Thiophene-core estrogen receptor ligands having superagonist activity

Article abstract of DOI:10.1021/jm400157e

To probe the importance of the heterocyclic core of estrogen receptor (ER) ligands, we prepared a series of thiophene-core ligands by Suzuki cross-coupling of aryl boronic acids with bromo-thiophenes and we assessed their receptor binding and cell biological activities. The disposition of the phenol substituents on the thiophene core, at alternate or adjacent sites, and the nature of substituents on these phenols, all contribute to binding affinity and subtype selectivity. Most of the bis(hydroxyphenyl)-thiophenes were ERβ selective, whereas the tris(hydroxyphenyl)-thiophenes were ERα selective; analogous furan-core compounds generally have lower affinity and less selectivity. Some diarylthiophenes show distinct superagonist activity in reporter gene assays, giving maximal activities 2-3 times that of estradiol, and modeling suggests that these ligands have a different interaction with a hydrogen-bonding residue in helix-11. Ligand-core modification may be a new strategy for developing ER ligands whose selectivity is based on having transcriptional activity greater than that of estradiol.

Full text of DOI:10.1021/jm400157e

Article
pubs.acs.org/jmc
Thiophene-Core Estrogen Receptor Ligands Having Superagonist
Activity
Jian Min,† Pengcheng Wang,† Sathish Srinivasan,‡ Jerome C. Nwachukwu,‡ Pu Guo,† Minjian Huang,†
Kathryn E. Carlson,§ John A. Katzenellenbogen,*,§ Kendall W. Nettles,‡ and Hai-Bing Zhou*,†
†Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, State Key
Laboratory of Virology, Wuhan University School of Pharmaceutical Sciences, Wuhan 430072, China
‡Department of Cancer Biology, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States
§Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: To probe the importance of the heterocyclic core
of estrogen receptor (ER) ligands, we prepared a series of
thiophene-core ligands by Suzuki cross-coupling of aryl boronic
acids with bromo-thiophenes and we assessed their receptor
binding and cell biological activities. The disposition of the
phenol substituents on the thiophene core, at alternate or
adjacent sites, and the nature of substituents on these phenols,
all contribute to binding affinity and subtype selectivity. Most of
the bis(hydroxyphenyl)-thiophenes were ERβ selective, whereas
the tris(hydroxyphenyl)-thiophenes were ERα selective; analo-
gous furan-core compounds generally have lower affinity and
less selectivity. Some diarylthiophenes show distinct superagonist activity in reporter gene assays, giving maximal activities 2−3
times that of estradiol, and modeling suggests that these ligands have a different interaction with a hydrogen-bonding residue in
helix-11. Ligand-core modification may be a new strategy for developing ER ligands whose selectivity is based on having
transcriptional activity greater than that of estradiol.
estrogen action without the increased risk of cancer from
stimulation of the breast and uterus.14−18 Despite the similarity
of their ligand-binding pockets (LBPs),19−21 considerable
success has been achieved in obtaining ER ligands having
affinity or potency preferences for one or the other ER subtype
(Figure 1),14 but thus far preferential efficacy or intrinsic activity
has been only in favor of ERα.22 The medical utility of ERβ
subtype-selective estrogens, however, is less clear.18
Prior to the discovery of ERβ, efforts to obtain tissue-
selective estrogens 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
was believed to arise from their differential engagement of
coregulator proteins in a target cell- and gene-specific
manner,23 and compounds such as tamoxifen (Figure 1), as
well as raloxifene, lasofoxifene, and basedoxifene, were found to
be largely agonistic in bone and antagonistic in breast but
having varying activity in the uterus.
INTRODUCTION
■
The beneficial physiological actions of estrogens on develop-
ment and functional maintenance of many target tissues
(reproductive system, bone,1 vasculature,2 brain,3 etc.4) are
well recognized, as are the pathological roles that they play in
promoting breast and uterine cancers.5−8 This spectrum of
effects in diverse tissues provides an intriguing opportunity for
the creation of tissue-selective estrogens that on balance have
net desirable vs undesirable activities such as ones that would
provide bone and cardiovascular protection without stimulation
of breast or uterus. The challenge of creating such selective
estrogens has, over time, been approached in different ways.
The actions of estrogens are mediated by two members of
the nuclear receptor superfamily,9 the estrogen receptor (ER)
subtypes, ERα and the more recently identified ERβ.10 These
subtypes have distinct tissue distributions (ERα mainly in the
female reproductive system and ERβ mainly in prostate, colon,
cardiovascular, and central nervous systems11), and 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.12,13 Thus, much
effort has been made to develop ER subtype-selective ligands
having tissue- and gene-selective biological activities. A major
focus has been on obtaining ERβ-selective compounds because
this ER subtype is considered more likely to mediate desirable
In the development of both SERMs and subtype-selective
ligands, extensive investigation has been made of nonsteroidal
compounds having heterocyclic cores. In fact, while the
Received: January 30, 2013
© XXXX American Chemical Society
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Figure 1. Estradiol and examples of some ERα (left) and ERβ-selective (right) ligands and a SERM.
Figure 2. Structure and binding affinity data of the most promising pyrazole, furan, and imidazole and the title thiophene compounds. Binding,
determined by competitive radiometric binding assays, is expressed as relative binding affinity (RBA) values, with estradiol (E2) as 100. α/β is the
ratio of ERα RBA to ERβ RBA.
peripheral substituents, typically phenols, simple alkyl groups,
and basic and polar phenyl substituents, have generally
remained the same, a wide variety of heterocyclic cores has
been explored. We and others have examined ER ligand cores
consisting of five-membered ring heterocycles5,24−37 and to a
lesser extent six-membered ring heterocycles.38 Some examples
of the five-membered ring heterocycle series are presented
below (Figure 2).
We noted that the highest affinity ligands had five-membered
heterocyclic cores of low polarity (furan > pyrazole >
imidazole), presumably reflecting both the penalty for desol-
vation to remove the ligand from water to bind to ER, as well as
an intolerance of the receptor ligand binding pockets for core
units having large dipole moments.22 Also, in the pyrazole and
furan series, we mapped quite extensively the requirements for
high ERα binding affinity and concluded that tri and even tetra
substitution was required.24,30 Thiophenes, by contrast, have
been relatively unexplored, and the only two prepared in our
prior studies had lower levels of substitution and quite low
affinities for ER.30 Hartmann and co-workers have reported on
certain disubstituted thiophenes as inhibitors of 17β-hydrox-
ysteroid dehydrogenase type1 (17β-HSD1), but they also
showed very low ER binding affinity,39,40 consistent with our
prior observations.
In connection with recent work on a series of very different
ER ligands, based on an inherently three-dimensional 7-thia-
bicyclo[2.2.1]hept-5-ene-7-oxide core, we prepared a series of
3,4-disubstituted thiophene precursors. These thiophenes were
used as diene components in an in situ oxidative Diels−Alder
reaction with various dienophiles to give the sulfoxide-bridged
bicyclic system.41 As part of that work, we tested the binding
affinity of some of the diaryl-substituted thiophene Diels−Alder
diene precursors (4a−d), and we were pleased to find that they
showed significant ER binding affinities, in some cases with
modest ERβ selectivity.
To pursue this lead further, we have now extended our
investigation of thiophene-core ER ligands by making system-
atic modifications of the number, nature, and orientation of
substituents on these ligands, examining their binding affinity
and cellular potencies for ERα and ERβ; some congeneric
furan-core ligands were also prepared for comparison. We have
been able to map structure−activity relationships (SARs) for
these compounds, and most intriguingly, we have identified
among these thiophene-core ligands, compounds that have
B
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a
Scheme 1. Synthesis of 2,5-Substituted Series Compounds 2a−m
a
Reagents and conditions: (a) [Pd] catalyst, Na2CO3, toluene/water (1:1), reflux, 24 h; (b) 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
thiophene core; locant positions on the thiophene core itself are given by numbers in italics.
striking superagonist activity. Superagonism, which is intrinsic
activity greater than that of a paradigmatic agonist, in this case
the endogenous hormone estradiol (E2), has only scant
precedent in the nuclear receptor field (see Discussion). The
enhanced intrinsic activity of ligands showing superagonism
might provide a novel mechanism for the development of
estrogens having desirable selective activity that is comple-
mentary to the reduced intrinsic activity of SERMs.42 In
addition, molecular modeling of these novel ligands complexed
to ER offers a framework for understanding the structural basis
for their superagonist activity and might reveal a general
strategy for obtaining superagonists for other members of the
nuclear receptor superfamily.
substituents on the phenyl groups. We also display these locant
positions clearly in indicator structures in the schemes and
tables. In addition, for clarity, the locant positions at which the
aryl groups are attached to the thiophene or furan core rings are
given in italic typeface.)
Chemical Synthesis. Thiophenes. In the synthesis of
compounds 2a−m (Scheme 1), key intermediates 1a−m were
obtained by a one-step double Suzuki cross-coupling
reaction.43,44 Compounds 1a−d and 1g−k were easily obtained
using Pd(OAc)2/PPh3 as catalyst. This reaction failed, however,
with compounds 1e and 1f, and attempts to employ the two-
step Suzuki coupling using Pd(OAc)2/PPh3 also gave very low
overall yields of 1e and 1f, accompanied by a large amount of
homo coupled diaryl byproducts. In a brief catalyst screen, we
found that while Pd2(dba)2, Pd(PPh3)Cl2, and Pd(CN)2Cl2 did
not give improved yields, Pd(dppf)Cl2 gave the products 1e
and 1f in 45% and 62% yields, respectively, with little
byproducts (Scheme 1A). We presume that this improvement
is due to the increased rate of reductive elimination resulting
from the increased bite angle of the dppf ligand.45 Unsym-
metrical compounds 1l and 1m were obtained by a two-step
Suzuki coupling (Scheme 1B). In the first step, 1 equiv of
commercially available 2,5-dibromothiophene reacted with 2
equiv of aryl boronic acid under standard conditions to give the
monosubstituted thiophene 19, and a second cross-coupling
with 2 equiv of aryl boronic acid gave the intermediates 1l−m.
Finally, treatment of the compounds 1a−m with boron
RESULTS
■
(We have deviated somewhat from the IUPAC rules in
specifying the locant positions of the hydroxyl, methyl, and
halogen substituents on the phenyl groups attached to the core
heterocycle (i.e., thiophene or furan): Strict application of
IUPAC rules is awkward because it causes locant positions on
the phenyl groups to change when the nature of the substituent
changes from −OMe (the thiophene or furan is the root) to
−OH (the phenol becomes the root). Thus, to facilitate
comparisons across compound series and discuss structure−
activity relationships in a more understandable way, we have
opted to use consistently the heterocyclic core (thiophene or
furan) as the root name, regardless of the nature of the
C
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a
Scheme 2. Synthesis of 3,4-, 2,4-, 2,3-Substituted Thiophenes 4a−f, 6a−b, and 8a−b
a
Reagents and conditions: (a) [Pd] catalyst, Na2CO3, toluene/water (1:1), reflux, 24 h; (b) 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
thiophene core; locant positions on the thiophene core itself are given by numbers in italics.
tribromide gave the final 2,5-disubstituted diphenolic thio-
phenes 2a−m in good yields.
coupling reactions (Scheme 3B). In the first step, 1 equiv of
commercially available 2,3,5-tribromothiophene was reacted
with 4 equiv of aryl boronic acid using standard conditions, and
the resulting 2,5-bis-substituted thiophenes 21 and 22 were
subsequently submitted to a second cross-coupling reaction
with 2 equiv of phenyl boronic acid to yield the intermediates
11a−b.
In the synthesis of 3,4-, 2,4-, 2,3-disubstituted thiophene
derivatives 4a−f, 6a−b, and 8a−b (Scheme 2), we used
Pd(OAc)2/PPh3 for the preparation of 3a−d but again turned
to Pd(dppf)Cl2 as catalyst for the preparation of compounds
3e−f, 5a−b, and 7a−b, which were obtained in moderate to
good yields from the appropriate dibromothiophene precursor.
Ether cleavage gave final products 4a−f, 6a−b, and 8a−b in
60−80% yields.
In our previous work on furan-core ER ligands,30 we found
that triaryl, especially trisphenol, furans showed higher binding
affinity and subtype selectivity than the corresponding
bisphenol analogues. Thus, we also wanted to introduce a
third phenol ring into the thiophene-core compounds. Key
intermediates 13a−c were prepared using 1 equiv of 2,3,5-
tribromothiophene with 5 equiv of aryl boronic acid using
standard conditions; ether cleavage then gave compounds 14a−
c in very good yields (Scheme 3C).
To investigate the effect of a third substituent on the
thiophene core, we synthesized a series of trisubstituted
thiophene compounds 10a−d, 12a−b, and 14a−c (Scheme
3). 3-Methyl-2,5-dibromothiophene (20) was first synthesized
by bromination of 3-methylthiophene using NBS.46 A one-step
Suzuki coupling with Pd(dppf)Cl2 afforded 9a−d, which gave
the products 10a−d in good yield after ether cleavage (Scheme
3A). 3-Phenyl bisphenol thiophenes 12a−b were obtained by
demethylation of 11a−b, prepared by two successive cross-
Furans. For comparison with selected thiophenes, we
prepared their furan-core counterparts (16a−d and 18a−d,
Scheme 4). 2,5-Dibromofuran (23) was synthesized by
D
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a
Scheme 3. Synthesis of Trisubstituted Compounds 10a−d, 12a−b, and 14a−c
a
Reagents and conditions: (a) [Pd] catalyst, Na2CO3, toluene/water (1:1), reflux, 24 h; (b) BBr3, CH2Cl2, −20 °C to rt, 4 h; (c) NBS, THF/AcOH
(1:1), rt, 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 thiophene core; locant positions on the thiophene core itself are given by numbers in italics.
bromination of furan using DMF as solvent with dropwise
addition of Br2 (Scheme 4A).47 3,4-Dibromofuran (24) was
synthesized by oxidation of commercially available (E)-2,3-
dibromo-2-butane-1,4-diol using chromosulfuric acid (Scheme
4B).48 Because 3,4-dibromofuran decomposes under the
reaction conditions, it was continuously removed from the
reaction mixture by steam distillation and stored below 0 °C as
a white solid. We first used Suzuki coupling reaction conditions
similar to those used for the thiophenes, but we failed to get to
corresponding furans. Considering the lower aromaticity of
furan compared to thiophene, we then switched to Pd(PPh3)4
as Pd(0) catalyst.49 The dibromofurans were coupled with 5
equiv of the aryl boronic acid and then deprotected with boron
tribromide to give the final products 16a−d and 18a−d in
moderate to good yields.
1
The identity of all final compounds were confirmed by H
NMR, 13C NMR, and mass spectroscopy (see Experimental
Section).
Biological Results. (See comment on nomenclature at the
start of the Results section)
Relative Binding Affinities. The relative binding affinities
were determined by a competitive radiometric receptor-binding
assay and are reported in Tables 1−3. These affinities are
presented as relative binding affinity (RBA) values, where
estradiol has an affinity of 100. For reference, the KD of
estradiol is 0.2 nM for ERα and 0.5 nM for ERβ. (The affinities
E
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a
Scheme 4. Synthesis of Corresponding Furan-Core Compounds 16a−d and 18a−d
a
Reagents and conditions: (a) Na2CO3, THF/water (1:1), Pd(PPh3)4, reflux, 12 h; (b)BBr3, CH2Cl2, −20 °C to rt, 4 h; (c) K2Cr2O7, H2SO4, H2O,
steam-distillation; (d) Br2, DMF, 30−35 °C, overnight. To simplify comparisons between compounds in closely related series, we designate locant
positions of the substituents on the phenyl groups with respect to the thiophene core; locant positions on the thiophene core itself are given by
numbers in italics.
for a subset of compounds representing the different modes of
diaryl core substitution are compared schematically in Figure
3.)
to the phenol) bound better than the corresponding non-
substituted compound 2a. The highest affinity ERβ ligand, 2,5-
bis(2-fluoro-4-hydroxyphenyl)thiophene (2e), binds to ERβ
with an affinity of 33 and a 16-fold ERβ binding affinity
preference (Table 1, entry 5). The highest affinity ERα ligand
in the series 2 was the 2-chloro analogue 2f, with an RBA of 6.7,
but it still showed ERβ selectivity (RBA of 10 for ERβ, Table 1,
entry 6).
The thiophenes having substituents at the C3 position (i.e.,
ortho to the phenol) all show dramatically decreased binding
affinities compared to the corresponding ones substituted at C2
(i.e., meta to the phenol). Their affinities are even lower than
that of the corresponding unsubstituted compound 2a (Table
1, entry 1), with the tetra ortho methyl compound 2d binding
particularly poorly (Table 1, entry 4). This is very likely due to
steric occlusion of the hydrogen bonding capacity of the
phenols by ortho substitution, which is known to greatly reduce
affinity in both steroidal and other nonsteroidal estrogens.50
The position of the hydroxyl group is also of great
importance, and when this group is moved from the para to
the meta position of the phenyl ring (e.g., 2i−k), binding
affinities are remarkably lowered (Table 1, entries 9−11), again
consistent with known trends in other ligand series. Unsym-
metrically substituted compounds (e.g., 2l and 2m) also had
somewhat lower binding affinities than their corresponding
symmetrical counterparts 2e and 2f (Table 1, entries 5 and 6).
Like of 2,5-disubstituted compounds (series 2), similar
trends were also observed for the 3,4-disubstituted (series 4),
2,4-disubstituted (series 6), and 2,3-disubstituted compounds
(series 8). The highest affinities were those substituted at C2,
meta to the phenol, and the lowest, those with C3 (ortho)
substitution or no substitution. Chlorine substitution gave the
highest affinity for both ERs (4f and 6b, Table 1, entries 19 and
Figure 3. Comparisons of ERα and ERβ binding affinities of
congeneric bisphenolic thiophene and furan-core ER ligands. In each
table, the RBA values are given for binding to ERα or [ERβ] for the
various biaryl thiophenes and furans we have studied. The top tables
are for the alternate-substituted systems and the bottom tables for the
adjacent-substituted systems. The disposition of the aryl groups with
respect to the heteroatom (S or O) is indicated by the numbers in
parentheses, which are color coded to enable reference to be made
between the structures and the appropriate columns in the tables.
A number of the bis(hydroxyphenyl)thiophenes (Table 1)
show moderate levels of ERβ selectivity, which is a general
characteristic of rather slender ligands.14 In terms of absolute
affinity, however, substituents on the phenol rings have major
effects on their binding affinity and selectivity. Among the 2,5-
bisphenol thiophenes (series 2), those substituted at C2 (meta
F
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a
Table 1. ERα and ERβ Relative Binding Affinity (RBAs) of Compounds 2a−m, 4a−f, 6a−b, and 8a−b
a
To simplify comparisons between compounds in closely related series, we designate locant positions of the substituents on the phenyl groups with
b
respect to the thiophene core; locant positions within the thiophene core itself are given by numbers in italics. Relative binding affinity (RBA)
values are determined by competitive radiometric binding assays and are expressed as IC50estradiol/IC50
× 100
the range or standard
compound
deviation (RBA, estradiol = 100%). In these assays, the KD for estradiol is 0.2 nM on ERα and 0.5 nM on ERβ.51 For details, see the Experimental
Section.
21). Other comparisons among the disubstituted thiophenes
(and furans) can be viewed more easily in Figure 3.
Thus, compounds 14a−c show ERα selectivity, with 14c having
an affinity for ERα of 86 and a selectivity of 14, which is similar
to that reported earlier for the triphenolic furans and
pyrazoles.24,30 This reversal of ER subtype binding preference
as additional substituents are added to the heterocycle core
likely reflects the fact that the ligand-binding pocket of ERα is
larger than in ERβ.24,30 Also, because members of the tris-
phenol series (series 14) bound better than the phenyl bis-
phenols (series 12), we inferred that the third phenolic
hydroxyl group might be involved in a hydrogen bond with
another residue in the ligand binding pocket, most likely T347
in ERα. Similar enhancement in binding with a third phenolic
To investigate the influence of an additional substituent on
the thiophene core, trisubstituted thiophenes 10a−d, 12a−b,
and 14a−c were prepared (Table 2). Introduction of a methyl
group at the 3-position of the thiophene core (Table 2, 10a−d)
did not cause a significant change in RBA but appeared to cause
a decrease in selectivity for ERβ (10a vs 2a, 10b vs 2b, 10c vs
2e, 10d vs 2f). When the methyl group was replaced by a
phenyl group, as with compounds 12a−b, there was a further
decrease in binding for ERβ (RBA of ERβ 12a 2.1 vs 2e 33, 12b
3.0 vs 2f 10), and when the third substituent is replaced by a
phenol moiety, the subtype selectivity was actually reversed.
G
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Table 2. ERα and ERβ Relative Binding Affinity (RBAs) of
Compounds 10a−d, 12a−b, and 14a−c
Table 3. ERα and ERβ Relative Binding Affinity (RBAs) of
Furan-Core Compounds 16a−d and 18a−b
a
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 thiophene core; locant positions within the
b
thiophene core itself are given by numbers in italics. Relative binding
a
affinity (RBA) values are determined by competitive radiometric
To simplify comparisons between compounds in closely related
compound
binding assays and are expressed as IC50estradiol/IC50
× 100
series, we designate locant positions of the substituents on the phenyl
groups with respect to the thiophene core; locant positions within the
thiophene core itself are given by numbers in italics. Relative binding
the range or standard deviation (RBA, estradiol = 100%). In these
assays, the KD for estradiol is 0.2 nM on ERα and 0.5 nM on ERβ.51
For details, see the Experimental Section.
b
affinity (RBA) values are determined by competitive radiometric
binding assays and are expressed as IC50estradiol/IC50
× 100
compound
the range or standard deviation (RBA, estradiol = 100%). In these
assays, the KD for estradiol is 0.2 nM on ERα and 0.5 nM on ERβ.51
For details, see the Experimental Section.
halogens, Cl and F, bind better than the proteo and Me
analogues, with Cl being better than F in most cases. Curiously,
despite their considerably different shapes, no clear pattern
emerges in comparing the alternate vs adjacent substitution
patterns (upper vs lower table), the two types of alternate or
adjacent substitutions (blue vs red in the thiophene tables), nor
are there systemic differences between the furans and the
congeneric thiophenes (right vs left). In addition to the
comparisons illustrated in this figure, other trends noted above
were the increased affinity and ERα preference that comes from
adding a third substituent, especially a phenol, onto the
thiophene and furan cores.
We will return to this type of comparative analysis where we
discuss the results of the cellular activity of these compounds
and when we present examples of docking representative
members into the ERα ligand-binding pocket and examine
ligand volumes, geometries, and conformations.
Transcriptional Activation Assays. The thiophene- and
furan-core compounds that have significant affinity for ERα or
ERβ were characterized for activation of an estrogen response
element (ERE)-driven luciferase reporter in HepG2 liver cells
cotransfected with ERα or ERβ; this is considered to be a
versatile cell system for characterizing the efficacy of ER
ligands.54 Dose−response curves were generated, and when full
curves were evident, potency (EC50) values and efficacy (Eff, %
of E2) values were determined. The results are summarized in
Table 4, and dose−response curves for representative ligands,
are shown in Figure 4.
Alternate-Position Aryl-Disubstituted Thiophenes (2,5-
Diaryl Series 2 and 2,4-Diaryl Series 6). The thiophenes in
these two regioisomeric series are congeneric except for the
position of the sulfur atom in the thiophene ring. In the
reporter gene assays, the series 2 (2,5-bisphenol-substituted)
hydroxyl group can be found in the furan and pyrazole systems
that we have previously investigated.24,30
Finally, to compare the affinity of thiophene-core ligands
with furan-core ligands, we prepared a limited series of
bis(hydroxylphenyl)furans (16a−d and 18a−d, Table 3).
Gratifyingly, most of these disubstituted furans retained the
ERβ selectivity of the corresponding thiophenes. The best
furan, compound 16c, had an RBA of 6.9 and 32 for ERα and
ERβ, respectively, giving a 4.7-fold ERβ selectivity (Table 3,
entry 3). Where direct comparisons can be made, however, the
furans (except for compound 16c) have lower affinity and
poorer selectivity than their corresponding thiophenes. This
might be due to the higher electron negativity of the oxygen
atom, making the furan core more polar than the thiophene
core, and/or the longer length of the C−S vs C−O bond,
which somewhat alters the geometric disposition of the
substituents.
Global comparisons of structure−affinity relationships
among the disubstituted thiophenes and furans can better be
made by reference to Figure 3, which displays an overlay of the
two core substitution patterns that unite congeneric (position-
ally isomeric) compounds, an “alternate” pattern, with the
phenols attached to nonadjacent positions (2,5- and 2,4-
patterns of substitution), and an “adjacent” pattern, with
phenols on neighboring positions (2,3- and 3,4- patterns of
substitution). RBA values for ERα and [ERβ] are shown to the
right. In all cases, the importance of C2 substitution on
enhancing affinity is evident. This has been noted in our prior
work on ER ligands with bicyclic cores41,52,53 and in a recent
report by Gust and colleagues on pyrroles.29 Beyond this, the
H
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a
Table 4. Potency and Intrinsic Activity of Thiophene and Furan Core Ligands
HepG2
Ishikawa
ERα
ERβ
ERα
compd
core
R
EC50 (μM)
Thiophenes
2.35
Eff (% E2)
EC50 (μM)
Eff (% E2)
198
Eff (% E2)
2a
2b
2e
2f
2,5-
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
H
125
4
1.54
6.60
4
111 32
148 23
315 125
169 14
2-Me
78
4
87 15
336 63
169 18
277 15
2-F
181 14
2-Cl
2.18
0.47
1.91
111
117
78
5
8
3
1.94
0.61
1.92
2l
2-Me, 2′-F
2,Me, 2′-Cl
8
5
7
2m
111
7
102
4a
4b
4c
4e
4f
3,4-
H
2.25
3.15
69
4
1.27
1.87
73
67
70
29
57
2
4
4
4
3
46
4
2-Me
3-Me
2-F
97 11
120 14
73
79
90
5
3
7
4
63
90
0
5
3
0.12
0.780
0.203
2-Cl
0.045
6a
6b
2,4-
2,3-
2,5-
2-F
1.06
123
6
1.34
177 17
117
201 13
2-Cl
0.563
87
5
0.660
2
81
5
8a
8b
2-F
65.9
69
82
3
8
33 10
67
6
6
2-Cl
1.16
5.70
116 14
114
10a
10b
10c
10d
3-Me
3-Me
3-Me
3-Me
H
1.52
116
70
5
5
1
1
1.23
2.46
200 10
106 23
284 44
225 27
56
260 53
72
2-Me
2-F
0.439
0.447
0.101
4
149
82
10.8
2-Cl
0.295
113
4
8
12a
12b
2,5-
3-Ph
3-Ph
2-F
0.615
0.066
70
72
1
2
39 11
91 15
2-Cl
1
1
92
4
14a
14b
14c
2,3,5-
H
0.485
0.387
0.009
82
84
52
6
5
5
2.23
−22
−1
−13
1
4
2
145
63
9
2
1
2-F
2-Cl
35
Furans
16a
16b
16c
16d
2,5-
3,4-
H
62
36
3
4
7
7
94 26
91 21
58
Me
F
3.79
3.89
8
6
8
154
82
159 13
61 48
Cl
106
3
118 11
18a
18b
18c
18d
H
10.6
81
97
73
86
2
5
2
6
37
56
5
3
82 25
Me
F
0.944
3.64
5.11
17.5
104
85 11
116
6
85 16
54
Cl
0.266
4
9
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 thiophene core; locant positions within the thiophene core itself are given by numbers in italics. Values indicating pronounced
superagonism (i.e., Eff values ≥120) are indicated in boldface type.
thiophenes mostly profiled as superagonists on both ERα and
ERβ, showing efficacies in ERβ 2−3 times greater than that of
estradiol (e.g., see compound 2l, Figure 4B). To test whether
the superagonist activity was cell-type specific, compounds were
also tested at a single, high dose (10 μM) for activation of the
ERE-luciferase reporter in Ishikawa endometrial cells that
contain native ERα, and here again most of the series 2 tested
profiled as superagonists (Table 4).
Although these compounds have affinities in the low
nanomolar range, most of their potencies in the HepG2 assays
were in the micromolar range, reflecting the fact that cellular
potency involves the affinities of the receptor both for the
ligand and for the recruited coregulators that provide the
essential signal for activation of transcription.55 In addition, the
liver-derived HepG2 cells are known to metabolize small
molecules, which sometimes reduces their cellular potency.56,57
With respect to the effects of substitutions on the phenolic
groups, the 2-methyl analogue 2b displayed lower potency than
2a despite its higher affinity. Thus, the lack of superagonist
activity for 2b in the HepG2 cells may reflect the fact that
maximal activity (i.e., saturation) was not reached at the doses
we tested. While the 2-Cl substitution in 2f had little effect on
activity, the 2-F containing analogue 2e displayed even greater
superagonist activity. The other alternate-position thiophene
regioisomers, series 6, differ only in the location of the sulfur
atom in the thiophene core from series 2. Nevertheless, the
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(Fulvestrant) does not have inverse agonist activity on ERβ
(Figure 4C, black line).
A summary of efficacy values for the alternate and adjacent
disubstituted thiophenes and furans are presented in Figure 5,
Figure 4. Activity profile of ERα selective compounds. HepG2 cells
were transfected with 3xERE-luc and expression plasmids for ERα or
ERβ and then dispensed in 384-well plates. The next day, cells were
treated with the indicated compounds for 24 h and assayed for
luciferase activity. Compound with ERα (red solid line), with ERβ
(blue solid line); estradiol with ERα (red dashed line, panel B only),
with ERβ (blue dashed line, panel B only); ICI with ERβ (black line,
panel C only).
Figure 5. Comparisons of ERα and ERβ intrinsic activities (Eff % of
E2) of congeneric bisphenolic thiophene and furan-core ER ligands.
Maximal activities above 120 are indicated in boldface. In each table,
the %Eff values are given for the reporter gene assays of the various
biaryl thiophenes and furans we have studied with ERα or [ERβ]. The
top tables are for the alternate-substituted systems and the bottom
tables for the adjacent-substituted systems. The disposition of the aryl
groups with respect to the heteroatom (S or O) is indicated by the
numbers in parentheses, which are color coded to enable reference to
be made between the structures and the appropriate columns in the
tables.
superagonist activity for the 2-F compound 6a was attenuated
with respect to the series 2 regioisomers, with the 2-Cl
substituted 6b having only a full agonist profile. Further
interpretation of the structural basis of superagonism is
presented in the Discussion.
according to the same layout as were the binding affinities
(Figure 3); efficacy values above 120 are noted in boldface. The
predominance of superagonism in the alternate compared to
the adjacent mode thiophenes and in the thiophenes compared
to the furans is very evident. To ascertain that the effects of
these compounds are ER mediated, we confirmed their activity
in a mammalian 2 hybrid assay of ER-coactivator binding and
also by the induction of a canonical ER target gene, GREB1
(see Supporting Information). A proposed structural model for
superagonist activity is presented in the Discussion.
The congeneric 2,5-disubstituted furan series 16 displayed
less agonist activity, but this may reflect the lower potency and
lack of curve saturation at the highest dose tested (10 μM).
Again, these differences in potency were not related to affinity,
as was the case with the corresponding compounds in series 2.
Adjacent-Position Aryl-Disubstituted Thiophenes (3,4-
Diaryl Series 4 and 2,3-Diaryl 8) and Trisubstituted
Thiophenes. Thiophenes in these series contain substitutions,
on adjacent positions on the thiophene, drastically changing the
overall shape of these ligands compared to those of the
alternate-position ligand. Also, compared to the planar ground-
state conformation of the alternate-position ligands, steric
interaction of the adjacent-disposed aryl groups also causes
substantial (ca. 30°) twisting of the phenols out of the plane of
the thiophene ring. These compounds displayed variable partial
to full agonist activity profiles, with little evidence of
superagonist activity. Surprisingly, 4e and 4f displayed >25-
fold better EC50 values on ERα and ERβ than the
corresponding 8a and 8b despite affinity differences of only
∼2-fold.
The effects of adding a third substituent onto the core were
very dramatic. For the methyl-substituted series 10, the
efficacies were similar to series 2, but with substantial increases
in potency for ERα. The phenyl 12 or phenol 14 series as well
displayed selective activation of ERα. In fact, compounds 12b
and 14c (Figure 4A,D) displayed low nM potency for activating
ERα. All of these changes in potency reflect the trends in
binding affinity. Notably, while 12b had no activity on ERβ up
to 10 μM, 14a and 14c displayed inverse agonist activity on
ERβ (Figure 4C,D), representing to the best of our knowledge
first-in-class ligands. Even the full antagonist ICI 182,780
DISCUSSION
■
The X-ray structures of many ER−ligand complexes show a
pocket volume that is considerably larger than the ligand
volume, with a ligand such as E2 appearing to be “suspended”
by close contacts and hydrogen bonding at its ends (the A and
D rings) but having relatively few close contacts at its middle
(B and C rings). The ER can form a variety of pocket shapes
upon binding structurally different ligands, and these shapes
relate to the overall conformation of the ER−ligand complex,
defining its interaction with coregulators, and ultimately
determining its biological output. To understand more fully
the connections between ligand shape, ligand-binding pocket
shape, and biological output of the ER, we have examined the
respective contributions that the core vs the peripheral
substituents of an ER ligand makes to its affinity, potency,
efficacy, and ER-subtype selectivity in a number of systems.
Reviving Thiophene-Core Ligands. In this work, we have
extended our examination of ER ligands having five-membered
ring heterocyclic cores such as isoxazoles,58 imidazoles,58
pyrazoles,23,41 and furans,30 to thiophenes. Previously, we
found that these five-membered, as well as some related six-
membered ring heterocyclic cores, afforded high affinity when
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the core had low polarity (or a small dipole moment) and when
there were a sufficient number of peripheral substituents.
Larger ligands showed distinct preferential affinity and potency
for ERα and smaller ligands for ERβ. However, our initial
efforts to prepare thiophene-core ligands from the furan 1,4-
diones precursors in the presence of thiolating agents were
largely thwarted by the strong tendency of the highly
substituted diones to form the furans in preference to the
thiophenes.30
Our interest in the thiophenes was revived when we prepared
some 3,4-diarylthiophenes to use as dienes for the preparation
of 7-thia-bicyclo[2.2.1]hept-5-ene-7-oxides through a Diels−
Alder reaction (using their corresponding monoxides).41 This
alternative approach of preparing the di- and trisubstituted
thiophenes from di- and tribromothiophene precursors using
palladium-mediated cross coupling with aryl boronic acids,
described here, works very well, and it enabled us to prepare
the larger series of thiophenes (as well as related furans) that
are examined herein.
Structural Trends in Binding Affinities and Estrogen
Receptor Subtype Selectivity in the Thiophenes and
Furans. From the binding data in Tables 1−3 and the
summary of the diaryl congeners (Figure 3), we can make some
novel observations and note a number of familiar trends.
With earlier biaryl-substituted heterocycle-core ER ligands,
we generally used simple 4-hydroxyphenyl (phenol) substitu-
ents and enhanced affinities by adding a third phenol and even
a fourth (alkyl) group. Here, we have enhanced affinities by
adding halogens (F, Cl) and small alkyl groups (Me) at C3,
meta to the phenolic OH. The boost in affinity can be quite
remarkable (>100-fold on ERα for compounds 2f vs 2a, and
>10-fold for many others) and presumably arises from a more
complete filling of the ligand-binding pocket, such that high
affinity binding can be obtained even with thiophenes and
furans containing only two substituted phenols. By contrast, as
has long been known in many ligand series, substitution at C3,
ortho to the phenol, greatly reduces affinity, presumably by
interfering with critical hydrogen bonding.20,50 It is of note that
in a series of B-seco or A-CD steroidal estrogens studied earlier,
a beneficial effect of meta but not ortho substitution was also
found.59,60 Similar observations of the beneficial effect of meta
substitution were made with a number of our bicyclo[2.2.1]-
heptane core ligands41,52,53 and have been noted recently by
Gust and colleagues in a series of pyrroles.29 Thus, filling voids
in ligand volume without interfering with critical receptor
interactions is an emerging strategy to generate high affinity ER
ligands. It was also surprising that in the bisaryl-substituted
thiophenes and furans, there are no great differences in the
affinity between members of the alternate-position vs adjacent-
position series, despite marked differences in ligand length and
shape, as well as phenol−heterocycle core torsional angles.
Other general trends are well-known. The ERβ preference of
smaller ligands (bisphenols or methyl-bisphenols) vs larger
ligands (trisphenols or phenyl bisphenols) is known from the
pyrazoles and furans24,30 and doubtless reflects the generally
smaller pockets that form around ERβ ligands.21 Bisphenol C3-
substituted ligands have high affinity and high ERβ selectivity;
the best bisphenolic compound is 2e, with an ERβ affinity of 33
and selectivity of 16; the best tris(hydroxyphenyl) thiophene is
compound 14c, with an ERα affinity of 85 and selectivity of 14.
Also, where they can be directly compared (2a/16a, 2b/16b,
2e/16c, 2f/16d, 4a/18a, 4b/18b, 4e/16c, and 4f/18d), the
thiophenes generally bind somewhat better than the furans
(though not by a large factor), perhaps because the thiophene
core is less polar than the furan core. This trend was also
evident in comparisons of the more polar core imidazoles with
the corresponding pyrazoles.24,58 The greater electron density
and higher polarizability of the sulfur atom as well as the
geometric changes due to the longer C−S bond vs C−O bond
could also be factors that enhance the affinity of the thiophenes
(see below).
Models for Thiophenes Binding in the Estrogen
Receptor α Ligand Binding Pocket and a Possible
Model for Superagonism. The most unusual finding in
this study is the pronounced superagonism shown by a number
of the alternate-substituted thiophenes. Ligands exhibiting
superagonistic activity have been described in the nuclear
receptor field. Gustafsson and others have reported that some
phytoestrogens gave a somewhat higher reporter gene maximal
transcriptional output than estradiol, especially on ERβ.61−64
Because the maximum transcriptional output from ERβ at
saturating concentrations of both expression plasmid and E2
ligand is only about 25−40% of that from ERα,62 one could
speculate that other ligands might be able to drive ERβ into a
yet more active conformation. Also, there is evidence that
steroidal ligands other than E2, notably 3β-adiol65 and Δ5-3β-
adiol,66 are likely the endogenous ligands for ERβ in some
tissue contexts. Nevertheless, we find that certain of the
thiophenes can drive superagonism from ERα as well as from
ERβ, so superagonism is not exclusively the domain of ERβ.
We have used molecular modeling to examine potential
modes of interaction of the thiophenes with ER and to explore
what might be the molecular basis for superagonism, which is
seen predominantly with the alternate-position thiophenes.
Crystal structures of ERα−ligand complexes show that the A-
ring phenolic hydroxyl of estradiol has hydrogen bonding
interactions with helix 3 residues Glu-353, Arg-394, and an
ordered water molecule, while the D-ring hydroxyl hydrogen
bonds with the helix 11 residue, His-524 (Figure 6A). This
stabilizes helices 3 and 11 and allows helix 12 to dock across
them. In this so-called active conformation, helix 12 forms one
side of a hydrophobic groove on the surface where transcrip-
tional coactivators bind. Hydrogen bonding to helix 3 via an A-
ring mimetic has been seen in every ligand-bound ER crystal
structure to date, while hydrogen bonding to helix 11 is more
variable.
Molecular modeling of complexes with ERα suggests that
thiophenes in the two alternate-substituted series bind in a
different manner compared to a typical steroidal agonist. It
seems reasonable to assume that the thiophenes in the 2,5- and
2,4-alternate-substituted series (2 and 6) would bind to ER in a
congruent fashion. The only expected difference would be in
the placement of the thiophene sulfur atom toward opposite
sides of the pocket, where it might engage in different
interactions with residues in the ligand-binding pocket.
However, there seem to be no pronounced differences between
the 2,5- and 2,4-bisphenolic thiophenes and furans in terms of
affinity and intrinsic activity, except the more pronounced
superagonism exhibited by the thiophenes compared to the
furans. The most likely polar interactions for the alternate-
pattern bisphenols then are with Glu-353 and Arg-394 on one
end and His-524 on the other (See Figure 6). While it is a
speculation, superagonism might be the consequence of the
increased O−O distance between the two phenols in the
thiophenes compared to other ligands. This distance is 10.9 Å
in E2 and 11.8 Å in diethylstilbestrol; however, in both alternate
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comprises part of the coactivator-binding site on the external
surface. The adjacent phenol thiophenes do not show
superagonism, and from this model, there is no apparent
reason why they should. All of these issues are being examined
further by X-ray crystallography.
CONCLUSION
■
In this paper, we present the synthesis of a novel series of
thiophenes and an investigation of their ER binding and
biological activity. Ligands of this series can be easily prepared
by Suzuki coupling, an approach that may prove convenient for
further library synthesis. These compounds show an interesting
binding affinity pattern: small substituents placed meta to the
phenols cause marked enhancements of binding affinity, and in
general, most of the bis(hydroxylphenyl) thiophenes were
selective for ERβ, whereas the tris(hydroxyphenyl) thiophenes
were selective for ERα. Reporter gene assays reflect these
trends but also reveal that a number of the alternate-pattern
thiophene bisphenols have marked superagonistic activity, more
so than the adjacent-pattern bisphenols or the corresponding
furans from either series. Molecular modeling suggests that the
1−2 Å increased O−O distance in the alternate-mode
thiophenes might be responsible for this superagonism. Further
studies of the cellular and in vivo biological activities of key
members of this new class of ER ligands, as well as X-ray
crystallographic analyses, which are underway, will be reported
in due course.
The generation of this new series of ER ligands provides
important insight into the diversity of structures that can
function as ligands for both ER subtype, and our findings may
open new strategies for the design of both ERα- and ERβ-
selective ligands. They also illustrate that changes to the ligand
core structure, although it is remote from close contacts with
residues in the ligand-binding pocket, can have important
effects on activity, in this case increased efficacy leading to
superagonism. When better understood at the level of
molecular interactions, superagonism itself might prove to be
a new mode for developing ER ligands having selective
activities.42
Figure 6. Model of thiophene ligands bound to ERα and comparisons
with estradiol and OBHS. A portion of the ligand binding pocket is
rendered as a ribbon, with selected amino acids shown as sticks. (A)
Structure of E2 bound ERα. The A ring of E2 forms hydrogen bonds
with E353 and R394, while the D ring bonds to H524 (PDB ID:
1ERE). (B) Structure of oxabicyclic heptane sulfonate (OBHS) bound
ERα.52 OBHS H-bonds to both the conserved R394/E353 pair and
also T347 on helix 3. The phenyl sulfonate binds extends between
helices 8 and 11. (C) Model of 2e bound to ERα with the conserved
H-bonding to R394/E353, and the other phenol extending between
helices 8 and 11. (D) Model of 4e with H-bonds to E353 and T347.
thiophene bisphenol,s it is 12.8 Å, that is, 1−2 Å longer. The
O−O distance in the thiophenes is also somewhat longer than
in the alternate-position furan bisphenols due to the greater
length of the C−S bonds (1.47 Å) vs the C−O bonds (1.24 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 bi-
sphenol thiophenes might be too long to form adequate
hydrogen bonds with His-524.
We recently published on a series of oxabicyclic compounds,
exemplified by OBHS, where the side group extends between
helices 8 and 11 (Figure 6B).52 Modeling predicts that the
thiophene 2e will bind similarly between helices 8 and 11
(Figure 6C), but this does not explain their superagonist
activity. We have found that ligand-specific “mis-positioning” of
helix 11 can interfere with the agonist conformation of helix
12,52 so we suggest that the superagonist activity may stem
from “improved” positioning of helix 11 that stabilizes helix 12
in the agonist conformation to an even greater degree than
does E2. This issue is being examined further by X-ray
crystallography.
The binding of thiophenes with adjacent phenols (series 4
and 8) was modeled with compound 4e based on the
assumption that the two hydroxyls will form hydrogen bonds
similar to the two phenols in OBHS by wrapping around helix
3. In this model (Figure 6D), the ligand makes no contacts with
helix 11, which would be a novel form of binding for an ER
ligand but one likely utilized by members of the cyclofenil class
of ligands. Changing the position of the sulfur (i.e., 2,3- vs 3,4-
substitution) could provide subtle shifts in helix 3, which
EXPERIMENTAL SECTION
■
General. Starting materials were purchased from Aldrich, Acros,
Aladin-reagent, and Alfa-Asar and were used without purification.
Tetrahydrofuran was freshly distilled from sodium/benzophenone
under argon. 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). Visual-
ization was achieved by UV light (254 nm).
1H NMR and 13C NMR spectra were measured on a Bruker Biospin
AV400 (400 MHz) 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. The purity of
all compounds for biological testing was determined by HPLC method
(see Supporting Information), confirming >95% purity.
General Procedure for Suzuki Coupling. Under Ar atmosphere, a
mixture of bromothiophene (1 equiv), arylboronic acid (2 equiv for
monosubstituted, 4 equiv for disubstituted, 5 equiv for trisubstituted
thiophenes), Pd catalyst, 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
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aqueous layer was extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over anhydrous MgSO4/Na2SO4
and then filtered and concentrated in vacuum. The product was
purified by column chromatography (CC).
General Procedure for Ether Cleavage. Under argon atmosphere,
to a solution of methoxyphenyl derivative (1 equiv) in dry
dichloromethane at −20 °C, boron tribromide (3 equiv per methoxy
function) was added dropwise. The reaction mixture was stirred at
room temperature. After 4 h, water was added to quench the reaction
and ethyl acetate was used to extract the aqueous layer. The combined
organic layers were washed with brine, dried over anhydrous MgSO4/
Na2SO4, and then filtered and concentrated in vacuum. The product
was purified by column chromatography (CC).
2,5-Bis(4-methoxyphenyl)thiophene (1a). Compound 1a was
prepared by reaction of 2,5-dibromothiophene and (4-methoxyphenyl)
boronic acid according to the general procedure for Suzuki coupling.
Purification by CC (petroleum ether:ethyl acetate = 98:2) gave the
title compound as a white solid (mp 94−98 °C). 1H NMR (400 MHz,
CDCl3) δ 7.57−7.51 (m, 4H), 7.15 (s, 2H), 6.95−6.89 (m, 4H), 3.84
(s, 6H). 13C NMR (101 MHz, CDCl3) δ 159.12, 144.51,126.84,
122.90, 114.31, 55.38.
2,5-Bis(3-chloro-4-methoxyphenyl)thiophene (1h). Compound
1h was prepared by reaction of 2,5-dibromothiophene and (3-
chloro-4-methoxyphenyl) boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a green solid
1
(mp 108−110 °C). H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 2.3
Hz, 2H), 7.46 (dd, J = 8.5, 2.3 Hz, 2H), 7.16 (s, 2H), 6.94 (d, J = 8.6
Hz, 2H), 3.94 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 147.35, 142.37,
142.17, 138.80, 123.65, 121.47, 113.80, 113.26, 56.38.
2,5-Bis(2-fluoro-5-methoxyphenyl)thiophene (1i). Compound 1i
was prepared by reaction of 2,5-dibromothiophene and (2-fluoro-5-
methoxyphenyl) boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a yellow solid (mp 102−104 °C). 1H
NMR (400 MHz, CDCl3) δ 7.38 (s, 2H), 7.06 (dd, J = 6.1, 3.0 Hz,
2H), 7.01−6.94 (m, 2H), 6.69 (dt, J = 8.9, 3.4 Hz, 2H), 3.74 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 155.82, 154.92, 152.51, 137.44,
127.16, 122.36, 117.04, 113.94, 55.84.
2,5-Bis(3-fluoro-5-methoxyphenyl)thiophene (1j). Compound 1j
was prepared by reaction of 2,5-dibromothiophene and (3-fluoro-5-
methoxyphenyl) boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
2,5-Bis(4-methoxy-2-methylphenyl)thiophene (1b). Compound
1b was prepared by reaction of 2,5-dibromothiophene and (4-
methoxy-2-methylphenyl) boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
98:2) gave the title compound as a yellow solid (mp 89−90 °C). H
NMR (400 MHz, CDCl3) δ 7.18 (s, 2H), 6.84 (dd, J = 7.9, 1.9 Hz,
4H), 6.51−6.45 (m, 2H), 3.76 (s, 6H). 13C NMR (101 MHz, CDCl3)
δ 159.86, 157.43, 155.99, 137.63, 131.12, 119.54, 102.13, 99.66, 95.60,
50.40.
1
(mp 72−74 °C). H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 8.4 Hz,
2H), 6.86 (s, 2H), 6.72 (d, J = 2.5 Hz, 2H), 6.68 (dd, J = 8.4, 2.6 Hz,
2H), 3.72 (s, 6H), 2.37 (s, 6H). 13C NMR (101 MHz, CDCl3) δ
159.11, 142.33, 137.55, 131.49, 126.87, 126.04, 116.24, 111.29, 55.29,
21.56.
2,5-Bis(4-fluoro-3-methoxyphenyl)thiophene (1k). Compound 1k
was prepared by reaction of 2,5-dibromothiophene and (4-fluoro-5-
methoxyphenyl) boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a yellow solid (mp 105−108 °C). 1H
NMR (400 MHz, CDCl3) δ 7.19 (m, 3H), 7.17−7.12 (m, 3H), 7.08
(dd, J = 10.7, 8.4 Hz, 2H), 3.96 (s, 6H). 13C NMR (101 MHz, CDCl3)
δ 153.40, 150.94, 147.91, 142.78, 130.87, 124.06, 118.29, 116.49,
56.35.
2,5-Bis(4-methoxy-3-methylphenyl)thiophene (1c). Compound
1c was prepared by reaction of 2,5-dibromothiophene and (4-
methoxy-3-methylphenyl) boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
(mp 78−81 °C). H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 8.4 Hz,
2-(2-Fluoro-4-methoxyphenyl)-5-(4-methoxy-2-methylphenyl)-
thiophene (1l). Compound 1l was prepared by reaction of compound
19 and (2-fluoro-4-methoxyphenyl) boronic acid according to the
general procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a yellow oil. 1H
NMR (400 MHz, CDCl3) δ 7.54 (t, J = 8.9 Hz, 1H), 7.37 (d, J = 8.4
Hz, 1H), 7.32 (d, J = 2.5 Hz, 1H), 6.97 (d, J = 3.6 Hz, 1H), 6.82 (d, J
= 2.4 Hz, 1H), 6.75 (ddd, J = 20.7, 10.9, 2.5 Hz, 3H), 3.83 (s, 6H),
2.45 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.92, 159.89, 159.20,
158.44, 142.34, 137.62, 136.51, 131.48, 129.08, 126.63, 125.17, 116.22,
115.03, 111.28, 110.51, 102.31, 102.05, 55.65, 55.28, 21.46.
2-(2-Chloro-4-methoxyphenyl)-5-(4-methoxy-2-methylphenyl)-
thiophene (1m). Compound 1m was prepared by reaction of
compound 19 and (2-chloro-4-methoxyphenyl) boronic acid accord-
ing to the general procedure for Suzuki coupling. Purification by CC
(petroleum ether:ethyl acetate = 98:2) gave the title compound as a
2H), 6.89 (s, 2H), 6.72 (d, J = 2.5 Hz, 2H), 6.58 (dd, J = 8.4, 2.6 Hz,
2H), 3.82 (s, 6H), 2.29 (s, 6H)..
2,5-Bis(4-methoxy-3,5-dimethylphenyl)thiophene (1d). Com-
pound 1d was prepared by reaction of 2,5-dibromothiophene and
(4-methoxy-3,5-dimethylphenyl) boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
(mp 99−101 °C). H NMR (400 MHz, CDCl3) δ 7.27 (s, 4H), 7.16
(s, 2H), 3.74 (s, 6H), 2.32 (s, 12H). 13C NMR (101 MHz, CDCl3) δ
156.63, 142.91, 131.36, 130.04, 126.07, 123.35, 59.83, 16.20.
2,5-Bis(2-fluoro-4-methoxyphenyl)thiophene (1e). Compound 1e
was prepared by reaction of 2,5-dibromothiophene and (2-fluoro-4-
methoxyphenyl) boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
1
98:2) gave the title compound as a yellow solid (mp 94−96 °C). H
1
yellow oil. H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 8.6 Hz, 1H),
NMR (400 MHz, CDCl3) δ 7.55 (t, J = 8.9 Hz, 2H), 7.34 (s, 2H),
6.78−6.67 (m, 4H), 3.83 (s, 6H). 13C NMR (101 MHz, CDCl3) δ
160.96, 158.47, 136.41, 129.07, 125.66, 114.84, 110.54, 102.30, 55.68.
2,5-Bis(2-chloro-4-methoxyphenyl)thiophene (1f). Compound 1f
was prepared by reaction of 2,5-dibromothiophene and (2-chloro-4-
methoxyphenyl) boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a green solid (mp 107−110 °C). 1H
NMR (400 MHz, CDCl3) δ 7.49 (d, J = 8.7 Hz, 2H), 7.26 (s, 2H),
7.03 (d, J = 2.6 Hz, 2H), 6.86 (dd, J = 8.7, 2.6 Hz, 2H), 3.84 (s, 6H).
2,5-Bis(3-fluoro-4-methoxyphenyl)thiophene (1g). Compound 1g
was prepared by reaction of 2,5-dibromothiophene and (3-fluoro-4-
methoxyphenyl) boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
7.27 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 3.6 Hz, 1H), 6.91 (d, J = 2.5 Hz,
1H), 6.86 (d, J = 3.6 Hz, 1H), 6.72 (dd, J = 8.8, 2.5 Hz, 2H), 6.66 (dd,
J = 8.4, 2.5 Hz, 1H), 3.69 (s, 3H), 3.70 (s, 3H), 2.35 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 158.22, 142.21, 138.25, 136.52, 131.78,
130.82, 130.45, 125.95, 125.58, 124.96, 124.71, 115.19, 114.59, 112.22,
110.23, 54.51, 54.18.
2,5-Bis-(4-hydroxyphenyl) thiophene (2a). Compound 2a was
prepared by 2,5-bis-(4-methoxyphenyl) thiophene (1a) and boron
tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the
1
title compound as a white solid (mp 168−170 °C). H NMR (400
MHz, acetone-d6) δ 8.50 (s, 1H), 7.42−7.33 (m, 4H), 7.08 (s, 2H),
6.81−6.71 (m, 4H). 13C NMR (101 MHz, acetone-d6) δ 158.18,
143.20, 127.51, 126.88, 123.52, 116.67. HRMS (MALDI/DHB) calcd
for C16H12O2S (M + H+) 268.0556, found 268.05525.
1
98:2) gave the title compound as a green solid (mp 92−97 °C). H
NMR (400 MHz, CDCl3) δ 7.35 (m, 4H), 7.17 (s, 2H), 7.00 (d, J =
2.6 Hz, 2H), 3.95 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 154.46,
141.69, 127.95, 127.38, 124.96, 123.65, 122.95, 112.31, 56.29.
2,5-Bis(4-hydroxy-2-methylphenyl)thiophene (2b). Compound 2b
was prepared by 2,5-bis(4-methoxy-2-methylphenyl)thiophene (1b)
M
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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 white solid (mp 172−175 °C). 1H NMR
(400 MHz, acetone-d6) δ 7.17 (t, J = 4.6 Hz, 2H), 6.92 (s, 2H), 6.74
(d, J = 12.5 Hz, 2H), 6.69−6.60 (m, 2H), 2.06 (s, 6H). 13C NMR (101
MHz, acetone-d6) δ 144.04, 136.74, 132.19, 131.69, 128.41, 126.86,
118.76, 115.40, 21.00. HRMS (MALDI/DHB) calcd for C18H16O2S
(M + H+) 296.0875, found 296.08655.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a yellow solid (mp 176−179 °C). 1H NMR (400 MHz,
acetone-d6) δ 7.39 (s, 2H), 7.07 (dd, J = 6.1, 2.8 Hz, 2H), 7.01−6.90
(m, 2H), 6.78−6.61 (m, 2H). 13C NMR (101 MHz, acetone-d6) δ
154.81, 152.50, 138.16, 127.77, 122.81, 117.92, 117.68, 114.74. HRMS
(MALDI/DHB) calcd for C16H10O2F2S (M + H+) 304.0361, found
304.03641.
2,5-Bis(3-fluoro-5-hyroxyphenyl)thiophene (2j). Compound 2j
was prepared by 2,5-bis(3-fluoro-5-methoxyphenyl)thiophene (1j)
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 (mp 212−214 °C). 1H NMR
(400 MHz, DMSO-d6) δ 7.55 (s, 2H), 7.01 (d, J = 9.9 Hz, 2H), 6.90
(s, 2H), 6.55 (d, J = 10.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ
164.57, 162.16, 159.35, 141.79, 135.74, 125.81, 108.29, 102.82, 102.59,
102.04, 101.81. HRMS (MALDI/DHB) calcd for C16H10O2F2S (M +
H+) 304.0361, found 304.03641.
2,5-Bis(4-hydroxy-3-methylphenyl)thiophene (2c). Compound 2b
was prepared by 2,5-bis(4-methoxy-3-methylphenyl)thiophene (1c)
and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 3:1)
1
gave the title compound as brown solid (mp187−189 °C). H NMR
(400 MHz, DMSO-d6) δ 7.38 (s, 2H), 6.95 (d, J = 1.2 Hz, 2H), 6.70
(dd, J = 8.2, 1.8 Hz, 2H), 6.64 (d, J = 8.2 Hz, 2H), 2.06 (s, 6H). 13C
NMR (101 MHz, DMSO- d6) δ 149.05, 142.28, 137.54, 131.46,
126.84, 125.99, 116.18, 111.24, 21.53. HRMS (MALDI/DHB) calcd
for C18H16O2S (M + H+) 296.0875, found 296.08655.
2,5-Bis(4-fluoro-3-hydroxyphenyl)thiophene (2k). Compound 2k
was prepared by 2,5-bis(4-fluoro-3-methoxyphenyl)thiophene (1k)
and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 3:1)
2,5-Bis(4-hydroxy-3,5-dimethylphenyl)thiophene (2d). Com-
pound 2d was prepared by 2,5-bis-(4-methoxy-3,5-dimethylphenyl)-
thiophene (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 brown solid (mp 201−203 °C). 1H
NMR (400 MHz, acetone-d6) δ 7.27 (s, 4H), 7.18 (s, 2H), 2.27 (s,
12H). 13C NMR (101 MHz, acetone-d6) δ 153.99, 143.32, 126.96,
126.36, 125.41, 123.27, 16.71. HRMS (MALDI/DHB) calcd for
C20H20O2S (M + H+) 324.1184, found 324.11785.
2,5-Bis(2-fluoro-4-hydroxyphenyl)thiophene (2e). Compound 2e
was prepared by 2,5-bis(2-fluoro-4-methoxyphenyl)thiophene (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 brown solid (mp 178−180 °C). 1H NMR
(400 MHz, acetone-d6) δ 7.59 (t, J = 8.9 Hz, 2H), 7.37 (s, 2H), 6.76
(ddd, J = 15.3, 10.8, 2.4 Hz, 4H). 13C NMR (101 MHz, acetone-d6) δ
161.72, 159.29, 137.16, 130.04, 126.03, 114.17, 113.13, 104.20. HRMS
(MALDI/DHB) calcd for C16H10O2F2S (M + H+) 304.0369, found
304.03641.
2,5-Bis(2-chloro-4-hydroxyphenyl)thiophene (2f). Compound 2f
was prepared by 2,5-bis(2-chloro-4-methoxyphenyl)thiophene (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 brown solid (mp 194−196 °C). 1H NMR
(400 MHz, acetone-d6) δ 7.50 (d, J = 8.5 Hz, 2H), 7.28 (s, 2H), 7.04
(d, J = 2.5 Hz, 2H), 6.91 (dd, J = 8.5, 2.5 Hz, 2H).13C NMR (101
MHz, acetone-d6) δ 141.00, 133.08, 133.06, 127.75, 124.89, 117.89,
117.81, 115.81, 115.73. HRMS (MALDI/DHB) calcd for
C16H10O2Cl2S (M + H+) 335.9788, found 335.97731.
2,5-Bis(3-fluoro-4-hydroxyphenyl)thiophene (2g). Compound 2g
was prepared by 2,5-bis(3-fluoro-4-methoxyphenyl)thiophene (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 green solid (mp 187−190 °C). 1H NMR
(400 MHz, acetone-d6) δ 7.47−7.37 (m, 2H), 7.37−7.31 (m, 2H),
7.28 (d, J = 8.3 Hz, 2H), 7.05 (t, J = 8.8 Hz, 2H). 13C NMR (101
MHz, acetone-d6) δ 133.04, 127.60, 124.87, 123.08, 122.51, 118.89,
114.81, 113.50. HRMS (MALDI/DHB) calcd for C16H10O2F2S (M +
H+) 304.0361, found 304.03641.
1
gave the title compound as a yellow solid (mp 169−172 °C) . H
NMR (400 MHz, acetone-d6) δ 8.74 (s, 2H), 7.16 (s, 4H), 6.98 (d, J =
5.3 Hz, 4H). 13C NMR (101 MHz, acetone-d6) δ 153.33, 150.93,
146.15, 143.19, 131.96, 125.22, 117.99, 115.71. HRMS (MALDI/
DHB) calcd for C16H10O2F2S (M + H+) 304.0361, found 304.03641.
2-(2-Fluoro-4-hydroxyphenyl)-5-(4-hydroxy-2-methylphenyl)-
thiophene (2l). Compound 2l was prepared by 2-(2-fluoro-4-
methoxyphenyl)-5-(4-methoxy-2-methylphenyl) thiophene (1l) 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 (mp 151−153 °C). 1H NMR (400 MHz,
acetone-d6) δ 7.57 (t, J = 8.9 Hz, 1H), 7.34 (d, J = 3.7 Hz, 1H), 7.27
(d, J = 8.3 Hz, 1H), 7.02 (d, J = 3.7 Hz, 1H), 6.84−6.68 (m, 4H), 2.38
(s, 3H). 13C NMR (101 MHz, acetone-d6) δ 161.67, 159.21, 158.11,
143.23, 138.12, 137.10, 132.17, 130.05, 127.22, 126.67−125.17,
118.32, 113.89, 113.04, 104.25, 104.00, 21.42. HRMS (MALDI/
DHB) calcd for C17H13O2FS (M + H+) 300.0607, found 300.06148.
2-(2-Chloro-4-hydroxyphenyl)-5-(4-hydroxy-2-methylphenyl)-
thiophene (2m). Compound 2m was prepared by 2-(2-chloro-4-
methoxyphenyl)-5-(4-methoxy-2-methylphenyl) thiophene (1m) 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 brown solid (mp 167−169 °C). 1H NMR (400 MHz,
acetone-d6) δ 7.49 (d, J = 8.5 Hz, 1H), 7.27 (dd, J = 7.4, 6.2 Hz, 2H),
7.02 (d, J = 3.5 Hz, 2H), 6.90 (dd, J = 8.5, 2.5 Hz, 1H), 6.81 (d, J = 1.7
Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 2.39 (s, 3H). 13C NMR (101 MHz,
acetone-d6) δ 158.65, 158.19, 144.22, 139.94, 138.12, 133.02, 132.20,
127.74, 126.74, 125.99, 125.17, 124.44, 118.36, 117.83, 115.73, 113.93,
21.44. HRMS (MALDI/DHB) calcd for C17H13O2ClS (M + H+)
316.0325, found 316.03193.
3,4-Bis(4-methoxyphenyl)thiophene (3a). Compound 3a was
prepared by reaction of 3,4-dibromothiophene and (4-
methoxyphenyl)boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a white solid (mp 105−106 °C). 1H
NMR (400 MHz, CDCl3) δ 7.23 (s, 2H), 7.12 (d, J = 8.8 Hz, 4H),
6.80 (d, J = 8.8 Hz, 4H), 3.80 (s, 6H). 13C NMR (100 MHz, CDCl3) δ
158.57, 141.32, 130.10, 129.22, 123.08, 113.57, 55.22.
3,4-Bis(4-methoxy-2-methylphenyl)thiophene (3b). Compound
3b was prepared by reaction of 3,4-dibromothiophene and (4-
methoxy-2-methylphenyl)boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
(mp 72−76 °C). 1H NMR (400 MHz, CDCl3) δ 7.15 (s, 2H), 6.94 (d,
J = 8.4 Hz, 2H), 6.64−6.58 (m, 4H), 3.75 (s, 6H), 2.03 (s, 6H). 13C
NMR (100 MHz, CDCl3) δ 158.46, 141.98, 137.67, 131.61, 128.98,
123.26, 115.26, 110.62, 55.08, 20.52.
2,5-Bis(3-chloro-4-hydroxyphenyl)thiophene (2h). Compound 2h
was prepared by 2,5-bis(3-chloro-4-methoxyphenyl)thiophene (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 green solid (mp 192−195 °C). 1H NMR
(400 MHz, DMSO-d6) δ 7.65 (s, 2H), 7.45 (d, J = 8.5 Hz, 2H), 7.39
(s, 2H), 7.02 (d, J = 8.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ
141.03, 138.97, 126.75, 126.48, 125.55, 124.53, 120.76, 117.50. HRMS
(MALDI/DHB) calcd for C16H10O2Cl2S (M + H+) 335.9774, found
335.97731.
2,5-Bis(2-fluoro-5-hydroxyphenyl)thiophene (2i). Compound 2i
was prepared by 2,5-bis(2-fluoro-5-methoxyphenyl)thiophene (1i) and
boron tribromide according to general procedure for ether cleavage.
3,4-Bis(4-methoxy-3-methylphenyl)thiophene (3c). Compound
3c was prepared by reaction of 3,4-dibromothiophene and (4-
N
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methoxy-3-methylphenyl)boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
(mp 77−78 °C). 1H NMR (400 MHz, CDCl3) δ 7.21 (s, 2H), 7.05 (s,
2H), 6.93 (dd, J = 8.4, 2.1 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 3.81 (s,
6H), 2.16 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 154.56, 139.23,
129.02, 125.14, 124.46, 120.58, 107.17, 53.00, 13.98.
3,4-Bis(4-methoxy-3,5-dimethylphenyl)thiophene (3d). Com-
pound 3d was prepared by reaction of 3,4-dibromothiophene and
(4-methoxy-3,5-dimethylphenyl) boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
(mp 92−96 °C). 1H NMR (400 MHz, CDCl3) δ 7.15 (s, 2H), 6.76 (s,
4H), 3.64 (s, 6H), 2.12 (s, 12H). 13C NMR (101 MHz, CDCl3) δ
156.01, 141.45, 132.13, 130.21, 129.49, 123.08, 59.76, 16.01.
3,4-Bis(2-fluoro-4-hydroxyphenyl)thiophene (4e). Compound 4e
was prepared by 3,4-bis(2-fluoro-4-methoxyphenyl)thiophene (3e)
and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 4:1)
gave the title compound as a white solid (mp 207−209 °C). 1H NMR
(400 MHz, acetone-d6) δ 8.91 (s, 1H), 7.45 (s, 2H), 6.97 (t, J = 8.6
Hz, 2H), 6.57 (ddd, J = 14.1, 10.0, 2.4 Hz, 4H). 13C NMR (101 MHz,
acetone-d6) δ 162.32, 159.09, 136.91, 132.62, 125.16, 116.35, 111.98,
103.59. HRMS (MALDI/DHB) calcd for C16H10O2F2S (M + H+)
304.0378, found 304.03641.
3,4-Bis(2-chloro-4-hydroxyphenyl)thiophene (4f). Compound 4f
was prepared by 3,4-bis(2-chloro-4-methoxyphenyl)thiophene (3f)
and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 4:1)
gave the title compound as a white solid (mp 179−183 °C). 1H NMR
(400 MHz, acetone-d6) δ 7.42 (s, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.84
(d, J = 2.5 Hz, 2H), 6.69 (dd, J = 8.4, 2.5 Hz, 2H). 13C NMR (101
MHz, acetone-d6) δ 158.20, 140.43, 134.40, 133.54, 127.38, 125.26,
116.85, 114.65. HRMS (MALDI/DHB) calcd for C16H10O2Cl2S (M +
H+) 335.9861, found 335.98513.
3,4-Bis(2-fluoro-4-methoxyphenyl)thiophene (3e). Compound 3e
was prepared by reaction of 3,4-dibromothiophene and (2-fluoro-4-
methoxyphenyl)boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
1
98:2) gave the title compound as a white solid (mp 88−90 °C). H
2,4-Bis(2-fluoro-4-methoxyphenyl)thiophene (5a). Compound 5a
was prepared by reaction of 2,4-dibromothiophene and (2-fluoro-4-
methoxyphenyl)boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
NMR (400 MHz, CDCl3) δ 7.27 (s, 2H), 6.94 (t, J = 8.7 Hz, 2H),
6.54−6.47 (m, 4H), 3.71 (s, 6H). 13C NMR (101 MHz, CDCl3) δ
161.47, 159.01, 135.52, 131.57, 124.49, 109.65, 101.81, 101.55, 55.49.
3,4-Bis(2-chloro-4-methoxyphenyl)thiophene (3f). Compound 3f
was prepared by reaction of 3,4-dibromothiophene and (2-chloro-4-
methoxyphenyl)boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a white solid (mp 121−125 °C). 1H
NMR (400 MHz, CDCl3) δ 7.24 (s, 2H), 6.92 (d, J = 8.5 Hz, 2H),
6.80 (d, J = 2.6 Hz, 2H), 6.59 (dd, J = 8.6, 2.6 Hz, 2H), 3.69 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 159.15, 139.18, 134.06, 132.37,
127.60, 124.54, 114.66, 112.49, 55.43.
1
98:2) gave the title compound as a white solid (mp 88−90 °C). H
NMR (400 MHz, CDCl3) δ 7.54−7.34 (m, 4H), 6.68−6.57 (m, 4H),
3.72 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 161.64, 161.01, 160.06,
159.17, 158.52, 137.12, 136.01, 129.78, 129.30, 125.32, 121.45, 116.33,
114.82, 110.55, 110.27, 102.30, 55.64, 55.59.
2,4-Bis(2-chloro-4-methoxyphenyl)thiophene (5b). Compound 5b
was prepared by reaction of 2,4-dibromothiophene and (2-chloro-4-
methoxyphenyl)boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a white solid (mp 135−138 °C). 1H
NMR (400 MHz, CDCl3) δ 7.47 (dd, J = 5.0, 3.6 Hz, 2H), 7.38 (dd, J
= 5.0, 3.6 Hz, 2H), 7.02 (dd, J = 4.3, 2.6 Hz, 2H), 6.85 (ddd, J = 8.7,
2.6, 0.6 Hz, 2H), 3.83 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 159.50,
159.26, 139.40, 139.02, 133.04, 132.93, 132.06, 131.64, 128.93, 127.81,
125.57, 123.51, 115.57, 115.34, 113.29, 113.15, 55.64, 55.61.
2,4-Bis(2-fluoro-4-hydroxyphenyl)thiophene (6a). Compound 6a
was prepared by 2,4-bis(2-fluoro-4-methoxyphenyl)thiophene (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 brown solid (mp 190−194 °C). 1H NMR
(400 MHz, acetone-d6) δ 9.26 (s, 1H), 9.12 (s, 1H), 7.62−7.60 (m,
4H), 6.75−6.73 (m, 4H). 13C NMR (101 MHz, acetone-d6) δ 161.79,
159.99, 159.33, 139.61, 138.21, 137.06, 131.00, 130.37, 125.81, 121.85,
114.25, 113.08, 112.74, 110.89, 110.09, 103.99. HRMS (MALDI/
DHB) calcd for C16H10O2F2S (M + H+) 304.0373, found 304.03641.
2,4-Bis(2-chloro-4-hydroxyphenyl)thiophene (6b). Compound 6b
was prepared by 2,4-bis(2-chloro-4-methoxyphenyl)thiophene (5b)
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 brown solid (mp 163−165 °C). 1H NMR
(400 MHz, acetone-d6) δ 7.56−7.47 (m, 3H), 7.41 (d, J = 8.5 Hz, 1H),
7.03 (dd, J = 11.6, 2.5 Hz, 2H), 6.90 (td, J = 8.3, 2.5 Hz, 2H). 13C
NMR (101 MHz, acetone-d6) δ 158.83, 158.72, 158.45, 158.34,
140.33, 140.08, 133.15, 132.88, 129.55, 127.36, 125.07, 124.20, 117.85,
117.55, 115.64, 115.48. HRMS (MALDI/DHB) calcd for
C16H10O2Cl2S (M + H+) 335.9784, found 335.97731.
3,4-Bis(4-hydroxyphenyl)thiophene (4a). Compound 4a was
prepared by 3,4-bis(4-methoxyphenyl) thiophene (3a) and boron
tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the
1
title compound as a white solid (mp 198−201 °C). H NMR (400
MHz, acetone-d6) δ 8.49 (s, 2H), 7.33 (s, 2H), 7.03 (d, J = 8.2 Hz,
4H), 6.75 (d, J = 8.1 Hz, 4H). 13C NMR (101 MHz, acetone-d6) δ
157.45, 142.46, 130.87, 129.04, 123.63, 115.86. HRMS (MALDI/
DHB) calcd for C16H12O2S (M + H+) 268.0563, found 268.05525.
3,4-Bis(4-hydroxy-2-methylphenyl)thiophene (4b). Compound 4b
was prepared by 3,4-bis(4- methoxy-2-methylphenyl)thiophene (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 white solid (mp 214−215 °C). 1H NMR
(400 MHz, DMSO-d6) δ 9.28 (s, 2H), 7.41 (s, 2H), 6.80 (d, J = 8.2
Hz, 2H), 6.57 (s, 2H), 6.50 (d, J = 8.2 Hz, 2H), 1.98 (s, 6H). 13C
NMR (100 MHz, DMSO-d6) δ 156.09, 141.68, 136.75, 131.19, 126.97,
123.24, 116.38, 112.20, 20.00. HRMS (MALDI/DHB) calcd for
C18H16O2S (M + H+) 296.0875, found 296.08655.
3,4-Bis(4-hydroxy-3-methylphenyl)thiophene (4c). Compound 4c
was prepared by 3,4-bis(4-methoxy-3-methylphenyl)thiophene (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 white solid (mp 183−184 °C). 1H NMR
(400 MHz, acetone-d6) δ 8.38 (s, 1H), 7.30 (s, 2H), 7.01 (s, 2H), 6.78
(d, J = 8.1 Hz, 2H), 6.70 (d, J = 8.2 Hz, 2H), 2.13 (s, 6H). 13C NMR
(101 MHz, acetone-d6) δ 155.40, 142.64, 132.15, 129.07, 128.18,
124.67, 123.35, 115.02, 16.23. HRMS (MALDI/DHB) calcd for
C18H16O2S (M + H+) 296.0875, found 296.08655.
3,4-Bis(4-hydroxy-3,5-dimethylphenyl)thiophene (4d). Com-
pound 4d was prepared by 3,4-bis(4-methoxy-3,5-dimethylphenyl)-
thiophene (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 brown solid (mp 193−194 °C). 1H
NMR (400 MHz, acetone-d6) δ 7.29 (s, 2H), 6.82 (s, 4H), 2.14 (s,
12H). HRMS (MALDI/DHB) calcd for C20H20O2S (M + H+)
324.1184, found 324.11785.
2,3-Bis(2-fluoro-4-methoxyphenyl)thiophene (7a). Compound 7a
was prepared by reaction of 2,3-dibromothiophene and (2-fluoro-4-
methoxyphenyl)boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
1
98:2) gave the title compound as a white solid (mp 76−78 °C). H
NMR (400 MHz, CDCl3) δ 7.32 (d, J = 5.2 Hz, 1H), 7.11−7.04 (m,
2H), 6.95 (t, J = 8.5 Hz, 1H), 6.58−6.49 (m, 4H), 3.72 (s, 3H), 3.71
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 159.79, 159.15, 137.60,
136.71, 135.01, 133.94, 133.58, 132.40, 129.86, 127.62, 125.01, 124.18,
114.80, 112.72, 55.46.
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2,3-Bis(2-chloro-4-methoxyphenyl)thiophene (7b). Compound 7b
was prepared by reaction of 2,3-dibromothiophene and (2-chloro-4-
methoxyphenyl)boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a white solid (mp 115−117 °C). 1H
NMR (400 MHz, CDCl3) δ 7.31 (d, J = 5.2 Hz, 1H), 7.08 (dd, J = 6.9,
1.6 Hz, 2H), 6.89 (d, J = 8.6 Hz, 1H), 6.83 (dd, J = 11.1, 2.6 Hz, 2H),
6.63 (dd, J = 8.6, 2.6 Hz, 1H), 6.57 (dd, J = 8.6, 2.6 Hz, 1H), 3.70 (s,
3H), 3.69 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 159.42, 140.20,
132.90, 131.97, 127.02, 125.52, 115.61, 113.30, 55.63.
2,3-Bis(2-fluoro-4-hydroxyphenyl)thiophene (8a). Compound 8a
was prepared by 2,3-bis(2-fluoro-4-methoxyphenyl)thiophene (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 brown solid (mp 158−162 °C). 1H NMR
(400 MHz, acetone-d6) δ 7.54 (d, J = 5.2 Hz, 1H), 7.15 (dd, J = 5.2,
1.7 Hz, 1H), 7.09 (t, J = 8.6 Hz, 1H), 6.98 (t, J = 8.5 Hz, 1H), 6.67−
6.54 (m, 4H). 13C NMR (101 MHz, acetone-d6) 166.73, 162.32,
160.05, 134.63, 133.66, 133.40, 132.91, 132.53, 131.23, 130.84, 130.16,
128.90, 125.51, 112.42, 103.96. HRMS (MALDI/DHB) calcd for
C16H10O2F2S (M + H+) 304.0377, found 304.03641.
2,3-Bis(2-chloro-4-hydroxyphenyl)thiophene (8b). Compound 8b
was prepared by 2,3-bis(2-chloro-4-methoxyphenyl)thiophene (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 white solid (mp 160−163 °C). 1H NMR
(400 MHz, acetone-d6) δ 9.16 (s, 1H), 8.98 (s, 1H), 7.53 (d, J = 5.2
Hz, 1H), 7.19−7.09 (m, 2H), 6.98−6.86 (m, 3H), 6.74 (dd, J = 8.4,
2.4 Hz, 1H), 6.66 (dd, J = 8.4, 2.4 Hz, 1H). 13C NMR (101 MHz,
acetone-d6) δ 159.09, 158.33, 135.31, 134.71, 134.29, 133.47, 130.69,
125.10, 124.58, 117.30,116.86, 114.95. HRMS (MALDI/DHB) calcd
for C16H10O2Cl2S (M + H+) 335.9769, found 335.97731.
(9a) 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 white solid (mp 160−163 °C). 1H NMR
(400 MHz, acetone-d6) δ 8.69 (s, 1H), 8.67 (s, 1H), 7.52−7.46 (m,
2H), 7.38−7.32 (m, 2H), 7.10 (s, 1H), 6.89 (dt, J = 4.8, 2.9 Hz, 4H),
2.27 (s, 3H). 13C NMR (101 MHz, acetone-d6) δ 157.79, 141.78,
136.80, 133.86, 130.76, 128.23, 116.77, 15.25. HRMS (MALDI/DHB)
calcd for C17H14O2S (M + H+) 282.1027, found 282.10260.
2,5-Bis(4-hydroxy-2-methylphenyl)-3-methylthiophene (10b).
Compound 10b was prepared by 2,5-bis(4-methoxy-2-methylphen-
yl)-3-methylthiophene (9b) 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 brown solid
(mp 158−163 °C). 1H NMR (400 MHz, acetone-d6) δ 8.68 (d, J = 9.1
Hz, 1H), 8.46 (s, 1H), 7.53−7.32 (m, 4H), 7.10 (s, 1H), 6.99−6.84
(m, 4H), 2.40 (s, 3H), 2.18 (s, 3H), 2.02 (s, 3H). 13C NMR (101
MHz, acetone-d6) δ 158.34, 157.77, 141.05, 139.24, 135.80, 137.90,
34.94, 133.15, 132.04, 129.23, 126.42, 124.59, 15.59, 117.66, 117.27,
113.97, 113.63, 29.91, 20.29, 14.49, 14.58. HRMS (MALDI/DHB)
calcd for C19H18O2S (M + H+) 310.1027, found 310.10220.
2,5-Bis(2-fluoro-4-hydroxyphenyl)-3-methylthiophene (10c).
Compound 10c was prepared by 2,5-bis(2-fluoro-4-methoxyphenyl)-
3-methylthiophene (9c) 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 brown solid
(mp 162−166 °C). 1H NMR (400 MHz, acetone-d6) δ 9.20 (d, J = 9.8
Hz, 2H), 7.55 (t, J = 8.9 Hz, 1H), 7.27 (dd, J = 19.0, 10.4 Hz, 2H),
6.84−6.67 (m, 4H), 2.16 (s, 3H). 13C NMR (101 MHz, acetone-d6) δ
162.46, 161.70, 160.00, 159.43, 159.07, 136.57, 136.31, 133.56, 130.86,
129.97, 128.71, 114.22, 113.53, 112.59, 104.42, 103.46, 14.89. HRMS
(MALDI/DHB) calcd for C17H12O2SF2 (M + H+) 318.0527, found
318.05206.
2,5-Bis(4-methoxyphenyl)-3-methylthiophene (9a). Compound
9a was prepared by reaction of 3-methyl-2,5-dibromothiophene and
4-methoxy-phenyl boronic acid according to the general procedure for
Suzuki coupling. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a white solid (mp 117−120 °C). 1H
NMR (400 MHz, CDCl3) δ 7.46−7.41 (m, 2H), 7.36−7.31 (m, 2H),
6.94 (s, 1H), 6.89−6.85 (m, 2H), 6.84−6.80 (m, 2H), 3.76 (s, 3H),
3.74 (s, 3H), 2.22 (s, 3H).
2,5-Bis(2-chloro-4-hydroxyphenyl)-3-methylthiophene (10d).
Compound 10d was prepared by 2,5-bis(2-chloro-4-methoxyphen-
yl)-3-methylthiophene (9d) 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
1
(mp 112−116 °C). H NMR (400 MHz, acetone-d6) δ 9.13 (s, 1H),
7.48 (d, J = 8.5 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.16 (s, 1H), 7.03
(dd, J = 11.0, 2.5 Hz, 2H), 6.90 (ddd, J = 8.7, 6.5, 2.5 Hz, 2H), 2.09 (s,
3H). 13C NMR (101 MHz, acetone-d6) δ 159.34, 158.60, 139.44,
136.23, 135.54, 135.26, 134.49, 133.32, 132.89, 130.22, 125.08, 124.40,
117.89, 117.25, 115.76, 115.25, 14.775. HRMS (MALDI/DHB) calcd
for C17H12O2SCl2 (M + H+) 349.9938, found 349.99296.
2,5-Bis(4-methoxy-2-methylphenyl)-3-methylthiophene (9b).
Compound 9b was prepared by reaction of 3-methyl-2,5-dibromo-
thiophene and (2-methyl-4-methoxy-phenyl) boronic acid according
to the general procedure for Suzuki coupling. Purification by CC
(petroleum ether:ethyl acetate = 98:2) gave the title compound as a
2,5-Bis(2-fluoro-4-methoxyphenyl)-3-phenylthiophene (11a).
Compound 11a was prepared by reaction of 3-bromo-2,5-bis(2-
fluoro-4-methoxyphenyl)thiophene (21) and phenylboronic acid
according to the general procedure for Suzuki coupling using
Pd(dppf)Cl2. Purification by CC (petroleum ether:ethyl acetate =
1
white solid (mp 112−114 °C). H NMR (400 MHz, CDCl3) δ 7.29
(d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.78−6.66 (m, 5H), 3.77
(s, 3H), 3.75 (s, 3H), 2.39 (s, 3H), 2.18 (s, 3H), 1.97 (s, 3H).
2,5-Bis(2-fluoro-4-methoxyphenyl)-3-methylthiophene (9c).
Compound 9c was prepared by reaction of 3-methyl-2,5-dibromo-
thiophene and (2-fluoro-4-methoxy-phenyl) boronic acid according to
the general procedure for Suzuki coupling. Purification by CC
(petroleum ether:ethyl acetate = 98:2) gave the title compound as a
white solid (mp 90−94 °C). 1H NMR (400 MHz, CDCl3) δ 7.49 (t, J
= 8.9 Hz, 1H), 7.30 (t, J = 8.7 Hz, 1H), 7.19 (d, J = 1.1 Hz, 1H),
6.78−6.64 (m, 4H), 3.80 (d, J = 8.5 Hz, 6H), 2.18 (d, J = 1.6 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 161.64, 161.04, 159.94, 159.17,
158.46, 136.06, 132.61, 130.08, 129.02, 128.38, 114.86, 114.11, 110.49,
110.05, 102.08, 55.64, 55.62, 14.89.
1
95:5) gave the title compound as a yellow oil. H NMR (400 MHz,
CDCl3) δ 7.48 (t, J = 8.9 Hz, 1H), 7.38 (s, 1H), 7.25−7.09 (m, 6H),
6.67 (d, J = 7.1 Hz, 2H), 6.55 (t, J = 9.7 Hz, 2H), 3.76 (s, 3H), 3.72 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 159.91, 159.22, 157.52, 139.37,
135.70, 131.79, 128.03, 127.31, 126.38, 125.84, 109.54, 109.09, 101.22,
101.11, 101.13, 54.58.
2,5-Bis(2-chloro-4-methoxyphenyl)-3-phenylthiophene (11b).
Compound 11b was prepared by reaction of 3-bromo-2,5-bis(2-
chloro-4-methoxyphenyl)thiophene (22) and phenylboronic acid
according to the general procedure for Suzuki coupling using
Pd(dppf)Cl2. Purification by CC (petroleum ether:ethyl acetate =
95:5) gave the title compound as a colorless oil. 1H NMR (400 MHz,
CDCl3) δ 7.44 (d, J = 8.6 Hz, 1H), 7.34 (s, 1H), 7.15 (td, J = 12.6, 6.4
Hz, 6H), 6.94 (d, J = 2.5 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 6.76 (dd, J
= 8.6, 2.5 Hz, 1H), 6.67 (dd, J = 8.6, 2.4 Hz, 1H), 3.73 (s, 3H), 3.70 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 160.13, 159.58, 140.12, 139.53,
136.55, 135.31, 134.83, 133.78, 132.92, 131.93, 128.60, 128.34, 127.06,
126.81, 125.37, 115.77, 115.23, 113.37, 113.01, 55.62.
2,5-Bis(2-chloro-4-methoxyphenyl)-3-methylthiophene (9d).
Compound 9d was prepared by reaction of 3-methyl-2,5-dibromo-
thiophene and (2-chloro-4-methoxy-phenyl) boronic acid according to
the general procedure for Suzuki coupling. Purification by CC
(petroleum ether:ethyl acetate = 98:2) gave the title compound as a
1
white solid (mp 122−124 °C). H NMR (400 MHz, CDCl3) δ 7.47
(d, J = 8.7 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.13 (s, 1H), 7.03 (d, J =
2.6 Hz, 1H), 7.00 (d, J = 2.6 Hz, 1H), 6.85 (dd, J = 8.1, 2.6 Hz, 2H),
3.83 (s, 3H), 3.81 (s, 3H), 2.11 (s, 3H).
2,5-Bis(2-fluoro-4-hydroxyphenyl)-3-phenylthiophene (12a).
Compound 12a was 2,5-bis(2-fluoro-4-methoxyphenyl)-3-phenylthio-
phene (11a) and boron tribromide according to general procedure for
2,5-Bis(4-hydroxyphenyl)-3-methylthiophene (10a). Compound
10a was prepared by 2,5-bis(4-methoxyphenyl)-3-methylthiophene
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ether cleavage. Purification by CC (petroleum ether:ethyl acetate =
2:1) gave the title compound as a green solid (mp 142−145 °C). H
8.7 Hz, 1H), 6.77 (ddd, J = 15.3, 10.7, 2.3 Hz, 2H), 6.70−6.57 (m,
4H). 13C NMR (101 MHz, acetone-d6) δ 162.36, 159.92, 158.23,
136.85, 135.35, 135.01, 134.97, 133.32, 132.58, 130.04, 128.31, 125,57,
124.45, 120.12, 113.10, 112.56, 112.50, 112.33, 104.28, 104.04. HRMS
(MALDI/DHB) calcd for C22H13O3SF3 (M + H+) 414.0541, found
414.05320.
2,3,5-Tris(2-chloro-4-hydroxyphenyl)thiophene (14c). Compound
14c was prepared by 2,3,5-tris(2-chloro-4-methoxyphenyl)thiophene
(13c) and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 2:1)
gave the title compound as a yellow solid (mp 185−187 °C). 1H NMR
(400 MHz, acetone-d6) δ 9.17 (s, 1H), 9.09 (s, 1H), 8.91 (s, 1H), 7.56
(d, J = 8.5 Hz, 1H), 7.36 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.05 (d, J =
2.5 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.95−6.89 (m, 3H), 6.77 (dd, J
= 8.4, 2.4 Hz, 1H), 6.68 (dd, J = 8.4, 2.4 Hz, 1H). 13C NMR (101
MHz, acetone-d6) δ 159.19, 159.07, 158.81, 158.44, 158.32, 138.51,
135.25, 134.27, 132.95, 127.25, 124.33, 118.00, 117.91, 117.25, 117.16,
117.07, 115.90, 115.86, 115.16, 115.07, 114.97, 114.88. HRMS
(MALDI/DHB) calcd for C22H13O3SCl3 (M + H+) 461.9662, found
461.96455.
1
NMR (400 MHz, acetone-d6) δ 9.22 (s, 1H), 7.67 (t, J = 8.9 Hz, 1H),
7.53 (s, 1H), 7.36−7.16 (m, 6H), 6.83−6.67 (m, 3H), 6.62 (dd, J =
11.6, 2.4 Hz, 1H). 13C NMR (101 MHz, acetone-d6) δ 159.19, 158.81,
158.32, 139.33, 138.51, 137.36, 135.25, 134.74, 134.27, 133.51, 132.95,
130.34, 127.25, 124.77, 124.33, 117.96, 117.16, 115.86, 115.02. HRMS
(MALDI/DHB) calcd for C22H14O2SF2 (M + H+) 380.0676, found
380.06771.
2,5-Bis(2-chloro-4-hydroxyphenyl)-3-phenylthiophene (12b).
Compound 12b was 2,5-bis(2-chloro-4-methoxyphenyl)-3-phenyl-
thiophene (11b) and boron tribromide according to general procedure
for ether cleavage. Purification by CC (petroleum ether:ethyl acetate =
2:1) gave the title compound as a yellow solid (mp 152−154 °C). 1H
NMR (400 MHz, acetone-d6) δ 7.58 (d, J = 8.5 Hz, 1H), 7.48 (s, 1H),
7.32−7.21 (m, 6H), 7.06 (d, J = 2.4 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H),
6.92 (dd, J = 8.7, 2.4 Hz, 1H), 6.84 (dd, J = 8.4, 2.4 Hz, 1H). 13C
NMR (101 MHz, acetone-d6) δ 158.83, 140.77, 135.61, 134.79,
133.17, 132.89, 129.29, 128.85, 127.72, 124.63, 117.87, 117.31, 115.78,
115.34. HRMS (MALDI/DHB) calcd for C22H14O2SCl2 (M + H+)
412.0103, found 412.00861.
2,5-Bis(4-methoxyphenyl)furan (15a). Compound 15a was
prepared by reaction of 2,5-dibromofuran and 4-methoxy-phenyl
boronic acid according to the general procedure for Suzuki coupling
using Pd(PPh)4. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a white solid (mp 113−114 °C). 1H
NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.8 Hz, 4H), 6.86 (t, J = 5.8
Hz, 4H), 6.50 (s, 2H), 3.77 (s, 6H). 13C NMR (101 MHz, CDCl3) δ
158.9, 152.8, 125.0, 124.0, 114.1, 105.6, 55.3.
2,3,5-Tris(4-methoxyphenyl)thiophene (13a). Compound 13a was
prepared by reaction of 2,3,5-tribromothiophene and 4-methoxy-
phenyl boronic acid according to the general procedure for Suzuki
coupling using Pd(dppf)Cl2. Purification by CC (petroleum ether:-
ethyl acetate = 95:5) gave the title compound as a red solid (mp 142−
145 °C). 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.7 Hz, 2H), 7.18
(dd, J = 8.1, 2.1 Hz, 2H), 7.13 (s, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.75
(dd, J = 11.4, 8.7 Hz, 4H), 3.77 (s, 3H), 3.74 (s, 3H), 3.73 (s, 3H).
2,3,5-Tris(2-fluoro-4-methoxyphenyl)thiophene (13b). Com-
pound 13b was prepared by reaction of 2,3,5-tribromothiophene and
(2-fluoro-4-methoxy-phenyl) boronic acid according to the general
procedure for Suzuki coupling using Pd(dppf)Cl2. Purification by CC
(petroleum ether:ethyl acetate = 95:5) gave the title compound as a
2,5-Bis(4-methoxy-2-methylphenyl)furan (15b). Compound 15b
was prepared by reaction of 2,5-dibromofuran and (4-methoxy-2-
methylphenyl) boronic acid according to the general procedure for
Suzuki coupling using Pd(PPh)4. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
(mp 106−108 °C). H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 8.4
1
Hz, 2H), 6.74 (d, J = 9.6 Hz, 4H), 6.45 (s, 2H), 3.76 (s, 6H), 2.46 (s,
6H). 13C NMR (101 MHz, CDCl3) δ 158.73, 152.21, 136.15, 128.32,
123.43, 116.50, 111.48, 108.98, 55.26, 22.21.
yellow oil. H NMR (400 MHz, CDCl3) δ 7.65 (dd, J = 9.3, 4.1 Hz,
1H), 7.34 (s, 1H), 7.10 (t, J = 8.7 Hz, 1H), 7.01 (t, J = 8.8 Hz, 1H),
6.71−6.61 (m, 2H), 6.54 (td, J = 10.0, 2.5 Hz, 4H), 3.76 (s, 3H), 3.72
(s, 6H). 13C NMR (101 MHz, CDCl3) δ 161.52, 161.01, 160.66,
160.05, 159.05, 158.53, 136.25, 133.87, 132.29, 131.61, 129.08, 127.92,
116.81, 114.57, 114.08, 110.57, 109.96, 102.31, 101.85, 55.80−55.40.
2,3,5-Tris(2-chloro-4-methoxyphenyl)thiophene (13c). Com-
pound 13c was prepared by reaction of 2,3,5-tribromothiophene and
(2-chloro-4-methoxy-phenyl) boronic acid according to the general
procedure for Suzuki coupling using Pd(dppf)Cl2. Purification by CC
(petroleum ether:ethyl acetate = 95:5) gave the title compound as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 8.7 Hz, 1H),
7.37 (s, 1H), 7.19 (d, J = 8.6 Hz, 1H), 7.05−6.98 (m, 2H), 6.91 (dd, J
= 11.9, 2.5 Hz, 2H), 6.85 (dd, J = 8.7, 2.6 Hz, 1H), 6.70 (dd, J = 8.6,
2.5 Hz, 1H), 6.64 (dd, J = 8.6, 2.5 Hz, 1H), 3.82 (s, 3H), 3.76 (d, J =
3.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 159.84, 159.44, 159.22,
138.59, 137.42, 136.63, 134.95, 133.97, 133.62, 132.84, 132.44, 131.82,
129.69, 127.76, 125.42, 124.94, 115.71, 114.94, 113.34, 112.74, 55.71.
2,3,5-Tris(4-hydroxyphenyl)thiophene (14a). Compound 14a was
prepared by 2,3,5-tris(4-methoxyphenyl)thiophene (13a) and boron
tribromide according to the general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 2:1) gave the title
compound as a brown solid (mp 285−288 °C). 1H NMR (400 MHz,
acetone-d6) δ 8.84 (s, 1H), 8.69 (s, 1H), 8.62 (s, 1H), 7.60−7.54 (m,
2H), 7.15−7.05 (m, 2H), 7.01−6.95 (m, 2H), 6.91−6.85 (m, 2H),
6.78−6.73 (m, 2H). 13C NMR (101 MHz, acetone-d6) δ 158.17,
138.99, 136.74, 136.43, 132.74, 131.35, 130.77, 128.07, 125.90, 125.39,
125.30, 116.66, 116.44, 116.19, 115.62, 111.18. HRMS (MALDI/
DHB) calcd for C22H16O3S (M + H+) 360.0819, found 360.08147.
2,3,5-Tris(2-fluoro-4-hydroxyphenyl)thiophene (14b). Compound
14b was prepared by 2,3,5-tris(2-fluoro-4-methoxyphenyl)thiophene
(13b) and boron tribromide according to general procedure for ether
cleavage. Purification by CC (petroleum ether:ethyl acetate = 2:1)
gave the title compound as a brown solid (mp 232−238 °C). 1H NMR
(400 MHz, acetone-d6) δ 9.21 (s, 1H), 9.14 (s, 1H), 8.98 (s, 1H), 7.63
(t, J = 8.9 Hz, 1H), 7.41 (s, 1H), 7.14 (t, J = 8.6 Hz, 1H), 7.05 (t, J =
2,5-Bis(2-fluoro-4-methoxyphenyl)furan (15c). Compound 15c
was prepared by reaction of 2,5-dibromofuran and (2-fluoro-4-
methoxy-phenyl) boronic acid according to the general procedure
for Suzuki coupling using Pd(PPh)4. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
(mp 95−97 °C). H NMR (400 MHz, CDCl3) δ 7.71 (t, J = 8.8 Hz,
2H), 6.74−6.65 (m, 2H), 6.61 (dd, J = 12.9, 2.4 Hz, 2H), 3.74 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 160.66, 159.79, 158.17, 146.90,
126.53, 112.06, 110.37, 102.19, 101.94, 55.63.
2,5-Bis(2-chloro-4-methoxyphenyl)furan (15d). Compound 15d
was prepared by reaction of 3,4-dibromofuran and (2-chloro-4-
methoxy-phenyl) boronic acid according to the general procedure for
Suzuki coupling using Pd(PPh)4. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
(mp 112−114 °C). H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.8
Hz, 2H), 7.07 (s, 2H), 7.00 (d, J = 2.6 Hz, 2H), 6.90 (dd, J = 8.8, 2.6
Hz, 2H), 3.84 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 158.99, 149.23,
131.01, 128.84, 122.17, 115.72, 113.46, 111.26, 55.59.
2,5-Bis(4-hydroxyphenyl)furan (16a). Compound 16a was pre-
pared by 2,5-bis(4-methoxyphenyl)furan (15a) 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
1
brown solid (mp 176−177 °C). H NMR (400 MHz, acetone-d6) δ
8.64 (s, 2H), 7.64 (d, J = 8.6 Hz, 4H), 6.90 (d, J = 8.7 Hz, 4H), 6.69
(s, 2H). 13C NMR (101 MHz, acetone-d6) δ 159.18, 145.20, 128.61,
126.88, 124.32, 116.68. HRMS (MALDI/DHB) calcd for C16H12O3
(M + H+) 252.0783, found 252.07810.
2,5-Bis(4-hydroxy-2-methylphenyl)furan (16b). Compound 16b
was prepared by 2,5-bis(4-methoxy-2-methylphenyl)furan (15b) and
boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 4:1) gave the title
compound as a brown solid (mp 184−187 °C). 1H NMR (400 MHz,
acetone-d6) δ 6.86−6.78 (m, 4H), 6.65 (d, J = 2.2 Hz, 2H), 6.55 (dd, J
Q
dx.doi.org/10.1021/jm400157e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
= 8.4, 2.4 Hz, 2H), 2.10 (s, 6H). 13C NMR (101 MHz, acetone-d6) δ
159.04, 146.02, 138.60, 132.18, 128.93, 123.75, 117.98, 113.29, 20.40.
HRMS (MALDI/DHB) calcd for C18H16O3 (M + H+) 280.1091,
found 280.10940.
2,5-Bis(2-fluoro-4-hydroxyphenyl)furan (16c). Compound 16c
was prepared by 2,5-bis(2-fluoro-4-methoxyphenyl)furan (15c) and
boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 4:1) gave the title
compound as a brown solid (mp 219−222 °C). 1H NMR (400 MHz,
acetone-d6) δ 9.16 (s, 1H), 7.81 (t, J = 8.8 Hz, 2H), 6.81 (dd, J = 8.6,
2.3 Hz, 2H), 6.75 (dd, J = 5.8, 2.5 Hz, 3H), 6.71 (d, J = 2.3 Hz, 1H).
13C NMR (101 MHz, acetone-d6) δ 161.52, 159.43, 147.91, 127.66,
112.87, 111.38, 110.53, 104.23, 103.99. HRMS (MALDI/DHB) calcd
for C16H16O3F2 (M + H+) 288.0595, found 288.05925.
2,5-Bis(2-chloro-4-hydroxyphenyl)furan (16d). Compound 16d
was prepared by 2,5-bis(2-chloro-4-methoxyphenyl)furan (15d) 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 (mp 194−198 °C). 1H NMR (400 MHz,
acetone-d6) δ 7.70 (d, J = 8.7 Hz, 2H), 6.91 (s, 2H), 6.88 (d, J = 2.5
Hz, 2H), 6.80 (dd, J = 8.7, 2.5 Hz, 2H). 13C NMR (101 MHz,
acetone-d6) δ 158.36, 150.23, 131.34, 130.08, 121.62, 118.03, 115.77,
111.54. HRMS (MALDI/DHB) calcd for C16H16O3Cl2 (M + H+)
320.0003, found 320.00015.
3,4-Bis(4-methoxyphenyl)furan (17a). Compound 17a was
prepared by reaction of 3,4-dibromofuran and 4-methoxy-phenyl
boronic acid according to the general procedure for Suzuki coupling
using Pd(PPh)4. Purification by CC (petroleum ether:ethyl acetate =
98:2) gave the title compound as a white solid (mp 144−148 °C). 1H
NMR (400 MHz, CDCl3) δ 7.42 (s, 2H), 7.08 (d, J = 8.8 Hz, 4H),
6.76 (d, J = 8.8 Hz, 4H), 3.73 (s, 6H).
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
compound as a brown solid (mp 170 °C). 1H NMR (400 MHz,
acetone-d6) δ 8.28 (s, 2H), 7.53 (s, 2H), 6.88 (d, J = 8.3 Hz, 2H), 6.64
(d, J = 2.3 Hz, 2H), 6.57 (dd, J = 8.2, 2.5 Hz, 2H), 2.00 (s, 6H). 13C
NMR (101 MHz, acetone-d6) δ 157.52, 141.30, 138.59, 132.41,
127.12, 124.25, 117.68, 113.31, 20.67. HRMS (MALDI/DHB) calcd
for C18H16O3 (M + H+) 280.1091, found 280.10940.
3,4-Bis(2-fluoro-4-hydroxyphenyl)furan (18c). Compound 18c
was prepared by 3,4-bis(2-fluoro- 4-methoxyphenyl)furan (17c) and
boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
1
compound as gray solid (mp 209−211 °C). H NMR (400 MHz,
acetone-d6) δ 9.03 (s, 1H), 7.71 (s, 2H), 7.00 (t, J = 8.6 Hz, 2H),
6.63−6.58 (m, 4H). 13C NMR (101 MHz, acetone-d6) δ 162.09,
159.70, 137.53, 130.41, 126.40, 114.54, 113.54, 104.61. HRMS
(MALDI/DHB) calcd for C16H16O3F2 (M + H+) 288.0595, found
288.05925.
3,4-Bis(2-chloro-4-hydroxyphenyl)furan (18d). Compound 18d
was prepared by 3,4-bis(2-chloro-4-methoxyphenyl)furan (17d) and
boron tribromide according to general procedure for ether cleavage.
Purification by CC (petroleum ether:ethyl acetate = 3:1) gave the title
1
compound as a gray solid (mp 179−183 °C). H NMR (400 MHz,
acetone-d6) δ 7.68 (s, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 2.5
Hz, 2H), 6.72 (dd, J = 8.4, 2.4 Hz, 2H). 13C NMR (101 MHz,
acetone-d6) δ 160.47, 140.17, 134.63, 133.47, 128.47, 125.58, 115.38,
113.44. HRMS (MALDI/DHB) calcd for C16H16O3Cl2 (M + H+)
320.0003, found 320.00015.
2-Bromo-5-(4-methoxy-2-methylphenyl)thiophene (19). Com-
pound 19 was prepared by reaction of 2,5-dibromofuran and (4-
methoxy-2-methylphenyl) boronic acid according to the general
procedure for Suzuki coupling using Pd(OAc)2. Purification by CC
(petroleum ether:ethyl acetate = 98:2) gave the title compound as a
3,4-Bis(4-methoxy-2-methylphenyl)furan (17b). Compound 17b
was prepared by reaction of 3,4-dibromofuran and (4-methoxy-2-
methylphenyl) boronic acid according to the general procedure for
Suzuki coupling using Pd(PPh)4. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
yellow oil. H NMR (400 MHz, CDCl3) δ 7.23 (d, J = 8.4 Hz, 1H),
6.98 (d, J = 3.7 Hz, 1H), 6.73 (m, 3H), 3.78 (s, 3H), 2.35 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 159.57, 144.81, 137.75, 133.82, 131.56,
129.96, 126.39, 125.98, 116.28, 111.38, 55.32, 21.28.
1
2,5-Dibromo-3-methylthiophene (20). Compound 20 was pre-
pared by bromination of 3-methyl thiophene using NBS. Distillation
gave the title compound as a yellow oil. 1H NMR (400 MHz, CDCl3)
δ 6.74 (s, 1H), 2.14 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 138.07,
131.90, 110.23, 108.47, 15.23.
3-Bromo-2,5-bis(2-fluoro-4-methoxyphenyl)thiophene (21).
Compound 21 was prepared by reaction of 2,3,5-dibromofuran and
(2-fluoro-4-methoxyphenyl) boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a yellow oil.
1H NMR (400 MHz, CDCl3) δ 7.44−7.33 (m, 2H), 7.23 (s, 1H),
6.73−6.60 (m, 4H), 3.77 (s, 3H), 3.75 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 161.51, 161.07, 160.62, 159.18, 158.58, 132.65, 128.79,
128.18, 110.70, 110.06, 102.37, 102.13, 101.90, 55.68.
3-Bromo-2,5-bis(2-chloro-4-methoxyphenyl)thiophene (22).
Compound 21 was prepared by reaction of 2,3,5-dibromofuran and
(2-chloro-4-methoxyphenyl) boronic acid according to the general
procedure for Suzuki coupling. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 8.7 Hz, 1H), 7.27 (d, J
= 8.6 Hz, 1H), 7.15 (s, 1H), 6.93 (dd, J = 13.3, 2.5 Hz, 2H), 6.76 (ddd,
J = 12.4, 8.6, 2.6 Hz, 2H), 3.74 (s, 3H), 3.72 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 160.63, 159.87, 140.52, 135.28, 133.48, 132.94,
131.68, 129.42, 124.35, 123.61, 115.79, 115.18, 113.39, 112.81, 110.66,
55.63.
(mp 102−106 °C). H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 1.7
Hz, 1H), 7.33 (d, J = 1.7 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.82 (d, J
= 2.4 Hz, 2H), 6.77 (dd, J = 8.4, 2.5 Hz, 2H), 3.82 (s, 6H), 2.24 (s,
6H).
3,4-Bis(2-fluoro-4-methoxyphenyl)furan (17c). Compound 17c
was prepared by reaction of 3,4-dibromofuran and (2-fluoro-4-
methoxy-phenyl) boronic acid according to the general procedure
for Suzuki coupling using Pd(PPh)4. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
1
(mp 117−119 °C). H NMR (400 MHz, acetone-d6) δ 7.75 (s, 2H),
7.10 (t, J = 8.8 Hz, 2H), 6.78−6.68 (m, 4H), 3.82 (s, 6H). 13C NMR
(101 MHz, acetone-d6) δ 162.57, 161.43, 160.13, 142.53, 132.02,
120.55, 110.92, 102.63, 102.37, 56.00.
3,4-Bis(2-chloro-4-methoxyphenyl)furan (17d). Compound 17d
was prepared by reaction of 3,4-dibromofuran and (2-chloro-4-
methoxy-phenyl) boronic acid according to the general procedure for
Suzuki coupling using Pd(PPh)4. Purification by CC (petroleum
ether:ethyl acetate = 98:2) gave the title compound as a white solid
(mp 137−140 °C). 1H NMR (400 MHz, acetone-d6) δ 7.84 (d, J = 8.8
Hz, 2H), 7.07 (s, 2H), 7.00 (d, J = 2.6 Hz, 2H), 6.90 (dd, J = 8.8, 2.6
Hz, 2H), 3.84 (s, 6H). 13C NMR (101 MHz, acetone-d6) δ 160.47,
140.17, 134.63, 133.47, 128.47, 125.58, 115.38, 113.44, 55.91.
4,4′-(Furan-3,4-diyl)diphenol (18a). Compound 18a was prepared
by 3,4-bis(4-methoxyphenyl) furan (17a) 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
2,5-Dibromofuran (23). Compound 23 was prepared by
1
1
brown solid (mp 172−174 °C). H NMR (400 MHz, acetone-d6) δ
bromination of furan using Br2 as a brown oil. H NMR (400 MHz,
7.63 (s, 2H), 7.10−7.05 (m, 4H), 6.78 (dd, J = 11.2, 4.5 Hz, 4H). 13C
NMR (101 MHz, acetone-d6) δ 157.53, 140.98, 130.45, 126.60,
124.21, 116.06. HRMS (MALDI/DHB) calcd for C16H12O3 (M + H+)
252.0782, found 252.07810.
CDCl3) δ 6.29 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 121.88,
114.24, 77.36, 77.04, 76.72.
3,4-Dibromofuran (24). Compound 24 was prepared through
oxidation of (E)-2,3-dibromo-2- 3-butane-1,4-diol using chromosulfu-
1
3,4-Bis(4-hydroxy-2-methylphenyl)furan (18b). Compound 18b
was prepared by 3,4-bis(4-methoxy-2-methylphenyl)furan (17b) and
boron tribromide according to general procedure for ether cleavage.
ric acid by steam-distillation as a colorless oil. H NMR (400 MHz,
CDCl3) δ 7.45 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 141.60,
103.97.
R
dx.doi.org/10.1021/jm400157e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Estrogen Receptor Binding Affinity. Relative binding affinities
were determined by a competitive radiometric binding assay as
previously described,51,67 using 2 nM [3H]estradiol as tracer
([2,4,6,7-3H]-estra-1,3,5(10)-triene-3,17β-diol, 70−115 Ci/mmol, Per-
kin-Elmer, Waltham, MA), and purified full-length human ERα and
ERβ were purchased from PanVera/Invitrogen (Carlsbad, CA).
Incubations were for 18−24 h at 0 °C. Hydroxyapatite (BioRad,
Hercules, CA) was used to absorb the receptor−ligand complexes, and
free ligand was washed away. The binding affinities are expressed as
relative binding affinity (RBA) values with the RBA of estradiol set to
100%. The values given are the average range or SD of two to three
independent determinations. Estradiol binds to ERα with a Kd of 0.2
nM and to ERβ with a Kd of 0.5 nM; these values were determined by
Scatchard analysis using the binding assay protocol described
previously.51
ACKNOWLEDGMENTS
■
Supported by grants from NSFC (20872116, 81172935,
91017005), the Program for New Century Excellent Talents
in University (NCET-10-0625), Key Project of Ministry of
Education of China (313040), the Research Fund for the
Doctoral Program of Higher Education of China
(20100141110021) and the Fundamental Research Funds for
the Central Universities (20113010202006). Research support
from the National Institutes of Health (PHS 5R37 DK015556
to J.A.K. and R01 DK077085 to K.W.N.) is gratefully
acknowledged. S.S. is supported by the Frenchman’s Creek
Women for Cancer Research. We are grateful to Teresa Martin
to help in the binding assays.
Gene Transcriptional Activity. Assays were performed as
previously described.52 HepG2 and Ishikawa cells cultured in
Dulbecco’s Minimum Essential Medium (DMEM) (Cellgro by
Mediatech, Inc. Manassas, VA) supplemented with 10% fetal bovine
serum (FBS) (Hyclone by Thermo Scientific, South Logan, UT), 1%
nonessential amino acids (Cellgro), penicillin−streptomycin−neo-
mycin antibiotic mixture, and Glutamax (Gibco by Invitrogen Corp.
Carlsbad, CA), were maintained at 37 °C and 5% CO2. HepG2 cells
were transfected with 10 μg of 3× ERE-luciferase reporter plus 1.6 μg
of ERα or ERβ expression vector per 10 cm dish using FugeneHD
reagent (Roche Applied Sciences, Indianapolis IN). The next day, the
cells were transferred to phenol-red-free growth media supplemented
with 10% charcoal-dextran sulfate-stripped FBS at a density of 20000
cells/well, incubated in 384-well plates overnight at 37 °C and 5%
CO2, and stimulated with various concentrations of compounds in
triplicate. Ishikawa cells grown in phenol-red-free growth media were
transfected with 10 μg of 3× ERE-luciferase reporter per 10 cm dish
using FugeneHD reagent. The next day, cells were plated at a density
of 20000 cells/well, incubated in 384-well plates overnight at 37 °C
and 5% CO2, and treated with compounds dissolved in DMSO using a
robotic pin-tool dispenser. Compounds were assayed in dose curves
ranging from 10 μM to 100 pM for ERE luciferase assays in HepG2
cells and at a single dose of 10 μM for ERE luciferase assays in
Ishikawa cells. Luciferase activity was measured after 24 h using
BriteLite reagent (Perkin-Elmer Inc., Shelton, CT) according to
manufacturer’s protocol. Raw data measured as relative light units were
normalized for each plate using the average of DMSO treated samples
as 0% and the average of top of the E2 curve as 100%.
ABBREVIATIONS USED
■
ER, estrogen receptor; CC, column chromatography; DCM,
dicholoromethane; E2, estradiol; HEC-1, human endometrial
cancer cells; HepG2, human hepatoma cells; LBD, ligand-
binding domain; LBP, ligand-binding pocket; RBA, relative
binding affinity; SAR, structure−activity relationship; THF,
tetrahydrofuran; TLC, thin layer chromatography
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Molecular Modeling. The thiophenes 2e or 4e were super-
positioned using68 COOT with OBHS in the OBHS-bound ER crystal
structure52 so that the A-ring mimetic formed the canonical hydrogen-
bonding pattern with R394 and E353. For 2e, the other phenol was
superposed with the phenyl-sulfonate substituent of OBHS, and for
4e, the second phenol was positioned to hydrogen bond with T347.
The models were transferred to CCP4MG for presentation.69
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC results and HPLC spectra for final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For J.A.K.: phone, 1 217 333 6310; E-mail, jkatzene@uiuc.
edu. For H.-B.Z.: phone, 1 86 27 6875 9586; E-mail, zhouhb@
whu.edu.cn.
Notes
The authors declare no competing financial interest.
S
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