In this work, we have developed a novel series of multi-target-directed ligands to address low levels of acetylcholine (ACh), oxidative stress, metal ion dysregulation, and the misfolded proteins. Novel apigenin-donepezil derivatives, naringenin-donepezil derivatives, genistein-donepezil derivatives and chalcone-donepezil derivatives have been synthesized, in vitro results showed that TM-4 was a reversible and potent huAChE (IC = 0.36 μM) and huBChE (IC = 15.3 μM) inhibitor, and showed potent antioxidant activity (ORAC = 1.2 eq). TM-4 could significantly inhibit self-induced Aβ aggregation (IC = 3.7 μM). TM-4 was also an ideal neuroprotectant, potential metal chelation agent, and it could inhibit and disaggregate huAChE-induced and Cu-induced Aβ aggregation. Moreover, TM-4 could activate UPS degradation pathway in HT22 cells and induce autophagy on U87 cells to clear abnormal proteins associated with AD. More importantly, TM-4 could cross BBB in vitro assay. In addition, in vivo assay revealed that TM-4 exhibited remarkable dyskinesia recovery rate and response efficiency on AlCl-induced zebrafish AD model, and TM-4 indicated surprising protective effect on Aβ-induced vascular injury. TM-4 presented precognitive effect on scopolamine-induced memory impairment. And the regulation of multi-targets for TM-4 were further conformed through transcriptome sequencing. More interesting, the blood, urine and feces metabolism in rat and rat/human liver microsome metabolism towards TM-4 were also investigated. Overall, TM-4 is a promising multi-function candidate for the development of drugs to Alzheimer’s disease.
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