Steric Effects in the Nitrosation of Piperidines1

[CANCER RESEARCH 34, 1079 1081, May 1974]

Brief Communication Steric Effects in the Nitrosation of Piperidines1

A. Russell Jones, William Lijinsky, and George M. Singer

Carcinogenesis Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

SUMMARY Milwaukee, Wis. They were purified by distillation, and purity was 99% or greater. The rates of reaction with nitrous acid of four methyl- Kinetics. The amine (1 mmole) was placed in a 5-ml substituted piperidines to form the corresponding N- volumetric flask. For the secondary amines, equimolar nitrosopiperidines were compared with that of piperidine. concentrations of nitrite and amine were found to result in The relative rates for piperidine, 2-methyl-, 2,6-dimethyl-, straight-line kinetic plots. An aliquot of sodium nitrite was and 2,2,6,6-tetramethyl-piperidine were approximately added to give a ratio of amine to nitrite of 1:1. A constant 100:20:10:1, showing considerable steric hindrance tp volume (250 n\) of glacial acetic acid in each reaction nitrosation by the Â«methylgroups. The rate of formation of mixture provided a pH of 4.1 to 4.2. The solutions were nitrosopiperidine from the tertiary amine /V-methylpiperi- diluted with water to 5 ml, stoppered, mixed, and immersed dine was about 10,000 times slower than that from piperi immediately in thermostatically controlled water baths. All dine. of the mixtures were investigated at 27Â°;mixtures contain ing 2,2,6,6-tetramethyl- and /V-methylpiperidine were also run at 57Â°. INTRODUCTION At intervals, the flasks were temporarily removed from the bath while 500-//1aliquots were withdrawn. The aliquots The possible formation in vivo of carcinogenic nitrosa- were added to 10-ml vials, each of which contained a mines by interaction of amines with nitrous acid in the magnetic stirring bar, excess calcium oxide (400 to 500 mg), stomach merits consideration as a cause of human cancer and 5.00 ml of a carbon disillude solution of n-tridecane or (4). It has been accepted that the extent of the nitrosation n-tetradecane (99 + %, 1 Â¿il/5.00ml). The vials were capped reaction depends on the nature of the amine (7). Many and stirred vigorously for 10 to 15 min. DÃ©cantationfrom hundreds of amines would qualify as progenitors of nitrosa- the sludge (calcium oxide, hydroxide, acetate, and dithiocar- mines in man (3), among them some drugs that are amines bamates) provided a clear carbon disulfide solution contain substituted on the carbon atoms neighboring the nitrogen ing only the internal standard alkane and the nitrosamine atom. There have been few studies of the kinetics of produced in the original mixture from the time of mixing to nitrosation of secondary amines (Refs. 1, 5, and 8; for the time the aliquot was added to the calcium oxide. review, see Ref. 6) and none of tertiary amines or of The /V-methylpiperidine mixtures also contained the sterically hindered secondary amines. Widely different rates original tertiary amine since, unlike secondary amines, of nitrosation of such amines would have a strong bearing tertiary amines cannot react with carbon disulfide in the on the significance of formation of the nitrosamines in the presence of calcium hydroxide to form insoluble dithiocar- genesis of cancer. bamate salts. For these cases, the carbon disulfide solutions As models for these studies we chose 4 readily available were decanted into a 2nd vial containing a magnetic stirring methyl piperidines, and we compared the rates of formation bar and 200 to 300 mg of potassium bisulfate. The vials were of the corresponding nitrosamines with that of ni capped and stirred for several min. DÃ©cantationagain trosopiperidine from piperidine under comparable condi provided a clear carbon disulfide solution containing only tions. the nitrosamine and the calibrating alkane. Measurement of the nitrosamines present in the carbon MATERIALS AND METHODS disulfide solutions was performed by injecting several ml of each solution directly into a gas Chromatographie column of Reagents. Piperidine, 2-methylpiperidine, 2,6-dimethyl- 15% polyphenyl ether on Chromosorb W, AW-DMCS, 60 piperidine, 2,2,6,6-tetramethylpiperidine, and /V-methyl- to 80 mesh (2-ft x '/4-inch glass, helium 60 ml/min, piperidine were obtained from Aldrich Chemical Company, fiame-ionization detector). The oven was operated isother- mally, but temperatures ranging from 150 170Â°wereused

1Research jointly sponsored by the Carcinogenesis Program of the in different experiments. The relative retention times of the nitrosamines are given National Cancer Institute and by the United States Atomic Energy Commission under contract with the Union Carbide Corporation. in Table 1. The areas under the nitrosamine and alkane Received December 26, 1973; accepted February 28, 1974. peaks were compared by means of response ratios derived

MAY 1974 1079

Table 1 at pH 4.1 and 27Â°,and the relative rates of reaction of the Retention times ofnilrosopiperidines on a gas Chromatographie column of secondary amines are shown graphically in Chart 1. The 15% polyphenyl ether on Chromosorb W The retention times of nitrosopiperidine and methyl-substituted ni- rate of formation of nitrosamine from the piperidine falls trosopiperidines are compared with those of saturated alkanes used as when one or more methyl groups is introduced a to the standards for calibration. nitrogen atom. The relative rates, as calculated from the pseudo 4th-order rate constants for piperidine, 2-methyl Retention time (min) at piperidine, 2,6-dimethylpiperidine, and 2,2,6,6-tetra- 150Â° 160Â° methylpiperidine, were approximately 100:20:10:1 (Ta Compound ble 2). There appeared to be considerable steric hindrance to TetradecanePentadecaneHexadecaneNitrosopiperidineNitroso-2-methylpiperidineNitroso-2nitrosation of the amine by the presence of even 1 methyl group adjacent to the nitrogen atom, and this hindrance

Table 2 ,6-dimethylpiperidineNitroso-2 Relative rale constants for nilrosalion of piperidines , 2, 6, 6-tetramethylpipe ridine2.23.35.22.94.54.71.63.92.32.9 The psuedo 4th-order rate constants for the reaction of piperidine and 4 methyl-substituted piperidines were calculated from the kinetic plots in Charts I to 3. 2000-

1500- constantperature[NO2 ]: rate AminePiperidine2-Methylpiperidine2 3hr~4)27Â°[amine] (I3mole

1000- :27Â° :27Â° ,6-Dimethylpiperidine2,2,6,6-TetramethylpiperidineiV-MelhylpiperidinePseudo4th-orderTem-:27Â° 500- :57Â° :57Â° 2-METHYLPIPERIDINE :2:4:O

LU i 250-

-19.23.11.20.13130.080.210.350.57

200- 2,6-DIMETHYLPIPERIDINE 300

150-

2,2,6,6-TETRAMETHYLPIPERIDINE

50 100 150 2,2,6,6 -TETRAMETHYLPIPERIDINE REACTION TIME (hr) Chart 1. Relative reaction rates of piperidine, 2-methylpiperidine, 2,6-dimethylpiperidine, and 2.2,6.6-tetramethylpiperidine with sodium nitrite in acetic acid at 27Â°.Initial concentrations: 0.2 Mamine as acetate salt, and 0.2 MNaNO2, pH 4.1. from appropriate blank runs. These nitrosamine values were used to calculate the amine concentrations for use in the kinetic calculations. The validity of this method was demonstrated by following to completion several reactions in which the final nitrosamine concentration was equal to the initial amine concentration, thus showing that no concurrent side reactions took place. The pH's of the reaction mixtures were measured and found to remain constant within 0.03 unit over 7 days, the period required for some of the experiments. 20 40 60 SO REACTION TIME(hr) RESULTS AND DISCUSSION Chart 2. Relative reaction rates of 2,2.6,6-tetramethvlpiperidine and A-methylpiperidine with sodium nitrite in acetic acid at 57Â°.Initial We used 0.2 Mamine 4- 0.2 Msodium nitrite in acetic acid concentrations: 0.2 Mamine as acetate salt, and 0.2 MNaNO2, pH 4.1.

1080 CANCER RESEARCH VOL. 34

the secondary amines at higher nitrite concentrations, but we found that increasing the ratio of nitrite to amine above 200- 1: 1 resulted in nonlinear pseudo 4th-order kinetic plots. This indicates that different mechanisms are involved in the nitrosation of secondary and tertiary amines. Only limited conclusions can be drawn about the rele vance of the results reported here to the problem of 180- formation of carcinogenic nitrosamines in the stomach from ingested amines and nitrite. The largest risk seems to be from unhindered, relatively weakly basic secondary amines, since the /V-nitroso derivatives of these are known to be strong carcinogens. The nitrosation of sterically hindered Â¿4 \160- secondary amines is likely to be less important, since the rate of reaction is slower and the /V-nitroso compounds formed might be less carcinogenic than the unsubstituted nitrosamines (2). Tertiary amines, although reacting much more slowly than secondary amines, do give rise to nitrosa 140- 0.2 M mines that are strong carcinogens. The increased rate of reaction with higher ratios of nitrite to amine is obviously important. The relative rates of reaction might be of less biological significance than it seems, since the residence time of amines and nitrite is measured in hours in the 120 stomach or in days in stored food. This could be sufficient 50 100 150 time for the formation of significant quantities of carcino REACTION TIME (hr) genic nitrosamines, even from amines, especially tertiary Chart 3. Relative reaction rates of /V-methy!piperidine with different amines, which react relatively slowly. concentrations of sodium nitrite in acetic acid at 57Â°.Initial concentra tions: 0.2 Mamine as acetate salt, and 0.2, 0.4, 0.8, and 1.6 MNaNO2, pH ACKNOWLEDGMENTS 4.1.

We thank David Podlecki for valuable assistance. increased with the number of methyl groups. There was no large difference in the pK of the secondary amines that could be responsible for these differences in nitrosation rates REFERENCES (e.g., the pK of piperidine is 11.12; the pK of tetramethylpiperidine is 11.07). 1. Fan, T. Y., and Tannenbaum, S. R. Factors Influencing the Rate of The formation of /V-nitrosopiperidine from /V-methyl- Formation of Nitrosomorpholine from Morpholine and Nitrite: Accel piperidine under the same conditions (at 27Â°)was so slow as eration by Thiocyanate and Other Anions. J. Agr. Food Chem., 21: to be unmeasurable. For comparison of the reaction with 237 240. 1973. nitrous acid of this compound with that of piperidine, a 2. Keefer, L.. Lijinsky, W.. and Garcia, H. Deuterium Isotope Effect on the Carcinogenicity of Dimethylnitrosamine in Rat Liver. J. Nati. similar study was carried out at a higher temperature. The Cancer Inst., 51: 299 302, 1973. formation of /V-nitrosopiperidine was compared with the 3. Lijinsky, W. In: H. R. Hearings, 92nd Congress, pp. 8 167, Stock formation of /V-nitroso-2,2,6,6-tetramethylpiperidine from the secondary amine at 57Â°.At this temperature, the Number 5270 1144. Washington, D. C.: United States Government Printing Office, 1971. reactions of both compounds were measurable, and the 4. Lijinsky, W., and Epstein, S. S. Nitrosamines as Environmental relative rates of formation of nitrosamine are shown in Carcinogens. Nature, 225: 21 23, 1970. Chart 2. It appears that the reaction rate of /V-methylpiperi- 5. Mirvish, S. S. Kinetics of Dimethylamine Nitrosation in Relation to dine is more than 2 orders of magnitude slower than that of Nitrosamine Carcinogenesis. J. Nati. Cancer Inst., 44:633 639, 1970. 2,2,6,6-tetramethylpiperidine and therefore is at least 6. Ridd, J. H. Nitrosation, Diazotisation and Deamination. Quart. Rev. 10,000 times slower than that of piperidine itself. London, 15: 418 441, 1961. 7. Sander, J., Schweinsberg, F., and Menz, H. P. Untersuchungen Ã¼berdie As shown in Chart 3, there is a considerable increase in the reaction rate of /V-methylpiperidine with an increasing Entstehung Cancerogener Nitrosamine in Magen. Z. Physiol. Chem.. 349: 1691 1697, 1968. ratio of nitrite to amine, the rate constant increasing 8. Taylor, T. W. J., and Price, L. S. The Action of Nitrous Acid on somewhat less than linearly with the nitrite ion concentra Amino-compounds. Part III. Dimethylamine, n-Propylamine, and tion. There is also an increase in the rate of nitrosation of Glycine Ethyl Ester. J. Chem. Soc., 2052 2059, 1929.

MAY 1974 1081

A. Russell Jones, William Lijinsky and George M. Singer

Cancer Res 1974;34:1079-1081.

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