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Isolation and Identification of a Metabolic Intermediate in the Selective Dechloroethylation of One of the Four Stereoisomers of 4-Methylcyclophosphamide1

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Isolation and Identification of a Metabolic Intermediate in the Selective Dechloroethylation of One of the Four Stereoisomers of 4-Methylcyclophosphamide1 [CANCER RESEARCH 38, 2592-2599, August 1978] 0008-5472/78/0038-0000$02.00 Isolation and Identification of a Metabolic Intermediate in the Selective Dechloroethylation of One of the Four Stereoisomers of 4-Methylcyclophosphamide1 George Abel, Peter J. Cox,2 Peter B. Farmer, Neville J. Haskins, Michael Jarman, Kanti Merai, and Wojciech J. Stec Chester Beatty Research Institute, Institute of Cancer Research: Royal Cancer Hospital, Fulham Road, London SW3 6JB England [G A P J C P B F M. J., K. M.¡:G. D. Searle & Co. Ltd., Lane End Road, High Wycombe, Buckinghamshire, HP12 4HL, England ¡N.J. H.]; and Centre 'tor Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-362 Lodz, Boczna 5, Poland [W. J. S.J ABSTRACT phenprocoumon is more effective than is the fî(+)-isomer (22). (+)-Methadone is inactive in the maintenance of opi The in vitro cytotoxicity in bioassay or cell culture ate-dependent patients (see Ref. 36). The analgesic activity assays of (-)-c/s-4-methylcyclophosphamide when me of propoxyphene resides largely in the (+)-isomer (see Ref tabolized by microsomes from the livers of male Wistar 5). rats was significantly less than that of metabolized (+)- c/s-, (+)-frans-, and (-)-rrans-4-methylcyclophosphamide. The metabolism of enantiomers can also differ substan tially. The influence of stereochemistry on drug metabolism However, metabolism of the individual Stereoisomers by has been reviewed by Jenner and Testa (23). Of the 4 liver microsomes from female BALB/c mice yielded stereoisomeric ephedrines (10), the isomers of absolute metabolite mixtures of approximately equal cytotc '¡city. configuration 2S-[(-)-ephedrine and (+)-pseudoephedrine] The extent of metabolism by rat liver microsomes f the were A/-demethylated more rapidly by rabbit liver micro four Stereoisomers was similar when assessed by sub somes than were the isomers of 2R geometry. The hydrox- strate disappearance, but the yield of alkylating metabo ylation and excretion of the antiinflammatory agent Ciclo- lites (expressed as phosphoramide mustard equivalents) profen in the rat proceeds more rapidly with the (+)-enan- was comparable in the case of three of the isomers but substantially less for the (-)-c/s-isomer. tiomer (11). In some cases enantiomeric interactions occur. For example, S(+)-amphetamine inhibits the metabolism of A new metabolite was isolated following the incubation fl(-) in the racemate (17, 39); the S-isomer of a psychoto- of (-)-cis-4-methylcyclophosphamide with rat liver micro mimetic amine, 1-(2,5-dimethoxy-4-methylphenyl)-2-amino- somes. It was identified by its breakdown products and its propane, inhibits the metabolism of the fl-isomer (26); and electron impact and chemical ionization mass spectra, the inactive (-)-propoxyphene increases plasma levels and especially by the presence in the latter of a pseudomo- enhances activity of the (+ )-antipode (28). lecular ion. Its structure was 2-[(2-chloroethyl)(2-chloro-1- hydroxyethyl)amino]tetrahydro-4-methyl-2H-1,3,2-oxaza- Species differences have also been recorded: (+ )- and (-)-hexobarbital are metabolized at different rates, and the phosphorine 2-oxide, an intermediate in dechloroethyla- ratio between the 2 varies between species, although sepa tion. rate investigations have revealed opposing results (12, 16, Species variation (mouse, rat, and rabbit) in the forma tion of this metabolite of low cytotoxicity (-1% of that of 4- 29). Striking species differences exist in the microsomal metabolism of the enantiomers of CP3-4which is chiral by hydroxy-4-methylcyclophosphamide) was noted and ac virtue of asymmetry at phosphorus. corded well with the observed species differences in In our studies of CP analogs, we found that the primary cytotoxicity assays. hydroxylation step in the metabolism by rat liver micro somes of 4-MeCP, as measured by substrate disappear INTRODUCTION ance, was only marginally affected by the configuration of the substrate, which can exist in 4 forms as a result of the The presence of 1 or 2 chiral centers in molecules of introduction of a second chiral center in the ring, and that pharmacological importance is frequently associated with the biological activities against the murine ADJ/PC6A tumor profound differences in the biological activities of the 2 or in vivo of the 4 compounds fell within a 2-fold range (15). 4 Stereoisomers. In the field of antiacetylcholine drugs, the However, an interesting observation, reported here, of (-)-enantiomers of those containing a single asymmetric the differences among the in vitro cytotoxicities of the 4 benzylic carbon atom show greater potency than do the different Stereoisomers led us to investigate further the (+)-enantiomers (21). The S(-)-form of the anticoagulant initial metabolic process. 1This investigation was supported by grants to the Chester Beatty 3The abbreviations used are: CP, cyclophosphamide; 4-MeCP, 4-methyl- Research Institute, Institute of Cancer Research:Royal Cancer Hospital, from cyclophosphamide (2-[bis(2-chloroethyl)amino]-tetrahydro-4-methyl-2H-1,3,2- the Medical Research Council (Grant G973/786-7K) and to the Polish Acad oxazaphosphorine 2-oxide); 4-OH-4-MeCP, 4-hydroxy-4-methylcyclophos- emy of Sciences, Centre for Molecular and Macromolecular Studies, from phamide; TLC, thin-layer chromatography; El, electron impact; CI, chemical the National Cancer Programme. ionization; NBP, 4-(p-nitrobenzyl)pyridine; ID5()medianinhibitory dose; AMU, 2To whom requests for reprints should be addressed. atomic mass unit. Received January 3, 1978; accepted April 25, 1978. 4 P. J. Cox, P. B. Farmer, and M. Jarman, manuscript in preparation. 2592 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. Isolation of an Intermediate in Dechloroethylation MATERIALS AND METHODS Cell Culture Assay. Walker ascites tumor cell lines were established and maintained as previously described (31). Metabolism. Washed liver microsomes were prepared (3) Cells from log-phase cultures were resuspended in fresh medium (Dulbecco's modified Eagle's medium plus 10% from male Wistar rats, female BALB/c mice, and female New Zealand White rabbits. Both rats and mice were pre- fetal calf serum containing penicillin and streptomycin) at a treated with sodium phénobarbital(1 g/liter drinking water) density of 2.78 x 104 (or 2.78 x 10s) cells/ml. The suspen for 10 days. The microsomal pellets were used immediately sion was dispensed in 4.5-ml (or 450-/U.I) portions into or stored at -30°until required. centrifuge tubes, and 0.5 ml (or 50 /¿I)oftest solution was The 4-MeCP stereoisomers (25, 35) were incubated (3) added. After 1 hr at 37°,the cells were harvested by with these microsomes; appropriate details are given with centrifugation (800 x g, 3 min) and resuspended in 5 ml of the relevant series of experiments. Substrate disappearance fresh medium. Portions (2 ml, 5 x 104 cells) of each cell was measured by stable isotope dilution (15). 4-OH-4-MeCP suspension were dispensed into 2 dishes of a Linbro multi- dish tray, which was then incubated at 37°under an atmos was prepared by KMn04 oxidation from the racemates and pure stereoisomers (6). phere of 10% CO2in air. Cell counts were made after 72 and Isolation and Identification of Metabolites. Incubations 96 hr from 1 dish of each duplicate each time. Comparison were terminated and worked up by the methods described of the growth of treated cells with that of appropriate previously (3). TLC was carried out on glass plates (20 x 20 controls was used to assess the percentage of inhibition of or 5 x 20 cm) coated with silicic acid (Merck Kieselgel G) cell growth. developed with CHCI:t:C2H5OH (9:1) unless otherwise Cells of the ascitic form of the ADJ/PC6A murine tumor stated. Separated components were located on the chro- were harvested freshly for each experiment from the peri matograms by exposure to iodine vapor or by a spraying toneal cavity of mice used for the routine maintenance of with acidic 2,4-dinitrophenylhydrazine (3). After removal of the tumor line in vivo. The cells and ascitic fluid were immediately diluted with Dulbecco's modified Eagle's me the major portion of each band, alkylating species were located with Epstein reagent (35). Materials were eluted dium containing 20% inactivated horse serum; glutamine (2 from the silicic acid with ethanol. Eluates were subjected to mM); alanine, asparagine, aspartate, glutamate, glycine, mass spectrometry or were reacted with acidic 2,4-dini proline, and serine (all 0.4 mM); and streptomycin and trophenylhydrazine by the published method (3, 6). penicillin. After being counted the cells were diluted to a Mass Spectrometry. The direct insertion technique was density of 1.11 x 106cells/ml. Portions (450 ¿¿I)weretreated used for all spectra. El mass spectra were determined with with test solution (50 /¿I)asdescribed above; the cells were an AEI-MS12 spectrometer with an ionizing voltage of 70 resuspended in 5 ml of medium and dispensed in 2-ml eV, a trap current of 100 ¿¿a,andan ¡onsource temperature portions (2 x 105cells). Cell counts were made after 72 hr. of -100°.Quantitation of 4-MeCP in samples containing the 4-MeCP isomers were activated either at a range of d.,-standard was achieved using the peak height ratio of the substrate concentrations (17.5 /J.Mto 10.7 mM) 30 times ions atm/e 225 and 229 ([M-CH2CI]+ for d„andd4 forms, those finally used to treat the cells (0.58 to 356 /^M) and respectively) (15). exactly as previously described (9) or in other experiments CI mass spectra were determined with a Finnigan 3200 at a single substrate concentration (0.57 mM) 10 times the spectrometer. Ammonia was introduced as the reactant gas maximum concentration (57 /J.M)used to treat the cells.
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