Inhibitors of Hepatitis C Virus NS3 . 4A Protease 1. Non-Charged Tetrapeptide Variants Robert B. Perni,* Shawn D. Britt, John C. Court, Lawrence F. Courtney, David D. Deininger, Luc J. Farmer, Cynthia A. Gates, Scott L. Harbeson, Joseph L. Kim, James A. Landro, Rhonda B. Levin, Yu-Ping Luong, Ethan T. O’Malley, Janos Pitlik, B. Govinda Rao, Wayne C. Schairer, John A. Thomson, Roger D. Tung, John H. Van Drie and Yunyi Wei Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, USA Received 13 June 2003; accepted 18 August 2003 Abstract—Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3 . 4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P 1 and P 2 inhibitor positions is discussed. # 2003 Elsevier Ltd. All rights reserved. HCV infection has reached epidemic proportion world- wide and to date therapy options are limited and clinical results are often unsatisfactory. Prevalence of the dis- ease and currently available treatments have been recently reviewed. 1 Inhibition of the hepatitis C virus NS3 . 4A protease has been an intense area of research since the mid-1990s and numerous groups have reported progress in the field. 24 The shallow, hydrophobic, highly flexible binding pocket of the NS3 . 4A protein represents a formidable challenge to drug design and only recently has a com- pound been reported to enter human testing. 5 Inclusion of terminal charged groups (e.g., carboxylates) on either or both sides of the enzyme active site has proven to provide substantial biochemical potency. Indeed, pro- duct inhibition of the NS3 . 4A proteolytic reaction have formed the basis of many design efforts. 6 However, charged groups often result in compromising cellular penetration and in vivo pharmacokinetics. 7 Reversible covalent binding to the catalytic serine is an alternative to electrostatic binding that has been utilized in other serine protease inhibitor series. 8 A series of peptide aldehydes have recently been reported which exploits this approach, but these compounds require carboxylate residues to attain reasonable potency. 9 Deletion of the acidic residues at P 5 and P 6 results in a dramatic decrease in binding affinity. We herein report a series of potent tetrapeptide inhibitors that do not require car- boxyl groups for binding potency and therefore offer improved potential for cell potency. We and others have recently described the effects of truncating substrates and substrate-derived competitive inhibitors from the optimal recognition length of ten 0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2003.08.050 Bioorganic & Medicinal Chemistry Letters 13 (2003) 4059–4063 *Corresponding author. Tel.: +1-617-444-6237; fax: +1-617-444- 6766; e-mail: robert_perni@vrtx.com