Metabolomics reveals a unique CYP3A-mediated C(sp3)-C(sp2) bond cleavage via ipso-addition reaction in drug metabolism

Cytochrome P450-mediated carbon–carbon (C-C) cleavages are unusual, especially for mammalian drug metabolizing enzymes. Revealing the unusual reactions in biological system is very arduous and selectively oxidative C-C cleavage is also a long-standing challenge in chemistry and biology. We herein present a rapid and efficient metabolomic-based approach to uncover human CYP3A-mediated non-polar, unstrained C(sp2)-C(sp3) bond cleavage in the CSF-1R inhibitor pexidartinib. Using synthetic metabolites, 18O2, and H218O, we demonstrate that one unique cleavage is via the ipso-addition reaction. This is the first report of CYP3A-mediated ipso-addition reaction to the 5-alkylated N-protected pyridin-2-amines. We have expanded the range of substrates undergoing CYP3A-mediated ipso-addition reactions beyond para-phenols to include N-protected alkylated pyridine-2-amines. Our metabolomic-based approach also successfully discovered the CYP3A-mediated C(sp2)-C(sp3) bond cleavage of PEX analogs as well as the antidepressant nefazodone. This work established an efficient strategy to identify the uncommon reactions in drug metabolism using a metabolomic strategy. More importantly, the environmentally friendly conditions of CYP3A-catalyzed unusual ispo-addition reactions hold the potential to inspire future exploration of biomimetic P450-reprogramming methods for addressing the challenging task of unactivated C-C bond cleavage in the field.