Design and synthesis of estrogen receptor degradation inducer based on a protein knockdown strategy

Article abstract of DOI:10.1016/j.bmcl.2011.11.086

We designed and synthesized estrogen receptor (ER) degradation inducers 5, 6, and 7, which crosslink the ER and the cellular inhibitor of apoptosis protein 1 (cIAP1). Compounds 5, 6, and 7 induced cIAP1-mediated ubiquitylation of ERa resulting in its proteasomal degradation.

Full text of DOI:10.1016/j.bmcl.2011.11.086

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1793–1796
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of estrogen receptor degradation inducer based
on a protein knockdown strategy
Yosuke Demizu a, Keiichiro Okuhira b, Hiromi Motoi a, Akiko Ohno a, Takuji Shoda a, Kiyoshi Fukuhara a,
a,
Haruhiro Okuda a, Mikihiko Naito b, , Masaaki Kurihara
⇑
⇑
a Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan
b Division of Biochemistry and Molecular Biology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, 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 estrogen receptor (ER) degradation inducers 5, 6, and 7, which crosslink the
Received 1 November 2011
Revised 18 November 2011
Accepted 21 November 2011
Available online 28 November 2011
ER and the cellular inhibitor of apoptosis protein 1 (cIAP1). Compounds 5, 6, and 7 induced cIAP1-med-
iated ubiquitylation of ERa resulting in its proteasomal degradation.
Ó 2012 Published by Elsevier Ltd.
Keywords:
Estrogen receptor
Protein knockdown
Tamoxifen
Ubiquitin-proteasome system
Breast cancer is the most common form of cancer in women,
and its incidence is increasing year by year. Estrogen receptors
(ERs) are often overexpressed in the tissues of breast cancer pa-
tients, which promotes the estrogen-dependent proliferation of
cancer cells.1–3 Therefore, ER antagonistic drugs, such as tamoxifen,
a non-steroidal selective estrogen receptor modulator, are effective
at treating breast cancer.4,5 Tamoxifen is metabolized by CYP2D6
and CYP3A4 into 4-hydroxytamoxifen, which has 30–100 times
more affinity for the estrogen receptor than tamoxifen itself.6
Tamoxifen is the most frequently prescribed drug for the treatment
of all stages of breast cancer,7 and it is also used to prevent the dis-
ease in women who are at high risk of developing breast cancer.8
However, tamoxifen has agonistic effects on ER in uterus cancer
cells and increases the risk of endometrial cancer.9,10 Furthermore,
tamoxifen activates the protein kinase B (Akt) signaling pathway
by binding to a particular ER variant, resulting in the inhibition
of apoptosis in cancer cells.11,12
Recently, we reported a protein knockdown strategy for inducing
the degradation of a target protein using the ubiquitin-proteasome
system (UPS).13–18 To degrade proteins of interest, we developed
SNIPER (Specific and Non-genetic IAP-dependent Protein Erasers),
hybrid molecules composed of bestatin (BS), an inhibitor of the
cellular apoptosis protein 1 (cIAP1), and a ligand for the target
protein.13–15 These molecules cross-linkcIAP1 and thetarget protein
to induce ubiquitylation and subsequent proteasomal degradation
of the target protein.
In this study, we applied this methodology to the selective
degradation of ERa in breast cancer cells. We designed SNIPER(ER)
hybrid molecules 5, 6, and 7, which induce the selective poly-ubiq-
uitylation of the ER to cause proteasomal degradation. Molecules 5,
6, and 7 contain two biologically active scaffolds: one is a tamoxi-
fen derivative, which is used to bind to the ER, and the other is a BS
moiety, which binds to cIAP1 to induce the ubiquitylation and sub-
sequent proteasomal degradation of the target protein (Fig. 1).
Molecules with different length linkers 5, 6, and 7 were designed
based on the X-ray structure (PDB ID: 3ERT) of the complex formed
between 4-hydroxytamoxifen and ERa,
19 and BS was ligated to the
dimethylamino moiety of 4-hydroxytamoxifen (red circle in
Fig. 2a) via an alkyl linker (Fig. 2b).
The ligated molecules 5, 6, and 7 were synthesized as follows
(Scheme 1). First, (E/Z)-endoxifen (1)20,21 was condensed with sev-
eral acids to afford amides 2, 3, and 4. After deprotection of one of
the N-Boc protecting groups of 2, 3, and 4, the generated amines
were reacted with N-Boc bestatin,11 and the subsequent deprotec-
tion of another of the molecules’ N-Boc protecting groups gave
ligated compounds 5, 6, and 7, respectively.22
Then, we evaluated the effects of compounds 5, 6, and 7 on ER
protein degradation in MCF-7 breast cancer cells by examining the
treatment dose (Fig. 3).23 The protein expression of ER
was in-
a
a
creased by treatment with (E/Z)-endoxifen (lane 3), which was
consistent with the findings of a previous report demonstrating
⇑
Corresponding authors. Tel.: +81 3 3700 1141; fax: +81 3 3707 6950.
E-mail addresses: miki-naito@nihs.go.jp (M. Naito), masaaki@nihs.go.jp (M.
that (E/Z)-endoxifen induced the accumulation of ER
a protein in
Kurihara).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.11.086

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Y. Demizu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1793–1796
Figure 1. Estrogen receptor degradation strategy using a ligated hybrid molecule.
Figure 2. (a) X-ray structure of the complex formed between 4-hydroxytamoxifen and ERa(3ERT). (b) Design of the SNIPER(ER) ER degradation inducer.
Boc
CO2H
n
Ph
N
H
Boc NH
Ph
n
HN
O
EDC, HOBt, DIPEA
CH2Cl2, rt, 12h
N
O
1) TFA, CH2Cl2, rt, 10min
O
2) EDC, HOBt, DIPEA,
CH2Cl2, rt, 12h
Ph
O
OH
OH
2 (n = 1)
3 (n = 3)
4 (n = 5)
Boc
OH
(E/Z)-endoxifen (1)
N
H
N
H
OH
O
3) 6N HCl aq., rt, 6h
Ph
H2N
O
HO HN
NH
O
Ph
5 (n = 1)
6 (n = 3)
7 (n = 5)
n
N
O
O
OH
Scheme 1. Synthesis of ligated compounds 5, 6, and 7.

Y. Demizu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1793–1796
1795
Figure 3. Dose-dependent ER
a
degradation responses induced by compounds 5, 6, and 7 and the effects of a protease inhibitor on these responses.
MCF-7 cells.24 Compared with (E/Z)-endoxifen (lane 3), a reduced
level of ER was observed in the cells treated with 10 or 30 lM
of compound 5, 6, or 7 (lanes 5, 6, and 7, respectively), and no
2. Doisneau-Sixou, S. F.; Cergio, C. M.; Carroll, J. S.; Hui, R.; Musgrove, E. A.;
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a
4. Deroo, B. J.; Korach, K. S. J. Clin. Invest. 2006, 116, 561.
apparent differences in the activities of them were observed during
the 6 h study period. On the other hand, the ERa level was not al-
tered by the combined use of (E/Z)-endoxifen and methyl bestatin
(MeBS)25 (lane 4). These results suggest that (E/Z)-endoxifen con-
jugated with BS as a single molecule (at a concentration of greater
5. Early Breast Cancer Trialists’ Collaborative Group Lancet 1998, 351, 1451.
6. Desta, Z.; Ward, B. A.; Soukhova, N. V.; Flockhart, D. A. J. Pharmacol. Exp. Ther.
2004, 310, 1062.
7. Early Breast Cancer Trialists’ Collaborative Group Lancet 1992, 339, 71.
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Cronin, W. M.; Vogel, V.; Robidoux, A.; Dimitrov, N.; Atkins, J.; Daly, M.;
Wieand, S.; Tan-Chiu, E.; Ford, L.; Wolmark, N. J. Natl. Cancer Inst. (Bethesda)
1998, 90, 1371.
9. Bernstein, L.; Deapen, D.; Cerhan, J. R.; Schwartz, S. M.; Liff, J.; McGann-
Maloney, E.; Perlman, J. A.; Ford, L. J. Natl. Cancer Inst. 1999, 91, 1654.
10. Shang, Y.; Brown, M. Science 2002, 295, 2465.
11. Arpino, G.; Wiechmann, L.; Osborne, C. K.; Schiff, R. Endocr. Rev. 2008, 29, 217.
12. Lin, S.-L.; Yan, L.-Y.; Zhang, X.-T.; Yuan, J.; Li, M.; Qiao, J.; Wang, Z.-Y.; Sheng, J.;
Sun, Q.-Y. PLoS One 2010, 5, e9013.
13. Itoh, Y.; Ishikawa, M.; Naito, M.; Hashimoto, Y. J. Am. Chem. Soc. 2010, 132,
5820.
14. Itoh, Y.; Ishikawa, M.; Kitaguchi, R.; Sato, S.; Naito, M.; Hashimoto, Y. Bioorg.
Med. Chem. 2011, 19, 3229.
15. Okuhira, K.; Ohoka, N.; Sai, K.; Nishimaki-Mogami, T.; Itoh, Y.; Ishikawa, M.;
Hashimoto, Y.; Naito, M. FEBS Lett. 2011, 585, 1147.
16. Sekine, K.; Takubo, K.; Kikuchi, R.; Nishimoto, M.; Kitagawa, M.; Abe, F.;
Nishikawa, K.; Tsuruo, T.; Naito, M. J. Biol. Chem. 2008, 283, 8961.
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than 10
protein. Furthermore, the suppressive effects of compounds 5, 6,
and 7 on ER were blocked by the addition of a proteasome inhib-
itor, MG132, indicating that compounds 5, 6, and 7 induced the
proteasomal degradation of ER
In summary, we used a protein knockdown method for the
selective degradation of ER and synthesized ER degradation
inducers 5, 6, and 7, which form crosslinks between ER and cIAP1.
Compounds 5, 6, and 7 were able to induce cIAP1-mediated ubiq-
uitylation and hence induce the proteasomal degradation of ER
lM) is required for the efficient degradation of the ERa
a
a
.
a
a
a
a
.
These molecules are novel candidates for therapeutic agents
against breast cancer, and further derivatization of these molecules
is currently underway.
18. Sato, S.; Aoyama, H.; Miyachi, H.; Naito, M.; Hashimoto, Y. Bioorg. Med. Chem.
Lett. 2008, 18, 3354.
Acknowledgments
19. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.; Agard, D. A.;
Greene, G. L. Cell 1998, 95, 927.
20. We used (E/Z)-endoxifen because (E)- and (Z)-4-hydroxytamoxifen derivatives
are isomerized in vivo: Crewe, H. K.; Notley, L. M.; Wunsch, R. M.; Lennard, M.
S.; Gillam, E. M. Drug Metab. Dispos. 2002, 30, 869.
This study was supported, in part, by Grants-in-Aid for Scien-
tific Research (C) (M.K.: 22590114) and Challenging Exploratory
Research (M.N.: 23659048) from the Japan Society for the Promo-
tion of Science, a research grant from the Astellas Foundation for
Research on Metabolic Disorders (M.N.), and a grant from the
Terumo Life Science Foundation (M.N.).
21. Preparation of (E/Z)-endoxifen: Fauq, A. H.; Maharvi, G. M.; Sinha, D. Bioorg.
Med. Chem. Lett. 2010, 20, 3036.
22. Spectroscopic data for compound 7: White solid; 1H NMR (400 MHz, CD3OD) d
6.91–7.35 (m, 13H), 6.40–6.76 (m, 5H), 3.98–4.34 (m, 4H), 3.66–3.83 (m, 3H),
2.91–3.18 (m, 7H), 2.30–2.47 (m, 4H), 1.28–1.84 (m, 11H), 0.89–0.99 (m, 9H);
13C NMR (100 MHz, CD3OD) 175.2, 173.1, 172.0, 142.9, 141.0, 138.5, 136.7,
136.4, 135.5, 135.1, 131.8, 130.4, 129.7, 129.3, 128.9, 128.6, 128.1, 127.7, 127.4,
125.8, 114.7, 113.9, 113.1, 68.6, 65.4, 55.1, 52.4, 40.9, 39.2, 35.3, 33.0, 32.5,
29.7, 29.2, 28.9, 26.4, 25.3, 24.9, 22.1, 21.2, 12.8; [HR-ESI(+)]: m/z calcd for
References and notes
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48H63N4O6 [M+H]+ 791.4748: found 791.4852.

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Y. Demizu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1793–1796
23. MCF-7 cells were treated with the test compounds at the indicated
concentrations in the presence or absence of 10 M of MG132 for 6 h, and
antibody (Santa Cruz), anti-b-actin mouse monoclonal antibody (SIGMA), and
anti-human cIAP1 goat polyclonal antibody (R&D systems).
24. Laïos, I.; Journe, F.; Laurent, G.; Nonclercq, D.; Toillon, R. A.; Seo, H. S.; Leclercq,
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25. MeBS interacts with the BIR3 domain of cIAP1 and induces the auto-
ubiquitylation of cIAP-1, resulting in the proteasomal degradation of cIAP1:
See Ref. 16.
l
then the cells were collected and extracted with lysis buffer (1% SDS, 0.1 M
Tris–HCl (pH 7.0), 10% glycerol) and boiled for 10 min. Protein concentrations
were determined by the BCA method, and equal amounts of protein lysate
were separated by SDS–PAGE, transferred to a PVDF membrane, and Western
blotted using the following antibodies: anti-human ER
a mouse monoclonal