New clicked full agonists of the estrogen receptor β

Article abstract of DOI:10.1039/c3ra00122a

A click chemistry approach was used to synthesize a series of 1,4-diaryl-substituted 1,2,3-triazoles designed to behave as estrogen receptor (ER) ligands. We studied their affinities for both receptors α and β, their agonist activities in a cell-based luciferase reporter assay and their effect on the proliferation of the hormone-dependent MCF-7 cell line. We found two compounds (3a and 3c) that behave as selective full agonists for ERβ at a 20 μM concentration, and one of them (3c) showed no proliferative effect on MCF-7 cells.

Full text of DOI:10.1039/c3ra00122a

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PAPER
New clicked full agonists of the estrogen receptor b3
Sebastian Demkowicz,ab Kamila Filipiak,ac Maciej Maslyk,ac Jakub Ciepielski,ad
Sonia de Pascual-Teresa,e Sonsoles Mart´ın-Santamar´ıa,a Beatriz de Pascual-Teresa*a
and Ana Ramos*a
Cite this: RSC Advances, 2013, 3, 3697
A click chemistry approach was used to synthesize a series of 1,4-diaryl-substituted 1,2,3-triazoles designed
to behave as estrogen receptor (ER) ligands. We studied their affinities for both receptors a and b, their
agonist activities in a cell-based luciferase reporter assay and their effect on the proliferation of the
hormone-dependent MCF-7 cell line. We found two compounds (3a and 3c) that behave as selective full
agonists for ERb at a 20 mM concentration, and one of them (3c) showed no proliferative effect on MCF-7
cells.
Received 9th January 2013,
Accepted 10th January 2013
DOI: 10.1039/c3ra00122a
www.rsc.org/advances
behave as ERb agonists and ERa antagonists,7 and present
interesting antitumor activity against two pancreatic cell
Introduction
lines.8 Introduction of a basic side chain in these scaffolds
has led to full antagonists of ERb, with potency in the low
micromolar concentration in a cell-based luciferase reporter
assay, and completely devoid of activity against the ERa at the
same concentration range.9
Click chemistry has had a profound effect on the design
and development of novel compounds for therapeutic applica-
tions.10 In particular, the Copper-Catalyzed reaction between
an Azide and an Alkyne (CuAAC) has been widely used in
fragment-based drug design, and target-guided synthesis (in
situ click chemistry).11
Tron and co-workers12 used CuAAC click chemistry to obtain a
series of 1,4-diaryl-substituted 1,2,3-triazoles 1 (Fig. 1) and
evaluated their effect on the proliferation of the hormone-
dependent MCF-7 cell line. The only active compound promoted
proliferation at a 100 pM concentration and possessed the two
hydroxy groups in the meta position. This compound was capable
of promoting transcriptional activation in HeLa cells expressing
higher levels of ERb than ERa at low concentrations. These data
suggest ER-b selectivity, but further studies on the affinity and
transcriptional response on both receptors are required to
establish the subtype selectivity of this type of compound.
In the search for new and selective ligands of both estrogen
receptor isoforms, we were interested in the observation of the
authors that the introduction of a triazole ring is compatible
with binding to the estrogen receptor, and the possibility to
apply this efficient synthetic procedure to obtain analogues
with additional substituents on the aromatic rings that could
favour the interactions with one of the receptor subtypes.
Thus, we have synthesized two series of 1,4-bis(hydrox-
yphenyl)-1H-1,2,3-triazoles 2a–h, 3a–f and 3h (Fig. 2), where
methyl, trifluoromethyl, fluoro, carboxy and methoxycarbonyl
groups have been introduced in ortho and meta positions of
The estrogen receptor (ER) is a member of the nuclear receptor
gene family binding the steroid hormone estradiol.1 Two
subtypes are known of ER, designated ERa and ERb. Both ER
subtypes have overlapping but also unique roles in estrogen-
dependent action and are important targets in the pharma-
ceutical industry. Additionally, ERa and ERb have different
transcriptional activities in certain ligand, cell-type, and
promoter contexts.
The low expression level of ERb in reproductive tissues such
as the uterus suggests that a selective ERb agonist may
maintain the beneficial effects of estrogen, without the
increased risk of breast and endometrial cancer. A number
of selective ligands have been already identified.2 ERb selective
agonist ERB-041 (226-fold selective for b) has been used to
demonstrate that this receptor may be a useful target for
certain inflammatory processes.3 Other non-steroidal scaffolds
which have been developed as ERb ligands are diarylpropane-
nitriles (DPN),4 2-phenylnaphthalenes (WAY-202196),2,5 and
aryl-2H-indazoles.6 We have described a series of benzo-
naphthofuran- and naphthothiophene-based ligands which
a
´
Departamento de Quımica, Facultad de Farmacia, Universidad CEU San Pablo,
28668-Boadilla del Monte, Madrid, Spain. E-mail: aramgon@ceu.es;
bpaster@ceu.es; Fax: (+34)913510496; Tel: (+34)913724796
bDepartment of Organic Chemistry, Gdansk University of Technology, 11/12 G.
´
Narutowicza St., 80-233 Gdansk, Poland
cDepartment of Molecular Biology, Faculty of Mathematics and Natural Sciences, The
John Paul II Catholic University of Lublin, 20-708 Lublin, Poland
dDepartment of Environmental Biochemistry and Chemistry, Faculty of Mathematics
and Natural Sciences, The John Paul II Catholic University of Lublin, 20-708 Lublin,
Poland
eInstitute of Food Science, Food Technology and Nutrition (ICTAN), Spanish National
´
Research Council (CSIC), Jose Antonio Novais 10, 28040-Madrid, Spain
Electronic supplementary information (ESI) available: NMR spectra of
3
compounds 2a–2h, 3a–3f and 3h. Predicted binding energies from the docking
studies. See DOI: 10.1039/c3ra00122a
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ligand binding domain (LBD) of both ERs. Several crystal-
lographic structures of the two receptor subtypes in complex
with several ligands have been considered: ERa in complex
with estradiol (PDB 1A52), raloxifene (PDB 1ERR), genistein
(PDB 1X7R), WAY-244 (PDB 1X7E), and 4-hydroxytamoxifen
(PDB 3ERT), and ERb in complex with genistein (PDB 1X7J),
THC (PDB 1L2J), WAY-202196 (PDB 1YYE), and 4-hydroxyta-
moxifen (PDB 2FSZ).
All the candidates to be synthesized (2 and 3) were able to
bind ERs, at least theoretically.
Regarding the predicted binding energies, these were more
favourable in the case of ERb–ligand complexes, as most of the
ligands led to values within the highest energy range (from
27.3 to 29.5 kcal mol21) together with estradiol-like poses.
However, in the case of ERa, only two compounds, 2a and 3a,
led to the best values in ERa (27.9 kcal mol21), although with
different docked poses: 2a presents a pose different to that for
estradiol, while 3a establishes interactions similar to those
found for estradiol, as will be discussed below.
Regarding the binding mode, the analysis of the docking
studies showed that all compounds could adopt estradiol-like
poses in ERb, while in ERa no estradiol-like poses were found
and nor they did show proper binding. Compounds 3a and 3d
were an exception because they were the only ligands
exhibiting estradiol-like poses when docked inside ERa
Fig. 1 Chemical structures of some known ERb-selective ligands.
one of the aromatic rings. We have evaluated the ERa and ERb
binding affinities of the synthesized compounds and studied
the transcriptional activation and proliferative activity in the
MCF-7 breast cancer line of the most interesting of these.
The best binders were compounds 2a and 3a, showing IC50
values of 8.4 and 8.00 mM, respectively, in ERa and a lower
binding affinity for ERb. Compound 3a emerged as a full ERb
agonist at 20 mM, while its effect on ERa was negligible at the
same concentration. This compound stimulated proliferation
of MCF-7 cells with maximal proliferation at 10 mM.
Interestingly, analogue 3c behaved as well as a full ERb
agonist at 20 mM, but showed no proliferative activity.
(predicted binding energies of 27.9 and 26.8 kcal mol21
,
respectively).
Overall, these results may point to a preference for ERb
binding, with the exception of compounds 2a and 3a, which
may present binding abilities for both receptors.
When studying ligand–receptor interactions in atomic
detail, it can be observed that compound 3a establishes
hydrogen bonds between the OH-1 group and Glu305-Arg346,
and the OH-399 group with His475 (ERb numbering), in an
estradiol-like fashion in both ER subtypes. Additionally, the
CF3 group is hosted in a hydrophobic cavity delimited by
Leu339, Met340, Leu343, and Leu380 (in ERa: Leu387, Met388,
Leu391, and Leu428). In the case of 3d, which also establishes
estradiol-like binding modes in both receptors, the presence of
the CH3 group in this position led to a decrease of 1.1
kcal mol21 in the docked binding energy, indicating the role of
Results and discussion
Molecular modelling
the CF group in the binding (see ESI ). The CF group is also
3
3
3
Preliminary docking studies were carried out in order to assess
the capability of the designed compounds to interact with the
present in 2a. In ERb, docking studies for this compound led
to an estradiol-like binding pose with the opposite orientation
to that found for 3a. So, hydrogen bonds were identified
between OH-1 and His-475; and between OH-499 and Glu305–
Arg346. The CF3 is accommodated in the vicinity of the
previously mentioned hydrophobic cavity, contacting Leu380,
Ile376, and Phe377 side chains (Fig. 3). In the case of ERa, this
compound is predicted to bind in a non estradiol-like pose.
These results suggest that the presence of a lipophilic R1 group
could favour the binding by the establishment of hydrophobic
interactions with the cavity delimited by Leu339, Met340,
Leu343, Ile376, Leu380 and Phe377 side chains (ERb number-
ing), and that scaffold 3 is more suitable for anchoring the
receptor (especially ERb).
Fig. 2 Structure of the synthesized 1,4-diaryl-substituted 1,2,3-triazoles.
3698 | RSC Adv., 2013, 3, 3697–3706
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Fig. 3 Docked binding modes obtained for compounds 2a and 3a in ERb.
Chemistry
As a result of the previous theoretical study, we decided to
synthesize compounds 2 and 3. The detailed synthesis of the
azide fragment is shown in Scheme 1. Some of the amines
used (4d–f) are commercially available, and some had to be
synthesized (4a–c) by Sn/HCl reduction of the corresponding
nitro compounds. After protection of the hydroxyl group
(together with the carboxylic groups in the case of 4e and 4f),
the TIPS-protected derivatives were transformed into azides 6
by reaction with tert-butyl nitrite and azidotrimethylsilane.
These azides were coupled to 3-ethynylphenol and 4-
((trimethylsilyl)ethynyl)phenol. The former is commercially
available, and the latter was obtained by Sonogashira coupling
between the commercially available 4-iodophenol and tri-
methylsilyl acetylene.
For the CuAAC reaction, we used CuSO4 (0.1 equiv.) and
ascorbic acid (0.2 equiv.) in the presence of tetrabutylammo-
nium fluoride to bring about the deprotection of 4-((trimethyl-
silyl)ethynyl)phenol in the reaction mixture and the
deprotection of the final triazoles.
We have chosen this synthetic pathway, involving the use of
protective groups, because, in our first attempt to carry out the
click reaction starting from unprotected azides, we observed a
low solubility of the reactants in the reaction medium, and the
formation of a complex mixture of compounds by TLC and 1H-
NMR analysis of the crude reaction.
Finally, acids 2e, 2f and 3f were transformed into the
corresponding methyl esters 2g, h and 3h by reaction with
MeOH in the presence of H2SO4.
Scheme 1 Synthesis of ligands 2 and 3. Reagents and conditions: (a) H2, Pd/C
for 4a, Sn/HCl for 4b and 4c; (b) 1 equiv. TIPSCl/Et3N for 5a–d, 2 equiv. TIPSCl/
Et3N for 5e–f; (c) t-BuONO/TMSN3; (d) TBFA, CuSO4?5H2O, sodium ascorbate,
DMF; (e) MeOH, H2SO4.
Estrogen receptor binding assays
The affinity for ERa and ERb of compounds 2 and 3 were
determined in an in vitro competitive binding assay, following
a reported method13 with some modifications. All compounds
were tested at 10 mM and only compounds 2a, 2e, 3a and 3d
presented enough affinity at this concentration to determine
the IC50 at least in one of the receptors. Table 1 shows a
summary of the results.
They were assayed for transcriptional activity through both
receptor subtypes, with estradiol as a reference. Reporter gene
assays were conducted in mammalian cells containing either
ERa- or ERb-responsive luciferase reporter gene. The luciferase
gene expression appears when ligand-bound ER undergoes
nuclear translocation, DNA binding, assembling of the co-
activators and other factors which are necessary to obtain a
functional transcription complex, in which the target gene is
expressed. The readout from those reporter cells fulfils similar
interaction demands to those which can be observed in vivo. In
the reporter gene assays on cells treated with saturating
Transcription assays
To characterize the agonist/antagonist profile we first sub-
mitted three compounds (2a, 2e and 3a) with the highest
binding affinity.
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Table 1 Affinitiesa of compounds 2 and 3 (IC50) and % of [3H]-estradiol binding
to ERa and ERb at 10 mMb
Ligand IC50 ERa/mM IC50 ERb/mM b/a ratio 10 mM ERa 10 mM ERb
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
3e
3f
8.4
NA
NA
NA
14.5
NA
NA
NA
8.00
NA
NA
61.4
NA
NA
NA
22.6
NA
NA
NA
130.0
NA
NA
NA
.100
NA
NA
2.6
—
—
—
8.9
—
—
—
47
98.5
100
75.3
100
100
87.3
79.7
100
94.4
100
100
100
100
100
100
100
100
76.9
60.9
94.8
83.9
98.6
50.7
100
100
72.5
100
97.8
78.3
—
—
—
—
—
—
Fig. 5 Transcriptional activation of ERa by estradiol (100 nM) and compounds
2a, 2e, 3a and 3c.
.100
NA
NA
3h
NA
a
Values are an average of at least 2 experiments with typical
14.74 of control in comparison to 1689.06 ¡ 74.17 for 100 nM
of estradiol). This result demonstrates that 3a is selective in its
transcriptional activity, behaving as a full agonist for ERb at 20
mM, while being a weak agonist for ERa at the same
concentration. It is of great interest to detect non-steroidal
molecules that display agonistic or antagonistic properties on
the ERs in a specific manner.
It should be noted that there is no correlation between
binding affinity and transcriptional potency for this com-
pound. This lack of correlation has been observed in other
cases,15 and could be explained by differences in the mode of
binding between the ligands, the ERs and various cellular co-
regulators, which could modulate the final ligand response in
the transcriptional assay.
b
standard errors below 15%. NA: not achieved. The percentage of
specific binding of [2,4,6,7,16,17-3H]-estradiol to ER.
concentrations of estradiol, ERb showed a maximal activity
which was 62% lower than the maximal activity observed on
ERa. This observation is in agreement with previously
published results.14
Compounds 2a and 3a showed agonistic effects in ERb
(Fig. 4). Thus, compound 2a induced a significant increase in
luciferase activity (414 ¡ 8.46% of control) at 10 mM, and
compound 3a showed a very high efficacy at 20 mM (633 ¡ 67.8
of control), which is comparable to the maximum response of
estradiol (694.38 ¡ 6.21 of control at 4 nM). Compound 2e
induced some increase in transcriptional activity but it was not
concentration-dependent.
In the case of ERa (Fig. 5), compound 2a was able to activate
the receptor with the maximum activity at 20 mM (859.56% of
control) in comparison to the maximum response of estradiol
in this receptor (1689.06 ¡ 74.17 at 100 nM). These results
suggest that 2a behaves as an agonist for both receptors with
similar efficacy.
Finally, no transcriptional effect was present on ERa at the
tested concentrations in the case of 2e. This compound could
be classified as a selective partial agonist of ERb. No
antagonistic activity was observed for all tested compounds.
Effect on the proliferation of a MCF-7 breast cancer cell line
We determined the proliferative activity of all the synthesized
compounds on the hormone-dependent MCF-7 breast cancer
line (Fig. 6 and Fig. 7).
More interestingly, only a slight activity on ERa was
observed in the case of compound 3a at 20 mM (320.42 ¡
Fig. 6 Concentration response curves of proliferation of MCF-7 treated with the
following compounds: 2a ($), 2e (.), and 3a (&), evaluated with crystal violet
assay. Data are mean ¡SD of at least 5 determinants in 2 independent
experiments.
Fig. 4 Transcriptional activation of ERb by estradiol (4 nM) and compounds 2a,
2e, 3a and 3c.
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Fig. 5). Thus, 3c is a full ERb agonist, without promoting the
proliferation of MCF-7 cells, a biological profile which holds
promise for the treatment of inflammatory diseases including
rheumatoid arthritis, and cardiovascular and CNS condi-
tions.14
Conclusions
A series of clicked 1H-1,2,3-triazoles have been synthesized,
and their affinity and transcriptional activation of both ERa
and ERb have been determined by means of in vitro assays.
This study has allowed us to detect two compounds (3a and 3c)
that behave as selective full agonists for ERb at 20 mM, while
their effect on ERa is considerably lower at the same
concentration. These compounds could be useful to gain
knowledge on the physiological role of ERb. While 3a induced
the proliferation of MCF-7 cells, its analogue 3c was
completely inactive in this breast cancer cell line. This
compound constitutes, therefore,
a new and promising
candidate for the development of ER binding agents useful
for the treatment of inflammatory, cardiovascular and CNS
diseases, without promoting undesired proliferative effects on
the breast.
Fig. 7 Concentration response curves of proliferation of MCF-7 treated with the
following compounds: (a) 2b ($), 2c (.), 2d (&), 2f (m), 2g (#), 2h (%), and (b)
3b (#), 3c (&), 3d ($), 3e (.), 3f (m), 3h (%). Data are mean ¡SD of at least 5
determinants in 2 independent experiments.
Experimental
General methods. Melting points (uncorrected) were deter-
mined on a Stuart Scientific SMP3 apparatus. Infrared (IR)
spectra were recorded with a Perkin-Elmer 1330 infrared
spectrophotometer. 1H and 13C NMR were recorded on a
Bruker 300-AC instrument. Chemical shifts (d) are expressed in
parts per million; coupling constants (J) are in Hertz. Mass
spectra were run on a Bruker Esquire 3000 spectrometer. Thin-
layer chromatography (TLC) was run on Merck silica gel 60
F-254 plates. Unless stated otherwise, starting materials used
were high-grade commercial products. For all tested com-
pounds .95% purity was confirmed by HPLC/MS using an
Esquire 3000 ion-trap mass spectrometer (Bruker-Daltronics,
Bremen, Germany) coupled to an Agilent HPLC system type HP
1100. A LiChroCART Supersphere100 RP-18 column (125 6 2
mm; 4 mm particle size) was used, eluting with H2O + 0.1%
TFA/MeOH + 0.1% TFA or acetonitrile + 0.1% TFA. Acid 2f was
obtained in a complex mixture with other unknown com-
pounds, and it was not possible to isolate it in pure form.
However, it could be transformed into the corresponding
methyl ester 2h.
Compound 3a induced an increase in proliferation, display-
ing a bell-shaped dose–response curve, with maximal prolif-
eration at 10 mM (1529.15% ¡ 99.6) of solvent control in
comparison to 446.17% ¡ 45.4 obtained for 1 pM estradiol.
The other two ER binders 2a and 2e also increased prolifera-
tion but with lower efficacy with a maximum at 10 mM (581.59
¡ 33.12) and 50 mM (483.35 ¡ 39.81) for 2a and 2e,
respectively.
This proliferative activity could be explained by its effect on
the ER pathway, as we proved that these compounds behave as
ER agonists.
The rest of the compounds presented different profiles that
did not correlate with their binding affinities (Fig. 7).
Interestingly compound 3c did not induce proliferation of
MCF7 cells, which is in agreement with its lack of affinity for
both receptors in the binding assay. However, taking into
account the observed lack of correlation between the binding
affinity and the functional activity of the previously studied
compounds (2a, 2e and 3a), we decided to study its
transcriptional activation capability (Fig. 4 and Fig. 5). We
found that 3c behaved as a full agonist at 20 mM, increasing
luciferase activity (634.1 ¡ 30.8 of control) to a similar extent
than estradiol at 4 nM (694.38 ¡ 6.21), while its activation of
ERa (455.2 ¡ 14.2 of control) was only 27% of the control
activity of estradiol (1689.06 ¡ 74.17 at 100 nM) (Fig. 4 and
General method for the synthesis of amines 4a–c
To a 100 mL round bottomed flask, containing a stirrer bar
and a reflux condenser, tin (1.65 g, 14 mmol) and the
corresponding phenol (10 mmol) were added. Then water (24
mL) followed by concentrated hydrochloric acid (9 mL) were
added, and the solution was heated under reflux for 1.5 h.
Then, the reaction mixture was cooled in an ice bath, a 5 M
NaOH solution (20 mL) was added, and the aqueous solution
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was extracted with AcOEt (3 6 50 cm3) and the combined
organic extracts were washed with saturated aqueous NaHCO3
and brine, dried (MgSO4) and concentrated to dryness to give
the desired amine 4.
Triisopropylsilyl 2-amino-5-((triisopropylsilyl)oxy)benzoate
5e. Yield 94%, oil; 1H NMR dH (300 MHz; CDCl3) 1.08 (18H,
d, J 7.3, 3CH3), 1.12 (18H, d, J 7.3, 3CH3), 1.12–1.27 (3H, m,
3CH), 1.28–1.45 (3H, m, 3CH), 5.00–5.85 (2H, brs, NH2), 6.56
(1H, d, J 8.7, ArH), 6.89 (1H, dd, J 8.8 and 2.9, ArH), 7.43 (1H, d,
J 2.9, ArH).
Triisopropylsilyl 5-amino-2-((triisopropylsilyl)oxy)benzoate
5f. Yield 86%, oil; 1H NMR dH (300 MHz; CDCl3) 1.00 (18H,
d, J 7.3, 6CH3), 1.03 (18H, d, J 7.3, 3CH3), 1.12–1.38 (6H, m,
6CH), 2.55–3.00 (2H, br s, NH2), 6.56–6.67 (2H, m, ArH), 7.02
(1H, d, J 2.1, ArH).
4-Amino-3-trifluoromethylphenol 4a. Yield 88%; 1H NMR dH
(300 MHz; DMSO) 4.88 (2H, s, NH2), 6.68–6.80 (3H, m, ArH),
8.92 (1H, s, OH).
4-Amino-2-fluorophenol 4b. Yield 93%; 1H NMR dH (300
MHz; CD3OD) 6.25–6.32 (1H, m, ArH), 6.40 (1H, dd, J 12.1, 2.4),
6.59 (1H, dd, J 9.6 and 8.6); 13C NMR dc (75.4 MHz, CD3OD)
105.3 (JCF 21 Hz), 112.9 (JCF 3 Hz), 119.3 (JCF 4 Hz), 138.0 (JCF 13
Hz), 141.6 (JCF 9 Hz) and 153.4 (JCF 238 Hz).
4-Amino-2-methylphenol 4c. Yield 90%; 1H NMR dH (300
MHz; CD3OD) 2.01 (3H, s, CH3), 6.34 (1H, dd, J 8.1 and 2.3,
ArH), 6.41–6.48 (2H, m, ArH); 13C NMR dc (75.4 MHz, CD3OD)
16.76, 116.20, 116.77, 120.68, 126.64, 140.40 and 149.90.
General method for the synthesis of azides
To an ice-cooled solution of the corresponding compound 6a–f
(1 g, 2.15 mmol) in CH3CN (5 mL), was added dropwise
t-BuONO (0.332 g, 381 mL, 3.22 mmol) followed by TMSN3
(0.301 g, 341 mL, 2.57 mmol) and the solution was stirred at
room temperature for 4 h. After concentrating under vacuum,
the crude product was purified by silica gel chromatography
(hexane) to give the desired product.
General method for the synthesis of the TIPS-protected
compounds 5a–d
To a solution of the corresponding phenol (2.83 mmol) in dry
DMF (2 mL) was added a solution of triisopropylsilyl chloride
(0.655 g, 3.40 mmol) and triethylamine (0.315 g, 3.11 mmol) in
dry DMF (1 mL) under N2, and the mixture was stirred at RT
for 24 h. The solution was filtered and the solvent was
evaporated. The resulting residue was purified by column
chromatography using hexane/AcOEt (15 : 1) as eluent to give
the desired product.
2-(Trifluoromethyl)-4-((triisopropylsilyl)oxy)aniline 5a. Yield
87%, oil; 1H NMR dH (300 MHz; CDCl3) 1.08 (9H, d, J 7.3, CH3),
1.12 (9H, d, J 7.3, 2CH3), 1.12–1.27 (3H, m, 3CH), 1.28–1.45
(3H, m, 3CH), 2.80–3.70 (2H, brs, NH2), 6.54 (1H, d, J 8.7, ArH),
6.77 (1H, dd, J 8.7 and 2.7, ArH), 6.88 (1H, d, J 2.7, ArH).
3-Fluoro-4-((triisopropylsilyl)oxy)aniline 5b. Yield 80%, oil;
1H NMR dH (300 MHz; CDCl3) 1.00 (18H, d, J 6.9, 6CH3), 1.12–
1.35 (3H, m, 3CH), 3.37 (2H, brs, NH2), 6.20–6.25 (1H, m, ArH),
6.32 (1H, dd, J 12.1, 2.8, ArH), 6.66 (1H, t, J 8.9, ArH).
3-Methyl-4-((triisopropylsilyl)oxy)aniline 5c. Yield 82%, oil;
1H NMR dH (300 MHz; CDCl3) 1.01 (18H, d, J 7.0, 6CH3), 1.10–
1.21 (3H, m, 3CH), 2.09 (3H, s, CH3Ar), 3.10–3.40 (2H, brs,
NH2), 6.29 (1H, dd, J 8.3, 2.9, ArH), 6.42 (1H, d, J 2.9, ArH), 6.50
(1H, d, J 8.3, ArH).
(4-Azido-3-(trifluoromethyl)phenoxy)triisopropylsilane 6a.
1
Yield 67%, oil; H NMR dH (300 MHz; CDCl3) 1.01 (18H, d, J
7.3, CH3 iPr), 1.10–1.21 (1H, m, CH iPr), 7.00 (1H, dd, J 8.6, 2.6,
ArH), 7.05–7.09 (2H, m, ArH).
(4-Azido-2-fluorophenoxy)triisopropylsilane 6b. Yield 71%,
1
oil; H NMR dH (300 MHz; CDCl3) 1.00 (18H, d, J 6.9, 6CH3),
1.09–1.28 (3H, m, 3CH), 6.55–6.63 (1H, m, ArH), 6.67 (1H, dd, J
11.1 and 2.6, ArH), 6.84 (1H, t, J 8.9, ArH); 13C NMR dC (75.4
MHz, CDCl3) 11.6, 16.7, 106.6 (JCF 22 Hz), 113.5 (JCF 3 Hz),
121.4 (JCF 3 Hz), 132.1 (JCF 9 Hz), 140.1 (JCF 12 Hz), 153.2 (JCF
246 Hz).
(4-Azido-2-methylphenoxy)triisopropylsilane 6c. Yield 64%,
1
oil; H NMR dH (300 MHz; CDCl3) 1.01 (18H, d, J 7.0, 3CH3),
1.10–1.21 (3H, m, CH), 2.15 (3H, s, CH3), 6.62 (1H, dd, J 8.5,
2.8, ArH), 6.67 (1H, dd, J 8.5, 6.7, ArH), 6.72 (1H, m, ArH); 13C
NMR dC (75.4 MHz, CDCl3) 12.99, 17.08, 17.99, 116.96, 118.82,
121.33, 130.27, 131.92, 151.73.
(4-Azido-3-methylphenoxy)triisopropylsilane 6d. Yield 70%,
1
oil; H NMR dH (300 MHz; CDCl3) 1.01 (18H, d, J 7.3, 3CH3),
1.10–1.21 (3H, m, 3CH), 2.08 (3H, s, CH3), 6.61 (1H, d, J 2.6,
ArH), 6.65 (1H, dd, J 8.5 and 2.6, ArH), 6.86 (1H, d, J 8.5, ArH);
13C NMR dC (75.4 MHz, CDCl3) 17.95, 18.31, 19.31, 119.74,
121.84, 124.70, 134.20, 134.79, 154.80.
Triisopropylsilyl 2-azido-5-((triisopropylsilyl)oxy)benzoate
6e. Yield 85%, oil; 1H NMR dH (300 MHz; CDCl3) 1.01 (18H,
d, J 7.3, 3CH3), 1.05 (18H, d, J 7.3, 3CH3), 1.10–1.21 (3H, m,
3CH), 1.23–1.38 (3H, m, 3CH), 6.99 (1H, dd, J 8.6, 2.6, ArH),
7.04 (1H, d, J 8.6, ArH), 7.42 (1H, d, J 2.6, ArH); 13C NMR dC
(75.4 MHz, CDCl3) 12.0, 12.6, 17.80, 17.82, 121.2, 123.11, 124.1,
125.2, 133.5, 152.7, 164.1.
2-Methyl-4-((triisopropylsilyl)oxy)aniline 5d. Yield 84%, oil;
1H NMR dH (300 MHz; CDCl3) 1.08 (9H, d, J 7.3, 3CH3), 1.12
(9H, d, J 7.3, 3CH3), 1.12–1.27 (3H, m, 3CH), 1.28–1.45 (3H, m,
3CH), 2.05 (3H, s, CH3), 2.60–3.40 (2H, brs, NH2), 6.46 (1H, d, J
8.3, ArH), 6.49 (1H, dd, J 8.3, 2.6, ArH), 6.55 (1H, d, J 2.6, ArH).
General method for the synthesis of the TIPS-protected
compounds 5e and 5f
To a solution of the corresponding acid (2.35 mmol) in dry
DMF (2 mL) was added a solution of triisopropylsilyl chloride
(1 g, 5.19 mmol) and triethylamine (0.500 g, 4.95 mmol) in dry
DMF (1 mL) under N2, and the mixture was stirred at RT for 24
h. The solution was filtered and the solvent was evaporated.
The resulting residue was purified by column chromatography
using hexane/AcOEt (15 : 1) as eluent to give the desired
product.
Triisopropylsilyl 5-azido-2-((triisopropylsilyl)oxy)benzoate 6f.
1
Yield 70%, oil; H NMR dH (300 MHz; CDCl3) 1.03 (18H, d, J
7.3, CH3 iPr), 1.06 (18H, d, J 7.3, 6CH3), 1.12–1.38 (6H, m,
6CH), 6.79 (1H, d, J 8.7, ArH), 6.90 (1H, dd, J 8.7 and 2.9, ArH),
7.38 (1H, d, J 2.9, ArH); 13C NMR dC (75.4 MHz, CDCl3) 12.04,
13.13, 17.88, 17.98, 121.76, 122.06, 123.24, 124.62, 131.86,
153.83, 163.39.
3702 | RSC Adv., 2013, 3, 3697–3706
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8.0, ArH), 6.95 (1H, d, J 8.5, ArH), 7.52 (1H, d, J 7.9, ArH), 7.62
(1H, s, ArH), 7.73 (2H, d, J 7.9, ArH), 8.89 (1H, s, triazole-H),
9.60 (1H, s, OH), 9.82 (1H, s, OH); 13C NMR dC (75.4 MHz,
DMSO) 15.9, 115.0, 115.6, 117.9, 118.7, 121.4, 122.5, 125.2,
126.6, 128.6, 147.2, 155.6, 157.3. MS (ESI) m/z: 268.00 [M + H]+.
4-[4-(4-Hydroxyphenyl)-1H-1,2,3-triazol-1-yl]-3-methylphenol
2d. From tetrabutylammonium fluoride (1.11 mL, 1 M solution
in THF); chromatography eluent: AcOEt/hexane 3 : 2; yield
4-((Trimethylsilyl)ethynyl)phenol. A solution of 4-iodophe-
nol (0.880 g, 4 mmol), trimethylsilylacetylene (0.855 mL, 6
mmol), PdCl2 (35.8 mg, 0.20 mmol, 5 mol%), Ph3P (0.106 g,
0.40 mmol, 10 mol%), CuI (19 mg, 0.10 mmol, 2.5 mol%), and
triethylamine (3.94 mL, 28.2 mmol) in dry acetonitrile (20 mL)
was heated at reflux under an argon atmosphere. After 3 h of
reflux, the reaction mixture was filtered through a Celite pad.
The filtrate was concentrated under reduced pressure and
chromatographed on silica gel using AcOEt/hexane (1 : 4) as
eluent to afford the desired compound (0.533 g, 70%) as a
brown solid; 1H NMR dH (300 MHz; CDCl3) 0.16 (9H, s, 3CH3),
4.60–5.50 (1H, brs, OH), 6.65 (2H, d, J 8.7, ArH), 7.27 (2H, d, J
8.7, ArH); 13C NMR dC (75.4 MHz, CDCl3) 0.15, 103.68, 106.71,
114.22, 116.85, 132.78, 158.43.
1
70%, mp 203 uC; nmax (KBr)/cm21 3425, 3186, 2537, 1612; H
NMR dH (300 MHz; CD3OD) 2.10 (3H, s, CH3), 5.00 (2H, br s,
2OH), 6.77–6.83 (2H, m, ArH), 6.89 (2H, d, J 8.5, ArH), 7.20 (1H,
d, J 8.5, ArH), 7.71 (2H, d, J 8.5, ArH), 8.25 (1H, s, triazole-H);
13C NMR dC (75.4 MHz, CD3OD) 17.9, 114.6, 116.9, 118.6,
122.9, 123.2, 128.4, 129.8, 136.7, 149.0, 159.1, 160.1. MS (ESI)
m/z: 267.98 [M + H]+.
General method for the click reactions
5-Hydroxy-2-(4-(4-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)ben-
zoic acid 2e. From tetrabutylammonium fluoride (1.65 mL, 1
M solution in THF); chromatography eluent: AcOEt/hexane
5 : 1; yield 63%, mp 122–128 uC; nmax (KBr)/cm21 3261, 2925,
A solution of 4-((trimethylsilyl)lethynyl)phenol (0.1 g, 0.53
mmol) or 3-ethynylphenol (0.063 g, 0.53 mmol) and the
corresponding azide (0.53 mmol) in DMF (10 mL) was treated
with a 1 M solution of tetrabutylammonium fluoride in THF
(see the amount below for each example) under argon. The
reaction mixture was stirred at 0 uC for 30 min. Then, a freshly
prepared 1 M aqueous solution of sodium ascorbate (0.2
equiv.) was added, followed by the addition of copper(II)
sulfate pentahydrate (0.1 equiv.). After stirring for 24 h under
an argon atmosphere, the reaction mixture was concentrated
under vacuum. Then AcOEt (50 mL) was added and the
solution was washed with 0.1 M HCl. The solvent was
evaporated and the resulting residue was purified by column
chromatography on silica using AcOEt/hexane as eluent, to
yield the desired product.
4-(4-(4-Hydroxyphenyl)-1H-1,2,3-triazol-1-yl)-3-(trifluoro-
methyl)phenol 2a. From tetrabutylammonium fluoride (1.11
mL, 1 M solution in THF); chromatography eluent: AcOEt/
hexane 3 : 2; yield 78%; mp 183–184 uC; nmax (KBr)/cm21 3500,
3037, 2917, 1619; 1H NMR dH (300 MHz; CD3OD) 5.08 (2H, brs,
2OH), 6.90 (2H, d, J 8.6, ArH), 7.18 (1H, dd, J 8.6, 2.6, ArH), 7.29
(1H, d, J 2.6, ArH), 7.43 (1H, d, J 8.6, ArH), 7.72 (2H, d, J 8.6,
ArH), 8.34 (1H, s, triazole-H); 13C NMR dC (75.4 MHz, CD3OD)
115.0 (JCF 5 Hz), 116.9, 120.5, 122.6, 124.1 (JCF 273 Hz), 124.2,
127.06, 128.0, 128.4, 128.7 (JCF 32 Hz), 131.9, 135.1, 149.0,
159.2, 161.0. MS (ESI) m/z: 344.00 [M + Na]+, 322.04 [M + H]+.
2-Fluoro-4-(4-(4-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)phenol
2b. From tetrabutylammonium fluoride (1.11 mL, 1 M solution
in THF); chromatography eluent: AcOEt/hexane 3 : 2; yield
76%, mp 258–259 uC; nmax (KBr)/cm21 3268, 3156, 2917, 1615;
1H NMR dH (300 MHz; CD3OD) 4.83 (2H, br s, 2OH), 6.86 (2H,
d, J 8.6, ArH), 7.09 (1H, t, J 8.9, ArH), 7.47–7.53 (1H, m, ArH),
7.62 (1H, dd, J 11.5 and 2.5, ArH), 7.69 (2H, d, J 8.6, ArH), 8.59
(1H, s, triazole-H); 13C NMR dC (75.4 MHz, CD3OD) 110.3 (JCF
23 Hz), 116.8, 117.9 (JCF 3 Hz), 119.3 (JCF 13 Hz), 119.44, 122.8,
128.3, 130.6, 147.1 (JCF 13 Hz), 152.8 (JCF 243 Hz), 159.2. MS
(ESI) m/z: 293.98 [M + Na]+, 271.99 [M + H]+.
1
1612; H NMR dH (300 MHz; CD3OD) 5.00 (2H, brs, OH), 6.87
(2H, d, J 8.4, ArH), 7.08 (1H, dd, J 8.7 and 3.0, ArH), 7.38 (2H,
dd J 8.7 and 3.0, ArH), 7.69 (2H, d, J 8.4, ArH), 8.34 (1H, s,
triazole-H); 13C NMR dC (75.4 MHz, CD3OD) 116.8, 118.2,
119.6, 123.0, 123.5, 128.3, 128.9, 129.4, 148.8, 159.0, 160.2,
169.4, 175.6. MS (ESI) m/z: 297.98 [M + H]+.
2-Hydroxy-5-(4-(4-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)ben-
zoic acid 2f. From tetrabutylammonium fluoride (1.65 mL, 1 M
solution in THF); chromatography eluent: AcOEt/hexane 5 : 1;
yield 71%, mp 272–273 uC (decomp.); nmax (KBr)/cm21 3395,
1
3141, 2917, 2850, 1675, 1612; H NMR dH (300 MHz; CD3OD)
4.86 (2H, brs, OH), 6.89 (2H, d, J 8.6, ArH), 6.99 (1H, d, J 8.7,
ArH), 7.71–7.79 (3H, m, ArH), 8.30 (1H, d, J 2.1, ArH), 8.58 (1H,
s, triazole-H); 13C NMR dC (75.4 MHz, CD3OD) 117.0, 118.7,
119.2, 121.1, 123.0, 124.0, 126.2, 128.3, 129.4, 149.7, 159.3,
163.63. MS (ESI) m/z: 297.99 [M + H]+.
4-(4-(3-Hydroxyphenyl)-1H-1,2,3-triazol-1-yl)-3-(trifluoro-
methyl)phenol 3a. From tetrabutylammonium fluoride (0.55
mL, 1 M solution in THF); chromatography eluent: AcOEt/
hexane 3 : 2; yield 82%, mp 182–183 uC; nmax (KBr)/cm21 3261,
3194, 2925, 2589, 1612; 1H NMR dH (300 MHz; CD3OD) 4.94
(2H, br s, 2OH), 6.80–6.84 (1H, m, ArH), 7.16 (1H, dd, J 8.6, 2.8,
ArH), 7.24 (1H, d, J 7.8, ArH), 7.29–7.37 (3H, m, ArH), 7.44 (1H,
d, J 8.6, ArH), 8.41 (1H, s, triazole-H); 13C NMR dC (75.4 MHz,
CD3OD) 113.7, 114.9, 116.7, 118.2, 120.5, 124.1 (JCF 273 Hz),
125.3, 127.0, 128.7 (JCF 37 Hz), 131.2, 131.9, 132.5, 148.8, 159.1,
161.0. MS (ESI) m/z: 344.01 [M + Na]+, 322.05 [M + H]+.
2-Fluoro-4-(4-(3-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)phenol
3b. From tetrabutylammonium fluoride (0.55 mL, 1 M solution
in THF); chromatography eluent: AcOEt/hexane 3 : 2; yield
81%, mp 225–226 uC; nmax (KBr)/cm21 3305, 2925, 2850, 1615;
1H NMR dH (300 MHz; CD3OD) 4.85 (2H, br s, 2OH), 6.79–6.82
(1H, m, ArH), 7.09 (1H, t, J 8.9, ArH), 7.24 (1H, t, J 7.9, ArH),
7.31–7.35 (2H, m, ArH), 7.48–7.52 (1H, m, ArH), 7.64 (1H, dd, J
11.4 and 2.5, ArH), 8.64 (1H, s, triazole-H); 13C NMR dC (75.4
MHz, CD3OD) 110.2 (JCF 23 Hz), 113.6, 116.6. 118.0 (JCF 4 Hz),
118.2, 119.4 (JCF 4 Hz), 120.3, 130.4 (JCF 8 Hz), 131.1, 132.6,
147.2 (JCF 13 Hz), 149.5, 152.8 (JCF 243 Hz), 159.1. MS (ESI) m/z:
293.97 [M + Na]+, 271.97 [M + H]+.
4-(4-(4-Hydroxyphenyl)-1H-1,2,3-triazol-1-yl)-2-methylphenol
2c. From tetrabutylammonium fluoride (1.11 mL, 1 M solution
in THF); chromatography eluent: AcOEt/hexane 3 : 2; yield
83%, mp 242–243 uC; nmax (KBr)/cm21 3335, 3149, 2917, 1612;
1H NMR dH (300 MHz; DMSO) 2.23 (3H, s, CH3), 6.86 (2H, d, J
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ArH), 7.62 (1H, d, J 8.7, ArH), 7.74 (2H, d, J 8.7, ArH), 9.01 (1H,
s, triazole-H); 13C NMR dC (75.4 MHz, CD3OD) 53.4, 115.8,
117.7, 119.3, 121.0, 127.4, 127.6, 129.2, 129.7, 130.3, 144.8,
161.7, 161.9, 165.6. MS (ESI) m/z: 312.03 [M + H]+.
3c. From tetrabutylammonium fluoride (0.55 mL, 1 M solution
in THF); chromatography eluent: AcOEt/hexane 3 : 2; yield
84%, mp 196–197 uC; nmax (KBr)/cm21 3318, 3141, 2925, 2850,
1
1615; H NMR dH (300 MHz; CD3OD) 2.28 (3H, s, CH3), 4.83
Methyl 2-hydroxy-5-(4-(4-hydroxyphenyl)-1H-1,2,3-triazol-1-
yl)benzoate 2h. Yield 86%, mp 211–213 uC; nmax (KBr)/cm21
3261, 3141, 2955, 2925, 1679, 1619; 1H NMR dH (300 MHz;
DMSO) 3.95 (3H, s, CH3), 6.88 (2H, d, J 8.4, ArH), 7.23 (1H, d, J
8.4, ArH), 7.75 (2H, d, J 8.6, ArH), 8.05 (1H, dd, J 8.9 and 2.8,
ArH), 8.25 (1H, d, J 2.8, ArH), 9.08 (1H, s, triazole-H), 9.68 (1H,
br s, OH), 10.60 (1H, br s, OH); 13C NMR dC (75.4 MHz, DMSO)
52.6, 114.3, 115.6, 118.8, 121.2, 121.3, 126.7, 126.9, 128.8,
147.5, 157.5, 159.2, 167.8. MS (ESI) m/z: 312.03 [M + H]+.
Methyl 2-hydroxy-5-(4-(3-hydroxyphenyl)-1H-1,2,3-triazol-1-
yl)benzoate 3h. Yield 72%, mp 202–203 uC; nmax (KBr)/cm21
3141, 2955, 1671, 1612; 1H NMR dH (300 MHz; DMSO) 3.95
(3H, s, CH3), 6.76–6.80 (1H, m, ArH), 7.25 (2H, t, J 8.9, ArH),
7.30–7.39 (2H, m, ArH), 8.07 (1H, dd, J 8.9, 2.8, ArH), 8.27 (1H,
d, J 2.8, ArH), 9.21 (1H, s, triazole-H), 9.59 (1H, br s, OH), 10.69
(1H, br s, OH); 13C NMR dC (75.4 MHz, DMSO) 52.6, 112.0,
114.3, 115.1, 116.2, 118.8, 119.5, 121.4, 127.0, 128.7, 129.9,
131.4, 147.3, 157.7, 159.2, 167.7. MS (ESI) m/z: 312.02 [M + H]+.
(2H, brs, OH), 6.78–6.82 (1H, m, ArH), 6.90 (1H, d, J 8.6, ArH),
7.25 (1H, t, J 7.9), 7.32–7.35 (3H, m, ArH), 7.47 (1H, dd, J 8.6
and 2.6), 7.55 (1H, d, J 2.5, ArH), 8.59 (1H, s, triazole-H); 13C
NMR dC (75.4 MHz, CD3OD) 16.3, 113.6, 116.1, 116.5, 118.2,
120.4, 120.5, 124.3, 127.4, 130.5, 131.1, 132.8, 149.3, 157.6,
159.1. MS (ESI) m/z: 268.00 [M + H]+.
4-(4-(3-Hydroxyphenyl)-1H-1,2,3-triazol-1-yl)-3-methylphenol
3d. From tetrabutylammonium fluoride (0.55 mL, 1 M solution
in THF); chromatography eluent: AcOEt/hexane 3 : 2; yield
80%, mp 210–211 uC; nmax (KBr)/cm21 3291, 2440, 1615; 1H
NMR dH (300 MHz; CD3OD) 2.12 (3H, s, CH3), 4.83 (2H, br s,
2OH), 6.76–6.83 (3H, m, ArH), 7.21–7.35 (4H, m, ArH), 8.35
(1H, s, triazole-H); 13C NMR dC (75.4 MHz, CD3OD) 17.8, 113.6,
114.6, 116.5, 118.2, 118.6, 124.3, 128.5, 129.8, 131.2, 132.8,
136.7, 148.8, 159.2, 160.2. MS (ESI) m/z: 289.98 [M + Na]+,
267.98 [M + H]+.
5-Hydroxy-2-(4-(3-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)ben-
zoic acid 3e. From tetrabutylammonium fluoride (1.11 mL, 1
M solution in THF); chromatography eluent: AcOEt/hexane
5 : 1; yield 71%, mp 165–166 uC; nmax (KBr)/cm21 3313, 2917,
Computational methods
To perform the theoretical study of the binding mode both
subtypes of ERa and ERb have been taken into account. As
macromolecules, several crystallographic structures of the two
receptor subtypes in complex with several ligands have been
considered: ERa in complex with estradiol (PDB 1A52),
raloxifene (PDB 1ERR), genistein (PDB 1X7R), WAY-244 (PDB
1X7E), and 4-hydroxytamoxifen (PDB 3ERT), and ERb in
complex with genistein (PDB 1X7J), THC (PDB 1L2J), WAY-
202196 (PDB 1YYE), and 4-hydroxytamoxifen (PDB 2FSZ).
Water molecules close to the amino acids Arg394 (ERb
Arg346) and Glu353 (Glu305 ERb) were kept for the docking
procedures. Thirteen ligands (compounds 2a–2g and 3a–3f)
were built using Maestro LigPrep module (www.schrodinger.
com). Gaussian 0316 at the B3LYP/3-21G* level was used to
optimize the geometries and to calculate point charges. Atom
types and bond types were assigned, and mol2 files were
generated. Macromolecule geometries were refined by using
Protein Preparation module in Maestro. The Glide module was
used to perform the docking calculations.17,18 The center of
the box was positioned on the center of the bound ligand
present in the crystallographic structure, and the box size was
set up to enclose the ligand binding domain. The docking
procedure was performed with XP (extra precision) mode, and
a van der Waals radii scale factor of 1.0/0.8 for receptor and
ligand, respectively. Induced Fit Docking was also used, and
contained constrained minimization of the receptor with an
RMSD cutoff of 0.18 Å, and Prime-side-chain prediction on
residues within a 5 Å of any ligand pose. Glide redocking was
performed into structures within 30 kcal mol21 of the lowest
energy structure with van der Waals scaling of 1.0/0.8 for
receptor and ligand, respectively. The best obtained result for
each ligand was considered for analysis of the ligand–receptor
interactions.
1
1705, 1589; H NMR dH (300 MHz; DMSO) 6.73–6.76 (1H, m,
ArH), 7.10 (1H, dd, J 8.6 and 2.8), 7.22–7.35 (4H, m, ArH), 7.44
(1H, d, J 8.5, ArH), 8.77 (1H, s, triazole-H), 9.54 (1H, brs, OH),
10.38 (1H, brs, OH), 12.92 (1H, brs, COOH); 13C NMR dC (75.4
MHz, DMSO) 111.8, 114.8, 116.0, 116.6, 118.6, 123.1, 127.1,
128.3, 129.7, 129.8, 131.8, 146.0, 157.7, 158.3, 166.1. MS (ESI)
m/z: 297.98 [M + H]+.
2-Hydroxy-5-(4-(3-hydroxyphenyl)-1H-1,2,3-triazol-1-yl)ben-
zoic acid 3f. From tetrabutylammonium fluoride (1.11 mL, 1 M
solution in THF); chromatography eluent: AcOEt/hexane 5 : 1;
yield 68%, mp 243–244 uC with decomposition; nmax (KBr)/
1
cm21 3134, 2917, 1675, 1619; H NMR dH (300 MHz; DMSO)
6.78 (1H, d, J 7.7, ArH), 7.20 (1H, d, J 8.9, ArH), 7.27 (1H, t, J
7.9, ArH), 7.34–7.39 (2H, m, ArH), 8.07 (1H, dd, J 8.9 and 2.6,
ArH), 8.29 (1H, d, J 2.6, ArH), 9.23 (1H, s, triazole-H), 9.57 (1H,
brs, OH); 13C NMR dC (75.4 MHz, DMSO) 112.0, 113.9, 115.1,
116.2, 118.5, 119.5, 121.5, 127.2, 128.5, 129.9, 131.4, 147.2,
157.7, 160.7, 170.8. MS (ESI) m/z: 297.99 [M + H]+.
General method for the synthesis of methyl esters 2g, 2h and
3h
The corresponding acid (2e, 2f, or 3f) (0.17 mmol) was
dissolved in MeOH (10 mL) and few drops of sulfuric acid
were added. The reaction mixture was heated under reflux for
6 h in an argon atmosphere. Then the reaction mixture was
concentrated under vacuum and the resulting residue was
purified by column chromatography on silica using AcOEt/
DCM (3 : 1) as eluent to yield the desired product.
Methyl 5-hydroxy-2-(4-(4-hydroxyphenyl)-1H-1,2,3-triazol-1-
yl)benzoate 2g. Yield 73%, mp 224–225 uC; nmax (KBr)/cm21
3373, 3059, 2925, 1723, 1686, 1619; 1H NMR dH (300 MHz;
CD3OD) 3.74 (3H, s, OCH3), 6.00 (2H, brs, OH), 6.97 (2H, d, J
8.7, ArH), 7.21 (1H, dd, J 8.7 and 2.7, ArH), 7.51 (1H, d, J 2.6
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Biological assays
Agonist profile. The agonistic and antagonistic activities of
compounds were evaluated using a commercially available
cell-based assay (INDIGO Bioscience’s ER Reporter Assay),
which allows the quantification of functional activities of the
test compounds against ERa and ERb. The system utilizes non-
human mammalian cells engineered to provide constitutive
high-level expression of ERa and ERb. Additionally, these cells
contain either ERa- or ERb-responsive luciferase reporter gene.
Thus, quantification of luciferase activity provides a surrogate
measure of ERa and ERb activity in the treated reporter cells.
Reporter cells were dispensed in 96-well plates and then
immediately dosed with the test compounds. Following
overnight incubation, the treatment media were discarded
and luciferase detection reagent was added. The intensity of
light emission from the ensuing luciferase reaction provides a
measure that is directly proportional to the level of ER
activation in the reporter cells. The assays were configured
to perform agonist and antagonist dose–response curves. In
order to obtain agonist dose–response curves ER reporter cells
were treated with media alone. Estradiol was used as a positive
control agonist. The EC50 values of estradiol for ERa and ERb
were 330 and 77 pM, respectively. The highest concentrations
of estradiol used in order to ensure the saturating conditions
were 100 nM (ERa) and 4 nM (ERb). The final solvent control
did not exceed 0.1% of DMSO. All measurements were
performed in triplicate.
Chemicals. 17-b-Estradiol, crystal violet, dextran-coated
charcoal, PSB, Tween-20, BSA, insulin were purchased from
Aldrich. Estradiol [2,4,6,7,16,17-3H(N)], and scintillation
counting liquid (Optifase HiSafe2) were obtained from
Perkin-Elmer, Salem, MA). Estrogen receptors
a and b
produced in insect cells and sodium pyruvate were purchased
from Invitrogen. Cell culture medium DMEM, FBS, antibiotics,
trypsin-EDTA, amino acids, and L-glutamine were purchased
from Lonza. DCC-FBS was obtained from Hyclone
(Erembodegem, Aalst, Belgium) and DMEM without phenol
red from Gibco.
Receptor binding studies. The ability of the compounds to
bind to ERs has been determined by competition assay
according to the method of Arcaro13 with some modifications.
Purified full-length human ERa and ERb have been incubated
for 4 h at 23 uC with different concentrations of compounds in
the presence of 5 nM [2,4,6,7,16,17-3H]-estradiol in total
volume 150 ml. The stock of tested compounds has been
prepared in DMSO. All these compounds, including
[2,4,6,7,16,17-3H]-estradiol and receptors were diluted in
Tween/PBS buffer (99.85 : 0.15 w/v). A vehicle control con-
tained 0.1% of DMSO. After incubation the non-bound
[2,4,6,7,16,17-3H]-estradiol has been removed by adding
mixture of 10% DCC and 2% bovine serum albumin,
incubating 15 min at 4 uC, followed by centrifugation at
6 000 g for 5 min at 4 uC. 150 ml of supernatant was added to 4
mL of scintillation liquid and the bound estradiol was
measured in liquid scintillation counter Beckman LS 6500
(Beckman Coulter, Inc.). Two independent experiments with
three repetitions for each compound were performed. Results
were expressed as a the percentage of the specific binding of
[2,4,6,7,16,17-3H]-estradiol to ER versus the log of competitor
concentration. Graph Pad Prism software (non-linear regres-
sion analysis) was used to calculate the concentration needed
to displace 50% of [2,4,6,7,16,17-3H]-estradiol (IC50). The
values of IC50 for estradiol were 8.98 and 6.87 nM for ERa
and ERb, respectively.
Proliferation assay. MCF-7 cells were seeded in 96-well
plates at 5 6 103 cells/well in DMEM containing 10% FBS, 0.01
mg mL21 insulin solution, 0.1 mM non-essential amino acids.
After 24 h the medium was changed for DMEM without phenol
red, containing 5% dextran-coated charcoal stripped FBS
(DCC-FBS), 0.1 mM non-essential amino acids, 1 mM sodium
pyruvate and 2 mM L-glutamine and pre-incubated 3 days
prior to treatment. Afterwards different concentrations of
assayed compounds (1–50 mM) were added to cells with/
without 1 pM estradiol in order to test the capacity to induce
or prevent the proliferation of MCF-7 cells. The final vehicle
concentration maximally 0.1% of DMSO (and 0.1% of ethanol
in the case of treatment with estradiol) served as a solvent
control. On day 4 the medium with compounds was refreshed.
On day 8 the media were removed and cells were fixed and
stained with a solution containing 1% ethanol and 0.5%
crystal violet. After rinsing and drying, the dye was solubilized
with 1% SDS and the absorbance was read at 570 nm (Biotec).
The viability was calculated considering controls without test
substance as 100% viable.
Abbreviations
DCM
THF
DMF
RT
dichloromethane
tetrahydrofuran
dimethylformamide
room temperature
ER
estrogen receptor
CuAAC
copper-catalyzed reaction between an azide
and an alkyne
TLC
thin layer chromatography
triisopropylsilyl chloride
azidotrimethylsilane
central nervous system
trifluoroacetic acid
TIPSCl
TMSN3
CNS
TFA
EDTA
DCC-FBS
ethylenediaminetetraacetic acid
dextran-coated charcoal-stripped fetal
bovine serum
Acknowledgements
This work was supported by the Spanish Ministry of Science
and Innovation (SAF2008-00945, CTQ2011-24741 and
´
AGL2012-30803) and Fundacion Universitaria San Pablo CEU
(USP-PC 13/10 and 15/11). We thank EADS-CASA for fellow-
ships to K.F., M.M., J.C. and S.D.
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 3697–3706 | 3705

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