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

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 coupled with 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 spectrometry method for quantification of TBE-31 was successfully developed using [¹³C₂¹⁵N₂]-TBE-31 to compensate for any variables encountered during sample processing and analysis.