[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.
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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. through the Finnigan manifold with the use of the calibra Quantification of Alkylating Activity. Alkylating activity tion gas inlet. The gas was obtained from a lecture bottle was measured by the reaction with Epstein reagent (NBP). (Cambrian Chemicals Ltd., Croydon, Surrey, England). Tri- Incubations were stopped by plunging the flasks into ice. deuteroammonia was introduced with the same inlet, also The microsomal protein was removed by centrifugation at from a lecture bottle (Merck, Sharpe and Dohme, Isotopes 65,000 x gav for 20 min at 2°.Portions (0.375 ml) of the Division, Hoddesdon, Hertfordshire, England). An ionizing supernatant were transferred to 10-ml conical glass centri voltage of 100 eV was used at a source temperature of 140°. fuge tubes; 0.2 ml of 0.5 M sodium acetate buffer, pH 4, and The indicated pressure of gas in the source was 200 to 230 0.2 ml of 2% NBP in ethylene glycol were added and mixed. /urn. Data obtained from the mass spectrometer were ana After the tubes were covered, they were incubated at 100° lyzed with an on-line Finningan 6000 data system. for 20 min and then cooled in ice. Color was developed by Bioassay. The procedure was that previously described the addition of 0.8 ml of triethylamine:acetone (1:1). After (9) except that substrate was metabolized in the presence being mixed the solution was centrifuged at 1500 x g for 2 of the cells. Thus to 8.9 ml Walker ascites cell suspension to 3 min, and the absorbance of the clear supernatant was (1.12 x 106 cells/ml) was added 0.1 ml substrate solution measured at 565 nm exactly 10 min after the addition of and 1.0 ml of a suspension containing microsomes equiva base. Phosphoramide mustard (obtained as its cyclohexyl- lent to 250 mg liver, 0.35 /¿molNADP+,6.9 /¿molglucose-6- ammonium salt from Dr. H. B. Wood, National Cancer phosphate, 6.25 jumol MgCI2-6H2O, and 0.875 unitglucose- Institute, Bethesda, Md.) was used as a standard. The 6-phosphate dehydrogenase, buffered at pH 7.4 with 100 absorbance due to the reaction of the remaining substrate jumol potassium phosphate or Tris-HCI. After incubation of with NBP was allowed for by using control incubations the cell suspension for 2 hr at 37°in 25-ml conical flasks lacking the cofactors required for the microsomal metabo under an Cvenriched atmosphere, female Wistar rats (in lism. Thus, when x is the concentration (/¿M)of total groups of 5) were given injections of 106 cells, and the alkylating metabolites formed (expressed as phosphoram- survival times of the animals were recorded. ide mustard equivalents), y is the substrate (initial concen-
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tration, ¿¿M),fis the substrate concentration :substrate con levels of growth inhibition by all 4 stereoisomers, as did the centration in phosphoramide mustard equivalents, and z is products of chemical (KMnO4) oxidation. the concentration (¿IM)of total phosphoramide mustard The extent of metabolism by rat and mouse liver micro equivalents measured, then somes as measured by substrate disappearance was com (y- x) pared to the extent of metabolism assessed by alkylating z =x +-1——¿ •¿ product appearance measured as phosphoramide mustard equivalents. The results are shown in Chart 1 (stored microsomes) and Table 3 (fresh microsomes). Additionally, RESULTS
Initial experiments were carried out with the bioassay 100-,^ system. When tested as described above with rat micro- somes suspended in phosphate buffer and with substrate 75-Ö —¿ .1. —¿ i"VN concentrations of 12 and 24 /¿g/ml,the (+ )-c/s- and (+ )- and (-)-frans-isomers were totally cytotoxic, whereas the 350-U (-)-c/s-enantiomer had little effect on increase in survival VNII time. Further experiments with rat microsomes that had been stored for a shorter period at -30° (2 to 3 weeks compared to 9 weeks) and in which Tris-HCI buffer was n—A—1iS. S^ used demonstrated the pure (-)-c/s-enantiomer to be some RAT MOUSE 6-fold less toxic than were the other forms (Table 1). 0.5-, These results were confirmed by the cell culture assay. i——+ Individual enantiomers were metabolized at a range of o substrate concentrations, and the inhibition of growth of oc Walker ascites cells by these metabolized samples was determined (Table 2). The (-)-c/s form metabolized by rat 5i—l~—+i - + * - -cis± - 4 ± cis-. * trans—— trans microsomes again showed toxicity 1 order of magnitude Chart 1. A. percentage of metabolism of individual 4-MeCP stereoi less than that of the other 3 forms. Metabolism with mouse somers and racemates in 45 min by microsomes (stored at -30° prior to liver microsomes, however, caused approximately equal use) from rat and mouse, expressed in terms of substrate disappearance (open bars) and alkylating product (phosphoramide mustard equivalents) appearance (hatched bars). B, ratio of percentage of metabolism by product Table 1 appearance to percentage of metabolism by substrate disappearance (from A). , 1 S.D. from either side of the mean of all 12 values. Only 2 Toxicity of activated 4-MeCP stereoisomers measured by bioassay values (') fall outside this range; that for the (-)-cis-stereoisomer differed The drug concentration necessary to cause 50% cell kill in the significantly from the mean (p < 0.05). The values shown are for a single presence of active rat liver microsomes in Tris-HCI was determined experiment. In this experiment the relative amounts of alkylating metabolites by bioassay (see "Materials and Methods"). Fifty % cell kill is formed from the 4 stereoisomers by rat liver microsomes were identical equivalent to 21% increase in survival time, taking the generation with the ratios found in 7 other experiments, irrespective of the length of time in vivo as 36 hr (9). Each value is derived from the survival storage of the microsomes, although absolute amounts varied between experiments. When the percentage of metabolism of (+)-c/s-4-MeCP judged times of 5 animals at each of 3 drug concentrations. All the values by alkylating metabolites was equated to 1, the relative values were 0.46 were obtained in parallel within 1 experiment. ±0.02 (S.E.) for (-(-efe-, 1.13 ±0.01 Jor (+)-trans-, and 1.09 ±0.01 for (¿tM)c/s-Configuration(+)Drug concentration (-)-trans-. Table 3 Percentage of metabolism of individual 4-MeCP stereoisomers 7.3 incubated with freshly prepared microsomes and an NADPH- (±) 12.4 6.6 generating system for 10 and 45 min (-) 43.8frans-Configuration5.5 5.1 The results in Columns A were calculated from substrate disap pearance. Columns B show the ratio of percentage of metabolism by product appearance:substrate disappearance. Table 2 Concentrations of 4-MeCP stereoisomers, activated by liver % of metabolism microsomes or by KMnOtoxidation, required to inhibit the growth Substrate 10-min incubation 45-min incubation of Walker ascites tumor cells in suspension culture by 50% Each value was obtained from a dose-response curve derived cition(+)-c/s-(-)-c;'s-(+)-trans-(-)-trans-(+)-cis-(-)-c/s-(+)-trans-(-)-frans-A27.525.547.532.574.072.088.093.0B0.650.18"0.700.780.800.620.910.810.6811ly u i from 5 to 8 substrate concentrations (individually metabolized). RatMouseMean (MM)Species Concentration (+)-c/s-Rat (+)-frans-50.7±6.26(2) 4.9±0.3a>6(2)c' Mouse 5.9 6.5 3.7 5.6 Chemical 3.4 2.73.6 1.7(-)-frans-4.33.4 oxidation(-)-c/s- " Mean ±S.E. 6 These values differ significantly (p < 0.01). 0 Numbers in parentheses, number of dose-response curves ±S.D.wi ±0.22A44.550.559.046.096.083.095.096.0B0.620.28"0.630.740.580.590.810.720.62±0.16 when more than 1. " These values differ significantly from the means.
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extracts of the microsomal metabolism mixtures were sub NH:tand ND3as the reactant gas are shown in Chart 3. jected to TLC, the results of which are shown diagrammati- The cytotoxicity of this new metabolite was estimated by cally in Chart 2, together with those for rabbit microsomal the Walker cell culture assay, quantification being made by the Epstein reaction. Four mg of (±)-c/'s-4-MeCP were metabolism. Also shown is the result of TLC of the KMnC^ oxidation products, which were identical for all 4 enantio- metabolized with rat liver microsomes, and the products mers. were extracted and subjected to TLC. Residual 4-MeCP, the The identity of the metabolite of RK0.44 (CHCI,:C,H5OH, new metabolite (RF0.44), 4-OH-4-MeCP, and a control band 9:1) was sought by El mass spectrometry. Four mg of (-)- of silicic acid were eluted and quantitated; racemic 4-MeCP c/s-4-MeCP were metabolized (at a concentration of 200 and phosphoramide mustard were used as standards. The ¿tg/ml)by rat liver microsomes, and the metabolites were amount of the new metabolite was approximated, as there extracted and chromatographed. The product at the appro was no synthetic material available, by reference to a priate RK(0.44) was eluted, the solution was concentrated standard curve one-half the slope of that for 4-MeCP (see to dryness, and the residue was dissolved in ethanol and "Discussion"). Recoveries and approximate IDr,„'s(1-hr subjected to rechromatography in the same solvent system. treatment) were: 4-MeCP (2.003 mg), ID5„,-2500 ¿¿g/ml; Two bands (RK0.44 and 0.34, staining equally in U vapor) new metabolite (-0.660 mg), ID.,,,, -550 /xg/ml; 4-OH-4- resulted; only that of the higher RKreacted positively, but MeCP (0.424 mg), ID.,,,,6 /ng/ml (expressed as phosphor- slowly, with acidic 2,4-dinitrophenylhydrazine. Both bands, amide mustard equivalents). when eluted and concentrated, gave signals appropriate for For determination of the relationship between metabo the monochloroethyl analog of 4-MeCP (6). In addition, the lism to alkylating products and cell growth inhibitory poten spectrum of the more mobile component contained large tial, further cell culture assays were conducted with serial peaks at mie 78 and 80, with appropriate intensities (3:1) dilutions of the drug metabolized at a single concentration. for an ion containing 1 chlorine atom. Table 4 shows the ID50'sagainst PC6 ascites cells, based on Reaction of the component of RF 0.44 to form a 2,4- substrate concentration and on alkylating activity of the dinitrophenylhydrazone yielded a product that was chro- metabolized stereoisomers. matographically identical (TLC in benzene, RK 0.36; cf., methylvinylketone 2,4-dinitrophenylhydrazone, RF 0.43) with synthetic 2-chloroacetaldehyde 2,4-dinitrophenylhy DISCUSSION drazone (2). The initial observation that, of the 4 stereoisomers of 4- The CI mass spectra of underivatized metabolite with both MeCP, the (-)-c/s form was almost devoid of toxicity in the rat microsomal metabolism bioassay was unexpected. Sub sequent experiments in which microsomes stored for a •¿ SOLVENTFRONT- shorter period were used and in which phosphate buffer was replaced by Tris-HCI [which may extend the half-life of the hydroxylated metabolites (9)] showed the (-)-c/s-isomer to have some cytotoxicity on metabolism, but it was still © <©> © less toxic than were its 3 stereoisomers. In contrast, the in vivo biological activity of this enantiomer was comparable with those of the other 3 isomers (15), and all 4 forms were metabolized in vitro at comparable rates. Moreover, metab olism of the parent compound, CP, by washed liver micro ABCDEABC (-f)-cis <-)-cis somes yielded predominantly 4-hydroxycyclophosphamide (3), and the toxicities of the individually metabolized enan- •¿ SOLVENTFRONT- tiomers of CP were not different despite their different activities in vivo (8). Metabolism of racemic 4-MeCP and 5,5-dimethylcyclophosphamide (6, 7) also yielded primarily
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70 100 150 200 250 300
'• 8 DEUTERORMMONIflCI RMP.:00004072 00BRMPIEi.
.l.i70 . .,i....ili...¡Ili..ii..,iii... 100 150 200 250 300 Chart 3. CI mass spectra of the new metabolite [R, 0.44, CHCIj^HjOH (9:1)] with the use of ammonia (A) and deuteroammonia (B) as reactant gas. The abscissas are graduated in units of mie, the left ordinales are graduated in percentage of relative intensity, and the right ordinates are graduated in percentage of total ion current.
Table 4 duced from the (—¿ )-c/s-enantiomer,measuredas phosphor- Concentrations of 4-MeCP stereo iso m er metabolite mixtures, amide mustard equivalents (Chart 1), was about one-half prepared with stored rat liver microsomes, required to inhibit the that from its 3 stereoisomers.The production of alkylating growth of ADJ/PC6 cells in suspension culture by 50% metabolites from the racemic (±)-c/s-isomer fell halfway The concentrations are based on initial substrate concentration between that measured for the (+)- and the (-)-c/s-isomers. (A) and alkylating products formed (phosphoramide mustard equiv TLC of extracts following incubation of the pure enantio- alents) (B). The concentrations were derived from a number of dose-response curves. mers revealed the presence of a new metabolite formed by rat but not mouse liver microsomes, which was clearly quan of (/¿M)A9.4 titatively important in the metabolism of the (-)-c/s-isomer dose-re sponse and which was also detectable in the extract derived from Configuration(+K/S- curves2 the metabolism of the (+)-c;s-isomer. 4-OH-4-MeCP was ±0.6a ±0.4 formed from each of the enantiomers by hepatic micro (->cis- 3 17.6 ±2.9* 4.2 ±0.6 0.24 somes of all species tested, but judged from the staining of (+)-trans- 3 10.1 ±1.8 5.8 ±0.9 0.57 the thin-layer chromatograms its formation was dramati (-)-trans-No. 2Concentration9. 5 ±2.9B4.9 5.2 ±1.4(B:A)0.520.55 cally reduced in the metabolism of (-)-c/s-4-MeCP by the a Mean ±S.E. rat. While stored microsomes were used for most of the 6 This value differs significantly (0.01 < p < 0.05) from those of work reported together with incubation times of up to 45 the other 3 stereoisomers. min, the pattern of metabolism was essentially the same when fresh microsomal preparations and 10- or 45-min reported similarity between the 4 stereoisomers (15). In the incubation times were used (see Charts 1 and 2 and Table experiments reported here, the frans compounds were 3). Two principal differences were observed. Firstly, the metabolized marginally more than were the cis forms, this levels of alkylating activity measured were higher; hence difference being more apparent with mouse than with rat the product appearance:substrate disappearance ratio is microsomes. However, the total alkylating metabolites pro larger for incubations with fresh microsomes than for those
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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. Isolation of an Intermediate in Dechloroethylation with stored preparations. Secondly, the more rapid metab amides (14) and since the additional use of trideuteroam- olism of 4-MeCP by female mouse microsomes compared monia gives complementary structural information (20). to male rat microsomes was more apparent with the shorter Thus ions formed by proton transfer ([M + H]+) occur at 1 incubation time. This species difference in metabolic rate AMU higher when ND;, is used. [M + NHj* ions are raised by 4 AMU's. In addition, labile protons (OH, NH) undergo has been noted for CP by Sladek (34). The new metabolite was reactive to 2,4-dinitrophenylhy- H—Dexchange in the presence 9f ND.,. In the ammonia CI drazine in acidic conditions, suggesting that it was or that mass spectrum of the new metabolite (Chart 3A), the most it released an aldehyde. Evidence from El mass spectrome- abundant ions were those corresponding to the monochlo- try showed the new metabolite to be closely related in roethyl derivative ([M + H]* atm/e 213; [M + NH.,]* atm/e structure to the product [2-(2-chloroethylamino)tetrahydro- 230); corresponding ions in the presence of ND.t(Chart 3B) 4-methyl-2H-1,3,2-oxazaphosphorine 2-oxide] of dechlo- were atm/e 216 and m/e 236, i.e., appropriately raised by 1 roethylation that had previously been identified (6) in ad and 4 AMU's, together with exchange of 2 N—Hprotons. mixture with 4-OH-4-MeCP as a microsomal metabolite of However, ions of higher m/e value were also abundant and racemic (c/s,frans)-4-MeCP. Thus the El mass spectrum of included m/e 308, attributable to [M + NH.,]* corresponding the new product contained all of the major signals attrib to the postulated structure and appropriately changed to uted to the N-monochloroethyl derivative, in particular mie m/e 314 in the presence of ND.,, and also m/e 290 and m/e 212 (M+) and m/e 163 ([M - Ch^CI]*). Also, after storage for 273, ascribed to losses of H2O from [M + NH.,]' and [M + several days, even at -30°,samples of the new metabolite H]+, respectively. The m/e's of 294 and 274 for correspond that had been eluted from thin-layer chromatograms ing ions in the NDa spectrum indicate the absence of the showed partial decomposition into the A/-monochloroethyl exchangeable protons of the NH and OH groups. Thus derivative. Thus further TLC revealed, in addition to the these ions may be ascribed to loss of D.f) from the corre unchanged metabolite, a component (detected by I2stain sponding ions at m/e 314 and 294. The pseudomolecular ing) that did not react with 2,4-dinitrophenylhydrazine and ion [M + H]+ (m/e 291) would be coincident with the that was identical in RK value and mass spectrometric isotopie (13C-containing) ion [M + NH, - H.,0]* and so was characteristics with the monodechloroethyl derivative (6). not independently discerned. The additional ion atm/e 298 The evidence thus far suggests that the new metabolite is a (->m/e 303 in the ND;ispectrum) was unassigned; it may be labile precursor thereof, and a probable candidate structure attributable to a decomposition product of the new metab is 2-[(2-chloroethyl)(2-chloro-1-hydroxyethyl)amino]tetra- olite since it was more abundant in the mass spectrum of a hydro-4-methyl-2H-1,3,2-oxazaphosphorine 2-oxide (Struc sample that had been stored for 1 week at -30°. In sum ture 1). This could yield the N-monochloroethyl derivative mary, the mass spectral evidence provides compelling sup by elimination of chloroacetaldehyde. port for the structure (Structure 1) proposed for the new metabolite. OH The approximate ID50value obtained for the new metabo I CH3 CHCH0CI lite indicated a cytotoxicity of about 1% of that of 4-OH-4- H CH- -N N';:",, MeCP. The compound was produced metabolically because NCH2CH2CI of its chemical inaccessibility and quantitated by the Ep stein reaction. The standard curve used was constructed on CH2—O "O the assumption that the new metabolite (Structure 1) af fords a color with one-half the absorbance of an equimolar
Structure 1. amount of 4-MeCP and was chosen by analogy to mono- dechloroethylcyclophosphamide, which has approximately For testing of this hypothesis, the 2 dinitrophenylhydra- half the alkylating potential of CP. Therefore the ID.â„¢forthe zine-reactive metabolites obtained after metabolism of (-)- new metabolite can only be regarded as an estimate. c/s-4-MeCP by rat liver microsomes were treated with acidic The (-)-c/'s-isomer yielded about 50% of the alkylating 2,4-dinitrophenylhydrazine, and the products were com metabolites formed by the other 3 isomers, but the apparent pared with the authentic 2,4-dinitrophenylhydrazones of reduction in toxicity was 6- to 10-fold. This discrepancy was methylvinylketone and chloroacetaldehyde. The product resolved by a final series of experiments with serial dilutions from the metabolite with the RF value of 4-OH-4-MeCP of a single substrate concentration (0.57 mM) rather than a corresponded with the former derivative, and the product range of higher concentrations of substrate separately from the new metabolite corresponded with the latter deriv metabolized (10.7 mw and downwards) (see "Materials and ative. Methods"). Under the latter conditions saturation of the The only direct evidence from its El mass spectrum for microsomal metabolic capacity and even substrate inhibi the proposed structure for the new metabolite was afforded tion of metabolism were observed. Thus several of the top by prominent signals atm/e 78/80 (1 chlorine), which could dose levels gave similar levels of cytotoxic products. Refer be ascribed to chloroacetaldehyde (M.W., 78/80) formed ence to Table 3 will show that, under the better metaboliz therefrom by thermal or El-induced elimination. There were ing conditions, the cytotoxicity of (-)-c/s-4-MeCP was only no signals of m le values higher than 212/214. However, the 2-fold less than that of the other isomers and that, when CI mass spectrum afforded additional evidence of structure compared on the basis of alkylating metabolites, this differ for the new metabolite. Ammonia was selected as the ence was abolished. reactant gas since it gives good pseudomolecular ions for Thus we have demonstrated the stereoselective dechlo-
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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. G. Abel et al. roethylation of 1 of the 4 enantiomers of 4-MeCP. The dimethylcyclophosphamide has also been isolated and influence of stereochemical factors on metabolism has characterized (7). These ring-hydroxylated products of the largely been studied with pairs of optical isomers and their cyclophosphamide series may be regarded as intermediates racemates, and this area has been comprehensively re in dealkylation. although ring opening does not result viewed by Jenner and Testa (23). The metabolic transfor directly in the loss of the alkyl group. Many examples exist mations of 4-MeCP described above do not fall under the in which the participation of carbinolamine intermediates usual definitions of substrate-product stereoselectivity as may be implicated (see Ref. 19). given by these authors; the substrates are oxidized at Dechloroethylation by microsomal enzymes has been similar rates, and the products are either unaltered in demonstrated as a metabolic pathway of varying impor configuration (4-hydroxylation) or have not been examined tance in the biotransformation of CP and isophosphamide for diastereoisomeric composition (1'-hydroxylation). In (3, 30), other CP analogs (6, 7), aniline mustard (4), and stead the stereochemical influence of the substrate is on chlorambucil (27). No intermediates were isolated. We the route of metabolism, examples of which are found in therefore believe that Structure 1 represents the first re the metabolism of warfarin (32) and norgestrel (33). The ported isolation and identification of an intermediate in case that we report is additionally unusual in that only 1 of dechloroethylation. Indeed, to our knowledge it is the first 4 stereoisomers is metabolized significantly by C-1'-oxida example of the mass spectral characterization of an unde tion. From consideration of molecular models of the iso rivatized carbinolamine intermediate in the mammalian N- mers (25), the following explanation may be suggested. The dealkylation of simple (i.e., not ring-closed) alkylamines. C—H bonds at C-4 and C-1', either of which may be oxidized to C—OH,are relatively closer together in the 2 c/s-isomers (2S.4S and 2R.4R) than they are in the frans ACKNOWLEDGMENTS compounds (2R.4S and 2S.4R). In the frans-configuration The authors wish to thank Professor AB. Foster for his interest and the oxazaphosphorine ring and the bis(2-chloroethyl)amino encouragement and M. Baker for determining the El mass spectra. substituent are nearly coplanar, whereas in the cis form the plane of the bis(2-chloroethyl)amino group is nearly at right REFERENCES angles to the N-3—C-4plane of the oxazaphosphorine ring. 1. Allen, J. G Blackburn, M. J., and Caldwell, S. M. The Metabolism by It is conceivable that the juxtaposition of the 2 brings the C- Man of an Anticonvulsant Oxadiazole. An Unusual Metabolic Route for 1'—Hbond close to the activated oxygen species when the Tertiary Amides. Xenobiotica, 1: 3-12, 1971. substrate is bound to cytochrome P-450 and hence makes 2. Böse,J. L., Foster, A. B.. and Stephens. R. W. Reaction of Periodate with Compounds Containing Active MéthylèneGroups.J. Chem. Soc , it more susceptible to oxidation. Quantitative differences in 3314-3321,1959. the side-chain oxidation of (+)- and (-)-c/s-enantiomers 3. Connors, T. A., Cox, P. J., Farmer. P. B., Foster, A. 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and Ãrans-4-Methylcyclophosphamide. Chem.-Biol. Interactions. 18: 47- Disposition of Chlorambucil in Rats. Xenobiotica, 7: 205-220. 1977. 57,1977. 28 Murphy, P. J., Nickander, R. C., Bellamy, G. M., and Kurtz, W. L. Effect 16. Furner, R. L., McCarthy, J. S., Stitzel. R. E., and Anders, M. W. of 7-Propoxyphene on Plasma Levels and Analgesic Activity of d-Propox- Stereoselective Metabolism of the Enantiomers of Hexobarbital. J. yphene in the Rat. J. Pharmacol. Exptl. Therap., J99: 415-422, 1976. Pharmacol. Exptl. Therap., 769: 153-158, 1969. 29 Noordhoek, J.. Van den Berg, A. P., Savenije-Chapel, E. M.. and 17. Gal, J., Wright, J., and Cho, A. K. In Vitro Metabolism of Amphetamine: Koopman-Kool. E. Metabolism of Hexobarbital Enantiomers and Inter An Apparent Enantiomeric Interaction. Res. Commun. Chem. Pathol. action with Cytochrome P-450 in Male and Female Mice and Rats. Z. Pharmacol.. 15: 525-540. 1976. Physiol. Chem.,357. 1045-1046, 1976. 18. Huang, M-T., West, S. B., and Lu. A. Y. H. Separation, Purification, and 30 Norpoth, K.. Addicks, H. W., Witting, U., Müller,G., and Raidt, H. Properties of Multiple Forms of Cytochrome P-450 from the Liver Quantitative Bestimmung von Cyclophosphamid, Ifosfamid und Trofos- Microsomes of Phenobarbital-Treated Mice. J. Biol. Chem., 251: 4659- famid sowie ihrer stabilen Metabolite auf der DC-Platte mit 4-Pyridinal- 4665,1976. dehyd-2-benzothiazolylhydrazon (PBH). Arzneimittel-Forsch., 25: 1331- 19. Hucker, H. B. Intermediates in Drug Metabolism Reactions. Drug Metab. 1336,1975. Rev., 2: 33-56. 1973. 31 Phillips, B. J. A Simple, Small Scale Cytotoxicity Test and Its Use in Drug 20. Hunt, D. F., McEwen, C. N., and Upham, R. A. Chemical Ionisation Mass Metabolism Studies. Biochem. Pharmacol., 23: 131-138. 1974. Spectrometry. II. Differentiation of Primary, Secondary and Tertiary 32 Pohl, L. R., Bales, R., and Trager, W. F. Warfarin: Stereochemical Amines. Tetrahedron Letters, 4539-4542, 1971. Aspects of Its Metabolism in Vivo in the Rat. Res. Commun. Chem. 21. Inch, T D., and Brimblecombe, R. W. Antiacetylcholine Drugs: Chemis Pathol. Pharmacol., 75. 233-256, 1976. try, Stereochemistry and Pharmacology. Intern. Rev. Neurobiol., 76. 67- 33 Sisenwine, S. F., Kimmel, H. B., Liu, A. L., and Ruelius, H. W. Excretion 144,1974. and Stereoselective Biotransformations of dl-, d- and 7-Norgestrel in 22. Jähnchen,E., Meinertz, T., Gilfrich, H-J., Groth, U., and Martini, A. The Women. Drug Metab. Disposition, 3: 180-188. 1975. Enantiomers of Phenprocoumon: Pharmacodynamic and Pharmacoki- 34 Sladek, N. E. Therapeutic Efficacy of Cyclophosphamide as a Function netic Studies. Clin. Pharmacol. Therap., 20: 342-349, 1976. of Its Metabolism. Cancer Res . 32. 535-542. 1972. 23. Jenner, P., and Testa, B. The Influence of Stereochemical Factors on 35 Struck, R. F., Thorpe, M. C.. Coburn, W. C., and Kirk, M. C. Isolation of Drug Disposition. Drug Metab. Rev.,2: 117-184, 1973. cis- and rrans-4-Methylcyclophosphamide and Antitumor Evaluation in 24. Kawalek, J. C., Levin, W., Ryan, D., Thomas, P., and Lu, A. Y. H. Vivo. Cancer Res., 35: 3160-3163, 1975. Purification of Liver Microsomal Cytochrome P-448 from 3-Methylchol- 36 Sullivan, H. R., Due, S. L., and McMahon, R. E. The Difference in anthrene-Treated Rabbits. Mol. Pharmacol., 11: 874-878, 1975. Activity between (+ ) and (-) Methadone Is Intrinsic and Not Due to a 25. Kinas, R., Pankiewicz, K., Stec, W. J., Farmer, P. B., Foster, A. B., and Difference in Metabolism. J. Pharm. Pharmacol., 27: 728-732, 1975. Jarman, M. Synthesis and Absolute Configuration of the Optically Active 37 Voelcker, G., Wagner, T., and Honors!, H-J. Identification and Pharma- Forms of 2-[Bis(2-chloroethyl)amino]-4-methyl-tetrahydro-2H-1,3,2-oxa- cokinetics of Cyclophosphamide (NSC-26271) Metabolites in Vivo. Can zaphosphorine 2-Oxide (4-Methylcyclophosphamide). J. Org. Chem., 42: cer Treat. Rept., 60: 415-422, 1976. 1650-1652,1977. 38 Wagner, T., Peter, G., Voelcker, G., and Hohorst. H-J. Characterization 26. McGraw, N. P., Gallery, P. S., and Castagnoli, N. In Vitro Stereoselective and Quantitative Estimation of Activated Cyclophosphamide in Blood Metabolism of the Psychotomimetic Amine, 1-(2,5-dimethoxy-4-methyl- and Urine. Cancer Res..37. 2592-2596, 1977. phenyl)-2-aminopropane. An Apparent Enantiomeric Interaction. J. Med. 39 Wright, J., Cho, A. K., and Gal. J. The Metabolism of Amphetamines in Chem. ,20: 185-189. 1977. Vitro by Rabbit Liver Preparations: A Comparison of R(-) and S(+ ) 27. Mitoma, C.. Onodera, T., Takegoshi, T., and Thomas. D. W. Metabolic Enantiomers. Xenobiotica, 7: 257-266, 1977.
AUGUST 1978 2599
Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1978 American Association for Cancer Research. Isolation and Identification of a Metabolic Intermediate in the Selective Dechloroethylation of One of the Four Stereoisomers of 4-Methylcyclophosphamide
George Abel, Peter J. Cox, Peter B. Farmer, et al.
Cancer Res 1978;38:2592-2599.
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