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Chem. Res. Toxicol. 2004, 17, 1217-1226

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1,3- vs 1,5-Intramolecular Alkylation Reactions in Isophosphoramide and Phosphoramide Mustards James B. Springer,† Young H. Chang,‡ Kyo I. Koo,§ O. Michael Colvin,† Michael E. Colvin,| M. Eileen Dolan,⊥ Shannon M. Delaney,⊥ James L. Flowers,† and Susan M. Ludeman*,† Duke Comprehensive Cancer Center and Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, Department of Chemistry, Korea Advanced Institute of Science and Technology, Taejeon 305-701, Korea, Department of Food and Nutrition, Hanyang University, Seoul 133-791, Korea, Schools of Natural Science and Engineering, University of California, Merced, California 95344, and Department of Medicine, University of Chicago, Chicago, Illinois, 60637 Received October 16, 2003

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by 31P NMR. In 0.33 M BisTris, pH 7.4, 37 °C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of δ 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of δ 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (δ 33.0) and reactivity (half-life 0.3 h at 25 °C) of the species attributed to 13 were consistent with the assigned structure. Resonances with 31P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 °C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 µM, while 12 (500 µM) was mutagenic with 246 mutant colonies/106 surviving cells.

Introduction Structural isomers ifosfamide and cyclophosphamide are clinically useful anticancer prodrugs that are metabolized to isophosphoramide mustard (IPM, 1) and phosphoramide mustard (PM, 2), respectively (1, 2). The cytotoxic effects of IPM and PM are believed to result from intermolecular, bisalkylation reactions involving the intermediacy of electrophilic aziridinyl species, as shown in Schemes 1 and 2 (1-8). For IPM, the formation of aziridine 3 is followed by nucleophilic attack (e.g., by DNA) to provide 4. The chloroethylamido functionality in 4 is then subject to aziridine formation (5) and reaction with a second nucleophile (e.g., a second strand of DNA) to give bisalkylation product 6 (e.g., cross-linked DNA). * To whom correspondence should be addressed. Tel: 919-681-2808. Fax: 919-668-3925. E-mail: [email protected]. † Duke University Medical Center. ‡ Korea Advanced Institute of Science and Technology. § Hanyang University. | University of California. ⊥ University of Chicago.

Similarly for PM, the formation of aziridinium ion 7 and subsequent attack by a nucleophile gives 8. Repetition of the sequence provides for bisalkylation (8 f 9 f 10). Although the intramolecular reactions of IPM and PM to give, respectively, 3 and 7 are well-established (3-6, 8), other intramolecular cyclizations to give phospholidines 11-13 are possible but have received less attention (Schemes 3 and 4). Zon and co-workers used 31P NMR and MS techniques to demonstrate that substitution of alkyl groups on the NH2 moiety in PM resulted in analogues that underwent intramolecular O-alkylation reactions to provide stable oxazaphospholidine products in significant yields at pH 7.4 (30-50%, δ 20-25) (9). In the same study, solutions of authentic PM were shown to give a persistent albeit low intensity signal with a chemical shift appropriate for an oxazaphospholidine (δ 22, 2% of the product distribution); however, the source of this resonance as a phospholidine was not established (and, as determined by the work reported herein, this resonance was not due to any of the phospholidines 11-

10.1021/tx030051k CCC: $27.50 © 2004 American Chemical Society Published on Web 08/05/2004

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Springer et al.

Scheme 1. Mechanism of Bisalkylation for IPM (1)

Scheme 2. Mechanism of Bisalkylation for PM (2)

Scheme 3. Intramolecular Alkylation Reactions Possible for IPM

Scheme 4. Intramolecular Alkylation Reactions Possible for PM

13). Investigations of oxaza- and/or diazaphospholidine formation from IPM have not been previously reported although an oxazaphospholidine has been proposed as a transient “downstream” intermediate following P-N bond hydrolysis in IPM (8). Modro and colleagues studied the formation of oxazaand diazaphospholidines using analogues of IPM and PM (10-13). While this work provided many insights into such intramolecular cyclizations, it is difficult to extrapolate the results and conclusions to the specific chemistry

of IPM and PM, particularly at pH 7.4. Many of the cyclizations reported by Modro et al. were examined using analogues with very different electronics and under conditions that included strong bases (e.g., sodium hydride, excess pyridine), high temperatures (80 °C), and/ or organic solvents. The aim of this investigation was the synthesis of oxaza- and diazaphospholidines 11-13 such that their physical, chemical, and biological characteristics could be established. These data would allow for a determina-

Isophosphoramide and Phosphoramide Mustards

tion of the significance, or lack thereof, of these compounds in the chemistry and biology of IPM and PM. The work is a continuation of our efforts to quantify the multiple pathways available to the PMs (14). It is also an extension of our investigations into the contributions of each of the cyclophosphamide and ifosfamide metabolites to the toxicity and mutagenicity of these oxazaphosphorines (15, 16). Compounds 11-13 would not be bisalkylating agents and, therefore, would not be expected to be of significant therapeutic value. As potential monoalkylators, however, these compounds could contribute to mutagenic effects and the resultant therapyinduced leukemia associated with oxazaphosphorines (17, 18).

Experimental Procedures Chemicals and solvents were generally purchased from Sigma-Aldrich or Fisher Scientific. Ether, CH2Cl2, and THF were dried over CaH2, CaCl2, or Na and distilled prior to use. PM (as the cyclohexylammonium salt) and IPM were gifts from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, The National Cancer Institute. Flash chromatography utilized silica gel (230-400 mesh) from ICN. NMR spectra were recorded on Varian Inova-400 and Unity-500 spectrometers. 1H and 13C Spectra were referenced to TMS (in CDCl3) or TSP (in D2O). Aqueous samples for 31P NMR were referenced to a capillary insert of 2% H3PO4; samples in CDCl3 were referenced to a capillary insert of 85% H3PO4. The values of pH correspond to observed readings and are uncorrected for D2O. The Duke Comprehensive Cancer Center Cell Culture Facility supplied the U937 and K562 cell lines. The I83-CLL cell line was a generous gift from Dr. Andrea Ko¨nig of the Memorial Sloan-Kettering Cancer Center. In general, synthetic methods were not optimized; the primary goal of this work was to obtain enough product simply to characterize that product chemically and biologically. 2-(2-Chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11). Chloroethylamine hydrochloride (11 mmol, 1.27 g) was added in solid form to a solution of POCl3 (11 mmol, 1.00 mL) in CH2Cl2 (45 mL) at -19 °C (ice/MeOH). A solution of Et3N (3.14 mL, 23 mmol) in CH2Cl2 (10 mL) was then added dropwise to the reaction mixture. The cold bath was removed, and the mixture was stirred overnight at room temperature. The solvent was evaporated at reduced pressure, and the residue was diluted with Et2O and filtered. The organic layer was dried (MgSO4), filtered, and concentrated to give a colorless oil, which exhibited two major resonances by 31P NMR [δ 14.9 and 9.9 (1:2 ratio, CDCl3)]. The desired intermediate, 2-chloroethylphosphoramidic dichloride [Cl2P(O)NHCH2CH2Cl], was assigned to the signal of lowest intensity based on a comparison of chemical shift values with bis(2-chloroethyl)phosphoramidic-15N dichloride [Cl2P(O)15N(CH2CH2Cl)2, δ 17.6 (CDCl3)] (14). (It has been noted by a reviewer that even “anhydrous” grade MgSO4 contains some contamination by water. Addition of this reagent to solutions of compounds containing water sensitive moieties such as P-Cl could contribute to lower yields.) A solution of crude 2-chloroethylphosphoramidic dichloride in CHCl3 (45 mL) was cooled to -15 °C (dry ice/ethylene glycol), and to this were added slowly and sequentially ethanolamine (11 mmol, 0.64 mL) and Et3N (24 mmol, 0.73 mL). Upon complete addition, the reaction mixture was stirred overnight at room temperature. The mixture was then filtered and concentrated at reduced pressure, and the residue was flash chromatographed [1.5 cm × 15 cm column, EtOH-EtOAc (1:9) eluent]. Compound 11 was obtained as an off-white solid [1.3 mmol, 0.24 g, 12% yield, mp 107-108 °C, Rf 0.14 (EtOH-EtOAc, 1:9) and 0.28 (MeOH-CH2Cl2, 1:9)]. 1H NMR (CDCl3): δ 4.444.29 and 4.29-4.15 (two m, 2H, CH2O), 3.60 (t, 3JHH ) 6 Hz, 2H, CH2Cl), 3.71-3.49 and 3.49-3.32 (two m, 4H, CH2N and

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1219 two NH), and 3.32-3.19 (m, 2H, CH2N). 13C NMR (CDCl3): δ 66.38, 45.56 (d, JCP ) 5 Hz), 43.48, and 42.42 (d, JCP ) 10 Hz). 31P NMR (CDCl ): δ 29.1. Anal. (C H ClN O P) C, H, N. 3 4 10 2 2 Compound 11 (C4H10ClN2O2P) theory found

carbon

hydrogen

nitrogen

26.03 26.12

5.46 5.38

15.18 15.22

2-Amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12). By analogy to a literature preparation of similar compounds (19, 20), a solution of 1-aziridineethanol (10.7 mmol, 0.86 mL) and Et3N (10.8 mmol, 1.5 mL) in CHCl3 (5 mL) was added dropwise via syringe to a cooled (0 °C) solution of POCl3 (10.7 mmol, 1.0 mL) in CHCl3 (15 mL). The syringe was rinsed with CHCl3 (5 mL), and this rinse was added to the reaction mixture. The ice bath was allowed to melt gradually, and the reaction mixture was then stirred overnight at room temperature. The mixture was again cooled (0 °C), and NH3 was then bubbled vigorously through the mixture for 1-2 min. The flask was capped tightly, and the mixture was stirred at room temperature for 3 h. The reaction mixture was directly applied to a column of silica gel (3.5 cm × 15 cm) and was flash chromatographed using MeOH-CH2Cl2 (1:9) as the eluent. This was essentially a filtration column used to remove noneluting salts. The material that eluted through this column was concentrated, and the residue was taken up in hot EtOAc and was flash chromatographed through a second column (3.5 cm × 15 cm column) using EtOH-EtOAc (1:9) to elute the product [Rf 0.31 in MeOH-CH2Cl2 (1:9)]. Compound 12 was obtained in 25% yield as a white solid (494 mg, 2.7 mmol, mp 108-110 °C). 1H NMR (CDCl3): δ 4.40-4.27 and 4.27-4.12 (two m, 2H, CH2O), 3.75-3.58 (m, 2H, CH2Cl), 3.58-3.23 (m, 4H, two NCH2), and 3.17-2.93 (br s, 2H, NH2). 13C NMR (CDCl3): δ 63.69, 47.02 (d, JCP ) 16 Hz), 46.24 (d, JCP ) 6 Hz), and 42.81. 31P NMR (CDCl ): δ 25.1. Anal. (C H ClN O P) C, H, N. 3 4 10 2 2 Compound 12 (C4H10ClN2O2P) theory found

carbon

hydrogen

nitrogen

26.03 26.31

5.46 5.48

15.18 14.90

N,N′-Di-tert-Butoxycarbonyl-2-(2-aminoethylamino)ethanol (15). A solution of di-tert-butyl dicarbonate [“(t-BOC)2O”; O[CO2C(CH3)3]2, 21.5 mmol, 4.69 g] in THF (2 mL) was added quickly and via syringe to a cooled (0 °C) solution of 2-(2aminoethylamino)ethanol (14: 9.8 mmol, 1.02 g; Aldrich) in THF (39 mL). Additional THF (2 mL) was used to rinse the syringe, and these washings were added to the reaction mixture. The ice bath was then removed, and the reaction was stirred overnight at room temperature. The reaction solution was decanted from the solid byproduct, the solids were washed with EtOAc, and the combined supernatants were concentrated at reduced pressure. The colorless residue was flash chromatographed (110 mL silica gel, 3.5 cm × 18 cm column) using EtOAc-hexanes (1:1) to elute faster eluting impurities and then EtOAc-hexanes (3:1) to elute 15 as a colorless oil [1.73 g, 5.7 mmol, 58% yield, Rf 0.18 (EtOAc-hexanes, 1:1)]. 1H NMR (CDCl3): δ 5.15-4.86 (br m, 1H, OH), 3.81-3.66 (br m, 2H, CH2O), 3.46-3.22 (br m, 7H, three NCH2 and NH), 1.47 (s, 9H, one tert-butyl), and 1.43 (s, 9H, one tert-butyl). N,N′-Di-tert-Butoxycarbonyl-2-(2-chloroethylamino)ethylamine (16). A solution of triphenylphosphine (6.3 mmol, 1.64 g) in THF (7 mL) was added slowly via syringe to a solution of N-chlorosuccinimide (6.3 mmol, 0.84 g) in THF (36 mL). Additional THF (6 mL) was used to rinse the syringe, and this was added to the reaction flask. The pale pink, turbid mixture was stirred at room temperature for several minutes and then to this was quickly added, via syringe, a solution of 15 (1.73 g, 5.7 mmol) in THF (7 mL). Additional THF (6 mL) was used to rinse the syringe, and this rinse was added to the reaction mixture, which then was stirred overnight at room temperature.

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The clear but dark-colored reaction mixture was concentrated on a rotary evaporator, and the residual dark oil was flash chromatographed (100 mL silica gel, 3.5 cm × 15 cm column) using EtOAc-hexanes (1:9) to elute faster eluting impurities and then EtOAc-hexanes (1:3) to elute 16 as a pale yellow oil [1.06 g, 3.3 mmol, 58% yield, Rf 0.38 (EtOAc-hexanes, 1:3)]. 1H NMR (CDCl ): δ 5.09-4.74 (br m, 1H), 3.72-3.47 (m, 4H), 3 3.47-3.34 (m, 2H), 3.34-3.21 (m, 2H), 1.50 (s, 9H), and 1.40 (s, 9H). 2-(2-Chloroethylamino)ethylamine Dihydrochloride (17). Compound 16 (3.3 mmol, 1.06 g) was dissolved in 25 mL of a saturated solution of HCl in MeOH (prepared by bubbling gaseous HCl through MeOH to generate a saturated solution, which was approximately 8 M in HCl). The flask was stoppered, and after the mixture was stirred overnight at room temperature, the mixture was concentrated on a rotary evaporator. CH2Cl2 (25 mL) was added to the residue, and the mixture was concentrated again. This was repeated once more with CH2Cl2 (25 mL) and then with ether (1 × 25 mL). After it was dried under high vacuum (2 h), the product was obtained as a white solid (0.58 g, 3.0 mmol, 91% yield). 1H NMR (D2O): δ 3.913.84 (m, 2H), 3.50-3.39 (m, 3H), 3.39-3.34 (br, 2H), and 3.303.24 (m, 1H). Anal. (C4H13Cl3N2) C, H, N. Compound 17 (C4H13Cl3N2) theory found

carbon

hydrogen

nitrogen

24.57 24.75

6.70 6.83

14.33 14.08

1-(2-Chloroethyl)-2-chloro-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (18). Dihydrochloride 17 (0.02 mmol, 4 mg) was placed into an NMR tube, and the tube was then flushed with N2 and fit with a septum. Dry CDCl3 (0.5 mL) was added, and the suspension was cooled to -25 °C (o-xylene/liquid N2 bath). POCl3 (0.02 mmol, 2 µL) and Et3N (0.09 mmol, 13 µL) were added, and then, the septum was quickly removed and replaced with an NMR cap. The mixture was shaken and allowed to sit overnight at room temperature. The 31P NMR spectrum of this reaction mixture displayed a single resonance at δ 29.8 (relative to an insert of 85% H3PO4). 1-(2-Chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13). An NMR sample of 18 (vide supra) was transferred to a vial, concentrated under a stream of N2, and then dried under high vacuum for 10 min. The residue was dissolved in 1.0 mL of BisTris (1.0 M in D2O, pD 7.0), and the pD was quickly readjusted to 7.0 using 40% NaOD/D2O. The sample was transferred to an NMR tube and stored on ice (∼10 min) until placed in the NMR probe. After the sample equilibrated for 5 min to an ambient probe temperature (∼25 °C), 31P NMR spectra were acquired at intervals of 10 min. As described in the text, the signal for 18 (δ 34.3 relative to external 2% H3PO4) decreased with the concomitant appearance of a resonance attributed to 13 at δ 30.4. 2-Benzyloxy-1-(2-chloroethyl)tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (19). Dihydrochloride 17 (3 mmol, 579 mg) was suspended in CH2Cl2 (12.5 mL), and the mixture was cooled to -29 °C (o-xylene/liquid N2 slush). To this was added POCl3 (3 mmol, 0.28 mL) and then, slowly, Et3N (12 mmol, 1.69 mL). Following complete addition, the reaction mixture was stirred at room temperature for 24 h. The mixture was then cooled to 0 °C and to this was added, slowly, benzyl alcohol (3 mmol, 0.31 mL) and then Et3N (3 mmol, 0.45 mL). The reaction mixture was stirred for 24 h at room temperature, and it was then washed with water (3 × 10 mL). The organic layer was dried (MgSO4) and concentrated on a rotary evaporator. The residual brown oil was flash chromatographed using a silica gel column (30 mL silica gel to a height of 7 in.). Fast eluting impurities were eluted with EtOAc, and the product was then eluted with EtOH-EtOAc (1:9). The product was obtained as a pale yellow oil [0.9 mmol, 243 mg, 30% yield, Rf 0.52 in EtOH-EtOAc (1:9)]. 1H NMR (CDCl3): δ 7.41-7.28 (m, 5H, aromatic), 5.02-4.97 (m, 2H, benzylic), 3.57-3.47 (m, 2H, CH2-

Cl), 3.41-3.12 (m, 6H, three NCH2), and 3.03 (d, J ) 11.5 Hz, 1H, NH). 13C NMR (CDCl3): δ 136.9, 128.5 (2C), 128.1, 127.6 (2C), 68.3 (d, J ) 6.5 Hz), 47.59 (d, J ) 17 Hz), 46.72 (d, J ) 5 Hz), 42.67 (d, J ) 4 Hz), and 39.31 (d, J ) 8 Hz). 31P NMR (CDCl3): δ 28.0. 31P NMR Kinetic Studies. General Procedure. NMR sample solutions were prepared immediately prior to use. A solution of 1.65 mL of BisTris buffer (1 M, pH 7.4), 2.85 mL of H2O, and 0.5 mL of D2O (NMR lock signal) was warmed to 37 °C and then poured rapidly into a vial containing the compound to be studied (0.11 mmol). The vial was shaken to effect dissolution, and then, the sample (22 mM compound in 0.33 M BisTris) was transferred to a 10 mm NMR tube. The pH value of the solution was checked both before and after the NMR experiment. The kinetic run was acceptable if the pH value varied by e0.2 pH units. A glass insert containing 2% H3PO4 was added to the NMR tube for use as a chemical shift standard, and the sample was kept in an ice bath during adjustment of field homogeneity (ca. 15-30 min). The sample was then placed in the NMR probe at 37 °C, and it was allowed to thermally equilibrate for 2-5 min prior to final adjustment of the magnetic field homogeneity and then the initiation of spectral acquisition. For the first kinetic analysis of a compound, the spectra were obtained every 10-20 min. In repeat kinetic analyses, the spectra were obtained at time intervals appropriate to the reactivity of the compound as indicated by the initial experiment. 31P NMR acquisitions at 202.4 MHz used a 10 kHz spectral window, 16k data points, a 60° pulse of 33.3 µs, gated 1H decoupling to suppress possible differential nuclear Overhauser effects, and a pulse recycle time of 2 s. The free induction decay (FID) signal was automatically stored, and the next spectral acquisition was initiated at time t, relative to the “zero” time (initiation of first acquisition). The stored FID signals were exponentially multiplied so as to result in an additional 2 Hz of line broadening in the frequency-domain spectra. Pseudo-firstorder kinetic plots were fit by linear least-squares analysis. The peak areas were used to measure relative concentrations. Cytotoxicity Assay. With minor modifications to a published procedure (21), the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; Cell Titer 96AQueous (Promega, Madison, WI)] assay was used to determine IC50 values against U937, K562, and I83CLL cell lines where IC50 was defined as the drug concentration required to inhibit MTS bioreduction to 50% of the control. The study compounds [4-hydroperoxyifosfamide (4-HO2-IF), 4-hydroperoxycyclophosphamide (4-HO2-CP), IPM, PM, and phospholidines 11 and 12] were dissolved in minimal 95% ethanol (∼1 mg per 25-100 µL 95% ethanol) and diluted with medium. The volume of ethanol in the final solutions did not exceed 0.5%. All solutions of test compounds were used immediately upon preparation and were not stored. The cytotoxicity assays included a control using ∼1% ethanol in medium. U937 and I83CLL cells were grown in RPMI-1640 medium (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 25 mM HEPES (Gibco), 2 mM L-glutamine (Gibco), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Gibco). K562 cells were grown in Iscove’s MEM (Gibco) supplemented with 10% FBS, 25 mM HEPES, and 2 mM L-glutamine. All studies were done in an incubator with a humidified atmosphere containing 5% CO2 at 37 °C. Briefly, 5.0 × 104 cells were seeded into each well of 96 well microtiter plates and exposed to varying concentrations of study agents for 48 h. To eliminate neighboring well toxicity observed with multiwell plate assays using PM or any compound that generates PM (22), each plate was sealed using sterile, adhesive plate sealers. The MTS reagent was added during the final 3 h of exposure time. The absorbance at 490 nm was measured in an EL-340 microplate reader (Bio-Tek, Winooski, VT), and the IC50 values were determined. A control experiment in the absence of cells was conducted to ensure that the study compounds did not produce any interfering absorbance at 490 nm. The study compounds were

Isophosphoramide and Phosphoramide Mustards prepared as for the cell experiments to a final concentration of 5 mM in medium (with minimal EtOH). MTS was added to wells containing these compounds as well as to control wells with media blank (with 1% EtOH). In a parallel experiment, wells were prepared with test compounds/media in the same manner but without the addition of MTS. The plates were incubated at 37 °C for 2 h. Relative to the controls, the test compounds gave no absorbance at 490 nm in the presence or absence of MTS. For Chinese hamster ovary (CHO) cells, the cytotoxicity induced by 11 and 12 was determined by the loss of colonyforming ability as previously described (16). Briefly, CHO cells were plated at a density of 3.5 × 105 cells/T25 flask. On the following day, the cells were treated with increasing concentrations of 11 or 12 in F12/HAM medium for one half-life of each compound (i.e., 3.3 and 26.9 h, respectively), and then, the medium was replaced with fresh, drug-free medium. Solutions of 11 or 12 were made fresh as needed and were used immediately upon preparation. The cells were replated at a density of 100, 200, or 250 cells/100 mm dish immediately following drug treatment. Cell colonies (>50 cells) were counted 7-9 days later after staining with methylene blue. The survival of colonyforming efficiency was expressed as a percent of the appropriate set of control cells exposed to vehicle alone. Complete cytotoxicity assays for 11 and 12 were done three and two times, respectively, with 6-9 replicates per concentration. Mutation Frequency. Two weeks prior to mutation studies, CHO cells were grown in hypoxanthine:thymidine:aminopterine medium to reduce the number of spontaneous mutations. The CHO cells were plated, treated as described above (cytotoxicity assay) in fresh medium, and maintained in exponential growth for an additional 7 day expression period before 1 × 105 cells were plated into 100 mm dish with 5 µg/mL 6-thioguanine (6TG). The cells were incubated for 12 days, stained as described above, and counted. The mutation frequency was determined by counting 6-TG resistant colonies and expressed as the number of 6-TG resistant colonies per 106 cells (16). Compounds 11 and 12 were analyzed two and three separate times, respectively, with 20-60 replicates per concentration.

Results and Discussion Intramolecular Alkylations in IPM and PM. There are three cyclization reactions possible for each IPM and PM as shown in Schemes 3 and 4, respectively. Intramolecular O-alkylation (path A) in IPM leads to oxazaphospholidine 11 while the same reaction in PM provides 12. Path B in each scheme depicts the primary reaction route of IPM and PM, namely, intramolecular 1,3-N-alkylation to give aziridines 3 and 7, respectively. As an alternative to aziridine formation, intramolecular 1,5-N-alkylation in either IPM or PM leads to diazaphospholidine 13 (path C). While it can also be imagined that aziridines 3 and 7 could act as branch points to the formations of phospholidines 11-13 (Schemes 3 and 4, paths D and E), such pathways are not favorable based on Baldwin’s rules of ring closure (23, 24). Modro and co-workers studied a number of N-phosphorylated aziridines under various conditions and found no evidence of diazaphospholidine formation by a mechanism paralleling that shown in Scheme 3, path E (intramolecular attack on an aziridinyl ring) (13). These researchers concluded that such a reaction corresponded to an unfavorable 5-endo-tet cyclization. Given (4) that the hybridization of the carbon atom in the aziridinyl ring in 3 is approximately sp2.4-2.5, we suggest that an intramolecular nucleophilic attack on the aziridinyl ring in 3 or 7 is most similar to either a 5-endo-tet or a 5-endo-trig process. Either pathway is incompatible with the steric constraints of 3/7 and an SN2

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1221 Table 1. 31P NMR-Derived Half-Lives and Chemical Shifts in BisTris at pH 7.4 ( 0.2 and 37 ( 2 °C compd

shift (ppm)a

half-life (h)

buffer concn (M)b

1 2 3 7 11 12 13

13.0 13.2 17.3 d 33.0 30.6 30.4

0.9 0.3 3.5c d 3.3e 26.9f 0.3 (∼25 °C)g

0.33 0.22 0.33 0.22 0.33 0.33 1.0 (pD 7.0)h

a Relative to a capillary insert or an external reference of 1-2% H3PO4. b Buffers can influence the reactions of IPM and PM (14); while BisTris produces minimal aberrations, it is desirable to use as low a buffer concentration as possible while still maintaining the desired pH. This consideration provided the basis for the buffer concentrations used in this study. c Time observed for a decrease of 50% in the signal intensity for 3 under the given conditions. Time measured after the complete disappearance of precursor 1. For the reactions of 3, second-order kinetics apply. Thus, the disappearance of 3 is not monoexponential and the value given is not truly a half-life by kinetic theory. d This short-lived species has never been directly observed; therefore, its half-life has not been determined (6, 7, 14). e Correlation coefficient 0.98. f Correlation coefficient 0.99. g Ambient probe temperature. h A pD value of 7.0 is approximately equal to a pH of 7.4 (28).

mechanism (13, 23, 24). As a cautionary note, it must be pointed out that Baldwin’s rules formally apply to rings containing atoms from the first row of the periodic table. The geometric constraints of unfavorable ring closures may be overcome by the presence of the larger phosphorus atom; however, there are multiple examples in the literature where cyclizations and reactivities of phosphoramides have proven consistent with Baldwin’s rules (14, 25). Thus, if 11-13 are formed from IPM and/or PM, it is likely that this occurs directly via paths A/C and not through the intermediacy of an aziridinyl moiety (paths D/E). Intramolecular O-Alkylation in IPM. Oxazaphospholidine 11 was synthesized, and its reactivity and spectral characteristics were studied by 31P NMR in 0.33 M BisTris at pH 7.4, 37 °C (Table 1). The 31P chemical shift of 11 did not match any signal detected in reaction solutions of IPM under the same conditions. Using peak height intensities as a measure of component concentration, the disappearance of 11 was found to follow firstorder kinetics with a half-life of 3.3 h (Table 1). The stability of 11 relative to that of IPM (54 min, Table 1) was such that if 11 had formed from IPM (Scheme 3, path A), it would have been observed by NMR (limits of detection ∼5%). As discussed in the preceding section, the formation of 11 from aziridine 3 would be unlikely based on steric constraints (Scheme 3, path D). Kinetic arguments could also be used to exclude any significant contribution of this pathway. In reaction solutions of IPM, the lifetime of 3 was measured using those 31P spectra where precursor IPM was no longer visible. As determined in this manner, the lifetime of 3 was essentially the same as that found for 11 under identical conditions (Table 1). Considering the kinetics of 3 and 11 as well as the limits of NMR detection, it is estimated that if at least 10% of 3 converted to 11 then a signal for the oxazaphospholidine would have been observed. Unexpectedly, the decomposition of 11 (0.33 M BisTris, pH 7.4, 37 °C) gave products with 31P NMR chemical shifts and sequences of reactivity (appearance and disappearance) that appeared to be the same as those provided by the decomposition of aziridine 3 under the same

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Scheme 5. Synthesis of Diazaphospholidine 13

conditions (Scheme 1). The sequence 11 f 3 (δ 17.34) f 4 (δ 13.85) f 5 (δ 18.01) f 6 (δ 14.69) (where Nu ) Nu′ ) OH) seemed unlikely, however, because the step 11 f 3 would be so energetically unfavorable. In addition, no signal for 3 was observed in solutions of 11, and given the kinetics of these compounds, after one half-life for 11, the ratio of peak heights for the resonances for 11 and 3 would have been approximately 2:1, respectively. Thus, the formation of 3 would have been detected if it had occurred. The direct conversion of 11 to 4 (Nu ) OH) would require ring opening through P-O bond scission. The incidence of P-O vs P-N bond cleavage is very pHdependent, and at pH 7.4, one would expect P-N bond scission to be predominant if not exclusive (12). On the other hand, one might assume that hydrolysis occurs via an addition-elimination mechanism involving the intermediacy of a pentacoordinate phosphorus. In this structure, five-membered rings prefer an apical-equatorial orientation with neutral nitrogen ligands assuming equitorial positions for electronic and steric reasons (26, 27a). This places the oxygen in the ring in an apical position, the position that favors a leaving group. By whatever mechanism of decomposition, it can be concluded that intramolecular O-alkylation in either IPM (Scheme 3, path A) or aziridine 3 (Scheme 3, path D) is not significant at pH 7.4. Intramolecular O-Alkylation in PM. Oxazaphospholidine 12 was synthesized, and its reactivity and spectral characteristics were studied by 31P NMR as described above for isomer 11. In 0.33 M BisTris, pH 7.4, 37 °C, the disappearance of 12 (δ 30.6) followed first-order kinetics and gave a half-life of 26.9 h (Table 1). The loss of 12 was linked to the formation of a compound with a chemical shift of δ 4.15, and this compound subsequently led to a species at δ 3.87. Neither species was due to phosphate or phosphoramidic diacid [(HO)2P(O)NH2] (28), as confirmed by spiking with authentic materials, and no other resonances were observed over the duration of the experiment (13 h). The chemical shifts of these upfield signals were in the range generally observed for phosphoester diacids [(HO)2P(O)OR] and phosphoramidic diacids [(HO)2P(O)NR2] (29), suggesting that the initial hydrolysis of 12 gave some transient intermediate(s) not detected in the 31P NMR spectra. The stability of 12 is such that its formation from PM (or aziridinyl 7) would be discernible by NMR (Table 1).

It can be concluded that intramolecular O-alkylation in either PM (Scheme 4, path A) or 7 (Scheme 4, path D) is not significant at pH 7.4. Intramolecular 1,5-N-Alkylation in IPM and PM. Diazaphospholidine 13 was synthesized in situ from 17 according to Scheme 5. In the past, we have had mixed results using thionyl chloride to chlorinate amino alcohols (30-32) and such was the case here where an attempt to directly convert 14 to 17 provided very impure product. In anticipation that the target compound 13 would also present purification challenges, it was reasoned that each starting material should be homogeneous. Thus, the alternative chlorination procedure shown in Scheme 5 (14 f 15 f 16 f 17) was pursued, and although it required additional steps, it provided 17 with a much higher purity than that given by the thionyl chloride reaction. An NMR sample of 17 in CDCl3 was allowed to react with POCl3 (1 equiv) and excess triethylamine. After sitting overnight at room temperature, the 31P NMR spectrum of this reaction mixture displayed a single resonance at δ 29.8 (relative to an insert of 85% H3PO4). No unreacted POCl3 was observed (δ ∼5). The chemical shift of δ 29.8 was consistent with that expected for a diazaphospholidine; therefore, it was attributed to the expected product 18. Further support for this assignment was based on the reactivity of this compound with water. Two equivalents of water (and one of triethylamine) were added to a CDCl3 solution of 18 (δ 29.8), and the reaction was monitored by 31P NMR at ambient probe temperature. In the first spectrum (taken 10 min after the addition of water), two signals were observed at δ 29.8 (unreacted 18) and 26.4. On the basis of peak areas (and without accounting for possible differences in relaxation times), the new signal at δ 26.4 accounted for 8% of the total phosphorus intensity. Over the next 20 min, the area of this new resonance did increase slightly (to 10%) but it was clear that the compound giving this signal was unstable as it quickly led to an unidentified decomposition product with a chemical shift of δ -3.2 (7% of the total phosphorus intensity). The addition of larger increments of water and triethylamine resulted in extensive conversion of phosphoryl chloride 18 to the transient intermediate with the shift at δ 26.4. Decomposition of this intermediate led to an increase in the resonance at δ -3.2 and, ultimately, to another signal

Isophosphoramide and Phosphoramide Mustards

at δ -2.7 (unidentified product). On the basis of chemical shift and reactivity patterns, these data were consistent with the conversion of chloro compound 18 (δ 29.8) to acid 13 (δ 26.4) followed by hydrolytic decomposition of 13 to acyclic phosphates and/or phosphoramidic acids [δ ∼ (-3)] (14, 28, 29). The NMR experiment described above indicated that a direct synthesis and isolation of 13 from 18 would be very difficult. It was decided, therefore, to continue to study the reactivity of 13 under in situ conditions. Thus, 18 was synthesized in CHCl3, and after evaporation of the organic solvent, the residue was dissolved in 1 M BisTris/D2O. The pD was adjusted to 7.0, which approximated a pH of 7.4 (33), and 31P NMR spectra were acquired at intervals of 10 min at ambient probe temperature. Under these conditions, the signal for 18 (δ 34.3 relative to external 2% H3PO4) decreased by 58% after 10 min and a resonance attributed to 13 was observed at δ 30.4. After the signal for 18 was no longer visible, the disappearance of 13 was monitored and its half-life was calculated to be approximately 20 min at ∼25 °C (Table 1). A cascade of other signals (δ ∼2-9) was observed throughout the experiment (160 min), and the ultimate end-point was inorganic phosphate (δ 2.3, verified by spiking with authentic material). On the basis of chemical shift, reactivity patterns, and the lack of any other obvious signals downfield from δ 9, assignment of the signals at δ 34.3 and 30.4 to 18 and 13, respectively, was relatively straightforward (albeit tentative because of the in situ generation of each compound). Given the short half-life (20 min, 25 °C) for the species attributed to 13, its formation from IPM or PM (or 3/7) could not be excluded based on the absence of an NMR signal for this compound in reaction mixtures of IPM or PM. On the other hand, the decomposition of 13 in BisTris led to an array of unidentified intermediates (δ 7.36, 7.74, 8.12, and 8.71), which 90 min after the disappearance of 13, still accounted for more than half of the total phosphorus intensity (with the remainder being the end product, phosphate). These intermediates, which would be relatively long-lived even at 37 °C, were not detected in BisTris reaction solutions of IPM or PM. This provided evidence that the precursor to these intermediates, i.e., 13, was not formed from IPM or PM (within the detection limits of the NMR experiment). We conclude, therefore, that intramolecular 1,5-N-alkylation in IPM or PM (or 3/7) to form 13 does not occur with any significance at pH 7.4. It would have been preferable to synthesize 13 or even a precursor to 13 in a manner that allowed for isolation and unambiguous identification. To this end, benzyl ester 19 was considered a viable precursor to 13 (Scheme 5). Previously, we had excellent success using catalytic transfer hydrogenolysis to effect the conversions of similar phosphoesters to compounds, which were generally unstable (e.g., IPM and PM) (4). While 19 was readily synthesized, isolated, and identified, attempts at catalytic debenzylation only led to decomposition products. It was thought that a simpler version of compound 13 might be easier to isolate; therefore, the dechloroethyl analogue of 13 was selected as a model of 1,5-Nalkylation in IPM and PM (2-hydroxy-tetrahydro-2H1,3,2-diazaphospholidine-2-oxide). For comparative purposes, the dechloroethyl analogue of 11/12 was also investigated (2-amino-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide). Neither model was ever isolated or

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1223 Table 2. IC50 Values (µM) Against Human Cancer Cell Linesa IC50 (µM) values compd

U937

K562

I83-CLL

4-HO2-IFb IPM (1) 11 4-HO2-CPb PM (2) 12

3 13 124 1 15 785

7 26 1972 3 16 3483

9 29 170 0.5 7 338

a IC 50 is defined here as the drug concentration required to inhibit MTS bioreduction to 50% of control with a 48 h exposure time. b 4-HO2-IF and 4-HO2-CP are 4-hydroperoxyifosfamide and 4-hydroperoxycyclophosphamide, respectively. See text for details.

characterized in an unambiguous manner despite dozens of synthetic attempts that included both “one-pot” and multistep reaction schemes. In Vitro Assays. Under physiological conditions, the formation of phospholidines 11 and 12 from IPM and PM was not significant within the limits of NMR detection. Nevertheless, it was desirable to examine the cytotoxicities and mutagenicities of these compounds such that the biological effects of even small concentrations could be determined. Direct comparisons of the relative activities of neutral species such as 11 and 12 with those of IPM and PM can be misleading, however, as the PMs are ionic under physiological conditions and are transported poorly across cell membranes (34, 35). Thus, it typically requires higher concentrations of IPM and PM to bring about intracellular effects than would be necessary when these alkylators are generated intracellularly through the normal metabolism of the parent ifosfamide and cyclophosphamide. In the absence of P450 activating enzymes, 4-HO2-IF and 4-HO2-CP are routinely used as preactivated forms of the corresponding prodrugs (1, 2). In aqueous solution, these preoxidized forms of the parent drugs spontaneously provide the metabolites of ifosfamide and cyclophosphamide. In cell culture studies, then, a better measure of the relative in vitro toxicities of 11 and 12 is a comparison of activities between the phospholidines and the preactivated oxazaphosphorines. A consideration of drug exposure times is another caveat that must be applied when drawing conclusions from cell culture assays. The compounds being studied here have widely varying half-lives that are dependent on conditions of pH, temperature, and buffer type, to name a few. Bisalkylation by IPM is much slower than that for PM (t1/2 values for sequential alkylations ) 81 and 167 min for IPM vs 19 and 19 min for PM; pH 7.4, 37 °C) (1). The half-life for 4-HO2-IF is about nine times longer than that of 4-HO2-CP (t1/2 ) 14 h for 4-HO2-IF vs 1.5 h for 4-HO2-CP in 1 M lutidine, pH 7.4, 37 °C) (2). Phospholidine 12 was much more stable than any of the other compounds studied (e.g., t1/2 ) 26.9 h for 12 vs 3.3 h for 11; pH 7.4, 37 °C; Table 1). With a 48 h drug exposure time, the cytotoxicity assays with the cancer cell lines likely allowed for at least about one half-life of each compound studied. Against various human cell lines, both 11 and 12 had higher IC50 values than their corresponding PMs (Table 2; 48 h drug exposure time). Compound 11 was approximately 20-280 times less toxic than 4-HO2-IF, and 12 was generally about 1000 times less toxic than 4-HO2CP. The cytotoxicity was also measured in CHO cells, and results showed 98 and 79% survival at 500 µM 11

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Figure 1. Effect of compounds 11 and 12 on cytotoxicity (top) and mutagenicity (bottom) in CHO cells. CHO cells were exposed to increasing concentrations of 11 or 12 for one half-life (3.3 and 26.9 h, respectively). The cytotoxicity induced by 11 and 12 was determined by loss of colony-forming ability and expressed as a percent of the appropriate set of control cells exposed to vehicle alone as previously described (16). The mutation frequency was determined by evaluating 6-thioguanine resistant colonies/106 cells at the HPRT locus in CHO cells as described previously (16). The cytotoxicity and mutation data represent an average from 2 to 3 separate experiments, and each point represents 6-9 replicate dishes for the cytotoxicity and 20-60 replicate dishes for the mutagenicity experiments.

and 12, respectively, following exposure for 3.3 or 26.9 h (the respective half-lives of the compounds). Thus, neither compound was cytotoxic against CHO cells (Figure 1). As shown in Figure 1, both 11 and 12 were evaluated for mutation frequency in CHO cells upon incubation at 37 °C for one half-life. Compound 11 (3.3 h of incubation at concentrations up to 500 µM) was not mutagenic as measured by TG resistant colonies per 106 cells at the HPRT locus in CHO cells. In contrast, 12 showed a dosedependent increase in mutations per 106 surviving cells with 246 ( 110 mutants/106 surviving cells (26.9 h of incubation at 500 µM). Under the same assay conditions but with a 1 h exposure time (roughly one half-life), a 50-fold lower concentration of 4-HO2-CP (10 µM) gave a mutation frequency of 70 mutants/106 surviving cells (16). Cyclizations in DMSO. Some years ago, we found that when a sample of PM (as the cyclohexylammonium salt) in DMSO was sonicated, an unusual decomposition was observed by 31P NMR (one not seen in aqueous solutions). For the study described herein, we revisited this reaction and found that the observed reaction was not related to the sonication but to the DMSO. Furthermore, the “unusual” product was oxazophospholidine 12 as determined by isolation from DMSO (concentration and chromatography) and comparison with authentic material (NMR, TLC). Specifically, anionic PM (cyclohexylammonium salt) in DMSO-d6 underwent intramolecular O-alkylation to give 12 in quantitative yield within 6 min at room temperature. The absence of label

scrambling when using anionic PM-β,β,β′,β′-d4 as the starting material (3) was consistent with the formation of 12 via path A as shown in Scheme 4. As a 5-exo-tet cyclization process, this conversion is in accord with Baldwin’s rules of ring closure (23, 24). Similarly, IPM (obtained as the free acid) in DMSOd6 plus 1 equiv of cyclohexylamine gave 11 as an isolable product, but cyclization was only 25% complete after 2 h at room temperature and 73% complete at the next observation point, 17 h after mixing. The use of IPMβ,β,β′,β′-d4 as a starting material (4) in DMSO-d6 with added cyclohexylamine gave 11 with what appeared to be some, but not complete, label scrambling. The data are not conclusive but suggest that relative to PM, the energy differences between intramolecular O-alkylation and 1,3-N-alkylation in IPM are smaller and these pathways compete in DMSO. The formation of 3 from IPM is a reversible reaction, and this is where label scrambling could occur, 3 a IPM f 11. Because this proposed reaction route does not involve the direct conversion of the aziridinium 3 to the phospholidine 11, Baldwin’s rules of ring closure do not have to be violated to account for label scrambling in 11. The exclusion of cyclohexylamine from the syntheses of PM and IPM gives the PMs as free acids (i.e., P-OH rather than P-O-). The NMR spectra of the free acids in DMSO-d6 were complex but indicated the slow formation of phospholidines as well as significant decomposition. In spectra of PM after 24 h in DMSO at room

Isophosphoramide and Phosphoramide Mustards

temperature, the phosphorus distribution suggested 10% formation of 12 and 25-50% of decomposition products. For IPM under similar conditions, the spectra after 11 h showed 72% unreacted IPM and no clear evidence of 11; however, after 8 days at room temperature, 40% of the phosphorus distribution was attributed to 11 with the remaining arising from decomposition products. As a control, authentic 11 and 12 were each dissolved in DMSO-d6 and followed by 31P NMR over time. After 8 days at room temperature, no decomposition was observed in either sample. That cyclohexylammonium salts of IPM and PM would be more susceptible to intramolecular O-alkylation reactions in DMSO is not surprising. DMSO is a polar, aprotic solvent that effectively solvates cations but is a poor solvator of anions. As a result, anions in DMSO generally exhibit increased nucleophilicity relative to when they are in water (36). Thus, in DMSO, the phosphate oxygen, which acts as an anionic nucleophile, would be expected to have increased reactivity while the charge neutral nitrogens should not exhibit increased reactivity.

Conclusion It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in IPM and PM produce the electrophilic alkylating agents 3 and 7, respectively (3, 4). The work described herein has investigated the role, if any, of 1,5-intramolecular cyclizations in the metabolisms of IPM and PM. The formation of phospholidines 11-13 would most likely require direct conversions from IPM and/or PM. The intermediacies of 3/7 would be contrary to the rules of ring closure, which suggest that 3/7 f 11-13 would require sterically unfavorable endotet/trig processes (13, 23, 24). The data support the conclusion that direct intramolecular O-alkylation in IPM to produce 11 (Scheme 3, path A) or in PM to give 12 (Scheme 4, path A) does not contribute to the chemistry of IPM or PM in aqueous solution at pH 7.4, 37 °C. In addition to the steric argument mentioned above, kinetic data have been presented, which support exclusion of the sequences 3 f 11 (Scheme 3, path D) and 7 f 12 (Scheme 4, path D). Contrary to tentative assignments in the literature (9), a resonance with a 31P NMR chemical shift indicative of 12 does not appear in reaction solutions of PM. Similarly, a 31P signal for 11 is not observed in reaction solutions of IPM. Such signals would be detected if the phospholidines were formed in any significant concentrations (>5%) because the lifetimes of the products would be comparable to or much greater than any proposed precursors. Our inability to isolate 13 coupled with its apparent instability make conclusions regarding intramolecular 1,5-N-alkylations in IPM and PM somewhat more tenuous. On the other hand, the spectral characteristics associated with the in situ formation and subsequent hydrolysis of 13 are consistent with those that would be expected for this compound. Because of the fact that the compound assigned as 13 gave hydrolysis products that were not found in solutions of IPM/3 or PM/7, we are confident that 1,5-N-alkylation is not a significant pathway at pH 7.4, 37 °C (Schemes 3 and 4, paths C/E). The cumulative results suggested that the lifetimes of 13 and related analogues are transient in the presence of even trace amounts of water. With the alleviation of ring strain

Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1225

as the driving force, the hydrolysis rates of phospholidines and similar cyclic compounds are often much greater (orders of magnitude) than those of acyclic counterparts (27b). Furthermore, hydrolysis is generally accelerated by the presence of P-NH2 and P-OH groups. The substitution of these groups with alkyl moieties generally affords stability to the ring system (10, 12, 27b). These generalities were found to apply to the phospholidines studied in this work. In contrast to the preferred formation of aziridinyl species under aqueous conditions, the production of phospholidines was favored in DMSO solutions of anionic IPM and PM. This was not surprising given that DMSO is known to increase the nucleophilicity of anions (in this case, the oxygen anion) (36). To avoid phospholidine formation, neither IPM nor PM, as salts or free acids, should be dissolved in DMSO. The chemical factors governing the rates and product distributions of intramolecular cyclization reactions have been the focus of extensive research (24, 37, 38). In particular, the factors determining the relative rates of intramolecular SN2 reactions to form three- and fivemembered rings have proven difficult to rationalize. Attempts to rationalize a particular ring formation in IPM and PM are further complicated by the fact that many variables exist among the possible cyclizations. Every reaction involves a different nucleophilic moiety and/or a product with a unique heteroatom bonding scheme. Ab initio calculations of the activation energies for all possible intramolecular cyclizations in IPM and PM may prove helpful in understanding the chemical bases of the experimental results of this study. Nevertheless, the kinetic data in aqueous solutions at pH 7.4 allow for the conclusion that phospholidine formation from IPM or PM (or an aziridinyl intermediate) does not contribute to the biological significance of these metabolites.

Acknowledgment. We thank Dr. Michael P. Gamcsik (Department of Medicine, Duke University Medical Center) for helpful suggestions and discussions. We also thank Dr. Ellen M. Shulman-Roskes (The Johns Hopkins Oncology Center, Baltimore, MD) for some spectral analyses obtained early in the study. We appreciate the help of Lynette R. Wilson (University of Chicago) in obtaining some early data on mutation frequencies in CHO cells. This work was supported in part by an award from the Korean Institute for Science and Technology (S.M.L.) and by Public Health Service Grants CA16783 (O.M.C.) and CA57725 (E.M.D.) from the National Cancer Institute (Department of Health and Human Services). NMR spectra were obtained at the Shared Instrumentation Facility of the Duke University Medical Center and the Duke Comprehensive Cancer Center.

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