Identification, Design and Computational Insights of Hybrid Thiadiazole Linked Fused Thiazole-Isoxazole as Potent Dual Inhibitor of AChE/BChE Alzheimer's Disease

Alzheimer’s disease (AD), involves progressive cognitive impairment primarily associated with cholinergic dysfunction. Dual inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) continue to represent a validated and clinically relevant therapeutic approach for alleviating cholinergic deficits in AD. In this study, a novel library of hybrid thiadiazole-linked fused thiazole-isoxazole derivatives (1-12) was rationally designed, synthesized, and fully characterized using 1 H-NMR, 13 C-NMR, FTIR, and HRMS spectroscopic techniques. Enzymatic inhibition assays demonstrated that several compounds exhibited potent dual inhibitory activity against both AChE and BuChE in the low micromolar range. The IC 50 values ranged from 8.42 ± 0.34 to 28.78 ± 0.91 μM for AChE and 9.17 ± 0.60 to 28.16 ± 0.98 μM for BuChE with reference drug donepezil (IC50 = 18.23 ± 0.93 μM for AChE and 20.38 ± 0.81 μM for BuChE). Compounds 6, 5, 3, and 10 emerged as the most promising dual inhibitors showed promising IC50 values of 8.42 ± 0.34 and 9.17 ± 0.60 μM (6), 9.03 ± 0.90 and 9.80 ± 0.84 μM (5), 9.76 ± 0.70 and 9.57 ± 0.63 μM (3), and 10.75 ± 0.19 and 10.28 ± 0.70 μM (10) against AChE and BuChE, respectively. Structure-activity relationship (SAR) analysis revealed that both electron-withdrawing (p-CF 3 , p-F, o,p-Cl, p-NO 3 ) and electron-donating (-OH) substituents on the phenyl ring significantly enhanced inhibitory potency. To gain molecular-level insight, docking studies, ADMET and DFT studies were performed to elucidate ligand-enzyme interactions within the active sites of AChE and BuChE. The results demonstrated favorable hydrogen-bonding and π-π stacking interactions within the catalytic gorge to support the observed experimental activities. Hence, this combined synthetic, biological, and computational investigation identifies the thiadiazole-linked fused thiazole-isoxazole scaffold as a promising lead for the development of next-generation dual cholinesterase inhibitors that offers potential for the rational design of multifunctional anti-Alzheimer’s therapeutics.