Design, synthesis and evaluation of 6-aryl-indenoisoquinolone derivatives dual targeting ERα and VEGFR-2 as anti-breast cancer agents

Article abstract of DOI:10.1016/j.ejmech.2016.04.029

The estrogen receptors have played important roles in breast cancer development and progression. Selective estrogen receptor modulators, such as Tamoxifen, have showed great benefits in the treatment and prevention of breast cancer. But the disadvantages of induction of endometrial cancer and drug resistance have limited their use. Multiple ligand which act at multiple biomolecular targets may exert favorable advantages of improved efficacy with lower incidence of side effects. In this work, we described the synthesis and evaluation of a series of 6-aryl-indenoisoquinolone derivatives as dual ERα and VEGFR-2 inhibitors. These compounds presented good ERα binding affinity and ERα antagonistic activity, as well as potent VEGFR-2 inhibitory potency. They also possessed excellent anti-proliferative activities against MCF-7, MDA-MB-231, Ishikawa and HUVEC cell lines. Further investigation of selective compound 21c showed that it was able to inhibit the activation of VEGFR-2 and the signaling transduction of Raf-1/MAPK/ERK pathway in MCF-7 cells.

Full text of DOI:10.1016/j.ejmech.2016.04.029

Accepted Manuscript
Design, synthesis and evaluation of 6-aryl-indenoisoquinolone derivatives dual
targeting ERα and VEGFR-2 as anti-breast cancer agents
Zhichao Tang, Chengzhe Wu, Tianlin Wang, Kejing Lao, Yejun Wang, Linyi Liu,
Moses Muyaba, Pei Xu, Conghui He, Guoshun Luo, Zhouyang Qian, Shaoxiong Niu,
Lijun Wang, Ying Wang, Hong Xiao, Qidong You, Hua Xiang
PII:
S0223-5234(16)30322-1
10.1016/j.ejmech.2016.04.029
EJMECH 8544
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2016
Revised Date: 10 April 2016
Accepted Date: 11 April 2016
Please cite this article as: Z. Tang, C. Wu, T. Wang, K. Lao, Y. Wang, L. Liu, M. Muyaba, P. Xu, C. He,
G. Luo, Z. Qian, S. Niu, L. Wang, Y. Wang, H. Xiao, Q. You, H. Xiang, Design, synthesis and evaluation
of 6-aryl-indenoisoquinolone derivatives dual targeting ERα and VEGFR-2 as anti-breast cancer agents,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.029.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
N
O
O
N
HO
OH
O
21c
ERα and VEGFR-2 dual inhibitor
ERα
IC50 = 7.2 ꢀM
VEGFR-2
IC50 = 0.099 ꢀM
Anti-proliferation against breast cancer
Raf-1/MAPK/ERK pathway inhibition

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of 6-aryl-indenoisoquinolone derivatives dual targeting
ERα and VEGFR-2 as anti-breast cancer agents
Zhichao Tanga,b, Chengzhe Wua,b, Tianlin Wanga,b, Kejing Laoa,b, Yejun Wanga,b, Linyi Liua,b,
Moses Muyabaa,b, Pei Xua,b, Conghui Hea,b, Guoshun Luoa,b, Zhouyang Qiana,b, Shaoxiong Niua,b,
Lijun Wangc, Ying Wangc, Hong Xiaoc, Qidong Youa,b, Hua Xianga,b,*.
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
cNanjing Brain Hospital Affiliated to Nanjing Medical University, 264 Guangzhou Road, Nanjing
210029, PR China
Corresponding author
*Tel.: +86 025 83271096; Fax: +86 025 83271096; E-mail: xianghua@cpu.edu.cn(H. Xiang).
Abstract
The estrogen receptors have played important roles in breast cancer development and
progression. Selective estrogen receptor modulators, such as Tamoxifen, have showed great
benefits in the treatment and prevention of breast cancer. But the disadvantages of induction of
endometrial cancer and drug resistance have limited their use. Multiple ligand which act at
multiple biomolecular targets may exert favorable advantages of improved efficacy with lower
incidence of side effects. In this work, we described the synthesis and evaluation of a series of
6-aryl-indenoisoquinolone derivatives as dual ERα and VEGFR-2 inhibitors. These compounds
presented good ERα binding affinity and ERα antagonistic activity, as well as potent VEGFR-2
inhibitory potency. They also possessed excellent anti-proliferative activities against MCF-7,
MDA-MB-231, Ishikawa and HUVEC cell lines. Further investigation of selective compound 21c
showed that it was able to inhibit the activation of VEGFR-2 and the signaling transduction of
Raf-1/MAPK/ERK pathway in MCF-7 cells.
Keywords: Estrogen receptor; VEGFR-2; Dual inhibitor; Breast cancer.
1. Introduction
Breast cancer (BC) is the most frequent cancer among women worldwide. It had been
estimated that 1.67million new cases of BC were diagnosed in 2012 over the world according to
WHO report [1]. The estrogen receptors ERα and ERβ which are ligand regulated transcription
factors that mediate estrogen activity in many important physiological processes play vital roles in
breast cancer development and progression [2,3]. More than 70% breast cancers are estrogen
receptor alpha (ERα) positive and estrogen dependent [4]. Endocrine therapy which aims to block
the ER transcription effect is regarded as an effective treatment. Thus ERα has provided an ideal
pharmaceutical target and a lot of ERα ligands have been developed as antagonists against ERα
positive breast cancer [5]. Selective estrogen receptor modulators (SERMs) are a special group of
ligands which act as antagonists in breast tissue but agonists in other tissues such as
cardiovascular system and bone [6,7]. Due to their special action mode, SERMs remain as
important anti-breast cancer agents with benefits in cardiovascular system and maintaining bone
density compared with aromatase inhibitors and pure anti-estrogens [8,9]. Tamoxifen is the first
1

ACCEPTED MANUSCRIPT
SERM and is currently widely used in the treatment and prevention of breast cancer with solid
efficacy documented in several randomized clinical trials [10,11]. Tamoxifen (Figure 1)
structurally contains a triphenylethylene scaffold and a basic side chain. Since then, a lot of
SERMs with various scaffolds mimicking Tamoxifen were developed for the treatment or
prevention of breast cancer [12,13]. Many alternative scaffolds for ER modulators have also been
reported,
e.g.,
the
heterocyclic
propylpyrazoletriol
(PPT),
exo-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester (OBHS)
and m-carborane-containing compound (Figure 1) [14-16].
Figure 1. Estrogen receptor ligands: (1) Tamoxifen; (2) Raloxifene; (3) PPT; (4) OBHS; (5)
m-carborane SERM.
Although SERMs have shown great benefits in treating ERα positive BC, they still remain
disadvantages. For example, long-term treatment of Tamoxifen increases the occurrence of
endometrial cancer [17]. And another common deficiency that limits the use of SERMs is intrinsic
and acquired drug resistance, in which breast tumors become refractory to endocrine therapies and
relapse [18,19]. Therefore, the project to develop a SERM with increased activity and less side
effects still draws much attention. Multiple ligand is a single chemical entity which acts at
multiple biomolecular targets. Designed multiple ligands may exert favorable advantages of
improved efficacy and lower incidence of side effects [20]. It has been an attractive way to design
multiple ligands in the development of anti-cancer agents [21]. Several studies have shown their
multiple ligands toward ER and another target (such as aromatase, HDAC and tubulin) in the
discovery of novel anti-breast cancer agents (Figure 2) [22-24].
Figure 2. Multiple ligands targeting ER.
We have previously reported a series of 2, 3-diaryl isoquinolinone derivatives as multiple
ligands of ERα and vascular endothelial growth factor receptor-2 (VEGFR-2) as anti-breast cancer
agents which showed inhibition on the both targets [25]. VEGFR-2 is a member of the receptor
2

ACCEPTED MANUSCRIPT
tyrosine kinase (RTK) family and the predominant effector of VEGF/VEGFR signaling in
promoting angiogenesis [26]. VEGFR-2 inhibitors were reported in the treatment of breast cancer
but are not sufficient as single therapy [27]. However, the combination of Tamoxifen with
Brivanib, a VEGFR-2 inhibitor, was reported not only to maximize therapeutic efficacy but also to
retard SERM resistant tumour growth [28]. The phosphorylation of VEGFR-2 will activate the
Raf-1/MAPK/ERK signaling which is very important for cell proliferation. Moreover, the
Raf-1/MAPK/ERK interplayes with ER in regulating estrogen-dependent gene expression and is
reported to be involved in the resistance of endocrine therapy [29-31]. It is supposed that a
multiple ligand of ER and VEGFR-2 may exhibite inhibition on both ER and MAPK pathways,
thus possessing enhanced activity against BC with less disadvantages.
As our ongoing interest to explore different scaffold structures as multiple ligands of ER and
VEGFR-2, we designed a series of 6-aryl-indenoisoquinolone derivatives with basic side chain
(Table 1). Herein, the synthesis and biological evaluation of the designed compounds were
described. They were evaluated for ERα binding affinity, cancer cell anti-proliferative activity,
VEGFR-2 inhibition and anti-angiogenesis activity, besides, a selective compound was further
investigated for its action mechanism and the molecular modeling was also performed to explore
the potential binding mode.
Table 1. Structures of designed compounds
R3
O
n
O
5
6
N
7
8
4
3
R1
R2
9
11
10
1
2
O
Compound
19a
R1
R2
n
2
R3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
19b
19c
19d
19e
19f
20a
20b
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
2
2
2
2
2
3
3
O
N
3

ACCEPTED MANUSCRIPT
20c
20d
20e
20f
21a
21b
21c
21d
21e
21f
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OH
3
3
3
3
2
2
2
2
2
2
O
N
OH
OH
OH
OH
OH
OH
O
OH
OH
N
OH
OH
2. Results and discussion
2.1. Chemistry.
The intermediate, 4-methoxyhomophthalic acid 12, was prepared from commercially
available 3-methoxybenzoic acid according to literature procedure in high yields [32]. The
synthesis of indenoisoquinolone is usually achieved through a key intermediate of
tetrahydroisoquinolone. The cyclodehydration of 12 in toluene and acetic anhydride gave 13. At
the same time, anisaldehyde 14 and p-aminophenol 15 were condensed in ethanol to afford imine
16. Anhydride 13 and imine 16 were then condensed in acetonitrile catalyzed by
KAl(SO4)2·12H2O to give tetrahydroisoquinoline-4-carboxylic acid 17 using reported conditions
[33]. Compound 18 was then prepared from 17 by chloridization-dehydrogenation and
intramolecular Friedel-Crafts reactions (Scheme 2). Combination of different side chains with
compound 18 afforded the target compounds 19a-f and 20a-f. Compounds 21a-f were prepared
from their corresponding 3,9-dimethoxy compounds 19a-f by demethylation with BBr3 (Scheme
3).
Scheme 1. Synthesis of compound 12. Reagents and conditions: (a) Chloral hydrate, conc. H2SO4,
rt, 12h, 83.6 %; (b) Zn, HOAc, rt, 30min; 93.6% (c) Conc. H2SO4, rt, 30min, 90%.
4

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of 18. Reagents and conditions: (d) acetic anhydride, toluene, reflux, 4h,
100%; (e) ethanol, reflux, 10h, 96.9%; (f) KAl(SO4)2·12H2O, acetonitrile, rt, 8h, 45.9%; (g) 1)
SOCl2, toluene, reflux, 2h; 2) AlCl3, CH2Cl2, rt, 10h, 86.7%.
Scheme 3. Synthesis of 19a-f, 20a-f and 21a-f. Reagents and conditions: (h) RCH2CH2Cl or
RCH2CH2CH2Cl, 20% sodium hydroxide, TBAB, THF, reflux, 4h, 37-72%; (i) BBr3, CH2Cl2, rt,
3h, 49-63%.
2.2. Biological evaluation.
2.2.1. ERα binding affinity assay.
The ERα binding affinities of synthesized compounds were initially assessed by following a
fluorescence polarization procedure. Tamoxifen was used as the positive control. Results are
shown in Table 2. The data can be analyzed in two groupings, compounds 19a-f and 20a-f, which
bearing two methoxyl group with variations in the basic side chain and compounds 21a-f with two
hydroxyl group. It’s obviously to note that compounds 21a-f possess better binding affinity than
the other two series. As is known that the hydrogen bonds formed between the two hydroxyl group
of estradiol and Glu353, Arg394 and His524 contribute a lot to keep the conformation stable
when estradiol binds to ERα [34]. The ability to form hydrogen bonds with these three key amino
acids is widely accepted to identify ERα ligands. The dihydroxyl compounds 21a-f showed about
3 fold greater binding affinity than the corresponding dimethoxyl compounds 19a-f. It could be
inferred that compounds 21a-f containing two hydroxyl groups to mimic estradiol were able to
form more hydrogen bonds with ERα than methoxyl substituted compounds 19a-f, thus presenting
5

ACCEPTED MANUSCRIPT
better binding affinities. The length of the basic side chain was taken into consideration to explore
the influence on binding affinity. In the antagonist binding mode, the terminal amino group
pointed toward and formed hydrogen bond with Asp351 to stabilize the conformation. Compounds
19a-f with two carbon atoms showed slightly better activity than 20a-f with three carbon atoms in
the side chain, which was consistent with the other known SERMs. Different amino substituents
were also investigated, among the more potent compounds 21a-f, it seemed that the bulkier group
(morpholinyl, N-methyl piperazinyl) were inferior to the others. Morpholinyl side chain
containing compound 21e showed the lowest binding affinity of 19.8 ꢀM, it was unexpected that
compound 19e and 20e which possessed two methoxyl group but contain morpholinyl exerted no
binding activity. Piperidyl, diethylamine and pyrrolidinyl were more preferable in the side chain.
21d with piperidyl as the basic group was the most potent compound with an IC50 of 3.4ꢀM which
is very close to Tamoxifen (IC50=1.0ꢀM).
Table 2 Binding affinity of synthesized compounds for ERα
compound
19a
IC50(ꢀM)
24.4
18.7
17.7
24.9
>100
23.3
39.4
39.8
40.3
1.0
compound
20d
IC50(ꢀM)
38.3
>100
35.2
7.6
19b
20e
19c
20f
19d
21a
19e
21b
6.7
19f
21c
7.2
20a
21d
3.4
20b
21e
19.8
10.6
20c
21f
Tamoxifen
2.2.2. Cancer cells anti-proliferative activity.
To evaluate the anticancer activity, all synthesized compounds were screened against human
breast cancer cell line MCF-7 (ER+), MDA-MB-231 (ER-) and human endometrial cancer cell
line Ishikawa. It is known that Tamoxifen will stimulate endometrial cell growth and induce
endometrial cancer after long term treatment, it is of concern to evaluate the influence of new
compounds targeting ER on endometrial cells. Results are summarized in Table 3. Tamoxifen was
used as the positive control in breast cancer cells .
As a global observation, most of the synthesized compounds presented significant
antiproliferative activity on these three cancer cell lines. Moreover, the active compounds
demonstrated better activity in breast cancer than in endometrial cancer cells, and most of them
were more active than Tamoxifen in the two breast cancer cells. As for ER+ and ER- breast cancer
cells, the compounds could inhibit their growth on the same degree which indicated that the
antiproliferative activity was acquired not only through ERα but a multi-target effect. Compounds
with morpholinyl group in the side chain which showed no binding affinity in ERα assay were
also not active in this test because the IC50 values of compound 19e, 20e and 21e were not
determined up to the highest concentrations tested, which indicated an ERα pathway on the
antiproliferative activity. The N-methyl piperazinyl compound 20f with three carbon atoms in the
side chain also showed no activity while compound 19f which has two carbon atoms in the side
6

ACCEPTED MANUSCRIPT
chain showed no activity only on MDA-MB-231, although compound 19f and 21f showed activity
but yet were less potent in their group. The bulkier basic group were still unfavourable in
suppressing cancer cell growth.
The most potent compound against MCF-7 was 19d with an IC50 of 0.3ꢀM, about 18 fold
more potent than Tamoxifen (5.3ꢀM), but only showed moderate activity compared with other
compounds against MDA-MB-231 and Ishikawa with the IC50 valued at 2.8ꢀM and 9.6ꢀM
respectively. It was interesting to find that dimethoxyl compounds (19a, 19b, 19c, 19d and 19f)
which were less potent in ERα binding assay manifested slightly greater potency than the
corresponding dihydroxyl compounds (21a, 21b, 21c, 21d and 21f) against MCF-7 which also
confirmed that the obtained antiproliferative activity was not only through ERα. However, this
preference was inversed in Ishikawa cells, the dihydroxyl group showed the best activity and the
most active compound 21b gave an IC50 of 4.0ꢀM. Similar to Ishikawa, the dihydroxyl group also
present better activity against MDA-MB-231 except 21a which showed a decrease compared with
19a and 20a. The most potent compound was 21c with an IC50 of 0.5ꢀM, exhibiting 27 fold
greater potency than Tamoxifen (13.9ꢀM).
Investigation on the length of the side chain revealed an unfavorite in longer side chain in
MCF-7 and a decrease in activity was observed (20a vs 19a, 20b vs 19b, 20c vs 19c and 20d vs
19d). Similar trend was seen in MDA-MB-231 (20b vs 19b, 20c vs 19c and 20d vs 19d) but no
such conclusion can be summarized based on the data of Ishikawa.
Analysis of the influence of different basic group on antiproliferative activity suggested a
preference for three medium size basic group, diethylamine, pyrrolidinyl and piperidyl (19b, 19c,
19d, 21b, 21c and 21d). For example, 19d, the most potent compound against MCF-7, was
piperidyl substituted, and 21c against MDA-MB-231 was pyrrolidinyl substituted while 21b
against Ishikawa was diethylamine substituted. Overall, compounds with shorter side chain and
the above three basic groups displayed the best activity against all of the three cancer cell lines.
Table 3 Cancer cells antiproliferative activity (IC50, ꢀM)
compound
19a
MCF-7
1.1
MDA-MB-231
1.1
Ishikawa
10.6
6.7
19b
19c
0.7
1.3
0.7
0.7
8.7
19d
19e
0.3
2.8
9.6
>100
1.1
>100
>100
0.6
>100
10.9
9.6
19f
20a
1.4
20b
20c
2.3
1.5
4.1
3.2
3.0
12.8
4.5
20d
20e
7.8
21.7
>100
>100
3.3
>100
>100
3.8
>100
>100
5.1
20f
21a
21b
21c
0.8
1.0
4.0
1.2
0.5
8.2
21d
1.1
0.8
4.3
7

ACCEPTED MANUSCRIPT
21e
21f
>100
2.5
>100
4.1
>100
6.1
Tamoxifen
5.3
13.9
6.0
2.2.3. Antiangiogenic activity evaluation.
This evaluation was carried out by assessing VEGFR-2 inhibitory activity and HUVEC
anti-proliferative activity. Results are summarized in Table 4 and Figure 1. As shown in Table 4,
six compounds manifested inhibition on VEGFR-2 (19a, 19b, 21a, 21b, 21c, 21d). The
dihydroxyl group compounds were also preferable for VEGFR-2 inhibition. The most potent
compounds 21b and 21c were equipotent to the positive control Sunitinib. The IC50 values of 21b
and 21c were 0.12ꢀM and 0.099ꢀM respectively while Sunitinib possessed an IC50 of 0.14ꢀM in
our test. These six compounds presented significant antiproliferative activity against HUVEC as
well. Compound 21c which showed best activity on VEGFR-2 was also the most potent one
against HUVEC with the IC50 of 0.8ꢀM, same with that of Sunitinib. When the dimethoxyl group
was changed into dihydroxyl group (19a vs 21a, 19b vs 21b), an increase on VEGFR-2 inhibition
by roughly ten fold was observed. It was found that medium size basic groups were superior to
others because when it changed from dimethylamine to diethylamine, the inhibition on VEGFR-2
was increased by about 3 fold (19a vs 19b, 21a vs 21b), this is especially true for 21c which
showed the greatest potency possesses an five-member ring pyrrolidinyl. As the direct effect of
VEGFR-2 inhibition, similar changes on activity against HUVEC proliferation were observed.
However, other five compounds (19f, 20a, 20b, 20c, 21f) which were inactive in VEGFR-2 assay
also showed noteworthy inhibition on HUVEC proliferation indicating another mechanism for
their acquired activity.
The inhibitory effect of the most potent compound 21c on VEGFR-2 was further determined
by assessing the phosphorylation level of VEGFR-2 in HUVEC cells using western blot. As
shown in Figure 3, 21c was able to block the phosphorylation of VEGFR-2 in a dose dependent
manner. Sunitinib was used as the positive control. At the concentration of 1ꢀM, 21c was
equipotent to Sunitinib and showed no statistically significant difference (P > 0.05).
Based on the above results, 21c showed excellent activities on the both targets and was
chosen as dual inhibitor candidate for further investigation.
Table 4 Antiangiogenic activity on VEGFR-2 and HUVEC
compound
19a
VEGFR-2
3.9
HUVEC
35.6
19b
1.03
NA
11.4
19c
>100
>100
>100
12.0
19d
NA
19e
NA
19f
NA
20a
NA
25.0
20b
NA
16.0
20c
NA
5.1
20d
NA
>100
>100
20e
NA
8

ACCEPTED MANUSCRIPT
20f
21a
NA
0.33
0.12
0.099
0.25
NA
>100
4.6
21b
1.8
21c
0.8
21d
1.3
21e
>100
1.1
21f
NA
Sunitinib
0.14
0.8
NA = not active.
Figure 3. 21c inhibits the phosphorylation of VEGFR-2 in HUVEC cells. Expression of
p-VEGFR-2 and VEGFR-2 in HUVEC cells were examined by western blots. Densitometric
analysis was performed to determine the phosphorylation rate of VEGFR-2. Values are mean ±SD
(n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control group.
2.2.4. Effect of 21c on the expression of progestrone receptor microRNA in MCF-7 cells.
To assess the pharmacological properties of the test compound on estrogen responsive genes,
we used real-time polymerase chain reaction (RT-PCR) in the ER positive MCF-7 cells to evaluate
the modulation of progesterone receptor (PgR). The progesterone receptor expression is
commonly used to assess estrogenic or antiestrogenic activity. As shown in Figure 4, presence of
10 nM estradiol (E2) was able to remarkably elevate the mRNA expression of PgR gene compared
to the vehicle control. The mRNA expression level of PgR with 10nM E2 was regard as 100%.
Tamoxifen was used as the positive control at the concentration of 1ꢀM in combination with
10nM E2 and could reduce the expression of PgR mRNA by about seventy percent. Compound
21c exhibited stronger antagonism against the expression of PgR mRNA than Tamoxifen at the
concentration of 1ꢀM in the presence of 10nM E2 and showed statistically significant difference
compared to Tamoxifen, indicating that 21c presented significantly antiestrogenic property.
9

ACCEPTED MANUSCRIPT
Figure 4. The increased mRNA expression of PgR induced by E2 was reversed by 21c in
MCF-7 cells. The mRNA expression of PgR was examined by Real-time PCR. Values are mean
##
±SD (n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. E2 group. # P < 0.05, P < 0.01,### P <
0.001.
2.2.5. 21c inhibits the MAPK signaling cascade as the result of VEGFR-2 phosphorylation
blockade in MCF-7 cells.
The Raf-1/MAPK/ERK pathway is an important cell signaling pathways and controls
multiple cellular processes, such as cell proliferation and differentiation [35]. Increased activity of
the Ras/Raf-1/MAPK pathway plays an important role in the development and progression and is
one of the hallmarks of more aggressive cancers. Over activation of Raf-1/MAPK pathway is also
involved in the endocrine resistance [31]. The inhibition of Raf-1/MAPK/ERK pathway is
supposed to enhance the effect of anti-hormonal therapy and retard endocrine resistance.
Compound 21c has shown its inhibition on VEGFR-2 in the antiagiogenesis evaluation, we made
further investigation of 21c on inhibiting VEGFR-2 activation and the Raf-1/MAPK/ERK
pathway transduction in MCF-7 cells to explore its dual-target effect. As shown in Figure 5 and
Figure 6, 21c was able to inhibit the activation of VEGFR-2 in MCF-7 cells as like in HUVEC.
We then investigated how 21c affected the Raf-1/MAPK/ERK pathway, two effectors, Raf-1 and
ERK were examined. As expected, result showed an inhibition on this pathway as their activation
was suppressed in the presence of 21c or Sunitinib. And there is no statistically significant
difference between 21c and Sunitinib at the concentration of 1ꢀM (P > 0.05). This synergetic
inhibitory effect of 21c on ERα and VEGFR-2 suggested it as an promising dual-target agent
against breast cancer.
10

ACCEPTED MANUSCRIPT
Figure 5. 21c inhibits the phosphorylation of VEGFR-2 in MCF-7 cells. (A) Expression of
p-VEGFR-2 and VEGFR-2 in MCF-7 cells were examined by western blots. (B) Densitometric
analysis was performed to determine the phosphorylation rate of VEGFR-2. Values are mean ±SD
(n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control group.
Figure 6. 21c inhibits the activity of the Raf-1/ERK pathway in MCF-7 cells. (A) Expression
of p-Raf-1, Raf-1, p-ERK1/2, and ERK1/2 were examined by western blots in MCF-7 cells. (B)
Densitometric analysis was performed to determine the phosphorylation rate of Raf-1 and
ERK1/2 . Values are mean ±SD (n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control group; #
P < 0.05, ## P < 0.01,### P < 0.001 vs. Control group.
2.3. Molecular modeling.
We performed docking studies to explore the potential binding mode of the dual targeting
compound 21c in ERα and VEGFR-2. Results are shown in Figure 7. 21c was favorably docked
into the binding site of ERα. The 9-OH of 21c interacted with Glu353 and Arg394 which mimic
the A-ring phenol of estradiol while the 3-OH interacted with His524 which mimic the 17-OH of
estradiol. The basic side chain of 21c pointed toward Asp351 to generate an antagonistic
conformation as that of 4-OHT. The binding orientation of 21c within the ligand binding domain
of VEGFR-2 showed a binding mode which was different with Sunitinib but similar to that of
diaryl urea VEGFR-2 inhibitors. The skeleton occupied the ATP region while 9-OH formed
hydrogen bonds with two amino acid residues in the hinge region, Glu915 and Cys917 which
played important roles in the interaction of VEGFR-2 and its ligands. The side chain of 21c
pointed toward a hydrophobic region which was commonly occupied by the diaryl urea fragment.
11

ACCEPTED MANUSCRIPT
Figure 7. (A) Predicted binding mode of 21c within the LBD of ERα (PDB ID: 3ERT). (B)
Binding mode of 4-OH-Tamoxifen within the LBD of ERα (PDB ID: 3ERT). (C) Predicted
binding mode of 21c within the LBD of VEGFR-2 (PDB ID: 1YWN).
3. Conclusion
To explore different scaffold structures as dual ligands of ERα and VEGFR-2, we have
designed and synthesized a series of 6-aryl-indenoisoquinolone derivatives with basic side chain.
Most of them presented good inhibitory activities toward ERα with excellent anti-proliferative
activities against three cancer cell lines, some of them showed potent VEGFR-2 inhibitory activity.
Compound 21c was found to be the most potential inhibitor on the both targets, further evaluation
revealed that 21c was able to inhibit the activation of VEGFR-2 and the signaling transduction of
Raf-1/MAPK/ERK pathway in MCF-7 cells. 21c was therefore a promising dual targeting
candidate for the development of anti-breast cancer agents. These results also suggested a new and
potential route in the discovery of drugs against breast cancer.
4. Experimental section
4.1. Chemistry
4.1.1. General.
Most chemicals and solvents were of analytical grade and, when necessary, were purified and
dried by standard methods. Reactions were monitored by thin-layer chromatography (TLC) using
precoated silica gel plates (silica gel GF/UV 254), and spots were visualized under UV light (254
nm). Melting points (uncorrected) were determined on a Mel-TEMP II melting point apparatus
and are uncorrected. Infrared (IR) spectra (KBr) were recorded on a Nicolet Impact 410
instrument (KBr pellet). 1H NMR and 13C NMR spectra were recorded with a Bruker Avance 300
MHz spectrometer at 300 K, using TMS as an internal standard. MS spectra were recorded on a
12

ACCEPTED MANUSCRIPT
Shimadzu GC–MS 2050 (ESI) or an Agilent 1946A-MSD (ESI) Mass Spectrum, or a LC/MSD
TOF HR-MS Spectrum. Column chromatography was performed with silica gel (100-200 mesh).
Chemical shifts (d) are expressed in parts per million relative to tetramethylsilane, which was used
as an internal standard, coupling constants (J) are in hertz (Hz), and the signals are designated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet.
4.1.2. General method I: SN2 reaction.
A suspension of compound 18 (0.4g, 1mmol), alkyl chlorides (2.5mmol), TBAB (0.1mmol)
in THF (5mL) was stirred at room temperature. A solution of NaOH (0.2g, 5mmol) in water (1mL)
was added. The mixture was heated to reflux for 4h under nitrogen. The reaction mixture was
poured into ice water (25mL). The precipitated product was collected by filtration, and further
purified by silica gel column chromatography to afford the final product.
4.1.3. General method II: demethylation of 6-arylindenoisoquinolone derivatives.
A solution of 6-arylindenoisoquinolone derivatives (0.4mmol) in dry CH2Cl2 (10mL) was
cooled to -30oC by cryotrap. A solution of BBr3 (2.4mmol) in dry CH2Cl2 (2.4mL) was added
dropwise under nitrogen. The mixture was gently warmed to room temperature and stirred for 3h.
The reaction mixture was quenched with H2O (50mL), then saturated sodium bicarbonate solution
was added to pH=8. The precipitate was collected by filtration, and further purified by silica gel
column chromatography to afford the demethylation product.
4.1.4. 3-Trichloromethyl-6-methoxy-1(3H)-isobenzofuranone (10). 3-Methoxylbenzonic acid (5g,
32.9mmol) was dissolved in concentrated sulfuric acid (20mL) and chloral hydrate (6.5g,
39.5mmol) was added. After reacting for 12h at room temperature, the mixture was poured onto
ice (100g), and the precipitated product was collected by filtration, washed with saturated sodium
bicarbonate solution and water to pH=7, air-dried to give crude product (9.1g) which was stirred
in ethanol (36.4ml) for 4 h, filtrated and air-dried to give pure compound 10 (7.7g, 83.6%); mp
128-130oC (ref: 132-134oC); MS (ESI, m/z): 282 [M+H]+.
4.1.5. 2-(β, β-Dichloroethenyl)-5-methoxybenzoic acid (11). Compound 10 (1g, 3.55mmol) was
firstly added in glacial acetic acid (5mL) and zinc dust (0.46g, 7.1mmol) was added in small
portions. The reaction mixture was stirred for 30 minutes at room temperature, and then heated to
reflux for 5minutes. The hot solution was immediately filtered and cooled to room temperature to
give white needle-shaped crystals. The crystals were collected by filtration and the filtrate was
poured into ice water (25mL). The precipitate was collected, air-dried and recrystallized with
acetic acid. Two parts of crystals were combined to give compound 11 (0.82g, 93.6%); mp
163-165oC (ref: 166-168oC); MS (ESI, m/z): 246 [M-H]-.
4.1.6. (2-Carboxy-4-methoxy)-phenyl acetic acid (12). Compound 11 (0.6g, 2.3mmol) was
dissolved in concentrated sulfuric acid (1.8mL). The mixture was stirred for 30 minutes at room
temperature and poured onto ice (5g). The precipitated product was filtered, washed with cold
water and purified by recrystallization from a mixed acetone/H2O solution ( acetone:H2O; 3:1) to
yield compound 12 as white solid (0.4g, 90%); mp 187-189oC; MS (ESI, m/z): 209 [M-H]-.
4.1.7. 7-Methoxyisochroman-1,3-dione (13). An mixture of compound 12 (2.0g, 11.1mmol), acetic
o
anhydride (2.1mL, 22.2mmol) and toluene (20mL) was refluxed at 120 C for 4 h. The solvent
was reduced in vacuo to give compound 13 as light yellow solid (1.82g, 100.0%); mp 136-138 oC;
MS (ESI, m/z): 193 [M+H]+.
4.1.8. 4-((4-Methoxybenzylidene)-amino)-phenol (16). A mixture of 4-aminophenol (5g,
o
45.4mmol), 4-methoxybenzaldehyde (5.6ml, 45.4mmol) in ethanol (50ml) was refluxed at 80 C
13

ACCEPTED MANUSCRIPT
for 2 h under an inert atmosphere. The reaction mixture was cooled to room temperature, and
precipitated product was collected by filtration to yield compound 16 as light yellow solid (10g,
96.9%); mp 182-184 oC; MS (ESI, m/z): 228 [M+H]+.
4.1.9.
(3S,4R)-2-(4-Hydroxyphenyl)-7-methoxy-3-(4-methoxyphenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoli
ne-4-carboxylic acid (17). Compound 13 (4.5g, 23.4mmol) was dissolved in acetonitrile (100ml),
then compound 16 (5.3g, 23.4mmol) and KAl(SO4)2·12H2O (5.54g, 11.7mmol) were added to the
solution. The mixture was stirred at room temperature for 8 h. Half of the acetonitrile was
evaporated in vacuo. The crude product was collected by filtration, washed successively with
acetonitrile and chloroform. The yellow product was stirred in water until it became a uniform
suspension, filtrated to afford light yellow solid. The pure compound 17 was prepared by stirring
in methanol to give a white solid (4.5g, 45.9%); mp 158-160 oC; 1H NMR (300MHz, DMSO-d6) δ
9.47 (s, 1H), 6.65-7.56 (m, 11H, Ar-H), 5.25 (d, J=5.7Hz, 1H), 4.83 (d, J=5.4Hz, 1H), 3.83 (s, 3H),
3.65 (s, 3H); MS (ESI, m/z): 418 [M-H]-.
4.1.10. 3,9-Dimethoxy-6-(4-hydroxyphenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (18).
Compound 17 (4.5g, 10.7mmol) was suspended in toluene (45 mL), and thionyl chloride (15.6ml,
214mmol) was added dropwise. Then the mixture was heated to reflux for 2h. The solvent was
removed in vacuo and the remaining was dissolved in dry dichloromethane (45ml). Aluminum
chloride (2.87g, 21.4mmol) was added into the solution after cooled to 0 oC by an external ice bath.
After reacting for 10 h at room temperature, the reaction mixture was poured into a solution of ice
water (200mL) and concentrated hydrochloric acid (9mL). The oil layer was separated and stirred
with ethanol (100mL). Compound 18 was given by filtration as red solid (3.7g, 86.7%); mp >300
1
oC; H NMR (300MHz, DMSO-d6) δ 5.40-8.45 (m, 10H, Ar-H), 3.86 (s, 3H), 3.74 (s, 3H); MS
(ESI, m/z): 400 [M+H]+.
4.1.11.
3,9-Dimethoxy-6-(4-(2-(dimethylamino)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-di
one (19a). Compound 19a was prepared from 18 and 2-chloro-N,N-dimethylethanamine
hydrochloride as red solid according to general method I (0.32g, 68.6%); mp 190-193 oC; IR (KBr)
1
3069, 2951, 2755, 1668, 1510, 1255, 1059, 788, 526 cm-1; H NMR (300MHz, DMSO-d6) δ
5.32-8.40 (m, 10H, Ar-H), 4.19 (t, J=5.5 Hz, 2H), 3.83 (s, 3H), 3.71 (s, 3H), 2.78 (t, 2H), 2.32 (s,
6H). 13C NMR (75MHz, DMSO-d6) δ 189.17, 162.43, 161.52, 159.12, 154.02, 136.83, 130.03,
129.77, 128.28, 126.37, 124.22, 124.10, 123.69, 123.18, 115.35, 115.12, 110.40, 108.81, 106.29,
65.78, 57.38, 55.75, 55.38, 45.26; MS (ESI, m/z): 471 [M+H]+; Anal. Calcd for C28H26N2O5: C,
71.47; H, 5.57; N, 5.95. Found: C, 71.09; H, 5.74; N, 5.64.
4.1.12.
3,9-Dimethoxy-6-(4-(2-(diethylamino)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dio
ne (19b). Compound 19b was prepared from 18 and 2-chloro-N,N-diethylethanamine
hydrochloride as red solid according to general method I (0.34g, 68.3%); mp 169-172 oC; IR (KBr)
3445, 2929, 1666, 1508, 1248, 1058, 1026,790 cm-1; 1H NMR (300MHz, CDCl3) δ 5.44-8.51 (m,
10H, Ar-H), 4.17 (t, J=6.0 Hz, 2H), 3.88 (s, 3H), 3.76 (s, 3H), 2.96 (t, J=6.0 Hz, 2H), 2.71 (q,
J=7.1 Hz, 4H), 1.13 (t, J=7.1 Hz, 6H). 13C NMR (75MHz, CDCl3) δ 189.52, 163.09, 161.26,
159.26, 157.93, 154.23, 136.84, 129.61, 128.98, 128.36, 126.51, 124.37, 124.10, 123.71, 122.86,
115.19, 114.62, 109.93, 108.23, 107.23, 66.67, 55.15, 55.00, 51.20, 47.52, 11.35; MS (ESI, m/z):
499 [M+H]+; Anal. Calcd for C30H30N2O5·0.5H2O: C, 70.98; H, 6.16; N, 5.52. Found: C, 70.53; H,
14

ACCEPTED MANUSCRIPT
6.14; N, 5.36.
4.1.13.
3,9-Dimethoxy-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-di
one (19c). Compound 19c was prepared from 18 and 1-(2-chloroethyl)pyrrolidine hydrochloride
as red solid according to general method I (0.36g, 72.6%); mp 171-174 oC; IR (KBr) 3432, 2926,
1663, 1509, 1246, 1057, 790, 600 cm-1; 1H NMR (300MHz, CDCl3) δ 5.42-8.50 (m, 10H, Ar-H),
4.24 (t, J=5.6 Hz, 2H), 3.87 (s, 3H), 3.75 (s, 3H), 3.00 (t, J=5.6 Hz, 2H), 2.70 (t, 4H), 1.86 (m,
4H). 13C NMR (75MHz, CDCl3) δ 189.53, 163.08 161.25, 159.22, 157.90, 154.20, 136.81, 129.68,
129.00, 128.37, 126.48, 124.35, 124.07, 123.69, 122.83, 115.24, 114.56, 109.96, 108.20, 107.21,
67.04, 55.15, 54.99, 54.59, 54.40, 23.02; MS (ESI, m/z): 497 [M+H]+; Anal. Calcd for
C30H28N2O5·0.4H2O: C, 71.53; H, 5.76; N, 5.56. Found: C, 71.36; H, 5.67; N, 5.53.
4.1.14.
3,9-Dimethoxy-6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dio
ne (19d). Compound 19d was prepared from 18 and 1-(2-chloroethyl)piperidine hydrochloride as
red solid according to general method I (0.33g, 64.7%); mp 98-100 oC; IR (KBr) 3437, 2943, 1663,
1509, 1246, 1057, 790, 600 cm-1; 1H NMR (300MHz,CDCl3) δ 5.43-8.49 (m, 10H, Ar-H), 4.23 (t,
J=5.9 Hz, 2H), 3.87 (s, 3H), 3.75 (s, 3H), 2.86 (t, J=5.9 Hz, 2H), 2.57 (brs, 4H), 1.65 (m, 4H),
1.49 (m, 2H). 13C NMR (75MHz, CDCl3) δ 189.50, 163.07 161.25, 159.21, 157.91, 154.19,
136.82, 129.67, 129.01, 128.34, 126.48, 124.35, 124.08, 123.67, 122.81, 115.25, 114.56, 109.93,
108.23, 107.29, 65.98, 57.43, 55.13, 54.98, 54.71, 25.42, 23.66; MS (ESI, m/z): 511 [M+H]+;
Anal. Calcd for C31H30N2O5·0.8H2O: C, 70.92; H, 6.07; N, 5.34. Found: C, 70.47; H, 6.05; N,
5.34.
4.1.15.
3,9-Dimethoxy-6-(4-(2-morpholinoethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(19e). Compound 19e was prepared from 18 and 4-(2-chloroethyl)morpholine hydrochloride as
red solid according to general method I (0.28g, 54.7%); mp 97-100 oC; IR (KBr) 3457, 2959, 1666,
1510, 1248, 1116, 1025, 572 cm-1; 1H NMR (300MHz,CDCl3) δ 5.46-8.57 (m, 10H, Ar-H), 4.25 (t,
J=5.6Hz, 2H), 3.90 (s, 3H), 3.78-3.81 (m, 7H), 2.90 (t, J=5.6Hz, 2H), 2.66 (t, J=4.3Hz, 4H). 13C
NMR (75MHz, CDCl3) δ 189.59, 163.13 161.33, 159.09, 158.01, 154.17, 136.90, 129.82, 129.05,
128.35, 126.54, 124.44, 124.14, 123.81, 122.78, 115.25, 114.65, 110.04, 108.27, 107.55, 66.43,
65.77, 57.14, 55.20, 55.05, 53.71; MS (ESI, m/z): 513 [M+H]+; HRMS (ESI) for C30H28N2O6+H
calcd 513.2020, found 513.2015.
4.1.16.
3,9-Dimethoxy-6-(4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,1
1(6H)-dione
(19f).
Compound
19f
was
prepared
from
18
and
1-(2-chloroethyl)-4-methylpiperazine hydrochloride as red solid according to general method I
o
(0.31g, 59.0%); mp 204-208 C; IR (KBr) 3477, 2934, 2791, 1664, 1509, 1305, 1245, 1058, 789,
606 cm-1; 1H NMR (300MHz,CDCl3) δ 5.41-8.46 (m, 10H, Ar-H), 4.23 (t, J=5.6Hz, 2H), 3.86 (s,
3H), 3.73 (s, 3H), 2.91 (t, J=5.6Hz, 2H), 2.69 (brs, 4H), 2.53 (brs, 4H), 2.32 (s, 3H). 13C NMR
(75MHz, CDCl3) δ 189.48, 163.06 161.23, 159.11, 157.88, 154.14, 136.78, 129.75, 129.04,
128.30, 126.45, 124.32, 124.04, 123.67, 122.78, 115.21, 114.52, 109.93, 108.18, 107.28, 65.85,
56.62, 55.13, 54.97, 54.51, 53.15, 45.54; MS (ESI,m/z): 526 [M+H]+; Anal. Calcd for C31H31N3O5:
C, 70.84; H, 5.94; N, 7.99. Found: C, 70.40; H, 5.99; N, 7.70.`
4.1.17.
3,9-
Dimethoxy
15

ACCEPTED MANUSCRIPT
-6-(4-(3-(dimethylamino)propoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(20a).
Compound 20a was prepared from 18 and 3-chloro-N,N-dimethylpropan-1-amine hydrochloride
as red solid according to general method I (0.21g, 43.4%); mp 178-180 oC; IR (KBr) 3415, 2934,
1
1665, 1407, 1247, 1057, 790, 605 cm-1; H NMR (300MHz, DMSO-d6) δ 5.34-8.42 (m, 10H,
Ar-H), 4.12 (t, J=6.3Hz, 2H), 3.84 (s, 3H), 3.72 (s, 3H), 2.46 (t, J=6.3Hz, 2H), 2.19 (s, 6H), 1.92
(m, 2H). 13C NMR (75MHz, DMSO-d6) δ 189.62, 162.43 161.54, 159.37, 157.90, 155.04, 136.84,
129.87, 129.75, 128.30, 126.39, 124.53, 124.12, 123.69, 123.195, 115.29, 115.11, 110.44, 108.84,
106.04, 66.21, 55.76, 55.57, 55.40, 45.08, 26.72; MS (ESI, m/z): 485 [M+H]+; Anal. Calcd for
C29H28N2O5: C, 71.88; H, 5.82; N, 5.78. Found: C, 71.57; H, 6.03; N, 5.54.
4.1.18.
3,9-
Dimethoxy
-6-(4-(3-(diethylamino)propoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(20b).
Compound 20b was prepared from 18 and 3-chloro-N,N-diethylpropan-1-amine hydrochloride as
o
red solid according to general method I (0.19g, 37.1%); mp 144-146 C; IR (KBr) 3412, 2934,
1
1667, 1510, 1249, 1058, 790, 604 cm-1; H NMR (300MHz,CDCl3) δ 5.42-8.51 (m, 10H, Ar-H),
4.17 (t, 2H), 3.87 (s, 3H), 3.74 (s, 3H), 2.89-2.99 (m, 6H), 2.19 (m, 2H), 1.26 (t, J=6.5Hz, 6H). 13C
NMR (75MHz, CDCl3) δ 189.56, 163.11, 161.28, 159.12, 157.93, 154.23, 136.79, 129.82, 129.10,
128.27, 126.50, 124.38, 124.06, 123.74, 122.79, 115.11, 114.48, 110.13, 108.20, 107.21, 65.54,
55.19, 55.01, 48.57, 46.14, 24.77, 9.65; MS (ESI, m/z): 513 [M+H]+; HRMS for C31H32N2O5+H
calcd 513.2384, found 513.2380.
4.1.19.
3,9-
Dimethoxy
(20c).
-6-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
Compound 20c was prepared from 18 and 1-(3-chloropropyl)pyrrolidine hydrochloride as red
o
solid according to general method I (0.21g, 41.2%); mp 200-204 C; IR (KBr) 3428, 2925, 1663,
1509, 1247, 1057, 790, 604 cm-1; 1H NMR (300MHz,CDCl3) δ 5.43-8.47 (m, 10H, Ar-H), 4.15 (t,
J=6.2Hz, 2H), 3.86 (s, 3H), 3.73 (s, 3H), 2.60-2.74 (m, 6H), 2.11 (m, 2H), 1.84 (m, 4H). 13C NMR
(75MHz, CDCl3) δ 189.51, 163.08, 161.21, 159.37, 157.86, 154.23, 136.78, 129.50, 129.10,
128.32, 126.46, 124.32, 124.04, 123.67, 122.89, 115.15, 114.49, 109.93, 108.15, 107.25, 66.26,
55.13, 54.98, 53.81, 52.63, 28.24, 22.96; MS (ESI,m/z): 511[M+H]+; Anal. Calcd for
C31H30N2O5·0.4H2O: C, 71.91; H, 5.99; N, 5.41. Found: C, 71.45; H, 5.71; N, 5.35.
4.1.20.
3,9-
Dimethoxy
-6-(4-(3-(piperidin-1-yl)propoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(20d).
Compound 20d was prepared from 18 and 1-(3-chloropropyl)piperidine hydrochloride as red solid
o
according to general method I (0.21g, 40%); mp 200-204 C; IR (KBr) 3415, 2938, 1163, 1509,
1
1246, 1058, 790, 604 cm-1; H NMR (300MHz,CDCl3) δ 5.45-8.51 (m, 10H, Ar-H), 4.14 (t,
J=6.2Hz, 2H), 3.88 (s, 3H), 3.75 (s, 3H), 2.47-2.59 (m, 6H), 2.09 (m, 2H), 1.64 (m, 4H), 1.45 (m,
2H). 13C NMR (75MHz, CDCl3) δ 189.55, 163.11, 161.26, 159.41, 157.92, 154.22, 136.84, 129.50,
129.09, 128.98, 126.50, 124.37, 124.10, 123.71, 122.83, 115.11, 114.54, 110.00, 108.22, 107.21,
66.43, 55.43, 55.16, 55.01, 54.18, 26.23, 25.40, 23.87; MS (ESI, m/z): 525 [M+H]+; HRMS for
C32H32N2O5+H calcd 525.2384, found 525.2381.
4.1.21.
3,9-
Dimethoxy
(20e)
-6-(4-(3-morpholinopropoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
.
Compound 20e was prepared from 18 and 4-(3-chloropropyl)morpholine hydrochloride as red
o
solid according to general method I (0.2g, 38.0%); mp 224-226 C; IR (KBr) 3049, 2943, 1662,
1508, 1246, 1056, 790, 603 cm-1; 1H NMR (300MHz,CDCl3) δ 5.45-8.50 (m, 10H, Ar-H) , 4.16 (t,
16

ACCEPTED MANUSCRIPT
J=6.2Hz, 2H), 3.88 (s, 3H), 3.75-3.78 (m, 7H,), 2.52-2.63 (m, 6H), 2.07 (m, 2H). 13C NMR
(75MHz, CDCl3) δ 189.52, 163.10, 161.26, 159.35, 157.93, 154.18, 136.84, 129.58, 129.02,
128.37, 126.49, 124.37, 124.09, 123.71, 122.79, 115.17, 114.57, 109.94, 108.22, 107.22, 66.49,
66.05, 55.15, 55.00, 54.91, 53.28, 25.90; MS (ESI, m/z): 527 [M+H]+; Anal. Calcd for
C31H30N2O6·1.2H2O: C, 67.92; H, 5.95; N, 5.11. Found: C, 67.46; H, 5.82; N, 4.80.
4.1.22.
3,9-
Dimethoxy
-6-(4-(3-(4-methylpiperazin-1-yl)propoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(20f). Compound 20f was prepared from 18 and 1-(3-chloropropyl)-4-methylpiperazine
hydrochloride as red solid according to general method I (0.21g, 39.0%); mp 179-182 oC; IR (KBr)
1
2959, 2800, 1666, 1510, 1252, 1056, 791, 605 cm-1; H NMR (300MHz,CDCl3) δ 5.41-8.44 (m,
10H, Ar-H) , 4.13 (t, J=6.2Hz, 2H), 3.86 (s, 3H), 3.71 (s, 3H), 2.56-2.63 (m, 10H), 2.32 (s, 3H),
2.06 (m, 2H). 13C NMR (75MHz, CDCl3) δ 189.42, 163.03, 161.19, 159.34, 157.83, 154.14,
136.77, 129.54, 129.01, 128.30, 126.42, 124.28, 124.02, 123.60, 122.77, 115.14, 114.46, 109.86,
108.15, 107.21, 66.15, 55.10, 54.95, 54.54, 52.59, 45.46, 26.20; MS (ESI, m/z): 540 [M+H]+;
HRMS for C32H33N3O5+H calcd 540.2493 , found 540.2505.
4.1.23.
3,9-Dihydroxy-6-(4-(2-(dimethylamino)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-di
one (21a). Compound 21a was prepared from 19a as red solid according to general method II
(0.1g, 56.4%); mp 186-190 oC; IR (KBr) 3073, 2922, 1650, 1509, 1246, 1056, 800 cm-1; 1H NMR
(300MHz, DMSO-d6) δ 10.25 (brs, 1H), 10.05 (s, 1H), 5.23-8.35 (m, 10H, Ar-H), 4.15 (t, J=5.3Hz,
2H), 2.67 (t, J=5.3Hz, 2H), 2.24 (s, 6H). 13C NMR (75MHz, DMSO-d6) δ 189.66, 162.47, 160.00,
159.10, 156.14, 155.03, 137.04, 130.01, 129.76, 126.83, 125.12, 124.27, 124.09, 123.77, 123.26,
116.64, 115.27, 111.71, 111.15, 106.04, 66.11, 57.59, 45.55; MS (ESI, m/z): 443[M+H]+; HRMS
for C26H22N2O5+H calcd 443.1601, found 443.1597.
4.1.24.
3,9-Dihydroxy-6-(4-(2-(diethylamino)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dio
ne (21b). Compound 21b was prepared from 19b as red solid according to general method II
o
1
(0.12g, 63.7%); mp 186-190 C; IR (KBr) 3070, 2918, 1650, 1507, 1241, 1050, 793 cm-1; H
NMR (300MHz, DMSO-d6) δ 10.09 (s, 1H), 5.24-8.36 (m, 10H, Ar-H), 4.26 (t, 2H), 3.11 (t, 2H),
2.83 (q, J=7.1Hz, 4H), 1.1 (t, J=7.1Hz, 6H). 13C NMR (75MHz, DMSO-d6) δ 189.65, 162.46,
160.00, 158.77, 156.15, 154.95, 137.02, 130.26, 129.82, 126.80, 125.10, 124.25, 124.11, 123.76,
123.21, 116.58, 115.36, 111.69, 111.17, 106.04, 65.52, 50.75, 47.07, 10.64; MS (ESI, m/z): 471
[M+H]+; HRMS for C28H26N2O5+H calcd 471.1914 , found 471.1917.
4.1.25.
3,9-Dihydroxy-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-di
one (21c). Compound 21c was prepared from 19c as red solid according to general method II
o
1
(0.13g, 64.0%); mp 186-190 C; IR (KBr) 3070, 2920, 1645, 1502, 1241, 1051, 795 cm-1; H
NMR (300MHz, DMSO-d6) δ 10.23 (brs, 2H), 5.25-8.37 (m, 10H, Ar-H), 4.19 (t, J=5.4Hz, 2H),
2.86 (t, J=5.4Hz, 2H), 2.57 (brs, 4H), 1.71 (brs, 4H). 13C NMR (75MHz, DMSO-d6) δ 189.69,
162.47, 160.15, 159.08, 156.19, 155.02, 137.04, 130.01, 129.76, 126.82, 125.10, 124.25, 124.07,
123.77, 123.25, 116.64, 115.24, 111.70, 111.19, 105.99, 67.04, 54.21, 54.02, 23.11; MS (ESI,
m/z): 469 [M+H]+; HRMS for C28H26N2O5+H calcd 469.2122, found 469.2117.
4.1.26.
3,9-Dihydroxy-6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dio
17

ACCEPTED MANUSCRIPT
ne (21d). Compound 21d was prepared from 19d as red solid according to general method II
o
1
(95mg, 49.2%); mp 186-190 C; IR (KBr) 3075, 2925, 1650, 1507, 1248, 1053, 801 cm-1; H
NMR (300MHz, DMSO-d6) δ 10.09 (brs, 1H), 5.26-8.36 (m, 10H, Ar-H), 4.20 (t, J=5.4Hz, 2H),
2.74 (t, J=5.4Hz, 2H), 1.35-1.53 (m, 6H). 13C NMR (75MHz, DMSO-d6) δ 189.63, 162.46, 160.00,
159.09, 156.14, 154.94, 137.02, 130.03, 129.75, 126.80, 125.12, 124.24, 124.08, 123.72, 123.22,
116.60, 115.31, 111.70, 111.14, 106.05, 65.84, 57.20, 54.36, 25.43, 23.79; MS (ESI, m/z): 483
[M+H]+; HRMS for C29H26N2O5+H calcd 483.1914 , found 483.1917.
4.1.27.
3,9-Dihydroxy-6-(4-(2-morpholinoethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(21e). Compound 21e was prepared from 19e as red solid according to general method II (0.11g,
58.1%); mp 186-190 oC; 1H NMR (300MHz, DMSO-d6) δ 10.29 (s, 1H), 10.06 (s, 1H), 5.26-8.37
(m, 10H, Ar-H), 4.22 (t, J=5.7Hz, 2H), 3.61 (t, J=4.5Hz, 2H), 2.76 (t, J=5.7Hz, 2H), 2.53 (brs,
4H). 13C NMR (75MHz, DMSO-d6) δ 189.65, 162.47, 159.98, 159.07, 156.14, 154.98, 137.03,
130.05, 129.76, 126.82, 125.12, 124.26, 124.09, 123.74, 123.24, 116.62, 115.31, 111.70, 111.15,
106.03, 66.14, 65.66, 56.94, 53.63; MS (ESI, m/z): 485 [M+H]+; HRMS for C29H27N3O5+H calcd
485.1912, found 485.1916.
4.1.28.
3,9-Dihydroxy-6-(4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-5H-indeno[1,2-c]isoquinoline-5,11
(6H)-dione (21f). Compound 21f was prepared from 19f as red solid according to general method
o
1
II (0.12g, 60.2%); mp 186-190 C; H NMR (300MHz, DMSO-d6) δ 5.24-8.36 (m, 10H, Ar-H),
4.19 (t, J=5.4Hz, 2H), 2.74 (t, J=5.4Hz, 2H), 2.39-2.50 (brs, 8H), 2.19 (s, 3H). 13C NMR (75MHz,
DMSO-d6) δ 189.66, 162.47, 160.02, 159.07, 156.14, 154.98, 137.00, 130.03, 129.75, 126.80,
125.11, 124.24, 124.09, 123.75, 123.24, 116.63, 115.30, 111.68, 111.16, 106.02, 65.85, 56.45,
54.52, 52.82, 45.50; MS (ESI, m/z): 498 [M+H]+; HRMS for C29H27N3O5+H calcd 498.2023,
found 498.2034.
4.2. Biological evaluation.
4.2.1. ERα Binding Affinity Assay.
The recombinant ERα (New Durg Screening Center, China Pharmaceutical University,
Nanjing, China) and the fluorescent estrogen ligands (self-made) were removed from the -80oC
freezer and thawed on ice for 1h prior to use. The fluorescent estrogen ligand was added to the
ERα and screening buffer (ES2 Screening Buffer, Invitrogen, USA) was added to make the final
concentration 9nM for fluorescent estrogen and 30nM for ERα. Test compounds were accurately
weighed and dissolved in DMSO, screening buffer was added to dilute to required concentration.
Test compound (1ꢀL) was added to 49ꢀL screening buffer in each well (384-well microplate,
Corning, USA). To this 50ꢀL of the fluorescent estrogen/ER complex was added to make up a
final volume of 100ꢀL. A positive control contained 50ꢀL estradiol buffer (1nM) and 50ꢀL
fluorescent estrogen/ER complex. A negative control contained 50ꢀL screening buffer and 50ꢀL
fluorescent estrogen/ER complex. The negative control was used to determine the polarization
value when no competitor was present (theoretical maximum polarization). The microplate was
incubated in the dark at room temperature for 2 h and shaken on a plate shaker. The polarization
values were read on a Safire microplate reader and used to calculate the IC50 values.
4.2.2. MTT Assay for Anti-proliferative Activities.
Cells were cultured in RPMI1640 medium (containing 10% (v/v) FBS, 100U/mL Penicillin
and 100ꢀg/mL Streptomycin) in a 5% CO2-humidified atmosphere at 37 oC. Cells were trypsinized
18

ACCEPTED MANUSCRIPT
and seeded at a density of 1×105/mL into a 96-well plate (100ꢀL/well) and incubated at 37oC, 5%
CO2 atmosphere for 24 h. After this time they were treated with 100ꢀL/well medium containing
test compounds which had been pre-prepared to provide the concentration range of 1×10-4mol/L,
1×10-5mol/L, 1×10-6mol/L and 1×10-7mol/L, and re-incubated for a further 48h. Control wells
were added the equivalent volume of medium containing 1% (v/v) DMSO. 20ꢀL MTT (5mg/mL)
was added and cells continued to incubate in darkness at 37oC for 4h. The culture medium was
then removed carefully and 150ꢀL DMSO was added. The cells were maintained at room
temperature in darkness for 20 min to ensure thorough color diffusion before reading the
absorbance. The absorbance values were read at 490nm for determination of IC50 values.
4.2.3. VEGFR-2 Kinase Inhibitory Assay.
VEGFR-2 kinase assay was conducted using homogeneous time resolved fluorescence
(HTRF) assay. The general procedures were as following: VEGFR-2 kinase (Invitrogen, USA),
substrates, ATP and test compounds were mixed and incubated in a final buffer with the total
volume of 10ꢀL in 384-well microplate. Wells containing the substrate and the kinase without
compound were used as total reaction control. The assay plate was incubated at 30oC in the dark
for 1 h. Detection was performed with HTRF module of Beckman Coulter detection platform to
get the fluorescence values which were further used to calculate the IC50 values.
4.2.4. Western Blots.
Cells with different treatments for 24h were washed twice with PBS, then collected and lysed
in lysis buffer (100 mM of Tris–Cl, pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 200 mM of
β-mercaptoethanol, 1mM of PMSF, 0.1 mM NaF and DTT) for 1 h on the ice. The lysates were
then subjected to centrifugation (13,000 rpm) at 4 °C for 20 min. Protein concentration in the
supernatants was detected by BCA protein assay (Thermo, Waltham, MA). Then equal amount of
protein was separated with 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF)
membranes (Millipore, Bedford, MA) using a semi-dry transfer system (Bio-rad, Hercules, CA).
Proteins were detected using specific antibodies overnight at 4 °C followed by HRP-conjugated
secondary antibodies for 1 h at 37 °C. All of the antibodies were diluted in PBST containing 1%
BSA. Enhanced chemiluminescent reagents (Beyotime, Jiangsu, China) were used to detect the
HRP on the immunoblots, and the visualized bands were captured by film. The bands were
quantified by Quantity One software (Vision 4.62, Bio-rad, Hercules, CA), and the relative protein
level were normalized to β-actin.
4.2.5. Real-Time Polymerase Chain Reaction (RT-PCR).
RNA samples were reverse transcribed to cDNA and the PCR reactions were performed
using TaKaRa SYBR Green Master Mix (Code. no. 638320) carried out in StepOnePlusTM
Real-Time PCR instrument (4376600, Life Technologies). The program for amplification was 1
cycle of 95°C for 2 min followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 95°C for 10 s.
The PCR results were normalized to GAPDH expression and were quantified by the ꢁꢁCT
method.
4.3. Molecular Modeling.
The molecular modeling was performed with Discovery Studio.2.5/CDOCK protocol
(Accelrys Software Inc.). The crystal structures of ERα complexed with 4-hydroxytamoxifen
(PDB code: 3ERT) and VEGFR-2 (PDB code: 1YWN) were downloaded from Protein Data Bank.
Compound 21c was drown and optimized using Hyperchem v7.0. The protein and ligand were
optimized and charged with CHARMm force field to perform docking. Up to 30 conformations
19

ACCEPTED MANUSCRIPT
were retained, and binding modes presented graphically are representative of the highest-scored
conformations.
Supporting Information
1H NMR and 13C NMR spectrum for compounds 19a-21f.
Acknowledgements
This work was supported by grants from the NSFC (81373279), the Twelfth Five-Year Plan Major
Project of Candidate Drugs (Ministry of National Science and Technology, 2012ZX09103101048),
the Jiangsu Province Science and Technology Support Program of Social Development Projects
(BE2012745) and the Innovation Project of Jiangsu Province.
Abbreviations
BC, breast cancer; ER, estrogen receptor; SERMs, selective estrogen receptor modulators; PPT,
propylpyrazoletriol;
OBHS,
exo-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester; AI,
aromatase inhibitor; HDAC, histone deacetylases; VEGF, Vascular endothelial growth factor;
VEGFR-2, Vascular endothelial growth factor receptor-2; RTK, receptor tyrosine kinase; MAPK,
mitogen-activated protein kinase; ERK, extracellular regulated kinases; HUVEC, human umbilical
vein endothelial cell; RT-PCR, real-time polymerase chain reaction; PgR, progesterone receptor;
E2, estradiol.
References
[1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global Cancer Statistics,
2012, CA-Cancer J. Clin. 65 (2015) 87-108.
[2] P. Ascenzi, A. Bocedi, M. Marino, Structure-function relationship of estrogen receptor alpha and
beta: impact on human health, Mol. Aspects Med. 27 (2006) 299-402.
[3] S. Sommer, S.A.W. Fuqua, Estrogen receptor and breast cancer, Semin. Cancer Biol. 11 (2001)
339-352.
[4] W.F. Anderson, N. Chatterjee, W.B. Ershler, O.W. Brawley, Estrogen receptor breast cancer
phenotypes in the surveillance, epidemiology, and end results database, Breast Cancer Res. Treat.
76 (2002) 27-36.
[5] E.A. Ariazi, J.L. Ariazi, F. Cordera, V.C. Jordan, Estrogen receptors as therapeutic targets in breast
cancer, Curr. Top. Med. Chem. 6 (2006) 181-202.
[6] V.C. Jordan, Antiestrogens and selective estrogen receptor modulators as multifunctional medicines.
1. Receptor interactions, J. Med. Chem. 46 (2003) 883-908.
[7] V.C. Jordan, Antiestrogens and selective estrogen receptor modulators as multifunctional medicines.
2. Clinical considerations and new agents, J. Med. Chem. 46 (2003) 1081-1111.
[8] B.S. Katzenellenbogen, J.A. Katzenellenbogen, Biomedicine - Defining the "S" in SERMs, Science
295 (2002) 2380-2381.
[9] V.C. Jordan, Chemoprevention of breast cancer with selective oestrogen-receptor modulators, Nat.
Rev. Cancer 7 (2007) 46-53.
[10] M. Clarke, R. Collins, C. Davies, J. Godwin, R. Gray, R. Peto, E.B.C.T.C. Grp, Tamoxifen for
early breast cancer: An overview of the randomised trials, Lancet 351 (1998) 1451-1467.
[11] B. Fisher, J.P. Costantino, D.L. Wickerham, C.K. Redmond, M. Kavanah, W.M. Cronin, V. Vogel,
A. Robidoux, N. Dimitrov, J. Atkins, M. Daly, S. Wieand, E. Tan-Chiu, L. Ford, N. Wolmark,
Tamoxifen for prevention of breast cancer: Report of the National Surgical Adjuvant Breast and
20

ACCEPTED MANUSCRIPT
Bowel Project P-1 study, J. Natl. Cancer Inst. 90 (1998) 1371-1388.
[12] T.L. Wang, Q.D. You, F.S.G. Huang, H. Xiang, Recent Advances in Selective Estrogen Receptor
Modulators for Breast Cancer, Mini-Rev. Med. Chem. 9 (2009) 1191-1201.
[13] J.A. Katzenellenbogen, The 2010 Philip S. Portoghese Medicinal Chemistry Lectureship:
addressing the "core issue" in the design of estrogen receptor ligands, J. Med. Chem. 54 (2011)
5271-5282.
[14] B.E. Fink, D.S. Mortensen, S.R. Stauffer, Z.D. Aron, J.A. Katzenellenbogen, Novel structural
templates for estrogen-receptor ligands and prospects for combinatorial synthesis of estrogens,
Chem. Biol, 6 (1999) 205-219.
[15] H.B. Zhou, J.S. Comninos, F. Stossi, B.S. Katzenellenbogen, J.A. Katzenellenbogen, Synthesis
and evaluation of estrogen receptor ligands with bridged oxabicyclic cores containing a
diarylethylene motif: Estrogen antagonists of unusual structure, J. Med. Chem. 48 (2005)
7261-7274.
[16] K. Ohta, T. Ogawa, A. Kaise, Y. Endo, Novel estrogen receptor (ER) modulators containing
various hydrophobic bent-core structures, Bioorgan. Med. Chem. 22 (2014) 3508-3514.
[17] F.E. Vanleeuwen, J. Benraadt, J.W.W. Coebergh, L.A.L.M. Kiemeney, C.H.F. Gimbrere, R. Otter,
L.J. Schouten, R.A.M. Damhuis, M. Bontenbal, F.W. Diepenhorst, A.W. Vandenbeltdusebout, H.
Vantinteren, Risk of Endometrial Cancer after Tamoxifen Treatment of Breast-Cancer, Lancet 343
(1994) 448-452.
[18] R. Clarke, M.C. Liu, K.B. Bouker, Z.P. Gu, R.Y. Lee, Y.L. Zhu, T.C. Skaar, B. Gomez, K. O'Brien,
Y. Wang, L. Hilakivi-Clarke, Antiestrogen resistance in breast cancer and the role of estrogen
receptor signaling, Oncogene 22 (2003) 7316-7339.
[19] A. Ring, M. Dowsett, Mechanisms of tamoxifen resistance, Endocr-Relat. Cancer 11 (2004)
643-658.
[20] R. Morphy, Z. Rankovic, Designed multiple ligands. An emerging drug discovery paradigm, J.
Med. Chem. 48 (2005) 6523-6543.
[21] N.M. O'Boyle, M.J. Meegan, Designed multiple ligands for cancer therapy, Curr. Med. Chem. 18
(2011) 4722-4737.
[22] W. Lv, J. Liu, T.C. Skaar, D.A. Flockhart, M. Cushman, Design and synthesis of norendoxifen
analogues with dual aromatase inhibitory and estrogen receptor modulatory activities, J. Med.
Chem. 58 (2015) 2623-2648.
[23] C. Tang, C. Li, S. Zhang, Z. Hu, J. Wu, C. Dong, J. Huang, H.B. Zhou, Novel Bioactive Hybrid
Compound Dual Targeting Estrogen Receptor and Histone Deacetylase for the Treatment of
Breast Cancer, J. Med. Chem. 58 (2015) 4550-4572.
[24] N.M. O'Boyle, J.K. Pollock, M. Carr, A.J. Knox, S.M. Nathwani, S. Wang, L. Caboni, D.M.
Zisterer, M.J. Meegan, beta-Lactam estrogen receptor antagonists and a dual-targeting estrogen
receptor/tubulin ligand, J. Med. Chem. 57 (2014) 9370-9382.
[25] Z.C. Tang, S.X. Niu, F. Liu, K.J. Lao, J.S. Miao, J.Z. Ji, X. Wang, M. Yan, L.Y. Zhang, Q.D. You,
H. Xiao, H. Xiang, Synthesis and biological evaluation of 2,3-diaryl isoquinolinone derivatives as
anti-breast cancer agents targeting ER alpha and VEGFR-2, Bioorg. Med. Chem. Lett. 24 (2014)
2129-2133.
[26] D.J. Hicklin, L.M. Ellis, Role of the vascular endothelial growth factor pathway in tumor growth
and angiogenesis, J. Clin. Oncol. 23 (2005) 1011-1027.
[27] M.E. Fratto, M. Imperatori, B. Vincenzi, F. Tomao, D. Santini, G. Tonini, New perspectives: role
21

ACCEPTED MANUSCRIPT
of Sunitinib in Breast Cancer, Clin. Ter. 161 (2010) 475-482.
[28] R.R. Patel, S. Sengupta, H.R. Kim, A.J. Klein-Szanto, J.R. Pyle, F. Zhu, T.Y. Li, E.A. Ross, S.
Oseni, J. Fargnoli, V.C. Jordan, Experimental treatment of oestrogen receptor (ER) positive breast
cancer with tamoxifen and brivanib alaninate, a VEGFR-2/FGFR-1 kinase inhibitor: A potential
clinical application of angiogenesis inhibitors, Eur. J. Cancer 46 (2010) 1537-1553.
[29] J.M.W. Gee, J.F.R. Robertson, I.O. Ellis, R.I. Nicholson, Phosphorylation of ERK1/2
mitogen-activated protein kinase is associated with poor response to anti-hormonal therapy and
decreased patient survival in clinical breast cancer, Int. J. Cancer 95 (2001) 247-254.
[30] Z. Madak-Erdogan, M. Lupien, F. Stossi, M. Brown, B.S. Katzenellenbogen, Genomic
collaboration of estrogen receptor alpha and extracellular signal-regulated kinase 2 in regulating
gene and proliferation programs, Mol. Cell. Biol. 31 (2011) 226-236.
[31] L.M. McGlynn, T. Kirkegaard, J. Edwards, S. Tovey, D. Cameron, C. Twelves, J.M. Bartlett, T.G.
Cooke, Ras/Raf-1/MAPK pathway mediates response to tamoxifen but not chemotherapy in breast
cancer patients, Clin. Cancer Res. 15 (2009) 1487-1495.
[32] R.A. Hill, S. Rudra, B. Peng, D.S. Roane, J.K. Bounds, Y. Zhang, A. Adloo, T.S. Lu,
Hydroxyl-substituted sulfonylureas as potent inhibitors of specific [H-3]glyburide binding to rat
brain synaptosomes, Bioorgan. Med. Chem. 11 (2003) 2099-2113.
[33] J. Azizian, A.A. Mohammadi, A.R. Karimi, M.R. Mohammadizadeh, A stereoselective
three-component reaction: KAl(SO4)2·12H2O, an efficient and reusable catalyst for the one-pot
synthesis of cis-isoquinolonic acids, J. Org. Chem. 70 (2005) 350-352.
[34] G.M. Anstead, K.E. Carlson, J.A. Katzenellenbogen, The estradiol pharmacophore: ligand
structure-estrogen receptor binding affinity relationships and a model for the receptor binding
site, Steroids 62 (1997) 268-303.
[35] L. Santarpia, S.M. Lippman, A.K. El-Naggar, Targeting the MAPK-RAS-RAF signaling pathway
in cancer therapy, Expert Opin. Ther. Tar. 16 (2012) 103-119.
22

ACCEPTED MANUSCRIPT
Captions for figuresꢀschemes and tablesꢁ
Captions for figuresꢁ
Figure 1. Estrogen receptor ligands: (1) Tamoxifen; (2) Raloxifene; (3) PPT; (4) OBHS; (5)
m-carborane SERM.
Figure 2. Multiple ligands targeting ER.
Figure 3. 21c inhibits the phosphorylation of VEGFR-2 in HUVEC cells. Expression of
p-VEGFR-2 and VEGFR-2 in HUVEC cells were examined by western blots. Densitometric
analysis was performed to determine the phosphorylation rate of VEGFR-2. Values are mean ±SD
(n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control group.
Figure 4. The increased mRNA expression of PgR induced by E2 was reversed by 21c in MCF-7
cells. The mRNA expression of PgR was examined by Real-time PCR. Values are mean ±SD
(n=5). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. E2 group. # P < 0.05, ## P < 0.01,### P < 0.001.
Figure 5. 21c inhibits the phosphorylation of VEGFR-2 in MCF-7 cells. (A) Expression of
p-VEGFR-2 and VEGFR-2 in MCF-7 cells were examined by western blots. (B) Densitometric
analysis was performed to determine the phosphorylation rate of VEGFR-2. Values are mean ±SD
(n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control group.
Figure 6. 21c inhibits the activity of the Raf-1/ERK pathway in MCF-7 cells. (A) Expression of
p-Raf-1, Raf-1, p-ERK1/2, and ERK1/2 were examined by western blots in MCF-7 cells. (B)
Densitometric analysis was performed to determine the phosphorylation rate of Raf-1 and
ERK1/2 . Values are mean ±SD (n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control group; #
P < 0.05, ## P < 0.01,### P < 0.001 vs. Control group.
Figure 7. (A) Predicted binding mode of 21c within the LBD of ERα (PDB ID: 3ERT). (B)
Binding mode of 4-OH-Tamoxifen within the LBD of ERα (PDB ID: 3ERT). (C) Predicted
binding mode of 21c within the LBD of VEGFR-2 (PDB ID: 1YWN).
Captions for schemesꢁ
Scheme 1. Synthesis of compound 12. Reagents and conditions: (a) Chloral hydrate, conc. H2SO4,
rt, 12h, 83.6 %; (b) Zn, HOAc, rt, 30min; 93.6% (c) Conc. H2SO4, rt, 30min, 90%.
Scheme 2. Synthesis of 18. Reagents and conditions: (d) acetic anhydride, toluene, reflux, 4h,
100%; (e) ethanol, reflux, 10h, 96.9%; (f) KAl(SO4)2·12H2O, acetonitrile, rt, 8h, 45.9%; (g) 1)
SOCl2, toluene, reflux, 2h; 2) AlCl3, CH2Cl2, rt, 10h, 86.7%.
Scheme 3. Synthesis of 19a-f, 20a-f and 21a-f. Reagents and conditions: (h) RCH2CH2Cl or
RCH2CH2CH2Cl, 20% sodium hydroxide, TBAB, THF, reflux, 4h, 37-72%; (i) BBr3, CH2Cl2, rt,
3h, 49-63%.
23

ACCEPTED MANUSCRIPT
Captions for tablesꢁ
Table 1. Structures of designed compounds
Table 2 Binding affinity of synthesized compounds for ERα
Table 3 Cancer cells antiproliferative activity (IC50, ꢀM)
Table 4 Antiangiogenic activity on VEGFR-2 and HUVEC
24

ACCEPTED MANUSCRIPT
Research Highlights
● Designed multiple ligands may exert improved efficacy with lower incidence of side
effects.
● A series of 6-aryl-indenoisoquinolone derivatives were described as dual ERα and
VEGFR-2 inhibitors.
● Compound 21c turned out to be a promising dual targeting candidate for breast cancer.