Discovery of Novel Nucleotide Prodrugs with Improved Potency Against HCV Variants Carrying NS5B S282T Mutation.

Resistant HCV variants carrying NS5B S282T mutation confer reduced sensitivity to sofosbuvir, the sole marketed NS5B polymerase inhibitor. On the basis of the finding that 2'-α-F-2'-ß-C-methylcytidine 5'-triphosphate (8) was more potent than sofosbuvir's active metabolite on inhibition of both wild-type and S282T mutant polymerase, a dual-prodrug approach has been established. Twenty-nine phosphoramidates with N4-modified cytosine were designed, synthesized, and evaluated for anti-HCV activity. The results showed that compounds 4c-4e and 4m (EC50 = 0.19-0.25 µM) exhibited comparable potency to that of sofosbuvir (EC50 = 0.15 µM) on inhibition of wild-type replicons. Notably, 4c (EC50 = 0.366 µM) was 1.5-fold more potent than sofosbuvir (EC50 = 0.589 µM) on inhibition of S282T mutant replicons. In vitro metabolic studies disclosed the possible metabolic pathways of 4c. The toxicity study results indicated a good safety profile of 4c. Together, 4c-4e and 4m hold promise for drug development for the treatment of HCV infection, especially the resistant variants with NS5B S282T mutation.