Background: Ensartinib (ESB) is a novel anaplastic lymphoma kinase inhibitor (ALK) with additional activity against Abelson murine leukemia (ABL), met proto-oncogene (MET), receptor tyrosine kinase (AXL), and v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1) and is considered a safer alternative for other ALK inhibitors. ESB chemical structure contains a dichloro-fluorophenyl ring and cyclic tertiary amine rings (piperazine) that can be bioactivated generating reactive intermediates.
Methods: In vitro metabolic study of ESB with human liver microsomes (HLMs) was performed and the hypothesis of generating reactive intermediates during metabolism was tested utilizing trapping agents to capture and stabilize reactive intermediates to facilitate their LC-MS/MS detection. Reduced glutathione (GSH) and potassium cyanide (KCN) were utilized as trapping agents for quinone methide and iminium intermediates, respectively.
Results: Four in vitro ESB phase I metabolites were characterized. Three reactive intermediates including one epoxide and one iminium intermediates were characterized. ESB bioactivation is proposed to occur through unexpected metabolic pathways. The piperazine ring was bioactivated through iminium ions intermediates generation, while the dichloro-phenyl group was bioactivated through a special mechanism that was revealed by LC-MS/MS.
Conclusion: These findings lay the foundations for additional work on ESB toxicity. Substituents to the bioactive centers (piperazine ring), either for blocking or isosteric replacement, would likely block or interrupt hydroxylation reaction that will end the bioactivation sequence.



中文翻译：

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使用 LC-MS/MS 表征人肝微粒体中抗癌药物 Ensartinib 的稳定和反应性代谢物：一种计算机和实用的生物活化方法

背景：恩沙替尼 (ESB) 是一种新型间变性淋巴瘤激酶抑制剂 (ALK)，对 Abelson 鼠白血病 (ABL)、met 原癌基因 (MET)、受体酪氨酸激酶 (AXL) 和 v-ros UR2 肉瘤病毒癌基因具有额外的活性同源物 1 (ROS1)，被认为是其他 ALK 抑制剂的更安全替代品。ESB 化学结构包含一个二氯氟苯环和环状叔胺环（哌嗪），它们可以被生物活化生成反应性中间体。
方法：进行了 ESB 与人肝微粒体 (HLM) 的体外代谢研究，并测试了在代谢过程中产生反应性中间体的假设，利用捕获剂捕获和稳定反应性中间体以促进其 LC-MS/MS 检测。还原型谷胱甘肽 (GSH) 和氰化钾 (KCN) 分别用作甲基醌和亚胺中间体的捕集剂。
结果：对四种体外 ESB I 期代谢物进行了表征。表征了三种反应性中间体，包括一种环氧化物和一种亚胺中间体。提出 ESB 生物活化是通过意想不到的代谢途径发生的。哌嗪环通过生成亚胺离子中间体进行生物活化，而二氯苯基通过 LC-MS/MS 揭示的特殊机制进行生物活化。
结论：这些发现为进一步研究 ESB 毒性奠定了基础。生物活性中心（哌嗪环）的取代基，无论是用于阻断或等排置换，都可能阻断或中断将结束生物活化序列的羟基化反应。