130
Chem. Res. Toxicol. 1992,5, 130-133
totoxicity in mice. Res. Commun. Chem. Pathol. Pharmacol. 58, 75-83. (14) Thorup, I., Wurtzen, G., Carstensen, J., and Olsen, P. (1983) Short term toxicity study in rats dosed with pulegone and menthol. Toxicol. Lett. 19,207-210. (15) Thomassen, D., Slattery, J. T., and Nelson, S. D. (1988)Contribution of menthofuran to the hepatotoxicity of pulegone: Assessment based on matched area under the curve and on matched time course. J. Pharmacol. Exp. Ther. 244,825-829. (16) Moorthy, B., Madyastha, P., and Madyastha, K. M. (1989) Hepatotoxicity of pulegone in rats: ita effects on microsomal enzymes, in vitro. Toxicology 55,327-337. (17) McClanahan, R. H., Huitric, A. C., Pearson, P. G., Desper, J. C., and Nelson, S. D. (1988)Evidence for a cytochrome P-450 catalyzed allylic rearrangement with double bond topomerization. J. Am. Chem. SOC. 110,1979-1981. (18) McClanahan, R. H., Thomaasen, D., Slattery, J. T., and Nelson, S. D. (1989)Metabolic activation of (R)-(+)-pulegoneto a reactive enonal that covalently binds to mouse liver proteins. Chem. Res. Toxicol. 2,349-355. (19) Madyastha, K. M., and Raj, C. P. (1990)Biotransformations of (R)-(+)-pulegoneand menthofuran in vitro: chemical basis for toxicity. Biochem. Biophys. Res. Commun. 173, 1086-1092. (20) Thomassen, D., Slattery, J. T., and Nelson, S. D. (1990)Menthofuran-dependent and independent aspects of pulegone hepatotoxicity. J. Pharmacol. Exp. Ther. 253,567-572. (21) Thomassen, D., Pearson, P. G., Slattery, J. T., and Nelson, S. D. (1991)Partial characterization of biliary metabolites of pulegone by tandem mass spectrometry: detection of glucuronide, glutathione, and glutathionyl glucuronide conjugates. Drug Metab. Dispos. 19,997-1003. (22) Takahashi, K.,Someya, T., Muraki, S., and Yoshida, T. (1980) A new keto-alcohol, (-)-mintlactone, (+)-isomintlactoneand minor components in peppermint oil. Agric. Bid. Chem. 44,1535-1543. (23) Woodward, R.B., and Eastman, R. H. (1950)The autoxidation 72,399-401. of menthofuran. J. Am. Chem. SOC. (24) Omura, T., and Sato, R. (1964)The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoproteinnature. J.Biol. Chem. 239,2370-2378. (25) Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R.
J. (1951)Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. (26) Rettie, A. E.,Rettenmeier, A. W., Howald, W. N., and Baillie, T. A. (1987)Cytochrome P-450-catalyzed formation of A4-VPA, a toxic metabolite of valproic acid. Science 235,890-893. (27) Rettie, A. E.,Eddy, A. C., Heimark, L. D., Gibaldi, M., and Trager, W. F. (1989)Characteristics of warfarin hydroxylation catalyzed by human liver microsomes. Drug Metab. Dispos. 17, 265-270. (28) Levisalles, J. (1957)La preparation des pyridazines B partir de derives du furanne. Bull. SOC. Chim. Fr., 997-1003. (29) Ruzo, L.O.,Casida, J. E., and Holden, I. (1985)Direct N.M.R. detection of an epoxy furan intermediate in peracid oxidation of the fungicide methfuroxam. J. Chem. Soc., Chem. Commun., 1642-1643. (30) Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davies, D. C., Gillette, J. R., and Brodie, B. B. (1973)Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J.Pharmucol. Exp. Ther. 187,185-194. (31) Hirsch, J. A., and Szur, A. J. (1972)The hydrolysis of a,a’-dimethoxydihydrofurans. J. Heterocycl. Chem. 9,523-529. (32) Manfredi, K. P., and Jennings, P. W. (1989)Effect of acid on the peracid oxidations of 3-methyItetrahydrobenzofuran.J. Org. Chem. 54,5186-5188. (33) Nelson, S.D., and Pearson, P. G. (1990)Covalent and non-covalent interactions in acute lethal cell injury caused by chemicals. Annu. Rev. Pharmacol. Toxicol. 30, 169-195. (34) Ravindranath, V.,Burka, L. T., and Boyd, M. R. (1984)Reactive metabolites from bioactivation of toxic methylfurans. Science 224,884-886. (35) Burka, L.T., and Boyd, M. R. (1985)Furans. In Bioactiuation of Foreign Compounds (Anders, M. W., Ed.) pp 243-257, Academic Press, Orlando. (36) Hanzlik, R. P., Hogberg, K., and Judson, C. M. (1984)Microsomal hydroxylation of specificallydeuterated monosubstituted benzenes. Evidence for direct aromatic hydroxylation. Biochemistry 23,3048-3055. (37) Korzekwa, K.,Trager, W., Gouterman, M., Spangler, D., and Loew, G. H. (1985)Cytochrome P-450mediated aromatic oxida107,4273-4279. tion: a theoretical study. J. Am. Chem. SOC.
Synthesis and Characterization of Monohydroxylated Derivatives of irH-Dibenzo[ c ,g ]carbazole Weiling Xue and David Warshawsky* Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056 Received August 12,1991 The synthesis of several monohydroxylated derivatives of the potent carcinogen 7H-dibenzo[cg]carbazole (DBC), including l-hydroxy-7H-dibenzo[cg]carbazole(1-OH-DBC), 13-chydroxydibenzo[cg]carbazole(13-c-OH-DBC), and 5-hydroxy-7H-dibenzo[cg]carbazole(5-0H-DBC), is described. 1-OH-DBC was prepared from 8-methoxy-2-tetralone and 2-naphthylhydrazine via Fischer indole synthesis followed by boron tribromide demethylation. The rearrangement and hydrolysis reactions to give 13-c-OH-DBC from DBC and benzoyl peroxide are discussed. The preparation and isolation of 5-OH-DBC, by hydrolysis of 5-acetoxy-Nacetyl-DBC, and the formation of its intermediate 5-acetoxy-DBC and its byproduct 6,6’-bis(5-OH-DBC) are described in detail.
Introduction 7H-Dibenzo[cg]carbazole(DBC)’ (I), found in a variety of complex mixtures (1-4),has demonstrated strong carcinogenic activities in animal tests (1, 5-7). StudiesLy this r&arch group (8-10)and others (il) have indicated that the metabolism of DBC is strictly analogous to that of neither polycyclic aromatic hydro-
* To whom correspondence should be addressed.
carbons nor the related N-heterocyclic polynuclear aromatic hydrocarbons. The primary metabolites of DBC using microsomal preparations of rat and mouse liver in have been identified monohydroXYlated derivatives Abbreviations: DBC, 7H-dibenzo[cg]carbazole;1-OH-DBC, 1hydroxy-DBC; 13-c-OH-DBC, 13-c-hydroxy-DBC; 5-OH-DBC, 5hydroxy-DBC; 6,6’-bis(5-OH-DBC),6,6’-bis(5-hydroxy-DBC);1-MeODBC, 1-methoxy-DBC;TMS, tetramethylsilane; MS, mass spectrum; TLC, thin-layer chromatography;CI, chemical ionization.
0893-228x/92/2705-0130$03.00/00 1992 American Chemical Society
Synthesis of 7H-Dibenzo[cc,g]carbazoleDerivatives rather t h a n dihydrodiols. N-Hydroxy-DBC (8),5hydroxy-DBC (10, 1 0 , and 3-hydroxy-DBC (9-11) have been reported as well as minor amounts of 2-hydroxy-DBC (9-11), 4-hydroxy-DBC (II), 6-hydroxy-DBC (II), and a few unidentified phenols. Most of these compounds have been synthesized and employed for identification of metabolites and DNA adducts (12).In this article, we describe the novel syntheses of l-hydroxy-DBC (1-OH-DBC), 13-c-hydroxy-DBC (13-c-OH-DBC), and 5-OH-DBC. 11-12
.w5 8
6
ti7
H 1
The synthesis of 1-OH-DBC, a new compound not reported in the literature, was achieved using 8-methoxy-2tetralone as the starting material according to published methods (9,11, 13) with modifications. Attempts were made to synthesize the N-hydroxy-DBC after the method of Jaccarini et al. (14) in which 2phenylindole, a structural analogue of DBC, was converted to N-hydroxy-2-phenylindoleb y oxidation with benzoyl peroxide followed by hydrolysis. However, in the case of DBC, a rearrangement product, 13-c-OH-DBC (2), was isolated and characterized. By the procedure of Stong (9), the product was found to be the intermediate, 13-cbenzoyloxy-DBC (3). 5-OH-DBC (4), an extremely unstable compound and the major metabolite of DBC from mouse and r a t liver microsomal fractions (10, 11), was synthesized by hydrolysis of 5-acetoxy-N-acetyl-DBC (5) (11). Exceptional attention and effort were paid in our investigation to the hydrolytic reaction conditions and the preparation and isolation of 5-OH-DBC (4); the intermediate, 5-acetoxyDBC (6), and the byproduct, 6,6'-bis(5-OH-DBC) in the form of 6,6'-bis(5-acetoxy-DBC) (7), were delineated. These newly synthesized compounds have been applied in our studies to t h e metabolism of DBC (10) and in t h e development of a nonradiometric method using fluorescence spectroscopy with HPLC for identifying and quantifying major metabolites of DBC.2
Experimental Section Infrared (IR) spectra (KBr) were obtained using a Perkin-Elmer 1600 FT-IR spectrophotometer. Proton NMR spectra were recorded on a Bruker NR/300 AF instrument in deuterated chloroform with TMS as the internal standard (0 ppm). The coupling constanta (Jvalue) were reported in hertz (Hz). High-resolution mass spectral (MS) data were determined on a Kratos Model MS 80 instrument using an internal standard of perfluorokerosine. Melting points were uncorrected. All chemicals, except those reported in the text, were purchased from Aldrich Chemical Co. (Milwaukee, WI). The purities of compounds synthesized were determined using HPLC peak area normalization (UV detector at 254 nm).Purity was determined to be >98% for all compounds except for the 5-OH-DBC. Because of the instability, the purity of 5-OH-DBC was found to vary with each synthesis but was greater than 95%. Because of the hazardous nature of the chemicals, all compounds were handled carefully. 8-Methoxy-2-tetralone was prepared from 1,7-dihydroxynaphthalene according to the literature (1516);mp 58-59 "C. 1-MeO-DBC. A mixture of 8-methoxy-2-tetralone(1.50 g, 8.5 "01) and 2-naphthylhydrazinehydrochloride (1.70 g, 8.7 mmol)
* W. Xue, J. Schneider, and D. Warshawsky, in preparation.
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 131 in anhydrous ethanol (40 mL) was heated to 50 "C, 3 drops of glacial acetic acid were added, and then the mixture was refluxed for 1h. After cooling, the reaction mixture was poured into cold water and the precipitate was filtered off, washed with water, dried, and then dissolved in glacial acetic acid (20 mL) presaturated with dry hydrogen chloride. The solution was heated at 105-110 "C with continuous bubbling of dry HC1 gas for 5-10 min. The cooled mixture was poured into water, and the precipitate was collected, washed, dried, and then chromatographed (50X 2.5 cm silica gel column, 70-230 mesh, 60 A, with chloroform as the eluant), giving the cyclized product, which was dissolved in toluene (30 mL) and dehydrogenated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone(1.97 g) at room temperature Overnight. The insoluble material was removed, the fiitrate was evaporated, and chromatography of the residue (silica gel column with chloroform as the eluant) afforded white crystalline 1-MeO-DBC (from acetone/water, 0.82 g, 34% yield): mp 243-245 "C; 'H NMR S 3.78 (a, 3 H, OCH3), 7.09 (d, 1 H, H2), 7.40-7.51 (m, 3 H, H3, H5, Hll), 7.84 (d, 1H, H4), 7.59-7.66 (m, 3 H, H6, H9, H12), 8.71 (s, 1 H, H7), 7.79 (d, 1 H, H8), 7.96 (d, 1 H, HlO), 8.26 (d, 1 H, H13), JZa = 7.7, J3,4 = 8.5, Jag = 8.4, Jlo,ll = 8.6, J12,13 = 8.9. MS: Calcd for Cz1Hl5NO,297.1154; found, 297.1145. 1-OH-DBC. To a solution of 1-MeO-DBC (79 mg) in anhydrous methylene chloride (15 mL) cooled to -60 "C was injected boron tribromide (2 mL, 1 M solution in methylene chloride) gradually through an airtight septum under nitrogen over a period of 5 min. After 2 h at this temperature and overnight at room temperature, 25 mL of water was carefully added with stirring. The organic phase was separated, washed twice with water, dried over MgS04, and evaporated to dryness under reduced pressure. From preparative thin-layer chromatography [TLC; plates prepared in this laboratory using silica gel 60 P F 254 366 (EM Laboratories, Elmsford, NY)on 20 X 20 cm X 1mm glass plates with chloroform as the developing solvent] 12 mg of 1-MeO-DBC was recovered and 15 mg of 1-OH-DBC (21%, from benzene/ hexane) was obtained mp 237-240 "C; 'H NMR S 5.59 (s, 1H, OH), 7.23 (d, 1H, H2), 7.49 (dd, 1H,H3),7.89 (d, 1H,H4),7.64 (d, 1 H, H5), 7.71 (d, 1 H, H6), 8.92 ( 8 , 1 H, H7), 7.83 (d, 1 H, H8), 7.64 (d, 1 H, H9), 8.01 (d, 1 H, HlO), 7.49 (dd, 1 H, H l l ) , 7.65 (dd, 1H, H12), 8.44 (d, 1H, H13), J2,3 = 7.7, J3,4 = 8.6, J5,6 = 8.7, Js,s = 8.7, Jlo,ll = 7.8, J11,12 = 8.3, J12,13 = 8.2. MS: Calcd for C20H13N0,283.0997; found, 283.0965. 13-c -(Benzoyloxy)-DBC (3). Benzoyl peroxide (0.65g, 2.7 mmol) in 60 mL of dry methylene chloride was dropped into a solution of DBC (0.24 g, 0.9 mmol) in 90 mL of dry methylene chloride. After 12 h of refluxing followed by cooling, the solution was washed with 5% aqueous sodium bicarbonate and water, dried over sodium sulfate, and evaporated to remove the solvent. Chromatography (silica gel column, 30 X 1.0 cm, methylene chloride to methylene chloride/ethyl acetate, 4/1, gradient) and crystallization from hexane/benzene (3/ 1)gave yellow crystals of 3 (0.15 g, 44%): mp 150-153 "C; 'H NMR 6 7.10 (dd, 1H, H3), 6.70 (d, 1 H, H5), 6.87 (d, 1 H, H6), 7.27-7.57 (m, 9 H, Ar), = 10.0, 7.86-7.98 (m, 4 H, Ar), 8.16 (d, 1H, H131, J 2 , 3 = 7.6, J5,6 J12,13 = 8.1. MS: Calcd for CnH1,N02, 387.126; found, 387.121. 13-c-OH-DBC (2). Compound 3 (100 mg) in 0.1 N NaOH solution of methanol/water @/I, 100 mL) was refluxed for 10 min under nitrogen. The solution was acidified with 0.1 N sulfuric acid and extracted with ethyl acetate (2 X 50 mL),and the organic layer was washed with water (3 x 10 mL) and dried over sodium sulfate. After evaporation of the solvent, the residue was chromatographed (silica gel column, 20 X 1.0 cm, methylene chloride to methylene chloride/ethyl acetate, 3/1, gradient) to give yellowish crystals of 2 (15 mg, 21%, from a hexane/methylene chloride solvent mixture): mp 140-144 "C; 'H NMR S 4.38 ( 8 , 1 H, OH), 7.90 (d, 1 H, Hl), 7.26 (dd, 1 H, H2), 7.04 (dd, 1 H, H3), 7.19 (d, 1H, H4), 6.26 (d, 1H, H5), 6.85 (d, 1 H, H6), 7.58 (d, 1 H, H8), 7.36 (d, 1 H, H9), 7.79 (d, 1 H, HlO), 7.48 (t, 1 H, H l l ) , 7.59 (t, 1H, H12), 8.37 (d, 1 H, H13), J1,2 = 8.2, J 2 , 3 = 7.1, J 3 , 4 = 6.7, J 5 , 6 = 9.7, Js,e = 7.4, J10,ll= 8.5, J11,12 = 7.2, J12,13 = 8.3. MS: Calcd for C2,,H13N0, 283.0997; found, 283.0998. 5-Acetoxy-N-acetyl-DBC (5) was prepared from N-acetylDBC (17) by the technique of Perin et al. (11): mp 158-159 "C. 5-OH-DBC (4). Compound 5 (10 mg) was stirred in 5 mL of 0.1 N NaOH in methanol/water (9/1) at ambient temperature under nitrogen in the dark for 6-10 min. The workup was the
+
Xue and Warshawsky
132 Chem. Res. Toxicol., Vol. 5, No. 1, 1992 Scheme I”
0
I
1
Ph-C=O
(2)
“Conditions: (I) benzoyl peroxide/CH,Cl,; (11) 0.1 N NaOH/MeOH
same as that described in the preparation of 13-c-OH-DBC,except TLC (silica gel, benzene/ethyl acetate/methanol, 20/2/1) was used in the dark under nitrogen instead of column chromatography, to give a light brown 4 ‘H NMR 6 9.16 (d, 1H, Hl), 8.02 (d, 1H, H4), 7.07 (8, 1H, H6), 8.63 (s,1H, H7), 7.82 (d, 1H, H8),
8.37 (d, 1H, HlO), 7.64-7.73 (m,3 H, H2, H9, and H12), 7.51-7.58 (m, 2 H, H3 and H l l ) , 9.19 (d, 1 H, H13), Jl,z= 9.7, 53,4= 8.0, Jlo,ll= 8.1, J1213 = 9.4. Ms: Calcd for Cd13NO, 283.0997; found, 283.0972. 5-Acetoxy-DBC(6). 1. Synthesis of 6 from 5. Compound 5 (10 mg) in methanol/water (95/5) was refluxed for 3 h. After cooling, the crystals were collected mp 260-262 O C ; IR (cm-’) 3354 (YN-H), 1735 (YC=O), 1384 (Y&, 1223 (VCW);‘H NMR 6 2.58 (8, 3 H, COCHS), 8.93 (d, 1 H, Hl), 7.67 (t, 1 H, H2), 8.00 (d, 1 H,H4), 7.46 (8, 1H,H6),8.86 (a, 1H,H7),7.77 (d, 1H,H8),8.06 (d, 1 H, HlO), 7.49-7.59 (m, 1 H, H3, H9, H11, and H12), 9.04 (d, 1 H, H13), J1.2 = 8.3, J3,4 = 8-09JB,S= 8.7, Jio,ii = 9.0, 5 1 2 ~ 3 = 8.5. MS: Calcd for CZ2HlSNO2, 325.1103; found, 325.1096. 2. Synthesis of 6 from 4. Compound 4 (2 mg) was dissolved in 15 drops of acetic anhydride and 2 drops of pyridine, and the solution was stirred at room temperature for 3 h. After neu-
tralization with ice-cold saturated aqueous sodium bicarbonate and extraction with ethyl acetate (3 X 0.5 mL), the organic phase was washed with water, dried over sodium sulfate, and evaporated to give 6 (from methanol): mp 258-260 “C. 6,6’-Bis(I-acetoxy-DBC)(7). Compound 5 (10 mg) in 5 mL of 0.1 N NaOH (MeOH/H20, 9/1) was refluxed for 15-20 min. After workup as described in the section on preparation of 4, the residue was acetylated in acetic anhydride (1mL) and pyridine (4 drops) at room temperature for 3 h and worked up as indicated in the above paragraph. TLC (silica gel, benzene/ethyl acetate/methanol, 20/2/1) and recrystallization from hexane gave
O C ; IR (cm-’13422 (w.~), 1763 (vc-o),1384 ( Y C N ) , 1194 (~c.0.c); ‘H NMR 6 2.05 ( ~ , H, 6 COCHJ, 9.24 (d, 2 H, H1, Hl’), 7.81 (t, 2 H, H2, H2’), 7.55 (t, 2 H, H3, H3’), 8.03 (d, 2 H, H4, H4’),8.69 (8, 2 H, H7,H7’), 7.86 (d, 2 H, H8, H8’), 7.46 (d, 2 H, H9, H9’), 8.07 (d, 2 H, H10, HW), 7.66 (t,2 H, H11, Hll‘), = 8.5,52,3 7.74 (t, 2 H, H12, H12’), 9.39 (d, 2 H, H13, H13’1, 51,~ = 7.4, J3,4= 8.0, J8,s = 8.8, Jlo,ll= 8.0, J12,13 = 8.5. Chemical ionization (CI) MS: 649 (M + 1).
7 mp 207-210
Results and Discussion The syntheses of 2-, 3-, and 4-methoxy-DBC were previously reported by this laboratory (9), using the Fischer indole synthesis adapted by Buu-Hoi et al. (18) and Perin et al. (11). Using similar approaches with modifications, 1-MeO-DBCwas afforded in an overall yield of 34% from 8-methoxy-2-tetralone. Conversion of 1-MeO-DBC to 1-OH-DBC by demethylation was difficult probably due to the steric hindrance that limits the access of the reagent to the methoxy group at position 1where it is near to ring E. The boron tribromide procedure, which was success-
+ H20. fully used in cleavage of 2-, 3-, and 4-methoxy-DBC to corresponding phenols in our laboratory3 (13),gave 1OH-DBC in only about 20% yield. Other methods tested such as pyridium chloride (19,20)and hydrogen bromide in water (21)or in acetic acid (21)did not cleave the methyl-oxygen bond or gave undesired side products. By the reaction of DBC with benzoyl peroxide, N(benzoyloxy)-DBCwas not isolated but rather 13-c-(benzoy1oxy)-DBC (3), which gave 13-c-OH-DBC (2) after basecatalyzed hydrolysis under mild conditions. Although the mechanism of the rearrangement is not very clear, DBC can be considered as a secondary arylamine. It has been reported that secondary aryl amines reacting with benzoyl peroxide do not give O-benzoylhydroxylamineabut rather hydroxybenzanilides through a nucleophilic ionic rearrangement (22,231.Therefore, it seems reasonable to propose that the conversion of DBC to 13-c-OH-DBC undergoes the similar mechanism shown in Scheme I. Spectral data support the structural assignment. High-resolution MS indicates the actual monohydroxyDBC after hydrolysis of the benzoate intermediate. The major mass fragment 254 (M’ - 291, which is also shown for other known phenolic derivatives of DBC, implies the loss of COH rather than NOH. The loss of symmetry displayed in the proton NMR spectrum also verifies the hydroxy moiety is not at the nitrogen of position 7 of the symmetrical molecule of DBC. The hydroxy group at the 13-c position, an annular carbon, creates a chiral carbon with an sp3hybridization at the site and reduces the aromatic character of the hydroxylated DBC. Reflecting the less aromatic character of this molecule, the entire spectrum is shifted upfield compared to other phenolic derivatives of DBC. Signals from the side of the molecule with the hydroxy moiety are shifted far upfield. In particular, positions 5 and 6 are more isolated from the large aromatic system that results in two clearly coupled and farther upfield doublets. Signals from position 8 through 13 more or less maintain the patterns seen in the parent compound. A broad singlet for a proton of the hydroxyl group is observed at 4.38 ppm, suggesting reasonably that the bond is a cyclic sp3 carbon. Because of the instability of 5-OH-DBC to air and light, no crystalline product or sufficient analytical data were reported. By our investigation through hydrolysis of 5AcO-N-Ac-DBC (5) under different conditions, three Xue,W. (1990) Syntheses of potential metabolites of 7H-dibenzo[cg]carbazole and dibenz[aj]acridine. Unpublished results.
Synthesis of 7H-Dibenzo[cc,g]carbazoleDerivatives
Chem. Res. Toxicol., Vol. 5, No. 1, 1992 133 tetralone, 5309-19-3; 2-naphthylhydrazine hydrochloride, 137668-36-1.
Scheme 11’
References li
a Conditions: (111) 0.1 N NaOH/MeOH + H20, room temperature; (IV) MeOH/H20, reflux; (V) 0.1 N NaOH/MeOH + HzO, reflux; (VI) acetic anhydride/pyridine.
compounds were isolated respectively. 5 reacted with wet methanol at a refluxing temperature for 3 h, giving a pure compound with a molecular formula of CzzHl5NO2,which suggests the cleavage of only one acetyl group in 5. IR and proton NMR clearly indicate ita ester characteristics rather than amide, which proves the structure of 5-AcO-DBC (6). Similarly to 5,6 reacted with the hydrolysis reagent (0.1 N NaOH in methanol/water 9/1)at room temperature for 5-10 min, and a chromatographically pure but not a crystalline 4 was also obtained. In turn, 4 is able to be converted to 6 by acetylation. When the reaction of 5 with the hydrolysis reagent was carried on at reflux for 15-20 min, another compound instead of 4 was formed. After stabilization of this compound by acetylation, the IR and proton NMR showed similarity to those of 6 and MS gave a molecular weight of 648 (7) that confirmed the existence of the previously reported oxidative dimer 6,6’-bis(5-OHDBC) (11) and excluded the possible products of aldol reaction. Interestingly, when 4 was reacted with the hydrolysis reagent at reflux and then acetylated, compound 7 was also produced. To summarize, Scheme I1 shows the relationships among 4, 5, 6, and 7. As more of these phenolic derivatives of DBC become available, a better understanding of the metabolism and DNA binding of DBC will be obtained in a number of biological systems.
Acknowledgment. This work was supported by NIEHS 04203. We thank Dm. K. Jayasimhulu and D. Liu for generating MS data and NMR data and Ms. L. Wilson for preparing the manuscript for publication. Registry NO. 1, 194-59-2; 2, 137668-30-5; 3, 137668-31-6; 4, 78448-06-3; 5, 137668-32-7; 6, 137668-33-8; 7, 137668-34-9; 1MeO-DBC, 137668-35-0; 1-OH-DBC, 110408-52-1;8-methoxy-2-
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