Design and synthesis three novel series of derivatives using natural acetylcholinesterase inhibitor-RLMS as template and in vitro, in vivo and in silico activities verification.

Acetylcholinesterase (AChE) is a pivotal enzyme in nerve conduction, controlling its activity with its inhibitor (AChEI) is crucial for the treatment of Alzheimer's disease (AD). However, current AChEIs are associated with considerable adverse effects. Previous work has identified 2α,3β,19α,23-tetrahydroxy-12-ene-28-oic acid (RLMS) as a promising natural AChEI. This study synthesized three novel series of AChEIs to elucidate the interaction mechanisms between the title enzyme and RLMS. Among the compounds, 1 and 22 emerged as the most potent and selective inhibitors exhibiting both irreversible and mixed competitive inhibition types against AChE. Molecular docking studies at the AChE active sites revealed binding modes that justify its potent enzyme inhibitory effects. Additionally, molecular dynamic simulations demonstrated robust and stable interactions of 1 and 22 with the binding sites of their target. In vitro assays showed derivates, especially 22, exhibited potential neuroprotective activities on HO-induced SH-SY5Y cell injury model. In vivo experiments showed that zebrafish models of AD treated with varying concentrations of 22 displayed obviously increased movement distance and speed, notably, at 25 μM level, 22 effectively reduced apoptosis in zebrafish brain cells. Collectively, this research delineates the intricate relationship between AChE and 22, suggesting its potential as a therapeutic agent for combating AD.