Potent Inhibitors of Subgenomic Hepatitis C Virus RNA Replication through Optimization of Indole-N-Acetamide Allosteric Inhibitors of the Viral NS5B Polymerase Steven Harper,* Salvatore Avolio, Barbara Pacini, Marcello Di Filippo, Sergio Altamura, Licia Tomei, Giacomo Paonessa, Stefania Di Marco, Andrea Carfi, Claudio Giuliano, Julio Padron, Fabio Bonelli, Giovanni Migliaccio, Raffaele De Francesco, Ralph Laufer, Michael Rowley, and Frank Narjes IRBM (Merck Research Laboratories, Rome), Via Pontina km 30,600, 00040 Pomezia, Rome, Italy Received January 19, 2005 Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off- target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC 50 ) 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog. Introduction Despite well over a decade having passed since hepatitis C virus (HCV) was identified as the pathogen responsible for most cases of non-A and non-B hepati- tis, 1 HCV infection continues to pose a significant world health problem. 2 There are around 170 million carriers of the virus worldwide, and increasingly, members of this community are facing medical complications that result from disease progression. HCV can lead to life- threatening liver disorders such as cirrhosis and hepa- tocellular carcinoma and is now recognized as the single leading cause of liver transplantation. 3 The gravity of HCV infection is compounded by the inadequacy of currently approved treatments for the disease. These are based on modified interferons that are expensive, poorly tolerated, and show variable success rates. 4 While new therapies to tackle HCV are urgently needed, efforts toward them have been impeded by the complex biology associated with the virus. HCV is not infectious in cell culture or in small animal models, presenting a significant challenge to the development of anti-HCV agents. However, the recent introduction of the HCV replicon assay, 5 a surrogate cell-based system in which replication of subgenomic viral RNA can be studied, has provided a crucial advance for drug discovery efforts targeting HCV proteins. This assay is validated by the observation that an inhibitor of a viral enzyme, the NS3 protease, 6 shows potency in this system and also elicits a strong reduction in viral titer in a clinical setting. 7 Consequently the search for compounds that efficiently block HCV replication in the replicon assay has become an intense area of pharma- ceutical research. In addition to NS3, the nonstructural region of the HCV genome encodes several additional enzymes that are believed to play fundamental roles in the viral life cycle and that are viable targets for drug discovery efforts. The NS5B protein is one of these and has been characterized as the RNA polymerase enzyme that catalyzes the synthesis of both a complementary (-)- stranded HCV RNA intermediate and the (+)-stranded viral genome itself. 8 In common with other nucleotide polymerizing enzymes, NS5B adopts a tertiary structure that resembles a right hand, with three constitutive peptide domains designating the palm, fingers, and thumb. 9 The catalytic action of the enzyme is mediated by two magnesium ions that are ligated in the palm domain and serve to activate the 3′-OH of the elongating RNA and to position the incoming nucleotide triphos- phate for nucleophilic attack. NS5B has emerged as an especially attractive target for drug discovery efforts toward antivirals for HCV and has been described as the most drugable HCV protein. 10 The absence of a functional counterpart to NS5B in mammalian cells, where DNA is usually the template during RNA transcription, may favor the development of selective and nontoxic inhibitors. Toward this goal several series of NS5B inhibitors that show activity in the replicon assay have been reported. Chain-terminat- ing nucleoside analogues that bind at the active site of NS5B have been found to show potency in the submi- cromolar range. 11 To date, no non-nucleoside inhibitors that both bind at the active site of NS5B and show cell- based activity have been reported. However, allosteric inhibition by small-molecule inhibitors of NS5B has emerged as a bona fide route toward inhibition of subgenomic HCV RNA replication, and a number of structurally diverse inhibitor classes have now been identified. 12-14 We recently reported a series of allosteric * To whom correspondence should be addressed. Phone: +39 06 91093444. Fax: +39 06 91093564. E-mail: steven_harper@merck.com. 4547 J. Med. Chem. 2005, 48, 4547-4557 10.1021/jm050056+ CCC: $30.25 © 2005 American Chemical Society Published on Web 06/11/2005