Acrolein mercapturates: Synthesis, characterization, and assessment of their role in the bladder toxicity of cyclophosphamide

Article abstract of 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- hydroxyPrMCA) 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-oxoPrMCAS-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 ~100% hydration of the aldehyde to the corresponding geminal diol was observed. When incubated at 25°C in 100 mM potassium phosphate buffer containing 1 M KCl, pH 8.0, 3-oxoPrMCA released ~6% and 3-oxoPrMCA S-oxide released ~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. These yields were unaffected by incubation in buffer for up to 2 h. Acrolein, 3-oxoPrMCA S-oxide, 3-oxoPrMCA and 3-oxoPrGSH, but not 3-hydroxyPrMCA, damaged the bladder dose-dependently when instilled intravesically in mice at concentrations of 10-20 mM. Potency was acrolein > 3-oxoPrMCA S-oxide > 3- oxoPrMCA > 3-oxoPrGSH. These data support the possibility that a mercapturic acid may be involved in the bladder toxicity of CP.