Design,Synthesis And Evaluation Of Novel Genistein Derivatives For The Treatment Of Alzheimer’s Disease

Alzheimer’s disease（AD）,the most common form of dementia in elderly people,is a progressive neurodegenerative disorder characterized by loss of cognitive ability,severe behavioral abnormalities,and ultimately death.Its pathogenesis is complex and its etiology is not still completely known,Current therapeutic options for the treatment of AD focuses on cholinesterases inhibitors and N-methyl-D-aspartate receptor antagonist,both of which can only be used to alleviate the symptoms of AD,and do not reverse or heal the disease.Therefore,it is particularly urgent to develop new anti-Alzheimer agents.Due to the complex pathological of AD,a single target can not prevent the progressive of neurodegeneration,multitarget-directed ligands design strategy had becomed a new direction in the research of anti-Alzheimer agents.In this article,we designed and synthesized four series of hybrids based on the multitarget-directed ligands design strategy.O-alkylbenzylamines derivatives of Apigenin,Naringenin,Genistein,and Chalcone.In this paper,9 intermediates and 37 target compounds were synthesized.The structures of all the compounds were confirmed by ¹H NMR and HRMS,and some of the compounds were characterized by ¹³C NMR.The in vitro results displayed that compound 7d was a reversible and highly selective h ACh E inhibitor(IC₅₀=0.53μM).Both the kinetic study and molecular docking showed that 7d was a mixed-type ACh E inhibitor and could bind simultaneously to the catalytic active site and peripheral anionic site of ACh E.Compound 7d also displayed good antioxidant activity（ORAC=1.1 eq）,neuroprotective effects and metal chelation property.Furthermore,compound 7d significantly inhibited self-induced,h ACh E-induced and Cu²⁺-induced Aβaggregation with 39.8%,42.1%and 74.1%,respectively,and remarkably disaggregated Cu²⁺-induced Aβ_1-42 aggregation（67.3%）.More importantly,7d cross blood-brain-barrier in vitro.Further,in vivo assay revealed that 7d significantly reversed scopolamine-induced memory deficit.Therefore,compound 7d was a promising multi-function agent for the development of anti-AD drugs and deserved further study.According to the biological evaluation data in vitro,compound TM-4 as a promising and balanced multi-function agent stood out from the crowd.TM-4 was a reversible and potent h ACh E and h Bu Ch E inhibitor IC₅₀ values of 0.36μM and 15.6μM,respectively,and showed potent antioxidant activity（ORAC=1.2 eq）.Compound TM-4 could significantly inhibit self-induced Aβ_1-42 aggregation(IC₅₀=3.7μM).And the molecular docking provided reasonable explain for its potent Ch Es inhibitory activity and Aβ_1-42 aggregation inhibitory potency.Compound TM-4 was also an ideal neuroprotectant,potential metal chelation agent,and it could inhibit and decompose h ACh E-induced and Cu²⁺-induced Aβaggregation.Moreover,compound TM-4 could activate UPS degradation pathway in HT22 cells and induce autophagy on U87 cells,which contributed to the clearance of the aggregated proteins associated with AD.More importantly,TM-4 could cross BBB in vitro assay,which supported our preliminary design.The in vivo assay displayed that compound TM-4 could remarkably improve dyskinesia recovery rate and response efficiency on Al Cl₃-induced zebrafish AD model,and it exhibited significantly potent neuroprotective effect on Aβ_1-40-induced vascular injury.Further,compound TM-4 did not develop any acute toxicity or mortality immediately at dose of 5000 mg/kg and presented better precognitive effects than donepezil,and the transcriptome sequencing were tested from the brain hippocampus after the above behavioral experiment completed,the results demonstrated that the up-regulated DEGs（such as Enpp2,Atp5h,Zdhhc17 and Eefla1）supported the multi-targets activity of TM-4,and the other up-regulated and down-regulated DEGs might be as a potential target for opening a new way to develop new drugs for the treatment of Alzheimer’s disease.More interesting,for TM-4,the metabolites（blood,urine and feces）in vivo,rat/human liver microsomal metabolism and intestinal flora（rat）metabolism in vitro could provide supports for the pharmacokinetic study of TM-4.This research has provided a good foundation for the development of novel multi-targets-directed anti-Alzheimer’s drugs.