Design,Synthesis And Evaluation Of Amino Acid Ester Prodrugs Of Liguzinediol

Liguzinediol,2,5-dihydroxymethyl-3,6-dimethylpyrazine,is a positive inotropic compound discovered by our laboratory through year s screening,targeting the sarcoplasmic reticulum(SR)Ca2+ ATPase to elevate SR Ca2+ transient,which circumvents ventricular arrhythmia occurred in currently inotropic agents.Langendorff perfusion conducted with normal isolated heart and various animal models of heart failure suggested that liguzinediol was with the marked inotropic effect without arrhythmia in extensive tests.Toxicity test indicated liguzinediol was of no acute and local toxicity,teratogenesis or mutagenicity.However,the study of metabolic mechanism proved that dihydroxymethyl in its structure was easily metabolized via sulfation or glucuronidation,which resulted in its short half-time.Besides,animal models of chronic heart failure showed significant prevention,making the attempt to abtain derivatives of liguzinediol with great significance.Furthermore,previous structure-activity relationship studies showed that the dihydroxylmethyl group of liguzinediol was necessary.Based on those,we propose the strategy of prodrugs where hydroxyl group in its structure is protect by amino acids to slow down the metabolism rate and prolong its half-time.The structure related with physicochemical properties,so we analysised and designed the structure of target compounds.Hydroxyl group provides excellent biocompatibility,contributing to the designing of protection by amino acids.The hydrophily of amino functional group could guarantee the solubility of prodrugs.In addition,considering the bad stability of amino acid ester prodrugs in water,we designed two series of prodrugs,amino acid ester and acetyl amino acid ester.In the design of acetyl amino acid ester,we made use of the p-π conjugated effect between the acetyl and lone pair electrons to reduce amino functional group’s effect to the hydrolyzing of ester bonds with the aim of improving the stability of prodrugs.According to the preliminary experiment,we found that it’s difficult for amino acid monoester to be separated and purified,combining of the considering of metabolic rate,we determined to design and synthesize dual ester prodrugs eventually.The ideal prodrugs should be equipped with appropriate stability,solubility,lipophilicity,bioconversion and pharmacokinetics.Therefore,we screened all synthesized compounds by the studies of chemical stability,physicochemical properties(solubility and lipophilicity),in vitro bioconversion and in vivo pharmacokinetics to obtain a favored prodrug.By the DMAP-catalyzed dicyclohexylcarbodiimide method,we synthesized eight acetyl amino acid dual ester prodrugs;with the deprotection by 15%TFA,we obtained an amino acid dual ester(all amino acids we used are of L configuration).All prodrugs were characterized by LC-MS,1H-NMR and 13C-NMR.The purity,stability.physicochemical properties,in vitro bioconversion and in vivo pharmacokinetics were investigated by HPLC.The results showed that the stability and liposolubility of compound 3 was poor;among those prodrugs,compound 2c,2d and 2e were too stable to be transformed in two kinds of bioconversion systems,which suggested that those prodrugs can’t release liguzinedial as quickly as we need.Finally,we determinated to make other five prodrugs(2a,2b,2f,2g and 2h)for the further studty.The in pharmacokinetics of liguzinediol following oral administrations of 2a,2b,2f,2g and 2h were tested and compared with that of liguzinediol dosed alone.The dection of liguzinediol was proformed following the establishment of HPLC;the results of this study were compared to approve a promising prodrug 2a.In this research subject,we examed stability,physiochemical properties,in vitro bioconversion and in vivo pharmacokinetics,indicating that the metabolic rate of prodrugs related with compounds’ structure,electronic effect and kinds of enzymes,which laids a solid foundation for further study of prodrugs.In addition,the using of in vitro systems in 80%human plasma and human liver microsome not only makes the evaluation direct but also saves the cost and workload.