X. Liu, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
afford an oily pure product of the title compound; yield: 0.14 g (67%);
1H NMR (600 MHz, CDCl3): δ 4.51–4.56 (q, J = 16.9 Hz, 1H), 5.56 (brs,
2H), 6.78–6.79 (d, J = 14.0 Hz, 4H), 7.19–7.20 (d, J = 12.0 Hz, 4H).
Elemental analysis: Calcd (%) for C14H13F3O2: C 62.69, H 4.13; and
Found C 62.54, H 4.17.
Fullerton, CA).
The data of receptor populations showing the appropriate dis-
sociation constant (Kd) and the receptor density (Bmax) were used for
the following competitive receptor-binding assay. The specific binding
data of [3H]E2 were first assessed by means of a Scatchard plot ana-
lysis30 to ensure that the binding of [3H]E2 to the GST-ER-LBDs is
merely one-site ligand binding. The data were then applied to a one-site
binding hyperbola nonlinear regression analysis using the software
package Prism 6 (GraphPad Software, La Jolla, CA) to measure changes
in the values of Bmax and Kd. The saturation receptor-binding assay was
performed at least three times for every preparation of GST-ER-LBDs for
the verification of the GST-ERα-LBD or GST-ERβ-LBD to warrant its use
in the next competitive binding assays.
4.3. Synthesis of BPE-Br
4.3.1. 2,2-bis(4-methoxyphenyl)-1,1,1-tribromoethane
To a mixture of H2SO4 and AcOH (total 8 ml, 1:1 v/v), tri-
bromoacetaldehyde CBr3CHO (3.0 mmol, 0.31 ml) and anisole
C6H5OCH3 (6.0 mmol, 0.65 ml) were added at 0 °C. The resulting
mixture was treated as described above for neutralization and extrac-
tion. The crude material obtained was purified by the silica gel column
chromatography eluted with petroleum ether/EtOAc (20:1, v/v) to give
the title compound with 15% yield. 1H NMR (600 MHz, CDCl3): δ 3.80
(s, 6H), 5.14 (s, 1H), 6.86–6.88 (d, J = 8.70 Hz, 4H), 7.61–7.63 (d,
J = 8.70, 4H). This compound was used for the next step without
further confirmation because of its low yield.
4.4.3. Radio-ligand binding assays for competitive binding
E2 and bisphenols were dissolved in N,N-dimethylsulfoxide (DMSO)
and diluted by 2 mg/mL γ-globulin with a half-logarithmic 3.16-fold
dilution method, keeping the DMSO concentration below 0.3%. γ-
Globulin was used as a blocker of nonspecific adsorption to the plas-
ticware. Bisphenols were examined for their ability to inhibit the
binding of [3H]E2 (final concentration: 1 nM) to GST-ERα-LBD (60 ng)
or GST-ERβ-LBD (60 ng). The assay solutions were incubated at 4 °C for
1 h, and the B/F separation was carried out by the DCC method as
described above. Radioactivity was determined on a liquid scintillation
counter (TopCount NXT; PerkinElmer Life Sciences Japan, Tokyo).
For the estimation of the binding affinity, the IC50 values (half
maximal inhibitory concentrations) were estimated from the dose-re-
sponse curves generated by the GraphPad Prism 6 software. Each assay
was performed in duplicate and repeated at least three times.
4.3.2. 2,2-bis(4-hydroxyphenyl)-1,1,1-tribromoethane (BPE-Br)
The obtained product of 2,2-bis(4-methoxyphenyl)-1,1,1-tri-
bromoethane (0.20 g, 0.42 mmol) dissolved in CH2Cl2 (20 ml) was
treated with BBr3 (1.3 mmol, 0.22 ml) as described above. The product
was purified by silica gel column chromatography eluted with petro-
leum ether/EtOAc (4:1, v/v) to afford an oily pure product of the title
compound. Yield, 0.12 g (64%); 1H NMR (600 MHz, CDCl3): δ 4.75 (brs,
2H), 5.10 (s, 1H), 6.79–6.80 (m, 4H), 7.54–7.56 (m, 4H). Elemental
analysis: Calcd (%) for C14H13Br3O2: C 37.29, H 2.46; and Found C
37.78, H 2.55.
4.4. Receptor binding assays for the estrogen receptors ERα and ERβ
4.5. In vitro biological assays for the estrogen receptors ERα and ERβ
4.4.1. Preparation of GST-fused estrogen receptor LBD proteins
The preparation of the glutathione S-transferase (GST)-fused re-
ceptor ligand-binding domain (LBD) of ERα and ERβ, namely, GST-
ERα-LBD and GST-ERβ-LBD, was carried out essentially as described
previously.23 These proteins were expressed in E. coli BL21α and pur-
ified on an affinity column (10 × 100 mm) of Glutathione-Sepharose
4B (GE Healthcare BioSciences, Piscataway, NJ) followed by gel fil-
tration on a column of Sephadex G-10 (15 × 100 mm; GE Healthcare
BioSciences). The purity was confirmed by SDS-PAGE using 12.5%
polyacrylamide gel and staining with Coomassie brilliant blue (CBB).
The protein concentrations were determined by the Bradford method.28
4.5.1. Luciferase reporter gene assay for transcription activation activity
HeLa cells were maintained in Eagle's Minimum Essential Medium
(Nissui, Tokyo) in the presence of 10% (v/v) fetal bovine serum at
37 °C. For luciferase assays, HeLa cells were seeded at 5 × 105 cells/6-
cm dish for 24 h and then transfected with 3 µg of reporter gene (pGL3/
3 × estrogen response element [ERE]) and 1 µg of ERα or ERβ ex-
pression plasmid (pcDNA3.1/ERs) by Lipofectamine LTX reagent
(Invitrogen Japan, Tokyo) according to the manufacturer's protocol.
Approximately 24 h after transfection, the cells were harvested and
plated into 96-well plates at 5 × 104 cells per well. The cells were then
treated with varying doses of chemicals diluted with 1% bovine serum
albumin (BSA)/PBS (v/v). BSA was used as a blocker of nonspecific
adsorption to the plasticware.
4.4.2. Radio-ligand binding assays for saturation binding
We performed saturation binding assays to ensure the quality of the
purified receptor proteins; these assays were conducted essentially as
described previously15,23,29 at 25 °C for 1 h. The assay conditions es-
tablished in this study were as follows. GST-ERα-LBD or GST-ERβ-LBD
(60 ng) was incubated with 1–10 nM tritiated ligand [3H]17β-estradiol
([3H]E2) (5.74 TBq/mmol; Amersham Biosciences, Buckinghamshire,
UK) in a final volume of 100 μl of binding buffer [10 mM Tris-HCl (pH
7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene
glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),
1 mM NaVO3, 10% glycerol, containing 2 mg/mL γ-globulin].
Twenty-four hours later, the luciferase activity was measured with
the appropriate reagent with the use of a Luciferase Assay System ac-
cording to the manufacturer's instructions (Promega, Madison, WI).
Light emissions were measured using a multilabel counter (Wallace
1420 ARVOsx; PerkinElmer). Cells treated with 1% BSA/PBS were used
as a vehicle control. Each assay was performed in triplicate and re-
peated at least three times.
Non-specific binding was evaluated using [3H]E2 together with non-
labeled E2 (final concentration: 10 μM) to quantify the specific binding
by subtracting the nonspecific binding from the total binding. For the
bound/free (B/F) separation, free [3H]E2 was removed by filtration
after incubation with 0.4% dextran-coated charcoal (DCC; Sigma-
Aldrich) in phosphate-buffered saline (PBS; pH 7.4) for 10 min at 4 °C.
The DCC-adsorbed free [3H]E2 was eliminated by the direct vacuum
filtration method using a 96-well filtration plate (MultiScreenHTS HV,
0.45-μm pore size; Millipore, Billerica, MA). Radioactivity was de-
termined on a liquid scintillation counter (LS6500; Beckman Coulter,
4.5.2. Transcriptional inhibitory activity in the luciferase reporter gene
assay
In the Schild plot pA2 analysis method, for the measurement of the
antagonistic activity of bisphenols for ERβ, four different concentra-
tions (0.01, 0.1, 1.0, and 10 μM) of the respective bisphenol were ex-
amined for a serial concentration of E2 (10−12 to 10−5 M in the final
solution). In the qualitative assay using agonist E2 at a fixed con-
centration, a serial concentration of bisphenols (10−12 to 10−5 M in the
final solution) was assayed in the presence of 10 nM E2, which elicits a
full activation of ERβ.
8