候选药物T-VA的质谱裂解规律及其在大鼠体内的代谢产物研究

Mass Fragmentation Pathway of a Candidate Drug T-VA and Its Metabolites in Rats

采用液相色谱-线性离子阱-静电场轨道阱高分辨质谱(LC/LTQ-Orbitrap MS)技术研究候选药物T-VA的裂解规律及体内代谢,建立了大鼠体内T-VA及其代谢产物的LC/MSn分析方法,分析讨论了各自的主要碎片离子峰、质谱特征与结构信息,发现了血浆中主要代谢物M1。借鉴药物化学的方法,合成得到了代谢产物实体M1-1,经1 HNMR、13 CNMR、HRMS确认其结构,根据其色谱、质谱特征进行验证,结果表明,合成得到的M1-1即为血浆主要代谢物M1。借助PC12细胞模型验证了代谢产物M1的神经保护活性,该结果可为进一步研究其生物转化过程与前药修饰提供重要信息。

The candidate drug T-VA（C24H28N4O4）was synthesized using two kind of neuroprotective ingredients from Chinese traditional medicinal herbs,and displayed promising protective effect on the injured PC12 cells.In previous study,this beneficial effect was due to the modulation of nuclear transcription factor-κB/p65（NF-κB/p65）and cyclooxygenase-2（COX-2）expressions.T-VA also exhibited neuroprotective effectin a rat model of ischemic stroke with concomitant improvement of motor functions.Understanding drug metabolites contributes to discovering and developing the novel drug from the metabolites possessed the pharmacological activities.The structure profile of the metabolites provides an essential perspective for the synthetic refinement and the candidates among an extensive series of potential structures,resulting in an optimum drug effectiveness and safety.Liquid chromatography with electrospray ionization mass spectrometric detection（LC-ESI-MS）has been extensively utilized for the online analysis and structural characterization of the active ingredients and metabolites.Thus,there is a need to determine the primary metabolites and mass fragmentation pathways of T-VA in order to understand its potential pharmacological applications.It is a pathway via LC/LTQ-Orbitrap MS to investigate the mass fragmentation of a candidate drug T-VA and study its metabolites in rats.As a result,a method of LC/MSn was established for the analysis of T-VA and its metabolites in rats.The fragmentation pathway of T-VA was explained using the Analyst V4.0software.By further analysis of main fragment ions（m/z317,285,135）and structural information（C17H21O4N2,RDB：8.5,delta ppm：-3.038ppm）,M1 [methyl-3-methoxy-4-（（3,5,6-trimethylpyrazin-2-yl）methoxy）benzoate]was discovered as one of the main metabolites.According to the suppositional structure of M1,M1-1was synthesized via condensation reaction,which was determined by nuclear magnetic resonance spectrum（1 H-NMR,13C-NMR）and HRMS.By comparing the mass spectrum characters and chromatographic features of M1-1and M1,it can confirm the exact structure of M1.Furthermore,the neuroprotective effect of M1 in differentiated PC12 cells were evaluated.As a result,M1 in different concentrations could protect PC12 cells injured by CoCl2（EC50 =16.01μmol/L）,which was close to T-VA.The result indicated that both T-VA and its main metabolite have neuroprotective effect,which provides references for further new drug design.In this study,metabolite of candidate drug T-VA was obtained by chemical synthesis and verified by MS technique,which provided a novel idea on the study of drug metabolism.M1 may become a potential neuroprotective agent and further studies are currently underway.