Identification of Amino Acid and Glutathione N-Conjugates of Toosendanin: Bioactivation of the Furan Ring Mediated by CYP3A4

Toosendanin (TSN) is a hepatotoxic triterpenoid extracted from Melia toosendan Sieb et Zucc. Considering that TSN contains the structural alert of the furan ring, it is believed that bioactivation of TSN may be responsible for its toxicity. Herein, the bioactivation potential and metabolism profiles of TSN were investigated. After an oral administration of 10 mg/kg TSN to rats, esterolysis and conjugation with amino acids were identified as the main metabolic pathways. The same types of conjugates were detected in liver microsomes in an NADPH-dependent manner. According to the remaining amount of the parent drug, the reactivity of trapping reagents with TSN reactive metabolites was sorted in a decreasing order of N-alpha-(tert-butoxycarbonyl)-l-lysine (Boc-Lys) > alanine, lysine, taurine, phenylalanine, serine, glutamic acid, glycine, and glutathione (GSH) > cysteine. No conjugates were observed in NADPH and N-acetyl cysteine (NAC)-supplemented human liver microsomal incubations. Further phenotyping studies and the chemical synthesis of the major conjugated standards proved that TSN was bioactivated by CYP3A4 and yielded a cis-butene-1,4-dial intermediate, which was prone to undergo 1,2-addition with the amino group of amino acids and GSH to form 3-pyrroline-2-one adducts. The sulfydryl group of GSH also attacked the intermediate and yielded S-conjugates by 1,4- or 1,2-addition, which would form pyrrole conjugates by further reacting with the amino group. Compared to the well-recognized S-conjugation of the furan ring, N-conjugation with multiple amino acids and GSH played a more important part in the elimination of reactive metabolites of TSN. The significance of these conjugates requires further investigation.