US007056947B2

(12) United States Patent (10) Patent N0.: US 7,056,947 B2 Powers et a]. (45) Date of Patent: Jun. 6, 2006

(54) AZA-PEPTIDE EPOXIDES 5,998,470 A * 12/1999 Halbert e161...... 514/482 6,331,542 B1 * 12/2001 Carr et a1...... 514/2378 (75) Inventors: James C. Powers, Atlanta, GA (US); 6,376,468 B1 4/2002 Overkleeft et a1. Juliana L. Asgian, Fullerton, CA (US); 6,387,908 B1 5/2002 Nomura et a1. Karen E. James, Cumming, GA (US); 6,462,078 B1 * 10/2002 Ono et a1...... 514/475 Zhao-Zhao Li, Norcross, GA (US) 6,479,676 B1 11/2002 Wolf 6,586,466 Bl* 7/2003 Yamashita ...... 5l4/483 (73) Assigneei Georgia Tech Research C0rp-, Atlanta, 6,689,765 B1 * 2/2004 Baroudy et a1...... 514/63 GA (Us) 6,831,099 B1* 12/2004 Crews e161...... 514/475 ( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS USC‘ 1540’) by 309 days‘ Kidwai et al, CA 125; 33462, 1996* (21) Appl. No.: 10/603,054 * Cited by examiner (22) Filed; Jun- 24, 2003 Primary ExamineriDeborah C. Lambkin _ _ _ (74) Attorney, Agent, or F irmiThomas, Kayden, (65) Pnor Pubhcatlon Data Horstemeyer & Risley LLP; Todd Deveau US 2004/0048327 A1 Mar. 11, 2004 (57) ABSTRACT Related US. Application Data

(60) Provisional application No. 60/394,221, ?led on Jul. ______5’ 2002’ provisional application NO_ 60/394,023’ ?led The present 1nvent1on prov1des compos1t1ons for 1nh1b1t1ng on ]u1_ 5’ 2002, provisional application NO 60/394, , methods for synthesizing the compositions, and 024, ?led on Jul, 5, 2002, methods of using the disclosed inhibitors. Aspects of the invention include aZa-peptide epoxide compositions (51) Int- 0- that inhibit proteases, for example cysteine proteases, either A61K 31/335 (2006-01) in vivo or in vitro. The disclosed compounds, pharmaceu C07D 303/38 (2006-01) tically acceptable salts, pharmaceutically acceptable deriva (52) US. Cl...... 514/475; 549/548 ?ves’ prodmgss or Combinations thereof can be used to treat (58) Field of Classi?cation Search ------549/ 548; disease or pathological conditions related to the activity of 514/475 proteases associated With a speci?c disease or condition. See application ?le for Complete Search history Such treatable conditions include viral infections, stroke, (56) References Cited neurodegenerative disease, and in?ammatory disease, among others. U.S. PATENT DOCUMENTS

5,770,732 A * 6/l998 Hirschmann et a1...... 544/141 50 Claims, N0 Drawings US 7,056,947 B2 1 AZA-PEPTIDE EPOXIDES E-64 CROSS-REFERENCED TO RELATED APPLICATIONS NH; ; o This application claims bene?t of US. Provisional Patent H o HZN A N /\/\/N N AA» on Application No. 60/394,221 ?led on Jul. 5, 2002, US. H H H\\ II,’ Provisional Patent Application No. 60/ 3 94,023, ?led on Jul. o I 5, 2002, and US. Provisional Patent Application No. Once the E-64 structure Was elucidated, the regearch 60/394,024 ?led on Jul. 5, 2002, all of Which are incorpo groups of Katunuma, Barrett, and others discovered E-64’s rated by reference in their entirety. inhibitory potency toWard a large number of other cysteine proteases. E-64 inhibits , ?cin, , BACKGROUND OF THE INVENTION B, H, F, K, L, O, S, V, X, , calpain II, cruzain, and other cysteine proteases. Cathepsin J and streptococcal cys teine protease are sloWly inhibited by E-64. 1. Field of the Invention Unlike many other microbial inhibitors, E-64 is a potent and speci?c irreversible inhibitor of cysteine proteases, and This invention relates generally to protease inhibitors and 20 is used as a diagnostic reagent for identi?cation of cysteine applications thereof, more speci?cally to peptide inhibitors proteases. The compound E-64 does not inhibit serine pro of cysteine proteases, even more speci?cally to aza-peptide teases, aspartic proteases, or metalloproteases. HoWever, not epoxides, methods of their use, and methods of their pro all cysteine proteases are inhibited by E-64. Examples of duction. non-inhibited cysteine proteases are legumain and . 25 Caspases and legumain are members of the CD clan of 2. Related Art cysteine proteases, While papain, , and Protease inhibitors are important therapeutics in the treat are members of clan CA. The folloWing table lists those ment of a variety of disease conditions including viral Which are inactivated by E-64 and those Which are infections such as HIV infection. Proteases are enzymes that not inactivated. cleave proteins or peptides and are classi?ed into several 30 Enzymes Inactivated or Not Inactivated by E-64 groups. For example, cysteine proteases form a group of enzymes involved in numerous disease states, and inhibitors of these enzymes can be used therapeutically for the treat enzymes inactivated rate (M7l s71) enzymes not inactivated ment of diseases involving cysteine proteases. 35 ?cin 0.084 (ID5O) trypsin To date, a structurally diverse variety of fruit bromelain 0.110 (ID50) Ot-chymotrypsin inhibitors have been identi?ed. Palmer, (1995) J. Med. stem bromelain 0.025 (ID5O) kallikrein Chem., 38, 3193, discloses certain vinyl sulfones Which act papain 0.104 (ID5O) pepsin as cysteine protease inhibitors for cathepsins B, L, S, O2 and 89,400 plasmin 40 4,000 elastase cruzain. Other classes of compounds, such as aldehydes, cathepsin L 96,250 mold acid protease 1.8 nM (Ki) LDH nitriles, ot-ketocarbonyl compounds, halomethyl ketones, 99,000 thennolysin diazomethyl ketones, (acyloxy)methyl ketones, ketomethyl cathepsin X 775 collagenase sulfonium salts and epoxy succinyl compounds have also cathepsin 0 >100 11M (IC5O) 1 (ICE) been reported to inhibit cysteine proteases. See Palmer, id, 45 cathepsin V >0.1 11M (IC5O) legumain and references cited therein. Many irreversible cysteine cathepsin J protease inhibitors have been described in the revieW by DPPI 100 streptococcal proteinase 624 PoWers, Asgian, Ekici, and James (2002) Chemical papaya proteinase IV 58,000 RevieWs, 102, 4639. See PoWers, id, and references cited calpain II 7,500 therein. HoWever, most of these knoWn inhibitors are not 50 bleomycin >160 11M (IC5O) cruzain 70,600 considered suitable for use as therapeutic agents in animals, vignain 32,500 especially humans, because they suffer from various short comings. These shortcomings include lack of selectivity, Therefore, because of the aforementioned de?ciencies in cytotoxicity, poor solubility, and overly rapid plasma clear 55 the art, there is a need for neW compounds and methods for ance. inhibiting proteases, in particular cysteine proteases. In addition, epoxides also have been shoWn to inhibit SUMMARY OF THE INVENTION cysteine proteases. The ?rst epoxysuccinyl peptide discov ered Was E-64, a natural inhibitor, Which Was initially Aspects of the present invention provide compositions for 60 isolated from Aspergillus japonicus by Hanada et al. in inhibiting proteases, methods for synthesizing the compo 1978. The chemical structure Was determined by optical sitions, and methods of using the disclosed protease inhibi tors. The compositions described herein can inhibit pro rotation, NMR, IR, MS, elemental analysis, and amino acid teases, for example cysteine proteases, either in vivo or in analysis to be N-(N-(L-3-trans-carboxyoxiran-2-carbonyl) vitro, by contacting a cysteine protease With an aza-peptide L-leucyl)agmatine. Hanada and his coworkers shoWed that 65 epoxide. The disclosed compounds, pharmaceutically E-64 Would inactivate the plant cysteine proteases papain, acceptable salts, pharmaceutically acceptable derivatives, ?cin, and bromelain. prodrugs, or combinations thereof can be used to treat US 7,056,947 B2 3 4 disease or pathological conditions related to the activity of aZa-peptide epoxides inhibit proteases such as gingipain, proteases associated With a speci?c disease or condition. separin, and clostripain. AZa-peptide epoxide inhibitors of Such treatable conditions include viral infections, stroke, gingipain can be used for treatment of periodontal diseases. neurodegenerative disease, and in?ammatory disease, AZa-peptide epoxide inhibitors of separin are useful for among others. Methods disclosed herein for treating dis stopping, modulating, or interfering With cell division. eases include administering an effective amount of an aZa Yet another aspect of the invention provides aZa-peptide peptide epoxide to a host in need thereof to inhibit or reduce epoxide protease inhibitors With hydrophobic amino acid protease activity in the host, particularly cysteine protease residues in the P2 site. These aZa-peptide epoxide protease activity, more particularly activity of caspases, calpains, inhibitors inhibit proteases such as cathepsins, including cathepins, papain, gingipain, clostripain, separin, or legu cathepsin B, and papain. AZa-peptide epoxide inhibitors of main. One or more aZa-peptide epoxides of the present cathepsin B are useful for treating hyperproliferative con invention can also be used alone or in combination With each ditions including cancer. other, other protease inhibitors, or another therapeutic agent Another aspect provides aZa-peptide epoxides having including anti-viral compounds such as anti-viral nucleo small hydrophobic alkyl amino acid residues at P2 are good sides including nucleoside analogs. inhibitors of calpain I and II. These inhibitors are useful as One aspect of the invention provides aZa-peptide epoxide neuroprotectants and can be used as therapeutics for the compositions, for example a compound or pharmaceutically treatment or prevention of neurodegeneration and stroke. acceptable salt or pharmaceutically acceptable derivative Exemplary neuodegenerative disorders that can be treated thereof according to Formula I beloW. In some aspects of the With the disclosed aZa-peptide epoxides include but are not present invention, aZa-peptide epoxide inhibitors are speci?c 20 limited to stroke, AlZheimer’s disease, Parkinson’s disease, for cysteine proteases and do not inhibit serine proteases or multiple sclerosis, neuropathies, Huntington’s disease, and aspartyl proteases. In another aspect of the present inven myotrophic lateral sclerosis (ALS). tion, these aZa-peptide epoxide compounds potently and In another aspect, this invention provides a method to speci?cally inhibit clan CD of cysteine proteases and are identify proteolytic enZymes and a method to prevent pro also inhibitors of clan CA. Exemplary differences between 25 teolysis. aZa-peptide epoxides disclosed herein and other cysteine proteases inhibitors include different mechanisms of inhibi DETAILED DESCRIPTION OF THE tion of the cysteine residue and the binding modes. INVENTION Some aZa-peptide epoxides of the present invention can be constructed to selectively inhibit individual cysteine The present invention may be understood more readily by proteases or groups of cysteine proteases. These aZa-peptide reference to the folloWing detailed description of the inven epoxides can, for example, contain acidic aZa-amino acid tion and the Examples included therein. residues in the Pl site. Such aZa-peptide epoxides are potent Before the present compounds, compositions and meth inhibitors of caspases. AZa-peptide epoxide caspase inhibi ods are disclosed and described, it is to be understood that tors are useful for the treatment of stroke and in?ammatory 35 this invention is not limited to speci?c synthetic methods, diseases, and as inhibitors of apoptosis. Thus, another aspect speci?c pharmaceutical carriers, or to particular pharmaceu provides a method of treating stroke, in?ammatory disease, tical formulations or administration regimens, as such may, or inhibiting apoptosis including administering an effective of course, vary. It is also to be understood that the termi amount of a aZa-peptide epoxide to a patient in need thereof. nology used herein is for the purpose of describing particular Such patients can include any mammal, for example a 40 embodiments only and is not intended to be limiting. mammal exhibiting symptoms characteristic protease Cysteine Proteases. The aZa-peptide epoxide composi related pathology or disease condition such as stroke, tions provided herein inhibit enZymatic cleavage of proteins in?ammatory disease, or pathology related to apoptosis. or peptides, or a combination thereof. Exemplary enZymes Another aspect of the present invention provides an inhibited by aZa-peptide epoxides include cysteine pro aZa-peptide epoxide composition containing an aZa-aspar 45 teases, for example, calpain. Calpain uses a cysteine residue agine residue at the Pl position. AZa-peptide epoxides in the catalytic mechanism in contrast to serine proteases having an aZa-asparagine residue at the Pl position inhibit Which utiliZe a serine residue. Exemplary cysteine proteases legumain and can, therefore, modulate the immune system include papain, cathepsin B, calpains, caspases, gingipain, through such inhibition. Cleavage of antigens by proteases clostripain, legumain, and several viral enZymes. such as legumain and related proteases is a step in antigen Caspases are a recently discovered family of cysteine presentation including the display of MHC class II peptides. endoproteases, Which are highly selective for Asp at the Pl Thus, another aspect of the invention provides a method of residue. As a result, this neWly emerging family of proteases modulating the immune system of a patient by administering has been called caspases (cysteinyl aspartate-speci?c pro to a host an effective amount of an aZa-peptide epoxide tease). All caspases contain the conserved pentapeptide composition. The aZa-peptide epoxide can modulate the motif Gln-Ala-Cys-X-Gly (QACXG)(SEQ.ID immune system by inhibiting the cleavage of antigens in the NO.l), Where XIArg, Gln, Gly (R, Q, G), and are synthe patient and thereby reducing the display of antigen peptides siZed as inactive proenZymes. The only other mammalian on cell surfaces. protease With speci?city for Asp is the lymphocyte serine Yet another aspect of the invention provides a method of protease, granZyme B. Many of the proteolytic cleavages treating autoimmune disease by administering an effective that are observed during apoptosis and cytokine maturation amount of an aZa-peptide epoxide to a host in need thereof. are due to the action of various caspases. Indeed, many of The host can be any mammal, including primates, Which the procaspases are activated by other caspases, Which demonstrate symptoms associated With any number of selectively cleave at P1 Asp residues in their recognition autoimmune diseases including but not limited to lupus, for sites. example lupus erythematosus, and cancers. 65 At present, there are 14 homologous members of the Another aspect of the invention provides aZa-peptide caspase family in humans. Some caspases are important epoxides containing basic residues at the Pl position. Such mediators of in?ammation, Where they are involved in the US 7,056,947 B2 5 6 production of in?ammatory cytokines, and others are “calpain I” and “calpain II” are used herein to refer to the involved in apoptosis, Where they participate in signaling micromolar and millimolar activated calpains, respectively, and effector pathways. Group I (1, 4, 5, 11, 12, 13, and 14) as described above. While calpains degrade a Wide variety of caspases are primarily mediators of in?ammation and are protein substrates, cytoskeletal proteins seem to be particu involved in proteolytic activation of proin?ammatory cytok larly susceptible to attack. In some cases, the products of the ines. Caspase-1 is also involved in the Fas and TNFR proteolytic digestion of these proteins by calpain are dis apoptotic pathWay. Group II (2, 3, and 7) caspases are late tinctive and persistent over time. Since cytoskeletal proteins phase effectors of apoptosis and are involved in the cleavage are major components of certain types of cells, this provides of key structural and homeostatic proteins. Caspase-3, also a simple method of detecting calpain activity in cells and knoWn as CPP32 (cysteine protease protein 32-kDa), Yama tissues. Activation of calpains and/or accumulation of break or apopain, is believed to be one of the major e?‘ectors in doWn products of cytoskeletal elements have been observed apoptosis. This is a key executioner because it is in neural tissues of mammals exposed to a Wide variety of responsible either partially or totally for proteolytic cleavage neurodegenerative diseases and conditions. For example, of key apoptotic proteins. It functions to decrease or destroy these phenomena have been observed folloWing ischemia in essential homeostatic pathWays during the effector phase of gerbils and rats, folloWing stroke in humans, folloWing apoptosis. Caspase-3 cleaves or activates nuclear enZymes, administration of the toxins kainate, trimethyltin, or colchi such as poly(ADP-ribose) polymerase (PARP), the 70 kDa cine in rats, and in human AlZheimer’s disease. subunit of the U1 small ribonucleoprotein, the catalytic Cathepsin B is involved in muscular dystrophy, myocar subunit of DNA-dependent protein kinase, and protein dial tissue damage, tumor metastasis, and bone resorption. kinase C6. Group III (6, 8, 9, 10) caspases are involved in 20 In addition, a number of viral processing enzymes, Which the upstream early activation of effector caspases. Studies are essential for viral infection, are cysteine proteases. have shoWn that caspase-8 and 10 can cleave radiolabeled Inhibitors of cysteine proteases Would have multiple thera precursors for caspase-3. Caspase-6 is the only knoWn peutic uses. caspase that cleaves the lamins, the major structural proteins Other important cysteine proteases are the bacterial in the nuclear envelope. Proteolysis of lamins is observed in 25 cells undergoing apoptosis. Caspase-8 (MACH/FLICE), enZymes clostripain and gingipain. Gingipain causes tissue Which can cleave all other knoWn caspases, is suggested to destruction during periodontal diseases. Legumain is a lie in the pinnacle of the apoptotic cascade, at least When related cysteine proteases Which is involved in in?ammatory apoptosis is initiated by some stimuli such as Fas-L and diseases. Separin is involved in separation of sister chroma tids during cell division. TNF. Accordingly, the present invention encompasses com 30 positions and methods of altering, inhibiting, or reducing the The present invention includes all hydrates, solvates, formation of enzymatic reaction products involving cysteine complexes and prodrugs of the compounds of this invention. proteases. Inhibiting the formation of cysteine protease The term prodrug refers to a pharmacologically inactive reaction products in vivo can provide therapeutic effects to compound that is converted to an active drug by a metabolic patients suffering from unregulated or 35 biotransformation. Prodrugs include compounds Wherein an Caspases have a speci?city for at least the four amino amino acid residue, or a polypeptide chain of tWo or more acids to the left of the cleavage site (P side). The S4 subsite (e.g., tWo, three or four) amino acid residues is covalently is the single most important determinant of speci?city joined through an amide or ester bond to a free amino, among caspases after the P1 Asp. The optimal sequences of hydroxy or carboxylic acid group of compounds of Formula the caspases Were obtained using a positional-scanning 40 I. Additional types of prodrugs are also encompassed. For combinatorial substrate library (PS-CSL). The optimal rec instance, free carboxyl groups can be derivatiZed as amides ognition sequences for these enZymes are closely related to or alkyl esters. The amide and ester moieties may incorpo the sequences found in knoWn macromolecular substrates. rate groups including but not limited to ether, amine and Group I caspases’ optimal sequence is Trp-Glu-His-Asp carboxylic acid functionalities. Free hydroxy groups may be (WEHD) (SEQ.ID N02) with S4 favoring hydrophobic 45 derivatiZed using groups including but not limited to amino acids. Group II caspases’ optimal sequence is Asp hemisuccinates, phosphate esters, dimethylaminoacetates, Glu-X-Asp (DEXD) (SEQ. ID N03) with a requirement for and phosphoryloxymethyloxycarbonyls, as outlined in D. Asp in S4. Group III caspases’ optimal sequence is N-Glu Fleisher, R. Bong, B. H. SteWart, Advanced Drug Delivery X-Asp Where N:Val or Leu and X can be any amino acid RevieWs (1996) 19, 115. Carbamate prodrugs of hydroxy ((V,L)EXD) (SEQ. ID N04) with a preference for 50 and amino groups are also included, as are carbonate pro branched, aliphatic side chains in S4. The S3 subsite prefers drugs and sulfate esters of hydroxy groups. DerivatiZation of glutamic acid (E) in most of the caspases Which could be hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl explained by the salt link betWeen Arg-341 (involved in ethers Wherein the acyl group may be an alkyl ester, option stabiliZation of the P1 aspartic acid) and the glutamic acid in ally substituted With groups including but not limited to P3. 55 ether, amine and carboxylic acid functionalities, or Where Neural tissues, including brain, are knoWn to possess a the acyl group is an amino acid ester as described above, are large variety of proteases, including at least tWo calcium also encompassed. Prodrugs of this type are described in R. stimulated proteases termed calpains. Calpains are present in P. Robinson et al., J. Medicinal Chemistry (1996) 39, 10. many tissues in addition to the brain. Calpain I is activated The subject invention also includes isotopically-labelled by micromolar concentrations of calcium While calpain II is 60 compounds, and the pharmaceutically acceptable salts activated by millimolar concentrations. In the brain, calpain thereof, Which are identical to those recited in Formula I, but II is the predominant form, but calpain I is found at synaptic for the fact that one or more atoms are replaced by an atom endings and is thought to be the form involved in long term having an atomic mass or mass number different from the potentiation, synaptic plasticity, and cell death. Other Ca2+ atomic mass or mass number usually found in nature. activated cysteine proteases may exist, and the term 65 Examples of isotopes that can be incorporated into com “calpain” is used to refer to all Ca2+ activated cysteine pounds of the invention include isotopes of hydrogen, car proteases, including calpain I and calpain II. The terms bon, nitrogen, oxygen, phosphorous, ?uorine and chlorine. US 7,056,947 B2 7 8 Compounds of the present invention, prodrugs thereof, and ally, have a non-peptide macromolecular carrier group pharmaceutically acceptable salts of said compounds or of covalently attached to their amino and/or carboxy termini. said prodrugs Which contain the aforementioned isotopes Such macromolecular carrier groups may include, for and/or other isotopes of other atoms are Within the scope of example, lipid-fatty acid conjugates, polyethylene glycol, or this invention. Certain isotopically-labelled compounds of carbohydrates. the present invention, for example those into Which radio The folloWing ?gure shoWs the structure of an aZa-peptide active isotopes such as 3H and 14C are incorporated, are epoxide. An aZa-amino acid residue is an alpha-amino acid useful in drug and/or substrate tissue distribution assays. residue Where the alpha-carbon has been replaced by a Tritiated, i.e., 3H, and carbon-l4, i.e., l4C, isotopes are nitrogen atom. It Will be abbreviated as the three letter code particularly preferred for their ease of preparation and for the amino acid preceded by an “A”. Therefore, aZa detectability. Further, substitution With heavier isotopes such alanine Will be abbreviated as AAla and aZa-aspartic acid as as deuterium, i.e., 2H, can afford certain therapeutic advan AAsp. The epoxide Will be abbreviated as EP for the C2H2O tages resulting from greater metabolic stability, for example residue. increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Iso topically labelled compounds of Formula 1 of this invention epoxide and prodrugs thereof can generally be prepared by carrying R, (EP) out the procedures disclosed in the Schemes and/or in the | O Examples and Preparations beloW, by substituting a readily N 2 available isotopically labelled reagent for a non-isotopically 20 Rl\ H / NW R3 labelled reagent. O If a chiral center or another form of an isomeric center is l—l present in a compound of the present invention, all forms of AZa-Amino such isomer or isomers, including enantiomers and diaste Acid Residue reomers, are intended to be covered herein. Inventive com 25 pounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the race The complete structures of several aZa-peptide epoxides mic mixture may be separated using Well-knoWn techniques and their abbreviated structures are shoWn in the folloWing and an individual enantiomer may be used alone. An enan ?gure. tiomerically enriched mixture means a mixture having 30 greater than about 50% of a single enantiomer. In cases in Which compounds have unsaturated carbon-carbon double O bonds, both the cis (Z) and trans (E) isomers are Within the scope of this invention. The compositions of the present o o NH2 invention can be substantially optically pure. Substantially 35 i NH N COOEt optically pure means a composition having greater than Ph/\O N N/ H H o 90%, preferably greater than 95%, most preferably greater o o than 98% of a single optical isomer. It must be noted that, as used in the speci?cation and the appended claims, the singular forms “a,” “an” and “the” 40 CbZ-Ala-Ala-AAsn-EP —COOEt include plural referents unless the context clearly dictates COOH otherWise. Thus, for example, reference to “an aromatic COOH o o f compoun ” includes mixtures of aromatic compounds, ref Ph 0 g g N COOEt erence to “a pharmaceutical carrier” includes mixtures of \/ T NH N’H WO tWo or more stitch carriers, and the like. 45 O O O In discussing the interactions of peptides With cysteine OH proteases, We have utiliZed the nomenclature of Schechter and Berger [Biochem Biophys. Res. Commun. 27, l57il62 (1967); incorporated herein by reference]. The individual CbZ-Leu-Glu-Thr-AAsp-EP —COOEt 0 amino acid residues of a substrate or inhibitor are designated 50 NH; P1, P2, etc. and the corresponding subsites of the enZyme are designated S1, S2, etc. The scissile bond of the substrate is o o Slil'. The most important recognition subsites of cysteine i H COOEt proteases are S1 and S2. Ph/\O N N/ N Amino acid residues and blocking groups are designated 55 m2 H o using standard abbreviations [see J. Biol. Chem. 260, l4i42 o 0 (1985) for nomenclature rules; incorporated herein by ref Ph erence]. An amino acid residue (AA) in a peptide or inhibitor CbZ-Leu-Phe-AGln-EP — COOEt structure refers to the part structure iNHiCHRliCOi, Where R1 is the side chain of the amino acid residue AA. It 60 Will be appreciated that at least one of the amino acid The R3 group Would be abbreviated as COZH, CO2Et, residues of the aZa-peptide epoxides of the invention may be COZR, CONHR, CONRR', or CO-AA-T if the aZa-peptide substituted by one of the Well knoWn non-naturally occur epoxide has an epoxysuccinate moiety in its structure. ring amino acid residues. Alterations such as these may Otherwise, the structure of the R3 group Would be draWn or serve to increase the stability, bioavailability and/or inhibi 65 abbreviated. tory action of the peptides of the invention. Moreover, any There are four structural isomers at the epoxide moiety, of the aZa-peptide epoxides described herein may, addition tWo trans isomers (2S,3S and 2R,3R) and tWo cis isomers US 7,056,947 B2 10 (2R,3S and 2S,3R). The numbering of the carbons of the sulfonate, p-toluenesulfonate and pamoate [i.e., l,l'-meth epoxide is shown above. The epoxide ring is also known as ylene-bis-(2-hydroxy-3-naphthoate)] salts. Those com an oxirane ring. pounds of the Formula I that are acidic in nature, are capable The term “amino,” as used herein, refers to iNHZ or of forming base salts With various pharmacologically derivatives thereof formed by independent replacement of acceptable cations. Examples of such salts include the alkali one or both hydrogen atoms thereon With a substituent or metal or alkaline earth metal salts and particularly, the substituents independently selected from alkyl, alkanoyl, sodium and potassium salts. aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, het The term “pharmaceutically acceptable derivative” refers eroarylalkyl, and an amino protecting group. to any homolog, analog, or fragment corresponding to the The term “Cl_1O acyl,” as used herein, refers to a Cl_1O aZa-peptide epoxides of the present invention provided alkyl group, as de?ned herein, having an attached carbonyl herein Which inhibits protease activity and is relatively group. non-toxic to the subject or host. The term “Cl_l0 alkoxy,” as used herein, refers to a Cl_1O The term “pharmaceutically acceptable” means a material alkyl group, as de?ned herein, attached to the parent that is not biologically or otherWise undesirable, i.e., the molecular group through an oxygen atom. material may be administered to an individual along With the The term “C 1_ 10 alkyl’’ as used herein refers to a branched selected bicyclic compound Without causing any undesirable or unbranched hydrocarbon group of carbon atoms, such as biological effects or interacting in a deleterious manner With methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, l-butyl, any of the other components of the pharmaceutical compo and the like or branched or unbranched hydrocarbon groups sition in Which it is contained. of carbon atoms that either contain double or triple carbon 20 As used herein, and Without limitation, the term “deriva bonds. tive” is used to refer to any compound Which has a structure The term “Cl_ 10 alkylamino,” as used herein, refers to a derived from the structure of the compounds of the present Cl’l0 alkyl group, as de?ned herein, to Which is attached at invention and Whose structure is suf?ciently similar to those least one amino substituent. disclosed herein and based upon that similarity, Would be The term “C3_ 15 cycloalkyl” as applied herein is meant to 25 expected, by one skilled in the art, to exhibit the same or include cyclic hydrocarbon chains. Examples of these cyclic similar activities and utilities as the claimed compounds. hydrocarbon chains include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, The folloWing abbreviations have also been used: AFC, cyclononane, cyclodecane, cycloundecane, etc. 7-amino-4-tri?uoromethylcoumarin; AAsp, aZa-aspartic acid residue; AAsn , aZa-asparagine; ALeu, aZa-leucine; The term “C2_ 12 dialkylamino,” as used herein, refers to 30 ALys, aZa-lysine residue; AHph, aZa-homophenylalanine tWo Cl_1O alkyl groups, as de?ned herein, that are attached to an amino substituent. residue; AOrn, aZa-omithine; AMC, 7-amino-4-methylcou The term “Cl_1O ?uoroalkyl,” as used herein, refers to a marin; CbZ, Ph-CHZOCOi; DCC, 1,3-dicyclohexylcarbo Cl’l0 alkyl group, as de?ned herein, to Which is attached at diimide; DMAP, 4-dimethylaminopyridine; DMF, N,N-dim ethylformamide; EDC, l -(3-dimethylaminopropyl) -3 - least one ?uorine substituent. 35 ethylcarbodiimide hydrochloride; EtOAc, ethyl acetate; The term “Cl_ 10 per?uoroalkyl,” as used herein, refers to HOBt, l-hydroxybenZotriaZole; HRMS, high resolution a C 1_ 10 alkyl group in Which all of the hydrogen atoms have mass spectrometry; IBCF, isobutyl chloroformate; NMM, 4 been replaced With ?uorine atoms. -methylmorpholine; Np2,2-naphthyl-alanyl; PhPr, Phenyl The term biotinyl, as use herein, refers to biotin Without propyl; TFA, tri?uoroacetic acid; THF, tetrahydrofuran; the biotin carboxyl hydroxyl group. 40 TLC, thin layer chromatography. By the term “effective amount” of a compound as pro vided herein is meant a nontoxic but suf?cient amount of the One embodiment of the present invention provides aZa compound to provide the desired utility. As Will be pointed peptide epoxides having the folloWing structural Formula I: out beloW, the exact amount required Will vary from subject to subject, depending on the species, age, and general 45 condition of the subject, the severity of the condition or disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” HoWever, an appropriate effective amount may be determined by one of 50 ordinary skill in the art using only routine experimentation. The term “pharmaceutically acceptable salt(s)”, as used herein, unless otherWise indicated, includes salts of acidic or wherein, basic groups Which may be present in the compounds of R1 is selected from the group consisting of M1, M2-AAl, Formula I. The compounds of Formula I that are basic in 55 M2-AA2-AAl, and M2-AA3-AA2-AAl; nature are capable of forming a Wide variety of salts With M1 is selected from the group consisting of NH24COi, various inorganic and organic acids. The acids that may be NH2%Si, NH2iSO2i, XiNH%Oi, X2N%Oi, used to prepare pharmaceutically acceptable acid addition XiNHiCSi, X2N%Si, XiNHisOzi, X2Ni salts of such basic compounds of Formula I are those that 802i, X%Oi, XiCSi, YisOzi, YiO%Oi, form non-toxic acid addition salts, i.e., salts containing 60 Y4OiCSi, phenyl substituted With K, phenyl disubsti pharmacologically acceptable anions, such as the hydrochlo tuted With K, and morpholine-COi; ride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, M2 is selected from the group consisting of H, NHZi phosphate, acid phosphate, isonicotinate, acetate, lactate, COi, NH2%Si, NHzisOzi, XiNH%Oi, salicylate, citrate, acid citrate, tartrate, TFA, pantothenate, X2N%Oi, XiNHiCSi, X2N%Si, XiNHi bitartrate, ascorbate, succinate, maleate, gentisinate, fuma 65 802i, XZNiSOZi, X%Oi, X%Si, YisOzi, rate, gluconate, glucaronate, saccharate, formate, benZoate, Y4OiCOi, Y4OiCSi, phenyl, phenyl substituted glutamate, methanesulfonate, ethanesulfonate, benZene With K, phenyl disubstituted With K, and morpholine-COi; US 7,056,947 B2 11 12 X is selected from the group-consisting of H, Cl_1O alkyl, alkyl substituted With CONH2, Cl_1O alkyl substituted With C3_l5 cycliZed alkyl, Cl_1O ?uoroalkyl, Cl_1O alkyl substi CONHR4, Cl_1O alkyl substituted With CO2H, Cl_1O alkyl tuted With J, C 1_ 1O ?uoroalkyl substituted With J, l-admantyl, substituted With CO2R4, CH2CH2SCH3, CH2-3-indolyl, 9-?uorenyl, phenyl, phenyl substituted With K, phenyl dis CH2-2-thienyl, CH2-2-furyl, CH2-3-furyl, CH2-2-imidaZyl, ubstituted With K, phenyl trisubstituted With K, naphthyl, Cl’l0 alkyl substituted With G, C1_ 10 alkyl With an attached naphthyl substituted With K, naphthyl disubstituted With K, phenyl substituted With G, Cl_1O alkyl With an attached naphthyl trisubstituted With K, Cl_1O ?uoroalkyl With an naphthyl substituted With G, phenyl substituted With G, and attached phenyl group, C1_ 10 alkyl With an attached phenyl naphthyl substituted With G; group, Cl_l0 alkyl With tWo attached phenyl groups, Cl_1O R4 is selected from the group consisting of C 1_ 10 alkyl and alkyl With an attached phenyl group substituted With K, Cl_1O alkyl substituted With phenyl; Cl’l0 alkyl With tWo attached phenyl groups substituted With Q is selected independently from the group consisting of K, Cl_1O alkyl With an attached naphthyl group, Cl_1O alkyl Cl_1O alkoxy, Cl_1O alkyl-Si, Cl_1O alkoxy substituted With With an attached naphthyl group substituted With K, Cl_1O phenyl, and Cl_1O alkyl-Si substituted With phenyl; alkyl With an attached phenoxy group, biotinyl, and Cl_1O G is selected independently from the group consisting of alkyl With an attached phenoxy group substituted With K on amidino (4C(=NH)NH2), guanidino (iNHC(=NH) the phenoxy group; NHZ), isothiureido (iS4C(=NH)NH2), amino, C1_6 alky Y is selected from the group consisting of Cl_l0 alkyl, lamino, C2_l2 dialkylamino, and imidaZyl; C37l5 cycliZed alkyl, C 1_ 1O ?uoroalkyl, C 1_ 10 alkyl substituted R3 is selected independently from the group consisting of With J, C1_ 10 ?uoroalkyl substituted With J, l-admantyl, R5, CO2H, CO2R5, CONHR6, CONR6R7, CO-AA4-T, 9-?uorenyl, phenyl, phenyl substituted With K, phenyl dis 20 ubstituted With K, phenyl trisubstituted With K, naphthyl, naphthyl substituted With K, naphthyl disubstituted With K, 0 naphthyl trisubstituted With K, Cl_1O ?uoroalkyl With an attached phenyl group, C1_ 10 alkyl With an attached phenyl group, Cl_l0 alkyl With tWo attached phenyl groups, Cl_1O 25 alkyl With an attached phenyl group substituted With K, 1O l, p Cl’l0 alkyl With tWo attached phenyl groups substituted With K, Cl_1O alkyl With an attached naphthyl group, Cl_1O alkyl 0 With an attached naphthyl group substituted With K, Cl_1O alkyl With an attached phenoxy group, biotinyl, and Cl_1O 30 1, AN alkyl With an attached phenoxy group substituted With K on the phenoxy group; J is selected from the group consisting of halogen, CO2H, OH, CN, NO2, NH2, Cl_1O alkoxy, Cl_1O alkylamino, C2_l2 Q5 35 dialkylamino, Cl_1O alkyl-OiCOi, Cl_1O alkyl-OiCOi 0 O NHi, and Cl_1O alkyl-Si; K is selected from the group consisting of halogen, Cl_1O alkyl, Cl_1O per?uoroalkyl, C 1_ 1O alkoxy, phenoxy, NO2, CN, k OH, CO2H, amino, Cl_1O alkylamino, C2_l2 dialkylamino, ,and ; Cl’l0 acyl, and Cl_l0 alkoXy-COi, and Cl_1O alkyl-Si; 40 AAl, AA2, and AA3 are side chain blocked or unblocked amino acids With the L con?guration, D con?guration, or no chirality at the ot-carbon selected from the group consisting of alanine, Valine, leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan, glycine, 45 R5 is selected independently from the group consisting of serine, threonine, cysteine, tyrosine, asparagine, glutamine, Cl_1O alkyl, C3_l5 cycliZed alkyl, Cl_1O alkyl With a phenyl aspartic acid, glutamic acid, lysine, arginine, histidine, phe group attached to the Cl_1O alkyl, C3_l5 cycliZed alkyl With nylglycine, beta-alanine, norleucine, norvaline, alpha-ami an attached phenyl group, Cl_l0 alkyl With an attached nobutanoic acid, epsilon-aminocaproic acid, citrulline, phenyl group substituted With K, Cl_1O alkyl With an hydroxyproline, ornithine, homoarginine, sarcosine, indo 50 attached phenyl group disubstituted With K, Cl_1O alkyl With line 2-carboxylic acid, 2-aZetidinecarboxylic acid, pipe an attached phenyl group trisubstituted With K, C3_l5 colinic acid (2-piperidine carboxylic acid), O-methylserine, cycliZed alkyl With an attached phenyl group substituted O-ethylserine, S-methylcysteine, S-ethylcysteine, S-benZyl With K, Cl_1O alkyl With a naphthyl group attached to the cysteine, NH2iCH(CH2CHEt2)4CO2H, alpha-aminohep Cl’l0 alkyl, C3_l5 cycliZed alkyl With an attached naphthyl tanoic acid, NH24CH(CH2-l-naphthyl)-CO2H, NH24CH 55 group, Cl_1O alkyl With an attached naphthyl group substi (CH2-2-naphthyl)-CO2H, NH2iCH(CH2-cycloheXyl) tuted With K, Cl_ 10 alkyl With an attached naphthyl group CO2H, NH2iCH(CH2-cyclopentyl)-CO2H, NH24CH disubstituted With K, Cl_1O alkyl With an attached naphthyl (CH2-cyclobutyl)-CO2H, NH24CH(CH2-cyclopropyl) group trisubstituted With K, and C3_l5 cycliZed alkyl With an CO2H, tri?uoroleucine, 4-?uorophenylalanine, lysine attached naphthyl group substituted With K; substituted on the epsilon nitrogen With a biotinyl group, and 60 T is selected independently from the group consisting of hexa?uoroleucine; OH, ORS, NHR9, and NR8R9; R2 is selected from the group consisting of Cl_1O alkyl, AA4 is a side chain blocked or unblocked amino acid With Cl’l0 alkyl substituted With Q, Cl_l0 alkyl substituted With the L con?guration, D con?guration, or no chirality at the phenyl, Cl_1O alkyl With an attached phenyl substituted With ot-carbon selected from the group consisting of alanine, K, Cl_1O alkyl substituted With naphthyl, Cl_1O alkyl With an 65 Valine, leucine, isoleucine, proline, methionine, methionine attached naphthyl substituted With K, phenyl, phenyl sub sulfoxide, phenylalanine, tryptophan, glycine, serine, threo stituted With K, naphthyl, naphthyl substituted With K, C 1_ 1O nine, cysteine, tyrosine, asparagine, glutamine, aspartic acid,

US 7,056,947 B2 15 16 PhPr-Val-Ala-AAsp-(R,R)-EP-CONHCH2CH2Ph, PhPr-Val-Ala-AAsp-(S,S)-EP-CONHCH2CH(OH)Ph, -C0minued H020 PhPr-Val-Ala-AAsp-(R,R)-EP-CONHCH2CH(OH)Ph, O PhPr-Val-Ala-AAsp-(S,S)-EP-CO-Ala-NHCH2Ph,PhPr-Val-Ala-AAsp-(R,R)-EP-CO-Ala-NHCH2Ph, 5 CbZ_ASp_Glu_Val\ g 0 U, PhPr-Val-Ala-AAsp-(S, S)-EP-CO-Leu-NH2, O PhPr-Val-Ala-AAsp-(R,R)-EP-CO-Leu-NH2, H020 PhPr-Val-Ala-AAsp-(S, S)-EP-CO-Phe-NH2, 10 W o PhPr-Val-Ala-AAsp-(R,R)-EP-CO-Phe-NH2, CbZ-Asp-Glu-Val \ N / NW N O PhPr-Val-Ala-AAsp-(S, S)-EP-CO-Tyr-NH2, H 0 AK), CbZ-Glu-Val-AAsp-(R,R)CbZ-Glu-Val-AAsp-(S,S)-EP-CONHCH2CH2Ph, -EP-CO-Phe-NH2, 15 O Q6\ CbZ-Asp-Glu-Val-AAsp-(S,S)-EP-CO-Phe-NH2, HOZC CbZ-Asp-Glu-Val-AAsp-(S,S)-EP-CONHCH2Ph, W O CbZ-Asp-Glu-Val-AAsp-(S,S)-EP-COOCH2Ph, CbZ_ASp_Glu_Val\ /N CbZ-Leu-Glu-Thr-AAsp-(S,S)-EP-CONHCH2CH2Ph, 20 g O N/\ CbZ-Leu-Glu-Thr-AAsp-(S,S)-EP-CO-Ala-NHCH2Ph, O K/O,

25 W O HOZC CbZ—Asp—Glu—Val\N/N N O W O H O I / , CbZ—Asp—Glu—Val\ /N O \ r N | O , o 30 / N HO C H020 o 2 W 0 CbZ-Asp-Glu-Val \g/ N MN CbZ-Asp-Glu-Val \g/ N N O O o , 35 O O | / , | \

/ N H020 H020 40 W O

W O ’ Cbz-Asp-Glu-Val \ N / NW N 0 Cbz _ Asp _ G111 _ Val\N/N N H O O | / , H O \ O 45 I H020 N /

EXEMPLARY METHODS OF PREPARATION H020W O 55 CbZ_ASp_Glu_Val\ /N , A. Preparation of the Epoxide Portion g N A Variety of epoxides can be synthesized by following the O O schemes shoWn below. Any aldehyde can be reacted With malonic acid to form the 0t,[3-unsaturated acid, Which can be H020 O 60 further transformed into an ethyl ester. The double bond is epoXidiZed using t-butyl peroxide and t-butyl lithium, fol

CbZ'ASP'Gm'Val\N/NW‘/V>)I\NH O I O/ , loWedsubstituted by theepoxide. deblocking This epoxide of the canethyl then ester be coupled to yield to the respective substituted hydraZide to yield an aZa-peptide 65 epoxide using peptide coupling procedures. \ O The diethyl ester epoxysuccinate can also be deblocked to yield the diacid, Which can be selectively coupled to differ US 7,056,947 B2 17 18 ent alcohols to yield a variety of epoxide monoesters. Hydrolysis of diethyl epoxysuccinate also yields the mono -continued ethyl ester, Which can be coupled With a variety of mono O COOEt KOH/EtOH substituted and disubstituted amines to form amide deriva tives of ethyl epoxysuccinate. The ethyl ester can then be A EtOOC hydrolyzed to the acid. These epoxides can then be coupled 0 COOH O COOR to substituted hydraZides to yield aZa-peptide epoxides. —> These processes are illustrated beloW. EDC, DMAP HOOC HOOC

COOR t-BuOOH/toluene B. Preparation of the Peptide Portion — —> t-BuLi/pentane The peptide portion of the aZa-peptide epoxide inhibitor ROOC THF can be prepared using standard peptide chemistry Which is Well described in publications such as The Peptides, Analy sis, Synthesis, Biology, Vol. li9, published in l979il987 by 0 COOR 0 COOH KOlUEtOH Academic Press; Houben-Weyl Methoden der Organischen —> Chemie, Vol. 15, Parts 1 and 2, Synthese von Peptiden, ROOC ROOC 20 published by Georg Thieme Verlag, Stuttgart in 1974; and (trans) Houben-Weyl Methods of Organic Chemistry, Vol. E22, Parts a, b, c, and d, Synthesis ofPeptides and Peptidomi metics published by Georg Thieme Verlag, Stuttgart COOEt COOEt 25 2000*2003 (references incorporated herein by reference). H——OH 33% H——OAc 33% The M1 group can be introduced using a number of HBr/AcOH HBr/AcOH different reaction schemes. First, it could be introduced HO H H Br EtOH directly on an amino acid as shoWn in the folloWing scheme cooEt cooEt hm (top), or the M 1 group could be introduced by reaction With 30 an amino acid ester, folloWed by removal of the ester group Diethyl L- (+)—ta.1trate to give the same product (bottom). COOEt

H —— OH NaOEt EtOH 35 H —— Br —> —> —> The techniques for introduction of the M1 group are Well LOOEt documented in The Peptides, Houben-Weyel, and many other textbooks on organic synthesis. For example reaction (R, R) With cyanate or p-nitrophenyl cyanate Would introduce a carbamyl group (MIINHZCOi). Reaction With H MeZNCOCl Would introduce the MeZNCOi group. Reac tion With p-nitrophenyl thiocarbamate Would introduce a HI | | I thio carbamyl group (MIINH2CSi). Reaction With NHZSOZCl Would introduce the NH2SO2i group. Reaction O With MeZNSOZCl Would introduce the MeZNSOZi group. Reaction With a substituted alkyl or aryl isocyanate Would i 4>CH2(COOH)2 COOH EtOH introduce the XiNHiCOi group Where X is a substi R H pyridine R/\/ DCC, DMAP tuted alkyl or aryl group. Reaction With a substituted alkyl 50 O or aryl isothiocyanate Would introduce the XiNH4CSi group Where X is a substituted alkyl or aryl group. Reaction COOE t —>l) t-BuOOH, t-BuLi OH R/\/ 2) KOlUEtOH R With xiso2ic1 Would introduce the XiSOZi group. Reaction With a substituted alkyl or aryl acid chloride Would O introduce an acyl group (MIXiCOi). For example, reac O COOEt 55 tion With MeO4COiCH2CH24COiCl Would give the KOlUEtOH 4' X4COi group Where X is a C2 alkyl substituted With a Cl alkyl-OCOi group. Reaction With a substituted alkyl or EtOOC aryl thioacid chloride Would introduce a thioacyl group 0 COOH (MIX4CSi). Reaction With a substituted alkyl or aryl RR'NH EDC, HOBt sulfonyl chloride Would introduce the xisozi group. For EtOOC example, reaction With dansyl chloride Would give the XiSOZi derivative Where X Was a naphthyl group mono O CONRR' O CONRR' KOlUEtOH substituted With a dimethylamino group. Reaction With a —> substituted alkyl or aryl chloroformate Would introduce the EtOOC HOOC X4OiCOi group. Reaction With a substituted alkyl or aryl chlorothioformate Would introduce the X4OiCSi. US 7,056,947 B2 19 20 There are many alternate reaction schemes Which could be used to introduce all of the above Ml groups to give either Ml-AA-OH or Ml-AA-OR'.

5 The Ml-AA-OH derivatives could then be used directly in the preparation of peptide hydraZides or could be converted into the dipeptides, tripeptides, and tetrapeptides Ml-AA AA-OH, Ml-AA-AA-AA-OH, or Ml-AA-AA-AA-AA-OH Which could then be converted to peptide hydraZides. The O substituted peptides Ml-AA-AA-OH, Ml-AA-AA-AA-OH, or Ml-AA-AA-AA-AA-OH could also be prepared directly H O 1. R1 i H from H-AA-AA-OH, H-AA-AA-AA-OH, or H-AA-AA Cbz—N N/NH2 2_ NaBH4 AA-AA-OH using the reactions described above for intro H or duction of the M1 group. Alternatively, the M1 group could E2 NaCNBH3 be introduced by reaction With carboxyl blocked peptides to R1 give Ml-AA-AA-OR', Ml-AA-AA-AA-OR', or Ml-AA 0 f AA-AA-AA-OR', folloWed by the removal of the blocking NH Cbz/ . N/ group R'. 20 - H C. Peparation of Peptide HydraZides Usually, peptide hydraZides are synthesiZed by reaction of an amino acid or peptide ester With hydraZine or by direct 25 The side chain of the aZa-amino acid residue can be coupling of an amino acid or peptide acid With hydraZine as introduced by reductive amination as shoWn speci?cally in shoWn in the folloWing tWo ?gures. They can also be the previous ?gure or by other methods knoWn by those synthesiZed directly by reaction of an amino acid or peptide skilled in the art or by alkylation as shoWn in the folloWing ester With hydrazine. ?gure.

o

1) BICHZCOOEt O NH2 NMM, DMF NH 2) NH3, NaCN R / _ N/ MeOH, DMF 1 H / R2

0

O BrCHZCOOtBu O OtBu NHZNHZ NH NMM’ DMF NH 2 Rl/ OMe MeOH RI/ Hi5 N/ Rl/ : N/ H 5 H

73"“2 film.. \ i2

0 NH2

0 o

\JkNH 2 H —>propanol Rl/N /NH US 7,056,947 B2 21 22 The precursors for basic side chain aZa-peptide epoxides Were prepared as shown in the following ?gure.

Rl/ H H 2 \\BOCBIWN ALys precursor O NH % R1/ 5 E/ W \Boc

2 AOrn precursor

20 D. Preparation of the AZa-peptide Epoxide l. EDC/HOBt Coupling Method -continued The epoxide portion of the aZa-peptide epoxide is coupled R2 to the substituted hydraZide by reacting the epoxide portion, 25 I 0 R1 N R3 the substituted hydraZide, EDC, and HOBt in DMF to form \ N/ the aZa-peptide epoxide (see the folloWing ?gure). H O

30 The folloWing ?gure shoWs hoW these methods are used 0 to build the AAsp and AAsn derivatives. Ho R3 R2 i or ii R I|\IH O 3 5 R1\ / NHZ 1\ N/ EDC, HOBt, DMF N H H R2 | 0 R1 N R3 R I peptidyl 40 O \N/H w/Q/ O 0 X HOZC A/Y5 R\ /NH iii, iv Methods for the protection and deprotection and replace 45 g ment of an amino protecting group With another moiety are Well knoWn. Deprotection of other side chain protecting 4 groups Were carried out by standard methods. aXIOEt;bXINH2; 2. The Mixed Anhydride Method cXIOt-Bu;dXIOH Another coupling method is the mixed anhydride method. 50 O In this method, the epoxide portion of the aZa-peptide epoxide is coupled to the substituted hydraZide by reacting X the epoxide portion (carboxylic acid) With NMM in DMF Y R1\ / N and IBCF folloWed by the substituted hydraZide to form the N 55 H O aZa-peptide epoxide (see the folloWing ?gure). Methods for O the protection and deprotection of side chain protecting groups are Well known. 6 Y I COOEt 7 Y I COOBZl 60 8 Y : CHZCHZPh 0 Ho R3 Reagents: (i) BrCH2COOEt, NMM, DMF; NH3/MeOH, 0.1 eq NaCN, DMF. (ii) BrCHZCOO-tBu, NMM DMF. (iii) R2| Wo Rl\N/NH NMM, IBCF, DMF 65 3, EDC, HOBt, DMF or NMM, IBCF, DMF. (iv) TFA (can H be used to deblock the t-Butyl Group in certain peptides Where XIO-tBu). US 7,056,947 B2 23 24 Examples of the preceding methods are exhibited below precipitated With 1:9 MeOH:CH2Cl2 and methanol to yield a and in the examples: White solid (68% yield). 1H NMR (DMSO-d6): 1.18 (d, 6H, E. Synthetic Procedures and Examples. CH3), 3.2 (d, 2H, NCH2CONH2), 4.044.12 (m, 1H, ot-H), 1. Material and Methods. Mono and dipeptidyl methyl 4.2 (m, 1H, ot-H), 5.03 (m, 2H, CbZ), 5.22 (m, 1H, NH), 7.18 esters Were purchased from Bachem Bioscience lnc., King (d, 1H, NH), 7347.5 (m, 6H, Ph and NH), 8.0 (m, 1H, NH), of Prussia, Pa. Tripeptides Were synthesized using standard 9.38 (m, 1H, NH). MS (FAB) m/Z 366 [(M+1)+]. HRMS coupling procedures such as the mixed anhydride method. (FAB) Calcd. For Cl6H24N5O5: 366.17774. Observed m/Z 366.17665. The 1H NMR spectra Were obtained using a Varian Mercury 5. Preparation of Peptidyl-AA2iNHiNH4CH2COO 300 MHZ spectrometer. Electrospray ioniZation (ESI), fast tBu (4c). Neat t-butyl bromoacetate (1 eq) Was added to a atom-bombardment (FAB) and high-resolution mass spec stirred solution of the peptide hydraZide (3) and NMM (1 eq) trometry Were performed using Micromass Quattro LC and in DMF pre-cooled at —100 C. The resulting solution Was VG Analytical 70-SE instruments. Elemental analysis Was performed by Atlantic Microlab lnc., Norcross, Ga. stirred for 30 min at —100 C., after Which the mixture Was alloWed to react at room temperature for 20 hours. The DMF 2. Preparation of Peptidyl HydraZides (3). Anhydrous Was removed by evaporation, and the resulting residue Was hydrazine (10 eq) Was added to a solution of the peptidyl Washed With Water, ?ltered, and dried in vacuo. Puri?cation methyl ester (1 eq) in MeOH at room temperature, and the on a silica gel column using the appropriate solvent gave 4c resulting mixture Was then stirred for 16 hours. As With most hydraZides, excess hydraZine and solvent Were removed by (yields:48465%). MS and 1H NMR (DMSO-d6 or CDCl3) Were consistent With the proposed structure. evaporation. The resulting residue Was Washed With ethanol 20 PhPr-Val-Ala-NHiNHiCH2COO-tBu Was puri?ed by and ether to give the desired peptidyl hydraZide (3) as a chromatography on silica gel column using 1:7 MeOH: White solid. MS and 1H NMR (CDCl3 or DMSO-d6) Were consistent With the proposed structures. CHZCl2 as the eluent; White solid, yield 56%. CbZ-Val-NHiNH4CH2COO-tBu Was puri?ed by col PhPr-Val-Ala-NHiNHZ, White solid, yield 75%. umn chromatography on silica gel using 1:20:4.2 MeOH: CbZ-Asp(O-tBu)-Glu(O-tBu)-Val-NHiNH2 Was puri 25 CH2Cl2:EtOAc as the eluent; White solid, yield 64%. ?ed by chromatography on a silica gel column using 1:9 1H NMR (DMSO-d6): 0.90 (t, 6H, Val), 1.40 (s, 9H, tBu), MeOH:CH2Cl2 as the eluent; White solid, yield 56%. 1.86 (m, 1H, Val), 3.37 (d, 2H, NHCHZCOOH), 3.72 (t, 1H, CbZ-Leu-Glu(O-tBu)-Thr-NHiNH2, White solid, yield ot-H), 4.99 (s, 2H, CbZ), 5.13 (d, 1H, NH), 7.30 (s, 5H, Ph), 97%. 9.38 (d, 1H, NH). N-BenZyloxycarbonylalanylalanyl HydraZide (CbZ-Ala 30 CbZ-Asp(O-tBu)-Glu(O-tBu)-Val-NHiNHiCH2COO Ala-NHiNHz) Was synthesiZed from CbZ-Ala-Ala-OMe tBu Was puri?ed by column chromatography on silica gel by hydraZinolysis; White solid (57% yield). 1H NMR using 221825 MeOH:CH2Cl2:EtOAc as the eluent; White (DMSO-d6): 1.1413 (d, CH3), 4.(L4.1 (m, 1H, ot-H), solid, yield 65%. MS (ESI) m/Z 736.6 [(M+1)+]. 1H NMR 4.1443 (m, 2H, ot-H and NH), 5.05 (s, 2H, CbZ), 7347.4 (m, (DMSO-d6): 0.90 (d, 6H, Val), 1.49 (s, 27H, tBu), 18542.20 5H, Ph), 7.5 (d, 1H, NH), 7.9 (d, 1H, NH), 9.05 (s, 1H, NH). 35 (m, 3H, Val and Glu), 2.21 (m, 2H, Glu), 24042.70 (m, 2H, CbZ-Leu-Leu-NHiNH2, White solid, yield 98%. Asp CH2), 3.30 and 3.38 (m, 3H, NHCH2 and NHCH2), Cbz-Val-NHiNHz, White solid, yield 92%. 4.054430 (m, 3H, ot-H), 5.05 (m, 2H, CbZ), 7.2(%7.40 (m, CbZ-Gl11(O-IB11)-V2ll-NHiNH2 Was puri?ed by chroma 5H, Ph), 7.604795 (m, 3H, NH), 9.2 (m, 1H, NH). tography on a silica gel column using 1:9 MeOH:CH2Cl2 as CbZ-Glu(O-tBu)-Val-NHiNH4CH2COO-tBu Was the eluent; White solid, yield 47453%. 40 puri?ed by column chromatography on silica gel using CbZ-lle-Glu(O-tBu)-Thr-NHiNH2, White solid, yield 2:18:5 MeOH:CH2Cl2:EtOAc as the eluent; White solid, 91%. yield 78%. MS (ESI) m/Z 565.3 [(M+1)+]. 1H NMR 3. Preparation of CbZ-Ala-Ala-NHiNHiCHZCOOEt (CDCl3): 0.95 (t, 6H, Val), 1.49 (s, 18H, tBu), 18542.20 (m, (4a). Ethyl bromoacetate (1.1 eq) Was added dropWise to a 3H, Val and Glu), 2.21 (m, 2H, Glu), 34543.70 (m, 3H, stirred solution of CbZ-Ala-Ala-NHiNH2 (1 eq) and NMM 45 NHCH2 and NHCH2), 4.254430 (m, 2H, ot-H), 5.05 (m, 2H, (1.1 eq) in DMF that Was cooled to —100 C. The resulting CbZ), 5.85 (d, 1H, NH), 7.05 (d, 1H, NH), 72047.40 (m, 5H, solution Was stirred for 30 min at —100 C., after Which the Ph), 8.00 (m, 1H, NH). mixture Was alloWed to react at room temperature for 36 CbZ-Leu-Glu(O-tBu)-Thr-NHiNH4CH2COO-tBu Was hours. The DMF Was evaporated, and the residue Was puri?ed by column chromatography on silica gel using 1:9 puri?ed on a silica gel column using 1:9 MeOH:CH2Cl2 as 50 MeOH:CH2Cl2 as the eluent; White solid, yield 34%. MS the eluting solvent system to a?‘ord 4a as a White solid (ESI) m/Z 680 [(M+1)+]. 1H NMR (DMSO-d6): 0.7409 (t, (yield:36%). 1H NMR (DMSO-d6): 1.18 (t, 9H, CH3), 3.5 6H, Leu CH3), 1.0 (d, 3H, Thr CH3), 141.3 (m, 2H, Leu (d, 2H, NCH2COOEt), 4.04.15 (m, 3H, ot-H and CH2), 1341.5 (m, 18H, tBu), 1.5418 (m, 2H, Leu CH and OCH2CH3), 4.2 (m, 1H, ot-H), 5.03 (m, 2H, CbZ), 5.18 (m, Glu CH2), 1.841.95 (m, 1H, Glu CH2), 2.1423 (m, 2H, Glu 1H, NH), 72247.40 (m, 5H, Ph), 7.4475 (d, 1H, NH), 7.9 55 CH2), 3.4 (d, 2H, NCH2), 3.9 (m, 1H, ot-H), 4.1 (m, 1H, ot-H (m, 1H, NH), 9.35 (m, 1H, NH). MS (FAB) m/Z 395 and Thr CHiOH), 4.3 (m, 1H, ot-H), 4.9 (d, 1H, NH), 5.03 [(M+1)+1 (m, 2H, CbZ), 7347.4 (m, 5H, phenyl), 7.5 (d, 1H, NH), 7.6 4. Preparation of N1-N-BenZyloxycarbonylalanylalanyl (d, 1H, NH), 8.05 (d, 1H, NH), 9.2 (d, 1H, NH). N2-carbamoylmethylhydraZine (CbZ-Ala-Ala-NHiNHi CbZ-lle-Glu(O-tBu)-Thr-NHiNHiCH2COO-tBu Was CHZCOiNHZ, 4b). The ethyl ester (4a) Was converted to 60 puri?ed by column chromatography on silica gel using 1:9 the amide (4b) by the method described by Hogberg et al. MeOH:CH2Cl2 as the eluent; White solid, yield 26%. MS The ethyl ester CbZ-Ala-Ala-NHiNH4CH2COOEt (4a, 1 (ESI) m/Z 680 [(M+1)+]. 1H NMR (DMSO-d6): 0.7409 (t, eq) Was dissolved in a 9 M solution of NH3 in methanol and 6H, lle CH3), 0941.0 (d, 3H, Thr CH3), 141.2 (m, 2H, lle a small amount of DMF, and alloWed to stir on an ice bath. CH2), 1341.5 (s, 18H, tBu), 1.6418 (m, 2H, lle CH and Glu To this solution Was added NaCN (0.1 eq). The ?ask Was 65 CH2), 1841.9 (m, 1H, Glu CH2), 2.1423 (m, 2H, Glu CH2), closed With a rubber septum and alloWed to stir at 00 C. for 3.4 (d, 2H, NCH2), 3.9 (m, 2H, ot-H), 4.1 (m, 1H, ot-H), 4.35 three days. The solvent Was evaporated and the product Was (m, 1H, Thr CH4OH), 4.8 (d, 1H, NH), 5.03 (s, 2H, CbZ), US 7,056,947 B2 25 26 5.05 (d, 1H, NH), 7347.4 (m, 5H, phenyl), 7.7 (d, 1H, NH), (150 mL), ?ltered and the solvent evaporated, to give 8.05 (d, 1H, NH), 9.2 (s, 1H, NH). trans-oxirane-2,3-dicarboxylic acid as a colorless semisolid 6. Preparation of CbZ-Leu-Leu-NHiNH4CH2iCHi (yield:97%). (CH3)2i Reductive Amination Procedure. lsobutyralde 10. Preparation of trans-Oxirane-2,3-dicarboxylic Acid hyde (1.1 eq) in THF Was added dropWise to a solution of the MonobenZyl Ester (5, YICOOBZl). The reagent EDC (2.32 peptidyl hydraZide CbZ-Leu-Leu-NHNH2 (1 eq) in THF. g, 11 mM) Was added to a stirred solution of trans-oxirane The reaction mixture Was stirred at room temperature for 16 2,3-dicarboxylic acid (1.32 g, 10 mM), benZyl alcohol (1.08 hours, and monitored by TLC. The solvent Was evaporated, g, 10 mM), and DMAP (122 mg, 1 mM) in DMF (100 mL), and the resulting hydraZone Was puri?ed by column chro Which has been cooled to 00 C. The resulting solution Was matography on silica gel using 1:9 MeOH:CH2Cl2 as the stirred for 15 h at room temperature. After removal of DMF, solvent system. The hydraZone (1 eq) in THF Was reduced the residue Was puri?ed by chromatography on tWo succes by the addition of NaBH4 (5 eq) in a small amount of MeOH, sive columns using 1:9 MeOH:CH2Cl2 as the eluent, fol and the mixture Was monitored by TLC. The solvent Was loWed by column chromatography using 1:4 MeOH:CH2Cl2 removed by evaporation and the residue Was dissolved in as the eluent to give 5 (Y :COOBZl), as a dark yelloW oil CH2Cl2. Successive Washings With saturated NH4Cl (yield:66%). 1H NMR: 3.60 (d, 1H, epoxy), 3.75 (d, 1H, removed the excess NaBH4. The organic layer Was Washed epoxy), 5.18 (s, 2H, PhCHZO), 7.38 (d, 5H, Ph). With saturated NaCl and dried over Mg8O4. The product 11. Preparation of trans-3-Phenethyloxirane-2-carboxylic CbZ-Leu-Leu-NHiNH4CHZiCHi(CH3)2 Was puri?ed Acid (5, Y:CH2CH2Ph). A stirred solution of 3-phenylpro by chromatography on a silica gel column using 1:1 EtOAc: pionaldehyde (6.7 g, 50 mM) and malonic acid (5.2 g, 50 CHZCl2 as the eluent, folloWed by recrystallization from 20 mM) in pyridine (4 mL) Was heated at 1004105° C. for 6 MeOH/CH2Cl2/hexane to give a White solid (yield 78%). hours. The reaction mixture Was poured onto ice/HCl. The MS and 1H NMR (DM8O-d6 or CDCl3) Were consistent solid precipitate Was Washed With Water suf?ciently to give With the proposed structure. 5-phenylpent-2-enoic acid (6.2 g, 35 mM, yield:70%). 7. Preparation of (28,38) and (2R,3R)-Oxirane-2,3-dicar The reagents DCC (33 mM) and DMAP (30 mM) Were boxylic Acid Monoethyl Esters (or Monoethyl Epoxysucci 25 added to a solution of 5-phenylpent-2-enoic acid (5.3 g, 30 nates, 5, YICOOEt). Enantiomerically pure diethyl mM) in EtOH (50 mL) and THE (20 mL), Which Was cooled epoxysuccinate esters (28,38) and (2R,3R) Were synthesiZed to 00 C. The resulting mixture Was stirred for 30 min at 0° from diethyl D-(—) and L-(+)-tartrate, respectively, folloW C., then alloWed to react for 15 h at room temperature. The ing the general method developed by Mori and lWasaWa. suspension Was ?ltered, and the ?ltrate Was concentrated. This procedure involved three steps including bromination, 30 The residue Was dissolved in EtOAc and Washed With 1 M elimination, and cycliZations. The selective hydrolysis of HCl (3><30 mL), H20 (10 mL), saturated NaHCO3 (3x30 one ester to yield monoethyl epoxysuccinates Was per mL), H20 (10 mL), saturated NaCl (20 mL), dried over formed by a method similar to that described previously by Mg8O4, and concentrated. Chromatography on a silica gel Rich and 8chaschke. column using 1:2 EtOAc:hexane as the eluent afforded 8. Preparation of trans-Oxirane-2,3-dicarboxylic Acid 35 5-phenyl-pent-2-enoic acid ethyl ester (3.75 g, yield:61%). Diethyl Ester (or Diethyl Epoxysuccinate). The trans-ox This compound Was epoxidiZed using the procedure irane-2,3-dicarboxylic acid diethyl ester Was synthesiZed described above in the synthesis of trans-oxirane-2,3-dicar using a general procedure for the stereocontrolled epoxida boxylic acid diethyl ester and puri?ed on a silica gel column tion of 0t,[3-unsaturated carbonyl compounds, Which Was With 1:1 EtOAc:hexanes as the eluent to afford trans-3 similar to the method previously described by Meth-Cohn. 40 phenethyloxirane-2-carboxylic acid ethyl ester as a light An anhydrous solution of t-butyl hydroperoxide in toluene yelloW oil (yield:46%). 1H NMR: 1.25 (t, 3H, Oet); 1.90 (t, (3.3 M solution, 46 mL, 1.5 eq), Was added to freshly 2H, PhCH2CH2), 2.80 (t, 2H, PhCH2), 3.2 (s, 2H, epoxy), distilled THE (240 mL) at —78° C. under argon. This Was 4.2 (q, 2H, Oet), 7.3 (m, 5Hb, Ph). folloWed by the addition of a solution of butyl lithium in A solution of 1 M NaOH (1.1 eq, 5.5 mL) Was added to pentane (1.7 M solution, 65 mL, 1.1 eq). The mixture Was 45 a stirred solution of trans-3-phenethyloxirane-2-carboxylic stirred at —78° C. for 5 min, then a solution of diethyl acid ethyl ester (1.1 g, 5 mM) in MeOH (10 mL) at room fumarate (17.2 g, 0.1 M, 1 eq) in THF (50 mL) Was added. temperature. After stirring for 1 h at room temperature, the The reaction mixture Was stirred at room temperature for 2 solution Was acidi?ed to pH 3, and extracted With EtOAc hours (monitored by TLC). 8odium sul?te (10 g) Was added (2x30 mL). The organic layer Was Washed With Water (2x30 and the mixture Was stirred for 20 min. The mixture Was 50 mL), saturated NaCl (20 mL), dried over Mg8O4, and concentrated to ca. 100 mL, diluted With ether (100 mL), concentrated to give trans-3-phenylethyloxirane-2-carboxy ?ltered through yiatomaceous earth (celite), and evaporated. lic acid (5, YICHZCHZPh) as a light yelloW oil (yield:72%). To the residue Was added 1 M HCl (100 mL). The product 1H NMR: 1.90 (t, 2H, PhCH2CH2), 2.80 (t, 2H, PhCH2), trans-oxirane-2,3-dicarboxylic acid diethyl ester Was 3.30 (s, 2H, epoxy), 7.30 (m, 5H, Ph). HRM8 (FAB) Calcd. extracted With EtOAc (3x100 mL), and the organic layer 55 For Cl2Hl2O5: 237. Observed m/Z 236.9. Was Washed With saturated NaCl (3x50 mL), dried over 12. General Procedure for Coupling Oxirane Dicarboxylic Mg8O4, and the solvent evaporated. Chromatography on a Acid Monoethyl Esters to Amines. The procedure used to silica gel column With 2:3 EtOAc:hexane afforded the synthesiZe amide derivatives of epoxysuccinate monoethyl product trans-oxirane-2,3-dicarboxylic acid diethyl ester esters Was similar to that of Therrien et al (Biochemistry (yield:52%). 60 2001 40 p 2702). To a solution of epoxysuccinate monoethyl 9. Preparation of trans-Oxirane-2,3-dicarboxylic Acid. A ester (1 g, 6.25 mM), amine/amino acid (1.2 eq) and HOBt solution of 1 M NaOH (98 mL, 1.9 eq) Was added to (1 eq) in CHCl3 (30 mL) at 0° C. Was added EDC (1.1 eq) trans-oxirane-2,3-dicarboxylic acid diethyl ester (9.8 g, 52 sloWly in 5 portions. The reaction Was stirred for one hour mM) in MeOH (30 mL) at 0° C. The resulting solution Was at 0° C. and then subsequently at room temperature for 16 stirred for 1 h at 0° C., then for 30 min at room temperature, 65 hours. The solvent Was evaporated and the residue Was after Which the solution Was acidi?ed to pH 3. Water and partitioned betWeen EtOAc (50 mL) and dH2O (5 mL). The MeOH Were evaporated. The residue Was treated With EtOH organic layer Was Washed With 0.5 M HCl (2x10 mL), sat US 7,056,947 B2 27 28 NaHCO3 (2x50 mL), dried over MgSO4 and concentrated. 1)’']. 1H NMR (CDCl3): 1241.4 (m, 3H, OCH2CH3), In general, the oxirane-2,3-dicarboxylic acid monoamides 2.4426 (m, 6H, NCH2 morph), 3.2434 (m, 2H, CON Were obtained in 43474% yield. The crude product Was HCH2), 3.45 and 3.65 (m, epoxy), 3.6438 (m, 4H, OCH2 puri?ed by chromatography on a silica gel column using 1:1 morph), 4.25 (m, 2H, OCH2CH3), 7.85 (m, 1H, NH). EtOAc:hexanes as the eluent to yield a White solid. Hydroly 19. (2S,3S)-Oxirane-2,3-dicarboxylic Acid MonobenZyl sis of the ester by KOH (1.2 eq) gave the desired amides Ester (HOOC-EP-COOCHZPh). 1H NMR (CDCl3): 3.70 (d, (yields:65495%). 2H, epoxy), 5.22 (d, 2H, CH2Ph), 7.35 (m, 5H, Ph). 13. (2S,3S)-3-(Dibutylcarbamoyl)oxirane-2-carboxylic 20. (2R,3R)-Oxirane-2,3-dicarboxylic Acid MonobenZyl Acid (HOOC-EP-CON(CH2CH2CH2CH3)2). This com Ester (HOOC-EP-COOCHZPh). 1H NMR (CDCl3): 3.65 (d, pound Was synthesiZed using the above procedure With 2H, epoxy), 5.17 (d, 2H, CH2Ph), 7.32 (m, 5H, Ph). dibutylamine as the starting material. 1H NMR (CDCl3): 21. (2S,3S)-Oxirane-2,3-dicarboxylic Acid Monophen 0.93 (t, 6H, CH3), 1.32 (m, 4H, CH2), 1.54 (m, 4H, CH2), ethyl Ester (HOOC-EP-COOCHZCHZPh). This epoxide Was 3.35 (m, 4H, CH2), 3.67 and 3.88 (d, 2H, epoxy). synthesiZed using the above procedure for trans-oxirane-2, 14. (2S,3S)-3-(N-Methyl-N-benZylcarbamoyl)oxirane-2 3-dicarboxylic acid monobenZyl ester by using (2S,3S) carboxylic Acid (HOOC-EP-CON(CH3)CH2Ph). This com oxirane-2,3-dicarboxylic acid and phenethyl alcohol as the pound Was synthesiZed using the above procedure With starting materials. 1H NMR (CDCl3): 2.98 (t, 2H, CH2), 3.65 NH(CH3)CH2Ph as the starting material. 1H NMR (CDCl3): (d, 2H, epoxy), 4.41 (m, 2H, OCHZ), 7.25 (m, 5H, Ph). 2.94 and 3.02 (d, 3H, CH3N), 3.72 and 3.96 (d, 2H, epoxy), 22. (2R,3R)-3-(2—Phenethylcarbamoyl)oxirane-2-car 4.78 (d, 2H, CH2Ph), 7.184732 (m, 5H, Ph). boxylic Acid (HOOC-(2R,3R)-EP-CONHCH2CH2Ph). 1H 15. (2S,3 S) -3 -(DibenZylcarbamoyl)oxirane-2 -carboxylic 20 NMR (CDCl3): 2.82 (t, 2H, CH2Ph), 3.35 and 3.69 (d, 2H, Acid (HOOC-EP-CON(CH2Ph)2). This compound Was syn epoxy), 3.54 (m, 2H, NCH2), 6.20 (b, 1H, NH), 7.154733 thesiZed using the above procedure With dibenZylamine as (m, 5H, Ph). the starting material. 1H NMR (CDCl3): 3.77 and 3.93 (d, 23. (2S,3S)-3-(3-BenZylcarbamoyl)oxirane-2-carboxylic 2H, epoxy), 4.58 (m, 4H, CHZPh), 7.144736 (m, 10H, Ph). Acid (HOOC-(2S,3S)-EP-CONHCH2Ph). This compound 16. Preparation of trans-3-(4-Chlorophenyl)oxirane-2 25 Was synthesiZed using PhCHZNH2 as the starting material. carboxylic Acid (HOOC-EP-Ph-4-Cl). The starting material, 1H NMR (CDCl3): 3.53 and 3.78 (d, 2H, epoxy), 4.45 (d, 4-chloro-trans-cinnamic acid (1 eq), Was cooled to —200 C. 2H, CH2Ph), 6.40 (b, 1H, NH), 7.254733 (m, 5H, Ph). in dry methanol. Thionyl chloride (3 eq) Was added drop 24. (2R,3R)-3-(3-BenZylcarbamoyl)oxirane-2-carboxylic Wise to the cooled solution over one hour. The mixture Was Acid (HOOC-(2R,3R)-EP-CONHCH2Ph). This compound stirred at —150 C. for an additional 30 minutes and subse 30 Was synthesiZed using PhCH2NH2 as the starting material. quently stirred at room temperature for 24 hours. Evapora 1H NMR (DMSO-d6): 3.50 and 3.60 (d, 2H, epoxy), 4.29 (d, tion of the volatiles yielded the methyl ester MeOOCi 2H, CH2Ph), 7.234733 (m, 5H, Ph), 8.89 (b, 1H, NH). CH=CH-Ph-4-Cl as a White solid (yield:89%). Without 25. (2S,3S)-3-(3-Ethylcarbamoyl)oxirane-2-carboxylic further puri?cation the methyl ester Was epoxidiZed. An Acid (HOOC-(2S,3S)-EP-CONHCH2CH3). This compound anhydrous solution of t-butyl hydroperoxide in toluene (3.3 35 Was synthesiZed using CH3CH2NH2 as the starting material. M, 1.5 eq) Was added to freshly distilled THF at —780 C. 1H NMR (CDCl3): 1.16 (t, 3H, CH3), 3.29 (q, 2H, CH2), under argon. A solution of butyllithium in pentane (1.7 M, 3.46 and 3.65 (d, 2H, epoxy), 6.10 (b, 1H, NH). 1.1 eq) Was added dropWise. The mixture Was stirred at —780 26. (2S,3S)-3-(2-Hydroxy-2-phenylethylcarbamoyl)ox C. for 5 minutes and then a solution of MeOOC4CH=CH irane-2-carboxylic Acid (PhCH(OH)CH2NHCO-(2S,3S) Ph-Cl (1 eq) in toluene Was added dropWise. The reaction 40 EP-COOH). This compound Was synthesiZed using PhCH Was stirred at —780 C. for 30 minutes then at room tempera (OH)CH2NH2 as the starting material. 1H NMR (CDCl3): ture for 16 hours. Solid sodium sul?te Was added and the 3.35 (m, 1H, NCH2), 3.54 (m, 2H, epoxy and NCH2), 3.63 mixture Was alloWed to stir for 20 minutes, then diluted With (s, 1H, epoxy), 4.80 (m, 1H, OCHZ), 7.20 (t, 1H, NH), ether and ?ltered over celite. The solvent Was evaporated, 73347.41 (m, 5H, Ph and NH). and the crude product Was puri?ed by column chromatog 45 27. (2R,3R)-3-(2-Hydroxy-2-phenylethylcarbamoyl)ox raphy on silica gel using 3:1 hexane/EtOAc as the eluent. 1H irane-2-carboxylic Acid (PhCH(OH)CH2NHCO-(2R,3R) NMR (CDCl3): 3.47 (d, 1H, epoxy), 3.83 (s, 3H, CH3), 4.08 EP-COOH). This compound Was synthesiZed using PhCH (d, 1H, epoxy), 7.21 (d, 2H, Ph), 7.33 (d, 2H, Ph). Standard (OH)CH2NH2 as the starting material. 1H NMR (CDCl3): deblocking of the methyl ester With sodium hydroxide (1 N) 3.37 (m, 1H, NCH2), 3.54 (m, 2H, epoxy and NCH2), 3.63 folloWed by acidic Workup (1 N HCl) produced the 3-(4 50 (s, 1H, epoxy), 4.80 (m, 1H, NCH2), 7.20 (t, 1H, NH), chlorophenyl) glycidic acid. 1H NMR (DMSO-d6): 3.65 (s, 73347.41 (m, 5H, Ph and NH). 1H, epoxy), 4.14 (s, 1H, epoxy), 7.37 (d, 2H, Ph), 7.43 (d, 28. (2S,3 S) -3 -(1—Benzylcarbamoylethylcarbamoyl)ox 2H, Ph). irane-2-carboxylic Acid (HOOC-(2S,3S)-EP-CO-Ala-NH 17. (2S,3S)-3-(3-Methoxypropylcarbamoyl)oxirane-2 BZl). This compound Was synthesiZed using NH2CH(CH3) carboxylic Acid Ethyl Ester (EtOOC-EP 55 CONHBZl as the starting material. 1H NMR (DMSO-d6): CONHCH2CH2CH2OCH3) Was synthesiZed using the 1.26 (d, 3H, Ala), 3.45 and 3.65 (d, 2H, epoxy), 4.26 (d, 2H, epoxide and amine coupling method With methoxypropy CH2Ph), 4.33 (m, 1H, ot-H), 7.19 and 7.29 (m, 5H, Ph), 8.51 lamine as the starting material. The crude product Was (t, 1H, NH), 8.67 (d, 1H, NH). puri?ed by column chromatography With 1:5 MeOH:CH2Cl2 29. (2R,3R)-3-(1—BenZylcarbamoylethylcarbamoyl)ox as the eluent. MS (ESl) m/Z 232 [(M+1)+]. 60 irane-2-carboxylic Acid (HOOC-(2R,3R)-EP-CO-Ala-NH 18. (2S,3S)-3-(3-(4-Morpholinyl)propylcarbamoyl)ox BZl). This compound Was synthesiZed using NH2CH(CH3) irane-2-carboxylic Acid Ethyl Ester (EtOOC-EP CONHBZl as the starting material. 1H NMR (DMSO-d6): CONHCH2CH2CH2morpholinyl) Was synthesiZed using the 1.25 (d, 3H, Ala), 3.49 and 3.65 (d, 2H, epoxy), 4.26 (d, 2H, epoxide and amine coupling method With 4-(3-aminopropyl) CH2Ph), 4.33 (m, 1H, ot-H), 7.19 and 7.29 (m, 5H, Ph), 8.51 morpholine as the starting material. The crude product Was 65 (t, 1H, NH), 8.67 (d, 1H, NH). puri?ed by column chromatography using 1:5 MeOH: 30. (2S,3S)-3-(1-Carbamoyl-3—methylbutylcarbamoyl) CHZCl2 as the eluent (7% yield). MS (ESl) m/Z 287 [(M+ oxirane-2-carboxylic Acid (HOOC-(2S,3S)-EP-CO-Leu US 7,056,947 B2 29 30 NH2). This compound Was synthesized using NHZCH Which renders it resistant to autolytic inactivation, but has no (CH2CH(CH3)2)CONH2 as the starting material. 1H NMR detectable affect on enzyme activity as compared to the (DMSO-d6): 0.84 (d, 6H, CH3), 1.46 (m, 2H, CH2), 1.55 (m, naturally occurring enzyme. The enzyme variant Was 1H, CH), 3.48 and 3.64 (d, 2H, epoxy), 4.20 (m, 1H, ot-H), expressed in E. coli, puri?ed ?rst by immobilized metal 7.00 (s, 1H, NH), 7.45 (s, 1H, NH), 8.50 (d, 1H, NH). chromatography via the N-terminal N-His tag, treated With 31 . (2R,3R) -3 -(1-Carbamoyl-3 -methylbutylcarbamoyl) excess oxidized glutathione to stabilize the reactive thiolate, oxirane-2-carboxylic Acid (HOOC-(2R,3R)-EP-CO-Leu and then re-puri?ed by size-exclusion chromatography. NH2). This compound Was synthesized using NHZCH Inhibition data Was measured using the progress curve (CH2CH(CH3)2)CONH2 as the starting material. 1H NMR assay method. Serial dilutions of each compound Were (DMSO-d6): 0.85 (d, 6H, CH3), 1.46 (m, 2H, CH2), 1.56 (m, prepared using an initial 8-fold dilution of a DMSO stock 1H, CH), 3.48 and 3.64 (d, 2H, epoxy), 4.20 (m, 1H, ot-H), into HGE (100 mM HEPES,20% glycerol v/v, 0.5 mM 7.00 (s, 1H, NH), 7.45 (s, 1H, NH), 8.50 (d, 1H, NH). EDTA), folloWed by seven serial tWo-fold dilutions into 32. (2S,3 S)-3-(1-Carbamoyl-2-phenylethylcarbamoyl) HGE and 12.5% DMSO, thus maintaining constant DMSO oxirane-2-carboxylic Acid (HOOC-(2S,3S)-EP-CO-Phe through the dilution series. Ten pL of diluted stocks or of NH2). This compound Was synthesized using NHZCH vehicle (HGE and 12.5% DMSO) Were placed in triplicate (CH2Ph)CONH2 as the starting material. 1H NMR (DMSO onto a 96-Well microtiter plate, alloWing several compounds d6): 2.78 (m, 1H, CH2Ph), 3.02 (m, 1H, CH2Ph), 3.25 and to be tested on each plate. The plate Was covered to 3.56 (d, 2H, epoxy), 4.45 (m, 1H, ot-H), 7.134725 (m, 6H, minimize evaporation, and the plate Was pre-Warmed to 300 Ph and NH), 7.57 (s, 1H, NH), 8.52 (d, 1H, NH). C. for 20 minutes. Enzyme Was diluted into 10.0 mL of assay 33. (2R,3R)-3-(1-Carbamoyl-2-phenylethylcarbamoyl) 20 buffer (HGE, 5 mM DTT, plus 15 uM Ac-YVAD-AMC, 2 oxirane-2-carboxylic Acid (HOOC-(2R,3R)-EP-CO-Phe nM approximate ?nal enzyme concentration), and this acti NH2). This compound Was synthesized using NHZCH vated reaction mixture Was added to the plate at 90 uL/Well. (CH2Ph)CONH2 as the starting material. 1H NMR (DMSO Progress of substrate hydrolysis Was monitored for 900 s in d6): 2.78 (m, 1H, CH2Ph), 3.02 (m, 1H, CH2Ph), 3.32 and a LabSystems (Needham, Mass.) Fluoroskan Ascent ?uo 3'.59 (d, 2H, epoxy), 4.42 (m, 1H, ot-H), 7.134 7.25 (m, 6H, 25 rescent plate-reader using 385 and 460 nm excitation and Ph and NH), 7.57 (s, 1H, NH), 8.60 (d, 1H, NH). emission ?lters, respectively, and a photomultiplier gain 33 . (2S,3S)-3 -(1-Carbamoyl-2(4-hydroxyphenyl)ethyl setting of 10. Triplicate curves Were averaged and ?t by carbamoyl)oxirane-2-carboxylic Acid (HOOC-(2S,3S)-EP nonlinear regression to the equation for irreversible inacti CO-Tyr-NHZ). This compound Was synthesized using vation shoWn beloW. NH2CH(CH2Ph-4-OH)CONH2 as the starting material. 1H 30 NMR (CD3COCD3): 2.85 (m, 1H, CH2Ph), 3.10 (m, 1H,

CHZPh), 3.40 and 3.59 (d, 2H, epoxy), 4.60 (m, 1H, ot-H), F = F 6.50 (b, 1H, OH), 6.74 and 7.07(d of d, 4H, Ph), 7.15 (s, 1H, (I) 0 + kobs NH), 7.35 (d, 1H, NH). 35 UTILITY OF THE PRESENT INVENTION Where F0 Was routinely ?xed to zero, since ?uorescence values Were alWays adjusted to an origin of 0. The second Peptide aza-peptide epoxides are irreversible inhibitors order rate constant k0” (M_ls_l) Was obtained from the for cysteine proteases. Peptide aza-peptide epoxides con slopes by linear regression, and errors represent the standard taining hydrophobic aza-amino acid residues in the P1 40 deviation of the regression slope. and/or P2 site have been found to be excellent inhibitors of Caspase-3, -6, and -8. Caspase-3, -6 and -8 Were cysteine proteases including cathepsin B and papain. We expressed in E. coli and puri?ed according to methods shoW that peptide aza-peptide epoxides containing aza previously described by Stennicke and Salvesen. Assays amino acid residues With anionic side chains in the P1 site using the ?uorogenic substrate Z-DEVD-AFC (kex:400 nm, are excellent inhibitors of caspases. Legumain is inhibited 45 KEMISOS nm) Were carried out on a Molecular Devices fMax by aza-peptide expoxides With a P1 aza-asparagine residue. ?uorescence microplate reader. Inhibition rates and equilib Clostripain and gingipain are inhibited by aza-peptide ria Were determined by the progress curve method. The epoxides With P1 basic side chains. Peptide aza-peptide standard 100 pL reaction Was started by adding enzyme to epoxides containing aza-amino acid residues With hydro a mixture of substrate (?nal concentration of Z-DEVD-AFC phobic side chains at the P2 site have also been found to be 50 100 uM) and various amounts of inhibitor in buffer (50 mM excellent inhibitors of several cysteine proteases including Hepes, 100 mM NaCl, 0.1% (W/v) CHAPS, sucrose 10% papain, cathepsin B, calpain I, and calpain II. These struc (W/v), and 10 mM DTT, at pH 7.4). The caspases Were tures may be used in vivo to treat diseases such as cancer and pre-activated for 10 min at 370 C. in the presence of 10 mM neurodegenerative diseases Which result from the uncon DTT. trolled proteolysis by cathepsin B, calpain, caspases, and 55 Legumain Assays With legumain Were performed as fol related cysteine proteases. These inhibitors may be used in loWs. A ?uorometric assay for legumain has been described vitro to prevent proteolysis Which occurs in the process of previously. Legumain, puri?ed from pig kidney tissue, Was production, isolation, puri?cation, storage, or transport of assayed at 300 C. in buffer (39.5 mM citric acid, 121 mM peptides and proteins. These inhibitors may be useful as NaZHPO4 at pH 5.8 containing 1 mM EDTA, 1 mM TCEP, therapeutic agents for treatment of neurodegeneration, viral 60 and 0.01% CHAPS) With Cbz-Ala-Ala-Asn-AMC as the infections, muscular dystrophy, myocardial tissue damage, substrate (10 uM ?nal concentration). The assays Were tumor metastasis, and bone resorption. carried out in a Perkin Elmer LS 3B ?uorescence spectrom eter (kex:360 nm, kem:460 nm) under the control of an 1. Enzyme Assays IBM-compatible computer running the FLUSYS softWare. Caspase-1. The preparation of the autolytically stable 65 The rate of substrate hydrolysis in the absence of inhibitor caspase-1 variant used in these studies has been described Was recorded, after Which the inhibitor Was added in a previously. Brie?y, the variant contains a mutation (D381E) negligible volume and the neW rate Was monitored. Rate US 7,056,947 B2 31 32 constants for irreversible inactivation Were found by non and added to 200 ML of a 0.1 M potassium phosphate buffer linear regression analysis of the pseudo ?rst-order curves containing 1.25 mM EDTA, 0.01% Brij 35 at pH 6.0, and the using the FLUSYS software, giving kobs. The second-order substrate Z-Arg-Arg-AMC (499 nM). The release of rate constant k2 Was calculated as kolnj [I], at [S] <10_3 the inhibition of papain, cathepsin B, and calpain by aza ug/uL) in the same potassium phosphate buffer containing 1 peptide epoxides. The inhibition constants are second-order mM DTT (freshly prepared). Aliquots (50 pL) from the rate constants and the inhibitors With the higher numbers are inhibition mixture Were WithdraWn at various time intervals more potent.

TABLE I

Inhibition of Papain Cathepsin B and Calpain by Aza-peptide Epoxides.

Inhibitor papain cathepsin B calpain Boc-Nva-AHph-trans-EP-COOEt 1.4 2.7 Boc-Nle-AHph-trans—EP—COOEt 1.6 1.3 Boc-Abu-AHph-trans—EP—CH2CH2Ph 0.2 NI Boc-Nle-AHph-trans—EP—CH2CH2Ph 0.5 NI Cbz-APhe-trans-EP-COOEt 8.5 1.8 Cbz-APhe-trans-EP-CH2CH2Ph 0.92 Cbz-ALeu-trans-EP-COOEt 16.1 4.43 1.75 Cbz-AHph-trans-EP-COOEt 18. 8 8.91 2.44 Ac-AHph-trans-EP-COOEt 26. 88 40.62 1.29 Cbz-Leu-ALeu-(ZS, 3S)-EP-COOEt 10.53 1.8 8.58 Cbz-Leu-ALeu-(ZR, 3R)-EP-COOEt 6.70 NI 1.45 Cbz-Leu-ALeu-trans-EP-COOEt 6.37 23 .32 3.09 Cbz-Leu-ALeu-cis-EP-COOEt 0.38 6.27 1.88 Cbz-Phe-ALeu-trans-EP-COOEt 0.91 3 .61 Cbz-Phe—ALeu—tra.ns—EP—CH2CH2Ph 0.7 NI Cbz-Phe-APhe-trans-EP-CH2CH2Ph 0.5 NI

US 7,056,947 B2 37 38 the interactions that are found in complexes of a particular TABLE VI enZyme With its substrates and/or inhibitors. Additional interactions With the enZyme can be obtained by tailoring the Inhibition of Legnnain by AZa-peptide Epoxides. R3 group of the inhibitor to imitate the amino acid residues Which are preferred by an individual protease at the S1‘ and S2 subsites. For example, aZa-peptide epoxides With R3 phenylalkyl groups Would interact effectively With caspase O O HLNHZ l, Which is shoWn to prefer such structures in alpha )L s N O R 3 o N N/ ketoamide peptide inhibitors. Likewise, the M1 group can H H interact With the S subsites of the target cysteine protease. Once a good inhibitor structure for a particular enzyme is found, it is then possible to change other characteristics such Schistosorne Legurnain Stereochern- IC5O as solubility or hydrophobicity by adding substituents to the inhibitor R3 istry (nM) M1 or R1 and R2 groups. The folloWing structures are inhibitors for the listed 12a COOEt 8,8 53 z 25 enZymes. The inhibitor sequences Were obtained from pep COOEt R,R 788 z 88 COOEt trans tide substrate and/or inhibitor sequences in the protease COOEt cis literature. 12b COOCH2Ph 8,8 47 z 33 COOCH2Ph trans 20 12c COOCH2CH2Ph 8,8 45 12d CONHCH2Ph S,S NI CliC6H4CH2OCO-Phe-AGly-EP-COOH for papain 12e CONHCH2CH2Ph S,S NI C6H5CH2NHCO—Gly—Phe—AGly—EP—COOH for cathepsin 12f CO-Ala-NH-Bzl R,R NI B 12g CON(nBu)2 8,8 68 z 4 Morpholine-CO-2-Naphthyl-AHph-EP-COOEt for cathepsin 12h CON(CH3)CH2Ph 8,8 63 z 11 25 S 12i CH2CH2Ph trans 70 z 14 2-Naphthyl—SO2—Ile—ATrp—EP—COOH for cathepsin 12j Ph-4-Cl trans 90 z 0 B l—Naphthyl—SO2—Val—ATrp—EP—COOH for cathepsin NI = no inhibition. B and L Pro-Phe-AArg-EP-COOH for cathepsin Legumain is inhibited by a variety of AAsn derivatives B and L Which are either not inhibitors for-caspases or very poor. CbZ—Ph6—L61l—L61l—AM6t(O2)—EP—COOH for cathepsin Both Schistosome and pig kidney legumain are inhibited. K Overall, the aZa-peptide epoxide inhibitors have Worked Ph—CH2iSO2—AAsp—EP—COOCH2Ph for caspase-l Ph—CH2CHFCO—Val—Ala—AAsp—EP—COOCH2Ph for caspase-l With every cysteine protease We have tested. The rates are 4-Cl-PhCHZCHZCO-Val-Ala-AAsp—EP—COOCH2Ph for caspase-l higher With cysteine proteases that belong to clan CD 4-NO2—PhCH2CH2CO-Val-Ala-AAsp—EP—COOCH2Ph for caspase-l compared to clan CA. 35 4-CH3O—PhCH2CH2CO—Val-Ala-AAsp—EP—COOCH2Ph for caspase-l 3—F—PhCH2CH2CO-Val-Ala—AAsp—EP-COOCH2Ph for caspase-l 3. Inhibition Mechanism 3,4-dichloro—PhCHZCHZCO-Val-Ala—AAsp—EP-COOCH2Ph for caspase-l The active site of cysteine proteases contains a cysteine Naphthyl—CH2OCO—Val—Ala-AAsp-EP—COOCH2Ph for caspase-l and a histidine residue. The proposed mechanism involves 4-CF3-PhCH2CH2CO—Val-Ala-AAsp-EP—COOCH2Ph for caspase-l 4-CH3—PhCHZCHZCO-Val-Ala—AAsp—EP—COOCH2Ph for caspase-l the attack of the active site cysteine residue on the epoxide PhCH2NHCO—Val—Ala—AAsp—EP—COOCH2Ph for caspase-l functional group to form a covalent adduct. An example of 4—HO—PhCH2CHZCO-Val-Ala-AAsp—EP—COOCH2Ph for caspase-l a caspase inhibitor is shoWn in the following ?gure. The 4-Cl-Ph-CH2OCO-Leu-Glu-Thr-AAsp-EP-COOEt for caspase-8 enZyme recognizes the Pl AAsp residue and inhibition 4-Cl-Ph-CH2OCO-Ile-Glu-Thr-AAsp-EP-COOEt for caspase-8 4-Cl-Ph-CH2OCO-Asp-Glu-Val-AAsp-EP-COOEt for caspase-3 occurs. Additional interactions Would occur betWeen the C5H9-OCO-Asp—Glu—Val—AAsp—EP-COOEt for caspase-3 extended substrate of the cysteine protease and 3-F-Ph—CH2OCO—Ala-Ala-AAsn-EP-COOEt for legurnain the inhibitor Which Would increase the binding affinity and 4-PhO—PhCH2OCO—Ala—Ala-AAsn-EP-COOEt for legurnain speci?city of the inhibitors. 3-F-Ph-CH2OCO—Leu—Glu—Thr—AAsp—EP—COOEt for caspase-6 PhCH2CH2CO-Val-Ala-AAsp-EP-COO(CH2)2Ph—4—Cl for caspase-l CbZ-Leu-Glu-Thr-AAsp—EP-COO(CH2)2Ph—4—CH3 for caspase-8 CbZ-Leu-Glu-Thr-AAsp-EP—COOCH2C6H1 1 for caspase-8 COOH CbZ-Asp-Glu-Val-AAsp-EP—COO(CH2)2C6H4—p—OCH3 for caspase-3 CbZ-Ala-Ala-AAsn-EP-COO(CH2)2—2—naphthyl for legurnain o f PhCH2CH2CO—Val—Ala—AAsp—EP—(CH2)2Ph—3—F for caspase-l N O PhCH2CHZCO-Val-Ala-AAsp-EP-COO(CH2)2—2—naphthyl for caspase-l R/ z N/ o CbZ-Leu-Glu-Thr-AAsp—EP—COO(CH2)2C6H4—p—NO2 for caspase-6 H l CbZ-Ala-Ala-AAsn-EP-COO(CH2)2C6H4—p—CN for legurnain PhCH2CHZCO-Val-Ala-AAsp-EP-COO(CH2)2C6H4—rn—OPh for caspase-l N ]\ 2,4-dinitrophenyl-Ahx-Gly-Phe-AAla-EP-COOH for cathepsin L CbZ—Leu—ALys—EP—COOEt gingipain CbZ-Leu-AOrn-EP-COOEt gingipain COOH CbZ—Leu—ALys—EP—COOEt clostripain CbZ-Leu-AOrn-EP-COOEt clostripain CbZ—Lys(Biotinyl)—Val—Ala—AAsp-EP—COOEt caspase-l

2 4. In Vitro Uses Cys — EnZ To use the above inhibitors in vitro, they are dissolved in 65 an organic solvent such as dimethylsulfoxide or ethanol, and The peptide and amino acid aZa-peptide epoxide deriva are added to an aqueous solution containing serine and/or tives, as shoWn above, bind to the enZymes using many of cysteine proteases. The ?nal concentration of the organic