Synthesis of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-11- methylbenzo[a]pyrene and its reaction with DNA

Article abstract of DOI:10.1021/tx980178n

Substitution of a methyl group in the bay region can enhance the tumorigenicity of polycyclic aromatic hydrocarbons such as chrysene, benz[a]anthracene, and others. This phenomenon has been related to facile DNA adduct formation of bay region diol epoxides with a methyl group and epoxide ring in the same bay. While anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene and its DNA adduct formation have been studied extensively, it is not known whether a methyl substituent in the bay region alters the reactivity of DNA in this system. This is of interest because 11- methylbenzo[a]pyrene, which has a bay region methyl group, is more tumorigenic than benzo[a]pyrene. To examine the question, we have devised and employed an efficient synthesis based on photochemical cyclization, and prepared anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-11- methylbenzo[a]pyrene, the likely ultimate carcinogen of 11- methylbenzo[a]pyrene. We have then reacted anti-7,8-dihydroxy-9,10- epoxy7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene with calf thymus DNA and found that it gives three major adducts. These were identified as having resulted from cis- and trans-ring opening of the (S,R,R,S)-enantiomer and from trans-ring opening of the (R,S,S,R)-enantiomer. The standard deoxyguanosine adduct markers were prepared, and their structures were tentatively assigned on the basis of their CD and ¹H NMR spectra. The adduct distribution of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-11- methylbenzo[a]pyrene) is quite different from that observed in the reaction of DNA with the corresponding diol epoxides of benzo[a]pyrene or with 5- methylchrysene. The heterogeneity of adducts obtained with anti-7,8- dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene thus may be related to the enhanced tumorigenicity of 11-methylbenzo[a]pyrene.

Full text of DOI:10.1021/tx980178n

Chem. Res. Toxicol. 1999, 12, 341-346
341
Syn th esis of a n ti-7,8-Dih yd r oxy-9,10-ep oxy-
7,8,9,10-tetr a h yd r o-11-m eth ylben zo[a ]p yr en e a n d Its
Rea ction w ith DNA
J yh-Ming Lin,*,† Dhimant Desai,† Lehua Chung,† Stephen S. Hecht,‡ and
Shantu Amin†
Organic Synthesis Facility, Naylor Dana Institute for Disease Prevention, American Health
Foundation, Valhalla, New York 10595, and University of Minnesota Cancer Center,
Minneapolis, Minnesota 55455
Received J uly 28, 1998
Substitution of a methyl group in the bay region can enhance the tumorigenicity of polycyclic
aromatic hydrocarbons such as chrysene, benz[a]anthracene, and others. This phenomenon
has been related to facile DNA adduct formation of bay region diol epoxides with a methyl
group and epoxide ring in the same bay. While anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene and its DNA adduct formation have been studied extensively, it is
not known whether a methyl substituent in the bay region alters the reactivity of DNA in this
system. This is of interest because 11-methylbenzo[a]pyrene, which has a bay region methyl
group, is more tumorigenic than benzo[a]pyrene. To examine the question, we have devised
and employed an efficient synthesis based on photochemical cyclization, and prepared anti-
7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene, the likely ultimate
carcinogen of 11-methylbenzo[a]pyrene. We have then reacted anti-7,8-dihydroxy-9,10-epoxy-
7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene with calf thymus DNA and found that it gives
three major adducts. These were identified as having resulted from cis- and trans-ring opening
of the (S,R,R,S)-enantiomer and from trans-ring opening of the (R,S,S,R)-enantiomer. The
standard deoxyguanosine adduct markers were prepared, and their structures were tentatively
1
assigned on the basis of their CD and H NMR spectra. The adduct distribution of anti-7,8-
dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene is quite different from that
observed in the reaction of DNA with the corresponding diol epoxides of benzo[a]pyrene or
with 5-methylchrysene. The heterogeneity of adducts obtained with anti-7,8-dihydroxy-9,10-
epoxy-7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene thus may be related to the enhanced
tumorigenicity of 11-methylbenzo[a]pyrene.
and 12 in 5-MeC), both adjacent to an unsubstituted
angular ring (3).
In tr od u ction
Polycyclic aromatic hydrocarbons (PAHs)1 with a meth-
yl group in the bay region adjacent to an unsubstituted
angular ring are frequently highly tumorigenic, and
usually more tumorigenic than other methyl isomers in
the same series, or than the parent hydrocarbon (1, 2).
We have studied this phenomenon with 5-methylchry-
sene (5-MeC) as a model carcinogen. Its tumorigenic
activity is greater than that of any of the other mono-
methylchrysenes or of chrysene itself (1). The presence
of a methyl group and epoxide ring in the same bay
region of a chrysene diol epoxide enhances the reactivity
with DNA in vitro and in vivo (1). These studies provide
biochemical support for the generalization that the
structural requirements favoring carcinogenicity of
methylated PAHs include the presence of a bay region
methyl group and a free peri position (e.g., positions 6
An extensive database regarding the metabolic activa-
tion and DNA binding properties of benzo[a]pyrene (BP)
is available. Iyer et al. have examined the tumor-
initiating activity on mouse skin of all possible mono-
methylated isomers of BP (4). In terms of tumor multi-
plicity, 11-methylBP (11-MeBP) was the most active com-
pound, its tumor initiating activity being approximately
3 times greater than that of BP. 1-, 3-, 4-, and 12-MeBP
had activities approximately equal to that of BP, while
the other isomers were less active, or inactive (4).
However, no studies have been reported on the meta-
bolic activation or macromolecular binding of 11-MeBP.
In this paper, we report our initial investigations on
comparative DNA adduct formation by 11-MeBPDE and
BPDE. We synthesized anti-7,8-dihydroxy-9,10-epoxy-
† American Health Foundation.
‡ University of Minnesota Cancer Center.
1
Abbreviations: 11-MeBPDE, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,-
10-tetrahydro-11-methylbenzo[a]pyrene; BPDE, anti-7,8-dihydroxy-9,-
10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BP, benzo[a]pyrene; 5-MeC,
5-methylchrysene; PAHs, polycyclic aromatic hydrocarbons; 11-MeBP,
11-methylbenzo[a]pyrene; 5-MeCDE, anti-1,2-dihydroxy-3,4-epoxy-
1,2,3,4-tetrahydro-5-methylchrysene; dG, 2′-deoxyguanosine; dA, 2′-
deoxyadenosine.
10.1021/tx980178n CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/16/1999

342 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
Lin et al.
7.4 (dd, 1H, H3, J 1,3 ) 2.9 Hz, J 3,4 ) 9.4 Hz), 7.49 (d, 1H, H1,
J 1,3 ) 2.9 Hz), 7.60-7.70 (m, 2H, H8 and H9), 7.90-8.00 (m +
s, 3H, H7, H10, and H6), 8.40 (s, 1H, H12), 8.78 (m, 1H, H4,
J 3,4 ) 9.4 Hz), 10.49 (s, 1H, CHO).
7,8,9,10-tetrahydro-11-methylbenzo[a]pyrene (11-MeB-
PDE) and examined its reaction with calf thymus DNA.
1-(2-Met h oxy-5-m et h ylch r ysen -11-yl)-2-m et h oxyet h yl-
en e (5). Under a N2 atmosphere, a solution of 1.8 M phenyl-
lithium (in 70:30 cyclohexane/ether, 0.90 mL, 1.60 mmol) was
added dropwise to a solution of (methoxymethyl)triphenylphos-
phonium chloride (0.69 g, 2.0 mmol) in anhydrous Et2O (15 mL)
at -78 °C. The mixture was stirred at -78 °C for 30 min, then
warmed to -20 °C for 30 min, and then again cooled to -78 °C.
A solution of carboxaldehyde 4 (0.4 g, 1.33 mmol) in anhydrous
THF (15 mL) was added. Stirring was continued for 1 h at -78
°C; then the reaction mixture was allowed to warm to room
temperature, and stirring was continued for 16 h. After the
usual workup, the crude olefin 5 was purified by chromatogra-
phy on silica gel with elution by hexane/CH2Cl2 (80:20) to give
5 (0.27 g, 61%) as a mixture of cis and trans isomers; it was
recrystallized from a mixture of hexane and Et2O: mp 121-
123 °C; 1H NMR (acetone-d6) δ 3.26 (s, 3H, CH3), 3.85 (s, 2.25H,
CHdCHOCH3), 3.95 (s, 0.75H, CHdCHOCH3), 4.10 (s, 3H,
OCH3), 5.90 (d, 0.25H, CHdCH, J ) 7.1 Hz), 6.60 (d, 0.75H,
CHdCH, J ) 12.6 Hz), 6.65 (d, 0.25H, CHdCH, J ) 7.1 Hz),
7.25 (dd, 1H, H3, J 2,3 ) 9.4 Hz, J 1,3 ) 2.8 Hz), 7.35 (d, 0.75H,
CHdCH, J ) 12.7 Hz), 7.40-7.60 (m, 3H, aromatic), 7.80-8.00
(m, 3H, aromatic), 8.75 (m, 1H), 9.05 (m, 1H).
Exp er im en ta l Section
1
Ap p a r a tu s. H NMR spectra were recorded in CDCl3 using
a Bruker model AM 360 WB spectrometer. Chemical shifts are
expressed in parts per million downfield from tetramethylsilane.
Mass spectra (MS) were recorded on a Hewlett-Packard model
5988A instrument. High-resolution MS were obtained with a
Finnigan Mat 95 instrument at the Mass Spectrometry Service
(University of Minnesota, Minneapolis, MN). Circular dichroism
(CD) spectra were determined in MeOH in an Aviv Associates
Inc. (Lakewood, NJ ) model 62ADS CD spectrometer. Thin-layer
chromatography (TLC) separations were performed on aluminum-
supported precoated silica gel plates from EM Industries
(Gibbstown, NJ ). Starting materials were obtained from Aldrich
Chemical Co. (Milwaukee, WI), unless stated otherwise. Ethyl
(3-methyl-1-naphthyl)acetate (1) was prepared in 50% yield as
reported in the literature (5).
1-Ca r bom eth oxy-1-(3-m eth yl-1-n a p h th yl)-2-(3-m eth oxy-
p h en yl)eth ylen e (2). To a solution of 1 (4.28 g, 20 mmol) being
stirred in anhydrous tetrahydrofuran (THF) (40 mL) at -78 °C
was added dropwise NaN[(CH3)3Si]2 (1.0 M, 25 mL, 25 mmol)
in anhydrous THF (100 mL). The mixture was stirred at -78
°C for 30 min and the reaction quenched with a solution of
m-anisaldehyde (2.72 g, 20 mmol) in anhydrous THF (20 mL).
After the mixture warmed to room temperature, it was stirred
for an additional 16 h, and then poured into a saturated solution
of NH4Cl. After extraction with CH2Cl2 (3 × 40 mL), the
combined organic layers were dried (MgSO4), filtered, and
evaporated to dryness to give crude 2. The crude compound was
purified by chromatography on silica gel with elution by hexane
to yield major trans olefin 2 as an oil (3.65 g, 55%): 1H NMR δ
1.20 (t, 3H, CH3CH2), 2.48 (s, 3H, CH3), 3.15 (s, 3H, OCH3), 3.81
(q, 2H, CH2), 6.3 (s, 1H, olefin), 6.65-6.75 (m, 2H), 7.25 (s, 1H),
7.35 (s, 1H), 7.4-7.6 (m, 2H), 7.65-7.95 (m, 3H), 8.05 (s, 1H).
11-Ca r boeth oxy-2-m eth oxy-5-m eth ylch r ysen e (3). A so-
lution of olefin 2 (2.7 g, 8.13 mmol) and iodine (5 mg) in
anhydrous benzene (1.2 L) was irradiated with a Pyrex-filtered
Hanovia 450 W medium-pressure UV lamp, while dry air was
bubbled through it. The reaction was stopped after 8 h; the
solvent was removed under reduced pressure, and the residue
was purified by chromatography on silica gel with elution by
EtOAc/hexane (1:9) to yield cyclic product 3 (1.0 g, 37%): 1H
NMR δ 1.25 (t, 3H, CH3CH2, J ) 7.2 Hz), 3.10 (s, 3H, CH3),
3.98 (s, 3H, OCH3), 4.42 (q, 2H, CH3CH2, J ) 7.2 Hz), 7.31 (dd,
1H, H3, J 3,4 ) 9.4 Hz, J 1,3 ) 2.8 Hz), 7.37 (d, 1H, H1, J 1,3 ) 2.8
Hz), 7.48-7.56 (m, 2H, H8 and H9), 7.82 (s, 1H, H6), 7.86 (dd,
1H, H7, J 7,8 ) 7.8 Hz and J 7,9 ) 1.3 Hz), 8.10 (d, 1H, H4 or
H10, J ) 8.5 Hz), 8.12 (s, 1H, H12), 8.73 (d, 1H, H4 or H10, J
) 9.4 Hz).
8-Meth oxy-11-m eth ylben zo[a ]p yr en e (6). Under
a N2
atmosphere, a stirred CH2Cl2 solution of 5 (0.25 g, 0.76 mmol)
was kept at 0 °C, while CH3SO3H (5 mL) was added dropwise
over the course of 10 min. The reaction mixture was then stirred
for an additional 6 h. The usual workup gave a crude product
which was purified by chromatography on silica gel with elution
by hexane/CH2Cl2 (4:1) to give 6 (0.17 g, 77%); it was recrystal-
1
lized from a mixture of hexane and Et2O: mp 152-153 °C; H
NMR δ 3.39 (s, 3H, CH3), 4.06 (s, 3H, OCH3), 7.41 (dd, 1H, H9,
J 9,10 ) 9.7 Hz, J 9,7 ) 2.9 Hz), 7.56 (d, 1H, H7, J 7,9 ) 2.9 Hz),
7.80-8.03 (m, 4H), 8.10-8.40 (m + s, 2H), 8.42 (s, 1H, H6), 9.21
(d, 1H, H10, J 9,10 ) 9.7 Hz).
8-Hyd r oxy-11-m eth ylben zo[a ]p yr en e (7). To a stirred
solution of BP derivative 6 (0.15 g, 0.51 mmol) in CH2Cl2 (150
mL) was added dropwise a solution of BBr3 in CH2Cl2 (0.55 mL,
0.55 mmol) at 0 °C under N2. The reaction mixture was then
stirred at room temperature for 12 h, and the excess reagent
was hydrolyzed with ice-cold H2O. The organic layer was washed
with H2O (3 × 50 mL), dried (MgSO4), filtered, and evaporated
to dryness to give crude 7 which was purified by chromatogra-
phy on silica gel with elution by hexane/CH2Cl2 to give 7 (0.12
g, 85%); it was recrystallized from a mixture of hexane and
1
Et2O: mp 222-224 °C; H NMR δ 3.39 (s, 3H, CH3), 7.45 (dd,
1H, H9, J 9,10 ) 9.6 Hz, J 9,7 ) 2.8 Hz), 7.65 (d, 1H, H7, J 7,9 ) 2.8
Hz), 7.80-8.10 (m, 4H), 8.10-8.45 (m + s, 2H), 8.46 (s, 1H, H6),
9.25 (d, 1H, H10, J 9,10 ) 9.6 Hz); high-resolution MS (positive
chemical ionization using ammonia as the reagent gas) calcd
for C21H15O 283.1125, found 283.1123.
11-Meth ylben zo[a ]p yr en e-7,8-d ion e (8). Adogen 464 (3
drops) was added to a mixture of 7 (0.1 g, 0.36 mmol), KH2PO4
(20 mL, 0.2 M solution), and Fremy’s salt (0.19 g, 0.72 mmol)
being stirred in CH2Cl2/benzene (20 mL, 5:1). Stirring was
continued for 3 h at room temperature. The organic layer was
collected, and the aqueous solution was extracted with benzene.
The benzene extract was washed with H2O and dried (MgSO4);
then the solvent was removed. The residue was purified by
chromatography on silica gel with elution by hexane/CH2Cl2 (50:
50) to give dione 8 (80 mg, 71%); it was recrystallized from a
2-Meth oxy-5-m eth ylch r ysen e-11-ca r boxa ld eh yd e (4). A
solution of 3 (0.90 g, 2.73 mmol) in 6.5 mL of anhydrous THF
was added dropwise to a solution of diisobutylaluminum hydride
(3 mL, 3 mmol) in 10 mL of anhydrous THF. The mixture was
stirred for 16 h at room temperature, poured into H2O, and
extracted with CH2Cl2. The organic layer was washed with H2O
and dried over MgSO4. Removal of the solvent gave crude
aldehyde 4 which was purified by chromatography on silica gel
with elution by hexane/CH2Cl2 (45:55) to give 4 (0.44 g, 54%);
it was recrystallized from a mixture of hexane and Et2O: mp
1
mixture of hexane and Et2O: mp 218-220 °C; H NMR δ 3.10
(s, 3H, CH3), 6.50 (d, 1H, H9, J 9,10 ) 10.8 Hz), 7.95-8.35 (m,
6H), 8.81 (d, 1H, H10, J 9,10 ) 10.8 Hz), 8.90 (s, 1H, H6); high-
resolution MS calcd for C21H12O2 296.0837, found 296.0836.
7,8-Dih ydr oxy-7,8-dih ydr o-11-m eth ylben zo[a ]pyr en e (9).
To a stirred suspension of dione 8 (60 mg, 0.20 mmol) in ethanol
(50 mL) was added NaBH4 (100 mg, 2.64 mmol) over the course
of 10 min. The reaction mixture was stirred for 24 h in an open
1
171-173 °C; H NMR δ 3.25 (s, 3H, CH3), 4.01 (s, 3H, OCH3),

11-Methylbenzo[a]pyrene Diol Epoxide-DNA Adduct
Chem. Res. Toxicol., Vol. 12, No. 4, 1999 343
Sch em e 1. Syn th esis of 11-MeBP DEa
a
(a) NaN[(CH3)3Si]2, m-anisaldehyde; (b) hν/I2, PhH; (c) DIBAL; (d) CH3OCH2P+(Ph)3Cl-, PhLi, THF; (e) CH3SO3H; (f) BBr3; (g) Fremy’s
salt; (h) NaBH4/MeOH; (i) mCPBA.
flask and was then poured into ice-cold H2O and extracted with
EtOAc (3 × 100 mL). The combined organic layers were washed
with H2O, dried (Na2SO4), and concentrated. The resulting
residue was purified by chromatography on Florisil with elution
by CH2Cl2/EtOAc (50:50) to give diol 9 (50 mg, 82%): 1H NMR
(acetone-d6) δ 3.08 (s, 3H, CH3), 4.52 (m, 1H, H8), 4.90 (d, 1H,
H7, J 7,8 ) 11.3 Hz), 6.22 (dd, 1H, H9, J ) 2.0 Hz, J 9,10 ) 10.3
Hz), 7.70 (dd, 1H, H10, J 8,9 ) 2.3 Hz, J 9,10 ) 10.4 Hz), 7.79-
7.80 (m, 2H), 8.05-8.18 (m, 4H), 8.51 (s, 1H, H6); high-
resolution MS (positive chemical ionization using ammonia as
the reagent gas) calcd for C21H17O2 301.1230, found 301.1228.
a n ti-7,8-Dih yd r oxy-9,10-ep oxy-7,8,9,10-t et r a h yd r o-11-
m eth ylben zo[a ]p yr en e (11-MeBP DE) (10). A mixture of diol
9 (30 mg, 0.1 mmol), m-chloroperbenzoic acid (85 mg, 0.5 mmol),
and anhydrous THF (20 mL) was stirred under N2 for 4 h. Then
the reaction mixture was diluted with Et2O (100 mL), washed
with cold 2% NaOH (3 × 20 mL) and H2O, and dried (K2CO3).
Removal of the solvent gave a pale yellow powder, which was
recrystallized from Et2O/CH2Cl2 to yield diol epoxide 10 (19 mg,
61%): mp 164-165 °C; 1H NMR (DMSO-d6) δ 3.19 (s, 3H, CH3),
3.87 (dd, 1H, H9, J 9,10 ) 4.5 Hz, J 8,9 ) 1.6 Hz), 4.04 (d, 1H, H8,
J 7,8 ) 8.5 Hz), 4.69 (bs, 1H, OH), 5.00 (d, 1H, H7, J 7,8 ) 8.6
Hz), 5.04 (bs, 1H, OH), 5.20 (d, 1H, H10, J 10,9 ) 4.5 Hz), 8.03
(dd, 1H, H2, J ) 7.6 Hz), 8.10 (s, 1H, H12), 8.16-8.19 (m, 3H,
H3, H4, and H5), 8.24 (dd, 1H, H1, J 1,2 ) 7.6 Hz, J 1,3 ) 1.1 Hz),
8.62 (s, 1H, H6).
P r ep a r a tion of DNA Ad d u cts. 11-MeBPDE (500 µg) in 500
µL of THF was added to a solution of calf thymus DNA (5 mg)
in 5 mL of 10 mM Tris-HCl buffer (pH 7). This mixture was
incubated at 37 °C overnight. Tetraols were removed from the
DNA solutions by three extractions with EtOAc (6 mL) and one
with Et2O (6 mL). The DNA was isolated and hydrolyzed
enzymatically to deoxyribonucleosides as described previously
(6). Unmodified deoxyribonucleosides were separated from
modified deoxyribonucleosides on Sep-Pak C18 cartridges (7).
The modified deoxyribonucleosides were analyzed by HPLC as
described below.
enzymatically hydrolyzed to deoxyribonucleosides as described
previously (6). A Beckman Ultrasphere ODS 5 µm column (4.6
mm × 250 mm; Beckman Instruments, Fullerton, CA) with the
following solvent system was used to separate the modified
deoxyribonucleosides: 46% MeOH in H2O for 34 min, then to
55% MeOH in H2O (linear gradient) over the course of 10 min,
and then 55% MeOH in H2O for 46 min. The flow rate was 1
mL/min. The three major peaks were collected, and the solvent
was evaporated; the resulting residues were redissolved in
MeOH, and a CD spectrum of each sample was determined.
P r ep a r a tion of P er a ceta tes of N2-d G-11-MeBP DE. The
three major N2-dG-11-MeBPDE adducts were converted to the
corresponding peracetates. The solvent was evaporated, and the
residue was dissolved in 3 mL of pyridine and mixed with 2
mL of Ac2O. The resulting mixture was stirred at room tem-
perature overnight. Excess reagent was removed in vacuo. The
residue was redissolved in THF and purified by HPLC on a
Beckman Ultrasphere ODS 5 µm column (4.6 mm × 250 mm).
The following solvent system was used for the separation of
modified deoxyribonucleosides at a flow rate of 1 mL/min: 15%
MeOH in H2O to 65% MeOH in H2O (linear gradient) over the
course of 15 min and then to 100% MeOH in 20 min. Each
sample was collected and its solvent evaporated, and the sample
was then redissolved in acetone-d6. 1H NMR spectra were
recorded at 360 MHz with a 5 mm 1H probe.
Mu ta gen icity Assa ys. Diol epoxides were dissolved in
DMSO, and Salmonella typhimurium strain TA 100 was used
for the assay performed as described with preincubation (8, 9).
Cytotoxicity was determined by reduction of the number of
colonies in nutrient agar plates. 11-MeBPDE was toxic above 1
nmol/plate. Reported mutagenicity values are the means of
triplicate assays. Background revertants (145 per plate) have
not been subtracted. The number of revertants per nanomole
was determined from the slopes of the linear portions of the
dose-response curves.
Resu lts a n d Discu ssion
P r ep a r a tion of Sta n d a r d 2′-d G or 2′-d A Ad d u cts fr om
11-MeBP DE. Either 2′-dG-5′-monophosphate (40 mg) or 2′-dA-
5′-monophosphate (40 mg) was dissolved in 1 mL of 10 mM Tris-
HCl buffer (pH 7.0). To the above solution was added 11-
MeBPDE (500 µg) in 0.45 mL of acetone, and the mixture was
incubated overnight at 37 °C. The reaction mixture was
extracted three times with equal volumes of EtOAc (saturated
with H2O) and once with Et2O. Finally, the remaining traces of
Et2O were evaporated from the aqueous layer with a gentle
stream of N2, and the modified deoxyribonucleotides were
Syn th esis of 11-MeBP DE. Our synthetic strategy
was based on photochemical cyclization of the appropri-
ately substituted ethylene as illustrated in Scheme 1. We
have found that the photochemical cyclization provides
a very efficient approach to the synthesis of dibenzo[a,l]-
pyrene diol epoxide and various substituted chrysene diol
epoxides (10, 11). The precursor for the photocyclization,
1-carbomethoxy-1-(3-methyl-1-naphthyl)-2-(3-methoxy-

344 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
Lin et al.
Ta ble 1. Ch em ica l Sh ifts a n d Cou p lin g Con sta n ts for th e Meth in e P r oton s of BP DE a n d 11-MeBP DE
methine protons
H7
H8
H9
H10
compound
δ (ppm)
δ (ppm)
coupling constant (Hz)
δ (ppm)
coupling constant (Hz)
δ (ppm)
BPDEa
4.71
5.00
3.92
4.04
J 7,8 ) 8.8
J 7,8 ) 8.5
3.86
3.87
J 9,10 ) 4.6
J 9,10 ) 4.5
5.21
5.20
11-MeBPDEa
a
NMR spectra were recorded in DMSO-d6 with a 5 mm 1H probe at 360 MHz on a Bruker AM 360 WB spectrometer.
phenyl)ethylene (2), was prepared by condensation of
ethyl (3-methyl-1-naphthyl)acetate (1) and m-methoxy-
benzaldehyde. Photochemical cyclization of 2 gave 2-meth-
oxy-5-methyl-11-carboethoxychrysene (3) in 37% yield. It
is interesting to note that the other possible structural
isomer, 4-methoxy-5-methyl-11-carboethoxychrysene, was
not detected. Conversion of the carboethoxy derivative 3
to aldehyde 4 was achieved in 54% yield with diisobutyl-
aluminum hydride, and the concentration of the corre-
sponding alcohol was determined to be <10% by chroma-
tography. One-carbon chain extension was accomplished
by means of the Wittig reaction with (methoxymethyl)-
triphenylphosphonium chloride to give 5 in 61% yield.
To construct the benzo[a]pyrene ring system, standard
acid-catalyzed cyclization was employed (10). Treatment
of 5 with methanesulfonic acid gave 8-methoxy-11-
methylBP (6) in 77% yield. Demethylation of 6 with boron
tribromide afforded 7 in 85% yield. 11-MeBPDE (10) was
prepared from 7 by application of well-established meth-
ods (12). The overall yield of 11-MeBPDE (10) from 7 was
35%. The presence of a methyl group in the bay region
may affect the conformation in the terminal ring. How-
1
ever, H NMR shows that chemical shifts and coupling
F igu r e 1. HPLC profile of the products formed upon reaction
of anti-11-MeBPDE with (A) 2′-dG-5′-monophosphate and (B)
2′-dA-5′-monophosphate.
constants of H7-H10 of BPDE and 11-MeBPDE are
almost identical (Table 1). These data suggest that 11-
MeBPDE and BPDE have similar conformations at the
terminal ring.
DNA Ad d u ct F or m a tion . To investigate DNA adduct
formation from 11-MeBPDE, the required nucleotide
markers were prepared by reacting the diol epoxide with
either 2′-dG-5′-monophosphate or 2′-dA-5′-monophos-
phate (13). The modified deoxyribonucleotides were
enzymatically hydrolyzed to deoxyribonucleosides which
were then analyzed by HPLC (Figure 1). Next, 11-
MeBPDE was incubated with calf thymus DNA. The
modified DNA was digested enzymatically to give nucleo-
side adducts that were analyzed by HPLC (Figure 2).
Peaks 1-3 were identified as adducts of dG and peaks
4-7 as adducts of dA by comparison of retention times
to those of the standard markers. The extent of total calf
thymus DNA adduct formation from 11-MeBPDE was
1.7-fold greater than that from BPDE. The extent of
formation of dG and dA adducts from 11-MeBPDE was
1.8 and 1.2 times greater than from BPDE, respectively.
Similar results were obtained when the anti-1,2-diol 3,4-
epoxides of 5-MeC and chrysene were compared (14). A
preliminary mutagenicity assay was conducted with
BPDE and 11-MeBPDE in S. typhimurium TA 100
(Figure 3). Although 11-MeBPDE is slightly more mu-
tagenic than BPDE at 0.5 and 1 nmol/plate, a more
detailed mutagenicity study will be required to fully
evaluate the comparative activities of those diol epoxides.
To determine the structure of the major adducts
formed by 11-MeBPDE with calf thymus DNA, three
deoxyguanosine adducts were collected from HPLC, and
F igu r e 2. HPLC profiles obtained upon analysis of enzymatic
hydrolysates of calf thymus DNA reacted with (A) BPDE and
(B) 11-MeBPDE.
NMR data are summarized in Table 2. The most intense
bands of the CD spectra of peak 2 and 3 have positive
signs; thus, these deoxyguanosine adducts must have the
S configuration at the point of attachment of the hydro-
carbon residue (15). The 1H NMR data in Table 2
1
their CD and H NMR spectra were determined. The CD
1
spectra are illustrated in Figure 4, and the relevant H

11-Methylbenzo[a]pyrene Diol Epoxide-DNA Adduct
Chem. Res. Toxicol., Vol. 12, No. 4, 1999 345
Ta ble 2. Ch em ica l Sh ifts a n d Cou p lin g Con sta n ts for th e Meth in e P r oton s of th e P er a ceta tes of Deoxyr ibon u cleosid e
Ad d u cts of a n ti-11-MeBP DEa
methine protons
C7-H
C8-H
C9-H
C10-H
peracetate
δ (ppm) coupling constant (Hz) δ (ppm) coupling constant (Hz) δ (ppm) coupling constant (Hz) δ (ppm)
peak 1, transb
7.00
7.00
6.83
6.80
6.62
J 7,8 ) 9.0
J 7,8 ) 8.9
J 7,8 ) 9.0
J 7,8 ) 9.3
J 7,8 ) 4.2
5.66
5.69
5.87
5.70
5.67
J 8,9 ) 1.9
J 8,9 ) 2.0
J 8,9 ) 2.1
J 8,9 ) 2.4
J 8,9 ) 2.6
6.53
6.36
6.48
5.87
5.92
J 9,10 ) 4.8
J 9,10 ) 4.8
J 9,10 ) 4.6
J 9,10 ) 3.7
J 9,10 ) 4.3
6.93
7.11
6.73
6.95
7.33
peak 2, cisb
peak 3, trans
trans-BP-tetraol
cis-BP-tetraol
a
b
NMR spectra were recorded in acetone-d6 with a 5 mm 1H probe at 360 MHz. Trans and cis refer to the relative configuration of the
purine and hydroxyl group at the C10 and C9 positions, respectively.
trans adduct of the (R,S,S,R)-enantiomer. These struc-
tures are illustrated in Figure 4. Figure 2A shows the
HPLC profile obtained after enzymatic hydrolysis of
BPDE-modified calf thymus DNA; the major peak has
been previously characterized as the trans dG adduct
derived from the (R,S,S,R)-enantiomer (13). Although the
formation of the cis adduct derived from the (R,S,S,R)-
enantiomer of 11-MeBPDE was not detected due to the
lack of a reference standard, the possibility that this
adduct coeluted with peak 1 or 2 cannot be excluded at
this time.
These results demonstrate that introduction of a meth-
yl group into the bay region of BPDE causes a change in
the pattern of reaction with DNA. While the reaction of
BPDE with DNA gives mainly the trans dG adduct of
F igu r e 3. Comparative mutagenicity of BPDE (]) and 11-
the (R,S,S,R)-enantiomer, reaction of 11-MeBPDE yields
MeBPDE (0) in S. typhimurium TA 100.
substantial amounts of products resulting from both cis
and trans addition of dG to the (S,R,R,S)-enantiomer,
as well as trans addition to the (R,S,S,R)-enantiomer.
This behavior is also different from that of racemic anti-
1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchry-
sene (5-MeCDE), which, in its reaction with DNA, gives
the trans adduct of the (R,S,S,R)-enantiomer as the major
product (16). At the present time, the significance of these
findings is unclear; however, it is clear that the bay
region methyl group affects the reactivity of 11-MeBPDE
with DNA, and this may be related to the tumorigenicity
of 11-MeBP being higher than that of BP or of the other
monomethyl isomers (4).
Ack n ow led gm en t. This study was supported by NCI
Contract NO1-CP-2115 and Grant CA-44377. We are
grateful to Professor Nicholas E. Geacintov who provided
the circular dichroism spectrometer for CD experiments
and to Mrs. Ilse Hoffmann for editing the manuscript.
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346 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
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