Selenophenes: Introducing a New Element into the Core of Non-Steroidal Estrogen Receptor Ligands

Article abstract of DOI:10.1002/cmdc.201600593

The importance of the heterocyclic core elements with peripheral phenolic and alkyl substituents as a dominant structural motif of ligands for the estrogen receptor (ER) has been well recognized. In this study we expanded the structural diversity of core

Full text of DOI:10.1002/cmdc.201600593

Accepted Article
Title: Selenophenes: Introducing a New Element into the Core of Non-
Steroidal Estrogen Receptor Ligands
Authors: Silong Zhang, Zhiyong Wang, Zhiye Hu, Changhao Li, Chu
Tang, Kathryn E. Carlson, Junjie Luo, Chune Dong, John A.
Katzenellenbogen, Jian Huang, and Haibing Zhou
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Selenophenes: Introducing a New Element into the Core of Non-
Steroidal Estrogen Receptor Ligands
Silong Zhang[a],‡, Zhiyong Wang[a],‡, Zhiye Hu[a], Changhao Li[b], Chu Tang[a], Kathryn E. Carlson[c],
Junjie Luo[a], Chune Dong[a], John A. Katzenellenbogen[c], Jian Huang[b],*, Hai-Bing Zhou[a], [d]
*
‡ These two authors contributed equally to this work
[a]
[b]
S. Zhang, Z. Wang, Z. Hu, C. Tang, J. Luo, Prof. Dr. C. Dong, Prof. Dr. H-B. Zhou
State Key Laboratory of Virology; Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, Wuhan
University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan 430071 (P.R. China)
E-mail: zhouhb@whu.edu.cn
C. Li, Prof. Dr. J. Huang
College of Life Sciences, Wuhan University;
No. 299 Bayi Road, Wuhan 430072 (P.R. China)
E-mail: jianhuang@whu.edu.cn.
[c]
[d]
Dr. K. E. Carlson, Prof. Dr. J. A. Katzenellenbogen
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801 (USA)
Prof. Dr. H-B. Zhou
Institute of Pharmacy & Pharmacology, University of South China
Hengyang 421001, China
Supporting information for this article is given via a link at the end of the document.
Abstract: The importance of the heterocyclic core elements with
peripheral phenolic and alkyl substituents as a dominant structural
motif of ligands for estrogen receptor (ER) has been well recognized.
Here, we expand the structural diversity of core elements by
preparing selenium-containing heterocycles and exploring the
activities of these selenophenes on the two ERs, ERα and ERβ.
Careful structure-activity relationship analysis of their ER binding
affinities showed that most selenophenes are ERβ-selective, with
the position of the phenol substituents on the selenophene-core and
the nature of these substituents having in a marked effect on their
novel agents leading to improved therapeutics that target the
estrogen receptor.
Introduction
Selenium is known to play important roles in the development
and maintenance of various physiological functions in humans,
and recent research on this trace element support its protective
effect against various types of cancer.[1-2] Recently, the biological
effects of organoselenium compounds have attracted much
attention,[3-5] as has their application as chiral catalysts.[6-8] In
particular, many diarylselenides possess anticancer, antiviral,
antimicrobial, as well as antioxidant properties.[9-11] In addition, a
number of biologically active selenaheterocycles, such as
cytoprotective agent ebselen, have recently been discovered.[12]
binding
affinities.
The
compound
bis(2-fluoro-4-
hydroxyphenyl)selenophene (2f) has the highest relative binding
affinity (RBA) of 24.3 for ERβ. In transcription assays, most of
selenophenes exhibit partial to full agonist activity for both ER
subtypes,
with
compounds
bis(2-methyl-4-
hydroxyphenyl)selenophene (2b), bis(4-fluoro-3-hydroxyphenyl) 3-
bromoselenophene (6f), 2,3,5-tris(hydroxyphenyl)-thiophenes (8b
and 8d) profiling as superagonists for ERα, but several compounds
display a range of ERα or ERβ antagonistic activities. A few
selenophenes exhibited antiproliferative activity, with compound 8c
showing antiproliferative effects comparable to that of 4OHT in
breast cancer MCF-7 cells while being nontoxic to normal VERO
cells. These new ligands could act as models for the development of
Moreover, we and other found that
compounds inhibited the motility and development of stages of
the comparable nematode Haemonchus contortus[13]
More
recently, fluselenamyl, novel planar benzoselenazole
a few selenophene
.
a
derivative, was used as a PET imaging agent for amyloid in
Alzheimer's disease (Figure 1). [14]
1
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Figure 1. Representative examples of selenium-containing bioactive agents.
Estrogens play important roles in the development and
maintenance of both reproductive and non-reproductive tissues
in both women and men.[15] Estrogens are required and can
provide health benefits in some tissues, such as the
reproductive system, bone, vasculature, and brain, but they can
also drive breast and endometrial cancers.[16-18] The broad
spectrum of estrogen effects in diverse tissues provides an
intriguing opportunity for the creation of tissue-selective estrogen
receptor ligands. The multiple actions of estrogens are known to
be mediated by two subtypes of the estrogen receptor (ER),
ERα and ERβ.[19] Although structurally related, these two
receptor subtypes have distinct tissue distributions; for example,
ERα is mainly found in the female reproductive system, and ERβ
is mainly expressed in prostate, colon, cardiovascular and
central nervous systems.[20] Even when both ER subtypes are
present in the same cells, estrogen action through these
receptors leads to differential gene regulation, resulting in
distinct physiological outcomes having non-overlapping and
even opposing functions.[21-24] Efforts to obtain tissue-selective
estrogens have focused on compounds, termed SERMs (for
selective estrogen receptor modulators), that showed different
levels of partial agonist/antagonist activity (i.e., intrinsic activity)
in different target tissues. The selectivity of SERMs is believed
to arise from their differential engagement of coregulator
proteins in a target cell- and gene-specific manner,[25-27] and
compounds such as tamoxifen, raloxifene, lasofoxifene, and
bazedoxifene were found to be largely agonistic in bone and
antagonistic in breast, but to have varying activity in the uterus.
However, the above-mentioned ER-targeted therapies are
limited by on-target side-effects, such as breast cancer risk with
hormone replacement therapy,[28] or thromboembolic events,
endometrial proliferation and hot flushes with the ERα ligand,
such as tamoxifen.[29]
In the development of both SERMs and subtype-selective
ligands, extensive investigation has been made of non-steroidal
compounds having heterocyclic cores. Typically, the peripheral
substituents, such as phenols, simple alkyl groups, and basic
and polar phenyl substituents, have generally remained the
same. By contrast, a wide variety of heterocyclic cores has been
explored, often in the context of SERM development in which
both five- and to a lesser extent six-membered ring heterocycles
have been used.[30-34] Some examples of the 5-membered ring
heterocycle series are presented below (Figure 2).
OH
HO
N
N
Propyl Pyrazole Triol (PPT)
Imidazole
ER = 2.12
ER
N
ER = 49
OH
N
ER
OH
HO
OH
HO
Se
R
R
OH
OH
substituent
HO
Cl
Selenophene-based ER ligands
Furan
ER = 140
ER
Thiophene
ER = 85.7
ER
S
O
OH
OH
Cl
HO
Cl
HO
Values shown are relative binding affinities (RBA) and relative to estradiol (E2 =100);
is ratio of ER /ER
Figure 2. Structure and binding affinity data of the most promising five-membered ring heterocycles and title selenophene compounds.
As part of our ongoing interest in ER ligands with different core
structures and biological activity,[35-38] we recently reported on a
series of novel ER ligands based on a thiophene core, and we
found that most of the 2,5- and 2,4-bis(hydroxyphenyl)-
thiophenes were ERβ selective, whereas the bulkier 2,3,5-
larger overall length of these thiophene core molecules
compared to heterocycles having only second row elements.[34]
ER ligands having heterocyclic cores have been built from
second row elements (O and N) and the third row element (S);
thus far, however, there are no reports of using the fourth row
element (Se). The well recognized importance of combining
heterocyclic core elements with phenolic and alkyl peripheral
substituents in the design of estrogen receptor (ER) ligands led
us to prepare ligands having selenophene cores. As relatively
unexplored structural elements of drugs, these selenium-
containing heterocycles expand the structure diversity of
heterocycle core elements, and we studied their binding affinity
tris(hydroxyphenyl)-thiophenes
were
ERα
selective.[34]
Analogous furan-core compounds, prepared for comparison,
generally had lower affinity and/or less selectivity than their
congeneric thiophenes. Intriguingly, some of the 2,5- and 2,4-
diarylthiophenes showed distinct superagonist activity in reporter
gene assays, giving maximal activities 2-3 times that of
estradiol; superagonist activity was ascribed to the somewhat
2
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and cellular potencies and efficacies for ERα and ERβ, finding
that some of them act as superagonists. We also investigated
their anti-proliferative activities in cancer and normal cell lines.
Our results, together with molecular modeling, offer a framework
for understanding the structural basis for superagonist activity,
and reveal what might be a general strategy for obtaining
superagonists for other members of the nuclear receptor
superfamily.
5%. Further studies with other palladium catalysts, including
Pd2(dba)2, Pd(CN)2Cl2, Pd(PPh3)Cl2, gave no improvement, an
exception being Pd(dppf)Cl2, which furnished modest to good
yields. Finally, treatment of the compounds 1a-j with boron
tribromide gave the final 2,5-disubstituted diphenolic
selenophenes 2a-j in good yields.[40]
The key intermediates, 3a-d, for the synthesis of 3,4-
disubstituted selenophene derivatives 4a-d (Scheme 1B) were
prepared by treating 3,4-dibromoselenophene 11 with aryl
boronic acid by using Pd(OAc)2/PPh3 instead of Pd(dppf)Cl2 as
the catalyst. The 3,4-dibromoselenophene intermediate (11)
cannot be obtained by bromination of selenophene, but it was
synthesized by debromination of 2,3,4,5-tetrabromoselenophene
Results and Discussion
Chemical Synthesis. Four series of novel selenophene-core
compounds, shown in Schemes 1-2, were prepared from
selenophene according to the synthetic procedures established
in our laboratory for producing thiophene-core ER ligands. In the
synthesis of compounds 2a-j (Scheme 1A), key intermediates
1a-j were obtained by the Suzuki cross-coupling of aryl boronic
acid with 2,5-dibromoselenophene 9 in the presence of sodium
carbonate and 1,1’-bis(diphenylphosphino)ferrocene (dppf)-
coordinated palladium chloride as a catalyst at reflux.[39] The key
intermediate selenophene precursor, 9, was prepared by
bromination of the selenophene using N-bromosuccinimide
(NBS). Initially, Suzuki cross-coupling was investigated using
Pd(OAc)2/PPh3 as the catalyst; however, the yield was below
(10)
using
Zn
and
AcOH
in
water.[41]
2,3,4,5-
Tetrabromoselenophene (10) was synthesized by bromination of
selenophene using CHCl3 and AcOH as solvent, with dropwise
addition of Br2.[42] The amount of bromine used and the rate of
addition proved to be important for the outcome of this reaction:
With large excess of bromine (>4 equiv.), 2,3,4,5-
tetrabromoselenophene (10) was the major product, whereas
with
less
than
2
equivalents
of
bromine,
2,5-
dibromoselenophene (9) could also be obtained in good yield.
Subsequent cleavage of the methoxyl groups of 3a-d by BBr3
yielded the desired products 4a-h in 51-68% yields.
Scheme 1. Synthesis of 2,5-substituted selenophene compounds. Reagents and conditions: (a) NBS, DMF; (b) [Pd] catalyst (Pd(dppf)Cl2 for Series I;
Pd(OAc)2/PPh3 for Series II), Na2CO3, toluene/water (1:1), reflux, 24 h; (c) BBr3, CH2Cl2, -20 °C to rt, 4 h; (d) Br2, AcOH, rt, 18h; (e) Zn, AcOH/water (1:1), 37 °C,
12 h. To simplify comparisons between compounds in closely related series, we designate locant positions of the substituents on the phenyl groups with respect
to the selenophene core; for clarity, locant positions on the selenophene core itself are given by numbers in italics.
With the aim of further investigating the effect of a third
substituent on the selenophene core, we prepared a series of
trisubstituted selenophene compounds 6a-h (Scheme 2A). First,
1 equiv of 2,3,5-tribromoselenophene was treated with 4 equiv
3
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of aryl boronic acid using Pd(dppf)Cl2 to afford 5a-h, followed by
ether cleavage to give compounds 6a-h directly.
with 5 equiv of aryl boronic acid using standard Suzuki coupling
conditions afforded the key esters 7a-d, which were
During our prior work on furan- and thiophene-core ER ligands, subsequently reacted with BBr3 at -20 °C to give the hydroxyl
we found that triaryl, especially triphenolic compounds, showed
higher binding affinity and subtype selectivity than the
corresponding bisphenolic compounds.[34] Thus, we also
attempted to introduce a third phenol ring into the selenophene
core to obtain triaryl-substituted selenophene analogues 8a-8d
(Scheme 2B). Reaction of 1 equiv of 2,3,5-tribromoselenophene
derivatives
8a-8d.
The
starting
material
2,3,5-
tribromoselenophene (12) was synthesized by bromination of
selenophene using Br2 in DMF.
The structures of compounds 1j and 7a were unambiguously
confirmed by X-ray crystallography.
Scheme 2. Synthesis of trisubstituted selenophene compounds. Reagents and conditions: (a) Br2, DMF; (b) [Pd] catalyst (Pd(dppf)Cl2 for Series III; Pd(PPh3)4) for
Series IV, Na2CO3, toluene/water (1:1), reflux, 24 h; (c) BBr3, CH2Cl2, -20 °C to rt, 4 h. To simplify comparisons between compounds in closely related series, we
designate locant positions of the substituents on the phenyl groups with respect to the selenophene core; for clarity, locant positions on the selenophene core
itself are given by numbers in italics.
Relative Binding Affinities. The binding affinities of
selenophene compounds for both ER and ER 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%.
for ERβ; β/ was 0.91), compounds 2f, 2g, 8b had high binding
affinity for ERβ and higher ERβ selectivity. Compound 6b also
retained high binding affinity for ER. Among the 2,5-bisphenol
selenophene (series I), those substituted at C2 (meta to the
phenol; 2b, 2e, 2f and 2g) demonstrate a better binding affinity
than the corresponding non-substituted compound 2a, with RBA
As an overall observation, it is notable that a large number of
the selenophene-core derivatives show moderate levels of ERβ
selectivity, which is agreement with our previous studies with
furan- and thiophene-core compounds.[34] In addition, compared
to furan- and thiophene-core compounds, these selenophene-
core derivatives generally demonstrate better binding affinity for
ERβ. Second, the addition of substituents in the phenol rings, as
well as the position of the hydroxyl group, have significant
effects on the binding affinity and selectivity of the ligands.
Compared to 4OHT (RBA values were 13.8 for ER[43] and 12.6
values ranging from
4
to 24. In fact, bis(2-fluoro-4-
hydroxyphenyl)selenophene (2f) has the highest binding affinity
for ERβ among all of the analogues, binding 8-fold better to ER
than does 2a; this increased affinity, however, comes at a cost in
ERβ subtype selectivity (Table 1, entries 2, 5-7 vs 1).
Interestingly, when the Me or Cl substituents are present in the
C3 position, next to the phenolic hydroxyl group, as in
compounds 2c, 2d and 2h, binding affinities for both ER
subtypes were dramatically decreased (Table 1, entries 2 vs 3,
4; 7 vs 8). Some interesting things were also observed with
4
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hydroxyl analogues 2i and 2j. The marked effect of the position
of the hydroxyl groups on ER binding affinities is evident from
entries 6 vs 9 and 10 (Table 1): when this group was moved
from the C4 to the C3-position of the phenyl ring, binding
affinities were also diminished.
were also observed for selenophene-core compounds (series II),
where affinities for ER were always less than 0.8%. Binding to
ERβ was mostly higher than to ERα, with again the C2-
substituted analog (4b) being the best.
We have previously observed that 3,4-disubstituted diphenolic
furan and thiophenes showed lower binding affinities than
corresponding 2,5-disubstituted compounds.[33] Similar trends
Table 1. Relative Binding Affinity (RBA) of series I (2a-j), series II (4a-e), series III (6a-g) and series IV (8a-d) for ER and ER.
5
4 OH
6
1
H (Br)
3
R
R
3
OH
HO
2
5
Se
2
core
core
Series I:
R
R
R
2a-h
R
R
2i-j
Series I:
Series II:
Series II:
Series III:
4a-d
6a-e
HO
OH
HO
Series IV: 8a-d
6f
b
b
Entry compound
Core
R
ER
ER
/
1
2
3
4
2a
2b
2c
H
2.87 ± 0.20
4.70
1.98
>94
0.61 ± 0.03
5.60 ± 0.43
2-Me
3-Me
11.1 ± 0.73
0.94 ± 0.05
1.21 ± 0.02
<0.01
<0.01
3,5-diMe
2d
>121
5
6
2e
2f
2,6-diMe
2-F
4.09 ± 0.05
5.90 ± 0.90
6.11 ± 0.05
0.29 ± 0.02
4.12 ± 0.15
24.3 ± 0.52
12.7 ± 3.66
1.00
4.11
2
Se
5
2.07
5.10
2g
2h
2-Cl
7
8
3-Cl
1.52 ± 0.19
1.83 ± 0.30
3.10 ± 0.73
9
2i
2j
2-F
4-F
2.02 ± 0.19
0.92 ± 0.07
0.90
3.37
6.28
10
4a
4b
4c
0.32 ± 0.04
0.27 ± 0.06
2.01 ± 0.11
6.70 ± 0.49
0.62 ± 0.12
H
11
24.81
0.87
12
13
14
15
2-Me
3-Me
3
4
0.71 ± 0.09
Se
1.56 ± 0.23
1.95 ± 0.58
11.7 ± 0.01
10.8 ± 0.77
4d
4e
0.58 ± 0.14
0.89 ±0.32
2.67
2.19
3-F
2-F
6a
6b
6c
2.32
0.74
4.40
0.43
0.25
0.28
1.30
5.04 ± 0.40
14.6 ± 2.84
H
2-Me
16
17
Br
3
3-Me
3,5-diMe
3-Cl
1.39 ± 0.71
2.17 ± 0.71
2.98 ± 0.24
1.31 ± 0.05
3.35 ± 0.83
6.12 ± 2.40
0.93 ± 0.07
0.74 ± 0.20
0.37 ± 0.03
4.36 ± 0.49
18
19
6d
Se
5
2
6e
6f
20
21
22
4-F
2-F
6g
H
8a
8b
7.39 ± 1.30
1.15
6.38 ± 0.23
1.25 ± 0.26
4.91 ± 0.34
23
24
3-Me
3,5-diMe
3-Cl
12.4 ± 1.47
2.73 ± 0.11
9.92
0.55
3
8c
8d
25
26
5
Se 2
1.71 ± 0.24
12.6 ± 1.7
1.06
0.91
1.62 ± 0.31
13.8 ± 0.2
27
4OHT
[a] To simplify comparisons between compounds in closely related series, we designate 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. [b] Relative Binding Affinity (RBA) values are determined by
compound
competitive fluorometric binding assays and are expressed as IC50estradiol/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.
To further assess the role of an additional substituent on the
selenophene core, we prepared the trisubstituted selenophene
analogues of 6a-g (series III, bromine substituted) and 8a-d
(series IV, arene substituted). The trisubstituted compounds
5
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showed an obvious increase in binding affinity for ER, with
RBA values ranging from 1 to 14, but by contrast, ERβ affinity
underwent no significant change. These trends are also similar
to those we reported earlier for furan- and thiophene-core
ligands.[34] Bromine substitution at the 3-position of the
selenophene core (6a) resulted in an 8-fold increase for ER
over 2a, and, as in series I and II, substituents at the C2-position
of phenol ring (6b, 6g) increased ER affinity, whereas those at
C3 (compounds 6c and 6e) or 3,5-disubstituted compound 6d
did the opposite, but less so than in the series I compounds. In
fact, the C2 methyl analog, 6b, had RBA values of 14.6 and 10.8
for ER and ERβ; it was the highest affinity ligand for ER,
binding more than 23 times better than that of the parent
compound 2a. The low binding affinity of compound 6f, which
had hydroxyl groups at C3-position instead of C4-position, had
lower ER binding affinities, as was the case in series I.
Figure 3. Graphical display of the RBA values for 2,5-disubstituted diphenolic
selenophenes (2a, 2c-d, 2h) and corresponding 2,3,5-triphenolic derivatives
(8a-d).
Compared to the compounds of series III, introduction of a
third phenol moiety on the selenophene core (series IV,
compounds 8a-d) did not cause a significant increase in binding
affinity for both ERα and ERβ. Nevertheless, compounds 8a-d,
with three phenolic groups on selenophene core, showed higher
binding affinity for both ERs than did the corresponding
bisphenolic group derivatives 2 (Figure 3). Overall then, the
positions of the substituent and hydroxyl group on the phenols
are of great importance for binding, with optimal affinity coming
with appropriate substitution at the C2-position on the phenols
and a hydroxyl group at the C4-position. In earlier work, we
suggested that the beneficial effect of small substituents at the
C-2 position of phenolic substituents on ligand binding was likely
due to a twisting of the phenol with respect to the ligand core, a
motion that increased the surface area of the ligand without
greatly increasing its steric size, thereby improving productive
contact between the ligand and residues in the ligand binding
pocket.[44]
Comparisons of congeneric selenophene, thiophene and
furan-core ER ligands in ERα and ERβ binding affinities have
been made in Table 2, which revealed the structure−affinity
relationships of the disubstituted selenophene, thiophenes and
furans. As
a global comparison, the importance of the
selenophene core on enhancing affinity is evident. In nearly all
cases, selenophenes show better binding affinity compared to
their corresponding thiophene and furan analogues, except
compound 2f, which still gives the high binding affinity for ERα
(5.9) and for ERβ (24.3). In addition, the importance of C2
substitution for enhancing affinity is evident (entries 2 vs 3, and
7 vs 8). This has been noted in our prior work on ER ligands,[34,
36-38]
and in a recent report by Gust and colleagues on
pyrroles.[33] Besides, the binding affinity of analogues containing
halogens (F and Cl) is better than the unsubstituted and Me
analogues (entries 2 vs 5, 3 vs 6). Other trends showed when
changing a substituent from C3- into C2-position, the binding
affinity will be better.
Table 2. Comparisons of congeneric selenophene, thiophene and furan-core ER ligands in ERα and ERβ binding affinitiesa.
6
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[a] In each table, the RBA values are given for binding to ERα and ERβ for the various biaryl selenophene, thiophenes and furans we have studied. The
disposition of the aryl groups with respect to the heteroatom (Se, S or O) is indicated by the numbers in parentheses.
The effects of the heterocycle geometry changes with the
nature of the heteroatom on the binding affinity of the ligands
were also investigated. Comparisons of bond lengths, atomic
distances and bond angles among the 2,5-disubstituted
selenophene, thiophenes and furans can be viewed more easily
in Figure 4. While it is a speculation, the higher binding affinity
might be the consequence of the increased HO−OH distance
between the two phenols in the selenophenes (13.5 Å)
compared to other ligands, which is 12.9 Å in the thiophenes
greater length of the C−Se bonds (1.9 Å) vs the C-S (1.47 Å)
and C−O bonds (1.2 Å) in the heterocyclic core (This bond
length difference also affects to a lesser degree, the display
angle of the two phenols on the heterocyclic core). Thus, the
alternate-position bisphenol selenophenes might be too long to
form adequate hydrogen bonds with His-524. When the hydroxyl
group was moved from the C4 to the C3-position of the phenyl
ring, the atomic distance of HO−OH is 11.7 Å, 11.2 Å and 10.5 Å
in selenophene, thiophene and furan, respectively (data not
shown), and the binding affinities of the corresponding ligands
were diminished (Figure 4), presumably because the atomic
HO−OH distances of HO−OH are too short.
and 12.0
Å in the furans. The O−Se distance in the
selenophenes is also considerably longer than in the alternate-
position in the thiophene and furan bisphenols, due to the
88.2°
103.8°
111.2°
F
F
F
F
F
F
S
Se
13.5
O
OH
OH
HO
OH
HO
HO
12.9
12.
0 Å
Å
Å
Thiophene
Selenophene
Furan
Bond Angle
C-Se-C
Bond Angle
C-S-C
Bond Angle
C-O-C
Bond Length
Bond Length
Bond Length
1.9 Å
1.5 Å
1.2 Å
C-X
HO OH
88.2°
103.8°
111.2°
13.5 Å
12.9 Å
12.0 Å
Figure 4. Comparisons of congeneric selenophene, thiophene and furan-core ER ligands in bond length, atomic distances and bond angle. The bond length
between the carbon and heteroatom in selenophene, thiophene and furan, and atomic distances between the two oxygen atoms in hydroxyl groups. The X is
representative of heteroatom (Se, S or O).
Transcription Activation Assays. Various selenophene-core
compounds, generally selected for their binding affinities or for
structural comparisons, were tested for their ability to stimulate
the transcriptional activities of ERα and ERβ in ER-responsive
luciferase reporter gene assays. These assays were conducted
in HEK293T cells transfected with an expression vector for
either ERα or ERβ, together with a widely used 3 × ERE-
luciferase reporter. With each compound, dose-response assays
were run in both an agonist mode (compound only) and an
antagonist mode (in the presence of 10 nM E2). The results from
both assays, summarized in Table 3, are expressed as EC50
(agonist mode) or IC50 (antagonist mode) values, together with
efficacies values (Eff (% E2)). The latter represents the ultimate
activity value at the highest concentration, compared to that for
E2; high Eff values indicate agonist activity, low values indicate
Figure 5. Illustrative dose-response curves for the ERβ agonist effects of E2,
antagonist character, and intermediate Eff values indicate partial
and three selenophene-core compounds 4b, 6a and 6b. Efficacy values (given
agonist/antagonist activity. (Note, it is difficult to establish EC50
in Table 3) are the mean ± SD from three experiments. For details, see the
values when the efficacy is low in agonist mode, and IC50 values
when efficacy is high in antagonist mode. Hence, some values
are missing) Dose-response curves for a few compounds are
shown as examples in Figure 5.
Experimental Section.
The interesting activities were seen in series I compounds, the
2,5-disubstituted diphenolic selenophenes. These ligands
displayed a wide range of activities at both ERα and ERβ.
Compound 2a was a partial antagonist of ERα and ERβ, with
nanomolar range IC50 values. Addition of a methyl group giving
2b, however, shifted activity substantially from an antagonist to
agonist (2b); in fact, 2b profiled as a superagonist on ERα and
7
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an agonist ERβ, showing efficacy on ERα 1.5 times greater than
that of estradiol (Table 3, entry 2). It is of note that we also
encountered superagonist activity in our earlier work on
thiophene-core ligands.[34] When the methyl group was shifted
from the C2- to C3-position (2c) or a second methyl group was
added (2d), the compounds are inactive on ER, as 2c and 2d
have low binding affinities to ERs.
Moreover, replacing the methyl group (2a) with a halogen
group also had obvious effects on the transcriptional activity of
the ER subtypes. The fluorine-substituted compound 2f profiled
as an ERα agonist, being about 6-fold more potent than 2b, but
with reduced efficacy. Also the chloro analogue 2g showed a
similar effect on ERα compared to 2b. However, when the chloro
was changed from the C2- to C3-position, compound 2h was still
an ERα partial agonist with higher potency compared to 2b
despite its lower affinity (Table 1, entries 8 vs 2). Surprisingly,
when the hydroxyl group was moved from the C4- to the C3-
position of the phenyl ring, compounds 2i, 2j acted as a partial
agonist at ERβ but showed less activity on ERα, with the
position of the substituent having only a minor effect on
transcriptional activity (Table 3, entries 9 and 10).
Comparisons of the position of phenolic groups in the
selenophene core indicated that the 3,4-disubstituted
compounds (series II) had decreased ER binding affinity
(Table 1, 4a-d) and also showed decreased efficacy as ER
agonists (Table 3, entries 11 vs 1; 12 vs 2). Interestingly, 4b had
improved ERβ binding affinity, as well as potency as an ERβ
agonist, whereas 4a, and 4c-d profiled as an ERβ antagonist
with IC50 values in the nanomolar (nM) range.
Addition of a third substituent onto the selenophene core also
had major effects on transcriptional activity. Introduction of the
bromine atom to 2a, converted it from antagonist into an ER full
agonist/ERβ partial agonist (compound 6a, Table 3, entries 1 vs
16). In fact, 6a displayed low nanomolar potency for activating
both ERs. However, when selenophenes had substituents on
the phenolic group, the trend of transcriptional activity is still
similar to compounds 2 (Table 3, entries 17 vs 2). For example,
2-methyl derivative 6b was also a substantial ERβ antagonist
activity, and analogues 6f also profiled as a very effective ERα
agonist, and analogues 6c-e also profiled as ERα antagonists.
Of note is that compound 6a showed about a 10-fold improved
binding affinity for ERα and increased efficacy, compared to 2a
and 4a. By contrast, the C3-hydroxyl analogue 6f showed
improved ERα binding affinity (Tables 1, entries 21 vs 10), as
well as efficacy; actually, 6f profiled as superagonist on ERα,
showing efficacy in ERα 2.2 times greater than that of estradiol
(Table 3, entry 21).
8
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Table 3. Effects of selenophene-core compounds on the transcriptional activities of Estrogen Receptora and antiproliferative activity in MCF-7 cells.
Agonist Modeb Antagonist Modec, d
ERα
ERβ
ERα
ERβ
series
MCF-7
VERO
entry
cmpd
2a
2b
2c
2d
2e
2f
EC50 (μM)
Eff (%E2)
16 ± 2
157 ± 12
33 ± 6
12 ± 4
-7 ± 3
EC50 (μM)
Eff (%E2)
11 ± 4
82 ± 7
24 ± 3
38 ± 6
60 ± 4
71 ± 1
14 ± 5
22 ± 8
55 ± 5
42 ± 10
IC50 (μM)
Eff (%E2)
44 ± 10
96 ± 13
103 ± 28
148 ± 2
-22 ± 8
59 ± 10
73 ± 8
IC50 (μM)
Eff (%E2)
Inhibition(%)e
IC50 (μM)f
-
-
0.52
0.028
35 ± 4
50 ± 11
98 ± 14
86 ± 13
66 ± 3
91 ± 6
74 ± 5
51 ± 16
89 ± 6
79 ± 3
0
16.6 ± 3.89
>50g
>50
1
2
3
4
5
6
7
8
9
1.36
0.079
-
0.3
3.4
9.3
0
-
-
-
-
-
-
-
-
>50
-
0.434
0.17
-
0.425
5.391
19.3
10.5
19.6
5.3
17.9
4.4
>50
I
0.23
1.28
0.04
-
68 ± 2
44 ± 13
49 ± 3
25 ± 8
25 ± 8
-
-
>50
2g
2h
2i
-
-
>50
-
-
-
100 ± 14
75 ± 7
-0.25
>50
0.13
0.15
-
-
>50
2j
-
0.72
68 ± 2
>50
10
11
12
13
14
15
4a
4b
4c
4d
4e
-
7 ± 3
19 ± 4
32 ± 8
21 ± 6
35 ± 5
-
16 ± 7
62 ± 8
54 ± 10
21 ± 3
17 ± 2
-
-
-
-
-
57 ± 2
80 ± 10
46 ± 9
33 ± 6
67 ± 3
0.475
49 ± 9
94 ± 10
50 ± 5
10 ± 7
58 ± 6
0
0
0
0
0
>50
>50
>50
>50
>50
-
0.034
-
0.32
1.147
0.341
0.001
-
II
-
-
-
-
6a
6b
6c
6d
6e
6f
0.015
69 ± 11
21 ± 5
0.013
40 ± 5
69 ± 9
46 ± 1
15 ± 6
27 ± 2
31 ± 7
50 ± 3
-
161 ± 22
93 ± 11
8 ± 1
2.53
75 ± 4
25 ± 8
0
>50
>50
>50
>50
>50
>50
>50
16
17
18
19
20
21
22
0.79
0.009
-
1.33
1.58
0.561
-
2.96
2.5
26.1
26.5
11.3
0
-
-3 ± 1
0.441
0. 51
21 ± 5
-
16 ± 4
-
-
26 ± 2
-
-
-
-
18 ± 13
125 ± 20
69 ± 16
59 ± 8
III
-
0.12
-
-23 ± 3
226 ± 28
73 ± 9
19 ± 2
0.3
-
127 ± 42
87 ± 6
6g
-
8.1
8a
8b
-
-4 ± 3
-
12 ± 3
77 ± 8
10.4
-
-7 ± 2
0.111
-
23 ± 3
82 ± 2
29.6
16.6
>50
>50
23
24
IV
0.32
112 ± 29
0.033
104 ± 29
9
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8c
8d
0.27
1.8
74 ± 4
148 ± 2
35 ± 3
0.017
0.061
-
69 ± 5
73 ± 14
-1 ± 1
0.87
-
27 ± 1
74 ± 19
35 ± 3
-
-
79 ± 7
91 ± 8
-20 ± 2
75.3
18.6
81.7
>50
>50
25
26
27
4OHT
0.008
0.003
0.001
15.1 ± 5.2
[a] Luciferase activity was measured in HEK293T cells transfected with 3 × ERE-driven luciferase reporter and expression vectors encoding ERα or ERβ and treated in triplicate. [b] In the agonist mode, increasing
concentrations of compounds alone (up to 10-5 M) are given. EC50 values and standard deviations (mean ± SD from three replicates) represent the concentrations producing 50% of the maximal response to the compound on
ERα or ERβ. Eff (% E2) is the maximal response to the compound as a percentage of the response to 10-8 M 17β-estradiol (E2). [c] In the antagonist mode, increasing concentrations of compounds (up to 10-5 M) are given
together with a maximally stimulating dose of E2 (10-8 M). IC50 values and standard deviations (mean ± SD from three replicates) represent the concentrations that reduce the E2 response by 50%. Eff (% E2) is the response to
the compound at the highest concentration as a percentage of the activity of 10-8 M 17β-estradiol (E2). [d] 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. [e] Relative inhibition rate of antiproliferative potencies determined by
MTT assays. The final concentration of the compounds is 10 μM. [f] IC50 values are an average of at least three independent experiments ± standard deviation (mean ± SD). [g] IC50 not determinable up to highest
concentrations tested.
10
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The most interesting activities were seen with the triaryl-
substituted selenophenes. When the bromo substituent was
replaced by a phenol moiety, the transcriptional activity was
residue Arg394, and more variably, through hydrogen bonding of
the D-ring hydroxyl group with helix 11 residue His524 (Figure
6A). This ligand-binding orientation allows helix 12 to dock
almost increased. Thus, 8a profiled as an antagonist on the ERs, across helices 3 and 11, where it forms one side of a
showing efficacy in ERα about 2 times greater than that of 2,5-
diphenolic analogue 2a (Table 3, entries 23 vs 1); 8b-d profiled
as agonists on both ERs, with increased efficacy compared to
corresponding the 2,5-diphenolic derivatives, with only 8c also
showing some antagonistic activity in ERα. 8b and 8d having
substituents (Me or Cl) at C3-position of phenolic group, were
more efficacious than estradiol, being ERα superagonists.
hydrophobic groove for the transcriptional coactivator binding
site on the surface that constitutes the functional core of AF2. In
contrast, SERMs such as 4-hydroxytamoxifen, directly or
indirectly obstruct the agonist position of helix 12, relocating it
out of this position and thereby blocking the recruitment of
transcriptional coactivators.
We recently reported on the crystal structure of oxabicyclic
Antiproliferative Activity on Breast Cancer and Normal Cells. compound OBHS, where one of phenols, which mimics the role
To evaluate the antiproliferative activity of these compounds,
most of the selenophene-core compounds were screened
against MCF-7 breast cancer cells, in which 4OH-tamoxifen acts
as an reference compound and also in normal (non-cancerous
and ER-negative) kidney epithelial cells (VERO[45]); the results
are summarized in Table 3.
In general, all the compounds in Table 3 that showed no
obvious antagonistic activities on ERα or ERβ on transcriptional
activities, exhibited weak or no inhibition of MCF-7 cells.
Compounds 2b and 2j, which displayed certain antagonist
activity on ERα, were essentially inactive, presumably due to
their poor binding affinities for ERα, with RBA values were less
than 1%. Trisubstituted selenophenes 6a-b, which possess high
binding affinities, however, show weak inhibition for MCF-7 cells.
This suggests that binding affinity and antiproliferative potency
are independent. Interestingly, modifications of position of
substituent (compound 6c) or addition of a second methyl group
of A-ring phenol of E2, engages in strong hydrogen bonding with
the helix 3 residue Glu353 and the helix 6 residue Arg394.
Beyond this, however, the large non-polar phenyl sulfonate
group in OBHS extends between helices 8 and 11, into a region
in which it makes strong steric clashes with helix 11 and
indirectly modulates the conformation of the critical helix 12
(Figure 6B), giving it partial antagonist activity, but limited
agonist activity.39
With reference to these modes of ligand binding, our modeling
shows that 2,5-disubstituted diphenolic selenophene 2f can
similarly form hydrogen bonds between one of the phenolic
hydroxyl groups with helix 3 residue Glu353 and helix 6 residue
Arg394; the second phenol also makes interaction with helix 11,
but it does so by forming hydrogen bond contacts with the helix
11 residue Gly521 (Figure 6C). In contrast to OBHS, however,
compound 2f lacks ER antagonist activity and is a full ER
agonist. Based on our model, we suggest that the hydrogen
(compound 6d) led to compounds having antiproliferative activity. bonding interaction with Gly521 constrains helix 11 in a manner
Furthermore, introduction of the third phenolic group
(compounds 8a-d) resulted in compounds having even more
promising antiproliferative activity. However, the most potent
compound in suppressing the proliferation of MCF-7 cell was 8c
with an inhibitory ratio of 75.3%, which was close to that of 4OH-
tamoxifen, probably due to its excellent antagonistic potency on
ERα.
Healthy VERO cells, commonly used for toxicity studies on
normal cells, were used for toxicity studies[46-48]; epithelial cells
from normal (non-cancerous) breast are very difficult to culture.
Notably, most of selenophene-core compounds, except 2a, were
nontoxic to healthy VERO cells, while 4OHT showed
considerable toxicity, consistent with the antiproliferative effect
of compound 8c being through ERα.
that does not further destabilize helix 12; rather, it mimics the
role of the D-ring phenol E2, which stabilizes helix 12 in the
agonist conformation. In support of this conjecture, we recently
published on a series of thiophene ligands, in which one of the
phenol groups also extended between helices 8 and 11. These
thiophenes showed pronounced agonist activity, which on
further study also appeared to stem from interactions with helix
11 that stabilized helix 12 in the agonist conformation.38
Interestingly, when a third phenol moiety is introduced into the
selenophene core, as in the triphenolic selenophene 8b (Figure
6D), our modeling reveals a possible novel mechanism of
agonism via the second phenol, which makes an interaction with
residue Gly420 of helix 8, thereby avoiding the clash with helix
11, but this does not explain its superagonist activity. We
presume that the superagonist activity of this compound was
due to an “improved” positioning of helix 8 that further stabilizes
helix 12 in the agonist conformation to an even greater degree
than does the binding of E2. Therefore, contact with helix 8 may
represent a novel epitope to generate an ERα superagonist.
Models for Selenophenes Binding to the Estrogen Receptor
α
Ligand Binding Pocket and a Possible Model for
Superagonism. E2 fits into the ERα ligand-binding pocket in an
orientation in which the A-ring phenolic hydroxyl has hydrogen
bonding interactions with helix 3 residue Glu353 and helix 6
11
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Figure 6. Model of selenophene ligands bound to ERα and comparisons with estradiol and OBHS. (A) Crystal structure of the ERα LBD in complex with E2 (PDB:
1ERE). The A-ring phenolic hydroxyl group of E2 forms hydrogen bonding interactions with helix 3 residue Glu353 and helix 6 residue Arg394, while the D-ring
17β alcohol interacts with the helix 11 residue His524. (B) Crystal structure of the ERα LBD in complex with oxabicyclic heptane sulfonate (OBHS) bound ERα.
OBHS H-bonds to the conserved Glu353 and Arg394 residues. The phenyl sulfonate extends outward between helices 8 and 11.39 (C) Computer-developed
model of 2f bound to ERα with conserved H-bonding to Glu353 and Arg394. The second phenolic group H-bonds to Gly521 on helix 11, which stabilizes helix 12
in the agonist conformation. (D) Computer-developed model of 8b bound to ERα with the conserved H-bonding to Glu353 and Arg394, and the second phenolic
group with H-bonds to Gly420 on helix 8, avoiding the clash with helix 11.
modeling suggests that interaction of one of the phenolic
hydroxyl groups with a residue in helix 8 might be responsible
Conclusions
for the superagonism of members of this ligand class, and this
represents a novel epitope for the design ERα superagonists.
Compared with the approved anti-breast cancer drug tamoxifen,
8c compounds exhibited substantial anti-proliferative potency in
breast cancer MCF-7 cell lines. Compound 2f has the highest
binding affinity for ERβ, but its anti-proliferative activity in breast
cancer MCF-7 cells is modest. Aside from compound 2a, no
compounds were toxic to non-transformed VERO cells, while
tamoxifen showed, at least to some extent, inherent toxicity on
these cells.
Our generation of this new selenophene series of ER ligands
provides important insights into the diversity of structures that
function as ER ligands and might lead to improved therapeutics
that target the estrogen receptor. While ER remains an
important pharmaceutical target for the treatment of breast
cancer, the quest for the “ideal” drugs for this disease will
certainly remain a challenge due to the fact that breast cancer is
a high complex disease.
To further explore anti-breast cancer drugs that might have
superior efficacy and fewer side effects than tamoxifen or other
therapeutic agents, we present the synthesis of a novel series of
selenophene-core compounds. This is the first report of
selenophene-core compounds as ER ligands, and we have
investigated their ER binding affinities, estrogen response
element-driven transcriptional activity, and cell antiproliferative
activity. Ligands in this selenophene-core compound class can
be easily prepared by Suzuki coupling reactions between
various bromoselenophenes and various aryl boronic acids.
Careful analysis of their ER binding affinity showed that the
selenophene core ligands are largely ERβ-selective, which is
similar to that reported earlier for ligands having thiophene and
furan cores. As before, the position of the phenolic group has a
marked effect on their binding affinity for ERβ, but in terms of
absolute affinity, most of the selenophenes have higher RBA
values for ER than those of the earlier classes. In transcription
assays, most of selenophenes exhibit partial or full ERβ agonist
activity, whereas on ERα, these ligands display a wide range of
activities, covering antagonists and agonists, with some
selenophenes even profiling as superagonists. Molecular
Experimental Section
12
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General. Starting materials were purchased from Aldrich, Acros,
Aladdin-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 argon atmosphere unless otherwise specified.
116.00, 16.27. HRMS (MALDI/DHB) calcd for C18H17O2Se (M + H+)
344.0444, found 344.0448.
2,5-Bis(4-hydroxy-3,5-dimethylphenyl)selenophene (2d). Compound
2d was prepared by 2,5-bis(4-methoxy-3,5-dimethylphenyl)selenophene
(1d) and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 3:1) gave
the title compound as a blue solid, 86% yield, mp 268-270 °C. 1H NMR
(400 MHz, Acetone-d6) δ 7.50 (s, 2H), 7.34 (s, 2H), 7.22 (s, 4H), 2.27 (s,
12H). 13C NMR (100 MHz, Acetone-d6) δ 154.14, 149.24, 129.00, 126.74,
125.39, 125.36, 16.67. HRMS (MALDI/DHB) calcd for C20H21O2Se (M +
H+) 372.0757, found 372.0761.
Reaction progress
was
monitored
by
analytical thin-layer
chromatography (TLC). Visualization was achieved by UV light (254 nm).
The characterization data for compounds 1, 3, 5, 7, and 9-12 were given
in Supporting Information.
1H NMR and 13C NMR spectra were measured on a Bruker Biospin
AV400 (400 MHz, 1H NMR; 100 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 an X-
4 Beijing Tech melting point apparatus, and the data were uncorrected.
The purity of all compounds for biological testing was determined by
HPLC method (see Supporting Information), confirming >95% purity.
2,5-Bis(4-hydroxy-2,6-dimethylphenyl)selenophene (2e). Compound
2e was prepared by 2,5-bis(4-methoxy-2,6-dimethylphenyl)selenophene
(1e) and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 3:1) gave
the title compound as a yellow solid, 85% yield, mp 213-215 °C. 1H NMR
(400 MHz, Acetone-d6) δ 8.31 (s, 2H), 6.90 (s, 2H), 6.63 (s, 4H), 2.18 (s,
12H). 13C NMR (100 MHz, Acetone-d6) δ 157.87, 149.82, 139.52, 129.88,
127.91, 115.07, 21.16. HRMS (MALDI/DHB) calcd for C20H21O2Se (M +
H+) 372.0754, found 372.0761.
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 (0.1
equiv), sodium carbonate (2 equiv) in an oxygen-free 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).
2,5-Bis(2-fluoro-4-hydroxyphenyl)selenophene (2f). Compound 2f was
prepared by 2,5-bis(2-fluoro-4-methoxyphenyl)selenophene (1f) and
boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a blue solid, 78% yield, mp 206-208 °C. 1H NMR (400 MHz,
Acetone-d6) δ 8.02 (s, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.8 Hz,
2H), 6.90 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, Acetone-d6) δ 159.26,
129.84, 129.78, 127.37, 127.73, 113.24, 104.21, 103.96. HRMS
(MALDI/DHB) calcd for C16H11O2F2Se (M + H+) 351.9942 found 351.9946.
General procedure for Ether Cleavage. Under Ar 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 reaction mixture was poured into water, and
extracted with ethyl acetate. The extracts were dried (Na2SO4) and
evaporated. The residue was purified by silica gel column
chromatography (CC).
2,5-Bis(2-chloro-4-hydroxyphenyl)selenophene (2g). Compound 2g
was prepared by 2,5-bis(2- chloro-4-methoxyphenyl)selenophene (1g)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a blue solid, 74% yield, mp 160-162 °C. 1H NMR (400 MHz,
Acetone-d6) δ 9.21 (s, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.44 (s, 2H), 7.03 (d,
J = 8.8 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, Acetone-d6)
δ 155.03, 153.51, 153.01, 149.92, 131.55, 129.93, 129.42, 128.54,
127.62, 126.83, 126.53, 126.13, 118.09, 117.64, 117.25. HRMS
(MALDI/DHB) calcd for C16H11F2O2Se (M + H+) 384.9351 found 384.9355.
2,5-Bis(4-hydroxyphenyl)selenophene (2a). Compound 2a was
prepared by 2,5-bis(4-methoxyphenyl)selenophene (1a) and boron
tribromide according to general procedure for ether cleavage. Purification
by CC (petroleum ether:ethyl acetate = 3:1) gave the title compound as a
blue solid, 72% yield, mp 137-139°C. 1H NMR (400 MHz, Acetone-d6) δ
7.46 (d, J = 8.8 Hz, 4H), 7.36 (s, 2H), 6.89 (m, 4H). 13C NMR (100 MHz,
Acetone-d6) δ 158.22, 149.14, 129.01, 127.89, 125.60, 116.70. HRMS
(MALDI/DHB) calcd for C16H13O2Se (M + H+) 316.0053, found 316.0057.
2,5-Bis(3-chloro-4-hydroxyphenyl)selenophene (2h). Compound 2h
was prepared by 2,5-bis(3-chloro-4-methoxyphenyl)selenophene (1h)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a blue solid, 73% yield, mp 168-170 °C. 1H NMR (400 MHz,
CD3OD) δ 7.46 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.30 (d, J =
8.4 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.21 (s, 2H),
6.87 (d, J = 8.4 Hz, 1H), 6.79 (t, J = 8.4 Hz, 2H). 13C NMR (100 MHz,
CD3OD) δ 158.63, 147.03, 132.73, 132.66, 129.62, 127.03, 117.84,
115.89. HRMS (MALDI/DHB) calcd for C16H11Cl2O2Se (M + H+) 384.1154
found 384.1156.
2,5-Bis(4-hydroxy-2-methylphenyl)selenophene (2b). Compound 2b
was prepared by 2,5-bis(4-methoxy-2-methylphenyl)selenophene (1b)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a yellow solid, 86% yield, mp 176-178 °C. 1H NMR (400
MHz, Acetone-d6) δ 8.48 (s, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.13 (s, 2H),
6.79 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 2.40 (d, J = 8.4 Hz, 6H).
13C NMR (100 MHz, Acetone-d6) δ 157.95, 149.73, 137.59, 132.39,
129.06, 128.40, 118.33, 113.92, 21.61. HRMS (MALDI/DHB) calcd for
C18H17O2Se (M + H+) 344.0444, found 344.0448.
2,5-Bis(2-fluoro-5-hydroxyphenyl)selenophene (2i). Compound 2i was
prepared by 2,5-bis(2-fluoro-5-methoxyphenyl)selenophene (1i) and
boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a yellow solid, 70% yield, mp 206-208 °C. 1H NMR (400
MHz, DMSO-d6) δ 9.65 (s, 2H), 7.15 (s, 2H), 7.14 (m, 4H), 6.75 (d, J =
8.8 Hz, 2H). 13C NMR (100 MHz, Acetone-d6) δ 154.82, 129.17, 124.79,
124.65, 117.85, 116.64, 116.55, 114.41. HRMS (MALDI/DHB) calcd for
C16H11F2O2Se (M + H+) 351.9947 found 351.9946.
2,5-Bis(4-hydroxy-3-methylphenyl)selenophene (2c). Compound 2c
was prepared by 2,5-bis(4-methoxy-3-methylphenyl)selenophene (1c)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a blue solid, 88% yield, mp 194-196 °C. 1H NMR (400 MHz,
Acetone-d6) δ 8.46 (s, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.33 (s, 2H), 7.27 (d,
J = 8.8 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 2.24 (s, 6H). 13C NMR (100 MHz,
Acetone-d6) δ 156.19, 149.19, 129.12, 129.05, 125.77, 125.43, 125.20,
2,5-Bis(4-fluoro-5-hydroxyphenyl)selenophene (2j). Compound 2j was
prepared by 2,5-bis(4-fluoro-5-methoxyphenyl)selenophene (1j) and
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201600593
ChemMedChem
FULL PAPER
boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a yellow solid, 72% yield, mp 175-177 °C. 1H NMR (400
MHz, Acetone-d6) δ 8.94 (s, 2H), 7.50 (m, 2H), 7.26 (d, J = 8.4 Hz, 2H),
7.17 (m, 4H). 13C NMR (100 MHz, Acetone-d6) δ 152.14, 149.24, 146.13,
134.02, 127.46, 118.48, 117.48, 115.93. HRMS (MALDI/DHB) calcd for
C16H11F2O2Se (M + H+) 351.9947 found 351.9946.
2H), 7.49 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 8.4 Hz,
2H), 6.87 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, CD3OD) δ 159.10,
158.82, 149.74, 141.81, 131.61, 131.42, 128.85, 128.10, 127.36, 116.88,
116.43, 116.33, 108.08. HRMS (MALDI/DHB) calcd for C16H12BrO2Se (M
+ H+) 394.9237 found 394.9240.
3-Bromo-2,5-bis(4-hydroxy-2-methylphenyl)selenophene
(6b).
Compound 6b was prepared by 3-bromo-2,5-bis(4-methoxy-2-
methylphenyl)selenophene (6b) and boron tribromide according to
general procedure for ether cleavage. Purification by CC (petroleum
ether:ethyl acetate = 3:1) gave the title compound as a yellow solid, 79%
yield, mp 52-54 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.59 (s, 2H), 7.26
(d, J = 8.4 Hz, 1H), 7.15 (d, J = 7.6 Hz, 2H), 6.81 (t, J = 8.8 Hz, 2H), 6.75
(d, J = 8.4 Hz, 2H), 2.41 (s, 3H), 2.23 (s, 3H). 13C NMR (100 MHz,
Acetone-d6) δ 154.89, 154.57, 149.50, 141.47, 129.88, 128.46, 127.44,
126.79, 126.68, 125.59, 125.07, 107.38, 16.67, 16.62. HRMS
(MALDI/DHB) calcd for C18H16BrO2Se (M + H+) 422.9548 found 422.9553.
3,4-Bis(4-hydroxyphenyl)selenophene (4a). Compound 4a was
prepared by 3,4-bis(4-methoxyphenyl)selenophene (3a) and boron
tribromide according to general procedure for ether cleavage. Purification
by CC (petroleum ether:ethyl acetate = 3:1) gave the title compound as a
blue solid, 61% yield, mp 144-147°C. 1H NMR (400 MHz, Acetone-d6) δ
8.24 (s, 2H), 7.76 (s, 2H), 6.85 (d, J = 8.4 Hz, 4H), 6.59 (d, J = 8.4 Hz,
4H). 13C NMR (100 MHz, Acetone-d6) δ 157.31, 144.98, 131.00, 128.54,
115.75. HRMS (MALDI/DHB) calcd for C16H13O2Se (M + H+) 317.2253,
found 317.2259.
3,4-Bis(4-hydroxy-2-methylphenyl)selenophene (4b). Compound 4b
was prepared by 3,4-bis(4-methoxy-2-methyl phenyl)selenophene (3b)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a yellow solid, 51% yield, mp 151-153 °C. 1H NMR (400
MHz, Acetone-d6) δ 8.20 (s, 2H), 7.84 (s, 2H), 6.83 (d, J = 8.0 Hz, 2H),
6.58 (d, J = 7.6 Hz, 2H), 6.52 (d, J = 7.2 Hz, 2H), 6.50 (d, J = 7.2 Hz, 6H),
2.01 (s, 6H). 13C NMR (100 MHz, Acetone-d6) δ 157.14, 145.56, 138.21,
132.37, 128.53, 117.31, 112.95, 20.63. HRMS (MALDI/DHB) calcd for
C18H17O2Se (M + H+) 345.0394, found 345.0398.
3-Bromo-2,5-bis(4-hydroxy-3-methylphenyl)selenophene
(6c).
Compound 6c was prepared by 3-bromo-2,5-bis(4-methoxy-3-
methylphenyl)selenophene (5c) and boron tribromide according to
general procedure for ether cleavage. Purification by CC (petroleum
ether:ethyl acetate = 3:1) gave the title compound as a blue solid, 82%
yield, mp 155-157 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 2H), 7.21
(d, J = 8.4 Hz, 2H), 7.10 (s, 1H), 6.72 (s, 2H), 6.68 (d, J = 8.0 Hz, 1H),
6.66 (d, J = 8.4 Hz, 1H), 2.36 (s, 3H), 2.17 (s, 3H). 13C NMR (100 MHz,
Acetone-d6) δ 156.93, 156.64, 149.47, 141.43, 132.30, 129.07, 128.63,
128.49, 128.41, 127.51, 126.05, 125.52, 125.24, 116.09, 115.62, 107.50,
16.27, 16.23. HRMS (MALDI/DHB) calcd for C18H16BrO2Se (M + H+)
422.9548 found 422.9553.
3,4-Bis(4-hydroxy-3-methylphenyl)selenophene (4c). Compound 4c
was prepared by 3,4-bis(4-methoxy-3-methylphenyl)selenophene (3c)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether : ethyl acetate = 3:1) gave the title
compound as a blue solid, 68% yield, mp 148-151 °C. 1H NMR (400 MHz,
Acetone-d6) δ 8.21 (s, 2H), 7.88 (s, 2H), 6.98 (s, 2H), 6.75 (d, J = 8.4 Hz,
2H), 6.69 (d, J = 8.4 Hz, 2H), 2.14 (s, 6H). 13C NMR (100 MHz, Acetone-
d6) δ 155.20, 145.11, 132.33, 130.84, 128.33, 128.21, 124.55, 114.97,
16.35. HRMS (MALDI/DHB) calcd for C18H17O2Se (M + H+) 345.0394,
found 345.0395.
3-Bromo-2,5-bis(4-hydroxy-3,5-dimethylphenyl)selenophene
(6d).
Compound 6d was prepared by 3-bromo-2,5-bis(4-methoxy-3,5-
dimethoxyphenyl)selenophene (5d) and boron tribromide according to
general procedure for ether cleavage. Purification by CC (petroleum
ether:ethyl acetate = 3:1) gave the title compound as a blue solid, 86%
yield, mp 199-201 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.64 (d, J = 8.4
Hz, 2H), 7.39 (s, 1H), 7.25 (s, 4H), 2.29 (s, 6H), 2.28 (s, 6H). 13C NMR
(100 MHz, Acetone-d6) δ 154.88, 154.56, 149.50, 141.48, 129.89, 128.46,
127.45, 126.69, 125.59, 125.07, 107.38, 16.70, 16.65. HRMS
(MALDI/DHB) calcd for C20H20BrO2Se (M + H+) 450.9864 found 450.9866.
3,4-Bis(3-fluoro-4-hydroxyphenyl)selenophene (4d). Compound 4d
was prepared by 3,4-bis(3-fluoro-4-methoxy phenyl)selenophene (3d)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a blue solid, 58% yield, mp 167-169 °C. 1H NMR (400 MHz,
CDCl3) δ 8.76 (s, 2H), 8.01 (s, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.91 (d, J =
7.6 Hz, 1H), 6.67 (d, J = 7.2 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H). 13C NMR
(100 MHz, CDCl3) δ 151.79, 144.66, 143.56, 131.39, 129.92, 126.19,
118.35, 117.38. HRMS (MALDI/DHB) calcd for C16H11F2O2Se (M + H+)
352.9892, found 352.9895.
3-Bromo-2,5-bis(3-chloro-4-hydroxyphenyl)selenophene
(6e).
Compound 6e was prepared by 3-bromo-2,5-bis(3-chloro-4-methoxy-
phenyl)selenophene (5e) and boron tribromide according to general
procedure for ether cleavage. Purification by CC (petroleum ether:ethyl
acetate = 3:1) gave the title compound as a gray solid, 72% yield, mp 98-
100 °C. 1H NMR (400 MHz, Acetone-d6) δ 9.33 (s, 2H), 7.64 (d, J = 8.8
Hz, 2H), 7.52 (s, 1H), 7.44-7.39 (m, 2H), 7.08 (d, J = 8.8 Hz, 2H). 13
NMR (100 MHz, Acetone-d6) δ 156.19, 149.19, 129.12, 129.05, 125.77,
125.43, 125.20, 116.00. HRMS (MALDI/DHB) calcd for C16H10Cl2O2Se
(M + H+) 462.8326 found 450. 8327.
C
3,4-Bis(2-fluoro-4-hydroxyphenyl)selenophene (4e). Compound 4e
was prepared from 3,4-bis(2-fluoro-4-methoxy phenyl)selenophene (3e)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a blue solid, 54% yield, mp 168-171 °C. 1H NMR (400 MHz,
Acetone-d6) δ 8.08 (s, 2H), 7.01 (s, 2H), 6.57 (d, J = 8.4 Hz, 1H), 6.55 (d,
J = 8.4 Hz, 1H), 6.52 (d, J = 8.4 Hz, 2H), 13C NMR (100 MHz, CDCl3) δ
163.73, 161.32, 155.95, 140.16, 132.01, 131.91, 105.39, 102.64. HRMS
3-Bromo-2,5-bis(4-fluoro-5-hydroxyphenyl)selenophene
Compound 6f was prepared by 3-bromo-2,5-bis(4-fluoro-5-
(6f).
methoxyphenyl)selenophene (5f) and boron tribromide according to
general procedure for ether cleavage. Purification by CC (petroleum
ether:ethyl acetate = 3:1) gave the title compound as a yellow solid, 70%
yield, mp 80-82 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 10.22
(s, 1H), 7.63 (s, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H),
7.16 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H),
7.01 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, Acetone-d6) δ 148.0, 145.7,
143.3, 141.0, 130.2, 121.5, 119.4, 118.4, 117.5, 117.3, 117.1, 116.9,
116.8, 116.6, 116.0. HRMS (MALDI/DHB) calcd for C16H10BrF2O2Se (M +
H+) 430.1023 found 430.1025.
(MALDI/DHB) calcd for C16H11F2O2Se (M
352.9895.
+
H+) 352.9892, found
3-Bromo-2,5-bis(4-hydroxyphenyl)selenophene (6a). Compound 6a
was prepared by 3-bromo-2,5-bis(4-methoxyphenyl)selenophene (5a)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a blue solid, 68% yield, mp 193-195 °C. 1H NMR (400 MHz,
DMSO-d6) δ 9.91 (s, 1H), 9.89 (s, 1H), 7.56 (s, 1H), 7.52 (d, J = 8.0 Hz,
3-Bromo-2,5-bis(2-fluoro-4-hydroxyphenyl)selenophene
Compound 6g was prepared from 3-bromo-2,5-bis(4-fluoro-5-
(6g).
14
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201600593
ChemMedChem
FULL PAPER
methoxyphenyl)selenophene (5g) and boron tribromide according to
general procedure for ether cleavage. Purification by CC (petroleum
ether:ethyl acetate = 3:1) gave the title compound as a yellow solid, 75%
yield, mp 80-82 °C. 1H NMR (400 MHz, CD3OD) δ 7.57 (m, 2H), 7.35 (d,
J = 8.4 Hz, 1H), 7.06 (d, J = 9.9 Hz, 2H), 6.93 (t, J = 8.3 Hz, 2H), 6.76 (d,
J = 10.0 Hz, 1H), 6.65 (m, 2H). 13C NMR (101 MHz, Acetone-d6) δ
161.10, 159.87, 159.18, 158.64, 132.56, 132.52, 128.94, 128.84, 112.48,
111.64, 103.26, 103.01. HRMS (MALDI/DHB) Calcd for C16H9BrF2O2Se
( M + H+) 430.9053 found 430.9052
icy bath, the supernatant was applied to a column of GSH-resin. The
collection was dialyzed in ice buffer for 4 h. After being checked by a
combination
of
sodium
dodecyl
sulfate–polyacrylamide
gel
electrophoresis and Western blotting, the protein was prepared as a 10-
mM stock in potassium phosphate and stored at −80 °C.[49]
Estrogen Receptor Binding Affinity. Relative binding affinities were
determined by a competitive fluorometric binding assay as previously
described. Briefly, 40 nM fluorescence tracer (coumestrol, Sigma-Aldrich,
MO) and 0.8 μM purified human ERα or ERβ ligand binding domain
(LBD) were diluted in 100 mM potassium phosphate buffer (pH 7.4),
containing 100 μg/mL bovine gamma globulin (Sigma-Aldrich, MO).
Incubations were for 2h at room temperature (25 °C). Fluorescence
polarization values were then measured. The binding affinities are
expressed as relative binding affinity (RBA) values with the RBA of 17-β
estradiol set to 100%. The values given are the average ± range of two
independent determinations. IC50 values were calculated according to
equations described previously.[50]
2,3,5-Tris(4-hydroxyphenyl)selenophene (8a). Compound 8a was
prepared by 2,3,5-tris(4-methoxyphenyl)selenophene (7a) and boron
tribromide according to general procedure for ether cleavage. Purification
by CC (petroleum ether:ethyl acetate = 3:1) gave the title compound as a
blue solid, 88% yield, mp 114-116 °C. 1H NMR (400 MHz, CD3OD) δ 7.42
(m, 2H), 7.36 (s, 1H), 7.10 (m, 4H), 6.80 (m, 2H), 6.71 (m, 4H). 13C NMR
(100 MHz, CD3OD) δ 158.43, 157.85, 157.38, 148.47, 142.57, 141.19,
131.43, 131.35, 130.84, 129.47, 129.33, 128.81, 128.12, 116.72, 116.22,
116.12. HRMS (MALDI/DHB) calcd for C22H17O3Se (M + H+) 407.0388
found 407.0397.
Gene Transcriptional Activity. The human embryonic kidney cell
lines, HEK 293T, was maintained in Dulbecco’s Minimum Essential
Medium (DMEM) (Gibco by Invitrogen Corp., CA) with 10% fetal bovine
serum (FBS) (Hylcone by Thermo Scientific, UT). Cells were plated in
phenol red-free DMEM with 10% FBS. HEK 293T cells were transfected
with 25 μL mixture per well, containing 300 ng of 3 × ERE-luciferase
reporter, 100 ng of ERα or ERβ expression vector, 125 mM calcium
chloride (GuoYao, China) and 12.5 μL 2 × HBS. The next day, the cells
were treated with increasing doses of ER ligands diluted in phenol red-
free DMEM with 10% FBS. After 24h, luciferase activity was measured
using Dual-Luciferase Reporter Assay System (Promega, MI) according
to the manufacturer’s protocol.
2,3,5-Tris(4-hydroxy-3-methylphenyl)selenophene (8b). Compound 8b
was prepared by 2,3,5-tris(4-methoxy-3-methylphenyl)selenophene (7b)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a black solid, 84% yield, mp 38-40 °C. 1H NMR (400 MHz,
Acetone-d6) δ 8.48 (s, 1H), 8.34 (s, 1H), 8.26 (s, 1H), 8.12 (s, 1H), 7.30
(d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 2H), 6.85
(d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 8.4 Hz, 2H), 2.25
(s, 3H), 2.16 (s, 3H), 2.14 (s, 3H). 13C NMR (100 MHz, Acetone-d6) δ
156.31, 155.70, 155.25, 147.91, 141.98, 140.70, 132.37, 132.25, 130.27,
129.10, 128.89, 128.84, 128.77, 128.42, 128.33, 125.77, 125.24, 125.15,
124.88, 116.01, 115.47, 115.34, 16.30, 16.25, 16.22. HRMS
(MALDI/DHB) calcd for C25H23O3Se (M + H+)450.0857 found 450.0866.
Cell Culture and Cell Viability Assay. The human breast cancer cell
lines MCF-7 was obtained from ATCC. Cells were maintained in DMEM
with 10% FBS. For all experiments, cells were grown in 96-well microtiter
plates (Nest Biotech Co., China) with appropriate ligand triplicate for 72h.
MTT colormetric tests (Biosharp, China) were employed to determine cell
viability per manufacturer instructions. IC50 values were calculated
according to the following equation using Origin software: Y = 100%
inhibition + (0% inhibition - 100% inhibition)/(1 + 10[(LogIC50-X)×Hillslope]),
where Y = fluorescence value, X = Log [inhibitor]. [50]
2,3,5-Tris(4-hydroxy-3,5-dimethylphenyl)selenophene (8c). Compound
8c
was
prepared
by
2,3,5-tris(4-methoxy-3,5-
dimethylphenyl)selenophene (7c) and boron tribromide according to
general procedure for ether cleavage. Purification by CC (petroleum
ether:ethyl acetate = 3:1) gave the title compound as a blue solid, 83%
yield, mp 76-78 °C. 1H NMR (400 MHz, Acetone-d6) δ 7.53 (s, 1H), 7.45
(s, 1H), 7.43 (s, 1H), 7.31 (s, 1H), 7.26 (s, 2H), 6.93 (d, J = 8.4 Hz, 4H),
2.28 (s, 6H), 2.17 (s, 6H), 2.14 (s, 6H). 13C NMR (100 MHz, Acetone-d6)
δ 167.50, 155.89, 153.51, 153.45, 131.03, 128.15, 126.23, 121.83,
121.73, 121.63, 112.56, 112.49, 112.40, 112.33, 35.49, 31.44. HRMS
(MALDI/DHB) calcd for C28H29O3Se (M + H+) 491.1328 found 491.1336.
Molecular Modeling. Crystal structure of ER LBD in complex with E2
was downloaded from the protein data bank (PDB ID: 1ERE).
Compounds 2f, and 8b were docked into the three-dimension structure of
ERα LBD with AutoDock software (version 4.2).[51-52] Crystallographic
coordinates of the 2f, and 8b were created by Biochemoffice. The crystal
structure of ERα LBD was obtained from the OBHS-bound ER crystal
structure and all water molecules were removed.[35] Preparations of all
ligands and the protein were performed with AutoDockTools (ADT). A
docking cube with the edge of 60 Å, 60 Å, 58 Å in X, Y, Z dimension
respectively (a grid spacing of 0.375 Å), which encompassed the whole
active site, was used throughout docking. On the basis of the Lamarckian
genetic algorithm (LGA), 80 runs were performed for each ligand with
500 individuals in the population. The figures were prepared using
PyMOL.
2,3,5-Tris(3-chloro-4-methoxyphenyl)selenophene (8d). Compound 8d
was prepared by 2,3,5-tris(3-chloro-4-methoxyphenyl)selenophene (7d)
and boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a yellow solid, 78% yield, mp 48-50 °C. 1H NMR (400 MHz,
Acetone-d6) δ 10.48 (s, 2H), 10.29 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.67
(s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.8
Hz, 1H), 7.10-7.06 (m, 2H), 7.05 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 8.4 Hz,
1H), 6.88 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, Acetone-d6) δ 153.76,
153.46, 153.02, 147.33, 141.84, 140.22, 131.13, 131.09, 130.91, 129.93,
129.79, 129.58, 127.79, 126.75, 121.82, 121.25, 121.06, 118.11, 117.75,
117.52. HRMS (MALDI/DHB) calcd for C22H14Cl3O3Se (M
510.9223 found 510.9228.
+
H+),
Acknowledgements
We are grateful to the NSFC (81373255, 31371331, 81573279),
Key Scientific Research Project of Ministry of Education of China
(313040), Hubei Province’s Outstanding Medical Academic
Leader Program, and Hubei Province Engineering and
Technology Research Center for Fluorinated Pharmaceuticals
Gene Clone and Protein Purification. Human ERα or ERβ ligand
binding domain (LBD) genes were amplified by PCR from plasmid pVP-
16-ERα and pVP-16-ERβ. The PCR product was cloned into plasmid,
and PGEx-KG E.coli BL21(DE3) used for the overexpression of ER-LBD.
The cells were induced by IPTG (10 μM) for 2 h, then cells were
harvested, frozen, and thawed in phosphate-buffered saline (PBS),
containing 1 mM EDTA and 1 mM DTT. After being ultrasonicated in an
15
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and to the National Institutes of Health (PHS 5R01 DK015556 to
J.A.K.) for support of this research.
Keywords: selenophenes
•
selective estrogen receptor
modulators• diversity of structure • relative binding affinity (RBA)
• breast cancer
16
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Entry for the Table of Contents
Add a New Element! This is the first report of selenophene-core compounds as ER ligands. In transcription assays, several
selenophenes profiling as superagonists for ERα; moreover, a few selenophenes exhibited antiproliferative activity comparable to that
of 4OHT in breast cancer MCF-7 cells while being nontoxic to normal VERO cells. These new ligands could act as models for the
development of novel agents leading to improved therapeutics that target the estrogen receptor.
18
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