Design, synthesis and evaluation of anti-proliferative activity of 2-aryl-4-aminoquinazoline derivatives as EGFR inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104848

A class of 2-aryl-4-aminoquinazoline derivatives (7a-7j, 8a-8h, 9a-9h and 10a-10k) were designed, synthesized and evaluated as EGFR inhibitors. The anti-proliferative activity of compounds in vitro showed that compound 9e was considered to be a promising derivative. Compared with the lead compound Angew2017-7634-1, 9e exhibited excellent inhibitory activity against A549, NCI-H460 and H1975 cell lines, with IC₅₀ values of 14.33 ± 1.16 μM, 17.81 ± 1.25 μM and 13.41 ± 1.14 μM, respectively. Moreover, 9e could effectively inhibit against Ba/F3-EGFR^{Del19/T790M/C797S} cell lines. In the kinase experiment, the most promising compound 9e exhibited excellent enzymatic inhibitory activity and selectivity for EGFR^L858R/T790M, with an IC₅₀ value of 0.74 μM. Further activity studies showed that 9e could not only induce remarkable cell-apoptosis of A549, but also block A549 cell lines in S-phase in a concentration-dependent manner. Furthermore, molecular docking study revealed the binding mode of 9e. All in all, we analyzed the structure–activity relationship of the target compounds, and explored their mechanism of action.

Full text of DOI:10.1016/j.bioorg.2021.104848

Bioorganic Chemistry 112 (2021) 104848
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and evaluation of anti-proliferative activity of 2-ary-
l-4-aminoquinazoline derivatives as EGFR inhibitors
a
a
a
a
b
a
Zhihui Zhou , Jie He , Feiyi Yang , Qingshan Pan , Zunhua Yang , Pengwu Zheng ,
a,
*
a,*
Shan Xu , Wufu Zhu
a
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, 605 Fenglin Road, Nanchang
3
b
30013, China
School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A class of 2-aryl-4-aminoquinazoline derivatives (7a-7j, 8a-8h, 9a-9h and 10a-10k) were designed, synthesized
and evaluated as EGFR inhibitors. The anti-proliferative activity of compounds in vitro showed that compound 9e
was considered to be a promising derivative. Compared with the lead compound Angew2017-7634-1, 9e
exhibited excellent inhibitory activity against A549, NCI-H460 and H1975 cell lines, with IC50 values of 14.33 ±
Synthesis
2
-aryl-4-aminoquinazoline derivatives
Anti-proliferative activity
Structure-activity relationship
1
.16
F3-EGFR
excellent enzymatic inhibitory activity and selectivity for EGFR
μ
M, 17.81 ± 1.25
μ
M and 13.41 ± 1.14
μ
M, respectively. Moreover, 9e could effectively inhibit against Ba/
Del19/T790M/C797S
cell lines. In the kinase experiment, the most promising compound 9e exhibited
L858R/T790M
, with an IC50 value of 0.74
μ
M.
Further activity studies showed that 9e could not only induce remarkable cell-apoptosis of A549, but also block
A549 cell lines in S-phase in a concentration-dependent manner. Furthermore, molecular docking study revealed
the binding mode of 9e. All in all, we analyzed the structure–activity relationship of the target compounds, and
explored their mechanism of action.
1
. Introduction
Lung cancer is one of the malignant tumors with the highest inci-
[8], 4G5P [9], 6JX0 [10] and 5ZWJ [11]) provided by worldwide Pro-
tein Data Bank (http://www.wwpdb.org/), we analyzed the binding
patterns of representative EGFR tyrosine kinase inhibitors (EGFR-TKIs).
The first-generation reversible EGFR-TKIs, such as 1 (Gefitinib) [12],
have been developed to address the problems associated with activation
mutations. However, after 9–14 months of treatment, a secondary drug
resistance mutation (T790M) appeared, resulting in the loss of the ac-
tivity of the first-generation inhibitors [13,14]. In order to overcome the
resistance caused by mutation of T790M, the second- and third-
generation irreversible EGFR-TKIs have been developed, such as 2
(Afatinib, BIBW2992) [15] and 3 (Osimertinib, AZD9291) [16]. The
main reason was that their acrylamide structure (Michael receptor)
formed a covalent bond with CYS797 residue to enhance their activity so
dence and mortality, accounting for about 1/4 of cancer mortality, and
its five-year relative survival rate is less than 20% [1]. Among them,
non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer
mortality rates, and its five-year survival rate is less than 15% [2]. Re-
searchers had found that epidermal growth factor receptor (EGFR) is
highly expressed in 20–80% of NSCLC patients [3,4]. EGFR, a trans-
membrane protein, belongs to the tyrosine kinase type receptor and is
a member of ErbB receptor family. It plays an important role in various
growth processes of cancer, including proliferation, differentiation,
migration, apoptosis and angiogenesis [5–7]. Studies had showed that
inhibited the tyrosine kinase activity of EGFR could effectively inhibit
tumor growth [7]. Therefore, the development of effective EGFR in-
hibitors has become one of the hot spots of targeted therapy for NSCLC.
So far, EGFR inhibitors have been developed to four generations, and
some of them have been approved by the FDA for the treatment of
NSCLC. As shown in Fig. 1, according to the PDB data (PDB code: 4I22
T790M
T790M/
as to overcome the resistance mediated by EGFR
or EGFR
L858R
mutation [17]. However, the third-generation EGFR-TKIs also had
drug resistance due to the emergence of the C797S mutation [18–20].
Therefore, there is an urgent need to develop new inhibitors that could
T790M/C797S
effectively inhibit the EGFR
mutation. Jia et al. [21] first
reported an allosteric inhibitor 4 (EAI045), which could effectively
*
Corresponding authors.
E-mail addresses: shanxu9891@126.com (S. Xu), zhuwufu-1122@163.com (W. Zhu).
https://doi.org/10.1016/j.bioorg.2021.104848
Received 8 November 2020; Received in revised form 17 March 2021; Accepted 18 March 2021
Available online 22 March 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
overcome the T790M/C797S mutation and was a non-ATP-competitive
2. Results and discussion
L858R/T797M/C797S
EGFR
inhibitor. Unfortunately, the efficacy of single
compound 4 was limited and required cooperation with Cetuximab
22]. In recent years, some fourth-generation EGFR inhibitors have been
2.1. Chemistry
[
reported [23–26], such as the 2-aryl-4-aminoquinazoline derivative
Angew2017-7634-1 [23]. Nevertheless, most of the compounds are still
in pre-clinical or clinical studies, and no drug on the market. Based on
above, this study analyzed and studied the molecular docking modes of
Angew2017-7634-1, and summarized its structure–activity relation-
ship to optimize its structure, hoping to obtain a batch of compounds
with excellent inhibitory activity against T790M/C797S mutation and
no side effects.
The synthetic route of the target compounds 7a-7j, 8a-8i, 9a-9h and
10a-10k were outlined in Scheme 1 and 2.
Firstly, the key intermediates 3a-3b were obtained by cyclization
and chlorination as revealed in Scheme 1. Taking commercially avail-
able 2-amino-4-fluorobenzoic acid (1a) or methyl 2-aminobenzoate (1b)
as starting material, compounds 2a-2b were synthesized by mechanical
◦
stirring with urea at 170 C for 12 h, which were then treated by POCl
3
via chlorination reaction with a few drops of DMF as catalyst to yield the
key intermediates 3a-3b. The intermediates 3a-3b with side chain
structure of different primary amines at room temperature for 3–4 h to
obtained intermediates 5a-5k by nucleophilic substitution reaction.
Finally, the target compounds 7a-7j, 8a-8i, 9a-9h and 10a-10k were
obtained by Suzuki reaction with side chains 6a-6e and intermediates
5a-5k as shown in Scheme 2. The expected structures of all target
According to research reports [23,27,28], the 2-aryl-4-aminoquina-
Del19/
zoline derivative Angew2017-7634-1 exhibited potent EGFR
T790M/C797S
kinase inhibitory activity with an IC50 value of 149 nM, and
WT
displayed approximately 163-fold selectivity over EGFR . Therefore,
in order to guide us to make a more reasonable transformation, we chose
T790M/C797S
EGFR
protein (PDB code: 5XGN [29]) to conduct molecular
1
13
1
docking study on Angew2017-7634-1. The results of the docking study
were showed in Fig. 2: (1) The hydroxyl group of phenol formed two
hydrogen bonds with MET793 and GLN791, while the aromatic ring of
phenol moiety made direct contact with the gatekeeper MET790. This
may be one of the reasons why the derivatives had high selectivity. (2)
The amino NH and nitrogen of the pendent pyridine group formed two
hydrogen bonds with the mutant SER797, which were necessary to
provide potent and selective EGFR triple mutation inhibition. In addi-
tion, as shown in Fig. 2C and 2D, the aromatic ring of phenol pointed to
the hydrophobic pocket produced by VAL726, LYS745, ASP855 and
compounds were confirmed by H NMR and C NMR. H NMR spectra
of all target compounds revealed an increase in the integration of the
aromatic protons indicating the presence of additional aromatic ring. In
addition, the target compounds (7b, 7g, 8b, 8g, 9a, 9c, 9e, 9g, 10b, 10f
2
and 10i) with hydroxyl group substituted R o-phenol showed the
singlet signals at 14.00–14.70 ppm corresponding to the -OH proton.
The target compounds (7d-7e, 7i-7j, 8d-8e, 8h-8i, 9b, 9d, 9f, 9h, 10c-
10d, 10g and 10j-10k) displayed the singlet signals at about 3.50–4.00
3
ppm assigned to the proton -OCH . Except for compounds 9e-9h and
10a-10k, the -CH- proton of other compounds appeared as a doublet
PHE856. Based on the analysis of the binding mode of Angew2017-
signals near 5.00 ppm.
T790M/C797S
7
634-1 with EGFR
, we simply drew a sketch of its binding
mode (Fig. 2A), and modified the compound on this basis.
2
2
.2. Biological evaluation
Accordingly, we optimized the structure of the lead compound
Angew2017-7634-1 to obtain a series of newly fourth-generation
EGFR-TKIs, and explored Angew2017-7634-1 active conformation,
and provided guidance for the design of fourth-generation EGFR-TKIs in
the future. The design strategy was showed in Fig. 3. Firstly, by
analyzing the binding mode of the hinge region, we retained its active
group and introduced some suitable hydrogen bond acceptor groups
.2.1. Anti-proliferative activity
The ant-proliferative activity of the target compounds 7a-7j, 8a-8h,
9
a-9h and 10a-10k were evaluated by MTT method against A549
(
human lung cancer cell lines), NCI-H460 (human lung cancer cell lines)
and H1975 (human lung adenocarcinoma cell lines) with AZD9291 as
positive control. Although the structure of some compounds had been
reported, the anti-proliferative activity in vitro had not been described in
detail [23,30]. Therefore, we also conducted in vitro anti-proliferative
assays on them. The ant-proliferative activity results of compounds
were showed as IC50 values were summarized in Tables 1–3. Moreover,
the structure–activity-relationship (SAR) of the target compounds was
discussed. As showed in Tables 1–3, most of the target compounds
exhibited more potent anti-proliferative activity against A549 cell lines
than NCI-H460 cell lines. The results revealed that most of the target
(
2 3
such as -NH , -OCH ). Meanwhile, the influence of phenolic hydroxyl
group’s position in the aromatic ring and whether there being nitrogen
in the benzene ring on the activity of the compounds were explored.
Secondly, in order to maintain the inhibitory activity of compounds, the
amino NH was retained to form a critical hydrogen bond with SER797.
Then, according to the bioisosterism, heterocyclic pyrazine and other
different five-membered heterocycles were introduced to replace 3-pyr-
idyl to form a hydrogen bond with SER797. Finally, the factors that
influence the formation of hydrogen bond in SER797 were discussed by
increasing the carbon chain length between the heterocyclic ring and the
amino NH. We appropriately increased the carbon chain length between
the heterocyclic ring and the amino NH. Based on the above, the target
compounds 7a-7j, 8a-8i, 9a-9h and 10a-10k were obtained.
compounds exhibited poor (IC50 > 100 or NA) inhibition against the
L858R / T790M
EGFR
double mutant cell lines H1975.
It has been reported that fluorine atom was widely used in medicinal
chemistry, which can enhance the biological activity of compounds
[
31]. Therefore, a fluorine atom was introduced to the 7 position of the
Fig. 1. The structure of representative EGFR inhibitors and their binding sites with proteins. (A) The binding site of the first-generation EGFR inhibitor Gefitinib and
protein (PDB code: 4I22). (B) The binding site of the second-generation EGFR inhibitor Afatinib and protein (PDB code: 4G5P). (C) The binding site of the third-
generation EGFR inhibitor Osimertinib and protein (PDB code: 6JX0). (D) The binding site of the fourth-generation EGFR inhibitor EAI045 and protein (PDB
code: 5ZWJ).
2

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
Fig. 2. Binding model of lead compound Angew2017-7634-1. (A) Simple diagram of the binding mode of Angew2017-7634-1 with amino acids. (B) The ribbon
T790M/C797S
T790M/C797S
binding diagram of Angew2017-7634-1 with EGFR
cavity binding diagram of Angew2017-7634-1 with EGFR
(PDB code: 5XGN). (C) The binding model of Angew2017-7634-1 bound to EGFR
. (D) The
T790M/C797S
.
Fig. 3. Design strategy for target compounds 7a-7j, 8a-8i, 9a-9h and 10a-10k.
◦
◦
Scheme 1. The synthetic route of key intermediates 3a-3b. Reagents and conditions: (a) urea, 170 C, 12 h; (b) POCl
3
, 110 C, 6 h.
quinazoline ring, which was expected to enhance the activity of the
target compound. The results of anti-proliferative activity in vitro were
showed in Table 1. Unfortunately, we found that the introduction of
fluorine atom decrease the inhibitory activity of target compounds
against the three cell lines, such as 7a and 7f, 7c and 7h. Then, we
investigated the biological activity effect of phenolic hydroxyl positions
on aromatic rings, and found that the o-phenolic hydroxyl group
derivatives on aryl groups exhibited stronger/more potent anti-
proliferative activity compared to meta-phenolic hydroxyl groups,
such as 7a and 7b (Angew2017-7634-1). It is worth noting that when
the position 7 of the quinazoline ring of the target compound was
replaced by a fluorine atom, the anti-proliferative activity of the com-
pound 7g (IC50 = 18.72
μ
M) was significantly improved against the
M). This may be the
A549 cell lines as compound 7b (IC50 = 49.89
μ
3

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
Scheme 2. The synthetic route of target compounds. Reagents and conditions: (a) THF, tri-ethylamine, r.t., 3–4 h; (b) 1,4-dioxane, Bis(tri-phenyl phosphine)
palladium (II) dichloride, boracic acid or 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, K
2
CO
3
, 80–90 ◦C, 5 h.
Table 1
Structures and activity of the target compounds 7a-7j and 8a-8i.
a
a
Compd.
R
1
R
2
X
IC50 (µM ± SD)
% Inhibition (10 µM)
L858R/T790M
Del19/T790M/C797S
A549
NCI-H460
H1975 (EGFR
)
Ba/F3-EGFR
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
H
H
H
H
H
F
3-OH
2-OH
2-NH
C
C
C
C
N
C
C
C
C
N
C
C
C
C
N
C
C
C
N
–
10.94 ± 1.04
49.89 ± 1.70
10.04 ± 1.00
15.47 ± 1.04
>100
23.21 ± 1.36
43.23 ± 1.64
59.03 ± 1.77
21.99 ± 1.34
28.29 ± 1.45
NA
1.52
94.19
1.85
NA
NA
2
23.32 ± 1.37
19.96 ± 1.30
>100
>100
NA
2-OCH
2-OCH
3-OH
3
3
2.77
NA
50.48 ± 1.70
18.72 ± 1.27
24.90 ± 1.40
NA
NA
g
h
i
F
2-OH
NA
NA
F
2-NH
2
48.03 ± 1.68
NA
>100
NA
F
2-OCH
2-OCH
3-OH
3
3
NA
NA
j
F
42.58 ± 1.63
28.90 ± 1.46
>100
NA
46.28 ± 1.67
>100
3.46
NA
a
b
c
d
e
f
H
H
H
H
H
F
>100
>100
NA
2-OH
59.22 ± 1.77
NA
98.93
13.84
NA
2-NH
2
61.18 ± 1.88
45.64 ± 1.66
NA
2-OCH
2-OCH
3-OH
2-OH
2-OCH
2-OCH
–
3
3
>100
NA
>100
NA
NA
>100
NA
>100
6.73
49.23
NA
g
h
i
F
37.60 ± 1.58
>100
NA
53.12 ± 1.37
>100
F
3
3
>100
NA
F
>100
NA
NA
b
AZD9291
–
0.66 ± 0.08
Nd
0.073 ± 0.15
–
NA: Not active.
Nd: Not done.
a
The values are an average of three separate determinations.
Used as a positive control.
b
result involved in the interaction of the fluorine atom with the o-
phenolic hydroxyl group on the aromatic ring. After introducing
different hydrogen bond acceptor groups to the side chain aryl group
and exploring the effect of the presence of nitrogen in the benzene ring
on the activity of the compound, we found that the anti-proliferative
activity of the target compounds 7c and 7d were increased against the
above three cell lines besides compound 7e. In particularly, compounds
heterocyclic rings (2-pyrazine, 2-thiophene, tetra-hydrofuran-3-yl) had a
great influence on the activity of the all target compounds. The possible
reason could be that the heteroatoms on these heterocyclic do not
interact with the amino acid residues of EGFR proteins. Fortunately, the
compounds 8a and 8g showed moderate inhibitory activity against
A549 cell lines, with IC50 values of 28.90 ± 1.46
μM and 37.60 ± 1.58
μ
M, respectively. With the replacement of a five-membered heterocyclic
7
c and 7d exhibited excellent anti-proliferative activity against H1975
M,
ring, the anti-proliferative activity of compounds 9b, 9e and 9f against
the three cell lines were better than that of the lead compound
Angew2017-7634-1 (7b). In particularly, compared with the lead
compound Angew2017-7634-1 (7b), compound 9e exhibited the most
excellent inhibitory activity against the three cell lines, with IC50 values
cell lines, with the IC50 values of 21.99 ± 1.34 μM and 28.29 ± 1.45 μ
respectively. Based on the bioisosterism, we introduced different six-
membered or five-membered heterocyclic rings to observe the change
in the activity of the target compounds. Therefore, the compounds 8a-8i
and 9a-9h were synthesized and tested for anti-proliferative activity,
and the anti-proliferative activity was showed in Tables 1 and 2. The
results showed that the introduction of six- and five-membered
of 14.33 ± 1.16
In addition, the IC50 values of compound 9b against A549 and NCI-H460
cell lines were 15.45 ± 1.19 M and 17.04 ± 1.23 M, respectively. As
μM, 17.81 ± 1.25
μM and 13.41 ± 1.14
μM, respectively.
μ
μ
4

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
Table 2
Structures and activity of the target compounds 9a-9h.
a
a
Compd.
R
1
R
2
X
IC50
(
μ
M ± SD)
% Inhibition (10 µM)
L858R/T790M
Del19/T790M/C797S
A549
NCI-H460
H1975 (EGFR
)
Ba/F3-EGFR
9
9
9
9
9
9
9
9
a
b
c
d
e
f
H
H
F
2-OH
2-OCH
2-OH
2-OCH
2-OH
2-OCH
2-OH
2-OCH
–
C
C
C
N
C
C
N
N
–
>100
>100
NA
3.21
56.64
19.55
19.76
91.00
7.52
24.15
NA
3
3
3
3
15.45 ± 1.19
NA
17.04 ± 1.23
NA
32.21 ± 1.51
>100
F
>100
>100
NA
H
H
F
14.33 ± ±1.16
24.04 ± 1.38
>100
17.81 ± ±1.25
34.50 ± 1.54
NA
13.41 ± ±1.14
37.88 ± 1,58
44.32 ± 1.67
>100
g
h
F
60.91 ± 1.79
0.66 ± 0.08
>100
b
AZD9291
–
Nd
0.073 ± 0.15
–
NA: Not active.
Nd: Not done.
a
The values are an average of three separate determinations.
Used as a positive control.
b
Table 3
Structures and activity of target compounds 10a-10k.
a
a
Compd.
R
1
R
2
X
IC50
(
μ
M ± SD)
% Inhibition (10 µM)
L858R/T790M
Del19/T790M/C797S
A549
NCI-H460
H1975 (EGFR
)
Ba/F3-EGFR
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
H
H
H
H
F
3-OH
2-OH
2-OCH
2-OCH
3-OH
2-OH
2-OCH
3-OH
2-OH
2-OCH
2-OCH
–
C
C
C
N
C
C
C
C
C
C
N
–
NA
10.17 ± 1.01
53.68 ± 1.73
51.63 ± 1.71
>100
7.14
42.37 ± 1.63
93.98 ± 1.98
41.45 ± 1.62
NA
>100
NA
30.02
49.59
42.27
33.29
28.78
25.82
60.68
68.55
37.63
5.76
3
3
NA
>100
90.11 ± 1.97
>100
NA
>100
F
NA
59.10 ± 1.84
NA
0g
0h
0i
F
3
>100
H
H
H
H
–
54.74 ± 1.16
>100
NA
>100
>100
NA
46.15 ± 1.54
>100
0j
3
3
NA
0k
NA
NA
>100
b
AZD9291
0.66 ± 0.08
Nd
0.073 ± 0.15
–
NA: Not active.
Nd: Not done.
a
The values are an average of three separate determinations.
Used as a positive control.
b
shown in Table 3, with the increasing length of the carbon chain, the
anti-proliferative activity of the target compounds against NCI-H460 cell
lines were decreased, except for the compound 10a with the IC50 value
compound Angew2017-7634-1 (7b), with an inhibition rate of 91.00%.
Interestingly, although compounds 10h and 10i basically lost their
inhibitory activity against these three types of cell lines, their inhibitory
Del19/T790M/C797S
of 10.17 ± 1.01
μM.
rates against EGFR
triple-mutant cell lines were reached
to 60.68% and 68.55%, respectively. Although most of the compounds
did not showed excellent inhibitory activity, compound 9e exhibited the
potential to inhibit the Del19/T790M/C797S triple-mutation at a con-
2
.2.2. Inhibitory activity of all target compounds against Ba/F3-EGFRDel19/
T790M/C797S
cells in vitro
In order to study the inhibitory ability of all target compounds
centration of 10 M.
μ
T790M/C797S
Del19/T790M/C797S
against EGFR
mutations, the Ba/F3-EGFR
cell lines were tested. The inhibitory activity of all target compounds on
2.2.3. Kinase inhibitory activity of partially preferred target compounds
Inspired by the excellent anti-proliferative activity of compounds 7b-
7d, 7h, 7j, 8g, 9b, 9e and 10a-10b, all of them were screened for further
kinase inhibition assays. As shown in Table 4, most IC50 values of
Ba/F3 (mouse pro-B) cell lines was showed in Tables 1–3. Among of
them, the inhibitory effect of target compound 9e was slightly lower
Del19/T790M/C797S
against EGFR
triple-mutant cell lines than that of lead
5

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
Table 4
WT
T790M/L858R
Activity of target compounds against EGFR and EGFR
.
a
c
a
c
Compd.
IC50
(μ
M)
Selectivity
Compd.
IC50
(μM)
Selectivity
WT
L8585R/T790M
WT
L8585R/T790M
EGFR
EGFR
EGFR
EGFR
7
7
7
7
7
8
b
c
d
h
j
>10
>10
>10
>10
>10
>10
1.58
>10
>10
>10
>10
2.55
>6.3
9b
>10
>10
>10
>10
>10
–
–
9e
0.74
>10
>13.5
–
–
10a
–
10b
Afatinib
>10
–
0.6
2.3
b
–
0.006
0.028
0.010
0.012
b
g
>3.9
AZD9291
Nd: Not done.
a
The values are an average of three separate determinations.
Used as a positive control.
b
c
WT
L8585R/T790M
IC50 (EGFR )/IC50 (EGFR
).
compounds were greater than 10
μ
M against EGFRWT and EGFRL858R/
kinase. It is worth noting that compounds 8g and 9e showed
percentage of 6.87%, while 9e showed a high percentage of 21.52% at
the concentration of 10 M. It is worth noting that compound 9e could
T790M
μ
L858R/T790M
strong inhibitory activity against EGFR
, with IC50 values of
also induce apoptosis of cells in the early stage, and the percentage of
apoptosis is as high as 21.85%, which further indicated that the target
compound 9e had a significant inhibitory effect on lung cancer cells.
2
.55 M and 0.74 M, respectively. In addition, the mutation selectivity
μ
μ
WT
of 9e was 13.5-times higher than that of EGFR . Compared with the
positive compound AZD9291, the weaker activity of the compound may
be due to the lack of covalent interaction with CYS797.
2.2.6. Cell cycle analysis of 9e
For the purpose of further studying the cell mechanism induced by
the optimized compound 9e, the cell cycle arrest experiment of 9e
blocking the cell cycle of A549 was carried out. We used flow cytometry
to test the cell cycle arrest of A549 cell lines after the 48 h treatment of
compound 9e. As shown in Fig. 6, compared with the blank control
group, the cell cycle of A549 was arrested in S-phase with the increased
concentration of compound, and showed significant concentration-
dependence manner.
2
.2.4. Morphology of cells by fluorescence microscopy of 9e.
The compound 9e with excellent activity was selected to conduct AO
fluorescence staining experiments for study the anti-proliferative and
apoptosis effects on A549 cell lines. As shown in Fig. 4, the control group
cells without any treatment showed a uniform and regular shape of
normal A549 cell lines, which were tightly arranged and orderly. After
cell lines were treated with compound 9e for 12 h, a series of apoptosis
was occurred. Compared with the control group, the number of A549
cell lines decreased, the fluorescence intensity increased significantly,
the edge of cells were sharp, the chromatin contracted significantly,
apoptotic bodies and cell fragments appeared, and the cell structure was
destroyed. With the concentration of compound 9e changed from 0.1
2.2.7. Docking study
In order to explore the binding mode of the target compound 9e and
the active site of EGFR, a molecular docking simulation study was car-
ried out using Auto-Dock and PYMOL software. The docking results were
μ
M to 1
μ
M, the apoptotic bodies was increased and chromatin
showed in Fig. 7. Fig. 7A and B showed that 9e could penetrate into the
L858R/T790M
contraction became more serious, which also indicated that 9e could
induce apoptosis of A549 cell lines in a concentration–dependent
manner.
EGFR
protein cavity, and the phenol structure formed a bi-
dentate hydrogen bond with the hinge region MET793, which maybe the
L858R/T790M
reason why 9e exhibited inhibitory activity against EGFR
mutation. Compared with the positive control AZD9291 (as shown in
Fig. 1C), compound 9e did not have any interaction with amino acid
residue CYS797, which may be the reason for its lower activity than
AZD9291. This docking result was consistent with our previous guess.
The compound 9e could bind closely with the protein, as shown in
Fig. 7C and 7D. The phenol substituted at position 2 of quinazoline faced
MET790 and extended into the hydrophobic cavity. It could not only
form a key hydrogen bond with the hinge region MET793, but also
interact with amino acid residues GLN791 to form a hydrogen bond. The
side chain amino part substituted at position 4 extended into the solvent
2
.2.5. Apoptosis result analysis of 9e.
In order to better accurately verify the apoptosis and cyto-
fluorimetric mechanism of 9e, 9e was carried out through the exposure
of Annexin V marker in the A549 cell lines. Fig. 5 showed the apoptosis
and cytofluorimetric situation of A549 cell lines treated with compound
9
e at the different concentrations of 2.5
μM, 5
μ
M and 10 M for 24 h.
μ
The results showed that compound 9e could induce apoptosis of cells in
a concentration-dependent manner. In addition, compared with the lead
compound Angew2017-7634-1 (7b), 9e induced late-apoptosis with a
Fig. 4. Morphology of cells by fluorescence microscopy of compound 9e.
6

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
Fig. 5. Cell-apoptosis result analysis of compound 9e. (Q2-1: mechanical damage; Q2-2: late apoptotic; Q2-3: living cells; Q2-4: early apoptotic).
Fig. 6. Cell cycle arrest of A549 cell lines by compound 9e.
region and formed two hydrogen bonds with amino acid residue
SER797, which was basically similar to the lead compound
Angew2017-7634-1 (7b). It is worth noting that Fig. 7D showed that
there was still a large cavity at the para-position of phenol, which could
accommodate a larger molecular structure, which will provide a new
design strategy for our subsequent structural transformation.
experiments showed that 9e could block A549 cell lines in the S-phase in
a concentration-dependent manner. Molecular docking study exhibited
that 9e could bind tightly to the EGFR protein, and the binding mode to
T790M/C797S
the EGFR
protein was basically the same as the lead com-
pound Angew2017-7634-1 (7b). All in all, it suggested that the target
compound 9e was expected to be a potential EGFR inhibitor to over-
L858R/T790M
Del19/T790M/C797S
come EGFR
and EGFR
mutations, which is
3
. Conclusion
worthy of our further study.
In this article, we designed and synthesized six series of 2-aryl-4-ami-
4. Experiment
noquinazoline derivatives, and evaluated their anti-proliferative activity
and enzyme inhibitory activity in vitro. Among them, compound 9e, as
the most promising derivative, not only exhibited excellent anti-
4.1. General
proliferative activity against A549, NCI-H460 and H1975 cell lines,
Unless otherwise required, all reagents used in the experiment were
purchased from commercial analytical grade and used without further
purification. Frequently used solvents (Ethanol, petroleum ether, ethyl
acetate and dichloromethane, etc.) were absolutely anhydrous. All ac-
tions were monitored through GF254 TLC plate and spots were observed
with iodine or light under 254 nm or 365 nm. Tetramethylsilane (TMS)
being an internal standard, the structures of the target compounds were
confirmed by 1H NMR and C NMR spectra on Bruker 400 MHz spec-
trometer (Bruker Bioscience Billerica, MA, USA) at room temperature.
Del19/T790M/C797S
but also inhibited EGFR
triple mutant Ba/F3 cell lines
M. In the kinase experiment, the
by 91.0% at the concentration of 10
μ
most promising compound 9e exhibited excellent enzymatic inhibitory
L858R/T790M
activity and selectivity for EGFR
, with an IC50 value of 0.74
μ
M. Therefore, we performed further pharmacological experiments to
compound 9e. Annexin V-FITC staining and acridine orange (AO)
staining tests showed that 9e could induce late-apoptosis of A549 cell
lines in a concentration-dependent manner. In addition, cell cycle
13
7

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
L858R/T790M
Fig. 7. Binding model of 9e. (A) The protein cavity binding diagram of 9e with EGFR
(PDB code: 6JX0). (B) The ribbon binding diagram of 9e with
L858R/T790M
T790M/C797S
T790M/C797S
EGFR
. (C) The ribbon binding diagram of 9e with EGFR
(PDB code: 5XGN). (D) The binding model of 9e bound to EGFR
.
Waters High Resolution Quadrupole Time of Flight Tandem Mass
Spectrometry (QTOF) was studied by Mass spectrometry (MS). The pu-
rity of the compounds was determined by Agilent 1260 liquid chro-
matograph fitted with an Inertex-C18 column. All target compounds
have purity greater than or equal to 95% [32,33]. All the materials were
obtained from commercial suppliers and used without purification,
unless otherwise specified. Compound 7a-7d and 8a-8d were previously
reported [23,30].
4.3. General procedure for preparation of key intermediates 3a-3b
The intermediates 2a-2b (25.3 mmol) were dissolved in phosphorus
oxychloride (30 mL). The reaction was catalyzed by N, N-dime-
◦
thylformamide (0.1 mL) and stirred for 6 h at 110 C. The completion of
the reaction was monitored by TLC. The reaction mixture was evapo-
rated under reduced pressure to afford viscous oil, which was used in the
next reaction. Afterward, the mixture was transferred to a beaker with
ice water acetate added slowly, and then filtered to obtain key in-
termediates 3a-3b.
4
.2. General procedure for preparation of key intermediates 2a-2b
-amino-4-fluorobenzoic acid 1a (10.0 g, 64.5 mmol) or methyl 2-
2
4.3.1. 2,4-dichloroquinazoline (3a)
aminobenzoate 1b (10.0 g, 65.4 mmol) was reacted with urea at
Light brown solid; Yield: 90.2%; 1H NMR (400 MHz, DMSO‑d
6
) δ
◦
1
70 C for 12 h under mechanical agitation. When TLC analysis indi-
(ppm) 8.30 (d, J = 8.4 Hz, 1H), 8.22–8.14 (m, 1H), 8.05 (d, J = 8.5 Hz,
1H), 7.96–7.88 (m, 1H).
cated that the reaction had gone to completion, the reaction mixture was
cooled to room temperature and was poured into saturated sodium bi-
carbonate (NaHCO
3
) aqueous solution. And then the reacted solution
4.3.2. 2,4-dichloro-7-fluoroquinazoline (3b)
was filtered after stirred for 0.5 h. After-ward, the filter residue was
added to aqueous solution of hydrochloric acid (HCl) and stirred for 3–4
h. The insoluble material was collected by suction filtration and dried to
give the key intermediates 2a-2b.
Brown solid; Yield: 93.5%; 1H NMR (400 MHz, DMSO‑d
6
) δ (ppm)
8.40 (dd, J = 9.2, 5.8 Hz, 1H), 7.92 (dd, J = 9.6, 2.5 Hz, 1H), 7.83 (td, J
= 8.9, 2.6 Hz, 1H).
4
.4. General procedure for preparation of intermediates 5a-5k
4
.2.1. 7-fluoroquinazoline-2,4-diol (2a)
1
A brown solid; Yield: 90.7%; H NMR (400 MHz, DMSO‑d
6
) δ (ppm)
The key intermediates 4a-4f (7.5 mmol) and tri-ethylamine (3 d)
1
1.36 (s, 1H), 11.26 (s, 1H), 7.93 (s, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.88
were dissolved in in THF (10 mL) stirred at room temperature for 10
min. Then, the compounds 3a-3b (5.0 mmol) dissolved in THF (15 mL)
were added dropwise to above mixture, and stirred for 3–4 h. Reaction
was monitored by thin-layer chromatography (TLC). The mixture was
concentrated under reduced pressure to obtain a solid. Finally, the solid
was dissolved an appropriate amount of water and then stirred and fil-
trated. Insoluble was collected and dried to obtain compounds 5a-5k.
(
d, J = 9.4 Hz, 1H).
4
.2.2. Quinazoline-2,4-diol (2b)
1
6
A yellow solid; Yield: 92.5%; H NMR (400 MHz, DMSO‑d ) δ (ppm)
1.20 (s, 2H), 7.87 (dd, J = 8.0, 1.3 Hz, 1H), 7.65–7.57 (m, 1H),
1
7
.19–7.10 (m, 2H).
4
.4.1. 2-chloro-N-(pyridin-3-ylmethyl)quinazolin-4-amine (5a)
1
A light yellow solid; Yield: 92.3%; H NMR (400 MHz, DMSO‑d
6
) δ
8

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
(
ppm) 9.31 (t, J = 5.6 Hz, 1H), 8.61 (s, 1H), 8.46 (d, J = 4.2 Hz, 1H),
7.62 (d, J = 8.3 Hz, 1H), 7.57–7.50 (m, 1H), 7.33 (d, J = 4.9 Hz, 1H),
6.99–6.91 (m, 2H), 3.76 (q, J = 6.6 Hz, 2H), 3.20 (t, J = 7.1 Hz, 2H).
8
7
.27 (d, J = 8.2 Hz, 1H), 7.80–7.74 (m, 2H), 7.62 (d, J = 8.4 Hz, 1H),
.54 (t, J = 7.6 Hz, 1H), 7.35 (dd, J = 7.8, 4.8 Hz, 1H), 4.74 (d, J = 5.6
Hz, 2H).
4.4.11. 2-chloro-7-fluoro-N-(2-(thiophen-2-yl)ethyl)quinazolin-4-amine
(
5k)
1
4
.4.2. 2-chloro-7-fluoro-N-(pyridin-3-ylmethyl)quinazolin-4-amine (5b)
A yellow solid; Yield: 90.6%; H NMR (400 MHz, DMSO‑d ) δ (ppm)
6
1
A yellow solid; Yield: 90.1%; H NMR (400 MHz, DMSO‑d
6
) δ (ppm)
8.98 (t, J = 5.4 Hz, 1H), 8.50–8.27 (m, 1H), 7.44 (dd, J = 16.7, 7.5 Hz,
2H), 7.33 (d, J = 4.9 Hz, 1H), 6.94 (d, J = 6.2 Hz, 2H), 3.93–3.67 (m,
2H), 3.19 (t, J = 7.1 Hz, 2H).
9
.42 (s, 1H), 8.63 (s, 1H), 8.48 (d, J = 4.8 Hz, 1H), 8.39 (dd, J = 9.1, 5.9
Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.42 (d, J =
1
0.1 Hz, 1H), 7.39–7.34 (m, 1H), 4.75 (d, J = 5.6 Hz, 2H).
4
.5. Preparation of target compounds 7a-7j, 8a-8i, 9a-9h and 10a-10k
4
.4.3. 2-chloro-N-(pyrazin-2-ylmethyl)quinazolin-4-amine (5c)
1
A light yellow solid; Yield: 94.2%; H NMR (400 MHz, DMSO‑d
6
) δ
Under the condition of nitrogen protection, compounds 5a-5k (0.4
(
ppm) 9.43 (t, J = 5.8 Hz, 1H), 8.73 (d, J = 1.4 Hz, 1H), 8.60 (s, 1H),
mmol), potassium carbonate solid (0.1 g, 0.7 mmol), bis(triphenyl-
phosphine) palladium (II) dichloride and compounds 6a-6e (0.6 mmol)
were added to the mixed solvent (1,4-dioxane:water = 4:1) in sequence
8
1
.55 (d, J = 2.6 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 7.82 (t, J = 7.7 Hz,
H), 7.65 (d, J = 8.3 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 4.88 (d, J = 5.6
◦
Hz, 2H).
and stirred for 5 h at 80–90 C. The reaction was monitored by thin-layer
chromatography (TLC). The reaction liquid was filtered, and then the
filtrate was evaporated under reduced pressure. The crude product was
purified by silica gel chromatography using MeOH/CH Cl (v/v, from
4
.4.4. 2-chloro-7-fluoro-N-(pyrazin-2-ylmethyl)quinazolin-4-amine (5d)
1
A yellow solid; Yield: 92.5%; H NMR (400 MHz, DMSO‑d
6
) δ (ppm)
2
2
9
.50 (t, J = 5.7 Hz, 1H), 8.73 (s, 1H), 8.60 (s, 1H), 8.55 (s, 1H), 8.41 (dd,
100:1 to 50:1) to obtain target compounds 7a-7j, 8a-8i, 9a-9h and 10a-
10k.
J = 9.1, 5.9 Hz, 1H), 7.50 (t, J = 8.9 Hz, 1H), 7.43 (d, J = 10.1 Hz, 1H),
4
.87 (d, J = 5.6 Hz, 2H).
4
.5.1. 3-(4-((pyridin-3-ylmethyl)amino)quinazolin-2-yl)phenol (7a)
A pale white solid; Yield: 60.5%; m.p.: 217.4–219.2 C; 1H NMR
◦
4
.4.5. 2-chloro-N-(thiophen-2-ylmethyl)quinazolin-4-amine (5e)
1
A yellow solid; Yield: 91.8%; H NMR (400 MHz, DMSO‑d
6
) δ (ppm)
(400 MHz, DMSO‑d ) δ (ppm) 9.50 (s, 1H), 8.95 (t, J = 5.7 Hz, 1H), 8.75
6
9
7
1
5
.39 (t, J = 5.9 Hz, 1H), 8.25 (d, J = 8.3 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H),
.64 (d, J = 8.3 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.40 (d, J = 5.0 Hz,
H), 7.11 (d, J = 3.5 Hz, 1H), 6.98 (dd, J = 5.1, 3.4 Hz, 1H), 4.89 (d, J =
.7 Hz, 2H).
(s, 1H), 8.46 (d, J = 3.8 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H), 7.95–7.86 (m,
4H), 7.77 (s, 1H), 7.50 (dd, J = 11.1, 5.1 Hz, 1H), 7.37 (dd, J = 7.7, 4.9
Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 6.86 (dd, J = 8.0, 1.8 Hz, 1H), 4.93 (d,
J = 5.6 Hz, 2H).
4
.4.6. 2-chloro-7-fluoro-N-(thiophen-2-ylmethyl)quinazolin-4-amine (5f)
4.5.2. 2-(4-((pyridin-3-ylmethyl)amino)quinazolin-2-yl)phenol (7b)
A pale white solid; Yield: 61.2%; m.p.: 222.8–225.1 C; 1H NMR
1
◦
A yellow solid; Yield: 89.9%; H NMR (400 MHz, DMSO‑d
6
) δ (ppm)
9
2
.47 (t, J = 5.9 Hz, 1H), 8.35 (dd, J = 9.1, 5.9 Hz, 1H), 7.48 (dd, J = 8.8,
(400 MHz, DMSO‑d ) δ (ppm) 14.67 (s, 1H, -OH), 9.29 (t, J = 5.8 Hz, 1H,
6
.7 Hz, 1H), 7.43 (d, J = 10.1 Hz, 1H), 7.40 (d, J = 5.5 Hz, 1H), 7.11 (d,
-NH-), 8.73 (s, 1H, pyridine, H ), 8.45 (d, J = 3.9 Hz, 1H, pyridine, H ),
2
6
J = 3.4 Hz, 1H), 7.00–6.95 (m, 1H), 4.88 (d, J = 5.7 Hz, 2H).
8.40 (dd, J = 8.2, 1.9 Hz, 1H, quinazoline, H ), 8.33 (d, J = 8.2 Hz, 1H,
8
pyridine, H
4
), 7.84 (dd, J = 15.4, 7.4 Hz, 2H, quinazoline, H
5 7
, H ), 7.77
4
.4.7. 2-chloro-N-((tetrahydrofuran-3-yl)methyl)quinazolin-4-amine (5
(d, J = 8.5 Hz, 1H, quinazoline, H ), 7.57 (d, J = 6.5 Hz, 1H, phenyl, H ),
6
6
g)
7.34 (t, J = 8.2 Hz, 2H, phenyl, H , pyridine, H ), 6.90 (t, J = 6.3 Hz, 2H,
4
5
A pale white solid; Yield: 90.5%; 1H NMR (400 MHz, DMSO‑d
ppm) 8.81 (t, J = 5.5 Hz, 1H), 8.27 (d, J = 8.3 Hz, 1H), 7.79 (t, J = 7.7
13
6
) δ
phenyl, H , H ), 4.93 (d, J = 5.7 Hz, 2H, -CH -). C NMR (101 MHz,
3
5
2
(
DMSO‑d ) δ 160.71, 160.65, 159.00, 148.64, 148.00, 146.57, 135.34,
6
Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 3.82–3.74 (m,
134.79, 133.75, 132.53, 128.94, 126.14, 126.01, 123.63, 122.71,
ꢀ
1
3
H), 3.73–3.67 (m, 1H), 3.63 (d, J = 7.4 Hz, 1H), 3.59–3.49 (m, 2H),
.49–3.44 (m, 1H), 2.66 (hept, J = 5.6 Hz, 1H), 2.04–1.92 (m, 1H), 1.65
118.95, 118.36, 117.17, 113.25, 41.79. TOF MS ES – (m/z): (Mꢀ H) ,
calcd for C19
15 5
H N O: 327.1246, found, 327.1246.
Angew2017-7634-1 (A1) [23,30]: A yellow solid; Yield: 74%; 1H
NMR (400 MHz, DMSO‑d
(
dq, J = 12.9, 6.6 Hz, 1H).
6
) δ (ppm) 14.68 (s, 1H), 9.31 (t, J = 5.8 Hz,
4
.4.8. 2-chloro-7-fluoro-N-((tetrahydrofuran-3-yl)methyl)quinazolin-4-
amine (5h)
A pale white solid; Yield: 87.9%; 1H NMR (400 MHz, DMSO‑d
1H), 8.73 (s, 1H), 8.46 (d, J = 4.5 Hz, 1H), 8.40 (dd, J = 8.2, 1.8 Hz, 1H),
8.34 (dd, J = 8.3, 1.3 Hz, 1H), 7.88–7.81 (m, 2H), 7.78 (dd, J = 8.4, 1.3
Hz, 1H), 7.57 (ddd, J = 8.2, 6.8, 1.4 Hz, 1H), 7.39–7.32 (m, 2H),
6
) δ
1
3
(
ppm) 8.87 (t, J = 5.3 Hz, 1H), 8.36 (dd, J = 9.1, 6.0 Hz, 1H), 7.45 (td, J
6.92–6.88 (m, 2H), 4.93 (d, J = 5.8 Hz, 2H). C NMR (150 MHz,
=
8.9, 2.8 Hz, 1H), 7.38 (dd, J = 10.2, 2.8 Hz, 1H), 3.82–3.74 (m, 1H),
DMSO‑d ) δ 160.8, 160.8, 159.0, 149.0, 148.2, 146.6, 135.2, 134.7,
6
3
1
2
.73–3.67 (m, 1H), 3.63 (q, J = 7.6 Hz, 1H), 3.54 (dd, J = 8.5, 4.9 Hz,
H), 3.49 (dd, J = 11.7, 4.4 Hz, 1H), 3.43 (dd, J = 13.3, 7.7 Hz, 1H),
.64 (p, J = 7.0, 6.2 Hz, 1H), 2.04–1.93 (m, 1H), 1.64 (dq, J = 12.3, 6.6
133.7, 132.5, 129.0, 126.1, 126.0, 123.6, 123.0, 119.0, 118.3, 117.2,
113.4, 41.8.
Hz, 1H).
4.5.3. 2-(2-aminophenyl)-N-(pyridin-3-ylmethyl)quinazolin-4-amine (7c)
◦
1
A faint yellow solid; Yield: 50.8%; m.p.: 201.3–203.8 C; H NMR
4
.4.9. 2-chloro-N-(2-(pyridin-3-yl)ethyl)quinazolin-4-amine (5i)
(400 MHz, DMSO‑d ) δ (ppm) 8.95 (s, 1H), 8.70 (s, 1H), 8.44 (d, J = 4.5
6
1
A yellow solid; Yield: 94.3%; H NMR (400 MHz, DMSO‑d
.86 (t, J = 5.5 Hz, 1H), 8.47 (s, 1H), 8.41 (d, J = 4.9 Hz, 1H), 8.22 (d, J
8.2 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.61 (d,
6
) δ (ppm)
Hz, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 7.83 (d, J =
8
8.0 Hz, 1H), 7.76 (d, J = 5.4 Hz, 2H), 7.67–7.59 (m, 1H), 7.55 (d, J = 6.9
Hz, 1H), 7.51–7.46 (m, 1H), 7.34 (dd, J = 7.7, 4.9 Hz, 1H), 7.10 (t, J =
7.5 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.54 (t, J = 7.4 Hz, 1H), 4.88 (d, J
= 5.7 Hz, 2H).
=
J = 8.3 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.30 (dd, J = 7.8, 4.8 Hz, 1H),
3
4
8
.76 (q, J = 6.6 Hz, 2H), 2.99 (t, J = 7.1 Hz, 2H).
.4.10. 2-chloro-N-(2-(thiophen-2-yl)ethyl)quinazolin-4-amine (5j)
4.5.4. 2-(2-methoxyphenyl)-N-(pyridin-3-ylmethyl)quinazolin-4-amine
(7d)
1
A yellow solid; Yield: 93.0%; H NMR (400 MHz, DMSO‑d
6
) δ (ppm)
A pale white solid; Yield: 60.2%; m.p.: 151.8–154.1 ◦C; 1H NMR
.89 (t, J = 5.6 Hz, 1H), 8.24 (d, J = 8.2 Hz, 1H), 7.84–7.76 (m, 1H),
9

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
(
400 MHz, DMSO‑d
6
) δ (ppm) 11.10 (s, 1H), 8.81–8.68 (m, 2H), 8.50 (d,
(d, J = 3.1 Hz, 1H), 8.41–8.34 (m, 1H), 8.25 (d, J = 2.9 Hz, 1H), 7.98 (d,
J = 5.3 Hz, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.52–7.42 (m, 2H), 7.38–7.32
(m, 1H), 7.08 (dd, J = 7.4, 5.0 Hz, 1H), 4.79 (d, J = 5.7 Hz, 2H), 3.83 (s,
J = 4.7 Hz, 1H), 8.08–7.98 (m, 2H), 7.93 (d, J = 7.9 Hz, 1H), 7.81 (dd, J
=
7.7, 1.9 Hz, 1H), 7.76 (ddd, J = 8.4, 5.6, 2.8 Hz, 1H), 7.65 (t, J = 7.9
Hz, 1H), 7.40 (dd, J = 7.9, 4.6 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.17 (t,
3H). 13C NMR (101 MHz, DMSO‑d
) δ 165.78, 163.30, 161.15, 159.11,
6
J = 7.5 Hz, 1H), 4.97 (d, J = 5.5 Hz, 2H), 3.90 (s, 3H). TOF MS ES + (m/
151.89, 149.25, 148.12, 147.16, 139.56, 135.40, 134.96, 125.80,
+
z): (M+H) , calcd for C21
18 4
H N O: 343.1599, found, 343.1599.
123.55, 123.49, 116.63, 115.17, 111.68, 110.44, 53.17, 41.44. TOF MS
ES
+
(m/z): (M+H)+, calcd for
20 5
C H16FN O: 362.1417, found,
4
.5.5. 2-(2-methoxypyridin-3-yl)-N-(pyridin-3-ylmethyl)quinazolin-4-
362.1418.
amine (7e)
A pale white solid; Yield: 55.2%; m.p.: 151.8–154.1 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 8.96 (t, J = 5.8 Hz, 1H), 8.67 (s, 1H), 8.46
4.5.11. 3-(4-((pyrazin-2-ylmethyl)amino)quinazolin-2-yl)phenol (8a)
A faint yellow solid; Yield: 46.4%; m.p.: 187.9–190.4 ◦C; 1H NMR
(
(
6
d, J = 4.7 Hz, 1H), 8.28 (d, J = 8.3 Hz, 1H), 8.26–8.22 (m, 1H),
(400 MHz, DMSO‑d
6
) δ (ppm) 9.49 (s, 1H), 9.13 (d, J = 22.4 Hz, 1H),
8
7
.00–7.96 (m, 1H), 7.81 (dd, J = 14.4, 7.4 Hz, 2H), 7.77–7.72 (m, 1H),
.58–7.52 (m, 1H), 7.35 (dd, J = 7.9, 4.8 Hz, 1H), 7.08 (dd, J = 7.2, 5.0
8.79 (s, 1H), 8.60 (s, 1H), 8.52 (s, 1H), 8.33 (d, J = 8.2 Hz, 1H), 7.84 (s,
1H), 7.78 (s, 3H), 7.53 (t, J = 7.3 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.84
1
3
Hz, 1H), 4.80 (d, J = 5.8 Hz, 2H), 3.84 (s, 3H). C NMR (101 MHz,
(d, J = 8.2 Hz, 1H), 5.01 (d, J = 5.4 Hz, 2H). TOF MS ES + (m/z):
+
DMSO‑d
6
) δ 160.88, 159.91, 159.30, 149.40, 148.84, 147.84, 146.99,
(M+H) , calcd for C19
15 5
H N O: 330.1355, found, 330.1354.
1
1
39.57, 135.65, 135.22, 133.01, 127.43 (2C), 126.06, 123.59, 122.27,
ꢀ
16.76, 113.14, 53.21, 41.39. TOF MS ES – (m/z): (Mꢀ H) , calcd for
O: 342.1355, found, 342.1353.
4.5.12. 2-(4-((pyrazin-2-ylmethyl)amino)quinazolin-2-yl)phenol (8b)
A pale white solid; Yield: 44.7%; m.p.: 175.1–179.6 ◦C; 1H NMR
20 17 5
C H N
(
400 MHz, DMSO‑d
6
) δ (ppm) 14.60 (s, 1H), 9.43 (d, J = 5.5 Hz, 1H),
4
.5.6. 3-(7-fluoro-4-((pyridin-3-ylmethyl)amino)quinazolin-2-yl)phenol
7f)
A faint yellow solid; Yield: 58.8%; m.p.: 230.5–233.1 ◦C; 1H NMR
400 MHz, DMSO‑d ) δ (ppm) 9.56 (s, 1H), 9.14 (s, 1H), 8.74 (s, 1H),
.47 (s, 1H), 8.42–8.36 (m, 1H), 7.92–7.85 (m, 3H), 7.52–7.47 (m, 1H),
8.81 (s, 1H), 8.61 (d, J = 1.6 Hz, 1H), 8.51 (d, J = 2.6 Hz, 1H), 8.38 (d, J
= 8.2 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 7.88–7.82 (m, 1H), 7.59 (dd, J =
15.7, 7.5 Hz, 2H), 7.36–7.30 (m, 1H), 6.91–6.83 (m, 2H), 5.02 (d, J =
(
(
6
5.6 Hz, 2H). 13C NMR (101 MHz, DMSO‑d
) δ 160.77, 159.14, 154.14,
6
8
7
146.61, 143.99, 143.66, 143.20, 133.69, 132.43, 131.50, 128.98,
.44 (d, J = 8.7 Hz, 1H), 7.39 (dd, J = 11.7, 7.0 Hz, 1H), 7.27 (t, J = 7.9
128.77, 126.07, 123.07, 118.95, 118.16, 117.15, 113.42, 44.25. TOF
ꢀ
Hz, 1H), 6.89 (d, J = 7.9 Hz, 1H), 4.92 (d, J = 5.4 Hz, 2H). TOF MS ES +
MS ES ꢀ (m/z): (Mꢀ H) , calcd for C19
15 5
H N O: 328.1198, found,
+
(
m/z): (M+H) , calcd for C20
H
15FN
4
O: 347.1308, found, 347.1308.
328.1193.
A40 [23,30]: A yellow solid; Yield: 72%; 1H NMR (400 MHz,
4
.5.7. 2-(7-fluoro-4-((pyridin-3-ylmethyl)amino)quinazolin-2-yl)phenol
7g)
A pale white solid; Yield: 59.3%; m.p.: 206.8–209.5 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 14.34 (s, 1H), 9.36 (t, J = 5.8 Hz, 1H),
.72 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 8.39 (dd, J = 10.1, 8.0 Hz, 2H),
.85 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 10.2 Hz, 1H), 7.45 (t, J = 8.8 Hz,
DMSO‑d
6
) δ 14.61 (s, 1H), 9.43 (d, J = 5.6 Hz, 1H), 8.81 (d, J = 1.5 Hz,
(
1H), 8.61 (dd, J = 2.6, 1.5 Hz, 1H), 8.52 (d, J = 2.6 Hz, 1H), 8.38 (dd, J
= 8.3, 1.3 Hz, 1H), 8.23 (dd, J = 7.9, 1.8 Hz, 1H), 7.85 (ddd, J = 8.3, 6.8,
1.3 Hz,1H), 7.78 (dd, J = 8.3, 1.3 Hz, 1H), 7.58 (ddd, J = 8.3, 6.8, 1.3
Hz, 1H),7.32 (ddd, J = 8.5, 7.1, 1.8 Hz 1H), 6.92–6.84 (m, 1H),
6.88–6.81 (m, 1H), 5.02 (d, J = 5.6 Hz, 2H).
(
6
8
7
1
1
3
H), 7.38–7.32 (m, 2H), 6.94–6.86 (m, 2H), 4.90 (d, J = 5.6 Hz, 2H).
) δ 166.20, 163.71, 161.77, 160.83, 158.72,
48.97, 148.26, 135.20, 134.51, 132.71, 129.21, 126.17, 123.55,
C
NMR (101 MHz, DMSO‑d
6
4.5.13. 2-(2-aminophenyl)-N-(pyrazin-2-ylmethyl)quinazolin-4-amine
(8c)
1
1
18.80, 118.32, 117.27, 115.19, 110.67, 110.48, 41.81. TOF MS ES +
A light yellow solid; Yield: 40.4%; m.p.: 172.6–174.3 ◦C; 1H NMR
+
(
m/z): (M+H) , calcd for C20
H
15FN
4
O: 347.1308, found, 347.1310.
(400 MHz, DMSO‑d
6
) δ (ppm) 9.04 (t, J = 5.9 Hz, 1H), 8.75 (s, 1H), 8.60
(
s, 1H), 8.51 (d, J = 2.6 Hz, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.16 (d, J =
4
.5.8. 2-(2-aminophenyl)-7-fluoro-N-(pyridin-3-ylmethyl)quinazolin-4-
8.0 Hz, 1H), 7.77 (d, J = 6.6 Hz, 2H), 7.50 (t, J = 7.2 Hz, 1H), 7.35 (s,
2H), 7.07 (t, J = 7.6 Hz, 1H), 6.71 (d, J = 8.2 Hz, 1H), 6.49 (t, J = 7.5 Hz,
1H), 4.97 (d, J = 5.6 Hz, 2H).
amine (7h)
A light yellow solid; Yield: 48.8%; m.p.: 190.8–194.5 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 8.96 (s, 1H), 8.68 (s, 1H), 8.44 (d, J = 4.0
Hz, 1H), 8.34 (dd, J = 14.3, 6.9 Hz, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.54
d, J = 10.4 Hz, 1H), 7.37 (dd, J = 15.9, 5.6 Hz, 4H), 7.10 (t, J = 7.5 Hz,
H), 6.74 (d, J = 8.1 Hz, 1H), 6.53 (t, J = 7.2 Hz, 1H), 4.86 (d, J = 5.3
Hz, 2H).
(
6
4.5.14. 2-(2-methoxyphenyl)-N-(pyrazin-2-ylmethyl)quinazolin-4-amine
(8d)
(
1
A pale white solid; Yield: 48.5%; m.p.: 180.4–184.1 ◦C; 1H NMR
(400 MHz, DMSO‑d
6
) δ (ppm) 9.00 (t, J = 5.9 Hz, 1H, -NH-), 8.72 (s, 1H,
pyrazine, H ), 8.58 (s, 1H, pyrazine, H ), 8.53 (s, 1H, pyrazine, H ), 8.32
5
4
3
4
.5.9. 7-fluoro-2-(2-methoxyphenyl)-N-(pyridin-3-ylmethyl)quinazolin-4-
(d, J = 8.2 Hz, 1H, quinazoline, H
8
), 7.84–7.77 (m, 1H, quinazoline, H
), 7.58–7.52 (m, 1H, quinazoline, H
5
),
),
amine (7i)
7.73 (d, J = 8.2 Hz, 1H, phenyl, H
6
7
A pale white solid; Yield: 52.7%; m.p.: 170.9–174.3 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 8.97 (t, J = 6.0 Hz, 1H), 8.64 (s, 1H), 8.45
7.43–7.34 (m, 2H, quinazoline, H6, phenyl, H
), 7.06 (d, J = 8.2 Hz, 1H,
), 4.88 (d, J = 5.8 Hz, 2H,
). C NMR (101 MHz, DMSO‑d ) δ
4
(
(
6
phenyl, H
-CH
3
), 6.97 (t, J = 7.4 Hz, 1H, phenyl, H
5
1
3
d, J = 4.8 Hz, 1H), 8.39–8.33 (m, 1H), 7.81 (d, J = 7.5 Hz, 1H),
2
-), 3.64 (d, J = 1.2 Hz, 3H, -CH
3
6
7
1
.50–7.43 (m, 3H), 7.41 (d, J = 7.3 Hz, 1H), 7.36 (dd, J = 11.2, 6.6 Hz,
H), 7.10 (d, J = 8.2 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H), 4.78 (d, J = 5.7
165.76, 163.28, 162.67, 159.00, 157.20, 148.89, 147.92, 135.60,
135.07, 130.54, 130.29, 125.60, 123.55, 120.07, 115.05, 112.22,
1
3
+
Hz, 2H), 3.71 (s, 3H). C NMR (101 MHz, DMSO‑d
6
) δ 165.74, 163.26,
111.35, 110.21, 55.62, 41.36. TOF MS ES + (m/z): (M+H) , calcd for
1
1
1
62.67, 159.00, 157.28, 151.75, 149.22, 148.10, 135.42, 135.00,
20 17 5
C H N O: 344.1511, found, 344.1511.
30.59, 130.10, 125.80, 123.43, 119.98, 114.71, 112.27, 111.48,
10.34, 55.67, 41.38.
4.5.15. 2-(2-methoxypyridin-3-yl)-N-(pyrazin-2-ylmethyl)quinazolin-4-
amine (8e)
4
.5.10. 7-fluoro-2-(2-methoxypyridin-3-yl)-N-(pyridin-3-ylmethyl)
A pale white solid; Yield: 48.1%; m.p.: 145.8–149.0 ◦C; 1H NMR
quinazolin-4-amine (7j)
(400 MHz, DMSO‑d
6
) δ (ppm) 9.14 (s, 1H), 8.74 (s, 1H), 8.56 (d, J =
A pale white solid; Yield: 53.1%; m.p.: 182.4–184.7 ◦C; 1H NMR
22.8 Hz, 2H), 8.29 (d, J = 47.2 Hz, 2H), 7.98–7.68 (m, 3H), 7.58 (s, 1H),
(
400 MHz, DMSO‑d
6
) δ (ppm) 9.03 (t, J = 5.9 Hz, 1H), 8.66 (s, 1H), 8.46
7.06 (s, 1H), 4.90 (s, 2H), 3.78 (s, 3H). 13C NMR (101 MHz, DMSO‑d
6
) δ
1
0

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
1
1
1
60.94, 159.71, 159.40, 154.41, 149.45, 147.03, 143.90, 143.85,
43.05, 139.52, 132.93, 127.53, 125.95, 123.57, 122.63, 116.62,
7.56–7.50 (m, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.36 (d, J = 5.0 Hz, 1H),
7.13 (d, J = 9.2 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 6.96 (t, J = 4.2 Hz, 1H),
4.95 (d, J = 5.5 Hz, 2H), 3.76 (s, 3H).
+
13.30, 53.14, 43.98. TOF MS ES + (m/z): (M+H) , calcd for
19 16 6
C H N O: 345.1464, found, 345.1463.
4
.5.22. 2-(7-fluoro-4-((thiophen-2-ylmethyl)amino)quinazolin-2-yl)
phenol (9c)
A faint yellow solid; Yield: 48.1%; m.p.: 240.1–242.4 ◦C; 1H NMR
(400 MHz, DMSO‑d
4
.5.16. 3-(7-fluoro-4-((pyrazin-2-ylmethyl)amino)quinazolin-2-yl)phenol
8f)
A faint yellow solid; Yield: 45.7%; m.p.: 204.8–206.5 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 9.52 (s, 1H), 9.17 (d, J = 5.9 Hz, 1H), 8.80
(
6
) δ (ppm) 14.40 (s, 1H), 9.42 (s, 1H), 8.55 (d, J =
(
(
6
8.2 Hz, 1H), 8.37 (dd, J = 9.1, 5.9 Hz, 1H), 7.66 (d, J = 10.2 Hz, 1H),
7.47 (t, J = 8.8 Hz, 1H), 7.38 (t, J = 7.4 Hz, 2H), 7.17 (d, J = 3.4 Hz, 1H),
s, 1H), 8.64–8.59 (m, 1H), 8.52 (d, J = 2.7 Hz, 1H), 8.46–8.39 (m, 1H),
7
7
.87–7.82 (m, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.52–7.41 (m, 2H),
6.99–6.91 (m, 3H), 5.05 (d, J = 5.7 Hz, 2H). TOF MS ES + (m/z):
1
3
+
.28–7.20 (m, 1H), 6.86 (d, J = 7.4 Hz, 1H), 5.01 (d, J = 5.6 Hz, 2H).
) δ 165.89, 163.40, 160.24, 159.28, 157.30,
54.42, 144.00, 143.68, 143.15, 139.36, 129.10, 125.97, 118.89,
C
(M+H) , calcd for C19
3
H14FN OS: 352.0920, found, 352.0921.
NMR (101 MHz, DMSO‑d
6
1
1
4.5.23. 7-fluoro-2-(2-methoxypyridin-3-yl)-N-(thiophen-2-ylmethyl)
quinazolin-4-amine (9d)
17.46, 114.73, 114.59, 111.68, 110.91, 43.99. TOF MS ES + (m/z):
+
A faint yellow solid; Yield: 45.4%; m.p.: 145.2–146.5 ◦C; 1H NMR
(
M+H) , calcd for C19
5
H14FN O: 348.1261, found, 348.1263.
(
400 MHz, DMSO‑d
6
) δ (ppm) 9.07 (t, J = 5.9 Hz, 1H), 8.36 (dd, J = 9.1,
4
.5.17. 2-(7-fluoro-4-((pyrazin-2-ylmethyl)amino)quinazolin-2-yl)phenol
6.0 Hz, 1H), 8.26 (dd, J = 4.9, 2.0 Hz, 1H), 8.09 (dd, J = 7.3, 2.1 Hz,
(
8g)
A pale white solid; Yield: 48.3%; m.p.: 220.4–223.9 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 14.27 (s, 1H), 9.45 (d, J = 6.3 Hz, 1H),
.80 (s, 1H), 8.60 (s, 1H), 8.52 (d, J = 2.6 Hz, 1H), 8.48–8.41 (m, 1H),
.22 (d, J = 7.5 Hz, 1H), 7.61 (dd, J = 10.2, 2.4 Hz, 1H), 7.47 (t, J = 8.9
1H), 7.53–7.42 (m, 2H), 7.37 (d, J = 4.8 Hz, 1H), 7.15–7.09 (m, 2H),
13
6.96 (dd, J = 5.1, 3.4 Hz, 1H), 4.94 (d, J = 5.6 Hz, 2H), 3.87 (s, 3H).
C
(
6
6
NMR (101 MHz, DMSO‑d ) δ 165.79, 163.31, 161.00, 158.87, 151.89,
8
8
147.17, 142.07, 139.62, 126.51, 125.97, 125.78, 125.12, 123.53,
116.62, 115.17, 111.68, 110.44, 53.18, 38.69. TOF MS ES + (m/z):
+
Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 6.90–6.82 (m, 2H), 5.00 (d, J = 5.4 Hz,
(M+H) , calcd for C19
4
H15FN OS: 367.1029, found, 367.1029.
1
3
2
1
1
6
H). C NMR (101 MHz, DMSO‑d ) δ 166.75, 164.25, 162.22, 161.28,
59.38, 154.46, 149.52, 144.48, 144.15, 143.73, 133.19, 129.66,
4.5.24. 2-(4-(((tetrahydrofuran-3-yl)methyl)amino)quinazolin-2-yl)
phenol (9e)
26.67, 119.27, 118.74, 117.72, 115.77, 111.17, 44.75. TOF MS ES ꢀ
ꢀ
A pale white solid; Yield: 44.4%; m.p.: 169.4–171.9 ◦C; 1H NMR
(
m/z): (Mꢀ H) , calcd for C19
5
H14FN O: 346.1104, found, 346.1103.
(
400 MHz, DMSO‑d
quinazoline, H
quinazoline, H
(t, J = 7.5 Hz, 1H, quinazoline, H
6.93 (t, J = 9.3 Hz, 2H, phenyl, H
hydrofuran, H -CH
-), 3.74 (t, J = 7.6 Hz, 1H, tetrahydrofuran, H
), 3.65 (dq, J = 14.6, 7.9, 7.3 Hz, 4H, -CH -, tetrahydrofuran, H
-CH -CH -), 2.02 (dq,
-), 2.76 (p, J = 6.5 Hz, 1H, tetrahydrofuran, H
J = 13.8, 7.8 Hz, 1H, tetrahydrofuran, H -CH -), 1.72 (dq, J = 13.0, 6.6
Hz, 1H, tetrahydrofuran, H ) δ
-CH-). 13C NMR (101 MHz, DMSO‑d
6
) δ (ppm) 14.78 (s, 1H, -OH), 8.80 (t, J = 5.4 Hz, 1H,
4
4
.5.18. 7-fluoro-2-(2-methoxyphenyl)-N-(pyrazin-2-ylmethyl)quinazolin-
-amine (8h)
7
), 8.45 (d, J = 7.8 Hz, 1H, -NH
), 7.85–7.73 (m, 2H, quinazoline, H
), 7.36 (t, J = 7.7 Hz, 1H, phenyl, H
, H
), 3.82 (q, J = 8.0 Hz, 1H tetra-
-CH
-CH
2
-), 8.31 (d, J = 8.2 Hz, 1H,
, phenyl, H ), 7.54
),
8
5
6
A pale white solid; Yield: 46.7%; m.p.: 193.2–195.6 ◦C; 1H NMR
6
4
(
400 MHz, DMSO‑d
6
) δ (ppm) 9.17 (s, 1H), 8.72 (s, 1H), 8.59 (s, 1H),
3
5
8
7
1
.54 (s, 1H), 8.43 (dd, J = 10.0, 5.9 Hz, 1H), 7.48 (d, J = 10.6 Hz, 2H),
5
2
2
2
-
.39 (d, J = 7.3 Hz, 2H), 7.07 (d, J = 8.5 Hz, 1H), 6.97 (t, J = 7.4 Hz,
2
2
2
-,
+
H), 4.88 (d, J = 5.5 Hz, 2H), 3.65 (s, 3H). TOF MS ES + (m/z): (M+H) ,
H
5
2
4
2
calcd for C20
H
16FN
5
O: 362.1417, found, 362.1416.
4
2
3
6
4
.5.19. 7-fluoro-2-(2-methoxypyridin-3-yl)-N-(pyrazin-2-ylmethyl)
161.39, 161.31, 159.74, 147.25, 134.03, 132.91, 129.49, 126.60,
quinazolin-4-amine (8i)
126.34, 123.47, 119.71, 118.77, 117.74, 113.91, 71.12, 67.35, 43.99,
A pale white solid; Yield: 45.9%; m.p.: 150.3–154.1 ◦C; 1H NMR
38.81, 30.14. TOF MS ES ꢀ (m/z): (Mꢀ H) , calcd for C19
-
19 3 2
H N O :
(
400 MHz, DMSO‑d
6
) δ (ppm) 9.17 (t, J = 5.9 Hz, 1H), 8.73 (d, J = 1.5
320.1399, found, 320.1398.
Hz, 1H), 8.62–8.57 (m, 1H), 8.53 (s, 1H), 8.43 (dd, J = 9.6, 6.1 Hz, 1H),
8
9
.23 (dd, J = 4.9, 1.9 Hz, 1H), 7.89 (dd, J = 7.4, 2.0 Hz, 1H), 7.49 (d, J =
.4 Hz, 2H), 7.05 (dd, J = 7.4, 5.0 Hz, 1H), 4.89 (d, J = 5.6 Hz, 2H), 3.77
4.5.25. 2-(2-methoxyphenyl)-N-((tetrahydrofuran-3-yl)methyl)
quinazolin-4-amine (9f)
(
s, 3H). 13C NMR (101 MHz, DMSO‑d
6
) δ 166.32, 163.84, 161.49,
A pale white solid; Yield: 45.8%; m.p.: 161.2–164.0 ◦C; 1H NMR
1
1
59.69, 154.76, 152.26, 147.69, 144.37 (2C), 143.56, 140.06, 126.36,
(400 MHz, DMSO‑d
6
) δ (ppm) 8.44 (s, 1H), 8.26 (d, J = 8.1 Hz, 1H), 7.77
23.82, 117.11, 115.80, 112.13, 110.95, 53.65, 44.41. TOF MS ES + (m/
(t, J = 7.6 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 8.2 Hz, 2H), 7.41
(t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 3.75
+
z): (M+H) , calcd for C18
6
H15FN O: 363.1370, found, 363.1370.
(
s, 4H), 3.71–3.65 (m, 1H), 3.63 (d, J = 7.4 Hz, 1H), 3.59–3.52 (m, 2H),
4
.5.20. 2-(4-((thiophen-2-ylmethyl)amino)quinazolin-2-yl)phenol (9a)
A faint yellow solid; Yield: 50.7%; m.p.: 220.4–222.5 ◦C; 1H NMR
400 MHz, DMSO‑d
3.50–3.45 (m, 1H), 2.77 (d, J = 7.0 Hz, 1H), 1.95 (dq, J = 13.4, 7.5 Hz,
13
1H), 1.66 (dq, J = 12.9, 6.6 Hz, 1H). C NMR (101 MHz, DMSO‑d
6
) δ
(
6
) δ (ppm) 14.74 (s, 1H, -OH), 9.37 (t, J = 6.0 Hz, 1H,
), 8.56 (d, J = 8.4 Hz, 1H, -NH-), 8.29 (d, J = 8.3 Hz, 1H,
), 7.81 (dt, J = 16.2, 8.0 Hz, 2H, quinazoline, H , phenyl,
), 7.37 (d, J = 5.4 Hz, 2H, qui-
), 7.18 (d, J = 3.5 Hz, 1H, thiophene, H ),
, H , thiophene, H ), 5.06 (d, J
-). TOF MS ES + (m/z): (M+H) , calcd for
OS: 334.1014, found, 334.1019.
161.45, 159.38, 157.31, 149.37, 132.51, 130.53, 129.83, 127.33,
quinazoline, H
quinazoline, H
7
125.33, 122.53, 120.03, 113.20, 112.33 (2C), 70.57, 66.79, 55.69,
43.22, 38.17, 29.54. TOF MS ES
+
(m/z): (M+H)+, calcd for
8
5
H
6
), 7.55 (t, J = 7.5 Hz, 1H, phenyl, H
4
20 21 3 2
C H N O : 336.1712, found, 336.1715.
nazoline, H , thiophene, H
6
5
3
6
.95 (dd, J = 16.2, 8.3 Hz, 3H, phenyl, H
3
5
4
+
4.5.26. 2-(7-fluoro-4-(((tetrahydrofuran-3-yl)methyl)amino)quinazolin-
2-yl)phenol (9g)
=
5.7 Hz, 2H, -CH
2
C
19
H
15
N
3
A pale white solid; Yield: 43.7%; m.p.: 170.9–173.1 ◦C; 1H NMR
(
400 MHz, DMSO‑d
6
) δ (ppm) 14.45 (s, 1H), 8.83 (t, J = 5.6 Hz, 1H),
4
.5.21. 2-(2-methoxyphenyl)-N-(thiophen-2-ylmethyl)quinazolin-4-amine
9b)
A faint yellow solid; Yield: 50.2%; m.p.: 200.3–201.5 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 9.00 (s, 1H), 8.27 (d, J = 8.3 Hz, 1H),
.84–7.76 (m, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H),
8.43 (d, J = 8.0 Hz, 1H), 8.38 (dd, J = 9.2, 6.0 Hz, 1H), 7.59 (dd, J =
10.2, 2.6 Hz, 1H), 7.44 (t, J = 8.8 Hz, 1H), 7.40–7.33 (m, 1H), 6.97–6.89
(m, 2H), 3.85–3.78 (m, 1H), 3.77–3.71 (m, 1H), 3.69–3.57 (m, 4H), 2.74
(p, J = 6.5 Hz, 1H), 2.01 (dt, J = 13.4, 6.6 Hz, 1H), 1.71 (dq, J = 12.8,
(
(
6
7
6.5 Hz, 1H). 13C NMR (101 MHz, DMSO‑d
) δ 166.15, 163.66, 161.73,
6
1
1

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
+
1
1
60.86, 158.86, 149.09, 132.67, 129.11, 126.18, 118.92, 118.34,
z): (M+H) , calcd for C20
H
18
N
4
OS: 363.1280, found, 363.1280.
17.30, 114.97, 110.47, 70.55, 66.80, 43.48, 38.20, 29.58. TOF MS ES
+
+
(m/z): (M+H) , calcd for C19
H18FN
3
O
2
: 340.1461, found, 340.1463.
4.5.32. 3-(7-fluoro-4-((2-(thiophen-2-yl)ethyl)amino)quinazolin-2-yl)
phenol (10e)
4
.5.27. 7-fluoro-2-(2-methoxypyridin-3-yl)-N-((tetrahydrofuran-3-yl)
A faint yellow solid; Yield: 37.9%; m.p.: 208.1–209.5 ◦C; 1H NMR
methyl)quinazolin-4-amine (9h)
(400 MHz, DMSO‑d
6
) δ (ppm) 9.53 (s, 1H), 8.57 (t, J = 5.5 Hz, 1H), 8.32
A pale white solid; Yield: 47.2%; m.p.: 145.6–146.8 ◦C; 1H NMR
(dd, J = 9.1, 6.0 Hz, 1H), 7.95 (d, J = 6.7 Hz, 2H), 7.47 (dd, J = 10.4, 2.6
Hz, 1H), 7.41 (dd, J = 8.8, 2.7 Hz, 1H), 7.35 (dd, J = 4.4, 2.0 Hz, 1H),
7.30 (t, J = 7.9 Hz, 1H), 6.97 (d, J = 4.6 Hz, 2H), 6.91–6.87 (m, 1H),
(
400 MHz, DMSO‑d
6
) δ (ppm) 8.54 (t, J = 5.6 Hz, 1H), 8.40–8.33 (m,
1
H), 8.26 (d, J = 3.0 Hz, 1H), 8.03 (d, J = 5.3 Hz, 1H), 7.44 (t, J = 8.8
Hz, 2H), 7.10 (dd, J = 7.3, 4.9 Hz, 1H), 3.87 (s, 3H), 3.81–3.74 (m, 1H),
3.90 (q, J = 6.7 Hz, 2H), 3.29 (t, J = 7.2 Hz, 2H). TOF MS ES + (m/z):
+
3
.73–3.67 (m, 1H), 3.63 (d, J = 7.5 Hz, 1H), 3.60–3.53 (m, 2H), 3.47
(M+H) , calcd for C20
3
H16FN OS: 366.1076, found, 366.1076.
(
dd, J = 13.9, 7.0 Hz, 1H), 2.77 (p, J = 6.7 Hz, 1H), 2.04–1.91 (m, 1H),
1
3
1
1
1
2
.67 (dq, J = 12.8, 6.5 Hz, 1H). C NMR (101 MHz, DMSO‑d
6
) δ 165.70,
4.5.33. 2-(7-fluoro-4-((2-(thiophen-2-yl)ethyl)amino)quinazolin-2-yl)
phenol (10f)
63.22, 161.19, 159.30, 151.76, 147.08, 139.47, 125.79, 123.71,
16.66, 114.91, 111.56, 110.44, 70.54, 66.78, 53.13, 43.23, 38.13,
A pale white solid; Yield: 44.1%; m.p.: 210.3–212.7 ◦C; 1H NMR
+
9.52. TOF MS ES + (m/z): (M+H) , calcd for C19
H19FN
4
O
2
: 355.1570,
(400 MHz, DMSO‑d
6
) δ (ppm) 14.42 (s, 1H), 8.92 (s, 1H), 8.42 (d, J =
found, 355.1568.
49.0 Hz, 2H), 7.61 (s, 1H), 7.41 (d, J = 32.2 Hz, 3H), 6.96 (d, J = 16.4
Hz, 4H), 3.90 (s, 2H), 3.34 (s, 2H). 13C NMR (101 MHz, DNSO-d
) δ
6
4
.5.28. 3-(4-((2-(thiophen-2-yl)ethyl)amino)quinazolin-2-yl)phenol
10a)
A faint yellow solid; Yield: 38.4%; m.p.: 186.5–188.1 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 9.65 (s, 1H), 8.77–8.65 (m, 1H), 8.32 (d, J
8.3 Hz, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 5.2 Hz, 2H), 7.47
dd, J = 8.3, 5.9 Hz, 1H), 7.34 (d, J = 5.1 Hz, 1H), 7.27 (t, J = 8.1 Hz,
165.80, 163.31, 160.65, 159.32, 157.01, 151.76, 141.43, 139.54,
129.33, 127.01, 125.24, 123.96, 119.18, 117.52, 114.85, 114.47,
(
ꢀ
111.41, 110.73, 42.24, 28.43. TOF MS ES ꢀ (m/z): (Mꢀ H) , calcd for
(
6
20 3
C H16FN OS: 364.0920, found, 364.0920.
=
(
4.5.34. 7-fluoro-2-(2-methoxyphenyl)-N-(2-(thiophen-2-yl)ethyl)
quinazolin-4-amine (10g)
1
2
1
1
1
H), 7.02–6.95 (m, 2H), 6.90 (d, J = 6.6 Hz, 1H), 3.89 (q, J = 6.8 Hz,
13
A pale white solid; Yield: 45.0%; m.p.: 170.6–173.3 ◦C; 1H NMR
H), 3.30 (t, J = 7.2 Hz, 2H). C NMR (101 MHz, DMSO‑d
6
) δ 160.88,
60.82, 159.04, 146.67, 141.34, 133.55, 132.44, 128.99, 127.01,
(400 MHz, DMSO‑d
6
) δ (ppm) 8.56 (t, J = 5.6 Hz, 1H), 8.35–8.28 (m,
26.10, 125.92, 125.36, 124.22, 122.91, 119.15, 118.21, 117.24,
1H), 7.56 (dd, J = 7.5, 1.8 Hz, 1H), 7.43 (t, J = 7.8 Hz, 3H), 7.33 (d, J =
5.0 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 6.97–6.93
+
13.46, 42.64, 28.38. TOF MS ES + (m/z): (M+H) , calcd for
C
20
H
17
3
N OS: 348.1171, found, 348.1171.
(m, 1H), 6.91 (d, J = 3.3 Hz, 1H), 3.75 (s, 5H), 3.26 (t, J = 7.1 Hz, 2H).
+
TOF MS ES + (m/z): (M+H) , calcd for C21
3
H18FN OS: 380.1233, found,
4
.5.29. 2-(4-((2-(thiophen-2-yl)ethyl)amino)quinazolin-2-yl)phenol
10b)
A pale white solid; Yield: 42.6%; m.p.: 190.3–194.1 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 14.75 (s, 1H, -OH), 8.87 (d, J = 5.7 Hz,
), 8.49 (d, J = 7.7 Hz, 1H, -NH-), 8.27 (d, J = 8.2 Hz,
), 7.86–7.79 (m, 1H, quinazoline, H ), 7.76 (d, J =
.4 Hz, 1H, phenyl, H ), 7.36 (dd, J
), 7.60–7.51 (m, 1H, quinazoline, H
11.6, 6.1 Hz, 2H, phenyl, H , thiophene, H
), 7.00–6.90 (m, 4H,
phenyl, H , H , thiophene, H , H -), 3.30
), 3.91 (q, J = 6.4 Hz, 2H, -CH
-thiophen-2-yl). C NMR (101 MHz, DMSO‑d ) δ
380.1234.
(
4.5.35. 3-(4-((2-(pyridin-3-yl)ethyl)amino)quinazolin-2-yl)phenol (10h)
A pale white solid; Yield: 39.8%; m.p.: 179.2–181.3 ◦C; H NMR
1
(
6
1
1
8
H, quinazoline, H
8
(400 MHz, DMSO‑d
), 8.47–8.39 (m, 2H, pyridine, H
quinazoline, H
), 7.95 (d, J = 9.0 Hz, 2H, quinazoline, H
J = 10.8 Hz, 3H, phenyl, H , pyridine, H , quinazoline, H
7.9 Hz, 1H, phenyl, H
), 7.30 (q, J = 8.2, 7.3 Hz, 2H, phenyl, H
idine, H ), 3.91 (q, J = 6.6 Hz, 2H,
), 6.88 (d, J = 7.3 Hz, 1H, phenyl, H
-CH -pyridin-3-yl). C NMR (101 MHz,
-), 3.09 (t, J = 7.1 Hz, 2H, -CH
DMSO‑d ) δ 159.57, 159.33, 157.32, 149.86 (2C), 147.39, 140.13,
6
) δ (ppm) 9.55 (s, 1H, -OH), 8.52 (s, 1H, pyridine,
H, quinazoline, H
5
7
H
2
6
, -NH
2
-), 8.20 (d, J = 8.2 Hz, 1H,
, H ), 7.73 (d,
), 7.48 (d, J =
, pyr-
6
6
8
6
7
=
4
5
6
4
5
3
5
3
4
2
2
5
1
3
(
t, J = 7.2 Hz, 2H, -CH
2
6
5
4
1
3
1
1
1
61.31, 159.55, 147.16, 141.83, 134.08, 132.97, 129.47, 127.51,
2
2
26.57, 126.44, 125.83, 125.24, 124.64, 123.32, 119.63, 118.75,
6
+
17.73, 113.91, 43.10, 28.86. TOF MS ES + (m/z): (M+H) , calcd for
136.28, 135.10, 132.60, 129.06, 127.75, 125.19, 123.42, 122.54,
C
20
H
17
3
N OS: 348.1171, found, 348.1171.
118.85, 117.09, 114.78, 113.79, 41.75, 31.64. TOF MS ES ꢀ (m/z):
-
(
Mꢀ H) , calcd for C21
18 4
H N O: 341.1402, found, 341.1404.
4
.5.30. 2-(2-methoxyphenyl)-N-(2-(thiophen-2-yl)ethyl)quinazolin-4-
amine (10c)
A pale white solid; Yield: 44.7%; m.p.: 175.1–177.8 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 8.46 (d, J = 8.2 Hz, 1H), 7.92 (t, J = 9.7
Hz, 1H), 7.86–7.76 (m, 2H), 7.68–7.62 (m, 1H), 7.58–7.52 (m, 1H), 7.46
t, J = 8.1 Hz, 1H), 7.32 (d, J = 4.7 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H),
.06 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 4.7 Hz, 2H), 3.79 (s, 5H), 3.28 (t, J
7.3 Hz, 2H). 13C NMR (101 MHz, DMSO‑d
) δ 159.41, 157.50, 141.70,
4.5.36. 2-(4-((2-(pyridin-3-yl)ethyl)amino)quinazolin-2-yl)phenol (10i)
A pale white solid; Yield: 41.7%; m.p.: 162.4–164.2 ◦C; H NMR
1
(
6
(400 MHz, DMSO‑d
6
) δ (ppm) 14.77 (s, 1H), 8.81 (t, J = 5.6 Hz, 1H),
8.53 (s, 1H), 8.46 (d, J = 7.8 Hz, 1H), 8.41 (d, J = 4.8 Hz, 1H), 8.25 (d, J
= 8.2 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.78–7.70 (m, 2H), 7.53 (t, J =
(
7
7.5 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.34–7.29 (m, 1H), 6.94 (dd, J =
13
=
6
12.0, 7.9 Hz, 2H), 3.92 (q, J = 6.6 Hz, 2H), 3.09 (t, J = 7.1 Hz, 2H).
C
1
1
2
34.10, 133.99, 133.00, 130.86, 130.58, 127.84, 126.93, 126.20,
6
NMR (101 MHz, DMSO‑d ) δ 161.30, 161.26, 159.55, 150.27, 147.91,
25.77, 125.13, 124.01, 123.12, 120.18, 113.16, 112.45, 55.93, 42.45,
147.18, 136.86, 135.39, 134.04, 132.92, 129.48, 126.59, 126.38,
-
8.42. TOF MS ES ꢀ (m/z): (Mꢀ H) , calcd for C21
H
19
N
3
OS: 360.1171,
123.98, 123.28, 119.64, 118.75, 117.71, 113.85, 42.39, 31.93. TOF MS
ꢀ
found, 360.1171.
ES ꢀ (m/z): (Mꢀ H) , calcd for C21
18 4
H N O: 341.1402, found, 341.1401.
4
4
.5.31. 2-(2-methoxypyridin-3-yl)-N-(2-(thiophen-2-yl)ethyl)quinazolin-
-amine (10d)
4.5.37. 2-(2-methoxyphenyl)-N-(2-(pyridin-3-yl)ethyl)quinazolin-4-
amine (10j)
A pale white solid; Yield: 43.9%; m.p.: 181.6–184.0 ◦C; 1H NMR
A pale white solid; Yield: 44.2%; m.p.: 176.4–178.1 ◦C; 1H NMR
(
400 MHz, DMSO‑d
6
) δ (ppm) 8.55 (s, 1H), 8.29–8.20 (m, 2H), 8.06 (d, J
(400 MHz, DMSO‑d
6
) δ (ppm) 8.44 (d, J = 2.8 Hz, 2H), 8.38 (d, J = 4.8
=
7.3 Hz, 1H), 7.76 (dt, J = 15.4, 8.1 Hz, 2H), 7.52 (t, J = 7.6 Hz, 1H),
Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.69 (d, J =
8.3 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.48 (t, J
= 7.5 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.31–7.25 (m, 1H), 7.11 (d, J =
7
3
.32 (d, J = 5.0 Hz, 1H), 7.14–7.07 (m, 1H), 6.98–6.89 (m, 2H), 3.88 (s,
H), 3.77 (q, J = 6.7 Hz, 2H), 3.29 (t, J = 7.2 Hz, 2H). TOF MS ES + (m/
1
2

Z. Zhou et al.
Bioorganic Chemistry 112 (2021) 104848
8
2
.3 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 3.73 (s, 5H), 3.05 (t, J = 7.1 Hz,
4.9. Annexin V-FITC staining
H).
The potent compound 9e was tested for its effect on cell cycle pro-
gression. The specific method of experiment referred to the previous
method of our research group [36]. A549 cells were seeded in 6-well
4
.5.38. 2-(2-methoxypyridin-3-yl)-N-(2-(pyridin-3-yl)ethyl)quinazolin-4-
amine (10k)
A pale white solid; Yield: 44.9%; m.p.: 165.4–166.8 ◦C; 1H NMR
400 MHz, DMSO‑d
) δ (ppm) 8.52 (d, J = 5.1 Hz, 1H), 8.47 (s, 1H), 8.40
plates at a density of 1 × 10 cells/well in RPMI 1640 medium and
6
(
(
6
were incubated for 24 h. The cancer cell lines were then treated with 10
s, 1H), 8.27 (d, J = 3.4 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.05 (d, J =
μ
M 7b and compound 9e at the different concentrations of 2.5
μ
M, 5 M
μ
◦
7
7
3
.2 Hz, 1H), 7.75 (dt, J = 15.5, 8.0 Hz, 2H), 7.65 (d, J = 7.8 Hz, 1H),
.51 (t, J = 7.4 Hz, 1H), 7.33–7.26 (m, 1H), 7.14–7.08 (m, 1H), 3.87 (s,
H), 3.77 (q, J = 6.6 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H).
and 10
μM for 48 h at 37 C in 5% CO
2
. Cultured cells stained with
◦
Annexin V-FITC and propidium iodide (PI) at 4 C for 0.5 h were tested
using the FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA)
by the Cell Quest software in the dark.
4
.6. In vitro anti-proliferative activity assays
4
.10. Molecular docking studies
The target compounds (7a-7j, 8a-8i, 9a-9h, 10a-10k) were evalu-
The three-dimensional structure of the EGFRT790M/C797S (PDB code:
ated for anti-proliferative activity against A549, NCI-H460 and H1975
cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) method according to the previous research by our groups [34].
5XGN) was obtained from RCSB Protein Data Bank (http://www.
wwpdb.org/). All the molecular docking simulations were performed
by the Auto-Dock 4.2 software. The preparation process of the protein
for docking mainly involves the addition of hydrogen atoms and
charges, elimination of unrelated water molecules, immobilization of
exact residues, and removal of endogenous ligands and etc. The prepa-
ration process of the molecules for docking mainly includes the addition
of hydrogen atoms and charges. Then the prepared molecules (target
compounds) were docked to the certified binding site of EGFR protein.
Subsequently, the genetic algorithm was used for energy optimization.
All the docking results were processed and modified by PYMOL 1.8.6
software (https://pymol.org) [37].
3
First, approximately 6 × 10 cells suspended in a minimum essential
medium (MEM) were plated onto each well in a 96-well plate and were
◦
incubated in 5% CO
2
at 37 C for 24 h. Next, the cancer cell lines adding
test compounds at concentrations of 1.23, 3.70, 11.11, 33.33 and 100
◦
μ
mol/L were continued for 72 h in 5% CO
2
at 37 C. Afterward, fresh
MTT was added to each well at a terminal concentration of 5 g/mL and
◦
was incubated with cells at 37 C for 4 h. Finally, after the removal of
MTT methoxypyrimidine, the cells were dissolved in 100 L dimethyl
μ
sulfoxide, and the data were recorded under an absorbance of 492 nm.
All compounds were tested three times under the same conditions, and
the test results were expressed by IC50 with the Bacus Laboratories
Incorporated Slide Scanner (Bliss) software.
Declaration of Competing Interest
4
.7. In vitro enzymatic activity assays
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
With AZD9291 and Afatinib as positive control, the potent com-
pounds were tested for their activities against EGFR enzyme using
Kinase-Glo Luminescent Kinase Assay (Promega, Madison, WI, USA)
according to the reported procedures [35]. The kinase assay was
implemented through Mobility shift assay. The Mixture the configured
available Brij-35 (0.0015%) and 50 mM HEPES (pH = 7.5) to get a ki-
nase buffer base. Then, compounds 7b-7d, 7h, 7j, 8g, 9b, 9e and 10a-
Acknowledgments
We gratefully acknowledge the generous support provided by Na-
tional Natural Science Foundation of China (82060629, 21967009),
Natural Science Foundation of Jiangxi, China (20202BABL216076,
20192BAB215061), Science and Technology Project Founded by the
Education Department of Jiangxi Province, China (GJJ190583), Youth
Top Talent Support Program of Jiangxi Science & Technology Normal
University (2019QNBJRC008), Nanchang Key Laboratory of Molecular
Targeted Anticancer Drug Design and Evaluation (2019-NCZDSY-007).
1
0b were configured to six concentrations using dimethyl sulfoxide. The
kinase buffer containing the compounds or dimethyl sulfoxide was
added to 96-well plates and mixed on the shaker for fifteen minutes.
Next, the solution was transferred in duplicate from a 96-well plate to a
3
84-well plate, and then an enzyme solution (EGFR kinase mixed with
kinase buffer) was added to each well. After incubation of the 384-well
plate for 10 min at room temperature, a 2.5x peptide solution (formed by
the addition of FAM-labeled peptide and ATP to the kinase base buffer)
Appendix A. Supplementary material
◦
was added to each well. After incubation at 28 C for a certain period of
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104848.
time, a stop buffer was added to each well for stop the reaction. The
inhibition value is obtained by converting the conversion data on the
caliper. The formula is that percent inhibition = (max-conversion)/
References
(
max–min)*100. Among them “max” stands for DMSO control, “min”
stands for low control. Finally, the IC50 values were obtained by fitting
the inhibition rate data using the XLFit excel add-in version software.
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[
2] C.E. DeSantis, J. Ma, A. Goding Sauer, L.A. Newman, A. Jemal, Breast cancer
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39–448.
3] P. Blume-Jensen, T. Hunter, Oncogenic kinase signaling, Nature 411 (2001)
55–365.
4
.8. Acridine orange staining
3
The potent compound 9e was tested for its ability of inducing cell
[4] G. Fontanini, M. De Laurentiis, S. Vignati, S. Chin `e , M. Lucchi, et al., Evaluation of
epidermal growth factor-related growth factors and receptors and of
neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer:
amphiregulin and microvessel count are independent prognostic indicators of
survival, Clin. Cancer Res. 4 (1998) 241–249.
apoptosis. The cells were seeded evenly in a 24-well plate. After 24 h,
compound 9e was added to 24-well plate at a concentration of 100
μ
g/
◦
mL and 1000
μg/mL. Plate was incubated in a 5% CO
2
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