Design and Synthesis of Novel Imidazo[4,5-c]pyridine Derivatives, Evaluation of Their Activity Against Hepatitis C Virus and In Silico Prediction of Their Binding Mode to NS4B Protein.

A series of suitably substituted imidazo[4,5-c]pyridine derivatives was synthesized, starting from 4-amino-2-chloropyridine, and their antiviral activity against hepatitis C virus (HCV) genotype 1b (GT1b) was evaluated in Huh5.2 replicon cells. Some of the N3-substituted analogs exhibited potent anti-HCV activity (EC50 = 6.61-47.24 μM), as determined by assessing their individual effects on viral RNA replication and protein expression levels. Among these, the most promising compound 30b demonstrated synergistic antiviral activity when combined with clinically approved direct-acting anti-HCV therapies, highlighting its potential utility in combination regimens. These new derivatives were rationally designed to target the nonstructural 4B (NS4B) protein of HCV. Our molecular docking studies reinforced the implication of this mechanism of action, suggesting a binding mode similar to the previously reported and structurally related inhibitors clemizole and anguizole. A conserved hydrophobic pocket within NS4B (encompassing TM1-3, the Walker A motif, and the amphipathic helix H1) was identified, supporting the validity of this scaffold for further structure-based optimization. Collectively, our results support further investigation of the imidazo[4,5-c]pyridine scaffold for the rational development of novel anti-HCV agents with potential application in combination therapies aimed at the effective elimination of chronic HCV infection.