Synthesis of anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene and its reaction with 2'-deoxyguanosine-5'-monophosphate,2'-deoxyadenosine-5'-monophosphate, and calf thymus DNA in vitro

Article abstract of DOI:10.1021/tx000104n

The remarkable carcinogenic activity of 6-nitrochrysene (6-NC) in several animal models, and its environmental presence, suggest its potential importance with regard to human cancer development. Depending on the bioassay model, 6-NC can be activated by si

Full text of DOI:10.1021/tx000104n

Chem. Res. Toxicol. 2000, 13, 1143-1148
1143
Syn th esis of
a n ti-1,2-Dih yd r oxy-3,4-ep oxy-1,2,3,4-tetr a h yd r o-6-n itr och r ysen e
a n d Its Rea ction w ith 2′-Deoxygu a n osin e-
5′-Mon op h osp h a te, 2′-Deoxya d en osin e-5′-Mon op h osp h a te,
a n d Ca lf Th ym u s DNA in Vitr o
J acek Krzeminski,† Dhimant Desai,† J yh-Ming Lin,† Vladimir Serebryany,†
Karam El-Bayoumy,‡ and Shantu Amin*,†
Organic Synthesis Facility and Division of Cancer Etiology and Prevention, Naylor Dana Institute
for Disease Prevention, American Health Foundation, Valhalla, New York 10595
Received May 8, 2000
The remarkable carcinogenic activity of 6-nitrochrysene (6-NC) in several animal models,
and its environmental presence, suggest its potential importance with regard to human cancer
development. Depending on the bioassay model, 6-NC can be activated by simple nitro
reduction, ring oxidation, or by a combination of ring oxidation and nitro reduction. Only the
first pathway has been clearly established. Thus, this study purports to unequivocally define
the other pathways. Toward this end, we report for the first time the synthesis of anti-1,2-
dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene (6-NCDE), a likely ultimate carcinogenic
metabolite of 6-NC. Also, we describe our initial investigation of its binding with calf thymus
DNA, 2′-deoxyguanosine-5′-monophosphate (2′-dGuo), and 2′-deoxyadenosine-5′-monophosphate
(2′-dAdo) in vitro. These adduct markers were then employed for comparison with those obtained
in the rat after in vivo treatment with 6-NC. On the basis of the results, it appears that the
major adduct formed in the liver of rats treated with 6-NC is not derived from 6-NCDE.
formation of adenomas and adenocarcinomas in the colon
(13). Because humans are exposed to these agents and
human tissues are capable of activating 6-NC to genotoxic
metabolites (14, 15), 6-NC may play a role in the
induction of certain human cancers (lung, breast, and
colon). Therefore, it is essential to delineate the mecha-
nisms of action that account for the carcinogenicity of
6-NC.
In tr od u ction
Humans are exposed to NO2-PAHs in cooked foods, and
in air pollution made up of combustion products (1-3).
6-Nitrochrysene (6-NC),1 one member of this class of
compounds, has been shown to be mutagenic in both
bacterial and mammalian cell systems (4, 5) and to be
tumorigenic in several rodent assays (6). Mutagenic NO2-
PAHs have been detected in tissues of lung cancer
patients who were nonsmokers, implicating these com-
pounds as possible initiators of lung tumors in humans
(7). Although 6-NC is present in the environment at
rather low levels, research on this compound has been
inspired because of its remarkable carcinogenic activity
in the newborn mouse lung adenoma assay (8, 9). In fact,
6-NC is the most potent lung tumorigen among all the
NO2-PAHs tested in this model assay, and its carcino-
genic potency approximates that of certain ultimate
carcinogenic metabolites of PAHs (8). In addition, intra-
mammary injection of 6-NC induced mammary tumors
in female CD rats whereby 6-NC was more potent than
the bay region diol epoxide derived from benzo[a]pyrene
(10, 11). We have also shown that oral administration of
6-NC elicits mammary cancer in female CD rats (12).
However, ip injection of 6-NC into CD rats leads to the
Studies in mice and rats and in vitro assays have
indicated that 6-NC can be activated by two major
pathways. The first pathway, involving the formation of
N-hydroxy-6-aminochrysene (N-OH-6-AC) by simple nitro
reduction, yields three major DNA adducts: N-(dG-8-yl)-
6-AC, N-(dI-8-yl)-6-AC, and 5-(dG-N2-yl)-6-AC (16). The
second pathway proceeds via the formation of the proxi-
mate carcinogen 1,2-dihydroxy-1,2-dihydro-6-nitrochry-
sene (1,2-DHD-6-NC) or 1,2-dihydroxy-1,2-dihydro-6-
aminochrysene (1,2-DHD-6-AC) by terminal ring oxidation
or by a combination of terminal ring oxidation and nitro
reduction, respectively (17); 1,2-DHD-6-NC or 1,2-DHD-
6-AC yields a major DNA adduct resulting from the
reaction with 2′-dGuo in the presence of rat liver mi-
crosomes in vitro (18). Levels of this adduct, and of those
derived from simple nitro reduction pathways, vary
depending on the animal model and route of administra-
tion of 6-NC (19-21). Unequivocal identification of the
adduct(s) derived from ring oxidation or from a combina-
tion of ring oxidation and nitro reduction has not been
achieved. We hypothesize that 6-NCDE or its amino
derivative is the ultimate carcinogenic metabolite of 6-NC
that is responsible for the formation of the unknown DNA
adduct detected in vivo (19-21). Thus, in the study
* To whom requests for reprints should be addressed.
† Organic Synthesis Facility.
‡ Division of Cancer Etiology and Prevention.
1
Abbreviations: 6-NC, 6-nitrochrysene; 6-NCDE, anti-1,2-dihy-
droxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene; 2′-dGuo, 2′-deoxy-
guanosine 5′-monophosphate; 2′-dAdo, 2′-deoxyadenosine 5′-monophos-
phate; N-OH-6-AC, N-hydroxy-6-aminochrysene; 1,2-DHD-6-NC, 1,2-
dihydroxy-1,2-dihydro-6-nitrochrysene; 6-AC, 6-aminochrysene; 1,2-
DHD-6-AC, 1,2-dihydroxy-1,2-dihydro-6-aminochrysene.
10.1021/tx000104n CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/05/2000

1144 Chem. Res. Toxicol., Vol. 13, No. 11, 2000
Krzeminski et al.
(m, 1H, CH2), 2.16-2.73 (m, 1H, CH2), 3.23-3.39 (m, 2H, CH2),
6.15 (bt, 1H, CHOAc), 7.68-7.77 (m, 2H, H8 and H9), 7.95-
7.98 (m, 1H, H7), 7.99 (d, 2H, H5 and H6, J 5,6 ) 9.19 Hz), 8.65
(d, 1H, H10, J 9,10 ) 7.87 Hz), 9.03 (s, 1H, H11).
(3) 3,4-Dih yd r o-6-n itr och r ysen e (3). To a solution of 2
(0.55 g, 1.73 mmol) in 10 mL of a dry THF/MeOH mixture (1:1)
was added NaOMe (150 mg, 2.77 mmol), and the resulting
mixture was refluxed for 10 min. This reaction mixture was
poured into H2O and extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with H2O (2 × 30 mL),
dried (MgSO4), and concentrated to afford crude 1-hydroxy-
1,2,3,4-tetrahydro-6-nitrochrysene.
presented here, we describe for the first time a convenient
synthesis of 6-NCDE and our initial investigation of its
binding with calf thymus DNA, 2′-dGuo, and 2′-dAdo in
vitro. These adduct markers were then employed for
comparison with those obtained in vivo after treatment
of rats with 6-NC.
Ma ter ia ls a n d Meth od s
Ca u tion : 6-Nitrochrysene, 1,2-DHD-6-NC, and 1,2-DHD-6-
AC are mutagenic in bacterial systems and tumorigenic in
rodents. Therefore, appropriate safety procedures must be fol-
lowed when working with these compounds.
To the above crude alcohol (0.41 g, 1.48 mmol) was added
p-toluenesulfonic acid (10 mg) in benzene (100 mL), and the
mixture was heated at reflux for 30 min in a Dean-Stark
apparatus. Workup, including chromatography on silica gel and
elution with a CH2Cl2/hexane mixture (30:70), gave olefin 3 in
Syn th esis. Melting points were recorded on a Fisher-J ohnson
melting point apparatus and are uncorrected. Proton NMR
spectra were recorded in CDCl3 using a Bruker AM 360WB
instrument. The chemical shifts are reported in parts per million
downfield from TMS. MS was carried out on a Hewlett-Packard
model 5988A instrument. High-resolution MS was carried out
on a Finnigan Mat95 instrument at the University of Minnesota
(Minneapolis, MN). Thin-layer chromatography (TLC) was
carried out on aluminum-supported, precoated silica gel plates
(EM Industries, Gibbstown, NJ ). All starting materials were
obtained from Aldrich Chemical Co. (Milwaukee, WI). 1,2,3,4-
Tetrahydrochrysene-1-one (22) was synthesized in three steps
from 2-(1-naphthyl)ethyl bromide by following a procedure
slightly modified from that described by Harvey et al. (23).
(1) 1-Acetoxy-1,2,3,4-tetr ah ydr och r ysen e (1). NaBH4 (1.32
g, 34.9 mmol) was added to a solution of the 1,2,3,4-tetrahy-
drochrysene-1-one (1.4 g, 5.70 mmol) in 14 mL of a THF/CH3-
OH mixture (1:1). The reaction mixture was then stirred at room
temperature for 2 h and poured into H2O. The reaction mixture
was extracted with EtOAc (3 × 2 mL), washed with H2O, and
dried (MgSO4). The workup gave the alcohol as a white solid
(1.40 g, 98%): mp 202-204 °C; MS m/z (relative intensity) 248
(M+, 100), 230 (80), 220 (60), 215 (40), 191 (45), 178 (20), 165
(10).
1
68% yield (0.32 g): mp 135-136 °C; H NMR δ 2.53-2.57 (m,
2H, CH2), 3.31 (t, 2H, CH2), 6.21-6.26 (m, 1H, olefin H2), 6.64
(d, 1H, olefin H1, J 1,2 ) 9.42 Hz), 7.52 (d, 1H, H12, J 11,12 ) 8.54
Hz), 7.72-7.76 (m, 2H, H8 and H9), 8.47 (dd, 1H, H7, J 7,8
)
6.89 Hz, J 7,9 ) 1.96 Hz), 8.51 (d, 1H, H11, J 11,12 ) 8.55 Hz),
8.72-8.75 (m and s, 2H, H5 and H10); MS m/z (relative
intensity) 275 (M+, 100), 245 (80), 228 (90), 202 (20); high-
resolution MS m/z calcd for C18H13NO2 275.0868, found 275.0956.
(4) tr a n s-1,2-Bis(ben zoyloxy)-1,2,3,4-tetr a h yd r o-6-n itr o-
ch r ysen e (4). A mixture of silver benzoate (0.80 g, 3.5 mmol)
and I2 (0.44 g, 1.77 mmol) in dry benzene (50 mL) was stirred
under reflux until the red color disappeared. A solution of olefin
3 (0.32 g, 1.16 mmol) in 10 mL of dry benzene was added, and
the resulting mixture was stirred under reflux for 12 h. The
reaction mixture was filtered while hot and washed with
benzene (2 × 25 mL). The filtrate was concentrated to give a
solid that was chromatographed on a silica gel column. Hexane
eluted the unreacted olefin 3 (60 mg). Further elution with a
hexane/EtOAc mixture (1:1) gave the pure dibenzoate derivative
4 (0.51 g, 85%): mp 205-207 °C; 1H NMR δ 2.41-2.51 (m, 1H,
CH2), 2.62-2.71 (m, 1H, CH2), 3.53-3.56 (m, 2H, CH2), 5.68-
5.70 (m, 1H, H2), 6.70 (d, 1H, H1, J 1,2 ) 5.56 Hz), 7.35-7.43
(m, 4H, from benzoyl ester), 7.45-7.56 (m, 2H, from benzoyl
ester), 7.78-7.83 (m, 3H, H8, H9, and H12), 7.94-7.97 (m, 2H,
from benzoyl ester), 8.07-8.09 (m, 2H, from benzoyl ester),
8.51-8.55 (m, 1H, H7), 8.62 (d, 1H, H11, J 11,12 ) 9.19 Hz), 8.75-
8.81 (m and s, 2H, H5 and H10); high-resolution MS m/z calcd
for C32H23NO6Na 540.1423, found 540.1433.
This white solid was dissolved in 30 mL of a benzene/pyridine
mixture (1:1), and DMAP (50 mg) and 5 mL of Ac2O were added.
The reaction mixture was stirred for 3 h at room temperature
and then evaporated to dryness. The residue was diluted with
H2O and extracted with EtOAc. The ethyl acetate extract was
dried (MgSO4) and concentrated to afford 1.35 g (81%) of
1
compound 1: mp 167-168 °C; H NMR δ 2.02-2.15 (m and s,
7H, CH3 and CH2), 3.10-3.15 (m, 1H, CH2), 3.35-3.41 (m, 1H,
CH2), 6.20 (bt, 1H, CHOAc), 7.56 (d, 1H, H12, J 11,12 ) 8.53 Hz),
7.58-7.66 (m, 2H, H8 and H9), 7.81 (d, 1H, H6, J 5,6 ) 9.19 Hz),
7.90 (dd, 1H, H7, J 7,8 ) 8.86 Hz, J 7,9 ) 1.31 Hz), 7.99 (d, 1H,
H5, J 5,6 ) 9.19 Hz), 8.58 (d, 1H, H11, J 11,12 ) 8.53 Hz), 8.69 (d,
1H, H10, J 9,10 ) 8.21 Hz); MS m/z (relative intensity) 290 (M+,
10), 230 (100), 215 (30), 202 (10), 189 (10); high-resolution MS
m/z calcd for C20H18O2Na 313.1212, found 313.1212.
(5) tr a n s-1,2-Bis(ben zoyloxy)-4-br om o-1,2,3,4-tetr a h y-
d r o-6-n itr och r ysen e (5). A suspension of N-bromosuccinimide
(0.1 g, 0.56 mmol), dibenzoate 4 (0.22 g, 0.43 mmol), and benzoyl
peroxide (5 mg) in CCl4 (6 mL) was heated under reflux in a N2
atmosphere for 30 min. The reaction mixture was cooled, and
the succinimide was removed by filtration. The filtrate was
concentrated and chromatographed on silica gel, and an EtOAc/
hexane mixture (1:1) eluted an unstable bromo compound 5 (180
mg, 71%): mp 195-197 °C; 1H NMR δ 3.15-3.22 (m, 2H, CH2),
5.79 (dd, 1H, H2, J 1,2 ) 3.28 Hz, J 2,3 ) 6.89 Hz), 6.18 (dd, 1H,
H4, J 3,4 ) 5.25 Hz, J 3′,4 ) 1.97 Hz), 6.69 (d, 1H, H1, J 1,2 ) 3.28
Hz), 7.35-7.59 (m, 6H), 7.80-7.85 (m, 2H), 7.91 (d, 1H), 8.00-
8.13 (m, 4H), 8.50-8.52 (m, 1H), 8.76-8.79 (m, 2H), 9.01 (s,
1H, H5). This bromo compound was used without further
purification for the next step.
(2) 1-Acetoxy-1,2,3,4-tetr a h yd r o-6-n itr och r ysen e (2). To
a stirred solution of 1 (1.35 g, 4.66 mmol) in 50 mL of dry CH2-
Cl2 was added a 1.3 M solution of N2O4 in CH2Cl2 (43 mL) over
the course of 5 min. Stirring was continued at room temperature
for 3 h. The excess N2O4 was removed by flushing a stream of
N2 through the reaction mixture. The reaction mixture was
poured into H2O, and the organic layer was washed with a
NaHCO3 solution (2 × 50 mL). The workup gave 1.2 g of a
mixture of two products. This was chromatographed on silica
gel and eluted with a hexane/EtOAc mixture (80:20) to give
compound 2 (870 mg, 59%): mp 56-60 °C; 1H NMR δ 2.06-
2.18 (m and s, 7H, OAc and CH2), 3.16-3.19 (m, 1H, CH2), 3.38-
3.43 (m, 1H, CH2), 6.18 (bt, 1H, CHOAc), 7.74 (d, 1H, H12, J 11,12
) 8.53 Hz), 7.76-7.81 (m, 2H, H8 and H9), 8.50-8.53 (m, 1H,
H7), 8.57 (d, 1H, H11, J 11,12 ) 8.53 Hz), 8.75 (s, 1H, H5), 8.77-
8.78 (m, 1H, H10); high-resolution MS m/z calcd for C20H17NO4-
Na 358.1055, found 358.1041.
(6) tr a n s-1,2-Bis(ben zoyloxy)-1,2-d ih yd r o-6-n itr och r y-
sen e (6). To a solution of bromide 5 (0.125 g, 0.02 mmol) in 20
mL of dry xylene was added 300 mg of NaHCO3. This mixture
was heated under reflux for 1 h. The product was filtered while
hot and washed with benzene (2 × 10 mL). The filtrate was
concentrated to yield a solid that was chromatographed on silica
gel. Elution with a hexane/CH2Cl2 mixture (1:1) gave 6 (67 mg,
62%): mp 215-217 °C; 1H NMR δ 6.17-6.20 (m, 1H, H2), 6.52
(dd, 1H, H3, J 2,3 ) 3.61 Hz, J 3,4 ) 10.17 Hz), 6.87 (d, 1H, H1,
J 1,2 ) 7.87 Hz), 7.26-7.57 (m, 7H), 7.78-7.79 (m, 2H), 7.89 (d,
1H, J ) 8.53 Hz), 7.98 (d, 1H, J ) 8.2 Hz), 8.08 (d, 1H, J ) 8.55
Hz), 8.48-8.53 (m, 1H), 8.67 (d, 1H, H11, J 11,12 ) 8.53 Hz), 8.74-
Further elution with a hexane/EtOAc mixture (1:1) gave
1-acetoxy-1,2,3,4-tetrahydro-12-nitrochrysene (300 mg, 14%):
1H NMR δ 2.0-2.04 (m and s, 5H, OAc and CH2), 2.08-2.14

Synthesis of 6-Nitrochrysene Diol Epoxide
Chem. Res. Toxicol., Vol. 13, No. 11, 2000 1145
8.77 (m, 1H, H10), 8.86 (s, 1H, H5); high-resolution MS m/z calcd
for C32H21NO6Na 538.1288, found 538.1288.
(7) tr a n s-1,2-Dih yd r oxy-1,2-d ih yd r o-6-n itr och r ysen e (7).
To a solution of 6 (51 mg, 0.1 mmol) in 6 mL of a dry THF/
MeOH mixture (1:1) was added NaOMe (11 mg, 0.2 mmol). This
mixture was stirred under reflux for 15 min. The reaction
mixture was concentrated, suspended in cold water, extracted
with EtOAc, then dried over MgSO4, and evaporated to dryness.
Crystallization of the crude product from MeOH gave 7 (15 mg,
48%) as yellow crystals: mp 230-232 °C dec; 1H NMR δ 4.31-
4.40 (m, 1H, H2), 4.77 (dd, 1H, H1, J 1,2 ) 11.16 Hz, J 1,OH
)
5.91 Hz), 5.39 (d, 1H, OH2, J 2,OH2 ) 5.95 Hz), 5.79 (d, 1H, OH1,
J H,OH1 ) 5.95 Hz), 6.25 (dd, 1H, H3, J 2,3 ) 2.3 Hz, J 3,4 ) 10.17
Hz), 7.38 (d, 1H, H4, J 3,4 ) 10.17 Hz), 7.81-7.89 (m, 2H, H8
and H9), 8.08 (d, 1H, H12, J 11,12 ) 8.56 Hz), 8.22 (d, 1H, H7,
J 7,8 ) 8.20 Hz), 8.89 (d, 1H, H11, J 11,12 ) 8.56 Hz), 8.94 (s, 1H,
H5), 9.01 (d, 1H, H10, J 9,10 ) 8.2 Hz); CIMS m/z (relative
intensity) 308 (M + 1, 40), 290 (M + 1 - H2O, 45), 260 (100),
244 (40); high-resolution MS m/z calcd for C18H13NO4 307.0844,
found 307.0832.
HPLC chromatographic characteristics (retention time of 74
min) and spectral data of this diol are identical to those of a
previously characterized metabolite of 6-NC (17).
(8) a n ti-1,2-Dih yd r oxy-3,4-ep oxy-1,2,3,4-t et r a h yd r o-6-
n itr och r ysen e (8). A mixture of dihydrodiol 7 (10 mg, 0.032
mmol) and recrystallized m-chloroperoxybenzoic acid (100 mg,
0.58 mmol) in 20 mL of dry tetrahydrofuran was stirred in a
N2 atmosphere for 5 h. This reaction mixture was then diluted
with diethyl ether (100 mL) and washed with a 2% sodium
hydroxide solution (3 × 20 mL) and then with H2O. The organic
layer was dried over K2CO3. Evaporation of the solvent gave
the light, cream-colored diol epoxide 8 (8 mg, 76%), which was
recrystallized from an ether/THF mixture: mp 238-240 °C dec;
1H NMR δ 3.88 (dd, 1H, H3, J 2,3 ) 0.96 Hz, J 3,4 ) 4.26 Hz),
4.03 (dd, 1H, H2, J 1,2 ) 8.53 Hz, J 2,3 ) 0.99 Hz), 4.78 (d, 1H,
H1, J 1,2 ) 8.53 Hz), 5.04 (d, 1H, H4, J 3,4 ) 4.26 Hz), 7.84-7.93
(m, 2H, H8 and H9), 8.29 (d, 1H, H12, J 11,12 ) 8.86 Hz), 8.33
(dd, 1H, H7, J 7,8 ) 7.86 Hz, J 7,9 ) 1.32 Hz), 9.00 (d, 1H, H11,
J 11,12 ) 8.86 Hz), 9.05 (dd, 1H, H10, J 8,10 ) 0.64 Hz, J 9,10 ) 9.51
F igu r e 1. UV spectra of 9-nitrophenanthrene (A) and 1,2,3,4-
tetrahydroxy-1,2,3,4-tetrahydro-6-nitrochrysene (B).
ribonucleosides as described previously (25-27). Unmodified
deoxyribonucleosides were separated from modified deoxyribo-
nucleosides on Sep-Pak C18 cartridges (25), and modified
deoxyribonucleosides were analyzed by HPLC as described
below.
HP LC An a lysis of Mod ified 2′-Deoxyr ibon u cleosid es.
HPLC on a Nucleosil C18 column (5 µm, 4.6 mm × 250 mm;
Phenomenex, Inc., Torrence, CA) utilized the following gradient
program to separate the modified deoxyribonucleosides: a linear
gradient of 30:70 MeOH/water to 85:15 MeOH/water over the
course of 55 min and then to 100% MeOH over the course of 9
min.
Hz), 9.51 (s, 1H, H5); high-resolution MS m/z calcd for C18H13
-
NO5 323.0816, found 323.0816.
Rea ction of 6-NCDE w ith Ca lf Th ym u s DNA. About 1
mg of 6-NCDE (8) in 1 mL of acetone was added gradually over
2 h to a solution of calf thymus DNA (10 mg) in 10 mL of 100
mM Tris-HCl buffer (pH 7). This mixture was incubated at 37
°C overnight. Tetraols were removed from the DNA solutions
by extraction with ethyl acetate (3 × 10 mL), followed by
extraction with diethyl ether (3 × 5 mL). One of the major
tetraols was isolated: 1H NMR (acetone-d6) δ 4.18 (dd, 1H, H2,
J 1,2 ) 8.64 Hz, J 2,3 ) 2.2 Hz), 4.39 (bs, 1H, H3), 4.95 (d, 1H,
H1, J 1,2 ) 8.64 Hz), 5.47 (bs, 1H, H4), 7.83-7.88 (m, 2H, H8
and H9), 8.17 (d, 1H, H12, J 11,12 ) 8.86 Hz), 8.38 (dd, 1H, H7,
J 7,8 ) 8.66 Hz, J 7,9 ) 2.05 Hz), 8.92 (d, 1H, H11, J 11,12 ) 8.86
Hz), 9.01 (dd, 1H, H10, J 8,10 ) 1.97 Hz, J 9,10 ) 9.57 Hz), 9.04 (s,
1H, H5). The UV spectrum of the tetraol was similar to that of
9-nitrophenanthrene (Figure 1). The retention time of the major
tetraol under HPLC conditions described below was 27.2 min.
A minor tetraol eluting after 31.8 min also exhibited a UV
spectrum similar to that of 9-nitrophenanthrene. The DNA was
isolated and hydrolyzed enzymatically to deoxyribonucleosides
(24). Modified deoxyribonucleosides were separated from un-
modified deoxyribonucleosides on Sep-Pak C18 cartridges (25),
and analyzed by HPLC as described below.
Resu lts a n d Discu ssion
Initially, we attempted to synthesize the diol epoxide
of 6-NC using 1,2,3,4-tetrahydrochrysene-1-one as the
starting material. However, nitration of 1,2,3,4-tetrahy-
drochrysene-1-one, using N2O4 in methylene chloride,
failed to yield a significant quantity of the desired
compound, 1,2,3,4-tetrahydro-6-nitrochrysene-1-one. In-
stead, on the basis of NMR analysis, 1,2,3,4-tetrahydro-
5-nitrochrysene-1-one was isolated as the major prod-
uct: 1H NMR δ 2.34-2.42 (m, 2H, CH2), 2.81 (dd, 2H,
CH2, J ) 6.24 Hz), 3.50 (dd, 2H, CH2, J ) 6.24 Hz), 7.81-
7.87 (m, 2H, H8 and H9), 8.47 (d, 1H, H12, J 11,12 ) 8.86
Hz), 8.52-8.55 (m, 1H, H7), 8.68 (d, 1H, H11, J 11,12
8.86 Hz), 8.78 (s, 1H, H6), 8.80-8.83 (m, 1H, H10).
)
Rea ction s of 6-NCDE w ith 2′-d Gu o or 2′-d Ad o. About 100
mg each of 2′-dGuo or 2′-dAdo were dissolved in 10 mL of 100
mM Tris-HCl buffer (pH 7.0). To the above solution was added
about 1 mg of 6-NCDE in 1 mL of acetone gradually over 2 h
before the mixture was incubated overnight at 37 °C. Samples
were extracted with ethyl acetate (3 × 10 mL) and with diethyl
ether (3 × 5 mL). A trace of ether in the aqueous layer was
flushed away with a stream of nitrogen, and the modified
deoxyribonucleotides were hydrolyzed enzymatically to deoxy-
An alternative approach was designed as outlined in
Scheme 1; here, 1-acetoxy-1,2,3,4-tetrahydro-6-nitrochry-
sene (2) is the key intermediate for the synthesis.
Reduction of 1,2,3,4-tetrahydrochrysene-1-one with so-
dium borohydride furnished the corresponding alcohol

1146 Chem. Res. Toxicol., Vol. 13, No. 11, 2000
Krzeminski et al.
Sch em e 1. Syn th esis of a n ti-1,2-Dih yd r oxy-3,4-ep oxy-1,2,3,4-tetr a h yd r o-6-n itr och r ysen e
which was acetylated with acetic anhydride in pyridine
to give 1-acetoxy-1,2,3,4-tetrahydrochrysene (1) in 81%
yield. Nitration of the acetoxy derivative 1 at room
temperature with N2O4 yielded two isomers. These two
isomers were separated by column chromatography, and
native synthetic approach was employed. Bromination
of trans-dibenzoate, 4, with NBS in carbon tetrachloride
led to an unstable trans-1,2-bis(benzoyloxy)-4-bromo-
1,2,3,4-tetrahydro-6-nitrochrysene (5) which was used
without further purification for the next step. Dehydro-
bromination of compound 5 with sodium bicarbonate in
xylene gave trans-1,2-bis(benzoyloxy)-1,2-dihydro-6-ni-
trochrysene (6). The dibenzoate-protecting group was
easily removed by treating compound 6 with sodium
methoxide to furnish trans-dihydrodiol 7 as a yellowish
solid in 48% yield. Compound 7 had chromatographic and
spectral properties that were identical to those of a
previously established metabolite of 6-NC (17). Finally,
epoxidation of dihydrodiol 7 with m-chloroperoxybenzoic
acid afforded anti-diol epoxide 8 as a creamy solid in 76%
yield.
1,2-DHD-6-NC (7) (or its amino derivative) and 6-NCDE
(8) (or its amino derivative) have been implicated as
proximate and ultimate carcinogens, respectively (17). To
prepare markers for the identification of unknown DNA
adducts that are formed in vivo, we have incubated
6-NCDE (8) with dGuo, dAdo, and calf thymus DNA. The
modified dGuo, dAdo, and DNA were isolated and enzy-
matically hydrolyzed to the corresponding modified nu-
cleosides. The HPLC profiles of the standard 6-NCDE-
modified 2′-deoxyguanosine and 2′-deoxyadenosine adducts
are illustrated in panels A and B of Figure 2, respectively.
Panel C represents the HPLC profile of the 6-NCDE-
modified calf thymus DNA; both modified 2′-deoxygua-
nosine and 2′-deoxyadenosine adducts had been formed.
In comparison, panel D represents the HPLC radiochro-
matogram profile of DNA isolated from the liver of rats
treated with [12-3H]-6-nitrochrysene (21). The retention
time of the major adduct formed in vivo is between 19
and 20 min (panel D). This clearly indicates that neither
6-NCDE-modified 2′-deoxyguanosine nor 6-NCDE-modi-
fied 2′-adenosine is involved in the formation of the DNA
adducts observed in the rat (21).
1
their structures were confirmed by H NMR. From the
1H NMR spectral analysis, we deduced that the major
isomer was 1-acetoxy-1,2,3,4-tetrahydro-6-nitrochrysene
(2), while the minor isomer was 1-acetoxy-1,2,3,4-tet-
rahydro-12-nitrochrysene. The evidence in support of our
assignment of the major isomer as 1-acetoxy-1,2,3,4-
tetrahydro-6-nitrochrysene (2) is the downfield shift of
H5 and the absence of its coupling with H6. In addition,
both H11 and H12 remained as two sets of doublets. On
the other hand, the 1-acetoxy-1,2,3,4-tetrahydro-12-ni-
trochrysene retains both H5 and H6 as a set of doublets,
whereas H11 appeared as a singlet and shifted downfield
due to the nitro substitution at a neighboring carbon.
The acetoxy group was removed by sodium methoxide
hydrolysis, and the resulting alcohol was dehydrated with
p-toluenesulfonic acid to the corresponding 3,4-dihydro-
6-nitrochrysene (3) in 68% yield. The structure of com-
pound 2 was further confirmed by reaction of nitro olefin
3 with DDQ to give 6-nitrochrysene, and the proton NMR
spectrum was identical to that of authentic 6-nitrochry-
sene. This confirmed that the nitro group was in the
6-position in the acetoxy derivative, 2. Compound 3 was
converted to the corresponding trans-dihydrodiol 7 and
anti-diol epoxide 8 by a procedure modified from the one
described by Fu et al. (28). Under standard Prevost
conditions, olefin 3 reacted with silver acetate to give the
desired diacetate derivative, but in poor yield. However,
substitution of silver acetate with silver benzoate in the
standard Prevost reaction gave trans-1,2-bis(benzoyloxy)-
1,2,3,4-tetrahydro-6-nitrochrysene (4) in 85% yield. De-
hydrogenation of 4 with DDQ yielded a complex mixture,
but the desired trans-1,2-bis(benzoyloxy)-1,2-dihydro-6-
nitrochrysene (6) was not detected. Therefore, an alter-

Synthesis of 6-Nitrochrysene Diol Epoxide
Chem. Res. Toxicol., Vol. 13, No. 11, 2000 1147
(4) El-Bayoumy, K., Delclos, K. B., Heflich, R. H., Walker, R., Shiue,
G.-H., and Hecht, S. S. (1989) Mutagenicity, metabolism and DNA
adduct formation of 6-nitrochrysene in Salmonella typhimurium.
Mutagenesis 4, 235-240.
(5) Delclos, K. B., and Heflich, R. H. (1989) Mutation induction and
DNA adduct formation in Chinese hamster ovary cells treated
with 6-nitrochrysene, 6-aminochrysene and their metabolites.
Mutat. Res. 279, 153-164.
(6) International Agency for Research on Cancer (1989) Diesel and
Gasoline Engine Exhausts and Some Nitroarenes. In IARC
Monographs on the Evaluation of Carcinogenic Risk to Humans,
Vol. 46, pp 1-458, International Agency for Research on Cancer,
Lyon, France.
(7) Tokiwa, H., Sera, N., Horikawa, K., Nakanishi, Y., and Shigemat,
N. (1993) The presence of mutagenic carcinogens in the excised
lung and analysis of lung cancer induction. Carcinogenesis 14,
1933-1938.
(8) Busby, W. F., J r., Garner, R. C., Chow, F. L., Martin, C. N.,
Stevens, E. K., Newberne, P. M., and Wogan, G. N. (1985)
6-Nitrochrysene is a potent tumorigen in newborn mice. Carcino-
genesis 6, 801-803.
(9) Wislocki, P. G., Bagan, E. S., Lu, A. Y. H., Dooley, K. L., Fu, P.
P., Han-Hsu, H., Beland, F. A., and Kadlubar, F. F. (1986)
Tumorigenicity of nitrated derivatives of pyrene, benz[a]an-
thracene, chrysene and benzo[a]pyrene in the newborn mouse
assay. Carcinogenesis 7, 1317-1322.
(10) El-Bayoumy, K., Rivenson, A., Upadhyaya, P., Chae, Y.-H., and
Hecht, S. S. (1993) Induction of mammary cancer by 6-nitrochry-
sene in female CD rats. Cancer Res. 53, 3719-3722.
(11) Hecht, S. S., El-Bayoumy, K., Rivenson, A., and Amin, S. (1994)
Potent mammary carcinogenicity in female CD rats of a fjord
region diol-epoxide of benzo[c]phenanthrene compared to a bay
region diol-epoxide of benzo[a]pyrene. Cancer Res. 54, 21-24.
(12) Rosa, J ., Boyiri, T., Amin, S., El-Bayoumy, K., Gu, X., and
Schwartz, J . (2000) The impact of a high-fat diet on the carcino-
genicity and levels of 8-hydroxydeoxyguanosine (8-OHdG) in the
mammary glands of rats treated with the environmental pollutant
6-nitrochrysene (6-NC): A comparison with 2-amino-1-methyl-
6-phenylimidazo[4,5-b]pyridine (PHIP). Proc. 91st Annual Meet.
Am. Assoc. Cancer Res. 41, abstract 5305.
F igu r e 2. HPLC profiles of the standard 6-NCDE-modified 2′-
deoxyguanosine and 2′-deoxyadenosine adducts (A and B,
respectively). HPLC profile of the 6-NCDE-modified calf thymus
DNA (C). HPLC radiogram profile of DNA isolated from the liver
of rats treated with [12-3H]-6-nitrochrysene (D).
(13) Imaida, K., Uneyama, C., Ogasawara, H., Hayashi, S., Fukuhara,
K., Miyata, N., and Takahashi, M. (1992) Induction of colon
adenocarcinomas in CD rats and lung adenomas in ICR mice by
6-nitrochrysene: comparison of carcinogenicity and aryl hydro-
carbon hydroxylase induction in the target organs of each species.
Cancer Res. 52, 1542-1545.
(14) Delclos, K. B., El-Bayoumy, K., Casciano, D. A., Walker, R. P.,
Kadlubar, F. F., Hecht, S. S., Shivapurkar, N., Mandal, S., and
Stoner, G. D. (1989) Metabolic activation of 6-nitrochrysene in
explants of human bronchus and in isolated rat hepatocytes.
Cancer Res. 49, 2909-2913.
(15) Chae, Y.-H., Yun, C.-H., Guengerich, F. P., Kadlubar, F. F., and
El-Bayoumy, K. (1993) Roles of human hepatic and pulmonary
cytochrome P450 enzymes in the metabolism of the environmental
carcinogen 6-nitrochrysene. Cancer Res. 53, 2028-2034.
(16) Delclos, K. B., Miller, D. W., Lay, J . O., J r., Casciano, D. A.,
Walker, R. P., Fu, P. P., and Kadlubar, F. F. (1987) Identification
of C8-modified deoxyinosine and N2- and C8-modified deoxygua-
nosine as major products of the in vitro reaction of N-hydroxy-
6-aminochrysene with DNA and the formation of these adducts
in isolated rat hepatocytes treated with 6-nitrochrysene and
6-aminochrysene. Carcinogenesis 8, 1703-1709.
In summary, we described for the first time the
synthesis of 1,2-DHD-6-AC, 6-NCDE, and 1,2,3,4-tet-
rahydroxy-1,2,3,4-tetrahydro-6-nitrochrysene. We have
also prepared highly useful markers, namely, 6-NCDE-
modified 2′-deoxyguanosine and 2′-deoxyadenosine ad-
ducts. Comparison of HPLC profiles of these markers
with that detected in liver DNA obtained from rats
treated with 6-NC showed that 6-NCDE is not involved
in the formation of the DNA adducts observed in vivo.
Therefore, we are currently testing alternative hypoth-
eses involving DNA adduct formation with either 1,2-
dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-aminochry-
sene or the hydroxylamine derived from the nitro reduction
of 1,2-DHD-6-NC or partial oxidation of the amino
functionality of 1,2-DHD-6-AC.
Ack n ow led gm en t. We thank Mr. J ohn Cunningham
from the American Health Foundation’s Instrumentation
Facility and acknowledge Dr. V. Reddy’s initial attempt
to synthesize 6-NCDE. This study was supported by NCI
Contract NCI-CB-77022-75, NCI Grant CA-35519, and
Cancer Center Support Grant CA-17613. We thank Ms.
Ilse Hoffmann for editing the manuscript.
(17) El-Bayoumy, K., Shiue, G.-H., and Hecht, S. S. (1989) Compara-
tive tumorigenicity of 6-nitrochrysene and its metabolites in
newborn mice. Carcinogenesis 10, 369-372.
(18) Li, E. E., Heflich, R. H., and Delclos, K. B. (1993) trans-1,2-
Dihydro-1,2-dihydroxy-6-aminochrysene is metabolized to form
a major adduct with deoxyguanosine and produces mutations in
the hprt gene of Chinese hamster ovary cells at G:C base pairs.
Carcinogenesis 14, 2109-2114.
(19) Delclos, K. B., El-Bayoumy, K., Hecht, S. S., Walker, R. P., and
Kadlubar, F. F. (1988) Metabolism of the carcinogen [3H]6-
nitrochrysene in the preweanling mouse: identification of 6-ami-
nochrysene-1,2-dihydrodiol as the probable proximate carcino-
genic metabolite. Carcinogenesis 9, 1875-1884.
(20) Delclos, K. B., Walker, R. P., Dooley, K. L., Fu, P. P., and
Kadlubar, F. F. (1987) Carcinogen-DNA adduct formation in the
lungs and livers of preweanling CD-1 male mice following
administration of [3H]-6-nitrochrysene, [3H]-6-aminochrysene, and
[3H]-1,6-dinitropyrene. Cancer Res. 47, 6272-6277.
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