stirred for 30 min at room temperature, then CuCl (498 mg, 5.0
mmol) was added in one portion, and the mixture was stirred at
room temperature for 1.5 h. Pyridine (2.8 mL, 34.3 mmol) and p-
iodoanisole (1.18 g, 5.0 mmol) were added in one portion, and
the resulting mixture was heated at 110oC for 34 h. After cooling,
the mixture was washed with 10% HCl aqueous solution, 10%
Na2S2O3 aqueous solution, water, and brine, dried over Na2SO4,
and concentrated. The residue was purified by silica gel column
chromatography (Et2O:n-hexane = 1:15 to 1:10) to give 42 mg
CO2 humidified incubator. On the day before an assay, MCF-7
cells were switched to DMEM (low glucose, phenol red-free)
supplemented with 5% dextran-coated charcoal-stripped FBS
(sFBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin.
Cells were trypsinized from the maintenance dish with phenol
red-free 0.25% trypsin-EDTA and seeded in a 96-well plate at a
density of 2000 cells per final volume of 100 µL DMEM (low
glucose, phenol red-free) supplemented with 5% sFBS, 100
IU/mL penicillin, and 100 µg/mL streptomycin. After 24 h, the
medium was replaced with 90 µL of fresh DMEM (Low glucose,
phenol red free) supplemented with 5% sFBS, 100 IU/mL
penicillin, and 100 µg/mL streptomycin, and 10 µL aliquots of
serial dilutions of test compounds or DMSO (dilution control),
were added to triplicate microcultures in the presence or absence
of 1 x 10-10 M estradiol. The cells were incubated for 5 days,
with one change of the medium after 3 days. Finally, the number
of cells was counted by adding WST-8 (10 µL) to the
microcultures followed by incubation for 2 h. The absorbance at
450 nm of each well was then determined as a measure of the
number of living cells in the culture. EC50 and IC50 values were
estimated with GraphPad Prism 4.
1
(5%) of the title compound as a colorless solid; H NMR (270
MHz, CDCl3) δ (ppm) 0.38 (s, 6H), 1.50-3.80 (brm, 8H), 3.77 (s,
6H), 6.75 (d, J = 9.1 Hz, 4H), 7.36 (d, J = 9.1 Hz, 4H); MS (EI)
m/z 384 (M+, 100%).
4.1.15. 1,7-Bis(4-hydroxyphenyl)-9, 10-dimethyl-m-
carborane (26)
To a solution of 25 (42 mg, 0.11 mmol) in 5 mL of CH2Cl2
was added 1 M BBr3 in CH2Cl2 (0.24 mL, 240 µmol) at -78oC,
and the mixture was stirred for 6 h at room temperature. The
mixture was poured onto ice and extracted with AcOEt. The
organic layer was washed with brine, dried over Na2SO4, and
concentrated. The residue was purified by silica gel column
chromatography (AcOEt:n-hexane = 1:2) to give 36 mg (92%) of
Acknowledgments
1
the title compound as a colorless solid; H NMR (270 MHz,
CDCl3) δ (ppm) 0.40 (s, 6H), 1.50-3.80 (brm, 8H), 5.15 (brs, 2H),
6.69 (d, J = 8.9 Hz, 4H), 7.31 (d, J = 8.9 Hz, 4H); MS (EI) m/z
356 (M+, 100%).
This research was supported by a Grant-in-Aid for High
Technology Research Program, a Grant-in-Aid for Scientific
Research (B) (No. 20390035), and a Grant-in-Aid for Young
Scientists (B) (No. 21790116) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
4.1.16. 1-[4-(2-Dimethylaminoethoxy)phenyl]-7-(4-
hydroxyphenyl)-9,10-dimethyl-m-carborane (9)
To
a solution of 26 (27 mg, 76 µmol) and 2-
References and notes
dimethylaminoethylchloride hydrochloride (14 mg, 99 µmol) in
0.5 mL of DMF and 0.5 mL of acetone was added K2CO3 (31 mg,
0.23 mmol), and the mixture was heated at 60oC for 5 h.
Saturated NaHCO3 aqueous solution was added, and the mixture
was extracted with Et2O. The organic layer was washed with
brine, dried over Na2SO4, and concentrated. The residue was
purified by silica gel column chromatography (MeOH:CHCl3 =
1:20 to 1:10) to give 4 mg (13%) of the title compound as a
1.
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4.
o
1
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colorless needles; mp 155.0-156.0 C (ether); H NMR (270
MHz, CDCl3) δ (ppm) 0.37 (s, 6H), 1.40-3.80 (brm, 8H), 2.39 (s,
6H), 2.80 (t, J = 5.4 Hz, 2H), 4.03 (t, J = 5.4 Hz, 2H), 6.56 (d, J
= 8.9 Hz, 2H), 6.61 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 8.9 Hz, 2H),
7.30 (d, J = 8.9 Hz, 2H); MS (EI) m/z 427 (M+), 59 (100%);
HRMS Calcd for C20H33B10NO2: 427.3514, Found: 427.3507.
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4.2.1. ERα-binding assay
Ligand-binding activity to estrogen receptor α (ERα) was
determined by means of the nitrocellulose filter binding assay
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assay buffer (20 mM Tris-HCl pH 8.0, 0.3 M NaCl, 1 mM
EDTA pH 8.0, 10 mM 2-mercaptoethanol, 0.2 mM
phenylmethylsulfonyl fluoride) and incubated with 4 nM [6,7-
3H]17β-estradiol in the presence or absence of an unlabeled
competitor at 4°C for 18 h. The incubation mixture was absorbed
by suction onto a nitrocellulose membrane that had been soaked
in binding assay buffer. The membrane was washed twice with
buffer (20 mM Tris-HCl pH 8.0, 0.15 M NaCl) and then with
25% ethanol in distilled water. Radioactivity that remained on
the membrane was measured in Atomlight (Perkin Elmer) by
using a liquid scintillation counter.
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4.2.2. MCF-7 Cell proliferation assay
Human breast adenocarcinoma cell line MCF-7 was routinely
cultivated in DMEM supplemented with 10% FBS and 100
IU/mL penicillin and 100 µg/mL streptomycin at 37oC in a 5%
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