We would like to thank Drs. Veeravalli and Dash for their concerns(1) regarding our article.(2) In the article, two metabolic activation pathways of tofacitinib (TFT) were identified, including oxidative decyanation and epoxidation. In addition, the resultant α-ketoaldehyde metabolite was proposed to be responsible for the observed mechanism-based inactivation of CYP3A4 induced by TFT. The major concern included the participation of the epoxidation pathway in TFT-induced CYP3A4 inactivation.(1) In our early study, analogue 3, a primary amine compound derived from TFT, was designed and synthesized to probe the roles of the oxidative decyanation and epoxidation in TFT-induced CYP3A4 inactivation.(2) The reduction of the cyanide group to the primary amine completely abolished the enzyme inhibitory property of the parent compound. Additionally, we succeeded in the detection of analogue 3-oxide (Scheme 1) arising from metabolic oxidation of analogue 3. This provides the key evidence for the unlikeliness of the involvement of the epoxide intermediate in TFT-induced CYP3A4 inactivation, although we cannot entirely exclude the participation of the epoxide metabolite. It is true that the saturation of carbons 3′ and 4′ (analogue 2) partially reduced the capability of the enzyme inactivation by TFT, which suggests the contribution of the epoxide metabolite to the TFT-induced enzyme inactivation. However, the observed reduction in the inhibitory effect was accompanied with a decreased formation of the corresponding aldehyde metabolite.(2) Thus, the observed partial loss of the inhibitory activity of analogue 2 most likely resulted from the decrease in the formation of the aldehyde metabolite and probably not from the blocking of the generation of the epoxide metabolite. Glutathione (GSH) is often employed to quench reactive metabolites that escape from the active sites of the host enzymes. Actually, both N-acetyl cysteine (NAC) and GSH were used to trap the epoxide intermediate of TFT, and both succeeded. Apparently, the resulting NAC conjugate showed a better peak shape, and that is why its ion chromatograph was included in the article. Three NAC conjugates derived from intermediates 6 and 8 were detected in microsomal incubations. Iminium 8 is proposed (Scheme 2) for the formation of the third NAC conjugate. The generation of the iminium ion is possibly initiated by the driving force from the lone pair electrons of N-a of TFT, along with the ring opening of the epoxide moiety. NAC conjugation may take place at C-1′ of TFT by Michael addition in the reaction of NAC with 8, followed by dehydration. This article references 2 other publications.

中文翻译：

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对Veeravalli和Dash致编辑的信的答复：“ Tofacitinib是一种基于机制的细胞色素P450 3A4灭活剂：重新探讨了环氧中间体和谷​​胱甘肽捕获的重要性”。

我们要感谢博士。Veeravalli和Dash对他们的关注（1）。（2）在本文中，确定了tofacitinib（TFT）的两个代谢活化途径，包括氧化脱氰和环氧化。另外，提出的所得α-酮醛代谢产物负责观察到的TFT诱导的CYP3A4的基于机理的失活。主要关注的问题包括环氧化途径参与TFT诱导的CYP3A4失活。（1）在我们的早期研究中，类似物3设计并合成了由TFT衍生的伯胺化合物，以探讨氧化脱氰和环氧化在TFT诱导的CYP3A4失活中的作用。（2）氰化物基团还原为伯胺完全消除了其对CYP3A4的酶抑制作用。母体化合物。此外，我们成功地检测类似物的3氧化物（方案1）从模拟的代谢氧化产生3。尽管我们不能完全排除环氧代谢物的参与，但这为环氧中间体参与TFT诱导的CYP3A4失活的可能性提供了关键证据。的确碳3'和4'的饱和度（模拟2）部分降低了TFT使酶失活的能力，这表明环氧化物代谢产物对TFT诱导的酶失活的贡献。然而，观察到的抑制作用降低伴随着相应的醛代谢产物的形成减少。（2）因此，观察到的类似物2抑制活性的部分损失最有可能是由于醛代谢产物形成的减少可能不是由于阻止环氧代谢物的产生。谷胱甘肽（GSH）通常用于淬灭从宿主酶活性位点逸出的反应性代谢产物。事实上，无论是ñ-乙酰半胱氨酸（NAC）和GSH用来捕获TFT的环氧中间体，两者均成功。显然，所得的NAC共轭物表现出更好的峰形，这就是为什么其离子色谱仪包含在文章中的原因。在微粒体温育中检测到三种来自中间体6和8的NAC缀合物。提议使用Iminium 8（方案2）来形成第三种NAC共轭物。亚胺离子的产生可能是由TFT Na的孤对电子的驱动力以及环氧化物部分的开环引发的。在NAC与8的反应中，通过迈克尔加成，可在TFT的C-1'处发生NAC共轭。，然后脱水。本文引用了其他2个出版物。