Synthesis of 13C215N2-labeled anti-inflammatory and cytoprotective tricyclic bis(cyanoenone) ([13C215N2]-TBE-31) as an internal standard for quantification by stable isotope dilution LC-MS method

Abstract

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. 13C215N2-labeled TBE-31 ([13C215N2]-TBE-31), which has two 13C and two 15N 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 coupledwith the use of a stable isotope-labeled internal standard. [13C215N2]-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 13C atoms with ethyl [13C]formate and two 15N atoms with hydroxyl[15N] amine. The stable isotope dilution liquid chromatography-mass spectrometrymethod for quantification of TBE-31 was successfully developed using [13C215N2]-TBE-31 to compensate for any variables encountered during sample processing and analysis.