Synthesis and evaluation of 17α-Arylestradiols as ligands for estrogen receptor α and β

Article abstract of DOI:10.1021/jm901898a

To identify novel estrogen receptor ligands a series of substituted 17α-arylestradiols were synthesized using the catalytic [2 + 2 + 2]cyclotrimerization of 17α-ethynylestradiol with various 1,7-diynes in the presence of Wilkinsons catalysts [Rh(PPh₃)₃Cl]. The compounds were subjected to competitive binding assays, cell-based luciferase reporter assays, and proliferation assays. These experiments confirmed their estrogenic character and revealed some interesting properties like mixed partial/full agonism for ERα/ERβ and different selectivity as a result of differing potencies for either ER.

Full text of DOI:10.1021/jm901898a

4290 J. Med. Chem. 2010, 53, 4290–4294
DOI: 10.1021/jm901898a
Synthesis and Evaluation of 17r-Arylestradiols as Ligands for Estrogen Receptor r and β
†
David Sedlak, Petr Novak, Martin Kotora,* and Petr Bartunek*
‡
,‡,§
,†
ꢀ
ꢀ
ꢁ
†
´
ꢁ ꢀ
Center for Chemical Genetics, Institute of Molecular Genetics, v.v.i., Academy of Sciences of the Czech Republic, Vıdenska 1083, 142 20 Prague,
Czech Republic, ‡Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague,
§
Czech Republic, and Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166
ꢀ
10 Prague, Czech Republic
Received December 28, 2009
To identify novel estrogen receptor ligands a series of substituted 17R-arylestradiols were synthesized
using the catalytic [2 þ 2 þ 2]cyclotrimerization of 17R-ethynylestradiol with various 1,7-diynes in the
presence of Wilkinson’s catalysts [Rh(PPh3)3Cl]. The compounds were subjected to competitive binding
assays, cell-based luciferase reporter assays, and proliferation assays. These experiments confirmed their
estrogenic character and revealed some interesting properties like mixed partial/full agonism for ERR/
ERβ and different selectivity as a result of differing potencies for either ER.
Introduction
complex with E2 and raloxifene in 19975 and ERβ LBD in
complex with genistein and raloxifene soon after cloning of
this gene in 1999.6
Estrogens are steroid hormones responsible for affecting a
wide range of biological functions. They predominantly act by
binding to their corresponding intracellular receptors, result-
ing in the downstream modulation of target gene transcrip-
tion. In 1996, a second gene encoding the estrogen receptor
(ERa) was identified.1 This discovery explained how estrogens
could be involved in mediating such diverse functions and
prompted researchers to define functions specific to ERR or
ERβ. While 17β-estradiol (E2) binds to both receptors with a
similar affinity,2 the tissue distribution of each receptor is
different. ERR mediates the action of estrogens in classical
tissues like the uterus and mammary gland. Given that ERR
has been shown to augment the proliferation of both healthy
and cancerous cells in a number of tissues, including the mam-
mary gland, ERR has traditionally been targeted in breast
cancer therapy.
17R-Arylestradiols have been shown to possess interesting
biological activities.7-14 Although they can be synthesized via
the reaction of estrone with the corresponding organolithi-
ums,7-14 this method lacks generality, requires the use of
protective groups, and often results in low yields of the desired
products. Given their interesting biological properties, the
development of a simple method for quickly generating a
series of variously modified 17R-arylestradiols is highly desir-
able. Considering the commercial availability of 17R-ethynyl-
estradiol, it is an ideal starting material for the synthesis of
17-arylestradiol derivatives that may later be screened as
potential estrogen receptor ligands.
Results and Discussion
ERβ functions in the ovary, brain, cardiovascular system,
and prostate. Additionally, it has been implicated by several
animal models to play a role in inflammation.3 Intriguingly, it
was shown that an increase in ERβ expression could inhibit
the growth of some estrogen-dependent cell lines.4 Moreover,
several clinical studies demonstrated an inverse correlation
between the level of ERβ expression and advancing grade of
breast cancer. Taken together, these findings led to the
hypothesis that ERβ may function as a potential tumor
suppressor.
From a general point of view, reactions that yield a signi-
ficant increase in molecular complexity are considered key
steps in organic synthesis. In this regard, a union of carbon-
carbon π bonds that create a carbocyclic system is especially
attractive. One such reaction is the [2 þ 2 þ 2]-cyclotrimeriza-
tion of alkynes into benzene derivatives. This reaction is
catalyzed by transition metal complexes such as Ni, Co, and
Rh under mild reaction conditions that are compatible with
the presence of various functional groups.15 This alkyne cyclo-
trimerization reaction has previously been used to synthesize
many natural products (steroids, alkaloids, terpenes, amino
acids, purine derivatives, etc.). Recently, we have successfully
used Ni16-18 and Rh19,20 based catalysts in the synthesis of
various potentially biologically active compounds. Given this
success, cyclotrimerization was chosen to synthesize a series of
17-arylestradiol derivatives.
The design of new synthetic ligands with modified proper-
ties and therefore effects on these receptors was facilitated by
the publication of the X-ray structure of ERR LBD in
*To whom correspondence should be addressed. For P.B.: phone,
þ420 241 063 117; fax, þ420 241 063 586; E-mail, bartunek@img.cas.cz.
For M.K.: phone, þ420 221 951 334; fax, þ420 221 951 326; E-mail,
kotora@natur.cuni.cz.
a Abbreviations: ER, estrogen receptor; LBD, ligand-binding
domain; E2, 17β-estradiol; 17EE, 17R-ethynylestradiol; RBA, relative
binding affinity; RTC, relative transactivation capacity; cLogP, computed
partition coefficient.
Because organic synthesis should be based on simple reac-
tion conditions that exploit readily available starting materi-
als, we wished to carry out the cyclotrimerization directly with
17R-ethynylestradiol possessing unprotected hydroxyl groups.
If successful, we would obtain in one-step the corresponding
r
pubs.acs.org/jmc
Published on Web 04/21/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4291
17R-arylestradiols, allowing us to avoid the use of protection
and deprotection protocols. To keep the reaction conditions
as simple as possible and circumvent the use of highly air sen-
sitive Ni(0) catalysts, we decided to use the readily available
and stable catalyst Wilkinson’s complex, [Rh(PPh3)3Cl],21 in
cyclotrimerization experiments. This complex was chosen as it
is one of the most general cyclotrimerization catalysts that
tolerates free OH groups.22
The cyclotrimerization of 17R-ethynylestradiol (1 equiv)
with diynes (1.2 equiv) was carried out in the presence of
[Rh(PPh3)3Cl] (10 mol %) in a mixture of toluene and MeCN
at 20 °C for 24 h (Scheme 1). The results obtained are summa-
rized in Table 1. Generally, the cyclotrimerization process
proceeded with reasonable efficiency giving rise to the corres-
ponding 17-arylestradiols 3a-3d in adequate isolated yields
(entries 1-4). Only in the case of diynes 2e and 2f were the
yields rather low (entries 5 and 6). Interestingly, attempts to
catalyze the reaction with [Cp*Ru(cod)Cl],23 which had
recently been shown to efficiently catalyze the cyclotrimeriza-
tion of alkynes bearing various functional groups failed,
and the formation of cyclotrimerization products were not
observed.
Initially, the newly prepared compounds were assayed for
their ability to specifically bind each ER. In vitro fluorescence
polarization-based competitive binding assays were carried
out andthe resulting data (showninTable2) demonstratethat
all compounds exhibit a high binding affinity for both ERs
with affinities ranging from 1 to 22% of the E2 binding
affinity. Substitution of the hydrogen atom at the 17R position
of E2 with an aryl group led to a decrease in ER binding
affinity. Because the overall lipophilicity of the compound
might also have influence on the binding to the receptor, we
compared theoretical partition coefficients to the compound
structures. Although an increased lipophilicity generally con-
tributes positively to the binding, we saw a decrease in binding
affinity in compounds with considerably higher cLogP com-
pared to both E2 and 17EE as probably a result of the
bulkiness of the substituent’s (3a, 3e, 3c). This effect was
especially pronounced for 3a, which is the most lipophilic of
the tested molecules but binds to both ERs with the lowest
affinity. Conversely, 17R-arylestradiols substituted with a
smaller aryl group (3d and 3f) bound better to the receptors
than compounds with larger and polar substituents.
To determine whether the ER binding affinities of each
compound correlated with their transactivation capacity, we
used a luciferase reporter assay in HEK293 cells. Dose-
response curves representing the activation of ERs in response
to each compound are shown in Figure 1 and the correspond-
ing analysis of the obtained data is summarized in Table 3.
Satisfyingly, the compounds that demonstrated the highest
binding affinities were also the most efficient activators of
transcription. For example, in the binding assay, compound
3f demonstrates a high affinity for both receptors and simi-
larly functions as a good agonist to activate both receptors
without preference in the reporter assays. Compound 3d
shows the highest affinity for both ERR (EC50 = 6.92 nM,
Log EC50=-8.16) and ERβ (EC50=22.7 nM, Log EC50=
-7.65) and similarly exhibits the highest transactivation
potency in both receptor reporter assays. Of interest, 3d is a
slightly more potent activator of ERR than ERβ.
As we successfully obtained a set of variously substituted
17R-arylestradiols 3, we decided to subject them to a range of
biochemical tests to assess their potential biological activity
with respect to ER signaling.
Scheme 1. Cyclotrimerization of 1 with Diynes 2 to 3
Interestingly, compound 3a is a poor agonist of ERβ
(EC50=174 nM, LogEC50=-6.76), however it preferentially
activates ERR. We measured by reporter assay that 3a is
13ꢀ more selective for ERR than ERβ. The decreased
potency of 3a for ERβ can be partially explained by the
fact that the binding cavity volume in the ERβ LBD is smaller
than in ERR (390 A3 versus 450 A3) as results from X-ray
structures of ER LBD complexes with corresponding
ligands.5,6 Thus, larger side chains at the 17R position of
estradiol mitigate efficient binding to ERβ but have less effect
on ERR binding.
Table 1. Cyclotrimerization of 17R-Ethynylestradiol 1 with Diynes 2
Catalyzed by [Rh(PPh3)3Cl]
entry
diyne 2
arylestradiol 3
yield (%)a
1
2
3
4
5
6
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
56
50
41
45
27
13
a Isolated yields.
Table 2. Binding Affinities of Compounds for Human ERR and ERβ
compd
Log IC50 ERR (M)a
Log IC50 ERβ (M)a
RBA ERR (%)b
RBA ERβ (%)b
cLogPc
E2
17EE
3a
-7.995 ( 0.081
-8.095 ( 0.070
-6.308 ( 0.033
-6.614 ( 0.094
-6.610 ( 0.095
-7.323 ( 0.121
-6.467 ( 0.131
-7.129 ( 0.227
-7.945 ( 0.053
-7.750 ( 0.047
-5.965 ( 0.218
-6.596 ( 0.102
-6.496 ( 0.063
-7.271 ( 0.199
-6.301 ( 0.078
-7.117 ( 0.191
100
126
100
3.78
3.86
6.36
4.77
5.57
5.20
6.84
5.01
63.2
1.05
4.48
3.55
21.2
2.27
14.9
2.06
4.16
4.13
21.2
2.97
13.6
3b
3c
3d
3e
3f
a Log IC50 values were determined by testing 14 different concentrations of each compound using the fluorescence polarization-based ER competitor
assay and plotting the resultant data using a nonlinear regression plot using GraphPad Prism 5.0 software. The experiment was performed in triplicate
and the error is reported as standard error of the mean (SEM). b Relative binding affinity (RBA) was calculated as a ratio of E2 and the competitor
concentration required to reduce the specific fluorescent ligand binding by 50%. c Computed partition coefficient (cLogP) was calculated using
ChemBioOffice2010 software suite (CambridgeSoft Corporation).

4292 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Sedlaꢀk et al.
Three out of the six tested compounds (3b, 3e, 3f) showed a
full agonistic profile for ERR. These compounds generally
reached at least 80% of the full activation level of E2, while for
ERβ, all compounds except one (3b) functioned as full
agonists. Moreover, 3d, 3e, and 3f tended to activate ERβ to
levels exceeding that of E2, however this supragonist property
did not correlate with the transactivation potency of the
compounds.
Steroid hormones exert their biological functions at nano-
and subnano-molar concentrations. One of the best estab-
lished in vitro assays for assessing the estrogenic properties of
ER ligands is the MCF-7 proliferation assay.24,25 For the
efficient growth of MCF-7 cells, only a low concentration of
estrogens in the medium is required. To analyze the estrogenic
properties of our compounds, we cultivated MCF-7 cells in
medium depleted of steroid hormones. The growth medium
was then reconstituted with varying concentrations of the
novel compounds and the cells assayed for proliferation. Data
collected from these experiments are summarized in Figure 2.
The proliferation rates measured are in agreement with the
results obtained from the ERR reporter assays and illustrate
clearly that the compounds possess estrogenic properties,
albeit with a potency that is approximately 2-3 orders lower
than E2. All of the compounds except 3b stimulated estrogen-
dependent growth at concentrations as low as 1 pM and
continued to augment proliferation at concentrations ranging
between 0.1 and 1 nM. Compound 3b did not potentiate any
estrogenic effect until it reached a concentration of 1 nM.
Maximal proliferative effects were observed at 100 nM (data
not shown). In agreement, out of all the compounds tested, 3b
also exhibited the weakest ability to function as an ERR
agonist in reporter assays.
Conclusion
In conclusion, we have prepared a series of 17R-arylestra-
diols and tested them using different biochemical assays such
as the fluorescence polarization-based competitive binding
assay, ERR and ERβ luciferase reporter assays, andthe MCF-
7 proliferation assay. In all of the assays performed, the
compounds clearly showed agonistic properties with one
compound displaying a 13ꢀ selectivity for ERR. Moreover,
we observed a variety of interesting biochemical properties
like mixed partial/full agonism in ERR/ERβ reporter assays
and differing affinities and potencies for ERR/ERβ as a result
of using different substituents at the 17R-position of estradiol.
Experimental Section
Figure 1. Transactivation of luciferase reporters by ERR and ERβ
in response to increasing concentrations of compound (b) or E2 (O)
in HEK293 reporter cells. The highest stimulation of the receptor in
response to E2 was arbitrarily set as100%. Each value is reported as
a mean ( standard error of the mean (SEM) from three experi-
ments.
All solvents were used as obtained unless otherwise noted.
Toluene was distilled from benzophenone/Na under Ar and
CH3CN was purchased from Sigma-Aldrich (St. Louis, MO).
Starting diynes 2 were prepared using standard procedures by the
reacting propargyl bromide with the corresponding C-acids
Table 3. HEK293 Reporter Assay Demonstrating the Effect of Compounds on the Transactivation Ability of ERR and ERβ
selectivity
for ERβd
compd Log EC50 ERR [M]a Log EC50 ERβ [M]a RTC ERR (%)b RTC ERβ (%)b efficacy ERR (%)c efficacy ERβ (%)c
E2
3a
3b
3c
3d
3e
3f
-10.19 ( 0.119
-7.852 ( 0.093
-6.474 ( 0.051
-7.783 ( 0.077
-8.160 ( 0.071
-7.036 ( 0.041
-7.444 ( 0.076
-10.21 ( 0.063
-6.756 ( 0.081
-6.655 ( 0.124
-7.461 ( 0.087
-7.645 ( 0.142
-6.616 ( 0.104
-7.574 ( 0.108
100
0.45
100
0.03
100 ( 7.7
55.2 ( 3.4
92.8 ( 4.3
64.5 ( 3.3
72.0 ( 3.2
87.2 ( 3.5
82.2 ( 4.2
100 ( 3.8
105 ( 5.1
65.5 ( 5.8
97.0 ( 5.0
112 ( 7.3
108 ( 5.1
111 ( 6.4
1.00
0.08
1.46
0.46
0.29
0.37
1.29
0.02
0.39
0.92
0.07
0.18
0.03
0.18
0.27
0.03
0.23
a Log EC50 values were generated by fitting data from the ERR and ERβ reporter assays in a nonlinear regression function using GraphPad Prism 5.0
software. The error of the determination is reported as standard error of the mean (SEM). b Relative transactivation capacity (RTC) of each compound
was calculated as a ratio of EC50 for E2 and for the corresponding compound. c Efficacy of each compound was calculated as a ratio of the highest
luciferase activity induced by the corresponding compound and E2. The highest luciferase activity induced by E2 was arbitrarily set as 100%. d Selectivity
of the compounds for ERβ was calculated as a ratio of relative transactivation capacity of the tested compound for ERβ and for ERR.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4293
Figure 2. Proliferation of estrogen-growth-dependent MCF-7 cells in response to differing concentrations of either E2 or compounds.
Previously starved cells were incubated in the presence of the tested compounds at the following concentrations: (a) 10-12 M; (b) 1.3 ꢀ 10-10 M;
(c) 3.2 ꢀ 10-9 M, and (d) 1.6 ꢀ 10-8 M. After 6 days of incubation, cell viability was measured as a capacity of cells to convert resazurin to
resorufin. Relative cell number was calculated as a ratio of number of viable cells treated with tested compounds and number of viable cells
treated with DMSO only. Values are reported as mean ( standard error of the mean (SEM) of three experiments.
1
underbasicconditions.17 RhCl(PPh3)3 was preparedaccording to
the previously reported procedures.26 Cp*RuCl(cod) was pur-
chased from Strem Ltd., and 17R-ethynylestradiol was purchased
from Fluka.
solid; mp 158 °C; [R]D =þ40.9 (c 0.0055 g/mL, acetone). H
NMR (400 MHz, C6D6) δ 0.71-0.78 (m, 1H), 1.04 (s, 3H),
1.05-1.19 (m, 1H), 1.30-1.40 (m, 4H), 1.62-1.65 (m, 2H), 1.69
(s, 3H), 1.70 (s, 3H), 1.72-1.77 (m, 2H), 1.90-1.96 (m, 2H),
2.21-2.27 (m, 1H), 2.60-2.70 (m, 2H), 3.22-3.34 (m, 4H), 4.46
(s, 1H), 6.39 (d, J=2.4 Hz, 1H), 6.47 (dd, J=8, 2.4 Hz, 1H), 6.92
(d, J=8 Hz, 1H), 6.99 (d, J=8 Hz, 1H), 7.18 (s, 1H) overlapped,
7.32 (s, 1H). 13C NMR (100 MHz, C6D6) δ 15.71, 25.08, 26.60,
26.66, 27.38, 28.50, 30.61, 34.74, 38.01, 38.39, 39.75, 40.52,
44.31, 47.87, 49.20, 75.75, 86.55, 113.69, 116.18, 124.00,
124.59, 127.37, 128.25, 129.24, 133.01, 138.53, 139.30, 140.03,
146.59, 154.96, 204.48 (2ꢀ). IR (ATR ZnSe) ν 3427, 2927, 2870,
1692, 1610, 1498, 1359, 1249, 1150 cm-1. MS (EI) 472 (9), 454
(12), 411 (100), 228 (14), 213 (13), 159 (17). HRMS (EI) cacld
for C31H36O4 472.261360, found 472.261119.
NMR spectra were recorded on a Varian UNITY 400 INOVA
instrument at 400 MHz (1H) and 100.6 MHz (13C) as solutions in
C6D6 and are referenced to the residual solvent signal. Infrared
spectra were recorded on a Bruker IFS 88 instrument. Mass
spectra were recorded on a FINNIGAN MAT INCOS 50
spectrometer. HR mass spectra were recorded on a ZAB-SEQ
VG Analytical spectrometer. HPLC analysis was carried out on
Supelcosil column (Si: 5 μm; 250 mm ꢀ 4.6 mm; heptane/iPrOH
98/2, 1 mL/min; detection: UV-210 nm). Purity of the prepared
compounds was determined by a combination of 1H NMR and
HLPC techniques and was found to be >95%.
Cyclotrimerization Reaction: General Procedure for RhCl-
(PPh3)3 Catalyzed Cyclotrimerization of 17r-Ethynylestradiol
with Diynes. 17R-Ethynylestradiol (0.5 mmol, 150 mg) was
placed into a solution of dry toluene (6 mL), and CH3CN (1
mL) was added to RhCl(PPh3)3 (0.05 mmol, 46 mg) and diyne 2
(0.6 mmol). The reaction mixture was stirred at 20 °C for 48 h or
until the consumption of the starting material (TLC). The
solvent was then evaporated under reduced pressure, and the
residue was subjected to column chromatography.
17r-[2,2-Bis(ethoxycarbonyl)-1,3-dihydro-2H-inden-5-yl]-
estradiol (3a). 1 (0.25 mmol, 74 mg), 2a (0.3 mmol, 70.8 mg),
RhCl(PPh3)3 (0.025 mmol, 23 mg). Column chromatography (2/
1 hexane/EtOAc) furnished 75 mg (56%) of the title compound
as a colorless solid; mp 164 °C; [R]D = þ35 (c 0.022 g/mL,
acetone). 1H NMR (400 MHz, C6D6) δ 0.71-0.79 (m, 1H), 0.87
(t, J = 6.8 Hz, 3H), 0.88 (t, J = 6.8 Hz, 3H), 1.06 (s, 3H),
1.03-1.12 (m, 1H), 1.29-1.42 (m, 4H), 1.61-1.75 (m, 4H),
1.88-2.00 (m, 2H), 2.18-2.23 (m, 1H), 2.58-2.70 (m, 2H),
3.73-3.85 (m, 4H), 3.91 (q, J=6.8 Hz, 2H), 3.92 (q, J=6.8 Hz,
2H), 5.14 (s, 1H), 6.47 (d, J=2.8 Hz, 1H), 6.57 (dd, J=8, 2.8 Hz,
1H), 6.94 (d, J=8 Hz, 1H), 7.04 (d, J=8 Hz, 1H), 7.164 (s, 1H)
signal overlapped, 7.37 (s, 1H). 13C NMR (100 MHz, C6D6) δ
14.60 (2ꢀ), 15.74, 25.05, 27.33, 28.46, 30.62, 34.67, 39.63, 40.46,
41.37, 41.76, 44.20, 47.83, 49.15, 61.66, 62.35 (2ꢀ), 86.64,
113.73, 116.23, 123.90, 124.49, 127.40, 129.23, 133.05, 138.56,
139.50, 140.19, 146.60, 155.06, 172.47 (2ꢀ). IR (ATR ZnSe) ν
3398, 2958, 2927, 2870, 1727, 1711, 1610, 1502, 1442, 1283, 1249,
1185, 1068, 1049, 1008 cm-1. MS (EI) 532 (8), 514 (72), 499 (17),
425 (17), 314 (12), 213 (56), 149 (77). HRMS (EI) cacld. for
C33H40O6 532.282489, found 532.283226.
Biological Testing: Materials. 17β-Estradiol (E2) was pur-
chased from Sigma-Aldrich (St. Louis, MO), phenol red-free
Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine
serum (FBS) were obtained from Invitrogen (Carlsbad, CA),
Hyclone charcoal/dextran treated fetal bovine serum (C/D FBS)
was purchased from Thermo Fisher Scientific Inc. (Waltham,
MA). One-Glo luciferase assay system and CellTiter-Blue cell
viability assay kits were obtained from Promega (Madison,
WI). Panvera’s fluorescence polarization-based ER compe-
titor assays, RED, for ERR and ERβ were purchased from
Invitrogen (Carlsbad, CA).
Fluorescence Polarization-Based Competitive Binding Assay.
The binding affinity of each compound for both ERs was
performed according to the manufacturer’s protocol. The ex-
periment was carried out in black 384-well plates (Corning Inc.,
NY) in a total volume of 40 μL. Fluorescent data were collected
on the EnVision (PerkinElmer, Inc., Waltham, MA) plate read-
er after a 3 h incubation at room temperature using optimized
BODIPY TMR FP Label consisting of 531 nm excitation filter,
595 nm S polarized emission filter, 595 nm P polarized emission
filter, and BODIPY TMR FP optical module. Collected data
were subsequently analyzed by GraphPad Prism 5.0 statistical
software and IC50 values were calculated from regression
function (dose-response, variable slope).
Reporter Assay. HEK293 cells were maintained in a mono-
layer in DMEM supplemented with 10% FBS, 2 mM glutamine,
and penicillin/streptomycin and incubated in a 5% CO2 humi-
dified atmosphere at 37 °C. To generate ERR and ERβ respon-
sive reporter cells, HEK293 cells were transfected with either
pCDNA3-hERR or pCDNA3-hERβ and the reporter vector
pGL4-3ERE-luc (Sedlak et al., unpublished results). Following
transfection, cells were maintained in phenol red-free DMEM
supplemented with 4% C/D FBS and 2 mM glutamine. Twenty-
four hours after transfection, cells were trypsinized, counted,
7r-(2,2-Diacetyl-1,3-dihydro-2H-inden-5-yl)-estradiol (3b). 1
(0.5 mmol, 150 mg), 2b (0.6 mmol, 105.6 mg), RhCl(PPh3)3 (0.05
mmol, 46 mg). Column chromatography (2/1 hexane/EtOAc)
furnished 119 mg (50%) of the title compound as a colorless

4294 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Sedlaꢀk et al.
and seeded at a density of 104 cells/well in white opaque cell
culture 384-well plates (Corning Inc., NY). Each compound
tested was serially diluted in DMSO and stored in 384-well
plates before being transferred by the JANUS Automated
Workstation (PerkinElmer, Inc.) equipped with a Pin Tool
(V&P Scientific, Inc., San Diego, CA). After a further 18 h
incubation, luciferase activity was determined using the One-
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Glo luciferase assay system according to the manufacturers
protocol (Promega Corp.). Luminescence was recorded on
the EnVision plate reader using 1s integration and data
were analyzed by GraphPad Prism 5.0 statistical software.
EC50 values were calculated from regression function (dose-
response, variable slope).
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Acknowledgment. This research was supported in part by
grant AV0Z50520514, Z40550506, MSM0021620857, and by
LC06077 of the Ministry of Education, Youth and Sports of
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Supporting Information Available: Spectral and analytical
data for 3a-3f. This material is available free of charge via the
Internet at http://pubs.acs.org.
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