Chem. Res. Toxicol. 1991,4, 373-381
373
Pyrroles as Effective Agents for Blocking Amine Nitrosation Allan L. Wilcox, Yen T. Bao, and Richard N. Loeppky* Department of Chemistry, University of Missouri, Columbia, Missouri 65211 Received August 6, 1990 The ability of 10 pyrroles to block the acidic nitrosation of morpholine has been determined by using an assay that measures their effectiveness in the presence of a 10-fold excess of amine. The log ('70 N-nitrosomorpholine) formed is a linear function of blocking agent concentrations ranging from 0.0 to 1.5 times the equivalents of initial nitrite. The negative slopes of these plots allow a ranking of the effectiveness of the blocking agent. Several of the pyrroles have been found to be much more effective than established blocking agents such as ascorbic acid. The following order of blocking ability has been determined: 2,5-dimethylpyrrole = l-benzyl-2,5dimethylpyrrole >> 4-methylcatechol > ascorbic acid = 1,2-phenylenediamine = pyrrole > 1,2,5-tribenzylpyrrole = 1-benzylpyrrole = octamethylporphine >> ammonium sulfamate = hydrazine = 2,5-diphenylpyrrole >>>> P-nicotyrine > 2-pyrrolecarboxylic acid. Pyrroles give complex mixtures devoid of N-nitroso compounds upon nitrosation.
Introductlon The nitrosamines constitute a family of powerful animal carcinogens (1) and there is evidence that they may be carcinogenic to humans as well (2).These compounds form easily from ubiquitous precursors. Amines or other nitrogen compounds react readily with nitrosating agents derived from nitrite or oxides of nitrogen to give nitrosamines, which are often adventitious contaminants of commercial substances. Nitrosamines also form naturally in humans who excrete small quantities of N-nitrosoproline in their urine daily (3). Much of this endogenous nitrosation takes place in the stomach. Nitrite, ingested in food and produced from the oral cavity reduction of dietary nitrate, reacts with stomach acid to produce nitrous acid (4). Although much of this nitrous acid is destroyed by reaction with other dietary constituents, some of it reacts to produce N-nitroso compounds. Mirvish has demonstrated that ascorbic acid is particularly effective in blocking gastric nitrosation (5). The blocking of nitrosamine formation and other Nnitrosation reactions has been a goal of numerous research efforts and is attached to significant practical consequences in the area of preventive chemical toxicology. In this paper we show that pyrroles are unusually effective at blocking nitrosamine formation, present a general assay for evaluating the blocking ability of amine-nitrosation inhibitors, demonstrate that phenolic compounds, some of which have been considered nitrosation catalysts,are effective blocking agents, and compare the blocking ability of a number of compounds (Chart I). Chemistry of Blocking Nitrosation. A large number of compounds, both synthetic and naturally occurring, have been shown to block nitrosation of amines by scavenging the nitrosating agent both in vitro (6) and in vivo (7).An excellent recent review which particularly emphasizes inhibition of endogenous nitrosation has been provided by Bartsch, Oshima, and Pignatelli (7).We briefly review here the significant chemical details of these processes. Although the principles to be discussed apply to N-nitrosoamides, N-nitrosoureas, and N-nitrosocarbamates, the chemistry of nitrosamine formation from secondary amines will serve as our model. Nitrosamines can form over the entire pH range and in nonaqueous as well as aqueous media (8-10). Their formation at high pH and under nonaqueous conditions is often the result of either nitrosation by oxides of nitrogen or a specific type of catalysis unique to the system. Nitrite ion itself does not nitrosate amines, but its dissolution in
Chart I
OH
HO PO
OH
PYC
cu I
CA
OH &OH
I
HY
PD
OH
CAT
He
HOH-CH,OH
OQC
HO NT
AS
OH
CH, ASC
YCAT
P h CCH- ,
i
H
PYR
CH,Ph
DYP
-
BDYP
TBP
Ph CH,Ph
H, P h CH,Ph
G
Ph
QCO,H
H
H
DPP
PYCA
\ I
NBP
OYP
acid results in several effective nitrosating agents as illustrated in eq l. In buffered acidic solutions, N203and NO2-
+ HX
-
HNOz
+ NzO3 + NOX + HZONO+
(1)
NOX are the principal nitrosating agents (IO). Their rate of reaction with an unprotonated amine is exceedingly fast and effectively encounter controlled (10). Two significant chemical reactions attenuate the rate of this process. Because of their strong basicity the concentration of the free base required for nitrosation of an amine is often very low in acidic media. Mirvish has shown that secondary amine nitrosation is effectively controlled by the amine pK, and the pH (11). NzO3 is thermally unstable and decomposes as shown in eq 2 at temperatures above 0 "C. NZ03 NO + NO2 (2) +
NO does not nitrosate amines, but it is readily air oxidized to NO2 (IO). The combination of NO with NOp generates the effective nitrosating agent NpO3 NOz can also dimerize
0~93-22a~/9~/2104-0373~02.50/0 0 1991 American Chemical Society
374 Chem. Res. Toxicol., Vol. 4, No. 3, 1991
Wilcox et al.
Table I. Nitrous Acid ConsumDtion of Psrroles equip ovrrole initial final % reacted 0.61 0.07 89 BDMP 90 1.04 0.11 BDMP 1.49 0.14 91 BDMP 2.04 0.48 76 BDMP 3.62 1.41 61 BDMP 0.40 0.00 100 NBP 0.51 0.00 100 NBP 0.68 0.04 NBP 95 0.80 0.02 97 NBP 0.85 0.13 85 NBP 1.27 0.34 73 NBP 1.59 0.53 67 NBP 3.03 1.66 45 NBP 1.50 0.25 DMP 83 2.25 1.52 32 DMP 3.00 2.81 6 DMP 0.64 0.00 100 PYR 98 PYR 1.29 0.02 1.78 0.29 84 PYR 64 PYR 2.50 0.90 3.57 2.23 37 PYR ~~
OH
OH
NO
Molar equivalents to [NOLI. Table 11. Blocking by DMP equiv of DMP 0.00 0.13 0.25 0.50 0.75 1.00
NMOR, mmol X 0.01 19.80 16.50 10.00 4.04 1.50 0.85
log (% NMOR) 2.00 1.92 1.70 1.31 0.88 0.63
%
NMOR 99.0 82.5 50.0
20.2 7.5 4.3
log (% NMOR) calcd" 2.06 1.86 1.68 1.29 0.90
0.52
OCalculated from the least-squaresline: log (% NMOR) = (2.06 0.05) - (1.54 0.09) (equiv of DMP); 9 = 0.990.
to form a nitrosating agent (9). Thus, NO in an oxygencontaining environment produces nitrosating species. These oxides of nitrogen easily partition into lipophilic media and result in nitrosation (12). A good blocking agent must effectively compete with the amine for the nitrosating agent. It must react more rapidly than the amine and not be converted to an inactive form (e.g., by protonation) under the reaction conditions. In terms of their mode of action, blocking agents can be divided into three categories: (1) Those that reduce the nitrosating agent to NO (e.g., ascorbic acid, a-tocopherol, hydroquinone, catechol, or thiols); (2) those that involve deamination of the amino group in YNH2 where Y is a carbon or another functional group and N2 or N20 is the product (e.g., urea, hydrazide, hydroxylamine, sulfamic acid, or 1,Cphenylenediamine); and (3) those compounds that covalently bind the nitrosating agent (e.g., certain phenols, anilines, or alkenes). The azide ion is an effective blocking agent (not within the classification above) which reacts to form the unstable nitrosyl azide which decomposes to Nz and NzO. Exemplary reactions of each type are given in eqs 3-5. HO.
OH ZHNO,
t
HO 08
0
wo t
HO 40x0 OH
2NO t
2H8O
(3)
0
"OH
Those blocking agents that reduce the nitrosating agent to NO have the problem of regeneration of nitrosating agents in the presence of oxygen. Nevertheless, numerous experiments have shown them to be effective under many circumstances. Mirvish has demonstrated the effectiveness of ascorbic acid in blocking the formation of nitrosamines produced from amines of several types (5). Under some conditions (low pH), ascorbic acid can catalyze nitrosamine formation through the generation of a reaction intermediate (5, 13). The ability of compounds having an NH2 group to block nitrosation depends upon its nucleophilicity. Primary amines, ammonia, hydrazine, and hydroxylamine are all strong bases, and their reactive unshared pair of electrons is reversibly protonated in acid, diminishing their effectiveness. When the NH2 group is bound to a carbonyl or sulfonyl group, such as it is in urea or sulfamic acid, basicity is significantly reduced but so is nucleophilicity (delocalization of the unshared pair), and these substances do not nitrosate as rapidly and must be used in large molar excess over the amine to be effective. Compounds that covalently bind the nitrosating agent would appear to have many attractive features as blocking agents. Many phenolic substances fall into this category, but a number of them have been found to catalyze the nitrosation of amines (14-22). For example, phenol is C-nitrosated to give 4-nitrosophenol. Tautomerism produces the oximinoquinone which can react a t the oxime group with more nitrosating agent to generate a reactive nitrosating agent. Catechol and hydroquinone phenols which possess two hydroxyl groups at the 1,2- or 1,Cpositions, respectively, are oxidized to quinones by nitrosating agents, but resorcinol (l,&hydroxyls) forms oximes and catalyzes nitrosamine formation (1S22). N-Alkylanilines form carcinogenic nitrosamines but rearrange (FischerHepp) in strong acid to the corresponding C-nitroso compounds (23). The kinetics and mechanism of this rearrangement have been extensively studied by Williams and utilized to determine the rate constants of "NO+"scavenging by several blocking agents (24-26). I t is evident that each class of blocking agent has some limitations and effectiveness is often a function of particular conditions. The discovery of new inhibitors of N-nitrosation is significant in the field of preventive toxicology.
Experimental Procedures Instrumentation. The following instrumentation and apparatus were utilized in this work melting points (uncorrected), Thomas-Hoover; UV-vis spectra, Perkin-Elmer 576 ST; IR spectra, Nicolet 20DXB FTIR. Proton and carbon NMR spectre were recorded on either a Nicolet NT (300 MHz for 'H,75 MHz for 'V),Jeol F X - W (90MHz for 'H, 22.5 MHz for '%), or V& EM-360L 60-MHz spectrometer in CDCIS,unless otherwise indicated. For TLC, EM Science aluminum plates precoated with 0.20-mm silica gel 60 F-254 or reversed-phaseWhatman MKCl$ 200-pm precoated glass plates were used. For GC, a HewlettPackard 588OA with a flame ionization or thermal energy analyzer
Chem. Res. Toxicol., Vol. 4, No. 3, 1991 375
Pyrroles as Amine Nitrosation Blocking Agents (TEA Model 502) detector with Supelco 0.25 mm X 30 m fused silica capillary columns was used. For GC-MS,a Hewlett-Packard 5890 series gas Chromatograph coupled to a HewletbPackard 5970 mass-selective detector and controlled with a Hewlett-Packard 59970 Chemstation computer was used. For high-resolution GC-MS, a K r a h MS-25 was used. For preparative-scale HPLC, two Waters Associatea Model 6OOOA p u m p controlled by a Waters Model 720 system controller with detection by a Waters Model 4.40 absorbance detector set at 254 nm were used. For analytical HPLC, a Waters chromatograph,consisting of a Waters Maxima 820 system controller, a Waters Model 490 programmable multiwavelength detector, a Waters Model 710B Wisp autosampler, and two Waters Model 510 pumps, was used. Materials. All solvents for chromatography were HPLC grade and were degassed before use. Solvents used for moisture-sensitive reactions (dimethoxyethane, diethyl ether) were distilled from sodium metal. Tetrahydrofuran used in moisture-sensitive reactions was passed through alumina, distilled from sodium metal, and redistilled from lithium aluminum hydride. Benzene was refluxed with a Dean-Stark trap and stored over molecular sieves. Common reagents from the shelves of the laboratory or the stockroom were used; other compounds were purchased from chemical distributors, unless noted below. Chemicals used were purified before use by normal procedures, if necessary. Preparation of l-Benzyl-2,5-dimethylpyrrole.The procedure described by Hazelwood and co-workers was followed (34) to give white crystals, mp 43.5-44 "C (lit. mp 48 "C). 90-MHz 'H NMR 6 7.310 (3 H, m), 7.249 (2 H, m), 5.854 (2 H, s), 5.004 (2 H, s), 2.136 (6 H, 8). 75MHz '% NMR: 6 138.48,128.64,127.91, 126.92, 125.57, 105.34, 46.64, 12.39. UV (7.744 X M in 209 nm, log t = 4.14. GC-MS m / z (re1 abunmethanol): X, dance): 186 (7), 185 (50), 184 (6), 91 (loo), 65 (13). Preparation of 1-Benzylpyrrole. The procedure of Papadopolous and co-workers (35)was followed to give an oil distilling at 130-130.5 "C/0.18 mmHg (lit. bp 276 "C/715 mmHg). 90-MHz 'H NMR: 6 7.210 - 7.088 (5 H, m), 6.626 (2 H, dd, J = 2.34, 1.56 Hz), 6.164 (2 H, dd, J = 2.34,1.57 Hz), 4.952 (2 H, 9). 22.5-MHz 13C NMR: 6 138.13, 128.50, 127.46, 126.88,120.96, 108.40,53.13. Preparation of 2$-Diphenylpyrrole. The procedure of Tedder and co-workers (36) was followed to give long white crystals, mp 143-144 "C (lit. mp 143-144 "C). 90-MHz 'H NMR: 6 8.554 (0.5 H, bs), 7.393 (10 H, m), 6.575 (2 H, d, J = 2.44 Hz). Preparation of l-Benzyl-2,5-diphenylpyrrole.The PaalKnorr procedure was used. 1,4-Diphenyl-l,4-butanedione (0.3178 g, 2.050 mmol) and benzylamine (0.2035 g, 1.900 mmol) were refluxed in 25 mL of glacial acetic acid for 1h. The solvent was removed in vacuo and the residue recrystallized from 95% ethanol to 0.2455 g (39%) of white crystals with mp 141-142 "C [lit. (37) mp 144 "C]. 90-MHz 'H NMR: 6 7.325 (14 H, m), 7.101 (2 H, m), 6.357 (2 H, s), 5.228 (2 H, s). 22.50-MHz '% NMR: 6 129.06, 128.36, 127.00, 125.97, 109.72, 48.83. Preparation of 6,6',12,12',18,18',24,24'-Octamethylporphine ('Acetonepyrrole"). The procedure of Rothemund and Gage (38)was followed to give a 95% yield of crystals, mp 298 "C dec (lit. mp 298 "C). 60-MHz 'H NMR: 6 7.0 (1H, bs), 5.92 (d, 1 H), 1.50 (3 H, s). Preparation of N-Methyl(3-pyridyl)-2-pyrrole (Nicotyrine). The procedure of Wickholm and co-workers (39) was followed to produce 21% pure 8-nicotyrine, bp 144-145 T / 1 4 "Hg (lit.bp 104-107 "C/1 "Hg). 90-MHz 'H NMR: 6 8.677 (1 H, d, J = 2.41 Hz), 8.514 (1 H, dd, J = 1.61, 4.82 Hz), 7.663 (1H, ddd, J = 8.05,2.41,1.61 Hz), 7.304 (1H, dd, J = 2.41,2.15 Hz), 6.255 (2 H, m), 3.671 (3 H, 8). 22.5-MHz '3c NMR: 6 149.18, 147.62, 135.27, 124.60, 123.04, 109.64, 108.08, 34.99. Preparation of 1,6-Diphenyl-2,5-hexynediol. The procedure of Sudweeks and Broadbent (40) for the preparation of 1,4-alkynediols was used. Ethyl bromide (65.70 g, 603.0 "01) was slowly added to magnesium metal (13.38 g, 550.5 mmol) in 200 mL of dry diethyl ether and the reaction allowed to go to completion. Dry benzene (800 mL) was then added, and acetylene (purified by bubbling through a dry ice/acetone bath, then passed through phosphorus pentoxide) was bubbled into the reaction mixture by a gas dispersion tube for 5 h. A grey precipitate formed during this time. The reaction was cooled to 0 "C and phenylacetaldehyde (67.78 g, 564.4 mmol) added dropwise over 15 min. The reaction was warmed to room temperature and stirred for 2 days. The
'
reaction was quenched with 200 mL of saturated ammonium chloride and the organic layer separated. The water layer was reextracted with ether, and the combined extracts were stripped of solvent. The residue was triturated twice with petroleum ether, leaving 64.40 g (83%) of off-white powder with mp 100-102 "C [lit. (40) mp 102-103 "C]. 90-MHz 'H NMR 6 7.322-7.173 (5 H, s), 4.545 (1H, t, J = 5.70 Hz),2.935 (2 H, d, J = 5.70 Hz), 2.055 (1 H, exchangeable 8 ) . 22.5-MHz lSC NMR 6 129.68, 128.24, 126.81, 86.10, 62.95, 43.71. Preparation of 1,6-Diphenyl-2,5-hexanediol. The procedure of Sudweeks and Broadbent for the preparation of other 1,4-&0h was used (40). 1,6-Diphenyl-2,5-hexynediol(l8.58 g, 69.85 "01) and 158 mg of prereduced platinum oxide were shaken in a Paar shaker in 250 mL of 95% ethanol under 60 psi hydrogen overnight. The solution was filtered and solvent removed in vacuo, leaving 20.67 g of residue. The residue was washed with diethyl ether, leaving 10.96 g (58%) of white crystals, mp 121.5-122 "C. Anal. Calcd for Cl8HzzO2:79.96; H, 8.20. Found: C, 79.66; H, 8.04. 90-MHz 'H NMR: 6 7.250 (5 H, s), 3.80 (1H, m), 2.724 (2 H, m), 2.304 (1H, exchangeable s), 1.672 (2 H, m). 75-MHz 13C NMR: 6 138.48,129.37,128.56,126.47,72.64,44.08,32.83. IR (Nujol mull) (cm-'): 3200 (8, br), 3052 (w), 2934 (w), 1590 (w), 1240 (m), 1184 (s), 1150 (m), 1139 (m), 1099 (w), 1076 (s), 1049 (s), 1028 (81,955 (s), 842,739,714 (s), 689 (9). GC-MS m / z (re1 abundance): 251 (0.2), 250 (0.3), 179 (16), 161 (60), 117 (57), 91 (loo), 65 (15). Preparation of 1,6-Diphenyl-2,5-hexanedione.The production of this dione by oxidation is fraught with problems, and numerous attempts were made before the one described here was found to be successful. The procedure of Giddings and Mills for oxidation of alcohols by in situ generation of ruthenium tetroxide (42) in a biphasic system was modified by conducting the reaction in a homogeneous medium. 1,6-Diphenyl-2,5-hexanediol(550 mg, 2.04 mmol), sodium bromate (300 mg, 2.05 mmol), and ruthenium tetroxide trihydrate (52.2 mg, 2.00 mmol) were stirred for 3 h in 600 mL of 70% acetonitrile/water. Additional sodium bromate (320 mg, 2.06 mmol) was added, and the solution was stirred for an additional 3 h. The reaction was extracted three times with 100 mL of methylene chloride, the combined extracts were dried over magnesium sulfate and filtered, and solvent was removed in vacuo, leaving 470 mg (87%) of white crystals, mp 61-63 "C [lit. (40) 63-64 "C]. 90-MHz 'H NMR 6 7.320-7.146 (5 H, m), 3.672 (2 H, s), 2.632 (2 H, s). 22.5-MHz '% NMR: 6 206.80,134.07, 129.54, 128.76, 127.07,49.95,35.64. GC-MS m/z (re1abundance): 267 (l),266 (3), 175 (NO),157 (14), 129 (22), 91 (99), 65 (19). Preparation of 1,2,5-Tribenzylpyrrole. The Pad-Knorr procedure was used for the preparation of this pyrrole. 1,6-Diphenyl-2,5-hexanedione (722.6 mg, 2.716 mmol) and benzylamine (298.2 mg, 2.787 mmol) were heated with stirring at 85 "C in 1.0 mL of glacial acetic acid for 3 h. The reaction was cooled and poured onto saturated sodium bicarbonate. The solution was extracted three times with diethyl ether, the combined extracts were dried over magnesium sulfate and filtered, and solvent was removed in vacuo. Flash chromatographyon silica gel gave 805.6 mg (88%)of a yellow viscous liquid which slowly crystallized. Recrystallization from diethyl ether gave 684.5 mg (75%) of white crystals with mp 69-70 "C. 90-MHz 'H NMR 6 7.307-6.736 (15 H, m), 5.883 (2 H, s), 4.799 (2 H, s), 3.789 (4 H, s). 75-MHz 13C NMR 6 139.46, 138.41, 131.58, 128.59, 128.47, 128.30, 127.98, 126.94, 126.08, 125.50,107.18,46.88,33.14. IR (KBr pellet) (cm-'1: 308 (w), 306 (w), 3027 (m), 2921 (w), 2851 (w), 1603 (w), 1493 (s), 1452 (51,1428(m), 1361 (w), 1304 (w), 1261 (w), 1073 (w), 1028 (w), 1016 (w), 750 (s), 728 (4, 695 (8). UV (6.04 X M in methanol): X, 212 nm, log c = 4.44; 240 nm (shoulder), log c = 4.04. High-resolution GC-MS: parent ion = 337.183, calcd 337.466 for C=HBN. GC-MS m/z (re1abundance): 338 (111,337 (66), 260 ( l l ) , 246 (42), 168 (ll),167 (14), 154 ( l l ) , 91 (loo), 65 (12). Nitrous Acid Consumption of Pyrroles. General Procedure. 2,6-Dimethyl-N-nitrosomorpholine (DMNM; internal standard), a variable amount of pyrrole, and 3.0 mL of glacial acetic acid were stirred for 5 min in a 5-mL reaction vial jacketed with water at 37 "C from a Haake Model A80 constant-temperature bath. Sodium nitrite solution (2.0 M, 100 pL, 2 x lo-' mol) was injected into the solution and the reaction stirred for 30 min. The reaction was poured onto saturated sodium carbonate mlution and extracted three times with 5 mL of methylene chloride. The
376 Chem. Res. Toxicol., Val. 4, No.3, 1991 combined extracts were filtered through magnesium sulfate and Whatman No. 1fiiter paper and diluted to 25 mL. The solution was analyzed by GC for pyrrole on an SPB-20 column. The results are shown in Table I. Assay of Nitrosation Blocking Abilities. (A) General Procedure. To 3.0 mL of glacial acetic acid were added 2.00 M aqueous morpholine solution (1.0 mL, 2.0 mmol) and 2,6-dimethyl-N-nitrosomorpholine(DMNM; as internal standard) in a 5-mL reaction vial jacketed with 37 OC water from a Haake Model A80 constant-temperature bath. The desired amount of blocking agent was added (0-1.5 equiv of sodium nitrite) with stirring. Solid compounds were added directly, and liquid compounds were washed into the reaction vial with the acetic acid solvent. After 5 min 2.00 M sodium nitrite solution (100 pL, 0.200 "01) was added. The mixture was stirred for 30 min and poured onto saturated potassium carbonate solution. This solution was extracted three times with 5 mL of methylene chloride. The combined extracts were dried over magnesium sulfate, filtered, and analyzed by gas chromatography for nitrosomorpholine (NMOR) and DMNM. GC analyses were performed on an SPB-20 column at 70 OC for 1min, then to 250 OC at 15 OC/min, or a similar program. Data are given in Table I1 for DMP and are typical. The data for each compound [log (% NMOR) vs nitrite equivalents of blocking agent] were subjected to linear regression, and the slopes are given in Table IV. The intercepts are 2. (B) Blocking Ability of l-Benzyl-2,5-dimethylpyrrole (BDMP). The ability of BDMP to block amine nitrosation was examined by the general procedure. These experiments were done with N-nitrosopiperidine as internal standard. The workup was as described under General Procedure with the exception that the reaction was poured onto an equimolar amount of 2,5-dimethylmorpholine prior to neutralization. No DMNM was detected in any of these experiments, which demonstrates the lack of artifactual formation of NMOR. "hatment of the data yielded the equation: log ('70 NMOR) = (1.97 f 0.04) - (1.52 f 0.05)(equiv of BDMP) r2 = 0.993
(C) Blocking Ability of 2,5-Dimethylpyrrole (DMP) in Buffer. The ability of DMP to block amine nitrosation was examined by the general procedure with the following changes. The assay was conducted in acetate buffer at pH 3.76 prepared by adjusting the pH of 60% aqueous acetic acid with sodium acetate. Morpholine was used neat, not as an aqueous solution. Treatment of the data gave the equation: log (% NMOR) = (2.1 f 0.1) - (1.7 f O.2)(equiv of DMP) (D) Effect of Concentration on the Blocking Ability of BDMP To Block Nitrosation. The general procedure is exemplified by the following: Morpholine solution (2.00 M, 1.00 mL, 2.00 mmol), DMNM solution (0.521 M in HOAc, 100 pL, 0.0521 mmol), and BDMP (11.17 mg, 0.06033 mmol) were stirred in 3.0 mL of glacial acetic acid for 5 min in a sealed reaction vial jacketed with water at 37 OC as in the preceding blocking experiments. Sodium nitrite solution (2.00 M, 100 pL, 2.00 X lo4 mol) was injected into the mixture and the reaction stirred for 30 min. The reaction was quenched and worked up as in the blocking experiments. GC analysis was performed on an SPB-5 column with a temperature program of 80 "C for 2 min, then to 250 OC at 15 OC/min. The retention times of NMOR and DMNM were 6.33 and 7.30 min, respectively. The amount of NMOR produced as a function of concentration of BDMP with BDMP:HN02 = 0.30 is shown in Figure 3. Characterization of BDMP Black. l-Benzyl-2,5-dimethylpyrrole (2.648 g, 1.430 X mol) was dissolved in 70 mL of glacial acetic acid and 3.0 M sodium nitrite (4.75 mL, 1.42 x lo-* mol). The reaction was stirred for 30 min at room temperature, then poured into 250 mL of water, and extracted three times with 50 mL of benzene. The combined extracts were washed with several portions of water and brine solution and dried over magnesium sulfate. The solvent was removed in vacuo, leaving 1.7218 g of black amorphous material. 'H NMR (major peaks, 90 MHz in methanol-d,): 6 7.2 (s, br) and 6.8 (s, br) (47 H), 4.904 (30 H, s),3.13 (2 H, s br), 2.5-1.65 (47 H, many peaks). IR (major bands,KBr pellet) (a&): 3054,3025,2917,1710,1630,1602,1580,
Wilcox et al. 1522,1486,1443,1414,1350,1233,1060,1024,973,737,698. The molecular weight of BDMP black was estimated by the freezing point depression of naphthalene following the experiment described by Roberts, Hollenburg, and Postma (43). The molecular weight of BDMP black is 360 f 40. UV-vis spectrum: A BDMP black solution (5.77 X M using 362 as molecular weight) showed absorptions at 205 and 210 nm of 0.35 and 0.705 absorbance unit, respectively. The path length was 1cm. The visible portion of the spectrum of a 7.21 X lo-' M solution showed an absorption at 350 nm of 0.76 absorption unit which slowly decayed to base line at approximately 710 nm. The magnetic susceptibility of BDMP black was determined by the NMR method of Evans (44) with dioxane as internal standard. 'H NMR spectra were recorded at 90 MHz on a stock solution of BDMP black in acetoned6 (2.14 X 1C2g/mL). No paramagnetic shifts were observed. Separation of BDMP Nitrosation Products. BDMP (0.9269 g, 5.01 x mol) was dissolved in 25 mL of glacial acetic acid at room temperature and sodium nitrite solution (2.0 M, 2.5 mL, 5.0 X mol) added dropwise over 3 min. The reaction was stirred for 30 min and then poured into 250 mL of water. An aliquot was neutralized with sodium carbonate and filtered through an Anotop 2-pm filter. The reaction mixture was extracted seven times with 50 mL of benzene. The combined benzene extracts (fraction AB) were extracted with 5 X 50-mL portions of 5% NaOH. The remaining benzene solution (fraction B) was dried over Na2S04and stripped of solvent to give 447 mg of material. The basic extract from AB was neutralized and extracted with 5 X 50-mL portions of ether which were combined, dried over Na$04, and stripped of solvent to give fraction A, w t 55 mg. The original aqueous extract was neutralized and extracted with 5 X 50-mL portions of ether. The combined ether extracts were dried over Na2S04and stripped of solvent to yield 56 mg of material (fraction C). A black amorphous material left after extractions was washed from the glassware with acetone and called fraction D (478 mg). HPLC analysis (reversed phase) of fractions A 4 showed each fraction to contain a number of components as did TLC. None of the TLC spots were positive to the Griess reagent. Fractions A-C were submitted to GC-MS analysis on a DB-5 column with temperature program 80-250 "C at 10 OC/min. The program was held at 80 O C for 2 min before the temperature was increased for sample B. In addition to solvent impurities and several unknowns, fractions A-C revealed the presence of 3-acetyl-l-benzyl-2,5-dimethylpyrrole [retention time 14.76min; MS m/z (re1abundance), 212 (36), 184 (9), 136 (5), 120 (2), 91 (loo), 65 (17), 51 (411 (structural assignment tentative, based on MS fragmentation) and the starting material (retention time 9.26 min). GS-MS analysis of fraction B showed the presence of benzyl alcohol (MS confirmed with library spectrum) among other substances. Similarly, fraction C was found to contain N-benzylidenebenzylamine [retention time 11.9 min; MS m/z (re1abundance), 194 (%), 117 (9),91 (100),89(lo),65 (201, which was in agreement with an authentic sample, and benzylamine (retention time 3.1 min) and N-benzylacetamide [retention time 8.2 min; MS m/z (re1 abundance), 107 (18), 106 (loo), 91 (40), 79 (23), 77 (21), 52 (8), 51 (19)],both of which were in agreement with library MS.
Results and Discussion Pyrroles as Blocking Agents. Gramine, which along with dimethylamine is t h e primary source of dimethylnitrosamine in malted beverages (271, contains a pyrrole ring fused to a benzene ring (indole system). We have investigated t h e mechanism of dimethylnitrosamine formation from a gramine model compound, 2-[(dimethylamino)methyl]pyrrole (28). This tertiary amine, like gramine, is unusually reactive toward nitrosation and reacts as shown in eq 6. The reaction proceeds by two competing pathways, one of which involves C-nitrosation of t h e pyrrole ring. Among other evidence this pathway was suggested by the isolation of maleimide oxime. Numerous other products are produced from the nitrosation of t h e pyrrole moiety but are unstable to either t h e reaction conditions or isolation.
Pyrroles as Amine Nitrosation Blocking Agents n
Chem. Res. Toxicol., Vol. 4, No. 3, 1991 377 100
CHs
(CH,),NNO
t
' p ro ot hd ue cr t s
0H O,&
(e)
H
These observations as well as those of Groenen (29) suggested that pyrroles may be effective in blocking amine nitrosation. Groenen determined that pyrrole was a natural constituent of tobacco which inhibited the nitrosation of tobacco alkaloids. Groenen reported in a conference abstract (and no subsequent publication to our knowledge) that at pH 3 pyrrole can completely block the nitrosation of morpholine. The ability of pyrrole to block nitrosation was compared with that of other nitrous acid scavengers, and the following order of ability was found pyrrole 1 4-methylcatechol > guaiacol > ascorbic acid > indole > isoamylmercaptan > phenol 1 ammonium chloride. Relatively little information is available in the literature on the nitrous acid chemistry of pyrroles (30). Pyrrole itself reacts with nitrous acid to produce a dark amorphous highly complex mixture called "nitrosopyrrole black", used as a dye in the early years of chemistry. The mono- and dioximes of maleimide have been isolated from it but little else (31). These products as well as subsequent studies on substituted pyrroles demonstrated that the compounds are readily C-nitrosated. No N-nitroso derivatives of pyrrole or its substituted derivatives are known (30). If reaction a t the nitrogen occurs, it is followed by rapid rearrangement to a ring carbon. In some cases nitrosation with a large excess of nitrous acid leads to diazo compound formation (30). Development of Assay. Nitrosamine contamination in commercial substances is usually encountered under circumstances where the nitrosamine is present as a small fraction of the amine from which it was derived. Under usual circumstances the nitrosamine is formed from small quantities of adventitious nitrosating agent and a large excess of amine or other nitrosatable compound. In order to approach these conditions and provide an experimental situation that facilitates a comparison of the ability of various nitrous acid scavengers to block amine nitrosation, a 10-fold excess of morpholine was reacted with nitrite in glacial acetic acid at 37 "C for 30 min. These conditions affected quantitative production of NMOR based on added nitrite. Initial experiments with pyrrole and its substituted derivatives demonstrate that these compounds are effective in blocking the nitrmtion of morpholine. Figure 1A shows how the percent NMOR produced is decreased by increasing nitrite equivalents of l-benzyl-2,5dimethylpyrrole (BDMP). In order to develop the best assay conditions, and because the reaction of pyrroles is complex, we examined the stochiometries of the reactions of pyrroles and nitrite. Assuming that pyrroles react with nitrite at a fixed stoichiometric ratio (alb), as illustrated in eq 7, the number of moles of pyrrole left after the reaction has gone to completion (Py,) is expressed in eq 8. The subscript i aN02- + bPy products (7)
-
mol of Py, = mol of Py, - (b/a)(mol of N02-)i (8) designates the initial values for pyrrole (Py) and NO2-. Equation 9 is obtained by dividing both sides of eq 8 by moles of Pyi and subsequent rearrangement Examination [(b)(mol of N02-)i]/[(a)(molof PyJ] = 1 - (mol of Py,)/(mol of Pyi) (9)
bp
u
0.0 0.0
40
0.2
0,4
0.6
0.1
1.0
Equivalents o f EDMP
30
0 0.0
0.2
0.4
0.6
0.8
I I
1.2
1.0
Equivalents BDMP
Figure 1. (A) Percent NMOR, based on NaN02, formed from the nitrosation of 2 mmol of morpholine with 0.2 mmol NaN02 in acetic acid in the presence of 0.0-1.5 nitrite equiv of the BDMP is plotted against equivalents of BDMP. (B) The same data are presented in logarithmic form, demonstrating the linear relationship from which -(slope) gives a measure of nitrosation blocking ability.
IZ0 100
i
I 601
I
20 0.0
I
I
05
10
I
I
1.5
2.0
I
25
30
Equivalents of NBP
Figure 2. Percent NBP reacted as a function of sodium nitrite (0.2 mmol) initial equivalents of NBP in acetic acid after 30 min. The slope of the linear portion gives the stoichiometricratio a / b . Table 111. Stoichiometries of Reactions of Pyrroles and Nitrite
pyrrole NBD BDMP PYR DMP a
aibo 2.2 0.2 1.35 f 0.02 0.8 0.1 0.433 0.002
* *
Nitrite per pyrrole.
of the right-hand side of eq 9 reveals that this is the ratio of the amount of pyrrole reacted Py, = (Py, - Py,) to the initial amount of pyrrole. Determination of the ratio @/a) is therefore possible by plotting the percent pyrrole reacted versus the initial molar ratio of nitrite to pyrrole, as shown in eq 10. The percent pyrrole reacted as a function of (100)(b/a)(mol of N02-)i/(mol of PyJ = % Py,
(10)
initial nitrite equivalents for several pyrroles was determined. The results for NBP are shown in Figure 2. The ratios b / a of the number of moles of pyrrole that react with 1mol of nitrite are determined from the slopes of the lines in the linear region. These values are given in Table 111.
Wilcox et al.
378 Chem. Res. Toxicol., Vol. 4, No. 3, 1991
NMOR Formation. Comparison of Various Compounds compound abbrev slopea errb equiV 2,5-dimethylpyrrole DMP 1.54 0.09 0.65 l-benzyl-2,5-dimethylpyrrole BDMP 1.52 0.05 0.66 MCAT 1.10 0.2 0.91 4-methylcatechol 1,4-phenylenediamine PD 1.00 0.2 1.00 ASC 1.00 0.2 1.00 ascorbic acid PYR 0.94 0.06 1.06 pyrrole 1,2,5-tribenzylpyrrole TBP 0.76 0.02 1.32 NBP 1-benzylpyrrole 0.75 0.08 1.33 6,6',12,12',18,18',24,24'-octamethyl- OMP 0.71 0.05 1.41 Table IV. Blocking
0
1
30
25 20
L
0
1
2
3
4
5
6
7
[BDMP] ( M x 0.01)
Figure 3. Ability of BDMP to block the formation of NMOR from morpholine (2 "01) is seen to decrease as the concentration of BDMP is increased with a fixed ratio of [NO,] of 0.3.
The values of Table I11 demonstrate the complexity of the reaction of pyrroles with nitrite. That the values are not integers implies the existence of two or more competing pathways in the reactions. Initial products of the reaction may react further with nitrite or react with other pyrrole molecules to dimeric intermediates. I t is interesting to note that >1 mol of nitrite is consumed by 1 mol of pyrrole for the N-substituted pyrroles whereas
