Design,Synthesis And Activity Exploration Of Multi-target Anti-AD Cheating Agents Based On The Coordination Theory Of Traditional Chinese Medicine With Emodin As The Lead Compound

Alzheimer’s Disease（AD）is the most common age-related neurodegenerative disease,which seriously threatens the health of the elderly.With the increasingly obvious trend of population aging in China,the number of AD patients is increasing year by year,which brings heavy medical and economic burdens to individuals,families and society.The exact pathogenesis of AD is not clear,and genetic,lifestyle and environmental factors may all be related to its occurrence and progression.Neurotransmitter deficiency,βamyloid（Aβ）aggregation,oxidative stress,inflammation and abnormal Tau protein phosphorylation are common pathogenic factors of AD.Drugs targeting acetylcholinesterase（ACh E）,which is a key enzyme in biological nerve conduction,are the main drugs for the treatment of AD.These drugs can improve patients’cognition,but the therapeutic effect is limited.Objective:Since it is difficult for a single target drug to deal with the complex pathogenesis of AD,the design and development of multi-target directed ligands（MTDLs）is a new strategy for the research and development of anti-AD drugs.More and more studies have shown that biometals(Cu²⁺,Zn²⁺,Fe²⁺/Fe³⁺)in the brain are involved in synaptic activities,and there is a certain connection between metal ion homeostasis and common targets of AD.Therefore,metal related therapeutics has developed rapidly,especially the research of metal chelators has become a hot spot in this field.According to the coordination chemistry theory of Traditional Chinese Medicine（TCM）,most active molecules of TCM contain coordination groups such as hydroxyl,carbonyl and carboxyl,which can be used as ligands to form complexes with metal ions and produce mutual synergy and antagonism.Using the natural chelating sites of active ingredients of TCM to form multi-target anti-AD chelating agents through computer-aided directional design is not only an effective means of new drug research and development of AD,but also an important way of new drug development of TCM.Emodin（1,3,8-trihydroxy-6-methylanthraquinone）is a natural anthraquinone containing polyphenol hydroxyl groups and has natural chelating sites.It mainly exists in rhubarb,polygonum cuspidatum,horseshoe flower,cassia seed and other TCM.Recent studies have shown that anthraquinone compounds have good antioxidant and neuroprotective effects,and can be used to treat neurodegenerative diseases such as AD.In this thesis,based on the coordination theory of TCM,emodin,a TCM component with a natural chelation site,was selected as the lead compound,aiming at the commonly used targets related to AD,using computer-aided design to carry out directional modification of emodin to synthesize a series of multiple targeted anti-AD chelators.And the anti-AD activities were evaluated in order to provide new ideas and insights for the research of new multi-target anti-AD drugs.Methods:1.The abilities of the designed emodin derivatives to inhibit Aβ_1-42 aggregation activities,inhibit cholinesterase activities,penetrate the blood-brain barrier（BBB）,drug induced liver injury and oral absorption availability were preliminarily judged by computer-aided prediction method.2.Taking emodin and tertiary amine salt as raw materials and potassium iodide as catalyst,the nucleophilic reaction took place in a suitable solvent system to obtain emodin derivatives and structural characterizations were carried out on these compounds.3.The complexation abilities and ratio between emodin derivatives and metal ions were studied by UV spectrophotometry.The inhibitory abilities of Aβ_1-42 aggregation of emodin derivatives were evaluated by sulfur T method（Th T）.The anti-ACh E activities of emodin derivatives were tested by Ellman’s colorimetry,and the anti-ACh E type of compounds was verified by kinetic method.The free radical scavenging abilities of emodin derivatives were evaluated by colorimetry and fluorescence probe method,and the antioxidant mechanisms of emodin derivatives were studied by cyclic voltammetry.Finally,the cytotoxicities of emodin derivatives and their protective effects on H₂O₂ induced cell injury were evaluated by MTT assay.Results:1.Through computer-aided prediction,the designed emodin derivatives could inhibit ACh E and could act on both catalytic active site（CAS）and peripheral anion binding site（PAS）;emodin derivatives could also inhibit Aβ_1-42 aggregation and had appropriate BBB permeability;emodin might cause drug induced liver injury,but the derivatization of emodin could effectively reduce the toxicity.2.A series of emodin derivatives were synthesized and characterized by melting point,NMR,MS and elemental analysis.3.Emodin and its derivatives had strong chelating abilities to Fe³⁺and Cu²⁺ions,poor chelating abilities to Al³⁺ions,compound a had good chelating abilities to Zn²⁺ions,and other compounds had weak chelating abilities to Zn²⁺ions.The coordination ratio between emodin derivatives and metal ions was 2:1.4.Compounds a,b and d had good inhibitory effects on Aβ_1-42 aggregation(self-aggregation,Cu²⁺-induced aggregation and ACh E-induced aggregation),and the inhibition rates of a and b were more than 65%.5.The activity of compounds a and b in inhibiting ACh E was comparable to that of the reference drug Tacrine.Among them,a had the strongest inhibitory effect on ACh E with a half inhibitory concentration(IC₅₀)of 0.067μM.Enzyme kinetic analysis showed that compounds a and b were mixed inhibition,which was consistent with the results of molecular docking.The complexes of emodin derivatives chelated with Cu²⁺and Zn²⁺showed better ACh E inhibitory activities and antioxidant activities.6.Emodin derivatives had strong antioxidant capacities.Compared with reference drugs,emodin derivatives had better scavenging capacities of hydroxyl radical（·OH）and peroxy radical,and the scavenging capacity of superoxide anion radical（.O₂^-）and ABTS.⁺radical were slightly weaker,but they were stronger than those of emodin.Cyclic voltammetry experiments showed that the scavenge of O₂^-radicals of emodin derivatives followed a single electron transfer mechanism that were concentration-dependent.7.The cytotoxicities of emodin derivatives were within the safe range.Emodin derivatives had protective effect on H₂O₂ induced cell damage.Conclusion:Four novel emodin derivatives were designed and synthesized.Combining various biological evaluation indicators,compounds a and b inhibited the self-induced aggregation,Cu²⁺-induced aggregation and ACh E-induced aggregation of Aβ_1-42 by more than65%.Compounds a and b could selectively inhibit ACh E and had higher ACh E inhibitory activities and antioxidant activities,and the metal chelating complexes showed better anti-ACh E activities and antioxidant activities.The toxicities of emodin derivatives were also within the safe range.From the above analysis of multiple dimensions,compounds a and b are in accordance with the design concept of multi-target anti-AD drugs,and are worthy of further study.