Design, synthesis, and biological activity of a novel series of benzofuran derivatives against oestrogen receptor-dependent breast cancer cell lines

Article abstract of DOI:10.1016/j.bioorg.2020.103566

A docking study of a novel series of benzofuran derivatives with ERα was conducted. In this study, we report the synthesis of a novel series of benzofuran derivatives and evaluation of their anticancer activity in vitro against MCF-7 human breast cancer cells, as well as their potential toxicity to ER-independent MDA-MB-231 breast cancer cells, human renal epithelial HEK-293 cells, and human immortal keratinocytes (HaCaT cells) by using the MTT colorimetric assay. The screening results indicated that the target compounds exhibited anti-breast cancer activity. The target compound 2-benzoyl-3-methyl-6-[2-(morpholin-4-yl)ethoxy]benzofuran hydrochloride (4e) exhibited excellent activity against anti-oestrogen receptor-dependent breast cancer cells and low toxicity. The preliminary structure-activity relationships of the target benzofuran derivatives have been summarised. In conclusion, the novel benzofuran scaffold may be a promising lead for the development of potential oestrogen receptor inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.103566

Journal Pre-proofs
Design, Synthesis, and Biological Activity of a Novel Series of Benzofuran
Derivatives Against Oestrogen Receptor-Dependent Breast Cancer Cell Lines
Li-Ping Jin, Qian Xie, Er-Fang Huang, Lin Wang, Bing-Qi Zhang, Jian-Shu
Hu, David Chi-Cheong Wan, Zhe Jin, Chun Hu
PII:
S0045-2068(19)31202-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103566
YBIOO 103566
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
26 July 2019
14 December 2019
2 January 2020
Please cite this article as: L-P. Jin, Q. Xie, E-F. Huang, L. Wang, B-Q. Zhang, J-S. Hu, D. Chi-Cheong Wan, Z.
Jin, C. Hu, Design, Synthesis, and Biological Activity of a Novel Series of Benzofuran Derivatives Against
Oestrogen Receptor-Dependent Breast Cancer Cell Lines, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.103566
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Design, Synthesis, and Biological Activity of a Novel Series of Benzofuran Derivatives
Against Oestrogen Receptor-Dependent Breast Cancer Cell Lines
Li-Ping Jin a, Qian Xie a, Er-Fang Huang a, Lin Wang a, Bing-Qi Zhang a, Jian-Shu Hu b, David Chi-Cheong Wan b,
Zhe Jin a,*, and Chun Hu a,*
a Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China;
b School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China.
Keywords:
ABSTRACT
A docking study of a novel series of benzofuran derivatives with ERα
was conducted. In this study, we report the synthesis of a novel series
of benzofuran derivatives and evaluation of their anticancer activity
in vitro against MCF-7 human breast cancer cells, as well as their
potential toxicity to ER-independent MDA-MB-231 breast cancer
cells, human renal epithelial HEK-293 cells, and human immortal
keratinocytes (HaCaT cells) by using the MTT colorimetric assay. The
screening results indicated that the target compounds exhibited anti-
breast cancer activity. The target compound 2-benzoyl-3-methyl-6-
[2-(morpholin-4-yl)ethoxy]benzofuran hydrochloride (4e) exhibited
excellent activity against anti-oestrogen receptor-dependent breast
cancer cells and low toxicity. The preliminary structure-activity
relationships of the target benzofuran derivatives have been
summarised. In conclusion, the novel benzofuran scaffold may be a
promising lead for the development of potential oestrogen receptor
inhibitors.
Breast cancer
Oestrogen receptor
Benzofuran derivatives
Inhibitor
Synthesis
Docking
Biological activity
* Corresponding authors.
E-mail address: jinzheln@163.com (Zhe Jin) and chunhu@syphu.edu.cn (Chun Hu).
https://doi.org/10.1016/j.bioorg.20xx.xx.xxx
Received 26 July 2019; Received in revised form xx xx 2019; Accepted xx xx 2019
Available online xx xx 2019
0045-2068/ © 2019 Elsevier Inc. All rights reserved.

expressed, and the use of ER inhibitors in
combination with GPER1 antagonists can achieve
better therapeutic effects [15].
1. Introduction
Oestrogen is an important female hormone,
Selective oestrogen receptor modulators
(SERMs) are structurally diverse drugs that
interact with intracellular oestrogen receptors in
target organs as oestrogen receptor agonists and
antagonists. SERMs exhibit oestrogenic activities
in bones and lipoprotein, but anti-oestrogenic
activities in breast and endometrial cells, which
can inhibit the hyperplasia of mammary glands
and endometrium [16-18]. Therefore, SERMs have
emerged a focus for research into avoiding or
decreasing the carcinogenic effect of hormone-
replacement therapy. In recent years, given the
progress and development of SERMs, they have
begun to supersede traditional hormone-
replacement therapy in the clinical setting [19].
Raloxifene, a second-generation SERMs, was the
first FDA-approved benzothiophene derivative to
treat osteoporosis and exerts lower endometrial
stimulation than tamoxifen [20,21] (Figure 1).
However, the long-term use of raloxifene can
cause some adverse effects, including the
development of tolerance and venous thrombosis
[20-24].
essential in many organs in women, and closely
related to the occurrence and development of
breast cancer [1,2]. At the cellular level, oestrogen
can mitigate the biological effects caused by the
oestrogen receptor (ER). In clinical practice, the ER
serves as a marker for clinical treatment and
prognosis estimation of many diseases, such as
breast cancer, prostate cancer, ovarian cancer,
ovarian cysts, and endometriosis [3-5]. ERs are
divided into classical nuclear receptors, which are
located in the nucleus and exert regulatory effects
through modulation of the transcription of specific
target genes, and membrane receptors, which
include G protein-coupled receptor 1 (GPER1),
previously known as GPR30 [6]. There are three
subtypes of ER: ERα, ERβ, and ERγ, which are
expressed differently in different tissues and even
within the same tissue [7,8]. In human breast
cancer cells, ERα and ERβ are highly expressed,
but the ratio of ERα and ERβ is different in benign
and malignant breast tumours. ERβ is
predominant in benign tissues, whereas ERα is
dominant in malignant tissues [9]. From a
functional perspective, ERα promotes breast
cancer cell growth and proliferation, whereas ERβ
promotes breast cancer cell proliferation and
suppresses tumour progression [10]. As a member
of the G protein-coupled receptor superfamily,
GPER1 is a membrane protein found on the cell
surface. GPER1 can associate specifically with
oestrogen and mediate cAMP activation and EGFR
transactivation. Compared with traditional ER,
GPER1 differs not only in interaction mode and
effect, but also in sequence homology. Therefore,
GPER1 serves as a separate oestrogen receptor and
plays an important role in oestrogen signal
transduction and the regulation of cell growth,
proliferation, and apoptosis; thus it is an attractive
target for breast cancer treatment [11-14]. Sirianni
et al. detected GPER1 mRNA expression in the
mouse spermatogonial cell line, GC-1, and RNA
interference of GPER1 found that the regulation of
oestrogen on differentiation of these cells was
dependent on the participation of GPER1. In breast
cancer cells, GPER1 and ERα are synchronously
N
O
O
OH
S
HO
raloxifene
O
N
tamoxifen
Figure 1. The structures of raloxifene and
tamoxifen
Benzofuran compounds are distributed
widely in higher plants such as Compositae,
Rutaceae, Liliaceae, and Cyperaceae; most have
strong biological activities and important
biological functions in the treatment of

Alzheimer’s disease, osteoporosis, arrhythmia,
Parkinson’s disease, and tumours [25,26]. Aslam et
al. reported the antibacterial and antifungal
activities of a series of 2-phenylbenzofuran
derivatives [27], and their results showed that
some compounds had strong inhibitory activity
against both the tested bacteria and fungi. Atta et
al. described the inhibition of growth of MCF-7
human breast cancer cells by a series of new
compounds containing benzofuran structures [28];
their results showed that most compounds exerted
strong cytotoxic activity (IC50 value of 5.56–20.8
μg/mL). Based on the structure-activity
relationship of these reported compounds,
including raloxifene and the active sites of ERα,
combining the principles of scaffold hopping,
hybridization, and bioisosterism, and using
O
O
R2NCH2CH2O
O
O
R2NCOCH2O
Figure 2. The structures of the designed
compounds
2. Results and Discussion
2.1 Docking studies
raloxifene
as
a
lead
compound,
the
In this study, the ERα selectivity and
structure-binding affinity correlations of the target
compounds were investigated through molecular
docking; in addition, the difference in anti-breast
cancer activity of the targets in the pharmacology
test was further researched by conformational
analysis and molecular graphics fit.
benzothiophene ring of raloxifene was replaced
with a benzofuran ring to yield the target 2-
arylcarbonyl-3-methylbenzofuran
skeleton.
Subsequently, a novel series of benzofuran
derivatives was designed as the target compounds
by using a molecular docking strategy, and nine
target compounds were synthesised and assayed
for inhibitory activities against MCF-7 and MDA-
MB-231 human breast cancer cells and HEK-293
human renal epithelial cells, which can stably
express GPER1 in vitro [29] (Figure 2).
The docking screening program AutoDock
Vina (version 4.0) was used for all the target
compounds in this study [30]. The default values
of the docking parameters in AutoDock Vina were
accepted. From the Protein Data Bank (PDB), the
crystal structure of ERα complexes with 4-
hydroxytamoxifen (PDB code: 3ERT) was selected
[31]. The grid box dimensions were 20 Å × 14 Å ×
12 Å around the active site. Docking was
performed using the empirical free energy
function and the binding results in our study were
visualised using the PyMOL Molecular Graphics
System (version 1.3) [32].

Figure 3. Docking model of 4a in the active site of ERα (3ERT)
Figure 4. Docking model of 4b in the active site of ERα (3ERT)
Figure 5. Docking model of 4e in the active site of ERα (3ERT)
Figure 6. Docking model of 4f in the active site of ERα (3ERT)

interactions with ARG394 with a distance of 2.9 Å
were found, as shown in Figure 5, which may
explain its increased inhibitory and selectivity
potency.
From the Autodock Vina results, the binding
free energies (ΔGb) of 4a, 4b, 4e, and 4f with ERα
(3ERT) were -8.2, -8.8, -8.5, and -8.2 kcal/mol,
respectively. In these complexes, there was
hydrogen bonding between oxygen atoms and
amino acid residues (Figures 3, 4, 5, and 6). The
docking simulation revealed that the oxygen
atoms on the 5-alkoxy chains of 4a and 4b formed
hydrogen bonds with THR347 with the distance of
3.1 Å and 2.9 Å. In addition, THR347 formed
hydrogen bonds with the oxygen atoms on both
the benzofuran ring and carbonyl group, as shown
in Figure 5. For the compound 4e, more H-bond
2.2 Chemistry
The parent 2-aroyl-3-methybenzofurans (3)
were
synthesised
from
or
2’,4’-
2’,5’-
dihydroxyacetophenone
dihydroxyacetophenone in a higher overall yield,
as shown in the synthetic routes described in
Scheme 1, by three steps: substitution, cyclization,
and debenzylation.
COCH3
OH
COCH3
OH
Ph
O
Ph
O
PhCOCH2Br
BBr3
-15oC
BnCl, KI
BnO
HO
K2CO3, TBAB
O
2
O
3
BnO
HO
1
Scheme 1. The synthetic route of 2-aroyl-3-methylbenzofurans
aroyl-3-methylbenzofuran derivatives (4a–4i)
could be obtained from an ordinary Williamson
reaction, as illustrated in Scheme 2.
The target compounds 5-alkoxy-2-aroyl- 3-
methylbenzofuran derivatives and 6-alkoxy- 2-
R2NCXCH2O
Ph
O
HO
K2CO3, CH3COCH3
reflux, 12h
Ph
O
ClCH2CXNR2
+
O
O
4a-4d
X=O, H2
3a
4a: X = O, NR2 = diethylamino
4b: X = H2, NR2 = piperin-1-yl
4c: X = O, NR2 = morpholin-4-yl
4d: X = O, NR2 = piperin-1-yl
Ph
Ph
K2CO3, CH3COCH3
reflux, 12h
ClCH2CXNR2
+
O
O
O
O
R2NCXCH2O
HO
X=O, H2
4e-4i
3b
4e: X = H2, NR2 = morpholin-4-yl
4f: X = H2, NR2 = pyrrolidin-1-yl
4g: X = O, NR2= piperin-1-yl
4h: X = O, NR2 = pyrrolidin-1-yl
4i: X = O, NR2 = morpholin-4-yl
Scheme 2. The synthetic route of 2-aroyl-3-methylbenzofuran derivatives

parent
hydroxybenzofuran
hydroxybenzofuran (3). The target compounds 4a–
4i were obtained by ordinary Williamson reaction.
compounds
or
2-aroyl-3-methyl-6-
2-aroyl-3-methyl-5-
In general, the target compounds 4a–4i were
synthesised via a four-step procedure as described
in
Scheme
1
and
Scheme
2.
2’,4’-
Dihydroxyacetophenone
or 2’,5’-dihydroxy-
acetophenone was chosen as the starting material
to synthesise the benzofuran derivatives. As the
hydroxy group at position 4’ or 5’ in the
acetophenone moiety had stronger reaction
activity than 2’-hydroxy group, which has an
intramolecular hydrogen bond; the 4’-hydroxy or
5’-hydroxy groups were protected by benzyl
chloride with potassium iodide as catalyst in dry
The structures and spectral characteristics of
the target compounds 3a–3b and 4a–4i are listed in
Table 1. All target compounds were characterised
by their mass spectra (MS), high-resolution mass
spectra (HR-MS), infrared spectra (IR), and proton
NMR spectra.
2.3 Inhibition of human breast cancer
acetone
benzyloxyacetophenone
benzyloxyacetophenone (1). After cyclization with
2-chloroacetophenone derivatives, the benzylation
protection was removed is to yield the target
to
yield
2‘-hydroxy-4’-
2’-hydroxy-5’-
All target compounds were initially tested for
inhibitory effects on MCF-7 human breast cancer
cells by means of the MTT assay [33]; the results
are displayed in Table 1.
or
Table 1. Inhibition of MCF-7 human breast cancer cells by the target compounds at 50 μM (n = 3).
4a 4b 4c 4d 4e 4f 4g 4h 4i
Inhibitory Ratio 41.03 68.15 26.21 25.06 64.23 60.78 34.64 35.71 37.82
(%) ±0.77 ±3.86 ±3.87 ±4.51 ±1.70 ±5.57 ±3.03 ±3.48 ±3.44
Note: MCF-7 human breast cancer cells (provided by the ATCC) were used. The data are expressed as x
± s, n = 3. The incubation time was 4 h and cells were protected from light during incubation.
cells, HEK-293 human renal epithelial cells, and
human immortal keratinocytes (HaCaT cells) by
Compounds 4a, 4b, 4e, and 4f, showing
greater inhibition of MCF-7 cells, were
means of MTT assay, and compared with
subsequently tested for the inhibitory effects on
tamoxifen and raloxifene. The results are
MCF-7 and MDA-MB-231 human breast cancer
presented in Table 2 and Table 3.
Table 2. Inhibition of MCF-7 and MDA-MB-231 human breast cancer cells and human immortal
keratinocytes (HaCaT cells) by the target compounds at 30 μM (n = 3).
Inhibitory Ratio (%)
MDA-MB-231
Ratio
No.
MCF-7/MDA-MB-231/
HaCaT
MCF-7
HaCaT
4a
4b
23.64±11.02
45.81±7.70
43.09±5.86
31.01±11.09
54.31±3.18
36.22±5.33
33.38±5.40
85.46±7.39
25.16±4.38
86.72±3.66
89.44±6.34
86.58±4.79
6.43±2.60
15.08±5.35
6.37±2.61
11.03±4.64
84.33±9.68
78.89±7.74
3.68: 5.19: 1
3.04: 5.67: 1
6.76: 3.95: 1
2.81: 7.86: 1
0.64: 1.06: 1
0.46: 1.10: 1
4e
4f
tamoxifen
raloxifene

Note: MCF-7 and MDA-MB-231 human breast cancer cells, human immortal keratinocytes (HaCaT
cells), and HEK293 human renal epithelial cells (provided by the ATCC) were used. The data are expressed
as x ± s, n = 3. The incubation time period was 4 h, and samples were protected from light.
Table 3. Inhibition of MCF-7 and MDA-MB-231 human breast cancer cells and HEK293 human renal
epithelial cells (n = 3).
IC50 (μM)
Ratio
No.
MCF-7/MDA-MB-231/ HEK-
MCF-7
MDA-MB-231
HEK-293
293
-
1.2:0.4:1
-
1.2:0.3:1
1.1:0.6:1
1.2:0.3:1
4a
4b
4e
>100
>100
11.54*
>100
8.36*
9.93*
9.83*
18.52*
31.25*
62.47*
33.13*
16.16*
32.78*
37.31*
42.23*
39.71*
17.66*
40.08*
4f
tamoxifen
raloxifene
Note: The final concentrations were 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 μm, respectively. * P<0.05
Based on the above pharmacological results,
the inhibitory activities against human breast
cancer of 6-alkoxy-2-benzoyl-3-methylbenzofuran
derivatives appeared to generally be stronger than
those of 5-alkoxy-2-benzoyl-3-methylbenzofuran
derivatives, and the inhibitory activities of the
target compounds with a 2-(alkylamino)ethyl
moiety, especially with 2-(morpholinyl)ethyl,
were stronger than that of the target compounds
containing 2-alkylaminocarbonylmethyl. This may
have been due to the target compounds with 2-
(alkylamino)ethyl, including 2-(morpholinyl)ethyl,
having a larger active binding site with ERα, such
as H-bond interactions and a hydrophilic group.
compounds showed the same inhibition in MCF-7
breast cancer cells and HEK-293 cells. As
mentioned above, GPER1 and ERα are
synchronously expressed in breast cancer cells [15],
such as MCF-7, and HEK-293 can stably express
GPER1 in the cell membrane in vitro [29]. There
was a difference in the expression of GPER1
between the membrane of HEK-293 and MDA-
MB-231, which corresponded with the inhibition
ratio [11]. Based on these biological activity results,
we speculated that the target compounds may also
have an influence on the GPER1-related pathway.
Based on the above results, we can conclude
that the target compounds probably act on ER.
As shown in Table 2, compared with the blank
control, all nine target compounds demonstrated
significant growth inhibition of ER-dependent
MCF-7 breast cancer cells, especially compound 4e.
The inhibitory effects of 4e on normal HaCaT cells
and ER-independent MDA-MB-231 breast cancer
cells were far lower than those of the positive
control, although they were slightly lower than the
positive controls tamoxifen and raloxifene. These
resulted indicated that 4e could effectively inhibit
the growth of ER-dependent MCF-7 breast cancer
cells, but had little effect on normal cells,
demonstrating better selectivity in the treatment of
breast cancer, most likely indicating that 4e acts
selectively on ER. Moreover, as shown in Table 3,
with the exception of compound 4a, the other
3. Conclusion
A novel series of benzofuran derivatives with
lower toxicity and fewer side-effects was
synthesised through structural modification of
raloxifene based on molecular docking. Further
efforts aimed at the development of potent ER
inhibitors, based on the modification of the target
compound 4e, will continue in our laboratory.
4. Experimental
4.1 Molecular docking
The protein structure was obtained from the
crystallographic complex of human ERα in

complex with 4-hydroxytamoxifen (RCSB PDB ID:
3ERT), the resolution value of the complex crystal
was 1.9 Å [31]. 4-Hydroxytamoxifen and other
non-protein molecules were deleted, all of the
hydrogen atoms were missed, and the heavy
atoms were added. The energy of docking was
optimised and the lowest energy conformations
were assigned. Autodock Vina software version
4.0 was used for the molecular docking process,
the interactions between ERα and the tested target
compounds were calculated using Lamarckian
Genetic Algorithm, the grid resolution was 0.3 Å,
the binding site radius was 15 Å, and other
parameters were the default values. All
compounds were docked into the active site of
3ERT. The complex of the ligand-receptor was
viewed by PyMOL Molecular Graphics System
(version 1.3).
4.2.1 General procedures for preparing
benzyloxyacetophenone derivatives (1)
In a 250 ml round-bottomed flask were placed
2’,4’-dihydroxyacetophenone
or
2’,5’-
dihydroxyacetophenone (0.10 mol), benzyl
chloride(0.105 mol), potassium iodide (0.01 mol),
anhydrous potassium carbonate (1 mol) and 100
ml dry acetone. The mixture was refluxed for 5 h
at 56-60 oC in oil bath. After reaction was finished,
the reaction liquid was cooled to room
temperature and filtered. The filtrate was
concentrated under reduced pressure to obtain the
crude. The solid was recrystallized from 100 ml
ethanol to obtain 1.
2’-Hydroxy-4’-benzyloxyacetophenone (1a)
A white powder, yield: 85.2 %, m.p: 101.6-
103.4 °C. EI-MS: 243.0([M+H]+), 264.9([M+Na]+); IR:
3030.4, 2940.4, 1622.9, 1569.9, 1507.3, 1494.2, 1466.2,
1452.7, 1430.5, 1388.2, 1364.6, 1247.1, 1183.2, 1132.7;
1H-NMR (400 MHz, CDCl3) δ 12.75 (1H, s), 7.64
(1H, d, J = 8.4 Hz), 7.44-7.32 (5H, m), 6.55-6.48 (2H,
m), 5.09 (2H, s), 2.56 (3H, s).
4.2 Synthesis
Melting points were determined using an X-4
digital melting point apparatus and were
uncorrected. IR spectra were recorded on KBr
discs by using a Bruker IFS55-IR spectrometer. 1H-
NMR spectra were recorded on Bruker ARX-300,
ARX-400 or ARX-600 MHz NMR spectrometers in
CDCl3 or DMSO-d6. The mass spectra (MS) were
obtained by electronic impact (EI) at 70 eV in an
Agilent spectrometer (with direct insertion probe)
or by electrospray (ESI) in a Waters spectrometer.
High-resolution mass spectrometry (HRMS)
analyses were recorded on an Agilent
Technologies 6530 Accurate-Mass Q-TOF Mass
Spectrometer.
2’-Hydroxy-5’-benzyloxyacetophenone (1b)
A bright yellow needle crystal, yield: 80.7 %,
m.p: 60.5-63.2 °C. EI-MS: 243.0([M+H]+),
265.0([M+Na]+); IR: 3068.2, 2929.8, 1643.7, 1619.0,
1583.2, 1492.9, 1469.9, 1384.3, 1341.5, 1297.2, 1220.5,
1199.5; 1H-NMR (400 MHz, CDCl3): δ 11.90 (1H, s),
7.47-7.34 (5H, m), 7.25 (1H, d, J = 3.0 Hz), 7.18 (1H,
dd, J = 9.0, 3.0 Hz), 6.93 (1H, d, J = 9.0 Hz), 5.04 (2H,
s), 2.58 (3H, s).
4.2.2 General procedures for preparing 2-benzoyl-3-
methyl-5-benzyloxybenzofuran derivatives and 2-
benzoyl-3-methyl-6-benzyloxybenzofuran derivatives
(2)
All commercial reagents and solvents were of
analytical grade and used without further
purification. 2-Chloro-4’-methoxyacetophenone
A
mixture
of
1
(0.10
(0.11
mol),
2-
mol),
and
2-chloro-4’-methylacetophenone
were
chloroacetophenone
synthesised in accordance with published
methods [30-32]. 4-Chloroacetylmorpholine, 1-
acetylpiperidine, N,N-diethyl-2-chloroacetamide,
4-(2-chloroethyl)morpholine hydrochloride, 1-(2-
chloroethyl)piperidine hydrochloride, and 2-
tetrabutylammonium bromide (TBAB, 0.05 mol),
50 ml 30 % aqueous potassium carbonate and 100
ml dichloromethane was refluxed at 40-45 oC in oil
bath for 12 h. Then the organic phase was
separated, washed with 1 mol/L hydrochloric acid
(30 ml×3) and water (30 ml), dried over Mg2SO4,
and concentrated under reduced pressure to
obtain solid. The solid was recrystallized from 80
ml ethanol to obtain crystal 2.
chloro-N,N-diethylethanamine
hydrochloride
were synthesised in accordance with published
methods [33-35].
2-Benzoyl-3-methyl-6-benzyloxybenzofuran (2a)

A white powder, yield: 69.6%, m.p: 91.4-
94.2°C. EI-MS: 343.1([M+H]+), 365.2([M+Na]+),
381.1([M+K]+); IR: 3070.63, 2916.3, 1635.6, 1597.0,
1560.4, 1498.5, 1470.9, 1448.1, 1385.6, 1368.7, 1216.8,
1501.2, 1474.3, 1446.7, 1377.1, 1353.5, 1311.9, 1242.3,
1224.1, 1162.2; H-NMR (400 MHz, CDCl3) δ 8.07-
8.02 (2H, m), 7.63-7.47 (4H, m), 6.99 (1H, d, J = 2.1
Hz), 6.90 (1H, dd, J = 8.5, 2.2 Hz), 2.59 (3H, s).
1
1
1201.7, 1183.6; H-NMR (400 MHz, CDCl3) δ 8.09
(2H, d, J = 7.2 Hz), 7.63-7.59 (1H, m), 7.55-7.48 (4H,
m), 7.46-7.39 (3H, m), 7.37 (1H, d, J = 7.2 Hz), 7.21
(1H, d, J = 2.5 Hz), 7.17 (1H, dd, J = 9.1, 2.5 Hz), 5.14
(2H, s), 2.62 (3H, s).
4.2.4 General procedure for preparing 2-benzoyl-3-
methylbenzofuran derivatives (4)
A mixture of 3 (2 mmol), 2-chloroacetamide or
2-chloroethanamine hydrochloride (2.2 mmol),
anhydrous potassium carbonate (20 mmol),
potassium iodide (1 mmol) and dry acetone 50 ml
was refluxed for 12 h, cooled to room temperature
and filtered. The filtrate was concentrated under
reduced pressure to obtain the crude and purified
by column chromatography to afford 4.
2-Benzoyl-3-methyl-5-benzyloxybenzofuran (2b)
Bright yellow needly crystal, yield: 78.3%;
m.p:
76.8-79.2°C.
EI-MS:
343.1([M+H]+),
365.1([M+Na]+); IR: 3060.5, 2923.0, 1628.2, 1598.7,
1
1563.3, 1500.9, 1447.7, 1383.0, 1268.5, 1167.9; H-
NMR (400 MHz, CDCl3) δ 8.06 (2H, d, J = 7.2 Hz),
7.61-7.55 (2H, m), 7.74-7.50(2H, m), 7.47 (2H, d, J =
7.2 Hz), 7.43-7.39(3H, m), 7.06 (2H, dd, J = 10.6, 1.8
Hz), 5.14 (2H, s), 2.61 (3H, s).
2-[(2-Benzoyl-3-methylbenzofuran-5-yl)oxy]-
N,N-diethylacetamide (4a)
A yellow powder, yield: 74.6%, m.p: 117.0-
118.0 °C. EI-MS: 365([M+]); HR-MS: calc. 366.1700,
found 366.1700; IR: 3050.2, 3010.9, 2858.8, 1657.1,
1608.9, 1586.2, 1586.9, 1442.8, 1351.5, 1301.4, 1266.5,
4.2.3 General procedures for preparing 2-benzoyl-3-
methyl-5-hydroxybenzofuran derivatives and 2-
benzoyl-3-methyl-6-hydroxybenzofuran derivatives (3)
1
1238.0, 1173.5; H-NMR (300 MHz, CDCl3) δ 8.06
To a solution of 2 (0.01 mol) in 30 ml
dichloromethane, was added dropwise slowly
boron tribromide (0.02 mol) in -15 oC ice bath. The
reaction continued for 10 min. After the reaction
was finished, poured the mixture and 30 ml water
into 125 ml separating funnel. The organic phase
was separated and the water phase was extracted
with 20 ml ethyl acetate. Combine organic phase,
dried over Mg2SO4 and concentrated under
reduced pressure to obtain crude. The crude was
washed with 10 ml petroleum ether and 3 ml
dichloromethane to obtain the solid 3.
(2H, d, J = 7.5 Hz,), 7.63-7.41 (4H, m), 7.21-7.11 (2H,
m), 4.75 (2H, s), 3.43 (4H, q, J = 6.9 Hz), 2.60 (3H, s),
1.20 (6H, dt, J = 26.6, 6.3 Hz); 13C NMR (150 MHz,
DMSO-d6) δ: 185.53, 166.65, 155.36, 149.35, 148.81,
137.91, 133.21, 129.71, 129.57, 128.97, 126.78, 119.21,
113.40, 104.66, 67.20, 41.15, 39.93, 14.68, 13.36, 10.29.
[3-Methyl-5-(piperidin-1-ylethoxy)benzofuran-2-
yl](phenyl)methanone (4b)
A white powder, yield: 78.9%, m.p: 52.2-56.4
°C. EI-MS: 363([M+]); HR-MS: calc. 364.1907, found
364.1902; IR: 3061.0, 2928.7, 2848.2, 1638.0, 1600.4,
1561.4, 1467.3, 1448.4, 1329.9, 1310.1, 1265.3, 1239.8,
1
2-Benzoyl-3-methyl-5-hydroxybenzofuran (3a)
1198.3, 1160.0; H-NMR (300 MHz, CDCl3) δ 8.11-
A yellow powder, yield: 87.0%, m.p: 164.0-
165.8°C. EI-MS: 253.1([M+H]+), 274.8([M+Na]+); IR:
3279.4, 2918.1, 1623.5, 1599.6, 1579.5, 1552.7, 1446.3,
8.04 (2H, m), 7.62-7.46 (3H, m), 7.42 (1H, d, J = 8.8
Hz), 7.13-7.08 (2H, m), 4.23 (2H, t, J = 5.9 Hz), 2.89
(2H, t, J = 5.9 Hz), 2.62-2.60 (7H, m), 1.68 (4H, m),
1.49 (2H, m); 13C NMR (150 MHz, DMSO-d6) δ:
185.44, 155.76, 149.14, 148.71, 137.92, 133.14, 129.68,
128.93, 126.99, 119.38, 113.35, 104.15, 66.81, 57.89,
54.88, 40.53, 26.06, 24.40, 10.28.
1
1377.6, 1307.5, 1237.6, 1245.8, 1204.8, 1181.2; H-
NMR (300 MHz, CDCl3) δ 8.10-8.05 (2H, m), 7.63-
7.48 (3H, m), 7.40 (1H, d, J = 8.8 Hz), 7.07-7.01 (2H,
m), 2.58 (3H, s).
2-[(2-Benzoyl-3-methylbenzofuran-5-yl)oxy]-1-
morpholinoethan-1-one (4c)
A white powder, yield: 70.0%, m.p: 159.5-
161.0. EI-MS: 380.2([M+H]+), 402.0([M+Na]+); HR-
MS: calc. 380.1492, found 380.1492; IR: 3066.5,
2-Benzoyl-3-methyl-6-hydroxybenzofuran (3b)
A yellow powder, yield: 94.0%, m.p: 150.8-
153.0°C. EI-MS: 253.0([M+H]+), 274.8([M+Na]+),
290.8([M+K]+); IR: 3228.8, 2923.2, 1612.6, 1549.1,

3009.5, 2961.3, 2856.6, 1663.0, 1637.6, 1560.2, 1440.2,
1475.0, 1313.4, 1234.2, 1210.8, 1183.9; 1H-NMR (300
MHz, CDCl3) δ 8.07 (2H, dd, J = 8.3, 1.3 Hz), 7.62-
7.57 (1H, m), 7.56-7.50 (2H, m), 7.46 (1H, d, J = 9.7
Hz), 7.16 (2H, dq, J = 5.2, 2.6 Hz), 4.78 (2H, s), 3.71-
3.64 (8H, m), 2.61 (3H, s); 13C NMR (150 MHz,
DMSO-d6) δ: 185.53, 166.41, 155.24, 149.37, 148.83,
137.91, 133.21, 129.71, 129.59, 128.97, 126.82, 119.20,
113.41, 104.84, 67.01, 66.56, 45.31, 42.09, 10.34.
2-[(2-Benzoyl-3-methylbenzofuran-5-yl)oxy]-1-
(piperidin-1-yl)ethan-1-one (4d)
A yellow powder, yield: 80.8%, m.p: 103.0-
105.0 °C. EI-MS: 377([M+]); HR-MS: calc. 378.1700,
found 378.1698; IR: 2918.1, 2853.2, 1646.6, 1561.9,
1485.9, 1469.5, 1437.7, 1364.6, 1326.9, 1239.0, 1210.6;
1H-NMR (300 MHz, CDCl3) δ 8.09-8.03 (2H, m),
7.64-7.57 (1H, m), 7.55-7.48 (2H, m), 7.44 (1H, d, J =
8.8 Hz), 7.20-7.13 (2H, m), 4.76 (2H, s), 3.55 (4H, s),
2.60 (3H, s), 1.74-1.51 (6H, m); 13C NMR (150 MHz,
DMSO-d6) δ: 185.48, 165.79, 155.30, 149.32, 148.79,
137.89, 133.18, 129.69, 129.56, 128.95, 126.83, 119.19,
113.36, 104.68, 67.26, 45.73, 42.63, 26.49, 25.78, 24.42,
10.32.
2-[(2-Benzoyl-3-methylbenzofuran-6-yl)oxy]-1-
(piperidin-1-yl)ethan-1-one (4g)
A white powder, yield: 70.8%, m.p: 100.9-
101.6 °C. EI-MS: 378.6([M+H]+), 400.5([M+Na]+),
416.5([M+K]+); HR-MS: calc. 378.1700, found
378.1698; IR: 3058.1, 2932.7, 2859.3, 1663.9, 1645.0,
1618.3, 1559.2, 1471.2, 1443.5, 1355.3,1320.2, 1269.4,
1
1252.5, 1176.5; H-NMR (600 MHz, CDCl3) δ 8.08-
8.04 (2H, m), 7.67-7.61 (2H, m), 7.53-7.50 (2H, m),
7.07 (1H, d, J = 2.1 Hz), 7.04 (1H, dd, J = 8.7, 2.2 Hz),
4.76 (2H, s), 3.60-3.53 (2H, m), 3.53-3.45 (2H, m),
2.61 (3H, s), 1.65 (2H, dt, J = 10.9, 5.6 Hz), 1.62-1.57
(2H, m), 1.57-1.50 (2H, m); 13C NMR (150 MHz,
DMSO-d6) δ: 185.02, 165.44, 160.38, 155.52, 147.87,
138.22, 132.91, 129.54, 128.90, 127.71, 122.71, 122.65,
114.70, 97.08, 66.92, 45.57, 42.62, 26.37, 25.72, 24.40,
10.35.
2-[(2-Benzoyl-3-methylbenzofuran-6-yl)oxy]-1-
(pyrrolidin-1-yl)ethan-1-one (4h)
A white powder, yield: 73.1%, m.p: 113.0-
113.5 °C. EI-MS: 364.6([M+H]+), 386.5([M+Na]+),
402.4([M+K]+); HR-MS: calc. 364.1543, found
364.1542; IR: 3059.7, 2969.1, 2869.9, 1660.3, 1642.8,
1617.7, 1593.9, 1557.3, 1443.2, 1353.1, 1269.5, 1239.0,
1169.5; 1H-NMR (300 MHz, CDCl3) δ 8.08-8.02 (2H,
m), 7.60-7.46 (4H, m), 7.06=7.02 (2H, m), 4.70 (2H,
s), 3.53 (4H, t, J = 6.8 Hz), 2.60 (3H, s), 1.97 (2H, dd,
J = 12.8, 6.4 Hz), 1.90-1.84 (2H,m); 13C NMR (150
MHz, DMSO-d6) δ: 185.04, 165.61, 160.44, 155.33,
147.84, 138.23, 132.90, 129.52, 128.90, 127.73, 122.68,
122.62, 114.74, 97.03, 66.99, 46.02, 45.08, 26.11, 23.97,
10.36.
[3-Methyl-6-(morpholinoethoxy)benzofuran-2-
yl](phenyl)methanone (4e)
A white powder, yield: 72.5%, m.p: 182.5-
184.0 °C. EI-MS: 366([M+]); HR-MS: calc. 366.1700,
found 366.1704; IR: 3038.9, 2936.2, 2866.4, 1642.8,
1616.2, 1558.8, 1447.5, 1356.3, 1267.7, 1236.6, 1163.7;
1H-NMR (300 MHz, CDCl3) δ 8.06 (2H, d, J = 7.6
Hz), 7.64-7.47 (4H, m), 7.05 (1H, s), 6.95 (1H, d, J =
8.6 Hz), 4.68 (2H, s), 4.30 (2H, t, J = 12.5 Hz), 4.01
(2H, d, J = 11.7 Hz), 3.72-3.43 (4H, m), 3.09 (2H, q),
2.61 (3H, s); 13C NMR (150 MHz, DMSO-d6) δ:
188.50, 161.67, 157.68, 149.87, 139.74, 135.84, 132.07,
132.03, 131.23, 125.47, 125.22, 116.32, 98.76, 66.53,
64.55, 58.50, 54.85, 12.57.
2-[(2-Benzoyl-3-methylbenzofuran-6-yl)oxy]-1-
morpholinoethan-1-one (4i)
A yellow powder, yield: 73.3%, m.p: 137.8-
138.6 °C. EI-MS: 380.4([M+H]+), 402.2([M+Na]+),
418.2([M+K]+); HR-MS: calc. 380.1492, found
380.1491; IR: 3055.7, 2959.3, 2922.6, 2857.2, 1662.6,
1640.5, 1618.1, 1554.1, 1473.9, 1440.4, 1357.5, 1269.0,
1238.1; 1H-NMR (300 MHz, CDCl3) δ 8.09-8.03 (2H,
m), 7.59 (2H, d, J = 8.4 Hz), 7.54-7.49 (2H, m), 7.08
(1H, d, J = 2.1 Hz), 7.02 (1H, dd, J = 8.7, 2.2 Hz), 4.78
(2H, s), 3.66-3.64 (8H, m), 2.61 (3H, s); 13C NMR
(150 MHz, DMSO-d6) δ: 185.03, 166.05, 160.30,
155.51, 147.87, 138.21, 132.90, 129.53, 128.88, 127.70,
122.69, 114.72, 97.10, 66.60, 66.50, 66.42, 45.08, 42.04,
10.34.
[3-Methyl-6-(pyrrolidin-1-
ylmethoxy)benzofuran-2-yl](phenyl)methanone (4f)
A white powder, yield: 72.5%, m.p: 209.2-
210.0 °C. EI-MS: 350([M+]); HR-MS: calc. 350.1751,
found 350.1752; IR: 2954.8, 1630.0, 1595.5, 1556.7,
1
1500.8, 1447.9, 1355.7, 1277.1, 1234.1, 1165.0; H-
NMR (300 MHz, CDCl3) δ 8.06 (2H, d, J = 7.6 Hz),
7.64-7.47 (4H, m), 7.05 (1H, s), 6.95 (1H, d, J = 8.6
Hz), 4.68 (2H, s), 4.30 (2H, t, J = 12.5 Hz), 4.01 (2H,
d, J = 11.7 Hz), 3.72-3.43 (4H, m), 3.09 (2H, q), 2.61
(3H, s).

4.3 Antitumor activity assays
3.
J. F. Couse, J. Lindzey, K. Grandien, J. A. Gustafsson,
K. S. Korach, Tissue distribution and quantitative
analysis of estrogen receptor-α (ERα) and estrogen
receptor-β (ERβ) messenger ribonucleic acid in the
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J. W. Woo, Y. R. Chung S. Ahn, E. Kang, E. K. Kim,
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in biomarker status in metastatic breast cancer and
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C. X. Ma, R. Bose, M. J. Ellis, Prognostic and
predictive biomarkers of endocrine responsiveness
for estrogen receptor positive breast cancer.
Advances in Experimental Medicine and Biology
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M. M. Liu, C. Albanese, C. M. Anderson, K. Hilty, P.
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Kushner, Opposing action of estrogen receptors
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S. Denger, G. Reid, H. Brand, M. Kos, F. Gannon,
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X. Wang, R. Sun, Y. Huang, Y. Yan, M. Gao, D. N.
Wang, D. Koirala, D. W. Li, C. Hu, Development of
thieno[3',2':5,6]thiopyrano[4,3-c]pyrazole-3-
carboxamide derivatives as the estrogen receptor
ligands: synthesis, characterization and biological
activity. Medicinal Chemistry 10 ( 2014 ) 836-842.
P. J. Balfe, A. H. McCann, H. M. Welch, M. J. Kerin,
Estrogen receptors β and breast cancer. European
Journal of Surgical Oncology 30 (2004) 1043-1050.
The inhibition of the target compounds
against ER-dependent breast cancer cells and their
potential toxicity were evaluated using the MTT
assay. MCF-7 cells were selected as an ER-
dependent breast cancer cell line, with MDA-MB-
231 ER-independent breast cancer cells, HEK-293
human renal epithelial cells, and human immortal
keratinocytes (HaCaT cells) examined for
comparison. Tamoxifen and raloxifene were used
as the positive control drugs. In brief, the above
strains were seeded respectively onto 96-well
plates at a density of 10,000 cells per well and
incubated at 37°C in a humidified atmosphere of
95% air and 5% CO2 for 24 h. The medium in each
well was replaced with 180 μL of culture medium
containing 30 μM of the compound of choice and
cultured for a further 48 h. To the incubation
medium, 20 μL of 5 mg/ml MTT reagent was
added and incubated for 4 h at room temperature
in the dark. The incubation medium was then
replaced with 150 μL DMSO. The absorbance
intensity at 570 nm was recorded using a
microplate reader, and the inhibition rate and IC50
values were calculated.
4.
5.
6.
7.
8.
Acknowledgement
9.
This work was supported by the National
Science Foundation of China (NSFC) by the grant
Nos. 21072130 and 21342006. The authors would
like to thank Autodock Vina for kindly providing
a free evaluation copy of their software package,
and Schrödinger, Inc. for allowing the use of
PyMOL Molecular Graphics System (version 1.3).
10. S. Saha Roy, R. K. Vadlamudi, Role of estrogen
receptor signaling in breast cancer metastasis.
International Journal of Breast Cancer, 2012 (2014)
654698.
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R. J. Weigel, Identification of a gene (GPR30) with
homology to the G-protein-coupled receptor
superfamily associated with estrogen receptor
expression in breast cancer. Genomics 45 (1997) 607-
617.
Conflict of interest
All authors declare that there are no conflicts
of interest associated with the publication of this
manuscript.
12. E. J. Filardo, J.A. Quinn, K. I. Bland, A. R.
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Graphical Abstract
Design, Synthesis, and Biological Activity
of a Novel Series of Benzofuran
Derivatives Against Oestrogen Receptor-
Dependent Breast Cancer Cell Lines
Li-Ping Jin, Qian Xie, Er-Fang Huang, Lin Wang, Bing-Qi
Zhang, Jian-Shu Hu, David Chi-Cheong Wan, Zhe Jin, and
Chun Hu
Docking model of 4e in the active site of ERα (3ERT)

Highlights
Design, Synthesis, and Biological Activity
of
a
Novel Series of Benzofuran
Derivatives Against Oestrogen Receptor-
Dependent Breast Cancer Cell Lines
Li-Ping Jin, Qian Xie, Er-Fang Huang, Lin Wang, Bing-Qi
Zhang, Jian-Shu Hu, David Chi-Cheong Wan, Zhe Jin, and
Chun Hu
1. A novel series of benzofuran
derivatives were designed and
synthesized through
structural modification of raloxifene
based on molecular docking.
2. The benzofuran derivatives were
evaluated for the anticancer activity in
vitro against breast cancer cell lines.
3. A target compound has exhibited
excellent anti-oestrogen receptor-
dependent breast cancer cell lines
activities and low toxicity.

Conflict of interest
Design, Synthesis, and Biological Activity
of a Novel Series of Benzofuran
Derivatives Against Oestrogen Receptor-
Dependent Breast Cancer Cell Lines
Li-Ping Jin, Qian Xie, Er-Fang Huang, Lin Wang, Bing-Qi
Zhang, Jian-Shu Hu, David Chi-Cheong Wan, Zhe Jin, and
Chun Hu
Conflict of interest
All authors declare that there are no conflicts
of interest associated with the publication of this
manuscript.