Reexamination of the Aqueous Chemistry of N-Nitroso-3

University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 ... Richard N. Loeppky, Sunil Sukhtankar, Feng Gu, and Mi...
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Chem. Res. Toxicol. 2003, 16, 715-720

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Reexamination of the Aqueous Chemistry of N-Nitroso-3-hydroxymorpholine, a Metabolite of the Carcinogen N-Nitrosomorpholine Hyun-Joong Kim and James C. Fishbein* Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 Received December 16, 2002

N-Nitroso-3-hydroperoxymorpholine was synthesized in 12 steps starting from a commercially available serine derivative and was subsequently characterized by elemental analysis, onedimensional 1H and 13C NMR spectroscopy, and homonuclear and heteronuclear twodimensional methods. Treatment of the hydroperoxide with triarylphosphine or trialkylphosphine in CD2Cl2 or CD3CN yields N-nitroso-3-hydroxymorpholine, a putative metabolite of the carcinogen N-nitrosomorpholine, as indicated by 1H and 13C NMR spectroscopy. A study of the decomposition of N-nitroso-3-hydroxymorpholine in aqueous buffer solution, ionic strength 1 M (KCl), at 25 °C is reported. The kinetics of decay obey a three term rate law, with terms for a hydrogen ion-dependent, pH-independent, and hydroxide ion-dependent reactions, similar to what has been reported for other R-hydroxynitrosamines. The stability of this compound is similar to that of R-hydroxynitrosopiperidine reported previously. The reaction of 3-hydroxymorpholine in aqueous solution, 25 °C, pH 6.6, yields (2-hydroxy-ethoxy)acetaldehyde (62%), acetaldehyde (26%), and glycolaldehyde (26%), based on analysis of the dintrophenylhydrazine derivatives. Both the stability and the products of decay of N-nitroso-3-hydroxymorpholine that are reported here contrast strongly with a previous description.

Introduction N-Nitrosomorpholine (1) is a powerful liver carcinogen in rats and mice and a respiratory tract carcinogen in the hamster (1-3). N-Nitrosomorpholine is found in foods, cosmetics, and toiletries (4), petroleum-based fluids, and some industrial workplace airspaces, particularly in the rubber industry (5-8). N-Nitrosomorpholine has been detected in human urine (9, 10) and human gastric juices (11), and levels are elevated in individuals who simultaneously consume nitrate-rich and proteinrich foods (10). In the rat, N-nitrosomorpholine is metabolized with the major isolated products as indicated in eq 1 (12, 13). The

nitrosamines presumably arise from 2′-hydroxylation and subsequent reduction or oxidation of the aldehyde. The biochemistry and chemistry of these nitrosamines, including the aldehyde and related compounds, have since been extensively investigated by the Loeppky group (1421). The 2-hydroxy-ethoxyacetic acid presumably arises from 3′-hydroxylation, ring opening of the R-hydroxynitrosamine with hydrolysis of the diazonium ion, and further enzymatic oxidation of the aldehyde. After these reports (12, 13), the synthesis of N-nitroso-3-hydroxy* To whom correspondence should be addressed. E-mail: jfishbei@ umbc.edu.

morpholine was reported (22). The purported R-hydroxynitrosamine was reported to decompose in phosphatebuffered aqueous media “almost entirely” to acetaldehyde with a 1% yield of the (2-hydroxy-ethoxy)acetaldehyde. This suggests that N-nitroso-3-hydroxymorpholine may not be a chemically competent intermediate in the formation of the 2-hydroxy-ethoxy-acetic acid product in eq 1. Perhaps alternatively, the N-nitroso-3-hydroxymorpholine in the biological experiments is further oxidized to the nitrosamide prior to ring opening. However, a number of concerns give pause to drawing firm conclusions from the solution chemistry of the purported N-nitroso-3-hydroxymorpholine. First, the identity of this compound was based solely on the assignation of a molecular ion in field desorption MS (22). Second, predominant fragmentation to acetaldehyde alone is difficult to reconcile. A ∼5% yield of glycolaldehyde (HOCH2CHO) was reported, but otherwise, exclusive acetaldehyde formation requires a net reduction that is, under these conditions, not well-precedented. Third, the isolation of the N-nitroso-3-hydroxymorpholine was effected by cold aqueous work up and extractionsconditions that seem unfavorable given the documented instability of R-hydroxynitrosamines in aqueous solutions. The first R-hydroxynitrosamines were synthesized and characterized by Okada, Mochizuki, and co-workers (23-26).

These R-hydroxmethylnitrosamines (3) had half-lives of about a minute at neutral pH. Subsequently, the

10.1021/tx020114j CCC: $25.00 © 2003 American Chemical Society Published on Web 05/06/2003

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syntheses and study of R-hydroxymethylene- (4) and an R-hydroxymethinyl-nitrosamine (5) have been reported, and these compounds are substantially more reactive, some with millisecond half-lives at neutral pH (27-30). Among these, the maximum half-life of R-hydroxynitrosopiperidine (6) is several seconds at 25 °C (30). In contrast, there is one report of surprisingly stable R-hydroxynitroso indoles (7); these are isolable solids (31). So, it is not beyond doubt that the morpholine derivative could indeed have been obtained. Given these uncertainties, we sought to generate and investigate the chemistry of N-nitroso-3-hydroxymorpholine more definitively. In contrast to the original reported synthesis of N-nitroso-3-hydroxymorpholine that involved an unconventional approach, nitrosation of the carbinolamine, we adopted a method similar in part to that employed in the synthesis of a variety of other R-hydroxynitrosamines, outlined in eq 2. A key step, originally

developed by Saavedra (32), involved the Pb(AcO-)4mediated oxidative decarboxylation to the R-acetoxy compound that was effected here in surprisingly good yield. Group exchange gave the stable R-hydroperoxy compound, which could be definitively characterized. As indicated in this paper, subsequent reduction of the hydroperoxide yields the R-hydroxynitrosomorpholine, whose stability and decomposition products both contrast with what was earlier reported but are quite typical for what has been reported for other aliphatic cyclic R-hydroxynitrosamines. The major product of decomposition, (2-hydroxy-ethoxy)acetaldehyde, unlike the previously reported acetaldehyde, is a signature fragment.

Experimental Section Caution: Many nitrosamines are powerful carcinogens. Precautions taken in handling include use of frequently changed double pairs of disposable gloves and a well-ventilated hood. Contaminated, and potentially contaminated materials, were treated with 50% aqueous sulfuric acid containing the commercially available oxidant “No Chromix” (Aldrich Chemical). Materials. Aprotic organic solvents were dried and purified by distillation with CaH before use. The chemicals for synthesis and kinetics were ACS grade or better. Organic chemicals were typically purified prior to use in synthesis or kinetic experiments. Water was deionized. Synthesis. Unless otherwise noted, NMR spectra were recorded under normal operating conditions, without control of the operating temperature of the sample compartment. Morpholine-3-carboxylic Acid. This was prepared essentially as described (33). N-Nitrosomorpholine-3-carboxylic Acid (8). Morpholine3-carboxylic acid was nitrosated following the general method of Lijinsky (34). Purification was effected by silica gel column chromatography using hexane/ethyl acetate 6/4. 1H NMR (CDCl3): (E) isomer 64.6%: 5.51 (1H, d), 4.71 (1H, d), 4.46 (1H, d), 4.00 (1H, dd), 3.52 (1H, dd), 3.32 (1H, td), 3.10 (1H, td); (Z) isomer 35.4%: 5.45 (1H, d), 4.62 (1H, d), 4.14 (2H, m), 3.84 (1H, dd), 3.67 (1H, dt). 13C NMR (CDCl3): δ 173.6, 171.6, 68.5, 67.5, 67.3, 66.1, 60.7, 51.0, 47.9, 38.9. 3-Acetoxy-N-nitrosomorpholine (9). The following adaptation of the published method was employed (32). To a solution of 100 mg (0.62 mmol) of N-nitrosomorpholine-3-carboxylic acid in 5 mL of benzene and 110 mg (1.38 mmol) of pyridine was

Kim and Fishbein added 350 mg (0.83 mmol) of lead tetraacetate at room temperature. The reaction mixture was stirred for 1.5 h at 80 °C and was cooled to room temperature and filtered through Celite. The solvent was removed with a stream of Ar. The product was purified by HPLC; yield, 65%. 1H NMR (CDCl3): (E) isomer (91%): 7.15 (1H, s), 4.58 (1H, dd), 4.25 (1H, d), 4.06 (1H, dd), 3.84 (1H, dd), 3.40 (1H, td), 3.01 (1H, td), 2.14 (3H, s); (Z) isomer (9%): 7.15 (1H, s), 4.78 (1H, d). 13C NMR (CDCl3): δ 170.4, 80.9, 70.5, 66.8, 38.8, 22.35. N-Nitroso-3-Hydroperoxymorpholine (10). The hydroperoxy compound was synthesized by group exchange analogous to what has been reported (29, 30, 35). To a solution of 9 (30 mg) in 2 mL of MeCN was added 2 mL of 50 wt % H2O2 in water. The reaction mixture was stirred for 4 h at 40 °C. The reaction mixture was extracted with methylene chloride. The organic layer was dried over MgSO4 and filtered, and the solvent was removed with a stream of Ar. The product was purified by recrystallization in CH2Cl2 and heptane 7/3. 1H NMR (CD2Cl2): isomer 1 (see Discussion: Structure) (95%): 6.14 (1H, d), 4.61 (1H, dd), 4.27 (1H, d), 4.00 (1H, dd), 3.86 (1H, dd), 3.40 (1H, td), 3.03 (1H, td); isomer 2 (5%): 6.15 (1H, s), 4.12(1H, d). 13C NMR (CD2Cl2): δ 89.4, 67.2, 65.4, 37.3. Preparation of 3-Hydroxy-N-nitrosomorpholine (11). The R-hydroxy compound was synthesized from the hydroperoxide 10 by reduction with triphenylphosphine, which facilitated spectral characterization. One equivalent was added to a stirred solution of 10 in CD2Cl2. Proton spectra were recorded immediately after addition of the phosphine. Compound 11 was not sufficiently stable to obtain 13C spectra at ambient temperature. To effect this, solid phosphene was added directly to 10 dissolved in CD2Cl2 contained in an NMR tube, and the tube was sealed, shaken to dissolve the phosphine, and then placed in the NMR sample compartment, which was maintained at -50 °C. Both 1H and 13C spectra were recorded under these conditions. 1H NMR (CD2Cl2, ambient temperature): (E) isomer (92%): 6.07 (1H, s), 4.51 (1H, dd), 4.16 (1H, d), 4.01 (1H, dd), 3.79 (1H, dd), 3.39 (1H, td), 3.07 (1H, td); (Z) isomer (8%): 5.92 (1H, s), 3.49 (1H, dd). 1H NMR (CD2Cl2, -50 °C): (Z) isomer (62%): 6.03 (1H, s), 4.56 (1H, dd), 4.15 (1H, d), 4.01 (1H, dd), 3.78 (1H, dd), 3.39 (1H, m), 3.02 (1H, m); (E) isomer (38%): 6.04 (1H, s), 4.52 (1H, dd), 4.20 (1H, d), 3.94 (1H, dd), 3.80 (1H, dd), 3.35 (1H, m), 2.98 (1H, m). 13C NMR (CD2Cl2 at -50 °C): δ 88.9, 79.4, 70.7, 67.2, 65.54, 65.48, 36.6. 2,3-Dioxene. 2,3-Dichloro-1,4-dioxane was prepared by the method of Bo¨eseken (36). To the 7.0 g of magnesium in ether was added 150 mL of a solution of 2,3-dichloro-1,4-dioxane (40 g, 0.237 mole) in ether at room temperature, and the reaction mixture was gently refluxed for 3 days. The product was purified by distillation, bp 93 °C, standard pressure. 1H NMR (CDCl3): 4.08 (4H, s), 5.97 (2H, s). 5-Hydroxy-3-oxapentanal 2,4-Dinitrophenylhydrazone (12). A mixture of 200 mg of 2,3-dioxene, 2 drops of concentrated hydrochloric acid, and 15 mL of H2O was refluxed with an oil bath. 2,4-Dinitrophenylhydrazine (560 mg, 1.98 mmol) was dissolved in 150 mL of 2 N HCl at 60 °C. After the mixture was cooled to room temperature, a cooled mixture of hydrolyzed 2,3dioxene was added. The reaction mixture was maintained at 0 °C for 30 min, filtered, and then rinsed with 40 mL of 1 N HCl and 50 mL of water. The product was purified by recrystallization in CH2Cl2. 1H NMR (CD2Cl2): 11.1 (1H, s), 9.08 (1H, d), 8.30 (1H, dd), 7.93 (1H, d), 7.58 (1H, t), 4.31 (2H, d), 3.72 (2H, m), 3.64 (2H, m), 2.00 (1H, t). 13C NMR (CD2Cl2): δ 148.5, 145.9, 139.2, 130.7, 130.50, 123.9, 117.3, 73.28, 70.61, 62.51. Glycolaldehyde 2,4-Dinitrophenylhydrazone (13). 2,4Dinitrophenylhydrazine (1.06 g, 3.7 mmol) was dissolved in 100 mL of 2 N HCl at 60 °C. After the mixture was cooled to room temperature, glycolaldehyde dimer (300 mg, 2.5 mmol) was added. The reaction mixture was cooled to 0 °C for 30 min, filtered, and rinsed with 50 mL of 2 N HCl and 50 mL of water. The product was purified by recrystallization in ethanol and water. 1H NMR (CDCl3): 11.2 (1H, s), 9.13 (1H, d), 8.35 (1H, dd), 7.93 (1H, d), 7.62 (1H, t), 4.50 (2H, m), 2.21 (1H, t).

Aqueous Chemistry of N-Nitroso-3-hydroxymorpholine Acetaldehyde-2,4-dinitrophenylhydrazone (14). This was synthesized analogous to 13, and the product was purified by recrystallization in ethanol. 1H NMR (CD2Cl2): 11.2 (1H, s), 9.09 (1H, d), 8.26 (1H, dd), 7.94 (1H, d), 7.59 (1H, t), 2.16 (1H, s). 1H and 13C NMR spectra of 10 and 11 and COSY, HETCOR and NOESY spectra for compound 11 are included in the Supporting Information. Kinetics. All kinetic runs were carried out using a Varian or Hewlett-Packard 8452A diode array spectrometer or an Applied Photophysics DX17MV stopped-flow spectrophotometer. In all cases, the temperature was maintained at 25 °C by means of an attached recirculating bath. The buffer solution in each reaction cell was made up by diluting a concentrated stock buffer solution. Reaction solutions were maintained at ionic strength µ ) 1.0 M, unless otherwise noted, using KCl. Values of pH were obtained using an Orion model SA 720 pH meter equipped with attached Corning combination electrode. Two point calibrations were done before recording pH values. Calibrations were carried out using commercially available standards or those prescribed by the Merck Index (37). Kinetics runs were initiated when solutions containing substrates dissolved in acetonitrile were injected into the cuvettes or injected mechanically into the observation cell of the stopped-flow instrument, to give a final substrate concentration of (1-2) × 10-4 M. Higher concentrations of substrate necessitated higher concentrations of the reductant phosphine, and the insolubility of the latter in predominantly aqueous media resulted in the appearance of endpoint drift in the reaction due to turbidity. The final acetonitrile concentration was 4 vol %. Products. Products were quantitated as 2,4-dinitrophenylhydrazones after separation by HPLC using Waters pumps and a UV/vis detector. The products of decomposition at pH 6.6 were quantitated after 5 half-lives of reaction of 3-hydroxy-N-nitrosomorpholine. To a solution of 3-hydroperoxy-N-nitrosomoropholine in MeCN was added 1.0 equiv of sodium bisulfite, and the reaction mixture was added to cacodylic acid buffer (pH 6.6). After 10 min, the solution was acidified (1 M HClO4) and 2,4dinitrophenylhydrazine was added. The reaction concentrations were typically in the range of 10-4-10-5. Separations were carried out with acetonitrile/H2O as eluent over a 25 cm Keystone C18 column. Peaks were identified initially by coelution and subsequently by spiking with an approximately 2-fold addition of an authentic standard. Quantitation of the products was accomplished using standard curves containing at least two points.

Results Kinetics. The decay of N-nitroso-3-hydroxymorpholine was studied by first carrying out reduction of the hydroperoxide by tri-n-butylphosphine in acetonitrile followed by mixing 1 vol of this with 25 vol of buffered aqueous reaction media. The N-nitroso-3-hydroxymorpholine was moderately stable in dry acetonitrile but decayed over a few hours at room temperature. The kinetics of disappearance of the N-NdO chromophore of N-nitroso-3hydroxymorpholine in aqueous media were generally monitored at 235 nm and exhibited good first-order kinetics over 3-5 half-lives of reaction. Experiments with acetic acid and cacodylic acid buffers indicated that a change in buffer concentration from 0.05 to 0.3 M changed the value of kobsd by less than 10%. Values of the rate constant ko for the buffer-independent reactions were obtained by either extrapolating plots of kobsd against buffer concentration to the zero intercept or using the value of kobsd obtained at a single buffer concentration. Values of kobsd obtained in unbuffered acidic solutions (pH e 2) were taken as the values of ko under these conditions. The values of ko varied as a function of pH in the range from pH 1 to 7 as indicated by the solid symbols in the plot of log ko against pH in Figure 1.

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Figure 1. Plots of log ko, the buffer independent rate constant for decay of cyclic R-hydroxynitrosamines, as a function of pH for reaction at 25 °C in aqueous solutions, ionic strength 1 M (KCl), 1% MeCN by volume. Data for N-nitroso-3-hydroxymorpholine in solid circles. For comparison, data are presented for the reactions of N-nitroso-2-hydroxypiperidine (open circles), N-nitroso-2-hydroxypyrolidine (open squares), and N-nitroso2-methyl-2-hydroxypyrolidine (open diamonds).

Products. 3-Hydroperoxy-N-nitrosomorpholine was decomposed in aqueous buffer containing 4 vol % acetonitrile using 1.1 equiv of the reductant sodium bisulfite. The aldehyde products were derivatized as dintirophenylhydrazones by acidification to pH ∼1 and addition of the hydrazine. The hydrazones were separately identified by coelution and spiking with authentic standards and quantified by HPLC. A control experiment with butyraldehyde (10-4 M) and 0.1 equiv NaHSO3 in cacodylate buffer (pH 6.10) gave a 96% yield of the butyraldehyde dinitrophenylhydrazone. Hydazones identified and quantified (% yield) were as follows: hydroxy-3-oxapentanal 2,4-dinitrophenylhydrazone (12; 62 ( 2%); 2-[(2,4-dinitrophenyl)hydrazono]ethanol (glycolaldehyde 2,4-dinitrophenylhydrazone; 13; 26 ( 2%); and N-(2,4-dinitrophenyl)N′-ethylidene-hydrazine (acetaldehyde 2,4-dinitrophenylhydrazone; 14; 26 ( 2%) . Two additional peaks were observed in the chromatogram that had UV/vis spectra expected for dintrophenylhydrazones, but these were not identified. Control experiments with authentic standards indicated that 12 and 13 slowly decompose in aqueous neutral media, but over 1 h, there was less than 5% decay. The yields reported above were determined within 20 min of acidification and addition of 2,4-dinitrophenylhydrazine to the reaction solutions.

Discussion Structure. Two-dimensional NMR analysis permitted the assignment of connectivity in the case of the 3-hydroperoxynitrosomorpholine as indicated in Figure 2, which presents the COSY spectrum and structure derived from this and other experiments. Each hydrogen in the morpholine structure exhibits a distinct resonance, and the HETCOR spectrum (Supporting Information) unambiguously establishes the C-H connectivities. Despite the fact that the downfield-most H3 is a singlet in the onedimensional spectrum, the weak interaction with H2 is detectable in the COSY spectrum of Figure 2. Identification of H2 combined with the HETCOR spectrum assigns

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Kim and Fishbein Table 1. Rate Constantsa for the Decay of Cyclic r-Hydroxynitrosamines in Aqueous Solutions at 25 °C, Ionic Strength 1 M (KCl)

Figure 2. COSY spectrum of N-nitroso-3-hydroperoxymorpholine (in CD2Cl2) and structural assignments based on the aggregate NMR spectral evidence (see text).

H2′. Identification of the hydrogens attached to C6, as distinct from C5, is assisted by the observation of interaction between H2 and H6 in the NOESY spectrum (Supporting Information). The single pair of triplets of doublets suggests that the conformation indicated in Figure 2 is predominant. The triplet of doublets arises from the coupling constants for H6-H5′ being comparable to the geminal H5-H5′ and H6-H6′ couplings and much larger than those for the H6-H5 and H5′-H6′ interactions. This is plainly evident in the spectrum of Figure 2. We are unable to unequivocally assign the major isomer for the hydroperoxide. The more downfield chemical shift of the 3H of relatively minor isomer is typical of the E isomer (38, 39). However, the Z isomer is typically not predominant, being so generally only in cases in which the other R-position is occupied by a sterically bulky substituent. Furthermore, a referee contends that the predominant isomer is E on the basis of the 13C NMR chemical shift for C5 is quite similar to that of C5 in the R-hydroxy compound, 11, for which all evidence is consistent with the predominant isomer being E (below). The difference between E and Z isomers in the 13C chemical shift of the carbon atoms R to the nitroso group can be as much as 10 ppm; the carbon syn to the nitroso oxygen is most upfield. The E form is predominant in the case of R-hydroxynitrosomorpholine; the assignment is made on the basis, first, of the largest 3H signal in this case being the most downfield of the two at room temperature (38, 39) and, second, on changes observed in the 1H NMR spectrum at low temperature. At -50 °C, a much larger fraction of the compound (62%) is in the Z form as compared to the room temperature spectrum. This is expected because of the greater importance of hydrogen bond stabilization of the Z form at the lower temperature. The one-dimensional proton spectra of N-nitroso-3hydroxymorpholine is otherwise qualitatively highly

a Rate constants defined in eq 3. b Standard error in parentheses. c The half-lives of R-hydroxy-cyclic compounds at pH 7.4. The value of kobsd was calculated at pH 7.4 using eq 3. d Determined in this work. e As reported in ref 30.

similar to that for the 3-hydroperoxynitrosomorpholine, but the former was not sufficiently stable, even at -50 °C in CaH-dried CD2Cl2, to obtain two-dimensional spectra. This instability contrasts with what had been reported previously in which putative N-nitroso-3-hydroxymorpholine was extracted from a cold aqueous work up, concentrated, and stored in the cold for weeks in a desiccator (22). Stability. Although N-nitroso-3-hydroxymorpholine is moderately stable in aprotic organic solvents, it decomposes rapidly in aqueous media with a maximum halflife, between pH 2 and pH 4, of less than 1 min. Figure 1 indicates (solid circles) the dependence of the rate constants for decay upon pH. Included are the profiles for other cyclic R-hydoxynitrosamines (open symbols) that have been previously published (30). The pH dependence is consistent with a three term rate law, indicated in eq 3.

kobsd ) k1 + kH+[H+] + kOH-[OH-]

(3)

The best fit to this rate law is indicated by the solid line in Figure 1, and the derived constants are included in Table 1, which for comparison contains those derived previously for the listed compounds. As can be seen by inspection of Figure 1 and Table 1, N-nitroso-3-hydroxymorpholine is similar in stability to R-hydroxynitrosopiperidine, and the slight differences are

Aqueous Chemistry of N-Nitroso-3-hydroxymorpholine

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not inconsistent with what might be expected from structure-reactivity correlations of acyclic R-hydroxynitrosamines (29). The rate constant kOH dominates the reaction in the physiological pH range and is about 2-fold larger for N-nitroso-3-hydroxymorpholine than for R-hydroxynitrosopiperidine (30). In the case of acyclic compounds, the mechanism for compounds of this reactivity appears to be as indicated in eq 4, involving rate-limiting

Scheme 1

expulsion of the diazoate leaving group. The endocyclic oxygen in the morpholine compound, which is electron withdrawing as compared to the carbon atom in the piperidine compound, makes the diazoate a better leaving group. In acyclic systems, there is a correlation between the pKa of the conjugate acid of the diazoate leaving group and the log of the rate constant kOH-, indicated in eq 5 (29).

log (kOH1/kOH2) ) -0.88(pKa1 - pKa2)

(5)

The difference in conjugate acid pKa values of the leaving groups is probably quite similar to the difference between (E)-methoxyethyldiazoic acid and butanediazoic acid, equal to 0.38 units (40). This and the relationship of eq 5 predicts a 2.1-fold greater rate constant for N-nitroso3-hydroxymorpholine, not dissimilar from what is observed. This consideration neglects the effect of the electron-withdrawing group on the countervailing effects of increasing the equilibrium constant in eq 4 while decreasing the driving force for leaving group expulsion. The balance appears from limited data to tip in favor of electron-withdrawing groups with a small net accelerating effect, so that a somewhat greater than 2-fold acceleration might have been predicted. However, in sum, the observed reactivity of N-nitroso-3-hydroxymorpholine is on the order of what might have been expected. The smaller value of kH+ for N-nitroso-3-hydroxymorpholine as compared to 2-hydroxynitrosopiperidine is also not unexpected. The transition state for this reaction bears a net positive charge as indicated in the mechanism of eq 6, so that the electron-withdrawing oxygen atom

would destabilize this relative to carbon (29). While in the case of acyclic systems, kH+ is insensitive to changes in the diazoic acid pKa, due to the compensating increase in electron density, and the overall positive charge in the transition state is destabilized to a small extent by the oxygen in the morpholine. It also has been established that the transition state in this reaction is mobile and there may in fact be some differences in the charge distribution in the transition states for cyclic vs acyclic reactions (29). The similar values of kHOH for the piperidine as compared to morpholine compounds are precisely what is expected for this reaction, which has been shown to be quite insensitive to electronic effects (29). Products. The aldehyde products identified from the decay of N-nitroso-3-hydroxymorpholine are those that

might be expected of typical diazonium ion chemistry. The alkane diazoate decays readily with loss of hydroxide ion to yield a diazonium ion, which undergoes substitution competitive with rearrangement, cation hydration, and hemiacetal decomposition, as indicated in Scheme 1. The rearrangement reaction and its consequences readily explain the essentially equal yields of the dinitrophenylhydrazones of acetaldehyde and glycoaldehyde. The equal yields observed here stand in sharp contrast to what was reported previously (22). Also at variance is the observation here that (2-hydroxy-ethoxy)acetaldehyde is the major product (62%), whereas it was previously claimed to be formed to the extent of ∼1%. The overall yield of the products observed in the present case, based on the processes of Scheme 1, approaches 90%; the remaining 10% is as yet unidentified. The observation that the major product is (2-hydroxy-ethoxy)acetaldehyde suggests that novel protein and DNA lesions may be anticipated from N-nitrosomorpholine exposure. These are currently under investigation.

Acknowledgment. We thank Prof. R. N. Loeppky (Missouri, Columbia) for sharing with us unpublished spectroscopic analyses of related compounds and Prof. Paul Smith (UMBC) for helpful discussions. This work was supported by Grant RO1-CA52881 from the National Cancer Institute of the NIH. Supporting Information Available: 1H NMR and 13C NMR spectra of 10 and 11 and COSY, HETCOR, and NOESY spectra of 10. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Lijinsky, W. (1992) Chemistry and Biolgy of N-Nitroso Compounds. Cambridge University Press, Cambridge, UK. (2) Druckrey, H., Preussmann, R., Ivankovic, S., and Schmahl, D. (1967) Organotropic carcinogenic effects of 65 various N-nitrosocompounds on BD rats. Z. Krebsforsch. 69, 103-201. (3) Mohr, U. (1979) Carcinogenesis of N-nitroso-morpholine and derivatives in Syrian golden hamsters. Prog. Exp. Tumor Res. 24, 235-244. (4) Spiegelhalder, B., and Preussmann, R. (1984) Contamination of toiletries and cosmetic products with volatile and nonvolatile N-nitroso carcinogens. J. Cancer Res. Clin. Oncol. 108, 160-163.

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