Design, synthesis, and biological evaluation of tetrahydroisoquinoline-based diaryl urea derivatives for suppressing VEGFR-2 signaling

Article abstract of DOI:10.1097/CAD.0000000000000718

A novel structural series of tetrahydroisoquinoline-based compounds that incorporate the diaryl urea moiety was designed, synthesized, and biologically evaluated as suppressors of VEFGR-2 signaling. As a consequence, compounds 9k and 9s exhibited comparable or superior cytotoxic activity to that of gefitinib against the tested three cell lines, including A549, MCF-7, and PC-3. Importantly, both of them downregulated the expression of VEGFR-2, and inhibited VEGFR-2 phosphorylation at the concentration of 0.5 or 1.0 μmol/l. Besides, they suppressed human umbilical vein endothelial cell tube formation at the concentration of 4.0 μmol/l. Considering their capability of down-regulating VEGFR-2 expression and inhibiting VEGFR-2 phosphorylation, 9k and 9s may serve as suppressors of angiogenesis for further investigation.

Full text of DOI:10.1097/CAD.0000000000000718

Preclinical report
1
Design, synthesis, and biological evaluation of
tetrahydroisoquinoline-based diaryl urea derivatives for
suppressing VEGFR-2 signaling
a
a
a,b
a,b
a
Yuanzheng Huang , Yang Zhang , Jiaming Li , Xiaodong Ma , Mengqi Hu ,
a
a
Yu Yang and Sufan Gao
A novel structural series of tetrahydroisoquinoline-based
compounds that incorporate the diaryl urea moiety was
designed, synthesized, and biologically evaluated as
suppressors of VEFGR-2 signaling. As a consequence,
compounds 9k and 9s exhibited comparable or superior
cytotoxic activity to that of gefitinib against the tested three
cell lines, including A549, MCF-7, and PC-3. Importantly,
both of them downregulated the expression of VEGFR-2,
and inhibited VEGFR-2 phosphorylation at the concentration
of 0.5 or 1.0 μmol/l. Besides, they suppressed human
umbilical vein endothelial cell tube formation at the
concentration of 4.0 μmol/l. Considering their capability of
down-regulating VEGFR-2 expression and inhibiting
VEGFR-2 phosphorylation, 9k and 9s may serve as
suppressors of angiogenesis for further investigation. Anti-
Cancer Drugs 00:000–000 Copyright © 2018 Wolters Kluwer
Health, Inc. All rights reserved.
Anti-Cancer Drugs 2018, 00:000–000
Keywords: angiogenesis, diaryl urea, immunofluorescence assay,
VEFGR-2 signaling
a
b
Department of Pharmaceutical Chemistry, School of Pharmacy and Department
of Medicinal Chemistry, Anhui Academy of Chinese Medicine, Hefei, China
Correspondence to Jiaming Li, PhD, School of Pharmacy, Anhui University of
Traditional Chinese Medicine, Hefei 230031, China
Tel: + 86 137 0569 4971; e-mail: lijiaming2017@sina.com
Received 7 May 2018 Revised form accepted 30 October 2018
Introduction
highlighting its importance as a structural element in
inhibitors of VEGFR-2 signaling.
Tumor angiogenesis plays a crucial role in malignant cell
proliferation and metastasis [1–3], which serves as a major
contributor to cancer-related death. Therefore, inhibition
of angiogenesis has been given increasing importance in
the treatment of cancers [4,5]. As a well-established and
specific angiogenic factor, VEGF regulates the angio-
genesis and vascular migration and is overexpressed in a
variety of malignant tumors, thereby being closely related
to the growth, metastasis, and poor prognosis of tumors.
VEGFR, belonging to receptor tyrosine kinase family,
mediates many biochemical and physiological processes
for neovascularization through binding to VEGF [6,7].
VEGFR mainly comprises three receptors: VEGFR-1
Tetrahydroisoquinoline alkaloids, as a class of naturally
occurring bioactive ingredients from medicinal plants, were
capable to suppress tumor proliferation through different
mechanisms, such as alkylation, regulation of growth-related
receptors and antiapoptotic genes, as well as inhibition of
angiogenesis [13–16]. Choquette et al. [17] found that some
tetrahydroisoquinolines can inhibit the formation of nascent
microcapillaries and prevent tumor cells from absorbing
nutrients by selectively suppressing VEGFR-2 signaling.
Our group previously designed and synthesized a series
of tetrahydroisoquinoline derivatives, among which com-
pounds 17d (IC =2.6 nmol/l) and 17e (IC =0.89 nmol/l)
50
50
(
Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4).
(Fig. 2) exhibited remarkable antiproliferative activity
Among them, VEGFR-2 has been identified as the
predominant mediator of tumor angiogenesis. Upon
responding to the stimulus of VEGF, VEGFR-2 triggers
the proliferation of vascular endothelial cell, thereby
facilitating the blood vessel growth, increasing the vas-
cular permeability and promoting tumor development
against MCF-7 cells [18].
In view of the importance of diary urea as structural element
of compounds that suppress VEGFR-2 signaling and the
antitumor activity of tetrahydroisoquinoline alkaloids, we
have therefore employed pharmacorphore-combination
strategy for designing suppressors of VEFGR-2 signaling.
On the basis of the tetrahydroisoquinolines discovered in our
previous study, a series of structurally novel hybrid molecules
were obtained by replacing (E)-3-(4-hydroxy-3-metho-
xyphenyl)acrylic acyl moiety of them with diaryl urea frag-
ments. Besides, the substituents on the phenyl attached to
the tetrahydroisoquinoline carbon and the terminal aryl of
the diaryl fragment were investigated.
[
8–10]. So far, numerous antiangiogenic drugs (Fig. 1)
that target VEGFR-2 signaling have been marketed,
including sorafenib [11], regorafenib, and linifanib [12].
In structure, all of them contain a diaryl urea moiety,
Supplemental Digital Content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions of this
article on the journal's website, www.anti-cancerdrugs.com.
0
959-4973 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.
DOI: 10.1097/CAD.0000000000000718
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2
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 1
The structures of sorafenib, regorafenib, and linifanib.
Fig. 2
The design ideas of novel diaryl urea compounds.
Materials and methods
Experimental section
measured with a Hewlett-Packard 1100 LC/MSD spec-
trometer (Agilent, Waldbronn, Germany).
General information
The reagents and solvents for reaction were purchased
from common commercial suppliers. If necessary, pur-
ification was carried out before use. Melting points
are determined on melting point apparatus (RDCSY-I)
and are uncorrected. H NMR and C NMR spectra
were recorded on 600 and 150 MHz instruments
General procedure for synthesis of compounds
4-(5-(3,4-dimethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]
isoquinolin-6(5H)-yl)aniline (8a)
1
13
5
-(3,4-dimethoxyphenyl)-5,6,7,8-tetrahydro-[1,3]diox-
olo[4,5-g]isoquinoline (5a) was prepared with our
previously reported method [18]. A mixture of Comp-
ound 5a (2.87 g, 9.2 mmol), p-fluoronitrobenzene
(
Bruker, Fallanden, Switzerland), respectively, with tet-
ramethylsilane as internal standard. MS spectra were
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A series compounds to inhibit VEGFR-2 Huang et al.
3
(
1.26 g, 11.4 mmol), and K CO (1.6 g, 11.6 mmol) in
Absorbance values were determined at 570 nm. The
IC50 values were calculated according to inhibition
ratios.
2
3
dimethylsulfoxide (DMSO) (20 ml) was stirred for 6 h
at 80°C and cooled to room temperature. Afterward,
the reaction mixture was diluted with aqueous solu-
tion and extracted with CH Cl . The organic layer was
washed with brine, dried with anhydrous sodium sul-
fate, filtered, and evaporated in vacuum. The crude
product was purified by column chromatography
OD controlꢀOD administration
Inhibition rate ð % Þ ¼
2
2
OD control
ꢁ100 % :
(
petroleum ether/EtOAc = 4 : 1 ∼ 2 : 1) to yield the
compound 7a. To the stirred solution of compound 7a
Immunofluorescence assay
Cells were washed with PBS and fixated in paraformalde-
hyde (4%). Samples were permeabilized and blocked in
in 90% ethanol (80 ml), Fe (1.2 g, 21.4 mmol) and
NH Cl (1.75 g, 32.7 mmol) were added. The mixture
was refluxed for 3 h and filtrated to give the crude
product after cooling to room temperature, which was
purified by column chromatography to give compound
4
0
.1% Triton X-100/10% goat serum in PBS for 1 h at room
temperature and incubated with primary antibodies
goat-anti-human VEGFR-2, rabbit-anti-human Phospho-
(
8
a as a yellow liquid.
VEGFR-2 (Tyr1175) overnight at 4°C. After washing with
PBS, samples were incubated with secondary antibody
(mouse anti-goat IgG-Cy3, mouse anti-rabbit IgG-FITC) for
30 min and DAPI for 5 min at 37°C. After discarding DAPI
and washing with PBS, photographs were taken under a
fluorescence microscope (Nikon Eclipse, TS100-FDH1;
Nikon Corporation, Tokyo, Japan).
Compound 8b was synthesized according to the synthetic
procedure given above (see details in supporting data,
Supplementary Data, Supplemental digital content 1,
http://links.lww.com/ACD/A276).
1
-(4-(5-(3,4-dimethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]
isoquinolin-6(5H)-yl)phenyl)-3-(4-methoxyphenyl)urea (9a)
A solution of 4-dimethoxybenzoic acid (0.42 g, 2.74 mmol),
Tube formation assay
Overall, 75 μl of growth factor-reduced Matrigel (Corning
Incorporated, Shanghai, China) was loaded into pre-
DPPA (0.6 ml, 2.8 mmol), and Et N (0.4 ml, 2.9 mmol) in
3
benzene (30 ml) was stirred under 40°C for 45 min, and then
refluxed for 2 h. The reaction mixture was concentrated under
reduced pressure and the residue was dissolved directly with
chilled 96-well tissue plates, and plates were placed at
4
3
7°C for 60 min. HUVECs (6 ∼ 8 × 10 cells) were added
CHCl (10 ml) for next step. Then a solution of 8a (1.10 g,
3
into each well and cultured in endothelial cell medium
on the gel for 4 ∼ 6 h. Different concentrations of test
compound and sofafenib were then added and incubated
for 8 h. The capillary networks were photographed by a
microscope.
2
.72 mmol) in CHCl (10 ml) was added into the aforemen-
3
tioned mixture. The resulting mixture was then stirred on oil
bath at 65°C for 4 h. After the reaction was completed, the
solvent was removed under reduced pressure, and the residue
was dissolved with CH Cl (30 ml), washed with brine
2
2
(
50 ml ×2), dried over anhydrous Na SO , filtered, and con-
2 4
centrated to afford the crude product, which was further
purified by silica gel flash chromatography (petroleum ether/
EtOAc =6 : 1∼4 : 1) to furnish 0.3 g of 9a as a white solid.
Results
Chemistry
The synthesis of target compounds 9a–9s was depicted
in Scheme 1. The tetrahydroisoquinoline intermediates
Compounds 9b ∼ 9s were synthesized according to the
synthetic procedure given above (see details in support-
ing data, Supplementary Data, Supplemental digital
content 1, http://links.lww.com/ACD/A276).
5
a–5b were prepared in accordance with our reported
procedure [18]. Nucleophilic Aromatic Substitution
reaction of 5a–5b with 1-fluoro-4-nitrobenzene furn-
ished 7a–7b, which were subsequently reduced to cor-
responding aniline derivatives 8a–8b. Meanwhile, the
aromatic acids 1a’–1s’ were converted into corre-
sponding isocyanates upon treatment with diphenyl
azidophosphate (DPPA). Finally, condensation of 8a–
Methyl thiazolyl tetrazolium assay
The antiproliferative activity of compounds against
A549, MCF-7, and PC-3 cell lines, as well as human
umbilical vein endothelial cells (HUVECs) was eval-
uated by methyl thiazolyl tetrazolium assay (MTT)
assay. Exponentially growing cells were harvested and
8
b with the newly afforded isocyanates provided the
tetrahydroisoquinoline-based diaryl urea derivatives
a–9s as the target compounds. All of them are struc-
9
1
13
3
turally determined by H NMR, C NMR, and MS.
plated in 96-well plates at a concentration of 5 × 10
cells/well. The cells in wells were treated with title
compounds, respectively, at various concentrations for
Reagents and conditions used were as follows: (a) SOCl2,
toluene, 77°C; (b) CH Cl , Et N, 0°C; (c) (1) POCl ,
2
2
3
3
4
8 h. Then, 22 ml of MTT (5 mg/ml) was added to each
toluene, 115°C; (2) NaBH , CH OH, 0°C; (d) K CO ,
4 3 2 3
well and incubated for 4 h at 37°C. Supernatant was
discarded, and DMSO was added to each well.
DMSO, 80°C; (e) Fe, NH Cl, 90% EtOH, 90°C; (f)
4
DPPA, Et N, benzene, reflux; and (g) CHCl , reflux.
3
3
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4
Anti-Cancer Drugs 2018, Vol 00 No 00
Scheme 1
Synthesis of compounds 9a–9s.
Biological activity
In-vitro antiproliferative activity
Compounds 9j, 9k, 9l, 9o, 9q, and 9s were comparable or
superior to gefitinib in cytotoxic activity against PC-3
cells. In general, compound 9s exhibited the most
attractive cytotoxic activity against all the tested three
cell lines throughout this series with the IC50 at the
single-digit micromolar level. Besides, compound 9k
exhibited acceptable antiproliferative activity against
A549 and MCF-7 cell lines. Compounds 9k and 9s, as
the representatives of this series, were subsequently
assayed against HUVECs for evaluating their cytotoxicity
toward normal cells with gefitinib as the reference. As
illustrated by Table 2, they displayed comparable cyto-
toxic profile toward HUVECs to that of gefitinib with
IC50 values at two-digit micromolar level.
All the target compounds were biologically evaluated for
their antiproliferative activities against A549 (lung can-
cer), MCF-7 (breast cancer), and PC-3 (prostate cancer)
cell lines with gefitinib as the reference using MTT
assay. As a result, a majority of compounds displayed
moderate antiproliferative activities (Table 1). Among
them, compounds 9j, 9o, and 9q are comparable to
gefitinib in the antiproliferative activity against A549 cell
line. Compounds 9k (IC = 3.24 ± 0.71 μmol/l) and 9s
5
0
(
IC = 2.81 ± 0.43 μmol/l) showed stronger inhibitory
50
activity than gefitinib against A549 cell line. Compounds
i, 9j, 9o, 9p, 9r, 9k, and 9s were superior to gefitinib in
the antiproliferative activity against MCF-7 cell line.
9
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A series compounds to inhibit VEGFR-2 Huang et al.
5
Table 1 The inhibitory activities of compounds against antiproliferation induced by A549, MCF-7, and PC-3 (n = 3)
IC50 (mean ± SD) (μmol/l)
Compounds
R
1
R
2
X, Y
A549
MCF-7
PC-3
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3,4-OCH
3
3
3
3
3
3
3
3
3
3
3
4-OCH
3
C, C
C, C
C, C
C, C
C, C
C, C
C, C
C, C
C, C
C, N
N, N
C, C
C, C
C, C
C, C
C, C
C, C
C, N
N, N
–
48.33 ± 5.04
> 100
> 100
> 100
49.92 ± 6.54
95.3 ± 6.67
38.21 ± 4.31
> 100
58.24 ± 4.53
25.61 ± 2.87
9.62 ± 2.78
9.76 ± 1.89
6.42 ± 0.63
16.96 ± 1.82
33.92 ± 2.56
48.64 ± 4.18
7.53 ± 0.97
4.96 ± 0.39
14.24 ± 2.08
9.64 ± 1.25
6.52 ± 0.75
10.56 ± 0.86
> 100
> 100
57.6 ± 4.63
> 100
4-CH
H
3
38.41 ± 3.73
57.63 ± 5.09
30.72 ± 2.46
86.40 ± 3.49
29.71 ± 2.53
34.56 ± 3.45
12.56 ± 3.52
7.23 ± 1.08
3.24 ± 0.71
11.21 ± 1.73
25.28 ± 2.44
28.82 ± 2.52
6.29 ± 0.39
9.63 ± 1.02
7.95 ± 0.91
12.32 ± 1.65
2.81 ± 0.43
5.62 ± 1.78
4-Cl
3-Cl
2-Cl
4-F
> 100
97.28 ± 5.45
58.83 ± 3.57
44.83 ± 4.69
23.68 ± 3.36
11.52 ± 2.03
16.64 ± 1.72
12.04 ± 0.77
62.10 ± 6.66
> 100
14.48 ± 1.75
28.86 ± 2.08
11.17 ± 1.55
22.41 ± 2.13
8.09 ± 0.93
14.38 ± 2.61
4-CF
3
4-NO
H
2
H
4-OCH
4-OCH
4-OCH
4-OCH
4-OCH
4-OCH
4-OCH
4-OCH
–
3
3,4-OCH
3
4-OCH
H
3
3
3
3
3
3
3
3
4-Cl
4-CF
3
4-NO
2
H
H
–
s
Gefitinib
The antiproliferative activities of compounds against all the tested cell lines were determined using the methyl thiazolyl tetrazolium assay, and gefitinib was employed as the
positive control.
The results were expressed as the IC50
.
Bold values represent the compounds synthesized.
As demonstrated in Fig. 4, phosphorylation of VEGFR-2
can be induced by hVEGF in HUVECs. Compared with
hVEGF (+ )/drug (−) group (Fig. 5b), compounds 9k and
Table 2 The cytotoxicity of compounds on human umbilical vein
endothelial cells
Compounds
IC50 (μmol/l)
9
s could down-regulate the expression of VEFGR-2 and
9
9
k
s
28.09 ± 2.01
14.89 ± 1.93
19.38 ± 1.95
effectively inhibit the phosphorylation of VEFGR-2 at
the concentrations of both 0.25 and 0.5 μmol/l. At the
concentration of 0.5 μmol/l, compound 9k exhibited
stronger suppressive efficacy against VEFGR-2 phos-
phorylation than that of sorafenib, whereas compound 9s
was comparable to that of sorafenib.
Gefitinib
The growth inhibitory activity of compounds against HUVECs was determined
using the MTT assay, and gefitinib was employed as the positive control.
The results were shown as the IC50 values (mean ± SD, four biological replicates).
HUVEC, human umbilical vein endothelial cell; MTT, methyl thiazolyl tetrazolium.
Bold values represent the compounds synthesized.
Tube formation assay
Effect of 9k and 9s on VEGFR-2 expression and
phosphorylation
Given the importance of VEGFR-2 in VEGF signaling
pathway, which mediates angiogenesis by its phosphor-
ylation, compounds 9k and 9s were investigated for their
effect on the expression and phosphorylation of VEGFR-
Compounds 9k and 9s were further evaluated for their
capability to suppress the tube formation. According to
the efficacy of them at the concentrations of 0.25, 1.0 and
4.0 μmol/l (Fig. 5), compounds 9k and 9s inhibited tube
formation in a dose-dependent manner in HUVECs.
They led to a dramatic inhibition of tube formation at the
concentration of 4.0 μmol/l; however, both compounds
were ineffective at the dosage of 0.25 μmol/l. Besides, at
the concentration of 4.0 μmol/l, their potency was slightly
inferior to sorafenib.
2
in A549 and HUVECs by immunofluorescence assay
with sorafenib as the positive control.
As shown in Fig. 3, both compounds 9k and 9s down-
regulated the expression of VEFGR-2 in A549 cells, and
effectively blocked the phosphorylation of VEFGR-2
at the concentration of 0.5 or 1.0 μmol/l. At the con-
centration of 1.0 μmol/l, the capability of compound 9s to
down-regulate VEFGR-2 expression was stronger than
that of sorafenib, and 9k was as effective as sorafenib.
Furthermore, when investigated at the same dosage,
both compounds led to more dramatic down-regulation
of VEGFR-2 phosphorylation in comparison with
sorafenib.
Conclusion
Tetrahydroisoquinoline serves as a well-established tem-
plate for developing anticancer agents, whereas the diaryl
urea moiety is a vital structural element of compounds
that suppress VEGFR-2 signaling. Thus, during our efforts
to explore novel modulators of VEGFR-2 signaling,
the pharmacorphore-combination strategy was introduc
ed by incorporating diaryl urea moiety to the tetra-
hydroisoquinoline scaffold. Among the obtained
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6
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 3
DAPI
VEGFR-2
Phospho-VEGFR-2
MERGE
(a)
(
(
b)
c)
(
(
(
d)
e)
f)
(
g)
h)
(
VEGFR-2 expression and phosphorylation in A549 cell
1
1
1000
0000
#
#
#
9
8
7
6
5
4
3
2
1
000
000
000
000
000
000
000
000
000
0
A(control)
#
#
#
#
#
##
B(0.5µM sofafenib)
C(1.0µM sofafenib)
D(0.5µM 9k)
a
##
#
*
aa
## ###
*
###
#
**
##
#
E(1.0µM 9k)
F(0.5µM 9s)
G(1.0µM 9s)
VEGFR2
Phospho-VEGFR2
The effect of compounds on VEGFR-2 expression and phosphorylation in A549 cells. The A549 cells were treated with sorafenib, 9k and 9s at the
concentration of 0.5 and 1.0 μmol/l, respectively. (a) The A549 cells without compound treatment; (b, c) the A549 cells treated with 0.5 or 1.0 μmol/l
sorafenib; (d, e) the A549 cells treated with 0.5 or 1.0 μmol/l 9k; (f, g) the A549 cells treated with 0.5 or 1.0 μmol/l 9s. Images were taken at a
magnification of × 200; (h) the bar chart presented the quantification of fluorescence intensity of each group (mean ± SD, three biological replicates).
#
##
###
a
aa
Compound-treated group versus control, P < 0.05, P < 0.01,
*P < 0.001.
P < 0.001; d and f versus b, P < 0.01, P < 0.001; e and g versus c,*P < 0.01,
*
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A series compounds to inhibit VEGFR-2 Huang et al.
7
Fig. 4
DAPI
VEGFR-2
Phospho-VEGFR-2
MERGE
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
VEGFR-2 expression and phosphorylation in HUVECs cells
1
1
1
4000
2000
0000
A(VEGF(-)/Drug(-))
#
B(VEGF(+)/Drug(-))
a
a
###
#
###
C(VEGF(+)/sofafenib(0.25umol/L))
D(VEGF(+)/sofafenib(0.5umol/L))
E(VEGF(+)/9k(0.25umol/L))
F(VEGF(+)/9k(0.5umol/L)
8
6
4
2
000
000
000
000
0
###
###
###
**
##
#
VEGFR2
Phospho-VEGFR2
The effect of compounds on VEGFR-2 expression and phosphorylation in HUVECs. The A549 cells were treated with compounds sorafenib, 9k, and
s. Each compound was divided into two concentration groups of 0.5 and 1.0 μmol/l, respectively. (a) The human umbilical vein endothelial cells
HUVECs) without treatment; (b) the HUVECs induced phosphorylation by hVEGF and without treatment; (c, d) the HUVECs induced
9
(
phosphorylation by hVEGF and treated with 0.25 or 0.5 μmol/l sorafenib; (e, f) the HUVECs induced phosphorylation by hVEGF and treated with 0.25
or 0.5 μmol/l 9k; and (g, h) the HUVECs induced phosphorylation by hVEGF and treated with 0.25 or 0.5 μmol/l 9s. Images were taken at a
magnification of × 200; (i) the bar chart presented the quantification of fluorescence intensity of each group (mean ± SD, three biological replicates).
#
###
a
Compound-treated group versus b, P < 0.05;
P < 0.001. e and g versus c, P < 0.05. f versus c, **P < 0.01.
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8
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 5
Tube formation assay
(a)
(
(
(
(
(
(
(
(
(
b)
c)
d)
e)
f)
g)
h)
i)
j)
(k)
(l)
1
50
00
1
***
*
**
*
**
*
**
5
0
0
*
**
*
**
*
**
(µmol/L)
The effect of compounds on tube formation in HUVECs. The human umbilical vein endothelial cells (HUVECs) were treated with sorafenib, 9k, and 9s.
Each compound was divided into three concentration groups of 0.25, 1.0 and 4.0 μmol/l, respectively. (a) The HUVECs without treatment; (b–d) the
HUVECs treated with 0.25, 1.0, or 4.0 μmol/l sorafenib; (e–g) the HUVECs treated with 0.25, 1.0, or 4.0 μmol/l 9k; and (g, h, j) the HUVECs treated
with 0.25, 1.0, or 4.0 μmol/l 9s. The density of tubules and tubular junctions elaborated by HUVEC were visualized in Matrigel culture. The dose-
dependent effect of compounds 9k and 9s on the total tube length (k) and number of branches (l) in HUVECs. Results are representative of three
independent experiments. Images were taken at a magnification of × 200. The treated group versus control, ***P < 0.01, n =4.
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A series compounds to inhibit VEGFR-2 Huang et al.
9
tetrahydroisoquinoline-based compounds, compounds 9k
and 9s exhibited comparable or superior antiproliferative
activity to that of gefitinib against the tested three cell lines,
including A549, MCF-7, and PC-3. To investigate their
cytotoxicity toward normal cells, both compounds were
assayed against HUVECs. Consequently, they displayed
comparable cytotoxicity against HUVECs to that of
gefitinib.
Conflicts of interest
There are no conflicts of interest.
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signaling plays an intimate role in tube formation.
Thus, 9k and 9s, with capability to down-regulate
VEGFR-2 signaling, were then evaluated for their
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at the concentration of 4.0 μmol/l. At the dosage, their
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k and 9s than sorafenib in tube formation may imply
sorafenib is capable to suppress tube formation
via modulating pathway (s) other than VEGFR-2
signaling.
1
1
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Taken together, owing to their favorable in-vitro perfor-
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Acknowledgements
This study was supported by grants from the National
innovative drug incubation base project (2012ZX09401066).
1
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