Molecular design, chemical synthesis, and biological evaluation of agents that selectively photo-degrade the transcription factor estrogen receptor-α

Article abstract of DOI:10.1039/c1ob05629h

2-Phenylquinoline (1) degraded proteins under photo-irradiation with long-wavelength UV light without additives and under neutral conditions. We designed and synthesized a 2-phenylquinoline-estrogen receptor-α (ER-α) agonist (hybrid 2) and a 2-phenylquino

Full text of DOI:10.1039/c1ob05629h

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 6357
www.rsc.org/obc
PAPER
Molecular design, chemical synthesis, and biological evaluation of agents that
selectively photo-degrade the transcription factor estrogen receptor-a†
Kana Tsumura,a Akane Suzuki,a Takeo Tsuzuki,a Shuho Tanimoto,a Hajime Kaneko,a Shuichi Matsumura,a
Masaya Imoto,b Kazuo Umezawa,a Daisuke Takahashia and Kazunobu Toshima*a
Received 20th April 2011, Accepted 15th June 2011
DOI: 10.1039/c1ob05629h
2-Phenylquinoline (1) degraded proteins under photo-irradiation with long-wavelength UV light
without additives and under neutral conditions. We designed and synthesized a
2-phenylquinoline-estrogen receptor-a (ER-a) agonist (hybrid 2) and a 2-phenylquinoline-ER-a
antagonist (hybrid 3) containing estradiol and 4-hydroxytamoxifen moieties, respectively. These
2-phenylquinoline hybrids effectively and selectively photo-degraded the target transcription factor,
ER-a, which has a high affinity for estradiol and 4-hydroxytamoxifen. Target-selective
photo-degradation was examined in both glass vessels and MCF-7 breast cancer cells, which are
dependent upon ER-a for growth. In addition, 2-phenylquinoline-estradiol hybrid 2 functioned as an
agonist of ER-a and promoted growth of MCF-7 in the absence of photo-irradiation, while it inhibited
the growth of MCF-7 cells upon photo-irradiation due to the photo-degradation of ER-a. In contrast,
2-phenylquinoline-4-hydroxytamoxifen hybrid 3 inhibited growth of MCF-7 cells in the absence of
photo-irradiation due to the antagonist effect of the 4-hydroxytamoxifen moiety against ER-a, and
upon photo-irradiation significantly inhibited cell growth due to the dual antagonist effect of the
4-hydroxytamoxifen moiety and photo-degradation of ER-a.
generally produced by the photo-excitation of a light-activated
Introduction
photo-sensitizer molecule. One of the most important benefits
The development of novel methods for selectively controlling
of PDT compared with other therapies is the ease of space and
specific protein functions is of considerable importance in the fields
time control for treatment. However, one of the drawbacks of
of chemistry, biology, and medicine. Protein degradation plays a
PDT is its potential for serious side effects, such as systemic
central role in many cellular functions. For example, misfolded and
toxicity and photosensitivity resulting from delocalization of a
damaged proteins are degraded and removed from cells to avoid
photo-sensitizer lacking molecular-targeted selectivity. The use
toxicity, and the cellular concentration of regulatory proteins is
of light-activated and molecular-targeted (LAMTA) molecules
maintained at optimal levels by degradation. In this context, much
may minimize the potential side effects associated with PDT by
attention has been given to development of an organic photo-
preventing delocalization of photo-sensitizers, thereby providing
activatable agent that can degrade proteins1 upon irradiation with
greater advantages than are achievable with conventional agents.
a specific wavelength of light under mild conditions and without
In addition, LAMTA molecules could potentially be a very useful
any additives (such as metals or reducing agents).
new class of pinpoint tools in a wide range of applications in the life
One of the most important applications of such selective photo-
sciences. However, to date there have been no reports of methods
degrading agents is in the field of photodynamic therapy (PDT).
employing light-activated agents for selective degradation of target
PDT is used to treat diseases characterized by neoplastic growth,
proteins. In this paper, we report the target-selective degradation
of a protein induced by a light-activated small organic molecule.3
We found that 2-phenylquinoline degrades proteins in solution
under long-wavelength UV photo-irradiation without additives
and under neutral conditions. Two 2-phenylquinoline synthetic
hybrids, 2-phenylquinoline-estradiol and 2-phenylquinoline-4-
hydroxytamoxifen, wereshown to effectively and selectively photo-
degrade recombinant estrogen receptor-a (ER-a), a key transcrip-
tion factor involved in the growth of MCF-7 breast cancer cells.
The target-selective degradation of ER-a led to the inhibition of
MCF-7 cell growth. To the best of our knowledge, this is the
first successful demonstration of target-selective degradation of a
including various cancers, age-related macular degeneration,
and actinic keratosis.2 Cell death is induced by the activity of
a reactive oxygen species (ROS) such as singlet oxygen 1O2,
aDepartment of Applied Chemistry, Faculty of Science and Technology, Keio
University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan
bDepartment of Bioscience and Informatics, Faculty of Science and Tech-
nology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522,
Japan. E-mail: toshima@applc.keio.ac.jp; Fax: +81-45-566-1576; Tel: +81-
45-566-1576
† Electronic supplementary information (ESI) available: 1H- and 13C-
NMR spectra of all new compounds. See DOI: 10.1039/c1ob05629h
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6357–6366 | 6357
©

View Article Online
protein in glass vessels and in cell culture by light switching under
neutral conditions.4 We anticipate that the present novel method
will be useful as a “smart” technology for selectively controlling
the function of target proteins. In addition, it should prove useful
in protein structure–activity studies, investigation of structural
domains, and the design of novel, protein-targeting therapeutic
drugs.
Results and discussion
Fig. 2 Photo-degradation of human estrogen receptor-a (ER-a) by
2-phenylquinoline (1). ER-a (1.0 mM) was incubated with 2-phenylquino-
line (1) in 20% acetonitrile/Tris-HCl buffer (pH 8.0, 50 mM) at 25 ◦C for
2 h under irradiation with a UV lamp (365 nm, 100 W) placed 10 cm from
the sample, and analyzed by tricine-SDS-PAGE. Lane 1: size marker; lane
2: ER-a alone; lane 3: ER-a with UV; lane 4: ER-a + 2-phenylquinoline
(1) (10 mM) without UV; lanes 5–8: ER-a + 2-phenylquinoline (1)
(concentrations 10, 3, 1, and 0.3 mM, respectively) with UV.
Investigation of 2-phenylquinoline as a protein photo-degrading
small organic molecule
In our previous work on DNA photocleavage, we found that
certain 2-phenylquinoline derivatives efficiently cleaved DNA
upon light activation.5 Based on these findings, we hypothesized
that if a 2-phenylquinoline derivative could be made to produce a
radical or ROS upon photo-excitation, this compound could also
be used for the degradation of protein molecules. To investigate
this hypothesis, we selected 2-phenylquinoline (1) (Fig. 1) as the
protein photo-degrading agent, and human ER-a as the target
protein. The 2-phenylquinoline scaffold is similar to estrogen in
terms of its affinity for the ER.6 Modulation of ER-a function
is an important factor in a variety of diseases, including breast
cancer and osteoporosis.7
To determine if 2-phenylquinoline is capable of modulating ER-
a function, we examined the photo-induced protein-degrading
activity of 2-phenylquinoline at concentrations of 10, 3.0, 1.0 and
0.1 mM against 1.0 mM ER-a in 20% acetonitrile/Tris-HCl buffer
(pH 8.0, 50 mM) using a long-wavelength UV lamp (365 nm,
100 W) for photo-irradiation. The extent of photo-degradation
was monitored by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE),8 and the results are shown in Fig. 2.
Comparison of lanes 3 and 4 with lane 2 shows that neither photo-
irradiation of ER-a in the absence of 2-phenylquinoline (lane 3)
nor treatment of ER-a with 2-phenylquinoline without photo-
irradiation (lane 4) resulted in a change in the SDS-PAGE profile.
In contrast, lane 5 shows fading of the band corresponding to
ER-a after exposure to 2-phenylquinoline with photo-irradiation,
indicating that degradation of ER-a took place. These results
show that 2-phenylquinoline degrades ER-a upon irradiation with
long wavelength UV light without the need of additives, although
protein degradation is incomplete.
The lack of ER-a degradation in the absence of light confirms
that UV light functions as a trigger to initiate protein degradation
by 2-phenylquinoline. In addition, it was confirmed that the
degradation ability was photo-dose-dependent. The SDS-PAGE
pattern obtained for 2-phenylquinoline-induced ER-a degrada-
tion contained faded and smeared bands, and no aggregation of
the protein was observed at the top of the gels. Additionally, no
Fig. 1 Chemical structures of 2-phenylquinoline (1), 2-phenylquinoline-estradiol (hybrid 2) (2), 2-phenylquinoline-4-hydroxytamoxifen (hybrid 3) (3),
estradiol (4), and 4-hydroxytamoxifen (5).
6358 | Org. Biomol. Chem., 2011, 9, 6357–6366
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
peaks corresponding to degraded peptide fragments were detected
by MALDI-TOF MS analysis. These results suggest that the
degradation reaction occurred in a non-site-specific manner, and
that ER-a was degraded into peptide fragments that were too small
for detection by SDS-PAGE and MALDI-TOF MS analyses.
Similar phenomena were reported by Jones9a and Nakanishi9b in
their protein cleavages by radical species. Therefore, we conclude
that 2-phenylquinoline degrades ER-a in a random fashion.
manner from the commercially available 9 and 11 of Scheme
1. 9 was amidated by the diamine derivative 12 in the presence
of O-(benzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluronium tetraflu-
oroborate (TBTU) and N-menthylmorpholine (NEM), yielding
derivative 13, which was then coupled with a 4-hydroxytamoxifen
analog 11 using K2CO3 and KI to produce hybrid 3 as a 1 : 1
mixture of E and Z isomers. These isomers could not be separated
by practical methods such as silica-gel column chromatography.
Design and synthesis of 2-phenylquinoline-estradiol and
2-phenylquinoline-4-hydroxytamoxifen hybrids
Selective affinity and photo-degradation capability of the hybrids
against ER-a
In order to improve the protein degrading ability and selectivity of
2-phenylquinoline, we designed two hybrid molecules, consisting
of 2-phenylquinoline conjugated with either estradiol (hereafter
referred to as hybrid (2)) or 4-hydroxytamoxifen (hereafter referred
to as hybrid (3)) (Fig. 1). Estradiol (4) and 4-hydroxytamoxifen (5)
(Fig. 1) have very strong and selective affinity for ER-a, and func-
tion as the agonist and antagonist of ER-a, respectively. Hybrid
molecules 2 and 3 were synthesized as shown in Scheme 1 below.
Hybrid 2 was synthesized using a modified version of the proce-
dure reported by Jones et al. in which an enediyne was employed
as a protein cleaver.10 Commercially available estradiol deriva-
tive 6 was regioselectively protected with a t-butyldimethylsilyl
(TBS) group to produce the silylated compound 7, which was
then treated with lithium diisopropylamide (LDA), followed by
addition of (CH2O)n to furnish the primary alcohol 8. Subsequent
esterification of 8 with 2-phenylquinoline-4-carboxylic acid (9) in
the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) and N,N-4-dimethylaminopyridine (DMAP) produced
derivative 10. Finally, deprotection of the TBS group in
10 using tetra-n-butylammonium fluoride (TBAF) and AcOH
yielded hybrid 2 (2-phenylquinoline-estradiol). 2-phenylquinoline-
4-hydroxytamoxifen (hybrid 3) was synthesized in a short-step
The binding affinity of synthetic hybrids 2 and 3 was examined us-
ing estradiol and 4-hydroxytamoxifen (Z isomer and a 1 : 1 mixture
of E and Z isomers) as reference samples. The method employed
uses fluorescence polarization to measure the capacity of a test
compound to displace a high affinity fluorescent ligand, in this case
R
fluorescent nonsteroidal estrogen, from ER-a. A Beacon 2000ꢀ
Fluorescence Polarization Instrument with a 490 nm excitation
filter and a 535 nm emission filter was utilized.11 The inhibitory
concentration (IC50) was determined by measuring polarization
intensity as a function of test compound concentration. Results
are shown in Fig. 3. The IC50 values were 151, 16.8, 2.8, 20.6, and
20.2 nM for hybrid 2, hybrid 3, estradiol, 4-hydroxytamoxifen
(Z), and 4-hydroxytamoxifen (E/Z = 1/1), respectively. There
was good agreement between the IC50 values determined for
estradiol and 4-hydroxytamoxifen (Z isomer) and those previously
reported.12 These results indicated that the ER-a binding affinity
of hybrid 2 is lower than that of estradiol, while the affinity of
hybrid 3 is similar to that of 4-hydroxytamoxifen. In addition,
we found that 4-hydroxytamoxifen (Z) and 4-hydroxytamoxifen
(E/Z = 1/1) had quite similar affinity for ER-a. Furthermore, it
was confirmed that the ER-a binding affinity of 1 was too low to
be detected by the competition binding assay.
Scheme 1 Synthesis of hybrid molecules. Reagents and conditions: (a) TBSOTf, 2,6-lutidine, CH2Cl2, 0 ◦C to rt, 2 h, 100%; (b) LDA, (CH2O)n, THF,
-78 to 0 ◦C, 17 h, 77%; (c) EDC, DMAP, CH2Cl2, 0 ◦C to rt, 2 h, 95%; (d) TBAF, AcOH, THF, 0 ◦C to rt, 1 h, 100%; (e) TBTU, NEM, DMF, rt, 20 h,
63%; (f) K2CO3, KI, DMF, 80 ◦C, 48 h, 21%.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6357–6366 | 6359
©

View Article Online
selective photo-degradation of proteins in glass vessels. Photo-
induced degradation of three types of protein (ER-a, bovine serum
albumin (BSA), and hen egg lysozyme (Lyso)) by hybrids 2 and
3 was evaluated by SDS-PAGE. The results are summarized in
Fig. 4. Significant degradation of ER-a occurred when exposed
to hybrids 2 and 3 under photo-irradiation (Fig. 4a and e).
Hybrids 2 and 3 degraded ER-a to a much greater extent than
2-phenylquinoline. These results were in sharp contrast to those
obtained with BSA and Lyso, which showed either no or slight
degradation under photo-irradiation with hybrid 2 or 3 (Fig. 4b,
c, f, and g). It was noteworthy that while Lyso was only slightly
degraded by hybrid 2 (Fig. 4c) in isolation, when ER-a and Lyso
were both present in the reaction mixture, only ER-a was degraded
(Fig. 4d). These results clearly indicate that 2-phenylquinoline-
estradiol and 2-phenylquinoline-4-hydroxytamoxifen promote se-
lective degradation only of the target protein, ER-a, upon photo-
irradiation, without any additives and under neutral conditions.
Fig. 3 Competition binding curves of hybrid 2, hybrid 3, estradiol
(4), and 4-hydroxytamoxifen (5) against the ER-a/fluorescent ligand
(FluormoneTM ES2) complex (ER-a/FluormoneTM ES2). Increasing con-
centrations of each competitor were incubated with 15 nM ER-a and 1 nM
FluormoneTM ES2 for 2 h at 25◦C, followed by measurement of fluorescence
polarization with a 490 nm excitation filter and a 535 nm emission filter.
The IC50 values were determined from non-linear regression analysis using
PrismꢀR (Graphpad Software, Inc.).
Mechanistic study of selective photo-degradation
Mechanistic studies of selective photo-degradation utilized 2-
From these results, we expect that although the binding affinity
of hybrid 2 was relatively low, this phenomenon might reduce
undesired agonist effects of hybrid 2 toward ER-a, and might
be beneficial for inhibition of cancer cell growth by selective
degradation of ER-a. We also expect that the high binding affinity
of hybrid 3, comparable to the antagonist 4-hydroxytamoxifen,
might result in a favorable dual effect; that is, the antagonist and
photo-degradation effects may act in concert to inhibit cancer
cell growth. Considering the results of binding studies involving 4-
hydroxytamoxifen (Z isomer and 1 : 1 mixture of E and Z isomers),
further studies involving hybrid 3 used a 1 : 1 mixture of E and Z
isomers.
phenylquinoline-estradiol hybrid 2. The activity of hybrid 2
∑
-
decreased in the presence of O2 and the H2O2 scavengers Tiron
∑
and KI, though these scavengers are not specific for O2 - and H2O2.
Furthermore, photo-irradiation of hybrid 2 in the presence of the
spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) produced
products with an ESR spectrum characteristic of the DMPO-
superoxide anion spin adduct DMPO/∑OOH and the DMPO-
hydroxyl radical spin adduct DMPO/∑OH. DMPO/∑OOH and
∑
DMPO/∑OH are products of the reaction of DMPO with O2
-
and the reaction of DMPO with ∑OH and/or the decay of
DMPO/∑OOH, respectively (Fig. 5).13 It was confirmed that no
peaks corresponding to DMPO/∑OOH and DMPO/∑OH were
detected by treatment of DMPO with hybrid 2 without photo-
irradiation or by photo-irradiation of DMPO in the absence of
Next, we evaluated 2-phenylquinoline-estradiol (hybrid 2)
and 2-phenylquinoline-4-hydroxytamoxifen (hybrid 3) for target-
Fig. 4 Photo-degradation of proteins by 2-phenylquinoline-estradiol (hybrid 2) and 2-phenylquinoline-4-hydroxytamoxifen (hybrid 3). Each protein
(1.0 mM) was incubated with hybrid 2 or 3 in 20% acetonitrile/Tris-HCl buffer (pH 8.0, 50 mM) at 25 ◦C for 2 h under irradiation with a UV lamp
(365 nm, 100 W) placed 10 cm from the sample, and the products were analyzed by tricine-SDS-PAGE. a), b), c), d), e) f) and g) represent a) ER-a with
hybrid 2, b) BSA with hybrid 2, c) Lyso with hybrid 2, d) ER-a + Lyso with hybrid 2, e) ER-a with hybrid 3, f) BSA with hybrid 3, and g) Lyso with hybrid
3. Lane 1: size marker; lane 2: protein alone; lane 3: protein with UV; lane 4: protein + compound (10 mM) without UV; lanes 5–8: protein + compound
(concentrations 10, 3, 1, and 0.3 mM, respectively) with UV.
6360 | Org. Biomol. Chem., 2011, 9, 6357–6366
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 5 ESR spectrum obtained during photo-irradiation of hybrid 2 in the presence of DMPO. Hybrid 2 (4 mM) and DMPO (100 mM) were dissolved
in 50% acetonitrile/Tris-HCl buffer (pH 8.0, 50 mM). The pink circles and the blue triangles represent the signals of DMPO/∑OOH and DMPO/∑OH,
respectively.
hybrid 2. Although a singlet oxygen-mediated degradation mech-
anism could not be ruled out, it was found that the degradation
ability of hybrid 2 in D2O (in which the lifetime of singlet oxygen
is extended) was similar to that in H2O. Furthermore, similar
results were obtained by using hybrid 3 in these mechanistic
studies. From these results, we conclude that ER-a degradation
by 2-phenylquinoline derivatives must be due to ROS produced
by photo-excitation of 2-phenylquinoline and O2.14 Importantly,
the life-time of ROS are very short; therefore, target selectivity
is generated by the location of the LAMTA molecules hybrids
2 and 3.
MCF-7 cells was investigated by western blotting with appropriate
monoclonal antibodies within the concentrations at which the
hybrid compounds showed no cytotoxicity. No degradation of
any of the proteins examined (ER-a, AR, ER-b, EGFR, cyclin
D1, cytochrome c, Hsp90, PARP, and a-tubulin) was observed
when hybrid 2 was not subjected to photo-irradiation (Fig. 7).
On the other hand, when hybrid 2 was subjected to 1 h of
photo-irradiation, only a dose-dependent degradation of ER-a
was observed (Fig. 7a). In addition, 2-phenylquinoline (1) itself
did not degrade ER-a even under photo-irradiation over a similar
concentration range (Fig. 7b). These results show the importance
of the hybrid structure for target-selective photo-degradation, in
which 2-phenylquinoline serves as the protein degradation moiety
and estradiol functions in protein recognition. Hybrid 3 exhibited
a similar target-selectivity (Fig. 7c). Thus, although a higher
concentration of hybrid 3 was required for the photo-degradation
of ER-a, under photo-irradiation hybrid 3 degraded ER-a in a
dose dependent manner but did not degrade the other proteins
tested. The difference in the strength of photo-degrading activity
between hybrids 2 and 3 may be a result of differences in cellular
uptake.
Target-selective photo-degradation of ER-a leading to inhibition of
the growth of MCF-7 breast cancer cells
We next evaluated the activity of our 2-phenylquinoline hybrids
at the cellular level. First, we utilized an MTT assay to evaluate
potential cytotoxic effects of hybrids 2 and 3. Breast cancer MCF-
7 cells, which are dependent upon ER-a for growth, were exposed
to each agent for 24 h with or without 1 h of photo-irradiation.
The IC50 values of hybrids 2 and 3 against MCF-7 cells without
photo-irradiation were >10 mM, and with photo-irradiation were
1.1 mM and >10 mM, respectively (Fig. 6). In the case of hybrid
3, no cytotoxicity was observed at a concentration of 100 mM with
or without photo-irradiation. These results indicate that hybrids
2 and 3 are in and of themselves non-toxic, while only hybrid
2 is cytotoxic upon photo-irradiation. Based on these results,
target-selective photo-degradation of ER-a by hybrids 2 and 3 in
Finally, we examined the effect of hybrids 2 and 3 on the growth
of MCF-7 breast cancer cells with and without photo-irradiation
under two different sets of culture conditions. Under culture con-
dition A, MCF-7 cells were maintained for 2 days in the absence
of phenol-red, a substance with a weak estrogenic activity,15 and
in the presence of charcoal-stripped fetal bovine serum (FBS).
Under these conditions, cell growth is estradiol dependent. Cells
Fig. 6 Effect of hybrids 2 and 3 on MCF-7 cell viability. Cells were seeded at 3000 per well in 10% FBS DMEM in a 96-well plate. After 24 h, samples
were incubated with the indicated concentrations of hybrids 2 or 3 at 25 ◦C for 1 h with or without photo-irradiation by a UV lamp (368 nm, 15 W)
placed 25 cm from the samples. Cells were then kept for 24 h at 37 ◦C and in 5% CO2 in air, and then MTT reagent wad added to each well and cells were
incubated for up to 3 additional hours. The absorbance at a single wavelength of 540 nm was read on a plate reader.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6357–6366 | 6361
©

View Article Online
Fig. 7 Western blot analysis of the effect of 2-phenylquinoline (1) and hybrids 2 and 3 with or without photo-irradiation on protein levels in MCF-7
cells. Cells were treated with each compound for 1 h at 25 ◦C with or without photo-irradiation using a UV lamp (368 nm, 15 W) placed 25 cm from the
samples. Each sample was analyzed using tricine-SDS-PAGE and immunoblotting with appropriate monoclonal antibodies. a-Tubulin levels are shown
as protein loading controls.
were cultured in this manner to enhance the assay sensitivity. Cells
were then treated with each hybrid photo-degrading agent for 3
In the present studies, we developed a new method for selective
days. Under the second set of culture conditions (condition B),
Conclusions
degradation of a target protein, ER-a, by photo-irradiation
using 2-phenylquinoline-estradiol and -4-hydroxytamoxifen hy-
brids under neutral conditions. Our results indicate that target-
selective photo-degradation of proteins by LAMTA molecules
may be a novel and effective way to control specific protein
functions as a means of controlling cell growth. In addition,
the results presented here will contribute to the molecular
design of novel artificial protein photo-degradation agents,
which should find wide applications in chemistry, biology, and
medicine.
estradiol was added before treatment with the hybrid compounds.
When MCF-7 cells were exposed to hybrid 2 (which contains
an estradiol moiety) under culture condition A without photo-
irradiation, cell growth was dose-dependent relative to hybrid 2.
These results indicate that hybrid 2 works as an agonist against
ER-a as a result of the estradiol component of its structure.
However, with 1 h of photo-irradiation under condition A, cell
growth was significantly depressed (Fig. 8a). These results clearly
show that photo-degradation of ER-a by hybrid 2 effectively
induces inhibition of the growth of MCF-7 cells. Under culture
condition B without photo-irradiation, MCF-7 cell growth was
independent of the concentration of hybrid 2, reaching the same
cell density at all concentrations of hybrid 2 due to the presence
of excess amounts of estradiol in the media. However, although
inhibitory activity was not particularly high, hybrid 2 did inhibit
MCF-7 cell growth with photo-irradiation due to degradation
of ER-a (Fig. 8b). On the other hand, when MCF-7 cells were
exposed to hybrid 3 (containing a 4-hydroxytamoxifen moiety)
under culture condition A with or without photo-irradiation, cells
did not increase in number with concentration of hybrid 3 because
hybrid 3 had no agonist activity against ER-a (Fig. 8c). Under
culture condition B without photo-irradiation, hybrid 3 inhibited
MCF-7 cell growth in a dose-dependent manner. These results
indicate that hybrid 3 functions as an antagonist of ER-a due
to its 4-hydroxytamoxifen structure. Furthermore, inhibition of
cell growth increased with photo-irradiation as the concentration
of hybrid 3 increased (Fig. 8d). These results clearly demonstrate
that hybrid 3 has dual antagonist and photo-degradation activity
toward ER-a and thereby effectively inhibits the growth of MCF-7
breast cancer cells.
Experimental section
General procedure for chemical synthesis
17a-ethynyl-b-estradiol (6), 2-phenylquinoline-4-carboxylic acid
(9), and (E/Z)-1-[4-chloroethoxy]phenyl]-1-(4-hydroxyphenyl)-2-
phenyl-1-butene (11) were purchased from Sigma Co., Aldrich
Co., and Santa Cruz Biotechnology, Inc. respectively. Melting
points were determined on a micro hot-stage (Yanako MP-S3)
and were uncorrected. Optical rotations were measured on a
JASCO DIP-370 photo-electric polarimeter. 1H-NMR spectra
were recorded on a Varian MVX-300 (300 MHz) or a JEOL ECA-
500 (500 MHz) spectrometer using trimethylsilane as internal
standard unless otherwise noted. ESI-TOF Mass spectra were
measured on a Waters LCT premier XE. Silica gel TLC and
column chromatography were performed on Merck TLC 60F-254
(0.25 mm), Silica Gel 60 N (spherical, neutral) (Kanto Chemical
Co., Inc.), and Wakosil 40C18 (Wako Pure Chemical Industries,
Ltd.), respectively. Air- and/or moisture-sensitive reactions were
6362 | Org. Biomol. Chem., 2011, 9, 6357–6366
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 8 Effect of hybrids 2 and 3 on MCF-7 cell growth. MCF-7 cells were maintained in culture in the presence 10% charcoal-stripped FBS for 2 days,
after which cells were seeded into 24-well culture plates (1 ¥ 104 per well) in the same media. After 24 h, samples were incubated with the indicated
concentrations of hybrids 2 or 3 with or without 1 h photo-irradiation using a UV lamp (368 nm, 15 W) placed 25 cm from the samples. a) Hybrid 2
grown under Condition A. b) Hybrid 2 grown under Condition B. c) Hybrid 3 grown under Condition A. d) Hybrid 3 grown under Condition B. For the
estradiol-treated groups b) and d), estradiol (1 nM) was added 30 min before treatment with hybrid 2 or 3. After treatment at 37 ◦C and in air maintained
at 5% CO2 for 3 days, cells were harvested and the cell number in each well was determined using a Coulter counter.
carried out under an atmosphere of argon using oven-dried glass-
ware. In general, organic solvents were purified and dried using an
appropriate procedure, and evaporation and concentration were
carried out under reduced pressure below 30 ◦C, unless otherwise
noted.
(¥ 2); Anal. Calcd for C26H38O2Si: C, 76.04; H, 9.33. Found: C,
75.67; H, 9.36%.
3-O-tert-Butyldimethylsilyl-17a-hydroxypropargyl-b-estradiol (8)
To a stirred solution of 7 (435 mg, 1.06 mmol) in dry THF
◦
(11 mL) was added LDA (1.77 mL, 3.18 mmol) at -78 C. The
3-O-tert-Butyldimethylsilyl-17a-ethynyl-b-estradiol (7)
reaction mixture was stirred for 0.5 h at -78 ◦C, and then (CH2O)n
(95.5 mg, 3.18 mmol) was added to the mixture. After stirring for
17 h at 25 ◦C, the mixture was poured into ice-cold water. The
resultant mixture was extracted with EtOAc and the extracts were
washed with brine, dried over anhydrous Na2SO4 and concentrated
in vacuo. Purification of the residue by column chromatography
(40 g of silica gel, 2/1 n-hexane/EtOAc) gave 8 (359 mg, 77%) as
white solids. Rf 0.30 (1/1 n-hexane/EtOAc); Mp. 178.0–179.0 ◦C;
To a stirred solution of 6 (309 mg, 1.04 mmol) in dry CH2Cl2 (15
mL) was added 2,6-lutidine (180 mL, 1.56 mmol) and T◦BSOTf
◦
(290 mL, 1.25 mmol) at 0 C. After stirring for 2 h at 25 C, the
mixture was poured into ice-cold water. The resultant mixture
was extracted with CHCl3 and the extracts were washed with
brine, dried over anhydrous Na2SO4 and concentrated in vacuo.
Purification of the residue by column chromatography (40 g of
silica gel, 3/1 n-hexane/EtOAc) gave 7 (428 mg, 100%) ◦as white
solids. Rf 0.80 (1/1 n-hexane/EtOAc); Mp. 122.5–123.5 C; [a]D29
+5.5 (c 1.22, CHCl3); 1H-NMR (300 MHz, CDCl3) d 7.18 (1H, d,
J = 8.4 Hz), 6.66 (1H, dd, J = 8.4 and 2.4 Hz), 6.58 (1H, d, J =
2.4 Hz), 2.83–2.78 (2H, m), 2.62 (1H, s), 2.38–2.12 (3H, m), 2.05–
1.96 (2H, m), 1.96–1.65 (5H, m), 1.55–1.23 (4H, m), 0.96 (9H, s),
0.89 (3H, s), 0.19 (6H, s); 13C-NMR (75 MHz, CDCl3) d 153.3,
137.8, 132.9, 126.1, 119.9, 117.2, 87.5, 79.9, 74.0, 49.5, 47.1, 43.6,
39.4, 39.0, 32.8, 29.6, 27.3, 26.3, 25.7 (¥ 3), 22.8, 18.1, 12.7, -4.4
1
[a]2D9 -3.3 (c 1.05, CHCl3); H-NMR (300 MHz, CDCl3) d 7.11
(1H, d, J = 8.4 Hz), 6.61 (1H, dd, J = 8.4 and 2.4 Hz), 6.55 (1H,
d, J = 2.4 Hz), 4.36 (2H, s), 2.83–2.78 (2H, m), 2.36–2.18 (3H,
m), 2.06–1.97 (1H, m), 1.88–1.61 (7H, m), 1.54–1.25 (4H, m), 0.97
(9H, s), 0.87 (3H, s), 0.19 (6H, s); 13C-NMR (75 MHz, CDCl3) d
153.3, 137.8, 132.9, 126.1, 119.9, 117.2, 89.3, 84.2, 79.9, 51.1, 49.6,
47.2, 43.6, 39.4, 38.9, 33.0, 29.6, 27.3, 26.3, 25.7 (¥ 3), 22.9, 18.1,
12.8, -4.4 (¥ 2);Anal. Calcd for C27H40O3Si: C, 73.59; H, 9.15.
Found: C, 73.41; H, 9.14%.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6357–6366 | 6363
©

View Article Online
3-O-tert-Butyldimethylsilyl-17a-hydroxypropargyl-b-estradiolyl
residue by reverse phase column chromatography (6 g of silica gel,
2-phenylquinoline-4-carboxylate (10)
0/100 to 100/0 MeOH/H2O) gave 13 (23.1 mg, 63%) as white
◦
1
solids. Rf 0.20 (6/1 CHCl3/MeOH); Mp. 149.0–150.0 C; H-
NMR (500 MHz, CDCl3) d 8.24–8.13 (4H, m), 7.93 (1H, s), 7.79–
7.73 (1H, m), 7.74–7.60 (4H, m), 6.75 (1H, m), 3.66 (2H, dt, J =
6.1 and 5.3 Hz), 2.90 (2H, t, J = 6.1 Hz), 2.46 (3H, s); 13C-NMR
(125 MHz, CDCl3) d 165.8, 156.9, 148.8, 143.1, 139.0, 130.2,
129.8, 129.0, 127.6, 127.3, 125.1, 123.5, 116.7, 50.4, 39.3, 36.1;
HRMS (ESI-TOF) m/z 306.1586 (306.1606 calcd. for C19H20N3O,
[M + H]+).
To a stirred solution of 8 (66.0 mg, 0.150 mmol) and 9 (45.0 mg,
0.181 mmol) in dry CH2Cl2 (1.5 mL) was added EDC (57.0 mg,
0.300 mmol) and DMAP (9.0 mg, 0.0749 mmol) at 0 ◦C. After
stirring for 2 h, the reaction mixture was poured into ice-cold
water. The resultant mixture was extracted with CHCl3 and the
extracts were washed with brine, dried over anhydrous Na2SO4
and concentrated in vacuo. Purification of the residue by column
chromatography (30 g of silica gel, 3/1 n-hexane/EtOAc) gave
10 (95.6 mg, 95%) as white solids. Rf 0.80 (1/1 n-hexane/EtOAc);
Mp. 86.0–87.0 ◦C; [a]D27 -5.1 (c 0.91, CHCl3); 1H-NMR (300 MHz,
CDCl3) d 8.75 (1H, dd, J = 8.4 and 1.0 Hz), 8.43 (1H, s), 8.26–
8.13 (3H, m), 7.81–7.43 (5H, m), 7.04 (1H, d, J = 8.4 Hz), 6.60
(1H, dd, J = 8.4 and 2.4 Hz), 6.54 (1H, d, J = 2.4 Hz), 5.16 (2H,
s), 2.78 (2H, m), 2.43–2.13 (3H, m), 2.11–1.91 (2H, m), 1.91–1.64
(5H, m), 1.55–1.22 (4H, m), 0.98 (9H, s), 0.89 (3H, s), 0.19 (6H, s);
13C-NMR (75 MHz, CDCl3) d 165.6, 156.7, 153.3, 149.3, 138.7,
137.7, 135.0, 132.8, 130.4, 130.0, 129.8, 128.9 (¥ 2), 127.9, 127.4 (¥
2), 126.1, 125.2, 123.9, 120.5, 119.9, 117.2, 91.3, 80.0, 79.5, 53.7,
49.7, 47.5, 43.6, 39.4, 38.9, 33.0, 29.6, 27.3, 26.3, 25.7 (¥ 3), 22.9,
18.2, 12.8, -4.4 (¥ 2); Anal. Calcd for C43H49NO4Si: C, 76.86; H,
7.35; N, 2.08. Found: C, 76.70; H, 7.63; N, 1.94%.
(E/Z)-N-(2-((2-(4-(1-(4-Hydroxyphenyl)-2-phenylbut-1-
enyl)phenoxy)ethyl)(methyl)amino)-ethyl)-2-phenylquinoline-4-
carboxamide (3)
To a stirred solution of 11 (11.2 mg, 0.0296 mmol) in dry DMF
(700 mL) were added K2CO3 (5.0 mg, 0.0362 mmol), KI (4.1 mg,
0.0247 mmol) and a solution of 13 (16.0 mg, 0.0524 mmol) in
dry DMF (700 mL) at room temperature. After stirring for 48 h
at 80 ◦C, the reaction mixture was quenched with water. The
resultant mixture was extracted with EtOAc and the extracts were
washed with brine, dried over anhydrous Na2SO4 and concentrated
in vacuo. Purification of the residue by column chromatography
(10 g of silica gel, 20/1 CHCl3/MeOH) gave 3 (4.1 mg, 21%,
1
E/Z = 1/1) as white solids. Rf 0.30 (20/1 CHCl3/MeOH); H-
NMR (500 MHz, CDCl3) d 8.29–8.22 (1/2H, m), 8.21–8.15 (3/2H,
m), 8.14–8.06 (2H, m), 7.96 (1/2H, s), 7.91 (1/2H, s), 7.75–7.66
(1H, m), 7.54–7.40 (4H m), 7.17–6.99 (6H, m), 6.94–6.92 (1H, m),
6.80–6.74 (1H, m), 6.66–6.61 (1H, m), 6.60–6.54 (2H, m), 6.46–
6.40 (1H, m), 6.29–6.23 (1H, m), 4.02 (1H, t, J = 5.5 Hz), 3.87
(1H, t, J = 5.5 Hz), 3.72 (1H, dt, J = 6.0 and 5.2 Hz), 3.67–
3.64 (1H, m), 2.90–2.71 (4H, m), 2.48–2.31 (5H, m), 0.91–0.84
(3H, m); HRMS (ESI-TOF) m/z 648.3223 (648.3226 calcd. for
C43H42N3O3, [M + H]+).
17a-Hydroxypropagyl-b-estradiolyl 2-phenylquinoline-4-
carboxylate (2)
To a stirred solution of 10 (84.0 mg, 0.125 mmol) in dry THF
(1.3 mL) was added AcOH (7.0 mL, 0.150 mmol) and TBAF
(150 mL, 0.150 mmol) at 0 ◦C. After stirring for 1 h, the reaction
mixture was poured into ice-cold water. The resultant mixture
was extracted with EtOAc and the extracts were washed with
brine, dried over anhydrous Na2SO4 and concentrated in vacuo.
Purification of the residue by column chromatography (30 g of
silica gel, 2/1 n-hexane/EtOAc) gave 2 (69.7 mg, 100%) ◦as white
solids. Rf 0.50 (1/1 n-hexane/EtOAc); Mp. 102.0–103.0 C; [a]D27
Protein photo-degradation
1
-6.5 (c 0.41, CHCl3); H-NMR (300 MHz, CDCl3) d 8.75 (1H,
Human recombinant estrogen receptor-a (ER-a), bovine serum
albumin (BSA) and hen egg lysozyme (Lyso) were purchased
from Sigma Co. A UV lamp (365 nm, 100 W, Blak-ray (B-100A),
UVP. Inc.) was used for the photo-irradiation. All the protein
degradation experiments were performed with ER-a, BSA or Lyso
(1.0 mM) in a volume of 10 mL containing 20% acetonitrile in
50 mM Tris-HCl buffer (pH 8.0) at 25 ◦C for 2 h under irradiation
of the UV lamp placed at 10 cm from the mixture. The protein-
sample levels were varied as indicated in the figure captions.
dd, J = 8.4 and 1.0 Hz), 8.43 (1H, s), 8.27–8.13 (3H, m), 7.82–7.43
(5H, m), 7.07 (1H, d, J = 8.4 Hz), 6.61 (1H, dd, J = 8.4 and 2.4 Hz),
6.55 (1H, d, J = 2.4 Hz), 5.16 (2H, s), 4.57 (1H, s), 2.80 (2H, m),
2.44–2.12 (3H, m), 2.11–1.91 (2H, m), 1.91–1.64 (5H, m), 1.50–
1.30 (4H, m), 0.89 (3H, s, Me-13); 13C-NMR (75 MHz, CDCl3) d
165.7, 156.8, 153.5, 149.1, 138.6, 138.0, 135.1, 132.1, 130.2, 130.0,
129.8, 128.9 (x 2), 127.9, 127.5 (x 2), 126.4, 125.2, 123.8, 120.6,
115.3, 112.7, 91.3, 80.0, 79.5, 53.8, 49.6, 47.5, 43.5, 39.4, 38.8, 32.9,
29.5, 27.2, 26.3, 22.9, 12.8; Anal. Calcd for C37H35NO4: C, 79.69;
H, 6.33; N, 2.51. Found: C, 79.72; H, 6.54; N, 2.77%.
Electrophoresis
SDS/polyacrylamide gel electrophoresis (SDS-PAGE) experi-
ments were performed as reported.8 After addition of a 4.8 mL
solution containing SDS (5%, wt/vol), glycerol (27%, vol/vol),
DTT (0.5%, wt/vol) and bromophenol blue (0.007%, wt/vol) to
the photoirradiated samples. Gels (8% for BSA and 12% for ER-a
and Lyso) were run by applying 110 V for 1.5 h for BSA or 2.5 h
for ER-a and Lyso. The gels were stained with SYPRO Ruby
Protein Gel Stain (Bio-Rad Lab. Inc.) for 3 h, destained in acetic
acid (7%, vol/vol) and methanol (10%, vol/vol) for 0.5 h, and
then washed with water. The gels were scanned with a Molecular
Imager FX (Bio-Rad Lab. Inc.) and images were processed using
N-(2-(Methylamino)ethyl)-2-phenylquinoline-4-carboxamide (13)
To a stirred solution of 9 (30.0 mg, 0.120 mmol) in dry DMF
(1.2 mL) was added NEM (22.8 mL, 0.180 mmol) and TBTU
(46.2 mg, 0.144 mmol) at 25 ◦C. The reaction mixture was stirred
for 1 h at 25 ◦C, and then a solution of 12 (31.5 mL, 0.360 mmol)
in dry DMF (1.2 mL) was added dropwise to the mixture over
a period of 15 min. After stirring for 20 h, the reaction mixture
was quenched with water. The resultant mixture was extracted
with EtOAc and the extracts were washed with brine, dried over
anhydrous Na2SO4 and concentrated in vacuo. Purification of the
6364 | Org. Biomol. Chem., 2011, 9, 6357–6366
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Adobe Photoshop software. Molecular weight markers were used
in each gel for calibration.
sodium orthovanadate, and 2 mM phenylmethylsulfonyl fluoride)
containing protease inhibitor cocktail (Roche Diagnostics Co.)
and homogenized with ULTRA SONIC HOMOGENIZER UH-
50 (STM◦Inc.). The lysate was centrifuged for 10 min at 14000
rpm at 4 C. Equal amounts of protein were separated by SDS-
PAGE in 10% polyacrylamide gels. Proteins were transferred onto
nitrocellulose membranes HybondTM -ECL (GE healthcare Japan
Co.). Membranes were blocked with Tris-buffered saline/0.1%
Tween 20 (TBST) containing 3% nonfat dry milk for 60 min
at room temperature. After washing five times with TBST,
membranes were incubated with appropriately diluted primary
antibodies at 4 ◦C overnight. After washing five times with TBST,
the blots were incubated with horseradish peroxidase-conjugated
specific secondary antibody for 1 h at room temperature and
then again washed five times. Then the complexes were visualized
in Medical Film Processer FPM100 (FUJIFILM holdings Co.)
using the enhanced chemiluminescence reagents ImmobilonTM
Western (MILLPORE Co.). The following primary antibodies
were used for the detection of the specific bands: ER-a (D-
12, Santa Cruz Biotechnology Inc.), a-Tubulin (DM1A, Santa
Cruz Biotechnology Inc.), AR (441, Santa Cruz Biotechnology
Inc.), EGFR (1005, Santa Cruz Biotechnology Inc.), PARP (H-
250, Santa Cruz Biotechnology Inc.), Hsp90 (H-114, Santa Cruz
Biotechnology Inc.), ER-b (H-150, Santa Cruz Biotechnology
Inc.), Cyclin D1 (M-20, Santa Cruz Biotechnology Inc.) and
Cytochrome c (556433, BD Biosciences).
ER-a competitor assay
Competition binding curves for the compounds 2–5 binding to
ER-a were obtained by using ER-a a competitor assay kits
purchased from PanVera Co. 2X Complex (15 nM ER-a/1 nM
FluormoneTM ES2) and the indicated concentrations of each test
compound were incubated in a volume of 100 mL containing
1% dimethylsulfoxide in ES2 Screening Buffer for 2 h at 25 ◦C.
Fluorescence polarization values were measured by the Beacon
R
2000ꢀ Fluorescence Polarization Instrument with a 490 nm
excitation filter and 535 nm emission filter, and plotted as a
function of the test compound concentration. The IC50 values were
R
obtained from non-linear regression using Prismꢀ from Graphpad
Software, Inc.
ESR spectrometry
ESR spectrum was recorded using a Bruker BioSpin EMX EPR
operating at 9.5 GHz with 100 kHz modulation. A mixture of 2
(4 mM) and DMPO (100 mM) in a volume of 1.0 mL containing
50% acetonitrile Tris-HCl buffer (pH 8.0, 50 mM) was placed in a
quartz flat cell and irradiated directly inside the microwave cavity
of the spectrometer using a UV lamp (365 nm, 100 W, Blak-ray
(B-100A), UVP. Inc.).
Cell culture
Growth inhibition assay
Human mammary cancer MCF-7 was grown at 37 ◦C in 5% CO2 in
air in DMEM medium supplemented with phenol red, L-glutamine
(2 mM), penicillin (100 Units/ml), kanamycin (5 mg ml-1) and 10%
fetal bovine serum.
MCF-7 cells were maintained in phenol red free DMEM contain-
ing 10% charcoal-stripped FBS (Hyclone Inc.) for 2 days, after
which cells were seeded at 1 ¥ 104 per well into 48-well culture
plates in the same medium. 24 h later, samples were incubated
with the indicated concentration of 2 or 3 with or without of 1 h of
photo-irradiation using a UV lamp (368 nm, 15 W, FL15BLB-368,
Sankyo Denki Co., Ltd.) placed at 25 cm from the samples. For the
estradiol-treated groups, estradiol (1 nM) was added 30 min before
the drug treatment. After 72 h treated, cell number in each well
was counted using a Coulter Counter (Beckman Coulter Inc.).
MTT Assay
Cells were seeded at 3 ¥ 103 per well in 96-well in 10% FBS
DMEM. After 24 h, samples were incubated with the indicated
concentration of 2 or 3 with or without of 1 h of photo-irradiation
using a UV lamp (368 nm, 15 W, FL15BLB-368, Sankyo Denki
Co., Ltd.) placed at 25 cm from the samples. Control cells were
treated with DMSO vehicle at a concentration equal to that in
drug-treated cells (final concentration, ꢀ1%). After 24 h treated,
MTT reagent was added to each well and cells were incubated for
up to 3 additional hours at 37 ◦C. The absorbances were measured
at 540 nm in a microplate reader SAFIRE (TECAN Inc.).
Acknowledgements
This research was supported in part by the 21st Century COE
Program “Keio Life-Conjugated Chemistry”, High-Tech Research
Center Project for Private Universities: Matching Fund Sub-
sidy, 2006-2011, and Scientific Research (B) (No. 20310140 and
23310153) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (MEXT).
Immunoblotting
4 ¥ 105 cells were plated on 60 mm dishes containing 10%
FBS DMEM. After 24 h, samples were incubated with the
indicated concentration of 1, 2 or 3 with or without 1 h of
photo-irradiation using a UV lamp (368 nm, 15 W, FL15BLB-
368, Sankyo Denki Co., Ltd.) placed at 25 cm from the samples.
After the incubation time, adherent cells were washed twice with
ice-cold PBS buffer, scraped with a rubber policeman, collected
in 1 ml PBS buffer, and centrifuged for 5 min at 3000 rpm at
Notes and references
1 (a) S. Pervaiz, A. Harriman and K. S. Gulliya, Free Radical Biol. Med.,
1999, 12, 389; (b) R. Miyake, J. T. Owens, D. Xu, W. M. Jackson and
C. F. Meares, J. Am. Chem. Soc., 1999, 121, 7453; (c) G. Plourde II,
A. El-Shafey, F. S. Fouad, A. S. Purohit and G. B. Jones, Bioorg. Med.
Chem. Lett., 2002, 12, 2985; (d) F. S. Fouad, J. M. Wright, G. Plourde
II, A. D. Purohit, J. K. Wyatt, A. El-Shafey, G. Hynd, C. F. Crasto,
Y. Lin and G. B. Jones, J. Org. Chem., 2005, 70, 9789; (e) A. Suzuki,
M. Hasegawa, M. Ishii, S. Matsumura and K. Toshima, Bioorg. Med.
4
◦C. The pellet was then resuspended in 100 ml lysis buffer
(20 mM Tris-HCl (7.5 pH), 1 mM EDTA, 150 mM NaCl, 0.5%
Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 1mM
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6357–6366 | 6365
©

View Article Online
Chem. Lett., 2005, 15, 4624; (f) S. Tanimoto, S. Matsumura and K.
Toshima, Chem. Commun., 2008, 3678–3680; (g) S. Tanimoto, S. Sakai,
S. Matsumura, D. Takahashi and K. Toshima, Chem. Commun., 2008,
5767–5769.
2 For selected reviews, see: (a) A. P. Castano, P. Mroz and M. R. Hamblin,
Nat. Rev. Cancer, 2006, 6, 535; (b) I. J. Macdonald and T. J. Dougherty,
J. Porphyrins Phthalocyanines, 2001, 5, 105.
3 For our preliminary communication, see: A. Suzuki, K. Tsumura, T.
Tsuzuki, S. Matsumura and K. Toshima, Chem. Commun., 2007, 4260–
4262.
9 For similar examples, see: (a) G. B. Jones, J. M. Wright, G. Hynd, J. K.
Wyatt, M. Yancisin and M. A. Brown, Org. Lett., 2000, 2, 1863; (b) T.
Furuta, M. Sakai, H. Hayashi, T. Asakawa, F. Kataoka, S. Fujii, T.
Suzuki, Y. Suzuki, K. Tanaka, N. Fishkin and K. Nakanishi, Chem.
Commun., 2005, 4575.
10 G. B. Jones, G. Hynd, J. M. Wright, A. Purohit, G. W. Plourde II, R. S.
Huber, J. E. Mathews, A. Li, M. W. Kilgore, G. J. Bubley, M. Yancisin
and M. A. Brown, J. Org. Chem., 2001, 66, 3688.
11 Estrogen receptor-a competitor assay, greenꢀR available from PanVera
Co. was used for the present study.
4 After our study, destruction of amyloid fibrils of a b2-microglobulin
fragment using thioflavin T by laser beam irradiation was reported,
see: D. Ozawa, H. Yagi, T. Ban, A. Kameda, T. Kawakami, H. Naiki
and Y. Goto, J. Biol. Chem., 2009, 284, 1009.
5 K. Toshima, R. Takano, Y. Maeda, M. Suzuki, A. Asai and S.
Matsumura, Angew. Chem., Int. Ed., 1999, 38, 3733.
12 R. Bolger, T. E. Wiese, K. Ervin, S. Nestich and W. Checovich, Environ.
Health Perspect., 1998, 106, 551.
13 (a) E. Wertz and B. Bolton, Electron Spin Resonance, McGraw-Hill,
New York, 1972; (b) H. M. Swartz, J. R. Bolton and D. C. Borg,
Biological Application of Electron Spin Resonance, Wiley-Interscience,
New York, 1972.
6 A. T. Vu, S. T. Cohn, E. S. Manas, H. A. Harris and R. E. Mewshaw,
Bioorg. Med. Chem. Lett., 2005, 15, 4520.
7 J. A. Scott and W. L. McGuire, In Endocrine-Dependent Tumors, K.
D. Voight and C. Knabbe, ed., Raven, New York, 1991, pp. 179–
196.
14 (a) B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology and
Medicine, Oxford University Press, Oxford, 1985; (b) K. J. A. Davies, J.
Biol. Chem., 1987, 262, 9895; (c) M. J. Davies, Biochem. Biophys. Res.
Commun., 2003, 305, 761.
15 Y. Berthois, J. A. Katzenellenbogen and B. S. Katzenellenbogen, Proc.
Natl. Acad. Sci. U. S. A., 1986, 83, 2496.
8 H. Scha¨gger and G. von Jagow, Anal. Biochem., 1987, 166, 368.
6366 | Org. Biomol. Chem., 2011, 9, 6357–6366
This journal is
The Royal Society of Chemistry 2011
©