Kyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan.
Bioorg Med Chem. 2011 Sep 15;19(18):5693-7. doi: 10.1016/j.bmc.2011.07.012. Epub 2011 Jul 23.
N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe(2+)-H(2)O(2) (Fenton's reagent) with Cu(2+) activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe(2+)-Cu(2+)-H(2)O(2) on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe(2+), Cu(2+), H(2)O(2) and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The (1)H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe(2+)-Cu(2+)-H(2)O(2)-NO.
N-亚硝胺在 Ames 试验中，在大鼠 S9 混合液的存在下形成α-羟亚硝胺，从而显示其诱变活性。Fe(2+)-H(2)O(2)(Fenton 试剂)衍生的羟基自由基与 Cu(2+)激活 N-亚硝胺，其烷基链长于丙基成分，成为直接作用诱变剂。Fe(2+)-Cu(2+)-H(2)O(2)对亚硝胺的反应与其代谢激活的关系尚未完全表征。在这里，我们报道了在 Fe(2+)、Cu(2+)、H(2)O(2)和一氧化氮(NO)存在下，N-亚硝基-N-甲基戊基胺(NMPe)衍生的直接作用诱变剂的鉴定，NO 是亚硝胺代谢的产物。NMPe 反应混合物的二氯甲烷提取物通过硅胶柱色谱法多次和制备高效液相色谱法(HPLC)进行分离；我们得到了作为产物的白色晶体。分离得到的直接作用诱变剂通过(1)H 和(13)C 核磁共振(NMR)光谱、红外(IR)光谱和 X 射线晶体学被临时鉴定为 5-乙基-5-硝基-1-吡唑啉 1-氧化物。为了确认诱变剂的结构，从 2-硝基丁烯和重氮甲烷合成了该纯品，然后用间氯过苯甲酸进行 N-氧化。从合成的直接作用诱变剂中获得的(1)H NMR 光谱数据与从分离的诱变剂中获得的数据相同。此外，5-乙基-5-硝基-1-吡唑啉 1-氧化物在鼠伤寒沙门氏菌 TA1535 中具有致突变性。结果表明，5-乙基-5-硝基-1-吡唑啉 1-氧化物是 NMPe 与 Fe(2+)-Cu(2+)-H(2)O(2)-NO 反应的直接作用诱变剂。
