Synthesis,Biological Activity Evaluation And Chromatographic Resolution Of Pyrrolidone Nerve Cell Protectants

Based on the related literature of the synthesis and biological activities of pyrrolidone neuroprotective agents,we found that it would be improved the neuroprotection of nerve cells if the polysubstituted pyrrolidone compound-optically active clausenamidone is connected to the more electronegative ester group at the C-3.Thus the optically active clausenamidone derivatives with the 3-ester substitued were designed and synthesized,and then they were evaluated in a model of glutamate-induced neuronal injury to determine the neuroprotective activity.At the same time,the research of structure-activity relationship of this kind of compounds shows that there are obvious stereochemical differences in pharmacological effects,those of the optical isomers of this kind of compounds in particular.Therefore,it is essential to determine the optical purity of pharmacological test compounds.In this paper,high performance liquid chromatography?HPLC?chiral stationary phase was used to investigate and separate the compounds.And the method of optical purity ee%was established to provide chromatography methodology basis for the compounds of this structural type.First,the optically active lead compound,clausenamidone,was synthesized according to the report:trans-cinnamic acid,the starting materials,underwent epoxidation to give the product epoxy cinnamic acid,and the product underwent revolution with optically activity?-methylbenzylamine to give an optically active epoxy cinnamic acid ammonium salt.Then the optically active epoxy cycinnamic acid and2-?methylamino?-1-phenyl-1-ethanone hydrochloride are subjected to condensation reaction to give a a linear amide,which underwent intramolecular cyclization with lithium hydroxide to give optically active multi-substituted pyrrolidone compounds clausenamidone and its geometrical isomer neoclausenamidone,which the ee%are>98%.Afteraward,the optically active clausenamidone and neoclausenamidone underwent esterification in a mix of 1-ethyl-?3-dimethylaminopropyl?carbodiimide hydrochloride?EDCI??which was dehydrating agent?,4-Dimethylaminopyridine?DMAP??which was a catalyst?and different carboxylic acid compounds to form 17 new pairs of enantiomers with highly electronegative ester group substituted pyrrolidone derivatives?34optical active compounds?.And the neuroprotective effect of these preparation of compounds was preliminarily evaluated in a model of glutamate-induced neuronal cell.The results show that?+?-N16 and?+?-N20 in polypyrrolidone derivatives have a good protective effect for the damage on glutamate-induced cerebellar granular nerve cell,and they can be used as a new kind of neuroprotective agent for further study.A total of 41 compounds including intermediates have been synthesized in this paper,34 of them have not been reported.All the new compounds have been identified by ¹H-NMR,¹³C-NMR and HRMS,and the enantiomeric excess were determined by HPLC chiral stationary phase method.All the ee%of optically activity compounds were greater than 98%.Based on the advantages and disadvantages of different liquid phase resolution methods,this paper chose a polysaccharide chiral stationary phase method to systematically investigate the chromatographic resolution of polysubstituted pyrrolidone compounds and clarify the resolution mechanism.First,the method of HPLC chiral stationary phase separation of clausenamidone and neoclausenamidone was established and all method validation parameters showed acceptable results.At the same time,thermodynamic experiment was conducted to investigate the chromatographic separation of compounds under different temperature conditions and molecular simulation was designed and established to realize the resolution mechanism.The ligands were clausenamidone and neoclausenamidone,while the receptors were the amylose and cellulose stationary phases with 12 units.Autodock was applied to obtain the image of molecular docking interaction.The results of the thermodynamic study showed that the???interactions and hydrogen bonding with the CSPs resulted in chiral separation,which corresponded to the results of docking modeling.Then,using the established chromatographic resolution method of the lead compound as a reference,the optical purity of the synthesized pyrrolidone ester derivatives were tested,and different chromatographic columns,different chromatographic mobile phases,different temperatures,and acid base mobile phase additive were examined.The influence of different chromatographic conditions,such as phase additives,on the resolution of derivatives with different stereostructure substituents,which lays a good experimental foundation for the subsequent research on the method of chromatographic resolution of polysubstituted pyrrolidone derivatives.