The escalating threat of antimicrobial resistance necessitates the urgent development of novel therapeutic agents targeting essential bacterial enzymes. In this study, a new series of 1,2,3-triazole-linked coumarin-vanillin hybrids was rationally designed and synthesized, integrating the bioactive moieties of coumarin and vanillin derivatives via the click chemistry technique. The synthesized compounds (AK1-AK30) were thoroughly characterized by 1H, 13C NMR, FT-IR, and HR-MS spectroscopic techniques. All the compounds exhibited broad-spectrum antibacterial efficacy against both gram-positive and gram-negative pathogens. AK19 demonstrated minimum inhibitory concentrations (MIC) of 1.9 μM against Bacillus subtilis and clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), and superior efficacy compared to ciprofloxacin against Pseudomonas aeruginosa and Shigella boydii. Additionally, it exhibited significant biofilm inhibition against B. subtilis and MRSA. Mechanistically, AK19 showed strong inhibition against Escherichia coli DNA gyrase and E. coli topoisomerase with IC50 values of 0.783 ± 0.04 μM and 7.136 ± 1.03 μM, respectively. Molecular docking and molecular dynamics simulations further substantiated these results by demonstrating stable binding of AK19 within the active sites of the targeted enzymes, with favourable binding free energies (ΔG = - 48 KJ/Mol and - 29 KJ/Mol, respectively). Additionally, bovine serum albumin (BSA) binding studies and ADME profiling indicated desirable pharmacokinetic characteristics. The overall results underscore the AK19 as a promising lead compound for the development of next-generation antibacterial agents targeting DNA gyrase and topoisomerase IV.
