Anti-hepatitis C virus activity of novel beta-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-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.
Rondla
Ramu
R
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA, USA.
Coats
Steven J
SJ
McBrayer
Tamara R
TR
Grier
Jason
J
Johns
Melissa
M
Tharnish
Phillip M
PM
Whitaker
Tony
T
Zhou
Longhu
L
Schinazi
Raymond F
RF
D000577:Amides; D000998:Antiviral Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003562:Cytidine / Q000031:analogs & derivatives; D016174:Hepacivirus / Q000187:drug effects*; D006801:Humans; D010756:Phosphoric Acids; D011355:Prodrugs / Q000494:pharmacology* / Q000627:therapeutic use; D011741:Pyrimidine Nucleosides / Q000494:pharmacology* / Q000627:therapeutic use; D014529:Uridine / Q000031:analogs & derivatives; D014779:Virus Replication / Q000187:drug effects
D000577:Amides; D000998:Antiviral Agents; D010756:Phosphoric Acids; D011355:Prodrugs; D011741:Pyrimidine Nucleosides; D003562:Cytidine; C011067:phosphoramidic acid; D014529:Uridine