Design, synthesis, and biological evaluation of novel 4-anilinoquinazoline derivatives bearing amino acid moiety as potential EGFR kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.02.061

In this study, a series of 4-anilinoquinazoline derivatives bearing amino acid moiety were designed, synthesized and evaluated for biological activities. The synthesized compounds were screened for anticancer activity against human hepatocellular carcinoma cell HepG2 using SRB assay. In?vitro cell growth inhibition assays indicated that compound 6m exhibited moderate inhibitory activities only against human hepatocellular carcinoma cells HepG2 with IC₅₀ of 8.3?μM. Synthetic derivatives showed excellent selectivity, such as compound 6m demonstrated a strong inhibition of EGFR (IC₅₀?=?0.0032?μM), with selectivity of over 2000-fold over other kinases. Apoptosis analysis revealed that compound 6m caused obvious induction of cell apoptosis. 6m significantly down-regulated the expression of Bcl-2 and up-regulated the expression of Bax, decreased mitochondrial membrane potential (ΔΨm), promoted the mitochondrial cytochrome c release into the cytoplasm, activated caspase-3, and finally induced apoptosis of HepG2 cells. Molecular docking indicated that compound 6m could bind well with EGFR. Therefore, compound 6m may be a potential agent for cancer therapy deserving further research.

Full text of DOI:10.1016/j.ejmech.2017.02.061

Accepted Manuscript
Design, synthesis, and biological evaluation of novel 4-anilinoquinazoline derivatives
bearing amino acid moiety as potential EGFR kinase inhibitors
You-Guang Zheng, Jun Su, Cai-Yun Gao, Ping Jiang, Lin An, Yun-Sheng Xue, Jian
Gao, Yi Liu
PII:
S0223-5234(17)30146-0
10.1016/j.ejmech.2017.02.061
EJMECH 9257
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2016
Revised Date: 24 February 2017
Accepted Date: 27 February 2017
Please cite this article as: Y.-G. Zheng, J. Su, C.-Y. Gao, P. Jiang, L. An, Y.-S. Xue, J. Gao, Y. Liu,
Design, synthesis, and biological evaluation of novel 4-anilinoquinazoline derivatives bearing amino
acid moiety as potential EGFR kinase inhibitors, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.02.061.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and biological evaluation of novel
4
-anilinoquinazoline derivatives bearing amino acid moiety as
potential EGFR kinase inhibitors
a, b, c
a, b
a, b
a, b
a, b
You-Guang Zheng
*, Jun Su , Cai-Yun Gao , Ping Jiang , Lin An
,
a, b
a, b
a, b
Yun-Sheng Xue , Jian Gao , Yi Liu *
a
College of Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China;
b
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou
Medical University, Xuzhou 221004, PR China;
c
Jiangsu Key Laborotary of Brain Disease Bioinformation, Xuzhou Medical
University, Xuzhou 221004, PR China;
You-Guang Zheng and Yi Liu are co-corresponding authors.
ABSTRACT
In this study, a series of 4-anilinoquinazoline derivatives bearing amino acid moiety
were designed, synthesized and evaluated for biological activities. The synthesized
compounds were screened for anticancer activity against human hepatocellular
carcinoma cell HepG2 using SRB assay. In vitro cell growth inhibition assays
indicated that compound 6m exhibited moderate inhibitory activities only against
human hepatocellular carcinoma cells HepG2 with IC50 of 8.3µM. Synthetic
derivatives showed excellent selectivity, such as compound 6m demonstrated a strong
inhibition of EGFR (IC =0.0032µM), with selectivity of over 2000-fold over other
5
0
kinases. Apoptosis analysis revealed that compound 6m caused obvious induction of
cell apoptosis. 6m significantly down-regulated the expression of Bcl-2 and
up-regulated the expression of Bax, decreased mitochondrial membrane potential
(∆Ψm), promoted the mitochondrial cytochrome c release into the cytoplasm,
activated caspase-3, and finally induced apoptosis of HepG2 cells. Molecular docking
indicated that compound 6m could bind well with EGFR. Therefore, compound 6m
may be a potential agent for cancer therapy deserving further research.
Keywords: 4-anilinoquinazoline derivatives, amino acid moiety, HepG2, apoptosis

ACCEPTED MANUSCRIPT
1
. Introduction
The heterocyclic compounds play a very important role in medicinal chemistry due to
their wide spectrum of biological properties. One class of the most important
heterocyclic compounds is quinazolines which have wide range of biological
properties such as the anticancer [1], antibacterial [2], anticonvulsant [3], antifungal
[
4], anti-HIV [5], antihypertensive [6], anti-inflammatory [7] and antimalarial [8]
activities. In the past 15 years, FDA has approved several anticancer drugs which have
quinazoline skeleton, such as gefitinib [9], erlotinib [10] and lapatinib [11]. In
addition, there are some quinazoline derivatives have been studied in clinical trials,
such as ZM447439 [12], AZD1152 [13], canertinib and so on [14] (Fig.1). Perusal of
literatures also revealed that quinazoline derivatives exhibited remarkable in vitro
anti-proliferative activity against the tumor cell lines，such as HepG2, A549, MCF-7
and so on[15-18]. Our research group has been focusing on the discovery of
4
-anilinoquinazoline derivatives as anticancer agents since 2006. Previously, we had
reported several 4-anilinoquinazoline derivatives [19, 20], which show significant
activity against cancer cells.
Fig.1. Quinazoline derivatives as anticancer agents

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As endogenous substance, amino acid could be used to modification of drug skeletons
to promote absorption of the drugs [21]. Furthermore, toxicity of drugs also could be
reduced via the introduction of amino acid [22].
In the light of our previous studies, we designed and synthesized a series of novel
4
-anilinoquinazoline derivatives through addition of various N-Boc protected amino
acids on position 6 of quinazoline ring (Fig.2)
Fig.2. design of novel 4-aminoquinazoline-amino acid derivatives
2
. Results and discussion
2
.1. Chemistry
The general synthetic strategy to obtain the target compounds is shown in Scheme 1
according to the literatures being reported [23-25]. The route began with
2
-amino-5-nitrobenzoic acid (1) as raw material, which was treated with formamidine
acetate in 2-methoxyethanol to obtain 6-nitro-3H-quinazolin-4-one (2). Compound (3),
which was prepared through chlorination of 6-nitro-3H-quinazolin-4-one (2), was
subjected to reflux with various substituted aniline in isopropanol to give the products
(
4). Reduction of the nitro group on compound (4) using iron powder gave
intermediates (5). The target compounds (6a-6r) were prepared by reacting compound
5) with various N-Boc protected amino acids.
(

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Scheme 1. The synthesis of 4-anilinoquinazoline-amino acid derivatives. Reagents
and conditions: (a)formamidine acetate, 2-methoxyethanol, reflux; (b)SOCl , DMF,
2
reflux; (c)substituted aniline, isopropanol, reflux; (d) iron powder, acetic acid/ethanol,
refiux; (e) N-Boc protected amino acids, HATU/DMF/ triethylamine, rt.
2
2
.2. Biological evaluation
.2.1. Anti-proliferation assay
To explore the potential antitumor effect, the synthetic derivatives were evaluated for
their in vitro anti-proliferative activities against human hepatocellular carcinoma cell
HepG2 by SRB assay. As shown in Table 1, several compounds demonstrated
moderate anti-proliferative activities against HepG2 (IC50 ranging from 4.3 to
3
4
4.8µM). Most of the target compounds with (3-bromophenyl) amino group on the
-position of quinazoline ring showed moderate potency against the cancer cell line,
such as compounds 6m, 6p and 6q (IC =8.3, 25.0 and 34.0µM, respectively). It is
5
0
worthy to note that compound 6i exhibited significant anti-proliferative effects against
HepG2 cell (IC =4.3µM) compared to other compounds bearing (3-(trifluoromethyl)
5
0
phenyl) amino moiety at C-4 position of quinazoline ring.
Table 1 In vitro anti-proliferative activity in tumor cell lines of target compounds
Compd
R1
R2
R3
HepG2 IC50 (µM)
6a
6b
6c
3-Cl
4-F
33±14.9
3-Cl
3-Cl
4-F
4-F
>50
>50

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6
d
e
3-Cl
3-Cl
3-Cl
4-F
13.8±5.5
6
4-F
4-F
>50
6
f
>50
6
6
g
h
3-CF3
3-CF3
3-CF3
4-H
4-H
4-H
>50
>50
6
6
i
j
4.3±1.8
3-CF3
3-CF3
3-CF3
4-H
4-H
4-H
>50
>50
>50
6
k
6
l

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6
m
3-Br
3-Br
3-Br
4-H
8.3±0.6
>50
6
n
o
4-H
4-H
6
>50
6
6
p
q
3-Br
3-Br
4-H
4-H
25±13.5
34±16.1
>50
6
r
3-Br
4-H
6
s
2-CH3
6-CH3
>50
ZM447439
1.4±0.2
2
.2.2. Kinases inhibitory activity of 4-anilinoquinazoline-amino acid derivatives
Several target compounds were assayed with the enzymatic activities against the
kinases that related to liver cancer, such as EGFR, Aurora B, VEGF2, MEK, RAF and
c-MET. Unfortunately, the result of kinases inhibitory activity indicated that most of
the target compounds exhibited poor activity against Aurora B, VEGF2, MEK, RAF
and c-MET, except compound 6m showed weak IC value at concentration of 6.2µM
50
(Aurora B), 18.7µM (VEGF2), 7.35µM (MEK), 17.30µM (RAF) and >500µM

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(c-MET)(Table 2). Meanwhile, several of the compounds were also tested for their
EGFR inhibitory activity. As shown in Table 2, all the tested compounds presented
stronger EGFR inhibitory activity (IC ranging from 0.0032 to 1.02µM) compared to
5
0
the other kinases. Especially, compound 6m was the most potent inhibitor of EGFR
(
IC = 0.0032µM), with selectivity of 1937-fold over aurora B (IC = 6.2µM),
50 50
5
5
843-fold over VEGF2 (IC = 18.7µM), 2296-fold over MEK (IC =7.25µM) and
50 50
406-fold over RAF (IC =17.30µM). Among the tested compounds, compound 6a
5
0
also exhibited potent EGFR inhibitory activity with IC of 0.017µM. It seems that
5
0
compounds (6a and 6m) bearing N-Boc protected glycine group at 6-position of
quinazoline skeleton have increased enzymatic inhibitory activity compared to the
compounds contained other N-Boc protected amino acids, such as N-Boc protected
L-alanine (6b, 6h and 6n). Based on the results of the anti-proliferative activity and
enzyme inhibition activity, compound 6m was chosen for further biological
evaluation in HepG2 cells.
Table2 The kinases enzymatic inhibition activity of target compounds
IC (µM)
5
0
Compd
EGFR
0.017±0.0014
>100
Aurora B
23.7±0.8
>100
VEGF2
MEK
RAF
c-MET
a
a
a
a
6a
6b
6c
6d
6e
NT
NT
NT
NT
a
a
a
a
NT
NT
NT
NT
a
a
a
a
a
NT
>100
NT
NT
NT
NT
a
a
a
a
0.14±0.0057
>100
NT
NT
NT
NT
a
a
a
a
a
NT
>100
NT
NT
NT
NT
a
a
a
a
a
6
f
NT
>100
NT
NT
NT
NT
a
a
a
a
a
6
6
g
h
NT
51.0±3.7
>100
NT
NT
NT
NT
a
a
a
a
>100
NT
NT
NT
NT
6
6
i
j
1.021±0.0891
>100
>100
>100
>100
>100
a
a
a
a
a
NT
>100
NT
NT
NT
NT
a
a
a
a
a
6
k
NT
>100
NT
NT
NT
NT
a
a
a
a
a
6
l
NT
>100
NT
NT
NT
NT
6
m
0.0032±0.0002
>100
6.2 ±0.3
>100
18.70±1.80 7.35±0.55 17.30±2.80
>100
a
a
a
a
a
a
a
a
a
a
6
n
o
p
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
a
a
6
NT
>100
NT
a
6
6
0.145±0.0127
>100
NT
0.0555±
a
a
a
a
q
>100
NT
NT
NT
NT
0
.0049
a
a
a
a
a
a
a
a
6
r
NT
>100
>100
NT
NT
NT
NT
NT
NT
NT
a
a
6
s
NT
NT

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a
a
a
a
ZM447439
0.11±0.0113
0.156±0.0007
NT
NT
NT
NT
a
Not Test
2
.2.3. Apoptosis analysis
The morphology of apoptosis was observed using hoechst 33258 staining. As shown
in Fig. 3, when treated with 2.0, 5.0, and 10.0 µM of compound 6m for 24 h, nuclei
appeared typical morphology of apoptosis, as condensed chromatin state, a high
degree of nuclear chromatin condensation and appearance of apoptotic bodies. This
result suggested that compound 6m induced apoptosis of HepG2 cells effectively.
Fig.3. Fluorescent staining of nuclei by Hoechst 33258 in ZM447439 (ZM) and 6m
treated or untreated cells followed by observation using fluorescence microscope
(×400).
To further confirm the induction of apoptosis by 6m, the HepG2 cells were treated
with various concentrations of 6m (2, 5, 10µM), then harvested and stained with
Annexin V, followed by PI staining in the dark. The samples were analyzed by flow
cytometry. As shown in Fig. 4, the percentages of apoptotic HepG2 cells was
increased from 4.86% (control) to 35.74% after 6m treatment, which suggested that
compound 6m caused obvious induction of cell apoptosis in
a
concentration-dependent manner.

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Fig.4. Apoptosis rates of HepG2 cells of the control and treated by ZM447439 (10
µM), 6m (2 µM, 5 µM, 10 µM) groups, measured using flow cytometry with double
staining of Annexin V and PI.
To explore the mechanism of compound 6m induced HepG2 cells death, the change
of mitochondrial membrane potential was investigated. After treatment of HepG2
cells with 6m at the concentrations of 2, 5, 10 µM, JC-1 staining solution was then
added, followed by photographing using fluorescence microscope. As shown in Fig. 5,
after 6m treatment for 24h, mitochondrial membrane potential of HepG2 cells was
decreased. These results suggested that 6m effectively decreased cells mitochondrial
membrane potential in a concentration-dependent manner.

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Fig.5. Effects of 6m induced a decrease in mitochondrial membrane potential.
Mitochondrial membrane potential of HepG2 cells was measured by JC-1, red
fluorescence represents the mitochondrial aggregate JC-1 and green fluorescence
indicates the monomeric JC-1(×200).
In order to further investigate the mechanism of the induction of cellular apoptosis,
we examined the expressions of proteins related to apoptotic pathway such as Bax,
Bcl-2, cyt c and cleaved-caspase-3 protein. Bcl-2 protein family (including
anti-apoptosis protein Bcl-2 and pro-apoptotic protein Bax), which locates on cell

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mitochondria and is associated with mitochondrial membrane, plays critical roles in
apoptosis of cells [26]. The HepG2 cells were treated with various concentrations of
6
m (2, 5, 10µM) for 24h, and then western blot was used to detect the expression of
Bax and Bcl-2. As shown in Fig. 7, 10µM of 6m treatment significantly increased the
expression of Bax (Fig. 6A, 6B), and decreased the levels of Bcl-2 (Fig. 6C, 6D).
Fig.6. Effect of 6m on the levels of Bax and Bcl-2 in HepG2 cells. 0.1% DMSO was
used as solvent control and 10µM ZM447439 (ZM) used as positive control (A)
Western blot analysis for Bax in HepG2 cell; (B) Densitometic analysis was
performed after normalization with β-actin.; (C) Western blot analysis for Bcl-2 in
HepG2 cell. (D) Densitometic analysis was performed after normalization with
β-actin.
These findings demonstrated that compound 6m decreased mitochondrial membrane
potential by decreasing the expression of Bcl-2 and increasing the expression of Bax.
We then examined the activation of mitochondrial cytochrome c (cyt c) to study the
effect of mitochondrial membrane potential on mitochondrial membrane channel. As
shown in Fig. 7, the treatment with 10µM of 6m increased the expression of cyt c,
which suggested that compound 6m induced cellular apoptosis by the mitochondrial
pathway.

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Fig.7. Effect of 6m on the levels of cytochrome c (cyt c) in HepG2 cells. 0.1% DMSO
as control group, 10 µM ZM447439 (ZM) group. (A) Western blot analysis for cyt c
in HepG2 cell. (B) Densitometic analysis was performed after normalization with
β-actin.
It has been recognized that caspase-3 expression is a critical initiator and executioner
of apoptosis [27]. By 24 h treatment with 6m at the concentrations of 2, 5, 10 µM, and
then the functional activity of cleaved-caspase-3 was monitored using western blot.
As shown in Fig. 8, the treatment with 5 and 10µM of 6m increased the expression
levels of cleaved-caspase-3, indicating that 6m induced apoptosis in HepG2 cells via
decreasing activation of caspase-3.

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Fig.8. Effect of 6m on the levels of caspase-3 in HepG2 cells. 0.1% DMSO as control
group, 10µM ZM447439 (ZM) group. (A) Western blot analysis for caspase-3 and
cleaved-caspase-3 in HepG2 cells. (B) Densitometic analysis was performed after
normalization with β-actin.
2
.3. Molecular docking studies
To understand the interaction between compound and kinases, the possible binding
modes of compound 6m on EGFR (PDB code: 2ITY) was explored using the
AutoDock 4.2. As shown in Fig 9, the quinazoline ring with the 3-N is oriented in the
back of the ATP-binding pocket, where its hydrogen bonds with the main chain amide
of Thr790 and Thr 854 through a well-defined water molecule. Besides, the amino
nitrogen atoms in the side chain amino acid of compound 6m forms another hydrogen
bond with the carbonyl oxygen atom of Leu718.

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Fig.9. Binding models of compound 6m target into active site of EGFR.
3
. Conclusions
In summary, a series of 4-anilinoquinazoline-amino acid derivatives, which exhibited
anti-proliferation potency against human hepatocellular carcinoma cell HepG2, were
synthesized. In vitro cell growth inhibition assays indicated that compound 6m
exhibited moderate inhibitory activities against a human hepatocellular carcinoma cell
HepG2 with IC of 8.3µM. Compound 6m demonstrated a strong inhibition of EGFR
5
0
(
IC = 0.0032µM), with selectivity of over 2000-fold over other kinases. Apoptosis
50
analysis revealed that compound 6m down-regulated the expression of Bcl-2 and
up-regulated the expression of Bax, decreased mitochondrial membrane potential
(∆Ψm), and then promoted the mitochondrial cytochrome c release into the cytoplasm,
activated caspase-3, induced apoptosis of HepG2 cells. Molecular docking of the
compound 6m into the active binding sites of EGFR, and the result indicated that
compound 6m could bind with EGFR.
4
. Experimental
4
.1. Chemistry
Unless otherwise noted, all reagents were purchased from commercial sources and
used without further purification. All compounds were routinely monitored by
thin-layer chromatography with silica gel GF-254 glass plates and viewed under UV
1
13
light at 254 nm. The H-NMR and C-NMR spectra were determined in DMSO-d or
6

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CDCl on a Bruker Avance 400 MHz spectrometer. Chemical shifts (δ) were reported
3
in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as an
internal standard. The mass spectra (MS) were obtained from Agilent 1100LC/MS
Spectrometry Services. HR-MS was obtained using a Q-tof high resolution mass
spectrometer.
4
.1.1. Synthesis of compounds (2-5)
Compounds 2, 3, 4 and 5 were prepared according to a similar procedure in reference
23-25].
.1.2 General procedure for the preparation of the compound 6
[
4
To a solution of N-Boc protected amino acids (1.2 mmol) in DMF (20 mL) was added
HATU (1.2 mmol) and triethylamine (1.2 mmol). The mixture was stirred at rt for 1.0
h, then compound 5 was added, and was monitored by thin layer chromatography.
After completion of the reaction, the reaction mixture was poured into ice water (200
mL) and extracted with ethyl acetate (3×50 mL). The combined organic extract was
washed with brine, dried over Na SO , filtered, concentrated and purified by a silica
2
4
gel column chromatography (8:1, EtOAc/petroleum ether) to afford compound 6.
.1.2.1 tert-butyl (2-((4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)amino)-2-
oxoethyl)carbamate (6a)
Off-white powder, yield: 43.5%. H NMR (400 MHz, CDCl ) δ 9.76 (s, 1H, NH),
4
1
3
8
7
4
1
.57 (s, 1H, ArH), 8.40 (s, 1H, NH), 8.12 (s, 1H, ArH), 7.76 (d, J = 8.0 Hz, 2H, ArH),
.55 (s, 1H, ArH), 7.48 (s, 1H, NH), 7.29 (s, 1H, ArH), 7.18 (t, J = 8.0 Hz, 1H, ArH),
1
3
.14 (s, 2H, CH ), 1.46 (s, 9H, CH );
C NMR (100 MHz, DMSO-d ) δ 169.06,
6
2
3
57.78, 156.43, 153.47, 147.02, 137.15, 128.98, 127.56, 124.25, 123.09, 119.27,
19.09, 117.03, 116.81, 115.81, 112.15, 78.58, 44.19, 28.69; LC-MS m/z: 446.1
1
+
+
[
M+H] ; HRMS(ESI) Calcd for C H ClFN NaO
3
([M+Na] ):468.1215,
2
1
21
5
found:468.1255.
4
.1.2.2 (R)-tert-butyl (1-((4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)amino)
-1-oxopropan-2-yl)carbamate(6b)
1
Off-white powder, yield: 47.6%. H NMR (400 MHz, CDCl ) δ 9.99 (s, 1H, NH),
3
8
.54 (s, 1H, ArH), 8.34 (s, 1H, NH), 8.20 (d, J = 4.0 Hz, 1H, ArH), 7.83 (d, J = 8.0

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Hz, 2H, ArH), 7.38 (d, J = 12.0 Hz, 1H, ArH), 7.31 (s, 1H, NH), 7.29 (s, 1H, ArH),
7
.20 (t, J = 8.0 Hz, 1H, ArH), 4.62(t, J = 8.0 Hz, 1H, CH), 1.62 (d, J = 8.0 Hz, 3H,
1
3
CH ), 1.44 (s, 9H, CH ); C NMR (100 MHz, DMSO-d ) δ 172.64, 170.79, 157.80,
3
3
6
1
1
55.70, 153.46, 147.03, 137.32, 128.93, 124.19, 119.27, 119.09, 117.04, 116.82,
+
15.81, 112.16, 78.58, 50.88, 28.70, 18.41; LC-MS m/z: 461.2 [M+H] ; HRMS(ESI)
+
Calcd for C H ClFN NaO ([M+Na] ):482.1371, found:482.1428.
2
2
23
5
3
4
.1.2.3 (R)-tert-butyl (1-((4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)amino)
-3-methyl-1-oxobutan-2-yl)carbamate(6c)
1
Off-white powder, yield: 36.3%. H NMR (400 MHz, CDCl ) δ 10.05 (s, 1H, NH),
3
8
.52 (s, 1H, ArH), 8.49 (s, 1H, NH), 8.20 (d, J = 4.0 Hz, 1H, ArH), 7.90-7.87 (m, 2H,
ArH), 7.37 (d, J = 8.0 Hz, 1H, ArH), 7.29 (s, 1H, NH), 7.22-7.18 (m, 2H, ArH), 4.36
t, J = 8.0 Hz, 1H, CH), 2.28-2.23 (m, 1H, CH), 1.42 (s, 9H, CH ), 1.25 (dd, J = 8.0
(
3
13
Hz, 6H, CH ); C NMR (100 MHz, DMSO-d ) δ 171.52, 157.82, 156.08, 154.99,
3
6
1
53.50, 147.12, 137.08, 128.98, 127.70, 124.30, 119.28, 119.09, 117.03, 116.82,
+
1
15.82, 78.59, 61.12, 30.79, 28.68, 19.72; LC-MS m/z: 489.2 [M+H] ; HRMS(ESI)
+
Calcd for C H ClFN NaO ([M+Na] ): 510.1684, found: 510.1765.
2
4
27
5
3
4
.1.2.4 (R)-tert-butyl (1-((4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)amino)
-4-methyl-1-oxopentan-2-yl)carbamate(6d)
1
Off-white powder, yield: 28.0 %. H NMR (400 MHz, DMSO-d ) δ 10.32 (s, 1H, NH),
6
9
.97 (s, 1H, ArH), 8.69 (s, 1H, NH), 8.57 (s, 1H, ArH), 8.13 (d, J = 8.0 Hz, 1H, ArH),
.92 (d, J = 12.0 Hz, 1H, ArH), 7.81 (s, 1H, NH), 7.79 (s, 1H, ArH), 7.44 (t, J = 8.0
7
Hz, 1H, ArH), 7.14 (d, J = 8.0 Hz, 1H, ArH), 4.26-4.20 (m, 1H, CH), 2.51 (s, 2H,
13
CH ), 1.72-1.68 (m, 1H, CH), 1.40 (s, 9H, CH ), 0.94 (dd, J = 4.0 Hz, 6H, CH ); C
2
3
3
NMR (100 MHz, DMSO-d ) δ 172.55, 157.80, 155.96, 153.48, 147.06, 137.30,
6
1
4
28.94, 127.75, 124.21, 119.28, 119.09, 117.05, 116.83, 115.80, 112.29, 78.55, 53.96,
+
+
1.06, 28.69, 23.46, 22.01; LC-MS m/z: 502.2 [M+H] ; LC-MS m/z: 518.3 [M+H] ;
+
HRMS(ESI) Calcd for C H ClFN NaO ([M+Na] ): 524.1841, found: 524.1883.
2
5
29
5
3
4
.1.2.5 (2R, 3S)-tert-butyl (1-((4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)
amino)-3-methyl-1-oxopentan-2-yl)carbamate(6e)

ACCEPTED MANUSCRIPT
1
Off-white powder, yield: 30.2%. H NMR (400 MHz, DMSO-d ) δ 10.83 (s, 1H, NH),
6
1
0.19 (s, 1H, ArH), 8.94 (s, 1H, ArH), 8.56 (s, 1H, ArH), 8.24 (s, 1H, NH), 8.11 (d, J
8.0 Hz, 1H, ArH), 7.90 (s, 1H, NH), 7.77 (d, J = 8.0 Hz, 1H, ArH), 7.46-7.41 (m,
=
1
1
0
1
2
H, ArH), 7.20 (d, J = 8.0 Hz, 1H, ArH), 4.05 (t, J = 8.0 Hz, 1H, CH), 2.04-1.95 (m,
H, CH), 1.56-1.51 (m, 2H, CH ), 1.40 (s, 9H, CH ), 0.91 (d, J = 8.0 Hz, 3H, CH ),
2
3
3
1
3
.86 (t, J = 8.0 Hz, 3H, CH ); C NMR (100 MHz, DMSO-d ) δ 171.66, 159.91,
3
6
46.76, 131.40, 126.29, 123.97, 123.52, 121.19, 115.19, 99.99, 79.72, 63.48, 37.07,
+
8.67, 25.14, 15.87, 11.27; LC-MS m/z: 502.2 [M+H] ; HRMS(ESI) Calcd for
+
C H ClFN NaO ([M+Na] ): 524.1841, found: 524.1830.
25
29
5
3
4
.1.2.6 (R)-tert-butyl (1-((4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)amino)
-4-(methylthio)-1-oxobutan-2-yl)carbamate(6f)
1
Off-white powder, yield: 46.7%. H NMR (400 MHz, DMSO-d ) δ 10.35 (s, 1H, NH),
6
9
.97 (s, 1H, ArH), 8.69 (s, 1H, NH), 8.57 (s, 1H, ArH), 8.13 (d, J = 4.0 Hz, 1H, ArH),
.92 (d, J = 12.0 Hz, 1H, ArH), 7.81 (s, 1H, NH), 7.79 (s, 1H, ArH), 7.44 (t, J = 8.0
7
Hz, 1H, ArH), 7.26 (d, J = 8.0 Hz, 1H, ArH), 4.26 (t, J = 12.0 Hz, 1H, CH), 2.57 (m,
13
2
H, CH ), 2.53-2.50 (m, 2H, CH ), 2.08 (s, 3H, CH ), 1.40 (s, 9H, CH ); C NMR
2 2 3 3
(
100 MHz, DMSO-d ) δ 171.67, 157.80, 156.03, 153.52, 147.09, 137.20, 128.93,
6
1
2
24.23, 119.28, 119.10, 117.05, 116.83, 115.78, 112.35, 78.71, 54.84, 31.98, 30.35,
+
8.68, 15.16; LC-MS m/z: 521.2 [M+H] ; HRMS(ESI) Calcd for C H ClFN NaO S
2
4
27
5
3
+
(
[M+Na] ):542.1405, found:542.1414.
4
.1.2.7 tert-butyl (2-oxo-2-((4-((3-(trifluoromethyl)phenyl)amino)quinazolin-6-yl)
amino)ethyl)carbamate(6g)
Off-white powder, yield: 38.5%. H NMR (400 MHz, CDCl ) δ 9.79 (s, 1H, NH),
1
3
8
.59 (s, 1H, ArH), 8.24 (s, 1H, ArH), 8.16 (d, J = 8.0 Hz, 1H, ArH), 7.77 (d, J = 4.0
Hz, 1H, ArH), 7.64 (s, 1H, NH), 7.50 (t, J = 8.0 Hz, 2H, ArH), 7.45 (s, 1H, NH), 7.38
d, J = 8.0 Hz, 1H, ArH), 7.28 (s, 1H, NH), 4.13 (d, J = 4.0 Hz, 2H, CH ), 1.45 (s, 9H,
(
2
13
CH ); C NMR (100 MHz, CDCl ) δ 157.05, 153.83, 146.62, 139.60, 135.31, 131.34,
3
3
1
8
31.03, 129.31, 128.51, 126.90, 125.45, 124.37, 122.74, 120.27, 118.11, 115.17,
+
1.42, 45.15, 28.29; LC-MS m/z: 462.2 [M+H] ; HRMS(ESI) Calcd for
+
C H F N NaO ([M+Na] ):484.1572, found:484.1615.
22
22
3
5
3

ACCEPTED MANUSCRIPT
4
.1.2.8 (R)-tert-butyl (1-oxo-1-((4-((3-(trifluoromethyl)phenyl)amino)quinazolin-6-yl)
amino)propan-2-yl)carbamate(6h)
1
Off-white powder, yield: 36.5%. H NMR (400 MHz, DMSO-d ): δ 10.31(s, 1H,
6
CONH), 10.10(s, 1H, CONH), 8.75(d, 1H, J=2.00Hz, NH), 8.59(s, 1H, Ar-H), 8.27(s,
1
H, Ar-H), 8.20(d, 1H, J=8.00Hz, Ar-H), 7.92(d, 1H, J=8.80Hz, Ar-H), 7.82(d, 1H,
J=8.80Hz, Ar-H), 7.62(t, 1H, J=8.00Hz, Ar-H), 7.46(d, 1H, J=8.00Hz, Ar-H), 7.20(d,
H, J=7.20Hz, Ar-H), 4.22(t, 1H, J=7.60Hz, CH), 1.40(s, 9H, CH ), 1.23(t, 3H,
1
3
1
3
J=7.20Hz, CH ); C NMR (100 MHz, CDCl ) δ 173.61, 156.96, 153.74, 146.69,
3
3
1
39.89, 135.80, 131.23, 130.91, 129.26, 128.55, 126.76, 125.52, 124.26, 122.81,
119.96, 117.98, 115.19, 111.45, 81.03, 52.78, 51.23, 45.88, 28.30, 18.08, 8.96;
+
+
ESI-MS 476.2 [M+1] . HRMS (ESI) Calcd for C H F N O ([M+Na] ):476.1904,
23
25
3
5
3
found:476.1913.
.1.2.9 (R)-tert-butyl
4
(3-methyl-1-oxo-1-((4-((3-(trifluoromethyl)phenyl)amino)
quinazolin-6-yl)amino)butan-2-yl)carbamate(6i)
1
Off-white powder, yield: 36. 0%. H NMR (400 MHz, CDCl ) δ 10.03 (s, 1H, NH),
3
8
.70 (s, 1H, ArH), 8.57 (s, 1H, ArH), 8.38 (d, J = 8.0 Hz, 1H, ArH), 8.31 (s, 1H, NH),
.87 (d, J = 8.0 Hz, 1H, ArH), 7.54 (t, J = 8.0 Hz, 1H, ArH), 7.40 (d, J = 12.0 Hz, 2H,
7
ArH), 7.33 (s, 1H, NH), 7.28 (s, 1H, ArH), 4.39 (t, J = 8.0 Hz, 1H, CH), 2.30-2.22 (m,
1
3
1
1
1
H, CH), 1.41 (s, 9H, CH ), 1.23-1.20 (m, 6H, CH ); C NMR (100 MHz, CDCl ) δ
3 3 3
57.51, 156.90, 153.76, 146.42, 139.97, 135.20, 131.28, 130.96, 129.32, 128.22,
26.84, 125.05, 124.09, 119.91, 115.07, 100.00, 81.27, 61.12, 30.82, 28.31, 19.28;
+
+
LC-MS m/z: 504.3 [M+H] ; HRMS(ESI) Calcd for C H F N NaO ([M+Na] ):
2
5
28
3
5
3
5
26.2024, found:526.2101.
.1.2.10 (R)-tert-butyl (4-methyl-1-oxo-1-((4-((3-(trifluoromethyl)phenyl)amino)
4
quinazolin-6-yl)amino)pentan-2-yl)carbamate(6j)
1
Off-white powder, yield: 40.0%. H NMR (400 MHz, CDCl ) δ 10.01 (s, 1H, NH),
3
8
.55 (d, J = 16.0 Hz, 2H, ArH), 8.36 (s, 1H, NH), 8.29 (d, J = 8.0 Hz, 1H, ArH), 7.86
d, J = 8.0 Hz, 1H, ArH), 7.54 (t, J = 8.0 Hz, 1H, ArH), 7.39 (d, J = 8.0 Hz, 2H, ArH),
.31 (s, 1H, NH), 7.28 (s, 1H, ArH), 4.63-4.58 (m, 1H, CH), 2.06 (t, J = 8.0 Hz, 2H,
(
7
1
3
CH ), 1.87-1.84 (m,1H, CH), 1.41 (s, 9H, CH ), 1.06 (dd, J = 8.0Hz, 6H, CH ); C
2
3
3

ACCEPTED MANUSCRIPT
NMR (100 MHz, CDCl ) δ 157.46, 156.78, 153.86, 146.76, 140.03, 135.29, 129.31,
3
1
26.81, 123.94, 119.84, 117.76, 115.06, 81.36, 54.38, 41.22, 28.33, 25.01, 21.42;
+
LC-MS m/z: 518.3 [M+H] ; HRMS(ESI) Calcd for C H F N NaO
2
6
30
3
5
3
+
(
[M+Na] ):540.2198, found:540.2285.
4
.1.2.11 (2R, 3S) -tert-butyl (3-methyl-1-oxo-1-((4-((3-(trifluoromethyl)phenyl)amino)
quinazolin-6-yl)amino)pentan-2-yl)carbamate(6k)
1
Off-white powder, yield: 37.7%. H NMR (400 MHz, CDCl ) δ 10.01 (s, 1H, NH),
3
8.65 (s, 1H, ArH), 8.57 (s, 1H, ArH), 8.37 (d, J = 8.0 Hz, 1H, ArH), 8.30 (s, 1H, NH),
7.87 (d, J = 8.0 Hz, 1H, ArH), 7.55 (t, J = 8.0 Hz, 1H, ArH), 7.43-7.40 (m, 2H, ArH),
7.36 (s, 1H, NH), 7.28 (s, 1H, ArH), 4.43 (t, J = 8.0 Hz, 1H, CH), 2.01-1.99 (m, 1H,
CH), 1.86-1.82 (m, 2H, CH ), 1.41 (s, 9H, CH ), 1.18 (d, J = 8.0 Hz, 3H, CH ), 1.04 (t,
2
3
3
1
3
J = 8.0 Hz, 3H, CH ); C NMR (100 MHz, CDCl ) δ 157.44, 156.94, 153.74, 146.35,
3
3
1
1
39.91, 135.20, 131.29, 130.97, 129.32, 128.19, 126.82, 125.54, 125.05, 124.16,
22.84, 117.95, 115.03, 99.99, 81.27, 60.23, 37.29, 28.30, 25.60, 15.66, 11.22;
+
+
LC-MS m/z: 518.3 [M+H] ; HRMS(ESI) Calcd for C H F N O ([M+H] ):518.2379,
2
6
31
3
5
3
found:518.2372.
.1.2.12 (R)-tert-butyl (4-(methylthio)-1-oxo-1-((4-((3-(trifluoromethyl)phenyl)amino)
4
quinazolin-6-yl)amino)butan-2-yl)carbamate(6l)
1
Off-white powder, yield: 42.0%. H NMR (400 MHz, CDCl ) δ 9.92 (s, 1H, NH),
3
8
8
7
.60 (s, 1H, ArH), 8.31-8.23 (m, 2H, ArH), 7.81 (d, J = 8.0 Hz, 1H, ArH), 7.54 (t, J =
.0 Hz, 2H, ArH), 7.47 (s, 1H, NH), 7.45 (s, 1H, NH), 7.40 (d, J = 8.0 Hz, 1H, ArH),
.28 (s, 1H, ArH), 4.70 (d, J = 4.0 Hz, 1H, CH), 2.78-2.73 (m, 2H, CH ), 2.32-2.27
2
1
3
(
m, 2H, CH ), 2.18 (s, 3H, CH ), 1.43 (s, 9H, CH ); C NMR (100 MHz, CDCl ) δ
2 3 3 3
1
1
1
57.25, 156.88, 153.92, 146.79, 139.77, 135.20, 131.02, 129.34, 128.69, 126.70,
25.04, 124.18, 122.79, 120.08, 117.90, 115.09, 81.50, 55.11, 32.21, 30.48, 28.35,
+
5.57; LC-MS m/z: 536.2 [M+H] ; HRMS(ESI) Calcd for C H F N NaO S
2
5
28
3
5
3
+
(
[M+Na] ):558.1763, found:558.1780.

ACCEPTED MANUSCRIPT
4
.1.2.13 tert-butyl (2-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-2-oxoethyl)
carbamate(6m)
1
Off-white powder, yield: 41.2%. H NMR (400 MHz, DMSO-d ): δ 10.27(s, 1H,
6
CONH), 9.94(s, 1H, CONH), 8.77(s, 1H, NH), 8.58(s, 1H, Ar-H), 8.16(t, 1H,
J=1.60Hz, Ar-H), 7.84-7.87(m, 2H, Ar-H), 7.81(d, 1H, J=9.20Hz, Ar-H), 7.28-7.37(m,
2
H, Ar-H), 7.15(t, 1H, J=6.00Hz, Ar-H), 3.81(d, 2H, J=6.40Hz, CH ), 1.41(s, 9H,
2
13
CH ); C NMR (100 MHz, CDCl ) δ 153.91, 146.79, 135.28, 130.08, 128.64, 126.74,
3
3
+
1
24.24, 122.45, 119.89, 115.21, 81.35, 28.33; ESI-MS 472.1 [M+1] ; HRMS(ESI)
+
Calcd for C H BrN O ([M+H] ):472.0979, found:472.0990.
2
1
23
5
3
4
.1.2.14 (R)-tert-butyl
(1-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-1-
oxopropan-2-yl)carbamate(6n)
1
Off-white powder, yield: 36.5%. H NMR (400 MHz, DMSO-d ): δ 10.35(s, 1H,
6
CONH), 10.27(s, 1H, CONH), 8.77(d, 1H, J=1.20Hz, NH), 8.15(s, 1H, Ar-H), 8.12(s,
1
2
1
1
1
H, Ar-H), 7.92(t, 1H, J=8.80Hz, Ar-H), 7.83(d, 2H, J=8.80Hz, Ar-H), 7.33-7.39(m,
H, Ar-H), 7.20(d, 1H, J=7.20Hz,Ar-H), 3.08-3.11(m, 1H CH), 1.40(s, 9H, CH3),
13
.32(d, 3H, J=7.20Hz, CH ); C NMR (100 MHz, CDCl ) δ 173.64, 156.89, 153.61,
3
3
46.19. 240.47, 135.66, 130.07, 128.24, 126.76, 126.69, 124.24, 122.43, 119.87,
+
15.04, 111.43, 81.23, 51.31, 28.33, 17.89, 8.60; ESI-MS 487.1 [M+1] ; HRMS(ESI)
+
Calcd for C H BrN O ([M+Na] ):486.1135, found:486.1148.
2
2
25
5
3
4
.1.2.15 (R)-tert-butyl
(1-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-3-
methyl-1-oxobutan-2-yl)carbamate(6o)
1
Off-white powder, yield: 36.2%. H NMR (400 MHz, DMSO-d ) δ 10.34 (s, 1H, NH),
6
9
.96 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.59 (s, 1H, ArH), 8.16 (s, 1H, NH), 7.91 (d, J =
.0 Hz, 1H, ArH), 7.85 (d, J = 8.0 Hz, 1H, ArH), 7.80 (d, J = 8.0 Hz, 1H, ArH), 7.35
8
(t, J = 8.0 Hz, 1H, ArH), 7.29 (d, J = 8.0 Hz, 1H, ArH), 6.99 (d, J = 8.0 Hz, 1H, ArH),
4
6
1
1
.02-3.98 (m, 1H, CH), 2.10-2.04 (m, 1H, CH), 1.41 (s, 9H, CH ), 0.96 (d, J = 4.0 Hz,
3
13
H, CH ); C NMR (100 MHz, DMSO-d ) δ 172.46, 171.52, 157.80, 156.08, 153.49,
3
6
47.14, 141.67, 137.09, 130.76, 128.94, 127.74, 126.34, 124.82, 121.59, 115.96,
+
12.38, 78.60, 63.95, 30.80, 28.69, 19.73; LC-MS m/z: 515.2 [M+H] ; HRMS(ESI)
+
Calcd for C H BrN NaO ([M+Na] ):538.1253, found:538.1260.
2
4
28
5
3

ACCEPTED MANUSCRIPT
4
.1.2.16
(R)-tert-butyl
(1-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-4-
methyl-1-oxopentan-2-yl)carbamate(6p)
1
Off-white powder, yield: 32.6%. H NMR (400 MHz, DMSO-d ) δ 10.31 (s, 1H, NH),
6
9
.95 (s, 1H, ArH), 8.69 (s, 1H, NH), 8.59 (s, 1H, ArH), 8.17 (s, 1H, NH), 7.94 (d, J =
.0 Hz, 1H, ArH), 7.86 (d, J = 8.0 Hz, 1H, ArH), 7.80 (d, J = 8.0 Hz, 1H, ArH), 7.35
8
(t, J = 8.0 Hz, 1H, ArH), 7.29 (d, J = 8.0 Hz, 1H, ArH), 7.13 (d, J = 8.0 Hz, 1H, ArH),
4
9
1
7
.23 (t, J = 12.0 Hz, 1H, CH), 2.52-2.50 (m, 2H, CH ), 1.73-1.68 (m, 1H, CH), 1.40 (s,
2
1
3
H, CH ), 0.94 (d, J = 8.0 Hz, 6H, CH ); C NMR (100 MHz, DMSO-d ) δ 172.46,
3
3
6
57.79, 155.96, 153.47, 137.32, 130.77, 126.31, 124.75, 121.59, 121.33, 115.95,
+
8.55, 53.96, 28.69, 23.46, 21.51; LC-MS m/z: 529.2 [M+H] ; HRMS(ESI) Calcd for
+
C H BrN NaO ([M+Na] ):550.1430, found:550.1423.
25
30
5
3
4
.1.2.17 (2R, 3S)-tert-butyl (1-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-3-
methyl-1-oxopentan-2-yl)carbamate(6q)
1
Off-white powder, yield: 28.0%. H NMR (400 MHz, DMSO-d ) δ 10.35 (s, 1H, NH),
6
9
.96 (s, 1H, ArH), 8.72 (s, 1H, NH), 8.59 (s, 1H, ArH), 8.16 (s, 1H, NH), 7.92 (d, J =
.0 Hz, 1H, ArH), 7.85 (d, J = 8.0 Hz, 1H, ArH), 7.80 (d, J = 8.0 Hz, 1H, ArH), 7.35
8
(t, J = 8.0 Hz, 1H, ArH), 7.29 (d, J = 8.0 Hz, 1H, ArH), 7.03 (d, J = 8.0 Hz, 1H, ArH),
4
.05 (t, J = 8.0 Hz, 1H, CH), 2.20-2.17 (m, 1H, CH), 1.56-1.53 (m, 2H, CH ), 1.40 (s,
2
1
3
9
H, CH ), 0.93 (d, J = 4.0 Hz, 3H, CH ), 0.87 (t, J = 8.0 Hz, 3H, CH ); C NMR (100
3
3
3
MHz, DMSO-d ) δ 171.66, 157.81, 155.98, 153.49, 141.68, 137.10, 130.76, 128.94,
6
1
1
26.33, 124.83, 121.59, 121.41, 115.97, 112.32, 78.57, 59.94, 36.74, 28.69, 25.13,
+
5.86, 11.23; LC-MS m/z: 529.2 [M+H] ; HRMS(ESI) Calcd for C H BrN NaO
2
5
30
5
3
+
(
[M+Na] ):550.1430, found:550.1427.
4
.1.2.18 (R)-tert-butyl
(1-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-4-
(methylthio)-1-oxobutan-2-yl)carbamate(6r)
1
Off-white powder, yield: 38.3%. H NMR (400 MHz, DMSO-d ) δ 10.34 (s, 1H, NH),
6
9.95 (s, 1H, ArH), 8.69 (s, 1H, NH), 8.59 (s, 1H, ArH), 8.17 (s, 1H, NH), 7.93 (d, J =
12.0 Hz, 1H, ArH), 7.86 (d, J = 8.0 Hz, 1H, ArH), 7.81 (d, J = 12.0 Hz, 1H, ArH),
7.35 (t, J = 8.0 Hz, 1H, ArH), 7.29 (d, J = 8.0 Hz, 1H, ArH), 7.25 (d, J = 8.0 Hz, 1H,

ACCEPTED MANUSCRIPT
ArH) 4.29-4.26 (m, 1H, CH), 2.60-2.56 (m, 2H, CH ), 2.52-2.50 (m, 2H, CH ), 2.09
2
2
1
3
(
s, 3H, CH ), 1.40 (s, 9H, CH ); C NMR (100 MHz, DMSO-d ) δ 172.46, 157.79,
3 3 6
1
56.03, 153.52, 147.14, 141.68, 137.21, 130.78, 128.90, 126.32, 124.76, 121.60,
115.93, 112.50, 99.99, 78.71, 63.95, 32.00, 30.36, 28.69, 15.16; LC-MS m/z: 547.2
+
+
[
M+H] ; HRMS(ESI) Calcd for C H BrN NaO S ([M+Na] ):570.0973,
24 28 5 3
found:570.0965.
4
.1.2.19
tert-butyl
(2-((4-((2,6-dimethylphenyl)amino)quinazolin-6-yl)amino)-2-
oxoethyl)carbamate(6s)
1
Off-white powder, yield: 50.9%. H NMR (400 MHz, DMSO-d ): δ 10.30(s, 1H,
6
CONH), 10.10(s, 1H, CONH), 9.54(s, 1H, NH), 8.67(s, 1H, Ar-H), 8.26(s, 1H, Ar-H),
7
2
.86(d, 1H, J=9.20Hz, Ar-H), 7.73(d, 1H, J=9.20Hz, Ar-H), 7.14(s, 4H, Ar-H), 3.83(d,
13
H, J=9.60Hz, CH ), 2.12(s, 6H, CH ), 1.40(s, 9H, CH ); C NMR (100 MHz,
2
3
3
CDCl ) δ 158.81, 154.26, 136.31, 128.26, 127.49, 126.51, 114.85, 45.97, 28.31, 18.42,
3
+
+
8
4
.64; ESI-MS 422.2 [M+1] ; HRMS(ESI) Calcd for C H N O ([M+Na] ):
23 28 5 3
22.2187, found: 422.2180.
4
.2 Cytotoxicity assay
The cytotoxicity of each compound was determined using sulforhodamine B assay.
Cells were seeded in 96-well plates and then treated with different concentrations of
drugs. After 72 h of incubation, cells were fixed with 10% trichloroacetic acid for 1 h
o
at 4 C, washed 5 times with tap water and air-dried. Cells that survived were stained
with 0.4% (w/v) sulforhodamine B (SRB) for 20 min at room temperature and washed
5
times with 1% acetic acid. Bound SRB was solubilized with 10mM Tris and
absorbance was measured at 540 nm.
.3 Kinase inhibition assays
4
Theses assays were carried out as described previously. All of the enzymatic reactions
o
were conducted at 30 C for 40 minutes. The 50µL reaction mixture contains 40mM
Tris, pH 7.4, 10mM MgCl , 0.1 mg/ml BSA, 1mM DTT, 10µM ATP, Kinase and the
2
enzyme substrate. The compounds were diluted in 10% DMSO and 5µl of the dilution
was added to a 50µL reaction so that the final concentration of DMSO is 1% in all of

ACCEPTED MANUSCRIPT
reactions. The assay was performed using Kinase-Glo Plus luminescence kinase assay
kit. It measures kinase activity by quantitating the amount of ATP remaining in
solution following a kinase reaction. The luminescent signal from the assay is
correlated with the amount of ATP present and is inversely correlated with the
amount of kinase activity. The IC values were calculated using nonlinear regression
5
0
with normalized dose-response fit using Prism GraphPad sofeware.
.4 Immunofluorescence assay
4
HepG2 cells were grown on glass cover slips sterilized and coated with poly-L-lysine.
The cells were treated with compound, and then were fixed with 4%
paraformaldehyde buffered in phosphate buffer solution (PBS) (15 min). After adding
5
5
4
00µL Hoechst 33258 staining solution, the cells were placed at room temperature for
min, washed by PBS. Fluorescence was determined using fluorescence microscopy.
.5 Flow cytometry analysis
5
Cells (5×10 cells/mL) were seeded in six-well plates and treated with compounds at
different concentrations for 72 h. The cells were then harvested by trypsinization and
washed twice with cold PBS. After centrifugation and removal of the supernatants,
cells were resuspended in 400µL of 1×binding buffer which was then added to 5µL of
annexin V-FITC and incubated at room temperature for 15 min. After adding 10µL of
PI the cells were incubated at room temperature for another 15 min in the dark. The
stained cells were analyzed by a flow cytometer.
4
.6 Mitochondrial membrane potential was observed under fluorescence microscopy
Cells were seeded in six-well plates and treated with compounds at different
concentrations. The cells were then harvested for 24h, and then were washed twice
with PBS (2 mL×2). After adding 1 mL JC-1 staining solution, the cells were
incubated at 37°C for 20 min, washed twice with JC-1 buffer solution, added 2mL
culture medium, and then were observed under fluorescence microscopy.
4
.7 Western blotting analysis
HepG2 cells were treated with compounds at different concentrations. Cells were then
harvested and washed in ice-cold PBS, cold RIPA buffer (contain 1% PMSF) for 30
min followed by sonication. After sonication cells were centrifuged at 14000 rpm at

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4
°C for 10 min and the supernatant was collected as whole cell lysate. Protein
concentration was determined by BCA method. Cell lysates containing equal amounts
of protein were separated by 12% SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose filter membrane. Membranes were blocked for 2 h at
room temperature, were incubated overnight at °C with primary antibodies in the
same buffer, and were then washed with washing buffer for 15 min. Membranes were
added secondary antibody, incubated for 2h, and then were then washed with washing
buffer for 15 min. Signal detection was accomplished by using chemiluminescence
(NBT/BCIP) kit.
4
.8 Docking studies
The X-ray crystal structure of EGFR (PDB code: 2ITY) was retrieved from protein
data bank (www.pdb.org). We used the AutoDock 4.2 Tools version 1.5.6 software
packages to perform the molecular docking experiment. Compound 6m was
energetically minimized by Amber 12 software before docking. Ligand docking was
carried out applying the Lamarckian genetic algorithm implemented in AutoDock 4.2.
The grid size was set to 40, 40 and 40 along the X-, Y- and Z-axis to recognize the
binding site. Spacing was set as 0.375 Å. Number of GA runs was set to 20.
Population size was set to 300. Maximum mumber of evals (medium) was set to
5
000000. The lowest binding energy conformers were selected out of 20 different
conformers for each docking simulation and resultant data was further analyzed.
Other miscellaneous parameters were assigned to the default values obtained from the
AutoDock 4.2 program.
Acknowledgment
The authors are grateful for the financial support by National Natural Science
Foundation of China (81602951); Natural Science Foundation of Jiangsu Province
(
BK20130216, BK20140225); Six talent peaks project in Jiangsu
Province(2016-SWYY-062); Science and technology project of Xuzhou City
KC16SG255); Jiangsu Key Laborotary of Brain Disease Bioinformation in Xuzhou
Medical College (Jsbl11103). Thank Dr. Xiao-Qing Wu (Departments of Molecular
(

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Biosciences and Radiation Oncology, University of Kansas) to edit for english
language.
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Highlights
ò We have devised and synthesized a series of novel 4-anilinoquinazoline
derivatives bearing amino acid moiety;
ò The synthesized compounds were screened for anticancer activity against human
hepatocellular carcinoma cell HepG2 using SRB assay. The target compounds
exhibited moderate inhibitory activities against human hepatocellular carcinoma
cells HepG2.
ò Synthetic derivatives showed excellent selectivity, such as compound 6m
demonstrated a strong inhibition of EGFR (IC =0.0032µM), with selectivity of
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0
over 2000-fold over other kinases, such as Aurora B, VEGF2, MEK, RAF and
c-MET;
ò
Apoptosis analysis revealed that compound 6m caused obvious induction of cell
apoptosis, decreased mitochondrial membrane potential through decreasing the
expression of Bcl-2 and increasing the expression of Bax, promoted the
mitochondrial cytochrome c release into the cytoplasm, activated caspase-3,
induced apoptosis of HepG2 cells..