Study of biotransformation of new selective carbonic anhydrase II inhibitor 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide

Abstract

The aim of the study was to determine biotransformation products of a new selective carbonic anhydrase II inhibitor – 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide.Materials and methods. The study was conducted on 3 Wistar rats and 3 rabbits of the Soviet Chinchilla breed. The suspension of the drug was administered intraperitoneally to rats at a dosage of 20 mg/kg, to rabbits - at a dosage of 1.6 mg/kg. The animal blood samples were collected before the administration and 1, 2, 4, 24 h after. Urine sampling was also performed in the rats before the administration and in the intervals of 0–4, 4–8, 8–24 h after. The identification of metabolites in blood, urine and plasma was carried out using HPLC-MS/MS. Poroshell 120 C 18 column (50×3.0 mm, 2.7 µm) with a Zorbax Eclipse Plus C18 pre-column (12.5×2.1 mm, 5.0 µm) was used for the chromatographic separation. The assumed metabolites were synthesized, their structure was confirmed by the NMR spectroscopy method and a high-resolution mass spectrometry. The obtained substances were compared with the substances identified in biological fluids by retention time, the main MRM-transitions and mass spectra.Results. The N-hydroxymetabolite was revealed in the analyses of plasma, blood and urine samples which had been formed by the addition of an oxygen atom to the drug molecule. Chromatographic peaks of this compound were identified at the MRM-transitions of 255→159, 255→117, 255→89 m/z at the 7.2nd min of the analysis. The N-oxide of 4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide and N-hydroxy-4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide were synthesized; potentially, they could have been obtained during the biotransformation. During the confirmatory HPLC-MS/MS tests based on the coincidence of the retention times, the main MRM transitions and mass spectra, the ratio of the peak areas at the identified metabolite it was established that an N-hydroxy derivative. Chromatographic peaks of the N-oxide detected in the analysis of the model mixtures of the standard substance at the MRM-transitions of 255→175, 255→133, 255→89 m/z at the retention time of 5.43 min, were absent in the animal samples.Conclusion. The studied drug is metabolized to form a single metabolite of N-hydroxy-4-(2-methyl-1,3-oxazole-5-yl)-benzenesulfonamide. This compound was found in freshly collected samples of biological fluids of both animal species. The structure of the metabolite was confirmed by the HPLC-MS/MS-method by comparison with the synthesized standard substance.