Discovery of Potent Hepatitis C Virus NS5A Inhibitors with Dimeric Structures

Abstract

The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translated into an in vivo effect in proof-of-concept clinical trials. Although the 50% effective concentration (EC 50 ) of the initial lead, the thiazolidinone BMS-824, was ϳ10 nM in the replicon assay, it underwent transformation to other inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, including the EC 50 , the drug concentration required to reduce cell growth by 50% (CC 50 ), and the resistance profile, however, remained unchanged, triggering an investigation to identify the biologically active species. Highperformance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated in medium revealed that the most active fractions could readily be separated from the parental compound and retained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes in compound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to be discussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors provided the foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in the identification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.Hepatitis C virus (HCV) is the major causative agent of non-A, non-B hepatitis worldwide, which affects more than 3% of the world's population. Of those infected with HCV, ϳ70% proceed to a chronic state which can lead to severe liver diseases, including fibrosis, cirrhosis, or hepatocellular carcinoma (1, 7). There is currently no vaccine against HCV and no generally effective therapy for all HCV genotypes. The current optimal therapy is pegylated alpha interferon in combination with ribavirin, a regimen associated with significant side effects and limited efficacy in the most prevalent patient population, consisting of genotype 1 (4). Therefore, there is an urgent need for the development of more effective, HCV-specific antiviral therapies with fewer side effects.In the search for more efficacious, safer HCV therapies, the most actively pursued antiviral targets have been the NS3 protease and NS5B RNA-dependent RNA polymerase, both essential enzymes for the replication of HCV (2,11,12). Exciting progress has been demonstrated in clinical trials with multiple HCV NS3 serine protease inhibitors, as well as with both nucleoside and nonnucleoside po...

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…Symmetry and dimeric structures are apparently important contributors to the antiviral activities of NS5A inhibitors (5,7). Biochemical studies have also suggested that these inhibitors directly bind to NS5A (5, 7).…”

“…Recent publications describe how small compounds, which apparently target NS5A, inhibit replication with extreme potency, highlighting the validity of NS5A as a target for drug development (5)(6)(7). NS5A is a nonstructural protein that has no defined role in the virus life cycle but is absolutely required for RNA replication (8 -10) and particle assembly (11)(12)(13)(14).…”

Inhibitors of HCV NS5A: From Iminothiazolidinones to Symmetrical Stilbenes

“…As a result of these findings, HPLC biogram fractionation experiments were performed, an exercise that traced potent HCV inhibitory activity to two minor components in the media identified as isomers of the dimeric species 48 (Figure 1). 17 The connectivity of the dimer was established by 1 H and 13 C NMR to be at the C-5 methine carbon, with the less mobile isomer on reversed phase LC analysis being the more potent compound, EC 50 = 0.6 nM, and which converted to the more mobile but less potent (EC 50 = 43 nM) isomer upon heating at 55°C. 17 In considering how compounds 46-48 might be generated, it was recognized that hydrogen abstraction at the C-5 methine carbon of 18 would form a classic captodative radical stabilized by the electron donating sulfur atom and the electron withdrawing carbonyl group.…”

“…17 The connectivity of the dimer was established by 1 H and 13 C NMR to be at the C-5 methine carbon, with the less mobile isomer on reversed phase LC analysis being the more potent compound, EC 50 = 0.6 nM, and which converted to the more mobile but less potent (EC 50 = 43 nM) isomer upon heating at 55°C. 17 In considering how compounds 46-48 might be generated, it was recognized that hydrogen abstraction at the C-5 methine carbon of 18 would form a classic captodative radical stabilized by the electron donating sulfur atom and the electron withdrawing carbonyl group. 18,19 A radical mechanism is consistent with the formation of 47 by a process involving combination of a C-5 radical with oxygen that would give hydroperoxide A (Scheme 2).…”

“…The first indication of an inherent chemical reactivity emerged when it was observed that, on standing in DMSO, compound 2 underwent an oxidative rearrangement to generate the thiohydantoin 46 (Scheme 2). 16,17 The structure of 46 was elucidated by correlation spectroscopy, which established the proximity of the furanyl-methylene protons (δ5.2, dd, 2H) to the amide carbonyl carbon (δ 171.4 ppm) and the thiourea carbon (δ 182.5 ppm) atoms. In addition, 16 Thiohydantoin 46, however, exhibited poor activity in the replicon assay, with an EC 50 = 13 μM, suggesting that other species in the cell culture media may be responsible for the observed inhibitory activity of 2, and this prompted a more detailed analysis of compound 18.…”