Direct detection of the intracellular formation of carboxyphosphamides using nuclear magnetic resonance spectroscopy.

31P nuclear magnetic resonance (NMR) spectroscopy was used in conjunction with cell perfusion techniques to monitor the intracellular chemistry of the cyclophosphamide (CP, CAS 6055-19-2) metabolites 4-hydroxy-cyclophosphamide (4-HO-CP) and aldophosphamide (AP) in U937 human histiocytic (CP-sensitive) and K562 human erythroleukemia (CP-resistant) cells. Similar experiments were carried out using the ifosfamide (IF, CAS3778-73-2) metabolites 4-hydroxyifosfamide (4-HO-IF) and aldoifosfamide (AIF). The hydroxy and aldehydic metabolites were generated by the triphenylphosphine reduction of 4-hydroperoxycyclophosphamide (4-HO2-CP) or 4-hydroperoxyifosfamide (4-HO2-IF) or by a spontaneous elimination/addition reaction involving water and 4-thiocyclophosphamide analogs 4-(2-hydroxyethyl) thiocyclophosphamide (4-ESCP) or mafosfamide. Cell death resulting from 4-HO-CP/AP perfusions was mimicked by perfusion with acrolein or an acrolein producing but non-alkylating, dechloro-CP analog. Acrolein toxicity was minimized by the presence of 2-mercaptoethanol or mesna (sodium 2-mercaptoethanesulfonate) in perfusion solutions as well as by fractional dose drug perfusions (sequential 2.5-3.0 h perfusions separated by cell washes with drug-free medium). The intracellular half-life for phosphoramide mustard (PM) at an intracellular pH value of 7.1 +/- 0.1 and an ambient probe temperature of 23 +/- 1 degree C in U937 cells was 2.1 h [k = (5.4 +/- 0.3) x 10(-3) min-1] and in K562 cells was 3.1 h [k = (3.7 +/- 0.4) x 10(-3) min-1]. Similar half-lives (2-4 h) were determined for intracellular isophosphoramide mustard (IPM). Fractional dose perfusion of U937 or K562 cells with 1.5 mmol/l 4-HO-CP/AP (generated from 4-HO2-CP) and 0.3 mmol/l mesna allowed for the observation of intracellular carboxyphosphamide (CBP); CBP was formed in higher concentrations in the CP-resistant K562 cells. Similar results were obtained using 4-ESCP and mafosfamide as sources of 4-HO-CP/AP. Identification of CBP was based on chemical shift, chemical stability, and membrane permeability studies of synthetic CBP. Concentrations of carboxyifosfamide (CBIF) formed in K562 cells were also greater than that in U937 cells.