Design and discovery of 4-anilinoquinazoline-urea derivatives as dual TK inhibitors of EGFR and VEGFR-2

Article abstract of DOI:10.1016/j.ejmech.2016.09.039

EGFR and VEGFR-2 are involved in pathological disorders and the progression of different kinds of tumors, the combined blockade of EGFR and VEGFR signaling pathways appears to be an attractive approach to cancer therapy. In this work, a series of 4-anilinoquinazoline derivatives containing substituted diaryl urea or glycine methyl ester moiety were designed and identified as EGFR and VEGFR-2 dual inhibitors. Compounds 19i, 19j and 19l exhibited the most potent inhibitory activities against EGFR (IC₅₀?=?1?nM, 78?nM and 51?nM, respectively) and VEGFR-2 (IC₅₀?=?79?nM, 14?nM and 14?nM, respectively), they showed good antiproliferative activities as well. Molecular docking established the interaction of 19i with the DFG-out conformation of VEGFR-2, suggesting that they might be type II kinase inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.09.039

Accepted Manuscript
Design and discovery of 4-anilinoquinazoline-urea derivatives as dual TK inhibitors of
EGFR and VEGFR-2
Hai-Qi Zhang, Fei-Hu Gong, Ji-Qing Ye, Chi Zhang, Xiao-Hong Yue, Chuan-Gui Li,
Yun-Gen Xu, Li-Ping Sun
PII:
S0223-5234(16)30769-3
10.1016/j.ejmech.2016.09.039
EJMECH 8905
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 July 2016
Revised Date: 10 September 2016
Accepted Date: 13 September 2016
Please cite this article as: H.-Q. Zhang, F.-H. Gong, J.-Q. Ye, C. Zhang, X.-H. Yue, C.-G. Li, Y.-G. Xu,
L.-P. Sun, Design and discovery of 4-anilinoquinazoline-urea derivatives as dual TK inhibitors of EGFR
and VEGFR-2, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.039.
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ACCEPTED MANUSCRIPT
Design and discovery of 4-anilinoquinazoline-urea derivatives
as dual TK inhibitors of EGFR and VEGFR-2
Hai-Qi Zhang1, Fei-Hu Gong1, Ji-Qing Ye, Chi Zhang, Xiao-Hong Yue, Chuan-Gui Li,
Yun-Gen Xu*, Li-Ping Sun*
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, PR China
1 These authors contributed equally to this work.
Graphical abstract

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Design and discovery of 4-anilinoquinazoline-urea derivatives
as dual TK inhibitors of EGFR and VEGFR-2
Hai-Qi Zhang1, Fei-Hu Gong1, Ji-Qing Ye, Chi Zhang, Xiao-Hong Yue, Chuan-Gui Li,
Yun-Gen Xu*, Li-Ping Sun*
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, PR China
1These authors contributed equally to this work.
ABSTRACT
EGFR and VEGFR-2 are involved in pathological disorders and the progression of different
kinds of tumors, the combined blockade of EGFR and VEGFR signaling pathways appears to be an
attractive approach to cancer therapy. In this work, a series of 4-anilinoquinazoline derivatives
containing substituted diaryl urea or glycine methyl ester moiety were designed and identified as
EGFR and VEGFR-2 dual inhibitors. Compounds 19i, 19j and 19l exhibited the most potent
inhibitory activities against EGFR (IC50 = 1 nM, 78 nM and 51 nM, respectively) and VEGFR-2
(IC50 = 79 nM, 14 nM and 14 nM, respectively), they showed good antiproliferative activities as
well. Molecular docking established the interaction of 19i with the DFG-out conformation of
VEGFR-2, suggesting that they might be type II kinase inhibitors.
Keywords: 4-Anilinoquinazoline-urea; EGFR; VEGFR-2; Tyrosine kinase inhibitor; Antitumor
1. Introduction
Protein kinases, especially receptor tyrosine kinases (RTKs), play a crucial role in signal
transduction pathways that regulate numerous cellular functions including proliferation,
differentiation, migration, and angiogenesis [1, 2]. The epidermal growth factor receptor (EGFR)
belongs to the ErbB family of receptor tyrosine kinase, which includes four members:
ErbB-1/EGFR, ErbB-2/HER-2/neu, ErbB-3/HER-3, and ErbB-4/HER-4 [3-5]. Binding of specific
ligands to the extracellular domain of EGFR induces dimerization and autophosphorylation,
activating the cytoplasmic tyrosine kinase domains and downstream signaling pathways [6]. EGFR
dysregulation by upactivation, overexpression or mutation can lead to tumorigenesis and metastasis,
via the activation of several signal transduction pathways (Ras/MAPK, PI3K/Akt, Jak/STAT) and
cellular processes, and is usually associated with a poor prognosis [7-9]. Therefore, EGFR
represents a valuable target for the design of anticancer agents [10-12].
Angiogenesis, the formation of new blood vessels from existing vasculature, is not only an
important event of normal physiological development but also a primary process for tumor growth
and metastasis [13]. VEGFR-2 (KDR) is a member of vascular endothelial growth factor receptors
(VEGFRs). Its activation by vascular endothelial growth factor (VEGF) initiates downstream

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signaling transduction, resulting in angiogenesis, vascular permeability enhancement and tumor
development [14-19]. Inhibition of VEGFR-2 has been considered as an effective approach for the
blockade of angiogenesis [20, 21].
EGFR and VEGFR-2 are involved in pathological disorders and the progression of different
kinds of tumors, they are closely related, sharing partial common downstream signaling pathways.
The functional relationship between EGFR and VEGFR-2 has been well-established: inhibition of
VEGFR-2 signaling pathway increases the antitumoral effect of EGFR inhibitors, while activation
of VEGFR-2 independent of EGFR signaling may give rise to the resistance of EGFR inhibitors
[6-8]. Thus, the simultaneous blockade of both EGFR and VEGFR signaling pathways appears to
be an attractive approach to cancer therapy [8, 22].
Quinazoline derivatives, especially 4-anilinoquinazolines, have attracted interests over the
years for their multiple biological activities, notably as EGFR inhibitors [23]. 4-Anilinoquinazoline
structure is present in six launched drugs consisting of gefitinib (Iressa), erlotinib (Tarceva),
lapatinib (Tykerb), vandetanib (Caprelsa), afatinib (Tovok) and icotinib (Conmana). Many
compounds which target the VEGFR-2 kinase are designed with diaryl urea or amide groups [7],
such as sorafenib (Nexavar), regorafenib (Stivarga), ponatinib (Iclusig), apatinib. Vandetanib is
considered to be a dual tyrosine kinase inhibitor targeting EGFR and VEGFR-2 [24] (Fig. 1).
In this paper, we employed substituted diaryl urea or glycine methyl ester moiety instead of
substituted aniline group (see e.g. structure of Vandetanib, Fig. 1) that attach the
4-anilinoquinazoline to give the novel hybrid scaffold (Fig. 2). It was documented that
4-anilinoquinazolines with basic side chains at C-7 position of the quinazoline nucleus can be
potent inhibitors of VEGFR-2 in both in vitro and in vivo assays [25]. Therefore the effect of
varying the O-substituent at the 7-position on the quinazoline core was also studied. In an attempt
to pursue new antitumor agents potently inhibited both EGFR and VEGFR-2, a series of novel
4-anilinoquinazoline-urea derivatives were designed and synthesized based on our previous work
[26]. The biological activities of these new compounds have been evaluated for their enzyme
inhibition and antiproliferative activities.

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Fig. 1. EGFR and/or VEGFR-2 inhibitors for clinical use.
Fig. 2. Design of the novel hybrid scaffold based upon the 4-anilinoquinazoline of vandetanib and
diaryl urea or glycine methyl ester moiety.
2. Results and discussion
2.1. Chemistry
The synthetic procedures were showed in Scheme 1-4. The key intermediate
4-chloro-6-methoxy-7-benzyloxyquinazoline (8) was obtained in seven steps (Scheme 1) [25], it
was then engaged in a nucleophilic substitution reaction in the presence of aniline (13a-k, 17).
As depicted in Scheme 2, the amino groups of commercial 4-nitroaniline derivatives (10) were
converted by condensation with commercial substituted aniline and triphosgene to obtain the

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compounds 12a-k. Then compounds 12a-k were reduced to their amino derivatives (13a-k) using
zinc powder.
4-Nitrobenzoyl glycine (15) was synthesized from 4-nitrobenzoyl chloride (14) and glycine in
the presence of aqueous NaOH solution. After compound 15 was converted into its methyl ester,
compound 16 was reduced to its amino derivative (17) using zinc powder (Scheme 3).
The synthesis of the final products is illustrated in Scheme 4. After taking off the benzyl of
compounds 9a-l, we get the desired products (19a-r) of various series through alkylation reaction in
the presence of appropriate commercial chloroalkane and potassium carbonate in
dimethylformamide.
H
N
H
NH2
X
H2N
N
Cl
O
Cl
i
R2
Cl
O
Cl
Cl
R2
+
+
O
Cl
O
O2N
O2N
X
11a-g
12a-k
10
H
H
N
N
ii
R2
O
H2N
X
13a-k
Scheme 2. Reagents and conditions: (i) TEA, DCM, 0-40oC; (ii) zinc powder, acetic acid glacial, MeOH/H2O, reflux.

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2.2. Biological evaluation
2.2.1. EGFR and VEGFR-2 inhibitory assay
All the synthesized quinazolin-4-amine derivatives 9a–f and 19a-r were evaluated for their
enzymatic activities against EGFR and VEGFR-2. The approved EGFR and VEGFR-2 inhibitor
drug vandetanib was used as a positive control. The results were summarized in Table 1. Among
these compounds, compound 19g and 19i displayed the most potent inhibitory activity against
EGFR with IC50 of 1 nM, while compound 19j and 19l showed the most potent activity with IC50 of
14 nM against VEGFR-2.
Most of the tested compounds 19a-r exhibited potent EGFR or VEGFR-2 inhibitory activities,
however 9a-f with a benzyloxy chain in C-7 position, showed a significant decrease in activity
against EGFR or VEGFR-2 (IC50 more than 10 µM). Introduction of chlorine in the ortho-position
of the urea group is a key modification to increase kinases inhibition of both EGFR and VEGFR-2
(compounds 19f-i versus 19a-e). Compounds 19f-g bearing 4-methylpiperazine group at 7-position
of quinazoline skeleton demonstrated higher potency against both EGFR and VEGFR-2 compared
to the corresponding compound 19k contained morpholine group and compound 19m-n contained
piperidine group at 7-position. However, introduction of morpholine (19l) or piperidine (19o) side
chains showed a significant decrease in activity against EGFR but increased inhibitory activity
against VEGFR-2 compared to compound 19i bearing 4-methylpiperazine group at 7-position, all
of them had meta-methyl and para-methyl substituents on the terminal phenyl ring. We also
discovered that compounds 19p-r with glycine methyl ester group showed moderate inhibition
against EGFR and VEGFR-2, which were comparable to compounds 19a-e.
2.2.2. Antiproliferation assay

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Antiproliferative activities of compounds were evaluated toward HT-29 (human colorectal
adenocarcinoma), MCF-7 (human breast cancer) and H460 (human lung cancer) cell lines by MTT
assay. As shown in Table 1, most of these compounds demonstrated moderate to good
anti-proliferative activities. Compounds 19i-j, 19l, 19n, 19o showed better activities against three
cell lines than the reference drug vandetanib, all of them had chlorine in the ortho-position of the
urea group. It is worthy to note that compound 19g only showed good activities against EGFR and
VEGFR-2, but it exhibited moderate anti-proliferative effects against HT-29, MCF-7 and H460
cells. Interestingly, compound 19o displayed the highest potency against three cancer cell lines
(HT-29, MCF-7 and H460) with IC50 of 1.85 µM, 1.27 µM and 2.90 µM, respectively, although the
compound showed moderate EGFR/VEGFR-2 inhibitory effects. Similarly, compound 19n bearing
piperidine group at 7-position of quinazoline skeleton and para-chlorine on the terminal phenyl
ring exhibited almost no potency against EGFR/VEGFR-2 (IC50 more than 10 µM), but showed
high antiproliferative activities against three cancer cell lines. Introduction of glycine methyl ester
group on compounds 19p-r resulted in a complete loss of cellular antiproliferative activities (IC50
more than 50 µM) except 19r (IC50 = 8.39 µM against MCF-7).
Table 1
Enzymatic and cellular results for 4-anilinoquinazoline derivatives.
Enzymatic inhibition
(IC50, nM)
Proliferative inhibition
Compd
X
R1
R2
(IC50, µM)
EGFR
VEGFR-2
HT-29
MCF-7
H460
H
H
H
H
H
H
2933
>10000
>50
>50
22.76
>50
38.75
>50
9a
9b
9c
9d
9e
p-Cl
>10000
4649
>10000
>10000
>10000
>10000
>50
>50
>50
>50
m-Cl
p-CH3
o-CH3
>50
>10000
>10000
>50
25.43
23.95
28.84

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H
H
m-CH3, p -CH3
>10000
194
235
500
502
668
14
>10000
576
>50
49.44
28.68
22.86
>50
>50
29.07
>50
49.57
28.39
17.80
8.16
22.29
22
9f
H
p-Cl
19a
19b
19c
19d
19e
19f
19g
19h
19i
H
815
H
m-Cl
588
24.72
32.17
36.47
>50
H
o-CH3
1986
214
H
m-CH3, p-CH3
H
25.94
17.33
20.65
4.81
Cl
Cl
Cl
Cl
261
33
p-Cl
1
279
>50
28
o-CH3
15
178
6.21
7.28
>50
26
m-CH3, p-CH3
1
79
1.76
Cl
Cl
Cl
Cl
Cl
Cl
m-Cl, p-F
p-Cl
78
17
14
528
6.41
21.99
7.29
>50
3.20
39.23
2.63
>50
12.10
28
19j
19k
19l
N
N
O
O
m-CH3, p-CH3
H
51
14
3.52
>50
5
212
>10000
817
611
1873
>10000
18
19m
19n
19o
19p
19q
p-Cl
4.38
1.85
>50
1.89
1.27
>50
m-CH3, p-CH3
2.90
>50
>50
275
N
763
404
>50
>50
O
9941
11
966
15
>50
8.39
>50
19r
18.95
11.83
37.10
vandetanib
2.3. Molecular docking studies
In order to understand the interaction between the synthesized compounds and kinases,

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molecular docking studies on the potent compound 19i were performed using the Schrodinger
software.
As shown in Fig. 3, the protonated piperazine nitrogen which connect methyl of compound 19i
interact with the amino acid Asp1003 through an ionic bond in the EGFR ATP binding site (PDB:
2ity). One hydrogen bond was formed between Met793 and N1 of the quinazoline, another
hydrogen bonds were formed between NHs of urea moiety and Asp855. Furthermore, hydrophobic
interactions were observed with amino acids in the active site of EGFR, including Val726, Leu718
and Leu844.
Docking study was also carried out for compound 19i into inactive DFG-out conformation of
VEGFR-2 (PDB: 2OH4) (Fig. 4). Compound 19i could be accommodated to the DFG-out
conformation of VEGFR-2 comfortably. The quinazoline nucleus is located in a hydrophobic
pocket lined with Leu838, Ala864, Phe916 and Leu1033. Quinazoline N1 forms a hydrogen bond
with the backbone amide NH of Cys917 in the hinge region. The central phenyl linking to the
quinazoline core through 4-NH occupies the hydrophobic pocket neighboring the ATP binding site.
Carbonyl and NHs of urea form hydrogen bond interactions with the Asp1044 backbone and
Glu883 side chain, respectively. Terminal 3,4-dimethylphenyl extends into a second hydrophobic
pocket created by the Leu887，Ile886，Leu1017 and Ile890.
Fig. 3. 3D model of the interaction between compound 19i and EGFR ATP binding site. The
hydrogen bonds are displayed as yellow dotted lines.

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Fig. 4. Molecular modeling of 19i bound to DFG-out conformation of VEGFR-2. The hydrogen
bonds are displayed as yellow dotted lines.
3. Conclusions
In summary, we have designed and synthesized a series of 4-anilinoquinazoline derivatives
containing substituted diaryl urea or glycine methyl ester moiety as EGFR and VEGFR-2 dual
inhibitors. Most of them exhibited good inhibitory potencies against EGFR and VEGFR-2 as well
as interesting antiproliferative activities. Molecular docking established the interaction of 19i with
the DFG-out conformation of VEGFR-2, suggesting that they might be type II kinase inhibitors.
Our results indicated that the functionalization of the terminal diaryl urea (-ArNHCONHAr) moiety
and chlorine in the ortho-position of the urea group lead to compounds (19i, 19j and 19l)
characterized by excellent inhibitory potencies.
4. Experimental section
4.1. Synthesis and characterization of compounds
4.1.1. General
All commercially available reagents and solvents were all analytical grade and used directly
without further purification. Melting points (uncorrected) were determined on a RY-1 MP apparatus.
1H nuclear magnetic resonance (NMR) spectra were determined in DMSO-d6 or CDCl3 solutions
using a Bruker AV-300 spectrometer. Chemical shifts were reported in ppm (δ). Infrared (IR)
spectra were recorded on Thermo FTIR spectrometer (KBr disks). Mass spectrometry was obtained
using a Q-tof high resolution mass spectrometer.
4.1.2. 3-methoxy-4-benzyloxybenzaldehyde (2)
To a solution of vanillin (1) (11.4 g, 75 mmol) in ethanol (100 mL), were added the benzyl

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chloride (13 mL, 112.5 mmol) and K2CO3 (13.5 g, 97.6 mmol). After 6 h at reflux, the mixture was
cooled to room temperature and filtered. The solvent was removed under reduced pressure and the
residue crystallization from EtOH gave pure 2 (17.3 g) as a light yellow solid with a yield of 95.7%.
1
Mp: 58~60°C; H NMR (CDCl3, 300 MHz): δ (ppm) 3.94 (s, 3H), 5.24 (s, 2H), 6.97-6.99 (d, J =
8.2 Hz, 1H), 7.25 (s, 1H), 7.29-7.45 (m, 6H), 9.83 (s, 1H).
4.1.3. 3-methoxy-4-benzyloxybenzoic acid (3)
The above benzaldehyde (6.05 g, 25 mmol) was dissolved in a mixture of acetone : H2O (75 :
62.5 mL) and to it KMnO4 (4.74 g, 30 mmol) was added and the mixture was stirred for 1 h at
reflux. The reaction mixture was filtered while hot. The filtrate was made alkaline (40% NaOH
solution) to PH 10-11 and filtered. Then the filtrate was acidified with HCl until no further
precipitate formed (PH 2-3). The resulting white precipitate was filtered, washed with water and
dried to afford 5.7 g of the product as a white solid with a yield of 89%. Mp: 173~175°C; 1H NMR
(CDCl3, 300 MHz): δ (ppm) 3.94 (s, 3H), 5.22 (s, 2H), 6.90-6.92 (d, J = 8.4 Hz, 1H), 7.31-7.44 (m,
5H), 7.60 (s, 1H), 7.65-7.68 (m, 1H).
4.1.4. Methyl 3-methoxy-4-benzyloxybenzoate (4)
A catalytic amount of concentrated H2SO4 was added to a solution of compound 3 (5.4 g, 20.9
mmol) in 50 mL of methanol and the mixture was refluxed for 10 h. It was allowed to cool. The
product crystallization from solution gave pure 4 (4.5 g) as a white solid with a yield of 80%. Mp:
1
70~72oC; H NMR (CDCl3, 300 MHz): δ (ppm) 3.90 (s, 3H), 3.95 (s, 1H), 5.23 (s, 2H), 7.32-7.57
(m, 5H), 7.58 (s, 1H), 7.60-7.61 (d, J = 2.0 Hz, 1H), 7.63-7.64 (d, J = 1.8 Hz, 1H).
4.1.5. Methyl 2-nitro-4-benzyloxy- 5-methoxybenzoate (5)
Fuming nitric acid (5.8 mL, 13.6 mmol) was added dropwise to a solution of methyl
3-methoxy-4-benzyloxybenzoate (4, 3.7 g, 13.3 mmol) in glacial acetic acid (34 mL) at 0-5°C and
this mixture was stirred at 0-5°C for 30 min and then for 6 h at room temperature. The reaction
mixture was poured on ice/water (120 mL) and the resulting precipitate was filtered and washed
with chill water, dried to afford 4.0 g of the product as a light yellow solid with a yield of 94.7%.
Mp: 128~130oC; 1H NMR (CDCl3, 300 MHz): δ (ppm) 3.90 (s, 3H), 3.95 (s, 3H), 5.23 (s, 2H), 6.89
(s, 1H), 7.28 (s, 1H), 7.33-7.50 (m, 5H).
4.1.6. Methyl 2-amino-4-benzyloxy-5-methoxybenzoate (6)
A mixture of compound 5 (2.0 g, 6.2 mmol), zinc powder (1.2 g, 19 mmol), glacial acetic acid
(3.6 ml, 63.3 mmol) in methanol : H2O (33 : 3 mL) was stirred at reflux for 3 h. The reaction
mixture was filtered while hot and the solid was washed with CH3OH. The solvent was removed
from filtrate and the resulting solid was diluted with water (50 mL) and subsequently extracted with
ethyl acetate. The extracts were combined, washed with saturated sodium bicarbonate and saturated

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saline solution, and dried over anhydrous Na2SO4. The solvent was removed under vacuum to give
6 as a light yellow solid (84.7%). Mp: 131~133°C; 1H NMR (CDCl3, 300 MHz): δ (ppm) 3.83-3.84
(d, J = 3.7 Hz, 6H), 5.14 (s, 2H), 5.52 (s, 2H), 6.16 (s, 1H), 7.26-7.42 (m, 6H).
4.1.7. 6-methoxy-7-benzyloxyquinazolin-4-ol (7)
To a solution of compound 6 (2.65 g, 9.23 mmol) in formamide (40 mL) was added
ammonium formate (0.53 g, 8.41 mmol). The reaction mixture was stirred for 3.5 h under reflux
conditions. Then cooled to room temperature. The precipitated crystals were collected by filtration,
washed with H2O and dried to afford 2.13 g of the product as a brown crystal solid with a yield of
1
82%. Mp: 127~129oC; H NMR (CDCl3, 300 MHz): δ (ppm) 4.01 (s, 3H), 5.29 (s, 2H), 7.30-7.55
(m, 5H), 7.61 (s, 1H), 7.99 (s, 1H), 8.20-8.25 (d, J = 13.8 Hz, 1H), 11.54 (s, 1H).
4.1.8. 4-chloro-6-methoxy-7-benzyloxyquinazoline (8)
Compound 7 (860 mg, 3.04 mmol) was added to thionyl chloride (9.5 mL) with magnetic
stirring. DMF (0.2 mL) and pyridine (0.23 mL) were then added dropwise and the mixture was
heated to reflux for 1 h. Isolation was performed by the slow addition of the reaction medium over
a mixture of ice and water with vigorous stirring. The resulting precipitate was filtered under a
1
vacuum and dried to give 8 (810 mg, 88.6%) as yellow powder. Mp: 132~134oC; H NMR
(DMSO-d6, 300 MHz): δ (ppm) 3.99 (s, 3H), 5.36 (s, 2H), 7.37-7.56 (m, 7H), 8.87 (s, 1H).
4.1.9. General procedure for 1-(4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)amino)phenyl)-3-
phenylurea derivatives (9a-f)
A mixture of compound 8 (178 mg, 0.59 mmol), aniline 13a-f (104 mg, 0.49 mmol) and
2-propanol (5 mL) was refluxed at 90oC with stirring in oil bath for 30 min. Upon completion of
the reaction, the reaction mixture was cooled to ambient temperature and the solid product
precipitated out was filtered, washed with cold 2-propanol and dried.
4.1.9.1. 1-(4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)amino)phenyl)-3-phenylurea (9a)
Yield, 64.5%; Mp: 244~246oC; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.99 (s, 3H), 5.32 (s,
2H), 6.92-6.97 (t, 1H), 7.24-7.29 (d, J = 15.6 Hz, 2H), 7.36-7.55 (m, 12H), 8.19 (s, 1H), 8.76 (s,
1H), 9.05 (s, 1H), 9.19 (s, 1H), 11.17 (s, 1H). ESI-MS: m/z 492 [M+H]+. HRMS (ESI): m/z calcd
for (C29H25N5O3+H)+: 492.203; found: 492.2024.
4.1.9.2. 1-(4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)amino)phenyl)-3-(4-chlorophenyl)urea
(9b)
1
Yield, 64%; Mp: 230~232oC; H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.96 (s, 3H), 5.28 (s,
2H), 7.28-7.60 (m, 14H), 8.00 (s, 1H), 8.61 (s, 1H), 8.97-9.02 (d, J = 15.0 Hz, 2H), 10.43(s, 1H).
ESI-MS: m/z 526 [M+H]+. HRMS (ESI): m/z calcd for (C29H24ClN5O3+H)+: 526.164; found:
526.1631.

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4.1.9.3. 1-(4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)amino)phenyl)-3-(3-chlorophenyl)urea
(9c)
Yield, 73.4%; Mp: 238~240oC; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.99 (s, 3H), 5.32 (s,
2H), 6.98 (s, 1H), 7.27-7.28 (d, J = 3.3 Hz, 2H), 7.37-7.55 (m, 10H), 7.70 (s, 1H), 8.18 (s, 1H),
8.75 (s, 1H), 9.33 (s, 1H), 9.41 (s, 1H), 11.12 (s, 1H). ESI-MS: m/z 526 [M+H]+. HRMS (ESI): m/z
calcd for (C29H24ClN5O3+H)+: 526.164; found: 526.1638.
4.1.9.4. 1-(4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)amino)phenyl)-3-(p-tolyl)urea (9d)
Yield, 93.3%; Mp: 235~237oC; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.23 (s, 3H), 3.98 (s,
3H), 5.31 (s, 2H), 7.05-7.08 (d, J = 8.1 Hz, 2H), 7.31-7.57 (m, 12H), 8.09 (s, 1H), 8.67 (s, 1H),
8.80 (s, 1H), 8.97 (s, 1H), 10.74 (s, 1H). HRMS (ESI): m/z calcd for (C30H27N5O3+H)+: 506.2187;
found: 506.2189.
4.1.9.5. 1-(4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)amino)phenyl)-3-(o-tolyl)urea (9e)
1
Yield, 67%; Mp: 236~238oC; H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.24 (s, 3H), 3.94 (s,
3H), 5.25 (s, 2H), 6.89-6.94 (t, 1H), 7.10-7.17 (m, 2H), 7.30-7.50 (m, 8H), 7.63-7.66 (d, J = 8.8 Hz,
2H), 7.83-7.89 (d, J = 6.2 Hz, 3H), 8.39 (s, 1H), 9.00 (s, 1H), 9.47 (s, 1H). ESI-MS: m/z 506
[M+H]+. HRMS (ESI): m/z calcd for (C30H27N5O3+H)+: 506.2187; found: 506.2188.
4.1.9.6.1-(4-((7-(benzyloxy)-6-methoxyquinazolin-4-yl)amino)phenyl)-3-(3,4-dimethylphenyl)urea
(9f)
Yield, 58.3%; Mp: 230~232oC; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.13 (s, 3H), 2.24 (s,
3H), 3.96 (s, 3H), 5.29 (s, 2H), 6.87-6.89 (d, J = 7.4 Hz, 1H), 6.96-7.04 (m, 1H), 7.30-7.59 (m,
12H), 7.97 (s, 1H), 8.59 (s, 1H), 9.03 (s, 1H), 10.28 (s, 1H). ESI-MS: m/z 520 [M+H]+. HRMS
(ESI): m/z calcd for (C31H29N5O3+H)+: 520.2343; found: 520.2349.
4.1.10. General procedure for 1-(4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-
4-yl)amino)phenyl)-3-phenylurea derivatives (19a-r)
The products of 9a-f taking off the benzyl (150 mg, 0.39 mmol) and potassium carbonate (325
mg, 2.35 mmol) were stirred together in DMF (5 mL) for 30 min. Then aminoalkyl chloride (118
mg, 0.47 mmol) and potassium iodide (65 mg, 0.39 mmol) were added and the mixture was
refluxed for 18 h. After cooling to room temperature, the mixture was diluted with water (30 mL)
and subsequently extracted with ethyl acetate. The extracts were combined, washed with saturated
sodium bicarbonate and saturated saline solution, and dried over anhydrous Na2SO4. Then the
solvent was evaporated in vacuum and the residue was recrystallised from ethyl acetate/petroleum
ether to give 19a-r as a light yellow solid.
4.1.10.1. 1-(4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)phenyl)-
3-phenylurea (19a)

ACCEPTED MANUSCRIPT
Yield, 11.1%; Mp: 128~130oC; IR (KBr): 3132, 2940, 2805, 1700, 1620, 1587, 1513, 1420, 1237,
1
1144, 1068, 926, 840, 751 cm-1; H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.89-1.97 (m, 2H),
2.04-2.48 (m, 13H), 3.93 (s, 3H), 4.13-4.17 (t, J = 5.3 Hz, 2H), 6.94-6.97 (d, J = 7.1 Hz, 2H), 7.13
(s, 1H), 7.24-7.34 (m, 3H), 7.43-7.46 (d, J = 8.9 Hz, 2H), 7.62-7.65 (d, J = 8.8 Hz, 2H), 7.81 (s,
1H), 8.26 (s, 1H), 8.38 (s, 1H), 8.64 (s, 1H), 9.38 (s, 1H). ESI-MS: m/z 542 [M+H]+. HRMS (ESI):
m/z calcd for (C30H35N7O3+H)+: 542.2874; found: 542.2872.
4.1.10.2. 1-(4-chlorophenyl)-3-(4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-
yl)amino)phenyl)urea (19b)
Yield, 21%; Mp: 132~134oC; IR (KBr): 3126, 2932, 2806, 1675, 1619, 1583, 1512, 1419, 1236,
1144, 1066, 930, 850, 830 cm-1; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.88-2.07 (m, 2H), 2.15
(s, 3H), 2.34-2.48 (m, 10H), 3.93 (s, 3H), 4.12-4.16 (t, J = 5.9 Hz, 2H), 7.12 (s, 1H), 7.28-7.31 (d, J
= 8.7 Hz, 2H), 7.38-7.50 (m, 4H), 7.63-7.66 (d, J = 8.8 Hz, 2H), 7.81 (s, 1H), 7.38 (s, 1H), 8.76 (s,
1H), 8.87 (s, 1H), 9.38 (s, 1H). ESI-MS: m/z 576 [M+H]+.
4.1.10.3. 1-(3-chlorophenyl)-3-(4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-
yl)amino)phenyl)urea (19c)
Yield, 16.8%; Mp: 118~120°C; IR (KBr): 3126, 2933, 2806, 1675, 1621, 1585, 1511, 1418,
1236, 1143, 1067, 925, 851, 777 cm-1; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.89-2.03 (m, 2H),
2.12 (s, 3H), 2.30-2.48 (m, 10H), 3.93 (s, 3H), 4.11-4.15 (t, J = 6.1 Hz, 2H), 7.12 (s, 1H), 7.22-7.31
(m, 3H), 7.38 (s, 1H), 7.45-7.48 (d, J = 7.4 Hz, 2H), 7.63-7.64 (d, J = 2.4 Hz, 2H), 7.72 (s, 1H),
7.82 (s, 1H), 8.26 (s, 1H), 8.38 (s, 1H), 9.41 (s, 1H). ESI-MS: m/z 576 [M+H]+.
4.1.10.4. 1-(4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)phenyl)-
3-(o-tolyl)urea (19d)
Yield, 20.5%; Mp: 138~140oC; IR (KBr): 3134, 2940, 2805, 1670, 1620, 1581, 1512, 1419, 1239,
1144, 1066, 923, 857, 753 cm-1; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.88-1.97 (m, 2H), 2.12
(s, 3H), 2.24 (s, 3H), 2.33-2.48 (m, 10H), 3.94 (s, 3H), 4.12-4.16 (t, J = 6.2 Hz, 2H), 7.12-7.25 (m,
4H), 7.47-7.48 (d, J = 1.5 Hz, 2H), 7.63-7.64 (d, J = 1.9 Hz, 2H), 7.85-7.96 (m, 3H), 8.38 (s, 1H),
8.98-9.03 (d, 1H), 9.37 (s, 1H). ESI-MS: m/z 556 [M+H]+. HRMS (ESI): m/z calcd for
(C31H37N7O3+H)+: 556.3031; found: 556.3038.
4.1.10.5. 1-(3,4-dimethylphenyl)-3-(4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)
quinazolin-4-yl)amino)phenyl)urea (19e)
Yield, 11.7%; Mp: 139~141oC; IR (KBr): 3129, 2937, 2802, 1685, 1621, 1576, 1503, 1400, 1279,
1
1145, 1072, 860 cm-1; H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.88-2.00 (m, 2H), 2.19 (s, 6H),
2.30 (s, 1H), 2.40-2.48 (m, 10H), 3.93 (s, 3H), 4.12-4.16 (t, J = 6.2 Hz, 2H), 7.12 (s, 1H), 7.18-7.26
(m, 2H), 7.44-7.47 (d, J = 8.8 Hz, 2H), 7.55-7.65 (m, 3H), 7.81 (s, 1H), 8.03 (s, 1H), 8.38 (s, 1H),

ACCEPTED MANUSCRIPT
8.96 (s, 1H), 9.38 (s, 1H). ESI-MS: m/z 570 [M+H]+. HRMS (ESI): m/z calcd for (C32H39N7O3+H)+:
570.3187; found: 570.3196.
4.1.10.6. 1-(2-chloro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)
phenyl)-3-phenylurea (19f)
Yield, 14%; Mp: 123~125oC; IR (KBr): 3128, 2940, 2806, 1675, 1621, 1598, 1501, 1417, 1285,
1144, 1066, 931, 856, 752 cm-1; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.88-1.91 (d, J = 10.1 Hz,
2H), 2.13 (s, 3H), 2.32-2.48 (m, 10H), 3.94 (s, 3H), 4.14-4.36 (s, 2H), 6.97-7.33 (m, 6H), 7.44-7.47
(d, J = 7.7 Hz, 1H), 7.68 (s, 1H), 7.80 (s, 1H), 8.05-8.11 (t, 2H), 8.27 (s, 1H), 8.46 (s, 1H), 9.47 (s,
1H). ESI-MS: m/z 576 [M+H]+. HRMS (ESI): m/z calcd for (C30H34ClN7O3+H)+: 576.2484; found:
576.2485.
4.1.10.7. 1-(2-chloro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)
phenyl)-3-(4-chlorophenyl)urea (19g)
Yield, 12%; Mp: 128~130oC; IR (KBr): 3126, 2941, 2807, 1675, 1620, 1576, 1505, 1400, 1284,
1145, 1091, 930, 857, 829 cm-1; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.91-1.96 (m, 2H), 2.15
(s, 3H), 2.34-2.56 (m, 10H), 3.96 (s, 3H), 4.14-4.18 (t, J = 6.2 Hz, 2H), 7.17 (s, 1H), 7.33-7.35 (d, J
= 8.7 Hz, 2H), 7.49-7.52 (d, J = 8.8 Hz, 2H), 7.70-7.73 (d, J = 8.9 Hz, 1H), 7.82 (s, 1H), 8.07-8.10
(d, J = 9.0 Hz, 2H), 8.30 (s, 1H), 8.48 (s, 1H), 9.45-9.49 (d, J = 10.1 Hz, 2H). ESI-MS: m/z 610
[M+H]+. HRMS (ESI): m/z calcd for (C30H33Cl2N7O3+H)+: 610.2095; found: 610.2098.
4.1.10.8. 1-(2-chloro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)
phenyl)-3-(o-tolyl)urea (19h)
Yield, 10.4%; Mp: 122~124oC; IR (KBr): 3134, 2941, 2806, 1671, 1622, 1577, 1505, 1420, 1283,
1144, 1068, 930, 856, 817 cm-1; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.88-1.96 (m, 2H), 2.13
(s, 3H), 2.26 (s, 1H), 2.34-2.48 (m, 10H), 3.95 (s, 3H), 4.13-4.17 (t, J = 5.8 Hz, 2H), 6.95-6.98 (d, J
= 7.6 Hz, 1H), 7.10-7.26 (m, 3H), 7.66-7.69 (d, J = 8.9 Hz, 1H), 7.76-7.86 (m, 2H), 8.03-8.08 (t,
2H), 8.46 (s, H), 8.52-8.58 (d, J = 16.9 Hz, 1H), 8.62-8.68 (d, J = 19.8 Hz, 1H), 9.47 (s, 1H).
ESI-MS: m/z 590 [M+H]+. HRMS (ESI): m/z calcd for (C31H36ClN7O3+H)+: 590.2641; found:
590.2644.
4.1.10. 9. 1-(2-chloro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)
phenyl)-3-(3,4-dimethylphenyl)urea (19i)
Yield, 25%; Mp: 130~132oC; IR (KBr): 3126, 2941, 2805, 1678, 1620, 1577, 1503, 1417, 1284,
1143, 1065, 930, 858, 818 cm-1; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.69-2.02 (m, 2H), 2.15
(s, 3H), 2.23-2.50 (m, 16H), 3.96 (s, 3H), 4.16-4.35 (s, 2H), 7.17-7.24 (m, 3H), 7.45-7.57 (m, 1H),
7.67-7.70 (d, J = 7.2 Hz, 1H), 7.89-7.91 (d, J = 4.3 Hz, 1H), 8.08 (s, 2H), 8.48 (s, 1H), 8.59 (s, 1H),
8.68 (s, 1H), 9.49 (s, 1H). ESI-MS: m/z 604 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.10.10. 1-(2-chloro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)
amino)phenyl)-3-(3-chloro-4-fluorophenyl)urea (19j)
1
Yield, 12%; Mp: 215~217°C; H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.07 (m, 2H), 2.47 (s,
3H), 2.82-3.00 (m, 10H), 3.98 (s, 3H), 4.22 (s, 2H), 7.20 (s, 1H), 7.25-7.27 (m, 1H), 7.32-7.38 (m,
1H), 7.58-7.61 (d, J = 8.9 Hz, 1H), 7.82-7.84 (d, J = 5.0 Hz, 1H), 7.90 (s, 1H), 8.01 (s, 1H),
8.18-8.21 (d, J = 9.0 Hz, 1H), 8.44 (s, 1H), 8.81 (s, 1H), 9.60 (s, 1H), 10.74 (s, 1H). HRMS (ESI):
m/z calcd for (C30H32Cl2FN7O3+H)+: 628.2; found: 628.2004.
4.1.10.11. 1-(2-chloro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)
-3-(4-chlorophenyl)urea (19k)
Yield, 26.7%; Mp: 213~215oC; IR (KBr): 3125, 2958, 2807, 1675, 1621, 1576, 1493, 1418, 1285,
1
1142, 1092, 930, 859, 829 cm-1; H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.79-2.12 (m, 2H),
2.36-2.63 (m, 6H), 3.46-3.69 (t, 4H), 3.94 (s, 3H), 4.15-4.38 (s, 2H), 7.16 (s, 1H), 7.31-7.33 (d, J =
7.8 Hz, 2H), 7.47-7.50 (d, J = 8.3 Hz, 2H), 7.67-7.70 (d, J = 7.8 Hz, 1H), 7.80 (s, 1H), 7.93-8.08 (t,
2H), 8.28 (s, 1H), 8.46 (s, 1H), 9.44-9.48 (d, J = 10.5 Hz, 2H). ESI-MS: m/z 619 [M+Na]+. HRMS
(ESI): m/z calcd for (C29H30Cl2N6O4+H)+: 597.1778; found: 597.1784.
4.1.10.12. 1-(2-chloro-4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)-
3-(3,4-dimethylphenyl)urea (19l)
Yield, 29%; Mp: 140~142°C; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.93 (m, 2H), 2.13-2.17
(d, 6H), 2.35-2.42 (m, 6H), 3.48-3.62 (m, 4H), 3.94 (s, 3H), 4.15 (s, 2H), 7.00-7.02 (d, J = 7.9 Hz,
1H), 7.15-7.23 (m, 3H), 7.66-7.68 (d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 8.06-8.09 (d, J = 11.5 Hz, 2H),
8.29 (s, 1H), 8.45 (s, 1H), 9.30 (s, 1H), 9.53 (s, 1H). HRMS (ESI): m/z calcd for
(C31H35ClN6O4+H)+: 591.2481; found: 591.2483.
4.1.10.13. 1-(2-chloro-4-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)quinazolin-4-yl)amino)phenyl)-
3-phenylurea (19m)
1
Yield, 19%; H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.36-1.48 (m, 6H), 2.36-2.54 (t, 4H),
2.61-2.80 (t, 2H), 3.91 (m, 3H), 4.19-4.21 (m, 2H), 6.97 (s, 1H), 7.14-7.34 (m, 3H), 7.44-7.47 (m,
1H), 7.62-7.75 (m, 1H), 7.80-7.90 (m, 1H), 8.05-8.11 (m, 2H), 8.20-8.30 (m, 2H), 8.46 (s, 1H),
9.33 (m, 1H), 9.48 (s, 1H). HRMS (ESI): m/z calcd for (C29H31ClN6O3+H)+: 547.2219; found:
547.2218.
4.1.10.14. 1-(2-chloro-4-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)quinazolin-4-yl)amino)phenyl)-
3-(4-chlorophenyl)urea (19n)
Yield, 26%; Mp: 142~144oC; IR (KBr): 3124, 2938, 2807, 1668, 1598, 1576, 1492, 1416, 1285,
1
1142, 1092, 933, 827, 689 cm-1; H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.36-1.60 (m, 6H),
2.70-2.75 (t, 4H), 2.86-2.92 (t, 2H), 3.94 (s, 3H), 4.20-4.24 (s, 2H), 7.04 (s, 1H), 7.20 (s, 1H),

ACCEPTED MANUSCRIPT
7.31-7.34 (d, J = 8.8 Hz, 2H), 7.47-7.50 (d, J = 8.34 Hz, 2H), 7.67-7.70 (d, J = 9.2 Hz, 1H), 7.79 (s,
1H), 7.93 (s, 1H), 8.05-8.06 (d, J = 2.1 Hz, 1H), 8.29 (s, 1H), 8.40-8.46 (d, J = 18 Hz, 1H),
9.43-9.49 (t, 1H). ESI-MS: m/z 581 [M+H]+. HRMS (ESI): m/z calcd for (C29H30Cl2N6O3+H)+:
581.1829; found: 581.1832.
4.1.10.15. 1-(2-chloro-4-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)quinazolin-4-yl)amino)phenyl)-
3-(3,4-dimethylphenyl)urea (19o)
Yield, 12%; Mp: 145~148°C; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.40 (m, 2H), 1.52 (m,
4H), 2.13-2.17 (d, 6H), 2.50-2.90 (m, 6H), 3.94 (s, 3H), 4.25 (s, 2H), 7.00-7.03 (d, J = 10.1 Hz, 1H),
7.15 (d, 1H), 7.21-7.23 (d, J = 7.6 Hz, 2H), 7.65 (d, 1H), 7.82 (s, 1H), 8.05-8.08 (m, 2H), 8.23 (s,
1H), 8.46 (s, 1H), 9.20 (s, 1H), 9.51 (s, 1H). HRMS (ESI): m/z calcd for (C31H35ClN6O3+H)+:
575.2532; found: 575.2531.
4.1.10.16. Methyl 2-(4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)
benzamido)acetate (19p)
1
Yield, 48.5%; Mp: 230~232°C; H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.03-2.05 (m, 2H),
2.48 (s, 3H), 2.62-3.16 (m, 10H), 3.65 (s, 3H), 3.98 (s, 3H), 4.00-4.02 (d, J = 5.8 Hz, 2H), 4.21 (t, J
= 3.0 Hz, 2H), 7.21 (s, 1H), 7.87-7.95 (m, 4H), 7.96 (s, 1H), 8.67 (s, 1H), 8.89-8.93 (t, 1H), 10.23
(s, 1H). HRMS (ESI): m/z calcd for (C27H34N6O5+H)+: 523.2663; found: 523.2671.
4.1.10.17. Methyl 2-(4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)benzamido)
acetate (19q)
1
Yield, 45%; Mp: 232~234°C; H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.93-1.97 (m, 2H),
2.40-2.48 (m, 6H), 3.51-3.61 (m, 4H), 3.64 (s, 3H), 3.96 (s, 3H), 3.99-4.01 (d, J = 5.5 Hz, 2H), 4.17
(t, J = 6.0 Hz, 2H), 7.19 (s, 1H), 7.85 (s, 1H), 7.87-7.98 (m, 4H), 8.51 (s, 1H), 8.82-8.86 (t, 1H),
9.62 (s, 1H). HRMS (ESI): m/z calcd for (C26H31N5O6+H)+: 510.2347; found: 510.2358.
4.1.10.18. Methyl 2-(4-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)quinazolin-4-yl)amino)
benzamido)acetate (19r)
1
Yield, 36.6%; Mp: 240~242°C; H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.30-1.40 (m, 2H),
1.42-1.53 (m, 4H), 2.35-2.47 (m, 4H), 2.71 (t, 2H), 3.64 (s, 3H), 3.95 (s, 3H), 3.98-4.00 (d, J = 5.5
Hz, 2H), 4.22 (s, 2H), 7.22 (s, 1H), 7.84 (s, 1H), 7.87-7.97 (m, 4H), 8.51 (s, 1H), 8.81-8.91 (t, 1H),
9.63 (s, 1H). HRMS (ESI): m/z calcd for (C26H31N5O5+H)+: 494.2398; found: 494.2407.
4.2. In vitro kinase assay
Materials: EGFR and VEGFR-2 were purchased from Carna. ATP, DMSO and EDTA were
purchased from Sigma. 96-well plate and 384-well plate were purchased from Corning.
A Caliper mobility shift assay was used to measure the potency of title compounds against
VEGFR-2 and EGFR. The compounds were dissolved to 10mM in DMSO. Dilute the compound to

ACCEPTED MANUSCRIPT
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 compound, transfer the compound in
tubes to one well on 96-well storage plate and serially dilute the compound by transferring 10µl to
90µl 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.
Kinase reaction: 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 28º
C for specified period of time. Add 25µl of stop buffer to stop reaction.
Collect data on Caliper. Copy conversion data from Caliper program. Convert conversion
values to inhibition values according to percent inhibition = (max-conversion)/(max-min)*100, in
which “max” stands for DMSO control; “min” stands for no enzyme activity control. Fit the data in
XLfit to obtain IC50 values. Equation used is
Y=Bottom + (Top-Bottom) / (1+(IC50/X)^HillSlope)
4.3. Cell proliferative assay
The antiproliferative activities of the prepared 4-anilinoquinazoline derivatives against HT-29,
MCF-7 and H460 cells were evaluated by MTT assay. Briefly, trypsinize disk and determine the
cell density, dilute the cell slurry to the required volume at optimized density, dispense 90 µl/well of
the cell slurry onto assay plates, then incubate the assay plates for 24 hours at 37oC, 5% CO2 under
humidified conditions. Prepare the reference and test compounds solution in 100% DMSO
according to the platemap (200× final concentration). Transfer 7 µl of the compound to 133 µl
complete medium (final conc: 10×), then transfer 10 µl of the compound to assay plate (final conc:
1×), incubate the plate at 37oC, 5% CO2 under humidified conditions. After 48 h exposure period,
20 µl of 5 mg/ml MTT was added to each well. Four hours later, 100 µl of testing solution (10%
SDS+5% isobutyl alcohol+0.010 M HCl) was added. After an overnight incubation at 37oC, 5%
CO2, the absorbance was measured at 580/680 nm on SpectraMax Plus 384 (MDC).
4.4. Molecular docking
Docking was carried out using Maestro 9.3. X-ray cocrystal structures of EGFR (PDB code
2TIY) and VEGFR-2 (PDB code 2OH4) were downloaded from RCSB Protein Data Bank and

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prepared using Protein Preparation Wizard Workflow provided in the Maestro graphical user
interface of the Schrödinger program suite, and the default settings were used. Compound 19i was
docked into the defined binding site without constraint and were ranked based on Glide-score. Top
ranking compounds were submitted and generated by Pymol.
Acknowledgments
We are grateful to the funds supported by the National Natural Science Foundation (No.
21172265 and No. 20872181), Qing Lan Project in Jiangsu Province and Graduate Education
Innovation Project in Jiangsu Province (CXZZ13_0322).This work was also supported by Excellent
Science and Technology Innovation Team of Jiangsu Province in 2015 China.
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Design and discovery of 4-anilinoquinazoline-urea derivatives
as dual TK inhibitors of EGFR and VEGFR-2
Hai-Qi Zhang1, Fei-Hu Gong1, Ji-Qing Ye, Chi Zhang, Xiao-Hong Yue, Chuan-Gui Li,
Yun-Gen Xu*, Li-Ping Sun*
1 These authors contributed equally to this work.
Highlights
ꢀ
ꢀ
Novel 4-anilinoquinazoline-urea derivatives as EGFR and VEGFR-2 dual inhibitors.
Most of them exhibited good inhibitory potencies against EGFR and VEGFR-2 as well as
interesting antiproliferative activities.
ꢀ
ꢀ
Molecular docking suggested that they might be type II kinase inhibitors.
Diaryl urea moiety and chlorine in the ortho-position of the urea group lead to high inhibitory
potencies.