Synthesis And Cholinesterase Activity Evaluation Of Piperonylic Acid Derivatives

Alzheimer's disease?AD?is a neurodegenerative disease.Its etiology and pathogenesis are still unclear.There are multiple hypotheses such as cholinergic hypothesis,?-amyloid hypothesis,tau protein hypothesis,etc.Up to now,acetylcholinesterase inhibitor?AChEI?,which is developed using acetylcholinesterase?AChE?as a target,has achieved significant efficacy and can improve cognitive impairment in patients with mild-to-moderate AD.However,the new anti-AD drugs developed with?-amyloid,BACE1??-secretase?,and tau protein hypothesis as targets have all failed.The existing AChEIs also have some problems.For example,Tacrine has hepatotoxicity,Rivastigmine has gastrointestinal adverse reactions,and Donepezil causes individual arrhythmia.Therefore,the development of new AChEI still has good research value and prospect.It is an important approach to obtain new AChEI from natural products.Natural products have a variety of biological activities and are compliant with humans.However,the structure of natural products is complex.It is difficult to mass produce for natural products.However,it can be obtained a large number of derivatives through developig simple natural product analogues in a short time.The possibility of obtaining the ideal drug can be greatly increased.In the previous,our group simplified the structure of Kivaconin B.The simple structure of chalcone skeleton was reserved.A series of chalcone and halochalcone derivatives,which contained tertiary amine side chains,was designed and obtained.Their inhibitory activity against AChE was strong.Based on the research of chalcone,a series of ferulic acid and cinnamic acid amide derivatives containing tertiary amine side chains had been designed and obtained as same as the structure of chalcone.They also had inhibitory activity against AChE.The research of the structure-activity relationship showed that the structure type of the tertiary amino group,the substitution position of the tertiary amino group,and the side chain length of the tertiary amine were all important factors affecting the AChE inhibitory activity.Piperic acid is a natural organic carboxylic acid containing methylenedioxy.At present,it has been found that piperonylic acid has antioxidant and antibacterial activity,and it can also inhibit the activity of tyrosinase.The project has designed and synthesized a series of piperonylic acid amide derivatives containing different side chain groups and different substitution positions.Then,it was further modified by introducing carbon-carbon double bonds and methoxy groups to explore the effect of methylenedioxy,carbon-carbon double bond,and methoxy groups on cholinesterase inhibitory activity.The purpose of the project is to obtain a class of new acetylcholinesterase inhibitors with good selectivity and high activity.The paper mainly includes the following aspects:?1?A series of piperonylic acid amide derivatives(A_2c–A_4j)were designed and synthesized to investigate the effect of the type of side chain group and its substitution position on cholinesterase activity.The research of the structure-activity relationship showed that the tertiary amine side chain was essential for anti-AChE activity.Simultaneously,para-substituted compounds had stronger inhibitory activity against AChE among them.The enhancement of inhibitory activity fllowed the order:dimethylamino>pyrrolyl>piperidyl>diethylamino.when the substitution position was the same,the compounds containing tertiary amine substituents were more sensitive to cholinesterase.Simultaneously,the substitution position of the tertiary amine side chain had stronger AChE inhibitory activity in the para-position and weaker AChE inhibitory activity in the ortho-position.Among them,compound A_4c had the strongest AChE inhibitory activity(IC₅₀=1.33±0.03?mol/L)and the highest AChE selectivity?selectivity=91.32?.The enzymatic kinetics of AChE were studied by compounds(A_4c and A_4e)which showed better inhibitory activity.The enzyme kinetics study showed that compound A_4c and compound A_4e belonged to the mixed inhibitor of AChE.They can combine with the catalytically active centers?CAS?and can the perioheral binding site?PAS?of AChE.?2?The structure of the para-substituted piperonylic acid amide derivatives containing tertiary amine side chain was further modified.A series of 3,4-?methylenedioxy?cinnamic acid amide derivatives(B_2c–B_2h),veratric acid amide derivatives(C_2c–C_2h)and anisic acid amide derivatives(D_2c–D_2e)were designed and synthesized to explore the effects of carbon-carbon double bonds and the mutual conversion between methylenedioxy and methoxyl on cholinesterase activity.The research of the structure-activity relationship showed that the introduction of carbon-carbon double bonds and methylenedioxy ring-opening analogues containing two methoxy groups can significantly increase the inhibitory activity anti-AChE.When the tertiary amine side chains were dimethylamino,piperidyl,and pyrrolyl substituents,the compounds had enhanced AChE inhibitory activity.The enhancement of inhibitory activity fllowed the order:dimethylamino>pyrrolyl>piperidyl.The ring-opening analogues have more significant than the introduction of carbon-carbon double bonds.Among them,compound C_2c had the strongest AChE inhibitory activity(IC₅₀=0.88±0.02?mol/L)and the highest AChE selectivity?selectivity=270.78?.?3?Molecular docking simulations was used to visually recognize the interplay of the compound with the surrounding residues near the active pocket of the protein.Molecular docking simulations showed that the compounds were able to interact with the amino acids of functional sites of AChE,such as His 440 in the active site of AChE,the choline binding site Phe 330,and the aromatic amino acid residue Phe 288on the inner surface of AChE valley.It plays a role in preventing the catalytic hydrolysis of acetylcholine?ACh?,reducing the affinity and binding between AChE and the substrate,,and reducing enzyme activity.?4?pKa and the logP were determined by potentiometric titration and shake flask method-UV spectrophotometry,respectively.The pKa of compound A_2c-A_4j ranged from 5.62 to 7.49.The logP values of the most compounds ranged from 0 to 5.Preliminary results indicated that most of piperonylic acid amide derivatives had good absorptive capacity.Stability experiment were studied by compound A_4c which showed better inhibitory activity.The results showed that the compound A_4c had good stability in the acid and alkali environment.However,compound A_4c had a certain degree of response to strong light.It needed to be stored in shading.