Development of a series of novel 4-anlinoquinazoline derivatives possessing quinazoline skeleton: Design, synthesis, EGFR kinase inhibitory efficacy, and evaluation of anticancer activities in?vitro

Article abstract of DOI:10.1016/j.ejmech.2017.07.005

4-anilinoquinazoline-based derivatives represent an attractive scaffold for small molecular EGFR-TKIs in the field of medicinal chemistry. A series of novel heterocyclic substituted derivatives have been designed, synthesized and evaluated their antitumor

Full text of DOI:10.1016/j.ejmech.2017.07.005

Accepted Manuscript
Development of a series of novel 4-anlinoquinazoline derivatives possessing
quinazoline skeleton: Design, synthesis, EGFR kinase inhibitory efficacy, and
evaluation of anticancer activities in vitro
Jin Chang, Hongyu Ren, Mingxia Zhao, Yan Chong, Wenwen Zhao, Yong He,
Yunling Zhao, Huabei Zhang, Chuanmin Qi
PII:
S0223-5234(17)30527-5
10.1016/j.ejmech.2017.07.005
EJMECH 9568
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 August 2016
Revised Date: 1 July 2017
Accepted Date: 3 July 2017
Please cite this article as: J. Chang, H. Ren, M. Zhao, Y. Chong, W. Zhao, Y. He, Y. Zhao, H. Zhang, C.
Qi, Development of a series of novel 4-anlinoquinazoline derivatives possessing quinazoline skeleton:
Design, synthesis, EGFR kinase inhibitory efficacy, and evaluation of anticancer activities in vitro,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.005.
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ACCEPTED MANUSCRIPT

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Development of a series of novel 4-anlinoquinazoline derivatives possessing
quinazoline skeleton: Design, synthesis, EGFR kinase inhibitory efficacy, and
evaluation of anticancer activities in vitro
Jin Chang†, Hongyu Ren†, Mingxia Zhao§, Yan Chong†, Wenwen Zhao†, Yong He‡,
Yunling Zhao‡, Huabei Zhang†, Chuanmin Qi†, *
†Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of
Chemistry, Beijing Normal University, Beijing, 100875, P. R. China.
‡Experimental Chemistry Center, College of Chemistry, Beijing Normal University,
Beijing Normal University, Beijing, 100875, P. R. China.
§Shanxi Institute of Technology, Yangquan, Shanxi, 045000, P. R. China.
Corresponding Author:
*To whom correspondence should be addressed: Phone: +86-13681380097, Fax:
+86-10-58802075. E-mail: qicmin@sohu.com

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ABSTRACT
4-anilinoquinazoline-based derivatives represent an attractive scaffold for small
molecular EGFR-TKIs in the field of medicinal chemistry. A series of novel
heterocyclic substituted derivatives have been designed, synthesized and evaluated
their antitumor bioactivities as potential EGFR-TKIs. Most of the new compounds
exhibited certain efficient inhibition potency for proliferation of a panel of five human
cancer cells with IC50 values at the low micromolar level, and some of them possessed
good broad-spectrum inhibition activities, compared to Gefitinib. Especially, the IC50
values of compound 21 against HepG2, A549, MCF-7, DU145 and SH-SY5Y cells
were 4.61, 9.50, 9.80, 6.79 and 7.77 µM, respectively, which were much lower than
those of Gefitinb. Furthermore, the highlighting compound 21 demonstrated excellent
inhibition activity against EGFR-TK with the IC50 value of 3.62 nM, similar to that of
Gefitinib(2.21 nM). The results of LDH release assay proved that compound 21 was
anti-proliferative rather than cytotoxicity on HepG2 cells. Compound 21 were able to
cause HepG2 cells to block in S phase and induce cell death mainly by apoptosis
through a mitochondrial dependent pathway. Moreover, the assessment of MMP, the
determination of intracellular free Ca2+ concentration, the production of ROS, and the
effects on the activity of caspase-3 in a dose-dependent manner demonstrated that
compound 21 induced cell apoptosis in HepG2 cells through the Ca2+/ROS-mediated
mitochondria/caspase-dependent apoptosis pathway largely. These preliminary results
evidenced that compound 21 could be a potential antitumor agent deserving further
study.
Key Words: 4-anilinoquinazoline derivatives; antitumor; anti-proliferative;
EGFR-TK inhibitory; cell cycle; cell apoptosis; mechanism of apoptosis
1. Introduction
Malignant tumor, also called cancer, is consider to be one of the most difficult to
cure diseases around the world today [1]. Surgery, radiotherapy and chemotherapy are
three major options for the cancer treatment up to present. Chemotherapy drugs
provide a unique method for systemic treatment of cancer [2, 3]. A major challenge
for antitumor drugs is to design new drugs that will more selectively inhibit cancer
cells in order to avoid undesirable side effects on normal cells. Targeted cancer
therapies may be more effective than other types of current treatments, due to less
harmful to normal cells and more safe and efficient than conventional cytotoxic
chemotherapies [4, 5]. Molecular targets for selective inhibitors that play pivotal roles
in the initiation and development of tumors may obtain the greatest clinical benefit
with minimal side effects [6, 7]. Protein tyrosine kinases (PTKs) are involved in the
regulation of almost all major cellular functions, such as cell proliferation, growth,
metabolism, survival, differentiation and apoptosis [8-10]. Several PTKs are known to
be activated in response to many tumor cell growth factors extensively and facilitate
tumor growth and progression, which have emerged as a new potential and effective
therapeutic approach in antitumor therapy. Hence, tumor growth inhibition (TGI) by

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directly targeting RTKs without side effects may provide an attractive new strategy
for cancer chemotherapy.
The epidermal growth factor receptor (EGFR) functions in mediating proliferation,
differentiation, and survival of normal as well as tumor cells. EGFR over-expression
or over-activity have been associated with many tumor types, including non-small cell
lung, breast, ovarian and squamous cell cancers, leading to uncontrolled proliferation
and insensitivity to apoptotic stimuli [11-14]. Most targeted drug therapies extensively
explored in clinical trials are either small molecule tyrosine kinase inhibitors or
monoclonal antibodies. Small molecule EGFR-TKIs have become a major molecular
target for selective anticancer drugs with a much lower risk of side effects[15, 16].
The most potential small molecule selective EGFR-TKIs are derived from currently
three series of organic small molecules, which include 4-anilinoquinazoline
derivatives,
4-phenylaminopyrrolopyrimidine
4-[ar(alk)ylamino]pyridopyrimidine
derivatives[3].
derivatives
Most of
and
the
4-anilinoquinazoline-type compounds including the marketing of antitumor agents
such as Gefitinib, Erlotinib，Lapatinib and Vandetanib etc.(Fig. 1) have been shown to
produce clinical therapeutic benefit against a variety of tumors. Numerous
4-anilinoquinazoline TKIs also have been described in the published literature and
generally exhibit potent inhibitory activities in vitro at nanomolar concentrations[17].
The co-crystal structures of EGFR-TK with Gefitinib and Erlotinib have provided a
rich set of structural information for drug discovery efforts on EGFR-TKIs. The ATP
binding modes of these EGFR-TKIs have been mostly well elucidated [18, 19]. The
binding mode analysis of Gefitinib indicates several key interactions with the ATP
pocket of EGFR, None of the X-ray studies have indicated that the amine function at
C-4 is involved in binding. The solvent region generally tolerates a variety of polar
functional groups without affecting the efficacy too much. Changes and modifications
of side chains in the solvent zone can alter and influence the physicochemical
properties of the inhibitors[14].
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Fig. 1
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Inspired by the great potential and importance of these compounds, much attention
has been paid to the design and synthesis of novel 4-anilinoquinazoline derivatives
and/or functionalized derivatives. More recently, we have previously reported the
synthesis of a series of novel quinazoline nitrogen mustard derivatives, and their
potential antitumor activities. Studies on molecular mechanisms demonstrated that it
can induce the S phase cell cycle arrest and cell apoptosis in HepG2 cells[20]. To
enhance EGFR targeted therapies and ameliorate drug properties, side chain
modification at C-6 and/or C-7 of the quinazoline scaffold may be able to improve
physic-chemical properties and offer a more beneficial pharmacokinetic profile. In the
present research, our purpose was to utilize the quinazoline as skeleton for
construction of EGFR-TKIs, as part of our follow-up drug research effort to explore
the optimal alkyl substitution at the C-7 position, the substituents present at the

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fragment of the heterocycle, often called solubilizing tail, are directed to the entrance
of the ATP binding site. We have designed and synthesized a novel series of
4-anilinoquinazoline substituted compounds containing different water-solubilizing
groups to help improving the pharmacokinetic properties of these inhibitors and
evaluated their anti-proliferative against a panel of five human cancer cell lines of
different types by MTT assay as well as EGFR-TK inhibition activities by ELISA
assay. And then we further have studied the mechanism of inhibiting tumor growth
and inducing apoptosis of cancer cells.
2. Results and discussion
2.1. Chemistry
To discover and develop suitable small molecular ATP-competitive EGFR-TKIs as
potential anticancer drugs containing the 4-anilinoquinazoline pharmacophore for the
tumor treatment of selective targeting, based on the structure-activity
relationships(SAR) or quantitative structure-activity relationships(QSAR) of the
4-anilinoquinazolines reported previously, and forasmuch as Vandetanib and
Cediranib as lead compounds, we have modified the quinazoline core by using
various substituents at C-7 position or C-4 position and designed a series of novel
analogues: replacement of the benzene ring at C-4 position with cyanobenzene,
retaining methoxy group at C-6 position and introduction of heterocycle-substituted
propoxy side chain at C-7 position of the quinazoline nucleus to determine whether
these derivatives possess greater inhibition of cell proliferation and higher induction
of cell apoptosis.
In order to synthesize all target compounds, we used commercially available
compound as the starting materials as outlined in Scheme 1[21, 22]. The general
process is depicted as follows: Methyl vanillate 1 was selected as the starting material
for the preparation of intermediate compounds 2-5 by alkylation, nitration, catalytic
hydrogenation, cyclization, respectively. Compound 5 was reacted with phosphorus
oxychloride to obtain the key intermediate 4-chloroquinazolines 6. The compounds
7a-c was synthesized using the intermediate 6 with different substituted anilines. The
final products 8-41 were obtained by reaction of 7a-c with different aliphatic amines
in high yields. All the target compounds are listed in Table 1.
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Scheme 1
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Table 1
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2.2. Biological evaluation
2.2.1. In vitro anti-proliferative activity screening of target compounds
It is accepted wisdom that cellular evaluations of RTK inhibitory activities provide

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more meaningful results for in vivo studies than direct measurements of enzyme
activity. The antitumor drug discovery screen in vitro has been designed to distinguish
between broad-spectrum antitumor and selective antitumor agents. In a first screening,
the target compounds were screened for the anti-proliferative activities against a panel
of five human cancer cell lines, belonging to different tumor types, namely human
liver cancer cell lines (HepG2), human lung carcinoma cell lines (A549), human
breast carcinoma cell lines (MCF-7), human prostate cancer cell lines (DU145) and
human neuroblastoma cell lines (SH-SY5Y) at a concentration of 0.1-100 µM.
Gefitinib and QWL-22b(our group have reported) were used as positive control drugs.
All the compounds were investigated for their anti-proliferative activities in vitro by
the MTT assay after 72 h. The inhibitory activities (IC50 values) are summarized in
Table 2. The IC50 values that were higher than 100 µM in all cell lines were not
included.
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Table 2
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As depicted in Table 2, the cellular results indicated that most of the active analogs
exhibited distinctive potential selective or broad-spectrum antitumor activities in five
cancer cell lines (viz. NCI, Merryland, USA) by introduction of aminoalkyl at C-7
position of quinazoline nucleus, however, compounds 12, 13, 19 and 22 were totally
inactive. Concerning selectivity against individual cancer cell line, most of the
compounds showed effectiveness against HepG2 cells, displaying IC50 values range
of 0.77 to 29.50 µM compared to Gefitinib and QWL-22b (29.79 and 3.06 µM,
respectively). Compounds 21, 24, 26, 29, 38 and 40 were found to possess significant
anti-proliferative activity against HepG2 cells with IC50 values of 4.61, 8.84, 9.58,
0.77, 4.03 and 2.11 µM, respectively. Similarly, most of the compounds overall
inhibited the growth of SH-SY5Y cells with IC50 values ranging from 5.44 to 28.92
µM in comparison with Gefitinib and QWL-22b (18.21 and 8.60 µM, respectively).
Compounds 14, 16, 21, 24, 26, 32, 33 and 34 showed higher anti-proliferative
activities with IC50 values of 8.08, 7.90, 7.77, 9.81, 5.44, 7.33, 6.89 and 6.27 µM,
respectively. Interestingly, compound 27 significantly and selectively inhibited the
growth of A549 cells with IC50 value of 9.82 µM, which was superior to
Gefitinib(12.08 µM).
Meanwhile, with regard to broad-spectrum antitumor activity, the results revealed
that the active compounds 21 and 40 have effective growth inhibition of all cancer
lines in the preliminary screening. Compound 21 displayed the strong and
broad-spectrum anti-proliferative activities against various types of cell lines with
IC50 values range of 4.61 to 9.80 µM, which were relatively higher than those of
Gefitinib. Besides, compound 40 showed evident activities against various different
cell lines with IC50 values range of 2.11 to 9.07 µM.
As mentioned above, compound 21 exhibited broad-spectrum potent antitumor
activities against the tested five human cancer cell lines. We further evaluated
compound 21 as potential antitumor drug. The MTT method was applied to further

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detect the anti-proliferation activities of compound 21 against HepG2 cells and A549
cells in vitro. The results of the effects of treatment time- and dosage-dependent on
cell lines were showed in Tables 3, 4 and Figs. 2, 3. It was found that compound 21
exerted antitumor effects in a time- and dosage-dependent manner and the IC50 values
of 21 were 13.02, 9.56 and 5.29 µM after 24, 48 and 72 h, respectively. Significant
differences between groups were considered statistically significant at *P < 0.05.
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Table 3 and Table 4
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Fig. 2 and Fig. 3
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As can be seen in Table 5, compound 21 treatment at less than 10 µM for 48 h on
HepG2 cells and less than 10 µM for 72 h on A549 cells began to exhibit significant
inhibition effect compared with control, and the inhibition activity increased with the
increasing treatment time and dosage of target compound which was confirmed by
synchronous morphological observation. On the basis of these results, compound 21
was chosen according to the distinctive effect for further biological studies, including
LDH activities assay, cell cycle analysis, cell apoptosis induction, and fluorescence
image assay in order to reveal a more detailed picture on the possible
anti-proliferative and cell apoptosis induction mechanisms. Significant differences
between groups were considered statistically significant at *P < 0.05.
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Table 5
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The excellent results obtained from compound 21 against human cancer cells
prompted us to study the selectivity on human normal cell lines and cancer cell lines
to evaluate the toxic side effects. The anti-proliferative effects of compound 21 were
evaluated on CCC-HPF-1 cells and A549 cells using standard cell viability protocol.
As described in Table 6, there was excellent selectivity in cell viability with IC50
values of 27.97 and 9.50 µM, respectively, which were similar to those of Gefitinib
(30.78 and 14.08 µM, respectively). Generally, it was evident compound 21 showed
selective anti-proliferative activities on human normal cells and cancer cells to some
extent. Significant differences between groups were considered statistically
significant at *P < 0.05.
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Table 6
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2.2.2. Structure-activity relationships and molecular docking
The changes of biological activities of the tested compounds could be correlated to

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the structure variations and/or modifications. Some general trends concerning
structure-activity relationships observed from the results on the title compounds are as
follows: With the introduction of a substitution at position 7- in the quinazoline
skeleton, we did obtain some active derivatives even if the IC50 values against five
tested cancer cells were much higher than those of Gefitinib. In particular, the
derivatives
bearing
a
4-phenyl-piperidinyl,
4-piperidinyl-piperidinyl,
4-ethyl-piperazinyl, and pyrrolyl in C-7 position quinazoline ring linked by propyl
side chain resulted the higher anti-proliferative activities on HepG2 cells. The
introduction of a cyano amino group at C-4 position quinazoline core led to some
compounds with IC50 values on HepG2 cells lower than those of Gefitinib. However,
no significant increase in the anti-proliferative activities was observed from
compounds 12, 13, 19 and 22 by introducing of methylbenzylamino,
hydroxyethylbenzylamino, 2-hydroxyethyl-piperidinyl and tetrahydroisoquinolyl
group in C-7 position quinazoline ring linked by propyl side chain respectively
exhibited greater reduction in the inhibitory activity. It may be due to the rigidity of
the benzene ring and the steric hindrance of the substituents. Compounds 17 and 18
bearing 4-hydroxypiperidinyl and 4-hydroxyethylpiperidinyl group at R2, exhibited
significant decrease in the anti-proliferative activities (IC50 values ranging from 21.62
µM to more than 100 µM toward HepG2 cells), which might be induced by the
electro-negativity of the oxygen atoms by our conjecture.
To explain the function of the 4-amino group in protein-ligand binding, the size and
electronic properties of substituent have been varied by cyano group. Moreover,
modifications of substituent position might reveal favorable interactions and size
restrictions in this part of the binding pocket. The test results were summarized in
Table 2. The initial plan to improve potency was to scan for possible hydrogen
bonding interactions and lipophilic contacts. Substituents varied in the 4-amino group
linked cyano group were to seek for possible lipophilic contacts. As compared to
Gefitinib, the potency of inhibitory activities against HepG2 cells was increased from
29.79 to 2.11 µM by introducing a cyano group including para-, meta- and ortho-
substituents in aniline ring. Compounds 26, 34 and 40 with cyano substituent at
different position showed distinct inhibitory activity against HepG2 cells. Compound
40 with ortho-cyano substituent showed more potent activity (IC50 = 2.11 µM) than
compound 26 with para-cyano substituent (IC50 = 9.58 µM). The activity of the latter
was higher than compound 34 with meta-cyano substituent (IC50 = 13.69 µM). The
results suggested that substituents in different positions led to different inhibitory
activities. This rule was also found in other substituted compounds. It also
demonstrated that introduction of certain substituents at para- or ortho- position is
more favorable than at meta- position. It may be related to whether it is more
beneficial to form a hydrogen bond with the amino acid residues of the skeleton of the
hydrophobic cavity. All the above SARs were also suitable for the inhibitory activities
of these compounds against A549 cells. However, the inhibitory activities against
A549 cells were much weaker than those of HepG2 cells.
For further elaboration of the SARs observed at EGFR-TK, molecular docking
study of compound 21 into the active site of EGFR-TK was performed using

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AutoDock 4.2.6 based on EGFR complex structure with Erlotinib (PDB code: 1M17).
As depicted in Fig. 4, compound 21 is nicely bound to the ATP binding site of
EGFR-TK. Compared to the binding model of Erlotinib, which N1of the quinazoline
accepted an H-bond from Met769 and N3 formed an H-bond bridge formation
through H2O with Thr776; compound 21 occupied the same EGFR-TK binding
pocked. Specially, there exists an H-bond with O atom at C-7 position interaction with
Cys773, which might be favor to the EGFR-TK inhibitory activity. The model also
showed that there is a π-π interaction and hydrophobic interaction between benzene
ring in the side chain in the equatorial position and Phe771 and Tyr777, which maybe
bring improved the EGFR-TK inhibitory activity.
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Fig. 4
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2.2.3 In vitro EGFR-TK inhibitory activity assay
Aiming to estimate the targeted effects of the designed compounds as TKIs, kinase
inhibitory activity of compound 21 was evaluated through EGFR-TK activity assay
by ELISA. Gefitinib, with high selectivity and potency to EGFR-TK, was chosen as a
positive drug in our kinase assay. The experimental results were showed in Table 7.
The inhibitory activities are given as OD values and percentage inhibition at various
concentrations of the compound 21. The concentration-dependent inhibitory curves
were shown in Fig. 5 and the IC50 values were listed in Table 8. Specifically speaking,
compound 21 displayed potent inhibitory activity at nanomolar concentration level
with a dose-dependent manner (IC50 = 3.62 nM for EGFR-TK), which is slightly
weaker comparable to the positive control Gefitinib (IC50 = 2.21 nM for EGFR-TK).
*
Differences between groups were considered statistically significant at P < 0.05.
Therefore, we could conclude that compound 21 can inhibit the function of EGFR-TK
and the anti-proliferative activity was produced partly by the interaction between
EGFR-TK and the tested compound.
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Table 7
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Fig. 5
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Table 8
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2.2.4 LDH activities detecting the cytotoxicity of compound 21

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The cytotoxicity of compound 21 against HepG2 cells was tested by LDH release
activities at 48 h, and Gefitinib was used as a positive control drug. The tested results
are shown in Table 9 and Fig.6. Comparing to full release group and natural release
group, it is found that the cytotoxicity on HepG2 cells of compound 21 was very low
at different concentrations, and the change of the value with the concentration is not
obvious, which is similar to that of Gefitinib. It was confirmed that compound 21 was
anti-proliferative rather than cytotoxicity on HepG2 cells and its mechanism of action
may be similar to Gefitinib. Significant differences between groups were considered
statistically significant at *P < 0.05.
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Table 9
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Fig. 6
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2.2.5 Cell cycle analysis on HepG2 cells by flow cytometry
In order to better elucidate that the mechanism of proliferation inhibition was
associated with cell cycle arrest, cell cycle distribution on HepG2 cells by treating
with various concentrations (0, 2.5, 5.0 and 10.0 µM) of compound 21 was performed.
As depicted in Table 10 and Fig. 7-8, the region marked with different colors
represents % population at different phases of the cell cycle. Significant changes in
cell cycle profile can be detected through a concentration-dependent manner increase
of cells in S phase together with a concomitant decrease of G0/G1 phase cells after 48
h of incubation, which is consistent with the one of the most efficient antitumor drug
Gefitinib, as a reference.
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Table 10
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Fig. 7 and Fig. 8
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Concretely speaking, compound 21 caused an increase in the proportion of cells in
S phase (from 21.46% in the control to 29.72% at 10.0 µM) in a
concentration-dependent manner with a concomitant decrease of cells in G0/G1 phase
of the cell cycle (from 69.40% in the control to 60.72% at 10.0 µM). These results
confirmed that compound 21 arrested cell cycle progression dramatically on HepG2
cells, which eventually inhibited tumor cell proliferation in a dose-dependent manner
by inducing S growth arrest and reducing the G0/G1 phase of the cell cycle and

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resulted in cell apoptosis.
2.2.6 Induction of apoptosis assay on HepG2 cells
There are three types of programmed cell death in biological systems, namely
autophagy, necrosis and apoptosis. Apoptosis is a series of biochemical events leading
to morphological changes and cell death. It has been observed that many cytotoxic
drugs show anticancer activities by inducing cell apoptosis. To further explain the
inhibition of cell growth, we first evaluated compound 21-induced apoptosis on
HepG2 cells using AnnexinV-FITC and propidium iodide (PI) double staining by flow
cytometry, which stain phosphatidylserine residues and DNA, respectively. The
results were shown in Table 11 and Fig. 9-10.
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Table 11
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Fig. 9 and Fig. 10
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As can be seen, compound 21 is effective in induction of cell apoptosis in a
dose-dependent manner. Treatment of HepG2 cells by 0, 2.5, 5.0 and 10.0 µM of
compound 21 for 48 h results in 52.4%, 82.8% and 98.7% of apoptotic cells (early +
late), as compared to 13.5% of apoptotic cells in an untreated control. This is
consistent with its nice binding affinity to EGFR-TK and potent activity in inhibition
of cell growth. These results revealed that compound 21 inhibited cell growth through
cell apoptosis induction.
2.2.7 Morphology of cells by Fluorescence microscopy
Flow cytometry alone cannot conclusively identify apoptotic cells, the results were
further corroborated by studying nuclear morphological changes of cells by
fluorescence microscopy. To further confirm the induction of apoptosis, we monitored
the morphological changes induced by compound 21 on HepG2 cells for Hoechst
33242 and PI double staining. After exposure to the various concentrations of
compound 21 for 48 h, the HepG2 cells showed significant evidence of apoptosis
mediated cell death, including chromatin condensation and nuclear fragmentation.
Cell morphology was investigated using fluorescence microscopy. As reflected in Fig.
11, it can be observed that the control cells showed the homogeneous blue
fluorescence in the nuclei without red-orange fluorescence as these cells cannot take
up PI staining solution, which suggested that there was no obvious nuclear
condensation and fragmentation in HepG2 cells. However, when the cells treated with
compound 21, the nuclei became convoluted and budded off into several fragments,
and nuclear condensation and fragmentation were observed in the pictures, indicating
a typical characteristic of apoptosis. Moreover, the treated cells exhibited red-orange
fluorescence due to staining with PI because apoptotic cells with ruptured membrane
allow the entry of PI to intercalate into DNA. Furthermore, it is found that more and

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more cells stained in dark blue were observed under the microscope as the increase of
concentration from 2.5 to 20 µM. It is suggested that compound 21 induce the cellular
apoptosis dose-dependently, which is consistent with the former anti-proliferation
activity assay.
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Fig. 11
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2.2.8 Effect on mitochondrial membrane potential loss
Impairment in mitochondrial function is an early event in the executive phase of
programmed cell death in various cell types, and appears as the result of a preliminary
decrease in the mitochondrial membrane potential(MMP), which is crucial for
caspase-dependent and caspase-independent apoptotic cell death pathways. The effect
of compound 21 on MMP loss in HepG2 cells was performed. The MMP loss in
untreated and treated groups is measured by Rhodamine-123 dye (Rh-123) which is
reduced by healthy mitochondria into fluorescent probe whose fluorescence is
measured by flow cytometry. As depicted in Table 12, Fig. 12-13, compound 21
treated HepG2 cells for 48 h showed loss of MMP with 2.1%, 3.4%, 6.4% and 34.5%
at 2.5, 5, 10 and 20 µM, respectively. The results indicated that the rationality and
possibility of mitochondria-mediated cell death. Significant differences between
groups were considered statistically significant at *P < 0.05, **P < 0.01.
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Table 12
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Fig. 12 and Fig. 13
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2.2.9 Detection of the intracellular level of Ca2+ concentration
Ca2+ as a second messenger for cellular functions may play an important role in the
regulation of apoptosis through mitochondria-mediated apoptosis pathway, which can
activate caspase-3 that direct executors of apoptosis, and induce cell apoptosis. The
intracellular free Ca2+ concentration was detected using the fluorescence Ca2+
indicator Fluo-3/AM, and the results were shown in Table 13 and Fig. 14. Statistical
analysis showed that the concentration of intracellular free Ca2+ in each drug
treatment group increased obviously compared with the negative control group (from
41.7% to 52.7% vs. 14.2% of the negative control group), indicating that compound
21 can cause the rise of intracellular free Ca2+ concentration in the early phase of
apoptosis with a concentration dependent manner, which can indicate the opening of
mitochondrial permeability transition pore. These results suggested that Ca2+ may be
involved in signal transduction in apoptotic cell death in HepG2 cells via
Ca2+-mediated mitochondrial damage. Significant differences between groups were

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considered statistically significant at *P < 0.05, **P< 0.01, ***P < 0.001.
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Table 13
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Fig. 14
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2.2.10 Detection of the intracellular level of ROS
Reactive oxygen species (ROS) may also function as secondary messengers in cell
signaling transduction pathways and cell cycle events, which are associated with the
apoptosis mediated by mitochondria, endoplasmic reticulum and death receptor. The
production of ROS was detected by the DCFH-DA capture agent to capture ROS. The
experimental results were presented in Fig. 15. The green fluorescence intensity of
DCF that was produced by DCFH through the oxidization by ROS was proportional
to the level of ROS. The results showed that the production of ROS levels were
significantly increased in HepG2 cells treated with different concentrations of the
target compound 21 for 48 h in comparison with the negative control group, and
indicated that compound 21 treated with HepG2 cells can induce the elevation of ROS
levels with a concentration-dependent manner, which may have an influence on the
changes of MMP, and may be involved in ROS-mediated cell apoptosis through
mitochondrial pathway.
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Fig. 15
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2.2.11 Detection of intracellular caspase-3 activity
Cysteinyl aspartate-specific proteases (caspase) show a crucial role in the process
of cell apoptosis. Particularly, caspase-3 is called executer of cell apoptosis and an
important part of the molecular mechanism of apoptosis, which is related to the
fragmentation of DNA, the accumulation of chromatin condensation and formation of
apoptosome. To further confirm the induction of apoptosis by compound 21, the
activation of caspase-3 in the apoptotic cells was carried out. In order to determine the
degree of activation of caspase-3, we used the caspase-3 sequence-specific peptide
substrate coupled to chromophore group, when the substrate was cut by caspase-3 in
the cells, the chromophore was free, the absorbance was measured by a microplate
reader, and the results were shown in Table 14 and Fig.16. Statistical analysis results
showed that caspase-3 activities were significantly enhanced by different
concentrations of compound 21 in HepG2 cells for 48 h in a concentration dependent
manner, compared with the negative control group. These results indicated caspase-3
is activated during apoptosis and compound 21 selectively inhibits proliferation of
HepG2 cells via activating caspase-3-mediated apoptosis. Significant differences

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between groups were considered statistically significant at *P< 0.05, **P < 0.01.
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Table 14
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Fig. 16
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3. Conclusions
In this work, a series of novel 4-aminoquinazoline derivatives as potent
EGFR-TKIs have been reported. The results showed that most compounds displayed
certain inhibition activities against the selected human cancer cells, and some
compounds exhibited better broad-spectrum inhibition activities than Gefitinb and
QWL-22b. It can be observed that the presence of substituents and their properties
strongly influenced the anti-proliferative activities. The IC50 values of compound 21
against HepG2, A549, MCF-7, DU145 and SH-SY5Y cells were much lower than
those of Gefitinb, however, the IC50 value of compound 21 against CCC-HPF-1 cells
was similar to that of Gefitinib. Compound 21 demonstrated more potent EGFR-TK
inhibitory activity with the IC50 of 3.62 nM, similar to that of Gefitinib (2.21 nM).
LDH release assay proved that compound 21 was anti-proliferative rather than
cytotoxicity in HepG2 cells. Compound 21 caused S phase arrest and induced cell
apoptosis to inhibit cell proliferation. Effects of MMP in HepG2 cells, and the
determination of intracellular free Ca2+ concentration, ROS and caspase-3 activity
indicated that compound 21 could induce apoptosis through mitochondria-mediated
apoptotic pathway and caspase-3-mediated mechanism. In general, compound 21
exerts potential antitumor activity through several mechanisms, including
anti-proliferation, cell cycle arrest and pro-apoptotic effects via the
mitochondrial-mediated caspase-dependent pathway, and may serve as model
molecule to help us to further design and develop more potent anticancer agents, and
also further investigate their mechanisms of action.
4. Experimental sections
4.1. Synthesis and characterization of target compounds
4.1.1. General methods/instruments
All commercially available starting materials, reagents and solvents were used
without further purification. All the reactions were routinely monitored by thin-layer
chromatography (TLC) in silica gel (GF254 Merck plates) and the products visualized
with ultraviolet lamp (254 nm and 365 nm). Column chromatography separations
were obtained on silica gel (200-300 mesh). Melting points were recorded on a RY-1
1
13
melting point apparatus and are uncorrected. H NMR and C NMR spectra were
determined in DMSO-d6 solutions using a Bruker Avance 400 MHz spectrometer with
TMS as the internal standard, and the values of the chemical shifts (δ) were given in

ACCEPTED MANUSCRIPT
ppm, coupling constants values (J) are given in Hz. Mass spectra (MS) were
performed on a LCT Premier XE (ESI) or 320-MS (EI) mass spectrometry. Chemical
names were created using ChemBioDraw Ultra 12.0 software.
4.1.2. Synthetic procedures
4.1.2.1. Synthesis of methyl 4-(3-chloropropoxy)-3-methoxybenzoate (2)
To a stirred solution of methyl vanillate 1 (10.0 g, 0.055 mol) in DMF (300 mL)
were added K2CO3 (10.4 g, 0.066 mol) and 1-bromo-3-chloropropane (11.4 g, 0.082
mol). The mixture was heated at 60 for 5 h. After the reaction mixture was cooled to
room temperature and poured into ice water (400 mL). After being stirred for 1 h, the
respective product was filtered off, washed with ice water and dried to obtain the
crude intermediate 2 as a white solid, which was used without purification. Yield:
97%, m.p.: 98-100 .
4.1.2.2. Synthesis of methyl 4-(3-chloropropoxy)-5-methoxy-2-nitrobenzoate (3)
To a stirred solution of crude 2 (10.0 g, 0.039 mol) in AcOH (35 mL) was slowly
added 65% HNO3 (8 mL) at 0-5 .Subsequently, the mixture was heated to 50 for 5
h, then cooled to room temperature and poured into ice water(300 mL). After being
stirred for 1 h, the product was extracted with ethyl acetate (3×25 mL), the combined
organic layer was washed with water, saturated sodium bicarbonate and saturated
sodium chloride, and then dried with anhydrous Na2SO4. The ethyl acetate was
removed under reduced pressure to afford the nitration product 3 as yellow oil. Yield:
88%, m.p.: 64-66 .
4.1.2.3. Synthesis of methyl 2-amino-4-(3-chloropropoxy)-5-methoxybenzoate (4)
A solution of 3 (6.0 g, 0.02 mol) in AcOH (35 mL) was stirred for 30 min at 80 ,
and powdered iron (3.5 g, 0.06 mol) was added. The mixture was stirred for another
40 min at 80 , and then the mixture was slowly poured into ice water and extracted
with ethyl acetate (3×30 mL). The organic layer was washed with water, saturated
sodium bicarbonate and saturated sodium chloride, and then dried with anhydrous
Na2SO4. The solvent was evaporated under reduced pressure to yield reduction
product 4. The brown solid residue was recrystallized from ethyl acetate/petroleum
ether to give a white solid. Yield: 82%, m.p.: 97-99 .
4.1.2.4. Synthesis of 7-(3-chloropropoxy)-6-methoxyquinazolin-4(3H)-one (5)
A mixture of formamidine acetate (5.0 g, 38 mmol), and crude 4 (5.0 g, 18 mmol)
in EtOH (40 mL) was heated at reflux for 6 h. The mixture was cooled to room
temperature, and then the precipitate was collected by filtration, washed with cold
ethanol and dried to get 5 as a white powder. Yield: 90%, m.p.:218-220 .
4.1.2.5. Synthesis of 4-chloro-7-(3-chloropropoxy)-6-methoxyquinazoline (6)
The mixture of 5 (2.0 g, 7.5 mmol) and POCl3 (10 mL) was stirred at reflux for 4 h.
The solution was concentrated under reduced pressure to dryness and excess ethyl
acetate was added. The solution was washed with water and brine, dried with
anhydrous Na2SO4 and concentrated to afford the pure compound 6 as a yellow solid.
Yield: 95%, m.p.:126-128 .
4.1.2.6. General process for the synthesis of 7a-c
To a stirred solution of 6 (1.0 g, 3.5 mmol) in isopropanol (20 mL) was added
aminobenzonitrile (0.5 g, 4.3 mmol). The mixture was heated under reflux for 2 h and

ACCEPTED MANUSCRIPT
cooled to room temperature. The light-yellow solid was filtered, washed and dried to
yield compounds 7a-c.
4.1.2.6.1. 4-((7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl)amino)benzonitrile (7a).
Yield: 95%, m.p.:126-128 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, NH),
8.58 (s, 1H, ArH), 8.13 (s, 1H, ArH), 8.11 (s, 1H, ArH), 7.85-7.82(m, 3H, ArH), 7.27
(s, 1H, ArH), 4.28 (t, J = 8.0 Hz, 2H, -OCH2CH2CH2Cl), 3.99 (s, 3H, -OCH3), 3.82 (t,
J = 8.0 Hz, 2H, -OCH2CH2CH2Cl), 2.30-2.24 (m, 2H, -OCH2CH2CH2Cl). ESI-MS:
+
m/z calcd found 370.13 [M+H] . Anal. calcd: C 61.84, H 4.63, Cl 9.63, N 15.21, O
8.69.
4.1.2.6.2. 3-((7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl)amino)benzonitrile (7b).
Yield: 93%, m.p.:126-128 . 1H NMR (400 MHz, DMSO-d6): δ 9.68 (s, 1H, NH),
8.55 (s, 1H, ArH), 8.37 (s, 1H, ArH), 8.13 (d, J = 8.1 Hz, 1H, ArH), 7.84 (s, 1H, ArH),
7.55(dd, J = 21.1, 7.8 Hz, 2H, ArH), 7.24 (s, 1H, ArH), 4.27 (s, 2H,
-OCH2CH2CH2Cl), 3.98 (s, 3H, -OCH3), 3.82 (s, 2H, -OCH2CH2CH2Cl), 2.26 (m, 2H,
+
-OCH2CH2CH2Cl). ESI-MS: m/z calcd found 370.15 [M+H] . Anal. calcd: C 61.83,
H 4.65, Cl 9.61, N 15.22, O 8.69.
4.1.2.6.3. 2-((7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl)amino)benzonitrile (7c).
Yield: 90%, m.p.:126-128 . 1H NMR (400 MHz, DMSO-d6): δ 9.65 (s, 1H, NH),
8.48 (s, 1H, ArH), 8.13 (d, J = 8.7 Hz, 1H, ArH), 8.06 (d, J = 8.04 Hz, 1H, ArH),
7.62-7.58 (m, 2H, ArH), 7.26 (s, 1H, ArH), 7.20 (s, 1H, ArH), 4.17 (s, 2H,
-OCH2CH2CH2Cl), 3.92 (s, 3H, -OCH3), 3.62 (s, 2H, -OCH2CH2CH2Cl), 2.12 (m, 2H,
+
-OCH2CH2CH2Cl). ESI-MS: m/z calcd found 370.13 [M+H] . Anal. calcd: C 61.89,
H 4.64, Cl 9.62, N 15.22, O 8.62.
4.1.2.7. General process for the synthesis of the title compounds (8-41)
To a stirred solution of compounds 7a-c (0.25 g, 0.68 mmol) in DMF (5 mL) was
added K2CO3 (1.5:1), KI (0.01:1) and various substituted aniline (1.5:1). The solution
was heated at 80 for 6-10 h. Then the reaction mixture was cooled to room
temperature, diluted with water (200 mL) and extracted with ethyl acetate (3×20 mL),
combined organic layer, washed with brine, dried with anhydrous Na2SO4 and
concentrated under vacuum to afford the target products as a solid. Compounds 8-41
were characterized as follows.
4.1.2.7.1.
4-((7-(3-(dimethylamino)propoxy)-6-methoxyquinazolin-4-yl)amino)benzonitrile (8)
Yield: 88%, m.p.:170-172 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, NH),
8.58 (s, 1H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 8.04 (m, 3H, ArH), 7.27 (s, 1H,
ArH), 4.28 (m, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 2.69 (m, 2H,
13
-NCH2CH2CH2O-), 2.18 (s, 6H, -CH3), 1.85 (m, 2H, -OCH2CH2CH2N-). C NMR
(100 MHz, DMSO-d6): δ 158.61, 156.59, 156.01, 149.85, 148.49, 144.85, 131.05,
126.71, 113.06, 108.81, 106.09, 102.76, 69.87, 56.06, 54.66, 49.24, 26.78. ESI-MS:
m/z calcd found 378.13 [M+H] +. Anal. calcd: C 66.85, H 6.16, N 18.53, O 8.46.
4.1.2.7.2.
4-((7-(3-(diethylamino)propoxy)-6-methoxyquinazolin-4-yl)amino)benzonitrile
(9)
Yield: 90%, m.p.:178-181 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, NH),
8.58 (s, 1H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 8.03 (m, 3H, ArH), 7.23 (s, 1H,

ACCEPTED MANUSCRIPT
ArH), 4.28 (m, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 2.89 (m, 4H,
-NCH2CH3), 2.59 (m, 2H, -NCH2CH2CH2O-), 1.87 (m, 2H, -OCH2CH2CH2N-), 1.01
(t, 6H, -NCH2CH3). 13C NMR (100 MHz, DMSO-d6): δ 158.82, 156.67, 156.53,
149.99, 148.87, 145.05, 131.65, 126.91, 113.24, 108.99, 106.19, 102.89, 71.09, 57.13,
54.86, 52.97, 26.78, 15.46. ESI-MS: m/z calcd found 406.33 [M+H] +. Anal. calcd: C
68.14, H 6.76, N 12.26, O 7.84.
4.1.2.7.3.
4-((7-(3-((2-hydroxyethyl)(methyl)amino)propoxy)-6-methoxyquinazolin-4-yl)amino)
benzonitrile (10) Yield: 83%, m.p.:218-220 . 1H NMR (400 MHz, DMSO-d6): δ 9.78
(s, 1H, NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.5 Hz, 2H, ArH), 8.03 (m, 3H, ArH), 7.23
(s, 1H, ArH), 4.18 (m, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 3.46 (t, 2H,
-CH2OH), 2.45-2.61 (m, 4H, -NCH2-), 2.21(s, 3H, -NCH3), 1.89 (m, 2H,
-OCH2CH2CH2N-). 13C NMR (100 MHz, DMSO-d6): δ 158.57, 156.48, 155.76,
149.54, 148.23, 144.35, 130.77, 126.15, 113.01, 108.62, 106.03, 102.21, 69.56, 58.89,
+
55.84, 54.04, 53.03, 48.77, 26.65. ESI-MS: m/z calcd found 408.23 [M+H] . Anal.
calcd: C 64.87, H 6.17, N 17.20, O11.76.
4.1.2.7.4.
4-((7-(3-(bis(2-hydroxyethyl)amino)propoxy)-6-methoxyquinazolin-4-yl)amino)benz
onitrile (11) Yield: 73%, m.p.:162-164 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s,
1H, NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.5 Hz, 2H, ArH), 8.03 (m, 3H, ArH), 7.23 (s,
1H, ArH), 4.18 (m, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 3.46-3.52 (m, 4H,
-CH2CH2OH), 2.45-2.63 (m, 6H, -NCH2-), 1.87 (m, 2H, -OCH2CH2CH2N-). 13C
NMR (100 MHz, DMSO-d6): δ 158.68, 156.56, 155.79, 149.68, 148.57, 144.51,
130.82, 126.35, 113.23, 108.77, 106.34, 102.45, 70.53, 58.09, 55.25, 54.87, 54.34,
+
26.89. ESI-MS: m/z calcd found 438.20 [M+H] . Anal. calcd: C 63.13, H 6.25, N
16.02, O 14.60.
4.1.2.7.5.
4-((7-(3-(benzyl(methyl)amino)propoxy)-6-methoxyquinazolin-4-yl)amino)benzonitri
le (12) Yield: 84%, m.p.:234-236 . 1H NMR (400 MHz, DMSO-d6): δ 9.71 (s, 1H,
NH), 8.48 (s, 1H, ArH), 8.03 (q, 2H, ArH), 7.79 (s, 1H, ArH), 7.73 (q, 2H, ArH), 7.42
(t, 2H, ArH), 7.34 (m, 2H, ArH), 7.26 (m, 2H, ArH), 5.21 (s, 2H, ArCH2-), 4.18 (m,
2H, -OCH2CH2CH2N-), 3.89 (s, 3H, -OCH3), 2.63 (m, 2H, -NCH2-), 2.56 (s, 3H,
-NCH3), 1.87 (m, 2H, -OCH2CH2CH2N-). 13C NMR (100 MHz, DMSO-d6): δ 158.76,
156.66, 156.01, 152.85, 148.49, 145.85, 139.18, 131.15, 128.71, 127.83, 127.56,
119.78, 113.86, 108.89, 106.27, 102.88, 69.87, 62.89, 55.03, 53.36, 42.34, 25.97.
ESI-MS: m/z calcd found 454.22 [M+H] +. Anal. calcd: C 71.52, H 6.01, N 15.42, O
7.05.
4.1.2.7.6.
4-((7-(3-(benzyl(2-hydroxyethyl)amino)propoxy)-6-methoxyquinazolin-4-yl)amino)b
enzonitrile (13) Yield: 87%, m.p.:215-218 . 1H NMR (400 MHz, DMSO-d6): δ 9.83
(s, 1H, NH), 8.48 (s, 1H, ArH), 8.03 (d, 2H, ArH), 7.83 (s, 1H, ArH), 7.73 (d, 2H,
ArH), 7.41 (t, 2H, ArH), 7.34 (m, 2H, ArH), 7.26 (m, 2H, ArH), 5.19 (s, 2H, ArCH2-),
4.18 (m, 2H, -OCH2CH2CH2N-), 3.89 (s, 3H, -OCH3), 2.45-2.63 (m, 4H, -NCH2-),
13
3.46-3.52 (m, 2H, -CH2CH2OH), 1.89 (m, 2H, -OCH2CH2CH2N-). C NMR (100

ACCEPTED MANUSCRIPT
MHz, DMSO-d6): δ 158.89, 156.76, 156.23, 152.88, 148.53, 145.88, 139.24, 131.27,
128.88, 127.87, 127.65, 119.74, 113.88, 108.94, 106.33, 102.92, 70.37, 63.89, 56.06,
55.66, 54.74, 52.82, 26.69. ESI-MS: m/z calcd found 484.21 [M+H] +. Anal. calcd: C
69.57, H 6.02, N 14.46, O 9.95.
4.1.2.7.7.
4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonitrile (14)
Yield: 83%, m.p.:160-162 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, NH),
8.58 (s, 1H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 8.04 (m, 3H, ArH), 7.23 (s, 1H,
ArH), 4.20 (m, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 2.58 (m, 2H, -NCH2-),
2.50 (m, 2H, -NCH2-), 2.10-1.93 (m, 2H, -NCH2CH2CH2O-), 1.91 (m, 2H,
-OCH2CH2CH2N-), 1.70 (m, 4H, -CH2CH2-). 13C NMR (100 MHz, DMSO-d6): δ
158.77, 156.68, 156.21, 149.89, 148.57, 144.89, 131.32, 126.86, 113.23, 108.88,
106.21, 102.87, 69.99, 56.28, 54.66, 53.87, 25.99, 22.78. ESI-MS: m/z calcd found
404.33 [M+H] +. Anal. calcd: C 68.49, H 6.23, N 13.38, O 7.91.
4.1.2.7.8.
4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonitrile (15)
Yield: 81%, m.p.:172-174 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, NH),
8.58 (s, 1H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 7.92-7.78 (m, 3H, ArH), 7.23 (s, 1H,
ArH), 4.19 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 1.95 (dd, J = 17.5, 10.8
Hz, 6H, -NCH2-), 1.60-1.38 (m, 8H, -OCH2CH2CH2N- and -CH2CH2CH2-). 13C NMR
(100 MHz, DMSO-d6): δ 158.75, 156.67, 156.31, 149.91, 148.53, 144.89, 131.17,
126.89, 113.33, 108.92, 106.22, 102.88, 69.76, 56.21, 55.34, 54.72, 26.89, 24.33,
+
23.28. ESI-MS: m/z calcd found 418.33 [M+H] . Anal. calcd: C 69.06, H 6.51, N
16.75, O 7.68.
4.1.2.7.9.
4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonit
rile (16) Yield: 85%, m.p.:170-172 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H,
NH), 8.58 (s, 1H, ArH), 8.13 (d, J = 8.6 Hz, 2H, ArH), 7.97-7.77 (m, 3H, ArH), 7.23
(s, 1H, ArH), 4.18 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 2.88 (t, J = 10.6
Hz, 2H, -NCH2CH2CH2O-), 1.58 (t, J = 12.6 Hz, 4H, -NCH2-), 1.38-1.13 (m, 10H,
-OCH2CH2CH2N- and piperidine-H, -CH3). 13C NMR (100 MHz, DMSO-d6): δ
158.77, 156.58, 156.33, 149.93, 148.52, 144.85, 131.27, 126.93, 113.45, 108.87,
106.32, 102.84, 69.88, 56.06, 54.34, 45.89, 31.33, 30.87, 26.65, 19.28. ESI-MS: m/z
calcd found 432.42 [M+H] +. Anal. calcd: C 69.56, H 6.79, N 16.20, O 7.45.
4.1.2.7.10.
4-((7-(3-(4-hydroxypiperidin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amino)benzon
itrile (17) Yield: 82%, m.p.:194-196 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H,
NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.6 Hz, 2H, ArH), 7.97 (m, 3H, ArH), 7.23 (s, 1H,
ArH), 4.18 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 3.67 (m, 1H, -CHOH),
2.82-2.77 (m, 4H, -NCH2-), 2.31-2.55 (m, 2H, -NCH2-), 1.91-1.61 (m, 6H,
-OCH2CH2CH2N- and -NCH2CH2-). 13C NMR (100 MHz, DMSO-d6): δ 158.86,
156.65, 156.43, 149.89, 148.68, 144.89, 131.43, 126.99, 113.61, 108.92, 106.45,
102.88, 70.87, 62.84, 55.86, 54.34, 42.78, 30.38, 25.88. ESI-MS: m/z calcd found
434.32 [M+H] +. Anal. calcd: C 66.48, H 6.29, N 16.14, O 11.09.

ACCEPTED MANUSCRIPT
4.1.2.7.11.
4-((7-(3-(4-(hydroxymethyl)piperidin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amin
o)benzonitrile (18) Yield: 87%, m.p.:262-264 . 1H NMR (400 MHz, DMSO-d6): δ
9.78 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.6 Hz, 2H, ArH), 7.97 (m, 3H, ArH),
7.23 (s, 1H, ArH), 4.18 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 3.63 (s, 2H,
-CH2OH), 2.89-2.77 (m, 6H, -NCH2-), 2.14 (m, 2H, -OCH2CH2CH2N-), 1.73-1.31 (m,
5H, -CHCH2OH and -NCH2CH2-). 13C NMR (100 MHz, DMSO-d6): δ 158.77,
156.67, 156.52, 149.81, 148.72, 144.85, 131.55, 126.83, 113.76, 108.89, 106.56,
102.82, 70.45, 62.02, 55.77, 54.65, 45.76, 32.38, 25.88, 24.02. ESI-MS: m/z calcd
found 448.32 [M+H] +. Anal. calcd: C 67.06, H 6.56, N 15.62, O 10.77.
4.1.2.7.12.
4-((7-(3-(2-(2-hydroxyethyl)piperidin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amin
o)benzonitrile (19) Yield: 84%, m.p.:194-196 . 1H NMR (400 MHz, DMSO-d6): δ
9.78 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.5 Hz, 2H, ArH), 7.96-7.78 (m, 3H,
ArH), 7.23 (s, 1H, ArH), 4.17 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 3.87 (t,
2H, -CH2CH2OH), 2.87-2.57 (m, 5H, -NCH2-), 1.99 (m, 2H, -OCH2CH2CH2N-),
1.65-1.37 (m, 8H, -CH2CH2OH and -CH2CH2CH2-). 13C NMR (100 MHz, DMSO-d6):
δ 158.89, 156.76, 156.65, 149.89, 148.84, 144.89, 131.64, 126.91, 113.88, 108.92,
106.66, 102.89, 70.89, 63.52, 60.17, 55.24, 54.58, 49.76, 32.88, 30.23, 25.88, 23.82,
+
21.03. ESI-MS: m/z calcd found 462.34 [M+H] . Anal. calcd: C 67.64, H 6.76, N
15.19, O 10.41.
4.1.2.7.13.
Ethyl
1-(3-((4-((4-cyanophenyl)amino)-6-methoxyquinazolin-7-yl)oxy)propyl)piperidine-4-
carboxylate (20) Yield: 80%, m.p.:192-194 . 1H NMR (400 MHz, DMSO-d6): δ 9.78
(s, 1H, NH), 8.58 (s, 1H, ArH), 8.13 (d, J = 8.6 Hz, 2H, ArH), 7.97-7.77 (m, 3H, ArH),
7.23 (s, 1H, ArH), 4.18 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 2.87 (m, 6H,
-NCH2CH2-), 2.28 (m, 1H, -CHCO-), 2.21-2.12 (m, 8H, -NCH2CH2-,
13
-OCH2CH2CH2N- and -OCH2CH3), 1.38 (t, 3H, -OCH2CH3). C NMR (100 MHz,
DMSO-d6): δ 168.35, 158.27, 156.62, 156.23, 149.88, 148.54, 144.77, 131.16, 126.78,
113.33, 108.89, 106.21, 102.85, 69.98, 59.06, 55.21, 54.68, 52.24, 39.67, 27.06, 25.78,
+
12.89. ESI-MS: m/z calcd found 490.24 [M+H] . Anal. calcd: C 66.23, H 6.37, N
14.29, O 13.11.
4.1.2.7.14.
4-((6-methoxy-7-(3-(4-phenylpiperidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonit
rile (21) Yield: 82%, m.p.:194-196 . 1H NMR (400 MHz, DMSO-d6): δ 9.69 (s, 1H,
NH), 8.47 (d, 1H, ArH), 8.03 (d, J = 8.7 Hz, 2H, ArH), 7.85-7.71 (m, 4H, ArH), 7.56
(m, 3H, ArH), 7.24-7,03 (m, 2H, ArH), 4.13 (t, 2H, -OCH2CH2CH2N-), 3.89 (s, 3H,
-OCH3), 2.85 (m, 1H, -CHAr), 2.12 (m, 2H, -OCH2CH2CH2N- ), 1.88-1.855 (m, 6H,
-NCH2-), 1.68–1.41 (m, 4H, -NCH2CH2-). 13C NMR (100 MHz, DMSO-d6): δ 158.76,
156.63, 156.11, 149.88, 148.57, 147.68, 144.85, 131.15, 128.71, 127.91, 126.89,
126.07, 113.36, 108.89, 106.14, 102.76, 69.97, 55.06, 53.66, 45.24, 39.03, 30.23,
+
26.58. ESI-MS: m/z calcd found 494.25 [M+H] . Anal. calcd: C 73.03, H 6.35, N
14.18, O 6.44.
4.1.2.7.15.

ACCEPTED MANUSCRIPT
4-((7-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propoxy)-6-methoxyquinazolin-4-yl)amino
)benzonitrile (22) Yield: 81%, m.p.:234-236 . 1H NMR (400 MHz, DMSO-d6): δ
9.78 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.6 Hz, 2H, ArH), 7.97-7.68 (m, 3H,
ArH), 7.23 (s, 1H, ArH), 7.03-7.12 (m, 4 H, ArH), 4.22 (t, 2H, -OCH2CH2CH2N-),
3.99 (s, 3H, -OCH3), 3.60 (s, 2H, -NCH2-), 3.17 (d, 2H, -NCH2CH2-), 2.85-2.70 (m,
4H, -NCH2-), 2.12-2.03 (m, 2H, -OCH2CH2CH2N-). 13C NMR (100 MHz, DMSO-d6):
δ 158.69, 156.67, 156.12, 149.89, 148.59, 144.97, 138.16, 137.65, 131.05, 128.47,
128.13, 127.87, 125.32, 113.23, 108.89, 106.21, 102.88, 70.87, 56.06, 53.56, 53.27,
50.87, 25.84, 25.08. ESI-MS: m/z calcd found 466.25 [M+H] +. Anal. calcd: C 72.23,
H 5.84, N 15.05, O 6.88.
4.1.2.7.16.
4-((7-(3-(4-(dimethylamino)piperidin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amino
)benzonitrile (23) Yield: 79%, m.p.:180-182 . 1H NMR (400 MHz, DMSO-d6): δ
9.78 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.6 Hz, 2H, ArH), 7.97 (m, 3H, ArH),
7.23 (s, 1H, ArH), 4.19 (t, 2H, -OCH2CH2CH2N-), 3.97 (s, 3H, -OCH3), 2.82-2.77 (m,
4H, -NCH2-), 2.27 (s, 6H, -NCH3), 2.21-2.05 (m, 3H, -NCH2- and -CH-), 1.81 (m, 4H,
-OCH2CH2CH2N- and -NCH2CH2-), 1.51 (m, 2H, -NCH2CH2-). 13C NMR (100 MHz,
DMSO-d6): δ 158.67, 156.64, 156.33, 149.88, 148.53, 144.91, 131.21, 126.81, 113.23,
108.91, 106.23, 102.88, 70.87, 68.89, 56.35, 53.86, 50.24, 40.89, 25.78, 25.67.
ESI-MS: m/z calcd found 461.42 [M+H] +. Anal. calcd: C 67.82, H 7.03, N 18.22, O
6.93.
4.1.2.7.17.
4-((7-(3-([1,4'-bipiperidin]-1'-yl)propoxy)-6-methoxyquinazolin-4-yl)amino)benzonitr
ile (24) Yield: 85%, m.p.:176-178 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H,
NH), 8.58 (s, 1H, ArH), 8.13 (d, J = 8.6 Hz, 2H, ArH), 7.91 (m, 3H, ArH), 7.23 (s, 1H,
ArH), 4.21 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 2.82-2.47 (m, 10H,
-NCH2-), 2.17-2.05 (m, 3H, -OCH2CH2CH2N- and -CH-), 1.81 (m, 2H, -NCH2CH2-),
1.61-1.51 (m, 8H, -CH2CH2CH2- and -NCH2CH2-). 13C NMR (100 MHz, DMSO-d6):
δ 158.89, 156.67, 156.57, 149.93, 148.63, 144.89, 131.63, 126.88, 113.16, 108.87,
106.23, 102.88, 71.02, 68.85, 56.01, 55.88, 53.66, 50.67, 27.59, 25.79, 24.74, 24.03.
ESI-MS: m/z calcd found 501.32 [M+H] +. Anal. calcd: C 69.58, H 7.26, N 16.79, O
6.37.
4.1.2.7.18.
4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)benzonit
rile (25) Yield: 83%, m.p.:162-164 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H,
NH), 8.58 (s, 1H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 7.84 (m, 3H, ArH), 7.23 (s, 1H,
ArH), 4.18 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 2.55-2.45 (m, 6H,
-NCH2-), 2.17 (s, 3H, -NCH3), 2.02-1.80 (m, 6H, -NCH2- and -OCH2CH2CH2N-). 13C
NMR (100 MHz, DMSO-d6): δ 158.89, 156.79, 156.33, 149.90, 148.65, 144.89,
131.31, 126.79, 113.76, 108.99, 106.32, 102.88, 70.87, 55.86, 54.57, 54.01, 52.23,
43.52, 25.88. ESI-MS: m/z calcd found 433.23 [M+H] +. Anal. calcd: C 66.63, H 6.53,
N 19.42, O 7.42.
4.1.2.7.19.
4-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amino)benzonitri

ACCEPTED MANUSCRIPT
le (26) Yield: 81%, m.p.:164-166 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H,
NH), 8.58 (s, 1H, ArH), 8.13 (d, J = 8.7 Hz, 2H, ArH), 7.89-7.81 (m, 3H, ArH), 7.23
(s, 1H, ArH), 4.19 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 1.95(m, 6H,
-NCH2-, -NCH2CH3, -OCH2CH2CH2N-), 1.41-1.07 (m, 6H, -NCH2-), 1.05 (m, 5H,
-NCH2- and -NCH2CH3). 13C NMR (100 MHz, DMSO-d6): δ 158.89, 156.79, 156.35,
149.91, 148.67, 144.89, 131.34, 126.81, 113.76, 108.97, 106.32, 102.86, 70.89, 55.86,
+
54.59, 54.27, 53.25, 47.78, 25.98, 12.89. ESI-MS: m/z calcd found 447.25 [M+H] .
Anal. calcd: C 67.23, H 6.73, N 18.85, O 7.19.
4.1.2.7.20.
4-((7-(3-(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amin
o)benzonitrile (27) Yield: 80%, m.p.:168-171 . 1H NMR (400 MHz, DMSO-d6): δ
9.78 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.11 (d, J = 8.7 Hz, 2H, ArH), 7.81 (m, 3H, ArH),
7.23 (s, 1H, ArH), 4.09 (t, 2H, -OCH2CH2CH2N-), 3.99 (s, 3H, -OCH3), 3.49 (t, 2H,
13
-NCH2CH2OH), 3.25-2.89 (m, 12H, -NCH2-), 1.95 (m, 2H, -OCH2CH2CH2N-). C
NMR (100 MHz, DMSO-d6): δ 158.88, 156.76, 156.43, 149.88, 148.56, 144.95,
131.43, 126.79, 113.37, 108.96, 106.17, 102.87, 71.04, 57.87, 57.78, 56.16, 55.96,
53.24, 26.38. ESI-MS: m/z calcd found 463.25 [M+H] +. Anal. calcd: C 64.95, H 6.52,
N 18.16, O 10.37.
4.1.2.7.21.
4-((6-methoxy-7-(3-(4-(pyridin-4-yl)piperazin-1-yl)propoxy)quinazolin-4-yl)amino)b
enzonitrile (28) Yield: 81%, m.p.:166-168 . 1H NMR (400 MHz, DMSO-d6): δ 9.78
(s, 1H, NH), 8.58 (s, 1H, ArH), 8.23 (d, 2H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 7.84
(m, 3H, ArH), 7.23 (s, 1H, ArH), 7.09 (d, 2H, ArH), 4.22 (t, 2H, -OCH2CH2CH2N-),
3.99 (s, 3H, -OCH3), 3.15–2.55 (m, 8H, -NCH2-), 2.37 (m, 2H, -NCH2-), 1.89 (m, 2H,
-OCH2CH2CH2N-). 13C NMR (100 MHz, DMSO-d6): δ 158.76, 156.65, 156.33,
150.67, 149.89, 149.01, 148.62, 144.88, 131.14, 126.84, 113.09, 108.89, 107.34,
106.21, 102.88, 70.81, 56.06, 54.09, 53.36, 49.24, 26.68. ESI-MS: m/z calcd found
496.25 [M+H] +. Anal. calcd: C 67.83, H 5.94, N 19.76, O 6.47.
4.1.2.7.22.
4-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)benzonitrile
(29)
Yield: 78%, m.p.:276-278 . 1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, NH),
8.58 (s, 1H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 7.92-7.78 (m, 3H, ArH), 7.23 (s, 1H,
ArH), 4.19 (t, 2H, -OCH2CH2CH2N-), 3.95 (s, 3H, -OCH3), 3.58 (m, 4H,
-OCH2CH2-), 2.47-2.32 (m, 6H, -NCH2-), 1.99 (m, 2H, -OCH2CH2CH2N-). 13C NMR
(100 MHz, DMSO-d6): δ 158.67, 156.55, 156.09, 149.56, 148.53, 144.89, 131.05,
126.73, 113.61, 108.81, 106.09, 102.79, 69.37, 66.06, 56.86, 54.97, 54.01, 26.88.
ESI-MS: m/z calcd found 419.32 [M+H] +. Anal. calcd: C 65.88, H 6.03, N 16.68, O
11.41.
4.1.2.7.23.
3-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonitrile (30)
Yield: 79%, m.p.:172-174 . 1H NMR (400 MHz, DMSO-d6): δ 9.68 (s, 1H, NH),
8.54 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.15 (d, J = 8.1 Hz, 1H, ArH), 7.83 (s, 1H, ArH),
7.67-7.44 (m, 2H, ArH), 7.21 (s, 1H, ArH), 4.19 (t, 2H, -OCH2CH2CH2N-), 3.98 (s,
3H, -OCH3), 2.58 (t, J = 7.1 Hz, 2H, -NCH2-), 2.51 (s, 2H, -NCH2-), 2.48 (s, 2H,

ACCEPTED MANUSCRIPT
13
-NCH2-), 2.04-1.93 (m, 2H, -OCH2CH2CH2N-), 1.70 (s, 4H, -CH2CH2-). C NMR
(100 MHz, DMSO-d6): δ 158.48, 156.35, 154.75, 149.86, 148.53, 147.89, 141.83,
130.05, 125.46, 119.67, 115.73, 110.61, 108.81, 106.41, 103.79, 69.87, 56.67, 54.83,
+
53.89, 25.86, 22.37. ESI-MS: m/z 404.30 [M+H] . Anal. calcd: C 68.44, H 6.24, N
17.38, O 7.94.
4.1.2.7.24.
3-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonitrile (31)
Yield: 81%, m.p.:146-148 . 1H NMR (400 MHz, DMSO-d6): δ 9.67 (s, 1H, NH),
8.55 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.15 (d, J = 8.0 Hz, 1H, ArH), 7.83 (s, 1H, ArH),
7.65-7.52 (m, 2H, ArH), 7.21 (s, 1H, ArH), 4.18 (s, 2H, -OCH2CH2CH2N-), 3.98 (s,
3H, -OCH3), 2.48-2.41 (m, 2H, -OCH2CH2CH2N-), 2.37 (s, 4H, -NCH2-), 1.94 (m,
4H, -NCH2-, -CH2CH2CH2-), 1.56-1.48 (m, 4H, -CH2CH2CH2-). 13C NMR (100 MHz,
DMSO-d6): δ 158.53, 156.39, 154.67, 149.89, 148.65, 147.88, 141.89, 130.06, 125.56,
119.76, 115.86, 110.77, 108.89, 106.43, 103.81, 69.72, 56.37, 55.44, 54.83, 26.87,
24.45, 23.37. ESI-MS: m/z 418.38 [M+H] +. Anal. calcd: C 69.03, H 6.53, N 16.74, O
7.70.
4.1.2.7.25.
3-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonit
rile (32) Yield: 83%, m.p.:142-144 . 1H NMR (400 MHz, DMSO-d6): δ 9.67 (s, 1H,
NH), 8.54 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.15 (dd, J = 5.1, 4.0 Hz, 1H, ArH), 7.83 (s,
1H, ArH), 7.67-7.49 (m, 2H, ArH), 7.20 (s, 1H, ArH), 4.17 (s, 2H, -OCH2CH2CH2N-),
3.98 (s, 3H, -OCH3), 2.85 (d, J = 11.2 Hz, 2H, -NCH2-), 2.53-2.50 (m, 2H,
-OCH2CH2CH2N-), 2.43 (t, J = 7.0 Hz, 2H, -NCH2-), 1.93 (ddd, J = 28.7, 17.0, 8.2
Hz, 4H, -OCH2CH2CH2N-, -NCH2CH2-), 1.57 (m, 3H, -NCH2CH2-, -CHCH3), 0.88
13
(d, J = 6.5 Hz, 3H, -CHCH3). C NMR (100 MHz, DMSO-d6): δ 158.56, 156.41,
154.69, 149.91, 148.67, 147.87, 141.84, 130.16, 125.77, 119.82, 115.86, 110.79,
108.91, 106.56, 103.87, 69.91, 56.32, 54.35, 45.87, 31.54, 30.88, 26.76, 19.43.
ESI-MS: m/z 432.32 [M+H] +. Anal. calcd: C 69.57, H 6.75, N 16.24, O 7.44.
4.1.2.7.26.
3-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)benzonit
rile (33) Yield: 86%, m.p.:152-154 . 1H NMR (400 MHz, DMSO-d6): δ 9.67 (s, 1H,
NH), 8.55 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.15 (d, J = 7.9 Hz, 1H, ArH), 7.83 (s, 1H,
ArH), 7.76-7.45 (m, 2H, ArH), 7.20 (s, 1H, ArH), 4.18 (s, 2H, -OCH2CH2CH2N-),
3.98 (s, 3H, -OCH3), 2.51 (s, 2H, -OCH2CH2CH2N-), 2.42 (dd, J = 28.5, 21.5 Hz, 8H,
-NCH2-), 2.16 (s, 3H, -NCH3), 2.03-1.89 (m, 2H, -OCH2CH2CH2N-). 13C NMR (100
MHz, DMSO-d6): δ 158.66, 156.47, 154.58, 149.84, 148.78, 147.88, 141.89, 130.26,
125.69, 119.82, 115.77, 110.83, 108.88, 106.69, 103.89, 70.86, 55.92, 54.59, 54.11,
+
52.36, 43.73, 25.85. ESI-MS: m/z 433.37 [M+H] . Anal. calcd: C 66.67, H 6.51, N
19.42,O 7.40.
4.1.2.7.27.
3-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amino)benzonitri
le (34) Yield: 83%, m.p.:142-144 . 1H NMR (400 MHz, DMSO-d6): δ 9.67 (s, 1H,
NH), 8.54 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.15 (d, J = 8.4 Hz, 1H, ArH), 7.83 (s, 1H,
ArH), 7.68-7.53 (m, 2H, ArH), 7.20 (s, 1H, ArH), 4.18 (t, J = 6.4 Hz, 2H,

ACCEPTED MANUSCRIPT
-OCH2CH2CH2N-), 3.98 (s, 3H, -OCH3), 2.53-2.49 (m, 2H, -NCH2-), 2.47-2.42 (m,
2H, -NCH2CH3), 2.31 (dt, J = 14.3, 7.2 Hz, 4H, -NCH2-), 1.94 (m, 2H,
-OCH2CH2CH2N-), 0.98 (t, J = 7.2 Hz, 3H, -NCH2CH3). 13C NMR (100 MHz,
DMSO-d6): δ 158.67, 156.45, 154.63, 149.89, 148.71, 147.85, 141.85, 130.21, 125.72,
119.79, 115.75, 110.81, 108.88, 106.63, 103.85, 70.85, 55.81, 54.54, 54.31, 53.29,
+
47.76, 25.89, 12.92. ESI-MS: m/z 447.30 [M+H] . Anal. calcd: C 67.25, H 6.78, N
18.81, O 7.16.
4.1.2.7.28.
3-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)benzonitrile
(35)
Yield: 80%, m.p.:266-268 . 1H NMR (400 MHz, DMSO-d6): δ 9.68 (s, 1H, NH),
8.58 (s, 1H, ArH), 8.38 (s, 1H, ArH), 8.15 (d, J = 8.4 Hz, 1H, ArH), 7.81 (s, 1H, ArH),
7.69-7.51 (m, 2H, ArH), 7.23 (s, 1H, ArH), 4.19 (m, 2H, -OCH2CH2CH2N-), 3.97 (s,
3H, -OCH3), 3.59 (m, 4H, -OCH2CH2-), 2.50-2.42 (m, 6H, -NCH2-), 1.99 (m, 2H,
-OCH2CH2CH2N-). 13C NMR (100 MHz, DMSO-d6): δ 158.67, 156.45, 154.63,
149.89, 148.71, 147.85, 141.85, 130.21, 125.72, 119.79, 115.75, 110.81, 108.88,
106.63, 103.85, 69.39, 66.84, 56.77, 54.97, 54.05, 26.92. ESI-MS: m/z calcd found
419.33 [M+H] +. Anal. calcd: C 65.86, H 6.02, N 16.67, O 11.45.
4.1.2.7.29.
2-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonitrile (36)
Yield: 78%, m.p.:142-144 . 1H NMR (400 MHz, DMSO-d6): δ 8.46 (s, 1H, ArH),
8.06 (d, J = 8.7 Hz, 2H, ArH), 7.92-7.87 (m, 2H, ArH), 7.27-7.23 (m, 2H, ArH), 4.12
(m, 2H, -OCH2CH2CH2N-), 3.81 (s, 3H, -OCH3), 2.65-2.21 (m, 6H, -NCH2-), 1.80 (m,
2H, -OCH2CH2CH2N-), 1.60 (m, 4H, -CH2CH2-). 13C NMR (100 MHz, DMSO-d6): δ
157.97, 156.27, 155.01, 149.85, 147.49, 141.38, 133.05, 128.71, 123.36, 118.01,
115.09, 110.76, 107.34, 102.89, 102.67, 69.89, 56.72, 54.86, 53.92, 25.88, 22.37.
ESI-MS: m/z calcd found 404.22 [M+H] +. Anal. calcd: C 68.46, H 6.25, N 17.36, O
7.93.
4.1.2.7.30.
2-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonitrile (37)
Yield: 80%, m.p.:156-158 . 1H NMR (400 MHz, DMSO-d6): δ 8.47 (s, 1H, ArH),
8.12 (d, J = 8.7 Hz, 1H, ArH), 8.06 (d, J = 8.7 Hz, 1H, ArH), 7.92 (m, 1H, ArH), 7.52
(m, 1H, ArH), 7.24 (s, 1H, ArH), 7.16 (m, 1H, ArH), 4.12 (m, 2H, -OCH2CH2CH2N-),
3.82 (s, 3H, -OCH3), 2.65-2.21 (m, 6H, -NCH2-), 1.95 (m, 2H, -OCH2CH2CH2N-),
1.32-1.30 (m, 6H, -CH2CH2CH2-). 13C NMR (100 MHz, DMSO-d6): δ 157.89, 156.33,
155.17, 149.88, 147.53, 141.39, 133.12, 128.79, 123.43, 118.07, 115.11, 110.91,
107.45, 102.90, 102.77, 69.78, 56.43, 55.55, 54.89, 26.91, 24.54, 23.43. ESI-MS: m/z
calcd found 418.24 [M+H] +. Anal. calcd: C 69.04, H 6.49, N 16.75, O 7.62.
4.1.2.7.31.
2-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinazolin-4-yl)amino)benzonit
rile (38) Yield: 84%, m.p.:162-164 . 1H NMR (400 MHz, DMSO-d6): δ 8.48 (s, 1H,
ArH), 8.13 (d, J = 8.7 Hz, 1H, ArH), 8.06 (d, J = 8.04 Hz, 1H, ArH), 7.62-7.58 (m, 2H,
ArH), 7.26 (s, 1H, ArH), 7.20 (s, 1H, ArH), 4.19 (m, 2H, -OCH2CH2CH2N-), 3.82 (s,
3H, -OCH3), 2.10 (m, 2H, -OCH2CH2CH2N-), 1.83 (m, 2H, -NCH2CH2-), 1.37 (m,
3H, -NCH2CH2-, -CHCH3), 0.74 (d, J = 6.5 Hz, 3H, -CHCH3). 13C NMR (100 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6): δ 157.94, 156.43, 155.22, 149.89, 147.55, 141.42, 133.15, 128.83, 123.46,
118.14, 115.18, 110.89, 107.51, 102.77, 102.87, 69.86, 56.37, 54.39, 45.88, 31.59,
+
30.92, 26.83, 19.46. ESI-MS: m/z 432.25 [M+H] . Anal. calcd: C 69.56, H 6.77, N
16.23, O 7.44.
4.1.2.7.32.
2-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-yl)amino)benzonit
rile (39) Yield: 81%, m.p.:154-157 . 1H NMR (400 MHz, DMSO-d6): δ 8.48 (s, 1H,
ArH), 8.13 (d, J = 8.7 Hz, 1H, ArH), 8.06 (d, J = 8.04 Hz, 1H, ArH), 7.91 (m, 1H,
ArH), 7.62-7.58 (m, 1H, ArH), 7.25 (s, 1H, ArH), 7.08 (s, 1H, ArH), 4.03 (m, 2H,
-OCH2CH2CH2N-), 3.87 (s, 3H, -OCH3), 2.51-2.22 (m, 8H, -NCH2-), 2.16 (s, 3H,
-NCH3), 2.08 (m, 2H, -OCH2CH2CH2N-), 1.80 (m, 2H, -OCH2CH2CH2N-). 13C NMR
(100 MHz, DMSO-d6): δ 157.98, 156.45, 155.27, 149.91, 147.59, 141.47, 133.19,
128.87, 123.49, 118.17, 115.19, 110.89, 107.53, 102.79, 102.89, 70.92, 55.97, 54.63,
54.18, 52.38, 43.78, 25.89. ESI-MS: m/z calcd found 433.25 [M+H] +. Anal. calcd: C
66.66, H 6.50, N 19.44, O 7.40.
4.1.2.7.33.
2-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinazolin-4-yl)amino)benzonitri
le (40) Yield: 83%, m.p.:160-163 . 1H NMR (400 MHz, DMSO-d6): δ 8.48 (s, 1H,
ArH), 8.13 (d, J = 8.7 Hz, 1H, ArH), 8.06 (d, J = 8.04 Hz, 1H, ArH), 7.91 (m, 1H,
ArH), 7.62-7.58 (m, 1H, ArH), 7.25 (s, 1H, ArH), 7.08 (s, 1H, ArH), 4.03 (m, 2H,
-OCH2CH2CH2N-), 3.87 (s, 3H, -OCH3), 2.51-2.22 (m, 8H, -NCH2-), 2.16 (s, 3H,
-NCH2CH3), 2.08 (m, 2H, -OCH2CH2CH2N-), 1.80 (m, 2H, -OCH2CH2CH2N-), 0.94
(t, J = 6.52 Hz, 3H, -NCH2CH3). 13C NMR (100 MHz, DMSO-d6): δ 157.97, 156.46,
155.26, 149.89, 147.59, 141.49, 133.21, 128.88, 123.51, 118.21, 115.23, 110.88,
107.57, 102.82, 102.88, 70.84, 55.87, 54.56, 54.37, 53.32, 47.77, 25.89, 12.93.
ESI-MS: m/z calcd found 447.26 [M+H] +. Anal. calcd: C 67.26, H 6.77, N 18.82, O
7.15.
4.1.2.7.34.
2-((6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)amino)benzonitrile
(41)
Yield: 81%, m.p.:272-274 . 1H NMR (400 MHz, DMSO-d6): δ 8.48 (s, 1H, ArH),
8.13 (d, J = 8.7 Hz, 1H, ArH), 8.06 (d, J = 8.7 Hz, 1H, ArH), 7.91 (m, 1H, ArH), 7.52
(m, 1H, ArH), 7.23 (s, 1H, ArH), 7.16 (m, 1H, ArH), 4.11 (m, 2H, -OCH2CH2CH2N-),
3.87 (s, 3H, -OCH3), 3.64-3.58 (m, 4H, -OCH2CH2-), 2.54-2.42 (m, 6H, -NCH2-),
1.98 (m, 2H, -OCH2CH2CH2N-). 13C NMR (100 MHz, DMSO-d6): δ 157.97, 156.49,
155.27, 149.92, 147.57, 141.53, 133.24, 128.89, 123.48, 118.19, 115.26, 110.89,
107.60, 102.87, 102.92, 69.46, 66.87, 56.87, 54.93, 54.12, 26.89. ESI-MS: m/z calcd
found 419.34[M+H] +. Anal. calcd: C 65.85, H 6.03, N 16.68, O 11.44.
4.2. Biochemical and biological assays
4.2.1. Cell lines culture and treatments
Human liver cancer cell line (HepG2), human lung carcinoma cell line (A549),
human breast carcinoma cell line (MCF-7), human prostate cancer cell line (DU145),
human neuroblastoma cancer cell line (SH-SY5Y) and human normal cell line
(CCC-HPF-1) were purchased from Cancer Hospital, Chinese Academy of Medical
Sciences, Beijing, P. R. China. All the cells were cultivated in DMEM/High Glucose

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(4.5 g/L glucose) medium or RPMI-1640 medium supplemented with 10% fetal
bovine serum (FBS), 4 mM glutamine, 50 units/mL of penicillin and 50 µg/mL of
streptomycin. All cell lines were routinely maintained at 37 in a humidified
incubator with 5% CO2 and 95% air.
4.2.2. In vitro anti-proliferative activities screening of target compounds assay
The anti-proliferative activities of all target compounds were determined using a
standard MTT-based colorimetric assay. For a typical screening experiment to
determine the cell proliferation, the tested cells were seeded in costar 96-well cell
culture clusters at a density of 2×103 cells/well in 100 µL of the proper culture
medium and performed of incubation at 37 with 5% CO2. After 24 h incubation
period，the medium was removed and the cells were treated with the indicated
concentrations of target compounds (0.1-100 µM) in 100 µL for 72 h. 100 µL of fresh
medium without cells was added as control. Triplicate wells were prepared for each
individual dose. After that, the cells were incubated with 10 µL MTT solution (5
mg/mL in PBS) for a further 4 h and then 100 µL of 10% SDS (w/v) with NH4Cl (1
mg/ml) was added to each well. The plates were kept for 10 h at 37℃with 5% CO2.
The purple color was produced. The absorbance of formazan was measured on a
BioTek microtiter plate reader at 570 nm with correction at 650 nm. At least three
independent experiments were performed. Survival ratios are expressed in
percentages with respect to untreated cells. The percent growth inhibition was
calculated
by
the
following
formula: %inhibition=[1-(ODtreated-ODblank)/(ODcontrol-ODblank)]×100. Then IC50 values
were calculated from the above data with SPSS16.0 software. The results were then
represented as obtained after averaging at least thrice independent experiments.
4.2.3. EGFR-TK binding inhibitory assay
The EGFR kinase assay was performed in order to determine the ability of
compound 21 to block the tyrosine kinase activity of EGFR-TK. Costar 96-well cell
culture clusters were pre-coated with 100 µL/well of 10 µg/mL streptavidin in
carbonate buffer at 4°C for 10 h. After the excess streptavidin solution was removed
by washing the wells thrice with wash buffer (0.05% Tween-20 in PBS). An aliquot
of solution (H2O/DMSO:99/1,v/v) (15 µL) containing various concentrations of a
screened inhibitor compound 21 was added to 15 µL DTT/kinase buffer (pH 7.5, CST)
containing 200 ng/µL EGFR-TK (Sino Biological Inc.), and then incubated at room
temperature (25℃) for 5 min, followed by addition of 30 µL mixed solution
containing 15 µL PTP1B (Tyr66) biotinylated peptide (CST), 1.2 µL ATP (CST) and
13.8 µL ultra-pure H2O. The resulting mixture was left for reaction at 37℃for 1 h,
and then added 60 µL stop buffer (50 mM EDTA, pH 8.0, CST) to stop the reaction.
25 µL/well of each enzymatic reaction mixture and 75 µL/well of ultra-pure H2O were
transferred to a 96-well streptavidin-coated cell culture cluster and incubated at 37℃
for 1 h. Following thrice of washing with PBS/T, 100 µL of primary antibody
(Phospho-Tyrosine Mouse mAb, 1:1000 in PBS/T with 1.5% BSA) was added to each
well and the plate was incubated at 37℃for another 1 h. The plate was again washed
thrice with PBS/T, and then 100 µL of secondary antibody (HRP-labeled Goat
Anti-Mouse IgG, 1:1000 in PBS/T with 1.5% BSA) was added to each well for 1 h of

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incubation at 37 , followed by thrice of washing with PBS/T. 100 µL of TMB
substrate system was added to each well and the plate was incubated at 37 for 15
min, and then the reaction was stopped by addition of 100 µL of 1 M H2SO4, and the
plate was read on the ELISA plate reader at 450 nm with correction at 650 nm. The
results were expressed as percent kinase activity of the vehicle control, and the IC50
value was defined as the compound concentration that resulted in 50% inhibition of
enzyme activity. The kinase assay was performed at least thrice independently.
4.2.4. LDH release assay
1.0×105 cells per well HepG2 cells were inoculated into a costar 24-well cell
culture cluster and incubated for 24 h at 37 in 5% CO2. Cytotoxicity was assessed
based on measuring the LDH release after 48 h of various dose compound 21
treatments. Prior to each assay, the cells were lysed with 2% (V/V) lysate in culture
media for 1 h at 37 to obtain a representative maximal LDH release as the positive
control with 100% cytotoxicity and the cells in culture medium alone was the low
control. The amounts of LDH in the supernatant were determined on a BioTek
microtiter plate reader and calculated as kit instructions. All tests were performed in
triplicate and the assay was repeated three times independently.
4.2.5. Flow cytometric analysis of cell cycle distribution assay
For flow cytometric analysis of DNA content, 5.0×105 cell/well HepG2 cells were
grown in a costar 6-well cell culture cluster and grown for 24 h at 37 in 5%CO2,
after the medium was removed and the cells were treated with different concentrations
of the test compound 21 for 48 h. Blank wells treated with medium only were also
included. After the incubation period, the HepG2 cells were collected, washed twice
with ice-cold PBS, centrifuged and then fixed with ice-cold ethanol (70%) for at least
24 h. Cells were then collected, washed twice with ice-cold PBS, and treated with 30
µL RNase A (1 mg/ml) in PBS at 37 for about 30 min, and then stained with 50 µL
propidium iodide (50 mg/mL) in PBS, the staining process lasted 30 min at 4 in
darkness. The cellular DNA content of the stained cells was then analyzed on
BD-FACS Aria flow cytometer and the cell cycle distribution was quantified.
4.2.6. Analysis of apoptosis induced in HepG2 cells
The HepG2 cells were seeded in a costar 6-well cell culture cluster at a seeding
density of 5.0×105 cells/mL and incubated for 24 h. When the cells were adhered,
various concentrations of compound 21 were added. The cells were treated with
compound 21 for 48 h and then collected, washed twice with ice-cold PBS, and cell
apoptosis was analyzed using AnnexinV-FITC (5 µL) and propidium iodide (5 µL)
double staining on BD-FACS Aria
flow cytometer. Early apoptotic cells were
defined as AnnexinV-FITC positive/PI negative, late apoptotic cells as
AnnexinV-FITC/PI double positive and necrotic cells as AnnexinV-FITC positive/PI
positive.
4.2.7. Morphological observation of HepG2 cells
Furthermore, dramatic alterations in the morphology of HepG2 cells were observed
by Hoechst 33342/propidium iodide dual staining under fluorescence microscopy to
determine whether the growth inhibitory activity of the selected compound 21 was
related to apoptosis induction. The HepG2 cells were cultivated in a costar 6-well cell

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culture cluster (5.0×104 cells per well) and maintained in DMEM medium with 10%
FBS at 37 for 24 h. After the cells attachment, the medium was replaced with new
medium containing varying concentrations of tested drugs. After 48 h treatment, the
medium was removed and the cells was washed twice with ice-cold PBS and then
stained with 1:1 ratio of Hoechst 33342/propidium iodide mixture at 37 for 30 min
in the darkness. The cells were washed with ice-cold PBS thrice. Stained cells of the
morphological observation was visualized using inverted the laser scanning confocal
microscope at 200× magnification.
4.2.8. Effect of compound 21 on mitochondrial membrane potential assay
Changes in mitochondrial membrane potential as a result of mitochondrial
perturbation were measured by staining with Rhodamine-123. The HepG2 cells were
inoculated in a costar 6-well cell culture cluster at a concentration of 5.0×105 cells/mL
and incubated with compound 21 at indicated concentrations for 48 h when all the
cells were adhered. After the cells were collected, washed with ice-cold PBS twice
and 500 µL Rhodamine-123 solution (5µg/mL) was added for 30 min before
termination of experiment. The HepG2 cells were trypsinized, collected, washed twice
with ice-cold PBS and resuspended in PBS. The fluorescence intensity of
mitochondrial membrane potential was measured in FL-1 channel vs counts on
BD-FACS Aria flow cytometer.
4.2.9. Determination of intracellular free Ca2+ using Fluo-3/AM assay
The HepG2 cells of 5.0×105 cells/mL were seeded in a costar 6-well cell culture
cluster and cultured for 24 h at 37 in 5% CO2. After then the medium was removed
and the cells were incubated with compound 21 at indicated concentrations for 48 h.
After the cells were collected, washed with ice-cold PBS twice and the fluorescence
Ca2+ indicator Fluo-3/AM solution (4 µM) was added for 30 min before termination of
experiment. The HepG2 cells were trypsinized, collected, washed twice with HEPES
and then resuspended in HEPES. The fluorescence intensity was measured in FL-1
channel vs counts on BD-FACS Aria flow cytometer.
4.2.10. Detection of intracellular reactive oxygen species (ROS) assay
The HepG2 cells of 5.0×105 cells/mL were grown in a costar 6-well cell culture
cluster and maintained for 24 h at 37 in 5% CO2. After then the medium was
removed and the cells were treated with compound 21 at indicated concentrations for
48 h. After the cells were collected, washed with ice-cold PBS twice and 1000 µL
DCFH-DA working solution (10 µM) was added at 37 for 30 min before
termination of experiment. The HepG2 cells were trypsinized, collected, washed twice
with ice-cold HBSS and resuspended in HBSS, the fluorescence was directly recorded
on FS5 fluorescence spectrometer.
4.2.11. Determination of caspase-3 activation assay
Caspase-3 activation in HepG2 cells treated with compound 21 at different doses
was determined following the instructions of caspase-3 activation colorimetric assay
kit (KeyGEN BioTECH). 48 h after treatment, the HepG2 cells were collected, rinsed
with ice-cold PBS and then lysed using lysis buffer contained 1% DTT on ice bath for
1-1.5 h. The cell lysate was centrifuged (10,000×g, 1 min, 4 ), and then the
supernatant were collected. Protein concentration in the supernatant was measured by

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Bradford protein quantitation assay. Finally, 50 µL of the supernatant and 50 µL of the
2×Reaction Buffer containing 1% DTT, and 5 µL of caspase-3 substrate were added to
each well on a costar 96-well cell culture cluster and the cluster was maintained for 4
h at 37 in 5% CO2. The absorbance was measured at 405 nm on a BioTek microtiter
plate reader.
4.2.12. Statistical analysis
All the statistical analyses were performed under SPSS16.0 software by a personal
computer. Each experiment was performed in triplicate, and all experiments were run
three times independently. Measurements from all the replicates were combined and
treatment effects analyzed. Unless otherwise indicated, the results are presented as the
mean ± SD. The differences between different treatments were analyzed using the
two-sided Student’s t test. P values of less than 0.05 or 0.01 were considered
significant or highly significant difference.
Acknowledgments
This work was supported by National Natural Science Foundation of China (No.
21071022) and the Fundamental Research Funds for the Central Universities. We
especially thank Huabei Zhang (Key Laboratory of Radiopharmaceuticals, Ministry of
Education, College of Chemistry, Beijing Normal University) for their kind help in
this paper.
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