两类抗HIV先导物的大鼠肝微粒体代谢产物筛查

Metabolite profiling of two anti-HIV lead compounds in rat liver microsomes

应用超高效液相色谱四极杆飞行时间质谱联用技术(UPLC-QTOF-MS/MS)并辅以代谢数据采集和处理软件,快速筛查了两类新型非核苷类逆转录酶抑制剂先导物DAPA-7012和DAAN-4442在大鼠肝微粒体的代谢产物,并分析结构中的代谢软点。应用质量亏损过滤技术在UPLC-QTOF-MS/MS上比较分析0 h与2 h-孵育样品的数据,在大鼠肝微粒体孵育液中分别筛查得到DAPA-7012的14个代谢产物和DAAN-4442的14个代谢产物。应用仪器的碰撞能量梯度功能(MSE)对部分产物进行MS2分析,获得代谢产物的碎片,推断碎裂途径及产物结构。结果显示,两个化合物的I相反应以氧化(羟基化)为主,Ⅱ相反应以谷胱甘肽结合为主。骨架结构上B环的差异(吡啶环和苯环)对两类先导物的肝微粒体代谢途径没有显著影响。化合物共同的易代谢位点是B环上的伯胺基与C环上的甲基。不同之处在于DAAN-4442 B环4-位上的硝基易发生还原反应,DAPA-7012C环的苄基碳原子(亚甲基)更易发生氧化反应。课题组前期构效关系(SAR)研究结果表明,伯胺基与甲基均为活性必需基团,有必要通过进一步结构优化来提高这些活性基团的稳定性。将先导物代谢产物信息与结构活性分析相结合,可以为先导物结构优化提供参考。

The metabolite profiling of DAPA-7012 and DAAN-4442, the lead compounds from two new kinds of non-nucleoside reverse transcriptase inhibitors （NNRTIs）, was performed using an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF-MS/MS）, with the assistance of a metabolite data processing software. By utilizing the mass defect filter （MDF） technique, the data acquired from the 0 h-incubation and the 2 h-incubation were compared and analyzed with the MetaboLynxTM software. After incubation, 14 metabolites of DAPA-7012 and 14 metabolites of DAAN-4442 were found in rat liver microsome. The MS2 spectra for some metabolites were obtained using the MSE technique to get fragment ions for structural elucidation. The results indicated that both compounds could undergo extensive metabolism in rat liver microsomes. The major phase I reaction was oxidation/hydroxylation. The major phase II reaction was S-glutathione conjugation. The metabolic pathways were similar between the two lead compounds, though they have different backbone structures. Besides, the 4-NO2 of ring B in DAAN-4442 was susceptible to reduction, the benzyl of ring C in DAPA-7012 was tend to be oxidized. The common metabolic soft spots were primary amine of ring B and two methyl groups of ring C. Early SAR results showed that the primary amine and methyl were necessary substituent groups. The stability of these active groups needs to be improved and optimized. The approach of combining metabolites information and structure-activity analysis can provide a reference for further structural optimization.