Discovery of chalcone-modified estradiol analogs as antitumour agents that Inhibit tumour angiogenesis and epithelial to mesenchymal transition

Article abstract of DOI:10.1016/j.ejmech.2019.04.071

Angiogenesis plays an essential role in tumourigenesis and tumour progression, and anti-angiogenesis therapies have shown promising antitumour effects in solid tumours. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, has been regarded as a potential antitumour agent mainly targeting angiogenesis. Here we synthesized a novel series of chalcones based on 2-methoxyestradiol and evaluated their potential activities against tumours. Compound 11e was demonstrated to have potent antiangiogenic activity. Further studies showed that 11e suppressed tumour growth in human breast cancer (MCF-7)xenograft models without obvious side effects. Evaluation of the mechanism revealed that 11e targeted the epithelial to mesenchymal transition (EMT)process in MCF-7 cells and inhibited HUVEC migration and then contributed to hindrance of angiogenesis. Thus, 11e may be a promising antitumour agent with excellent efficacy and low toxicity.

Full text of DOI:10.1016/j.ejmech.2019.04.071

Accepted Manuscript
Discovery of chalcone-modified estradiol analogs as antitumour agents that Inhibit
tumour angiogenesis and epithelial to mesenchymal transition
Cong Wang, Leilei Li, Dongyang Fu, Tiantian Qin, Yange Ran, Feng Xu, Xinrui Du,
Haiying Gao, Shuaijun Sun, Tengjiao Yang, Xueyan Zhang, Junfeng Huo, Wen Zhao,
Zhenzhong Zhang, Xiufang Shi
PII:
S0223-5234(19)30399-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.071
EJMECH 11306
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 December 2018
Revised Date: 29 April 2019
Accepted Date: 29 April 2019
Please cite this article as: C. Wang, L. Li, D. Fu, T. Qin, Y. Ran, F. Xu, X. Du, H. Gao, S. Sun, T. Yang,
X. Zhang, J. Huo, W. Zhao, Z. Zhang, X. Shi, Discovery of chalcone-modified estradiol analogs as
antitumour agents that Inhibit tumour angiogenesis and epithelial to mesenchymal transition, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.071.
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ACCEPTED MANUSCRIPT
Discovery of Chalcone-Modified Estradiol Analogs as Antitumour
Agents that Inhibit Tumour Angiogenesis and Epithelial to
Mesenchymal Transition
a
a
a
a
a
a
b
Cong Wang , Leilei Li , Dongyang Fu
, Tiantian Qin ,Yange Ran , Feng Xu , Xinrui Du ,
a,c
a,d
e
a
a
a
Haiying Gao , Shuaijun Sun , Tengjiao Yang
, Xueyan Zhang , Junfeng Huo , Wen Zhao ,
a
a
Zhenzhong Zhang , Xiufang Shi *
a
Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province;
Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education; Key
Laboratory of Henan Province for Drug Quality and Evaluation; School of Pharmaceutical
Sciences, Zhengzhou University, Zhengzhou 450001, P. R. China
b
Department of Clinical Medicine，Zhengzhou University, 40 Daxue Road, Zhengzhou, Henan
4
c
50052, China
Department of Pharmacy, People's Hospital of Daqing, 241 Jianshe Road, Development District,
Daqing 163316, Heilongjiang, China.
d
Department of Pharmacy, Zhengzhou Central Hospital Affiliated to Zhengzhou University, 195
Tongbai Road, Zhengzhou 450053, Henan, China
e
HeNan No.3 Provincial People’s Hospital, Funiu Road, Zhongyuan District, Zhengzhou 450000,
Henan, China
*Corresponding author at: School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue
Avenue, Zhengzhou, Henan 450001, PR China. E-mail address: xfxiu@zzu.edu.cn (X.Shi),
wangcong@zzu.edu.cn (C Wang)
Abstract
Angiogenesis plays an essential role in tumourigenesis and tumour progression, and
anti-angiogenesis therapies have shown promising antitumour effects in solid tumours.
2
-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, has been regarded
as a potential antitumour agent mainly targeting angiogenesis. Here we synthesized a
novel series of chalcones based on 2-methoxyestradiol and evaluated their potential
activities against tumours. Compound 11e was demonstrated to have potent
antiangiogenic activity. Further studies showed that 11e suppressed tumour growth in
human breast cancer (MCF-7) xenograft models without obvious side effects.
Evaluation of the mechanism revealed that 11e targeted the epithelial to mesenchymal
transition (EMT) process in MCF-7 cells and inhibited HUVEC migration and then
contributed to hindrance of angiogenesis. Thus, 11e may be a promising antitumour
agent with excellent efficacy and low toxicity.
Keywords: 2-methoxyestradiol, antitumour, angiogenesis, EMT, migration
1

ACCEPTED MANUSCRIPT
Graphical abstract
Introduction
Angiogenesis, the formation of new blood vessels from the pre-existing vessels, has
been described as one of the hallmarks of cancer. It plays an essential role in tumour
[
1-3]
growth, invasion, and metastasis . Tumour angiogenesis occurs when the cancer
cells stimulate new blood vessel growth in order to bring oxygen and nutrients to a
[4, 5]
tumour . As a tumour grows in size, diffusion is no longer sufficient to oxygenate the
[
6, 7]
cells at the centre of the mass and a hypoxic environment is created . Cancer cells
secrete a variety of growth factors and cytokines that stimulate the classical angiogenic
signalling pathways, extracellular matrix remodelling, and an inflammatory response
[
8-10]
that lead to new blood vessel formation
VEGF) is known as the most important growth factor that binds to VEGFR2,
. Vascular endothelial growth Factor
(
VEGFR1, and VEGFR3 tyrosine kinase receptors on the endothelial cell surface,
2

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[
11, 12]
which promotes proliferation and migration of endothelial cells
. Some signalling
pathways also take part in angiogenic response including PI3K/Akt, Wnt, and
[
12-14]
Notch
.
Extensive efforts are directed towards antiangiogenic therapies that combat cancer
by preventing it from accessing the blood supply that is critical for tumour growth and
[
15-17]
survival
. The antiangiogenic inhibitors approved by the FDA can be divided into 3
categories: (1) monoclonal antibodies designed to target specific pro-angiogenic
factors and receptors, e.g. Bevacizumab directed against vascular endothelial growth
[
18]
factor (VEGF) used for colorectal and lung cancer ; (2) tyrosine kinase
inhibitors(TKI) to target receptors of multiple proangiogenic factors, e.g. sorafenib
[
19]
treatment for advanced renal cell carcinoma ; (3) mTOR inhibitors (mammalian
target of rapamycin inhibitors), e.g. everolimus reduced tumor volume and
[20, 21]
angiogenesis in Hepatocellular carcinoma (HCC) mouse models
. Although
antiangiogenic therapy made certain progress in prolonging the survival of cancer
patients, there are still only a few satisfactory results because of certain limitations
including insignificant clinical effects, concerns about safety, tumour recurrence and
[
22, 23]
drug resistance
inhibitors is urgently required.
ME2 is derived from the NADPH-dependent cytochrome P450 metabolism of
7β-estradiol. 2ME2 is an endogenous metabolite with antiangiogenic and antitumour
. Therefore, the development of novel, safer and multi-targeted
2
1
[
24]
effects . The antiangiogenic effect is mainly due to inhibition of cell adhesion and
migration, induction of apoptosis of vascular endothelial cell and inhibition of vascular
[
25]
endothelial growth factors and hypoxic-inducible factor (HIF) . Additionally, 2ME2
combats cancer by targeting tubulin polymerization via binding at the
colchicine-binding site and arresting cell cycle at G2/M-phase. Several phase Ⅰand Ⅱ
clinical studies have been conducted to evaluate 2ME2 under the name of Panzem®, a
formulation in a nanocrystal colloidal dispersion (NCD®), in multiple myeloma,
[
26-28]
glioblastoma ovarian and prostate cancers
. However, the short half-life and poor
[24, 29]
bioavailability of 2ME2 limited its clinical applications
. New analogs STX140
and ENMD-1198 were demonstrated to have potentially higher anticancer activities
than that of 2ME2. These compounds are being evaluated in Phase I/Phase Ⅱclinical
[
30, 31]
trials
. Research demonstrated STX140 inhibited the proliferation of MCF-7 cells
[
32]
and caused arrest in the G2/M phase of the cell cycle , and latest research shows that
STX140 can target CA Ⅸ which is regarded as a potential treatment target in
[
33]
triple-negative breast cancer (TNBC) , ENMD-1198 inhibited HIF1-a, but it also
[
34]
decreased STAT3 and NF-kB levels in breast and prostate cancer . Chalcones are
the derivatives of (E)-1,3-diphenyl-2-propene-1-one and represent a class of
compounds consisting of two aryl rings linked by an α,β-unsaturated ketone moiety.
It’s reported that chalcones possessed anticancer activity by preventing tubulin
[
35]
polymerization . Various chalcone-modified steriod derivatives were synthesized
and some of the derivatives showed potent anticancer activity against certain human
[
36]
cancer cell lines , for example the chalcone with a trimethoxyphenyl unit (1) was
considered as potential cytotoxic effect and antitubulin activity, 2-chalcone substituted
1
7-methylene estratriene (2) exhibited highly antiproliferative activity against colon
3

ACCEPTED MANUSCRIPT
HCT-116, lung NCIH-460, glioma U-251 and breast MDA-MB-435 cancer cells
[37]
(
GI =1.6 µM, 2.6 µM, 1.4 µM, 1.6 µM) , 16-E-arylidene and rostenes (3) were
50
demonstrated potential anticancer agent against CCRF-CEM, K-562, RPMI-8226 and
[
38]
SR leukemia cell lines (GI =3.94 µM, 2.61 µM, 6.90 µM, 1.79 µM) . 17-chalconyl
5
0
pregnenolones exhibited the highly activity against MCF-7 and MDA-MB-231
[
39]
(
IC =2 µM) . and estrone-16-oxime (4) showed highly cytotoxic effect against HeLa
50
[40]
cells (IC =4.41 µM) . (Fig. 1)
5
0
However, synthesis of chalcones on 2-methoxyestradiol (2ME2) has not been
studied so far. In order to further explore the SAR of 2ME2 and enrich the structure
types of the chalconyl steroid compounds, we first synthesized 2-chalconyl estradiol
analogs using 2-acetyl-estradiol-17β-acetate as the steroid precursor. The 2-acetyl
group of estradiol was used to form α,β-unsaturated carbonyl moiety with different
aldehydes according to Claisen-Schmidt condensation, which chalcone unit differs
[
26]
from the reported lies in the positions of carbonyl and double bond . We also
prepared a novel class of D-ring chalcone-modified 2-methoxyestradiol analogs and
evaluated the antiproliferative activity, antiangiogenic activity and anti-metastasis
effect on MCF-7. We found that compound 11e is a multi-target compound, which has
a good effect on inhibiting angiogenesis and anti-migration of MCF-7 in breast cancer.
Fig. 1. Colchicine, 2ME2 and 2ME2 analogs, chalcone and chalcone steroid analogs
4

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Results
Chemistry.
The synthesis route of the target 2-benzylideneacetyl substituted 17β-estradiol
compounds is depicted in Scheme 1. Chalcone moiety was synthesized from aldehyde
[
41]
and acetophenone according to Claisen-Schmidt condensation. Saxena and
[
37]
Panchapakesan reported 2-aroylvinyl-substituted 17β-estradiol synthesis from a
2
2
-formyl estradiol substrate and various acetophenones. Here,
-acetyl-estradiol-17β-acetate (6) was prepared by esterification using the Fries
[
42]
rearrangement with estradiol as the starting material based on Rao’s method . Then,
different substituted aromatic aldehydes were employed in the Claisen-Schmidt
condensation to obtain various 2-benzylideneacetyl-substituted 17β-estradiol
compounds. The overall yield of 2-chalcone estradiol (7) from estradiol was
5
2%~64%.
The 2-chalcone moiety was shown to be in the trans-configuration of the double bond
1
according to the coupling constant (J) values of 15.4-15.7 Hz. In the H NMR spectrum
of compounds, two protons of the propenone moiety were observed as two doublets at δ
1
3
7
.81~8.26 (CH=C-CO) and 7.47~7.76 ppm (=CH-CO). In the C NMR spectrum, the
ketone carbonyl groups were detected at 192.90-193.99 ppm and the vinylic carbons
C-20 and C-21 were observed as the chemical shift at 118.21-123.51 and
1
39.93-147.83 ppm, respectively, indicating the presence of α,β-unsaturated ketones.
The E-conformation in the α,β-unsaturated ketone in the 2-chalcone steroid were
further supported by X-ray crystal structure of 7e (Fig. 2a). The α,β-unsaturated ketone
system is characterized by C19=O2 and C20-C21 bond lengths of 1.243(3) and 1.319(4)
Å and a torsion angle O2-C19-C20-C21 of 4.6(5)˚, H20-C20-C21-H21 of
1
76.67˚(Mercury version 3.10). The date are consistent with the studies of other
[
41, 43]
chalcone derivatives reported previously
.
The synthesis of various 16E-arylidenoestraene derivatives has been carried out as
depicted in Scheme 2. 2ME2 was synthesized from commercially available estradiol as
[
42]
described in a literature . The C3-OH of 2ME2 was then selectively acetylated by
using acetic anhydride in isopropanol to afford 8 with 95% yield. Subsequent Jones
oxidation followed by base hydrolysis gave 2-methoxyestrone (10) at 82% overall yield.
Finally, the Claisen-Schmidt reaction of 2-methoxyestrone with the corresponding
benzaldehyde derivatives at room temperature in the alkaline medium afforded
1
8
1
6
6E-benzylidene-substituted 2-methoxyestrone derivatives 11a-l. The intermediates
-10 can be conveniently purified by crystallization.The overall yield of
6E-benzylidene 2-methoxyestrone (11) from 2-methoxyestradiol was approximately
8%. We adopted a one-pot reaction; compound 10 did not need separation and
following Claisen-Schmidt reaction was performed immediately using benzaldehyde to
obtain16E-benzylidene 2-methoxyestrone (11). The overall yield of 11 was increased
by 78%.
5

ACCEPTED MANUSCRIPT
1
The H NMR signal of the methine-H was found the downfield region at 7.36 to 7.81
ppm for the E-isomer 11 as expected due to deshielding caused by proximity to the
[
37]
1
carbonyl group in agreement with the literature . In the H NMR spectrum of
compounds 11a~11l, 16-arylidene hydrogen is a singlet at approximately 7.48 ppm, but
1
3
it becomes a doublet in 11c due to the splitting of fluorine. In the C NMR spectrum,
the expected signal for a ketone carbonyl at 208.81–209.74 and the vinylic carbons
C-16 and C-20 at 131.25–141.02 ppm indicated the presence of α,β-unsaturated
ketones. This observation has been confirmed by the X-ray diffraction analysis of 11c
(Fig. 3b). The α,β-unsaturated ketone system is characterized by C17=O3 and
C16-C20 bond lengths of 1.207(4) and 1.341 Å (5) Å, respectively, and a torsion angle
C17-C16-C20-C21 of -179.0˚ (3). These properties are very similar to those observed
[
44]
in two related arylidene derivatives of estrone
.
compound
Ar
compound
Ar
7
a
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
4-benzyloxyphenyl
7e
7f
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2,4-dichlorophenyl
7
b
7
c
7g
7h
7
d
Scheme 1 Synthesis of 2-chalconyl substituted estradiol derivatives.
Reagents and conditions: (a) Ac O, Pyridine, 60 ℃; (b) ZrCl , CH Cl , N , rt; (c)
2
4
2
2
2
aromatic aldehyde, 95% EtOH, 10% NaOH, 60 ℃.
compound
1a
1b
1c
1d
1e
1f
Ar
compound
11g
Ar
1
phenyl
4-bromophenyl
3-nitryphenyl
1
4-fluorophenyl
3,4-difluorophenyl
3-chlorophenyl
4-chlorophenyl
2,4-dichlorophenyl
11h
1
11i
4-methylsulfonatephenyl
4-methylphenyl
1
11j
1
11k
2,3-bimetyoxyphenyl
3,4,5-trimethoxyphenyl
1
11l
6

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of 16-benzylidene-substituted 2-methoxyestrone derivatives
Reagents and conditions: (a) Ac O, isopropanol, rt; (b) Jones reagents, 0 ℃ ; (c) 10%
2
NaOH, MeOH; (d) aromatic aldehyde, EtOH, 10% KOH, rt; (e) aromatic aldehyde,
EtOH, 10% KOH.
a
b
Fig. 2. X-ray crystal structure of 7e (a) and 11c (b)
Biological Testing
The effect of 2ME2 derivates on cytotoxicity in vitro and angiogenesis in vivo
All 20 analogs were screened for antitumour activities against six different cancer
cell lines including SKN-SH (Neuroblastoma), B16 (Melanoma, skin), A549 (Lung
adenocarcinoma), MGC-803 (Gastric cancer), EC109 (Esophageal carcinoma) and
SMMC-7721 (Hepatocellular carcinoma) cells in vitro, The percentage of growth
inhibition was assessed using SRB assay. The 50% growth inhibitory concentration
(
IC ) were determined and listed in Table 1. The results indicated that most of the
50
synthesized compounds showed moderate cytotoxic activity in all six cell lines.
Table 1 Cytotoxic activity of 2- or 16- chalcone substituted estradiol analogs
Drug
IC (µΜ)
50
SKN-SH
1.6±1.2
22.2±1.3
4.5±0.6
18.9±1.2
>128
B16
EC-109
4.1±0.8
26.5±1.4
5.6±0.7
18.2±1.3
>128
MGC-803 A549
SMMC-7721
8.4±0.9
2
7
7
7
7
7
7
ME2
3.4±0.6
16.5±1.2
8.9±0.9
17.4±1.2
>128
4.0±0.9
9.8±1.0
4.0±0.6
12.2±1.1
>128
1.1±0.0
a
b
c
d
e
f
5.9±0.8
5.5±0.8
8.6±0.9
52.6±1.7
15.5±1.2
10.9±1.0
0.6±0.3
26.1±1.5
16.1±1.0
>128
16.4±1.2
10.3±1.0
15.9±1.2
12.4±1.1
13.2±1.1
8.5±0.9
8.3±0.9
8.0±0.9
11.3±1.1
6.3±0.8
7

ACCEPTED MANUSCRIPT
7
7
g
15.0±1.1
22.7±1.4
22.4±1.4
27.0±1.4
8.0±1.3
15.3±1.2
13.1±1.1
17.8±1.3
16.8±1.2
14.2±1.3
10.1±1.0
17.9±1.3
16.5±1.2
2.6±0.4
14.0±1.2
19.4±1.3
22.8±1.6
19.9±1.3
16.2±1.2
24.3±1.3
14.7±1.2
15.3±1.4
17.6±1.2
23.3±1.4
18.2±1.3
22.5±1.4
10.5±1.1
13.9±1.1
15.7±1.1
12.5±1.1
17.7±1.2
13.5±1.1
10.7±1.0
12.3±1.1
14.9±1.2
15.8±1.2
14.1±1.1
12.7±1.1
10.5±1.0
20.5±1.1
18.3±1.3
18.7±1.3
12.1±1.1
13.7±1.1
19.4±1.1
25.3±1.4
15.0±1.2
22.6±1.3
23.2±1.4
15.5±1.2
39.4±1.6
21.2±1.2
5.9±0.8
44.9±1.1
14.5±1.2
17.2±1.5
8.2±1.0
5.1±1.7
16.9±1.1
4.3±1.0
1.7±0.2
0.1±0.9
8.1±1.0
37.3±1.5
20.8±1.3
8.6±0.7
2.8±0.5
h
1
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1
1
1
21.0±1.3
24.7±1.4
17.8±1.2
42.0±1.6
24.5±1.4
20.5±1.3
20.1±1.3
18.2±1.5
14.3±1.8
1
1
1
1
6.7±0.8
1
7.7±0.9
1
6.0±0.8
53.0±1.7
20.4±1.3
14.5±1.2
1
12.7±1.3
18.8±1.3
1
IC values were obtained from three independent repeats and represented as mean ± SD.
5
0
Among the 2-chalcone substituted derivatives tested, the compounds with electron
donor substituents in the chalcone B-ring had a better antiproliferative activity than the
compounds with the electron acceptor substituents. The compounds carrying a
methoxy group on the chalcone B-ring displayed higher antiproliferative activities than
compounds 7e, 7f, 7g,7h carrying an electron-withdrawing chlorine atom. Compound
7
d, carrying no substituents on the B-ring, was inactive. This is consistent with
[
37]
Panchapakesan’s studies
.
The 16-benzylidene substituted 2-methoxyestradiol derivatives had moderate
antiproliferative activity in six cancer cell lines tested. We found that
1
6-benzylidene-substituted estradiol derivatives with an electron-withdrawing halogen
in the chalcone B-ring have enhanced the antiproliferative potential against the
SMMC-7721 cells excluding 11d. However, the only electron donor-substituted
1
6-benzylidene derivative 11l containing 3,4,5-trimethoxy group had better
antiproliferative activities.
Due to haemolysis induced by high concentrations of DMSO, we chose compounds
7
b, 7d, 11b, 11d, 11e, 11g, 11h and 11k with high water solubility for further
screening to investigate the antiangiogenic effects in the chick embryo chorioallantoic
membrane model. The chicken embryo provides a unique model for investigating the
blood vessel response to antiangiogenic agents. The control chicken chorioallantoic
membranes showed well-developed neovascular zones under the filter saturated with
carrier buffer after 3 days’ culture, whereas the neovascularization in the chicken
chorioallantoic membranes treated with 11b, 11e, 11g and 11d were significantly
suppressed; however, 7b and 7d showed less significant difference. In addition, we
evaluated the toxicity of these compounds against HEK-293 cells (human embryonic
kidney cell line), the IC was assessed by MTT method and the results were shown in
5
0
Table 2, these compounds showed low toxicity on normal cell line except 7b.
The 16-benzylidene-modified 2-methoxyestradiol analog 11e significantly reduced
the number of blood vessels and vascular area resembling the antiangiogenic effect of
8

ACCEPTED MANUSCRIPT
2
ME2.
Fig 3 Screening compounds by chick chorioallantoic membrane assay. (A) Antiangiogenic activities of
different drugs were measured using CAM assay. Control (normal saline 50 µg/mL), E2 (17β-estradiol
5
0 µg/mL), DXMX (dexamethasone 50 µg/mL), 2ME2 (50 µg/mL), and the compounds (50 µg/mL).
Statistical analysis of percentage of the vascular counts (B) and the number of blood vessels (C). Data
9

ACCEPTED MANUSCRIPT
are presented as the means ± SD, n=3, *p < 0.05 and **p < 0.01 versus Control.
Table 2 Cytotoxic activity against HEK-293 cells (IC )
5
0
Compounds
7b
7d
11b
11d
IC (µΜ)
Compounds
13.1±0.7
11e
＞100
11g
87.8±5.5
11i
77.1±12.4
11k
5
0
IC (µΜ)
＞100
48.9±7.1
＞100
51.0±1.4
5
0
Compound 11e inhibits tumour growth in the human breast cancer (MCF-7)
xenograft models
On basis of our results, we chose 11e for further study due to its potent
antiangiogenic and moderate cytotoxic activity in cancer cells. 2ME2 is an
endogenous metabolite of estradiol that is regarded as a potential compound for the
treatment of breast cancer, and the MCF-7 cell is a breast cancer cell line with strong
angiogenesis and tumour growth abilities. Hence, the antitumour activities of 2ME2
and 11e were examined in the nude mice subcutaneously implanted with the MCF-7
cells. The body weights and tumor volumes were measured and recorded every other
day. As shown in Fig 4B, C, after 21-days’ treatment, the tumour weights and tumour
volumes were apparently decreased in the mice treated with 11e. Meanwhile, the body
weights of nude mice in control group and 11e group gradually increased during the
treatment period, while the body weights of nude mice in 2ME2 group significantly
lower than that in the other groups, suggesting 11e possessed a lower toxicity than
2
ME2 (Fig 5D). The results indicated that 11e may be a more effective and safer
antitumour agent than 2ME2.
1
0

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Fig 4 Compound 11e inhibited the tumour growth in vivo. The nude mice were dosed by an
intraperitoneal injection with Control (normal saline, 0.2 mL/day), 2ME2 (45 mg/kg/day), 11e-L (22.5
mg/kg/day) and 11e-H (45 mg/kg/day) for 21 days (n = 5 mice/group). (A) Nude mice and tumours at
the end of therapy (day 21). (B) Tumour weight at the end of treatment (day 21). (C) Tumour volumes
and (D) Body weights for the athymic nude mice during 21 days’ treatment. Data are presented as the
#
means ± SD, n=5/group. *p < 0.05 and **p< 0.01 versus Control group, p< 0.05 versus 2ME2 group
.
Toxicity evaluation of compound 11e in vivo and in vitro
To evaluate the toxicity of 11e, we collected main organs including heart, liver,
spleen, lung, and kidney in nude mice and used Hematoxylin and eosin staining (HE
staining) to examine the tissue damage. As shown in Fig 5A, there was obvious liver
cell edema in the 2ME2 group, while the 11e group showed lower liver cell edema,
other organs were not significantly affected. To further evaluate the liver damage, we
examined the levels of the enzymes in the serum associated with the liver function
including alanine transaminase (ALT), aspartate transaminase (AST) and alkaline
phosphatase (ALP). As shown in Fig 5B, the levels of the three markers were
increased in the 2ME2 group; however, the 11e group had relatively small changes,
the mean levels of the three enzymes of 11e group were lower than that of 2ME2
group, indicating that 11e is safer than 2ME2.
11

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Fig 5 Histopathological examination and biochemical serum analysis in xenograft models. (A) HE
staining for major organs (heart, liver, spleen, lung, kidney) in each group. The scale bars
represent 100 µm. (B) Concentrations of ALT, AST and ALP in the serum of the mice. Control
(
normal saline, 0.2 mL/day), 2ME2 (45 mg/kg/day), 11e-L (22.5 mg/kg/day) and 11e-H (45 mg/kg/day)
#
Data are presented as the mean ± SD. *p < 0.05 and **p < 0.01 versus Control group. p<.0.05
versus 2ME2 group.
Additionally, we evaluated the cytotoxicity of 11e and 2ME2 in normal human cells
and in cancer cells, including HUVEC (human umbilical vein endothelial cells),
GES-1 (human immortalized gastric epithelial cells), MCF-7 (breast cancer),
MGC-803 (gastric cancer), PC-3 (prostate cancer). As shown in Table 1, the IC of
5
0
11e is higher than that of 2ME2. As shown in Fig 6, cell viabilities were gradually
reduced when given increasingly dose of 2ME2 and 11e while at the same dose the
cytotoxicity of 11e was lower than that of 2ME2, indicating that the powerful effect of
11e on inhibition of tumour growth is not dependent on a direct cytotoxicity effect.
1
2

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Fig 6 Compound 11e had low cytotoxicity in normal and cancer cells. Cell viability curves were
measured after 11e or 2ME2 treatment for 48 h or 72 h in (A-E) HUVEC, GES-1, MCF-7,
MGC-803, PC-3 cell lines respectively. Data are presented as the mean±SD of 3 independent
tests.
Compound 11e inhibits the migration of HUVEC cells in vitro.
Compound 11e inhibited the migration of HUVEC cells in vitro. Migration of human
endothelial cells is prerequisite for angiogenesis. Thus, we evaluated whether 11e could
inhibit the migration of endothelial cells. HUVECs were treated with 0 µM (Control), 2
µM (low dose group) or 10 µM (high dose group) of 11e, and the migration ability was
1
3

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evaluated by the wound healing assay. The results showed that the migration rate of 11e
treatment group is lower than that of control group, which demonstrated 11e could
inhibit cell migration（Fig.7A B）. Similar results were observed in transwell
assay(Fig.7C D).
The VEGF/VEGFR signaling pathway plays an important role in angiogenesis.
Hence, we examined the expression of VEGFR. Western blotting analysis showed that
11e reduced the expression of VEGFR in HUVECs. FAK is a protein tyrosine kinase
associated with cell migration and the SHC adapter protein acts as a signal transporter
in the regulation of endothelial cell migration. The expression of SHC and
phosphorylated FAK was lower in 11e treatment group while the total FAK levels
were unchanged (Fig.7E, F). These data indicated that 11e suppressed blood vessel
formation which may relate to the reduction of VEGFR expression and phosphorylated
FAK and SHC.
Fig 7 The effect and mechanism of 11e on HUVEC cells migration in vitro. (A, B) Effect and statistical
analysis of 11e on HUVECs migration in wound healing assay. (C, D) Effect and statistical analysis of
11e on HUVECs migration in transwell assay. (E, F) Expression and statistical analysis of VEGFR,
FAK, P-FAK, SHC in HUVECs treated with different concentrations of 11e for 48 h. Control: treated
with no drug, 11e-L: treated with 2 µM 11e, 11e-H: treated with 10 µM 11e. All data are represented as
1
4

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the mean ± SD of 3 independent tests. *p < 0.05 and **p < 0.01 versus Control.
Compound 11e inhibits the migration of MCF-7 cells in vitro.
The cell viability was tested, as is listed in Table 3, 11e showed little toxicity both
on breast cancer cells (MCF-7, MDA-MB-231), healthy human embryonic kidney
cells (HEK-293) and human esophageal epithelial cells (Het-1A). Migration is
characteristic for endothelial and cancer cells and is essential for angiogenesis and
tumour metastasis. We evaluated whether 11e can suppress migration in cancer cells.
MCF-7 breast cancer cells were selected due to their high metastatic potential and the
effect of 11e on tumour cell migration was studied in vitro by wound healing assay. The
11e-L (2 µM) and 11e-H (10 µM) groups had lower wound healing rate of the MCF-7
cells compared with that in the control group (Fig 8A-B). As shown in Fig 8C-D, the
transwell assay showed cell number from the upper chamber to the bottom chamber
were reduced with the treatment of 11e. Compared with the control group, compound
11e significantly inhibited MCF-7 cell migration.
EMT plays an important role in the process of tumor invasion and metastasis. To
elucidate the possible mechanism of inhibition on migration, we detected the
expression of ZO-1, E-cadherin, N-cadherin, and Vimentin by the Western blotting
assay. The results indicated that 11e increased the expression of the epithelial marker
proteins ZO-1 and E-cadherin and the expression of the mesenchymal markers
N-cadherin and Vimentin were decreased, which indicated that 11e can inhibit the EMT
process in MCF-7 cells.
Table 3 Cytotoxic activity of 11e (IC )
5
0
MCF-7
MDA-MB-231
Het-1A
HEK-293
11e (µΜ)
77.0±2.7
78.6±8.3
＞100
＞100
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5

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Fig 8 The effect and mechanism of 11e on MCF-7 cells migration in vitro. (A, B) Effect and statistical
analysis of 11e on MCF-7 cell migration in wound healing assay. (C, D) Effect and statistical analysis
of 11e on MCF-7 cell migration in transwell assay. (E, F) Expression and statistical analysis of ZO-1,
E-cadherin, N-cadherin and vimentin in MCF-7 cells treated with different concentrations of 11e for 48
h. Control: treated with no drug, 11e-L: treated with 2 µM 11e, 11e-H: treated with 10 µM 11e. Data are
represented as the means ± SD of 3 independent tests. *p < 0.05 and **p < 0.01 versus Control.
Discussion
Breast cancer is the most common cancer in women and is the leading cause of
[
45]
death among women . Accumulating evidence indicates that the development of
breast cancer is predominantly associated with increased lifetime exposure to
[
46]
endogenous and exogenous hormones . 2ME2 is a potent antitumour and
[
29, 47,
antiangiogenic compound for the treatment of breast cancer and prostate cancer
4
8]
.
Some progress has made for the treatment of breast cancer with 2ME2 structural
modification, for example, isoform-specific sulfamate derivatives are responsible for
[
33]
the high specificity on Carbonic anhydrase IX (CA IX) , quinazolinone-modified
[49]
2
-methoxyestradiol showed antiproliferative activity against breast cancer cells
.
Here, we first synthesized chalconyl estradiol analogs and evaluated the
1
6

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antiproliferative activity. The results showed chalconyl substituted derivatives played
an insignificant cytotoxic effect on SKN-SH, B16, EC-109, MGC-803, A549,
SMMC-7721 cell lines. And following, we evaluated the anti-angiogenic efficacy of
these compounds using the chorioallantoic assay, the potent inhibition efficacy on
angiogenesis of 11e was observed. Our results showed that 11e decreased the
expression of VEGFR, the signal transporter SHC and thus downregulated the
activation of FAK, these changes may associate with the inhibition efficacy on
angiogenesis and cell migration of 11e. 11e had a similar efficacy as 2ME2 on
angiogenesis while the antitumour effect of 11e in vivo was superior to that of 2ME2
suggesting that 11e had other anticancer targets. Then we tested the influence of 11e
on cell migration in MCF-7 cells, and the results showed that 11e increased the
epithelial marker proteins (ZO-1 and E-cadherin), while the mesenchymal markers
proteins (N-cadherin and Vimentin) were decreased, which illustrated 11e targets
epithelial to mesenchymal transition (EMT).
EMT is the transition from an epithelial-like phenotype into a mesenchymal
phenotype adopted early during metastatic progression to permit invasion and
[
50]
migration of the metastatic cells to secondary site within the body . In carcinomas,
the difference between cancer stem cells (CSCs) and non℃ CSCs is likely to be
[
51, 52]
attributable largely to the cellular biological programme EMT
. Activation of the
EMT programme in the neoplastic cells was demonstrated to be closely associated
with the CSC state revealing multifaceted effects of the EMT programme in driving
[
53, 54]
the malignant phenotype of carcinoma cells
. CSCs are a fundamental component
of the metastatic cascade and will lead to novel therapeutic strategies against
[
55-57]
metastatic cancer
. In addition, CSCs are particularly adept in stimulating
angiogenesis to promote tumour growth and to increase overall tumour aggressiveness
[
58, 59]
before and after the therapy
, It is reported that CSCs generate proangiogenic
factors to stimulate angiogenesis and at the same time, the tumour vasculature assists
[
60]
in maintenance of CSC self-renewal . Our results showed that 11e plays potent
effects on angiogenesis and EMT, and because the two have close connection with
CSCs, we speculate that 11e may target CSCs and then exhibit a potent efficacy on
antitumor. That is what we are searching for in the following study.
Conclusion
In this study, we synthesized a series of 2ME2 analogs containing the chalcone
moiety that had antiangiogenic effects. 11e was identified as the most potent derivate
using the CAM assay. The growth of MCF-7 xenografted tumour in vivo was
significantly inhibited by 11e and there were less obvious adverse effects on major
organs in nude mice. Further mechanistic studies indicated that the antiangiogenic
effect of 11e was mediated by inhibition of migration and downregulating of VEGFR
expression in human endothelial cells. The migration of cancer cells was also
suppressed by 11e which may relate to the inhibition of EMT. Our preclinical results
suggest that 11e is a promising compound for the treatment of breast carcinoma.
1
7

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Experimental section
General chemistry
All the chemicals and solvent were purchased from commercial sources. Solvents
and reagents were dried and purified according to the methods described in the
literature. Column chromatography was carried out using silica gel (200-300 mesh);
analytical thin-layer chromatography (TLC) was performed on silica gel GF
2
54
1
13
(
Qingdao Haiyang Chemical Co, Ltd). H and C NMR spectra were recorded on a
Bruker AVANCE DPX-400 NMR spectrometer (operating at 400 MHz for H and 101
1
1
3
MHz for C). The numbering used for assignment of the NMR signals is reported in
supplement material. High resolution mass spectra (HRMS) were recorded on an
Esquire 3000 mass spectrometer by electrospray ionization (ESI) after dissolving the
compounds in methanol. Melting points were determined using a Beijing Keyi melting
point apparatus XT5A and were uncorrected. Estradiol was purchased from Zhejiang
Xianju Pharmaceutical Co., Ltd. All solvents were of analytical grade unless stated
otherwise.
General procedure for the synthesis of compound
General procedure for the synthesis of 2-chalconyl substituted estradiol
derivatives
We followed the method described by Rao et al. to prepare compounds 5 and 6. Then
2
-acetyl-estradiol-17β-acetate (6) reacted with corresponding aldehyde through the
Claisen-Schmidt reaction to provide (E)-Estradiol-2-yl-3'-arylprop-2'-en-1'-one
derivatives.
1
. (2'E)-1-Estra-1,3,5(10)-trien-3,17β-dihydroxy-2yl-3'-arylprop-2'-en-1'-one
To a stirred solution of arylaldehyde (0.65 mmol) in 95% ethanol (10 mL), an
aqueous solution of 10% NaOH (5 mL) was added, then 2-Acetyl-estradiol-17β-acetate
(0.2 g, 0.56 mmol) in 95% ethanol (5 mL) was slowly added. The reaction mixture
was further stirred at 60 ℃ for 2 h. After completion, the reaction mixture was cooled to
ºC and neutralized with 10% HCl aqueous solution. When the solution was adjusted to
6
0
a pH ∼ 5 (pH paper), an abundant yellow precipitate was formed. The precipitate was
filtered and purified by chromatography (PE: EA=3:1) to give following compounds
1
8

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1
.1.
2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(2"-methoxyphenyl)-prop-2'-e
n-1'-one (7a)
(
1
Yellow solid. 80% yield, mp. 155.6-156.6 ℃ ; H NMR (400 MHz, CDCl ) δ 12.69 (s,
3
1
7
7
2
2
3
1
H, 3-OH), 8.19 (d, J = 15.7 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.80 (s, 1H, 1-CH),
.73 (d, J = 15.7 Hz, 1H, 20-vinylic-H, =CH-CO), 7.65 (dd, J = 7.7, 1.6 Hz, 1H, 27-CH),
.44 – 7.36 (m, 1H, 25-CH), 7.02 (t, J = 7.8 Hz, 1H, 26-CH), 6.96 (d, J = 8.3 Hz, 1H,
4-CH), 6.73 (s, 1H, 4-CH), 3.93 (s, 3H, 23-OCH ), 3.75 (t, J = 7.6 Hz, 1H, 17α-CH),
3
.89 (ddd, J = 15.2, 7.4, 3.3 Hz, 2H, 6-CH ), 2.43~1.17(m, 13H, rest of the 5×CH and
2
2
1
3
×CH of steroidal ring), 0.81 (s, 3H, 18-CH3). C NMR (101 MHz, CDCl ) δ
3
94.0(CO), 161.2 (C23), 158.9 (C3), 147.0 (C5), 140.4 (C21), 132.0 (C27), 131.3
(C10), 129.4 (C25), 126.3 (C1), 123.8 (C22), 121.2 (C20), 120.8 (C26), 118.3 (C2),
1
17.7 (C24), 111.3 (C4), 81.8 (C17), 55.6 (OCH ), 50.1 (C14), 43.6 (C9), 43.2 (C13),
3
3
8.6 (C8), 36.6 (C12), 30.6 (C16), 30.0 (C6), 26.9 (C7), 26.4 (C11), 23.1 (C15), 11.1
-
(C18). ES(-) (MeOH): (m/z) calculated for C H O ([M-H] ), 431.2228; found
28
31
4
4
31.2227.
1
.2.
(2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(3"-methoxyphenyl)-prop-2'-e
n-1'-one (7b)
1
Yellow solid. 79% yield, mp. 189.6-191.6 ℃ ; H NMR (400 MHz, CDCl ) δ 12.59 (s,
3
1
7
H, 3-OH), 7.85 (d, J = 15.5 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.76 (s, 1H, 1-CH),
.58 (d, J = 15.5 Hz, 1H, 20-vinylic-H, =CH-CO), 7.36 (t, J = 7.9 Hz, 1H, 26-CH), 7.27
(d, J = 7.8 Hz, 1H, 27-CH), 7.20 – 7.14 (m, 1H, 23-CH), 7.03 – 6.94 (m, 1H, 25-CH),
6
.73 (s, 1H, 4-CH), 3.87 (s, 3H, 24-OCH ), 3.75 (t, J = 8.5 Hz, 1H, 17α-CH), 2.87 (dd,
3
J = 10.5, 4.8 Hz, 2H, 6-CH ), 2.46~1.16(m, 13H, rest of the 5×CH and 3×CH of
2
2
1
3
steroidal ring), 0.81 (s, 3H, 18-CH ). C NMR (101 MHz, CDCl ) δ 193.3 (CO), 161.3
3
3
(C24), 160.0 (C3), 147.4 (C5), 144.8 (C21), 136.2 (C22), 131.5 (C10), 130.0 (C26),
1
8
3
26.2 (C1), 121.1 (C27), 120.8 (C20), 118.1 (C2), 117.8 (C4), 116.0 (C25), 114.3 (C23),
1.8 (C17), 55.4 (OCH ), 50.1 (C14), 43.6 (C9), 43.2 (C13), 38.6 (C8), 36.6 (C12),
3
0.6 (C16), 30.0 (C6), 26.8 (C7), 26.4 (C11), 23.1 (C15), 11.1 (C18). ES(-) (MeOH):
-
(m/z) calculated for C H O ([M-H] ), 431.2228; found 431.2225.
2
8
31
4
1
.3.
(2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(4"-methoxyphenyl)-prop-2'-e
n-1'-one（7c）
1
Yellow solid. 77% yield, mp. 213.5-214.9 ℃; H NMR (400 MHz, CDCl ) δ 12.72 (s,
3
1
H, 3-OH), 7.88 (d, J = 15.4 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.78 (s, 1H, 1-H), 7.64
(d, J = 8.8 Hz, 2H, 23- and 27-CH), 7.50 (d, J = 15.4 Hz, 1H, 20-vinylic-H, =CH-CO),
6
.97 (t, J = 5.7 Hz, 2H, 24- and 26-CH), 6.73 (s, 1H, 4-CH), 3.87 (s, 3H, 25-OCH3),
1
9

ACCEPTED MANUSCRIPT
3
1
.76 (t, J = 8.4 Hz, 1H, 17α-CH), 2.88 (dd, J = 10.5, 4.8 Hz, 2H, 6-CH ), 2.46~1.16(m,
2
1
3
3H, rest of the 5×CH and 3×CH of steroidal ring)), 0.81 (s, 3H, 18-CH ). C NMR
2
3
(
101 MHz, CDCl ) δ 193.3(C19, CO), 161.9 (C25), 161.2(C3), 147.1 (C21), 144.8 (C5),
3
1
31.4 (C10), 130.5 (C23 and C27), 127.5 (C1), 126.0 (C22), 118.2 (C20), 117.9 (C2),
1
17.8 (C24 and C26), 114.5 (C4), 81.8 (C17), 55.5 (OCH ), 50.1 (C14), 43.6 (C9), 43.2
3
(
1
C13), 38.6 (C8), 36.6 (C12), 30.7 (C16), 30.0 (C6), 26.9 (C7), 26.4 (C11), 23.1 (C15),
-
1.1 (C18). ES(-) (MeOH): (m/z) calculated for C H O ([M-H] ), 431.2228; found
28
31
4
4
31.2226.
1
.4.
(
2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(4"-benzyloxyphenyl)-prop-2'
en-1'-one（7d）
-
1
Yellow solid. 75% yield, mp. 222.8-226.8 ℃; H NMR (400 MHz, CDCl ) δ 12.72 (s,
3
1
7
H, 3-OH), 7.88 (d, J = 15.4 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.78 (s, 1H, 1-CH),
.63 (d, J = 8.7 Hz, 2H, 23- and 27-CH), 7.51 (d, J = 15.4 Hz, 1H, 20-vinylic-H,
=CH-CO), 7.47~7.32(m, 5H, benzylic-5×CH), 7.04 (d, J = 8.8 Hz, 2H, 24- and 26-CH),
6
.74 (s, 1H, 4-CH), 5.13 (s, 2H, benzylic-CH ), 3.76 (t, J = 8.2 Hz, 1H,17α-CH), 2.88
2
(
dd, J = 10.7, 4.7 Hz, 2H, 6-CH ), 2.45~1.14(m, 13H, rest of the 5×CH and 3×CH of
2
2
1
3
steroidal ring), 0.81 (s, 3H, 18-CH ). C NMR (101 MHz, CDCl ) δ 193.3 (CO), 161.2
3
3
(C3), 161.0(C25), 147.1 (C21), 144.7(C5), 136.4 (benzylic-C), 131.4 (C10), 130.5
(C23) and C27), 128.7 (benzylic-m-CH), 128.2 (benzylic-p-CH), 127.7
(benzylic-o-CH), 127.5 (C1), 126.1 (C22), 118.2 (C20), 118.0 (C2), 117.8 (C24 and
C26), 115.4 (C4), 81.9 (C17), 70.2 (benzylic-CH ), 50.1 (C14), 43.6 (C9), 43.2 (C13),
2
3
8.7 (C8), 36.6 (C12), 30.7 (C16), 30.0 (C6), 26.9 (C7), 26.5 (C11), 23.1 (C15), 11.1
-
(C18). ES(-) (MeOH): (m/z) calculated for C H O ([M-H] ), 507.2541; found
34 35 4
5
07.2539.
1
.5.
(2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(2"-chlorophenyl)-prop-2'-en-
1
'-one（7e）
1
Yellow solid. 78% yield, mp. 166.0-168.2 ℃; H NMR (400 MHz, CDCl ) δ 12.49 (s,
3
1
H, 3-OH), 8.26 (d, J = 15.6 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.83 – 7.73 (m, 2H, 1-
and 24-CH), 7.58 (d, J = 15.6 Hz, 1H, 20-vinylic-H, =CH-CO), 7.50 – 7.43 (m, 1H,
2
7-CH), 7.40 – 7.32 (m, 2H, 25- and 26-CH), 6.75 (s, 1H, 4-CH), 3.75 (t, J = 8.5 Hz, 1H,
1
7α-CH), 2.88 (dd, J = 9.4, 6.3 Hz, 2H, 6-CH2), 2.44~1.14(m, 13H, rest of the 5×CH
2
1
3
and 3×CH of steroidal ring), 0.80 (s, 3H, 18-CH ). C NMR (101 MHz, CDCl ) δ
3
3
1
(
1
93.2 (CO), 161.3 (C3), 147.6 (C21), 140.6 (C5), 135.6 (C10), 133.2 (C23), 131.6
C22), 131.4 (C24), 130.4 (C25), 128.0 (C1), 127.1 (C27) ,126.3 (C26), 123.2 (C20),
18.0 (C2), 117.9 (C4), 81.8 (C17), 50.01 (C14), 43.6 (C9), 43.2 (C13), 38.6 (C8), 36.6
(
C12), 30.6 (C16), 30.0 (C6), 26.8 (C7), 26.4 (C11), 23.1 (C15), 11.1 (C18). ES(-)
-
(MeOH): (m/z) calculated for C H ClO ([M-H] ), 435.1732; found 435.1730.
2
7
28
3
2
0

ACCEPTED MANUSCRIPT
1
.6.
(2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(3"-chlorophenyl)-prop-2'-en-
1
'-one（7f）
1
Yellow solid. 80% yield, mp. 161.0-162.3 ℃; H NMR (400 MHz, CDCl ) δ 12.52 (s,
3
1
7
H, 3-OH), 7.80 (d, J = 15.5 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.74 (s, 1H, 1-CH),
.63 (s, 1H, 23-CH), 7.58 (d, J = 15.5 Hz, 1H, 20-vinylic-H, =CH-CO), 7.52 (d, J = 6.6
Hz, 1H, 27-CH)), 7.44 – 7.34 (m, 2H, 25- and 26-CH) ), 6.74 (s, 1H, 4-CH), 3.76 (t, J =
.5 Hz, 1H, 17α-CH), 2.96 – 2.81 (m, 2H, 6-CH ), 2.48~1.19(m, 13H, rest of the 5×CH
8
2
2
1
3
and 3×CH of steroidal ring), 0.81 (s, 3H, 18-CH ). C NMR (101 MHz, CDCl ) δ
3
3
1
(
1
93.0 (CO), 161.3 (C3), 147.7 (C21), 143.2 (C5), 136.6 (C22), 135.0 (C10), 131.7
C24), 130.5 (C26), 130.3 (C1), 128.1 (C25), 126.9 (C27), 126.1 (C23), 121.7 (C20),
18.0 (C2), 117.9 (C4), 81.8 (C17), 50.1 (C14), 43.6 (C9), 43.2 (C13), 38.6 (C8), 36.6
(
C12), 30.6 (C16), 30.1 (C6), 26.8 (C7), 26.5 (C11), 23.1 (C15), 11.1 (C18). ES(-)
-
(MeOH): (m/z) calculated for C H ClO ([M-H] ), 435.1732; found 435.1731.
2
7
28
3
1
.7.
(2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(4"-chlorophenyl)-prop-2'-en-
1
'-one（7g）
1
Yellow solid. 79% yield, mp. 206.8-210.0 ℃; H NMR (400 MHz, CDCl ) δ 12.55 (s,
3
1
7
H, 3-OH), 7.83 (d, J = 15.5 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.75 (s, 1H, 1-CH),
.58 (dd, J = 12.0, 7.1 Hz, 3H, 20- vinyl-H, 23- and 27-CH), 7.41 (d, J = 8.5 Hz, 2H, 24-
and 26-CH), 6.74 (s, 1H, 4-CH), 3.76 (t, J = 8.5 Hz, 1H, 17α-CH), 2.94 – 2.83 (m, 2H,
6
1
-CH ), 2.45~1.15(m, 13H, rest of the 5×CH and 3×CH of steroidal ring), 0.81 (s, 3H,
2 2
1
3
8-CH ). C NMR (101 MHz, CDCl ) δ 193.0 (CO), 161.3 (C3), 147.6 (C21), 143.4
3
3
(
C5), 136.7 (C10), 133.3 (C25), 131.6 (C22), 129.8 (C23 and C27), 129.3 (C24 and
C26), 126.1 (C1), 120.8 (C20), 118.1 (C2), 117.9 (C4), 81.8 (C17), 50.1 (C14), 43.6
C9), 43.2 (C13), 38.6 (C8), 36.6 (C12), 30.6 (C16), 30.0 (C6), 26.8 (C7), 26.4 (C11),
(
2
-
3.1 (C15), 11.1 (C18). ES(-) (MeOH): (m/z) calculated for C H ClO ([M-H] ),
27 28 3
4
35.1732; found 435.1730.
1
.8.
(2'E)-1-(3,17β-dihydroxy-1,3,5(10)-estratrienyl)-3'-(2",4"-bichlorophenyl)-prop-
2
'-en-1'-one（7h）
1
Yellow solid. 81% yield, mp. 206.8-210.0 ℃; H NMR (400 MHz, CDCl ) δ 12.43 (s,
3
1
7
H, 3-OH), 8.18 (d, J = 15.5 Hz, 1H, 21-vinylic-H, CH=C-CO), 7.73 (s, 1H, 1-CH),
.70 (d, J = 8.5 Hz, 1H, 24-CH), 7.56 (d, J = 15.5 Hz, 1H, 20-vinylic-H, =CH-CO), 7.49
(d, J = 2.1 Hz, 1H, 27-CH), 7.33 (dd, J = 8.4, 2.0 Hz, 1H, 26-CH), 6.75 (s, 1H, 4-CH),
3
2
1
.75 (dd, J = 12.8, 8.3 Hz, 1H, 17α-CH), 2.89 (dd, J = 9.5, 6.3 Hz, 2H, 6-CH2),
.42~1.14(m, 13H, rest of the 5×CH and 3×CH of steroidal ring) , 0.80 (s, 3H,
2
1
3
8-CH ). C NMR (101 MHz, CDCl ) δ 192.9 (CO), 161.3 (C3), 147.8 (C21), 139.3
3
3
2
1

ACCEPTED MANUSCRIPT
(C5), 136.7 (C23), 136.2 (C10), 131.8 (C22), 131.7 (C27), 130.2 (C24), 128.7 (C1),
1
4
27.6 (C26), 126.2 (C25), 123.5 (C20), 118.0 (C2), 117.9 (C4), 81.8 (C17), 50.1 (C14),
3.6 (C9), 43.2 (C13), 38.6 (C8), 36.6 (C12), 30.6 (C16), 30.0 (C6), 26.8 (C7), 26.4
(
C11), 23.1 (C15), 11.1 (C18). ES(-) (MeOH): (m/z) calculated for
-
C H Cl O ([M-H] ), 469.1343; found 469.1340.
27
27
2
3
General procedure for the synthesis of 11a~k
We followed the method described by Rao et al. to get compound 8, 9, and 10. Then
2
-methoxy-3-acetyloxyestrone (9) or 2-methoxyestrone (10) react with corresponding
aldehyde through the Claisen-Schmidt reaction to provide16E-benzylidene
2
-methoxyestrone derivatives.
2
. (E)-16-(arylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10) trien-17-one (11)
To a stirred solution of 10 (0.2 g, 0.67 mmol) [or 9(0.23 g, 0.67 mmol)] in ethanol (20
mL), was added an aqueous solution of 10% KOH (2 mL), then corresponding
benzaldehyde (7.50 mmol, 1.5 equiv) was added. The reaction mixture was stirred for 3
h in the room temperature and then poured into cold water. When the resulting solution
was neutralized with 10% HCl aqueous solution, a lot of yellow precipitation formed.
The precipitates were filtered under reduced pressure, washed with water, dried and
crystallized from methanol to obtain compound 11a~k.
2
.1. (E)-16-(benzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10) trien-17-one (11a)
1
Yellow solid. yield 91%. mp. 202.5-206.4 ℃ ; H NMR (400 MHz, CDCl ) δ 7.56 (d,
3
J = 7.4 Hz, 2H, 22 and 26-CH), 7.48 (s, 1H, 16-arylidene-CH), 7.43 (t, J = 7.3 Hz, 2H,
2
5
2
3
3 and 25-CH), 7.40–7.34 (m, 1H, 24-CH), 6.80 (s, 1H, 4-CH), 6.67 (s, 1H, 1-CH),
.52 (s, 1H, 3-OH), 3.87 (s, 3H, 2-OCH ), 2.99 (dd, J = 15.7, 5.6 Hz, 1H, 15b-CH),
3
.92–2.79 (m, 2H, 6-CH ), 2.60–2.49 (m, 1H, 15a-CH), 2.46–1.39 (m, 9H, rest of the
2
13
×CH and 3×CH of steroidal ring), 1.01 (s, 3H, 18-CH ). C NMR (101 MHz, CDCl )
2
3
3
δ 209.7 (CO), 144.8 (C2), 143.7 (C3), 136.0 (C16), 135.6 (C20), 133.3 (C10), 131.2
C21), 130.4 (C5), 129.3 (C22 and C26), 129.2 (C23 and C25), 128.7 (C24), 114.7 (C4),
(
1
2
08.0 (C1), 56.1 (OCH ), 48.6 (C14), 47.9 (C13), 44.4 (C9), 38.0 (C8), 31.8 (C12),
3
9.1 (C6), 28.9 (C7), 26.9 (C11), 26.3 (C15), 14.6(C18). ES(-) (MeOH): (m/z)
-
calculated for C H O ([M-H] ), 387.1966; found 387.1965.
2
6
27
3
2
.2.
(E)-16-(4'-flurobenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11b)
1
Light yellow solid. yield 93%. mp. 231.2-233.5 ℃ ; H NMR (400 MHz, CDCl ) δ
3
7
.55 (dd, J = 8.0, 5.7 Hz, 2H, 22 and 26-CH ), 7.44 (s, 1H, 16-arylidene-CH), 7.12 (t, J
=
8.5 Hz, 2H, 23 and 25-CH), 6.80 (s, 1H, 4-CH), 6.67 (s, 1H, 1-CH), 5.49 (s, 1H,
2
2

ACCEPTED MANUSCRIPT
3
2
3
-OH), 3.88 (s, 3H, 2-OCH ), 2.95 (dd, J = 15.6, 6.4 Hz, 1H, 15b-CH), 2.92–2.81 (m,
3
H, 6-CH ), 2.51 (t, J = 14.1 Hz, 1H, 15a-CH), 2.47 -1.39 (m, 9H, rest of the 3×CH and
2
2
1
3
×CH of steroidal ring), 1.01 (s, 3H, 18-CH ). C NMR (101 MHz, CDCl ) δ 209.5
3
3
(CO), 164.3 (C24), 144.8 (C2), 143.7 (C3), 135.6 (C16), 132.2 (C20), 132.2 (C10),
1
32.1(C21), 131.8 (C5), 131.2 (C22), 129.2 (C26), 116.0 (C23) , 115.8 (C25), 114.7
(
C4), 108.0 (C1), 56.1 (OCH ), 48.6 (C14), 47.8 (C13), 44.4 (C9), 38.0 (C8), 31.7
3
(
C12), 29.0 (C6), 28.8 (C7), 26.9 (C11), 26.3 (C15), 14.6 (C18). ES(-) (MeOH): (m/z)
-
calculated for C H FO ([M-H] ), 405.1871; found 405.1867.
2
6
26
3
2
.3.
(E)-16-(3',5'-difluorobenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11c)
1
Yellow solid. yield 95%. mp. 234.1-237.6 ℃ ; H NMR (400 MHz, CDCl ) δ 7.42–
3
7
.37 (m, 1H, 26-CH), 7.36 (d, J = 1.9 Hz, 1H, 16-arylidene-CH), 7.29 (dd, J = 8.4, 4.2
Hz, 1H, 25-CH), 7.21 (dt, J = 9.7, 8.2 Hz, 1H, 22-CH), 6.80 (s, 1H, 4-CH), 6.68 (s, 1H,
1
1
2
-CH), 5.47 (s, 1H, 3-OH), 3.87 (s, 3H, 2-OCH ), 2.94 (ddd, J = 15.7, 6.4, 1.5 Hz, 1H,
3
5b-CH), 2.90-2.79 (m, 2H, 6-CH ), 2.51 (ddd, J = 15.6, 12.7, 2.9 Hz, 1H, 15a-CH),
2
.45-1.38(m, 9H, rest of the 3×CH and 3×CH of steroidal ring), 1.00 (s, 3H, 18-CH ).
2
3
1
3
C NMR (101 MHz, CDCl ) δ 209.2 (CO), 144.7 (C23), 143.7 (C24), 136.8 (C2),
3
1
32.8 (C3), 131.0 (C16), 131.0 (C20), 129.2 (C10), 127.0 (C21), 118.6 (C5), 118.4
(C26), 117.8 (C25), 117.6 (C22), 114.7 (C4), 107.9 (C1), 56.1 (OCH3), 48.4 (C14),
4
7.9 (C13), 44.3 (C9), 37.9 (C8), 31.7 (C12), 28.9 (C6), 28.8 (C7), 26.9 (C11), 26.2
-
(C15), 14.6 (C18). ES(-) (MeOH): (m/z) calculated for C H F O3([M-H] ), 423.1777;
26 25 2
found 423.1775.
2
.4.
(E)-16-(3'-chlorobenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11d)
1
Light yellow solid. yield 93%. mp. 212.7-216.0 ℃ ; H NMR (400 MHz, CDCl ) δ
3
7
7
3
.53 (s, 1H, 16-arylidene-CH), 7.45–7.41 (m, 1H, 26-CH ), 7.39 (m, 1H, 25-CH), 7.37–
.32 (m, 2H, 22 and 24-CH), 6.80 (s, 1H, 4-CH), 6.68 (s, 1H, 1-CH)), 5.45 (s, 1H,
-OH), 3.87 (s, 3H, 2-OCH ), 2.96 (ddd, J = 15.9, 6.4, 1.6 Hz, 1H, 15b-CH), 2.91 – 2.79
3
(
m, 2H, 6-CH), 2.54 (ddd, J = 15.7, 12.6, 3.0 Hz, 1H, 15a-CH), 2.47 –1.40(m, 9H, rest
1
3
of the 3×CH and 3×CH of steroidal ring), 1.01 (s, 3H, 18-CH ). C NMR (101 MHz,
2
3
CDCl ) δ 209.2 (CO), 144.8 (C2), 143.7 (C3), 137.4 (C16), 134.7 (C20 and C21), 131.7
3
(
(
(
C10), 131.1 (C23), 129.9 (C5 and C25), 129.8 (C22), 129.2 (C24), 128.5 (C26), 114.7
C4), 108.0 (C1), 56.1 (OCH3), 48.5 (C14), 47.9 (C13), 44.4 (C9), 38.0 (C8), 31.7
C12), 29.0 (C6), 28.8 (C7), 26.9 (C11), 26.3 (C15), 14.6 (C18). ES(-) (MeOH): (m/z)
-
calculated for C H ClO ([M-H] ), 421.1576; found 421.1576.
2
6
26
3
2
.5.
(E)-16-(4'-chlorobenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11e)
1
Light yellow solid. yield 92%. mp. 226.8-230.4 ℃ ; H NMR (400 MHz, CDCl ) δ
3
2
3

ACCEPTED MANUSCRIPT
7
1
3
2
.49 (d, J = 8.5 Hz, 2H, 22 and 26-CH), 7.45-7.40 (m, 2H, 23 and 25-CH), 7.38 (s, 1H,
6-arylidene-CH), 6.80 (s, 1H, 4-CH), 6.67 (s, 1H, 1-CH), 5.49 (s, 1H, 3-OH), 3.87 (s,
H, 2-OCH ), 2.94 (ddd, J = 15.7, 6.3, 1.5 Hz, 1H, 15b-CH), 2.86 (dd, J = 11.3, 5.7 Hz,
3
H, 6-CH ), 2.52 (ddd, J = 15.6, 12.6, 2.9 Hz, 1H, 15a-CH), 2.43 -1.40 (m, 9H, rest of
2
1
3
the 3×CH and 3×CH of steroidal ring) 1.01 (s, 3H, 18-CH3). C NMR (101 MHz,
2
CDCl ) δ 209.4 (CO), 144.8 (C2), 143.7 (C3), 136.5 (C16), 135.2 (C20), 134.1 (C10),
3
1
1
3
31.9 (C24), 131.5 (C21), 131.1 (C25), 129.2 (C22 and C26), 129.0 (C23 and C25),
14.7 (C4), 108.0 (C1), 56.1 (OCH3), 48.5 (C14), 47.9 (C13), 44.4 (C9), 38.0 (C8),
1.7 (C12), 29.1 (C6), 28.8 (C7), 26.9 (C11), 26.3 (C15), 14.6 (C18). ES(-) (MeOH):
-
(m/z) calculated for C H ClO ([M-H] ), 421.1576; found 421.1575.
26 26 3
2
.6.
(E)-16-(2',4'-dichlorobenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11f)
1
Yellow solid. yield 95%. mp. 225.1-228.7 ℃ ; H NMR (400 MHz, CDCl ) δ 7.73 (s,
3
1
H, 23-CH), 7.50 (d, J = 8.4 Hz, 1H, 26-CH), 7.47 (s, 1H, 16-arylidene-CH), 7.29 (d, J
=
(
8.2 Hz, 1H, 25-CH ), 6.80 (s, 1H, 4-CH), 6.66 (s, 1H, 1-CH), 5.49 (s, 1H, 3-OH), 3.87
s, 3H, 2-OCH ), 2.99–2.71 (m, 3H, 15b-CH and 6-CH , 2.49 (t, J = 14.0 Hz, 1H,
3
2)
1
1
5a-CH), 2.42-1.36 (m, 9H, rest of the 3×CH2 and 3×CH of steroidal ring), 1.02 (s, 3H,
13
8-CH ). C NMR (101 MHz, CDCl ) δ 208.6 (CO), 144.8 (C2), 143.7 (C3), 138.7
3
3
(C16), 136.4 (C22), 135.3 (C20), 132.4 (C10), 131.0 (C21), 130.5 (C26), 129.9 (C5),
1
29.2 (C23), 128.3 (C25), 127.0 (C24), 114.7 (C4), 108.0 (C1), 56.1 (OCH ), 48.5
3
(
2
C14), 48.1 (C13), 44.3 (C9), 37.9 (C8), 31.7 (C12), 28.9 (C6), 28.8 (C7), 26.9 (C11),
6.2 (C15), 14.5 (C18). ES(-) (MeOH): (m/z) calculated for C H Cl O ([M-H] ),
26 25 2 3
-
4
55.1186; found 455.1186.
2
.7. (E)-16-(4'-bromobenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11g)
1
Light yellow solid. yield 92%. mp. 246.5-249.0 ℃ ; H NMR (400 MHz, CDCl ) δ
3
7
2
3
2
.55 (d, J = 8.5 Hz, 2H, 22 and 26-CH), 7.43 (s, 1H, 16-arylidene-CH), 7.42-7.38 (m,
H, 23 and 25-CH ), 6.80 (s, 1H, 4-CH), 6.67 (s, 1H, 1-CH), 5.50 (s, 1H, 3-OH), 3.87 (s,
H, 2-OCH , 2.93 (ddd, J = 15.7, 6.4, 1.5 Hz, 1H, 15b-CH), 2.86 (dd, J = 11.4, 5.6 Hz,
3
)
H, 6-CH ), 2.50 (ddd, J = 15.7, 12.6, 3.0 Hz, 1H, 15a-CH), 2.46-1.42 (m, 9H, rest of
2
1
3
the 3×CH and 3×CH of steroidal ring), 1.00 (s, 3H, 18-CH ). C NMR (101 MHz,
2
3
CDCl ) δ 209.3 (CO), 144.8 (C2), 143.7 (C3), 136.7 (C16), 134.5 (C20 and C10), 132.0
3
(C21), 131.7 (C23 and C25), 131.1 (C22 and C26), 129.2 (C5), 123.5 (C24), 114.7 (C4),
1
08.0 (C1), 56.1 (OCH ), 48.5 (C14), 47.9 (C13), 44.3 (C9), 38.0 (C8), 31.7 (C12),
3
2
9.1 (C6), 28.8 (C7), 26.9 (C11), 26.3 (C15), 14.6 (C18). ES(-) (MeOH): (m/z)
-
calculated for C H ClO ([M-H] ), 465.1071; found 465.1072.
2
6
26
3
2
4

ACCEPTED MANUSCRIPT
2
.8.
(E)-16-(3'-nitrobenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11h)
1
Light yellow solid. yield 94%. mp. 234.1-238.5 ℃ ; H NMR (400 MHz, CDCl3) δ
.41 (s, 1H, 22-CH), 8.22 (d, J = 8.0 Hz, 1H, 24-CH), 7.84 (d, J = 7.5 Hz, 1H, 26-CH),
.61 (t, J = 8.0 Hz, 1H, 25-CH), 7.49 (s, 1H, 16-arylidene-CH), 6.80 (s, 1H, 4-CH), 6.68
8
7
(
s, 1H, 1-CH), 5.49 (s, 1H, 3-OH), 3.88 (s, 3H, 2-OCH , 2.99 (dd, J = 15.8, 6.2 Hz, 1H,
3)
1
9
5b-CH), 2.95-2.82 (m, 2H, 6-CH ), 2.62 (t, J = 13.5 Hz, 1H, 15a-CH), 2.48-1.40 (m,
2
13
H, rest of the 3×CH and 3×CH of steroidal ring), 1.03 (s, 3H, 18-CH ). C NMR
2
3
(
101 MHz, CDCl ) δ 208.8 (CO), 148.5 (C23), 144.8 (C2), 143.8 (C3), 138.9 (C16),
3
1
37.3 (C21), 136.0 (C20), 130.9 (C10), 130.4 (C26), 129.7 (C5), 129.2 (C25), 124.2
(C24), 123.6 (C22), 114.7 (C4), 108.0 (C1), 56.1 (OCH3), 48.4 (C14), 48.0 (C13), 44.4
(
C9), 37.9 (C8), 31.7 (C12), 29.0 (C6), 28.8 (C7), 26.9 (C11), 26.2 (C15), 14.5 (C18).
-
ES(-) (MeOH): (m/z) calculated for C H NO ([M-H] ), 433.1889; found 433.1887.
2
6
27
5
2
.9. (E)-16-(4'-methylsulfonylbenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11i)
1
Light yellow solid. yield 93%. mp. 235.4-237.3 ℃ ; H NMR (400 MHz, CDCl ) δ
3
7
1
.99 (d, J = 8.1 Hz, 2H, 22 and 26-CH), 7.72 (d, J = 8.2 Hz, 2H, 23 and 25-CH), 7.48 (s,
H, 16-arylidene-CH), 6.80 (s, 1H, 4-CH), 6.67 (s, 1H, 1-CH), 5.50 (s, 1H, 3-OH), 3.87
(
s, 3H, 2-OCH ), 3.09 (s, 3H, 24-SO CH ), 2.97 (dd, J = 15.9, 5.9 Hz, 1H, 15b-CH),
3 2 3
2
.91-2.80 (m, 2H, 6-CH ), 2.59 (t, J = 14.3 Hz, 1H, 15a-CH), 2.48 -1.40 (m, 9H, rest of
2
1
3
the 3×CH and 3×CH of steroidal ring), 1.03 (s, 3H, 18-CH ). C NMR (101 MHz,
2
3
CDCl ) δ 208.85 (CO), 144.8 (C2), 143.8 (C3), 141.0 (C16), 140.4 (C24), 139.6 (C20),
3
1
30.9 (C10), 130.7 (C21 and C25), 129.1 (C23 and C25), 127.7 (C22 and C26), 114.7
(
(
(
C4), 108.0 (C1), 56.1 (OCH3), 48.4 (C14), 48.0 (C13), 44.5 (SO CH ), 44.3 (C9), 38.0
2
3
C8), 31.7 (C12), 29.2 (C6), 28.8 (C7), 26.9 (C11), 26.2 (C15), 14.5 (C18). ES(-)
-
MeOH): (m/z) calculated for C H O S([M-H] ), 465.1741; found 465.1740.
2
7
29
5
2
.10.
(E)-16-(4'-methylbenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11j)
1
Light yellow solid. Yield: 89%. mp. 251.2-253.7 ℃ ; H NMR (400 MHz, CDCl ) δ
3
7
.48 (s, 1H , 16-arylidene-CH), 7.46 (s, 2H, 22 and 26-CH), 7.23 (d, J = 7.9 Hz, 2H, 23
and 25-CH), 6.80 (s, 1H, 4-CH), 6.67 (s, 1H, 1-CH), 5.48 (s, 1H, 3-OH), 3.87 (s, 3H,
2
6
-OCH ), 2.98 (dd, J = 15.6, 5.2 Hz, 1H, 15b-CH), 2.85 (dt, J = 17.0, 8.3 Hz, 2H,
3
-CH2), 2.60-2.51 (m, 1H, 15a-CH), 2.39 (s, 3H, 24-CH ), 2.49 -1.39 (m, 9H, rest of
3
13
the 3×CH and 3×CH of steroidal ring)1.00 (s, 3H, 18-CH ). C NMR (101 MHz,
2
3
CDCl ) δ 209.7 (CO), 144.8 (C2), 143.7 (C3), 139.7 (C16), 135.1 (C24), 133.3 (C20),
3
1
32.9 (C10), 131.3 (C21), 130.4 (C5), 129.5 (C23 and C25), 129.3 (C22 and C26),
1
14.7 (C4), 108.0 (C1), 56.1 (OCH ), 48.7 (C14), 47.8 (C13), 44.4(C9), 38.0(C8),
3
3
1.8(C12), 29.2(C6), 28.9(C7), 26.9(C11), 26.3 (C15), 21.5 (24-CH ), 14.6 (C18). ES(-)
3
2
5

ACCEPTED MANUSCRIPT
-
(
MeOH): (m/z) calculated for C H O ([M-H] ), 401.2122; found 401.2122.
2
7
29
3
2
.11. (E)-16-(2',3'-dimethoxy benzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11k)
1
Yellow solid. Yield: 89%. mp. 185.2-188.6 ℃ ; H NMR (400 MHz, CDCl ) δ 7.81 (s,
3
1
6
3
H, 16-arylidene-CH), 7.16 (d, J = 7.2 Hz, 1H, 26-CH), 7.10 (t, J = 8.0 Hz, 1H, 25-CH),
.95 (d, J = 7.3 Hz, 1H, 24-CH), 6.80 (s, 1H, 4-CH), 6.66 (s, 1H, 1-CH), 5.49 (s, 1H,
-OH), 3.89 (s, 3H, 22-OCH ), 3.87 (s, 3H, 2-OCH ), 3.85 (s, 3H, 23-OCH ), 2.89 (dd,
3
3
3
J = 15.3, 7.1 Hz, 1H, 15b-CH), 2.85–2.77 (m, 2H, 6-CH ), 2.50 (ddd, J = 15.7, 12.8, 3.0
2
Hz, 1H, 15a-CH), 2.43-1.36(m, 9H, rest of the 3×CH and 3×CH of steroidal ring), 1.01
2
1
3
(
s, 3H, 18-CH ). C NMR (101 MHz, CDCl ) δ 209.5 (CO), 153.0 (C22), 149.1 (C23),
3
3
1
44.7 (C2), 143.7 (C3), 137.1 (C16), 131.3 (C20), 129.9 (C10), 129.3 (C5), 128.0
(C25), 123.7 (C26), 121.4 (C21), 114.7 (C24), 113.4 (C4), 108.0 (C1), 61.4 (OCH3),
5
6.1 (OCH ), 55.9 (OCH ), 48.5 (C14), 47.9 (C13), 44.3 (C9), 38.0 (C8), 31.8 (C12),
3 3
2
9.2 (C6), 28.9 (C7), 26.9 (C11), 26.3 (C15), 14.6 (C18). ES(-) (MeOH): (m/z)
-
calculated for C H O ([M-H] ), 447.2177; found 447.2176.
2
8
31
5
2
.12.
E)-16-(3',4',5'-trimethoxybenzylidene)-3-hydroxy-2-methoxy-estra-1,3,5(10)
trien-17-one (11l)
(
1
Yellow solid. Yield: 82%. mp. 191-198 ℃ ; H NMR (400 MHz, CDCl ) δ 7.40 (s, 1H,
3
1
3
3
1
1
1
6-arylidene-CH), 6.81 (s, 3H, 4, 22 and 26-CH), 6.67 (s, 1H, 1-CH), 5.48 (s, 1H,
-OH), 3.91 (s, 6H, 23 and 25-OCH ), 3.90 (s, 3H, 24-OCH ), 3.87 (s, 3H, 2-OCH ),
3
3
3
.07-2.94 (m, 1H, 15b-CH), 2.86 (m, 2H, 6-CH), 2.52 (ddd, J = 15.4, 12.6, 2.8 Hz, 1H,
5a-H), 2.46-1.40 (m, 9H, rest of the 3×CH and 3×CH of steroidal ring), 1.01 (s, 3H,
2
13
8-CH ). C NMR (101 MHz, CDCl ) δ 209.5 (CO), 153.3 (C23 and C25), 144.8 (C2),
3
3
43.7 (C3), 139.5 (C16), 135.1 (C24), 133.5 (C20), 131.1 (C10), 129.2 (C5), 114.7
C22 and C26), 108.0 (C4), 107.8 (C1), 61.0 (OCH ), 56.3 (OCH ), 56.1 (OCH ), 48.6
(
(
3
3
3
C14), 47.8 (C13), 44.4 (C9), 37.9 (C8), 31.8 (C12), 28.9 (C6), 28.8 (C7). 26.9 (C11),
-
2
6.3 (C15), 14.6 (C18). ES(-) (MeOH): (m/z) calculated for C H O ([M-H] ),
29 33 6
4
77.2283; found 477.2284.
Cell line and cell culture
The human breast cancer cell line MCF-7 was purchased from Chinese Academy of
Sciences, and the human umbilical vein endothelial cells (HUVECs) were purchased
from Jiangsu KeyGEN Biotechology Corporation. All cells were cultured in RPMI
1
640 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL
penicillin, and 100 µg/mL streptomycin. The cells were incubated in a humidified
atmosphere of 5% CO at 37 ˚C.
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Cytotoxicity assay in vitro
SRB assay
Cytotoxic activity was evaluated by colorimetric sulforhodamine B (SRB) assay.
Briefly, cancer cells in the exponential growth phase were treated with trypsin and
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diluted to 5×10 cells/mL. Then, 100 µL of the cell suspension was transferred into 96
well plates. After cells were cultured overnight, the synthesized derivatives were added
at indicated concentrations and the results were compared with the control group. After
treatment for 72 h, the cells were fixed with 10% TCA for 1 h, stained with SRB for 15
min, and dissovled with Tris-HCl. The absorbance of each hole in the 96 well plates
was measured by a Multiskan Spectrum reader. The concentration causing 50% cell
growth inhibition (IC ) was calculated and analyzed by the SPSS 15.0 software.
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MTT assay
Cytotoxicity was determined by the MTT assay. Briefly, the cells were seeded in
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6-well plates at a density of 3×10 cells per well and incubated overnight. Then, the
cells were changed to fresh medium containing various concentrations of drugs. After
8 h or 72 h, 20 µL MTT was added to each well at a final concentration of 500 µg/mL
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and cells were incubated for 4 h. Then, the precipitate was completely dissolved by 150
µL of DMSO and the absorbance was measured at 570 nm.
Chick chorioallantoic membrane (CAM) assay
Inhibition effect of angiogenesis in vivo was measured using CAM assay. Briefly,
fertilized chicken eggs were incubated at 37˚C for 6 days. Then, a small hole was
punched in the broad side of an egg and a window was carefully created through the
eggshell. The CAM of the embryo was treated with E2, 2ME2, DXMX and compounds
we synthesized. The eggs were returned to a humidified egg incubator and chicken
chorioallantoic membranes were fixed and harvested after 72h. Angiogenesis was
quantified by counting the number of blood vessel numbers and imaged the
neovascularization zones. Ten eggs were used per group to ensure the reproducibility of
the assay.
Wound healing assay
Cells were seeded in 6-well plates and then scraped off in each well using a sterile
1
00 µL pipette tip. Subsequently, cells were washed twice to remove detached cells.
Then, cells were incubated in medium containing 2% FBS. The wound gaps were
imaged using a microscope connected to a digital camera and then drugs were added.
After 48 h incubation, the wound gaps were once again imaged and the migration rates
were examined.
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Transwell assay
Cells were seeded on the top side of the polycarbonate transwell filters in the
serum-free medium. The complete serum-containing medium was used as a
chemoattractant in the bottom chamber. After 24 h, migrated cells were fixed in 4%
paraformaldehyde for 20 min, stained with crystal violet for 20 min, and imaged by a
Nikon fluorescence microscope.
Western blotting
Cells were lysed by RIPA lysis buffer containing 1% protease and phosphatase
inhibitor cocktail on ice for 30 min. The lysates were then centrifuged at 12,000 g for 10
min at 4 ºC and supernatant were collected. The total protein concentrations were
determined by a BCA Kit. The proteins were added with proper loading buffer and
boiled to denature. These proteins were resolved by 8% SDS-PAGE and transferred to
polyinylidene difluoride membranes. After blocking the nonspecific binding sites with
5
% non-fat milk, the membranes were incubated with primary antibodies overnight at 4
˚C and then incubated with the secondary antibodies for 2 h at room temperature. The
protein bands were visualized by an enhanced chemiluminescence (ECL) detection
system. The densitometric analysis were performed using Image J software.
Animals and tumour xenograft model
All animal experiments were conducted in accordance with the ethical standards and
have been approved by the Ethics Committee of Zhengzhou University. Male/Female
nude mice (aged 4-6 week, perchased from Silikejingda, Hunan) were raised in
individually ventilated cages and received adequate sterile water and food. For the
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xenograft models, MCF-7 cells (5×10 in 200 µL saline) were subcutaneously injected
into the right forelimb of nude mice. When the average tumour volume reached
3
1
00mm , the animals were randomized and assigned to the following groups: control
(nomal saline, 0.2 mL/day), 2ME2 (45 mg/kg/day), 11e-L (22.5 mg/kg/day) and 11e-H
(45 mg/kg/day) ( n=5 mice/group); corresponding drugs were dosed by an
intraperitoneal injection to each group and lasted 21 days. Body weights and tumour
volumes were measured every other day. Tumour volume was calculated using the
3
following formula: tumour volume (mm ) = length ×width ×width/2. 21 days’
treatment, the nude mice were killed and major organs were collected, fixed in 4%
buffered paraformaldehyde and paraffin-embedded for hematoxylin and eosin (HE)
staining for toxicity assessment.
Biochemical serum analysis
Toxicity and adverse effects were observed by assessing animal body weight, hepatic
serum marker and damage to major organs. Blood samples were collected. Let the
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samples sit at room temperature for 30 min, and centrifuge the blood sample at 4000
rpm for 4 min, collect the supernatant which is serum. According to the instructions to
prepare the relevant solution and add it into the sample bottle of the automatic
biochemical analyzer, set the relevant procedures to start the determination for
biochemical serum analysis of alkaline phosphatase (ALP), alanine transaminase
(ALT), and aspartate transaminase (AST).
Histological analysis
Hematoxylin and eosin (HE) staining was used to assess cell necrosis. Major organs
(heart, liver, spleen, lung and kidney) were collected, fixed in 4% buffered
paraformaldehyde and paraffin-embedded for HE staining. We prepared 5 µm tumour
sections from paraffin-embedded tumour tissue samples. The sections were
deparaffinized in xylene, rehydrated in ethanol, rinsed in distilled water, and then fixed
with 4% formaldehyde. After fixed with formaldehyde, sections were stained with
hematoxylin and eosin followed by dehydration in graded alcohol. The sections were
mounted on glass slides previously treated with poly-L-Lysine and imaged using a
microscope.
Statistical analysis
All experiments were repeated three times independently. Statistical analysis was
performed using One-Way ANOVA for in vitro assay, and individual differences
between the treatments were analyzed using the unpaired t-test by the Graphpad Prism
software (All groups were compared in pairs). All comparisons are made relative to the
untreated controls and significance of difference is indicated as *p < 0.05 and **p <
0
.01.
Acknowledgments
This work was supported by the Science and Technology Research Key Project in the
Henan province Department of Education (Project No. 18A350013 for Cong Wang).
Project supported by the Henan foundation and frontier technology research program.
the National Natural Foundation of China (Grant No. 81573364), the Science and
technology key project of Henan province (No. 172102310171), and Zhengzhou
University graduate student independent innovation project. Science and Technology
Innovation Talents of Henan Provincial Education Department (19IRTSTHN001, by
W.Z).
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