Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics. A significant route of phase II drug metabolism is conjugation with glutathione (GSH), which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes. The aim of this work was to investigate the GSH-mediated metabolic pathway of a representative UA, C-2028. GSH-supplemented incubations of C-2028 with rat, but not with human, liver cytosol led to the formation of a single GSH-related metabolite. Interestingly, it was also revealed with rat liver microsomes. Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole. Therefore, the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate. In turn, incubations of C-2028 and GSH with human recombinant glutathione S-transferase (GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form, respectively. These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule. Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction. Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry. Mechanisms for their formation were proposed. The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important. This may affect the patient's drug clearance due to GST activity, loss of GSH, or the interactions with GSH-conjugated drugs. Moreover, GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

不对称双吖啶 (UAs) 是一类新型有效的抗肿瘤活性药物。II 期药物代谢的一个重要途径是与谷胱甘肽 (GSH) 结合，它可以是非酶促的和/或由 GSH 依赖性酶催化的。这项工作的目的是研究具有代表性的 UA C-2028 的 GSH 介导的代谢途径。GSH 补充的 C-2028 与大鼠而非人类肝细胞质的孵育导致单一 GSH 相关代谢物的形成。有趣的是，大鼠肝微粒体也发现了这一点。它的形成不依赖于 NADPH，并且不受与细胞色素 P450 (CYP450) 抑制剂 1-氨基苯并三唑共同孵育的抑制。因此，直接缀合途径发生在没有事先 CYP450 催化的底物生物活化的情况下。反过来，C-2028 和 GSH 与人重组谷胱甘肽 S-转移酶 (GST) P1-1 或与热/依他尼酸灭活的肝细胞溶质酶一起孵育分别导致 GSH 结合形式的存在或缺乏。这些发现证明了 GST 必须参与 GSH 硫醇基团的初始活化，以实现对底物分子的亲核攻击。在非酶促反应过程中也形成了另一种 C-2028-GSH S-缀合物。两种GSH S-缀合物均通过组合液相色谱/串联质谱进行表征。提出了它们的形成机制。C-2028 与 GST 介导和/或直接 GSH 结合的能力被怀疑在临床上很重要。由于 GST 活性、GSH 丢失或与 GSH 结合药物的相互作用，这可能会影响患者的药物清除率。