Synthesis Of The Fluorinated Internal Standard And Its Application In The Pharmacokinetic Study Of The Clopidogrel Deuterated Derivative CDD01

Cardiovascular disease is the leading cause of death in humans,and thrombosis is one of the main pathogenesis of cardiovascular disease.The formation of arterial thrombosis depends on the activation and aggregation of platelet,and the platelet membrane P2Y₁₂ receptor plays a key role in the process of platelet activation and aggregation.Clopidogrel,a clinically widely used irreversible inhibitor of the P2Y₁₂receptor,is a prodrug that is inactive in itself and requires two steps of metabolic activation in vivo by CYP450 enzymes,of which CYP2C19 has genetic polymorphisms that lead to individual differences in its therapeutic effects.In addition,the activation efficiency of clopidogrel is low.The later marketed prasugrel addressed the shortcomings of clopidogrel but introduced the risk of fatal bleeding.In view of the shortcomings of clopidogrel and prasugrel,with the aim of reducing the risk of bleeding and increasing efficacy,based on clopidogrel,my group has developed a series of clopidogrel deuterated derivatives by deuteration of the hydrogen of methyl ester at the 7-position and introduction of an acyl group at the 2-position to form an ester.In order to support their further development,the pharmacokinetics of such compounds needs to be investigated in depth.Therefore,this thesis developed a liquid chromatography-tandem mass spectrometry（LC-MS/MS）method for the analysis of the active metabolite MP-DH4（Due to the instability of DH4,it was derivatised to form the derivative MP-DH4）and its closely related two inactive metabolites MP-DH3（Due to the instability of DH3,it was derivatised to form the derivative MP-DH3）and DSM3 of these compounds in vivo.In order to maximise the elimination of errors during sample handling and instrumental analysis and to ensure the accuracy and precision of the analytical method,for the determination of target compounds in biological samples,ICH-M10 and the Guidelines for the Validation of Methods for the Quantitative Analysis of Biological Samples in the Pharmacopoeia of the People’s Republic of China（2020 Edition）require the use of internal standard methods for quantitative analysis.Usually,the ideal internal standard is an isotopic internal standard or a structural analogue of the target compound.In the past,when developing LC-MS/MS methods for the metabolites of clopidogrel,MP-H3,MP-H4 and SM3,the trideuterated structures of the three metabolites were used as internal standards.The three target compounds in this thesis,MP-DH3,MP-DH4 and DSM3,are all trideuterated and contain a chlorine atom,which have high abundance of stable isotopes(³⁵Cl 76%,³⁷Cl24%),resulting in the high abundance of the isotopic excimer ion peaks[M+1+H]⁺,[M+2+H]⁺and[M+3+H]⁺for the target compounds in the Electron spray Ionization.In order to ensure that the difference in molecular weight between the internal and external standards can reach the resolution of the mass spectra,a deuterated internal standard would require at least four hydrogen atoms to be deuterated on the basis of the structure of the substance to be measured,but it is difficult to synthesis.If a non-deuterated structure is used as the internal standard,the non-deuterated structure also contains a chlorine atom and its isotopic peak[M+3+H]⁺will interfere with the[M+H]⁺peak of the external standard.How to solve the above contradictions and challenges then becomes the key scientific problem in the design and synthesis of the internal standard in this thesis.After comparison,the following strategy for the selection and synthesis of the internal standard was proposed:the fluorine atom and the chlorine atom are both halogens with similar properties,and there is no stable isotope of fluorine atom with high abundance.For this reason,the molecular weight of the internal standard is 19 Da less than that of the test by replacing the chlorine atom in the non-deuterated structure of the test with a fluorine atom,allowing the internal standard to be distinguished from the external standard in the mass spectrum.In order to make the properties of the internal standard close to those of the external standard,minor structural changes are made to the target compound,achieving the objective of the internal standard method to eliminate errors in the sample pre-treatment and instrumental analysis process.In this thesis,the clopidogrel deuterated derivative CDD01 was used as the object.A precise LC-MS/MS analytical method was developed for the simultaneous analysis of the three metabolites in rat plasma,beagle dog plasma and rat tissues using the synthesized fluorinated structural analogues as internal standards.The plasma pharmacokinetic properties of the three metabolites of CDD01 were evaluated,and the tissue distribution of the active and related inactive metabolites of thienopyridines was studied for the first time.（1）Synthesis of the fluorinated internal standard and development of the LC-MS/MS analytical methodThe fluorostructural analogue F-MP-H3 of MP-DH3,an in vitro derivative of sulfhydryl metabolite DH3,and F-SM3,a fluorostructural analogue of in vivo metabolite DSM3,were synthesized and structurally characterized,respectively for the first time.The LC-MS/MS method was established for the analysis of three matrices,rat plasma,Beagle dog plasma and rat tissue,using F-MP-H3 as a common internal standard for MP-DH3 and MP-DH4 and F-SM3 as an internal standard for DSM3.The method validation was performed according to the requirements of ICH-M10 and the Guidelines for the Validation of Methods for the Quantitative Analysis of Biological Samples in the Pharmacopoeia of the People’s Republic of China（2020 edition）,and there was no mutual interference between the internal standards and external standards in rat plasma,and the recoveries of the internal and external standards were close to each other and the matrix effects were close to each other.In addition,the results of selectivity,precision and accuracy in all three matrices met the requirements.This indicates that the fluorinated substructure of the target compound synthesized in this thesis is an ideal internal standard.（2）Pharmacokinetic study of the CDD01 metabolites DH3,DH4 and DSM3 in rat plasmaAfter single intragastric administration of the CDD01 1 mg/kg,3 mg/kg and 10mg/kg in rats,the mean T_max of the metabolites DH3,DH4 and DSM3 in plasma ranged from 0.222 h to 0.250 h,0.417 h to 0.542 h and 0.375 h to 0.458 h,respectively.DH3had the shortest peak time,while DH4 and DSM3 had close peak times.The mean t_1/2values for DH3 ranged from 0.320 h to 0.822 h,for DH4 from 2.07 h to 6.34 h and for DSM3 from 12.0 h to 20.9 h.The elimination of DH3 was the fastest,followed by DH4and DSM3 is the slowest.The C_max and AUC_0-t of DH3 were the lowest among the three metabolites.The C_max of DH4 was higher compared to DSM3,while the AUC_0-t was lower compared to DSM3,which was related to the slow elimination rate of DSM3.The exposure for DH3 and DH4 after medium dose and high dose administration differed between males and females,with females being more exposed than males.In the dose range of 1-10 mg/kg administered,the AUC_0-t and C_max of the active metabolite DH4 didn’t show a trend of linear kinetic characteristics.After single intravenous administration of the CDD01 1 mg/kg to rats,plasma concentrations of three metabolites,DH3,DH4 and DSM3,were detectable at 5 min.This indicated that the CDD01 can be rapidly distributed to the liver or intestine where metabolic activation occurs.（3）Pharmacokinetic study of the CDD01 metabolites DH3,DH4 and DSM3 in the plasma of beagle dogsAfter single oral administration of the CDD01 0.3 mg/kg,1 mg/kg and 3 mg/kg in beagle dogs,the mean T_max values of plasma metabolites DH3 were 0.500 h,0.0833 h and 0.625 h,DH4 were 0.625 h,0.167 h and 0.750 h,DSM3 were 1.00 h,0.375 h and4.38 h.DH3 had the shortest peak time,followed by DH4,and DSM3 had the longest peak time.Of the three metabolites,DH4 had the lowest AUC_0-t and DH3 had the highest C_max.（4）Tissue distribution study of the CDD01 metabolites DH3,DH4 and DSM3 in ratsAfter single intragastric administration of the compound CDD01 3 mg/kg to rats,tissue samples were analysed at 15 min,30 min and 6 h after administration.DSM3 and DH4 were distributed in all tissues 15 min after administration,suggesting a rapid distribution of DSM3 and DH4.In all tissues,DSM3 concentrations decreased slightly or increased at 6 h,suggesting a slow elimination of DSM3 in these tissues while the concentration of DH4 decreased significantly at 6 h,suggesting that DH4 was eliminated rapidly in these tissues.The concentration of DH3 was near or below the lower limit of quantification in all tissues except for the small intestine,liver and kidney,which was associated with low plasma exposure of DH3 itself.The higher concentrations of the three compounds in the small intestine and liver compared to other tissues may be related to the absorption and metabolism in the small intestine and the metabolism in the liver.