Highly Potent Non-peptidic Inhibitors of the HCV NS3/NS4A Serine Protease David Sperandio, a, * Anthony R. Gangloff, a Joane Litvak, a Richard Goldsmith, a,y Jason M. Hataye, a Vivian R. Wang, a Emma J. Shelton, a Kyle Elrod, a James W. Janc, a James M. Clark, a Ken Rice, a,{ Steve Weinheimer, b Kap-Sun Yeung, b Nicholas A. Meanwell, b Dennis Hernandez, b Andrew J. Staab, b Brian L. Venables b and Jeffrey R. Spencer a a Celera, 180 Kimball Way, South San Francisco, CA 94080, USA b Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, PO Box 5100, Wallingford, CT 06492, USA Received 29 May 2002; accepted 29 July 2002 Abstract—Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A ledtotheidentificationofapotentZn 2+ -dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn 2+ conditions (K i =27nM). This compound (46) binds also to NS3/NS4A in a Zn 2+ independent fashion (K i =1 mM).TheSARofthisclassofcompoundsunderZn 2+ conditionsishighlydivergentcomparedtotheSARintheabsenceof Zn 2+ , suggesting two distinct binding modes. # 2002 Elsevier Science Ltd. All rights reserved. The prevalence of chronic Hepatitis C virus (HCV) infections in the US was recently determined to be around 1.8% (2.7 million people), with about 15,000 new cases every year. 1 Worldwide, HCV is estimated to infect 170 million people. 1 Over 85% of all cases become chronic. 2,3 Current therapies of HCV infections with interferons show only modest results and have ser- ious side effects such as depression, fever, and fatigue. Recently, combination treatment with ribavarin, 4 a broad spectrum antiviral and pegylated interferon 5 has proven to be superior to monotherapy. Despite these advances, a broadly effective antiviral therapy for HCV is still elusive. An excellent review by Dymock summarizes emerging therapies for HCV. 6 Significant research efforts are currently directed towards targeting viral enzymes. The HCV RNA gen- ome is translated into a 9.5 kb polyprotein (C-E1-E2- P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which is then processedintotheactiveviralproteins.Hostcellproteases are responsible for cleavages in the C-E1-E2-P7-NS2 region. 7 The NS2-NS3 cleavage is performed by NS2, most likely a Zn 2+ containing metalloprotease. 8 The remaining processings of the NS3-NS4A-NS4B-NS5A- NS5B fragment are all dependent on the NS3/NS4A protease, which makes this chymotrypsin-like serine protease a promising target. 7 Highly potent substrate- based peptidic inhibitors for the HCV NS3/NS4A pro- tease were developed (Ac-Asp-d-Gla-Leu-Ile-Cha-Cys- OH, K i =1.5nM). 9 Non peptidic small molecule inhibi- tors for NS3/NS4A were published by Rational Drug Design Laboratories 10 and Schering-Plough 11 with activities in the low micromolar range. We recently published a new class of highly potent, reversible and selective serine protease inhibitors. 12 The binding of these new inhibitors, which are derived from a bis-benzimidazolemethane fragment, is dependent on the presence of Zn 2+ . X-ray structure determination revealed a Zn 2+ ion at the catalytic site, which is coor- dinated by His 57 and Ser 195 of the enzyme, and by two imidazole-nitrogens of the bidentate ligand bis- benzimidazole. 12 We explored this binding motif for the 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(02)00680-7 Bioorganic & Medicinal Chemistry Letters 12 (2002) 3129–3133 *Corresponding author. Tel.: +1-650-866-6544; fax +1-650-866- 6655; e-mail: david.sperandio@celera.com y Present address: Genentech, MS 18, 1 DNA Way, South San Fran- cisco, CA 94080, USA { Present address: Exelixis, 170 Harbor Way, South San Francisco, CA 94083, USA