group 50 EtOH; 5. LAH or suitable reducing agent, THF, reflux; 6. and M is an amino acid. R is either hydrogen or an amino HCl/dioxane, CHCl/ether; 7. pyr, TC, THF; 8. compound acid Side chain, and R2 is either hydrogen or an amino acid 1, BSA, TEA, THF; 9. H/Pd/C or suitable catalyst, HCl/ Side chain. However, either both R and R2 are hydrogen, or dioxane, EtO; 10. RSOCl, TEA, THF. one of R or R is an amino acid side chain and the other one DETAILED DESCRIPTION OF THE of R and R is hydrogen. R and R are hydrogen, or are 55 bonded together to form ethylene or substituted ethylene. INVENTION -SO-Ro is a Sulfonyl moiety. Additionally, the dissociation The present invention relates to novel cysteine protease constant for inhibition, K, of a protease with the inhibitor, inhibitors. It is believed that the enzyme is reversibly is no greater than about 100 uM. inhibited by means of interactions between the N-blocking A further aspect of the invention provides methods of 60 groups and the R or R group of the inhibitor and the inhibiting a cysteine protease inhibitor comprising revers Surface of the binding Sites of the enzyme, via hydrogen ibly binding a cysteine protease inhibitor of the invention to bonding and hydrophobic interactions. a cysteine protease. Generally, the inhibitors of the present invention inhibit An additional aspect provides cysteine proteases inhibited cysteine proteases and do not inhibit Serine, aspartyl, and by the cysteine protease inhibitors of the invention. 65 Zinc proteases. The mechanisms of Serine protease action Further, the invention provides methods of treating cyS have been described by Walsh, C., in “Enzymatic Reaction teine protease associated disorders comprising administer Mechanisms' pp. 94-97, W.H. Freeman and Co., San 6,030.946 S 6 Francisco, 1979. The serine at the active site reacts with the proteases is undesirable. Alternatively, the cysteine protease carbonyl of the Substrate, forming a tetrahedral intermediate. inhibitors of the present invention may be bound to a The inhibitors of this invention have no carbonyl at the site chromatographic Support, using techniques well known in of nucleophilic attack, and are not Susceptible to attack by the art, to form an affinity chromatography column. A Serine proteases. Sample containing an undesirable cysteine protease is run However, in Some embodiments, the protease inhibitors through the column to remove the protease. Alternatively, of the present invention may have activity against other the same methods may be used to identify new proteases. types of proteases, Such as Serine, aspartyl or other In a preferred embodiment, the cysteine protease inhibi metalloproteases, but to a lesser extent. In particular, the tors are useful for inhibiting cysteine proteases implicated in protease inhibitors of the invention may have activity a number of diseases. In particular, B, L, and S, against Serine proteases, for example, they may have activity cruZain, I and II, and interleukin 1B converting against chymotrypsin. enzyme are inhibited. These are examples of lySOSomal cysteine proteases implicated in a wide spectrum Cysteine proteases are a family of proteases that bear a thiol group at the active site. These proteases are found in of diseases characterized by tissue degradation. Such dis 15 eases include, but are not limited to, arthritis, muscular bacteria, Viruses, eukaryotic microorganisms, plants, and dy Strophy, inflammation, tumor in Va Sion, animals. Cysteine proteases may be generally classified as glomerulonephritis, parasite-borne infections, Atlzheimer's belonging to one of four or more distinct Superfamilies. disease, periodontal disease, and cancer metastasis. For Examples of cysteine proteases that may be inhibited by the example, mammalian lySOSomal thiol proteases play an novel cysteine protease inhibitors of the present invention important role in intracellular degradation of proteins and in include, but are not limited to, the plant cysteine proteases the processing of Some peptide hormones. Enzymes Similar Such as , ficin, aleurain, ory Zain and actinidin; mam malian cysteine proteaseS Such as cathepsins B, H, J, L, N, to cathepsins B and L are released from tumors and may be S, T, O, O2 and C, ( is also known as dipeptidyl involved in tumor metastasis. Cathepsin L is present in peptidase I), interleukin converting enzyme (ICE), calcium diseased human Synovial fluid and transformed tissues. 25 Similarly, the release of cathepsin B and other lySOSomal activated neutral proteases, I and II, bleomycin proteases from polymorphonuclear granulocytes and mac , Viral cysteine proteases Such as picomian 2A and 3C, aphthovirus endopeptidase, cardiovirus endopeptidase, rophages is observed in trauma and inflammation. comovirus endopeptidase, potyvirus endopeptidases I and II, The cysteine protease inhibitorS also find application in a adenovirus endopeptidase, the two endopeptidases from multitude of other diseases, including, but not limited to, chestnut blight virus, togavirus cysteine endopeptidase, as gingivitis, malaria, leishmaniasis, filariasis, Osteoporosis and well as cysteine proteases of the polio and rhinoviruses, and Osteoarthritis, and other bacterial and parasite-borne infec cysteine proteases known to be essential for parasite tions. The compounds also offer application in Viral lifecycles, such as the proteases from Species of Plasmodia, diseases, based on the approach of inhibiting proteases Entamoeba, Onchocera, Trypansoma, Leishmania, necessary for viral replication. For example, many picor Haemonchus, Dictyostelium, Therileria, and Schistosoma, 35 noviruses including poliovirus, foot and mouth disease Such as those associated with malaria (P falciparium), Virus, and rhinovirus encode for cysteine proteases that are trypanoSomes (T. Cruzi, the enzyme is also known as cruzain essential for cleavage of Viral polyproteins. or ), murine P vinckei, and the C. elegans cysteine Additionally, these compounds offer application in disor protease. For an extensive listing of cysteine proteases that ders involving interleukin-1? converting enzyme (ICE), a may be inhibited by the cysteine protease inhibitors of the 40 cysteine protease responsible for processing interleukin 1B: for example, in the treatment of inflammation and immune present invention, see Rawlings et al., Biochem. J. based disorders of the lung, airways, central nervous System 290:205-218 (1993), hereby expressly incorporated by ref and Surrounding membranes, eyes, ears, joints, bones, con CCCC. nective tissues, cardiovascular System including the Accordingly, inhibitors of cysteine proteases are useful in 45 pericardium, gastrointestinal and urogenital Systems, the a wide variety of applications. For example, the inhibitors of skin and the mucosal membranes. These conditions include the present invention are used to quantify the amount of infectious diseases where active infection exists at any body cysteine protease present in a Sample, and thus are used in Site, Such as meningitis and Salpingitis, complications of assays and diagnostic kits for the quantification of cysteine infections including Septic Shock, disseminated intravascu proteases in blood, lymph, Saliva, or other tissue samples, in 50 lar coagulation, and/or adult respiratory distress Syndrome; addition to bacterial, fungal, plant, yeast, Viral or mamma acute or chronic inflammation due to antigen, antibody lian cell cultures. Thus in a preferred embodiment, the and/or complement deposition; inflammatory conditions Sample is assayed using a Standard protease Substrate. A including arthritis, chalangitis, colitis, encephalitis, known concentration of cysteine protease inhibitor is added, endocarditis, glomerulonephritis, hepatitis, myocarditis, and allowed to bind to a particular cysteine protease present. 55 pancreatitis, pericarditis, reperfusion injury and Vasculitis. The protease assay is then rerun, and the loSS of activity is Immune-based diseases include but are not limited to con correlated to cysteine protease activity using techniques well ditions involving T-cells and/or macrophages Such as acute known to those skilled in the art. and delayed hyperSensitivity, graft rejection, and graft Thus, methods of inhibiting a protease are provided, Versus-host disease, auto-immune diseases including Type I wherein the cysteine protease inhibitors of the invention 60 diabetes mellitus and multiple Sclerosis. Bone and cartilage may be added to a Sample of cysteine protease, to form a reabsorption as well as diseaseS resulting in excessive depo cysteine protease/cysteine protease inhibitor complex. Sition of extracellular matrix Such as interstitial pulmonary Additionally, the cysteine protease inhibitors are also fibrosis, cirrhosis, Systemic Sclerosis, and keloid formation useful to remove, identify or inhibit contaminating cysteine may also be treated with the inhibitors of the present proteases in a Sample. For example, the cysteine protease 65 invention. The inhibitors may also be useful in the treatment inhibitors of the present invention are added to Samples of certain tumors that produce IL 1 as an autocrine growth where proteolytic degradation by contaminating cysteine factor and in preventing the cachexia associated with certain 6,030.946 7 8 tumors. Apoptosis and cell death are also associated with methyl, ethyl, prop-yl, isopropyl, butyl, Sec-butyl, isobutyl, ICE and ICE-like activities and may be treated with the tert-butyl, Vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, inhibitors of the present invention. 2-butenyl, 3-butenyl, 2-methylallyl, ethynyl, 1-propynyl, Furthermore, the cysteine protease inhibitors of the 2-propynyl, etc.). present invention find use in drug potentiation applications. “Cycloalkyl', as in cycloalkyl and cycloalkylalkyl, means For example, therapeutic agents Such as antibiotics or anti a Saturated or unsaturated, monocyclic or polycyclic hydro tumor drugs can be inactivated through proteolysis by carbon radical containing 3 to 20 carbon atoms or the endogeneous cysteine proteases, thus rendering the admin number of carbon atoms indicated, wherein the carbon atom istered drug less effective or inactive. For example, it has with the free Valence is a member of a non-aromatic ring, been shown that bleomycin, an antitumor drug, can be and any carbocyclic ketone and thioketone derivative hydrolyzed by , a cysteine protease (see thereof (e.g., the term cycloalkyl is meant to include Sebti et al., Cancer Res. January 1991, pages 227-232). cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, Accordingly, the cysteine protease inhibitors of the inven cyclohexenyl, bicyclo2.2.2]octyl, 1,2,3,4-tetrahydro-1- tion may be administered to a patient in conjunction with a naphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, therapeutic agent in order to potentiate or increase the 15 9-fluorenyl, etc.). activity of the drug. This co-administration may be by “Heterocycloalky 1’, as in he terocycloalkyl, Simultaneous administration, Such as a mixture of the cyS heterocycloalkylalkanoylamino, heterocycloalkylcarbonyl, teine protease inhibitor and the drug, or by Separate Simul heterocycloalkylcarbonyl, and the like, means cycloalkyl as taneous or Sequential administration. defined above wherein 1 to 5 of the indicated carbon atoms In addition, cysteine protease inhibitors have been shown is replaced by a heteroatom chosen from N, O, S, P or AS, to inhibit the growth of bacteria, particularly human patho wherein the atom with the free valence is a member of a genic bacteria (see Bjorck et al., Nature 337:385 (1989)). non-aromatic ring, and any heterocyclic ketone, thioketone, Accordingly, the cysteine protease inhibitors of the present Sulfone or Sulfoxide derivative thereof, (e.g., the term het invention may be used as antibacterial agents to retard or erocycloalkyl is meant to include piperidyl, pyrrolidinyl, inhibit the growth of certain bacteria. 25 pyrrolinyl, imidazolidinyl, indolinyl, quinuclidinyl, The cysteine protease inhibitors of the invention also find morpholinyl, piperazinyl, N-methylpiperazinyl, piperadinyl, use as agents to reduce the damage of bacterial cysteine 4,4-dioxo-4-thia pipe ridinyl, 1,2,3,4-tetrahydro-3- proteases to host organisms. For example, StaphylococcuS isoquinolyl, 2,4-diaza-3-oxo-7-thia-6-bicyclo[3.3.0octyl, produces a very active extracellular cysteine protease which etc.). Thus, hetero(C)cycloalkyl includes the radicals degrades insoluble elastin, possibly contributing to the con morpholinyl, piperazinyl, piperidinyl and the like. nective tissue destruction Seen in bacterial infections Such as "Aryl” means an aromatic monocyclic or polycyclic Septicemia, Septic arthritis and otitis. See Potempa et al., J. hydrocarbon radical containing 6 to 14 carbon atoms or the Biol. Chem. 263(6):2664-2667 (1988). Accordingly, the number of carbon atoms indicated and any carbocylic ketone cysteine protease inhibitors of the invention may be used to or thioketone derivative thereof, wherein the carbon atom treat bacterial infections to prevent tissue damage. 35 with the free Valence is a member of an aromatic ring, (e.g., The present invention generally provides new peptide aryl includes phenyl, naphthyl, anthracenyl, phenanthrenyl, based and peptidomimetic cysteine protease inhibitors for 1,2,3,4-tetrahydro-5-naphthyl, 1-oxo-1,2-dihydro-5- use as reversible cysteine protease inhibitors. By "cysteine naphthyl, 1-thioxo-1,2-dihydro-5-naphthyl, etc.). For the protease inhibitor” herein is meant an inhibitor which inhib purposes of this application aryl includes heteroaryl. "Het its cysteine proteases. In a preferred embodiment, the cyS 40 eroaryl' means an aromatic monocyclic or polycyclic hydro teine protease inhibitors are specific to cysteine proteases, carbon radical containing overall from 5 to 14 atoms or the that is, they do not inhibit other types of protease Such as number of atoms indicated, wherein 1 to 5 of the indicated Setnne, aspartyl, or other metalloproteases. However, in carbon atoms are replaced by a heteroatom chosen from N, alternative embodiments, the cysteine protease inhibitors of O, S, P or AS, wherein the atom with the free valence is a the invention may inhibit other types of proteases as well; 45 member of an aromatic ring, and any heterocyclic ketone for example, they may have activity against Serine proteases. and thioketone derivative thereof (e.g., the term heteroaryl is By “reversible” herein is meant that the inhibitor binds meant to include thienyl, furyl, pyrrolyl, pyrimidinyl, non-covalently to the enzyme, and is to be distinguished isoxazolyl, oxa Xoly 1, indolyl, ben Zob thienyl, from irreversible inhibition. See Walsh, Enzymatic Reaction 50 isobenzofuranyl, purinyl, isoquinoly 1, pterdinyl, Mechanisms, Freeman & Co., N.Y., 1979. “Reversible” in pyrimidinyl, imidazolyl, pyridyl, pyrazolyl, pyrazinyl, this context is a term understood by those skilled in the art. 4-oxo-1,2-dihydro-1-naphthyl, 4-thioxo-1,2-dihydro-1- In addition, the reversible cysteine protease inhibitors are naphthyl, etc.). Thus, hetero(C)aryl includes the radicals competitive inhibitors, that is, they compete with Substrate pyridyl, pyrimidinyl, and the like. in binding reversibly to the enzyme, with the binding of 55 “1,2-Phenylenedimethylene' means a divalent radical of inhibitor and Substrate being mutually exclusive. In addition, the formula -CH2CHCH-. For example, the group the Stoichiometry of inhibition is 1:1; that is, a single R Y-Z X- in which Y is -N(R), Z is -CH inhibitor molecule is Sufficient to inhibit a single enzyme (R)-, X is carbonyl and R together with R forms molecule. 1,2-diphenylenedimethylene' is 1,2,3,4-tetrahydro-3- Unless otherwise Stated, the following terms used in the 60 isoquinolylcarbonyl and Substituted derivatives and indi Specification and claims are defined for the purposes of this vidual Stereoisomers and mixture of Stereoisomers thereof. application and have the meanings given below: Substituted derivatives of the 1,2-phenylenedimethylene “Alkyl', as in alkyl, alkyloxy, alkylthio, alkylsulfonyl, divalent radical may contain a hydroxy group on any carbon alkylcarbamoyl, dialkylcarbamoyl, heteroarylalkyl, within the ring System or an OXO group on either of the arylalkyl, and the like, means a Straight or branched, Satu 65 unsaturated ring carbon atoms. rated or unsaturated hydrocarbon radical having from 1 to 10 “Methylene” as in “(Cl)methylene” and “(C-7) carbon atoms or the number of carbon atoms indicated (e.g., methylene' mean a Straight, Saturated divalent radical hav 6,030.946 9 10 ing the number of carbon atoms indicated. For example, Heterocycloalkyl (forming heterocycloalkylcarbonyl) is “(C)methylene' includes trimethylene (-(CH2) ) and also preferred, optionally Substituted with a radical Selected tetramethylene (-(CH2)4-). Thus, the group R-Y- from hydroxy, alkyl, heterocycloalkyl, alkanoyl, Z-X- in which Y is -N(R), Z is -CH(R)-, X is alkyloxycarbonyl, arylalkyloxycarbonyl and heterocy carbonyl and R together with R forms trimethylene is cloalkylcarbonyl. Aryl preferably is phenyl. Aralkyl prefer 2-pyrrolidinylcarbonyl and the individual Stereoisomers and ably is phenylalkyl of 7 to 12 carbon atoms, particularly mixtures of stereoisomers thereof. Substituted derivatives of benzyl. The optional Substituents of an aryl or aralkyl moiety the trimethylene and tetramethylene divalent radicals may preferably are one or two groupS alkyl of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms, halogen of atomic contain a hydroxy group, or a protected derivative thereof, number of from 9 to 35, hydroxy and/or amino, preferably or an OXO group on any of the ring carbon atoms. Suitable one or two groups methyl, methoxy, chlorine, bromine, hydroxy protective groups are defined below. fluorine, hydroxy or amino, particularly one hydroxy, amino, “Oxa(C7)methylene' and “aza(C7)methylene' mean chlorine, bromine, or fluorine, optionally in protected form methylene as defined above wherein one of the indicated where appropriate, nitro, alkyl or arylsulfonyl, or halogen carbon atoms is replaced by an oxygen or nitrogen atom, substituted alkyl of 1 to 5 carbon atoms, particularly trif respectively. For example, "Oxa(Cs)methylene' includes 15 luoromethyl. Also included are perfluoro groups, Such as 3-oxapentamethylene (-CHCHOCH2CH2-) and perfluoro alkyl, aryl, and aralkyl. 2-oxapentamethylene (-CHOCH2CHCH-). Thus, Particularly preferred in regards to R, are: (1) (C1-C5) -C(O)NRoR means the radical 4-morpholinylcarbonyl alkyl, preferably ethyl; (2) (C3-C7)cycloalkyl, preferably when Ro and R together form 3-oxapentamethylene and cyclopentyl or cyclohexyl; (3) (C3-C7)cycloalkyl-(C1-C5) the radical 1-piperazinylcarbanoyl when Ro and R. alkyl, especially (C5-C6)cycloalkyl-methyl; (4) (C3-C7) together form 3-aZapentamethylene. cycloalkyl-(C1-C5)alkenyl, especially (C5-C6)cycloalkyl “Adjacent, as use in the phrase “R together with an methylene; (5) phenyl; (6) (C7-C12)phenylalkyl, especially adjacent R', means that the atoms to which the Re and benzyl, (7) aryl or aralkyl Substituted by one or two groups R groups are respectively attached are in turn attached to of C1-C5 alkyl, C1-C5 alkoxy, halogen, hydroxy or amino, 25 with one or two groups of methyl, methoxy, chlorine, one another. bromine, fluorine, hydroxy or amino being preferred and In the broadest embodiment, the cysteine protease inhibi hydroxy, amino, chlorine, bromine or fluorine being particu tors of the present invention comprise two N-Substituents larly preferred; (11) aryl or aralkyl substituted by one or two linked via an ethylenediamine or piperazine group. groups of halogen-Substituted C1-C5 alkyl, especially trif Suitable N-Substituents include, but are not limited to, luoromethyl, nitro, Sulfonyl, or arylsulfonyl, in protected acyl, alkyloxycarbonyl, Sulfonyl, Sulfamoyl, peptidyl, form where appropriate; (12) perfluoro groups, Such as carbamoyl, Sulfinyl, aralkyl, or hydrogen, or combinations perfluoro alkyl, aryl, and aralkyl. Most preferred R, acyl thereof, including, but not limited to, acyl peptidyl, alky groups include phenyl and 4-morpholinyl. loxycarbonyl peptidyl, Sulfonyl peptidyl, Sulfamoyl Thus, preferred acyl group S C peptidyl, carbamoyl peptidyl and Sulfinyl peptidyl. 35 alkyloxycarbonylalkanoyl, arylalkanoyl, aroyl and alkanoyl In a preferred embodiment, the reversible cysteine pro groups (optionally Substituted with a radical Selected from tease inhibitors of the present invention comprise composi carbo Xy, alkyl oxy carbonyl and tions having the formula depicted in Formula 1: heterocycloalkylalkanoylamino), cycloalkylcarbonyl, het erocycloalkylcarbonyl (optionally Substituted with a radical Formula 1 40 Selected from hydroxy, alkyl, heterocycloalkyl, alkanoyl, R1 R2 alkyl oxycarbonyl, arylalkyl oxy carbonyl and heterocycloalkylcarbonyl), arylalkanoyl, and aroyl. By “acyl peptidyll” or “peptidyl acyl herein is meant a A -x peptidyl group linked to an acyl group. It is to be understood R R4 45 that the peptidyl group is linked to one of the nitrogens of the ethylenediamine or piperazine moieties, and the acyl group is linked to the peptidyl group. Thus, the free terminal In this embodiment, A and X are N-Substituents selected functionality of an acyl peptidyl group is the R7 group. from the group consisting of acyl, acyl peptidyl, Accordingly, the order of the functionalities differS depend alkyloxycarbonyl, alkyloxycarbonyl peptidyl, Sulfonyl, Sul 50 ing on whether it is the A group or the X group which is the fonyl peptidyl, peptidyl, Sulfamoyl, Sulfamoyl peptidyl, acyl peptidyl, the group is called an acyl peptidyl moiety carbamoyl, carbamoyl peptidyl, Sulfinyl, and Sulfinyl pepti when describing the A group and a peptidyl acyl moiety dyl. R and R2 are hydrogen or an amino acid Side, wherein when describing the X group. Formula 2 shows a cysteine only 1 of R or R2 is an amino acid Side chain and the other protease inhibitor with an acyl peptidyl group at the A and one of R or R is hydrogen. R and R are hydrogen, or are 55 a peptidyl acyl group at the X position, wherein n is from 1 bonded together to form ethylene or substituted ethylene. to 10. By “acyl herein is meant a -COR, group. Suitable R, groups include, but not limited to, an alkyl (forming Formula 2 alkanoyl), a cycloalkyl (forming cycloalkylcarbonyl), a R1 R2 cycloalkylalkyl, a cycloalkylalkenyl, an aryl (forming 60 aroyl), or an aralkyl. In Such an instance, alkyl is preferably of 1 to 5 carbon atoms, especially ethyl. Cycloalkyl prefer R- c-ever tw: C-R, ably is of 3 to 7 carbon atoms, preferably cyclopentyl or cyclohexyl. Cycloalkylalkyl or cycloalkylalkenyl preferably R R4 is of 3 to 7 carbon atoms in the cycloalkyl, particularly 5 or 65 6 carbon atoms, and of 1 to 5 carbon atoms, particularly 1 AS is described below, the choice of the peptidyl groups carbon atom, in the alkyl or alkylene moieties thereof. will depend on the protease to be inhibited. 6,030.946 11 12 By "peptidyll” herein is meant a peptide or peptidomi from 9 to 35, hydroxy and/or amino, preferably one or two metic Structure. In Some embodiments, peptidyl is a single groups methyl, methoxy, chlorine, bromine, fluorine, amino acid; in other embodiments, the peptidyl group com hydroxy or amino, particularly one hydroxy, amino, prises at least two amino acids linked via a peptide bond or chlorine, bromine, or fluorine, optionally in protected form isostere. The peptidyl group may include up to about 10 where appropriate, halogen-Substituted alkyl of 1 to 5 car amino acids, with 1 to 7 being preferred, although cysteine bon atoms, particularly trifluoromethyl. protease inhibitors are generally from about 1 to about 4 Specifically excluded from the definition of amino acid amino acids in length, Since Smaller inhibitors are usually Side chain is OXO. Thus, the ethylenediamine does not desired in therapeutic applications. contain peptide bonds. The peptidyl group may comprise naturally occurring The peptidyl functionality may also be depicted as shown amino acids and peptide bonds, or Synthetic peptidomimetic in Formulae (a) and (b): Structures, i.e. “analogs”. Thus "amino acid”, “peptide residue', or "peptidyll” as used herein means both naturally (a) occurring and Synthetic amino acids, i.e. amino acid analogs. O For example, homo-phenylalanine is considered an amino 15 acid for the purposes of the invention. “Amino acid” also Ny1/yx1'N,Z. X ls includes imino acid residues Such as proline and hydrox yproline. The term as used herein also refers to portions of (b) an amino acid, Such as an amino acid Side chain. Thus, R, O O R2, Rs, Re and R, for example, may be amino acid Side chains, which includes naturally occurring amino acid side Ny-s-s-sZ. X ls ls chains as well as non-naturally occurring Side chain analogs as described herein. The side chains may be in either the (R) or the (S) configuration. In a preferred embodiment, the in which n is 0 to 9; X-X represents a linkage Selected amino acids are in the (S) or L-configuration. 25 from -C(O)NR-, -CHNR-, -C(O)CH- and If non-naturally occurring Side chains are used, i.e. amino -NRC(O)-; Y is -CH(R)- or -NR-; and Z is acid analogs, non-amino acid Substituents may be used, for -(CH-)-, -C(R)(R)- or -N(R)-; wherein R example to prevent or retard in Vivo degradations. Such is hydrogen or as defined below, R is hydrogen or methyl non-amino acid Substituents will normally include, but are and each R is an amino acid Side chain. Thus each R is not limited to, an alkyl (optionally Substituted with a radical independently hydrogen, alkyl (optionally Substituted with a Selected from hydroxy, alkyloxy, amino, alkylamino, radical Selected from hydroxy, alkyloxy, amino, alkylamino, dialkylamino, uriedo, alkyluriedo, mercapto, alkylthio, dialkylamino, uriedo, alkyluriedo, mercapto, alkylthio, carboxy, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, carboxy, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, alkylsulfinyl, alkylsulfonyl, guanidino, -P(O)(OR), alkylsulfinyl, alkylsulfonyl, guanidino, -P(O)(OR), -OP(O)(OR) or -OP(O)(R), wherein each R is 35 -OP(O)(OR) or -OP(O)(R), wherein each R is independently hydrogen or alkyl, or a protected derivative independently hydrogen or alkyl, or a protected derivative thereof), cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, a thereof), cycloalkyl cycloalkylalkyl, a group selected from group Selected from aryl (including heteroaryl) and arylalkyl aryl and arylalkyl (which group is optionally Substituted at (including heteroarylalkyl and heteroarylalkenyl), (which its aryl ring with one to three radicals Selected from hydroxy, group is optionally Substituted at its aryl ring with one to 40 amino, guanidino, halo, optionally halo-Substituted alkyl, three radicals Selected from hydroxy, amino, guanidino, alkyloxy and aryl, or a protected derivative thereof) or halo, optionally halo-Substituted alkyl, alkyloxy and aryl, or together with an adjacent R forms a divalent radical a protected derivative thereof), alkoxy, cyano, carboxy, Selected from (C) methylene and 1,2- alkyloxycarbonyl, alkanoyl, carbamoyl, alkylcarbamoyl, phenylenedimethylene (which radical is optionally Substi dialkylcarbamoyl, alkyloxy(alkyl)carbamoyl, or aminoalky 45 tuted with hydroxy, or a protected derivative thereof, or lcarbamoyl. In Such an instance, alkyl is preferably of 1 to OXO). 5 carbon atoms, preferably branched, particularly isobutyl. The peptidyl group may also contain additional functional Cycloalkyl preferably is of 3 to 7 carbon atoms, preferably groups, as depicted by “PG” of Formula 3 below. Thus, the cyclopentyl or cyclohexyl. Cycloalkylalkyl or cycloalkyla protecting group, "PG", of Formula 3 may be a peptide lkenyl preferably is of 3 to 7 carbon atoms in the cycloalkyl, 50 amino end blocking group or a label, as these terms are particularly 5 or 6 carbon atoms, and of 1 to 5 carbon atoms, defined below. By the term “peptide amino end blocking particularly 1 carbon atom, in the alkyl or alkylene moieties group” herein is meant, for example, groups including, but thereof. Aryl preferably is phenyl or naphthyl, especially not limited to alkyloxycarbonylalkanoyl (preferably of over 2-naphthyl. Heteroaryl preferably is pyridinyl, thienyl, espe all 2 to 10 carbon atoms), alkyloxycarbonyl (preferably of cially 2-thienyl, or furyl, especially 2-furyl. Heteroarylalkyl 55 overall 2 to 10 carbon atoms and particularly tert and heteroalkenyl preferably has 1 to 6 carbon atoms, butoxycarbonyl (BOC) or benzyloxycarbonyl (CBZ, Z)), especially 1 carbon atom in the alkyl or alkylene moieties alkanoyl (preferably of overall 2 to 10 carbon atoms and thereof. The heteroaryl moiety of heteroarylalkyl and het optionally Substituted with a radical Selected from carboxy, eroarylakylene preferably has the Significances indicated alkyloxycarbonyl and heterocycloalkylalkanoylamino), above as preferred for heteroaryl. Aralkyl preferably is 60 cycloalkylcarbonyl (preferably of overall 4 to 8 carbon phenylalkyl of 7 to 12 carbon atoms, particularly atoms), heterocycloalkylcarbonyl (preferably of overall 6 to naphthylmethyl, benzyl and phenylethyl. Alkoxy preferably 10 atoms and optionally Substituted with a radical Selected is of 1 to 5 carbon atoms, preferably methoxy. Acyloxy from hydroxy, alkyl, heterocycloalkyl, alkanoyl, preferably is of 2 to 6 carbon atoms, preferably acetoxy. The alkyloxycarbonyl, arylalkyloxycarbonyl and heterocy optional Substituents of an aryl or aralkyl moiety preferably 65 cloalkylcarbonyl and particularly a heterocycloalkylcarbo are one to three radicals of alkyl of 1 to 5 carbon atoms, nyl group of the formula-C(O)NROR wherein Rio and alkoxy of 1 to 5 carbon atoms, halogen of atomic number of R together form aza(C)methylene, Oxa(C)methylene 6,030.946 13 14 or (C7)methylene, particularly oxapentamethylene to form will be appreciated, additional amino acids may be added in 4-morpholinylcarbonyl (Mu)), arylalkyloxycarbonyl the same manner. Similarly, it should be understood that (preferably comprising aryl of 6 to 10 carbon atoms and these Structures do not accurately reflect the Situation where alkyloxy of 1 to 5 carbon atoms), carbamoyl, alkylcarbam- one or more of the amino acids is a proline. oyl (preferably of overall 2 to 6 carbon atoms), dialkylcar- 5 In general, Suitable amino acids of the peptidyl groups of bamoyl (preferably of overall 2 to 11 carbon atoms, aryl- the invention are initially chosen on the basis of the rules carbamoyl (preferably of overall 7 to 11 carbon atoms), governing Substrate Specificity in cysteine proteases. That is, arylalkylcarbamoyl (preferably comprising aryl of 6 to 10 combinations of amino acids that confer Specificity to the enzyme to be inhibited will be used. carbon atoms and alkyl of 1 to 5 carbon atoms), arylalkanoyl It is to be understood that the order of the amino acid side (preferably comprising aryl of 6 to 10 carbon atoms and 10 chains within the inhibitor is significant in conferring inhibi alkanoyl of overall 1 to 6 carbon atoms), aroyl (preferably tor targeting. Thus, as is additionally described below, the of overall 7 to 11 carbon atoms and particularly benzoyl), amino acid Side chain attached to the ethylenediamine or alkylsulfonyl (preferably of 1 to 10 carbon atoms), aryla- piperazine Structure of the inhibitor, generally referred to lkylsulfonyl (preferably comprising aryl of 6 to 10 carbon herein as “R” or “R”, will occupy the S position of the atoms and alkyl of 1 to 5 carbon atoms), alkylsulfamoyl 15 enzyme’s substrate when the inhibitor is bound (preferably of 1 to 5 carbon atoms), dialkylsulfamoyl to the enzyme. That is, for example, the “R” amino acid (preferably of 2 to 10 carbon atoms), arylsulfonyl (of 6 to 10 Side chain of the targeting group is the Presidue of the carbon atoms, including heteroarylsulfonyl, preferably com- inhibitor. Similarly, if there is a peptidyl group in the A prising heteroaryl of overall 4 to 8 atoms), arylsulfamoyl position, the amino acid Side chain of the peptidyl group (preferably of 6 to 10 carbon atoms, including heteroaryl- 20 which is closest to the ethylenediamine moiety will occupy Sulfamoyl preferably comprising heteroaryl of 4 to 8 atoms), the S. position of the enzyme’s Substrate binding site when alkylsulfinyl (preferably of 1 to 5 carbon atoms), dialky- the inhibitor is bound to the enzyme, and thus is the P laminosulfinyl (preferably of 2 to 10 carbon atoms), aryl- residue. If present, additional amino acid Side chains of the Sulfinyl (preferably comprising aryl of 6 to 10 carbon atoms peptidyl will occupy the Ps, P, etc. positions. including heteroarylsulfinyl, preferably comprising het- 25 Conversely, if the peptidyl group occupies the X position, eroaryl of 4 to 8 atoms). Temporary protecting groups are the amino acid Side chains occupy the S', S', etc. position known in the art, See Greene, Protective Groups in Organic of the enzyme’s Substrate binding Site. These amino acid Synthesis, John Wiley & Sons, 1991, hereby incorporated by Side chains are considered the P', P," etc. residues of the reference. inhibitor. As will be appreciated by those in the art, it is Formula 3 depicts a cysteine protease inhibitor with 30 possible to have peptidyl groups at both the A and X peptidyl groups in both the A and X positions, wherein n is positions, to confer increased specificity on the inhibitor for from 1 to 5: the particular cysteine protease to be inhibited. It should be understood that the inhibitors of the present Formula 3 invention potentially have a certain Symmetry which could R1 R2 35 effect nomenclature. Thus, for example, if the A and the X groups are identical, the inhibitor can be thought of as having an R group in one conformation or an R group in re-ever terro the opposite conformation. Without being bound by theory, R R4 the A group is assumed to be binding in the P positions, and 40 the X group to be binding to the P' positions of the enzyme. The choice of the amino acid Side chains of the R, R2, Rs, The amino acids, or peptide residues, are normally linked RandR group, and the amino acids of the A and X groups via a peptide bond or linkage, i.e. a peptidic carbamoyl will be made using the available information about the group, i.e. -CONH-. In a preferred embodiment, the bond Substrate Specificity of the protease, and is routine to those between the nitrogens of the ethylenediamine or piperazine 45 skilled in the art using commercially available substrates. moiety and the A and X group is a peptide bond. Similarly, For example, interleukin-1B converting enzyme displays the when the peptidyl group contains two or more amino acids, greatest Specificity demonstrated for a cysteine protease the bond between the two is preferably a peptide bond. toward a Substrate, requiring an aspartyl side chain in the P. However, the bond between the amino acids of the peptidyl position. The papain Superfamily of cysteine proteases have group may be a peptide isosteric or peptidomimetic bond 50 an extended specificity site containing five to seven signifi such as CO-CH, CH-NH, azapeptide and retro-inverso cant SubSites, with the dominant one being S., which is a bonds, as is depicted by X-X in Formulae (a) and (b). hydrophobic pocket that binds phenylalanyl-like Sidechains

Formula 4 R1 R2 ro-o------R R O )-(s------o-ra O R R l,

Formula 4 depicts a cysteine protease inhibitor with very well. Cathepsin B, Similar to papain, accepts a pheny peptidyl groups comprising two amino acids as both the A lalanine side chain in S, as well as an arginyl sidechain. For and the X group. Thus, in formula 4, the A-B linkage is a 65 a general review, see “Proteinase Inhibitors', in Research peptide or peptidomimetic bond. PG is a protecting group, as Monographs in Cell and Tissue Physiology (1986), ed. defined herein, and Q may be either carbon or nitrogen. AS Barrett et al., Vol. 12, Chapter 4: Inhibitors of Cysteine 6,030.946 15 16 Proteinases, Daniel Rich, Elsevier, New York, hereby chlorine, bromine, fluorine, hydroxy or amino, with expressly incorporated by reference. In addition, the Speci hydroxy, amino, chlorine, bromine or fluorine being most ficity of the interleukin 1? converting enzyme (ICE), was preferred; (8) C1-C5 alkyl substituted with nitro, alkyl or explored in Thornberry et al., Supra, also expressly incor arylsulfonyl, optionally protected where appropriate; (9) porated by reference herein. Table 1 lists some of the favored C1-C5 alkyl substituted with halogen, preferably trifluo amino acid side chains for the P and P (R or R) positions romethyl; (10) aryl or aralkyl substituted by one or two for a number of cysteine proteases. groups of C1-C5 alkyl, C1-C5 alkoxy, halogen, hydroxy or amino, with one or two groups of methyl, methoxy, chlorine, TABLE 1. bromine, fluorine, hydroxy or amino being preferred and hydroxy, amino, chlorine, bromine or fluorine being particu enzyme P, P (R or R2) larly preferred; (11) aryl or aralkyl substituted by one or two papain Phe, Tyr, 2-napthyl, Leu, Arg, Lys, Lys(e-Z), groups of halogen-Substituted C1-C5 alkyl, especially trif Nle, Ile, Ala guanidino-phenylalanine, luoromethyl, nitro, Sulfonyl, or arylsulfonyl, in protected Hph, Nile cathepsin B Phe, Tyr, Tyr(I), 2-napthyl, Arg, Lys, Lys(e-Z), form where appropriate. Arg, guanidino- guanidino 15 Preferred alkyloxycarbonyls include, but are not limited phenylalanine, Cit phenylalanaine, to, Boc, CBZ and Z. Hph, Cit, Nile By "peptidyl alkyloxycarbonyl' or “alkyloxycarbonyl cathepsin L or Phe, Tyr, 2-napthyl Arg, Lys, Lys(e-Z), cruzain guanidino peptidyl herein is meant a peptidyl group linked to a phenylalanaine, alkyloxycarbonyl group. It is to be understood that the Hph, Cit, Nile peptidyl group is linked to one of the nitrogens of the cathepsin S Phe, Tyr, 2-napthyl, Val, Arg, Lys, Lys(e-Z), ethylenediamine or piperazine moieties, and the alkyloxy Leu, Nile, Ile, Ala guanidino phenylalanaine, carbonyl group is linked to the peptidyl group. Thus, the free Hph, Cit, Nile terminal functionality of a alkyloxycarbonyl peptidyl group DPP-1 Gly, Ala Phe, Tyr is the Rs group of Formula 5. As described above for acyl calpain Val, Leu, Nle, Ile, Phe Tyr, Phe, Met, 25 peptidyl, the order of the functionalities differS depending on Met(O), Val ICE Ala, Val, His Asp whether it is the A group or the X group which is the cathepsin O2 Leu, Met, Nile Arg, Lys, Lys(e-Z), alkyloxycarbonyl peptidyl, the group may be called a alky guanidino-phenylalanine, loxycarbonyl peptidyl moiety when describing the A group Hph, Nile and a peptidyl alkyloxycarbonyl moiety when describing the Xgroup. Formula 5 shows a cysteine protease inhibitor with *citruline alkyloxycarbonyl peptidyl groups at both the A and X AS will be appreciated by those in the art, when non positions, wherein n is from 1 to 5, and AA is an amino acid: naturally occuring amino acid Side chain analogs are used, they will be chosen initially by steric and biochemical Formula 5 Similarities to the naturally occuring Side chains. 35 R1 R2 By “alkyloxycarbonyl herein is meant a -COORs group, wherein C is carbon and O is oxygen. In this )-( embodiment, Suitable Rs groups include, but are not limited so-c-ever twic-os, to, an alkyl, a cycloalkyl, a cycloalkylalkyl, an aryl, or an R R4 aralkyl (forming an arylalkyloxycarbonyl). In Such an 40 instance, alkyl is preferably of 1 to 5 carbon atoms, espe cially ethyl. Cycloalkyl preferably is of 3 to 7 carbon atoms, By “sulfonyl' herein is meant an -SOR group, preferably cyclopentyl or cyclohexyl. Cycloalkylalkyl pref wherein S is sulfur, and O is oxygen. The Sulfur atom is erably is of 3 to 7 carbon atoms in the cycloalkyl, particu attached to one of the nitrogens of the ethylenediamine or larly 5 or 6 carbon atoms, and of 1 to 5 carbon atoms, 45 piperazine moieties, and thus the Sulfonyl group is also a particularly 1 carbon atom, in the alkyl moieties thereof. Sulfonamide because of the attachment to the nitrogen Aryl preferably is phenyl. Aralkyl preferably is phenylalkyl group. The Ro moiety of the Sulfonyl group may include, but of 7 to 12 carbon atoms, particularly benzyl. The optional is not limited to, an alkyl (forming alkylsulfonyl), a Substi Substituents of an aryl or aralkyl moiety preferably are one tuted alkyl, a cycloalkyl, a cycloalkylalkyl, a or two groups independently Selected from: alkyl of 1 to 5 50 cycloalkylalkenyl, an aryl (forming arylsulfonyl, including carbon atoms, alkoxy of 1 to 5 carbon atoms, halogen of heteroaryl (forming heteroarylsulfonyl)), or an aralkyl atomic number of from 9 to 35, hydroxy and/or amino, (forming arylalkylsulfonyl). In Such an instance, alkyl is preferably one or two groups methyl, methoxy, chlorine, preferably of 1 to 5 carbon atoms, especially methyl. Sub bromine, fluorine, hydroxy or amino, particularly one Stituted alkyl is preferably of 1 to 5 carbon atoms, bearing hydroxy, amino, chlorine, bromine, or fluorine, optionally in 55 Substitutions of alkoxy of 1 to 5 carbon atoms, halogen of protected form where appropriate, nitro, alkyl or atomic number of from 9 to 35, hydroxy and/or amino, arylsulfonyl, or halogen-substituted alkyl of 1 to 5 carbon preferably one or two groups methyl, methoxy, chlorine, atoms, particularly trifluoromethyl. bromine, fluorine, hydroxy or amino, particularly one Particularly preferred are: (1) C1-C5 alkyl, especially hydroxy, amino, chlorine, bromine, or fluorine, optionally in ethyl; (2) C3-C7 cycloalkyl, preferably cyclopentyl or 60 protected form where appropriate, nitro, alkyl or cycolhexyl; (3) C3-C7(cycloalkyl)-C1-C5 alkyl, preferably arylsulfonyl, or halogen-substituted alkyl of 1 to 5 carbon C5-C6(cycloalkyl)methyl; (4) C3-C7(cycloalkyl-alkenyl)- atoms, particularly trifluoromethyl. Cycloalkyl preferably is C1-C5 alkyl, preferably C5-C6(cycloalkylalkenyl)methyl; of 3 to 7 carbon atoms, preferably cyclopentyl or cyclo (5) phenyl; (6) C7-C12 phenylalkyl, preferably benzyl; (7) hexyl. Cycloalkylalkyl or cycloalkylalkenyl preferably is of C1-C5 alkyl substituted by C1-C5 alkyoxy, halogen, 65 3 to 7 carbon atoms in the cycloalkyl, particularly 5 or 6 hydroxy or amino, with C1-C5 alkyl preferably substituted carbon atoms, and of 1 to 5 carbon atoms, particularly 1 by one or two groups Selected from methyl, methoxy, carbon atom, in the alkyl or alkylene moieties thereof. Aryl 6,030.946 17 18 preferably is phenyl, pentafluorophenyl or naphthyl. Aralkyl Suitable -NHRio and -NRoR groups include, but preferably is phenylalkyl of 7 to 12 carbon atoms, particu are not limited to, an NH, or an NH-alkyl (forming larly benzyl and phenethyl. The optional substituents of an alkylsulfamoyl), an NH-cycloalkyl, an NH-cycloalkylalkyl, aryl or aralkyl moiety preferably are one or two groups alkyl an NH-aryl (forming arylsulfamoyl or heteroarylsulfamoyl), of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms, 5 an NH-aralkyl, N-dialkyl (forming dialkylsulfamoyl) and halogen of atomic number of from 9 to 35, hydroxy and/or N-alkylaralkyl. Cycloalkyl preferably is of 3 to 7 carbon amino, preferably one or two groups methyl, methoxy, atoms, preferably cyclopentyl or cyclohexyl. Cycloalkyla chlorine, bromine, fluorine, hydroxy or amino, particularly lkyl preferably is of 3 to 7 carbon atoms in the cycloalkyl, one hydroxy, amino, chlorine, bromine, or fluorine, option ally in protected form where appropriate, nitro, alkyl or particularly 5 or 6 carbon atoms, and of 1 to 5 carbon atoms, arylsulfonyl, or halogen-substituted alkyl of 1 to 5 carbon particularly 1 carbon atom, in the alkyl moieties thereof. atoms, particularly trifluoromethyl. Aryl preferably is phenyl or heteroaryl. Aralkyl preferably is Particularly preferred Ro moieties of the Sulfonyl group phenylalkyl of 7 to 12 carbon atoms, particularly benzyl. include (1) C1-C5 alkyl, especially methyl; (2) C3-C7 The optional Substituents of an aryl or aralkyl moiety cycloalkyl, preferably cyclopentyl or cyclolhexyl; (3) preferably are one or two groupS alkyl of 1 to 5 carbon C3-C7(cycloalkyl)-C1-C5 alkyl, preferably C5-C6 15 atoms, alkoxy of 1 to 5 carbon atoms, halogen of atomic (cycloalkyl)methyl; (4) C3-C7(cycloalkylalkenyl)-C1-C5 number of from 9 to 35, hydroxy and/or amino, preferably alkyl, preferably C5-C6(cycloalkylalkenyl)-methyl; (5) one or two groups methyl, methoxy, chlorine, bromine, phenyl, preferably pentafluorophenyl or naphthyl, (6) fluorine, hydroxy or amino, particularly one hydroxy, amino, C7-C12 phenylalkyl, preferably benzyl; (7) C1-C5 alkyl chlorine, bromine, or fluorine, optionally in protected form Substituted by C1-C5 alkyOXy, halogen, hydroxy or amino, where appropriate, nitro, alkyl or arylsulfonyl, or halogen with C1-C5 alkyl preferably substituted by one or two substituted alkyl of 1 to 5 carbon atoms, particularly trif groupS Selected from methyl, methoxy, chlorine, bromine, luoromethyl. Especially preferred is when both Ro and R1 fluorine, hydroxy or amino, with hydroxy, amino, chlorine, are methyl. bromine or fluorine being most preferred; (8) C1-C5 alkyl In a preferred embodiment, the Ro and R groups of a Substituted with nitro, alkyl or arylsulfonyl, optionally pro 25 -NRR, are bonded together to form 5 or 6 membered tected where appropriate; (9) C1-C5 alkyl substituted with alicyclic or heteroalicyclic ring moieties. Preferred are halogen, preferably trifluoromethyl; (10) aryl or aralkyl piperidine, morpholine, pyrrolidine, piperazine, or Substi substituted by one or two groups of C1-C5 alkyl, C1-C5 tuted piperazine. alkoxy, halogen, hydroxy or amino, with one or two groups By "peptidyl Sulfamoyl herein is meant a peptidyl group of methyl, methoxy, chlorine, bromine, fluorine, hydroxy or linked to a Sulfamoyl group. AS above, the peptidyl group is amino being preferred and hydroxy, amino, chlorine, bro linked to one of the nitrogens of the ethylenediamine or mine or fluorine being particularly preferred; (11) aryl or piperazine moieties, and the Sulfamoyl group is linked to the aralkyl Substituted by one or two groups of halogen peptidyl group. Thus, the free terminal functionality of a substituted C1-C5 alkyl, especially trifluoromethyl; nitro; Sulfamoyl peptidyl group is the Rio group. AS described Sulfonyl, or arylsulfonyl, in protected form where appropri 35 above for acyl peptidyl, the order of the functionalities ate. differS depending on whether it is the Agroup or the X group Especially preferred are phenyl, naphthyl, and benzyl. which is the Sulfamoyl peptidyl; the group may be called a By "peptidyl sulfonyl' or “sulfonyl peptidyll” herein is Sulfamoyl peptidyl moiety when describing the A group and meant a peptidyl group linked to a Sulfonyl group. AS above, a peptidyl Sulfamoyl moiety when describing the X group. the peptidyl group is linked to one of the nitrogens of the 40 Formula 7 shows a cysteine protease inhibitor with a sul ethylenediamine or piperazine moieties, and the Sulfonyl famoyl peptidyl group at the A position and a peptidyl group is linked to the peptidyl group. Thus, the free terminal sulfamoyl at the X position, wherein n is from 1 to 10, and functionality of a Sulfonyl peptidyl group is the Rogroup. AS AA is an amino acid. described above for acyl peptidyl, the order of the function alities differS depending on whether it is the A group or the 45 Formula 7 X group which is the Sulfonyl peptidyl, the group may be R1 R2 called a Sulfonyl peptidyl moiety when describing the A group and a peptidyl Sulfonyl moiety when describing the X ( )-( group. Formula 6 shows a cysteine protease inhibitor with Ro-N-S-AA-n-N N-AA in S-N-R Sulfonyl peptidyl groups at both the A and X positions, 50 y R R4 O wherein n is from 1 to 5, and AA is an amino acid.

Formula 6 By “sutffinyl' herein is meant a -SOR, group, where S R1 R2 is Sulfur, O is oxygen, and Ro is a group as defined herein. 55 The Sulfur and oxygen atoms are double bonded together. )-( The sulfur atom is attached to one of the nitrogens of the ethylenediamine or piperazine moieties. Preferred Sulfinyl R-itself twri groups include alkylsulfinyl, dialkylaminoSulfinyl, and O R R4 O arylsulfinyl, including heteroarylsulfinyl. 60 By "Sulfinyl peptidyll” herein is meant a peptidyl group By "Sulfamoyl herein is meant an -SO2NRoR group, linked to a Sulfinyl group. AS above, the peptidyl group is wherein S is Sulfur, O is oxygen, and N is nitrogen. The linked to one of the nitrogens of the ethylenediamine or Sulfur atom of the Sulfone is linked to one of the nitrogens piperazine moieties, and the Sulfinyl group is linked to the of the ethylenediamine or piperazine moieties. In Some peptidyl group. Thus, the free terminal functionality of a embodiments, the sulfamoyl comprises an -SONHR 65 Sulfinyl peptidyl group is the Ro moiety. AS described above, group, wherein H is hydrogen, and in other embodiments it the order of the functionalities differs depending on whether comprises an -SO2NRR, group. it is the Agroup or the X group which is the Sulfinyl peptidyl, 6,030.946 19 20 the group may be called a Sulfinyl peptidyl group when describing the A moiety, and a peptidyl Sulfinyl when Formula 11 describing the X group. Formula 8 depicts a cysteine pro tease inhibitor with a Sulfinyl peptidyl group at the A position and a peptidyl Sulfinyl at the X position, wherein n 5 is from 1 to 10, and M is an amino acid. It should be understood that the depiction of the ethyl Formula 8 enediamine group in Formula 11 and others as having a R1 R2 certain conformation is merely pictorial. Thus, Formula 11, and the other ethylenediamine moieties may be depicted as ( )-( shown in Formula 1 1A: i-ster ters Formula 11A R R4 15 A. n1)n-1 N Yx H By “carbamoyl” herein is meant an-CONRR group, His hydrogen, C is carbon and O is oxygen. Ro and R are In a preferred embodiment, the N-substituents are linked defined as above. via a substituted ethylenediamine moiety. By “substituted ethylenediamine' herein is meant an ethylenediamine group By “carbamoyl peptidyl” herein is meant a carbamoyl which has one or more of the hydrogen atoms replaced by group linked to a peptidyl group. The term “carbamoyl a Substituent group. It should be understood that the Substi peptidyll” includes both “carbamoyl peptidyll” and “peptidyl tuted ethylenediamine does not contain a peptide bond; that carbamoyl”. That is, in one embodiment the carbamoyl is 25 is, neither the R or the R2 positions may be a carbonyl. attached to one of the nitrogens of the ethylenediamine or In a preferred embodiment, the ethylenediamine is Sub piperazine moieties, and the peptidyl moiety is attached to stituted with an R group on either one of the carbons of the ethylene. Thus, as shown in Formula 12, R and R are either the carbamoyl. In this embodiment, the terminal function hydrogen or an amino acid Side chain. It is to be understood ality is the peptidyl. Alternatively, the peptidyl moiety may that only one of R and R is an amino acid Side, and the be attached to the ethylenediamine or piperazine moiety and other one of R and R is hydrogen. Preferably, R is an the carbamoyl moiety attached to the peptidyl. In this amino acid Side chain and R is hydrogen. embodiment, the terminal functionality is the carbamoyl. These two embodiments are depicted below in Formulas 9 Formula 12 (carbamoyl peptidyl) and 10 (peptidyl carbamoyl) as com R1 R2 prising the X moiety. It should be understood that Formulas 35 9 and 10 do not accurately reflect the structure where one of A-N N-X the amino acids is a proline. H H

Formula 9 40 R1 R2 AS will be appreciated by those in the art, there are a large number of possible A and X combinations which can be )-( O| R| O|| made. Any A group may be combined with any X group. In A N-C-NC-C-N PG one embodiment, the A and the X group are the same; for example, the A group may be a Sulfonyl peptidyl group and R3 R4 45 the X group may be a peptidyl Sulfonyl group. However, it should be understood that even if both the A and X groups are Sulfonyls, for example, the Ro groups of each Sulfonyl Formula 10 need not be the same. This is similarly true for the other R1 R2 50 embodiments. In an alternative embodiment, the A group O R O and the X group are different. When R is an amino acid side chain and R. is hydrogen, AN N !-- | \'" preferred A and X group combinations are depicted in Table 2: R R4 " YR 55 TABLE 2 The two N-Substituents of the invention are linked via an Preferred A group Preferred X group ethylenediamine or Substituted ethylenediamine group. By PAEBG-amino acid: sulfonyl “ethylenediamine group” herein is meant a -NH-CH PAEBG-peptidyl sulfonyl 60 alkyloxycarbonyl peptidyl sulfonyl CH-NH-group, wherein N is nitrogen, C is carbon, and sulfonyl peptidyl sulfonyl H is hydrogen. The two N-Substituents, depicted herein as carbamoyl peptidyl sulfonyl “A” and “X”, are each linked to one of the nitrogens of the sulfamoyl peptidyl sulfonyl acyl peptidyl sulfonyl ethylenediamine. "Linked herein means a covalent attach sulfinyl peptidyl sulfonyl ment. Thus, in one embodiment, the cysteine protease 65 PG-amino acid peptidyl-PAEBG inhibitors of the invention have the structure shown in alkyloxycarbonyl peptidyl peptidyl-PAEBG Formula 11: 6,030.946 21 22

TABLE 2-continued Formula 15

Preferred A group Preferred X group A-NA wN-X sulfonyl peptidyl peptidyl-PAEBG 5 carbamoyl peptidyl peptidyl-PAEBG sulfamoyl peptidyl peptidyl-PAEBG R5 R6 acyl peptidyl peptidyl-PAEBG sulfinyl peptidyl peptidyl-PAEBG *PAEBG = peptide amino end blocking group; or PG 1O Formula 16 When R is an amino acid side chain and R is hydrogen, R1 preferred A and X group combinations are depicted in Table ) 3: AN N-X TABLE 3 15 \-( Preferred A group Preferred X group R6 sulfonyl peptidyl-PAEBG sulfonyl peptidyl alkyloxycarbonyl sulfonyl peptidyl sulfonyl 2O sulfonyl peptidyl carbamoyl Formula 17 sulfonyl peptidyl sulfamoyl R2 sulfonyl peptidyl acyl sulfonyl peptidyl sulfinyl PAEBG peptidyl amino acid-PG A-N N-X PAEBG peptidyl peptidyl alkyloxycarbonyl 25 PAEBG peptidyl peptidyl sulfonyl PAEBG peptidyl peptidyl carbamoyl PAEBG peptidyl peptidyl sulfamoyl R5 PAEBG peptidyl peptidyl acyl PAEBG peptidyl peptidyl sulfinyl 30 In Formulas 16 and 17, it is to be understood that the two Substitution groups of the Six-membered heterocyclic ring of The preferred A and X groups listed in Tables 2 and 3 piperazine are in para position to each other; that is, there apply to the piperazine moieties described below as well. may be Substitution groups at R and R or R and Rs. R, In al preferred embodiment, the ethylenediamine S sub- R, R and R are independently hydrogen or an amino acid Stituted on the nitrogens of the ethylenediamine, as shown in Side chain. Formula 1. In this embodiment, R, and R are either both 35 a. preferred embodiment, the cysteine protease inhibi hydrogen or are bonded together to form ethylene of Sub- tors of the present invention have a peptidyl moiety as the stituted ethylene. By “ethylene or. ethylene group” herein “A” group and a Sulfonyl moiety as the “X” group, and an is meant a (-CHCH-) group, i.e. a saturated carbon- R group, and thus have the general formula shown in carbon bond, Serving to connect two Sp-hybridized carbon Formula 18 and Formula 18A. As will be appreciated by atoms; that is, the two carbons of the ethylene group are 40 those in the art, Formula 18 does not accurately represent the double bonded together. By substituted ethylene' herein is Structure when one or more of the amino acids of the meant an ethylene group which has one of the hydrogens of peptidyl group are proline. the ethylene replaced by a Substitutent group, i.e. a (-CHCHR-) group, where R is Rs or R, as depicted Formula 18 below. When Rs and R form ethylene or substituted as R1 R2 Ola ethylene, the Substituted ethylenediamine moiety is a pip erazine moiety, which in turn may be Substituted or unsub stituted. That is, the carbon atOmS of the piperazine moiety PG--N-C-C-HN N-S-R. may have no Substituent groups, i.e. only hydrogen, as "| | || shown in Formula 13, an R or R group as shown in 5 R R4 O Formula 14, an Rs or R group as shown in Formula 15, or O both an R and R or an R and Rs group (Formulas 16 and 17). Formula 18A Formula 13 R1 R2 / \ 55 ) ( O A-N. N-X ro-ever i-R Formula 14 R R4 O R1 R2 60 )-( When the cysteine protease inhibitors have the formula A-N N-X depicted in Formulas 18 and 18A, preferably the inhibitors M / have an R group. In addition, preferably n=1 or 2. In one preferred embodiment, R and R are hydrogen; in an 65 alternate preferred embodiment, R and R together form In a preferred embodiment, R is an amino acid Side chain unsubstituted ethylene, to form a piperazine which has a and R is hydrogen. Single Substituent group at R. 6,030.946 23 24 In a preferred embodiment, the cysteine protease inhibi Thus, preferred embodiments include compounds of For tors have a Sulfonyl peptidyl moiety as the “A” group and a mula I: Sulfonyl moiety as the “X” group, and an R group, and thus have the general formula shown in Formula 19 and 19A (where the amino acid is not proline):

Formula 19 R1)-(i. R2 R-itar i in which: O R R4 O A and X are independently R-X-, wherein R is hydrogen, alkyl oxy carbonyl alkanoyl, 15 alkyloxycarbonyl, alkanoyl (optionally Substituted with a radical Selected from carboxy, alkyloxycarbonyl and Formula 19A heterocycloalkylalkanoylamino), cycloalkylcarbonyl, R1 R2 heterocycloalkylcarbonyl (optionally substituted with a O| R O )-( O radical Selected from hydroxy, alkyl, heterocycloalkyl, R- N-C-C-HN N-S-R. alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and " heterocycloalkylcarbonyl), arylalkyloxycarbonyl, O R R4 O carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, arylalkylcarbamoyl, arylalkanoyl, aroyl, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, 25 alkylsulfamoyl, dialkylsulfamoyl, arylsulfamoyl, When the cysteine protease inhibitors have the formula heteroarylsulfamoyl, alkylsulfinyl, dialkylaminoSulfi depicted in Formula 19 and 19A, preferably the inhibitors nyl or arylsulfinyl and X is a bond or a divalent radical have an R group. In addition, preferably n=1 or 2. AS above, of Formulae (a) or (b): this structure is not accurate when one or more of the amino acids of the peptidyl are proline. In one embodiment, R and (a) R are hydrogen; in a preferred embodiment, R and R. together form unsubstituted ethylene, to form a piperazine which has a single Substituent group at R. (b) 35 In a preferred embodiment, the cysteine protease inhibi tors of the invention have an acyl peptidyl moiety as the “A” group and a Sulfonyl moiety as the “X” group as depicted as Formula 20 and Formula 20A (when the amino acid is not 40 proline): in which n is 0 to 9; X-X represents a linkage Selected from -C(O)NR-, -CHNR-, -C(O)CH- and Formula 20 -NRC(O)-; Y is -CH(R)- or -NR-; and Z is R1 R2 -(CH2)2-, -C(Rs)(R)- or -N(R)-; wherein R 45 is hydrogen or as defined below, R is hydrogen or methyl )-( and each R is independently hydrogen, alkyl (optionally Substituted with a radical Selected from hydroxy, alkyloxy, R-c-ever i-R amino, alkylamino, dialkylamino, uriedo, alkyluriedo, R R4 O mercapto, alkylthio, carboxy, carbamoyl, alkylcarbamoyl, 50 dialkylcarbamoyl, alkylsulfinyl, alkylsulfonyl, guanidino, -P(O)(OR), -OP(O)(OR) and -OP(O)(R), Formula 20A wherein each R is independently hydrogen or alkyl, or a R1 R2 protected derivative thereof), cycloalkyl, cycloalkylalkyl, a O R O )-( O group Selected from aryl and arylalkyl (which group is | 55 optionally Substituted at its aryl ring with one to three R- N-C-C7 N N-S-Ro radicals Selected from hydroxy, amino, guanidino, halo, optionally halo-Substituted alkyl, alkyloxy and aryl, or a l, l, O protected derivative thereof) or together with an adjacent R forms a divalent radical Selected from (Cl)methylene 60 and 1,2-phenylenedimethylene (which radical is optionally substituted with hydroxy, or a protected derivative thereof, When the cysteine protease inhibitors have the formula or oxo), with the proviso X and A are not both hydrogen; depicted in Formula 20 and 20A, preferably the inhibitors R and R are both hydrogen or one of R or R is cyano, have a R group. Preferably n=1 or 2. In one embodiment, carboxy, alkyloxycarbonyl, alkanoyl, carbamoyl, R and R are hydrogen; in an alternative embodiment R. 65 alkylcarbamoyl, dialkylcarbamoyl, alkyloxy (alkyl) and R together form unsubstituted ethylene, to form a carbamoyl, aminoalkylcarbamoyl, R., as defined piperazine which has a Single Substituent group at R. above, or R-X-, wherein R is as defined above 6,030.946 25 26 and X is a divalent radical of Formulae (a) or (b), as 2-thienyl, 2-furyl, 4-imidazolyl, 3-indolyl, 3-pyridylmethyl, defined above; and 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, RandR together form optionally Substituted ethylene or 3-indolylmethyl, methoxy, acetoxy, (Cs)alkyl (optionally are independently R1, as defined above; and the phar Substituted with a radical Selected from mercapto, carboxy, maceutically acceptable Salts, individual isomers and 5 amino, methylthio, methylsulfonyl, carbamoyl, mixtures of isomers thereof. dimethylcarbamoyl, guanidino and hydroxy, or a protected Preferred Formula I compounds include compounds derivative thereoftl, a group Selected from phenyl, wherein A is R-X-, wherein R is hydrogen, alky 1-naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, loxycarbonylalkanoyl of overall 3 to 10 carbon atoms, 2-naphthylmethyl and 2-phenylethyl (which group is option (Co.)alkoxycarbonyl, (C-o)alkanoyl (optionally Substi ally Substituted at its aryl ring with one radical Selected from tuted with a radical Selected from carboxy, (C) hydroxy, amino, chloro, bromo and fluoro, or a protected alkyloxycarbonyl and hetero(Cs)cycloalkyl (C-o) form thereof) or together with an adjacent R forms a alkanoylamino), (C)cycloalkylcarbonyl, hetero(C) divalent radical Selected from (Cl)methylene and 1,2- cycloalkylcarbonyl (optionally Substituted with a radical phenylenedimethylene (which radical is optionally Substi Selected from hydroxy, (C-5)alkyl, hetero(Cs)cycloalkyl, 15 tuted with hydroxy, or a protected derivative thereof, or (C-5)alkanoyl, (C-5)alkyloxycarbonyl, (C-o)aryl(C-5) OXO); and R is (Cs)alkyl (optionally Substituted with one alkyloxycarbonyl and hetero(Cs)cycloalkylcarbonyl), (C- or two radicals Selected from amino, chloro, bromo, fluoro 1O)aryl (Cs) alkyloxycarbonyl, carbamoyl, (C-5) and hydroxy, or a protected derivative thereof), perfluoro alkylcarbamoyl, di(Cs) alkylcarbamoyl, (C-o) (Cs)alkyl, (Cs)cycloalkyl, (Cs)cycloalkylmethyl or a arylcarbamoyl, (C-o)aryl(Cs)alkylcarbamoyl, (C-o)aryl group Selected from phenyl, naphthyl and benzyl (which (C-5)alkanoyl, (C7-1)aroyl, (C-5)alkylsulfonyl, di(C-5) group is optionally Substituted with one radical Selected alkylsulfamoyl, (C-o)arylsulfonyl, (C-o)aryl(C-5) from amino hydroxy, chloro, bromo or fluoro, or a protected alkylsulfonyl or hetero(Cs)arylsulfonyl; and X is a derivative thereof). divalent radical of Formula (a), wherein n is 0 to 5; X-X Particularly preferred compounds of this embodiment represents a linkage selected from -C(O)NR-; Y is 25 include n is 0 to 1; R is butyl, 2-phenylethyl, N(R)-; Z is -CH(R)-; R is hydrogen or as 2-methylsulfonylethyl, 2-tert-butoxycarbonylethyl, 2-tert defined below; and R' is (Cl)cycloalkyl, (C)cycloalkyl butoxycarbonylmethyl, 4-tert-butoxycarbonylaminobutyl, (Cs)alkyl, pyridyl, thienyl, furyl, imidazolyl, indolyl, 4-benzoylaminobutyl or benzyloxymethyl, R is hydrogen, pyridyl(C1-)alkyl, thienyl(C1-)alkyl, furyl(Ce)alkyl, tert-butoxycarbonyl, ben Zyloxycarbonyl, acetyl, imidazolyl (C1-)alkyl, indolyl(Ce) alkyl, (Cs)alkyl 3-carboxypropionyl, 3-methoxycarbonylpropionyl, (optionally substituted with a radical selected from biotiny laminohe Xanoyl, phenylacetyl, benzoyl, mercapto, carboxy, amino, methylthio, methylsulfonyl, dimethylsulfamoyl, benzylsulfonyl, 1-piperizinylcarbonyl, carbamoyl, dimethylcarbamoyl, guanidino and hydroxy, or a 4-methyl-1-piperazinylcarbonyl or 4-morpholinylcarbonyl; protected derivative thereof), a group Selected from phenyl, R is 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, naphthyl, phenyl(Ce)alkyl, naphthyl(C)alkyl, (which 35 4-imidazolylmethyl, 3-indolylmethyl, (Cs) alkyl group is optionally Substituted at its aryl ring with one to (optionally substituted with a radical selected from three radicals Selected from amino, hydroxy, chloro, bromo, mercapto, carboxy, amino, methylthio, methylsulfonyl, fluoro, iodo, methyl, trifluoromethyl, methoxy and phenyl, carbamoyl, dimethylcarbamoyl, guanidino and hydroxy, or a or a protected derivative thereof) or together with an adja protected derivative thereof), a group Selected from benzyl, cent R forms a divalent radical Selected from (C) 40 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl methylene and 1,2-phenylenedimethylene (which radical is (which group is optionally Substituted at its aryl ring with optionally Substituted with hydroxy, or a protected deriva one radical Selected from hydroxy, amino, chloro, bromo tive thereof, or oxo), X is -S(O)-Ro, wherein Ro is (C-5) and fluoro, or a protected form thereof) or together with an alkyl (optionally substituted with one or two radicals adjacent R or R forms a divalent radical Selected from Selected from amino, chloro, bromo, fluoro, hydroxy and 45 (C)methylene and 1,2-phenylenedimethylene (which methoxy, or a protected derivative thereof), perhalo(Cs) radical is optionally Substituted with hydroxy, or a protected alkylsulfonyl, (C-7)cycloalkyl, (C-7)cycloalkyl (Cs)alkyl derivative thereof, or oxo); and R is methyl, or a group Selected from phenyl, pentafluorophenyl, naph trifluoromethyl, optionally Substituted phenyl, 2-naphthyl or thyl and phenyl(Ce)alkyl (which group is optionally Sub 2-phenylethyl. Stituted at its aryl ring with one to two radicals Selected from 50 Especially preferred embodiments include n is 0; R is amino, chloro, bromo, fluoro, hydroxy, methoxy and option butyl, 2-phenylethyl or 2-methylsulfonylethyl; R is ally halo-Substituted methyl, or a protected derivative hydrogen, tert-butxoy carbonyl, benzyloxycarbonyl, thereof); biotinylaminohexanoyl, benzoyl, 1-piperiziny-carbonyl, R is hydrogen and R2 is Re, as defined above; and 4-methyl-1-piperazinylcarbonyl or 4-morpholinylcarbonyl, R and R are each hydrogen or together form optionally 55 R is (Cs) alkyl, optionally Substituted benzyl, Substituted ethylene. 1-naphthylmethyl, 2-naphthylmethyl, 3-pyridinylmethyl or Further preferred compounds of this embodiment include 2-methylsulfonylethyl, and Ro is phenyl, 1-naphthyl or n is 0 to 2, R2 is Re, as defined below; R and R together 2-phenylethyl. form optionally Substituted ethylene; R is hydrogen, (C- As are depicted in FIG. 1, preferred inhibitors of the s)alkoxycarbonyl, (C)alkanoyl (optionally Substituted 60 invention include (abbreviation first): Mu-Phe-retro-(D.L)- with a radical Selected from carboxy, (C-5) PheSOPh: 2-benzyl-4-(morpholinecarbonylphenylalanyl)- alkyloxycarbonyl and hetero(Cas)cycloalkyl-(Ce) 1-phenylsulfonylethylenediamine; Mu-Phe-retro-(D.L)- alkanoylamino), -C(O)NRR, wherein Ro and R. Le uSO Ph: 2-isobutyl-4- together form aza(C)methylene, Oxa(C)methylene or (morpholine carbonyl phenylala nyl)-1- (C7) methylene, (Cs) cycloalkyl carbonyl, 65 phenylsulfonylethylenediamine; Mu-Tyr-retro-(D.L)- benzyloxycarbonyl, acetyl, benzoyl or dimethylsulfamoyl; LeuSOPh: 2-isobutyl-4-(morpholinecarbonyltyrosyl)-1- R is (Cs)cycloalkyl, (Cs)cycloalkylmethyl, 3-pyridyl, phenylsulfonylethylene diamine; Mu-Phe-retro-(D,L)

6,030.946 29 30 Behavior and analysis of rapid equilibrium and Steady-state Intermediates useful for the preparation of compounds of enzyme systems, 1975, Wiley-lnterscience Publication, John the invention in which R or R is carboxy, carbamoyl, Wiley & Sons, New York, or for competitive binding inhibi alkylcarbamoyl, dialkylcarbamoyl, alkoxy(alkyl)carbamoyl tors from the following calculation: or aminoalkylcarbamoyl are commercially available or can be readily prepared by one of ordinary skill in the art. For Equation 2 example, reactive piperazine intermediates in which R1 or R forms an amide derivative can be prepared by acylation V is the rate of Substrate hydrolysis in the absence of of an appropriate amine, or protected derivative thereof, inhibitor, and V is the rate in the presence of competi with an appropriate protected carboxylic acid (e.g. tive inhibitor. N-protected 2-piperazinecarboxylic acid) and then depro It is to be understood that dissociation constants are a tecting. The protected piperazine carboxylic acid is prepared particularly useful way of quantifying the efficiency of an from commercially available Starting material via Standard enzyme with a particular Substrate or inhibitor, and are protection chemistry. frequently used in the art as such. If an inhibitor exhibits a AS will be appreciated in the art, the above Synthetic very low K, it is an efficient inhibitor. Accordingly, the 15 techniques may be used to Synthesize the cysteine protease cysteine protease inhibitors of the present invention have inhibitors of the invention. Representative examples of Such dissociation constants, K, of at most about 100 uM. Pre Syntheses are outlined in the Examples. ferred embodiments have inhibitors that exhibit dissociation In one embodiment, the cysteine protease inhibitors of the constants of at most about 10 uM, at most about 1 uM, with present invention are further purified if necessary after the most preferred embodiments having dissociation con Synthesis, for example to remove unreacted materials. For stants of at most about 100 nM. example, the cysteine protease inhibitors of the present In the preferred embodiment, the cysteine protease inhibi invention may be crystallized, or passed through chroma tors are chiral. By the term "chiral” or grammatical equiva tography columns using Solvent mixtures to elute the pure lents herein is meant a compound that exhibits asymmetry. inhibitors. That is, the chiral compound is not identical with its mirror 25 Once produced, the cysteine protease inhibitors of the image. Thus in the preferred embodiment, the compounds of present invention may be easily Screened for their inhibitory the present invention are pure diasteromers. Chiral effect. The inhibitor is first tested against the cysteine compounds, and particularly chiral cysteine protease protease for which the targeting group of the inhibitor was inhibitors, are useful in the present invention because bio chosen, as outlined above. Alternatively, many cysteine logical Systems, and enzymes in particular, are proteases and their corresponding chromogenic Substrates Stereospecific, preferring the (S) or L-form of amino acids. are commercially available. Thus, a variety of cysteine Thus in the preferred embodiment, the A and X groups of the proteases are routinely assayed with Synthetic chromogenic cysteine protease inhibitors of the present invention will Substrates in the presence and absence of the cysteine have amino acid Side chains in the (S) or L-configuration, protease inhibitor, to confirm the inhibitory action of the although Some inhibitors may utilize amino acid Side chains 35 compound, using techniques well known in the art. The in the (R) or D-configuration. effective inhibitors are then Subjected to kinetic analysis to In alternative embodiments, the compositions of the calculate the K values, and the dissociation constants deter present invention are not pure epimers, but are mixtures that mined. contain both epimerS. If a compound inhibits at least one cysteine protease, it is The synthesis of the cysteine protease inhibitors of the 40 a cysteine protease inhibitor for the purposes of the inven present invention proceeds as follows. tion. Preferred embodiments have inhibitors that exhibit the Synthesis of cysteine protease inhibitors with A and X correct kinetic parameters against at least the targeted cyS groups linked via Substituted ethylenediamine linkages is teine protease. depicted generally in FIGS. 2 and 3. For unsubstituted In Some cases, the cysteine protease is not commercially ethylenediamine linkages, R and R2 are hydrogen. FIG. 3 45 available in a purified form. The cysteine protease inhibitors depicts the R. Synthesis, FIG. 4 depicts the R. Synthesis. of the present invention may also be assayed for efficacy Both Schemes are depicted using a peptidyl group as a using biological assayS. For example, the inhibitors may be representative “A” moiety and a Sulfonyl group as a repre added to cells or tissues that contain cysteine proteases, and sentative “X” moiety; however, those in the art will appre the biological effects measured. ciate that these Schemes may be used to Synthesize cysteine 50 In one embodiment, the cysteine protease inhibitors of the protease inhibitors with other A and X groups. present invention are Synthesized or modified Such that the Synthesis of cysteine protease inhibitors with R- and R. in vivo and in vitro proteolytic degradation of the inhibitors Substituted piperazine as the linkage is depicted generally in is reduced or prevented. Generally, this is done through the FIG. 4. For unsubstituted piperazine, R and Rs are hydro incorporation of Synthetic amino acids, derivatives, or Sub gen. AS above, a peptidyl group is shown as a representative 55 stituents into the cysteine protease inhibitor. Preferably, only “A” group and a Sulfonyl group as the representative “X” one non-naturally occurring amino acid or amino acid Side group, as above, those in the art will appreciate that this chain is incorporated into the cysteine protease inhibitor, Scheme may be used to Synthesize cysteine protease inhibi Such that the targeting of the inhibitor to the enzyme is not tors with other A and X groups. Significantly affected. However, Some embodiments that use Synthesis of cysteine protease inhibitors with R and R. 60 longer cysteine protease inhibitors containing a number of Substituted piperazine as the linkage is depicted generally in targeting residues may tolerate more than one Synthetic FIG. 5. For unsubstituted piperazine, R and R are hydro derivative. In addition, non-naturally occurring amino acid gen. AS above, a peptidyl group is shown as a representative Substituents may be designed to mimic the binding of the “A” group and a Sulfonyl group as the representative “X” naturally occurring Side chain to the enzyme, Such that more group, as above, those in the art will appreciate that this 65 than one Synthetic Substituent is tolerated. Alternatively, Scheme may be used to Synthesize cysteine protease inhibi peptide isosteres are used to reduce or prevent inhibitor tors with other A and X groups. degradation. 6,030.946 31 32 In this embodiment, the resistance of the modified cys Thus the methods are applicable to both human therapy and teine protease inhibitors may be tested against a variety of Veterinary applications. For example, the veterinary appli known commercially available proteases in vitro to deter cations include, but are not limited to, canine, bovine, feline, mine their proteolytic Stability. Promising candidates may porcine, caprine, equine, and Ovine animals, as well as other then be routinely Screened in animal models, for example domesticated animals including reptiles, Such as iguanas, using labelled inhibitors, to determine the in vivo stability turtles and Snakes, birds Such as finches and members of the and efficacy. parrot family, lagomorphs. Such as rabbits, rodents Such as rats, mice, guinea pigs and hamsters, amphibians, fish, and In one embodiment, the cysteine protease inhibitors of the arthropods. Valuable non-domesticated animals, Such as Zoo present invention are labelled. By a “labelled cysteine pro animals, may also be treated. In the preferred embodiment tease inhibitor herein is meant a cysteine protease inhibitor the patient is a mammal, and in the most preferred embodi that has at least one element, isotope or chemical compound ment the patient is human. attached to enable the detection of the cysteine protease The administration of the cysteine protease inhibitors of inhibitor or the cysteine protease inhibitor bound to a the present invention can be done in a variety of ways, cysteine protease. In general, labels fall into three classes: a) including, but not limited to, orally, Subcutaneously, isotopic labels, which may be radioactive or heavy isotopes, intravenously, intranasally, transdermally, intraperitoneally, b) immune labels, which may be antibodies or antigens, and 15 intramuscularly, intrapulmonary, vaginally, rectally, or c) colored or fluorescent dyes. The labels may be incorpo intraocularly. In Some instances, for example, in the treat rated into the cysteine protease inhibitor at any position. ment of wounds and inflammation, the cysteine protease Examples of useful labels include ''C, H, biotin, and inhibitors may be directly applied as a Solution or Spray. fluorescent labels as are well known in the art. The pharmaceutical compositions of the present invention In the preferred embodiment, the cysteine protease inhibi comprise a cysteine protease inhibitor in a form Suitable for tors of the present invention are administered to a patient to administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water Soluble form, treat cysteine protease-associated disorders. By "cysteine Such as being present as pharmaceutically acceptable Salts, protease-associated disorders' or grammatical equivalents which is meant to include both acid and base addition Salts. herein is meant pathological conditions associated with “Pharmaceutically acceptable acid addition salt” refers to cysteine proteases. In Some disorders, the condition is asso 25 those salts that retain the biological effectiveness of the free ciated with increased levels of cysteine proteases, for bases and that are not biologically or otherwise undesirable, example, arthritis, muscular dystrophy, inflammation, tumor formed with inorganic acids Such as hydrochloric acid, invasion, and glomerulonephritis are all associated with hydrobromic acid, Sulfuric acid, nitric acid, phosphoric acid increased levels of cysteine proteases. In other disorders or and the like, and organic acids Such as acetic acid, propionic diseases, the condition is associated with the appearance of acid, glycolic acid, pyruvic acid, Oxalic acid, maleic acid, an extracellular cysteine protease activity that is not present malonic acid, Succinic acid, fumaric acid, tartaric acid, citric in normal tissue. In other embodiments, a cysteine protease acid, benzoic acid, cinnamic acid, mandelic acid, methane is associated with the ability of a pathogen, Such as a virus, Sulfonic acid, ethaneSulfonic acid, p-tolueneSulfonic acid, to infect or replicate in the host organism. Salicylic acid and the like. "Pharmaceutically acceptable Specific examples of cysteine protease associated disor 35 base addition Salts' include those derived from inorganic derS or conditions include, but are not limited to, arthritis, bases Such as Sodium, potassium, lithium, ammonium, muscular dystrophy, inflammation, tumor invasion, calcium, magnesium, iron, Zinc, copper, manganese, alumi glomerulonephritis, malaria, Alzheimer's disease, disorders num salts and the like. Particularly preferred are the asSociated with autoimmune System breakdowns, periodon ammonium, potassium, Sodium, calcium, and magnesium Salts. Salts derived from pharmaceutically acceptable tal disease, cancer metastasis, trauma, inflammation, 40 organic non-toxic bases include Salts of primary, Secondary, gingivitis, leishmaniasis, filariasis, osteoporosis and and tertiary amines, Substituted amines including naturally osteoarthritis, and other bacterial and parasite-borne occurring Substituted amines, cyclic amines and basic ion infections, and others outlined above. eXchange resins, Such as isopropylamine, trimethylamine, In particular, disorders associated with interleukin 11t3 diethylamine, triethylamine, tripropylamine, and ethanola converting enzyme (ICE) are included, as outlined above. 45 mine. In this embodiment, a therapeutically effective dose of a The pharmaceutical compositions may also include one or cysteine protease inhibitor is administered to a patient. By more of the following: carrier proteins Such as Serum “therapeutically effective dose” herein is meant a dose that albumin; buffers, fillerS Such as microcrystalline cellulose, produces the effects for which it is administered. The exact lactose, corn and other Starches, binding agents, Sweeteners dose will depend on the disorder to be treated and the 50 and other flavoring agents, coloring agents, and polyethyl amount of cysteine protease to be inhibited, and will be ene glycol. Additives are well known in the art, and are used ascertainable by one skilled in the art using known tech in a variety of formulations. niques. In general, the cysteine protease inhibitors of the The following examples serve to more fully describe the present invention are administered at about 1 to about 1000 manner of using the above-described invention, as well as to mg per day. For example, as outlined above, Some disorders 55 Set forth the best modes contemplated for carrying out are associated with increased levels of cysteine proteases. various aspects of the invention. It is understood that these Due to the 1:1 stoichiometry of the inhibition reaction, the examples in no way Serve to limit the true Scope of this dose to be administered will be directly related to the amount invention, but rather are presented for illustrative purposes. of the excess cysteine protease. In addition, as is known in All references cited herein are incorporated by reference. the art, adjustments for inhibitor degradation, Systemic ver 60 EXAMPLES SuS localized delivery, and rate of new protease Synthesis, as well as the age, body weight, general health, Sex, diet, time Example 1 of administration, drug interaction and the Severity of the Synthesis of Cysteine Protease Inhibitor with a disease may be necessary, and will be ascertainable with Carbamoyl Peptidyl A Group, a Sulfonyl X Group, routine experimentation by those skilled in the art. 65 and Ethylenediamine Backbone A "patient' for the purposes of the present invention Unless otherwise indicated, all reactions were performed includes both humans and other animals and organisms. under an inert atmosphere of argon at room temperature. 6,030.946 33 34 THF was distilled from sodium benzophenone ketyl. All ethylenediamine hydrochloride (0.5 g, 1.7 mmol, prepared other Solvents and commercially available material were by conversion of the N-phenylsulfonylnorleucine methyl used without further purification. Unless otherwise noted, all ester to its corresponding acid, by aqueous Sodium hydrox reagents were obtained from Aldrich, Inc. ide treatment; amide formation, CD1/NH; reduction of the (4-morpholine carbonylphenylalanyl)-2-phenethyl-1- carbonyl amide to its corresponding amine; followed by HCl phenylsulfonyl ethylenediamine (Mu-Phe-retro-(D.L)-Hph dioxane treatment) was added. After 1 hour, the reaction was SOPh) diluted with EtOAc (100 mL) and washed with 1M HCl (50 To a Suspension of homophenylalanine (Synthetech) (7 g, mL), saturated aqueous NaHCO3 (50 mL), dried over 39.1 mmol) in distilled water (21.5 mL) was added a 2 M MgSO, and filtered. The solvent was removed under aqueous NaOH (21.5 mL). After 5 minutes, the suspension reduced pressure, triturated with hexane (15 mL) and dried had cleared and phenylsulfonyl chloride (5.48 mL, 43 in vacuo, giving 0.40 g (76%) of Boc-Np2-retro-(D.L)- mmol) was added. After 2 hours, the reaction mixture's pH NleSOPh. was adjusted to 12 with 2 M aqueous NaOH (5 mL), and To a solution of HCI*Np2-retro-(D.L)-NleSOPh (0.979, extracted with EtO (2x100 mL). The aqueous layer's pH 1.98 mmol, prepared by conversion of Boc-Np2-retro-(D, was adjusted to 1 with 6 M HCl (10 mL), and the product 15 L)-NleSOPh to HCl*Np2-retro-(DL)-NleSOPh via HC1/ was extracted with EtOAc (100 mL), dried over MgSO dioxane) in THF (25 mL) at -10° C. were added pivaloyl filtered, and the Solvent was removed under reduced pres chloride (0.27 mL, 2.2 mmol) and 4-methylmorpholine sure. The residue was triturated with EtO (2x100 mL), (0.48 mL, 4.4 mmol). After 1 hour, the reaction mixture was filtered, and pumped to dryness, giving 8.74 g (70%) of diluted with CHCl (100 mL) and washed with 1M HCl phenylsulfonylhomophenylalanine. (100 mL), saturated aqueous NaHCO(100 mL), dried over To a Solution of 4-morpholinecarbonylphenylalanine MgSO, and filtered. The solvent was removed under (Mu-Phe(OH, 0.14g, 0.48 mmol, prepared according to the reduced pressure yielding 0.66 g (66%) of Piv-Np2-retro method described in Esser, R. et.al., Arthritis & Rheumatism (D.L)-NleSOPh TLC: Rf=0.3 (20% EtOAc/CH,Cl). (1994), 37, 236) in THF (10 mL)) at -10° C. were added 4-methylmorpholine (0.11 mL, 0.96 mmol), followed by 25 Example #3 isobutyl chloroformate (64 ul, 0.48 mmol). After 5 minute activation, 2-phenethyl-1-phenylsulfonylethylenediamine Synthesis of a Cysteine Protease Inhibitor hydrochloride (0.15g, 0.44 mmol, prepared by conversion containing a Sulfonyl Peptidyl A Group, a Sulfonyl of the N-phenylsulfonylhomophenylalanine to the amide via X Group, and a Piperazine Backbone CD1/NH/THF reaction, then by reduction of the carbonyl Synthesis of 2-butyl-1-(2-naphthylsulfonyl)-4- amide to its corresponding amine with lithium aluminium (phenylsulfonyl-2-naphthylalanyl)-1,4-piperazine, abbrevi hydride, followed by treatment with HCl in dioxane) was ated PhSO-Np2-pip-retro-(D.L)-NleSO2Np. added. After 1 hour, the reaction mixture was diluted with CHCl (50 mL) and washed with 1 M HCl (50 mL) To a solution, Z-Nle-OH (10g, 38 mmol) in THF (100 followed by saturated aqueous NaHCO (50 mL), dried over 35 mL) at -10°C. were added pyridine (6.7 mL, 83 mmol), and MgSO, filtered, and the solvent was removed under thionyl chloride (3.02 mL, 42 mmol). After 30 minutes, reduced pressure. The residue was diluted with CHCl (5 triethylamine (11.6 mL, 83 mmol) and N-benzylglycine mL) and precipated from hexane (200 mL), filtered, and ethyl ester (5.4 mL, 42 mmol) were added. After 2 hours, the dried in vacuo yielding 0.22 g (89%) of the desired product. reaction mixture was diluted with EtOAc (200 mL) and Thin-layer chromatography (TLC) was performed on 40 washed with 1M HCl (150 mL), saturated aqueous NaHCO each Sample. Visualization was accomplished by means of a (150 mL), dried over MgSO, and filtered. The solvent was UV light at 254 nm, followed by ninhydrin, bromocreosol removed under reduced pressure, yielding 16.61 (100%) of green, or p-anisaldehyde stain. The retention factor (R.) of Z-norleucyl-N-benzylglycine ethyl ester. the Mu-Phe-retro-(D.L)-Hph-SOPh was 0.67 (10% To a solution of HBr notreucyl-N-benzylglycine ethyl MeOHt/CH,Cl). 45 ester (14.6 9, 38 mmol, prepared by conversion of the Z-norleucyl-N-benzylglycine ethyl ester to its HBr salt with Example 2 30% HBr in AcOH.) in EtOH (50 mL) was added triethy Synthesis of a Cysteine Protease Inhibitor with an lamine (13.2 mL, 94 mmol). After 2 hours the solvent was Acyl Peptidyl A Group, a Sulfonyl X Group, and removed under reduced pressure. The residue was dissolved 50 in EtOAc (100 mL) and washed with 1M HCl (100 mL), an Ethylenediamine Backbone saturated aqueous NaHCO (100 mL), dried over MgSO, Synthesis of 2-butyl-4-(pivaloyl-2-naphthylalanyl)-1- and filtered. Diluted with EtO (300 mL) and placed at 0° phenylsulfonyl ethylenediamine (abbreviated Piv-Np2 C. for 2 hours. The Solution was filtered and the Solid was retro-(D.L)-NleSOPh). To a solution of HCl*norleucine dried in vacuo yielding 6.3 g (64%) 1-benzyl-3-butyldiketo methyl ester (4.87 9, 26.8 mmol) in THF (20 mL) were 55 1,4-piperazine. added triethylamine (8.22 mL, 59 mmol) and phenylsulfonyl To a solution of 20HCl)*1-benzyl-3-butyl-1,4-piperazine chloride (3.76 mL, 30 mmol). After 16 hours, the solvent (2.2 g, 8.2 mmol, prepared by conversion of 1-benzyl-3- was removed under reduced pressure. The residue was butyldiketo-1,4-piperazine to 4-benzyl-3-butyl-1,4- dissolved in EtOAc (100 mL) and washed with 1M HCl, piperazine via reduction with lithium aluminum hydride saturated aqueous NaHCO, dried over MgSO, and filtered. 60 followed by HCl/dioxane treatment) in THF (50 mL) were The Solvent was removed under reduced pressure giving added, BSA (4.4 mL, 18 mmol), triethylamine (2.5 mL, 18 6.04 g (79%) of N-phenylsulfonylnorleucine methyl ester. mmol), and 2-maphthylsulfonyl chloride (2g, 9 mmol). After To a solution of t-butoxycarbonyl-2-naphthylalanine 16 hours, the reaction mixture was diluted with EtOAc (100 (Synthetech) (0.599, 1.88 mmol) in THF (25 mL) at -10° mL) and washed with 1M HCl (100 mL), saturated aqueous C. were added 4-methylmorptnoline (0.41 mL, 3.8 mmol), 65 NaHCO (100 mL), dried over MgSO, and filtered. The followed by isobutyl chloroformate (0.25 mL, 1.88 mmol). Solvent was removed under reduced pressure and the residue After 5 minute activation, 2-(D.L)-butyl-1-phenyl sulfonyl was diluted with CHCI (100 mL). A mixture of MgSO/ 6,030.946 35 36 activated carbon/silica (1:1:1, 5 g) was added to the Solution, Example #5 heated to reflux for 5 minutes, and filtered. The Solvent was removed under reduced pressure yielding 2.7 g (80%) of Synthesis of Cysteine Protease Inhibitor Containing 4-benzyl-2-butyl-1-(2-naphthylsulfonyl)-1,4-piperazine. an Acyl Peptidyl A Group, a Sulfonyl X Group, To a solution of Z-(2-naphthylalanine) (2.1 g, 5.9 mmol, and a Piperazine Backbone prepared by conversion of 2-naphthylalanine (Synthetech) Synthesis of 1-benzylsulfonyl-4-(phenylacetyl-(2-naphthyl to its Z-derivative with benzyloxycarbonyl chloride.) in alanyl))-1,4-piperazine, abbreviated Phac-Np2-pip THF (50 mL) at -10°C. were added pyridine (0.96 mL, 12 GlySOBzl. mmol), thionyl chloride (0.43 mL, 5.9 mmol). After 30 To a suspension of piperazine (4.g., 46 mmol) in THF (100 minutes, a Solution of 2-butyl-1-(2-naphthylsulfonyl)-1,4- mL) at -10° C. were added BSA (11.5 mL, 46 mmol) and piperazine hydrochloride (2.4 g, 5.4 mmol, prepared by C-toluenesulfonyl chloride (3 g, 15 mmol). After 1 hour the conversion of 1-benzyl-3-butyl-1-(2-naphthylsulfonyl)-1,4- reaction was filtered, diluted with EtOAc (100 mL), washed piperazine to 2-butyl-1-(2-naphthylsulfonyl)-1,4-piperazine with saturated aqueous NaHCO, dried over MgSO, and with H/5% Pd/C/IPA (isopropanol) in a Parr apparatus, and filtered. The Solvent was removed under reduced pressure, followed by HCl/dioxane treatment) BSA (1.5 mL, 5.9 15 and the residue was triturated with Et2O (20 mL)/hexane (20 mmol), and triethylamine (1.7 mL, 12 mmol) in CHCl (50 mL), and pumped to a Solid giving 2.5 g (67%) of mL) was added. After 1 hour, the reaction mixture was 1-benzylsulfonyl-1,4-piperazine. diluted in CHCl (100 mL) and washed with 1M HCl (100 To a solution of Z-(2-naphthylalanine) (0.81 g, 6.9 mmol) mL), saturated aqueous NaHCO, dried over MgSO, and in THF (50 mL) were added pyridine (1.12 mL, 13.7 mmol) filtered. The Solvent was removed under reduced pressure, and thionyl chloride (0.5 mL, 6.9 mmol). After 30 minutes, diluted with EtO (10 mL), precipitated from hexane (100 a Solution of 1-benzylsulfonyl-1,4-piperazine (1.5 g., 6.2 mL), filtered, and dried in vacuo giving 1.7 g (43%) of mmol), BSA (3.4 mL, 13.7 mmol), and triethylamine (1.9 4-(Z-(2-naphthyl-alanyl)-2-butyl-1-(2-naphthylsulfonyl)-1, mL, 13.7 mmol) in THF (50 mL) was added. After 2 hours, 4-piperazine. the reaction mixture was diluted with EtOAc (200 mL), To a solution of 4-(2-naphthylalanyl)-2-butyl-1-(2- 25 washed with 1M HCl (200 mL), saturated aqueous NaHCO naphthylsulfonyl)-1,4-piperazine hydrobromide (0.2g, 0.35 (200 mL) dried over MgSO, and filtered. The solvent was mmol prepared from the Z derivative via 30% HBr in removed under reduced pressure. The residue was dissolved AcOH) in THF (20 mL) at -10° C. were added BSA (0.19 in CHCl (30 mL), precipitated from hexane (200 mL), mL, 0.76 mmol), phenylsulfonyl chloride (50 ul, 0.38 filtered, and dried in vacuo giving 1.5 g (70%) of mmol), and triethylamine (0.11 mL, 0.76 mmol). After 1 4-(ben Zyl oxy carbonyl-(2-naphthylala nyl) hour, the reaction mixture was diluted with EtOAc (50 mL), -1-benzylsulfonyl-1,4-piperazine, Z-Np2-pip-GlySOBZl. washed with 1M HCl (50 mL), saturated aqueous NaHCO To a solution of 4-(2-napthylalanyl)-1-benzylsulfonyl-1, (100 mL), dried over MgSO, and filtered. The solvent was 4-piperazine (0.40 g, 0.77 mmol, prepared by conversion of removed under reduced pressure, and the residue was diluted Z-Np2-pip-GlySOBzl to its HBr salt with 30% HBr in with CHCl (5 mL), precipitated with diisopropyl ether (50 35 AcOH) in THF (50 mL) at -10°C. were added phenylacetyl mL), filtered, and dried in vacuo giving 0.1 g (43%) of final chloride (0.11 mL, 0.84 mmol) and triethylamine (0.23 mL, 1.7 mmol). After 1 hour, the reaction was diluted with product. TLC: R=0.78 (50% EtOAc/CH,Cl) EtOAc (100 mL), washed with 1M HCl (100 mL), saturated Example #4 aqueous NaHCO (100 mL), dried over MgSO, and fil Synthesis of Cysteine Protease Inhibitor Containing 40 tered. The Solvent was removed under reduced preSSure, and a Alkyloxycarbonyl Peptidyl A Group, a Sulfonyl the residue was dissolved in CHCl (10 mL), precipitated X Group, and a Piperazine Backbone from hexane (100 mL), filtered, and dried in vacuo yielding Synthesis of 4-(benzyloxycarbonyl-2-naphthylalanyl)-1-(2- 0.28 g (65%) of the desired product. TLC: R=0.39 (20% naphthylsulfonyl)-1,4-piperazine, abbreviated Z-Np2-pip EtOAC/CHCl) GlySONp. 45 The remainder of the compounds depicted in FIG. 1 were To a suspension of piperazine (3.4g, 40 mmol) in the THF Synthesized using the above techniques. (100 mL) at 0°C. were added BSA (9.8 mL, 40 mmol) and 2-naphthylsulfonyl chloride (3 g, 13 mmol). After 30 Example 6 minutes, the reaction was filtered and the Solvent was Inhibition Kinetics removed under reduced pressure. The residue was diluted in 50 CHCl (100 mL), washed with saturated aqueous NaHCO Conditions for cathepsin B: 50 mM phosphate, pH 6.0, (100 mL), dried over MgSO, and filtered. The solvent was 2.5 mM EDTA, 2.5 mM DTT Substrate: Z-Arg-Arg removed under reduced pressure, triturated with EtO (20 AMC=50 uM (Km=190 uM). The assay at 250 was started mL)/hexane (20 mL), and dried in vacuo giving 3.4 g (92%) by the addition of cat B (final concentration approx 10 nM) 1-(2-naphthylsulfonyl)-1,4-piperazine. 55 and the increase in fluorescence at 450 nm with excitation at To a solution of Z-(2-naphthylalanine) (2.1 g, 5.8 mmol) 380 nm was followed over 2 min. The depression in the rate in THF (50 mL) at 0°C. were added pyridine (0.97 mL, 11 of Substrate hydrolysis following addition of varying con mmol) and thionyl chloride (0.43 mL, 6 mmol). After 30 centrations of inhibitors was noted. The assay was linear minutes, 1-(2-naphthylsulfonyl)-1,4-piperazine (1.5 g, 5.4 throughout the range observed. Duplicate runs were mea mmol) and triethylamine (1.7 mL, 12 mmol) were added. 60 Sured. After 1 hour the reaction was diluted with EtOAc (100 mL), Conditions for cathepsin L: 50 mM acetate, pH 5.5, 2.5 washed with 1M HCl (100 mL), saturated aqueous NaHCO mM EDTA, 2.5 mM DTT Substrate: Z-PhteArg-AMC=5 (100 mL), dried over MgSO, filtered The solvent was uM (Km=2 uM). The assay at 25 was started by the addition removed under reduced pressure and diluted with CH-Cl of cat L (final concentration approx 1 nM) and the increase (20 mL), precipated from hexane (200 mL), filtered, and 65 in fluorescence at 450 nm with excitation at 380 nm was dried in vacuo giving 1.8 g (55%) of the desired product. followed over 2 min. The depression in the rate of substrate TLC: R=0.39 (50% EtOAc/hexane). hydrolysis following addition of varying concentrations of 6,030.946 37 38 inhibitors was noted. The assay was linear throughoutt-the 3. A reversible cysteine protease inhibitor according to range observed. Duplicate runs were measured. claim 2 wherein R is an amino acid side chain. Conditions for cathepsin S: 50 mM phosphate, pH 6.5, 2.5 4. A reversible cysteine protease inhibitor according to mM EDTA, 2.5 mM DTT Substrate: Z-Val-Val-Arg claim 2 having the formula: AMC=10 uM (Km=18 uM). The assay at 25 was started by 5 the addition of cat S (final concentration approx. 30 pMO and R2 the increase in fluorescence at 450 nm with excitation at 380 nm was followed over 2 min. The depression in the rate of Substrate hydrolysis following addition of varying concen A-N N-X trations of inhibitors was noted. The assay was linear throughout the range observed. Duplicate runs were mea Sured. Rs Conditions for cruZain were the same as for cathepsin L with the exception that the Km for the Substrate was 1 uM. The respective K values were estimated by using the wherein Dixon plot as described by Irwin Segel in : 15 Rs is (a) an amino acid side chain or (b) hydrogen, Behavior and analysis of rapid equilibrium and Steady-state wherein either both R- and Rs are hydrogen, or one of enzyme systems, 1975, Wiley-lnterscience Publication, John R or Rs is an amino acid side chain and the other one Wiley & Sons, New York, or for competitive binding inhibi of R and Rs is hydrogen. tors from the following calculation: 5. A cysteine protease inhibitor according to claim 2 having the formula:

R1 R2

25 )-( V is the rate of Substrate hydrolysis in the absence of re-ever i-R inhibitor, whereas V is the rate in the presence of R3 R4 O competitive inhibitor. We claim: 1. A reversible cysteine protease inhibitor having two wherein N-Substituents linked via an ethylenediamine or a substi n is from 1 to 10; tuted ethylenediamine, wherein the dissociation constant for PG is a protecting group; inhibition, K, of a protease with the inhibitor, is no greater than about 100 uM, and wherein said N-substituents are Ro is selected from the group consisting of alkyl Selected from the group consisting of acyl, acyl peptidyl, (optionally Substituted with a member of the group alkyloxycarbonyl, alkyloxycarbonyl peptidyl, Sulfonyl, 35 consisting of hydroxy, alkoxy, amino, and halogens), peptidyl, Sulfamoyl, Sulfamoyl peptidyl, Sulfinyl, Sulfinyl cycloalkyl, cyloalkylalkyl, cyloalkylalkenyl, aryl, Sub peptidyl, carbamoyl, and carbamoyl peptidyl, wherein Said Stituted aryl, aralkyl, and Substituted aralkyl, wherein ethylenediamine Substituent is an amino acid Side chain. Said Substituents of Said Substituted aralkyl, and Sub 2. A reversible cysteine protease inhibitor having the Stituted aralkyl are 1 or 2 members of the group formula: 40 consisting of alkyl, alkoxy, halogens, hydroxy, and amino; and R1 R2 AA is an amino acid. 6. A cysteine protease inhibitor according to claim 2 having the formula: A - 45 R R4 R1)-( R2 wherein A and X are N-Substituents Selected from the group 50 R-c-ever i consisting of acyl, acyl peptidyl, alkyloxycarbonyl, R R4 O alkyloxycarbonyl peptidyl, Sulfonyl, peptidyl, Sulfamoyl, Sulfamoyl peptidyl, Sulfinyl, Sulfinyl wherein peptidyl, carbamoyl, and carbamoyl peptidyl, 55 n is from 1 to 10; R is (a) an amino acid side chain or (b) hydrogen; AA is an amino acid; R is (a) an amino acid side chain or (b) hydrogen, R, is selected from the group consisting of alkyl, wherein either (1) both R and R2 are hydrogen, or (2) cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, one of R or R is an amino acid side chain and the heterocylcoalkyl, aryl, and aralkyl, wherein Said aryl other one of R and R is hydrogen; and 60 and aralkyl moieties are optionally Substituted with one R and R are hydrogen, or are bonded together to form or two groups of alkyl of 1 to 5 carbon atoms, alkoxy ethylene or substituted ethylene; of 1 to 5 carbon atoms, halogen of atomic number of wherein Said ethylene Substituent is an amino acid Side from 9 to 35, hydroxy and/or amino, optionally pro chain; tected with nitro, alkyl or arylsulfonyl, or halogen wherein the dissociation constant for inhibition, K, of a 65 Substituted alkyl of 1 to 5 carbon atoms, or perfluoro protease with the inhibitor, is no greater than about 100 group; wherein Said heterocycloalkyl is optionally Sub luM. Stituted with a radical Selected from hydroxy, alkyl, 6,030.946 39 40 heterocycloalkyl, alkanoyl, alkyloxycarbonyl, aryla lkyloxycarbonyl and heterocycloalkylcarbonyl; and (a) Ro is selected from the group consisting of alkyl O (optionally Substituted with a member of the group consisting of hydroxy, alkoxy, amino, and halogens), Ny1/yx^n,Z. X ls cycloalkyl, cyloalkylalkyl, cyloalkylalkenyl, aryl, Sub (b) Stituted aryl, aralkyl, and Substituted aralkyl, O O wherein said substituents of said substituted aryl and Substituted aralkyl are 1 or 2 members of the group consisting of alkyl, alkoxy, halogens, hydroxy, and 1O N17-14. l ls amino. 7. A pharmaceutical composition comprising a reversible cysteine protease inhibitor having the formula: in which n is 0 to 9; X-X represents a linkage Selected from -C(O)NR-, -CHNR-, -C(O)CH- and 15 -NRC(O)-; Y is -CH(R)- or -NR-; and Z is R1 R2 -(CH2)2-, -C(Rs)(R)- or -N(R)-; wherein R is hydrogen or as defined below; R is hydrogen or methyl, and each Re is independently hydrogen, alkyl (optionally -x Substituted with a radical Selected from hydroxy, alkyloxy, R R4 amino, alkylamino, dialkylamino, ureido, alkylureido, mercapto, alkylthio, carboxy, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, alkylsulfinyl, alkylsulfonyl, guanidino, wherein -P(O)(OR), -OP(O)(OR) or -OP(O)(R), A and X are N-Substituents Selected from the group wherein each R is independently hydrogen or alkyl, or a consisting of acyl, acyl peptidyl, alkyloxycarbonyl, 25 protected derivative thereof), cycloalkyl, cycloalkylalkyl, alkyloxycarbonyl peptidyl, Sulfonyl, peptidyl, aryl or arylalkyl (wherein said aryl ring is optionally Sub Sulfamoyl, Sulfamoyl peptidyl, Sulfinyl, Sulfinyl Stituted with one to three radicals Selected from hydroxy, peptidyl, carbamoyl, and carbamoyl peptidyl, amino, guanidino, halo, optionally halo-Substituted alkyl, R is (a) an amino acid side chain or (b) hydrogen; alkyloxy and aryl, or a protected derivative thereof) or R is (a) an amino acid side chain or (b) hydrogen, together with an adjacent R forms a divalent radical wherein either both R and R are hydrogen, or one of Selected from (C) methylene and 1,2- R or R2 is an amino acid Side chain and the other one phenylenedimethylene (which radical is optionally Substi of R and R is hydrogen; and tuted with hydroxy, or a protected derivative thereof, or R and R are hydrogen, or are bonded together to form OXO), with the proviso X and A are not both hydrogen; ethylene or substituted ethylene; 35 R and R2 are both hydrogen or one of R or R is cyano, wherein Said ethylene Substituent is an amino acid Side carboxy, alkyloxycarbonyl, alkanoyl, carbamoyl, chain; alkylcarbamoyl, dialkylcarbamoyl, alkyloxy (alkyl) wherein the dissociation constant for inhibition, K, of a carbamoyl, aminoalkylcarbamoyl, R., as defined protease with the inhibitor, is no greater than about 100 above, or R-X-, wherein R is as defined above luM; and 40 and X is a divalent radical of Formulae (a) or (b), as a pharmaceutically acceptable carrier. defined above; and 8. A cysteine protease inhibitor of Formula I: R and R are hydrogen or together form -CHRs, -CHR, wherein Rs and R are both hydrogen or one of Rs or R is R or R-X-, wherein Re, R, and 45 X- are as defined above, with the proviso that Rs is hydrogen, when R is R or R-X-, and R is hydrogen when R is R or R-X-, and the pharmaceutically acceptable Salts, individual isomers and mixtures of isomers thereof. 50 9. The compound of claim 8 in which A is R-X-; wherein R is hydrogen, alkyloxycarbonylalkanoyl of over in which: all 3 to 10 carbon atoms, (C-o)alkoxycarbonyl, (C-o) A and X are independently R-X-, wherein R is alkanoyl (optionally Substituted with a radical Selected from hydrogen, alkyl oxy carbonylalkanoyl, carboxy, (C-o)alkyloxycarbonyl and hetero(Cs)cycloalkyl alkyloxycarbonyl, alkanoyl (optionally Substituted with 55 (C-o)alkanoylamino), (C)cycloalkylcarbonyl, hetero a radical Selected from carboxy, alkyloxycarbonyl and (Cs)cycloalkylcarbonyl (optionally Substituted with a radi heterocycloalkylalkanoylamino), cycloalkylcarbonyl, cal Selected from hydroxy, (Cs)alkyl, hetero(Cs) heterocycloalkylcarbonyl (optionally substituted with a cycloalkyl, (C-5)alkanoyl, (C-5)alkyloxycarbonyl, (C-o) radical Selected from hydroxy, alkyl, heterocycloalkyl, aryl (Cs) alkyl oxy carbonyl and he tero (Cs) alkanoyl, alkyloxycarbonyl, arylalkyloxycarbonyl and 60 cycloalkylcarbonyl), (C-o)aryl(Cs)alkyloxycarbonyl, heterocycloalkylcarbonyl), arylalkyloxycarbonyl, carbamoyl, (C-5)alkylcarbamoyl, di(Cs)alkylcarbamoyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, (C-o)arylcarbamoyl, (C-o)aryl(Cs)alkylcarbamoyl, (C- arylcarbamoyl, arylalkylcarbamoyl, arylalkanoyl, 10)aryl(C-5)alkanoyl, (C7-)aroyl, (C-o)alkylsulfonyl, aroyl, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, di(Cs)alkylsulfamoyl, (C6-10)arylsulfonyl, (C-o)aryl(C. alkylsulfamoyl, dialkylsulfamoyl, arylsulfamoyl, 65 5)alkylsulfonyl or hetero(Cs)arylsulfonyl; and X is a alkylsulfinyl, dialkylaminoSulfinyl or arylsulfinyl; and divalent radical of Formula (a), wherein n is 0 to 5; X-X X is a bond or a divalent radical of Formulae (a) or (b): represents -C(O)NR ; Y is -N(R)-; Z is -CH 6,030.946 41 42 (R)-; each R is independently hydrogen, (C-7) phenylacetyl, benzoyl, dimethylsulfamoyl, benzylsulfonyl, cycloalkyl, (C-7)cycloalkyl (Cs)alkyl, pyridyl, thienyl, 1-piperizinylcarbonyl, 4-methyl-1-piperazinylcarbonyl or furyl, imidazolyl, indolyl, pyridyl(Ce)alkyl, thienyl(C) 4-morpholinylcarbonyl; R is 3-pyridylmethyl, alkyl, furyl(C)alkyl, imidazolyl(C)alkyl, indolyl(C) 2-thienylmethyl, 2-furylmethyl, 4-imidazolylmethyl, alkyl, (C-5)alkyl (optionally Substituted with a radical 5 Selected from mercapto, carboxy, amino, methylthio, 3-indolylmethyl, (Cs)alkyl (optionally substituted with a methylsulfonyl, carbamoyl, dimethylcarbamoyl, guanidino radical Selected from mercapto, carboxy, amino, methylthio, and hydroxy, or a protected derivative thereof) phenyl, methylsulfonyl, carbamoyl, dimethylcarbamoyl, guanidino naphthyl, phenyl(Ce)alkyl, naphthyl(C)alkyl, (which and hydroxy, or a protected derivative thereof), benzyl, group is optionally Substituted at its aryl ring with one to 1-naphthylmethyl, 2-naphthylmethyl and 2-phenylethyl three radicals Selected from amino, hydroxy, chloro, bromo, (which group is optionally Substituted at its aryl ring with fluoro, iodo, methyl, trifluoromethyl, methoxy and phenyl, one radical Selected from hydroxy, amino, chloro, bromo or a protected derivative thereof) or together with an adja and fluoro, or a protected form thereof) or together with an cent R forms a divalent radical Selected from (C) adjacent R or R forms a divalent radical Selected from methylene and 1,2-phenylenedimethylene (which radical is 15 optionally Substituted with hydroxy, or a protected deriva (C)methylene and 1,2-phenylenedimethylene (which tive thereof, or oxo); radical is optionally Substituted with hydroxy, or a protected X is -S(O).R., wherein Ro is (Cs)alkyl (optionally derivative thereof, or oxo); and Ro is methyl, Substituted with one or two radicals selected from trifluoromethyl, optionally Substituted phenyl, 2-naphthyl or amino, chloro, bromo, fluoro, hydroxy and methoxy, or 2-phenylethyl. a protected derivative thereof), perhalo(Cs) 12. The compound of claim 11 in which n is 0; R is butyl, alkylsulfonyl, (C-7)cycloalkyl, (C-7)cycloalkyl (Cs) 2-phenylethyl or 2-methylsulfonylethyl; R is hydrogen, alkyl, phenyl, pentafluorophenyl, naphthyl or phenyl tert-but Xoy carbonyl, ben Zylo Xy carbonyl, (Ce)alkyl (which group is optionally Substituted at its biotinylaminohexanoyl, benzoyl, 1-piperiziny-carbonyl, aryl ring with one to two radicals Selected from amino, 25 4-methyl-1-piperazinylcarbonyl or 4-morpholinylcarbonyl, chloro, bromo, fluoro, hydroxy, methoxy and option R is (Cs) alkyl, optionally Substituted benzyl, ally halo-Substituted methyl, or a protected derivative 1-naphthylmethyl, 2-naphthylmethyl, 3-pytrainylmethyl or thereof); R is hydrogen and R2 is Re, as defined above. 2-methylsulfonylethyl, and Ro is phenyl, 1-naphthyl or 10. The compound of claim 9 wherein n is 0 to 2; R is 2-phenylethyl. Reas defined below; R is hydrogen, (Cs) 13. A cysteine protease inhibitor according to claim 2 alkoxycarbonyl, (C)alkanoyl (optionally Substituted with wherein at least one of R and R is an amino acid side a radical Selected from carboxy, (Cs)alkyloxycarbonyl and chain. hetero(Cs)cycloalkyl(C)alkanoylamino), or -C(O) 14. A compound according to claim 8 wherein at least one NRR, wherein Ro and R together form aza(C) 35 of R and R is cyano, carboxy, alkyloxycarbonyl, alkanoyl, methylene, Oxa(C2-)methylene, (C-7)methylene, (C-8) carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, alkyloxy cycloalkylcarbonyl, benzyloxycarbonyl, acetyl, benzoyl or (alkyl)carbamoyl, aminoalkylcarbamoyl, Re, as defined dimethylsulfamoyl, Re (Cs)cycloalkyl, (Cs) above, or R-X-, wherein R is as defined above and cycloalkyl methyl, 3-pyridyl, 2-thienyl, 2-furyl, X is a divalent radical of Formulae (a) or (b). 4-imidazolyl, 3-indolyl, 3-pyridylmethyl, 2-thienylmethyl, 40 2-furylmethyl, 4-imidazolylmethyl, 3-indolylmethyl, 15. A reversible cysteine protease inhibitor having the methoxy, acetoxy, (Cs)alkyl (optionally Substituted with a formula: radical Selected from mercapto, carboxy, amino, methylthio, methylsulfonyl, carbamoyl, dimethylcarbamoyl, guanidino R1 R2 and hydroxy, or a protected derivative thereof), phenyl, 45 1-naphthyl, 2-naphthyl, benzyl, 1-naphthylmethyl, 2-naphthylmethyl, 2-phenylethyl (which group is optionally A-N N-X Substituted at its aryl ring with one radical Selected from hydroxy, amino, chloro, bromo and fluoro, or a protected form thereof) or together with an adjacent R forms a 50 wherein divalent radical Selected from (Cl)methylene and 1,2- phenylenedimethylene (which radical is optionally Substi A is a N-Substituent Selected from the group consisting of tuted with hydroxy, or a protected derivative thereof, or acyl peptidyl, alkyloxycarbonyl peptidyl, peptidyl, Sul OXO); and R is (Cs)alkyl (optionally Substituted with one famoyl peptidyl, Sulfinyl peptidyl, and carbamoyl pep or two radicals Selected from amino, chloro, bromo, fluoro 55 tidyl; X is a N-Substituent selected from the group and hydroxy, or a protected derivative thereof), perfluoro consisting of acyl, acyl peptidyl, alkyloxycarbonyl, (Cs)alkyl, (C5-6)cycloalkyl, (C5-6)cycloalkylmethyl, alkyloxycarbonyl peptidyl, Sulfonyl, Sulfonyl peptidyl, phenyl, naphthyl or benzyl (which group is optionally Sub peptidyl, Sulfamoyl, Sulfamoyl peptidyl, Sulfinyl, Sulfi Stituted with one radical Selected from amino hydroxy, nyl peptidyl, carbamoyl, and carbamoyl peptidyl, chloro, bromo and fluoro, or a protected derivative thereof). 60 R is hydrogen; 11. The compound of claim 10 in which n is 0 to 1; R is butyl, 2-phenylethyl, 2-methylsulfonylethyl, 2-tert R is an amino acid side chain; butoxycarbonylethyl, 2-tert-butoxycarbonylmethyl, 4-tert wherein the dissociation constant for inhibition, K, of a butoxycarbonylaminobutyl, 4-benzoylaminobutyl or benzy protease with the inhibitor, is no greater than about 100 loxymethyl, R is hydrogen, tert-butoxycarbonyl, 65 tM. benzyloxycarbonyl, acetyl, 3-carboxypropionyl, 16. A pharmaceutical composition comprising a reversible 3-methoxycarbonylpropionyl, biotinylaminohexanoyl, cysteine protease inhibitor having the formula: 6,030.946 43 44 (Cs-1)aryl, (C7-12)aralkyl, Substituted(C7-12)aralkyl, R1 R2 di(Cs)alkyl, (C-s)alkyl(C7-)aralkyl, or Rio and R. are bonded to form a 5 or 6 membered alicyclic or heteroalicylic ring moieties, Peptidyl is 1-10 amino acids; said substituents of said substituted aryl and substituted aralkyl are 1 or 2 members of the group consisting of wherein (Cs)alkyl, (Cs)alkoxy, halogens of atomic number A is a N-Substituent Selected from the group consisting of 9-35, hydroxy, and amino; acyl peptidyl, alkyloxycarbonyl peptidyl, peptidyl, Sul R is (a) an amino acid Side chain or (b) hydrogen; famoyl peptidyl, Sulfinyl peptidyl, and carbamoyl pep R is (a) an amino acid side chain or (b) hydrogen, tidyl; X is a N-substituent selected from the group wherein either (1) both R, and R are hydrogen, or (2) consisting of acyl, acyl peptidyl, alkyloxycarbonyl, one of R or R is an amino acid Side chain and the alkyloxycarbonyl peptidyl, Sulfonyl, Sulfonyl peptidyl, 15 other one of R and R is hydrogen; peptidyl, Sulfamoyl, Sulfamoyl peptidyl, Sulfinyl, Sulfi R and R are hydrogen, or are bonded together to form nyl peptidyl, carbamoyl, and carbamoyl peptidyl; ethylene or substituted ethylene wherein said ethylene R is hydrogen; Substituent is an amino acid Side chain; and R is an amino acid Side chain; wherein the dissociation constant for inhibition, K, of a wherein the dissociation constant for inhibition, K, of a protease with the inhibitor, is no greater than about 100 protease with the inhibitor, is no greater than about 100 tM. luM; and 18. A pharmaceutical composition comprising a reversible a pharmaceutically acceptable carrier. cysteine protease inhibitor having the formula: 17. A reversible cysteine protease inhibitor having the formula: 25 R1 R2 R1 R2 -x -x R R4 R R4 wherein A and X are N-substituents selected from the group wherein consisting of C(O)R7 (acyl), acyl peptidyl, C(O)ORs 35 A and X are N-Substituents Selected from the group (alkyloxycarbonyl), alkyloxycarbonyl peptidyl, S(O) consisting of C(O)R7 (acyl), acyl peptidyl, C(O)ORs Ro (Sulfonyl), peptidyl, S(O)NRoR (Sulfamoyl), (alkyloxycarbonyl), alkyloxycarbonyl peptidyl, S(O) Sulfamoyl peptidyl, S(O)R (sulfinyl), sulfinyl peptidyl, 2Ro (Sulfonyl), peptidyl, S(O)NRoRi, (Sulfamoyl), C(O)NRoR (carbamoyl), and carbamoyl peptidyl; Sulfamoyl peptidyl, S(O)R (sulfinyl), sulfinyl peptidyl, R7 is selected from the group consisting of (Cs)alkyl, C(O)NRoR (carbamoyl), and carbamoyl peptidyl; 40 (C-7)cycloalkyl, (C-7)cycloalkyl(C-5)alkyl, (C-7) R7 is selected from the group consisting of (C-5)alkyl, cycloalkyl (Cs)alkenyl, hetero(C-7)cycloalkyl, (C-7)cycloalkyl, (C-7)cycloalkyl(Cs)alkyl, (C-7) (Cs-1)aryl, Substituted(C5-1)aryl, (C7-12)aralkyl, cycloalkyl (Cs)alkenyl, hetero(C-7)cycloalkyl, Substituted(C7-)aralkyl, wherein said hetero group is (Cs)aryl, Substituted(Cs)aryl, (C7-)aralkyl, and Selected from the group consisting of hydroxy, (C-5) Substituted(C7)aralkyl, wherein said hetero group is 45 alkyl, hetero(C7)cycloalkyl, (C-s)alkanoyl, (C-5) Selected from the group consisting of hydroxy, (C-5) alkoxycarbonyl, (Cs)aryl(Cs)alkoxycarbonyl, and alkyl, hetero(C-7)cycloalkyl, (C-5)alkanoyl, (C-5) hetero(C7)cycloalkylcarbonyl, alkoxycarbonyl, (Cs)aryl(Cs)alkoxycarbonyl, and Rs is Selected from the group consisting of (Cs)alkyl hetero(C7)cycloalkylcarbonyl, 50 (optionally Substituted with a member of the group Rs is selected from the group consisting of (Cs)alkyl consisting of hydroxy, (C-5)alkoxy, amino, and halo (optionally Substituted with a member of the group gens of atomic number 9–35), (C-7)cycloalkyl, (C-7) consisting of hydroxy, (C-5)alkoxy, amino, and halo cycloalkyl(Cs)alkyl, (C-7)cycloalkyl (Cs)alkenyl, gens of atomic number 9–35), (C-7)cycloalkyl, (C-7) (Cs)aryl, Substituted(Cs)aryl, (C7-)aralkyl, and cycloalkyl(Cs)alkyl, (C-7)cycloalkyl (Cs)alkenyl, 55 Substituted(C7-)aralkyl, (Cs-1)aryl, Substituted(Cs-1)aryl, (C7-12)aralkyl, and Ro is Selected from the group consisting of (Cs)alkyl Substituted(C7-)aralkyl, (optionally Substituted with a member of the group Ro is selected from the group consisting of (Cs)alkyl consisting of hydroxy, (C-5)alkoxy, amino, and halo (optionally Substituted with a member of the group gens of atomic number 9–35), (C)cycloalkyl, (C-7) consisting of hydroxy, (Cs)alkoxy, amino, and halo 60 cycloalkyl(Cs)alkyl, (C-7)cycloalkyl (Cs)alkenyl, gens of atomic number 9–35), (C-7)cycloalkyl, (C-7) (Cs-1)aryl, Substituted(Cs-1)aryl, (C7-12)aralkyl, and cycloalkyl(Cs)alkyl, (C-7)cycloalkyl (Cs)alkenyl, Substituted(C7-)aralkyl, (Cs-1)aryl, Substituted(Cs-1)aryl, (C7-12)aralkyl, and Ro and R are independently Selected from the group Substituted(C7)aralkyl, consisting of hydrogen, (C-5)alkyl, (C-7)cycloalkyl, Ro and R are independently selected from the group 65 (C-7)cycloalkyl (Cs)alkyl, (Cs)aryl, Substituted consisting of hydrogen, (C-5)alkyl, (C-7)cycloalkyl, (Cs-1)aryl, (C7-12)aralkyl, SubstitutedC7-12)aralkyl, (C-7)cycloalkyl (Cs)alkyl, (Csa)aryl, Substituted di(Cs)alkyl, (C-s)alkyl(C7-)aralkyl, or Rio and R. 6,030.946 45 46 are bonded to form a 5 or 6 membered alicyclic or Y is -CH(R)- or -NR-; heteroalicylic ring moieties, Z is -(CH2), —C(Rs)(R)- or -N(R)-; Peptidyl is 1-10 amino acids; R is hydrogen or as defined below; said substituents of said substituted aryl and substituted Rs is hydrogen or methyl, aralkyl are 1 or 2 members of the group consisting of 5 (Cs)alkyl, (C-5)alkoxy, halogens of atomic number each Re is independently hydrogen, (C-5)alkyl 9-35, hydroxy, and amino; (optionally substituted with a radical selected from R is (a) an amino acid side chain or (b) hydrogen; hydroxy, (Cs)alkyloxy, amino, (Cs)alkylamino, R is (a) an amino acid side chain or (b) hydrogen, di(Cs) alkylamino, uriedo, (Cs) alkyluriedo, wherein either (1) both R and R2 are hydrogen, or (2) mercapto, (Cs)alkylthio, carboxy, carbamoyl, (C-5) one of R or R is an amino acid side chain and the alkylcarbamoyl, di(Cs) alkylcarbamoyl, (C-5) other one of R and R is hydrogen; alkylsulfinyl, (C-5)alkylsulfonyl, guanidino, -P(O) R and R are hydrogen, or are bonded together to form (OR), -OP(O)(OR) or -OP(O)(R), (C) ethylene or substituted ethylene wherein said ethylene cycloalkyl, (C-7)cycloalkyl(CIs)alkyl, (C5-1)aryl, Substituent is an amino acid Side chain; 15 (Cs)aryl(Cs)alkyl (which group is optionally Sub wherein the dissociation constant for inhibition, K, of a Stituted at its aryl ring with one to three radicals protease with the inhibitor, is no greater than about 100 Selected from hydroxy, amino, guanidino, a halogen, luM; and optionally halogen Substituted (Cs)alkyl, (C-5) a pharmaceutically acceptable carrier. alkyloxy and (Cs)aryl, or a protected derivative 19. A cysteine protease inhibitor of Formula I: thereof) or together with an adjacent R forms a divalent radical Selected from (Cl)methylene and 1,2-phenylenedimethylene (which radical is optionally Substituted with hydroxy, or a protected derivative thereof, or oxo), with the proviso X and Aare not both hydrogen; 25 each R2 is independently hydrogen or (Cs)alkyl or a protected derivative thereof; R and R2 are both hydrogen or one of R or R is cyano, carboxy, (Cs) alkyloxycarbonyl, (Cs)alkanoyl, wherein carbamoyl, (Cs) alkylcarbamoyl, di(C-5) A and X are independently R-X-; alkylcarbamoyl, (Cs) alkyloxy((Cs) alkyl) R is selected from the group consisting of hydrogen, carbamoyl, amino(Cs) alkylcarbamoyl, R, as alkyloxycarbonylalkanoyl of overall 3-10 carbon atoms, (C-o)alkyloxycarbonyl, (C-o) alkanoyl defined above, or R-X-, wherein R is as defined (optionally substituted with a radical selected from above and X is a divalent radical of Formulae (a) or carboxy, (C-o) alkyloxycarbonyl and hetero(Cs) 35 (b), as defined above; cy cloalkyl (C-o) alkanoylamino), (Cas) R and R are hydrogen or together form optionally cycloalkylcarbonyl, hetero(Cs)cycloalkylcarbonyl Substituted ethylene, wherein Said ethylene Substituent (optionally substituted with a radical selected from is an amino acid Side chain or are independently R1, as hydroxy, (C-5)alkyl, hetero(Cas)cycloalkyl, (C-5) defined above; and the pharmaceutically acceptable alkanoyl, (C-5)alkyloxycarbonyl, (C-o)aryl(C-5) 40 Salts, individual isomers and mixtures of isomers alkyloxycarbonyl and hetero(Cs)cycloalkylcarbonyl), thereof. (C-o)aryl(C-5)alkyloxycarbonyl, carbamoyl, (C-5) 20. The compound of claim 19, wherein alkylcarbamoyl, di(Cs)alkylcarbamoyl, (C-o) A is R-X-; arylcarbamoyl, (C6-10)aryl (Cs)alkylcarbamoyl, R is hydrogen, alkyloxycarbonylalkanoyl of overall 3 to (C-o)aryl(CIs) alkanoyl, (C7-11)aroyl, (C-o) 45 10 carbon atoms, (C-o)alkoxycarbonyl, (C-o) alkylsulfonyl, (C-o)arylsulfonyl, (C-o)aryl(C-5) alkanoyl (optionally Substituted with a radical Selected alkylsulfonyl, (Cs) alkylsulfamoyl, di(C-5) from carboxy, (C-o)alkyloxycarbonyl and hetero(Cs) alkylsulfamoyl, (C-o) arylsulfamoyl, (C-5) cy cloalkyl (C-o) alkanoylamino), (Cas) alkylsulfinyl, di(Cs)alkylaminosulfinyl, and (C-o) cycloalkylcarbonyl, hetero(Cs)cycloalkylcarbonyl arylsulfinyl; 50 (optionally substituted with a radical selected from hydroxy, (C-5)alkyl, hetero(Cas)cycloalkyl, (C-5) X is a bond or a divalent radical of Formulae (a) or (b): alkanoyl, (C-5)alkyloxycarbonyl, (C-o)aryl(C-5) alkyloxycarbonyl and hetero(C)cycloalkylcarbonyl), (a) (C-o)aryl(CIs)alkyloxycarbonyl, carbamoyl, (C-5) 55 alkylcarbamoyl, di(Cs)alkylcarbamoyl, (C-o) arylcarbamoyl, (C-o)aryl(Cs) alkylcarbamoyl, Ny1/yx-S,Z. X ls (Co-o)aryl(C1-s)alkanoyl, (C7-11)aroyl, (C-10) (b) alkylsulfonyl, di(Cs) alkylsulfamoyl, (C-o) O O arylsulfonyl, (C-o)aryl(Cs)alkylsulfonyl or hetero 60 (Cs)arylsulfonyl, Ny-S-S-NsZ. X ls ls X is a divalent radical of Formula (a); H n is 0 to 5; X-X represents -C(O)NR-; n is 0 to 9; 65 Y is -N(R)-; X-X represents a linkage Selected from-C(O)NR-, Z is -CH(R)-; -CHNR-, -C(O)CH- and -NRC(O)-; R is hydrogen or as defined below; 6,030.946 47 48 each Re is independently hydrogen, (C-7)cycloalkyl, R and R together form optionally Substituted ethylene; (C-7)cycloalkyl(Cs)alkyl, pyridyl, thienyl, furyl, Ra is hydrogen, (Cs)alkoxycarbonyl, (C2-)alkanoyl imidazolyl, indolyl, pyridyl(C)alkyl, thienyl(C) alkyl, furyl(C)alkyl, imidazolyl(C)alkyl, indolyl (optionally substituted with a radical selected from (Ce)alkyl, (Cs)alkyl (optionally Substituted with a carboxy, (Cs) alkyloxycarbonyl and hetero(Cs) radical Selected from mercapto, carboxy, amino, cycloalkyl(C)alkanoylamino), -C(O)NRoR, methylthio, methylsulfonyl, carbamoyl, Ro and R together form aza(C-)methylene, dimethylcarbamoyl, guanidino and hydroxy, or a pro Oxa(C-)methylene or (C-7)methylene, (Cas) tected derivative the reof), phenyl, naphthyl, cycloalkylcarbonyl, benzyloxycarbonyl, acetyl, ben phenyl(C)alkyl, naphthyl(C)alkyl, (which group 1O Zoyl or dimethylsulfamoyl; is optionally Substituted at its aryl ring with one to three R16 is (C5-6)cycloalkyl, (C5-6)cycloalkylmethyl, radicals Selected from amino, hydroxy, chloro, bromo, 3-pyridyl, 2-thienyl, 2-furyl, 4-imidazolyl, 3-indolyl, fluoro, iodo, methyl, trifluoromethyl, methoxy and 3-pyridylmethyl, 2-thienylmethyl, 2-furylmethyl, phenyl, or a protected derivative thereof) or together 4-imidazolylmethyl, 3-indolylmethyl, methoxy, with an adjacent R forms a divalent radical Selected 15 acetoxy, (Cs)alkyl (optionally Substituted with a radi from (Cl)methylene and 1,2-phenylenedimethylene cal Selected from mercapto, carboxy, amino, (which radical is optionally substituted with hydroxy, methylthio, methylsulfonyl, carbamoyl, or a protected derivative thereof, or oxo); dimethylcarbamoyl, guanidino and hydroxy, or a pro X is -S(O).R.; tected derivative thereof), phenyl, 1-naphthyl, Ro is (Cs)alkyl (optionally Substituted with one or two 2-naphthyl, ben Zyl, 1-naphthyl methyl, radicals Selected from amino, chloro, bromo, fluoro, 2-naphthylmethyl, 2-phenylethyl (which group is hydroxy and methoxy, or a protected derivative optionally Substituted at its aryl ring with one radical thereof), perhalo(Cs)alkylsulfonyl, (C-7)cycloalkyl, Selected from hydroxy, amino, chloro, bromo and (C-7)cycloalkyl(Cs)alkyl or a group selected from fluoro, or a protected form thereof) or together with an phenyl, pentafluorophenyl, naphthyl and phenyl(C) 25 adjacent R forms a divalent radical Selected from alkyl (which group is optionally Substituted at its aryl (C)methylene and 1,2-phenylenedimethylene (which ring with one to two radicals Selected from amino, radical is optionally Substituted with hydroxy, or a chloro, bromo, fluoro, hydroxy, methoxy and option protected derivative thereof, or oxo), and ally halo-Substituted methyl, or a protected derivative Ro is (Cs)alkyl (optionally Substituted with one or two thereof); radicals Selected from amino, chloro, bromo, fluoro and R is hydrogen; hydroxy, or a protected derivative thereof), perfluoro (Cs)alkyl, (C)cycloalkyl, (C)cycloalkylmethyl R2 is R., and or a group Selected from phenyl, naphthyl and benzyl R and R are each hydrogen or together form optionally (which group is optionally Substituted with one radical Substituted ethylene. 35 Selected from amino hydroxy, chloro, bromo and 21. The compound of claim 20 wherein fluoro, or a protected derivative thereof). n is 0 to 2; R is Re, as defined below; k k k k k