Design, synthesis, in silico modelling, and in vitro neuroprotective evaluation of Benzo[D]oxazole–1,3,4-oxadiazole hybrids

Benzoxazoles are privileged scaffolds with diverse pharmacological applications. To identify novel neuroprotective agents, benzoxazole–1,3,4-oxadiazole hybrids were synthesized and evaluated through integrated computational and pharmacological approaches. In this study we reported nine substituted 1,3,4-oxadiazolyl benzoxazole derivatives were synthesized via a one-pot reaction from benzoxazole carbohydrazide using phosphoryl chloride, carbon disulfide, bromocyanide, and mild base conditions in ethanol and 1,4-dioxane. The synthesized compounds were purified and structurally confirmed by ¹H-NMR, ESI-MS, and HPLC. In silico evaluation of biological activity potential, drug-likeness, and toxicity profiles was performed using freely available computational tools. Molecular docking was conducted against the GABA A A receptor benzodiazepine-binding site (PDB ID: 6 × 3X) using AutoDock, with diazepam as positive control. Selected ligand–protein complexes were further redocked and refined using the Glide module of the Schrödinger Suite, followed by molecular dynamics simulations using the Desmond package to assess binding stability and conformational dynamics. Neuroprotective activity was evaluated in SH-SY5Y human neuroblastoma cells (ATCC-CRL-2266) subjected to pentylenetetrazole (PTZ)-induced neurotoxicity, with cell viability determined by the MTT assay. The computational studies showed Compound-2 had a binding affinity of –8.3 kcal/mol, stronger than diazepam (–6.8 kcal/mol), with interactions involving Phe144, Lys143/162, Glu211, And Tyr212. In vitro, Compound-2 improved SH-SY5Y viability to 59.1%, while Compound-9 restored viability to 94.3%, indicating near-complete reversal of PTZ-induced cytotoxicity. The pronounced effect of Compound-9 correlated with its favorable receptor-binding profile, suggesting GABAergic modulation as the probable mechanism. Benzoxazole–1,3,4-oxadiazole hybrids, particularly Compounds 2 and 9, demonstrated strong GABA_A receptor binding and significant protection against PTZ-induced cytotoxicity. These findings support their potential as neuroprotective leads, warranting further evaluation of mechanisms, in vivo efficacy, and safety for translational application.