Synthesis and Identification of Major Metabolites of Environmental

Aug 18, 2005 - Penn State Milton S. Hershey Medical Center, Penn State College of Medicine, Department of Pharmacology, H078, 500 University Drive, ...
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Chem. Res. Toxicol. 2005, 18, 1438-1443

Synthesis and Identification of Major Metabolites of Environmental Pollutant Dibenzo[c,mno]chrysene Arun K. Sharma* and Shantu Amin Penn State Milton S. Hershey Medical Center, Penn State College of Medicine, Department of Pharmacology, H078, 500 University Drive, Hershey, Pennsylvania 17033 Received April 22, 2005

Dibenzo[c,mno]chrysene commonly known as naphtho[1,2-a]pyrene (N[1,2-a]P) is an environmental pollutant, recently identified in coal tar extract, in air-borne particulate matter, in marine sediment, and in cigarette-smoke condensate. We recently reported an efficient synthesis of N[1,2-a]P and examined its in vitro metabolism by male Sprague Dawley rat liver S9 fraction, which resulted in a number of dihydrodiol and phenolic metabolites. The synthesis of 10-hydroxy-N[1,2-a]P and fjord region N[1,2-a]P trans-9,10-dihydrodiol, which were identified among the various metabolites, was assigned earlier by comparing with the synthetic standards. The other major metabolites were separated by HPLC and, based on the 1H NMR analysis, were tentatively suggested to be the two K-region dihydrodiols, that is, N[1,2-a]P trans-4,5dihydrodiol (6) and N[1,2-a]P trans-7,8-dihydrodiol (7), and the hydroxy derivatives of N[1,2a]P. To unequivocally assign the structure to each of the peaks and to have them in larger amounts for toxicological studies, we have now synthesized the two K-region dihydrodiols and the 1-/3-hydroxy-N[1,2-a]P, short-listed based on the proton NMR of the collected peaks. The K-region dihydrodiols 6 and 7 were prepared by the treatment of N[1,2-a]P with OsO4 to give a mixture of cis dihydrodiols 2 and 3, followed by pyridinium chlorochromate-assisted oxidation to quinones 4 and 5, and finally reduction with NaBH4 to afford the dihydrodiols 6 and 7 with the desired trans stereochemistry. The 1-hydroxy-N[1,2-a]P (22) and 3-hydroxy-N[1,2-a]P (23) were synthesized using a photochemical approach. As expected, all the synthesized dihydrodiol and phenolic derivatives of N[1,2-a]P identified with those obtained from in vitro metabolism enabling the assignment of all the major metabolites.

Introduction The mutagenicity/carcinogenicity of polycyclic aromatic hydrocarbons (PAHs)1 in general has been found to depend on their structural features. Diol epoxides derived from PAHs that contain a fjord region have been shown to be more potent carcinogens than those derived from bay region (1-4). The strong steric interactions of the fjord region force the molecule to distort from planarity. These nonplanar diol epoxides react more favorably with deoxyadenosine (dA) than deoxyguanosine (dG) in DNA (5-12), and the fjord-region modified-DNA adducts have been found to be more difficult to repair than a bay-region adduct (12-15). Dibenzo[a,l]pyrene (DB[a,l]P), having a fjord region in the molecule, is by far the most potent mammary gland (16, 17), skin (16-18), lung (19), and * To whom correspondence should be addressed. Arun K. Sharma, Penn State Milton S. Hershey Medical Center, Penn State College of Medicine, Department of Pharmacology, H078, 500 University Drive, Hershey, PA 17033. Phone: 717-531-0003, ext 285016. Fax: 717-5315013. E-mail: [email protected]. 1 Abbreviations: PAHs, polycyclic aromatic hydrocarbons; N[1,2a]P, dibenzo[c,mno]chrysene (naphtho[1,2-a]pyrene); N[1,2-a]P trans9,10-dihydrodiol, (() trans-9,10-diydroxy-9,10-dihydronaphtho[1,2-a]pyrene; N[1,2-a]P cis-4,5-dihydrodiol, (() cis-4,5-dihydroxy-4,5-dihydronaphtho[1,2-a]pyrene; N[1,2-a]P cis-7,8-dihydrodiol, (() cis-7,8dihydroxy-7,8-dihydronaphtho[1,2-a]pyrene; N[1,2-a]P trans-4,5-dihydrodiol, (() trans-4,5-dihydroxy-4,5-dihydronaphtho[1,2-a]pyrene; N[1,2a]P trans-7,8-dihydrodiol, (() trans-7,8-dihydroxy-7,8-dihydronaphtho[1,2-a]pyrene; N[1,2-a]PDE, (() anti-9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydro-N[1,2-a]P; 1-OH-N[1,2-a]P, 1-hydroxynaphtho[1,2-a]pyrene; 3-OH-N[1,2-a]P, 3-hydroxynaphtho[1,2-a]pyrene; DB[a,l]P, dibenzo[a,l]pyrene.

oral (20) tumorigen known. It metabolizes to anti- and syn-11,12-diol-13,14-epoxides (21) which are potent tumorigens. The K-region dihydrodiol metabolite, that is, DB[a,l]P trans-8,9-dihdyrodiol, is found to metabolize further to active intermediates and bind to DNA to give stable adducts (22). Dibenzo[c,mno]chrysene (Naphtho[1,2-a]pyrene, N[1,2-a]P), an environmental pollutant (23, 24), also contains a fjord region and is a structural isomer of DB[a,l]P. Its metabolism using S9 fraction from phenobarbital/β-naphthoflavone-induced rat liver homogenate showed, apart from phenolic derivatives, the formation of a fjord region N[1,2-a]P trans-9,10-dihydrodiol and two K-region dihydrodiols (25), which can metabolize further to corresponding diol epoxides and cause DNA lesions. Because of these similarities in structure and metabolism pattern, we anticipate N[1,2a]P to be an equally potent carcinogen as DB[a,l]P. Our ongoing investigations have supported this fact and revealed that N[1,2-a]P is highly toxic in the AJ mouse model assay even at very low doses (unpublished results) just like DB[a,l]P. However, its carcinogenic potential

10.1021/tx050109q CCC: $30.25 © 2005 American Chemical Society Published on Web 08/18/2005

Synthesis and Identification of N[1,2-a]P Metabolites

remained unexplored essentially because of the lack of synthetic routes available for the synthesis of its potential metabolites. We had previously synthesized the N[1,2-a]P trans-9,10-dihydrodiol and 10-hydroxy-N[1,2-a]P which were identified as in vitro metabolites (25, 26). To unequivocally assign the structure of the remaining phenolic and dihydrodiol metabolites and to have them in larger amounts for toxicological studies and as markers for various biological studies, chemical standards are required. On the basis of these considerations, we developed convenient methods for their syntheses that led to the identification of all the major metabolites formed in vitro.

Materials and Methods Melting points were recorded on a Fischer-Johns melting point apparatus and are uncorrected. Unless otherwise stated, NMR spectra were recorded using a Bruker AM 360WB 360 MHz NMR spectrometer in CDCl3 with tetramethylsilane (TMS) as an internal standard. Chemical shifts were recorded in ppm downfield from the internal standard. MS were run on a Hewlett-Packard model 5988A instrument. High-resolution MS (EI) were determined at the Chemistry Instrumentation Center, State University of New York at Buffalo. Thin-layer chrtomatography (TLC) was developed on aluminum-supported precoated silica gel plates (EM industries, Gibbstown, NJ). Ethyl 5-methoxy-1-naphthaleneacetate and ethyl 7-methoxy-1-naphthaleneacetate were prepared from 5-methoxy tetralone and 7-methoxy tetralone, respectively, using literature methods (27). In vitro metabolism of N[1,2-a]P with phenobarbital/β-naphthoflavone-induced Male Sprague Dawley Rat S9 Liver Homogenate was carried out as reported earlier (25). The metabolites of N[1,2-a]P were analyzed by HPLC on a 4.6 mm × 250 mm (5 µM) Vydac C18 reverse-phase column (Separation Group Hesperia, CA) with solvent A (H2O) and solvent B (methanol), using a gradient program from A-B (50:50) to A-B (0:100) over 40 min. cis-4,5-Dihydroxy-4,5-dihydronaphtho[1,2-a]pyrene (2)/ cis-7,8-Dihydroxy-7,8-dihydronaphtho[1,2-a]pyrene (3). To a solution of 1 (106 mg, 0.35 mmol) in pyridine (3 mL) was added a solution of OsO4 (178 mg, 0.70 mmol) in pyridine (1 mL), and the mixture was stirred at room temperature under nitrogen for 18 h. The reaction mixture was quenched with aqueoussaturated NaHSO3 solution and extracted with EtOAc. The organic layer was washed with water and dried (MgSO4), and the solvent was removed. The crude mixture was chromatographed over a silica gel column (EtOAc/CH2Cl2 1:9) to afford a mixture (91 mg, 77%) of N[1,2-a]P cis-4,5-dihydrodiol 2 and N[1,2-a]P cis-7,8-dihydrodiol 3. 1H NMR (acetone-d6) of 2/3 δ: 4.92-4.95 (m, 1H), 5.04-5.08 (m, 1H), 5.22-5.25 (m, 1H), 5.305.33 (m, 1H), 7.42-7.48 (m, 1H), 7.51-7.57 (m, 1H), 7.66-7.77 (m, 4H), 7.88 (d, 1H, J ) 6.9 Hz), 7.99-8.29 (m, 13H), 8.46 (br s, 1H), 8.78 (d, 1H, J ) 9.5 Hz), 9.07-9.12 (m, 2H). Naphtho[1,2-a]pyrene-4,5-dione (4)/Naphtho[1,2-a]pyrene-7,8-dione (5). The mixture of 2/3 (50 mg, 0.15 mmol) was dissolved in CH2Cl2 (6 mL) and THF (0.2 mL), and to it was added PCC (97 mg, 0.45 mmol). The reaction mixture was stirred at room temperature for 2 h, diluted with CH2Cl2, and washed with water. The organic layer was dried over MgSO4 and filtered, and the solvent was removed to give a mixture which was purified by tituration with an ether/hexanes (1:9) to yield a mixture of diones 4/5 (44 mg, 88%) as a dark red solid. 1H NMR δ: 7.48 (t, J ) 7.2 Hz, 1H), 7.70-8.20 (m, 17H), 8.47 (d, 1H, J ) 7.2 Hz), 8.54 (d, 1H, J ) 9.5 Hz), 8.67-8.68 (m, 2H), 8.87-8.90 (m, 2H). trans-4,5-Dihydroxy-4,5-dihydronaphtho[1,2-a]pyrene (6)/trans-7,8-Dihydroxy-7,8-dihydronaphtho-[1,2a]pyrene (7). To a suspension of 4/5 (33 mg, 0.1 mmol) in EtOH (60 mL) was added NaBH4 (38 mg, 1.0 mmol) portion-wise, and the mixture was stirred at room temperature for 4 h. It was

Chem. Res. Toxicol., Vol. 18, No. 9, 2005 1439 then concentrated under reduced pressure to one-fourth of its original volume, diluted with water, and extracted with EtOAc. The organic layer was dried (MgSO4), filtered, and concentrated to afford the mixture of 6/7 (32 mg, 95%) that was separated by HPLC using a Hiber Si 60 column using a mixture of EtOAc/ hexanes (1:1) as solvent system. trans-4,5-Dihydroxy-4,5-dihydronaphtho[1,2-a]pyrene (6). Mp 185-187 °C. 1H NMR (acetone-d6) δ: 5.14 (d, 1H, J ) 10.1 Hz), 5.19 (d, 1H, J ) 10.1 Hz), 6.67-7.80 (m, 3H), 7.98-8.03 (m, 4H), 8.09-8.14 (m, 2H), 8.36 (s, 1H), 9.09-9.14 (m, 2H). High-resolution MS calcd for C24H16O2, 336.1145; found, 336.1151. trans-7,8-Dihydroxy-7,8-dihydronaphtho[1,2-a]pyrene (7). Mp 207-209 °C. 1H NMR (acetone-d6) δ: 4.73 (d, 1H, J ) 10.2 Hz), 4.80 (d, 1H, J ) 10.2 Hz), 7.47-7.57 (m, 2H), 7.92 (d, 1H, J ) 7.2 Hz), 8.03-8.08 (m, 2H), 8.17-8.29 (m, 5H), 8.61 (s, 1H), 8.78 (d, 1H, J ) 9.2 Hz). High-resolution MS calcd for C24H16O2, 336.1145; found, 336.1151. 1-Methoxy-5-ethoxycarbonylbenzo[c]chrysene (12). To a solution of ethyl 5-methoxy-1-naphthaleneacetate (8) (1.22 g, 5.0 mmol) in THF (30 mL) at 0 °C was added LDA (2 M solution in THF, heptane, and ethylbenzene, 4.5 mL, 9.0 mmol) dropwise and the mixture was stirred at 0 °C for 2 h. A solution of 2-naphthaldehyde (0.78 g, 5.0 mmol) in THF (25 mL) was then added dropwise. The reaction mixture was warmed to room temperature and then refluxed for 6 h. It was then cooled to room temperature; ice-cold water was added, then it was acidified with dilute HCl and extracted with EtOAc. The organic layer was washed with water and dried over anhydrous MgSO4. Concentration in vacuo gave a residue which was purified on a silica gel column with EtOAc/hexanes (3:97) to yield a mixture of E and Z forms of olefin 10 (1.09 g, 57%) as determined by its proton spectrum. A stirred solution of 10 (0.99 g, 2.6 mmol) and I2 (cat.) in benzene (1.0 L) was then irradiated with a Hanovia 450W medium-pressure lamp, with a Pyrex filter, for 8 h, while the dry air was bubbled through the solution. Removal of solvent gave a residue which was purified by chromatography on silica gel with EtOAc/hexanes (3:97) to give 12 (0.80 g, 81%) as a pale yellow crystalline solid. Mp 144-146 °C. 1H NMR δ: 1.36 (t, 3H, J ) 7.2 Hz), 4.09 (S, 3H), 4.51 (q, 2H, J ) 7.2 Hz), 7.04 (d, 1H, J ) 8.1 Hz), 7.51 (dd, 1H, J ) 8.1 and 8.2 Hz), 7.64-7.71 (m, 2H), 7.85-8.03 (m, 3H), 8.02 (d, 1H, J ) 7.5 Hz), 8.16 (s, 1H), 8.44 (d, 1H, J ) 9.5 Hz), 8.96 (d, 1H, J ) 9.5 Hz), 9.00 (d, 1H, J ) 8.2 Hz). High-resolution MS calcd for C26H20O3, 380.1407; found, 380.1411. 3-Methoxy-5-ethoxycarbonylbenzo[c]chrysene (13). Mp 113-114 °C. 1H NMR δ: 1.38 (t, 3H, J ) 7.2 Hz), 3.98 (S, 3H), 4.55 (q, 2H, J ) 7.2 Hz), 7.31 (dd, 1H, J ) 8.9 and 2.3 Hz), 7.66-7.69 (m, 2H), 7.76 (d, 1H, J ) 2.3 Hz), 7.84-7.93 (m, 4H), 8.01-8.03 (m, 1H), 8.13 (s, 1H), 8.85 (d, 1H, J ) 9.2 Hz), 9.00 (d, 1H, J ) 8.9 Hz). High-resolution MS calcd for C26H20O3, 380.1407; found, 380.1411. 1-Methoxy-5-hydroxymethylbenzo[c]chrysene (14). To a well-stirred suspension of LiAlH4 (0.42 g, 11.1 mmol) in anhydrous ether (30 mL) at 0 °C was added a solution of 12 (0.7 g, 1.85 mmol) in anhydrous Et2O (30 mL) dropwise. After the addition was complete, the reaction mixture was warmed to room temperature and stirred for another 1 h. It was then diluted with ether, poured into ice-cold water, and acidified with 10% HCl. The organic layer was separated, and the aqueous layer was extracted again with ether. The combined etherextract was washed with water and dried over anhydrous MgSO4. Removal of solvent afforded a crude residue which was purified by silica gel column chromatography (EtOAc/hexanes 20:80) to give 14 (0.55 g, 88%) as a pale yellow solid. Mp 187188 C. 1H NMR δ: 4.10 (s, 3H), 5.43 (s, 2H), 7.10 (d, 1H, J ) 7.9 Hz), 7.60-7.67 (m, 3H), 7.85 (d, 1H, J ) 8.5 Hz), 7.91(d, 1H, J ) 8.5 Hz), 8.00-8.03 (m, 1H), 8.11 (s, 1H, H6), 8.36 (d, 1H, J ) 9.2 Hz), 8.48 (d, 1H, J ) 8.5 Hz), 8.88-8.94 (m, 2H). High-resolution MS calcd for C24H18O2, 338.1301; found, 338.1310. 3-Methoxy-5-hydroxymethylbenzo[c]chrysene (15). Mp 161-163 °C. 1H NMR δ: 4.03 (s, 3H), 5.35 (s, 2H), 7.33 (dd,

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1H, J ) 8.9 and 2.3 Hz), 7.62-7.66 (m, 2H), 7.77-7.80 (m, 2H), 7.88 (d, 1H, J ) 9.2 Hz), 7.91(d, 1H, J ) 8.9 Hz), 7.97 (s, 1H, H6), 7.99-8.01 (m, 1H), 8.60 (d, 1H, J ) 2.3 Hz), 8.76 (d, 1H, J ) 8.9 Hz), 8.92 (d with fine splitting, 1H, J ) 8.9 Hz). Highresolution MS calcd for C24H18O2, 338.1301; found, 338.1312. 1-Methoxy-5-formylbenzo[c]chrysene (16). To a stirred suspension of PCC (0.42 g, 1.95 mmol) in CH2Cl2 (50 mL) at room temperature was added a solution of alcohol 14 (0.44 g, 1.3 mmol) in CH2Cl2 (25 mL) dropwise. The resulting mixture was stirred for 4 h, diluted with CH2Cl2, washed with 10% HCl and water, and dried (MgSO4). Concentration in vacuo provided a residue that was purified on a silica gel column with EtOAc/ hexanes (5:95) to yield 0.38 g (86%) of 16 as a pale yellow crystalline solid. Mp 185-186 °C. 1H NMR δ: 4.13 (s, 3H), 7.15 (dd, 1H, J ) 7.5 and 1.0 Hz), 7.62-7.71 (m, 4H), 7.96 (s, 2H), 8.03-8.06 (m, 1H), 8.44-8.47 (m, 2H), 8.96-9.01 (m, 2H), 10.66 (s, 1H). High-resolution MS calcd for C24H16O2, 336.1145; found, 336.1154. 3-Methoxy-5-formylbenzo[c]chrysene (17). Mp 124-125 °C. 1H NMR δ: 4.00 (s, 3H), 7.39 (dd, 1H, J ) 8.5 and 2.3 Hz), 7.46 (d, 1H, J ) 2.3 Hz), 7.67-7.70 (m, 2H), 7.89-7.92 (m, 3H), 7.98 (d, 1H, J ) 8.5 Hz), 8.01-8.04 (m, 1H), 8.39 (s, 1H), 8.83 (d, 1H, J ) 9.2 Hz), 8.96-8.99 (m, 1H), 10.70 (s, 1H). Highresolution MS calcd for C24H16O2, 336.1145; found, 336.1153. 1-Methoxynaphtho[1,2-a]pyrene (20). A suspension of anhydrous (methoxymethyl)triphenylphosphonium chloride (1.54 g, 4.5 mmol) in diethyl ether (freshly distilled over sodium, 30 mL) was cooled to -78 °C under N2. To this mixture was added PhLi (1.8 M in cyclohexane/ether 70:30, 1.8 mL, 3.3 mmol), dropwise, and the mixture was stirred for 30 min. The reaction mixture was then warmed to -30 °C, stirred for another 30 min, and again cooled to -78 °C. A solution of 16 (0.3 g, 0.9 mmol) in THF (20 mL) was then added dropwise; the mixture was allowed to warm to room temperature and was stirred overnight. The reaction was quenched with 1N HCl; the mixture was washed several times with water and extracted with ethyl acetate. The combined organic layers were dried (MgSO4), filtered, and concentrated. The resulting residue was purified on a silica gel column (EtOAc/hexanes 1:99) to yield a mixture of cis- and trans-isomers of olefin 18 (0.25 g, 83%) as determined by its proton spectrum. To a solution of 18 (0.20 g, 0.6 mmol) in anhydrous CH2Cl2 (20 mL) at room temperature was added CH3SO3H (2 mL) dropwise over a period of 10 min. The reaction mixture was stirred at room temperature for 3 h, poured into ice-cold water, and extracted with CH2Cl2. The organic layer was washed with 10% NaHCO3 solution and then with water and dried over over MgSO4. The crude viscous mass obtained after the removal of solvent was purified by silica gel column (EtOAc/hexanes 1:99) to give 0.17 g (85%) of 20 as a pale yellow solid. Mp 186-187 °C. 1H NMR δ: 4.22 (s, 3H), 7.54 (d, 1H, J ) 8.2 Hz), 7.66-7.71 (m, 1H), 7.73-7.78 (m, 1H), 7.92-7.95 (m, 3H), 8.04-8.08 (m, 2H), 8.11 (d, 1H, J ) 8.2 Hz), 8.46 (s, 1H), 8.68 (d, 1H, J ) 9.5 Hz), 9.27 (d, 1H, J ) 8.5 Hz), 9.40 (d, 1H, J ) 9.5 Hz). High-resolution MS calcd for C25H16O, 332.1196; found, 332.1199. 3-Methoxynaphtho[1,2-a]pyrene (21). Mp 214-215 °C. 1H NMR δ: 4.22 (s, 3H), 7.65-7.77 (m, 3H), 7.93 (d, 1H, J ) 8.9 Hz), 8.06 (d, 2H, J ) 8.9 Hz), 8.12 (d, 1H, J ) 9.2 Hz), 8.22 (d, 1H, J ) 8.9 Hz), 8.24 (d, 1H, J ) 7.6 Hz), 8.47 (d, 1H, J ) 9.2 Hz), 8.50 (s, 1H), 9.26 (d with fine splitting, 2H, J ) 8.9 Hz). High-resolution MS calcd for C25H16O, 332.1196; found, 332.1203. 1-Hydroxynaphtho[1,2-a]pyrene (22). To a stirred solution of 20 (0.1 g, 0.3 mmol) in CH2Cl2 (20 mL) at room temperature, a solution of BBr3 (1 M solution in CH2Cl2, 0.6 mL, 0.6 mmol) was added dropwise under N2 over 10 min. The reaction mixture was then refluxed for 20 h, cooled, and quenched with ice-cold water. The organic layer was washed twice with water and dried over MgSO4. Removal of solvent gave the crude solid that was purified by silica gel column chromatography (EtOAc/hexanes 6:94) to yield 22 (71 mg, 74%) as a crystalline solid. Mp 202203 °C. 1H NMR (acetone-d6) δ: 7.66 (d, 1H, J ) 8.2 Hz), 7.717.76 (m, 1H), 7.80-7.85 (m, 1H), 7.99-8.06 (m, 3H), 8.05-8.17

Sharma and Amin

Figure 1. HPLC profile of in vitro metabolites of N[1,2-a]P. Metabolites identified: peak 1, N[1,2-a]P trans-4,5-dihydrodiol; peak 2, N[1,2-a]P trans-7,8-dihydrodiol; peak 3, N[1,2-a]P trans9,10-dihydrodiol; peak 4, 3-hydroxy-N[1,2-a]P; peak 5, 10hydroxy-N[1,2-a]P; peak 6, 1-hydroxy-N[1,2-a]P; and peak 7, N[1,2-a]P. (m, 3H), 8.60 (s, 1H), 8.72 (d, 1H, J ) 9.5 Hz), 9.32 (d, 1H, J ) 8.2 Hz), 9.40 (d, 1H, J ) 9.5 Hz). High-resolution MS calcd for C24H14O, 318.1040; found, 318.1044. 3-Hydroxynaphtho[1,2-a]pyrene (23). Mp 184-185 °C. 1H NMR (acetone-d6) δ: 7.69-7.82 (m, 3H), 8.01 (d, 1H, J ) 8.9 Hz), 8.13 (d, 3H, J ) 9.2 Hz), 8.23 (d, 1H, J ) 8.2 Hz), 8.30 (d, 1H, J ) 9.5 Hz), 8.46 (d, 1H, J ) 9.2 Hz), 8.58 (s, 1H, H6), 9.24 (d, 1H, J ) 9.2 Hz), 9.29 (d, 1H, J ) 8.2 Hz). High-resolution MS calcd for C24H14O, 318.1040; found, 318.1045.

Results and Discussion The carcinogenic potential of a hydrocarbon can be assessed from its metabolism pattern, that is, the type of metabolites formed. For example, the formation of dihydrodiol metabolites, which can activate further to the active intermediates, may lead to the formation of stable DNA lesions causing mutations. Our recent in vitro metabolism study on N[1,2-a]P exhibited the formation of a series of dihydrodiol and phenolic metabolites as shown in Figure 1 (25). The identity of peak 3 and peak 5 have been confirmed earlier (25) by independent syntheses, as N[1,2-a]P trans-9,10-dihydrodiol and 10hydroxy-N[1,2-a]P, respectively. The other peaks were collected by HPLC and were tentatively assigned as dihydrodiol (peaks 1 and 2) and phenolic derivatives (peaks 4 and 6) based on their 1H NMR and MS. After confirmation of N[1,2-a]P trans-9,10-dihydrodiol, the electron-rich 4,5-K region and 7,8-K region are the most likely sites for the formation of dihydrodiol metabolites. Thus, their synthesis was the first on our list and the synthetic sequence is outlined in Scheme 1. Treatment of N[1,2-a]P with OsO4 gave a mixture of N[1,2a]P cis-4,5-dihydrodiol (2) and N[1,2-a]P cis-7,8-dihydrodiol (3). The mixture was practically inseparable at this stage and so was treated as such with pyridinium chlorochromate (PCC) to result in a mixture of diones 4 and 5. This mixture on reaction with NaBH4 in EtOH afforded the mixture of N[1,2-a]P trans-4,5-dihydrodiol (6) and N[1,2-a]P trans-7,8-dihydrodiol (7). These two dihydrodiols could now be easily separated by HPLC using a Hibar Si60 column and a mixture of EtOAc/ hexane (1:1) solvent system to give pure N[1,2-a]P trans-

Synthesis and Identification of N[1,2-a]P Metabolites

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Scheme 1. Synthesis of N[1,2-a]P trans-4,5-Dihydrodiol (6) and N[1,2-a]P trans-7,8-Dihydrodiol (7)

Scheme 2. Synthesis of 1-Hydroxy-N[1,2-a]P (22) and 3-Hydroxy-N[1,2-a]P (23)

4,5-dihydrodiol (6) and N[1,2-a]P trans-7,8-dihydrodiol (7). The structures were assigned on the basis of their 1 H NMR and MS data. When these synthetic compounds were used, peak 1 was identified as N[1,2-a]P trans-4,5dihydrodiol and peak 2 was identified N[1,2-a]P trans7,8-dihydrodiol (Figure 1). The structural assignment was based on the identical NMR and MS data of the dihydrodiols 6 and 7 with the metabolite peak 1 and peak 2, respectively. The synthetic compounds 6 and 7 are particularly important for their further metabolism and DNA binding studies. After having all the major dihydrodiols assigned, our next goal was to identify the phenolic derivatives. From synthetic point of view, a hydroxy group can be introduced to any of the 14 carbon positions of N[1,2-a]P, thus, leading to a possibility of formation of 14 different phenolic derivatives during metabolism of N[1,2-a]P. The 10-hydroxy-N[1,2-a]P, obtained en route to the synthesis of N[1,2-a]P trans-9,10-dihydrodiol (Scheme 1) corresponded to peak 5 in the metabolism trace (Figure 1).

That left us with the 13 more possible phenols, and it was necessary to rule out the formation of some and tentatively identify those formed during the in vitro metabolism before initiating the syntheses. The structure of N[1,2-a]P consists of a pyrene moiety and a naphthalene moiety fused together. Carbon positions 1, 2, and 3 and the 4,5- and 13,14-K regions can be considered as typical of a pyrene moiety. However, the fusion of a naphthalene ring gives rise to another 7,8-K region. As observed from the reaction with OsO4 (Scheme 1), 4,5and 7,8-K regions are the most reactive sites. Thus, to start with, we carried out the dehydration of N[1,2-a]P trans-4,5-dihydrodiol and N[1,2-a]P trans-7,8-dihydrodiol under acidic conditions which led to the formation of a mixture of 4-/5-hydroxy and 7-/8-hydroxy-N[1,2-a]P. However, none of these phenols corresponded to those obtained in the metabolism. 9-Hydroxy-N[1,2-a]P obtained, along with 10-hydroxy-N[1,2-a]P, after the dehydration of N[1,2-a]P trans-9,10-dihydrodiol also did not match with any of the phenolic peaks. The 1H NMR of the

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collected peaks 4 and 6 showed the presence of both two downfield doublets typical of fjord-region protons H12 and H13, thus, eliminating the possibility of 12- and 13hydroxy-N[1,2-a]P. The appearance of these signals as doublets also indicated the neighboring positions 11 and 14 do not bear a hydroxy group. The singlet signals for H-6 ruled out the formation 6-OH derivative. Further, the lack of any singlets/meta-coupled doublets in the proton NMR spectra, possibly due to protons at 1-position and 3-position, eliminated the possibility of 2-hydroxyN[1,2-a]P. Thus, 1-OH- and 3-OH-N[1,2-a]P were thought to be the most likely phenolic metabolites formed during the in vitro metabolism. To unequivocally confirm our assumption, and to assign the individual peaks, we carried out the syntheses of these hydroxy derivatives. The method used for the synthesis of 1-/3-hydroxyN[1,2-a]P follows a well-established photochemical approach (Scheme 2). The key intermediate 5-ethoxycarbonyl-1-methoxy-B[c]C (12), for the synthesis of 1-hydroxyN[1,2-a]P, was prepared by the photocyclization of olefin 10 which was obtained by LDA-assisted condensation (28)2 of ethyl 5-methoxy-1-naphthaleneacetate (8) with 2-naphthaldehyde. On reduction with LiAlH4, compound 12 resulted in the 5-hydroxymethyl derivative 14, which was oxidized to the corresponding aldehyde 16 by treatment with PCC in methylene chloride. The Wittig reaction of aldehyde 16 with (methoxymethyl)triphenylphosphonium chloride in the presence of phenyllithium gave the olefin 18 which was cyclized using methanesulfonic acid to yield 1-methoxy-N[1,2-a]P (20). Demethylation of methoxy group with BBr3 afforded the desired 1-hydroxyN[1,2-a]P (22). The structure of phenol 22 was determined on the basis of 1H NMR and MS. Its 1H NMR was identical to that of the highest intensity metabolite peak 6 in the metabolism trace (Figure 1), and also their retention times in HPLC were the same. 3-HydroxyN[1,2-a]P (23) was synthesized using a similar synthetic sequence (Scheme 2) starting from ethyl 7-methoxy-1naphthaleneacetate (9). Again, the structure of 23 was established based on 1H NMR and MS, which were identical to the peak 4 in the metabolism trace (Figure 1). Their retention times in HPLC were also identical. In summary, convenient methods for the synthesis of various dihydrodiol and phenolic metabolites of N[1,2a]P have been developed, and the structures of all the major metabolites of N[1,2-a]P obtained in vitro have been confirmed (Figure 1). These synthetic standards will also serve as markers for identifying the metabolites of N[1,2-a]P in vivo. The phenols will be useful toward identifying the glucocronide metabolites in vivo. The dihydrodiol metabolites, specially the formation of fjordregion N[1,2-a]P trans-9,10-dihydrodiol indicates that it can further metabolize to the corresponding diol epoxide which can bind covalently to DNA causing mutations and leading ultimately to the tumor induction. The K-region dihydrodiols 6 and 7, which can metabolize further to electrophilic active metabolites and give stable DNA adducts, will be useful standards to study their further metabolism pattern.

Acknowledgment. This work was supported by NCI Contract NO2-CB-37025-48. Further support has been 2 The reaction did not go to completion. However, the olefin mixture was separated easily from the unreacted starting materials by silica gel column chromatography.

Sharma and Amin

provided by the Penn State Cancer Institute of the Penn State College of Medicine. Supporting Information Available: Experimental procedures for compounds 13, 15, 17, 21, and 23, and a figure showing the 1H NMR spectra of peak 4 and peak 6 collected from HPLC. This material is available free of charge via the Internet at http://pubs.acs.org.

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