Design, Synthesis And Activity Study Of Ligustrazine Derivatives Based On Early Drugability Evaluatio

Background:With the application of compatibility theory and combination principle,we had chosen several active ingredients to synthesize a series of ligustrazine-phenolic acid derivatives and ligustrazine-cinnamic acid derivatives in previous research.The results showed that ligustrazine derivatives presented neuroprotective effects on injured differentiated PC 12 cells,of which,T-VA（C24H28N4O4）displayed promising protective effect.However,because of carboxyl groups in phenolic acids and cinnamic acids,all these ligustrazine derivatives have ester bonds.Compounds with ester or amide bonds like T-VA,are liable to enzymatic hydrolysis in vivo such as the action of plasma esterases.Optimization of druggability is of great significance to improve the efficiency of new drug research and development.Research purpose:With the strategy of druggability optimization,to design and investigate nerve protection activity,metabolic stability and cardiac toxicity of different kinds of ligustrazine derivative,and provide new ideas of the discovery of efficient,more stabilized and safer ligustrazine derivatives.Research methods:Via LC/LTQ-Orbitrap MS to investigate the mass fragmentation of T-VA and study its metabolites in rats.According to the suppositional structure,via condensation reaction synthesized the main metabolite,and evaluated its neuroprotective effect.A series of ligustrazine derivatives were synthesized by conjoining ligustrazine and phenols（4-Hydroxybenzyl alcohol,tyrosol,vanillyl alcohol,4-Hydroxybenzylamine,tyramine,vanillylamine）with ester,ether and amide bonds.Those designed compounds were evaluated for their neuroprotective effects against CoCl₂ injured differentiated PC 12 cells.The structure-activity relationships of these novel compounds are also briefly discussed.To verify potential druggability of ligustrazine derivatives with ether,amide and ester bond（2c,4c,T-VA）,their plasma stability and liver microsomal metabolic stability was preliminarily evaluated in vitro.Based on high content analysis technology,the real-time multi-parameter myocardial toxicity evaluation model of H9c2 was established and the cardiotoxicity of 2c,4c,T-VA were evaluated.Research results:The main metabolite of T-VA（M1）was found Via LC/LTQ-Orbitrap MS,and the structure of M1 showed that the ester bond is the liable metabolism site of T-VA.According to the suppositional structure of M1,M1-1 was synthesized via condensation reaction.By comparing the mass spectrum characters and chromatographic features of M1-1 and M1,it can confirm the exact structure of M1.Furthermore,the neuroprotective effect of M1 in differentiated PC 12 cells was evaluated.As a result,M1 in different concentrations could protect PC 12 cells injured by CoCl₂(EC₅₀=16.01 μM).21 compounds were designed and synthesized by conjoining ligustrazine and phenols with ester,ether and amide bonds.The structures of the entire ligustrazine derivatives were confirmed by ¹H-NMR,¹³C-NMR and HRMS.The results showed that TMP and its derivatives presented protective effects on injured differentiated PC 12 cells and most of the ligustrazine derivatives were more active than TMP,among them 2c was the most active compound,with an EC₅₀ value of 1.07 μM.Pretreatment with 2c dramatically alleviated morphological manifestations of cell damage and led to an increase in neurite-bearing cells compared to model cells.The metabolic stability of ligustrazine derivatives with ether,amide and ester bond（2c,4c,T-VA）in rat plasma and liver microsomal showed that 2c and 4c were stable during metabolism;and because of its ester bond,T-VA was liable to enzymatic hydrolysis in rat plasma and liver microsomal.The effectiveness of the real-time multi-parameter myocardial toxicity evaluation model,which based on high content analysis technology,was verified by Dox.The results of high content analysis showed that 2c without apparent cardiac toxicity,and T-VA can causes a decline in the number of cells,shrinking nucleus,reducing mitochondrial membrane potential and disturbing the calcium balance,has a certain cardiac toxicity.4c merely can cause a decline of the H9c2 cell number,and the toxicity is inferior to T-VA,it may cause myocardial cell damage by other pathway.Conclusion:The results of metabolic studie showed that T-VA is liable to hydrolysis in vivo.The neuroprotective effect of the main metabolite is weaker than T-VA.These results mean that the activity of T-VA is influenced by the metabolic stability.Optimization of druggability is of great significance to improve the efficiency of new drug research and development.Ligustrazine derivatives containing ether bonds exhibited high potency,with EC₅₀ values below 15 μM;among them 2c was the most active compound,with an EC₅₀ value of 1.07 μM.Furthermore,we found these ether conjuncted derivatives which containing bis-ligustrazine substituents showed better neuroprotective activities than the single-ligustrazine componds.Base on the above evidence,the additive methoxy moiety might enhance the protective activity.The metabolic stability of ligustrazine derivatives with ether,amide and ester bond（2c,4c,T-VA）in rat plasma and liver microsomal showed that change the metabolic site by structural modification can improve the metabolic stability.This study provides useful information for future design of analogous ligustrazine derivatives.The results of high content analysis showed that ligustrazine derivatives may have cardiac toxicity at the the largest drug concentration of activity evaluation,and can affect its subsequent development.These prove that evaluate cardiac toxicity in the early stages of ligustrazine derivatives research,can help identify potential toxicity is not suitable to continue to develop,is of great significance to improve the efficiency of new drug research and development.