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 (IC₅₀ = 0.36 μM) and huBChE (IC₅₀ = 15.3 μM) inhibitor, and showed potent antioxidant activity (ORAC = 1.2 eq). TM-4 could significantly inhibit self-induced Aβ_1-42 aggregation (IC₅₀ = 3.7 μM). TM-4 was also an ideal neuroprotectant, potential metal chelation agent, and it could inhibit and disaggregate huAChE-induced and Cu²⁺-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 AlCl₃-induced zebrafish AD model, and TM-4 indicated surprising protective effect on Aβ_1-40-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.

在这项工作中，我们已经开发了一系列新的多目标导向的配体，以解决低水平的乙酰胆碱（ACh），氧化应激，金属离子失调和蛋白质折叠错误的问题。合成了新的芹菜素-多奈哌齐衍生物，柚皮苷-多奈哌齐衍生物，染料木素-多奈哌齐衍生物和查尔酮-多奈哌齐衍生物，体外结果表明TM-4是可逆的有效的hu AChE（IC ₅₀  = 0.36μM）和hu BChE（ IC ₅₀  = 15.3μM）抑制剂，并显示有效的抗氧化活性（ORAC = 1.2 eq）。TM-4能显著抑制自感应的β _1-42聚集（IC ₅₀  = 3.7μM）。TM-4也是一个理想的神经保护剂，潜在的金属螯合剂，故能抑制和集计胡胆碱酯酶诱导和Cu ²⁺诱导的阿β聚集。此外，TM-4可以激活HT22细胞中的UPS降解途径，并诱导U87细胞自噬以清除与AD相关的异常蛋白。更重要的是，TM-4可以通过BBB体外测定。此外，体内试验表明，TM-4在AlCl ₃诱导的斑马鱼AD模型中表现出显着的运动障碍恢复率和响应效率，并且TM-4表示在一个令人惊讶的保护作用β _1-40诱导的血管损伤。TM-4表现出对东pol碱引起的记忆障碍的认知前作用。通过转录组测序进一步证实了TM-4对多靶点的调控。更有趣的是，还研究了大鼠和大鼠/人肝脏微粒体对TM-4的血液，尿液和粪便的代谢。总体而言，TM-4是开发针对阿尔茨海默氏病的药物的有希望的多功能候选产品。