663
J . Med. Chem. 1994,37, 563-564
PI Aspartate-Based Peptide a-((2,6-Dichlorobenzoy1)oxy)methyl Ketones as Potent Time-Dependent Inhibitors of Interleukin-l@-Converting Enzyme Roland E. Dolle,'*t Denton Hoyer,t C. V. C. Prasad,t Stanley J. Schmidt,? Carla T. Helaszek,* Robert E. Miller,* and Mark A. Ator* Departments of Medicinal Chemistry a n d Enzymology and Receptor Biochemistry, Sterling Winthrop Pharmaceuticals Research Division, 1250 South Collegeuille Road, P.O. Box 5000, Collegeville, Pennsylvania 19426 Received October 12, 1993
Interleukin-10-convertingenzyme (ICE) is the processing protease responsible for the production of the potent inflammatory mediator interleukin-10 (IL-lB).l Inhibitors of this intracellular protease are viewed as potential therapeutic agents for the treatment of a variety of chronic inflammatory disease states.2 The detection and characterization of ICE as the IL-10 processing enzyme was independently reported by research groups at Immunexla4 and Merck.leIf The human enzyme has subsequently been purified from THP-1cells,the cDNA cloned,and the amino acid sequence determined.lcpd*fThe enzyme is a heterodimer consisting of 20- and 10-kD noncovalently bound subunits and has been characterized as a thiol (cysteine) protease.s Interestingly, the primary structure of ICE bears no sequence homology with any known member of the cysteine (or serine) protease ~uperfamily.~ ICE cleaves the biologically inactive human IL-la precursor protein (pro-IL-10) between residues Asp116and Ala117 to release the biologicallyactive mature cytokine.letfJ Peptide-based a-((2,6-dichlorobenzoyl)oxy)methyl ketones6 1-3 which contain a P1 L-aspartic acid residue were synthesized and evaluated as time-dependent inhibitors of the enzyme. The selection of the P2 Val and Ala in inhibitors 2 and 3 as hydrophobic residue replacements for the native P2 His is based on previously reported peptide substrate data.lfV6
... - P4 .P3 .P2 - P1 - P1' - P2' - P3 '- P4' - ..
...-Tyr-Val-His-A~p"~-Ala"~-Pro-Val-Arg-.,.
t
ICE cleavage site
on pro-IL-lp
The synthesis of the requisite peptide a-((arylacy1)oxy)methyl ketones was carried out using methodology described by Krantz6 and is exemplified by the synthesis of inhibitor 1 (Scheme 1). N-(Benzyloxycarbony1)aspartic acid 0-tert-butyl ester 4 (Z-Asp(0tBu)-OH;Bachem) was converted to Z-Asp(OtBu)-CH2Br5 upon (1)activation of the carboxylate as a mixed anhydride (1.4 equiv of N-methylmorpholine, 1.3 equiv of ethyl chloroformate, THF,-15 "C); (2)reaction of the mixed anhydride with diazomethane (2 equiv of CHzN2 in Et20, -15 "C 25 "C); and (3) HBr-catalyzed decomposition of the in situ generated diazo ketone (excess glacial HOAc-45 % HBr (l:l), 0 "C) in 75% yield. Noteworthy is the survival of the tert-butyl (side chain) ester during the acid-catalyzed decomposition of the diazo ketone. Treatment of 5 with
-
t Department of 1 Department of
Medicinal Chemistry. Enzymology and Receptor Biochemistry.
Table 1. Results of ICE and Cathepsin B Assays with Inhibitors 1-3 and 7
inhibitor Z-Asp-CH2DCBC (1) Z-Val-Asp-CH2DCB (2) Z-Val-Ala-AspCH2DCB (3) Z-G~U-CH~DCB (7)
cathepsin selectivity ICEo kobJII1 Bb k04,11 1CE:cathepein (M-18-1) (M-1s-1) B >700:1 7,100 i 200
