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Further Studies on the Conversion of 4-Hydroxyoxazaphosphorines to Reactive Mustards and Acrolein in Inorganic Buffers¹

Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455

The rates at which the 4-hydroxyoxazaphosphorines, 4-hydroxycyclophosphamide and 4-hydroxyifosfamide, are converted to reactive mustards and acrolein in phosphate and bicarbonate buffers were determined as a function of pH, ionic strength, temperature, and buffer concentration. Conversion was firstorder with respect to both the 4-hydroxyoxazaphosphorine and phosphate or carbonate serving as a catalyst. The catalytic constant for dianionic phosphate-catalyzed conversion of 4-hydroxyifosfamide to isophosphoramide mustard and acrolein at 37° was 0.189 min^-1 M^-1; a value of 0.194 min^-1 M^-1 was obtained when dianionic phosphate-catalyzed conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was examined. A catalytic constant of 3.09 min^-1 M^-1 was obtained for carbonate-catalyzed conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein. Hydroxyl ion and water also catalyzed the reaction; catalytic constants were 2710 and 0.000006 min^-1 M^-1, respectively. The rate at which the 4-hydroxyoxazaphosphorines were converted to reactive mustards and acrolein in phosphate buffer increased as the pH, ionic strength, and temperature increased. The energy of activation was about 20 kcal/mol. Dianionic phosphate, carbonate, hydroxyl ion, and water were apparently acting as general base catalysts of the rate-limiting step (probably the conversion of the intermediate aldehyde to the corresponding reactive mustard and acrolein) of the overall reaction, although specific base-general acid catalysis could not be ruled out. Bifunctional catalysis of the rate-limiting step did not appear to be significant and certainly was not obligatory as concluded previously by our laboratory. Assuming that the oncotoxic specificity of the oxazaphosphorines resides with the 4-hydroxyoxazaphosphorines and that their cytotoxic effect at therapeutic doses is largely mediated by the reactive mustards released within cells, these observations offer the possibility that intracellular general base catalytic activity serves as an important determinant with regard to the oncotoxic potential and specificity of the oxazaphosphorines. General base catalytic activity varies directly with pH, ionic strength, temperature, and the concentration of the base. The influence of some of these factors on the development of cyclophosphamide-induced bladder toxicity has already been demonstrated.

¹ Supported by USPHS Grant CA 21737. This is Paper 13 in a series of papers on cyclophosphamide metabolism.

² A description of this investigation appears in a thesis submitted in 1983 to the Department of Pharmacology, University of Minnesota, Minneapolis, in partial fulfillment of the requirement for the Ph.D. degree.

³ Present address: Department of Pharmacology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, N. Y. 14642.

⁴ To whom requests for reprints should be addressed, at the University of Minnesota, Department of Pharmacology, 3-260 Millard Hall, 435 Delaware Street S.E., Minneapolis, Minn. 55455.

Received 5/18/83. Accepted 9/ 2/83.