Rational design and optimization of selenophenes with basic side chains as novel potent selective estrogen receptor modulators (SERMs) for breast cancer therapy

Article abstract of DOI:10.1039/c7md00163k

To increase the diversity of estrogen receptor (ER) ligands having novel structures and activities, series of selenophene derivatives with a basic side chain (BSC) were synthesized and their biological activity as subtype-selective antagonists for the ER was explored. Compared with the selenophenes without a BSC, most compounds showed an increase in binding affinity, and several compounds displayed enhanced antagonist potency and antiproliferative activity. Especially, compound 16c exhibited excellent transcriptional activity for ERα (IC₅₀ = 13 nM) which made this compound the most potent antagonist for ERα of the whole series and is 66-fold better than the best selenophene compound without a BSC. Moreover, several compounds showed values of IC₅₀ better than that of 4-hydroxytamoxifen in breast cancer MCF-7 cells. The modeling study indicated that the basic side chain might contribute to their increased antagonist potency and antiproliferative activity. These new ligands have the potential to be further developed as novel agents to improve therapeutics that target the estrogen receptor.

Full text of DOI:10.1039/c7md00163k

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Rational Design and Optimization of Selenophenes with Basic Side
Chains as Novel Potent Selective Estrogen Receptor Modulators
(SERMs) for Breast Cancer Therapy†
Received 00th January 20xx,
Accepted 00th January 20xx
Junjie Luo†a, Zhiye Hu†a, Yuan Xiaoa, Tongxin Yanga, Chune Donga,c, Jian Huangb, Hai-Bing Zhoua,*
DOI: 10.1039/x0xx00000x
To increase the diversity of estrogen receptor (ER) ligands having novel structures and activities, series of selenophene
derivatives with a basic side chain (BSC) were synthesized and their biological activity as subtype-selective antagonist for
the ER was explored. Compared with the selenophenes without a BSC, most compounds showed an increase in binding
affinity, and several compounds displayed enhanced antagonist potency and antiproliferative activity. Especially
compound 16c exhibited excellent transcriptional activity for ERα (IC50 = 13 nM) which made this compound the most
potent antagonist for ERα of the whole series and is 66-fold better than the best selenophene compound without a BSC.
Moreover, several compounds showed the values of IC50 better than that of 4-hydroxytamoxifen in breast cancer MCF-7
cells. The modeling study indicated that the basic side chain might contribute to their increased antagonist potency and
antiproliferative activity. These new ligands have the potential to be further developed as novel agents to improve
therapeutics that target the estrogen receptor.
www.rsc.org/
antitumor, antiviral, antimicrobial, and antioxidant properties.15-17
Various biologically active selenaheterocycles such as ebselen have
been discovered in recent years.18 Furthermore, anticancer
Introduction
Selenium (Se) is an important nutritional trace element involved
in different physiological functions, such as antioxidative, antitumor
and chemopreventive properties. Dietary Se has been inversely
associated with the risk of cancer, and substantial evidence shows
that selenium has a significant influence on the incidence of many
cancers.1 Epidemiological studies reveal that low Se status may
contribute to the etiology of different diseases, for example, viral
infections, reproductive deficiencies, loss of immunocompetence,
thyroid and cardiovascular diseases, and pancreatitis.2 Selenium is
present in more than twenty five human selenoproteins, and most
of them have been involved in anti-oxidant defence systems and
cancer prevention etc.3-4 The anti-carcinogenic potential of Se has
also been reported in geographical studies during the last 40 years.5
A variety of selenium-containing compounds with diverse chemical
structures are known to inhibit cell proliferation in vitro, such as
mechanisms of MSeA have been hypothesized to involve estrogen
receptor (ER) stress signal mediators and apoptosis.19, 20
As we all known, estrogens are important regulators of many
physiological functions related to the reproductive and non
reproductive tissues in both women and men.21, 22 The effects of
estrogens are mediated via two estrogen receptor subtypes, ERα
and ERβ,23 which are ligand-regulated transcription factors that
regulate many physiological and pathological processes. The
estrogen receptors have different tissue distributions and
significant differences in their ligand binding preferences.24, 25 While
estrogens are necessarily benefit in some tissues, including
reproductive system,26 skeletal,27 cardiovascular,28 and central
nervous systems,29 inappropriate or over-expression of ER is
associated with a number of endocrine disorders. For example, the
estrogen receptors play a predominant role in breast cancer growth
because of the proproliferative effect.30 Thus, developing ER
subtype-selective ligands with tissue- and gene-selective biological
activities is a critical clinical objective. In order to discover “ideal”
selective estrogen receptor modulators (SERMs), extensive
investigation has been made to increase the chemical diversity of
these compounds, especially the non-steroidal ones. Apart from the
preserved peripheral substituents, e.g. phenols, simple alkyl groups,
and polar phenyl substituents, a wide variety of heterocyclic cores
have been explored,31 from the five-membered ring heterocycles to
a lesser extent six-membered ring heterocycles as well as fused
heterocycles. Some examples of the five-membered ring
heterocycles are presented below (Figure 1), including furan 1,31
inorganic
selenium
salts,6-11
selenoamino
acids,5
methylselenocyanate,12, 13 as well as benzyl and phenyl selenium
derivatives.14 In particular, many diarylselenides possess anticancer,
a Hubei Provincial Key Laboratory of Developmentally Originated Disease; Hubei
Province Engineering and Technology Research Center for Fluorinated
Pharmaceuticals; State Key Laboratory of Virology; Wuhan University School of
Pharmaceutical Sciences, Wuhan 430071, China
b College of Life Sciences, Wuhan University, Wuhan 430072, China
c Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
†These two authors contributed equally to this work.
* Corresponding author: E-mail: zhouhb@whu.edu.cn
Electronic Supplementary Information (ESI) available: [1H NMR and 13C NMR
spectra of final compounds]. See DOI: 10.1039/x0xx00000x
† The authors declare no compeꢀng interests.
This journal is © The Royal Society of Chemistry 20xx
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33
imidazole 2,32,
propyl pyrazole triol 3,34-36 thiophene 4,37 act as models with improved biological activity for the development
selenophene 538 and raloxifene (RAL) etc. Because activity profiles of novel estrogen receptor ligands. It was proved that several
of the current SERMs e.g. tamoxifen are not ideal and resistance to compounds (e.g. 16c) showed the value of IC50 better than that of
their effectiveness as antitumor agents can develop with time 4-hydroxytamoxifen in breast cancer MCF-7 cells.
(Figure 1), there has been interest in finding new SERMs that might
prove more effective as hormonal or therapeutic agents.
Figure 1. Examples of the five-membered ring heterocycles 1-5 as
ER ligands, structures of known SERMs Raloxifene and 4-
Figure 2. Rational for design of novel potent selenophenes with a
Hydroxytamoxifen.
BSC for breast cancer therapy.
As part of our long-term interest in the development of ligands
40
for the ERs having novel structures and activities,39, we have
2. Results and Discussion
2.1. Chemical Synthesis
recently described a new series of analogues based on selenohene
Three series of novel selenophene derivatives, as depicted in
scaffold that showed good binding affinity to ERs. The 2,5-bis(2-
Schemes 1, were prepared from selenophenes according to the
fluoro-4-hydroxylphenyl)selenophene 8c showed the highest
synthetic procedures established in our laboratory (described in the
relative binding affinity (RBA, Estradiol = 100) of 24.3 for ERβ. In
Experimental Section).
transcription assays, most of selenophenes exhibited partial to full
In the synthesis of compounds 9a-g (Scheme 1A), key
agonist activity for both ER subtypes, but several compounds
displayed a range of ERα or ERβ antagonistic activities.38 A few
intermediates 7a-g were obtained using the Suzuki cross-coupling
reaction of aryl boronic acids with 2,5-disubstituted selenophene
6a.38 42 Then, compounds 7a-g were treated with boron tribromide
selenophenes exhibited antiproliferative activity comparable to that
of 4OHT in breast cancer MCF-7 cells. Interestingly, from the
affording the corresponding 2,5-disubstituted diphenolic
modeling study of the complex of ERα-8c, it is observed that one of
selenophenes 8a-g. Finally, the target products 9a-g were obtained
phenolic group of 8c interacts with helix 11 through H-bonds and
from corresponding phenols and chloroethyl pyrrolidine or
does not further destabilize helix 12; rather, it mimics the role of
piperidine by Williamson ether synthesis under microwave
the D-ring phenol of E2 and stabilizes helix 12 in the agonist
conditions. This step produced
a mixture of monoalkylated
conformation.
products 9a-g (yield 60-70%) and was accompanied by about 30-
40% of dialkylated byproducts (Scheme 1A).
Therapeutic targeting of the estrogen receptor has traditionally
involved direct disruption of the surface coactivator binding sites by
a basic side chain (BSC) that is a characteristic structural feature of
SERMs, such as tamoxifen and raloxifene (Figure 1). The nature and
spatial orientation of the basic side chains in SERMs can influence
their tissue selectivity and affect the balance of desired and
undesired activities.41 Thus, we wondered whether the introduction
of basic side chains into selenophene-based core structure (e.g., a
pyrrolidine or piperidine side chain) might provide ER ligands with
interesting antagonistic activities (Figure 2). Herein, we introduced
two different types of aminoethoxy moieties into the selenophene
core system, placing these at different positions of the phenolic
groups. We expected that these selenophene-core derivatives could
In the synthesis of 3,4-disubstituted selenophene derivatives
12a-i (Scheme 1B), 10a-i served as the key intermediates, which
were prepared by treating 3,4-dibromoselenophene 6b38 with aryl
boronic acids by using Pd(OAc)2/PPh3 as the catalyst. Subsequent
ether cleavage of 10a-i by pyridine hydrochloride yielded the
intermediates 11a-i. Finally, treatment of intermediates 11a-i with
chloroethyl pyrrolidine or piperidine afforded the desired products
12a-i in good yields.
In our previous work on furan or thiophene derived ER ligands,37
triphenol furans and thiophenes were proved to be more effective
than the corresponding bisphenol analogues with higher binding
affinity and subtype selectivity. In particularly,
a few triaryl
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selenophenes exhibited substantial antiproliferative activity in
breast cancer MCF-7 cells.38 Thus, we wanted to introduce a basic 2.2. Relative Binding Affinities.
side chain into the triphenol selenophene scaffold, as such
selenophenes 16a-c were obtained by demethylation of 15a-c,
which were prepared through Suzuki cross-coupling reactions
followed by installation of a BSC (Scheme 1C). In the first step, 1
equiv of 2,3,5-tribromoselenophene 6c38 was reacted with 4 equiv
of aryl boronic acid under standard conditions, and the resulting
2,5-bis-substituted selenophenes 13 were subsequently submitted
to cross-coupling reaction with 2 equiv of phenyl boronic acid to
yield the intermediates 14a-c. Installation of the two types of basic
side chains in the free phenolic positions in 14a-c was effected by a
Williamson reaction, and the remaining methyl group was then
selectively cleaved with boron tribromide, leaving the basic side
chain unaffected. By this approach, we prepared the analogues
containing the two types of basic side chains at the para position of
the C-3 phenyl group, 16a-16c.
The binding affinities of selenophene compounds for both ERα
and ERβ were determined by a competitive fluorometric receptor-
binding assay and are summarized in Table 1. These affinities are
presented as relative binding affinity (RBA) values, where estradiol
(E2) has an affinity of 100%.
As a global observation, it is noteworthy that the position of basic
side chains in the phenyl ring of selenophene derivatives has very
significant effects on the binding affinity of conjugates. In general,
most of the compounds in series I (except 9c and 9f) that possess
the basic side chain in the 2-phenol moiety exhibit better binding
affinity for ERα. On the contrary, the vast majority of series II (apart
from 12b and 12c) that located the basic side chain in the 3-phenol
moiety display moderate levels of ERβ selectivity. The compound
that shows the highest binding affinity for ERβ and moderate ER
subtype selectivity of all of ligands is 9f, which possesses a basic
side chain on the 2-phenol unit. The RBA values of this compound
are 4.59 and 23.6 for ERα and ERβ, respectively; and it has an
ERα/ERβ selectivity as low as 0.19 (Table 1, entry 6). Compared to
the
parent
compound
2,5-bis(2-chloro-4-hydroxylphenyl)-
selenophene38 (RBA values were 6.11 for ERα and 12.7 for ERβ; α/β
was 0.48), 9f still retained high binding affinity for ERβ and
displayed higher selectivity. The compound that has the highest
ERα/ERβ selectivity is 12h (ERα/ERβ ratio < 0.1), which also shows
high RBA value for ERβ (23.3). As we expected, these halogens, Cl
and F, bind better than the other ligands, with Cl being better than
F in most cases (9e, 9f, and 12g-i).
Compared to the diphenolic selenophene compounds previously
reported by our group, the introduction of the basic side chains can
alter the ER subtype selectivity through changing substitution
position. Moreover, introduction of substituent in the phenol rings,
has significant effects on the binding affinity and selectivity. Among
the series I, the nonsubstituted compound 9a showed a good
binding affinity (12.4 and 1.02 for ERα and ERβ respectively). When
the substitution occurred at meta position of the phenol ring (9b-
9g), moderate affinity, ranging from 0.1 to 23.6 was observed.
Interestingly, most of the compounds in series I show better
selectivity for ERα, and when basic side chains were introduced at
C-3 position of the selenophene ring (series II), most of the
compounds show good selectivity for ERβ (Table 1, entries 8-16).
For the disubstituted selenophene derivatives, when the
substitution occurred at 3- and 4-positions (12a-12i), lower binding
affinities were observed. Similar trend was also observed for furan
and thiophene derivatives.38 Nevertheless, it should be pointed out
that these compounds also show a moderate affinity for ERβ.
Moreover, the binding affinities and selectivities for ERβ
dramatically changed when the substitution was moved from C-2
position to C-3 position (12c-12f, entries 10-13). For comparison,
trisubstituted selenophene analogues with basic side chain were
also prepared (series III), except 16b with undetectable ERβ binding
affinity of less than 0.1, all showed good binding affinity for both
ERα and ERβ (entries 17-19). The desired compound showing
Scheme 1. Synthesis of substituted selenophene derivatives.
Reagents and conditions: (a) [Pd] catalyst, Na2CO3, toluene/water
(1:1), reflux, 24 h; (b) BBr3, CH2Cl2, -20 °C to rt, 4 h; (c) N-(2-
chloroethyl)pyrrolidine or N-(2-chloroethyl)piperidine, KOH, K2CO3,
DMF, microwave, 120 °C, 45 min; (d) Pyridine hydrochloride, 190 °C,
3 h.
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highest binding affinity for ERα was 16c (13.8 for ERα), which also
displayed a moderate ER subtype selectivity.
Table 1. Relative Binding Affinity (RBA) of compounds 9a-g, 12a-i,
16a-c for ERα and ERβα.
2.3. Transcription Activation Assays
The effects of these selenophene derivatives on ER
transcriptional activity were determined using an ER responsive
luciferase reporter gene. HEK 293 cells were seeded in 24-cell plates
at a concentration of 2 × 106 cells/plates and allowed 24 hours to
settle. Then the cells were transfected with a widely used 3 × ERE-
luciferase reporter and an ERα or ERβ expression plasmid for
agonist activity (% efficacy) and potency (EC50) determinations.
These cells were stimulated with increasing concentrations of 17β-
estradiol (E2) or these compounds. For antagonist mode assays (%
efficacy and IC50), cells were stimulated with a combination of
estradiol (10 nM) and an increasing concentration of the various
selenophene compounds. The luciferase activity was measured next
day. These results are summarized in Table 3, and dose-response
curves for representative samples are shown in Figure 3.
aFor simplify comparisons the compounds in related series, we
designated locant positions of the substituents on the phenyl
groups with respect to the selenophene core; locant positions on
the selenophene core itself are given by numbers in italics. bRelative
Binding Affinity (RBA) values are determined by competitive
estradiol
fluorometric binding assays and are expressed as IC50
/
compound
IC50
× 100 ± the range (RBA, estradiol = 100%). In these
assays, the Kd value of estradiol is 3.1 nM for ERα and 3.4 nM for
ERβ, respectively. For details, see Experimental Section.
Overall, we found that the compounds synthesized by N-(2-
chloroethyl)pyrrolidine might display a better binding affinity for ER
than those obtained from N-(2-chloroethyl)piperidine.
Compared with our previous work, it was interesting to find that
most of the biaryl substituted selenophene compounds with basic
side chains exhibited an equivalent or better binding affinity for ER
than the corresponding parent selenophenes except 9b (Table 2).
Table 2. A direct comparison of RBA values to ERα and ERβ for the
various biaryl selenophene compounds with a BSC in this work and
the corresponding ones without a BSC studied previously.38
Figure 3. Illustrative dose-response curves for the ERα antagonist
effects of 4TOH, and two selenophene-core compounds 12g and
16c. Efficacy values are the mean ± SD from three experiments. For
details, see the Experimental Section.
The interesting activities are seen in compounds of series I, 2,5-
disubstituted selenophenes 9a-g. These ligands displayed a wide
range of activities at both ERα and ERβ, most of which show potent
and highly efficacious ERα agonists and ERβ antagonists
respectively. Compound 9a with pyrrolidinyl side chain was a weak
agonist of ERα and antagonist of ERβ with nanomolar to micromolar
range IC50, however, addition of a methyl group to 9a converted it
from agonist into antagonist for ERα and a strong antagonist for
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ERβ (9b, IC50 = 0.24 and 0.146 μM for ERα and ERβ, respectively). binding affinity and antiproliferative potency should be
Compared with 9b, compound 9c prepared by N-(2-chloroethyl) independent. Furthermore, introduction of the basic side chains in
piperidine didn’t have obvious influence on the activity (Table 3, compounds demonstrated more promising antiproliferative activity.
entries 2 and 3). Moreover, replacing the methyl group (9b) with a
halogen group also had significant effects on the transcriptional
activity of the ER subtypes. The fluorine-substituted compounds 9d
and 9e profiled as ERα agonist, being about 4 to 6-fold more potent
than 9b, whereas the chloro analogues 9f and 9g had little effect on
ERα (Table 3, entries 4, 5 and 6, 7). However, when the halogen
group was introduced into these compounds, the antagonistic
activity for ERβ significantly increased (Table 3, entries 4 and 6).
Comparisons of the position of basic side chains in selenophene
core indicate that 3,4-disubstituted compounds (series II) with
decreased ERα binding affinity (Table 1, 12a-i), which also
demonstrate decreased efficacy as ERα agonists in general (Table
3). Interestingly, these 3, 4-disubstituted ligands show improved
ERβ binding affinity, also display increased potency as ERβ
antagonist, whereas 12b profiled as the moderate ERβ agonist with
μM range IC50 but had low efficacy. When the methyl group was
changed from the C2- to C3-position (12e, 12f), these compounds
profiled as antagonist on ERβ, and were more efficacious compared
to 12c and 12d (Table 3, entries 12 vs 10; 13 vs 11). It was
remarkable that 12e could act as a potent ERβ antagonist with a
nanomolar IC50 value (5 nM).
Addition of a third substituent onto the selenophene core also
having drastic effects on transcriptional activity. We prepared three
compounds 16a-c that showed potent and highly efficacious ERα
and ERβ antagonists, especially for compound 16c, which gave the
low IC50 values of 0.013 and 0.016 μM for ERα and ERβ,
respectively. This makes 16c the most potent antagonist for ERα of
the whole series, and the transcription activity of 16c is 66-fold
better than the best compound in selenophenes without a BSC.38
The trend keeps consistent with the RBA of these compounds.
Again, there was also a trend that most compounds with pyrrolidine
side chains displayed better activities than those with piperidine
side chains.
2.4. Antiproliferative activity on breast cancer cells. To evaluate
antiproliferative activity of this type of compounds, all
selenophene-core compounds were screened against MCF-7, and
the results were summarized in Table 3.
Overall, most of selenophene derivatives were effective in
inhibiting the MCF-7 breast cancer cells. Among them, six
compounds, 9a, 12h-i and 16a-c, exhibited potent antiproliferative
activity with the value of IC50 better than that of 4-
hydroxytamoxifen in breast cancer MCF-7 cells. Generally, the
compounds that displayed potent and highly efficacious
antiproliferative activity on MCF-7 breast cancer cells, also showed
moderate to good binding affinities for ERα and ERβ; meanwhile,
those two compounds (9g and 12f) had low antiproliferative activity
due to their unobvious RBA values (less than 1%). However, the
compounds 9d, 9e, and 12a possessing high binding affinities
showed weak inhibition for MCF-7 cells, which suggested that the
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Table 3. Effects of selenophene-core compounds on the transcriptional activities of Estrogen Receptors α and β and antiproliferative activity in MCF-7 cells a.
Agonist Modeb
Antagonist Modec, d, e
MCF-7
R
n
ERα
EC50 (μM)
ERβ
ERα
ERβ
IC50 (μM)
IC50 (μM)e
Entry
cmpd
9a
Eff (%E2)
EC50 (μM)
Eff (%E2)
IC50 (μM)
Eff (%E2)c
Eff (%E2)
83.9 ± 7.7
4.6 ± 15
1
2
H
1
0.219
-
15.3 ± 4.8
-
-
1.213
0.874
0.041
-
71.5 ± 16.7
1.266
0.146
1.235
1.120
1.945
0.299
2.96
5.77 ± 0.06
53.8± 4.24
25.3 ± 0.55
>100
9b
2-Me
1
-
0.24
72.3 ± 12.5
78.3 ± 27.8
3
9c
2-Me 2
2.369
0.136
0.204
-
11.2 ± 17.2
-
-
52.3 ± 7.7
78.3 ± 29.4
92.1 ± 10.9
75.4 ± 4.2
49.4 ± 11.1
24.9 ± 14.4
46.7 ± 12.3
22.8 ± 8.9
66.7 ± 3.6
4
9d
2-F
2-F
2-Cl
2-Cl
H
1
2
86.9 ± 26.6
-
-
-
5
9e
68.4 ± 13.5
-
-
-
-
>100
6
9f
1
-
-
-
0.415
-
69.7 ± 21.9
7.81 ± 0.13
>100
7
9g
2
0.911
1.003
-
19.9 ± 5.0
-
-
-
8
12a
12b
12c
1
45.9 ± 6.3
-
1.147
-
-
-
-
0.102
0.451
0.043
>100
9
H
2
-
-
53.5 ± 11
-
1.589
0.179
89.2 ± 8.3
21.4 ± 1.7
8.13 ± 0.46
16.6 ± 1.34
10
2-Me
1
-
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84.6 ± 21.3
11
12
13
14
15
16
17
18
19
20
12d
12e
12f
2-Me
3-Me
3-Me
2-F
2
1
2
0.158
23.6 ± 19.7
-
-
-
-
-
-
-
-
-
-
-
0.237
0.836
-
1.335
0.005
0.415
0.54
43.4 ± 10.2
34.1 ± 5.8
30.3 ± 12.2
45.0 ± 1.2
68.1 ± 19.6
37.7 ± 11.0
49.2 ± 9.9
33.8 ± 14.2
170.0 ± 1.0
-20 ± 2
15.8 ± 0.98
12.8 ± 2.40
>100
0.116
36.5 ± 4.9
20.8 ± 3
87.4 ± 5.6
-
0.721
30.3 ± 12.2
-
12g
12h
12i
2
-
-
-
0.069
0.769
0.413
0.974
0.779
0.013
0.003
15.8 ± 2.6
38.5 ± 16.4
77.8 ± 25.7
56.2 ± 9.4
91.2 ± 10.4
24.8 ± 4.5
35 ± 3
15.6 ± 2.41
10.9 ± 1.12
14.1 ± 2.27
4.95 ± 0.26
7.04 ± 0.49
7.05 ± 0.82
15.6 ± 1.77
2-Cl
2-Cl
H
1
2
-
-
-
1.159
0.331
0.475
5.391
0.016
0.001
-
-
-
16a
16b
16c
1
-
-
9.2 ± 3.3
-
-
H
2
1.831
-
-
-
2-Me
1
4OHT
0.008
35 ± 3
-1 ± 1
aLuciferase activity was measured in HEK293T cells transfected with 3 × ERE-driven luciferase reporter and expression vectors encoding ERα or ERβ and treated in triplicate with increasing
b
c
doses (up to 10-5 M) of the compounds. EC50 and standard deviation (mean ± SD), shown as a percentage of 10-8 M 17β-estradiol (E2), were determined. IC50 and standard deviation (mean ±
SD) were determined in the percentage of 10-8 M 17β-estradiol (E2) on ERα or ERβ. ERs have considerable basal activity in HEK293T cells; compounds with inverse agonist activity are given
negative efficacy values. Omitted EC50 or IC50 values were too high to be determined accurately; Omitted Eff (%E2) values were too low to be accurately determined. eIC50 values are an average
d
of at least three independent experiments ± standard deviation (mean ± SD).
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In order to determine that the antiproliferative activity of
2.5. Structural analysis of the origin of the enhanced antagonist
character of these ligands.
these compounds arises from their antiestrogenicity on ER, the
cells were incubated with 50 μM of 9a, 12b and 16a in the
presence and absence of 10 nM E2. As shown in Figure 4, after
incubation with different doses of ligands, relative cell activities
decreased substantially. Exogenous estradiol could also restore
part of the MCF-7 cells, which demonstrated that these
selenophene derivatives inhibited the MCF-7 cells by interacting
with ER. As such, those potent compounds in suppressing the
proliferation of MCF-7 cell showed moderate to excellent
antiproliferative activity, which might be attributed to their
antagonistic potency on ER.
Estradiol supports transcriptional activation of ERα and ERβ
by stabilizing helix 12 in a position where it forms a hydrophobic
groove for binding transcriptional coactivators. The traditional
SERMs or full antagonists have typically been developed by
adding a bulky side group that directly obstructs the agonist
position of helix 12, relocating it out of this position and thereby
blocking the recruitment of transcriptional coactivators.43
Consistent with this model, we find that 11c (Figure 5A) can
similarly form a hydrogen bond with residue Glu353 on helix 3
and residue Arg394 on helix 6 respectively.44 In contrast, the
SERM with a close structure, raloxifene (Figure 5C), has a bulky
side chain that projects between helices 3 and 11, directly
displacing helix 12 from its active conformation and destroying
the transcriptional coactivator binding site; moreover, for
compound 12e (Figure 5B) mimics the binding orientation of
raloxifene, with the BSC on one of the phenol group directly
interacting with any helix 12 residues, which is consistent with
raloxifene, thus giving 12e potent ERα antagonist activity. For
triaryl substituted selenophene 16c, the ligand is consistent with
raloxifene: the similar hydrogen bonds and directly displacing
helix 12 with BSC to those of raloxifene and 12e, the second
phenolic hydroxy of 16c also forms hydrogen bond with the
residue His524 on helix 11 (Figure 5D), which makes the 16c the
most potent antagonist for ERα of the whole series.
Figure 4. Effect of E2, 9a, 12b and 16a on the proliferation of
hormone-dependent breast cancer MCF-7 cells after 5 days of
culture. Nontreated MCF-7 cells are used as the control set at
100 %. Mean of three separate experiments ± range.
Figure 5. Model of selenophene ligands 11c, 12e and 16c bound to ERα and comparisons with raloxifene. (A) Computer-developed
model of 11c bound to the ERα (PDB: 1ERR). The phenolic hydroxyl group of 11c forms hydrogen bonding interactions with residue
Glu353 on helix 3 and residue Arg394 on helix 6 respectively. (B) Computer-developed model of 12e bound to the ERα with the
conserved H-bonds to Glu353 and Arg394 residues. The basic side chain of the 6e is oriented toward helix 12. (C) Crystal structure of
the ERα LBD in complex with raloxifene. Raloxifene forms H-bonds to the conserved Glu353, Arg394, Asp351, and His524 residues. The
basic side chain displaces helix 12. (D) Computer-developed model of the ERα LBD in complex with 16c. Consistent with the complex of
raloxifene, the second phenol forms a hydrogen bond with the residue His524 on helix 11, and the basic side chain displaces helix 12.
8 | J. Name., 2012, 00, 1-3
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Journal Name
3. Conclusions
reaction mixture was poured into water, and extracted with ethyl
Therapeutic targeting of the estrogen receptor has traditionally acetate. The extracts were dried (Na2SO4) and evaporated. The
involved direct disruption of the surface coactivator binding sites by residue was purified by silica gel column chromatography (CC).
a basic side chain that is a characteristic structural feature of
Method B: a mixture of methoxyphenyl selenophene derivative
SERMs, such as tamoxifen and raloxifene. In this study, we (1 equiv) and pyridine hydrochloride (10 equiv) was stirred at 190 oC
sequentially introduced different basic side chains into the for 3 h, after which, the reaction mixture was cooled to room
selenophene scaffold to form new subtype-selective antagonists for temperature. The aqueous layer was extracted with ethyl acetate.
the estrogen receptor. Interestingly, most of the compounds The combined organic layers were washed with dilute hydrochloric
displayed good binding affinity and increased antagonistic activity acid, dried over anhydrous Na2SO4 and then filtered and
for both ERs in comparison with the parent selenophenes without a concentrated in vacuum. The product was purified by column
BSC. Several compounds showed the value of IC50 better than that chromatography (CC).
of 4-hydroxytamoxifen in breast cancer MCF-7 cells. The
General procedure for the installation of Basic Side Chain by
preliminary mechanistic study indicated the antiproliferative Williamson ether synthesis. To a mixture of phenolic selenophene
activity arises from their antiestrogenicity; the modeling study derivative (1 equiv) and N-(2-chloroethyl)pyrrolidine or N-(2-
showed that the introduction of basic side chain had a significant chloroethyl)piperidine (1.1 equiv) in DMF was added K2CO3 (4
effect on antiproliferative activity of these compounds via directly equiv) and KOH (4 equiv). The reaction mixture was stirred for 45
interacting with helix 12. These new ligands could act as models for mins under microwave conditions. The reaction mixture was poured
the development of novel agents to improve therapeutics that into water, and extracted with ethyl acetate. The extracts were
target the estrogen receptor.
dried (Na2SO4) and evaporated. The residue was purified by silica
gel column chromatography (CC).
4. Experimental Section
4.1. Chemistry
2-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-5-(4-hydroxylphenyl)-
selenophene 9a. Compound 9a was prepared by 2,5-bis(4-
hydroxylphenyl)selenophene (8a) and N-(2-chloroethyl)pyrrolidine
according to general procedure for Williamson ether synthesis.
Purification by CC (petroleum ether:ethyl acetate = 1:1) gave the
title compound as a yellow solid (65% yield; mp 89-92 °C); 1H NMR
(400 MHz, Acetone-d6) δ 7.97 (dd, J = 10.2, 2.8 Hz, 2H), 7.07 (d, J =
8.8 Hz, 2H), 7.01 – 6.95 (m, 2H), 6.82 (d, J = 8.8 Hz, 2H), 6.72 (t, J =
10.1 Hz, 2H), 4.15 – 4.04 (m, 2H), 2.90 (t, J = 5.9 Hz, 2H), 2.65 (t, J =
6.0 Hz, 4H), 1.82 – 1.71 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ
157.69, 156.76, 143.96, 143.58, 131.09, 130.26, 129.37, 129.29,
129.01, 127.71, 115.40, 114.48, 66.62, 54.65, 54.47, 23.54. HRMS
(ESI) calcd for C22H23NO2Se [M + H]+, 414.0972; found 414.0976.
4.1.1. General
Starting materials were purchased from Aldrich, Acros, Aladin-
reagent, and Alfa-Asar and were used without purification. Toluene
was freshly distilled from sodium, and dichloromethane was
distilled from anhydrous CaH2. Glassware was oven-dried,
assembled while hot, and cooled under an inert atmosphere. Unless
otherwise noted, all reactions were conducted in an inert
atmosphere. All reactions were performed under an Ar atmosphere
unless otherwise specified. Reaction progress was monitored by
analytical thin-layer chromatography (TLC). Visualization was
achieved by UV light (254 nm).
1H NMR and 13C NMR spectra were measured on a Bruker
AVANCE III 400 (400 MHz, 1H NMR; 101 MHz, 13C NMR) instrument.
Chemical shifts are reported in ppm (parts per million) and are
referenced to either tetramethylsilane or the solvent. Melting
points were determined on a X-4 Beijing Tech melting point
apparatus, and the data were uncorrected.
2-(2-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5-(2-methyl-4-
hydroxylphenyl)selenophene 9b. Compound 9b was prepared by
2,5-bis(2-methyl-4-hydroxylphenyl)selenophene (8b) and N-(2-
chloroethyl)pyrrolidine according to general procedure for
Williamson ether synthesis. Purification by CC (petroleum
ether:ethyl acetate = 1:1) gave the title compound as a yellow solid
(67% yield; mp 82-86 °C); 1H NMR (400 MHz, Acetone-d6) δ 7.30 (d,
J = 8.5 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 7.10 (dd, J = 7.9, 3.8 Hz, 2H),
General procedure for Suzuki Coupling. Under Ar atmosphere, a
mixture of bromoselenophene (1 equiv), arylboronic acid (3 equiv
for disubstituted, 4 equiv for trisubstituted selenophenes), Pd
catalyst, sodium carbonate (2 equiv) in an oxygen-free 6.85 (d, J = 2.4 Hz, 1H), 6.79 – 6.75 (m, 2H), 6.71 (dd, J = 8.3, 2.4 Hz,
1H), 4.14 (dd, J = 7.7, 4.2 Hz, 2H), 2.92 – 2.88 (m, 2H), 2.67 – 2.62
(m, 4H), 2.41 (s, 3H), 2.36 (s, 3H), 1.75 (dd, J = 6.8, 3.2 Hz, 4H). 13C
NMR (101 MHz, Acetone-d6) δ 159.31, 158.18, 150.15, 149.24,
137.57, 137.50, 132.38, 132.28, 129.58, 129.27, 129.02, 128.09,
118.43, 117.69, 114.01, 112.81, 67.68, 55.49, 55.15, 24.20, 21.80,
21.70. HRMS (ESI) calcd for C24H27NO2Se [M + H]+, 442.1285; found
442.1286.
toluene/water (1:1) solution was stirred at 120 °C for 24 h, after
which, the reaction mixture was cooled to room temperature. The
aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4 and then filtered and concentrated in vacuum. The product
was purified by column chromatography (CC).
General procedure for ether cleavage. Method A: under Ar
2-(2-Methyl-4-(2-(piperidin-1-yl)ethoxy)phenyl)-5-(2-methyl-4-
hydroxylphenyl)selenophene 9c. Compound 9c was prepared by
2,5-bis(2-methyl-4-hydroxylphenyl)selenophene (8b) and N-(2-
chloroethyl)piperidine according to general procedure for
atmosphere, to
a solution of methoxyphenyl selenophene
derivative (1 equiv) in dry dichloromethane, boron tribromide (3
equiv per methoxy function) was added dropwise at -20 °C. The
mixture was allowed to stir for 4 h, and quenched with MeOH. The
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Journal Name
Williamson ether synthesis. Purification by CC (petroleum 6.93 (m, 2H), 6.82 (dt, J = 4.6, 2.3 Hz, 1H), 4.11 (t, J = 5.7 Hz, 2H),
2.81 (t, J = 5.8 Hz, 2H), 2.58 – 2.52 (m, 4H), 1.70 – 1.63 (m, 4H).13C
NMR (101 MHz, DMSO-d6) δ 158.27, 157.95, 145.89, 144.80,
ether:ethyl acetate = 1:1) gave the title compound as a yellow solid
1
(65% yield; mp 156-158 °C); H NMR (400 MHz, Acetone-d6) δ 7.30
131.55, 131.36, 131.11, 130.92, 128.95, 128.53, 126.51, 124.72,
116.71, 115.84, 115.18, 114.37, 66.96, 53.90, 53.83, 23.05. HRMS
(t, J = 6.8 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.12 (q, J = 3.8 Hz, 2H),
(ESI) calcd for C22H21Cl2NO2Se [M + H]+, 482.2830; found 482.2830.
6.86 (d, J = 2.5 Hz, 1H), 6.79 (dt, J = 5.5, 2.8 Hz, 2H), 6.72 (dd, J = 8.3,
2.6 Hz, 1H), 4.12 (s, 2H) , 2.72 (t, J = 6.0 Hz, 2H), 2.50 (s, 4H), 2.42 (s,
3H), 2.38 (s, 3H), 1.56 (dt, J = 10.9, 5.6 Hz, 4H), 1.42 (dd, J = 11.1, 6.0
Hz, 2H). 13C NMR (101 MHz, Acetone-d6) δ 159.41, 158.01, 150.07,
2-(2-Chloro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-5-(2-chloro-4-
hydroxylphenyl)selenophene 9g. Compound 9g was prepared by
149.31, 137.56, 136.73, 132.39, 132.26, 129.50, 129.26, 129.06,
2,5-bis(2-fluoro-4-hydroxylphenyl)selenophene (8d) and N-(2-
128.23, 118.36, 117.70, 113.94, 112.86, 66.85, 58.69, 55.74, 26.76,
chloroethyl)piperidine according to general procedure for
25.05, 21.77, 21.66. HRMS (ESI) calcd for C25H29NO2Se [M + H]+,
Williamson ether synthesis. Purification by CC (petroleum
454.1450; found 454.1452.
ether:ethyl acetate = 1:1) gave the title compound as a yellow solid
(65% yield; mp 92-95 °C); 1H NMR (400 MHz, DMSO-d6) δ 7.59 (d, J =
2-(2-Fluoro-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5-(2-fluoro-4-
8.7 Hz, 1H), 7.54 – 7.45 (m, 3H), 7.16 (d, J = 2.5 Hz, 1H), 7.01 – 6.94
hydroxylphenyl)selenophene 9d. Compound 9d was prepared by
(m, 2H), 6.83 (dt, J = 11.9, 5.9 Hz, 1H), 4.15 – 4.04 (m, 2H), 2.65 (t, J
2,5-bis(2-fluoro-4-hydroxylphenyl)selenophene (8c) and N-(2-
= 5.8 Hz, 2H), 2.42 (s, 4H), 1.48 (dd, J = 10.8, 5.4 Hz, 4H), 1.37 (d, J =
4.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 158.40, 157.91, 145.84,
chloroethyl)pyrrolidine according to general procedure for
Williamson ether synthesis. Purification by CC (petroleum
144.83, 131.60, 131.36, 131.09, 130.91, 128.97, 128.58, 126.43,
ether:ethyl acetate = 1:1) gave the title compound as a yellow solid
124.74, 116.68, 115.87, 115.18, 114.50, 66.10, 57.13, 54.28, 25.48,
(70% yield; mp 162-165 °C); 1H NMR (400 MHz, CD3OD) δ 7.65 (t, J =
8.7 Hz, 1H), 7.56 (dd, J = 5.5, 3.2 Hz, 2H), 7.52 (dd, J = 4.3, 1.6 Hz,
1H), 6.87 (dd, J = 8.0, 6.0 Hz, 2H), 6.65 (ddd, J = 15.5, 10.8, 2.5 Hz,
2H), 4.21 (t, J = 5.5 Hz, 2H), 3.05 (t, J = 5.4 Hz, 2H), 2.80 (t, J = 6.7 Hz,
4H), 1.95 – 1.85 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 159.33 (d,
J = 247.1 Hz), 159.06 (d, J = 247.3 Hz), 141.32 (d, J = 29.0 Hz), 140.02
(d, J = 29.7 Hz), 129.17 (d, J = 12.1 Hz), 129.01 (d, J = 11.4 Hz),
127.35, 126.71, 116.05, 114.37, 113.07, 112.33, 103.67, 103.42,
103.04, 102.78, 67.84, 54.56, 54.42, 23.60. HRMS (ESI) calcd for
C22H21F2NO2Se [M + H]+, 450.0784; found 450.0781.
23.84. HRMS (ESI) calcd for C23H23Cl2NO2Se [M + H]+, 496.0349;
found 496.0341.
3,4-Bis(2-chloro-4-methoxylphenyl)selenophene 10e. Compound
10e was prepared by 3,4-dibromoselenophene (6b) and 2-chloro-4-
methoxylphenylboronic acid according to general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
1
98:2) gave the title compound as a yellow solid; H NMR (400 MHz,
CDCl3) δ 7.71 (s, 2H), 7.09 (t, J = 14.5 Hz, 2H), 6.74 (dt, J = 17.4, 14.8
Hz, 2H), 6.67 – 6.56 (m, 2H), 3.77 – 3.63 (m, 6H).
2-(2-Fluoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-5-(2-fluoro-4-
3,4-Bis(2-chloro-4-hydroxylphenyl)selenophene 11e. Compound
hydroxylphenyl)selenophene 9e. Compound 9e was prepared by 11e
was
prepared
by
3,4-bis(2-chloro-4-
2,5-bis(2-fluoro-4-hydroxylphenyl)selenophene (8c) and N-(2- methoxylphenyl)selenophene (10e) using boron tribromide
chloroethyl)piperidine according to general procedure for according to general procedure for ether cleavage. Purification by
Williamson ether synthesis. Purification by CC (petroleum CC (petroleum ether:ethyl acetate = 4:1) gave the title compound as
1
ether:ethyl acetate = 1:1) gave the title compound as a yellow solid a yellow solid; H NMR (400 MHz, Acetone-d6) δ 8.00 (d, J = 4.9 Hz,
1
(65% yield; mp 158-162 °C); H NMR (400 MHz, DMSO-d6) δ 10.38 2H), 7.01 – 6.93 (m, 2H), 6.82 (d, J = 2.5 Hz, 2H), 6.71 – 6.63 (m, 2H).
(s, 1H), 7.76 – 7.68 (m, 1H), 7.68 – 7.58 (m, 2H), 6.99 (dd, J = 13.6,
2.5 Hz, 1H), 6.86 (dd, J = 8.8, 2.4 Hz, 2H), 6.75 – 6.64 (m, 2H), 4.11 3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-4-(4-hydroxylphenyl)-
selenophene 12a. Compound 12a was prepared by 3,4-bis(4-
hydroxylphenyl)selenophene (11a) and N-(2-chloroethyl)pyrrolidine
according to general procedure for Williamson ether synthesis.
Purification by CC (petroleum ether:ethyl acetate = 1:2) gave the
title compound as a yellow solid (65% yield; mp 89-92 °C); 1H NMR
(400 MHz, Acetone-d6) δ 7.96 (dd, J = 10.2, 2.8 Hz, 2H), 7.06 (d, J =
8.8 Hz, 2H), 7.00 – 6.94 (m, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.71 (t, J =
10.1 Hz, 2H), 4.14 – 4.03 (m, 2H), 2.89 (t, J = 5.9 Hz, 2H), 2.64 (t, J =
6.0 Hz, 4H), 1.81 – 1.70 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ
157.62, 156.69, 143.89, 143.51, 131.02, 130.19, 129.30, 129.22,
128.94, 127.44, 115.33, 114.41, 66.55, 54.58, 54.40, 23.47. HRMS
(ESI) calcd for C22H23NO2Se [M + H]+, 414.0972; found 414.0970.
(t, J = 5.9 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.43 (s, 4H), 1.50 (dt, J =
10.8, 5.5 Hz, 4H), 1.38 (d, J = 5.1 Hz, 2H). 13C NMR (101 MHz, DMSO-
d6) δ 159.28 (d, J = 247.2 Hz), 159.05 (d, J = 249.1 Hz), 141.30 (d, J =
31.4 Hz), 140.06 (d, J = 32.3 Hz), 129.16 (d, J = 11.3 Hz), 128.96 (d, J
= 12.1 Hz), 127.32, 126.72, 116.04, 114.44, 113.06, 112.34, 103.67,
103.42, 103.06, 102.80, 66.65, 57.60, 54.79, 25.96, 24.33. HRMS
(ESI) calcd for C23H23F2NO2Se [M + H]+, 464.0940; found 464.0948.
2-(2-chloro-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5-(2-chloro-4-
hydroxylphenyl)selenophene 9f. Compound 9f was prepared by
2,5-bis(2-chloro-4-hydroxylphenyl)selenophene (8d) and N-(2-
chloroethyl)pyrrolidine according to general procedure for
Williamson ether synthesis. Purification by CC (petroleum
ether:ethyl acetate = 1:1) gave the title compound as a yellow solid
(55% yield; mp 137-140 °C); 1H NMR (400 MHz, DMSO-d6) δ 7.56 (t,
J = 7.0 Hz, 1H), 7.51 – 7.42 (m, 3H), 7.13 (d, J = 2.6 Hz, 1H), 6.98 –
3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-4-(4-hydroxylphenyl)-
selenophene 12b. Compound 12b was prepared by 3,4-bis(4-
hydroxylphenyl)selenophene (11a) and N-(2-chloroethyl)piperidine
according to general procedure for Williamson ether synthesis.
10 | J. Name., 2012, 00, 1-3
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Purification by CC (petroleum ether:ethyl acetate = 1:2) gave the 5.6 Hz, 2H), 2.76 (d, J = 20.0 Hz, 4H), 2.01 (s, 3H), 1.96 (s, 3H), 1.84
title compound as a yellow solid (72% yield; mp 159-163 °C); 1H (s, 4H). 13C NMR (101 MHz, Acetone-d6) δ 156.86, 155.31, 145.22,
NMR (400 MHz, Acetone-d6) δ 8.01 – 7.90 (m, 1H), 7.98 – 7.93 (m, 144.92, 132.27, 132.02, 131.61, 130.62, 128.48, 128.32, 128.27,
1H), 7.08 – 7.04 (m, 1H), 7.09 – 6.92 (m, 2H), 6.99 – 6.94 (m, 1H), 128.25, 126.49, 124.54, 114.88, 111.19, 68.03, 55.53, 55.26, 24.22,
6.83 – 6.78 (m, 1H), 6.75 (ddd, J = 11.3, 6.7, 2.3 Hz, 2H), 6.74 – 6.70 16.55, 16.31. HRMS (ESI) calcd for C24H27NO2Se [M + H]+, 442.1285;
(m, 1H), 4.06 (t, J = 6.0 Hz, 2H), 2.72 – 2.66 (m, 2H), 2.48 (s, 4H), found 442.1283.
1.54 (dt, J = 10.9, 5.5 Hz, 4H), 1.41 (d, J = 2.5 Hz, 2H). 13C NMR (101
MHz, DMSO-d6) δ 157.70, 156.63, 143.83, 143.47, 130.84, 130.13, 3-(3-Methyl-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(3-methyl-4-
130.10, 129.18, 129.16, 128.85, 115.27, 114.36, 65.84, 57.80, 54.79, hydroxylphenyl)selenophene 12f. Compound 12f was prepared by
25.94, 24.31. HRMS (ESI) calcd for C23H25NO2Se [M + H]+, 428.1129; 3,4-bis(3-methyl-4-hydroxylphenyl)selenophene (11c) and N-(2-
found 428.1120.
chloroethyl)piperidine
according to general procedure for
Williamson ether synthesis. Purification by CC (petroleum
3-(2-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4-(2-methyl-4-
ether:ethyl acetate = 1:2) gave the title compound as a yellow solid
hydroxylphenyl)selenophene 12c. Compound 12c was prepared by (65% yield; mp 85-89 °C); 1H NMR (400 MHz, Acetone-d6) δ 7.92
3,4-bis(2-methyl-4-hydroxylphenyl)selenophene (11b) and N-(2- (dd, J = 9.9, 2.8 Hz, 2H), 7.02 – 6.95 (m, 2H), 6.86 (dd, J = 8.4, 2.0 Hz,
chloroethyl)pyrrolidine according to general procedure for 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 2.0 Hz, 2H), 4.15 (t, J = 5.7
Williamson ether synthesis. Purification by CC (petroleum Hz, 2H), 2.92 (dd, J = 10.9, 5.3 Hz, 2H), 2.70 (s, 4H), 2.15 – 2.08 (m,
ether:ethyl acetate = 1:2) gave the title compound as a yellow solid 6H), 1.62 (dt, J = 11.0, 5.6 Hz, 4H), 1.49 – 1.39 (m, 2H). 13C NMR (101
(67% yield; mp 97-101 °C); 1H NMR (400 MHz, Acetone-d6) δ 7.90 MHz, Acetone-d6) δ 159.39, 158.19, 150.12, 149.26, 137.58, 137.53,
(dd, J = 7.9, 2.8 Hz, 2H), 6.98 (dd, J = 11.8, 1.2 Hz, 2H), 6.86 (dd, J = 132.39, 132.27, 129.52, 129.28, 129.06, 128.12, 118.41, 117.68,
8.4, 1.8 Hz, 1H), 6.70 (dt, J = 18.3, 8.3 Hz, 3H), 4.09 (t, J = 5.8 Hz, 113.99, 112.87, 66.75, 58.59, 55.66, 26.67, 24.97, 21.75, 21.65.
2H), 2.93 (t, J = 5.7 Hz, 2H), 2.67 (s, 4H), 2.11 (d, J = 4.7 Hz, 6H), 1.78 HRMS (ESI) calcd for C25H29NO2Se [M + H]+, 454.1450; found
– 1.73 (m, 4H). 13C NMR (101 MHz, Acetone-d6) δ 159.22, 158.08, 454.1448.
150.06, 149.15, 137.47, 137.41, 132.29, 132.19, 129.49, 129.17,
128.92, 128.00, 118.33, 117.60, 113.92, 112.71, 55.40, 55.06, 29.84, 3-(3-Fluoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(3-fluoro-4-
24.11, 21.71, 21.60. HRMS (ESI) calcd for C24H27NO2Se [M + H]+, hydroxylphenyl)selenophene 12g. Compound 12g was prepared by
442.1285; found 442.1283.
3,4-bis(3-fluoro-4-hydroxylphenyl)selenophene (11d) and N-(2-
chloroethyl)piperidine according to general procedure for
Williamson ether synthesis. Purification by CC (petroleum
3-(2-Methyl-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(2-methyl-4-
hydroxylphenyl)selenophene 12d. Compound 12d was prepared by ether:ethyl acetate = 1:2) gave the title compound as a yellow solid
3,4-bis(2-methyl-4-hydroxylphenyl)selenophene (11b) and N-(2- (74% yield; mp 89-93 °C); 1H NMR (400 MHz, DMSO-d6) δ 7.63 (t, J =
chloroethyl)piperidine
according to general procedure for 9.1 Hz, 1H), 7.59 – 7.55 (m, 2H), 7.54 – 7.51 (m, 1H), 6.90 (dd, J =
Williamson ether synthesis. Purification by CC (petroleum 13.6, 2.5 Hz, 1H), 6.78 (dd, J = 8.8, 2.4 Hz, 1H), 6.65 – 6.58 (m, 2H),
ether:ethyl acetate = 1:2) gave the title compound as a yellow solid 4.03 (t, J = 5.9 Hz, 2H), 2.57 (t, J = 5.8 Hz, 2H), 2.34 (s, 4H), 1.41 (dt, J
1
(55% yield; mp 91-95 °C); H NMR (400 MHz, Acetone-d6) δ 7.93 (d, = 10.8, 5.5 Hz, 4H), 1.30 (d, J = 5.1 Hz, 2H). 13C NMR (101 MHz,
J = 2.8 Hz, 1H), 7.91 (d, J = 2.8 Hz, 1H), 7.00 (d, J = 1.6 Hz, 1H), 6.97 DMSO-d6) δ 159.22 (d, J = 241.9 Hz), 158.99 (d, J = 233.1 Hz), 141.24
(s, 1H), 6.86 (dd, J = 8.4, 2.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.70 (d, (d, J = 25.6 Hz), 140.00 (d, J = 27.4 Hz), 129.10 (d, J = 14.0 Hz),
J = 2.0 Hz, 2H), 4.15 (t, J = 5.7 Hz, 2H), 2.94 – 2.87 (m, 2H), 2.70 (s, 128.90 (d, J = 15.9 Hz), 127.26, 126.66, 115.98, 114.38, 113.00,
4H), 2.16 – 2.08 (m, 6H), 1.65 – 1.60 (m, 4H), 1.49 – 1.42 (m, 2H). 112.28, 103.61, 103.36, 103.00, 102.74, 66.59, 57.54, 54.73, 25.90,
13C NMR (101 MHz, Acetone-d6) δ 159.38, 158.17, 150.11, 149.25, 24.27. HRMS (ESI) calcd for C23H23F2NO2Se [M + H]+, 462.0948;
137.56, 137.52, 132.38, 132.26, 129.51, 129.27, 129.04, 128.11, found 462.0942.
118.39, 117.67, 113.98, 112.86, 66.74, 58.58, 55.65, 26.66, 24.96,
21.74, 21.64. HRMS (ESI) calcd for C25H29NO2Se [M + H]+, 454.1450; 3-(2-chloro-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4-(2-chloro-4-
found 454.1455.
hydroxylphenyl)selenophene 12h. Compound 12h was prepared by
3,4-bis(2-chloro-4-hydroxylphenyl)selenophene (11e) and N-(2-
chloroethyl)pyrrolidine according to general procedure for
3-(3-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4-(3-methyl-4-
hydroxylphenyl)selenophene 12e. Compound 12e was prepared by Williamson ether synthesis. Purification by CC (petroleum
3,4-bis(3-methyl-4-hydroxylphenyl)selenophene (11c) and N-(2- ether:ethyl acetate = 1:2) gave the title compound as a yellow solid
chloroethyl)pyrrolidine according to general procedure for (73% yield; mp 79-83 °C); 1H NMR (400 MHz, CD3OD) δ 7.82 (dt, J =
Williamson ether synthesis. Purification by CC (petroleum 8.3, 4.1 Hz, 2H), 6.93 (dd, J = 8.4, 3.3 Hz, 1H), 6.83 (ddd, J = 6.4, 5.5,
ether:ethyl acetate = 1:2) gave the title compound as a yellow solid 1.7 Hz, 1H), 6.66 (dd, J = 8.6, 2.6 Hz, 2H), 6.59 (t, J = 2.3 Hz, 1H), 6.44
(72% yield; mp 84-88 °C); 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 4.8 (dd, J = 8.4, 2.5 Hz, 1H), 4.13 – 4.08 (m, 2H), 3.31 – 3.27 (m, 2H),
Hz, 2H), 7.73 (d, J = 4.8 Hz, 1H), 6.89 – 6.76 (m, 1H), 6.52 (dd, J = 8.1, 3.12 (dd, J = 12.4, 5.9 Hz, 4H), 1.90 (td, J = 6.8, 2.9 Hz, 4H). 13C NMR
5.4 Hz, 2H), 6.48 – 6.42 (m, 2H), 4.01 (t, J = 5.6 Hz, 2H), 2.96 (t, J = (101 MHz, DMSO-d6) δ 158.00, 157.36, 141.15, 140.89, 132.98,
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132.60, 132.36, 132.30, 130.16, 130.03, 128.76, 126.85, 115.66, 139.30, 130.91, 130.48, 130.43, 127.99, 127.42, 127.14, 126.96,
114.69, 113.84, 113.02, 66.83, 59.73, 53.89, 23.06. HRMS (ESI) calcd 116.32, 116.01, 114.98, 63.93, 54.19, 53.30, 23.09. HRMS (ESI) calcd
for C22H21Cl2NO2Se [M + H]+, 482.0193; found 482.0190.
for C28H27NO3Se [M + H]+, 506.1234; found 506.1231.
3-(2-Chloro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(2-chloro-4-
3-(4-(2-(Piperidin-1-yl)ethoxy)phenyl)-2,5-bis
(4-
hydroxylphenyl)selenophene 12i. Compound 12i was prepared by hydroxylphenyl)selenophene 16b. Compound 16b was obtained
3,4-bis(2-chloro-4-hydroxylphenyl)selenophene (11e) and N-(2- from the demethylation of compound 15b using boron tribromide,
chloroethyl)piperidine
Williamson ether synthesis. Purification by CC (petroleum methoxylphenyl)selenophene
ether:ethyl acetate = 1:2) gave the title compound as a yellow solid chloroethyl)pyrrolidine according to general procedure for
(70% yield; mp 109-113 °C); 1H NMR (400 MHz, CD3OD) δ 7.93 – Williamson
ether synthesis. Purification by CC
according to general procedure for the latter was prepared by 3-(4-hydroxylphenyl)-2,5-bis(4-
(14a) and N-(2-
7.89 (m, 2H), 7.03 – 6.99 (m, 1H), 6.91 (dt, J = 6.6, 3.3 Hz, 2H), 6.71 (dichloromethane:ethyl acetate = 2:3) gave the title compound 16b
(dd, J = 6.3, 4.2 Hz, 2H), 6.54 (dd, J = 8.4, 2.3 Hz, 1H), 4.12 (t, J = 5.3 as a yellow solid (62% yield; mp 142-146 °C); 1H NMR (400 MHz,
Hz, 2H), 2.97 (t, J = 5.2 Hz, 2H), 2.76 (s, 4H), 1.68 (dd, J = 10.9, 5.4 DMSO-d6) δ 7.52 – 7.42 (m, 3H), 7.21 (d, J = 8.7 Hz, 2H), 7.04 (d, J =
Hz, 4H), 1.53 (d, J = 4.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 8.6 Hz, 2H), 6.84 (dd, J = 19.9, 8.7 Hz, 2H), 6.70 (d, J = 8.6 Hz, 2H),
158.06, 157.30, 141.14, 140.90, 132.97, 132.60, 132.36, 132.26, 4.09 (t, J = 5.6 Hz, 2H), 2.80 (t, J = 5.3 Hz, 2H), 2.55 (d, J = 19.8 Hz,
130.16, 130.03, 128.72, 126.90, 115.65, 114.75, 113.83, 113.08, 4H), 1.54 (dt, J = 10.8, 5.5 Hz, 4H), 1.40 (d, J = 4.4 Hz, 2H). 13C NMR
65.81, 57.22, 54.30, 25.47, 23.85. HRMS (ESI) calcd for (101 MHz, DMSO-d6) δ 157.92, 157.62, 157.36, 147.33, 141.43,
C23H23Cl2NO2Se [M + H]+, 496.0349; found 496.0346.
139.39, 130.41, 130.34, 128.15, 128.03, 127.40, 127.21, 127.06,
116.32, 115.97, 114.80, 65.28, 57.39, 54.54, 25.41, 23.86. HRMS
3-(4-hydroxylphenyl)-2,5-bis(4-methoxylphenyl)selenophene 14a. (ESI) calcd for C29H29NO3Se [M + H]+, 520.1391; found 520.1398.
Compound 14a was prepared by 3-bromo-2,5-bis(4-
methoxylphenyl)selenophene (13a) and 4-hydroxylphenylboronic 3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-2,5-bis
acid according to general procedure for Suzuki Coupling. hydroxylphenyl)selenophene 16c. Compound 16c was obtained
(2-methyl-4-
Purification by CC (petroleum ether:ethyl acetate = 30:1) gave the from the demethylation of compound 15c using boron tribromide,
title compound as a yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.51 – the latter was prepared by 3-(4-hydroxylphenyl)-2,5-bis(4-
7.48 (m, 1H), 7.51 – 7.48 (m, 1H), 7.42 – 7.40 (m, 1H), 7.22 – 7.16 methoxylphenyl)selenophene
(14b)
and
N-(2-
(m, 4H), 6.93 – 6.89 (m, 2H), 6.81 – 6.72 (m, 4H), 3.84 (s, 3H), 3.79 chloroethyl)pyrrolidine according to general procedure for
(s, 3H).
Williamson
ether
synthesis.
Purification
by
CC
(dichloromethane:ethyl acetate = 2:3) gave the title compound 16c
(2-methyl-4-methoxylphenyl)- as a yellow solid (52% yield; mp 89-93 °C); 1H NMR (400 MHz,
3-(4-hydroxylphenyl)-2,5-bis
selenophene 14b. Compound 14b was prepared by 3-bromo-2,5- DMSO-d6) δ 9.78 (s, 1H), 9.63 (s, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.33
bis(2-methyl-4-methoxylphenyl)selenophene (13b) and 4- (d, J = 1.9 Hz, 1H), 7.28 (dd, J = 8.2, 2.3 Hz, 1H), 7.24 – 7.12 (m, 2H),
hydroxylphenylboronic acid according to general procedure for 6.96 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.73 (dd, J = 8.3, 2.2
Suzuki Coupling. Purification by CC (petroleum ether:ethyl acetate = Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 4.45 – 4.33 (m, 2H), 3.50 (d, J = 38.9
1
30:1) gave the title compound as a yellow solid; H NMR (400 MHz, Hz, 2H), 3.38 (s, 4H), 2.17 (s, 3H), 2.01 (d, J = 11.8 Hz, 3H), 1.93 (d, J
CDCl3) δ 7.48 – 7.33 (m, 3H), 7.21 – 7.16 (m, 2H), 6.88 (d, J = 2.6 Hz, = 4.9 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 156.40, 155.93,
1H), 6.86 – 6.73 (m, 5H), 3.86 – 3.84 (t, J = 8.9 Hz, 6H), 2.53 (d, J = 153.82, 145.42, 141.29, 137.38, 135.30, 132.37, 131.94, 131.86,
8.1 Hz, 3H), 2.08 (d, J = 13.6 Hz, 3H).
131.66, 128.17, 127.54, 125.84, 125.46, 124.78, 124.65, 115.17,
115.03, 113.62, 110.61, 110.42, 65.65, 54.98, 53.45, 23.12, 16.46,
(4- 16.43. HRMS (ESI) calcd for C30H31NO3Se [M + H]+, 534.1537; found
3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-2,5-bis
hydroxylphenyl)selenophene 16a. Compound 16a was obtained 534.1539.
from the demethylation of compound 15a using boron tribromide,
the latter was prepared by 3-(4-hydroxylphenyl)-2,5-bis(4-
methoxylphenyl)selenophene (14a) and N-(2- ligand binding domain (LBD) genes were amplified by PCR from
chloroethyl)pyrrolidine according to general procedure for plasmid pVP-16-ERα and pVP-16-ERβ. The PCR product was cloned
Williamson ether synthesis. Purification by CC into plasmid, and PGEx-KG E. coli BL21 (DE3) used for the
(dichloromethane:ethyl acetate = 2:3) gave the title compound 16a overexpression of ER-LBD. The cells were induced by IPTG (10 μM)
as a yellow solid (56% yield; mp 105-109 °C); 1H NMR (400 MHz, for
h, then cells were harvested, frozen, and thawed in
4.2. Gene Clone and Protein Purification. Human ERα or ERβ
2
DMSO-d6) δ 9.79 (d, J = 23.2 Hz, 2H), 7.57 – 7.47 (m, 3H), 7.31 – 7.27 phosphate-buffered saline (PBS), containing 1 mM EDTA and 1 mM
(m, 2H), 7.10 – 7.07 (m, 4H), 6.99 (d, J = 8.8 Hz, 1H), 6.85 (t, J = 8.0 DTT. After being ultrasonicated in an icy bath, the supernatant was
Hz, 1H), 6.78 – 6.71 (m, 2H), 4.36 (t, J = 4.9 Hz, 2H), 3.53 (d, J = 19.2 applied to a column of GSH-resin. The collection was dialyzed in ice
Hz, 2H), 3.33 (d, J = 3.5 Hz, 4H), 1.97 (d, J = 3.5 Hz, 4H). 13C NMR buffer for 4 h. After being checked by a combination of sodium
(101 MHz, DMSO-d6) δ 157.93, 157.40, 156.97, 147.39, 141.56, dodecyl sulfate–polyacrylamide gel electrophoresis and Western
12 | J. Name., 2012, 00, 1-3
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blotting, the protein was prepared as a 10 mM stock in potassium Acknowledgements
phosphate and stored at −80 °C.45
We are grateful to the NSFC (81573279, 81373255), Major
4.3 Estrogen Receptor Binding Affinity. Relative binding affinities Project of Technology Innovation Program of Hubei Province
were determined by a competitive fluorometric binding assay as (2016ACA126), Hubei Province’s Outstanding Medical Academic
previously described. Briefly, 40 nM fluorescence tracer Leader Program, Hunan Province Cooperative Innovation Center for
(coumestrol, Sigma-Aldrich, MO) and 0.8 μM purified human ERα or Molecular Target New Drug Study, and the Fundamental Research
ERβ ligand binding domain (LBD) were diluted in 100 mM potassium Funds for the Central Universities of China (2015306020201) for
phosphate buffer (pH 7.4), containing 100 μg/mL bovine gamma support of this research. We thank Dr. Chu Tang for help with the
globulin (Sigma-Aldrich, MO). Incubations were for 2h at room Molecular Modeling.
temperature (25 °C). Fluorescence polarization values were then
measured. The binding affinities are expressed as relative binding Conflict of Interest
affinity (RBA) values with the RBA of 17β-estradiol set to 100%. The
values given are the average
± range of two independent The authors declare no competing interests.
determinations. IC50 values were calculated according to equations
described previously.46
Notes and References
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Table of Contents
Selenophenes with basic side chains as novel estrogen receptor ligands showed excellent antagonist
activity and better antiproliferative activity than that of 4-hydroxytamoxifen in breast cancer MCF-7
cells.