Rančić Aleksandra, Babić Nikola, Orio Maylis, Peyrot Fabienne
Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, Université Paris Cité, CNRS, F-75006 Paris, France.
iSm2, Aix-Marseille University, CNRS, Centrale Marseille, F-13397 Marseille, France.
Antioxidants (Basel). 2023 Feb 7;12(2):402. doi: 10.3390/antiox12020402.
Nitroxides are potent tools for studying biological systems by electron paramagnetic resonance (EPR). Whatever the application, a certain stability is necessary for successful detection. Since conventional tetramethyl-substituted cyclic nitroxides have insufficient in vivo stability, efforts have recently been made to synthesize more stable, tetraethyl-substituted nitroxides. In our previous study on piperidine nitroxides, the introduction of steric hindrance around the nitroxide moiety successfully increased the resistance to reduction into hydroxylamine. However, it also rendered the carbon backbone susceptible to modifications by xenobiotic metabolism due to increased lipophilicity. Here, we focus on a new series of three nitroxide candidates with tetraethyl substitution, namely with pyrrolidine, pyrroline, and isoindoline cores, to identify which structural features afford increased stability for future probe design and application in in vivo EPR imaging. In the presence of rat liver microsomes, pyrrolidine and pyrroline tetraethyl nitroxides exhibited a higher stability than isoindoline nitroxide, which was studied in detail by HPLC-HRMS. Multiple metabolites suggest that the aerobic transformation of tetraethyl isoindoline nitroxide is initiated by hydrogen abstraction by P450-Fe = O from one of the ethyl groups, followed by rearrangement and further modifications by cytochrome P450, as supported by DFT calculations. Under anaerobic conditions, only reduction by rat liver microsomes was observed with involvement of P450-Fe.
氮氧化物是通过电子顺磁共振（EPR）研究生物系统的有力工具。无论应用如何，成功检测都需要一定的稳定性。由于传统的四甲基取代环状氮氧化物在体内稳定性不足，最近人们致力于合成更稳定的四乙基取代氮氧化物。在我们之前关于哌啶氮氧化物的研究中，在氮氧化物部分周围引入空间位阻成功提高了其还原为羟胺的抗性。然而，由于亲脂性增加，它也使碳骨架容易受到外源性代谢的修饰。在这里，我们专注于一系列新的具有四乙基取代的三种氮氧化物候选物，即具有吡咯烷、吡咯啉和异吲哚啉核心的化合物，以确定哪些结构特征能为未来的探针设计和体内EPR成像应用提供更高的稳定性。在大鼠肝微粒体存在的情况下，吡咯烷和吡咯啉四乙基氮氧化物表现出比异吲哚啉氮氧化物更高的稳定性，通过HPLC-HRMS对其进行了详细研究。多种代谢物表明，四乙基异吲哚啉氮氧化物的需氧转化是由P450-Fe = O从其中一个乙基上夺取氢引发的，随后进行重排并由细胞色素P450进一步修饰，DFT计算支持了这一观点。在厌氧条件下，仅观察到大鼠肝微粒体参与的还原反应，涉及P450-Fe。
