Design and synthesis of nonsteroidal progesterone receptor antagonists based on C,C′-diphenylcarborane scaffold as a hydrophobic pharmacophore

Article abstract of DOI:10.1016/j.ejmech.2014.07.034

The progesterone receptor (PR) plays important roles in multiple physiological processes, including female reproduction. Here, we report the synthesis of nonsteroidal PR antagonists containing a boron cluster as the hydrophobic core. We found that 1,7-diphenyl-meta-carborane was the preferred substructure among the three carborane isomers. Compound 39 was the most potent PR antagonist (IC₅₀: 29 nM). Compound 41 also exhibited potent activity (IC₅₀: 93 nM), and did not bind to androgen receptor, glucocorticoid receptor or mineralocorticoid receptor. These compounds may be useful for investigating potential clinical applications of PR modulators.

Full text of DOI:10.1016/j.ejmech.2014.07.034

European Journal of Medicinal Chemistry 84 (2014) 264e277
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of nonsteroidal progesterone receptor
antagonists based on C,C0-diphenylcarborane scaffold as a
hydrophobic pharmacophore
,
Shinya Fujii a b, Ayumi Yamada a, Eiichi Nakano a, Yuki Takeuchi a, Shuichi Mori a,
,
*
Hiroyuki Masuno a, Hiroyuki Kagechika a
a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
b Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The progesterone receptor (PR) plays important roles in multiple physiological processes, including fe-
male reproduction. Here, we report the synthesis of nonsteroidal PR antagonists containing a boron
cluster as the hydrophobic core. We found that 1,7-diphenyl-meta-carborane was the preferred sub-
Received 24 April 2014
Received in revised form
8 July 2014
Accepted 10 July 2014
Available online 10 July 2014
structure among the three carborane isomers. Compound 39 was the most potent PR antagonist (IC50
:
29 nM). Compound 41 also exhibited potent activity (IC50: 93 nM), and did not bind to androgen receptor,
glucocorticoid receptor or mineralocorticoid receptor. These compounds may be useful for investigating
potential clinical applications of PR modulators.
Keywords:
Carborane
© 2014 Elsevier Masson SAS. All rights reserved.
Progesterone receptor (PR)
Hydrophobic pharmacophore
Scaffold
1. Introduction
done on potential clinical applications of PR antagonists. Mife-
pristone (RU-486: 3) is a representative PR antagonist with a ste-
Progesterone receptor (PR) is a member of the nuclear receptor
superfamily of ligand-inducible transcription factors [1] and has
roles in multiple physiological processes, including female repro-
duction. The endogenous PR ligand progesterone (P4, 1) is involved
in regulation of uterine cell proliferation/differentiation, implan-
tation, ovulation, and mammary gland growth/differentiation
[2e5]. Various synthetic PR agonists have been developed and are
used clinically for contraception, hormone replacement therapy to
reduce estrogen-mediated endometrial cancer risk, and treatment
of gynecological disorders [6e8]. The PR agonists currently in
clinical use have a steroidal skeleton, but this is associated with
various side effects due to cross-binding affinity for other nuclear
steroid receptors, including androgen receptor (AR), glucocorticoid
receptor (GR), and mineralocorticoid receptor (MR). Therefore,
several nonsteroidal PR agonists have been developed in order to
minimize such side effects [9]. Tanaproget (2) is one of the most
investigated nonsteroidal PR agonists [10], and clinical application
is expected in due course. On the other hand, little work has been
roidal skeleton, and is used to induce abortion in some European
countries [11]. The results of investigation of 3 suggested that PR
antagonists might be effective not only as contraceptive agents, but
also in the treatment of endometriosis [12], uterine leiomyoma
[13], and breast cancer [14,15]. Thus, development of potent and
selective nonsteroidal PR antagonists is required for detailed
investigation of the pharmaceutical potential of PR modulation.
Several nonsteroidal PR antagonists, such as 4a and 5a, have been
developed based on the structure of 2 [16,17]. However, it was
revealed that quite small modifications of these compounds can
cause switching of agonistic/antagonistic activity of the com-
pounds. For example, modification of the carbonyl group of 4a to a
thiocarbonyl group yielded agonist 4b [18]. Modification of the
hydrophobic part of 5a to 5b also dramatically changed the activity
[17]. We recently developed coumarin derivatives, such as 6, as
novel nonsteroidal PR antagonists [19]. However, the pharmaco-
phore of these coumarin derivatives is common to that of tanap-
roget (2) and other derivatives (Fig. 1).
On the basis of these considerations, we planned to develop
novel nonsteroidal PR antagonists based on novel nonsteroidal
scaffold structures. For this purpose, we focused on carboranes,
which are icosahedral boron clusters. Carboranes (dicarba-closo-
* Corresponding author.
E-mail address: kage.chem@tmd.ac.jp (H. Kagechika).
http://dx.doi.org/10.1016/j.ejmech.2014.07.034
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
265
Fig. 1. Structures of PR endogenous ligand P4 (1) and synthesized agonists and antagonists.
dodecaboranes; Fig. 2) are polyhedral carbon-containing boron
clusters that have a bulky spherical structure [20,21]. There are
three isomeric carboranes, differing in the position of the two
carbon atoms. They all exhibit high hydrophobicity, as well as
having high thermal and chemical stability. We have already
applied the carborane cage as a hydrophobic structural moiety for
nuclear receptor ligands, and developed novel carborane-based li-
gands for vitamin D receptor (VDR) [22,23], androgen receptor (AR)
[24,25], and estrogen receptor (ER) [26,27]. These carborane-based
nonsteroidal ligands are advantageous for separation of pleiotropic
receptor functions, as well as having excellent chemical stability.
Herein, we describe the development of non-secosteroidal PR an-
tagonists based on a carborane scaffold.
phenyl group of 3. Two substituents can be introduced on the two
carbon atoms of each carborane isomer, so we investigated three
different diphenylcarborane scaffolds, namely 1,2-diphenyl-o-car-
borane, 1,7-diphenyl-m-carborane, and 1,12-diphenyl-p-carborane
structure. As a bulky substituent corresponding to the 11b-phenyl
group of 3, we chose a substituted phenyl group.
2.2. Synthesis
Scheme 1 shows the synthesis of 1,2-diphenyl-o-carborane de-
rivatives. Copper (I)-mediated Ullmann-type coupling using C-
lithiated o-carborane and 4-iodobenzonitrile gave 1-(4-
cyanophenyl)-o-carborane (10) [32], and SNAr reaction using the
sodium salt of 10 and 4-fluoronitrobenzene afforded compound 11
with the 1,2-diphenyl-o-carborane core structure. Catalytic hydro-
genation of the nitro group using Pd-carbon under a hydrogen at-
mosphere afforded amine 12. Methylation of amine 12 with
iodomethane gave monomethyl derivative 13 and dimethyl deriv-
ative 14. Acylation or sulfonylation of 12 gave the corresponding
amide or sulfonamide. Carbamate 17 was also prepared from amine
12 by using chloroformate. N-Methylated derivatives 19e22 were
prepared by methylation of 15e18, respectively.
Scheme 2 shows the synthesis of 1,7-diphenyl-m-carborane
derivatives. Ullmann-type coupling using C-lithiated m-carborane
and iodobenzene gave 1-phenyl-m-carborane 23, and further
Ullmann-type coupling using 4-iodobenzonitrile afforded com-
pound 24 bearing the 1,7-diphenyl-m-carborane core structure.
Nitration with mixed acid gave 40-nitrated product 26 as a major
product together with 30-nitrated compound 25. After separation of
the isomers, reduction of the nitro group of each compound under a
hydrogen atmosphere afforded amines 27 and 28, respectively.
Acetylation or methanesulfonylation of 30-amino derivative 27 gave
amide 29 and sulfonamide 30. N-Methylation of 29 and 30 gave 31
2. Chemistry
2.1. Molecular design
Our previous work on nuclear receptor ligands bearing a car-
borane cage as a hydrophobic pharmacophore demonstrated that
the C-phenylcarborane framework functions as an effective alter-
native structure to a steroidal skeleton [24e27]. We also estab-
lished that C-cyanophenylcarborane is a privileged structure for AR
antagonists [24], and this finding suggested that the C-cyanophe-
nylcarborane substructure might also be useful for development of
ligands for other nuclear receptors specific for 3-ketosteroids,
including PR. Therefore, we chose the C-cyanophenylcarborane
framework as a core structure for nonsteroidal PR antagonists.
In the ligand-dependent activation of nuclear receptors, ago-
nists induce a drastic conformational change of the receptors to the
active form, including folding of the helix (12th helix, H12 in PR) at
the terminal of the ligand-binding domain (LBD) of the receptors
[28,29]. Full antagonism toward nuclear receptors can be obtained
by inhibiting the folding of H12, and therefore introduction of a
bulky substituent into a ligand to inhibit formation of the active
conformation of the receptor is an effective design approach for
nuclear receptor antagonists [30]. Structurally, the steroidal PR
antagonist mifepristone (3) has an aryl substituent at the 11
sition in addition to the standard steroidal skeleton. X-ray analysis
suggested that the aryl substituent at 11 -position would disturb
b-po-
b
the proper folding of H12 of PR, and this is considered to be the
reason for the antagonistic activity of 3 toward PR [31].
Based on these considerations, we designed folding inhibitor-
type nonsteroidal PR antagonists using cyanophenylcarborane as
the core structure and an aryl group corresponding to the 11
b
-
Fig. 2. Structures of carboranes.

266
S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
Scheme 1. Synthesis of 1,2-diphenyl-o-carborane derivatives. Reagents and conditions: (a) n-BuLi, CuCl, DME, 0 ꢀC, then 4-iodobenzonitrile, pyridine, 80 ꢀC, 49%; (b) NaH, 4-
fluoronitrobenzene, DME, 80 ꢀC, 51%; (c) Pd(OH)2/C, H2, EtOH-THF, rt, 64%; (d) MeI, DMF, rt, 20% (13), 53% (14); (e) acyl chloride, solvent, 85%e98%; (f) NaH, MeI, DMF, rt, 85%-quant.
Scheme 2. Synthesis of 1,7-diphenyl-m-carborane derivatives. Reagents and conditions: (a) n-BuLi, CuCl, DME, 0 ꢀC, then iodobenzene, pyridine, 80 ꢀC, 66%; (b) n-BuLi, CuCl, DME,
0 ꢀC, then 4-iodobenzonitrile, pyridine, 80 ꢀC, 68%; (c) HNO3, H2SO4, CH2Cl2, rt, 27% (15), 69% (16); (d) (for 27) Pd/C, H2, MeOH, rt, 74%; (for 28) Pd(OH)2/C, H2, EtOH-THF, rt, 64%; (e)
acetyl chloride, pyridine, CH2Cl2, rt, 78% (for 29); methanesulfonyl chloride, pyridine, CH2Cl2, rt, 70% (for 30); (f) NaH, MeI, DMF, rt, 65% (for 31), 66% (for 32); (g) MeI, DMF, rt, 17%
(33), 22% (34); (e) acyl chloride, base, solvent, 33%e94%; (f) NaH, MeI, DMF, rt, 63%-quant.
and 32, respectively. As for the 40-substituted compounds,
methylation of amine 28 with iodomethane gave monomethyl
derivative 33 and dimethyl derivative 34. Acylation or sulfonylation
of 28 gave amides or sulfonamides (35e38), respectively. N-
Methylated derivatives of amide and sulfonamide were also pre-
pared by methylation of 39e42.
derivatives. Ullmann-type coupling using C-lithiated p-carborane
and iodobenzene gave 1-phenyl-p-carborane (43), and further
Ullmann-type coupling using 4-iodobenzonitrile afforded com-
pound 44 bearing the 1,12-diphenyl-p-carborane core structure.
Nitration with mixed acid gave 40-nitrated product 45 as a mixture
with 30-nitrated minor product (ca: 4:1). Reduction of the nitro
group of the isomeric mixture under a hydrogen atmosphere
afforded amine 46 together with the 30-amino isomer (ca: 3:1).
1,12-Diphenyl-p-carborane derivatives were also synthesized in
a similar manner to that used for preparation of m-carborane

S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
267
Table 1
Acetylation or methanesulfonylation of 46 gave acetoamide 47 or
sulfonamide 48, respectively. The 30-isomer was separated in this
step. N-Methylated acetoamide 49 and sulfonamide 50 were pre-
pared by methylation with iodomethane (Scheme 3).
PR-antagonistic activity of 1,2-diphenyl-o-carborane derivatives in T-47D alkaline
phosphatase assay.a
3. Results
3.1. PR antagonistic activity
PR-agonistic and -antagonistic activities of synthesized com-
pounds were evaluated by means of alkaline phosphatase assay
using T-47D human breast carcinoma cell line, in which alkaline
phosphatase expression is regulated by PR [33]. None of the syn-
thesized carborane derivatives exhibited activity alone (data not
shown), i.e., they did not act as PR agonists. PR-antagonistic activity
was examined in the presence of 1 nM P4 (1), and the IC50 values
were calculated (Tables 1e3).
Several 1,2-diphenyl-o-carborane derivatives exhibited moder-
ate PR-antagonistic activity (Table 1). Compound 14 bearing a
dimethylamino group, which corresponds to the substituent of
mifepristone (3), exhibited PR-antagonistic activity with the IC50
Compd
R1
R2
T-47D IC50 (m
M)a
13
14
15
16
17
18
19
20
21
22
Me
Me
H
Me
H
H
H
3.9 0.32
3.5 0.43
2.2 0.20
>10
>10
>10
>10
>10
6.5 0.43
>10
COMe
COPh
COOBn
SO2Me
COMe
COPh
COOBn
SO2Me
H
Me
Me
Me
Me
a
Expression of alkaline phosphatase was induced with 1 nM progesterone.
value of 3.5
antagonistic activity among the 1,2-diphenyl-o-carborane de-
rivatives with the IC50 value of 2.2 M. N-Methylation of amide
mM, and acetoamide 15 exhibited the most potent
m
exhibited lower affinity than that of the corresponding acetoamide
or methanesulfonamide. These results suggested that the potent
PR-antagonistic activity of acetyl and methanesulfonyl derivatives
in T-47D alkaline phosphatase assay was indeed mediated by
binding of the compound with PR.
Next, we examined the biological activity of 1,12-diphenyl-p-
carborane derivatives bearing an acetyl or a methanesulfonyl
moiety (Table 3). Secondary acetoamide 47 and sulfonamide 48 did
not exhibit PR-antagonistic activity. Tertiary amide 49 and sulfon-
amide 50 exhibited PR-antagonistic activity with submicromolar
derivatives resulted in decreased activity.
In contrast to the o-carborane derivatives, the 1,7-diphenyl-m-
carborane derivatives exhibited potent PR-antagonistic activity
(Table 2). Many of the tested m-carborane derivatives exhibited
submicromolar IC50 values. Among the 40-substituted derivatives,
amine derivatives 33 and 34 exhibited significant PR-antagonistic
activity, while amide and sulfonamide derivatives 33e38 exhibi-
ted potent activity. Secondary acetoamide 35, secondary meth-
anesulfonamide 37 exhibited potent activity, with IC50 values
around 0.1 mM. Further, N-methylation of these amide and sulfon-
IC50 values (0.56
tencies were lower than those of the corresponding 1,7-diphenyl-
m-carborane derivatives 39 and 41 (0.029
respectively).
mM and 0.65 mM, respectively), though their po-
amide derivatives increased their potency, and N-methyl-
acetoamide 39 exhibited the most potent activity with the IC50
value of 29 nM. Though the 30-substituted compounds 29e32 also
exhibited potent PR-antagonistic activity, their potency was
somewhat lower than that of the corresponding 40-substituted
compounds. We also determined binding affinity of the m-carbor-
ane derivatives using human PR-LBD and [3H]-labeled P4 (1).
Binding affinity to PR-LBD was roughly correlated with the PR
antagonistic activity evaluated by T-47D alkaline phosphatase
assay. 4-Substituted N-Acetyl and N-methanesulfonyl derivatives
35, 37, 39 and 41 exhibited potent binding affinity (binding
mM and 0.093 mM,
3.2. Binding affinity toward other steroid hormone receptors
To investigate selectivity of the synthesized PR antagonists, we
examined the binding affinity of the most potent compound 39
and related sulfonamide 41 toward other 3-ketosteroid hormone
nuclear receptors; androgen receptor (AR), glucocorticoid receptor
(GR), and mineralocorticoid receptor (MR). The binding affinities
IC50 < 0.1
mM), and benzoyl and benzenesulfonyl derivatives
Scheme 3. Synthesis of 1,12-diphenyl-p-carborane derivatives. Reagents and conditions: (a) n-BuLi, CuCl, DME, 0 ꢀC, then iodobenzene, pyridine, 80 ꢀC, 51%; (b) n-BuLi, CuCl, DME,
ꢀC, then 4-iodobenzonitrile, pyridine, 80 ꢀC, 76%; (c) HNO3, H2SO4, CH2Cl2, rt, 52%; (d) Pd(OH)2/C, H2, EtOH-THF, rt, 66%; (e) acetyl chloride, pyridine, CH2Cl2, rt, 65% (for 47);
methanesulfonyl chloride, pyridine, CH2Cl2, rt, 77% (for 48); (f) NaH, MeI, DMF, rt, 71% (for 49), 50% (for 50).
0

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S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
Table 2
4. Discussion
PR-antagonistic activity of 1,7-diphenyl-m-carborane derivatives in T-47D alkaline
phosphatase assay and PR binding affinity using hPR-LBD.
Several nonsteroidal PR antagonists that are considered to have
potential clinical utility have been developed, though all have a
similar backbone or pharmacophore. Tanaproget (2) and its de-
rivatives show potent PR ligand activities, but their ago-
nisteantagonistic activities are strongly substituent-dependent. In
this study, we designed novel nonsteroidal PR ligand candidates
based on the structure of the steroidal PR antagonist mifepristone
(3). We focused on two key structural parts of 3, that is, the
cyclohexenone ring as significant group for PR binding, and the
dimethylaminophenyl group at the 11-position as
a critical
component for antagonistic activity. We arranged two aromatic
rings corresponding to these key structures on a carborane moiety
as a hydrophobic core structure. As shown in Schemes 1e3, it is
straightforward to construct C,C0-diarylcarboranes with different
orientation and distance between two aromatic rings by using the
three isomeric carboranes. In other words, carboranes are useful
building blocks that can be used to arrange two or more sub-
stituents in well-defined, distinct spatial positions.
The PR-dependent alkaline phosphatase assay revealed that
none of the carborane derivatives examined exhibited PR-agonistic
activity, though some of them showed potent PR-antagonistic ac-
tivity. Among the three types of carborane derivatives, 1,7-
diphenyl-m-carborane derivatives exhibited significant PR-
antagonistic activity, while 1,2-diphenyl-o-carborane derivatives
and 1,12-diphenyl-p-carborane derivatives exhibited no or lower
PR antagonistic activity. These results suggested that the 1,7-
diphenyl-m-carborane core structure is a versatile scaffold for PR
full antagonists.
Compd
Substituent
Position
30
T-47D
IC50
Binding
IC50
M)b
(
m
M)a
(m
R1
R2
29
30
31
32
33
34
35
36
37
38
39
40
41
42
COMe
SO2Me
COMe
SO2Me
Me
H
H
0.098 0.018
0.27 0.018
0.049 0.003
0.34 0.031
0.28 0.047
0.61 0.077
0.067 0.008
0.20 0.019
0.13 0.038
0.43 0.12
0.067
0.079
0.11
7.1
0.34
25
0.022
6.6
0.043
0.095
0.042
2.1
Me
Me
H
Me
H
H
H
H
Me
Me
Me
Me
40
Me
COMe
COPh
SO2Me
SO2Ph
COMe
COPh
SO2Me
SO2Ph
0.029 0.005
0.24 0.062
0.093 0.034
1.3 0.36
0.087
1.4
a
Expression of alkaline phosphatase was induced with 1 nM progesterone.
The concentration of [3H]progesterone was 4 nM.
b
We previously showed that the cyanophenylcarborane moiety
can function as an alternative structure to 3-ketosteroids such as
testosterone [24], and thus we considered that the second phenyl
ring of the 1,7-diphenyl-m-carborane derivatives (i.e., the 4-
acetoaminophenyl group of 39) functionally corresponds to the
were evaluated using the receptor ligand-binding domains and
radiolabelled ligands (Table 4). Compound 39 exhibited moderate
binding affinities toward AR and GR, though these affinities were
quite low in comparison with that towards PR. In contrast to 39,
compound 41 bearing tertiary sulfonamide in place of acetoamide
of 39 did not exhibit binding affinity toward AR, GR, or MR. These
results suggest that the 1,7-diphenyl-m-carborane core structure is
a versatile scaffold for development of PR antagonists, and also
that simple structural modification by introducing substituents
might effectively enhance the PR selectivity. The potent and se-
lective nonsteroidal PR antagonists 39 and 41 are expected to be
useful in the process of developing clinical applications of PR
antagonists.
11b-phenyl group of the steroidal PR antagonist 3, serving to
disturb the proper folding of H12 of PR. Docking simulation using
co-crystal structure of PR LBD and compound 39 supported the
concept of compound design. Since the overall molecular structure
of 1,7-diphenyl-m-carborane derivatives is considerably different
from that of 3, our results suggest that the ligand-binding pocket of
PR can accept a variety of compounds having different frameworks,
at least as antagonists. Docking study suggested the difference in
location of phenyl groups of 3 and m-carborane derivative 39 that
interfere the folding of H12, and this was a possible reason why the
acetyl and methanesulfonyl derivatives were more potent than the
N,N-dimethyl derivative 34. Though the N,N-dimethyl functionality
is of relevance for the high activity of 3, structureeactivity rela-
tionship of 1,7-diphenyl-m-carborane derivatives as for phenyl
substituents could be different from that of steroidal compounds.
Compounds bearing an acetoamide group (35, 39) or a meth-
anesulfonamide group (37, 41) exhibited potent antagonistic ac-
tivity, whereas benzoyl derivatives (36, 40) or benzensulfonyl
derivatives (38, 42) exhibited comparatively weak activity. N-
Methylated compounds 37 and 41 were more potent than the
parent compounds 35 and 39, respectively. X-ray crystallographic
3.3. Docking simulation
In order to estimate the binding mode of m-carborane de-
rivatives, docking simulation using the co-crystal structure of hu-
man PR LBD with mifepristone (3) (PDB ID: 2W8Y) [31] was
performed. Fig. 4 shows the docking model of compound 39 with
PR-LBD as superimposition of co-crystal structure of 3. In the
calculated structure, cyano group of 39 interacts with Gln725 and
Arg766; these residues are interacting with 3-carbonyl group of 3.
The carborane moiety of 39 binds at hydrophobic region of the PR
LBD, where the CD ring of 3 is located. The acetamidophenyl group
analysis of the PR-LBD complex with 3 revealed that the 11b-sub-
stituent of 3 interacts with the hydrophobic pocket lined by four
of compound 39 is oriented in direction different from that of 11
b
-
hydrophobic amino acid residues (Gly722, Trp755, Met759 and
phenyl group of 3, interacting with side chain of Met909 in H12.
Formation of hydrogen-bonding between acetamide and the re-
ceptor was not observed. This study suggested that the m-carbor-
ane derivatives function as PR antagonist in the designed manner as
illustrated in Fig. 3, whereas the second phenyl group destabilize
the active folding of H12 in different manner from that of 3.
Met909), and also that slight steric interference between the 11b-
substituent and Met909 side chain could disturb the active folding
of PR H12 [31]. It is speculated that increasing the hydrophobicity
by introduction of a methyl group at the 40-substiuent of m-car-
borane derivatives increases the PR-antagonistic activity, and also
that a benzoyl or benzenesulfonyl group is too large to be

S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
269
Table 3
compatible with the slight steric interference required for antago-
nistic activity. Our present results indicate that the substituent on
the phenyl group has a critical role in binding to PR and in PR-
antagonist activity.
PR-antagonistic activity of 1,12-diphenyl-p-carborane derivatives in T47D alkaline
phosphatase assay.
5. Conclusion
We designed and synthesized three series of C,C0-diphe-
nylcarborane derivatives as candidate nonsteroidal PR antagonists.
Biological evaluation revealed that, among the three diphe-
nylcarborane frameworks, the 1,7-diphenyl-m-carborane core
structure functions as a versatile scaffold for PR antagonists.
Structural development and structureeactivity relationship studies
suggested that one of the aryl groups of 1,7-diphenyl-m-carborane
derivatives is critical for PR antagonism, serving to inhibit the active
Compd
R1
R2
T-47D IC50 (m
M)a
47
48
49
50
COMe
SO2Me
COMe
SO2Me
H
H
Me
Me
>10
>10
0.56 0.032
0.65 0.010
a
folding of PR in a manner slightly different to the 11b-substituent of
Expression of alkaline phosphatase was induced with 1 nM progesterone.
3. The most potent compound 39 exhibited potent PR-antagonistic
activity and high binding affinity for PR, and the related compound
41 also exhibited potent activity with high selectivity for PR over
AR, GR and MR. The present results are expected to contribute to
clinical development of PR antagonists.
Table 4
Binding affinity of compounds 39 and 41 toward 3-ketosteroid nuclear receptors.
Compd
PR
AR
GR
MR
IC50
(
m
M)a
IC50
(
m
M)b
IC50
(
m
M)c
IC50 (m
M)d
N.De
N.De
6. Experimental
39
41
0.042
0.087
4.5
2.9
N.De
N.De
a
b
c
6.1. Chemistry
The concentration of [3H]progesterone was 4 nM.
The concentration of [3H]DHT was 4 nM.
The concentration of [3H]dexamethasone was 1 nM.
The concentration of [3H]aldosterone was 2 nM.
No detectable binding was observed.
All reagents were purchased from SigmaeAldrich Chemical Co.,
Tokyo Kasei Kogyo Co., Wako Pure Chemical Industries, and Kanto
Chemical Co., INC. NMR spectra were recorded on Bruker AVANCE
d
e
Fig. 3. Design of novel PR antagonists based on carboranes. A) Design scheme for cyanophenylcarborane-based folding inhibitor-type PR antagonists. B) Structures of the three
designed series of C,C0-diphenylcarborane derivatives.

270
S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
NMR (400 MHz, Acetone-d6)
d
8.11 (d, 2H, J ¼ 9.1 Hz), 7.93 (d, 2H,
J ¼ 9.0 Hz), 7.86 (d, 2H, J ¼ 8.8 Hz), 7.70 (d, 2H, J ¼ 8.8 Hz), 3.41e1.72
(m, 10H); 13C NMR (125 MHz, Acetone-d6)
d
150.05, 136.62, 134.95,
133.50, 133.26, 132.63, 124.54, 118.00, 115.58, 84.85, 84.37.
6.1.1.3. 1-(4-Aminophenyl)-2-(4-cyanophenyl)-1,2-dicarba-closo-
dodecaborane (12). Compound 11 (50 mg, 0.14 mmol) was dis-
solved in ethanol (1 mL) and THF (0.5 mL), and palladium hy-
droxide on carbon (5 mg) was suspended in the solution and stirred
under hydrogen atmosphere. After 3 h, the reaction mixture was
filtered through Celite and evaporated. The crude product was
purified by silica gel column chromatography (eluent: n-hexane/
ethyl acetate 5:1) and 28.8 mg of 12 (0.09 mmol, 64%) was obtained.
The compound was recrystallized from CH2Cl2/n-hexane. Pale yel-
low needle; mp: 198.2e199.9 ꢀC; 1H NMR (400 MHz, Acetone-d6)
d
7.79 (d, 2H, J ¼ 8.7 Hz), 7.69 (d, 2H, J ¼ 8.8 Hz), 7.23 (d, 2H,
J ¼ 8.8 Hz), 6.46 (d, 2H, J ¼ 8.8 Hz), 5.11 (br s, 2H), 3.40e1.62 (m,
10H); 13C NMR (125 MHz, Acetone-d6)
151.83, 136.20, 133.06,
d
132.88, 132.73, 118.21, 118.03, 114.96, 114.20, 89.91, 85.27.
Fig. 4. Docking model of m-carborane derivative 39 with hPR-LBD by a docking pro-
gram AutoDock [34]. Superimposition of PR LBD binding to mifepristone (3) (blue) and
docking model of 39 (orange) is displayed.
6.1.1.4. 1-(4-Cyanophenyl)-2-(4-methylaminophenyl)-1,2-dicarba-
closo-dodecaborane (13) and 1-(4-cyanophenyl)-2-(4-
dimethylaminophenyl)-1,2-dicarba-closo-dodecaborane
(14).
400 or AVANCE 500 spectrometers. Chemical shifts are reported in
Under argon atmosphere, iodomethane (94 L, 1.53 mmol) was
m
ppm as
d values from tetramethylsilane. Data are reported as fol-
added to a solution of 12 (100 mg, 0.30 mmol) in DMF (3 mL) at 0 ꢀC
and stirred at 40 ꢀC. After 2 days, the solvent was removed under
reduced pressure then the residue was diluted with ethyl acetate.
The organic layer was washed with water and brine, dried with
sodium sulfate, and evaporated. The crude product was purified by
flush silica gel column chromatography (eluent: n-hexane/ethyl
acetate 5:1) and 22.0 mg of 13 (0.06 mmol, 20%) and 57.3 mg
(0.16 mmol, 53%) of 14 was obtained. The compounds were
recrystallized from CH2Cl2/n-hexane. 13; Yellow block; mp:
lows; chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q
quartet; br, broad; m, multiplet), coupling constants (Hz), integra-
tion. Mass Spectra were collected on Bruker Daltonics microTOF-
2focus or JEOL AX505H in the positive and negative ion modes.
Melting points were obtained on a Yanagimoto micro melting point
apparatus without correction. The purity of compounds was
determined by elemental analysis or HPLC analysis confirming
ꢁ95% purity (see Supporting Information).
146.3e148.0 ꢀC; 1H NMR (400 MHz, CDCl3)
d
7.55 (d, 2H, J ¼ 8.6 Hz),
6.1.1. Preparation of compounds
7.45 (d, 2H, J ¼ 8.7 Hz), 7.18 (d, 2H, J ¼ 8.9 Hz), 6.28 (d, 2H,
6.1.1.1. 1-(4-Cyanophenyl)-1,2-dicarba-closo-dodecaborane
(10).
J ¼ 8.8 Hz), 3.91 (br s, 1H), 3.20e1.79 (m, 10H), 2.76 (d, 3H,
J ¼ 5.0 Hz); 13C NMR (125 MHz, Acetone-d6)
d 152.62, 136.25,
Under argon atmosphere, n-butyllithium (1.65 mol/L in n-hexane,
13.9 mL, 22.9 mmol) was added dropwise to a solution of o-car-
borane (3.00 g, 20.8 mmol) in dimethoxyethane (20 mL) at 0 ꢀC. The
reaction mixture was stirred for 30 min, then copper (I) chloride
(2.68 g, 27.0 mmol) was added to the reaction vessel and stirred at
room temperature. After 1 h, pyridine (7 mL) and 4-iodobenzonitrile
(5.24 g, 22.9 mmol) was added and stirred at 80 ꢀC for overnight. The
reaction mixture was cooled at room temperature then diluted with
diethyl ether, filtered through Celite. The mixture was washed with
5% aqueous solution of sodium thiosulfate, 2 M hydrochloric acid,
water and brine then dried with sodium sulfate and evaporated. The
crude product was purified by flush silica gel column chromatog-
raphy (eluent: n-hexane/CH2Cl2, 2:1) and 2.49 g of 10 (10.1 mmol,
133.06, 132.81, 132.72, 118.21, 117.48, 114.94, 111.83, 90.16, 85.38,
39.84: 14; Yellow needle; mp: 181.0e182.3 ꢀC; 1H NMR (400 MHz,
CDCl3)
J ¼ 9.1 Hz), 6.37 (d, 2H, J ¼ 9.1 Hz), 3.29e1.68 (m, 10H), 2.91 (s, 6H);
13C NMR (125 MHz, Acetone-d6)
152.48, 136.23, 133.08, 132.72,
d
7.56 (d, 2H, J ¼ 8.8 Hz), 7.45 (d, 2H, J ¼ 8.8 Hz), 7.21 (d, 2H,
d
132.61, 118.20, 117.27, 114.96, 111.85, 89.88, 85.40, 39.86.
6.1.1.5. 1-(4-Acetamidophenyl)-2-(4-cyanophenyl)-1,2-dicarba-
closo-dodecaborane (15). Under argon atmosphere, acetyl chloride
(24
mL, 0.34 mmol) was added to a solution of 12 (100 mg,
0.30 mmol) in pyridine (3 mL) at 0 ꢀC and stirred at room tem-
perature. After 30 min, the reaction was quenched with water and
diluted with ethyl acetate. The organic layer was washed with 2 M
hydrochloric acid and brine, dried with sodium sulfate, and evap-
orated. The crude product was purified by silica gel column chro-
matography (eluent: n-hexane/ethyl acetate 1:1) and 108 mg of 15
(0.28 mmol, 93%) was obtained. The compound was recrystallized
from CH2Cl2/n-hexane. Yellow needle; 1H NMR (400 MHz, Acetone-
49%) was obtained. 1H NMR (400 MHz, CDCl3)
d 7.66 (d, 2H,
J ¼ 8.9 Hz), 7.61 (d, 2H, J ¼ 8.8 Hz), 3.97 (br s,1H), 2.91e1.75 (m,10H).
6.1.1.2. 1-(4-Cyanophenyl)-2-(4-nitrophenyl)-1,2-dicarba-closo-
dodecaborane (11). Under argon atmosphere, sodium hydride (60%
in oil, 25 mg, 0.61 mmol) was added to a solution of 10 (100 mg,
0.41 mmol) in DMF (4 mL) and stirred at 0 ꢀC. After 10 min, 4-
d6)
7.52 (d, 2H, J ¼ 9.4 Hz), 7.49 (d, 2H, J ¼ 9.4 Hz), 3.19e1.96 (m, 10H),
2.03 (s, 3H); 13C NMR (125 MHz, Acetone-d6)
169.31, 142.72,
d
9.26 (br s, 1H), 7.82 (d, 2H, J ¼ 8.8 Hz), 7.69 (d, 2H, J ¼ 8.8 Hz),
fluoronitrobenzene (52 mL, 0.49 mmol) was added to the reaction
mixture and stirred at 0 ꢀC. After 1 h, the reaction was quenched
with saturated aqueous solution of ammonium chloride and
diluted with ethyl acetate. The organic layer was washed with
water and brine, dried with sodium sulfate, and evaporated. The
crude product was purified by flush silica gel column chromatog-
raphy (eluent: n-hexane/ethyl acetate 10:1) and 77.5 mg of 11
(0.21 mmol, 51%) was obtained. The compound was recrystallized
from CH2Cl2/n-hexane. Yellow needle; mp: 179.2e180.0 ꢀC; 1H
d
135.64, 133.17, 132.67, 132.34, 124.77, 119.09, 118.09, 115.08, 87.34,
84.80, 24.28.
6.1.1.6. 1-(4-Benzoylaminophenyl)-2-(4-cyanophenyl)-1,2-dicarba-
closo-dodecaborane (16). Under argon atmosphere, triethylamine
(30
mL, 0.22 mmol) and benzoyl chloride (20 mL, 0.22 mmol) was
added to a solution of 12 (66.7 mg, 0.20 mmol) in CH2Cl2 (1 mL) at

S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
271
0
ꢀC and stirred at room temperature. After 30 min, the reaction
washed with water and brine, dried with sodium sulfate, and
evaporated. The crude product was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate 1:1) and 41.5 mg
of 19 (0.11 mmol, 85%) was obtained. The compound was recrys-
tallized from CH2Cl2/n-hexane. White block; mp: 187.2e188.2 ꢀC;
was quenched with water and diluted with CH2Cl2. The organic
layer was washed with 2 M hydrochloric acid and brine, dried with
sodium sulfate, and evaporated. The crude product was purified by
silica gel column chromatography (eluent: n-hexane/ethyl acetate
3:1) and 74.1 mg of 16 (0.17 mmol, 85%) was obtained. The com-
pound was recrystallized from CH2Cl2/n-hexane. White powder;
1H NMR (400 MHz, Acetone-d6)
d
7.82 (d, 2H, J ¼ 8.8 Hz), 7.69 (d, 2H,
J ¼ 8.8 Hz), 7.65 (d, 2H, J ¼ 8.8 Hz), 7.24 (d, 2H, J ¼ 8.8 Hz), 3.56e1.69
mp: 250.7e252.3 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.81 (dd, 2H,
(m, 10H), 3.12 (s, 3H), 1.69 (br s, 3H); 13C NMR (125 MHz, Acetone-
J ¼ 7.9, 1.4 Hz), 7.73 (br s, 1H), 7.57 (d, 2H, J ¼ 8.8 Hz), 7.57e7.55 (m,
1H), 7.49 (dd, 2H, J ¼ 7.8, 7.6 Hz), 7.49 (d, 2H, J ¼ 9.2 Hz), 7.47 (d, 2H,
J ¼ 8.1 Hz), 7.42 (d, 2H, J ¼ 9.0 Hz), 3.62e1.82 (m, 10H); 13C NMR
d6) d 148.02, 135.45, 133.18, 132.83, 132.68, 127.83, 118.05, 115.22,
86.22, 84.69, 54.97, 36.85, 22.42.
(125 MHz, Acetone-d6)
d
165.72, 140.15, 135.32, 134.30, 132.42,
6.1.1.10. 1-(4-Cyanophenyl)-2-{4-(N-methylbenzamido)phenyl)}-1,2-
dicarba-closo-dodecaborane (20). Under argon atmosphere, sodium
hydride (60% in oil, 5 mg, 0.12 mmol) was added to a solution of 16
(44.9 mg, 0.10 mmol) in DMF (1 mL) and stirred at 0 ꢀC. Then
132.11, 131.56, 131.37, 129.00, 127.03, 125.75, 119.33, 117.53, 114.34,
85.42, 83.09.
6.1.1.7. 1-(4-Benzyloxycarbonylaminophenyl)-2-(4-cyanophenyl)-
1,2-dicarba-closo-dodecaborane (17). Under argon atmosphere, so-
dium hydrogen carbonate (28.3 mg, 0.34 mmol) was added to a
solution of 12 (100 mg, 0.30 mmol) in THF (2 mL) then benzyl
iodomethane (30 mL, 0.51 mmol) was added to the reaction mixture
at 0 ꢀC and stirred at room temperature. After 10 min, the reaction
was quenched with saturated aqueous solution of ammonium
chloride and diluted with ethyl acetate. The organic layer was
washed with brine, dried with sodium sulfate, and evaporated. The
crude product was purified by silica gel column chromatography
(eluent: n-hexane/CH2Cl2, 3:1 to ethyl acetate only) and 46.6 mg of
20 (0.10 mmol, quant.) was obtained. The compound was recrys-
tallized from CH2Cl2/n-hexane. White plate; mp: 175.3e176.3 ꢀC;
chloroformate (48
m
L, 0.34 mmol) was added dropwise to the re-
action and stirred at 0 ꢀC. After 3 h, benzyl chloroformate (48
mL,
0.34 mmol) was added dropwise to the reaction and stirred at 0 ꢀC.
After 1 h, the reaction mixture was heated to room temperature.
After 14 h, benzyl chloroformate (excess) was added dropwise to
the reaction. After 6 h, the reaction was quenched with water and
diluted with ethyl acetate. The organic layer was washed with
brine, dried with sodium sulfate, and evaporated. The crude
product was purified by silica gel column chromatography (eluent:
n-hexane/ethyl acetate 3:1 to 4:1) and 137 mg of 17 (0.29 mmol,
98%) was obtained. The compound was recrystallized from CH2Cl2/
n-hexane. White block; mp: 173.4e173.8 ꢀC; 1H NMR (400 MHz,
1H NMR (400 MHz, Acetone-d6)
d
7.49 (d, 2H, J ¼ 8.8 Hz), 7.43 (d, 2H,
J ¼ 8.8 Hz), 7.36 (tt, 1H, J ¼ 6.8, 2.0 Hz), 7.27 (d, 2H, J ¼ 8.8 Hz),
7.14e7.01 (m. 4H), 6.85 (d, 2H, J ¼ 8.8 Hz), 3.74e1.65 (m, 10H), 3.40
(s, 3H); 13C NMR (125 MHz, Acetone-d6)
d 170.37, 148.53, 137.03,
135.38, 133.33, 132.58, 132.44, 130.45, 129.29, 128.58, 128.11, 127.72,
118.34, 115.29, 86.29, 84.76, 37.88.
Acetone-d6)
d
8.93 (br s, 1H), 7.82 (d, 2H, J ¼ 8.6 Hz), 7.69 (d, 2H,
6.1.1.11. 1-{4-(Benzyloxycarbonyl)(methyl)aminophenyl}-2-(4-
cyanophenyl)-1,2-dicarba-closo-dodecaborane (21). Under argon
atmosphere, sodium hydride (60% in oil, 13 mg, 0.36 mmol) and
J ¼ 8.6 Hz), 7.52 (d, 2H, J ¼ 8.9 Hz), 7.46 (d, 2H, J ¼ 9.0 Hz), 7.41e7.31
(m, 5H), 5.14 (s, 2H), 3.42e1.64 (m, 10H); 13C NMR (125 MHz,
Acetone-d6)
d
154.00, 142.65, 137.48, 135.73, 133.22, 132.71, 132.52,
iodomethane (68 mL, 1.12 mmol) was added to a solution of 17
129.31, 129.03, 128.98, 124.51, 118.41, 118.13, 115.16, 87.46, 84.89,
67.24.
(100 mg, 0.21 mmol) in DMF (3 mL) and stirred at 0 ꢀC. After 3 h, the
reaction was quenched with saturated aqueous solution of
ammonium chloride and diluted with ethyl acetate. The organic
layer was washed with water and brine, dried with sodium sulfate,
and evaporated. The crude product was purified by silica gel col-
umn chromatography (eluent: n-hexane/CH2Cl2, 1:1) and 111.3 mg
of 21 (0.23 mmol, quant.) was obtained. The compound was
recrystallized from CH2Cl2/n-hexane. White plate; mp:
6.1.1.8. 1-(4-Cyanophenyl)-2-(4-methanesulfonylaminophenyl)-1,2-
dicarba-closo-dodecaborane (18). Under argon atmosphere, meth-
anesulfonyl chloride (70 mg, 0.61 mmol) was added to a solution of
12 (100 mg, 0.30 mmol) in CH2Cl2 (3 mL) and pyridine (37
mL,
0.46 mmol) at 0 ꢀC then stirred at room temperature. After 4 h,
methanesulfonyl chloride (excess) was added to the reaction and
stirred. After 17 h, the reaction was quenched with water and
diluted with ethyl acetate. The organic layer was washed with 2 M
hydrochloric acid and brine, dried with sodium sulfate, and evap-
orated. The crude product was purified by silica gel column chro-
matography (eluent: n-hexane/ethyl acetate 2:1) and 113.0 mg of
18 (0.27 mmol, 90%) was obtained. The compound was recrystal-
lized from ethyl acetate/n-hexane. White powder; mp:
158.1e158.9 ꢀC; 1H NMR (400 MHz, CDCl3)
d
7.52 (d, 2H, J ¼ 8.7 Hz),
7.39 (d, 2H, J ¼ 8.7 Hz), 7.40e7.27 (m, 5H), 7.36 (d, 2H, J ¼ 9.1 Hz),
7.12 (d, 2H, J ¼ 8.8 Hz), 5.14 (s, 2H), 3.25 (s, 3H), 3.12e1.63 (m, 10H);
13C NMR (125 MHz, Acetone-d6)
d 155.15, 146.71, 137.63, 135.69,
133.20, 132.65, 131.97, 129.31, 128.87, 128.69, 127.00, 125.28, 118.10,
115.20, 86.70, 84.78, 67.97, 37.05.
6.1.1.12. 1-(4-Cyanophenyl)-2-{4-(N-methylmethylsulfonamido)
phenyl)}-1,2-dicarba-closo-dodecaborane (22). Under argon atmo-
sphere, sodium hydride (60% in oil, 7.3 mg, 0.18 mmol) and iodo-
252.7e253.7 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.56 (d, 2H, J ¼ 8.7 Hz),
7.48 (d, 2H, J ¼ 8.8 Hz), 7.39 (d, 2H, J ¼ 8.9 Hz), 6.97 (d, 2H,
J ¼ 8.9 Hz), 6.52 (br s, 1H), 3.27e1.86 (m, 10H), 3.01 (s, 3H); 13C NMR
methane (50 mL, 0.76 mmol) was added to a solution of 18 (63 mg,
(125 MHz, Acetone-d6)
d
141.93, 135.55, 133.16, 132.96, 132.61,
0.15 mmol) in DMF (2 mL) at 0 ꢀC and stirred at room temperature.
After 4 h, iodomethane (excess) was added to the reaction mixture
and stirred at room temperature. After 13 h, the reaction was
quenched with saturated aqueous solution of ammonium chloride
and diluted with ethyl acetate. The organic layer was washed with
brine, dried with sodium sulfate, and evaporated. The crude
product was purified by silica gel column chromatography (eluent:
n-hexane/ethyl acetate 2:1) and 60.9 mg of 22 (0.14 mmol, 93%)
was obtained. The compound was recrystallized from CH2Cl2/n-
hexane. White needle; mp: 196.6e197.2 ꢀC; 1H NMR (400 MHz,
125.56, 118.99, 118.05, 115.13, 86.87, 84.74.
6.1.1.9. 1-(4-Cyanophenyl)-2-{4-(N-methylacetamido)phenyl)}-1,2-
dicarba-closo-dodecaborane (19). Under argon atmosphere, sodium
hydride (60% in oil, 7 mg, 0.16 mmol) was added to a solution of 15
(50 mg, 0.13 mmol) in DMF (1.5 mL) and stirred at 0 ꢀC. Then
iodomethane (41 mL, 0.67 mmol) was added to the reaction mixture
at 0 ꢀC and stirred at room temperature. After 30 min, the reaction
was quenched with saturated aqueous solution of ammonium
chloride and diluted with ethyl acetate. The organic layer was
Acetone-d6)
d
7.81 (d, 2H, J ¼ 8.7 Hz), 7.69 (d, 2H, J ¼ 8.8 Hz), 7.60 (d,

272
S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
2H, J ¼ 8.9 Hz), 7.34 (d, 2H, J ¼ 8.9 Hz), 3.34e1.96 (m, 10H), 3.24 (s,
3H), 2.81 (s, 3H); 13C NMR (125 MHz, Acetone-d6)
143.99, 134.96,
131.96, 131.31, 131.21, 127.91, 124.13, 117.27, 114.21, 84.39, 82.86,
37.17, 35.68.
6.1.1.16. 1-(3-Aminophenyl)-7-(4-cyanophenyl)-1,7-dicarba-closo-
dodecaborane (27). Compound 25 (120 mg, 0.33 mmol) was
hydrogenerated in ethyl acetate (0.5 mL) and methanol (0.3 mL)
with 10% palladium on carbon (12 mg) under atmospheric pressure
of H2. The mixture was stirred at room temperature for 3 h. After
removal of catalyst by filtration, the filtrate was concentrated and
gave to 27 (80 mg, 74%) as white solid. The compound 27 was
recrystallized from n-hexane/CHCl3: mp: 153e155 ꢀC; 1H NMR
d
6.1.1.13. 1-Phenyl-1,7-dicarba-closo-dodecaborane
(23). Under
argon atmosphere, n-butyllithium (1.65 mol/L in n-hexane,13.8 mL,
22.9 mmol) was added dropwise to a solution of m-carborane
(3.00 g, 20.8 mmol) in dimethoxyethane (20 mL) at 0 ꢀC. The re-
action mixture was stirred for 30 min, then copper (I) chloride
(2.68 g, 27.0 mmol) was added to the reaction vessel and stirred at
room temperature. After 1 h, pyridine (7 mL) and iodobenzene
(2.0 mL, 18.4 mmol) was added and stirred at 85 ꢀC. After 3 h, the
reaction mixture was cooled at room temperature then diluted
with diethyl ether, filtered through Celite. The mixture was washed
with 5% aqueous solution of sodium thiosulfate, 2 M hydrochloric
acid, water and brine then dried with sodium sulfate and evapo-
rated. The crude product was purified by silica gel column chro-
matography (eluent: n-hexane only and n-hexane/ethyl acetate
20:1) and 3.03 g of 23 (13.7 mmol, 66%) was obtained. 1H NMR
(500 MHz, CDCl3)
d
7.58 (br s, 4H), 7.04 (t, 1H, J ¼ 8.0 Hz), 6.82 (ddd,
1H, J ¼ 7.5 Hz, 2.0 Hz, 0.5 Hz), 6.74 (t, 1H, J ¼ 2.0 Hz), 6.61 (ddd, 1H,
J ¼ 7.5 Hz, 2.0 Hz, 0.5H z), 3.5e2.0 (br m, 10H); 13C NMR(500 MHz,
CDCl3)
113.0.
d 146.5, 140.1, 135.9, 132.3, 129.5, 128.9, 118.1, 115.6, 114.6,
6.1.1.17. 1-(4-Aminophenyl)-7-(4-cyanophenyl)-1,7-dicarba-closo-
dodecaborane (28). Compound 26 (50 mg, 0.15 mmol) was dis-
solved in methanol (1.5 mL) and THF (1 mL). Palladium hydroxide
on carbon (5 mg) was suspended in the solution and stirred under
hydrogen atmosphere. After 1.5 h, the reaction mixture was filtered
through Celite and evaporated. The crude product was purified by
silica gel column chromatography (eluent: n-hexane/ethyl acetate
5:1) and 29.0 mg of 28 (0.09 mmol, 60%) was obtained. The com-
pound was recrystallized from CH2Cl2/n-hexane. Yellow plate; mp:
(400 MHz, CDCl3)
3.77e1.67 (m, 10H), 3.07 (br s, 1H).
d
7.43 (dd, 2H, J ¼ 8.2, 1.9 Hz), 7.30e7.25 (m, 3H),
160.7e162.0 ꢀC; 1H NMR (400 MHz, Acetone-d6)
(d, 2H, J ¼ 8.7 Hz), 6.59 (d, 2H, J ¼ 8.7 Hz), 4.93 (br s, 2H), 4.06e1.71
(m, 10H); 13C NMR (125 MHz, Acetone-d6)
150.34, 140.49, 133.36,
d 7.79 (s, 4H), 7.21
6.1.1.14. 1-(4-Cyanophenyl)-7-phenyl-1,7-dicarba-closo-dodecabor-
ane (24). Under argon atmosphere, n-butyllithium (1.65 mol/L in n-
hexane, 9.15 mL,15.1 mmol) was added dropwise to a solution of 23
(3.03 g, 13.7 mmol) in dimethoxyethane (14 mL) at 0 ꢀC. The re-
action mixture was stirred for 30 min, then copper (I) chloride
(1.76 g, 17.8 mmol) was added to the reaction vessel and stirred at
d
129.78, 129.32, 123.07, 118.48, 114.44, 113.77, 80.92, 77.26.
6.1.1.18. 1-(3-Acetamidophenyl)-7-(4-cyanophenyl)-1,7-dicarba-
closo-dodecaborane (29). Acetyl chloride (9
added to a solution of 27 (40 mg, 0.12 mmol) in CH2Cl2 (0.5 mL) and
pyridine (10
L, 0.13 mmol) under Ar at 0 ꢀC. The mixture was
mL, 0.13 mmol) was
room temperature. After
1
h, pyridine (5 mL) and 4-
iodobenzonitrile (3.45 g, 15.1 mmol) was added and stirred at
85 ꢀC. After 5 h, the reaction mixture was cooled at room temper-
ature then diluted with diethyl ether, filtered through Celite. The
mixture was washed with 5% aqueous solution of sodium thiosul-
fate, 2 M hydrochloric acid, water and brine then dried with sodium
sulfate and evaporated. The crude product was purified by silica gel
column chromatography (eluent: n-hexane only and n-hexane/
acetone 50:1) and flush silica gel column chromatography (eluent:
n-hexane/acetone 50:1 and 100:1) then 2.98 g of 24 (9.27 mmol,
m
stirred at room temperature for 1 h. Then the reaction was
quenched with water and extracted by CH2Cl2, The organic layer
was washed with 2 M HCl, 2 M NaOH, and brine, dried over Na2SO4,
filtered and concentrated. Purification by silica gel column chro-
matography (n-hexane/ethyl acetate, 2:1) gave 29 (35 mg, 78%) as a
yellow solid. Compound 29 was recrystallized with n-hexane and
ethyl acetate. mp: 202e204 ꢀC; 1H NMR (500 MHz, CDCl3)
s, 1H), 7. 58 (br s, 4H), 7.45 (d, 1H, J ¼ 7.5 Hz), 7.21 (m, 3H), 2.18 (s,
3H), 3.5e2.0 (br m, 10H); 13C NMR (125 MHz, CDCl3)
168.5, 140.0,
d 7.7 (br
68%) was obtained. 1H NMR (400 MHz, Acetone-d6)
7.58 (dd, 2H, J ¼ 8.0, 1.4 Hz), 7.43e7.35 (m, 3H), 3.41e2.25 (m, 10H);
13C NMR (125 MHz, CDCl3)
140.06, 134.85, 132.33, 129.11, 128.89,
d 7.81 (s, 4H),
d
138.2, 135.8, 132.4, 129.4, 129.0, 123.8, 120.4, 118.1, 113.1, 24.8.
d
128.65, 127.90, 118.08, 113.07, 78.45, 76.58.
6.1.1.19. 1-(4-Cyanophenyl)-7-(3-methylsulfonamidophenyl)-1,7-
dicarba-closo-dodecaborane (30). Methanesulfonyl chloride (10
0.13 mmol) was added to a solution of 27 (40 mg, 0.12 mmol) in
CH2Cl2 (0.5 mL) and pyridine (10
L, 0.13 mmol) under Ar at 0 ꢀC.
mL,
6.1.1.15. 1-(4-Cyanophenyl)-7-(3-nitrophenyl)-1,7-dicarba-closo-
dodecaborane (25) and 1-(4-cyanophenyl)-7-(4-nitrophenyl)-1,7-
dicarba-closo-dodecaborane (26). A mixture of nitric acid (12 mL)
and sulfuric acid (48 mL) was added dropwise to a solution of 24
(2.76 g, 8.59 mmol) in CH2Cl2 (90 mL) at 0 ꢀC and stirred at room
temperature. After 30 min, the reaction mixture was quenched
with iced water and diluted with CH2Cl2. The organic layer was
washed with saturated aqueous solution of sodium hydrogen car-
bonate and brine, dried with sodium sulfate, and evaporated. The
crude product was purified by silica gel column chromatography
(eluent: n-hexane/ethyl acetate 15:1) and 854.9 mg of 25
(2.33 mmol, 27%) and 2.17 g of 26 (5.92 mmol, 69%) were obtained.
m
The mixture was stirred at room temperature for 1.5 h. Then the
reaction was quenched with water and extracted by CH2Cl2,
washed with 2 M HCl, 2 M NaOH, and brine, dried over Na2SO4,
filtered and concentrated. Purified by silica gel column chroma-
tography (n-hexane/ethyl acetate, 2:1) gave 30 (34 mg, 70%) as a
white solid. 30 was recrystallized with n-hexane and ethyl acetate.
208e210 ꢀC; 1H NMR (500 MHz, CDCl3)
7.18 (m, 1H), 6.37 (br s, 1H), 3.03 (s, 3H), 3.5e2.0 (br m, 10H); 13C
NMR(125 MHz, CDCl3) 139.7, 137.1, 136.7, 132.4, 130.1, 128.9, 124.7,
d 7.58 (s, 4H), 7.29(m, 3H),
d
120.7, 119.9, 118.0, 113.2, 39.9.
25; 1H NMR (400 MHz, Acetone-d6)
d
8.34 (dd, 1H, J ¼ 2.0, 1.8 Hz),
8.21 (ddd, 1H, J ¼ 8.2, 2.2, 0.9 Hz), 7.79 (ddd, 1H, J ¼ 8.0, 2.0, 0.9 Hz),
7.60 (s, 4H), 7.50 (dd, 1H, J ¼ 8.1, 8.1 Hz), 3.47e1.76 (m, 10H): 26 was
recrystallized from CH2Cl2/n-hexane. 26; White powder; mp:
6.1.1.20. 1-(3-N-Methylacetamidophenyl)-7-(4-cyanophenyl)-1,7-
dicarba-closo-dodecaborane (31). NaH (8 mg, 0.21 mmol) was
added to a solution of 29 (27 mg, 0.07 mmol) in DMF (0.5 mL) under
189.1e189.6 ꢀC; 1H NMR (400 MHz, Acetone-d6)
d
8.24 (d, 2H,
J ¼ 9.1 Hz), 7.91 (d, 2H, J ¼ 9.1 Hz), 7.82 (s, 4H), 3.44e1.53 (m, 10H);
13C NMR (125 MHz, Acetone-d6)
148.42, 140.73, 138.98, 132.59,
129.38, 128.91, 123.70, 117.49, 113.24, 77.47, 77.00.
Ar at 0 ꢀC. Then iodomethane (13
mL, 0.21 mmol) was added to the
mixture and stirring was continued at room temperature for 12 h.
Then the reaction was quenched with water and extracted by ethyl
acetate, washed with aqueous NH4Cl, and brine, dried over Na2SO4,
d

S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
273
filtered and concentrated. Purification by silica gel column chro-
matography (n-hexane/ethyl acetate, 2:1) gave 31 (18 mg, 65%) as a
benzoyl chloride (23
(60 mg, 0.18 mmol) in pyridine (2 mL) at 0 ꢀC and stirred at room
temperature. After 2 h, benzoyl chloride (23 L, 0.20 mmol) and
mL, 0.20 mmol) was added to a solution of 28
white solid. mp: 152e155 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.59 (s,
7.43 (br d,1H, J ¼ 7.5 Hz), 7. 35(t,1H, J ¼ 8.0 Hz), 7.27 (br s,1H),
7.17 (br d, J ¼ 8.0 Hz, 1H), 3.25 (s, 3H), 3.2e2.0 (br m, 10H), 1.85(s,
3H); 13C NMR (125 MHz, CDCl3)
170.0, 144.9, 139.7, 136.7, 132.4,
m
4H),
d
CH2Cl2 (6 mL) was added to the reaction mixture and stirred at
room temperature. After 14 h, the reaction was quenched with
water and diluted with CH2Cl2. The organic layer was washed with
2 M hydrochloric acid and brine, dried with sodium sulfate, and
evaporated. The crude product was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate 2:1) and 76.3 mg
of 36 (0.17 mmol, 94%) was obtained. The compound was recrys-
tallized from CH2Cl2/n-hexane. White plate; mp: 200.8e201.5 ꢀC;
d
130.0, 128.9, 127.6, 127.1, 126.8, 118.0, 113.2, 37.3, 22.6.
6.1.1.21. 1-(3-N-Methylmethylsulfonamidophenyl)-7-(4-
cyanophenyl)-1,7-dicarba-closo-dodecaborane (32). NaH (7 mg,
0.18 mmol) was added to a solution of 30 (25 mg, 0.06 mmol) in
DMF (0.3 mL) under Ar at 0 ꢀC, then iodomethane (11
mL,
1H NMR (400 MHz, Acetone-d6)
J ¼ 8.5 Hz), 7.86 (d, 2H, J ¼ 8.8 Hz), 7.82 (s, 4H), 7.59e7.50 (m, 5H),
3.39e1.58 (m, 10H); 13C NMR (125 MHz, Acetone-d6)
166.50,
d 9.67 (br s, 1H), 7.98 (d, 2H,
0.18 mmol) was added to the mixture and stirring was continued at
room temperature for 4 h. The reaction was quenched with water
and extracted by CHCl3, washed with aqueous NH4Cl and brine,
dried over Na2SO4, filtered and concentrated. Purification by silica
gel column chromatography (n-hexane/ethyl acetate, 4:1) gave 32
(17 mg, 66%) as a yellow solid. 32 was recrystallized with n-hexane
d
141.38,140.34,135.95, 133.44,132.58,130.45,129.85,129.34,129.09,
128.39, 120.68, 118.48, 113.97, 79.79, 77.79.
6.1.1.25. 1-(4-Cyanophenyl)-7-(4-methanesulfonylaminophenyl)-1,7-
dicarba-closo-dodecaborane (37). Under argon atmosphere, meth-
anesulfonyl chloride (70 mg, 0.61 mmol) was added to a solution of
and ethyl acetate. 1H NMR (500 MHz, CDCl3)
1H), 7.38 (m, 1H), 7.31 (m, 2H), 3.32 (s, 3H), 2.83 (s, 3H), 3.2e2.0 (br
m, 10H); 13C NMR (125 MHz, CDCl3)
141.8, 139.8, 136.1, 132.4,
d 7.58 (s, 4H), 7.49(br s,
d
28 (100 mg, 0.30 mmol) in CH2Cl2 (3 mL) and pyridine (37 mL,
129.5, 128.9, 126.6, 126.3, 126.0, 118.0, 113.2, 38.3, 35.5.
0.46 mmol) at 0 ꢀC then stirred at room temperature. After 1 h, the
reaction was quenched with water and diluted with ethyl acetate.
The organic layer was washed with 2 M hydrochloric acid and
brine, dried with sodium sulfate, and evaporated. The crude
product was purified by silica gel column chromatography (eluent:
n-hexane/ethyl acetate 2:1). The crude product was washed with
saturated aqueous solution of sodium hydrogen carbonate, dried
with sodium sulfate, and evaporated. 103.5 mg of 37 (0.25 mmol,
83%) was obtained. Yellow needle; mp: 238.0e239.0 ꢀC; 1H NMR
6.1.1.22. 1-(4-Cyanophenyl)-7-(4-methylaminophenyl)-1,7-dicarba-
closo-dodecaborane (33) and 1-(4-cyanophenyl)-7-(4-
dimethylaminophenyl)-1,7-dicarba-closo-dodecaborane
(34).
Under argon atmosphere, iodomethane (56 L, 0.92 mmol) was
m
added to a solution of 28 (60 mg, 0.18 mmol) in DMF (2 mL) at 0 ꢀC
and stirred at room temperature. After 2 days, the reaction mixture
was quenched with water and diluted with ethyl acetate. The
organic layer was washed with brine, dried with sodium sulfate,
and evaporated. The crude product was purified by silica gel col-
umn chromatography (eluent: n-hexane/ethyl acetate 20:1) and
12.2 mg of 33 (0.03 mmol, 17%) and 16.3 mg (0.04 mmol, 22%) of 34
was obtained. The compounds were recrystallized from CH2Cl2/n-
hexane. 33; White solid; mp: 143.9e145.0 ꢀC; 1H NMR (400 MHz,
(400 MHz, Acetone-d6)
J ¼ 8.9 Hz), 7.30 (d, 2H, J ¼ 8.9 Hz), 3.40e1.42 (m, 10H), 3.04 (s, 3H);
13C NMR (125 MHz, Acetone-d6)
140.52, 140.22, 133.40, 130.75,
d 8.80 (br s, 1H), 7.81 (s, 4H), 7.55 (d, 2H,
d
129.77, 129.75, 119.90, 118.43, 113.95, 79.38, 77.81, 60.51, 39.83.
6.1.1.26. 1-(4-Benzenesulfonamidophenyl)-7-(4-cyanophenyl)-1,7-
Acetone-d6)
d
7.79 (s, 4H), 7.26 (d, 2H, J ¼ 8.8 Hz), 6.52 (d, 2H,
dicarba-closo-dodecaborane (38). Under argon atmosphere, benze-
J ¼ 8.8 Hz), 5.27 (br s, 1H), 4.08e1.61 (m, 10H), 2.77 (d, 3H,
nesulfonyl chloride (20
mL, 0.23 mmol) was added to a solution of 28
J ¼ 5.2 Hz); 13C NMR (125 MHz, Acetone-d6)
d
151.54,140.58,133.38,
(60 mg, 0.18 mmol) in CH2Cl2 (2 mL) and triethylamine (50 m
L,
129.82, 129.31, 122.63, 118.50, 113.85, 112.18, 81.08, 77.34, 60.53;
Anal. Calcd. for C16H22B10N2$1/4H2O: C, 54.14; H, 6.39; N, 7.89.
Found C, 54.19; H, 6.28, N; 7.90: 34; Yellow needle; Mp:
0.37 mmol) at 0 ꢀC then stirred at room temperature. After 12 h,
benzenesulfonyl chloride (excess) was added to the reaction and
stirred at room temperature. After 25 h, the reaction was quenched
with water and diluted with ethyl acetate. The organic layer was
washed with 2 M hydrochloric acid and brine, dried with sodium
sulfate, and evaporated. The crude product was purified by silica gel
column chromatography (eluent: n-hexane/CH2Cl2 5:1) and
30.8 mg of 38 (0.06 mmol, 33%) was obtained. Colorless block;
202.2e203.2 ꢀC; 1H NMR (400 MHz, CDCl3)
d
7.79 (s, 4H), 7.33 (d,
2H, J ¼ 9.1 Hz), 6.65 (d, 2H, J ¼ 9.1 Hz), 3.20e1.47 (m, 10H), 2.95 (s,
6H); 13C NMR (125 MHz, Acetone-d6)
151.80, 140.55, 133.39,
d
129.82, 129.19, 122.59, 118.49, 113.87, 112.49, 80.43, 77.40, 40.21.
6.1.1.23. 1-(4-Acetamidophenyl)-7-(4-cyanophenyl)-1,7-dicarba-
mp:195.0e196.0 ꢀC; 1H NMR (400 MHz, CDCl3)
d 7.80 (d, 2H,
closo-dodecaborane (35). Under argon atmosphere, acetyl chloride
J ¼ 8.7 Hz), 7.58 (m, 5H), 7.48 (t, 2H, J ¼ 7.5 Hz), 7.32 (d, 2H, J ¼ 7.5 Hz),
(14
mL, 0.20 mmol) was added to a solution of 28 (60 mg,
6.95 (d, 2H, J ¼ 8.9 Hz), 6.52 (s, 1H), 3.2e1.7 (br m, 10H); 13C NMR
0.18 mmol) in pyridine (2 mL) at 0 ꢀC and stirred at room tem-
perature. After 10 min, the reaction was quenched with water and
diluted with ethyl acetate. The organic layer was washed with 2 M
hydrochloric acid and brine, dried with sodium sulfate, and evap-
orated. The crude product was purified by silica gel column chro-
matography (eluent: n-hexane/ethyl acetate 1:1) and 59.8 mg of 35
(0.16 mmol, 89%) was obtained. The compound was recrystallized
from CH2Cl2/n-hexane. White plate; mp: 211.5e212.0 ꢀC; 1H NMR
(125 MHz, CDCl3) d 139.88, 139.13, 137.44, 133.49, 132.34, 131.46,
129.39, 129.00, 128.84, 127.29, 120.20, 118.05, 113.08, 78.01, 76.65.
6.1.1.27. 1-(4-Cyanophenyl)-7-{4-(N-methylacetamido)phenyl)}-1,7-
dicarba-closo-dodecaborane (39). Under argon atmosphere, sodium
hydride (60% in oil, 3.6 mg, 0.09 mmol) was added to a solution of
35 (29.1 mg, 0.08 mmol) in DMF (1 mL) and stirred at 0 ꢀC. Then
iodomethane (24 mL, 0.39 mmol) was added to the reaction mixture
(400 MHz, Acetone-d6)
J ¼ 8.9 Hz), 7.30 (d, 2H, J ¼ 8.9 Hz), 3.32e1.50 (m, 10H), 3.04 (s, 3H);
13C NMR (125 MHz, Acetone-d6)
169.17, 141.36, 140.25, 133.41,
d
8.80 (br s, 1H), 7.81 (s, 4H), 7.55 (d, 2H,
at 0 ꢀC and stirred at room temperature. After 5 h, the reaction was
quenched with saturated aqueous solution of ammonium chloride
and diluted with ethyl acetate. The organic layer was washed with
water and brine, dried with sodium sulfate, and evaporated. The
crude product was purified by silica gel column chromatography
and 20.1 mg of 39 (0.05 mmol, 63%) was obtained. The compound
was recrystallized from CH2Cl2/n-hexane. White powder; mp:
d
129.78, 129.04, 119.56, 118.47, 118.46, 113.88, 79.72, 77.66, 24.30.
6.1.1.24. 1-(4-Benzoylaminophenyl)-27-(4-cyanophenyl)-1,7-
dicarba-closo-dodecaborane (36). Under argon atmosphere,

274
S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
162.1e162.8 ꢀC; 1H NMR (400 MHz, Acetone-d6)
(d, 2H, J ¼ 8.4 Hz), 7.34 (d, 2H, J ¼ 8.7 Hz), 3.42e1.65 (m, 10H), 3.20
(br s, 3H), 1.82 (br s, 3H); 13C NMR (125 MHz, Acetone-d6)
169.43,
146.68, 140.07, 134.17, 133.42, 129.77, 128.20, 118.42, 113.94, 78.99,
77.91, 36.86, 36.77, 22.50.
d
7.81 (s, 4H), 7.64
carborane (0.60 g, 4.16 mmol) in dimethoxyethane (3.6 mL) at 0 ꢀC.
The reaction mixture was warmed to room temperature for 30 min,
then copper (I) chloride (535.1 mg, 5.40 mmol) was added to the
reaction vessel. After the reaction mixture was stirred for 1 h,
pyridine (2.3 mL) and iodobenzene (50 mL, 14.60 mmol) was added
and stirred at 80 ꢀC. After 3 h, the reaction mixture was cooled at
room temperature then diluted with diethyl ether, filtered through
Celite. The mixture was washed with 5% aqueous solution of so-
dium thiosulfate, 2 M hydrochloric acid, water and brine, dried with
sodium sulfate and evaporated. 794 mg of 43 (mixture with 29% of
iodobenzene and 8% of p-carborane, 2.12 mmol, 51% yield) was
obtained. The crude product was recrystallized from n-hexane. 1H
d
6.1.1.28. 1-(4-Cyanophenyl)-7-{4-(N-methylbenzamido)phenyl)}-
1,7-dicarba-closo-dodecaborane (40). Under argon atmosphere,
sodium hydride (60% in oil, 5 mg, 0.13 mmol) was added to a so-
lution of 36 (41 mg, 0.09 mmol) in DMF (1 mL) and stirred at 0 ꢀC.
Then iodomethane (29 mL, 0.47 mmol) was added to the reaction
mixture at 0 ꢀC and stirred at room temperature. After 1 h, the
reaction was quenched with saturated aqueous solution of
ammonium chloride and diluted with ethyl acetate. The organic
layer was washed with water and brine, dried with sodium sulfate,
and evaporated. The crude product was purified by silica gel col-
umn chromatography (eluent: n-hexane/ethyl acetate 1:1) and
49.5 mg of 40 (0.11 mmol, quant.) was obtained. mp:
NMR (400 MHz, CDCl3)
(br s, 1H).
d 7.24e7.15 (m, 5H), 3.14e1.65 (m,10H), 2.90
6.1.1.32. 1-(4-Cyanophenyl)-12-phenyl-1,12-dicarba-closo-dodeca-
borane (44). Under argon atmosphere, n-butyllithium (1.65 mol/L
in n-hexane, 6.30 mL, 10.34 mmol) was added dropwise to a solu-
tion of 43 (included 29% of iodobenzene and 8% of p-carborane,
1.47 g, 4.18 mmol) in dimethoxyethane (4.3 mL) at 0 ꢀC. The reac-
tion mixture was warmed to room temperature for 30 min, then
copper (I) chloride (1.03 g, 10.44 mmol) was added to the reaction
vessel. After the reaction mixture was stirred for 1 h, pyridine
(1.5 mL) and 4-iodobenzonitrile (2.87 g, 12.54 mmol) was added
and stirred at 80 ꢀC. After 15 h, the reaction mixture was cooled at
room temperature then diluted with diethyl ether, filtered through
Celite. The mixture was washed with 5% aqueous solution of so-
dium thiosulfate, 2 M hydrochloric acid, water and brine, dried with
sodium sulfate and evaporated. The crude product was purified by
flush silica gel column chromatography (eluent: n-hexane/ethyl
acetate 50:1) and 2.62 g of 44 (included 61% of p-iodobenzonitrile,
3.18 mmol, 76% yield) was obtained. The product was purified by
preparative thin-layer chromatography (eluent: n-hexane/ethyl
acetate 20:1) and recrystallized from n-hexane. 1H NMR (400 MHz,
175.3e176.3 ꢀC; 1H NMR (400 MHz, Acetone-d6)
d 7.79 (s, 4H), 7.46
(d, 2H, J ¼ 8.8 Hz), 7.31 (tt, 1H, J ¼ 7.5, 1.4 Hz), 7.30 (d, 2H, J ¼ 7.0 Hz),
7.23 (dd, 2H, J ¼ 7.8, 7.4 Hz), 7.16 (d, 2H, J ¼ 8.8 Hz), 4.19e1.50 (m,
10H), 3.41 (s, 3H); 13C NMR (125 MHz, Acetone-d6)
d 170.44, 147.05,
140.15, 137.27, 133.42, 133.03, 130.50, 129.80, 129.39, 129.30, 128.61,
127.74, 118.43, 114.00, 79.11, 77.89, 38.18.
6.1.1.29. 1-(4-Cyanophenyl)-7-(4-N-methylmethylsulfonamidophenyl)-
1,7-dicarba-closo-dodecaborane (41). Under argon atmosphere, so-
dium hydride (60% in oil, 5 mg, 0.13 mmol) was added to a solution of
37 (50 mg, 0.11 mmol) in DMF (1 mL) and stirred at 0 ꢀC. Then
iodomethane (32 mL, 0.53 mmol) was added to the reaction mixture
at 0 ꢀC and stirred at room temperature. After 10 min, the reaction
was quenched with saturated aqueous solution of ammonium chlo-
ride and diluted with ethyl acetate. The organic layer was washed
with water and brine, dried with sodium sulfate, and evaporated. The
crude product was purified by silica gel column chromatography
(eluent: n-hexane/CH2Cl2 1:1) and 44.7 mg of 41 (0.10 mmol, 91%)
was obtained. The compound was recrystallized from CH2Cl2/n-
hexane. White needle; mp: 171.1e171.6 ꢀC; 1H NMR (400 MHz,
CDCl3)
d
7.50 (d, 2H, J ¼ 8.8 Hz), 7.36 (d, 2H, J ¼ 8.8 Hz), 7.25e7.17
(m, 5H), 3.24e2.06 (m, 10H).
6.1.1.33. 1-(4-Cyanophenyl)-12-(4-nitrophenyl)-1,12-dicarba-closo-
dodecaborane (45). A mixture of nitric acid (0.4 mL) and sulfuric
acid (1.6 mL) was added dropwise to a solution of 44 (included 61%
of 4-iodobenzonitrile, 250.9 mg, 0.31 mmol) in CH2Cl2 (3 mL) and
stirred at 0 ꢀC. After 1 h, the reaction mixture was quenched with
iced water and diluted with CH2Cl2. The organic layer was washed
with saturated aqueous solution of sodium hydrogen carbonate,
water and brine, dried with sodium sulfate, and evaporated. The
crude product was purified by flush silica gel column chromatog-
raphy (eluent: n-hexane/ethyl acetate 50:1 to 25:1) and 79.1 mg of
45 (mixture with 23% of 3-nitrated isomer (0.05 mmol, 16%),
0.16 mmol, 52% yield). The compound was recrystallized from
CH2Cl2/n-hexane. 45; White block; 1H NMR (400 MHz, CDCl3)
Acetone-d6)
d
7.81 (s, 4H), 7.60 (d, 2H, J ¼ 8.9 Hz), 7.45 (d, 2H,
J ¼ 8.8 Hz), 3.48e1.54 (m, 10H), 3.33 (s, 3H), 2.91 (s, 3H); 13C NMR
(125 MHz, Acetone-d6) d 143.94,140.10,133.43,129.78,129.31,126.54,
118.43, 113.94, 79.00, 77.91, 37.85, 35.43.
6.1.1.30. 1-(4-Cyanophenyl)-7-(4-N-methylbenzenesulfonamido-
phenyl)-1,7-dicarba-closo-dodecaborane (42). Sodium hydride (60%
in oil, 3 mg, 0.08 mmol) was washed with n-hexane and added DMF
(1 mL). The reaction vessel was displaced argon atmosphere then
added 38 (30 mg, 0.06 mmol) in DMF (1 mL) and iodomethane
(19 m
L, 0.32 mmol) at 0 ꢀC and stirred at room temperature. After
1 h, the reaction was quenched with saturated aqueous solution of
ammonium chloride and diluted with ethyl acetate. The organic
layer was washed with brine, dried with sodium sulfate, and
evaporated. The crude product was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate 4:1) and 22.7 mg
of 42 (0.05 mmol, 83%) was obtained. The compound was recrys-
tallized from CH2Cl2/n-hexane. White powder; mp: 59.1e60.2 ꢀC;
d
8.06 (d, 2H, J ¼ 9.1 Hz), 7.51 (d, 2H, J ¼ 8.8 Hz), 7.41 (d, 2H,
J ¼ 9.1 Hz), 7.35 (d, 2H, J ¼ 8.8 Hz), 3.28e2.05 (m, 10H).
6.1.1.34. 1-(4-Aminophenyl)-12-(4-cyanophenyl)-1,12-dicarba-closo-
dodecaborane (46). Compound 45 (mixture with 13% of 3-isomer,
285.8 mg, 0.68 mmol) was dissolved in ethyl acetate (16 mL).
Palladium on carbon (34.0 mg) was suspended in the solution and
stirred under hydrogen atmosphere. After 4 h, the reaction mixture
was filtered through Celite and evaporated. The crude product was
purified by silica gel column chromatography (eluent: n-hexane/
ethyl acetate 5:1) and 161.9 mg of 46 (mixture with 30% of 3-NH2
derivatives, 0.45 mmol, 66%) was obtained. 1H NMR (400 MHz,
1H NMR (400 MHz, Acetone-d6)
d 7.81 (s, 4H), 7.22e7.68 (m, 1H),
7.60e7.56 (m, 4H), 7.54 (d, 2H, J ¼ 8.8 Hz), 7.17 (d, 2H, J ¼ 8.9 Hz),
3.37e1.58 (m, 10H), 3.20 (s, 3H); 13C NMR (125 MHz, Acetone-d6)
d
143.55, 140.10, 137.37, 134.05, 133.80, 133.44, 129.95, 129.79,
129.12, 128.42, 126.86, 118.43, 113.95, 79.00, 77.91, 38.07.
6.1.1.31. 1-Phenyl-1,12-dicarba-closo-dodecaborane
(43). Under
argon atmosphere, n-butyllithium (1.65 mol/L in n-hexane,
2.77 mL, 4.57 mmol) was added dropwise to a solution of p-
CDCl3)
d
7.49 (d, 2H, J ¼ 8.8 Hz), 7.35 (d, 2H, J ¼ 8.7 Hz), 6.99 (d, 2H,
J ¼ 8.7 Hz), 6.46 (d, 2H, J ¼ 8.7 Hz), 3.68 (s, 2H), 3.5e1.5 (m, 10H).

S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
275
6.1.1.35. 1-(4-Acetamidophenyl)-12-(4-cyanophenyl)-1,12-dicarba-
closo-dodecaborane (47). Acetyl chloride (300 L, 4.2 mmol) was
compound was recrystallized from CH2Cl2/n-hexane. Colorless
plate; 1H NMR (400 MHz, Acetone-d6)
m
d
7.72 (d, 2H, J ¼ 8.8 Hz), 7.53
added to a solution of 46 (mixture with 30% of 3-NH2 derivatives,
56.7 mg, 0.17 mmol) in CH2Cl2 (3 mL) and pyridine (2 mL) at 0 ꢀC
and stirred at room temperature. After 1 h, the reaction was
quenched with water and diluted with CH2Cl2. The organic layer
was washed with 5% aqueous solution of potassium hydrogen
sulfate, saturated aqueous solution of sodium hydrogen carbonate,
water and brine, dried with sodium sulfate, and evaporated. The
crude product was purified by silica gel column chromatography
(eluent: n-hexane/ethyl acetate 3:1, 1:1 to ethyl acetate only) and
42.4 mg of 47 (0.11 mmol, 65%) was obtained. The compound was
washed with n-hexane, CH2Cl2 and methanol and recrystallized
from ethyl acetate. White powder; 1H NMR (400 MHz, CDCl3)
(d, 2H, J ¼ 8.8 Hz), 7.37 (d, 2H, J ¼ 9.1 Hz), 7.32 (d, 2H, J ¼ 9.1 Hz),
3.29 (s, 3H), 2.88 (s, 3H), 3.41e1.95 (m, 10H); HRMS(ESIþ) Calcd. for
C
17H25B10N2O2S [MþH]þ: 431.2567. Found 431.2362.
6.2. Biological evaluation
6.2.1. Alkaline phosphatase assay
T-47D breast-carcinoma cells were cultured in RPMI 1640 me-
dium with 10% (v/v) fetal bovine serum and Penicillin-Streptomycin
Mixed Solution. Cells were plated in 96-well plates at 1 ꢃ 104 cell/
well and incubated overnight (37 ꢀC, 5% CO2 in air). After 24 h, cells
were treated with fresh medium containing test compound in the
presence or absence of progesterone (1 nM), and further incubated
for 24 h. The medium was aspirated and the cells were fixed with
d
10.02 (br s,1H), 7.76 (d, 2H, J ¼ 8.7 Hz), 7.46 (d, 2H, J ¼ 8.8 Hz), 7.44
(d, 2H, J ¼ 8.7 Hz), 7.16 (d, 2H, J ¼ 8.9 Hz), 2.01 (s, 3H), 3.5e1.5 (m,
10H); HRMS(ESIꢂ) Calcd. for C17H21B10N2O [MꢂH]ꢂ: 379.2584.
Found 379.2588.
100
mL of 1.8% formalin (in PBS). The fixed cells were washed with
PBS and 75
mL of assay buffer (1 mg/mL p-nitrophenol phosphate in
diethanolamine water solution, pH 9.0, 2 mM MgCl2) was added.
The mixture was incubated at room temperature with shielding
from light for 2 h, then the reaction was terminated by the addition
6.1.1.36. 1-(4-Cyanophenyl)-12-(4-methylsulfonamidophenyl)-1,12-
dicarba-closo-dodecaborane (48). Methanesulfonyl chloride (40 mL,
0.52 mmol) was added to a solution of 46 (mixture with 30% of 3-
of 100 mL of NaOH per each well. The absorbance at 405 nm was
NH2 derivatives 81.0 mg, 0.17 mmol) in CH2Cl2 (3.5 mL) and pyri-
measured. All experiments were performed in triplicate or more.
dine (30 m
L) at 0 ꢀC and stirred at room temperature. After 2.5 h, the
reaction was quenched with water and diluted with CH2Cl2. The
organic layer was washed with 5% aqueous solution of potassium
hydrogen sulfate, saturated aqueous solution of sodium hydrogen
carbonate, water and brine, dried with sodium sulfate, and evap-
orated. The crude product was washed with ethyl acetate and
58.1 mg of 48 (0.14 mmol, 77%) was obtained. The compound was
recrystallized from chloroform. White powder; 1H NMR (400 MHz,
6.2.2. hPR binding assay
hPR-binding assay was performed using recombinant hPR-LBD
purchased from Invitrogen. hPR-LBD was diluted with buffer
(20 mM TriseHCl, 300 mM NaCl, 1 mM EDTA, 5 mM DTT, pH 8.0) to
5 nM and 300 m
L aliquots were incubated in the dark at 4 ꢀC with
4 nM [1,2,6,7-3H]progesterone (Perkin Elmer) and reference or test
compounds (dissolved in DMSO; final concentration of DMSO was
3%). Nonspecific binding was assessed by addition of a 200-fold
CDCl3)
d
7.50 (d, 2H, J ¼ 8.7 Hz), 7.35 (d, 2H, J ¼ 8.7 Hz), 7.21 (d, 2H,
J ¼ 8.8 Hz), 7.02 (d, 2H, J ¼ 8.8 Hz), 6.26 (br s, 1H), 3.01 (s, 3H),
3.5e1.5 (m, 10H); HRMS(ESI-) Calcd. for C16H21B10N2O2S [MꢂH]ꢂ:
415.2554. Found 415.2263.
excess of nonradioactive progesterone. After 24 h, 30
Dextran T-70/ -globulin-coated charcoal suspension was added to
the ligand/protein mixtures (1% activated charcoal, 0.05% -glob-
mL of
g
g
ulin, 0.05% Dextran 70, final concentrations) and incubated at 4 ꢀC
for 5 min. The charcoal was removed by centrifugation for 5 min at
1300 g, and the radioactivity of the supernatant was measured in
Ultima Gold scintillation cocktail (Perkin Elmer) by using a liquid
scintillation counter. All experiments were performed in duplicate.
6.1.1.37. 1-(4-Cyanophenyl)-12-(4-N-methylacetamidophenyl)-1,12-
dicarba-closo-dodecaborane (49). Sodium hydride (60% in oil,
12.3 mg, 0.31 mmol) was added to a solution of 47 (65.8 mg,
0.17 mmol) in DMF (4.0 mL) and stirred at 0 ꢀC. After 15 min,
iodomethane (140
mL, 2.94 mmol) was added to the reaction
mixture and stirred at room temperature. After 1.5 h, the reaction
was quenched with saturated aqueous solution of ammonium
chloride and diluted with ethyl acetate. The organic layer was
washed with water and brine, dried with sodium sulfate, and
evaporated. The crude product was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate 2:1) and 46.6 mg
of 49 (0.12 mmol, 71%) was obtained. The compound was purified
by silica gel column chromatography (eluent: n-hexane/ethyl ace-
tate 2:1) and recrystallized from CH2Cl2/n-hexane. White powder;
6.2.3. hAR binding assay
A hAR-LBD expression plasmid vector which encodes GST-
hARLBD (627-919 aa, EF domain) fusion protein under the lac
promoter was transfected into Escherichia coli strain HB-101. An
overnight culture (10 mL) of the bacteria was added to 1 L of LB
medium and incubated at 27 ꢀC until the optical density at 600 nm
reached 0.6e0.7. Following the addition of IPTG to a concentration
of 1 mM, incubation was continued for an additional 4.5 h. Cells
were harvested by centrifugation at 4000 g at 4 ꢀC for 15 min and
stored at ꢂ80 ꢀC until use. All subsequent operations were per-
formed at 4 ꢀC. The bacterial pellet obtained from 40 mL of culture
was resuspended in 1 mL of ice-cold TEGDM buffer (10 mM
TriseHCl pH 7.4, 1 mM EDTA, 10% glycerol, 10 mM DTT, 10 mM
sodium molybdate). The suspension was subjected to sonication
using 10 ꢃ 10 s bursts on ice, and crude GST-hARLBD fraction was
prepared by centrifugation of the suspension at 12,000 g for 30 min
at 4 ꢀC. The crude receptor fraction was diluted with buffer (20 mM
TriseHCl pH 8.0, 0.3 M KCl, 1 mM EDTA) to a protein concentration
of 0.3e0.5 mg/mL and used in binding assays as hAR-LBD fraction.
Aliquots of the hAR-LBD fraction were incubated in the dark at 4 ꢀC
with [3H]DHT (Perkin Elmer, 4 nM final concentration) and refer-
ence or test compounds (dissolved in DMSO, final concentration of
DMSO was 2%). Nonspecific binding was assessed by addition of a
1H NMR (400 MHz, Acetone-d6)
d
7.72 (d, 2H, J ¼ 8.8 Hz), 7.54 (d, 2H,
J ¼ 8.8 Hz), 7.37 (d, 2H, J ¼ 8.6 Hz), 7.25 (d, 2H, J ¼ 8.8 Hz), 3.26e1.99
(m, 10H), 3.16 (br s, 3H), 1.79 (br s, 3H); HRMS(ESIþ) Calcd. for
C
18H25B10N2O [MþH]þ: 395.2897. Found 395.2892.
6 . 1 . 1 . 3 8 . 1 - ( 4 - C y a n o p h e n y l ) - 1 2 - ( 4 - N - m e t h y l -
methylsulfonamidophenyl)-1,12-dicarba-closo-dodecaborane (50).
Sodium hydride (60% in oil, 2.5 mg, 0.06 mmol) was added to a
solution of 48 (15.1 mg, 0.04 mmol) in DMF (0.5 mL) and stirred at
0
ꢀC. After 15 min, iodomethane (40
mL, 0.84 mmol) was added to
the reaction mixture and stirred at room temperature. After 2.5 h,
the reaction was quenched with saturated aqueous solution of
ammonium chloride and diluted with ethyl acetate. The organic
layer was washed with water and brine, dried with sodium sulfate,
and evaporated. 10.4 mg of 50 (0.02 mmol, 50%) was obtained. The
200-fold excess of nonradioactive DHT. After 18 h, a Dextran 70/g-

276
S. Fujii et al. / European Journal of Medicinal Chemistry 84 (2014) 264e277
globulin-coated charcoal suspension was added to the ligand/pro-
tein mixture (1% activated charcoal, 0.05% -globulin, 0.05%
Dextran, 70 final concentrations) and the whole was incubated at
ꢀC for 10 min. The charcoal was removed by centrifugation for
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Acknowledgments
This work described in this paper was partially supported by
Grants in-Aid for Scientific Research from the Ministry of Educa-
tion, Science, Sports and Culture, Japan (grant no. 25460146 to S.F.).
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