Design and synthesis of tamoxifen derivatives as a selective estrogen receptor down-regulator

Article abstract of DOI:10.1016/j.bmcl.2013.11.078

We designed and synthesized an estrogen receptor (ER) down-regulator (5), which is a derivative of tamoxifen with a long alkyl side chain. Compound 5 effectively reduced ER protein levels in MCF-7 cells and had an antagonistic effect.

Full text of DOI:10.1016/j.bmcl.2013.11.078

Bioorganic & Medicinal Chemistry Letters 24 (2014) 87–89
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of tamoxifen derivatives as a selective estrogen
receptor down-regulator
Takuji Shoda a, , , Keiichiro Okuhira b, , Masashi Kato a,c, Yosuke Demizu a, Hideshi Inoue c,
⇑
Mikihiko Naito b, Masaaki Kurihara a,d,
⇑
a Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
b Division of Biochemistry and Molecular Biology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
c School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
d Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized an estrogen receptor (ER) down-regulator (5), which is a derivative of
tamoxifen with a long alkyl side chain. Compound 5 effectively reduced ER protein levels in MCF-7 cells
and had an antagonistic effect.
Received 14 November 2013
Revised 23 November 2013
Accepted 27 November 2013
Available online 4 December 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Breast cancer
Estrogen receptor
SERD
Tamoxifen
R1
O
N
Estrogen receptor (ER) belongs to the nuclear receptor
superfamily and is often overexpressed in the tissues of breast
cancer patients, which promotes the estrogen-dependent prolifer-
ation of cancer cells.1–4 Tamoxifen (Fig. 1) is an orally available ER
antagonist, which competitively blocks the binding of estrogen,
such as 17b-estradiol (E2), to the receptor and is effective at treat-
ing breast cancer in pre- and post-menopausal women.5,6 Tamox-
ifen is one of the selective estrogen receptor modulators (SERMs),
which act as either agonists or antagonists depending on the target
tissue. Therapy using tamoxifen is often limited because tamoxifen
possesses agonistic effects in uterine cancer cells and increases the
risk of endometrial cancer.7–9 Another class of currently available
antagonists is selective estrogen receptor down-regulators
(SERDs).10,11 This class of compounds not only interferes with the
binding of E2 to ER but also induces the rapid down-regulation
of ER.10–13 The latter property causes no agonistic activity in any
tissues. SERDs are structurally divided into two groups. One is ste-
roidal compounds, such as fulvestrant, which is a steroidal ana-
R2
Tamoxifen (R1 = H, R2 = CH3)
4-OHT (R1= OH, R2 = CH3)
Endoxifen (R1 = OH, R2 = H)
OH
H
O
S
F
F
H
H
F
F
HO
F
Fulvestrant
O
OH
logue with a long alkyl side chain from the 7
a position of E2
(ICI182,780, Fig. 1). Although clinical studies have shown some
success with fulvestrant,13–15 its poor pharmacodynamic proper-
ties and lack of oral bioavailability have limited its clinical
GW5638
⇑
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).
Figure 1. Structures of tamoxifen, tamoxifen active metabolites, fulvestrant, and
These authors equally contributed to this work.
GW5638.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.078

88
T. Shoda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 87–89
O
Cl
O
OH
O
SOCl2
reflux
2-phenoxyethyl chloride
AlCl3, CH2Cl2
BrMg
Et2O, rt, N2
OTHP
conc. HCl
MeOH, rt
1
2, 88%
HO
O
R1
HO
O
Cl
N
R2
R1R2NH
MeOH, TEA, 120 o
C
3, 83%
4, R1 = (CH2)5CH3, R2 = H, 41%
5, R1 = (CH2)11CH3, R2 = H, 40%
6, R1 = (CH2)17CH3, R2 = H, 39%
7, R1 = (CH2)11CH3, R2 = CH3, 26%
8, R1 = (CH2)11CH3, R2 = (CH2)11CH3, 12%
Scheme 1. Synthesis of tamoxifen derivatives.
utility.13–16 The other group is non-steroidal compounds, such as
GW5638 (Fig. 1). The structure of GW5638 is similar to tamoxifen
and contains an acrylic acid side chain extending from the triphen-
ylethylene core.17 GW5638 showed no agonistic effects in the
uterus17 due to a decrease in stability of the ER.18 Additionally, it
has been shown that GW5638 is useful for inhibiting the growth
of tamoxifen-resistant breast tumors.19
site, and physically prevents H12 interaction.20 We thought that
this long alkyl side chain might play a key role in down-regulation
of the ER. Therefore, we designed tamoxifen derivatives into which
simple alkyl chains were introduced on the amine moiety of
tamoxifen. The alkyl chains is expected to be outside the ER, as
determined from the X-ray structure (PDB ID: 3ERT),21 as shown
in our previous report.22
Thus, the concept of SERDs has provided very important infor-
mation; however, it is clear that there is an unmet medical need
for SERDs with improved pharmaceutical properties. In this Letter,
we report the design and synthesis of tamoxifen derivatives that
induced down-regulation of the ER. One of the compounds showed
the ability to down-regulate the ER in MCF-7 cells and antagonistic
activity against ER-mediated gene expression.
Tamoxifen is metabolized by metabolizing enzymes into 4-
hydroxytamoxifen (4-OHT) and endoxifen (Fig. 1),23,24 which
have 100 times more affinity for the estrogen receptor than
tamoxifen itself.25,26 The synthetic route to these compounds
is shown in Scheme 1. We synthesized (E/Z)-4-[1-{4-(2-chloro-
ethoxy)phenyl}-2-phenylbut-1-en-1-yl]phenol (3) according to a
previous report27 with slight modification. Then, compounds
4–8 were synthesized from corresponding amines at moderate
yields.28
Fulvestrant has a long alkyl side chain, which protrudes from
the ligand binding pocket, binds along the coactivator recruitment
Figure 2. Western blot analyses of ER
a
protein levels in MCF-7 cells. Cells were incubated with DMSO or compound (10
l
M) for 6 h. Whole proteins were extracted and ER
a
a
protein levels were analyzed by Western blotting. (A) Effects of the length of the alkyl side chain. (B) Effects of the substitution of amine moiety. (C) Dose dependence of ER
degradation induced by compounds 5 and 4, and the effects of a protease inhibitor on these responses.

T. Shoda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 87–89
89
We examined the effects of the length of the long alkyl side
chain on reducing ER protein levels in MCF-7 breast cancer cells.
MCF-7 cells were treated with these compounds, whole protein
was extracted, and ER protein levels were analyzed by Western
blotting, as reported previously.22 As shown in Figure 2A, reduction
of the ER protein level was observed in the cells treated with
10 M 5 (lane 5), but no significant differences in the activities of
4 and 6 were observed under these conditions (lanes 4 and 6,
respectively). The protein level of ER was slightly increased by
is due to proteasomal degradation. This molecule may be included
in a novel SERD against breast cancer. Further optimization and
experiments are currently ongoing in our lab.
a
a
Acknowledgment
a
l
This study was supported, in part, by a Research Fund from the
Japan Health Sciences Foundation.
a
treatment with 10 lM 4-OHT and endoxifen (lanes 2 and 3), which
References and notes
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7, a methyl derivative of 5, and 8, a dodecyl derivative of 5, did not
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has the ability to induce the proteasomal degradation of ER
a pro-
tein in MCF-7 cells.
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a
In summary, we designed and synthesized new tamoxifen
derivatives with a simple long alkyl side chain, which down-regu-
late the ERa protein level in MCF-7 cells. The reduction of ERa by 5
24. Teft, W. A.; Mansell, S. E.; Kim, R. B. Drug Metab. Dispos. 2011, 39, 558.
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28. Experimental method and spectroscopic data for compound 5: Compound 3
(0.11 mmol), dodecylamine (0.63 mmol), and triethylamine (0.3 mmol) were
dissolved in methanol. The mixture was heated in a pressure vessel at 120 °C
until the reaction was completed. The mixture was evaporated in vacuo and
the products were purified by column chromatography (23.3 mg, 40%). Clear
viscous compound; 1H NMR (400 MHz, CDCl3) d 8.49 (s, 1H), 7.65 (br, 1H),
6.37–7.13 (m, 13H), 3.86–4.05 (m, 4H), 3.07–3.18 (m, 2H), 2.87–2.94 (m, 2H),
2.40–2.47 (m, 2H), 1.64–1.68 (m, 2H), 1.25 (s, 18H), 0.86–0.91 (m, 6H); 13C
NMR (100 MHz, CDCl3) d 156.4, 156.0, 155.6, 155.1, 142.9, 142.8, 141.3, 141.0,
138.0, 137.7, 137.2, 135.2, 134.8, 132.3, 132.2, 130.9, 130.8, 129.9, 128.1, 128.0,
126.1, 115.5, 114.8, 114.2, 113.5, 63.4, 63.2, 50.8, 48.1, 46.6, 46.5, 32.1, 29.9,
29.8, 29.7, 29.6, 29.4, 29.3, 27.9, 26.9, 26.4, 26.3, 22.9, 14.4, 13.9; [HR-ESI(+)]:
m/z calcd for C36H49NO2 [M+H]+ 528.3836, found 528.3820.
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Figure 3. Antagonistic effect of ERa-mediated gene expression in MCF-7 cells.