Aliphatic substitution of o-carboranyl phenols enhances estrogen receptor beta selectivity

Article abstract of DOI:10.1248/cpb.c13-00796

The two subtypes of estrogen receptor (ER), ERα and ERβ, differ greatly in expression pattern and biological functions, and ERβ-selective ligands candidates to treat immune-related disorders. ERβ-selective ligands have mostly been designed based the idea of introducing a substituent that interferes sterically with the ligand's interaction with Met421 to selectively decrease the affinity for ER a? (the equivalent residue in ERβ is Ile373). Therefore, we designed and synthesized a series of carboranyl phenol derivatives bearing an aliphatic substituent as candidate ERβ-selective ligands. Introduction of a longer aliphatic substituent into the carboranyl moiety enhanced the ERβ selectivity of o-carboranyl phenol derivatives 4, but not m-carboranyl bisphenol derivatives 5. Compound 4c showed 7.4-fold ERβ selectivity in ER-binding assay and exhibited moderate estrogenic activity in cell proliferation assay using MCF-7 cell line.

Full text of DOI:10.1248/cpb.c13-00796

386
Note
Chem. Pharm. Bull. 62(4) 386–391 (2014)
Vol. 62, No. 4
Aliphatic Substitution of o-Carboranyl Phenols Enhances Estrogen
Receptor Beta Selectivity
,a
Kiminori Ohta,a Takumi Ogawa,a Asako Kaise,a Akifumi Oda,b and Yasuyuki Endo*
a Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University; 4–4–1 Komatsushima, Aoba-ku, Sendai
981–8558, Japan: and b Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa
University; Kakuma-machi, Kanazawa, Ishikawa 920–1192, Japan.
Received October 10, 2013; accepted January 20, 2014
The two subtypes of estrogen receptor (ER), ERα and ERβ, differ greatly in expression pattern and
biological functions, and ERβ-selective ligands candidates to treat immune-related disorders. ERβ-selective
ligands have mostly been designed based the idea of introducing a substituent that interferes sterically with
the ligand’s interaction with Met421 to selectively decrease the affinity for ERα (the equivalent residue in
ERβ is Ile373). Therefore, we designed and synthesized a series of carboranyl phenol derivatives bearing an
aliphatic substituent as candidate ERβ-selective ligands. Introduction of a longer aliphatic substituent into
the carboranyl moiety enhanced the ERβ selectivity of o-carboranyl phenol derivatives 4, but not m-carbora-
nyl bisphenol derivatives 5. Compound 4c showed 7.4-fold ERβ selectivity in ER-binding assay and exhibited
moderate estrogenic activity in cell proliferation assay using MCF-7 cell line.
Key words carborane; estrogen receptor; subtype selectivity; estrogenic activity; docking study
We have shown that the carborane cage is available as a may be therapeutically useful to treat chronic intestinal and
novel hydrophobic pharmacophore for drug development,1) joint inflammation by modulating the immune response.27)
and since then, various carborane-containing bioactive com- Thus, ERβ-selective ligands are of interest as potential clini-
pounds have been reported.2,3) Prior to that, carboranes had cal candidates and also as probes for ERβ-related molecular
been used only as boron carriers for boron neutron capture biology.
therapy (BNCT), based on their high boron content.4) We
The X-ray crystal structures of receptor-ligand complexes
also designed and synthesized various carborane-containing of ERα and ERβ show that their binding pockets are quite
bioactive compounds as candidate nuclear receptor ligands.5–8) similar.28) Binding of 2 to the ligand binding domains (LBDs)
One derivative, 1-(4-hydroxyphenyl)-12-hydroxymethyl-p-car- of the two ERs is illustrated in Fig. 2. The same amino acid
borane (BE120, 1), shows more potent estrogenic activity than residues are involved in hydrogen bonding with the two hy-
the endogenous female hormone estrogen, estradiol (E2, 2, droxyl groups of E2 in the LBDs of ERα and ERβ, and thus
Fig. 1).1,9,10) Subsequently, many carboranyl phenol derivatives E2 is accommodated equally well by the two LBDs. However,
have been synthesized and their estrogen-related biological in the hydrophobic pocket of the LBDs, Leu384 and Met421
activities examined.11–18) Compounds that either induce or of ERα are substituted by Met336 and Ile373 in ERβ, respec-
inhibit cellular estrogenic responses are used for estrogen re- tively (Fig. 2). Compound 3 is an ERβ-selective ligand which
placement therapy after menopause or the treatment of breast was designed based the idea of decreasing the affinity for ERα
cancer, respectively.19–21) Tissue-selective ligands, which act by introducing a n-butyl group that interferes sterically with
as agonists for bone metabolism and as antagonists for the Met421 of ERα29) (Fig. 3).
female reproductive systems (known as selective estrogen re-
ceptor modulators, SERMs) are widely used for the treatment rivatives and have discovered various types of ER modulators,
of osteoporosis.22) partial agonists, super-agonists, antagonists, and SERMs.11–18)
We have synthesized a range of carboranyl phenol de-
The estrogen receptor subtypes ERα23) and ERβ24) are ex- In addition, our quantitative structure–activity relationship
pressed in breast and uterus and in bone, colon, endothelial (QSAR) studies of very simple carboranyl phenols showed
cells, bladder, and areas of the brain important for cognition, that the hydrophobicity of the carborane cage is significantly
respectively. Most ER modulators are non-selective for ER correlated with ERα-binding affinity.30,31) Although carbora-
subtypes, but it has been proposed that compounds with
ER subtype selectivity would be useful as third-generation
SERMs.25,26) Harris et al. showed that ERβ-selective ligands
Fig. 1. Structures of Carborane-Containing Estrogen BE120 1 and Es-
tradiol 2
The authors declare no conflict of interest.
Fig. 2. Differences of Amino Acid Residues around the ER LBDs
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: yendo@tohoku-pharm.ac.jp

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387
nyl phenol is a privileged structure as an ER ligand, little is was synthesized by the literature procedure, was reacted with
known about the effect of aliphatic substituents on the carbo- ethyl iodide or allyl bromide in the presence of NaH as a base
rane cage. Therefore, we designed and synthesized carboranyl to afford C-ethyl or C-allyl intermediates in 77% or 50%
phenol derivatives 4 and 5 with aliphatic substituents on the yield, respectively. Demethylation of the methoxy group of 7
carborane cage for examination of their ERβ selectivity and with BBr3 afforded the target compounds 4a and b in 96% and
estrogenic activities. From the viewpoints of ease of synthesis 60% yields, respectively. The allyl group of compound 4b was
and the directions of the aliphatic substituents, we focused reduced by catalytic hydrogenation using Pd/C to afford the
on o-carboranyl phenol and m-carboranyl bisphenol as being corresponding C-n-propyl derivative 4c in 80% yield.
most relevant to drug design.
Chart 2 summarizes the synthesis of m-carboranyl bis-
Synthesis of o-carboranyl phenol derivatives 4a–c is sum- phenol derivatives 5a and b. 7-Iodo-m-carborane 8 was ob-
marized in Chart 1. 4-Methoxyphenyl-o-carborane 6, which tained by iodination of m-carborane.32) Grignard reagents,
which were freshly prepared from Mg turnings and the
corresponding halide, were reacted with 8 by Pd-catalyzed
coupling reaction to afford B-allyl and B-n-butyl m-carborane
in 99% and 61% yields, respectively.33) They were treated
with n-butyl lithium to obtain the lithium salts, which were
transformed into copper salts with Cu(I)Cl, and converted to
the diaryl-m-carboranes 10a and b in 37% and 39% yields,
respectively.34,35) B-n-Propyl derivative 5a was synthesized by
catalytic hydrogenation of the allyl group of 10a, followed by
deprotection of the phenol group with BBr3 in 45% yield over
Fig. 3. Structure of ERβ Selective Ligand 3
Reagents and conditions: a) NaH, ethyl iodide or allyl bromide, DMF, rt; b) BBr3, CH2Cl2, rt; c) Pd/C, H2, MeOH/AcOEt, rt.
Chart 1. Synthesis of o-Carborane Derivatives 4a–c
Reagents and conditions: a) Grignard reagents, Pd(PPh3)2Cl2, Cu(I)I, THF, reflux; b) i) n-BuLi, DME, rt, ii) Cu(I)Cl, pyridine, 4-iodoanisole, 100°C; c) Pd/C, H2, MeOH/
AcOEt, rt; d) BBr3, CH2Cl2, rt.
Chart 2. Synthesis of m-Carborane Derivatives 5a and b

388
Vol. 62, No. 4
the 2 steps. Compound 5b was obtained by deprotection of the the n-propyl group and Met421 of hERα. A docking study
phenol group of 10b in 82% yield.
of 5b with hERs showed that the second phenol group is ac-
The ER binding affinities of the synthesized compounds commodated in the secondary hydrophobic pocket of both
were evaluated by means of competitive ER binding assays ER LBDs (data not shown). The decrease of binding affinity
using [2,4,6,7-3H]17β-estradiol and human recombinant ERα and the poor selectivity of 5b for ERs may suggest that the
and ERβ.36) Table 1 summarized the relative binding affinity direction of n-butyl group is not suitable for steric repulsion to
(RBA) values, estimated based on the RBA of estradiol 2, occur with Met421 of ERα.
taken as 100. All the test compounds 4 and 5 bound to the
Next, we evaluated the agonist and antagonist activities
ERs in a dose-dependent manner. Introduction of substituents of the most ERβ-selective compound 4c by means of cell
onto the carborane cage decreased the binding affinity for proliferation assay using the MCF-7 cell line, which shows
both ERs. As the substituent was lengthened, the o-carboranyl estrogen-dependent growth.36) Compound 4c promoted cell
phenol derivatives 4 showed a decrease of binding affinity for proliferation in a dose-dependent manner and the EC50 value
both ERs, but the decrease was more marked for ERα. Com- was 3.0×10−9 M (Fig. 5). When the maximum potency (Emax
)
pound 4a showed weak binding affinity for ERα but its bind- of E2 was taken as 100%, that of 4c was 110%. Thus, com-
ing affinity for ERβ was higher than that of E2. This may sug- pound 4c is simply a weaker ER full agonist.
gest that ethyl group has an unfavorable steric interaction with
In conclusion, we examined the effect of introducing an ali-
the hydrophobic pocket of ERα, but not that of ERβ. The RBA phatic substituent into the carborane cage of o- or m-carbora-
values of allyl derivative 4b were decreased for both ERs. The nyl phenol derivatives based on the structure of a well-known
allyl group is more polar than a n-propyl group and would ERβ-selective ligand 3. Interestingly, the ERβ selectivity of
be disfavored in a hydrophobic environment. Compound 4c o-carboranyl phenol derivatives 4 was enhanced as the length
showed a high RBA value with 7.4-fold selectivity for ERβ. of the substituent was increased, whereas m-carboranyl phenol
These results show that introduction of an alkyl group onto derivatives 5 showed little ERβ selectivity. The C-n-propyl
the carborane cage markedly influences the ERβ selectivity. o-carboranyl phenol 4c showed 7.4-fold ERβ selectivity in ER-
On the other hand, m-carborane bisphenol derivatives 5a and binding assay and moderate estrogenic activity in MCF-7 cell
b showed little ER-subtype selectivity. We previously found proliferation assay. These results provide useful information
that non-substituted m-carborane bisphenol had a similar RBA on substituent effects to guide research aimed at discovery of
value to E2, while introduction of an alkylamino group into selective carborane-containing estrogen modulators.
the second phenol group greatly decreased the binding affin-
ity and changed the functional activity from ER agonist to ER
Experimental
General Considerations Melting points were determined
antagonist.37) It seems that the phenol group is accommodated
in a secondary hydrophobic pocket and forms hydrogen bonds with a Yanaco micro melting point apparatus and were not
1
in both ER LBDs.
corrected. H- and 13C-NMR spectra were recorded with JEOL
A docking simulation of hERα (PDB: 1ERE) and hERβ JNM-EX-270 and JNM-LA-400 spectrometers. Chemical
1
(PDB: 2I0G) with the carborane containing ligands, 4c and shifts of H-NMR signals were referenced to tetramethylsilane
5b, was performed with an automatic docking program (0.0ppm) as an internal standard. Chemical shifts of 13C-NMR
(GOLD 5.2).38) A docking mode of 4c in the hERβ LBD is signals were referenced to residual 13C present in deuterated
shown in Fig. 4a. The highly hydrophobic o-carborane cage solvents. The splitting patterns are designated as follows: s
and n-propyl group are located in the hydrophobic pocket of (singlet), d (doublet), t (triplet), m (multiplet), q (quartet), and
the receptor, but the o-carborane cage of 4c is close to Ile373 br (broad). Mass spectra were recorded on a JEOL JMS-
of hERβ, contrary to our hypothesis (Fig. 4a). Interestingly, DX-303 spectrometer. Column chromatography was car-
the arrangement of 4c in the LBDs of ERα is different from ried out using Fuji Silysia BW80s and TLC was performed
that in ERβ (Fig. 4b) and n-propyl group of 4c is close to on Merck silica gel F254. Carboranes were purchased from
Met421 in the hERα LBD (Fig. 4a). We suggested that the Katchem s.r.o. Other chemicals were purchased from Aldrich
ERβ selectivity of 4c is caused by the steric repulsion between or Tokyo Kasei Ltd. and used without further purification, or
Table 1. Binding Affinity for ERα and ERβ and Subtype Selectivity of Compounds 4 and 5
RBAa)
Compound
R
Selectivity ERβ/ERα
ERα
43.7
9.3
ERβ
140.7
44.9
4a
4b
4c
5a
Ethyl
3.2
4.8
7.4
0.8
Allyl
n-Propyl
n-Propyl
n-Butyl
11.8
27.6
32.4
87.0
21.7
5b
43.5
100
1.3
1.0
E2 (1)
100
a) Relative binding affinity with respect to that of estradiol 2 taken as 100%. All binding assays were performed in duplicate (n=2) and the numbers are average values.

April 2014
389
Fig. 4. Docking Model of 4c to the LBDs of ERα and ERβ
(a) Docking mode of 4c in the hERβ LBD (atom colors: H (white) B (green), C (gray), N (blue), O (red) and S (yellow)). The corresponding amino acid residues of hERα
are colored orange. Both Ile373 of ERβ and Met421 of ERα are displayed with a ball and stick model. (b) The arrangements of 4c in the LBDs of ERα (brown) and ERβ
(aqua).
δ (ppm): 0.97 (t, J=7.7Hz, 3H), 1.60–3.60 (brm, 10H), 1.85 (q,
J=7.7Hz, 2H), 3.82 (s, 3H), 6.87 (d, J=9.2Hz, 2H), 7.53 (d,
J=9.2, 2H); MS (electron ionization (EI)) m/z 278 (M+, 100%).
1-(4-Methoxyphenyl)-2-allyl-1,2-dicarba-closo-dodecabo-
1
rane: Colorless solid; 50% yield; H-NMR (400MHz, CDCl3)
δ (ppm): 1.60–3.60 (brm, 10H), 2.53 (d, J=7.7Hz, 2H), 3.83 (s,
3H), 4.73–4.79 (m, 1H), 5.03 (d, J=10.1Hz, 1H), 5.56–5.68 (m,
1H), 6.88 (d, J=8.7Hz, 2H), 7.54 (d, J=8.7, 2H); MS (EI) m/z
290 (M+, 100%).
General Procedure of Demethylation of 7 To a solu-
tion of 6 in CH2Cl2 was added a solution of 1M BBr3 (1.2eq)
in CH2Cl2 at −78°C, and the mixture was stirred at room
temperature for 30h, then poured onto ice and extracted with
AcOEt. The organic layer was washed with brine, dried over
Fig. 5. Dose–Response Curve of Compound 4c in MCF-7 Cell Prolif-
Na2SO4, and concentrated. The residue was purified by silica
gel column chromatography with 1:5 AcOEt–n-hexane to give
the phenol derivative 4.
eration Assay
MCF-7 cells were incubated with 4c (1×10−6 to 1×10−12 M) for 4d, and the results
are shown as relative cell number, with the value for 0.1nM estradiol taken as 1.
Dashed line shows Emax of E2. Cell proliferation assays was performed in triplicate
(n=3). Values are means S.D. for separate experiments.
1-(4-Hydroxyphenyl)-2-ethyl-1,2-dicarba-closo-dodecabo-
rane (4a): 96% yield; colorless needles (AcOEt–n-hexane); mp
1
were prepared according to procedures described in the litera- 106.5–108.0°C; H-NMR (400MHz, CDCl3) δ (ppm): 0.97 (t,
ture. All solvents were commercial products of reagent grade, J=7.7Hz, 3H), 1.60–3.60 (brm, 10H), 1.85 (q, J=7.7Hz, 2H),
and were used without further purification.
5.25 (brs, 1H), 6.81 (d, J=9.2Hz, 2H), 7.50 (d, J=8.7, 2H).
General Procedure of C-Alkylation of 6 To a solution MS (EI) m/z 264 (M+, 100%); high resolution (HR)-MS Calcd
of 6 in dry N,N-dimethylformamide (DMF) was added NaH for C10H20B10O: 264.2517, Found: 264.2521.
(1.2eq) at 0°C under an argon (Ar) atmosphere, and the mix-
1-(4-Hydroxyphenyl)-2-allyl-1,2-dicarba-closo-dodecabo-
ture was stirred for 30min at the same temperature. Alkyl rane (4b): 60% yield; colorless needles (CH2Cl2–n-hexane); mp
1
halide (1.2eq) was added, and stirring was continued at room 96.0–97.5°C; H-NMR (400MHz, CDCl3) δ (ppm): 1.60–3.60
temperature for 2.5h. The reaction mixture was poured onto (brm, 10H), 2.54 (d, J=7.2Hz, 2H), 4.72–4.79 (m, 1H), 5.03
ice and extracted with Et2O. The organic layer was washed (d, J=10.1Hz, 1H), 5.34 (brs, 1H), 5.56–5.68 (m, 1H), 6.81
with brine, dried over Na2SO4, and concentrated. The residue (d, J=8.7Hz, 2H), 7.50 (d, J=8.7, 2H); MS (EI) m/z 276
was purified by silica gel column chromatography with 1:20 (M+, 100%); HR-MS Calcd for C11H20B10O: 276.2517, Found:
AcOEt–n-hexane to give the corresponding C-alkylated com- 276.2516.
pound.
1-(4-Hydroxyphenyl)-2-n-propyl-1,2-dicarba-closo-dodeca-
1-(4-Methoxyphenyl)-2-ethyl-1,2-dicarba-closo-dodecabo- borane (4c): To a solution of 4b (25mg) in AcOEt (5mL) and
1
rane: Colorless solid; 77% yield; H-NMR (400MHz, CDCl3) MeOH (5mL) was added Pd on carbon (5mg) and the mixture

390
Vol. 62, No. 4
was stirred at room temperature for 30min under a hydrogen 1H-NMR (400MHz, CDCl3) δ (ppm): 0.92 (t, J=8.3Hz, 2H),
atmosphere. The mixture was filtered, the residue was washed 0.97 (t, J=7.3Hz, 3H), 1.43 (sext, J=7.8Hz, 2H), 1.60–3.60
with AcOEt, and the organic layer was concentrated. The resi- (brm, 9H), 3.77 (s, 6H), 6.76 (d, J=8.8Hz, 4H), 7.37 (d,
due was purified by silica gel column chromatography with J=8.8Hz, 4H).
1:5 AcOEt–n-hexane to give 4c (21mg, 80%) as colorless
1,7-Bis-(4-hydroxyphenyl)-9-n-propyl-1,7-dicarba-closo-do-
1
1
solid. Colorless oil; H-NMR (400MHz, CDCl3) δ (ppm): 0.74 decaborane (5a): 82% yield; colorless oil; H-NMR (400MHz,
(t, J=7.2Hz, 3H), 1.60–3.60 (brm, 10H), 1.41 (sext, J=7.7Hz, CDCl3) δ (ppm): 0.91 (t, J=7.7Hz, 2H), 0.96 (t, J=7.2Hz, 3H),
2H), 1.73 (t, J=8.7Hz, 2H), 5.45 (brs, 1H), 6.82 (d, J=8.7Hz, 1.42 (sext, J=7.2Hz, 2H), 1.60–3.60 (brm, 9H), 5.32 (brs,
2H), 7.49 (d, J=8.7, 2H); MS (EI) m/z 278 (M+, 100%); 2H), 6.69 (d, J=8.7Hz, 4H), 7.31 (d, J=8.7Hz, 4H); MS (EI)
HR-MS Calcd for C11H22B10O: 278.2674, Found: 278.2678.
General Procedure of Synthesis of 9 A solution of Found: 370.2943.
m/z 370 (M+, 100%); HR-MS Calcd for C17H26B10O2: 370.2936,
Grignard reagent (4eq) was added dropwise to a stirred dry
1,7-Bis-(4-hydroxyphenyl)-9-n-butyl-1,7-dicarba-closo-do-
tetrahydrofuran (THF) solution (10mL) of 8 (1.0g, 3.70mmol) decaborane (5b): Quant; colorless oil; 1H-NMR (400MHz,
at 0°C under an Ar atmosphere, and stirring was continued CDCl3) δ (ppm): 0.86–0.96 (m, 5H), 1.30–1.43 (m, 4H),
at room temperature for 30min. Bis(triphenylphosphine)- 1.60–3.60 (brm, 9H), 5.82 (brs, 2H), 6.69 (d, J=8.7Hz, 4H),
palladium(II) dichloride (260mg, 0.37mmol) and cupper(I) 7.31 (d, J=8.7Hz, 4H); MS (EI) m/z 384 (M+, 100%); HR-MS
iodide (70.5mg, 0.37mmol) were added in one portion, and Calcd for C18H28B10O2: 384.3092, Found: 384.3094.
the reaction mixture was refluxed for 60h. After removal of
Competitive Binding Assay for ERs Ligand binding to
the solvent, Et2O was added to the residue and the excess estrogen receptors α (ERα) and β (ERβ) was determined by
Grignard reagent was destroyed by slow addition of 10% HCl the nitrocellulose filter binding assay method. ER (0.5µg/
aqueous solution. The organic phase was separated from the tube, PanVera Co., Ltd.) was diluted with binding assay buffer
mixture, and the aqueous layer was extracted with Et2O. The (20mM Tris–HCl pH 8.0, 0.3M NaCl, 1mM ethylenediamine-
combined organic phase was washed with water and brine, tetraacetic acid (EDTA) pH 8.0, 10mM 2-mercaptoethanol,
dried over Na2SO4, and concentrated. The residue was purified 0.2mM phenylmethylsulfonyl fluoride) and incubated with
by column chromatography on silica gel with n-hexane to af- 4nM [6,7-3H]17β-estradiol in the presence or absence of an
ford the corresponding 9.
unlabeled competitor at 4°C for 18h. The incubation mixture
9-Allyl-1,7-dicarba-closo-dodecaborane (9a): 99% yield; was absorbed by suction onto a nitrocellulose membrane that
1H-NMR (400MHz, CDCl3) δ (ppm): 1.40–3.50 (brm, 9H), had been soaked in binding assay buffer. The membrane was
1.78 (brs, 2H), 2.88 (brs, 2H), 4.75–4.90 (m, 2H), 5.86 (m, washed twice with buffer (20mM Tris–HCl pH 8.0, 0.15M
1H); MS (EI) m/z 184 (M+, 100%).
NaCl) and then with 25% ethanol in distilled water. Radioac-
9-n-Butyl-1,7-dicarba-closo-dodecaborane (9b): 61% yield; tivity that remained on the membrane was measured in Atom-
1H-NMR (400MHz, CDCl3) δ (ppm): 0.80–0.95 (m, 5H), light (NEN) by using a liquid scintillation counter.
1.30–1.40 (m, 5H), 1.40–3.50 (brm, 9H), 2.85 (brs, 2H); MS
Cell Culture At 80% confluence, cells of the human
breast adenocarcinoma cell line MCF-7 were trypsinized from
(EI) m/z 200 (M+, 100%).
General Procedure of Synthesis of 10 To a solution of the maintenance dish with 0.25% trypsin–EDTA and collected
9 (350mg) in 1,2-dimethoxyethane (DME) (3mL) was added by centrifugation (4°C, 1500rpm, 5min). The supernatant
dropwise a solution of 2.66M n-BuLi in hexane (2.2eq) at was removed, 1mL of Dulbecco’s modified Eagle’s medium
0°C under an Ar atmosphere, and the mixture was stirred for (DMEM) supplemented with 10% fetal bovine serum (FBS),
30min. Cu(I)Cl (2.2eq) was added in one portion, and stirring 100IU/mL penicillin and 100mg/mL streptomycin was added,
was continued at room temperature for 1h. Pyridine (15eq) and cell aggregates were broken up by thorough pipetting.
and p-iodoanisole (2eq) were added in one portion, and the Cells were seeded in a dish at a concentration of 4×104 cell/
mixture was refluxed for 20h, then cooled to room tempera- mL or 8×104 cell/mL, and cultivated at 37°C in a 5% CO2
ture, and diluted with AcOEt. Insoluble materials were filtered humidified incubator. Cells were routinely cultivated 2d or 3d
off through Celite. The filtrate was washed with 10% HCl later.
aqueous solution, 10% Na2S2O3 aqueous solution, water and
Cell Proliferation Assay Cells of the human breast
brine, dried over Na2SO4, and concentrated. The residue was adenocarcinoma line MCF-7 were routinely cultivated in
purified by silica gel column chromatography with 1:30 to DMEM supplemented with 10% FBS, 100IU/mL penicillin
1:20 AcOEt–n-hexane to give the corresponding 10.
and 100mg/mL streptomycin at 37°C in an incubator under
1,7-Bis-(4-methoxyphenyl)-9-allyl-1,7-dicarba-closo-dode- an atmosphere of 5% CO2 in air. On the day before assay,
caborane (10a): 37% yield; 1H-NMR (400MHz, CDCl3) δ MCF-7 cells were switched to DMEM (low glucose phenol
(ppm): 1.60–3.60 (brm, 9H), 1.86 (d, J=7.2Hz, 2H), 3.76 (s, red-free supplemented with 5% sFBS, 100IU/mL penicillin
6H), 4.80–4.93 (m, 2H), 5.91 (m, 1H), 6.75 (d, J=8.7Hz, 4H), and 100mg/mL streptomycin). Cells were trypsinized from
7.36 (d, J=9.2Hz, 4H); MS (EI) m/z 396 (M+, 100%).
the maintenance dish into phenol red-free trypsin–EDTA
1,7-Bis-(4-methoxyphenyl)-9-n-butyl-1,7-dicarba-closo-do- and seeded in a 96-well plate at a density of 2×103 cells per
1
decaborane (10b): 39% yield; H-NMR (400MHz, CDCl3) δ final volume of 100µL DMEM supplemented with 5% sFBS,
(ppm): 0.87–0.96 (m, 5H), 1.31–1.41 (m, 4H), 1.60–3.60 (brm, 100IU/mL penicillin and 100mg/mL streptomycin. After 24h,
9H), 3.78 (s, 6H), 6.76 (d, J=8.7Hz, 4H), 7.37 (d, J=8.7Hz, the medium was replaced with 90µL of fresh DMEM and
4H); MS (EI) m/z 412 (M+, 100%).
10 µL of drug solution, supplemented with serial dilutions of
1,7-Bis-(4-methoxyphenyl)-9-n-propyl-1,7-dicarba-closo- 4-OH-Tam or DMSO as the dilution control in the presence
dodecaborane The title compound was prepared by the or absence of 1×10−11 M E2, was added to triplicate microcul-
same method as described for the synthesis of 4c. 55% yield; tures. Cells were incubated for 4d, and medium with 4-OH-

April 2014
391
Tam in the presence or absence of 1×10−11 M E2 was replaced
once after 3d. At the end of the incubation time, proliferation
was evaluated by using a cell counting kit. WST-8 (10µM) was
added to microcultures and cells were incubated for 2h. The
absorbance at 450nm was measured. This parameter is related
to the number of living cells in the culture.
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Acknowledgments This research was supported by a
Grant-in-Aid from the Strategic Research Program for Pri-
vate Universities (2010–2014), 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 of Japan.
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