Identification and synthesis of metabolites of 4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide

Introduction: 4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide is a new selective type II carbonic anhydrase inhibitor with local action through instillation into the eyes. For a complete pharmacokinetic study of this drug, it is necessary to detect and synthesize its metabolites for their systemic exposure evaluation. Materials and Methods: The investigation was performed on 6 Wistar rats and 6 Soviet Chinchilla rabbits. The drug in the form of a 1% suspension was administered by intraperitoneal injection. Blood was sampled in a volume of 0.2 mL at the following time points: before administration and 1 h, 2 h, 4 h, 24 h after administration. Then 150 µL of each sample was centrifuged to produce plasma. Urine was simultaneously sampled in rats using metabolic cells: before administration and at intervals of 0-2 h, 2-4 h, 4-6 h, 6-24 h after administration of the drug. The identification of metabolites in these objects was performed using HPLC-MS/MS. Then the detected biotransformation products were synthesized. The structure of the obtained substances was confirmed by NMR spectroscopy and high-resolution mass spectrometry. At the final stage, animal biological fluids and model samples with the addition of the synthesized compounds were analyzed using HPLC-MS/MS to establish the structure of metabolites.Results and Discussion: N-hydroxy-4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide, 4-(5-(hydroxymethyl)-1,3,4-oxadiazole-2-yl)-benzenesulfonamide and 4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonic acid were identified and synthesized. The complete coincidence of the structure of metabolites and the synthesized substances was established as a result of their comparison in retention time, the ratio of the areas of chromatographic peaks at the main MRM transitions, as well as mass spectra.Conclusion: N-hydroxy-4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide and 4-(5-(hydroxymethyl)-1,3,4-oxadiazole-2-yl)-benzenesulfonamide are products of biotransformation of the studied drug. It was found that 4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonic acid is formed by decomposition of N-hydroxymetabolite in urine samples during the collection process.