Studies on the Chemical and Biological Properties of Antitumour Triazenes

Abstract

Chemical and biological properties of l-aryl-3,3-dialkyltriazenes were studied with the aim of elucidating their mode of action and the identity of the active antitumour species. Photolysis of DTIC at alkaline pH afforded 2-azahypoxanthine, and at mildly acidic pH (2 - 6) 4-carbomoylimidazolium-5-olate. 5-Diazoimidazole-4-carboxamide, an intermediate in the photodecomposition of OTIC, was responsible for the interaction of DTIC with nucleophiles such as thiol compounds in the presence of sunlight. Another dimethyltriazene, 1-(4-acetylphenyl)-3,3-dimethyltriazene (AcDMT) was photostable, but subject to protolysis at pH< 5.2. The stability of monomethylbut not hydroxymethyl-triazenes (both dimethyltriazene metabolites) at physiological pH, measured by UV spectroscopy, appeared great enough to allow distribution in vivo to a distant tumour site. Certain properties of N-demethylation of AcDMT such as species differences, investigated by normal phase HPLC, differed from the N-demethylation of aminopyrine, a model substrate of cytochrome P-450 N-demethylation. The metabolism of dialkyltriazenes under conditions of the bioassay was studied by reverse phase HPLC and monomethyltriazenes were identified as metabolic products of active antitumour derivatives. Monoalkyltriazenes were themselves metabolised, probably yielding a product other than the corresponding arylamine. 1-(4-Acetylphenyl)-3-methyltriazene (AcMMT) was significantly less toxic to hepatocytes than AcDMT when cell viability was assayed by the trypan blue dye exclusion test. Incubation of TLX5 lymphoma cells with AcMMT resulted in a significantly greater reduction in viability of the triazene sensitive than the resistant cell lines. Neither TLX5 cell line was able to activate AcDMT, or to metabolise AcMMT. There was no significant difference between the two TLX5 cell lines in their total glutathione content. Monoalkyltriazenes, but not arylamines, prevented the oxidation of glutathione in mouse liver homogenate in Earl's buffer. AcMMT depleted total glutathione levels in mouse hepatocytes, and the increase in extracellular total glutathione could not account for intracellular loss. AcMMT and AcDMT did not inhibit the enzyme glutathione reductase.