Synthesis, Characterization, and Biological Evaluation of Novel Azo-Based Imidazo[1,2- <i>a</i> ]pyridine Derivatives: In Vitro and In Silico Investigations

In this study, a series of azo-linked imidazo-[1,2-<i>a</i>]-pyridine derivatives (<b>4a</b>-<b>f</b>) was synthesized and fully characterized using FTIR, NMR (¹H and ¹³C), and mass spectrometry. Structural and electronic properties were investigated through Density Functional Theory (DFT) at the B3LYP/6-311++G-(d,p) level, revealing key insights into charge distribution, molecular orbitals, and reactivity indices. The antibacterial and antibiofilm activities were assessed in vitro against Gram-positive and Gram-negative strains, including multidrug-resistant isolates. <b>4e</b> exhibited the most potent antibacterial activity, with minimum inhibitory concentrations (MICs) of 0.5-1.0 mg/mL, and retained activity against resistant strains, showing MICs of 0.5-0.7 mg/mL against <i>Escherichia coli</i> <i>CTXM</i> and <i>Klebsiella pneumoniae</i> <i>NDM</i>. <b>4b</b> and <b>4c</b> notably inhibited biofilm formation at concentrations as low as 0.4 mg/mL against <i>E. coli</i>. Molecular docking studies indicated strong binding affinities for the active compounds, with <b>4b</b> achieving the most favorable docking score (-10.4 kcal/mol) against the bacterial target GyrB. These binding interactions were further corroborated by molecular dynamics simulations, which demonstrated stable protein-ligand complexes over a 100 ns trajectory with minimal structural deviations. Additionally, ADME-T and drug-likeness evaluations predicted favorable pharmacokinetic profiles for the majority of the compounds, highlighting their viability for further development. Overall, this study establishes azo-based imidazo-[1,2-<i>a</i>]-pyridine derivatives as promising scaffolds for future antibacterial drug discovery.