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

Abstract

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 (IC₅₀ ~26 nM) with favorable hepatocytotoxicity (CC₅₀ ~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 (IC₅₀ = 153.7 nM), entecavir (ETV)-resistant HBV carrying L180M/S202G/M204V substitutions (HBV_ETV^R; IC₅₀ = 373.2 nM), and adefovir dipivoxil (ADV)-resistant HBV carrying A181T/N236T substitutions (HBV_ADV^R; IC₅₀₌192.6 nM), whereas ETV and ADV were less potent against HBV_ETV^R and HBV_ADV^R (IC₅₀: >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 (IC₅₀: 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.

Keywords: Adefovir; Anti-HBV drugs; Drug resistance; Entecavir; HBV; Nucleoside/nucleotide reverse transcriptase inhibitors.