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A medicinal chemistry campaign thatwas conducted to address apotential genotoxic liability associated with an aniline-derived scaffoldin a series of HCV NS5A inhibitors with dual GT-1a/-1b inhibitoryactivity is described. Anilides 3band 3cwere used as vehicles to explore structural modifications that retainedantiviral potency while removing the potential for metabolism-basedunmasking of the embedded aniline. This effort resulted in the discoveryof a highly potent biarylimidazole chemotype that established a potencybenchmark in replicon assays, particularly toward HCV GT-1a, a strainwith significant clinical importance. Securing potent GT-1a activityin a chemotype class lacking overt structural liabilities was a criticalmilestone in the effort to realize the full clinical potential oftargeting the HCV NS5A protein. [ABSTRACT FROM AUTHOR]

The biphenyl derivatives 2and 3areprototypes of a novel class of NS5A replication complex inhibitorsthat demonstrate high inhibitory potency toward a panel of clinicallyrelevant HCV strains encompassing genotypes 1–6. However, thesecompounds exhibit poor systemic exposure in rat pharmacokinetic studiesafter oral dosing. The structure–activity relationship investigationsthat improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification ofthe highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was therealization that the arylglycine cap of 2could be replacedwith an alkylglycine derivative and still maintain the high inhibitorypotency of the series if accompanied with a stereoinversion, a findingthat enabled a rapid optimization of exposure properties. Compound 33had EC50values of 50 and 9 pM toward genotype-1aand -1b replicons, respectively, and oral bioavailabilities of 38–108%in preclinical species. Compound 33provided clinicalproof-of-concept for the NS5A replication complex inhibitor class,and regulatory approval to market it with the NS3/4A protease inhibitorasunaprevir for the treatment of HCV genotype-1b infection has recentlybeen sought in Japan. [ABSTRACT FROM AUTHOR]

Abstract: The hepatitis C virus NS5A protein is an established and clinically validated target for antiviral intervention by small molecules. Characterizations are presented of compounds identified as potent inhibitors of HCV replication to provide insight into structural elements that interact with the NS5A protein. UV-activated cross linking and affinity isolation was performed with one series to probe the physical interaction between the inhibitors and the NS5A protein expressed in HCV replicon cells. Resistance mapping with the second series was used to determine the functional impact of specific inhibitor subdomains on the interaction with NS5A. The data provide evidence for a direct high-affinity interaction between these inhibitors and the NS5A protein, with the interaction dependent on inhibitor stereochemistry. The functional data supports a model of inhibition that implicates inhibitor binding by covalently combining distinct pharmacophores across an NS5A dimer interface to achieve maximal inhibition of HCV replication. [Copyright &y& Elsevier]

Abstract: In a recent disclosure,1 we described the discovery of dimeric, prolinamide-based NS5A replication complex inhibitors exhibiting excellent potency towards an HCV genotype 1b replicon. That disclosure dealt with the SAR exploration of the peripheral region of our lead chemotype, and herein is described the SAR uncovered from a complementary effort that focused on the central core region. From this effort, the contribution of the core region to the overall topology of the pharmacophore, primarily vector orientation and planarity, was determined, with a set of analogs exhibiting <10nM EC50 in a genotype 1b replicon assay. [Copyright &y& Elsevier]

Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ~5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects. [ABSTRACT FROM AUTHOR]