Anti-hepatitis C Virus Activity of Novel β-D-2′-C-methyl-4′-azido Pyrimidine Nucleoside Phosphoramidate Prodrugs

Background: 2′-C-methyl and 4′-azido nucleosides have previously demonstrated inhibition of hepatitis C virus (HCV) replication by targeting the RNA-dependent RNA polymerase NS5B. In an effort to discover new and more potent anti-HCV agents, we envisioned synthesizing nucleoside analogues by combining the 2′-C-methylmoiety with the 4′-azido-moiety into one molecule. Methods: 2′-C-methyl-4′-azido pyrimidine nucleosides were synthesized by first converting 2′-C-methyl ribonucleosides to the corresponding 4′-exocyclic methylene nucleosides. Treatment with iodine azide, benzoylation of the 2′- and 3′-hydroxy groups, oxidative displacement of the 5′-iodo group with meta-chloroperoxybenzoic acid, and debenzoylation gave the desired 2′-C-methyl-4′-azido uridine and thymidine analogues in good yield. Standard conversion of uridine to cytidine via the 4-triazole yielded 2′-C-methyl-4′-azido cytidine. In addition, 5′-phosphoramidate derivatives of 2′-C-methyl-4′-azido uridine and cytidine were synthesized to bypass the initial phosphorylation step. Results: The prepared nucleosides and their 5′-monophosphate prodrugs were evaluated for their ability to inhibit replication of the hepatitis C virus in a subgenomic replicon cell based assay. Cytotoxicity in Huh7 cells was determined simultaneously with anti-HCV activity by extraction and amplification of both HCV RNA and ribosomal RNA. Among the newly synthesized compounds, only the 5′-monophosphate nucleoside prodrugs had modest and selective anti-HCV activity. All prepared pyrimidine nucleosides and 5′-monophosphate nucleoside prodrugs displayed no evidence of cytotoxicity at high concentrations. Conclusions: This work is the first example of both inactive uridine and cytidine analogues of a nucleoside being converted to active anti-HCV nucleosides via 5′-monophosphate prodrugs.