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Reactions of the melatonin metabolite AMK (N1-acetyl-5-methoxykynuramine) with reactive nitrogen species: Formation of novel compounds, 3-acetamidomethyl-6-methoxycinnolinone and 3-nitro-AMK


Address reprint requests to Dr Rüdiger Hardeland, Institute of Zoology, Anthropology and Developmental Biology, University of Göttingen, Berliner Str. 28, D-37073 Göttingen, Germany.


Abstract:  The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) was found to be unstable in air when adsorbed on a thin-layer silica gel chromatography plate, a result that is in good agreement with the relatively high reactivity of this compound. Three novel main products were separated from the reaction mixture and identified by mass spectrometry and nuclear magnetic resonance data as: (i) 3-acetamidomethyl-6-methoxycinnolinone (AMMC), (ii) 3-nitro-AMK (AMNK, N1-acetyl-5-methoxy-3-nitrokynuramine), and (iii) N-[2-(6-methoxyquinazolin-4-yl)-ethyl]-acetamide (MQA). AMMC and AMNK are shown to be nonenzymatically formed also in solution, by nitric oxide (NO) in the first case, and by a mixture of peroxynitrite and hydrogen carbonate, in the second one. The use of three different NO donors, PAPA-NONOate, S-nitroso-N-acetylpenicillamine and sodium nitroprussiate led to essentially the same results, with regard to a highly preferential formation of AMMC; AMNK was not detected in these reaction systems. Competition experiments with the NO scavenger N-acetylcysteine indicate a somewhat lower reactivity compared with the competitor. Peroxynitrite led to AMNK formation in the presence of physiological concentrations of hydrogen carbonate at pH 7.4, but not in its absence, indicating that nitration involves a mixture of carbonate radicals and NO2, formed from the peroxynitrite-CO2 adduct. No AMMC was detected after AMK exposure to peroxynitrite. Both AMNK and AMMC exhibited a much lower reactivity toward 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radicals than did AMK. In a competition assay for hydroxyl radicals, AMMC showed prooxidant properties, whereas AMNK was a moderate antioxidant. AMMC and AMNK should represent relatively stable physiological products, although their rates of synthesis are still unknown and may be low. Formation of these compounds may contribute to the disappearance of AMK from tissues and body fluids.