Synthesis of (C2N2)-C-13-N-15-labeled anti-inflammatory and cytoprotective tricyclic bis(cyanoenone) ([(C2N2)-C-13-N-15]-TBE-31) as an internal standard for quantification by stable isotope dilution LC-MS method

摘要

Tricyclic bis(cyanoenone), TBE-31, one of the most potent activators of the Keap1/Nrf2/antioxidant response element pathway, has been developed as a new anti-inflammatory and cytoprotective agent. (C2N2)-C-13-N-15-labeled TBE-31 ([(C2N2)-C-13-N-15]-TBE-31), which has two C-13 and two N-15 atoms in two cyano groups, was designed to develop a method for quantification of cell, tissue, and plasma levels of TBE-31 that involves chromatography/mass spectrometry coupled with the use of a stable isotope-labeled internal standard. [(C2N2)-C-13-N-15]-TBE-31 was successfully synthesized in four steps from a previously reported intermediate, which is prepared in 11 steps from cyclohexanone, by introduction of two C-13 atoms with ethyl [C-13]formate and two N-15 atoms with hydroxyl[N-15]amine. The stable isotope dilution liquid chromatography-mass spectrometry method for quantification of TBE-31 was successfully developed using [(C2N2)-C-13-N-15]-TBE-31 to compensate for any variables encountered during sample processing and analysis.