Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris Descartes, Sorbonne Paris Cité, 45 rue des Saints-Pères, 75270 Paris, France.
Chem Res Toxicol. 2013 Oct 21;26(10):1561-9. doi: 10.1021/tx400250a. Epub 2013 Sep 26.
Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals, such as 1a ("Finland" radical), are useful EPR probes for oximetry. However, they are rapidly metabolized by liver microsomes in the presence of NADPH, with the formation of diamagnetic quinone-methide metabolites resulting from an oxidative decarboxylation of one of their carboxylate substituents. In an effort to obtain TAM derivatives potentially more metabolically stable in vivo, we have synthesized four new TAM radicals in which the carboxylate substituents of 1a have been replaced with esters groups bearing various alkyl chains designed to render them water-soluble. The new compounds were completely characterized by UV-vis and EPR spectroscopies, high resolution mass spectrometry (HRMS), and electrochemistry. Two of them were water-soluble enough to undergo detailed microsomal metabolic studies in comparison with 1a. They were found to be stable in the presence of the esterases present in rat liver microsomes and cytosol, and, contrary to 1a, stable to oxidation in the presence of NADPH-supplemented microsomes. A careful study of their possible microsomal reduction under anaerobic or aerobic conditions showed that they were more easily reduced than 1a, in agreement with their higher reduction potentials. They were reduced into the corresponding anions not only under anaerobic conditions but also in the presence of dioxygen. These anions were much more stable than that of 1a and could be characterized by UV-vis spectroscopy, MS, and at the level of their protonated product. However, they were oxidized by O₂, giving back to the starting ester radicals and catalyzing a futile cycle of O₂ reduction. Such reactions should be considered in the design of future stable EPR probes for oximetry in vivo.
三（对羧基三噻吩基甲基）（TAM）自由基，如 1a（“芬兰”自由基），是用于血氧测定的有用的 EPR 探针。然而，在存在 NADPH 的情况下，它们会被肝微粒体迅速代谢，形成顺磁性醌甲化物代谢物，这是由于其羧酸盐取代基之一发生氧化脱羧反应所致。为了获得体内代谢更稳定的 TAM 衍生物，我们合成了四个新的 TAM 自由基，其中 1a 的羧酸盐取代基被带有各种烷基链的酯基取代，旨在使其水溶性更强。新化合物通过紫外可见和 EPR 光谱、高分辨率质谱（HRMS）和电化学完全表征。其中两种具有足够的水溶性，可以与 1a 进行详细的微粒体代谢研究。它们在大鼠肝微粒体和胞质中存在的酯酶存在下稳定，与 1a 不同，在补充 NADPH 的微粒体存在下稳定，不易被氧化。仔细研究它们在厌氧或有氧条件下可能的微粒体还原表明，它们比 1a 更容易还原，这与它们较高的还原电位一致。它们不仅在厌氧条件下，而且在氧气存在下被还原为相应的阴离子。这些阴离子比 1a 的阴离子更稳定，可以通过紫外可见光谱、MS 和质子化产物的水平进行表征。然而，它们被 O₂ 氧化，还原回到起始的酯基自由基，并催化 O₂ 还原的无效循环。在设计用于体内血氧测定的未来稳定的 EPR 探针时，应考虑到这些反应。
