2,4-Disubstituted quinazolines targeting breast cancer cells via EGFR-PI3K

Article abstract of DOI:10.1016/j.ejmech.2019.03.030

A series of novel 2,4-disubstituted quinazolines were synthesized and evaluated for their anti-tumor activity against five human cancer cells (MDA-MB-231, MCF-7, PC-3, HGC-27 and MGC-803) using MTT assay. Among them, compound 9n showed the most potent cytotoxicity against breast cancer cells. Compound 9n also significantly inhibited the colony formation and migration of MDA-MB-231 and MCF-7 cells. Meanwhile, compound 9n induced cell cycle arrest at G1 phase and cell apoptosis, as well as increased accumulation of intracellular ROS. Furthermore, compound 9n exerted anti-tumor effects in vitro via decreasing the expression of anti-apoptotic protein Bcl-2 and increasing the pro-apoptotic protein Bax and p53. Mechanistically, compound 9n markedly decreased p-EGFR and p-PI3K expression, which revealed that compound 9n targeted breast cancer cells via interfering with EGFR-PI3K signaling pathway. Molecular docking suggested that compound 9n could indeed bind into the active pocket of EGFR. All the findings suggest that compound 9n might be a valuable lead compound for anti-tumor agents targeting breast cancer cells.

Full text of DOI:10.1016/j.ejmech.2019.03.030

Accepted Manuscript
2,4-Disubstituted quinazolines targeting breast cancer cells via EGFR-PI3K
Er-dong Li, Qiao Lin, Ya-qi Meng, Lu-ye Zhang, Pan-pan Song, Na Li, Jing-chao Xin,
Peng Yang, Chong-nan Bao, Dan-qing Zhang, Yang Zhang, Ji-kuan Wang, Qiu-rong
Zhang, Hong-min Liu
PII:
S0223-5234(19)30248-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.030
EJMECH 11200
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 January 2019
Revised Date: 12 March 2019
Accepted Date: 13 March 2019
Please cite this article as: E.-d. Li, Q. Lin, Y.-q. Meng, L.-y. Zhang, P.-p. Song, N. Li, J.-c. Xin, P. Yang,
C.-n. Bao, D.-q. Zhang, Y. Zhang, J.-k. Wang, Q.-r. Zhang, H.-m. Liu, 2,4-Disubstituted quinazolines
targeting breast cancer cells via EGFR-PI3K, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.03.030.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
,4-Disubstituted quinazolines targeting breast cancer cells via
EGFR-PI3K
2
a,b
a,b
a,b
a,b
a,b
a,b
a,b
Er-dong Li , Qiao Lin , Ya-qi Meng , Lu-ye Zhang , Pan-pan Song , Na Li , Jing-chao Xin ,
a,b
a,b
a,b
a,b
a,b
Peng Yang , Chong-nan Bao , Dan-qing Zhang , Yang Zhang , Ji-kuan Wang , Qiu-rong
*
,a,b
*,a,b
Zhang , Hong-min Liu
a
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China
b
Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou 450001,
PR China
*Corresponding Authors: Qiu-rong Zhang, Ph.D & Hong-Min Liu, Ph.D
Zhengzhou University, Zhengzhou, 450001, PR China
E-mail: zqr409@yeah.net (Qiu-rong Zhang) & liuhm@zzu.edu.cn (Hong-min Liu)

ACCEPTED MANUSCRIPT
Abstract
A series of novel 2,4-disubstituted quinazolines were synthesized and evaluated for their
anti-tumor activity against five human cancer cells (MDA-MB-231, MCF-7, PC-3, HGC-27 and
MGC-803) using MTT assay. Among them, compound 9n showed the most potent cytotoxicity against
breast cancer cells. Compound 9n also significantly inhibited the colony formation and migration of
MDA-MB-231 and MCF-7 cells. Meanwhile, compound 9n induced cell cycle arrest at G1 phase and
cell apoptosis, as well as increased accumulation of intracellular ROS. Furthermore, compound 9n
exerted anti-tumor effects in vitro via decreasing the expression of anti-apoptotic protein Bcl-2 and
increasing the pro-apoptotic protein Bax and p53. Mechanistically, compound 9n markedly decreased
p-EGFR and p-PI3K expression, which revealed that compound 9n targeted breast cancer cells via
interfering with EGFR-PI3K signaling pathway. Molecular docking suggested that compound 9n could
indeed bind into the active pocket of EGFR. All the findings suggest that compound 9n might be a
valuable lead compound for anti-tumor agents targeting breast cancer cells.
Key words: Quinazoline, Breast cancer, EGFR-PI3K

ACCEPTED MANUSCRIPT
1
. Introduction
The epidermal growth factor receptor (EGFR) plays an important role in cellular functions [1-4],
and it is often over-expressed and uncontrolled in a variety of human tumor cells [5]. PI3K especially
as a downstream protein of EGFR signaling pathway is dysregulated in breast cancer, non-small-cell
lung cancer, colorectal cancer and prostate cancer [6-10]. The EGFR-PI3K signaling pathway not only
affects the proliferation, differentiation, invasion and metastasis of tumor cells, but also plays a key
role in the treatment of tumors [11-14]. Therefore, targeting EGFR-PI3K pathway is one of the
effective ways to treat tumors.
Quinazoline derivatives are valued for their diverse pharmacological properties such as anti-tumor,
antimalarial, antibacterial, anti-inflammatory, anti-convulsant and anti-diabetic activities [15-19]. In
recent years, many quinazoline-based compounds have been approved for clinical use by US Food and
Drug Administration (FDA) including Gefitinib, Erlotinib and Lapatinib [20-24] (Fig. 1). Based on the
study of the structure-activity relationship of quinazoline derivatives [25], the 4-aminoquinazoline was
selected as the scaffold to study.
Meanwhile, it has been reported that compound 1, compound 2, and compound 3 have superior
anti-tumor effects on tumor cells, and all of them contain 4-trifluoromethylbenzyloxy group [26-28]
(
Fig. 1). Inspired by the results of the above studies, 4-trifluoromethylbenzyl was linked to the
sulfhydryl group at position 2 of the quinazoline by the principle of molecular hybridization. Then, we
synthesized a series of 2,4-disubstituted quinazolines and evaluated their bioactivity in vitro. Finally,
western blotting and molecular docking were performed to explore the possible mechanism of activity
of the compounds on breast cancer cells.
Fig.1 Anti-tumor agents and design of 2,4-disubstituted quinazolines.

ACCEPTED MANUSCRIPT
2
2
. Results and discussion
.1. Chemistry
The synthetic strategy to prepare the target compounds is depicted in Scheme 1. Compound 5 was
prepared by the reaction of 2-anthranilamide (compound 4) with carbon disulfide in the presence of
KOH in ethanol for 36h. Then, compound 6 was acquired from the reaction of compound 5 with
4
8
8
-trifluoromethylbenzyl chloride under basic condition in the mixture of H O and 1,4-dioxane for 4h in
2
4.5% yield, that was further reacted with phosphorus oxychloride at 65 � for 1h to get compound 7 in
3% yield. Next, the compound 7 was reacted with different amines to obtain compound 8a-e and 9a-v
1
13
in 54.7-84.2% yield. Finally, all the target compounds were fully characterized by H NMR, C NMR
and HRMS.
Scheme 1. Reagents and conditions: (a) CS , KOH, EtOH, reflux, 36h; (b) 4-trifluoromethylbenzyl chloride, KOH,
2
H O, 1,4-dioxane, 90 � , 4h, HCl; (c) POCl , 65 � , 1h; (d) Alkyl amines, Isopropanol, reflux, 6h. (e) Aromatic
2
3
amines, DMF, 90 � , 4h.
2
2
.2. Evaluation of biological activity
.2.1 Antiproliferative activity
In order to explore the anti-tumor effect of the target compounds, compounds 8a-e and 9a-v were
evaluated against five human cancer cells, including MDA-MB-231 (Human breast cancer cell line),
MCF-7 (Human breast cancer cell line), PC-3 (Human prostate cancer cell line), HGC-27 (Human
gastric carcinoma cell line) and MGC-803 (Human gastric carcinoma cell line) using MTT assay.
Gefitinib was employed as the positive control. The results are shown in Table 1.

ACCEPTED MANUSCRIPT
Table 1 Cytotoxicity of 2,4-disubstituted quinazolines 8a-e and 9a-v against five human cancer cells.
a
IC50(µM)
Compound
R
MDA-MB-231 MCF-7
PC-3
HGC-27
23.88±1.37
13.40±1.12
38.67±1.58
>50
MGC-803
13.11±1.11
12.63±1.10
>50
8
8
a
-(CH
-(CH
-(CH
-(CH
-(CH
2
2
2
2
2
)
)
)
)
)
5
CH
3
3
20.2±1.03
22.34±1.34
>50
24.30±1.38
19.04±1.28
10.46±1.02
26.04±1.28
>50
b
7
CH
26.14±1.41
>50
8
c
11CH
3
8
d
13CH
3
>50
>50
>50
>
50
8
9
e
5
28.38±1.36
18.41±1.26
>50
25.52±1.40
20.01±1.24
34.81±1.54
20.43±1.31
14.14±1.23
26.23±1.41
36.94±1.56
12.94±1.02
13.42±0.37
8.68±0.77
13.53±1.13
7.07±0.63
7.11±0.42
7.90±0.75
7.88±0.86
21.12±1.32
16.70±1.22
32.05±1.62
32.48±151
>50
>50
18.60±1.27
22.23±1.34
>50
a
C
6
H
5
-
44.01±1.64
>50
26.35±1.42
>50
9
b
2-CF
3
-C
6
H
4
-
9
c
2-Cl-C
6
H
4
-
>50
>50
>50
>50
9
d
2-OCH
3
2
-C
6
H
4
-
>50
>50
>50
>50
9
e
2-OCH
CH
3
-C
6
H
4
-
>50
>50
>50
>50
9
f
2-CH
3-CF
3-Cl-C
3-Br-C
3-NO -C
4-F-C
4-Cl-C
4-NO -C
-C
-C
3
-C
6
H
4
-
27.05±1.43
11.14±1.04
18.02±1.25
22.12±1.34
31.54±1.49
15.02±1.17
15.05±1.56
8.67±0.93
5.46±1.02
>50
37.53±1.57
15.32±1.18
25.12±1.40
25.57±1.40
>50
27.53±1.14
11.68±0.45
19.89±1.17
18.31±1.26
>50
24.36±1.38
15.06±1.17
14.56±1.16
23.68±1.37
>50
9
9
g
3
-C
6
H
4
-
h
6 4
H -
9
i
6 4
H -
9
j
2
6
H
4
-
9
k
6
H
4
-
36.29±1.56
21.49±1.33
17.92±1.25
5.10±0.85
>50
19.53±1.29
14.12±1.28
16.62±1.22
7.56±0.05
34.56±1.53
23.49±1.37
33.03±1.59
>50
9.62±0.98
16.76±1.27
8.17±0.91
6.17±0.79
36.11±1.27
13.79±1.14
36.98±1.56
>50
9
l
6
H
4
-
9
m
2
6
H
4
-
9
n
o
p
q
4-CF
3
6 4
H -
9
4-CH
3
6 4
H -
9
9
4-OCH
3
2
-C
6
H
4
-
15.73±1.19
23.03±1.36
>50
47.19±1.67
>50
4-OCH
CH
3
-C
-C
-C
-C
6
H
4
-
9
r
2,4-OCH
2,5-OCH
3,4-OCH
3
3
3
6
6
6
H
3
-
-
-
>50
9
s
t
H
3
3
16.12±1.20
18.85±1.27
14.51±1.14
>50
6.99±0.84
>50
>50
8.58±0.93
16.26±1.21
>50
9
H
14.08±1.14
18.60±1.27
7.37±0.86
17.94±1.25
14.66±1.16
13.99±1.14
9
u
v
3-Cl-4-F-C
6
H
3
-
22.97±1.36
>50
9
3,4,5-OCH
3
-C
6
H
2
-
8.24±0.91
b
Gefitinib
-
8.72±1.10
7.34±0.86
7.99±0.54
12.44±0.87
8.82±0.63
a
Cells were treated with different concentrations of the compounds for 72h. IC50 values are the Mean ± SD of three
b
separate experiments. Positive control.
In order to explore the structure-activity relationship, different substituents were introduced to
quinazoline scaffold. As shown in Table 1, most of compounds exerted moderate to excellent cytotoxic
activity against five human cancer cells. Especially, compound 9n showed the most excellent
cytotoxicity against the tested cells (MDA-MB-231, MCF-7, PC-3, HGC-27 and MGC-803) with IC50
values of 5.46±1.02µM, 5.10±0.85µM, 7.88±0.86µM, 7.56±0.05µM and 6.17±0.79µM, respectively.
Comparing 9k-n with 9o-q, we concluded that compounds with electron-withdrawing groups at
4
-position of benzene exhibited better bioactivity than compounds with electron-donating groups. From

ACCEPTED MANUSCRIPT
the bioactivity data of compounds 9b-f, the results revealed that the substituent at 2-position of benzene
had little cytotoxic activity for cancer cells. At the same time, comparing 8a-e with 9k-n, the results
indicated that electron-withdrawing groups at 4-position of benzene was favorable for the antitumor
activity. Among all target compounds, compound 9n exhibited the most excellent growth inhibition
activity against breast cancer cells. Therefore, in order to investigate the mechanism of these series of
compounds, compound 9n and breast cancer cells (MDA-MB-231 and MCF-7) were selected for
further study.
2
.2.2 Compound 9n suppressed the proliferation of MDA-MB-231and MCF-7 cells
We firstly investigated the impact of compound 9n on cell viability in MDA-MB-231 and MCF-7
cells by MTT assay for 24, 48 and 72h, respectively. As displayed in Fig. 2A, the cell viability
significantly decreased with the delay of time and the increase of compound concentration. The results
suggested that compound 9n could inhibit cell proliferation in a time-dependent and dose-dependent
manner. Then, we explored the effect of compound 9n on the cell proliferation by colony formation
assay. As shown in Fig. 2B, the cells treated with 9µM of compound 9n formed fewer and smaller
colonies than the control group. It is demonstrated that compound 9n inhibited the proliferation of
MDA-MB-231 and MCF-7 cells in a dose-dependent manner. As described in Fig. 2C and 2D, the
percentage of MDA-MB-231 cells in G1 phase increased from 49.17% to 69.54% after treatment of
compound 9n with 3, 6 and 12µM for 24h. The percentage of MCF-7 cells in G1 phase increased from
5
2.21% to 75.03%, indicating that compound 9n inhibited the proliferation of cells by inducing cell
cycle arrest in G1 phase. Finally, scratch assay was used to explore whether compound 9n can inhibit
the migration of tumor cells. As presented in Fig. 2E, a marked decrease in scratch� repaired capacity
of the cells was observed as the concentration of the compound 9n increased, which revealed that
migration of MDA-MB-231 and MCF-7 cells was inhibited in a dose-dependent manner after 72h of
compound 9n administration.

ACCEPTED MANUSCRIPT
Fig. 2 Compound 9n inhibited the proliferation of MDA-MB-231 and MCF-7 cells. (A) The viability of
MDA-MB-231 and MCF-7 cells after treatment with different concentration of compound 9n for 24, 48 and 72h,
respectively. (B) MDA-MB-231 and MCF-7 cells were treated with compound 9n at concentrations of 0, 3, 6 and
9
µM for 7 days; (C) Effect of compound 9n on cell cycle in MDA-MB-231 and MCF-7 cells; (D) Quantitative
analysis of the percentage of MDA-MB-231 and MCF-7 cells cycle phase distribution; (E) Scratch assay of
MDA-MB-231 and MCF-7 cells with different concentrations of compound 9n (magnification, 100). Three
individual experiments were performed for each group. The data was expressed as the Mean ± SD.
2
.2.3 Compound 9n induced MDA-MB-231 and MCF-7 cells apoptosis
Apoptosis is considered a major way that most of the anti-cancer drugs kill tumor cells. Therefore,
we determined the effect of compound 9n on apoptosis of MDA-MB-231 and MCF-7 cells using DAPI
staining. The number of apoptotic morphological changes was significantly increased after treatment
with the indicated concentration of compound 9n for 48h (Fig. 3A). Then, in order to better
characterize the mode of apoptosis induced by compound 9n, we performed a biparametric
cytofluorimetric analysis using cell apoptosis detection kit, Propidium Iodide (PI) and Annexin V-FITC.
The results showed that the percentage of apoptotic cells in MDA-MB-231 cells increased from 6.71%

ACCEPTED MANUSCRIPT
to 31.1% and the percentage of apoptotic cells in MCF-7 cells increased from 7.33 % to 27.7 % after
incubation with compound 9n for 48h (Fig. 3B & 3C), suggesting that compound 9n induced the
cellular apoptosis in a dose dependent manner. Meanwhile, western blot analysis was performed to
examine the expression of apoptosis related proteins (Fig. 3D). After treatment with compound 9n, the
expression of anti-apoptotic protein Bcl-2 was significantly decreased and the pro-apoptotic proteins
Bax and p53 were up-regulated both in MDA-MB-231 and MCF-7 cells (Fig. 3E). Finally, it was
noteworthy that accumulation of intracellular reactive oxygen species (ROS) can lead to apoptotic cell
death, and have direct effect on cellular structure and function [29]. Therefore, we detected the
production of intracellular ROS using a fluorescent probe, DHE. The results revealed that the
accumulation of ROS increased markedly with the increase of compound 9n concentration for 48h (Fig.
3
F). These data suggested that compound 9n induced MDA-MB-231 and MCF-7 cells apoptosis
through the activation of intrinsic apoptotic pathway.

ACCEPTED MANUSCRIPT
Fig. 3 Compound 9n induced apoptosis in MDA-MB-231 and MCF-7 cells. (A) The morphology of the nuclei of
MDA-MB-231 and MCF-7 cells was analyzed by DAPI staining (magnification, 100). Yellow arrow indicates
nuclear lysis; (B) MDA-MB-231 and MCF-7 cells apoptosis was detected by Annexin V-FITC/PI on flow
cytometry after treatment with compound 9n at different concentrations; (C) Statistical histogram of apoptotic cells
of MDA-MB-231 and MCF-7 cells. The data was expressed as the Mean ± SD. *p < 0.05, **P < 0.01, ***p <
0
.001 compared with the control group; (D) Western blotting analysis of the effect of compound 9n intervention on
the expression of Bcl-2, Bax and p53 in MDA-MB-231 and MCF-7 cells after treatment with compound 9n at a
concentration of 3, 6, 12µM for 48h. GAPDH was used as control; (E) Histograms of statistical analysis of
expression levels of Bax, Bcl-2 and p53 in MDA-MB-231 and MCF-7 cells. (*p < 0.05, **P < 0.01, ***p < 0.001
compared with the control group). (F) Detection of intracellular reactive oxygen species (ROS) in MDA-MB-231
and MCF-7 cells (magnification, 100). Results were expressed as percentages of control and presented as
Mean±SD for three independent experiments.
2
.2.4 The effect of compound 9n on EGFR-PI3K signaling pathways
To further explore the mechanism of cytotoxicity of compound 9n in MDA-MB-231 and MCF-7

ACCEPTED MANUSCRIPT
cells, the effect of compound 9n on the expression of EGFR and its downstream protein was
investigated (Fig. 4A and 4B). As the concentration of compound 9n applied to human breast cancer
cells (MDA-MB-231 and MCF-7) increased, the expression of phospho-EGFR decreased in a
dose-dependent manner, but the total amount of EGFR protein expression was unchanged. Similarly,
the expression levels of phospho-PI3K and phospho-AKT were also down-regulated in a
dose-dependent manner, while the total PI3K and AKT protein expression levels did not change. These
data indicated that compound 9n regulated MDA-MB-231 and MCF-7 cells via the EGFR-PI3K
signaling pathway.
Fig. 4 The effect of compound 9n on the EGFR-PI3K signaling pathway. (A)The MDA-MB-231 and MCF-7 cells
were stimulated with different concentration (0, 3, 6, 12µM) of compound 9n for 8h and then subjected to western
blot analysis using the indicated antibodies. (B) Quantitative analysis of the proteins level. (*p < 0.05, **P < 0.01,
***p < 0.001 compared with the control group). Results were expressed as percentages of control and presented as
Mean±SD for three independent experiments.
2
.2.5 Molecular docking study
In order to predict the possible binding mode of compound 9n with EGFR and PI3K and to
explain the activity discrepancy of compounds (8a-e and 9a-v), molecular docking study was carried
out using Molecular Operating Environment (MOE) 2014. EGFR (PDB code: 2ITO) and PI3K (PDB
code: 3APC) were retrieved from the Protein Data Bank (http://www.rcsb.org/pdb) for the docking
calculations. Compounds 9n (the most potent compound), 8a (the moderately potent) and 9r (least
potent compound) were selected as ligands. To verify the reliability of the docking, we first re-docked

ACCEPTED MANUSCRIPT
the co-crystallized into the original protein (2ITO). The RMSD between crystal structure and docked
structure was 1.0 Å, which indicated that the docking was reliable (Fig. S1). As shown in Fig. 5A,
compound 9n interacted with residues Lys745, Cys797 and Leu781 by π-H interaction, respectively.
The NH of compound 9n formed a hydrogen bond with Asp855 (2.06 Å), indicating that compound 9n
could tightly contact with EGFR. The docking result (Fig. 5B) showed that compound 9n formed
hydrogen bonds with Lys 833 (2.36 Å), Lys 890 (2.30 Å) and Asp 836 (2.63 Å), respectively. The
benzene ring of aniline interacted with Tyr 867 through π-H interaction. Comparing the docking results
of compounds 8a (Fig. 5C) and 9n with PI3K, we found that compound 9n interacted with Tyr 867
through π-H interaction and 8a did not. This computational result gave a reasonable explanation for the
lack of activity of compound 8a and also indicated that the aniline with an electron-withdrawing group
at 4-position was slightly favorable than fatty amine for the antitumor activity. The lacking interaction
with Lys 890 and Tyr 867, which may result in the poor antitumor activity of compound 9r (Fig. 5D).
The docking results were consistent with the biological results.
Fig. 5 (A) Predicted binding model of compound 9n (dark red) with EGFR (Docking score S is -10.2104). (B)

ACCEPTED MANUSCRIPT
Predicted binding model of compound 9n (green) with PI3K (Docking score S is -11.1432). (C) Predicted binding
model of compound 8a (orange) with PI3K (Docking score S is -10.0910). (D) Predicted binding model of
compound 9r (cyan) with PI3K (Docking score S is -9.7324). The interacting residues were displayed in sticks.
Hydrogen bonds and π-H interaction were shown in green dashed lines.
3
. Conclusion
In conclusion, a series of novel 2,4-disubstituted quinazolines were synthesized and evaluated for
their cytotoxic activity against MDA-MB-231, MCF-7, PC-3, HGC-27 and MGC-803 cells using MTT
assay. Among all the tested compounds, compound 9n showed the most potent anti-proliferative
activity against the tested cells, especially for breast cancer cells (MDA-MB-231 and MCF-7).
Moreover, cell survival assay, clone formation assay and scratch assay analysis indicated that
compound 9n inhibited cell proliferation and migration, Furthermore, cell cycle experiments showed
that compound 9n induced cell cycle arrest at G1 phase. Meanwhile, DAPI staining, apoptosis,
detection of apoptotic protein expression and ROS experiments together suggested that compound 9n
induced apoptosis of MDA-MB-231 and MCF-7 cells through the activation of intrinsic apoptotic
pathway. Finally, western blot and molecular docking study analysis indicated that compound 9n
regulated MDA-MB-231 and MCF-7 cells function via the EGFR-PI3K signaling pathway. All the
findings suggest that compound 9n might be a valuable lead compound for anti-tumor agents targeting
breast cancer cells.
4
4
. Experimental section
.1. General procedure
Reagents and solvents were purchased from commercial sources and were used without further
1
13
purification. H NMR and C NMR spectra were recorded on a Bruker 400 MHz and 101 MHz
spectrometer respectively. High resolution mass spectra (HRMS) were recorded on a Waters
Micromass Q-T of Micromass spectrometer by electrospray ionizaton (ESI). Melting points were
determined on an X-5 micromelting apparatus. Reactions were monitored by thin-layer
chromatography (TLC) on 0.25mm silicagel plates (GF254).
4
.2 General procedure for the synthesis of compounds 5 and 6
Compound 5 and 6 were synthesized according to the literature [30, 31], H NMR and C NMR
spectra of compounds (5,6) were in accordance with the published data.
.3 General procedure for the synthesis of compound 7
1
13
4

ACCEPTED MANUSCRIPT
Compound 6 (1.5 mmol) was added to 5 mL of phosphorus oxychloride and then heated to 65 �
for 1h. Then, the mixture was concentrated under reduced pressure and purified by column
chromatography (PE:EA = 2:1) to give compound 7.
4
-Chloro-2-((4-(trifluoromethyl)benzyl)thio)quinazoline (7). White solid, 65.2% yield, Mp
1
1
00.7-101.2 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 8.04 (dd, J = 7.9, 1.2 Hz, 1H, Ar-H), 7.80 (d, J
6
=
7.4 Hz, 1H, Ar-H), 7.74 (d, J = 8.2 Hz, 2H, Ar-H), 7.68 (d, J = 7.8 Hz, 2H, Ar-H), 7.62 (d, J = 8.1 Hz,
1
3
1
1
H, Ar-H), 7.44 (t, J = 7.4 Hz, 1H, Ar-H), 4.59 (s, 2H SCH ). C NMR (101 MHz, DMSO-d ) δ (ppm)
2 6
61.09, 154.76, 148.08, 142.79, 134.62, 129.95, 127.90, 127.58, 126.00, 125.75, 125.15, 122.83,
+
1
19.97, 32.76. HR-MS (ESI) calcd for C H ClF N S [M+H] : 355.0284, found: 355.0282.
1
6
10
3
2
4
.4 General procedure for the synthesis of compounds 8a-e
Compound 7 (1.04 mmol, 2.00 eq) was dissolved in isopropanol (10 ml), then an appropriate
amount of amine (1.00 eq) was added, and the mixture was heated to 85 °C for 6h. When the reaction
was completed, filtered, dried and the crude product was purified by column chromatography (PE:EA =
1
:1) to give compound 8a-e in 54.7-64.5% yield.
4
.4.1. N-hexyl-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (8a)
1
White solid, 57.7% yield, Mp 91.7-92.0 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 8.38 (t, J =
6
5
.2 Hz, 1H, NH), 8.17 (d, J = 8.2 Hz, 1H, Ar-H), 7.74 – 7.63 (m, 5H, Ar-H), 7.56 (d, J = 8.3 Hz, 1H,
Ar-H), 7.39 (t, J = 7.6 Hz, 1H, Ar-H), 4.52 (s, 2H, SCH ), 3.46 (dd, J = 12.9, 6.6 Hz, 2H, CH ), 1.56
2
2
(
dd, J = 13.9, 6.9 Hz, 2H, CH ), 1.25 (d, J = 3.0 Hz, 6H, (CH ) ), 0.84 (dd, J = 12.5, 6.4 Hz, 3H, CH ).
2 2 3 3
1
3
C NMR (101 MHz, DMSO-d ) δ (ppm) 165.58, 158.78, 149.56, 144.34, 132.85, 129.41, 127.49,
6
1
27.17, 126.05, 125.00, 124.31, 122.88, 113.05, 40.53, 33.35, 30.96, 28.35, 26.15, 22.00, 13.78.
+
HR-MS (ESI) calcd for C H F N S [M+H] : 420.1721, found: 420.1724.
2
2
24
3
3
4
.4.2. N-octyl-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (8b)
1
White solid, 64.5% yield, Mp 92.1-92.6 ℃. H NMR (400 MHz, DMSO-d ) δ (ppm) 8.37 (t, J =
6
5
1
2
.4 Hz, 1H, NH), 8.18 (d, J = 7.6 Hz, 1H, Ar-H), 7.67 (dd, J = 18.9, 8.4 Hz, 5H, Ar-H), 7.59 – 7.53 (m,
H, Ar-H), 7.42 – 7.36 (m, 1H, Ar-H), 4.52 (s, 2H, SCH ), 3.47 (dd, J = 13.0, 6.7 Hz, 2H, CH ), 3.35 (s,
2
2
H, CH ), 1.57 (dd, J = 13.9, 7.0 Hz, 2H, CH ), 1.22 (dd, J = 12.5, 5.9 Hz, 8H, (CH ) ), 0.82 (t, J = 6.8
2
2
2 4
1
3
Hz, 3H, CH3). C NMR (101 MHz, DMSO-d ) δ (ppm) 165.57, 158.77, 149.57, 144.35, 132.85,
6
1
2
4
29.41, 127.49, 127.17, 126.05, 125.00, 124.31, 122.88, 113.04, 40.51, 33.34, 31.18, 28.68, 28.60,
+
8.36, 26.46, 22.00, 13.82. HR-MS (ESI) calcd for C H F N S [M+H] : 448.2034, found: 448.2032.
24
28
3
3
.4.3. N-dodecyl-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (8c)
1
White solid, 58.3% yield, Mp 71.8-72.0 ℃. H NMR (400 MHz, DMSO-d ) δ (ppm) 8.40 (s, 1H,
6

ACCEPTED MANUSCRIPT
NH), 8.17 (d, J = 8.1 Hz, 1H, Ar-H), 7.67 (dd, J = 12.1, 6.7 Hz, 5H, Ar-H), 7.55 (d, J = 8.3 Hz, 1H,
Ar-H), 7.39 (t, J = 7.6 Hz, 1H, Ar-H), 4.52 (s, 2H, SCH ), 3.47 (dd, J = 12.9, 6.6 Hz, 2H, CH ), 1.57
2
2
13
(
dd, J = 13.7, 6.8 Hz, 2H, CH ), 1.22 (d, J = 21.9 Hz, 18H, (CH ) ), 0.83 (t, J = 6.8 Hz, 3H, CH ). C
2 2 9 3
NMR (101 MHz, DMSO-d ) δ (ppm) 165.54, 158.76, 149.43, 144.30, 132.89, 130.02, 129.41, 127.35,
6
1
25.95, 125.00, 124.34, 122.89, 113.01, 40.50, 33.34, 31.23, 28.93, 28.62, 28.33, 26.43, 22.02, 13.85.
+
HR-MS (ESI) calcd for C H F N S [M+H] : 504.2660, found: 504.2662.
2
8
36
3
3
4
.4.4. N-tetradecyl-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (8d)
1
White solid, 59.1% yield, Mp 76.4-76.9 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 8.38 (t, J =
6
5
.0 Hz, 1H, NH), 8.17 (d, J = 8.2 Hz, 1H, Ar-H), 7.66 (dd, J = 20.7, 8.4 Hz, 5H, Ar-H), 7.55 (d, J = 8.3
Hz, 1H, Ar-H), 7.38 (t, J = 7.5 Hz, 1H, Ar-H), 4.52 (s, 2H, SCH ), 3.47 (dd, J = 12.7, 6.4 Hz, 2H, CH ),
2
2
13
1
.64 – 1.50 (m, 2H, CH ), 1.21 (d, J = 25.5 Hz, 22H, (CH ) ), 0.83 (t, J = 6.6 Hz, 3H, CH ). C NMR
2 2 11 3
(
101 MHz, DMSO-d ) δ (ppm) 165.56, 158.77, 149.52, 144.25, 132.76, 129.35, 127.53, 127.21, 125.97,
6
1
24.95, 124.22, 122.86, 113.05, 40.50, 33.37, 31.23, 28.95, 28.65, 28.36, 26.46, 22.02, 13.78. HR-MS
+
(
ESI) calcd for C H F N S [M+H] : 532.2973, found: 532.2975.
3
0
40
3
3
4
.4.5. N-cyclopentyl-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine(8e)
1
Oily liquid, 54.7% yield. H NMR (400 MHz, DMSO-d ) δ (ppm) 8.29 (d, J = 7.6 Hz, 1H, NH),
6
8
1
1
1
.12 – 8.05 (m, 1H, Ar-H), 7.73 – 7.69 (m, 3H, Ar-H), 7.66 (d, J = 8.4 Hz, 2H, Ar-H), 7.60 – 7.54 (m,
H, Ar-H), 7.42 – 7.35 (m, 1H, Ar-H), 4.52 (s, 2H, SCH ), 3.37 (s, 1H, CH), 1.99 – 1.90 (m, 2H, CH ),
2
2
13
.79 – 1.68 (m, 2H, CH ), 1.67 – 1.49 (m, 4H, (CH ) ). C NMR (101 MHz, DMSO-d ) δ (ppm)
2
2 2
6
65.55, 158.47, 149.56, 144.32, 132.82, 129.42, 127.47, 127.15, 125.98, 125.03, 124.16, 123.23,
+
1
4
4
13.02, 52.17, 33.44, 31.79, 23.65. HR-MS (ESI) calcd for C H F N S [M+H] : 404.1408, found:
21
20
3
3
04.1407.
.5 General procedure for the synthesis of compounds 9a-v
Compound 7 (2.00 eq) was dissolved in N,N-Dimethylformamide(DMF), then an appropriate
amount of aromatic amines (1.50 eq) was added, and the mixture was heated to 90 °C for 4h. When the
reaction was completed, filtered, dried and the crude product was purified by column chromatography
(
PE:EA = 1:1 ~ 1.5:1) to give compound 9a-v in 55.3-84.2% yield.
4
.5.1 N-phenyl-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9a)
1
White solid, 63.8% yield, Mp 144.2-144.9 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 9.91 (s, 1H,
6
NH), 8.48 (d, J = 8.1 Hz, 1H, Ar-H), 7.81 (t, J = 7.6 Hz, 1H, Ar-H), 7.75 (d, J = 7.7 Hz, 2H, Ar-H), 7.67
d, J = 8.2 Hz, 1H, Ar-H), 7.60 (q, J = 8.6 Hz, 4H, Ar-H), 7.52 (t, J = 7.2 Hz, 1H, Ar-H), 7.40 (t, J = 7.9
(
13
Hz, 2H, Ar-H), 7.16 (t, J = 7.4 Hz, 1H, Ar-H), 4.47 (s, 2H, SCH2). C NMR (101 MHz, DMSO-d ) δ
6
(
ppm) 165.26, 157.29, 150.18, 143.96, 138.60, 133.49, 129.57, 128.45, 127.51, 127.19, 126.35, 125.58,

ACCEPTED MANUSCRIPT
+
1
25.01, 124.27, 123.26, 122.97, 113.11, 33.34. HR-MS (ESI) calcd for C H F N S [M+H] : 412.1095,
22 16 3 3
found: 412.1097.
4
.5.2. 2-((4-(Trifluoromethyl)benzyl)thio)-N-(2-(trifluoromethyl)phenyl)quinazolin-4-amine (9b)
1
White solid, 60.2% yield, Mp 116.4-117.1 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 10.02 (s,
6
1
7
H, NH), 8.40 (d, J = 8.1 Hz, 1H, Ar-H), 7.87 (d, J = 8.2 Hz, 1H, Ar-H), 7.82 – 7.76 (m, 2H, Ar-H),
.66 – 7.61 (m, 3H, Ar-H), 7.52 (d, J = 8.1 Hz, 3H, Ar-H), 7.29 (d, J = 8.0 Hz, 2H, Ar-H), 4.20 (s, 2H,
1
3
SCH2). C NMR (101 MHz, DMSO-d ) δ (ppm) 165.32, 159.43, 150.18, 144.13, 136.30, 134.89,
6
1
33.66, 133.26, 131.96, 129.25, 127.83, 127.34, 126.91, 126.73, 126.19, 125.10, 124.81, 123.22,
+
1
15.28, 112.53, 33.09. HR-MS (ESI) calcd for C H F N S [M+H] : 480.0969, found: 480.0967.
23
15
6
3
4
.5.3. N-(2-chlorophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9c)
1
White solid, 63.4% yield, Mp 163.6-163.9 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.92 (s,
6
1
7
1
1
1
4
4
H, NH), 8.81 (d, J = 8.2 Hz, 1H, Ar-H), 8.01 (t, J = 7.7 Hz, 1H, Ar-H), 7.88 (d, J = 8.3 Hz, 1H, Ar-H),
.72 (t, J = 8.0 Hz, 2H, Ar-H), 7.67 (d, J = 7.7 Hz, 1H, Ar-H), 7.60 – 7.41 (m, 4H, Ar-H) ,7.13 (t, J =
13
7.2 Hz, 2H, Ar-H), 4.29 (s, 2H, SCH2). C NMR (101 MHz, DMSO-d ) δ (ppm) 164.16, 158.55,
6
42.30, 136.03, 134.22, 131.11, 130.00, 129.83, 129.56, 129.30, 128.01, 127.80, 127.48, 127.08,
+
25.45, 124.96, 124.83, 119.72, 111.34, 32.97. HR-MS (ESI) calcd for C H ClF N S [M+H] :
22
15
3
3
46.0706, found: 446.0698.
.5.4. N-(2-methoxyphenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9d)
1
White solid, 69.5% yield, Mp 152.5-153.5 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 9.61 (s, 1H,
6
NH), 8.40 (d, J = 7.9 Hz, 1H, Ar-H), 7.81 – 7.75 (m, 1H, Ar-H), 7.62 (d, J = 7.8 Hz, 1H, Ar-H), 7.59 –
.52 (m, 3H, Ar-H), 7.51 – 7.46 (m, 1H, Ar-H), 7.38 (d, J = 8.1 Hz, 2H, Ar-H), 7.34 – 7.27 (m, 1H,
Ar-H), 7.18 (d, J = 7.4 Hz, 1H, Ar-H), 7.04 (td, J = 7.6, 1.1 Hz, 1H, Ar-H), 4.30 (s, 2H, SCH ), 3.80 (s,
7
2
1
3
3
1
H, OCH3). C NMR (101 MHz, DMSO-d ) δ (ppm) 165.40, 158.20, 153.90, 150.15, 144.34, 133.37,
6
29.51, 127.73, 127.27, 127.02, 126.61, 126.19, 125.60, 124.88, 124.85, 123.23, 120.22, 112.89,
+
1
11.93, 55.56, 33.17. HR-MS (ESI) calcd for C H F N OS [M+H] : 442.1201, found: 442.1200.
23
18
3
3
4
.5.5. N-(2-ethoxyphenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9e)
1
White solid, 70.4% yield, Mp 159.8-160.3 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.33 (s,
6
1
7
8
H, NH), 8.68 (d, J = 8.3 Hz, 1H, Ar-H), 7.98 (t, J = 7.7 Hz, 1H, Ar-H), 7.80 (d, J = 8.4 Hz, 1H, Ar-H),
.69 (t, J = 7.6 Hz, 1H, Ar-H), 7.53 – 7.47 (m, 3H, Ar-H), 7.42 (t, J = 7.8 Hz, 1H, Ar-H), 7.27 (d, J =
.2 Hz, 1H, Ar-H), 7.11 (dd, J = 14.4, 7.6 Hz, 3H, Ar-H), 4.30 (s, 2H, SCH ), 4.11 (q, J = 6.9 Hz, 2H,
2
1
3
OCH ), 1.21 (t, J = 6.9 Hz, 3H, CH ). C NMR (101 MHz, DMSO-d ) δ (ppm) 163.78, 158.13, 153.46,
2
3
6
1
1
4
42.48, 135.72, 129.49, 129.03, 128.08, 127.72, 127.41, 126.93, 125.45, 125.22, 124.91, 124.66,
+
22.75, 120.37, 113.20, 111.42, 63.87, 32.94, 14.55. HR-MS (ESI) calcd for C H F N OS [M+H] :
24
20
3
3
56.1357, found: 456.1354.

ACCEPTED MANUSCRIPT
4
.5.6. N-(o-tolyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9f)
1
White solid, 73.0% yield, Mp 179.4-180.2 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.65 (s,
6
1
7
7
1
1
H, NH), 8.79 (d, J = 8.2 Hz, 1H, Ar-H), 7.99 (t, J = 7.7 Hz, 1H, Ar-H), 7.81 (d, J = 8.3 Hz, 1H, Ar-H),
.69 (t, J = 7.7 Hz, 1H, Ar-H), 7.47 (t, J = 10.1 Hz, 4H, Ar-H), 7.42 – 7.34 (m, 2H, Ar-H), 7.03 (d, J =
13
.8 Hz, 2H, Ar-H), 4.24 (s, 2H, SCH ), 2.25 (s, 3H, CH ). C NMR (101 MHz, DMSO-d ) δ (ppm)
2
3
6
63.92, 158.40, 142.34, 135.83, 135.39, 135.04, 130.75, 129.42, 128.07, 127.82, 127.44, 126.95,
26.52, 125.46, 124.99, 124.87, 122.75, 119.08, 111.48, 32.91, 17.65. HR-MS (ESI) calcd for
+
C H F N S [M+H] : 426.1252, found: 426.1253.
2
3
18
3
3
4
.5.7. 2-((4-(Trifluoromethyl)benzyl)thio)-N-(3-(trifluoromethyl)phenyl)quinazolin-4-amine (9g)
1
White solid, 74.0% yield, Mp 155.5-155.8 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.71 (s,
6
1
7
H, NH), 8.87 (d, J = 8.3 Hz, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.07 (d, J = 7.9 Hz, 1H, Ar-H), 7.99 (t, J =
.7 Hz, 1H, Ar-H), 7.85 (d, J = 8.3 Hz, 1H, Ar-H), 7.75 – 7.66 (m, 2H, Ar-H), 7.64 (d, J = 7.8 Hz, 1H,
13
Ar-H), 7.55 (d, J = 8.1 Hz, 2H, Ar-H), 7.39 (d, J = 8.0 Hz, 2H, Ar-H), 4.50 (s, 2H, SCH2). C NMR
(
101 MHz, DMSO-d ) δ (ppm) 164.27, 157.81, 142.03, 138.01, 135.65, 129.90, 129.43, 129.23, 128.52,
6
1
27.90, 127.59, 126.74, 125.44, 125.26, 125.02, 124.83, 122.73, 122.56, 121.24, 112.08, 33.19.
+
HR-MS (ESI) calcd for C H F N S [M+H] : 480.0969, found: 480.0969.
2
3
15
6
3
4
.5.8. N-(3-chlorophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9h)
1
White solid, 70.1% yield, Mp 156.6-157.2 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.43 (s,
6
1
H, NH), 8.77 (d, J = 7.9 Hz, 1H, Ar-H), 7.96 (dd, J = 15.4, 7.8 Hz, 2H, Ar-H), 7.81 (d, J = 8.3 Hz, 1H,
Ar-H), 7.68 (dd, J = 12.0, 8.0 Hz, 2H, Ar-H), 7.59 (d, J = 8.1 Hz, 2H, Ar-H), 7.49 (d, J = 8.1 Hz, 1H,
13
Ar-H),7.46 – 7.40 (m, 2H, Ar-H), 7.35 (d, J = 8.0 Hz, 1H, Ar-H), 4.51 (s, 2H, SCH2). C NMR (101
MHz, DMSO-d ) δ (ppm) 164.09, 157.76, 141.97, 138.39, 135.79, 132.85, 130.29, 129.48, 127.94,
6
1
27.62, 126.85, 126.16, 125.45, 125.06, 124.72, 123.52, 122.75, 119.82, 111.93, 33.19. HR-MS (ESI)
+
calcd for C H ClF N S [M+H] : 446.0706, found: 446.0705.
2
2
15
3
3
4
.5.9. N-(3-bromophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9i)
1
White solid, 71.3% yield, Mp 160.3-161.2 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.62 (s,
6
1
7
H, NH), 8.88 – 8.79 (m, 1H, Ar-H), 8.08 (d, J = 1.7 Hz, 1H, Ar-H), 7.98 (t, J = 7.7 Hz, 1H, Ar-H),
.83 (d, J = 8.3 Hz, 1H, Ar-H), 7.74 (d, J = 8.1 Hz, 1H, Ar-H), 7.68 (t, J = 7.7 Hz, 1H, Ar-H), 7.58 (d, J
=
8.1 Hz, 2H, Ar-H), 7.50 (d, J = 8.1 Hz, 1H, Ar-H), 7.44 (d, J = 8.0 Hz, 1H, Ar-H), 7.42 – 7.38 (m, 2H,
1
3
Ar-H), 4.52 (s, 2H, SCH2). C NMR (101 MHz, DMSO-d ) δ (ppm) 164.12, 157.73, 142.04, 138.58,
6
1
1
4
4
35.73, 130.58, 129.52, 128.98, 127.93, 127.48, 126.81, 125.46, 125.08, 124.94, 123.83, 122.76,
+
21.11, 120.05，111.97, 33.23. HR-MS (ESI) calcd for C H BrF N S [M+H] : 490.0200, found:
22
15
3
3
90.0201.
.5.10. N-(3-nitrophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9j)

ACCEPTED MANUSCRIPT
1
White solid, 55.3% yield, Mp 218.8-219.5 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.57 (s,
6
1
7
7
H, NH), 8.95 – 8.78 (m, 2H, Ar-H), 8.24 (d, J = 8.1 Hz, 1H, Ar-H), 8.08 (d, J = 8.2 Hz, 1H, Ar-H),
.98 (t, J = 7.7 Hz, 1H, Ar-H), 7.83 (d, J = 8.3 Hz, 1H, Ar-H), 7.70 (dd, J = 17.3, 8.5 Hz, 2H, Ar-H),
13
.59 (d, J = 8.2 Hz, 2H, Ar-H), 7.48 (d, J = 8.0 Hz, 2H, Ar-H), 4.56 (s, 2H, SCH ). C NMR (101 MHz,
2
DMSO-d ) δ (ppm) 164.38, 157.72, 147.71, 141.84, 138.52, 135.60, 130.31, 129.93, 129.47, 127.92,
6
1
27.60, 126.71, 125.45, 125.10, 124.84, 122.75, 120.19, 118.68, 112.19, 33.50. HR-MS (ESI) calcd for
+
C H F N O S [M+H] : 457.0946, found: 457.0945.
2
2
15
3
4
2
4
.5.11. N-(4-fluorophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9k)
1
White solid, 71.9% yield, Mp 170.6-171.0 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 9.95 (s, 1H,
6
NH), 8.45 (d, J = 7.7 Hz, 1H, Ar-H), 7.84 – 7.79 (m, 1H, Ar-H), 7.77 – 7.72 (m, 2H, Ar-H), 7.68 –7.65
(
m, 1H, Ar-H), 7.60 (q, J = 8.6 Hz, 4 H, Ar-H), 7.55 – 7.50 (m, 1H, Ar-H), 7.26 – 7.19 (m, 2H, Ar-H),
1
3
4
1
.46 (s, 2H, SCH2). C NMR (101 MHz, DMSO-d ) δ (ppm) 165.23, 160.00, 157.60, 157.34, 150.12,
6
43.93, 134.89, 133.52, 129.56, 127.52, 126.35, 125.05, 124.97, 123.21, 115.19, 114.97, 113.01, 33.35.
+
HR-MS (ESI) calcd for C H F N S [M+H] : 430.1001, found: 430.1003.
2
2
15
4
3
4
.5.12. N-(4-chlorophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9l)
1
White solid, 73.0% yield, Mp 180.0-181.7 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.70 (s,
6
1
7
=
H, NH), 8.86 (d, J = 8.3 Hz, 1H, Ar-H), 7.98 (t, J = 7.6 Hz, 1H, Ar-H), 7.84 (d, J = 8.3 Hz, 1H, Ar-H),
.75 (d, J = 8.4 Hz, 2H, Ar-H), 7.68 (t, J = 7.6 Hz, 1H, Ar-H), 7.58 (d, J = 7.9 Hz, 2H, Ar-H), 7.51 (d, J
13
8.4 Hz, 2H, Ar-H), 7.39 (d, J = 7.8 Hz, 2H, Ar-H), 4.51 (s, 2H, SCH2). C NMR (101 MHz,
DMSO-d ) δ (ppm) 164.14, 157.69, 142.08, 135.88, 135.65, 130.38, 129.47, 128.59, 128.21, 127.89,
6
1
26.77, 126.59, 125.03, 124.87, 124.50, 122.77, 111.98, 33.27. HR-MS (ESI) calcd for C H ClF N S
22 15 3 3
+
[
M+H] : 446.0706, found: 446.0706.
4
.5.13. N-(4-nitrophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9m)
1
White solid, 58.1% yield, Mp 219.7-220.3 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.29 (s,
6
1
7
H, NH), 8.80 (d, J = 7.9 Hz, 1H, Ar-H), 8.26 (d, J = 9.1 Hz, 2H, Ar-H), 8.11 (d, J = 9.1 Hz, 2H, Ar-H),
.97 (t, J = 7.5 Hz, 1H, Ar-H), 7.83 (d, J = 8.3 Hz, 1H, Ar-H), 7.66 (d, J = 7.4 Hz, 1H, Ar-H), 7.60 (dd,
13
J = 17.7, 8.2 Hz, 4H, Ar-H), 4.58 (s, 2H, SCH2). C NMR (101 MHz, DMSO-d ) δ (ppm) 164.54,
6
1
62.78, 157.51, 150.24, 140.84, 135.39, 134.86, 129.53, 126.87, 126.54, 125.06, 124.74, 124.21,
+
1
23.54, 122.23, 115.29, 114.28, 33.54. HR-MS (ESI) calcd for C H F N O S [M+H] : 457.0946,
22
15
3
4
2
found: 457.0945.
4
.5.14. 2-((4-(Trifluoromethyl)benzyl)thio)-N-(4-(trifluoromethyl)phenyl)quinazolin-4-amine (9n)
1
White solid, 72.7% yield, Mp 163.5-163.9 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.45 (s,
6
1
8
H, NH), 8.81 (d, J = 8.3 Hz, 1H, Ar-H), 7.98 (s, 1H, Ar-H), 7.95 (d, J = 6.2 Hz, 2H, Ar-H), 7.82 (d, J =
.3 Hz, 1H, Ar-H), 7.76 (d, J = 8.5 Hz, 2H, Ar-H), 7.67 (t, J = 7.6 Hz, 1H, Ar-H), 7.56 (d, J = 8.1 Hz,

ACCEPTED MANUSCRIPT
1
3
2
1
1
4
4
H, Ar-H), 7.44 (d, J = 8.0 Hz, 2H, Ar-H), 4.52 (s, 2H, SCH ). C NMR (101 MHz, DMSO-d ) δ (ppm)
2 6
64.39, 157.77, 142.10, 140.93, 135.59, 129.37, 128.17, 127.85, 127.53, 126.68, 125.65, 125.46,
+
25.00, 124.87, 124.73, 122.76, 120.79, 112.19, 33.37. HR-MS (ESI) calcd for C H F N S [M+H] :
23
15
6
3
80.0969, found: 480.0970.
.5.15. N-(p-tolyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9o)
1
White solid,73.5% yield, Mp 165.1-166.0 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.61 (s,
6
1
7
H, NH), 8.82 (d, J = 8.3 Hz, 1H, Ar-H), 7.96 (t, J = 7.7 Hz, 1H, Ar-H), 7.83 (d, J = 8.3 Hz, 1H, Ar-H),
.67 (t, J = 7.6 Hz, 1H, Ar-H), 7.58 – 7.55 (m, 2H, Ar-H), 7.54 (s, 1H, Ar-H), 7.32 (d, J = 8.0 Hz, 2H,
Ar-H), 7.28 (d, J = 8.2 Hz, 2H, Ar-H), 7.22 – 7.16 (m, 1H, Ar-H), 4.48 (s, 2H, SCH ), 2.33 (s, 3H, CH ).
2
3
1
3
C NMR (101 MHz, DMSO-d ) δ (ppm) 163.91, 157.59, 150.23, 142.06, 140.84, 136.09, 135.65,
6
1
34.84, 134.07, 129.52, 129.09, 126.85, 125.03, 122.23, 115.30, 114.27, 111.80, 33.18, 20.58. HR-MS
+
(
ESI) calcd for C H F N S [M+H] : 426.1252, found: 426.1253.
2
3
18
3
3
4
.5.16. N-(4-methoxyphenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9p)
1
White solid, 73.0% yield, Mp 180.2-181.2 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.48 (s,
6
1
7
=
H, NH), 8.73 (d, J = 8.3 Hz, 1H, Ar-H), 7.96 (t, J = 7.7 Hz, 1H, Ar-H), 7.78 (d, J = 8.3 Hz, 1H, Ar-H),
.58 (d, J = 4.5 Hz, 2H, Ar-H), 7.55 (d, J = 3.7 Hz, 2H, Ar-H), 7.32 (d, J = 8.0 Hz, 2H, Ar-H), 7.19 (d, J
13
7.9 Hz, 1H, Ar-H), 7.02 (d, J = 8.9 Hz, 2H, Ar-H), 4.46 (s, 2H, SCH ), 3.78 (s, 3H, OCH ). C NMR
2
3
(
101 MHz, DMSO-d ) δ (ppm) 163.95, 162.79, 157.82, 157.59, 150.25, 140.82, 134.86, 129.53, 126.87,
6
1
26.55, 124.96, 124.62, 122.24, 115.29, 114.28, 113.90, 111.77, 55.31, 33.19. HR-MS (ESI) calcd for
+
C H F N OS [M+H] : 442.1201, found: 442.1201.
2
3
18
3
3
4
.5.17. N-(4-ethoxyphenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9q)
1
White solid, 65.6% yield, Mp 167.0-167.8 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.62 (s,
6
1
7
H, NH), 8.78 (d, J = 8.3 Hz, 1H, Ar-H), 7.97 (t, J = 7.7 Hz, 1H, Ar-H), 7.80 (d, J = 8.3 Hz, 1H, Ar-H),
.67 (t, J = 7.5 Hz, 1H, Ar-H), 7.56 (dd, J = 8.5, 3.6 Hz, 4H, Ar-H), 7.31 (d, J = 8.0 Hz, 2H, Ar-H), 7.01
(
d, J = 8.9 Hz, 2H, Ar-H), 4.46 (s, 2H, SCH ), 4.07 – 4.01 (m, 2H, OCH ), 1.32 (t, J = 6.9 Hz, 3H, CH ).
2 2 3
1
3
C NMR (101 MHz, DMSO-d ) δ (ppm) 163.80, 157.59, 157.12, 150.23, 142.14, 135.68, 134.86,
6
1
29.57, 129.15, 126.86, 126.69, 125.01, 124.90, 122.23, 115.30, 114.35, 111.75, 63.25, 33.11, 14.48.
+
HR-MS (ESI) calcd for C H F N OS [M+H] : 456.1357, found: 456.1355.
2
4
20
3
3
4
.5.18. N-(2,4-dimethoxyphenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9r)
1
White solid, 63.8% yield, Mp 132.8-133.4 � . H NMR (400 MHz, DMSO-d ) δ (ppm) 11.38 (s,
6
1
7
H, NH), 8.71 (d, J = 8.2 Hz, 1H, Ar-H ), 7.97 (t, J = 7.7 Hz, 1H, Ar-H), 7.83 (d, J = 8.3 Hz, 1H, Ar-H),
.68 (t, J = 7.7 Hz, 1H, Ar-H), 7.52 (d, J = 8.1 Hz, 2H, Ar-H), 7.39 (d, J = 8.6 Hz, 1H, Ar-H), 7.15 (d, J
=
8.0 Hz, 2H, Ar-H), 6.82 (d, J = 2.4 Hz, 1H, Ar-H), 6.68 (dd, J = 8.6, 2.4 Hz, 1H, Ar-H), 4.32 (s, 2H,
1
3
SCH ), 3.81 (s, 6H, (OCH ) ). C NMR (101 MHz, DMSO-d ) δ (ppm) 164.08, 158.54, 155.22,
2
3 2
6

ACCEPTED MANUSCRIPT
1
42.78, 135.37, 129.48, 128.83, 127.71, 127.39, 126.58, 125.50, 124.90, 124.46, 122.79, 120.28,
+
1
4
4
18.04, 111.65, 104.73, 99.26, 55.75, 55.49, 33.03. HR-MS (ESI) calcd for C H F N O S [M+H] :
24
20
3
3
2
72.1307, found: 472.1306.
.5.19. N-(2,5-dimethoxyphenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9s)
1
White solid, 63.8% yield，Mp 176.0-176.9 ℃. H NMR (400 MHz, DMSO-d ) δ (ppm) 9.56 (s, 1H,
6
NH), 8.37 (d, J = 8.0 Hz, 1H, Ar-H), 7.78 (dd, J = 11.3, 4.1 Hz, 1H, Ar-H), 7.64 – 7.61 (m, 1H, Ar-H),
7
3
.55 (d, J = 8.2 Hz, 2H, Ar-H), 7.51 – 7.47 (m, 1H, Ar-H), 7.42 (d, J = 8.1 Hz, 2H, Ar-H), 7.26 (d, J =
.1 Hz, 1H, Ar-H), 7.10 (d, J = 9.0 Hz, 1H, Ar-H), 6.86 (dd, J = 9.0, 3.1 Hz, 1H, Ar-H), 4.33 (s, 2H,
13
SCH ), 3.76 (s, 3H, OCH ), 3.73 (s, 3H, OCH ). C NMR (101 MHz, DMSO-d ) δ (ppm) 165.40,
2
3
3
6
1
1
57.97, 152.96, 150.13, 147.75, 144.26, 133.40, 129.95, 129.55, 127.47, 126.24, 126.00, 124.93,
24.87, 123.14, 113.21, 112.92, 112.70, 111.36, 56.11, 55.42, 33.24. HR-MS (ESI) calcd for
+
C H F N O S [M+H] : 472.1307, found: 472.1307
2
4
20
3
3
2
4
.5.20. N-(3,4-dimethoxyphenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9t)
1
White solid, 84.2% yield, Mp 174.1-175.1 ℃. H NMR (400 MHz, DMSO-d ) δ (ppm) 11.53 (s,
6
1
7
1
4
1
1
H, NH), 8.77 (d, J = 8.2 Hz, 1H, Ar-H), 7.97 (t, J = 7.7 Hz, 1H, Ar-H), 7.81 (d, J = 8.3 Hz, 1H, Ar-H),
.67 (t, J = 7.6 Hz, 1H, Ar-H), 7.54 (d, J = 8.1 Hz, 2H, Ar-H), 7.35 (d, J = 2.0 Hz, 1H, Ar-H), 7.32 (s,
H, Ar-H), 7.30 (s, 1H, Ar-H), 7.21 (dd, J = 8.6, 2.0 Hz, 1H, Ar-H), 7.05 (d, J = 8.7 Hz, 1H, Ar-H),
13
.48 (s, 2H, SCH ), 3.78 (s, 3H, OCH ), 3.75 (s, 3H, OCH ). C NMR (101 MHz, DMSO-d ) δ (ppm)
2
3
3
6
63.90, 157.70, 148.66, 147.63, 142.18, 135.69, 129.59, 127.88, 127.56, 126.84, 125.46, 124.99,
24.85, 122.75, 119.17, 117.57, 111.75, 111.50, 109.91, 55.69, 55.64, 33.14. HR-MS (ESI) calcd for
+
C H F N O S [M+H] : 472.1307, found: 472.1304.
2
4
20
3
3
2
4
.5.21. N-(3-chloro-4-fluorophenyl)-2-((4-(trifluoromethyl)benzyl)thio)quinazolin-4-amine (9u)
1
White solid, 80.0% yield, Mp 146.1-146.4 ℃. H NMR (400 MHz, DMSO-d ) δ (ppm) 10.07 (s,
6
1
H, NH), 8.45 (d, J = 8.2 Hz, 1H, Ar-H), 8.10 (dd, J = 6.8, 2.6 Hz, 1H, Ar-H), 7.83 (t, J = 7.6 Hz, 1H,
Ar-H), 7.76 – 7.72 (m, 1H, Ar-H), 7.69 (d, J = 8.0 Hz, 1H, Ar-H), 7.62 (s, 4H, Ar-H), 7.55 (t, J = 7.3 Hz,
1
3
1
1
1
H, Ar), 7.44 (t, J = 9.1 Hz, 1H, Ar), 4.49 (s, 2H, SCH2). C NMR (101 MHz, DMSO-d ) δ (ppm)
6
65.07, 157.13, 149.83, 143.64, 135.88, 135.85, 133.77, 129.58, 127.61, 127.29, 126.19, 125.27,
25.05, 124.36, 123.26, 123.03, 118.94, 116.66, 112.94, 33.41. HR-MS (ESI) calcd for
+
C H ClF N O S [M+H] : 464.0611, found: 464.0610.
2
2
14
4
4
2
4
.5.22. 2-((4-(Trifluoromethyl)benzyl)thio)-N-(3,4,5-trimethoxyphenyl)quinazolin-4-amine (9v)
1
White solid, 74.7% yield, Mp 162.6-163.1 ℃. H NMR (400 MHz, DMSO-d ) δ (ppm) 10.68 (s,
6
1
7
H, NH), 8.68 (d, J = 8.0 Hz, 1H, Ar-H), 7.88 (t, J = 7.5 Hz, 1H, Ar-H), 7.75 (d, J = 8.2 Hz, 1H, Ar-H),
.62 – 7.54 (m, 3H, Ar-H), 7.48 (d, J = 7.4 Hz, 2H, Ar-H), 7.22 (s, 2H, Ar-H), 4.50 (s, 2H, SCH ), 3.79
2
1
3
(
s, 6H, (OCH ) ), 3.65 (s, 3H, OCH ). C NMR (101 MHz, DMSO-d ) δ (ppm) 164.74, 157.39, 152.58,
3 2 3 6

ACCEPTED MANUSCRIPT
1
43.35, 134.88, 134.26, 134.05, 129.68, 127.67, 127.36, 125.66, 125.52, 125.02, 124.17, 122.82,
+
1
5
5
12.71, 101.71, 60.10, 55.90, 33.28. HR-MS (ESI) calcd for C H F N O S [M+H] : 502.1412, found:
25
22
3
3
3
02.1413.
. Materials and Methods
5
.1 Materials
All compounds ware dissolved in DMSO to make a 10µM stock solution. RPMI-1640 and MTT
were purchased from Solarbio. Fetal Bovine Serum (FBS) was obtained from Tianhang Biotechnology
Co. Annexin V-FITC/PI Apoptosis Detection Kit and Cell Cycle Detection Kit were purchased from
Keygen Biotech. Dihydroethidium kit was purchased from Beyotime Biotechnology. Bax and p53
polyclonal antibodies were purchased from Proteintech; Bcl-2 monoclonal antibodies and
Phospho-EGF Receptor Antibody Sampler Kit were purchased from CST. PI3 Kinase Monoclonal
Antibody and PI3 Kinase (phospho-Tyr467/199) Antibody were purchased from SAB. Phospho-AKT
(
Ser473) rabbit polyclonal antibody and AKT rabbit polyclonal antibody were purchased from SAB.
GAPDH rabbit monoclonal antibody was purchased from Good-Here Biotechnology. All the other
reagents used were of analytical grade.
5
.2 Cell culture and treatment
Human cancer cells MDA-MB-231, MCF-7, PC-3, HGC-27 and MGC-803 were purchased from
American Type Culture Collection (ATCC, Shanghai, China). All of them were cultured in RPMI-1640
with 10% FBS. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO and were
2
stimulated with different concentrations of 9n. Cells were fed every 2–3 days and sub-cultured when
they reached 70–80% confluence.
5
.3 MTT assay
Cells in the logarithmic growth phase were seeded in 96-well plates at 3,000-5,000 cells per well.
After the cells were cultured for 24h, different concentrations of compound 9n were treated for 24, 48
and 72h, respectively. MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, Solarbio)
was added to each well at a final concentration of 0.5 mg/ml. After 4h in a 37 °C incubator, the
medium was aspirated. 150µl DMSO was then added to each well to dissolve the formazan, and the
plate was shaken on a shaker for 10 minutes. The absorbance was measured by an enzyme-linked
immunosorbent assay reader (BioTek, USA) at a wavelength of 490 nm, and the cell survival rate was
measured. Viability rate = Abs 490 treated cells/Abs 490 control cells × 100%. The
concentration-response curve generated by SPSS 16.0 software was used to determine the
concentration of compound (IC ) required to inhibit cell growth by 50%. Cell viability curves were
5
0

ACCEPTED MANUSCRIPT
generated using GraphPad Prism 7.0 software at various concentrations of all compounds. Results were
Mean ± SD of three independent experiments.
5
.4 Colony formation assay
MDA-MB-231 and MCF-7 cells in the logarithmic growth phase were plated in a 6-well plate at a
density of 500 cells per well, and then the cells were cultured at 37 � in a 5%CO humidified
2
atmosphere. After the cells were cultured for 24h, different concentrations (0, 3, 6, 9µM) of compound
9
0
n were treated for 7 days. The cells were incubated with ice methanol for 10 minutes and stained with
.1% crystal violet solution. After the plate is naturally dried, images were photographed.
Morphological analysis was performed through microscopy (Leica EZ4W, China). The experiment was
repeated three times.
5
.5 Cell cycle analysis
MDA-MB-231 and MCF-7 cells in the logarithmic growth phase were plated in a six-well plate at
5
a density of 2×10 cells per well. After the cells were cultured for 24h, different concentrations (0, 3, 6,
1
2µM) of compound 9n were administered for 24h. The cells were harvested with trypsin, washed with
PBS (phosphate-buffered saline) and incubated in pre-cooled 70% ethanol overnight at 4 � . The cells
were collected and detected by the cell cycle detection kit (Keygen Biotech, China), according to the
manufacturer's protocol. Briefly, cells were washed with ice-cold PBS, and resuspended in RNase A
solution and propidium iodide (PI) solution. Then the mixture was incubated at room temperature for
3
0 minutes in the dark. Ten thousand cells were collected and analyzed. Cell cycle progression of each
sample was measured by FAC-SCalibur flow cytometer (BD, Biosciences, San Jose, CA, USA) and
data was processed using ModFit LT software.
5
.6 Scratch assay
MDA-MB-231 and MCF-7 cells in the logarithmic growth phase were collected and seeded in
5
6
-well plates at 1.5×10 cells per well. After the cells were cultured for 24h, "#" was drawn at the
bottom of the plate. Cells were cultured in RPMI-1640 medium containing 2% FBS. Photos were taken
with a microscope as control groups. Different concentrations (0, 2, 4, 6µM) of compound 9n were
treated for 72h. Take photos again as experimental groups. Three fields were randomly selected from
each sample. Images were processed with Photoshop and Illustartor software.
5
.7 Cell morphology analysis and DAPI staining
MDA-MB-231 and MCF-7 cells in the logarithmic growth phase were plated in a 6-well plate at a
5
density of 2×10 cells per well. After the cells were cultured for 24h, different concentrations (0, 2, 4,
8
µM) of compound 9n were treated for 48h. Then, the cells were incubated with pre-cooled methanol
at 4 °C for 10 min, and resuspended in the DAPI dye solution in the dark. Three fields of each sample

ACCEPTED MANUSCRIPT
were selected and photographed with a fluorescence microscope (Nikon Eclipse Ti-S, Nikon Ltd,
Japan), and the images were processed with Photoshop and Illustartor software.
5
.8 Analysis of apoptosis by flow cytometry
MDA-MB-231 and MCF-7 cells in the logarithmic growth phase were plated in a six-well plate at
5
a density of 2×10 cells per well. After the cells were cultured for 24h, different concentrations (0, 2, 4,
6
µM) of compound 9n were treated for 48h. The cells were collected and measured with the Annexin
V-FITC/PI Apoptosis Detection Kit (Keygen Biotech, China), according to the manufacturer's protocol.
Briefly, cells were washed with ice-cold PBS, and resuspended in Annexin V-FITC solution and
propidium iodide (PI) solution. The mixture was incubated at room temperature for 30 minutes in the
dark. The FAC-SCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) was used to detect
apoptosis in each sample, and data was analyzed using Flow Jo software.
5
.9 Intracellular ROS assay
MDA-MB-231 and MCF-7 cells in the logarithmic growth phase were plated in a six-well plate at
5
a density of 2×10 per well. After the cells were cultured for 24h, different concentrations (0, 2, 4, 8µM)
of compound 9n were administrated for 48h. Then, the cells were incubated in DHE solution at 37 °C
for 30 min in the dark. Three fields were selected for each sample and photographed with a
fluorescence microscope (Nikon Eclipse Ti-S, Nikon Ltd, Japan). Process images with Photoshop and
Illustartor software
5
.10 Western blot analysis
Western blot were performed as described in the previous study [32]. The cells were treated with
compound 9n at 0, 3, 6 and 12µM. The intracellular proteins were extracted the RIPA lysate buffer
Beyotime Biotechnology) and the protein contents were determined by the BCA protein quantification
(
kit (Beyotime Biotechnology). Total proteins were separated by the SDS-PAGE according to the
molecular weight. Blots were blocked at room temperature for 2h in 5% nonfat dry milk-PBS-0.05%
Tween 20. The primary antibody (1:1000) was incubated overnight at 4 °C and then washed three times
with PBS-Tween20 (0.05%) for 10 min/time. The horseradish peroxidase-conjugated secondary
antibody (1:10,000) was incubated for 2h at room temperature and then washed three times with
PBS-Tween20 (0.05%) for 10 min/time. Blots exposure was performed with an ECL
chemiluminescence solution. All bands were analyzed Image J software. All the above experimental
results were repeated three times.

ACCEPTED MANUSCRIPT
5
.11 Molecular docking
Molecular docking study was performed using MOE 2014. The protein of EGFR (PDB code:2ITO)
and PI3K (PDB code:3APC) was downloaded from RCSB Protein Data Bank. The crystal structure of
EGFR and PI3K were selected as acceptor and prepared using default parameters in the QuickPrep
module. The protonation states of the ionizable residues were adjusted at pH=7. The 3D structures of
all compounds were prepared by taking energy minimization and conformational search. The pose with
the best score was used to explain the binding model of compound 9n in the active site of protein.
5
.12 Statistical analysis
All experimental data are the results of the experiment repeated three times. Statistical analysis
operations and mapping were done using Photoshop, ImageJ, SPSS, Graph Pad, etc. All experimental
results are expressed as Mean ± SD. The mean comparison between groups was performed by
ONE-WAY ANOVA and the Turkey method. P<0.05 was considered statistically significant.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81430085),
and Natural Science Foundation of Henan (No. 182300410321).
Conflicts of Interest
There are no conflicts of interest to report.
References：
[
1] Y. Ji, Q. Sun, J. Zhang, H. Hu, MiR-615 inhibits cell proliferation, migration and invasion by targeting EGFR in
human glioblastoma, Biochem Biophys Res Commun, 499 (2018) 719-726.
2] H. You, K. Meng, Z.Y. Wang, The ER-alpha36/EGFR signaling loop promotes growth of hepatocellular carcinoma
cells, Steroids, 134 (2018) 78-87.
[
[
[
3] M.A. Lemmon, J. Schlessinger, Cell signaling by receptor tyrosine kinases, Cell, 141 (2010) 1117-1134.
4] X. Qu, L. Yang, Q. Shi, X. Wang, D. Wang, G. Wu, Lidocaine inhibits proliferation and induces apoptosis in
colorectal cancer cells by upregulating mir-520a-3p and targeting EGFR, Pathol Res Pract, 214 (2018) 1974-1979.
5] R.B. David S. Salomon, Fortunato Ciardiello, Nicola Normanno, Epidermal growth factor-related peptides and
their receptors in human malignancies, Critical Reviews in Oncology/Hematology 19 (1994) 183-232.
6] W. Li, Q. Sun, L. Song, C. Gao, F. Liu, Y. Chen, Y. Jiang, Discovery of 1-(3-aryl-4-chlorophenyl)-3-(p-aryl)urea
derivatives against breast cancer by inhibiting PI3K/Akt/mTOR and Hedgehog signalings, Eur J Med Chem, 141
[
[
(
2017) 721-733.
[
7] X. Yang, Y. Ding, M. Xiao, X. Liu, J. Ruan, P. Xue, Anti-tumor compound RY10-4 suppresses multidrug resistance
in MCF-7/ADR cells by inhibiting PI3K/Akt/NF-kappaB signaling, Chem Biol Interact, 278 (2017) 22-31.
8] Z. Liu, Q. Zheng, W. Chen, M. Wu, G. Pan, K. Yang, X. Li, S. Man, Y. Teng, P. Yu, W. Gao, Chemosensitizing effect
[
of Paris Saponin I on Camptothecin and 10-hydroxycamptothecin in lung cancer cells via p38 MAPK, ERK, and Akt
signaling pathways, Eur J Med Chem, 125 (2017) 760-769.

ACCEPTED MANUSCRIPT
[
9] D.A. Fruman, H. Chiu, B.D. Hopkins, S. Bagrodia, L.C. Cantley, R.T. Abraham, The PI3K Pathway in Human
Disease, Cell, 170 (2017) 605-635.
10] S. Liebscher, L. Koi, S. Lock, M.H. Muders, M. Krause, The HIV protease and PI3K/Akt inhibitor nelfinavir does
[
not improve the curative effect of fractionated irradiation in PC-3 prostate cancer in vitro and in vivo, Clin Transl
Radiat Oncol, 2 (2017) 7-12.
[
[
11] D. Hanahan, R.A. Weinberg, Hallmarks of Cancer: The Next Generation, Cell, 144 (2011) 646-674.
12] K. Okkenhaug, M. Graupera, B. Vanhaesebroeck, Targeting PI3K in Cancer: Impact on Tumor Cells, Their
Protective Stroma, Angiogenesis, and Immunotherapy, Cancer Discov, 6 (2016) 1090-1105.
[
(
[
13] Y. Yarden, G. Pines, The ERBB network: at last, cancer therapy meets systems biology, Nat Rev Cancer, 12
2012) 553-563.
14] N. Golob-Schwarzl, S. Krassnig, A.M. Toeglhofer, Y.N. Park, M. Gogg-Kamerer, K. Vierlinger, F. Schroder, H.
Rhee, R. Schicho, P. Fickert, J. Haybaeck, New liver cancer biomarkers: PI3K/AKT/mTOR pathway members and
eukaryotic translation initiation factors, Eur J Cancer, 83 (2017) 56-70.
[
15] C. Mendoza-Martinez, J. Correa-Basurto, R. Nieto-Meneses, A. Marquez-Navarro, R. Aguilar-Suarez, M.D.
Montero-Cortes, B. Nogueda-Torres, E. Suarez-Contreras, N. Galindo-Sevilla, A. Rojas-Rojas, A. Rodriguez-Lezama,
F. Hernandez-Luis, Design, synthesis and biological evaluation of quinazoline derivatives as anti-trypanosomatid
and anti-plasmodial agents, Eur J Med Chem, 96 (2015) 296-307.
[
16] T.S. Patel, S.F. Vanparia, S.A. Gandhi, U.H. Patel, R.B. Dixit, C.J. Chudasama, B.C. Dixit, Novel stereoselective
,3-disubstituted quinazoline-4(3H)-one derivatives derived from glycine as a potent antimalarial lead, New
Journal of Chemistry, 39 (2015) 8638-8649.
17] A.M. Alafeefy, A.A. Kadi, O.A. Al-Deeb, K.E. El-Tahir, N.A. Al-Jaber, Synthesis, analgesic and anti-inflammatory
evaluation of some novel quinazoline derivatives, Eur J Med Chem, 45 (2010) 4947-4952.
18] H.M. Patel, M.N. Noolvi, A.A. Shirkhedkar, A.D. Kulkarni, C.V. Pardeshi, S.J. Surana, Anti-convulsant potential
of quinazolinones, RSC Advances, 6 (2016) 44435-44455.
19] S. Lin, Y. Li, Y. Zheng, L. Luo, Q. Sun, Z. Ge, T. Cheng, R. Li, Design, synthesis and biological evaluation of
quinazoline-phosphoramidate mustard conjugates as anticancer drugs, Eur J Med Chem, 127 (2017) 442-458.
20] A. Inoue, K. Kobayashi, K. Usui, M. Maemondo, S. Okinaga, I. Mikami, M. Ando, K. Yamazaki, Y. Saijo, A.
2
[
[
[
[
Gemma, H. Miyazawa, T. Tanaka, K. Ikebuchi, T. Nukiwa, S. Morita, K. Hagiwara, First-line gefitinib for patients
with advanced non-small-cell lung cancer harboring epidermal growth factor receptor mutations without
indication for chemotherapy, J Clin Oncol, 27 (2009) 1394-1400.
[
21]C.E. Geyer, J. Forster, D. Lindquist, S. Chan, C.C. Romieu, T. Pienkowski, A.J. Gruszfeld, J. Crown, A. Chan, B.
Kaufman, D. Skarlos, M. Campone, N. Davidson, M. Berger, C. Oliva, S.D. Rubin, S. Stein, D. Cameron, Lapatinib
plus Capecitabine for HER2-Positive Advanced Breast Cancer, N Engl J Med, 355 (2006) 2733-2743.
[
22] M.J. Moore, D. Goldstein, J. Hamm, A. Figer, J.R. Hecht, S. Gallinger, H.J. Au, P. Murawa, D. Walde, R.A. Wolff,
D. Campos, R. Lim, K. Ding, G. Clark, T.V. Nomikos, M. Ptasynski, W. Parulekar, Erlotinib plus gemcitabine
compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National
Cancer Institute of Canada Clinical Trials Group, J Clin Oncol, 25 (2007) 1960-1966.
[
23] M.H. Cohen, J.R. Johnson, Y.F. Chen, R. Sridhara, R. Pazdur, FDA Drug Approval Summary: Erlotinib (Tarceva®)
Tablets, The Oncologist, 10 (2005) 416-466.
24] M.H. Cohen, G.A. Williams, G.C. Rajeshwari Sridhara, J. W. David McGuinn, D. Morse., S. Abraham, A. Rahman,
[
C. Liang, R. Lostritto, A. Baird, R. Pazdur, United States Food and Drug Administration Drug Approval Summary:
Gefitinib(ZD1839; Iressa) Tablets, Clinical Cancer Research, 10 (2004) 1212–1218.
[
25] V. Alagarsamy, K. Chitra, G. Saravanan, V.R. Solomon, M.T. Sulthana, B. Narendhar, An overview of
quinazolines: Pharmacological significance and recent developments, Eur J Med Chem, 151 (2018) 628-685.

ACCEPTED MANUSCRIPT
[
26] K. Kohda, I. Terashima, K. Koyama, K. Watanabe, K. Minerura, Potentiation of the Cototoxicity of
6
Chlorethylnitrosourea by O -Arylmethylguanines, Biol Pharm Bull, 18 (1995) 424-430.
[
27] S. Hanessian, L. Auzzas, G. Giannini, M. Marzi, W. Cabri, M. Barbarino, L. Vesci, C. Pisano, Omega-alkoxy
analogues of SAHA (vorinostat) as inhibitors of HDAC: a study of chain-length and stereochemical dependence,
Bioorg Med Chem Lett, 17 (2007) 6261-6265.
[
28] F. Thuaud, S. Kojima, G. Hirai, K. Oonuma, A. Tsuchiya, T. Uchida, T. Tsuchimoto, M. Sodeoka, RE12 derivatives
displaying Vaccinia H1-related phosphatase (VHR) inhibition in the presence of detergent and their
anti-proliferative activity against HeLa cells, Bioorg Med Chem, 22 (2014) 2771-2782.
[
29] B. Wei, Q. Lin, Y.G. Ji, Y.C. Zhao, L.N. Ding, W.J. Zhou, L.H. Zhang, C.Y. Gao, W. Zhao, Luteolin ameliorates rat
myocardial ischaemia-reperfusion injury through activation of peroxiredoxin II, Br J Pharmacol, 175 (2018)
315-3332.
30] S. Chou, W.K. Yin, Y.S. Chung, L.S. Chang, C.W. Liu, S.F. Chen, K.S. Shih, Kilogram-Scale Synthesis of a Highly
Selective r1-Adrenoceptor Antagonist (DL-028A), Organic Process Research & Development, 6 (2002) 273-278.
31] M.A. Qhobosheane, A. Petzer, J.P. Petzer, L.J. Legoabe, Synthesis and evaluation of 2-substituted
3
[
[
4
5
(3H)-quinazolinone thioether derivatives as monoamine oxidase inhibitors, Bioorg Med Chem, 26 (2018)
531-5537.
[
32] Q. Lin, X.-Y. Chen, J. Zhang, Y.-L. Yuan, W. Zhao, B. Wei, Upregulation of SIRT1 contributes to the
cardioprotective effect of Rutin against myocardial ischemia-reperfusion injury in rats, Journal of Functional Foods,
6 (2018) 227-236.
4

ACCEPTED MANUSCRIPT
Highlights
‹
The 2,4-disubstituted quinazolines showed moderate to good growth inhibition against the tested
five human cancer cells.
‹
Among them, compound 9n exerted the most excellent anti-proliferative activities against breast
cancer cells.
‹
‹
Compound 9n inhibited the colony formation and migration of MDA-MB-231 and MCF-7 cells.
Compound 9n caused cell cycle arrest at G1 phase and induced apoptosis of MDA-MB-231 and
MCF-7 cells.
‹
Compound 9n inhibited tumor growth through EGFR-PI3K signaling pathway.