Novel nucleoside analogues targeting HCV replication through an NS5A-dependent inhibition mechanism.

A series of new tricyclic nucleosides were synthesized and evaluated as hepatitis C virus (HCV) replication inhibitors. Initial screening in a HCV replicon system, derived from a genotype 1b isolate, identified 9-benzylamino-3-(β-D-ribofuranosyl)-3H-imidazo[4',5':5,6]pyrido[2,3-b]pyrazine (15d) as the most potent analogue. Comparative assessment of 15d activity against HCV full-length viruses or subgenomic replicons derived from genotypes 1 to 4 revealed a specificity of the compound for genotypes 1 and 3. Surprisingly, resistance mutations selected against 15d were mapped to domains II and III of the non-structural protein 5A (NS5A), but not to the RNA-dependent RNA polymerase residing in NS5B. These results argue that compound 15d might represent a lead for the development of a novel class of NS5A inhibitors.