Synthesis and reactivity of the catechol metabolites from the equine estrogen, 8,9-dehydroestrone

Article abstract of DOI:10.1021/tx010049y

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with an increased cancer risk. Previously, we showed that the equine estrogens equilin and equilenin, which are major components of the widely prescribed estrogen replacement formulation Premarin, are metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. Chem. Res. Toxicol. 1998, 11, 1113-1127]. In this study, we have synthesized 8,9-dehydroestrone (a third equine estrogen component of Premarin) and its potential catechol metabolites, 4-hydroxy-8,9-dehydroestrone and 2-hydroxy-8,9-dehydroestrone. Both 2-hydroxy-8,9-dehydroestrone and 4-hydroxy-8,9-dehydroestrone were oxidized by tyrosinase or rat liver microsomes to o-quinones which reacted with GSH to give one mono-GSH conjugate and two di-GSH conjugates. Like endogenous estrogens, 8,9-dehydroestrone was primarily converted by rat liver microsomes to the 2-hydroxylated rather than the 4-hydroxylated o-quinone GSH conjugates; the ratio of 2-hydroxy-8,9-dehydroestrone versus 4-hydroxy-8,9-dehydroestrone was 6:1. Also in contrast to experiments with equilin, 4-hydroxyequilenin was not observed in microsomal incubations with 8,9-dehydroestrone or its catechols. The behavior of 2-hydroxy-8,9-dehydroestrone was found to be more complex than 4-hydroxy-8,9-dehydroestrone as GSH conjugates resulting from 2-hydroxy-8,9-dehydroestrone were detected even without oxidative enzyme catalysis. Under physiological conditions, 2-hydroxy-8,9-dehydroestrone isomerized to 2-hydroxyequilenin to form the very stable 2-hydroxyequilenin catechol; however, 4-hydroxy-8,9-dehydroestrone was found to be stable under similar conditions. Finally, preliminary studies conducted with the human breast tumor S-30 cell lines demonstrated that the catechol metabolites of 8,9-dehydroestrone were much less toxic than 4-hydroxyequilenin (20-40-fold). These results suggest that the catechol metabolites of 8,9-dehydroestrone may have the ability to cause cytotoxicity in vivo primarily through formation of o-quinones; however, most of the adverse effects of Premarin estrogens are likely due to formation of 4-hydroxyequilenin o-quinone from equilin and equilenin.

Full text of DOI:10.1021/tx010049y

754
Chem. Res. Toxicol. 2001, 14, 754-763
Syn th esis a n d Rea ctivity of th e Ca tech ol Meta bolites
fr om th e Equ in e Estr ogen , 8,9-Deh yd r oestr on e
Fagen Zhang, Dan Yao, Yousheng Hua, Richard B. van Breemen, and
J udy L. Bolton*
Department of Medicinal Chemistry and Pharmacognosy (M/ C 781), College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois, 60612-7231
Received March 1, 2001
The risk factors for women developing breast and endometrial cancers are all associated
with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been
correlated with an increased cancer risk. Previously, we showed that the equine estrogens
equilin and equilenin, which are major components of the widely prescribed estrogen
replacement formulation Premarin, are metabolized to highly cytotoxic quinoids which caused
oxidative stress and alkylation of DNA in vitro [Bolton, J . L., Pisha, E., Zhang, F., and Qiu, S.
Chem. Res. Toxicol. 1998, 11, 1113-1127]. In this study, we have synthesized 8,9-dehy-
droestrone (a third equine estrogen component of Premarin) and its potential catechol
metabolites, 4-hydroxy-8,9-dehydroestrone and 2-hydroxy-8,9-dehydroestrone. Both 2-hydroxy-
8,9-dehydroestrone and 4-hydroxy-8,9-dehydroestrone were oxidized by tyrosinase or rat liver
microsomes to o-quinones which reacted with GSH to give one mono-GSH conjugate and two
di-GSH conjugates. Like endogenous estrogens, 8,9-dehydroestrone was primarily converted
by rat liver microsomes to the 2-hydroxylated rather than the 4-hydroxylated o-quinone GSH
conjugates; the ratio of 2-hydroxy-8,9-dehydroestrone versus 4-hydroxy-8,9-dehydroestrone was
6:1. Also in contrast to experiments with equilin, 4-hydroxyequilenin was not observed in
microsomal incubations with 8,9-dehydroestrone or its catechols. The behavior of 2-hydroxy-
8,9-dehydroestrone was found to be more complex than 4-hydroxy-8,9-dehydroestrone as GSH
conjugates resulting from 2-hydroxy-8,9-dehydroestrone were detected even without oxidative
enzyme catalysis. Under physiological conditions, 2-hydroxy-8,9-dehydroestrone isomerized to
2-hydroxyequilenin to form the very stable 2-hydroxyequilenin catechol; however, 4-hydroxy-
8,9-dehydroestrone was found to be stable under similar conditions. Finally, preliminary studies
conducted with the human breast tumor S-30 cell lines demonstrated that the catechol
metabolites of 8,9-dehydroestrone were much less toxic than 4-hydroxyequilenin (20-40-fold).
These results suggest that the catechol metabolites of 8,9-dehydroestrone may have the ability
to cause cytotoxicity in vivo primarily through formation of o-quinones; however, most of the
adverse effects of Premarin estrogens are likely due to formation of 4-hydroxyequilenin
o-quinone from equilin and equilenin.
tissue selectivity in postmenopausal women, and a
distinct pharmacological profile that results in significant
clinical activity in vasomotor, neuroendocrine, and bone
preservation parameters (10, 11).
Despite the numerous benefits of estrogen replacement
therapy, there have been troubling, controversial reports
concerning the increase in risk of developing breast and
endometrial cancers, particularly for women on long-term
high dose estrogen replacement therapy (12-18). The
exact mechanism(s) by which estrogens cause these
hormone-dependent cancers is unknown. One mechanism
In tr od u ction
There are many benefits of estrogen replacement
therapy including relief of menopausal symptoms, pre-
vention of osteoporosis, and cardiovascular disease (1).
In addition, there is some evidence that estrogen replace-
ment therapy can protect women from Alzheimer’s
disease (2-5) and stroke (6, 7), and improve motor
disability in Parkinsonian postmenopausal women with
motor fluctuations (8). Equine estrogens make up ap-
proximately 50% of the estrogens in the widely prescribed
estrogen replacement therapy marketed under the name
of Premarin (Wyeth-Ayerst). One of these equine estro-
gens, 8,9-dehydroestrone differs from endogenous human
estrogens in that it contains a 8,9-double bond in the B
ring. The presence of 8,9-dehydroestrone1 in Premarin
is the primary reason that there has never been a generic
replacement for Premarin (9). Recent studies have shown
that 8,9-dehydroestrone has estrogenic activity, novel
1
Abbreviations: 8,9-dehydroestrone, 3-hydroxy-1,3,5(10),8,9-es-
tratetraen-17-one; 2-hydroxy-8,9-dehydroestrone, 2,3-dihydroxy-1,3,5-
(10),8,9-estratetraen-17-one; 4-hydroxy-8,9-dehydroestrone, 3,4-dihy-
droxy-1,3,5(10),8,9-estratetraen-17-one;equilenin,3-hydroxy-1,3,5(10),6,8-
estrapentaen-17-one; 4-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8-
estrapentaen-17-one; 2-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8-
estrapentaen-17-one; 4-hydroxyequilin, 3,4-dihydroxy-1,3,5(10),7(8)-
estratetraen-17-one; 2-hydroxyequilin, 2,3-dihydroxy-1,3,5(10),7(8)-
estratetraen-17-one; 4-hydroxyestrone, 3,4-dihydroxy-1,3,5(10)-estratrien-
17-one; estrone, 3-hydroxy-1,3,5(10)-estratrien-17-one; equilenin,
1,3,5(10),6,8-estrapentaen-3-ol-17-one; equilin, 1,3,5(10),7-estratetraen-
3-ol-17-one; LC-MS, liquid chromatography-mass spectrometry; ER,
estrogen receptor.
* To whom correspondence should be addressed. Fax: (312) 996-
7107. E-mail: J udy.Bolton@UIC.edu.
10.1021/tx010049y CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/18/2001

Catechol Metabolites from 8,9-Dehydroestrone
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 755
Sch em e 1. Syn th esis of 8,9-Deh yd r oestr on e
Sch em e 2. Syn th esis of 2-Hyd r oxy-8,9-d eh yd r oestr on e
might involve metabolism of estrogens to catechols, which
2-hydroxylation as observed for endogenous estrogens to
mainly 4-hydroxylation for equilenin. Recently, we have
found that the equine catechol estrogens, 4-hydrox-
yequilenin and 4-hydroxyequilin, form unusual cyclic
adducts with DNA in vitro (32-34). If the similar adducts
are formed in vivo, which are not repaired efficiently,
mutations could result leading to initiation of the carci-
nogenic process in hormone sensitive tissues.
There has only been one report on the metabolism of
8,9-dehydroestrone which suggested that the only me-
tabolite was 17â-hydroxy-8,9-dehydroestradiol (35). To
accurately study the potential for catechol formation from
8,9-dehydroestrone, we synthesized 8,9-dehydroestrone
(Scheme 1), 4-hydroxy-8,9-dehydroestrone (Scheme 2),
and 2-hydroxy-8,9-dehydroestrone (Scheme 3). We then
examined the metabolism of 8,9-dehydroestrone in rat
liver microsomes and showed that both 2-hydroxylated
and 4-hydroxylated GSH conjugates were formed (Scheme
4). Finally, we tested the relative toxicity of 8,9-dehy-
are then oxidized to redox active/electrophilic o-quinones
that could initiate the carcinogenic process by binding
to cellular macromolecules. For example, it is well
established that the endogenous estrogens, estrone and
17â-estradiol, are metabolized by P450 enzymes to 2- and
4-hydroxylated catechols (19, 20). The o-quinones formed
from peroxidase/P450-catalyzed oxidation of these cat-
echols have previously been implicated as the ultimate
carcinogens. Redox cycling between the catechols and
their quinones generates reactive hydroxyl radicals which
cause oxidation of the purine/pyrimidine residues of DNA
(21). The o-quinones could also alkylate DNA to form
adducts which have been detected both in vitro (22-24)
and in vivo (25-27). There have also been a few studies
on the potential for equine estrogens to be metabolized
to reactive catechol intermediates (28-31). It has been
demonstrated that increasing unsaturation in the B ring
leads to a change in product distribution from primarily

756 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Sch em e 3. Syn th esis of 4-Hyd r oxy-8,9-d eh yd r oestr on e
Zhang et al.
Sch em e 4. Meta bolism of 8,9-Deh yd r oestr on e to o-Qu in on es a n d Rea ction w ith GSH
on Hewlett-Packard (Palo Alto, CA) UV-vis spectrophotometer.
1H NMR spectra were obtained with a Bruker (Billerica, MA)
Avance DPX300 spectrometer at 300 MHz. Positive ion electro-
spray mass spectra were obtained using a Hewlett-Packard
5989B MS Engine quadrupole mass spectrometer equipped with
a ChemStation data system and high-flow pneumatic nebulizer-
assisted electrospray LC-MS interface. The mass spectrometer
was interfaced to a Hewlett-Packard 1050 gradient HPLC
system. The quadrupole analyzer was maintained at 120 °C and
unit resolution was used for all measurements. Nitrogen at a
pressure of 80 psi was used for nebulization of the HPLC
effluent, and nitrogen bath gas at 300 °C and a flow rate of 10
L/min were used for evaporation of solvent from the electro-
spray. A mass range from 300 to 1050 mass units were scanned
every 1.5 s (1.5 s/scan). Tandem MS (MS-MS) spectra were
obtained using a Micromass (Manchester, U.K.) Quattro II triple
quadrupole mass spectrometer equipped with an electrospray
ionization source. Collision-induced dissociation was carried out
using a range of collision energy from 25 to 70 eV and argon
collision gas pressure of 2.7 µbar.
droestrone and its catechol metabolites in breast cancer
cells. The data suggest that although catechols were
formed from 8,9-dehydroestrone, they were considerably
less toxic then 4-hydroxyequilenin which implies that
quinoids formed from equilin and equilenin are more
likely to contribute to the adverse effects of Premarin.
Ma ter ia ls a n d Meth od s
Ca u tion : The catechol estrogen o-quinones were handled in
accordance with NIH guidelines for the Laboratory Use of
Chemical Carcinogens (36). All chemicals were purchased from
Aldrich (Milwaukee, WI), Fisher Scientific (Itasca, IL), or Sigma
(St. Louis, MO) unless stated otherwise. (3H)-GSH (glycine-2-
3H) was obtained from NEN Life Science Products, Inc. (Boston,
MA) and diluted to a specific activity of 40 mCi/mmol. 2-Hy-
droxyequilenin was synthesized as described previously (37).
In str u m en ta tion . HPLC experiments were performed on a
Shimadzu (Columbia, MD) LC-10A gradient HPLC equipped
with a SIL-10A auto injector, SPD-M10AV UV-vis photodiode
array detector, and SPD-10AV detector. Peaks were integrated
with Shimadzu EZ-Chrom software. UV spectra were measured
HP LC Met h od ology. Two general methods were used to
analyze and separate the various metabolites and GSH conju-

Catechol Metabolites from 8,9-Dehydroestrone
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 757
Sch em e 5. Mech a n ism of Isom er iza tion of 2-Hyd r oxy-8,9-d eh yd r oestr on e to 2-Hyd r oxyequ ilen in
gates. All retention times reported in the text were obtained
using Method A.
and the solvent removed in vacuo to give a yellow liquid which
was purified by column chromatography (silica gel) using ethyl
acetate/hexane (1:8 v/v) as eluent to give 3 (2.5 g, 52% yield):
1H NMR (CDCl3) δ 0.23 (s, 6H, Si(CH3)2), 0.99 (s, 9H, C(CH3)3),
2.30 (m, 2H, CH2), 2.69 (t, 2H, CH2), 5.15 (dd, 1H, J cis ) 10.6
Hz, J gem ) 1.8 Hz, Hcis of CH2dCH), 5.50 (dd, 1H, J trans ) 10.6
Hz, J gem ) 1.8 Hz, Htrans of CH2dCH), 6.08 [m, 1H, H-C(2)],
6.56 (m, 1H, CH2dCH), 6.66 (m, 2H, ArH), 7.20 (d, 1H, J ) 9
Hz, ArH); CI-MS m/z 287 (100%) [M + H]+.
Method A. Analytical HPLC analysis was performed using a
4.6 × 250 mm Ultrasphere C-18 column (Beckman) on the
Shimadzu HPLC system described above. The mobile phase
consisted of 20% acetonitrile in 0.25% perchloric acid/0.25%
acetic acid (pH 3.5) at a flow rate of 1.0 mL/min for 5 min,
increased to 18% acetonitrile over 5 min, isocratic for 50 min,
increased to 45% over the next 10 min, and increased to 90%
acetonitrile over the last 5 min of the run.
(3) 3-tert-Butyldimethylsiloxy-8,9-dehydroestrone, 4. The cy-
cloaddition reaction procedure described in the literature was
followed (39). To a 100 mL, three-necked, round-bottom flask,
a solution of TiCl4 (1 mL, 9.15 mmol) in dry CH2Cl2 (15 mL)
was added to a solution of 2-methylcyclopent-2-en-1-one (0.32
g, 3.33 mmol) at -78 °C. The yellow mixture was stirred for 15
min and a solution of 6-tert-butyldimethylsiloxy-1-vinyl-3,4-
dihydronaphathalene 3 (2.32 g, 8.40 mmol) in dry CH2Cl2 (15
mL) was added dropwise over 1 h. The mixture was stirred for
another 5 h at -78 °C until 2-methylcyclopent-2-en-1-one could
not be detected by TLC (silica gel, hexane/ethyl acetate ) 8:1).
The mixture was warmed to 0 °C and 1 M HCl (70 mL) was
added. The final mixture was extracted with ethyl ether (2 ×
250 mL), washed with brine (2 × 250 mL), dried over sodium
sulfate, filtered, and evaporated to give a deep yellow liquid
which was further purified by flash column chromatography
(silica gel) using ethyl acetate/hexane (1:8 v/v) as eluent to give
4 as a yellow power (0.66 g, 7.2% yield): 1H NMR (acetone-d6)
δ 0.21 (s, 6H, Si(CH3)2), 0.99 (s, 9H, C(CH3)3), 1.02 (s, 3H, CH3),
1.41 (m, 1H), 1.77 (m, 2H), 2.12 (m, 1H), 2.36 (m, 5H), 2.47 (m,
1H), 2.70 (m, 2H), 2.80 (m, 1H), 6.63 (s, 1H), 6.68 (d, 1H, J )
8.3 Hz, ArH), 6.88 (d, 1H, J ) 8.3 Hz, ArH); 13C NMR (acetone-
d6) δ -4.25 [Si(CH3)2], 18.7 [C(CH3)3], 20.7, 22.5, 26.2, 26.4, 27.4,
28.1 29.5, 37.0, 47.4, 49.2, 118.2, 119.8, 123.9, 126.8, 130.6,
133.4, 137.9, 154.8, 221.7 (C17); CI-MS (positive ion, methane)
m/z 383 (100%) [M + H]+.
Method B. For LC-MS-MS analysis of GSH conjugates of
catechol metabolites from 8,9-dehydroestrone, analytical HPLC
analysis was performed using a 4.6 × 250 mm Ultrasphere C-18
column (Beckman) on the above-mentioned Hewlett-Packard
HPLC system, and tandem mass spectra were obtained using
the Micromass system interfaced with the Hewlett-Packard
HPLC system. The mobile phase consisted of 20% acetonitrile
in 0.5% ammonium acetate (pH 3.5) at a flow rate of 1.0 mL/
min for 5 min, increased to 18% acetonitrile over 5 min, isocratic
for 50 min, increased to 45% over the next 10 min, and increased
to 90% acetonitrile over the last 5 min of the run.
Syn th esis of 8,9-Deh yd r oestr on e (Sch em e 1). (1) 6-tert-
Butyldimethylsiloxytetralone, 2. 6-tert-Butyldimethylsiloxyte-
tralone was synthesized as described in the literature (38).
Briefly, to a stirred mixture of 6-hydroxy-1-tetralone 1 (2.5 g,
15.4 mmol) and imidazole (2.6 g, 38.5 mmol) in DMF (14 mL)
under a stream of nitrogen at room temperature was added tert-
butyldimethylsilyl chloride (2.9 g, 18.7 mmol). After stirring for
18 h, the reaction mixture was diluted with diethyl ether (280
mL). The resulting mixture was then washed with 1 M HCl (200
mL) and brine (2 × 50 mL), dried over MgSO4, filtered, and the
solvent removed en vacuo. The residue obtained was purified
by flash chromatography (silica gel, hexane/ethyl acetate, 3:1)
to give 2 as a pale yellow liquid (4.6 g, 96% yield): 1H NMR
(CDCl3) δ 0.23 (s, 6H, Si(CH3)2), 0.97 (s, 9H, C(CH3)3), 2.09 (m,
2H, CH2), 2.61 (t, 2H, J ) 6.3 Hz, CH2), 2.90 (t, 2H, J ) 6.3 Hz,
CH2), 6.65 (s, 1H, ArH), 6.74 (d, 1H, J ) 8.4 Hz, ArH), 7.96 (d,
2H, J ) 8.4 Hz, ArH); 13C NMR (CDCl3) δ -4.09, 18.4, 23.6,
25.8, 30.1, 39.1, 118.9, 119.3, 127.1, 129.7, 147.0, 160.5, 197.5;
CI-MS m/z 277 (100%) [M + H]+.
(4) 8,9-Dehydroestrone, 5. To a flame dried flask, 6-tert-
butyldimethylsiloxy-8,9-dehydroestrone 4 (300 mg, 0.785 mmol),
dry THF (15 mL), and Bu4NF (1.5 mL, 1 M in THF) were added.
The mixture was stirred for 20 min at room temperature,
followed by addition of THF (50 mL). This solution was washed
with brine (2 × 50 mL), dried over Na2SO4, filtered, and the
solvent was removed en vacuo to give a yellow residue which
was further purified by flash chromatography (silica gel, hexane:
acetone ) 2:1 + 1% MeOH) to give 8,9-dehydroestrone 5 (160
mg, 76% yield). Complete characterization was accomplished
by 1D and 2D NMR experiments including 1H, 13C, HMQC,
HMBC, and COSY: 1H NMR (acetone-d6) δ 1.01 (s, 3H, 18-CH3),
1.41 (m, 1H, H11), 1.74 (m, 2H, H12), 2.12 (m, 1H, H12), 2.27 (m,
5H, 2 × H15), 2.49 (m, 1H, H16), 2.4 (m, 2H, H11), 2.67 (m, 1H,
H14), 6.63 (m, 2H, H4), 7.10 (s, 1H, J ) 8.3 Hz, H1), 8.18 (bs,
1H, OH3); 13C NMR (acetone-d6) δ 20.7 (C18), 22.6 (C15), 26.2
(2)
6-tert-Butyldimethylsiloxy-1-vinyl-3,4-dihydronaph-
athalene, 3. To a flame dried flask, 6-tert-butyldimethylsilox-
ytetralone 2 (4.6 g, 16.6 mmol) and anhydrous THF (50 mL)
were added. Vinylmagnesium bromide (50 mL, 1 M in THF) was
added to this mixture through a dropping funnel at room
temperature. After stirring for 16 h, the mixture was refluxed
for 40 min, cooled to room temperature, and HCl (60 mL, 1 M)
was added through
a dropping funnel. The final reaction
mixture was extracted with diethyl ether (2 × 100 mL). The
combined organic layers were washed with saturated NaHCO3
(2 × 60 mL) and brine (2 × 60 mL), dried over Na2SO4, filtered,

758 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Zhang et al.
(C6), 27.5 (C11,), 28.1 (C12), 29.4 (C16), 37.0 (C9), 47.4 (C13), 49.2
(C14), 113.4 (OCH3), 115.1 (C1), 124.1 (C4), 126.9 (C7), 128.8 (C5),
132.2 (C10), 137.9 (C8), 146.3 (C2), 156.7(C3), 221.8 (C17); UV
4-(2′,3′-dimethoxyphenyl)butanoic acid 14 (42). Compound 14
was cyclized with polyphosphoric acid (PPA) to produce 5,6-
dimethoxy-1-tetralone 15 (42). Demethylation of 5,6-dimethoxy-
1-tetralone 15 using anhydrous AlCl3 /benzene gave 5,6-
dihydroxy-1-tetralone 16: 1H NMR (acetone-d6) δ 2.07 (m, 2H,
CH2), 2.49 (t, 2H, J ) 6.3 Hz, CH2), 2.97 (t, 2H, J ) 6.3 Hz,
CH2), 6.81 (d, 1H, J ) 8.4 Hz, ArH), 7.46 (d, 1H, J ) 8.4 Hz,
ArH), 9.17 (b, 2H, OH); 13C NMR (CDCl3) δ 23.5, 23.7, 39.1,
113.8, 120.2, 126.8, 132.9, 142.3, 149.9, 197.1; CI-MS m/z 179
(100%) [M + H]+.
(2) 5,6-Di-tert-butyldimethylsiloxytetralone, 17. 5,6-Di-tert-
butyldimethylsiloxytetralone 17 was prepared by reacting 5,6-
dihydroxy-1-tetralone 16 (2.6 g, 14.5 mmol) with tert-butyldim-
ethylsilyl chloride (7.2 g, 46.44 mmol) in DMF (50 mL) and
imidazole (6.8 g, 100.8 mmol) according to the same procedure
described above (6.4 g, 94% yield): 1H NMR (acetone-d6) δ 2.05
(m, 2H, CH2), 2.52 (m, 2H, J ) 7.0 Hz, CH2), 2.93 (m, 2H, CH2),
6.92 (d, 1H, J ) 8.4 Hz, ArH), 7.58 (d, 1H, J ) 8.4 Hz, ArH);
CI-MS m/z 407 (100%) [M + H]+.
(CH3OH), 220, 286 nm; EI-MS m/z 268 (100%) [M]+•
.
Syn th esis of 2-Hyd r oxy-8,9-d eh yd r oestr on e (Sch em e 2).
(1) 6,7-Dihydroxy-1-tetralone, 7. To a flame dried flask, 6,7-
dimethoxy-1-tetralone 6 (5.0 g, 24 mmol), anhydrous AlCl3 (25.5
g, 192 mmol), and benzene (100 mL) were added under a stream
of nitrogen. The mixture was refluxed for 2.5 h, cooled to room
temperature, and 3 N HCl (100 mL) was added at 0 °C. After
filtration, the deep yellow solid was collected and recrystallized
from hot water to gave 6,7-dihydroxy-1-tetralone 7 (1.5 g, 35%
yield): 1H NMR (acetone-d6) δ 2.04 (m, 2H, CH2), 2.44 (t, 2H, J
) 6.3 Hz, CH2), 3.11 (t, 2H, J ) 6.3 Hz, CH2), 6.71 (s, 1H, ArH),
7.35 (s, 1H, ArH), 8.44 (b, 2H, OH); 13C NMR (CDCl3) δ 23.8,
25.8, 39.1, 119.1, 119.7, 127.3, 129.9, 147.2, 152.5, 196.5; CI-
MS m/z 179 (100%) [M + H]+.
(2) 6,7-Di-tert-butyldimethylsiloxytetralone, 8. 6,7-Di-tert-
butyldimethylsiloxytetralone 8 (3.2 g, 94% yield) was prepared
by reacting 6,7-dihydroxy-1-tetralone 7 (1.5 g, 8.42 mmol) with
tert-butyldimethylsilyl chloride (3.17 g, 20.5 mmol) in DMF (20
mL) and imidazole (2.84 g, 42.1 mmol) according to the same
procedure described above: 1H NMR (acetone-d6) δ 0.24 (s, 6H,
Si(CH3)2), 0.27 (s, 6H, Si(CH3)2), 1.01 (s, 18H, C(CH3)3), 2.05
(m, 2H, CH2), 2.50 (t, 2H, J ) 7.0 Hz, CH2), 2.84 (t, 2H, J ) 7.0
Hz, CH2), 6.81 (s, 1H, ArH), 7.46 (d, 1H, ArH); 13C NMR
(acetone-d6) δ -4.50, 18.3, 18.4, 23.6, 25.5, 25.6, 118.2, 120.3,
139.3, 145.7, 151.8,195.6; CI-MS m/z 407 (100%) [M + H]+.
(3) 6,7-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphath-
alene, 9. 6,7-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaph-
athalene 9 (1.08 g, 31% yield) was obtained by reacting 6,7-di-
tert-butyldimethylsiloxytetralone 8 (3.5 g, 8.6 mmol) with vi-
nylmagnesium bromide (50 mL, 1 M in THF) following the same
procedure as described above for 6-tert-butyldimethylsiloxy-1-
vinyl-3,4-dihydronaphathalene 3: 1H NMR (CDCl3) δ 0.23 (s,
12H, Si(CH3)2), 0.99 (s, 18H, C(CH3)3), 2.21 (m, 2H, CH2), 2.62
(m, 2H, CH2), 5.15 (dd, 1H, J cis ) 10.6 Hz, J gem ) 1.8 Hz, Hcis of
CH2dCH), 5.51 (dd, 1H, J trans ) 10.6 Hz, J gem ) 1.8 Hz, Htrans
of CH2dCH), 6.07 [m, 1H, H-C(2)], 6.56 (m, 1H, CH2dCH), 6.64
(s, 1H, ArH), 6.89 (d, 1H, ArH); CI-MS m/z 417 (100%) [M +
H]+.
(4) 2,3-Di-tert-butyldimethylsiloxy-8,9-dehydroestrone, 10. Ac-
cording to the same cycloaddition reaction procedure described
above, 6,7-di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphath-
alene 9 (1.8 g, 4.3 mmol) was converted into 2,3-di-tert-
butyldimethylsiloxy-8,9-dehydroestrone 10 (250 mg, 11% yield)
by reaction with 2-methylcyclopent-2-en-1-one (0.21 g, 2.18
mmol) in the presence of TiCl4 (6.4 mmol): 1H NMR (acetone-
d6) δ 0.22 (s, 12H, Si(CH3)2), 0.99 (s, 18H, C(CH3)3), 1.02 (s, 3H,
CH3), 1.41 (m, 1H), 1.73 (m, 2H), 2.15 (m, 1H), 2.30 (m, 5H),
2.40 (m, 1H), 2.65 (m, 2H), 2.81 (m, 1H), 6.67 (s, 1H), 6.74 (d,
1H, ArH); 13C NMR (acetone-d6) δ -3.69, 19.14, 20.8, 22.6, 26.3,
27.4, 28.1, 28.4, 29.5, 37.0, 47.4, 49.4, 116.3, 121.0, 126.7, 129.6,
130.6, 133.8,145.6, 147.5, 221.7 (C17); APCI-MS (positive ion,
methane) m/z 513 (100%) [M + H]+.
(3) 5,6-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphath-
alene, 18. 5,6-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaph-
athalene 18 (1.1 g) was obtained by reacting 5.6-di-tert-bu-
tyldimethylsiloxytetralone 17 (4.21 g, 10.4 mmol) with vinyl-
magnesium bromide (70 mL, 1 M in THF) following the same
procedure as described above for 6-tert-butyldimethylsiloxy-1-
vinyl-3,4-dihydronaphathalene.
(4) 3,4-Di-tert-butyldimethylsiloxy-8,9-dehydroestrone, 19. Ac-
cording to the same cycloaddition reaction procedure described
above, 5,6-di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphath-
alene 18 (1.1 g, 2.64 mmol) was converted into 3,4-di-tert-
butyldimethylsiloxy-8,9-dehydroestrone 19 (30 mg, 2.5% yield)
by reaction with 2-methylcyclopent-2-en-1-one (0.16 g, 1.66
mmol) in the presence of TiCl4 (6.4 mmol): 1H NMR (ace-
tone-d6) δ 6.71 (d, 1H, J ) 8.4 Hz, ArH), 6.76 (d, 1H, J )
8.4 Hz, ArH); APCI-MS (positive ion) m/z 513 (100%) [M +
H]+.
(5) 4-Hydroxy-8,9-dehydroestrone, 20. 3,4-Di-tert-butyldi-
methylsiloxy-8,9-dehydroestrone 19 (25 mg, 1.95 mmol) was
deprotected using Bu4NF to give 4-hydroxy-8,9-dehydroestrone
20 (7 mg, 51% yield): 1H NMR (acetone-d6) δ 1.02 (s, 3H, CH3),
1.42 (m, 1H), 1.80 (m, 2H), 2.10 (m, 1H), 2.30 (m, 5H), 2.45 (m,
1H), 2.55 (m, 2H), 2.80 (m, 1H), 6.61 (d, 1H, J ) 8.4 Hz, ArH),
6.66 (d, 1H, J ) 8.4 Hz, ArH), 7.11 (b, 1H, OH), 8.24 (b, 1H,
OH), Negative APCI-MS m/z 283 (100%) [M - H]-.
GSH Con ju ga tes of 2-Hyd r oxy-8,9-d eh yd r oestr on e-o-
qu in on e a n d 4-Hyd r oxy-8,9-d eh yd r oestr on e-o-qu in on e.
The o-quinone GSH conjugates of 2-hydroxy-8,9-dehydroestrone
and 4-hydroxy-8,9-dehydroestrone were prepared by incubating
the corresponding catechol (0.5 mM) with 5.0 mM GSH and
tyrosinase (0.4 µg/mL) in 2 mL of 50 mM sodium phosphate
buffer (pH 7.4), 37 °C for 30 min. The conjugates were isolated
from the aqueous phase using solid-phase extraction cartridges
(Oasis; Waters Corporation, Milford, MA) and eluted with
methanol. The methanol extract was concentrated to 150 µL
and aliquots (25 µL) were analyzed directly by HPLC. Two di-
GSH conjugates and one mono-GSH conjugates (Scheme 4) were
obtained with both 2-OHDHES and 4-OHDHES. Since the
tandem mass spectrometry of the di-SG and mono-SG conju-
gates showed a similar pattern for each GSH conjugate, MS-
MS data are only reported for the 4-OHDHES-diSG and
4-OHDHES-SG (method A): 4-OHDHES-diSG1, UV (CH3OH/
H2O) 256 nm, positive ion electrospray MS m/z 895 [M + H]+
(100%); MS-MS with CID of m/z 895 gave m/z 877 [MH - H2O]+
(10%), 820 [MH - Gly]+ (5%), 766 [MH - Glu]+ (30%), 637 [MH
- 2Glu]+ (20%), retention time 20 min; 4-OHDHES-diSG2, UV
(CH3OH/H2O) 262 nm, positive ion electrospray MS m/z 895 [M
+ H]+ (100%); CID MS-MS pattern same as 4-OHDHES-diSG1,
retention time 21 min; 4-OHDHES-SG, UV (CH3OH/H2O) 255
nm, positive ion electrospray MS m/z 590 [M + H]+ (100%); MS-
MS with CID of m/z 590 gave m/z 515 [MH - Gly]+ (20%), 461
[MH - Glu]+ (40%), 358 [MH - 2Glu]+ (60%), 315 [MH - SG +
32]+, retention time 55 min; 2-OHDHES-diSG1, UV (CH3OH/
(5) 2-Hydroxy-8,9-dehydroestrone, 11. 2-Hydroxy-8,9-dehy-
droestrone 11 (8 mg, 37% yield) was obtained by deprotection
of 2,3-di-tert-butyldimethylsiloxy-8,9-dehydroestrone 10 (50 mg,
0.097 mmol) with Bu4NF (0.5 mL, 1.0 M in THF) according to
the same procedure described above: 1H NMR (acetone-d6) δ
1.01 (s, 3H, CH3), 1.41 (m, 1H), 1.74 (m, 2H), 2.15 (m, 1H), 2.26
(m, 5H), 2.40 (m, 1H), 2.55 (m, 2H), 2.91 (m, 1H), 6.61 (s, 1H,
ArH), 6.72 (s, 1H, ArH), 7.60 (b, 2H, OH); CI-MS m/z 285 (100%)
[M + H]+.
Syn th esis of 4-Hydr oxy-8,9-Deh ydr oestr on e (20) (Sch em e
3). (1) 5,6-Dihydroxy-1-tetralone, 16. 2-Carboxyethyltriphe-
nylphosphonium bromide was obtained by refluxing triph-
enylphosphine with 3-bromopropanoic acid in the presence of
benzene (40). 2,3-Dimethoxybenzaldehyde 12 was treated with
2-carboxyethyltriphenylphosphonium bromide in the presence
of NaH and DMSO to give 4-(3′,5′-dimethoxyphenyl)-3-butenoic
acid bromide 13 (41). Hydrogenation of 13 using 5% Pd/C gave

Catechol Metabolites from 8,9-Dehydroestrone
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 759
H2O) 260, 334 nm; positive ion electrospray MS m/z 895 [M +
H]+ (100%); MS-MS pattern same as 4-OHDHES-diSG1, reten-
tion time 24 min; 2-OHDHES-diSG2, UV (CH3OH/H2O) 257,
335 nm, positive ion electrospray MS m/z 895 [M + H]+ (100%),
retention time 25 min; 2-OHDHES-SG, UV (CH3OH/H2O) 272,
337 nm, positive ion electrospray MS m/z 590 [M + H]+ (100%);
retention time 51 min.
obtained by regression and linear estimation analysis. The data
represent the mean ( SD of triplicate determinations.
Resu lts a n d Discu ssion
Syn th esis of 8,9-Deh yd r oestr on e. There are reports
in the literature on the synthesis of 8,9-dehydroestrone
and 3-methoxy-8,9-dehydroestrone (39, 47-49). One
method involved isomerization of equilin to 8,9-dehy-
droestrone in the presence of glacial acetic acid and
hydrochloric acid (47). However, this study did not report
sufficient spectral data in order to unequivocally confirm
the structure of 8,9-dehydroestrone. When this method
was attempted, only a mixture of products resulted which
could not be separated by chromatography. Other litera-
ture synthetic methods included multistep reactions (39,
48, 49). However, all of these methods concentrated on
the synthesis of 3-methoxy-8,9-dehydroestrone which
could not be easily converted into 8,9-dehydroestrone.
Demethylation of 3-methoxy-8,9-dehydroestrone was at-
tempted by using mild demethylation agents, such as
BBr3 (50), BBr3‚SMe2 (51), AlCl3/C2H5SH (52), and BCl3
(53); however, these experiments were not successful due
to the instability of the conjugated 8,9-double bond in the
B ring. Therefore, an alternative method for the synthesis
of 8,9-dehydroestrone was developed (Scheme 1). In this
methodology, the commercially available 6-hydroxy-1-
tetralone 1 was used as starting material. After protec-
tion of the 6-hydroxy group with tert-butyldimethylsilyl
chloride, 6-tert-butyldimethylsiloxy-1-tetralone 2 was
obtained. The protected tetralone was converted into
1-vinyl-6-tert-butyldimethylsiloxy-3,4-dihydronaphatha-
lene 3 by reacting compound 2 with commercially avail-
able vinylmagnesium bromide. Catalytic cycloaddition of
1-vinyl-6-tert-butyldimethylsiloxy-3,4-dihydronaphatha-
lene with 2-methylcyclopent-2-en-1-one in the presence
of TiCl4 gave 3-tert-butyldimethylsiloxy-8,9-dehydroestro-
ne 4. Removal of the protecting group under mild
conditions with Bu4NF gave 8,9-dehydroestrone 5. On the
basis of this methodology, the potential catechol metabo-
lites of 8,9-dehydroestrone, 4-hydroxy-8,9-dehydroestrone
(Scheme 3) and 2-hydroxy-8,9-dehydroestrone (Scheme
2), were also synthesized.
Kin etic Stu d ies. The rate of formation 2-hydroxyequilenin
from the autoxidation of 2-hydroxy-8,9-dehydroestrone (0.5 mM)
in potassium phosphate buffer (pH 7.4, 37 °C) was determined
by monitoring product formation by UV-visible spectroscopy
using a Hewlett-Packard HP8452 diode array spectrophotom-
eter. After 1.5 h, the pH was adjusted to 4 and the solution was
extracted with CHCl3 (2 mL). The organic layer was concen-
trated and analyzed by positive ion CI-MS (methane). The [M
+ H]+ ion of 2-hydroxy-8,9-dehydroestrone (285) was reduced
95% compared to the starting material and the [M + H]+ ion of
2-hydroxyequilenin (283) was the base peak in the spectrum.
In cu ba tion s. Female Sprague-Dawley rats (180-200 g)
were obtained from Sasco Inc. (Omaha, NE). The rats were
pretreated with dexamethasone to induce P450 3A isozymes
(43). Dexamethasone was administered by i.p. injections of 100
mg/kg in corn oil daily for 4 days and the animals were sacrificed
on day 5. Microsomes were prepared from rat liver, and protein
and P450 concentrations determined as described previously
(44). Incubations containing microsomal protein were conducted
for 10-30 min at 37 °C in 50 mM phosphate buffer (pH 7.4,
500 µL total volume). Substrates were added as solutions in
dimethyl sulfoxide, and (3H)-GSH (specific activity of 40 mCi/
mmol) was added in phosphate buffer to achieve final concen-
trations of 0.5 and 1.0 mM, respectively. A NADPH generating
system consisting of 1.0 mM NADP+, 5 mM isocitric acid, and
0.2 units of isocitric dehydrogenase/mL were used together with
5.0 mM MgCl2. For control incubations NADP+ was omitted.
The reactions were initiated by the addition of NADP+, and
terminated by chilling in an ice bath followed by the addition
of perchloric acid (25 µL).
Ad d u ct Qu a n tifica tion . The reaction mixtures were cen-
trifuged at 13 000 rpm for 6 min to remove precipitated
microsomal protein. Aliquots of the supernatant (100 µL) were
analyzed directly by HPLC as described above for the GSH
conjugates. For quantification of GSH conjugates, 0.3 mL
aliquots of the column effluent were collected every 18 s during
each run, and radioactivity was measured with a Beckman
model LS 5801 liquid scintillation counter. Concentrations of
the GSH conjugates were calculated by summing the radioactiv-
ity associated with each peak and converting the data to
nanomolar amounts using the specific activity of the (3H)-GSH.
Each analysis was performed immediately after the incubation
in order to limit degradation of the o-quinone GSH conjugates.
GSH Con ju ga tes of 2-Hyd r oxy-8,9-d eh yd r oestr o-
n e- a n d 4-Hyd r oxy-8,9-d eh yd r oestr on e-o-qu in on e.
Previous studies have shown that enzymatic oxidation
of endogenous catechol estrogens generated o-quinones
which could be trapped in situ by GSH (54, 55). In
addition, we have shown that 4-hydroxyequilenin and
4-hydroxyequilin readily autoxidized to o-quinones which
reacted with GSH to form the corresponding GSH
conjugates at physiological pH and temperature (50, 56).
In contrast, 4-hydroxy-8,9-dehydroestrone was found to
be very stable under physiological conditions; however,
it could be oxidized to an o-quinone by oxidative enzymes.
This o-quinone was trapped with GSH to form one mono-
GSH conjugate (retention time 55 min) and two di-GSH
conjugates eluting at 20 and 21 min (HPLC method A).
Similar to other equine catechol estrogen GSH conju-
gates, these GSH conjugates were unstable and we could
not obtain sufficient material for NMR characterization,
and as a result their structures could not be determined
unambiguously. However, based on the LC-MS and MS-
MS data, the structures of the GSH conjugates (mono or
di) are consistent with reaction of the o-quinone of
4-hydroxy-8,9-dehydroestrone with one or two GSH
Cytotoxicity Exp er im en ts in S-30 Br ea st Ca n cer Cells.
Cell viability was determined by Trypan blue exclusion (45, 46).
The S-30 cell line was a generous gift from V. C. J ordan
(Northwestern University, Evanston, IL). Briefly, S-30 cells were
maintained in MEME supplemented with 1% penicillin, strep-
tomycin, fungizome, 10 mM nonessential amino acids, and 5%
2× stripped bovine serum. The medium was changed 24 h before
beginning cytotoxicity assays to maintain logarithmic growth.
The cells were treated with 2-OHDHES, 4-OHDHES, 4-OHEN,
8,9-dehydroestrone, and equilenin for 18 h. The test samples
were assayed in triplicate, and final concentrations ranged from
1.6 to 500 µM. Each assay included negative controls (cells
treated with DMSO only) that were used to define 100% cell
viability. After treatment, floating cells were collected by
centrifugation at 3000 rpm for 5 min, and attached cells were
first trypsinized and then harvested by centrifugation. Floating
and attached cells were combined, washed with PBS, and
stained with 0.4% Trypan blue. A drop of cell suspension was
placed on a hemocytometer and the cell numbers were deter-
mined under a microscope. The dead cells were stained blue
while viable cells remained unstained. The LC50 values were

760 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Zhang et al.
MS pattern as that of the 4-hydroxy-8,9-dehydroestrone
GSH conjugates. However, incubation of 2-hydroxy-8,9-
dehydroestrone in phosphate buffer (pH 7.4) gave a
distinctive time-dependent UV absorbance change (Fig-
ure 3). The new species formed at the end of the
incubation had the same UV absorbance pattern as that
of 2-hydroxyequilenin (Figure 3). This transformation
was further confirmed by HPLC with a co-injection of an
authentic sample 2-hydroxyequilenin. In addition, ex-
traction of the final incubation solution with CHCl3 and
analysis by CI-MS showed an ion at 283 corresponding
to the protonated molecule of 2-hydroxyequilenin. It is
likely that the transformation of 2-hydroxy-8,9-dehy-
droestrone to 2-hydroxyequilenin under these conditions
was accomplished through a analogous o-quinone me-
thide mechanism (Scheme 5) by which 4-hydroxyequilin
isomerized to 4-hydroxyequilenin (34). The GSH conju-
gates of 2-hydroxy-8,9-dehydroestrone and 4-hydroxy-8,9-
dehydroestrone imply that quinones were formed prior
to nucleophilic GSH addition. These o-quinones might
produce toxicity through depletion of cellular GSH. Once
GSH is depleted, the o-quinones could alkylate cysteine
residues on cellular proteins, resulting in toxic effects (59,
60).
F igu r e 1. Positive ion electrospray MS/MS with CID of the
protonated molecule (m/z 590) of 4-hydroxy-8,9-dehydroestrone
mono-GSH conjugate.
Oxid a tion of Ca tech ol Estr ogen s a n d 8,9-Deh y-
d r oestr on e by Ra t Liver Micr osom es. We examined
the oxidation of 2-hydroxy-8,9-dehydroestrone, 4-hy-
droxy-8,9-dehydroestrone, and 8,9-dehydroestrone to
quinoid metabolites in rat liver microsomes by trapping
these reactive species with [3H]GSH (Table 1). The
trapping reaction should be very efficient because of the
high concentration of GSH in the incubation medium and
the relatively fast rate of GSH addition as compared to
amino and hydroxyl groups (61, 62). However, a small
amount of binding to the microsomal protein by the
quinoids is possible and as a result conjugate formation
shown in Table 1 should be considered a lower limit for
the generation of quinoids. Microsomal incubations with
the catechols showed that 2-hydroxy-8,9-dehydroestrone
was a slightly better substrate than 4-hydroxy-8,9-
dehydroestrone in terms of o-quinone formation (Table
1). Incubations with 8,9-dehydrodroestrone also showed
production of mono-GSH conjugates from both catechols
although considerably less o-quinone derived GSH con-
jugates were produced in incubations with the parent
phenol as compared to similar experiments with the
catechols. These data differ from what has been reported
for the metabolism of 8,9-dehydroestrone in female dogs
where only the 17â-hydroxy-8,9-dehydroestradiol was
detected (35). It is possible that these catechols were
formed in the female dogs which were not detected
because of the instability of the catechols and o-quinones.
Regioselectivity was observed in the P-450-catalyzed
hydroxylation of 8,9-dehydroestrone since 2-hydroxy-8,9-
dehydroestrone GSH conjugates predominated over 4-hy-
droxy-8,9-dehydroestrone GSH conjugates by a factor of
6. Previously, we studied the metabolism of estrone,
equilin, and equilenin to catechol o-quinone GSH conju-
gates in rat liver microsomes (50, 54). It was shown that
estrone was primarily metabolized to 2-hydroxyestrone
(2:4 hydroxylation ratio ) 6:1), equilin was primarily
metabolized to 4-hydroxyequilin (2:4 hydroxylation ratio
) 1:6), and equilenin was exclusively metabolized to
4-hydroxyequilenin. The present data showed that the
ratio of 2:4 hydroxylation of 8,9-dehydroestrone was 6:1
F igu r e 2. Positive ion electrospray MS/MS with CID of the
protonated molecule (m/z 895) of 4-hydroxy-8,9-dehydroestrone
di-GSH conjugate.
molecules (Figure 1, ref 2). The molecule at m/z 590 was
consistent with the protonated molecule of the mono-
GSH conjugate (Figure 1). The product ion spectrum of
this ion of m/z 590 gave abundant fragment ions at m/z
515 [MH - 75]+ (loss of glycine), m/z 461 [MH - 129]+
(loss of pyroglutamic acid), and m/z 315. The two GSH
conjugates of 4-hydroxy-8,9-dehydroestrone had similar
MS-MS patterns. The MS-MS pattern of 4-hydroxy-8,9-
dehydroestrone-diSG 1 is shown in Figure 2. The product
ion spectrum of the ion at m/z 895 gave abundant
fragment ions at m/z 820 [MH - 75]+ (loss of glycine),
m/z 766 [MH - 129]+ (loss of pyroglutamic acid), and m/z
637 [MH - 258]+ (loss of two pyroglutamic acid moieties).
Loss of 129 units (pyroglutamic acid) from the protonated
molecule is a characteristic fragment ion of GSH conju-
gates formed during MS-MS (57, 58). The behavior of
2-hydroxy-8,9-dehydroestrone was found to be different
from 4-hydroxy-8,9-dehydroestrone under similar condi-
tions. This catechol formed one mono-GSH conjugate at
a retention time of 51 min and two di-GSH conjugates
at 24 and 25 min. These conjugates were formed either
in the absence or in the presence of oxidative enzymes
(P450 or tyrosinase). Like the GSH conjugates of 4-hy-
droxy-8,9-dehydroestrone, these GSH conjugates were
also unstable and only LC-MS-MS spectra were obtained.
The mono- and di-GSH conjugates showed the same MS-

Catechol Metabolites from 8,9-Dehydroestrone
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 761
Ta ble 1. Con ver sion of 8,9-Deh yd r oestr on e a n d Its Ca tech ol Meta bolites to o-Qu in on e-Der ived GSH Con ju ga tes by Ra t
Liver Micr osom esa
retention
rate of formation
substrate
conjugate
time (min)
(nmol/nmol of P450‚10 min)
8,9-dehydroestrone
2-OHDHES-SG
4-OHDHES-SG
51
55
0.037 ( 0.003
0.007 ( 0.001
tota l ) 0.044 ( 0.004
0.17 ( 0.02
2-hydroxy-8,9-dehydroestrone
4-hydroxy-8,9-dehydroestrone
2-OHDHES-diSG 1
2-OHDHES-diSG 2
2-OHDHES-SG
24
25
51
0.23 ( 0.01
4.50 ( 0.03
tota l ) 4.90 ( 0.06
0.31 ( 0.01
4-OHDHES-diSG 1
4-OHDHES-diSG 2
4-OHDHES-SG
20
21
55
0.48 ( 0.02
2.07 ( 0.03
tota l ) 2.86 ( 0.06
a
Incubations were conducted for 30 min with 0.5 mM substrate, rat liver microsomes (1.0 nmol P450/mL) in the presence of an NADPH-
generating system and [3H]GSH, at 37 °C. Radioactivity eluting from the HPLC column was measured in fractions collected at 18-s
intervals. Results are the average ( SD of three incubations. Background radioactivity in the control (-NADP+) samples have been
subtracted from each peak.
Ta ble 2. Cytotoxicity of Equ in e Estr ogen s a n d Th eir
Ca tech ol Meta bolites in S-30 Br ea st Ca n cer Cellsa
substrate
LC50 (µM)
2-hydroxy-8,9-dehydroestrone
4-hydroxy-8,9-dehydroestrone
4-hydroxyestrone
4-hydroxyequilenin
8,9-dehydroestrone
equilenin
84 ( 7
147 ( 7
251 ( 9
4.0 ( 0.1b
126 ( 7
155 ( 8
a
Cell viability was assessed by Trypan blue exclusion as
described in Materials and Methods. Values are expressed as the
b
mean ( SD of at least three determinations. From ref 63.
8,9-dehydroestrone in rat liver microsomes and the
relative cytotoxicity of these catechols in breast cancer
cells. We have found that 8,9-dehydroestrone was pri-
marily metabolized to 2-hydroxy-8,9-dehydroestrone in
rat liver microsomes and that this catechol can autoxi-
dize, isomerize, and aromatize giving 2-hydroxyequilenin.
In contrast, 4-hydroxy-8,9-dehydroestrone is a stable
catechol under physiological conditions. These data might
explain the enhanced toxicity of 2-hydroxy-8,9-dehy-
droestrone compared to 4-hydroxy-8,9-dehydroestrone in
breast cancer cells. In should be noted that both catechol
metabolites of 8,9-dehydroestrone were much less toxic
(20-40-fold) than 4-hydroxyequilenin which is the major
metabolite of both equilin and equilenin (34, 50). These
results may suggest that the catechol metabolites of 8,9-
dehydroestrone might have the ability to cause cytotox-
icity in vivo primarily through formation of catechol
quinoids; however, most of the adverse effects of Pre-
marin estrogens are likely due to formation of quinoids
from equilin and equilenin.
F igu r e 3. (A) UV-vis spectral analysis of the conversion of
2-hydoxy-8,9-dehydroestrone to 2-hydroxyequilenin. Incubations
contained 2-hydroxy-8,9-dehydroestrone (0.15 mM) in potassium
phosphate buffer (50 mM, pH 7.4, 37 °C). Scans were taken
every 5 min for 1.5 h. (B) UV spectrum of 2-hydroxyequilenin.
which was quite similar to that of the endogenous
estrogen, estrone.
Cytotoxicity of 8,9-Deh yd r oestr on e a n d Ca tech ol
Meta bolites in Br ea st Ca n cer Cells (S-30). Prelimi-
nary studies conducted with the ERR stably transfected
human breast tumor cell line S-30, demonstrated that
the cytotoxicity of the catechol metabolites from 8,9-
dehrdroestrone were much higher than that of 4-hydrox-
yestrone, but less than that of 4-hydroxyequilenin (63).
The cytotoxicity of 2-hydroxy-8,9-dehydroestrone is al-
most double that of 4-hydroxy-8,9-dehydroestrone which
had similar cytotoxic effects as 8,9-dehrydroestrone and
equilenin (Table 2). The higher cytotoxicity of 2-hydroxy-
8,9-dehydroestrone might result from the fact that this
catechol can autoxidize to 2-hydroxy-8,9-dehydroestrone
o-quinone under physiological conditions without oxida-
tive enzymatic catalysis (see above). In addition, the
microsomal incubations suggest that the amount of
quinoids formed in the S30 cells is likely higher for
2-hydroxy-8,9-dehydroestrone as compared to 4-hydroxy-
8,9-dehydroestrone.
Ack n ow led gm en t. This research was supported by
NIH Grant CA73638 (J .L.B.) and CA83124 (R.B.v.B.). We
are grateful to Dr. V. C. J ordan (Northwestern Univer-
sity) for the gift of the S-30 cell line.
Refer en ces
(1) Grodstein, F., Stampfer, M. J ., Colditz, G. A., Willett, W. C.,
Manson, J . E., J offe, M., Rosner, B., Fuchs, C., Hankinson, S. E.,
Hunter, D. J ., Hennekens, C. H., and Speizer, F. E. (1997)
Postmenopausal hormone therapy and mortality. New Eng. J .
Med. 336, 1769-1775.
(2) Wickelgren, I. (1997) Estrogen: A new weapon against Alzhe-
imer’s. Science 276, 676-677.
In conclusion, data have been presented on the forma-
tion of GSH conjugates from the catechol metabolites of
(3) Henderson, V. W. (1997) The epidemiology of estrogen replace-
ment therapy and Alzheimer’s disease. Neurology 48, S27-S35.

762 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Zhang et al.
(4) Henderson, V. W., Paganini-Hill, A., Miller, B. L., Elble, R. J .,
Reyes, P. F., Shoupe, D., McCleary, C. A., Klein, R. A., Hake, A.
M., and Farlow, M. R. (2000) Estrogen for Alzheimer’s disease in
women: randomized, double-blind, placebo-controlled trial. Neu-
rology 54, 295-301.
(5) Mulnard, R. A., Cotman, C. W., Kawas, C., van Dyck, C. H., Sano,
M., Doody, R., Koss, E., Pfeiffer, E., J in, S., Gamst, A., Grundman,
M., Thomas, R., and Thal, L. J . (2000) Estrogen replacement
therapy for treatment of mild to moderate Alzheimer disease: a
randomized controlled trial. Alzheimer’s Disease Cooperative
Study. J . Am. Med. Assoc. 283, 1007-1015.
(6) Paganinihill, A. (1995) Estrogen replacement therapy and stroke.
Prog. Cardio. Dis. 38, 223-242.
(7) Clark, K. E., Baker, R. S., and Lang, U. (2000) Premarin-induced
increases in coronary and uterine blood flow in nonpregnant
sheep. Am. J . Obstet. Gynecol. 183, 12-17.
(8) Tsang, K. L., Ho, S. L., and Lo, S. K. (2000) Estrogen improves
motor disability in Parkinsonian postmenopausal women with
motor fluctuations. Neurology 54, 2292-2298.
(9) Thayer, A. (1997) Thumbs-down on generic estrogen. Chem. Eng.
News 75, 13.
(10) Baracat, E., Haidar, M., Lopez, F. J ., Pickar, J ., Dey, M., and
Negro-Vilar, A. (1999) Estrogen activity and novel tissue selectiv-
ity of delta 8,9-dehydroestrone sulfate in postmenopausal women.
J . Clin. Endocrinol. Metab. 84, 2020-2027.
(11) Dey, M., Lyttle, C. R., and Pickar, J . H. (2000) Recent insights
into the varying activity of estrogens. Maturitas. 34 (Suppl. 2),
S25-S33.
(28) Purdy, R. H., Moore, P. H., Williams, M. C., Goldzheher, H. W.,
and Paul, S. M. (1982) Relative rates of 2- and 4-hydroxyestrogen
synthesis are dependent on both substrate and tissue. FEBS Lett.
138, 40-44.
(29) Li, S. A., Klicka, J . K., and Li, J . J . (1985) Estrogen 2- and
4-hydroxylase activity, catechol estrogen formation, and implica-
tions for estrogen carcinogenesis in the hamster kidney. Cancer
Res. 45, 181-185.
(30) Li, J . J ., Li, S. A., Oberley, T. D., and Parsons, J . A. (1995)
Carcinogenic activities of various steroidal and nonsteroidal
estrogens in the hamster kidney: relation to hormonal activity
and cell proliferation. Cancer Res. 55, 4347-4351.
(31) Sarabia, S. F., Zhu, B. T., Kurosawa, T., Tohma, M., and Liehr,
J . G. (1997) Mechanism of cytochrome P450-catalyzed aromatic
hydroxylation of estrogens. Chem. Res. Toxicol. 10, 767-771.
(32) Shen, L., Qiu, S., van Breemen, R. B., Zhang, F., Chen, Y., and
Bolton, J . L. (1997) Reaction of the Premarin metabolite 4-hy-
droxyequilenin semiquinone radical with 2′-deoxyguanosine: For-
mation of unusual cyclic adducts. J . Am. Chem. Soc. 119, 11126-
11127.
(33) Shen, L., Qiu, S., Chen, Y., Zhang, F., van Breemen, R. B., Nikolic,
D., and Bolton, J . L. (1998) Alkylation of 2′-deoxynucleosides and
DNA by the Premarin metabolite 4-hydroxyequilenin semi-
quinone radical. Chem. Res. Toxicol. 11, 94-101.
(34) Zhang, F., Chen, Y., Pisha, E., Shen, L., Xiong, Y., van Breemen,
R. B., and Bolton, J . L. (1999) The major metabolite of equilin,
4-hydroxyequilin autoxidizes to an o-quinone which isomerizes
to the potent cytotoxin 4-hydroxyequilenin-o-quinone. Chem. Res.
Toxicol. 12, 204-213.
(35) Chandrasekaran, A., Osman, M., Raveendranath, P., Chan, K.,
Scatina, J . A., and Sisenwine, S. F. (1995) Characterization of
metabolites of delta(8,9)-dehydroestrone in dogs. J . Mass Spec-
trom. 30, 1505-1511.
(36) NIH Guidelines for the Laboratory Use of Chemical Carcinogens.
Washington, DC, U.S. Government Printing Office, 1981.
(37) Zhang, F., and Bolton, J . L. (1999) Synthesis of the equine
estrogen metabolites 2-hydroxyequilin and 2- hydroxyequilenin.
Chem. Res. Toxicol. 12, 200-203.
(12) Steinberg, K. K., Smith, S. J ., Thacker, S. B., and Stroup, D. F.
(1994) Breast cancer risk and duration of estrogen use - The role
of study design in meta-analysis. Epidemiology 5, 415-421.
(13) Davidson, N. E. (1995) Hormone-replacement therapy - Breast
versus heart versus bone. New Engl. J . Med. 332, 1638-1639.
(14) Colditz, G. A., Hankinson, S. E., Hunter, D. J ., Willett, W. C.,
Manson, J . E., Stampfer, M. J ., Hennekens, C., Rosner, B., and
Speizer, F. E. (1995) The use of estrogens and progestins and the
risk of breast cancer in postmenopausal women. New Eng. J . Med.
332, 1589-1593.
(38) Woo, L. W., Howarth, N. M., Purohit, A., Hejaz, H. A. M., Reed,
M. J ., and Potter, B. V. L. (1998) Steroidal and nonsteroidal
sulfamates as potent inhibitors of steroid sulfatase. J . Med. Chem.
41, 1068-1083.
(39) Quinkert, G., Grosso, M. D., Doring, A., Doring, W., Schenkel, R.
I., Bauch, M., Dambacher, G., Bats, J . W., Zimmermann, G., and
Durner, G. (1995) Total synthesis with a chrogenic opening move
demonstrated on steroids with estrane or 18a-homoestrane
skeleton. Helv. Chim. Acta 78, 1345-1391.
(15) Lupulescu, A. (1995) Estrogen use and cancer incidence: A review.
Cancer Invest. 13, 287-295.
(16) Zumoff, B. (1998) Does postmenopausal estrogen administration
increase the risk of breast cancer? Contributions of animal,
biochemical, and clinical investigative studies to a resolution of
the controversy. Proc. Soc. Exp. Biol. Med. 217, 30-37.
(17) Service, R. F. (1998) New role for estrogen in cancer? Science 279,
1631-1633.
(18) Henderson, B. E., and Feigelson, H. S. (2000) Hormonal carcino-
genesis. Carcinogenesis 21, 427-433.
(19) Martucci, C. P., and Fishman, J . (1993) P450 enzymes of estrogen
metabolism. Pharmacol. Ther. 57, 237-257.
(20) Liehr, J . G. (1994) Mechanism of metabolic activation and
inactivation of catecholestrogens: A basis of genotoxicity. Polycycl.
Aromat. Compd. 6, 229-239.
(21) Yager, J . D., and Liehr, J . G. (1996) Molecular mechanisms of
estrogen carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 36, 203-
232.
(40) Findlay, J ., and Kwan, D. (1973) Metabolites from a scytalidium
species. Can. J . Chem. 51, 3299-3301.
(41) Beetz, T., Meuleman, D. G., and Wieringa, J . H. (1982) Inhibitors
of blood platelet aggregation. Activity of some 1H-benz[de]-
isoquinolinecarboximidamides on the in vivo blood platelet ag-
gregation induced by collagen. J . Med. Chem. 25, 714-719.
(42) Oka, Y., Motohash, M., Sugihara, H., Miyashita, O., Itoh, K.,
Nishikawa, M., and Yurugi, S. (1977) Syntheses of conformation-
ally rigid catecholamine derivatives. Chem. Pharm. Bull. 25, 632-
639.
(22) Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and
Cavalieri, E. (1992) Synthesis and characterization of estrogen
2,3- and 3,4-quinones. Comparison of DNA adducts formed by the
quinones versus horseradish peroxidase-activated catechol estro-
gens. Chem. Res. Toxicol. 5, 828-833.
(23) Stack, D. E., Byun, J ., Gross, M. L., Rogan, E. G., and Cavalieri,
E. L. (1996) Molecular characteristics of catechol estrogen quino-
nes in reactions with deoxyribonucleosides. Chem. Res. Toxicol.
9, 851-859.
(24) Akanni, A., and Abul-Hajj, Y. J . (1997) Estrogen-nucleic acid
adducts: reaction of 3,4-estrone-o-quinone radical anion with
deoxyribonucleosides. Chem. Res. Toxicol. 10, 760-766.
(25) Liehr, J . G., Avitts, T. A., Randerath, E., and Randerath, K. (1986)
Estrogen-induced endogenous DNA adduction: possible mecha-
nism of hormonal cancer. Proc. Natl. Acad. Sci. U.S.A. 83, 5301-
5305.
(43) Okey, A. B. (1990) Enzyme induction in the cytochrome P-450
system. Pharmac. Ther. 45, 241-298.
(44) Thompson, J . A., Malkinson, A. M., Wand, M. D., Mastovich, S.
L., Mead, E. W., Schullek, K. M., and Laudenschlager, W. G.
(1987) Oxidative metabolism of butylated hydroxytoluene by
hepatic and pulmonary microsomes from rats and mice. Drug
Metab. Dispos. 15, 833-840.
(45) Andrews, D. L., Williams, G. S., Manoney, J . C., and West, J . R.
(1999) DNA fragmentation during exposure of rat cerelella to
ethanol under hypoxia imposed in vitro. J . Neurobiol. 7, 82-92.
(46) Sandoval, M., Zhang, X. J ., Liu, X., Mannick, E. E., Clark, D. A.,
and Miller, M. J . (1997) Peroxynitrite-induced apoptosis in T84
and RAW 264.7 cells: attenuation by L-ascorbic acid. Free Radical
Biol. Med. 22, 489-495.
(47) Hirschmann, H., and Wintersteiner, O. (1938) Isoequilin A. J .
Biol. Chem. 126, 737-748.
(26) Han, X., Liehr, J . G., and Bosland, M. C. (1995) Induction of a
DNA adduct detectable by 32P-postlabeling in the dorsolateral
prostate of NBL/Cr rats treated with estradiol-17 beta and
testosterone. Carcinogenesis 16, 951-954.
(27) Cavalieri, E. L., Stack, D. E., Devanesan, P. D., Todorovic, R.,
Dwivedy, I., Higginbotham, S., J ohansson, S. L., Patil, K. D.,
Gross, M. L., Gooden, J . K., Ramanathan, R., Cerny, R. L., and
Rogan, E. G. (1997) Molecular origin of cancer: Catechol estrogen-
3,4,-quinones as endogenous tumor initiators. Proc. Natl. Acad.
Sci. U.S.A. 94, 10937-10942.
(48) J ung, M. E., and Halweg, K. M. (1984) Stereoselectivity in
intramolecular Diels-Alder reactions: A short total synthesis of
estrone. Tetrahedron Lett. 25, 2121-2124.
(49) Stein, R. P., Buzby, G. C., and Smith, H. (1970) Totally synthetic
steroid hormones - XX. Novel total synthesis of equilin and
equilenin. Tetrahedron. 26, 1917-1933.
(50) Chang, M., Zhang, F., Shen, L., Pauss, N., Alam, I., van Breemen,
R. B., Blond-Elguindi, S., and Bolton, J . L. (1998) Inhibition of
glutathione S-transferase activity by the quinoid metabolites of
equine estrogens. Chem. Res. Toxicol. 11, 758-765.

Catechol Metabolites from 8,9-Dehydroestrone
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 763
(51) Williard, P. G., and Fryhle, C. B. (1980) Boron trihalide-methyl
sulfide complexes as convenient reagents for dealkylation of aryl
ethers. Tetrahedron Lett. 21, 3731-3734.
(52) Node, M., Nishide, K., Fuji, K., and Fujita, E. (1980) Hard acid
and soft nucleophile system. 2. Demethylation of methyl ethers
of alcohol and phenol with an aluminum halide-thiol system. J .
Org. Chem. 45, 4275-4277.
(53) Teitel, S., and O’Brien, J . P. (1976) Selective removal of an
aromatic methylenedioxy group. J . Org. Chem. 41, 1657-1658.
(54) Iverson, S. L., Shen, L., Anlar, N., and Bolton, J . L. (1996)
Bioactivation of estrone and its catechol metabolites to quinoid-
glutathione conjugates in rat liver microsomes. Chem. Res.
Toxicol. 9, 492-499.
(55) Butterworth: M., Lau, S. S., and Monks, T. J . (1996) 17â-Estradiol
metabolism by hamster hepatic microsomes: Implications for the
catechol-O-methyl transferase-mediated detoxication of catechol
estrogens. Drug Metab. Dispos. 24, 588-594.
(56) Shen, L., Pisha, E., Huang, Z., Pezzuto, J . M., Krol, E., Alam, Z.,
van Breemen, R. B., and Bolton, J . L. (1997) Bioreductive
activation of catechol estrogen-ortho-quinones: Aromatization of
the B ring in 4-hydroxyequilenin markedly alters quinoid forma-
tion and reactivity. Carcinogenesis 18, 1093-1101.
(57) Kassahum, K., Davis, M., Hu, P., Martin, B., and Baillie, T. (1997)
Biotransformation of the naturally occurring isothiocyanate sul-
foraphane in the rat: Indentification of phase I metabolitea and
glutathione conjugates. Chem. Res. Toxcol. 10, 1228-1233.
(58) Ramanathan, R., Cao, K., Cavalieri, E., and Gross, M. L. (1998)
Mass spectrometric methods for distinguishing structural isomers
of glutathione conjugates of estrone and estradiol. J . Am. Soc.
Mass Spectrom. 9, 612-619.
(59) Dehal, S. S., and Kupfer, D. (1996) Evidence that the catechol
3,4-dihydroxytamoxifen is a proximate intermediate to the reac-
tive species binding covalently to proteins. Cancer Res. 56, 1283-
1290.
(60) Dehal, S. S., and Kupfer, D. (1999) Cytochrome P-450 3A and
2D6 catalyze ortho hydroxylation of 4-hydroxytamoxifen and
3-hydroxytamoxifen (droloxifene) yielding tamoxifen catechol:
involvement of catechols in covalent binding to hepatic proteins.
Drug Metab. Dispos. 27, 681-688.
(61) Bolton, J . L., Valerio, L. G. J ., and Thompson, J . A. (1992) The
enzymatic formation and chemical reactivity of quinone methides
correlate with alkylphenol-induced toxicity in rat hepatocytes.
Chem. Res. Toxicol. 5, 816-822.
(62) Kalyanaraman, B., Premovic, P. I., and Sealy, R. C. (1987)
Semiquinone anion radicals from addition of amino acids, pep-
tides, and proteins to quinones derived from oxidation of catechols
and catecholamines: An ESR stabilization study. J . Biol. Chem.
262, 11080-11087.
(63) Chen, Y., Liu, X., Pisha, E., Constantinou, A. I., Hua, Y., Shen,
L., van Breemen, R. B., Elguindi, E. C., Blond, S. Y., Zhang, F.,
and Bolton, J . L. (2000) A metabolite of equine estrogens,
4-hydroxyequilenin, induces DNA damage and apoptosis in breast
cancer cell lines. Chem. Res. Toxicol. 13, 342-350.
TX010049Y