New bromisoval (bromural) metabolites in human urine: a-(cystein-S-yl)isovalerylurea, a-(N-acetylcystein-S-yl)isovalerylurea and a-(cysteamin-S-yl)isovaleric acid

Abstract

Large amounts of a-(N-acetylcystein-S-yl)isovalerylurea (AcCVU) and a-(cystein-S-yl)isovalerylurea (CVU) and trace amounts of isovalthine (Ivt), N-acetylisovalthine (AcIvt) and a-(cysteamin-S-yl)isovaleric acid (CAVA) were found in human urine after administration of a-bromoisovalerylurea (Bromisoval, Bromural). AcCVU and CVU were isolated from urine and identified by gas chromatography—mass spectrometry (GC—MS) as trimethylsilyl (TMS) derivatives, by MS of the methyl esters, by chemical degradation and by NMR (CVU only), IR and chromatographic comparison with synthetic reference compounds. Ivt, AcIvt and CAVA were identified by co-chromatography with pure reference compounds in several chromatographic systems. Syntheses of CVU, βCVU, AcCVU and CAVA are described. CVU is hydrolysed under mildly alkaline conditions to give urea and a cyclic intermediate, 2-isopropyltetrahydro-1,4-thiazin-3-one-5-carboxylic acid (CITO), which was identified by GC—MS as the bis-TMS derivative. Under more drastic alkaline or acidic conditions, CITO, CVU and AcCVU are hydrolysed to give Ivt, an acidic sulphur amino acid. Urinary excretion of CVU was measured quantitatively on an amino acid analyser. The sum of AcCVU and AcIvt was measured as N-acetylalanine (after desulphuration of the sample) by selected ion monitoring using isobutane chemical ionization and trideutero-AcCVU as an internal standard. After a single oral dose of 0.9 g of Bromisoval, two healthy adults excreted in urine 64 and 43 mole-%, respectively, in the form of the mercapturic acids AcCVU + AcIvt (mainly AcCVU, with only trace amounts of AcIvt) and 4.6 and 5.9 mole-% as CVU within 24 h. The excretion rate was maximal at about 5 h after drug intake with concentrations up to 12.5 mmole/1 of AcCVU (+AcIvt) and 1.1 mmole/l of CVU found in a 3/2-6-h urine sample. Hence, the dominating metabolic pathway of the xenobiotic Bromisoval is debromination by liver glutathione-S-transferases. The corresponding glutathione and cysteinylglycine derivatives can be expected to be present in bile. We detected the urinary Bromisoval metabolites by chance in a woman who claimed not to have taken any drugs. It is possible that the freely available sedative a-bromoisovalerylurea is present in mixtures that are not signified as being drugs. Our findings make necessary the re-investigation of Japanese studies on isovalthinuria, which has been claimed to be induced by many compounds related to the cholesterol metabolism.