Synthesis and evaluation of tamoxifen derivatives with a long alkyl side chain as selective estrogen receptor down-regulators

Article abstract of DOI:10.1016/j.bmc.2015.05.002

Abstract Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. An antagonist that acts as not only an inhibitor of ligand binding but also an inducer of the down-regulation of ER would be useful for the treatment for ER-po

Full text of DOI:10.1016/j.bmc.2015.05.002

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of tamoxifen derivatives with a long alkyl
side chain as selective estrogen receptor down-regulators
Takuji Shoda a, , Masashi Kato , Rintaro Harada , Takuma Fujisato , Keiichiro Okuhira ,
⇑
a,b
a
a,b
c
a
b
c
a,d,
⇑
Yosuke Demizu , Hideshi Inoue , Mikihiko Naito , Masaaki Kurihara
a
Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Division of Biochemistry and Molecular Biology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. An antagonist that
acts as not only an inhibitor of ligand binding but also an inducer of the down-regulation of ER would be
useful for the treatment for ER-positive breast cancer. We previously reported the design and synthesis of
a selective estrogen receptor down-regulator (SERD), (E/Z)-4-(1-{4-[2-(dodecylamino)ethoxy]phenyl}-2-
phenylbut-1-en-1-yl)phenol (C12), which is a tamoxifen derivative having a long alkyl chain on the
Received 24 March 2015
Revised 30 April 2015
Accepted 2 May 2015
Available online xxxx
amine moiety. This compound induced degradation of ER
a via a proteasome-dependent pathway and
Keywords:
showed an antagonistic effect in MCF-7 cells. With the aim of increasing the potency of SERDs, we
designed and synthesized various tamoxifen derivatives that have various lengths and terminal groups
of the long alkyl side chain. During the course of our investigation, C10F having a 10-fluorodecyl group
on the amine moiety of 4-OHT was shown to be the most potent compound among the tamoxifen deriva-
tives. Moreover, computational docking analysis suggested that the long alkyl chain interacted with the
hydrophobic region on the surface of the ER, which is a binding site of helix 12 and coactivator. These
results provide useful information to develop promising candidates as SERDs.
Breast cancer
Estrogen receptor
SERD
Tamoxifen
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
region of genes and transcriptional pathways are activated.
Therefore, the proliferation of ER-positive breast cancer cells is
9
Estrogen receptors (ERs) are a class of the nuclear receptor fam-
ily divided into two subtypes, ER and ERb, which are derived from
promoted by estrogens. Inhibition of ER activation is thus impor-
–3
a
tant for the development of anti-breast cancer drugs.1
distinct genes but display a high degree of structural conservation
in their DNA and ligand binding domains (LBDs).1 These two ER
isoforms are expressed at similar low levels in normal breast. At
least 70% of breast cancer patients are categorized as having ER-
Tamoxifen (Fig. 1) is the most widely used drug for hormonal
therapy of ER-positive breast cancer. Tamoxifen is a triph-
enylethylene derivative that is metabolized by drug-metabolizing
enzymes into 4-hydroxytamoxifen (4-OHT) and endoxifen
–5
positive cancer because growth of the tumor cells occurs by ER
a
(Fig. 1), which have 100 times more affinity for ER than tamoxifen
6
–8
10–13
activation.
ERs are activated by estrogen, such as 17b-estradiol
itself.
The antagonistic mechanism of 4-OHT was suggested by
(
E2), which is produced by the ovaries. Once estrogen binds to
the X-ray structure of 4-OHT and human ERa
LBD.14 The triph-
the ER, the conformation of the ER is changed and coactivator
can bind to it. The ER-coactivator complex binds to the promoter
enylethylene core was shown to be buried in the ligand binding
pocket and the dimethylamino group of 4-OHT projected out of
the ligand binding pocket. Therefore, the conformation of helix
1
4
1
2 is changed to an antagonistic formation.
Fulvestrant (ICI 182,780) and ICI 164,384 (Fig. 2), which are E2
derivatives with a long alkyl side chain from the 7 -position of E2,
2
Abbreviations: (Boc) O, di-tert-butyl dicarbonate; E2, 17b-estradiol; ER, estrogen
receptor; DIEA, N,N-diisopropylethylamine; DMAP, N,N-dimethyl-4-aminopyridine;
DMSO, dimethylsulfoxide; 4-OHT, 4-hydroxytamoxifen; SERD, selective estrogen
receptor down-regulator; TBAF, tetra-n-butylammonium fluoride; THF, tetrahydro-
furan; TsCl, p-toluenesulfonyl chloride.
a
are known as pure antagonists. These compounds not only inter-
fere with the binding of E2 to ER but also induce the rapid
⇑
Corresponding authors. Tel.: +81 3 3700 1141; fax: +81 3 3707 6950.
E-mail addresses: tsho@nihs.go.jp (T. Shoda), masaaki@nihs.go.jp (M. Kurihara).
15,16
down-regulation of ER.
Owing to this latter property, these
http://dx.doi.org/10.1016/j.bmc.2015.05.002
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
T. Shoda et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
CH
3
CH3
R2
N
H
N
CH3
R1
O
CH
3
HO
O
n
1
2
C6 (n = 5)
Tamoxifen (R = H, R = CH3)
1
2
C12 (n = 11)
C18 (n = 17)
4
3
-OHT (R = OH, R = CH )
1
2
Endoxifen (R = OH, R = H)
Figure 3. Structures of tamoxifen derivatives.18
Figure 1. Structures of tamoxifen, 4-OHT and endoxifen.
compounds are called selective estrogen receptor down-regulators
SERDs). The X-ray structure of ICI 164,384 and rat ERb complex
and 10-fluorodecyl (C10F) amines. The routes of synthesis of these
compounds are summarized in Schemes 1 and 2. These compounds
(
17
suggested the pure antagonistic mechanism of SERDs. The alkyl
side chain of ICI 164,384 was shown to protrude from the ligand
binding pocket and the terminal of the alkyl side chain is thrust
into a small hydrophobic pocket, which is formed by the side
chains of Leu261, Met264, Ile265 and Leu286 in helices 3 and
were prepared by using a microwave apparatus from (E/Z)-4-[1-{4-
18,19
(
2-chloroethoxy)phenyl}-2-phenylbut-1-en-1-yl]phenol
(1)
and the corresponding alkyl amines with a base. We obtained all
compounds at moderate yields.
The synthesis of C10F is depicted in Scheme 2. It was synthe-
sized in 4 steps. The amine moiety of 10-amino-1-decanol was pro-
tected by the Boc group and the hydroxyl moiety was tosylated to
obtain 7. Treatment with TBAF followed by HCl resulted in 9.
Finally, C10F was obtained from 1 and 9 by using a microwave
apparatus at a sufficient yield.
1
7
5
.
The protruding alkyl side chain of ICI 164,384 sterically pre-
vents both the positioning of helix 12 and coactivator binding,
resulting in destabilization of the ER.17
We previously reported the design and synthesis of tamoxifen
derivatives with a long alkyl side chain on the amine moiety of
1
8
4
-OHT (Fig. 3). We synthesized triphenylethylene derivatives in
which various lengths of alkyl chain had been introduced, such
as hexyl (C6), dodecyl (C12) and octadecyl (C18) (Fig. 3). We
expected that a long alkyl chain on the amine moiety might pro-
trude from the ligand binding pocket and prevent helix 12 interac-
tion and coactivator binding, resulting in destabilization of the ER.
Among these compounds, C12 showed the ability to down-regulate
the ER and an antagonistic effect in MCF-7 cells.18
In the present study, in order to obtain more potent SERDs and to
clarify the mechanismof down-regulation of ER, we sought to design
and synthesize six tamoxifen derivatives by modification of the alkyl
chain. We evaluated the ability of ER degradation in MCF-7 cells and
the binding affinity for ER. After optimization of the length of the
alkyl chain, we performed modification of its terminal.
Furthermore, we performed a computational study to predict the
binding mode of our compound to ER. The result indicated that the
interaction between the alkyl chain and the hydrophobic surface
of ER is important for the down-regulation of ER.
2
.2. Down-regulation of ER
First, we examined the effects of the length of the long alkyl
chain on reducing ER
MCF-7 cells were treated with these compounds, whole protein
was extracted and ER protein levels were analyzed by Western
blotting, as reported previously.
tions of the ER protein levels were observed in the cells treated
with 10 M C8, C10 and C12 (lanes 5–7) compared with the control
lane 1). C10 was the most effective compound among the long
alkyl chain derivatives because the band of ER had almost disap-
a protein levels in MCF-7 breast cancer cells.
a
18,20
As shown in Figure 4, reduc-
a
l
(
a
peared in association with it (lane 6). No significant differences in
the treatment of C6, C14, C16 and C18 were observed under these
conditions (lanes 4, 8, 9 and 10, respectively). The protein level of
ER
a was slightly increased by treatment with 10 lM 4-OHT and
endoxifen (lanes 2 and 3), which was identical to findings reported
previously.
21,22
Next, we examined the effects of the terminal of the long alkyl
2
2
. Results
chain in terms of reducing ER
induced the reduction of ER
a
a
protein levels in MCF-7 cells. C10F
in the cells compared with C10
.1. Chemistry
(
Fig. 5, lanes 3 and 4). On the other hand, treatment with C10OH
did not reduce the ER protein level (lane 5). The reduction of
ER by C10F occurred in a dose-dependent manner (lanes 8–10)
a
We designed six novel 4-OHT derivatives with octyl (C8), decyl
a
(
C10), tetradecyl (C14), hexadecyl (C16), 10-hydroxydecyl (C10OH)
and was inhibited by a proteasome inhibitor, MG132 (lane 11).
These results show that C10F has the ability to induce the protea-
somal degradation of ER
the terminal group are important for the down-regulation of ER.
a in MCF-7 cells and that the length and
CH3 OH
H
O
S
2.3. ER binding affinity
F
F
H
H
F
HO
To evaluate the ability of compounds to bind to ERa, a fluores-
F
F
Fulvestrant (ICI 182,780)
cence polarization-based competitive binding assay was con-
ducted. The mean IC50 and RBA for the compounds tested in the
ER competitive binding assay are presented in Table 1. The relative
binding affinity (RBA) value for each competing compound was
then calculated according to the IC50 of E2 (0.17 nM). The IC50 value
CH3 OH
H
CH3
N
H
H
of C18 was estimated to be over 100 lM because C18 had difficulty
CH
3
HO
dissolving in the buffer solution and the binding curve of C18 did
not saturate in the range that we examined. The IC50s of C6 and
C8 were 151 and 53 nM, respectively. The IC50s of C10–C14 were
in the range of 3.6–14 nM. Therefore, the binding affinities of C6,
O
ICI 164,384
Figure 2. Structures of fulvestrant and ICI 164,384.
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T. Shoda et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
CH
3
CH
3
a
H
N
Cl
HO
O
R
HO
O
n
1
2
3
4
5
6
: n = 7, R = H, (80%, C8)
: n = 9, R = H, (67%, C10)
: n = 13, R = H, (36%, C14)
: n = 15, R = H, (43%, C16)
: n = 9, R = OH, (43%, C10OH)
Scheme 1. Synthesis of long alkyl chain derivatives of 4-OHT. Reagents and conditions: (a) CH
20 °C.
3
(CH
)
2 n
2
NH or 10-amino-1-decanol, triethylamine, methanol, microwave,
1
a, b
H
N
c
H
d
H2N
• HCl
N
F
H2N
Boc
F
Boc
e
OTs
9
OH
9
9
9
7
(29%)
8 (75%)
9 (99%)
CH3
H
N
HO
O
F
9
10 (35%, C10F)
Scheme 2. Synthesis of C10F. Reagents and conditions: (a) (Boc)
rt, 2 h, (e) 1, DIEA, CH OH, microwave, 120 °C, 6 h.
2 2
O, CH Cl
2
, rt, 5 h, (b) TsCl, DMAP, pyridine, CH
2
Cl
2
, rt, 2 days, (c) TBAF, THF, reflux, 7 h, (d) HCl, 1,4-dioxane,
3
Table 1
a
Estrogen receptor binding affinities of compounds
IC50 (nM)
RBA (%)
4
C6
C8
C10
C12
C14
C16
C18
C10OH
C10F
-OHT
5.6
151
53
3.6
14
11
4.8
>100,000
210
3.4
3.0
0.11
0.3
4.7
1.2
1.5
3.5
Figure 4. Reduction of endogenous ER
compounds (C6–C18). MCF-7 cells were incubated with 10
a
by 4-OHT, endoxifen and synthesized
M of the indicated
n.d.b
l
0.08
5.0
compounds for 6 h. Shown are immunoblots of cell lysates stained with the
indicated antibodies.
a
The IC50 value for each competing ligand was calculated according to the sig-
moidal inhibition curve. The relative binding affinity (RBA) values were calculated
against E2 by using the following equation: RBA (%) = 100 Â (the IC50 for E2/the IC50
for the test compound).
C8 and C18 are weaker than that of 4-OHT (5.6 nM), while those of
C10–C16 are close to that of 4-OHT. The IC50 values of C10F and
C10OH were 3.4 and 210 nM, respectively. The binding affinity of
C10F was close to that of C10, whereas the binding affinity of
C10OH was about 50-fold weaker than that of C10. These results
indicate that the nature of the terminal of the alkyl chain plays
an important role in the binding affinity for ER. Taking these find-
ings together with the results of the Western blotting analysis
b
n.d. = not determined.
(Figs. 4 and 5), the length of the alkyl chain is important for binding
and down-regulation of the ER and the introduction of a fluoro
group at the terminal of the alkyl chain effectively increases the
down-regulation without decreasing the binding activity of the
compounds.
2
.4. Computational modeling study
To predict the binding mode of the compound in the ER binding
pocket, we conducted a computational modeling study. Figure 6
shows the docking models of C10 and human ER LBD in which
a
helix 12 is eliminated (PDB ID: 3ERT). In the calculated structure,
the triphenylethylene core is located in the ligand binding pocket
in a similar manner to 4-OHT. We also calculated the docking
Figure 5. (Left) Reduction of endogenous ER
MCF-7 cells were incubated with 10 M of the indicated compounds for 6 h. (Right)
Dose-dependent response of ER reduction induced by C10F and proteasome
inhibitor MG132 inhibited ER degradation. MCF-7 cells were incubated with the
indicated concentrations of C10F for 6 h in the presence or absence of a proteasome
inhibitor, MG132 (10 M). Shown are immunoblots of cell lysates stained with
antibodies mentioned in the Section 4.
a by 4-OHT, C10, C10F and C10OH.
l
model of ICI 164,384 for ERa LBD. As shown in Figure 6, the side
a
chains of both C10 and ICI 164,384 extend in the same direction
and bind along the shallow groove that is formed by helices 3
and 5. The terminal of the alkyl chain reaches the hydrophobic
region formed by the side chains of Leu354, Met357, Ile358,
a
l
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4
T. Shoda et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
helix 12 and coactivator in the agonist conformation.23 This alkyl
side chain sterically inhibited the helix 12 interaction and coactiva-
tor binding, resulting in destabilization of the ER . In the case of C6
or C16, which bound to ER but did not induce down-regulation of
ER , too short as well as too long alkyl chains are unfavorable for
the down-regulation of ER
In summary, we designed and synthesized new tamoxifen
derivatives with a long alkyl chain, which down-regulate the ER
a
a
a
a.
a
protein level in MCF-7 cells. The most effective length for down-
regulation of the ER was determined by performing Western
blotting and binding assay and introducing a fluoro group at the ter-
minal of the alkyl chain that maintained high binding affinity for
ERa and increased the potency for SERD activity. Computational
modeling study showed the binding mode of our compound. The
terminal alkyl group interacted with the hydrophobic surface of
the ER and the position of the terminal alkyl chain sterically inhib-
ited the helix 12 interaction and the coactivator binding, resulting
in destabilization of the ER. Our findings provide useful information
to design a promising candidate for SERDs.
Figure 6. Computational model of human ER
a LBD and C10. The hydrophobic
region (formed by Leu354, Met357, Ile358, Leu379 and Trp383), C10 and ICI
64,384 are colored green, yellow and cyan, respectively.
1
4
4
. Experimental section
.1. Chemistry
Leu379 and Trp383 (Fig. 6, green). The positioning of the alkyl
chain of our compound can sterically interfere with helix 12 inter-
action and the coactivator binding.
4
-Hydroxytamoxifen was purchased from Fluka. All other
reagents and solvents were purchased from Sigma–Aldrich, Wako
Pure Chemical and Tokyo Chemical Industry and were used with-
out purification. Analytical TLC was carried out using Merck silica
gel 60 F254 pre-coated plates and visualized using a 254 nm UV
lamp, phosphomolybdic acid, p-anisaldehyde or ninhydrin stain-
ing. Column chromatography was performed with silica gel (spher-
ical, neutral) purchased from Kanto Chemical. The microwave
3
. Discussion
SERD is a compound that can inhibit estrogen action by reduc-
ing ER protein levels in breast cancer cells. It is clear that this type
of compound has particular promise for effective endocrine thera-
pies in breast cancer. We previously reported that a long alkyl
chain derivative of 4-OHT induced degradation of the ER
by a proteasome pathway and showed antagonistic activity in
a protein
1
reactions were carried out using Biotage Initiator. H NMR and
1
3
1
8
C NMR spectra were obtained on a Varian AS 400 Mercury spec-
MCF-7 cells. Herein, we performed optimization studies to obtain
more potent compounds.
1
13
trometer (400 MHz for H and 100 MHz for C). Chemical shifts
are expressed as ppm downfield from a solvent residual peak or
internal standard tetramethylsilane (TMS). FT-IR spectra were
First, we synthesized various lengths of long alkyl chain deriva-
tives of 4-OHT to clarify the optimal length of the alkyl chain.
Western blotting analysis showed that C10, which has a decyl
group on the amine moiety of 4-OHT, is more potent among
derivatives that have a simple alkyl chain on the amine moiety
recorded on JASCO FT-IR 4100 equipped with an ATR unit as a sam-
À1
pling module and are expressed in
m
(cm ). High-resolution mass
spectra were obtained on a Shimadzu IT-TOF MS equipped with an
electrospray ionization source. The syntheses of (E/Z)-4-(1-{4-[2-
(Fig. 4). The length of the alkyl chain is important for the down-
(
(
hexylamino)ethoxy]phenyl}-2-phenylbut-1-en-1-yl)phenol (C6),
E/Z)-4-(1-{4-[2-(dodecylamino)ethoxy]phenyl}-2-phenylbut-1-
regulation of ER.
Furthermore, we designed and synthesized C10F, which has a
fluoro group at the terminal of the alkyl chain of C10. The design
concept of this compound was based on the following findings:
X-ray crystallographic analysis of the complex of rat ERb and ICI
en-1-yl)phenol (C12) and (E/Z)-4-(1-{4-[2-(octadecylamino)ethox
y]phenyl}-2-phenylbut-1-en-1-yl)phenol (C18) were as described
in our previous report.
1
7
1
64,384 (PDB ID: 1HJ1), the alkyl side chain of ICI 164,384 pro-
4
1
.1.1. (E/Z)-4-(1-{4-[2-(Decylamino)ethoxy]phenyl}-2-phenylbut-
-en-1-yl)phenol (3, C10)
trudes from the ligand binding pocket and the terminal of alkyl
side chain is thrust into a small hydrophobic pocket, which is
formed by the side chains of Leu261, Met264, Ile265, Leu286 and
Trp290 (all residue numbering is based on 1HJ1; these residues
are Leu354, Met357, Ile358, Leu379 and Trp383, respectively, in
To a solution of decylamine (99.4 mg, 0.63 mmol, 3.2 equiv) in
methanol
(0.3 mL),
(E/Z)-4-[1-{4-(2-chloroethoxy)phenyl}-2-
1
8
phenylbut-1-en-1-yl]phenol (1, 74.7 mg, 0.20 mmol, 1.0 equiv)
and triethylamine (29.1 mg, 0.28 mmol, 1.5 equiv) were added.
The mixture was stirred at 120 °C for 4 h by microwave-assisted
synthesis (TLC monitoring: dichloromethane/MeOH, 9:1). The mix-
ture was concentrated in a vacuum. The crude product was puri-
3
ERT), within the shallow hydrophobic groove that is formed by
helices 3 and 5. Therefore, we expected that the modification of
the terminal of alkyl group would perturb the interaction between
the terminal alkyl group and ER protein surface and influence the
ability to degrade ER. As we expected, the binding affinity of
C10F for ER
of ER was more effective than for C10 (Fig. 5). In the case of
C10OH, the hydrophilic substitute of C10, the ability to down-reg-
ulate the ER protein level was decreased (Fig. 5). This is attributed
to a decrease of the binding affinity of C10OH for ER (Table 1).
fied using
a silica gel column (8 g, dichloromethane/MeOH,
9
.5:0.5). The fractions containing the product were combined and
a was close to that of C10 (Table 1) and the reduction
concentrated under reduced pressure to give clear viscous C10.
a
The NMR spectrum shows a nearly 1:1 mixture of E and Z isomers.
1
Yield = 65.6 mg, 67%. H NMR (400 MHz, CDCl
3
) d 7.17–7.06
a
(
6
(
12H, m), 7.00 (2H, d, J = 8.4 Hz), 6.81 (2H, d, J = 8.4 Hz), 6.74–
.72 (4H, m), 6.65 (2H, d, J = 8.4 Hz), 6.46 (2H, d, J = 8.4 Hz), 6.41
2H, d, J = 8.4 Hz), 4.73 (2H, br), 4.09 (2H, t, J = 5.0 Hz), 3.93 (2H,
t, J = 5.0 Hz), 3.04 (2H, t, J = 5.0 Hz), 2.94 (2H, t, J = 5.0 Hz), 2.71
a
Computational modeling study showed the predicted binding
mode of C10 (Fig. 6). The alkyl side chains of C10 and ICI 164,384
interacted with the same hydrophobic surface that is covered with
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T. Shoda et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
(
1
(
1
1
1
2
1
2H, t, J = 7.4 Hz), 2.65 (2H, t, J = 7.4 Hz), 2.51–2.44 (4H, m), 1.58–
3
CDCl ) d 157.5, 156.6, 155.8, 154.8, 143.0, 142.9, 141.0, 140.9,
1
3
.50 (4H, m), 1.29–1.25 (28H, m), 0.93–0.86 (12H, m); C NMR
) d 157.5, 156.6, 155.8, 154.9, 143.0, 142.9,
138.2, 137.0, 136.5, 135.5, 135.2, 132.3, 132.2, 130.9, 130.8,
130.0, 128.1, 128.0, 126.1, 115.5, 114.8, 114.2, 113.5, 66.8, 66.5,
51.0, 49.9, 48.8, 48.7, 32.2, 30.0, 29.9, 29.8, 29.7, 29.6, 29.3, 29.2,
27.5, 22.9, 14.4, 13.9; FT-IR (ATR) 3729, 3597, 2924, 2854, 1603,
100 MHz, CDCl
3
41.0, 140.9, 138.2, 137.0, 136.5, 135.4, 135.1, 132.3, 132.2,
30.9, 130.8, 130.0, 128.1, 128.0, 126.1, 115.5, 114.8, 114.2,
13.5, 66.7, 66.5, 49.9, 48.8, 48.7, 32.1, 29.8, 29.7, 29.6, 29.3,
1507, 1458, 1372, 1224, 1172, 1108, 1045, 830, 771, 691, 606,
À1
9.2, 27.5, 22.9, 14.4, 13.9; FT-IR (ATR) 3725, 3603, 2925, 2857,
546, 528, 518, 513 cm ; HR-MS (ESI+): m/z calcd for C40
H58NO
2
À1
+
603, 1507, 1456, 1374, 1224, 1173, 1045, 830, 770, 688 cm
;
[M+H] 584.4462, found 584.4402.
+
2
HR-MS (ESI+) m/z calcd for C34H46NO [M+H] 500.3523, found
5
00.3529.
4.1.5. (E/Z)-4-[1-(4-{2-[(10-Hydroxydecyl)amino]ethoxy}phenyl)-
2-phenylbut-1-en-1-yl]phenol (6, C10OH)
4
1
.1.2. (E/Z)-4-(1-{4-[2-(Octylamino)ethoxy]phenyl}-2-phenylbut-
-en-1-yl)phenol (2, C8)
The same procedure as for C10 was used, starting from 1 and
The same procedure as for C10 was used, starting from 1 and
10-amino-1-decanol.
Yield = 30.6 mg, 43%. Viscous compound. 1H NMR (400 MHz,
octylamine.
Yield = 74.4 mg, 80%. Clear viscous compound; 1H NMR
400 MHz, CDCl ) d 7.17–7.05 (12H, m), 7.00 (2H, d, J = 8.8 Hz),
.81 (2H, d, J = 8.8 Hz), 6.74–6.72 (4H, m), 6.65 (2H, d, J = 8.8 Hz),
.46 (2H, d, J = 8.8 Hz), 6.41 (2H, d, J = 8.8 Hz), 4.09 (2H, t,
3
CD OD) d 7.20–7.06 (12H, m), 7.03 (2H, d, J = 8.5 Hz), 6.85 (2H, d,
J = 8.7 Hz), 6.78 (2H, d, J = 8.6 Hz), 6.74 (2H, d, J = 8.7 Hz), 6.67
(2H, d, J = 8.5 Hz), 6.50 (2H, d, J = 8.9 Hz), 6.45 (2H, d, J = 8.7 Hz),
4.17 (2H, t, J = 5.0 Hz), 4.00 (2H, t, J = 5.0 Hz), 3.67–3.59 (4H, m),
3.12 (2H, t, J = 5.0 Hz), 3.02 (2H, t, J = 5.0 Hz), 2.91–2.70 (4H, m),
2.52–2.42 (4H, m), 1.75–1.10 (32H, m), 0.91 (6H, t, J = 7.4 Hz);
(
6
6
3
J = 5.0 Hz), 3.92 (2H, t, J = 5.0 Hz), 4.40–3.60 (2H, br), 3.03 (2H, t,
J = 5.0 Hz), 2.93 (2H, t, J = 5.0 Hz), 2.71 (2H, t, J = 7.6 Hz), 2.65 (2H,
t, J = 7.4 Hz), 2.51–2.44 (4H, m), 1.57–1.48 (4H, m), 1.31–1.27
1
3
3
C NMR (100 MHz, CD OD) d 157.70, 156.20, 155.46, 154.51,
142.88, 142.83, 141.20, 138.01, 137.20, 136.74, 135.32, 132.27,
132.23, 130.89, 130.86, 129.93, 128.09, 128.05, 126.12, 115.40,
114.69, 114.28, 113.50, 63.17, 49.25, 47.89, 41.11, 37.77, 35.71,
32.91, 32.16, 29.41, 28.25, 27.13, 25.89, 24.38, 15.41, 13.94,
13
(
20H, m), 0.93–0.85 (12H, m);
3
C NMR (100 MHz, CDCl ) d
1
1
1
4
1
1
57.5, 156.6, 155.8, 154.8, 143.0, 142.9, 141.0, 140.9, 138.2,
37.0, 136.5, 135.4, 135.2, 132.3, 132.2, 130.9, 130.8, 130.0,
28.1, 128.0, 126.1, 115.5, 114.8, 114.2, 113.5, 66.8, 66.5, 49.9,
8.9, 48.8, 32.0, 29.8, 29.7, 29.6, 29.5, 29.3, 29.2, 27.5, 22.9, 14.4,
13.88; FT-IR (ATR) 3643, 3471, 2927, 2857, 1726, 1671, 1603,
À1
1508, 1455, 1372, 1239, 1173, 1105, 1046, 911, 830, 731 cm
;
+
3.9; FT-IR (ATR) 3729, 3597, 2925, 2859, 1603, 1506, 1458,
3
HR-MS (ESI+) m/z calcd for C34H46NO [M+H] 516.3472, found
À1
372, 1237, 1174, 1109, 1044, 831, 769, 700, 607, 527, 514 cm
;
516.3448.
+
2
HR-MS (ESI+) m/z calcd for C32H42NO [M+H] 472.3210, found
4
72.3215.
4
.1.6. 10-[(tert-Butoxycarbonyl)amino]decyl 4-methylbenzene-
sulfonate (7)
4
.1.3. (E/Z)-4-(1-{4-[2-(Tetradecylamino)ethoxy]phenyl}-2-
Di-t-butyl dicarbonate (2.5 g, 12 mmol, 1.0 equiv) was added to
a solution of 10-amino-1-decanol (2.0 g, 12 mmol, 1.0 equiv) in
phenylbut-1-en-1-yl)phenol (4, C14)
The same procedure as for C10 was used, starting from 1 and
CH
CH
2
Cl
Cl
2
(40 ml) at room temperature. After stirring for 5 h, the
was removed in vacuo. The crude product was dissolved
tetradecylamine.
Yield = 38.0 mg, 36%. Clear viscous compound; 1H NMR
2
2
in CH
2
2
Cl (16 ml) and pyridine (4.5 g, 57 mmol, 5.0 equiv). TsCl
(
6
6
400 MHz, CDCl
3
) d 7.16–7.06 (12H, m), 7.00 (2H, d, J = 8.4 Hz),
.81 (2H, d, J = 8.4 Hz), 6.74–6.72 (4H, m), 6.65 (2H, d, J = 8.4 Hz),
.46 (2H, d, J = 8.4 Hz), 6.40 (2H, d, J = 8.4 Hz), 4.51 (2H, br), 4.09
(
4.5 g, 24 mmol, 2.0 equiv) and DMAP (141 mg, 1.2 mmol,
0
.1 equiv) were added and stirred for 6 h at room temperature.
The reaction mixture was washed with brine, dried with sodium
sulfate and then evaporated. The residue was purified by column
chromatography (EtOAc/hexane, 1:4). Yield = 1.41 g, 29%.
(
2
2H, t, J = 5.0 Hz), 3.93 (2H, t, J = 5.0 Hz), 3.03 (2H, t, J = 5.0 Hz),
.94 (2H, t, J = 5.0 Hz), 2.71 (2H, t, J = 7.6 Hz), 2.65 (2H, t,
J = 7.6 Hz), 2.51–2.44 (4H, m), 1.58–1.48 (4H, m), 1.30–1.26 (44H,
) d 157.5,
56.6, 155.8, 154.8, 143.0, 142.9, 141.0, 140.9, 138.2, 137.0,
36.5, 135.5, 135.2, 132.3, 132.2, 130.9, 130.8, 130.0, 128.1,
28.0, 126.1, 115.5, 114.8, 114.2, 113.5, 66.8, 66.5, 50.0, 49.9,
8.9, 48.8, 32.2, 29.9, 29.8, 29.7, 29.6, 29.3, 29.2, 27.5, 22.9, 14.4,
1
m), 0.93–0.86 (12H, m); 1 C NMR (100 MHz, CDCl
3
Colorless oil. H NMR (400 MHz, CD
3
OD) d 7.79 (2H, d, J = 8.0 Hz),
3
7
3
1
1
2
.35 (2H, d, J = 8.0 Hz), 4.53 (1H, s), 4.01 (2H, t, J = 6.0 Hz), 3.10–
.07 (2H, m), 2.45 (3H, s), 1.65–1.59 (2H, m), 1.44 (9H, s), 1.26–
.22 (14H, m); C NMR (100 MHz, CD OD) d 156.25, 144.86,
3
33.83, 130.03, 128.11, 79.02, 70.93, 40.81, 30.26, 29.59, 29.50,
1
1
1
4
1
1
1
3
9.43, 29.10, 29.01, 28.65, 26.98, 25.52, 21.88; FT-IR (ATR) 3406,
3.9; FT-IR (ATR) 3729, 3597, 2925, 2855, 1604, 1507, 1459,
372, 1226, 1174, 1108, 1046, 830, 771, 694, 528, 516, 513 cm
À1
À1
2928, 2858, 1703, 1516, 1456, 1361, 1220, 1174, 952, 772 cm
MS (ESI+) m/z calcd for C22H37NNaO S [M+Na] 450, found 450.
5
;
;
+
+
2
HR-MS (ESI+): m/z calcd for C38H54NO [M+H] 556.4149, found
5
56.4121.
4
.1.7. tert-Butyl (10-fluorodecyl)carbamate (8)
Compound 7 (278 mg, 0.65 mmol, 1.0 equiv) was dissolved in
4
.1.4. (E/Z)-4-(1-{4-[2-(Hexadecylamino)ethoxy]phenyl}-2-
phenylbut-1-en-1-yl)phenol (5, C16)
THF (5 ml). A TBAF solution (1.0 M in THF, 1.5 ml, 1.5 mmol,
2.3 equiv) was added dropwise to the reaction mixture at room
temperature and refluxed for 7 h. The solvent was evaporated
and the residue was purified by column chromatography
The same procedure as for C10 was used, starting from 1 and
hexadecylamine.
Yield = 45.3 mg, 43%. Clear viscous compound; 1H NMR
1
(
6
400 MHz, CDCl
.82 (2H, d, J = 8.4 Hz), 6.73 (4H, d, J = 8.4 Hz), 6.65 (2H, d,
J = 8.4 Hz), 6.47 (2H, d, J = 8.4 Hz), 6.41 (2H, d, J = 8.4 Hz), 4.09
3
) d 7.17–7.08 (26H, m), 7.01 (2H, d, J = 8.4 Hz),
(EtOAc/hexane, 1:6). Yield = 134 mg, 75%. Colorless oil. H NMR
(400 MHz, CD OD) d 4.53 (1H, br), 4.44 (2H, dt, J = 47.2, 6.4 Hz),
3
3.13–3.08 (2H, m), 1.76–1.62 (2H, m), 1.44 (9H, s), 1.40–1.28
1
3
(
(
2H, t, J = 5.0 Hz), 3.93 (2H, t, J = 5.0 Hz), 4.13–3.91 (2H, br), 3.03
2H, t, J = 5.0 Hz), 2.93 (2H, t, J = 5.0 Hz), 2.71 (2H, t, J = 7.4 Hz),
(14H, m); C NMR (100 MHz, CD OD) d 156.2, 84.50, 45.43,
3
40.81, 30.71, 30.51, 30.26, 29.64, 29.42, 28.64, 27.00, 25.78,
2
1
.65 (2H, t, J = 7.4 Hz), 2.50–2.46 (4H, m), 1.58–1.50 (4H, m),
25.33; FT-IR (ATR) 3357, 2929, 2855, 1700, 1518, 1458, 1364,
.32–1.26 (52H, m), 0.93–0.88 (12H, m); 13C NMR (100 MHz,
À1
1249, 1173, 1040, 1002, 913, 868, 791, 744 cm
.
Please cite this article in press as: Shoda, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.002

6
T. Shoda et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4
.1.8. 10-Fluorodecan-1-amine hydrochloride (9)
Compound 8 (142 mg, 0.52 mmol) was dissolved in a HCl solu-
instructions. Fluorescence polarization signals (mP values) were
then measured using EnVision multiple plate reader (Perkin
Elmer) with a 470 nm excitation filter and a 535 nm emission filter.
tion (ca. 4 M in 1,4-dioxane) and stirred for 2 h at room tempera-
ture. The solvent was evaporated to give compound 9.
The fraction of compound bound to ERa was correlated to the mP
1
Yield = 111 mg, 99%. Colorless viscous compound.
H
NMR
value and plotted against values of competitor concentrations. The
IC50 values were obtained by fitting the data using GraphPad Prism
6 J. Relative binding affinity (RBA) was calculated by dividing the
IC50 of E2 (0.17 nM) by the IC50 of the compound and is expressed
as a percent (E2 = 100).
(
2
3
400 MHz, CD OD) d 4.43 (2H, dt, J = 47.6, 6.0 Hz), 3.23 (2H, br),
13
.70 (2H, br), 1.75–1.63 (2H, m), 1.52–1.25 (14H, m); C NMR
OD) d 84.45, 45.40, 40.30, 30.61, 29.61, 29.50,
9.40, 29.19, 26.75, 25.36; HR-MS (ESI+) m/z calcd for C10
(
100 MHz, CD
3
2
H23FN
+
[
M+H] 176.1736, found 176.1767.
4
.5. Computational modeling studies
4
2
.1.9. (E/Z)-4-[1-(4-{2-[(10-Fluorodecyl)amino]ethoxy}phenyl)-
-phenylbut-1-en-1-yl]phenol (10, C10F)
The crystal structure of the complex of human ERa and 4-OHT
The same procedure as for C10 was used, starting from 1 and 9.
(PDB code 3ERT), from which helix 12 was deleted, was used as a
template for the computational study. Molecular modeling and
graphics manipulations were performed using the Molecular
Operating Environment (MOE, Chemical Computing Group) and
1
Yield = 24 mg, 35%. Colorless viscous compound.
400 MHz, CD
H NMR
(
7
6
3
OD) d 7.21–7.05 (12H, m), 7.01 (2H, d, J = 8.3 Hz),
.01 (2H, d, J = 8.3 Hz), 6.77–6.70 (4H, m), 6.66 (2H, d, J = 8.5 Hz),
.47 (2H, d, J = 8.6 Hz), 6.41 (2H, d, J = 8.5 Hz), 4.43 (2H, dt,
2
4
UCSF-Chimera software packages.
J = 47.4, 6.2 Hz,), 4.09 (2H, t, J = 5.0 Hz,), 3.93 (2H, t, J = 5.0 Hz),
3
2
1
1
1
1
5
3
1
1
7
5
.04 (2H, t, J = 5.0 Hz), 2.94 (2H, t, J = 5.0 Hz), 2.77–2.61 (4H, m),
Acknowledgments
.55–2.39 (4H, m), 1.82–1.61 (4H, m), 1.59–1.47 (4H, m), 1.46–
.20 (24H, m), 0.99–0.86 (6H, m); 13C NMR (100 MHz, CD
OD) d
We thank Mariko Seki for technical advice regarding cell culture
and the Western blotting analysis and Dr. Tomoko Nishimaki-
Mogami for help to set up the fluorescent multiple plate reader.
This work was supported, in part, by JSPS KAKENHI Grant
Number 26860085 (TS).
3
58.60, 157.74, 157.05, 156.07, 144.17, 144.13, 142.14, 142.01,
39.37, 138.20, 137.69, 133.52, 132.36, 131.15, 129.27, 127.26,
16.70, 116.70, 116.03, 115.38, 114.61, 86.51, 84.89, 67.85, 67.55,
1.08, 51.04, 49.99, 49.91, 40.11, 31.92, 31.73, 30.85, 30.64,
0.52, 30.42, 30.34, 28.67, 28.63, 26.60, 26.54, 25.14, 24.42,
5.52, 15.13, 15.09; FT-IR (ATR) 3779, 3549, 3047, 2926, 2857,
727, 1602, 1505, 1457, 1379, 1240, 1172, 1120, 1043, 910, 824,
References and notes
À1
+
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; HR-MS (ESI+) m/z calcd for C H45NO F [M+H]
2
3
4
.
.
.
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9.
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.3. Western blot analysis
1
MCF-7 cells were treated with the compounds at the indicated
concentrations in the presence or absence of 10 lM MG132 (pur-
chased from Peptide Institute) for 6 h, and then the cells were col-
lected and extracted with lysis buffer (1% SDS, 0.1 M Tris–HCl, pH
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6. Carlson, R. W. Clin. Breast Cancer 2005, 6, S5.
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7
.0, 10% glycerol) and boiled for 10 min. Protein concentrations
were determined by the BCA method, and equal amounts of pro-
tein lysate were separated by SDS–PAGE, transferred to a PVDF
membrane and subjected to Western blotting using the following
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2
2
2
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4
.4. Fluorescence polarization assay
Test compounds were dissolved in DMSO to prepare a stock
solution (10 mM). Fluorescent polarization-based competition
binding assays were conducted to determine the relative binding
affinity of compounds for ER
a using commercially available kits
(
P2698, Life Technologies) according to the manufacturer’s
Please cite this article in press as: Shoda, T.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.002