Synthesis, Biological Evaluation, And Pharmacokinetic Study Of Novel Liguzinediol Derivatives

Liguzinediol (LZDO) is a para-dihydroxy derivative of ligustrazine (1,which was isolated from the traditional Chinese medicine herb Chuanxiong), and its chemical name is 2,5-dihydroxymethyl-3,6-dimethylpyrazine. We recently found that LZDO had potent positive inotropic effect and diastolic function on the myocardium without arrhythmia risk, and its positive inotropic effect in isolated rat hearts was mediated through an elevation of transient SR Ca2+,which may act on SR Ca2+ ATPase. LZDO thus has a unique biological mechanism that may prove effective in treating heart failure clinically. Moreover, pharmacokinetic study of LZDO showed that its half-life was about 1.6 h and its clearance was approximately 0.9 L/h·kg after intragastric administration, suggesting that elimination or biotransformation of LZDO was relatively quick in rats. The plasma concentration would be too low and has not a very good therapeutic effect when the interval time using medicine is too long. Administration of the drug frequently would lead to poison and reduce treatment compliance of the patients.Cardiotonic agents on the market at present exert their therapeutic activity by inducing positive inotropic effect on the myocardium cell membrane. But this mechanism of action is inherently responsible for the heart side effects observed in patients undergoing a long-term treatment. LZDO act on SR Ca2+ ATPase inside myocardium cells with high safety. The half life of drug reflects the speed of excretion, biotransformation, and storage in vivo, and it decides to the dosage and frequency of administration. It is necessary to modify molecular structure of LZDO and study pharmacodynamics of the derivatives, in order to satisfy different drug delivery and dosage form clinically.Structure-activity relationship (SAR) studies indicated that para-dihydroxy is very important for positive inotropic effect.In this paper, LZDO was taken as the lead compound, three prodrugs were prepared by esterification of LZDO and two LZDO secondary alcohol derivatives were prepared through increasing the stereospecific blockade of substituted groups of LZDO, the compounds were evaluated for drug application, in order to satisfy different drug delivery and dosage form clinically.. The inotropic effects on the myocardium in normal isolated rat hearts, metabolic stability of prodrugs and pharmacokinetic of the parent compound in rat plasma after intragastric administration of 2,5-diacetoxymethyl-3,6-dimethylpyrazine and pharmacokinetic of 2,5-di-(1-hydroxyl ethyl)-3,6-dimethylpyrazine, 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine in rat plasma have been studied in comparison with the parent drug LZDO. The results showed that prodrugs and LZDO secondary alcohol derivatives exerted positive inotropic effects in a dose-dependent manner, 2,5-diacetoxymethyl-3,6-dimethylpyrazine was highly metabolized and converted to LZDO quickly and the half-life of LZDO strung out to 4 h after intragastric administering 2,5-diacetoxymethyl-3,6-dimethylpyrazine, the half life of 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine were prolonged compared to LZDO, and their pharmacokinetic parameters profiles were improved. On the basis of in vitro and in vivo studies, LZDO prodrug and LZDO secondary alcohol derivatives were found to be a superior candidate for increasing myocardial contractility.This thesis includes two parts:1 Synthesis, Biological Evaluation, and Pharmacokinetic Study of Novel Liguzinediol Prodrugs1.1 Synthesis of LZDO Prodrugs2, 5-Diacetoxymethyl - 3, 6- dimethylpyrazine, 2, 5- di- iso- butyryloxymethyl - 3, 6- dimethyl pyrazine, 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazine and LZDO secondary alcohol derivatives 2, 5 - di-(1- hydroxylethyl) -3,6- dimethylpyrazine, 2,5 -di- (1-hydroxylpropyl) -3,6-dimethylpyrazine were prepared from Ligustrazine. The structrues were comfimed by UV, IR,NMR and MS.1.2 Bioactive Screening of LZDO ProdrugsInotropic actions of LZDO prodrug in normal isolated rat hearts were investigated by Langendorff technique. The results showed that the positive inotropic effect was impaired when LZDO was esterified. 2,5-Diacetoxymethyl-3,6-dimethylpyrazine was the most potent prodrug for a positive inotropic effect on the myocardium,it increased LVDP by 15.0 ± 5.5 %,26.3 ± 7.7%,and 44.7 ± 6.6 % at 1 ?M,10 ?M,and 100?M,respectively; increased +dP/dtmax by 1.9 ± 0.5%,9.8 ± 7.0 %,and 31.8 ± 5.3 % at 1 ?M, 10 ?M,and 100 ?M,respectively; and increased-dP/dtmax by 4.9±5.9%,9.0±5.1%,and 20.9±5.1%at 1?M,10?M,and 100?M,respectively.1.3 Pharmacokinetic Study of LZDO Prodrug1.3.1 Metabolic Stability Analysis.Prodrugs and parent compound LZDO were subjected to metabolic stability in the presence of rat liver microsomes and rat plasma. LZDO was fairly stable in rat liver microsomes and rat plasma; prodrugs were highly metabolized and converted to desirable parent compound in 1-3 h;The corresponding loss of prodrug compounds and formation of parent drug LZDO was determined by HPLC.1.3.2 Identification of Intermediate from Rat Liver Microsomes and Rat PlasmaThe structure of intermediate was identified by HPLC-MS, The results showed that prodrug take off a esterfunction to become intermediate, and the intermediate took off the other esterfunction to become LZDO.1.3.3 Determination of LZDO and 2,5-Diacetoxymethyl-3,6-dimethylpyrazine in Rat Plasma by Liquid Chromatography SpectrometryA liquid chromatography spectrometry method has been developed and validated for the quantification of LZDO and 2,5-diacetoxymethyl-3,6-dimethylpyrazine in rat plasma. Caffeine was selected as the internal standard (IS, National Institute for the Control of Pharmaceutical and Biological Products). Analytes were extracted from rat plasma by liquid/liquid extraction using ethanol-ethyl acetate (1 :4) with high efficiency. The chromatographical separation was performed on Phecda-C18 ( 4.6 mm × 250 mm, 5 ?m) with a gradient elution of methanol and deionized water. The chromatographic run time was 19 min per injection and flow rate was 1 mL/min. The retention time was 6.4,12.3, and 16.4 min for LZDO, IS, and 2,5-diacetoxymethyl-3,6-dimethylpyrazine, respectively. The method was sensitive with the limit of quantitation at 200 ng/mL for LZDO and 2,5-diacetoxymethyl-3,6-dimethylpyrazine in 100 ?L of rat plasma,respectively. Good linearity (r = 0.9995 for LZDO and r = 0.9996 for 2,5-diacetoxymethyl-3,6-dimethylpyrazine) was obtained covering the concentration of 0.2-100 ?g/mL (LZDO and 2,5-diacetoxymethyl-3,6-dimethylpyrazine). The intra-and inter-day assay precision ranged from 2.4 to 6.7% and 2.4 to 3.6% for LZDO, respectively. The intra-and inter-day assay precision ranged from 3.3 to 8.6% and 1.7 to 6.1% for 2,5-diacetoxymethyl- 3,6-dimethylpyrazine,respectively. In addition, the stability, extraction recovery and matrix effect involved in the method were also validated.Compared to LZDO, the AUC of 2,5-diacetoxymethyl-3,6-dimethylpyrazine after intragastric dose are comparable to LZDO, the Tmax and Cmax of 2,5-diacetoxymethyl-3,6-dimethylpyrazine are lower than those of LZDO, suggesting that 2,5-diacetoxymethyl-3,6-dimethylpyrazine converted into LZDO in vivo. Moreover, by comparison, the half-life (h) of LZDO strung out to 4.01, 4.24,4.39 for 10,20,40 mg/kg, respectively, the clearance (L/h-kg) of LZDO was reduced to 0.40, 0.52, 0.41 for 10,20, 40 mg/kg, respectively, suggesting that elimination of LZDO after taking 2,5-diacetoxymethyl-3,6-dimethylpyrazine was relatively more slowly than taking LZDO in rats.1.3.4 Determination of 2,5-Diacetoxymethyl-3,6-Dimethylpyrazine and LZDO in Rat Tissues by HPLC and Its application to Tissues Distribution StudyAfter ig 2,5-diacetoxymethyl-3,6-dimethylpyrazine (20 mg·kg-1) to rats, LZDO can be found in all determined tissues and pancreas received more drug than any other tissues. And 2,5-diacetoxymethyl-3,6-dimethylpyrazine can not be detected in all determined tissues, it indicated that 2,5-diacetoxymethyl-3,6-dimethylpyrazine could convert to LZDO quickly in vivo.The concentration of LZDO in all determined tissues fallen-off 4 h after ig administration and LZDO can hardly be found in some rat tissues 8 h after ig administration, it shows that LZDO can rapidly spread and eliminate.1.3.5 Excretion and Metabolites of LZDO ProdrugTo analysis of LZDO prodrug and its metabolites in rat urine and feces after ig administration,an TOF MS-IDA-MS/MS method was applied to identify the metabolites after a single ig administration of 20 mg-kg-1 2,5-diacetoxymethyl-3,6-dimethylpyrazine to rats. Urine and feces were collected and analyzed by HPLC-MS. The result showed that 2,5-diacetoxymethyl-3,6-dimethylpyrazine could not be detected in the urine and feces. Compared to excretion and metabolites of LZDO, there is no new metabolite was detected in the urine and feces after ig 2,5-diacetoxymethyl-3,6-dimethylpyrazine.In conclusion, the positive inotropic effect on the myocardium of 2,5-diacetoxymethyl-3,6-dimethylpyrazine was evaluated by known experimental techniques. 2,5-Diacetoxymethyl-3,6-dimethylpyrazine was transformed enzymatically to the parent drug LZDO in rats.2,5-Diacetoxymethyl-3,6-dimethylpyrazine emerged as a novel cardiotonic agent with prolonging the half-life and effective drug duration of LZDO in vivo. On the basis of in vitro and in vivo studies, 2,5-diacetoxymethyl-3,6-dimethylpyrazine emerged as a potent and an alternative drug for the treatment of chronic heart failure.2 Synthesis, Biological Evaluation, and Pharmacokinetic Study of Novel Liguzinediol Derivatives2.1 Synthesis of LZDO Secondary Alcohol DerivativesLZDO secondary alcohol derivatives 2,5-di-(1-hydroxyl ethyl)-3,6-dimethylpyrazine and 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine were prepared from LZDO. The structrues were comfimed by UV, IR, NMR and MS.2.2 Bioactive Screening of LZDO Secondary Alcohol DerivativesInotropic actions of LZDO secondary alcohol derivatives in normal isolated rat hearts were investigated by Langendorff technique. The results showed that secondary alcohol derivatives produced the positive inotropic effect in normal isolated rat hearts, and the positive inotropic effect of compounds was impaired when the stereospecific blockade of substituted groups of LZDO was increased.2.3 Pharmacokinetic Study of LZDO Secondary Alcohol Derivatives2.3.1 Determination of 2,5-Di-(1-hydroxylpropyl)-3,6-dimethylpyrazine in Rat Plasma by Liquid Chromatography SpectrometryA liquid chromatography spectrometry method has been developed and validated for the quantification of 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine in rat plasma. Caffeine was selected as the internal standard (IS, National Institute for the Control of Pharmaceutical and Biological Products). Analytes were extracted from rat plasma by liquid/liquid extraction using ethanol with high efficiency. The chromatographical separation was performed on Phecda-C18(4.6 mm x 250 mm, 5 ?m) with a gradient elution of methanol and deionized water. The chromatographic run time was 11 min per injection and flow rate was 1 mL/min. The retention time was 6.4 and 9.4 min for IS,and 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine,respectively. The method was sensitive with the limit of quantitation at 280 ng/mL for 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine in 100 ?L of rat plasma, respectively. Good linearity (r=0.9999) was obtained covering the concentration of 0.28-73 ?g/mL. The intra-and inter-day assay precision ranged from 3.4 to 5.2% and 1.3 to 4.6% for 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine, respectively. In addition, the stability, extraction recovery and matrix effect involved in the method were also validated.Compared to LZDO, the half-life (h) of LZDO strung out 3.6, the clearance (L/h-kg) of LZDO was reduced to 0.3mg/kg, respectively, suggesting that elimination of 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine was relatively more slowly than LZDO in rats. The relative bioavailability was 90 %.2.3.2 Determination of 2,5-Di-(1-hydroxyl propyl)-3,6-dimethylpyrazine in Rat Tissues by HPLC and Its Application to Tissues Distribution StudyAfter ig 2,5-di-(1-hydroxyl propy l)-3,6-dimethylpyrazine (20 mg·kg-1) to rats,2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine can be found in all determined tissues and muscle received more drug than any other tissues. The concentration of 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine in all determined tissues fallen-off 4 h after ig administration and 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine can hardly be found in some rat tissues 8 h after ig administration, it shows that 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine can rapidly spread and eliminate.2.3.3 Excretion and Metabolites of 2,5-Di-(1-hydroxyl propyl)-3,6-dimethylpyrazineTo analysis of 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine and its metabolites in rat urine and feces after ig administration, an TOF MS-IDA-MS/MS method was applied to identify the metabolites after a single ig administration of 20 mg·kg-1 2,5-di-(1-hydroxylpropyl)-3,6-dimethylpyrazine to rats. Urine and feces were collected and analyzed by HPLC-MS. The result showed that The result is that 7 metabolites were detected in the feces, metabolite was not detected except 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine in the urine, and they were tentatively identified. Moreover, we have find out the possible metabolic pathway of 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine in rat.In conclusion, 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine emerged as a novel cardiotonic agent with prolonging the half-life and effective drug duration in vivo. On the basis of in vitro and in vivo studies, 2,5-di-(1-hydroxyl propyl)-3,6-dimethylpyrazine emerged as a potent and an alternative drug for the treatment of chronic heart failure.