Design, synthesis, and in vitro and in vivo anti-angiogenesis study of a novel vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor based on 1,2,3-triazole scaffold

Article abstract of DOI:10.1016/j.ejmech.2020.113083

In the past five years, our team had been committed to click chemistry research, exploring the biological activity of 1,2,3-triazole by synthesizing different target inhibitors. In this study, a series of novel indole-2-one derivatives based on 1,2,3-triazole scaffolds were synthesized for the first time, and their inhibitory activity on vascular endothelial growth factor receptor-2 (VEGFR-2) was tested. Most of the compounds had shown promising activity in the VEGFR-2 kinase assay and had low toxicity to human umbilical vein endothelial cells (HUVECs). The compound 13d (IC₅₀ = 26.38 nM) had better kinase activity inhibition ability than sunitinib (IC₅₀ = 83.20 nM) and was less toxic to HUVECs. Moreover, it had an excellent inhibitory effect on HT-29 and MKN-45 cells. On the one hand, by tube formation assay, transwell, and Western blot analysis, compound 13d could inhibit VEGFR-2 protein phosphorylate on HUVECs, thereby inhibiting HUVECs migration and tube formation. In vivo study, the zebrafish model with VEGFR-2 labeling also verified that compound 13d had more anti-angiogenesis ability than sunitinib. On the other hand, molecular docking and molecular dynamics (MD) simulation results showed that compound 13d could stably bind to the active site of VEGFR-2. Based on the above findings, compound 13d could be considered an effective anti-angiogenesis drug and has more development value than sunitinib.

Full text of DOI:10.1016/j.ejmech.2020.113083

Journal Pre-proof
Design, synthesis, and in vitro and in vivo anti-angiogenesis study of a novel vascular
endothelial growth factor receptor-2 (VEGFR-2) inhibitor based on 1,2,3-triazole
scaffold
De-pu Wang, Kai-li Liu, Xin-yang Li, Guo-qing Lu, Wen-han Xue, Xin-hua Qian,
Kamara Mohamed O, Fan-hao Meng
PII:
S0223-5234(20)31055-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.113083
EJMECH 113083
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 August 2020
Revised Date: 1 December 2020
Accepted Date: 1 December 2020
Please cite this article as: D.-p. Wang, K.-l. Liu, X.-y. Li, G.-q. Lu, W.-h. Xue, X.-h. Qian, K. Mohamed
O, F.-h. Meng, Design, synthesis, and in vitro and in vivo anti-angiogenesis study of a novel vascular
endothelial growth factor receptor-2 (VEGFR-2) inhibitor based on 1,2,3-triazole scaffold, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113083.
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2020 Published by Elsevier Masson SAS.

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Design, synthesis, and in vitro and in vivo anti-angiogenesis
study of a novel vascular endothelial growth factor
receptor-2 (VEGFR-2) inhibitor based on 1,2,3-triazole
scaffold
a,1
a,1
a, b
a
a
De-pu Wang , Kai-li Liu , Xin-yang Li , Guo-qing Lu , Wen-han Xue ,
a
a
a,*
Xin-hua Qian , Kamara Mohamed O , Fan-hao Meng
a
School of Pharmacy, China Medical University, Shenyang, 110122,
China.
b
Department of Pharmacy, Shengjing Hospital of China Medical
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University, Shenyang, 110004, China.
*
Correspondence: Professor Fan-hao Meng, School of pharmacy, China
Medical University, Shenyang, 77 Puhe Road, 110122, P. R. China.
E-mail: fhmeng@cmu.edu.cn Fax: +86-24-31939448
1
The contributions of these authors are equal to this work.
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Abstract
In the past five years, our team had been committed to click chemistry
research, exploring the biological activity of 1,2,3-triazole by
synthesizing different target inhibitors. In this study, a series of novel
indole-2-one derivatives based on 1,2,3-triazole scaffolds were
synthesized for the first time, and their inhibitory activity on vascular
endothelial growth factor receptor-2 (VEGFR-2) was tested. Most of the
compounds had shown promising activity in the VEGFR-2 kinase assay
and had low toxicity to human umbilical vein endothelial cells
(HUVECs). The compound 13d (IC = 26.38 nM) had better kinase
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activity inhibition ability than sunitinib (IC = 83.20 nM) and was less
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toxic to HUVECs. Moreover, it had an excellent inhibitory effect on
HT-29 and MKN-45 cells. On the one hand, by tube formation assay,
transwell, and western blot analysis, compound 13d could inhibit
VEGFR-2 protein phosphorylate on HUVECs, thereby inhibiting
HUVECs migration and tube formation. In vivo study, the zebrafish
model with VEGFR-2 labeling also verified that compound 13d had more
anti-angiogenesis ability than sunitinib. On the other hand, molecular
docking and molecular dynamics (MD) simulation results showed that
compound 13d could stably bind to the active site of VEGFR-2. Based on
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the above findings, compound 13d could be considered an effective
anti-angiogenesis drug and has more development value than sunitinib.
Keywords: 1,2,3-Triazole; Anti-angiogenesis; VEGFR-2; Zebrafish.
1. Introduction
Angiogenesis is the formation of new blood vessels by sprouting or
splitting from pre-existing blood vessels [1-4]. It plays a critical role in
the pathogenesis of various disorders, most notably growth and metastasis
of solid tumors [5]. The growth and metastasis of tumors require new
blood vessels to transport nutrients and oxygen [6]. Vascular endothelial
growth factor (VEGF) is a highly specific vascular endothelial growth
factor that is overexpressed in most solid tumors [7]. Excessive VEGF in
solid tumors promotes the angiogenesis of adjacent blood vessels, and the
excessive production of blood vessels can cause the imbalance of the
metabolic microenvironment and accelerate tumor invasion and
metastasis [6,8,9]. Therefore, anti-angiogenesis is an essential form of
inhibiting tumor growth and metastasis [10-12].
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Vascular endothelial growth factor receptor-2 (VEGFR-2)，as the vital
receptor of VEGF, which activated by VEGF initiates downstream signal
transduction, ultimately leads to angiogenesis, tumor proliferation, and
migration [7,13]. Therefore, a long-held concept that inhibition of
VEGFR-2 would cause efficient anti-angiogenesis and antitumor
response [14,15]. At present, the small molecule VEGFR-2 inhibitors
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have emerged as promising anti-angiogenesis agents against a wide
variety of cancers, such as sorafenib, cabozantinib, and sunitinib, and
applied in clinical cancer therapy (Fig. 1) [16-18]. However,
chemotherapy drugs can cause specific adverse side effects and affect
patients' health [19]. Therefore, it is necessary to find low toxicity
VEGFR-2 inhibitors with a novel structure to treat cancer and enrich
VEGFR-2 inhibitors [7].
In the course of identifying various chemical fragments that could
serve as a scaffold for novel anti-angiogenesis agents, our programs
began with the indolin-2-one derivative sunitinib, a potent multitargeted
kinase inhibitor of VEGFR-2, PDGFR, and c-KIT kinases [20-22]. The
privileged indolin-2-one scaffold (red dashed rectangle in Fig. 1) was
regarded as the most promising pharmacophore binding to the active site
of VEGFR-2 kinase [23,24]. Moreover, it should be pointed out that
numerous indolin-2-one derivatives have been reported as potent
antitumor agents [23,25-28]. Therefore, the indolin-2-one scaffold was
selected as a nucleus to develop a new VEGFR-2 inhibitor. The
combination principle of multiple pharmacophores for developing a
single molecule usually may enhance biological activity and improve
bioavailability [29]. In our previous work, inspired by various biological
properties of 1,2,3-triazole (blue dashed rectangle in Fig. 1), which could
bind with biomolecular targets to fulfill its envisaged role as potential
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pharmacophore [30-32], two series of 1,2,3-triazole derivatives (TS-13n,
B-10n in Fig. 1) had been independently reported as potent antitumor
agents in our laboratory [33-35].
Based on the findings mentioned earlier, we have been inspired to
design and synthesize a series of novel derivatives derived by insertion of
a 1,4-diphenyl-1H-1,2,3-triazole moiety on the active nucleus of
indolin-2-one through an ethylene bridge (Fig. 1). Herein, twenty-eight
novel indolin-2-one derivatives bearing 1,2,3-triazole moiety and their
VEGFR-2 kinase inhibitory activity were reported. These derivatives
exhibited potent inhibition of VEGFR-2 tyrosine kinase activity and
efficiently inhibited angiogenesis with less toxicity in vitro and in vivo,
suggesting that these derivatives could be considered promising lead
compounds for further development of novel anti-angiogenesis agents.
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Fig. 1. Structures of sorafenib, cabozantinib, sunitinib, TS-13n, B-10n
and design of the target compounds.
2. Results and discussion
2.1. Chemistry
The
synthetic
pathway
adopted
to
prepare
novel
indole-2-one-1,2,3-triazole derivatives (13a-14n) was depicted in
Scheme 1. First, the preparation of different substituted azide benzene
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(2a-2n) involved diazo-reaction and displacement reaction with sodium
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azide (NaN ) (synthetic detail procedure is given in the Experimental
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section) [35]. Click chemistry is a practical approach for generating a
1,2,3-triazole unit [34]. The novel intermediates (4a-4n) were obtained in
high yields via Cu (I)-catalyzed azide-alkyne cycloaddition (CuAAC)
between aryl-azides (2a-2n) and commercial 4-ethynylbenzaldehyde (3)
in the presence of sodium ascorbate and CuSO ·5H O as a catalyst in
4
2
DMF and water mixture as the solvent system [35]. The compounds 9-10
were prepared from compounds 5-6 via Sandmeyer's method described in
the literature [37,38], reduced which using hydrazine hydrate to give
compound 11-12 [39]. Finally, the title compounds (13a-14n) were
accomplished by employing Claisen-Schmidt condensation reaction
between indolin-2-ones (11-12) and various 1,2,3-triazole aromatic
aldehydes (4a-4n) with a catalytic amount of piperidine as a base [40,
41].
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Scheme 1. Reagents and conditions: (i) NaNO , HCl, NaN , CH Cl , H O,
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°
°
0-5 C, 3-5 h; (ii) CuSO ·5H O, ascorbic acid, KI, DMF, H O, 50 C, 6-10
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2
2
°
h; (iii) chloral hydrate, Na SO , NH OH·HCl, HCl, H O, 85 C, 3 h; (iv)
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2
°
°
°
conc H SO , 60 C, 0.5 h, 90 C 1.5 h; (v) N H ·H O, EtOH, H O, 100 C,
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°
10 h; (vi) EtOH, piperidine, 80 C, 4-8 h.
The known intermediates 9-12 and novel intermediates 4a-4n were
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characterized by melting points and spectroscopic techniques ( H NMR,
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ESI-MS), and the target compounds (13a-14n) also were characterized by
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melting points and spectroscopic techniques ( H NMR, C NMR, HRMS,
and FT-IR). The melting points of intermediates 9-12 approximately
matched with melting points data in existing literatures [42,43].
Since the indole-2-one derivative has an exocyclic double bond, the
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target compounds could exist in the form of E or Z isomers. As for
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compound 13m, 14a, 14b, 14j, 14k, and 14m, the H NMR spectra
revealed the existence of their inseparable E/Z mixtures, even the E/Z
ratios using the corresponding chemical shifts and integrals. However,
because we could not separate them from the silica gel column
13
chromatography or conventional method, the C NMR spectra of these
mixtures were not shown.
Although several documents had reported that the analogs of title
compounds obtained in the similar synthetic method were
Z-configurations [44,45], confirmation of the stereochemistry of
indole-2-one-1,2,3-triazole derivatives still be requisite. Thus, the most
potency compound 13d, as the representative compound, was chosen to
undergo the NOESY experiment. The NOE correlation (Fig. 2) between
H (δ=7.73 ppm) and H (δ=7.85 ppm) suggested that the isomer
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11
obtained correspond to Z-configuration (Z-13d), which should not exist in
E-configuration (E-13d). This result also illustrated that other compounds
(except 13m, 14a, 14b, 14j, 14k, and 14m) also were Z isomers.
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Fig. 2. NOESY of the representative compound 13d.
2.2. Biological evaluation
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2.2.1. VEGFR-2 inhibitory assay in vitro and structure-activity
relationship study (SAR)
To evaluate the inhibitory effects of sunitinib and all indole-2-one
derivatives based on 1,2,3-triazole scaffolds (13a-14n) on VEGFR-2
kinase activity, VEGFR-2 inhibitory assay was carried out and the results
were summarized in Table 1. Based on the VEGFR-2 kinase activity
evaluation, the preliminary structure-activity relationship (SAR) in this
work was summarized.
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Table 1. The IC value of compounds (13a-14n) and sunitinib against
50
VEGFR-2, HT-29 cells, MKN-45 cells, and HUVECs.
a
IC50
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Compd R
NO.
3a
R
VEGFR-2
(nM)
HT-29
(µM)
MKN-45(µM) HUVECs
(µM)
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H
93.11±1.95 7.07±1.10
110.60±2.37 8.71±0.48
11.59±0.81 12.68±1.69
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
H
H
H
9.77±0.86
4.69±0.24
1.92±0.37
14.69±0.93
13.70±0.13
7.94±0.36
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44.67±0.94 3.94±0.38
26.38±1.09 1.61±0.45
115.42±2.37 10.53±0.79 12.54±0.97 19.26±1.07
>200
>20
>20
>20
3g
3h
3i
198.54±1.37 17.73±3.80 16.70±0.82 14.71±0.23
>200
>200
>20
>20
>20
>20
>20
>20
3j
162.81±2.36 13.99±0.41 16.21±0.99 18.24±0.58
169.42±1.45 14.39±1.44 14.63±0.53 19.72±1.02
3k
3l
>200
>20
>20
>20
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3m
3n
4a
4b
4c
4d
4e
4f
H
H
F
F
F
F
F
F
F
F
F
F
F
F
F
178.91±1.36 15.78±2.55
108.36±0.88 9.67±0.39
99.67±0.73 8.12±1.29
71.55±0.16 5.87±0.32
89.16±0.63 7.32±0.64
36.90±0.54 3.98±0.61
139.50±1.65 8.98±0.42
171.12±1.09 12.97±0.72
>20
>20
>20
8.89±0.42
11.13±0.17 13.70±0.13
7.21±1.90
6.81±1.49
8.63±0.27
9.88±0.76
34.62±0.34 13.83±0.42
8.51±0.54
8.19±1.30
>20
14.36±1.99
17.25±1.22
>20
4g
4h
4i
192.51±0.47
>20
119.93±1.36 5.95±0.27
7.54±0.55
18.24±0.58
186.44±1.02 10.46±0.43 14.08±0.88 19.42±0.59
170.18±2.19 11.24±0.86 15.81±0.52 19.58±2.14
4j
4k
4l
187.24±2.88 13.98±0.43 15.21±0.73
>20
>20
>20
193.41±0.97
190.07±1.98
>20
>20
>20
>20
4m
1
2

1
4n
F
168.89±2.34 16.52±0.77
83.20±1.36 10.34±0.96
>20
>20
sunitinib -
-
9.25±0.77
6.37±0.59
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a ：IC50 values are presented as mean values of at least three independent
determinations.
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We found that compared to the positive drug (sunitinib IC = 83.20
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nM), most compounds exhibited excellent inhibitory activity against
VEGFR-2. The semi-inhibitory concentration IC50 value of seven
compounds (13a, 13c, 13d, 14a-14d) was less than 100 nM, which all
showed excellent inhibition to VEGFR- 2. Furthermore, 13d (IC =
5
0
26.38 nM) and 14d (IC = 36.9 nM) exhibited the best activities among
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them.
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Moreover, we analyzed the effect of two substitutions of the
indolin-2-one, 13a-13n (R = H) and 14a-14n (R = F) possessed similar
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IC values for each comparison. It showed that the substitution effect of
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the indole-2-one part might not have too obvious value on the
structure-activity relationship. Then, concerning the activity of
derivatives with an unsubstituted or substituted phenyl group attached to
triazole scaffold, incorporation of unsubstituted phenyl group or phenyl
substituted by electron-donating groups led to compounds 13a-13d and
14a-14d with the better inhibitory action on VEGFR-2 relative to the
electron-withdrawing groups substituted analogs (13e-13n and 14e-14n).
Furthermore, the activity of tolyl substituted and unsubstituted derivatives
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was 4-CH >3-CH >2-CH >H. Besides, there was no significant
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difference in the inhibitory activity between electron-withdrawing groups.
The results showed that the phenyl side groups substituted with
electron-donor groups might significantly influence indole 1,2,3-triazole
scaffolds' increased potency. In contrast, electron-withdrawing groups
might have a common effect or decrease the inhibitory activities of
scaffolds.
2.2.2. HT-29 cell lines, MKN-45 cell lines, HUVECs proliferation assay
in vitro
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CCK-8 assay was used to explore the effects of target compounds on
the cell proliferation of human colon cancer (HT-29) cell lines, human
gastric cancer (MKN-45) cell lines, and human umbilical vein endothelial
cells (HUVECs). The positive control sunitinib was also conducted to
assess the inhibitory abilities of cell proliferation of all compounds,
shown as IC values, and summarized in Table 1. Including the most
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potency compound 13d verified by the VEGFR-2 kinase inhibition assay,
the target compounds' overall toxicity to HUVECs was lower than that of
sunitinib. Furthermore, compound 13d had a good effect on inhibiting cell
viability for HT-29 and MKN-45 cells. Interestingly, combined with the
VEGFR-2 kinase inhibition assay results, the compound 13d could inhibit
the kinase activity of VEGFR-2 more effectively than sunitinib in vitro
and simultaneously had less toxic to HUVECs.
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2.2.3. Anti-angiogenesis effect in vitro
Based on the above experimental results, compound 13d had the most
potential to become a VEGFR-2 inhibitor. Therefore, the inhibitory effect
of compound 13d on angiogenesis was further explored. Transwell was
used to explore the inhibitory ability of compound 13d on HUVEC cell
migration, and the results were shown in Fig. 3A. As the compound
concentration increased, the migration ability of HUVECs decreased,
signified that the changes have occurred in a concentration-dependent
manner. Since compound 13d was less toxic to HUVECs at low
concentrations, the facts excluded the migratory inhibition of compound
13d caused by toxic effects. At the same time, HUVECs were treated
with the same concentration of compound 13d to explore its effect on
HUVECs' ability to form tubules, and the results were shown in Fig. 3B.
Similarly, the tube formative length decreased with the concentration of
13d increased.
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These data indicated that compound 13d could inhibit HUVECs
migration and tube formation in vitro, which was an essential step in
inhibiting angiogenesis.
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Fig. 3. Compound 13d inhibited HUVECs migration and the tube
formation. (A) Transwell analyzed the inhibitory effects of compound
13d on HUVECs migration. (B) Effects of compound 13d on the tube
formation of HUVECs in vitro.
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2.2.4. Western blot analysis and anti-angiogenesis research in vivo.
Furthermore, Western blot was carried out to analyze VEGFR-2
protein's phosphorylation on HUVECs cell membranes at different
compound concentrations. The results were shown in Figure 4A. As the
concentration of compound 13d increased, the phosphorylation of
VEGFR-2 in HUVECs decreased. That is, the activation of VEGFR-2
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decreased. Simultaneously, to verify the effect of compound 13d on
inhibiting angiogenesis in vivo, four groups of blood vessel-specific
fluorescent transgenic zebrafish, in a total of 40, were used for
anti-angiogenesis research. Zebrafish is currently the ideal vascular
biology research and anti-tumor angiogenesis drug evaluation model,
which can intuitively observe angiogenesis. The results were shown in
Fig. 4B. At a 40 mg/L concentration, compound 13d had a better
inhibitory effect on zebrafish internode vascular (ISV) angiogenesis than
sunitinib. Also, compound 13d even effectively inhibited zebrafish ISV
regeneration at a lower concentration (20 mg/L).
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In general, compound 13d could effectively inhibit the phosphorylation
of VEGFR-2 with better anti-angiogenesis ability than sunitinib in vivo.
These results were significantly different.
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Fig. 4. The compound 13d could inhibit the phosphorylation of VEGFR-2
and the angiogenesis of zebrafish microvessels. (A) Western blot analysis
of the phosphorylation changes of VEGFR-2 with increasing compound
13d concentration. (B) The effects of compound 13d and sunitinib on the
neovascularization of zebrafish in vivo.
2.3. Molecular modeling
2.3.1. Molecular docking study
Molecular docking simulations were performed using MOE (Molecular
Operating Environment, version 2016.08, Chemical Computing Group
Inc., Canada) software to study the possible binding pattern of target
compounds in the active site of VEGFR-2. Moreover, the crystal structure
of VEGFR-2 in complex with sunitinib (PDB ID: 4AGD) was adopted in
the docking calculations. The best docking conformation of the potent
compound 13d based on the compound activity against VEGFR-2 kinase
was selected as the most probable binding conformation. The positive
drug sunitinib, as the co-crystallized ligand, was re-docked using the
same procedure as 13d.
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The re-docking result demonstrated that sunitinib (Fig. 5B) could stack
well with the original ligand (Fig. 5A), two H-bonds predicted by the
simulation of sunitinib were exactly the same to those existed in the
crystal structure, and the Arene-H conjugates were roughly the same.
These data strongly certified the feasibility of the simulation and
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rationalized the predictive interactions of the compound 13d with
VEGFR-2 (Fig. 5C).
The docking conformation of compound 13d in the binding site
showed that carbonyl on the indole fragment formed a strong H-bond
with Phe1047, the lateral phenyl and the phenyl group linked to the
indole formed Arene-H conjugates with Val848 and Leu840, respectively.
By contrast, either H-bonds or the Arene-H interactions of 13d were less
than sunitinib. Simultaneously, as a whole molecule, 13d spatially
extended more in-depth into the pocket of protein; this provided a
reasonable explanation that compound 13d had better kinase activity
inhibition ability than sunitinib, which was confirmed by the VEGFR-2
kinase assay.
1
1
1
2
0

1
2
Fig. 5. The 2D diagram and 3D representation of original ligand (A),
sunitinib (B) and compound 13d (C) showing their interactions with the
VEGFR-2 active site (PDB code: 4AGD).
3
4
5
6
7
2.3.2. Molecular dynamics (MD) simulation
Based on the compound activity against VEGFR-2 kinase, the docking
conformation of the potent compound 13d obtained in MOE (Molecular
2
1

1
2
3
4
5
6
7
8
9
0
1
2
Operating Environment) was selected to conduct molecular dynamics
(MD) simulation in-depth the binding pose. MD simulation was then
carried out in explicit aqueous solution for 10 ns. For comparison, MD
simulation of positive control sunitinib in complex with VEGFR-2 was
also performed.
The stability of two systems under simulation was evaluated by the
root-mean-square deviation (RMSD) of the backbone atoms related to the
starting structures (Fig. 6). As can be seen in the plots, all systems were
stable during the 10-ns MD simulation; the values of RMSD of
compound 13d remained between 2 and 2.5 Å after reaching the summit,
which demonstrated that the hit compound 13d was stabilized in the
active site.
1
1
1
1
3
2
2

1
2
3
4
Fig. 6. RMSD tendency of two systems (13d and sunitinib) versus time in
the 10 ns MD simulation.
2.4. Toxicity prediction
Table 2. The pharmacokinetic studies of 13d and sunitinib.
Compound
NO.
Pharmacokinetic studies
Water
solubility
-3.051
Plasma protei Acute Oral
Tetrahymena
pyriformis
1.299
1.653
b
c
d
e
binding
0.831
Toxicity
1.773
1
3d
Sunitinib
-3.217
0.914
2.683
b
c
d
e
5
6
unit: logS; unit: 100%; unit: kg/mol; unit: pIGC (ug/L).
5
0
As a comprehensive and open-source tool, admetSAR was used to
7
8
9
0
1
2
3
4
5
6
7
8
9
0
predict 13d and sunitinib's toxicity, and the results were shown in Table 2
[46]. Since the acute oral toxicity units and Tetrahymena pyriformis are
kg/mol and pIGC (ug/L), respectively (the lower value represents the
50
1
1
1
1
1
1
1
1
1
1
2
lower toxicity), compound 13d had low toxicity than sunitinib in both
toxicity prediction assays, which inconsistent with the result of HUVECs
proliferation assay.
3. Conclusion
The newly synthesized indolin-2-one derivatives based on
1,2,3-triazole scaffolds, evaluated as VEGFR-2 inhibitors, showed
promising activity in the VEGFR-2 kinase inhibition assay. Among them,
compound 13d showed better activity inhibition on VEGFR-2 and
lowered toxicity to HUVECs, and had an excellent inhibitory effect on
HT-29 and MKN-45 cells than sunitinib. Transwell and tube formation
experiments showed that compound 13d inhibited HUVECs migration
2
3

1
2
3
4
5
6
7
8
9
0
1
2
3
and tube formation ability in a concentration-dependent manner.
Furthermore, western blot analysis manifested compound 13d could
decrease the phosphorylation of VEGFR-2 in HUVECs. At the same time,
by using VEGFR-2 specific fluorescent transgenic zebrafish, the
inhibitory effect of compound 13d on angiogenesis was verified in vivo.
Besides, the rationality and scientificity of molecular design were verified
by docking research and molecular dynamics simulation.
In conclusion, we designed and synthesized compound 13d as a novel
VEGFR-2 inhibitor that is more effective, less toxic than sunitinib.
Compound 13d provided a better choice for drug research and the
development of new VEGFR-2 inhibitors.
1
1
1
1
4. Experimental
4.1. Chemistry
1
1
4
5
4.1.1. General methods
Unless otherwise stated, all chemical reagents and solvents were
1
1
1
1
2
2
2
6
7
8
9
0
1
2
purchased from commercial sources and can be used without further
purification. Melting points were measured on a capillary electrothermal
melting point apparatus without calibration. By thin layer
chromatography (TLC), using GF254 silica gel from Qingdao Ocean
Chemical Company (Qingdao, China), the reaction progress was
monitored with a fluorescent indicator on a 254nm glass plate and
visualized by ultraviolet light. Column chromatography was performed
2
4

1
2
3
4
5
6
7
8
using silica gel (200-300 mesh) from Qingdao Ocean Chemical Company
1
13
(Qingdao, China), recorded H NMR and C NMR spectra on a Bruker
1
13
AMX500 ( H at 500 MHz, C at 126 MHz) magnetic resonance
spectrometer at ambient temperature. All NMR spectra were recorded
using DMSO-d as a solvent, and chemical shifts were reported in ppm
6
(parts per million) relative to tetramethylsilane (TMS) as an internal
standard.
4.1.2. The synthesis of substituted azido benzenes (2a-2n)
9
0
1
2
3
4
5
6
7
8
9
0
1
2
Compound 2a was synthesized by using the following procedure: To a
stirred solution of aniline (1a) (3.00 g, 32.21 mmol) in dichloromethane
1
1
1
1
1
1
1
1
1
1
2
2
2
(120 mL) were added 30 mL of aqueous NaNO (2.67 g, 38.66 mmol)
2
solution and 37% concentrated hydrochloric acid (HCl) (3 mL)
°
successively. After 0.5 h of stirring at 0 C, a solution of sodium azide
(NaN ) (2.93 g, 45.10 mmol) dissolved in water (20 mL) was added
3
°
dropwise, and the reaction mixture was kept stirring at 0-5 C for 3-5 h.
The progress of the reaction was monitored through the TLC test. After
the reaction was completed, the organic phase was separated and washed
with brine, dried over anhydrous Na SO , filtered, and the solvent was
2
4
removed under reduced pressure. The residue was purified by column
chromatography on silica gel using petroleum ether to afford the title
compound as pale yellow oil liquid.
Compounds 2b-2n were prepared using the identical synthetic
2
5

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
procedure as 2a.
4.1.3. The synthesis of substituted azido benzenes (4a-4n)
Compound 4a was synthesized by using the following procedure:
Reaction of commercially 4-ethynylbenzaldehyde (3) (1.00 g, 7.68 mmol)
with azidobenzene (2a) (1.01 g, 8.45 mmol) in 15 mL of DMF proceeded
°
at 50 C for 5 h in the presence of 4 mL of aqueous copper sulfate
pentahydrate (CuSO •5H O) (0.15 g, 0.60 mmol), ascorbic acid (0.15 g,
4
2
0.85 mmol) and a catalytic amount of KI. The progress of the reaction
was monitored through the TLC test. After the reaction was completed,
the reaction was added 100 mL of water and extracted with ethyl acetate
(3×100 mL). The combined extracts were washed with brine, dried over
anhydrous Na SO , filtered, and the solvent was removed under reduced
1
1
1
1
1
1
1
1
1
1
2
2
2
2
4
pressure. The crude residue was purified by column chromatography on
silica gel using ethyl acetate/petroleum ether to afford the title compound
as pale yellow solid.
Compounds 4b-4n were prepared using the identical synthetic
procedure as 4a.
4.1.3.1. 4-(1-phenyl-1H-1,2,3-triazol-4-yl)benzaldehyde (4a)
°
1
Light yellow solid, yield: 49.52%, mp: 187.3 C. H NMR (500 MHz,
DMSO-d ) δ 10.04 (s, 1H, CHO), 9.50 (s, 1H, H-triazole), 8.18 (d, J = 7.6
6
Hz, 2H, Ar-H), 8.05 (d, J = 7.5 Hz, 2H, Ar-H), 7.97 (d, J = 7.8 Hz, 2H,
Ar-H), 7.65 (t, J = 7.4 Hz, 2H, Ar-H), 7.54 (t, J = 7.3 Hz, 1H, Ar-H).
2
6

+
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
ESI-MS [M+H] m/z: 250.13.
4.1.3.2. 4-[1-(o-tolyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4b)
°
1
Light yellow solid, yield: 45.15%, mp: 119.4 C. H NMR (500 MHz,
DMSO-d ) δ 10.04 (s, 1H, CHO), 9.16 (d, J = 1.5 Hz, 1H, H-triazole),
6
8.18 (s, 2H, Ar-H), 8.04 (d, J = 3.1 Hz, 2H, Ar-H), 7.52 (s, 3H, Ar-H),
+
7.46 (s, 1H, Ar-H), 2.23 (s, 3H, CH ). ESI-MS [M+H] m/z: 264.15.
3
4.1.3.3. 4-[1-(m-tolyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4c)
°
1
Light brown solid, yield: 44.12%, mp: 174.4 C. H NMR (500 MHz,
DMSO-d ) δ 10.04 (s, 1H, CHO), 9.48 (s, 1H, H-triazole), 8.17 (d, J = 7.7
6
1
1
1
1
1
1
1
1
1
1
2
2
2
Hz, 2H, Ar-H), 8.04 (d, J = 7.7 Hz, 2H, Ar-H), 7.80 (s, 1H, Ar-H), 7.75 (d,
J = 7.5 Hz, 1H, Ar-H), 7.52 (t, J = 7.7 Hz, 1H, Ar-H), 7.35 (d, J = 7.0 Hz,
+
1H, Ar-H), 2.44 (s, 3H, CH ). ESI-MS [M+H] m/z: 264.15.
3
4.1.3.4. 4-[1-(p-tolyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4d)
°
1
Light brown solid, yield: 46.14%, mp: 189.4-193.3 C. H NMR (500
MHz, DMSO-d ) δ 10.04 (s, 1H, CHO), 9.46 (d, J = 1.8 Hz, 1H,
6
H-triazole), 8.17 (d, J = 6.4 Hz, 2H, Ar-H), 8.04 (d, J = 6.4 Hz, 2H, Ar-H),
7.84 (d, J = 6.4 Hz, 2H, Ar-H), 7.45 (d, J = 6.6 Hz, 2H, Ar-H), 2.40 (s, 3H,
+
CH ). ESI-MS [M+H] m/z: 264.16.
3
4.1.3.5. 4-[1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4e)
°
1
Light yellow solid, yield: 46.26%, mp: 126.8 C. H NMR (500 MHz,
DMSO-d ) δ 10.05 (s, 1H, CHO), 9.30 (s, 1H, H-triazole), 8.20 (d, J = 7.1
6
Hz, 2H, Ar-H), 8.04 (d, J = 7.2 Hz, 2H, Ar-H), 7.93 (t, J = 7.6 Hz, 1H,
2
7

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
Ar-H), 7.64 (d, J = 10.4 Hz, 2H, Ar-H), 7.49 (t, J = 6.8 Hz, 1H, Ar-H).
+
ESI-MS [M+H] m/z: 268.10.
4.1.3.6. 4-[1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4f)
°
1
Light brown solid, yield: 44.25%, mp: 179.9 C. H NMR (500 MHz,
DMSO-d ) δ 10.03 (s, 1H, CHO), 9.53 (s, 1H, H-triazole), 8.15 (d, J = 7.8
6
Hz, 2H, Ar-H), 8.04 (d, J = 7.8 Hz, 2H, Ar-H), 7.90 – 7.83 (m, 2H, Ar-H),
7.72 – 7.66 (m, 1H, Ar-H), 7.39 (t, J = 8.4 Hz, 1H, Ar-H). ESI-MS
+
[M+H] m/z: 268.09.
4.1.3.7. 4-[1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4g)
°
1
1
1
1
1
1
1
1
1
1
1
2
2
2
Yellow solid, yield: 46.54%, mp: 203.5 C. H NMR (500 MHz,
DMSO-d ) δ 10.04 (s, 1H, CHO), 9.47 (s, 1H, H-triazole), 8.16 (d, J = 6.8
6
Hz, 2H, Ar-H), 8.07 – 7.94 (m, 4H, Ar-H), 7.51 (dd, J = 9.1, 4.8 Hz, 2H,
+
Ar-H). ESI-MS [M+H] m/z: 268.10.
4.1.3.8. 4-[1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4h)
°
1
Light yellow solid, yield: 42.05%, mp: 121.3 C. H NMR (500 MHz,
DMSO-d ) δ 10.04 (s, 1H, CHO), 9.26 (s, 1H, H-triazole), 8.19 (d, J = 7.8
6
Hz, 2H, Ar-H), 8.04 (d, J = 7.8 Hz, 2H, Ar-H), 7.82 (t, J = 8.0 Hz, 2H,
Ar-H), 7.68 (t, J = 7.6 Hz, 1H, Ar-H), 7.63 (t, J = 7.5 Hz, 1H, Ar-H).
+
ESI-MS [M+H] m/z: 284.03.
4.1.3.9. 4-[1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl]benzaldehyde (4i)
°
1
Light yellow solid, yield: 46.15%, mp: 191.4 C. H NMR (500 MHz,
DMSO-d ) δ 10.04 (s, 1H, CHO), 9.57 (s, 1H, H-triazole), 8.15 (d, J = 6.1
6
2
8