7-Deaza-7-fluoro modification confers on 4′-cyano-nucleosides potent activity against entecavir/adefovir-resistant HBV variants and favorable safety

We designed, synthesized and identified a novel nucleoside derivative, 4′-C-cyano-7-deaza-7-fluoro-2′-deoxyadenosine (CdFA), which exerts potent anti-HBV activity (IC_{50} ~26 nM) with favorable hepatocytotoxicity (CC_{50} ~56 μM). Southern blot analysis using wild-type HBV (HBV_{WT})-encoding-plasmid-transfected HepG2 cells revealed that CdFA efficiently suppresses the production of HBV_{WT} (IC_{50} = 153.7 nM), entecavir (ETV)-resistant HBV carrying L180M/S202G/M204V substitutions (HBV_{ETV}^{R}; IC_{50} = 373.2 nM), and adefovir dipivoxil (ADV)-resistant HBV carrying A181T/N236T substitutions (HBV_{ADV}^{R}; IC_{50=}192.6 nM), whereas ETV and ADV were less potent against HBV_{ETV}^{R} and HBV_{ADV}^{R} (IC_{50}: >1,000 and 4,022.5 nM, respectively). Once-daily peroral administration of CdFA to human-liver-chimeric mice over 14 days (1 mg/kg/day) comparably blocked HBV_{WT} and HBV_{ETV}^{R} viremia by 0.7 and 1.2 logs, respectively, without significant changes in body-weight or serum human-albumin levels, although ETV only slightly suppressed HBV_{ETV}^{R} viremia (CdFA vs ETV; p = 0.032). Molecular modeling suggested that ETV-TP has good nonpolar interactions with HBV_{WT} reverse transcriptase (RT_{WT})’s Met204 and Asp205, while CdFA-TP fails to interact with Met204, in line with the relatively inferior activity against HBV_{WT} of CdFA compared to ETV (IC_{50}: 0.026 versus 0.003 nM). In contrast, the 4′-cyano of CdFA-TP forms good nonpolar contacts with RT_{WT}’s Leu180 and RT_{ETV}^{R}’s Met180, while ETV-TP loses interactions with RT_{ETV}^{R}’s Met180, explaining in part why ETV is less potent against HBV_{ETV}^{R} than CdFA. The present results show that CdFA exerts potent activity against HBV_{WT}, HBV_{ETV}^{R} and HBV_{ADV}^{R} with enhanced safety and that 7-deaza-7-fluoro modification confers potent activity against drug-resistant HBV variants and favorable safety, shedding light to further design more potent and safer anti-HBV nucleoside analogs.