phosphorus-radioisotopes and phosphoramide-mustard

Development of resistance of cancer cells against cyclophosphamide (CP) is probably associated with an increased conjugation with glutathione. 31P NMR spectroscopy was used to monitor the time courses for the chemical conjugation with glutathione of the CP metabolites 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PM) at 24 degrees C. PM incubated with a 10-fold molar excess of glutathione showed a disappearance of the PM signal (t1/2 = 112 min), accompanied by an increase of two signals, attributed to the intermediate PM monoglutathione conjugate and the PM diglutathione conjugate. After 680 min, only a signal assigned to the PM diglutathione conjugate was found. This conjugate was relatively stable. The formation of the PM diglutathione conjugate was confirmed with fast atom bombardment mass spectrometry (FAB-MS). The rate constant for the disappearance of the PM signal in incubations with glutathione was 6.2 x 10(-3) min-1, and was 5.4 x 10(-3) min-1 in incubations without glutathione, indicating that the rate-limiting step in both reactions in the formation of aziridinium ions. When 4-OHCP was incubated with a 10-fold molar excess of glutathione, six signals was found which were not present in spectra of incubations without glutathione. In addition to the signals assigned to the mono- and diglutathionyl conjugates of PM, four signals were found of which the pattern of formation in time was identical. These four signals correspond to the four stereoisomers of 4-glutathionylcyclophosphamide (4-GSCP). The formation of 4-GSCP was confirmed with FAB-MS. Within 120 min after the start of the reaction no free 4-OHCP or aldophosphamide signals were found in the spectra. Free PM was detected in all spectra indicating that degradation of 4-GSCP gives rise to PM, the ultimate cytotoxic metabolite of CP, 4-GSCP therefore appears an important pool of phosphoramide mustard, which in turn can be deactivated by glutathione.

Topics: Cyclophosphamide; Glutathione; Magnetic Resonance Spectroscopy; Phosphoramide Mustards; Phosphorus Radioisotopes; Spectrometry, Mass, Fast Atom Bombardment

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.

Topics: Alkylation; Animals; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; DNA; Female; Magnetic Resonance Spectroscopy; Mice; Mice, Inbred C57BL; Phosphoramide Mustards; Phosphorus Radioisotopes; Spectrophotometry, Ultraviolet

31P nuclear magnetic resonance spectroscopy was used to measure the pKa (4.28 +/- 0.2) of isophosphoramide mustard (IPM) at 20 degrees C and to study the kinetics and products of the decomposition of IPM at a solution pH value of ca. 7.4 and at temperatures between 20 and 47 degrees C in the presence of nucleophilic trapping agents. At 37 degrees C, the half-life for the first alkylation was ca. 77 min and ca. 171 min for the second alkylation; these data may be compared with those for phosphoramide mustard (Engle, T.W.; Zon, G.; Egan, W.J. Med. Chem. 1982, 25, 1347), wherein the half-lives for the first and second alkylations are approximately the same (18 min). The rate of fragmentation of aldoifosfamide to IPM and acrolein was also studied by NMR spectroscopy (pH 7.0; 37 degrees C; 0.07 M phosphate); under the noted conditions, the half-life of aldoifosfamide was found to be ca. 60 min.

Topics: Alkylating Agents; Antineoplastic Agents; Chemical Phenomena; Chemistry; Half-Life; Ifosfamide; Kinetics; Magnetic Resonance Spectroscopy; Phosphoramide Mustards; Phosphorus Radioisotopes