Construction of benzofuranone library: via a metal-free, one-pot intermolecular condensation, and their application as efficient estrogen receptor β modulators

Article abstract of DOI:10.1039/c9cc05756k

Facile synthesis of benzofuranone was achieved through a metal-free, one-pot intermolecular condensation between α-hydroxy aryl ketones and resorcinol derivatives. A library of 20 compounds with moderate to good overall yields was prepared. These compounds showed strong binding toward estrogen receptors along with good selectivity for ERβ (>190-fold over ERα). Anti-proliferative activity on DU-145, U-87, and MCF-7 cells gave inhibition IC₅₀ values in the low μM range, which suggested the promising potential therapeutic applications of these new classes of benzofuranones.

Full text of DOI:10.1039/c9cc05756k

Volume 55 Number 97 18 December 2019 Pages 14545–14702
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Xiaodong Shi, Hai-Bing Zhou et al.
Construction of benzofuranone library via a metal-free,
one-pot intermolecular condensation, and their application
as efficient estrogen receptor β modulators

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Construction of benzofuranone library via a
metal-free, one-pot intermolecular condensation,
and their application as efficient estrogen
receptor b modulators†
Cite this: Chem. Commun., 2019,
55, 14570
Received 25th July 2019,
Accepted 2nd October 2019
Xueke Peng,‡a Zhiye Hu,‡a Jing Zhang,‡a Wentao Ning,a Silong Zhang,a
DOI: 10.1039/c9cc05756k
a
a
Chune Dong,
Xiaodong Shi *b and Hai-Bing Zhou
*
rsc.li/chemcomm
Facile synthesis of benzofuranone was achieved through a metal-
free, one-pot intermolecular condensation between a-hydroxy aryl
ketones and resorcinol derivatives. A library of 20 compounds with
moderate to good overall yields was prepared. These compounds
showed strong binding toward estrogen receptors along with good
selectivity for ERb (4190-fold over ERa). Anti-proliferative activity
on DU-145, U-87, and MCF-7 cells gave inhibition IC50 values in the
low lM range, which suggested the promising potential therapeutic
applications of these new classes of benzofuranones.
Estrogens play a predominant role in regulating human physio-
logical functions, including reproductive tissues and nonreproduc-
tive tissues in both males and females.1 The estrogen receptors (ER)
generally contain two major subtypes, ERa and ERb, which display
different tissue distribution and biological performance.2 The ERa
Scheme 1 Representative ERb-selective ligands.
mainly expresses in the reproductive tissues3 such as breasts and only a few compounds have been identified with ERb binding
and uterus, while ERb exists in the nonreproductive tissues such selectivity.8 Three reported ERb-selective compounds are shown in
as skeletal, cardiovascular, and central nervous systems.4 In the Scheme 1A.
reproductive tissues, there will be an increased risk of cancer if
Isoflavone genistein, as a phytoestrogen, was the first ERb-
the levels of ERa are over-expressed or abnormal, and ERb selective compound identified and isolated from natural products.9
displays an anti-proliferative effect.5 Therefore, ERb is considered ERB-04110 and indazoles11 are the other two examples with good
as an important pharmaceutical target for the potential treatment of ERb selectivity over ERa (4200-fold). All these compounds have
various related diseases and symptoms.6
been considered as important pharmacophores for therapeutic
The ligand-binding pockets (LBPs) of ERa and ERb are very applications. Therefore, identifying novel building blocks with
similar with differences only on two amino acids: Met421 and effective ER binding and high selectivity is always attractive, and
Leu384 in ERa and Ile373 and Met336 in ERb.7 Therefore, of great importance in biomedical research. Herein, we report
achieving the selective binding of these two targets is extremely our discovery of 3,3-disubstituted benzofuranone derivatives as
challenging. Most estrogens have good selectivity to ERa, a novel class of ER modulators. With the successful development
of a facile synthesis of these compounds via a metal-free, one-pot
intermolecular condensation between a-hydroxy ketone and
resorcinol, a small library of 20 compounds was rapidly prepared.
a State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis
and Drug Discovery (Wuhan University), Ministry of Education, Hubei Province
Competitive binding and anti-proliferative activity assays revealed
that several compounds have activity toward estrogen receptors
with excellent selectivity for ERb (Scheme 1B).
Over the past decade, our group has been focused on studying
the biological activity of estrogen receptor and developing
effective small molecules that regulate the process as potential
drug candidates.12 With some success in understanding how
Engineering and Technology Research Center for Fluorinated Pharmaceuticals,
Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China.
E-mail: zhouhb@whu.edu.cn; Tel: +86 2768759586
b Department of Chemistry, University of South Florida, Tampa, FL 33620-5250,
USA. E-mail: xmshi@usf.edu; Tel: +1 813 974 7249
† Electronic supplementary information (ESI) available. CCDC 1918835. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c9cc05756k
‡ These three authors contributed equally to this work.
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at 120 1C with 2.0 equiv. NaOH was identified as the optimal
condition, which yielded 3a in a moderate yield (51%). It is
noteworthy that the amount of base significantly influences the
reaction performance. Only trace amounts of 3a were detected
with 1.0 equiv. of NaOH. When increasing the amount of base to
3.0 equiv., a reduced yield was also observed, likely resulting
from the decomposition of lactone. Other tested bases (KOH,
Na2CO3, tBuOK, Cs2CO3) all resulted in lower yields. Moreover,
inferior yields were always obtained on either increasing or
decreasing the reaction temperature. With the obtained optimal
conditions, a series of benzofuranones 3 was prepared. The
substrate scope is shown in Table 1.
As indicated above, the phenolic group is crucial for further
biological evaluation. Therefore, a sequential synthetic approach
was then applied. The crude products with the anisole motif from
the condensation step were directly charged to demethylation
Scheme 2 Synthetic design for the benzofuranone library.
small molecules bind in the active pocket of the estrogen receptor, conditions. Phenyl rings containing different electron-donating
we recently put more effort into the design and evaluation of the or electron-withdrawing groups could all be readily prepared
heterocycle building blocks as potential modulators for ER.13 As using this protocol (4a–4n). A library of 20 compounds with
shown above, all three examples with good ERb-binding selectivity meta-phenolic (MP) groups was prepared and isolated. The
(Scheme 1A) contain meta-hydroxy phenol as a common moiety, feasibility to access a library of 3,3-diaryl benzofuranone deriva-
which is linked to other aromatic units. Based on this analysis, we tives greatly highlighted the value of this reported protocol in
wondered whether this general structural design concept can practical synthesis. With all these compounds in hand, their
be further extended to other heterocycles bearing similar meta- binding affinities toward both ERa and ERb were determined
hydroxy phenol. One proposed heterocycle building block was using a standard competitive fluorometric receptor-binding
benzofuranone 3, as shown in Scheme 2A.
assay.17 The binding comparison of this new class of compounds
Notably, 3-mono-substituted benzofuranones are not stable 3 and 4 over estradiol was performed. The Relative Binding
due to the more acidic proton on C3, and its potential resonance Affinity (RBA) values are shown in Table 2.
formation.14 Thus, 3,3-disubstituted benzofuranone derivatives
To our great delight, this new class of compounds 3 showed
become more promising candidates to evaluate our hypothesis. a superior binding ability towards ERb and ERa. The structure–
According to literature, the general strategies to synthesize these activity relationship study indicated that for compounds containing
compounds are Pd-catalyzed C–H lactonization,15 and Lewis two substituted phenyl groups, the position of the hydroxyl group of
acid-catalyzed a-keto-ester condensation with phenol.16 There-
fore, a feasible synthetic route with readily available starting
materials is highly desirable. From the retro-synthetic design
point of view, a reaction between resorcinol and the corres-
ponding diarylketones would be an ideal pathway. The synthetic
route was designed based on the base-mediated aryl rearrange-
ment and sequential intramolecular cyclization, as shown in
Scheme 2B. This new route is very attractive since (A) the overall
transformation is based on intermolecular condensation for a
diverse substrate modification and (B) transition metal catalysts
are not needed for easy purification and library construction. To
test this new route, both diarylketone 20 and a-hydroxy ketone 2
were used to react with resorcinol 1a under basic conditions.
To facilitate the proposed rearrangement, a high temperature
is generally preferred. With NaOH (2 eq.) as the base, the reaction
of 1a with 2a0 was tested under different solvents, such as DMSO,
DME, ethylene glycol, or butanol at 110 1C. However, no desired
product 3a was observed with a messy reaction in all the cases.
Interestingly, 3a was obtained in 23% yield on replacing 2a0 with
the less reactive hydroxy ketone 2a in glycerol. The structure of
compound 3a was unambiguously confirmed by X-ray diffraction
analysis. Encouraged by this result, extensive screening of
conditions was conducted with regards to the solvents, base,
Table 1 Reaction substrate scopea
a
Reaction condition: a-hydroxyl ketone derivative (1.0 equiv.), resorcinol
(1.0 equiv.), and sodium hydroxide (2.0 equiv.) in glycerol was stirred at
and temperature (see ESI†). Conducting the reaction in glycerol 120 1C for 12 h; isolated yield.
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Table 2 Relative binding affinity (RBA) of 3 and 4 for ERa and ERba
activity of these compounds are provided in ESI†). In terms of
agonist activity, compound 4e showed 2.3-fold more potency for
ERb over ERa, while 4n exclusively exhibited ERb agonist
activity. For compounds 4i and 4m, ERa selectivity was observed
(4.8 and 1.8-fold, respectively). Moreover, the compound 4i
exclusively exhibited ERb antagonist activity without observing
obvious ERa antagonist activity.
To evaluate the antiproliferative activity of these types of
compounds, the above four compounds were then screened
against DU-145 metastatic prostate cancer cells, U-87 glioma cells,
and MCF-7 breast cancer cells, and the results are summarized
in Fig. 1. Overall, 4e, 4m, and 4n showed a moderate effect in
inhibiting all three testing cells, while 4i only displayed an
inhibition on DU-145 cells. Among them, compound 4e showed
obvious anti-proliferative activity towards DU-145 cells, and
compound 4n displayed excellent anti-proliferative activity
towards U-87 cells and MCF-7 cells. Moreover, these four
compounds are not toxic to normal Vero cell lines (Fig. 1).
Furthermore, annexin V-FITC/PI staining assays showed that
Compound
ERa
ERb
9.01 ꢀ 0.75
b/a
5.0
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
1.81 ꢀ 0.30
0.15 ꢀ 0.18
1.24 ꢀ 0.17
1.71 ꢀ 0.26
2.04 ꢀ 0.06
1.22 ꢀ 0.13
3.51 ꢀ 0.47
3.45 ꢀ 0.56
0.38 ꢀ 0.18
0.62 ꢀ 0.10
0.11 ꢀ 0.04
4.17 ꢀ 0.36
2.72 ꢀ 0.34
3.84 ꢀ 0.53
0.75 ꢀ 0.11
0.09 ꢀ 0.02
o0.01
—
—
0.81 ꢀ 0.18
0.65
0.7
1.4
2.3
0.64
1.9
2.6
2.4
1.2 ꢀ 0.34
2.77 ꢀ 0.06
2.76 ꢀ 0.31
2.25 ꢀ 0.22
6.41 ꢀ 0.59
0.97 ꢀ 0.24
1.45 ꢀ 0.04
21.17 ꢀ 0.21
0.86 ꢀ 0.09
3.19 ꢀ 0.22
0.41 ꢀ 0.07
14.08 ꢀ 0.28
0.41 ꢀ 0.06
o0.01
192.5
0.21
1.2
0.11
18.8
4.6
—
—
17.9
12.5
0.12 ꢀ 0.05
0.4 ꢀ 0.01
0.09 ꢀ 0.06
—
4m
4n
7.15 ꢀ 0.27
1.13 ꢀ 0.12
a
Relative Binding Affinity (RBA) values are determined by competitive
fluorometric binding assays and are expressed as IC50 estradiol/IC50 the effect of compound 4n on U87 cells is associated with
compound ꢁ 100 ꢀ the range (RBA, estradiol = 100%).
apoptosis (see ESI† for details). These results indicate that the
benzofuranone derivatives showed extensive inhibitory activity
phenyl has a significant effect on the RBA of the compounds (4a–4f).
Most of the compounds exhibit good ERb selectivity when the
hydroxyl group is at the meta-position of the phenyl group. In
particular, compound 4e gave the best ERb binding affinity and
selectivity. The relative binding towards ERb is 4190-fold
higher than that towards ERa. Notably, 4e showed a better or
comparable ERb binding selectivity compared to the three
reported ERb selective compounds, namely, Genistein, ERB-041,
and Indazole. The ERb selectivity of Genistein is only 7.2.9 The
ERb selectivity of compound 4e is approximately twice higher
than that of Indazole (ERb selectivity was about 100)11 and
slightly lower than that of ERB-041, which has the ERb selectivity
of 226.10 Interestingly, compound 4i with an isopropyl group
instead of diaryl also showed good selectivity to ERb, revealing
another potential design direction for selective ER binding. Four
compounds (4e, 4i, 4m, and 4n) with the best selectivity for ERb
binding were selected to further evaluate their transcriptional
activities of ERs compared to 17b-estradiol (E2). The results are
summarized in Table 3.
against cancer cells, and the anti-proliferative activity of MCF-7
cells is mainly derived from the antagonist activity for ERa,
while the anti-proliferative activity of DU-145 cells and U-87 cells
are mainly produced from the agonist activity for ERb.
To verify the potential interaction between benzofuranone
compounds with ERa and ERb, the molecular modelling of 4f
and 4e was performed because the two compounds showed
high ERa and ERb binding affinities, respectively (Fig. 2).
Fig. 2A shows the docking mode of compound 4f binding to
ERa. The phenolic hydroxyl group of the compound 4f that
mimics the A ring of E2 can form a hydrogen bond with Glu353
on the helix 3. Meanwhile, the benzofuran core also displays
interactions with ERa, which can form a hydrogen bond with
Met421. On the other hand, compound 4e adopted different
binding orientations as shown in Fig. 2B. The hydroxyl group
on the benzofuran, instead of the phenolic hydroxyl group,
forms a hydrogen bond with Glu305. The interactions of the
As shown in Table 3, all compounds showed moderate to
good transcriptional antagonist activity for ERa and agonist
activity for ERb (the ERa agonist activity and ERb antagonist
Table 3 Transcriptional activity of 4e, 4i, 4m and 4na
ERa (antagonist mode)
ERb (agonist mode)
Compound
EC50 (mM)
Eff (% E2)
EC50 (mM)
Eff (% E2)
4e
4i
4m
4n
0.725
—
0.728
0.628
33 ꢀ 11
92 ꢀ 11
39 ꢀ 2
21 ꢀ 3
1.28
1.39
0.935
0.775
46 ꢀ 9
51 ꢀ 4
62 ꢀ 11
79 ꢀ 5
a
Fig. 1 Anti-proliferative activities on DU-145, U-87 and MCF-7 cells and
toxicity in normal Vero cell lines for benzofuranone derivatives (IC50, mM).
IC50 values are an average of at least three independent experiments ꢀ
standard deviation (mean ꢀ SD), # represents the IC50 4100 mM.
Luciferase activity was measured in HEK293T cells transfected with
3 ꢁ ERE-driven luciferase reporter and expression vectors encoding ERa
or ERb and treated in triplicate with increasing doses (up to 10ꢂ5 M) of
the compounds.
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We are grateful to the NSFC (81773557, 81573279 and
81373255), Major Project of Technology Innovation Program
of Hubei Province (2016ACA126), NSFHP (2017CFA024), and
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Conflicts of interest
There are no conflicts to declare.
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