Synthesis of Triphenylethylene Bisphenols as Aromatase Inhibitors That Also Modulate Estrogen Receptors

Article abstract of DOI:10.1021/acs.jmedchem.5b01677

A series of triphenylethylene bisphenol analogues of the selective estrogen receptor modulator (SERM) tamoxifen were synthesized and evaluated for their abilities to inhibit aromatase, bind to estrogen receptor α (ER-α) and estrogen receptor β (ER-β), and antagonize the activity of β-estradiol in MCF-7 human breast cancer cells. The long-range goal has been to create dual aromatase inhibitor (AI)/selective estrogen receptor modulators (SERMs). The hypothesis is that in normal tissue the estrogenic SERM activity of a dual AI/SERM could attenuate the undesired effects stemming from global estrogen depletion caused by the AI activity of a dual AI/SERM, while in breast cancer tissue the antiestrogenic SERM activity of a dual AI/SERM could act synergistically with AI activity to enhance the antiproliferative effect. The potent aromatase inhibitory activities and high ER-α and ER-β binding affinities of several of the resulting analogues, together with the facts that they antagonize β-estradiol in a functional assay in MCF-7 human breast cancer cells and they have no E/Z isomers, support their further development in order to obtain dual AI/SERM agents for breast cancer treatment.

Full text of DOI:10.1021/acs.jmedchem.5b01677

Article
pubs.acs.org/jmc
Synthesis of Triphenylethylene Bisphenols as Aromatase Inhibitors
That Also Modulate Estrogen Receptors
Wei Lv,† Jinzhong Liu,‡ Todd C. Skaar,‡ Elizaveta O’Neill,† Ge Yu,† David A. Flockhart,‡
and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and The Purdue University Center for
Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States
‡Division of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202,
United States
S
* Supporting Information
ABSTRACT: A series of triphenylethylene bisphenol analogues of the selective estrogen receptor modulator (SERM) tamoxifen
were synthesized and evaluated for their abilities to inhibit aromatase, bind to estrogen receptor α (ER-α) and estrogen receptor
β (ER-β), and antagonize the activity of β-estradiol in MCF-7 human breast cancer cells. The long-range goal has been to create
dual aromatase inhibitor (AI)/selective estrogen receptor modulators (SERMs). The hypothesis is that in normal tissue the
estrogenic SERM activity of a dual AI/SERM could attenuate the undesired effects stemming from global estrogen depletion
caused by the AI activity of a dual AI/SERM, while in breast cancer tissue the antiestrogenic SERM activity of a dual AI/SERM
could act synergistically with AI activity to enhance the antiproliferative effect. The potent aromatase inhibitory activities and
high ER-α and ER-β binding affinities of several of the resulting analogues, together with the facts that they antagonize β-estradiol
in a functional assay in MCF-7 human breast cancer cells and they have no E/Z isomers, support their further development in
order to obtain dual AI/SERM agents for breast cancer treatment.
reduction in risk of distant metastasis when compared to
tamoxifen alone.4 The use of AIs is also reported to cause fewer
vaginal bleeding events, thromboembolic events, and endo-
metrial cancer occurrences than tamoxifen.4−6 However, the
use of AIs is associated with serious side effects. Since AIs
nonselectively deplete estrogen in the whole body, they lead to
severe musculoskeletal pain, reduction of bone density, and an
increased frequency of bone fractures and cardiovascular
events.7−11 According to the 5-year ATAC trial, anastrozole
treatment led to a higher incidence of bone fractures (11% vs
7.7%) and arthralgia (35.6% vs 29.4%) than tamoxifen.4 The
increased musculoskeletal pain caused by AIs negatively
impacts patient compliance. Reported AI discontinuation
rates attributed to severe musculoskeletal symptoms range
from 13% to 52%.12−14 Nonadherence rates are also high.15,16
For example, observations from three data sets indicate that
INTRODUCTION
■
Aromatase (also known as CYP19) is a member of the general
class of cytochrome P450 enzymes. It catalyzes the conversion
of androgens to estrogens, which is a crucial step in the
biosynthesis of estrogens in the human body.1 Aromatase
inhibitors (AIs) have been widely used for treatment of
hormone-receptor-positive breast cancer in postmenopausal
women. Currently, three AIs [letrozole (1), anastrozole (2),
and exemestane (3), Figure 1] have been approved by the
FDA. Comparative clinical trials involving postmenopausal
women with breast cancer have demonstrated that AIs are
superior to the selective estrogen receptor modulator (SERM)
tamoxifen (4) (Figure 1), which, as with other SERMs, blocks
estrogen receptors in breast cancer tissue while stimulating
them in a variety of normal tissues.2−6 In the 5-year ATAC
(arimidex, tamoxifen, alone or in combination) trial, the use of
anastrozole alone resulted in a 13% improvement of disease-
free survival, 21% reduction in the time-to-recurrence, 42%
reduction in occurrence of contralateral breast cancer, and 14%
Received: August 3, 2015
© XXXX American Chemical Society
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Figure 1. Structures of the aromatase inhibitors letrozole, anastrozole, exemestane, and the selective estrogen receptor modulator tamoxifen.
Figure 2. Structures and biological activities of (E,Z)-norendoxifen, Z-norendoxifen, E-norendoxifen, and 4′-hydroxynorendoxifen.
only 62−79% of women adhere (take anastrazole more than
80% of the days during the treatment period) after 3 years.16
One possible approach to improve the efficacy and decrease
the side effects associated with AIs is to build SERM activity
into them. The estrogenic activity of a dual AI/SERM due to
binding to and stimulation of estrogen receptors (ERs) in
noncancerous musculoskeletal tissue could counteract some of
the negative effects of the dual AI/SERM that result from
global estrogen loss due to aromatase inhibition. On the other
hand, the antagonistic blockade of ERs in breast cancer cells by
a dual AI/SERM might act synergistically with the decrease in
estrogen concentration due to aromatase inhibition, assuming
that the inhibition of estrogen production is not totally
complete. In other words, as with the SERMs, the ER agonist
effect of a dual AI/SERM would be beneficial in normal,
noncancerous musculoskeletal tissue relative to an AI alone by
decreasing the side effects that result from estrogen depletion,
while the ER antagonist effects of a dual AI/SERM would be
beneficial in breast cancer cells by blocking the effect of residual
estrogen resulting from incomplete aromatase inhibition. In
fact, according to Brodie et al., a combination of the aromatase
inhibitor letrozole and the estrogen receptor antagonist/down-
regulator fulvestrant was more effective than either letrozole or
fulvestrant alone in suppressing breast tumor growth and in
delaying the development of tumor resistance.17−19 In that case,
the delay in development of resistance was thought to result
from down-regulation of the ER by fulvestrant and an
associated down-regulation of signaling proteins that play a
role in the maintenance of hormonal resistance.19 Meanwhile, it
is also possible that the estrogenic component of the SERM
activity of a dual AI/SERM agents could stimulate estrogen
receptors in noncancerous musculoskeletal tissues and
ameliorate the side effects caused by estrogen depletion of
conventional AIs (e.g., osteoporosis, musculoskeletal pain, and
bone fractures). In fact, according to the ATAC trail, a
combination of anastrozole with tamoxifen resulted in fewer
fractures than when anastrozole was used alone.20 As expected,
the combination resulted in more fractures than tamoxifen
alone, indicating that the fractures were the result of the AI and
not the SERM.20 For these reasons, dual AI/SERM agents
could possibly have superior efficacy and decreased side effects
compared to conventional AIs alone. However, the combina-
tion of anastrozole with tamoxifen resulted in a greater
incidence of endometrial cancer (0.3%) than anastrozole
alone (0.1%), and in this regard it may be better to combine
raloxifene-type SERM activity than tamoxifen-type SERM
activity with an AI (different SERMs produce unique spectra
of agonist activities in different normal tissues because they
have different affinities for ER-α and ER-β, which are expressed
to different extents in various tissues, and the consequences of
binding to ER-α and ER-β are different).21 Although the ATAC
trial showed no therapeutic advantage of a specific tamoxifen
plus anastrozole combination with regard to anticancer
activity,20 this result should not be generalized to all SERM
plus AI combinations or to a hypothetical dual AI/SERM agent.
Of note, the serum concentration of anastrozole, a relatively
weak aromatase inhibitor, was 27% lower in the combination
arm of the ATAC trial, although it was claimed that this was of
no pharmacological significance.22
Norendoxifen is a metabolite of tamoxifen, and it is also a
potent aromatase inhibitor.23 The synthesis of (E,Z)-
norendoxifen (5) was reported in 2013.24 Biological testing
results confirmed the aromatase inhibitory activity of (E,Z)-
norendoxifen and further established high affinity for both ER-
α and ER-β (Figure 2), establishing (E,Z)-norendoxifen as the
first substance with potential dual AI and ER binding
activity.24,25 The E- and Z-norendoxifen isomers (E-5 and Z-
5) were also prepared via stereoselective synthetic routes, and
their biological activities revealed that E-norendoxifen is the
more potent aromatase inhibitor, while Z-norendoxifen
displayed greater affinity for both ER-α and ER-β.24 To
optimize efficacy and CYP selectivity, a series of norendoxifen
analogues were subsequently designed and prepared using a
structure-based drug design approach. This led to the discovery
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a
Table 1. Aromatase Inhibitory Activity and Estrogen Receptor Binding Affinities of Triphenylethylene Bisphenols
a
The values are mean values of at least three experiments. Percent ER competition was determined at the concentration of 100 μM for each
compound. EC50 values were determined only for compounds that displayed >90% competition.
Figure 3. Design strategy for triphenylethylene bisphenol analogues.
of 4′-hydroxynorendoxifen (6), which has elevated potency
against aromatase and higher affinity for ER-α and ER-β. It is
also a more potent antagonist of estradiol-stimulated
progesterone receptor mRNA expression in MCF-7 cells
compared to norendoxifen.26
Like 4-hydroxytamoxifen, most norendoxifen analogues
undergo facile E/Z isomerization in solution.24 Although the
detailed mechanism is still controversial, the E/Z isomerization
is considered to be facilitated by the presence of a phenolic
hydroxyl group in one of the para positions.27 The rate of
isomerization speeds up as the number of para phenolic
hydroxyl group increases, and the rate is also dependent on the
solvent and temperature.28 The E/Z isomerization makes the
preparation of pure E and Z isomers of norendoxifen analogues
difficult. Moreover, since isomerization happens both in stock
solutions and during biological testing, it also influences the
accuracy of the biological testing results for pure E and Z
isomers. To develop more promising norendoxifen analogues
for treatment of breast cancer, it is important to prepare
analogues as pure E and Z isomers. A mixture of E and Z
isomers would complicate the pharmacological profiles and
limit the use of the drugs because the E and Z isomers would be
expected to have different biological activities against
aromatase, ERs, and other CYPs. This argument is in fact
supported by results derived from testing tamoxifen, which
document different relative binding affinities of the two isomers
for the ER, as well as different agonist vs antagonist properties
as monitored by uterine weight.29 Furthermore, the isomer-
ization of (Z)-4-hydroxytamoxifen to its less active isomer is
thought to contribute to tamoxifen resistance.30
To overcome the problem presented by E/Z isomerization, a
series of triphenylethylene bisphenol analogues were designed
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a
Scheme 1. Synthesis of Analogues 11−13
a
Reagents and conditions: (a) 4,4′-dihydroxybenzophenone, Zn, TiCl4, THF; (b) NaH, MOMCl, THF; (c) NBS, CCl4; (d) KCN, THF, H2O; (e)
methanol, HCl; (f) NaH, imidazole, THF; (g) NaH, 1,2,4-triazole, THF.
a
Scheme 2. Synthesis of Analogues 17−20
a
Reagents and conditions: (a) NaH, PvCl, THF; (b) 4,4′-dihydroxybenzophenone, Zn, TiCl4, THF; (c) NaH, MOMCl, THF; (d) NBS, CCl4; (e)
KCN, THF, H2O; (f) methanol, HCl; (g) NaH, imidazole, THF; (h) KOH, THF, H2O; (i) NaH, 1,2,4-triazole, THF.
by eliminating the aminoethoxy side chain of norendoxifen,
thus resulting in two identical substituents on one end of the
double bond and eliminating the possibility of E/Z isomers.
This allowed a straightforward evaluation of the aromatase
inhibitory activities, ER-α and ER-B binding affinities, and
abilities of the compounds to antagonize β-estradiol-stimulated
transcriptional activity in MCF-7 human breast cancer cells.
The results of these studies will facilitate the development of a
new generation of dual AI/SERM agents for breast cancer
treatment.
groups) on the meta or para positions of the “A” ring; (2)
introduction of iron-coordinating groups (nitrile, imidazole, or
triazole groups) in the location of the ethyl group. Hydrogen
bond donors on the “A” ring can be expected to form hydrogen
bonds with aromatase and the ERs, while iron-coordinating
groups could improve aromatase inhibitory activity by
coordinating to the iron of aromatase.
Synthesis. A short and efficient synthetic route was
established to prepare analogues with an iron-coordinating
group in the location of the ethyl side chain (Scheme 1). The
bisphenol 9 was first prepared by McMurry cross-coupling of
acetophenone (8) with 4,4′-dihydroxybenzophenone as
described.25 The bisphenol 9 was treated with an excess of
MOMCl to afford the diprotected product 10 in good yield.
The protected intermediate 10 underwent a series of reactions,
including bromination with NBS, alkylation of potassium
cyanide, and deprotection of the MOM groups with HCl to
afford the nitrile 11 in very good yield. The imidazole product
12 and triazole compound 13 were also obtained in good yield
by treating 10 with similar sequential reactions, including
RESULTS AND DISCUSSION
■
Design of Triphenylethylene Bisphenol Analogues.
Compound 7 is a weak aromatase inhibitor, and it also shows
moderate binding affinities to ER-α and ER-β (Table 1).24 This
substance is also a good ER antagonist without significant
agonistic side effects in MCF-7 cells.31 On the basis of the
structure of compound 7, the following structural modifications
were explored to improve the potency (Figure 3): (1)
incorporation of hydrogen bond donors (hydroxyl or amino
D
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a
Scheme 3. Synthesis of Analogues 25−28
a
Reagents and conditions: (a) Zn, TiCl4, THF; (b) Boc2O, dioxanes; (c) NBS, CCl4; (d) KCN, H2O, THF; (e) HCl, methanol; (f) NaH, imidazole,
THF; (g) KOH, H2O, THF; (h) NaH, 1,2,4-triazole, THF.
a
Scheme 4. Synthesis of Analogue 32
a
Reagents and conditions: (a) NaH, PvCl, THF; (b) 4,4′-dihydroxybenzophenone, Zn, TiCl4, THF; (c) NaH, MOMCl, THF; (d) NBS, CCl4; (e)
NaH, imidazole, THF; (f) KOH, H2O, methanol; (g) HCl, methanol.
a
Scheme 5. Synthesis of Analogue 35
a
Reagents and conditions: (a) 22, Zn, TiCl4, THF; (b) Boc2O, dioxanes; (c) NBS, CCl4; (d) NaH, imidazole, THF; (e) KOH, H2O, methanol; (f)
HCl, methanol.
bromination with NBS, alkylation of imidazole or 1,2,4-triazole,
and cleavage of the phenols.
protecting groups were removed with HCl to directly provide
the products 17a,b. To prepare the imidazole products 18a,b,
compounds 16a,b underwent a series of sequential reactions
including bromination with NBS, alkylation of imidazole,
deprotection of the pivaloyl group with KOH, and removal
of the MOM groups under acidic conditions to afford 18a,b in
good yield. Interestingly, subjection of 16a,b to a similar
sequence of reactions incorporating 1,2,4-triazole instead of
imidazole led to the production of two isomers in each case
(i.e., 19a and 20a were obtained from 16a, and 19b and 20b
were obtained from 16b) due to the presence of two
nonequivalent nucleophilic nitrogens in the 1,2,4-triazole
system vs only one for the imidazole case. Compounds 19a
and 19b were isolated as the major products, and compounds
20a and 20b were the minor products.
Analogues 17a, 18a, 19a, and 20a were designed by
incorporating a hydroxyl group on the “A” ring to probe the
importance of a hydrogen bond donor in the para position. For
analogues 17b, 18b, 19b, and 20b, a fluorine atom was
introduced ortho to the “A” ring hydroxy group. The presence
of the electronegative fluorine atom would increase the acidity
of the hydroxy group and enable stronger hydrogen bonds to
be formed. To prepare analogues 17−20, the corresponding
hydroxylated acetophenones 14a,b were first protected with a
pivaloyl group and the product was reacted with 4,4′-
dihydroxybenzophenone under the McMurry cross-coupling
conditions to provide the bisphenols 15a,b (Scheme 2). The
phenolic hydroxyl groups were protected by MOM groups to
afford 16a,b. Compounds 16a,b were brominated with NBS,
followed by alkylation of KCN, to install the nitrile group.
Unexpectedly, the pivaloyl group was also cleaved under the
alkylation reaction conditions. In the next step, the MOM
In order to prepare analogues with an amino group in the
para position of the “A” ring, 4-aminoacetophenone (21) was
reacted with the diprotected 4,4′-dihydroxybenzophenone 22
under McMurry cross-coupling reaction conditions to afford 23
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(Scheme 3). The amino group was protected with a Boc group,
and the product 24 was subjected to a series of sequential
reactions, including bromination with NBS, alkylation of KCN
(both pivaloyl groups were cleaved under these conditions),
and removal of the Boc group with HCl to afford the product
25 in very good yield. The imidazole product 26 and triazole
products 27 and 28 were also obtained by subjecting 24 to a
similar set of reactions.
Analogue 32 was designed to probe the effect of introducing
a hydroxyl group in the meta position of the “A” ring. The
synthesis of 32 is outlined in Scheme 4. The phenolic hydroxyl
group of 29 was first protected with a pivaloyl group. The
product 30 reacted with 4,4′-dihydroxybenzophenone under
McMurry cross-coupling reaction conditions, followed by
protection of the phenolic hydroxyl groups with MOMCl, to
afford 31. Compound 31 underwent bromination with NBS,
alkylation of imidazole, and removal of the pivaloyl group and
MOM groups to afford 32 in good yield.
To synthesize analogue 35 with a meta amino group in the
“A” ring, 3-aminoacetophenone (33) was reacted with the
diprotected 4,4′-dihydroxybenzophenone 22 under McMurry
cross-coupling reaction conditions, followed by protection of
the amino group with a Boc group to afford 34 (in Scheme 5).
Then, compound 34 underwent bromination with NBS,
alkylation with imidazole, and cleavage of the pivaloyl group
and Boc group to provide 35 in good yield.
uniformly unfavorable for ER binding affinity. The imidazole 26
displayed much weaker binding affinities with ER-α (EC50
=
1830 nM) and ER-β (EC50 = 296 nM) compared with
compound 12, while compounds 25, 27, and 28 all have weak
binding affinities with ER. Rotating the “A” ring para hydroxyl
group to the meta position (32 vs 18a) did not influence
aromatase inhibitory activity, but it decreased the binding
affinities with ER-α and ER-β significantly. Rotating the “A”
ring para amino group to the meta position (35 vs 26)
decreased both aromatase inhibitory activity and ER binding
affinities.
Transcriptional Activities in MCF-7 Human Breast
Cancer Cells. To investigate the effects of ligand binding on
ER-mediated transcriptional activities, the triphenylethylene
bisphenols were tested for their abilities to antagonize β-
estradiol (E2) in a functional assay. Four compounds (12, 18a,
18b, and 26) were selected for this test because of their high
binding affinities to both ER-α and ER-β. In this commonly
used assay, the progesterone receptor (PGR) mRNA
expression is used for assessing estrogenic or antiestrogenic
activity in MCF-7 human breast cancer cells.32 As shown in
Figure 4, E2 (10 nM) was able to significantly increase the PGR
Biological Activities. The aromatase inhibitory activities
and ER-α/ER-β binding affinities of the bisphenols are
summarized in Table 1. Compound 11 with a nitrile side
chain showed slightly improved aromatase inhibitory activity
(IC50 = 12 800 nM) when compared with compound 7 (IC50
=
24 900 nM), but it only displayed very weak binding affinity for
both ER-α and ER-β. The imidazole compound 12 was the
most potent aromatase inhibitor (IC50 = 4.77 nM), and it also
retained high binding affinities with both ER-α (EC50 = 27.3
nM) and ER-β (EC50 = 40.9 nM). Compound 13 with the
triazole side chain was also a good aromatase inhibitor (IC50
=
137 nM) but had weak ER binding affinity. Similar structure−
activity relationships were also observed for compound series
17a−20a and series 17b−20b. The nitrile compounds (17a
and 17b) are weak aromatase inhibitors (IC50 = 15200−17200
nM), and they showed no binding affinity for ER-α and ER-β.
The triazole compounds (19a,b and 20a,b) are moderate
aromatase inhibitors (IC50 = 2980−14200 nM), but they only
showed weak binding affinities for ER-α (EC50 ≥ 943 nM) and
ER-β (EC50 ≥ 1080 nM). The imidazole compounds (18a and
18b) are very potent aromatase inhibitors (IC50 = 60.0−94.4
nM), and they also displayed good binding affinities for ER-α
(IC50 = 85.2−97.8 nM) and ER-β (IC50 = 56.3−73.6 nM).
Compared with the “A” ring unsubstituted analogues 11−13,
introducing a hydroxyl group in the para position of the “A”
ring (analogues 17a−20a) unexpectedly resulted in moderate
decreases in aromatase inhibitory activity or ER binding
affinities. A comparison of series 17b−20b with series 17a−
20a reveals that incorporating a fluorine atom ortho to the
hydroxyl group produced minor effects on aromatase inhibitory
activity and ER-α/ER-β binding affinity; except in the case of
the two triazole systems, it significantly decreased ER-α/ER-β
affinity. The introduction of an amino group in the para
position of the “A” ring (analogues 25−28) produced minor
effects on aromatase inhibitory activity, or in the case of the
nitriles 11 vs 25, it increased the inhibitory activity dramatically
(IC50 of 12 800 vs 36.3 nM). However, the para amino group is
Figure 4. Abilities of compounds 12, 18a, 18b, 26, endoxifen, and
(E,Z)-norendoxifen (1 μM) to antagonize β-estradiol (E2, 10 nM)-
stimulated progesterone receptor (PGR) mRNA expression in MCF-7
cells.
mRNA expression compared to the control, which contained
only 0.1% methanol (vehicle). The PGR mRNA expression
level with 10 nM E2 stimulation alone was set as 100%, and the
antiestrogenic effects of the compounds were monitored by the
reduction of stimulated mRNA levels. Endoxifen (positive
control) can antagonize the PGR mRNA expression in the
presence of 10 nM E2 to 10%, which is consistent with the
published result.32 (E,Z)-Norendoxifen can also antagonize the
stimulatory effects of E2, as PGR mRNA expression level was
reduced to 33% as we previously reported.25 All of the tested
triphenylethylene bisphenol analogues (12, 18a, 18b, and 26)
were able to antagonize the ER-stimulated PGR mRNA
expression to the levels of 26−31%, regardless of their different
binding affinities with estrogen receptors. The antiestrogenic
effects of the tested compounds as monitored by the reduction
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Figure 5. (a) The hypothetical binding mode of 12 (green) in the active site of aromatase (PDB code 3s79)33 overlapped with E-norendoxifen
(pink). (b) The hypothetical binding mode of 12 (green) in the active site of ER-α (PDB code 3ert)34 overlapped with Z-norendoxifen (pink).
Figure 6. Hypothetical binding mode of compound 36 (cyan) in the active site of aromatase (PDB code 3s79)33 overlapped with E-norendoxifen
(pink), and a comparison of aromatase inhibitory activities of compounds 7, 5, 12, and 36.
of estradiol-stimulated mRNA levels were weaker than
endoxifen but very similar to (E,Z)-norendoxifen.
result that is not surprising since the imidazole fragment was
installed in 12 in a location for iron binding based on the
structure calculated for the E-norendoxifen−aromatase com-
plex. The imidazole group faces toward the heme and
coordinates with the iron. One of the phenolic hydroxy groups
forms a hydrogen bond with the backbone carbonyl group of
Leu372, which is similar to E-norendoxifen binding. A notable
difference between 12 and E-norendoxifen can be observed in
their interaction with Ser478 and Asp309. For E-norendoxifen,
a hydrogen bond was observed between the ether oxygen and
the side chain hydroxyl group Ser478. For compound 12,
because of the size of the imidazole group, the whole molecule
moves “up” (further away from the heme) when compared with
E-norendoxifen. Due to this move, the other phenolic hydroxy
group moves away from Ser478 and approaches Asp309 with
the formation of a hydrogen bond with the backbone carbonyl
group of Asp309.
Molecular Modeling. Molecular docking studies were
performed in order to investigate the possible binding mode of
the triphenylethylene bisphenol analogues with aromatase and
ER-α. Compound 12 was docked into the active site of
aromatase (PDB code 3s79)33 with GOLD 3.0. The high
potency of 12 compared with 7 (>5000-fold improvement in
aromatase inhibitory activity) indicates imidazole−iron bond-
ing. Therefore, a distance constraint (1.5−3.5 Å) was imposed
between the imidazole nitrogen and iron during docking. The
best docking pose of 12 (shown in Figure 5a) was overlapped
with the hypothetical binding mode of E-norendoxifen (E-5)
that was previously reported (see Supporting Information for
more detailed molecular modeling results, including stereo-
views, hydrogen bond angles, and distances calculated between
hydrogens and hydrogen bond acceptors).24 The binding mode
of 12 is in general very similar to that of E-norendoxifen, a
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To explore the binding mode with ER-α, compound 12 was
docked into the active site of ER-α (PDB code 3ert)34 with
GOLD 3.0. The best docking pose of 12 (shown in Figure 5b)
was overlapped with the published hypothetical binding mode
of Z-norendoxifen (Z-5). According to the docking results, the
binding mode of 12 is nearly identical to that of Z-
norendoxifen. The imidazole group is situated in the ethyl
binding pocket surround by Met421, Met388, and Leu428. One
of the phenolic hydroxy groups forms bifurcated hydrogen
bonds with Arg394 and Glu353. The other phenolic hydroxy
group projects toward the outside of the ligand binding pocket
and forms a hydrogen bond with Thr347.
Our previous testing results showed that the aminoethoxyl
side chain of (E,Z)-norendoxifen (5) is favorable for aromatase
inhibitory activity (>240-fold increase compared with 7, Figure
6).24 In this report, the imidazole group of compound 12 is also
demonstrated to be optimal for aromatase inhibitory activity
(>5000-fold increase compared with 7). Unfortunately,
combining the aminoethoxyl side chain and imidazole group
in one molecule (compound 36) did not result in a more
potent aromatase inhibitor than 12.26 The failure of combining
optimal substitutions to afford the most potent compound can
be attributed to the hypothetical imidazole-induced binding
mode movement as noted for compound 12. The hypothetical
binding mode of compound 36 overlapped with the hypo-
thetical binding mode of E-norendoxifen (E-5) is shown in
Figure 6. Similar to compound 12, the imidazole nitrogen of 36
coordinates with the iron. Because of the size of the imidazole
group, the whole molecule 36 also moves “up” (further away
from the heme) when compared with E-norendoxifen. This
movement pushes the ether oxygen away from Ser478
(distance 3.7 Å vs 3.3 Å of E-norendoxifen) and weakens the
hydrogen bond between the ether oxygen and the side chain of
Ser478. This movement also pushes the aminoethoxy side
chain away from Asp309, resulting in the loss of the salt bridge
interaction (between the protonated amino group and the
carboxyl group of Asp309) and hydrogen bond (between the
protonated amino group and the backbone carbonyl group of
Asp309). Therefore, the aminoethoxy side chain of compound
36 cannot contribute positively to the aromatase inhibitory
activity.
therapeutically useful dual AI/SERM agents. Any future
biological work with these compounds should provide a
detailed profile of their agonistic and antagonistic activities in
various organs and tissues in animal models. The facts that
compounds 12, 18a, and 18b have high binding affinity to ER-α
and ER-β and are analogues of the SERM tamoxifen argue in
favor of the possibility of them being estrogen antagonists in
breast tissue and estrogenic agonists in other tissues.
EXPERIMENTAL SECTION
■
General. Melting points were determined using capillary tubes with
a Mel-Temp apparatus and are uncorrected. The nuclear magnetic
resonance (1H and 13C NMR) spectra were recorded using a Bruker
ARX300 spectrometer (300 MHz) with a QNP probe or a Bruker
DRX-2 spectrometer (500 MHz) with a BBO probe. High-resolution
mass spectra were recorded on a double-focusing sector mass
spectrometer with magnetic and electrostatic mass analyzers. The
purities of biologically important compounds were determined by
HPLC or elemental analyses. For elemental analyses, the observed
percentages differ less than 0.40% from the calculated values. For
HPLC, the major peak accounted for ≥95% of the combined total
peak area when monitored by a UV detector at 254 nm. The HPLC
analyses were performed on a Waters 1525 binary HPLC pump/
Waters 2487 dual λ absorbance detector system using a 5 μm C18
reversed phase column. The cytochrome P450 (CYP) inhibitor
screening kit for aromatase (CYP19) was purchased from BD
Biosciences (San Jose, CA). Estrogen receptor α and β competitor
assay kits were purchased from Invitrogen (Carlsbad, CA).
General Procedure for the McMurry Cross-Coupling
Reaction. Zinc powder (653 mg, 10 mmol) was suspended in dry
THF (8 mL), the mixture was cooled to 0 °C, and then TiCl4 (0.55
mL, 5 mmol) was added dropwise under argon. When the addition
was complete, the mixture was warmed to room temperature and then
heated at reflux for 2 h. After cooling down, a solution of the
corresponding benzophenone (1 mmol) and ketone (3 mmol) in dry
THF (8 mL) was added, and the mixture was heated at reflux in the
dark for 3 h. After being cooled to room temperature, THF was
evaporated. The residue was dissolved in saturated NH4Cl aqueous
solution (20 mL) and extracted with ethyl acetate (20 mL × 4). The
organic layers were combined, dried over Na2SO4, concentrated in
vacuo, and further purified by silica gel column chromatography to
provide the McMurry cross-coupling product.
4-(1,1-Bis(4-hydroxyphenyl)prop-1-en-2-yl)phenyl Pivalate
(15a). A suspension of 14a (678 mg, 4.98 mmol) and NaH (206
mg, 95%, 8.15 mmol) in dry THF (5 mL) was stirred under argon for
10 min, and then pivaloyl chloride (0.90 mL, 7.31 mmol) was added.
The solution was stirred at room temperature for 3 h and quenched
with water (2 mL). The solvent was evaporated, and the residue was
dissolved with water (15 mL) and extracted with ethyl acetate (15 mL
× 4). The organic layers were combined, dried over Na2SO4, and
concentrated. The concentrated product was reacted with 4,4′-
dihydroxylbenzophenone (1.98 g, 9.24 mmol) according to the
general McMurry cross-coupling reaction procedure. The product was
purified by silica gel column chromatography (2:1 hexanes−ethyl
acetate) to afford the product 15a as a white solid (1.55 g, 77%): mp
138−140 °C. 1H NMR (300 MHz, methanol-d4 and CDCl3) δ 7.11−
7.08 (m, 2 H), 7.03−7.00 (m, 2 H), 6.80−6.74 (m, 4 H), 6.69−6.66
(m, 2 H), 6.48−6.45 (m, 2 H), 2.07 (s, 3 H), 1.29 (s, 9 H); 13C NMR
(75 MHz, methanol-d4 and CDCl3) δ 179.3, 156.7, 156.0, 150.1, 143.7,
140.7, 136.6, 136.3, 134.0, 133.5, 132.6, 131.7, 122.1, 116.1, 115.6,
40.4, 28.3, 24.5; ESIMS m/z (relative intensity) 425 (MNa+, 100);
HRESIMS m/z calcd for C26H27O4 (MH+) 403.1909, found 403.1916.
4-(1,1-Bis(4-hydroxyphenyl)prop-1-en-2-yl)-2-fluorophenyl
Pivalate (15b). A suspension of 14b (187 mg, 1.21 mmol) and NaH
(70.7 mg, 95%, 2.42 mmol) in dry THF (5 mL) was stirred at room
temperature under argon. Pivaloyl chloride (0.22 mL, 1.8 mmol) was
added dropwise. The mixture was stirred at room temperature for 3 h
and quenched with water (2 mL). The THF was evaporated, and the
residue was dissolved with 20% K2CO3 solution (20 mL) and
CONCLUSION
■
A series of triphenylethylene bisphenol analogues were
designed and synthesized by eliminating the aminoethoxy
side chain of (E,Z)-norendoxifen. The biological testing results
showed that an imidazole group in the location of the ethyl
group was optimal for both aromatase inhibitory activity and
ER binding affinities. The imidazole compounds 12, 18a, 18b,
and 26 displayed superior aromatase inhibitory activity
compared with (E,Z)-norendoxifen, while 12 also showed ER
binding affinities comparable with (E,Z)-norendoxifen and 18a
and 18b were slightly less active. These four imidazoles also act
as antagonists in the ER transcriptional assays in MCF-7 breast
cancer cells with activity similar to (E,Z)-norendoxifen. The
possible binding modes of the most potent compound 12 with
aromatase and ER-α were also investigated by molecular
modeling, and the results were used to rationalize the observed
activity trends. Since these triphenylethylene bisphenol
analogues have no possibility for the E/Z isomerization
problems encountered with the norendoxifen analogues that
were previously published, they introduce a superior class of
compounds that can be developed toward the goal of obtaining
H
DOI: 10.1021/acs.jmedchem.5b01677
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
extracted with ethyl acetate (15 mL × 3). The organic layers were
combined and dried over Na2SO4 and concentrated. The residue was
combined with 4 4′-dihydroxybenzophenone (0.80 g, 3.73 mmol) and
reacted according to the general McMurry cross-coupling reaction
procedure. The product was further purified by silica gel column
chromatography (7:3 hexanes−ethyl acetate) to afford the product
4-(1,1-Bis(4-(methoxymethoxy)phenyl)prop-1-en-2-yl)-2-flu-
orophenyl Pivalate (16b). The crude product was purified by silica
gel column chromatography (85:15 hexanes−ethyl acetate) to provide
the product 16b as a colorless oil (194 mg, 49%). 1H NMR (300 MHz,
CDCl3) δ 7.15−7.11 (m, 2 H), 7.02−6.99 (m, 2 H), 6.97−6.87 (m, 3
H), 6.82−6.79 (m, 2 H), 6.74−6.71 (m, 2 H), 5.19 (s, 2 H), 5.08 (s, 2
H), 3.50 (s, 3 H), 3.43 (s, 3 H), 2.10 (s, 3 H), 1.36 (s, 9 H);
MALDIMS m/z (relative intensity) 508 (M+, 80), 379 (100);
HRESIMS m/z calcd for C30H33FO6Na (MNa+) 531.2159, found
531.2143.
1
15b as yellow oil (348 mg, 68%). H NMR (300 MHz, CDCl3) δ
7.07−7.04 (m, 2 H), 7.04−6.84 (m, 3 H), 6.79−6.76 (m, 2 H), 6.74−
6.71 (m, 2 H), 6.52−6.49 (m, 2 H), 2.09 (s, 3 H), 1.35 (s, 9 H); EIMS
m/z (relative intensity) 420 (M+, 16), 57 (100); negative ion
HRESIMS m/z calcd for C26H24FO4 (M − H+)¯ 419.1659, found
419.1665.
General Procedure for the Preparation of 11, 17a,b, and 25.
A solution of 10 or 16a,b or 24 (0.169 mmol) and N-
bromosuccinimide (30.8 mg, 0.173 mmol) in CCl4 (5 mL) was
heated at reflux under argon for 3 h. After cooling down, the solid was
removed by filtration and the filtrate was concentrated in vacuo. The
residue was dissolved in THF (4.5 mL), and a solution of KCN (46.2
mg, 0.709 mmol) in water (1.5 mL) was added. The mixture was
stirred at room temperature overnight. The solvent was evaporated,
and the residue was dissolved with water (10 mL) and extracted with
ethyl acetate (10 mL × 3). The organic layers were combined, dried
over Na2SO4, and concentrated in vacuo. The residue was dissolved
with methanol (4.5 mL), and concentrated HCl (1 mL) was added.
The solution was stirred at room temperature overnight. The solvent
was removed, and the residue was dissolved with water (10 mL),
neutralized with NaHCO3, and extracted with ethyl acetate (10 mL ×
3). The organic layers were combined, dried over Na2SO4, and
concentrated in vacuo to provide the crude product 11 or 17a,b or 25.
4,4-Bis(4-hydroxyphenyl)-3-phenylbut-3-enenitrile (11). The
crude product was purified by silica gel column chromatography (2:1
hexanes−ethyl acetate) to provide the pure product 11 as an orange
solid (61.7 mg, 51%): mp 198−200 °C. 1H NMR (300 MHz,
methanol-d4 and CDCl3) δ 7.19−7.10 (m, 5 H), 7.09−7.05 (m, 2 H),
6.83−6.79 (m, 2 H), 6.69−6.65 (m, 2 H), 6.47−6.43 (m, 2 H), 3.50
(s, 2 H); 13C NMR (75 MHz, methanol-d4 and CDCl3) δ 157.9, 157.0,
145.9, 141.5, 134.7, 134.3, 133.3, 132.1, 130.9, 129.7, 128.5, 127.2,
120.0, 116.8, 115.8, 26.8; EIMS m/z (relative intensity) 327 (M+,
100); HREIMS m/z calcd for C22H17NO2 (M+) 327.1254, found
327.1264. Anal. Calcd for C22H17NO2·CH3OH: C, 76.86; H, 5.89; N,
3.90. Found: C, 76.88; H, 5.57; N, 3.93.
(2-(4-Aminophenyl)prop-1-ene-1,1-diyl)bis(4,1-phenylene)
Bis(2,2-dimethylpropanoate) (23). Zinc powder (1.12 g, 17.1
mmol) was suspended in dry THF (10 mL), and the mixture was
cooled to 0 °C. TiCl4 (0.8 mL, 7.2 mmol) was added dropwise under
argon. When the addition was complete, the mixture was warmed to
room temperature and then heated at reflux for 2 h. After cooling
down, a solution of 2235 (501 mg, 1.31 mmol) and 21 (175 mg, 1.29
mmol) in dry THF (10 mL) was added, and the mixture was heated at
reflux in the dark for 3 h. After the reaction mixture was cooled to
room temperature, THF was carefully evaporated. The residue was
dissolved with saturated ammonium chloride aqueous solution (20
mL) and extracted with ethyl acetate (20 mL × 4). The organic layers
were combined, dried over Na2SO4, concentrated in vacuo, and further
purified by silica gel column chromatography, eluting with 3:1
dichloromethane−hexanes, followed by 2:1 hexanes−ethyl acetate, to
afford the product 23 as yellow solid (284 mg, 45%): mp 208−210 °C.
1H NMR (300 MHz, CDCl3) δ 7.23−7.20 (m, 2 H), 7.05−7.02 (m, 2
H), 6.95−6.92 (m, 2 H), 6.91−6.88 (m, 2 H), 6.76−6.73 (m, 2 H),
6.51−6.48 (m, 2 H), 2.10 (s, 3 H), 1.37 (s, 9 H), 1.31 (s, 9 H); 13C
NMR (75 MHz, CDCl3) δ 177.1, 176.9, 149.5, 148.8, 144.8, 141.1,
140.7, 136.2, 136.1, 133.6, 131.8, 131.1, 130.3, 121.0, 120.4, 114.6,
39.1, 39.0, 27.1, 27.0, 23.3; ESIMS m/z (relative intensity) 508
(MNa+, 100); HRESIMS m/z calcd for C31H36NO4 (MH+) 486.2645,
found 486.2648.
General Procedure for the Preparation of 10 and 16a,b. A
solution of bisphenol (9 or 15a,b, 0.502 mmol) and NaH (53.0 mg,
95%, 2.10 mmol) in dry THF (5 mL) was stirred under argon for 10
min, and then methyl chloromethyl ether (0.16 mL, 2.10 mmol) was
added. The mixture was stirred at room temperature for 3 h and
quenched with saturated aqueous NaHCO3 solution (2 mL). The
solvent was evaporated, and the residue was dissolved in saturated
aqueous NaHCO3 solution (15 mL) and extracted with ethyl acetate
(15 mL × 4). The organic layers were combined, dried over Na2SO4,
and concentrated in vacuo to provide the crude product 10 or 16a,b.
4,4′-(2-Phenylprop-1-ene-1,1-diyl)bis((methoxymethoxy)-
benzene) (10). The crude product was purified by silica gel column
chromatography (9:1 hexanes−ethyl acetate) to provide the pure
3,4,4-Tris(4-hydroxyphenyl)but-3-enenitrile (17a). The crude
product 17a was purified by silica gel column chromatography (1:1
hexanes−ethyl acetate) to provide the pure product as red foam
1
(83%). H NMR (300 MHz, methanol-d4) δ 7.07−7.00 (m, 4 H),
6.84−6.80 (m, 2 H), 6.73−6.69 (m, 2 H), 6.66−6.62 (m, 2 H), 6.50−
6.46 (m, 2 H), 3.53 (s, 2 H); 13C NMR (75 MHz, methanol-d4) δ
158.2, 157.6, 157.2, 144.5, 135.1, 134.8, 133.1, 132.7, 132.0, 131.8,
127.7, 120.1, 116.4, 116.2, 115.4, 25.8; ESIMS m/z (relative intensity)
366 (MNa+, 100); HRESIMS m/z calcd for C22H17NO3Na (MNa+)
366.1106, found 366.1120. HPLC purity 99.8% (C-18 reverse phase,
methanol−H2O, 90:10).
1
product 10 as a colorless oil (69%). H NMR (300 MHz, CDCl3) δ
3-(3-Fluoro-4-hydroxyphenyl)-4,4-bis(4-hydroxyphenyl)but-
3-enenitrile (17b). The crude product was purified by silica gel
column chromatography (1:1 hexanes−ethyl acetate) to provide the
7.18−7.07 (m, 7 H), 7.04−7.00 (m, 2 H), 6.82−6.79 (m, 2 H), 6.71−
6.68 (m, 2 H), 5.21 (s, 2 H), 5.07 (s, 2 H), 3.52 (s, 3 H), 3.43 (s, 3 H),
2.14 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 155.9, 155.2, 144.3,
138.2, 137.3, 136.9, 134.7, 132.0, 131.2, 129.3, 127.9, 126.0, 115.7,
115.0, 94.4, 94.3, 56.0, 55.9, 23.4; ESIMS m/z (relative intensity) 413
(MNa+, 100); HRESIMS m/z calcd for C25H26O4Na (MNa+)
413.1729, found 413.1731.
4-(1,1-Bis(4-(methoxymethoxy)phenyl)prop-1-en-2-yl)-
phenyl Pivalate (16a). The crude product was purified by silica gel
column chromatography (85:15 hexanes−ethyl acetate) to provide the
pure product 16a as a colorless oil (81%). 1H NMR (300 MHz,
CDCl3) δ 7.16−7.13 (m, 4 H), 7.02−6.99 (m, 2 H), 6.88−6.85 (m, 2
H), 6.82−6.79 (m, 2 H), 6.73−6.69 (m, 2 H), 5.19 (s, 2 H), 5.08 (s, 2
H), 3.51 (s, 3 H), 3.43 (s, 3 H), 2.12 (s, 3 H), 1.34 (s, 9 H); 13C NMR
(75 MHz, CDCl3) δ 177.0, 155.9, 155.3, 149.1, 141.6, 138.5, 137.2,
136.7, 133.8, 132.0, 131.1, 130.2, 120.8, 115.7, 115.2, 94.4, 56.0, 39.0,
27.1, 23.4; ESIMS m/z (relative intensity) 513 (MNa+, 100);
HRESIMS m/z calcd for C30H34O6Na (MNa+) 513.2253, found
513.2272.
1
pure product 17b as white solid (70%): mp 180−183 °C. H NMR
(300 MHz, methanol-d4) δ 7.07−7.04 (m, 2 H), 6.91−6.87 (m, 1 H),
6.84−6.79 (m, 3 H), 6.78−6.75 (m, 1 H), 6.73−6.70 (m, 2 H), 6.53−
6.49 (m, 2 H), 3.55 (s, 2 H); EIMS m/z (relative intensity) 361 (M+,
100); negative ion HRESIMS m/z calcd for C22H15FNO3 (M − H+)¯
360.1036, found 360.1033. HPLC purity 99.3% (C-18 reverse phase,
methanol−H2O, 90:10).
3-(4-Aminophenyl)-4,4-bis(4-hydroxyphenyl)but-3-eneni-
trile (25). The crude product 25 was purified by silica gel column
chromatography (1:1 hexanes−ethyl acetate) to provide the pure
1
product 25 as light yellow solid (70%): mp 235−238 °C. H NMR
(300 MHz, methanol-d4) δ 7.06−7.03 (m, 2 H), 6.96−6.93 (m, 2 H),
6.82−6.79 (m, 2 H), 6.72−6.69 (m, 2 H), 6.59−6.56 (m, 2 H), 6.47−
6.44 (m, 2 H), 3.52 (s, 2 H); 13C NMR (75 MHz, methanol-d4) δ
158.1, 157.1, 147.9, 143.9, 135.3, 135.0, 133.1, 131.8, 131.6, 131.1,
128.0, 120.2, 116.4, 116.3, 115.3, 25.7; EIMS m/z (relative intensity)
342 (M+, 100); HRESIMS m/z calcd for C22H19N2O2 (MH+)
I
DOI: 10.1021/acs.jmedchem.5b01677
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
343.1447, found 343.1446. HPLC purity 97.8% (C-18 reverse phase,
methanol−H2O, 90:10).
with water (10 mL), neutralized with NaHCO3, and extracted with
ethyl acetate (10 mL × 3). The organic layers were combined, dried
over Na2SO4, concentrated in vacuo, and further purified by silica gel
column chromatography (9:1 dichloromethane−methanol) to provide
the product 18a,b or 26.
4,4′,4″-(3-(1H-Imidazol-1-yl)prop-1-ene-1,1,2-triyl)triphenol
(18a). Red glass (58%). 1H NMR (300 MHz, methanol-d4) δ 7.35 (s,
1 H), 7.06−7.03 (m, 2 H), 6.91 (s, 1 H), 6.88−6.79 (m, 5 H), 6.77−
6.73 (m, 2 H), 6.58−6.54 (m, 2 H), 6.49−6.45 (m, 2 H), 4.88 (s, 2
H); 13C NMR (75 MHz, methanol-d4) δ 158.0, 157.3, 157.1, 145.2,
138.3, 134.9, 133.0, 132.4, 132.1, 131.7, 128.6, 120.5, 116.4, 116.1,
115.4, 52.2; negative ion ESIMS m/z (relative intensity) 383 [(M −
H+)¯, 8], 315 (100); negative ion HREIMS m/z calcd for C24H19N2O3
(M − H+)¯ 383.1396, found 383.1398. HPLC purity 97.6% (C-18
reverse phase, methanol−H2O, 90:10).
General Procedure for the Preparation of 12 and 13. A
solution of 10 (186 mg, 0.476 mmol) and N-bromosuccinimide (81.7
mg, 0.459 mmol) in CCl4 (5 mL) was heated at reflux under argon for
3 h. After cooling down, the solid was removed by filtration and the
filtrate was concentrated in vacuo. The residue was dissolved in dry
THF (8 mL), and the solution was added to a solution of imidazole or
1,2,4-triazole (1.07 mmol) and NaH (30.1 mg, 1.25 mmol) in THF (4
mL) at 0 °C. The mixture was warmed to room temperature and
stirred under argon overnight. The reaction was quenched with
saturated NH4Cl aqueous solution (1 mL). The solvent was
evaporated, and the residue was dissolved with saturated NH4Cl
aqueous solution (25 mL) and extracted with ethyl acetate (25 mL ×
4). The organic layers were combined, dried over Na2SO4, and
concentrated in vacuo. The residue was dissolved with methanol (5
mL), concentrated HCl (0.4 mL), and heated at reflux for 0.5 h. The
solvent was removed, and the residue was neutralized with saturated
NaHCO3 solution (15 mL) and extracted with ethyl acetate (15 mL ×
4). The organic layers were combined, dried over Na2SO4,
concentrated in vacuo, and further purified by silica gel column
chromatography (95:5 dichloromethane−methanol) to provide the
product 12 or 13.
4,4′-(2-(3-Fluoro-4-hydroxyphenyl)-3-(1H-imidazol-1-yl)-
prop-1-ene-1,1-diyl)diphenol (18b). Orange foam (49%). 1H
NMR (300 MHz, methanol-d4) δ 7.41 (s, 1 H), 7.08−7.05 (m, 2
H), 6.97 (s, 1 H), 6.85 (s, 1 H), 6.82−6.79 (s, 2 H), 6.77−6.74 (m, 2
H), 6.73−6.65 (m, 3 H), 6.51−6.48 (m, 2 H), 4.92 (s, 2 H); ESIMS
m/z (relative intensity) 403 (MH+, 13), 335 (100); HRESIMS m/z
calcd for C24H20FN2O3 (MH+) 403.1458, found 403.1459. HPLC
purity 98.9% (C-18 reverse phase, methanol−H2O, 90:10).
4,4′-(3-(1H-Imidazol-1-yl)-2-phenylprop-1-ene-1,1-diyl)-
diphenol (12). The purified product was dissolved in DMSO (1 mL)
and then diluted with water (10 mL). The solid was collected by
filtration and dried in vacuo to provide the product 12 as a yellow solid
4,4′-(2-(4-Aminophenyl)-3-(1H-imidazol-1-yl)prop-1-ene-
1
1,1-diyl)diphenol (26). Yellow foam (59%). H NMR (300 MHz,
methanol-d4) δ 7.40 (s, 1 H), 7.05−7.01 (m, 2 H), 6.94 (s, 1 H),
6.84−6.73 (m, 7 H), 6.52−6.49 (m, 2 H), 6.47−6.43 (m, 2 H), 4.90
(s, 2 H); 13C NMR (75 MHz, methanol-d4) δ 158.0, 157.1, 147.6,
144.8, 138.2, 135.1, 133.2, 133.0, 131.7, 130.8, 128.3, 120.6, 116.3,
115.3, 52.2; ESIMS m/z (relative intensity) 384 (MH+, 18), 316
(100); HRESIMS m/z calcd for C24H22N3O2 (MH+) 384.1712, found
384.1725. HPLC purity 99.4% (C-18 reverse phase, methanol−H2O,
90:10), 99.8% (C-18 reverse phase, methanol−H2O, 85:15).
1
(150 mg, 89%): mp 225−227 °C. H NMR (300 MHz, DMSO-d6) δ
7.41 (s, 1 H), 7.10−6.99 (m, 8 H), 6.79−6.76 (m, 3 H), 6.69−6.66
(m, 2 H), 6.44−6.41 (m, 2 H), 4.89 (s, 2 H); 13C NMR (75 MHz,
DMSO-d6) δ 157.6, 156.8, 144.8, 140.8, 133.6, 132.7, 132.3, 131.0,
130.4, 128.8, 127.4, 119.9, 116.2, 115.3, 51.1; ESIMS m/z (relative
intensity) 369 (MH+, 33), 301 (100); HRESIMS m/z calcd for
C24H20N2O2Na (MNa+) 391.1422, found 391.1414. Anal. Calcd for
C24H20N2O2·1.3H2O: C, 73.56; H, 5.81; N, 7.15. Found: C, 73.47; H,
5.83; N, 6.90.
General Procedure for the Preparation of 19a,b, 20a,b, 27,
and 28. A solution of 16a,b or 24 (0.201 mmol) and N-
bromosuccinimide (35.8 mg, 0.201 mmol) in CCl4 (5 mL) was
heated at reflux under argon for 3 h. After cooling down, the solid was
removed by filtration and the filtrate was concentrated in vacuo. The
residue was dissolved in dry THF (6 mL) and the solution was added
to a solution of 1,2,4-triazole (42.6 mg, 0.62 mmol) and NaH (32 mg,
95%, 1.27 mmol) in THF (3 mL). The mixture was stirred at room
temperature overnight. The reaction was quenched with saturated
NH4Cl aqueous solution (1 mL). The solvent was evaporated, and the
residue was dissolved in saturated ammonium chloride aqueous
solution (10 mL) and extracted with ethyl acetate (10 mL × 3). The
organic layers were combined, dried over Na2SO4, and concentrated in
vacuo. The product was dissolved in THF (2.5 mL), and 2 N KOH
solution (2.5 mL) was added. The mixture was stirred at room
temperature overnight. The solvent was evaporated, and the residue
was dissolved with saturated aqueous ammonium chloride solution (15
mL) and extracted with ethyl acetate (10 mL × 3). The organic layers
were combined, dried over Na2SO4, concentrated in vacuo, and further
purified by silica gel column chromatography (95:5 dichloromethane−
methanol). The purified product was dissolved in methanol (4.5 mL),
and concentrated HCl (1 mL) was added. The mixture was stirred at
room temperature overnight. The solvent was evaporated, and the
residue was dissolved in water (10 mL), neutralized with NaHCO3,
and extracted with ethyl acetate (10 mL × 3). The organic layers were
combined, dried over Na2SO4, concentrated in vacuo, and further
purified by silica gel column chromatography (9:1 dichloromethane−
methanol) to first provide 19a,b or 27 and then 20a,b or 28.
4,4′-(2-Phenyl-3-(1H-1,2,4-triazol-1-yl)prop-1-ene-1,1-diyl)-
1
diphenol (13). Yellow solid (54 mg, 54%): mp 143−145 °C. H
NMR (300 MHz, methanol-d4 and CDCl3) δ 7.98 (s, 1 H), 7.90 (s, 1
H), 7.19−7.15 (m, 2 H), 7.08−7.02 (m, 5 H), 6.80−6.77 (m, 2 H),
6.73−6.69 (m, 2 H), 6.46−6.43 (m, 2 H), 5.20 (s, 2 H); 13C NMR (75
MHz, methanol-d4 and CDCl3) δ 158.0, 157.1, 147.3, 140.9, 134.5,
133.4, 132.1, 130.8, 129.7, 128.2, 116.6, 115.7, 56.0; EIMS m/z
(relative intensity) 369 (M+, 53), 300 (100); HREIMS m/z calcd for
C23H19N3O2 (M+) 369.1472, found 369.1484. Anal. Calcd for
C23H19N3O2·1.6CH3OH: C, 70.23; H, 6.09; N, 9.99. Found: C,
70.48; H, 5.70; N, 9.59.
General Procedure for the Preparation of 18a,b and 26. A
solution of 16a,b or 24 (0.127 mmol) and N-bromosuccinimide (24.8
mg, 0.139 mmol) in CCl4 (5 mL) was heated at reflux under argon for
3 h. After cooling down, the solid was removed by filtration and the
filtrate was concentrated in vacuo. The residue was dissolved in dry
THF (5 mL), and the solution was added to a solution of imidazole
(35.4 mg, 0.52 mmol) and NaH (27.1 mg, 95%, 1.07 mmol) in THF
(3 mL). The mixture was stirred at room temperature overnight. The
reaction was quenched with saturated NH4Cl aqueous solution (1
mL). The solvent was evaporated, and the residue was dissolved with
saturated NH4Cl aqueous solution (10 mL) and extracted with ethyl
acetate (10 mL × 3). The organic layers were combined, dried over
Na2SO4, and concentrated in vacuo. The product was dissolved in
THF (2.5 mL), and 2 N KOH solution (2.5 mL) was added. The
mixture was stirred at room temperature overnight. The solvent was
evaporated, and the residue was dissolved with saturated NH4Cl
aqueous solution (15 mL) and extracted with ethyl acetate (10 mL ×
3). The organic layers were combined, dried over Na2SO4,
concentrated in vacuo, and further purified by silica gel column
chromatography (95:5 dichloromethane−methanol). The purified
product was dissolved with methanol (4.5 mL), and concentrated
HCl (1 mL) was added. The mixture was stirred at room temperature
overnight. The solvent was evaporated, and the residue was dissolved
4,4′,4″-(3-(1H-1,2,4-Triazol-1-yl)prop-1-ene-1,1,2-triyl)-
1
triphenol (19a). Yellow oil (93.1 mg, 60%). H NMR (300 MHz,
methanol-d4) δ 8.00 (s, 1 H), 7.87 (s, 1 H), 7.28−7.25 (m, 2 H),
6.90−6.87 (m, 2 H), 6.81−6.74 (m, 4 H), 6.55−6.52 (m, 2 H), 6.49−
6.46 (m, 2 H), 5.18 (s, 2 H); 13C NMR (75 MHz, methanol-d4) δ
158.0, 157.3, 157.1, 151.8, 145.3, 135.2, 134.8, 133.2, 132.2, 132.1,
131.9, 116.2, 116.1, 115.4, 55.0; negative ion ESIMS m/z (relative
intensity) 384 [(M − H+)¯, 5], 315 (100); negative ion HREIMS m/z
J
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Journal of Medicinal Chemistry
Article
calcd for C23H18N3O3 [(M − H+)¯] 384.1348, found 384.1353. HPLC
purity 98.8% (C-18 reverse phase, methanol−H2O, 90:10).
4,4′,4″-(3-(4H-1,2,4-Triazol-4-yl)prop-1-ene-1,1,2-triyl)-
residue was dissolved with H2O (20 mL) and extracted with ethyl
acetate (3 × 10 mL). The organic layers were combined, dried over
Na2SO4, concentrated in vacuo, and further purified by silica gel
chromatography (4:1 hexanes−ethyl acetate) to provide 30 as pale
yellow oil (541 mg, 95%). 1H NMR (300 MHz, CDCl3) δ 7.74 (d, J =
7.7 Hz, 1 H), 7.59 (s, 1 H), 7.40 (t, J = 7.9 Hz, 1 H), 7.21 (d, J = 8.1
Hz, 1 H), 2.53 (s, 3 H), 1.32 (s, 9 H).
1
triphenol (20a). Yellow oil (32.5 mg, 21%). H NMR (300 MHz,
methanol-d4) δ 8.23 (s, 2 H), 7.08−7.05 (m, 2 H), 6.95−6.92 (m, 2
H), 6.83−6.80 (m, 2 H), 6.78−6.75 (m, 2 H), 6.60−6.57 (m, 2 H),
6.48−6.45 (m, 2 H), 5.03 (s, 2 H); 13C NMR (75 MHz, methanol-d4)
δ 158.2, 157.7, 157.4, 146.0, 144.5, 134.7, 134.5, 133.0, 132.1, 131.8,
131.5, 116.5, 116.4, 115.4, 50.8; negative ion ESIMS m/z (relative
intensity) 384 [(M − H+)¯, 3], 315 (100); negative ion HREIMS m/z
calcd for C23H18N3O3 (M − H+)¯ 384.1348, found 384.1359. HPLC
purity 96.1% (C-18 reverse phase, methanol−H2O, 90:10).
3-(1,1-Bis(4-(methoxymethoxy)phenyl)prop-1-en-2-yl)-
phenyl Pivalate (31). The acetophenone 30 (1.47 g, 6.67 mmol) and
4,4′-dihydroxylbenzophenone (0.953 g, 4.4 mmol) were reacted
according to the general McMurry cross-coupling reaction procedure.
The product was purified by silica gel column chromatography (2:1
hexanes−ethyl acetate) to provide impure bisphenol intermediate
which was dissolved in dry THF (20 mL) and treated with NaH
(0.231 g, 95%, 9.14 mmol). The mixture was stirred 30 min under
argon, and then chloromethyl methyl ether (2.0 mL, 9.0 mmol) was
added dropwise. After stirring 3 h, the reaction was quenched with
saturated NaHCO3 (10 mL) and the solvent was evaporated. The
organic products were extracted from the aqueous phase using ethyl
acetate (3 × 15 mL). The organic layers were combined, dried over
Na2SO4, concentrated, and purified by silica gel column chromatog-
raphy, eluting with 6:1 hexanes−ethyl acetate to provide product 31 as
pale yellow oil (0.912 g, 43%). 1H NMR (300 MHz, CDCl3) δ 7.19−
7.09 (m, 3 H), 7.06−7.00 (m, 2 H), 6.95 (dt, J = 7.7, 1.3 Hz, 1 H),
6.89 (t, J = 2.0 Hz, 1 H), 6.86−6.78 (m, 3 H), 6.76−6.71 (m, 2 H),
5.20 (s, 2 H), 5.08 (s, 2 H), 3.51 (s, 3 H), 3.43 (s, 3 H), 2.15 (s, 3 H),
1.34 (s, 9 H); 13C NMR (75 MHz, CDCl3) δ 176.92, 155.92, 155.45,
150.75, 145.63, 138.87, 137.06, 136.52, 133.56, 131.88, 131.09, 128.66,
126.78, 122.19, 119.07, 115.71, 115.18, 94.40, 55.91, 38.97, 27.08,
23.17; ESIMS m/z (MNa+) 513; HRESIMS m/z calcd for C30H34O6
(MNa+) 513.2253, found 513.2234.
4,4′-(2-(3-Hydroxyphenyl)-3-(1H-imidazol-1-yl)prop-1-ene-
1,1-diyl)diphenol (32). A solution of 31 (0.68 g, 1.39 mmol) and N-
bromosuccinimide (247 mg, 1.39 mmol) in CCl4 (30 mL) was heated
at reflux under argon for 2 h. After cooling down, the solid was filtered
off, and the solvent was evaporated. The residue was dissolved in dry
THF (10 mL) and added to a solution of NaH (67 mg, 95%, 2.78
mmol) and imidazole (143 mg, 2.1 mmol) in dry THF (10 mL). The
mixture was stirred at room temperature overnight. The reaction was
then quenched with saturated NH4Cl (4 mL) solution, and the solvent
was evaporated. The product was extracted from saturated NH4Cl (15
mL) solution using ethyl acetate (3 × 15 mL). The organic layers were
combined, dried over Na2SO4, and concentrated. The product was
dissolved in methanol (5 mL) and treated with 2 N KOH to bring the
pH above 12. After the reaction mixture was stirred overnight, it was
quenched with saturated NH4Cl (10 mL) solution and the solvents
were evaporated. The product was extracted from saturated NH4Cl
(10 mL) solution using ethyl acetate (4 × 10 mL). The organic layers
were combined, dried over Na2SO4, and concentrated. The product
was dissolved in methanol (10 mL) and treated with concentrated HCl
(1 mL). After stirring overnight, the reaction was neutralized using
NaHCO3 and methanol was evaporated. Saturated NH4Cl (10 mL)
solution was added, and the product was extracted using ethyl acetate
(3 × 10 mL). The organic layers were combined, washed with brine,
dried over Na2SO4, concentrated, and further purified using silica gel
column chromatography, eluting with 10:1 dichloromethane−
methanol to provide 32 (103 mg, 21%) as white glass. 1H NMR
(300 MHz, methanol-d4) δ 7.35 (s, 1 H), 7.09−7.04 (m, 2 H), 7.01−
6.90 (m, 2 H), 6.86−6.74 (m, 5 H), 6.56 (dd, J = 2.0, 1.1 Hz, 1 H),
6.52 (dq, J = 4.2, 1.6 Hz, 2 H), 6.49−6.43 (m, 2 H), 4.89 (s, 2 H); 13C
NMR (75 MHz, methanol-d4) δ 160.80, 160.65, 159.84, 148.40,
145.46, 140.76, 137.12, 137.01, 135.68, 135.39, 134.14, 132.82, 131.11,
124.67, 123.00, 120.24, 118.87, 117.84, 117.40, 54.78; ESIMS m/z
(relative intensity) 385 (MH+, 16), 317 (100); HRESIMS m/z cacld
for C24H20N2O3 (MH+) 385.1552, found 385.1556. HPLC purity:
100% (C-18 reverse phase, methanol−H2O, 90:10).
4,4′-(2-(3-Fluoro-4-hydroxyphenyl)-3-(1H-1,2,4-triazol-1-yl)-
prop-1-ene-1,1-diyl)diphenol (19b). Orange foam (37%). 1H
NMR (300 MHz, methanol-d4) δ 8.07 (s, 1 H), 7.90 (s, 1 H),
7.29−7.26 (s, 2 H), 6.83−6.61 (m, 7 H), 6.52−6.47 (m, 2 H), 5.20 (s,
2 H); ESIMS m/z (relative intensity) 426 (MNa+, 100); HRESIMS
m/z calcd for C23H18FN3O3Na (MNa+) 426.1230, found 426.1243.
HPLC purity 99.6% (C-18 reverse phase, methanol−H2O, 90:10).
4,4′-(2-(3-Fluoro-4-hydroxyphenyl)-3-(4H-1,2,4-triazol-4-yl)-
prop-1-ene-1,1-diyl)diphenol (20b). Yellow foam (21%). 1H NMR
(300 MHz, methanol-d4) δ 8.31 (s, 2 H), 7.11−7.06 (m, 2 H), 6.86−
6.66 (m, 7 H), 6.53−6.49 (m, 2 H), 5.06 (s, 2 H); ESIMS m/z
(relative intensity) 426 (MNa+, 100); HRESIMS m/z calcd for
C23H18FN3O3Na (MNa+) 426.1230, found 426.1244. HPLC purity
98.0% (C-18 reverse phase, methanol−H2O, 90:10).
4,4′-(2-(4-Aminophenyl)-3-(1H-1,2,4-triazol-1-yl)prop-1-ene-
1
1,1-diyl)diphenol (27). Orange foam (24%). H NMR (300 MHz,
methanol-d4) δ 8.02 (s, 1 H), 7.87 (s, 1 H), 7.26−7.23 (m, 2 H),
6.85−6.82 (m, 2 H), 6.79−6.73 (m, 4 H), 6.50−6.43 (m, 4 H), 5.19
(s, 2 H); 13C NMR (75 MHz, methanol-d4) δ 158.0, 157.1, 151.7,
147.5, 145.2, 144.8, 135.4, 135.0, 133.1, 132.1, 131.7, 130.3, 116.3,
116.1, 115.3, 54.9; ESIMS m/z (relative intensity) 407 (MNa+, 100);
HRESIMS m/z calcd for C23H20N4O2Na (MNa+) 407.1484, found
407.1490. HPLC purity 97.6% (C-18 reverse phase, methanol−H2O,
90:10).
4,4′-(2-(4-Aminophenyl)-3-(4H-1,2,4-triazol-4-yl)prop-1-ene-
1
1,1-diyl)diphenol (28). Yellow foam (20%). H NMR (300 MHz,
methanol-d4) δ 8.24 (s, 2 H), 7.06−7.03 (m, 2 H), 6.89−6.86 (m, 2
H), 6.83−6.80 (m, 2 H), 6.79−6.76 (m, 2 H), 6.54−6.51 (m, 2 H),
6.47−6.44 (m, 2 H), 5.03 (s, 2 H); 13C NMR (75 MHz, methanol-d4)
δ 158.2, 157.3, 148.0, 145.4, 144.5, 134.9, 134.7, 133.0, 132.0, 131.7,
131.5, 129.7, 116.5, 116.4, 115.3, 50.8; ESIMS m/z (relative intensity)
407 (MNa+, 100); HRESIMS m/z calcd for C23H20N4O2Na (MNa+)
407.1484, found 407.1498. HPLC purity 99.2% (C-18 reverse phase,
methanol−H2O, 90:10).
(2-(4-((tert-Butoxycarbonyl)amino)phenyl)prop-1-ene-1,1-
diyl)bis(4,1-phenylene) Bis(2,2-dimethylpropanoate) (24). A
solution of 23 (284 mg, 0.585 mmol) and Boc2O (207 mg, 0.948
mmol) in dry dioxane (10 mL) was heated at reflux under argon for 5
h. After cooling down, the solvent was evaporated, and the residue was
dissolved with 10% K2CO3 solution (20 mL) and extracted with ethyl
acetate (20 mL × 3). The organic layers were combined, dried over
Na2SO4, concentrated, and further purified by silica gel column
chromatography (85:15 hexanes−ethyl acetate) to provide 24 as white
solid (274 mg, 80%): mp 187−190 °C. 1H NMR (300 MHz, CDCl3)
δ 7.23−7.20 (m, 2 H), 7.18−7.15 (m, 2 H), 7.06−7.02 (m, 4 H),
6.89−6.86 (m, 2 H), 6.75−6.72 (m, 2 H), 2.10 (s, 3 H), 1.50 (s, 9 H),
1.36 (s, 9 H), 1.30 (s, 9 H); 13C NMR (75 MHz, CDCl3) δ 177.0,
176.9, 149.6, 149.0, 146.7, 140.7, 140.2, 138.1, 137.2, 136.6, 135.7,
131.8, 131.0, 129.8, 121.1, 120.4, 117.8, 80.4, 39.1, 39.0, 27.4, 27.1,
27.0, 23.3; EIMS m/z (relative intensity) 585 (M+, 0.6), 57 (100);
HRESIMS m/z calcd for C36H43NO6Na (MNa+) 608.2988, found
608.3009.
3-Acetylphenyl Pivalate (30).36 A solution of compound 29
(421 mg, 3.09 mmol) in dry THF (7 mL) was stirred under argon.
NaH (111 mg, 95%, 4.39 mmol) was added portionwise. The solution
was stirred for 30 min, and then trimethylacetyl chloride (0.6 mL, 4.87
mmol) was added dropwise. After stirring for 2 h, the reaction was
quenched with H2O (2 mL) and the solvent was evaporated. The
(2-(3-((tert-Butoxycarbonyl)amino)phenyl)prop-1-ene-1,1-
diyl)bis(4,1-phenylene) Bis(2,2-dimethylpropanoate) (34). The
acetophenone 33 (0.201 g, 1.49 mmol) and benzophenone 22 (0.682
g, 1.78 mmol) were reacted according to the general McMurry cross-
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Journal of Medicinal Chemistry
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coupling reaction procedure. The product was purified by silica gel
chromatography (4:1 hexanes−ethyl acetate) to provide the impure
intermediate, which was then treated with di-tert-butyl dicarbonate
(0.312 g, 1.43 mmol) in THF (20 mL). The reaction mixture stirred
under argon for 36 h. The solvent was evaporated and the residue was
purified by silica gel chromatography (4:1 hexanes−ethyl acetate) to
provide 34 as cloudy oil (0.685 g, 79%). 1H NMR (300 MHz, CDCl3)
δ 7.25−7.13 (m, 4 H), 7.11−7.02 (m, 3 H), 6.92−6.86 (m, 2 H), 6.78
(s, 1 H), 6.76−6.71 (m, 2 H), 6.42 (brs, 1 H), 2.12 (s, 3 H), 1.51 (s, 9
H), 1.37 (s, 9 H), 1.30 (s, 9 H); 13C NMR (75 MHz, CDCl3) δ
177.01, 152.65, 149.69, 149.06, 144.51, 140.44, 139.98, 138.03, 137.55,
136.06, 131.65, 130.96, 128.56, 124.21, 121.09, 120.40, 119.08, 116.68,
80.31, 39.06, 28.31, 27.05, 23.42; HRESIMS m/z (relative intensity)
calcd for C36H43NO6 (MNa+) 608.2988, found 608.2999.
12 was docked into the ligand binding pocket of ER-α using the
GOLD 3.0 program. The best docking solution according to GOLD
fitness score was selected.
Inhibition of Recombinant Human Aromatase (CYP19) by
Microsomal Incubations. The activity of recombinant aromatase
(CYP19) was determined by measuring the conversion rate of the
fluorometric substrate 7-methoxy-4-trifluoromethylcoumarin (MFC)
to its fluorescent metabolite 7-hydroxytrifluoromethylcoumarin
(HFC). Experimental procedures were consistent with the published
methodology.37 All of the incubations were performed using
incubation times and protein concentrations that were within the
linear range for reaction velocity. The fluorometric substrate, MFC,
was dissolved in acetonitrile with the final concentration of 25 mM. All
tested samples were dissolved in either methanol or DMSO. The
sample solutions (2 μL) were mixed well with 98 μL of NADPH-
cofactor mix (16.25 μM NADP+, 825.14 μM MgCl2, 825.14 μM
glucose 6-phosphate, and 0.4 U/mL glucose 6-phosphate dehydrogen-
ase) and were prewarmed for 10 min at 37 °C. Enzyme/substrate mix
was prepared with fluorometric substrate, recombinant human
aromatase (CYP19), and 0.1 M potassium phosphate buffer (pH
7.4). Reactions were initiated by adding enzyme/substrate mix (100
μL) to bring the incubation volume to 200 μL, and the mixture was
incubated for 30 min. All the reactions were stopped by adding 0.1 M
Tris base dissolved in acetonitrile (75 μL). The amount of fluorescent
product was determined immediately by measuring fluorescent
response using a BioTek (Winooski, VT) Synergy 2 fluorometric
plate reader. Excitation−emission wavelengths for MFC metabolite
were 409 and 530 nm. The standard curve for MFC metabolite was
constructed using the appropriate fluorescent metabolite standards.
Quantification of samples was performed by applying the linear
regression equation of the standard curve to the fluorescence response.
The limit of quantification for the metabolites of MFC was 24.7 pmol
with intra- and interassay coefficients of variation less than 10%. The
rates of metabolite formation in the presence of the test inhibitors
were compared with those in the control, in which the inhibitor was
replaced with vehicle. The extent of enzyme inhibition was expressed
as the percentage of remaining enzyme activity compared to the
control. IC50 values were determined as the inhibitor concentrations
that brought about half reduction in enzyme activity by fitting all the
data to a one-site competition equation using GraphPad Prism 5.0
(GraphPad Software Inc., San Diego, CA).
Binding Affinities for Recombinant Human ER-α and ER-β.
The binding affinities of ER-α and ER-β were determined by
measuring the change of polarization value when the fluorescent
estrogen ligand, ES2, was displaced by the tested compounds.
Experimental procedures were consistent with the protocol provided
by Invitrogen. The fluorescent estrogen ligand, ES2, was provided in
methanol/water (4:1, v/v) at a concentration of 1800 nM.
Recombinant human ER-α and ER-β were provided in buffer (50
mM Bis-Tris propane, 400 mM KCl, 2 mM DTT, 1 mM EDTA, and
10% glycerol), at concentrations of 734 and 3800 nM, respectively. All
tested samples were dissolved in either methanol or DMSO. The
sample solutions (1 μL) were mixed well with 49 μL of ES2 screening
buffer (100 mM potassium phosphate, 100 μg/mL BGG, and 0.02%
NaN3). The ER-α/ES2 complex was prepared with the fluorescent
estrogen ligand ES2, human recombinant ER and ES2 screening buffer
at concentrations of 9 nM ES2 and 30 nM ER-α. The ER-β/ES2
complex was prepared with the fluorescent estrogen ligand ES2,
human recombinant ER-β and ES2 screening buffer at concentrations
of 9 nM ES2 and 20 nM ER-β. Reactions were initiated by adding ER/
ES2 complex (50 μL) to bring the incubation volume to 100 μL and
incubated for 2 h avoiding light. The polarization value was
determined by measuring fluorescent response using a BioTek
(Winooski, VT) Synergy 2 fluorometric plate reader. Excitation−
emission wavelengths for fluorescence polarization were 485 and 530
nM. The polarization values in the presence of the test competitors
were compared with those of the control, in which the competitor was
replaced with vehicle. The extent of competition was expressed as the
percentage of remaining polarization compared to the control. EC50
values were determined as the competitor concentrations that brought
4,4′-(2-(3-Aminophenyl)-3-(1H-imidazol-1-yl)prop-1-ene-
1,1-diyl)diphenol (35). A solution of 34 (0.341 g, 0.58 mmol) and
N-bromosuccinimide (83 mg, 0.46 mmol) in CCl4 (30 mL) was
heated at reflux under argon for 2 h. After cooling down, the solid was
filtered off, and the solvent was evaporated. The residue was dissolved
in dry THF (10 mL) and added to a solution of NaH (16 mg, 95%,
0.63 mmol) and imidazole (39 mg, 0.58 mmol) in dry THF (15 mL).
The mixture was stirred at room temperature overnight. The reaction
was then quenched with saturated NH4Cl (3 mL) solution, and the
solvent was evaporated. The residue was dissolved in saturated NH4Cl
(10 mL), and the product was extracted using ethyl acetate (3 × 15
mL). The organic layers were combined, washed with brine (10 mL),
dried over Na2SO4, and concentrated. The product was dissolved in
methanol−THF (7:3, 10 mL) and treated with 1 N KOH (2 mL).
After the reaction mixture stirred 1 h, it was quenched with saturated
NH4Cl (10 mL), and the solvent was evaporated. The product was
extracted using ethyl acetate (3 × 15 mL), and the combined extract
was dried over Na2SO4, concentrated, dissolved in methanol (8 mL),
and the solution was treated with concentrated HCl (1.5 mL). After
stirring 2 h at 50 °C, the reaction was neutralized using NaHCO3 and
methanol was evaporated. Saturated NH4Cl (10 mL) solution was
added, and the product was extracted using ethyl acetate (3 × 15 mL).
The organic layers were combined, washed with brine, dried over
Na2SO4, concentrated, and purified using silica gel column
chromatography, eluting with 5:1 dichloromethane−methanol, to
provide 35 as white glass (55 mg, 25%). 1H NMR (300 MHz,
methanol-d4) δ 7.36 (s, 1 H), 7.09−7.01 (m, 2 H), 6.97−6.88 (m, 2
H), 6.84 (dq, J = 9.4, 2.6 Hz, 3 H), 6.81−6.73 (m, 2 H), 6.51−6.55
(m, 2 H), 6.50−6.44 (m, 2 H), 6.41 (dt, J = 7.7, 1.3 Hz, 1 H), 4.89 (s,
2 H); 13C NMR (75 MHz, methanol-d4) δ 158.13, 157.30, 148.54,
145.57, 142.46, 134.75, 134.59, 133.51, 132.90, 131.65, 130.01, 121.14,
117.94, 116.32, 115.26, 52.44; ESIMS m/z (relative intensity) 384
(MH+, 28), 316 (100); negative ion HRESIMS m/z calcd for
C24H21N3O2 [(M − H+)−] 382.1555, found 382.1563. HPLC purity:
100% (C-18 reverse phase, methanol−H2O, 90:10).
Molecular Docking of Compounds 12 and 36 in the Active
Site of Aromatase. The structures of compounds 12 and 36 were
constructed with Sybyl 7.1 software and energy minimized to 0.01
kcal/mol by the Powell method using Gasteiger−Huckel charges and
the Tripos force field. The crystal structure of aromatase was obtained
from the Protein Data Bank (PDB code 3s79),33 and the natural ligand
(androstenedione) and all crystal water molecules were removed.
Compounds 12 and 36 were docked into the androstenedione binding
pocket in aromatase using the GOLD 3.0 program. A distance
constraint was added between the imidazole nitrogen of 12 or 36 and
the iron to confine the distance within 1.5−3.5 Å during docking. The
best docking solutions according to the GOLD fitness scores were
selected.
Molecular Docking of Compound 12 in the Active Site of
ER-α. The structure of compound 12 was constructed with Sybyl 7.1
software and energy minimized to 0.01 kcal/mol by the Powell
method using Gasteiger−Huckel charges and the Tripos force field.
The crystal structure of ER-α was obtained from the Protein Data
Bank (PDB code 3ert),34 and the natural ligand (4-hydroxy tamoxifen)
and all crystal water molecules (except the water that forms bifurcated
hydrogen bonds with Glu353 and Arg394) were removed. Compound
L
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Journal of Medicinal Chemistry
Article
about half reduction in polarization value by fitting all the data to a
one-site competition equation using GraphPad Prism 5.0 (GraphPad
Software Inc., San Diego, CA).
ACKNOWLEDGMENTS
■
This research was supported by the Purdue University Center
for Cancer Research and the Indiana University Cancer Center
Joint Funding Award 206330, by the Purdue Center for Cancer
Research Grant P30 CA023168, by a Purdue Research
Foundation research grant, and by an award from the Floss
Endowment, provided to the Department of Medicinal
Chemistry and Molecular Pharmacology, Purdue University.
W.L. also thanks Dr. Charles Paget for providing a graduate
travel award.
Cell Culture and Test Compound Treatment. Estrogen
receptor-positive human breast carcinoma cell line (MCF-7 cells)
were seeded at a density of 105 cells/well in six-well plates and
maintained at 37 °C under a humidified atmosphere of 5% CO2 and
95% air in minimum essential media (MEM) supplemented with 10%
fetal bovine serum (FBS). Before the test compound treatments, the
cells were preconditioned in charcoal-stripped FBS for 72 h to remove
the estrogens from the growth medium containing 10% FBS. The cells
were treated with vehicle (0.1% methanol) alone, 1 μM test
compound, or 1 μM endoxifen (positive control) for 24 h in the
presence of 10 nM β-estradiol (E2) dissolved in MEM supplemented
with 10% charcoal-stripped FBS.
Ribonucleic Acid (RNA) Extraction and Concentration
Measurement. The MCF-7 cells treated with test compounds or
experimental controls for 24 h were harvested for progesterone
receptor (PGR) messenger ribonucleic acid (mRNA) extraction.
Before ribonucleic acid (RNA) extraction, genomic DNA was
eliminated. RNA was extracted from approximately 3 × 105 cells by
RNeasy Plus Mini Kit (Qiagen Inc., Valencia, CA, USA). The RNA
concentration was measured using the Qubit RNA BR assay (Life
Technologies Corp., Carlsbad, CA) for the Qubit 2.0 fluorometer (Life
Technologies Corp., Carlsbad, CA). The RNA was stored at −80 °C
before further use.
ABBREVIATIONS USED
■
AI, aromatase inhibitor; SERM, selective estrogen receptor
modulator; ATAC, arimidex, tamoxifen, alone or in combina-
tion; ER, estrogen receptor; FDA, Food and Drug Admin-
istration; MOMCl, methyl chloromethyl ether; NBS, N-
bromosuccinimide; CYP, cytochrome P450; E2, β-estradiol;
PGR, progesterone receptor; PDB, Protein Data Bank
REFERENCES
■
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01677.
Molecular models of compound 12 in the active sites of
aromatase and ER-α, and associated tables listing
hydrogen bond angles (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@
purdue.edu.
■
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Notes
The authors declare no competing financial interest.
M
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