Synthesis of anti-1, 2-Dihydroxy-3, 4-epoxy-1, 2, 3, 4-tetrahydro-6

Karam El-Bayoumy, Arun K. Sharma, Jyh-Ming Lin, Jacek Krzeminski, Telih Boyiri, Leon C. King, Guy Lambert, William Padgett, Stephen Nesnow, and Shantu...
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Chem. Res. Toxicol. 2000, 13, 1143-1148

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Synthesis of anti-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene and Its Reaction with 2′-Deoxyguanosine5′-Monophosphate, 2′-Deoxyadenosine-5′-Monophosphate, and Calf Thymus DNA in Vitro Jacek Krzeminski,† Dhimant Desai,† Jyh-Ming Lin,† Vladimir Serebryany,† Karam El-Bayoumy,‡ and Shantu Amin*,† Organic Synthesis Facility and Division of Cancer Etiology and Prevention, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595 Received May 8, 2000

The remarkable carcinogenic activity of 6-nitrochrysene (6-NC) in several animal models, and its environmental presence, suggest its potential importance with regard to human cancer development. Depending on the bioassay model, 6-NC can be activated by simple nitro reduction, ring oxidation, or by a combination of ring oxidation and nitro reduction. Only the first pathway has been clearly established. Thus, this study purports to unequivocally define the other pathways. Toward this end, we report for the first time the synthesis of anti-1,2dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene (6-NCDE), a likely ultimate carcinogenic metabolite of 6-NC. Also, we describe our initial investigation of its binding with calf thymus DNA, 2′-deoxyguanosine-5′-monophosphate (2′-dGuo), and 2′-deoxyadenosine-5′-monophosphate (2′-dAdo) in vitro. These adduct markers were then employed for comparison with those obtained in the rat after in vivo treatment with 6-NC. On the basis of the results, it appears that the major adduct formed in the liver of rats treated with 6-NC is not derived from 6-NCDE.

Introduction Humans are exposed to NO2-PAHs in cooked foods, and in air pollution made up of combustion products (1-3). 6-Nitrochrysene (6-NC),1 one member of this class of compounds, has been shown to be mutagenic in both bacterial and mammalian cell systems (4, 5) and to be tumorigenic in several rodent assays (6). Mutagenic NO2PAHs have been detected in tissues of lung cancer patients who were nonsmokers, implicating these compounds as possible initiators of lung tumors in humans (7). Although 6-NC is present in the environment at rather low levels, research on this compound has been inspired because of its remarkable carcinogenic activity in the newborn mouse lung adenoma assay (8, 9). In fact, 6-NC is the most potent lung tumorigen among all the NO2-PAHs tested in this model assay, and its carcinogenic potency approximates that of certain ultimate carcinogenic metabolites of PAHs (8). In addition, intramammary injection of 6-NC induced mammary tumors in female CD rats whereby 6-NC was more potent than the bay region diol epoxide derived from benzo[a]pyrene (10, 11). We have also shown that oral administration of 6-NC elicits mammary cancer in female CD rats (12). However, ip injection of 6-NC into CD rats leads to the * To whom requests for reprints should be addressed. † Organic Synthesis Facility. ‡ Division of Cancer Etiology and Prevention. 1 Abbreviations: 6-NC, 6-nitrochrysene; 6-NCDE, anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene; 2′-dGuo, 2′-deoxyguanosine 5′-monophosphate; 2′-dAdo, 2′-deoxyadenosine 5′-monophosphate; N-OH-6-AC, N-hydroxy-6-aminochrysene; 1,2-DHD-6-NC, 1,2dihydroxy-1,2-dihydro-6-nitrochrysene; 6-AC, 6-aminochrysene; 1,2DHD-6-AC, 1,2-dihydroxy-1,2-dihydro-6-aminochrysene.

formation of adenomas and adenocarcinomas in the colon (13). Because humans are exposed to these agents and human tissues are capable of activating 6-NC to genotoxic metabolites (14, 15), 6-NC may play a role in the induction of certain human cancers (lung, breast, and colon). Therefore, it is essential to delineate the mechanisms of action that account for the carcinogenicity of 6-NC. Studies in mice and rats and in vitro assays have indicated that 6-NC can be activated by two major pathways. The first pathway, involving the formation of N-hydroxy-6-aminochrysene (N-OH-6-AC) by simple nitro reduction, yields three major DNA adducts: N-(dG-8-yl)6-AC, N-(dI-8-yl)-6-AC, and 5-(dG-N2-yl)-6-AC (16). The second pathway proceeds via the formation of the proximate carcinogen 1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (1,2-DHD-6-NC) or 1,2-dihydroxy-1,2-dihydro-6aminochrysene (1,2-DHD-6-AC) by terminal ring oxidation or by a combination of terminal ring oxidation and nitro reduction, respectively (17); 1,2-DHD-6-NC or 1,2-DHD6-AC yields a major DNA adduct resulting from the reaction with 2′-dGuo in the presence of rat liver microsomes in vitro (18). Levels of this adduct, and of those derived from simple nitro reduction pathways, vary depending on the animal model and route of administration of 6-NC (19-21). Unequivocal identification of the adduct(s) derived from ring oxidation or from a combination of ring oxidation and nitro reduction has not been achieved. We hypothesize that 6-NCDE or its amino derivative is the ultimate carcinogenic metabolite of 6-NC that is responsible for the formation of the unknown DNA adduct detected in vivo (19-21). Thus, in the study

10.1021/tx000104n CCC: $19.00 © 2000 American Chemical Society Published on Web 10/05/2000

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presented here, we describe for the first time a convenient synthesis of 6-NCDE and our initial investigation of its binding with calf thymus DNA, 2′-dGuo, and 2′-dAdo in vitro. These adduct markers were then employed for comparison with those obtained in vivo after treatment of rats with 6-NC.

Materials and Methods Caution: 6-Nitrochrysene, 1,2-DHD-6-NC, and 1,2-DHD-6AC are mutagenic in bacterial systems and tumorigenic in rodents. Therefore, appropriate safety procedures must be followed when working with these compounds. Synthesis. Melting points were recorded on a Fisher-Johnson melting point apparatus and are uncorrected. Proton NMR spectra were recorded in CDCl3 using a Bruker AM 360WB instrument. The chemical shifts are reported in parts per million downfield from TMS. MS was carried out on a Hewlett-Packard model 5988A instrument. High-resolution MS was carried out on a Finnigan Mat95 instrument at the University of Minnesota (Minneapolis, MN). Thin-layer chromatography (TLC) was carried out on aluminum-supported, precoated silica gel plates (EM Industries, Gibbstown, NJ). All starting materials were obtained from Aldrich Chemical Co. (Milwaukee, WI). 1,2,3,4Tetrahydrochrysene-1-one (22) was synthesized in three steps from 2-(1-naphthyl)ethyl bromide by following a procedure slightly modified from that described by Harvey et al. (23). (1) 1-Acetoxy-1,2,3,4-tetrahydrochrysene (1). NaBH4 (1.32 g, 34.9 mmol) was added to a solution of the 1,2,3,4-tetrahydrochrysene-1-one (1.4 g, 5.70 mmol) in 14 mL of a THF/CH3OH mixture (1:1). The reaction mixture was then stirred at room temperature for 2 h and poured into H2O. The reaction mixture was extracted with EtOAc (3 × 2 mL), washed with H2O, and dried (MgSO4). The workup gave the alcohol as a white solid (1.40 g, 98%): mp 202-204 °C; MS m/z (relative intensity) 248 (M+, 100), 230 (80), 220 (60), 215 (40), 191 (45), 178 (20), 165 (10). This white solid was dissolved in 30 mL of a benzene/pyridine mixture (1:1), and DMAP (50 mg) and 5 mL of Ac2O were added. The reaction mixture was stirred for 3 h at room temperature and then evaporated to dryness. The residue was diluted with H2O and extracted with EtOAc. The ethyl acetate extract was dried (MgSO4) and concentrated to afford 1.35 g (81%) of compound 1: mp 167-168 °C; 1H NMR δ 2.02-2.15 (m and s, 7H, CH3 and CH2), 3.10-3.15 (m, 1H, CH2), 3.35-3.41 (m, 1H, CH2), 6.20 (bt, 1H, CHOAc), 7.56 (d, 1H, H12, J11,12 ) 8.53 Hz), 7.58-7.66 (m, 2H, H8 and H9), 7.81 (d, 1H, H6, J5,6 ) 9.19 Hz), 7.90 (dd, 1H, H7, J7,8 ) 8.86 Hz, J7,9 ) 1.31 Hz), 7.99 (d, 1H, H5, J5,6 ) 9.19 Hz), 8.58 (d, 1H, H11, J11,12 ) 8.53 Hz), 8.69 (d, 1H, H10, J9,10 ) 8.21 Hz); MS m/z (relative intensity) 290 (M+, 10), 230 (100), 215 (30), 202 (10), 189 (10); high-resolution MS m/z calcd for C20H18O2Na 313.1212, found 313.1212. (2) 1-Acetoxy-1,2,3,4-tetrahydro-6-nitrochrysene (2). To a stirred solution of 1 (1.35 g, 4.66 mmol) in 50 mL of dry CH2Cl2 was added a 1.3 M solution of N2O4 in CH2Cl2 (43 mL) over the course of 5 min. Stirring was continued at room temperature for 3 h. The excess N2O4 was removed by flushing a stream of N2 through the reaction mixture. The reaction mixture was poured into H2O, and the organic layer was washed with a NaHCO3 solution (2 × 50 mL). The workup gave 1.2 g of a mixture of two products. This was chromatographed on silica gel and eluted with a hexane/EtOAc mixture (80:20) to give compound 2 (870 mg, 59%): mp 56-60 °C; 1H NMR δ 2.062.18 (m and s, 7H, OAc and CH2), 3.16-3.19 (m, 1H, CH2), 3.383.43 (m, 1H, CH2), 6.18 (bt, 1H, CHOAc), 7.74 (d, 1H, H12, J11,12 ) 8.53 Hz), 7.76-7.81 (m, 2H, H8 and H9), 8.50-8.53 (m, 1H, H7), 8.57 (d, 1H, H11, J11,12 ) 8.53 Hz), 8.75 (s, 1H, H5), 8.778.78 (m, 1H, H10); high-resolution MS m/z calcd for C20H17NO4Na 358.1055, found 358.1041. Further elution with a hexane/EtOAc mixture (1:1) gave 1-acetoxy-1,2,3,4-tetrahydro-12-nitrochrysene (300 mg, 14%): 1H NMR δ 2.0-2.04 (m and s, 5H, OAc and CH ), 2.08-2.14 2

Krzeminski et al. (m, 1H, CH2), 2.16-2.73 (m, 1H, CH2), 3.23-3.39 (m, 2H, CH2), 6.15 (bt, 1H, CHOAc), 7.68-7.77 (m, 2H, H8 and H9), 7.957.98 (m, 1H, H7), 7.99 (d, 2H, H5 and H6, J5,6 ) 9.19 Hz), 8.65 (d, 1H, H10, J9,10 ) 7.87 Hz), 9.03 (s, 1H, H11). (3) 3,4-Dihydro-6-nitrochrysene (3). To a solution of 2 (0.55 g, 1.73 mmol) in 10 mL of a dry THF/MeOH mixture (1:1) was added NaOMe (150 mg, 2.77 mmol), and the resulting mixture was refluxed for 10 min. This reaction mixture was poured into H2O and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with H2O (2 × 30 mL), dried (MgSO4), and concentrated to afford crude 1-hydroxy1,2,3,4-tetrahydro-6-nitrochrysene. To the above crude alcohol (0.41 g, 1.48 mmol) was added p-toluenesulfonic acid (10 mg) in benzene (100 mL), and the mixture was heated at reflux for 30 min in a Dean-Stark apparatus. Workup, including chromatography on silica gel and elution with a CH2Cl2/hexane mixture (30:70), gave olefin 3 in 68% yield (0.32 g): mp 135-136 °C; 1H NMR δ 2.53-2.57 (m, 2H, CH2), 3.31 (t, 2H, CH2), 6.21-6.26 (m, 1H, olefin H2), 6.64 (d, 1H, olefin H1, J1,2 ) 9.42 Hz), 7.52 (d, 1H, H12, J11,12 ) 8.54 Hz), 7.72-7.76 (m, 2H, H8 and H9), 8.47 (dd, 1H, H7, J7,8 ) 6.89 Hz, J7,9 ) 1.96 Hz), 8.51 (d, 1H, H11, J11,12 ) 8.55 Hz), 8.72-8.75 (m and s, 2H, H5 and H10); MS m/z (relative intensity) 275 (M+, 100), 245 (80), 228 (90), 202 (20); highresolution MS m/z calcd for C18H13NO2 275.0868, found 275.0956. (4) trans-1,2-Bis(benzoyloxy)-1,2,3,4-tetrahydro-6-nitrochrysene (4). A mixture of silver benzoate (0.80 g, 3.5 mmol) and I2 (0.44 g, 1.77 mmol) in dry benzene (50 mL) was stirred under reflux until the red color disappeared. A solution of olefin 3 (0.32 g, 1.16 mmol) in 10 mL of dry benzene was added, and the resulting mixture was stirred under reflux for 12 h. The reaction mixture was filtered while hot and washed with benzene (2 × 25 mL). The filtrate was concentrated to give a solid that was chromatographed on a silica gel column. Hexane eluted the unreacted olefin 3 (60 mg). Further elution with a hexane/EtOAc mixture (1:1) gave the pure dibenzoate derivative 4 (0.51 g, 85%): mp 205-207 °C; 1H NMR δ 2.41-2.51 (m, 1H, CH2), 2.62-2.71 (m, 1H, CH2), 3.53-3.56 (m, 2H, CH2), 5.685.70 (m, 1H, H2), 6.70 (d, 1H, H1, J1,2 ) 5.56 Hz), 7.35-7.43 (m, 4H, from benzoyl ester), 7.45-7.56 (m, 2H, from benzoyl ester), 7.78-7.83 (m, 3H, H8, H9, and H12), 7.94-7.97 (m, 2H, from benzoyl ester), 8.07-8.09 (m, 2H, from benzoyl ester), 8.51-8.55 (m, 1H, H7), 8.62 (d, 1H, H11, J11,12 ) 9.19 Hz), 8.758.81 (m and s, 2H, H5 and H10); high-resolution MS m/z calcd for C32H23NO6Na 540.1423, found 540.1433. (5) trans-1,2-Bis(benzoyloxy)-4-bromo-1,2,3,4-tetrahydro-6-nitrochrysene (5). A suspension of N-bromosuccinimide (0.1 g, 0.56 mmol), dibenzoate 4 (0.22 g, 0.43 mmol), and benzoyl peroxide (5 mg) in CCl4 (6 mL) was heated under reflux in a N2 atmosphere for 30 min. The reaction mixture was cooled, and the succinimide was removed by filtration. The filtrate was concentrated and chromatographed on silica gel, and an EtOAc/ hexane mixture (1:1) eluted an unstable bromo compound 5 (180 mg, 71%): mp 195-197 °C; 1H NMR δ 3.15-3.22 (m, 2H, CH2), 5.79 (dd, 1H, H2, J1,2 ) 3.28 Hz, J2,3 ) 6.89 Hz), 6.18 (dd, 1H, H4, J3,4 ) 5.25 Hz, J3′,4 ) 1.97 Hz), 6.69 (d, 1H, H1, J1,2 ) 3.28 Hz), 7.35-7.59 (m, 6H), 7.80-7.85 (m, 2H), 7.91 (d, 1H), 8.008.13 (m, 4H), 8.50-8.52 (m, 1H), 8.76-8.79 (m, 2H), 9.01 (s, 1H, H5). This bromo compound was used without further purification for the next step. (6) trans-1,2-Bis(benzoyloxy)-1,2-dihydro-6-nitrochrysene (6). To a solution of bromide 5 (0.125 g, 0.02 mmol) in 20 mL of dry xylene was added 300 mg of NaHCO3. This mixture was heated under reflux for 1 h. The product was filtered while hot and washed with benzene (2 × 10 mL). The filtrate was concentrated to yield a solid that was chromatographed on silica gel. Elution with a hexane/CH2Cl2 mixture (1:1) gave 6 (67 mg, 62%): mp 215-217 °C; 1H NMR δ 6.17-6.20 (m, 1H, H2), 6.52 (dd, 1H, H3, J2,3 ) 3.61 Hz, J3,4 ) 10.17 Hz), 6.87 (d, 1H, H1, J1,2 ) 7.87 Hz), 7.26-7.57 (m, 7H), 7.78-7.79 (m, 2H), 7.89 (d, 1H, J ) 8.53 Hz), 7.98 (d, 1H, J ) 8.2 Hz), 8.08 (d, 1H, J ) 8.55 Hz), 8.48-8.53 (m, 1H), 8.67 (d, 1H, H11, J11,12 ) 8.53 Hz), 8.74-

Synthesis of 6-Nitrochrysene Diol Epoxide 8.77 (m, 1H, H10), 8.86 (s, 1H, H5); high-resolution MS m/z calcd for C32H21NO6Na 538.1288, found 538.1288. (7) trans-1,2-Dihydroxy-1,2-dihydro-6-nitrochrysene (7). To a solution of 6 (51 mg, 0.1 mmol) in 6 mL of a dry THF/ MeOH mixture (1:1) was added NaOMe (11 mg, 0.2 mmol). This mixture was stirred under reflux for 15 min. The reaction mixture was concentrated, suspended in cold water, extracted with EtOAc, then dried over MgSO4, and evaporated to dryness. Crystallization of the crude product from MeOH gave 7 (15 mg, 48%) as yellow crystals: mp 230-232 °C dec; 1H NMR δ 4.314.40 (m, 1H, H2), 4.77 (dd, 1H, H1, J1,2 ) 11.16 Hz, J1,OH ) 5.91 Hz), 5.39 (d, 1H, OH2, J2,OH2 ) 5.95 Hz), 5.79 (d, 1H, OH1, JH,OH1 ) 5.95 Hz), 6.25 (dd, 1H, H3, J2,3 ) 2.3 Hz, J3,4 ) 10.17 Hz), 7.38 (d, 1H, H4, J3,4 ) 10.17 Hz), 7.81-7.89 (m, 2H, H8 and H9), 8.08 (d, 1H, H12, J11,12 ) 8.56 Hz), 8.22 (d, 1H, H7, J7,8 ) 8.20 Hz), 8.89 (d, 1H, H11, J11,12 ) 8.56 Hz), 8.94 (s, 1H, H5), 9.01 (d, 1H, H10, J9,10 ) 8.2 Hz); CIMS m/z (relative intensity) 308 (M + 1, 40), 290 (M + 1 - H2O, 45), 260 (100), 244 (40); high-resolution MS m/z calcd for C18H13NO4 307.0844, found 307.0832. HPLC chromatographic characteristics (retention time of 74 min) and spectral data of this diol are identical to those of a previously characterized metabolite of 6-NC (17). (8) anti-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6nitrochrysene (8). A mixture of dihydrodiol 7 (10 mg, 0.032 mmol) and recrystallized m-chloroperoxybenzoic acid (100 mg, 0.58 mmol) in 20 mL of dry tetrahydrofuran was stirred in a N2 atmosphere for 5 h. This reaction mixture was then diluted with diethyl ether (100 mL) and washed with a 2% sodium hydroxide solution (3 × 20 mL) and then with H2O. The organic layer was dried over K2CO3. Evaporation of the solvent gave the light, cream-colored diol epoxide 8 (8 mg, 76%), which was recrystallized from an ether/THF mixture: mp 238-240 °C dec; 1H NMR δ 3.88 (dd, 1H, H3, J 2,3 ) 0.96 Hz, J3,4 ) 4.26 Hz), 4.03 (dd, 1H, H2, J1,2 ) 8.53 Hz, J2,3 ) 0.99 Hz), 4.78 (d, 1H, H1, J1,2 ) 8.53 Hz), 5.04 (d, 1H, H4, J3,4 ) 4.26 Hz), 7.84-7.93 (m, 2H, H8 and H9), 8.29 (d, 1H, H12, J11,12 ) 8.86 Hz), 8.33 (dd, 1H, H7, J7,8 ) 7.86 Hz, J7,9 ) 1.32 Hz), 9.00 (d, 1H, H11, J11,12 ) 8.86 Hz), 9.05 (dd, 1H, H10, J8,10 ) 0.64 Hz, J9,10 ) 9.51 Hz), 9.51 (s, 1H, H5); high-resolution MS m/z calcd for C18H13NO5 323.0816, found 323.0816. Reaction of 6-NCDE with Calf Thymus DNA. About 1 mg of 6-NCDE (8) in 1 mL of acetone was added gradually over 2 h to a solution of calf thymus DNA (10 mg) in 10 mL of 100 mM Tris-HCl buffer (pH 7). This mixture was incubated at 37 °C overnight. Tetraols were removed from the DNA solutions by extraction with ethyl acetate (3 × 10 mL), followed by extraction with diethyl ether (3 × 5 mL). One of the major tetraols was isolated: 1H NMR (acetone-d6) δ 4.18 (dd, 1H, H2, J1,2 ) 8.64 Hz, J2,3 ) 2.2 Hz), 4.39 (bs, 1H, H3), 4.95 (d, 1H, H1, J1,2 ) 8.64 Hz), 5.47 (bs, 1H, H4), 7.83-7.88 (m, 2H, H8 and H9), 8.17 (d, 1H, H12, J11,12 ) 8.86 Hz), 8.38 (dd, 1H, H7, J7,8 ) 8.66 Hz, J7,9 ) 2.05 Hz), 8.92 (d, 1H, H11, J11,12 ) 8.86 Hz), 9.01 (dd, 1H, H10, J8,10 ) 1.97 Hz, J9,10 ) 9.57 Hz), 9.04 (s, 1H, H5). The UV spectrum of the tetraol was similar to that of 9-nitrophenanthrene (Figure 1). The retention time of the major tetraol under HPLC conditions described below was 27.2 min. A minor tetraol eluting after 31.8 min also exhibited a UV spectrum similar to that of 9-nitrophenanthrene. The DNA was isolated and hydrolyzed enzymatically to deoxyribonucleosides (24). Modified deoxyribonucleosides were separated from unmodified deoxyribonucleosides on Sep-Pak C18 cartridges (25), and analyzed by HPLC as described below. Reactions of 6-NCDE with 2′-dGuo or 2′-dAdo. About 100 mg each of 2′-dGuo or 2′-dAdo were dissolved in 10 mL of 100 mM Tris-HCl buffer (pH 7.0). To the above solution was added about 1 mg of 6-NCDE in 1 mL of acetone gradually over 2 h before the mixture was incubated overnight at 37 °C. Samples were extracted with ethyl acetate (3 × 10 mL) and with diethyl ether (3 × 5 mL). A trace of ether in the aqueous layer was flushed away with a stream of nitrogen, and the modified deoxyribonucleotides were hydrolyzed enzymatically to deoxy-

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Figure 1. UV spectra of 9-nitrophenanthrene (A) and 1,2,3,4tetrahydroxy-1,2,3,4-tetrahydro-6-nitrochrysene (B). ribonucleosides as described previously (25-27). Unmodified deoxyribonucleosides were separated from modified deoxyribonucleosides on Sep-Pak C18 cartridges (25), and modified deoxyribonucleosides were analyzed by HPLC as described below. HPLC Analysis of Modified 2′-Deoxyribonucleosides. HPLC on a Nucleosil C18 column (5 µm, 4.6 mm × 250 mm; Phenomenex, Inc., Torrence, CA) utilized the following gradient program to separate the modified deoxyribonucleosides: a linear gradient of 30:70 MeOH/water to 85:15 MeOH/water over the course of 55 min and then to 100% MeOH over the course of 9 min.

Results and Discussion Initially, we attempted to synthesize the diol epoxide of 6-NC using 1,2,3,4-tetrahydrochrysene-1-one as the starting material. However, nitration of 1,2,3,4-tetrahydrochrysene-1-one, using N2O4 in methylene chloride, failed to yield a significant quantity of the desired compound, 1,2,3,4-tetrahydro-6-nitrochrysene-1-one. Instead, on the basis of NMR analysis, 1,2,3,4-tetrahydro5-nitrochrysene-1-one was isolated as the major product: 1H NMR δ 2.34-2.42 (m, 2H, CH2), 2.81 (dd, 2H, CH2, J ) 6.24 Hz), 3.50 (dd, 2H, CH2, J ) 6.24 Hz), 7.817.87 (m, 2H, H8 and H9), 8.47 (d, 1H, H12, J11,12 ) 8.86 Hz), 8.52-8.55 (m, 1H, H7), 8.68 (d, 1H, H11, J11,12 ) 8.86 Hz), 8.78 (s, 1H, H6), 8.80-8.83 (m, 1H, H10).

An alternative approach was designed as outlined in Scheme 1; here, 1-acetoxy-1,2,3,4-tetrahydro-6-nitrochrysene (2) is the key intermediate for the synthesis. Reduction of 1,2,3,4-tetrahydrochrysene-1-one with sodium borohydride furnished the corresponding alcohol

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Krzeminski et al.

Scheme 1. Synthesis of anti-1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene

which was acetylated with acetic anhydride in pyridine to give 1-acetoxy-1,2,3,4-tetrahydrochrysene (1) in 81% yield. Nitration of the acetoxy derivative 1 at room temperature with N2O4 yielded two isomers. These two isomers were separated by column chromatography, and their structures were confirmed by 1H NMR. From the 1 H NMR spectral analysis, we deduced that the major isomer was 1-acetoxy-1,2,3,4-tetrahydro-6-nitrochrysene (2), while the minor isomer was 1-acetoxy-1,2,3,4-tetrahydro-12-nitrochrysene. The evidence in support of our assignment of the major isomer as 1-acetoxy-1,2,3,4tetrahydro-6-nitrochrysene (2) is the downfield shift of H5 and the absence of its coupling with H6. In addition, both H11 and H12 remained as two sets of doublets. On the other hand, the 1-acetoxy-1,2,3,4-tetrahydro-12-nitrochrysene retains both H5 and H6 as a set of doublets, whereas H11 appeared as a singlet and shifted downfield due to the nitro substitution at a neighboring carbon. The acetoxy group was removed by sodium methoxide hydrolysis, and the resulting alcohol was dehydrated with p-toluenesulfonic acid to the corresponding 3,4-dihydro6-nitrochrysene (3) in 68% yield. The structure of compound 2 was further confirmed by reaction of nitro olefin 3 with DDQ to give 6-nitrochrysene, and the proton NMR spectrum was identical to that of authentic 6-nitrochrysene. This confirmed that the nitro group was in the 6-position in the acetoxy derivative, 2. Compound 3 was converted to the corresponding trans-dihydrodiol 7 and anti-diol epoxide 8 by a procedure modified from the one described by Fu et al. (28). Under standard Prevost conditions, olefin 3 reacted with silver acetate to give the desired diacetate derivative, but in poor yield. However, substitution of silver acetate with silver benzoate in the standard Prevost reaction gave trans-1,2-bis(benzoyloxy)1,2,3,4-tetrahydro-6-nitrochrysene (4) in 85% yield. Dehydrogenation of 4 with DDQ yielded a complex mixture, but the desired trans-1,2-bis(benzoyloxy)-1,2-dihydro-6nitrochrysene (6) was not detected. Therefore, an alter-

native synthetic approach was employed. Bromination of trans-dibenzoate, 4, with NBS in carbon tetrachloride led to an unstable trans-1,2-bis(benzoyloxy)-4-bromo1,2,3,4-tetrahydro-6-nitrochrysene (5) which was used without further purification for the next step. Dehydrobromination of compound 5 with sodium bicarbonate in xylene gave trans-1,2-bis(benzoyloxy)-1,2-dihydro-6-nitrochrysene (6). The dibenzoate-protecting group was easily removed by treating compound 6 with sodium methoxide to furnish trans-dihydrodiol 7 as a yellowish solid in 48% yield. Compound 7 had chromatographic and spectral properties that were identical to those of a previously established metabolite of 6-NC (17). Finally, epoxidation of dihydrodiol 7 with m-chloroperoxybenzoic acid afforded anti-diol epoxide 8 as a creamy solid in 76% yield. 1,2-DHD-6-NC (7) (or its amino derivative) and 6-NCDE (8) (or its amino derivative) have been implicated as proximate and ultimate carcinogens, respectively (17). To prepare markers for the identification of unknown DNA adducts that are formed in vivo, we have incubated 6-NCDE (8) with dGuo, dAdo, and calf thymus DNA. The modified dGuo, dAdo, and DNA were isolated and enzymatically hydrolyzed to the corresponding modified nucleosides. The HPLC profiles of the standard 6-NCDEmodified 2′-deoxyguanosine and 2′-deoxyadenosine adducts are illustrated in panels A and B of Figure 2, respectively. Panel C represents the HPLC profile of the 6-NCDEmodified calf thymus DNA; both modified 2′-deoxyguanosine and 2′-deoxyadenosine adducts had been formed. In comparison, panel D represents the HPLC radiochromatogram profile of DNA isolated from the liver of rats treated with [12-3H]-6-nitrochrysene (21). The retention time of the major adduct formed in vivo is between 19 and 20 min (panel D). This clearly indicates that neither 6-NCDE-modified 2′-deoxyguanosine nor 6-NCDE-modified 2′-adenosine is involved in the formation of the DNA adducts observed in the rat (21).

Synthesis of 6-Nitrochrysene Diol Epoxide

Figure 2. HPLC profiles of the standard 6-NCDE-modified 2′deoxyguanosine and 2′-deoxyadenosine adducts (A and B, respectively). HPLC profile of the 6-NCDE-modified calf thymus DNA (C). HPLC radiogram profile of DNA isolated from the liver of rats treated with [12-3H]-6-nitrochrysene (D).

In summary, we described for the first time the synthesis of 1,2-DHD-6-AC, 6-NCDE, and 1,2,3,4-tetrahydroxy-1,2,3,4-tetrahydro-6-nitrochrysene. We have also prepared highly useful markers, namely, 6-NCDEmodified 2′-deoxyguanosine and 2′-deoxyadenosine adducts. Comparison of HPLC profiles of these markers with that detected in liver DNA obtained from rats treated with 6-NC showed that 6-NCDE is not involved in the formation of the DNA adducts observed in vivo. Therefore, we are currently testing alternative hypotheses involving DNA adduct formation with either 1,2dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-aminochrysene or the hydroxylamine derived from the nitro reduction of 1,2-DHD-6-NC or partial oxidation of the amino functionality of 1,2-DHD-6-AC.

Acknowledgment. We thank Mr. John Cunningham from the American Health Foundation’s Instrumentation Facility and acknowledge Dr. V. Reddy’s initial attempt to synthesize 6-NCDE. This study was supported by NCI Contract NCI-CB-77022-75, NCI Grant CA-35519, and Cancer Center Support Grant CA-17613. We thank Ms. Ilse Hoffmann for editing the manuscript.

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