Synthesis and antitumor evaluation of 2,3-diarylbenzofuran derivatives on HeLa cells

Article abstract of DOI:10.1016/j.bmcl.2017.03.010

2,2-Dihydroxyarylethanones, readily prepared from the commercially available aromatic ethyl ketones, were reacted with resorcinol, 3-methoxyphenol or 2-methoxyphenol in multi steps one-pot manner promoted by trifluoroacetic acid to furnish the 2,3-diarylbenzofuran derivatives in 22–95% yield. Sixteen targeted compounds were synthesized and characterized by ¹H NMR, ¹³C NMR and HRMS. MTT assay indicated that most compounds possessed effectively inhibitory activities against the proliferation of HeLa cell. Among them, 4f had the highest inhibitory activities, with the IC₅₀ being 13.40?±?2.04?μmol/L. Cell cycle analysis, Annexin V-FITC/propidium iodide dual staining assay and western blotting analysis revealed that 4f inhibited the proliferation of Hela cell through apoptosis induction in a dose-dependent manner via obviously up-regulated the levels of Bak and Bim, while striking down-regulated the level of Bcl-2 and Bcl-xL protein.

Full text of DOI:10.1016/j.bmcl.2017.03.010

Accepted Manuscript
Synthesis and antitumor evaluation of 2,3-diarylbenzofuran derivatives on HeLa
cells
Guo-Xue He, Jing-Mei Yuan, Hai-Miao Zhu, Kai Wei, Ling-Yun Wang, Shi-
Lin Kong, Dong-Liang Mo, Cheng-Xue Pan, Gui-Fa Su
PII:
S0960-894X(17)30245-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.010
BMCL 24762
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 November 2016
19 February 2017
6 March 2017
Please cite this article as: He, G-X., Yuan, J-M., Zhu, H-M., Wei, K., Wang, L-Y., Kong, S-L., Mo, D-L., Pan, C-
X., Su, G-F., Synthesis and antitumor evaluation of 2,3-diarylbenzofuran derivatives on HeLa cells, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.010
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and antitumor evaluation of
2,3-diarylbenzofuran derivatives on HeLa cells
Guo-Xue He†, Jing-Mei Yuan†, Hai-Miao Zhu, Kai Wei, Ling-Yun Wang, Shi-Lin Kong, Dong-Liang Mo,
Cheng-Xue Pan∗ and Gui-Fa Su∗
Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Pharmaceutical Sciences, Guangxi Normal
University, 15 Yu Cai Road, Guilin 541004, P.R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
2,2-Dihydroxyarylethanones, readily prepared from the commercially available aromatic ethyl
ketones, were reacted with resorcinol, 3-methoxyphenol or 2-methoxyphenol in multi steps one-
pot manner promoted by trifluoroacetic acid to furnish the 2,3-diarylbenzofuran derivatives in
Revised
Accepted
22~95% yield. Sixteen targeted compounds were synthesized and characterized by 1H NMR, 13
NMR and HRMS. MTT assay indicated that most compounds possessed effectively inhibitory
activities against the proliferation of Hela cell. Among them, 4f had the highest inhibitory
activities, with the IC50 being 13.40 2.04 µmol/L. Cell cycle analysis, Annexin V-
FITC/propidium iodide dual staining assay and western blotting analysis revealed that 4f
inhibited the proliferation of Hela cell through apoptosis induction in a dose-dependent manner
via obviously up-regulated the levels of Bak and Bim, while striking down-regulated the level of
Bcl-2 and Bcl-xL protein.
C
Available online
Keywords:
2,3-diarylbenzofuran
multistep one-pot reaction
antitumor activity against HeLa cells
2016 Elsevier Ltd. All rights reserved.
Benzofuran scaffold presents as a key structural unit in
numerous bioactive natural products and synthetic
pharmaceutical compounds.1 Many benzofuran derivatives
exhibit a broad range of biological activities such as anticancer,2
antimicrobial,3 antifungal,4 antioxidative,5 anti-HIV activities,6
some of which were served as acetylcholinesterase inhibitors7
and histone deacetylase inhibitors.8
Among the structurally diverse benzofuran derivatives, the
2,3-diarylbenzo[b]furan has gained considerable attention.9
Structurally, its framework was the combination of three
pharmacologically important subunits of benzofuran,
diarylmethane10 and 1,2-diphenylethylene (stilbene).11 Many
bioactive natural products also had been found possessing a 2,3-
diarylbenzo[b]furan scalfold.9a,9g,9l-9n In particular, the derivatives
present one or more unprotected hydroxyl groups occupying an
especially important position, since this functional group was
widely found in natural medicines9a,9e,9g,9l-9n and some
Fig. 1. Selective examples for bioactive natural products containing the 2,3-
diarylbenzo[b]-furan core
representative compounds were depicted in Fig. 1.
-furan derivatives with unprotected hydroxyl groups. Therefore,
to develop an efficient, material readily available and easy-to-
handle procedure for synthesis of 2,3-diaryl benzo[b]furans,
especially those can directly access the products with unprotected
hydroxyl groups, are still very much desired.
Up to now, many synthetic methodologies developed to access
to these important heterocyclic compounds were focused on the
transition metal-catalyzed protocol which was usually required
for expensive metal catalysts and complex ligands.9a-9k And most
of the available protocols were difficult to enlarge to provide
enough samples for pharmacological studies. What's more, very
few of them could provide direct synthesis of 2,3-diarylbenzo[b]-
Herein, we would like to report a simple and efficient protocol
for the synthesis of 2,3-diarylbenzofuran derivatives with at least
one unprotected hydroxyl group from readily available aromatic
———
∗ Corresponding author. Tel. & fax: +86-773-582-6869; e-mail: chengxuepan@163.com (C.-X. PAN)
∗ Corresponding author. Tel. & fax: +86-773-582-6869; e-mail: gfysglgx@163.com (G.-F. SU)
† These authors equally contributed to this work.

O
O
HO
OH
ethyl ketones and phenols, whose inhibitory activities against the
proliferation of HeLa cells in vitro were investigated.
O
R
O
O
H+
H+
H2O
OH
OH
Ar1
3
Ar1
Ar1
H2O
Inspired by the reactions of ninhydrin with phenols reported
by Kim and Jung12 (Scheme 1A, see Supporting Information), we
considered that a novel route for the synthesis of benzoins
(Scheme 1B, see SI) might be developed based on the
indeno[1,2-b]benzofuranones synthesis procedure. Prompted by
this idea, the 2,2-dihydroxy-1-phenylethanone 2a, readily
prepared from acetophenone followed the literature procedure13
with slight modification, was mixed with resorcinol (3a) and
subjected to AcOH, BF3-Et2O, H2SO4, CF3CO2H (TFA) from
room temperature to reflux conditions. When H2SO4 was used,
the reaction was complicated and the reactants easily carbonized.
With AcOH or BF3-Et2O, the reaction was incomplete and a lot
of reactants were recovered. TFA gave the best result and a new
product could be isolated in 38% yield, and structural analysis
indicated that it was not benzoin derivative but 2,3-
O
+
H
2
M1
MP1
+
H
O
HO
R
R
R
H2O
O
3
Ar1
Ar1
Ar1
Ar2
Ar2
MP2
M2
4
Scheme 3. Plausible mechanism for the formation of 4
Since the 2,3-diarylbenzofuran derivatives 4 possess the
structural characteristic of the anti-estrogens agents used to treat
the estrogen receptor-dependent cancer, we evaluated their
inhibitory activities against the proliferation of HeLa cell lines in
vitro by using MTT assay and the results were summarized in
Table 2.
diarylbenzofuran derivative.
After further optimizing reaction conditions (Scheme 2),
different 2,2-dihydroxy-1-aryl-ethanones 2 reacted with phenols
to afford sixteen 2,3-diarylbenzofuran derivatives 4 in 22-95%
yield (Table 1). The 3D structure of 4f indicated that the dihedral
angle between Ar2 and benzofuran ring is about 45º (see SI).
Table 2. Anticancer activities of 4 against HeLa cell lines
Compd.
4a
IC50/(µM) a
Compd.
4i
IC50/(µM) a
29.70
28.88
57.78
47.54
>100
13.40
26.87
29.91
10.04
29.21
28.31
32.74
36.72
32.02
13.71
19.89
21.15
4b
4j
4c
4k
4d
4l
4e
4m
4n
4f
4g
4o
4h
4p
HCPT
aEach IC50 value was calculated from three independent experiments
performed in triplicate. Errors were within the range of 5~10% of the reported
values.
As shown in Table 2, most compounds exhibited cytotoxicity
activities against the proliferation of HeLa cell with IC50 values
of micromole level, among which 4f had the highest inhibitory
activities, with the IC50 being 13.40 µM, almost similar potency
to the positive control HCPT. Among the natural benzofuran
derivatives, only the anti-cancer activities of Diptoindonesin G
and Anigopreissin A derivatives were reported.15 Diptoindonesin
G15a showed anti-proliferation effect against P-388 cells (IC50
=13.2 1.1µM) and Permethylated Anigopreissin A exhibited
cytotoxicity towards HepG2 and MCF7 cells, with the IC50 being
0.24 0.05 and 0.85 0.04 µM, respectively.15b
Scheme 2. General methods to prepare 2,3-diarylbenzofuran derivatives 4.
Reagents and conditions: (i) SeO2 (2 mmol), 1,4-dioxane (1 mL), H2O (2
drops), 80 °C, 7 h, 61~82%; (ii) phenols 3 (2.5 mmol, 3 equiv.), CF3CO2H (1
mL), THF (1 mL), 80~100 °C, 18~24 h, 22~95%.
The plausible mechanism14 for the formation of 4, might start
from the protonation of 2 to form the carbocation M1, which
could undergo a Friedel-Crafts reaction with 3 to give the
intermediate MP1. Then the MP1 was further protonated and
converted to carbocation M2, followed by another Friedel-Crafts
reaction with 3 to afford the intermediate MP2. MP2 continued
to undergo an intramolecular condensation, with the driving force
being aromatized to the benzofuran ring and afforded the target
compound 4 (Scheme 3).
To investigate the molecular mechanism of the targeted
compounds underlying the inhibitory effect on HeLa cell, 4f was
chosen to conduct further bioactive investigation.
Table 1. Chemical structure and yield of synthesized compounds 4a-4p
Yield (%) a
Compd.
Ar1
Compd.
Ar1
Yield (%) a
4a
Ph
4i
4-CH3-C6H4
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
4-HO-C6H4
4-MeO-C6H4
3-pyridinyl
2-naphthyl
60
36
22
74
95
44
94
65
57
68
79
94
66
72
24
73
4b
Ph
4j
4c
Ph
4k
4d
2-CH3-C6H4
2-Br-C6H4
3-CH3-C6H4
3-Br-C6H4
3-MeO-C6H4
4l
4e
4m
4n
4f
4g
4o
4h
4p
a Isolated yield

Firstly, HeLa cell lines were treated with different
concentrations of 4f and the cell inhibition was measured by
MTT assay. The results in Fig. 2 revealed that the cell inhibition
increased remarkably when the concentrations of 4f changed
from 10 µM to 20 µM, the cell inhibition would increase from
about 30% to 65%. These data indicated that compound 4f might
be a potential anticancer agent in a dose-dependent manner.
Fig. 4. Induction of apoptosis by 4f in HeLa cells with the density of 0 µM 10
µM, 20 µM, 30 µM, for 24 h, then stained with fluorescence FITC and PI,
analyzed by flow cytometer.
Fig. 2. Dose-dependent manner effect of 4f on HeLa cells
Finally, the western blot analysis results in Fig. 5 revealed that
4f could obviously up-regulate the levels of Bak and Bim protein,
while striking down-regulated the level of Bcl-2 and Bcl-xL
protein, which further confirmed the underlying mechanism of
the inducing apoptosis by 4f.
Then, the effect of 4f on the cell cycle distribution was
investigated. As shown in Fig. 3, the populations of every period
had no obvious change with an increasing concentration of 4f
after 48 h, which indicated that 4f might inhibit the cell
proliferation via other signaling pathways rather than by blocking
the function of the cell cycle.
Fig. 5. Western blot analysis of the HeLa cells treated with 4f (0 µM, 10 µM,
20 µM, 30 µM, respectively) on the cell apoptosis proteins. β-actin served as
the internal control.
In conclusion, we developed a novel protocol for synthesis of
2,3-diarylbenzofuran derivatives in one-pot two steps starting
from readily available aromatic ethyl ketone and resorcinol, 3-
methoxyphenol or 2-methoxyphenol. This protocol provides an
efficient, material readily available and easy-to-handle procedure
to afford the 2,3-diarylbenzo[b]furans with bioactive important
unprotected hydroxyl group in moderate to high yield. The
results of antitumor activity evaluation in vitro against HeLa cells
indicated that most of the targeted compounds exhibit potent
cytotoxicity with IC50 values at micromole level. They could
inhibit the proliferation of HeLa cell through apoptosis inducing
in a dose-dependent manner by up-regulating the levels of Bak
and Bim, and down-regulating the level of Bcl-2 and Bcl-xL
protein, and 4f could serve as a potential anticancer reagent for
further investigation.
Fig. 3. Results of cell cycle distribution by flow cytometry. Cells was treated
with increasing concentrations of 4f (10 µM, 20 µM, 30 µM, respectively) for
48 h and untreated HeLa as a control.
Thirdly, the Annexin V-FITC/propidium iodide assay to
determine the mode of cancer cells death induced by 4f was
conducted. As shown in Fig. 4, the apoptosis ratios of the HeLa
cell treated with 4f in different concentration were found
obviously increasing from 15.3% (10 µM of 4f), 27.5% (20 µM
of 4f) to 46.5% (30 µM of 4f). These results indicated that the
cell death induced by the 4f was mainly caused by the apoptotic
pathway.

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Acknowledgments
Financial support by National Natural Science Foundation of
China (21162002, 21462008), Guangxi Natural Science
Foundation (2014GXNSFAA118053, 2015GXNSFDA139009),
and State Key Laboratory for Chemistry and Molecular
Engineering of Medicinal Resources, Ministry of Science and
Technology of China (CMEMR2011-17, CMEMR2012-A014,
CMEMR2014-A01) were greatly appreciated.
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Synthesis and antitumor evaluation of 2,3-diarylbenzofuran
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Guo-Xue He†, Jing-Mei Yuan†, Hai-Miao Zhu, Kai Wei, Ling-Yun Wang, Shi-Lin Kong, Dong-Liang Mo,
Cheng-Xue Pan*, Gui-Fa Su*
OH
O
O
+
R
O
Ar1
OH
one-pot
Ar1
OH
OH
OH
4f
IC50 = 13.40 ¦ÌM
OH
HO
R
R
altered.