Chem. Res. Toxicol. 1990,3,93-97
93
Articles Synthesis and Mutagenicity of trans-Dihydrodiol Metabolites of Benzo[ b ]naphtha[ 2,l-dlthiophene Bijay Misra and Shantu Amin* Division of Chemical Carcinogenesis, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595 Received October 25, 1989
The syntheses of potentially important metabolites of benzo[b]naphtho[2,1-d]thiophene ( [ 2,1]BNT)-trans- 1,2-dihydroxy-1,2-dihydrobenzo[b] naphtho [ 2,l-d]thiophene ( [2,1] BNT-
1,2-diol) and trans-3,4-dihydroxy-3,4-dihydrobenzo[b]naphtho[2,1-d]thiophene ([2,1]BNT-3,4diol)-are described. The syntheses involved preparation of the appropriate 1-(3-benzo[b]thiopheneyl)-2-(methoxyphenyl)ethylenesfollowed by photocyclization to methoxy- [2,1]BNTs, hydrolysis to hydroxy-[2,1]BNTs, oxidation to [2,1]BNT-diones, and NaBH, reduction. The dihydrodiols were tested for mutagenicity in Salmonella typhimurium TA 100 with activation; [2,1]BNT-3,4-diol, which can form a bay region diol epoxide, was as mutagenic as [2,1]BNT whereas [2,1]BNT-1,2-diol was inactive. These results suggest that the metabolic activation of [2,1]BNT proceeds partially via formation of a bay region diol epoxide.
Introduction Benzo[blnaphtho [2,l-d]thiophene ([ 2,1]BNT)' (I),a sulfur analogue of chrysene, is known to be present in crude oil (I), shale oil (2),fresh and used engine oil (3,4), diesel exhaust (5),and several coal-derived products (6-8) and, hence, is a widely distributed environmentalpollutant. It is also a potential carcinogen. Studies have clearly demonstrated that the carcinogenic effects of polynuclear aromatic hydrocarbons are mainly due to bay region oxidation products formed metabolically by monooxygenases and hydrolases (9, 10). However, a recent report (11) indicated that [2,1]BNT was metabolized primarily to sulfoxide (2) and sulfone (3). Nevertheless, [2,1]BNT is mutagenic (12, 13) toward Salmonella typhimurium TA 100 and TA 98 and is known to induce monooxygenase (14, 15) activity in rat liver. For the past several years, our interests have centered on the synthesis and biological activity of the proximate and ultimate metabolites of various PAH carcinogens (16-19). In order to study the metabolic pathways of [2,1]BNT in detail and to investigate the applicability of the bay region oxidation theory to thiaarenes, we have undertaken an investigation of the in vitro metabolism of [2,1]BNT with rat liver homogenate (20) in conjuction with mutagenicity assays of its major metabolites in S. typhimurium TA 100. Preliminary results from our metabolism study of [2,1]BNT prompted u s to synthesize t h e postulated metabolites, and in this report we describe the synthesis of trans-1,2-dihydroxy1,2-dihydrobenzo[b] naphtho[ 2,l-d]thiophene ([ 2,1]BNT1,2-diol) (17) and trans-3,4-dihydroxy-3,4-dihydrobenzoAbbreviations: [2,1]BNT,benzo[b]naphtho[2,1-d]thiophene; [2,1]BNT-1,2-diol, trans-1,2-dihydroxy-1,2-dihydrobenzo[b]naphtho[2,l-d]thiophene: [2,1]BNT-3,4-diol, trans-3,4-dihydroxy-3,4-dihydrobenzo[b]naphtho[?,l-d]thiophene: chrysene-l,2-diol, trans-1,2-dihydroxy-1,2-dihydrochrysene.
0893-228~/90/2703-0093$02.50/0
[b]naphtho[2,1-d]thiophene([2,1]BNT-3,4-diol) (18). Syntheses of 17 and 18 enabled two [2,1]BNT metabolite structures to be confirmed and have allowed mutagenicity and carcinogenicity testing to commence. 5
4
io
\O 1
2
3
Experimental Section Melting points are uncorrected. Proton NMR spectra were recorded on a Bruker AM 360-MHz instrument. T h e proton chemical shifts are reported in ppm downfield from TMS at 0 ppm. High-resolution mass spectra (MS) were determined on a VG 70-250 double-focusing magnetic sector instrument, a t Rockefeller University. 3-Methylbenzo[b] thiophene was purchased from Lancaster Synthesis. All other reagents used were procured from Aldrich Chemical Co. 1- (3-Benzo[ b Ithiopheney1)-2-(2-methoxypheny1)et hylene (5a). 3-(Bromomethyl)benzo[b]thiophenewas synthesized from 3-methylbenzo[ blthiophene according to the literature (21). [ (3-Benzo[b]thiopheneyl)methyl]triphenylphosphonium bromide (8.8 g) was prepared (22) by refluxing 2-(bromomethy1)benzo[blthiophene (4.5 g, 0.02 mol) and triphenylphosphine (5.2 g, 0.02 mol) in benzene (200 mL) for 2 h in a 90% yield, mp 291-292 "C. A mixture of [ (3-benzo[b]thiopheneyl)methyl]triphenylphosphonium bromide (4) (4.9 g, 0.01 mol), 2-anisaldehyde (1.36 g, 0.01 mol), and sodium methoxide (1.1g, 0.02 mol) in MeOH (150 mL) was stirred at ambient temperature for 24 h. After the addition of 150 mL of HzO, the organic layer was extracted into CH,Cl, (2 X 150 mL). The solvent was removed and the resulting residue was filtered through a silica gel column with elution by hexane. T h e filtrate was concentrated under reduced pressure to afford 5a as a thick oil (1.3 g, 50%): 'H NMR (CDCl,) b 3.8 (s, 3 H, OCHJ, 6.9-7.0 (d, 2 H, vinylic), 7.1-7.6 (m, 7 H), 7.88
0 1990 American Chemical Society
94 Chem. Res. Toxicol., Vol. 3, No. 2, 1990 (d, 1 H), 8.01 (d, 1 H); MS, m/e (relative intensity) 266 (M+, 100), 235 (39), 234 (IO). Analogously, l-(3-benzo[b] thiopheneyl)-2-(3-methoxypheny1)ethylene (5b) and 1-(3-benzo[b]thiopheneyl)-2-(4-methoxypheny1)ethylene (5c) were prepared from 4 and 3- or 4-anisaldehyde in 62% and 58% yields, respectively. 5b: 'H NMR 6 3.8 (s, 3 H, OCH,), 6.8-7.0 (d, 2 H, vinylic), 7.1-7.6 (m, 7 H, aromatic), 7.8-8.2 (m, 2 H, aromatic); MS, m / e (relative intensity) 266 (M+, 100). 5c: 'H NMR 6 3.85 (s, 3 H, OCH,), 6.92 (d, 2 H, vinylic), 7.1-7.3 (m, 2 H), 7.35-7.55 (m, 5 H), 7.9 (d, 1 H), 8.1 (d, 1 H); MS, m / e (relative intensity) 266 (M+, 100). 4-Methoxybenzo[ blnapht ho[2,1-dIt hiophene (9). A solution of 5a (0.53 g, 2 mmol) and iodine ( 5 mg) in dry benzene (1 L) was irradiated with a Pyrex filtered Havonia 450-W mediumpressure UV lamp while dry air was bubbled into the solution. The cyclization of 5a was monitored by TLC. After 7 h, the solvent was removed under reduced pressure and the yellow residue was recrystallized from ethanol to yield 9 (0.3 g, 57%): mp 128-130 "C; 'H NMR (CDC13)6 4.05 (s, 3 H, OCH,) 6.95 (dd, 1 H, H,, J2,3 = 8.7 Hz,J3,1 = 2.4 Hz), 7.48-7.58 (m, 3 H, Hz, Ha, and Hg), 7.7 (dd, 1 H , H,, J1,,= 8.2 Hz, J1,3 = 2.4 Hz), 7.9-8.1 (m, 1 H , H7),8.15 (d, 1 H, H6, J6,5 = 8.4 Hz), 8.28 (m, 1 H, Hlo), 8.31 (d, 1 H, H,, J5,6 = 8.4 Hz); MS, m / e (relative intensity) 265 (M+ + 1, 100). Conversion of 5c to 2-methoxybenzo[b]naphtho[2,1-d]thiophene (7) was similarly achieved in 60% yield: mp 92-93 "C; 'H NMR (CDC13) 6 4.0 (s, 3 H, OCH,), 7.2 (dd, 1 H, H3, 53.4 = 8.9 Hz, 53,' = 2.4 Hz), 7.38 (d, 1 H, H,, J1,3 = 2.4 Hz), 7.48-7.52 (m, 2 H, HE = and Hg),7.82 (d, 1 H, H5, J 5 , 6 = 8.5 Hz), 7.85 (d, 1 H , H,, J4,, 8.9 Hz), 7.96 (dd, 1 H, H7, J 7 , a = 8.9 Hz, J 7 , g = 2.2 Hz), 8.08 (d, 1 H, He, J6,5 = 8.5 Hz), 8.21 (dd, 1 H , HI,, J10,g = 7.8 Hz, JIO,E = 1.8 Hz); MS, m / e (relative intensity) 264 (M', 100). Photocyclization of 5b, however, gave a mixture of l-methoxyand 3-methoxybenzo[b]naphtho[2,1-d]thiophenes(0.4 g, 70% ). The mixture of methoxybenzo[b]naphtho[2,1-d]thiophenes was used in the subsequent step without further purification. 4-Hydroxybenzo[ b]naphtho[2,1-d]thiophene (13). T o a stirred solution of 9 (0.26 g, 1mmol) in CH2C12(100 mL), a solution of boron tribromide (1 mL, 1 M) in CH2ClZwas added a t 0 "C under nitrogen over a period of 5 min. After 12 h a t room temperature, the mixture was hydrolyzed with ice-cold HzO, the organic layer was washed several times with HzO and dried (MgSO,), and the solvent was removed to yield crude 13, which was recrystallized from CH,Cl,/hexane (2:8) (0.18 g, 72%): mp 18C-182 OC; 'H NMR (CDC13) 6 6.92 (dd, 1 H, H3,J3,2 = 8.1 Hz, J 3 , 1 = 2.4 Hz), 7.4-7.6 (m, 3 H, H,, Ha, and Hg), 7.7-7.8 (m, 2 H, H1and H7),8.1-8.3 (m, 3 H, H6, Hlo, and H5);MS, m/e (relative intensity) 250 (M+, loo), 221 (39). High-resolution MS: calcd for M+, 250.042; found, 250.044. 2-Hydroxybenzo[b]naphtho[2,1-d]thiophene (1 1) was secured in 67% yield from 7 by the same procedure as described for 13: mp 171-172 "C; 'H NMR (CDCl,) 8 7.18 (dd, 1 H, H,, J,,, = 8.73 Hz, J 3 , 1 = 2.4 Hz), 7.42 (d, 1 H, H1, J1,3 = 2.4 Hz), 7.48-7.5 (m, 2 H, H8 and Hg),7.8 (d, 1 H, H5, J5,6 = 8.4 Hz), 7.9 (d, 1 H , H4, J4,,= 8.7 Hz), 7.95 (m, 1 H, H7, J7,8 = 8.8 Hz), 8.05 (d, 1 H , H6, J6:5 = 8.4 Hz), 8.2 (dd, 1 H, HI,, J10,g = 8.7 Hz, J l o , ~ = 2.7 Hz). High-resolution MS: calcd for M+, 250.042; found, 250.043. 1-OH- and 3-OH[2,1]BNT (10 and 12) were similarly prepared from the mixture of 6 and 8 by reaction with boron tribromide. The products were separated by silica gel column chromatography with elution by hexane/CH,Cl, (7:3), yielding 10 (0.1 g, 20%): mp 170-171 "C; 'H NMR (CDCl,) 6 7.0 (dd, 1 H, Hz, J2,3= 7.9 Hz, J 2 , 4 = 2.2 Hz), 7.4 (overlapped dd, 1 H, H,, J3,4= J3,2 = 7.9 Hz), 7.48-7.58 (m, 2H, H8 and Hg), 7.62 (d, 1 H, H4,J4,3= 7.9 Hz), 7.82 (d, 1 H , Hg, J 5 , 6 = 8.4 Hz), 8.0 (dd, 1 H, H7, J 7 , 8 = 7.9 Hz, J7,9= 2.4 Hz), 8.2-8.3 (m, 2 H, Hlo and H6); MS, m / e (relative intensity) 250 (M', 1001, 221 (30.5). High-resolution MS: calcd for M+, 250.042; found, 250.048. Elution with hexane/CH,CI, (1:l) yielded 12 (0.2 g, 55%): mp 232-234 "C; 'H NMR (CDC1,) 6 7.35 (dd, 1 H, H2, Jz,1 = 8.7 Hz, J2,4 = 2.4 Hz), 7.41 (d, 1 H , H4, J 4 , z = 2.4 Hz), 7.48-7.58 (m, 3 H, H1, Ha, and H&, 7.8 (d, 1 H , = 8.7 Hz), 8.05 (m,1 H , H7), 8.28 (d, 1 H, He, J6.5 = 8.7 H5, J5,6 = 8.3 Hz); MS, m/e (relative intensity) Hz), 8.31 (d, 1 H, Hlo, Jlo,g 250 (M+, 100). High-resolution MS: calcd for M', 250.042; found, 250.042.
Misra and Amin [2,1]BNT-3,4-dione (15). To a stirring mixture of 12 (0.12 g, 0.5 mmol) were added KH2P04(20 mL, 0.17 M) and Fremy's salt (0.32 g, 1.1mmol) in 75 mL of CHzClz/benzene (1684) and Adogen 464 (5 drops), and the stirring was continued for 2 h a t 25 "C. The contents were hydrolyzed with H 2 0 (200 mL), and the organic portion was extracted into benzene (2 X 100 mL). The benzene layer was washed with H 2 0 , dried (MgSO,), and concentrated under reduced pressure. The resulting dark residue was recrystallized from CHzC12/hexane to yield 15 as a bright red crystalline solid (70 mg, 53%): mp 178-180 "C dec; 'H NMR (CDCl,) 6 6.78 (d, 1 H, H,, J2,1= 10.2 Hz), 7.55-7.65 (m, 2 H , HB and Hg), 7.7 (d, 1 H, H1, J,,, = 10.2 Hz), 7.92 (d, 1 H , H6, J6,s = 8.4 Hz), 8.2-8.3 (m, 3 H, H5, Hlo, and H7); MS, m / e (relative intensity) 264 (M+, 26.5), 236 (100). High-resolution MS: calcd for M+, 264.024; found, 264.026. Similarly, [2,1]BNT-l,2-dione (14) was prepared from 10 (50 mg, 0.2 mmol) in 60% yield: mp 108-110 "C; 'H NMR (CDC13) 6 6.45 (d, 1 H, H3, J3,4= 10.5 Hz), 7.35-7.6 (m, 4 H, H4, H5, H8, and Hg),7.95 (m, 1 H , Hlo), 8.18 (d, 1 H, H7, J7,8= 7.3 Hz), 8.35 (d, 1 H, H6, J 6 , 5 = 7.8 Hz); MS, m/e (relative intensity) 264 (M+, 18.5), 236 (100). High-resolution MS: calcd for M+, 264.024; found, 264.029. [2,1]BNT-l,4-dione (16) was isolated from oxidation of 13 (50 mg, 0.2 mmol) by the same procedure as described above in 60% yield: mp 170-172 "C; 'H NMR (CDCl,) 6 6.68 (d, 1 H, H3, J3,2 = 10.2 Hz), 6.92 (d, 1 H, H,, J2,3= 10.2 Hz), 7.5-7.6 (m, 2 H , H8 and Hg), 7.98 (dd, 1 H, H7, J7,8= 7.3 Hz, J7,9= 1.4 Hz),8.21 (d, 1 H, H6, J 6 , 5 = 8.1 Hz), 8.25 (dd, H , Hi,, J10,g = 7.8 Hz, Jlo.8 = 1.8 Hz), 8.48 (d, 1 H, H,, 55,6 = 8.1 Hz); MS, m / e (relative intensity) 264 (M+, 26.51, 236 (100). High-resolution MS: calcd for M+, 264.024; found, 264.026. trans - 1,2-Dihydroxy-1,2-dihydrobenzo[ b Inapht ho[2,1dlthiophene (17). Sodium borohydride (0.5 g, 14 mmol) was added to a stirred suspension of 14 (70 mg, 2.6 mmol) in ethanol (150 mL), and the reaction mixture was stirred a t room temperature for 12 h. The resulting light yellow solution was diluted with H 2 0 (300 mL) and extracted into ethyl acetate (2 X 100 mL). The organic phase was washed with HzO, dried (MgSO,), and concentrated, and the resulting residue was separated by column chromatography on Florisil. The fraction eluted with CHzClz/ ethyl acetate (65:35) contained the diol 17 (35 mg, 40% yield): 'H NMR ( a ~ e t 0 n e - d 6~ 4.4 ) (d, 1 H, OH,, J0HZ.z = 5.4 Hz), 4.5-4.6 (m, 1 H, HJ, 5.0 (d, 1 H, OH,, JoH',' = 5.4 Hz), 5.05 (m, 1 H, Hl), 6.1 (dd, 1 H, H3,J3,4 = 10.0 Hz, J3,2 = 2.7 Hz), 6.6 (dd, 1 H, H4, J 4 , 3 = 10.0 Hz, J4,2 = 2.0 Hz), 7.3 (d, 1 H, H5, J5,e = 7.6 Hz), 7.44-7.55 (m, 2 H, H8 and Hg), 7.9-8.0 (m, 1 H , H7), 8.18 (d, 1 H , H6, J 6 , 5 = 7.6 Hz), 8.2-8.3 (m, 1 H, Hlo); MS, m/e (relative intensity) 268 (M+, 100), 250 (70), 222 (100). High-resolution MS: calcd for M+, 268.056; found, 268.056. trans -3,4-Dihydroxy-3,4-dihydrobenzo[b ]naphtho[$,ldlthiophene (18). [2,1]BNT-3,4-diol was prepared from 15 in 50% yield as described for 17: 'H NMR (acetone-ds) 8 4.3-4.4 (m, 1 H , H3), 4.07 (dd, 1 H, H4, J4,3= 10.7 Hz, J4,OH4 = 5.0 Hz), 5.4 (d, 1 H, OH,, JOH3,3 = 5.1 Hz), 5.65 (d, 1 H, OH4, JOH4,4 = 5.0 Hz), 6.18 (dd, 1 H, Hz, J2,l = 9.0 Hz, J2,3 = 3.0 Hz), 6.6 (d, 1 H, H1, J1,, = 9.0 Hz, J1,3 = 2.0 Hz), 7.5-7.6 (m, 2 H, Ha and Hg), 7.68 (d, 1 H, H,,J 5 , 6 = 8.4 Hz), 8.0-8.1 (m, 1 H, H7), 8.25 (d, 1 H, H6, 4 5 = 8.4 Hz), 8.35-8.45 (m, 1 H, Hlo); MS, m/e (relative intensity) 268 (M+, loo), 250 (70), 222 (100). High-resolution MS: calcd for M+, 268.056; found, 268.062. Mutagenicity Assays. S. typhimurium strain TA 100 was kindly provided by Dr. Bruce N. Ames, University of California, Berkeley. [2,1]BNT and [2,1]BNT-diols were dissolved in dimethyl sulfoxide, and assays were performed as described with preincubation (23,24). The purities of BNT-1,Zdiol and BNT3,4-diol were greater than 99% as analyzed by HPLC. In these studies 100 pL of S-9 mix (32 mg of protein/mL) was used per plate, and 2-aminoanthracene a t a dose of 5 kg/plate was employed as positive control. Reported mutagenicity values are the means of triplicate assays. Background revertants (150/plate) have not been subtracted.
Results and Discussion Several m e t h o d s h a v e been r e p o r t e d for the s y n t h e s e s of dihydrodiols and hydroxy derivatives of PAH (25,26).
Chem. Res. Toricol., Vol. 3, No. 2, 1990 95
[2,l]BNT-diols
Scheme I. Synthesis of [2,1]BNT-1,2-dlol and [2,1]BNT-3,4-diola
a) R = p O M e b) R = B O M e c) R : k O M e I
6
R 7 R
5R
3R 19 R 11 R 12 R R
E
= = I
=
1-OMe 2-OMe 3-OMe 4-OMe 1-OH 2-OH 3-OH 4-OH
1p
Lb
HO'*'& OH
-
17
14
0
14 (I) o-/m-lp-anisaldehyde, CH30Na/CH30H; (11) hv; (111) BBr3/CHzC12;(IV) (KSO,),NO; (V) NaBH,.
One procedure for the synthesis of non-K-region dihydrodiols involves oxidation of terminal ring phenols to quinones followed by reduction. We have used this approach for the syntheses of 17 and 18. The syntheses of four isomeric terminal ring phenols1-hydroxybenzo[b]naphtho[ 2,l-d]thiophene (1-OH[2,1]BNT) (lo),2-OH[2,1]BNT (ll),3-OH[2,1]BNT (12),and 4-OH[2,1]BNT (13)-and the two isomeric dihydrodiols 17 and 18 are outlined in Scheme I. Preparation of hydroxy[2,1]BNTs began with (3-benzo[b] thiopheney1)methyltriphenylphosphonium bromide (4). Reaction of 4 with o-anisaldehyde and p-anisaldehyde yielded the olefins 5a and 5c in 50% and 58% yields, respectively. Photolysis of 5a and 5c gave 4-methoxy[2,1]BNT (9)and 2-methoxy[2,1]BNT (7),respectively. However, reaction of 4 with m-anisaldehyde followed by photolysis of the resulting olefin (5b)afforded a mixture of l-methoxy[2,1]BNT (6)and 3-methoxy[2,1]BNT (8).Hydrolysis of 6 and 8 with boron tribromide gave a mixture of 10 and 12,which were separated by column chromatography. The structures of 10 and 12 were confirmed by proton NMR. In the proton spectrum of l-OH[B,l]BNT (lo), H2 appeared at 7.0 ppm as a doublet of doublets, whereas in 3-OH[2,1]BNT (12),H2was seen at 7.35 ppm as a doublet of doublets. H4 of 12 appeared 0.2 ppm upfield compared to H4 of 10 as expected. The synthesis of 17 was readily accomplished in good yield from 10 by oxidation with Fremy's salt to [2,1]BNT-1,2-dione (14) followed by NaBH, reduction. Oxidation of 4-OH[2,1]BNT (13), with an a-naphthol moiety, gave mostly [S,l]BNT-l,Cdione (16)along with a small amount of [2,1]BNT-3,4-dione(15). These results were expected on the basis of previous studies-e.g., oxidation of a-naphthols generally gives 1,4-diones (27). In contrast, oxidation of l-OH[2,1]BNT (10)gave mainly [2,1]BNT-1,2-dione (141,which is an exception to the general rule of oxidation by Fremy's salt. We made similar observations in the oxidation of 1-hydroxy-5-methylwhich both chrysene and l-hydroxy-6-methylchrysene, have an a-naphthol moiety; the products were 5-methyl(28). chrysene-1,Zdione and 6-methylchrysene-l,2-dione It may further be noted that the hydroxyl group in 10 is located in the bay region of [2,1]BNT and bay region hydroxyl functions generally resist oxidation by Fremy's
9 10
B
8.5
8.0
7.5
7.0
6.5 PPM
6.0
5.5
5.0
4.5
Figure 1. Proton NMR spectra (in acetone-d,) of (A) [2,1]BNT-1,2-diol and (B) [2,1]BNT-3,4-diol.
salt apparently due to steric interaction (29).The oxidation of phenol 10 to 14 in good yield indicates that the bay region in [2,1]BNT is less susceptible to steric interactions. Oxidation of 3-OH[2,1]BNT (12) exclusively yielded [2,1]BNT-3,4-dione (151,and reduction of 15 by NaBH, afforded 18 in 27% overall yield. The structures of the diols, 17 and 18, were confirmed by proton NMR and
96
Misra and Amin
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 8001
A
(2) Willey, C., Iwo, M., Castle, R. N., and Lee, M. L. (1981) Deter-
[2,1]BNT-3.4-DIOL
0 [2,1lBNT
n
4!Izl
5 ‘
300
8
200
2
100 0
0
40
80
120
160 200 240 Fg/Plate
280 320
Figure 2. Mutagenicity toward S. typhimurium T A 100 of
[2,1]BNT (e),[2,l]BNT-1,2-diol (m), and [2,1]BNT-3,4-diol (A). Assays were carried out in the presence of 9OOOg supernatant from the livers of rats pretreated with Aroclor 1254. Background revertants have not been subtracted.
high-resolution MS. The proton NMR spectra of 17 and 18 are displayed in Figure 1. The conformations of the hydroxyl groups in PAH dihydrodiols can be determined from the coupling constants of their vicinal hydrogens (30,31). The coupling constant between HI and H2 in 17 was 9.0 Hz, indicating a predominant pseudodiaxial orientation; i.e., the two hydroxyl groups in 17 are quasi-diequatorial. This appears to be a unique feature in [2,1]BNT, since hydroxyl groups of bay region dihydrodiols usually orient in a pseudodiaxial conformation. A quasi-diequatorial orientation of the hydroxyl groups in 18 was also assigned on the basis of the coupling constant of H3 and H4(10.7 Hz). The results of the mutagenicity assays of [2,1]BNT, [2,1]BNT-1,2-diol (17), and [2,1]BNT-3,4-diol (18) are shown in Figure 2. [2,1]BNT and [2,1]BNT-3,4-diol had comparable mutagenic activities at doses up to 40 pg/plate. At higher doses, [2,1]BNT appeared to be toxic. [2,1]BNT-1,Zdiol was less mutagenic than the other two compounds. These results suggest that [2,1]BNT-3,4-diol could be one proximate mutagen of [2,1]BNT, via conversion to the corresponding bay region diol epoxide. Our ongoing metabolism studies have demonstrated that both [2,1]BNT-3,4-diol and [2,1]BNT-l,2-diolare formed when [2,1]BNT is incubated with liver 9OOOg supernatant obtained from rats pretreated with Aroclor (20). These diols account for approximately 20% of the metabolites formed under these conditions. These results suggest that metabolic activation of [2,1]BNT may occur in part through a bay region diol epoxide, as observed for chrysene.
Acknowledgment. This study was supported by Grant 44377 from the National Cancer Institute. We thank Dr. Stephen Hecht for his critical comments and helpful suggestions. We thank Dr. Sharon Murphy for providing the preliminary results on BNT metabolism. We also thank Barbara Way for performing mutagenicity assays and Beth Appel for final preparation of the manuscript. Registry No. 1, 239-35-0; 4, 125847-33-8; 5a, 125847-34-9; 6, 125847-35-0; 7, 125847-36-1; 8, 31247-68-4; 9, 125847-37-2; 10, 125847-38-3; 11, 125847-39-4; 12, 125847-40-7; 13, 125847-41-8; 14,125847-42-9; 15, 125847-43-0; 16,125847-44-1; 17, 125847-45-2; 18,125847-46-3;2-(bromomethyl)benzo[b]thiophene,10133-20-7; triphenylphosphine, 603-35-0; 2-anisaldehyde, 135-02-4; 3-anisaldehyde, 591-31-1; 4-anisaldehyde, 123-11-5.
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