Novel benzamide derivatives with indole moiety as dual-target antiviral agents: Rational design, efficient synthesis, and potent anti-influenza activity through concurrent binding to PAC terminal domain and viral nucleoprotein

In this study, a series of benzamide derivatives with an indole moiety as dual-target inhibitors were designed, synthesized and evaluated against the RNA-dependent RNA polymerase (RdRp) complex of influenza viruses. The target compounds can simultaneously disrupt two key molecular interactions: the PA_C terminal domain and the nucleoprotein (NP) oligomerization. Through efficient synthesis and structure-activity relationship (SAR) analysis, compounds 8e and 8f as highly potent inhibitors were identified. Both compounds (8e and 8f) exhibited submicromolar EC₅₀ values (1.64 ± 0.05 μM and 1.41 ± 0.27 μM) against influenza A virus (H1N1, A/WSN/33) and broad-spectrum activity against other influenza strains, including influenza B virus and multiple subtypes of influenza A. Notably, their cytotoxicity was significantly reduced compared to previous benzofurazan derivatives, with CC₅₀ values exceeding 100 μM. Surface plasmon resonance (SPR) experiments confirmed that 8e and 8f bound strongly to the PA C-terminal domain (KD = 8.90 μM and 4.82 μM) and NP (KD = 52.5 μM and 3.13 μM). Computational modeling approaches, including molecular docking, molecular dynamics (MD) simulations, and dynamical cross-correlation matrix (DCCM) analysis, principal component analysis (PCA) analysis and density functional theory (DFT) calculations, were employed to elucidate the putative binding modes and delineate critical interaction sites between the ligands and target proteins. These insights not only modulated subsequent structure-based lead optimization but also strengthened our understanding of the molecular determinants governing antiviral activity. This research provides a promising scaffold for developing dual-target antiviral agents with enhanced potency and safety, offering new strategies to combat influenza viruses.