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United States Patent (10) Patent N0.: US 7,056,947 B2 Powers Et A]

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United States Patent (10) Patent N0.: US 7,056,947 B2 Powers Et A] 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, proteases, methods for synthesizing the compositions, and 024, ?led on Jul, 5, 2002, methods of using the disclosed protease 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 papain, ?cin, bromelain, cathepsin BACKGROUND OF THE INVENTION B, H, F, K, L, O, S, V, X, calpain, 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 caspases. 25 Caspases and legumain are members of the CD clan of 2. Related Art cysteine proteases, While papain, cathepsins, and calpains 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 enzymes 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 cysteine protease 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 cathepsin B 89,400 plasmin 40 cathepsin H 4,000 elastase cruzain. Other classes of compounds, such as aldehydes, cathepsin L 96,250 mold acid protease cathepsin K 1.8 nM (Ki) LDH nitriles, ot-ketocarbonyl compounds, halomethyl ketones, cathepsin S 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) clostripain cathepsin F caspase 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 hydrolase >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
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