Oxidative transformation of 2-hydroxyestrone. Stability and reactivity of 2,3-estrone quinone and its relationship to estrogen carcinogenicity

Article abstract of DOI:10.1021/tx950205z

The carcinogenicity of estrogens in rodents and man has been attributed to either alkylation of cellular macromolecules and/or redox-cycling, generation of active radicals, and DNA damage. Metabolic activation of estradiol leading to the formation of catechol estrogens is believed to be a prerequisite for its genotoxic effects. 4-Hydroxyestradiol, although not 2- hydroxyestradiol, is a potent inducer of tumors in hamsters. Previous studies have shown that 3,4-estrone quinone can redox-cycle and is capable of inducing exclusively single strand DNA breaks in MCF-7 breast cancer cells, as well as react with various nucleophiles (thiol, imidazole, amino, phenolate, and acetoxy) to give Michael addition products. These results support the possible involvement of 3,4-catechol/quinone estrogens in estrogen's carcinogenicity. To explain the decreased carcinogenicity of 2- hydroxyestrogens, the reactions of 2,3-estrone quinone (2,3-EQ) with nucleophiles were investigated. Reactions of 4-methylimidazole with 2,3-EQ gave a complex mixture of products leading to the formation of the catechol, C-O dimerization product, and a 1,6-Michael addition product identified as the 1-(4-methylimidazolo)-2-hydroxyestrone. Reactions of 2,3-EQ under mildly basic conditions with either ethyl phenolate or acetate gave several products which were characterized as the C-O and C-C dimers, catechol, and 3,5- dihydroxy-1(10),3-estradiene-2,17-dione. No Michael addition products were detected under these experimental conditions. The same products were also observed during the synthesis of 2,3-EQ, which led us to postulate that the lack of carcinogenicity of 2-hydroxyestrogens may be related to the increased reactivity and decreased stability of the quinone under physiological conditions. These results are contrasted with those obtained with 3,4-EQ which is much more stable and therefore could diffuse from the site of formation to the target tissue. These results along with rapid methylation and clearance may be very likely explanations for the decreased carcinogenicity of 2-hydroxyestrogens.

Full text of DOI:10.1021/tx950205z

8
60
Chem. Res. Toxicol. 1996, 9, 860-865
Oxid a tive Tr a n sfor m a tion of 2-Hyd r oxyestr on e.
Sta bility a n d Rea ctivity of 2,3-Estr on e Qu in on e a n d Its
Rela tion sh ip to Estr ogen Ca r cin ogen icity
†
†,‡
‡
Katmerka Tabakovic, William B. Gleason, William H. Ojala, and
,
†
Yusuf J . Abul-Hajj*
Department of Medicinal Chemistry, Biomedical Engineering Center, and Department of Laboratory
Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455
Received December 8, 1995X
The carcinogenicity of estrogens in rodents and man has been attributed to either alkylation
of cellular macromolecules and/or redox-cycling, generation of active radicals, and DNA damage.
Metabolic activation of estradiol leading to the formation of catechol estrogens is believed to
be a prerequisite for its genotoxic effects. 4-Hydroxyestradiol, although not 2-hydroxyestradiol,
is a potent inducer of tumors in hamsters. Previous studies have shown that 3,4-estrone
quinone can redox-cycle and is capable of inducing exclusively single strand DNA breaks in
MCF-7 breast cancer cells, as well as react with various nucleophiles (thiol, imidazole, amino,
phenolate, and acetoxy) to give Michael addition products. These results support the possible
involvement of 3,4-catechol/quinone estrogens in estrogen’s carcinogenicity. To explain the
decreased carcinogenicity of 2-hydroxyestrogens, the reactions of 2,3-estrone quinone (2,3-
EQ) with nucleophiles were investigated. Reactions of 4-methylimidazole with 2,3-EQ gave a
complex mixture of products leadng to the formation of the catechol, C-O dimerization product,
and a 1,6-Michael addition product identified as the 1-(4-methylimidazolo)-2-hydroxyestrone.
Reactions of 2,3-EQ under mildly basic conditions with either ethyl phenolate or acetate gave
several products which were characterized as the C-O and C-C dimers, catechol, and 3,5-
dihydroxy-1(10),3-estradiene-2,17-dione. No Michael addition products were detected under
these experimental conditions. The same products were also observed during the synthesis of
2
,3-EQ, which led us to postulate that the lack of carcinogenicity of 2-hydroxyestrogens may
be related to the increased reactivity and decreased stability of the quinone under physiological
conditions. These results are contrasted with those obtained with 3,4-EQ which is much more
stable and therefore could diffuse from the site of formation to the target tissue. These results
along with rapid methylation and clearance may be very likely explanations for the decreased
carcinogenicity of 2-hydroxyestrogens.
In tr od u ction
Recent studies have shown a connection between
excessive exposure to estrogens and the development of
cancer in several tissues including breast, endometrium,
liver, and kidney in humans and experimental animals
inducing exclusively single strand DNA breaks in a
human breast cancer cell line (12). Studies in our
laboratory are continuing to investigate the importance
of redox-cycling in the carcinogenicity of estrogen o-
quinones.
Covalent binding of proteins may inhibit essential
functions of enzymes or regulatory proteins which may
ultimately lead to their toxic effects. Thus, it is quite
possible that the carcinogenicity/toxicity of estrogen
o-quinones may be, in part, due to their ability to react
with proteins. It has been well documented by us and
others that thiols react with estrogen o-quinones by
Michael addition (13-15). Furthermore, addition reac-
tions between lysine and quinones have been implicated
in several protein-quinone interactions. For example,
the decrease in lysine content of casein following incuba-
tion of caffeoquinone and chlorogenquinone has been
attributed to the addition of the ꢀ-amino group of lysine
to the respective quinone moiety (16, 17). Studies in our
laboratory showed that lysine can form Michael addition
products with 3,4-EQ as well as form the iminoquinone
products (18). To further our understanding of the
chemistry of conjugate addition to estrogen o-quinones,
our laboratory has carried out extensive studies on the
reaction of 3,4-EQ with several model compounds rep-
resenting the amino acid side chains of cysteine, lysine,
histidine, aspartic acid, and tyrosine in which catechol
(
1-7). Although the exact mechanism for carcinogenesis
induced by estrogenic compounds is not fully understood,
it is generally believed that the estrogen o-quinones
produced by the oxidation of catechol estrogens by phenol
oxidase (8), prostaglandin H synthase (9), and cytochrome
P-450 (10) have the potential to be cytotoxic and/or
genotoxic. The molecular mechanisms for o-quinone
cytotoxicity have been attributed to the alkylation of
cellular macromolecules and/or redox cycling generating
reactive oxygen species which oxidize essential cellular
components. Previous studies from this laboratory have
1
shown that 3,4-estrone o-quinone (3,4-EQ) can redox-
cycle leading to the formation of hydrogen peroxide,
hydroxyl radical, and the semiquinone of 3,4-EQ (11).
Furthermore, we have shown that 3,4-EQ is capable of
*
Author to whom correspondence should be addressed.
Department of Medicinal Chemistry.
Biomedical Engineering Center and Department of Laboratory
†
‡
Medicine and Pathology.
X
Abstract published in Advance ACS Abstracts, J une 1, 1996.
Abbreviations: 2,3-estrone quinone, 2,3-EQ; 3,4-estrone quinone,
1
3
,4-EQ; 2-hydroxyestrone, 2-OHE
1
; 4-hydroxyestrone, 4-OHE
N.
1
; 4-hy-
droxyestradiol, 4-OHE ; triethylamine, Et
2
3
S0893-228x(95)00205-0 CCC: $12.00 © 1996 American Chemical Society

2
,3-Estrone Quinone
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 861
addition products were obtained (19). On the other hand,
amino acid side chain mimics of arginine, serine, tryp-
tophan, and asparagine did not lead to adduct formation.
The reactivity and stability of 3,4-EQ have been well
documented; however, very little is known about 2,3-EQ
even though the 2,3-catechol is the major metabolite of
estrogens (20-22). The present study investigates the
reactivity of the catechol and the 2,3-estrogen quinone
with various amino acid nucleophiles. The results from
previous studies and this investigation show that 2,3-
EQ is considerably less stable than 3,4-EQ and, while it
forms addition products with thiols and imidazole, it
undergoes extensive transformations under mildly basic
conditions to give several products.
Ta ble 1. Cr ysta l Da ta for Com p ou n d 7
parameter
compound
empirical formula
formula wt (amu)
crystal color and habit
cryst dimensions (mm)
cryst system
a (Å)
C23H30O6
402.49
colorless, needle
0.64 × 0.11 × 0.11
monoclinic
12.573(3)
7.042(2)
12.882(2)
1050.5(3)
2
b (Å)
c (Å)
3
V (Å )
Z
space group
calcd density (g/cm )
P21 (#4)
1.272
3
F000
432
7.47
µ(Cu KR) (cm-1)
no. of measured reflections
total
unique
4234
2017
2967
0.032
0.044
0.045
Exp er im en ta l P r oced u r es
no. of obsd refl. (I > 3.00σ(I))
Rint
R
Ch em ica ls. Estrone was purchased from Steraloids (Wilton,
NH). 4-Ethylphenol and 4-methylimidazole were obtained from
Aldrich Chemical Co. (Milwaukee, WI). Deuterated solvents
were purchased from Aldrich Chemical Co.. The synthesis of
the catechol estrogens was carried out as described by Stuben-
rauch and Knuppen (23). The estrogen o-quinones were syn-
thesized by the oxidation of the catechol estrogens by activated
wR
-
-
3
3
max e in final difference map
+0.22 e /Å
-0.22 e /Å
-
-
min e in final difference map
2
2 2
,3-EQ (1, 500 mg, 1.76 mmol) in CH Cl (10 mL), and the
2
MnO (24) or by oxidation of the 2-amino estrone (23). Ca u tion :
reaction mixture was stirred at room temperature under
nitrogen. The quinone was consumed within 1 h (checked by
TLC), after which the products were isolated and purified.
The estrogen o-quinones are potentially hazardous and were
handled in accordance with NIH Guidelines for the Laboratory
Use of Chemical Carcinogens (25).
Ch a r a cter iza tion of Com p ou n d s. All of the compounds
were characterized by nuclear magnetic resonance ( H-NMR),
ultraviolet (UV), infrared (IR), and mass spectrometry (MS).
Melting points (uncorrected) were taken on a Fisher-J ohns
apparatus. H-NMR spectra were obtained with a GE-300 MHz
2 2
Evaporation of the CH Cl showed the presence of five spots
which were separated on a silica gel column. Elution of the
column with 25% EtOAc in benzene gave compound 2. Further
elution of the column yielded a light orange solid of 5: mp 105-
07 °C dec; R
max (nm) 230, 318, 466; H-NMR (CDCl
.15 (s, 1H, H-4), 6.06 (s, 1H, H-4′), 5.43 (s, 1H, OH, D
1
1
λ
6
f
) 0.23 in 25% benzene in EtOAc; UV (CH
2
Cl
2
)
1
1
3
) δ 6.32 (s, 1H, H-1),
NMR spectrometer, and the chemical shift data are reported in
parts per million (δ) downfield from tetramethylsilane as an
internal standard. Nuclear Overhauser effect (NOE) difference
spectra were recorded by applying a presaturation pulse with
a decoupler on resonance and subtracting the trace from the
corresponding reference spectra recorded under identical condi-
tions but with the decoupler off-resonance. Typical spectra were
obtained from 64 transients. The UV spectra were obtained
using a Beckman DU-70. The IR spectra were obtained on a
Nicolet 5 DXC FT-IR spectrometer. Mass spectral data were
determined on an AEI MS-30, a VG 7070 E-HF, and a Finnigan
MAT 95.
X-r a y Cr ysta llogr a p h y. Colorless crystals of 7 were grown
in acetone/hexane. All measurements were made on a Rigaku
AFC6S X-ray diffractometer with graphite monochromated Cu
KR radiation (l ) 1.54178 Å) using the w - 2q scan mode. The
crystal temperature was maintained at 173(1) K by using a
Molecular Structure Corp. low temperature device. Intensities
were corrected for Lorentz and polarization effects. Equivalent
reflections were merged, and absorption effects were corrected
using the ψ scan technique (transmission factors: 0.96-1.00)
as described by North et al. (26). The structure was solved by
direct methods using SHELXS86 (27) and refined and finished
using the TEXSAN structure analysis package (28). All non-
hydrogen atoms were refined anisotropically. Hydrogens bonded
to carbon atoms were placed in calculated positions (0.95 Å) and
were not refined. Hydrogens bonded to heteroatoms were
located in difference Fourier maps, and their positional param-
eters were refined. Crystal data are given in Table 1.
2
O
exchangeable), 1.21 (s, 3H, 18′-CH
3
+
), 0.85 (s, 3H, 18-CH
3
); EI
+
+
MS m/z 570 (M + 2, 18.6), 568 (M , 26.5), 511 (M - C
3
H
5
O,
+
+
+
2
-
.3), 483 (M - C
5
H
9
O, 2.3), 470 (M - C
6
H
10O, 1.6), 455 (M
+
C
7
H
13O, 1.5), 444 (M - C
8
H
12O, 1.4), 286 (100), in agreement
with the proposed structure of 5. Continued elution of the
column gave 17.6% yield of a white solid of 3. This compound
was found to be unstable; it undergoes oxidation first to the
quinone adduct followed by decomposition to 2 and methylimi-
dazole.
In a separate experiment, the reaction mixture was acetylated
immediately prior to separation and purification over a silica
gel column. Acetylation using a 1:1 mixture of pyridine/acetic
anhydride followed by the usual workup gave a mixture of the
acetates of 2, 3, and 5. Separation on silica gel by elution with
a mixture of benzene/EtOAc/MeOH (6.5:2.0:1.5) gave the ac-
etates of 2 and 5 which were not purified any further. Contin-
ued elution of the column yielded the 2,3-diacetylated 1-(4-
methylimidazolo)estrone 4: mp 152 °C dec; Rf ) 0.35 (25%
EtOAC in benzene); UV λmax (nm) 220, 240, 318; IR (KBr) cm-1
1
2931, 2918, 1766, 1740, 1487, 1380, 1372, 1206, 1130, 1015; H-
NMR (CDCl ) δ 7.36 (s, 1H, H-2 imidazole), 6.91 (s, 1H, H-4),
6.19 (s, 1H, H-5 imidazole), 2.89 (m, 2H, H-6), 2.28 (s, 3H, OAc),
2.27 (s, 1H, OAc), 2.17 (s, 3H, CH -imidazole), 0.91 (s, 3H, 18-
3
3
+
CH ); ES MS m/z 450 (M , 8.5).
3
Rea ction of 2,3-Estr on e Qu in on e (1) w ith a n d w ith ou t
4-Eth ylp h en ol or Acetic Acid . To a solution of 4-ethylphenol
(0.35 mmol) in CH Cl (30 mL) was added three drops of Et N
and 2,3-estrogen o-quinone (1, 100 mg, 0.35 mmol), and the
reaction mixture was stirred at room temperature. The quinone
was totally consumed within 30 min (checked by TLC). Evapo-
ration of CH Cl left a solid that showed several spots on TLC
2
2
3
Syn th esis of Ca tech ol Estr ogen s a n d Estr ogen Qu in o-
n es. Synthesis of the 2-OHE
1
and 2,3-EQ was carried out as
described by Stubenrauch and Knuppen (23) and by our
2
2
1
laboratory (24). 2,3-EQ (1): H-NMR (CDCl
3
) δ 6.25 (d, 1H, H-4,
). 2-OHE
) δ 6.81 (s, 1H, H-1), 6.61 (s, 1H, H-4), 2.8
m, 2H, H-6), 0.86 (s, 3H, 18-CH ).
Rea ction of 2,3-Estr ogen o-Qu in on e (1) w ith 4-Meth -
ylim id a zole. 4-Methylimidazole (160 mg, 1.94 mmol) in CH
Cl (50 mL) was added to a solution of freshly prepared (in situ)
which were separated on a silica gel column. Elution of the
column with 25% EtOAc in benzene yielded compounds 2 and
5 which were characterized as described above. Product 9 was
f
J ) 2.4 Hz), 6.18 (s, 1H, H-1), 0.91 (s, 3H, 18-CH
3
1
1
(
(
2): H-NMR (CDCl
3
3
obtained in 5% yield: mp 210 °C dec; R ) 0.49 in 25% EtOAc
3
in benzene; UV (CH OH) λmax (nm) 222, 241, 278, 420; IR (KBr)
-
1
1
2
-
cm 3409, 2961, 2927, 2850, 1739, 1384; H-NMR (CDCl
7.72 (d, 1H, H-6′, J ) 8.3 Hz), 7.34 (s, 1H, H-1′), 7.20 (s, 1H,
3
) δ
2

8
62 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Tabakovic et al.
F igu r e 1. Products from the reaction of 2,3-estrone o-quinone with 4-methylimidazole.
H-4), 7.14 (d, 1H, H-6, J ) 8.3 Hz), 5.92 (bs, 1H, OH, D
2
O
Previous studies have shown that thiols react with 2,3-
EQ by 1,6-Michael addition, leading to the formation of
both the 1- and 4-substituted adducts (14), while reac-
tions with amine nucleophiles resulted in 1,2-additions,
leading to the formation of the monoiminoquinone and
bisiminoquinone products (31). To further our studies
on the reactivity of 2,3-EQ toward other nucleophiles and
to compare these results with 3,4-EQ, the present
investigation reports the results of the reactions of 2,3-
EQ with methylimidazole, acetate, and ethyl phenolate.
exchangeable), 5.82 (bs, 1H, OH, D O exchangeable), 1.26 (t,
2
+
3
H, 18′-CH
3
), 0.80 (s, 3H, 18-CH
3
); EI MS m/z 566.8 (M + H) ;
+
FAB MS (glycerol/TFA matrix 2000Res) m/z 567.2 (M + H ),
5
quantities of the unstable compound 6 for partial characteriza-
tion: R
6
2
0
5
+
65.2 (M - H ). Continued elution of the column gave sufficient
1
f
) 0.26 (25% EtOAc in benzene); H-NMR (CDCl
.34 (bs, 1H, 3-OH, D O exchangeable), 6.10 (d, 1H, H-4, J )
.8 Hz), 6.06 (s, 1H, H-1), 2.17 (s, 1H, 5-OH, D O exchangeable),
); EI MS m/z 302.2 (M , 87.6), 286 (M - 16,
3
) δ
2
2
+
.92 (s, 3H, 18-CH
3
5), 284 (M - 18, 38), 163 (18), 145 (46), 139 (100), 107 (22).
Compound 7 was identified as the monoacetate: mp 103-
In the reaction of 2,3-EQ with 4-methylimidazole, three
major products were isolated and purified using a com-
bination of column chromatography and preparative TLC
1
05 °C; R
f
3
) 0.23 (25% EtOAc in benzene); UV (CH OH) λmax
-1
(nm) 202, 240; IR (KBr) cm 3410, 2990, 2930, 2890, 1771, 1740,
1
1
3
675, 1650, 1371, 1134; H-NMR (CDCl ) δ 6.49 (s, 1H, H-4), 6.02
(Figure 1). Spectroscopic data for compound 2 were
(
s, 1H, H-1), 2.83 (s 1H, OH, D
2
O exchangeable), 2.26 (s, 3H,
+
found to be identical to those of an authentic sample of
CH
3
+
CO), 0.88 (s, 3H, 18-CH
3
); ES MS m/z 344.2 (M , 10), 367.2
2-OHE which may be formed by reduction of 2,3-EQ by
1
(M
+ Na, 100).
Compound 8 had the following characteristics: mp 210-212
either the catechol adduct 3 or the catechol precursors
°
(
1
C dec; R ) 0.44 (25% EtOAc in benzene); UV (CH
f
3
OH) λmax
to 5. The structure of the dimeric product 5a /5b was
deduced essentially from UV, MS, and NMR data. The
mass spectral data showed a molecular ion at 568
corresponding to the molecular mass of the proposed
structure. The products from oxidative phenolic coupling
can be formed through a C-C or C-O bond (33).
Confirmation of the presence of the C-O bond comes
from the NMR spectrum which shows one singlet at 6.06
ppm attributed to one proton of the o-quinone moiety,
and two other singlets at 6.32 and 6.15 ppm which are
assigned to the two aromatic protons of the catechol
moiety. However, on the basis of NMR spectra, it is not
possible to discriminate between two possible isomers
which could arise through a C-O coupling reaction.
Formation of 5 is postulated to occur either by a one-
electron transfer via a charge transfer complex (34), or
by reduction of 2,3-EQ by the Michael addition product
-1
nm) 206, 237; IR (KBr) cm 2980, 2930, 2870, 1773, 1734, 1676,
1
3
652, 1647, 1384, 1201, 1138; H-NMR (CDCl ) δ 6.59 (s,1H,
3
H-4), 6.20 (s,1H, H-1), 2.46-2.48 (m, 4H), 2.27 (s, 3H, 3-CH -
CO), 2.06 (s, 3H, 5-CH
3
CO), 0.91 (s, 3H, 18-CH
3
); ES MS m/z
+
+
3
2
86 (M , 70); EI MS m/z 386 (M , 0.28), 344 (21), 302 (100),
86 (17.7), 43 (19.8).
Resu lts a n d Discu ssion
Although the underlying mechanisms of estrogen car-
cinogenesis are as yet unresolved, it has been proposed
that metabolic activation of estradiol leading to the
formation of catechol estrogens is a prerequisite for its
genotoxic activity (7). Whether arylation of macromol-
ecules or redox-cycling of catechols/quinones is involved
in estrogen carcinogenesis is the subject of intensive
investigations in several laboratories. 2-OHE
predominant metabolite of estrogens in most tissues, yet
-hydroxyestradiol has been found to be a much more
2
is the
3
leading to the formation of 2, which could Michael add
to 2,3-EQ leading to the formation of the catechol
precursor to 5 followed by oxidation to 5 by 2,3-EQ, to
regenerate 2. It is interesting to note that the C-C
coupling products from this reaction were not detected.
4
potent inducer of tumors (29). The differences in activi-
ties between the two catechols have been ascribed to the
rapid methylation and metabolic clearance of 2-OHE
as well as inhibition of O-methylation of 4-OHE
-OHE (30). Because of these results, our laboratory has
2
(29)
2
by
Compound 3 showed a singlet at 6.88 ppm assigned to
an aromatic proton and other peaks corresponding to the
structure of 4-methylimidazole, suggesting a Michael
addition product. However, full characterization of 3
could not be carried out due to the fact that it undergoes
air oxidation to the quinonoid adduct of 3 followed by
2
2
conducted extensive studies on the reactions of 3,4-EQ
with several nucleophiles to determine the nature of the
chemical interactions between the estrogen quinones and
macromolecules (13, 18, 19, 31, 32).

2
,3-Estrone Quinone
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 863
decomposition to 2-OHE
1
and methylimidazole as de-
tected on TLC and by NMR. To further characterize the
structure of 3, the reaction mixture between 2,3-EQ and
methylimidazole was acetylated immediately following
2 2
evaporation of CH Cl and the acetylated product 4 was
obtained as a stable crystalline material. Proton NMR
showed the presence of a singlet at 6.91 ppm assigned
to the aromatic ring of the steroid, three singlets at 7.36
(
1H), 6.19 (1H), and 2.17 (3H) ppm assigned to the
methylimidazole moiety, and two singlets at 2.28 and
.27 ppm assigned to the acetates at C-2 and C-3. Mass
2
spectral data (ES) revealed a molecular ion peak at 450
corresponding to the proposed structure of 4. The as-
1
signed regiochemistry of 4 was supported by H NOE
difference experiments. Irradiation of the resonance at
6
.81 ppm in 2 assigned to the C-1 proton in ring A
resulted in a significant enhancement of the signals at
.3 ppm protons for C-11 while irradiation of the singlet
2
at 6.61 assigned to the C-4 proton gave significant
enhancement of the signals at 2.8 ppm for C-6 protons.
Thus, when the resonance at 6.91 ppm in 4 was irradi-
ated, enhancement of the signal at 2.89 ppm for C-6 was
observed. These results strongly suggest that the me-
thylimidazole reacts with 2,3-EQ by 1,6-Michael addition
resulting in the formation of the C-1 adduct.
F igu r e 2. Products obtained from the reaction of 2,3-estrone
o-quinone with ethylphenol or acetic acid in the presence of
Et N, as well as in the presence or absence of Et N.
3 3
°C gave about 70% of the quinone which underwent
transformation within a few minutes to the same prod-
ucts as the temperature increased. Thus, it seems that
during the synthesis of 2,3-EQ a considerable amount of
chemistry takes place that leads to the formation of
several products including 2, 5, 6, and 9. The structures
of 2 and 5 were found to be identical to those obtained
from the reaction of methylimidazole with 2,3-EQ.
Compound 9 was found to be dimeric, and its structure
was deduced from UV, MS, and NMR data. The mass
spectrum of 9 showed a molecular ion at 566 (ES)
corresponding to a molecular mass of the proposed
structure and is consistent with 1,4 C-C coupling of
estrone quinone. Further support for this was obtained
It is interesting to note that, under the conditions used
in the present study, no C-4 imidazolo addition product
was observed. It is quite likely that the C-4 adduct was
formed but is sufficiently labile that the reaction may be
readily reversible. In contrast, the C-1 adduct is readily
isolable and is relatively stable toward retro-Michael
reactions, although we ultimately had to stabilize the
compound as the diacetate. Alternatively, the C-4 adduct
may not be formed due to differences in electrostatic
factors leading to increased reactivity at the C-1 relative
to the C-4 position even though C-1 is more sterically
hindered. At present, we cannot give a satisfactory
explanation for these observations. Furthermore, while
it is quite possible that either the N-1 or N-3 of meth-
ylimidazole adds to the C-1 position of 2,3-EQ, we propose
that the N-1 position is involved in bond formation as
shown for compound 3. This rationale is based on earlier
studies on the reaction of the N-1 methylimidazole
position and the C-1 position of 3,4-EQ as fully charac-
terized using X-ray analysis of 1-(4-methylimidazolo)-4-
hydroxyestrone (19).
+
from FAB MS which showed peaks at 567 (FAB ) and
-
565 (FAB ). In a separate experiment with a crude
+
sample of 9, FAB MS showed the presence of an
additional small peak at 849 indicating the presence of
a trimer, which lends further support to 1,4 C-C coupling
of the quinone. Proton NMR of 9 showed the presence
of two singlets and two doublets in the aromatic region.
The singlets at 7.34 and 7.20 ppm were assigned to the
C-1′ and C-4 protons, respectively, whereas the doublets
at 7.52 and 7.14 ppm were assigned to the C-6′ and C-6
protons, respectively. The NMR also showed two ex-
changeable protons at 5.92 and 5.82 ppm assigned to the
two phenolic -OH groups. The UV spectrum showed
absorption bands at 222, 278, and 420 nm which are
consistent with the structure of compound 9. Formation
of this compound probably involved 1,4 C-C coupling
leading to the quinone intermediate which undergoes
isomerization to give ultimately the quinone methide
product 9. While previous studies in our laboratory failed
to show the formation of quinone methide from either
3,4-EQ or 2,3-EQ, it is quite possible that they may have
been formed but were undetected. Recent studies by
Bolton’s group (35) have shown the formation of quinone
methide intermediates from oxidation of the 2,3-dihy-
droxy-5,6,7,8-tetrahydronaphthalene, which represent
the A/B ring skeleton of 2,3-catechol estrogen.
In the reaction of 2,3-EQ with ethylphenol (Figure 2),
a trace amount of triethylamine was used to ionize the
phenolate as previously used in our studies with 3,4-EQ
(19). Although several products were formed, isolation
and identification of the products using spectral methods
showed no Michael addition of the phenolate. When
acetic acid was reacted with 2,3-EQ in the presence of
Et
3
N, the same products were obtained as in the pheno-
late reaction. These results suggested that the reactions
did not involve the nucleophiles, which led us to inves-
tigate further the reactivity and stability of 2,3-EQ and
2
,3-catechol estrone under different experimental condi-
tions. Thus, when 1 was treated with trace amounts of
Et N in CH Cl , the same reaction products were ob-
3
2
2
served as detected by TLC analysis. During the synthe-
sis of 2,3-EQ from oxidation of 2-aminoestrone as de-
scribed by Stubenrauch and Knuppen (23) or by oxidation
Compound 6 was isolated using a combination of
column chromatography and TLC, and even though it
was not very stable, sufficient quantities were available
to carry out preliminary characterization of the com-
pound. Mass spectral data showed a compound that has
of 2-OHE
sentially the same reaction products were formed, albeit
with different yields. Furthermore, oxidation of 2-OHE
with MnO in extremely dry CH Cl conditions at -70
1 2
with MnO (24) at room temperature, es-
1
2
2
2

8
64 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Tabakovic et al.
MS and EI) which is consistent with the proposed
structure of 8.
In the present investigation, we used model reactions
carried out in CH
2 2
Cl to delineate possible modes of
reaction of nucleophiles with 2,3-EQ. Since it is quite
possible that the behavior of o-quinones may be different
under physiological conditions, we carried out studies on
the reaction of 2,3-EQ with several nucleophiles, includ-
ing thiol, amine, carboxylate, and phenolate, in a mixture
2 2 2
of CH Cl /DMF/H O (1:1:1). The product profiles for
these reactions were compared using TLC and were
found to be essentially similar to those obtained in
reactions in a methylene chloride solution. Furthermore,
these observations are consistent with our previous
studies on reaction of 3,4-EQ with lysine in an aqueous
solution in which no differences were observed between
the product profiles in organic and aqueous systems (18).
Covalent binding of 2,3-EQ to thiol and imidazole
nucleophiles is consistent with 1,6-Michael addition. On
the other hand, formation of 3 suggests 1,4-Michael
addition of the hydroxyl nucleophile to a methine posi-
tion. This is a unique reaction especially since the
hydroxyl nucleophile could also add to either the C-1 or
C-4 positions. To our knowledge, this constitutes the first
observation for addition of a nucleophile to a ring
junction. Thus, the formation of this intermediate may
provide an additional explanation for the transformation
of the quinone to the catechol as evidenced by the
transformation of 3 to the catechol under mild aqueous
conditions. Attempts at showing a similar reaction with
F igu r e 3. ORTEP drawing of 7 with crystallographic number-
ing system. Ellipsoids representing the non-hydrogen atoms
are shown at the 50% probability level.
a molecular weight of 302, suggesting the addition of a
water molecule to the estrogen quinone. NMR data
showed the presence of a singlet at 6.06 ppm and a
doublet at 6.10 ppm assigned to the C-1 and C-4 protons,
respectively. However, this compound was found to
decompose even in the NMR tube. Thus, in order to
obtain a pure sample of 6, we carried out a separate study
in which the reaction mixture was acetylated immedi-
ately using pyridine/acetic anhydride and the mixture
worked up in the usual manner. Isolation and purifica-
tion of the acetylated mixture gave two acetylation
products of 6. Compound 7 was obtained as a white solid
which was crystallized from acetone/hexane to provide
the analytically pure compound which was characterized
3
,4-EQ have not been successful. Whether this observa-
tion is unique to 2,3-EQ remains to be elucidated.
It is worthwhile here to analyze the differences be-
tween the two catechol estrogens. Exogenous 4-hydroxy-
estrogens are more carcinogenic than 2-hydroxyestrogens
in hamsters (29). This observation has been proposed
to be not due to differences in the reactivities of the
catechol estrogen toward cellular nucleophiles, since their
respective reactivities in chemical or biological systems,
such as alkylation, oxidation to quinones, or formation
of conjugates, appear to be comparable (36), but rather
due to reduced bioavailability of 2-hydroxyestrogens to
the target tissues as a result of increased metabolic
methylation and clearance (29). While these observations
may partly explain the differences between the catechol
estrogens, other factors obtained from more recent stud-
ies may equally play an additional role in these differ-
ences. Although previous reports have suggested no
differences in reactivities between the catechol estrogens
(36), recent results by Bolton’s group (37) showed that
2,3-EQ was found to be 17-fold more reactive than 3,4-
EQ, thus confirming previous qualitative reports of
reactivity differences between the two o-quinones (38).
These results as well as those reported in this and
previous investigations lead us to suggest that a relation-
ship may exist between the stability and reactivity of the
estrogen quinones and their carcinogenic potential. It
is tempting to postulate that the lack of carcinogenicity
1
using UV, IR, H-NMR, and mass spectral studies. The
proton NMR spectrum of 7 revealed the presence of a
quinol product with two singlet peaks in the aromatic
region at 6.49 and 6.02 ppm assigned to the C-4 and C-1
protons, respectively. The assigned positions for the C-4
1
and C-1 protons were supported by H NOE difference
studies. Irradiation of the resonance at 6.49 ppm as-
signed to the C-4 proton showed a positive NOE effect
on the signal at 2.83 ppm, while irradiation of the
resonance at 6.02 ppm assigned to the C-1 proton showed
no enhancement of the signal at 2.83 ppm. The NMR
also showed an exchangeable proton at 2.83 ppm as-
signed to an OH group and a singlet at 2.26 ppm assigned
to the methyl protons of the acetate group. These results
suggested the presence of a secondary acetoxy group, two
aromatic protons, and another hydroxyl group which was
assigned to the C-5 position. These studies, however,
could not differentiate between the R or â orientation of
the C-5 OH group. Conclusive evidence for the structure
of 7 was obtained from X-ray crystallographic analysis
performed on a single crystal of 7. Figure 3 is an ORTEP
drawing of 7 which shows the results of 1,4-Michael
addition in which the hydroxyl nucleophile attacks the
C-5 position of 2,3-EQ from the R-face. The X-ray
structural data for 7 showed that this product crystallizes
as an acetone solvate, which in fact gave us considerable
difficulty in the interpretation of the NMR data of the
crystalline product obtained following recrystallization
from acetone/hexane. Crystallographic data for 7 are
summarized in Table 1 (see Experimental Procedures).
Compound 8 was identified as the diacetyl derivative of
of 2,3-OHE may be related to the increased reactivity
2
and decreased stability of the quinone under physiological
conditions. In contrast, the greater stability of 3,4-EQ,
as evidenced by our ability to obtain this compound in
crystalline form (24), may allow more time for diffusion
of the reactive intermediate away from the site of
formation to interact with vital cellular macromolecules,
leading to its high carcinogenicity relative to 2,3-EQ.
Thus, a complex interaction among many factors, includ-
6
. The presence of the two acetyl moieties was demon-
strated by the signals at 2.27 ppm for the C-3 acetyl
group and at 2.06 ppm for the C-5 acetyl group. The
mass spectral data gave a molecular ion peak at 386 (ES

2
,3-Estrone Quinone
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 865
ing the rate of formation of reactive intermediates, their
bioavailability, stability, and inherent reactivity, deter-
mines the extent of an estrogen’s cytotoxicity. Conse-
quently, the ability of a reactive metabolite to diffuse
could be one important factor leading to its direct
carcinogenicity.
(14) Abul-Hajj, Y. J ., and Cisek, P. L. (1986) Regioselective reaction
of thiols with catechol estrogens and estrogen-o-quinones. J .
Steroid Biochem. 25, 245-247.
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4
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Adv. Exp. Med. Biol. 177, 423-434.
In conclusion, data have been presented concerning the
reactivity of 2-OHE and 2,3-EQ leading to the formation
1
(
17) Hurrell, R. F., Finot, P. A., and Cuq, J . L. (1982) Protein-
polyphenol reactions. I. Nutritional and metabolic consequences
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of Michael addition adducts in reactions of 2,3-EQ
involving imidazole. On the other hand, reactions under
mildly basic conditions lead to dimerization and addition
of hydroxyl nucleophile to the C-5 position of 2,3-EQ. Our
results suggest that the increased reactivity and de-
creased stability of 2,3-catechol/quinone estrogens may
provide an additional explanation for their decreased
carcinogenicity.
(18) Tabakovic, K., and Abul-Hajj, Y. J . (1994) Reaction of L-lysine
with estrone-3,4-o-quinone. Chem. Res. Toxicol. 7, 696-701.
(19) Abul-Hajj, Y. J ., Tabakovic, K., Gleason, W. B., and Ojala, W. H.
1996) Reactions of 3,4-estrone quinone with mimics of amino acid
(
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(
20) Purdy, R. H., Moore, P. H., J r., Williams, M. C., Goldzieher, J .
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3
differentiated metabolism of [6,7- H] 17â-oestradiol in rats: Male-
Ack n ow led gm en t. This research was supported in
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(
(
(
Su p p or tin g In for m a tion Ava ila ble: The X-ray crystal-
lographic data for compound 7 (24 pages), contained in many
libraries on microfiche, immediately follows this article in the
microfilm version of the journal, and can be ordered from the
ACS (see any current masthead page for ordering information).
(25) NIH Guidelines for the Laboratory Use of Chemical Carcinogens
1981) NIH Publication No. 81-2385, U.S. Government Printing
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Office, Washington, DC.
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