Synthesis and biological evaluation of p-carborane bisphenols and their derivatives: Structure-activity relationship for estrogenic activity

Article abstract of DOI:10.1016/j.bmc.2008.12.044

p-Carborane bisphenols and their derivatives were prepared and evaluated for binding affinity to estrogen receptor α. Their estrogenic activity was evaluated by means of transcriptional assay and cell proliferation assay using MCF-7 cell lines. 1,12-Bis(4

Full text of DOI:10.1016/j.bmc.2008.12.044

Bioorganic & Medicinal Chemistry 17 (2009) 1109–1117
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of p-carborane bisphenols and their
derivatives: Structure–activity relationship for estrogenic activity
a
a
b
c
c
a,*
Takumi Ogawa , Kiminori Ohta , Toru Iijima , Tomoharu Suzuki , Shigeru Ohta , Yasuyuki Endo
a
Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Graduate School of Medical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
p-Carborane bisphenols and their derivatives were prepared and evaluated for binding affinity to estro-
gen receptor . Their estrogenic activity was evaluated by means of transcriptional assay and cell prolif-
Received 10 December 2008
Revised 16 December 2008
Accepted 17 December 2008
Available online 25 December 2008
a
eration assay using MCF-7 cell lines. 1,12-Bis(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane 4a
showed potent estrogenic activity, approaching that of 17b-estradiol, in transactivation assay. The activ-
ity of isomers 5a and 6a was drastically affected by the change in the position of one of the hydroxyl
groups; 6a (ortho-OH in one ring) was about 1000 times less potent than 4a. Modification of this hydroxyl
group with alkyl groups decreased the estrogenic activity in all isomers. Compound 4a also showed
potent MCF-7 cell proliferation-enhancing activity.
Keywords:
Estrogen receptor
Agonists
Ó 2008 Elsevier Ltd. All rights reserved.
Carborane
Phenols
Structure–activity relationship
1
. Introduction
in the central nervous system, and in the cardiovascular system.
7
(Fig. 1) The first step in the appearance of estrogenic activity is
The carboranes (dicarba-closo-dodecaboranes) are chemical
mediated by the binding of agonist ligands to estrogen receptors
(ER) and b, resulting in a conformational change. The ligand-
bound ER dimerizes, forms huge complexes with various cofactors,
building blocks of remarkable thermal stability and high boron
content; they are resistant to attack by most types of reagent,
a
1
and are generally inactive toward biological systems. One of their
and binds to specific promoter elements of DNA to initiate gene
7
most striking features is the ability of the two carbon atoms and 10
boron atoms to adopt icosahedral geometry in which the carbon
and boron atoms are hexa-coordinated. This feature of the struc-
ture gives rise to the unusual properties of such molecules and
their carbon and boron derivatives. Their properties make them
uniquely suitable for various specialized applications, including
synthesis of polymers for high temperature use and neutron
transcription. Compounds that either induce or inhibit cellular
estrogen responses have potential value as biochemical tools and
candidates for drug development. Since the discovery of the non-
steroidal estrogens, many estrogen agonists and antagonists have
been developed as agents for regulating fertility, preventing and
controlling hormone-responsive breast cancer, and post-meno-
8
pausal hormone replacement.
2
shielding. In the fields of medicinal chemistry and pharmaceutical
Binding of ligands to the ER ligand binding domain (LBD) pri-
marily requires a phenolic ring, which hydrogen-bonds with
sciences, incorporation of large numbers of boron atoms into tu-
mor calls for boron neutron capture therapy (BNCT) has become
9
Glu353 and Arg394 amino acid residues of the hER LBD. It is also
3
of interest in recent years. Most carborane-containing compounds
well-known that the secondary alcohol group of E2 interacts with
9
that have so far been synthesized are composed of cellular building
His524 of hER. The hydrophobic group should closely match the
4
5
6
blocks (nucleic acids, amino acids, sugars, and so on), to which
carborane units are added.
hydrophobic surface of the ER, so as to increase the binding affin-
ity. The hydrophobic structure also plays a role as a scaffold, fixing
the spatial positions of the hydrogen-bonding functional groups. In
our studies to develop new hydrophobic structures for use in drug
design, we have focused on the exceptional hydrophobic character
and spherical geometry of carboranes, and utilized them as a
hydrophobic component of biologically active molecules.10
The steroid hormone estrogen influences the growth, differenti-
7
ation, and functioning of many target tissues. 17b-Estradiol (E2) is
an endogenous estrogen that plays important roles in the female
and male reproductive systems, as well as in bone maintenance,
We have reported a potent estrogen agonist bearing a carborane,
-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-dodeca-
*
Corresponding author. Tel.: +81 22 727 0142; fax: +81 22 275 2013.
E-mail address: yendo@tohoku-pharm.ac.jp (Y. Endo).
1
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.044

1
110
T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
OH
logical activities and the structure–activity relationships of these
designed molecules 4–7.
OH
HO
2. Results and discussion
2.1. Chemistry
1
HO
potent agonist
estradiol (E2)
native ligand
(
in vitro and in vivo)
OH
The designed molecules 4a–4d and 5a–5d were synthesized
from p-carborane as summarized in Scheme 1. p-Carborane
was treated with n-BuLi and Cu(I)Cl, and reacted with 4-iodo-
HO
HO
1
5
1
-(tert-butyldimethylsiloxy)benzene,
which was prepared
from 4-iodophenol, under Ullmann coupling conditions, to afford
mono-substituted compound 8 and di-substituted compound 9
in 31% and 36% yield, respectively. Compound 4a was syn-
thesized by deprotection of 9 with tetrabutylammonium fluoride
(TBAF) in quantitative yield, and then treated with alkyl iodide
to afford compound 4b–4d. Compound 8 was easily transformed
1
6
17
2
3
HO
potent partial agonist (in vitro)
SERM (in vivo)
potent agonist (in vitro)
into diaryl p-carborane 10 by Ullmann coupling with 3-iodoani-
sole in 74% yield. Compound 10 was treated with BBr to af-
3
Figure 1. Structure and properties of estradiol (E2) and carborane derivatives as
estrogen receptor modulators. Compounds 2 and 3 have carborane bisphenol
structure.
16
ford demethylated compound 11 as a major product in 42%
yield, bisphenol derivative 5a in 30% yield, and desilylated com-
pound 5b as a by-product in 16% yield, respectively. The phenol
group of intermediate 11 was reacted with alkyl iodide, followed
by desilylation with 10% HCl aqueous solution to afford com-
pounds 5c and 5d. The other designed molecules 6a–6d and 7
were synthesized in line with Scheme 2. Compound 13 was syn-
borane (1),11 which has an activity greater than that of E2 in ER
transcriptional assay and ER
binding assay. (Fig. 1) 12 The com-
a
a
pound also showed potent in vivo effects on the recovery of uterine
weight and bone loss in OVX mice.12 These results suggested that
hydrophobic interaction along the spherical carborane cage pro-
duces a stronger interaction with the receptor than that in the case
of E2. We have utilized the strong binding of the spherical carbo-
rane cage to the cavity of ER LBD to design and synthesize various
carborane bisphenol derivatives as candidate ER modulators. We
found that bis(4-hydroxyphenyl)-o-carborane (2) exhibited anti-
11
thesized by a stepwise Ullmann coupling via intermediate 12
1
6
from p-carborane. Demethylation of compound 13 with BBr3
afforded bisphenol derivative 6a in 78% yield and methoxy
derivative 6b in 5% yield, respectively, because there was a small
steric effect with the p-carborane cage. The steric effect made it
easy to selectively protect one of the two phenol groups of com-
pound 6a with tert-butyldimethylsilyl (TBS) chloride to afford
compound 14 in 44% yield, and this was easily transformed into
compounds 6c and 6d by usual alkylation and the subsequent
desilylation. Compound 8 was reacted with iodobenzene under
Ullmann coupling conditions, followed by desilylation with TBAF
to afford compound 7a. Compound 7b was prepared by means of
aromatic nucleophilic substitution reaction of compound 12
1
3
estrogenic activity. Furthermore, in an in vivo evaluation, com-
pound 2 exhibited estrogenic action in bone, preventing bone loss
without inducing estrogenic action in the uterus; it is therefore a
candidate for application as a new type of selective estrogen recep-
tor modulator (SERM) to treat osteoporosis.13 We also found that
bis(4-hydroxyphenyl)-m-carborane (3) has estrogenic activity
somewhat weaker than that of E2 in transcriptional assay, and its
binding affinity for ER
a
is similar to that of E2.14 It is a remarkable
with hexafluorobenzene to afford 15, followed by demethyla-
18
result that o- and m-carborane derivatives exhibited different
activities. In other words, the carborane cage appears to be a suit-
able hydrophobic pharmacophore for ER, with a great deal of po-
tential for modulating the activity.
Therefore, we were interested in the activity of bisphenol deriv-
atives bearing p-carborane, another isomer of carboranes. We de-
signed and synthesized p-carborane bisphenol derivatives 4–6
Fig. 2. Compounds 7a and 7b were also synthesized in order to
understand the effects of hydroxyl or alkoxy substituents on the
activity and the relationship between the electronic properties of
benzene ring and the activity. In this article, we describe the bio-
tion with BBr3.
2.2. Competitive binding assay with ER
a
3
A
competitive binding assay using [6,7- H]17b-estradiol
(K = 0.4 nM) and human recombinant ER
d
a
was employed for
1
2
initial screening of the synthesized compounds. Table 1 sum-
marizes the binding affinity data for tested compounds as rela-
tive values to that of estradiol. The bisphenol derivatives, 4a,
5a, and 6a showed strong binding affinity for ER
E2), and compound 7a also strongly bound to the ER
a
(stronger than
LBD. In the
a
OR
R
OR
RO
R
R
R
R
HO
HO
HO
HO
R = H (4a)
CH3 (4b)
n-C H (4c)
R = H (5a)
CH3 (5b)
n-C H (5c)
R = H (6a)
CH3 (6b)
n-C H (6c)
R = H (7a)
F (7b)
3
7
3
7
3 7
n-C H (4d)
n-C H (5d)
n-C H (6d)
5 11
5
11
5
11
Figure 2. Molecules designed as candidate estrogen receptor ligands based upon p-carborane bisphenol structure.

T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
1111
OTBS
OH
c
OR
H
H
a
b
4
b (37%)
4c (24%)
d (17%)
+
H
TBSO
8
TBSO
9
HO
4a
100%
HO
4
31%
36%
d
OCH
3
OH
OH
OCH
3
e
+
HO
+
TBSO
10
TBSO
11
42%
5a
30%
HO
5b
7
4%
16%
OR
f
5c (48%)
HO
5d (44%)
Scheme 1. Synthesis of compounds 4a–4d and 5a–5d. Reagents: (a) n-BuLi, DME then Cu(I)Cl, 4-iodo-1-(tert-butyldimethylsiloxy)benzene, pyridine; (b) TBAF, THF; (c) alkyl
iodide, K
2
CO
3
, DMF; (d) n-BuLi, DME then Cu(I)Cl, 3-iodoanisole, pyridine; (e) BBr
3
, CH
2
Cl
2
; (f) alkyl iodide, K
2
CO
3
, DMF, then 10% HCl.
H CO
HO
H CO
3
3
H
H
a
b
c
+
H
H CO
12
H CO
13
HO
6a
HO
6b
3
3
68%
36%
78%
5%
HO
RO
d
e
6
a
6
c (44%)
1
4
TBSO
HO
6d (43%)
44%
F
5
F5
f, g
h
c
8
12
7
3%
a
H CO
15
28%
7b
93%
HO
3
HO
3
Scheme 2. Synthesis of compounds 6a–6d, 7a, and 7b. Reagents: (a) n-BuLi, DME then CuCl(I), 4-iodoanisole, pyridine; (b) n-BuLi, DME then CuCl(I), 2-iodoanisole, pyridine;
c) BBr , CH Cl ; (d) TBSCl, triethylamine, CH Cl ; (e) alkyl iodide, K CO , DMF, then 10% HCl; (f) n-BuLi, DME then CuCl(I), iodobenzene, pyridine; (g) TBAF, THF; (h) n-BuLi,
ether, then C
(
3
2
2
2
2
2
3
6 6
F .
derivatives with an alkoxy group, the binding affinity to ER
a
de-
character of the pentafluorophenyl group apparently impairs
the binding of 7b to the ER ligand binding pocket.
creased as the carbon chain of the alkoxy group was lengthened,
except for p-substituted derivatives. The binding affinity of the
compounds with a n-pentyl group was markedly decreased
a
2.3. Transcriptional activation assay of synthesized compounds
(
4d, 5d, and 6d). The binding of the derivatives to the ERa ligand
binding pocket is impaired by the steric effect of the alkyl side
chains. However, it is not known exactly why compounds 4b
To evaluate the activity of the synthesized compounds as ER
agonists and antagonists, transcriptional assay was done with
ERE/Luci (firefly luciferase) and phRL/CMV (Renilla luciferase)
plasmids, which were transiently transfected into MCF-7 cells.19
Figure 3 summarizes the results of the transcriptional activation
and 4c exhibited higher binding affinity for ER
compound 4a. Introduction of the pentafluorophenyl group led
to a decrease of the binding affinity for ER . The electrostatic
a than the parent
a

1
112
T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
Table 1
a
4b
4c
Binding affinity of test compounds to human ER
a
8
6
4
0
0
0
Compound
R
Binding affinityb
4
d
4
4
4
4
5
5
5
5
6
6
6
6
7
7
a
b
c
d
a
b
c
d
a
b
c
d
a
b
p-
OH
OCH
149
212
165
38
144
95
5b
5c
3
O-n-C
O-n-C
OH
3
H
H
7
5
6
6
6
d
b
c
d
5
11
m-
o-
OCH
3
O-n-C
O-n-C
OH
3
H
H
7
73
45
5
11
126
125
112
61
130
61
OCH
3
20
0
O-n-C
O-n-C
H
3
H
H
7
5
11
F(5)
1
0-12
10-11 10-10
10-9
10-8
10-7
10-6
a
All binding assays were performed in duplicate (n = 2).
Values are average of percentage displacement of [ H]estradiol (4 nM) specific
Concentration (M)
b
3
binding to human recombinant ER
affinity of estradiol is taken as 100.
a
by each compound at 4 nM. The binding
Figure 4. Transcriptional assay with the alkoxy derivatives, 4b–4d, 5b–5d, and 6b–
6d. All experiments were performed using the same method as described in Figure
3. Solid lines (4b–4d) indicate para-substituted compounds. Wide dashed lines (5b–
5d) and fine dashed lines (6b–6d) indicate meta- and ortho-substituted compounds,
respectively.
80
60
40
20
0
The estrogenic activity of alkylated compounds is summarized
4
5
6
7
7
a
a
a
a
b
in Figure 4. None of the alkylated compounds exhibited anti-estro-
genic activity (data not shown). Introduction of an alkyl group onto
the hydroxyl group led to a decrease of estrogenic activity. How-
ever, para-substituted compounds retained moderate estrogenic
activity regardless of the length of the alkyl chain (Fig. 4, 4b–4d).
Among meta- and ortho-substituted compounds, methyl and
n-propyl groups were compatible with estrogenic activity, but
the n-pentyl group abrogated both estrogenic activity and binding
E2
to ER
a LBD (Fig. 4, 5b–5d and 6b-6d). The activities of compounds
5
d and 6d, which have an n-pentyl group at the meta and ortho po-
sition, respectively, were markedly decreased compared with those
of the methyl derivatives 5b and 6b, respectively. Sterically bulky
substituents around the phenol group adversely affect the estro-
genic activity, and these results are in good accordance with the ef-
10-12
10-11
10-10
10-9
10-8
10-7
1
0-14
10-13
Concentration (M)
fects on binding to ERa.
Figure 3. Transcriptional activation by the bisphenol derivatives 4a, 5a, and 6a, and
a and 7b. MCF-7 cells were transfected with ERE (SV-40)-LUC and phRL/CMV, and
7
À7
À14
2.4. Cell proliferation assay using MCF-7 cell line
incubated with test compounds (1 Â 10 –1 Â 10 ). EtOH was used as a standard
(
control). Results are shown as means ± SD for triplicate transfections.
The estrogenic activities of bisphenol compounds 4a, 5a, and
6
a and monophenol 7a were evaluated by cell proliferation assay
assay of bisphenol compounds 3a, 4a, and 5a, and compounds 6
and 7, which have no anti-estrogenic activity (data not shown).
20
with human breast cancer cell lines, MCF-7 (Table 2). The order
of cell proliferation-inducing activity, 4a > 5a > 6a, was consistent
with those of binding and transactivation assays. In addition, the
activities of the potent estrogen agonist 1 and o- and m-carbo-
rane-based bisphenol derivatives 2 and 3, which were developed
in our previous studies, were evaluated and compared with that
of 4a. The cell proliferation-inducing activity of 4a was less than
1
7b-Estradiol and the test compounds induced the expression of
À7
À14
luciferase in dose-dependent manner at 1 Â 10 –1 Â 10
M.
p-Carborane bisphenol 4a showed by far the most potent estro-
genic activity, which was similar to that of E2. The activity of
other bisphenol isomers 5a and 6a was strongly dependent on
the position of the hydroxyl group. Interestingly, the activity of
6
a was about 1000 times weaker than that of 4a. The order of
estrogenic activity was 4a > 5a > 6a, which is the same as that
of the binding affinity for ER . The estrogenic activity of non-
Table 2
EC50 values of bisphenol and monophenol derivatives in cell proliferation assay using
MCF-7 cells
a
a
b
substituted compound 7a is higher than that of 6a and similar
Compound
EC50 (M)
to that of 5a. The activity of the pentafluorophenyl derivative,
À11
À10
À10
À10
À13
À12
À10
À9
4
5a
a
2.57 ± 1.61 Â 10
1.95 ± 0.59 Â 10
2.71 ± 0.95 Â 10
1.09 ± 0.40 Â 10
5.42 ± 1.50 Â 10
3.04 ± 1.72 Â 10
9.44 ± 3.98 Â 10
3.78 ± 1.41 Â 10
7
b, is weaker than that of 7a. It seems that the electrostatic char-
6
7
a
a
acter of the pentafluoro group or the electron-deficient benzene
ring of 7b negatively affects estrogenic activity, in accordance
with the binding assay.
In view of the very high estrogenic activity of 4a compared to
that of 5a and 6a, both hydroxyl groups of 4a are likely to interact
E2
1
2
3
strongly with the primary amino acid residues of the ER
a
ligand
a
MCF-7 cells were incubated with the test compounds for 5 days. Cell prolifer-
binding pocket, as the phenolic hydroxyl group and the secondary
alcoholic hydrogen of E2 interact with Glu353 and Arg394, and
His524.
ation assays were performed in triplicate (n = 3).
EC50 values of the test compounds were estimated from the sigmoidal dose–
response curves using GraphPad Prism 4 software.
b

T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
1113
that of E2 and 1, but greater than that of bisphenol derivatives 2
and 3.
Elmer 2400 CHN spectrometer. Column chromatography was car-
ried out using Merck Silica Gel 60 (0.063–0.200 m) and TLC was
l
The activities of other bisphenol isomers 5a and 6a were dras-
tically affected by the position of the hydroxyl group, and the
estrogenic activity of 6a was about 1000 times weaker than that
of 4a. One phenolic OH group, which is common to all of designed
derivatives, most probably interacts with Glu353 and Arg394 of
hER LBD, with which E2 and 1 form hydrogen bonds. The other
phenolic OH group seems to modulate the potency of the estro-
genic activity. Compound 4a showed potent binding affinity, more
potent estrogenic activity than any of the other designed com-
pounds in transactivation assay, and the most potent MCF-7 cell
proliferation-inducing activity. The potency was markedly de-
creased by the introduction of an alkyl group onto the phenolic hy-
droxyl group. Since the estrogenic activity of o-phenol derivative
performed on Merck Silica Gel F254. p-Carborane was purchased
from Katchem s.r.o. (Prague, Czech Republic). Other reagents were
purchased from Wako Pure Chemical Industries, Ltd, Sigma–Al-
drich Co., and Tokyo Chemical Industry, Ltd (TCI). All solvents were
commercial products of reagent grade, and were used without fur-
ther purification.
4
4
.2. Synthesis
.2.1. 4-Iodo-1-(tert-butyldimethylsiloxy)benzene14
A mixture of 4-iodophenol (10.0 g, 45.5 mmol), tert-butyldi-
methylsilyl chloride (TBSCl) (8.22 g, 54.5 mmol) and triethylamine
7.6 mL, 54.5 mmol) in 20 mL of CH Cl was stirred for 1.5 h at
(
2
2
6
a was less than that of m-phenol derivative 5a or the unsubstitut-
room temperature. The reaction mixture was poured into water
and extracted with AcOEt. The organic layer was washed with
ed benzene derivative 7a in both cell assays, the OH group of 6a
may disturb the interaction with the hER LBD. The position of
the OH group thus appears to be very important for modulating
the potency of estrogenic activity. The OH group of 4a presumably
has a significant interaction with the hER LBD. The His524 amino
acid residue of hER interacts with the secondary alcohol of E2
and the primary alcohol of 1, and probably compound 4a would
bind to the hER LBD with the same binding mode as compound
brine, dried over Na
purified by column chromatography on silica gel with n-hexane
to give the title compound (15.4 g, 100%) as a colorless oil; 1
NMR (270 MHz, CDCl ) d (ppm) 0.18 (6H, s), 0.97 (9H, s), 6.61
2H, dd, J = 2.1, 6.8 Hz), 7.50 (2H, dd, J = 2.1, 6.6 Hz); MS (EI) m/z
2 4
SO , and then concentrated. The residue was
H
3
(
3
+
34 (M ), 227 (100%); HRMS calcd for C12
H19IOSi: 334.0250, found:
1
2
334.0231.
1.
However, there is a possibility that the phenolic OH of 4a inter-
acts with some other amino acid residue located further in the
binding pocket because the distance between two phenolic OH
groups of 4a is greater than that between the phenolic OH group
and the alcoholic groups of E2 or 1. If this is so, it will be an impor-
tant consideration in the design of novel ER modulators.
4.2.2. 1-{4-(tert-Butyldimethylsiloxy)phenyl}-1,12-dicarba-closo-
dodecaborane (8) and 1,12-bis{4-(tert-butyldimethylsiloxy)
phenyl}-1,12-dicarba-closo-dodecaborane (9)
To a solution of p-carborane (4.0 g, 27.7 mmol) in 15 mL of DME
was added dropwise 1.54 M n-BuLi in hexane (37.8 mL, 58.2 mmol)
at 0 °C under an Ar atmosphere, and the mixture was stirred for
3
. Conclusion
3
0 min at the same temperature. CuCl (5.77 g, 58.2 mmol) was
In conclusion, we designed and synthesized a series of p-carbo-
added in one portion and the mixture was stirred at room temper-
ature for 1 h. Pyridine (33.6 mL, 416 mmol) and 4-iodo-1-(tert-
butyldimethylsiloxy)benzene (13.9 g, 41.6 mmol) were added in
one portion, and the mixture was heated at 110 °C for 21 h. After
cooling to room temperature, the mixture was diluted with AcOEt
and insoluble materials were removed by filtration through Celite.
The filtrate was washed with water and brine, dried over Na SO ,
2 4
and then concentrated. The residue was purified by column chro-
matography on silica gel with n-hexane to give 8 (3.05 g, 31%) as
rane bisphenol derivatives utilizing p-carborane as a hydrophobic
pharmacophore. ER binding ability was assessed by means of
a
receptor binding assay and estrogenic activity by means of transac-
tivation and cell proliferation assays. p-Carborane bisphenol 4a
showed high binding ability to ERa and potent estrogenic activity.
The activities of other bisphenol isomers 5a and 6a were drastically
influenced by the position of the second hydroxyl group: the estro-
genic activity of 6a in transactivation assay was about 1000 times
weaker than that of 4a. Conversion of the hydroxyl group to alkoxy
decreased the estrogenic activity. However, the para-substituted
compounds retained moderate estrogenic activity regardless of
the length of the alkyl chain. Both hydroxyl groups of 4a appear
the first eluted compound and 9 (5.56 g, 36%) as the second eluted
1
compound; Compound 8: H NMR (270 MHz, CDCl
3
) d (ppm) 0.15
(6H, s), 0.94 (9H, s), 1.60–3.60 (10H, br m), 2.73 (1H, br m), 6.60
+
(2H, d, J = 8.9 Hz), 7.04 (2H, d, J = 8.9 Hz); MS (EI) m/z 350 (M ),
1
to interact strongly with amino acid residues of the ER
a
ligand
293 (100%); Compound 9: H NMR (270 MHz, CDCl
3
) d (ppm)
binding pocket. The cell proliferation-inducing activity of 4a was
less than that of E2 and 1, but greater than that of bisphenol deriv-
atives 2 and 3.
0.16 (12H, s), 0.95 (18H, s), 1.60–3.60 (10H, br m), 6.61 (4H, d,
+
J = 8.7 Hz), 7.07 (4H, d, J = 8.9 Hz); MS (EI) m/z 556 (M ), 500
(100%).
4
.2.3. 1,12-Bis(4-hydroxyphenyl)-1,12-dicarba-closo-dodeca-
4
4
. Experimental
borane (4a)
To a solution of 9 (4.85 g, 8.7 mmol) in 20 mL of THF was
added 1 M tetrabutylammonium fluoride (TBAF) in THF
.1. General consideration
(
18.3 mL, 18.3 mmol) at room temperature, and the mixture
Melting points were determined with a Yanaco micro melting
was stirred for 30 min at the same temperature. The mixture
was poured into water and extracted with AcOEt. The organic
layer was washed with water and brine, dried over Na SO , and
2 4
then concentrated. The residue was purified by column chroma-
tography on silica gel with 1:5 AcOEt/n-hexane to give 4a
point apparatus and were not corrected. 1H NMR and C NMR
spectra were recorded with JEOL JNM-EX-270, JNM-LA-400 and
JNM-LA-600 spectrometers. Chemical shifts for 1H NMR spectra
were referenced to tetramethylsilane (0.0 ppm) as an internal stan-
13
13
dard. Chemical shifts for C NMR spectra were referenced to resid-
(2.85 g, 100%) as a colorless solid; Colorless needles (AcOEt/n-
1
3
1
ual C present in deuterated solvents. The splitting patterns are
designated as follows: s (singlet), d (doublet), t (triplet) and m
hexane); mp 292–294 °C;
6
H NMR (270 MHz, DMSO-d ) d
(ppm) 1.60–3.60 (10H, br m), 6.60 (4H, d, J = 8.7 Hz), 7.00 (4H,
1
3
(
multiplet). Mass spectra were recorded on a JEOL JMS-DX-303
spectrometer. Elemental analyses were performed with a Perkin
d, J = 8.9 Hz), 9.71 (2H, br s); C NMR (100 MHz, CD
3
OD) d
+
(ppm) 83.7, 115.7, 128.6, 129.3, 159.0; MS (EI) m/z 328 (M ,

1
114
T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
1
5
00%); Anal. Calcd for C14
1.23; H, 6.12.
H
20
B
10
O
2
: C, 51.20; H, 6.14. Found: C,
J = 8.9 Hz), 6.75 (1H, dd, J = 1.1, 2.3 Hz), 6.76–6.82 (1H, m), 6.84
(1H, dd, J = 1.1, 1.9 Hz), 7.07 (2H, d, J = 8.9 Hz), 7.09 (1H, dd,
+
J = 7.7 Hz, 8.6 Hz); MS (EI) m/z 457 (M ), 400 (100%).
4
1
.2.4. 1-(4-Hydroxyphenyl)-12-(4-methoxyphenyl)-1,
2-dicarba-closo-dodecaborane (4b)
To a mixture of 4a (200 mg, 0.61 mmol) and K CO (110 mg,
2 3
4.2.8. 1-{4-(tert-Butyldimethylsiloxy)phenyl}-12-(3-
methoxyphenyl)-1,12-dicarba-closo-dodecaborane (11),
1-(3-hydroxyphenyl)-12-(4-hydroxyphenyl)-1,12-dicarba-closo-
dodecaborane (5a) and 1-(4-hydroxyphenyl)-12-(3-methoxy-
phenyl)-1,12-dicarba-closo-dodecaborane (5b)
0
0
.79 mmol) in 3 mL of DMF was added iodomethane (0.049 mL,
.73 mmol), and the mixture was stirred for 2.5 h at room temper-
ature. The mixture was poured into water and extracted with Et
The organic layer was washed with 10% Na aqueous solution,
water and brine, dried over Na SO , and then concentrated. The
residue was purified by column chromatography on silica gel with
:10 AcOEt/n-hexane to give 4b (77 mg, 37%) as a colorless solid;
2
O.
S
2 2
O
3
To a solution of 10 (900 mg, 1.97 mmol) in 5 mL of CH
2 2
Cl was
2
4
added dropwise 1 M BBr in CH Cl (2.52 mL, 2.52 mmol) at 0 °C
3
2
2
under an Ar atmosphere, and the mixture was stirred for 1 h at
room temperature. The mixture was poured onto ice and extracted
with AcOEt. The organic layer was washed with water and brine,
dried over Na SO , and then concentrated. The residue was purified
2 4
by column chromatography on silica gel with 1:5 AcOEt/n-hexane
to give 11 (365 mg, 42%) as the first eluted compound, 5b (104 mg,
1
1
Colorless cubes (AcOEt/n-hexane); mp 197–198.5 °C; H NMR
270 MHz, CDCl ) d (ppm) 1.60–3.60 (10H, br m), 3.75 (3H, s),
.87 (1H, br s), 6.63 (2H, d, J = 8.7 Hz), 6.70 (2H, d, J = 9.1 Hz),
(
3
4
7
1
3
.10 (2H, d, J = 8.9 Hz), 7.15 (2H, d, J = 9.1 Hz);
C NMR
(
1
100 MHz, CDCl
3
) d (ppm) 55.3, 82.0, 82.1, 113.3, 114.8, 128.3,
16%) as the second eluted compound and 5a (197 mg, 42%) as the
+
1
28.6, 128.8, 129.0, 155.7, 159.6; MS (EI) m/z 342 (M , 100%); Anal.
: C, 52.61; H, 6.48. Found: C, 52.53; H, 6.39.
third eluted compound; Compound 11: H NMR (270 MHz, CDCl
3
)
Calcd for C15
H
22
B
10
O
2
d (ppm) 0.16 (6H, s), 0.94 (9H, s), 1.60–3.60 (10H, br m), 5.16 (1H,
br s), 6.62 (2H, d, J = 8.7 Hz), 6.68 (1H, dd, J = 0.8, 2.3 Hz), 6.71 (1H,
ddd, J = 0.8, 2.3, 8.4 Hz), 6.80 (1H, ddd, J = 0.8, 1.8, 7.3 Hz), 7.04 (1H,
4
1
.2.5. 1-(4-Hydroxyphenyl)-12-{4-n-propoxyphenyl)-1,
2-dicarba-closo-dodecaborane (4c)
+
dd, J = 7.9, 8.1 Hz, 1H), 7.07 (2H, d, J = 8.9 Hz); MS (EI) m/z 442 (M ),
Compound 4c was prepared by the same method as described
for the synthesis of 4b, but with n-propyl iodide in place of iodo-
386 (100%); Compound 5b: colorless flakes (AcOEt/n-hexane); mp
1
161–163 °C; H NMR (270 MHz, CDCl
3
) d (ppm) 1.60–3.60 (10H, br
methane; 24% yield; colorless cubes (AcOEt/n-hexane); mp
m), 3.76 (3H, s), 5.73 (1H, s), 6.64 (2H, d, J = 8.9 Hz), 6.74–6.85 (3H,
m), 7.09 (1H, t, J = 7.9 Hz), 7.10 (2H, d, J = 8.7 Hz); C NMR
1
13
1
3
84.5–186 °C; H NMR (270 MHz, CDCl ) d (ppm) 0.99 (3H, t,
J = 7.4 Hz), 1.60–3.60 (10H, br m), 1.76 (2H, sextet, J = 7.4 Hz),
(100 MHz, CDCl
3
) d (ppm) 55.23, 82.0, 82.7, 113.4, 113.6, 114.8,
3
6
.85 (2H, t, J = 6.6 Hz), 5.52 (1H, br s), 6.63 (2H, d, J = 8.9 Hz),
.68 (2H, d, J = 9.1 Hz), 7.10 (2H, d, J = 9.1 Hz), 7.13 (2H, d,
119.6, 128.5, 128.8, 129.0, 137.7, 155.8, 159.0; MS (EI) m/z 342
+
(M , 100%); Anal. Calcd for C15
22 10 2
H B O : C, 52.61; H, 6.48. Found:
1
3
J = 9.2 Hz); C NMR (100 MHz, CDCl
8
MS (EI) m/z 370 (M ), 328 (100%); Anal. Calcd for C17
5
3
) d (ppm) 10.5, 22.5, 69.5,
C, 52.27; H, 6.15; Compound 5a: colorless cubes (AcOEt/n-hexane);
1
0.2, 82.0, 113.8, 114.8, 128.2, 128.5, 128.7, 129.1, 155.6, 159.2;
3
mp 262–263.5 °C; H NMR (270 MHz, CDCl ) d (ppm) 1.60–3.60
+
H
26
B
10
O
2
: C,
(10H, br m), 4.77 (1H, s), 4.81 (1H, s), 6.63 (2H, d, J = 8.9 Hz),
6.68 (1H, dd, J = 0.7 Hz, 2.3 Hz), 6.70–6.75 (1H, m), 6.81 (1H, ddd,
J = 0.8, 1.8, 8.6 Hz), 7.05 (1H, dd, J = 7.4, 7.7 Hz), 7.10 (2H, d,
5.11; H, 7.07. Found: C, 55.19; H, 7.09.
1
3
4
1
.2.6. 1-(4-Hydroxyphenyl)-12-(4-n-pentoxyphenyl)-1,
2-dicarba-closo-dodecaborane (4d)
Compound 4d was prepared by the same method as described
J = 8.9 Hz); C NMR (100 MHz, CD
3
OD) d (ppm) 83.3, 84.5, 115.4,
115.8, 116.4, 119.3, 128.5, 129.3, 130.2, 138.7, 158.3, 159.1; MS
+
20 10 2
(EI) m/z 328 (M , 100%); Anal. Calcd for C14H B O : C, 51.20; H,
for the synthesis of 4b, but with n-pentyl iodide in place of iodo-
6.14. Found: C, 50.95; H, 5.95.
methane; 17% yield; colorless leaflets (AcOEt/n-hexane); mp
1
1
37–139.5 °C; H NMR (270 MHz, CDCl
3
) d (ppm) 0.99 (3H, t,
4.2.9. 1-(4-Hydroxyphenyl)-12-{3-n-propoxyphenyl)-1,
12-dicarba-closo-dodecaborane (5c)
J = 6.9 Hz), 1.30–1.45 (4H, m), 1.60–3.60 (10H, br m), 1.74 (2H,
quintet, J = 6.6 Hz), 3.88 (2H, t, J = 6.4 Hz), 5.37 (1H, br s), 6.62
2 3
To a mixture of 11 (15 mg, 0.034 mmol) and K CO (5.2 mg,
(
2H, d, J = 8.6 Hz), 6.68 (2H, d, J = 8.9 Hz), 7.10 (2H, d, J = 8.6 Hz),
0.037 mmol) in 0.5 mL of DMF was added n-propyl iodide (6 mg,
0.037 mmol), and the mixture was stirred for 1.5 h at room tem-
perature. Then, 1 mL of 10% HCl aqueous solution was added,
and stirring was continued for 30 min at the same temperature.
7
2
1
.13 (2H, d, J = 8.7 Hz); 13C NMR (100 MHz, CDCl
3
) d (ppm) 14.0,
2.4, 28.1, 28.8, 68.0, 81.9, 82.2, 113.8, 114.8, 128.2, 128.5, 128.6,
+
29.0, 155.6, 159.1; MS (EI) m/z 398 (M ), 328 (100%); Anal. Calcd
for C19
H
30
B
10
O
2
: C, 57.26; H, 7.59. Found: C, 57.26; H, 7.69.
The mixture was extracted with Et
washed with 10% Na aqueous solution, water and brine, dried
over Na SO , and then concentrated. The residue was purified by
column chromatography on silica gel with 1:7 Et O/n-hexane to
give 5c (6 mg, 48%) as a colorless solid; colorless cubes (AcOEt/n-
2
O, and the organic layer was
2 2 3
S O
4
.2.7. 1-{4-(tert-Butyldimethylsiloxy)phenyl}-12-(3-methoxy-
2
4
phenyl)-1,12-dicarba-closo-dodecaborane (10)
2
To a solution of 8 (1.0 g, 2.85 mmol) in 5 mL of DME was added
dropwise 1.54 M n-BuLi in hexane (2.2 mL, 3.42 mmol) at 0 °C un-
der an Ar atmosphere, and the mixture was stirred for 30 min at
the same temperature. CuCl (311 mg, 3.14 mmol) was added in
one portion and the mixture was stirred at room temperature for
1
3
hexane); mp 145.5–147 °C; H NMR (270 MHz, CDCl ) d (ppm)
1.02 (3H, t, J = 7.4 Hz), 1.60–3.60 (10H, br m), 1.78 (2H, quintet,
J = 7.4 Hz), 3.86 (2H, t, J = 6.6 Hz), 5.52 (1H, br s), 6.64 (2H, d,
J = 8.9 Hz), 6.68–6.83 (3H, m), 7.04–7.15 (1H, m), 7.10 (2H, d,
1
3
1
2
h. Pyridine (3.5 mL, 42.7 mmol) and 3-iodoanisole (0.38 mL,
.85 mmol) were added in one portion, and the mixture was
3
J = 8.9 Hz); C NMR (100 MHz, CDCl ) d (ppm) 10.5, 22.4, 69.5,
81.6, 82.8, 113.8, 114.6, 115.3, 119.7, 128.2, 128.5, 129.2, 138.0,
+
heated at 110 °C for 24 h. After cooling to room temperature, the
mixture was diluted with AcOEt and insoluble materials were re-
moved by filtration through Celite. The filtrate was washed with
155.1, 159.1; MS (EI) m/z 370 (M ), 328 (100%); HRMS calcd for
17 26 10 2
C H B O : 370.2936, found: 370.2946.
water and brine, dried over Na
residue was purified by column chromatography on silica gel with
2
SO
4
, and then concentrated. The
4.2.10. 1-(4-Hydroxyphenyl)-12-(3-n-pentoxyphenyl)-1,
12-dicarba-closo-dodecaborane (5d)
n-hexane to 1:20 AcOEt/n-hexane to give 10 (960 mg, 74%) as a
Compound 5d was prepared by the same method as described
for the synthesis of 5c, but with n-pentyl iodide in place of n-pro-
pyl iodide; 44% yield; colorless cubes (AcOEt/n-hexane); mp 116–
1
colorless solid; H NMR (270 MHz, CDCl
3
) d (ppm) 0.16 (6H, s),
0
.95 (9H, s), 1.60–3.60 (10H, br m), 3.76 (3H, s), 6.62 (2H, d,

T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
1115
1
1
4
7
2
1
17 °C; 1H NMR (270 MHz, CDCl
3
) d (ppm) 0.86 (3H, t, J = 7.1 Hz),
C, 52.61; H, 6.48. Found: C, 52.69; H, 6.25; Compound 6a: colorless
plates (AcOEt/n-hexane); mp 201–202.5 °C; H NMR (270 MHz,
CDCl ) d (ppm) 1.60–3.60 (10H, br m), 4.85 (1H, s), 5.56 (1H, s),
3
6.64 (2H, d, J = 8.9 Hz), 6.70 (1H, dd, J = 1.3, 8.1 Hz), 6.78 (1H,
1
.60–1.71 (6H, m), 1.60–3.60 (10H, br m), 3.82 (2H, t, J = 6.6 Hz),
.78 (1H, br s), 6.55 (2H, d, J = 8.6 Hz), 6.55–6.80 (3H, m), 6.90–
1
3
.10 (1H, m); C NMR (100 MHz, CDCl
3
) d (ppm) 14.0, 22.4, 28.1,
8.8, 68.0, 81.6, 82.9, 113.8, 114.6, 115.3, 119.8, 128.2, 128.5,
29.3, 138.0, 155.0, 159.2; MS (EI) m/z 398 (M ), 328 (100%); HRMS
ddd, J = 1.3, 7.6, 8.1 Hz), 7.11 (2H, d, J = 8.7 Hz), 7.11 (1H, ddd,
+
13
J = 1.3, 7.5, 7.7 Hz), 7.31 (1H, dd, J = 1.6, 8.2 Hz);
(100 MHz, CDCl
28.5, 128.9, 130.1, 130.9, 152.8, 155.7; MS (EI) m/z 328 (M ,
C NMR
calcd for C19
H
30
B
10
O
2
: 398.3249, found: 398.3251.
3
) d (ppm) 79.7, 84.9, 114.8, 118.1, 120.5, 120.7,
+
1
4
(
.2.11. 1-(4-Methoxyphenyl)-1,12-dicarba-closo-dodecaborane
12)
To a solution of p-carborane (2.0 g, 13.7 mmol) in 10 mL of DME
100%); Anal. Calcd for C14
51.30; H, 5.90.
20 10 2
H B O : C, 51.20; H, 6.14. Found: C,
was added dropwise 1.54 M n-BuLi in hexane (19.0 mL, 29.1 mmol)
at 0 °C under an Ar atmosphere, and the mixture was stirred for
4.2.14. 1-{4-(tert-Butyldimethylsiloxy)phenyl}-12-(2-hydroxy-
phenyl)-1,12-dicarba-closo-dodecaborane (14)
3
0 min at the same temperature. CuCl (311 mg, 3.14 mmol) was
A
mixture of 6a (400 mg, 1.22 mmol), TBSCl (202 mg,
1.34 mmol) and triethylamine (0.19 mL, 1.34 mmol) in 5 mL of
CH Cl was stirred for 3 h at room temperature. The reaction mix-
ture was poured into water and extracted with AcOEt. The organ-
ic layer was washed with brine, dried over Na SO , and then
added in one portion and the mixture was stirred at room temper-
ature for 1.5 h. Pyridine (17 mL, 208 mmol) and 4-iodoanisole
2
2
(
3.89 g, 16.6 mmol) were added in one portion, and the mixture
was heated at 110 °C for 18 h. After cooling to room temperature,
the mixture was diluted with AcOEt and insoluble materials were
removed by filtration through Celite. The filtrate was washed with
2
4
concentrated. The residue was purified by column chromatogra-
phy on silica gel with 1:10 AcOEt/n-hexane to give 14 (238 mg,
44%) as a colorless solid; H NMR (270 MHz, CDCl
0.17 (6H, s), 0.95 (9H, s), 1.60–3.60 (10H, br m), 5.57 (1H, s),
6.62 (2H, d, J = 8.9 Hz), 6.70 (1H, dd, J = 1.3, 8.1 Hz), 6.77 (1H,
ddd, J = 1.5, 7.0, 8.3 Hz), 7.08 (2H, d, J = 8.9 Hz), 7.11 (1H, ddd,
J = 1.6, 7.3, 8.9 Hz), 7.31 (1H, dd, J = 1.6, 8.2 Hz); MS (EI) m/z
1
water and brine, dried over Na
residue was purified by column chromatography on silica gel with
:50 to 1:20 AcOEt/n-hexane to give 12 (2.37 g, 68%) as a colorless
2
SO
4
, and then concentrated. The
3
) d (ppm)
1
1
solid; H NMR (270 MHz, CDCl
3
) d (ppm) 1.60–3.70 (10H, br m),
2
.74 (1H, br s), 3.74 (3H, s), 6.68 (2H, d, J = 9.1 Hz), 7.12 (2H, dd,
+
+
J = 9.1 Hz); MS (EI) m/z 250 (M , 100%).
442 (M ), 386 (100%).
4
1
.2.12. 1-(2-Methoxyphenyl)-12-(4-methoxyphenyl)-1,
2-dicarba-closo-dodecaborane (13)
4.2.15. 1-(4-Hydroxyphenyl)-12-{2-n-propoxyphenyl)-1,
12-dicarba-closo-dodecaborane (6c)
To a solution of 12 (1.5 g, 6.0 mmol) in 5 mL of DME was added
Compound 6c was prepared by the same method as described
dropwise 1.54 M n-BuLi in hexane (4.2 mL, 6.6 mmol) at 0 °C under
an Ar atmosphere, and the mixture was stirred for 30 min at the
same temperature. CuCl (650 mg, 6.6 mmol) was added in one por-
tion and the mixture was stirred at room temperature for 1.5 h.
Pyridine (7.3 mL, 90 mmol) and 2-iodoanisole (0.86 mL, 6.6 mmol)
were added in one portion, and the mixture was heated at 110 °C
for 42 h. After cooling to room temperature, the mixture was di-
luted with AcOEt and insoluble materials were removed by filtra-
tion through Celite. The filtrate was washed with water and
for the synthesis of 5c; 44% yield; Colorless needles (AcOEt/n-hex-
1
3
ane); mp 107.5–108.5 °C; H NMR (270 MHz, CDCl ) d (ppm) 1.12
(3H, t, J = 7.4 Hz), 1.60–3.60 (10H, br m), 1.91 (2H, sextet,
J = 7.1 Hz), 3.88 (2H, t, J = 6.4 Hz), 5.07 (1H, br s), 6.62 (2H, d,
J = 8.7 Hz), 6.73–6.80 (2H, m), 7.11 (2H, d, J = 8.7 Hz), 7.13–7.20
1
3
3
(1H, m), 7.38 (1H, dd, J = 1.6 Hz, 8.4 Hz); C NMR (100 MHz, CDCl )
d (ppm) 11.1, 22.3, 70.4, 80.1, 84.6, 112.6, 114.7, 119.8, 123.2,
+
128.5, 129.4, 129.8, 131.1, 155.5, 156.1; MS (EI) m/z 370 (M ),
328 (100%); Anal. Calcd for C17
C, 55.12; H, 7.08.
26 10 2
H B O : C, 55.11; H, 7.07. Found:
2 4
brine, dried over Na SO , and then concentrated. The residue was
purified by column chromatography on silica gel with 1:20
1
AcOEt/n-hexane to give 13 (771 mg, 36%) as a colorless solid;
NMR (270 MHz, CDCl ) d (ppm) 1.60–3.70 (10H, br m), 3.74 (3H,
s), 3.75 (3H, s), 6.70 (2H, d, J = 8.9 Hz), 6.75–6.83 (2H, m), 7.16
2H, d, J = 8.9 Hz), 7.15–7.25 (1H, m), 7.37 (1H, dd, J = 1.5,
H
4.2.16. 1-(4-Hydroxyphenyl)-12-{2-n-pentoxyphenyl)-1,
12-dicarba-closo-dodecaborane (6d)
3
Compound 6d was prepared by the same method as de-
(
scribed for the synthesis of 5d; 43% yield; colorless needles
+
1
7
.7 Hz); MS (EI) m/z 356 (M , 100%).
(AcOEt/n-hexane); mp 66–67 °C; H NMR (270 MHz, CDCl
3
) d
(
ppm) 1.21 (3H, t, J = 7.1 Hz), 1.35–1.60 (4H, m), 1.60–3.60
4
1
1
.2.13. 1-(2-Hydroxyphenyl)-12-(4-hydroxyphenyl)-1,
2-dicarba-closo-dodecaborane (6a) and 1-(4-hydroxyphenyl)-
2-(2-methoxyphenyl)-1,12-dicarba-closo-dodecaborane (6b)
(10H, br m), 1.89 (2H, quintet, J = 8.2 Hz), 3.90 (2H, t,
J = 6.6 Hz), 4.97 (1H, br s), 6.62 (2H, d, J = 8.9 Hz), 6.70–6.80
(2H, m), 7.12 (2H, d, J = 9.1 Hz), 7.12–7.20 (1H, m), 7.38 (1H,
1
3
To a solution of 13 (771 mg, 2.17 mmol) in 5 mL of CH
added 1 M BBr in CH Cl
Ar atmosphere, and the mixture was stirred for 2 h at room tem-
perature. The mixture was poured onto ice and extracted with
AcOEt. The organic layer was washed with water and brine, dried
2
Cl
2
was
3
dd, J = 1.6, 8.4 Hz); C NMR (100 MHz, CDCl ) d (ppm) 14.1,
3
2
2
(3.68 mL, 3.68 mmol) at À78 °C under an
22.5, 28.5, 28.6, 68.7, 80.0. 84.6, 112.6, 114.7, 119.8, 123.2,
+
128.5, 129.4, 129.8, 131.1, 155.5, 156.1; MS (EI) m/z 398 (M ),
328 (100%); Anal. Calcd for C19
7.67. Found: C, 55.94; H, 7.74.
H
30
B
10
O
2
Á0.5 H
2
O: C, 55.99; H,
2 4
over Na SO , and then concentrated. The residue was purified by
column chromatography on silica gel with 1:5 AcOEt/n-hexane to
give 6b (40 mg, 5%) as the first eluted compound and 6a
4.2.17. 1-(4-Hydroxyphenyl)-12-phenyl-1,12-dicarba-closo-
dodecaborane (7a)
(
556 mg, 78%) as the second eluted compound; Compound 6b: col-
To a solution of 8 (500 mg, 1.43 mmol) in 3 mL of DME was
added dropwise 1.58 M n-BuLi in hexane (1.0 mL, 1.58 mmol) at
0 °C under an Ar atmosphere, and the mixture was stirred for
30 min at the same temperature. CuCl (155 mg, 1.57 mmol) was
added in one portion and the mixture was stirred at room temper-
ature for 1 h. Pyridine (1.7 mL, 21.4 mmol) and iodobenzene
1
orless cubes (AcOEt/n-hexane); mp 170.5–172 °C;
(
4
H
NMR
) d (ppm) 1.60–3.60 (10H, br m), 3.75 (3H, s),
.76 (1H, br s), 6.63 (2H, d, J = 8.9 Hz), 6.75–6.83 (2H, m), 7.12
270 MHz, CDCl
3
(
1H, d, J = 8.7 Hz), 7.19 (2H, ddd, J = 1.6, 7.4, 7.8 Hz), 7.37 (1H, dd,
1
3
J = 1.4, 7.9 Hz); C NMR (100 MHz, CDCl
3
) d (ppm) 55.2, 79.8,
8
1
4.6, 112.6, 114.7, 120.3, 123.6, 128.5, 129.4, 129.9, 131.1, 155.5,
56.7; MS (EI) m/z 342 (M , 100%); Anal. Calcd for C15H B O :
22 10 2
(159
lL, 1.43 mmol) were added in one portion, and the mixture
+
was heated at 110 °C for 24 h. After cooling to room temperature,

1
116
T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
the mixture was diluted with AcOEt and insoluble materials were
removed by filtration through Celite. The filtrate was washed with
was measured in Atomlight (NEN) by using a liquid scintillation
counter.
water and brine, dried over Na
residue was diluted with 3 mL of THF, and then 1 M TBAF in THF
2.14 mL, 2.14 mmol) was added. The mixture was stirred for 1 h
at room temperature, poured into water, and extracted with AcOEt.
The organic layer was washed with brine, dried over Na SO , and
2 4
SO , and then concentrated. The
4.4. Transcriptional activation assay using human breast cancer
cell lines, MCF-7 cells
(
2
4
Human breast cancer cell line MCF-7 cells were maintained in
DMEM (Sigma Chemical Co.) containing penicillin and streptomy-
cin with 5% fetal bovine serum (FBS; Life Technologies, Rockville,
MD). ERE-luciferase reporter assay using MCF-7 cells was per-
formed according to the reported method. Briefly, transient trans-
concentrated. The residue was purified by column chromatography
on silica gel with 1:5 AcOEt/n-hexane to give 7a (148 mg, 33%) as a
colorless solid; colorless needles (AcOEt/n-hexane); mp 218–
1
2
19.5 °C; H NMR (270 MHz, CDCl
3
) d (ppm) 1.60–3.60 (10H, br
TM
m), 5.81 (1H, br s), 6.64 (2H, dd, J = 2.3, 6.1 Hz), 7.10 (2H, dd,
fections in MCF-7 cells were performed using Transfast (Promega
J = 2.1, 6.6 Hz), 7.13–7.25 (5H, m); 13C NMR (100 MHz, CDCl
Co., Madison, WI), according to the manufacturer’s protocol. Trans-
3
) d
(
1
5
ppm) 82.2, 82.6, 114.8, 127.1, 128.1, 128.3, 128.6, 129.1, 136.3,
fections were done in 96-well plates at 8000 cells/well with 0.1
lg
+
55.6; MS (EI) m/z 312 (M , 100%); Anal. Calcd for C14
H
20
B10O: C,
of p(ERE) -SV40-luc and 0.3 ng of phRL/CMV (Promega Co.) as
3
3.82; H, 6.45. Found: C, 53.88; H, 6.35.
internal standards. Twenty-four hours after addition of the sample,
TM
the assay was performed with a Dual Luciferase assay kit (Prome-
ga Co.). For the assay of anti-estrogens, the inhibitory effect of test
4
1
.2.18. 1-(4-Methoxyphenyl)-12-pentafluorophenyl-1,
2-dicarba-closo-dodecaborane (15)
To a solution of 12 (500 mg, 2.00 mmol) in 10 mL of Et
compounds on the estrogenic activity of E2 at the concentration of
À10
1
4
4
 10
M was examined.
2
O was
added dropwise 2.67 M n-BuLi in hexane (0.9 mL, 2.40 mmol) at
.5. Cell proliferation assay using MCF-7 cell lines
0
3
°C under Ar atmosphere, and the mixture was stirred for
0 min at the same temperature. To a reaction mixture was added
.5.1. Cell culture
hexafluorobenzene (458 lg, 3.99 mmol) in one portion, and the
mixture was stirred for 48 h at room temperature. The mixture
was poured into 10% HCl aqueous solution and extracted with
2 2 4
Et O. The organic layer was washed with brine, dried over Na SO ,
At 80% confluence, cells of the human breast adenocarcinoma
cell line MCF-7 was trypsinized from the maintenance dish with
0.25% trypsin-EDTA and collected by centrifugation (4 °C,
1500 rpm, 5 min). The supernatant of medium was removed,
1 mL of DMEM supplemented with 10% FBS, 100 IU/mL penicillin
and then concentrated. The residue was purified by column chro-
matography on silica gel with 1:7 AcOEt/n-hexane to give 15
233 mg, 28%) as colorless solid; 1H NMR (270 MHz, CDCl
and 100 mg/mL streptomycin was added, and cell aggregates
were broken up by thorough pipetting. Cells were seeded in a
(
(
3
) d
ppm) 1.50–3.80 (10H, br m), 3.75 (3H, s), 6.71 (2H, dd, J = 2.1,
4
4
+
dish at a concentration of 4 Â 10 cell/mL or 8 Â 10 cell/mL,
and cultivated at 37 °C in a 5% CO humidified incubator. Cells
were routinely cultivated two days or three days later.
6
1
.8 Hz), 7.13 (2H, dd, J = 2.1, 6.8 Hz, 2H); MS (EI) m/z 416 (M ,
00%).
2
4
1
.2.19. 1-(4-Hydroxyphenyl)-12-pentafluorophenyl-1,
2-dicarba-closo-dodecaborane (7b)
4
.5.2. Cell proliferation assay
Cells of the human breast adenocarcinoma line MCF-7 were
To a solution of 15 (30 mg, 0.072 mmol) in 1 mL of CH
was added 1 M BBr in CH Cl
der an Ar atmosphere, and the mixture was stirred for 6 h at
room temperature, then poured onto ice and extracted with
AcOEt. The organic layer was washed with water and brine,
dried over Na SO , and then concentrated. The residue was puri-
2 4
fied by column chromatography on silica gel with 1:7 AcOEt/n-
hexane to give 7b (27 mg, 93%) as a colorless solid; Colorless
2 2
Cl
routinely cultivated in DMEM supplemented with 10% FBS, 100
IU/mL penicillin and 100 mg/mL streptomycin at 37 °C in a 5%
CO humidified incubator. On the day before an assay, MCF-7
2
cells were switched to DMEM (low glucose phenol red-free sup-
plemented with 5% sFBS, 100 IU/mL penicillin and 100 mg/mL
streptomycin) Cells were trypsinized from the maintenance dish
with phenol red-free trypsin-EDTA and seeded in a 96-well plate
3
2
2
(0.5 mL, 0.5 mmol) at À78 °C un-
3
3
at a density of 1 Â 10 or 2 Â 10 cells per final volume of 100
DMEM supplemented with 5% sFBS, 100 IU/mL penicillin and
00 mg/mL streptomycin. After 24 h, the medium was replaced
with 90 L of fresh DMEM and 10 L of drug solution, supple-
lL
1
needles (AcOEt/n-hexane); mp 233.5–235 °C;
270 MHz, CDCl
s), 6.64 (2H, dd, J = 2.3, 6.8 Hz), 7.08 (2H, dd, J = 2.1, 6.6 Hz);
H
NMR
(
3
) d (ppm) 1.50–3.80 (10H, br m), 5.37 (1H, br
1
l
l
1
3
C NMR (100 MHz, CDCl
28.7, 156.0; MS (EI) m/z 402 (M , 100%); HRMS calcd for
: 402.2046, found: 402.2054.
3
) d (ppm) 70.8, 88.0, 114.9, 128.3,
mented with serial dilutions of 4-OH-Tam or DMSO as the dilu-
+
1
C
À10
tion control in the presence or absence of 1 Â 10
M or 1 Â 10
14
H
15
B
10
F
5
O
2
À11
M E2, was added to triplicate microcultures. Cells were incu-
bated for 5 days, and medium with 4-OH-Tam in the presence or
À10
À11
4
.3. Competitive binding assay to ER
a
absence of 1 Â 10
after 3 days. At the end of the incubation time, proliferation
was evaluated by using WST-8. 10 M of WST-8 was added to
microcultures and cells were incubated for 2 h. The absorbance
at 450 nm was measured. This parameter is related to the num-
ber of living cells in the culture.
M or 1 Â 10
M E2 was replaced once
The ligand binding activity of estrogen receptor
determined by the nitrocellulose filter binding assay method.
ER (0.5 g/tube, PanVera Co., Ltd) was diluted with a binding
a
(ER
a
) was
l
a
l
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 phenylmethylsul-
3
fonyl fluoride) and incubated with 4 nM [6,7- H]17b-estradiol
Acknowledgments
in the presence or absence of an unlabeled competitor at 4 °C
for 16 h. The incubation mixture was absorbed by suction onto
a nitrocellulose membrane that had been soaked in binding as-
say buffer. The membrane was washed twice with buffer
This research was supported by a Grant-in-Aid for High Tech-
nology Research Program, a Grant-in-Aid for Scientific Research
(B) (No. 16390032) and a Grant-in-Aid for Young Scientists (B)
(No. 18790089) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
(
20 mM Tris–HCl pH 8.0, 0.3 M NaCl) and then with 25% ethanol
in distilled water. Radioactivity that remained on the membrane

T. Ogawa et al. / Bioorg. Med. Chem. 17 (2009) 1109–1117
1117
K.; Kawachi, E.; Shudo, K. Chem. Pharm. Bull. 1999, 47, 585; (c) Endo, Y.;
Yaguchi, K.; Kawachi, E.; Kagechika, H. Bioorg. Med. Chem. Lett. 2000, 10, 1733;
References and notes
(
d) Endo, Y.; Iijima, T.; Yaguchi, K.; Kawachi, E.; Inoue, N.; Kagechika, H.; Kubo,
1
.
(a) Bregradze, V. I. Chem. Rev. 1992, 92, 209; (b) Valliant, J. F.; Guenther, K. J.;
King, A. S.; Morel, P.; Schaffer, P.; Sogbein, O. O.; Stephenson, K. A. Coord. Chem.
Rev. 2002, 232, 173.
A.; Itai, A. Bioorg. Med. Chem. Lett. 2001, 11, 1307; (e) Ohta, K.; Iijima, T.;
Kawachi, E.; Kagechika, H.; Endo, Y. Bioorg. Med. Chem. Lett. 2004, 14, 5913; For
androgen: (f) Fujii, S.; Hashimoto, Y.; Suzuki, T.; Ohta, S.; Endo, Y. Bioorg. Med.
Chem. Lett. 2005, 15, 227; (g) Fujii, S.; Goto, T.; Ohta, K.; Hashimoto, Y.; Suzuki,
T.; Ohta, S.; Endo, Y. J. Med. Chem. 2005, 48, 4654; (h) Goto, T.; Ohta, K.; Suzuki,
T.; Ohta, S.; Endo, Y. Bioorg. Med. Chem. 2005, 13, 6414; (i) Ohta, K.; Goto, T.;
Fujii, S.; Suzuki, T.; Ohta, S.; Endo, Y. Bioorg. Med. Chem. 2008, 16, 8022.
1. (a) Endo, Y.; Iijima, T.; Yamakoshi, Y.; Yamaguchi, M.; Fukasawa, H.; Shudo, K.
J. Med. Chem. 1999, 42, 1501; (b) Endo, Y.; Iijima, T.; Yamakoshi, Y.; Kubo, A.;
Itai, A. Bioorg. Med. Chem. Lett. 1999, 9, 3313.
2
3
.
.
Plešek, J. Chem. Rev. 1992, 92, 269.
(a) Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F.-G.; Barth, R. F.; Codogni, I.
M.; Wilson, J. G. Chem. Rev. 1998, 98, 1515; (b) Hawthorne, M. F.; Maderna, A.
Chem. Rev. 1999, 99, 3421; (c) Armstrong, A. F.; Valliant, J. F. Dalton Trans. 2007,
4
240; (d) Yamamoto, T.; Nakai, K.; Matsumura, A. Cancer Lett. 2008, 262, 143.
(a) Lesnikowski, Z. J.; Shi, J.; Schinazi, R. F. J. Organomet. Chem. 1999, 581, 156;
b) Todd, J. A.; Rendina, L. M. Inorg. Chem. 2002, 41, 3331; (c) Lesnikowski, Z. J.
1
4
.
(
Eur. J. Org. Chem. 2003, 4489; (d) Olejniczak, A. B.; Semenuc, A.; Kwiatkowski,
M.; Lesnikowski, Z. J. J. Organomet. Chem. 2003, 680, 124; (e) Tjarks, W.; Tiwari,
R.; Byun, Y.; Narayanasamy, S.; Barth, R. F. Chem. Commun. 2007, 4978.
(a) Wyzlic, I. M.; Soloway, A. H. Tetrahedron Lett. 1992, 33, 7489; (b) Kahl, S. B.;
Kasar, R. A. J. Am. Chem. Soc. 1996, 118, 1223; (c) Naeslund, C.; Ghirmal, S.;
Sjoeberg, S. Tetrahedron 2005, 61, 1181.
1
1
1
1
2. Endo, Y.; Iijima, T.; Yamakoshi, Y.; Fukasawa, H.; Miyaura, C.; Inada, M.; Kubo,
A.; Itai, A. Chem. Biol. 2001, 8, 341.
3. (a) Endo, Y.; Yoshimi, T.; Miyaura, C. Pure Appl. Chem. 2003, 75, 1197; (b) Endo,
Y.; Yoshimi, T.; Ohta, K.; Suzuki, T.; Ohta, S. J. Med. Chem. 2005, 48, 3941.
4. Ogawa, T.; Ohta, K.; Yoshimi, T.; Yamazaki, H.; Suzuki, T.; Ohta, S.; Endo, Y.
Bioorg. Med. Chem. Lett. 2006, 16, 3943.
5. (a) Figadère, B.; Norman, A. W.; Henry, H. L.; Koeffler, H. P.; Zhou, J.-Y.;
Okamura, W. H. J. Med. Chem. 1991, 34, 2452; (b) Yashima, E.; Huang, S.;
Matsushima, T.; Okamoto, Y. Macromolecules 1995, 28, 4184.
6. (a) Coult, R.; Fox, M. A.; Gill, W. R.; Herbertson, P. L.; MacBride, J. A. H.; Wade, K.
J. Organomet. Chem. 1993, 462, 19; (b) Gill, W. R.; Herbertson, P. L.; MacBride, J.
A. H.; Wade, K. J. Organomet. Chem. 1996, 507, 249.
5
.
.
6
(a) Thimon, C.; Panza, L.; Morin, C. Synlett 2003, 1399; (b) Morandi, S.; Ristori,
S.; Berti, D.; Panza, L.; Becciolini, A.; Martini, G. Biochim. Biophys. Acta 2004,
1664, 53; (c) Bonechi, C.; Ristori, S.; Martini, S.; Panza, L.; Martini, G.; Rossi, C.;
Donati, A. Biophys. Chem. 2007, 125, 320.
1
7.
8.
9.
(a) Green, S.; Walter, P.; Kumar, V.; Krust, A.; Bornert, J. M.; Argos, P.; Chambon,
P. Nature 1986, 320, 134; (b) Kuiper, G. G. M. J.; Enmark, E.; Pelto-Huikko, M.;
Nilsson, S.; Gustafsson, J. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5925.
(a) Constantine, G. D.; Pickar, J. H. Curr. Opin. Pharmacol. 2003, 3, 626; (b) Leng,
X.-H.; Bray, P. F. Drug Discovery Today 2005, 2, 85; (c) Motivala, A.; Pitt, B. Drugs
1
1
1
2
7. Endo, Y.; Yoshimi, T.; Iijima, T.; Yamakoshi, Y. Bioorg. Med. Chem. Lett. 1999, 9,
3
387.
8. Batsanov, A. S.; Fox, M. A.; Howard, J. A. K.; Wade, K. J. Organomet. Chem. 2000,
97, 157.
2
007, 67, 647.
5
Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard, R. E.; Bonn, T.; Engstrom,
O.; Ohman, L.; Green, G. L.; Gustafsson, J.; Carlquist, M. Nature 1997, 389, 753.
9. Kitamura, S.; Ohmegi, M.; Sanoh, S.; Sugihara, K.; Yoshihara, S.; Fujimoto, N.;
Ohta, S. Environ. Health Perspect. 2003, 111, 329.
0. Ohta, K.; Chiba, Y.; Ogawa, T.; Endo, Y. Bioorg. Med. Chem. Lett. 2008, 18, 5050.
1
0. For retinoid: (a) Iijima, T.; Endo, Y.; Tsuji, M.; Kawachi, E.; Kagechika, H.; Shudo,
K. Chem. Pharm. Bull. 1999, 47, 398; (b) Endo, Y.; Iijima, T.; Kagechika, H.; Ohta,