Dipeptidyl a-fluorovinyl Michael acceptors: Synthesis and activity against cysteine proteases Koen Steert, a Ibrahim El-Sayed, a Pieter Van der Veken, a Alisa Krishtal, b Christian Van Alsenoy, b Gareth D. Westrop, c Jeremy C. Mottram, d Graham H. Coombs, c Koen Augustyns a and Achiel Haemers a, * a Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, BE-2610 Antwerp, Belgium b Department of Chemistry, University of Antwerp, Universiteitsplein 1, BE-2610 Antwerp, Belgium c Division of Infection & Immunity, Institute of Biomedical and Life Sciences, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, UK d Wellcome Centre for Molecular Parasitology and Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Received 23 August 2007; revised 21 September 2007; accepted 23 September 2007 Available online 23 October 2007 Abstract—The synthesis of novel dipeptidyl a-fluorovinyl sulfones using a Horner–Wadsworth–Emmons approach on N-Boc-L- phenylalaninal is described. Inhibitory assays against a Leishmania mexicana cysteine protease (CPB2.8DCTE) revealed low biolog- ical activity. Relative rates of Michael additions of 2 0 -(phenethyl)thiol with vinyl sulfone and a-fluorovinyl sulfone were determined, and ab initio calculations on several Michael acceptor model structures were performed; both were in agreement with the biological testing results. Ó 2007 Elsevier Ltd. All rights reserved. Cysteine proteases are peptide bond-cleaving enzymes. They make use of a nucleophilic thiol group that attacks the peptide carbonyl group, giving rise to the free amine and a peptidyl thioester—which after hydrolysis affords the carboxylic acid part. They have been divided into several clans, which are subdivided into several families (http://merops.sanger.ac.uk/). Cysteine proteases are very widespread in nature and hold cornerstone posi- tions in the metabolism of both eukaryotic and prokary- otic organisms. Several inhibitors are involved in drug development programmes targeting human proteases for diseases such as osteoporosis, arterial thrombosis, rheumatoid arthritis, tumour invasion and metastasis and Alzheimer’s disease. 1,2 Cysteine proteases are also considered as vital for protozoa such as Plasmodium, Trypanosome and Leishmania and inhibitors of falcipain and cruzain, two major parasitic cysteine proteases, are under investigation as possible treatment of malaria and Chagas’ disease. 3 Most of these inhibitors are derived from the corre- sponding peptide substrate of the target enzyme. They are modified at the P1 position where the amide group is substituted for a so-called warhead: an electrophilic group with high affinity for the active site thiol group. Frequently used warheads include ketones and nitriles affording reversible tight binding inhibitors or epoxides and Michael acceptors capable of reacting covalently with the thiolate affording irreversible inhibitors. 1 One of the most interesting Michael acceptor groups are peptide vinyl sulfones and their analogues such as vinyl sulfonamides and vinyl sulfonate esters. 4 Vinyl sulfones selectively inhibit several cysteine proteases in a low nM range. Compounds such as 1 inhibit falcipains and cruz- ain and are important tools in antiprotozoal drug design and development (Fig. 1). 5–7 Very few Michael acceptor inhibitors with substitutions on the a-vinyl carbon have been reported. An a-acet- oxymethyl group (Fig. 2) affords compounds showing activity in an in vitro malaria model (IC 50 = 10 nM). A double addition–elimination mechanism of action is claimed but detailed kinetic analysis of enzyme inhibi- tion to verify this claim is not reported. 8 0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2007.09.075 Keywords: Cysteine proteases; a-Fluorovinyl Michael acceptors; Inhibitors. * Corresponding author. Tel.: +32 38202717; fax: +32 38202739; e-mail: achiel.haemers@ua.ac.be Available online at www.sciencedirect.com Bioorganic & Medicinal Chemistry Letters 17 (2007) 6563–6566