Reactions of 3,4-estrone quinone with mimics of amino acid side chains

Article abstract of DOI:10.1021/tx950146p

Reaction of 3,4-estrone o-quinone (3,4-EQ) with several amino acid side chain mimics, including 4-ethylphenol, 4-methylimidazole, acetic acid, and propanethiol, gave a mixture of several products including the catechol, Michael addition products, and dime

Full text of DOI:10.1021/tx950146p

434
Chem. Res. Toxicol. 1996, 9, 434-438
Rea ction s of 3,4-Estr on e Qu in on e w ith Mim ics of Am in o
Acid Sid e Ch a in s
Yusuf J . Abul-Hajj,*,† Katmerka Tabakovic,† William B. Gleason,†,‡ and
William H. Ojala‡
Department of Medicinal Chemistry and Biomedical Engineering Center and Department of
Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455
Received August 21, 1995X
Reaction of 3,4-estrone o-quinone (3,4-EQ) with several amino acid side chain mimics,
including 4-ethylphenol, 4-methylimidazole, acetic acid, and propanethiol, gave a mixture of
several products including the catechol, Michael addition products, and dimeric products of
the catechol. On the other hand, several other amino acid side chain mimics, including ethanol,
acetamide, 1-ethylguanidine, and 3-methylindole, did not result in any addition products or
catechol formation. Michael additions to 3,4-EQ with 4-methylimidazole, acetate, and 4-ethyl
phenoxide resulted in 1,4-addition, leading to C-1 adducts while reaction with propanethiol
gave the C-2 addition product.
radical, and the semiquinone of 3,4-EQ. Although pro-
duction of reactive oxygen species (ROS) through redox
cycling has been implicated in damage to macromol-
ecules, including DNA, RNA, and proteins (15), the
assignment of the actual ROS by 3,4-EQ-induced DNA
damage awaits further study.
In tr od u ction
Most chemical carcinogens exert their activity either
through covalent interaction of a reactive metabolite with
DNA in the target organ (1) or by modulating one or more
of a variety of biochemical and biological steps related
to the process of tumor formation. Carcinogenic hor-
mones are widely believed to belong to the latter group
of chemicals because of their proliferative effect on target
cells. Estrogens have been shown to induce mammary,
pituitary, cervical, and uterine tumors in rats, mice, and
guinea pigs (2). Estradiol and other estrogens induce
renal carcinoma in 80-100% of Syrian hamsters within
6-8 months (3). Although the exact mechanism for
carcinogenesis induced by estrogenic compounds is not
fully understood, it is generally believed that metabolic
activation of estradiol leading to the formation of catechol
estrogens is a prerequisite for its genotoxic activity (4).
It has been proposed that the steroid estrogens may
generate reactive intermediates, particularly arene ox-
ides (5-8) and quinones/semiquinones, during their
metabolism, in analogy to the metabolism of aromatic
polycyclic hydrocarbons which are known to be implicated
in carcinogenesis (9).
The estrogen o-quinones produced by the oxidation of
catechol estrogens by phenol oxidase (10), prostaglandin
H synthase (11), and cytochrome P-450 oxidase (12) have
the potential to be cytotoxic and genotoxic. These
compounds can undergo one-electron redox cycling re-
sulting in the formation of semiquinone, superoxide
anion, and hydroxyl radical. Studies in our laboratories
have shown that 3,4-EQ1 was capable of inducing specific
DNA damage in a human breast cancer cell line (13).
These studies showed that 3,4-EQ is unique among
o-quinones in that it causes exclusively single strand
DNA breaks/alkali-labile sites. Furthermore, studies by
Nutter et al. (14) provided conclusive evidence document-
ing the production of hydrogen peroxide, the hydroxyl
Another potential mechanism which may be involved
in the carcinogenicity/toxicity of estrogens involves ad-
duct formation with DNA and/or proteins. Estrogen
o-quinones/semiquinones and estrogen 1,2-epoxide have
been proposed to be responsible for estradiol’s genotoxic
activities. Studies in our laboratory showed that the
major pathway for irreversible binding of estrogens to
protein involves the estrogen o-quinones/semiquinones
and not the estrogen arene oxides (16). Thus, it is quite
possible that the carcinogenicity/toxicity of estrogens may
be in part due to estrogen o-quinones/semiquinones which
are capable of arylation of protein nucleophiles. Although
our knowledge of conjugation of estrogen o-quinones with
thiols (17-19) and primary amines (20, 21) is well
documented, very little is known about the reactions of
amino acid side chain nucleophiles with estrogen qui-
nones. Considering the many different cellular proteins,
nucleophilic side chains, and reactive estrogen metabo-
lites which potentially contribute to total covalent binding
of estrogens, it seems quite likely that some other
nucleophilic centers may be involved in protein arylation
which could change protein structure and cellular func-
tion, resulting in a toxic response. This paper reports
on the reactions of estrogen o-quinones with amino acid
side chain mimics to provide us with an understanding
of the chemistry of conjugate addition of various nucleo-
philes to estrogen quinones.
Exp er im en ta l P r oced u r es
Ch em ica ls. Estrone was purchased from Steraloids (Wilton,
NH). 4-Ethylphenol, 4-methylimidazole, 1-propanethiol, 3-meth-
ylindole, and 1-ethylguanidine sulfate were obtained from
Aldrich Chemical Co. (Milwaukee, WI). Acetamide was obtained
from Sargent-Welch Scientific Co. (Skokie, IL). Deuterated
solvents were purchased from Aldrich Chemical Co. The
synthesis of the catechol estrogens was carried out as described
by Stubenrauch and Knuppen (22). The estrogen o-quinones
* 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, February 1, 1996.
1
Abbreviations: 3,4-estrone quinone, 3,4-EQ; reactive oxygen spe-
cies, ROS; 4-hydroxyestrone, 4-OHE1; triethylamine, Et3N.
0893-228x/96/2709-0434$12.00/0 © 1996 American Chemical Society
+
+

3,4-Estrone Quinone
Ta ble 1. Cr ysta l Da ta for Com p ou n d 3
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 435
in CH2Cl2 (10 mL) and stirred at room temperature. After 30
min there was no quinone left (checked by TLC), and product 3
precipitated as a white solid. The solid was filtered off, washed
with CH2Cl2, and recrystallized from THF: mp 288-290 °C dec;
Rf 0.04 in 25% EtOAc in benzene and Rf ) 0.17 in 25% benzene
in EtOAc; UV (CH2Cl2) λmax (nm) 241, 286; IR (KBr) cm-1 3400,
2935, 2927, 2890, 1736, 1617, 1617, 1507, 1405, 1384; 1H-NMR
(CDCl3) δ 7.93 (s, 1H, H-2 imidazole), 6.85 (s, 1H, H-2), 6.65 (s,
1H, H-5 imidazole), 5.30 (s, 2H, OH, D2O exchangeable), 2.28
(s, 3H, CH3 imid.), 0.85 (s, 3H, 18-CH3); EI MS m/ z 366.2 (M+,
100), 351 (2.55), 325 (22.6), 310 (10.8). These data are in
agreement with the proposed structure of 3. The mother liquid
was evaporated to dryness and applied to a silica gel column.
Elution of the column with 25% EtOAc in benzene gave
4-hydroxyestrone 2: mp 263-265 °C [lit. mp 260-265 °C (22)],
Rf ) 0.56 in 25% EtOAc in benzene. Further elution of the
column gave trace amounts of an orange-yellow solid 4: mp
242-245 °C dec; Rf ) 0.34 in 25% EtOAc in benzene; UV
(CH2Cl2) λmax (nm) 229, 466; IR (KBr) cm-1 3440, 2962, 2926,
1740, 1735, 1660, 1364, 1261, 1097, 1023, 902; 1H-NMR (CDCl3)
δ 7.39 (s, 1H, H-2′), 7.32 (d, 1H, H-2, J ) 8 Hz), 7.22 (d, 1H,
H-1, J ) 8 Hz), 5.32 (bs, 1H, OH), 3.00-1.42 (30 H, m,
methylene and methine protons), 1.22 (s, 3H, 18′-CH3), 0.92 (s,
3H, 18-CH3); high resolution EI MS m/ z (obsd) 568.2836 (M+,
9), 286 (100), 284 (83.6), 269 (31), 267 (11), 226 (19.3); (calcd)
568.7160.
parameter
empirical formula
compound
C26H34O4N2
438.57
formula wt (amu)
crystal color and habit
cryst dimensions (mm)
cryst system
a (Å)
colorless needle
0.50 × 0.10 × 0.10
orthorhombic
10.386(2)
22.577(3)
10.177(2)
2386.5(6)
4
b (Å)
c (Å)
V (Å3)
Z
space group
calcd density (g/cm3)
F000
P212121 (#19)
1.220
944
µ(Cu KR) (cm-1
)
6.23
no. of measured reflections
total
5053
unique
4398
no. of observed refl. (I > 3.00σ(I))
Rint
2011
0.047
R
0.048
wR
0.051
max e- in final difference map
min e- in final difference map
+0.21 e-/Å3
-0.26 e-/Å3
were synthesized by the oxidation of the catechol estrogens by
activated MnO2 as described previously in our laboratory (23).
Ca u tion : The estrogen o-quinones are potentially hazardous and
were handled in accordance with NIH Guidelines for the
Laboratory Use of Chemical Carcinogens (24).
Rea ction of 3,4-Estr ogen o-Qu in on e (1) w ith 1-P r op a n e-
th iol. 1-Propanethiol (31.8 µL, 0.35 mmol) was added to a
solution of quinone (1, 100 mg, 0.35 mmol) in CH2Cl2 (20 mL)
which resulted in decoloration of the solution in 20 min.
Evaporation of the CH2Cl2 left a solid that showed the presence
of three spots on TLC which were separated using preparative
TLC (25% EtOAc in benzene). The spot having the lower Rf
value was identified as 4-OHE1. The spot with the higher Rf
value gave a light yellow solid of 5: mp 102-103 °C dec; 1H-
NMR (CDCl3) δ 6.96 (s, 1H, H-1), 6.42 (s, 1H, OH, D2O
exchangeable), 5.38 (s, 1H, OH, D2O exchangeable), 2.63 (t, 2H,
-SCH2-), 1.56 (q, 2H, -CH2CH3), 1.00 (t, 3H, CH3), 0.91 (s, 3H,
18-CH3); EI MS m/ z 360.2 (100), 155 (4), 97(3.7).
Ch a r a cter iza tion of Com p ou n d s. All of the compounds
were characterized by nuclear magnetic resonance ([1H]-NMR),
ultraviolet (UV), infrared (IR), and mass spectrometry (MS).
Melting points (uncorrected) were taken on a Fisher-J ohns
apparatus. [1H]-NMR spectra were obtained with a GE-300
MHz NMR spectrometer, and the chemical shift data are
reported in parts per million (δ) downfield from tetramethyl-
silane as an internal standard. Nuclear Overhauser effect
(NOE) difference spectra were recorded by applying a presatu-
ration pulse with a decoupler on resonance and subtracting the
trace from the corresponding reference spectra recorded under
identical conditions but with the decoupler off-resonance. Typi-
cal 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 3 were grown
in tetrahydrofuran. All measurements were made on a Rigaku
AFC6S X-ray diffractometer with graphite monochromated Cu
KR radiation (λ ) 1.54178 Å) using the ω - 2θ 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: 0.94-1.00)
as described by North et al. (25). The structure was solved by
direct methods using SHELXS86 (26) and refined and finished
using the TEXSAN structure analysis package (27). 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.
Syn t h esis of Ca t ech ol E st r ogen s a n d E st r ogen Qu i-
n on es. Synthesis of the 4-OHE1 was carried out as described
by Stubenrauch and Knuppen (22) and the quinone as described
in our laboratory (23). 3,4-EQ (1): 1H-NMR (CDCl3) δ 0.85 (s,
3H, 18-CH3), 6.21 (d, 1H, H-2), 7.16 (d, 1H, H-1). 4-OHE1 (2):
1H-NMR (CDCl3) δ 0.89 (s, 3H, 18-CH3), 6.71-6.75 (dd, 2H, H-1
and H-2).
Rea ction of 3,4-Estr ogen o-Qu in on e (1) w ith 4-Eth yl-
p h en ol. To a solution of 4-ethylphenol (43.01 mg, 0.35 mmol)
in CH2Cl2 (30 mL) was added three drops of (Et)3N and 3,4-
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). Evaporation
of CH2Cl2 left a solid that showed several spots on TLC that
were separated on a silica gel column. Elution of the column
with 25% EtOAc in benzene yielded red-orange product 6, 36
mg: mp 223 °C dec; Rf ) 0.53 in 25% EtOAc in benzene; UV
(CH2Cl2) λmax (nm) 233, 270; IR (KBr) cm-1 3400, 2940, 2926,
1
2840, 1739, 1505, 1363, 1211; H-NMR (CDCl3) δ 7.28 (d, 2H,
arom.), 7.25 (s, 1H, H-2), 6.97 (d, 2H, arom.), 5.40 (s, 2H, OH,
D2O exchangeable), 2.69 (q, 2H, CH2), 1.24 (t, 3H, CH3), 0.97
(s, 3H, 18-CH3); EI MS m/ z 406 (M+, 100), 321 (12), 282 (18),
107 (96). The data are in agreement with the structure of the
proposed compound. Further elution of the column gave 2
followed by compound 4.
R ea ct ion of 3,4-E st r ogen o-Qu in on e (1) w it h Acet ic
Acid . To a solution of quinone (1, 100 mg, 0.35 mmol) in CH2Cl2
(10 mL) and acetic acid (105 µL, 1.75 mmol) was added five
drops of (Et)3N in CH2Cl2 (10 mL), and the mixture was stirred
at room temperature. After 45 min, the reaction was complete
(absence of quinone checked by TLC). The methylene chloride
solution was evaporated to dryness, which showed several spots
on TLC that 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 dark-red product 7: mp
138-140 °C dec; Rf ) 0.38 in 25% EtOAc in benzene; UV
(CH2Cl2) λmax (nm) 232, 274; IR (KBr) cm-1 3420, 2928, 2810,
1739, 1734, 1653, 1190; 1H-NMR (CDCl3) δ 7.28 (s, 1H, H-2),
5.02 (bs, 2H, OH, D2O exchangeable) 2.53 (s, 3H, -COCH3), 0.93
(s, 3H, 18-CH3); high resolution EI MS m/ z (observed) 344.16
Rea ction of 3,4-Estr ogen o-Qu in on e (1) w ith 4-Meth -
ylim id a zole. 4-Methylimidazole (28.90 mg, 0.35 mmol) in
CH2Cl2 (15 mL) was added to quinone (1, 100 mg, 0.35 mmol)
31 (M+, 19.5), 303 (M+ + 2H+ - CH3CO, 19), 302 (M+ + H+
-
+
+

436 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Abul-Hajj et al.
F igu r e 1. Products from the reaction of 3,4-estrone-o-quinone with 4-methylimidazole, 4-ethylphenol, 1-propylthiol, and acetic
acid. *Reagents: a ) ethanol; b ) acetamide; c ) 1-ethylguanidine; d ) 3-methylindole.
CH2CO, 100), 286 (M+ - CH3COO, 21); (calcd) 344.4120. Con-
tinued elution of the column gave a dimeric product of 3,4-
estrogen o-quinone, which gave 1H-NMR and EI MS data
consistent with the structure of 4.
Resu lts a n d Discu ssion
The reactions of 3,4-EQ with several amino acid side
chain mimics gave different levels of complexities in the
spectrum of reaction products. All reactions were nor-
mally complete within 1 h as determined by loss of
quinone. The reaction products were isolated and puri-
fied by a combination of TLC and column chromatogra-
phy and characterized by UV, IR, NMR, and mass
spectral data.
The reaction of 3,4-EQ with 4-methylimidazole was
complete within 1 h and resulted in the formation of one
major compound identified as the Michael addition
product 3 (Figure 1). Trace amounts of compound 2 were
isolated and found to be identical to an authentic sample
of 4-hydroxyestrone, and its formation may involve
reduction of 3,4-EQ either by the catechol adduct 3 or
by the catechol precursors to 4 and its isomers. Com-
pound 3 was formed as a white precipitate, and its
purification involved recrystallization from tetrahydro-
furan and full characterization by UV, IR, NMR, and MS.
The proton NMR spectrum of 3 indicates the presence of
a hydroquinone adduct which showed a singlet peak in
the aromatic region at 6.85 ppm, two OH protons, D2O
exchangeable, as well as two other peaks at 7.93 and 6.65
ppm characteristic of the imidazole group. The appear-
ance of a singlet at 6.85 ppm could not differentiate
between the C-1 and C-2 protons, since the chemical
shifts in CDCl3 for both C-1 and C-2 protons in 4-hy-
droxyestrone (2) are observed at 6.71-6.75 ppm. Thus,
the downfield shift that is observed following addition
at either the C-1 or C-2 position would be difficult to
interpret. Conclusive evidence for the structure of the
imidazolo adduct was obtained from X-ray crystallo-
F igu r e 2. ORTEP drawing of 3 with crystallographic number-
ing system. Ellipsoids representing the non-hydrogen atoms are
shown at the 50% probability level.
graphic analysis performed on a single crystal of 3.
Figure 2 is an ORTEP drawing of 3 which clearly shows
the result of a 1,4-Michael addition in which the N-1
nitrogen of 4-methylimidazole attacks the C-1 position
of 3,4-EQ. The X-ray structural data show that this
product crystallizes as a tetrahydrofuran solvate. Crys-
tallographic data for 3 are summarized in Table 1.
Compound 4a /4b was found to be dimeric, and its
structure was deduced from UV, MS, and NMR data.
This compound was found to be identical to that obtained
previously from reactions of 3,4-EQ with lysine (21).
Products of oxidative coupling can be formed through
either a C-C or C-O bond formation as described
previously (28). Confirmation of the presence of a C-O
+
+

3,4-Estrone Quinone
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 437
bond comes from the NMR spectrum showing one singlet
at 6.06 ppm, which can be attributed to one proton of
the quinone moiety, and two other singlets at 6.13 and
6.32 ppm which are attributed to two aromatic protons
of the catechol moiety. On the basis of the NMR spectra,
it is not possible to discriminate between 4a and 4b
which could arise through C-O coupling reactions. It is
interesting to note that 4 was formed in all reactions in
which addition products and catechol are formed. How-
ever, compound 4 was not obtained following reaction of
3,4-EQ with ethanol, acetamide, ethylguanidine, and
3-methylindole in which no reaction products were
observed. While the exact reasons for this are not fully
understood, it is quite likely that a small amount of
reduction of 3,4-EQ by any of the Michael adduct cat-
echols would give 2, which under the reaction conditions
would partly Michael add to 3,4-EQ to give the catechol
precursors to 4, which in turn would be oxidized to 4 by
3,4-EQ, thereby regenerating 2. This redox interchange
chemistry is consistent with the effect of heteroatom
substituents on the quinone/catechol redox potential.
Alternatively, a one-electron transfer from the reactant
via a charge-transfer complex will result in oxidative
phenolic coupling through a C-C or C-O bond formation
(29). Studies by Murty and Penning (30) on reactions of
1,2-naphthoquinone with cysteine showed that none of
their products corresponded to either the bis-thiol ad-
ducts or coupled phenol products, although they indicated
that it is possible that they were formed in minor
quantities. Therefore, it is quite conceivable that the
differences in these results may be related to the struc-
tural differences and/or the reactivities of the estrogen
o-quinones and the o-naphthoquinone.
In the reaction of 3,4-EQ with propanethiol, one major
product 5 and two minor products 2 and 4 were isolated
and identified using UV, IR, NMR, and MS data. Com-
pound 5 was isolated, purified, and recrystallized to give
a light yellow solid. The proton NMR spectrum of 5
showed the presence of the propyl group, two OH protons
at 6.42 and 5.38 ppm, D2O exchangeable, and a singlet
at 6.96 for the C-1 proton which shifted to a lower
magnetic field due to the presence of an adjacent thiol
group. Support for 1,6-Michael addition of thiols to 3,4-
EQ is based on earlier studies by J ellinck (19) and us
(17) in which thiols were found unequivocally to add at
C-2 using specifically labeled [1-3H]- and [2-3H]-4-hy-
droxyestrogens.
found in high enough purity to permit further charac-
terization. Spectral data obtained for the red-brown
adduct 7 are consistent with a 1,4-Michael addition of
the acetate nucleophile. Proton NMR showed the pres-
ence of a singlet at 7.28 ppm attributed to the C-2 proton
(supported by NOE studies as discussed in the next
paragraph), a broad singlet at 5.02 for OH, D2O exchange-
able, and a singlet at 2.53 ppm corresponding to the
carbonyl methyl.
In order to identify the regiochemistry of adduction to
3,4-EQ by acetate and phenoxide, we carried out detailed
NOE difference spectra on 3, 5, 6, and 7. Since the
structures of 3 and 5 are unequivocally established, NOE
studies on these two compounds were found extremely
helpful in assigning the regiochemistry of acetate and
phenoxide addition to 3,4-EQ. Irradiation of the reso-
nance at 6.85 ppm in 3 assigned to the C-2 proton in ring
A did not enhance the signal for protons at C-11, which
is consistent with C-1 substituted 3,4-catechol estrogens.
On the other hand, when the singlet at 6.96 ppm in 5
was irradiated, a significant enhancement of the signals
at 2.37 ppm for C-11 protons was observed. These
studies support C-2 addition of thiols and are consistent
with our previous results using tritium labeling experi-
ments (17). When the singlet resonances at 7.25 and 7.28
ppm in 6 and 7, respectively, were irradiated, no en-
hancement of the C-11 protons was observed. These
results suggest that the acetate and phenoxide react with
3,4-EQ by 1,4-Michael addition, resulting in the forma-
tion of the C-1 adducts. While the exact reasons for the
differences in addition of various nucleophiles are not yet
fully understood, the results obtained from this investi-
gation showed that “hard” nucleophiles add to C-1 of 3,4-
EQ while “soft” nucleophiles add to the C-2 position of
3,4-EQ. These results are supported by our preliminary
molecular orbital calculation studies, which showed that
charge density data predict that C-1 is a “hard” electro-
phile while C-2 is a “soft” electrophile. These observa-
tions are essentially in agreement with other reports on
the addition of nucleophiles to o-quinones (31-33).
It is interesting to note that while reactions of 3,4-EQ
with phenol, acetate, thiol, and imidazole lead to addition
products, several other amino acid side chain mimics,
including ethanol, acetamide, ethylguanidine, and meth-
ylindole, did not result in any addition products or
catechol formation (Figure 1) which may be explained by
the decreased nucleophilicity of these groups.
Reaction of 3,4-EQ with 4-ethylphenol was extremely
slow, and only traces of products were observed. Since
the phenolic group in tyrosine may be in the ionized form
in biological systems, reactions of 3,4-EQ with 4-eth-
ylphenol were carried out in the presence of triethylamine
and were complete within 30 min. Three products, 6, 2,
and 4a /4b, were isolated and characterized. Proton NMR
of 6 showed two AB doublets at 7.28 and 6.97 ppm
assigned to the aromatic phenol, a triplet at 1.24 ppm
corresponding to the methyl group in the ethyl side chain
and a singlet at 7.25 ppm attributed to the C-2 proton of
the steroid. Support for 1,4-Michael addition of the
phenoxide is obtained from NOE studies (see next
paragraph). Mass spectra showed a molecular ion (100%)
at m/ z 406 which supports the structure proposed for 6.
Reactions with 3,4-EQ and acetic acid were also con-
ducted in a manner identical to that described for
4-ethylphenol in which triethylamine was used in the
reaction mixture. As before, a number of reaction
products including 2, 7, and 4a /4b were isolated and
It is now well established that reactive intermediates
play an important role in the cytotoxicity of chemicals
and drugs. The fact that estrogens are metabolized to
catechols (34), which are in turn oxidized to the o-
quinones/semiquinones (35), may provide an explanation
for their toxicity/carcinogenicity by a number of mecha-
nisms including redox cycling and the generation of ROS
as well as arylation of macromolecules (36). Studies in
our laboratory are continuing to investigate the impor-
tance of redox cycling in the carcinogenicity of 3,4-EQ
(13, 14, 37). Covalent binding of estrogen quinones to
proteins may inhibit essential functions of the enzyme(s)
or regulatory protein(s), which may ultimately lead to
their toxic effect. Preliminary studies in our laboratory
showed that estrogen quinones react with bovine serum
albumin by covalent binding to possibly several nucleo-
philic centers. Previous studies have shown that the
primary sites of covalent modification of proteins by
alkylating agents include cysteine SH, histidine N (of the
imidazole), and the N-terminal NH2 group and the
+
+

438 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Abul-Hajj et al.
(14) Nutter, L. M., Wu, Y.-Y., Ngo, E. O., Sierra, E. E., Gutierrez, P.
L., and Abul-Hajj, Y. J . (1994) An o-quinone form of estrogen
produces free radicals in human breast cancer cells: Correlation
with DNA damage. Chem. Res. Toxicol. 7, 23-28.
(15) Liehr, J . G., and Roy, D. (1990) Free radical generation by redox
cycling of estrogens. Free Radical Biol. Med. 8, 415-423.
(16) Abul-Hajj, Y. J . (1985) Estrogen 1,2-epoxides or estrogen quinones/
semiquinones. Biochem. Biophys. Res. Commun. 133, 1078-1085.
(17) Abul-Hajj, Y. J ., and Cisek, P. L. (1988) Catechol estrogen adducts.
J . Steroid Biochem. 31, 107-110.
(18) 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.
(19) J ellinck, P. H. (1988) 4-Hydroxyestradiol is conjugated with thiols
primarily at C-2: Evidence from regio-specific displacement of
tritium by cat liver microsomes or tyrosinase. Steroids 51, 395-
409.
(20) Khasnis, D., and Abul-Hajj, Y. J . (1994) Estrogen quinones:
Reaction with propylamine. Chem. Res. Toxicol. 7, 68-72.
(21) Tabakovic, K., and Abul-Hajj, Y. J . (1994) Reaction of lysine with
estrone 3,4-o-quinone. Chem. Res. Toxicol. 7, 696-701.
(22) Stubenrauch, G., and Knuppen, R. (1976) Convenient large scale
preparation of catechol estrogens. Steroids 28, 733-741.
(23) Abul-Hajj, Y. J . (1984) Synthesis of 3,4-estrogen-o-quinones. J .
Steroid Biochem. 21, 621-622.
ꢀ-amino group of lysine (38). Whether the estrogen
quinones can also modify proteins at these sites as well
as other sites remains to be elucidated.
The results obtained from this investigation as well as
earlier studies show that model compounds representing
the amino acid side chains of cysteine, lysine, histidine,
aspartic acid, glutamic acid, and tyrosine have been found
to react with estrogen o-quinones, leading to the forma-
tion of catechol estrogen adducts. On the other hand,
amino acid side chain mimics of arginine, serine, tryp-
tophan, asparagine, and glutamine were found not to be
involved in adduct formation. Since it is quite possible
that the cytotoxicity of the estrogens could depend to a
large degree on metabolic conversion to the reactive
o-quinone/semiquinone intermediates, the reactions of
amino acid side chain mimics described in the present
in vitro study might well occur as well in vivo. Studies
with model proteins containing a representative array
of amino acids are currently being conducted.
(24) NIH Guidelines for the Laboratory Use of Chemical Carcinogens
(1981) NIH Publication No. 81-2385, U.S. Government Printing
Office, Washington, DC.
Ack n ow led gm en t. This research was supported in
part by the National Cancer Institute, Grant CA57615.
(25) North, A. C. T., Phillips, D. C., and Mathews, F. S. (1968) A
semiempirical method of absorption correction. Acta Crystallogr.,
Sect. A 24, 351-359.
Refer en ces
(26) SHELXS 86 (1985) Program for the solution of crystal structures,
University of Go¨ttingen, Germany.
(27) TEXSAN-TEXRAY (1985) Structure Analysis Package, Molecular
Structure Corp., The Woodlands, TX.
(1) Lutz, W. K. (1979) In vivo covalent binding of organic chemicals
to DNA as a quantitative indicator in the process of chemical
carcinogenesis. Mutat. Res. 65, 289-356.
(28) Ins, A., Meroni, G., and Ferrara, L. (1977) Two dimers, 4:4′- and
2:2′-di(estradiol), obtained by chemical oxidative coupling of
estradiol. J . Steroid Biochem. 8, 1259-1261.
(29) Becker, H. D., and Turner, A. B. (1988) Quinones as oxidants and
dehydrogenation agents. In The Chemistry of Quinonoid Com-
pounds (Patai, S., and Rappaport, Z., Eds.) Part 2, pp 1351-1385,
Wiley, New York.
(2) IARC Group (1979) IARC Monographs on Evaluation of Car-
cinogenic Risk of Chemicals to Humans, Sex Hormones (II), Vol.
21, pp 173-221, International Agency for Research on Cancer,
Lyon, France.
(3) Kirkman, H. (1984) Estrogen-induced tumors of the kidney in the
Syrian hamster. III. Growth characteristics in the Syrian ham-
ster. Natl. Cancer Inst. Monogr. 1, 1-57.
(4) Li, J . J ., and Li, S. A. (1984) Estrogen induced tumorigenesis in
the Syrian hamster: roles for hormonal and carcinogenic activi-
ties. Arch. Toxicol. 55, 110-118.
(5) Nambara, A., and Honma, S. (1971) C-2 hydroxylatin of 2-deu-
terioestrogen in the rat. Lack of “NIH Shift.” Chem. Pharm. Bull.
(Tokyo) 19, 1727-1730.
(6) LeQuesne, P. W., Durga, A. V., Subramanyan, V., Soloway, A.
H., Hart, R., and Purdy, R. H. (1980) Biomimetic synthesis of
catechol estrogens: Potentially mutagenic arene oxide intermedi-
ates in estrogen metabolism. J . Med. Chem. 23, 239-240.
(7) Metzler, M. (1974) Metabolism of stilbene estrogens and steroidal
estrogens in relation to carcinogenicity. Arch. Toxicol. 55, 104-
109.
(8) Gladek, A., and Liehr, J . G. (1989) Mechanism of genotoxicity of
diethylstilbesterol in vivo. J . Biol. Chem. 264, 16847-16852.
(9) Slaga, T. J ., Bracken, W. J ., Gleason, G., Levin, W., Yagi, H.,
J erina, D. M., and Conney, A. H. (1979) Marked differences in
the skin tumor-initiating activities of the optical enantiomers of
the diastereomeric benzo(a)pyrene 7,8-diol-9,10-epoxide. Cancer
Res. 39, 67-71.
(10) Kalyanaraman, B., Felix, C. C., and Sealy, R. C. (1985) Semi-
quinone radicals of catecholamines, catecholestrogens, and their
metal ion complex. Environ. Health Perspect. 64, 185-198.
(11) Degen, G. H. (1990) Role of prostaglandin-H synthase in mediat-
ing genotoxic and carcinogenic effects of estrogens. Environ.
Health Perspect. 88, 217-223.
(12) Capdevila, J ., Sack, Y., and Falck, J . R. (1984) The mechanistic
plurality of cytochrome P-450 and its biological ramifications.
Xenobiotica 14, 105-118.
(30) Murty, V., and Penning, T. M. (1992) Polycyclic aromatic hydro-
carbon (PAH) ortho-quinone conjugate chemistry: Kinetics of thiol
addition to PAH ortho-quinones and structures of thioether
adducts of naphthalene-1,2-dione. Chem. Biol. Interact. 84, 169-
188.
(31) Penning, T. M. (1993) Dihydrodiol dehydrogenase and its role in
polycyclic aromatic hydrocarbon metabolism. Chem. Biol. Interact.
89, 1-34.
(32) Monks, T. J ., Hanzlik, R. R., Cohen, G. M., Ross, D., and Grahams,
D. G. (1992) Contemporary issues in toxicology. Quinone chem-
istry and toxicology. Toxicol. Appl. Pharmacol. 112, 2-16.
(33) Tse, D. C. S., McCreery, R. L., and Adams, R. N. (1976) Potential
oxidative pathways of brain catecholamines. J . Med. Chem. 19,
37-40.
(34) Fishman, J . (1963) Role of 2-hydroxyestrone in estrogen metabo-
lism. J . Clin. Endocrinol. Metab. 23, 207-210.
(35) Marks, F., and Hecker, F. (1969) Metabolism and mechanism of
action of estrogens. XII. Structure and mechanism of formation
of water-soluble and protein-bound metabolites of estrone in rat-
liver microsomes in vitro and in vivo. Biochim. Biophys. Acta 187,
250-265.
(36) O’Brien, P. J . (1991) Molecular mechanisms of quinone toxicity.
Chem.-Biol. Interact. 80, 1-41.
(37) Nutter, L. M., Zhou, B., Sierra, E. E., Wu, Y.-Y., Rummel, M. M.,
Gutierrez, P., and Abul-Hajj, Y. J . (1994) Cellular biochemical
determinants modulating the metabolism of estrone 3,4-quinone.
Chem. Res. Toxicol. 7, 609-613.
(38) Ellis, M. K., Golding, B. T., and Watson, W. P. (1984) Intrinsic
reactivities in the alkylation of protected amino acids by (R)- and
(S)-methyl-oxirane. J . Chem. Soc., Perkin Trans. 2, 1737-1743.
(13) Nutter, L. M., Ngo, E. O., and Abul-Hajj, Y. J . (1991) Character-
ization of DNA damage induced by 3,4-estrone-o-quinone in
human cells. J . Biol. Chem. 226, 16380-16386.
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