Identification of two new types of S-linked conjugates of Etoc in rat

Abstract

1. Two major metabolites of ¹⁴C-labelled (4S, 1R)-trans-Etoc[(S)-2-methyl-4-oxo-3-(2-propynyl)cyclopent-2-enyl (1R)-trans-chrysanthemate] were purified using a combination of chromatographic techniques and identified by spectroanalysis (nmr(HMBC) and FAB-, TSP-MS). These were established as new types of S-linked conjugates (sulphonic acid and mercapturic acid types).

2. To examine the mechanism of formation of the sulphonic acid and mercapturic acid conjugates, sodium sulphate or glutathione labelled with ³⁵S were administered to rat along with unlabelled trans-Etoc. Both sulphonic acid and mercapturic acid conjugates were found in the excreta, more of the former being yielded with ³⁵S-sodium sulphate than with ³⁵S-glutathione, implying that a sulphonic acid was incorporated into the double bond of a possible intermediate after reduction of sulphate to sulphite. The mercapturic acid conjugate was produced only with ³⁵S-glutathione, implying incorporation of glutathione into the triple bond before subsequent generation of mercapturic acid from the glutathione conjugate.

3. Additional investigation of whether or not the mercapturic acid conjugate was produced by mixing the alcohol moiety of Etoc®, PGL (4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one) and N-acetyl-L-cysteine under alkaline conditions. However, spectral data for the synthesized compound were not the same as those of the metabolite generated in vivo. That is, the addition reaction appeared to proceed by anti-Markownikov's rule, whereas the in vivo metabolite was apparently formed according to Markownikov's rule. Addition of glutathione at a triple bond has not been reported to our knowledge for any other foreign compounds in mammalian species.