Chemical synthesis of two novel diaryl ether dimers of estradiol-17β

Article abstract of DOI:10.1016/j.steroids.2003.10.003

We recently detected the formation of estradiol-17β (estradiol) dimers, linked together through a diaryl ether bond between the C-3 phenolic oxygen of one estradiol molecule and the 2- or 4-position aromatic carbon of another estradiol, following incubations of [³H]estradiol with human liver microsomes or cytochrome P450 enzymes in the presence of NADPH. Using estradiol as the starting material, we designed a four-step method for the chemical synthesis of these two estrogen dimers with the Ullmann condensation reaction as a key step. Step 1: Synthesis of 2- or 4-bromoestradiol from estradiol. Step 2: Protection of the C-3 phenolic hydroxyl group of the 2- or 4-bromoestradiol. Step 3: The Ullmann condensation reaction between the phenol-protected bromoestradiol and the estradiol potassium salt under our modified reaction conditions (with a 41% product yield). Step 4: Removal of the C-3 benzyl group by catalytic hydrogenation. The chromatographic and various spectrometric properties of the two synthesized compounds were identical to those metabolically formed by human cytochrome P450 3A4.

Full text of DOI:10.1016/j.steroids.2003.10.003

Steroids 69 (2004) 61–65
Chemical synthesis of two novel diaryl ether dimers of estradiol-17␤
Anthony J. Leea, J. Walter Sowella, William E. Cothamb, Bao Ting Zhua,∗
Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA
a
b
Department of Chemistry and Biochemistry, College of Science and Mathematics, University of South Carolina, Columbia, SC 29208, USA
Received 28 August 2003; accepted 8 October 2003
Abstract
We recently detected the formation of estradiol-17␤ (estradiol) dimers, linked together through a diaryl ether bond between the C-3 phe-
nolic oxygen of one estradiol molecule and the 2- or 4-position aromatic carbon of another estradiol, following incubations of [3H]estradiol
with human liver microsomes or cytochrome P450 enzymes in the presence of NADPH. Using estradiol as the starting material, we designed
a four-step method for the chemical synthesis of these two estrogen dimers with the Ullmann condensation reaction as a key step. Step 1:
Synthesis of 2- or 4-bromoestradiol from estradiol. Step 2: Protection of the C-3 phenolic hydroxyl group of the 2- or 4-bromoestradiol. Step
3: The Ullmann condensation reaction between the phenol-protected bromoestradiol and the estradiol potassium salt under our modified
reaction conditions (with a 41% product yield). Step 4: Removal of the C-3 benzyl group by catalytic hydrogenation. The chromatographic
and various spectrometric properties of the two synthesized compounds were identical to those metabolically formed by human cytochrome
P450 3A4.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Estradiol; Nonpolar estrogen metabolites; Estrogen dimers
1. Introduction
these polar estrogen metabolites, 2-methoxyestradiol and
estradiol-17-fatty acid esters are two examples of nonpo-
lar estrogen metabolites that also have unique biological
properties. It is known that 2-methoxyestradiol has strong
growth-inhibitory, apoptotic, and antiangiogenic actions [7].
Recent results have suggested that the naturally occurring
lipoidal estradiol-17-stearate appeared to have a strong,
preferential growth-stimulatory and carcinogenic activity
in the fat-rich mammary tissues over other target organs
(such as the uterus and pituitary), which is different from
the parent hormone estradiol [8,9].
During our recent study of the NADPH-dependent
metabolism of [3H]estradiol by human liver microsomes
and fifteen selectively expressed human CYP isoforms,
we detected ≥20 nonpolar radioactive metabolite peaks
(designated as M1–M20), in addition to a large num-
ber of hydroxylated or keto metabolites (Lee et al., sub-
mitted for publication). Among ∼20 nonpolar estradiol
metabolites detected, M15 and M16 were only selectively
formed by a few of the human CYP isoforms (predom-
inantly CYP3A4 and CYP3A5). The formation of these
two representative nonpolar estrogen metabolites by hu-
man CYP isoforms did not correlate with their overall
catalytic activity for the oxidative metabolism of estra-
diol. The structures of the metabolically formed M15 and
M16 were identified to be the dimers of estradiol, linked
The endogenous estrogens such as estradiol-17␤ (estra-
diol) and estrone are important female gonadal hormones
that have very diverse physiological and pathophysiolog-
ical actions. Although many of the biological actions of
endogenous estrogens are believed to be directly mediated
by the estrogen receptors (namely, the ER-␣ and/or ER-␤
subtypes), there is also mounting experimental evidence
suggesting that some of the unique biological actions of
the endogenous estrogens are exerted by their metabolites.
For instance, 4-hydroxyestradiol is a genotoxic/mutagenic
estrogen metabolite [1–3], and it also appears to have its
own signal-transduction pathway that is different from the
classical ER␣-mediated pathways [4]. This catechol estro-
gen metabolite has been suggested to play an important role
in hormonal carcinogenesis in animal models and humans
[3]. In addition, a few other hydroxyestrogen metabo-
lites (such as 2-hydroxyestradiol, 15␣-hydroxyestradiol,
and 16␣-hydroxyestrone) have also been suggested to
have unique biologic properties [5,6]. In addition to
Abbreviations: Estradiol, estradiol-17␤; HPLC, high-pressure liq-
uid chromatography; TLC, thin-layer chromatography; CYP, cytochrome
P450; NMR, nuclear magnetic resonance
∗
Corresponding author. Tel.: +1-803-777-4802; fax: +803-777-8356.
E-mail address: BTZhu@cop.sc.edu (B.T. Zhu).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2003.10.003

62
A.J. Lee et al. / Steroids 69 (2004) 61–65
2.2. Spectrometric analyses
HO
16'
17'
18'
15'
Mass spectra were recorded by using a VG70S analytical
mass spectrometer. An aliquot of the ethanol solution of the
test compound was used for the direct-probe mass analysis.
The NMR spectra were recorded on a Varian Inova 500 spec-
trometer operating at a proton frequency of 500.21 MHz and
a carbon frequency of 125.79 MHz. Chemical shifts were
given as δ values with reference to CD3OD as an internal
standard.
13'
12'
11'
14'
8'
M15
7'
6'
9'
10'
1'
2'
5'
4'
18
OH
12
17
11
9
3'
13
14
16
1
4
O
10
5
2
15
8
2.3. Synthesis
3
7
HO
6
2.3.1. 4-Bromoestradiol (compound 2) and
2-bromoestradiol (compound 3) [10]
18
OH
12
A solution of estradiol (1.5 g, 5.5 mmol) in chloroform
(250 ml) was treated with N-bromosuccinimide (1.05 g,
5.9 mmol) dissolved in chloroform (150 ml). The mixture
was refluxed for an hour and the solvent was then removed
in vacuo to yield a solid. The crude product was dissolved
in methanol (25 ml) and precipitated by addition of water
(200 ml). The solid was filtered and dried in vacuo. Upon
crystallization from ethanol, 4-bromoestradiol was separated
as short needles which, after recrystallization, afforded the
product with a melting point (m.p.) of 209–211 ◦C in 34%
(yield 654 mg) (literature m.p. value: 213.5–215 ◦C [9]).
After separation of 4-bromoestradiol, the ethanol solution
was dried and the residues were dissolved in acetonitrile.
2-Bromoestradiol was obtained as long rods in 23% yield
(437 mg) with an m.p. of 200–207 ◦C (literature value:
197–198 ◦C [11]).
17
11
13
14
16
1
9
10
2
15
8
7
3
6
5
HO
4
M16
2'
O
3'
1'
11'
10'
4'
9'
12'
5'
6'
18'
8'
13'
OH
14'
15'
7'
17'
16'
Fig. 1. Structures of M15 and M16, two novel diaryl ether dimers of
estradiol-17␤ that were metabolically formed by human CYP enzymes in
the presence of NADPH.
together through a diaryl ether bond between a pheno-
lic oxygen atom of one estradiol molecule and the 2- or
4-position aromatic carbon of another estradiol (struc-
tures shown in Fig. 1). We report here an efficient method
for the chemical synthesis of these two novel estradiol
dimers.
2.3.2. 4-Bromoestradiol 3-O-benzyl ether (compound 4)
A 50-ml round-bottom flask was charged with 4-bromoe-
stradiol (527 mg, 1.5 mmol), anhydrous K2CO3 (1.382 g,
10 mmol) and 25 ml acetonitrile, and then 180 ␮l of benzyl
bromide (1.5 mmol) was also added. The reaction mix-
ture was refluxed with stirring for 1 h. The hot reaction
mixture was then filtered under a reduced pressure, and
the filtrate was concentrated. 4-Bromoestradiol 3-O-benzyl
ether was crystallized as long needles: 463 mg (70%
yield); m.p. 163–164 ◦C; Rf of 0.47 on Silica gel TLC
(toluene/chloroform/ethyl acetate, 1/10/2); molecular weight
(m/z) 440.1342 (440.1351 calculated for C25H29BrO2, with
2.0 ppm error); 1H NMR (CD3OD): 2nd order phenyl (ben-
zyl) multiplets at 7.473 (2H, for H-ortho), 7.355 (2H, for
H-meta), 7.283 (1H, for H-para), 7.228 (d, J = 8.5 Hz, 1H,
for H-1), and 6.683 (d, J = 8.5 Hz, 1H, for H-2) ppm (δ).
2. Experimental
2.1. Chemicals and reagents
N-Bromosuccinimide, benzyl bromide, potassium car-
bonate (K2CO3, anhydrous), 4-picoline (4-methylpyridine),
copper (II) oxide (CuO), anhydrous sodium sulfate
(Na2SO4), 10% palladium on carbon (Pd-C), and Celite
were of ACS grades and purchased from ACROS (through
Fisher Scientific, Atlanta, GA). Estradiol (in ∼50 g of
quantity, used as the starting material for chemical syn-
thesis) and very small amounts (5 mg) of 2-bromoestradiol
and 4-bromoestradiol (used as reference standards) were
purchased from Steraloids (Newport, RI). Methanol-d4
(CD3OD) was obtained from Cambridge Isotope Laborato-
ries, Inc. (Andover, MA).
2.3.3. M16 3-O-benzyl ether (compound 5) [12]
A mixture of 4-bromoestradiol 3-O-benzyl ether (441 mg,
1.0 mmol), estradiol (272 mg, 1.0 mmol), anhydrous K2CO3
(290 mg, 2.1 mmol) in 4-picoline (5 ml) was incubated at
130 ◦C for 3 h, and then CuO (40 mg, 0.5 mmol) was added.
The reaction mixture was heated at 155–160 ◦C with stirring
under argon for 72 h, cooled to room temperatures, diluted

A.J. Lee et al. / Steroids 69 (2004) 61–65
63
with 50 ml ethyl acetate, and vacuum-filtered through Celite.
The filtrate was extracted with 1 N HCl (50 ml) twice to
remove 4-picoline. The organic layer was dried with an-
hydrous Na2SO4 and concentrated under reduced pressure.
The residue was purified by column chromatography eluting
with a gradient from 33% ethyl acetate in hexanes to 100%
ethyl acetate. The fractions were combined, concentrated,
and further purified by column chromatography eluting with
33% ethyl acetate in hexanes. The fractions were combined
and concentrated and the residues were purified by crystal-
lization from ethanol. M16 3-benzyl ether was obtained as
colorless needles: 262 mg (41% yield); m.p. 142–144 ◦C;
Rf of 0.22 on Silica gel TLC (50% ethyl acetate/hexanes);
molecular weight (m/z) 632.3864 (632.3866 calculated for
C43H52O4, 0.3 ppm error); 1H NMR (CD3OD): overlapped
region 7.100–7.197 (5H, for H-1ꢀ, H-meta, and H-ortho),
overlapped region 6.999–7.022 (2H, for H-1 and H-para),
6.903 (d, J = 8.6 Hz, 1H, for H-2), 6.517 (dd, J = 8.6 and
2.6 Hz, 1H, for H-2ꢀ), and 6.444 (d, J = 2.6 Hz, 1H, for
H-4ꢀ) ppm (δ).
2.3.6. M15 3-O-benzyl ether (compound 7) [12]
Into a 50-ml round-bottom flask, 441 mg of crude 2-
bromoestradiol 3-O-benzyl ether (containing 2,4-dibromoe-
stradiol 3-O-benzyl ether), estradiol (272 mg, 1.0 mmol), and
anhydrous K2CO3 (290 mg, 2.1 mmol) in 5 ml 4-picoline
were added. The mixture was first incubated at 130 ◦C for
3 h, and then CuO (40 mg, 0.5 mmol) was added. The reac-
tion mixture was heated at 155–160 ◦C with stirring under ar-
gon for 72 h, cooled to room temperatures, diluted with ethyl
acetate, and vacuum-filtered through Celite. The filtrate was
extracted with 1 N HCl (50 ml) twice to remove 4-picoline.
The organic layer was dried with anhydrous Na2SO4 and
concentrated under a reduced pressure. The residue was pu-
rified by column chromatography eluting with 50% ethyl
acetate in hexanes. The residue was purified again by col-
umn chromatography eluting with 33% ethyl acetate in hex-
anes. The fractions were combined and concentrated and the
yellow-colored residues were not further purified.
2.3.7. M15
Into a Parr flask, crude M15 3-O-benzyl ether in 100 ml
ethanol and Pd-C (500 mg) were added. The reaction mix-
ture was subjected to hydrogen at 40 psi for 4 h and the
catalyst was removed by vacuum filtration through Celite.
The filtrate was concentrated and the residue was dissolved
in hot acetonitrile. During the concentration of acetonitrile
solution with heating, white amorphous powder was ob-
tained as M15: 39 mg; Rf of 0.25 on Silica gel TLC (50%
ethyl acetate/hexanes); molecular weight (m/z) 542.3407
2.3.4. M16
Into a Parr flask, M16 3-benzyl ether (240 mg, 0.38 mmol)
in 100 ml ethanol and Pd-C (500 mg) were added. The re-
action mixture was subjected to hydrogen at 40 psi for 4 h
and the catalyst was removed by vacuum filtration through
Celite. The filtrate was concentrated and the residue was
dissolved in hot acetonitrile. During the concentration of the
acetonitrile solution with heating, white amorphous pow-
der was obtained as M16: 136 mg (66% yield); Rf of 0.22
on Silica gel TLC (50% ethyl acetate/hexanes); molecular
weight (m/z) 542.3387 (542.3396 calculated for C36H46O4,
1.7 ppm of error); 1H NMR (CD3OD): 7.140 (d, J = 8.5 Hz,
1H, for H-1ꢀ), 7.040 (d, J = 8.5 Hz, 1H, for H-1), 6.740 (d,
J = 8.5 Hz, 1H, for H-2), 6.530 (dd, J = 8.5 and 2.5 Hz,
1H, for H-2ꢀ), and 6.470 (d, J = 2.5 Hz, 1H, for H-4ꢀ)
ppm (δ).
1
(542.3396 calculated for C36H46O4, 2.0 ppm of error); H
NMR (CD3OD): 7.178 (d, J = 8.5 Hz, 1H, for H-1ꢀ), 6.773
(s, 1H, for H-1), 6.639 (dd, J = 8.5 and 2.3 Hz, 1H, for
H-2ꢀ), 6.612 (s, 1H, for H-4), and 6.564 (d, J = 2.3 Hz,
1H, for H-4ꢀ) ppm (δ).
3. Results and discussion
2.3.5. 2-Bromoestradiol 3-O-benzyl ether (compound 6)
A 50-ml round-bottom flask was charged with 2-bromoe-
stradiol (527 mg, 1.5 mmol) and anhydrous K2CO3 (1.382 g,
10 mmol) in 25 ml acetonitrile, and then benzyl bromide
(180 ␮l, 1.5 mmol) was added. The reaction mixture was re-
fluxed with stirring for 1 h. The hot reaction mixture was then
filtered under a reduced pressure, and the filtrate was con-
centrated. 2-Bromoestradiol 3-O-benzyl ether was obtained
as white powder (539 mg, 82%). Although TLC analysis
showed a single spot (Silica gel, toluene/chloroform/ethyl
acetate, 1/10/2, Rf = 0.49), further mass spectrometric
analysis showed two molecular weights: one correspond-
ing to 2-bromoestradiol 3-O-benzyl ether (compound 6),
with a molecular weight (m/z) of 440.1339 (440.1351 cal-
culated for C25H29BrO2, 2.7 ppm error), and the other one
corresponding to 2,4-dibromoestradiol 3-O-benzyl ether,
with m/z 520 (its high-resolution mass was not deter-
mined).
The key structural characteristic of M15 and M16 (struc-
tures shown in Fig. 1) is the presence of a diaryl ether bond
between two estradiol moieties. The method commonly used
for preparation of a diaryl ether linkage has been the reaction
between an alkali metal phenolate and a halogenated ben-
zene in the presence of copper (or a copper salt) as catalyst,
which was originally developed by Ullmann and is widely
known as the Ullmann condensation reaction [13–15]. Ac-
cordingly, a four-step synthetic scheme (depicted in Fig. 2)
for the synthesis of M15 and M16 was designed with the
Ullmann condensation reaction as a key step. Step 1: Syn-
thesis of 2- or 4-bromoestradiol from estradiol. Step 2: Pro-
tection of the C-3 phenolic hydroxyl group of the 2- or
4-bromoestradiol. Step 3: The Ullmann condensation reac-
tion of the phenol-protected bromoestradiol and the estradiol
potassium salt under our modified reaction conditions (with
a 41% product yield). Step 4: Removal of the C-3 benzyl
group by catalytic hydrogenation.

64
A.J. Lee et al. / Steroids 69 (2004) 61–65
OH
tected in Step 2. We selected benzyl ether as the protecting
group for the following reasons: (i) its stability under the
alkaline conditions required for the Ullmann condensation
reaction; (ii) its easy removal; and (iii) that the reaction
condition has little or no effect on other parts of the prod-
uct. It should be noted that the hydroxyl group at the C-17
position of 2- or 4-bromoestradiol was not protected since
it is known that the secondary alkyl hydroxyl group is not
ionized during the Ullmann condensation reaction. For pro-
tection, benzyl bromide (in equivalent amount) and K2CO3
(in excess) were added to an acetonitrile solution containing
2- or 4-bromoestradiol and refluxed. Upon recrystallization,
3-O-benzyl ethers of 2- or 4-bromoestradiol (compound 6
or 4) were obtained as powder or long needles, respectively,
and their yields were 70–81%. The structures of the prod-
ucts were confirmed by analyzing their high resolution mass
HO
1
NBS, CHCl3, reflux
OH
OH
Br
HO
HO
3
2
Br
Benzyl bromide, K2CO3,
CH3CN, reflux
OH
OH
Br
O
O
Br
6
4
1
and H NMR spectra (aromatic protons). 4-Bromoestradiol
E2, K2CO3, picoline,
130ºC, 3h then 150ºC, 72h
OH
3-O-benzyl ether (compound 4) was highly pure according
HO
1
to TLC, mass, and H NMR analyses. However, the mass
and NMR spectra of 2-bromoestradiol 3-O-benzyl ether
(compound 6) showed that there were small amounts of
2,4-dibromoestradiol 3-O-benzyl ether present as impurity.
In Step 3, 3-O-benzyl ethers of M16 or M15 (compound
5 or 7) were synthesized by using the Ullmann conden-
sation reaction between estradiol 3-potassium salt and the
3-O-benzyl ether of 4- or 2-bromoestradiol. It has been doc-
umented that the Ullmann condensation usually requires
high temperatures (up to 300 ◦C), long reaction time (up to
3 days), and strong polar solvents [16,17]. Notably, these
harsh reaction conditions often produce competitive side
products such as dehalogenated arene and homocoupled di-
aryls [17]. Although the overall yield of this reaction usu-
ally is around 40–50%, much lower yields have often been
reported in cases involving substituted reactants [12]. Since
the reactants used for chemical synthesis of M15 and M16
are bulky (2- or 4-bromoestradiol 3-O-benzyl ether and the
potassium salt of estradiol), we had chosen to carry out the
Ullmann condensation reaction at 150 ◦C for 3 days in the
presence of CuO as a catalyst [12]. This modified procedure
was adopted after review of the literature on the synthe-
sis of other similar compounds. Also, 4-picoline was used
here as a solvent since it has a higher boiling point (145 ◦C)
and basicity (pKa = 7.96) than the commonly used pyri-
dine (boiling point = 115 ◦C and pKa = 5.19). Under these
reaction conditions and with 4-bromoestradiol 3-O-benzyl
ether (compound 4) as the starting material, a benzyl deriva-
tive of M16 (compound 5) was obtained with a moderate
yield (41%) even after two steps of column chromatogra-
phy and recrystallization. The structure of the product was
O
O
7
5
OH
O
O
OH
H2, Pd-C, EtOH
OH
HO
HO
O
OH
M16
O
HO
OH
M15
Fig. 2. Synthetic scheme for M15 and M16.
In Step 1, estradiol was brominated at the C-2 or C-4
position by reaction of estradiol with N-bromosuccinimide
[10]. Briefly, the chloroform solution of estradiol and
N-bromosuccinimide was refluxed and the solvent was
evaporated to yield a white solid. This solid was then dis-
solved in methanol and precipitated upon addition of wa-
ter. After two times of recrystallization, 4-bromoestradiol
(compound 2) was obtained first as short needles. The
resulting ethanol solution was dried and the residue was
redissolved in acetonitrile. 2-Bromoestradiol (compound
3) was crystallized as long rods. Both compounds were
identified by comparing their m.p. and Rf values on the
TLC with those of the reference standards. The reaction
of estradiol with N-bromosuccinimide for the preparation
of 2- and 4-bromoestradiol was repeated multiple times to
obtain sufficient quantities for the next step of synthesis.
To prevent the formation of side products during the Ull-
mann condensation reaction, the phenolic hydroxyl group
(i.e. at the C-3 position) of 2- or 4-bromoestradiol was pro-
1
confirmed by high resolution mass and H NMR (aromatic
protons). The benzyl derivative of M15 (compound 7) was
synthesized under the same reaction conditions.
In the final procedure (Step 4), the benzyl group was re-
moved by catalytic hydrogenation in the presence of Pd-C.
The reaction mixture was subjected to hydrogen at 40 psi for
4 h and the catalyst was then removed by vacuum filtration

A.J. Lee et al. / Steroids 69 (2004) 61–65
65
condensation reaction (with 4-picoline as a solvent) was
used to generate the corresponding diaryl ether product,
with a moderate yield of 41%. The chromatographic and
spectrometric properties of the two synthesized compounds
(M15 and M16) were identical to the enzymatically formed
M15 and M16. The ready availability of sufficient amounts
of these estrogen dimers makes it possible for us to further
determine any biological activities that may be associated
with this novel class of nonpolar estrogen metabolites.
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Fig. 3. Gradient-enhanced heteronuclear multiple quantum coherence-
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CD3OD and DMSO-d6 (for M15) as internal standard. Parameters for the
two-dimensional experiments included 4000 Hz 1H (F2) spectral window
collected with 2K complex data points. A 200 ppm F1 window for 13C
was used for the gHMQC experiments.
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through Celite. Both M15 and M16 were obtained as powder
from acetonitrile solution upon concentration. Their struc-
tures were confirmed through analysis of their high resolu-
tion mass and the 1H NMR spectra of their aromatic protons.
The 1H and 13C NMR spectra of M15 and M16 were shown
in Fig. 3 as gHMQC spectra. It is of note that the chemically
synthesized M15 and M16 showed identical 1H NMR peaks
to those of enzymatically formed M15 and M16 (data not
shown). HPLC analysis of the enzymatically and chemically
prepared M15 or M16 showed the same retention times, and
co-injection of metabolically formed and chemically syn-
thesized M15 or M16 into HPLC showed a single peak.
In summary, two novel diaryl ether dimers of estradiol
were chemically synthesized by using estradiol as the start-
ing material. After appropriate protection of the phenolic
hydroxyl groups of 2- or 4-bromoestradiol, the Ullmann