3′-Deoxy Phosphoramidate Dinucleosides as Improved Inhibitors of Hepatitis C Virus Subgenomic Replicon and NS5B Polymerase Activity

Abstract

Phosphoramidate dinucleosides named "GC 3'-OH" series, carrying various phosphoramidate linkages have been previously reported as hepatitis C virus (HCV) inhibitors. To enhance the efficacy of these dinucleotides, we synthesized a novel "GC 3'-H" series as potential chain terminators. We showed that their inhibition potency is strongly increased by the introduction of novel neutral and bis-negatively charged phosphoramidate side chains. Their inhibitory effect on HCV NS5B polymerase was evaluated in vitro and in HCV subgenomic replicon containing Huh-6 cells. As expected, 3'-H compounds are more potent than their 3'-OH counterparts to inhibit HCV polymerase activity. The most potent inhibitor, a 5'-phosphorylated dinucleotide bearing a bis-negatively charged amino side chain (7), exhibits an IC(50) value of 8 μM in vitro and EC(50) value of 2.6 μM in the HCV subgenomic replicon system. A molecular structure model is presented to propose an interpretation of the gain afforded by the of 3'-H-cytidine modification.