Discovery of 4,6-Disubstituted Pyrimidine Derivatives as Novel Dual VEGFR2/FGFR1 Inhibitors

Article abstract of DOI:10.1002/cbdv.202100095

Abnormalities in the FGFRs signaling pathway and VEGFR2 amplification often occur in a variety of tumors, and they synergistically promote tumor angiogenesis. Studies have shown that the up-regulation of FGF-2 is closely related to the resistance of VEGFR2 inhibitors. Activation of the FGFRs signal is a signal of compensatory angiogenesis after VEGFR2 resistance. Dual VEGFR2/FGFR1 inhibitors contribute to overcoming the resistance of VEGFR2 inhibitors and inhibit tumor growth significantly. Based on this, we designed and synthesized a series of 4,6-disubstituted pyrimidine derivatives as dual VEGFR2/FGFR1 inhibitors by the molecular hybridization strategy. 3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)amino]pyrimidin-4-yl}-1-methylurea (8b) had the best inhibitory activities against VEGFR2 and FGFR1 at 10 μM (82.2 % and 101.0 %, respectively), it showed moderate antiproliferative activities against A549 and KG-1 cell lines as well. Besides, molecular docking was also carried out to study the binding mode of 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)-amino]-pyrimidin-4-yl}-1-methylurea (8b) with VEGFR2 and FGFR1. These studies reveal that this series of compounds deserve further optimization.

Full text of DOI:10.1002/cbdv.202100095

DOI: 10.1002/cbdv.202100095
FULL PAPER
Discovery of 4,6-Disubstituted Pyrimidine Derivatives as Novel Dual
VEGFR2/FGFR1 Inhibitors
Jin-Yang Zhang,a Wen-Jun Xue,a Min Wang,a Wen Li,a Ru Dong,a Ming-Tao Li,a and Li-Ping Sun*a
a
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, P. R. China, e-mail: chslp@cpu.edu.cn
Abnormalities in the FGFRs signaling pathway and VEGFR2 amplification often occur in a variety of tumors,
and they synergistically promote tumor angiogenesis. Studies have shown that the up-regulation of FGF-2 is
closely related to the resistance of VEGFR2 inhibitors. Activation of the FGFRs signal is a signal of compensatory
angiogenesis after VEGFR2 resistance. Dual VEGFR2/FGFR1 inhibitors contribute to overcoming the resistance of
VEGFR2 inhibitors and inhibit tumor growth significantly. Based on this, we designed and synthesized a series of
4,6-disubstituted pyrimidine derivatives as dual VEGFR2/FGFR1 inhibitors by the molecular hybridization strategy.
3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)amino]pyrimidin-4-yl}-1-methylurea (8b) had the
best inhibitory activities against VEGFR2 and FGFR1 at 10 μM (82.2% and 101.0%, respectively), it showed
moderate antiproliferative activities against A549 and KG-1 cell lines as well. Besides, molecular docking was also
carried out to study the binding mode of 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)-amino]-
pyrimidin-4-yl}-1-methylurea (8b) with VEGFR2 and FGFR1. These studies reveal that this series of compounds
deserve further optimization.
Keywords: VEGFR2, FGFR1, dual inhibitors, 4,6-disubstituted pyrimidine, antitumor activity.
effect with VEGFs in promoting tumor blood vessel
Introduction
growth.[7] The activation of FGFs signaling participates
Vascular Endothelial Growth Factor Receptor
2
in the resistance to VEGFs pathway inhibition (Fig-
(VEGFR2) plays a crucial role in promoting angio- ure 2).[8] Among them, FGF-2 plays a particularly
genesis and increasing vascular permeability as a prominent role in angiogenesis. FGF-2 is bound to
receptor tyrosine kinase, and inhibiting VEGFR2 has Fibroblast Growth Factor Receptors (FGFRs) and
become an effective strategy for tumor treatment.[1] induces endothelial cell proliferation. FGF-2 promotes
The FDA has approved several small-molecule VEGFR2 the expression of VEGF by enhancing the signal of the
inhibitors for tumor treatment, such as Sorafenib,[2] PI3K pathway, and VEGF also needs to be in the
Pazopanib,[3] and Lenvatinib (Figure 1).[4] As an increas- presence of FGF-2 to promote angiogenesis. When
ing number of VEGFR2 inhibitors were used clinically, VEGF and FGF-2 are expressed simultaneously, the
drug resistance has gradually become prominent.[5]
density and permeability of blood vessels will be
It was reported that FGF-2 overexpression is one of improved, and blood vessels will grow rapidly.[9]
the mechanisms of resistance to VEGFR2 inhibitors.[6] Furthermore, alterations of the FGFR gene have been
Fibroblast Growth Factors (FGFs) are division promot- detected in a variety of tumor types.[10] FGFR gene
ing factors and chemokine in vascular endothelial cells, alterations and VEGFR up-regulation are commonly
which can stimulate the migration, proliferation, and found in the same types of cancers, such as lung
differentiation of vascular cells, and have a synergistic cancer,[11,12] gastric cancer,[13,14] and breast cancer.[15]
For the above reasons, selective targeting FGFR1
and VEGFR2 has potential therapeutic advantages for
tumors, also can overcome VEGFR2 inhibitors resist-
ance. Furthermore, the aberrant tumor vasculature
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202100095
Chem. Biodiversity 2021, 18, e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
Figure 1. Structure of representative VEGFR and FGFR inhibitors.
dual VEGFR/FGFR inhibitor (Figure 1).[18] To avoid the
toxicities and side effects of drug interactions that may
be caused by the combination of single-target drugs,
we designed a series of dual VEGFR2/FGFR1 inhibitors
based on Pazopanib and NVP-BGJ398. Pazopanib, a
multitargeted kinase inhibitor, was approved by the
FDA in 2009 and 2012 for the treatment of renal cell
carcinoma and soft tissue sarcoma, respectively. Its
IC50 value is 30 nM for VEGFR2. According to reports,
the NH on aniline and the N on pyrimidine formed
hydrogen bonds with Cys919 of the hinge region, and
benzopyrazole filled the hydrophobic pocket.[3] NVP-
BGJ398, a potently pan-FGFR inhibitor with a pyrimi-
dine core, inhibited FGFR1-3 with the respective IC50
values of 0.9 nM, 1.4 nM, and 1.0 nM. The cocrystal
structure indicated that the NH on the aniline and the
N on the pyrimidine formed two key hydrogen bonds
with residue Ala564 of the hinge. The 3,5-dimeth-
oxyaniline fragment occupied the hydrophobic pocket,
Figure 2. VEGFR-FGFR signaling pathways.
mediated by pro-angiogenic factors enhanced tumor and its O formed a hydrogen bond with Asp641.[20]
potential by supporting immunosuppressive tumor Based on the study of the above binding mode, we
microenvironment
to
escape
host
immune designed a series of 4,6-disubstituted pyrimidine
surveillance.[16,17] Hence targeting the VEGFR and FGFR derivatives as dual VEGFR2/FGFR1 inhibitors using the
signaling pathway can also improve the tumor molecular hybridization strategy (Scheme 1).
immune microenvironment.[18]
At present, several FGFR inhibitors have been
approved by the FDA, such as Erdafitinib.[19] And some
FGFR inhibitors are in clinical studies, such as NVP-
BGJ398.[20] Besides, ODM-203 has been reported as a
www.cb.wiley.com
(2 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
Scheme 1. Design of target dual VEGFR2/FGFR1 inhibitors.
Results and Discussion
4,6-dichloropyrimidine (1) as the starting material, 4-
chloro-6-methylaminopyrimidine (2) was prepared by
amination reaction with methylamine. The intermedi-
Chemistry
The synthesis route of the target compounds 8a–8t ate 2 was acylated by triphosgene (BTC) with pyridine
was outlined in Scheme 2. All target compounds were as base to (6-chloropyrimidin-4-yl)(methyl)carbamic
synthesized according to the following route: Using chloride (3). Then, the intermediate 3 reacted with 3,5-
Scheme 2. Synthesis of target compounds 8a–8t. Reagents and condition: (a) Methylamine in ethanol (30–33%), i-PrOH, r.t.,1 h; (b)
°
°
BTC, pyridine, dry DCM, 0 C, 1 h; (c) 4 (3,5-dimethoxyaniline), Et3N, dry DCM, 0 C, 2 h; (d) NCS, dry CH3CN, reflux; (e) 7 (anilines),
°
concentrated HCl, i-PrOH, 90 C, 12 h.
www.cb.wiley.com
(3 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
dimethoxyaniline (4) in the presence of triethylamine was substituted by 4-methyl, the FGFR1 inhibitory
to
form
1-(6-chloropyrimidin-4-yl)-3-(3,5-dimeth- activities of compounds 8f, 8l, and 8s were better
oxyphenyl)-1-methylurea (5). The intermediate 5 and (99.9%, 86.2%, and 69.6%, respectively), and the VEGFR2
NCS were heated to reflux in dry acetonitrile to obtain inhibitory activities of 8f and 8l were also better (63.1%
1-(6-chloropyrimidin-4-yl)-3-(2,6-dichloro-3,5-dimeth-
oxyphenyl)-1-methylurea (6a), 1-(6-chloropyrimidin-4- para-position showed good inhibitory activities for
yl)-3-(2,4-dichloro-3,5-dimethoxyphenyl)-1-methylurea
VEGFR2. Compound 8b (R4 =4-methoxy, 82.2%) showed
and 67.8%, respectively). The compounds with R4 at
(6b), and 3-(2-chloro-3,5-dimethoxyphenyl)-1-(6-chlor- better inhibitory activity against VEGFR2 than compound
opyrimidin-4-yl)-1-methylurea (6c). Finally, intermedi- 8e (R4 =2-methoxy, 46.6%). Overall, compound 8b
ates 6a–6c were substituted with various anilines (7) exhibited the strongest potency against VEGFR2 and
under the condition of concentrated hydrochloric acid FGFR1 (82.2% and 101.0%, respectively), which were
to obtain the target compounds 8a–8t.
comparable to the reference drug Nintedanib (97.7%
and 101.1%, respectively).
Kinase Profiling
Antiproliferative Activity Study
Based on our previous research, the activities of
compounds 8a–8t against FGFR1 and VEGFR2 were FGFR gene alterations and VEGFR up-regulation were
determined at the concentration of 10 μM. The results frequently found in lung cancer and breast cancer, so
were shown in Table 1. In general, the inhibition rates of the antiproliferative activities of the compounds were
the target compounds 8a–8t on FGFR1 were slightly detected in human lung cancer A549 cell lines and
better than VEGFR2. The inhibition rates of compounds human breast cancer MCF-7 cell lines by MTT assays.
8a, 8b, 8f, and 8l for VEGFR2 and FGFR1 were above Besides, we chose the human liver cancer HepG-2 cell
60% and 85%, respectively. By analyzing the lines and leukemia KG-1 cell lines (FGFR1-translocated)
structureÀ activity relationship, we found that the posi- for antiproliferation assay by MTT or CCK-8 assays. As
tion of the chlorine atoms had a great influence on the shown in Table 2, the compounds showed better
potency. The compounds with both chlorine substituents activities against A549 cell lines and MCF-7 cell lines
at R1 and R3 showed good inhibitory activities. When R4 than HepG-2 cell lines in general. The compound 8b
Table 1. Kinase inhibition (%, 10 μM) against VEGFR2 and FGFR1.
Compound
R1
R2
R3
R4
Inhibition%[a] at 10 μM
VEGFR2
FGFR1
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
81.1
82.2
40.8
23.5
46.6
63.1
22.0
48.1
42.3
27.6
40.6
67.8
<0
30.4
31.2
<0
8.6
6.0
47.3
16.8
97.7
99.8
101.0
100.7
95.6
98.0
99.9
96.2
103.7
97.6
41.9
81.3
86.2
1.6
47.3
49.3
<0
27.2
43.7
69.6
33.9
101.1
4-OMe
2,6-di-F
4-CF3
2-OMe
4-Me
2-F
3-F
3-Cl-2-F
H
4-OMe
4-Me
8l
8m
8n
8o
8p
8q
8r
4-CF3
2,6-di-F
3-Cl-2-F
4-CF3
2,6-di-F
2-OMe
4-Me
8s
8t
H
H
3-Cl-2-F
Nintedanib
[a]
Assays were performed in replicate (n=2).
www.cb.wiley.com
(4 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
Table 2. In vitro antiproliferative activity in different tumor cell lines.
Compound
Antitumor cell proliferation
A549
MCF-7
HepG-2
(IC50, μM)
KG-1
(% at 10 μM)
(IC50,[a] μM)
(IC50, μM)
(% at1 μM)
8a
8b
8d
8f
8g
8h
8i
8j
>200
>200
>200
>200
N.A.[b]
>200
70.6
70.0
–
79.5
–
71.7
67.8
–
23.5
23.6
28.9
74.9
65.1
67.0
–
74.0
–
54.4
18.6
–
11.7
7.1
15.7
71.3
28.4�6.3
56.9�8.2
45.8�5.9
18.9�6.0
>200
92.6�31.7
28.2�0.6
51.3�6.0
22.8�9.2
>200
149.1�7.6
106.3�15.2
176.8�36.9
56.0�7.9
90.6�10.2
57.7�22.8
>200
>200
>200
23.2�3.2
11.0�1.5
113.6�6.4
83.4�19.5
2.4�0.8
46.0�2.6
21.2�2.8
>200
>200
4.7�0.2
8k
8l
8s
Nintedanib
2.8�0.5
[a]
[b]
Results are expressed as Mean�SD from three independent experiments. No activity. –, not tested.
exhibited a moderate antiproliferative effect on Cys919 residue in the hinge region of VEGFR2 (2.2 Å and
A549 cell lines (IC50 =28.4�6.3 μM), although it had 2.2 Å, respectively), and the 4-methoxyaniline fragment
both good potencies against VEGFR2 and FGFR1. of 8b forming a πÀ π stacking interaction with the
Moreover, the inhibition rate of compound 8b against Phe918 residue. The molecular docking results showed
KG-1 cell lines was 70.0% at 10 μM, which was similar that the interaction between compound 8b and FGFR1
to the reference drug Nintedanib (74.9% at 10 μM). was more strongly than that between compound 8b
Although 8k and 8g showed only medium potency and VEGFR2, which could explain that the inhibition
against VEGFR2, compound 8k had a better antiproli- activity of the compounds 8b against FGFR1 was slightly
ferative activity for MCF-7 cell lines (IC50 =11.0� better than the activity of VEGFR2. In general, molecular
1.5 μM), and compound 8g had a better antiprolifer- docking showed that compound 8b could bind to
ative activity for A549 cell lines (IC50 =18.9�6.0 μM). VEGFR2 and FGFR1 tightly. The specific structureÀ activity
Therefore, compound 8b was selected for subsequent relationship was shown in Figure 4.
molecular docking studies.
Molecular Docking
Conclusions
We clarified the binding mode of the effective inhibitor In summary, we designed and synthesized a series of
8b with VEGFR2 and FGFR1 by molecular docking. As 4,6-disubstituted pyrimidine derivatives as novel dual
shown in Figure 3, compound 8b could bind closely to VEGFR2/FGFR1 inhibitors, and then, evaluated their
FGFR1 through three hydrogen bonds, one cation-π VEGFR2/FGFR1 inhibitory activities and tumor cell
interaction, several electrostatic interactions, and many antiproliferation activities in vitro. Most of them
Van der Waals interactions. The 3,5-dimethoxyaniline showed good potency against FGFR1 and VEGFR2 and
fragment of 8b occupied a hydrophobic pocket, formed moderate antiproliferative activities. The inhibition
a cation-π interaction with the Lys514 residue. At the rates of 8b against VEGFR2 and FGFR1 were 82.2%
same time, one of the O of the methoxy group formed a and 101.0% at 10 μM, respectively. Besides, antiprolif-
hydrogen bond with the Asp641 residue (2.6 Å). The NH eration assay in vitro showed that compound 8b had
of aniline and the N of pyrimidine formed two key moderate inhibitory activity against human lung
hydrogen bonds with the Ala564 residue in the hinge cancer A549 cell lines with an IC50 of 28.4 μM.
region of FGFR1 (2.4 Å and 2.6 Å, respectively). Besides, Furthermore, molecular docking explained the inter-
the docking results showed that two hydrogen bonds actions of 8b with the VEGFR2 and FGFR1. Our results
and several electrostatic interactions were formed would provide the basis for the design and discovery
between 8b and VEGFR2. The NH of aniline and the N of of new dual VEGFR2/FGFR1 inhibitors.
pyrimidine formed two key hydrogen bonds with the
www.cb.wiley.com
(5 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
Figure 3. The docking model of compound 8b with VEGFR2 and FGFR1. The hydrogen bonds are displayed as yellow dotted lines.
(A) 3D model of 8b bound to VEGFR2 (PDB code: 3CJF). (B) Compound 8b bound to the ATP binding pocket of VEGFR2. (C) 3D
model of 8b bound to FGFR1 (PDB code: 3TT0). (D) Compound 8b bound to the ATP binding pocket of FGFR1.
Figure 4. SAR of compound 8.
www.cb.wiley.com
(6 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
Experimental Section
added to stir and dissolve under the ice bath
protected by nitrogen, then, the dry DCM solution of
compound 3 prepared in the previous step was added
Chemical Synthesis
The materials involved were purchased from commer- slowly. After the addition, dry triethylamine (8 mmol,
cial suppliers, the reagents and solvents are analyti- 1.2 mL) was added to the mixture slowly and stirred in
cally graded and can be used directly without further the ice bath for 1 h. At the end of the reaction, 10%
purification. All reactions were monitored by thin-layer diluted hydrochloric acid was added and stirred for
chromatography (TLC) (purchased from Qingdao Ma- 5 min, followed by DCM extraction (40 mL×3). The
rine Chemical Plant). Flash column chromatography organic phase was washed with brine, combined, and
was performed with silica gel (200–300 mesh, pur- dried over Na2SO4. After filtration and concentration in
1
chased from Qingdao Marine Chemical Plant). H- and vacuo, the residues were purified by silica gel flash
13C-NMR spectra were determined in (D6)DMSO or chromatography (DCM/methanol 400:1) to the ob-
CDCl3 solutions using the Bruker AV-300 spectrometer. tained white solid 5 with a two-step yield of 25.3% in
Q-tof high-resolution mass spectrometer was used for total.
mass spectrometry analysis. The melting points were
measured on the RY-1 MP device. The IR spectra were
1-(6-Chloropyrimidin-4-yl)-3-(2,6-dichloro-3,5-di-
recorded on a Nicolet iS10 infrared spectrometer.
methoxyphenyl)-1-methylurea
(6a)
and
1-(6-Chloropyrimidin-4-yl)-3-(2,4-dichloro-3,5-di-
methoxyphenyl)-1-methylurea (6b)
6-Chloro-N-methylpyrimidin-4-amine (2)
4,6-Dichloropyrimidine (3.45 g, 24 mmol) and 60 mL Compound 5 (1.5 mmol, 483.1 mg) was added to the
isopropanol were added to a reaction bottle, stirred, flask. Under the nitrogen atmosphere, dry acetonitrile
and 18 mL methylamine ethanol solution was slowly was added to the flask for stirring and dissolution.
added to the reaction bottle. The reaction solution Then, the solution of NCS (6 mmol, 801 mg) in DCM
°
was stirred for 1 h at room temperature. After the was slowly added and refluxed at 80 C for 1.5 h. The
reaction was completed, the reaction solution was mixture was washed with brine, combined, and dried
concentrated under reduced pressure. Next, purifica- over Na2SO4. After filtration and concentration in
tion of the crude residue by flash column chromatog- vacuo, the residues were purified by silica gel flash
raphy on silica gel (ethyl acetate/petroleum ether 4:1) chromatography (petroleum ether/ethyl acetate/DCM
afforded the white 2 (3.18 g, 92.3%).
8:1:1) to give 6a (330 mg, 56.4%) and 6b (250 mg,
42.7%).
(6-Chloropyrimidin-4-yl)methylcarbamic Chloride
(3)
3-(2-Chloro-3,5-dimethoxyphenyl)-1-(6-chloro-
pyrimidin-4-yl)-1-methylurea (6c)
In the atmosphere of nitrogen, triphosphamide
(4 mmol, 1.18 g) was added to the flask, and 20 mL dry Compound 5 (0.5 mmol, 161 mg) was added to the
DCM was added for stirring and dissolving. Anhydrous flask. Under the nitrogen atmosphere, dry acetonitrile
pyridine (8 mmol, 1.5 mL) was slowly added in an ice was added to the flask for stirring and dissolution.
bath. After the drip was completed, compound 2 Then, a solution of NCS (0.6 mmol, 80.2 mg) in DCM
°
(8 mmol, 1.15 g) in DCM was slowly added to the flask. was slowly added and heated to 80 C for 5 h. The
After stirring for 1 h in an ice bath, 10% diluted mixture was washed with brine, collect the organic
hydrochloric acid was added and stirred for 5 min. phase, and dried over Na2SO4. After filtration and
Then, 40 mL DCM was used to extract the organic concentration in vacuo, the residues were purified by
phase 3 times. The organic phase was washed with silica gel flash chromatography (petroleum ether/DCM
brine, combined, dried by Na2SO4, and then, concen- 10:1) to give 6c (86 mg, 48.3%).
trated.
3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-methyl-1-
[6-(phenylamino)pyrimidin-4-yl]urea (8a)
1-(6-Chloropyrimidin-4-yl)-3-(3,5-dimethoxy-
phenyl)-1-methylurea (5)
Compounds 6a and 7a (aniline) were successively
Compound 4 (3.2 mmol, 489.9 mg) was added to a added to a reaction tube containing 2 ml isopropanol
two-neck flask, and 15 mL dry DCM solution was and stirred. Then, drops of concentrated
3
www.cb.wiley.com
(7 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
°
hydrochloric acid were added and heated to 90 C
Caliper reading: Read and collected data on Caliper.
overnight. The reaction solution was concentrated Converted the converted values to inhibition rates.
under reduced pressure and dissolved in DCM again. Percent inhibition=(maxÀ conversion)/(maxÀ min)×
The mixture was washed with brine, combined organic 100, where ‘max’ stands for DMSO control and ‘min’
phase, and dried over Na2SO4. After filtration and stands for low control.
concentration in vacuo, the residues were purified by
TLC on silica gel (DCM/methanol 80:1) to give 8a as
pale yellow solid (5 mg, 11.2%). The preparation of the
Cell Proliferative Assay
other title compounds (8b–8t) was accomplished by a The antiproliferative activities of the compounds
similar synthetic procedure. All the spectroscopic data against A549, MCF-7, and HepG-2 cells were evaluated
of all the compounds can be found in Supporting by MTT assay. The cells (A549, MCF-7, and HepG-2) at
Information.
the logarithmic growth stage were inoculated in a 96-
well plate with 4000 cells per well and incubated at
°
5% CO2 and 37 C for 24 h. After adherent to the wall,
In vitro Kinase Assay
°
the cells were cultured for 72 h at CO2 and 37 C in
The potencies of the compounds against VEGFR2 and different concentration of compounds (10À 3 μM,
FGFR1 were measured using the caliper mobility shift 10À 2 μM, 10À 1 μM, 10 μM, 102 μM). The supernatant
assay in vitro. All compounds were separately dis- was discarded, and the serum-free medium containing
solved in DMSO to prepare the solution with a 0.5 mg/mL MTT of fresh preparation was added to
concentration of 10 mM.
each well with 20 μL. After the cultivation, the super-
natant was discarded and 150 μL DMSO was added to
Preparation of source plate: The compounds were dissolve the MTT precipitate per well. After it was
diluted to 50 times of the final desired maximum oscillated and mixed on a micro oscillator, the optical
inhibitory concentration in DMSO, and transferred density was measured at a wavelength of 570 nm on a
100 μL of this solution to the wells of a 96-well plate. microplate reader.
Took 100 μL of pure DMSO and added it to two vacant
wells of the same 96-well plate as a no enzyme control
The antiproliferative activities of the compounds
and a no compound control. The plate was a source against KG-1 cells were evaluated by CCK-8 assay. The
plate.
cells were digested and counted, and 3.0×104 cells
per mL cell suspension were prepared. The 100 μL cell
Preparation of intermediate plate: Took 10 μL of suspension was added to each well of the 96-well
°
the compound solution in the source plate and plate and incubated at 5% CO2 and 37 C for 24 h.
transferred it to an intermediate plate, it was a new Dilute the concentration of the compounds to 10 μM,
96-well plate. Took 90 μL of kinase base buffer and add 100 μL corresponding drug-containing medium to
add it to each well of the intermediate plate. Shaked each well, then, the cells were cultured for 72 h at CO2
°
the compounds in the intermediate plate for 10 min.
and 37 C. The 10 μL CCK-8 was added to each well
and incubated for 2 h. After it was oscillated and
Preparation of assay plate: In duplicate, took 5 μL mixed on a micro oscillator, the optical density was
of liquid from each well of the intermediate plate and measured at a wavelength of 450 nm on a microplate
transferred it to a new 384-well plate as an assay plate. reader.
Kinase reaction: Added kinase, FAM-labeled pep-
Molecular Docking
tide, and ATP in kinase base buffer in sequence. 5 μL
of compounds were already present in the 10% DMSO Molecular docking of the compound 8b into the X-ray
of the assay plate. Took 10 μL of 2.5 times enzyme structure of VEGFR2 (PDB code: 3CJF) and FGFR1 (PDB
solution and added it to each well of the assay plate. code: 3TT0) were carried out using the Maestro 11.8,
°
Incubated at 25 C for 10 min. Then, took 10 μL of 2.5 respectively. The crystal structure of VEGFR2 and
times peptide solution and added it to each well of FGFR1 were downloaded from RCSB Protein Data
°
the assay plate. Incubated at 28 C for a while. Took Bank. Unless otherwise specified, the default parame-
25 μL of stop buffer and added it to each well to stop ter settings were used. The steps of the docking
the reaction.
process are as follows: 1) Preparation of protein:
Proteins prepared using the tool of Protein Preparation
www.cb.wiley.com
(8 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
presses lymph node and lung metastases of human
Wizard in Maestro 11.8. The missing atoms and polar
hydrogens were added. Automatically optimized
hydroxy, Asn, Gln, and His states using ProtAssign in
the H-bond assignment. Deleted all water molecules.
The force field applied OPLS3e. 2) Receptor grid
generation: The whole protein was defined as the
receptor and the ATP binding site of the kinase was
selected as the binding site. The grid box was centered
on the ligand and defined to dock ligands similar in
size to the workspace ligand. 3) Preparation of ligand:
Compounds were processed using LigPrep of the
Maestro 11.8 with default settings. 4) Molecular
docking: Molecular docking was performed by Ligand
Docking of Glide. After the molecular docking, the
types of proteinÀ ligand interaction were analyzed. The
image files were generated using pymol.
mammary breast tumor MDA-MB-231 via inhibition of
vascular endothelial growth factor-receptor (VEGF-R) 2 and
VEGF-R3 kinase’, Clin. Cancer Res. 2008, 14, 5459–5465.
[5] N. Ferrara, ‘Pathways mediating VEGF-independent tumor
angiogenesis’, Cytokine Growth Factor Rev. 2010, 21, 21–
26.
[6] D. Ribatti, A. Vacca, M. Rusnati, M. Presta, ‘The discovery of
basic fibroblast growth factor/fibroblast growth factor-2
and its role in haematological malignancies’, Cytokine
Growth Factor Rev. 2007, 18, 327–334.
[7] A. Bikfalvi, S. Klein, G. Pintucci, D. B. Rifkin, ‘Biological roles
of fibroblast growth factor-2’, Endocrine Reviews 1997, 18,
26–45.
[8] O. Casanovas, D. J. Hicklin, G. Bergers, D. Hanahan, ‘Drug
resistance by evasion of antiangiogenic targeting of VEGF
signaling in late-stage pancreatic islet tumors’, Cancer Cell
2005, 8, 299–309.
[9] S. Javerzat, P. Auguste, A. Bikfalvi, ‘The role of fibroblast
growth factors in vascular development’, Trends Mol. Med.
2002, 8, 483–489.
[10] M. Touat, E. Ileana, S. Postel-Vinay, F. Andre, J. C. Soria,
‘Targeting FGFR Signaling in Cancer’, Clin. Cancer Res.
2015, 21, 2684–2694.
Acknowledgments
The authors appreciate the financial support from the
State Key Laboratory for the Chemistry and Molecular
Engineering of Medicinal Resources of Guangxi Normal
University (CMEMR2014-CMEMR2018).
[11] E. Carrillo de Santa Pau, F. C. Arias, E. Caso Pelaez, G. M.
Munoz Molina, I. Sanchez Hernandez, I. Muguruza Trueba,
R. Moreno Balsalobre, S. Sacristan Lopez, A. Gomez Pinillos,
M. del Val Toledo Lobo, ‘Prognostic significance of the
expression of vascular endothelial growth factors A, B, C,
and D and their receptors R1, R2, and R3 in patients with
non-small cell lung cancer’, Cancer 2009, 115, 1701–1712.
[12] N. Cihoric, S. Savic, S. Schneider, I. Ackermann, M. Bichsel-
Naef, R. A. Schmid, D. Lardinois, M. Gugger, L. Bubendorf, I.
Zlobec, ‘Prognostic role of FGFR1 amplification in early-
stage non-small cell lung cancer’, Br. J. Cancer 2015, 110,
2914–2922.
[13] S. Juttner, C. Wissmann, T. Jons, M. Vieth, J. Hertel, S.
Gretschel, P. M. Schlag, W. Kemmner, M. Hocker, ‘Vascular
endothelial growth factor-D and its receptor VEGFR-3: two
novel independent prognostic markers in gastric adenocar-
cinoma’, J. Clin. Oncol. 2006, 24, 228–240.
Author Contribution Statement
Jin-Yang Zhang and Wen-Jun Xue designed and
synthesized these compounds and wrote the article.
Ru Dong, Ming-Tao Li, Min Wang, and Wen Li
performed the experiments, analyzed the data, and Li-
Ping Sun designed and conceived the experiments.
[14] H. Murase, M. Inokuchi, Y. Takagi, K. Kato, K. Kojima, K.
Sugihara, ‘Prognostic significance of the co-overexpression
of fibroblast growth factor receptors 1, 2 and 4 in gastric
cancer’, Mol. Clin. Oncol. 2014, 2, 509–517.
[15] S. Ghosh, C. A. W. Sullivan, M. P. Zerkowski, A. M. Molinaro,
D. L. Rimm, R. L. Camp, G. G. Chung, ‘High levels of vascular
endothelial growth factor and its receptors (VEGFR-1,
VEGFR-2, neuropilin-1) are associated with worse outcome
in breast cancer’, Hum. Pathol. 2008, 39, 1835–1843.
[16] Y. Huang, S. Goel, D. G. Duda, D. Fukumura, R. K. Jain,
‘Vascular normalization as an emerging strategy to
enhance cancer immunotherapy’, Cancer Res. 2013, 73,
2943–2948.
[17] C. Salem, M. Yosra, C. Sophie, E. Bernard, H. Meriem, N. M.
Zaeem, ‘Hypoxia Promotes Tumor Growth in Linking
Angiogenesis to Immune Escape’, Front. Immunol. 2012, 3,
21–30.
[18] T. H. Holmstrom, A. M. Moilanen, T. Ikonen, M. L. Bjorkman,
T. Linnanen, G. Wohlfahrt, S. Karlsson, R. Oksala, T. Korjamo,
S. Samajdar, S. Rajagopalan, S. Chelur, K. Narayanan, R. K.
References
[1] G. Limaverde-Sousa, C. Sternberg, C. G. Ferreira, ‘Antiangio-
genesis beyond VEGF inhibition: a journey from antiangio-
genic single-target to broad-spectrum agents’, Cancer
Treat. Rev. 2014, 40, 548–557.
[2] L. Adnane, P. A. Trail, I. Taylor, S. M. Wilhelm, ‘Sorafenib
(BAY 43-9006, Nexavar), a dual-action inhibitor that targets
RAF/MEK/ERK pathway in tumor cells and tyrosine kinases
VEGFR/PDGFR in tumor vasculature’, Methods Enzymol.
2006, 407, 597–612.
[3] P. A. Harris, A. Boloor, M. Cheung, R. Kumar, R. M. Crosby,
R. G. Davis-Ward, A. H. Epperly, K. W. Hinkle, R. N. Hunter,
J. H. Johnson, ‘Discovery of 5-[[4-[(2,3-dimethyl-2H-indazol-
6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzene-
sulfonamide (Pazopanib), a novel and potent vascular
endothelial growth factor receptor inhibitor’, J. Med. Chem.
2008, 51, 4632–4640.
[4] J. Matsui, Y. Funahashi, T. Uenaka, T. Watanabe, A.
Tsuruoka, M. Asada, ‘Multi-kinase inhibitor E7080 sup-
www.cb.wiley.com
(9 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2100095
Ramachandra, J. Mani, R. Nair, N. Gowda, T. Anthony, S.
(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-
methyl-urea (NVP-BGJ398), a potent and selective inhibitor
of the fibroblast growth factor receptor family of receptor
tyrosine kinase’, J. Med. Chem. 2011, 54, 7066–7083.
Dhodheri, S. Mukherjee, R. K. Ujjinamatada, N. Srinivas, M.
Ramachandra, P. J. Kallio, ‘ODM-203, a Selective Inhibitor of
FGFR and VEGFR, Shows Strong Antitumor Activity, and
Induces Antitumor Immunity’, Mol. Cancer Ther. 2019, 18,
28–38.
[19] A. Markham, ‘Erdafitinib: First Global Approval’, Drugs
2019, 79, 1017–1021.
[20] V. Guagnano, P. Furet, C. Spanka, V. Bordas, D. G. Porta,
‘Discovery of 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-
Received February 5, 2021
Accepted April 6, 2021
www.cb.wiley.com
(10 of 10) e2100095
© 2021 Wiley-VHCA AG, Zurich, Switzerland