Mechanistic exploration of 4-bromobenzyloxy-1,2,3-triazole derivatives as dual cholinesterase inhibitors: Kinetic, molecular dynamics, and halogen-contact insights

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) regulate cholinergic neurotransmission, and their partial inhibition is an established symptomatic strategy in Alzheimer's disease; clarifying how individual ligands engage these enzymes is therefore central to inhibitor design. To probe these cholinesterase-ligand interaction mechanisms, a series of eight 4-bromobenzyloxy-1,2,3-triazole derivatives (10a-10 h) was synthesized and evaluated as AChE and BChE inhibitors. All compounds displayed concentration-dependent inhibition of both enzymes at micromolar concentrations (IC50 = 1.52-4.05 µM for AChE and 2.18-7.83 µM for BChE). Compound 10e was the most effective AChE inhibitor and compound 10 g the most effective BChE inhibitor. Detailed kinetic analysis, with inhibition constants obtained by global nonlinear regression and the mechanism assigned by Akaike-based model selection, revealed predominantly mixed-type inhibition (Ki = 0.95 µM, α = 2.10 for 10e-AChE; Ki = 1.55 µM, α = 2.14 for 10g-BChE) and predominantly noncompetitive inhibition for 10d, indicating simultaneous or allosteric engagement of the catalytic and peripheral sites. These mechanisms were corroborated by molecular docking, 100 ns molecular dynamics simulations, and MM/GBSA analysis, and the docking binding energies correlated closely with experimental potency across the series (r2 > 0.99). Geometric analysis showed that 10 g engages the BChE gorge through an additional short, non-linear C-F···O contact rather than a classical halogen bond. In silico DFT, ADMET, and toxicity predictions indicated generally drug-like, CNS-penetrant profiles for most derivatives, although 10d was flagged for potential genotoxic liability and deprioritized. Collectively, these findings characterize the dual cholinesterase inhibitory activity of the 4-bromobenzyloxy-1,2,3-triazole scaffold and position 10e and 10 g as effective lead-like compounds warranting further optimization.