Design and discovery of quinazoline- and thiourea-containing sorafenib analogs as EGFR and VEGFR-2 dual TK inhibitors

Article abstract of DOI:10.3390/molecules23010024

Both EGFR and VEGFR-2 play a critical role in tumor growth, angiogenesis and metastasis, and targeting EGFR and VEGFR-2 simultaneously represents a promising approach to cancer treatment. In this work, a series of novel quinazoline- and thiourea-containing sorafenib analogs (10a–v) were designed and synthesized as EGFR and VEGFR-2 dual TK inhibitors. Their in vitro enzymatic inhibitory activities against EGFR and VEGFR-2, and antiproliferative activities against HCT-116, MCF-7 and B16 cell lines were evaluated and described. Most of the compounds showed potent activities against both cell lines and TK kinases. Compounds 10b and 10q which exhibited the most potent inhibitory activities against EGFR (IC₅₀ = 0.02 μM and 0.01 μM, respectively), VEGFR-2 (IC₅₀ = 0.05 μM and 0.08 μM, respectively), and good antiproliferative activities, also displayed competitive anti-tumor activities than sorafenib in vivo by B16 melanoma xenograft model test.

Full text of DOI:10.3390/molecules23010024

molecules
Article
Design and Discovery of Quinazoline- and
Thiourea-Containing Sorafenib Analogs as
EGFR and VEGFR-2 Dual TK Inhibitors
Shaofeng Sun †, Jingwen Zhang †, Ningning Wang, Xiangkai Kong, Fenghua Fu,
ID
Hongbo Wang *
and Jianwen Yao *
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation ( Yantai University),
Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs
in Universities of Shandong, Yantai University, Yantai 264005, China; chemsunshaofeng@sina.com (S.S.);
zhangjingwenydyy@163.com (J.Z.); wn573963424@163.com (N.W.); jwyao405@163.com (X.K.);
fenghua@luye.com (F.F.)
* Correspondence: hongbowangyt@gmail.com (H.W.); jwyao@ytu.edu.cn (J.Y.);
Tel.:+86-535-6706025 (H.W. & J.Y.)
† These authors contributed equally to this work.
Received: 2 December 2017; Accepted: 21 December 2017; Published: 23 December 2017
Abstract: Both EGFR and VEGFR-2 play a critical role in tumor growth, angiogenesis and metastasis,
and targeting EGFR and VEGFR-2 simultaneously represents a promising approach to cancer
treatment. In this work, a series of novel quinazoline- and thiourea-containing sorafenib analogs
(
10a–v) were designed and synthesized as EGFR and VEGFR-2 dual TK inhibitors. Their in vitro
enzymatic inhibitory activities against EGFR and VEGFR-2, and antiproliferative activities against
HCT-116, MCF-7 and B16 cell lines were evaluated and described. Most of the compounds showed
potent activities against both cell lines and TK kinases. Compounds 10b and 10q which exhibited the
most potent inhibitory activities against EGFR (IC50 = 0.02
µM and 0.01 µM, respectively), VEGFR-2
(IC50 = 0.05 M and 0.08 M, respectively), and good antiproliferative activities, also displayed
µ
µ
competitive anti-tumor activities than sorafenib in vivo by B16 melanoma xenograft model test.
Keywords: quinazoline; thiourea; sorafenib; TK inhibitor; molecular docking
1. Introduction
The epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor
(VEGFR-2), as potent targets for cancer therapy, have been proved to be crucial in signal transduction
pathways involved in tumor cell proliferation, differentiation, migration and angiogenesis [1–4]. As a
complicated signal network of interconnected circuits, EGFR and VEGFR-2 usually share common
downstream signaling pathways. EGFR inhibition can decrease VEGF expression and attenuate
angiogenesis, in the meantime leading to VEGFR-2 upregulation, and ultimately giving rise to the
resistance of EGFR inhibitors [
5,
6]. Therefore, the combined inhibition of both EGFR and VEGFR-2 has
thereby simultaneously become an efficient approach to cancer treatment with a synergistic effect [7,8
].
EGFR and VEGFR-2 share a catalytic domain that contains a cleft where adenosine triphosphate
(ATP) binds. Based on this catalytic cleft, some small-molecule TK inhibitors have been developed
and approved by the US Food and Drug Administration (FDA) in the past decade (Figure 1),
which include gefitinib (AstraZeneca, London, UK, 2003), erlotinib (Genentech, South San Francisco,
CA, USA and OSIP, Melville, NY, USA, 2004), sorafenib (Bayer, Leverkusen, Germany and Onyx,
South San Francisco, CA, USA, 2005), vandetanib (Bristol-Myers Squibb, New York, NY, USA, 2006),
lapatinib (GlaxoSmithkline, London, UK, 2007), regorafenib (Bayer, Leverkusen, 2012), and Afatinib
Molecules 2018, 23, 24; doi:10.3390/molecules23010024
www.mdpi.com/journal/molecules

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(Boehringer Ingelheim, Ingelheim, Germany, 2013) [
9
–
13]. Most of these tyrosine kinase inhibitors
contain various quinazoline scaffolds that can interact with the ATP-binding pocket of EGFR
and VEGFR-2.
Cl
Br
F
F
HN
HN
N
O
HN
N
O
N
N
O
O
N
O
O
N
O
N
O
O
N
Vandetanib
Erlotinib
Gefitinib
Cl
Cl
Cl
F
O
F
O
N
HN
HN
H
N
HN
H
N
N
HN
S
O
N
N
F
N
O
O
O
O
O
N
N
O
N
Lapatinib
Afatinib
Dacomitinib
Cl
F
O
N
N
N
HN
N
HN
N
NH
O
O
O
O
O
O
N
N
F
O
HN
O
O
N
Cediranib
Canertinib
Icotinib
R
N
N
H
N
H
Cl
N
N
N
H
H
N
N
O
N
O
N
F3C
O
O
O
O
O
R = H, Sorafenib
R = F, Regorafenib
Tandutinib
Figure 1. EGFR and/or VEGFR-2 tyrosine kinase inhibitors.
Sorafenib, a bisaryl ureas multikinase inhibitor, has been developed and launched for the
treatment of renal cell carcinoma (RCC) and unresectable hepatocellular carcinoma (HCC). Due to
its significant antitumor activities, more and more attention has been focused on the modification of
sorafenib for years [14–16]. In our previous work [17–20], several series of diaryl-thiourea-containing
sorafenib derivatives were designed and synthesized as antitumor agents (Figure 2); most compounds
showed moderate to stronger in vitro antiproliferative activities, and some compounds exhibited
potent inhibitory activities against the phosphorylation of VEGFR and the anti-angiogenic activities,
which suggested that the thiourea moiety may improve the cytotoxicity of these compounds to some
extent. Interestingly, compounds with 1,3-substitution on the B ring (Figure 2C) showed stronger
anti-angiogenic activities than those with 1,4-substitution [18].
In an attempt to develop potent and selective antitumor agents, a series of quinazoline- and
thiourea-containing sorafenib derivatives (as shown in Figure 2) were designed and synthesized,
and their inhibitory activities against EGFR, VEGFR-2, and three cancer cell lines were evaluated.
Moreover, the compounds 10b, 10m and 10q were chosen to estimate their in vivo antitumor activity
by a B16 melanoma xenograft model test. The compounds 10b and 10q exhibited the most potent
inhibitory activities and better in vivo antitumor activities than sorafenib. Docking simulation was
used to illustrate a common mode of interaction at the ATP binding site of EGFR and VEGFR-2.

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Figure 2. Modifications of sorafenib.
2. Results and Discussion
2.1. Chemistry
The synthetic route to the target compounds was illustrated as outlined in Scheme 1.
2-Amino-4,5-dimethoxy-benzoic acid (Compound ) was synthesized from methylvanillin
(Compound ) by nitration, oxidation and reduction, which was further treated with formamidine
4
1
◦
acetate in 140 C to give 6,7-dimethoxy-3H-quinazolin-4-one (Compound
5
5
) in 95.7% yield. Compound
) as a key
was reacted with SOCl2 to afford 4-chloro-6,7-dimethoxyquinazolin (Compound
6
intermediate of target compounds, which was condensed with 3-aminophenol or 3-aminothiophenol
to give corresponding compounds 7a and 7b respectively. Furthermore, various substituted anilines
(4-fluoro-3-trifluoromethylaniline, 4-chloro-3-trifluoromethylaniline, 4-bromo-3-trifluoromethylaniline,
3,5-di(trifluoromethyl)aniline, 3-trifluoromethylaniline, 4-trifluoromethoxyanoline, 2,4-dichloroaniline,
3,4-difluoroaniline, 4-chloroaniline, 4-fluoroaniline and 4-methylaniline) (8a
CS2 and then treated with BTC to generate corresponding phenyl isothiocyanates (9a
to good yields. Finally, these phenyl isothiocyanates were reacted with each compound (7a or 7b
in DCM at room temperature to afford target compounds (10a ) in the total yields of 40.1% to 74.7%.
–
k) were reacted with
–k) in moderate
)
–v
The final products were purified by column chromatography and their structures were characterized
by 1H-NMR and HRMS.
Scheme 1. Reagents and conditions: (a) nitric acid (b) potassium permanganate, NaOH (c) H2; Pd/C
(d) formamidine acetate (e) SOCl2; DMF (f) 3-amino phenol/3-amino thiophenol, KOBu-t, K2CO3;
DMF (g) Dabco; CS2, toluene (h) BTC; CHCl3 (i) DCM.

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2.2. Biology
2.2.1. EGFR and VEGFR-2 Inhibitory Assay
All the synthesized compounds (10a
–v
) were assayed with the enzymatic activities against EGFR
and VEGFR-2 using sorafenib as a positive control. As shown in Table 1, most of tested compounds
showed potent inhibitory activities against EGFR and VEGFR-2. Among them, compounds 10m and
10q displayed the most potent activity with IC50 of 0.01
µM against EGFR, and compound 10b showed
the most potent activity with IC50 of 0.05 M against VEGFR-2, which is comparable to sorafenib with
µ
IC50 of 0.02 µM against EGFR and 0.08 µM against VEGFR-2 respectively.
Structure–activity relationships (SARs) were inferred from the data of the enzymatic experiment
reported in Table 1. Compounds (10a, 10b, 10c, 10m, 10o and 10q), containing two strong electron-
withdrawing groups on the terminal aromatic ring, exhibited potent inhibitory activities against
EGFR with IC50 values ranging from 0.01 to 0.05 µM, and against VEGFR-2 with IC50 values ranging
from 0.05 to 0.19
showed an obvious decrease of activities (IC50 more than 10
µ
M. However, compounds 10f
,
10k
,
10n and 10v bearing electron-donating groups
M), The reasons may be as follows:
µ
(a) the compounds with electron-deficient terminal aromatic rings exist hydrophobic interaction
with specific amino acid residues; (b) some electron-withdrawing groups (such as -F, -Cl, -Br, -CF3)
on terminal aromatic rings can form hydrogen bonds by amino acid residues. Most of compounds
10l–
v, bearing diaryl thioether fragment, showed more potent activity against both EGFR and VEGFR-2
compared to the corresponding diaryl ether compounds 10a
–
k, which suggested that the thioether
moiety may improve the enzymatic inhibitory activity of these compounds. Moreover, the introduction
of chlorine substituent at ortho-position of the thiourea group displayed weaker activity against both
EGFR and VEGFR-2 (compounds 10g versus 10i, 10r versus 10t).
Table 1. Enzymatic and cellular results of the target compounds.
S
Ar
X
N
N
H
H
O
O
N
N
Comp
NO.
Subsituent
Ar
IC50 (µM) a
IC50 (µM) a
X
EGFR
VEGER-2
HCT116
MCF-7
B16
CF3
10a
10b
10c
O
0.04
0.19
37.36
38.15
17.72
22.36
16.95
F
CF3
Cl
O
O
0.02
0.05
0.05
0.18
9.13
6.11
9.68
Br
10.03
CF3
CF3
10d
O
N.D.
N.D.
15.02
18.82
8.49
CF3
10e
10f
O
O
0.14
>10
0.35
>10
12.16
58.61
13.30
18.79
14.76
53.95
CF3
OCF3
Cl
10g
O
7.71
>10
19.83
17.09
24.47
Cl

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Table 1. Cont.
IC50 (µM) a
Comp
NO.
Subsituent
Ar
IC50 (µM) a
X
EGFR
VEGER-2
HCT116
MCF-7
B16
F
10h
O
4.88
7.06
49.51
15.99
19.38
F
Cl
10i
10j
O
O
O
S
4.17
5.90
>10
>10
7.11
>10
>100
89.66
>100
8.79
22.14
23.59
26.48
24.96
20.91
32.43
27.35
34.13
>100
9.33
F
10k
10l
CF3
N.D.
0.01
>10
N.D.
0.07
>10
F
CF3
10m
10n
S
8.11
7.89
Cl
OCF3
S
64.47
68.37
CF3
10o
S
0.02
0.09
9.57
19.02
12.25
CF3
10p
10q
S
S
0.07
0.01
0.27
0.08
9.03
8.35
21.04
15.66
12.94
5.57
CF3
Br
CF3
Cl
10r
S
3.56
>10
63.10
14.79
27.39
Cl
F
10s
10t
S
S
S
S
3.17
2.86
5.35
5.14
6.93
6.62
>100
23.09
27.26
30.53
26.40
21.31
42.16
57.61
61.23
F
Cl
F
10u
10v
>10
0.02
>10
0.08
83.25
10.55
28.87
17.87
>100
9.29
sorafenib
N.D. not determined; a Assays were performed in triplicate (n ≥ 3).
2.2.2. In Vitro Antiproliferative Activity Assay
In vitro cell cytotoxicities of all the new compounds were initially evaluated against HCT116,
MCF-7 and B16 cell lines by MTT assay using sorafenib as a positive control. The results were also
summarized in Table 1. Most of the target compounds exhibited potent antiproliferative activities
against all three cell lines. Among the tested compounds, compounds 10b, 10c, 10e, 10l, 10m, 10o
and 10q showed comparable antiproliferative activities to that of sorafenib and selective inhibitory
activities against different cell lines. Compounds 10b and 10q, with the most potent EGFR/VEGFR-2
inhibitory activities, also displayed better potent antiproliferative activities against HCT-116, MCF-7

Molecules 2018, 23, 24
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and B16 cell lines than sorafenib. The antiproliferative activities of the compounds were influenced by
substituents on the terminal aromatic ring: (1) Compounds 10b 10c 10e 10l 10m 10o and 10q with
strong electron-withdrawing groups (such as -F, -Cl, -Br, -CF3) on terminal aromatic ring exhibited
potent antiproliferative activities against all three cell lines; (2) Compounds 10f 10k 10n and 10v
,
,
,
,
,
,
,
containing electron-donating group (such as -CH3, -OCF3) showed relative weaker activity against the
cancer cell lines. It indicated that the electron-withdrawing group on the terminal aromatic ring is also
essential for the antiproliferative activities, and the antiproliferative activity of the title compounds is
related to their dual EGFR/VEGFR-2 inhibitory activities.
2.2.3. In Vivo Antitumor Activity Assay
The C57BL/6J mice were employed to establish the xenograft model of B16 melanoma, and the
compounds 10b
as a positive control. As shown in Table 2, compounds 10b
tumor regression, the growth of B16 tumors were inhibited at 31.25%, 49.22%, 20.31% and 64.06% by
administering orally with sorafenib, 10b 10m, and 10q at 90 mg/kg, respectively. Compounds 10b
,
10m and 10q were chosen to test their in vivo antitumor activity using sorafenib
,
10m 10q, and sorafenib can cause
,
,
and 10q displayed better inhibitory activities against B16 melanoma than that of sorafenib. No obvious
weight loss was observed in all treated groups.
Table 2. The effect of 10b, 10m, 10q and sorafenib on the growth of B16 xenograft model.
Body Weight (g)
Groups
Dosage (mg/kg)
Tumor Weight (g)
Inhibition Rate (%)
Number Initial/End
Initial
End
Control
Sorafenib
10b
10m
10q
0
6/6
6/6
6/6
6/6
6/6
19.6 ± 2.3
20.7 ± 2.1
18.9 ± 1.8
19.2 ± 2.1
19.1 ± 2.7
21.8 ± 2.3
21.3 ± 2.4
19.5 ± 3.0
20.9 ± 1.6
20.2 ± 2.7
1.28 ± 0.56
0.88 ± 0.46
0.65 ± 0.22 *
1.02 ± 0.36 *
0.46 ± 0.14 *
90
90
90
90
31.25%
49.22%
20.31%
64.06%
Antitumor effect is assessed by i.p. treatment of C57BL/6J mice carrying B16 tumor xenografts with 90 mg/kg of
test compounds, sorafenib and CMC-Na as control. * p < 0.05, compared with sorafenib.
2.3. Molecular Docking Studies
In order to better understand the interaction between the title compounds and kinases, molecular
docking studies on the potent representative compound 10q were performed using the Tripos
Sybyl-x2.0 software (2.0, Tripos Inc., St. Louis, MO, USA).
As shown in Figure 3, compound 10q could be accommodated with EGFR comfortably (PDB: 2ity).
the protonated N3 of quinazoline interacted in the EGFR ATP binding site with the amino acid Met-793
through an ionic bond. The NH of the thiourea were capable of forming hydrogen bonds with the
amino acid residues Pro-794 and Met-793 respectively. Another hydrogen bond was formed between
Lys-745 and the oxygen atoms of 6,7-position substituents of quinazoline. Hydrophobic interactions
were observed with amino acid residues in the active site of EGFR, including Phe-795, Met-793
and Leu-718.
The binding model of compound 10q into the ATP-binding cavity of VEGFR-2 kinase (PDB: 4asd)
is also depicted in Figure 3. In this docking model, compound 10q could be accommodated to the
inactive DFG-out conformation of VEGFR-2 comfortably. The quinazoline and meta-disubstituted
central phenyl were positioned in a hydrophobic pocket lined with Phe-795, Leu-844, Met-793, Val-726
and Leu-718. The hydrogen bond was formed between NH of the thiourea group and the amino acid
residue Asp-800. In addition, 6,7-methoxy group of quinazoline could form another hydrogen bond
with Lys-745.

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(A)
(B)
Figure 3.
(
A) 3D molecular docking model of compound 10q with EGFR active site; (B) 3D model
of the interaction between compound 10q and VEGFR-2 ATP binding site. The hydrogen bonds are
displayed as yellow dotted lines.
3. Experimental Section
3.1. General Information
All the materials used were purchased from commercial suppliers and without further purification.
Solvents were distilled before using and flash chromatography was performed using silica gel (60 Å,
200–300 mesh). All reactions were monitored by thinlayer chromatography on 0.25 mm silica gel
plates (60GF-254) and visualized with UV light. Melting points were determined on an electrothermal
melting point apparatus and were uncorrected. Proton nuclear magnetic resonance spectra were
obtained on a Brucker Avance 400 spectrometer using TMS as an internal standard in DMSO-d6 or
CDCl3 solutions. Chemical shifts were reported in delta (δ) units, parts per million (ppm) downfield
from trimethylsilane. ESI-MS were determined on an API 4000 spectrometer. High-resolution mass
spectral (HRMS) data were conducted by the Shandong Analysis and Test Center, and are reported as
m/z (relative intensity).
3.1.1. 4,5-Dimethoxy-2-nitrobenzaldehyde (2)
4,5-Dimethoxy benzaldehyde (16.6 g, 0.1 mol) was slowly added to 65% (g/g) nitric acid (100 mL)
under stirring at a temperature range of 20–25 ◦C. The mixture was stirred for another 1 h and slowly
added to 400 mL of ice water, a large amount of yellow solid was formed. The crude product was got
by filtration and recrystallized in ethanol to get 4,5-Dimethoxy-2-nitrobenzaldehyde (Compound
16.0 g as a yellow solid, yield 78.2%, m.p. 133–135 ◦C.
2)
3.1.2. 4,5-Dimethoxy-2-nitrobenzoic acid (3)
Compound 2 (33.0 g, 156.3 mmol) was suspended in 0.6 M NaOH (500 mL) under stirring, KMnO4
(19.7 g, 125.0 mmol) was slowly added to the reaction mixture. After 7 h of stirring, the initial purple
color went black, then a yellow solution was got after filtration. The pH value of the solution was
adjusted to 2–3 by 10% HCl and large amounts of yellow solid was got. 4,5-Dimethoxy-2-nitro-benzoic
acid (Compound 3) was obtained after filtration as a yellow solid, yield: 83.0%, m.p. 193.2–195.0 ◦C.
3.1.3. 2-Amino-4,5-dimethoxy-benzoic acid (4)
Compound 3 (10 g, 44.02 mmol), Pd/C (0.5 g) and 200 mL methanol were added to the 500 mL
high pressure reactor, the air in the reactor was replaced by H2 (0.1 MPa) three times and shut down
◦
the vent valve. The mixture was stirred at 45 C and 0.4 MPa of H2 for 2 h, after the reaction was over,

Molecules 2018, 23, 24
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Pd/C was filtered out and the solution was distilled under vacuum as soon as possible. The resulti◦ng
residue was filtered and washed to give a yellow solid 7.2 g with a yield of 83.7%, m.p. 170.4–172.0 C.
3.1.4. 6,7-Dimethoxy-3H-quinazolin-4-one (5)
Compound
4
(5.0 g, 25.38 mmol) and formamidine acetate (4.0 g, 38.83 mmol) were dissolved in
◦
5 mL of DMSO and heated to 140–170 C under stirring. The initial black color went brown and some
solid was formed. The reaction mixture was constantly stirred for 4h and then cooled to 25 ◦C. 50 mL
H2O was added to the mixture and the product was obtained by filtration as a light yellow solid 5.0 g
with a yield of 95.7%, m.p. 295.5–297.0 ◦C.
3.1.5. 4-Chloro-6,7-dimethoxy-quinazoline (6)
A solution of compound
5 (1.7 g, 8.2 mmol) in SOCl2 (15 mL) containing 0.05 mL of
N,N-dimethylformamide (DMF) were heated to reflux in 100 mL flask for 1 h. Excess SOCl2 was
distilled in vacuum and the resulting residue was adjusted to pH value 8–10 with aqueous Na2CO3.
1.53 g of product was got after filtered as yellow solid, yield: 82.2%, m.p. 180.4–182.0 ◦C.
3.1.6. 3-(6,7-Dimethoxyquinazolin-4-yloxy)-benzenamine (7a)
A solution of 3-aminophenol (0.35 g, 3.2 mmol) in dry DMF (20 mL) was treated with potassium
tert-butoxide (0.44 g, 3.9 mmol), and the mixture was stirred at room temperature for 1 h under
nitrogen atmosphere, then a solution of
were added respectively. The mixture was heated to 80–85 C for 2 h. After the temperature was
cooled to room temperature, the mixture was diluted with water (100 mL) and extracted with EtOAc
6
(0.76 g, 3.4 mmol) in DMF (50 mL) and potassium carbonate
◦
(3
×
100 mL). The extract was washed with brine (2
×
100 mL), dried over anhydrous sodium
sulfate, and concentrated under reduced pressure to give light-brown solid 7a 0.74 g, yield: 77.9%,
m.p. 183.5–185.0 C. H-NMR (400 MHz, DMSO-d6)
◦
1
δ 3.97 (s, 3H, O-CH3), 3.98 (s, 3H, O-CH3),
5.28 (s, 2H, -NH2), 6.98 (dd, J = 1.6 Hz, 9.6 Hz, 1H, Ph-H-6), 7.13 (d, J = 7.6 Hz, 1H, Ph-H-4),
7.23 (d, J = 1.6 Hz, 1H, Ph-H-2), 7.43 (t, J = 7.8 Hz, 1H, Ph-H-5), 7.55 (s, 1H, quinazoline H-5), 7.63 (s, 1H,
quinazoline H-8), 8.56 (s, 1H, quinazoline H-2). HRMS (AP-ESI) m/z: calcd. for C16H15N3O3 [M + H]+
298.1192, found 298.1199.
3.1.7. 3-(6,7-Dimethoxyquinazolin-4-ylsulfanyl)-benzenamine (7b)
The synthesis is analogous to 7a with 3-aminethiophenol as the starting material. The product
was obtained as light yellow solid, yield: 74.0%, m.p. 172.4–175.2 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
3.97 (s, 3H, O-CH3), 3.98 (s, 3H, O-CH3), 5.33 (s, 2H, -NH2), 6.68 (dd, J = 1.6 Hz, 9.6 Hz, 1H,
Ph-H-6), 6.73 (d, J = 7.6 Hz, 1H, Ph-H-4), 6.80 (d, J = 1.6 Hz, 1H, Ph-H-2), 7.13 (t, J = 7.8 Hz, 1H,
Ph-H-5), 7.31 (s, 1H, quinazoline H-5), 7.33 (s, 1H, quinazoline H-8), 8.70 (s, 1H, quinazoline H-2).
HRMS (AP-ESI) m/z: calcd. for C16H15N3O2S [M + H]+ 314.0963, found 314.0958.
3.1.8. Substituted Phenyl Isothiocyanates (9a–k)
The synthesis of compounds (9a–k) were the same as the method described in our previous
article [20], the crude products were purified by chromatography (ethyl acetate/petroleum ether = 1:1)
on silica gel to afford corresponding phenyl isothiocyanates in moderate to good yields.
3.1.9. Quinazoline and thiourea-containing sorafenib analogs (10a–v)
1-[3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl]-3-(4-fluoro-3-trifluoromethylphenyl)thiourea (10a). Compound 7a
(0.50 g,◦1.65 mmol) was dissolved in 10 mL of dichloromethane (DCM) and the solution was cooled
to 0–5 C in ice bath under stirring, a solution of 9a (0.38 g, 1.76 mmol) in DCM were added
dropwise. The reaction mixture was stirred at room temperature for 16 h and the precipitation
appeared. The suspension was filtered and washed with DCM to give light grey solid 10a 0.51 g,

Molecules 2018, 23, 24
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◦
1
yield: 59.1%, m.p. 130.5–133.0 C, purity: 98.0% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.98
(s, 3H, quinazoline -OCH3-6), 3.99 (s, 3H, quinazoline -OCH3-6), 7.11 (d, J = 7.8 Hz, 1H, Ph-H-6),
7.39–7.51 (m, 5H), 7.55 (s, 1H, quinazoline H-8), 7.77–7.81 (m, 1H, Ph’-H-2), 7.95 (dd, J = 2.5, 6.4 Hz, 1H,
Ph’-H-6), 8.56 (s, 1H, quinazoline H-2), 10.16 (s, 1H, -NHCS-), 10.28 (s, 1H, -NHCS-). HRMS(AP-ESI)
m/z: calcd. for C24H19F4N4O3S [M + H]+ 519.1114, found 519.1121.
The other compounds of this series were synthesized following the general procedure as
described above.
1-(4-Chloro-3-trifluoromethylphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10b). Off white
solid, yield: 65.5%, m.p. 140.1–141.0 ◦C, purity: 98.1% (HPLC). 1H-NMR (400 MHz, DMSO-d6) δ 3.96
(s, 3H, quinazoline -OCH3-6), 3.97 (s, 3H, quinazoline -OCH3-7), 7.11 (d, J = 7.8 Hz, 1H), 7.33–7.48
(m, 3H), 7.52 (d, J = 4.9 Hz, 2H), 7.65 (d, J = 8.7 Hz, 1H), 7.79 (dd, J = 2.6, 8.4 Hz, 1H), 8.07 (d, J = 2.6 Hz,
1H), 8.54 (s, 1H), 10.20 (s, 1H, -NHCS-), 10.27 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for
C24H19ClF3N4O3S [M + H]+ 535.1418, found 535.0925.
1-(4-Bromo-3-trifluoromethylphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10c). Off white
◦
solid, yield: 65.6%, m.p. 186.3–188.0 C, purity: 98.0% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ 3.96 (s,
3H, quinazoline -OCH3-6), 3.97 (s, 3H, quinazoline -OCH3-7), 7.11 (d, J = 7.8 Hz, 1H), 7.36 (s, 1H),
7.39–7.48 (m, 2H), 7.50–7.59 (m, 2H), 7.71 (dd, J = 8.5, 2.6 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 8.09
(d, J = 2.5 Hz, 1H), 8.55 (s, 1H), 10.29 (s, 1H, -NHCS-), 10.34 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z:
calcd. for C24H19BrF3N4O3S [M + H]+ 580.0313, found 580.0318.
1-(3,5-Ditrifluoromethylphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10d). Off white
solid, yield: 47.6%, m.p. 122.8–125.6 ◦C, purity: 98.4% (HPLC). 1H-NMR (400 MHz, DMSO-d6) δ 3.98
(s, 3H, quinazoline -OCH3-6), 3.99 (s, 3H, quinazoline -OCH3-7), 7.15 (d, J = 7.8 Hz, 1H, Ph-H-6), 7.40
(s, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.53 (s, 1H), 7.55 (s, 1H), 7.81 (s, 1H), 8.26
(s, 2H), 8.56 (s, 1H), 10.42 (s, 1H, -NHCS-), 10.49 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for
C25H19F6N4O3S [M + H]+ 569.1082, found 569.1078.
1-(3-Trifluoromethylphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10e). White solid, yield:
◦
1
71.7%, m.p. 147.5–149.6 C, purity: 98.7% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.96 (s, 3H,
quinazoline -OCH3-6), 3.97 (s, 3H, quinazoline -OCH3-7), 7.07–7.11 (m, 1H), 7.38 (d, J = 4.8 Hz, 2H),
7.42–7.47 (m, 2H), 7.52–7.58 (m, 3H), 7.74 (dd, J = 2.4, 7.4 Hz, 1H), 7.94 (s, 1H), 8.55 (s, 1H), 10.12 (s, 1H,
-NHCS-), 10.17 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C24H20F3N4O3S [M + H]+ 501.1208,
found 501.1210.
1-(4-Trifluoromethoxyphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10f). Off white solid,
yield: 54.5%, m.p. 171.5–174.0 ◦C, purity: 98.7% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ 3.97 (s, 6H,
quinazoline -OCH3), 7.06 (dd, J = 2.1, 7.8 Hz, 1H), 7.32 (d, J = 8.6 Hz, 2H), 7.37 (s, 1H), 7.42 (t, J = 8.2 Hz,
1H), 7.53 (d, J = 10.8 Hz, 2H), 7.66–7.73 (m, 3H), 8.55 (s, 1H), 10.75 (s, 1H, -NHCS-), 10.81 (s, 1H,
-NHCS-). HRMS (AP-ESI) m/z: calcd. for C24H20F3N4O4S [M + H]+ 517.1157, found 517.1151.
1-(2,4-Dichlorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10g). Off white solid, yield:
◦
1
65.5%, m.p. 187.4–188.2 C, purity: 98.3% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.98 (s, 3H,
quinazoline -OCH3-6), 3.99 (s, 3H, quinazoline -OCH3-7), 7.09-7.13 (m, 1H), 7.40 (s, 1H), 7.41–7.48
(m, 3H), 7.56 (s, 1H), 7.59 (s, 1H), 7.62 (s, 1H), 7.69 (d, J = 2.3 Hz, 1H), 8.56 (s, 1H), 9.60 (s, 1H, -NHCS-),
10.19 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C23H19Cl2N4O3S [M + H]+ 501.0555, found
501.0550.
1-(3,4-Difluorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10h). Light grey solid, yield:
◦
1
47.6%, m.p. 181.3–184.1 C, purity: 98.0% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.96 (s, 3H,
quinazoline -OCH3-6), 3.97 (s, 3H, quinazoline -OCH3-7), 7.08 (dd, J = 2.5, 8.4 Hz, 1H), 7.22 (ddt, J = 2.2,
4.3, 8.6 Hz, 1H), 7.33–7.47 (m, 4H), 7.50–7.60 (m, 2H), 7.69 (dd, J = 2.5, 8.6 Hz, 1H), 8.55 (s, 1H), 10.06

Molecules 2018, 23, 24
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(s, 1H, -NHCS-), 10.12 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C23H19F2N4O3S [M + H]+
469.1146, found 469.1149.
1-(4-Chlorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10i). Off white solid, yield:
◦
1
74.7%, m.p. 198.3–201.0 C, purity: 98.1% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.97 (s, 3H,
quinazoline -OCH3-6), 3.98 (s, 3H, quinazoline -OCH3-7), 7.05 (dd, J = 2.5, 8.3 Hz, 1H), 7.34–7.45
(m, 4H), 7.46–7.51 (m, 1H), 7.53–7.61 (m, 3H), 7.64 (d, J = 2.5 Hz, 1H), 8.55 (s, 1H), 10.55 (s, 1H, -NHCS-),
10.62 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C23H20ClN4O3S [M + H]+ 467.0945, found
467.0948
1-(4-Fluorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10j). Off white solid, yield:
◦
1
53.0%, m.p. 168.5–170.0 C, purity: 98.3% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.96 (s, 3H,
quinazoline -OCH3-6), 3.98 (s, 3H, quinazoline -OCH3-7), 7.05 (dd, J = 2.0, 8.1 Hz, 1H), 7.12–7.20
(m, 2H), 7.38 (s, 1H), 7.39–7.46 (m, 2H), 7.48–7.49 (m, 2H), 7.54 (s, 1H), 7.62 (d, J = 2.4 Hz, 1H), 8.55
(s, 1H), 10.21 (s, 1H, -NHCS-), 10.32 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C23H20FN4O3S
[M + H]+ 451.1240, found 451.1244.
1-p-tolyl-3-[3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl]thiourea (10k). Off white solid, yield: 40.1%,
m.p. 138.4–140.0 ◦C, purity: 99.2% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ 2.28 (s, 3H, Ph’-CH3),
3.99 (s, 6H, -OCH3), 7.04–7.08 (m, 1H), 7.14 (d, J = 8.2Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 7.38 (s, 1H), 7.41
(s, 1H), 7.43 (s, 1H), 7.55 (s, 1H), 7.59 (s, 1H), 8.56 (s, 1H), 9.80 (s, 1H, -NHCS-), 9.81 (s, 1H, -NHCS-).
HRMS(AP-ESI) m/z: calcd. for C24H23N4O3S [M + H]+ 447.1491, found 447.1497.
1-[3-(6,7-Dimethoxyquinazolin-4-ylsulfanyl)phenyl]-3-(4-fluoro-3-trifluoromethylphenyl) thiourea
White solid, yield: 70.1%, m.p. 128.4–130.0 ◦C, purity: 98.2% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
3.99 (s, 6H, -OCH3), 7.33 (s, 1H), 7.35 (s, 1H), 7.42 (d, J = 7.8Hz, 1H), 7.46–7.52 (m, 2H), 7.55
(10l).
δ
(d, J = 7.8Hz, 1H), 7.76–7.80 (m, 2H), 7.92 (dd, J = 2.6, 6.4Hz, 1H), 8.68 (s, 1H), 10.04 (s, 1H, -NHCS-),
10.16 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C24H19F4N4O2S2 [M + H]+ 535.0886, found
535.0882.
1-(4-Chloro-3-trifluoromethylphenyl)-3-[3-(6,7-dimeth◦oxyquinazolin-4-ylsulfanyl)phenyl] thiourea (10m).
1
Off white solid, yield: 54.1%, m.p. 162.2–164.0 C, purity: 98.8% (HPLC). H-NMR (400 MHz,
DMSO-d6)
δ 3.97 (s, 6H, -OCH3), 7.32 (d, J = 8.8 Hz, 2H), 7.41 (dd, J = 1.4, 7.8 Hz, 1H), 7.49 (t, J = 7.9 Hz,
1H), 7.60–7.70 (m, 2H), 7.74–7.83 (m, 2H), 8.07 (d, J = 2.6 Hz, 1H), 8.67 (s, 1H), 10.24 (s, 1H, -NHCS-),
10.29 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C24H19ClF3N4O2S2 [M + H]+ 552.0590, found
552.0593.
1-(4-Trifluoromethoxyphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10n). Off white
solid, yield: 47.2%, m.p. 138.0–139.3 ◦C, purity: 98.3% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ 3.97
(s, 6H, -OCH3), 7.29–7.34 (m, 4H), 7.36 (dd, J = 1.4, 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.69–7.76
(m, 2H), 7.82 (dd, J = 2.2, 8.4 Hz, 1H), 7.96 (s, 1H), 8.68 (s, 1H), 11.09 (s, 1H, -NHCS-), 11.15 (s, 1H,
-NHCS-). HRMS(AP-ESI) m/z: calcd. for C24H20F3N4O3S2 [M + H]+ 533.0929, found 533.0931.
1-(3,5-Ditrifluoromethylphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10o). Light
yellow solid, yield: 43.8%, m.p. 168.2–171.0 ◦C, purity: 98.5% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ
3.98 (s, 6H, -OCH3), 7.32 (s, 1H), 7.34 (s, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.66
(d, J = 7.8 Hz, 1H), 7.79–7.81 (m, 2H), 8.26 (s, 2H), 8.69 (s, 1H), 10.41 (s, 1H, -NHCS-), 10.46 (s, 1H,
-NHCS-). HRMS(AP-ESI) m/z: calcd. for C25H19F6N4O2S2 [M + H]+ 585.0854, found 585.0857.
1-(3-Trifluoromethylphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10p). Off white
solid, yield: 49.4%, m.p. 127.5–131.0 ◦C, purity: 98.1% (HPLC). 1H-NMR (400 MHz, DMSO-d6) δ 3.98
(s, 6H, -OCH3), 7.32 (s, 1H), 7.34 (s, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.46–7.51 (m, 2H), 7.56 (t, J = 7.9 Hz,
1H), 7.66 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.80 (t, J = 1.7 Hz, 1H), 7.95 (s, 1H), 8.69 (s,
1H), 10.17 (s, 1H, -NHCS-), 10.21 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C24H20F3N4O2S2
[M + H]+ 517.0980, found 517.0983.

Molecules 2018, 23, 24
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1-(4-Bromo-3-trifluoromethylphenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl] thiourea (10q).
◦
1
Off white solid, yield: 52.3%, m.p. 150.3–152.0 C, purity: 98.4% (HPLC). H-NMR (400 MHz,
DMSO-d6) 3.98 (s, 6H, -OCH3), 7.33 (s, 1H), 7.35 (s, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 7.8 Hz,
δ
1H), 7.64 (d, J = 7.8 Hz, 1H), 7.71 (dd, J = 2.2, 8.8 Hz, 1H), 7.77 (s, 1H), 7.83 (d, J = 8.8 Hz, 1H), 8.07 (d,
J = 2.2 Hz, 1H), 8.69 (s, 1H), 10.19 (s, 1H, -NHCS-), 10.26 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd.
for C24H19BrF3N4O2S2 [M + H]+ 596.0085, found 596.0080.
1-(2,4-Dichlorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10r). White solid, yield:
◦
48.1%, m.p. 172.4–174.0 C, purity: 98.5% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ 3.99 (s, 6H,
-OCH3), 7.33 (s, 1H), 7.35 (s, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.43 (dd, J = 2.4, 8.6 Hz, 1H), 7.49 (s, 1H),
7.60 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.85 (t, J = 1.7 Hz, 1H), 8.68 (s,
1H), 9.60 (s, 1H, -NHCS-), 10.19 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C23H19Cl2N4O2S2
[M + H]+ 518.0326, found 518.0322.
1-(3,4-Difluorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10s). Off white solid,
yield: 45.4%, m.p. 128.2–130.4 ◦C, purity: 98.0% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ 3.97 (s, 6H,
-OCH3), 7.31 (t, J = 4.7 Hz 3H), 7.35–7.42 (m, 2H), 7.46 (d, J = 7.9 Hz, 1H), 7.80 (dd, J = 2.1, 7.8 Hz, 1H),
7.97–7.88 (m, 2H), 8.68 (s, 1H), 10.11 (s, 2H, -NHCS-). HRMS (AP-ESI) m/z: calcd. for C23H19F2N4O2S2
[M + H]+ 485.0917, found 485.0918.
1-(4-Chlorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10t). Off white solid, yield:
◦
1
47.1%, m.p. 124.8–126.1 C, purity: 98.1% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.98 (s, 6H,
-OCH3), 7.30 (d, J = 8.6 Hz, 2H), 7.35–7.38 (m, 3H), 7.43–7.53 (m, 3H), 7.65 (dd, J = 2.3, 8.4 Hz, 1H), 7.79
(t, J = 2.4 Hz, 1H), 8.67 (s, 1H), 10.01 (s, 1H, -NHCS-), 10.04 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd.
for C23H20ClN4O2S2 [M + H]+ 484.0716, found 484.0712.
1-(4-Fluorophenyl)-3-[3-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10u). Off white solid, yield:
◦
1
40.6%, m.p. 152.0–153.2 C, purity: 98.0% (HPLC). H-NMR (400 MHz, DMSO-d6)
δ 3.97 (s, 6H,
-OCH3), 7.12–7.21 (m, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.34–7.40 (m, 1H), 7.41–7.51 (m, 3H), 7.62–7.69 (m,
1H), 7.78 (t, J = 1.8 Hz, 1H), 8.67 (s, 1H), 9.88 (s, 1H, -NHCS-), 9.95 (s, 1H, -NHCS-). HRMS(AP-ESI)
m/z: calcd. for C23H20FN4O2S2 [M + H]+ 467.1012, found 467.1015
1-p-tolyl-3-[3◦-(6,7-dimethoxyquinazolin-4-ylsulfanyl)phenyl]thiourea (10v). White solid, yield: 43.9%, m.p.
163.1–165.0 C, purity: 98.2% (HPLC). 1H-NMR (400 MHz, DMSO-d6)
δ 2.26 (s, 3H, Ph-CH3), 3.97
(s, 6H, -OCH3), 7.13 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 3.2 Hz, 2H), 7.32 (d, J = 3.1 Hz, 2H), 7.34–7.39
(m, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.66 (dt, J = 1.4, 8.2 Hz, 1H), 7.80 (t, J = 2.0 Hz, 1H), 8.67 (s, 1H),
9.84 (s, 1H, -NHCS-), 9.85 (s, 1H, -NHCS-). HRMS(AP-ESI) m/z: calcd. for C24H23N4O2S2 [M + H]+
463.1262, found 463.1263.
1H-NMR of compound 10a–v are shown in Supplementary Materials.
3.2. EGFR and VEGFR-2 Inhibitory Assay
Materials: EGFR and VEGFR-2 were purchased from Carna Biosciences (Framingham, MA, USA).
ATP, DMSO and EDTA were purchased from Sigma (CA, USA). 96-well plate and 384-well plate were
purchased from Corning.
In vitro enzymatic activities of all the synthesized compounds against VEGFR-2 and EGFR
were evaluated by a caliper mobility shift assay using sorafenib as a positive control. Each of these
compounds was dissolved in DMSO at 10 mM, diluted to 50
concentration in reaction by 100% DMSO, and 100 L of each dilution was added to one well on 96-well
storage plate, and diluted by transferring 10 L to 90 L of 100% DMSO in the next well. Besides,
100 L of 100% DMSO was added to two empty wells for no compound control and no enzyme control
in the same 96-well plate respectively. The plate was marked as source plate. 10 L of each compound
was transferred from the source plate to a new 96-well plate, which was marked as the intermediate
×
of the final desired highest inhibitor
µ
µ
µ
µ
µ

Molecules 2018, 23, 24
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plate. Next, 90
by shaking vigorously for 10 min. A total of 5
transferred to a 384-well plate in duplicate.
µ
L of 1
×
kinase base buffer was added to each well of the intermediate plate, and mixed
µL of each well from the 96-well intermediate plate was
Kinase reaction: kinase in 1
base buffer were added, then 10
assay plate. After incubating at room temperature for 10 min, 10
added to each well of the 384-well assay plate. The reaction was incubated at 28 ◦C for a specified
period of time. Finally, the kinase reactions were quenched with 25 L of EDTA and the plates were
×
kinase base buffer, FAM-labeled peptide and ATP in the 1
L of 2.5 enzyme solution was added to each well of the 384-well
L of 2.5 peptide solution was
×
kinase
µ
×
µ
×
µ
washed. The dates were collected and converted with the caliper program.
3.3. In Vitro Antiproliferative Activity Assay
HCT116, MCF-7 and B16 cell lines were plated on 96-well plates at a density of 5000 per well and
incubated overnight. The cells were treated with compounds and sorafenib at final concentrations
ranging from 0.5 to 200 µM, while control cells were treated with equal volume DMSO. After 48 h,
0.5% MTT (Amresco, Solon, OH, USA) solution was added to each well, and further incubation for 4 h,
then cells were centrifuged at 2500 rpm for 15 min and removed from the culture medium. And add
150 µL DMSO to dissolve the formazan. After mixing for 5 min, optical density was detected at 570 nm
on a microplate reader (Thermo, Waltham, MA, USA).
3.4. In Vivo Antitumor Activity Assay
Male C57BL/6J mice were employed to establish the xenograft tumor model by subcutaneous
injection (S.C.) of 6
with compounds 10b
×
106 B16 cells. Twenty-four hours after inoculation, the animal were administrated
,
10m, 10q, sorafenib or 0.5% CMC-Na orally successively for 14 days. In the end
of treatment, the mice were sacrificed and the tumor were peeled off and weighted. The inhibition rate
of tumor growth was calculated. All animal protocols were conformed to the Guidelines for the Care
and Use of Laboratory Animals approved by the Animal Care and Use Committee of Yantai University.
3.5. Molecular Docking Study
Molecular docking was carried out using a Sybyl/Surflex dock based on the crystal structures of
EGFR (PDB ID: 2ITY) and VEGFR-2 (PDB ID: 4ASD), which were downloaded from the RCSB Protein
Data Bank and prepared using Docking Suite. Hydrogen was added and minimized using the Tripos
force field and Pullman charges, and the default setting were used. Compound 10q was depicted with
the Sybyl/Skeetch module (Tripos Inc.) and docked into the defined binging site without constraint.
Molecular docking results were submitted and generated with Pymol.
4. Conclusions
In summary, a series of novel quinazoline- and thiourea-containing sorafenib analogs (10a–v)
were designed and synthesized as EGFR and VEGFR-2 dual TK inhibitors. Most of the compounds
exhibited good enzymatic activities and potent antiproliferative activities against HCT-116, MCF-7
and B16 cell lines, which suggested that the introduction of electron-withdrawing groups on terminal
phenyl ring was more favorable for enzyme inhibitory activities and antiproliferative activities.
Compounds 10b and 10q, which exhibited the most potent inhibitory activities against EGFR (IC50
=
0.02 M and 0.01 M, respectively), VEGFR-2 (IC50 = 0.05 M and 0.08 M, respectively), and good
µ
µ
µ
µ
antiproliferative activities, also displayed better anti-tumor activities than sorafenib in vivo by B16
melanoma xenograft model test. Furthermore, molecular docking of the most potent inhibitor 10q into
the ATP-binding site of EGFR and VEGFR-2 was performed and the result suggested that compound
10q could bind well with the active site of EGFR and VEGFR-2. Therefore, compounds 10b and 10q
could be developed as potent anticancer agents in the future.

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Supplementary Materials: The supplementary materials are available online, 1H-NMR of compound 10a–v.
Acknowledgments: This work was supported by Taishan Scholar Project, National Natural Science Foundation of
China (No. 81202038, 81728020), Key Research Project of Shandong Province (2017GSF18177) and Ph.D. Programs
Foundation of Yantai University (No. YX13B03-2013).
Author Contributions: J.Y. and H.W. conceived and designed the experiments; S.S., X.K. and J.Z. performed
the experiments; N.W. and J.Z. analyzed the data; F.F. contributed reagents/materials/analysis tools; J.Y. wrote
the paper.
Conflicts of Interest: The author declare no conflicts of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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