Hepatitis C virus (HCV) infection is a global health problem that impacts approximately 180 million individuals. Until recently the current therapy for treating HCV infection has been regular injections of pegalated α-interferon (PEG-IFN) with daily oral administration of ribavirin (RBV). However, PEG-IFN/RBV treatment is only effective for only 50% of genotype 1 patients and associated with significant adverse effects including fatigue, hemolytic anemia, depression, and flulike symptoms. Therefore, the search for direct acting antivirals (DAAs) that are safe and effective has become an urgent endeavor. HCV NS5B polymerase, an essential enzyme for the HCV RNA replication, has emerged as an attractive and validated target for the direct HCV therapeutic intervention. Since NS5B polymerase needs a divalent metal ion as a cofactore in the active site for its catalytic function, the metal chelation motif-containing quinolone-3-carboxylic scaffold has been explored as a new class of non-nucleoside NS5B inhibitors. Two groups have recently reported a preliminary structure-activity relationship (SAR) study on the 4-quinolone-3-carboxylic acids as HCV NS5B inhibitors, just focused on the N-1, C-3 and C-6/7 substitutions. Based on the binding mode revealed by the cocrystal structure of the quinolone inhibitor bound to the NS5B enzyme, for the first time we proposed to introduce a hydrophobic group at C-2 position on the quinolone ring to improve the anti-HCV potency. By making use of the new method to synthesize 2-substituted quinolone-3-carboxylic acid derivatives recently developed by our group, we conducted a comprehensive SAR study on the 2-aryl-3-carbonylquinolone-based non-nucleoside inhibitors of HCV NS5B polymerase. Starting from the readily accessible amides and 3-oxo-3-arylpropanoates, structurally diverse 2-substituted quinolone-3-carboxylic acid derivatives were efficiently furnished by a tandem addition-elimination reaction/nucleophilic aromatic substitution reaction via an imine-enamine intermediate. The anti-HCV potency and cytotoxicity were evaluated in the HCV-infected host cells Huh7.5.1 assay system. To our delight, the incorporation of a hydrophobic aryl group into 2-position of the quinolone core really enhanced the inhibitory activity against the HCV replication in the host cells with a 2-fold selectivity over the cytotoxicity. Meanwhile, a small size hydrophobic group at N-1 position was favored for the 2-arylquinolone-derived NS5B inhibitors. Further structural variation was investigated on the C-3 and C-7 substituents, with an aromatic ester and an N-methyl piperazine being an optimal moiety, respectively. The global structural optimization at positions N-1, C-2, C-3 and C-7 resulted in the discovery of novel 2-aryl substituted quinolone inhibitors with low micromolar EC₅₀ values to inhibit the replication of the HCV RNA in the host cell Huh7.5.1 and therapeutic indices of 2～6, providing a new promising lead for the further development into anti-HCV drug candidates.

Key words: HCV NS5B polymerase, 2-substituted quinolone-3-carboxylic acid, non-nucleoside inhibitor, direct acting antiviral, allosteric site