50
Chem. Res. Toxicol. 1993,6, 5G58
Caffeine-Derived N-Nitroso Compounds. 11. Synthesis and Characterization of Nitrosation Products from Caffeidine and Caffeidine Acid Rajiv Kumar,? Claus-D. Wacker,j Peter Mende,l Bertold Spiegelhalder,i Rudolf Preussmann,* and Maqsood Siddiqi’pt Department of Pharmaceutical Sciences and Department of Biochemistry, University of Kashmir, Srinagar, J&K, India, and Department of Environmental Carcinogens, German Cancer Research Centre, Heidelberg, Germany Received July 10, 1992
Caffeine on alkaline hydrolysis produces caffeidine [l-methyl-4-(methylamino)-5-(N-methylcarbamoyl)imidazole] and caffeidine acid [ N -[4-(5-carboxy-l-methylimidazolyl)]-N,”-dimethylurea]. We now report the synthesis and chemical characterization of mononitrosocaffeidine [l-methyl-4-(N-methyl-N-nitrosoamino)-5-(N-methylcarbamoyl)imidazole] ,dinitroso, caffeidine [1-methyl-4(N-methyl-N-nitrosoaino)-5-(N-methyl-N-nitrosocarbamoyl)imidazole] and mononitrosamidocaffeidine [l-methyl-4-(methyl~mino)-5-(N-methyl-N-nitrosocarbamoyl)imidazole] based on spectral analysis. The characterization of nitrosated byproducts obtained during the synthesis of these compounds is also presented. Caffeidine is shown to undergo rapid nitrosation in acidic medium to form mononitrosocaffeidine (MNC), an asymmetric N-nitrosamine, and dinitrosocaffeidine (DNC), a N-nitrosamide. Although the reaction proceeds with preferential nitrosation of the amino group in caffeidine, the results also support partial involvement of a mononitrosamide intermediate in the formation of MNC and DNC through transnitrosation of the amino group. The stability data suggest that the nitroso group a t the amino nitrogen in DNC influences the reactivity of amide nitroso group. The presence of a trisubstituted ureide moiety in caffeidine acid has been confirmed by NMR nuclear Overhauser effect experiments. Nitrosation of caffeidine acid under acidic conditions produced NNdimethylparabanic acid (DMPA, NN-dimethylimidazolidinetrione) as a major product with low amounts of mononitrosocaffeidine and NJV’-dimethyl-N-nitrosourea, whereas nitrosation with NOBFl/pyridine in aprotic medium gave rise to an anhydride, 1,4-dimethyl-4,5-dihydro5,7-dioxo-1H,7H-imidazo[4,5-d1[1,31oxazine. The nitrosation of methyl ester of caffeidine acid resulted in the formation of a N-nitrosourea derivative, N-[4-(5-carboxy-l-methylimidazolyl)]N’-nitroso-N,”-dimethylurea. On the basis of these results a scheme of reactions has been proposed where nitrosation of ureide moiety in caffeidine acid is postulated to initiate a cascade of reactions, involving imidazole ring opening and recyclization, leading to the formation of DMPA. Intr5ition Caffeine (1) is known to be hydrolyzed to caffeidine (4) and caffeidine acid (2) in alkaline conditions (I, 2). However, the possibility of human exposure to these or related compounds and their potential for endogenous nitrosation have not been taken into consideration while assessing human risk from environmentalN-nitroso compounds (3, 4 ) . The commonly consumed salted tea in Kashmir, a highincidence area for esophageal and gastric cancers (51, is prepared by boiling green tea leaves in the presence of sodium bicarbonate and NaCl (6). In a recent study we have demonstrated high levels of methylating activity on in vitro nitrosation of the salted tea (7). Experiments for identifying precursors for the observed activity in the beverage showed that the main constituent of tea leaves, caffeine (11, when treated under conditions of tea preparation gave rise to caffeidine (4) and caffeidine acid (2)
Caffeidine (4), an imidazolederivativewith methylamino and N-(methylcarbamoyl) substituents, is a weakly basic compound which readily undergoes nitrosation in acidic conditions, producing mononitrosocaffeidine (MNC,’ 5) and dinitrosocaffeidine(DNC, 6) (7). On the other hand, caffeidine acid (2), an imidazole derivative with a trisubstituted urea moiety, reacts with nitrite in acidic medium, forming a symmetrical cyclic triketo compound, NJPdimethylparabanic acid (DMPA, 131, NJP-dimethyl& nitrosourea (DMNU, 14), and MNC (5) (7). In the present study we report (i) synthesis and spectral characterization of nitrosation products of caffeidine (4) and caffeidine acid (21, (ii) evidence for the structure of caffeidine acid (2) by NMR NOE experiments, (iii) the stability o€ various nitroso derivatives of caffeidine (4) and caffeidine acid (2) at different pHs, and (iv)the effects of pH and nitrite variation on the formation of MNC (6) from caffeidine (4) and DMPA (13) from caffeidine acid
(7). To whom correspondence should be addressed at the Department of Environmental Carcinogens, German Cancer Research Centre, Im Neuenheimer Feld 280, Heidelberg, Germany. + University of Kashmir. 1 German Cancer Research Centre.
.~
.,
Abbreviations: MNC, mononitrosocaffeidine; DNC, dinitrosocaffeidine; MNDNC, mononitrodinitrosocaffeidine; MNAC, mononitrmamidocaffeidine; DMPA, dimethylparabanic acid; DMNU, dimethylnitrosourea; t-BOC, (tert-buty1oxy)carbonyl;NOE, nuclear Overhauser effect; TLC, thin-layer chromatography.
0 1993 American,Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 51
Caffeine-Derived N-Nitroso Compounds
Table I. Chemical Shifts in 'HNMR of Caffeidine and Its Nitrosation Products compound no.
aromatic CH
group (ppmimultiplicity) amino aminoNCH3 NH
ring CH3
7.03 (q,'J = 0.6 Hz) 3.70 (d, 'J = 0.6 Hz) 3.9513.88" (2 d, 4J= 0.6 Hz) 7.50/nd0 (q,'J = 0.5 Hz) 7.6317.56' (2 q, 4J = 0.4 Hz) 3.9513.900 (2 d, 'J = 0.4 Hz) 4.20 (a) 7.34 (br 3.64 (br 7.46 (q,'J = 0.5 Hz) 3.87 (d, 'J = 0.5 Hz) 4.18 (a) 4.19 (a) 3.56 (d, 'J = 0.6 Hz) 7.31 (q,'J = 0.6 Hz)
4 5
6 7 8 9 10 11 12
amido NH
amido NCH3
2.75 (br a) 4.05 (br a) 2.77 (d, 3J = 4.8 Hz) 3.5014.16" (2 a) 2.8712.64°(2 d, 35 = 4.9 Hz) 3.3414.15"(2 a) 3.1313.20" (2 a) 3.3814.19" (2 a) 3.1813.26"(2 a) 2.90 (d, 35 = 4.9 Hz) 3.20 (a) 2.93 (d, 3J = 4.8 Hz) 3.25 (br 8) 3.06 (d, 3J = 5.92 (br a) 3.25 (a) 4.9 Hz)
urethane NCH3
BOCCH,
3.20 (a) 3.20 (a) 3.22 (8) 3.21 (a)
1.49 (a) 1.35 (8) 1.51 ( 8 ) 1.36 ( 8 )
6.87 (br 8 ) 7.34 (br 8 ) 7.45 (bra) 8.03 (bra)
Major/minor isomer due to E / Z isomerism of the NNO group; nd = not detected. 4J< 0.4 Hz. Table 11. Chemical Shifts in '3c NMR of Caffeidine and Its Nitrosation Products aromatic aromatic c-4 C-5
group (ppm) amino amido ringCH3 NCH3 C=Oa
amido urethane BOC BOC BOC NCH3 NCH3 C=Oa CO CH3 aromatic C-2 4 136.99 (CH) 151.89 109.74 34.36 31.91 162.02 25.30 5 137.95 (CH) 140.33 118.99 35.04 33.33 159.68 26.23 6 139.27/138.40 (CH)b 145.71 114.92 34.26/38.1g6 31.16 164.13 26.30/26.06b 7 143.40 (CN02) 141.02 117.60 36.12/38.04* 30.40/36.23b 163.50 26.30/26.11b 8 137.25 (CHI 140.88 120.05 36.90 160.57 25.77 34.10 155.87 81.59 28.00 9 138.26 (CH) 145.80 115.04 34.19 164.45 26.17 34.66 153.54 80.85 27.97 10 137.83 (CN02) 142.85 125.96 37.12 158.70 26.31 35.87 156.32 82.96 28.08 11 ndc (CN02) nd nd 35.88 nd 26.07 34.49 nd 82.18 27.88 12 143.47 (CH) 160.67 106.25 35.43 29.33 161.46 27.76 a In case of two carbonyl functions in the molecule, the higher 6 values were assigned to the carboxamide group according to reference data in the literature; experimentally not checked. Major/minor isomer due to EIZ isomerism of the NNO group. c nd = not detected due to low amount. compound no.
Table 111. Chemical Shifts in 'H NMR of Caffeidine Acid and Its Nitrosation Products group (ppm/multiplicity) compound no. aromatic CH ring CH3 urea NCH3 urea NHCH3 urea NH amido NCH3 COOH 2 8.46 (br s ) O 3.87 (br s ) 4 2.75 (s) 2.54 ( 8 ) 6.25 (br s) 12.08 (br s) 13 14 16 17 18 0
45 C
7.61 (q, 4 5 = 0.6 Hz) 3.95 (d, 4J= 0.6 Hz) 7.49 (q, 4 5 = 0.6 Hz) 3.82 (d, 4J= 0.6 Hz) 7.42 (q,4J = 0.6 Hz) 3.82 (d, 4J= 0.6 Hz)
3.18 ( 8 ) 2.80 ( 8 )
2.78 (s)
3.21 (e) 3.10 (s)
2.76 (d, 3J= 4.6 Hz) 5.22 (br s) 3.4gb(s)
______
OCH3
4.34 (br s) 3.55 (8) 3.88 (s) 3.82 ( 8 )
0.4 Hz. N(NO)CH3.
Table IV. Chemical Shifts in
compound no. 2 13 16 17 18
NMR of Caffeidine Acid and I t s Nitrosation Products
aromatic C-2 (CHI
aromatic C-4
aromatic C-5
ring CH3
138.06
142.28
117.11
35.52
143.52 138.99 139.61
149.11 147.55 148.46
104.13 114.96 114.00
33.58 35.24 35.27
group (ppm) urea urea amido NCH3 NHCH3 NCH3 35.90 24.77
27.05 30.38
NC=Oa
OC=On
156.73 153.97 150.85 157.38 155.24
159.62
-
O=CC=Oa
OCH3
156.83 152.84 159.74 159.21
36.57 27.20 51.70 28.80 38.27b 51.74 In case of two carbonyl functions in the molecule, the lower 6 values were assigned to the NC=O group according to reference data in
the literature; experimentally not checked, except for compound 17. N(NO)CH3.
(2) and propose (v) a mechanism for the formation of DMPA (13) from caffeidine acid (2).
Experimental Section (1) General. Caffeine was purchased from Aldrich-Chemie
(Steinheim, Germany). Caffeidine nitrate was a generous gift from Boehringer Ingelheim KG (Ingelheim, Germany). Nitrosyl tetrafluoroborate and di-tert-butyl dicarbonate were from Merck (Darmstadt, Germany). All other reagents were of analytical grade and used without further purification. Diazomethane was prepared from N-methyl-N-nitrosourea. lH NMR and l3C NMR were recorded in CDCl3 on Bruker AC 250 spectrometer a t 250 and 62.89 MHz (bb-decoupled, DEPT-90, DEPT-135) and are reported in parts per million (ppm) downfield from internal Me4Si. Mass spectra were taken on Finnigan MAT 711, EI, 70 eV, HR, in combination with Finnigan MAT 95, CI-positive (meth-
ane), 150 eV, ion source temperature 150 OC. IR spectra were recorded on Perkin Elmer 580B. HPLC was carried out on a Hewlett Packard 1090 liquid chromatograph interfaced with HP Chem Station with a diode-array detector. Spectrophotometric measurements were made on Perkin-Elmer Lambda 5 model UV/ vis spectrophotometer. Melting points were determined in open capillaries and are uncorrected. lH and 13CNMR chemical shifts for caffeidine and its nitrosation products are given in Tables I and 11, and for caffeidine acid and its nitrosation products in Tables I11 and IV. (2) S t u d y on Caffeidine. (A) Synthesis of Caffeidine (4). Caffeidine (4) was synthesized by hydrolysis of caffeine (1) according to previously described procedure (8)and purified by column chromatography on silica gel using CH2ClZ-MeOH as solvent. I t was crystallized from diisopropyl ether-CHzC12 (yield 28%): mp 84-85 "C [lit. mp 83-85 "C @)I; MS m/z (relative
52 Chem. Res. Tonicol., Vol. 6, No. 1, 1993 intensity) 168 (M+,loo), 153 (M+- CH3,0.3), 138 (M+- NHCH3, 25.1);IR (KBr) 3380, 1610, 1575, 1535 cm-'. (B) Nitrosation of Caffeidine (4) under Acidic Conditions. An aqueous solution of caffeidine (4)(0.33g, 2mmol) was adjusted to pH 2with HCl and nitrosated by slow addition of NaNOz (0.55 g, 8 mmol) with stirring and cooling. The pH of the reaction was checked and maintained a t 2.0 during the incubation (1 h) a t room temperature. Nitrosation was terminated by addition of ammonium sulfamate (1 9). The reaction mixture was extracted with CHzCl2(3X 30 mL), washed with sodium chloride-saturated water, and dried over sodium sulfate. On concentration, the CHzCl2yielded crystalline mononitrosocaffeidine (MNC, 5),(0.34 g, 87.8%): mp 152-153 "C; MS m/z (relative intensity) 167 (M+ - NO, 66.7),139 (M+-CHz=NNO, 34.3),42 (NN=O+, 100);IR (KBr) 3300, 1650, 1590, 1555, 1500 cm-l. Anal. Calcd for C7HllN502: C, 42.62;H,5.62;N, 35.52. Found: C,42.68;H, 5.52; N, 35.42. The CHzClz extract from the above reaction showed a minor product in TLC in addition to MNC (5). The extract was analyzed on HPLC (Hypersil ODS 5-pm column; water-methanol (3070) as mobile phase run isocratically for 10 min followed by a linear gradient reaching 70:30 in the next 10 min and read a t 240 nm). Compound 5 eluted a t 4.2 min, and the minor product, whose isolation could not be achieved due to ita low yield, a t 15.3 min. However, it was later identified as dinitrosocaffeidine (6)and was synthesized from 5 (see below). Using purified synthetic compound as reference, the yield of 6 in the above reaction was found to be 0.2% . (C) Nitrosation of MNC (5) with Nitrosyl Tetrafluoroborate. Compound 5 (2.4g, 12 mmol) was added to a stirring suspension of NOBF4 (2g, 17 mmol) in CH3CN (30mL) a t 0 "C. Pyridine (1.3g, 17 mmol) in 5 mL of CH3CN was added dropwise to the reaction mixture. The reaction was continued for 1 h; solvent was removed under vacuum and the yellowish residue dissolved in CHzCl2(10mL). The whitish precipitate of pyridine salt was filtered, and the TLC of filtrate showed the presence of a major and a minor product. The mixture was resolved on a silica gel column where the minor product, mononitrodinitrosocaffeidine (MNDNC, 7), eluted first with CHzClz and was crystallized from diisopropyl ether-CHzClz (yield 0.2 g, 7.2% 1: mp 150 "C; MS m/z (relative intensity) 271 (M+,0.3),255 (M+ - 0 from NOz,O.l), 241 (M+- NO, loo),213 (M+- CHz=NNO, 17.5);IR (KBr) 1690,1545,1525, 1510 cm-l. Anal. Calcd for C7H9N,05: C 31.0;H, 3.35;N, 36.0. Found: C, 31.18;H,3.27;N, 35.90. Pure dinitrosocaffeidine (6) was obtained on further elution with CHzClz-diethylether and crystallized from diisopropyl ether (yield 2.1 g, 77.3%): mp 91 "C; MS m/z (relative intensity) 226 (M+,0.45),196 (M+ - NO, 1001,168(M+ - C H p N N O , 9.3);IR (KBr) 1690,1570,1510,1470 cm-I. Anal. Calcd for C7H10N603: C, 37.16;H, 4.42;N, 37.16. Found: C, 37.55;H, 4.63;N, 36.96. (D)Synthesis of t-BOC-caffeidine (8). Di-tert-butyl dicarbonate (10.4g, 60 mmol) in 15 mL of dioxane was added to a solution of caffeidine (4)(4g, 30 mmol) in water (15mL) and dioxane (15mL). The pH was adjusted to 9.0 with NaOH and the reaction continued for 48 h with continuous stirring a t room temperature. At the end of reaction, this solution was concentrated in vacuo and extracted with CHzClz (30 X 3 mL). The extract was dried over sodium sulfate and concentrated to 10 mL. On elution with CHZClz-MeOH (9:l)on silica gel, a pure t-BOC derivative of caffeidine (8)was obtained (yield 6g, 94.0% ). It was crystallized from diisopropyl ether-hexane a t -22 "C: MS m/z (relative intensity) 268 (M+,60.3),212 (M+- C4H8,7.9), 195 (M+- OC4Hg133.6),168 (M+- C4Hs- COz, 100);IR (KBr) 3240, C, 1710,1650,1570,1510 cm-I. Anal. Calcd for CIZHZON~O~: 53.72;H, 7.51;N, 20.88. Found: C, 53.90;H, 7.55;N, 20.88. (E) Nitrosation of t-BOC-caffeidine (8) with NOBFl and Pyridine. t-BOC-caffeidine(8) (4g, 14.9mmol) was slowly added to the suspension of NOBF4 (2g, 17 mmol) in CHsCN (20 mL) a t -30 "C. Pyridine (1.35 g, 17 mmol) in CH3CN was added dropwsie to the reaction mixture and stirred for 1 h. The solvent was evaporated in vacuo and the semisolid residue dissolved in CHZC12 (10 mL). The precipitated salt of pyridine was removed
Kumar et al. by filtration. The filtrate on TLC examination showed the presence of three major producta which were separated by silica gel chromatography. Eluting first with CH2C12, provided the C-nitro-N-nitroso derivative of t-BOC-caffeidine (1 1) which was crystallized from diisopropyl ether-hexane (yield 0.05 g, 1.0% ): mp 87-88 "C; MS m/z (relative intensity) 342 (M+, 23.4),314 (M+- 28,11.4),286 (M+- ChH8, 8.5),258 (M+- C4He - COz, 9.51, 241 (M+- C4HgOCO,23.0),227 (M+-C4Hs-CH3NN=O, 89.6); IR (KBr) 1710,1540,1500cm-l. Anal. Calcd for C&&&: C, 42.11;H,5.30;N, 24.55. Found: C, 42.31;H, 5.40; N, 24.15. Further elution of the column with CHzC12-MeOH provided a mixture of two compounds, which were sequentially separated by CHzClz-diethyl ether on silica gel, as the C-nitro derivative of t-BOC-caffeidine(10) (yield0.2g, 4.2% ) and mononitrosamidct-BOC-caffeidine (9)(yield 2g, 45.1% ). Bothof these compounds were crystallized from diisopropyl ether-CHzClz. Compound 10: mp 145-147 "C; MS m/z (relative intensity) 313 (M+, 41), 240 (M+ - OC4H9,51.6), 213 (M+ - C4Hs - COz, 100);IR (KBr) 1710, 1670,1540,1495 cm-l. Anal. Calcd for C1zHlsN605: C, 46.0; H, 6.11;N, 22.35. Found: C, 45.92;H, 6.12;N, 21.90. Mononitrosamido-t-BOC-caffeidine(9):mp 75-77 OC; MS m/z (relative intensity) 297 (M+, 17.6),268 (M+ - 29,4.7),241 (M+ - C4Hs,6.9),182 (M+- C4H8 - CH3NN=O, 100);IR (KBr) 1710, 1690,1550, 1510,1490 cm-I. Anal. Calcd for ClzH19Nb04: C, 48.48;H, 6.44;N, 23.56. Found: C, 48.65;H, 6.46;N, 23.46.
(F) Synthesis of Mononitrosamido Derivative of Caffeidine (12) from 9. The mononitrosamido-t-BOC-caffeidine (9) (2g, 6.7 mmol) was stirred a t 0 "C in 15 mL of HC1-formic acid for 40 min. The product was extracted with CHzClZ (10X 3 mL), dried over sodium sulfate, and concentrated under nitrogen. In TLC, the CHC12 extract showed the presenceof mononitrosamido caffeidine (MNAC, 12) along with compounds 5 and 6 and traces of 4. However, pure 12 was obtained by repeated crystallization from diisopropyl ether-CHzCl2 (yield 50 mg, 3.7% ): mp 106-107 "C; MS m/z (relative intensity) 197 (M+, 23.1),167 (M+ - NO, 72.3),139(Mt-CH2NN=O,47.8),138(M+-CH3NN=O,31.9), 42 (CHzNz+, 100);IR (KBr) 3390,1700,1595, 1470,1460,1440 cm-1. Anal. Calcd for C7HllN50z: C, 42.62;H, 5.62;N, 35.52. Found: C, 42.70;H, 5.62;N, 35.52. (G) Stability Measurements of Nitrosated Compounds. The stability of MNC (5), DNC (6),MNDNC (7), mononitrosamido-t-BOC-caffeidine (9), and MNAC (12) was measured in buffered solution (10 or 20 pg/mL; O.D. 0.5-1.0; pH 1-9) by monitoring absorbance between 200 and 300 nm a t appropriate intervals depending on the rate of degradation of the compound a t a particular pH. Readings were repeated three times for each compound. (H) Formation of MNC (5) from Caffeidine (4) as a Function of pH. Caffeidine (4)(0.1mM) was nitrosated with or 10 mM) in 20 mL of citrate buffer (1 sodium nitrite (0.1,0.2, M; pH 1-6) for 1 h a t 37 "C. The reaction was stopped by slowly pouring the reactants into 3.6 M HzSO4 (1 mL) saturated with ammonium sulfamate and made up to a final volume of 40 mL with water. Aliquots (20mL) were loaded onto Extrelut (Merck, Darmstadt, Germany), eluted with CHzClz (40 mL), and concentrated to 5 mL a t 80 "C which was further reduced to 1 mL under nitrogen. Aliquots were measured on HPLC as described earlier. Compound 5 was detected a t 4.2 min and quantitated from the standard curve of the purified compound. The recovery percentage of 5 was calculated (>95%), and corrections were incorporated in the results. (3) Study on Caffeidine Acid. (A) Preparation of Caffeidine Acid (2) from Caffeine (1). Caffeidine acid (2) was prepared by treating caffeine (1) (10g, 51.5 mmol) with 2 M NaOH (100mL) by modification of previously described methods (1, 2, 9). Compound 2 was purified by silica gel column chromatography using CHzClz-MeOH as solvent and crystallized in MeOH (yield 4.2 g, 38.4%): mp 164-165 "C; MS m/z(relative intensity) 212 (M+, 8.9),194 (M+ - HzO, 4.71, 168 (M+ - COz, 41.2),155 (M+ - COz - CH3,74.7), 110 (M+ - COz - CHaNHCO, 100);IR (KBr) 3600, 2300, 3260, 1725, 1715, 1660, 1630 cm-l. Anal. Calcd for C8H12N403: C, 45.28;H, 5.70;N, 26.40. Found: C, 45.25;H, 5.81;N, 26.14.
Caffeine-Derived N-Nitroso Compounds In addition to compounds 2 and 4, the alkaline hydrolysis of caffeine also yielded three minor products. One of these (70.0 4.0 11.1 23.5 4.0 >70.0 5.0 14.3 >70.0 14.9 3.6 6.0 13.0 8.1 0.3 16.4 7.0 3.5 3.4 6.2 8.0 0.8 0.1 1.0 1.2 0.02 9.0 0.05 0 nd = not determined due to conversion to 5.
18
9.0 14.2 27.5 38.3 >70.0 >70.0 16.4 6.0
2.3
Dinitrosocaffeidine (6) is a new nitroso compound with N-nitrosoamino as well as N-nitrosamide functions within the same molecule. It exhibits characteristic N-nitrosamide property in its stability at different pHs (Table VI. Results on mutagenicity using Salmonella typhimurium where 6 showed genotoxic action without metabolic activation (unpublished data) also confirm nitrosamide being the characteristic functional group in the molecule. The N-nitroso compound with blocked amino group (9), as well as the mononitrosamido caffeidine (12),showed a higher stability at various pHs compared to 6 (Table VI, implicating a yet not so clear but probably important role of N-nitrosamino group on the reactivity and stability of the nitrosamide group in the molecule. The formation of a C-nitro compound (7) in the above reaction, though unusual, as the nitrosation was carried out in aprotic medium, is not entirely unexpected since NO+is a powerful oxidant (19)and nitration at C = C bonds with NOCl/pyridinethrough 1,2-addition-eliminationhas been previously reported (20). Compound 7 , a byproduct of forced nitrosation, was mainly studied, being a new nitro-nitroso compound. Its reduced stability at different pHs compared to 6 is presumably due to the presence of a nitro group on the imidazole ring. Although nitrosation of caffeidine (4) by NaN02/H+ mainly produced 5 with low amounts of 6, the question remained whether a mononitrosamide derivative could also be a possible intermediate in the reaction, since amides are known to undergo reversible nitrosation with a strong capability to transnitrosate an amine or amino group independent of any nucleophilic catalyst (21,221. In order to check this possibility, the MNAC (12)was synthesized using di-tert-butyl dicarbonate for blocking the more reactive amino group, before introducing nitroso function at the amide nitrogen. The protective tert-butoxycarbonyl group (t-BOC)has the advantageof being removable under acidic conditions where N-nitrosamidesare relatively more stable compared to basic conditions (23,241. The 'H NMR of t-BOC-caffeidine (8) displayed signals for tertiary methyl groups at 6 1.49ppm. All other signals were similar to 4. The nitrosation of 8 yielded an N-nitroso derivative as major product (9) and a C-nitro-N-nitroso (11) and C-nitro (10)derivative as minor products (Scheme 111). For compound 9, the signal at 6 7.46 ppm in 'H NMR indicated the presence of a proton at C-2, but the signal due to amide proton was absent. The MS fragmentation pattern with M+ 297 confirmed the assigned structure. The 'H NMR of 10 did not show any signal for aromatic proton indicating substitution at C-2, and a broad singlet at 6 8.03 ppm indicated the presence of an amide H. The MS data with M+ at mlz 313 confirmed it to be a nitro derivative, which was supported by a strong IR band at 1670cm-l. 'Hand 13CNMR data of 11 showed substitution
at both C-2 and amide nitrogen. The structure of 11 was confirmed by MS, which showed M+ at mlz 342, and a characteristic band for NO2 group in the IR spectrum. It is interesting to note that, analogous to the nitrosation of 5 with NOBF4 (Scheme 111, in addition to the formation of nitrosamide derivative (9) the above reaction alsoyielded C-nitro (10) and C-nitro-N-nitroso (11)derivatives. The C-nitro substitution can be explained by the increased reactivity of C-2 of the imidazole ring, which can undergo electrophilic substitution under conditions used for the nitrosation reaction. The existence of a C-nitro derivative (lo), without nitrosation at amide nitrogen, is indicative of the steric hindrance caused by the t-BOC moiety on the reactivity of the amide group. On removal of the t-BOC group from 9 by HC1-formic acid, mononitrosamidocaffeidine(12)was obtained. Under these conditions, however, compound 12 was accompanied by compounds 5 and 6. A careful crystallization of 12 from the mixture resulted in purification of the compound. The 13C NMR spectra of 12 showed signals for C-4 and C-5 carbons at 6 106.25 and 160.67 ppm, respectively. These shifts were distinct from those observed for corresponding carbons in analogous compounds. The upfield shift for C-4 and downfield shift for C-5 are explained by the upush-pullweffect exerted by the electrondonating methylamino group and the electron-withdrawing nitrosamide function, respectively. On the basis of spectra data, 12 was identified as l-methyl-4-(methylamino)-5-(N-methyl-N-nitrosocarbamoyl)imidazole. The presence of 5 and 6 in the reaction mixture containing the mononitrosamidocaffeidine (12)strongly suggeststhat the nitroso group at amide nitrogen is capable of both intra- and intermolecular transnitrosation, leading to the formation of 5 and 6. Thus, transitory existence of a direct methylating compound other than 6 is implicit in this reaction. The presence of small amounta of caffeidine (4) in the reaction mixture is suggestive of denitrosation of 12. The stability of different nitrosated derivatives of caffeidine was investigated in order to determine their spontaneous degradation at different pHs. As shown in Table V, DNC (6) shows a typical nitrosamide pattern of degradation with increasing stability through decrease in acidity whereas reduced stability at alkaline pH (25,26). MNDNC (7) was less stable at all pHs as compared to DNC (6). The mononitroso-t-BOC derivativeof caffeidine (9) showed increasing stability with decrease in acidity, whereas the half-life ( t 1 4 between pH 4 and 6 was >70 h. The mononitrosamido derivative of caffeidine (12) showed a much higher stability at all pHs compared to its dinitroso analogue 6. It was, however, less stable as compared to 9 where the amino nitrogen was blocked with t-BOC. These observations suggest a destabilizing role of the nitroso group at the amino nitrogen in dinitrosocaffeidine (6). Nitrosation of Caffeidine Acid. Caffeidine acid (2) is the major product of alkaline hydrolysis of caffeine (1). The formation of 2 was first reported in 1928, and the structure then suggested on the basis of chemical and physical data was that of an iminocarboxylic acid (3) (2). In a later study based on NMR and MS data, a trisubstituted urea derivative of imidazolecarboxylic acid was proposed (1). However, the supporting data could not provide convincing evidence for the assigned structure, as the spectral information could as well fit into the iminocarboxylic acid form (Scheme I). Therefore, in order
Kumar et al.
56 Chem. Res. Toxicol., Vol.6,No.1, 1993
Scheme 111. Synthesis of Mononitrosamidocaffeidine
tsoc,o H 4
9
HsC-HN -C
H3C-
B
p"s
Jp I
-
1H+
No
H
5
12
to assign an unambiguous molecular structure of caffeidine acid (2), we conducted NMR nuclear Overhauser effect (NOE) experiment on the methyl ester of caffeidine acid (17) (Scheme V). Methylation of 2 by diazomethane at 0 "C does not affect its structure due to the mild nature of the reaction. The NOE showed increased intensity of peak due to NH upon irradiation of NHCH3 which was less pronounced when NCH3 was irradiated. However, no effect was observed when OCH3 and ring NCH3 were irradiated. These results proved the presence of a substituted urea derivative in the molecular structure of caffeidine acid (2). Scheme IV. Nitrosation of Caffeidine Acid in Acidic Medium
11
The nitrosation of 2 by NaN02 under acidic conditions yielded a cyclic triketo compound as the major product which was characterized as N,"-dimethylparabanic acid (13,45 % ). Other products of the reaction included N,"dimethyl-N-nitrosourea (14,0.5 % ) and mononitrosocaffeidine (5,0.4%), in addition to several yet unidentified compounds which could not be separated due to low yields. The DMPA (13) was identified by mass fragmentation with M+ at m/z 142 and on comparison of chemical and physical data with reported values for the compound (27). Similarly,the identity of 14 and 5 was also confirmed using reference compounds. The formation of DMPA (13) from 2 takes place between pH 1and 4 with different nitrite concentrations (Figure 2). At pH 1-2 the reaction was completed within 15 min. The acid-catalyzed nitrosation reaction, however, did not show formation of the N-nitrosourea derivative of 2, c nrmt. 0.1 mM
I
8
CH,
60
2
20
! 0
1
2
4
3 PH
Figure 2. Formation of DMPA a t different DH and nitrite 13
14
5
concentrations from caffeidine acid (0.1 mM).
Chem. Res. Toxicol., Vol. 6, No. I, 1993 87
Caffeine-Derived N-Nitroso Compounds
Scheme V. Nitrosation of Caffeidine Acid and Its Methyl Ester ma
I
___+
I
2
15
17
18
16
Scheme VI. Proposed Mechanism for the Formation of Dimethylparabanic Acid on Nitrosation of Caffeidine Acid in Acidic Medium
2
1s
HO
I
20
21
22
13
whereas when 2 was treated with acid in the absence of NaN02, no reaction took place. This clearly meant that either nitrite itself acta as acatalyst or nitrite acts through an unstable nitrosated intermediate in the reaction producing DMPA (13). The nitrosation of caffeidine acid (2) was, therefore, carried out using NOBFJpyridine in aprotic medium. However,this also did not form a nitroso derivative and instead gave an anhydride, l,4-dimethyl4,5-dihydro-5,7-dioxo-1H,7H-imidazo[4,5-d] [1,3]oxazine (16), with the pyridine salt of nitrosated caffeidine acid (15) as an intermediate. Compound 16 showed signals for two C = O groups at 6 150.85 and 152.84 ppm in 13C NMR supported by strong IR bands at 1780 and 1730 cm-1. It was further characterized by comparison of 'H NMR, MS, and IR data with those reported in the
literature (9). The formation of 16 suggested that N-nitroso substitution on the urea moiety renders C=O susceptible to a nucleophilic attack by COO- due to the electron-withdrawingproperties of the nitroso group and makes the N-nitrosomethylamino moiety a facile leaving group. The methyl ester of caffeidine acid, l-methyl-54methoxycarbonyl)-4-[N-(methylcarbamoyl)-N-methylamino] imidazole (171, was synthesizedwith two objectives. First, to obtain a derivative which could be used for the elucidation of structure of caffeidine acid (2) by NMR NOE and, second, for obtaining a nitroso derivative to study its possible role as an intermediate to DMPA (13) formation.
-
58 Chem. Res. Toxicol., Vol. 6, No. 1, 1993 The nitrosation of the methyl ester of caffeidine acid (17) under acidic conditions (NaNOdH+) and also with NOBFr/pyridine in CH3CN produced the N-nitrosourea derivative of the methyl ester (18) with 94% yield. The lH NMR spectrum of 18 showed a signal due to N(N0)CH3 at 6 3.49 ppm as against at 6 2.76 ppm in the parent methyl ester. However, the signal due to the methyl ester group showed a slight upfield shift and appeared at 6 3.82 ppm along with a doublet (4J= 0.6 Hz) due to ring CH3 coupling with aromatic proton. The MS spectrum of 18 showed M+ at mlz 255. The formation of a stable compound 18 under acidic conditions suggests a similar reaction occurring at the ureide moiety of caffeidine acid (2). This in turn may initiate a cascade of reactions leading to the formation of DMPA (13) as proposed in Scheme VI. The proposed mechanism takes into account the nitrosation of the ureide moiety and the possibility of an electrophilic addition of the NO group at C-5 of imidazole ring followed by elimination of COOH group under acidic conditions. Displacement of COOH by a nitroso group via an A - S E ~ mechanism is known in aromatic systems (28) which in the present reaction would be facilitated by proton acceptor C=O and NO groups of the ureide moiety at C-4. The presence of a strong electron-withdrawing NO group at C-5 polarizes the double bond between C-4 and C-5 even more than in compound 2 (strong push-pull effect). This may result in a more olefinic character of this double bond and the imidazole ring and should favor a hydrolytic cleavage of the imidazole ring leading to the formation of 21. The tautomeric forms of 21 can be hydrolyzed to an open-chain diketo compound 22 in one or more steps followed by cyclization to DMPA (13) with release of methyl diazotate, a facile leaving group. Although at this stage supporting evidence for the proposed mechanism is lacking, further work is in progress toward isolation and characterization of intermediates formed during the reaction. In view of the known toxicological relevance of N-nitrosamines and N-nitrosamides (29),the mutagenic potential of several of the compounds presented here is being assessed in S. typhimurium as well as in mammalian cells. The carcinogenicity of asymmetric nitrosamine MNC (5) and the nitrosamide DNC (6) in rats is also underway in order to evaluate their tumorigenicity and organotropy.
Acknowledgment. We would like to thank Prof. Dr. M. Wiessler for helpful discussions; Dr. N. Frank for advice on stability studies; and Dr. W. Hull and Ms. E. Reitmann for NMR analysis (all from DKFZ, Heidelberg). Thanks are also due to Mr. Dehio and Dr. D. Reichert of Boehringer Ingelheim KG, Ingelheim, Germany, for their gift of caffeidine nitrate and the HPLC method for the measurement of caffeidine. M.S. and R.K. would like to thank the German Cancer Research Center, Heidelberg, Germany, for providing financial support during their stay as guest scientists. The support received from Indian Council of Medical Research, New Delhi, and G.S.F., Munich, under the Indo-German scientific cooperation program is gratefully acknowledged. References Kigasawa,K., Ohkubo, K., Shimizu, H., Kohagizawa,T., and Shoji, R. (1974) Decomposition and stabilization of drugs X. A new decomposition route of caffeine in alkaline solution. Chem. Pharm. Bull. 22, 2448-2451. (2) Blitz, H., and Rakett, H. (1928) Caffeidine and caffeidine carboxylic acid (in German). Chem. Ber. 61, 1409-1422. (1)
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Registry Numbers Supplied by Author. 1,58-08-2; 2, 554536-15-1; 4, 20041-90-1; 13, 5176-82-9; 16, 10626059-7; 17, 20041-83-2.
