Synthesis and biological evaluation of 2,3-diaryl isoquinolinone derivatives as anti-breast cancer agents targeting ERα and VEGFR-2

Article abstract of DOI:10.1016/j.bmcl.2014.03.042

The estrogen receptor α is recognized as important pharmaceutical target for breast cancer therapy, and vascular endothelial growth factor receptors (VEGFRs) play important roles in tumor angiogenesis including breast cancer. A series of 2,3-diaryl isoqui

Full text of DOI:10.1016/j.bmcl.2014.03.042

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2129–2133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 2,3-diaryl isoquinolinone
derivatives as anti-breast cancer agents targeting ER
a and VEGFR-2
Zhichao Tang a,b, , Shaoxiong Niu a,b, , Fei Liu a,b, Kejing Lao a,b, Jingshan Miao c, Jinzi Ji c, Xiang Wang c,
Ming Yan c, Luyong Zhang c, Qidong You a,b, Hong Xiao d, , Hua Xiang a,b,
⇑
⇑
a Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
c New Drug Screening Center, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
d Nanjing Brain Hospital Affiliated to Nanjing Medical University, 264 Guangzhou Road, Nanjing 210029, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The estrogen receptor
a is recognized as important pharmaceutical target for breast cancer therapy, and
Received 5 December 2013
Revised 12 March 2014
Accepted 14 March 2014
Available online 22 March 2014
vascular endothelial growth factor receptors (VEGFRs) play important roles in tumor angiogenesis
including breast cancer. A series of 2,3-diaryl isoquinolinone derivatives were designed and synthesized
targeting both estrogen receptor
7c, 7d and 7f exhibited significant anti-proliferative and anti-angiogenesis activities via ER
a (ERa) and VEGFR-2. Bioactivity evaluation showed that compounds
a and
VEGFR-2 dependent mechanisms.
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
2,3-Diaryl isoquinolinone derivatives
Synthesis
Anti-breast cancer
ER
a
VEGFR-2
Breast cancer (BC) is the most common malignancy and the
leading cause of cancer death in women worldwide.1 Estrogen
receptor (ER ), a member of the large superfamily of nuclear
receptors, is overexpressed and predominantly involved in more
than 70% breast cancer patients.2 ER
is responsible for estrogen-
resistance is incompletely clear, it had been proved that the activa-
tion of Ras/Raf-1/Mitogen-activated protein kinase (MAPK) signal
pathway is involved in Tamoxifen resistance.4 The MAPK pathway
a
a
can phosphorylate and activate ERa in a ligand-independent
a
manner, resulting in transcription of estrogen-regulated genes
induced proliferation in breast cancer. In this case, tumors depend
on estrogens for their survival and endocrine therapy is currently
used to inhibit ER signaling by competitively binding to ER with
anti-estrogens or estrogen deprivation. Selective estrogen receptor
modulators (SERMs) are non-steroidal agents which act as anti-
estrogens in breast tissue and are widely used in the treatment
and prevention of ER positive breast cancer.3 Despite their great
benefits in treating BC, SERMs may cause negative side effects
due to their estrogenic activity in other tissues. For example,
stimulation in the uterus would increase risk of endometrial
cancer. Another common side effect in endocrine therapy is drug
resistance. Whatever the endocrine treatment used, resistance
may occur. This is especially true with Tamoxifen which is never
given more than 5 years. Although the molecular mechanism of
and cell proliferation.5 Study also showed that MAPK pathway
collaborates with ERa in exerting direct genomic actions in breast
cancer cells through extracellular signal-regulated kinase 2 (ERK2),
a downstream effector.6
Angiogenesis plays an important role in both local tumor
growth and distant metastasis in many cancers as well as breast
cancer.7 Vascular endothelial growth factor receptor-2 (VEGFR-2,
or kinase insert domain receptor, KDR) is a member of the receptor
tyrosine kinase (RTK) family and is proposed to function as a
dominant receptor of VEGF/VEGFR signaling in the angiogenesis
pathway.8 The Ras/MAPK pathway which is very important in
promoting cell proliferation, is also activated in VEGF/VEGFR signal
transduction and plays important role in promoting tumor
angiogenesis.9,10 VEGFR-2 inhibitors were reported in treating
breast cancer but are not sufficient when used as mono-
therapy.11,12 But a combination of Tamoxifen and a low dose of a
VEGFR-2 inhibitor, Brivanib alaninate, was reported not only to
maximize therapeutic efficacy but also to retard SERM resistant
tumour growth.13
⇑
Corresponding authors. Tel./fax: +86 025 83271096 (H.Xiang).
E-mail addresses: xiaohong63xx@163.com (H. Xiao), xianghua@cpu.edu.cn
(H. Xiang).
Same contribution to this paper.
http://dx.doi.org/10.1016/j.bmcl.2014.03.042
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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Z. Tang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2129–2133
Table 1
In the research to find novel and effective anti-breast cancer
Structures of synthesized compounds
agents, the cross-talk between ER and receptor tyrosine kinase sig-
naling aroused our interests. Based on the above facts, VEGFR-2
inhibitors can not only inhibit angiogenesis in breast cancer but
may also retard SERMs resistance in BC through inhibition of
Ras/MAPK pathway. So we hypothesized that if a compound could
serve as an anti-estrogen as well as a VEGFR-2 inhibitor, there
should be better effect in treating BC. Through review of literature,
we found that VEGFR-2 inhibitors with scaffold of indol-2-one,
such as Sunitinib and YM231146, bear some structural similarities
with SERMs (Fig. 1). All of them contain aromatic scaffold and flex-
ible side chain with tertiary amine substituent in the end. Based on
these findings, we proposed to design and synthesize a series of
compounds with characteristics of both SERMs and VEGFR-2 inhib-
itors. It was expected for these compounds with dual targets to
gain more satisfactory effects for anti-breast cancer with fewer
side effects. A series of 2,3-diaryl isoquinolinone derivatives with
flexible basic side chains were synthesized and evaluated, the car-
bonyl of isoquinolinone was expected to function as in indol-2-
one, the length of the side chain was also taken into consideration
to evaluate its influence on activities (Table 1).
O
R1
O
N
n
R3
R2
N
R4
Compound
R3
R4
n
R1, R2
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
OH
OH
OH
OH
OH
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
OH
OH
OH
OH
OH
2
2
2
2
2
2
3
3
2
2
2
2
3
3
–CH3, –CH3
–(CH2)4–
–(CH2)5–
–CH2CH2N(CH3)CH2CH2–
–CH2CH2OCH2CH2–
–CH2CH3, –CH2CH3
–(CH2)4–
–(CH2)5–
–CH3, –CH3
–(CH2)4–
–(CH2)5–
–CH2CH2N(CH3)CH2CH2–
–(CH2)4–
–(CH2)5–
OH
OH
The synthetic route of target 2-(4-alykoxylphenyl)-isoquinoli-
none derivatives is shown in Scheme 1. The key intermediate,
4-methoxyhomophthalic acid (3), was prepared from the commer-
cially available 3-methoxybenzoic acid and chloral hydrate
according to literature procedure in high yields.14 In this procedure,
m-methoxybenzoic acid was condensed with chloral hydrate to
obtain the lactone 1. This lactone was then reduced by zinc in
acetic acid to give dichlorovinyl derivative 2, and hydrolyzation
of 2 in concentrated sulfuric acid finally gave 3. The 4-methoxy-
homophthalic acid was then condensed with anisoyl chloride by
heating the intimate mixture of the two at 200 °C to obtain
compound 4. Reflux of compound 4 and p-aminophenol in acetic
acid for 4 h produced compound 5. The nucleophilic substitution
reaction between compound 5 and various alkyl chlorides in
acetone afforded compounds 6a–h in the presence of K2CO3.
Compounds 7a–f were obtained by demethylation in acetic acid
and 40% aqueous hydrobromide acid from compounds 6a–h. The
structures of synthesized compounds have been confirmed by IR,
NMR and mass spectrometry.
indicates that the skeleton of 2,3-diaryl isoquinolinone could
favorably mimic that of extradiol. The dihydroxyl compounds
7a–f possessed apparently better affinities with ERa than dimeth-
oxyl compounds 6a–h. It was supposed that the dihydroxyl group
had formed more hydrogen bonds with related amino acids than
dimethoxyl group in the ligand binding domain of ERa. Among
the dihydroxyl compounds, 7b, 7e and 7f all held the inhibition
rate of more than 95% which were very close to that of Tamoxifen.
The IC50 of 7b, 7e and 7f valued at 3.1
respectively, while the positive control Tamoxifen valued at
1.9 M.
Once the binding affinity of our compounds with ER
lM, 2.3 lM and 1.3 lM
l
a
was
confirmed, we conducted the VEGFR-2 kinase inhibition assay.
The results are shown in Table 2. From the results, we can see that
synthesized compounds exhibited from moderate to strong
inhibitory activities compared with Sunitinib. When R3 and R4
were changed from methoxyl to hydroxyl (6a and 7a, 6c and 7c,
6d and 7d), there was an increase in inhibitory activity except
for compounds 6b and 7b where a decrease in inhibition was
observed. There was also an increase in inhibition when the length
of side chain was increased by one carbon atom (6c and 6g, 6d and
6h, 7c and 7f). But a slight decrease was also observed for 7b and
The binding affinities of synthesized compounds with ER
a
were initially assessed by following a fluorescence polarization
procedure15 with Tamoxifen as the positive control. The results
are displayed in Table 2. Most of the target compounds showed
good binding affinities with ER
a
compared with Tamoxifen which
O
N
N
H
N
O
N
F
H
O
N
O
R1
H
O
N
n
Sunitinib
R2
N
R3
O
N
Tamoxifen
R4
O
OH
O
diaryl isoquinolinones
NH
O
HO
O
N
N
H
Acolbifene
YM231146
Figure 1. Chemical structures of SERMs, VEGFR-2 inhibitors and designed compounds.

Z. Tang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2129–2133
2131
CCl3
Cl
Cl
COOH
COOH
COOH
a
c
b
O
O
O
COOH
O
O
O
3
1
2
O
Cl
O
O
d
O
O
O
O
O
e
f
O
4
N
N
O
O
OH
O
R
O
OH
6
5
H2N
OH
g
N
HO
R
O
O
7
Scheme 1. Reagents and conditions: (a) Chloral hydrate, concd H2SO4, rt 12 h; (b) Zn, HOAc, rt 30 min; (c) Concd H2SO4, rt 30 min; (d) 200 °C, 6 h; (e) HOAc, reflux, 4 h; (f)
R(CH2)nCl, K2CO3, acetone, reflux 12 h; (g) HOAc, 40% HBr, reflux, 24 h.
Table 2
Bioactivities of synthesized compounds
Compound
ERa
MCF-7
VEGFR-2
Inh% (0.1 mg/ml)
IC50
(l
M)
Inh% (0.1 mg/ml)
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
63.12
63.60
72.83
62.09
64.84
69.10
57.57
59.65
88.57
95.93
87.14
87.94
95.70
99.89
100.0
—
N/A
863
322
429
N/A
383
479
N/A
18.9
11.8
3.63
9.45
12.7
2.73
1.89
—
66.07
83.80
65.30
79.43
51.19
92.54
98.46
72.75
79.43
49.87
84.83
103.34
48.33
100.26
—
PBS
Sunitinib(3µM)
Sunitinib(10µM) Sunitinib(30µM)
Tamoxifen
Sunitinib
100
N/A = not active, the IC50 is more than 1000 lM.
7e. Interestingly, 6b, 7b and 7e all have a pyrrolidinyl group in the
end of side chain while 7b and 7e hold the lowest inhibition rate
which is less than 50%. Compound 6e with morpholinyl group
which is same with YM231146 had an inhibition rate of only
51.19% while 6f with diethylamino group that is same with Suniti-
nib held the inhibition rate of 92.54%. There seems to be big influ-
ence on activity by different basic group. Among these compounds,
7d and 7f were shown to be the most potent. Their activities were
at the same level with Sunitinib, the IC50 value of 7d and 7f was
7d (3µM)
7d (10µM)
7d (30µM)
1.9
lM and 1.4
lM respectively with Sunitinib valued at 1.03
lM.
The results in ER
a
binding and VEGFR-2 kinase inhibition tests
7f (3µM)
7f (10µM)
7f (30µM)
have confirmed our idea of designing dual-targeted compounds.
We then tested their anti-proliferative activities in vitro against
MCF-7 breast cancer cells with Tamoxifen as the positive control.16
The results are summarized in Table 2. Synthesized compounds
demonstrated anti-proliferative activities from close to Tamoxifen
Figure 2. Results of CAM assay.
compounds led to notably better anti-proliferative activities than
dimethoxyl compounds. It seemed that there is no significant
influence on activity caused by different length of side chain.
to inactive. The activity differences (7a–f vs 6a–h) in ER
affinity assay were also observed in this test. Dihydroxyl
a binding

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Z. Tang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2129–2133
Figure 3. (A) A1: Docking poses of 7f (purple) within LBD of ERa, A2: Superimposed poses of 7f and 4-OH-Tamoxifen (white), PDB ID: 3ERT. (B) B1: Docking poses of 7f
(purple) within LBD of VEGFR-2, B2: Superimposed poses of 7f and Sunitinib (yellow), PDB ID: 4AGD.
Compound 7f was found to be the most active inhibitor with the
IC50 value of 2.73 M which was slightly greater than that of
Tamoxifen (IC50 = 1.89 M), following by 7c with IC50 value of
3.63 M. It was also observed that compound 6e with morpholinyl
in side chain had no activity against MCF-7 breast cancer cells
while 7f and 7c with piperidyl group turned to be the most potent.
points toward His524, while the antiestrogenic side chain is pro-
l
jected toward Asp351 (Fig. 3, A1 and A2). In the binding pocket of
VEGFR-2, the indol-2-one skeleton of Sunitinib is located into the
binding pocket and the side chain stretches toward the edge of
the pocket. The hydrogen bond interaction of 1-NH with Glu915
and 2-carbonyl with Cys917 play important role in stabilizing the
binding mode. Compound 7f could enter the binding pocket and
form hydrogen bonds with Glu915 and Cys917 with its 7-OH but
not 1-carbonyl which was out of our expectation (Fig. 3, B1 and
B2). It was supposed that the 3-phenyl become a hindrance for
compound to enter the pocket deeply. The side chain of 7f also
stretched toward the edge of the pocket like Sunitinib. Docking sim-
ulation suggested possible basis for the observed activities.
l
l
Considering the role in ERa binding, basic group as an important
bonding site made great contribution to the activity of the
compound.
Based on the preliminary results of VEGFR-2 kinase inhibitory
activity, compounds 7d and 7f were selected to perform chicken
chorioallantoic membrane (CAM) assay to investigate their inhibi-
tion of angiogenesis in vivo. Test compounds and the positive con-
trol Sunitinib dissolved in DMSO were placed on sterile methyl
In conclusion, we have designed and synthesized a series of
cellulose filter papers at 3
l
M, 10
l
M and 30
l
M with phosphate
‘-diaryl isoquinolinones targeting both ER
compounds were expected to perform more effective anti-ER
positive breast cancer effects through inhibition of ER and
VEGFR-2 simultaneously. Biological evaluation showed that
most of the synthesized compounds exert ER binding affinity
and VEGFR-2 inhibition. Further investigation also showed they
possessed good anti-proliferation effects against MCF-7 breast
cancer cells and potential anti-angiogenesis effects in vivo.
Compound 7f was found to be the most potential dual inhibitor
and deserved further study. This work may provide a new and
potential route to develop effective drugs for breast cancer.
a and VEGFR-2. These
buffered saline (PBS) as the blank control. Results are shown in
Figure 2. Compared with blank control group, compounds 7d and
7f could significantly inhibit angiogenesis. And the inhibitory
ability was proportional to the concentration. Compared with Sun-
itinib group, 7d and 7f presented comparable inhibitory activity.
Overall, compounds showed potential anti-angiogenesis activities
in vivo.
To further rationalize the prospective activities of 2,3-diaryl iso-
quinolinone derivatives against ERa and VEGFR-2, molecular docking
studies were performed using the Discovery Studio 2.5/CDOCKER pro-
a
a
a
tocol. The docking orientation and interactions of 7f within the ligand
binding domain (LBD) of ERa and VEGFR-2 are shown in Figure 3. In
Acknowledgments
the docking study, the core skeleton of 2,3-diaryl isoquinolinone is
favorably positioned similar to 4-hydroxytamoxifen (OHT). The
7-OH of isoquinolinone plays the role of 4-OH of OHT and forms
hydrogen bonds with Glu353 and Arg394. The other OH group
This work was supported by grants from the National Natural
Science Foundation of China (81373279), the Twelfth Five-Year

Z. Tang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2129–2133
2133
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03.
042. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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