Synthesis, characterization and biological evaluation of formononetin derivatives as novel EGFR inhibitors: Via inhibiting growth, migration and inducing apoptosis in breast cancer cell line

Article abstract of DOI:10.1039/c7ra09825a

Over the past few decades, the human epidermal growth factor receptor (EGFR) has been established as an attractive target for non-small cell lung cancer (NSCLC) therapy. Nevertheless, the approved EGFR inhibitors, gefitinib or erlotinib have shown minimum clinical activity to breast cancer patients, who also highly expressed EGFR. In this study, we designed and synthesized a series of novel formononetin derivatives by reference to the binding mode of lapatinib to EGFR. In vitro EGFR and cell growth inhibition assay demonstrated that compound 4v exhibited the most potent anti-EGFR (IC₅₀ = 14.5 nM) and anti-proliferation activity (IC₅₀ = 5.44 ± 1.28 μM) against MDA-MB-231 cell line, which was comparable to that of lapatinib (EGFR, IC₅₀ = 5.6 nM; MDA-MB-231, IC₅₀ = 2.48 ± 1.04 μM). Further biological experiment results demonstrated that 4v could effectively induce apoptosis, inhibit proliferation and migration in MDA-MB-231 cells through targeting EGFR and then blocking the downstream signaling pathways, EGFR/PI3K/Akt/Bad, EGFR/ERK and EGFR/PI3K/Akt/β-catenin, respectively. However, it had no significant influence on cell cycle distribution and the related proteins (Cyclin A, Cyclin D1, CDK4) expression. In vivo anti-tumor results also preliminarily confirmed the effectiveness of 4v in tumor chemotherapy in mice and indicated its potential as a new EGFR inhibitor in the treatment of MDA-MB-231 malignant tumor.

Full text of DOI:10.1039/c7ra09825a

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PAPER
Synthesis, characterization and biological
evaluation of formononetin derivatives as novel
EGFR inhibitors via inhibiting growth, migration and
inducing apoptosis in breast cancer cell line†
Cite this: RSC Adv., 2017, 7, 48404
ab
ab
ab
ab
Hong-Yan Lin,‡ Wen-Xue Sun,‡ Chao-Sai Zheng, Hong-Wei Han,
ab
ab
ab
ab
ab
Xue Wang, Ya-Han Zhang, Han-Yue Qiu, Cheng-Yi Tang, Jin-Liang Qi,
Gui-Hua Lu, Rong-Wu Yang,* Xiao-Ming Wang
ab
ab
ab
and Yong-Hua Yang *ab
*
Over the past few decades, the human epidermal growth factor receptor (EGFR) has been established as an
attractive target for non-small cell lung cancer (NSCLC) therapy. Nevertheless, the approved EGFR
inhibitors, gefitinib or erlotinib have shown minimum clinical activity to breast cancer patients, who also
highly expressed EGFR. In this study, we designed and synthesized a series of novel formononetin
derivatives by reference to the binding mode of lapatinib to EGFR. In vitro EGFR and cell growth
inhibition assay demonstrated that compound 4v exhibited the most potent anti-EGFR (IC50 ¼ 14.5 nM)
and anti-proliferation activity (IC50 ¼ 5.44 ꢀ 1.28 mM) against MDA-MB-231 cell line, which was
comparable to that of lapatinib (EGFR, IC50 ¼ 5.6 nM; MDA-MB-231, IC50 ¼ 2.48 ꢀ 1.04 mM). Further
biological experiment results demonstrated that 4v could effectively induce apoptosis, inhibit
proliferation and migration in MDA-MB-231 cells through targeting EGFR and then blocking the
downstream signaling pathways, EGFR/PI3K/Akt/Bad, EGFR/ERK and EGFR/PI3K/Akt/b-catenin,
respectively. However, it had no significant influence on cell cycle distribution and the related proteins
Received 4th September 2017
Accepted 9th October 2017
(
Cyclin A, Cyclin D1, CDK4) expression. In vivo anti-tumor results also preliminarily confirmed the
DOI: 10.1039/c7ra09825a
effectiveness of 4v in tumor chemotherapy in mice and indicated its potential as a new EGFR inhibitor in
rsc.li/rsc-advances
the treatment of MDA-MB-231 malignant tumor.
cellular migration, apoptosis and mitogen-activated protein
kinase signalling, Ras/Raf/MEK/ERK, as implicated in cell
The epidermal growth factor receptor (EGFR) is a trans- proliferation. It is well known that inhibition of EGFR kinase-
membrane protein, belonging to the family of tyrosine mediated signal is a well-established strategy for the treatment
kinases and involved in cell proliferation and signal trans- of advanced stage non-small cell lung cancer (NSCLC) and also
Introduction
7,8
1
duction. It has been conrmed that EGFR is highly or abnor- correlated well with the proliferation and migration of aggres-
9,10
mally expressed in many solid tumors and is correlated to sive triple negative breast cancer, MDA-MB-231.
tumor cell proliferation, angiogenesis, tumor invasion, metas-
Studies have shown that triple-negative breast cancer was
2,3
tasis, and inhibition of apoptosis. Over-expression of EGFR is considered another potential indication for EGFR kinase
also crucial for the evolution of malignant tumors, such as inhibitors because they are not amenable to endocrine or HER-2
10,11
glioma cell, kidney, lung, breast, prostate and pancreatic targeted therapies, while they highly-express EGFR kinase.
4–6
cancer. The activation of EGFR phosphorylates its down- Some literatures have suggested a crucial role of EGFR in breast
stream signalling molecules PI3K and Akt, which are involved in cancer proliferation and migration. However, inhibition of
EGFR by getinib or erlotinib showed low efficacy in arresting
tumor cell growth at the cell line level and minimum clinical
a
State Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of
Plant Molecular Biology, Nanjing University, Nanjing, 210023, China. E-mail:
Yangyh@nju.edu.cn; Wangxm07@nju.edu.cn; Robertyang@nju.edu.cn; Fax: +86-25-
7,12
activity.
Such extraordinary phenomenon always attracts us
to explore the underlying mechanism and discover new potent
EGFR inhibitors that are naturally derived chemicals as breast
cancer therapeutics.
8
9681381; Tel: +86-25-89681381
b
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry
University, Nanjing, 210037, China
Natural products are a major source of drug discovery and
†
1
‡
Electronic supplementary information (ESI) available. See DOI: cancer therapy. Formononetin (Fig. 1) is a bioactive isoavone
0.1039/c7ra09825a
found in diverse Chinese medicine herbals, such as Amorpha
These two authors contribute equally to this article.
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Fig. 1 Formononetin derivatives with anticancer activity.
1
3
13,14
13,14
fruticose, Glycyrrhiza,
Trifolium pretense,
Astragalus docking was conducted to calculate the binding energy
15
membranaceus, which belonging to the family of Leguminosae. (CDOCKER INTERACTION ENERGY in CDOCKER protocol in
It has attracted broad attention because of multiple pharma- Discovery Studio 3.5) between compounds and EGFR complex
1
6,17
17
cological activities, such as anti-bacterial,
oxidant, anti-diabetic
anti-cancer, anti- structure (1XKK.pdb, downloaded from the PDB). Finally, 22
18
19,20
21
and anti-hyperlipidemia. Currently, compounds were picked to continue assays subsequently and
many formononetin analogues (Fig. 1) were designed as anti- their binding energy were shown in Table S1 (ESI†).
cancer agents and explored the biological mechanism against In view of the binding energy, 4v is the best one among them.
various human cancer cell lines. As reported that formononetin For better understanding of the potential of 4v, we further
can induce cell apoptosis via PI3K/AKT signaling pathway in examined the interaction of 4v with EGFR (PDB code 1XKK) with
22
human nasopharyngeal and cervical carcinoma cells. For- lapatinib as the positive control. All the amino acid residues
mononetin–dithiocarbamate hybrids 8i could inhibit growth which had interactions with lapatinib were exhibited and the
and migration of PC-3 cells via MAPK/Wnt signaling pathway results were depicted in Fig. 2A. In the binding model, lapatinib
23
and cause cell cycle arrest in G1 phase. While its 7-phos- bound well to EGFR mainly via three hydrogen bonds with ARG
phoramidate derivative 8d and nitrogen-mustard derivative 6n 803, ASP 800 and PHE856, a p-cation bond with LYS 745 and
could induce cell cycle arrest in G2/M phase and cell a p–p bond with PHE856. In the binding model of 4v with EGFR
24,25
apoptosis.
In addition, Manupati et al. reported a chrysin (Fig. 2C), there formed a hydrogen bond with MET 793, a p-
analog CHM-09 which potentiated the mesenchymal–epithelial cation bond with LYS 745, a p-anion bond with ASP 800, a p–p
transition of breast cancer stem cells through inhibiting bond with PHE856 and a p-sigma bond with MET766 which are
10
EGFR. In the light of previous studies, we reported here the the main contributors for the effective combination between
designation of novel formononetin derivatives as potent anti- them. The 3D models of the interaction between lapatinib, 4v
cancer agents through targeting EGFR and further investiga- and EGFR were also depicted in Fig. 2B and D. For Fig. 2E, we
tion of their underlying biological mechanisms.
further compared the structure and target combine similarity of
v and lapatinib. The result demonstrated that 1c might be
4
a potential EGFR inhibitor.
The synthetic routes of novel formononetin derivatives are
outlined in Scheme 1, and structure of derivatives 4a–4v are
Results and discussion
Chemistry
In this study, we hypothesize that the design of synthetic shown in Table 1. These compounds were obtained by three
derivatives of formononetin against EGFR with lapatinib as steps which were elucidated in the Experimental section. All of
1
13
positive control may improve its anticancer efficacy. Before them are rst reported and characterized by H NMR, C NMR,
synthesis, a molecular screening process was performed by elemental analysis, melting test and mass spectroscopy, and
computer assist drug design (CADD) method. Molecular results are in full accordance with their depicted structures.
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Fig. 2 Molecular docking analysis of 4v and lapatinib, showing proposed binding modes with EGFR (PDB code 1XKK). (A) Interactions of lapatinib
with the amino acid residues at the binding pocket of EGFR (carbon atom, gray; oxygen atom, red; hydrogen atom, white; nitrogen atom, purple;
chlorine atom, bright green; fluorine atom, light green; sulphur atom, orange). (B) Binding pose of lapatinib in the protein surface of EGFR (carbon
atom, blue; oxygen atom, red; hydrogen atom, white; nitrogen atom, purple; chlorine atom, bright green; fluorine atom, sulphur atom, orange).
Hydrogen bonds were displayed as green dashed lines. (C) Interactions of 4v with the amino acid residues at the binding pocket of EGFR (carbon
atom, gray; oxygen atom, red; hydrogen atom, white; nitrogen atom, purple; sulphur atom, orange). (D) Binding pose of 4v in the protein surface
of EGFR (carbon atom, blue; oxygen atom, red; hydrogen atom, white; nitrogen atom, purple; sulphur atom, orange). Hydrogen bond was
displayed as green dashed lines. (E) 3D mode of the interaction between the compounds (lapatinib, brown; 4v, blue) and the EGFR binding site.
The protein is represented by molecular surface.
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Scheme 1 Chemical synthesis routine of 4a–4v. Reagents and conditions: (a) ethyl bromoacetate, acetone, K
hydrazine hydrate, ethanol, reflux, 10 hours; (c) benzaldehyde, glacial acetic acid, ethanol, reflux, 6 hours.
2 3
CO , reflux, 10 hours; (b)
Table 1 Chemical structures of 4a–4v
compounds presented stronger EGFR inhibitory activity
compared to formononetin itself. Especially, compound 4v was
the most potent inhibitor of EGFR (IC50 ¼ 14.5 nM), which was
at least 10-fold stronger than formononetin (IC50 > 150 nM) and
almost comparable to getinib (IC50 ¼ 6.2 nM) and lapatinib
(
IC50 ¼ 5.6 nM). Among them, compound 4n also showed
potent EGFR inhibitory activity with an IC50 value of 20.6 nM.
Generally, electron-donating group improved the enzymatic
inhibitory activity more signicantly than the electron-
withdrawing group, especially when the substitution appeared
at the R3-position of benzene ring of formononetin skeleton.
Compound
R
1
R
2
R
3
R
4
R
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
H
F
H
H
F
H
F
H
Cl
H
Cl
H
Br
OH
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Table 2 The EGFR inhibitory activity of 4a–4v, formononetin, gefitinib
and lapatinib
H
H
H
Cl
H
H
Cl
Cl
H
H
H
H
CF
H
H
EGFR kinase
F
Compound
% inhibition at 150 nM
IC50 (nM)
H
H
Cl
H
H
H
H
OH
NH
H
CF
CH
H
OCH
OCH
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
54.7
51.6
33.5
70.3
68.4
52.3
50.2
72.8
60.1
58.5
56.2
50.9
73.5
75.7
43.1
66.8
70.4
62.9
68.3
80.6
76.9
87.3
26.4
91.5
94.2
120.4
131.6
>150
52.6
66.2
142.2
146.3
30.7
98.4
105.8
112.5
139.5
33.1
20.6
>150
48.9
30.4
56.4
40.6
47.8
35.5
14.5
>150
6.2
2
3
3
3
OCH
H
H
H
H
3
s
t
u
v
3
3
OCH
H
H
3
CH(CH
OC
3 2
)
7 7
H
Biological studies
4s
4
4
4
t
u
v
In order to screen out the optimal active compounds from all
synthesized formononetin derivatives, a comparative study was
carried out to evaluated their in vitro EGFR inhibition activities. Formononetin
Getinib
Lapatinib
As shown in Table 2, we clearly found that most of the tested
5.6
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Table 3 In vitro cell proliferation of compounds 4a–4v, formononetin and lapatinib against MCF-7, MDA-231 H460, H1650, L02 and VERO cell
lines
IC50 (mM)
Compound
MCF-7
MDA-231
H460
H1650
L02
VERO
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
23.7 ꢀ 1.87
24.5 ꢀ 1.98
42.6 ꢀ 0.25
21.5 ꢀ 0.91
30.7 ꢀ 1.42
23.9 ꢀ 1.12
41.5 ꢀ 2.01
17.8 ꢀ 1.64
25.4 ꢀ 1.73
25.7 ꢀ 1.53
21.5 ꢀ 0.63
21.1 ꢀ 2.76
20.9 ꢀ 2.24
19.5 ꢀ 1.73
31.3 ꢀ 1.93
24.6 ꢀ 2.52
17.2 ꢀ 1.93
15.9 ꢀ 3.42
29.1 ꢀ 1.27
12.3 ꢀ 1.11
25.9 ꢀ 1.94
11.5 ꢀ 1.52
36.4 ꢀ 3.01
13.5 ꢀ 3.08
7.10 ꢀ 2.81
15.1 ꢀ 4.13
20.7 ꢀ 1.62
33.6 ꢀ 1.83
18.3 ꢀ 1.03
27.8 ꢀ 0.75
17.9 ꢀ 2.25
19.5 ꢀ 1.52
8.30 ꢀ 1.23
16.3 ꢀ 1.24
18.7 ꢀ 1.02
23.2 ꢀ 2.19
16.1 ꢀ 2.75
9.35 ꢀ 1.02
9.25 ꢀ 1.05
33.8 ꢀ 2.15
12.3 ꢀ 1.49
15.4 ꢀ 1.03
13.1 ꢀ 1.84
10.1 ꢀ 1.55
8.42 ꢀ 1.21
13.2 ꢀ 4.02
5.44 ꢀ 1.28
26.6 ꢀ 2.84
12.5 ꢀ 2.13
2.48 ꢀ 1.04
21.5 ꢀ 2.04
18.5 ꢀ 2.33
16.2 ꢀ 2.05
16.5 ꢀ 1.22
22.1 ꢀ 2.87
24.3 ꢀ 1.46
16.5 ꢀ 5.49
8.02 ꢀ 1.02
58.1 ꢀ 3.54
19.6 ꢀ 1.55
22.7 ꢀ 2.28
16.3 ꢀ 1.99
14.2 ꢀ 1.69
18.2 ꢀ 2.24
42.5 ꢀ 6.42
15.5 ꢀ 1.95
16.7 ꢀ 2.49
24.6 ꢀ 3.39
21.58 ꢀ 2.09
18.6 ꢀ 2.23
14.3 ꢀ 1.84
6.36 ꢀ 1.55
27.8 ꢀ 2.22
3.02 ꢀ 0.89
1.22 ꢀ 0.35
11.9 ꢀ 1.18
38.6 ꢀ 3.56
17.8 ꢀ 2.13
49.6 ꢀ 3.65
33.4 ꢀ 3.59
13.9 ꢀ 1.44
12.9 ꢀ 2.58
9.45 ꢀ 1.08
19.7 ꢀ 2.27
29.7 ꢀ 2.59
10.8 ꢀ 1.77
12.4 ꢀ 1.68
22.5 ꢀ 2.68
15.6 ꢀ 2.07
10.1 ꢀ 1.46
19.3 ꢀ 2.04
8.79 ꢀ 1.08
40.2 ꢀ 3.88
15.3 ꢀ 2.31
24.3 ꢀ 1.89
12.2 ꢀ 1.45
7.26 ꢀ 1.02
29.5 ꢀ 3.25
2.64 ꢀ 0.98
1.64 ꢀ 0.98
43.5 ꢀ 3.64
>100
>100
25.3 ꢀ 1.74
>100
>100
65.8 ꢀ 4.62
62.5 ꢀ 2.01
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
41.6 ꢀ 2.48
>100
29.3 ꢀ 1.94
57.9 ꢀ 1.42
78.8 ꢀ 2.23
36.5 ꢀ 3.14
>100
>100
67.3 ꢀ 1.52
>100
>100
>100
55.7 ꢀ 2.05
s
t
u
v
>100
>100
>100
95.2 ꢀ 4.72
89.5 ꢀ 5.82
38.6 ꢀ 2.56
35.6 ꢀ 2.25
>100
Formononetin
Getinib
Lapatinib
82.6 ꢀ 5.73
35.2 ꢀ 3.08
42.5 ꢀ 2.59
Then we further evaluated their anti-proliferation activities than formononetin itself with the standard of IC50 values. To be
against four cancer cell lines, human breast cancer cells (MCF-7, specic, most compounds showed better anti-proliferation activity
MDA-MB-231), human lung carcinoma cells (H460, H1650), as against MDA-MB-231 than MCF-7, which might because of the
well as two non-cancer cell lines, normal human liver cells (L02) high expression of EGFR in the former. For MDA-MB-231 cell line,
and African green monkey kidney cell (VERO) by the cell based 4h (IC50 ¼ 8.30 ꢀ 1.23), 4m (IC50 ¼ 9.35 ꢀ 1.02 mM), 4n (IC50 ¼ 9.25
MTT assay. The results are summarized in Table 3 and expressed ꢀ 1.05 mM), 4t (IC50 ¼ 8.42 ꢀ 1.21 mM) and 4v (IC50 ¼ 5.44 ꢀ 1.28
by the half maximal inhibitory concentration (IC50) values. As mM) showed relatively better anti-proliferation activities than other
shown in Table 3, all compounds showed good anti-proliferation compounds and their inhibitory efficacy against MDA-MB-231
activity against the cancer cells, and most of them even better were double stronger than against MCF-7 cell line (4h, IC50
¼
Fig. 3 The effects of 4v on phosphorylation of EGFR and intracellular kinases in MDA-MB-231 cells. (A) Representative image of immunoblot
analysis of EGFR signaling in MDA-MB-231 cells treated with 4v. (B) Relative band intensity was determined by Image J software. GAPDH served
as a loading control. Images are representative of three independent experiments. Data are means ꢀ S.E.M. from three independent experiments
(*P < 0.05; **P < 0.01).
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7.8 ꢀ 1.64 mM, 4m, IC50 ¼ 20.9 ꢀ 2.24 mM, 4n, IC50 ¼ 19.5 ꢀ 1.73 of getinib against breast cancer cell lines (MCF-7, IC50 ¼ 13.5 ꢀ
mM, 4t, IC50 ¼ 12.3 ꢀ 1.11 mM and 4v, IC ¼ 11.5 ꢀ 1.52 mM). 3.08 mM, MDA-MB-231, IC50 ¼ 12.5 ꢀ 2.13 mM). Generally
50
Combined with the data of two human lung cancer cell lines, we speaking, for comprehensive consideration anti-proliferation
clearly found that 4h and 4v showed much stronger inhibition activities and the impact on log P (Table S2, ESI†), 4v possessing
effect than others, especially for 4v (MCF-7, IC50 ¼ 11.5 ꢀ 1.52 mM, 4-benzyloxy can be considered the optimal one in all newly
MDA-MB-231, IC50 ¼ 5.44 ꢀ 1.28 mM, H460, IC50 ¼ 6.36 ꢀ 1.55 mM, synthetic compounds, and appeared to be a more efficient inhib-
H1650, IC50 ¼ 7.26 ꢀ 1.02 mM), which effect is almost comparable itor of MDA-MB-231 cell viability (IC50 ¼ 5.44 ꢀ 1.28 mM) when
to that of lapatinib (MCF-7, IC50 ¼ 7.1 ꢀ 2.81 mM, MDA-MB-231, compared with formononetin (IC50 ¼ 26.6 ꢀ 2.84 mM).
IC50 ¼ 2.48 ꢀ 1.04 mM, H460, IC50 ¼ 1.22 ꢀ 0.35 mM, H1650,
EGFR activation has been associated with multiple biological
IC50 ¼ 1.64 ꢀ 0.98 mM) and much better than the inhibitory effect processes in cancer progression, such as cell proliferation,
Fig. 4 The effects of 4v on cell cycle distribution in MDA-MB-231 cells. (A) Cell cycle analysis of MDA-MB-231 cells treated with different
concentrations of 4v. (B) Cell cycle analysis of MDA-MB-231 cells treated with 4v for different time (G1 phase, green; S phase, yellow and G2/M
phase, blue). (C) Statistic analysis of dose-dependent assay. (D) Statistic analysis of time-dependent assay. (E) Representative image of immu-
noblot analysis of cell cycle related proteins in MDA-MB-231 cells treated with 4v. (F) Relative band intensity was determined by Image J software.
b-Actin served as a loading control. Images are representative of three independent experiments. Data are means ꢀ S.E.M. from three inde-
pendent experiments (*P < 0.05; **P < 0.01).
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migration and metastasis through different signaling path- MB-231 cells, well-polarized mitochondria were marked by red
26
ways. To investigate the effect of 4v on signal transduction uorescent staining. Aer treated with 2 or 4 mM of 4v, this
mechanism, we performed immunoblot analysis of EGFR and pattern was replaced by green uorescence in MDA-MB-231
its downstream signaling pathways: extracellular signal regu- cells. CCCP was used as the positive control here. Addition-
lated kinase (ERK) and Akt. As shown in Fig. 3, MDA-MB-231 ally, ow cytometry results also showed that untreated cells with
cells that express high EGFR were treated with increasing well-polarized, red-emitting mitochondria localized in the
concentrations of 4v and demonstrated a dose-dependent upper region of the plot. Cells exposed to 4v for 12 hours
decrease in phosphorylation of EGFR, ERK and Akt.
underwent a progressive loss of red uorescence, indicated by
To further substantiate the anti-proliferative effect of 4v on a downward in 4v treated groups. The ratio of cells with high
MDA-MB-231 cells, we evaluated its impact on the cell cycle membrane potential decreased from 85.2% to 80.8% aer
arrest. As illustrated in Fig. 4A–D, 4v treated cells demonstrated exposed to 2 mM 4v for 12 hours and even decreased to 56.8% in
no obvious cell cycle arrest versus the control groups. Then, we 4 mM 4v treated group. Likewise, the loss of mitochondrial
performed immunoblot analysis of cell cycle related proteins, potential in MDA-MB-231 cells caused by 4v also showed time-
Cyclin A, Cyclin D1 and CDK4. And the results shown in Fig. 4E dependent manner.
and F indicated that the expression of the cell cycle related
proteins in MDA-MB-231 cells did not increase aer treated with populations of MDA-MB-231 cells treated with 4v using ow
compound 4v. cytometry analysis. As shown in Fig. 6A, cells treated with
Based on the above results, we also evaluated the apoptotic
The transmembrane potential (DJ ) is an important increasing concentrations of 4v and demonstrated a dose-
m
parameter for mitochondrial functionality and its dissipation dependent increase in apoptotic populations. In addition,
27
means the involvement of mitochondria in early apoptosis. As cells treated with 4v for increasing time also demonstrated
shown in Fig. 5, microscopy revealed that in untreated MDA- a time-dependent manner in apoptotic populations and
Fig. 5 4v decreased the mitochondrial membrane potential of MDA-MB-231 cells. (A) Cytofluorimetric analysis of mitochondrial membrane
m m
potential (DJ ) by JC-1 staining. (B) Flow cytometry analysis of MDA-MB-231 cells with high DJ (%) by JC-1 staining. Each point represents the
mean ꢀ S.E.M. from three replicates (*P < 0.05, **P < 0.01).
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Fig. 6 The effects of 4v on cell apoptosis in MDA-MB-231 cells. (A) Annexin V/PI double staining and cytometry analysis of 4v-induced apoptosis
in a dose-dependent manner. (B) Annexin V/PI double staining and cytometry analysis of 4v-induced apoptosis in a time-dependent manner. (C)
Western blot analyses of apoptosis-related proteins (Bax, Bcl2, cleaved caspase 3, cleaved PARP and cytochrome c) separated by SDS-PAGE. (D)
Relative band intensity was determined by Image J software. GAPDH served as a loading control. Images are representative of three independent
experiments. Data are means ꢀ S.E.M. from three independent experiments (*P < 0.05; **P < 0.01).
a
signicant high percentage (approximately 90.4%) of activated, b-catenin disassembles from complex comprised of
apoptotic cells were observed in 36 hours-treated cells Axin, APC and GSK3b and translocates into nuclear. Then it
compared to vehicle control (Fig. 6B). This observation was interacts with TCF4 which promotes the downstream target
further substantiated by concomitant increased expression of gene transcription. On the basis of the wound healing assay, we
pro-apoptotic proteins, Bax, cytochrome c, cleaved caspase-3, subsequently investigated the inuence of 4v on Wnt signaling
cleaved PARP and decreased expression of anti-apoptotic Bcl2 pathway in MDA-MB-231 cells. Western bolt analysis results
aer exposure to 4v (Fig. 6C).
shown in Fig. 7C indicated that treatment of MDA-MB-231 cells
Tumor cell migration has been identied as one hallmark of with 4v resulted in the decrease expression of b-catenin and the
malignant tumor progression, particularly for the establish- increase expression of Axin-2 in dose-dependent manners.
ment of lethal secondary metastases at distant organs. To These results collectively suggested that 4v might inhibit
investigate whether 4v could inhibit the migratory capacity of migration of MDA-MB-231 cells via Wnt signaling pathway.
MDA-MB-231 cells, the conventional in vitro wound healing
To evaluate the anti-tumor effects of 4v in vivo, we subcuta-
assay was also conducted. As shown in Fig. 7A and B, the control neously inoculated MDA-MB-231 cells in to the right ank of
group cells gradually occupied the cell-free space within the female nude mice. Two weeks later, all the mice got visible
channel over time and the cells almost ll the channel aer 48 tumors. The mice were randomized into 3 groups, vehicle (olive
ꢁ
ꢁ
1
ꢁ1
hours. Whereas, cells migration capacity of 4v-treated group oil), 4v (5 mg kg ) and positive control (getinib, 5 mg kg
;
1
was greatly inhibited and the inhibition was dose-dependent. lapatinib, 5 mg kg ). As shown in Fig. 8A, the administration of
The Wnt signaling pathway is crucial for multiple cellular 4v greatly inhibited the tumor growth compared with the
processes including cell proliferation and migration. Once vehicle group and the inhibition effect was similar to that of
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Fig. 7 The effects of 4v on cell migration in MDA-MB-231 cells. (A) Cell migration was detected by the wound scrape assay. Representative
images of cell migration in the wound scrape model at 0, 24 and 48 hours are shown. (B) Statistical analysis of the wound scrape assay. (C)
Western blot analyses of Wnt signaling pathway proteins (b-catenin and Axin-2) separated by SDS-PAGE. (D) Relative band intensity was
determined by Image J software. GAPDH served as a loading control. Images are representative of three independent experiments. The data
represent the means ꢀ S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.
lapatinib, while the traditional EGFR inhibitor getinib showed a XT4 MP Apparatus (Taike Corp., Beijing, China) and uncor-
extremely low therapeutic efficiency on MDA-MB-231 cells. Aer rected. TLC was carried out on the glass-backed silica gel sheets
˚
the last time of treatment on day 21, the average weight of (silica gel 60 A GF254) and visualized in UV light (254 nm).
tumors from 4v-treated mice was nearly 2-fold less than the
Cell culture reagents were purchased from Gibco Life Tech-
olive oil treated group (Fig. 8C). Meanwhile, 4v has no signi- nology (Grand Island, NY, USA). MTT, PMSF, and RIPA lysis
cant inuence on the body weight of the mice (Fig. 8B).
buffer (#P0013B) were purchased from Beyotime Institute of
Biotechnology (Haimen, China). Cell Cycle Detection Kit and
Annexin V-FITC/PI apoptosis detection kit were purchased from
KeyGEN Biotech Co. Ltd (Nanjing, China). Mitochondrial
membrane potential assay kit with JC-1 was purchased from
Beyotime Biotech Co. Ltd (Haimen, China). BCA protein assay
kit (#23227) was purchased from Pierce (Rockford, IL, USA).
PVDF membranes were purchased from Biosharp (Hefei,
China). Anti-Axin-2 (20540-1-AP) was purchased from Pro-
teintech (Wuhan, China); anti-cleaved PARP-1 (#sc-56196) was
purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas,
USA). Anti-EGFR (WL0682a), anti-ERK (WL01864), anti-p-ERK
Experimental
General information and materials
Formononetin (purity $ 99%), ethyl bromoacetate, hydrazine
hydrate, a series of benzaldehyde with different substituents,
acetic acid, acetone, ethanol, etc. According to experimental
conditions, all reagents used were of analytical reagent grade
(
AR) except ethanol which was puried and purchased from
Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Getinib
purity $ 99%) and lapatinib (purity $ 99%) were purchased
(
(
WLP1512), anti-Akt (WL0003b), anti-p-Akt (WLP001a), anti-
CDK4 (WL02143), anti-Cyclin A (WL01753), anti-Cyclin D1
WL01435a), anti-b-Actin (WL0002c), anti-Bax (WL01637), anti-
1
13
from J&K Scientic Ltd. (Shanghai, China). H NMR and
C
NMR spectra of all intermediate and nal products were
(
recorded on a DRX 600 spectrometer and DMSO-d6 as solvent.
Bcl2 (WL01556), anti-cleaved caspase-3 (WL01992), anti-
cytochrome c (WL01571) and anti-b-catenin (WL0962a) were
purchased from Wanlei Biotech Co. Ltd (Shenyang, China).
GAPDH antibody (#E1A7021) and secondary antibody (anti-
1
13
Chemical shis (d) for H NMR and C NMR spectra were re-
ported in ppm (d). The ESI-MS spectra were obtained on
a Mariner Biospectrometry Workstation (ESI-TOF) mass spec-
trometer. Melting points of products were determined using
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Fig. 8 4v suppresses tumor growth of MDA-MB-231 xenografts in nude mice. (A) Tumor volumes of the mice. (B) Body weight of the mice. (C)
Tumor weight of mice. Data are the mean ꢀ S.E.M. of 8 mice per group (*P < 0.05; **P < 0.01 versus vehicle (olive oil)).
mouse or anti-rabbit IgG) (#E1WP319 or #E1WP318) were reaction mixture was stirred under the condition of reux at
ꢂ
purchased from Enogene (Nanjing, China). ECL Kit (#34077) 80 C. Hydrazine hydrate (0.075 mol, 3.9 g) was added and
were purchased from Thermo Scientic (USA).
stirred for a further 10 hours with the reaction system getting
claried gradually. While stirring, the product was checked with
TLC continually to choose the right point in time to stop the
reaction. Aer the system temperature cooling to room
temperature, the compound 3 which is white was isolated by
ltration when precipitated. The product was dried in the oven
weighing 7.45 g (84%). White powder, yield 87%; mp: 200.4–
General procedure for preparation of compound 2
A mixture of formononetin (0.04 mol, 11.44 g) and potassium
carbonate (0.08 mol, 22.48 g) were stirred in acetone under the
ꢂ
condition of reux at 60 C for 3 hours to get dissolved and
claried. Meanwhile, proportionable ethyl bromoacetate
ꢂ
1
2
7
01.7 C; H NMR (300 MHz, DMSO) d 9.32 (s, 1H), 7.78 (d, J ¼
0.9 Hz, 1H), 7.18 (d, J ¼ 8.2 Hz, 2H), 7.07–6.68 (m, 3H), 6.43 (d,
(0.04 mol, 4.9 g) was prepared which dissolved in appropriate
acetone and added to the system dropwise to react another 10
hours. And then reaction system was ltered in hot to obtain
J ¼ 34.4 Hz, 2H), 4.42 (s, 2H), 4.31 (s, 2H), 3.71 (s, 3H). ESI-TOF,
+
calcd for C17
calcd for C17
C, 61.94; H, 4.41; N, 8.66; O, 24.59.
H
14
N
2
O
5
([M + Na] ) 349.31, found 349.09. Anal.

ltrate. When the ltrate tended to be cooling, a large amount
of compound 2 separated out and washed with petroleum ether,
% sodium hydroxide and distilled water successively. The
product was dried in the oven weighing 9.97 g (87%). White
H
14
N
2
O
5
: C, 62.57; H, 4.32; N, 8.59; O, 24.52. Found:
1
ꢂ
1
powder, yield 95%; mp: 188.5–189.3 C; H NMR (300 MHz, General procedure for preparation of compounds 4a–4v
CDCl ) d 8.24 (d, J ¼ 8.9 Hz, 1H), 7.92 (s, 1H), 7.53–7.46 (m, 2H),
3
Compounds 4a–4v were synthesized by reacting the compound
7
2
1
.03 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.00–6.94 (m, 2H), 6.84 (d, J ¼
3
(0.0008 mol) with the different substituted ethyl benzaldehyde
.4 Hz, 1H), 4.73 (s, 2H), 4.31 (q, J ¼ 7.1 Hz, 2H), 3.84 (s, 3H),
(
0.0008 mol) in a Schiff base condensation reaction. The reac-
.25 (dd, J ¼ 8.4, 5.6 Hz, 3H). ESI-TOF, calcd for C19
H
16
O
6
([M +
ꢂ
tion was stirred at 80 C for 8 hours in the presence of a bit of
glacial acetic acid (4–8 drops) in ethanol. The compounds 4a–4v
were obtained by ltering aer precipitation and all structure
+
Na] ) 363.33, found 363.09. Anal. calcd for C19
H
16
O
6
: C, 67.06;
H, 4.74; O, 28.21. Found: C, 66.87; H, 4.92; O, 28.29.
shown in Table 1.
General procedure for preparation of compound 3
0
(Z)-N -Benzylidene-2-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-
Compound 2 (0.025 mol, 8.85 g) was added to a clean round- 7-yl)oxy)-acetohydrazide (4a). White powder, yield 75%; mp:
ꢂ
1
bottom ask which contained 30–40 mL of ethanol. The 250.5–252.3 C; 96.5% purity (HPLC); H NMR (300 MHz, DMSO)
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d 11.60 (d, J ¼ 7.3 Hz, 1H, –CH]N), 8.32–8.00 (m, 1H, Ar-H), 7.90– 7.90–7.64 (m, 2H, N-NH, Ar-H), 7.64–7.40 (m, 2H, Ar-H), 7.19 (d,
.62 (m, 3H, N-NH, Ar-H), 7.44 (d, J ¼ 3.6 Hz, 3H, Ar-H), 7.27–7.11 J ¼ 8.6 Hz, 2H, Ar-H), 7.01 (t, J ¼ 8.6 Hz, 1H, Ar-H), 6.84 (d, J ¼
m, 2H, Ar-H), 7.03 (t, J ¼ 8.6 Hz, 1H, Ar-H), 6.85 (d, J ¼ 8.1 Hz, 2H, 7.0 Hz, 2H, Ar-H), 6.45 (t, J ¼ 20.6 Hz, 2H, Ar-H), 5.15–4.60 (m,
Ar-H), 6.60–6.24 (m, 2H, Ar-H), 5.09–4.62 (m, 2H, –O–CH –), 3.72 2H, –O–CH –), 3.71 (s, 3H, –OCH ). ESI-TOF, calcd for
7
(
2
2
3
13
+
(
(
s, 3H, –OCH
3
). C NMR (75 MHz, DMSO) d 169.43 (s, 14C), 164.73
s, 18C), 159.64 (s, 11C), 158.04 (s, 6C), 157.08 (s, 16C), 148.40 (s, for C25
C
25
H
19
F
H
2
N
2
O
5
([M + Na] ) 487.1184, found 487.1173. Anal. calcd
: C, 64.66; H, 3.91; F, 8.18; N, 6.03; O, 17.22.
19FN
2
O
5
9
1
1
5
C), 144.35 (s, 19C), 134.56 (s, 20C), 134.32 (s, 23C), 130.68 (s, 2C), Found: C, 64.58; H, 3.99; F, 8.23; N, 6.11; O, 17.31.
0
30.46 (s, 4C), 129.32 (s, 21C, 25C), 129.29 (s, 22C, 24C), 127.62 (s,
(Z)-N -(2-Chlorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H-
3C), 127.40 (s, 3C), 126.75 (s, 8C), 119.13 (s, 15C), 114.32 (s, 1C, chromen-7-yl)oxy)acetohydrazide (4f). White powder, yield 75%;
ꢂ
1
C), 105.83 (s, 12C), 102.47 (s, 10C), 66.88 (s, 17C), 55.45 (s, 7C). mp: 216.2–217.3 C; 95.8% purity (HPLC); H NMR (300 MHz,
+
ESI-TOF, calcd for C H N O ([M + Na] ) 451.1372, found DMSO) d 11.82 (d, J ¼ 20.7 Hz, 1H, –CH]N), 8.78–8.32 (m, 1H,
25 20 2 5
451.1351. Anal. calcd for C H N O : C, 70.09; H, 4.71; N, 6.54; O, Ar-H), 8.00 (m, 2H, Ar-H), 7.78 (s, 1H, N-NH), 7.57–7.37 (m, 2H,
25 20 2 5
1
8.67. Found: C, 69.58; H, 4.75; N, 6.61; O, 18.74.
Ar-H), 7.21 (dd, J ¼ 7.0, 1.4 Hz, 2H, Ar-H), 7.09–6.95 (m, 1H, Ar-
0
(Z)-N -(2-Fluorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H- H), 6.86 (d, J ¼ 8.5 Hz, 2H, Ar-H), 6.59–6.31 (m, 2H, Ar-H), 5.20–
chromen-7-yl)oxy)acetohydrazide (4b). White powder, yield 4.48 (m, 2H, –O–CH
6
MHz, DMSO) d 11.71 (s, 1H, –CH]N), 8.62–8.19 (m, 1H, Ar-H), for C25
2
–), 3.73 (s, 3H, –OCH
3
). ESI-TOF, calcd for
19ClN ([M + Na] ) 485.0982, found 485.0974. Anal. calcd
2 5
19ClN O : C, 64.87; H, 4.14; Cl, 7.66; N, 6.05; O, 17.28.
ꢂ
1
+
3%; mp: 245.7–247.1 C; 95.3% purity (HPLC); H NMR (300
C
25
H
2 5
O
H
7
.91 (dt, J ¼ 15.0, 7.6 Hz, 1H, Ar-H), 7.76 (s, 1H, N-NH), 7.55–7.39 Found: C, 64.69; H, 4.27; Cl, 7.74; N, 6.12; O, 17.32.
0
(m, 1H, Ar-H), 7.35–7.22 (m, 2H, Ar-H), 7.21–7.13 (m, 2H, Ar-H),
(Z)-N -(3-Chlorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H-
7
6
.02 (t, J ¼ 9.1 Hz, 1H, Ar-H), 6.84 (d, J ¼ 8.0 Hz, 2H, Ar-H), 6.62– chromen-7-yl)oxy)acetohydrazide (4g). White powder, yield
ꢂ
1
.22 (m, 2H, Ar-H), 5.15–4.60 (m, 2H, –O–CH –), 3.71 (s, 3H, 78%; mp: 231.8–232.6 C; 96.5% purity (HPLC); H NMR (400
2
1
3
–
1
1
3
OCH ). C NMR (75 MHz, DMSO) d 169.54 (s, 14C), 164.86 (s, MHz, DMSO) d 11.76 (s, 1H, –CH]N), 8.35–7.95 (m, 1H, Ar-H),
8C), 161.96 (s, 11C), 160.31 (s, 6C), 157.05 (s, 25C), 149.08 (s, 7.94 (s, 1H, N-NH), 7.85–7.72 (m, 1H, Ar-H), 7.68 (d, J ¼ 3.2 Hz,
6C), 145.26 (s, 9C), 141.19 (s, 19C), 137.13 (s, 23C), 132.38 (d, J 1H, Ar-H), 7.48 (dd, J ¼ 13.8, 7.9 Hz, 2H, Ar-H), 7.21 (s, 2H, Ar-
¼
8.5 Hz, 21C), 126.90 (d, J ¼ 15.3 Hz, 2C, 4C), 125.39 (s, 13C), H), 7.04 (s, 1H, Ar-H), 6.87 (d, J ¼ 22.1 Hz, 2H, Ar-H), 6.58–6.27
1
2
22.11 (s, 3C), 121.97 (s, 22C), 119.14 (s, 15C), 116.40 (s, 8C, (m, 2H, Ar-H), 5.20–4.60 (m, 2H, –O–CH –), 3.71 (s, 3H, –OCH ).
2 3
+
0C), 116.53 (s, 24C), 114.31 (s, 1C, 5C), 105.81 (s, 12C), 102.48 ESI-TOF, calcd for C H ClN O ([M + Na] ) 485.0982, found
2
5
19
2 5
(
C
s, 10C), 65.12 (s, 17C), 55.45 (s, 7C). ESI-TOF, calcd for 485.0974. Anal. calcd for C H ClN O : C, 64.87; H, 4.14; Cl,
25 19 2 5
+
25
H19FN
2 5
O ([M + Na] ) 469.1278, found 469.1263. Anal. calcd 7.66; N, 6.05; O, 17.28. Found: C, 64.69; H, 4.27; Cl, 7.74; N, 6.12;
for C25
H
19FN
2
O
5
: C, 67.26; H, 4.29; F, 4.26; N, 6.28; O, 17.92. O, 17.32.
0
Found: C, 67.14; H, 4.34; F, 4.31; N, 6.35; O, 18.02.
(Z)-N -(4-Chlorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H-
0
(Z)-N -(3-Fluorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H- chromen-7-yl)oxy)acetohydrazide (4h). White powder, yield
ꢂ
1
chromen-7-yl)oxy)acetohydrazide (4c). White powder, yield 59%; mp: 213.8–215.2 C; 97.3% purity (HPLC); H NMR (300
ꢂ
1
7
3%; mp: 220.1–223.9 C; 95.5% purity (HPLC); H NMR (300 MHz, DMSO) d 11.67 (s, 1H, –CH]N), 8.35–8.10 (m, 1H, Ar-H),
MHz, DMSO) d 11.69 (s, 1H, –CH]N), 8.39–7.98 (m, 1H, Ar-H), 7.74 (dd, J ¼ 8.5, 4.8 Hz, 2H, Ar-H), 7.51 (dd, J ¼ 8.4, 5.0 Hz, 2H,
.72 (s, 1H, N-NH), 7.63–7.37 (m, 3H, Ar-H), 7.31–7.11 (m, 3H, Ar-H), 7.21 (d, J ¼ 8.6 Hz, 2H, Ar-H), 7.08–6.75 (m, 7.5 Hz, 3H, Ar-
–), 3.72 (s,
7
Ar-H), 7.08–6.92 (m, 1H, Ar-H), 6.84 (d, J ¼ 7.9 Hz, 2H, Ar-H), H), 6.59–6.27 (m, 2H, Ar-H), 5.15–4.55 (m, 2H, –O–CH
2
+
6
3
4
6
4
.46 (t, J ¼ 21.5 Hz, 2H, Ar-H), 5.15–4.60 (m, 2H, –O–CH
2
–), 3H, –OCH
([M + Na] ) 485.0982, found 485.0979. Anal. calcd for C25
3
). ESI-TOF, calcd for C25
H
19ClN
2
O
5
([M + Na] )
19ClN : C,
+
.71 (s, 3H, –OCH
3
). ESI-TOF, calcd for C25
H19FN
2
O
H
5
H
2 5
O
69.1278, found 469.1263. Anal. calcd for C25
7.26; H, 4.29; F, 4.26; N, 6.28; O, 17.92. Found: C, 67.14; H, 4.27; Cl, 7.74; N, 6.12; O, 17.32.
.34; F, 4.31; N, 6.35; O, 18.02.
2 5
19FN O : C, 64.87; H, 4.14; Cl, 7.66; N, 6.05; O, 17.28. Found: C, 64.69; H,
0
(Z)-N -(2,3-Dichlorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-
0
(Z)-N -(4-Fluorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H- 4H-chromen-7-yl)oxy)acetohydrazide (4i). White powder, yield
ꢂ
1
chromen-7-yl)oxy)acetohydrazide (4d). White powder, yield 82%; mp: 214.2–215.6 C; 96.5% purity (HPLC); H NMR (300
ꢂ
1
6
7%; mp: 261.9–263.1 C; 96.6% purity (HPLC); H NMR (300 MHz, DMSO) d 11.89 (d, J ¼ 20.9 Hz, 1H, –CH]N), 8.80–8.35 (m,
MHz, DMSO) d 11.60 (s, 1H, –CH]N), 8.35–7.96 (m, 1H, Ar-H), 1H, Ar-H), 8.04–7.87 (m, 1H, Ar-H), 7.86–7.59 (m, 2H, N-NH, Ar-
.89 (s, 1H, N-NH), 7.75 (dt, J ¼ 10.2, 5.3 Hz, 2H, Ar-H), 7.25 H), 7.43 (td, J ¼ 7.9, 4.1 Hz, 1H, Ar-H), 7.19 (d, J ¼ 8.4 Hz, 2H, Ar-
ddd, J ¼ 21.7, 13.2, 6.1 Hz, 4H, Ar-H), 7.00 (s, 1H, Ar-H), 6.83 (s, H), 7.07–6.94 (m, 1H, Ar-H), 6.84 (d, J ¼ 8.2 Hz, 2H, Ar-H), 6.46
H, Ar-H), 6.50 (d, J ¼ 16.2 Hz, 2H, Ar-H), 5.1–4.55 (m, 2H, –O– (t, J ¼ 23.2 Hz, 2H, Ar-H), 5.15–4.60 (m, 2H, –O–CH –), 3.71 (s,
CH –), 3.70 (s, 3H, –OCH ). ESI-TOF, calcd for C H FN O ([M 3H, –OCH ). ESI-TOF, calcd for C H Cl N O ([M + Na] )
7
(
2
2
+
2
3
25 19
2
5
3
25 18
2 2 5
+
+
Na] ) 469.1278, found 469.1263. Anal. calcd for C H FN O : 519.0593, found 519.0582. Anal. calcd for C H Cl N O : C,
25 19 2 5 25 18 2 2 5
C, 67.26; H, 4.29; F, 4.26; N, 6.28; O, 17.92. Found: C, 67.14; H, 60.38; H, 3.65; Cl, 14.26; N, 5.63; O, 16.08. Found: C, 60.29; H,
4
.34; F, 4.31; N, 6.35; O, 18.02.
3.69; Cl, 14.30; N, 5.74; O, 16.15.
0
0
(Z)-N -(3,4-Diuorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-
(Z)-N -(2,6-Dichlorobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-
4
H-chromen-7-yl)oxy)acetohydrazide (4e). White powder, yield 4H-chromen-7-yl)oxy)acetohydrazide (4j). White powder, yield
ꢂ
1
ꢂ
1
74%; mp: 238.5–239.8 C; 96.3% purity (HPLC); H NMR (300 77%; mp: 181.6–182.7 C; 95.6% purity (HPLC); H NMR (300
MHz, DMSO) d 11.70 (s, 1H, –CH]N), 8.34–7.94 (m, 1H, Ar-H), MHz, DMSO) d 11.86 (s, 1H, –CH]N), 8.55–8.22 (m, 1H, Ar-H),
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7
7
3
.95–7.60 (m, 1H, N-NH), 7.55 (d, J ¼ 7.9 Hz, 2H, Ar-H), 7.48– 14.1, 7.0 Hz, 2H, Ar-H), 7.00 (t, J ¼ 8.3 Hz, 1H, Ar-H), 6.82 (t, J ¼
.35 (m, 1H, Ar-H), 7.18 (d, J ¼ 8.0 Hz, 2H, Ar-H), 7.10–6.75 (m, 10.8 Hz, 2H, Ar-H), 6.56 (d, J ¼ 8.0 Hz, 2H, Ar-H), 6.47–6.28 (m, 2H,
H, Ar-H), 6.50 (d, J ¼ 29.2 Hz, 2H, Ar-H), 5.05–4.60 (m, 2H, –O– Ar-H), 5.61 (d, J ¼ 9.2 Hz, 2H, –NH ), 5.55–4.51 (m, 2H, –O–CH –),
2
2
1
3
+
CH –), 3.70 (s, 3H, –OCH ). C NMR (75 MHz, DMSO) d 169.71 3.70 (s, 3H, –OCH ). ESI-TOF, calcd for C H N O ([M + Na] )
2
3
3
25 21
3 5
(
s, 14C), 165.00 (s, 18C), 159.57 (s, 11C), 158.11 (s, 6C), 157.12 (s, 466.1481, found 466.1473. Anal. calcd for C25
H N O : C, 67.71; H,
21 3 5
1
6C), 143.84 (s, 9C), 139.31 (s, 19C), 134.43 (s, 21C, 25C), 134.39 4.77; N, 9.48; O, 18.04. Found: C, 67.65; H, 4.82; N, 9.53; O, 18.14.
0
(Z)-2-((3-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)-N -(2-
(s, 20C), 131.59 (s, 13C), 129.93 (s, 2C, 4C), 129.87 (s, 23C),
1
1
1
5
6
3
29.54 (s, 22C, 24C), 128.12 (s, 8C), 126.77 (s, 3C), 119.14 (s, (triuoromethyl)benzylidene)acetohydrazide
5C), 114.31 (s, 1C, 5C), 105.72 (s, 12C), 102.33 (s, 10C), 66.86 (s, powder, yield 73%; mp: 203.1–204.5 C; 96.7% purity (HPLC);
(4o).
White
ꢂ
+
1
7C), 55.45 (s, 7C). ESI-TOF, calcd for C H Cl N O ([M + Na] )
H NMR (300 MHz, DMSO) d 11.90 (d, J ¼ 35.0 Hz, 1H, –CH]N),
2
5
18
2 2 5
19.0593, found 519.0582. Anal. calcd for C H Cl N O : C, 8.75–8.32 (m, 1H, Ar-H), 8.19 (dd, J ¼ 16.1, 7.6 Hz, 1H, Ar-H),
2
5
18
2 2 5
0.38; H, 3.65; Cl, 14.26; N, 5.63; O, 16.08. Found: C, 60.29; H, 7.85–7.52 (m, 4H, N-NH, Ar-H), 7.19 (d, J ¼ 7.3 Hz, 2H, Ar-H),
.69; Cl, 14.30; N, 5.74; O, 16.15.
7.09–6.91 (m, 1H, Ar-H), 6.84 (d, J ¼ 7.5 Hz, 2H, Ar-H), 6.62–
–), 3.70 (s, 3H,
0
(
Z)-N -(3-Bromobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H- 6.22 (m, 2H, Ar-H), 5.21–4.60 (m, 2H, –O–CH
2
+
chromen-7-yl)oxy)acetohydrazide (4k). White powder, yield –OCH
3
). ESI-TOF, calcd for C26
H
19
F
H
3
N
2
O
5
([M + Na] ) 519.1246,
: C, 62.90; H, 3.86;
ꢂ
1
82%; mp: 224.0–225.2 C; 97.8% purity (HPLC); H NMR (300 found 519.1209. Anal. calcd for C26
19
F
3
N
2
O
5
MHz, DMSO) d 11.69 (s, 1H, –CH]N), 8.30–7.95 (m, 1H, Ar-H), F, 11.48; N, 5.64; O, 16.11. Found: C, 62.82; H, 3.94; F, 11.53; N,
.90 (d, J ¼ 9.7 Hz, 1H, Ar-H), 7.69 (d, J ¼ 7.7 Hz, 1H, Ar-H), 7.59 5.72; O, 16.24.
(Z)-2-((3-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)-N -(4-
7
0
(
7
6
d, J ¼ 6.6 Hz, 1H, Ar-H), 7.39 (dd, J ¼ 14.5, 6.7 Hz, 1H, Ar-H),
.19 (d, J ¼ 8.4 Hz, 2H, Ar-H), 6.99 (d, J ¼ 8.1 Hz, 1H, Ar-H), (triuoromethyl)benzylidene)acetohydrazide
(4p).
White
ꢂ
.84 (s, 2H, Ar-H), 6.56–6.22 (m, 2H, Ar-H), 5.15–4.59 (m, 2H, powder, yield 68%; mp: 176.7–178.2 C; 97.5% purity (HPLC);
1
–
O–CH
2
–), 3.70 (s, 3H, –OCH
3
). ESI-TOF, calcd for
H NMR (300 MHz, DMSO) d 11.79 (s, 1H, –CH]N), 8.43–8.05
+
C
25
H
19BrN
2 5
O
([M + Na] ) 529.0477, found 529.0438. Anal. calcd (m, 1H, Ar-H), 7.92 (d, J ¼ 8.1 Hz, 2H, Ar-H), 7.78 (t, J ¼ 7.2 Hz,
for C25
H19BrN
2
O
5
: C, 59.19; H, 3.77; Br, 15.75; N, 5.52; O, 15.77. 3H, N-NH, Ar-H), 7.19 (dd, J ¼ 8.7, 2.1 Hz, 2H, Ar-H), 7.02 (t, J ¼
Found: C, 59.10; H, 3.83; Br, 15.82; N, 5.59; O, 15.80.
8.7 Hz, 1H, Ar-H), 6.84 (d, J ¼ 8.3 Hz, 2H, Ar-H), 6.61–6.29 (m,
0
13
(
Z)-N -(3-Hydroxybenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo- 2H, Ar-H), 5.17–4.61 (m, 2H, –O–CH –), 3.71 (s, 3H, –OCH ).
C
2
3
4
H-chromen-7-yl)oxy)acetohydrazide (4l). White powder, yield NMR (75 MHz, DMSO) d 169.73 (s, 14C), 165.08 (s, 18C), 159.79–
ꢂ
1
82%; mp: 294.1–295.7 C; 95.2% purity (HPLC); H NMR (400 159.10 (m, 11C), 158.05 (s, 6C), 157.08 (s, 16C), 146.63 (s, 9C),
MHz, DMSO) d 11.59 (s, 1H, –CH]N), 8.25–7.90 (m, 1H, Ar-H), 142.67 (s, 19C), 138.56 (s, 20C), 138.39 (s, 23C), 130.18 (s, 2C,
7
8
2
.68 (s, 1H, N-NH), 7.28–6.95 (m, 6H, Ar-H), 6.83 (d, J ¼ 4C), 128.20 (s, 21C, 25C), 128.01 (s, 13C), 126.74 (s, 22C, 24C),
.1 Hz, 3H, Ar-H), 6.52 (d, J ¼ 25.9 Hz, 2H, Ar-H), 5.15–4.60 (m, 126.16 (d, J ¼ 4.7 Hz, 3C), 125.46 (s, CF ), 123.66 (s, 8C), 119.13
). C NMR (100 MHz, DMSO) (s, 15C), 114.31 (s, 1C, 5C), 105.79 (s, 12C), 102.54 (s, 10C), 66.84
3
1
3
2 3
H, –O–CH –), 3.72 (s, 3H, –OCH
d 169.31 (s, 14C), 164.66 (s, 18C), 158.14 (s, 14C), 158.10 (s, 6C), (s, 17C), 55.44 (s, 7C). ESI-TOF, calcd for C26
H
19
F
3
N
2
O
5
([M +
+
1
1
1
1
1
C
57.08 (s, 24C), 148.44 (s, 9C), 144.54 (s, 19C), 135.83 (s, 20C), Na] ) 519.1246, found 519.1209. Anal. calcd for C H F N O :
35.66 (s, 22C), 130.35 (s, 2C, 4C), 128.09 (s, 13C), 126.74 (s, 3C), C, 62.90; H, 3.86; F, 11.48; N, 5.64; O, 16.11. Found: C, 62.82; H,
19.33 (s, 8C), 118.85 (s, 21C), 118.02 (s, 23C), 117.76 (s, 14C), 3.94; F, 11.53; N, 5.72; O, 16.24.
2
6
19 3 2 5
0
14.32 (s, 25C, 1C, 5C), 113.23 (s), 113.12 (s), 105.81 (s, 12C),
(Z)-2-((3-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)-N -(4-
02.82 (s, 10C), 66.87 (s, 17C), 55.45 (s, 7C). ESI-TOF, calcd for methylbenzylidene)acetohydrazide (4q). White powder, yield
+
ꢂ
1
25
H N O ([M + Na] ) 467.1321, found 467.1314. Anal. calcd 62%; mp: 202.1–203.3 C; 96.2% purity (HPLC); H NMR (300
20 2 6
for C25
H
20
N
2
O
6
: C, 67.56; H, 4.54; N, 6.30; O, 21.60. Found: C, MHz, DMSO) d 11.52 (d, J ¼ 6.4 Hz, 1H, –CH]N), 8.32–7.9 (m,
67.47; H, 4.61; N, 6.39; O, 21.65.
1H, Ar-H), 7.74 (s, 1H, N-NH), 7.58 (d, J ¼ 7.8 Hz, 2H, Ar-H),
0
(
Z)-N -(4-Hydroxybenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo- 7.35–7.10 (m, 4H, Ar-H), 7.01 (t, J ¼ 8.6 Hz, 1H, Ar-H), 6.84 (d,
4
8
H-chromen-7-yl)oxy)acetohydrazide (4m). White powder, yield J ¼ 7.6 Hz, 2H, Ar-H), 6.45 (dd, J ¼ 28.6, 17.3 Hz, 2H, Ar-H), 5.17–
ꢂ
2%; mp: 248.3–249.5 C; 95.3% purity (HPLC); H NMR (300 4.51 (m, 2H, –O–CH –), 3.71 (s, 3H, –OCH ), 2.32 (s, 3H, –CH ).
2 3 3
1
+
MHz, DMSO) d 11.38 (d, J ¼ 8.6 Hz, 1H, –CH]N), 10.01 (s, 1H, ESI-TOF, calcd for C26
H
22
N
2
O
5
([M + Na] ) 465.1529, found
–
8
8
OH), 8.25–7.85 (m, 1H, Ar-H), 7.76 (s, 1H, N-NH), 7.51 (d, J ¼ 465.1513. Anal. calcd for C26
22 2 5
H N O : C, 70.58; H, 5.01; N, 6.33;
.4 Hz, 2H, Ar-H), 7.19 (dd, J ¼ 8.8, 2.6 Hz, 2H, Ar-H), 7.01 (t, J ¼ O, 18.08. Found: C, 70.14; H, 5.16; N, 6.42; O, 18.14.
0
.5 Hz, 1H, Ar-H), 6.89–6.73 (m, 4H, Ar-H), 6.54–6.25 (m, 2H, Ar-
(Z)-N -(2-Methoxybenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-
H), 5.10–4.55 (m, 2H, –O–CH
2 3
–), 3.70 (s, 3H, –OCH ). ESI-TOF, 4H-chromen-7-yl)oxy)acetohydrazide (4r). White powder, yield
+
ꢂ
1
calcd for C H N O ([M + Na] ) 467.1321, found 467.1314. 86%; mp: 218.5–219.9 C; 97.1% purity (HPLC); H NMR (400
2
5
20 2 6
Anal. calcd for C H N O : C, 67.56; H, 4.54; N, 6.30; O, 21.60. MHz, DMSO) d 11.62 (d, J ¼ 17.4 Hz, 1H, –CH]N), 8.71–8.32 (m,
2
5
20 2 6
Found: C, 67.47; H, 4.61; N, 6.39; O, 21.65.
1H, Ar-H), 7.84 (ddd, J ¼ 16.7, 7.7, 1.5 Hz, 2H, N-NH, Ar-H), 7.47–
7.34 (m, 1H, Ar-H), 7.21 (dd, J ¼ 8.8, 2.4 Hz, 2H, Ar-H), 7.09 (t, J ¼
0
(Z)-N -(4-Aminobenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-4H-
chromen-7-yl)oxy)acetohydrazide (4n). Orange powder, yield 69%; 9.1 Hz, 1H, Ar-H), 7.02 (dt, J ¼ 10.7, 8.2 Hz, 2H, Ar-H), 6.86 (d, J ¼
ꢂ
1
mp: 247.6–248.3 C; 95.4% purity (HPLC); H NMR (300 MHz, 8.0 Hz, 2H, Ar-H), 6.60–6.27 (m, 2H, Ar-H), 5.15–4.55 (m, 2H, –O–
), 3.72 (s, 3H, –OCH ). ESI-
DMSO) d 11.23 (d, J ¼ 5.6 Hz, 1H, –CH]N), 8.14–7.78 (m, 1H, Ar- CH
2
–), 3.86 (d, J ¼ 3.2 Hz, 3H, –OCH
3
3
+
H), 7.75 (s, 1H, N-NH), 7.34 (d, J ¼ 8.5 Hz, 2H, Ar-H), 7.17 (dt, J ¼ TOF, calcd for C26
H N O
22 2 6
([M + Na] ) 481.1478, found 481.1463.
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Paper
22 2 6
Anal. calcd for C26H N O : C, 68.11; H, 4.84; N, 6.11; O, 20.94. Cell lines and culture conditions
Found: C, 68.02; H, 4.92; N, 6.23; O, 20.98.
The cell lines used in this study were MCF-7, MDA-MB-231,
H460, H1650, L02 and VERO. They were obtained from State
Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University. MCF-7, MDA-MB-231, L02 and VERO cells were
maintained in DMEM/high glucose supplemented with 10%
0
(Z)-N -(4-Methoxybenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-
4
H-chromen-7-yl)oxy)acetohydrazide (4s). White powder, yield
ꢂ
1
75%; mp: 193.5–194.5 C; 95.6% purity (HPLC); H NMR (400
MHz, DMSO) d 11.52 (s, 1H, –CH]N), 8.29–7.95 (m, 1H, Ar-H),
.75–7.56 (m, 2H, Ar-H), 7.21 (d, J ¼ 7.5 Hz, 2H, Ar-H), 7.01
dd, J ¼ 8.7, 6.8 Hz, 3H, Ar-H), 6.85 (s, 2H), 6.63–6.22 (m, 2H, Ar-
H), 5.12–4.59 (m, 2H, –O–CH ),
7
(
FBS, L-glutamine, and penicillin/streptomycin and incubated at
ꢂ
3
7 C in humidied atmosphere containing 5% CO . H460 and
2
2
–), 3.80 (d, J ¼ 2.8 Hz, 3H, –OCH
3
+
H1650 cells were maintained in RPMI.1640 medium supple-
3
4
.72 (s, 3H, –OCH ). ESI-TOF, calcd for C H N O ([M + Na] )
3 26 22 2 6
mented with 10% FBS and penicillin/streptomycin and incu-
81.1478, found 481.1463. Anal. calcd for C H N O : C, 68.11;
ꢂ
2
6
22 2 6
bated at 37 C in humidied atmosphere containing 5% CO
2
.
H, 4.84; N, 6.11; O, 20.94. Found: C, 68.02; H, 4.92; N, 6.23; O,
0.98.
Z)-N -(3,4-Dimethoxybenzylidene)-2-((3-(4-methoxyphenyl)-4-
2
Animals feeding
0
(
oxo-4H-chromen-7-yl)oxy)acetohydrazide (4t). White powder, Female nude mice (5–7 weeks old) were obtained from Model
ꢂ
1
yield 63%; mp: 140.7–141.8 C; 96.7% purity (HPLC); H NMR Animal Research Center of Nanjing University (Nanjing, China).
(
400 MHz, DMSO) d 11.52 (d, J ¼ 20.7 Hz, 1H, –CH]N), 8.28– Briey, mice were fed with free access to pellet food and water in
ꢂ
7
1
8
2
6
.90 (m, 1H, Ar-H), 7.73 (s, 1H, Ar-H), 7.32 (dd, J ¼ 9.1, 1.6 Hz, plastic cages at 21 ꢀ 2 C and kept on a 12 hours light–dark
H, Ar-H), 7.21 (dd, J ¼ 8.4, 2.6 Hz, 3H, Ar-H), 7.03 (dd, J ¼ 16.7, cycle. All animal procedures were performed in accordance with
.3 Hz, 2H, Ar-H), 6.86 (d, J ¼ 6.8 Hz, 2H, Ar-H), 6.59–6.31 (m, the Guidelines for Care and Use of Laboratory Animals of
H, Ar-H), 5.18–4.60 (m, 2H, –O–CH
H, –OCH ), 3.72 (s, 3H, –OCH ). ESI-TOF, calcd for C27
Na] ) 511.1584, found 511.1558. Anal. calcd for (2006). All efforts were made to minimize animal suffering and
: C, 66.39; H, 4.95; N, 5.73; O, 22.93. Found: C, 66.24; to reduce the number of animals used.
2
–), 3.81 (dd, J ¼ 8.5, 6.6 Hz, Nanjing University and approved by the Animal Ethics
H
24
N
2
O
7
Committee of the Ministry of Science and Technology of China
3
3
+
(
[M
C
+
27 24 2 7
H N O
H, 5.03; N, 5.86; O, 23.06.
0
(
Z)-N -(4-Isopropylbenzylidene)-2-((3-(4-methoxyphenyl)-4-oxo-
EGFR kinase phosphorylation assay
4
H-chromen-7-yl)oxy)acetohydrazide (4u). White powder, yield
3
,28
The assays were carried out as described previously.
Phos-
ꢂ
1
86%; mp: 198.2–199.6 C; 97.2% purity (HPLC); H NMR (300
phorylation assays were performed in a nal volume of 20 mL
MHz, DMSO) d 11.52 (d, J ¼ 11.9 Hz, 1H, –CH]N), 8.31–7.95 (m,
containing 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 10 mM MnCl ,
2
1
H, Ar-H), 7.75 (s, 1H, N-NH), 7.61 (d, J ¼ 8.2 Hz, 2H, Ar-H), 7.30
ꢁ
1
2
00 mM substrate peptide, 0.25 mM DTT, 0.1 mg mL BSA, 10
(
7
dd, J ¼ 8.0, 5.1 Hz, 2H, Ar-H), 7.19 (dd, J ¼ 8.8, 2.5 Hz, 2H, Ar-H),
ng of wild-type EGFR kinase (catalogue no. 40187, BPS Biosci-
.02 (t, J ¼ 8.6 Hz, 1H, Ar-H), 6.84 (d, J ¼ 8.1 Hz, 2H, Ar-H), 6.56–
.30 (m, 2H, Ar-H), 5.12–4.55 (m, 2H, –O–CH –), 3.71 (s, 3H,
3
2
ence), 10 mM magnesium acetate, 100 mM g-[ P] ATP, and
inhibitors or DMSO control (1.25% v/v). For IC50 curves with the
wild-type enzyme, the following concentrations of the
compounds were tested in triplicate: 150, 100, 50, 25, 15, 10, 7.5,
, and 2.5 nM. Reactions were started by the addition of the
magnesium acetate/ATP mixture. Aer 30 minutes incubation
6
2
–
OCH ), 2.90 (m, 1H, ꢁ CH ðCH3Þ ), 1.20 (dd, J ¼ 6.9, 1.8 Hz, 6H,
3
2
+
ꢁ
CHðCH3 Þ ). ESI-TOF, calcd for C H N O ([M + Na] )
2
28 26 2 5
4
93.1842, found 493.1837. Anal. calcd for C H N O : C, 71.48;
2
8
26 2 5
5
H, 5.57; N, 5.95; O, 17.00. Found: C, 71.31; H, 5.62; N, 5.995; O,
7.12.
Z)-N -(4-(Benzyloxy)benzylidene)-2-((3-(4-methoxyphenyl)-4-
oxo-4H-chromen-7-yl)oxy)acetohydrazide (4v). White powder,
1
ꢂ
at 30 C, 5 mL of each reaction was spotted on phosphocellulose
0
(
P81 paper (Whatman). The P81 paper was then washed 5 times
with 50 mM phosphoric acid for 15 minutes, dried, and exposed
to a phosphorimager screen (Storm, GE Healthcare), which was
scanned and densitometrically analyzed the next day. The
sequence of the substrate peptide was derived from phospho-
lipase C-g1. The IC50 values were calculated using nonlinear
regression with normalized dose response t using Prism
GraphPad soware.
ꢂ
1
yield 69%; mp: 182.4–183.5 C; 97.2% purity (HPLC); H NMR
(
300 MHz, DMSO) d 11.46 (d, J ¼ 8.5 Hz, 1H, –CH]N), 8.30–7.90
(m, 1H, Ar-H), 7.80 (d, J ¼ 8.4 Hz, 1H, N-NH), 7.63 (d, J ¼ 8.3 Hz,
2
8
2
4
H, Ar-H), 7.38 (ddd, J ¼ 19.5, 13.1, 6.8 Hz, 5H, Ar-H), 7.19 (d, J ¼
.0 Hz, 2H, Ar-H), 7.05 (dd, J ¼ 15.8, 7.5 Hz, 3H, Ar-H), 6.85 (s,
H, Ar-H), 6.58–6.22 (m, 2H, Ar-H), 5.14 (s, 2H, Ph-CH
2
), 5.10–
1
3
.55 (m, 2H, –O–CH –), 3.70 (s, 3H, –OCH ). C NMR (75 MHz,
2 3
DMSO) d 169.17 (s, 14C), 164.43 (s, 18C), 160.48 (s, 11C), 160.26
Anti-proliferation assay
(s, 23C), 148.21 (s, 6C), 144.14 (s, 16C), 137.21 (s, 9C), 129.24 (s,
1
3
1
1
1
5
9C), 128.99 (s, 27C), 128.94 (s, 2C, 4C, 21C, 25C), 128.42 (s, 29C, The anti-proliferation activity of the prepared compounds
1C), 128.25 (s, 13C), 127.31 (s, 28C, 32C), 127.23 (s, 31C), against four cancer cell lines, MCF-7, MDA-MB-231, H460,
15.62 (s, 20C, 3C), 114.32 (s, 1C, 5C, 22C, 24C), 105.80 (s, 8C, H1650 and two normal cell lines, L02 and VERO were evaluated
29
2C), 102.86 (s, 15C), 102.45 (s, 10C), 69.81 (s, 26C), 66.89 (s, as described elsewhere. Four kinds of cancer cell lines and two
+
7C), 55.45 (s, 7C). ESI-TOF, calcd for C32
57.1791, found 557.1805. Anal. calcd for C32
26 2 6
H N O ([M + Na] ) normal cell lines were plated in 96-well plates at the appropriate
3
H
26
N
2
O
6
: C, 71.90; density (2.0 ꢃ 10 cells per well) with maintained in DMEM (or
H, 4.90; N, 5.24; O, 17.96. Found: C, 71.83; H, 4.98; N, 5.31; O, 1640 medium) with 10% FBS and cultivated for 12 hours at
1
ꢂ
8.03.
37 C, 5% CO
2
atmosphere. All cells treated with different
48416 | RSC Adv., 2017, 7, 48404–48419
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concentrations (0, 0.1, 1, 10 and 100 mM) of each compound were counted using the Flowjo 7.6.1 soware excluding cell
with formononetin and lapatinib as positive references. Aer 24 debris.
ꢁ
1
hours, 20 mL PBS containing of MTT solution (2.5 mg mL ) was
added to each well and incubated for a further 4 hours. Then Wound-healing assay
1
50 mL of DMSO was added to each well to dissolve purple
MDA-MB-231 cells were seeded in a tissue culture 6-well plate at
crystal. The plates were shaken to ensure the completion solu-
bility for 10 minutes at room temperature. The values of
absorbance were recorded on ELISA reader (ELx800, BioTek,
USA) at a wavelength of 570 nm. All experiments were per-
formed in three replicate wells for each concentration of all
compounds including formononetin, and all assay experiments
were carried out three times. The IC50 values dened as the
concentrations that cause a 50% loss of cell viability were
calculated by Origin 7.5 and exhibited in Table 3.
5
2
an initial density of 2 ꢃ 10 cells per cm overnight. A micro-
pipette tip was used to create a wound in the monolayer by
scraping aer cells seeding and incubation for 24 hours. Iden-
tical cell-free space was observed by phase-contrast microscopy
(NIKON, Japan) and digital images were taken by cells at 0, 24
and 48 hours. Subsequently, the NIH Image J image analysis
soware was used to outline the wound areas and analysis
quanties the reduced cell-free space, n ¼ 3.
Western blot analysis
Apoptosis analysis by ow cytometry
Total proteins were extracted from MDA-MB-231 cells which were
Cell apoptosis assay of compound 4v for MDA-MB-231 cell was treated with compound 4v at different concentrations (0, 2, 4 and
carried out by Annexin V/PI assays and monitored by ow 8 mM). Cells obtained were lysed in RIPA lysis buffer containing
3
cytometry. Briey, 5 ꢃ 10 cells per well were seeded in 6-well protease inhibitor phenylmethanesulfonyl uoride (PMSF,
plates for 12 hours and then treated with 4v (0, 2, 4 and 8 mM) PMSF : RIPA ¼ 1 : 100) on ice for 20–30 minutes. Protein content
for 24 hours or treated with 4 mM 4v for 0, 12, 24 and 36 hours. was measured using a Pierce BCA protein assay kit. Protein
Next, cells were collected and washed twice with PBS and then samples were separated by sodium dodecyl sulphate-
centrifuged to obtain sedimentation. 500 mL 1ꢃ binding buffer polyacrylamide gel electrophoresis (SDS-PAGE) and then trans-
(
10 mM HEPES, PH 7.4, 140 mM NaOH, 2.5 mM CaCl ) was ferred onto polyvinylidene diuoride (PVDF) membranes which
2
added to each EP tube to make cells resuspended and stained were blocked with 5% non-fat-milk for 1 hour. All membranes
with 5 mL of Annexin V-FITC and 5 mL of PI for 30 minutes at were separated and incubated with primary antibodies including
room temperature in the dark. Apoptotic cells were quantied EGFR (rabbit polyclonal IgG, 1 : 1000 dilution), p-EGFR (rabbit
using a BD Accuri C6 Flowjo Cytometer (BD, USA). Statistical polyclonal IgG, 1 : 1000 dilution), ERK (rabbit polyclonal IgG,
analysis was done using Flowjo 7.6.1 soware.
1 : 500 dilution), p-ERK (rabbit polyclonal IgG, 1 : 500 dilution),
Akt (rabbit polyclonal IgG, 1 : 500 dilution), p-Akt (rabbit poly-
clonal IgG, 1 : 500 dilution), GAPDH (rabbit polyclonal IgG,
Mitochondrial membrane potential staining
1
: 1000 dilution), CDK4 (rabbit polyclonal IgG, 1 : 500 dilution),
Cyclin A (rabbit polyclonal IgG, 1 : 500 dilution), Cyclin D1
rabbit polyclonal IgG, 1 : 500 dilution), b-actin (rabbit polyclonal
JC-1 is an ideal uorescent probe which is widely used in the
detection of mitochondrial membrane potential (DJ ).
m
(
According to the requirements, MDA-MB-231 cells were
IgG, 1 : 500 dilution), Bax (rabbit polyclonal IgG, 1 : 500 dilution),
Bcl2 (rabbit polyclonal IgG, 1 : 500 dilution), cleaved caspase-3
3
cultured in 6-well plates (5 ꢃ 10 cells per well) for 12 hours at
ꢂ
3
7 C, 5% CO
2
atmosphere and then treated with compound 4v
(rabbit polyclonal IgG, 1 : 500 dilution), cleaved-PARP (mouse
(
0, 2 and 4 mM) for 12 hours and with positive control drug,
monoclonal IgG, 1 : 600 dilution), cytochrome c (rabbit poly-
clonal IgG, 1 : 500 dilution), b-catenin (rabbit polyclonal IgG,
ꢂ
CCCP in another well for 30 minutes at 37 C. Cell in all wells
were collected and rinsed with PBS. Then 500 mL JC-1 staining
1
: 500 dilution) and Axin-2 (rabbit polyclonal IgG, 1 : 1000 dilu-
ꢂ
solution (5ꢃ) was added to each EP tube and incubated at 37 C
ꢂ
tion) at 4 C shaking overnight. Aer the membrane was washed
three times with tris-buffered saline buffer, HRP-conjugated
secondary antibody (diluted 1 : 50 000) was used at room
temperature. Detection was performed by an enhanced chemi-
luminescent reagent (Thermo Fisher Scientic, USA) according to
the manufacturer's instructions. Bands were then recorded by
a digital camera (Tanon 5200, Tanon, China). Finally, the results
were analyzed with Image J Soware (National Institutes of
Health, BetheSEMa, Maryland, USA), and all the targeted protein
were normalized to GAPDH or b-actin.
for 20 minutes. Finally, cells were washed twice with JC-1
staining solution (1ꢃ) and then resuspended to detect by BD
Accuri C6 Flowjo Cytometer and Olympus confocal microscope,
and data was analyzed using FV-10-ASW 1.7 viewer.
Cell cycle arrested analysis by ow cytometry
3
Aer incubation in 6-well plates (5 ꢃ 10 cells per well), MDA-
MB-231 cells were treated with compound 4v (0, 2, 4 and 8
mM) for 24 hours or treated with 4 mM 4v for 0, 24 and 36 hours.
Aer that, cells were collected and washed twice with PBS and
ꢂ
Transplantation of MDA-MB-231 cells into nude mice
then xed in 70% ethanol at 4 C for at least 2 hours. Next, cells
ꢁ
1
were resuspended in PBS with 0.1 mg mL RNase A and 5 mg Cultured MDA-MB-231 cells were washed with and resuspended
ꢁ1
6
mL propidium iodide (PI) in the dark condition. The measure in ice-cold PBS. Portions of the suspension (6 ꢃ 10 cells in 0.1
was performed by BD Accuri C6 Flowjo Cytometer (BD, USA). mL) were injected into the right anks of female nu/nu mice.
The date of cells in the G1, S and G2/M phases of the cell cycle Two weeks aer the injection, the mice bearing tumors (an
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average size of 90 mm ) were distributed into 3 groups (n ¼ 8
Paper
3
Abbreviations
ꢁ1
mice per group) according to tumor volumes. 4v (5 mg kg ) and
ꢁ
1
lapatinib (5 mg kg ) were dissolved in olive oil and adminis-
tered once every two days for 11 times by intraperitoneal CADD Computer assist drug design
injection. Vehicle (olive oil)-treated group was included as CCCP Carbonylcyanidem-chlorophen-ylhydrazone
EGF
Epidermal growth factor
control. Body weight and tumor volumes were measured and
EGFR Epidermal growth factor receptor
recorded every two days. Long diameter (L) and short diameter
ESI-
MS
Electrospray ionization-mass spectrometry
(S) of a tumor were measured with a vernier caliper and the
tumor volume was calculated using the following formula: L ꢃ
2
HER2 Human epidermal growth factor receptor-2
HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
MAPK Mitogen-activated protein kinase
(
S) /2. Twenty-one days aer injection, the mice were killed and
tumors were separated. Tumor weight was measured and tumor
sections were xed in paraformaldehyde. The rest of the
MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
ꢂ
sections were frozen in liquid nitrogen and stored at ꢁ80 C.
NSCLC Non-small-cell carcinoma
PARP Poly-ADP ribose polymerase
Data and statistical analysis
PI
Propidium iodide
All experiments were repeated at least three times. Data are
presented as mean ꢀ S.E.M. and statistical comparisons
between the treated and untreated groups were performed
using one-way ANOVA using GraphPad PRISM5 (Graphpad Inc.,
La Jolla, USA). P-Values of <0.05 were considered statistically
signicant; P-values of <0.01 were considered statistically very
signicant.
SAR
TLC
UV
Structure–activity relationship
Thin layer chromatography
Ultraviolet
Acknowledgements
Conclusion
The authors are grateful to the Program for Changjiang Scholars
and Innovative Research Team in University (IRT_14R27), the
National Natural Science Foundation of China (31470384,
In this study, a series of novel formononetin derivatives tar-
geting EGFR kinase were designed, synthesized and evaluated
for their potential anti-proliferation activities against human
breast and lung cancer cell lines. Among them, compound 4v
31171161, and 31670298), and the Fundamental Research
Funds for the Central Universities (020814380002).
(
IC50 ¼ 14.5 nM) showed potent EGFR inhibitory activity which
Notes and references
was almost comparable to that of getinib (IC50 ¼ 6.2 nM) and
lapatinib (IC50 ¼ 5.6 nM). For in vitro anti-proliferation activi-
ties, 4v exhibited potent inhibitory activity against MDA-MB-231
cell line (IC50 ¼ 5.44 ꢀ 1.28 mM) which was also comparable to
lapatinib (IC50 ¼ 2.48 ꢀ 1.04 mM) but much better than getinib
1
A. Olayioye, R. M. Neve and N. E. Hynes, EMBO J., 2000, 17,
159–3167.
I. Okamoto, FEBS J., 2010, 277, 309–315.
3
2
3 M. M. Hamed, S. S. Darwish, J. Herrmann, A. H. Abadi and
M. Engel, J. Med. Chem., 2017, 60, 2853–2868.
(IC50 ¼ 12.5 ꢀ 2.13 mM) and formononetin (IC ¼ 26.6 ꢀ 2.84
50
mM) itself. The structure–activity relationship (SAR) analyzed by
molecular docking showing that 4v could bind well to the active
site of EGFR as lapatinib. Flow cytometry analysis and mito-
chondrial membrane potential staining results demonstrated
that 4v could effectively cause loss of mitochondrial membrane
potential and then induce apoptosis on MDA-MB-231 cells, but
had no signicant inuence on cell cycle distribution. Western
blot results further conrmed that 4v inhibited MDA-MB-231
cell proliferation by blocking EGFR downstream Ras/Raf/MEK/
ERK signaling pathway and induced MDA-MB-231 cell
apoptosis by regulating EGFR/PI3K/Akt/Bad pathway. In addi-
tion, 4v could also effectively inhibit migration of MDA-MB-231
cells involving to Wnt signaling pathway. The preliminary in
vivo anti-tumor results demonstrated the effectiveness of 4v in
tumor chemotherapy in mice and indicated its possibility as
a new EGFR inhibitor in curing MDA-MB-231 malignant tumor.
4
5
6
Y. Yamanaka, H. Friess, M. Kobrin, M. Buchler, H. Beger and
M. Korc, Anticancer Res., 1992, 13, 565–569.
J. M. Rae and M. E. Lippman, Breast Cancer Res. Treat., 2004,
83, 99–107.
R. E. Sobol, R. W. Astarita, C. Hofeditz, H. Masui,
R. Fairshter, I. Royston and J. Mendelsohn, J. Natl. Cancer
Inst., 1987, 79, 403–407.
7
8
9
S. Balakrishnan, S. Mukherjee, S. Das, F. A. Bhat, P. Raja
Singh, C. R. Patra and J. Arunakaran, Cell Biochem. Funct.,
2017, 35, 217–231.
K. Takasawa, A. Takasawa, M. Osanai, T. Aoyama, Y. Ono,
T. Kono, Y. Hirohashi, M. Murata and N. Sawada, Cancer
Lett., 2017, 403, 66–73.
A. Sgambato, F. Casaluce, P. Maione, A. Rossi, E. Rossi,
A. Napolitano, G. Palazzlol, M. A. Bareschion, C. Schettino,
P. C. Sacco, F. Ciadiello and C. Gridelli, Curr. Med. Chem.,
2012, 19, 3337–3352.
1
0 K. Manupati, N. R. Dhoke, T. Debnath, R. Yeeravalli,
K. Goguloth, S. Saeidpour, U. C. De, S. Debnath and
A. Das, FEBS J., 2017, 284, 1830–1854.
Conflicts of interest
The authors declare no conict of interest.
48418 | RSC Adv., 2017, 7, 48404–48419
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
1
1 M. J. Duffy, P. M. Mcgowan and J. Crown, Int. J. Cancer, 2012, 21 S. Medjakovic and A. Jungbauer, J. Steroid Biochem., 2008,
31, 2471–2477. 108, 171–177.
2 D. Zhang, A. M. Tari, U. Akar, B. K. Arun, T. A. LaFortune, 22 Y. M. Jin, T. M. Xu, Y. H. Zhao, Y. C. Wang and M. H. Cui,
1
1
R. Nieves-Alicea, G. N. Hortobagyi and N. T. Ueno, Mol.
Tumor Biol., 2014, 35, 2279–2284.
Cancer Ther., 2010, 9, 3090–3099.
23 D. J. Fu, L. Zhang, J. Song, R. W. Mao, R. H. Zhao, Y. C. Liu,
Y. H. Hou, J. H. Li, J. J. Yang, C. Y. Jin, P. Li, X. L. Zi,
H. M. Liu, S. Y. Zhang and Y. B. Zhang, Eur. J. Med. Chem.,
2016, 127, 87–99.
24 Y. Q. Li, F. Yang, L. Wang, Z. Cao, T. J. Han, Z. A. Duan, Z. Li
and W. J. Zhao, Eur. J. Med. Chem., 2016, 112, 196–208.
ˇ
1
1
1
3 O. Lap ˇc ı
́
k, M. Hill, I. Cern ´y , J. Lachman, N. Al-Maharik,
H. Adlercreutz and R. Hampl, Plant Sci., 1999, 148, 111–119.
4 L. Hutabarat, H. Greeneld and M. Mulholland, J.
Chromatogr. A, 2000, 886, 55–63.
5 X. Y. Wu, H. Xu, Z. F. Wu, C. Chen, J. Y. Liu, G. N. Wu,
X. Q. Yao, F. K. Liu, G. Li and L. Shen, Oncotarget, 2015, 6, 25 J. Ren, H. J. Xu, H. Cheng, W. Q. Xin, X. Chen and K. Hu, Eur.
4563–44578. J. Med. Chem., 2012, 54, 175–187.
6 X. Yu, W. Wang and M. Yang, Food Chem., 2007, 104, 715– 26 P. V. M. D. Alan, Cancer, 2005, 103, 2435–2446.
4
1
1
1
1
2
7
20.
27 X. J. Zhu, Y. Shi, J. Peng, C. S. Guo, N. N. Shan, P. Qin, X. B. Ji
and M. Hou, Blood, 2009, 114, 5362–5367.
7 T. Fukai, A. Marumo, K. Kaitou, T. Kanda, S. Terada and
T. Nomura, Life Sci., 2002, 71, 1449–1463.
8 Y. Ungar, O. F. Osundahunsi and E. Shimoni, J. Agric. Food
Chem., 2003, 51, 4394–4399.
9 S. Sato, J. Takeo, C. Aoyama and H. Kawahara, Bioorg. Med.
Chem., 2007, 15, 3445–3449.
28 Y. G. Zheng, J. Su, C. Y. Gao, P. Jiang, L. An, Y. S. Xue, J. Gao
and Y. Liu, Eur. J. Med. Chem., 2017, 130, 393–405.
29 H. Y. Lin, Z. K. Li, L. F. Bai, S. K. Baloch, F. Wang, H. Y. Qiu,
X. Wang, J. L. Qi, R. W. Yang, X. M. Wang and Y. H. Yang,
Biochem. Pharmacol., 2015, 96, 93–106.
0 Z. N. Ji, W. Zhao, G. Liao, R. Choi, C. K. Lo, T. T. X. Dong and
K. W. K. Tsim, Gynecol. Endocrinol., 2006, 22, 578–584.
This journal is © The Royal Society of Chemistry 2017
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