Design,Synthesis,Single Crystal Analysis And Activity Studies Of Multi-target Anti-AD Lead Compound Of Chrysin In Traditional Chinese Medicine Alpinia Oxyphylla Miq.Fructus

Alzheimer’s disease（AD）is the most common neurodegenerative brain disease.With further exploration of the pathogenesis of AD,researchers have found that the occurrence and pathological changes of AD are not caused by a single cause.It has extremely complex pathological mechanisms,such as cholinergic loss,b-Amyloid（Ab）deposition,hyperphosphorylation of tau,oxidative stress and metal ion homeostasis imbalance.Based on the characteristics of multi-target and multi-factor of AD,therapeutic drugs targeting only a single target cannot fundamentally solve or delay the pathological process of AD.Therefore,"one molecule,multiple targets"multi-target-directed Ligands（MTDLs）therapy strategy emerged.This strategy can modulate multiple biological targets simultaneously,and has the advantages of higher efficacy,better safety and simpler drug administration.Studies have shown that it is a potential method to screen active small molecules for the treatment of AD from natural products.Alpinia oxyphylla Miq.Fructus,a traditional Chinese medicine,has been regarded as a classic Chinese medicine for the treatment of dementia since ancient times and has the function of neuroprotection and intelligence enhancement.Chrysin（5,7-dihydroxyflavone）is one of the important natural flavonoids in this traditional Chinese medicine,which has the characteristics of abundant sources,easy access and high feasibility of study.Several studies have shown that chrysin has a variety of biological activities,including anti-inflammatory,antioxidant and neuroprotective effects.In this work,we planned to construct the directed ligand based on the MTDLs strategy with chrysin as the lead compound,and preliminarily evaluate its synergistic anti-AD activities,in order to obtain the candidate drug of multi-target anti-AD.In this study,the natural active small molecule—chrysin in Alpinia oxyphylla Miq.Fructus was screened out based on literature investigation and research.Based on the MTDLs strategy,the orientation design of chrysin was carried out,and series of amines derivatives（1～5）and amides derivatives（6～9）were synthesized by nucleophilic substitution reaction.The characterization data is consistent with the design goals.The selective chelating ability of the derivatives to various metal ions was investigated by UV–vis spectrophotometric titrations.The research results showed that compounds 1～5 can selectively chelate Cu²⁺,Fe²⁺,Zn²⁺and Al³⁺,especially for Cu²⁺.The copper complexes were further synthesized.Two single crystals of copper complexes,[Cu(C₁₉H₁₈NO₄)₂]and[Cu C₂₁H₂₂Cl₃NO₅],were prepared for the first time by solvent evaporation method.X-ray crystal structure analysis provided a reliable theoretical basis for their biological activity at the molecular level.The inhibitory activity and type of action of chrysin derivatives and their copper complexes on cholinesterase（Ch E）were studied by Ellman method.The research results shown that the derivatives were potent inhibitors of acetylcholinesterase（ACh E）and butylcholinesterase（Bu Ch E）.Compound 1 and8 showed the highest Bu Ch E selective inhibition activity.Kinetic experiments show that they are mixed inhibitors（competitive inhibition+non-competitive inhibition）.The hydrolysis of Bu Ch E was detected by high performance liquid chromatography（HPLC）in vitro under simulated physiological conditions.Compound 8 could release the small molecule of chrysin,indicating that compound 8 had the characteristics of"hidden"multi-target ligand,which was in line with the design strategy.The antioxidant activities of the derivatives and metal complexes were studied by Fenton reaction,cyclic voltammetry（CV）and oxygen radical absorbance capacity-fluorescein（ORAC-FL）.The research results showed that the chrysin derivatives had certain scavenging activity against·OH and·O₂^-,especially the scavenging activity against·OH was more than 300 times that of vitamin C.Compounds 1～5 could form complexes with Cu²⁺and their antioxidant activities of complexes were significantly increased.The aggregation inhibition activity of the derivatives on Ab_1-42was further investigated.The results showed that the derivatives had certain inhibitory effects on self-induced,Cu²⁺-induced and ACh E-induced Aβ_1-42 aggregation.The neurocytotoxicity of representative derivatives was investigated by MTT assay.Finally,the binding modes of the derivatives with ACh E,Bu Ch E and Ab_1-42 were studied by semi-flexible molecular docking method.The results showed that the chrysin derivatives had certain affinity for ACh E,Bu Ch E and Aβ_1-42,and could bind to the catalytic active sites（CAS）and peripheral binding site（PAS）of ACh E and Bu Ch E,which verified the results of kinetic experiments.The prediction results showed that the derivatives had good blood-brain barrier（BBB）penetration and drug-like properties.In this study,nine novel chrysin derivatives were synthesized based on MTDLs strategy,and most of them showed Ch E inhibitory activity,strong antioxidant activity and Aβ_1-42aggregation inhibitory activity.Compound 1 and 8 showed the best selective inhibition of Bu Ch E,which may play a therapeutic role in treating the high Bu Ch E activity in the brain of patients with advanced AD.X-ray single crystal analysis of metal complex provides reliable basis for structural activity relationship analysis at molecular level.In addition,the prediction results of drug-like properties showed that these compounds have strong BBB penetration and drug-like properties.These results will provide new ideas and methods for the research of innovative traditional Chinese medicine drugs based on chrysin,and also provide certain molecular models and theoretical basis for the design and development of multi-targeted anti-AD drugs.