Design And Synthesis Of Phenolic Acid Amide Derivatives And Their Anti-α-synuclein Aggregation Activity

Parkinson’s disease(PD)is a neurodegenerative disease.The formation of intracellular protein inclusion bodies are the characteristics of neuropathology in PD,which is mainly composed of misfolded and aggregated of α-Synuclein(α-Syn).More and more data suggest that there is a close relationship between α-Syn and the pathogenesis of PD.The aggregation of α-Syn has become the most important participant in the neurodegenerative process that occurred in the context of PD and other neurodegenerative diseases(collectively referred to as synucleinopathy).Therefore,controlling the aggregation of α-Syn is an effective way to PD therapeutic intervention.Up to date,a number of natural and synthesized small molecular compounds have been reported as the optional inhibitor of α-Syn aggregation.Especially,the phenolic compounds,which includes many natural products such as protocatechuic acid,gallic acid,caffeic acid,ferulic acid,rosmarinic acid,chlorogenic acid,etc,have significantly inhibitory effects on the formation of α-Syn oligomers and fibrils.1.For the main goal of inhibiting aggregation of α-Syn,the gallic acid(GA)is effectively modified by enhancing its lipophilicity,permeability of the blood-brain barrier and improving its anti-aggregation performance of α-Syn.These analogs can reversibly binding to the α-Syn β-sheet structure and disturbing the early formation of oligomers,thereby preventing the development of α-Syn fibrillation.In this paper,some small molecules with planar conjugated sheet-like characteristics were designed and synthesized,include amide derivatives of phenolic acids and amide derivatives of GA as following:A total of 23 compounds was synthesized.Using p-phenylenediamine,benzenesulfonic acid and benzoic acid compounds as raw materials,N-acyl-p-phenylenediamine compounds were synthesized by HATU acylation,and then N-acyl-p-phenylenediamine compounds with carboxylic acid and sulfonic acid compounds were further acylated to obtain N,N’-diacyl-p-phenylenediamine compounds.The GA derivatives were obtained by demethylation of the methyl-protected GA acyl-p-phenylenediamines under boron tribromide.These compounds were all characterized by NMR and HRMS.2.All synthetic compounds were evaluated for inhibitory activities against α-Syn aggregation(inhibition rate).The thioflavin T(Th T)fluorescence data shown that the inhibition rate of most GA amide derivatives was more than 80%,up to 94.4%,which was a significant improvement over GA(78.2%).The GA amide derivatives display excellent anti-α-Syn-aggregation activities.3.Some representative GA amide derivatives with better inhibition rate were selected to study their IC50 and anti-aggregating kinetic.The IC50 values of these compounds were all in micromolar range,down to 0.98 μM,further indicated that the GA amide derivatives have good inhibitory activities.The kinetic results showed that the aggregation of α-Syn included three phases: the initial phase,the exponential phase and the equilibrium phase,and the addition of compounds 3bb,3be or 4ba performed inhibitory effects on the initial stage of α-Syn fibril formation and throughout the entire aggregation process.4.Transmission Electron Microscope(TEM)analysis.The aggregation form of individual α-Syn was a typical network and bundled fibrils,the addition of compounds 3bb,3be or 4ba made the aggregated form of α-Syn fibrils appear sparse,shorter and finer,which intuitively shown that these compounds effectively inhibit α-Syn aggregation.5.Circular Dichroism(CD)detection.The changes of α-Syn secondary structure were monitored at initial state(0 day)and end state(3 days)of incubation.At the initial state,the spectrum of un-incubated α-Syn showed a random coil conformation with a highly negative ellipticity at 198 nm.At the end state,the negative ellipticity at198 nm decreases,which indicated a decrease in the number of random curl conformations,this result was corresponded to the formation of α-Syn fibrils.The addition of compounds 3bb,3be and 4ba significantly reduced the degree of negative ellipticity at 198 nm,which indicatied these compounds inhibited the conformational transition of α-Syn.This result was also corresponded to the inhibitory activity onα-Syn aggregation.A series of phenolic acid amide derivatives including GA were designed and synthesized in this paper,most of which have suitable molecular polarity and strong blood-brain barrier permeability.Various methods were used to evaluate and analyze its inhibitory activity on α-Syn aggregation and some novel GA amide derivatives display better anti-α-Syn-aggregation activity than GA,which provide the practical and theoretical supports on the candidate drugs development for prevention and treatment of PD.