Synthesis and anti-viral activity of a series of D- and l-2′-deoxy-2′-fluororibonucleosides in the subgenomic HCV replicon system

Based on the discovery of (2′R)-D-2′-deoxy-2′- fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of D- and L-2′-deoxy-2′-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2′-fluoro group, was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2′R)-D-2′-deoxy-2′,5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N⁴-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N⁴-hydroxyl and the 2′-fluoro into one molecule, resulting (2′R)-D-2′-deoxy- 2′-fluoro-N⁴-hydroxycytidine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.