Hybrid molecules with dual inhibition of acetylcholinesterase and tau hyperphosphorylation: design, inhibitory activity evaluation, apoptosis assessment, and mechanistic exploration.

A comprehensive therapeutic strategy for Alzheimer's disease (AD) requires simultaneous inhibition of acetylcholinesterase (AChE) and targeting of hyperphosphorylated Tau (P-Tau)-mediated pathogenesis. To address this need, the present study designed a series of hybrid molecules by integrating three pharmacophoric scaffolds with established P-Tau-modulating activity (phenothiazine, dibenzazepine and benzothiazepinones) into AChE-inhibiting frameworks: indanone (derived from the clinical AChE inhibitor Donepezil) or 9-chloro-1,2,3,4-tetrahydroacridine (derived from Tacrine, another clinically approved AChE inhibitor). Following preliminary in silico evaluations including druggability predictions and absorption, distribution, metabolism, excretion, toxicity (ADMET) profiling, twelve compounds (C1-C12) with potential AChE/P-Tau dual-target binding affinity were identified and subsequently synthesized. Among these, four compounds (C5, C6, C7, and C11) exhibited significant AChE inhibitory activity, with IC50 values ranging from 205.3 to 257.1 nM, comparable to that of tacrine (226.0 nM). Notably, the indanone-phenothiazine hybrid compound C11 stood out as the most promising candidate, it achieved the lowest P-Tau/total Tau (T-Tau) ratio (5.30 × 10-6) in okadaic acid (OA)-induced SH-SY5Y cells, outperforming hydromethylthionine mesylate (5.40 × 10-6), a leading clinical candidate for Tau aggregation inhibition. Beyond its dual inhibitory activities, C11 ameliorated OA-induced cell apoptosis, further supporting its potential as anti-AD agent. Subsequent mechanistic explorations confirmed that C11 alleviated oxidative stress and downregulated Tau phosphorylation at specific pathogenic sites (Ser396, Ser262, Thr181). Concurrently, C11 modulated the expression of glycogen synthase kinase-3β (GSK-3β), a critical kinase driving P-Tau formation. In conclusion, this study identifies novel dual-target inhibitors against AChE and P-Tau, and provides new therapeutic insights into AD treatment.