Design,Synthesis And Evaluation Of Novel Apigenin Derivatives As Multi-target Agents For The Treatment Of Alzheimer's Disease

Alzheimer's disease?AD?is a chronic,progressive brain degenerative disease common in the elderly,which is charactered by memory loss,language disorders,and behavioral disorders.Up to now,the pathogenesis of AD has not been fully understood,and its main pathological features include loss of acetylcholine?ACh?level,the deposition of?-amyloid?A??and excessive tau phosphorylation.The currently marketed drugs mainly include cholinesterase inhibitors?donepezil,rivastigmine and galantamine?and N-methyl-D-aspartate?NMDA?receptor antagonists?memantine?.Long-term clinical uses show that these drugs can alleviate the symptoms of AD patients in a short time,but it can not prevent and reverse the course of AD.Therefore,the development of new AD treatment drugs is urgent.In view of the complex pathogenesis of AD,the single-target drug can not prevent the progression of the disease,and multi-targeted drugs targeting AD pathological networks have become a new field of developing anti-AD drugs.Apigenin,a natural flavonoid with multiple pharmacological activities,such as antioxidant activity and neuroprotective effect,but the low bioavailability limits its clinical use in AD.Rivastigmine is a cholinesterase inhibitor approved by FDA for the treatment of mild to moderate AD,and the carbamate fragment is the key pharmacophore for the cholinesterase inhibition.Therefore,accoding to the multi-target directed ligands strategy,apigenin was used as the lead compound,and carbamate fragment of rivastigmine was introduced to apigenin to get novel series of apigenin derivatives.Based on the inverse synthesis analysis of the target compound,apigenin,as the starting material,we reacted with different carbamoyl chloride in the presence of K₂CO₃ in CH₃CN.Finally,seven double substituted and four trisubstituted apigenin-O-carbamate derivatives were obtained by controlling the reaction ratio and reaction time,and the structures of target compounds were characterized by ¹H NMR,¹³C NMR,HR-ESI-MS and HPLC.The cholinesterase inhibitory activity of all target compounds was tested and the structure-activity relationship was discussed.The results showed that the target compounds3a³k had moderate to good acetylcholinesterase inhibitory activity,and the trisubstituted apigenin-O-carbamate showed better acetylcholinesterase inhibitory activity than the double-substituted target compounds.In addition,the carbamate substituent showed obvious effect on the cholinesterase inhibitory activity,and the N-methyl-N-ethyl substituted carbamoyl group showed better AChE inhibitory activity than the other substituents.Compounds 3d and 3i showed the best AChE inhibitory activity with IC₅₀values of 6.8?M and 9.2?M,respectively.The reversibility experiments showed that compound 3d was a reversible AChE inhibitor.In this paper,the antioxidant activity of all target compounds was tested by ORAC method.The results showed that the target compounds 3a³g with bis-carbamate showed better antioxidant activity than the trisubstituted carbamate compound 3i³k,indicating the hydroxyl group contributed to the antioxidant activity.The inhibition and disaggregation activities of A?_1-42-42 self-aggregation towards compounds 3d and 3i were tested by thioflavin T method and transmission electron microscopy.The results showed that the compound 3d showed good inhibition activity?77.9%?and disaggregation activity?66.2%?,and compound 3i showed appropriate inhibition and disaggregation with 35.2%and 40.4%,respectively.In addition,the docking study provided a reasonable explanation for the inhibition of compound 3d.The neuroprotective effects of compounds 3d and 3i on H₂O₂-induced PC12 inJury were tested by MTT assay.The results showed that compound 3d possessed better neuroprotective effect than 3i,and showing that the hydroxyl group of compound 3d played an important factor.In this paper,the chelation property of compounds 3d and 3i was tested using UV-Vis spectroscopy.The results showed that compound 3d was a selective metal chelating agent,while compound 3i did nto show any chelation property.Further,the results of the Thioflavin T method and the transmission electron microscopy test showed that compound3d could inhibit?78.9%?and disaggregate?64.6%?Cu²⁺-induced A?_1-42-42 aggregation.In this paper,the parallel artificial membrane permeation-blood-brain barrier model?PAMPA-BBB?was used to evaluate blood-brain barrier permeability in vitro of compounds 3d and 3i.The results showed that the permeabilities of compounds 3d and 3i were 15.49×10^-66 cm/s and 19.16×10^-66 cm/s,respectively,indicating that compounds 3d and 3i could penetrate the blood-brain barrier in vitro.Further,3d indicated remarkable dyskinesia recovery rate and response efficiency on AlCl₃-induced zebrafish AD model,and exhibited surprising protective effect on A?_1-40-induced vascular injury.The above results indicated that compound 3d was a highly efficient and balanced multi-target candidate compound,which provides important clues and theoretical basis for the development of multi-target anti-AD drugs.