Development of photoswitchable estrogen receptor ligands

Article abstract of DOI:10.1248/cpb.c19-01108

Photopharmacology has attracted attention as an approach for the development of novel therapeutics because it allows regulation of the bioactivity of compounds based on their conformational change by photo-irradiation. Previously, we have reported several types of selective estrogen receptor (ER) modulators based on diphenylmethane skeleton. To develop novel photopharmacological reagents, we designed and synthesized a set of ER ligands based on azobenzene skeleton, which can switch its conformation following UV irradiation. Our results showed that after UV irradiation, the Z-form of the synthesized compound 9 interacted with ERα, with a K_D value of 2.5μM, whereas the E-form of compound 9 did not bind ability to ERα at 10μM.

Full text of DOI:10.1248/cpb.c19-01108

398
Chem. Pharm. Bull. 68, 398–402 (2020)
Vol. 68, No. 4
Note
Development of Photoswitchable Estrogen Receptor Ligands
Keisuke Tsuchiya,a,b,# Tomohiro Umeno,c,# Genichiro Tsuji,b Hidetomo Yokoo,b,d
,b,d
,b
Masakazu Tanaka,c Kiyoshi Fukuhara,a Yosuke Demizu,* and Takashi Misawa*
a Graduate School of Pharmacy, Showa University; 1–5–8 Hatanodai, Shinagawa-ku, Tokyo 142–0064, Japan:
b National Institute of Health Sciences; 3–25–26 Tonomachi, Kawasaki-ku, Kawasaki 210–9501, Japan:
c Graduate School of Biomedical Sciences, Nagasaki University; 1–14 Bunkyo-machi, Nagasaki 852–8521, Japan: and
d Graduate School of Medical Life Science, Yokohama City University; 2–10–1 Wakaba, Mihama-ku, Kanagawa
261–0014, Japan.
Received December 23, 2019; accepted January 15, 2020
Photopharmacology has attracted attention as an approach for the development of novel therapeutics be-
cause it allows regulation of the bioactivity of compounds based on their conformational change by photo-ir-
radiation. Previously, we have reported several types of selective estrogen receptor (ER) modulators based on
diphenylmethane skeleton. To develop novel photopharmacological reagents, we designed and synthesized a
set of ER ligands based on azobenzene skeleton, which can switch its conformation following UV irradiation.
Our results showed that after UV irradiation, the Z-form of the synthesized compound 9 interacted with
ERα, with a KD value of 2.5µM, whereas the E-form of compound 9 did not bind ability to ERα at 10µM.
Key words estrogen receptor; photopharmacology; diazo moiety; fluorescent polarization assay
priate wavelength.8) Owing to its photochemical properties,
Introduction
Estrogen receptor (ER), which is a superfamily of nuclear the azobenzene moiety has been broadly utilized to change the
receptors, plays pivotal roles in physiological actions, such as conformation of compounds and control their bioactivities.11)
cell growth and bone marrow homeostasis.1,2) 17β-Estradiol, an
With the aim of development of novel ER ligands, we hy-
endogenous ligand for ERα, binds to the ligand binding domain pothesized that photopharmacological ER ligands are a prom-
of ERα and promotes its transactivation, resulting in target gene ising strategy to overcome the organ selectivity of ER ligands
transcription. It has been reported that ERα is overexpressed in the treatment of breast cancer. Recently, photoswitchable
in 70% of breast cancer cells and that it plays an important Farnesoid X receptor (FXR) ligands based on GW4064, a
role in promoting tumor progression.3) Therefore, ERα may be representative FXR agonist, have been reported.12) The com-
a promising target for breast cancer therapy, and the several pounds have a diazo moiety instead of the unsaturated double
ERα antagonists have been developed4–6) (Fig. 1). Among these, bond of GW4064, and their FXR activity is regulated by UV
tamoxifen and raloxifen are well known as selective ER modu- irradiation in cells. These findings suggested that photoswitch-
lators and broadly used for breast cancer therapy.6)
able reagents are applicable for intracellular proteins. Previ-
Although tamoxifen shows promising antitumor effects ously, Hashimoto and colleagues reported that diphenylmeth-
against breast cancer cells, it also acts as an agonist against ane (DPM) skeleton can be a bioisoster of steroid skeleton,
in the uterus ERα,7) and such poor organ selectivity become a and appropriate modifications of DPM skeleton can be applied
problem in its clinical use. To overcome poor organ selectiv- to develop several nuclear receptor ligands, such as vitamin
ity, innovative strategies have been urgently required for de- D receptor and ERs.13,14) Among these ligands, PBP showed
velopment of novel ligands. Recently, photopharmacology has potent antagonistic activity against ERα, with IC50 value of
garnered attention as a promising strategy to regulate the bio- 4.9nM.13) Furthermore, its diphenylsilane derivative also ex-
activity of compounds under UV irradiation. To data, several hibits transactivation activity against ERα, with IC50 value of
photopharmaceutical agents containing diazo, spiropyran, and 29.7 nM,15) indicating that substitution of heptane chain with
diarylethene moieties have been reported8–10) (Fig. 2). Espe- other atoms is applicable in the development of ER ligands.
cially, compounds that contain a diazo moiety undergo trans- Hence, we designed and synthesized a set of photoswitchable
to-cis isomerization under irradiation with light of an appro- ER ligands containing a diazo moiety (Fig. 3). Moreover, we
Fig. 1. Chemical Structures of 17β-Estradiol, Tamoxifen, and Raloxifen
# These authors contributed equally to this work.
*To whom correspondence should be addressed. e-mail: demizu@nihs.go.jp; misawa@nihs.go.jp
© 2020 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
399
Fig. 2. Examples of Photochemical Moieties: Azobenzene, Spiropyran,
and Diarylethene
Chart 1. Synthesis of Compounds 1–10
Next, we evaluated the conformational changes in com-
pounds 1–10 by measuring the UV spectra and the E/Z ratio
of the compounds were determined using HPLC analysis.
The UV spectra of compounds 1–10 are shown in Fig. 4.
Compound 1 did not exhibit any conformational change after
UV irradiation at 254 or 365nm, whereas compounds 2, 6,
and 7 showed dramatic conformational changes from E- to
Z-form after UV irradiation at 365nm. These results showed
that alkylation of the phenol group may exert an advantage
to their conformational change. However, further investiga-
tion revealed that the Z-isomer of compounds 2–7 was rapidly
reversed to the E-form at room temperature (25°C) even in the
dark; therefore, the effects of these conformational changes
on the binding affinity of the compounds to ERα could not
Fig. 3. Chemical Structures of Photoswitchable FXR Ligands Based on
GW4064, ER Ligands Based on PBP Skeleton, and the Designed Photo-
switchable PBP Derivatives in This Study
evaluated their UV spectra to confirm their conformational be investigated. It has been reported that o-fluorination of
change after UV irradiation, and we investigated the binding diphenyldiazo compounds can stabilize and prolong the E/Z-
of the synthesized compounds to ERα.
conformation via their steric effect.21) On the basis of this re-
port, we employed tetra-fluorinated diphenyldiazo compounds
at the o-position, and confirmed their conformational change
Results
Chemistry Initially, we prepared a set of azobenzene after UV irradiation. Compound 8 did not show any confor-
derivatives 1–10, via coupling and modification of aryl diazo- mational change after UV irradiation, whereas compounds
nium species with phenolic compounds (Chart 1). Azobenzene 9 and 10, which had four fluorine atoms at the o-position
1, 2, 5, 8 and 9 were synthesized according to a previously showed conformational changes after UV irradiation (Table 1;
reported method.16–20) Next, the phenolic hydroxy group on E/Z ratio: 9, 13/87; 10, 11/89), and their half-life (t1/2) in the
1 and 8 was alkylated to afford 2–6 and 9, and both hydroxy E-form was prolonged because of their steric effects (Table 1;
groups in 1 were acetylated (7). To synthesize tetrafluoro- E/Z ratio after 16h incubation at 37°C: 9, 28/72; 10, 32/68).
azobenzene 8 and 9, 2,6-difluoroaniline and (4-amino-3,5- These data revealed that the phenolic hydroxy group at the p-
difluorophenyl)methanol were converted to diazonium salt via position took disadvantages on conformational control by UV
treatment with NaNO2 in conc. HCl. Next, the resultant diazo- irradiation, and the compounds 9, and 10 could be a candidate
nium salt was subjected to a coupling reaction with 3,5-difluo- as photoswitchable ERα ligands.
rophenol in the presence of aqueous NaOH, thus yielding
Finally, to investigate the effects of conformational changes
tetrafluoroazobenzene with or without a hydroxymethyl group; on the binding affinity of the compounds to ERα, the binding
methylation of a phenolic hydroxy group of compound 8 pro- of compounds 9 and 10 to ERα was examined. The binding
vided 9 as a mixture of E- and Z-isomers, but isomerization of affinity of 9 and 10 was evaluated using fluorescent polar-
Z- to E-isomer occurred during column chromatography.
ization (FP) assay kit following the manufacturer’s instruc-

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Fig. 4. UV-Vis Spectra of Compounds 1–10 (Solid Line)
Samples were irradiated with 365nm light for 3min (dotted line), or subsequently irradiated with 254nm for 3min (broken line). Each compound was dissolved in etha-
nol to a concentration of 1mM.
tion (Thermo Fisher Scientific, U.S.A.). As shown in Fig. 5, ER binding affinity was controlled by UV irradiation, sug-
Z-isomer of compound 9 showed moderate binding affinity gesting that compound 9 can be a promising candidate of a
to ERα, whereas its E-isomer had no binding affinity even photoswitchable ERα ligand. The detailed structure–activity
at 10µM. Moreover, the Z- and E-isomers of compounds 10 relationship (SAR) analysis and ERα-transactivation activity
bound to ERα, but the binding affinity of compounds 10 was of compound 9 in the cells are in progress.
lower than that of 9. These results showed that the Z-isomer of
compounds 9 and 10 exhibited the higher binding affinity to
ERα than did the E-isomer of both compounds, and that intro-
Experimental
General All chemicals were purchased from Sigma-
duction of a hydroxymethyl group at the p-position decreased Aldrich (U.S.A.), FUJIFILM Wako Pure Chemical Corpo-
the binding affinity of the compounds to ERα.
ration (Japan), and Tokyo Chemical Industry (Japan) and
In conclusion, we designed and synthesized a set of photo- were used without further purification. TLC analysis was
switchable ER ligands based on diphenyldiazo skeleton. Intro- conducted using Merck silica gel 60 F254 pre-coated plates
duction of fluorine at the o-position prolonged their half-life and visualized using a 254nm/365nm UV lamp, and iodine
(t1/2) as a Z-form by its steric effects. Moreover, the binding af- stain. Column chromatography was performed using silica gel
1
finity of compounds to ERα was investigated using FP assay, (spherical, neutral) purchased from Kanto Chemical. H- and
and the results revealed that the Z-isomer of compound 9 13C-NMR spectra were recorded on a JEOL ECZ600 spec-
showed higher binding affinity to ERα than did the E-isomer. trometer, and measurements were carried out in CDCl3 with
Although the binding affinity of compound 9 was moderate 0.03% tetramethylsilane or dimethyl sulfoxide (DMSO)-d6.
compared to that of known ER ligands, such as tamoxifen, the High-resolution mass spectra (HR-MS) were measured using

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
401
a Shimadzu IT-TOF MS equipped with an electrospray ioniza- s), 6.99 (2H, d, J=8.4Hz), 7.45 (2H, d, J=8.4Hz), 7.84 (2H,
tion source.
d, J=7.2Hz), 7.89 (2H, d, J=7.2Hz). 13C-NMR (151MHz,
(4-((4-Ethoxyphenyl)diazenyl)phenyl)methanol (3) To a CDCl3) δ: 10.45, 22.47, 64.79, 69.79, 114.67, 122.68, 124.69,
solution of 4-(4-hydroxyphenyl)azobenzyl alcohol (1, 57.1mg, 127.38, 143.07, 146.78, 152.16, 161.67.; HR-MS (ESI) m/z:
0.250mmol) in N,N-dimethylformamide (DMF) (0.5mL) [M+H]+ Calcd. for C16H19N2O2, 271.1441. Found, 271.1434.
were added K2CO3 (69.1mg, 0.500mmol) and iodoethane
(4-((4-(2-(Dimethylamino)ethoxy)phenyl)diazenyl)phe-
(22.2 µL, 0.275mmol) at room temperature, and the mixture nyl)methanol (6) To a solution of 4-(4-hydroxyphenyl)-
was warmed to 70°C. After being stirred for 4h, the reaction azobenzyl alcohol (1, 113mg, 0.499mmol) and K2CO3
mixture was diluted with EtOAc/n-hexane (1:3), and washed (1.43g, 10.3mmol) in acetone/water (9:1, 5mL) were
successively with 1M HCl and brine. The organic layer was added 2-(dimethylamino)ethyl chloride hydrochloride (262mg,
dried over Na2SO4 and concentrated in vacuo to give a crude 1.82mmol) and K2CO3 (390mg, 2.82mmol) at 0°C, and the
product, which was purified by flash column chromatography mixture was stirred at the same temperature for 30min. Then,
on silica gel (50% EtOAc in n-hexane) to give title compound the mixture was refluxed for 2h. After cooling to room tem-
1
3 (44.1mg, 69%) as an orange solid. mp 126–128°C; H-NMR perature, the solution was filtered, and the filtrate was con-
(600MHz, CDCl3) δ: 1.45 (2H, t, J=7.8Hz), 2.03 (1H, brs, centrated in vacuo. To the residue was added Et2O, and the
1H), 4.10 (2H, q, J=7.8Hz, 2H), 4.74 (2H, s), 6.99 (2H, d, suspension was filtered to afford the title compound 6 (102mg,
1
J=8.4Hz), 7.46 (2H, d, J=8.4Hz), 7.85 (2H, d, J=6.6Hz), 68%) as an orange solid. mp 142–144°C; H-NMR (600MHz,
7.90 (2H, d, J=6.6Hz). 13C-NMR (151MHz, CDCl3) δ: 14.72, DMSO-d6) δ: 2.19 (6H, s), 2.61 (2H, t, J=6.0Hz), 4.11 (2H, t,
63.81, 64.85, 114.66, 122.71, 124.72, 127.41, 143.08, 146.83, J=6.0Hz), 4.55 (2H, s), 5.35 (1H, s), 7.09 (2H, d, J=8.4Hz),
152.20, 161.48. HR-MS (electrospray ionization (ESI)) m/z: 7.46 (2H, d, J=8.4Hz), 7.78 (2H, d, J=7.2Hz), 7.83 (2H,
[M+H]+ Calcd for C15H17N2O2, 257.1285. Found, 257.1276.
d, J=7.2Hz). 13C-NMR (151MHz, DMSO-d6) δ: 45.54,
(4-((4-n-Propylphenyl)diazenyl)phenyl)methanol (4) To 57.56, 62.45, 66.27, 115.05, 122.18, 124.44, 127.08, 145.65,
a
solution of 4-(4-hydroxyphenyl)azobenzyl alcohol (1, 146.09, 150.89, 161.15. HR-MS (ESI) m/z: [M+H]+ Calcd for
57.1mg, 0.250mmol) in DMF (0.5mL) were added C17H22N3O2, 300.1707. Found, 300.1714.
K2CO3 (69.1mg, 0.500mmol) and 1-iodopropane (26.8µL, 4-((4-(Acetoxymethyl)phenyl)diazenyl)phenyl
0.276mmol) at room temperature, and the mixture was (7) To solution of 4-(4-hydroxyphenyl)azobenzyl al-
Acetate
a
warmed to 70°C. After being stirred for 4h, the reaction cohol (1, 50.0mg, 0.219mmol) in pyridine (2.2mL) were
mixture was diluted with EtOAc/n-hexane (1:3), and washed added Et3N (61.4µL, 0.441mmol), N,N-dimethyl-4-amino-
successively with 1M HCl and brine. The organic layer was pyridine (13.5mg, 0.111mmol) and acetic anhydride (23.0µL,
dried over Na2SO4 and concentrated in vacuo to give a crude 0.243mmol) at 0°C, and the mixture was gradually warmed
product, which was purified by flash column chromatography to room temperature. After being stirred for 6h, the reaction
on silica gel (50% EtOAc in n-hexane) to give title compound mixture was diluted with EtOAc, and washed successively
1
4 (44.7mg, 66%) as an orange solid. mp 130°C; H-NMR with 1M HCl and brine. The organic layer was dried over
(600MHz, CDCl3) δ: 1.05 (3H, t, J=6.6Hz), 1.84 (2H, sextet, Na2SO4 and concentrated in vacuo to give a crude product,
J=6.6Hz), 2.16 (1H, brs), 3.98 (2H, t, J=6.6Hz), 4.72 (2H, which was purified by flash column chromatography on
silica gel (5–20% EtOAc in n-hexane) to give title compound
1
7 (11.6mg, 17%) as an orange solid. mp 100–102°C; H-NMR
(600MHz, CDCl3) δ: 2.07 (3H, s), 2.27 (3H, s), 5.11–4.96 (2H,
Table 1. E/Z Ratio of Compounds 9 and 10
m), 7.18 (2H, d, J=8.4Hz), 7.43 (2H, d, J=8.4Hz), 7.83 (2H,
E/Z-Area ratio
d, J=6.6Hz), 7.89 (2H, d, J=6.6Hz). 13C-NMR (151MHz,
E-Isomer
Z-Isomer
CDCl3) δ: 20.98, 21.16, 65.69, 122.23, 124.10, 123.02, 128.76,
138.85, 150.17, 152.23,152.71, 169.10, 170.79. HR-MS (ESI)
m/z: [M+H]+ Calcd for C17H17N2O4, 313.1183. Found,
313.1189.
(E)-9 Nature
89%
13%
28%
94%
11%
32%
11%
87%
72%
6%
(Z)-9 365nm 3min
(Z)-9 37°C 16h
(E)-10 Nature
(4-((1,6-Difluro-4-methoxyphenyl)diazenyl)-3,5-difluo-
(Z)-10 365nm 3min
(Z)-10 37°C 16h
89%
68%
rophenyl)methanol (10; E/Z-Isomer) To
a solution of
(4-amino-3,5-difluorophenyl)methanol (32.7mg, 0.205mmol)
Fig. 5. Binding Affinity of Each E/Z-Isomer of Compounds 9 and 10 to ERα as Examined by FP Assay

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Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
and conc. HCl (100µL) in water (750µL) was added dropwise for the Promotion of Science (KAKENHI, Grants 15K18905,
NaNO2 (15.6mg, 0.226mmol) in water (450µL) at 0°C. Then, 18K14880 to T. M., 19K16333 to G. T, 17k08385 to Y. D.
a cooled solution of 3,5-difluorophenol (29.3mg, 0.225mmol) 18H05502 to Y. D.), the Terumo Foundation for Life Sciences
in 1M NaOH aq was added dropwise to the reaction mix- and Arts (to T. M. and Y. D.), the Takeda Science Founda-
ture, and the mixture was stirred at the same temperature for tion (to T. M., and Y. D.), the Naito Foundation (to Y. D.), the
1h. After acidified with 1M HCl, the solution was extracted NOVARTIS Foundation (Japan) for the Promotion of Science
with EtOAc, and the combined organic phase was dried over (to Y. D.), and Grant for Basic Science Research Project from
MgSO4. Removal of the solvent afforded a crude product, the Sumitomo Foundation (to Y. D.).
which was purified by column chromatography on silica
gel (40% EtOAc in n-hexane) to give 3,5-difluoro-4-(2,6-
Conflict of Interest The authors declare no conflict of
difluoro-4-hydroxyphenyl)azobenzyl alcohol (S-1, 15.8mg, interest.
26%). To a solution of S-1 (15.8mg, 52.6µmol) in DMF
(0.5mL) were added K2CO3 (14.5mg, 0.105mmol) and iodo-
methane (4.26µL, 68.4µmol) at room temperature, and the
mixture was warmed to 70°C. After being stirred for 5h, the
reaction mixture was diluted with EtOAc/n-hexane (1:2), and
washed successively with 1M HCl and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuo to
give a crude product, which was purified by flash column
chromatography on silica gel (25% EtOAc in n-hexane) to
give title compound 10 (E,Z-mixture: 8.0mg, 48%, NOTE: A
part of Z-isomer isomerized to E-isomer during flash column
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Acknowledgments This work was supported in part by
Grants from AMED under Grant Number 19mk0101120j0002
(to Y. D.) and 19mk0101166j0001 (to T. M.), the Japan Society