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Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors

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Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 282 Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors BY ANJA JOHANSSON ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2003 Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Medicinal Chemistry presented at Uppsala University in 2003. ABSTRACT Johansson, A. 2003. Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 282. 79 pp. Uppsala. ISBN 91-554- 5538-7. Hepatitis C Virus (HCV) is the leading cause of chronic liver disease worldwide as well as the primary indication for liver transplantation. More than 3% of the world’s population is chronically infected with HCV and there is an urgent need for effective therapy. NS3 protease, a viral enzyme required for propagation of HCV in humans, is a promising target for drug development in this area. This thesis addresses the design, synthesis and biochemical evaluation of new HCV NS3 protease inhibitors. The main objective of the thesis was the synthesis of peptide-based protease inhibitors of the bifunctional full-length NS3 enzyme (protease-helicase/NTPase). Three types of inhibitors were synthesized: i) classical serine protease inhibitors with electrophilic C-terminals, ii) product-based inhibitors with a C-terminal carboxylate group, and iii) product-based inhibitors with C-terminal carboxylic acid bioisosteres. The developmental work included the establishment of an improved procedure for solid-phase peptide synthesis (SPPS) in the N-to-C direction, in contrast to the C-to-N direction of classical SPPS methods. This inverse method facilitated synthesis of the peptides modified at the C-terminal. The potency of more than seventy newly synthesized inhibitors was assessed in an in vitro assay using the native form of the protease, i.e. the full-length NS3. The structure-activity relationship (SAR) data achieved was different from SAR data obtained from the more widely used truncated NS3 (protease domain) assay, indicating that the helicase domain of NS3 participates in the binding of the inhibitors. The most potent inhibitors identified in this study contained a C-terminal phenyl acyl sulfonamide moiety, i.e. a carboxylic acid bioisostere. It is concluded that the acyl sulfonamide moiety is a promising P1-P1´ spanning entity, which may have potential for use in the development of more drug-like HCV protease inhibitors. Anja Johansson, Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Box 574, SE-751 23, Uppsala, Sweden Anja Johansson 2003 ISSN 0282-7484 ISBN 91-554-5538-7 Printed in Sweden by Tryck & Medier, Universitetstryckeriet, Uppsala 2003 Till min familj ♥ PAPERS INCLUDED IN THE THESIS This thesis is based on the following papers, which are referred to in the text by their Roman numerals: I. Johansson Anja, Åkerblom Eva, Ersmark Karolina, Lindeberg Gunnar, Hallberg Anders. (2000). An Improved Procedure for N- to C-Directed (Inverse) Solid-Phase Peptide Synthesis. J. Comb. Chem. 2(5): 496-507. II. Johansson Anja, Hubatsch Ina, Åkerblom Eva, Lindeberg Gunnar, Winiwarter Susanne, Danielson U. Helena, Hallberg Anders. (2001). Inhibition of Hepatitis C Virus NS3 Protease Activity by Product- Based Peptides is Dependent on Helicase Domain. Bioorg. Med. Chem. Lett. 11(2): 203-206. III. Johansson Anja, Poliakov Anton, Åkerblom Eva, Lindeberg Gunnar, Winiwarter Susanne, Samuelsson Bertil, Danielson U. Helena, Hallberg Anders. (2002). Tetrapeptides as Potent Protease Inhibitors of Hepatitis C Virus Full-Length NS3 (Protease-Helicase/NTPase). Bioorg. Med. Chem. 10(12): 3915-3922. IV. Johansson Anja, Poliakov Anton, Åkerblom Eva, Wiklund Karin, Lindeberg Gunnar, Winiwarter Susanne, Danielson U. Helena, Samuelsson Bertil, Hallberg Anders. Acyl Sulfonamides as Potent Protease Inhibitors of the Hepatitis C Virus Full-Length NS3 (Protease-Helicase/NTPase). A Comparative Study of Different C- Terminals. (Submitted) Reprints were made with permission from the publishers; the American Chemical Society and Elsevier Science. Contents 1 Introduction ............................................................................................9 1.1 Hepatitis C Virus (HCV) Infection....................................................9 1.1.1 Transmission, Prevalence and Consequences ....................................9 1.1.2 Therapy ............................................................................................11 1.2 The HCV Genome and Life Cycle ..................................................12 1.2.1 Polyprotein Processing.....................................................................13 1.2.2 Antiviral Targets ..............................................................................14 1.2.3 HCV NS3 Protease...........................................................................14 1.3 Serine Proteases...............................................................................15 1.3.1 Mechanism of Action.......................................................................15 1.3.2 Development of Inhibitors ...............................................................16 1.4 HCV NS3 Protease Inhibitors..........................................................19 1.4.1 Peptidic/Peptidomimetic Inhibitors..................................................19 1.4.1.1 Electrophilic Inhibitors........................................................20 1.4.1.2 Product-Based Inhibitors.....................................................21 1.4.1.3 Noncleavable Substrate Inhibitors.......................................23 1.4.1.4 Prime-Side Inhibitors ..........................................................24 1.4.2 Miscellaneous...................................................................................24 1.5 Biological Evaluation of HCV NS3 Protease Inhibitors..................25 1.5.1 Animal Models.................................................................................25 1.5.2 Cell Culture System .........................................................................25 1.5.3 HCV NS3 Protease In Vitro Assay...................................................26 2 Aims of the Present Study....................................................................28 3 Design and Strategy..............................................................................29 4 Solid-Phase Peptide Synthesis (SPPS) ................................................31 4.1 Inverse SPPS....................................................................................32 4.2 Improvements in Inverse SPPS Methodology (Paper I)..................34 4.2.1 Investigation of coupling conditions ................................................36 5 Synthesis of HCV NS3 Protease Inhibitors (Papers II-IV) ...............42 5.1 Synthesis of P1 Building Blocks (Paper IV)....................................42 5.1.1 Pentafluoroethyl Ketone Precursors.................................................42 5.1.2 α-Ketotetrazole Precursors...............................................................43 5.1.3 α-Keto Acid Precursor .....................................................................45 5.1.4 Tetrazoles .........................................................................................45 5.1.5 Phenyl Acyl Sulfonamides ...............................................................46 5.2 Synthesis of Peptide-Based Inhibitors .............................................47 5.2.1 Classical SPPS (Papers II-IV) ..........................................................47 5.2.2 Inverse SPPS (Paper IV) ..................................................................48 6 Structure-Activity Relationships.........................................................52 6.1 Full-Length HCV NS3 In Vitro Assay ............................................52 6.2 Full-Length Versus Truncated NS3 Assays (Paper II) ....................53 6.3 Tetra- and Tripeptide Library (Paper III).........................................56 6.4 P1 Modifications (Paper IV)............................................................61 7 Concluding Remarks............................................................................64 8 Acknowledgements ...............................................................................65 9 References..............................................................................................67 Abbreviations and Definitions AA or aa amino acid Abu L-2-aminobutyric acid Ac acetyl ACE angiotensin converting enzyme ACPC 1-aminocyclopropane-1-carboxylic acid AIDS acquired immune deficiency syndrome Boc tert-butoxycarbonyl Cha β-cyclohexylalanine D-Gla D-γ-carboxyglutamic acid DIEA N,N-diisopropylethylamine Dif 3,3-diphenylalanine DMAP 4-(dimethylamino)pyridine DMF N,N-dimethylformamide EC50 inhibitor concentration giving 50% inhibition of replication in a cell culture system EDC N-ethyl-N´-(3-dimethylaminopropyl)-carbodiimide hydrochloride enzymes biological catalysts Fm 9-fluorenylmethyl Fmoc 9-fluorenylmethyloxycarbonyl Glt glutaryl/4-carboxy-butanoyl HATU N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl- methylene]-N-methylmethanaminium hexafluorophosphate N-oxide HBTU N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N- methylmethanaminium hexafluorophosphate N-oxide HCV hepatitis C virus helicase enzyme unwinding duplex DNA or RNA hepatitis liver inflammation HIV human immunodeficiency
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