Synthesis, Microsome-Mediated Metabolism, and Identification of

Apr 18, 2008 - Department of Pharmacology and Chemical Carcinogenesis and Chemoprevention Program of Penn State Cancer Institute, H072, Penn State ...
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Chem. Res. Toxicol. 2008, 21, 1154–1162

Synthesis, Microsome-Mediated Metabolism, and Identification of Major Metabolites of Environmental Pollutant Naphtho[8,1,2-ghi]chrysene Arun K. Sharma,*,†,‡ Krishnegowda Gowdahalli,‡ Melissa Gimbor,‡ and Shantu Amin†,‡ Department of Pharmacology and Chemical Carcinogenesis and ChemopreVention Program of Penn State Cancer Institute, H072, Penn State College of Medicine, 500 UniVersity DriVe, Hershey, PennsylVania 17033 ReceiVed January 25, 2008

Naphtho[8,1,2-ghi]chrysene, commonly known as naphtho[1,2-e]pyrene (N[1,2-e]P) is a widespread environmental pollutant, identified in coal tar extract, air borne particulate matter, marine sediment, cigarette smoke condensate, and vehicle exhaust. Herein, we determined the ability of rat liver microsomes to metabolize N[1,2-e]P and an unequivocal assignment of the metabolites by comparing them with independently synthesized standards. We developed the synthesis of both the fjord region and the K-region dihydrodiols and various phenolic derivatives for metabolite identification. The 12-OH-N[1,2-e]P, fjord region dihydrodiol 14 and diol epoxide 15 were synthesized using a Suzuki cross-coupling reaction followed by the appropriate manipulation of the functional groups. The K-region trans-4,5-dihydrodiol (18) was prepared by the treatment of N[1,2-e]P with OsO4 to give cis-dihydrodiol 16, followed by pyridinium chlorochromate oxidation to quinone 17, and finally reduction with NaBH4 to afford the dihydrodiol 18 with the desired trans stereochemistry. The 9-OH-N[1,2-e]P (30) and N[1,2-e]P trans9,10-dihydrodiol (32) were also synthesized following a Suzuki cross-coupling approach starting from 1,2,3,6,7,8-hexahydropyrene-4-boronic acid. The metabolism of N[1,2-e]P with rat liver microsomes led to several dihydrodiol and phenolic metabolites as assessed by the HPLC trace. The 11,12-dihydrodiol and 4,5-dihydrodiol were identified as major dihydrodiol metabolites. The synthesized 9,10-dihydrodiol, on the other hand, did not match with any of the peaks in the metabolism trace. Among the phenols, only 12-OH-N[1,2-e]P was identified in the metabolism. The other phenolic derivatives synthesized, that is, the 4-/5-, 9-, 10-, and 11-hydroxy derivatives, were not detected in the metabolism trace. In summary, N[1,2-e]P trans-11,12-dihydrodiol was the major metabolite formed along with N[1,2-e]P 4j,5-transdihydrodiol and 12-OH-N[1,2-e]P on exposure of rat liver microsomes to N[1,2-e]P. The presence of N[1,2-e]P in the environment and formation of fjord region dihydrodiol 14 as a major metabolite in in vitro metabolism studies strongly suggest the role of N[1,2-e]P as a potential health hazard. Introduction Peri-condensed hexacyclic PAHs,1 the widespread environmetal contaminants, are structurally diverse molecules (1) having carcinogenicity that range from highly potent, for example, dibenzo[a,l]pyrene (DB[a,l]P), to noncarcinogenic compounds. Among PAHs, DB[a,l]P, with a fjord region in the molecule, is by far the most potent mammary gland (2, 3), skin (2-4), lung (5), and oral (6) tumorigen known. It is metabolized to anti- and syn-11,12-diol-13,14-epoxides (7), which are potent tumorigens. The only two other hexacyclic PAHs in this class * To whom correspondence should be addressed. Tel: 717-531-0003ext. 285016. Fax: 717-531-0244. E-mail: [email protected]. † Chemical Carcinogenesis and Chemoprevention Program of Penn State Cancer Institute. ‡ Department of Pharmacology. 1 Abbreviations: PAHs, polycyclic aromatic hydrocarbons; N[1,2-e]P, naphtho[8,1,2-ghi]chrysene (naphtho[1,2-e]pyrene); N[1,2-e]P cis-4,5-dihydrodiol, (()-cis-4,5-dihydroxy-4,5-dihydronaphtho[1,2-e]pyrene; N[1,2e]P trans-4,5-dihydrodiol, (()-trans-4,5-dihydroxy-4,5-dihydronaphtho[1,2e]pyrene; N[1,2-e]P trans-9,10-dihydrodiol, (()-trans-9,10-dihydroxy-9,10dihydronaphtho[1,2-e]pyrene; N[1,2-e]P trans-11,12-dihydrodiol, (()-trans11,12-dihydroxy-11,12-dihydronaphtho[1,2-e]pyrene; N[1,2-e]PDE; 4/5hydroxynapththo[1,2-e]pyrene, 4/5-OH-N[1,2-e]P, (()-anti-11,12-dihydroxy13,14-epoxy-11,12,13,14-tetrahydro-N[1,2-e]P;9-OH-N[1,2-e]P,9-hydroxynapththo[1,2-e]pyrene; 10-OH-N[1,2-e]P, 10-hydroxynapththo[1,2-e]pyrene; 11OH-N[1,2-e]P, 11-hydroxynapththo[1,2-e]pyrene; 12-OH-N[1,2-e]P, 12hydroxynapththo[1,2-e]pyrene.

Figure 1. Structures of N[1,2-e]P (1), N[1,2-a]P (2), and DB[a,l]P (3).

of compounds that possess a fjord region and a pyrene moiety are N[1,2-a]P and N[1,2-e]P; the latter actually contains both a fjord region and a bay region similar to DB[a,l]P (Figure 1). Both of the compounds have recently been synthesized in our laboratory (8-10) and have been detected in coal tar extract, air borne particulate matter, and marine sediment (11); cigarette smoke and vehicle exhaust (12); and urban dust (13). Our initial metabolism studies by male Sprague-Dawley rat liver S9 fraction showed that, just like DB[a,l]P, both N[1,2a]P and N[1,2-e]P lead to the formation of corresponding fjord region dihydrodiol metabolites, which can be metabolized further to electrophilic diol epoxides that efficiently bind to DNA (8, 10). Interestingly, our investigation of the formation of DNA adducts in C3H10T1/2C18 mouse embryo fibroblasts exposed to N[1,2-a]P and N[1,2-e]P and their corresponding dihydrodiols

10.1021/tx8000384 CCC: $40.75  2008 American Chemical Society Published on Web 04/18/2008

Identification of Naphtho[1,2-e]pyrene Metabolites

revealed that although both N[1,2-a]P and its fjord region 9,10dihydrodiol lead to the formation of a comparable number of DNA adducts (33.3 and 22.2 pmol DNA adducts/µg DNA, respectively), the difference in DNA adduct formation was extremely high between N[1,2-e]P and its fjord region 11,12dihydrodiol (1.47 and 105.9 pmol DNA adducts/µg DNA, respectively) (14). This indicated the lack of efficient metabolism of N[1,2-e]P in the used mouse embryo fibroblasts but also clearly demonstrated that once N[1,2-e]P is metabolized to dihydrodiol, the amount of DNA damage was much higher than dihydrodiols derived from both N[1,2-a]P and DB[a,l]P. This suggests that N[1,2-e]P might be an even more serious health hazard than DB[a,l]P, subject to its capability to metabolize to N[1,2-e]P trans-11,12-dihydrodiol. We have previously reported an efficient metabolism of N[1,2-e]P using S9 fraction from phenobarbital/β-naphthoflavone-induced rat liver homogenate (10). The major metabolites were separated by HPLC, and 1H NMR analysis suggested the formation of N[1,2-e]P trans-11,12-dihydrodiol (14) along with K-region dihydrodiol, that is, N[1,2-e]P trans-4,5-dihydrodiol (18). None of the phenolic metabolites could be assigned. To establish further that N[1,2-e]P is a good substrate for the metabolism, we have now exposed it to rat liver microsomes that led to the formation of several dihydrodiol and phenolic metabolites, including fjord region N[1,2-e]P trans-11,12dihydrodiol (14) as a major metabolite. The formation of fjord region dihydrodiol in these metabolism studies and its ability to cause substantial DNA lesions (14) suggest that N[1,2-e]P may be a more powerful carcinogen than DB[a,l]P or N[1,2a]P. However, its carcinogenic potential remained unexplored essentially due to the lack of synthetic routes available for the synthesis of its potential metabolites. In this paper, we describe the preparation of N[1,2-e]P trans-dihydrodiol and phenolic metabolites by independent syntheses and use them for identification of metabolites formed from rat liver microsomal metabolism of N[1,2-e]P.

Experimental Procedures Caution: N[1,2-e]P and its deriVatiVes described in this paper may be carcinogenic. Therefore, appropriate safety procedures should be followed when working with these compounds. Melting points were recorded on a Fischer-Johns melting point apparatus and are uncorrected. NMR spectra were recorded using a Bruker AM 360WB 360 MHz NMR spectrometer or a Bruker Avance 500 MHz spectrometer. 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, NY. TLC was developed on aluminum-supported precoated silica gel plates (EM industries, Gibbstown, NJ). 4-Pyreneboronic acid was prepared according to the literature method (15). 4-Bromo-1,2,3,6,7,8-hexahydropyrene (23), precursor for the corresponding boronic acid, was synthesized by the bromination of 1,2,3,6,7,8-hexahydropyrene (24), according to a literature method (16). Microsomes were prepared from livers of adult male Sprague-Dawley rats as previously described (17). The protein concentration was determined to be 10 mg/mL using Biorad protein concentration assay (Biorad Laboratories, CA). The metabolites of N[1,2-e]P were analyzed by HPLC (HP 1100 series) 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 linear gradient program from A:B (40:60) to A:B (0:100) over 60 min (solvent system 1). The purity of N[1,2e]PDE (15) was determined by a normal-phase HPLC with LiChrosorb Si 60 10 mm (250 mm × 4 mm) column at 1.0 mL min-1 using a mixture of THF/hexanes (25:75) as a solvent (solvent system 2).

Chem. Res. Toxicol., Vol. 21, No. 5, 2008 1155 4-(2-Formylphenyl)pyrene (7). A mixture of 4-pyrene boronic acid 4 (1.35 g, 5.5 mmol), 2-bromobenzaldehyde (0.925 g, 0.58 mL, 5.0 mmol), cesium fluoride (1.67 g, 11.0 mmol), and Pd(PPh3)4 (0.22 g, 0.19 mmol) in anhydrous DME (30 mL) was heated to reflux under N2 for 17 h. The reaction mixture was cooled to room temperature, and water was added and extracted with CH2Cl2. The organic layer was washed with 5% NaOH and water and dried over MgSO4. The solvent was removed to give the crude mixture, which was purified on a silica gel column (eluent: EtOAc:hexanes/1:99) to give 1.1 g (72%) of 7 as a crystalline solid; mp 147-148 °C. 1 H NMR (300 MHz, CDCl3): δ 7.61-7.69 (m, 2H), 7.76 (dd, 1H,J ) 7.5 and 1.6 Hz), 7.81 (d, 1H, J ) 7.8 Hz), 7.93 (dd, 1H, J ) 7.9 and 7.8 Hz), 8.04 (s, 1H), 8.06 (d, 1H, J ) 7.6 Hz), 8.14 (s, 2H), 8.19-8.27 (m, 4H), 9.82 (s, 1H, CHO). MS m/z 306 (M+). 4-(2-Formyl-4-methoxyphenyl)pyrene (8). A mixture of boronic acid 4 (2.35 g, 9.55 mmol), 2-bromo-5-methoxybenzaldehyde (1.87 g, 8.68 mmol), cesium fluoride (2.90 g, 19.1 mmol), and Pd(PPh3)4 (0.37 g, 0.32 mmol) in anhydrous DME (45 mL) was heated to reflux for 18 h. The crude 8 obtained by following a similar workup, as in case of 7, was purified on a silica gel column (eluent: EtOAc: hexanes/1:99) to give 2.1 g (72%) of 6 as a crystalline solid; mp 125-126 °C. 1H NMR (300 MHz, CDCl3): δ 3.99 (s, 3H, OCH3), 7.33 (dd, 1H, J ) 8.5 and 2.6 Hz), 7.53 (d, 1H, J ) 8.5 Hz), 7.69 (d, 1H, J ) 2.6 Hz), 7.82 (d, 1H, J ) 7.9 Hz), 7.93 (dd, 1H, J ) 7.9 and 7.9 Hz), 8.02 (s, 1H), 8.03-8.08 (m, 1H), 8.13 (s, 2H), 8.18-8.26 (m, 3H), 9.76 (s, 1H, CHO). MS m/z 336 (M+). 4-[2-(β-Methoxyethylene)phenyl]pyrene (9). To the (methoxymethyl)triphenylphosphonium chloride (4.50 g, 13.15 mmol), well-dried under vacuum with careful heating, was added Et2O (freshly distilled over sodium, 80 mL). To the resulting suspension, cooled to -78 °C under N2, was added PhLi (1.8 M in 70:30/ cyclohexane:ether, 5.48 mL, 9.86 mmol) dropwise, and the mixture was stirred for 30 min at the same temperature. The reaction mixture was then warmed to -30 °C and stirred for another 30 min. It was then cooled again to -78 °C, and a solution of 7 (0.80 g, 2.63 mmol) in THF (50 mL) was added dropwise. The reaction mixture was left overnight, allowing the temperature to rise gradually to room temperature. It was quenched with dilute HCl, washed with water, and extracted with ethyl acetate. The organic layer was dried over MgSO4, and the solvent was removed to give the crude product, which was purified on a silica gel column (eluent: EtOAc: hexanes/3:97) to give olefin 9 as a mixture of cis:trans/56:44 isomers as determined by 1H NMR; yield, 0.72 g (82%); viscous oil. 1H NMR (300 MHz, CDCl3): δ 3.17 (s, 1.35H, OCH3, trans), 3.72 (s, 1.65H, OCH3, cis), 4.96 (d, 0.56H, J ) 7.2 Hz, olefinic, cis), 5.57 (d, 0.44H, J ) 12.8 Hz, olefinic, trans), 5.83 (d, 0.56H, J ) 7.2 Hz, olefinic, cis), 7.04 (d, 0.44H, J ) 12.8 Hz, olefinic, trans), 7.35-7.56 (m, 3H), 7.64 (d, 0.44H, J ) 7.9 Hz), 7.86-7.94 (m, 2H), 8.01-8.07 (m, 2H), 8.13 (s, 2H), 8.20-8.24 (m, 3H), 8.38 (d, 0.56H, J ) 7.9 Hz). MS m/z 334 (M+). 4-[2-(β-Methoxyethylene)-4-methoxyphenyl]pyrene (10). (Methoxymethyl)triphenylphosphonium chloride (9.08 g, 26.5 mmol), Et2O (160 mL), PhLi (1.8 M in 70: 30/cyclohexane:ether, 11.03 mL, 19.87 mmol), aldehyde 8 (1.78 g, 5.3 mmol),and THF (100 mL) were mixed according to the earlier described procedure for 9. A similar workup gave the crude mixture,which was purified on a silica gel column using a mixture of EtOAc and hexane (3:97) to give 1.53 g (79%) of product 10 as a mixture of cis:trans/47:53 isomers as determined by 1H NMR; clear viscous oil. 1H NMR (300 MHz, CDCl3): δ 3.19 (s, 1.59H, OCH3, trans), 3.73 (s, 1.41H, OCH3, cis), 3.97 (s, 1.59H, OCH3, trans), 4.00 (s, 1.41H, OCH3, cis), 4.95 (d, 0.47H, J ) 7.2 Hz, olefinic, cis), 5.56 (d, 0.53H, J ) 12.8 Hz, olefinic, trans), 5.84 (d, 0.47H, J ) 7.2 Hz, olefinic, cis), 6.94-6.97 (m, 1H), 7.07 (d, 0.53H, J ) 12.8 Hz, olefinic, trans), 7.18-7.20 (m, 0.53H), 7.37-7.40 (m, 1H), 7.93-7.96 (m, 2H), 8.01-8.07 (m, 2.47H), 8.14 (s, 2H), 8.20-8.23 (m, 3H). MS m/z 364 (M+). Naphtho[1,2-e]pyrene (1). To a solution of 9 (0.65 g, 1.95 mmol) in anhydrous CH2Cl2 (40 mL) at room temperature was added CH3SO3H dropwise over a period of 10 min. The reaction mixture was stirred at room temperature for 3 h, poured into ice

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Sharma et al.

Scheme 1. Synthesis of 12-OH-N[1,2-e]P (12), N[1,2-e]P trans-11,12-Dihydrodiol (14), and N[1,2-e]PDE (15)

cold water, and extracted with CH2Cl2. The organic layer was washed with 10% NaHCO3 solution and then with water (2×) and dried over over MgSO4. The crude viscous mass obtained after the removal of solvent was purified on a silica gel column (eluent, hexanes) to give 0.48 g (81%) of 1 as a pale yellow solid; mp 112-113 °C, lit. (18) 109-110 °C, lit. (19) mp 278-280 °C. 1H NMR (300 MHz, CDCl3): δ 7.62-7.70 (m, 2H), 8.00-8.10 (m, 6H), 8.20-8.22 (m, 2H), 8.78 (d, 1H, J ) 8.9 Hz), 8.87 (d, 1H, J ) 7.9 Hz), 9.17 (d, 1H, J ) 8.5 Hz), 9.23 (d, 1H, J ) 7.9 Hz). HRMS calcd for C24H14, 302.1093; found, 302.1097. 12-Methoxynaphtho[1,2-e]pyrene (11). To a solution of 10 (1.36 g, 3.74 mmol) in anhydrous CH2Cl2 (100 mL) at room temperature was added CH3SO3H (12 mL), and the reaction mixture was stirred for 2 h. Following a similar workup as in the case of 1 gave the crude mixture, which was purified on a silica gel column (eluent: EtOAc:hexanes/5:95) to give 1.16 g (93%) of 11 as a pale yellow crystalline solid; mp 170-172 °C. 1H NMR (300 MHz, CDCl3): δ 4.03 (s, 3H), 7.32 (dd, 1H, J ) 9.2 and 2.6 Hz), 7.38 (d, 1H, J ) 2.6 Hz), 7.97-8.11 (m, 5H), 8.18-8.22 (m, 2H), 8.78 (d, 1H, J ) 9.2 Hz), 8.87 (d, 1H, J ) 7.5 Hz), 9.08 (d, 1H, J ) 9.2 Hz), 9.17 (d, 1H, J ) 8.2). HRMS calcd for C25H16O, 332.1196; found, 332.1201. 12-Hydroxynaphtho[1,2-e]pyrene (12). To a stirred solution of 11 (1.06 g, 3.2 mmol) in CH2Cl2 (90 mL) at room temperature under N2 was added BBr3 (1.0 M solution in CH2Cl2, 6.4 mL, 6.4 mmol) dropwise over a period of 10 min. After stirring for 5 h, the mixture was quenched with ice-cold water. The organic layer was washed with water (3×) and dried over MgSO4. Removal of the solvent gave the crude mixture, which was purified by silica gel column chromatography using EtOAc:hexanes (1:9) as an eluent to yield 12 (0.91 g, 89%) as a pale yellow solid; mp 215-217 °C. 1 H NMR (300 MHz, DMSO-d6): δ 7.31 (dd, 1H, J ) 9.2 and 2.6 Hz), 7.43 (d, 1H, J ) 2.6 Hz), 8.04 (d, 1H, J ) 8.8 Hz), 8.10-8.15 (m, 2H), 8.16-8.21 (m, 2H), 8.30-8.36 (m, 2H), 8.90 (d, 1H, J ) 8.8 Hz), 8.99 (d, 1H, J ) 9.2 Hz), 9.05 (d, 1H, J ) 7.9 Hz), 9.17 (d, 1H, J ) 7.9 Hz), 10.07 (s, 1H). HRMS calcd for C24H14O, 318.1040; found. 318.1043. Naphtho[1,2-e]pyrene-11,12-dione (13). To a stirred solution of 12 (0.87 g, 2.74 mmol) in CH2Cl2:C6H6:THF/(45:135:5) (195 mL) at room temperature were added 0.17 M KH2PO4 solution (150 mL), Fremy’s salt (2.2 g, 8.22 mmol), and 4 drops of Adogen. The reaction mixture was stirred at room temperature for 3 h and then diluted with CH2Cl2. The organic layer was separated, washed with water (4×), dried (MgSO4), and filtered. The crude residue obtained after removal of solvent was triturated with a mixture of acetone:hexane (2:8), filtered, and washed thoroughly with the same mixture to give 0.76 g (83%) of 13 as a dark red solid; mp 253-255

°C. 1H NMR (300 MHz, CDCl3): δ 6.48 (d, 1H, H-13, J ) 10.5 Hz), 7.95 (t, 1H, J ) 7.9 Hz), 8.01-8.06 (m, 3H), 8.20-8.29 (m, 5H), 8.71 (d, 1H, J ) 8.2 Hz), 8.76 (d, 1H, J ) 7.9 Hz). HRMS calcd for C25H16O, 332.0841; found, 332.0836. trans-11,12-Dihydroxy-11,12-dihydronaphtho[1,2-e]pyrene (14). To a suspension of dione 13 (0.33 g, 1.0 mmol) in EtOH (350 mL) was added NaBH4 (1.13 g, 30.0 mmol) in portions over a period of 10 min. The mixture was stirred at room temperature while a stream of oxygen was bubbled through the solution. After 24 h, the EtOH solution was concentrated under reduced pressure to about one-fourth of its original volume, diluted with water, and extracted with EtOAc (4 × 50 mL). The organic layer was dried (MgSO4), filtered, and concentrated. The resulting residue was titurated with ether:hexane (2:1) mixture, kept in the freezer overnight, and filtered to yield 0.30 g (89%) of dihydrodiol 14 as a pale yellow solid; mp 209-210 °C. 1H NMR δ (500 MHz, DMSO-d6): 4.55 (ddd, 1H, J ) 10.5, 4.5, and 2.5 Hz), 4.60 (dd, 1H, J ) 10.5 and 5.5 Hz), 5.41 (d, 1H, J ) 4.5 Hz), 5.75 (d, 1H, J ) 5.5 Hz), 6.27 (dd, 1H, J ) 10.5 and 2.5 Hz), 7.20 (dd, 1H, J ) 10.5 and 2.5 Hz), 7.87 (d, 1H, J ) 8.5 Hz), 7.98-8.10 (m, 2H), 8.17 (s, 2H), 8.26-8.29 (m, 2H), 8.65 (d, 1H, J ) 8.0 Hz), 8.92 (d, 1H, J ) 8.5 Hz), 9.01 (d, 1H, J ) 8.0 Hz). HRMS m/z calcd for C24H16O2, 336.1145; found, 336.1150. trans-11,12-Dihydroxy-13,14-epoxy-11,12,13,14-tetrahydronaphtho[1,2-e]pyrene (15). A solution of dihydrodiol 14(40 mg, 0.12 mmol) and m-CPBA (0.20 g, 1.2 mmol) in freshly distilled THF (40 mL) was stirred at room temperature under N2 and monitored by normal-phase HPLC using analytical Licrosorb Si60 column (5 µm) (solvent system 2). After 2 h of stirring, the mixture was diluted with 150 mL of ether, washed with cold 2% NaOH (2 × 100 mL) and water (3 × 150 mL), and was then dried (K2CO3), filtered, and concentrated at room temperature. The concentrate was washed with a mixture of hexane:ether (3:1, 50 mL) and dried in vacuo to yield diol epoxide 15 (30 mg, 71%) as a pale yellow solid; mp 196-198 °C. 1H NMR (500 MHz, DMSO-d6): δ 3.76 (dd, 1H, J ) 4.0 and 1.5 Hz), 3.83-3.86 (ddd, 1H, J ) 8.5, 5.0, and 1.5 Hz), 4.71 (dd, J ) 8.5 and 6.0 Hz), 4.77 (d, 1H, J ) 4.0 Hz), 5.68 (d, 1H, J ) 5.0 Hz), 5.87 (d, 1H, J ) 6.0 Hz), 8.04 (d, 1H, J ) 8.5 Hz), 8.10 (m, 2H), 8.19 (d, 2H, J ) 1.5 Hz), 8.31 (d, 1H, J ) 7.5 Hz), 8.34 (d, 1H, J ) 7.5 Hz), 8.80 (d, 1H, J ) 8.0 Hz), 9.01 (d, 1H, J ) 8.5 Hz), 9.03 (d, 1H, J ) 8.0 Hz). HRMS m/z calcd for C24H16O3, 352.1099; found, 352.1102. cis-4,5-Dihydroxy-4,5-dihydronaphtho[1,2-e]pyrene (16). To a solution of 1 (0.12 g, 0.4 mmol) in pyridine (3 mL) was added a solution of OsO4 (0.20 g, 0.8 mmol) in pyridine (1 mL). The reaction mixture was stirred under nitrogen for 24 h and then quenched with saturated NaHSO3 solution. After extraction with

Identification of Naphtho[1,2-e]pyrene Metabolites

Chem. Res. Toxicol., Vol. 21, No. 5, 2008 1157

Figure 3. Comparison of the 1H NMR spectra of peak 3 collected from the metabolism trace (A) and synthetically obtained N[1,2-e]P trans11,12-dihydrodiol (B). Both of the spectra were recorded at 500 MHz using DMSO-d6 as a solvent.

Figure 2. HPLC profile of in vitro metabolites of N[1,2-e]P using rat liver microsomes (solvent system 1). Metabolites identified are as follows: peak 1, N[1,2-e]P trans-4,5-dihydrodiol; peak 2, unidentified; peak 3, N[1,2-e]P trans-11,12-dihydrodiol; peak 4, unidentified; peak 5, 12-OH-N[1,2-e]P; and peak 6, N[1,2-e]P. The retention times of other synthesized dihydrodiol and phenolic derivatives not detected in the rat liver microsomal metabolism are as follows: N[1,2-e]P cis-4,5dihydrodiol, 22.29 min; N[1,2-e]P trans-9,10-dihydrodiol, 20.47 min; 4- or 5-OH-N[1,2-e]P, 38.82 min; 9-OH-N[1,2-e]P, 32.69 min; 10OH-N[1,2-e]P, 28.91 min; and 11-OH-N[1,2-e]P, 47.36 min.

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 using a mixture of EtOAc:CH2Cl2 (1:9) to give the dihydrodiol 16 (81 mg, 60%) as a white solid; mp 222-224 °C. 1H NMR (300 MHz, acetone-d6): δ 5.14 (d, 1H, H-4/5, J ) 3.8 Hz), 5.21 (d, 1H, H-5/4, J ) 3.8 Hz), 7.66-7.80 (m, 4H), 7.88-7.94 (m, 2H), 8.14 (d, 2H, J ) 8.5 Hz), 8.73 (d, 1H, J ) 7.9 Hz), 8.80 (d, 1H, J ) 8.9 Hz), 8.90 (d, 1H, J ) 7.9 Hz), 9.02 (d, 1H, J ) 8.5 Hz). HRMS calcd for C24H16O2, 336.1145; found, 336.1148. Naphtho[1,2-e]pyrene-4,5-dione (17). To a solution of 16 (60 mg, 0.18 mmol) in CH2Cl2 (7 mL) and THF (0.2 mL) was added PCC (116 mg, 0.54 mmol), and the reaction mixture was stirred at room temperature for 2 h. It was then diluted with CH2Cl2 and washed with water (2×). The organic layer was dried over MgSO4 and filtered, and the solvent was removed to give nearly pure dione, which was further purified by trituration with a ether:hexane (1:9) mixture to afford 17 (54 mg, 90%) as a dark red solid; mp 232-233 °C. 1H NMR (300 MHz, CDCl3): δ 7.65-7.80 (m, 4H), 8.02-8.05 (m, 2H), 8.44-8.49 (m, 3H), 8.66-8.68 (m, 1H), 8.82 (d, 1H, J ) 7.5 Hz), 9.02 (d, 1H, J ) 7.5 Hz). HRMS calcd for C25H16O, 332.0841; found, 332.0839. trans-4,5-Dihydroxy-4,5-dihydronaphtho[1,2-e]pyrene (18). To a suspension of 17 (40 mg, 0.12 mmol) in EtOH (60 mL) was added NaBH4 (45 mg, 1.2 mmol), and the mixture was stirred at room temperature for 8 h. Following a similar workup as in the case of 14 gave the crude dihydrodiol 18, which was further purified on a silica gel column (eluent: EtOAc:CH2Cl2/15:85) to yield 33 mg (82%) of 18 as a white solid; mp 196-198 °C. 1H NMR (500 MHz, DMSO-d6): δ 4.87 (dd, 1H, J ) 9.0 and 5.0 Hz), 4.96 (dd, 1H, J ) 9.0 and 5.0 Hz), 5.80 (d, 2H, J ) 5.0 Hz), 7.67-7.79 (m, 4H),

Scheme 2. Synthesis of N[1,2-e]P trans-4,5-Dihydrodiol (18)

7.87-7.90 (m, 2H), 8.15-8.17 (m, 2H), 8.73 (d, 1H, J ) 8.5 Hz), 8.78 (d, 1H, J ) 8.5 Hz), 8.81 (d, 1H, J ) 9.0 Hz), 8.94 (d, 1H, J ) 8.5 Hz). HRMS calcd for C24H16O2, 336.1145; found, 336.1149. Methyl-[2-(pyren-4-yl)phenyl]acetate (19). A mixture of pyrene4-boronic acid (4) (0.88 g, 3.6 mmol), methyl-2-bromophenylacetate (0.76 g, 3.3 mmol), cesium fluoride (1.0 g, 6.6 mmol), and Pd(PPh3)4 (0.15 g, 0.13 mmol) in anhydrous DME (25 mL) was heated to reflux for 12 h. The crude 19 obtained by following a similar workup, as in case of 7, was purified by silica gel column chromatography (eluent: EtOAc:hexanes/2:98) to give 1.09 g (95%) of 19 as a colorless viscous oil. 1H NMR (300 MHz, CDCl3): δ 3.38 (s, 3H), 3.45 (d, 1H, J ) 16.1 Hz), 3.54 (d, 1H, J ) 16.1 Hz), 7.47-7.55 (m, 4H), 7.75 (d, 1H, J ) 7.9 Hz), 7.91 (dd, 1H, J ) 7.9 and 7.5 Hz), 7.99 (s, 1H), 8.04 (dd, 1H, J ) 7.9 and 7.5 Hz), 8.13 (s, 2H), 8.18-8.25 (m, 3H). MS m/z350 (M+). 2-(Pyren-4-yl)phenylacetic Acid (20). To a solution of 19 (1.05 g, 3.0 mmol) in EtOH (150 mL) was added a saturated aqueous solution of NaOH (7.5 mL), and the resulting mixture was heated to reflux for 1 h. The solvent was removed, water was added, and the basic aqueous layer was extracted with CH2Cl2 to remove impurities. The aqueous layer was then acidified with concentrated HCl and extracted with CH2Cl2. The organic layer was dried over MgSO4, and the solvent was removed to give 0.92 g (91%) of acid 20 as a white solid; mp 95-96 °C. 1H NMR (300 MHz, CDCl3): δ 3.43 (d, 1H, J ) 16.4 Hz), 3.52 (d, 1H, J ) 16.4 Hz), 7.46-7.52 (m, 4H), 7.70 (d, 1H, J ) 7.9 Hz), 7.85 (dd, 1H, J ) 7.9 and 7.5

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Figure 4. Comparison of the 1H NMR spectra of peak 1 collected from the metabolism trace (A) and synthetically obtained N[1,2-e]P trans4,5-dihydrodiol (B). Both of the spectra were recorded at 500 MHz using DMSO-d6 as a solvent.

Scheme 3. Attempted Synthesis of 9-OH-N[1,2-e]P Starting from 4-Bromopyrene: The Cyclization of Acid Derivative 20 Using MeSO3H Actually Led to the Formation of Seven-Membered Ketone Derivative 21 Instead of the Desired N[1,2-e]P-9-one (22)

Hz), 7.95 (s, 1H), 7.98 (dd, 1H, J ) 7.9 and 7.5 Hz), 8.10 (s, 2H), 8.10-8.21 (m, 3H). MS m/z 336 (M+). Benzocyclohepten-6-(5H)-one[7,8,9-cd]pyrene (21). Method 1. To a refluxing solution of 20 (0.50 g, 1.5 mmol) in CH2Cl2 (15 mL) was added dropwise CH3SO3H (4 mL), and the refluxing was continued for 12 h under N2. Following a similar workup as described above for 1 yielded the crude ketone 21, which was purified by silica gel column chromatography with a mixture of ethyl acetate and hexane (3:97) to give 0.34 g (71%) of 21 as a yellow solid.

Sharma et al. Method 2. To a solution of 20 (0.10 g, 0.30 mmol) in CH2Cl2 (10 mL) containing a drop of DMF was added oxalyl chloride (0.19 g, 0.13 mL, 1.5 mmol) in CH2Cl2 (5 mL) dropwise at room temperature, and the reaction mixture was stirred for 2 h. The solvent was removed, and dry CS2 (7 mL) was added. This mixture was cooled to 0 °C, and AlCl3 (0.08 g, 0.6 mmol) was added. The reaction mixture was heated to reflux for 1 h, cooled, poured into ice-cold solution of water and HCl, extracted with CH2Cl2, washed with water (2×), and dried over MgSO4. Removal of the solvent gave the nearly pure 21, which was further purified as before to afford 93 mg (97%) of 21 as a yellow solid; mp 233-235 °C. 1H NMR (300 MHz, CDCl3): δ 3.94 (br s, 1H), 4.45 (br s, 1H), 7.39-7.52 (m, 3H), 7.62 (d, 1H, J ) 7.2 Hz), 8.11-8.16 (m, 2H), 8.22 (d, 1H, J ) 8.8 Hz), 8.25 (d, 1H, J ) 8.2 Hz), 8.33 (d, 1H, J ) 7.9 Hz), 8.39 (d, 1H, J ) 7.9 Hz), 8.52 (s, 1H), 8.68 (d, 1H, J ) 8.2 Hz). MS m/z 318 (M+). (1,2,3,6,7,8-Hexahydropyren-4-yl)boronic Acid (24). To a solution of 4-bromo-1,2,3,6,7,8-hexahydropyrene (23) (2.87 g, 10 mmol) at -78 °C under N2 was added n-BuLi (2.5 M in hexanes, 8.8 mL, 22 mmol) dropwise, and the reaction mixture was stirred at the same temperature for 1 h. Triisopropylborate (3.76 g, 4.6 mL, 20 mmol) was then added in one portion. The reaction was stirred at -78 °C for 30 min, warmed to room temperature, and stirred for another 1 h. The reaction mixture was then diluted with ether (200 mL) and acidified with 10% HCl. The ether extract was washed with water and dried over MgSO4. Removal of solvent gave a white solid, which was filtered and washed with hexanes/ether (9/1) to give 24 (2.1 g, 83%); mp 283-285 °C. 1H NMR (500 MHz, DMSO-d6): δ 1.20-1.34 (m, 4H), 1.91-1.96 (m, 3H), 2.98-3.03 (m, 4H), 3.15 (t, 1H, J ) 6.0 Hz), 7.32 (s, 2H), 8.04 (s, 1H). Methyl-[2-(1,2,3,6,7,8-hexahydropyren-4-yl)phenyl]acetate (25). A mixture of 1,2,3,6,7,8-hexahydropyrene-4-boronic acid 24 (1.16 g, 4.6 mmol), methyl-2-bromophenylacetate (0.96 g, 4.2 mmol), cesium fluoride (1.28 g, 8.4 mmol), and Pd(PPh3)4 (0.19 g, 0.17 mmol) in anhydrous DME (35 mL) was heated to reflux for 12 h. The crude 25, obtained by following a similar workup as compound 7, was purified by silica gel column chromatography with a mixture of ethyl acetate and hexane (1:99) to give 1.35 g (90%) of 25 as a white solid; mp 85-86 °C. 1H NMR (300 MHz, CDCl3): δ 1.89-1.99 (m, 2H), 2.01-2.13 (m, 2H), 2.56-2.67 (m, 1H), 2.72-2.80 (m, 1H), 3.05-3.14 (m, 6H), 3.43 (d, 1H, J ) 16.1 Hz), 3.52 (d, 1H, J ) 16.1 Hz), 3.57 (s, 3H), 7.01 (s, 1H), 7.19 (s, 2H), 7.22-7.26 (m, 1H), 7.32-7.43 (m, 3H). MS m/z 356 (M+). 2-(1,2,3,6,7,8-Hexahydropyren-4-yl)phenylacetic Acid (26). To a solution of 25 (1.25 g, 3.5 mmol) in EtOH (200 mL) was added a saturated aqueous solution of NaOH (10 mL), and the resulting mixture was heated to reflux for 30 min. A similar workup as described above for acid 20 gave 1.1 g (92%) of acid 26 as a white solid; mp 155-157 °C. 1H NMR (300 MHz, CDCl3): δ 1.81-1.96 (m, 2H), 2.01-2.10 (m, 2H), 2.55-2.63 (m, 1H), 2.67-2.75 (m, 1H), 3.00-3.10 (m, 6H), 3.42 (d, 1H, J ) 16.4 Hz), 3.52 (d, 1H, J ) 16.4 Hz), 6.96 (s, 1H), 7.16 (s, 2H), 7.20-7.23 (m, 1H), 7.31-7.40 (m, 3H). MSm/z342 (M+). 1,2,3,6,7,8-Hexahydronaphtho[1,2-e]pyrene-10-(9H)-one (27). To a refluxing solution of 26 (0.99 g, 2.9 mmol) in CH2Cl2 (15 mL) was added dropwise MeSO3H (7 mL), and the reaction mixture was heated to reflux for 12 h under N2. Following a similar workup as described above for 1 yielded the crude ketone, which was purified by silica gel column chromatography with a mixture of ethyl acetate and hexane (2:98) to give 0.62 g (66%) of 27 as a yellow solid; mp 175-177 °C. 1H NMR (500 MHz, CDCl3): δ 2.01-2.06 (m, 4H), 3.06 (t, 2H, J ) 6.0 Hz), 3.16 (t, 2H, J ) 6.0 Hz), 3.41-3.47 (m, 4H), 3.87 (s, 2H), 7.20 (d, 1H, J ) 7.0 Hz), 7.27-7.34 (m, 4H), 7.71 (d, 1H, J ) 7.5 Hz). MSm/z324 (M+). 9-Acetoxy-1,2,3,6,7,8-hexahydronaphtho[1,2-e]pyrene (28). A solution of 27 (0.13 g, 4.0 mmol) in isopropenyl acetate (15 mL) and acetic anhydride (2 mL) was heated to reflux under N2 atmosphere for 24 h. The reaction mixture was cooled, poured into water, and extracted with ethyl acetate. The EtOAc extract was dried over MgSO4, and the solvent was removed to give the crude product, which was purified by silica gel column chromatography

Identification of Naphtho[1,2-e]pyrene Metabolites

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Scheme 4. Synthesis of 9-OH-N[1,2-e]P (30) and N[1,2-e]P trans-9,10-Dihydrodiol (32)

using EtOAc:hexanes (3:97) to yield 28 (133 mg, 91%) as a pale yellow solid; mp 102-104 °C. 1H NMR (300 MHz, CDCl3): δ 1.98-2.09 (m, 4H), 2.42 (s, 3H), 3.17 (t, 2H, J ) 5.9 Hz), 3.23 (t, 2H, J ) 5.9 Hz), 3.58 (t, 2H, J ) 5.9 Hz), 3.85 (t, 2H, J ) 5.9 Hz), 7.17 (s, 1H), 7.16 (s, 2H), 7.21 (d, 1H, J ) 7.0 Hz), 7.24 (d, 1H, J ) 7.0 Hz), 7.44-7.53 (m, 2H), 7.71 (dd, 1H, J ) 7.5 and 2.0 Hz), 8.42 (d, 1H, J ) 8.2 Hz). MSm/z364 (M+). 9-Acetoxynaphtho[1,2-e]pyrene (29). To a solution of 28 (0.12 g, 0.33 mmol) in toluene (20 mL) was added DDQ (400 mg, 1.76 mmol), and the mixture was heated to reflux for 24 h. The reaction mixture was cooled, filtered, and purified by silica gel column chromatography (eluent: EtOAc:hexanes/3:97) to give 0.11 g (93%) of 29 as a pale yellow solid; mp 50-52 °C. 1H NMR (500 MHz, CDCl3): δ 2.53 (s, 3H), 7.65-7.67 (m, 2H), 7.75 (s, 1H), 8.01-8.14 (m, 5H), 8.24 (t, 2H, J ) 8.0 Hz), 9.03-9.06 (m, 1H), 9.15 (d, 1H J ) 8.0 Hz), 9.32 (d, 1H, J ) 8.5 Hz). MSm/z358 (M+). 9-Hydroxynaphtho[1,2-e]pyrene (30). To a solution of 29 (30 mg, 0.11 mmol) in methanol (10 mL) was added 200 µL of concentrated HCl, and the reaction mixture was stirred at room temperature for 24 h. Methanol was removed under reduced pressure and extracted with EtOAc, washed with water and brine, and dried with MgSO4.The crude residue was purified by silica gel column chromatography (eluent: hexanes:EtOAc, 90:10) to yield 30 (24 mg, 88%); mp 146-148 °C. 1H NMR (500 MHz, DMSOd6): δ 7.71 (t, 1H, J ) 7.5 Hz), 7.92 (t, 1H, J ) 8.0 Hz), 8.10 (d, 1H, J ) 6.5 Hz), 8.19-8.24 (m, 3H), 8.29 (s, 2H), 8.32 (d, 1H, J ) 2.5 Hz), 8.42 (d, 1H, J ) 7.5 Hz), 8.59 (d, 1H, J ) 7.5 Hz), 8.92 (d, 1H, J ) 8.0 Hz), 9.67 (d, 1H, J ) 8.0 Hz). HRMS calcd for C24H14O, 318.1040; found, 318.1046. Naphtho[1,2-e]pyrene-9,10-dione (31). To a solution of 30 (80 mg, 0.36 mmol) in 30 mL of benzene/CH2Cl2 (5:1) at room temperature were added Fremy’s salt (0.29 g, 1.08 mmol), 0.17 M KH2PO4 (15 mL), and Adogen (2 drops). The mixture was stirred for 6 h and worked up as described above for 13. The crude product was purified by silica gel column chromatography (EtOAc:hexanes/ 9:1) to yield 31 (96 mg, 81%) as a dark red solid; mp 202-204 °C.1H NMR (500 MHz, CDCl3): δ 7.61 (t, 1H, J ) 7.0 Hz), 7.77 (t, 1H, J ) 7.0 Hz), 8.04-8.23 (m, 6H), 8.27 (d, 1H, 7.0 Hz), 8.39 (d, 1H, J ) 7.5 Hz), 8.86 (d, 1H J ) 8 Hz), 9.80 (d, 1H, J ) 8.5 Hz). HRMS calcd for C25H16O, 332.0841; found, 332.0843. trans-9,10-Dihydroxy-9,10-dihydronaphtho[1,2-e]pyrene (32). To a solution of 31 (46 mg, 0.14 mmol) in ethanol was added NaBH4 (54 mg, 1.4 mmol), and the reaction mixture was stirred at room temperature for 5 h. A similar workup as described above for 14 gave the crude solid, which was triturated with ether:hexane (2:1) mixture and filtered to yield 0.37 g (78%) of dihydrodiol 32 as a pale yellow solid; mp 178-180 °C. 1H NMR (500 MHz, DMSOd6): δ 4.95-4.97 (m, 1H), 5.43 (t, 1H, J ) 4.0 Hz), 7.42-7.53

(m, 2H), 7.83 (d, 1H, J ) 7.5 Hz), 7.99 (d, 1H, J ) 7.0 Hz), 8.11-8.18 (m, 2H), 8.23 (s, 2H), 8.33 (d, 1H, J ) 7.0 Hz), 8.37 (d, 1H, J ) 7.5 Hz), 8.65 (d, 1H, J ) 8.0 Hz), 8.87 (d, 1H, J ) 8.0 Hz). HRMS calcd for C24H16O2, 336.1145; found, 336.1146. Preparation of Microsomes. Microsomes were prepared from livers of adult male Sprague-Dawley rats as previously described (17). Briefly, whole liver was homogenized in 3 volumes of 0.25 M potassium phosphate/0.15 M KCl/10 mM EDTA buffer pH 7.25 using electric homogenizer and five passes with a hand homogenizer. The homogenate was centrifuged at 10000g for 25 min. The supernatant was filtered through glass wool and ultracentrifuged at 105000g for 90 min. The supernatant was discarded, and the pellet was resuspended in 0.10 M sodium pyrophosphate/10 mM EDTA buffer pH 7.25 for wash and ultracentrifuged at 105000g for 60 min. The supernatant was discarded, and the pellet was resuspended in 0.10 M potassium phosphate/10 mM EDTA/0.10 mM DTT buffer pH 7.25 with 20% glycerol and immediately stored at -80 °C. All buffers also contained 0.25 mM PMSF. The protein concentration was determined using Biorad protein concentration assay (Biorad Laboratories, CA) to be 10 mg/mL. Metabolism of N[1,2-e]P with Rat Liver Microsomes. N[1,2e]P (2.0 mg) was dissolved in 200 µL of DMSO and added to 10 mL of pH 7.4 buffer (0.1 M K-PO4, 3 mM MgCl2, 5 mM KCl, 2 mM NADP+, 5 mM glucose-6-phosphate, 5 units of glucose-6phosphate dehydrogenase, and 5 mg of microsomes) with a final microsomal protein concentration of 0.5 mg/mL. The reaction mixture was incubated shaking at 37 °C for 45-90 min. Metabolism was stopped by adding 2 mL of acetone, and metabolites were extracted with EtOAc (3×), dried (MgSO4), filtered, and concentrated in vacuo. The residue was dissolved in 500 µL of MeOH and analyzed by HPLC using solvent system 1.

Results and Discussion N[1,2-e]P, required for metabolism, was synthesized using a Suzuki cross-coupling approach, which is well-established in our laboratory (8-10, 20-23) and a widely used method (18, 24-28) for constructing PAH skeletons and is generally preferred over the photochemical approach since it allows for a larger scale. The other method reported earlier for the synthesis of N[1,2-e]P 1 is through oxidative photocyclizations leading to low overall yield (19). The synthesis of N[1,2-e]P carried out using a Suzuki cross-coupling approach is outlined in Scheme 1. The palladium-catalyzed Suzuki cross-coupling of 4-pyreneboronic acid (4) with 2-bromobenzaldehyde (5) gave the aldehyde 7, which on treatment with methoxymethyl(triphenylphosphonium) chloride in the presence of PhLi resulted in

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the formation of olefin 9. Compound 9 cyclized readily in the presence of MeSO3H to give N[1,2-e]P (1) in high yield, which was characterized on the basis of NMR and high-resolution MS. The 1H NMR spectrum exhibited two downfield doublets (δ 9.17 and 9.23) typical of fjord region protons (H1 and H14). The metabolism of N[1,2-e]P with rat liver microsomes was carried out according to a literature method (17). Rat liver microsomes were incubated with N[1,2-e]P at 37 °C for 1 h in the presence of cofactors. The metabolites were extracted with EtOAc and concentrated in vacuo, the residue was dissolved in MeOH, and the metabolite generation was determined by HPLC analysis (solvent system 1). The profile of the metabolites by reverse-phase HPLC is shown in Figure 2. On the basis of our earlier metabolism studies (8, 10, 21) for various other PAHs, we expect the retention time (Rt) of peaks 1-3 (23.47, 27.89, and 29.35 min) as typical of dihydrodiol metabolites and peaks 4 and 5 (44.63 and 46.82 min) as possible hydroxyl metabolites (Figure 2). To unequivocally assign the structures, we synthesized various possible dihydrodiol and phenolic metabolites. The three likey dihydrodiol metabolites for N[1,2-e]P are fjord region 11,12-dihydrodiol and two K-region dihydrodiols: 4,5and 9,10-dihydrodiol. The expected proximate metabolite N[1,2e]P trans-11,12-dihydrodiol (14), which can be metabolized further to the ultimate carcinogen (14), the corresponding diol epoxide 15, was synthesized using a similar Suzuki crosscoupling approach as adapted for N[1,2-e]P (1) (Scheme 1). 12-Methoxy-N[1,2-e]P (11) was obtained from 4-pyreneboronic acid (4) following similar reaction steps as for the N[1,2-e]P synthesis. Compound 11 on treatment with BBr3 in CH2Cl2 afforded the 12-OH-N[1,2-e]P (12), which was oxidized by Fremy’s salt [(SO3K)2NO] to give quinone 13. Reduction of 13 with NaBH4 in EtOH gave the desired N[1,2-e]P trans-11,12dihydrodiol (14) in good yield (Scheme 1). The structure of 14 was characterized by 1H NMR and high-resolution MS data. HPLC analysis of the synthetic dihydrodiol 14 (Rt ) 29.35 min) identified it with peak 3 in the metabolism trace (Figure 2). To further confirm the identity of this metabolite, a sufficient amount of peak 3 was collected by HPLC and its 1H NMR spectrum was recorded. The 1H NMR spectra of the collected peak 3 from the metabolite mixture and the synthetic dihydrodiol 14 were identical (Figure 3) and confirmed the assignment of N[1,2-e]P trans-11,12-dihydrodiol to peak 3 in the metabolism trace. The dihydrodiol 14 was further converted to N[1,2-e]PDE (15) by treatment with m-CPBA using THF as a solvent (Scheme 1). The structure of 15 was assigned on the basis of 1 H NMR and high-resolution MS analysis. The purity of diol epoxide 15 was further determined by normal-phase HPLC analysis (solvent system 2). The diol epoxide 15 is a critical intermediate for the synthesis of DNA adduct markers and oligonucleotides and to subsequently determine the mutagenic potential of N[1,2-e]P. The other expected dihydroxy derivative from metabolism of N[1,2-e]P, the K-region N[1,2-e] P trans-4,5-dihydrodiol (18), was synthesized starting from the parent N[1,2-e]P as depicted in Scheme 2. Reaction of N[1,2-e]P (1) with OsO4 in pyridine furnished the N[1,2-e]P cis-4,5-dihydrodiol 16, which on treatment with PCC in CH2Cl2 gave the quinone 17 in quantitative yield. Reduction of quinone 17 with NaBH4 resulted in the desired N[1,2-e]P trans-4,5-dihydrodiol 18. The structure was assigned based on the proton NMR and MS analysis. The HPLC analysis of the synthetic compound 18 (Rt ) 23.47 min) revealed it to be identical to peak 1 in the metabolism trace (Figure 2). The assignment was further confirmed based on the

Sharma et al.

identical 1H NMR data and by HPLC coinjection of 18 with the mixture obtained from metabolism. The comparison of 1H NMR spectra of collected peak 1 and the synthetic standard 18 (Figure 4) clearly demonstrated N[1,2-e]P trans-4,5-dihydrodiol as a major content of peak 1. The extra signals in 1H NMR spectrum of collected peak 1 may be due to the presence of a small hidden peak overlapping with peak 1 or it may be due to some impurities emerging due to small quantities of the material collected for NMR analysis. The N[1,2-e]P cis-4,5-dihydrodiol (16, Rt ) 22.29 min) did not match with any of the peaks in the metabolism trace. Apart from its potential use as a synthetic marker, compound 18 is particularly useful for further metabolism and DNA-binding studies. Peak 2 (Figure 2) could not be isolated in a sufficient amount to get a good NMR spectrum. However, the retention time (Rt ) 27.89 min) was in the range of dihydrodiol metabolites and was thus expected to be the only other possible K-region N[1,2e]P trans-9,10-dihdyrdiol. A synthetic standard was thus required to determine the identity of peak 2. The synthesis of 9,10-dihydrodiol was first attempted starting from Suzuki crosscoupling reaction between pyrene-4-boronic (4) acid and 2-bromophenylmethyl acetate (Scheme 3). The reaction worked extremely well, and the resulting ester 19 was converted to acid derivative 20 by refluxing in ethanol in the presence of KOH. Interestingly, the attempts to obtain the keto derivative 22, the key intermediate for the synthesis of 9,10-dihydrodiol, failed, and treatment of 20 with methanesulfonic acid actually led to the cyclization at C-3 in the pyrene ring to form a sevenmembered keto derivative 21. The structure of 21 was assigned based on 1H NMR spectrum, which exhibited a singlet at δ 8.52 for H-14 and clearly ruled out the formation of 22 in which no singlet signals were expected. The formation of 22 was further dismissed by the lack of two downfield doublet signals expected for fjord region protons of 22. In addition, a doublet (δ 8.68), typical of H-7 in 21, appeared downfield due to the neighboring carbonyl group and further supported the structure 21. We thus changed the reaction conditions and first converted the acid derivative 20 to acid chloride with oxalyl chloride followed by in situ cyclization using AlCl3. However, this strategy again led to the formation of seven-membered keto derivative 21. It was interesting to note that in case of olefinic intermediates 9 and 10 (Scheme 1), cyclization occurred favorably at C-5 position to form compounds 1 and 11, respectively, while the acid intermediate 20 preferred to cyclize at C-3 position to form 21 (Scheme 3). Similar cyclization to seven-membered ring, under intramolecular Friedel-Craft’s acylation conditions on a pyrene moiety, has been documented earlier in the literature (29). The mechanism for this discrepancy pattern in cyclization remains unclear. To avoid this undesired cyclization, we coupled 1,2,3,6,7,8hexahydropyrene-4-boronic acid (24), instead of pyrene-4boronic acid, with 2-bromophenylmethyl acetate (Scheme 4). The resulting ester derivative 25 was then converted to the corresponding acid 26, which cyclized efficiently on treatment with methanesulfonic acid at the only possible C-5 position of the hexahydropyrene moiety. The resulting cyclized product could exist as a keto or enol tautomer. A singlet at δ 3.87 typical of CH2protons adjacent to keto group in the 1H NMR spectrum suggested it to be the keto tautomer 27. On the basis of a literature report (30), the exclusive formation of keto tautomer may be attributed to some remote steric factors that force 27 to exist as a ketone. This way planarization associated with ketone to phenol tautomerization is avoided, resulting in less steric clash in the fjord region.

Identification of Naphtho[1,2-e]pyrene Metabolites

Attempts to dehydrogenate 27 by treatment with DDQ to obtain 9-OH-N[1,2-e]P actually led to the aromatization of pyrene moiety but failed to enolize to phenol. The ketone 27 was thus esterified with isopropenyl acetate followed by aromatization with DDQ to give 9-acetoxy-N[1,2-e]P (29). Methanolysis of 29 in the presence of concentrated HCl furnished 9-OH-N[1,2-e]P (30), which was then converted to N[1,2-e]P-9,10-dione (31) by treating with Fremy’s salt. Quinone (31) was finally transformed to the desired N[1,2-e]P trans9,10-dihydrodiol (32) by reduction with NaBH4 in ethanol. The dihydrodiol 32 was characterized by 1H NMR and highresolution MS data, and its purity was further determined by HPLC. The HPLC retention time (20.47 min) of 32 did not match with any of the peaks in the metabolism trace. Peak 2 (Rt ) 27.89, Figure 2), which was thought to be trans-9,10dihydrodiol, thus remained unidentified. After confirming the identity of dihydrodiol metabolites, our next goal was to identify the phenolic metabolites of N[1,2e]P. The 12-OH-N[1,2-e]P (12) (Rt ) 46.82 min), obtained en route to the synthesis of N[1,2-e]P trans-11,12-dihydrodiol (Scheme 1), corresponded to peak 5 in the metabolism trace (Figure 2). Because of the low intensity of peak 5, it could not be collected by HPLC in sufficient amount to obtain a clean NMR spectrum for comparison with the synthetic standard. Peak 5 was assigned as 12-OH-N[1,2-e]P based on the identical Rt (46.82 min) and HPLC co-injection with the synthetic standard. 9-OH-N[1,2-e]P (30) (Rt ) 32.69 min), obtained en route to the synthesis of N[1,2-e]P trans-9,10-dihydrodiol (Scheme 4), did not match with any of the peaks in the metabolism trace. As observed from the reaction with OsO4 (Scheme 2), 4,5K-region is the most reactive site and is likely to have a hydroxyl substitution during metabolism. Thus, to start with, we carried out the dehydration of N[1,2-e]P trans-4,5-dihydrodiol (18) under acidic conditions, which led to the formation of exclusively one of the 4- or 5-OH-N[1,2-e]P as determined by HPLC analysis of the crude reaction mixture. The only major peak that appeared at Rt ) 38.82 min in HPLC (solvent system 1) was collected and characterized on the basis of 1H NMR spectrum. The spectrum exhibited four downfield doublets due to fjord (δ 9.27 and 9.31) and bay region (δ 8.91 and 8.97) protons and all other protons signals expected of 4- or 5-OHN[1,2-e]P. The exact position (4- or 5-) of the hydroxyl group could not be determined from the data. However, this phenolic derivative (Rt ) 38.82 min) did not correspond to any of the major peaks in the metabolism trace. Furthermore, 10-OHN[1,2-e]P (Rt ) 28.91 min), obtained along with a minor 9-OHN[1,2-e]P after the dehydration of N[1,2-e]P trans-9,10dihydrodiol (32), and 11-OH-N[1,2-e]P (Rt ) 47.36 min), obtained along with 12-OH-N[1,2-e]P after the dehydration of N[1,2-e]P trans-11,12-dihydrodiol (14), also did not match with any of the peaks in the metabolism HPLC trace. Thus, peak 5 (12-OH-N[1,2-e]P) was the only phenolic metabolite assigned among the hydroxy derivatives synthesized. In summary, the metabolism of N[1,2-e]P using rat liver microsomes led to both dihydrodiol and phenolic metabolites. Convenient methods for the syntheses of various dihydrodiol and phenolic metabolites of N[1,2-e]P were developed, and the structures of three major metabolites of N[1,2-e]P obtained by treatment with rat liver microsomes (Figure 2) were confirmed by comparison with the synthetic standards: peak 1 (trans-4,5dihydrodiol), peak 3 (trans-11,12-dihydrodiol), and peak 5 (12OH-N[1,2-e]P). These synthetic standards will also serve as markers for identifying the metabolites of N[1,2-e]P in vivo and are useful compounds for determining genotoxicity in

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animals. The phenols will be useful toward identifying the glucuronide metabolites in vivo. Among the dihydrodiol metabolites identified, the K-region dihydrodiol, that is, N[1,2e]P trans-4,5-dihydrodiol (18), will be a useful standard to study its further metabolism pattern. Above all, the formation of fjord region N[1,2-e]P trans-11,12-dihydrodiol, which can further metabolize to the corresponding diol epoxide and bind covalently to DNA, causing mutations and leading ultimately to the tumor induction, as the major metabolite, indicates the potential risk of the presence of N[1,2-e]P in the environment. Acknowledgment. This study was supported by NCI Contract NO2-CB-56603 and by the Penn State Cancer Institute of the Penn State College of Medicine. We thank Dr. Jyh-Ming Lin, Solution Phase NMR Facility at Core Research Facilities of the Penn State College of Medicine, for recording the NMR spectra. Supporting Information Available: Copies of the 1H NMR spectra for new compounds 1, 7-16, 19-21, and 24-32. This material is available free of charge via the Internet at http:// pubs.acs.org.

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