Design, synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmcl.2017.04.029

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC₅₀ value of 4.52?±?2.47?μM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure–activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.

Full text of DOI:10.1016/j.bmcl.2017.04.029

Accepted Manuscript
Design, synthesis, biological evaluation and molecular docking studies of novel
3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast
cancer agents
Guoshun Luo, Mingqi Chen, Weiting Lyu, Ruheng Zhao, Qian Xu, Qidong You,
Hua Xiang
PII:
S0960-894X(17)30405-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.029
BMCL 24882
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 January 2017
16 March 2017
11 April 2017
Please cite this article as: Luo, G., Chen, M., Lyu, W., Zhao, R., Xu, Q., You, Q., Xiang, H., Design, synthesis,
biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives
targeting ERα as anti-breast cancer agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/
10.1016/j.bmcl.2017.04.029
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Design, synthesis, biological evaluation and molecular docking studies of novel
3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents
Guoshun Luoa,#, Mingqi Chenb,#, Weiting Lyua, Ruheng Zhaoa, Qian Xua, Qidong Youa, Hua Xianga*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
bLaboratory of Biology, School of Higher Vocational Education, China Pharmaceutical University, 639
Longmian Avenue, Nanjing, 211198, PR China
* Corresponding author. Tel.: +86 025 83271096; Fax: +86 025 83271096(H. Xiang).
E-mail addresses: xianghua@cpu.edu.cn (H. Xiang)
# Same contribution to this paper.
Abstract
The estrogen receptor (ER) has played an important role in breast cancer development and progression
and is a central target for anticancer drug discovery. In order to develop novel selective ERα
modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one
derivatives based on previously reported lead compounds. The biological results indicated that most of
the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities
against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted
compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent
anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity
with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells
with IC50 value of 4.52
2.47 µM. Further molecular docking studies were also carried out to
investigate binding pattern of the newly synthesized compounds with ERα. All these results together
with the structure–activity relationships (SARs) indicated that these
3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for
further optimization as novel SERMs.
Keywords: 3-aryl-4-anilino-2H-chromen-2-one derivatives; synthesis; anti-breast cancer; ERα;
docking studies.
Breast cancer (BC) is the most frequent cancer among women worldwide. It had been estimated

that 1.67 million new cases of BC were diagnosed in 2012 over the world according to a recent report.1
Estrogen receptor α (ERα), a member of the large superfamily of nuclear receptors, is overexpressed
and predominantly involved in more than 70% breast cancer patients.2
The estrogen receptor (ERα) has been an oncology target for the treatment of breast cancer since
the 1960s.3 Since then, a number of selective ER modulators (SERMs) and down regulators (SERDs)
have been discovered, and this field has been extensively reviewed.4-6 SERMs are a class of
nonsteroidal compounds which act as antagonists in breast tissue but agonists in other tissues such as
cardiovascular system and bone.7, 8 Due to their special action mode, SERMs remain as important
anti-breast cancer agents with benefits in cardiovascular system and maintaining bone density
compared with aromatase inhibitors and pure anti-estrogens.9, 10 Among them, tamoxifen (a SERM, 1a),
containing a triphenylethylene scaffold and a basic side chain, was the first SERM approved in the US
for the adjunctive treatment of metastatic breast cancer and have provided invaluable treatments for a
number of patients.11-13 The search for new antiestrogen agents is still continuing, however, due to
emerging drug-resistance mechanisms.14 More specifically, there is still an unmet medical need for a
SERM with increased activity and less side effects.
The coumarin scaffold has been known to medicinal chemists for about 50 years for its
antagonistic properties against the ER.15, 16 In recent years, 7-hydroxycoumarins, 7-hydroxychromenes
and 7-hydroxychromanes, have been reported as selective ER ligands or modulators (SERMs) by many
academic groups and pharmaceutical companies.17-21 SP500263 (3), a coumarin-based SERM, binds
with high affinity to ERα and potently inhibits estrogen-dependent MCF-7 proliferation with similar
IC50 value to tamoxifen (Fig. 1).22 Recently, SS5020 (4) had been reported to downregulate the
estrogen receptor with antitumor effects against chemically induced mammary tumors.23 Structurally
(Fig. 1), compound 4 can be viewed as a hybrid of the known SERM SP500263 (3) and SERD
GW7604 (2b),24 the latter of which is a descendant of 4-hydroxytamoxifen (1b). Furthermore, a related
analogue of SS5020, compound 5 was also reported to be a selective ER down regulator (SERD) by the
company AstraZeneca (Fig. 1).25 However, 3-aryl-4-anilino-2H-chromen-2-one derivatives bearing
basic side chain haven’t been investigated as SERMs till now.

Figure 1. Chemical structures of reported SERMs and SERDs
The molecular basis of ER modulators and down regulators has been reported in a diverse variety
of crystal structure determinations.26, 27 The crystal structure of GW5638 (2a) and compound 5, two
SERDs, in complex with ERα shows that common phenyl acrylic acids overlay whereas the
3-aryl-2H-chromen-2-one skeleton overlay with the triphenylethylene scaffold (Fig. 2A) (PDB ID code:
1R5K). We thus hypothesized that replacing the phenyl acrylic acid of compound 5 with phenyl alkyl
amine should be a reasonable approach to design novel coumarin containing SERMs whose basic side
chain might overlay with the corresponding part of 4-hydroxyl tamoxifen.
In our process research of coumarin derivatives as SERMs, we assumed that it is possible to
combine the hybrid fragments of compounds
3 and 5 to design a series of novel
3-aryl-4-anilino-2H-chromen-2-one derivatives bearing flexible basic side chains as ligands of ER.
Further modification will focus on the substituents at C-7 position of coumarin skeleton and flexible
basic side chains to obtain diverse derivatives (Fig. 2B). All the title compounds were evaluated for
their ERα binding affinities and anti-proliferative activities against two human cancer cell lines
(MCF-7 and Ishikawa). Several molecules exhibited comparable ERα binding affinity and
anti-proliferative potency with Tamoxifen. Furthermore, molecular docking has been carried out. The
possible binding modes with ERα and preliminary structure–activity relationships were also discussed.

Figure 2. (A) Superimposed pose of 2a (green) with 5 (violet) binding to ERα, the protein was shown
in surface; (B) Chemical structures of reported SERMs, SERDs and designed compounds. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
The synthesis of the 4-[(2-aminoethoxy)]-substituted anilines 11a-f and the desired
4-anilino-3-aryl-2H-chromen-2-ones 15a-f, 16a-f and 18a-f is outlined in Scheme 1. The
4-[(2-aminoethoxy)]-substituted anilines 11a-f were obtained in four steps involving: i) reaction of
p-nitrophenol 6 with 2-chloroethanol; ii) chlorination of the resulting 2-hydroxyethyl ether 8; iii)
treatment of the 2-chloroethyl derivative 9 with selected amines; and, finally iv) reduction of the nitro
group in the aminated derivatives 10a-f. As for intermediates 12-14, the synthesis procedure has been
previously reported by our group.28 Target compounds 15a-f, 16a-f and intermediates 17a-f were then
furnished from their intermediates 12-14 by nucleophilic substitution with a variety of anilines 11a-f.
Finally, compounds 18a-f were prepared from their corresponding intermediates 17a-f via
Bn-deprotection in the presence of Pd-C in THF (Scheme 1).

Scheme 1. Reagents and conditions: (a) NaOH, H2O, 80°C, 8 h; (b) pyridine, SOCl2, 1,4-dioxanert,
40°C, 2 h; (c) RCl or RCl·HCl, K2CO3, KI, DMF, 100°C, 4 h; (d) Pd-C, H2, MeOH, rt, 8 h; (e)
corresponding anilines, K2CO3, EtOH, 75oC, 3 h; (f) Pd-C, H2, THF, rt, 12 h.
The ERα binding affinities of test compounds were determined by a fluorescence polarization
displacement assay using commercial estrogen receptor competitor assay kits with Tamoxifen as the
positive control.29-31 The binding affinities are listed in Table 1-3 and are expressed as relative binding
affinity (RBA) values, that is, relative to the affinity of estradiol (E2), which is set at 100%. The data
can be analyzed in three groupings, compounds 15a-f, which bearing an H at C-7 position with
variations in the basic side chain, compounds 16a-f with a methoxy group and 18a-f with a hydroxyl
group. As shown in Table 1, most of title compounds 15a-f exhibited fair binding affinities towards
ERα in comparison with Tamoxifen. Compounds 15c and 15d exhibited relatively higher potency with
RBA values of 0.48% and 052% on ERα, respectively. The results indicated that the skeleton of
3-aryl-4-anilino-2H-chromen-2-one could favorably mimic that of estradiol while H substitution at C-7
position is unfavorable for ERα binding affinity.

Table 1
Structures and relative binding affinities (RBA) of synthesized compounds 15a-f
RBAa (%) or percent competition b
compound
15a
R
H
H
H
H
H
H
R1
ERα
0.35
0.17
15b
0.38
15c
0.52
17% competition at 100 µMc
0.087
15d
15e
15f
-
-
-
-
100
E2
Tamoxifen
7.64
aRelative binding affinity (RBA) values were calculated by EC5032[E2]/EC50[test compound] × 100
(RBA of E2 = 100%), shown as mean of two experiments.
bPercent competition of each compound was calculated from the polarization values.33
cEC50 is not determined (more than 100 µM).
To prove the previous hypothesis, we subsequently introduced a methoxy group into C-7 position
of coumarin skeleton and evaluated the binding affinities of 16a-f. The results are shown in Table 2.
Among compounds 16a-f, 16d displayed the most potent ERα binding affinity activity with RBA value
of 0.95% while morpholinyl side chain containing compound 16e showed the lowest binding affinity in
accordance with previous 15f. The improvement of the binding affinity on ERα was observed in this
series compared with 15a-f (16a vs 15a, 16b vs 15b and 16d vs 15d). It was supposed that these ring
A-expanded analogues 16a-f were assumed to be more efficiently filling the larger cavity of ERα

Table 2
Structures and relative binding affinities (RBA) of synthesized compounds 16a-f
RBAa (%) or percent competitionb
compound
16a
R
H
H
H
H
H
H
R1
ERα
0.72
0.46
16b
0.44
16c
0.75
24% competition at 100 µMc
0.12
16d
16e
16f
-
-
-
-
100
7.64
32
E2
Tamoxifen
aRelative binding affinity (RBA) values were calculated by EC50 [E2]/EC50[test compound] × 100
(RBA of E2 = 100%), shown as mean of two experiments.
bPercent competition of each compound was calculated from the polarization values 33
c EC50 is not determined (more than 100 µM).
As is known that the hydrogen bonds formed between the two hydroxyl group of estradiol and
Glu353, Arg394 and His524 contribute a lot to keep the conformation stable when estradiol binds to
ERα.34 As the ability to form hydrogen bonds with these key amino acids is widely accepted to identify
ERα ligands, we next evaluated the hydroxyl containing compounds 18a-f. Expectedly, it’s obviously
to note that compounds 18a-f possess better binding affinity than the other two series. It could be
inferred that compounds 18a-f containing hydroxyl groups on the C-7 position to mimic estradiol were
able to form more hydrogen bonds with ERα than H substituted compounds 15a-f and methoxyl
substituted compounds 16a-f, thus presenting better binding affinities.
Table 3
Structures and relative binding affinities (RBA) of synthesized compounds 18a-f

RBAa (%)
compound
18a
R
H
H
H
H
H
H
R1
ERα
2.02
1.86
1.72
2.83
0.053
0.48
18b
18c
18d
18e
18f
-
-
-
-
100
E2
Tamoxifen
7.64
aRelative binding affinity (RBA) values were calculated by EC5032[E2]/EC50[test compound] × 100
(RBA of E2 = 100%), shown as mean of two experiments.
Different amino substituents were also investigated, among all the compounds 15a-f, 16a-f and
18a-f, it seemed that the bulkier group (morpholinyl, N-methyl piperazinyl) were inferior to the others.
It was unexpected that compound 15e and 16e which possessed morpholinyl exerted no binding
activity. Dimethylamine, pyrrolidinyl and piperidyl were more preferable in the side chain. 18d with
piperidyl as the basic group was the most potent compound with RBA value of 2.83% which is close to
Tamoxifen (RBA= 7.64%).
To evaluate the anticancer activity, all synthesized compounds were screened against human
breast cancer cell line MCF-7 (ER+) and human endometrial cancer cell line Ishikawa by MTT method
with Tamoxifen as positive control. It is known that Tamoxifen will stimulate endometrial cell growth
and induce endometrial cancer after long term treatment. Therefore it is of concern to evaluate the
influence of new compounds targeting ER on endometrial cells. Results are summarized in Table 4.
As a global observation, most of the title compounds presented significant anti-proliferative
activity on these two cancer cell lines. Moreover, the active compounds demonstrated better activity in
breast cancer than in endometrial cancer cells, and most of them were more active than Tamoxifen in

the two breast cancer cells. Compounds 16e and 18e with morpholinyl group in the side chain which
showed no binding affinity in ERα assay were also not active in this test with IC50 values of more than
100 µM, which indicated an ERα pathway on the anti-proliferative activity. However compound 15e
exhibited moderate anti-proliferative activity on these two cancer cell lines but showed no binding
affinity in ERα assay, whose anti-proliferative activity maybe acquired not only through ERα but a
multi-target effect. Compounds 15f, 16f and 18f with N-methyl piperazinyl showed moderate activity
on MCF-7 and Ishikawa but yet were less potent in their respective groups, which indicated the bulkier
basic group was unfavorable in suppressing cancer cell growth.
The most potent compound against MCF-7 was 16d with an IC50 of 4.52 µM, about 3 fold more
potent than Tamoxifen (11.35 µM), but only showed moderate activity compared with other
compounds against Ishikawa with the IC50 valued at 11.58 µM. It was interesting to find that
dimethoxy compounds (16a, 16c, 16d and 16f) which were less potent in ERα binding assay
manifested slightly greater potency than the corresponding 7-hydroxyl compounds (18a, 18c, 18d and
18f) against MCF-7 which may also confirm that the obtained anti-proliferative activity was not only
through ERα.
Analysis of the influence of different basic group on anti-proliferative activity suggested a
preference for three medium size basic group, diethylamine, pyrrolidinyl and piperidyl (15b, 15c, 15d,
16b, 16c, 16d, 18b, 18c and 18d). For example, 16d, the most potent compound against MCF-7 and
18d against Ishikawa were both piperidyl substituted. Overall, compounds with the above three basic
groups displayed the best activity against all of the two cancer cell lines.
Table 4
Cancer cells anti-proliferative activity
(IC50, µM)a, b
compound
MCF-7
Ishikawa
15a
15b
15c
15d
15e
15f
8.76 2.23
7.57 2.56
6.21 0.82
5.86 0.94
47.45 7.05
15.63 1.37
8.32 1.17
10.21 3.75
6.51 0.94
11.76 4,67
10.83 3.84
9.3 1.21
8.74 3.46
49.53 8.34
23.42 6.96
12.23 3.09
8.84 1.36
9.39 2.82
16a
16b
16c

16d
16e
4.52 2.47
>100
11.58 3.81
>100
16f
12.39 3.38
9.53 0.73
8.81 1.08
8.42 2.27
7.31 2.12
>100
15.24 4.07
13.25 2.16
14.83 4.14
11.75 3.47
8.43 1.06
>100
18a
18b
18c
18d
18e
18f
17.59 2.03
11.35 3.13
19.36 4.68
16.47 2.04
Tamoxifen
aAnti-proliferative effect (IC50) of MCF-7 (ER+) and Ishikawa (endometrial cancer) cell lines, the
concentration of compound that inhibit 50% of the cell growth after 48 h of drug exposure measured by
MTT assay.
bEach value was shown as mean SD of three experiments.
In summary, replaced the H or OMe on C-7 position of coumarin framework with OH, most of the
title compounds slightly decreased cell growth inhibitory potency in MCF-7 cell line. However,
completely reverse trend was seen in ERα binding affinity. Interestingly, introduction of medium size
basic groups such as pyrrolidinyl and piperidyl on terminal aniline could increase both the ERα binding
affinity and anti-proliferative activities against two cancer cell lines. Furthermore, a significant
decrease of anti-proliferative activity was observed by introduction of morpholinyl group indicating
that this group is unfavourable in these series. The preliminary structure–activity relationship (SAR)
results were summarized in Fig. 3.
Figure 3. SARs of amino substituted 3-aryl-4-anilino-2H-chromen-2-ones 15a-f, 16a-f and 18a-f

The docking study was carried out using the Discovery Studio 3.0/CDOCKER protocol. The
X-ray crystallographic structure of ERα complexed with 4-hydroxytamoxifen (OHT) was obtained
from the RCSB Protein Data Bank (http://www.pdb.org/pdb/home/home.do) with PDB code 3ERT. The
details about molecular docking process are described in the experimental section.
From the previous work, we hypothesized that the basic side chains of our target compounds and
OHT should overlay whereas coumarin core should overlay with the triphenylethylene scaffold of OHT.
To test this hypothesis, representative compounds 15d, 16d and 18d were docked into the active site of
ERα. As is shown in Fig. 4, in the binding pocket of ERα, the triphenylethylene scaffold of
4-hydroxytamoxifen (red stick) is located into the binding pocket and the side chain stretches toward
the edge of the pocket. Furthermore, an excellent superimposition of 15d, 16d, and 18d over the
structure of OHT was observed, which suggested that the designed compounds exhibit the same
binding patterns as that of OHT.
Figure 4. Superimposed poses of OHT (red) with representative derivatives 15d (blue), 16d (green)
and 18d (yellow) binding to ERα are shown in Fig. 5A, 5B and 5C, receptively, the protein was shown
in surface. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
To further support our structural modifications, a deeper binding mode analysis was performed on
the promising compounds 15d, 16d and 18d with structural diversities on the C-7 position of coumarin
skeleton. In the docking study, the core skeleton of 3-aryl-4-anilino-2H-chromen-2-one is favorably
positioned similar to 4-hydroxytamoxifen (OHT). The basic side chains of 15d, 16d and 18d pointed
toward Asp351 to generate an antagonistic conformation as that of 4-OHT. The docking orientation and
interactions of OHT within the ligand binding domain (LBD) of ERα are shown in Figure 5A. The
hydrogen bond interaction of 4-OH of OHT with Glu353 and Arg394 plays important role in stabilizing
the binding mode. Compound 15d with H on the C-7 position of coumarin skeleton didn’t form any
hydrogen bond with ERα, which is in accordance with its low binding affinity (Fig. 5B). However, the
two methoxys containing 16d formed two hydrogen bonds with Arg394 and His524 respectively (Fig.
5C). Furthermore, these results also confirmed that the obtained anti-proliferative activities of 15d and

16d were not only through ERα but by a multiple way.
Figure 5. Selected results from molecular modelling: Only the most important interacting amino acid
residues and the most favored docking arrangements to ERα of each ligand are shown. The ligands are
highlighted by green carbon atoms: a) detailed interaction of cocrystal ligand OHT with ERα; b)
detailed interaction of 15d with ERα; c) detailed interaction of 16d with ERα; d) detailed interaction of
18d with ERα. Red dashed lines indicate potential hydrogen bonds to key residues. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The plausible binding mode of 18d in the active site of ERα was shown in Figure 6D. As we
expected, the 7-OH of 18d formed hydrogen bonds with two amino acid residues in the hinge region,
Glu353 and Arg394. It is noting that these two hydrogen bonds were consistent with OTH and
contributed significantly to the good binding affinity with ERα. In addition, the p-methoxy of 18d
interacted with His524 and formed a hydrogen bond. Molecular docking simulation supported the
initial design strategy and suggested possible basis for the observed activities. Both biological date and
docking results demonstrated that compounds 15d, 16d and 18d could be promising ERα inhibitors and
their other potential mechanisms deserve further exploration.
By structural modifications on the coumarin and basic side chain moieties in prior leads 3 and 5, a
series of 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain have been designed,
synthesized and evaluated in ERα binding and anti-proliferative activity assays. Most of them

presented moderate to excellent ERα binding affinities and better anti-proliferative effects in
comparison with the positive control tamoxifen. The hydroxy containing compound 18d with piperidyl
was found to be the most potential inhibitor on ERα while the methoxy containing compound 16d
exhibited the best in vitro proliferation inhibition activities in cellular assays with IC50 value of 4.52
µM on the MCF-7 cell line, which suggested that the obtained anti-proliferative activity might not be
only through ERα. All these results together with molecular docking analysis led to a new class of
promising ERα targeting candidates for the development of anti-breast cancer agents. Further
derivatization of this series of compounds and investigation of multi-targeted mechanisms will appear
in due course.
Supplementary data
Supplementary data (the 1H NMR, 13C NMR and HRMS spectra of target compounds 15a-f, 16a-f and
18a-f) associated with this article can be found, in the online version.
Acknowledgements
This work was supported by grants from the NSFC (81373279), the Twelfth Five-Year Plan Major
Project of Candidate Drugs (Ministry of National Science and Technology.
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32. The percent competition values are plotted against the concentration of test compounds and
analyzed by a non-linear regression curve fitting. The concentration of the test compounds needed
to displace half of the bond ligand equals EC50 of the test compound.
33. Three controls were included in this assay: (1) assay maximum polarization control (10 µL of
ER/Fluoromone TM complex solution and 10 uL of ER red assay buffer with 4% DMSO, provides
maximum polarization value for assay); (2) assay minimum polarization control (10 µL of
ER/Fluromone TM complex solution and 10 µL of E2 (20 µM), provides bottom baseline); (3) free
TM
TM
Fluoromone
tracer control (10 µL Fluoromone
tracer and 10 µL of ER Red assay buffer
with 4% DMSO, provides absolute minimum polarization value). The polarization values were
normalized to percent competition using the following equation: C% = (A0 – A) / (A0 – A100
)
C%= the percent competition, A0 = A at 0% competition, A100 = A at 100% competition, and A =
observed A value.
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Graphical abstract