Deroussent Alain, Ré Micheline, Hoellinger Henri, Vanquelef Enguerran, Duval Olivier, Sonnier Michèle, Cresteil Thierry
IFR54, Institut Gustave Roussy, Pavillon de recherche, 94805 Villejuif, France.
Rapid Commun Mass Spectrom. 2004;18(4):474-82. doi: 10.1002/rcm.1357.
Ethoxidine (N-methyl-12-ethoxy-2,3,8,9-tetramethoxybenzo[c]phenanthridinium methylsulfonate salt) is a synthetic 2-methoxy-12-ethoxy derivative of the natural alkaloid fagaronine. This new inhibitor of DNA-topoisomerase I is considered as a potential antitumor agent with higher in vitro activity than fagaronine. In order to further improve the efficiency of ethoxidine, its in vitro biotransformation by hepatic monooxygenases and the structures of its metabolites were investigated by high-performance liquid chromatography (HPLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) and accurate mass measurement by time-of-flight mass spectrometry (TOFMS). When ethoxidine was incubated with BNF-treated rat liver microsomes or with cells expressing different recombinant human cytochrome P450, the same four ethoxidine metabolites (m(1)-m(4)) were detected and were formed exclusively by CYP1A1. The structures of these metabolites were assigned from ESI-MS/MS mass spectra and compared with those of ethoxidine derivatives. Accurate mass measurements of in-source ESI-TOFMS fragment ions exhibited successive neutral losses of C(2)H(4) and CO for ethoxidine and its metabolites. Whereas a 15 Da loss (methyl radical) was observed for the metabolites m(1)-m(4) containing a quaternary ammonium group, a 16 Da loss (methane) was observed for ethoxidine and could have resulted from the presence of two methoxy groups at adjacent positions (C-2 and C-3). The proposed oxidative modifications of ethoxidine were further confirmed by determination of the number of exchangeable hydrogen atoms and by the proposed elemental compositions of the metabolites based on accurate mass measurements by TOFMS. Two major metabolites resulted from O-demethylation of ethoxidine; one was tentatively identified as 12-ethoxyfagaronine (m(3)) and the second as an O-demethylated ethoxidine isomer (m(4)). Two polar metabolites were shown to be O-demethylated (m(1)) and hydroxylated (m(2)) derivatives of 12-ethoxyfagaronine. When 12-ethoxyfagaronine was incubated under the same conditions as ethoxidine, m(2) was formed, thus supporting the proposal that 12-ethoxyfagaronine is the primary oxidative product of ethoxidine.
乙氧啶（N - 甲基 - 12 - 乙氧基 - 2,3,8,9 - 四甲氧基苯并[c]菲啶甲基磺酸盐）是天然生物碱法卡林二醇的一种合成2 - 甲氧基 - 12 - 乙氧基衍生物。这种新型DNA拓扑异构酶I抑制剂被认为是一种潜在的抗肿瘤药物，其体外活性高于法卡林二醇。为了进一步提高乙氧啶的效率，通过高效液相色谱（HPLC）结合电喷雾电离串联质谱（ESI - MS/MS）以及飞行时间质谱（TOFMS）进行精确质量测量，研究了其在肝单加氧酶作用下的体外生物转化及其代谢产物的结构。当乙氧啶与经β - 萘黄酮处理的大鼠肝微粒体或与表达不同重组人细胞色素P450的细胞一起孵育时，检测到相同的四种乙氧啶代谢产物（m(1) - m(4)），且它们仅由CYP1A1形成。这些代谢产物的结构通过ESI - MS/MS质谱确定，并与乙氧啶衍生物的结构进行比较。源内ESI - TOFMS碎片离子的精确质量测量显示，乙氧啶及其代谢产物相继发生C(2)H(4)和CO的中性丢失。对于含有季铵基团的代谢产物m(1) - m(4)，观察到15 Da的质量损失（甲基自由基）；对于乙氧啶，观察到16 Da的质量损失（甲烷），这可能是由于相邻位置（C - 2和C - 3）存在两个甲氧基所致。通过测定可交换氢原子的数量以及基于TOFMS精确质量测量得出的代谢产物的推测元素组成，进一步证实了乙氧啶的氧化修饰。乙氧啶的O - 去甲基化产生了两种主要代谢产物；一种初步鉴定为12 - 乙氧基法卡林二醇（m(3)），另一种为O - 去甲基化乙氧啶异构体（m(4)）。两种极性代谢产物被证明是12 - 乙氧基法卡林二醇的O - 去甲基化（m(1)）和羟基化（m(2)）衍生物。当12 - 乙氧基法卡林二醇在与乙氧啶相同的条件下孵育时，形成了m(2)，从而支持了12 - 乙氧基法卡林二醇是乙氧啶主要氧化产物的提议。
