Nemr_2026_Drug.Dev.Res_87_e70259

Multi-target directed ligands (MTDLs) advocate for a novel therapeutic framework for Alzheimer's disease (AD) owing to its complicated multifactorial nature. Therefore, the present study focuses on designing and synthesizing pyrazolo[3,4-d]pyrimidine derivatives, followed by biological evaluation as MTDLs for AD. Most of the obtained derivatives showed potent AChE inhibitory efficacy, significant suppression of Abeta, and pronounced antioxidant activity in vitro. Compound 8b (IC(50) = 0.346 microM) and 9a (IC(50) = 0.168 microM) are the most potent inhibitors of AChE relative to donepezil (IC(50) = 0.213 microM). Furthermore, both 8b (IC(50) = 1.475 microM) and 9a (IC(50) = 1.060 microM) exhibited remarkable Abeta inhibitory activity, surpassing that of donepezil (IC(50) = 7.944 microM). Also, they demonstrated remarkable antioxidant capacity, with an ORAC index of 1.66 and 0.90 Trolox equivalents. The in vivo investigation of the most potent compounds (8b and 9a) revealed a significant improvement in scopolamine-induced cognitive deficits in animals. The molecular docking of compounds 8b and 9a within the human acetylcholinesterase (hAChE) active site showed auspicious binding with both catalytic active site (CAS) and peripheral anionic site (PAS). In addition, molecular dynamics (MD) simulations were employed to investigate the stability of compounds 8b and 9a within the hAChE active site and to support the reliability of the docking analysis. Finally, In silico physicochemical and pharmacokinetics prediction analyses were performed, and the findings were well aligned with their corresponding in vitro results. Overall, the results highlight 8b and 9a as potential multifunctional candidates for AD.