Structure-based studies led to the identification of a constrained derivative of S-trityl-L-cysteine (STLC) scaffold as a candidate inhibitor of hepatitis C virus (HCV) NS5B polymerase. A panel of STLC derivatives were synthesized and investigated for their activity against HCV NS5B. Three STLC derivatives, 9, F-3070, and F-3065, were identified as modest HCV NS5B inhibitors with IC50 values between 22.3 to 39.7 μM. F-3070 and F-3065 displayed potent inhibition of intracellular NS5B activity in the BHK-NS5B-FRLuc reporter and also inhibited HCV RNA replication in the Huh7/Rep-Feo1b reporter system. Binding mode investigations suggested that the STLC scaffold can be used to develop new NS5B inhibitors by further chemical modification at one of the trityl phenyl group.

Hepatitis C virus (HCV) NS5B polymerase is an important and attractive target for the development of anti-HCV drugs. Here we report on the design, synthesis and evaluation of twenty-four novel allosteric inhibitors bearing the 4-thiazolidinone scaffold as inhibitors of HCV NS5B polymerase. Eleven compounds tested were found to inhibit HCV NS5B with IC50 values ranging between 19.8 and 64.9 μM. Compound 24 was the most active of this series with an IC50 of 5.6 μM. A number of these derivatives further exhibited strong inhibition against HCV 1b and 2a genotypes in cell based antiviral assays. Molecular docking analysis predicted that the thiazolidinone derivatives bind to the NS5B thumb pocket-II (TP-II). Our results suggest that further optimization of the thiazolidinone scaffold may be possible to yield new derivatives with improved enzyme- and cell-based activity.

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of anti-HCV drugs. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening and in vitro NS5B inhibition assays. One hundred and sixty thousand compounds from the Otava database were virtually screened against the thiazolone inhibitor binding site on NS5B (thumb pocket-2, TP-2), resulting in a sequential down-sizing of the library by 2.7 orders of magnitude to yield 59 NS5B non-nucleoside inhibitor (NNI) candidates. In vitro evaluation of the NS5B inhibitory activity of the 59 selected compounds resulted in a 14% hit rate, yielding 8 novel structural scaffolds. Of these, compound 1 bearing a 4-hydrazinoquinazoline scaffold was the most active (IC50 = 16.0 µM). The binding site of all 8 NNIs was mapped to TP-2 of NS5B as inferred by a decrease in their inhibition potency against the M423T NS5B mutant, employed as a screen for TP-2 site binders. At 100 µM concentration, none of the eight compounds exhibited any cytotoxicity, and all except compound 8 exhibited between 40–60% inhibition of intracellular NS5B polymerase activity in BHK-NS5B-FRLuc reporter cells. These inhibitor scaffolds will form the basis for future optimization and development of more potent NS5B inhibitors.

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC50 = 7.7 µM) and 2 (IC50 = 10.6 µM) as represented by hybrid compound 27 (IC50 = 6.7 µM). We have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene moiety in 27, by generating a set of six analogs. This resulted in the identification of compound 34 with an IC50 of 2.6 µM. To probe the role of stereochemistry towards the observed biological activity, we synthesized and evaluated the D-isomers 41 (IC50 = 19.3 µM) and 45 (IC50 = 5.4 µM) as enantiomers of the L-isomers 27 and 34, respectively. The binding site of compounds 32 and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Here we report the synthesis and biological evaluation of a new series of α,γ-diketo acids (DKAs) as NS5B polymerase inhibitors. We initiated structure-activity relationship (SAR) optimization around the furan moiety of compound 1a [IC50 = 21.8 μM] to achieve more active NS5B inhibitors. This yielded compound 3a [IC50 = 8.2 μM] bearing the 5-bromobenzofuran-2-yl moiety, the first promising lead compound of the series. Varying the furan moiety with thiophene, thiazole and indazole moieties resulted in compound 11a [IC50 = 7.5 μM] bearing 3-methylthiophen-2-yl moiety. Finally replacement of the thiophene ring with a bioisosteric phenyl ring further improved the inhibitory activity as seen in compounds 21a [IC50 = 5.2 μM] and 24a [IC50 = 2.4 μM]. Binding mode of compound 24a using glide docking within the active site of NS5B polymerase will form the basis for future SAR optimization.