Synthesis and characterization of hydrogen peroxide activated estrogen receptor beta ligands

Article abstract of DOI:10.1016/j.bmc.2019.04.003

The development and evaluation of selective estrogen receptor modulators (SERMs) is of interest because of the complex and significant role of estrogen receptors in normal tissues as well as disease states. In neurodegenerative disorders such as Alzheimer

Full text of DOI:10.1016/j.bmc.2019.04.003

Accepted Manuscript
Synthesis and characterization of hydrogen peroxide activated estrogen receptor
beta ligands
Hyejin Park, Joseph D. McEachon II, Julie A. Pollock
PII:
S0968-0896(18)32079-0
https://doi.org/10.1016/j.bmc.2019.04.003
BMC 14857
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 December 2018
18 March 2019
2 April 2019
Please cite this article as: Park, H., McEachon, J.D. II, Pollock, J.A., Synthesis and characterization of hydrogen
peroxide activated estrogen receptor beta ligands, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.04.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and characterization of hydrogen
peroxide activated estrogen receptor beta
ligands
Leave this area blank for abstract info.
Hyejin Park, Joseph D. McEachon II, and Julie A. Pollock
Department of Chemistry, University of Richmond, 138 UR Drive, Richmond, VA 23173, USA

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis and characterization of hydrogen peroxide activated estrogen receptor beta
ligands
Hyejin Park, Joseph D. McEachon II, and Julie A. Pollock*
Department of Chemistry, University of Richmond, 138 UR Drive, Richmond, VA 23173, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The development and evaluation of selective estrogen receptor modulators (SERMs) is of
interest because of the complex and significant role of estrogen receptors in normal tissues as
well as disease states. In neurodegenerative disorders such as Alzheimer’s disease and multiple
sclerosis, estrogen receptor beta (ERβ) seems to provide a protective anti-inflammatory
response. Due to the increase in reactive oxygen species (ROS) in these diseases, we have
masked ERβ ligands, including diarylproprionitrile (DPN), as boronate esters that release the
active estrogen in the presence of H2O2. Here we demonstrate their synthesis, decreased binding
affinities, kinetics of release, and selectivity toward ROS. The most promising ligand can be
unmasked in the presence of pathological H2O2 to modulate ERβ transcription in cells.
Available online
Keywords:
Estrogen
Pro-drug
Estrogen receptor beta
Boronic acid pinacol ester
Hydrogen peroxide
2009 Elsevier Ltd. All rights reserved.
1. Introduction
The idea of boron-based pro-estrogens has been utilized
before; in previous studies, the selective estrogen receptor
In response to sensing infections and injury, the immune
system initiates and amplifies expression of innate immunity
genes and adaptive immune responses. This burst of
inflammatory activity is important in surmounting the infection
or repairing the injury, but it is intended to be a local and time-
limited event that normally undergoes abatement. When
resolution is limited or incomplete, inflammation becomes
chronic. Within the central nervous system, this deregulation of
inflammation can be linked to neurodegenerative diseases such as
Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease1.
Within these inflammatory environments, there is typically an
overproduction of reactive oxygen species (ROS) including
hydrogen peroxide (H2O2), hydroxyl radical (·OH), superoxide
modulators (SERMs) tamoxifen and endoxifen have been
masked as boronate esters13-15. Tamoxifen and endoxifen both
target ERα as therapies for breast cancer. The previous work
focused mostly on the cellular consequences of these boronate
ester SERMs observing similar or enhanced effects to the
unmodified anti-estrogens in breast cancer cells. They were able
to observe the presence of the SERMs after incubation of the
boronate esters with MCF7 or T47D cells, presumably due to
increased levels of ROS13,15. In addition, they showed increased
bioavailability and drug accumulation at tumor sites within
mouse xenograft models16.
Due to the successes of tamoxifen and endoxifen boronate
esters, we set out to mask alternative estrogens. In particular, we
focused on the potent and selective ERβ agonist
diarylpropionitrile (DPN, Fig. 1a)16,17. ERβ selective ligands,
such as DPN, have shown significant promise in exhibiting
-
(O2 ·), and nitric oxide (NO)2.
The role of estrogens and their receptors – estrogen receptor
alpha (ERα) and estrogen receptor beta (ERβ) – in biology,
including inflammation, is complex.Estrogen appears to provide
neuroprotection through its actions as a potent anti-oxidant, anti-
apoptotic, and anti-inflammatory agent3. Therefore, we set out to
mask ER ligands for release in the presence of ROS. Boronate
esters have been used extensively for the protection or masking
of phenols due to their rapid and quantitative release in aqueous
neuroprotective effects in a number of neurological diseases18-24
.
In addition, molecules that activate ERβ repress transcription of
proinflammatory genes; specifically, DPN has been shown to be
effective in a model of inflammation following lung injury25. Due
to the increased levels of ROS and pathological oxidative stress
within neurodegenerative disorders and inflammatory diseases,
we hypothesize that appending a boronate ester to the ERβ ligand
will allow for consumption H2O2 and simultaneous release of the
active ligand achieving dual results. In addition, the masking of
the active phenol would allow for selective release of the
estrogen in the presence of H2O2, providing site-selective
modulation of ER. Herein, we report the synthesis of boronic
4
H2O2 . They have been developed for the selective detection and
5
imaging of H2O2 and since been utilized as pro-drugs of matrix
metalloproteinases6, masked chelators of metal ions7,8, selectively
activatable DNA cross-linking agents9,10, cytotoxic agents11, and
pro-drugs of histone deacetylase inhibitors12.
*Corresponding author. Tel.: +1-804-484-1578; fax: +1-804-289-8233; email: jpollock@richmond.edu

acid pinacol ester pro-estrogens of endogenous estrogens and the
ERβ-selective agonist DPN. The boronate esters have decreased
affinity for both ERα and ERβ and are converted into the active
phenol in the presence of H2O2 in vitro and in cells.
structures of estradiol29 and genistein30 bound into the ligand
binding domain (LBD) of ERβ indicate that the A-ring phenol of
the ligands forms hydrogen bonds with glutamate 305 and
arginine 346 (Fig. S1). When we performed docking studies of
DPN into ERβ LBD (PDB: 1X7J), there was no surprise that the
best scoring hit placed the A-ring of DPN in the same position
(Fig. 1b). Therefore, we set out to block the phenols on DPN as
well as estradiol. Although a variety of boronate esters have been
used as H2O2 activatable groups, we selected the boronic acid
pinacol ester for the large size that could cause steric clashes to
inhibit interaction with the ER binding pocket.
2.2. Chemistry
Our initial efforts focused on masking the endogenous
steroidal estrogen, 17β-estradiol as a boronic acid pinacol ester
on the A-ring of the steroid as a proof of principle. A previous
study had examined boronated estrone derivatives and their
oxidation to estrone in the presence of H2O231. However, since
our route to the boronated estradiol (3) required synthesis of the
corresponding estrone (2), we evaluated both protected
endogenous estrogens. These compounds were obtained through
a straightforward modification of estrone (Scheme 1). The
phenol of estrone was activated as the corresponding triflate (1)
and palladium-mediated cross-coupling allowed installation of
the boronic acid pinacol ester (2) in good yield. Reduction of the
ketone resulted in the corresponding boronic acid pinacol ester
17β-estradiol (3) in good yield.
Next, we turned to the non-steroidal ERβ selective agonist,
DPN. Since DPN contains two phenolic groups, we synthesized
three derivatives with either one or both phenols masked as
boronic acid pinacol esters. We generated DPN following
literature precedent and the boronate ester DPNs (6a-6c) with a
modified protocol (Scheme 2)16. First, compounds 4a-4c were
prepared through a base-mediated aldol condensation of the
appropriate arylaldehydes and phenylacetonitriles in excellent
yields. The alkenes were reduced using sodium borohydride to
generate compounds 5a-5c in moderate to good yields. Removal
of the methyl protecting groups on 5a and 5b was successful
using aluminum chloride and 1-dodecanethiol. Finally,
palladium-mediated cross-coupling of the bromide with
bis(pinacolato)diboron allowed the installation of the boronic
acid pinacol esters (6a-6c) albeit with poor yields.
Figure 1. a) Structures of endogenous ER ligands, E2 and E1, and ERβ-
selective agonists, genistein and DPN. b) DPN docked into the binding
pocket of ERβ ligand binding domain (LBD). Only key residues are shown.
2. Results and discussion
2.1. Design of masked estrogens
It is well established in ER literature that the presence of a
phenol is preferred for receptor binding (see reviews26-28). Indeed,
endogenous estrogens such as estradiol (E2) and estrone (E1) as
well as phytoestrogens such as genistein and the synthetic ERβ
selective ligand DPN contain A-ring phenols (Fig. 1a). Crystal
O
O
OH
O
a
b
c
O
O
TfO
B
B
O
O
HO
1
2
3
Scheme 1. Synthesis of boronic acid pinacol ester estrone (2) and estradiol (3). Reagents and Conditions (a) trifluoromethane
sulfonic anhydride, 2,6-lutidine, dimethylaminopyridine, CH2Cl2, 0 °C, 1 h (70 %); (b) pinacolborane, Pd(dppf)Cl2,
triethylamine, dioxane, 100 °C, 18 h (82 %); (c) NaBH4, methanol/tetrahydrofuran, 0 °C (78 %).
R2
O
Scheme 2. Synthesis of boronic acid pinacol ester DPNs (6a-
6c). Reagents and Conditions (a) 40% KOH, EtOH, rt. 1.5 h
(4a, 95%; 4b, 100%; 4c, 100%) (b) NaBH4, EtOH, 70 °C, 18
h (5a, 83%; 5b, 82%; 5c, 34%) (c) AlCl3, 1-dodecanethiol,
CH2Cl2 (34%) (d) bis(pinacolato)diboron, KOAc,
NC
a
b
H
+
R2
CN
R1
R1
4a:
4b:
4c:
R
1 = Br, R2 = OCH3
R1 = OCH3, R2 = Br
R1, R2 = Br
Pd(dppf)Cl2, 80 °C, 18 h (6a, 34%; 6b, 35%; 6c, 17%).
2.3. Estrogen receptor binding affinity
R2
R2
c, d
The boronate ester pro-estrogens were evaluated in
competitive fluorescence polarization binding assays to
CN
O
B
O
CN
1 = Br, R2 = OCH3
R1 = OCH3, R2 = Br
R1, R2 = Br
R1
Bpin =
R1
5a:
5b:
5c:
6a:
6b:
6c:
R
R1 = Bpin, R2 = OH
R1 = OH, R2 = Bpin
R1, R2 = Bpin

determine their affinities for full length ERα and ERβ (Fig. S2).
Binding affinities shown in Table 1 are expressed as relative
binding affinity (RBA) values (estradiol = 100%) and were
calculated using previously described methods32. The binding
affinity of the phenolic estradiol, estrone, and DPN matched
previous literature16,33. As expected, the binding affinities
decreased, although in most cases moderately, when the phenols
were replaced with boronic acid pinacol esters. For both ERα and
ERβ, the RBA of 2 was approximately 6-fold less than estrone
and the RBA of 3 was approximately 3-fold less than estradiol.
As noted above, DPN is selective for ERβ over ERα; however,
we observed a decrease in binding affinity for 6a-6c for both
receptors. For ERα, 6a and 6b had 4.5- and 6-fold decreased
affinity while 6c did not compete productively. For ERβ, the
binding affinity of 6a and 6b decreased approximately 5-fold
while 6c decreased 20-fold in comparison to DPN. We were
surprised that placement of the boronic ester in either of the R1
(6a) or R2 (6b) positions essentially resulted in the same
decreased binding affinity; from previous studies of DPN and
derivatives as well as our docking results (Fig. 1b), we expected
replacement of the A-ring phenol (6a) to have more of an effect
on binding16. The moderate decrease in binding affinities for
compounds 6a and 6b is most likely because they still contain
one free phenol and are capable of fitting into the flexible binding
pocket of ERβ. Not surprisingly, however, was the fact that
replacement of both phenols resulted in the poorest binder, 6c.
Table 1. Estrogen receptor relative binding affinities
Compound
RBAa
ERα
12.8 ± 4.0
100
ERβ
Estrone (E1)
14.5 ± 11.6
100
Estradiol (E2)
DPN
2
0.392 ± 0.062
2.2 ± 1.1
7.7 ± 4.4
2.5 ± 1.4
34.0 ± 10.1
1.58 ± 0.36
1.68 ± 1.22
0.39 ± 0.26
3
33.0 ± 12.5
0.087 ± 0.023
0.063 ± 0.024
< 0.001
6a
6b
6c
aRBA = relative binding affinity, where estradiol = 100%. Data are presented
as mean ± standard deviation of three replicates for E1, 2, 3, DPN, 6a-6c and
six replicates for E2.
Figure 1. Kinetics of reaction of 3 with H2O2. a) Absorbance spectra of 3 (75 μM) with 10-fold H2O2 (750 µM) over 4 hours in PBS at 37 °C. Blue decreased in
shade over time; scans taken every minute for the first 30 minutes, then every 5 minutes until 1 hour and every hour until 4 hours. 3 is black; E2 is dashed black.
inset: data fit to a pseudo-first order model for reactant consumption during the reaction. b) Linear fit of kobs and [H2O2]for conversion of 3 to E2 gives the
second-order rate constant. Data presented as mean ± standard deviation of three replicates.
2.4. Kinetics of H2O2-mediated conversion to phenol
absorbance at 235 nm, produced E1 at a similar rate with a
second order rate constant of 4.81 ± 0.86 M-1sec-1 (Fig. S3). It is
important to note that incubation of 3 or 2 alone in PBS for 4
hours at 37 °C did not result in a change in the UV spectra (Fig.
S5a).
We first investigated the boronate ester estradiol 3 as our
model system to evaluate H2O2-activation. The reaction was
monitored in real time by UV spectroscopy in PBS (pH 7.4) at 37
°C for four hours to mimic a biological environment (Fig. 2). As
illustrated in Figure 2a, pro-estrogen 3 exhibits moderate
absorbance at 235 nm while E2 does not. Upon addition of 10
equivalents of H2O2, the peak at 235 nm decreases overtime
eventually producing an absorbance spectrum equal to that of E2.
Kinetic analysis of the data collected under pseudo-first order
conditions (Fig. 2a, inset) gave a second order rate constant of
5.57 ± 0.23 M-1sec-1 for disappearance of the starting material
(Fig. 2b). The conversion of 3 to E2 was confirmed by GC-MS
analysis of the reaction mixture after 30 minutes of incubation;
the retention time moved from 15.7 min to 12.5 min and the
masses corresponded to the expected compounds (Fig. S4). In
addition, reaction of pro-estrogen 2 with H2O2, when monitored
by UV spectroscopy and quantified by consumption of
Next, we examined the boronic acid pinacol ester masked
DPN derivatives 6a-6c in similar conditions. DPN exhibited a
stronger absorbance at 275 nm than any of the boronate
derivatives. Therefore, we monitored the conversion of each
compound to DPN using UV spectroscopy in PBS (pH 7.4) at 37
°C for four hours (Fig. 3). Similar to the estrone and estradiol
pro-estrogens, there was complete conversion in approximately
10 minutes after addition of 10 equivalents of H2O2 (Fig. 3a-c).
Kinetic analysis of the data at various concentrations of peroxide
revealed that the second order rate constants for appearance of
the product DPN were fairly consistent for each derivative 6a-6c
with second order rate constants of 6.46 ± 1.01 M-1sec-1, 5.92 ±
0.57 M-1sec-1, and 5.14 ± 0.29 M-1sec-1, respectively (Fig. S6,
3d). Not surprisingly, 6c, which contains two boronic acid

DPN gives the second-order rate constant. Data presented as mean ± standard
deviation of three replicates.
pinacol esters, was slightly slower than 6a and 6b. We were
unable to uncouple the conversion of one pinacol boronate to the
phenol from the other because there was not a differential
wavelength to monitor; however, DPN was observed as the
product at the end of the reaction time (Fig. 3c). As above,
incubation of 6a-6c in PBS for 4 hours at 37 °C did not result in
changes in the UV spectra (Fig. S5b). The second order rate
constants for the conversion of any of the boronate ester
compounds here is consistent with previously presented
systems6,8.
2.5. ROS Selectivity
Since H2O2 is not the only ROS in biological systems, we
examined the reactivity of the boronate esters toward other ROS,
including tert-butylhydroperoxide (TBHP), hypochlorite (OCl–),
nitric oxide (NO), hydroxyl radical (·OH), and superoxide (O2–·).
We chose to focus on the DPN pro-estrogens 6a-6c because of
the selectivity for ERβ and therapeutic implications in
neuroinflammatory disorders. Due to background absorbance
issues with the alternative ROS, we analyzed the end points of
these reactions using HPLC (Fig. 4a-c). The retention time for
DPN was 19 min while the retention time for 6a, 6b, and 6c were
22 min, 23 min, and 28 min, respectively. As expected after 1
hour of exposure to 5-fold H2O2, all of the pro-estrogens were
converted to DPN. Alternatively, incubation with 5-fold TBHP,
NO, and hydroxyl radical did not convert 6a-6c into DPN. In
addition, there was approximately 22 % conversion of the
boronate esters in the presence of 5-fold superoxide and 50-80 %
consumption in the presence of 5-fold hypochlorite (Fig. 4d). We
were able to observe small conversion of 6c to 6a and 6b with
both superoxide and hypochlorite, indicating oxidation of one
boronate ester (Fig. 4c). There were approximately equal
amounts of 6a and 6b in those cases indicating no selectivity for
one boronate ester over the other. Some boronate ester peaks
were observed to broaden or misshapen (most evident in Fig. 4b
with NO trace) that we attribute to artifacts of the HPLC. When
we examined the UV spectra recorded by the photodiode array,
the spectra aligned well with 6b across the full retention time,
changing only in absorptivity and not shape; in addition, purity
measurements by the photodiode array indicated that the purity
threshold was met along the peak (Fig. S7). The DPN boronate
esters showed less selectivity, specifically against OCl–, in
comparison to previous experimentation on the estrone boronate
ester 2 that showed minimal reactivity in the presence of
hypochlorite23. However, other groups have observed this
phenomena indicating that hypochlorite is another promising
Figure 3. Kinetics of reaction of 6a-6c with H2O2. a) Absorbance spectra of
6a (75 μM) with 10-fold H2O2 (750 μM) over 4 hours in PBS at 37 °C. Green
decreases in shade over time; scans taken every minute for the first 30
minutes, then every 5 minutes until 1 hour and every hour until 4 hours. 6a is
black; DPN is dashed black. b) Absorbance spectra of 6b (75 μM) with 10-
fold H2O2 (750 μM) over 4 hours in PBS at 37 °C. Orange decreases in shade
over time; scans taken every minute for the first 30 minutes, then every 5
minutes until 1 hour and every hour until 4 hours.vv 6b is black; DPN is
dashed black. c) Absorbance spectra of 6c (75 μM) with 10-fold H2O2 (750
μM) over 4 hours in PBS at 37 °C. Blue decreases in shade over time; scans
taken every minute for the first 30 minutes, then every 5 minutes until 1 hour
and every hour until 4 hours. 6c is black; DPN is dashed black. d) ) Linear fit
of kobs and [H2O2]for conversion of 6a (green), 6b (orange), and 6c (blue) to
ROS that can convert boronate esters into phenols34,35
.
Figure 4. HPLC monitoring of reaction of 6a-6c with various reactive oxygen species. a) Reaction of 6a (150 μM, gray) with 5-fold of each ROS for 1 hour in
PBS at 37 °C. b) Reaction of 6b (150 μM, gray) with 5-fold of each ROS for 1 hour in PBS at 37 °C. c) Reaction of 6c (150 μM, gray) with 5-fold of each ROS
for 1 hour in PBS at 37 °C. Each line is representative of one replicate. d) Quantification of consumption of 6a-6c (green, orange, blue, respectively) incubated
for 1 hour with 5-fold of different reactive oxygen species (ROS). Data is presented as mean ± standard deviation of three replicates. NO was generated from
NOC5, superoxide was generated from a xanthine oxidase reaction, and hydroxyl radical was generated from Fe2+ and H2O2. (See experimental for complete
reaction conditions)

This lack of selectivity is not a downfall for compounds 6a-6c
because increased levels of the oxidant hypochlorite have also
been linked to a variety of inflammatory diseases such as
Alzheimer’s disease36. Therefore, release of the active ERβ
ligand in the presence of multiple ROS could allow for
advantageous protective effects.
presence of the boronic acid pinacol ester decreases the binding
affinity for both ERα and ERβ by 3-20 fold depending on the
ligand. The ERβ-selective ligand DPN was masked as three
different boronate esters 6a-6c, which have similar functionality
and reactivity in vitro. The integration of a ROS reactive group
into a functional estrogen has implications in a variety of
neurodegenerative and inflammatory diseases. The pro-estrogens
6a-6c could have dual effects in absorbing H2O2 and releasing an
ERβ-selective ligand with therapeutic potential. In particular,
pro-estrogen 6c, that masks both phenolic rings, exhibits
increased selectivity for H2O2, and diminishes the binding
affinity for the receptors most significantly. In addition, the ERβ
agonist activity is masked by the boronate esters of 6c in a
cellular environment; however, when H2O2 is at a pathological
level, the agonist activity is restored.
2.6. ERβ transcriptional activation
In order to probe the therapeutic potential of the boronate ester
masked DPN ligands, we examined the ability to activate ERβ
transcriptional activity in a cellular environment using a
luciferase-based assay. We chose to focus on the most promising
compound, 6c, which was rapidly converted to DPN in the
presence of excess H2O2 (Fig. 3c) but also resulted in the most
detrimental effect on ER binding (Table 1). As seen in Figure
5a, E2 and DPN exhibited potencies (EC50 = 0.020 nM and 0.72
This work expands upon previous studies with boronate ester
linked SERMs in breast cancer contexts illustrating together that
the installation of a boronate ester is a promising pro-drug
strategy for ER modulation. Due to the complexity of ER
signaling, context specific estrogens (e.g. SERMs and subtype-
selective ligands) are constantly being developed. The release of
an active ER ligand in the presence of H2O2 provides an
alternative approach to developing context specific estrogens or
anti-estrogens.
mM, respectively) consistent with literature as expected (EC50
=
0.030 mM and 0.89 nM, respectively)16. However, treatment of
the cells for 24 hours with 6c less potent (Fig. 5b, EC50 = 470
nM). As pathological levels of H2O2 in Alzheimer’s disease
models and other inflammatory disorders have been reported to
be at least 100 µM37-39, we added exogenous H2O2 at 50 µM and
100 µM to mimic the inflammatory environment. The potency of
E2 and DPN did not change upon addition of H2O2; however, the
transcriptional potency of 6c increased (Fig. 5b, EC50 = 6.1 nM
and 1.2 nM, respectively). At the highest concentration of H2O2
used, 6c behaved most similarly to DPN, indicating that the
active ligand was released in the cellular environment at these
pathological levels.
4. Experimental
4.1. Synthesis
Reagents and solvents for synthesis were purchased from
commercial sources and used without further purification. 1H and
13C NMR were recorded on a 500 MHz spectrometer or a 300
MHz spectrometer and are reported in ppm using solvent as an
internal standard. Preparative column chromatography was
performed on silica gel (230-400 mesh) and thin-layer
chromatography (TLC) was carried out using pre-coated silica
gel plates.
4.1.1. Synthesis of boronic ester estrone and
estradiol
4.1.1.1. Synthesis of triflate estrone (1)
Dimethylaminopyridine (0.058 g, 0.45 mmol), 2,6-lutidine
(0.46 mL, 3.48 mmol) and trifluoromethane sulfonic anhydride
(0.35 mL, 2.23 mmol) were added to a solution of estrone (0.5 g,
1.85 mmol) in dichloromethane (15 mL). The reaction mixture
was stirred at 0 °C for 1 h and was washed with 10 % HCl, 10 %
NaHCO3 and brine. The resulting solution was dried with Na2SO4
and concentrated under vacuum. The product was purified using
silica gel column, eluted with dichloromethane/hexane (9:1) to
1
obtain estrone triflate (1) as a white crystal (0.41 g, 56 %): H
NMR (300 MHz, CDCl3) δ 7.33 (dd, J = 8.6, 1.0 Hz, 1 H), 7.07 –
6.96 (m, 2 H), 2.93 (dd, J = 8.7, 4.3 Hz, 2 H), 2.50 (dd, J = 18.2,
8.4 Hz, 1 H), 2.45 – 2.34 (m, 1 H), 2.29 (td, J = 10.5, 9.5, 3.7 Hz,
1 H), 2.22 – 1.89 (m, 4 H), 1.73 – 1.33 (m, 6 H), 0.91 (s, 3 H);
13C NMR (75 MHz, CDCl3) δ 220.2, 147.6, 140.3, 139.3, 127.2,
125.1, 121.2, 120.9, 118.3, 116.6, 112.4, 50.4, 47.8, 44.1, 37.8,
35.7, 31.5, 29.3, 26.1, 25.7, 21.5, 13.8.
Figure 5. Transcriptional activation by ERβ in response to ligands after 24
hours treatment at the indicated concentrations. a) Control experiments with
E2 and DPN. Values are given as mean ± standard deviation of four
replicates. b) Experiments with 6c without H2O2 (squares, n = 6), 6c with 50
µM H2O2 (diamonds, n =4), and 6c with 100 µM H2O2 (circles, n = 6). Values
are given as mean ± standard deviation of replicates as indicated.
4.1.1.2. Synthesis of boronic ester estrone (2)
3. Conclusions
To a mixture of 1 (0.5 g, 1.28 mmol) and Pd(dppf)Cl2 (41.8
mg, 0.049 mmol) under nitrogen were added 1,4-dioxane (6.2
mL, 72.7 mmol), triethylamine (1 mL, 7.5 mmol) and
pinacolborane (0.5 mL, 3.58 mmol). The reaction mixture was
heated to 100 °C and stirred overnight. After cooling to room
In summary, we have prepared several steroidal and non-
steroidal boronate esters as pro-estrogens for the estrogen
receptors. They are converted rapidly and completely to the
phenolic estrogens when reacted with H2O2. In addition, the

temperature, the crude reaction mixture concentrated in vacuo,
and purified with silica gel column using ethyl acetate/hexane
(1:3) to afford boronic ester estrone (2) as a white solid (0.37 g,
76 %): 1H NMR (300 MHz, CDCl3) δ 7.70 – 7.53 (m, 2 H), 7.33
(d, J = 7.7 Hz, 1 H), 3.05 – 2.83 (m, 2 H), 2.61 – 2.40 (m, 2 H),
2.35 (td, J = 10.4, 4.2 Hz, 1 H), 2.27 – 1.89 (m, 5 H), 1.73 – 1.44
(m, 4 H), 1.36 (s, 12 H), 0.93 (d, J = 2.8 Hz, 3 H); 13C NMR (75
MHz, CDCl3) δ 220.7, 143.1, 135.8, 135.6, 132.2, 124.8, 83.7,
50.6, 48.0, 44.7, 38.1, 35.8, 31.6, 29.1, 26.5, 25.6, 24.8, 24.8,
21.6, 13.8. HRMS (ESI-TOF) exact mass calcd for C24H35O3BNa
[M+Na]+ 405.2576, found 405.2594.
Prepared from 4-bromobenzaldehyde (406 mg, 2.2 mmol) and
4-bromophenylacetonitrile (389 mg, 2.0 mmol) to produce
yellow solid (0.726 g, 100 %): H NMR (300 MHz, CDCl3) δ
7.75 (d, J = 8.5 Hz, 2 H), 7.63 – 7.56 (m, 4 H), 7.54 (s, 2 H), 7.44
(s, 1 H); 13C NMR (75 MHz, CDCl3) δ 141.0, 133.1, 132.3,
130.7, 127.5, 125.2, 123.8, 117.3, 111.4.
1
4.1.2.2. General procedure for reduction
NaBH4 (1.0 equiv.) was added slowly to a solution of
diarylpropionitrile (1.0 equiv.) in EtOH and pyridine under N2
atmosphere. The reaction was stirred at 70 °C overnight, then
cooled to room temperature and quenched with water. The
reaction mixture was diluted with 100 mL H2O and acidified with
6 M HCl. The organic layers were extracted with ether (3 x 50
mL), washed with brine and water, dried with Na2SO4, and
concentrated in vacuo.
4.1.1.3. Synthesis of boronic ester estradiol (3)
2 (0.35 mg, 0.92 mmol) was dissolved in a mixture of 1:1 v/v
methanol/tetrahydrofuran (6 mL) and was cooled in an ice bath.
Sodium borohydride (148 mg, 3.83 mmol) was added portion
wise and stirred until the starting material was consumed as
indicated by TLC analysis. Brine (20 mL) and excess water was
added to the reaction mixture and extracted with dichlormethane
(2 x 20 mL). The combined organic layers were washed with
brine and dried with Na2SO4. The solution was concentrated
under vacuum and purified using silica gel chromatography using
CH2Cl2/hexane (9:1) to yield boronic ester estradiol (3) as a white
4.1.2.2.1. Compound 5a
Prepared as above with NaBH4 (43 mg, 1.3 mmol) and 4a
(0.400 g, 1.3 mmol) in EtOH (4 mL) and pyridine (1 mL) to yield
1
white solid (0.335 g, 83 %): H NMR (300 MHz, CDCl3) δ 7.40
(d, J = 8.4 Hz, 2 H), 7.14 (d, J = 8.8 Hz, 2 H), 6.98 (d, J = 8.3
Hz, 2 H), 6.87 (d, J = 8.7 Hz, 2 H), 3.96 (s, 1 H), 3.80 (s, 3 H),
3.08 (dd, J = 7.2, 4.7 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ
159.5, 135.3, 131.7, 131.0, 128.6, 126.7, 121.4, 120.4, 114.5,
55.4, 41.5, 38.6.
1
solid (0.27 g, 78 %): H NMR (300 MHz, CDCl3) δ 7.61 – 7.55
(m, 2 H), 7.34 – 7.31 (m, 1 H), 3.77 – 3.71 (m, 1 H), 2.90 (dd, I =
8.7, 4.3 Hz, 2 H), 2.46 – 2.23 (m, 2 H), 2.23 – 2.05 (m, 1 H), 2.05
– 1.83 (m, 2 H), 1.86 – 1.63 (m, 1 H), 1.36 (s, 12 H), 0.81 (dd, J
= 3.0, 0.6 Hz, 3 H); 13C NMR (75 MHz, CDCl3) δ 143.8, 136.1,
135.6, 132.0, 124.8, 83.2, 81.9, 50.3, 44.7, 43.2, 38.5, 36.8, 31.9,
30.6, 29.7, 29.7, 29.4, 29.3, 27.2, 26.0, 24.8, 24.8, 23.2, 22.7,
14.1, 11.0. HRMS (ESI-TOF) exact mass calcd for C24H33O3BNa
[M+Na]+ 403.2419, found 403.2413.
4.1.2.2.2. Compound 5b
Prepared as above with NaBH4 (60.4 mg, 1.6 mmol) and 4b
(0.500 g, 1.6 mmol) in EtOH (5 mL) and pyridine (1 mL) to yield
1
white solid (0.414 g, 82 %): H NMR (300 MHz, CDCl3) δ 7.47
(d, J = 8.4 Hz, 2 H), 7.10 (d, J = 8.4 Hz, 2 H), 7.01 (d, J = 8.6
Hz, 2 H), 6.82 (d, J = 8.6 Hz, 2 H), 3.93 (dd, J = 7.7, 6.6 Hz, 1
H), 3.79 (s, 3 H), 3.09 (dd, J = 8.9, 7.1 Hz, 2 H); 13C NMR (75
MHz, CDCl3) δ 159.0, 134.2, 132.1, 130.3, 129.2, 127.8, 122.2,
120.0, 114.01, 55.2, 41.1, 39.4.
4.1.2. Synthesis of boronic ester DPNs
4.1.2.1. General procedure for
arylacetonitrile/aldehyde condensation
A solution of 40% aqueous KOH (0.23 mL/mmol nitrile) was
diluted with EtOH (0.46 mL/mmol nitrile) and was added to the
solution of appropriate arylaldehyde (1.1 equiv.) and
arylacetonitrile (1.0 equiv.) in EtOH (0.35 mL/mmol nitrile) at
room temperature, which resulted in immediate formation of
white precipitate. The reaction mixture was stirred for 1.5 hours
and the precipitate was collected through vacuum filtration. The
residue was washed with water and cold EtOH.
4.1.2.2.3. Compound 5c
Prepared as above with NaBH4 (53 mg, 1.4 mmol) and 4c
(0.500 g, 1.4 mmol) in EtOH (5 mL) and pyridine (1 mL) to yield
white solid (0.171 g, 34 %): H NMR (300 MHz, CDCl3) δ 7.48
(d, J = 8.5 Hz, 2 H), 7.41 (d, J = 8.4 Hz, 2 H), 7.09 (d, J = 8.4
Hz, 2 H), 6.96 (d, J = 8.4 Hz, 2 H), 3.96 (t, J = 7.1 Hz, 1 H), 3.09
(dd, J = 7.2, 5.1 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ 134.6,
133.7, 132.3, 131.8, 131.0, 129.2, 122.5, 121.7, 119.6, 41.2, 38.9.
1
4.1.2.1.1. Compound 4a
4.1.2.3. General deprotection and coupling methods
Prepared from 4-bromobenzaldehyde (406 mg, 2.2 mmol) and
4-methoxyphenylacetonitrile (0.272 mL, 2.0 mmol) to produce
yellow solid (0.592 g, 95 %): 1H NMR (300 MHz, CDCl3) δ 7.71
(d, J = 8.5 Hz, 2 H), 7.58 (dd, J = 8.7, 8.0 Hz, 4 H), 7.33 (s, 1 H),
6.95 (d, J = 8.9 Hz, 2 H), 3.85 (s, 3 H); 13C NMR (75 MHz,
CDCl3) δ 160.7, 138.5, 132.9, 132.2, 130.5, 127.4, 126.7, 124.4,
117.9, 114.6, 112.1, 55.5.
AlCl3 (5 equiv.) and dodecanethiol (2 equiv.) were added to
CH2Cl2 (2 mL) and cooled to 0 °C. Methyl ether (1 equiv.) in
CH2Cl2 (1mL) was added to the AlCl3/thiol solution. The mixture
was allowed to warm up to room temperature and stirred
overnight. The reaction mixture was then poured into ice water
(50 mL) and the product was extracted with EtOAc (3 x 20 mL).
The organic layers were combined and washed with water and
brine. The organic layer was dried over Na2SO4 and concentrated
under reduced pressure to yield crude product, which was
purified by silica gel column chromatography using 1:5
EtOAc/hexane.
4.1.2.1.2. Compound 4b
Prepared from 4-methoxybenzaldehyde (0.260 mL, 2.2 mmol)
and 4-bromophenylacetonitrile (0.392 g, 2.0 mmol) to produce
1
yellow solid (0.653 g, 100 %): H NMR (300 MHz, CDCl3) δ
7.88 (d, J = 8.7 Hz, 2 H), 7.59 – 7.48 (m, 4 H), 7.44 (s, 1 H), 6.98
(d, J = 8.9 Hz, 2 H), 3.87 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ
161.8, 142.3, 134.0, 132.3, 131.4, 127.4, 126.4, 123.0, 118.3,
114.6, 107.6, 55.6.
To a mixture of bis(pinacolato)diboron (1.1 equiv.), KOAc (3
equiv.) and Pd(dppf)Cl2 (0.03 equiv.) in dioxane was added a
solution of bromide (1 equiv.) in dioxane under N2 atmosphere.
The mixture was stirred at 80 °C overnight and the completion of
the reaction was confirmed by TLC. The volatiles from the
reaction mixture were removed under reduced pressure. The
4.1.2.1.3. Compound 4c

residue was diluted with ethyl acetate (10 mL) and washed with
water. The organic layers were combined, washed with brine, and
dried over Na2SO4. The crude product was concentrated under
reduced pressure and purified by silica gel column
chromatography using 1:3 EtOAc/hexane.
The X-ray structure of the ligand binding domain (LBD) of
ERβ bound to genistein (PDB: 1X7J)30 was used to dock DPN
using Swissdock40,41. The analysis of the data and alignment of
the structures was performed in VMD42.
4.3. Estrogen receptor binding assays
4.1.2.3.1. Compound 6a
The relative binding affinity (RBA) of the compounds were
assessed using PolarScreenTM ERα and ERβ Competitor Assays,
Green (Life Technologies). Briefly, stock solutions of the ligands
(10 mM in DMSO) were serially diluted and added to premixed
solutions of full length human recombinant ERα or ERβ with the
fluormone tracer at concentrations suggested by the manufacturer
in ES2 screening buffer (0.1 mg/mL acetylated BGG, 0.1 M
potassium phosphate, pH 7.4, 0.02% sodium azide). The black
384-well plate was covered and incubated at room temperature
for 2 hours and the fluorescence polarization was subsequently
read using a SpectraMax i3 plate reader (ex. 485 nm, em. 535
nm). The polarization data was fit using GraphPad Prism 7 and
IC50 values calculated. The RBA values were calculated from the
IC50 values relative to that of estradiol for each replicate.
Prepared from 5a (0.200 g, 0.64 mmol), AlCl3 (0.423 g, 3.2
mmol) and dodecanethiol (0.30 mL, 1.3 mmol) according to the
procedure above. After purification, the intermediate compound a
1
was obtained as a white solid (0.066 g, 34 %): H NMR (300
MHz, Acetone-d6) δ 8.47 (s, 1 H), 7.46 (d, J = 8.3 Hz, 2 H), 7.26
– 7.12 (m, 4 H), 6.85 (d, J = 8.5 Hz, 2 H), 4.26 (t, J = 7.5 Hz, 1
H), 3.17 (d, J = 7.5 Hz, 2 H); 13C NMR (75 MHz, Acetone-d6) δ
206.2, 158.1, 137.5, 132.3, 132.2, 129.7, 127.4, 121.4, 121.4,
116.6, 41.6, 38.6. Prepared from intermediate compound a (93.5
mg, 0.31 mmol), bis(pinacolato)diboron (86.6 mg, 0.34 mmol),
KOAc (88.2 mg, 0.93 mmol), Pd(dppf)Cl2 (6.8 mg, 0.009 mmol)
in dioxane (1.6 mL) as described above to afford compound 6a
1
(0.0364 g, 34 %): H NMR (300 MHz, Acetone-d6) δ 8.42 (s, 1
H), 7.68 (d, J = 8.1 Hz, 2 H), 7.27 (d, J = 8.0 Hz, 2 H), 7.22 (d, J
= 8.6 Hz, 2 H), 6.85 (d, J = 8.6 Hz, 2 H), 4.28 (t, J = 7.6 Hz, 1
H), 3.20 (d, J = 7.7 Hz, 2 H), 1.33 (s, 12 H); 13C NMR (75 MHz,
Acetone-d6) δ 206.1, 158.1, 141.5, 135.6, 132.4, 129.6, 127.7,
121.5, 116.6, 84.5, 42.6, 38.8, 25.2. HRMS (ESI-TOF) exact
mass calcd for C21H24NO3BNa [M+Na]+ 372.1745, found
372.1746.
4.4. UV spectroscopy kinetic studies
Boronic ester solutions used for UV spectroscopy were
prepared in 1 mL volumes at a final concentration of 75 μM in
PBS with the indicated concentration of H2O2. Absorbance
measurements were collected in a Cary 100 Bio UV-visible
spectrophotometer. For the kinetic studies, absorbance at a range
of 400-200 nm were measured once per minute for the first 30
minutes, every five minutes for 30‒60 min, and then every half-
hour for the remaining 3 hours for a simulated-first order boronic
4.1.2.3.2. Compound 6b
Prepared from 5c (0.150 g, 0.48 mmol), AlCl3 (0.314 g, 2.4
mmol) and dodecanethiol (0.29 mL, 1.2 mmol) according to the
procedure above. After purification the intermediate compound b
was obtained as white solid (0.049 g, 34 %): 1H NMR (300 MHz,
Acetone-d6) δ 8.24 (s, 1 H), 7.56 (d, J = 8.5 Hz, 2 H), 7.32 (d, J =
8.4 Hz, 2 H), 7.06 (d, J = 8.5 Hz, 2 H), 6.77 (d, J = 8.5 Hz, 2 H),
4.31 (t, J = 7.5 Hz, 1 H), 3.12 (d, J = 7.4 Hz, 2 H); 13C NMR (75
MHz, Acetone-d6) δ 206.0, 132.7, 132.1, 131.3, 131.1, 130.9,
130.6, 115.8, 32.6, 23.3, 14.3. Prepared from the intermediate
compound b (64 mg, 0.21 mmol), bis(pinacolato)diboron (60 mg,
0.23 mmol), KOAc (60.5 mg, 0.63 mmol) and Pd(dppf)Cl2 (4.7
mg, 0.006 mmol) in dioxane (1.1 mL) as described above to
acid pinacol ester conversion. For determination of kobs
,
absorbance values at 235 nm (for compound 2 or 3) or 275 nm
(for compounds 6a-6c) were converted to boronic ester estrogen
concentration at time, t, using the following equation:
[BEE]/[BEE]0 = (At – Ainf) / (A0 – Ainf)
(1)
where [BEE]0 is the initial concentration of boronic ester
estrogen prior to reaction with H2O2 and A0 and Ainf are the
absorbances measured respectively at reaction time t = 0, and t =
infinity (full conversion to phenol). Concentration values were
then entered into GraphPad Prism 7 and tested for mathematical
fits. The model that provided the best fit for the data was an
exponential decay model described by the equation:
1
afford compound 6b (0.0256 g, 35 %): H NMR (300 MHz,
Acetone-d6) δ 8.19 (s, 1 H), 7.76 (d, J = 8.2 Hz, 2 H), 7.40 (d, J
= 8.1 Hz, 2 H), 7.08 (d, J = 8.5 Hz, 2 H), 6.76 (d, J = 8.6 Hz, 2
H), 4.32 (t, J = 7.5 Hz, 1 H), 3.13 (d, J = 7.5 Hz, 2 H), 1.34 (s, 12
H); 13C NMR (75 MHz, Acetone-d6) δ 206.1, 157.5, 140.3,
136.0, 131.3, 128.6, 127.9, 121.2, 116.1, 84.7, 41.6, 40.3, 25.2.
HRMS (ESI-TOF) exact mass calcd for C21H24NO3BNa [M+Na]+
372.1745, found 372.1723.
[BEE] = [BEE]0*ekt
(2)
where k is the observed rate constant kobs
.
4.5. Gas Chromatography-Mass Spectrometry (GCMS) analysis
Solutions of 3 used for GCMS were prepared in 50 µL
volumes at a final concentration of 500 µM in PBS. H2O2 was
added in 10-fold excess to a final concentration of 5 mM before
incubation at 37 °C for 1 hour. After incubation, the organic
products were extracted with 50 µL of ethyl acetate before
injection of 5 µL on the GCMS (Shimadzu GCMS-QP5050,
RTX5 column: 30 meters, 0.25 mm ID, 0.25 µm film thickness).
4.1.2.3.3. Compound 6c
Prepared from 5c (70 mg, 0.19 mmol), bis(pinacolato)diboron
(108 mg, 0.42 mmol), KOAc (55 mg, 0.58 mmol) and
Pd(dppf)Cl2 (4.7 mg, 0.006 mmol) in dioxane (1.1 mL) according
to the procedure above to afford compound 6c (0.0145 g, 17 %):
1H NMR (300 MHz, CDCl3) δ 7.78 (d, J = 8.1 Hz, 2 H), 7.73 (d,
J = 8.0 Hz, 2 H), 7.25 (d, J = 7.7 Hz, 2 H), 7.12 (d, J = 8.0 Hz, 2
H), 4.01 (dd, J = 7.9, 6.7 Hz, 1 H), 3.17 (dd, J = 10.3, 7.4 Hz, 2
H), 1.35 (s, 12 H), 1.34 (s, 12 H); 13C NMR (75 MHz, CDCl3) δ
139.2, 138.0, 135.4, 135.1, 128.6, 126.8, 120.0, 84.0, 83.8, 42.2,
39.7, 24.9. HRMS (ESI-TOF) exact mass calcd for
C27H35NO4B2Na [M+Na]+ 482.2654, found 482.2663.
4.6. ROS selectivity test
Solutions of 6a-6c used for HPLC were prepared in 50 μL
volumes at a final concentration of 150 μM in PBS. Each ROS
was added in a 5‒fold excess to a final concentration of 750 μM
before incubation at 37 °C for 1 hour. After incubation, the
solutions were centrifuged (6000 rpm, 2 min) prior to injection
on the HPLC (30 minutes isocratic 50:50 MeOH: H2O, Kromasil
100-5-C18, 4.6 x 250 mm, 0.5 mL/min, followed at 226 nm).
H2O2, hypochlorite (NaOCl), and tert-butylhydroperoxide
4.2. DPN docking

17. Carroll, V.M.; Jeyakumar, M.; Carlson, K.E.; Katzenellenbogen,
J.A. J. Med. Chem. 2012, 55, 528-537.
18. Brown, C.M.; Mulcahey, T.A.; Filipek, N.C.; Wise, P.M.
Endocrinol. 2010, 151, 4916-4925.
19. Crawford, D.K.; Mangiardi, M.; Song, B.; Patel, R.; Du, S.;
Sofroniew, M.V.; Voskuhl, R.R.; Tiwari-Woodruff, S.K. Brain
2010, 133, 2999-3016.
20. Kumar, S.; Patel, R.; Moore, S.; Crawford, D.K.; Suwanna, N.;
Mangiardi, M.; Tiwari-Woodruff, S.K. Neurobiol. Dis. 2013, 56,
131-144.
(TBHP) were delivered from 30%, 70%, and 10% aqueous
solutions, respectively. Superoxide (O2 ·) was generated from the
-
enzymatic reaction of xanthine oxidase (2 units, 0.5 μL) and
hypoxanthine (5 μL of 10 mM stock) in the presence of 5 units of
catalase to quench any H2O2. Nitric Oxide (NO) was generated
from NOC5 (stock solution 1 mM in 0.1 M NaOH); degradation
of 1050 μM NOC5 in aqueous solution will generate 750 μM
after 60 min. Hydroxyl Radical (OH·) was generated by mixing
25 μL of 30 mM H2O2 with 25 μL of 150 mM Fe2+.
21. Lewis, D.K.; Johnson, A.B.; Stohlgren, S.; Harms, A.; Sohrabji, F.
J. Neuroimmunol. 2008, 195, 47-59.
22. Saijo, K.; Collier, J.G.; Li, A.C.; Katzenellenbogen, J.A.; Glass,
C.K. Cell 2011, 145, 584-595.
4.7. Luciferase assay
The transcriptional activation of the ligands were assessed
using the Human Estrogen Receptor Beta reporter assay system
(Indigo Biosciences). ERβ reporter cells consisting of an ERβ-
responsive promoter gene linked to the luciferase gene were
defrosted and seeded into a 96-well plate. The cells were treated
with the ligands at the indicated concentrations according to the
manufacturer’s protocol. After 24 hours of incubation, the
treatment media was discarded and the luciferase detection
reagent added. The luminescence of each sample well was
quantified using a SpectraMax i3 plate reader after 10 minutes of
incubation at room temperature.
23. Smith, J.A.; Das, A.; Butler, J.T.; Ray, S.K.; Banik, N.L.
Neurochem. Res. 2011, 36, 1587-1593.
24. Suwanna, N.; Thangnipon, W.; Kumar, S.; de Vellis, J. EXCLI J.
2014, 13, 1097-1103.
25. Yu, H.P.; Hsieh, Y.C.; Suzuki, T.; Shimizu, T.; Choudhry, M.A.;
Schwacha, M.G.; Chaudry, I.H. Am. J. Physiol. Lung Cell Mol.
Physiol. 2006, 290, L1004-1009.
26. Mohler, M.L.; Narayanan, R.; Coss, C.C.; Hu, K.; He, Y.; Wu, Z.;
Hong. S.S.; Hwang, D.J.; Miller, D.D.; Dalton, J.T. Expert Opin.
Ther. Pat. 2010, 20, 507-534.
27. Paterni, I.; Granchi, C.; Katzenellenbogen, J.A.; Minutolo, F.
Steroids 2014, 90, 13-29.
28. Farooq, A. Curr. Top. Med. Chem. 2015, 15, 1372-1384.
29. Pike, A.C.W.; Brzozowski, A.M.; Hubbard, R.E.; Walton, J.;
Bonn, T.; Thorsell, A.-G.; Engstrom, O.; Ljunggren, J.;
Gustaffson, J.-A.; Cariquist, M. PDB: 2J7X
Acknowledgments
30. Manas, E.S.; Xu, Z.B.; Unwalla, R.J.; Somers, W.S. Structure,
2004, 12, 2197-2207.
31. Govan, J.M.; McIver, A.L.; Riggsbee, C.; Deiters, A. Angew.
Chem. Int. Ed. Engl. 2012, 51, 9066-9070.
32. Carlson, K.E.; Choi, I.; Gee, A.; Katzenellenbogen, B.S.;
Katzenellenbogen, J.A. Biochemistry 1997, 36, 14897-14905.
33. Blair, R.M.; Fang, H.; Branham, W.S.; Hass, B.S.; Dial, S.L.;
Moland, C.L.; Tong, W.; Shi, L.; Perkins, R.; Sheehan, D.M.
Toxicol. Sci. 2000, 54, 138-153.
34. Sikora, A.; Zielonka, J.; Lopez, M.; Joseph, J.; Kalyanaraman, B.
Free Rad. Biol. Med. 2009, 47, 1401-1407.
35. Sun, X.; Xu, Q.; Kim, G.; Flower, S.E.; Lowe, J.P.; Yoon, J.;
Fossey, J.S.; Qian, X.; Bull, S.D.; James, T.D. Chem. Sci. 2014, 5,
3368-3373.
36. Wyatt, A.R.; Kumita, J.R.; Mifsud, R.W.; Gooden, C.A.; Wilson,
M.R.; Dobson, C.M. Proc. Nat. Ac. Sci. 2014, 111, E2081-E2090.
37. Coombes, E.; Jiang, J.; Chu, X.-P.; Inoue, K.; Seeds, J.; Branigan,
D.; Simon, R.P.; Xiong, Z.-G. Antioxid. Redox Signal. 2011, 14,
1815-1827.
38. Mayes, J.; Tinker-Mill, C.; Kolosov, O.; Zhang, H.; Tabner, B.J.;
Allsop, D. J. Biol. Chem. 2014, 289, 12052-12062.
39. Yang, J.; Yang, J.; Liang, S.G.; Xu, Y.; Moore, A.; Ran, C. Sci.
Rep. 2016, 6, 35613.
This work was supported by start-up funds from the
University of Richmond (J.A.P.). H.P. and J.D.M. were
recipients of Puryear-Topham-Pierce fellowships from the
chemistry department at the University of Richmond and
acknowledge support from the University of Richmond Arts and
Sciences Undergraduate Research Committee. The authors would
like to thank Prof. Michelle Hamm and Dr. Diane Kellogg for
their help with instrumentation and Phil Joseph and Robert
Plymale who are essential to the success of the undergraduate
research program at the University of Richmond.
References and notes
1.
2.
Glass, C.K.; Saijo, K.; Winner, B.; Marchetto, M.C.; Gage, F.H.
Cell 2010, 140, 918-934.
Uttara, B.; Singh, A.V.; Zamboni, P.; Mahajan, R.T. Curr.
Neuropharmacol. 2009, 7, 65-74.
3.
4.
Straub, R.H. Endocrine Rev. 2007, 28, 521-574.
Kuivila, H.G.; Armour, A.G. J. Am. Chem. Soc. 1957, 79, 5659-
5662.
5.
6.
7.
Albers, A.E.; Okreglak, V.S.; Chang, C.J. J. Am. Chem. Soc. 2006,
128, 9640-9641.
Major Jourden, J.L.; Cohen, S.M. Angew. Chem. Int. Ed. Engl.
2010, 49, 6795-6797.
Charkoudian, L.K.; Pham, D.M.; Franz, K.J. J. Am. Chem. Soc.
2006, 128, 12424-12425.
40. Grosdidier, A.; Zoete, V.; Michielin, O. Nucleic Acids Res. 2011,
39, W270-277.
41. Grosdidier, A.; Zoete, V.; Michielin, O. J. Comput. Chem. 2011,
32, 2149-2159.
42. Humphrey, W.; Dalke, A.; Schulten, K. J. Molec. Graphics, 1996,
14, 33-38.
8.
9.
Franks, A.T.; Franz, K.J. Chem. Comm. 2014, 50, 11317-11320.
Chen, W.; Balakrishnan, K.; Kuang, Y.; Han, Y.; Fu, M.; Gandhi,
V.; Peng, X. J. Med. Chem. 2014, 57, 4498-4510.
Supplementary Material
10. Kuang, Y.; Balakrishnan, K.; Gandhi, V.; Peng, X. J. Am. Chem.
Soc. 2011, 133, 19278-19281.
11. Vadukoot, A.K.; AbdulSalam, S.F.; Wunderlich, M.; Pullen, E.D.;
Landero-Figueroa, J.; Mulloy, J.C.; Merino, E.J. Bioorg. Med.
Chem Lett. 2014, 22, 6885-6892.
Supplementary data associated with this article can be found
in the online version.
12. Liao, Y.; Xu, L.; Ou, S.; Edwards, H.; Luedtke, D.; Ge, Y.; Qin,
Z. ACS Med. Chem. Lett. 2018, 9, 635-640.
13. Jiang, Q.; Zhong, Q.; Zhang, Q.; Zheng, S.; Wang, G. ACS Med.
Chem. Lett. 2012, 3, 392-396.
14. Zhang, C.; Zhong, Q.; Zhang, Q.; Zheng, S.; Miele, L.; Wang, G.
Breast Cancer Res. Treat. 2015, 152, 283-291.
15. Zhong, Q.; Zhang, C.; Zhang, Q.; Miele, L.; Zheng, S.; Wang, G.
BMC Cancer, 2015, 15, 625.
16. Meyers, M.J.; Sun, J.; Carlson, K.E.; Marriner, G.A.;
Katzenellenbogen, B.S.; Katzenellenbogen, J.A. J. Med. Chem.,
2001, 44, 4230-4251.