Increased hydrophobicity and estrogenic activity of simple phenols with silicon and germanium-containing substituents

Article abstract of DOI:10.1021/jm3013757

Here, we report the systematic synthesis and characterization of simple phenols bearing a trialkyl(aryl)silyl or trialkyl(aryl)germyl functional group as a hydrophobic substituent. These silicon and germanium analogues exhibited higher hydrophobicity than

Full text of DOI:10.1021/jm3013757

Article
pubs.acs.org/jmc
Increased Hydrophobicity and Estrogenic Activity of Simple Phenols
with Silicon and Germanium-Containing Substituents
Shinya Fujii, Yu Miyajima, Hiroyuki Masuno, and Hiroyuki Kagechika*
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo
101-0062, Japan
ABSTRACT: Here, we report the systematic synthesis and
characterization of simple phenols bearing a trialkyl(aryl)silyl
or trialkyl(aryl)germyl functional group as a hydrophobic
substituent. These silicon and germanium analogues exhibited
higher hydrophobicity than the corresponding carbon
analogues, with a difference in log P value of approximately
0.6, independent of the alkyl(aryl) species. Trimethylsilylphe-
nol and trimethylgermylphenol exhibited smaller pKa values
than the corresponding carbon analogue or unsubstituted
phenol, indicating that trialkylsilyl and trialkylgermyl func-
tional groups have a negative substituent constant (σ). The
trialkylsilyl- and trialkylgermylphenols exhibited more potent estrogenic activity as compared with the carbon analogues. The
substituent parameters and structure−activity relationship reported here may be helpful for drug discovery utilizing the heavier
group 14 elements.
INTRODUCTION
■
Hydrophobic interaction, as well as hydrogen bonding and
other polar interactions, plays an essential role in the
interactions between bioactive compounds and their target
proteins.1 Thus, the application of novel hydrophobic
structures is expected to be useful in the development of
bioactive compounds. For example, we have demonstrated the
utility of carboranes (carbon-containing boron clusters)2 as the
hydrophobic pharmacophore of specific ligands for several
nuclear receptors, including retinoid receptors,3 vitamin D
receptor,4 estrogen receptor (ER),5 and androgen receptor
(AR).6 In these examples, the boron cluster interacts with
hydrophobic amino acid residues of the ligand-binding pocket
of the receptors.4,5 Thus, we considered that the introduction
Figure 1. Examples of sila-substituted bioactive substances.
of other hydrophobic moieties might elicit distinctive chemical
and pharmacological properties.
One strategy for the development of drug candidates bearing
corresponding carbon analogues,7,11 there have been few
novel hydrophobic substructures is sila-substitution of hydro-
carbons.7 Several silicon-containing analogues of bioactive
investigations to compare the hydrophobicity of alkylsilicon
compounds, such as retinoids Am555S (1)8 and sila-Am80
and alkylgermanium substituents. Because systematic determi-
nation of hydrophobicity parameters of these heavier group 14
atom-containing substituents could be useful for the design of
novel drug candidates, we set out to synthesize a series of
(2)9 and the topoisomerase inhibitor karenitecin (BNP1350)
3,10 have been reported, and these compounds exhibit unique
properties distinct from those of the parent carbon analogues
simple phenols bearing a trialkyl(aryl)silyl or trialkyl(aryl)-
(Figure 1). Germanium, one of the heavier group 14 atoms, has
germyl functional group and to compare their hydrophobicity,
also been considered as a possible component of bioactive
substituent constant, and biological activity with those of the
compounds, but there are only a few reports describing
carbon analogues.
As silicon and germanium-containing compounds, we
biologically active germanium-containing molecules. The
characteristic properties of these silicon- and germanium-
designed 4-trialkyl(aryl)silylphenols and 4-trialkyl(aryl)-
containing derivatives result at least partly from the change of
their inherent hydrophobicity compared with the correspond-
ing carbon analogues. Although it is known that the
hydrophobicity of silicon analogues is higher than that of
Received: September 22, 2012
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
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gemylphenols (Figure 2). In addition, because 4-substituted
phenols are pharmacophores for estrogen receptor (ER)
hydroxyl group was protected with a TBS group and then the
triphenylsilyl group was introduced. The carbon analogue 7
corresponding to triethylsilylphenol 8 and triethylgermylphenol
9 was synthesized from phenol by alkylation under acidic
conditions. Other carbon analogues 4, 10, 13, and 15 are
commercially available and were purchased.
Hydrophobicity. The hydrophobicity of compounds 4−16
was determined as the octanol−water partition coefficient (P)
using an HPLC method.13 Table 1 summarizes the log P values
Table 1. Hydrophobicity Parameter of 4-Substituted Phenols
Figure 2. Structures of compounds investigated in this study.
ligands,12 we aimed to investigate the structure (element)−
estrogenic activity relationship of the designed compounds.
RESULTS AND DISCUSSION
Synthesis. Synthesis of the silicon and germanium-
containing phenols is summarized in Scheme 1. Lithiation of
a
■
compd
R1, R2, R3
Me3
El
log P
π
phenol
4
1.46
3.50
4.12
4.11
5.03
5.69
5.71
b
b
C
+2.04 (+1.98)
+2.66 (+2.59)
+2.65
5
Si
a
Scheme 1. Synthesis of the Designed 4-Substituted Phenols
6
Ge
C
7
Et3
+3.57
8
Si
+4.23
9
Ge
C
+4.25
c
10
11
12
13
14
15
16
Ph3
6.25
+4.79
c
Si
6.83
+5.37
c
Ge
C
6.97
+5.51
Me2Et
Me2Ph
4.04
4.64
4.31
4.91
+2.58
Si
+3.18
C
+2.85
Si
+3.45
a
Hansch−Fujita hydrophobicity parameter of each substituent.
b
c
Values cited from ref 11. These log P values are tentative because
the hydrophobicity was beyond the range of determination of log P by
the HPLC method (0 < log P < 6).
of the compounds and the Hansch−Fujita hydrophobicity
parameter π of the substituents,14 which was deduced by
subtraction of the log P value of phenol (1.46) from the
observed log P value. Among the trimethyl derivatives 4−6,
silicon analogue 5 exhibited a log P value of 4.12, which is 0.62
larger than that of carbon analogue 4. The hydrophobicity
parameter π of the trimethylsilyl group was 2.66, which is close
to the reported value.11 Trimethylgermylphenol 6 exhibited a
log P value of 4.11, which is almost the same as that of 5 and
0.61 larger than that of 4. As for the other derivatives, such as
triethyl and triphenyl derivatives, the silicon analogues also
exhibited larger log P values than the corresponding carbon
analogues with differences in the log P value of 0.5−0.7. The
differences of log P values between the silicon analogues and
corresponding germanium analogues were again small. It is
interesting that the differences of log P values between silicon
analogues and the corresponding carbon analogues were
approximately the same, independent of the substituents on
these elements. The difference of hydrophobicity can be mainly
attributed to difference of bond length of the central atom, and
so it is reasonable that the germanium analogues exhibited log
P values similar to those of corresponding silicon analogues
(covalent radii: C, 77 pm; Si, 117 pm; Ge, 122 pm).
a
Conditions: (a) n-BuLi, trialkylsilyl chloride, or trialkylgermyl
chloride, THF, −78 to 0 °C, 31% quant; (b) t-BuLi, trialkylsilyl
chloride, or trialkylgermyl chloride, THF, −78 to 0 °C, 59% for 6; (c)
TBAF, THF, 0 °C; (d) TBSCl, imidazole, DMF, rt, 78%; (e) t-BuLi,
triphenylsilyl chloride, THF, −78 °C to rt; (f) TBAF, THF, 0 °C, 50%
from 18; (g) 3-ethyl-3-pentanol, TFA, rt, 86%.
4-bromophenol 17 followed by reaction with trialkylsilyl
chloride or trialkylgermyl chloride gave the desired 4-
trialkylsilylphenols and 4-trialkylgermylphenols. A silyl or
germyl group was also introduced at the phenolic oxygen of
17, but this was usually removed under aqueous workup
conditions. However, in the case of triethylsilylphenol 8, the
triethylsilyl group on the phenolic oxygen remained after
workup and had to be removed by hydrolysis. Introduction of
the triphenylsilyl group into 17 did not proceed, so first the
Acidity. The acidity of the phenolic hydroxyl group was also
determined for selected compounds (Table 2). The pKa values
B
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Journal of Medicinal Chemistry
Article
and triethylgermylphenol 9 exhibited potent activity with EC50
values of 3.4 and 2.1 nM, respectively, being more potent than
the corresponding carbon analogue 7 (EC50 = 23 nM).
Regarding other trialkyl derivatives, the silyl and germyl
derivatives also exhibited more potent activity than the
corresponding carbon analogues. The triphenyl derivatives 10,
11, and 12 all exhibited only moderate MCF-7 cell
proliferation-promoting activity. These results suggest that
increase of hydrophobicity of the trialkyl substructure (R3El−)
increases affinity for ER, but a large increase of hydrophobic
volume results in loss of full ER agonistic activity.
Table 2. pKa Values of Trimethylsilyl- and
Trimethylgermylphenols
compd
El
pKa
σa
phenol
10.44
10.54
10.06
10.20
Next, we examined transcriptional activity of compounds 7,
8, and 9 with potent activities in MCF-7 cell assay, using T47D-
Kbluc cell line that expresses an estrogen-responsive luciferase
reporter (Figure 3A).20 Triethylsilyl derivative 8 exhibited the
4
5
6
C
+0.10
−0.38
−0.24
Si
Ge
of silicon analogue 5 (10.06) and germanium analogue 6
(10.20) were smaller than that of phenol (10.44), indicating
that these substituents behave as electron-withdrawing groups.
It is well-known that pKa values and substituent constants σ are
affected by the solvent system used and depend on the
structure of compounds.15 Our results obtained in 20%
aqueous methanol were consistent with reported results for
benzoic acid derivatives and phenol derivatives in aqueous
ethanol.15,16
Estrogenic Activity. Estrogen receptor (ER) is a member
of the nuclear receptor superfamily, and its ligand is the
endogenous estrogen estradiol (E2).17 We have previously
reported that simple phenols bearing a hydrophobic substituent
at the 4-position act as ER ligands,18 and here we compared the
estrogenic activities of the silicon and germanium-containing
phenols with those of the parent phenols by assay of their
ability to promote estrogen-dependent proliferation of human
breast cancer cell line MCF-7 (Table 3).19 The trialkyl
derivatives exhibited marked MCF-7 cell proliferation-promot-
ing activity. Among the tested compounds, triethylsilylphenol 8
Table 3. Proliferation-Promoting Activity of 4-Substituted
Phenols toward MCF-7 Cells
Figure 3. (A) Luciferase assay of compounds 7−9 using T47D-Kbluc
cell line. (B) Competitive binding assay of compounds 7−9 using
human ERα and [3H]E2. The concentration of [3H]-E2 was 1.0 × 10−8
M.
most potent ER agonistic activity, and triethylgermyl derivative
9 also exhibited higher potency than that of carbon analogue 7.
We also examined the ER-binding affinity of the triethyl
derivatives. The binding affinity was examined by competitive
binding assay using [3H]E2 and human ERα (Figure 3B).21
Triethylsilylphenol 8 and triethylgermylphenol 9 exhibited
dose-dependent binding, and the affinities of these compounds
exceeded that of the carbon analogue 7 by approximately 1
order of magnitude. These results suggest that the increased
MCF-7 cell proliferation-promoting activity of these com-
pounds is induced by stronger binding to and activation of ER.
To understand the interaction between the hydrophobic
substructure of these simple phenols and the receptor surface,
we conducted docking simulations using the cocrystal structure
of hERα-LBD with estradiol (E2) (PDB ID: 1G50)22 by a
docking program AutoDock.23 Figure 4 shows the docking
models of silyl analogue 8. In the calculated structure, phenolic
hydroxyl group of 8 forms hydrogen bonds to residues of ER
(E353 and R394) in a similar manner to that in case of E2. The
a
compd
R
El
EC50 [nM]
4
Me3
C
870
63
5
Si
6
Ge
C
100
23
7
Et3
8
Si
3.4
2.1
9
Ge
C
b
10
11
12
13
14
15
16
Ph3
(16%)
b
Si
(29%)
b
Ge
C
(47%)
Me2Et
Me2Ph
190
41
Si
C
38
Si
16
a
EC50 values for MCF-7 cell proliferation-promoting activity were
evaluated. Cell proliferation assay was performed in triplicate (n = 3).
b
Maximum response in comparison with E2.
C
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Journal of Medicinal Chemistry
Article
1
Industries, and Kanto Chemical Co., Inc. H and 13C NMR spectra
were recorded on a Bruker AVANCE 400 spectrometer or a Bruker
1
AVANCE 500 spectrometer. Chemical shifts for H NMR and 13C
NMR are reported as parts per million (ppm) relative to chloroform
1
(7.26 ppm for H NMR and 77.00 ppm for 13C NMR). Data are
reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t,
triplet; q quartet; br, broad; and m, multiplet), coupling constants
(Hz), and integration. Melting points were taken on a Yanagimoto
micro melting point apparatus. Mass spectra were obtained with a
Daltonics microTOF-2focus and a JEOL AX-505 spectrometer. The
purity of compounds was determined by elemental analysis,
confirming ≥95% purity. Elemental analyses were carried out by
Yanaco MT-6 CHNCORDER spectrometer.
Preparation of Compounds. General Procedure A for Silyl
Compounds 5, 14, and 16. Under argon atmosphere, a solution of n-
BuLi in n-hexane (3.0 equiv for p-bromophenol) was added dropwise
to a solution of p-bromophenol in THF at −78 °C, and the mixture
was stirred at room temperature for 1 h. Trialkyl(aryl)silyl chloride
(3.0 equiv for p-bromophenol) was added dropwise at −78 °C, and
the mixture was stirred at room temperature for several hours. The
reaction was quenched with saturated aqueous ammonium chloride,
and then the mixture was extracted with ethyl acetate and dried with
sodium sulfate and evaporated. The residue was purified by column
chromatography (hexane/EtOAc) and gave the desired compound.
General Procedure B for Germyl Compounds 6, 9, and 12. Under
argon atmosphere, a solution of t-BuLi in n-pentane (3.0 equiv for p-
bromophenol) was added dropwise to a solution of p-bromophenol in
THF at −78 °C. The mixture was stirred at room temperature for 1 h.
Trialkyl(aryl)lgermyl chloride (2.0−10 equiv) was added dropwise at
−78 °C. The mixture was stirred at room temperature for 4.5 h, and
then the reaction was quenched with saturated aqueous ammonium
chloride. The solvent was extracted with ethyl acetate and dried with
sodium sulfate and evaporated. The residue was purified by column
chromatography (hexane/EtOAc) and gave the desired compound.
4-Trimethylsilylphenol (5). Prepared by the general procedure A
(quant). The compound was recrystallized by water to afford colorless
Figure 4. Docking model of triethylsilylphenol 8 and hER-LBD. Side
chains of residues near the ligand (<4 Å) are displayed. Protein surface
is indicated as a light-blue mesh.
hydrophobic triethylsilyl moiety at 4-position of 8 binds at the
hydrophobic region of ER-LBD and is surrounded by many
hydrophobic amino acid residues (Figure 4). This calculation
suggests that hydrophobicity of 4-position of phenol is essential
to ER binding and also supports the idea that increase of
hydrophobicity of the substituent at the 4-position increases the
affinity for ER. Many structure−activity relationship studies
about ER ligands revealed that various factors affect their ligand
potency, in particular, location and direction of the second
hydroxyl group corresponding to 17-hydroxyl group of E2 that
interacts with H524 of ER.12 In this study, interestingly,
increase of hydrophobicity without significant change of
molecular shape significantly increases the affinity to ER and
the estrogenic activity. These findings are helpful for develop-
ment of novel ER ligands. These results further suggest that
simple sila-substitution or germa-substitution of bioactive
compounds may result in significant enhancement of biological
activity.
1
crystals. H NMR (400 MHz, CDCl3) δ 0.25 (s, 9 H), 4.70 (s, 1 H),
6.84 (d, J = 8.5 Hz, 2 H), 7.41 (d, J = 8,5 Hz, 2 H). 13C NMR (125
MHz, CDCl3) δ −0.8, 115.9, 135.7, 135.4, 138.3, 141.0, 159.1. Anal.
Calcd for C9H14OSi + 1/8H2O: C, 64.14; H, 8.52. Found: C, 64.17; H,
8.46.
4-Trimethylgermylphenol (6). Prepared by the general procedure B
(59%). Distillation under reduced pressure gave the title compound as
a colorless solid. 1H NMR (500 MHz, CDCl3) δ 0.36 (s, 9 H), 4.63 (s,
1 H), 6.84 (d, J = 8.4 Hz, 2 H), 7.35 (d, J = 8.5 Hz, 2 H). 13C NMR
(125 MHz, CDCl3) δ −1.66, 115.5, 133.5, 134.4, 155.7. Anal. Calcd
For C9H14OGe + 1/8H2O: C, 50.73; H, 6.74. Found: C, 50.87; H,
6.78.
CONCLUSION
■
We prepared various phenols bearing a trialkylated group 14
element substituent at the 4-position and determined the
hydrophobicity of the functional groups. Trialkylsilyl and
trialkylgermyl groups exhibited higher hydrophobicity than
the corresponding carbon analogues, with a difference in log P
value of approximately 0.6 in both cases, and the difference in
log P value versus the carbon analogue was independent of the
substituents on these elements. We also determined the
substituent constant (σ value) of the trimethyl derivatives and
demonstrated that trialkylsilyl and trialkylgermyl substituents
function as electron-withdrawing groups in terms of their effect
on the phenolic hydroxyl group. Sila-substitution and germa-
substitution of the simple phenols examined here resulted in
significant enhancement of estrogenic activity. This approach
may also be applicable to various ER ligands or other bioactive
compounds. Further, the parameters and structure−activity
relationship presented here should be helpful in application of
the heavier group 14 elements for drug development.
4-(1,1-Diethylpropyl)phenol (7). A solution of phenol (211 mg,
2.24 mmol) in TFA (4.0 mL) was treated with 3-ethyl-3-pentanol
(0.34 mL, 2.46 mmol). Stirring was continued at room temperature for
16 h. The solution was concentrated, and the residue was diluted with
CH2Cl2. The organic layer was washed with water, saturated aqueous
sodium bicarbonate, and brine. The combined organic layer was dried
with sodium sulfate, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography (hexane/EtOAc,
1
10:1) and gave 340 mg (79%) of 7 as colorless solid. H NMR (500
MHz, CDCl3) δ 0.65 (t, J = 7.4 Hz, 9 H), 1.63 (q, J = 7.4 Hz, 6 H),
6.77 (d, J = 8.8 Hz, 2 H), 7.16 (d, J = 8,8 Hz, 2 H). 13C NMR (125
MHz, CDCl3) δ 7.9, 28.7, 73.0, 114.6, 128.0, 139.5, 152.8. Anal. Calcd
for C10H20O: C, 81.20; H, 10.48. Found: C, 81.23; H, 10.47. Melting
point 78.9−79.5 °C.
4-Triethylsilylphenol (8). Under argon atmosphere, a solution of n-
BuLi in n-hexane (5.5 mL, 8.7 mmol) was added dropwise at −78 °C
to a solution of p-bromophenol (502 mg, 2.9 mmol) in 3.0 mL of
THF. The mixture was stirred at room temperature. Triethylchlor-
osilane (1.5 mL, 8.7 mmol) was added dropwise at −78 °C, and then
THF (4 mL) added. The mixture was stirred at room temperature for
2 h, and then the reaction was quenched with saturated aqueous
ammonium chloride. The solvent was extracted with ethyl acetate and
EXPERIMENTAL SECTION
Chemistry. All reagents were purchased from Sigma-Aldrich
Chemical Co., Tokyo Kasei Kogyo Co., Wako Pure Chemical
■
D
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Journal of Medicinal Chemistry
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dried with sodium sulfate and evaporated. The residue was purified by
column chromatography (hexane) to yield 806 mg (86%) of 8b as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 0.76 (q, J = 7.8 Hz, 6 H),
0.76 (q, J = 7.9 Hz, 6 H), 0.96 (t, J = 7.8 Hz, 9 H), 1.01 (t, J = 7.9 Hz,
9 H), 6.84 (d, J = 8.4 Hz, 2 H), 7.34 (d, J = 8,4 Hz, 2 H). A
tetrabutylammonium fluoride solution (1.0 M in THF, 1.6 mL, 1.6
mmol) was added dropwise to a solution of 8b (758 mg, 2.35 mmol)
in 7.0 mL of THF at 0 °C, and then the mixture was stirred for 20 min.
The mixture was poured into 100 mL of water and extracted with ethyl
acetate. The organic layers were dried with sodium sulfate and
concentrated. The product was purified by silica gel chromatography
(hexane/EtOAc, 8:1) and distillation under reduced pressure gave 130
4-Dimethylphenylsilylphenol (16). Prepared by the general
procedure A (39%). Distillation under reduced pressure gave colorless
1
oil. H NMR (400 MHz, CDCl3) δ 0.53 (s, 6 H), 4.73 (s, 1 H), 6.84
(d, J = 8.5 Hz, 2 H), 7.33−7.39 (m, 3 H), 7.41 (d, J = 8.4 Hz, 2 H),
7.50−7.55 (m, 2 H). 13C NMR (125 MHz, CDCl3) δ −2.23, 114.9,
127.8, 129.0, 129.3, 134.1, 135.9, 138.6, 156.4. HRMS Calcd for
C14H16OSi [M + H]: 228.0970. Found: 228.0976.
Determination of log P. The 1-octanol/water partition
coefficient, log P, was determined by HPLC method based on the
OECD Guideline for Testing Chemicals 117.13 the measurement was
performed on a Mightysil RP-18 GP 250−4.6 (5 μm) (Kanto
Chemical Co. Inc., Japan) by using an HPLC instrument (UV/vis
detector (UV-2077, JASCO), pump (PU-2089, JASCO), and oven
(CO-965, JASCO)). The injection volume was 10 μL, and the flow-
rate was 1.0 mL/min in all cases. The compounds were detected by
measuring UV absorption at 240 and 230 nm. The temperature of the
column was kept at 40.0 ( 0.1) °C during the measurement. The
mobile phase was methanol−aqueous 0.1 M phosphoric acid system,
changing the methanol concentration from 80% (v/v) to 60% (v/v) by
5% (v/v) concentration steps. The dead time t0 was measured with
thiourea as the unretained compound. Each measurement was
performed in triplicate, and the mean was used for the further
calculations. The capacity factor of each compound in 100% aqueous
eluent, log kw, was calculated by extrapolation of the line fitted on the
measured log k values of methanol−aqueous mobile phase. For the
reference compounds (phenol, p-cresol, 4-chlorophenol, 4-phenyl-
phenol, 2,4,6-trichlorophenol, diphenylether, and pentachlorophenol),
the calculatd log kw values were plotted against log P values, and the
calibration graph was determined (log P = 1.1369 + 0.1886, R2 =
0.9966). The log P values of tested phenols were obtained by
interpolation of the calculated capacity factors log kw on the calibration
graph.
Determination of pKa. The pKa values were determined by
measurement of the change of absorbance at λmax in the pH-dependent
UV spectra of ionic species in 20:80 (w/w) methanol−phosphate
buffer (in the pH range of 7.0−12.0). The values of pH obtained with
the pH meter were corrected by using the equation: pH* = pH
(recorded) − d (d = 0.01).24 The pH values at which the
concentration of free phenol and ionic phenoxide is equal are
determined as pKa values.
Cell Proliferation Assay Using MCF-7 cell lines. Cells of the
human breast adenocarcinoma line MCF-7 were routinely cultivated in
DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100
mg/mL streptomycin at 37 °C in a 5% CO2 humidified incubator. On
the day before an assay, MCF-7 cells were switched to DMEM (low
glucose, phenol red-free supplemented with 5% stripped FBS, 100 IU/
mL penicillin, and 100 mg/mL streptomycin) Cells were trypsinized
from the maintenance dish with phenol red-free trypsin-EDTA and
seeded in a 96-well plate at a density of 1.2 × 103 cells per final volume
of 100 μL of DMEM supplemented with 5% stripped FBS, 100 IU/mL
penicillin, and 100 mg/mL streptomycin. After 24 h, the medium was
1
mg (27%) of 8 as a colorless solid. H NMR (400 MHz, CDCl3) δ
0.77 (q, J = 7.8 Hz, 6 H), 0.96 (t, J = 7.8 Hz, 9 H), 4.65 (s, 1 H), 6.84
(d, J = 8.5 Hz, 2 H), 7.38 (d, J = 8,4 Hz, 2 H). 13C NMR (125 MHz,
CDCl3) δ 3.5, 7.4, 114.9, 128.5, 135.8, 156.1. Anal. Calcd for
C12H20OSi: C, 69.17; H, 9.67. Found: C, 69.00; H, 9.49. Melting point
30.8−32.2 °C.
4-Triethylgermylphenol (9). Prepared by the general procedure B
(54%). Distillation under reduced pressure gave the title compound as
a colorless solid. 1H NMR (400 MHz, CDCl3) δ 0.93−0.99 (m, 6 H),
1.04−1.08 (m, 9 H), 4.62 (s, 1H), 6.84 (d, J = 8.5 Hz, 2 H), 7.31 (d, J
= 8.4 Hz, 2 H). 13C NMR (125 MHz, CDCl3) δ 4.24, 8.91, 115.03,
130.56, 135.3, 155.6. Anal. Calcd for C12H20OGe: C, 56.98; H, 7.97.
Found: C, 56.75; H, 7.90.
4-Bromo(t-butyldimethylsilyloxy)benzene (18). Imidazole (469
mg, 6.92 mmol) and TBSCl (781 mg, 5.19 mmol) were added to a
solution of p-bromophenol (599 mg, 3.46 mmol) in DMF (10 mL)
and then stirred for 1 h at 0 °C. The reaction was quenched with
addition of water. The mixture was extracted with ethyl acetate and
dried with sodium sulfate and then evaporated to yield 779.9 mg
(78.2%) of the title compound. 1H NMR (500 MHz, CDCl3) δ 0.2 (s,
6 H), 0.99 (s, 9 H), 6.72 (d, J = 8.9 Hz, 2 H), 7.32 (d, J = 8.8 Hz, 2 H).
4-Triphenylsilylphenol (11). Under argon atmosphere, a solution of
t-BuLi in n-pentane (2.5 mL, 3.9 mmol) was added dropwise at −78
°C to a solution of 18 (564 mg, 1.95 mmol) in 10 mL of THF. The
mixture was stirred at room temperature for 1 h. Chlorotriphenylsilane
(1.15 g, 3.9 mmol) in 3 mL of THF was added dropwise at −78 °C.
The mixture was stirred at room temperature for 2.5 day, and then the
mixture was quenched with saturated aqueous ammonium chloride.
The solvent was extracted with ethyl acetate and dried with sodium
sulfate and evaporated. The residue was purified by column
chromatography (hexane/EtOAc, 8:1) to yield 596 mg of the
intermediate 19. A TBAF solution (1.0 M in THF, 1.6 mL, 1.6
mmol) was added dropwise to the imtermediate 19 (55.6 mg, 0.12
mmol) in 2 mL of THF at 0 °C and stirred for 15 min. The reaction
mixture was poured into water and extracted with ethyl acetate. The
organic layer was dried with sodium sulfate and concentrated. The
product was purified by silica gel chromatography (hexane/EtOAc,
9:1) to give 27 mg (50%, 2 steps) of the title compound as a colorless
crystals. Recrystallization from dichloromethane/hexane gave colorless
1
crystals. H NMR (500 MHz, CDCl3) δ 4.8 (s, 1 H), 6.86 (d, J = 8.5
replaced with 90 μL of fresh DMEM and 10 μL of drug solution. Final
Hz, 2 H), 7.36−7.56 (m, 15 H), 7.57 (d, J = 8.0 Hz, 2 H). 13C NMR
(125 MHz, CDCl3) δ 115.1, 125.2, 127.8, 129.5, 134.5, 136.3, 138.2,
156.9. Anal. Calcd for C24H20OSi: C, 81.77; H, 5.72. Found: C, 81.54;
H, 5.99. Melting point 236.7−237.6 °C.
−6
concentration of compounds was 10−12 M to
M. Cells were
incubated for 7 days, and medium was replaced once after 4 days. At
the end of the incubation time, proliferation was evaluated by using
WST-8. Then 10 μL of WST-8 was added to microcultures and cells
were incubated for 2 h. The absorbance at 450 nm was measured. This
parameter is related to the number of living cells in the culture. The
number of living cells was plotted against the logarithm of the
concentration, and then the sigmoidal fit was carried out using the
KaleidaGraph4.01 to calculate EC50 values.
T47D-Kbluc ER Reporter Assay. T47D-Kbluc cells were
routinely cultivated in RPMI1640 medium supplemented with 10%
FBS at 37 °C in a 5% CO2 humidified incubator. Cells were
trypsinized from the maintenance dish with phenol red-free trypsin
and seeded in a 96-well plate at a density of 2.0 × 104 cells per final
volume of 90 μL of DMEM (low glucose, phenol red-free
supplemented with 8% stripped FBS). After incubation at 37 °C for
24 h, 10 μL of drug solution (1% DMSO in medium) was added. Final
concentration of compounds was 10−14 M to 10−7 M, and final
4-Triphenylgermylphenol (12). Prepared by the general procedure
B (54%). Recrystallization from hexane/EtOAc gave a colorless solid.
1H NMR (500 MHz, CDCl3) δ 4.75 (s, 1 H), 6.88 (d, J = 8.5 Hz, 2
H), 7.38−7.54 (m, 15 H), 7.5 (d, J = 7.6 Hz, 2 H). 13C NMR (125
MHz, CDCl3) δ 115.5, 127.0, 128.2, 129.1, 135.3, 136.3, 136.9, 156.4.
Anal. Calcd for C24H20OGe: C, 72.60; H, 5.08. Found: C, 72.35; H,
5.24. Melting point 228.3−229.4 °C.
4-Ethyldimethylsilylphenol (14). Prepared by the general proce-
dure A (31%). Distillation under reduced pressure gave colorless
solids. 1H NMR (500 MHz, CDCl3) δ 0.22 (s, 6 H), 0.7 (q, J = 7.9 Hz,
2 H), 0.94 (t, J = 7.9 Hz, 3 H), 4.67 (s, 1 H), 6.83 (d, J = 8.5 Hz, 2 H),
7.39 (d, J = 8.5 Hz, 2 H). 13C NMR (125 MHz, CDCl3) δ −3.38, 7.39,
7.59, 114.9, 130.6, 135.2, 156.1. Anal. Calcd for C10H16OSi: C, 66.61;
H, 8.94. Found: C, 66.65; H, 8.66.
E
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Journal of Medicinal Chemistry
Article
concentration of DMSO was 0.1%. After incubation for 24 h, half
volume of medium was removed and 50 μL of Steady-Glo (Promega
Co.) was added. After 5 min, the chemiluminescence was measured
with microplate reader.
potent retinoid antagonistic activity. Bioorg. Med. Chem. Lett. 2000, 10,
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retinoid agonists bearing substituted dicarba-closo-dodecaborane.
Relation between retinoidal activity and conformation of two aromatic
nuclei. Bioorg. Med. Chem. Lett. 2001, 11, 1307−1311.
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potent nonsecosteroidal vitamin D receptor ligands: direct observation
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S.; Ito, S.; Kano, A.; Taoda, Y.; Kawachi, E.; Kagechika, H.; Tanatani,
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boron cluster derivatives. Bioorg. Med. Chem. Lett. 2012, 22, 1756−
1760.
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Carborane-based androgen antagonists active in LNCaP cells with a
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Kagechika, H. Development of androgen receptor ligands by
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hER Binding Assay. The recombinant human ER was purchased
from Wako Pure Chemical Industries, Ltd. Bio-Gel HT hydrox-
ylapatite (Bio-Rad, Hercules, CA) was washed five times with the
buffer (50 mM Tris-HCl, 1 mM KH2PO4, pH 7.2). The
hydroxylapatite slurry was adjusted to 50% (by volume) hydrox-
ylapatite in the suspension. The ER was diluted with binding buffer
(50 mM Tris, pH 7.5, 10% glycerol, 0.1 mM butylated hydroxyanisole,
10 mM mercaptoethanol, 0.5% yeast extract) to a protein
concentration of 2−4 nM. Then 2 μL of 10−6 M [3H]estradiol
solution in DMSO was added to each tube, followed by 2 μL aliquots
of the competitor solutions in DMSO. After addition of 200 μL of ER
solution to each tube, the tubes were placed in the refrigerator. The
final incubation conditions were the following: 10−8 M [3H]-estradiol,
10−4−10−8 M competitor. After 14−18 h, the bound ligand was
assayed by adsorption on hydroxylapatite for 15 min at 0 °C, followed
by three washes with 1 mL of 0.05 M Tris, pH 7.3. After the last wash,
the hydroxylapatite pellet was resuspended in 0.3 mL of EtOH and
radioactivity was counted in 7 mL of scintillation cocktail (ACS II). All
experiments were performed in duplicate.
Molecular Modeling. Structure of LBD of human ER alpha was
prepared from the Protein Data Bank accession 1g50. The structure
added for polar hydrogens, and partial atomic charges were assigned
using AutoDockTools (ADT).23 Structures of ligand were optimized
using MOPAC 2012 (Stewart J. J. P., Stewart Computational
Chemistry, Colorado Springs, CO, USA, HTTP://OpenMOPAC.net
(2012)) with PM3 parameters and partial atomic charges of them were
assigned using ADT. Molecular docking was performed using
AutoDock 4.2 with Genetic Algorithm. Autodock parameters for
silicon atom Rii = 1.60 and εii = 0.875 were used.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-3-5280-8032. Fax: +81-3-5280-8127. E-mail:
kage.chem@tmd.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work described in this paper was partially supported by
Grants in-Aid for Young Scientist from the Ministry of
Education, Science, Sports and Culture, Japan (grant no.
23790128 to S.F.).
(7) (a) Showell, G. A.; Mills, J. S. Chemistry challenges in lead
optimization: silicon isosteres in drug discovery. Drug Discovery Today
2003, 8, 551−556. (b) Mills, J. S.; Showell, G. A. Exploitation of
silicon medicinal chemistry in drug discovery. Expert Opin. Investig.
Drugs 2004, 13, 1149−1157. (c) Gately, S.; West, R. Novel
therapeutics with enhanced biological activity generated by the
strategic introduction of silicon isosteres into known drug scaffolds.
Drug Dev. Res 2007, 68, 156−163.
ABBREVIATIONS USED
ER, estrogen receptor; TBAF, tetra-n-butylammonium fluoride;
TBS, t-butyldimethylsilyl
■
(8) (a) Yamakawa, T.; Kagechika, H.; Kawachi, E.; Hashimoto, Y.;
Shudo, K. Retinobenzoic acids. 5. Retinoidal activities of compounds
having a trimethylsilyl or trimethylgermyl group(s) in human
promyelocytic leukemia cells HL-60. J. Med. Chem. 1990, 33, 1430−
1437. (b) Higginbotham, K. B.; Lozano, R.; Brown, T.; Patt, Y. Z.;
Arima, T.; Abbruzzese, J. L.; Thomas, M. B. A phase I/II trial of TAC-
101, an oral synthetic retinoid, in patients with advanced
hepatocellular carcinoma. J. Cancer Res. Clin. Oncol. 2008, 134,
1325−1335.
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