Design and synthesis of silicon-containing steroid sulfatase inhibitors possessing pro-estrogen antagonistic character

Article abstract of DOI:10.1016/j.bmc.2014.02.025

Steroid sulfatase (STS) is a potential target for treatment of postmenopausal hormone-dependent breast cancer. Several steroidal STS inhibitors have been reported, but steroidal compounds are difficult to optimize and may interact with other targets. On t

Full text of DOI:10.1016/j.bmc.2014.02.025

Bioorganic & Medicinal Chemistry 22 (2014) 2244–2252
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of silicon-containing steroid sulfatase
inhibitors possessing pro-estrogen antagonistic character
Daisuke Kajita a, , Masaharu Nakamura a, , , Yotaro Matsumoto b, Makoto Makishima c, Yuichi Hashimoto a
⇑
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Faculty of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan
c Department of Biochemistry, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Steroid sulfatase (STS) is a potential target for treatment of postmenopausal hormone-dependent breast
cancer. Several steroidal STS inhibitors have been reported, but steroidal compounds are difficult to
optimize and may interact with other targets. On the other hand, we have shown that diphenylmethane
(DPM) derivatives act as estrogen receptor (ER) agonists and antagonists. Here, we aimed to design and
synthesize non-steroidal DPM-type STS inhibitors that would also serve as pro-estrogen antagonists,
Received 22 January 2014
Revised 10 February 2014
Accepted 10 February 2014
Available online 28 February 2014
releasing a metabolite with ERa-antagonistic activity upon hydrolysis by STS. We synthesized a series
of compounds and evaluated their biological activities by means of STS-inhibitory activity assay and
ER reporter gene assay. Among them, silicon-containing compound 16a showed strong STS-inhibitory
Keywords:
Steroid sulfatase inhibitor
Estrogen antagonist
Silicon
Diphenylmethane
Breast cancer
activity (IC50 = 0.17
(IC50 = 29.7 nM).
lM). Further, its putative metabolite (12a) exhibited potent ERa-antagonistic activity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
In addition to aromatase, steroid sulfatase (STS) is involved in
estrogen synthesis in breast cancer. STS catalyzes the hydrolysis
of steroid-3-sulfates to 3-hydroxysteroids. Since the systemic
circulating precursors for estrogens and androgens are predomi-
nantly sulfates such as dehydroepiandrosterone sulfate (DHEAS)
and estrone sulfate (E1S), STS plays a crucial role in production
of androgens and estrogens [estrone (E1) and estradiol (E2)].6
Further, the activity of STS in cancer tissue is several hundred times
higher than that of aromatase.7 Since STS produces estrogens
through a different route from aromatase, it is expected that simul-
taneous administration of STS inhibitors and aromatase inhibitors
might show an additive or synergistic anti-tumor effect. Therefore,
STS is considered to be a promising target for anticancer drug
development.8 Several STS inhibitors have been reported (Fig. 1).
Among them, estrone 3-O-sulfamate (EMATE) was evaluated in a
clinical trial. However, it was found that metabolites of EMATE ex-
hibit potent estrogenic activity9, and so might promote tumor
growth. Therefore, the development of EMATE was discontinued.
Since steroidal compounds are difficult to optimize, and they or
their metabolites might also act on other targets, non-steroidal
STS inhibitors have attracted attention. For example, STX64, which
has a coumarin skeleton, shows potent STS-inhibitory activity and
a clinical trial has been carried out.10
Breast cancer is one of the most common types of cancer in
women; 70–80% of these cancers are hormone-dependent1 and
more than 70% express high levels of estrogen receptor alpha
(ERa
).2 Endocrine therapies are standard treatments for
hormone-dependent breast cancer, based on the use of an anti-
estrogen or estrogen antagonist to block production or utilization
of estrogens or to inhibit their functional effects.3,4 In premenopau-
sal women, estrogens promoting breast tumor growth are mainly
synthesized in the ovaries, and therefore, a luteinizing hormone-
releasing hormone (LH-RH) agonist5 that suppresses the function
of pituitary hormone to promote estrogen synthesis in the ovaries
may be particularly useful. On the other hand, in postmenopausal
women, whose ovarian function is lowered, estrogens are mainly
synthesized from androgen secreted from the adrenal glands. Since
the conversion of androgen to estrogen is performed by aromatase,
aromatase inhibitors are often used to treat postmenopausal
hormone-dependent breast cancer. However, the therapeutic
effect of aromatase inhibitors is not always sufficient, and drugs
with another mechanism of action may also be required.
The non-steroidal diphenylmethane (DPM) derivative 1 has also
been reported as an STS inhibitor (Fig. 2).11 However, its putative
metabolite 1m has ER-agonistic activity.12 Therefore, compound
⇑
Corresponding author. Tel.: +81 3 5841 7847; fax: +81 3 5841 8495.
E-mail address: nakamura-nk@iam.u-tokyo.ac.jp (M. Nakamura).
The first two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.02.025
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
2245
antagonist upon hydrolysis by STS. The biological activities of these
inhibitors were examined by means of STS-inhibitory activity
assay and ER reporter gene assay.
2. Results and discussion
Figure 1. Structures of EMATE (estrone 3-O-sulfamate) and STX64.
2.1. Molecular design and computational studies
It has been reported that the STS-inhibitory activity of sulfur
derivative 2 was stronger than that of carbon derivative 1.11
Generally, bonds involving sulfur are longer than those involving
carbon. The single-bond covalent radii of carbon and sulfur are
75 pm and 103 pm, respectively, whereas that of silicon is
116 pm.19 Therefore, we hypothesized that replacement of quater-
nary carbon in the DPM skeleton with silicon might also result in
enhancement of STS-inhibitory activity. Based on this hypothesis,
we designed silicon-containing compound 3a. We next calculated
the distances between the two benzene ring (d) in compounds 1,
2 and 3a. Structure optimizations were performed at the MP2/
6-31+G⁄ level of theory with the Gaussian 09 program package.20,21
The results are illustrated in Figure 4. As expected, distance (d) in
silicon compound 3a was longer than those in carbon compound
1 and sulfur compound 2.
O2
O2
S
S
H2N
O
O
NH2
HO
OH
1
1m
1 and its putative
Figure 2. Structures of diphenylmethane (DPM) derivative
metabolite 1m.
1 was considered unsuitable for use as a therapeutic agent for the
same reason as EMATE.
Recently, we found that although some DPM derivatives have
ER-agonistic activity, others have ER-antagonistic activity.12 There-
fore, we set out to design novel non-steroidal DPM-type STS
inhibitors that would release ER antagonist metabolites upon
hydrolysis by STS (Fig. 3). A similar approach has already been
reported for steroid analogs.13
In recent years, various silicon-containing bioactive compounds
have been reported.14,15 The introduction of silicon into
compounds can improve various biological properties, including
selectivity, potency, pharmacokinetics, pharmacodynamics and
cell penetration. Indeed, several silicon-containing agents have
advanced to clinical trials.16,17 Also, we have developed silicon-
containing tubulin polymerization inhibitors.18 It has already been
reported that replacement of quaternary carbon in DPM with
sulfur increased the STS-inhibitory activity. Since the silicon atom
is larger than carbon, like sulfur, we speculated that introduction of
silicon could also enhance STS-inhibitory activity.
2.2. Chemistry
Based on our hypothesis, we planned to prepare silicon-
containing compound 3a and its derivatives 8, 10 and 15a–18a.
These compounds were prepared by usual organic synthetic meth-
ods as illustrated in Scheme 1. Compounds 9a, 11a, 12a and 14a
were prepared according to the reported method.22 Briefly,
bromophenols (5a–b) were reacted with dichlorodialkylsilane in
the presence of n-butyllithium (n-BuLi) in tetrahydrofuran (THF).
Compounds 3a, 15a, 16a and 18a were synthesized by reaction
of 9a, 11a, 12a and 14a with excess sulfamoyl chloride.23 Com-
pound 17a was prepared in an analogous manner, starting from
dichlorodipropylisilane 4d and bromophenol 5c. Mono sulfamate
In this paper, we describe the design and synthesis of non-
steroidal, silicon-containing STS inhibitors that release an ER
Dehydroepiandrosterone sulfate
(DHEAS)
Estrone sulfate (E1S)
R1
R1
Si
O2
S
O2
S
STS
STS
H2N
O
O
NH2
R2
R2
S TS inhibitor
Estrone (E1)
Dehydroepiandrosterone
(DHEA)
Metabolism by STS
R1
R1
Si
Estradiol (E2)
Androstenediol
HO
OH
R2
R2
E R α agonist
Antagonistic
Agonistic activity
Agonistic activity
activity
ERα
Promotes growth
of breast cancer cells
Figure 3. Schematic illustration of the expected action mechanisms of our DPM-type STS inhibitors with pro-estrogen antagonistic character.

2246
D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
H
H
S
Si
O2
S
O2
S
O2
S
O2
S
O2
S
O2
S
H2N
O
O
NH2
H2N
O
O
NH2
H2N
O
O
NH2
2.56Å
1
2.81Å
2
3.08 Å
3a
Figure 4. Distances (d) between the two benzene rings in compounds 1, 2 and 3a.
R1
R1
Br
R1
R1
Si
Si
a
b
R1
Cl
R1
Cl
O2
S
O2
S
+
Si
HO
HO
OH
H2N
O
O
NH2
R2
R2
R2
R2
R2
4a R1 = Me
4b R1 = Et
5a R2 = H
3a R1 = Me, R2 = H
15a R1 = Et, R2 = H
16a R1 = Et, R2 = Me
9a R1 = Me, R2 = H
11a R1 = Et, R2 = H
5b R2 = Me
4c R1 = n-Bu
12a R1 = Et, R2 = Me
18a R1 = n-Bu, R2 = Me
14a R1 = n-Bu, R2 = Me
n-Pr
n-Pr
n-Pr
n-Pr
Br
Si
Si
c
d
n-Pr
Cl
n-Pr
Cl
Si
+
BnO
BnO
OBn
HO
OH
4d
5c
19
13a
n-Pr
n-Pr
Si
e
O2
S
O2
S
H2N
O
O
NH2
17a
Si
f
g
Si
O2
O2
O2
9a
3a
S
S
S
N
O
O
N
H2N
O
OH
8
10
Scheme 1. Synthesis of silicon-containing compounds 3a, 15a–18a, 8 and 10. Reagents and conditions: (a) n-BuLi/hexane, THF, ꢀ78 °C to rt, 2–45%; (b) excess sulfamoyl
chloride, N,N-dimethylacetamide (DMA), rt, 25–47%; (c) n-BuLi/hexane, THF, 1 h at 0 °C, 5 h at rt, 4.3%; (d) Pd/C, ethanol, rt at 3 days 3.8%; (e) excess sulfamoyl chloride, DMA,
rt, 1.4%; (f) sulfamoyl chloride (1.2 equiv), DMA, 4 h at rt, 13%; (g) NaH, CH3I, N,N-dimethylformamide (DMF), 19 h at rt, 16%.
8 was synthesized by reaction of 9a with 1.2 equiv of sulfamoyl
chloride. Compound 10 was prepared by methylation of compound
3a.
Carbon and sulfur derivatives 1, 3b, 15b–18b and 2 were
synthesized according to the procedure described for silicon-
containing compounds, as shown in Scheme 2.
exception of 17a and 18a, silicon derivatives showed greater
STS-inhibitory activity than the corresponding carbon derivatives.
However, the STS-inhibitory activity of the silicon derivatives did
not exceed that of sulfur derivative 2. Since 15a is more potent
than 3a, it appears that replacement of the methyl groups on
silicon with ethyl groups increased the activity. A similar tendency
was confirmed in carbon derivatives 3b and 15b. On the other
hand, the STS-inhibitory activity of 15a was greater than that of
16a. Therefore, introduction of methyl groups at the R2 position
2.3. STS-inhibitory activity evaluation
STS-inhibitory activity was determined by measuring total la-
beled estrone ([3H]E1) and estradiol ([3H]E2) formed from labeled
estrone sulfate ([3H]E1S) in MCF-7 breast cancer cells.24 STX64 was
used as a positive control.
First, we evaluated the STS-inhibitory activity of 3a and 8–10
(Table 1). As expected, 3a, which has two sulfamate substituents,
exhibited potent STS-inhibitory activity (81.9% inhibition at
R1
R1
R1
R1
a
O2
S
O2
S
HO
OH
H2N
O
O
NH2
R2
R2
R2
R2
1
3b
6
R1 = H, R2 = H
R1 = Me, R2 = H
R
1 = H, R2 = H
1
l
M). The activity of mono sulfamate 8 was slightly weaker than
that of 3a (76.4% inhibition at 1 M). Bisphenol 9a, which is
expected to be a metabolite of 3a, was inactive in the concentra-
tion range examined (<10% inhibition at 1 M). A comparison of
9b
R
R
1 = Me, R2 = H
1 = Et, R2 = H
l
15b
16b
17b
11b
12b
13b
R1 = Et, R2 = H
R1 = Et, R2 = Me
R1 = Et, R2 = Me
R
1 = n-Pr, R2 = Me
R1 = n-Pr, R2 = Me
l
18b R1 = n-Bu, R2 = Me
14b R1 = n-Bu, R2 = Me
3a, 8 and 9a indicated that the STS-inhibitory activity was attenu-
ated as the number of sulfamate substituents was decreased.
Methylation of the amino group of sulfamate 3a, that is, compound
10, resulted in loss of activity (<10% inhibition at 1 lM). Sulfamate
seems to be essential for STS-inhibitory activity in this series of
S
S
b
O2
S
O2
S
HO
OH
H2N
O
O
NH2
7
2
compounds.
Scheme 2. Synthesis of carbon and sulfur derivatives 1, 3b, 15b–18b and 2.
Reagents and conditions: (a) excess sulfamoyl chloride, DMA, 6 h to 2 days at rt,
7.4–82%; (b) excess sulfamoyl chloride, DMA, 5 days at rt, 4.6%.
Next, we evaluated the STS-inhibitory activity of various silicon,
carbon and sulfur derivatives of 3a as shown in Table 2. With the

D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
2247
Table 1
order is similar to the order of the distance (d) between the
aromatic rings. Therefore, the electropositivity on the central atom
may also play a role in the STS-inhibitory activity. Thus, introduc-
tion of electropositive atoms into the methylene moiety might en-
hance the STS-inhibitory activity.
Results of STS-inhibitory activity assay of the DPM derivatives
Compound
R1
R2
STS inhibition (%)
0.1 1 lM
2.5. ER reporter gene assay
l
M
3a
8
9a
10
OSO2NH2
OSO2NH2
OH
OSO2NMe2
—
OSO2NH2
OH
OH
OSO2NMe2
—
35.8
25.2
<10
<10
—
81.9
76.4
<10
<10
100
The ER-agonistic and antagonistic activities of selected putative
metabolites were evaluated by means of reported methods.25,26 As
shown in Table 3, ER-agonistic activity was calculated as EC50 (nM)
and ER -antagonistic activity as IC50 (nM). Some of the assay data in
the table were taken from our previous paper.12
STX64
As previously reported11, the carbon derivative 6 and sulfur
derivative 7 exhibited ER-agonistic activity. Compounds 9a and
9b, which have methyl groups on the silicon atom, showed
Table 2
Results of STS-inhibitory activity assay of the DPM derivatives
ER
weak ER
a
-agonistic activity, and compounds 11a and 13a exhibited
-agonistic activity. Therefore, compounds that might give
a
rise to these metabolites are not appropriate as anti-cancer agents,
since the metabolites might promote the growth of breast cancer.
On the other hand, in the assay of ER-antagonistic activity, com-
Compound
X
R1
R2
STS inhibition (%)
0.1
pounds 11a–13a and 11b–14b showed potent ER
activity. The ER -antagonistic activity of 12a was greater than that
of 11a, in accordance with the results for the corresponding carbon
compounds 11b and 12b. This result suggests that ER -antagonis-
a-antagonistic
l
M
1
lM
a
1
2
3a
3b
15a
15b
16a
16b
17a
17b
18a
18b
C
S
Si
C
Si
C
Si
C
Si
C
Si
C
H
—
Me
Me
Et
Et
Et
Et
n-Pr
n-Pr
n-Bu
n-Bu
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
<10
57.1
35.8
<10
62.0
11.0
22.6
<10
<10
<10
<10
10.3
50.1
89.7
81.9
<10
96.4
29.9
82.6
25.4
18.7
21.1
16.4
28.0
a
tic activity was increased by the introduction of methyl groups at
the R2 position. The silicon and carbon derivatives both showed
the tendency that ERa-antagonistic activity was improved by
the introduction of bulky substituents on the central atom X.
However, the introduction of n-butyl groups slightly reduced the
ERa-antagonistic activity. On the other hand, the ERb-antagonistic
activity of the silicon-containing compounds was weaker than that
of the corresponding carbon compounds. In other words, the
silicon-containing compounds showed higher ER
did the corresponding carbon compounds.
a selectivity than
We next evaluated the agonistic and antagonistic activities of
the sulfamate derivatives, as shown in Table 4. Compounds 15a,
of the aromatic rings appears to reduce the activity of 16a. A
similar tendency was seen in the carbon derivatives 15b and
16b. The order of STS-inhibitory activity of 16a–18a was
16a ꢁ 17a > 18a. The STS-inhibitory activity of 17a bearing
n-propyl groups on silicon was much less than that of 16a, while
18a bearing n-butyl groups on silicon was even less potent. These
results suggest that replacement of a small alkyl group on silicon
with a bulkier substituent may reduce the STS-inhibitory activity.
b and 16a, b showed no ER
compound 2 showed strong ER
compounds exhibited ER -antagonistic activity at
bis-phenol derivatives exhibiting ER -antagonistic activity might
a
-agonistic activity. Interestingly, sulfur
-agonistic activity at 10 M. Some
M. The
a
l
3 l
a
a
be formed by hydrolysis of the sulfonamide moieties of the
sulfamate derivatives in the medium.
The experimental results are summarized in Table 5. The lead
compounds, that is, carbon derivative 1 and sulfur derivative 2,
showed strong STS-inhibitory activity, but the putative metabolites
2.4. Calculation of the charge distribution
exhibited ER
containing compound 16a also exhibited potent STS-inhibitory
activity (IC50 = 0.17 M), comparable to that of the lead
compounds, whereas its putative metabolite 12a showed potent
ER -antagonistic activity (IC50 = 29.7 nM).
a-agonistic activity. On the other hand, the silicon-
To interpret the order of STS-inhibitory activity (sulfur > sili-
con > carbon derivatives), we examined the effect of introduction
of each atom in silico.
l
a
As shown in Figure 4, there is a difference in the distance (d)
between the aromatic rings depending upon the nature of the
introduced atom: the distance decreased in the order of 3a
(d = 3.08 Å) > 2 (d = 2.81 Å) > 1 (d = 2.56 Å) > 3b (d = 2.52 Å, figure
not shown). However, the order of STS-inhibitory activity was
2 > 3a > 1 > 3b. The STS-inhibitory activity of quaternary carbon
derivative 3b was considerably weaker than that of carbon deriva-
tive 1, and we speculated that the introduction of methyl groups at
the methylene moiety of 1 might be the reason for this. Based on
this assumption, we thought that the presence of the methyl
groups on silicon in compound 3a might be the reason why the
inhibitory activity of 3a was less than that of 2.
3. Conclusion
We designed and synthesized
a series of non-steroidal
DPM-type STS inhibitors possessing pro-estrogen antagonistic
character, that is, that would generate a metabolite with ER-antag-
onistic activity upon hydrolysis by STS. In STS-inhibitory activity
assay, we found that silicon derivatives showed greater STS-
inhibitory activity than the corresponding carbon derivatives.
Among them, silicon-containing compound 16a exhibited potent
STS-inhibitory activity (IC50 = 0.17
compounds 1 and 2. Further the putative metabolite of 16a, that is,
12a, showed potent ER -antagonistic activity (IC50 = 29.7 nM) and
lacked ER -agonistic activity. Compounds with these properties
lM), comparable to that of lead
Next, we calculated the charge distributions of 1, 2, 3a and 3b.
As shown in Figure 5, the order of electropositivity of the central
atom between the two benzene rings was 3a > 2 > 3b > 1. This
a
a

2248
D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
Figure 5. Calculated charge distributions of 1, 2, 3a and 3b.
Table 3
Results of ER reporter gene assay of DPM derivatives
Compound
X
R1
R2
ER
a
ERb
a
b
a
b
EC50 (nM)
IC50 (nM)
EC50 (nM)
IC50 (nM)
6
7
9a
9b
11a
11b
12a
12b
13a
13b
14a
14b
C
S
Si
C
Si
C
Si
C
Si
C
Si
C
H
—
Me
Me
Et
Et
Et
Et
n-Pr
n-Pr
n-Bu
n-Bu
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
909c
22.6%d
N.A.
1910c
63.8%c
33.5%e
1115c
11.9%e
9.3%e
N.A.c
N.A.
N.A.
N.A.
37.8%e
25.3%e
28.7%e
7.1%e
N.A.
50.9%d
36.1%d
115
N.A.
53.0%f
93c
118
29.7
3.53
26.2
N.A.c
N.A.
53.5%d
264c
804
N.A.
5.6%e
N.A.
4.9%e
6.1%e
N.A.c
4.92
140c
N.A.c
146c
N.A.c
N.A.
30%c,d
14a
N.A.
N.A.: no activity.
a
ER-agonistic activity.
b
ER-antagonistic activity.
c
Data from our previous paper.12
d
e
f
% inhibition at 3
% of maximal activation of E2 at 10
% inhibition at 1 M.
lM.
l
M.
l
are candidate therapeutic agents for postmenopausal breast
cancer.
NMR are reported in parts per million (ppm) downfield from
tetramethylsilane (d) and coupling constants are given in hertz
(Hz). The following abbreviations are used for spin multiplicity:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad. 13C NMR chemical shifts are reported in ppm relative
to the centerline of the triplet at 77.0 ppm of deuteriochloroform.
Mass spectra were recorded on a JEOL JMS-HX110 spectrometer.
Melting points (mp) were determined on a MP-J3 melting point
apparatus (Yanaco, Japan). Routine thin layer chromatography
(TLC) and preparative TLC (PTLC) were performed on silica gel 60
4. Experimental
4.1. Chemistry
4.1.1. General
1H NMR spectra were recorded on JEOL JNM-GX500 and
JNME-CA-500 (500 MHz) spectrometers. Chemical shifts for 1H

D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
2249
Table 4
Results of ER reporter gene assay of DPM derivatives
Compound
X
R1
R2
ER
a
ERb
Antagonistic activity (%) at 3 lM
Agonistic activity (%) at 10
l
M
Antagonistic activity (%) at 3
lM
Agonistic activity (%) at 10
l
M
1
2
3a
3b
15a
15b
16a
16b
17a
17b
18a
C
S
H
—
H
H
H
H
H
H
—
55.3
37.5
N.A.
6.63
100
53.2
94.8
36.7
87.1
54.9
16.4
—
2.25
N.A.
N.A.
7.79
62.8
49.3
47.2
49.3
18.0
43.9
N.A.
100
10.8
9.50
N.A.
N.A.
82.8
16.7
N.A.
1.10
N.A.
N.A.
N.A.
—
Si Me
Me
Si Et
Et
Si Et
Et
Si n-Pr
n-Pr
C
C
Me N.A.
Me N.A.
Me
Me
Me
C
—
—
—
C
—
—
Si n-
Bu
18b
C
n-
Me
—
42.5
—
N.A.
Bu
N.A.: no activity
Table 5
Summary of experimental results
Compound
X
R1
R2
STS inhibition IC50
(lM)
Activity of the metabolite on ERa
1
2
16a
C
S
Si
H
—
Et
H
H
Me
1.024
0.088
0.170
Agonist
Agonist
Antagonist (ERa: IC50 = 29.7 nM)
F254 plates (Merck, Germany). Flash column chromatography was
4.1.1.3.
(12a).
4,40-(Diethylsilanediyl)bis(2-methylphenol)
To a solution of 4-bromo-o-cresol (6.0 g, 32 mmol) in
performed on Silica gel 60 (spherical, particle size 40–100
Kanto).
lm;
dry tetrahydrofuran (36 mL) was added n-butyl lithium in
n-hexane (2.69 M, 27.6 mL, 75.6 mmol) under an Ar atmosphere
at ꢀ78 °C. The mixture was stirred at the same temperature for
4.1.1.1. 4,40-(Dimethylsilanediyl)diphenol (9a).
To a solution
of 4-bromophenol (3.82 g, 22.1 mmol) in dry tetrahydrofuran
(25 mL) was added n-butyl lithium in n-hexane (2.69 M, 19 mL,
51.5 mmol) under an Ar atmosphere at ꢀ78 °C. The mixture was
stirred at room temperature for 1 h. To this was added dichlorodi-
methylsilane (0.88 mL, 7.3 mmol) at ꢀ78 °C, and stirring was con-
tinued at room temperature for 5 h. The reaction was quenched
with NH4Cl aq, and the whole was extracted with ethyl acetate.
The extract was successively washed with water and brine, and
then dried over MgSO4. The solvent was evaporated, and the resi-
due was purified by means of silica gel column chromatography
(hexane/ethyl acetate = 9:1 to 2:1) to give 9a (0.81 g, 45.4%) as a
white solid. Mp: 172–174 °C (lit. 174 °C). 1H NMR (500 MHz,
CD3OD): d 7.30 (4H, d, J = 8.6 Hz), 6.76 (4H, d, J = 8.6 Hz), 0.43
(6H, s). 13C NMR (500 MHz, CD3OD): d 159.41, 136.63, 129.22,
115.90. MS (FAB+) m/z: 245 (M+H+).
1 h, and then added to a solution of dichlorodiethylsilane
(1.6 mL, 10.6 mmol) in dry tetrahydrofuran (1.6 mL) at ꢀ78 °C.
Stirring was continued at room temperature for 5.5 h. The reaction
was quenched with NH4Cl aq, and the whole was extracted with
ethyl acetate. The extract was successively washed with water
and brine, and then dried over MgSO4. The solvent was evaporated,
and the residue was purified by means of silica gel column
chromatography (hexane/ethyl acetate = 9:1 to 5:1) to give 12a
(1.24 g, 39.1%) as
a
white solid. Mp: 110–112 °C. 1H NMR
(500 MHz, CD3OD): d 7.15 (2H, s), 7.11 (2H, d, J = 7.9 Hz), 6.73
(2H, d, J = 7.9 Hz), 2.16 (6H, s), 0.96–0.94 (10H, m). 13C NMR
(500 MHz, CD3OD): d 157.51, 138.54, 134.85, 127.19, 124.92,
115.21, 16.22, 7.85, 5.32. MS (FAB+) m/z: 300 (M+).
4.1.1.4.
(19).
Bis(4-(benzyloxy)-3-methylphenyl)dipropylsilane
The title compound was prepared according to the
4.1.1.2. 4,40-(Diethylsilanediyl)diphenol (11a).
The title
procedure described for 9a, starting from dichlorodi-n-propylsi-
lane (4d) and 1-(benzyloxy)-4-bromo-2-methylbenzene (5c) (y.
4.29%). Yellow oil. 1H NMR (500 MHz, CDCl3): d 7.45 (4H, d,
J = 7.4 Hz), 7.39 (4H, t, J = 7.4 Hz), 7.34–7.31 (2H, m), 7.27 (4H,
m), 6.88 (2H, d, J = 8.0 Hz), 5.08 (4H, s), 2.27 (6H, s), 1.44–1.34
(4H, m), 1.03–0.95 (10H, m). 13C NMR (500 MHz, CDCl3): d
157.75, 137.49, 137.18, 133.89, 128.49, 127.79, 127.72, 127.11,
compound was prepared according to the procedure described
for 9a, starting from dichlorodiethylsilane (y. 11.8%). Colorless
oil. 1H NMR (500 MHz, CDCl3): d 7.36 (2H, d, J = 7.5 Hz), 6.82 (2H,
d, J = 7.5 Hz), 4.88 (2H, s), 1.02–0.97 (10H, m). 13C NMR
(500 MHz, CDCl3): d 156.44, 136.64, 127.63, 115.02, 7.51, 4.30.
MS (FAB+) m/z: 272 (M+).

2250
D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
126.25, 110.66, 69.52, 18.58, 17.47, 16.50, 15.75. HRMS (FAB+) m/z:
0.17 mmol) in dry N,N-dimethylformamide (1 mL) was added so-
dium hydride (60% suspension in mineral oil, 34.4 mg, 0.79 mmol)
at room temperature, and the mixture was stirred at the same tem-
perature for 1 h. To this was added methyl iodide (0.054 mL,
0.87 mmol), and stirring was continued at room temperature for
19 h. The reaction was quenched with H2O, and the whole was
extracted with ethyl acetate. The extract was successively washed
with water and brine, and then dried over MgSO4. The solvent was
evaporated, and the residue was purified by means of silica gel
column chromatography (hexane/ethyl acetate = 4:1) to afford a
white solid. This was recrystallized from hexane/ethyl acetate
to give 10 (12.9 mg, 16.1%) as colorless needles. Mp: 128–129 °C.
calcd for C34H40O2Si 508.2798, found 508.2758 (M+).
4.1.1.5. 4,40-(Dipropylsilanediyl)bis(2-methylphenol) (13a).
To a mixture of bis(4-(benzyloxy)-3-methylphenyl)dipropylsilane
(19) (747 mg, 1.47 mmol), ethanol (0.5 mL) and ethyl acetate
(1.5 mL) was added 5% Pd/C (37.4 mg) at room temperature, and
the mixture was stirred under an H2 atmosphere at the same tem-
perature for 18 h. The reaction mixture was filtered through a Cel-
ite pad and the solvent was evaporated. The residue was purified
by PTLC (hexane/ethyl acetate = 4:1) to give 13a (18.4 mg, 3.80%)
as a white solid. Mp: 99–101 °C. 1H NMR (500 MHz, CD3OD): d
7.04 (2H, s), 6.99 (2H, d, J = 8.0 Hz), 6.62 (2H, d, J = 8.0 Hz), 2.06
(6H, s), 1.28–1.19 (4H, m), 0.85 (10H, m). 13C NMR (500 MHz, CD3-
OD): d 157.50, 138.49, 134.79, 127.46, 124.88, 115.15, 18.88, 18.57,
16.98, 16.27. MS (FAB+) m/z: 328 (M)+. HRMS (FAB+) m/z: calcd for
1H NMR (500 MHz, CDCl3):
d 7.51 (4H, d, J = 8.6 Hz), 7.27
(4H, d, J = 8.6 Hz), 2.98 (12H, s), 0.55 (6H, s). 13C NMR (500 MHz,
CDCl3): d 151.33, 136.46, 135.81, 121.20, 38.86, -2.25. HRMS (FAB+)
m/z: calcd for C18H27N2O6S2Si 459.1080, found 459.1077 (M+H+).
C
20H28O2Si 328.1859, found 328.1851 (M+).
4.1.1.10. (Diethylsilanediyl)bis(4,1-phenylene) bis(sulfamate)
4.1.1.6.
4,40-(Dibutylsilanediyl)bis(2-methylphenol)
(14a).
(15a).
cedure
The title compound was prepared according to the pro-
described for 3a, starting from
The title compound was prepared according to the procedure de-
scribed for 9a, starting from 4-bromo-o-cresol and dichlorodibu-
tylsilane (y. 2.00%). Colorless oil. 1H NMR (500 MHz, CDCl3): d
6.93 (2H, s), 6.87 (2H, d, J = 8.0 Hz), 6.67 (2H, d, J = 8.0 Hz), 4.61
(2H, s), 2.50 (4H, t, J = 8.0 Hz), 2.22 (6H, s), 1.58–1.51 (4H, m),
1.38–1.29 (4H, m), 0.91 (6H, t, J = 7.4 Hz). 13C NMR (500 MHz,
CDCl3): d 151.64, 135.13, 130.97, 126.78, 123.33, 114.67, 34.73,
33.95, 22.32, 15.73, 13.94. HRMS (FAB+) m/z: calcd for C22H32O2Si
356.2172, found 356.2151 (M+).
4,40-(diet-
hylsilanediyl)diphenol (11a) (y. 24.8%). Colorless crystals. Mp:
175–176 °C. 1H NMR (500 MHz, CD3OD): d 7.55 (4H, d, J = 8.6 Hz),
7.32 (4H, d, J = 8.6 Hz), 1.11 (4H, t, J = 7.7 Hz), 0.98 (6H, q,
J = 7.7 Hz). 13C NMR (500 MHz, CD3OD): d 153.17, 137.33, 135.52,
122.83, 7.57, 4.64. HRMS (FAB+) m/z: calcd for C16H23N2O6S2Si
431.0769, found 431.0767 (M+H+).
4.1.1.11.
bis(sulfamate) (16a).
(Diethylsilanediyl)bis(2-methyl-4,1-phenylene)
The title compound was prepared
4.1.1.7. (Dimethylsilanediyl)bis(4,1-phenylene) bis(sulfamate)
(3a).
To a solution of 4,40-(dimethylsilanediyl)diphenol (9a)
according to the procedure described for 3a, starting from
4,40-(diethylsilanediyl)bis(2-methylphenol) (12a) (y. 25.1%). White
(200 mg, 0.82 mmol) in N,N-dimethylacetamide (1 mL) was added
sulfamoyl chloride (283 mg, 2.46 mmol) at 0 °C. The mixture was
stirred at room temperature for 3 h. The reaction was quenched
with H2O aq, and the whole was extracted with ethyl acetate.
The extract was successively washed with water and brine, and
then dried over MgSO4. The solvent was evaporated, and the resi-
due was purified by means of silica gel column chromatography
(hexane/ethyl acetate = 2:1 to 1:1) to afford a white solid. This
was recrystallized from chloroform/hexane to give 3a (154.4 mg,
46.8%) as colorless crystals. Mp: 154–155 °C. 1H NMR (500 MHz,
CD3OD): d 7.07 (4H, d, J = 8.6 Hz), 6.81 (4H, d, J = 8.6 Hz), 0.56
(6H, s). 13C NMR (500 MHz, CD3OD): d 153.13, 137.66, 136.63,
122.84. HRMS (FAB+) m/z: calcd for C14H19N2O6S2Si 403.0454,
found 403.449 (M+H+).
solid. Mp: 155–156 °C. 1H NMR (500 MHz, CD3OD):
d 7.37
(2H, s), 7.34–7.32 (4H, m), 2.32 (6H, s), 1.09–0.99 (4H, m), 0.98–
0.96 (6H, m). 13C NMR (500 MHz, CD3OD): d 151.68, 138.99,
135.57, 134.65, 132.15, 122.67, 16.65, 7.62, 1.62. HRMS (FAB+) m/z:
calcd for C18H26N2O6S2SiNa 481.0899 found 481.0906 (M+Na+).
4.1.1.12.
bis(sulfamate) (17a).
(Dipropylsilanediyl)bis(2-methyl-4,1-phenylene)
The title compound was prepared
according to the procedure described for 3a, starting from 4,40-
(diethylsilanediyl)diphenol (13a) (y. 1.35%). White solid. Mp:
100–102 °C. 1H NMR (500 MHz, CD3OD): d 7.27 (2H, d, J = 8.0 Hz),
7.25 (2H, s), 7.23 (2H, dd, J = 8.0, 1.1 Hz), 2.24 (6H, s), 1.36 (4H,
m), 1.09 (4H, m), 0.98 (6H, t, J = 7.2 Hz). 13C NMR (500 MHz, CD3-
OD): d 149.68, 138.21, 135.70, 133.72, 130.60, 121.11, 29.69,
18.44, 17.23, 16.59, 15.05. HRMS (FAB+) m/z: calcd for C20H30N2O6-
S2Si 486.1315, found 486.1329 (M+).
4.1.1.8. 4-((4-Hydroxyphenyl)dimethylsilyl)phenyl sulfamate
(8).
To a solution of 4,40-(dimethylsilanediyl)diphenol (9a)
(24 mg, 0.98 mmol) in N,N-dimethylacetamide (1 mL) was added
sulfamoyl chloride (135.9 mg, 1.18 mmol) at 0 °C. The mixture
was stirred at room temperature for 4 h. The reaction was
quenched with H2O aq, and the whole was extracted with ethyl
acetate. The extract was successively washed with water and brine,
and then dried over MgSO4. The solvent was evaporated, and the
residue was purified by means of silica gel column chromatogra-
phy (chloroform/methanol = 40:1) and PTLC (hexane/ethyl ace-
tate = 2:1) to give 8 (42.7 mg, 13.4%) as a colorless oil. 1H NMR
(500 MHz, CDCl3): d 7.54 (1H, dd, J = 6.6, 2.0 Hz), 7.39 (1H, dd,
J = 6.6, 2.0 Hz), 7.29 (1H, dd, J = 6.6, 2.0 Hz), 6.84 (1H, dd, J = 6.6,
2.0 Hz), 4.90 (3H, br s), 0.52 (6H, s). 13C NMR (500 MHz, CDCl3): d
156.78, 150.87, 138.54, 135.93, 135.89, 128.46, 121.41, 115.22, -
2.16. HRMS (FAB+) m/z: calcd for C14H17NO4SSiNa 346.0545, found
346.0558 (M+Na+).
4.1.1.13. (Dibutylsilanediyl)bis(2-methyl-4,1-phenylene) bis
(sulfamate) (18a).
The title compound was prepared
according to the procedure described for 3a, starting from 4,40-
(dibutylsilanediyl)bis(2-methylphenol) (14a) (y. 33.8%). White
solid. Mp: 55–56 °C. 1H NMR (500 MHz, CD3OD): d 7.22 (2H, d,
J = 8.6 Hz), 7.07 (2H, s), 7.01 (2H, d, J = 8.6 Hz), 2.57 (4H, t,
J = 7.7 Hz), 1.59–1.58 (4H, m), 1.37–1.30 (4H, m), 0.93 (4H, t,
J = 7.4 Hz). 13C NMR (500 MHz, CD3OD): d 148.58, 142.49, 132.41,
132.40, 127.78, 123.01, 35.97, 34.97, 23.30, 16.67, 14.25. MS
(FAB+) m/z: not detected.
4.1.1.14.
(1).
Methylenebis(4,1-phenylene)
The title compound was prepared according to the proce-
bis(sulfamate)
dure described for 3a, starting from 4,40-methylenediphenol (6) (y.
81.6%). Colorless crystals. Mp: 173–175 °C. 1H NMR (500 MHz, CD3-
OD): d 7.26 (4H, dd, J = 6.6, 2.3 Hz), 7.22 (4H, dd, J = 6.6, 2.3 Hz),
3.99 (2H, s). 13C NMR (500 MHz, CD3OD): d 150.49, 141.10,
131.00, 123.45, 41.44. MS (FAB+) m/z: not detected.
4.1.1.9.
ethylsulfamate)
(Dimethylsilanediyl)bis(4,1-phenylene)
(10). To solution
bis(dim-
a
of
(dim-
ethylsilanediyl)bis(4,1-phenylene) bis(sulfamate) (3a) (70.2 mg,

D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
2251
4.1.1.15. Propane-2,2-diylbis(4,1-phenylene) bis(sulfamate)
(3b). The title compound was prepared according to the pro-
the combined cell pellets from several culture flasks, followed by
homogenization in a homogenizer. The homogenate was diluted
with Tris–EDTA (TE) buffer and centrifuged (15,000g, 4 °C,
15 min). We quantified the protein concentration of the superna-
tant by the Bradford method, and adjusted the concentration to
0.2 mg/mL by adding TE buffer. We used this solution as the STS
solution.
cedure described for 3a, starting from 4,4’-(propane-2,2-
diyl)diphenol (9b) (y. 26%). White solid. Mp: 199–201 °C. 1H
NMR (500 MHz, CD3OD): d 7.30 (8H, dd, J = 6.8, 2.2), 7.22 (8H, dd,
J = 6.8, 2.2), 1.69 (6H, s). 13C NMR (500 MHz, CD3OD): d 148.85,
148.71, 127.74, 121.56, 42.25, 29.89. HRMS (FAB+) m/z: calcd for
C
15H18N2O6S2 386.0606, found 386.0621 (M+).
STS activity was determined by measuring the total of labeled
estrone ([3H]E1) and estradiol ([3H]E2) formed from labeled
estrone sulfate ([3H]E1S, PerkinElmer Co., Ltd). Mixtures of STS
4.1.1.16. Pentane-3,3-diylbis(4,1-phenylene) bis(sulfamate)
(15b). The title compound was prepared according to the
solution (300
(10 L) were incubated at 37 °C for 5 h. Then, 1 mL of toluene
was added to the reaction mixture. Each mixture was shaken and
l lCi) and each inhibitor
L), [3H]E1S (final: 10 nM, 1
procedure described for 3a, starting from 4,4’-(pentane-3,3-diyl)
l
diphenol (11b) (y. 26%). White solid. Mp: 230–232 °C. 1H NMR
(500 MHz, CD3OD):
d
7.24 (4H, d, J = 9.2 Hz), 7.21 (4H, d,
centrifuged to allow phase separation. The organic layer (600 lL)
J = 9.2 Hz), 2.17 (4H, q, J = 7.3 Hz), 0.64 (6H, t, J = 7.3 Hz). 13C NMR
(500 MHz, CD3OD): d 147.89, 146.12, 128.91, 121.39, 48.86, 28.41,
8.26. HRMS (FAB+) m/z: calcd for C17H22N2O6S2 414.0919, found
414.0894 (M+).
containing [3H]E1 and [3H]E2 was added to 4 mL of scintillation
cocktail. The radioactivity of each solution was measured in a
liquid scintillator. The inhibitory activity in the presence of
1 lM STX64 was defined as 100%. The assay was performed in
triplicate.
4.1.1.17. Pentane-3,3-diylbis(2-methyl-4,1-phenylene) bis(sul-
famate) (16b).
The title compound was prepared according
4.3. Reporter gene assay
to the procedure described for 3a, starting from 4,4’-(pentane-3,3-
diyl)bis(2-methylphenol) (12b) (y. 45%). Yellow solid. Mp: 140–
142 °C. 1H NMR (500 MHz, CD3OD): d 7.14 (2H, d, J = 8.6 Hz),
6.98–6.92 (4H, m), 2.20 (6H, s), 2.03 (4H, q, J = 7.3 Hz), 0.52 (6H, t,
J = 7.3 Hz). 13C NMR (500 MHz, CD3OD): d 176.57, 176.24, 160.20,
159.97, 155.82, 150.56, 78.46, 58.15, 45.07, 36.81. HRMS (FAB+)
m/z: calcd for C19H26N2O6S2 442.1232, found 442.1255 (M+).
ER-agonistic and antagonistic activities of compounds were
evaluated by means of reporter gene assay using a Gal4-human
ER
serted into the pCMX-GAL4 vector to obtain pCMX-GAL4-hER
(pCMX-flag vector to make pCMX-ER /b). GAL4-responsive
a a/b was in-
or ERb reporter system.25 A fragment of human ER
a
MH100 (USA)x4-tk-LUK reporter was used. Human embryonic kid-
ney (HEK 293) cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) without Phenol Red containing 5% fetal bovine
serum (FBS) and antibiotic–antimycotic (Nacalai) at 37 °C in a
humidified atmosphere of 5% CO2 in air. Transfections were per-
formed by the calcium phosphate coprecipitation method. Test
compounds were added at 8 h after transfection. Cells were har-
vested 16–20 h after the treatment, and luciferase and b-galactosi-
dase activities were assayed using a luminometer and a microplate
reader. DNA cotransfection experiments were done with 50 ng of
reporter plasmid, 15–20 ng pCMX-s-galactosidase, 10–15 ng of
each receptor expression plasmid and pGEM carrier DNA to make
a total of 150 ng DNA per well in a 96-well plate. Luciferase data
were normalized to an internal b-galactosidase control, and re-
ported values are means of triplicate assays. Antagonist activity
was measured in the presence of 0.3 nM 17b-estradiol.
4.1.1.18. Heptane-4,4-diylbis(2-methyl-4,1-phenylene) bis(sul-
famate) (17b).
The title compound was prepared according to
the procedure described for 3a, starting from 4,4’-(heptane-4,4-
diyl)bis(2-methylphenol) (13b) (y. 7.4%). White solid. Mp: 180–
182 °C. 1H NMR (500 MHz, CD3OD): d 7.14 (2H, d, J = 8.6 Hz), 6.98
(2H, s), 6.93 (2H, d, J = 8.6 Hz), 2.20 (6H, s), 1.97–1.93 (4H, m),
0.91–0.86 (4H, m), 0.77 (6H, t, J = 6.9 Hz). 13C NMR (500 MHz, CD3-
OD): d 148.53, 148.40, 131.82, 131.79, 127.47, 122.44, 49.87, 41.06,
18.29, 16.92, 14.99. HRMS (FAB+) m/z: calcd for C21H30N2O6S2
470.1545, found 470.1541 (M+).
4.1.1.19. Nonane-5,5-diylbis(2-methyl-4,1-phenylene) bis(sulfa-
mate) (18b).
The title compound was prepared according to
the procedure described for 3a, starting from 4,4’-(nonane-5,5-
diyl)bis(2-methylphenol) (14b) (y. 31%). White solid. Mp: 75–
77 °C. 1H NMR (500 MHz, CD3OD): d 7.23 (2H, d, J = 8.6 Hz), 7.07
(2H, d, J = 2.3 Hz), 7.02 (2H, d, J = 8.6, 2.3 Hz), 2.29 (6H, s), 1.27
(4H, m), 0.97–0.91 (4H, m), 0.83 (6H, t, J = 7.2). 13C NMR
4.4. Computational methods
All calculations were carried with the Gaussian 09 program
package.26 The molecular structures and harmonic vibrational
frequencies were obtained using the hybrid density functional
method based on Becke’s three-parameter exchange function and
the Lee–Yang–Parr nonlocal correlation functional (B3LYP).27–29
We used 6-31+G⁄ basis set for the all atoms. Geometry optimiza-
tion and vibrational analysis were performed at the same level.
All stationary points were optimized without any symmetry
assumptions, and characterized by normal coordinate analysis at
the same level of the theory (the number of imaginary frequency,
NIMAG, 0 for minima). The natural charges were calculated with
the NBO 5.9 program package30,31 on Gaussian 09 at the same level
as used for geometry optimization.
(500 MHz, CDCl3):
d 147.73, 146.51, 130.89, 130.33, 126.69,
120.96, 48.49, 37.14, 25.99, 23.27, 16.88, 14.01. HRMS (FAB+) m/
z: calcd for C23H34N2O6S2 498.1858, found 498.1880 (M+).
4.1.1.20. Thiobis(4,1-phenylene) bis(sulfamate) (2).
The ti-
tle compound was prepared according to the procedure described
for 3a, starting from 4,40-methylenediphenol (7) (y. 4.6%). White so-
lid. Mp: 150–152 °C. 1H NMR (500 MHz, CD3OD): d 7.30 (4H, dd,
J = 6.3, 2.0 Hz), 7.19 (4H, dd, J = 6.3, 2.0 Hz). 13C NMR (500 MHz,
CD3OD): d 151.49, 135.21, 133.51, 124.43. HRMS (FAB+) m/z: calcd
for C12H12N2O6S3 375.9858, found 375.9897 (M+).
4.2. STS-inhibitory activity assay
Acknowledgments
The STS-inhibitory activity assay was performed essentially
according to the reported method.24 Briefly, MCF-7 cells were cul-
tured in D-MEM (Minimum Essential Medium) supplemented with
10% FBS at 37 °C under 5% CO2 in air. Homogenate of MCF-7 cells
was prepared by repeated (five times) freezing and thawing of
The work described in this paper was partially supported by a
Grant-in-aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Sciences and Technology, Japan, and a grant
from the Japan Society for the Promotion of Science.

2252
D. Kajita et al. / Bioorg. Med. Chem. 22 (2014) 2244–2252
Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.;
Supplementary data
Li, X.; Knox, J.E.; Hratchian, H.P.; Cross, J.B.; Bakken, V.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratmann, R.E.; Yazyev, O.; Austin, A.J.; Cammi, R.; Pomelli, C.;
Ochterski, J.W.; Ayala, P.Y.; Morokuma, K.; Voth, G.A.; Salvador, P.;
Dannenberg, J.J.; Zakrzewski, V.G.; Dapprich, S.; Daniels, A.D.; Strain, M.C.;
Farkas, O.; Malick, D.K.; Rabuck, A.D.; Raghavachari, K.; Foresman, J.B.; Ortiz, J.
V.; Cui, Q.; Baboul, A.G.; Clifford, S.; Cioslowski, J.; Stefanov, B.B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R.L.; Fox, D.J.; Keith, T.; Al-
Laham, M.A.; Peng, C.Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.M. W.;
Johnson, B.; Chen, W.; Wong, M.W.; Gonzalez, C.; and Pople, J.A.; Gaussian Inc,
Wallingford CT, Gaussian 09 (Revision-A.01), Gaussian: Pittsburgh, PA, 2009.
21. Møller, C.; Plesset, M. S. Phys. Rev. 1934, 46, 618.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.025.
References and notes
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