Synthesis, Biological Evaluation, and Molecular Dynamics Studies of Novel 1,2,4-Triazole-Functionalized Benzene-Sulfonate Derivatives as Potential Enzyme Inhibitors and Cytotoxicity in HepG2 Cells

Background/Objectives: In this study, novel benzenesulfonate derivatives containing a 1,2,4-triazole ring were synthesized. Methods: Their molecular structures were characterized by IR, 1H NMR, and 13C NMR spectroscopy, together with Q-TOF LC/MS analyses. For compounds 5b and 5d, the available mass spectrometric data were not used for structural confirmation, and their structural assignments were based primarily on the spectroscopic data. The inhibitory effects of the synthesized compounds (3–5) against acetylcholinesterase (AChE) and glutathione S-transferase (GST) enzymes were evaluated. Results: Comparison of the results with the reference inhibitors, donepezil and ethacrynic acid, showed that compound 3e exhibited the highest AChE inhibitory activity among the tested compounds, with an IC50 value of 1.60 ± 0.198 μM. In addition, compound 4a showed remarkable GST inhibitory activity, with an IC50 value of 1.95 ± 0.492 μM, demonstrating greater potency than the reference inhibitor. The cytotoxic effects of the selected compounds were further assessed against the HepG2 cell line after 24 and 48 h of treatment. Overall, the synthesized molecules exhibited potent enzyme inhibitory activities together with promising cytotoxic effects, highlighting their potential as lead candidates for further biological investigations. In silico analyses were performed to elucidate the molecular basis of the experimentally observed acetylcholinesterase (AChE) inhibitory activity of the synthesized derivatives. Molecular docking against human AChE (PDB ID: 4EY7) identified compound 3e as the most favorable binder among the synthesized series, while 3f also exhibited a strong binding profile. Subsequent 100 ns molecular dynamics simulations of the hAChE–3e, hAChE–3f, and hAChE–donepezil complexes demonstrated stable protein conformations and persistent ligand accommodation within the catalytic gorge. MM/GBSA calculations further indicated a more favorable binding-energy profile for 3e than for 3f, whereas donepezil showed the most favorable overall calculated binding energy. Per-residue decomposition highlighted key gorge residues, including Trp86, Trp286, Tyr337, Tyr341, Phe295, and Phe297, as important contributors to ligand stabilization. Conclusions: Collectively, these computational findings provide structural and energetic support for the experimentally observed AChE inhibitory activity of the lead derivatives.