An Aziridinium Ion Intermediate in the Nitrosation of a Hexetidine

Richard N. Loeppky, Sukhjeet P. Singh, Saleh Elomari, Riley Hastings, and Thomas E. Theiss. Journal of the American Chemical Society 1998 120 (21), 51...
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Chem. Res. Toxicol. 1994, 7, 861-867

861

An Aziridinium Ion Intermediate in the Nitrosation of a Hexetidine Modell Richard N. Loeppky* and Jae-Young Bae Department of Chemistry, University of Missouri, Columbia, Missouri 65211 Received June 8, 1994@

The nitrosation chemistry of 1,3,5-trimethyl-5-aminohexahydropyrimidine (2) has been investigated as a model for the behavior of the antimicrobial agent hexetidine (1)under similar conditions. The reaction of 2 with sodium nitrite in glacial acetic acid gives 4-methyl-4[(methylnitrosamino)methyll-3-nitroso-1,3-oxazolidine (4) a s the major nitrosamine. This compound arises from a molecular rearrangement which proceeds through the diazotization of the primary amino group followed by intramolecular displacement of nitrogen to generate an aziridinium ion. The N-nitrosooxazolidine 4 forms from the nitrosation o f a n imidazolidine produced from the aziridinium ring hydrolytic opening. The N-nitrosooxazolidine 4, an isomer, 5-methyl-5-[(methylnitrosamino)methyll-3-nitroso-l,3-oxazolidine (14),which is not formed in the nitrosation of 2, and an analog 4-methyl-4-[[(2-ethylhexyl)nitrosaminolmethyll-3-nitroso1,3-oxazolidine (22)have been independently synthesized. The N-nitrosooxazolidine 22 which would be formed from hexetidine is not present in its nitrosation mixture, suggesting the absence of reactive aziridinium ions in that case. The dissimilar nitrosation chemistry of 2 and 1 are discussed.

Introduction It has been known for many years that carcinogenic nitrosamines can form in the stomach from the reaction of secondary amines with nitrous acid arising from either consumed or endogenously produced nitrite (1). All humans excrete N-nitrosoproline in their urine, much of which is generated by in vivo nitrosation (2). With only a few exceptions, it has been generally assumed that tertiary nitrogen compounds nitrosate too slowly to be significant generators of carcinogenic nitrosamines in vivo (3-6). Much of our research has been directed at testing the validity of this assumption by elucidating the structural characteristics which render tertiary nitrogen compounds highly reactive toward nitrosamine formation (4-9). We define rapid nitrosamine formation from such compounds as occurring in those cases where nitrosamine formation can be easily detected after several minutes exposure to nitrosating agents at ambient temperatures. Our prior work has led to the hypothesis that geminal diamines should be substrates for rapid nitrosation as is shown in Scheme 1or produce nitrosamines following

The following paper describes the characterization of the major nitrosamine produced from the nitrosation of 1 or 3 (9).

n

J

A significant problem encountered in elucidating the structure of this major product, which we call HEXN02 (21),is the presence of the chiral N-bound 2-ethylhexyl side chains in hexetidine and its nitrosation products.

Scheme 1

R R fi + HNO,

H+

NO R

R

Yo dJ\R

+

H,d%

acid catalyzed fragmentation to secondary amines. We have tested this hypothesis by investigating the nitrosation chemistry of hexetidine (11,hexedine (31, common commercial antimicrobial agents, and related model compounds (5,9). Preliminary investigation showed that hexetidine nitrosates rapidly at room temperature to give more than eight N-nitroso compounds. The characterization of these substances has been undertaken both by our group and by Preussmann’s group in Heidelberg (10).

Commercial hexetidine is a mixture of diastereoisomers, and the 13CNMR spectrum of hexetidine shows 3-4 lines for many of the carbons in the molecule. The problem is even more severe in the nitrosation products because of the existence of Z I E isomers resulting from N-nitroso groups. In our attempt to overcome this difficulty, we

@Abstractpublished in Advance ACS Abstracts, October 15, 1994. Dedicated to Prof. R. Preussmann on the occasion of his 65th birthday.

1-(2-ethylhe~l~-3-nitroso-4-methyl-4-[[~-(2-ethylhe~l)-~-nitrosoaminolmethyllimidazolidine;MPLC, medium pressure liquid chromatog-

Abbreviations: FID, flame ionization detector; TEA, thermal energy analyzer;HRMS, high resolution mass spectrometry; HEXNO, raphy; LDA, lithium diisopropylamide.

Q893-228X/94/27Q7-~861~Q4.50/0 0 1994 American Chemical Society

862 Chem. Res. Toxicol., Vol. 7, No. 6,1994

synthesized the model amine, 5-amino-l,3,5-trimethylhexahydropyrimidine (2), and investigated its nitrosation chemistry. We made the reasonable assumption that it would react like hexetidine and that an elucidation of the structure of its major nitrosation products would permit the synthesis of the corresponding compounds with the methyl groups replaced with 2-ethylhexyl chains. We report here that hexetidine and 2 produce different nitrosation products. The nitrosation of 2 leads to an aziridinium ion, a potent alkylating agent. We have probed the nitrosation products from hexetidine to determine whether similar aziridinium ions are formed and a t present have no evidence supporting their formation.

Loeppky and Bae trimethyl-5-aminohexahydropyrimidine(2), from 1,3,5-trimethyl5-nitrohexahydropyrimidine.The product distilled a t 69-70 "C, 10 mmHg [lit. (11) bp 69 "C, 10 mmHg1, to produce 9.3 g of a colorless liquid (65% conversion): IR (neat, cm-l): 3450-3120 (m), 2970-2600 (s); lH NMR (90 MHz, CDC13): d 3.43 (d, J = 9 Hz, 2H), 2.40 and 1.79 (two d, J = 10 Hz, 4H), 2.22 (s, 6H), 1.02 (s,3H); 13C NMR (22.5 MHz, CDC13): 6 79, 66, 47, 42, 25.

Nitrosation of 1,3,5-Trimethyl-5-aminohexahydropyrimidine (2). Four nitrosation reactions were performed employ-

ing 7.2 mg of compound 2 each dissolved in 1mL of glacial AcOH (50 mM, pH 4-5) and containing either 1, 2, 4, or 8 equiv of aqueous NaNOz (from 0.2 M stock solution). Each mixture was allowed to stir for 1 h a t room temperature. The reaction mixtures were neutralized by adding saturated aqueous NazCOS solution, and the pH of each solution was adjusted to 11 with additional NaZC03. The product mixtures were extracted Experimental Section into benzene. The organic layers were washed with brine, dried (MgSOr), and concentrated to 2 mL. Each analytical sample Caution:Since many nitrosamines are known to be carcinowas finally subjected to GC study (GC-FID, MS, and TEA). In genic, nitrosation mixtures and speeific nitrosamines should be order to isolate the major N-nitroso product (corresponding t o handled with extreme care. the largest peak on GC-TEA chromatogram), the nitrosation General. Melting points were determined on a Thomasprocedure was scaled up. Thus reaction of 3.4 g of 2 with a Hoover capillary-tube apparatus and are uncorrected. Flash single molar equivalent of acidic nitrite (to maximize the yield chromatography was carried out on E. Merck silica gel 60 (230of the major product) was carried out to produce 2.6 g of a crude 400 mesh), and medium pressure liquid chromatography (MF'LC), mixture, which was subjected to flash chromatography (CHZon E. Merck silica gel 60 (25-310 mesh). Thin layer chromaClz to 5% EtOAc in CH2C12). The relatively nonpolar fraction tography (TLC) was performed on precoated silica gel 60 F-254 (Rf = 0.72, 100% EtOAc) turned out to be the major N-nitroso plates (20 cm x 20 cm, layer thickness 0.2 mm) manufactured product as determined by GC-TEA. A similar workup procedure by E. Merck. TLC plates were visualized by UV irradiation (254 to that used for the previous analytical samples provided 0.63 nm) and developed by the Griess reagent for N-nitroso comg of pale-yellow solid: IR (KBr, cm-l): 2973 (m), 2890 (m), pounds. High pressure liquid chromatography (HPLC) was 1460-1410 (s), 1380-1210 (s); 'H NMR (500 MHz, CDC13): 6 performed on a Waters chromatograph equipped with a M-510 5.17-5.02 (two d, J = 10 Hz; two d, J = 8.5 Hz, 2H), 4.62-4.54 solvent delivery system, a n automatic sampler (WISP Model and 3.86-3.79 (two d, J = 15 Hz; two d, J = 10 Hz, 2H), 4.31710B), a multiwavelength detector, and a MAXIMA 820 data 3.85 (four d, J = 10 Hz, 2H), 3.74 and 2.98 (two s, 3H), 1.72 and system. Reversed phase columns were used: a Zorbax analyti1.58 (two S, 3H); I3C NMR (125 MHz, CDC13): 6 80.2, 79.9, 76.7, cal column (ODS, 25 cm x 4.6 mm i d . ) and an Alltech 75.8, 63.9, 63.4, 59.7, 50.2, 41.1, 34.4, 22.7, 21.8. HRMS for semipreparative column (ODS, 25 cm x 10 mm i.d.1. GC-MS CeH12N302 (M - NO): calcd 158.0929, found 158.0927 (M+ not was run on a Hewlett-Packard Model 5890 gas chromatograph seen). Anal. Calcd for C~H12N403:C, 38.28; H, 6.43; N, 29.78. equipped with a Supelco SPB-1 fused silica capillary column Found: C, 38.94; H, 6.34; N, 29.74. (30 m x 0.25 mm i d . ) connected to a Hewlett-Packard 5970 Synthesis of 4-(Hydroxymethyl)-4-methyl-3-nitroso1,3mass selective detector. GC-FID was performed on a Hewlettoxazolidine (6). 4-(Hydroxymethyl)-4-methyl-3-nitroso-1,3Packard Model 5890-11 with a Supelco SPB-1 column (30 m x oxazolidine (6)was prepared by the method of Eiter (12) from 0.25 mm id.). GC-TEA was run on a Hewlett-Packard Model 2-amino-2-methyl-1,3-propanediol, formaldehyde, and sodium 5880A gas chromatograph equipped with an Alltech SE-30 fused nitrite in acetic acid to give 11.4 g (78%)of a light yellow solid: silica capillary column (30 m x 0.54 mm i d . ) and a TEA mp 43-44 "C [lit. (12) 44 "C]; IR (KBr, cm-'1: 3620-3100 (s), analyzer (Model 502). FT-IR spectra were recorded on a Nicolet 2950-2750 (m), 1450-1220 (s); 'H NMR (300 MHz, CDC13): 6 20 DXB spectrophotometer. 1H and 13C NMR spectra (CDCl3) 5.22 and 5.13 (two d, J = 9 Hz, 2H), 4.34 and 3.92 (two d, J = were recorded on a Joel FX-SOQ, a Nicolet NT-300, or a Bruker 9 Hz, 2H), 3.99 and 3.83 (two d, J = 11 Hz, 2H), 1.63 (s, 3H); AMX-500 spectrometer. Medium resolution mass spectra were 13C NMR (75 MHz, CDC13): 6 80.4, 74.9, 65.7, 64.7, 20.6. obtained on a Kratos MS-25 spectrometer (E1 mode). High resolution mass spectra (HRMS) were determined at the Synthesis of 4-Formyl-4-methyl-3-nitroso-1,3-oxazoliMidwest Center for Mass Spectrometry, Lincoln, NE. Elemental dine (7).An adaptation of the Swern oxidation procedure was analysis was conducted by Desert Analytics, Tucson, AZ. utilized (13). To a solution of dry (distilled over PzOd CHzClz Reagents were obtained from commercial suppliers (reagent (40 mL) and freshly distilled oxalyl chloride (5 mL, 55 mmol) grades) and used as received unless otherwise specified. Solwas added dropwise at -78 "C 8.5 mL of dry DMSO (110 mmol, vents were distilled according to conventional procedure shortly distilled over CaHz) dissolved in 15 mL of CHzClz. The mixture before use. The temperature programs for gas chromatography was stirred for 5 min a t -78 "C, and then the dried alcohol 6 and HPLC conditions for analysis of the nitrosation products (7.3 g, 50 mmol) dissolved in a minimum amount of CHzClz was are as follows: GC-FID: He carrier, 100 "C for 3 min to 250 "C added dropwise. Stirring was continued for 30 min a t -78 "C. for 12 min a t 10 "C/min; GC-MS: He carrier 100 "C for 3 min Distilled triethylamine (35 mL, 250 mmol) was added a t -78 to 250 "C for 12 min at 10 "C/min; GC-TEA He carrier, 100 "C "C to stop the reaction. The quenched reaction mixture was for 3 min to 250 "C for 12 min a t 10 Wmin, pyrolyzer at 450 stirred for 10 min and then allowed to warm to room tempera"C, interface a t 200 "C; HPLC: CH30H/Hz0 (90:10), 1 mumin ture. Distilled water (100 mL) was added, and the separated for 30 min a t 250 nm. aqueous layers were reextracted with additional CHzC12. The Synthesis of lY3,5-Trimethyl-5-nitrohexahydropyrimi- organic layers were combined, washed with aqueous LiCl dine. The method of Senkus (11) was used to prepare 1,3,5solution (to remove excess of DMSO), and dried over anhydrous trimethyl-5-nitrohexahydropyrimidine(87 g, 72%): mp 49-50 MgS04. The filtrate was evaporated to dryness and subjected "C [lit. (11) 48.6 "C]; IR (KBr, cm-'): 1549 (SI, 1351 (m), 1044 to gradient flash chromatography (CHzClz to 1:l CHzClfitOAc). (m); 'H NMR (300 MHz, CDC13): 6 3.45 (d, J = 12 Hz, 2H), The aldehyde product was not easily separated from the 3.36 (d, J = 8.5 Hz, lH),2.59 (d, J = 8.5 Hz, lH), 2.23 (d, J = unreacted starting material 6. Distillation of the mixture a t 12 Hz, 2H), 2.28 (s, 6H), 1.52 (s, 3H); 13C NMR (75 MHz, 80 "C (0.75 mmHg) produced light-yellow crystals upon cooling, CDC13): 6 84, 78, 60, 42, 24. which were further recrystallized from hexane to give the pure Synthesis of 1,3,5-Trimethyl-5-aminohexahydropyrimi- aldehyde product 7 (1.4 g, 19%): mp 58-59 "C; IR (KBr, cm-l): 2993-2715 (m), 1774-1710 ( s ) ,1465-1220 (s); 'H NMR (500 dine (2). Senkus's method (11) was utilized to prepare 1,3,5-

Chem. Res. Toxicol., Vol. 7, No. 6,1994 863

Aziridinium Intermediate from Amine Nitrosation MHz, CDCl3): 6 9.75 and 9.31 (two s, lH), 5.92-5.16 (two d, J = 5.5 Hz; two d, J = 10 Hz, 2H), 4.59-3.88 (four d, J = 10 Hz, 2H), 1.82 and 1.55 (two S, 3H); 13C NMR (125 MHz, CDCl3): 6 195.5, 193.6,81.5,80.8, 74.7, 73.8,69.5,68.4, 18.1, 15.6. Anal. Calcd for C5H!&03: C, 41.65; H, 5.60; N, 19.44. Found: C, 41.75; H, 5.67; N, 19.35.

Synthesis of 4-Methyl-4-[(methylamino)methyll-3nitroso-l,3-oxa~olidine (8). Methylamine gas (0.61 g, 19.7 mmol estimated) available from a commercial supplier was dissolved in 5 mL of absolute methanol, followed by slow addition of a minimum amount of glacial AcOH to achieve pH 6 in the solution. To this methylammonium acetate solution (6-fold excess) was added the aldehyde 7 (0.48 g, 3.3 mmol), followed by addition of 0.22 g (3.3 mmol) of NaBH3CN (95% purity). The reaction mixture was stirred a t room temperature for 24 h. Methanol and excess of CH3NH2 were removed in vacuo, and the residue was taken up in 10 mL of distilled water. The aqueous solution was brought to pH 11 using saturated aqueous Na2C03. The organic mixture was extracted into diethyl ether, and the organic phase was washed with brine solution, dried (MgS04), and concentrated. The crude mixture was flash-chromatographed (EtOAc to 2% MeOH in EtOAc) t o afford light-yellow oil 8 (0.14 g, 27%): IR (neat, cm-l): 36003100 (m), 2980-2790 (m), 1480-1250 ( 8 ) ; 'H NMR (500 MHz, CDC13): 6 5.08 and 5.01 (two d, J = 10 Hz, 2H), 4.16 and 3.77 (two d, J = 10 Hz, 2H), 2.92 and 2.83 (two d, J = 15 Hz, 2H), 2.32 (9, 3H), 1.51 (9, 3H), 1.19 (br s, 1H); 13C NMR (125 MHz, CDC13): 6 80.1, 76.2, 63.8, 57.9, 36.9, 22.0; HRMS for CsH13N302: calcd 159.1007, found 159.1006. (The supplementary material includes the 13C and lH NMR spectra of 8, which attest to its purity.)

Synthesis of 4-Methyl-4-[(methylnitrosamino)methyl]3-nitroso-l,3-oxazolidine (4). An aqueous solution (3 mL) of a %fold excess of sodium nitrite (104 mg, 1.5 mmol) was added slowly to 80 mg (0.5 mmol) of the synthesized amine 8 dissolved in 5 mL of glacial AcOH (pH 4-5). The mixture was stirred a t room temperature for an hour and then made alkaline (pH 11). The reaction mixture was extracted into benzene and dried over anhydrous MgS04. The filtrate was concentrated and subjected to flash chromatography (CHZClz to 5% EtOAc in CHzClz). The major solid product was further purified by recrystallization from hexane to give light-yellow crystals 4 (63 mg, 67%), mp 48-49 "C. The infrared spectrum (KBr) and NMR spectra ('H and 13C,CDCl3) of this synthetic product were exactly identical to those of the major product from the nitrosation of model compound 2. HRMS for C6H12N30~(M - NO): calcd 158.0929, found 158.0926 (M+ not seen). Anal. Calcd for CgH12N403: C, 38.28; H, 6.43; N, 29.78. Found C, 38.45; H, 6.59; N, 30.04. Synthesisof N-Acetonylphthalimide.N-Acetonylphthalimide was prepared by the method of Lancaster and VanderWerf (14) to give 14 g (54%)of white crystalline material: mp 120123 "C [lit. (14)124 "C]; IR (KBr, cm-'): 1770 ( s ) , 1720 (9); lH NMR (300 MHz, CDCl3): 6 7.86-7.71 (m, 4H), 4.52 (s,2H), 2.27 ( s , ~ H )13C ; NMR (75 MHz, CDCl3): 6 200.0, 167.5,134.6, 132.7, 123.9, 47.3, 27.4.

in 10 mL of THF at -78 "C to quench the reaction. THF was stripped off using a rotary evaporator, and the reaction mixture was extracted into methylene chloride. The organic layers were washed with brine solution and dried over anhydrous MgS04. The filtrate was concentrated to give a complex mixture, which was subjected to repeated flash chromatography (2% MeOH in CHzC12) to produce 564 mg of very hygroscopic yellow crystals of 2-methyl-3-(methylnitrosamino)-l-phthalimido-2-propanol (14%): mp 58-60 "C; IR (Nujol, cm-l): 3600-3010 (51, 17001660 (s), 1470-1250 (s); 'H NMR (300 MHz, CDC13): 6 7.627.41 (m, 4H), 5.73-5.03 (br s, lH), 5.23-3.06 (m, 4H), 3.052.42 (four s, 3H), 1.12-0.98 (four s, 3H); 13C NMR (75 MHz, CDCl3): 6 168.7,168.6, 168.3, 144.3, 132.9,130.3, 129.7, 123.5, 122.6, 89.0, 88.8, 88.7, 74.4, 74.3, 73.3, 73.2, 61.6, 60.4, 57.4,

57.2,57.1,53.4,49.2,48.7,41.9,41.8,35.0,34.9,33.7,33.6,24.4,

23.0, 22.8; HRMS for C13Hlfi303 (M - OH): calcd 260.1035, found 260.1033 (M+ not seen).

Synthesisof l-Amino-2-methyl-3-(methylnitrosamino)2-propanol (17). 2-Methyl-3-(methylnitrosamino)-l-phthalimido-2-propanol(275 mg, 1"01) and 0.29 mL of 55%aqueous NH2NH2 (5 mmol) were mixed together in 10 mL of ethanol. The mixture was heated a t reflux for 48 h, and the reaction was stopped by addition of cold distilled water (3 mL). Upon lowering the temperature of the mixture to 0 "C, a white precipitate (phthalhydrazide) formed as a byproduct. After filtering off phthalhydrazide and removing ethanol, the reaction mixture was extracted into ethyl acetate. The organic phase was washed with brine solution and dried (MgSO4). The filtrate was concentrated, and the resulting residue was subjected to flash chromatography (3:2 CHZClfieOH) to give 100 mg of light-yellow oil, l-amino-2-methyl-3-(methylnitrosamino)-2propanol: IR (CC4, cm-l): 3590-3140 (m), 2976 (m), 1550 (s); 'H N M R (500 MHz, CDCl3): 6 4.19-3.53 (four d, J = 15 Hz, 2H), 3.91 and 3.19 (twos, 3H), 2.71-2.64 (m, 2H), 1.16 and 1.07 (two 9, 3H); 13CNMR (125 MHz, CDCl3): 6 72.5, 71.8,61.1,60.8, 52.2,48.9,48.4,41.6, 34.5,23.7,23.2; HRMS for C5H1fi302 (M 1): calcd 148.1086, found 148.1088.

+

Synthesis of 6-Methyl-6-[(methylnitrosamino)methyl]3-nitroso-1,3-oxazolidine(14). Fifty milligrams of l-amino2-methyl-3-(methylnitrosamino)-2-propanol (0.34 mmol) was

dissolved in 1.5 mL of glacial AcOH (pH 4-51, followed by addition of 43 pL of 36% aqueous formaldehyde (0.51 mmol). After stirring a t room temperature for 3 h, 46 mg of NaNOz (0.66 mmol) in 2 mL of distilled water was added to nitrosate the cyclized secondary amine intermediate. The mixture was stirred a t room temperature for a n hour. The reaction was stopped by neutralization with saturated aqueous NazC03; the pH of the solution was adjusted to 11 with additional Na~C03. The reaction mixture was extracted into CHzC12, and the organic phase was washed with brine solution, dried, and evaporated. The resulting residue was subjected to flash chromatography (1% MeOH in CHzC12) to give 43 mg of light-yellow liquid product 14 (67%): IR (Cc4, cm-l): 2980-2850 (m), 1550 (s), 1250 (s); lH NMR (500 MHz, CDC13): 6 5.83-5.04 (four d, J = 6 Hz; four d, J = 10 Hz, 2H), 4.38-4.17 (m, 2H), 3.89-3.22 (m, Synthesisof 2-Methyl-3-(methylnitrosamino)-l-phthal- 2H), 3.84,3.83,3.07, and 3.06 (fours, 3H), 1.29, 1.28, 1.17, and 1.16 (four S, 3H); 13CNMR (125 MHz, CDCl3): 6 83.0,82.7,82.6, imido-2-propanol(16). The method of Seebach et al. was 82.3,79.5,79.0, 77.8, 77.4,58.9,58.3,54.4,53.9,50.7,50.2,49.2, utilized (15). Lithium diisopropylamide (LDA) was generated 48.9, 41.1, 40.9, 33.9, 33.8, 30.8, 29.6, 21.8, 21.6, 20.9, 20.8; in the following manner. Freshly distilled diisopropylamine(1.5 HRMS for C$€1&02 (M - NO): calcd 158.0929,found 158.0928 g, 15 mmol) and 25 mL of dry THF were placed in a flame(M+ not seen). dried 100 mL three-necked round-bottomed flask under nitroSynthesisof 4Methyl4[[(2ethyl-l-hexyl)amino]methyl]gen. To this solution was added, dropwise a t -78 "C, 6 mL of 3-nitroso-1,3-oxazolidine.(Ethylhexy1)ammonium acetate 2.5 M n-BuLi solution (15 mmol) using a syringe; appearance was prepared by adding glacial AcOH (1 mL) to 930 mg of of milky color was observed upon addition. The mixture was racemic 2-ethylhexylamine (reagent grade, 7.2 mmol) in 5 mL brought to room temperature for 5 min and then back to -78 of methanol until pH 6 was achieved. To this solution was "C. To this LDA solution was added dropwise at -78 "C 1.1g added in one portion 170 mg (1.2 mmol) of 4-formyl-4-methylof freshly distilled N-nitrosodimethylamine (15 mmol) which 3-nitroso-l,3-oxazolidine (7)dissolved in 5 mL of methanol, was obtained from the nitrosation of dimethylamine; upon followed by the addition of 75 mg of NaBH3CN (95% purity, 1.2 addition, a cloudy peach color had appeared. ARer 10 min of mmol). The reaction mixture was stirred for 24 h at room stirring, 3.0 g of dry N-acetonylphthalimide (15 mmol) in 20 mL temperature. Methanol was removed in vacuo, and the resultof dry THF was slowly added a t -78 "C; upon addition, a dark ing residue was brought to pH 11with saturated aqueous Nazgreen color had appeared. The mixture was stirred for 1.5 h at C03 solution. The product was extracted into benzene, and the -78 "C, followed by addition of 2 mL of glacial AcOH (15 mmol)

864 Chem. Res. Toxicol.,Vol. 7, No. 6, 1994

Loeppky and Bae

H, 11.61; N, 5.83. HRMS for C13H26N02 (M - H): calcd organic phase was dried over anhydrous MgS04 and filtered. 228.1964, found 228.1957 (M+ not observed). The concentrated residue was subjected to flash chromatography (4:l hexane/EtOAc) to give 262 mg of a light-yellow viscous oil, Synthesis of 3-(2-Ethyl-l-hexyl)-4-[[(2-ethyl-l-hexyl)4-methyl-4-[[(2-ethyl-l-hexyl)am~o~methyll-3-~troso1,3-oxazoamino]methyll-4-methyl-1,3-oxazolidine (24). 3-(2-Ethyllidine (85%): bp 154-155 "C, 1mm Hg; IR (neat, cm-'): 3400l-hexyl)-4-(hydroxymethyl)-4-methyl-1,3-oxazolidine (9.7 g, 43 3200 (w), 2970-2840 (4,1460 (m), 1320 (m);lH NMR (500 MHz, mmol) in 20 mL of CHzCl2 was added dropwise to a mixture CDCl3): 6 5.21 and 5.10 (two d, J = 8 Hz, ZH), 4.28 and 3.84 prepared from 20 mL of dry CHzClz and oxalyl chloride (5.6 mL, (two d, J = 9 Hz, ZH), 3.05 and 2.92 (two d, J = 12 Hz, 2H), 64 mmol) to which had been added dropwise, at -78 "C, 6 mL 2.48-2.47 (d, J = 6 Hz, 2H), 1.60 (s, 1 CHd, 1.38-1.20 (br m, of dry DMSO (85 mmol) in 30 mL of CHzC12. Stirring was 4 CH2 and 1 CH), 0.86-0.79 (m, 2 CH3); 13C NMR (125 MHz, continued for 1h at -78 "C, and freshly distilled triethylamine CDCl3): 6 80.4, 76.4, 64.3,55.9,53.8,39.4,31.1,28.9,24.3,23.0, (30 mL, 213 mmol) was added to stop the reaction. The 22.3, 14.0, 10.8; LR-FAB (NaI matrix) for C13H27N30~(M+): quenched mixture was stirred for 10 min and then allowed to calcd 257.2, found 257.2. warm to room temperature. Distilled HzO (100 mL) was added, Synthesis of 4-Methyl-4-[ [(2-ethyl-l-hexyl)nitrosaminol- and the layers were separated. The aqueous layer was extracted several more times with CH2C12, and the combined extracts were methyl]-3-nitroso-1,3-oxazolidine (22). 4-Methyl-4-(2-ethylwashed with aqueous LiCl solution to remove the excess DMSO 1-hexylaminomethyl)-3-nitroso-1,3-oxazolidine (143 mg, 0.56 and were dried over MgS04. After evaporation to dryness, the mmol) was dissolved in 2 mL of glacial AcOH. To this solution residue from the CHzCl2 extracts was subjected to flash chrowas added dropwise a t room temperature 120 mg of NaNO2 matography (7:3 hexane/ethyl acetate). The crude aldehyde (1.73 mmol, 3-fold excess) in 2 mL of distilled water. The [3-(2-ethyl-1-hexyl)-4-formyl-4-methyl-1,3-oxazolidinel was conmixture was stirred for 2 h, and the reaction was stopped by taminated with approximately 30% side products and starting adding saturated aqueous Na2C03 solution (pH 11). The reacmaterials and was used in the next step without further tion mixture was extracted into benzene, and the organic phase purification. The lH NMR clearly indicated the CHO. was dried over anhydrous MgS04, filtered, and concentrated. The resulting residue was applied to flash chromatography (1:l To a solution of (2-ethyl-1-hexy1)ammoniumacetate, prepared hexane/EtOAc) to give 150 mg of light-yellow viscous oil 20 by the acidification of 4.2 g (33 mmol) of racemic (2-ethylhexy1)(94%): bp 182-183 "C, 1 mm Hg; IR (neat, cm-'): 2965-2860 amine in 20 mL of methanol containing 1.5 mL of glacial acetic (s), 1460 (s),1291 (s);'H NMR (500 MHz, CDC13): 6 5.21-5.02 acid, was added the impure aldehyde (2.5 g), from the preceding (four d, J = 8 Hz, ZH), 4.56 and 3.87 (two s, ZH), 4.37-4.30 (m, experiment, in 10 mL of methanol, and this was followed by lH), 4.14-4.06 (m, lH), 3.94-3.85 (m, lH), 3.58-3.34 (m, lH), the addition of 1.1 g (16 mmol) of NaBH3CN. The reaction 1.76 and 1.62 (two s, IC&), 1.77-1.52 (m, lCH), 1.36-1.08 (br mixture was stirred at room temperature for 48 h and then m, 4CH2), 0.90-0.81 (m, 2CH3);13CNMR (125 MHz, CDCl3): 6 stripped of methanol. Water (15 mL) was added to the residue, 80.3,79.9,77.4,76.3,76.2,64.2,63.8,58.1,56.7,56.6,49.2,49.1, and the mixture was brought to pH 11by the addition of 30% 48.0,47.9,37.4,37.3,36.5,30.5,30.1,30.0,28.4,28.3,28.2,23.8, NaOH. The resulting mixture was extracted with ether, and 23.5, 23.3, 23.1, 22.8, 22.2, 22.1, 13.9, 10.3, 10.1. Anal. Calcd the ether extracts were washed with water and brine and then for C13H~6N403:C, 54.51; H, 9.16; N, 19.57. Found: C, 54.36; dried (NazS04) and concentrated. The crude mixture was H, 9.43; N, 19.68. repeatedly subjected to flash chromatography (1:1hexanelethyl Synthesis of 3-(2-ethyl-l-hexyl)-4-(hydroxymethyl)-4- acetate) to give a colorless viscous oil (260 mg), 342-ethyl1,3-oxazol-hexyl)-4-[[(2-ethyl-1-hexyl)aminolmethyll-4-methylmethyl-1,3-oxazolidine.A homogeneous solution of 92.2 g lidine: IR (neat, cm-I): 3200-3400 (w); lH NMR (500 MHz, in 450 mL of (0.87 mol) of 2-amino-2-methyl-1,3-propanediol CDC13): 6 4.35-4.25 (br s, lH), 3.74 (dd, J = 4.2, 1.3 Hz, lH), methanol was acidified with 65 mL of glacial acetic acid to pH 3.42 (t,J = 10 Hz, lH), 3.36-3.31 (m, 2H), 2.46-2.26 (m, 5H), 6. To this acidic solution was added at 0 "C 37.8 g (0.29 mol) of 1.46-1.26 (m, 18H), 0.97-0.96 (two apparent singlets, 3H), 2-ethylhexanal in 100 mL of methanol over the course of 10 min. 0.94-0.82 (m, 12H); 13CNMR (125 MHz, CDCl3): 6 76.1, 66.6, To this mixture was added 19.1 g of NaBH3CN in 5 mL portions 66.3,63.9,63.8,63.7,62.9,58.9,58.8,50.9,50.8,50.7,38.6,38.5, a t 0 "C, and after the addition was complete, stirring was 38.0,37.9,31.2,31.1,30.9,30.8,28.8,28.7,28.6,24.4,24.3,24.1, continued for 10 h a t room temperature. The methanol was 23.9, 23.2, 23.0, 19.9, 18.5, 18.4, 14.1, 10.7, 10.6, 10.5, 10.3. removed under reduced pressure, and 200 mL of 30% NaOH HRMS for C21H43N20 (M - H): calcd 339.3376,found 339.3395 was added to the residue. The intermediate secondary amine (M+ not observed). was extracted into ether, and the combined extracts were washed with water and brine, dried over Na2S04, and stripped Nitrosation of 3-(2-Ethyl-l-hexyl)-4-[ [(2-ethyl-l-hexyl)of ether to give 36.3 g of a viscous oil. amino]methyl]-4-methyl-1,3-oxazolidine. To a glacial acetic acid solution (5 mL) of 140 mg (0.4 mmol) of 3-(2-ethyl-l-hexyl)The crude secondary amine was used directly in the next step was 44 [(2-ethyl-l-hexyl)amino]methyll-4-methyl-1,3-oxazolidine without further purification by adding it (14.6 g, 0.067 mol) to added slowly a n aqueous sodium nitrite solution (110 mg, 1.6 100 mL of methanol containing 8 mL of acetic acid. To this mmol). The mixture was stirred a t room temperature for 2 h acidic solution was added 36% aqueous formaldehyde (8.2 mL, and then made alkaline (pH 11)with dilute NaOH solution. The 0.1 mol), and the resulting solution was stirred a t 25 "C for 4 h. mixture was extracted into ether, dried over NazS04, and Methanol and the excess formaldehyde were removed under concentrated. The residue was subjected to flash chromatogvacuum, and 50 mL of saturated NaHC03 solution was added raphy (10%ethyl acetatehexane) to give a light yellow oil (109 to the residue which was extracted with ether. The combined mg, 69%)as the major product, which was identified as 2,3-bisether extracts were washed with water and brine, dried over [(2-ethyl-1-hexyl)nitrosamino]-2-methyl-l-propanol(25): lR (neat, Na2S04, and stripped of solvent under vacuum to give a colorless cm-1): 3150-3700 (m), 1250-1480 (s); lH NMR (500 MHz, viscous oil which was subjected to flash chromatography (1:l CDC13): 6 4.56-4.49 and 4.17-4.11 (m, lH), 4.01-3.77 (m, 3H), hexane/ethyl acetate) to give the desired 3-(2-ethyl-l-hexyl)-43.64-3.39 (m, 2H), 3.38-3.28 (m, 2H), 3.07-3.02 (br s, lH), (hydroxymethyl)-4-methyl-1,3-oxazolidine (16.6 g, 0.066 mol, 1.75-1.55 (m, 2H), 1.46 and 1.34 (two apparent singlets, 3H), 99%), which was further purified by distillation: bp 138-142 1.31-0.86 (m 16H), 0.83-0.74 (m, 12H); 13C NMR (125 MHz, "C, 1 Torr; IR (neat, cm-I): 3200-3600; lH NMR (500 MHz, CDC13): 6 67.9,67.8,66.6,66.4,56.8,56.7,56.1,49.3,46.6,46.5, CDC13): 6 4.63 (d, J = 2.5 Hz, lH), 4.11 (dd, J = 3.3, 2.6 Hz, 46.4,46.3, 38.9, 38.8, 38.7, 37.5,36.4,36.3,30.8,30.7,30.4,30.3, lH), 3.96 (d, J = 10 Hz, lH), 3.57 (d, J = 8 Hz, lH), 3.30 (d, J 30.0,29.9, 28.5, 28.4, 28.3, 28.2,28.1, 24.2,24.1,23.7,23.6,23.4, = 11 Hz, lH), 3.25 (d, J = 11 Hz, lH), 2.98-2.89 (br s, lH), 23.2, 22.9,22.8,22.7, 20.8,20.7,20.3, 20.2,13.9,13.8,10.6, 10.2, 2.33-2.27 (m, 2H), 1.53-1.43 (m, lH), 1.35-1.22 (m, 8H), 0.99 10.1, 9.9. Anal. Calcd for C20H42N403: C, 62.12; H, 10.96; N, (s, 3H), 0.88-0.81 (m, 6H); 13CNMR (125 MHz, CDC13): 6 85.5, 14.49. Found: C, 62.05; H 10.93; N, 14.20. HRMS for 85.4, 75.1, 63.3,63.2, 62.6,49.1,48.9,38.7,38.3,31.4,30.7,29.1, C20H42N302 (M - NO): calcd 356.3277,found 356.3264 (M+not 28.2,24.8,23.6,23.1,23.0, 15.8,14.1,14.0,11.0,9.8. Anal.Calcd observed). for C13H27N02: C, 68.06; H, 11.87; N, 6.11. Found: C, 68.46;

Chem. Res. Toxicol., Vol. 7, No. 6, 1994 866

Aziridinium Intermediate from Amine Nitrosation

Scheme 3

75.8 4 . 5 5 d,J=15 4 . 6 1 d,J=15 3.88 d,J=lO 4 . 2 8 d,J=lO 3 4 . 4 -CH, 2.98 s

-80.2 5.06 d.J=lO 5 . 1 6 d,J-10

/

2 1-H'

'63.4

21 . 8 1.72 s

Figure 1. NMR assignments are given for 4 [E,E (major isomer)]. The top number is the 1% chemical shift, and numbers directly below it are thelH data. l1

Scheme 2

\

10

1.oH 5 (HOAcI

.

1 CH,NH,*AcO.

CH,N

2 . NaBH,CN 8

.

7

-Y. Results and Discussion

Like hexetidine, 2 is easily synthesized. The condensation of nitroethane and methylamine with formaldehyde yields the parent ring, and reduction with H2/Ni(R) gives 2. The nitrosation of 2 (HOAdNaN02, 1:l) is also similar to that of hexetidine in that it gives a complex mixture of N-nitroso compounds. Silica gel chromatography gives a homogeneous yellow oil having the formula C6H12N403. Both the NMR spectra of this material and its TLC chromatographic characteristics indicated that it was a mixture of two ZIE isomers, as anticipated for a compound having at least one N-nitroso group. On the basis of its NMR spectral characteristics (Figure 11, we have assigned the structure 4 to the major isomer of this mixture. The presence of the N-nitrosooxazolidine is indicated by the isolated CH2 (13C, 6 80.2; lH, 6 5.16). The fact that only two isomers were present, rather than the four expected from a compound containing two nitrosamine functional groups, indicates that the ring nitrogen is attached to quaternary carbon whose steric bulk does not permit the nitroso oxygen to lie on its side of the molecule. ZIE isomer signals are clearly seen for the methylnitrosamine group as well as its attached CH, and several other atoms. The structural assignment was confirmed by unambiguous synthesis of 4 as shown in Scheme 2. The parent N-nitrosooxazolidine 6 was produced by nitrosation of 2-amino-2-methy1-l73-propanediol (6),employing literature conditions for the synthesis of N-nitrosooxazolidines (12). Swern oxidation gave the aldehyde 7,which yielded the amine 8 upon reductive amination with methylamine.

Nitrosation gave 4, identical in all respects with the major nitrosamine from the nitrosation of 2. Reductive amination of 7 was chosen when the condensation of methylamine with the tosylate of 6 failed, probably as a result of the steric bulk of this neopentyl-like system. The bonding of the nitrogen to the quaternary carbon in 4 indicates that its production has involved a skeletal rearrangement of 2. The rearrangement is well rationalized as shown in Scheme 3 through the production of an aziridinium ion 9. Nitrosation a t the primary amino group precedes nitrosation at one of the ring nitrogens and generates an unstable diazonium ion which suffers intramolecular attack to give 9. Precedent for the generation of an aziridinium ion within a 6-membered ring (piperidine) exists (16). This facile alkylating agent suffers rapid nucleophilic ring opening to the alcohol 10. It undergoes nitrogen assisted nitrosative cleavage to the imminium ion 11, which cyclizes to 12, a reaction intermediate for which we have GC-MS evidence, but which we have not completely characterized. The nitrosative dealkylation of 12 gives 4. It is striking that this process occurs so rapidly even in the presence of limited amounts of nitrous acid. The formation of an isomeric N-nitrosooxazolidine 14 from the nitrosation of 2 has also been considered. Diazotization of the NHz group of 2 followed by hydrolysis would produce the tertiary alcohol 13. Nitrosative ring opening followed by condensation of the OH group with the formaldimminium ion as shown in Scheme 4 would produce, after nitrosative dealkylation, the nitrosamine 14. The alternative N-nitrosooxazolidine 14 was synthesized by an unambiguous route as shown in Scheme 5. Condensation of the ketone of 16 with lithiodimethylnitrosamine according to the procedure of Seebach et al. (15) led to the nitrosamino alcohol 16. The phthalimido blocking group was removed by hydrazine treatment to give the amino alcohol 17. Reaction of this intermediate with formaldehyde and nitrous acid in a single step using the method of Eiter et al. (12) gave the desired N-nitrosooxazolidine 14 in good yield. However, chromatographic comparison of nitrosation mixtures from 2 and spiking experiments with 14 failed to reveal its presence in the nitrosation product mixture. Another interesting feature of the nitrosation of the model compound 2 is shown in Scheme 6. The imidazolidine 10 produced from the hydrolytic ring opening of the aziridinium ion can be nitrosated at either of the two

Loeppky and Bae

866 Chem. Res. Toxicol.,Vol. 7, No. 6, 1994 Scheme 7

Scheme 4

% cH%

k0

R = -CH2CHIC2H51C,H9

in a maximum yield of 60% (9). Because of its potent alkylating characteristics, we have sought to establish whether an aziridinium ion is also formed in the nitrosation of hexetidine. Using the reductive amination chemistry depicted in Scheme 2, we have synthesized the 2-ethyl-1-hexyl analog of 4, 4-methyl-4-[[(2Scheme 5 ethyl-l-hexyl)nitrosamino]methyl]-3-nitroso-l,3-oxa0 zolidine (22), by replacing methylamine with 2-ethyl-lhexanamine. Comparison of the HPLC chromatograms of the hexetidine nitrosation mixture with that of 22 and 0 spiking experiments failed to detect the presence of 22 CH,O 15 2 s CH,i& in the reaction mixture. The bis-nitrosamine 22, however, would be the result CH OH of both an aziridinium ion based rearrangement and a nitrosative dealkylation of 23 (see Scheme 7). Attempts to synthesize 23 have, to date, been unsuccessful. Nitrosation of the amine precursor 24 results in opening of the oxazolidine ring to give the bis-nitrosamino alcohol 25. Chromatographic comparisons also failed to reveal Scheme 6 its presence in the hexetidine nitrosation mixture. Thus, NO a t this point in time we have no evidence for the formation of aziridinium ions in the nitrosation of hexetidine, although our data are not sufficient to completely rule out the participation of such an intermediate. Numerous products are formed in the nitrosation, and we have concentrated on the N-nitroso compounds. This investigation has concentrated on the characterization of the major nitrosamine from 2 and a search for other nitrosamines analogous to those formed from hexetidine. While a thorough mechanism determination awaits further work, the product studies do date permit some discussion. Why do hexetidine and its methyl 12 analog 2 give rise to such different nitrosation chemistry? The key appears to lie in the relative reactivity of the nitrogens to give the nitrosammonium ions 18 or 19. The primary and tertiary amino groups in the two molecules. opening of the ring through the assistance of the other In normal aliphatic tertiary amines the formation of the nitrogen will give different products. The pathway quaternary nitrosammonium ion is reversible. In the observed involves the conversion of 19 to 12 which is, in case of primary and secondary amines nitrosammonium turn, converted into the product 4 by nitrosative dealkyion formation is probably not significantly reversible lation. An N-nitrosooxazine 20 or a product derived from because rapid proton loss from these ions is possible and it by nitrosative dealkylation would arise from the leads either to stable nitrosamines (secondary amines) nitrosammonium ion 18. This nitrosammonium ion must or rapid diazonium ion formation (primary amines). not form in significant concentration because no NBecause of the electron withdrawing effect of the adjacent nitrosooxazines are observed in the product mixture. The nitrogen in the geminal diamine structure of the hexaabsence of these products is probably due to the steric hydropyrimidines (1 and 2), these tertiary amines are factors centered at the quaternary carbon which impede less basic than the primary amino group in each comthe formation of the crowded nitrosammonium ion 18. pound (9) and are, as a result, free for nitrosammonium In contrast to 2, the nitrosation of hexetidine produces ion formation through nitrosation. If the nitrosammothe N-nitrosoimidazolidine 21 (9). The other nitrosnium ion derived from hexetidine is less stable than that amines produced from the hexetidine nitrosation are well derived from its methyl analog 2, because of steric factors, rationalized as arising from its acid catalyzed hydrolysis N,W-bis(2-ethyl-l-hexy1)-1,2,3-triamino-2- and it rapidly ring opens through the assistance of the product adjacent nitrogen (Scheme 8, path a) to give a nitrosmethylpropane (10)and constitute a maximum of 20% amine before it can undergo reversal of nitrosation, then of the reaction mixture, while HEXNO (21) is generated

1

Aziridinium Intermediate from Amine Nitrosation

Chem. Res. Toxicol., Vol. 7, No. 6, 1994 867 Supplementary Material Available: lH and 13C NMR spectra of compound 8, 4-methyl-4-[(methylamino)methyll-3nitroso-l,3-oxazolidine(2 pages). Ordering information is given on any current masthead page.

References (1) Sander, J., Schweinsberg, F., and Menz, H. P. (1968) Formation of carcinogenic nitrosamines in the stomach. Hoppe Seyler's Z . Physiol. Chem. 349, 1691-1697.

cH3v A+

A

a possible understanding of the differences in the nitrosation chemistry of hexetidine and 2 is at hand. The less reactive nitrosammonium ion derived from 2 can undergo reversible denitrosation (hydrolysis), and this can allow for the nearly irreversible nitrosation of the primary amino group which leads to the diazonium ion and the aziridinium ion (Scheme 8, path c). The nitrosammonium ion derived from 2 is also more conformationally flexible due to the smaller methyl groups and can possibly undergo an intramolecular transfer of the nitrosonium ion from the tertiary to the primary amino group through a five-membered ring transition state (Scheme 8, path b). Perhydropyrimidines with larger alkyl groups on N undergo more rapid acid catalyzed ring opening (17). These data are consistent with our hypothesis regarding the relative reactivities of the nitrosammonium ions. Because of the possible deleterious health effects which could arise from the formation of aziridinium ions derived from the nitrosation of hexetidine and related compounds, further research into their possible role in these nitrosation reactions is warranted. The product studies performed here, however, do not suggest the formation of aziridinium ions from the nitrosation of hexetidine. On the other hand, the very rapid formation of nitrosamines from both 1 and 2 shows that compounds of this type react rapidly enough to form significant quantities of possibly carcinogenic nitrosamines in vivo. Structurally related compounds must also be considered good candidates for in vivo nitrosation and nitrosamine formation.

Acknowledgment. The support of this research by a grant from the National Cancer Institute, R37 CA 26914, is gratefully acknowledged.

(2) Ohshima, H., and Bartsch, H. (1981) Quantitative estimation of endogenousnitrosation in humans by monitoringN-nitrosoproline excreted in the urine. Cancer Res. 41, 3658-3662. (3) Smith, P. A. S., and Loeppky, R. N. (1967) Nitrosative cleavage of tertiary amines. J. Am. Chem. SOC.89, 1147-1157. (4) Gowenlock, B., Hutcheson, R. J., Little, J., and Pfab, J. (1979) Nitrosative dealkylation of some symmetrical tertiary amines. J . Chem. SOC.,Perkin Trans. 2, 1110-1114. ( 5 ) Loeppky, R. N., Shevlin, G., and Yu, L. (1990)Rapid nitrosamine formation from tertiary nitrogen compounds: An overview. In Significance of N-Nitrosation of Drugs (Eisenbrand, G., Bolzer, G., and Nicolai, H. v, Eds.) Drug Development and Evaluation, Vol. 16, pp 253-266, Gustav Fischer Verlag, New York. (6) Loeppky, R. N., Bao, Y. T., Bae, J. Y., Yu, L., and Shevlin, G. (1994)Blocking nitrosamine formation: Understanding the chemistry of rapid nitrosation. In Nitrosamines and Related N-Nitroso Compounds: Chemistry and Biochemistry (Loeppky,R. N., and Michejda, C. J., Eds.) pp 52-65, American Chemical Society, Washington, DC. (7) Loeppky, R. N., Outram, J. R., Tomasik, W., and Faulconer, J. M. (1983) Rapid nitrosamine formation from a tertiary amine: the nitrosation of 2-(N,N-dimethylaminomethyl)pyrrole.Tetrahedron Lett. 24,4271-4274. (8) Loeppky,R. N., and Yu, L. (1990)Nitrosamines, N-nitrosoamides, and diazonium ions f "tri-N-substituted amidines. Tetrahedron Lett. 31,3263-3266. (9) Bae, J. Y., Mende, P., Shevlin, G., Spiegelhalder, B., Preussmann, R., and Loeppky, R. N. (1994) The nitrosation of hexetidine and hexedine: Characterization of the major nitrosamine from common antimicrobial agents. Chem. Res. Toxicol. (following paper in this issue). (10) Mende, P., Wacker, C.-D., Preussmann, R., and Spiegelhalder, B. (1993) Nitrosation of the antimicrobial drug hexetidine: Nitrosamines derived from a triamine decomposgion product. Food Chem. Tozicol. 31,53-58. (11) Senkus, M. (1946)The preparation of some hexahydropyrimidines from nitroparaffins. J . Am. Chem. SOC.68,1611-1613. Eiter, K., Hebenbrock, K. F., and Kabbe, H. J. (1972) New openchain and cyclic a-nitrosaminoalkyl ethers. Justus Liebigs Ann. Chem. 766,55-77. Mancuso, A. J., Huang, S.-L., and Swern, D. (1978) Oxidation of long-chain and related alcohols to carbonylsby dimethyl sulfoxide "activated" by oxalyl chloride. J. Org. Chem. 43, 2480-2482. Lancaster, R. E., and VanderWerf, C. A. (1958) Improved synthesis of 3-methylpyrrole. J. Org. Chem. 23, 1208-1209. Seebach, D., Enders, D., and Renger, B. (1977) Lithiation and electrophilic substitution at a-methylene groups of nitrosamines. Reactivity umpolungof secondary amines. Chem. Ber. 110,18521865. Hammer, C. F., Heller, S. R., and Craig, J. H. (1972) Reactions of ,5-substituted amines. 11. Nucleophilic displacement reactions on 3-chloro-1-ethylpiperidine. Tetrahedron 28, 239-253. Evans, R. F. (1967)Hydropyrimidines.Aust. J . Chem. 20, 16431661.