DIRECT-DETECTION OF THE INTRACELLULAR FORMATION OF CARBOXYPHOSPHAMIDES USING NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

P-31 nuclear magnetic resonance (NMR) spectroscopy was used in conjunc tion with cell pei;fusion techniques to monitor the intracellular chem istry of the cyclophosphamide (CP, CAS 6055-19-2) metabolizes 4-hydrox y-cyclophosphamide (4-HO-CP) and aldophosphamide (AP) in U937 human hi stiocytic (CP-sensitive) and K562 human erythroleukemia (CP-resistant) cells. Similar experiments were carried out using the ifosfamide (IF, CAS 3778-73-2) metabolites 4-hydroxyifosfamide (4-HO-IF) and aldoifos famide (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 elim ination/addition reaction involving water and 4-thiocyclophosphamide a nalogs 4-(2-hydroxyethyl)thiocyclophosphamide (4-ESCP) or mafosfamide. Cell death resulting from 4-HO-CP/AP perfusions was mimicked by perfu sion with acrolein or an acrolein producing but non-alkylating, dechlo ro-CP analog. Acrolein toxicity was minimized by the presence of 2-mer captoethanol 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). Th e intracellular half-life for phosphoramide mustard (PM) at an intrace llular pH value of 7.1 +/- 0.1 and an ambient probe temperature of 23 +/- 1 degrees C in U937 cells was 2.1 h [k = (5.4 +/- 0.3) x 10(-3) mi n(-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 isopho sphoramide mustard (IPM). Fractional dose perfusion of U937 or K562 ce lls 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 K5 62 cells. Similar results were obtained using 4-ESCP and mafosfamide a s sources of 4-HO-CP/AP. Identification of CBP was based on chemical s hift, chemical stability: and membrane permeability studies of synthet ic CBP. Concentrations of carboxyifosfamide (CBIF) formed in K562 cell s were also greater than that in U937 cells.