Inhibitors of hepatitis C virus NS3 . 4A protease 2. Warhead SAR and optimization RobertB.Perni,* JanosPitlik,ShawnD.Britt,JohnJ.Court, LawrenceF.Courtney,DavidD.Deininger,LucJ.Farmer,CynthiaA.Gates, ScottL.Harbeson,RhondaB.Levin,ChaoLin,KaiLin,Young-ChoonMoon, Yu-PingLuong,EthanT.O’Malley,B.GovindaRao,JohnA.Thomson, RogerD.Tung,JohnH.VanDrieandYunyiWei Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, USA Received 14 October 2003; revised 12 January 2004; accepted 14 January 2004 Abstract—The a-ketoamide warhead (e.g., 15)wasfoundtobeapracticalreplacementforaliphaticaldehydesinaseriesofHCV NS3 . 4A protease inhibitors. Structure–activity relationships and prime side optimization are discussed. # 2004ElsevierLtd.Allrightsreserved. A replacement for the prototype aldehyde covalent warhead was sought for our evolving HCV NS3 . 4A proteaseinhibitorseries.WedescribetheSARofaseries of warheads situated on a previously described tetra- peptide scaffold. An unusual a-ketoamide active-site binding motif has been identified and is discussed in detail. The attractiveness of the NS3 . 4A protease as a target for inhibiting hepatitis C viral replication has been dis- cussed often over the last several years 1a b and the first protease inhibitor, BILN 2061, has recently entered clinical trials. 1c d We have recently reported on the structure–activity relationships of a series of tetrapep- tide aldehyde inhibitors (e.g., 1)ofthehepatitisCvirus NS3 . 4A protease culminating in the identification of a sub-mM inhibitor, 2 (K i =0.89 mM). 2 Although useful tools, aliphatic aldehyde warheads are relatively unstable and consequently unlikely to be suitable as drugs.Wenowdescribeeffortsaimedatreplacingalde- hyde warheads with functionality that allows for the requisite covalent binding to the catalytic serine while maintaining the necessary chemical stability. The a- ketoamide warhead was found to possess the necessary physicochemical and biochemical characteristics. Ithasbeenestablished 3 thatthepreferredP 1 substituent for three of the four natural substrate cleavage sites of the NS3 . 4A is cysteine. 4 A simple cysteine residue is incompatible with an electrophilic warhead due to intramolecular or intermolecular reactions. We chose aminobutyric (Abu) acid as the initial cysteine replace- ment providing an ethyl group at P 1 . A survey of potential covalent warheads was subsequently per- formed based on this Abu scaffold. The inhibitory properties of these compounds are summarized in Table1. Syntheses of various tetrapeptide inhibitors possessing different electrophilic warheads are described in Schemes1and2. The protected Abu-OH (3) was converted to the corresponding ‘reduced’ warheads using standard methodologies from aldehyde 4. 5 12 and the resulting reagents (5a–h) were used for subsequent coupling reactions(Scheme2). 0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2004.01.022 Bioorganic & Medicinal Chemistry Letters 14 (2004) 1441–1446 Keywords: Hepatitis C; Protease inhibitors. *Corresponding author. Tel.: +1-617-444-6237; fax: +1-617-444- 6766; e-mail: robert_perni@vrtx.com