Ramu K, Fraiser L H, Mamiya B, Ahmed T, Kehrer J P
Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin 78712-1074, USA.
Chem Res Toxicol. 1995 Jun;8(4):515-24. doi: 10.1021/tx00046a005.
Acrolein is the metabolite of cyclophosphamide (CP) believed to be involved in the bladder toxicity associated with this anticancer drug. The mechanism by which this extremely reactive intermediate is delivered to the bladder is not known. Glutathione (GSH) readily conjugates with acrolein, and the acrolein mercapturate S-(3-hydroxypropyl)-N-acetylcysteine (3-hydroxy-PrMCA) has been found in the urine of animals and man given CP. The objectives of this study were to prepare and characterize synthetic standards of the GSH acrolein adduct (3-oxopropyl)glutathione (3-oxoPrGSH), the acrolein mercapturates S-(3-oxopropyl)-N-acetylcysteine (3-oxoPrMCA) and 3-hydroxyPrMCA, and the S-oxidation product of 3-oxoPrMCA (3-oxoPrMCA S-oxide). In addition, the release of acrolein from, and the bladder toxicity of, these conjugates was determined. 3-OxoPrGSH and 3-oxoPrMCA were prepared with a 99% yield by condensing acrolein with GSH and N-acetylcysteine, respectively. 3-HydroxyPrMCA was prepared with a 63% yield by refluxing 3-chloropropanol and N-acetylcysteine in a basic medium. Oxidation of 3-oxoPrMCA with H2O2 was used to prepare 3-oxoPrMCA S-oxide. By decreasing the reaction time to 1 h, and adjusting the ratio of 3-oxoPrMCA to H2O2, the yield of 3-oxoPrMCA S-oxide was increased to 96%. The anhydrous aldehyde, 3-oxoPrMCA, afforded characteristic aldehydic proton resonances (1H NMR) in deuterated dimethyl sulfoxide. New resonances were observed in deuterated water, indicating a 75% hydration of the aldehyde to the corresponding geminal diol. This phenomenon was enhanced with 3-oxoPrMCA S-oxide where approximately 100% hydration of the aldehyde to the corresponding geminal diol was observed. When incubated at 25 degrees C in 100 mM potassium phosphate buffer containing 1 M KCl, pH 8.0, 3-oxoPrMCA released approximately 6% and 3-oxoPrMCA S-oxide released approximately 16-18% of the theoretical maximum yield of acrolein after 30 min, as indicated by an increase in absorbance at 210 nm and confirmed by trapping this aldehyde as a semicarbazone. There was less than a 2% yield of acrolein from 3-hydroxyPrMCA or 3-oxoPrGSH under similar conditions. At pH 7.4 the release of acrolein from 3-oxoPrMCA and 3-oxoPrMCA S-oxide was decreased by 50%. An assay where aldehydes are reacted with m-aminophenol in acid media produced fluorescence consistent with 72%, 46%, 23%, and 1% yields of acrolein from 3-oxoPrMCA S-oxide, 3-oxoPrMCA, 3-oxoPrGSH, and 3-hydroxyPrMCA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
丙烯醛是环磷酰胺（CP）的代谢产物，被认为与这种抗癌药物相关的膀胱毒性有关。这种极具反应性的中间体输送至膀胱的机制尚不清楚。谷胱甘肽（GSH）能轻易与丙烯醛结合，并且在给予CP的动物和人的尿液中发现了丙烯醛的硫醚氨酸S-（3-羟丙基）-N-乙酰半胱氨酸（3-羟基-PrMCA）。本研究的目的是制备并表征谷胱甘肽-丙烯醛加合物（3-氧代丙基）谷胱甘肽（3-氧代PrGSH）、丙烯醛硫醚氨酸S-（3-氧代丙基）-N-乙酰半胱氨酸（3-氧代PrMCA）和3-羟基PrMCA以及3-氧代PrMCA的S-氧化产物（3-氧代PrMCA S-氧化物）的合成标准品。此外，还测定了这些共轭物中丙烯醛的释放情况以及膀胱毒性。3-氧代PrGSH和3-氧代PrMCA分别通过丙烯醛与GSH和N-乙酰半胱氨酸缩合制备，产率为99%。3-羟基PrMCA通过在碱性介质中回流3-氯丙醇和N-乙酰半胱氨酸制备，产率为63%。用H2O2氧化3-氧代PrMCA制备3-氧代PrMCA S-氧化物。通过将反应时间缩短至1小时，并调整3-氧代PrMCA与H2O2的比例，3-氧代PrMCA S-氧化物的产率提高到了96%。无水醛3-氧代PrMCA在氘代二甲亚砜中给出了特征性的醛基质子共振（1H NMR）。在氘代水中观察到了新的共振，表明醛向相应偕二醇的水合率为75%。这种现象在3-氧代PrMCA S-氧化物中更为明显，其中观察到醛向相应偕二醇的水合率约为百分之百。当在含有1 M KCl、pH 8.0的100 mM磷酸钾缓冲液中于25℃孵育时，3-氧代PrMCA在30分钟后释放出约6%的理论最大产率的丙烯醛，3-氧代PrMCA S-氧化物释放出约16 - 18%的理论最大产率的丙烯醛，这通过210 nm处吸光度的增加得以表明，并通过将该醛捕获为半卡巴腙得以证实。在类似条件下，3-羟基PrMCA或3-氧代PrGSH的丙烯醛产率低于2%。在pH 7.4时，3-氧代PrMCA和3-氧代PrMCA S-氧化物的丙烯醛释放量降低了50%。一种醛在酸性介质中与间氨基酚反应的测定方法产生的荧光分别与3-氧代PrMCA S-氧化物、3-氧代PrMCA、3-氧代PrGSH和3-羟基PrMCA的丙烯醛产率72%、46%、23%和1%一致。（摘要截于400字）
