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378

Chem. Res. Toxicol. 2004, 17, 378-382

2,6-Diarylaminotetrahydropyrans from Reactions of Glutaraldehyde with Anilines: Models for Biomolecule Cross-Linking Alistair P. Henderson,*,† Christine Bleasdale,† William Clegg,‡ and Bernard T. Golding† School of Natural SciencessChemistry and Chemical Crystallography Unit, School of Natural Sciences, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, United Kingdom Received August 27, 2003

Glutaraldehyde reacts with weakly nucleophilic anilines, e.g., 3-fluoro-4-nitroaniline, which are models for amino groups in DNA, to give meso-2,6-disubstituted tetrahydropyrans, e.g., meso-2,6-di-(3-fluoro-4-nitroanilino)tetrahydropyran, that were characterized spectroscopically and by X-ray crystal structure analysis. This contrasts with the outcome of reactions with more strongly nucleophilic amines, which give rise to N-substituted 1,4-dihydropyridines. The mechanism of formation of the tetrahydropyrans is proposed to involve initial attack of the amine on one of the aldehyde groups of glutaraldehyde to give a carbinolamine intermediate. The ensuing cyclization to a tetrahydropyran, rather than dehydration to an imine leading to a dihydropyridine, is explained as a result of a competition between the lone pair of the amino function of the carbinolamine and the two lone pairs of the hydroxyl group. The formation of the tetrahydropyran is more likely with an amino function of low nucleophilicity, whereas dehydration to an imine leading to a dihydropyridine is favored with an amino function of higher nucleophilicity. The formation of tetrahydropyrans may be relevant to the toxicology of glutaraldehyde by providing a mechanistic basis for DNA adduction or DNA-protein crosslinking.

Introduction Glutaraldehyde (1) is a bioactive agent that is toxic to both Gram-positive (e.g., Mycobacterium tuberculosis) and Gram-negative bacteria, fungi, and viruses, including human immunodeficiency virus (1, 2). The preservative and antimicrobial properties of glutaraldehyde have made the substance a speciality chemical with applications in the cosmetic and toiletry industries (2). However, reports of adverse health effects from human exposures to glutaraldehyde led to concerns over the widespread applications of this substance. An additional reason for this alarm stemmed from the known toxicity and carcinogenicity of other aldehydes such as formaldehyde and malonaldehyde (3, 4). Glutaraldehyde is now regarded as highly hazardous to health, having sensitizing effects on the skin, eyes, and respiratory system of exposed persons. The main symptoms of exposure to glutaraldehyde are skin discoloration and ulceration, contact dermatitis, eye irritation, rhinitis, and occupational asthma (5). To understand the toxicology of glutaraldehyde, information is required on its reactions with proteins and DNA. To provide a mechanistic and structural basis for studies with biomolecules, we have performed model studies in which glutaraldehyde was reacted with a series of amines of varying nucleophilicity. These included * To whom correspondence should be addressed. Email: alistair@ email.unc.edu. Current address: Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7431. † School of Natural SciencessChemistry. ‡ Chemical Crystallography Unit, School of Natural Sciences.

amines 2a-d, some of which were selected on account of the similarity of their amino groups to those in adenine, cytosine, and guanine, using the pKa of the conjugate acid of each amine as a guide to its nucleophilic reactivity. The products previously reported from the reactions of glutaraldehyde with amino functions were primarily 1,1,5,5-tetraaminopentanes (6), 1,5-bis-imines (7), 1,5-bisamines (8), and N-alkyl-1,4-dihydropyridines (9, 10). The latter compounds are difficult to isolate, due to their low stability, and were normally reduced in situ to the corresponding piperidine (9). Lubig et al. (10) reported the formation of N-alkyl-1,4-dihydropyridines from reactions of glutaraldehyde and amines (e.g., n-pentylamine) at pH > 7. Below this pH, 1,5-bis-imines and polymeric material were obtained (10). There is also a report of the formation of a 2,6-disubstituted tetrahydropyran from the reaction of glutaraldehyde with benzotriazole (11), although this product was not well-characterized. The cross-linking potential of glutaraldehyde needs to be better understood in the context of DNA and proteins adducts. We have found that the outcome of the reaction of glutaraldehyde with an amine depends critically on the nucleophilicity of the amine. In this paper, we show that amines of relatively low nucleophilicity (e.g., 3-fluoro4-nitroaniline) exhibit a propensity to form 2,6-disubstituted tetrahydropyrans (model for a glutaraldehyde cross-link), whereas amines of higher nucleophilicity (e.g., L-valine methyl ester) give N-substituted 1,4-dihydropyridines.

10.1021/tx034177t CCC: $27.50 © 2004 American Chemical Society Published on Web 02/11/2004

Reactions of Glutaraldehyde with Anilines

Materials and Methods Hazardous Materials: Glutaraldehyde must be handled in a well-ventilated hood by an operator wearing appropriate protective clothing. Instruments. 1H and 13C NMR spectra were run on a Bruker WP-200E or a JEOL-JNM-LA spectrometer. Residual proton signals from the deuterated solvents were used as references [acetonitrile (1.95 ppm), D2O (4.81 ppm), d6-DMSO (2.50 ppm), and chloroform (7.25 ppm)] for 1H spectra. The residual 13C signal from the deuterated solvent was used as a reference [acetonitrile (1.2 and 117.8 ppm), d6-DMSO (39.7 ppm), and chloroform (77.0 ppm)] for 13C spectra. All coupling constants were measured in Hertz. Infrared spectra were recorded on a Nicolet 20-PC Fourier Transform IR spectrophotometer as either potassium bromide disks or Nujol mulls. Mass spectra were recorded on a Kratos MS80 RF spectrophotometer using MNBA in FAB mode or in +EI mode. Combustion analysis was performed using a Carlo Erba 1106 CHN analyzer. Chemicals. Chemicals and solvents were either AnalarR grade, which were used directly, or laboratory reagent grade purified further where appropriate. 3-Fluoro-4-nitroaniline and 3-fluoro-6-nitroaniline were synthesized according to the literature (12). Preparation of 1. Ozone (ca. 10-4 mol min-1) was bubbled through a solution of cyclopentene (2.0 g, 29 mmol) in dichloromethane (50 mL) at -78 °C. The reaction was stirred during the addition of ozone, and after 4.5 h, a blue color due to excess ozone was apparent. Triphenylphosphine (7.7 g, 29 mmol) was added, and the resulting solution was allowed to warm to room temperature. The resulting pale orange solution was concentrated in vacuo, and ether (50 mL) was added. The mixture was filtered, and the filtrate was evaporated to dryness. This process was repeated to yield an orange oil of glutaraldehyde and triphenylphosphine residues. The oil was subjected to Kugelro¨hr distillation (oven temperature, 55 °C; 10 mmHg) to give a colorless oil (1.54 g, 52%). 1H NMR (200 MHz, CDCl3): δ 9.50 (2H, b H-1), 2.29 (4H, t, J 7.2 Hz, H-2 and H-4), 1.69 (2H, q, J 7.1 Hz, H-3). 13C NMR (50.3 MHz, CDCl3): δ 201.59 (CdO), 42.75 (CH2), 14.44 (CH2) (13). Reactions of Glutaraldehyde with Amino Acids. To glutaraldehyde (10 mg) in d3-acetonitrile (0.7 mL) was added the amino acid (2 mol equiv), and the reaction was monitored by 1H NMR. The products were not isolated. 1. Valine Methyl Ester. 1H NMR (500 MHz, d3-acetonitrile): δ 5.80 (2H, dt, J 1.4 and 8.3 Hz, H-2, H-6), 4.37 (2H, m, H-3, H-5), 3.72 (3H, s, OMe), 3.50 (2H, m, H-4), 3.23 (1H, d, J 5.3 Hz, CH-N), 1.99 (1H, m, CH), 0.88 (6H, m, 2 × Me). 2. Valine Diethylamide. 1H NMR (500 MHz, d3-acetonitrile): δ 5.90 (2H, dt, J 1.4 and 8.0 Hz, H-2, H-6), 4.33 (2H, m, H-3, H-5), 3.28 (6H, m, 2 × CH2 and H-4), 3.01 (2H, d, J 5.7 Hz, CH-N), 2.21 (1H, m, CH), 1.15 (12H, m, 4 × Me). Synthesis of N-(4-Chlorophenyl)-1,4-dihydropyridine (4c). To pyridine (2.34 g, 2.38 mL, 29.6 mmol) was added 1-chloro-2,4-dinitrobenzene (5.0 g, 24.7 mmol), and the solution was heated at 65 °C for 1 h. The resulting cream-colored solid was washed with dichloromethane and dried in vacuo (0.01 mmHg) to yield N-(2,4-dinitrophenyl)pyridinium chloride (14, 15) as a cream-colored solid (6.1 g, 88%); mp 195-197 °C, lit. (16) 191 °C. 1H NMR (200 MHz, D2O): δ 9.12 (1H, s, ArH-3), 9.09 (2H, m, pyH-2, pyH-6), 8.87 (2H, m, pyH-3, pyH-5), 8.30 (2H, m, ArH-5, ArH-6), 8.18 (1H, m, pyH-4). To 4-chloroaniline (0.26 g, 2.0 mmol), in dry DMF (4 mL), was added N-(2,4-dinitrophenyl)pyridinium chloride (0.50 g, 2.0 mmol), and the mixture was heated at 100 °C for 24 h. The resulting solution was concentrated in vacuo (0.01 mmHg) to give a brown oil that was partitioned between dichloromethane (40 mL) and water (40 mL). The aqueous layer was concentrated to give N-(4-chlorophenyl)pyridinium chloride (14) as a brown solid (0.35 g, 77%); mp 116-119 °C, lit. (14) 123-124 °C. 1H NMR (200 MHz, d6-DMSO): δ 9.06 (2H, d, J 6.8 Hz, pyH-2, pyH-6), 8.69 (1H, m, pyH-4), 8.19 (2H, m, pyH-3, pyH-5), 7.69 (4H, s, ArH).

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 379 To N-(4-chlorophenyl)pyridinium chloride (0.23 g, 1.08 mmol) in a heterogeneous mixture of ether (15 mL) and water (15 mL) was added sodium dithionite (1.13 g, 6.5 mmol) and potassium carbonate (0.90 g, 6.5 mmol). The mixture was heated at 100 °C for 1 h. The ethereal layer was separated and washed with saturated aqueous sodium bicarbonate (30 mL), dried (Na2SO4), and filtered, and the filtrate was concentrated to give N-(4chlorophenyl)-1,4-dihydropyridine (4c) (17, 18) as an orange solid (0.15 g, 73%); mp 75-77 °C, lit. (19) 70-78 °C. 1H NMR (200 MHz, d3-acetonitrile): δ 7.32 (2H, d, J 9.1 Hz, ArH-3, ArH-5), 7.06 (2H, d, J 9.1 Hz, ArH-2, ArH-6), 6.47 (2H, dt, J 1.4 and 8.5 Hz, H-2, H-6), 4.78 (2H, m, H-3, H-5), 3.01 (2H, m, H-4). Synthesis of N,N-Diethyl-3-methyl-2-(4H-pyridin-1-yl)butyramide (4b). To valine (10.0 g, 85 mmol) was added trifluoroacetic anhydride (35.9 g, 23.9 mL, 171 mmol), and the solution was stirred for 24 h. The resulting mixture was extracted into ether (50 mL), and the ethereal extract was filtered. The filtrate was extracted with 20% aqueous sodium hydrogen carbonate, and the resulting aqueous solution was acidified with dilute hydrochloric acid. The product was extracted into ethyl acetate (3 × 50 mL). The combined extracts were dried (Na2SO4) and concentrated in vacuo (0.01 mmHg) to yield N-trifluoroacetylvaline (5a) (20) as a white solid (5.19 g, 29%); mp 88-90 °C, lit. (21) 86-88 °C. 1H NMR (200 MHz, d6-DMSO): δ 13.09 (1H, s, b, OH), 10.75 (1H, d, J 6.8 Hz, NH), 4.24 (1H, m, CH-N), 2.25 (1H, m, CH), 1.01 (6H, d, J 6.8 Hz, 2 × Me). To N-trifluoroacetylvaline (0.65 g, 3.3 mmol) and triethylamine (0.40 g, 0.55 mL, 4.0 mmol) in dichloromethane (5 mL) at 0 °C was added i-butyl chloroformate (0.54 g, 0.51 mL, 4.0 mmol), and the resulting solution was stirred for 30 min. After this time, diethylamine (0.29 g, 0.41 mL, 4.0 mmol) was added and the mixture was stirred for a further 30 min. The solution was allowed to warm to room temperature and stirred for a further 16 h. The volatiles were removed in vacuo (0.01 mmHg) to give a solid that was washed with petrol followed by water and then dried in vacuo (0.01 mmHg) to yield N-trifluoroacetylvaline diethylamide (5b) as a white solid (0.89 g, 91%); mp 130-132 °C. Found: C, 49.46; H, 6.98; N, 10.73%, C11H19N2O2F3 requires C, 49.25; H, 7.14; N, 10.44. 1H NMR (200 MHz, d6-DMSO): δ 9.83 (1H, d, J 7.0 Hz, NH), 4.57 (1H, dd, J 6.3 and 8.8 Hz, CH-N), 2.96 (4H, m, 2 × CH2), 2.21 (1H, m, CH), 1.21 (3H, t, J 7.0 Hz, Me), 1.11 (3H, t, J 7.0 Hz, Me), 0.95 (3H, d, J 6.7 Hz, Me), 0.91 (3H, d, J 6.7 Hz, Me). 13C NMR (50.3 MHz, d6-DMSO): 194.10 (CF3CdO), 167.23 (CdO), 115.16 (CF3), 56.67 (CH), 32.23 (CH), 29.75 (CH2), 20.15 (Me), 16.97 (Me). MS (+EI): 268 (M+, 8%), 183 (2), 168 (5), 100 (100), 72 (53). To N-trifluoroacetylvaline diethylamide (0.51 g, 2.02 mmol) in methanol (10 mL) was added aqueous ammonia (10 mL), and the reaction was stirred at 50 °C for 2 days. The solution was concentrated and redissolved in acetonitrile (20 mL), dried (Na2SO4), filtered, and concentrated to give valine diethylamide (5c) (22) as a colorless oil (0.32 g, 92%). 1H NMR (200 MHz, d3-acetonitrile): δ 6.65 (2H, b, NH2) 4.21 (1H, b, d, J 3.8 Hz, CH-N), 3.40 (4H, m, 2 × CH2), 2.18 (1H, m, CH), 1.13 (12H, m, 4 × Me). To valine diethylamide (86 mg, 0.50 mmol) in dry DMF (5.0 mL) was added N-(2,4-dinitrophenyl)pyridinium chloride (140 mg, 0.50 mmol), and the mixture was stirred at 100 °C for 24 h. The resulting mixture was concentrated in vacuo, and the oil was partitioned between water (10 mL) and dichloromethane (10 mL). The aqueous layer was lyophilized to give N,N-diethyl3-methyl-2-pyridin-1-yl-butyramide chloride (5d) as a brown oil (63 mg, 47%). 1H NMR (300 MHz, D2O): δ 9.14 (2H, d, J 5.6 Hz, pyH-2, py-H6), 8.65 (1H, t, J 7.8 Hz, pyH-4), 8.14 (2H, t, J 7.2 Hz, pyH-3, pyH-5), 5.53 (1H, d, J 10.7 Hz, CH-N), 3.62 (4H, m, 2 × CH2), 2.72 (1H, m, CH), 1.26 (3H, t, J 7.2 Hz, Me), 1.12 (6H, m, 2 × Me), 0.79 (3H, d, J 6.6 Hz, Me). To N,N-diethyl-3-methyl-2-pyridin-1-yl-butyramide chloride (39 mg, 0.15 mmol) in water/ether (1:1 v/v, 10 mL) were added potassium carbonate (0.12 g, 0.87 mmol) and sodium dithionite

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Henderson et al.

Scheme 1. Synthesis of 4b

(0.15 g, 0.87 mmol). The resulting heterogeneous mixture was stirred and heated at reflux for 1 h. After this time, the ethereal layer was washed with aqueous sodium bicarbonate, dried (Na2SO4), filtered, and concentrated to give 4b as a sticky orange solid (45 mg). 1H NMR (200 MHz, d3-acetonitrile): δ 5.98 (2H, dt, J 1.4 and 8.3 Hz, H-2, H-6), 4.31 (2H, m, H-3, H-5), 3.37 (5H, m, 2 × CH2 and CH-N), 2.94 (2H, m, H-4), 2.28 (1H, m, CH), 1.22 (12H, m, 4 × Me). General Procedure for Reactions of Glutaraldehyde with Anilines. To 1 (1 mol equiv) in dry acetonitrile was added the aniline 2a-d (2 mol equiv), and the reaction was stirred under N2 for 24 h. The resulting solid was filtered off, recrystallized from petrol/acetone, and dried in vacuo (0.01 mmHg) to give the product. 1. meso-2,6-Di(4-cyanoanilino)tetrahydropyran (3a). White crystals (46%); mp 165-166 °C. Found: C, 71.77; H, 5.59; N, 17.64%, C19H18N4O requires C, 71.66; H, 5.70; N, 17.61. 1H NMR (200 MHz, d6-DMSO): δ 7.62 (4H, d, J 8.6 Hz, ArH-3, ArH-5), 7.36 (2H, d, J 8.4 Hz, 2 × NH), 6.95 (4H, d, J 8.8 Hz, ArH-2, ArH-6), 5.12 (2H, m, N-CH-O), 1.84 (6H, m, 3 × CH2). 13C NMR (50.3 MHz, d -DMSO): δ 155.69 (ArC-4), 138.45 (ArC6 3), 125.55 (ArC-1), 118.61 (ArC-2), 102.81 (CN), 84.28 (C-2, C-6), 34.76 (C-3, C-5), 26.87 (C-4). υmax cm-1 (KBr): 3400 (NH), 3158 (Ar-H), 2907 (C-H), 2210 (CN), 1609, 1582, 1527 (Ar), 840 (4Ar). MS (+EI): 336 ([M+ + H2O], 46%), 201 (M+ - 4-cyanoaniline, 32). 2. meso-2,6-Di(4-nitroanilino)tetrahydropyran (3b). Yellow crystals (61%); mp 143-145 °C. Found: C, 56.47; H, 4.99; N, 15.27%, C17H18N4O5 requires C, 56.98; H, 5.06; N, 15.63. 1H NMR (200 MHz, d6-DMSO): δ 8.17 (4H, d, J 9.1 Hz, ArH-3, ArH-5), 7.85 (2H, d, J 8.2 Hz, 2 × NH), 6.98 (4H, d, J 9.1 Hz, ArH-2, ArH-6), 5.22 (2H, m, N-CH-O), 1.88 (6H, m, 3 × CH2). 13C NMR (50.3 MHz, d -DMSO): δ 153.20 (ArC-4), 137.64 (ArC6 1), 126.20 (ArC-3) 112.77 (ArC-2), 79.54 (C-2, C-6), 29.78 (C-3, C-5), 21.81 (C-4). υmax cm-1 (KBr): 3373 (NH), 3134 (Ar-H), 2928 (C-H), 1602, 1547, (Ar), 1502, 1374 (C-NO2), 846 (4-Ar). MS (+EI): 267 (M+ - 2 × NO2, 9%), 220 (26), 138 (89). 3. meso-2,6-Di(3-fluoro-4-nitroanilino)tetrahydropyran (3c). Yellow crystals (76%); mp 184-186 °C. Found: C, 51.55; H, 3.96; N, 12.72%, C17H16N4O5F2 requires C, 51.63; H, 4.34; N, 14.18. 1H NMR (200 MHz, d6-DMSO): δ 8.06 (4H, m, ArH5, ArH-6, 6.98 (2H, d, J 15.1, H-2), 6.80 (2H, d, J 8.8, 2 × NH), 5.28 (2H, m, N-CH-O), 1.89 (6H, m, 3 × CH2). 13C NMR (75.5 MHz, d6-DMSO): δ 157.6 (ArC-3, d, J 259 Hz), 154.3 (ArC-4, d, J 12 Hz), 127.9 (ArC-5, b), 125.8 (ArC-6, d, J 5.8 Hz), 109.5 (ArC1), 100.0 (ArC-2, d, J 24 Hz), 79.0 (C-2, C-6), 29.2 (C-3, C-5), 21.3 (C-4). υmax cm-1 (KBr): 3366 (NH), 3083 (Ar-H), 2956 (CH), 1618, 1599, 1542 (Ar), 1507 (C-NO2), 1440 (C-F), 843 (4Ar). MS (+EI): 395 (MH+, 28%), 239 (26). 4. meso-2,6-Di(3-fluoro-6-nitroanilino)tetrahydropyran (3d). Yellow crystals (15%); mp 188-190 °C. Found: C, 52.28; H, 3.79; N, 13.75%, C17H16N4O5F2 requires C, 51.76; H, 4.09; N, 14.21. 1H NMR (200 MHz, d6-DMSO): δ 8.26 (4H, m, ArH4, ArH-5), 7.54 (2H, d, J 9.0 Hz, 2 × NH), 6.79 (2H, m, ArH-2), 5.55 (2H, m, N-CH-O), 2.06 (6H, m, 3 × CH2). 13C NMR (75.5 MHz, d6-DMSO): δ 166.9 (ArC-3, d, J 253), 145.4 (ArC-6, d, J 7 Hz), 143.4 (ArC-1), 129.6 (ArC-2, d, J 23 Hz), 106.0 (ArC-5, d,

Scheme 2. Reaction of 1 with 4a-c, and 2e

J 8 Hz), 102.3 (ArC-4, J 29 Hz), 78.8 (C-2, C-6), 29.6 (C-3, C-5), 21.2 (C-4). υmax cm-1 (KBr): 3367 (NH), 3098 (Ar-H), 2957 (C-H), 1633, 1582, 1542 (Ar), 1510 (C-NO2), 1443 (C-F). MS (+EI): 394 (M+, 28%), 239 (26), 156 (14).

Results and Discussion Reactions of Valine Methyl Ester, Valine Diethylamide, NE-Carbobenzoxylysine Methyl Ester, and 4-Chloroaniline with Glutaraldehyde. Reaction of L-valine methyl ester with glutaraldehyde in acetonitrile gave the N-alkyl-1,4-dihydropyridine (4a) as the primary product. A reference compound for 4a, 4b, was independently synthesized and characterized spectroscopically (Scheme 1). Reaction of the valine diethylamide directly with 1 yielded the corresponding N-substituted 1,4dihydropyridine (4b). Similarly, reactions of N-carbobenzoxylysine methyl ester and 4-chloroaniline (2e) with glutaraldehyde each gave an N-substituted 1,4-dihydropyridine, 4c and 4d, according to NMR analysis of the crude reaction product (Scheme 2). It was not possible to isolate pure samples of these 1,4-dihydropyridines because of their instability and the formation of other products (see below for 2e). However, a reference sample of pure 4d was synthesized by reduction of N-(4-chlorophenyl)pyridinium chloride (14) with sodium dithionite (17). Reactions of Amines 2a-d with Glutaraldehyde. Reaction of glutaraldehyde with 4-cyanoaniline (2a) gave 3a (isolated yield 46% from a reaction in which 2 mol equiv of amine were used). The 1H NMR spectral data were consistent with the assignment of the structure of the adduct as a 2,6-di(4-cyanoanilino)tetrahydropyran. The H-2/H-6 multiplet resonance observed at δ 5.12 ppm in the 1H NMR spectrum of 3a collapsed to a broadened doublet (J 8 Hz) in the presence of D2O, while the NH resonance (doublet at δ 7.36 ppm, J 8 Hz) disappeared. The stereochemistry of 3a was assigned as cis (meso) by a crystal structure analysis. Although the precision of the crystal structure is limited by extensive whole molecule disorder, the molecular configuration and conformation are clear. The molecule has exact mirror symmetry, with the reflection plane bisecting the tetrahydropyran ring and containing the O atom and the opposite CH2 group.

Reactions of Glutaraldehyde with Anilines

Figure 1. Molecular structure of 3c in the crystalline state, with 50% probability ellipsoids for nonhydrogen atoms; the minor disorder component [placing the F atom on C(8) instead of C(6)] is not shown. The left- and right-hand sides of the molecule are related by a crystallographic mirror plane.

Scheme 3. Reaction of 1 with Anilines 2a-e

In a similar manner, reactions of glutaraldehyde with 4-nitroaniline (2b), 3-fluoro-4-nitroaniline (2c), and 3-fluoro-6-nitroaniline (2d) yielded the corresponding 2,6-di(anilino)tetrahydropyran 3b-d. The structure of 3c was secured by a crystal structure analysis. As for 3b, the molecule has exact mirror symmetry. There is minor disorder involving the F atoms, corresponding to superposition of two alternative orientations for the substituted phenyl rings, and also disorder of acetone solvent molecules in partially occupied sites, but this does not affect the overall molecular configuration, which is cis (meso) by crystallographic symmetry. The molecular structure, which is similar to that of 3b with different substituents, is shown in Figure 1. The structures of 3b,d were assigned by comparison of their NMR spectral data with the data for 3a,c (Scheme 3). Examination of the crude product from reaction of 4-chloroaniline with glutaraldehyde (see above) indicated the presence of 2,6-di(4-chloroanilino)tetrahydropyran 3e in addition to the 1,4-dihydropyridine 4c, in a 1:1 ratio by comparison of NMR resonances with those for 3a,c. A preliminary experiment in which glutaraldehyde was reacted with O2′,3′,5′-tri(4-tert-butylbenzoyl)guanosine in dichloromethane gave an adduct in

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low yield that was tentatively identified from its 1H NMR spectrum (δ 5.20 ppm for 2- and 6-H) as a tetrahydropyran analogous to 3a-d. Previous attempts to react glutaraldehyde with nucleosides or DNA (23) did not identify any adducts. Mechanism of Formation of Tetrahydropyrans 3. Reaction of an amine with glutaraldehyde initially yields a carbinolamine, which can dehydrate to an imine or cyclize to either a tetrahydropyran or tetrahydropyridine (Scheme 4). The relative extents of these pathways will be determined by the reactivity of the lone pair of the amine vs the two lone pairs of the hydroxyl group, the formation of the tetrahydropyran being more likely with an amine of low basicity/nucleophilicity, whereas formation of the imine and tetrahydropyridine will be favored with amines of higher basicity/nucleophilicity. The relative reactivities of the amino and hydroxyl lone pairs in the possible intermediates will also be influenced by steric factors, which will be more significant for the amino groups. The intermediate tetrahydropyran could afford the product 2,6-disubstituted tetrahydropyran by elimination of hydroxyl, followed by addition of a second molecule of the amine. Alternatively, the initial carbinolamine could react with a second molecule of amine to give a bis-carbinolamine that affords the 2,6-disubstituted tetrahydropyran via an intermediate imine. For reactions of glutaraldehyde with amines in acetonitrile, the 2,6-disubstituted tetrahydropyran products 3 precipitated from the reaction mixtures. In this case, the preferential formation of the 2,6-disubstituted tetrahydropyran might have been the result of Le Chatelier’s principle driving an equilibrium to the insoluble product. However, the 2,6-disubstituted tetrahydropyrans were also dominant products from reactions in dichloromethane and methanol, where there was no precipitation. Also, reactions with 1:1 stoichiometry of amine to glutaraldehyde gave predominately 2,6-disubstituted tetrahydropyran, although the yield of this product was improved by using 2:1 stoichiometry. The pKa values for amines 2a-c,e are 1.71, 1.18, -0.30, and 4.16 respectively (cf. pKa -2 for a typical alcohol) (24). A pKa value for 2d has not been reported, but it is expected to be similar to that of 2c. Hence, for the amines of pKa e 1.71, only a tetrahydropyran product was observed. With 2e, the nucleophilicity of the amino function is sufficient that both tetrahydropyran and dihydropyridine products were observed. With amines of relatively high nucleophilicity, e.g., 5c, only the dihydropyridine product 4b was observed.

Conclusions We have fully characterized a number of meso-2,6disubstituted tetrahydropyrans from the reactions of weakly nucleophilic anilines with glutaraldehyde. Previous studies have primarily observed N-substituted dihy-

Scheme 4. Mechanism of Formation of Tetrahydropyrans 3

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dropyridines from reactions of glutaraldehyde with amines, but we now have shown that this is a consequence of using relatively nucleophilic amines. The model studies described provide a mechanistic and structural basis for understanding the reactions of glutaraldehyde with biomolecules and the associated complex toxicology.

Acknowledgment. We thank the University of Newcastle upon Tyne and the EPSRC for support of personnel and equipment.

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