First unequivocal identification of the critical acyl radicals from the anti-tuberculosis drug isoniazid and its hydrazide analogs by complementary applications of ESR spin-trapping and HPLC/MS methods

Abstract

The carbon-centered isonicotinic acyl radical of isoniazid (INH), a widely-used frontline anti-tuberculosis drug, has been considered to play a critical role in inhibiting Mycobacterium tuberculosis, but not fully identified. Here we show that this radical intermediate can be unequivocally characterized by complementary applications of ESR spin-trapping and HPLC/MS methods by employing N-tert-butyl-α-phenylnitrone (PBN) as the suitable spin-trapping agent, which can form the most stable radical adduct. More importantly, for the first time, analogous carbon-centered acyl radicals and their respective NAD+ adducts have also been detected and identified from its two isomers (nicotinic acid hydrazide and 2-pyridinecarbohydrazide) and benzhydrazide which are structurally-related to INH, but not by 2-chloroisonicotinohydrazide. This study represents the first unequivocal identification of the carbon-centered acyl radicals of INH and other hydrazide analogs by both ESR spin-trapping and HPLC/MS methods, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti-tuberculosis drugs.