Synthesis of novel estrogen receptor antagonists using metal-catalyzed coupling reactions and characterization of their biological activity

Article abstract of DOI:10.1021/jm3013773

Estrogen receptor (ER) antagonists are valuable in the treatment of ER-positive human breast cancer. In this study, we designed and synthesized nine new derivatives of 17β-estradiol (E₂) with a bulky side chain attached to its C-7α position, and determined their ER antagonistic activity using in vitro bioassays. Four of the derivatives showed a strong inhibition of ERα transactivation activity in a luciferase reporter assay and blocked ERα interactions with coactivators. Similarly, these derivatives also strongly inhibited the growth of the ERα-positive human breast cancer cells. Computational docking analysis was conducted to model the interaction of these antagonists with the human ERα and showed that they could tightly bind to the ERα in a manner similar to that of ICI-182,780, a pure ER antagonist. These results provide an example that attachment of a bulky side chain to the C-7α position of E₂ can produce ER antagonists with ER affinity comparable to that of ICI-182,780.

Full text of DOI:10.1021/jm3013773

Article
pubs.acs.org/jmc
Synthesis of Novel Estrogen Receptor Antagonists Using Metal-
Catalyzed Coupling Reactions and Characterization of Their
Biological Activity
Xiang-Rong Jiang,† Pan Wang,† Carolyn L. Smith,‡ and Bao Ting Zhu*,†,§
†Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160,
United States
‡Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, United States
§Department of Biology, South University of Science and Technology of China, Shenzhen 518055, China
ABSTRACT: Estrogen receptor (ER) antagonists are valuable
in the treatment of ER-positive human breast cancer. In this
study, we designed and synthesized nine new derivatives of 17β-
estradiol (E2) with a bulky side chain attached to its C-7α
position, and determined their ER antagonistic activity using in
vitro bioassays. Four of the derivatives showed a strong
inhibition of ERα transactivation activity in a luciferase reporter
assay and blocked ERα interactions with coactivators. Similarly,
these derivatives also strongly inhibited the growth of the ERα-
positive human breast cancer cells. Computational docking
analysis was conducted to model the interaction of these
antagonists with the human ERα and showed that they could
tightly bind to the ERα in a manner similar to that of ICI-
182,780, a pure ER antagonist. These results provide an example
that attachment of a bulky side chain to the C-7α position of E2
can produce ER antagonists with ER affinity comparable to that
of ICI-182,780.
attached to the C-7α position. The 7α-substituted 17β-estradiol
derivatives are a class of compounds of considerable
pharmaceutical interest because they can serve as pure ER
antagonists, and the design and synthesis of these compounds
have been a main goal of many investigations.5−11 At the
molecular level, it is known that the binding of a pure
antagonist to the ERα protein interferes with receptor
dimerization and particularly its interaction with coactivators,
consequently blocking the transcriptional activity.12 In the
present study, we sought to design and synthesize several
representative E2 derivatives with a bulky ring-based structure
attached to the C-7α position. A number of in vitro bioassays
have been used to test the biological functions of these E2
derivatives to determine whether they could function as
effective ER antagonists. Out of a total of nine compounds
synthesized in this study, four showed an effective ER
antagonist activity with a rather high binding affinity for the
human ERα and ERβ. The results of this study provide an
example that attachment of a bulky structure to the C-7α
position of E2 can produce ER antagonists with comparable
INTRODUCTION
■
Since the 1970s, the incidence of breast cancer remains highest
among all cancers for women living in the United States, and at
present, it is one of the leading causes of cancer-related
mortality for women in this country.1 The use of estrogen
receptor (ER) antagonists such as tamoxifen has become a
valuable strategy as an adjuvant hormonal therapy for ERα-
positive human breast cancer.2 In addition, these antiestrogens
are also effective for the prevention of estrogen-inducible breast
cancer in high risk populations.3 Tamoxifen, a well-known
partial agonist of ERs, has a predominant antiestrogenic activity
(i.e., ER antagonist activity) under most conditions, but it also
has estrogenic activity (i.e., ER agonist activity), and this is of
particular concern relative to tamoxifen-resistant breast cancer.
Pure ER antagonists, such as ICI-182,780 (fulvestrant)4a and
ICI-164,3844b that are devoid of ER agonistic activity, have
been developed as alternatives to tamoxifen.4 Studies have
shown that the ER-positive human breast cancer cells that
become resistant to tamoxifen often are still sensitive to the
anticancer effect of fulvestrant, which has been approved for
clinical use in the United States.
Structurally, most of the pure ER antagonists contain the
core structure of 17β-estradiol (E2) with a long side chain
Received: September 23, 2012
© XXXX American Chemical Society
A
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Scheme 1. Flow Chart for the Synthesis of 3,17β-Bis(hydroxy)-7α-(6-hexynyl)-estra-1,3,5(10)-trien (Compound 7) Using E2
a
(Compound 1) as the Starting Material
a
The reagents and the reaction conditions used are summarized below: (a) DHP, pyridinium p-toluenesulfonate, DCM, reflux. (b) (1) LDA, t-
BuOK, THF, −78°C. (2) B(OMe)3, 0°C. (3) H2O2, H2O, 25°C. c. Swern oxidation. (d) t-BuOK, THF, 0°C, compound 9, −78°C. (e) 6 N HCl,
THF. (f) (Et)3SiH, BF3·Et2O, DCM. (g) NaI, acetone, reflux, 12 h.
a
Scheme 2
a
For the reaction conditions, refer to Schemes 3 and 4.
receptor binding affinity as ICI-182,780. Studies are ongoing to
further test these ER antagonists for their potential usefulness
and efficacy in the treatment and prevention of ER-positive
breast cancer using in vivo models.
ranging from 54 to 85% (Schemes 2 and 3). Using the “click
reaction” (typically, compound 7, azide, sodium ascorbate, and
CuSO4 in water and ethyl alcohol, v/v = 1:1), compounds 17
and 18 were synthesized in 81% and 74% yield, respectively
(Schemes 2 and 4).
DESIGN AND CHEMICAL SYNTHESIS OF NOVEL
ESTROGEN RECEPTOR ANTAGONISTS
■
BIOLOGICAL ACTIVITY STUDY
■
After adequate amount of the new E2 derivatives had been
synthesized and purified, we performed a series of in vitro
experiments to test their biological activity, which included the
ERα/ERβ binding assays, cell proliferation assay for both ER-
positive and ER-negative human breast cancer cell lines, and the
reporter assays for ERα trans-activation and for receptor
interaction with coactivators. The data are summarized below.
New E2 Derivatives Retain High Binding Affinity for
Human ERα and ERβ. First, we determined the relative
binding affinity (RBA) of each newly synthesized E2 derivative
for human ERα and ERβ in vitro by using the radioligand−
receptor competition assay. In this in vitro assay, the
recombinant human ERα and ERβ proteins were used as the
receptor proteins, and a final concentration of 10 nM [3H]E2
was used the radioligand. Each of the competing ligands (i.e.,
As depicted in Scheme 1, compound 4 was prepared from E2
according to the procedures previously established in our
laboratory (described in the Experimental Section). Compound
9 was prepared from compound 8 by reacting with sodium
iodide while refluxing in acetone overnight, in 98% yield.
Compound 4 was then reacted with compound 9 to give
compound 5 in 60% yield using potassium tert-butoxide in
THF.6,7,9 Deprotection of THP at the C-3 and C-17β positions
from compound 5 using HCl (6 N) in THF gave compound 6
in 91% yield. Then compound 6 was reduced to compound 7
using triethylsilane in the presence of boron trifluoride etherate
in methylene chloride in 75% yield. From this rather versatile
intermediate (compound 7), the palladium-catalyzed coupling
reaction (typically, compound 7, aryl iodide, PdCl2(PPh3)2,
CuI, and TEA in DMF) gave compounds 10−16 in yields
B
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a
Scheme 3
a
Typical conditions: compound 7, aryl iodide, PdCl2(PPh3)2, CuI, and TEA in DMF.
a
Scheme 4
a
Typical conditions: compound 7, azide, sodium ascorbate, and CuSO4 in water and ethyl alcohol (v/v = 1:1).
the new E2 derivatives) was tested at a wide range of final
concentrations (0, 0.24, 0.98, 3.9, 15.6, 62.5, 250, and 1000
nM). The RBA value for each competing estrogen derivative
was then calculated according to the RBA of E2 (see the
Experimental Section for details).
Several New E2 Derivatives Can Inhibit the Estrogen-
Dependent Growth of ER-Positive Human Breast Cancer
Cells. It is known that the growth of ERα-positive MCF-7 cells
can be stimulated by ER agonists and inhibited by ER
antagonists.13,14 Of the nine derivatives made in this study,
four of them (i.e., J3, J4, J7, and J9) showed a concentration-
dependent inhibition of the growth of MCF-7 cells, effective at
as low as 1 or 10 nM concentrations (Figure 3A). The IC50
values of J3, J4, and J9 for growth inhibition were
approximately 50 nM, and the IC50 value for J7 is
approximately 100 nM. Although these compounds are not as
potent as ICI-182,780 (IC50 of approximately 2 nM), their
inhibitory potency is higher than that of tamoxifen (IC50 of
approximately 200 nM) (Figure 3A). Using J3 and J4 as
representative compounds, we also tested their activity in the
ER-negative MDA-MB-231 cells. As predicted, no appreciable
effect (inhibition or stimulation) on the growth of these ER-
negative cells was observed when J3 or J4 was present (Figure
3B). However, the other five compounds (J1, J2, J5, J6, and J8)
showed either no effect or a stimulatory effect on MCF-7 cell
growth (Figure 3C).
Although each of the E2 derivatives has a rather bulky
structure (Figure 1) attached to the C-7α position, the binding
affinity of these derivatives is very high (see Figure 2 and Table
1). It was predicted beforehand that these compounds most
likely would still retain the ability to interact with the ligand-
binding domains of human ERα and ERβ in similar ways as
would E2 by forming hydrogen bonds between the C-3 and C-
17β hydroxyl groups of the ligand molecules and the amino
acid residues in the binding domains of the receptors.
Experimental data showed that while each of the nine newly
synthesized E2 derivatives did not show significant preference
for binding to human ERα vs ERβ, significant differences were
noted in the binding affinities of different derivatives for the
human receptors. While J1 and J9 showed the highest binding
affinity for human ERs (their RBAs > 20% of E2), the RBAs of
J2, J5, J7, and J8 were 3−5% of E2, and the RBAs of J3, J4, and
J6 were only approximately 1% of E2.
Upon comparing the structures of J3, J4, J7, and J9 with
those of the other five chemicals, we noticed that the two
C
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Figure 1. Structures of the newly synthesized E2 derivatives. This figure shows the structures of all nine E2 derivatives that were synthesized in this
study. The abbreviations are given for convenience. J1 or compound 14, 3,17β-bis(hydroxy)-7α-[6-(4-pyrrol-1-yl-phenyl)-hex-5-ynyl]-estra-
1,3,5(10)-trien; J2 or compound 16, 3,17β-bis(hydroxy)-7α-(6-naphthalen-2-yl-hex-5-ynyl)-estra-1,3,5(10)-trien; J3 or compound 11, 3,17β-
bis(hydroxy)-7α-[6-(4-Isoxazol-5-yl-phenyl)-hex-5-ynyl]-estra-1,3,5(10)-trien; J4 or compound 10, 4-{4-[6-((7α,17β)-3,17-dihydroxyestra-
1,3,5(10)-trien-7-yl)-hex-1-ynyl]-phenyl}-piperazine-1-carboxylic acid tert-butyl ester; J5 or compound 12, 3-{4-[6-((7α,17β)-3,17-dihydroxyestra-
1,3,5(10)-trien-7-yl)-hex-1-ynyl]-phenyl}-3-oxo-propionic acid ethyl ester; J6 or compound 15, 3,17β-bis(hydroxy)-7α-[6-(4-piperidin-1-ylmethyl-
phenyl)-hex-5-ynyl]-estra-1,3,5(10)-trien; J7 or compound 17, 3-{4-[4-((7α,17β)-3,17-dihydroxyestra-1,3,5(10)-trien-7-yl)-butyl]-1,2,3-triazol-1-yl}-
7-hydroxy-1-benzopyran-2-one; J8 or compound 13, 2-{4-[6-((7α,17β)-3,17-dihydroxyestra-1,3,5(10)-trien-7-yl)-hex-1-ynyl]-benzyl}-malonic acid
diethyl ester; and J9 or compound 18, 3,17β-bis(hydroxy)-7α-{4-[1-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-1H-1,2,3-triazol-4-yl]-butyl}-estra-
1,3,5(10)-trien.
closely placed rings and the presence of nitrogen and oxygen
atoms in the side chain seemed important for the antagonist
activity. Therefore, the precursors in Scheme 4 appear to be
more promising for the design and synthesis of pure ER
antagonists than the precursors in Scheme 3.
cells suggests that these compounds do not block this ligand-
independent activity. The compounds that exerted the least
agonist activity (J3, J4, J7, and J9) were tested for their ability
to block E2-induced ERα function in HeLa cells (Figure 4D and
Table 2). Compounds J7 and J9 were the strongest antagonists
of the E2-stimulated luciferase expression, while J3 and J4 were
less effective inhibitors, consistent with their partial agonist
activity observed in Figure 4C.
Regulation of ERα Transcriptional Activity by the New
E2 Derivatives. While E2 is known to be an agonist for ERα in
all cell environments, other compounds, particularly partial
agonists/antagonists vary in their ability to stimulate ERα
transcriptional activity depending upon cell types. In this study,
the ability of the new E2 derivatives to affect the transcriptional
activity of ERα was tested in three different cell lines. In the
MCF-7 human breast cancer cells, we observed that 100 nM of
J1, J2, or J5 exerted a similar activation of the ERα-based
transcription of a target gene as did 1 nM E2 but that J6 and J8
at the same concentration showed a markedly weaker effect
(Figure 4A). In comparison, J3 and J4 were very poor agonists,
and J7 and J9 had no agonist activity. While the profile of the
transcriptional activity of these E2 derivatives in HeLa cells
(Figure 4C) was very similar to that obtained for MCF-7 cells,
several of the derivatives produced more agonist activity in
HepG2 cells than in HeLa or MCF-7 cells. For instance, J3 and
J9 are poor agonists in the latter cell types but exert
approximately 50% of the agonist activity of E2 in HepG2
cells (Figure 4B), suggesting that these compounds may
regulate some of the ERα functions in a cell-type dependent
manner. The ER activity in HepG2 cells is positively influenced
by the activity of the N-terminal portion of the receptor, which
encompasses the activation function 1 (AF-1) domain,12 and
the ability of J3 and J9 to stimulate luciferase activity in these
The transcriptional activity of ERα is dependent upon
coactivators such as SRC-1 which bind to ERα in an agonist-
dependent manner and help to remodel chromatin via their
intrinsic or associated histone acetyltransferase activities.15 To
determine if the transcriptional activity induced by each of the
E2 derivatives was reflected in their ability to regulate the
recruitment of coactivators to ERα, the mammalian two-hybrid
assays were performed. As shown in Figure 5A, the new
derivatives with the greatest agonist activity (i.e., J1, J2, J5, J6,
and J8) effectively promoted the interaction between the ER
and the SRC-1 coactivator, while those with antagonist activity
(i.e., J3, J4, J7, and J9) did not induce significant SRC-1
binding. As expected, the pure ER antagonist ICI-182,780 also
did not promote detectable ERα-SRC-1 interaction. Thus, the
relative ability of these new E2 derivatives to stimulate ERα
transcriptional activity correlates well with their ability to
promote ERα interaction with the SRC-1 coactivator. Finally, a
prior report had demonstrated that pure antagonists such as
ICI-182,780 can strongly promote ERα interaction with the
CBP coactivator at a domain distinct from the agonist binding
site.16 As these new compounds are C7α side-chain derivatives
of E2 as is ICI-182,780, the ability of these derivatives to
D
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Figure 2. Comparison of the relative binding affinities (RBAs) of nine new E2 derivatives (J1 to J9) synthesized in this study. Eight concentrations
(0.06, 0.24, 0.98, 3.9, 15.6, 62.5, 250, and 1000 nM) of each competing ligand were tested to determine the concentration-dependent inhibition of
the binding of 10 nM [3H]E2 to the recombinant human ERα and ERβ. Each point was the mean of duplicate measurements (average variations
were <5%). The incubation mixture in the absence of a competing chemical was set as the control. The IC50 values were calculated according to the
inhibition curves, and their respective RBAs were then calculated according to the method described in the Experimental Section.
important determinant of whether the CH3 domain of CBP
Table 1. IC50 and RBA Values of the New E2 Derivatives for
ERα and ERβ
can be recruited to ERα.17
a
ERα
IC50 (nM)
ERβ
IC50 (nM)
COMPUTATIONAL MOLECULAR MODELING
STUDY
■
compds
RBA (%)
RBA (%)
E2
6.0
28.3
100.0
21.2
3.4
8.7
19.1
100.0
45.7
7.2
The three-dimensional structures of the ERα LBD in
association with both an agonist and an antagonist have been
resolved using X-ray crystallography.18 The most notable
difference between these two conformations is that in the
antagonist-binding conformation, the helix-12 (H12), which is
critical for formation of the docking site for the LXXLL-
containing coactivators, is forced to adapt an alternative
position.18 With this conformation, the ERα lacks transcrip-
tional activity. Using the ERα LBD in complex with raloxifene
(an ER antagonist) as a template, we analyzed the binding
interaction of the ERα LBD with the new ER antagonists
developed in this study by using computational docking tools.
As shown in Figure 6 (A and B), both ICI-182,780 and J9
can bind inside the binding pocket of the human ERα in a very
similar manner, and their steroidal rings, especially the C-3 and
C-17β hydroxyl groups, overlapped with each other extensively.
Their hydroxyl groups form hydrogen bonds with the amino
acid residues E353, R394, and H524 of ERα in a similar fashion
as those formed with E2. In addition to the formation of
hydrogen bonds between the C-3 or C-17β hydroxyl groups of
J9 and ERα, it appears that its side chain also forms an
additional hydrogen bond with ERα-N532, although it would
J1 (compd 14)
J2 (compd 16)
J3 (compd 11)
J4 (compd 10)
J5 (compd 12)
J6 (compd 15)
J7 (compd 17)
J8 (compd 13)
J9 (compd 18)
175.7
481.8
567.4
96.5
120.7
331.1
436.4
185.0
565.9
111.9
150.6
41.2
1.2
2.6
1.1
2.0
6.2
4.7
637.8
116.0
126.4
24.4
0.9
1.5
5.2
7.8
4.7
5.8
24.6
21.1
a
The IC50 values for each competing ligand was calculated according
to the sigmoidal inhibition curves as shown in Figure 2, and the
relative binding affinity (RBA) values were calculated against E2 by
using the following equation: RBA = The IC50 for E2/The IC50 for the
test compound.
promote CBP binding to ERα was determined. In comparison
to ICI-182,780, none of the derivatives effectively promoted the
binding of the CH3 domain of CBP to the ERα LBD (Figure
5B). This effectively distinguishes the molecules with
prominent antagonist activity (J3, J4, J7, and J9) from the
pure ER antagonist ICI-182,780 and thereby reinforces the
concept that the chemical nature of the C7α side chain is an
E
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Figure 3. Effect of the new E2 derivatives on the proliferation of human breast cancer cell lines. The ERα-positive MCF-7 and the ERα-negative
MDA-MB-231 human breast cancer cell lines were cultured and seeded in 96-well plates. Different final concentrations of the ER ligands (at 0, 0.1,
0.32, 1.6, 8, 40, 200, and 1000 nM) were added to the cell culture medium for 6 days with one medium change at the end of the third day. The cell
density was determined by using the crystal violet staining method as described in the Experimental Section. Each point was the mean SEM (N =
6). Note that for the y-axis, 100% refers to the cell density after 6 days of culture in the presence of vehicle only, and 0% means the initial seeding
density of the cells on day 0. In these experiments, since no additional estrogen was added to the cell culture medium, all the estrogens came with the
10% FBS contained in the cell culture medium. (A) Four of the E2 derivatives (J3, J4, J7, and J9) showed a concentration-dependent inhibition of
the growth of ER-positive MCF-7 cells, and their effect was compared with ICI-182,780 and tamoxifen. (B) Two representative ER antagonists (J3
and J4) were further tested in the ER-negative MDA-MB-231 cells, and they did not show an appreciable effect on the growth of these cells. (C) Five
of the other E2 analogues (J1, J2, J5, J6, and J8) did not show an appreciable effect on the growth of the ER-positive MCF-7 cells.
be difficult to estimate its relative contribution to the overall
binding interaction. Notably, the high degree of overall
similarity between the binding modes of ICI-182,780 and J9
inside the ERα binding pocket as predicted by computational
docking analysis is consistent with their relative binding
affinities for the human ERα (the RBA of ICI-182,780 is 45%
of E2, and the RBA of J9 is 24.6%). Using the same method, we
also docked J3, J4, and J7 into the ERα LBD, and similar
binding interactions between the ligands and the receptor were
observed (data not shown).
It is of note that based on the docking models developed in
this study, we noticed that the pure antagonists (ICI-182,780
and the newly synthesized J3, J4, J7, and J9) adopt a unique
binding mode compared to E2 by flipping their core steroidal
structure 180° along the C-3 hydroxyl and C-17β hydroxyl axis.
In this way, the long C-7α side chain was positioned in the C-
11β channel and then exited the ligand binding pocket in a
manner similar to that of raloxifene. However, the characteristic
hydrogen bonds of C-3 and C-17β hydroxyl groups with the
receptor binding pocket basically remain unchanged. This
predicted binding mode is rather intriguing, and it is consistent
with the reported crystal structure of human ERβ in complex
with ICI-164,384 (an analogue of ICI-182,780).19
In conclusion, we have designed and synthesized in this study
nine E2-based new derivatives with a bulky side chain attached
to its C-7α position. While all these derivatives have
considerable binding affinity for human ERα and ERβ, only
four of them have pure ER antagonistic activity, based on in
vitro analyses using the ERα transactivation activity assay and
the proliferation assay in the ER-positive human breast cancer
cells. Molecular computational modeling analysis showed that
these new ER antagonists can bind inside the ERα binding
pocket in a way similar to that of other known ERα antagonists
such as ICI-182,780. The results of this study showed that these
new ER antagonists are good candidates for further structural
modifications as well as testing of their usefulness and efficacy
in treating and preventing ERα-positive breast cancers.
F
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Figure 4. Assessment of the ERα agonist and antagonist activities of the new E2 derivatives. (A) MCF-7 cells were transfected with the ERE-Elb-Luc
reporter plasmid and treated with the vehicle (0.1% ethanol), 1 nM E2, 100 nM ICI-182,780, or 100 nM of each of the new E2 derivatives. HepG2
(B) and HeLa (C) cells were transfected with the expression vector pCR3·1-ERα and the reporter plasmid ERE-Elb-Luc, and they were also treated
with the ligands as described for panel A. (D) HeLa cells were transfected as described above and were treated in the absence (white bars) or
presence (stripped bars) of 1 nM E2 with the ligands as indicated. For each of these experiments, the luciferase values are normalized to those
obtained for E2 treatment. Bars represent the average SEM of at least 3 experiments.
In all of the in vitro bioactivity assays, the new E2 derivatives
Table 2. Computed Binding Energy Values (ΔEbinding) for
a
synthesized in this study were first purified and then dissolved in 200-
the Binding of the E2 Derivatives with Human ERα
proof ethanol to a stock concentration of 1 mM. They were then
ligand
Ecomplex
Eligand
Ereceptor
ΔEbinding
further diluted to desired concentrations with the binding buffer (for
ER binding assays) or with the cell culture medium (for treating
cultured cells).
ICI-182,780
−8924.06
−9575.35
−9630.81
−8020.15
−8924.53
103.58
42.78
26.26
84.23
88.85
−8834.09
−9464.53
−9468.65
−7936.76
−8828.70
−193.57
−153.60
−188.42
−167.60
−164.69
J3 (compd 11)
J4 (compd 10)
J7 (compd 17)
J9 (compd 18)
Spectrometric Analyses. Mass spectra were determined 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 nuclear magnetic resonance (NMR) spectra of all test
compounds were determined in deuterochloroform or dimethyl
sulfoxide-d6 solution using a Varian Mercury/VX 300 spectrometer
with tetramethylsilane (TMS) as an internal standard (δ = 0) unless
noted otherwise. The purity of all synthesized final products was
determined to be ≥95% using a Waters analytical HPLC unit (model
Alliance 2695) coupled with a photodiode array detector (model
2996) and a Restek Pinnacle II reverse phase analytical column (C18,
5 μm, 150 × 4.6 mm). The HPLC elution included a linear gradient of
5−95% methanol in H2O + 0.1% formic acid over a 30 min period
with a flow rate of 1.5 mL/min.
Synthesis of 3,17β-Bis(tetrahydropyranyloxy)-7α-(6-hexyn-
yl)-estra-1,3,5(10)-trien-6-one (Compound 5). The synthesis of
compound 4 was described in our recent study.21,22 For the synthesis
of compound 5, a 1.0 M solution of t-BuOK in THF (5.35 mL, 5.35
mmol) was added to a cooled solution (at 0 °C) of compound 4 (1.2
g, 2.67 mmol) in THF (20 mL). The reaction mixture was stirred at 0
a
The ligand−ERα interaction energy values (ΔEbinding) were calculated
using the following equation: ΔEbinding = Ecomplex − (Ereceptor + Eligand),
where Ecomplex is the potential energy for ERα in complex with a ligand,
Ereceptor is the potential energy of ERα alone, and Eligand is the potential
energy for the ligand alone.
EXPERIMENTAL SECTION
■
Chemicals and Reagents. Most of the commercially available
reagents were obtained from Sigma-Aldrich (St. Louis, MO) or
ACROS (through Fisher Scientific, Atlanta, GA). 17β-Estradiol (E2)
was purchased from Steraloids (Newport, RI). Tetrahydrofuran
(THF) was distilled over sodium benzophenone ketyl and dichloro-
methane distilled over calcium hydride in our laboratory prior to use.
Most of the chemicals and solvents used in this study were of ACS
grade and used directly without additional steps of purification.
G
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Figure 5. Use of the mammalian two hybrid assay to examine the interactions between the ERα LBD and SRC-1 or the CBP CH3 domain. The
luciferase activity of HeLa cells transfected with the expression vectors for GAL-SRC-1 (A) or GAL-CBP-CH3 (B) in the presence of the VP16-ERα-
LBD expression vector and the pG5-Luc reporter plasmid. Cells were treated with vehicle (0.1% ethanol), 1 nM E2, 100 nM ICI-182,780, or 100 nM
of each of the E2 derivatives. The values are normalized to those obtained for VP16-ERα-LBD and the respective GAL-coactivator in the presence of
E2. Bars represent the average SEM of at least 3 experiments.
Figure 6. Molecular docking of representative E2 derivatives with the ligand binding domain of human ERα. (A) The structures of the ERα ligand
binding domain in complex with ICI-182,780 and J9. (B) Formation of hydrogen bonds between several amino acid residues in the ERα ligand
binding domain and ICI-182,780 or J9. The protein structure was shown in colored ribbons according to the secondary structures (red for α-helix,
blue for β-sheets, green for turns, and white for coils). Amino acids and ligands are shown as sticks and balls. Oxygen, nitrogen, carbon, and fluorine
atoms are colored red, blue, gray, and cyan, respectively. Hydrogens are omitted from all structures.
°C for 40 min and then cooled to −78 °C. 6-Iodo-1-hexyne
(compound 9, 1.48 g, 7.1 mmol) was added dropwise to the solution.
The reaction mixture was allowed to warm to 0 °C and stirred for 2 h,
and then to room temperature, and stirred overnight. The reaction was
quenched with water and extracted with ethyl acetate. The organic
phase was dried over Na2SO4, and the solvent was evaporated in vacuo.
Flash chromatography (hexane/ethyl acetate = 4:1) afforded
compound 5 in 60% yield (0.86 g). Data from spectrometric analyses
of the afforded compound are summarized below. 1H NMR (300
MHz, CDCl3): 7.61 (1H, m), 7.23 (1H, m), 7.13 (1H, dd, J = 2.7, 8.7
Hz), 5.38 (1H, m), 4.57 (1H, m), 3.82 (2H, m), 3.69 (1H, t, J = 8.1
Hz), 3.53 (1H, m), 3.41 (1H, m), 2.61 (1H, m), 2.31 (2H, m), 2.07−
1.72 (10H, m), 1.61−1.18 (20H, m), 0.73 (3H, s). MS (TOF MS ES+,
C34H46O5, M 534): 535 (M + 1) (base peak). HRMS [535 (M + 1)]:
535.3423 (calculated) and 535.3422 (observed).
Synthesis of 3,17β-Bis(hydroxy)-7α-(6-hexynyl)-estra-
1,3,5(10)-trien-6-one (Compound 6). A solution containing
compound 5 (0.86 g, 1.6 mmol), HCl (15 mL, 6 M), and THF (15
mL) was stirred at room temperature overnight. The reaction mixture
was poured into water and then extracted with ethyl acetate (4 × 50
mL). The combined organics were dried over Na2SO4, concentrated,
and then chromatographed (hexane/ethyl acetate = 2:1) to afford
compound 6 (0.54 g, 91% yield). Data from spectrometric analyses of
1
the afforded compound are summarized below. H NMR (300 MHz,
DMSO-d6): 9.56 (1H, s), 7.26 (2H, m), 6.96 (1H, dd, J = 2.7, 8.1 Hz),
3.54 (1H, t, J = 8.1 Hz), 2.59 (1H, m), 2.48 (2H, m), 2.31 (2H, m),
2.08 (2H, m), 1.85 (4H, m), 1.47−1.13 (11H, m), 0.73 (3H, s). MS
(TOF MS ES+, C24H30O3, M 366): 367 (M + 1) (base peak). HRMS
[367 (M + 1)]: 367.2277 (calculated) and 367.2273 (observed).
Synthesis of 3,17β-Bis(hydroxy)-7α-(6-hexynyl)-estra-
1,3,5(10)-trien (Compound 7). Compound 6 (0.26 g, 0.70 mmol)
H
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was dissolved in methylene chloride (20 mL). To this solution was
added triethylsilane (3.5 mL), followed by boron trifluoride etherate
(12.5 mL) dropwise. The reaction was stirred for 2 days at room
temperatures, cooled to 0 °C, and quenched with 10% potassium
carbonate (20 mL). The heterogeneous solution was filtered through a
short silica plug and the organic layer separated. The aqueous layer
was extracted twice with methylene chloride (20 mL). The organic
fractions were combined, dried over sodium sulfate, and evaporated in
vacuo to a yellow solid. Purification by flash chromatography (hexane/
ethyl acetate = 2:5) provided compound 7 (0.185 g, 75%). Data from
spectrometric analyses of the afforded compound are summarized
below. 1H NMR (300 MHz, DMSO-d6): 8.96 (1H, s), 7.02 (1H, d, J =
8.4 Hz), 6.48 (1H, dd, J = 2.7, 8.4 Hz), 6.40 (1H, d, J = 2.7 Hz), 4.46
(1H, d, J = 5.1 Hz), 3.51 (1H, m), 2.75 (1H, dd, J = 5.1, 16.5 Hz), 2.70
(1H, t, J = 2.7 Hz), 2.57 (1H, d, J = 16.5 Hz), 2.48 (2H, m), 2.24−2.07
(4H, m), 1.85 (1H, m), 1.76 (1H, m), 1.62 (2H, m), 1.47−1.13 (11H,
m), 0.64 (3H, s). MS (C24H32O2, M 352): 157 (base peak), 269, 352.
HRMS [352 (M)]: 352.2402 (calculated) and 352.2398 (observed).
Synthesis of 6-Iodo-1-hexyne (Compound 9). Under nitrogen,
5 mL (41.3 mmol) of 6-chloro-1-hexyne (compound 8) was added
into a mixture of 39 g (262 mmol) of sodium iodide and 100 mL of
acetone. The mixture was reflux overnight. After completion, the
reaction mixture was allowed to cool to room temperature. The
organic solvent was removed in vacuo. Then, to the residue 150 mL of
hexane was added. After the solid was removed by filtration, the filtrate
was concentrated. Flash chromatography (hexane as the eluent)
afforded compound 9 (8.4 g, 98%). Data from spectrometric analyses
of the afforded compound are summarized below. 1H NMR (300
MHz, CDCl3): 3.21 (2H, t, J = 6.9 Hz), 2.33 (2H, t-d, J = 2.7, 6.9 Hz),
1.97 (2H, m), 1.64 (2H, m).
Experimental Procedures for the Palladium-Catalyzed
Coupling Reaction. Under nitrogen protection, a mixture of
compound 7 (20 mg, 0.057 mmol) and R-I (0.063 mmol) was
dissolved in dry DMF (1 mL). PdCl2(PPh3)2 (2 mg, 2.85 μmol) and
CuI (1.08 mg, 5.7 μmol) were added to the mixture solution. The
resulting mixture was cooled down to −20 °C and TEA (48 μL, 0.342
mmol) was added. After 15 min, the cold bath was removed, the
reaction solution was warmed to room temperatures for another 24 h.
Water (10 mL) was added to the reaction mixture. The aqueous layer
was extracted with ether acetate (3 × 20 mL). The combined organic
layer was dried over Na2SO4, concentrated in vacuo. Flash
chromatography (ether acetate/hexene = 1:2) gave the desired
products. Data from spectrometric analyses of the afforded
compounds are summarized below.
4-{4-[6-((7α,17β)-3,17-Dihydroxyestra-1,3,5(10)-trien-7-yl)-hex-1-
ynyl]-phenyl}- piperazine-1-carboxylic Acid tert-butyl ester (Com-
pound 10). Yield, 85%. 1H NMR (300 MHz, CDCl3): 7.21 (1H, d, J =
8.4 Hz), 7.07 (1H, d, J = 8.4 Hz), 6.75 (2H, d, J = 8.4 Hz), 6.56 (1H,
dd, J = 2.7, 8.4 Hz), 6.45 (1H, d, J = 2.7 Hz), 4.87 (1H, s), 3.66 (1H,
brs), 3.45 (4H, t, J = 5.1 Hz), 3.08 (4H, t, J = 5.1 Hz), 2.81 (1H, dd, J
= 4.8, 16.5 Hz), 2.65 (1H, d, J = 16.5 Hz), 2.29 (2H, m), 2.03 (1H,
m), 1.85 (1H, m), 1.68 (1H, m), 1.55−1.16 (23H, m), 0.71 (3H, s).
MS (TOF MS ES+, C39H52N2O4, M+1 613): 613 (base peak). HRMS
[(M+1, 613)]: 613.4005 (calculated) and 613.4017 (observed).
3,17β-Bis(hydroxy)-7α-[6-(4-isoxazol-5-yl-phenyl)-hex-5-ynyl]-
estra-1,3,5(10)-trien (Compound 11). Yield, 82%. 1H NMR (300
MHz, CDCl3): 8.27 (1H, d, J = 2.1 Hz), 7.70 (2H, d, J = 8.4 Hz), 7.42
(2H, d, J = 8.4 Hz), 7.13 (1H, d, J = 8.4 Hz), 6.61 (1H, dd, J = 2.7, 8.4
Hz), 6.52 (1H, d, J = 2.7 Hz), 6.49 (1H, d, J = 2.1 Hz), 4.78 (1H, brs),
3.72 (1H, t, J = 7.8 Hz), 2.87 (1H, dd, J = 5.4, 16.8 Hz), 2.72 (1H, d, J
= 16.8 Hz), 2.39 (2H, t, J = 6.9 Hz), 2.29 (2H, m), 2.09 (1H, m), 1.90
(1H, m), 1.59−1.21 (15H, m), 0.76 (3H, s). MS (TOF MS ES+,
C32H37NO3, M 495): 496 (M + 1) (base peak). HRMS [496 (M +
1)]: 496.2851 (calculated) and 496.2849 (observed).
16.2 Hz), 2.66 (1H, d, J = 16.2 Hz), 2.35 (2H, m), 2.24 (2H, m), 2.03
(1H, m), 1.83 (1H, m), 1.69 (1H, m), 1.56−1.16 (16H, m), 0.71 (3H,
s). MS (C35H42O5, M 542): 542, 496, 466, 189 (base peak). HRMS
[542 (M)]: 542.3032 (calculated) and 542.3010 (observed).
2-{4-[6-((7α,17β)-3,17-Dihydroxyestra-1,3,5(10)-trien-7-yl)-hex-1-
ynyl]-benzyl}- malonic Acid Diethyl Ester (Compound 13). Yield,
1
76%. H NMR (300 MHz, CDCl3): 7.25 (2H, d, J = 8.1 Hz), 7.12
(3H, m), 6.62 (1H, dd, J = 2.7, 8.4 Hz), 6.45 (1H, d, J = 2.7 Hz), 5.51
(1H, brs), 4.15 (4H, m), 3.72 (1H, t, J = 7.2 Hz), 3.62 (1H, t, J = 8.1
Hz), 3.17 (2H, d, J = 7.2 Hz), 2.85 (1H, dd, J = 5.1, 16.5 Hz), 2.70
(1H, d, J = 16.5 Hz), 2.35 (2H, t, J = 6.9 Hz), 2.29 (2H, m), 2.09 (1H,
m), 1.89 (1H, m), 1.62−1.11 (21H, m), 0.76 (3H, s). MS (C35H42O5,
M 600): 600 (base peak). HRMS [600 (M)]: 600.3451 (calculated)
and 600.3471 (observed).
3,17β-Bis(hydroxy)-7α-[6-(4-pyrrol-1-yl-phenyl)-hex-5-ynyl]-
estra-1,3,5(10)-trien (Compound 14). Yield, 75%. 1H NMR (300
MHz, CDCl3): 7.36−7.19 (4H, m), 7.10 (1H, d, J = 8.7 Hz), 7.02
(2H, t, J = 2.4 Hz), 6.56 (1H, m), 6.48 (1H, m), 6.28 (2H, t, J = 2.4
Hz), 4.59 (1H, brs), 3.67 (1H, brs), 2.83 (1H, dd, J = 3.9, 16.2 Hz),
2.67 (1H, d, J = 16.2 Hz), 2.33 (2H, m), 2.24 (2H, m), 2.06 (1H, m),
1.81 (1H, m), 1.69 (1H, m), 1.57−1.09 (14H, m), 0.71 (3H, s). MS
(C34H39NO2, M 493): 493 (base peak), 272, 157. HRMS [493 (M)]:
493.2981 (calculated) and 493.2977 (observed).
3,17β-Bis(hydroxy)-7α-[6-(4-piperidin-1-ylmethyl-phenyl)-hex-5-
ynyl]-estra-1,3,5(10)-trien (Compound 15). Yield, 54%. 1H NMR
(400 MHz, CDCl3): 7.36 (2H, d, J = 8.0 Hz), 7.25 (2H, d, J = 8.0 Hz),
7.14 (1H, d, J = 8.4 Hz), 6.63 (1H, dd, J = 2.8, 8.4 Hz), 6.55 (1H, d, J
= 2.8 Hz), 3.66 (1H, t, J = 6.8 Hz), 2.77 (1H, dd, J = 5.2, 16.8 Hz),
2.60 (1H, d, J = 16.8 Hz), 2.31−2.21 (4H, m), 2.16 (2H, t, J = 6.8 Hz),
2.07 (2H, m), 1.84 (2H, m), 1.64−1.11 (24H, m), 0.71 (3H, s). MS
(TOF MS ES+, C36H47NO2, M+1 526): 526 (base peak). HRMS [(M
+1, 526)]: 526.3685 (calculated) and 526.3691 (observed).
3,17β-Bis(hydroxy)-7α-(6-naphthalen-2-yl-hex-5-ynyl)-estra-
1
1,3,5(10)-trien (Compound 16). Yield, 82%. H NMR (300 MHz,
CDCl3): 8.24 (1H, m), 7.75 (2H, m), 7.53−7.32 (4H, m), 7.07 (1H, d,
J = 8.4 Hz), 6.56 (1H, dd, J = 2.7, 8.4 Hz), 6.42 (1H, d, J = 2.7 Hz),
3.62 (1H, t, J = 8.7 Hz), 2.81 (1H, dd, J = 5.4, 16.8 Hz), 2.68 (1H, d, J
= 16.8 Hz), 2.48 (2H, t, J = 5.7 Hz), 2.24 (2H, m), 1.99 (1H, m), 1.79
(1H, m), 1.66−1.15 (16H, m), 0.70 (3H, s). MS (C34H38O2, M 478):
478 (base peak), 157. HRMS [478 (M)]: 478.2872 (calculated) and
478.2866 (observed).
Experimental Procedures for the “Click Reaction” (Scheme
4). In a mixture of compound 7 (20 mg, 0.057 mmol) and sodium
azide (0.063 mmol) in water and ethyl alcohol (v/v = 1:1, 2 mL),
sodium ascorbate (17 μL), 0.017 mmol of freshly prepared 1 M
solution in water was added, followed by the addition of copper(II)
sulfate pentahydrate 7.5% in water (14 μL, 0.0042 mmol). The
mixture was stirred vigorously at room temperature for 24 h. Ethanol
was removed, and the residue was diluted with water, cooled in ice,
and then the precipitate was collected by filtration. After washing the
precipitate with cold water, it was dried under vacuum to afford the
desired product. Data from spectrometric analyses of the afforded
compounds are summarized below.
3-{4-[4-((7α,17β)-3,17-Dihydroxyestra-1,3,5(10)-trien-7-yl)-butyl]-
1,2,3-triazol-1-yl}-7-hydroxy-1-benzopyran-2-one (Compound 17).
Yield, 81%. 1H NMR (300 MHz, DMSO-d6): 8.51 (1H, s), 8.24 (1H,
s), 7.69 (1H, d, J = 8.7 Hz), 7.03 (1H, d, J = 8.4 Hz), 6.85 (1H, dd, J =
2.4, 8.7 Hz), 6.78 (1H, d, J = 2.4 Hz), 6.48 (1H, dd, J = 2.4, 8.4 Hz),
6.40 (1H, d, J = 2.4 Hz), 4.46 (1H, brs), 3.41 (1H, t, J = 6.9 Hz), 2.67
(2H, m), 2.25 (2H, m), 1.88−1.15 (20H, m), 0.64 (3H, s). MS
(C33H78N3O5, M 555): 555 (base peak). HRMS (TOF MS ES+) [556
(M+1)]: 556.2811(calculated) and 556.2811(observed).
3,17β-Bis(hydroxy)-7α-{4-[1-(2,3-dihydro-1,4-benzodioxin-2-yl-
methyl)-1H-1,2,3-triazol-4-yl]-butyl}-estra-1,3,5(10)-trien (Com-
1
pound 18). Yield, 74%. H NMR (300 MHz, CDCl3): 7.99 (1H, s),
3-{4-[6-((7α,17β)-3,17-Dihydroxyestra-1,3,5(10)-trien-7-yl)-hex-1-
7.29 (1H, s), 7.09 (1H, d, J = 8.4 Hz), 6.86 (4H, m), 6.64 (1H, m),
6.53 (1H, m), 4.57 (1H, m), 4.29 (1H, m), 3.83 (1H, m), 3.75 (1H, t,
J = 8.4 Hz), 2.79 (1H, m), 2.67 (3H, m), 2.26 (2H, m), 2.10 (2H, m),
1.90 (1H, m), 1.71−1.10 (15H, m), 0.75 (3H, s). MS (C33H41N3O4,
M 543): 543 (base peak, direct exposure probe). HRMS [543 (M),
ynyl]-phenyl}-3-oxo-propionic Acid Ethyl Ester (Compound 12).
1
Yield, 64%. H NMR (300 MHz, CDCl3): 7.80 (2H, d, J = 8.4 Hz),
7.36 (2H, d, J = 8.4 Hz), 7.08 (1H, d, J = 8.4 Hz), 6.56 (1H, dd, J =
2.7, 8.4 Hz), 6.45 (1H, d, J = 2.7 Hz), 5.58 (1H, s), 4.77 (1H, s), 4.14
(2H, q, J = 7.2 Hz), 3.90 (2H, s), 3.67 (1H, brs), 2.82 (1H, dd, J = 4.8,
I
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direct exposure probe]: 543.3097(calculated) and 543.3088-
(observed).
The binding buffer consisted of 10% glycerol, 2 mM dithiothreitol, 1
mg/mL BSA, and 10 mM Tris-HCl at pH 7.5. The ERα washing buffer
contained 40 mM Tris-HCl and 100 mM KCl (pH 7.4), but the ERβ
washing buffer contained only 40 mM Tris-HCl (adjusted to pH 7.4).
The 50% hydroxylapatite slurry was prepared first by vigorously mixing
10 g of hydroxylapatite with 60 mL of the Tris-HCl solution (50 mM,
pH 7.4). Hydroxylapatite was then allowed to settle for 20 min at
room temperature, and the supernatant was decanted. The above
procedures were repeated 10 times, and afterward, hydroxylapatite was
kept in the 50 mM Tris-HCl solution overnight at 4 °C.
Hydroxylapatite slurry (EMD Biosciences, Inc. San Diego, CA) was
then adjusted to an approximate final concentration of 50% (v/v)
using the same Tris-HCl solution and stored at 4 °C.
Synthesis of 2-Azidomethyl-2,3-dihydro-1,4-benzodioxine
(Compound 22). To a solution of 2-hydroxymethyl-1,4-benzodiox-
ane (compound 20; 20 mmol) in toluene (150 mL) were added, with
vigorous stirring, imidazole (22 mol), triphenylphosphine (21 mmol),
and iodine (22 mmol). After 2 h at ambient temperature, the solution
was decanted from the gummy precipitate. The decantate was
decolorized by shaking it with aqueous Na2S2O3 and water, then
drying (Na2SO4) and evaporating to dryness to afford the crude
product 2-iodomethyl-1,4-benzodioxane (compound 21). This crude
product was used in the next step without further purification.
On the day of performing the ER binding assay, the [3H]E2 solution
was freshly diluted in the binding buffer, and an aliquot (45 μL) of the
[3H]E2 solution was added to a 1.5 mL microcentrifuge tube, intended
for a final [3H]E2 concentration at 10 nM. Each of the competing
ligands (in 50 μL volume of the binding buffer) was then added to the
mixture for the intended final concentrations at 0, 0.24, 0.98, 3.9, 15.6,
62.5, 250, and 1000 nM. Immediately before the addition of the ERα
or ERβ protein, it was diluted in the binding buffer and mixed gently
with repetitive pipeting. An aliquot (5 μL) of the diluted ERα or ERβ
solution was precisely added to the mixture containing 45 μL of the
[3H]E2 and 50 μL of the competing ligand and then mixed with
repetitive pipeting. Nonspecific binding (NSB) by the [3H]E2 was
determined in separate tubes by inclusion of a 400-fold concentration
of the nonradioactive E2 (at a final concentration of 4 μM). The
binding mixture was incubated at 4 °C for overnight.
To a solution of 2-iodomethyl-1,4-benzodioxane (compound 21; 10
mmol) in 40 mL of DMF was added sodium azide (40 mmol). The
mixture was heated to 40 °C overnight, then the resulting solid was
removed by filtration, and the filtrate was poured into a mixture of
hexane and ethyl acetate (400 mL; 3:1). The solid that formed was
filtered, and the filtrate was concentrated in vacuo. The crude product
was purified by a silica gel column (eluting with a mixture of hexane
and ethyl acetate (15−40%) to afford 2-azidomethyl-2,3-dihydro-1,4-
benzodioxine (compound 22) in 62% yield (over two steps). Data
from spectrometric analyses of compound 22 are summarized below.
CDCl3, 400 MHz, 6.91 (4H, m), 4.36 (1H, dd, J = 2.0, 11.2), 4.27
(1H, m), 4.14 (1H, dd, J = 6.0, 11.2 Hz), 3.32 (2H, m).
Synthesis of 3-Azido-7-hydroxycoumarin (Compound 26). 3-
Azido-7-hydroxycoumarin was prepared according to the published
procedures.23 Typical procedures are as follows: A mixture of 2,4-
dihydroxy benzaldehyde (2.76 g, 20 mmol), N-acetylglycine (2.34 g,
20 mmol), and anhydrous sodium acetate (60 mmol) in acetic
anhydride (100 mL) was refluxed under stirring for 4 h. The reaction
mixture was poured onto ice to give a yellow precipitate. After
filtration, the yellow solid was washed by ice water before it was
refluxed in a solution of concentrated HCl and ethanol (2:1, 30 mL)
for 1 h, then ice water (40 mL) was added to dilute the solution. The
solution was then cooled in an ice bath, and NaNO2 (40 mmol) was
added. The mixture was stirred for 10 min, and NaN3 (60 mmol) was
added in portions. After stirring for another 15 min, the resulting
precipitate was filtered off, washed with water, and dried under
reduced pressure to afford a brown solid, 3-azido-7-hydroxycoumarin
(compound 26), 2.2 g (54% overall yield).
At the end of the incubation, 100 μL of the hydroxylapatite slurry
was added to each tube, and the tubes were incubated on ice for 15
min with 3 times of brief vortexing. An aliquot (1 mL) of the
appropriate washing buffer was added, mixed, and centrifuged at
10,000g for 5 min, and the supernatants were discarded. This wash
step was repeated twice. Hydroxylapatite pellets were then
resuspended in 200 μL of ethanol (followed by another rinse with
200 L of ethanol), and then the content was transferred to scintillation
3
vials (containing 3 mL of scintillation fluid) for measurement of H-
radioactivity with a liquid scintillation counter (Packard Tri-CARB
2900 TR; Downers Grove, IL).
The specific binding (pmol/mL) of the human ERα or ERβ protein
at each concentration point was calculated using the following
equation:
ERα or ERβ
(d.p.m. for total binding − d.p.m. for NSB) × dilution factor
=
final volume of the mixture × (d.p.m/pmol [3H]E2)
The IC50 value for each competing estrogen was calculated according
to the four parameter sigmoidal inhibition curve in regression wizard
of the SigmaPlot software (all the curve regressions had a Rsqr higher
than 0.95) by setting the y value in the four parameter sigmoidal
equation to 50. Relative binding affinity (RBA) was calculated using
the following equation:
IC50 for E2
RBA =
IC50 for the test compound
ERα and ERβ Binding Assays. For the in vitro ERα and ERβ
radioligand binding assays, [2,4,6,7,16,17-3H]E2 (specific activity of
110 Ci/mmol) was used as the radioligand. It was obtained from
PerkinElmer (Waltham, MA) and was purified in our laboratory using
a high-pressure liquid chromatography (HPLC)-based method20
before it was used in the binding assays. The recombinant human
ERα and ERβ proteins were obtained from PanVera (Madison, WI).
According to the supplier, the recombinant human ERα and ERβ were
produced in a baculovirus-mediated expression system, and they were
soluble and functionally active, with post-translational modifications
similar to those found in mammalian cells.
In Vitro Cell Proliferation Assay to Determine the
Antiestrogenic Activity in Human Breast Cancer Cells. The
ER-positive MCF-7 and ER-negative MDA-MB-231 human breast
cancer cells were obtained from the American Type Culture Collection
(ATCC, Manassas, VA). The culture conditions for these two cell lines
were described in detail in our earlier study.24 Note that in these
experiments, no additional estrogen was added to the cell culture
medium, and all the estrogens came from the 10% FBS contained in
the cell culture medium. The human breast cancer cells were first
propagated in the 75 cm2 flasks to 80% confluence under 37 °C air
with 5% CO2 and 95% humidity to 80% confluence. They were then
detached from the flask by treatment with 3 mL of the trypsin-EDTA
solution for 5 min. Cell suspensions were centrifuged, and the cell
The following buffer solutions were used in the ERα and ERβ
binding assay, and they were prepared beforehand and stored at 4 °C.
J
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sediments were resuspended in phenol red-free culture medium
containing 10% FBS at the desired density of 105 cells/mL. A 0.1 mL
aliquot of the cell suspension was then added to each well of the 96-
well microplates usually at a final density of 104 cells per well. After the
cells were allowed to attach and grow for 24 h, the cell culture medium
was changed, and different drug treatments were introduced at that
time. In most experiments, the drug treatment lasted for 6 days with
one medium change on the fourth day following the initial drug
treatment.
The cell density in each well was determined by using the crystal
violet staining method.24 Briefly, the culture medium in the
microplates was first removed by aspiration, and then the cells in
each well were fixed with 1% glutaraldehyde for 15 min. After
removing the fixation solution, each well was rinsed with PBS buffer
and allowed to dry at room temperature. The cells in each well were
then stained with 50 μL of 0.1% crystal violet (dissolved in 20%
methanol and 80% deionized water) for 15 min at room temperature,
and the plates were rinsed carefully with tap water to remove residual
crystal violet. The stained dye was then dissolved in 100 μL of 0.5%
Triton X-100 for two hours. After the addition of 50 μL of 200-proof
ethanol, the absorbance values of each well were measured at 560 and
405 nm with a UVmax microplate reader (Molecular Device, Palo
Alto, CA), and the difference in the absorbance values at these two
wavelengths were used to represent the cell density.
ERα Trans-Activation Assays. The HeLa (human cervical
cancer), HepG2 (human hepatoma), and MCF-7 cell lines employed
for these experiments were maintained in DMEM supplemented with
10% FBS. For ERα trans-activation assays, cells were plated in a 6-well
plate and grown overnight in phenol-red free DMEM containing 10%
FBS, followed by transfection with the indicated plasmids using
lipofectamine-2000 (Invitrogen). Thereafter, cells were treated with
vehicle or the indicated concentrations of the ERα ligands, and 20−24
h later cells were harvested for luciferase activity assay using a
Luciferase Assay Kit (Promega) and a Luminoskan Ascent Thermo
Labsystems apparatus (Thermo Electron, Milford, MA). Relative
luciferase units were normalized to total protein content of the cell
lysate measured by the Bio-Rad protein assay (Bio-Rad, Hercules,
CA).
ERα-Coactivator Interaction Assays. Interactions between the
ERα ligand binding domain (LBD) and either the SRC-1 or CBP
coactivators were assessed by mammalian two-hybrid assay essentially
as described previously.16,25 HeLa cells were transfected with 100 ng of
the expression vectors for the GAL4 DNA binding domain fused to
either SRC-1 (pBIND-SRC-1e) or the CBP CH3 domain (pBIND-
CBP). Along with pBIND expression vectors, 1000 ng of the
expression vector for VP16-ERα-LBD (pACT-hERα-LBD) and 1000
ng of the pG5-Luc reporter plasmid were used. Control experiments
employed equivalent amounts of the pACT and pBIND empty
vectors. Cells were treated with either 0.1% ethanolic vehicle or the
indicated concentrations of ligands for 20−24 h prior to cell harvest
and assay as described above.
pocket was defined to include all residues within the 7-Å reach of the
original ligand raloxifen. A combination of Monte Carlo and simulated
annealing methods was used to explore all possible conformations of
the ligands. One hundred conformations were obtained, and the one
with the lowest potential binding energy was chosen for further
minimization using the Standard Dynamics Cascade protocol in the
Discovery Studio. The backbone of the protein and its key residues in
the binding pocket (namely, E353, R394, and H524) were fixed during
the docking procedures.
For energy minimizations, the steepest descent method was
employed first to 10 kcal/(molÅ) root-mean-square (RMS) energy
gradient and followed by the Polak and Ribiere conjugate gradient
method until the final convergence criterion reached 0.01 kcal/(molÅ)
RMS energy gradient. Then, the whole system was heated from 100 to
300 K in 2 ps and equilibrated in 300 K for 100 ps. One hundred
conformations were collected in 20 ps production phase at 300 K. The
conformation with the lowest potential energy was further minimized
and used for the Binding Energy Calculation protocol. The backbone
of the protein and its key residues in the binding pocket (namely,
E353, R394, and H524) were constrained during the simulation
process.
AUTHOR INFORMATION
Corresponding Author
■
*Department of Pharmacology, Toxicology and Therapeutics,
University of Kansas Medical Center, MS-1018, room HLSIC-
4061, 2146 W. 39th Ave., Kansas City, KS 66160. Phone: 913-
588-9842. Fax: 913-588-7501. E-mail: BTZhu@kumc.edu.
Author Contributions
X.-R.J. and P.W. contributed equally to this study.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by grants from the National Institutes
of Health (Grant No. CA097109 and ES015242 to B.T.Z. and
DK53002 to C.L.S.).
ABBREVIATIONS USED
■
ER, estrogen receptor; ERα and ERβ, the α and β subtype of
ER, respectively; E2, 17β-estradiol; RBA, relative binding
affinity; LBD, ligand binding domain
REFERENCES
■
(1) Cancer Facts & Figures 2009; American Cancer Society. http://
www.cancer.org/docroot/STT/content/STT_1x_Cancer_Facts_
Figures_2009.asp?from=fast.
(2) Jordan, V. C. Antiestrogens and selective estrogen receptor
modulators as mutifunctional medicines. 1. Receptor interactions. J.
Med. Chem. 2003, 46, 883−908.
(3) Fisher, B.; Costantino, J. P.; Wickerham, D. L.; Cecchini, R. S.;
Cronin, W. M.; Robidoux, A.; Bevers, T. B.; Kavanah, M. T.; Atkins, J.
N.; Margolese, R. G.; Runowicz, C. D.; James, J. M.; Ford, L. G.;
Wolmark, N. Tamoxifen for the prevention of breast cancer: current
status of the National Surgical Adjuvant Breast and Bowel Project P-1
study. J. Natl. Cancer Inst. 2005, 97, 1652−1662.
Computational Modeling. A docking study was performed with
the InsightII modeling software (Version 2005, Accelrys Inc. San
Diego, CA) installed in a Dell Precision 690 workstation with the Red
Hat Enterprise Linux WS4.0 operating system (Red Hat Inc. Raleigh,
NC). Energy minimization and molecular dynamics simulation were
performed with the Discovery Studio modeling program (Version 1.7,
Accelrys Inc. San Diego, CA) installed on the same computer. The
CHARMm force field in DiscoveryStudio was used for molecular
dynamics simulation.
(4) (a) Howell, A.; Osborne, C. K.; Morris, C.; Wakeling, A. E. ICI
182,780 (Faslodex): Development of a novel, “pure” antiestrogen.
Cancer 2000, 89, 817−825. (b) Wakeling, A. E.; Bowler, J. Steroidal
pure antioestrogens. J. Endocrinol. 1987, 112, R7−R10.
(5) Lin, H. R.; Safo, M. K.; Abraham, D. J. Identification of a series of
tetrahydroisoquinoline derivatives as potential therapeutic agents for
breast cancer. Bioorg. Med. Chem. Lett. 2007, 17, 2581−2589.
(6) Hussey, S. L.; He, E.; Peterson, B. R. Synthesis of chimeric 7α-
substituted estradiol derivatives linked to cholesterol and cholesteryl-
amine. Org. Lett. 2002, 4, 415−418.
Because the crystal structure of human ERα in complex with a full
antagonist is presently not available, the crystallographic structure of
ERα in complex with raloxifen (PDB code: 1ERR26), which is a partial
agonist/antagonist, was used as a template for the docking study of
ICI-182,780 and the newly synthesized ER antagonists. The structures
of ICI-182,780 and ER antagonists were first built using the Builder
module and then minimized with the Discover module of InsightII.
The docking method was described in our earlier study.14 In brief, the
flexible docking was carried out using the Simulated Annealing
Docking method in the Affinity module of InsightII. The binding
K
dx.doi.org/10.1021/jm3013773 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(7) Marc, B. S.; Frank, R. W.; John, A. K. Synthesis and binding
affinities of novel Re-containing 7α-substituted estradiol complexes:
models for breast cancer imaging agents. J. Org. Chem. 1999, 64,
8108−8121.
Carlquist, M. Molecular basis of agonism and antagonism in the
oestrogen receptor. Nature 1977, 389, 753−758.
(8) Termblay, M. R.; Simard, J.; Poirier, D. Parallel solid-phase
synthesis of a model library of 7α-alkylamide estradiol derivatives as
potential estrogen receptor antagonists. Bioorg. Med. Chem. Lett. 1999,
9, 2827−2832.
(9) Tedesco, R.; Katzenellenbogen, J. A.; Napolitano, E. An
expeditious route to 7α-substituted estradiol derivatives. Tetrahedron
Lett. 1997, 38, 7997−8000.
(10) French, A. N.; Wilson, S. R.; Welch, M. J.; Katzenellenbogen, J.
A. A synthesis of 7α-subatituted estradiols: synthesis and biological
evaluation of a 7α-pentyl-substituted BODIPY fluorescent conjugate
and a fluorine-18-labeled 7α-pentylestradiol analog. Steroids 1993, 58,
157−169.
(11) Bowler, J.; Lilley, T. J.; Pittam, J. D.; Wakeling, A. E. Novel
steroidal pure antiestrogens. Steroids 1989, 54, 71−99.
(12) Tzukerman, M. T.; Esty, A.; Santiso-Mere, D.; Danielian, P.;
Parker, M. G.; Stein, R. B.; Pike, J. W.; McDonnell, D. P. Human
estrogen receptor transactivational capacity is determined by both
cellular and promoter context and mediated by two functionally
distinct intramolecular regions. Mol. Endocrinol. 1994, 8, 21−30.
(13) Lippman, M. E.; Bolan, G. Oestrogen-responsive human breast
cancer in long-term tissue culture. Nature 1975, 256, 592−593.
(14) Lippman, M. E.; Bolan, G.; Huff, K. The effects of estrogens and
antiestrogens on hormone-responsive human breast cancer in long-
term tissue culture. Cancer Res. 1976, 36, 4595−4601.
(15) Chakravarti, D.; LaMorte, V. J.; Nelson, M. C.; Nakajima, T.;
Schulman, I. G.; Juguilon, H.; Montminy, M.; Evans, R. M. Role of
CBP/P300in nuclear receptor signalling. Nature 1996, 383, 99−103.
(16) Jaber, B. M; Gao, T.; Huang, L.; Karmakar, S.; Smith, C. L. The
pure estrogen receptor antagonist ICI 182,780 promotes a novel
interaction of estrogen receptor-α with the 3′,5′-cyclin adenosine
monophosphate response element-binding protein-binding protein/
p300 coactivators. Mol. Endocrinol. 2006, 20, 2695−2710.
(17) Hoffman, K. L.; Foster, E. A.; Smith, C. L. The terminal
substituents of 7α,6-hexanyl derivatives of estradiol determine their
selective estrogen receptor modulator versus agonist activities. Steroids
2012, 77, 496−503.
(18) Zhu, B. T.; Shim, J. Y.; Nagai, M.; Bai, H. W. Molecular
modelling study of the mechanism of high-potency inhibition of
human catechol-O-methyltransferase by (-)-epigallocatechin-3-O-
gallate. Xenobiotica 2008, 38, 130−146.
(19) Pike, A. C.; Brzozowski, A. M.; Walton, J.; Hubbard, R. E.;
Thorsell, A. G.; Li, Y. L. Structural insights into the mode of action of a
pure antiestrogen. Structure 2001, 9, 145−153.
(20) Lee, A. J.; Cai, M. X.; Thomas, P. E.; Conney, A. H.; Zhu, B. T.
Characterization of the oxidative metabolites of 17β-estradiol and
estrone formed by fifteen selectively-expressed human cytochrome
P450 isoforms. Endocrinology 2003, 144, 3382−3398.
(21) Jiang, X. R.; Wang, P.; Fu, X.; Zhu, B. T. Chemical synthesis and
biochemical characterization of a biotinylated derivative of 17β-
estradiol with a long side chain covalently attached to its C-7alpha
position. Steroids 2008, 73, 1252−1261.
(22) Jiang, X. R.; Sowell, J. W.; Zhu, B. T. Synthesis of 7α-substituted
derivatives of 17β-estradiol. Steroids 2006, 71, 334−342.
(23) Krishnamoorthy, S.; Fang, X.; Brandom, M. C.; Su, L.; Hannah,
N. B.; Qian, W. A fluorogenic 1,3-dipolar cycloaddition reaction of 3-
azidocoumarins and acetylenes. Org. Lett. 2004, 6, 4603−4606.
(24) Wang, P.; Wen, Y.; Han, G. Z.; Sidhu, P. K.; Zhu, B. T.
Characterization of the oestrogenic activity of non-aromatic steroids:
are there male-specific endogenous oestrogen receptor modulators? Br.
J. Pharmacol. 2009, 158, 1796−1807.
(25) Jaber, B.; Mukopadhyay, R.; Smith, C. L. Estrogen receptor-α
interaction with the CREB bind protein coactivator is regulated by the
cellular environment. J. Mol. Endocrinol. 2004, 32, 307−323.
(26) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.;
Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G. L.; Gustafsson, J. A.;
̈
L
dx.doi.org/10.1021/jm3013773 | J. Med. Chem. XXXX, XXX, XXX−XXX