Synthesis, characterization, and in vitro and in silico antibacterial evaluation of 5-(3-nitrophenyl)-1, 3, 4-thiadiazol-2-amine

Introduction – This study reports the synthesis, structural characterization, and antibacterial evaluation of 5-(3-nitrophenyl)-1, 3, 4-thiadiazol-2-amine (NPTA), a commercially available thiadiazole derivative with potential antimicrobial activity. Methods – NPTA was synthesized through a green, one-pot condensation reaction between thiosemicarbazide and 3-nitrobenzoic acid in absolute ethanol, affording a pale-yellow crystalline solid with a melting point of 110 °C–112 °C. The compound was characterized using Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, confirming its structure. In vitro antibacterial assays, in silico ADMET and toxicity profiling, molecular docking, frontier molecular orbital (FMO) analysis, and 200 ns molecular dynamics simulations were performed. Results – In vitro antibacterial assays revealed significant activity against Klebsiella pneumoniae ATCC 13883, Acinetobacter baumannii, and Listeria monocytogenes ATCC 19114, with growth inhibition zones of 25.63 ± 0.17 mm, 29.09 ± 1.31 mm, and 26.65 ± 0.19 mm, respectively, and minimum inhibitory concentrations (MICs) ranging from 50 to 100 μg/mL. In silico ADMET and toxicity profiling predicted favourable drug-likeness, absorption, and safety. Molecular docking indicated strong binding affinities (−6.2 to −7.0 kcal/mol) with key bacterial targets, i.e., DNA gyrase subunit B (PDB: 1KZN) and penicillin-binding protein 4 (PDB: 3HUN). Frontier molecular orbital (FMO) analysis revealed a HOMO-LUMO energy gap of 3.79 eV, suggesting high electronic stability and reactivity. Furthermore, 200 ns molecular dynamics simulations confirmed the temporal stability of NPTA–protein complexes, particularly with DNA gyrase subunit B. Discussion – These results demonstrate the promising antibacterial potential of NPTA and support its further development as a multifunctional thiadiazole-based antimicrobial candidate.