Phenyl and Diaryl Ureas with Thiazolo[5,4-d]pyrimidine Scaffold as Angiogenesis Inhibitors: Design, Synthesis and Biological Evaluation

Article abstract of DOI:10.1002/cbdv.201800493

Angiogenesis is crucial for tumor growth and inhibition of angiogenesis has been regarded as a promising approach for cancer therapy. Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important factor in angiogenesis. In this work, a novel series of thiazolo[5,4-d]pyrimidine derivatives inhibiting angiogenesis were rationally designed and synthesized. Their inhibitory activities against human umbilical vein endothelial cells (HUVEC) were investigated in vitro. 1-(4-Fluorophenyl)-3-{4-[(5-methyl-2-phenyl[1,3]thiazolo[5,4-d]pyrimidin-7-yl)amino]phenyl}urea (19b) and 1-(3-Fluorophenyl)-3-{4-[(5-methyl-2-phenyl[1,3]thiazolo[5,4-d]pyrimidin-7-yl)amino]phenyl}urea (19g) exhibited the most potent inhibitory effect on HUVEC proliferation (IC₅₀=12.8 and 5.3 μm, respectively). Compound 19g could inhibit the migration of human umbilical vein endothelial cells. These results support the further investigation of these compounds as potent anticancer agents.

Full text of DOI:10.1002/cbdv.201800493

Title: Phenyl and Diarylureas with Thiazolo [5,4-d]pyrimidine Scaffold
as Angiogenesis Inhibitors: Design, Synthesis and Biological
Evaluation
Authors: Li-Ping Sun, Wen-jun Xue, Ya-hui Deng, Ji-ping Liu, Yu Liu,
and Zhong-hui Yan
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201800493
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800493

10.1002/cbdv.201800493
Chemistry & Biodiversity
Phenyl and Diarylureas with Thiazolo [5,4-d]pyrimidine Scaffold as
Angiogenesis Inhibitors: Design, Synthesis and Biological Evaluation
Wen-jun Xue1, Ya-hui Deng1, Zhong-hui Yan1, Ji-ping Liu2, Yu Liu2,*, Li-ping Sun1,**
1Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing, P. R. China
2School of Life Science and Technology, China Pharmaceutical University, Nanjing, P. R. China
*Author for correspondence: liuyu@cpu.edu.cn
**Author for correspondence: chslp@cpu.edu.cn
Abstract: Angiogenesis is crucial for tumor growth and inhibition of angiogenesis has been
regarded as a promising approach for cancer therapy. Vascular endothelial growth factor
receptor-2 (VEGFR-2) is an important factor in angiogenesis. In this work, a novel series of
thiazolo[5,4-d]pyrimidine derivatives inhibiting angiogenesis were rationally designed and
synthesized. Their inhibitory activities against human umbilical vein endothelial cells (HUVEC)
were investigated in vitro. Compound 19b and 19g exhibited the most potent inhibitory effect on
HUVEC proliferation (IC50 = 12.8 and 5.3μM respectively). Compound 19g could inhibit the
migration of human umbilical vein endothelial cells. These results support the further
investigation of these compounds as potent anticancer agents.
Keywords: thiazolopyrimidine, Angiogenesis, Anticancer agents
Graphical abstract:
Introduction
Angiogenesis is regulated by many growth factors. Among these growth factors, vascular
endothelial growth factor (VEGF) is very important in the direct regulation of angiogenesis[1]. The
VEGF family is a highly specific and potent proangiogenic factor, which can promote the
formation of tumor blood vessels. The VEGF family, which consists of six groups including
VEGF-A, -B, -C, -D, -E and the placental growth factor (PlGF), binds in an overlapping pattern to
different but structurally correlated VEGF receptors (VEGFRs)[2]. The three main members
(VEGFR1-3) of VEGFR belongs to the tyrosine kinase receptor superfamily (RTKs), playing an
important role in neovascularization and tumor growth[3]. The binding of VEGFs to VEGFRs
induces receptor dimerization, leads to phosphorylation of the intracellular kinase domain of
VEGFRs and triggers activation of downstream cell signaling cascades, including PI3K/AKT,
PKC, Raf/Ras and ERK pathways[4]. The activation of the VEGF-VEGFR signaling pathway
results in vascular/lymphatic endothelial cell proliferation and formation of new vessel branches.
Of the three members of the VEGFR family, VEGFR-2 is critical for vascular endothelial cell
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10.1002/cbdv.201800493
Chemistry & Biodiversity
development[5]. Inhibition of VEGFR-2 has been demonstrated as an attractive treatment method
in cancer. Various derivatives of phthalazines[6], anthranilamides[7], quinazolines[8], quinolones[9],
2-oxindoles[10] and pyrimidines[11] have been disclosed as potent inhibitors of VEGFR-2. We have
previously disclosed the synthesis and the evaluation of oxazolopyrimidines, exemplified by the
general structure 1 [12]. Based on this, we want to improve the physical and chemical properties of
these compounds to find more potent VEGFR-2 inhibitors. With the aim to enhance their affinity
with VEGFR-2, we envisioned the replacement of oxazolopyrimidine with thiazolopyrimidine to
provide the general structure series I and series II (Figure 1). We hypothesized that the
thiazolopyrimidine ring would bind to the hinge region of the kinase and designed series I
compounds. The structure of bis-aryl urea appears frequently in VEGFR-2 inhibitors[13-17]. Hence,
we introduced the urea moiety and envisioned that it would be a key recognition element to form
addition interaction with the back pocket of the kinase[18]. Then the series II compounds were
designed. In this paper, we reported the synthesis and evaluation of the two series compounds.
Figure 1. Replacement of the oxazolopyrimidine scaffold in the oxazolopyrimidine- based
VEGFR2 kinase inhibitors 1 by a thiazolopyrimidine moiety.
Results and discussion
Chemistry
The synthetic method of the target compounds 11a-11j is outlined in Scheme 1. The reaction
of diethyl malonate and acetamidine hydrochloride generated compound 4. After introducing a
nitro group into 4, intermediate 6 was obtained by chlorination of 5 by reacting with phosphorus
oxychloride[19]. Then compound 6 was converted to the corresponding amine 7 by reduction[20, 21]
.
The reaction of compound 7 with a substituted aromatic amine gave the amination product. The
amino group at the 5-position of the compound 9a-9j[19] reacted with an acid chloride to obtain the
intermediate 10a-10j[23]. After the action of Lawson's reagent with compound 10a-10j[22], the target
compounds were generated by cyclization in the presence of cesium carbonate.
The synthesis of 19a-19m was depicted in Scheme 2. Compound 14a was prepared by
reacting diethylaminomalonate with benzoyl chloride. The reaction of compound 14a with
Lawsson’s reagent gave the compound 15a. Compound 15a reacted with acetamidine
hydrochloride to give intermediate 16a and then it could produce the important intermediate 17a
under the action of phosphorus oxychloride.
The reaction of 4-nitroaniline with aromatic amines and triphosgene in toluene could give the
compound 22a. Then compound 22a was converted to corresponding amine 18a by reduction in
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10.1002/cbdv.201800493
Chemistry & Biodiversity
hydrogen[18]. The intermediate 17a-m reacted with 18a-m in isopropanol to afford the target
compounds in good yields.
(a) EtONa/EtOH, 85°C; (b) HNO3, AcOH, 10°C; (c) POCl3, PhNEt2, 115°C; (d) Fe, HCl, EtOH,
reflux; (e) EtOH/H2O, reflux; (f) ArCOCl, THF, 66°C; (g) Lawsson’s reagent,Cs2CO3, xylene,
110°C.
Scheme 1. Synthesis of compounds 11a-11j
(a) TEA, DCM, rt.; (b) Lawsson’s reagent, THF, reflux; (c) Acetamidine hydrochloride, NaOMe/
MeOH, reflux; (d)POCl3, N,N-diethylaniline, 110 °C ; (e) Isopropanol, HCl, 85°C .
(a) BTC, Et3N, toluene, 110°C; (b) H2/Pd, MeOH or ethyl acetate, r.t.
Scheme 2. Synthesis of compounds 19a-19m
Anti-proliferation assay
The anti-proliferative activity of the two series compounds against VEGF-induced HUVEC
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10.1002/cbdv.201800493
Chemistry & Biodiversity
proliferation was determined (shown in Table 1). Generally, moderate inhibitory activity was
shown in series I compounds with IC50 values at 10-5 to 10-6M. The compound 11h showed the
best antiproliferative activity with IC50 value of 21.28μM and was slightly better than the positive
control sorafenib. (sorafenib as a positive control: 73.16μM). Initial efforts were made to explore
substitutions on the two benzene rings. Based on the anti-proliferative activity, we found that the
substituents of the two benzene rings greatly affected the anti-proliferative activity of the
compounds in this series. According to the activities of the compounds 19a-19m, it can be seen
that the introduction of electron withdrawing group (4-Cl) to the A-ring has proven to be
detrimental to antiproliferative activity. When the Cl atom was replaced by a 4-MeO group in
series I, the VEGF-HUVEC inhibitory activity improved a little. Comparing the results of 11b-11d
and 11h-11j in series I compounds, the introduction of electron withdrawing group to the B-ring,
such as 4-Cl, resulted in the improvement of inhibitory activity.
After the introduction of urea group, the cellular activity of series II compounds was
significantly improved when compared to series I compounds. Compound 19b and 19g showed
good inhibitory activity (IC50 value of 12.8 and 5.3μM respectively) and the activity of compound
19g was comparable to the positive control sunitinib. When the hydrogen atom was replaced by a
chlorine atom in A-ring, the VEGF-HUVEC inhibitory activity decreased a little. Substituents of
the B-ring also had some effect on the anti-proliferative activity. The results were consistent with
that of series I compounds. Comparing the results of compounds 19a-19g and 19h-19m, it was
shown that when the electron withdrawing group was replaced by an electron donating group, the
VEGF-HUVEC inhibitory activity decreased. This may suggest that electron withdrawing groups
were more favorable for binding. A fluorine atom at 3-position on the B ring (compound 19g)
seems to be the optimal substituent and maintained strong inhibitory activity (IC50 = 5.3μM). The
information of SAR provided us a guideline in further evaluation of the compounds.
Some compounds of series I were determined the kinase inhibition rate of VEGFR (shown in
Table 2). Among them, the inhibition rate compound 11g was best and was only 37.3% at 50 μM.
At first, we envisioned that the replacement of oxazolopyrimidine with thiazolopyrimidine could
provide more potent VEGFR-2 inhibitors. These results suggested that series I compounds could
inhibit the proliferation of HUVEC and had some anti-angiogenetic effect. But the
anti-angiogenetic activity of these compounds was not due to VEGFR-2 competitive inhibition
and may have another unknown mechanism of action.
Table 1. Inhibition activity of thiazolo[5,4-d]pyrimidine derivatives on HUVEC proliferation and
VEGFR-2
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10.1002/cbdv.201800493
Chemistry & Biodiversity
Entry
Series
Compound
R1
R2
IC50/μM
[a]
1
11a
11b
I
I
H
H
H
4-Cl
\
2
41.14
58.71
292.4
\
3
11c
I
H
4-CH3
4-OCH3
4-Cl
4
11d
I
H
5
11e
I
4-Cl
4-Cl
4-Cl
4-OCH3
4-OCH3
4-OCH3
H
6
11f
I
4-CH3
4-OCH3
4-Cl
65.04
\
7
11g
I
8
11h
I
21.28
67.44
101.6
24.3
12.8
20.7
50.4
51.4
52.6
5.3
9
11i
I
4-CH3
4-OCH3
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
11j
I
19a
II
II
II
II
II
II
II
II
II
II
II
II
II
\
19b
H
4-F
19c
H
4-Cl
19d
H
3,4-diCH3
4-OCH3
4-CH3
3-F
19e
H
19f
H
19g
H
19h
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
\
H
91.7
50.3
66.5
59.4
66.26
57.6
73.16
1.16
19i
4-F
19j
4-Cl
19k
3,4-diCH3
4-OCH3
4-CH3
\
19l
19m
Sorafenib
Sunitinib
\
\
\
[a]: Not determined
Table 2. Inhibition activity of thiazolo[5,4-d]pyrimidine derivatives on VEGFR2 (percentage
inhibition at 50 μM)
%inhibition on VEGFR2
SD
Compound
11c
R1
H
R2
4-CH3
4-OCH3
4-Cl
-1.3
-12.5
35.4
15.5
37.3
7.7
0.9
3.8
1.3
7
11d
H
11e
4-Cl
4-Cl
4-Cl
4-OCH3
4-OCH3
4-OCH3
\
11f
4-CH3
4-OCH3
4-Cl
11g
1.1
0.7
3.4
0.1
0.4
11h
11i
4-CH3
4-OCH3
\
22.5
23.4
99.0
11j
Sorafenib
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10.1002/cbdv.201800493
Chemistry & Biodiversity
Wound Healing Assay
Based on the data obtained on VEGF-HUVEC anti-proliferation assay, the activity of compound
19g was best and was chosen to go on further evaluation considering the physicochemical
property of the compound. Endothelial cell migration plays an essential role in angiogenesis and
the inhibition of this process will block the formation of new blood vessels. Therefore, compound
19g was tested to assess its ability in inhibiting HUVEC migration. As illustrated in Figure 2,
compound 19g have some effect on the inhibition of HUVEC migration. In the negative control
group, the HUVECs migrated into the denuded area after 48h incubation. In the compound 19g
treated group, the HUVEC migration which was induced by VEGF was reduced to about 80%
(percentage number of migrated cells over the negative group) at the different concentration. It
could be shown that compound 19g could weakly suppress VEGF-induced migration of HUVEC
in vitro. Combined with the results of anti-proliferation assay, it demonstrated that compound 19g
was able to inhibit HUVEC proliferation but suppress HUVEC migration weakly.
6μM
3μM
1.5μM
NC
0h
48
h
Figure 2. Compound 33 inhibited the chemotactic invasion of HUVECs.
Conclusion
In summary, a novel series of thiazolo[5,4-d]pyrimidine derivatives inhibiting VEGFR were
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10.1002/cbdv.201800493
Chemistry & Biodiversity
rationally designed, synthesized, and identified. To our regret, the VEGFR-2 inhibition activity of
the new compounds was not improved by replacing oxazolopyrimidine[12] with thiazolopyrimidine.
The activity of aniline-substituted thiazolopyrimidine derivatives (series I) against VEGFR is not
satisfactory. Series I compounds had some effect on the inhibition of HUVEC proliferation but the
anti-angiogenetic effect of these compounds is not due to VEGFR-2 competitive inhibition. After
the replacement of benzene ring with the urea group, the activity of series II compounds were
significantly improved compared to series I compounds. Among them, compound 19b and 19g
exhibited the most potent inhibitory effect on HUVEC proliferation (IC50 = 12.8 and 5.3μM
respectively). Compound 19g could inhibit VEGF-stimulated migration weakly. Compound 19g was
able to inhibit HUVEC proliferation but suppress HUVEC migration weakly in an unknown
mechanism. In conclusion, the above results demonstrated that these compounds can be used as
candidate compounds for further investigation of angiogenesis inhibitors.
Experimental Section
General
Melting points (MP) were determined with XT-4 apparatus and are reported without correction.
Infrared spectra were obtained with Nicolet Impact 410 spectrophotometer. 1H nuclear magnetic
resonance (NMR) spectra were collected on a Brucker AMX 300 MHz spectrometer using CDCl3
or DMSO-d6 as the solvent with TMS as the internal reference. Mass spectra (EI) were obtained
on SHIMADZU GCMS-QP2010 system. All reactions were monitored by HPLC or by thin-layer
chromatography (TLC) in silica gel (purchased from Qingdao Haiyang chemical plant). All
products visualized with ultraviolet lamp (254 and 365 nm).
2-methylpyrimidine-4,6-diol (4)
In a three-necked flask, sodium (4.14 g, 180 mmol) was slowly added in portions to anhydrous
ethanol at 0°C. It was stirred until the sodium disappeared to obtain a sodium alcohol system.
Then acetamidine hydrochloride (6.24 g, 66 mmol) was slowly added. After stirring for about ten
minutes, diethyl malonate (9.6 g, 60 mmol) was added and the mixture was refluxed at 85 °C for
three hours. The reaction was cooled to room temperature and filtered. The residue was washed
with methanol and dissolved in hot water. pH was adjusted to 5 and then the filtered precipitate
was washed with ice water and anhydrous methanol twice and dried to give the title compound as
a white solid (6.42 g, 84.9%).
2-methyl-5-nitropyrimidine-4,6-diol (5)
Nitric acid (756mg, 0.5 mmol) was added to glacial acetic acid (360 mg, 6 mmol) at 0°C and
stirred. Then 2-methyl-4, 6-dihydroxypyrimidine (252 mg, 2 mmol) was slowly added into the
mixture in portions and reacted for 2 hours at 0°C . The reaction mixture was diluted with 0.5 mL
of ice water and stirred for five minutes. It was filtered and the filtered precipitate washed with ice
water to give a pink solid (230mg, 67.3%).
4, 6-dichloro-2-methyl-5-nitropyrimidine (6)
To a solution of 2-methyl-5-nitro-4,6-dihydroxypyrimidine (256 mg, 1.5 mmol) in phosphorus
oxychloride (1.5 ml), diethylaniline (317 mg, 2.6 mmol) was slowly added and the mixture was
heated to 115 °C for 3 hours. After cooled to room temperature, the mixture was add about 15 mL
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10.1002/cbdv.201800493
Chemistry & Biodiversity
of ice water and stirred for 30 min. The precipitate was collected by filtration and washed with ice
water to give a dark yellow solid (180 mg, 57.7%).
4, 6-dichloro-2-methylpyrimidin-5-amine (7)
2-methyl-5-nitro-4, 6-dichloropyrimidine (104 mg, 0.5 mmol) was added to a solution of
methanol/water (3ml, v:v = 9:1) and stirred. Iron powder (112 mg, 2 mmol) and a drop of
hydrochloric acid were added to the reaction mixture. The mixture was heated to 75 °C for five
hours. After cooled to room temperature, the mixture was filtered with celite and the precipitate
was washed with methanol repeatedly. The filtrate was concentrated under reduced pressure and
then extracted with dichloromethane. It was purified by flash chromatography on silica gel (ethyl
acetate/petroleum ether 1:3 to 1:2) to yield the title compound (60mg, 67.8%).
6-chloro-2-methyl-N4-phenylpyrimidine-4,5-diamine (9a)
4, 6-dichloro-2-methylpyrimidin-5-amine (340 mg, 1.9 mmol) was added to a solution of
ethanol/water (5ml, v:v = 1:8) and stirred. Then aniline (552 mg, 2 mmol) and a drop of
hydrochloric acid were added slowly. The reaction mixture was heated to 82 °C for 24 hours. The
reaction mixture was cooled and dried. It was purified by flash chromatography on silica gel (ethyl
acetate/petroleum ether 1:8) to yield the title compound (367 mg, 83.1%).
N-(4-chloro-2-methyl-6-(phenylamino)pyrimidin-5-yl)benzamide (10a)
2-methyl-5-amino-4-anilino-6-chloropyrimidine
(340
mg,
1.45
mmol)
and
4-dimethylaminopyridine (265 mg, 2.17 mmol) were added to a solution of anhydrous
tetrahydrofuran. A solution of benzoyl chloride (507 mg, 2.9 mmol) in tetrahydrofuran was slowly
added to the mixture. The reaction mixture was heated to 67 °C for 12 hours. After cooled to room
temperature, the reaction mixture was dissolved in ethyl acetate. Then it was washed with 5%
hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride. The organic layer
was dried over anhydrous sodium sulfate and filtered. It was dried to give a light brown solid (446
mg, 91.2%).
Diethyl benzamidomalonate (14a)
To a solution of diethylaminomalonate (1.05 g, 5 mmol) in anhydrous dichloromethane， the
benzoyl chloride (770 mg, 5.5 mmol) was added slowly. Then triethylamine (1.06g, 10.5 mmol)
was added dropwise to the system. The reaction mixture was stirred at room temperature for 4
hours. The reaction mixture was washed with 5% aqueous sodium hydroxide solution, 5%
hydrochloric acid and saturated sodium chloride. The organic layer was dried and was purified by
flash chromatography on silica gel (ethyl acetate / petroleum ether 1:4) to yield the title compound
as a white solid (1.19 g, 85%).
Diethyl 2-phenylthioamidomalonate (15a)
Benzoylamide malonate (2.23 g, 8 mmol) and Lawson's reagent (2.26 g, 5.6 mmol) was added to
anhydrous tetrahydrofuran. The mixture was stirred at 70°C for 8 hours. The reaction solution
was cooled and dried under vacuum. It was purified by flash chromatography on silica gel (ethyl
acetate / petroleum ether 1:8) and was placed in a refrigerator to be solidified. Then it was dried to
obtain a yellow powder (2.2 g, 93%). 1H-NMR (300 MHz, DMSO-d6 ): δ 1.22 (t, J = 7.1 Hz, 6H),
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10.1002/cbdv.201800493
Chemistry & Biodiversity
4.16-4.26 (m, 4H), 5.89 (s, 1H), 7.42-7.47 (m, 2H), 7.51-7.56 (m, 1H), 7.75-7.78 (m, 2H),10.96 (s,
1H).
N-(4, 6-dihydroxy-2-methylpyrimidin-5-yl) benzothioamide (16a)
In a three-necked flask, sodium (593 mg, 23.4 mmol) was slowly added in portions to anhydrous
methanol at 0°C. It was stirred until the sodium disappeared to obtain a sodium alcohol system.
Then acetamidine hydrochloride (725 mg, 7.8 mmol) was slowly added. After stirring for about
ten minutes, diethyl 2-phenylthioamidomalonate (2.09 g, 7.1 mmol) was added and the mixture
was refluxed at 75 °C for three hours. The reaction was cooled to room temperature and the pH
was adjusted to 3. It was filtered after 30 minutes and washed with a small amount of ice water
two times. It was dried to yield the title compound as a pale yellow solid (1.12 g, 60.5%).
7-chloro-5-methyl-2-phenylthiazolo[5,4-d]pyrimidine (17a)
To a solution of N-(4,6-dihydroxy-2-methylpyrimidin-5-yl) benzothioamide in phosphorus
oxychloride (4 ml), diethylaniline (845 mg, 5.7 mmol) was slowly added and the mixture was
heated to 110 °C for 12 hours. After cooled to room temperature, the mixture was added to 10
times of ice water and stirred. The precipitate was collected by filtration and dissolved in
dichloromethane. It was purified by flash chromatography on silica gel (ethyl acetate / petroleum
ether 1:20) to give a dark yellow solid (198 mg, 27.1%). 1H-NMR (300 MHz, DMSO-d6): δ 2.72
(s, 3H), 7.60-7.72 (m, 3H), 8.11-8.14 (m, 1H), 8.20-8.23 (m, 1H).
1-(4-nitrophenyl)-3-phenylurea (22a)
To a solution of triphosgene（178mg, 0.6 mmol） in toluene, aniline (177 mg, 1.65 mmol) and
Et3N（243 mg, 2 mmol）in toluene (3 mL) was added at 0 °C. After being stirred for 30min at room
temperature, the mixture was refluxed at 115°C. 4-nitroaniline (208 mg, 1.5 mmol) was added to
the reaction mixture. The reaction mixture was stirred for 12h at 115°C. Then the mixture was
cooled and filtered. The residue was washed with ethyl acetate and methanol, dried under vacuum
to give the title compound as a yellow solid (261 mg, 64%). ESI-MS: m/z 256.1[M-H]-.
1-(4-aminophenyl)-3-phenylurea (18a)
A mixture of 1-(4-nitrophenyl)-3-phenylurea (264 g, 0.97 mmol), ethyl acetate (5 mL), and 10%
Pd/C was hydrogenated in a round-bottomed flask for 24 hours, filtered through Celite,
concentrated to dryness under reduced pressure, and dried under vacuum to give
1-(4-aminophenyl)-3-phenylurea as a yellow solid (205 mg, 80%). ESI-MS: m/z 250.1[M+Na]+.
When the same procedure for preparing compound 26 was used, thirteen substituted diaryl ureas
were obtained.
1-(4-((5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)-3-phenylurea (19a)
7-chloro-5-methyl-2-phenylthiazolo[5,4-d]pyrimidine
(77
mg,
0.3
mmol)
and
1-(4-aminophenyl)-3-phenylurea（67 mg, 0.3 mmol）were added in to a round bottom bottle. Then
2ml isopropanol and a drop of hydrochloric acid were added. The mixture was refluxed overnight
at 82°C. It was then cooled and filtered. The residue was washed with methanol and ethyl acetate,
recrystallized in methanol to give the title compound (103 mg, 74.1%). m.p 230～231°C. IR
(KBr)： 3127.1, 1671.2, 1693.3, 1598.2, 1583.5, 1541.5, 1509.6, 1441.8, 1401.4, 1313.3, 1238.6,
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10.1002/cbdv.201800493
Chemistry & Biodiversity
1
1133.4, 907.2, 835.6, 735.7, 689.4, 555.9, 522.9 cm-1；H-NMR (300 MHz, DMSO-d6 ): δ 2.53 (s,
3H), 6.75 (d, J = 8.8 Hz, 2H), 7.23-7.30 (m, 3H), 7.45-7.53 (m, 5H), 7.64-7.70 (m, 2H), 8.02-8.06
(m, 2H), 8.63 (s, 1H), 8.72 (s, 1H), 10.09 (s, 1H). ESI-MS: m/z 453.1[M+H]+.
5-methyl-N,2-diphenylthiazolo[5,4-d]pyrimidin-7-amine (11a)
N-(4-chloro-2-methyl-6-(phenylamino)pyrimidin-5-yl)benzamide (67.6 mg, 0.2 mmol) and
Lawson's reagent (64.6 mg, 0.16 mmol) were added into a solution of anhydrous xylene. The
reaction mixture was heated to 110 °C for 2 hours. Then cesium carbonate (130 mg, 0.4 mmol)
was added and stirred for 5 hours. After cooled to room temperature, 10 mL dichloromethane was
added and the mixture was washed with 10 mL water. It was dried and the precipitate was washed
with anhydrous ethanol and dried to give a white solid (48.3 mg, 73.1%). m.p 190-192 °C. IR
(KBr): 3394.2, 3127.4, 1611.0, 1580.8, 1541.9, 1494.9, 1468.3, 1441.6, 1401.2, 1268.4, 1236.2,
1125.8, 906.2, 752.1, 686.5, 577.4 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 2.54 (s, 3H), 7.09 (t,
J = 7.3 Hz, 1H), 7.34-7.39 (m, 2H), 7.56-7.59 (m, 3H), 7.96 (d , J = 7.7 Hz, 2H), 8.10-8.14 (m,
2H), 9.81 (s, 1H). ESI-MS: m/z 317.1[M-H]-.
N-(4-chlorophenyl)-5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-amine (11b)
The title compound was obtained as a solid. Yield: 53%. m.p 185～187°C. IR（KBr）： 3126.7,
1610.7, 1576.9, 1544.1, 1490.1, 1466.5, 1400.9, 1124.6, 1029.5, 986.8, 907.9, 555.8, 430.2 cm-1；
1H-NMR (300 MHz, DMSO-d6): δ 2.56 (s, 3H), 7.43 (d, J = 8.8 Hz, 2H), 7.58-7.60 (m, 3H), 8.03
(d, J = 8.8 Hz, 2H), 8.11-8.15 (m, 2H), 9.95 (s, 1H). ESI-MS: m/z 351.1[M-H]-.
5-methyl-2-phenyl-N-(p-tolyl)thiazolo[5,4-d]pyrimidin-7-amine (11c)
The title compound was obtained as a solid. Yield: 38%. m.p 192～193°C. IR（KBr）： 3395.4,
3126.9, 1610.7, 1576.2, 1545.2, 1519.8, 1468.1, 1401.1, 1355.5, 1329.0, 1268.3, 1237.1, 1123.1,
1
906.2, 811.4, 761.3, 687.2, 629.3, 573.4, 508.0 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.30 (s,
3H), 2.53 (s, 3H), 7.18 (d, J = 8.2 Hz, 2H), 7.58-7.60 (m, 3H), 7.82 (d, J = 8.2 Hz, 2H), 8.11-8.15
(m, 2H), 9.74 (s, 1H). ESI-MS: m/z 333.1[M+H]+.
N-(4-methoxyphenyl)-5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-amine (11d)
The title compound was obtained as a solid. Yield: 44.1%. m.p 152～154°C. IR（KBr）： 3126.9,
1618.0, 1581.7, 1542.7, 1517.4, 1467.2, 1400.6, 1249.5, 1124.8, 1030.1, 905.1, 821.9, 576.3,
1
554.9, 522.0 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.51 (s, 3H), 3.77 (s, 3H), 6.96 (d, J = 9.0
Hz, 2H), 7.57-7.60 (m, 3H), 7.82 (d, J = 9.0 Hz, 2H), 8.10-8.14 (m, 2H), 9.74 (s, 1H). ESI-MS:
m/z 349.1[M+H]+.
N,2-bis(4-chlorophenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-amine (11e)
The title compound was obtained as a solid. Yield: 56.8%. m.p 318～320°C. IR（KBr）： 3127.1,
1
1655.1, 1617.9, 1400.4, 1133.2, 907.2, 554.3, 524.2 cm-1； H-NMR (300 MHz, DMSO-d6): δ
2.56 (s, 3H), 7.43 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 8.9 Hz, 2H), 8.14 (d, J
= 8.4 Hz, 2H), 10.00 (s, 1H). ESI-MS: m/z 403.0[M+OH]-.
2-(4-chlorophenyl)-5-methyl-N-(p-tolyl)thiazolo[5,4-d]pyrimidin-7-amine (11f)
The title compound was obtained as a solid. Yield: 46.8%. m.p 182～184°C. IR（KBr）： 3127.0,
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10.1002/cbdv.201800493
Chemistry & Biodiversity
1608.1, 1577.5, 1543.2, 1518.6, 1400.6, 1263.6, 1124.8, 1087.2, 1029.6, 987.3, 904.8, 832.3,
1
813.6, 734.9, 556.9, 495.4 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.28 (s, 3H), 2.51 (s, 3H),
7.16 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.3 Hz, 2H), 8.11 (d, J = 8.5 Hz,
2H), 9.77 (s, 1H). ESI-MS: m/z 365.1[M-H]-.
2-(4-chlorophenyl)-N-(4-methoxyphenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-amine (11g)
The title compound was obtained as a solid. Yield: 49%. m.p 172～174°C. IR（KBr）： 3397.9,
3126.8, 1610.6, 1580.1, 1542.4, 1507.2, 1465.7, 1400.5, 1355.9, 1264.4, 1246.9, 1171.7, 1121.2,
1
1088.2, 1031.5, 836.5, 815.8, 577.9, 558.3 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.51 (s, 3H),
3.77 (s, 3H), 6.96 (dd, J = 9.0 Hz, J = 1.9 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 9.0 Hz,
2H), 8.13 (d, J = 8.6 Hz, 2H), 9.77 (s, 1H). ESI-MS: m/z 381.0[M-H]-.
N-(4-chlorophenyl)-2-(4-methoxyphenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-amine (11h)
The title compound was obtained as a solid. Yield: 67%. m.p 253～255°C. IR（KBr）： 3128.6,
1605.1, 1571.9, 1540.0, 1491.4, 1466.2, 1402.6, 1327.1, 1308.8, 1255.9, 1180.8, 1119.1, 1029.8,
835.0, 561.0, 508.7 cm-1； 1H-NMR (300 MHz, DMSO-d6): δ 2.56 (s, 3H), 3.86 (s, 3H), 7.15 (d, J
= 8.8 Hz, 2H), 7.43 (d, J = 8.9 Hz, 2H), 8.03 (d, J = 9.0 Hz, 2H), 8.09 (d, J = 8.8 Hz, 2H), 9.89 (s,
1H). ESI-MS: m/z 381.1[M-H]-.
2-(4-methoxyphenyl)-5-methyl-N-(p-tolyl)thiazolo[5,4-d]pyrimidin-7-amine (11i)
The title compound was obtained as a solid. Yield: 66.1%. m.p 350～352°C. IR（KBr）： 3127.9,
1642.9, 1604.4, 1572.9, 1540.9, 1513.0, 1468.0, 1400.7, 1253.6, 1176.3, 1124.9, 1029.8, 906.8,
1
835.1, 554.0 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.30 (s, 3H), 2.52 (s, 3H), 3.86 (s, 3H),
7.12~7.19 (m, 4H), 7.81 (d, J = 8.7 Hz, 2H), 8.07 (d, J = 8.4 Hz, 2H), 9.65 (s, 1H). ESI-MS: m/z
361.1[M-H]-.
N,2-bis(4-methoxyphenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-amine (11j)
The title compound was obtained as a solid. Yield: 30.7%. m.p 177～178°C. IR (KBr)： 3127.2,
1609.3, 1582.5, 1537.9, 1511.0, 1465.7, 1400.5, 1353.7, 1326.3, 1300.4, 1255.6, 1229.8, 1172.2,
1
1112.4, 1029.6, 831.2, 630.7, 571.4 cm-1； H-NMR (300 MHz, DMSO-d6 ): δ 2.50 (s, 3H), 3.75
(s, 3H), 3.84 (s, 3H), 6.94 (d, J = 8.9 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.9 Hz, 2H),
8.03 (d, J = 8.7 Hz, 2H), 9.61 (s, 1H). ES-MS: m/z 379.2[M+H]+.
1-(4-fluorophenyl)-3-(4-((5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)urea
(19b)
The title compound was obtained as a yellow solid. Yield: 28.5%. m.p 221～222°C. IR (KBr)：
3126.8,1670.9, 1628.7, 1588.5, 1551.1, 1507.3, 1473.5, 1443.9, 1400.5, 1614.7, 1208.5, 1183.6,
1
904.9, 835.8, 763.9, 687.4, 554.7, 518.4 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.53 (s, 3H),
6.95 (t, J = 7.3 Hz, 1H), 7.25-7.32 (m, 2H), 7.44-7.47 (m, 4H), 7.57-7.60 (m, 3H), 8.11-8.13 (m,
2H), 8.70-8.72 (m, 2H), 9.81 (s, 1H). ESI-MS: m/z 505.2[M+Cl]-.
1-(4-chlorophenyl)-3-(4-((5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)ure
a (19c)
The title compound was obtained as a green solid. Yield: 66.5%. m.p 235～236°C. IR (KBr):
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10.1002/cbdv.201800493
Chemistry & Biodiversity
3128.2, 1617.3, 1541.7, 1508.4, 1400.8, 1312.9, 1126.3, 1029.4, 986.9, 908.1, 834.9, 734.9, 689.4,
1
555.4 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.56 (s, 3H), 7.23-7.31 (m, 2H), 7.44-7.49 (m,
3H), 7.55-7.60 (m,4H), 7.78 (dd, J = 8.9 Hz, J = 3.2 Hz, 2H), 8.13 (s, 2H), 9.12 (s, 1H), 9.40 (s,
1H), 10.19 (s, 1H). ESI-MS: m/z 487.1[M+H]+.
1-(3,4-dimethylphenyl)-3-(4-((5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-yl)amino)
phenyl)urea (19d)
The title compound was obtained as a yellow-green solid. Yield: 59%. m.p 278～279°C. IR (KBr)：
3126.5, 1618.8, 1585.2, 1541.6, 1505.9, 1400.9, 1313.8, 1124.7, 908.1, 835.1, 763.9, 735.0, 688.3,
1
554.5, 522.3 cm-1； H-NMR (300 MHz, DMSO-d6 ): δ 2.48 (s, 6H), 2.55 (s, 3H), 7.30-7.33 (m,
3H), 7.49 (d, J = 8.9 Hz, 2H), 7.57-7.60 (m, 3H), 7.79 (d, J = 8.6 Hz, 2H), 8.13 (s, 2H), 9.65 (s,
2H), 10.10 (s, 1H). ESI-MS: m/z 502.2[M-H+Na]-.
1-(4-methoxyphenyl)-3-(4-((5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)u
rea (19e)
The title compound was obtained as a bright-yellow solid. Yield: 71.4%. m.p 268～269°C. IR
（KBr）： 3127.9, 2535.8, 2361.0, 1700.8, 1620.4, 1586.4, 1560.8, 1541.1, 1508.1, 1466.8, 1401.0,
1314.2, 1227.8, 1178.5, 1125.4, 1030.2, 910.4, 832.2, 763.0, 687.33, 634.1, 555.85, 521.8 cm-1；
1H-NMR (300 MHz, DMSO-d6): δ 2.54 (s, 3H), 3.68 (s, 3H), 6.85 (d, J = 8.9 Hz, 2H), 7.35 (d, J =
8.9 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.57-7.59 (m, 3H), 7.77 (d, J = 8.8 Hz, 2H), 8.11-8.14 (m,
2H), 8.72 (s, 1H), 8.84 (s, 1H), 10.06 (s, 1H). ESI-MS: m/z 483.2[M+H]+.
1-(4-((5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)-3-(p-tolyl)urea (19f)
The title compound was obtained as a yellow-green solid. Yield: 32.2%. m.p 232～234°C. IR
（KBr）： 3126.7, 1618.2, 1508.2, 1400.6, 1125.0, 987.0, 908.0, 734.9, 554.9 cm-1； 1H-NMR (300
MHz, DMSO-d6): δ 2.48 (s, 3H), 2.55 (s, 3H), 7.06 (d, J = 8.7 Hz, 1H), 7.27-7.34 (m, 2H),
7.45-7.49 (m, 2H), 7.53-7.59 (m, 4H), 7.76-7.81 (m, 2H), 8.13 (s, 2H), 8.96 (s, 1H), 9.17 (s, 1H),
9.51 (s, 1H). ESI MS: m/z 501.2[M+Cl]-.
1-(3-fluorophenyl)-3-(4-((5-methyl-2-phenylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)urea
(19g)
The title compound was obtained as a solid. Yield: 71.8%. m.p 293～295°C. IR（KBr）： 3375.2,
3129.0, 2473.1, 1702.7, 1618.8, 1590.2, 1561.3, 1540.2, 1512.6, 1490.1, 1440.6, 1400.9, 1312.6,
1
1267.4, 1253.6, 1201.6, 1183.7, 1137.1, 911.7, 836.4, 764.1, 687.0, 555.6, 522.0 cm-1； H-NMR
(300 MHz, DMSO-d6): δ 2.56 (s, 3H), 6.72-6.78 (m, 1H), 7.10 (d, J = 8.1 Hz, 1H), 7.24-7.32 (m,
1H), 7.45-7.51 (m, 3H), 7.58-7.60 (m, 3H), 7.79 (d, J = 9.0 Hz, 2H), 8.11-8.15 (m, 2H), 9.16 (s,
1H), 9.34 (s, 1H), 10.21 (s, 1H). ESI-MS: m/z 471.1[M+H]+.
1-(4-((2-(4-chlorophenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)-3-phenylure
a (19h)
The title compound was obtained as an orange solid. Yield: 48.9%. m.p 358～360°C. IR（KBr）：
3131.5, 1630.3, 1543.9, 1511.8, 1401.0, 1313.0, 1133.4, 908.1, 838.3, 735.2, 555.7, 523.1 cm-1；
1H-NMR (300 MHz, DMSO-d6): δ 2.52 (s, 3H), 6.94 (t, J = 7.3 Hz, 1H), 7.24-7.31 (m, 2H),
7.40-7.46 (m, 4H), 7.67(t, J = 8.3 Hz, 2H), 6.94 (t, J = 7.3 Hz, 2H),7.76-7.82 (m, 2H), 8.10-8.14
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10.1002/cbdv.201800493
Chemistry & Biodiversity
(m, 2H), 8.61-8.67 (m, 2H),9.80 (s, 1H), 10.13 (s, 1H). ESI-MS: m/z 521.1[M+Cl]-.
1-(4-((2-(4-chlorophenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)-3-(4-fluorop
henyl)urea (19i)
The title compound was obtained as a reddish brown solid. Yield: 19.8%. m.p 324～325°C. IR
（KBr）： 3129.8, 1629.8, 1561.0, 1508.9, 1400.8, 1124.7, 986.9, 907.3, 835.6, 733.6, 555.1,
1
522.3, 406.7 cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.53 (s, 3H), 7.07-7.13 (m, 2H), 7.39-7.46
(m, 4H), 7.64-7.69 (m, 2H), 7.75-7.80 (m, 2H), 8.09-8.14 (m, 2H), 10.19 (s, 1H), 10.29 (s, 1H),
10.37 (s, 1H). ESI-MS: m/z 550.2[M+HCOOH]-.
1-(4-chlorophenyl)-3-(4-((2-(4-chlorophenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-yl)amino)p
henyl)urea (19j)
The title compound was obtained as a red solid. Yield: 38.7%. m.p 295～297°C. IR（KBr）：
3127.1, 1629.3, 1545.1, 1509.0, 1400.7, 1311.6, 1124.8, 987.0, 907.1, 833.4, 734.8, 555.5, 523.3
1
cm-1； H-NMR (300 MHz, DMSO-d6): δ 2.53 (s, 3H), 7.29 (d, J = 8.9 Hz, 2H), 7.40-7.47 (m,
2H), 7.53-7.57 (m, 2H), 7.65-7.70 (m, 2H), 7.76-7.80 (m, 2H), 8.10-8.15 (m, 2H), 10.19 (s, 1H),
10.29 (s, 1H), 10.37 (s, 1H). ESI-MS: m/z 520.1[M+H]+.
1-(4-((2-(4-chlorophenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)-3-(2,3-dimet
hylphenyl)urea (19k)
The title compound was obtained as a dark red solid. Yield: 72.1%. m.p 295～297°C. IR（KBr）：
1
3126.9, 1624.4, 1508.9, 1400.7, 1125.6, 987.2, 911.7, 833.1, 733.7, 556.3, 522.7 cm-1； H-NMR
(300 MHz, DMSO-d6): δ 2.42 (s, 6H), 2.53 (s, 3H), 7.18 (d, J = 8.7 Hz, 2H), 7.42-7.48 (m, 3H),
7.66 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H), 8.13 (d, J = 8.6 Hz, 2H), 9.65 (s, 2H), 10.10 (s,
1H). ESI-MS: m/z 515.2[M+H]+.
1-(4-((2-(4-chlorophenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)-3-(4-methox
yphenyl)urea (19l)
The title compound was obtained as a solid. Yield: 47.6%. m.p 278～280°C. IR（KBr）： 3126.9,
1629.9, 1560.8, 1509.8, 1400.8, 1126.5, 1029.9, 987.3, 911.1, 834.4, 732.7, 555.6 cm-1； 1H-NMR
(300 MHz, DMSO-d6): δ 2.52 (s, 3H), 3.70 (s, 3H), 6.85 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.9 Hz,
2H), 7.39-7.45 (m, 2H), 7.65-7.70 (m, 2H), 7.74-7.80 (m, 2H), 8.10-8.15 (m, 2H), 8.61 (s, 1H),
8.70 (s, 1H), 10.12 (s, 1H). ESI-MS: m/z 517.1[M+H]+.
1-(4-((2-(4-chlorophenyl)-5-methylthiazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)-3-(mtolyl)ur
ea (19m)
The title compound was obtained as a brown solid. Yield: 31%. m.p 318～319°C. IR（KBr）：
3127.9, 1629.6, 1542.8, 1509.9, 1401.2, 1311.4, 1221.9, 1124.9, 1091.4, 1021.3, 905.6, 831.7,
731.1, 556.2, 521.5 cm-1；1H-NMR (300 MHz, DMSO-d6 ): δ 2.25 (s, 3H), 2.56 (s, 3H), 7.07-7.15
(m, 1H), 7.30-7.38 (m, 2H), 7.30-7.38 (m, 2H), 7.45-7.47 (m, 2H), 7.78-7.87 (m, 2H), 8.14-8.18
(m, 2H), 8.57-8.62 (m, 1H), 10.18 (s, 1H). ESI-MS: m/z 502.2[M+H]+.
VEGFR-2 Kinase Assay
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10.1002/cbdv.201800493
Chemistry & Biodiversity
Materials: VEGFR-2 was purchased from Invitrogen. ATP, MgCl2, MnCl2, DTT were purchased
from Sigma. 384-well plate was purchased from Corning. HTRF KinEASE-TK was purchased
from Cisbio.
Dilute the compound to 50X of the final desired highest inhibitor concentration in reaction by
100% DMSO. Transfer 100µl of this compound dilution to a well in a 96-well plate. For all
compounds, transfer the compound in tubes to one well on 96-well storage plate and serially dilute
the compound by transferring 10 μl to 40 μl of 100% DMSO in the next well. Add 100μl of 100%
DMSO to two empty wells for no compound control and no enzyme control in the same 96-well
plate. Mark the plate as source plate.
Transfer 10μl of compound from source plate to a new 96-well plate as the intermediate plate.
Add 90μl of 1x Kinase base buffer to each well of the intermediate plate. Mix the compounds in
intermediate plate for 10 min on shaker. Transfer 5μl of each well from the 96-well intermediate
plate to a 384-well plate in duplicates.
Add kinase in 1x kinase base buffer. Add FAM labeled peptide and ATP in the 1x kinase base
buffer. Assay plate already contains 5μl of compound in 10% DMSO. Add 10μl of 2.5x enzyme
solution to each well of the 384-well assay plate. Incubate at room temperature for 10 min. Add
10μl of 2.5x peptide solution to each well of the 384-well assay plate. Incubate at 28°C for
specified period of time. Add 25μl of stop buffer to stop reaction.
Copy conversion data from Beckman Coulter program. Convert conversion values to
inhibition values according to percent inhibition ¼ (max-conversion)/(max-min)*100, in which
“max” stands for DMSO control; “min” stands for no enzyme activity control.
Cell Proliferation Assay
HUVECs were added onto 96-well microculture plates at a concentration of 4 · 104 cells/ml.
Then, they were incubated for 24h at 37°C under 5% CO2. The compounds were diluted to the
corresponding concentration using 5% FBS and appropriate amount of VEGF. The mediums with
different compound concentrations were added onto 96-well microculture plates. Cells were
continued to incubate for 48 h at 5% CO2, 37°C. After 48 h of drug treatment, cells were exposed
to 20μL MTT (0.5%) and were incubated at 37°C for further 4 h. After removing the medium,
100μL DMSO were added to each well and the optical density was measured at 490 nm. Average
IC50 values were determined by MTT tetrazolium dye assay. Each experiment was performed
three times.
Wound Healing Assay
HUVECs were added onto 6-well plates and the cells were confluent to form a monolayer
after overnight incubation. Use a sterile tip to draw a 1 to 2 mm wide of cell-free "scratch" area.
PBS buffer were used to wash away the free cells 3 times. DMEM cell culture mediums
containing different concentrations of the compounds were added to the cells. Low concentration
serum (3% FBS) was added to the culture medium to maintain normal cell growth and avoid the
effect of serum on cell migration. The scratched area was observed under an inverted microscope
at 0 h, 24 h and 48 h after dosing and photographed. After 48 h, the cell migration rate at each
compound concentration was calculated by comparing with 0 h. The effect of the compounds on
the migration ability of HUVEC was evaluated by the migration of endothelial cells in the
scratched area.
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10.1002/cbdv.201800493
Chemistry & Biodiversity
Acknowledgments
This work was supported by the National Natural Science Foundation of China (NO. 20872181,
NO. 21172265) and Innovation Program for the Postgraduates in Jiangsu Province (NO.
02705116).
Conflict of Interest: We declare that we have no conflicts of interest.
Author Contribution Statement
Wen-jun Xue, Ya-hui Deng designed and synthesized these compounds. Zhong-hui Yan, Ji-ping
Liu performed the VEGFR-2 Kinase Assay, Cell Proliferation Assay and Wound Healing Assay. Yu
Liu and Li-ping Sun conceived and designed the experiments.
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