Analytical Studies on Antileprous Drugs

VI. On the Analysis of Sulfone Drugs and their Metabolites by Thin-layer Chromatography

Abstract

To find out a favorable method for the analysis of various sulfone drugs and their metabolites, the separation of 55 compounds relating to the present drugs was studied by thin-layer chromatography (TLC).
The separation of DDS, DDSO, 4, 4'-Diaminodiphenyl sulfide (DDSD) and their mono-N-acetylates could easily be achieved on a thin-layer plate. When benzene/ethylacetate (2/3) was used as a developing system, the order of their Rf values was S>SO₂>SO and NH₂>NHCOCH₃. Also, the order was found to be R, R': NO₂>H>OCH₂CH₂OC₂ H₅>NH2NH>OH, NH₂NHCOCH3, NHCH₂CH₂OH>COOH, NHCH₂COOH in the case of 4-R-C₆H_4/SO₂C₆H₄-R'-4'. When the same solvent system was used, no clear difference was found between the Rf value of a diphenyl sulfone compound (DS) and that of the corresponding diphenyl sulfonamide (DSA) or the diphenyl ethyl sulfonamide (DESA), though the Rf value of the former was somewhat higher than that of the latter two.
Applying this method, a sample of human urine was examined after the oral intake of DDSD. The metabolites identified were DDSD, the mono-N-acetylate (MAcDDSD) and DDS (all of them faintly detected), and MAcDDS, unchanged DDSO, and MAcDDSO (all of them markedly detected).
It was also coincident with the result of the experiment, that the MAcDDSO was more labile under auto-oxidation than DDSO when they were allowed to stand in ethylacetate. Therefore, it can be said that this experiment suggests the reason why a considerable portion of DDSO is found to be existent as MAcDDS in the urine sample, even though this oxidation is presumed to be accelerated by TPN-linked liver microsomal function. However, in the urine sample of a rabbit, DDS, MAcDDS, MAcDDSO, and unchanged DDSO could merely be detected.
Several authors had already reported the reduction of some dialkyl sulfoxides to the corresponding sulfides by an enterobacteria^{5a, b)} or their oxidation by liver microsomes^5c) However, no report hitherto mentioned the reduction of diaryl sulfoxdes in vivo.
In the second step, the separation of some water-soluble labile derivatives or conjugates of DDS was carried out to certify the main metabolites of Promin by TLC. A compound named Promin A (PrA) was prepared for this purpose, together with ¹⁴C-PrA, and intraveneously injected into several rabbits, which were synthesized by sodium glucuronate (GNa), glucuronic amide (GNH₂) or GK-6-¹⁴C respectively, instead of glucose in the case of Promin. All of them were more labile than Promin in aqueous solution.
As the result, it was found that the main metabolites in the rabbit urine were DDS and mono-N-glucosiduronate (DDSG). This result suggested that a main metabolite of Promin might be DDS mono-N-glucoside, especially if the Rf values of DDS mono-and di-N-glucoside were compared with those of the corresponding mono- and di-N-glucosidu-ronate on a TLC plate, even though DDS N-glucosides were more labile than the N-glucosiduronates, and also the mono-N-glucoside as a standard material could not be isolated yet.
In spite of the report by Dr. Sweet^6a) and Dr. Bushby^6b) who had respectively presumed and maintained the existence of DDS mono-N-glucose sulfonite (mono-Promin) as a main metabolite of Promin, this predominant desulfinition was again certified in the case of PrA.
In addition, because the measured radioactivity in the rabbit urine and the plasma was calculated to be too low to explain the total activity in ¹⁴C-PrA injected into the rabbit, the GK-6-¹⁴C freed from ¹⁴C-PrA was supposed to be separately catabolized in vivo, though the relation between the xylulose cycle could not be explained.