Anti-hepatitis C virus activity of novel β-D-2′-C-methyl-4′-azido pyrimidine nucleoside phosphoramidate prodrugs

• Authors: Ramu Rondla, Emory University; Steven Coats, Emory University; Tamara R. McBrayer, RFS Pharma, LLC; Jason Grier, Emory University; Melissa Johns, Emory University; Phillip M. Tharnish, RFS Pharma, LLC; Tony Whitaker, RFS Pharma, LLC; Longhu Zhou, Emory University; Raymond Schinazi, Emory University
• Journal Title: Antiviral Chemistry and Chemotherapy
• Volume: Volume 20, Number 2
• Publisher: Sage Journals | 2009-11-17, Pages 99-106
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.3851/IMP1400
• Subjects: Biology, Cell; Biology, Virology
• Keywords: Amides; Antiviral Agents; Cell Line; Cell Survival; Cytidine; Hepacivirus; Humans; Phosphoric Acids; Prodrugs; Pyrimidine Nucleosides; Uridine; Virus Replication

Abstract

Background: 2′-C-methyl 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-methyl-moiety 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. © 2009 International Medical Press.