Development of boron-cluster-based progesterone receptor antagonists bearing a pentafluorosulfanyl (SF5) group

Article abstract of DOI:10.1248/cpb.c19-00522

The progesterone receptor (PR) plays an important role in various physiological processes, especially in the female reproductive system, and abnormalities of PR function are associated with several diseases, including some types of cancer. Non-steroidal P

Full text of DOI:10.1248/cpb.c19-00522

1
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Chem. Pharm. Bull. 67, 1278–1283 (2019)
Vol. 67, No. 12
Regular Article
Development of Boron-Cluster-Based Progesterone Receptor Antagonists
Bearing a Pentafluorosulfanyl (SF ) Group
5
Shuichi Mori, Nozomi Tsuemoto, Tomoya Kasagawa, Eiichi Nakano, Shinya Fujii, and
Hiroyuki Kagechika*
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU); 2–3–10 Kanda-
Surugadai, Chiyoda-ku, Tokyo 101–0062, Japan.
Received June 23, 2019; accepted September 6, 2019
The progesterone receptor (PR) plays an important role in various physiological processes, especially
in the female reproductive system, and abnormalities of PR function are associated with several diseases,
including some types of cancer. Non-steroidal PR ligands are of interest as candidate drugs for treatment
of PR-related diseases without the serious adverse effects that may be caused by steroidal ligands. For the
development of non-steroidal PR ligands, both a hydrophobic backbone and a polar functional group corre-
sponding to the 3-carbonyl group of progesterone, which interacts with Gln725 and Arg766 of the PR-ligand
binding domain, are critically important. We previously showed that carborane is a useful hydrophobic
pharmacophore for PR antagonists, and in this work, we introduced the pentafluorosulfanyl (SF ) group as
5
a novel polar functional group of carborane-based non-steroidal PR antagonists. All the synthesized SF5-
containing carborane derivatives exhibited PR-antagonistic activity at micromolar or submicromolar con-
centration. Among them, compounds 11 are potent progesterone antagonists with submicromolar IC50 values.
Key words progesterone receptor; antagonist; carborane; pentafluorosulfanyl group
Introduction
clusters with an icosahedral cage structure, and there are three
The progesterone receptor (PR) is a member of the nuclear isomers, depending on the positions of the two carbon atoms,
21)
receptor superfamily. Its endogenous agonist, progesterone (1, that is, ortho-, meta-, and para-carborane. Carboranes have
Fig. 1), binds to the PR ligand-binding domain (PR-LBD), re- high thermal and chemical stability, and exceptionally high
2
2–24)
sulting in the activation of transcription of target genes, whose hydrophobicity,
and we have applied them as hydrophobic
1)
promoters contain PR-response elements. PR is involved in core structures of various bioactive compounds, especially
the regulation of various physiological processes, especially ligands of various nuclear receptors, such as retinoid recep-
those involved in the female reproductive system, including tors, androgen receptor, and vitamin D receptor. Among
2
5)
26)
27)
2
,3)
regulation of uterine cell proliferation/differentiation, mam- the synthesized compounds, we found that the phenyl carbo-
4
)
5)
mary gland growth/differentiation, the ovulation cycle,
rane derivatives 5–7 (Fig. 2) exhibited PR-antagonistic activ-
A docking study of 5 with the PR-LBD indicated that
for contraception, the carborane moiety of 5 occupies the space corresponding
6
)
18,20)
and sexual behaviors. Various synthetic PR agonists and an- ity.
tagonists have been clinically employed
7
–9)
18)
induction of abortion, hormone replacement therapy, and treat- to the steroid CD ring of progesterone (1). In addition, the
ment of gynecological disorders. The synthetic PR antagonist cyano group of 5 was suggested to interact with Gln725 and
mifepristone (2, Fig. 1) is a representative abortifacient, and Arg766 in the PR-LBD, which also interact with 3-carbonyl
10)
11)
18)
is also effective in the treatment of endometriosis, uterine group of progesterone (1). On the other hand, compound 7
12)
13)
leiomyoma, and breast cancer. However, all PR ligands in having an m-carborane core exhibited bidirectional PR ligand
clinical use, including 2, possess a steroidal structure, which activity, namely, it showed PR-antagonistic activity in the low
may cause significant adverse effects due to cross-activity concentration range, and PR-agonistic activity in the high con-
14)
20)
with other steroid hormone receptors. To avoid these adverse centration range. Compound 8 having a 3-trifluoromethyl
effects, several non-steroidal PR ligands have been developed, substituent on the phenyl group showed only antagonistic ac-
15)
16)
20)
such as tanaproget (3) and sulfonamide derivative 4 (Fig. tivity. These results indicate that the mode of PR ligand ac-
). In the design of such ligands, a polar functional group and tivity is affected by the nature and position of the polar func-
1
a hydrophobic pharmacophore that mimics the steroid skeleton tional group on the phenyl group. Therefore, it is important to
are critically important for the interaction of the ligands with investigate the characteristics of a variety of polar functional
the PR-LBD. In the case of 3, X-ray crystallographic analysis groups of PR ligands.
of the complex of 3 with the PR-LBD revealed that the cyano
The pentafluorosulfanyl (SF ) group is known as a “super
5
group of 3 interacts with Gln725 and Arg766 of PR-LBD, trifluoromethyl group,” because it possesses larger volume,
which are the same amino acid residues that interact with the stronger electronegativity, and higher lipophilicity than the
1
5)
28–30)
3
-carbonyl group of 1.
In the previous study, we developed non-steroidal PR an- tion of high polarity and lipophilicity, together with chemical
tagonists bearing a boron-cluster carborane as the hydrophobic stability, it is attracting considerable interest in the field of
trifluoromethyl group.
Because of its unique combina-
17–20)
29–31)
core structure.
Carboranes, or more precisely dicarba- medicinal chemistry.
Indeed, many investigations involv-
closo-dodecaboranes (C B H ), are carbon-containing boron ing the replacement of the trifluoromethyl group in bioactive
2
10 12
*
To whom correspondence should be addressed. e-mail: kage.chem@tmd.ac.jp
©
2019 The Pharmaceutical Society of Japan

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1279
Fig. 1. Progesterone 1 and Synthetic PR Ligands 2–4
Fig. 2. Carborane-Based Non-steroidal PR Antagonists
Chart 1. Synthesis of Pentafluorosulfanyl (SF )-Containing Carborane Derivatives 9–11
5
32–34)
molecules with a SF group have been reported.
The SF5 group. Synthesis of 9–11 is illustrated in Chart 1. Sandmeyer
5
group has five fluorine atoms in a pyramidal structure, and iodination of 4- or 3-pentafluorosulfanylanilines (12 and 13)
might interact in a distinctive way with proteins. Therefore, in afforded iodobenzenes 14 and 15, respectively. Ullmann-type
this work, we investigated the utility of the SF group as the coupling of m- or p-carborane with 14 and 15 gave phenylcar-
5
polar functional group of PR ligands.
borane derivatives 16–18. The unsubstituted carbon atom of
The phenyl carborane derivatives 5–8 were used as the the carboranes of 16–18 was lithiated with lithium diisopro-
lead compounds because the substituent on their phenyl group pylamide and then reacted with several aldehydes, affording
would act as the key polar functional group to bind PR-LBD. the corresponding secondary alcohols 9, 10, and 11 as racemic
Based on the structures of 5–8, we designed the m- and p- mixtures.
carborane derivatives 9–11 having a SF group on the phenyl
The PR-ligand activities of 9–11 were evaluated by means
5

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Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
Table 1. PR-Antagonistic Activity of SF -Containing Carborane Deriva-
5
tives
Compound
Carborane
X
R
IC50 (µM)
9
9
9
9
9
a
b
c
m
m
m
m
m
m
p
4-SF5
4-SF5
4-SF5
4-SF5
4-SF5
3-SF5
3-SF5
3-SF5
Me
Et
1.9± 0.07
2.9± 0.23
3.1± 0.26
4.3± 0.11
2.9± 0.10
1.7± 0.18
0.37± 0.027
0.42± 0.054
n-Pr
d
e
n-Bu
Cyclopropyl
Cyclopropyl
Me
1
1
1
0e
1a
1e
p
Cyclopropyl
Fig. 3. Competitive Binding Assay of the SF -Containing Carborane
5
Derivatives
3
Table 2. PR-Antagonistic Activity of p-Carborane Derivatives Having
Different Functional Groups
The concentration of [ H]progesterone was 4nM. Mifepristone (MP) was used as
the positive control.
than the corresponding m-carborane derivatives 9 and 10.
To evaluate the effect of polar functional groups, we syn-
thesized compounds 19, 20, and 21, having different polar
functional groups on the same skeletal structure of 11e,
18)
according to our previous report, and examined their PR-
antagonistic activities (Table 2). Among them, 19 with a cyano
group and 20 with a nitro group showed potent PR-antagonis-
Compound
X
IC50 (µM)
1
1
2
2
1e
SF5
CN
0.42± 0.054
0.045± 0.005
0.051± 0.004
0.35± 0.054
9
tic activity. Compound 11e with a SF group and 21 with a
5
0
NO2
CF3
trifluoromethyl group showed relatively low PR-antagonistic
activity compared to 19 and 20. These results suggest that
functional groups having π electron system such as nitro and
cyano group could interact more strongly with PR-LBD than
1
of alkaline phosphatase assay using the T-47D human breast functional groups having multiple fluorine atoms. Compounds
cancer cell line, in which alkaline phosphatase expression is 11e and 21 showed comparable antagonistic activity, while SF5
regulated by PR. The PR-agonistic activity was evaluated in group is stronger electron-withdrawing than trifluoromethyl
35)
terms of the increase ratio of alkaline phosphatase activity in group, which suggests that the electron density of the phenyl
the presence of test compound alone, while the PR-antagonis- ring of the carborane derivatives would not be important for
tic activity was evaluated in terms of inhibition of coexisting the interaction with PR-LBD, and that the multiple fluorines
progesterone (1nM)-induced alkaline phosphatase activity by of SF and trifluoromethyl group might play similar function
5
the test compound.
in PR-LBD.
All of the synthesized SF -containing carborane derivatives
Finally, we examined the PR-binding affinity of selected
5
exhibited PR-antagonistic activity (Table 1). None of the test compounds 10e, and 11e (Fig. 3). The binding affinities were
compounds exhibited PR-agonistic activity. Although com- evaluated by means of competitive binding assay using the
3
pound 7 bearing a p-cyanophenyl moiety showed bidirectional PR-LBD and [1,2,6,7- H]progesterone. Both 10e and 11e
PR ligand activity (antagonistic activity at low concentration, bound to the PR-LBD at submicro- to micromolar concentra-
and agonistic activity at high concentration), compounds tions. Compound 11e that exhibited higher PR-antagonistic ac-
9
a–e having the same skeletal structure as 7 did not act as tivity than 10e showed higher binding affinity to the PR-LBD
PR agonists even in the high concentration range. This result than 10e. These results suggest that the PR-antagonistic activ-
indicates that the mode of PR ligand activity can be changed ity of these compounds evaluated by T-47D alkaline phospha-
depending upon the nature of the polar functional group on tase assay was mediated by binding of the compounds to PR.
the phenyl group. The antagonistic activity of 9a–e having
We designed and synthesized a series of carborane-based
an m-carborane core and a 4-pentafluorosulfanylphenyl group PR antagonists having a pentafluorosulfanyl group as the
was moderate (IC50 approx. 2–4µM), and the length of the polar functional group. All of the synthesized compounds
alkyl chain (R group) of the secondary alcohol did not greatly 9–11 exhibited PR-antagonistic activity, and in particular, the
affect the potency. The potency of 10e (IC =1.7 µM) having p-carborane derivatives 11 showed potent antagonistic activi-
5
0
a 3-pentafluorosulfanylphenyl group on m-carborane was sim- ties at submicromolar concentration. The results suggest that
ilar to that of 9a–e. On the other hand, 11a (IC =0.37 µM) the pentafluorosulfanyl group is an effective polar functional
5
0
and 11e (IC =0.42µM) having a p-carborane and a 3-SF
group that can interact with the PR-LBD as an alternative to
group, exhibited about 10-fold higher antagonistic activities the 3-carbonyl group of progesterone. Although the antago-
5
0
5

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1281
nistic activities of SF -containing carborane derivatives 9–11 for 20h. After cooling, the reaction mixture was diluted with
5
were lower than that of 19 or 20 having a cyano group or a ethyl acetate, and insoluble materials were filtered off through
nitro group, respectively, further structural optimization might celite. The filtrate was washed successively with 5% sodium
increase the activity. The carborane structure and pentaf- thiosulfate, 2M hydrochloric acid, and brine, dried over
luorosulfanyl group may prove to be valuable building blocks sodium sulfate, and evaporated. The residue was purified by
for the design of ligands not only for PR, but also for other silica gel column chromatography (eluent:n-hexane) to give
nuclear receptors.
the products 16–18.
-(4-Pentafluoro-λ -sulfanylphenyl)-1,7-dicarba-closo-
dodecaborane (16)
Yellow solid (27% yield): H-NMR (400MHz, CDCl ) δ:
6
1
Experimental
1
General SF -containing anilines 12 and 13 were sup-
5
3
plied from UBE Industries, LTD., Japan. Other reagents were 7.68 (d, J=7.2Hz, 2 H), 7.53 (d, J=6.8Hz, 2 H), 3.12 (brs, 1
19
purchased from Sigma-Aldrich (U.S.A.), Tokyo Chemical In- H), 3.5–1.5 (brm, 10 H); F-NMR (376MHz, CDCl ) δ: 83.41
3
dustry (Japan), Wako Pure Chemical Industries, Ltd. (Japan), (quin, J=150.0Hz, 1F), 62.48 (d, J=150.0Hz, 4F).
6
and Kanto Chemicals (Japan) and were used without further
purification. NMR spectra were recorded on Bruker AVANCE dodecaborane (17)
00 or Bruker AVANCE 500 spectrometers. Chemical shifts Colorless solid (25% yield): H-NMR (400MHz, CDCl ) δ:
1-(3-Pentafluoro-λ -sulfanylphenyl)-1,7-dicarba-closo-
1
4
3
for NMR are reported as parts of per million (ppm) rela- 7.81 (t, J=2.0Hz, 1 H), 7.71 (ddd, J=8.0, 4.0, 0.8Hz, 1 H),
1
tive to chloroform (7.27ppm for H-NMR and 77.23ppm for 7.59 (d, J=8.8Hz, 1 H), 7.39 (t, J=8.0Hz, 1 H), 3.13 (brs, 1
13
19
19
C-NMR) and benzotrifluoride (−63.72ppm for F-NMR) as H), 4.0–1.5 (brm, 10 H); F-NMR (376MHz, CDCl ) δ: 83.13
3
an external standard. Coupling patterns are denoted as fol- (quin, J=150.1Hz, 1F), 62.53 (d, J=150.0Hz, 4F).
6
lows: singlet (s), doublet (d), doublet of doublets (dd), doublet
of doublets of doublets (ddd), doublet of triplets (dt), triplet (t), dodecaborane (18)
quartet (q), quintet (quin), multiplet (m), broad singlet (brs), Colorless solid (45% yield): H-NMR (400MHz, CDCl ) δ:
1-(3-Pentafluoro-λ -sulfanylphenyl)-1,12-dicarba-closo-
1
3
and broad multiplet (brm). MS were collected on a Bruker 7.62 (dt, J=8.4, 2.0Hz, 1 H), 7.59 (dd, J=2.0, 1.6Hz, 1 H),
Daltonics microTOF-2focus spectrometer in the negative ion 7.37 (d, J=8.0Hz, 1 H), 7.30 (dd, J=8.4, 8.0Hz, 1 H), 2.85
19
mode. Melting points were obtained on a Yanagimoto micro (brs, 1 H), 3.5–1.5 (brm, 10 H) ; F-NMR (376MHz, CDCl3)
melting point apparatus without correction. Elemental analy- δ: 83.10 (quin, J=150.1Hz, 1F), 62.48 (d, J=150.1Hz, 4F).
ses were carried out by using Yanaco MT-6 CHNCORDER
spectrometer.
General Procedure for Synthesis of 9–11 A solution
of lithium diisopropylamide in a mixture of n-hexane and
General Procedure for Synthesis of 14 or 15 A cold tetrahydrofuran (1.1M, 0.14mL, 0.16mmol) was added to a
solution of NaNO (102mg, 1.48mmol) in water (2.5mL) was solution of 16–18 (48mg, 0.14mmol) in THF (2mL) at −78°C
2
added slowly to a solution of 12 or 13 (306mg, 1.40mmol) in under an Ar atmosphere, and the mixture was stirred at
conc. HCl (1.5mL) and ice (2.5g). The mixture was stirred −78°C for 20min. The appropriate aldehyde (2.0–10 equiv.)
at 0°C for 2min and then added slowly to a solution of KI was added to the reaction mixture at −78°C. The resulting
(220mg, 1.33mmol) in water (20mL) at 0°C. The resulting mixture was stirred at −78°C for 1h, then poured into satu-
mixture was stirred for 30min on ice and then allowed to rated ammonium chloride, and extracted with ethyl acetate.
warm to r.t. for 1h, and the product was extracted with di- The organic layer was washed with water and brine, dried
chloromethane. The organic layer was washed with saturated over sodium sulfate, and evaporated. The residue was purified
aqueous NaHCO3 solution, dried over sodium sulfate, and by silica gel column chromatography (eluent:n-hexane/ethyl
concentrated. Purification by silica gel column chromatogra- acetate, 10/1) to give the racemic alcohols 9–11.
6
phy (eluent:n-hexane) gave iodinated products 14 or 15.
-Iodophenylsulfur Pentafluoride (14)
Colorless oil (87% yield): H-NMR (400MHz, CDCl ) δ:
1-(4-Pentafluoro-λ -sulfanylphenyl)-7-(1-hydroxyethyl)-1,7-
3
dicarba-closo-dodecaborane (9a)
Colorless oil (86% yield): H-NMR (400MHz, CDCl ) δ:
1
1
3
3
8
.09 (t, J=2.0Hz, 1 H), 7.86 (d, J=8.0Hz, 1 H), 7.75 (dd, 7.65 (d, J=7.2Hz, 2 H), 7.54 (d, J=6.8Hz, 2 H), 4.14–4.07
19
J=8.4, 2.0Hz, 1 H), 7.22 (dd, J=8.8, 8.0Hz, 1 H); F-NMR (m, 1 H), 3.5–1.5 (brm, 10 H), 1.89 (d, J=4.8Hz, 1 H), 1.34
19
(376MHz, CDCl ) δ: 82.81 (quin, J=150.1Hz, 1F), 62.53 (d, (d, J=5.2Hz, 3 H); F-NMR (376MHz, CDCl ) δ: 83.10
3
3
J=150.1Hz, 4F).
-Iodophenylsulfur Pentafluoride (15)
(quin, J=150.1Hz, 1F), 62.48 (d, J=150.0Hz, 4F); high
−
4
resolution (HR)MS electrospray ionization (ESI ) Calcd for
1
−
Colorless solid (82% yield): H-NMR (400MHz, CDCl ) δ: C H B F OS [M−H] : 391.1929. Found: 391.1937.
.82 (d, J=7.2Hz, 2 H), 7.49 (d, J=7.2Hz, 2 H); F-NMR
3
10 18 10 5
19
6
7
(
1-(4-Pentafluoro-λ -sulfanylphenyl)-7-(1-hydroxypropyl)-1,7-
376MHz, CDCl ) δ: 83.21 (quin, J=150.3Hz, 1F), 62.55 (d, dicarba-closo-dodecaborane (9b)
3
1
J=150.1Hz, 4F).
Colorless oil (62% yield): H-NMR (400MHz, CDCl ) δ:
3
General Procedure for Synthesis of 16–18 A solution of 7.66 (d, J=7.2Hz, 2 H), 7.53 (d, J=6.8Hz, 2 H), 3.76–3.72
n-butyllithium in n-hexane (2.67M, 0.8mL, 2.14mmol) was (m, 1 H), 3.0–1.5 (brm, 10 H), 1.88 (d, J=5.2Hz, 1 H),
added to a solution of m- or p-carborane (211mg, 1.46mmol) 1.75–1.70 (m, 1 H), 1.43–1.37 (m, 1 H), 1.02 (t, J=5.6Hz, 3
1
9
in 1,2-dimethoxyethane (10mL) at 0°C under an Ar atmo- H); F-NMR (376MHz, CDCl ) δ: 83.12 (quin, J=150.2Hz,
3
−
sphere, and the mixture was stirred at 0°C for 30min. Copper 1F), 62.49 (d, J=150.1Hz, 4F); HRMS (ESI ) Calcd for
−
(
I) chloride (162mg, 1.64mmol) was added to the reaction C H B F OS [M−H] : 405.2086. Found: 405.2094.
11
20 10 5
6
mixture at room temperature. After 1h, pyridine (1.5mL)
1-(4-Pentafluoro-λ -sulfanylphenyl)-7-(1-hydroxybutyl)-1,7-
and pentafluorosulfanylated iodobenzene 14 or 15 (500mg, dicarba-closo-dodecaborane (9c)
1
1.51mmol) were added, and the mixture was stirred at 80°C
Colorless oil (71% yield): H-NMR (400MHz, CDCl ) δ:
3

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Chem. Pharm. Bull.
Vol. 67, No. 12 (2019)
7
.66 (d, J=7.2Hz, 2 H), 7.53 (d, J=8.0Hz, 2 H), 3.85–3.82 H), 2.9–1.6 (brm, 10 H), 2.88 (dd, J=8.3, 1.2Hz, 1 H) 1.70 (s,
(
1
m 1H), 3.0–1.5 (brm, 10 H), 1.86 (d, J=4.8Hz, 1 H), 1 H), 0.83–0.76 (m, 1 H), 0.60–0.59 (m, 2 H), 0.34–0.25 (m,
.61–1.57 (m, 1 H), 1.42–1.35 (m, 3 H), 0.92 (t, J=5.8Hz, 3 2 H); C-NMR (126MHz, CDCl ) δ, 137.9, 132.0, 131.7, 131.0,
3
19
13
−
H); F-NMR (376MHz, CDCl ) δ: 83.12 (quin, J=150.1Hz, 129.2, 118.3, 112.7, 87.0, 81.5, 17.9, 4.8, 3.2; HRMS (ESI )
1
3
−
−
F), 62.49 (d, J=150.8Hz, 4F); HRMS (ESI ) Calcd for Calcd for C H B F O [M+HCOO] : 362.2530. Found:
14 22 10 3 3
−
C H B F OS [M−H] : 419.2242. Found: 419.2250.
362.2534.
1
2
22 10 5
6
1
-(4-Pentafluoro-λ -sulfanylphenyl)-7-(1-hydroxypentyl)-1,7-
1-(3-Nitrophenyl)-12-(1-cyclopropyl-1-hydroxymethyl)-1,12-
dicarba-closo-dodecaborane (9d)
Colorless oil (56% yield): H-NMR (400MHz, CDCl ) δ:
dicarba-closo-dodecaborane (20)
Colorless solid: H-NMR (400MHz, CDCl ) δ: 8.09 (m, 2
1
1
3
3
7
.65 (d, J=7.2Hz, 2 H), 7.53 (d, J=7.2Hz, 2 H), 3.84–3.82 H), 7.54 (ddd, J=8.0, 3.2, 1.8Hz, 1 H), 7.37 (t, J=8.0Hz, 1
(
m, 1 H), 3.0–1.5 (brm, 10 H), 1.87 (d, J=4.8, 1 H), 1.67–1.62 H), 3.3–2.3 (brm, 10 H), 2.87 (dd, J=8.3, 5.3Hz, 1 H), 1.68
19
(m, 1 H), 1.43–1.32 (m, 5 H), 0.92 (t, J=5.8, 3 H); F-NMR (d, J=5.0Hz, 1 H), 0.82–0.75 (m, 1 H), 0.64–0.54 (m, 2 H),
13
(376MHz, CDCl ) δ: 83.16 (quin, J=150.3Hz, 1F), 64.30 (d, 0.32–0.27 (m, 2 H); C-NMR (126MHz, CDCl ) δ, 148.1,
3
3
−
J=150.0Hz, 4F); HRMS (ESI ) Calcd for C H B F OS 138.4, 133.3, 129.3, 123.5, 122.5, 17.9, 5.0, 3.2. Anal Calcd for
13
25 10 5
−
[
M−H] : 433.2399. Found: 433.2407.
C B H NO : N, 4.18; C, 42.97; H, 6.31. Found: N, 4.08; C,
12 10 21 3
6
1
-(4-Pentafluoro-λ -sulfanylphenyl)-7-(1-cyclopropyl-1- 42.71; H, 6.15.
hydroxymethyl)-1,7-dicarba-closo-dodecaborane (9e)
Colorless oil (68% yield): H-NMR (400MHz, CDCl ) δ: hydroxymethyl)-1,12-dicarba-closo-dodecaborane (21)
1-(3-Trif luoromethylphenyl)-12-(1-cyclopropyl-1-
1
3
1
7.67 (d, J=7.2Hz, 2 H), 7.55 (d, J=7.2Hz, 2 H), 3.20 (q,
Colorless solid: mp 76–78°C; H-NMR (500MHz, CDCl )
3
J=3.5Hz, 1 H), 3.0–1.5 (brm, 10 H), 1.93 (d, J=3.6Hz, 1 δ: 7.48 (d, J=7.7Hz, 1 H), 7.46 (s, 1 H) 7.41 (d, J=8.4Hz, 1
H), 1.28–0.98 (m, 1 H), 0.73–0.60 (m, 2 H), 0.45–0.38 (m, 2 H), 7.31 (d, J=7.9Hz, 1 H), 3.3–2.3 (br, m, 10 H), 2.87 (dd,
1
9
H); F-NMR (376MHz, CDCl ) δ: 83.12 (quin, J=149.8Hz, J=8.3, 4.9Hz, 1 H) 1.67 (d, J=5.0Hz, 1 H), 0.83–0.75 (m, 1
3
−
1
F), 62.49 (d, J=150.1Hz, 4F); HRMS (ESI ) Calcd for H), 0.64–0.56 (m, 1 H), 0.55–0.49 (m, 1 H), 0.35–0.22 (m, 2
−
13
C H B F OS [M−H] : 417.2086. Found: 417.2094.
H); C-NMR (126MHz, CDCl ) δ: 137.5, 130.8 (q, J=31.5),
1
2
20 10
5
3
6
1
-(3-Pentafluoro-λ -sulfanylphenyl)-7-(1-cyclopropyl-1- 130.8, 128.9, 125.5 (q, J=3.8), 124.2 (q, J=3.8), 123.9 (q,
hydroxymethyl)-1,7-dicarba-closo-dodecaborane (10e)
Colorless oil (79% yield): H-NMR (400MHz, CDCl ) δ: C H B F O [M+HCOO] : 405.2451. Found: 405.2451.
.82 (dd, J=2.0, 1.6Hz, 1 H), 7.71 (ddd, J=8.4, 2.4, 1.2Hz,
−
J=273.4), 86.8, 82.4, 17.9, 5.0, 3.3; HRMS (ESI ) Calcd for
14 22 10
1
−
3
3
3
7
Alkaline Phosphatase Assay Using T-47D Cells T-47D
1
H), 7.61 (d, J=8.8Hz, 1 H), 7.39 (dd, J=8.4, 7.6Hz, 1 breast-carcinoma cells were cultured in RPMI 1640 me-
H), 3.20 (q, J=4.4Hz, 1 H), 3.0–1.5 (brm, 10 H), 1.95 (d, dium with 10% (v/v) fetal bovine serum. Cells were plated in
4
J=4.8Hz, 1 H), 1.04–0.97 (m, 1 H), 0.74–0.63 (m, 2 H), 96-well plates at 10 cell/well and incubated overnight (37°C,
13
0
.54–0.48 (m, 2 H); C-NMR (126MHz, methanol-d ) δ: 5% CO in air). The next day, the medium was replaced with
4 2
155.0 (quin, J=17.6Hz), 138.2, 132.7, 130.8, 127.8 (quin, fresh medium containing test compound, and incubation was
J=4.5Hz), 126.4 (quin, J=4.8Hz), 85.4, 77.1, 76.8, 19.0, 5.47, continued for 24h. Then, the medium was aspirated and the
1
9
4
1
.03; F-NMR (376MHz, CDCl ) δ: 81.64 (quin, J=148.0Hz, cells were fixed with 100µL of 1.8% formalin (in phosphate
3
−
F), 60.83 (d, J=147.9Hz, 4F); HRMS (ESI ) Calcd for buffered saline (PBS)). The fixed cells were washed with PBS
−
C H B F OS [M−H] : 417.2086. Found: 417.2090.
and 75µL of assay buffer (1mg/mL p-nitrophenol phosphate
12
20 10 5
6
1
- ( 3 - P e n t a f l u o r o - λ - s u l f a n y l p h e n y l ) -1 2 - (1- in diethanolamine water solution, pH 9.0, 2mM MgCl ) was
2
hydroxyethyl)-1,12-dicarba-closo-dodecaborane (11a)
added. The mixture was incubated at room temperature with
1
Colorless oil (90% yield): H-NMR (400MHz, CDCl3) shielding from light for 2h, and then the reaction was termi-
δ: 7.63 (ddd, J=8.0, 2.0, 0.8Hz, 1 H), 7.59 (t, J=2.0Hz, 1 nated by the addition of 100µL of NaOH. The absorbance at
H), 7.38 (d, J=8.0Hz, 1 H), 7.30 (dd, J=8.0, 7.2Hz, 1 H), 405nm was measured.
3
.77 (m, 1 H), 3.5–1.5 (br, m, 10 H), 1.65 (d, J=6Hz, 1 H),
hPR-Binding Assay hPR-Binding assay was performed
13
1.13 (d, J=6.4Hz, 1 H); C-NMR (126MHz, methanol-d4) using recombinant hPR-LBD purchased from Invitrogen
δ: 154.9 (quin, J=17.4Hz), 138.9, 132.0, 130.5, 127.5 (quin, (A15672). hPR-LBD was diluted with buffer (20mM Tris–
J=4.5Hz), 125.8 (quin, J=4.7Hz), 90.2, 82.6, 69.9, 23.6; HCl, 300mM NaCl, 1mM EDTA, 5mM DTT, pH 8.0) to
1
9
F-NMR (376MHz, methanol-d ) δ: 81.74 (quin, J=148.0Hz, 14 µg/mL of total protein and 300µL aliquots were incu-
4
−
3
1
F), 60.78 (d, J=148.0Hz, 4F); HRMS (ESI ) Calcd for bated in the dark at 4°C with 4nM [1,2,6,7- H]progesterone
1
−
C H B F OS [M−H] : 391.1929. Found: 391.1938.
(PerkinElmer, Inc., U.S.A.) and reference or test compounds
-(3-Pentafluoro-λ -sulfanylphenyl)-12-(1-cyclopropyl-1- (dissolved in dimethyl sulfoxide (DMSO); final concentration
hydroxymethyl)-1,12-dicarba-closo-dodecaborane (11e) of DMSO was 3%). Nonspecific binding was assessed by ad-
Colorless oil (92% yield): H-NMR (400MHz, CDCl ) δ: dition of a 200-fold excess of nonradioactive progesterone.
0
18 10 5
6
1
1
3
7
.63–7.60 (m, 2 H), 7.39 (d, J=6.4Hz, 1 H), 7.30 (dd, J=6.8, After 24h, 30µL of Dextran T-70/c-globulin-coated charcoal
6.0Hz, 1 H), 2.88 (q, J=3.5Hz, 1 H), 3.5–1.5 (br, m, 10 suspension was added to the ligand/protein mixtures (1%
H), 1.69 (d, J=4.0Hz, 1 H), 0.83–0.76 (m, 1 H), 0.64–0.51 activated charcoal, 0.05% γ-globulin, 0.05% Dextran 70, final
−
(
m, 2 H), 0.34–0.25 (m, 2 H); HRMS (ESI ) Calcd for concentrations) and incubated at 4°C for 5min. The charcoal
−
C H B F OS [M−H] : 417.2086. Found: 417.2087.
was removed by centrifugation for 5min at 1300×g, and the
1
2
20 10 5
1
-(3-Cyanophenyl)-12-(1-cyclopropyl-1-hydroxy- radioactivity of the supernatant was measured in Ultima Gold
scintillation cocktail (PerkinElmer, Inc.) by using a liquid
Colorless solid: mp 145–147°C; H-NMR (500MHz, CDCl ) scintillation counter. All experiments were performed in du-
methyl)-1,12-dicarba-closo-dodecaborane (19)
1
3
δ: 7.52 (m, 2H), 7.45 (t, J=1.6Hz, 1 H), 7.31 (t, J=7.6Hz, 1 plicate.

Vol. 67, No. 12 (2019)
Chem. Pharm. Bull.
1283
3
74–400 (2007).
Acknowledgments This research was partly supported by
JSPS KAKENHI Grant Number 18K06546 (to SM), Sasagawa
Scientific Research Grant from The Japan Science Society,
and JSPS Core-to-Core Program, A, and Advanced Research
Networks the Platform Project for Supporting Drug Discovery
and Life Science Research funded by Japan Agency for Medi-
cal Research and Development (AMED).
1
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V., Malakian K., Lockhead S., Olland A., Svenson K., Terefenko E.
A., Unwalla R. J., Wilhelm J. M., Wolfrom S., Zhu Y., Zhang Z.,
Zhang P., Winneker R. C., Wrobel J., J. Med. Chem., 48, 5092–5095
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Conflict of Interest The authors declare no conflict of
Kagechika H., Eur. J. Med. Chem., 84, 264–277 (2014).
interest.
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