| Objective:To synthesize dihydroartemisinin derived-dimer(DHA dimer)and to confirm its absolute configuration.To investigate the stability in vitro of DHA dimer in different media and identify the main degradation products.To determine the equilibrium solubility and apparent partition coefficient in octanol-water/buffer solution systems of the dimer.To investigate the antimalarial activity in vitro of the dimer to Plasmodium falciparum 3D7.To establish the model of mice infected with Plasmodium yoelii,and then investigate the antimalarial activity in vivo of the dimer to Plasmodium yoelii rodent malaria.To primarily investigate the possible metabolites and major metabolic pathways of the dimer in rats.Methods:1.Synthesis and physicochemical characterization of dimerDihydroartemisinin derived-dimer was synthesized from DHA by etherification,with BF3?Et3O as catalyzer.The product was determined by LC-MS/MS,and its purity was calculated by area normalization method.The final product was characterized by HR-MS and 1H-NMR.At room temperature,product was recrystallizated in petroleum ether to obtain single crystal,and its absolute configuration was confirmed by SXRD.An LC-MS/MS method was established to investigate the stability of the dimer in pH 1.2-8.0 buffer solutions,blood and medium(37 oC for 24 h).The equilibrium solubility of dimer in pH 4.0-8.0buffer solutions and its apparent partition coefficient in octanol-water/buffer solution systems were determined.Meanwhile,HPLC-HRMS method was used to identifythe degradation products in pH 1.2 buffer solution.2.Investigation on antimalarial activity of dimer in vitroPlasmodium falciparum 3D7 was used as the model.Sorbitol synchronization method was used to synchronize the growth cycle of Plasmodium.With DHA as positive control group,SYBR Green?was used to determine the inhibition to proliferation of Plasmodium falciparum 3D7 treated by dimer.The values of IC50(50% inhibitory concentration)and IC95(95% inhibitory concentration)were used to evaluate the antimalarial activity of drug in vitro.3.Pharmacodynamics study of dimer in vivoMice infected with Plasmodium yoelii was used as the model.In four-day inhibition test,mice were received dimer of different doses respectivly after intragastric administration and tail intravenous injection,with DHA as positive control group.A day after drug withdrawal,by means of blood collection and microscopic examination,the infection ratios,inhibition ratios,ED50(50%effective dose)values,ED90(90% effective dose)values and negative conversion ratios were estimated.A week after drug withdrawal,the infection ratios among different dose groups were calculated and compared.A month after drug withdrawal,the recrudescence ratios,cure ratios,mortality and the average survival days between different dose groups were compared.The pathological change of the spleen and liver tissues in survival mice were observed.4.Primary study on metabolism in vivo of dimerHPLC-HRMSn method was used to primarily study on metabolism in vivo of dimer and DHA.the possible metabolites and metabolic pathways of the dimer in rats were inferred by comparing with blank group and DHA group.Results:1.Synthesis and physicochemical characterization of dimerThe chemical structure of dimer was confirmed by HR-MS and 1H-NMR.The yield was about 75%.The melting point was 160.5 oC-161 oC.And the purity of dimerwas greater than 98%.The results of SXRD showed that the structure of compound was asymmetric dimer,that was a dimer of ?-and ?-DHA,bis(3,6,9-trimethyl-3,12-epidioxy-3,4,5,5a,6,7,8,8a,9,10-decahydro-12H-pyrano [4,3-j] [1,2]benzodioxepin-10-yl)ether.A LC-MS/MS method was developed for the quantitative analysis of dimer.The precision of LC-MS/MS content determination method of dimer was less than 5.58%,the accuracy was 98.54%-105.70%,the matrix effect and the recovery rate of extraction were ranged from 96.21% to 104.11%.The quantitative results showed that after incubation at 37 oC for 24 h in different biological medium and buffer solutions,the percentages of residual concentration were 69.94% ± 3.37%(blood),85.54% ± 1.40%(medium),76.95% ± 0.61%(pH 4.0),76.86% ± 1.75%(pH 6.8),80.94% ± 0.65%(pH 7.4)and 85.75% ± 0.68%(pH 8.0),respectively.And dimer was fully degraded after 6 h(37 oC)in pH 1.2 buffer solution.The main degradation products were identified as ?-DHA and ?-DHA.The equilibrium solubility of dimer were(54.02 ± 3.36)?g/ml(water),(128.79 ± 9.31)?g/ml(pH 4.0),(230.33 ± 17.79)?g/ml(pH 5.0),(313.76 ± 17.32)?g/ml(pH 6.8),(425.94 ± 35.07)?g/ml(pH 7.4)and(466.06 ± 21.76)?g/ml(pH 8.0),respectively.The partition coefficient of dimer were 1.55 ± 0.19(water),1.12 ± 0.05(pH 4.0),1.41± 0.28(pH 5.0),1.48 ± 0.20(pH 6.8),1.36 ± 0.02(pH 7.4)and 1.22 ± 0.26(pH 8.0).2.Antimalarial activity of dimer in vitroThe inhibition effect of administration groups to proliferation of Plasmodium falciparum 3D7 increased with the increase of drug concentration.The IC50((0.51 ±0.12)nmol/L)of dimer was significantly lower than this of DHA((1.81 ± 0.70)nmol/L)(P < 0.001).And the IC95((4.72 ± 1.86)nmol/L)of dimer was lower than that of DHA((6.40 ± 2.05)nmol/L)(P < 0.05).3.Pharmacodynamics study of dimer in vivoAt one day after the last intragastric administration,the ED50((0.44 ± 0.03)mg/kg/d)and ED90((1.95 ± 0.10)mg/kg/d)of dimer were both lower than those of DHA,which were(0.76 ± 0.03)mg/kg/d and(4.15 ± 0.32)mg/kg/d,respectively(P <0.001).In tail intravenous injection groups,the ED50((0.18 ± 0.03)mg/kg/d)and ED90((0.83 ± 0.08)mg/kg/d)of dimer were lower than those of DHA,which were(0.32 ± 0.03)mg/kg/d and(1.87 ± 0.11)mg/kg/d,respectively(P < 0.001).After treated by two administration routes,the inhibition effect of administration groups to Plasmodium yoelii rodent malaria increased with the dose-dependence.The body weight of mice gradually decreased.The greater dose increased,the less body weight reduced.After intragastric administrations(7.80 or 15.6 mg/kg/d),the negative conversion ratios of dimer were 75% or 100%,which were both bigger than those of DHA(37.5% or 62.5%),respectively(P < 0.001).After intravenous administration(3.0 or 6.0 mg/kg/d),the negative conversion ratios of dimer(62.5% or 100%)were both bigger than those of DHA(37.5% or 50%),respectively(P < 0.001).At one week after the last dose,the infection ratios of dimer were lower than DHA in some dose(P < 0.001).At one month after the last intragastric administration of dimer(15.6 mg/kg/d),the recrudescence ratio and survival ratio of mice were 50% and 75%,respectively.After intravenous administration of dimer(3.0,6.0 mg/kg/d),the recrudescence ratios of mice respectively were accordingly 60%,25%,the survival ratios were62.5%,100%,respectively.After intragastric or intravenous administration of DHA,the recrudescence ratios and survival ratios of mice were 100% and 0.And the body weight of survival mice were all markedly increased.Any lesions in liver and spleen of survival mice were not observed.4.Primary study on metabolism of dimer in rats18 possible metabolites were observed after intragastric administered with DHA,included 11 phase ? metabolites and 7 phase ? metabolites.Phase ?metabolites were formed mainly by hydroxylation,deoxygen,desaturation.And phase ? metabolites were glucuronidation metabolites.The metabolites mainly appeared in urine and bile.Meanwhile,27 possible metabolites were detected in rats after intragastric administration of dimer,including DHA and 11 relevant metabolites of DHA.The metabolic pathways of the other 15 possible metabolites were deoxygen,hydroxylation,hydroxylation with dehydration.The metabolites mainly excreted from faeces,urine and bile.Conclusion:1.Asymmetric dimer was successfully synthesized and purified.The chemical name of the product was a dimer of ?-and ?-DHA,bis(3,6,9-trimethyl-3,12-epidioxy-3,4,5,5a,6,7,8,8a,9,10-decahydro-12H-pyrano [4,3-j] [1,2]benzodioxepin-10-yl)ether.The dimer had a poor water-solubility.In pH 4.0-8.0buffer solutions,the dimer were relatively stable,and the stability and equilibrium solubility enhanced with the increase of pH.The apparent partition coefficient firstly increased(pH 4.0-6.8)and then decreased(pH 6.8-8.0).The dimer was stable in culture medium,and was easily degraded in plasma.Dimer was rapidly degraded into?-DHA and ?-DHA in pH 1.2 buffer solution.2.The results of pharmacodynamics study in vitro showed that the inhibition effect of drug treated groups to Plasmodium falciparum 3D7 increased with the increase of drug concentration.The antimalarial efficacy of dimer was greater than DHA,according to the IC50 and IC95 of two drugs.3.The results of pharmacodynamics study in vivo showed that the inhibition effect of dose groups to Plasmodium yoelii rodent malaria presented dose-dependence for both administration routes.A day after drug withdrawal,the antimalarial efficacy of dimer was greater than DHA.A mouth after drug withdrawal,the recrudescence ratio of dimer was lower than DHA,the negative conversion ratio and cure ratio of dimer were superior to DHA.4.The results of primary study on metabolism in rats showed that metabolites and metabolic pathways of DHA conformed to literature reported.The dimer was metabolized to DHA and 11 relevant metabolites of DHA,also metabolized to 15 other possible metabolites.The metabolic pathways were mainly deoxygen,hydroxylation,hydroxylation with dehydration,desaturation and glucuronidation.The metabolites mainly excreted from faeces,urine and bile. |
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