Design, synthesis and biological evaluation of novel 4-anlinoquinazoline derivatives as EGFR inhibitors with the potential to inhibit the gefitinib-resistant nonsmall cell lung cancers

Article abstract of DOI:10.1080/14756366.2018.1518957

A series of quinazoline derivatives with benzylidene hydrazine carboxamide were designed and synthesised as EGFR inhibitors. Most compounds exhibited exceptional anti-proliferative activity against A549, HepG2, MCF-7 and H1975 cells. Furthermore, six compounds demonstrated excellent inhibition activity against EGFR^WT with the IC₅₀ value both less than 2 nM. Among the six compounds, 44 exhibited the strongest activity (0.4 nM) and potently inhibited EGFR^L858R/T790M (0.1 μM). Excitingly, the most potent compound 14 showed excellent enzyme inhibitory activity with 6.3 nM and 8.4 nM for both EGFR^WT and EGFR^T790M/L858R. The result of AO single staining and Annexin V/PI staining showed that the compound 14 and 44 could induce remarkable apoptosis of A549 cells. The compound 14 arrested the cell cycle at the S phase and compound 44 arrested the cell cycle at the G0 phase in A549 cells. These preliminary results demonstrate that compound 14 and 44 may be promising lead compound-targeting EGFR.

Full text of DOI:10.1080/14756366.2018.1518957

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation
of novel 4-anlinoquinazoline derivatives as
EGFR inhibitors with the potential to inhibit the
gefitinib-resistant nonsmall cell lung cancers
Caolin Wang, Shan Xu, Liang Peng, Bingliang Zhang, Hong Zhang, Yingying
Hu, Pengwu Zheng & Wufu Zhu
To cite this article: Caolin Wang, Shan Xu, Liang Peng, Bingliang Zhang, Hong Zhang, Yingying
Hu, Pengwu Zheng & Wufu Zhu (2019) Design, synthesis and biological evaluation of novel 4-
anlinoquinazoline derivatives as EGFR inhibitors with the potential to inhibit the gefitinib-resistant
nonsmall cell lung cancers, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 204-218,
DOI: 10.1080/14756366.2018.1518957
To link to this article: https://doi.org/10.1080/14756366.2018.1518957
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UK Limited, trading as Taylor & Francis
Group
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
018, VOL. 34, NO. 1, 204–218
2
https://doi.org/10.1080/14756366.2018.1518957
Design, synthesis and biological evaluation of novel 4-anlinoquinazoline
derivatives as EGFR inhibitors with the potential to inhibit the gefitinib-resistant
nonsmall cell lung cancers
ꢀ
ꢀ
Caolin Wang , Shan Xu , Liang Peng, Bingliang Zhang, Hong Zhang, Yingying Hu, Pengwu Zheng and Wufu Zhu
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University,
Nanchang, Jiangxi, China
ABSTRACT
ARTICLE HISTORY
A series of quinazoline derivatives with benzylidene hydrazine carboxamide were designed and synthes-
ised as EGFR inhibitors. Most compounds exhibited exceptional anti-proliferative activity against A549,
HepG2, MCF-7 and H1975 cells. Furthermore, six compounds demonstrated excellent inhibition activity
Received 23 June 2018
Revised 18 August 2018
Accepted 28 August 2018
WT
against EGFR with the IC50 value both less than 2 nM. Among the six compounds, 44 exhibited the
L858R/T790M
KEYWORDS
strongest activity (0.4 nM) and potently inhibited EGFR
(0.1 lM). Excitingly, the most potent com-
Quinazoline derivatives;
benzylidene hydrazine;
NSCLC; EGFR; inhibitors
WT
pound 14 showed excellent enzyme inhibitory activity with 6.3 nM and 8.4 nM for both EGFR
and
T790M/L858R
EGFR
. The result of AO single staining and Annexin V/PI staining showed that the compound 14
and 44 could induce remarkable apoptosis of A549 cells. The compound 14 arrested the cell cycle at the
S phase and compound 44 arrested the cell cycle at the G0 phase in A549 cells. These preliminary results
demonstrate that compound 14 and 44 may be promising lead compound-targeting EGFR.
Introduction
A recent study exposed the occurrence of the tertiary point
1
2
mutation C797S in 40% of the patients treated with AZD9291 .
This mutation causes the drug molecules to lose covalent interac-
tions, thereby resulting in a decrease in inhibitory activity. These
data indicated that there is a great need to solve the mutation
problem without relying on covalent reaction with Cys797.
Therefore, developing noncovalent inhibitors strategy to overcome
Epidermal growth factor receptor (EGFR) is overexpressed in sev-
eral human tumours including non-small cell lung cancer
1
(
NSCLC) . As a target of NSCLC treatment, EGFR has become a
2
–4
hotspot in anti-tumour research in past few years . Some EGFR
small molecule inhibitors have yielded good results in tumour tar-
geted therapies. The first generation of EGFR inhibitors erlotinib
and gefitinib (Figure 1) has achieved remarkable benefits in
1
3,14
the T790M mutation attracted the researchers’ attention
. Chen
1
5
et al. reported that compound BMC2009069-9a is a novel non-
covalent inhibitors candidate with the potency to overcome the
problem of T790M.
patients carrying “sensitizing mutations” such as L858R and exon-
5
1
9 deletions . Unfortunately, most patients develop resistance
after 10–14 months of treatment with gefitinib and erlotinib, and
the statistics show that gatekeeper residue (T790M) mutation was
detected in 50%
In our previous study, a series of quinazoline derivatives were
6
,7
16
.
designed and synthesised as noncovalent EGFR inhibitors . The
For this mutation, covalent inhibitors were developed to over- most promising compound BMC201725-9o exhibited inhibitory
come these resistances. Afatinib, a typical covalent inhibitor and activity against EGFR (IC50 ¼ 56 nM). Further research shows that
an ATP-competitive anilinoquinazoline derivative, was approved BMC201725-9o can induce apoptosis in A549 cells, but shows
L858R/T790M
by the US FDA in 2013 for advanced NSCLC patients with active weak inhibitory activity against EGFR
(IC50 > 1000 nM).
8
mutant EGFR . However, afatinib contains a reactive “warhead” In this study, in order to enhance the inhibitory activity of EGFR
which can irreversibly bind to proteins other than the target, against L858R/T790M mutation and improve the target com-
6
,8,9
resulting in a toxic burden that limits its clinical utility
.
pounds properties, further structural modifications were mainly
AZD9291, a third-generation irreversible inhibitor and approved focused on the position C-7 of quinazoline. The goal of structural
by the FDA in November 2015 for the treatment of EGFR T790M modification was to increase inhibitory activity against T790M by
mutation in NSCLC patients, has a 200-fold selectivity for the increasing the interaction of the compound with the ATP binding
10
T790M/L858R double mutant over wild-type EGFR . These inhibi- site and reduce oxidation of phenolic hydroxyl groups. Following
tors contain an electrophilic Michael addition receptor moiety that this method, we synthesised one novel quinazoline derivatives by
can covalently bind to the conserved cysteine residue (Cys797) of three modification strategies: (a) The introduction of a polar flex-
EGFR at the lip of the ATP binding cleft of EGFR to facilitate the ible chain on the oxygen atom allows the solubilised tail to better
occupation of the EGFR ATP binding site and overcome the resist- reach the solvent zone. (b) According to the principle of bio-elec-
11
ance caused by the T790M .
tronic isosteres, oxygen atom is replaced by a more stable carbon
CONTACT Wufu Zhu
zhuwufu-1122@163.com, zhuwf@jxstnu.edu.cn; Pengwu Zheng
zhengpw@126.com
Jiangxi Provincial Key Laboratory of Drug Design
and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, 605 Fenglin Road, Nanchang, Jiangxi 330013, China
ꢀ
These authors contribute equally to this work.
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
205
Figure 1. The chemical structures of several small molecule EGFR inhibitors.
Figure 2. Structures and design strategy for target compounds 9–54.
1
atom. (c) Adding heteroatoms on benzene rings to increase hydro- and UV light visualisation. H NMR and11C NMR spectra were
gen bonding receptors. The design strategy is shown in Figure 2. recorded on a Brucker AV-400 spectrometer at 400 MHz and
Herein we assessed antitumour activity of all target compounds Brucker AV-500 spectrometer at 125 MHz using deuterated sol-
against A549, HepG2, MCF-7, H1975 cancer cell lines. In addition, vents as an internal standard. Coupling constants (J) are expressed
WT
L858R/T790M
the EGFR
and EGFR
kinase inhibitory activities of in hertz (Hz). Chemical shifts (d) are given in parts per million
some potential compounds were evaluated. Among these (ppm). High-resolution ESI- MS were recorded on an Applied
designed compounds, compounds 14 and 44 showed the poten- Biosystems Q-STAR Elite ESI- LC- MS/MS mass spectrometer. The
tial against cell and mutant EGFR kinases, which was demon- purity of compounds was determined with reverse-phase HPLC
strated the potential for overcoming the T790M mutant. In analysis to be over 95% (see Supporting information). HPLC instru-
addition, this article further disclosed studies data on docking ment: Dionex Summit HPLC (Column: Diamonsil C18, 5.0 mm, 4.6
studies, AO single staining, cell cycle and apoptosis of 14 and 44.
250 mm (Agilent Technologies); detector: PDA-100 photodiode
array; injector: ASI-100 autoinjector; pump: p-680A). A flow rate of
1
0
.0 ml/min was used with mobile phase of MeOH in H O with
.1% modifier (ammonia v/v).
2
Materials and methods
Reagents and general methods
All reagents and solvents used were purchased from commercial
sources without further purification. Flash chromatography was
Preparation of compounds 1–6
performed using silica gel (200–300 mesh). All reactions were Compounds 1–6 were synthesised according to the procedures in
1
6
monitored by TLC, using silica gel plates with fluorescence F254 our previous group research .

2
06
C. WANG ET AL.
Preparation of (S)-phenyl (4-((3-chloro-4-fluorophenyl) mixture was refluxed for 9–10 h until TLC showed the completion
amino)-7-((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl) carbamate of the reaction. After cooling to room temperature, the precipitate
7a). To the mixture of phenyl chloroformate (13.8 g, 88.5 mmol) was filtered and dried to yield 9–54 which were purified by
(
1
6
and DIPEA (11.5 g, 88.5 mmol) in anhydrous 1,4-dioxane (80 ml), a isopropanol .
solution of compound 6a (13.5 g, 35.4 mmol) in anhydrous 1,4-
dioxane (60 ml) was slowly added at 10 C. After the addition was 6-yl)-2-(4-(dimethylamino)benzylidene)hydrazine-1-carboxamide
(E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
ꢁ
completed, the mixture was warmed to room temperature for (9). This compound was obtained as white solid in 88% yield. Mp
ꢁ
þ
1
another 1.5 h, and the solvent was evaporated under reduced 236.4–239.4 C. ESI-MS m/z: [M þ H] 507.1. H NMR (400 MHz,
pressure. The residue was dissolved in dichloromethane (60 ml), DMSO-d ) d 10.84 (s, 1H), 9.83 (s, 1H), 9.07 (s, 1H), 8.92 (d,
6
and washed with water (3 ꢂ 20 ml), dried over anhydrous Na SO , J ¼ 8.4 Hz, 1H), 8.51 (s, 1H), 8.10 (d, J ¼ 6.6 Hz, 1H), 7.88 (d,
2
4
concentrated under reduced pressure to afford (S)-phenyl(4-((3- J ¼ 11.7 Hz, 1H), 7.79 (s, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H), 7.46–7.37 (m,
chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-yl)oxy) quinazo- 1H), 7.32 (s, 1H), 6.79 (d, J ¼ 8.5 Hz, 2H), 4.13 (s, 3H), 2.98 (s, 6H).
lin-6-yl) carbamate 7a as yellow oil (15.8 g, 90.3%), which were
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
yl)oxy)quinazolin-6-yl)-2-(4-(dimethylamino)benzylidene)hydrazine-
immediately used in the next step without further purification.
þ
ESI-MS m/z: [M þ H] 496.1.
1
7
(
1
-carboxamide (10). This compound was obtained as yellow solid in
ꢁ
þ
1
Preparation of phenyl (4-((3-chloro-4-fluorophenyl) amino)-
6% yield. Mp 238.4–241.2 C. ESI-MS m/z: [M þ H] 563.1. H NMR
7
-methoxy quinazolin-6-yl)carbamate (7b). To the mixture of
phenyl chloroformate (13.8 g, 88.5 mmol) and DIPEA (11.5 g,
8.5 mmol) in anhydrous 1,4-dioxane (60 ml), a solution of com-
400 MHz, DMSO-d ) d 10.89 (s, 1H), 9.86 (s, 1H), 9.13 (s, 1H), 9.00 (s,
6
H), 8.50 (s, 1H), 8.08 (d, J ¼ 6.1 Hz, 1H), 7.89 (s, 1H), 7.78 (s, 1H), 7.54
8
(
d, J ¼ 8.1 Hz, 2H), 7.41 (t, J ¼ 9.0 Hz, 1H), 7.32 (s, 1H), 6.73 (d,
pound 6 b (11.3 g, 35.4 mmol) in anhydrous 1,4-dioxane (30 ml)
J ¼ 8.3 Hz, 2H), 5.45 (s, 1H), 4.04 (s, 2H), 4.01–3.95 (m, 1H), 3.89 (d,
ꢁ
was slowly added at 10 C. After the addition was completed, the
J ¼ 4.5 Hz, 1H), 2.98 (s, 6H), 2.43 (dd, J ¼ 13.8, 6.7 Hz, 1H), 2.18 (s, 1H).
mixture was warmed to room temperature for another 1.5 h, and
the solution was poured into water with stirring for 15 min. The
precipitate was filtered and washed with water, dried to furnish
phenyl (4-((3-chloro-4-fluorophenyl) amino)-7-methoxy quinazolin-
(E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
6
-yl)-2-(4-(diethylamino)benzylidene)hydrazine-1-carboxamide (11).
This compound was obtained as white solid in 86% yield. Mp
ꢁ
þ
1
2
26.1–229.4 C. ESI-MS m/z: [M þ H] 535.1. H NMR (400 MHz,
1
4
6
-yl) carbamate 7 b as light yellow solid (14.2 g, 91.6%) , which
DMSO-d ) d 10.84 (s, 1H), 9.83 (s, 1H), 9.07 (s, 1H), 8.92
6
were immediately used in the next step without further purifica-
(
(
(
(
d, J ¼ 8.4 Hz, 1H), 8.51 (s, 1H), 8.10 (d, J ¼ 6.6 Hz, 1H), 7.88
þ
1
tion. ESI-MS m/z: [M þ H] 439.1. H NMR (400 MHz, DMSO-d
6
) d
d, J ¼ 11.7 Hz, 1H), 7.79 (s, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H), 7.46–7.37
m, 1H), 7.32 (s, 1H), 6.79 (d, J ¼ 8.5 Hz, 2H), 4.13 (s, 3H), 2.98
s, 6H).
9
7
6
.36 (d, J ¼ 14.2 Hz, 2H), 8.37 (s, 1H), 8.18 (d, J ¼ 5.0 Hz, 1H),
.86–7.74 (m, 1H), 7.38 (d, J ¼ 4.2 Hz, 2H), 7.15 (s, 2H), 7.10 (s, 1H),
.75 (d, J ¼ 7.7 Hz, 3H), 3.96 (s, 3H).
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
Preparation of N-(4-((3-chloro-4-fluorophenyl)amino)-7-
yl)oxy)quinazolin-6-yl)-2-(4-(diethylamino)benzylidene)hydrazine-1-
(
cyclopentyloxy)quinazolin-6-yl) hydrazinecarboxamide (8a). A
mixture of 7a (4.2 g, 8.40 mmol) and 80% hydrazine monohydrate
3 ml) in 1,4-dioxane (20 ml) was refluxed for 2 h and monitored
carboxamide (12). This compound was obtained as white solid in
ꢁ
þ
1
8
(
1
7
(
4% yield. Mp 236.4–238.9 C. ESI-MS m/z: [M þ H] 591.2. H NMR
(
400 MHz, DMSO) d 10.85 (dd, J ¼ 48.5, 28.5 Hz, 1H), 10.13–9.58 (m,
by TLC. After cooling to room temperature, the yellow solid was
filtered off and washed with 1,4-dioxane and water, and dried to
afford the (S)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydro-
furan-3 -yl)oxy)quinazolin-6-yl)hydrazine carboxamide 8a as a
H), 9.09 (s, 1H), 8.98 (s, 1H), 8.40 (s, 1H), 8.02 (s, 1H), 7.87 (s, 1H),
.68 (s, 1H), 7.51 (d, J ¼ 8.7 Hz, 2H), 7.36 (s, 1H), 7.24 (s, 1H), 6.68
d, J ¼ 8.8 Hz, 2H), 5.42 (s, 1H), 4.05 (dt, J ¼ 15.6, 6.6 Hz, 2H),
4
(
.00–3.94 (m, 1H), 3.88 (dt, J ¼ 12.7, 6.4 Hz, 1H), 3.40 (s, 4H), 2.42
dd, J ¼ 13.9, 6.2 Hz, 1H), 2.22–2.12 (m, 1H), 1.12 (t, J ¼ 6.9 Hz, 6H).
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
1
6
ꢁ
white solid (2.4 g, 65.2%) . Mp 262.3–263.7 C. ESI-MS m/z:
[
þ
M þ H] 434.1. 1H NMR (400 MHz, DMSO-d
6
) d 9.78 (s, 1H), 9.28 (s,
(
1
7
1
3
7
H), 8.92 (s, 1H), 8.43 (d, J ¼ 12.7 Hz, 1H), 8.07 (d, J ¼ 6.6 Hz, 1H),
yl)oxy)quinazolin-6-yl)-2-((5-morpholinothiazol-2-yl)methylene)hy-
.92 (s, 1H), 7.76 (d, J ¼ 8.6 Hz, 1H), 7.40 (t, J ¼ 9.1 Hz, 1H), 7.24 (s,
H), 5.36 (s, 1H), 4.68 (s, 2H), 4.01 (dd, J ¼ 10.2, 4.0 Hz, 1H),
.98–3.86 (m, 2H), 3.82 (dd, J ¼ 12.4, 7.9 Hz, 1H), 2.37 (td, J ¼ 12.2,
.5 Hz, 1H), 2.15–1.84 (m, 1H).
drazine-1-carboxamide (13). This compound was obtained as
ꢁ
þ
white solid in 86% yield. Mp 236.4–238.9 C. ESI-MS m/z: [M þ H]
1
6
9
(
12.1. H NMR (400 MHz, DMSO-d ) d 11.04 (s, 1H), 9.85 (s, 1H),
6
.02 (s, 1H), 8.86 (s, 1H), 8.50 (s, 1H), 8.08 (d, J ¼ 6.8 Hz, 2H), 7.77
N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxy quinazo-
lin-6-yl)hydrazine carboxamide (8b). The synthesis of compound
dt, J ¼ 7.4, 3.5 Hz, 1H), 7.58 (s, 1H), 7.42 (t, J ¼ 9.1 Hz, 1H), 7.30 (s,
1
H), 5.43 (s, 1H), 4.15–4.05 (m, 2H), 3.96 (q, J ¼ 8.0 Hz, 1H), 3.88 (td,
J ¼ 8.2, 4.2 Hz, 1H), 3.74 (t, J ¼ 4.9 Hz, 4H), 3.51 (t, J ¼ 4.9 Hz, 4H),
.40 (dt, J ¼ 14.4, 7.1 Hz, 1H), 2.25–2.14 (m, 1H).
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
8
9
b was similar to the compound 8a. A mixture of 7 b (4.0 g,
.12 mmol) and 80% hydrazine monohydrate (5 ml) in 1,4-dioxane
2
(20 ml) was refluxed for 2 h and monitored by TLC. After cooling to
(
room temperature, the white solid was filtered off and washed with
water, and dried to afford the N-(4-((3-chloro-4-fluorophenyl)a-
mino)-7-methoxy quinazolin-6-yl) hydrazine carboxamide 8 b as a
yl)oxy)quinazolin-6-yl)-2-((2-(pyrrolidin-1-yl)pyrimidin-5-yl)methyle-
ne)hydrazine-1-carboxamide (14). This compound was obtained as
ꢁ
þ
ꢁ
yellow solid in 85% yield. Mp 236.4–238.9 C. ESI-MS m/z: [M þ H]
white solid (2.4 g, 68.5%) [16]. Mp 263.8–264.6 C. ESI-MS m/z:
1
þ
591.1. H NMR (400 MHz, DMSO-d
6
) d 11.09 (s, 1H), 10.05 (s, 1H),
[
M þ H] 376.1. 1H NMR (400 MHz, DMSO-d ) d 9.76 (s, 1H), 9.20 (s,
6
9
7
(
(
2
.01 (s, 2H), 8.66 (s, 2H), 8.55 (s, 1H), 8.08 (dd, J ¼ 6.8, 2.6 Hz, 1H),
1
7
4
H), 8.91 (s, 1H), 8.47 (s, 1H), 8.07 (d, J ¼ 4.9 Hz, 1H), 7.90 (s, 1H),
.81–7.69 (m, 1H), 7.40 (t, J ¼ 9.1 Hz, 1H), 7.27 (s, 1H), 4.67 (s, 2H),
.04 (s, 3H).
.89 (s, 1H), 7.77 (dd, J ¼ 8.8, 4.0 Hz, 1H), 7.44 (t, J ¼ 9.1 Hz, 1H), 7.32
s, 1H), 5.43 (d, J ¼ 5.3 Hz, 1H), 4.02 (dd, J ¼ 10.1, 3.8 Hz, 2H), 3.98
d, J ¼ 9.2 Hz, 1H), 3.82 (dt, J ¼ 8.4, 4.1 Hz, 1H), 3.56–3.52 (m, 4H),
.47–2.39 (m, 1H), 2.16 (dd, J ¼ 13.1, 6.4 Hz, 1H), 1.99–1.93 (m, 4H).
General procedure for the preparation of compounds 9–54
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
To a solution of 8a–b (0.46 mmol) in Ethanol/DMF (4 ml), 1.1 equiv yl)oxy)quinazolin-6-yl)-2-((5-(pyrrolidin-1-yl)furan-2-yl)methylene)hy-
of aldehydes and 98% sulfuric acid (1 drop) were added, and the drazine-1-carboxamide (15). This compound was obtained as

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
207
ꢁ
þ
ꢁ
þ
1
yellow solid in 78% yield. Mp 236.4–238.9 C. ESI-MS m/z: [M þ H]
yield. Mp 235.4–237.5 C. ESI-MS m/z: [M þ H] 561.2. H NMR
1
5
79.1. H NMR (400 MHz, DMSO) d 11.07 (s, 1H), 10.06 (s, 1H), 9.09 (400 MHz, DMSO-d ) d 11.14 (s, 1H), 9.86 (s, 1H), 9.08 (s, 1H), 8.93
6
(
(
(
s, 1H), 8.65 (s, 1H), 8.55 (s, 1H), 8.08 (dd, J ¼ 6.8, 2.6 Hz, 1H), 7.89 (s, 1H), 8.53 (s, 1H), 8.10 (s, 1H), 8.02 (s, 1H), 7.79 (s, 1H), 7.68 (d,
s, 1H), 7.77 (dd, J ¼ 8.8, 4.0 Hz, 1H), 7.44 (t, J ¼ 9.1 Hz, 1H), 7.32 J ¼ 7.8 Hz, 2H), 7.43 (s, 1H), 7.40 (s, 1H), 7.34 (s, 2H), 4.13 (d,
s, 1H), 7.01 (s, 1H) 6.41 (s, 1H),5.43 (d, J ¼ 5.3 Hz, 1H), 4.02 (dd, J ¼ 2.3 Hz, 3H), 3.46 (s, 2H), 2.34 (s, 4H), 1.51 (s, 4H), 1.40 (s, 2H).
J ¼ 10.1, 3.8 Hz, 2H), 3.98 (d, J ¼ 9.2 Hz, 1H), 3.82 (dt, J ¼ 8.4, 4.1 Hz,
(E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
1
1
H), 3.42–3.62 (m, 4H), 2.47–2.39 (m, 1H), 2.16 (dd, J ¼ 13.1, 6.4 Hz, 6-yl)-2-(4-(morpholinomethyl)benzylidene)hydrazine-1-carboxamide
H), 1.93–1.89 (m, 4H).
(23). This compound was obtained as white solid in 87% yield. Mp
ꢁ
þ
1
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3- 236.4–239.5 C. ESI-MS m/z: [M þ H] 563.1. H NMR (400 MHz,
yl)oxy)quinazolin-6-yl)-2-((5-(dimethylamino)thiophen-2-yl)methyle-
ne)hydrazine-1-carboxamide (16). This compound was obtained as
6
DMSO-d ) d 11.15 (s, 1H), 9.87 (s, 1H), 9.10 (s, 1H), 8.93 (s, 1H),
8
.56 (s, 1H), 8.12 (s, 1H), 8.03 (s, 1H), 7.80 (s, 1H), 7.78 (d,
ꢁ
þ
white solid in 89% yield. Mp 236.4–238.9 C. ESI-MS m/z: [M þ H]
J ¼ 7.8 Hz, 2H), 7.42 (s, 3H), 7.35 (s, 1H), 4.13 (d, J ¼ 2.3 Hz, 3H),
1
5
9
69.1. H NMR (400 MHz, DMSO-d
6
) d 10.78 (s, 1H), 9.84 (s, 1H),
.03 (s, 1H), 8.89 (s, 1H), 8.49 (s, 1H), 8.09 (s, 1H), 7.98 (s, 1H), 7.78
3
.66 (s, 2H), 3.45 (s, 4H), 2.44 (s, 4H).
E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
-yl)-2-(4-((4-methylpiperazin-1-yl)methyl)benzylidene)hydrazine-1-
(
(
s, 1H), 7.40 (d, J ¼ 9.1 Hz, 1H), 7.29 (s, 1H), 7.10 (s, 1H), 5.88 (s,
H), 5.41 (s, 1H), 4.10 (s, 2H), 3.99 (s, 1H), 3.86 (s, 1H), 2.99 (d,
J ¼ 9.6 Hz, 6H), 2.44–2.36 (m, 1H), 2.28–2.18 (m, 1H).
E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
-yl)-2-((6-(pyrrolidin-1-yl)pyridin-3-yl)methylene) hydrazine-1-car-
boxamide (17). This compound was obtained as yellow solid in
6
1
carboxamide (24). This compound was obtained as yellow solid in
ꢁ
þ
1
8
(
(
1% yield. Mp 231.4–235.8 C. ESI-MS m/z: [M þ H] 576.2. H NMR
(
400 MHz, DMSO-d ) d 11.12 (s, 1H), 9.84 (s, 1H), 9.07 (s, 1H), 8.93
s, 1H), 8.52 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.80 (s, 1H), 7.68 (d,
6
6
J ¼ 8.2 Hz, 2H), 7.44 (s, 1H), 7.41 (d, J ¼ 7.5 Hz, 2H), 7.34 (s, 1H),
ꢁ
þ
1
7
7% yield. Mp 236.4–238.9 C. ESI-MS m/z: [M þ H] 534.1. H NMR
400 MHz, DMSO-d ) d 10.87 (s, 1H), 9.81 (s, 1H), 9.00 (s, 1H), 8.90
s, 1H), 8.50 (s, 1H), 8.24 (s, 1H), 8.09 (s, 1H), 7.89 (s, 2H), 7.78 (s,
H), 7.45–7.36 (m, 1H), 7.30 (s, 1H), 6.59 (d, J ¼ 9.1 Hz, 1H), 4.10 (d,
J ¼ 4.9 Hz, 3H), 3.43 (s, 4H), 1.95 (s, 4H).
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
4
4
.09 (d, J ¼ 26.9 Hz, 3H), 3.49 (s, 2H), 2.53 (s, 4H), 2.39–2.30 (m,
(
(
1
6
H), 2.15 (s, 3H).
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
yl)oxy)quinazolin-6-yl)-2-(4-((dimethylami no)methyl)benzylidene)-
hydrazine-1-carboxamide (25). This compound was obtained as
(
ꢁ
þ
white solid in 86% yield. Mp 236.4–239.5 C. ESI-MS m/z: [M þ H]
yl)oxy)quinazolin-6-yl)-2-((6-(pyrrolidin-1-yl)pyridin-3-yl)methylene)-
hydrazine-1-carboxamide (18). This compound was obtained as
1
5
9
8
77.2. H NMR (400 MHz, DMSO-d ) d 11.19 (s, 1H), 9.86 (s, 1H),
6
.15 (s, 1H), 9.01 (s, 1H), 8.51 (s, 1H), 8.09 (dd, J ¼ 6.9, 2.6 Hz, 1H),
ꢁ
þ
white solid in 79% yield. Mp 236.4–238.9 C. ESI-MS m/z: [M þ H]
.02 (s, 1H), 7.80–7.75 (m, 1H), 7.69 (d, J ¼ 7.9 Hz, 2H), 7.40 (d,
1
5
9
90.2. H NMR (400 MHz, DMSO-d
.08 (s, 1H), 8.99 (s, 1H), 8.50 (s, 1H), 8.23 (d, J ¼ 2.1 Hz, 1H), 8.09
6
) d 10.95 (s, 1H), 9.84 (s, 1H),
J ¼ 9.1 Hz, 1H), 7.37 (s, 1H), 7.34 (d, J ¼ 6.3 Hz, 2H), 5.45 (s, 1H),
4
3
.05 (d, J ¼ 3.3 Hz, 2H), 3.97 (q, J ¼ 7.7 Hz, 1H), 3.91–3.83 (m, 1H),
.41 (s, 2H), 2.47–2.39 (m, 1H), 2.18 (d, J ¼ 6.7 Hz, 1H), 2.1 (s, 6H)
(
7
dd, J ¼ 6.9, 2.6 Hz, 1H), 7.94 (d, J ¼ 9.8 Hz, 1H), 7.90 (s, 1H),
.80–7.75 (m, 1H), 7.41 (t, J ¼ 9.0 Hz, 1H), 7.31 (s, 1H), 6.50 (d,
(
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
J ¼ 8.8 Hz, 1H), 5.44 (s, 1H), 4.02 (d, J ¼ 3.2 Hz, 2H), 4.00–3.93 (m,
yl)oxy)quinazolin-6-yl)-2-(4-((diethylamino) methyl)benzylidene)hy-
drazine-1-carboxamide (26).
1
2
H), 3.86 (d, J ¼ 5.0 Hz, 1H), 3.44 (s, 4H), 2.46–2.36 (m, 1H),
.20–2.11 (m, 1H), 1.96 (s, 4H).
This compound was obtained as yellow solid in 81% yield. Mp
(E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
ꢁ
þ
1
2
26.4–231.5 C. ESI-MS m/z: [M þ H] 605.2. H NMR (400 MHz,
6
-yl)-2-(4-((dimethylamino)methyl)benzylidene)hydrazine-1-carbox-
DMSO-d ) d 11.19 (s, 1H), 9.86 (s, 1H), 9.15 (s, 1H), 9.01 (s, 1H),
6
amide (19). This compound was obtained as white solid in 84%
ꢁ
þ
1
8.51 (s, 1H), 8.09 (dd, J ¼ 6.9, 2.6 Hz, 1H), 8.02 (s, 1H), 7.80–7.75 (m,
yield. Mp 226.5–229.4 C. ESI-MS m/z: [M þ H] 521.1. H NMR
1
H), 7.69 (d, J ¼ 7.9 Hz, 2H), 7.40 (d, J ¼ 9.1 Hz, 1H), 7.37 (s, 1H),
.34 (d, J ¼ 6.3 Hz, 2H), 5.45 (s, 1H), 4.05 (d, J ¼ 3.3 Hz, 2H), 3.97 (q,
(
(
1
6
400 MHz, DMSO-d ) d 11.12 (s, 1H), 9.84 (s, 1H), 9.08 (s, 1H), 8.92
7
s, 1H), 8.52 (s, 1H), 8.11 (d, J ¼ 6.7 Hz, 1H), 8.02 (s, 1H), 7.78 (s,
J ¼ 7.7 Hz, 1H), 3.91–3.83 (m, 1H), 3.41 (s, 2H), 2.54 (s, 4H),
H), 7.68 (d, J ¼ 7.8 Hz, 2H), 7.40 (d, J ¼ 7.2 Hz, 3H), 7.33 (s, 1H),
2
.47–2.39 (m, 1H), 2.18 (d, J ¼ 6.7 Hz, 1H), 1.51 (s, 6H).
4
.12 (s, 3H), 3.42 (s, 2H), 2.16 (s, 6H).
(
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
(
E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
-yl)-2-(4-((diethylamino)methyl)benzylidene)hydrazine-1-carboxa-
yl)oxy)quinazolin-6-yl)-2-(4-(pyrrolidin-1-y lmethyl)benzylidene)hy-
6
drazine-1-carboxamide (27). This compound was obtained as
mide (20). This compound was obtained as white solid in 86%
ꢁ
þ
ꢁ
þ
1
white solid in 83% yield. Mp 235.4–239.5 C. ESI-MS m/z: [M þ H]
yield. Mp 226.1–229.6 C. ESI-MS m/z: [M þ H] 549.2. H NMR
1
6
9
03.2. H NMR (400 MHz, DMSO-d ) d 11.18 (s, 1H), 9.86 (s, 1H),
.14 (s, 1H), 9.01 (s, 1H), 8.50 (s, 1H), 8.09 (s, 1H), 8.01 (s, 1H), 7.78
(
(
1
400 MHz, DMSO-d ) d 11.12 (s, 1H), 9.84 (s, 1H), 9.08 (s, 1H), 8.92
6
6
s, 1H), 8.52 (s, 1H), 8.11 (d, J ¼ 6.7 Hz, 1H), 8.02 (s, 1H), 7.78 (s,
(
s, 1H), 7.67 (d, J ¼ 7.8 Hz, 2H), 7.38 (dt, J ¼ 21.0, 11.0 Hz, 4H), 5.45
H), 7.68 (d, J ¼ 7.8 Hz, 2H), 7.40 (d, J ¼ 7.2 Hz, 3H), 7.33 (s, 1H),
(
2
s, 1H), 4.05 (s, 2H), 3.97 (d, J ¼ 7.3 Hz, 1H), 3.87 (s, 1H), 3.59 (s,
4
.12 (s, 3H), 3.42 (s, 2H), 2.46–2.54(m, 4H) 1.46 (s, 6H).
H), 2.49 (s, 1H), 2.43 (s, 4H), 2.17 (s, 1H), 1.70 (s, 4H).
(E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
6
-yl)-2-(4-(pyrrolidin-1-ylmethyl)benzylidene)hydrazine-1-carboxa-
yl)oxy)quinazolin-6-yl)-2-(4-(piperidin-1-yl methyl)benzylidene)hy-
drazine-1-carboxamide (28). This compound was obtained as yel-
400 MHz, DMSO-d ) d 11.13 (s, 1H), 9.85 (s, 1H), 9.08 (s, 1H), 8.93 low solid in 87% yield. Mp 236.4–238.5 C. ESI-MS m/z: [M þ H]
mide (21). This compound was obtained as yellow solid in 89%
ꢁ
þ
1
yield. Mp 231.4–234.5 C. ESI-MS m/z: [M þ H] 547.1. H NMR
ꢁ
þ
(
(
6
1
s, 1H), 8.52 (s, 1H), 8.10 (s, 1H), 8.01 (s, 1H), 7.79 (s, 1H), 7.67 (d, 617.2. H NMR (400 MHz, DMSO-d
6
) d 11.16 (s, 1H), 9.85 (s, 1H),
J ¼ 8.2 Hz, 2H), 7.42 (s, 3H), 7.33 (s, 1H), 4.12 (s, 3H), 3.60 (s, 2H), 9.13 (s, 1H), 9.00 (s, 1H), 8.49 (s, 1H), 8.07 (d, J ¼ 4.4 Hz, 1H), 8.00
2
.44 (s, 4H), 1.70 (s, 4H).
E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-
-yl)-2-(4-(piperidin-1-ylmethyl)benzylidene)hydrazine-1-carboxa-
(s, 1H), 7.75 (s, 1H), 7.66 (d, J ¼ 8.0 Hz, 2H), 7.43–7.31 (m, 4H), 5.43
(s, 1H), 4.04 (s, 2H), 3.95 (dd, J ¼ 15.7, 7.9 Hz, 1H), 3.85 (dd,
J ¼ 13.1, 8.3 Hz, 1H), 3.44 (s, 2H), 2.40 (dd, J ¼ 14.1, 8.0 Hz, 1H), 2.31
(
6
mide (22). This compound was obtained as yellow solid in 92% (s, 4H), 2.19–2.10 (m, 1H), 1.48 (d, J ¼ 5.2 Hz, 4H), 1.38 (s, 2H).

2
08
C. WANG ET AL.
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3- (s, 1H), 8.52 (s, 1H), 8.11 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 7.66 (d,
yl)oxy)quinazolin-6-yl)-2-(4-(morpholinomethyl)benzylidene)hydra-
J ¼ 7.8 Hz, 2H), 7.45–7.38 (m, 1H), 7.33 (s, 1H), 7.06 (d, J ¼ 8.0 Hz,
zine-1-carboxamide (29). This compound was obtained as white 2H), 4.14 (s, 2H), 4.12 (s, 3H), 3.58 (s, 4H), 2.71 (s, 2H), 2.49–2.43
ꢁ
þ
solid in 86% yield. Mp 232.4–236.8 C. ESI-MS m/z: [M þ H] 619.2. (m, 4H).
1
H NMR (400 MHz, DMSO-d
6
) d 11.28 (s, 1H), 9.97 (s, 1H), 9.24 (s,
(E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6-
1
7
1
3
2
H), 9.11 (s, 1H), 8.60 (s, 1H), 8.18 (d, J ¼ 4.7 Hz, 1H), 8.11 (s, 1H), yl)-2-(4-(2-(4-methylpiperazin-1-yl)ethoxy) benzylidene)hydrazine-1-
.87 (d, J ¼ 7.8 Hz, 1H), 7.77 (d, J ¼ 7.9 Hz, 2H), 7.47 (dd, J ¼ 20.2, carboxamide (36). This compound was obtained as white solid in
ꢁ
þ
1
2.3 Hz, 4H), 5.55 (s, 1H), 4.15 (s, 2H), 4.08–4.03 (m, 1H), 3.99 – 91% yield. Mp 215.2–220.5 C. ESI-MS m/z: [M þ H] 606.2. H NMR
.93 (m, 1H), 3.69 (s, 2H), 2.53 (s, 4H), 2.48 (d, J ¼ 9.4 Hz, 1H), (400 MHz, DMSO-d
.30–2.18 (m, 1H), 1.80 (s, 4H).
8.86 (s, 1H), 8.46 (s, 1H), 8.04 (d, J ¼ 4.9 Hz, 1H), 7.90 (s, 1H), 7.71
6
) d 10.99 (s, 1H), 9.80 (s, 1H), 9.01 (s, 1H),
(
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3- (s, 1H), 7.60 (d, J ¼ 8.4 Hz, 2H), 7.36 (t, J ¼ 9.3 Hz, 1H), 7.27 (s, 1H),
yl)oxy)quinazolin-6-yl)-2-(4-((4-methylpipe razin-1-yl)methyl)benzyli- 7.00 (d, J ¼ 8.4 Hz, 2H), 4.07 (s, 2H), 4.05 (s, 3H), 2.68 (s, 2H), 2.44
dene)hydrazine-1-carboxamide (30). This compound was obtained (s, 8H), 2.28 (s, 3H).
ꢁ
as white solid in 85% yield. Mp 233.4–237.6 C. ESI-MS m/z:
(S,E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-((tetrahydrofuran-3-
ino)ethoxy)benzylide-
þ
1
[
(
1
7
2
2
M þ H] 632.2. H NMR (400 MHz, DMSO-d
6
) d 11.28 (s, 1H), 9.97 yl)oxy)quinazolin-6-yl)-2-(4-(2-(dimethylam
s, 1H), 9.24 (s, 1H), 9.11 (s, 1H), 8.60 (s, 1H), 8.18 (d, J ¼ 4.7 Hz, ne)hydrazine-1-carboxamide (37). This compound was obtained as
ꢁ
þ
H), 8.11 (s, 1H), 7.87 (d, J ¼ 7.8 Hz, 1H), 7.77 (d, J ¼ 7.9 Hz, 2H), white solid in 90% yield. Mp 221.5–231.8 C. ESI-MS m/z: [M þ H]
1
.47 (dd, J ¼ 20.2, 12.3 Hz, 4H),d 5.36 (s, 1H), 4.03 (d, J ¼ 3.3 Hz, 607.2. H NMR (400 MHz, DMSO-d
) d 11.09 (s, 1H), 9.87 (s, 1H),
6
H), 3.96 (d, J ¼ 7.8 Hz, 1H), 3.86 (dd, J ¼ 8.4, 4.7 Hz, 1H), 3.48 (s, 9.14 (s, 1H), 9.01 (s, 1H), 8.51 (s, 1H), 8.10 (s, 1H), 7.97 (s, 1H), 7.78
H), 2.66–2.51 (m, 4H), 2.38 (s, 4H), 2.15 (s, 3H).
(s, 1H), 7.67 (d, J ¼ 8.3 Hz, 2H), 7.42 (t, J ¼ 9.1 Hz, 1H), 7.33 (s, 1H),
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6- 7.00 (d, J ¼ 8.4 Hz, 2H), 5.45 (s, 1H), 4.10 (s, 2H), 4.04 (s, 2H), 3.98
yl)-2-(4-(2-(dimethylamino)ethoxy)benzylid ene)hydrazine-1-carbox- (s, 1H), 3.89 (s, 1H), 2.63 (s, 2H), 2.22 (s, 6H), 2.18–2.13 (m, 2H).
amide (31). This compound was obtained as white solid in 76%
(S,E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-((tetrahydrofuran-3-
(
ꢁ
þ
1
yield. Mp 205.2–209.5 C. ESI-MS m/z: [M þ H] 551.18. H NMR yl)oxy)quinazolin-6-yl)-2-(4-(2-(diethylamin o)ethoxy)benzylidene)-
(
(
7
(
4
(
400 MHz, DMSO-d
) d 10.98 (s, 1H), 9.80 (s, 1H), 9.02 (s, 1H), 8.88 hydrazine-1-carboxamide (38). This compound was obtained as
6
ꢁ
þ
d, J ¼ 4.0 Hz, 1H), 8.47 (d, J ¼ 4.3 Hz, 1H), 8.06 (d, J ¼ 4.2 Hz, 1H), yellow solid in 89% yield. Mp 210.1–213.9 C. ESI-MS m/z: [M þ H]
1
.93 (d, J ¼ 11.1 Hz, 1H), 7.74 (s, 1H), 7.61 (d, J ¼ 8.5 Hz, 2H), 7.39 635.2. H NMR (400 MHz, DMSO-d
6
) d 11.10 (s, 1H), 9.87 (s, 1H),
d, J ¼ 9.2 Hz, 1H), 7.27 (d, J ¼ 9.0 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 2H), 9.14 (s, 1H), 9.01 (s, 1H), 8.51 (s, 1H), 8.09 (dd, J ¼ 6.8, 2.3 Hz, 1H),
.07 (s, 3H), 4.06 (d, J ¼ 5.3 Hz, 2H), 2.61 (d, J ¼ 5.3 Hz, 2H), 2.18 7.97 (s, 1H), 7.80–7.75 (m, 1H), 7.67 (d, J ¼ 8.5 Hz, 2H), 7.41 (d,
s, 6H).
(
J ¼ 9.1 Hz, 1H), 7.33 (s, 1H), 7.04–6.97 (m, 2H), 5.46 (s, 1H), 4.04 (s,
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6- 2H), 3.97 (d, J ¼ 7.6 Hz, 2H), 3.88 (d, J ¼ 4.4 Hz, 2H), 2.84 (s, 2H),
yl)-2-(4-(2-(diethylamino)ethoxy)benzyliden e)hydrazine-1-carboxa- 2.61 (s, 4H), 2.43 (dd, J ¼ 13.8, 6.0 Hz, 1H), 2.20–2.12 (m, 1H), 1.00
mide (32). This compound was obtained as white solid in 79% (t, J ¼ 6.7 Hz, 6H).
ꢁ
þ
1
yield. Mp 208.5–210.2 C. ESI-MS m/z: [M þ H] 579.22. H NMR
(S,E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-((tetrahydrofuran-3-
(
400 MHz, CDCl
) d 11.01 (s, 1H), 9.84 (s, 1H), 9.06 (s, 1H), 8.92 (s, yl)oxy)quinazolin-6-yl)-2-(4-(2-(pyrrolidin-1 -yl)ethoxy)benzylidene)-
3
1
H), 8.51 (s, 1H), 8.09 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 7.65 (d, hydrazine-1-carboxamide (39). This compound was obtained as
ꢁ
þ
J ¼ 8.3 Hz, 2H), 7.40 (d, J ¼ 9.1 Hz, 1H), 7.32 (s, 1H), 7.04 (d, white solid in 88% yield. Mp 216.2–218.9 C. ESI-MS m/z: [M þ H]
1
J ¼ 8.9 Hz, 2H), 4.12 (s, 3H), 4.06 (d, J ¼ 6.0 Hz, 2H), 2.78 (s, 2H), 633.2. H NMR (400 MHz, DMSO-d
) d 11.07 (s, 1H), 9.86 (s, 1H),
6
2
.55 (d, J ¼ 7.0 Hz, 4H), 0.97 (t, J ¼ 7.0 Hz, 6H).
9.14 (s, 1H), 9.01 (s, 1H), 8.50 (s, 1H), 8.09 (dd, J ¼ 6.8, 2.4 Hz, 1H),
(
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6- 7.97 (s, 1H), 7.80–7.75 (m, 1H), 7.67 (d, J ¼ 8.4 Hz, 2H), 7.40 (d,
yl)-2-(4-(2-(pyrrolidin-1-yl)ethoxy)benzylid ene)hydrazine-1-carboxa- J ¼ 9.2 Hz, 1H), 7.33 (s, 1H), 6.99 (d, J ¼ 8.5 Hz, 2H), 5.45 (s, 1H),
mide (33). This compound was obtained as yellow solid in 84% 4.09–4.02 (m, 4H), 3.96 (t, J ¼ 7.8 Hz, 1H), 3.91–3.84 (m, 1H),
ꢁ
þ
1
yield. Mp 203.2–208.8 C. ESI-MS m/z: [M þ H] 577.2. H NMR 3.63–3.53 (m, 4H), 2.43 (t, J ¼ 7.2 Hz, 2H), 2.37 (s, 4H), 2.16 (d,
(
(
(
400 MHz, DMSO-d ) d 11.03 (s, 1H), 9.85 (s, 1H), 9.06 (s, 1H), 8.91 J ¼ 7.2 Hz, 1H), 1.91–1.87 (m, 1H).
6
s, 1H), 8.51 (s, 1H), 8.10 (dd, J ¼ 7.0, 2.6 Hz, 1H), 7.96 (s, 1H), 7.78
(S,E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-((tetrahydrofuran-3-
d, J ¼ 8.9 Hz, 1H), 7.65 (d, J ¼ 8.5 Hz, 2H), 7.42 (t, J ¼ 9.2 Hz, 1H), yl)oxy)quinazolin-6-yl)-2-(4-(2-(piperidin-1-yl)ethoxy)benzylidene)hy-
7
.32 (s, 1H), 7.05 (d, J ¼ 8.3 Hz, 2H), 4.12 (d, J ¼ 6.7 Hz, 5H), 2.79 (d, drazine-1-carboxamide (40). This compound was obtained as yel-
ꢁ
þ
J ¼ 7.1 Hz, 2H), 2.52 (s, 4H), 1.68 (d, J ¼ 5.5 Hz, 4H).
low solid in 86% yield. Mp 213.4–218.4 C. ESI-MS m/z: [M þ H]
1
(
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6- 647.2. H NMR (400 MHz, DMSO-d ) d 11.05 (s, 1H), 9.87 (s, 1H),
6
yl)-2-(4-(2-(piperidin-1-yl)ethoxy)benzylide ne)hydrazine-1-carboxa- 9.08 (s, 1H), 8.92 (s, 1H), 8.52 (s, 1H), 8.11 (d, J ¼ 6.7 Hz, 1H), 7.96
mide (34). This compound was obtained as white solid in 75% (s, 1H), 7.78 (s, 1H), 7.66 (d, J ¼ 8.4 Hz, 2H), 7.43 (t, J ¼ 9.1 Hz, 1H),
ꢁ
þ
1
yield. Mp 205.6–208.4 C. ESI-MS m/z: [M þ H] 591.22. H NMR 7.33 (s, 1H), 7.06 (d, J ¼ 8.5 Hz, 2H), 5.55 (s, 1H), 4.02–3.98 (m, 4H),
(
400 MHz, DMSO-d
6
) d 11.01 (s, 1H), 9.84 (s, 1H), 9.07 (s, 1H), 8.92 3.96–3.94 (t, J ¼ 7.8 Hz, 1H), 3.91–3.84 (m, 1H), 2.89 (s, 2H),
(
s, 1H), 8.52 (s, 1H), 8.09 (d, J ¼ 2.8 Hz, 1H), 7.96 (s, 1H), 7.76 (d, 2.53–2.48 (m, J ¼ 7.2 Hz, 1H), 2.44 (s, 4H), 2.16–2.08 (m, J ¼ 5.7 Hz,
J ¼ 8.8 Hz, 1H), 7.65 (d, J ¼ 8.5 Hz, 2H), 7.41 (d, J ¼ 6.6 Hz, 1H), 7.33 1H),1.50 (s, 4H), 1.39 (s, 2H).
s, 1H), 7.05 (d, J ¼ 8.5 Hz, 2H), 4.12 (s, 3H), 4.04 (s, 2H), 2.66 (t,
(S,E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-((tetrahydrofuran-3-
J ¼ 5.8 Hz, 2H), 2.43 (s, 4H), 1.53–1.46 (m, 4H), 1.38 (d, yl)oxy)quinazolin-6-yl)-2-(4-(2-morpholinoe thoxy)benzylidene)hy-
(
J ¼ 5.0 Hz, 2H).
drazine-1-carboxamide (41). This compound was obtained as
ꢁ
þ
(
E)-N-(4-((4-chloro-3-fluorophenyl)amino)-7-methoxyquinazolin-
-yl)-2-(4-(2-morpholinoethoxy)benzylidene) hydrazine-1-carboxa- 649.2. H NMR (400 MHz, DMSO-d
white solid in 70% yield. Mp 214.2–216.5 C. ESI-MS m/z: [M þ H]
1
6
6
) d 11.08 (s, 1H), 9.86 (s, 1H),
mide (35). This compound was obtained as white solid in 79 yield. 9.14 (s, 1H), 8.98 (d, J ¼ 18.5 Hz, 1H), 8.50 (s, 1H), 8.09 (d, J ¼ 7.0 Hz,
ꢁ
þ
1
Mp 210.2–215.3 C. ESI-MS m/z: [M þ H]
593.20.
H
NMR 1H), 7.97 (s, 1H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.67 (d, J ¼ 8.4 Hz, 2H),
(
400 MHz, DMSO-d
6
) d 11.02 (s, 1H), 9.84 (s, 1H), 9.07 (s, 1H), 8.91 7.42 (t, J ¼ 9.2 Hz, 1H), 7.33 (s, 1H), 7.01 (d, J ¼ 8.4 Hz, 2H), 5.42

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
209
(
d, J ¼ 22.1 Hz, 1H), 4.15 (d, J ¼ 5.3 Hz, 2H), 4.04 (s, 2H), 3.97 (d,
J ¼ 8.0 Hz, 1H), 3.88 (d, J ¼ 5.1 Hz, 1H), 3.58 (s, 4H), 2.76–2.67 (m, yl)-2-(4-(3-(4-methylpiperazin-1-yl)propoxy) benzylidene)hydrazine-
H), 2.56–2.51 (m, J ¼ 5.7 Hz, 1H),2.42 (d, J ¼ 13.7 Hz, 4H), 2.26–2.18 1-carboxamide (48). This compound was obtained as white solid
(E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6-
2
ꢁ
þ
1
(m, J ¼ 5.7 Hz, 1H).
in 88% yield. Mp 216.5–218.2 C. ESI-MS m/z: [M þ H] 620.2.
H
(S,E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-((tetrahydrofuran-3-
NMR (400 MHz, DMSO-d ) d 11.02 (s, 1H), 9.85 (s, 1H), 9.06 (s, 1H),
6
yl)oxy)quinazolin-6-yl)-2-(4-(2-(4-methylpi perazin-1-yl)ethoxy)ben- 8.91 (s, 1H), 8.51 (s, 1H), 8.09 (d, J ¼ 6.6 Hz, 1H), 7.96 (s, 1H), 7.77
zylidene)hydrazine-1-carboxamide (42). This compound was (s, 1H), 7.65 (d, J ¼ 8.6 Hz, 2H), 7.42 (t, J ¼ 9.1 Hz, 1H), 7.32 (s, 1H),
ꢁ
obtained as white solid in 78% yield. Mp 206.210.2 C. ESI-MS m/z:
7
2
.04 (d, J ¼ 8.7 Hz, 2H), 4.11 (s, 3H), 4.05 (s, 2H), 2.49–2.49 (m, 8H),
.40 (d, J ¼ 7.2 Hz, 2H), 2.13 (s, 3H), 1.88 (d, J ¼ 7.2 Hz, 2H).
þ
1
[
(
M þ H] 662.2. H NMR (400 MHz, DMSO-d
6
) d 11.08 (s, 1H), 9.86
s, 1H), 9.14 (s, 1H), 8.98 (d, J ¼ 18.5 Hz, 1H), 8.50 (s, 1H), 8.09 (d,
4
.(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-
J ¼ 7.0 Hz, 1H), 7.97 (s, 1H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.67 (d,
J ¼ 8.4 Hz, 2H), 7.42 (t, J ¼ 9.2 Hz, 1H), 7.33 (s, 1H), 7.01 (d,
J ¼ 8.4 Hz, 2H), 5.42 (d, J ¼ 22.1 Hz, 1H), 4.15 (d, J ¼ 5.3 Hz, 2H), 4.04
3
-yl)oxy)quinazolin-6-yl)-2-(4-(3-(diethylamin o)propoxy)benzylide-
ne)hydrazine-1-carboxamide (49)/NMR (400 MHz, DMSO-d6)
1
8
d
1.08 (s, 1H), 9.86 (s, 1H), 9.13 (s, 1H), 9.00 (s, 1H), 8.50 (s, 1H),
.08 (d, J ¼ 6.9 Hz, 1H), 7.98 (s, 1H), 7.78 (d, J ¼ 10.0 Hz, 1H), 7.66
(
2
4
s, 2H), 3.97 (d, J ¼ 8.0 Hz, 1H), 3.88 (d, J ¼ 5.1 Hz, 1H), 3.58 (s, 4H),
.76–2.67 (m, 2H), 2.58–2.56 (m, J ¼ 5.7 Hz, 1H),2.42 (d, J ¼ 13.7 Hz,
H), 2.35–2.08 (m, 2H). 2.21–2.18 (m, J ¼ 5.7 Hz, 1H).
(
d, J ¼ 8.4 Hz, 2H), 7.41 (t, J ¼ 9.1 Hz, 1H), 7.32 (s, 1H), 6.98 (d,
J ¼ 8.5 Hz, 2H), 5.44 (s, 1H), 4.04 (d, J ¼ 4.8 Hz, 4H), 3.96 (t,
(
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6-
J ¼ 7.6 Hz, 1H), 3.89–3.85 (m, 1H), 2.44 (s, 1H), 2.37 (t, J ¼ 7.1 Hz,
yl)-2-(4-(3-(dimethylamino)propoxy)benzyl idene)hydrazine-1-car-
boxamide (43). This compound was obtained as yellow solid in
2
H), 2.27 (s, 1H), 2.15 (s, 6H), 1.85 (d, J ¼ 6.8 Hz, 2H).
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
ꢁ
þ
1
7
6% yield. Mp 209.3–213.5 C. ESI-MS m/z: [M þ H] 565.2. H NMR
yl)oxy)quinazolin-6-yl)-2-(4-(3-(diethylamin o)propoxy)benzylidene)-
hydrazine-1-carboxamide (50) This compound was obtained as
(
(
(
400 MHz, DMSO-d ) d 11.10 (s, 1H), 9.93 (s, 1H), 9.16 (s, 1H), 9.01
6
s, 1H), 8.61 (s, 1H), 8.19 (dd, J ¼ 6.9, 2.7 Hz, 1H), 8.06 (s, 1H), 7.88
ꢁ
þ
yellow solid in 86% yield. Mp 208.2–213.5 C. ESI-MS m/z: [M þ H]
dt, J ¼ 9.1, 3.4 Hz, 1H), 7.74 (d, J ¼ 8.1 Hz, 2H), 7.51 (t, J ¼ 9.2 Hz,
1
6
9
7
49.2. H NMR (400 MHz, DMSO-d
6
) d 11.06 (s, 1H), 9.85 (s, 1H),
1
H), 7.42 (s, 1H), 7.13 (d, J ¼ 8.3 Hz, 2H), 4.21 (s, 3H), 4.14 (t,
.13 (s, 1H), 9.01 (s, 1H), 8.50 (s, 1H), 8.09 (dd, J ¼ 6.9, 2.6 Hz, 1H),
J ¼ 6.4 Hz, 2H), 2.46 (s, 2H), 2.24 (s, 6H), 2.01–1.93 (m, 2H).
E)-N-(4-((4-chloro-3-fluorophenyl)amino)-7-methoxyquinazolin-
-yl)-2-(4-(3-(diethylamino)propoxy)benzylid ene)hydrazine-1-car-
boxamide (44). This compound was obtained as white solid in
.96 (d, J ¼ 6.4 Hz, 1H), 7.78 (dt, J ¼ 8.8, 3.4 Hz, 1H), 7.66
(
(
d, J ¼ 8.4 Hz, 2H), 7.41 (t, J ¼ 9.1 Hz, 1H), 7.33 (s, 1H), 6.98 (d,
6
J ¼ 8.5 Hz, 2H), 5.45 (s, 1H), 4.05 (dd, J ¼ 8.3, 4.5 Hz, 4H), 3.97
q, J ¼ 7.7 Hz, 1H), 3.87 (td, J ¼ 8.4, 4.5 Hz, 1H), 2.54 (s, 2H), 2.46 (d,
J ¼ 7.0 Hz, 4H), 2.43–2.36 (m, 1H), 2.20–2.11 (m, 1H), 1.83
q, J ¼ 6.7 Hz, 2H), 0.94 (t, J ¼ 7.1 Hz, 6H).
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
(
ꢁ
þ
1
7
9% yield. Mp 221.5–229.3 C. ESI-MS m/z: [M þ H] 593.2. H NMR
(
(
(
400 MHz, DMSO-d ) d 11.10 (s, 1H), 9.93 (s, 1H), 9.16 (s, 1H), 9.01
6
(
s, 1H), 8.61 (s, 1H), 8.19 (dd, J ¼ 6.9, 2.7 Hz, 1H), 8.06 (s, 1H), 7.88
dt, J ¼ 9.1, 3.4 Hz, 1H), 7.74 (d, J ¼ 8.1 Hz, 2H), 7.51 (t, J ¼ 9.2 Hz,
(
yl)oxy)quinazolin-6-yl)-2-(4-(3-(pyrrolidin-1-yl)propoxy)benzylidene)-
hydrazine-1-carboxamide (51). This compound was obtained as
1
H), 7.42 (s, 1H), 7.13 (d, J ¼ 8.3 Hz, 2H), 4.21 (s, 3H), 4.14 (t,
J ¼ 6.4 Hz, 2H), 2.46 (s, 2H), 2.24 (s, 6H), 2.01–1.93 (m, 2H).
ꢁ
þ
white solid in 83% yield. Mp 223.1–230.5 C. ESI-MS m/z: [M þ H]
(
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6-
1
6
9
47.2. H NMR (400 MHz, DMSO-d ) d 11.08 (s, 1H), 9.87 (s, 1H),
6
yl)-2-(4-(3-(pyrrolidin-1-yl)propoxy)benzyli dene)hydrazine-1-carbox-
amide (45). This compound was obtained as yellow solid in 77%
yield. Mp 216.7–219.6 C. ESI-MS m/z: [M þ H] 591.2. H NMR
400 MHz, DMSO-d ) d 10.91–10.70 (m, 1H), 9.87 (s, 1H), 8.96 (s,
H), 8.77 (s, 1H), 8.49 (s, 1H), 8.07 (s, 1H), 7.94 (s, 1H), 7.80–7.69
.14 (s, 1H), 9.00 (s, 1H), 8.50 (s, 1H), 8.08 (d, J ¼ 4.8 Hz, 1H), 7.96
ꢁ
þ
1
(s, 1H), 7.77 (s, 1H), 7.66 (d, J ¼ 8.5 Hz, 2H), 7.41 (t, J ¼ 9.0 Hz, 1H),
7
(
2
.32 (s, 1H), 6.98 (d, J ¼ 8.3 Hz, 2H), 5.45 (s, 1H), 4.04 (s, 4H), 3.96
d, J ¼ 7.7 Hz, 1H), 3.87 (d, J ¼ 4.3 Hz, 1H), 2.53 (s, 2H), 2.43 (s, 4H),
.14 (s, 2H), 1.93–1.85 (m, 2H), 1.68 (s, 4H).
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
(
6
1
(
1
4
m, 1H), 7.34 (d, J ¼ 42.0 Hz, 2H), 7.51 (t, J ¼ 9.2 Hz, 1H), 7.42 (s,
(
H), 7.13 (d, J ¼ 32.2 Hz, 2H),4.35 (s, 2H), 4.06 (s, 3H), 2.38–2.32 (m,
H), 1.83 (s, 2H), 1.05 (t, J ¼ 7.0 Hz, 2H), 0.83 (t, J ¼ 6.9 Hz, 6H).
yl)oxy)quinazolin-6-yl)-2-(4-(3-(piperidin-1-yl)propoxy)benzylidene)-
hydrazine-1-carboxamide (52). This compound was obtained as
(
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6-
ꢁ
þ
white solid in 81% yield. Mp 222.5–226.5 C. ESI-MS m/z: [M þ H]
yl)-2-(4-(3-(piperidin-1-yl)propoxy)benzylid ene)hydrazine-1-carbox-
amide (46). This compound was obtained as white solid in 84%
1
6
61.2. H NMR (400 MHz, DMSO-d
6
) d 11.07 (s, 1H), 9.86 (s, 1H),
ꢁ
þ
1
9.13 (s, 1H), 8.99 (s, 1H), 8.49 (s, 1H), 8.07 (dd, J ¼ 6.9, 2.6 Hz, 1H),
yield. Mp 218.5–221.5 C. ESI-MS m/z: [M þ H] 605.2. H NMR
7
1
.95 (s, 1H), 7.76 (s, 1H), 7.65 (d, J ¼ 8.7 Hz, 2H), 7.41 (t, J ¼ 9.1 Hz,
H), 7.31 (s, 1H), 6.97 (d, J ¼ 8.8 Hz, 2H), 5.45 (s, 1H), 4.04 (d,
(
(
400 MHz, DMSO-d ) d 11.02 (s, 1H), 9.85 (s, 1H), 9.06 (s, 1H), 8.92
6
s, 1H), 8.52 (s, 1H), 8.10 (d, J ¼ 6.9 Hz, 1H), 7.96 (s, 1H), 7.77 (s,
J ¼ 6.6 Hz, 4H), 3.94 (s, 1H), 3.86 (d, J ¼ 4.5 Hz, 1H), 2.41–2.33 (m,
1
H), 7.65 (d, J ¼ 8.7 Hz, 2H), 7.42 (s, 1H), 7.33 (s, 1H), 7.04 (d,
4
1
H), 2.31 (s, 2H), 2.14 (s, 2H), 1.87 (d, J ¼ 7.9 Hz, 2H), 1.48 (s, 4H),
J ¼ 8.7 Hz, 2H), 4.12 (s, 3H), 4.05 (t, J ¼ 6.2 Hz, 2H), 2.38 (t,
.37 (s, 2H).
J ¼ 6.7 Hz, 2H), 2.33 (s, 4H), 1.91–1.87 (m, 2H), 1.48 (d, J ¼ 4.9 Hz,
(S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3-
4
H), 1.37 (s, 2H).
yl)oxy)quinazolin-6-yl)-2-(4-(3-morpholino propoxy)benzylidene)hy-
drazine-1-carboxamide (53). This compound was obtained as
(
E)-N-(4-((3-chloro-4-fluoropheny)amino)-7-methoxyquinazolin-6-
yl)-2-(4-(3-morpholinopropoxy)benzylidene)hydrazine-1-carboxa-
ꢁ
þ
mide (47). This compound was obtained as white solid in 79% white solid in 82% yield. Mp 226.5–229.5 C. ESI-MS m/z: [M þ H]
þ
1
1
ꢁ
yield. Mp 226.5–230.2 C. ESI-MS m/z: [M þ H] 607.2. H NMR 663.2. H NMR (400 MHz, DMSO-d
) d 10.97 (s, 1H), 9.84 (s, 1H),
6
(
(
7
7
2
400 MHz, DMSO-d
6
) d 11.02 (s, 1H), 9.84 (s, 1H), 9.06 (s, 1H), 8.89 9.10 (s, 1H), 8.98 (s, 1H), 8.43 (s, 1H), 8.03 (s, 1H), 7.95 (s, 1H), 7.70
d, J ¼ 26.4 Hz, 1H), 8.52 (s, 1H), 8.10 (d, J ¼ 6.7 Hz, 1H), 7.96 (s, 1H), (s, 1H), 7.65 (d, J ¼ 8.4 Hz, 2H), 7.36 (t, J ¼ 9.1 Hz, 1H), 7.26 (s, 1H),
.78 (s, 1H), 7.66 (d, J ¼ 7.4 Hz, 2H), 7.41 (dd, J ¼ 18.2, 9.1 Hz, 1H), 6.97 (d, J ¼ 8.4 Hz, 2H), 5.41 (d, J ¼ 8.2 Hz, 1H), 4.06 (d, J ¼ 6.2 Hz,
.33 (s, 1H), 7.04 (d, J ¼ 7.4 Hz, 2H), 4.12 (s, 3H), 4.07 (d, J ¼ 5.4 Hz, 2H), 4.02 (d, J ¼ 3.4 Hz, 2H), 3.94 (t, J ¼ 7.7 Hz, 1H), 3.87 (dd, J ¼ 8.4,
H), 3.58 (s, 4H), 2.43 (t, J ¼ 6.5 Hz, 2H), 2.37 (s, 4H), 1.94–1.85 4.7 Hz, 1H), 3.56 (t, J ¼ 4.6 Hz, 4H), 2.43 (s, 1H), 2.41 (d, J ¼ 7.1 Hz,
(m, 2H).
2H), 2.35 (s, 4H), 2.14 (d, J ¼ 8.8 Hz, 1H), 1.90–1.84 (m, 2H).

2
10
C. WANG ET AL.
(
S,E)-N-(4-((3-chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-3- Cell apoptosis assay by flow cytometry
6
yl)oxy)quinazolin-6-yl)-2-(4-(3-(4-methylpi perazin-1-yl)propoxy)ben- A549 cells were seeded in 16-well plates at a density of 1 ꢂ 10
zylidene)hydrazine-1-carboxamide (54). This compound was cells/well in RPMI 1640 medium and treated with 0.5, 1 mM 10 d
ꢁ
obtained as yellow solid in 81% yield. Mp 216.5–219.5 C. ESI-MS for 48 h. Cultured cells were stained with Annexin V-FITC and pro-
þ
1
ꢁ
m/z: [M þ H] 676.2. H NMR (400 MHz, DMSO-d ) d 11.07 (s, 1H), pidium iodide (PI) in the dark at 4 C for 30 min and analysed by
6
FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA)
using Cell Quest software.
9
.85 (s, 1H), 9.12 (s, 1H), 8.99 (s, 1H), 8.48 (s, 1H), 8.06 (d,
J ¼ 7.1 Hz, 1H), 7.95 (s, 1H), 7.75 (s, 1H), 7.64 (d, J ¼ 8.2 Hz, 2H),
7
1
6
.40 (t, J ¼ 9.0 Hz, 1H), 7.30 (s, 1H), 6.96 (d, J ¼ 8.3 Hz, 2H), 5.43 (s,
H), 4.13–4.00 (m, 4H), 3.95 (q, J ¼ 7.5 Hz, 1H), 3.86 (dt, J ¼ 12.2,
.1 Hz, 1H), 2.60 (s, 2H), 2.45 (s, 1H), 2.43–2.35 (m, 4H), 2.35–2.22
Flow cytometric analysis of cell cycle distribution assay
5
For flow cytometric analysis of DNA content, 5.0 ꢂ 10 cell/well
(
m, 4H), 2.15 (s, 1H), 2.12 (s, 3H), 1.96–1.77 (m, 2H).
A549 cells were grown in a costar 6-well cell culture cluster and
ꢁ
grown for 24 h at 37 C in 5% CO
2
, after the medium was
removed and the cells were treated with specific concentrations
of the test compounds 14 and 44 for 48 h. Blank wells treated
with medium only were also included. After the incubation period,
Biological evaluation
Cytotoxicity assay in vitro
The cytotoxic activities of target compounds 9–54 were evaluated the A549 cells were collected, washed twice with ice-cold PBS,
with HepG2, A549, MCF-7 and H1975 cell lines by the standard centrifuged and then fixed with ice-cold ethanol (70%) for at least
MTT assay in vitro, with compounds EGFR inhibitors afatinib as a 24 h. Cells were then collected, washed twice with ice-cold PBS,
ꢁ
positive control. The cancer cell lines were cultured in minimum and treated with 30 ml RNase A (1 mg/mL) in PBS at 37 C for
about 30 min, and then stained with 50 ml propidium iodide
essential medium (MEM) supplement with 10% fetal bovine serum
ꢁ
(50 mg/mL) in PBS, the staining process lasted 30 min at 4 C in
(
FBS). Approximately 4 ꢂ 103 cells, suspended in MEM medium,
darkness. The cellular DNA content of the stained cells was then
analysed on BD-FACS Aria III flow cytometer and the cell cycle dis-
tribution was quantified.
were plated onto each well of a 96-well plate and incubated in
5
ꢁ
% CO2 at 37 C for 24 h. The test compounds at indicated final
concentrations were added to the culture medium and the cell
cultures were continued for 72 h. Fresh MTT was added to each
well at a terminal concentration of 5 lg/mL and incubated with
Docking studies
ꢁ
cells at 37 C for 4 h. The formazan crystals were dissolved in
For docking purposes, the three-dimensional structure of the
EGFR PDB code: 4G5P was obtained from RCSB Protein Data Bank.
Autodock 4.2 (The Scripps Research Institute, La Jolla, CA, USA),
Discover Studio 4.5 visualiser and Open Babel (Biovia Dassault
System, Accelrys, San Diego, CA, USA) were used for the docking
study. Firstly, using the Discover Studio 4.5 visualiser to prepare
ligands (14 and 44) and acceptor protein (PDB code: 4G5P), then
saved as pdb format after energy minimisation for ligand and
1
00 lL DMSO each well, and the absorbency at 492 nm (for
absorbance of MTT formazan) and 630 nm (for the reference wave-
length) was measured with the ELISA reader. All of the com-
pounds were tested three times in each of the cell lines. The
results expressed as inhibition rates or IC50 (half-maximal inhibi-
tory concentration) were the averages of two determinations and
calculated by using the Bacus Laboratories Incorporated Slide
Scanner (Bliss) software (Bacus Laboratories Inc, Lombard, IL, USA). clean protein, defined the activity site for acceptor protein.
Secondly, the pdb file of ligand and the acceptor protein were
converted to the pdbqt format by the Open Babel tool. Thirdly,
docking was carried out by uploading the pdbqt file to the
Autodock 4.5, and the result was named out.pdbq. Moreover,
EGFR kinases assay in vitro
The selected compounds with excellent anti-proliferative activities
were tested for their activity against EGFR kinases through the out.pdbpt file was uploaded to the Discover Studio 4.5 visualiser
mobility shift assay. All kinases assays were performed in 96-well for analysing the result. All calculations were performed on Silicon
plates in a 50 lL reaction volume. The kinase buffer contains Graphics workstation. Detailed user guidance and basic docking
5
0 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM operations can be found on AutoDock official website http://auto-
DTT. The stop buffer contains 100 mM HEPES, pH 7.5, 0.015% Brij- dock.scripps.edu/faqs-help/tutorial.
5, 0.2% Coating Reagent #3 and 50 mM EDTA. The compounds
3
were diluted to 500 lM by 100% DMSO, then 10 lL of compound
was transferred to a new 96-well plate as the intermediate plate,
Results and discussion
and 90 lL kinase buffer was added to each well. Then 5 lL of Chemistry
each well of the intermediate plate was transferred to 384-well
The structure and synthetic route of 9–54 are shown by Table 1
plates. The following amounts of enzyme and substrate were used
per well: kinase base buffer, FAM-labeled peptide, ATP and
enzyme solution. Wells containing the substrate, enzyme, DMSO
without compound were used as DMSO control. Wells containing
just the substrate without enzyme were used as a low control.
Incubate at room temperature for 10 min. Add 10 lL peptide solu-
and Scheme 1, respectively. The key intermediates 8a–b was syn-
thesised from commercially available 2-amino-4-fluorobenzoic acid
through eight steps. Finally, condensation of 8a–b with aromatic
aldehydes in ethanol in the presence of a catalytic amount of gla-
cial acetic acid yielded target compounds 9–54 respectively.
Commercially available heterocyclic aldehydes reacted with 8a–b
produced compounds 9–18, and side chains 57a-1 and 60a–f
reacted with 8a–b produced compounds 18–54.
ꢁ
tion to each well. Incubate at 28 C for specified period of time
and stop reaction by 25 lL stop buffer. At last data was collected
on Caliper program and conversion values were converted to
The general strategy for the synthesis of compounds 57a-1
inhibition
max ꢃ min) ꢂ 100. “max” stands for DMSO control; “min” stands cially available 4-hydroxybenzaldehyde (55) with 1-bromo-2-
for low control. chloroethane or 1-bromo-2-chloropropane produced intermediates
values.
Percent
inhibition ¼ (max ꢃ conversion)/ and 60a–f is shown in Scheme 2. The reaction of the commer-
(

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
211
Table 1. Chemical structures of the target compounds 9–54.
Cpd. No
R
1
R
2
Yield (%)
88
Cpd. No
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
R
1
R
2
Yield (%)
79
9
Me
Me
Me
Me
Me
Me
Me
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
76
84
86
75
84
79
86
91
85
90
78
89
89
88
Me
77
86
79
70
Me
Me
Me
Me
Me
Me
84
78
86
Me
Me
Me
Me
Me
Me
76
89
79
92
77
87
84
81
79
86
88
81
81
83
86
87
83
86
81
85
82
Me
76
81

2
12
C. WANG ET AL.
Scheme 1. Synthetic route of target compounds 9–54.
Scheme 2. Synthetic route of side chains 57a-l and 60a–f.
56a–56b. Nucleophilic aromatic substitution of the position of panel of four human cancer cell lines, belonging to different
chlorine in 56a–58b with small molecule amines in DMF at micro- tumour types, namely human liver cancer cell lines (HepG2),
ꢁ
wave 125 C yielded compounds 57a-l. Commercially available ter- human breast carcinoma cell lines (MCF-7), human lung carcinoma
ephthalaldehyde (58) reacted with triethyl orthoformate to obtain cell lines (A549), Human lung adenocarcinoma cell lines (H1975)
the crude product and purified by column chromatography (PE/ at a concentration of 0.1–100 mM. HepG2, A549, and MCF-7 were
EA, 5.95v/v) to afford the intermediate 59. Compound 59 was
reductively aminated in the presence of sodium borohydride to
give the crude product. Finally, a solution of the crude product in
methanol reacted with a hydrochloric acid-methanol solution to
afford the intermediate 60a–f.
selected to test the broad-spectrum anti-cell proliferative activity
of compounds on tumour cells. H1975 was used to verify whether
anti-cell proliferative activity was also observed for gefitinib-resist-
ant cell lines. The results expressed as IC50 values were summar-
ised in Table 2 and the values were the average of at least two
independent experiments. The IC50 values that were higher than
Reagents and conditions: (a) EtOH, Formamidine acetate, 24h;
(
b) Con.H SO , fuming HNO , 2h; (c) SOCl , DMF(cat.), 4h; (d) 3-
2 4 3 2
1
00 lM in all cell lines were not included.
chloro-4-fluoroaniline, Isopropanol, Triethylamine, 1.5h; (e) (S)-
As shown in Table 2, the results of the cells showed that
tetrahydrofuran-3-ol, 60% NaH, THF, 3h; CH OH, NaOH; (f) 80%
3
after the C-7 position of the quinazoline nucleus was modified,
the anti-proliferative activity of H1975 against most of the target
compounds was significantly increased. However, compounds 23,
hydrazine monohydrate, FeCl
phenyl chloroformate, DIPEA, 1,4-dioxane, 10 C to rt, 1.5h; (h)
3
, activated carbon, EtOH, 1h; (g)
ꢁ
8
0% hydrazine monohydrate, 1,4-dioxane, reflux; (i) commercially
24, 29, 38, 39, 43, 49 were showed no activity on some cell
available Heterocyclic aldehydes, acetic acid(Cat.), EtOH, reflux.
Reagents and conditions: (a) acetonitriles, anhydrous, K CO , 1-
lines. The potential compounds well inhibited the growth of
H1975 cells with IC50 values ranging from 0.83 ± 0.17 to
2
3
bromo-2-chloroethane or 1-bromo-2-chloropropane, 1 h, rt; (b)
DMF, microwave, Small molecule amines, 1 h, rt. (c) triethyl ortho-
formate, ammonium chloride, r.t.; (d) 1, methanol water, sodium
borohydride; 2, diluted hydrochloric acid, heated. 3, hydrogen
1
6.58 ± 1.39 lM. Notably, the anti-proliferative activity of com-
pounds 14, 28, 44, 46, 50, 52 and 54 against H1975 cells was a
single-digit level with IC50 values of 1.72 ± 0.85, 3.97 ± 0.64,
1.03 ± 0.17, 3.10 ± 0.49, 1.53 ± 0.52, 2.20 ± 0.37 and 6.23 ± 0.99 lM,
respectively. Likewise, compounds 10, 12, 13, 19, 21, 24, 37, 42
and 48 showed moderate inhibitory activity. Unfortunately, com-
pounds 9, 15, 16, 20, 25, 32, 34, 36, 38 and 49 had poorer cel-
ꢁ
chloride in methanol, T ¼ 0–20 C.
In vitro anti-proliferative activity screening of compounds 9–54
Taking afatinib as reference compound, the target compounds lular activity of H1975 despite their very good broad
9–54) were evaluated for the anti-proliferative activity against a antitumour activity.
(

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
213
Table 2. In vitro anti-proliferative activities against different cancer cell lines for 72 h of the target compounds.
a
Anti-proliferative activities in different cancer cell lines (IC50 , lM)
Cpd. No
HepG
2
A549
MCF-7
H1975
9
0.72 ± 0.19
0.14 ± 0.08
11.45 ± 1.16
0.31 ± 0.13
1.35 ± 0.31
4.63 ± 0.36
8.17 ± 0.98
2.43 ± 0.45
13.22 ± 1.31
5.81 ± 0.61
9.89 ± 0.96
9.49 ± 0.47
21.34 ± 1.47
59.48 ± 4.75
>100
2.86 ± 0.13
3.76 ± 0.21
53.61 ± 4.75
>100
3.13 ± 0.45
>100
17.49 ± 1.62
36.33 ± 2.57
13.24 ± 1.03
46.72 ± 4.46
12.72 ± 1.25
16.51 ± 1.42
10.18 ± 1.09
8.20 ± 0.81
1.91 ± 0.34
67.27 ± 6.24
17.39 ± 1.24
40.25 ± 3.04
13.40 ± 0.54
>100
0.02 ± 0.01
24.47 ± 1.27
9.58 ± 0.94
28.4 ± 1.78
14.50 ± 1.27
5.04 ± 0.64
1.08 ± 0.17
16.99 ± 1.04
2.28 ± 0.26
>100
2.84 ± 0.22
1.51 ± 0.13
31.4 ± 2.12
0.28 ± 0.12
1.48 ± 0.19
5.9 ± 0.25
2.63 ± 0.16
0.61 ± 0.12
15.4 ± 1.14
0.35 ± 0.09
1.35 ± 0.14
2.37 ± 0.34
14.01 ± 0.78
3.68 ± 0.49
12.89 ± 1.12
12.43 ± 1.25
3.80 ± 0.22
4.14 ± 0.57
8.90 ± 0.48
31.13 ± 2.55
>100
3.12 ± 0.38
4.41 ± 0.28
36.91 ± 3.54
>100
4.30 ± 0.63
>100
21.01 ± 1.68
37.25 ± 2.89
12.51 ± 1.24
44.23 ± 3.75
9.21 ± 1.16
21.8 ± 1.34
6.2 ± 0.81
11.72 ± 1.34
1.99 ± 0.68
64.82 ± 5.35
24.15 ± 1.23
28.82 ± 1.29
10.82 ± 0.68
>100
0.32 ± 0.02
16.88 ± 1.34
6.85 ± 0.65
27.52 ± 1.29
12.23 ± 0.89
6.21 ± 0.68
1.23 ± 0.37
28.54 ± 1.27
2.56 ± 0.46
>100
38.42 ± 1.26
0.91 ± 0.29
2.63 ± 0.16
/
40.72 ± 2.24
12.32 ± 1.65
40.27 ± 3.24
10.59 ± 0.65
16.32 ± 1.34
1.72 ± 0.85
32.27 ± 2.64
60.27 ± 5.24
35.26 ± 2.35
51.59 ± 4.35
15.65 ± 1.49
76.27 ± 6.27
16.58 ± 1.39
63.35 ± 6.13
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
10.51 ± 0.77
2.5 ± 0.36
14.56 ± 1.21
19.2 ± 1.46
7.99 ± 0.82
8.71 ± 0.54
17.82 ± 1.22
24.22 ± 2.27
>100
4.32 ± 0.61
4.13 ± 0.54
23.14 ± 1.54
>100
15.26 ± 1.65
40.27 ± 3.27
62.57 ± 5.48
>100
2.25 ± 0.32
>100
3.97 ± 0.64
65.24 ± 5.54
36.27 ± 2.54
45.27 ± 3.34
32.56 ± 3.45
49.27 ± 4.68
61.57 ± 5.67
25.17 ± 1.35
24.65 ± 1.86
10.16 ± 1.12
>100
24.95 ± 1.66
40.12 ± 4.03
8.62 ± 1.13
36.42 ± 2.78
10.85 ± 1.43
19.54 ± 1.35
11.38 ± 1.07
14.66 ± 1.35
3.01 ± 0.54
63.86 ± 5.35
24.22 ± 1.24
37.76 ± 2.54
2.52 ± 0.54
43.07 ± 3.34
0.41 ± 0.01
23.19 ± 1.47
4.01 ± 0.56
34.82 ± 1.26
2.96 ± 0.36
9.48 ± 0.86
1.11 ± 0.14
33.84 ± 1.14
4.65 ± 0.39
>100
>100
35.28 ± 2.35
25.47 ± 1.44
11.17 ± 1.34
>100
1.03 ± 0.17
38.24 ± 1.23
3.10 ± 0.49
41.23 ± 2.07
10.17 ± 1.26
32.83 ± 1.27
1.53 ± 0.52
18.56 ± 1.35
2.20 ± 0.37
24.83 ± 1.08
6.23 ± 0.99
>100
41.19 ± 2.04
0.07 ± 0.61
1.40 ± 0.08
/
39.99 ± 2.14
1.32 ± 0.38
1.33 ± 0.09
2.17 ± 0.14
b
BMC201725-9o
Afatinib
Gefitinib
b
0.49 ± 0.08
12.70 ± 2.98
b
The value “>100” indicates that no inhibitory effect at 100 lM compound concentration.
a
The values are an average of two separate determinations.
Used as a positive control.
b
Interestingly, compound 14 showed a slight selectivity for 44 and 50 show the most prominent activity, they all have the
H1975 cells. It is noteworthy that compound 44 have inhibitory characteristics of a tricarbon chain linker and diethylamine. In the
activity against H1975 cells with an IC50 value of 1.03 ± 0.17 lM, c strategy, the activity on H1975 is significantly increased and the
which was lower than that of afatinib (0.49 ± 0.08 lM). However, activity on other cells remained since the hydroxyl group is
the IC50 values of other cells were 0.02 ± 0.01, 0.41 ± 0.01 and replaced by an amine, such as compounds 9–12. When the aro-
0
.32 ± 0.02 lM, respectively, which were slightly higher than that matic ring is replaced with an aromatic heterocycle, the activity
of afatinib (1.40 ± 0.83, 1.33 ± 1.28, 2.63 ± 1.06 lM).
against all cells slightly decreases. Same as we expected, com-
pounds 13 and 14 in which the aromatic rings contain two heter-
oatoms, exhibit very prominent inhibitory activity against H1975.
Structure-activity relationship analysis
In combination with the activity of all compounds on the four
cells, we found that most of the compounds’ activity is reduced in
Kinase inhibitory activity
the b strategy. Affected by the 4 position of the aromatic ring, the In order to find inhibitors targeting L858R/T790M, some com-
density of the electron cloud of the aromatic ring is reduced. In pounds with significant inhibitory activity against H1975 cells
strategy a, the cell activity of the three carbon chains is apparently were selected. The in vitro enzymatic inhibitory activities against
L858R/T790M
WT
stronger than that of the two carbons. Among them, compounds EGFR
and EGFR
were evaluated by using the

2
14
C. WANG ET AL.
Table 3. Inhibitory activity of selected compounds against different types of EGFRs in vitro.
EGFR IC50 (nM)
Cpd. No
R
1
R
2
WT
16.0
2.5
10.8
6.3
2.1
3.8
4.0
4.0
1.2
2.3
0.4
1.9
1.8
1.3
1.7
2.6
56
L858R/T790M
>1000
>1000
>1000
8.4
LogD7.4a
5.9
5.8
4.3
4.2
3.7
3.6
4.2
4.1
3.4
4.1
3.3
4.0
4.0
3.2
3.9
3.9
4.7
2.3
2.5
1
1
1
1
1
2
2
2
3
4
4
4
4
5
5
5
0
2
3
4
9
1
4
8
7
2
4
6
8
0
2
4
Me
Me
Me
Me
508.4
482.4
>1000
>1000
248.1
213.5
107.4
320.5
596.8
151.9
561.6
187.9
>1000
3.8
Me
Me
Me
BMC201725-9ob
Afatinibb
/
/
/
/
/
/
4.0
3.25
Gefitinibb
823.7
The value “>1000” indicates that no inhibitory effect at 100 lM compound concentration.
a
The result is predicted by https://chemicalize.com/#/calculation.
Used as a positive control.
b
well-established ELISA-based assay, and afatinib was employed as afatinib (IC 50 ¼ 3.8 nM). Among the compounds tested, most of
positive controls (Table 3). Notably, compound 14 (IC50 ¼ 8.4 nM) the compounds showed single-digit nanomolar levels of wild-type
with a 2- (pyrrolidin-1-yl) pyrimidine side chain at the 7-position kinase activity. However, most compounds had low inhibitory
of the quinazoline ring had almost the same inhibitory activity as activity against the L858R/T790M mutant. It is noteworthy that

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
215
when the C-7 side chain introduced a flexible hydrophobic chain, acridine orange (AO) and the shape of the cell was full and the
the inhibitory activity of these compounds on L858R/T709M kinase edge was clear. But in the other pictures (Figure 3(b,c,d)), the cell
was significantly enhanced. Among them, compound 44 was the showed an identical phenomenon, the shape of which was abnor-
best representative. And the most potent compound 14 exhibited mal with cell shrinkage, chromatin condensation or decomposition
WT
L858R/T790M
excellent inhibition against EGFR
and EGFR
with an into fragments of varying sizes. This phenomenon indicated that
IC50 at 6.3 nM and 8.4 nM, respectively.
compounds 14 and 44 can induce A549 cell apoptosis.
Morphologic changes of A549 cells under inverted microscopy
and fluorescence microscopy
Induction of apoptosis assay on A549 cells
Apoptosis refers to the autonomous and orderly death of cells
To explain the inhibition of cell growth, the apoptotic experiment controlled by genes. Furthermore, its startup is affected by many
by acridine orange (AO) single staining would be carried out to external factors. To further elucidate the relationship between
exam the effect of compounds 14 and 44 on A549 cell. As shown apoptosis and compounds 14 and 44, Annexin V-FITC and propi-
in Figure 3, the control group cell (Figure 3(a)) was stained with dium iodide (PI) double staining flow cytometry was used to
Figure 3. Morphologic changes of A549 cells under inverted microscopy and fluorescence microscopy. 3(a) the control group cell treated with nothing; 3(b)
Experimental group treated with 1.33lM concentration of afatinib. 3(c) Experimental group treated with 1.59 lM concentration of 14. 3(d) Experimental group treated
with 0.41 lM concentration of 44.
Figure 4. Cell apoptosis analysis on A549 cells treated with compounds 14 and 44 at 2.5lM detected by FCM.

2
16
C. WANG ET AL.
evaluate the effect of compounds 14 and 44 on apoptosis of Molecular docking study
A549 cells at specific concentrations influences. The results are
shown in Figure 4.
To better understand how the 14 and 44 contributed to the EGFR
kinase inhibitory activities, molecular docking simulation studies
were carried out by using AutoDock 4.2, Open Babel and Discover
Studio 4.5 visualizer. And based on the in vitro inhibition results,
we selected compounds 14 and 44, our best EGFR inhibitors in
According to the results of Figure 4, compounds 14 and
44 effectively induced cell apoptosis at a concentration of
2
.5 lM. Treatment of A549 cells with compounds 14 and 44
for 48 h resulted in 34.9% and 72.76% of apoptotic cells (ear-
ly þ late), respectively, compared to 12.18% of apoptotic cells
in the untreated control. These results revealed that com-
pounds 14 and 44 inhibited cell growth through cell apoptosis
induction.
this study, as the ligand examples. And the co-crystal structure of
T790M
EGFR
in complex with afatinib was obtained from the RSC
Protein Data Bank (PDB code: 4G5P). The four selected com-
pounds (afatinib, BMC201725-9o, 14 and 44) occupied the ATP
site of EGFR kinase (Figure 6(a)). The binding patterns of com-
pounds 14 and 44 to EGFR kinase are shown in detail in
Figure 6(b,c,d).
Cell cycle analysis on A549 cells by flow cytometry
As clearly shown in Figure 6(b), the two compounds 14 and 44
Aiming to better elucidate the relationship between the mechan- all formed a hydrogen bond from the N1 of quinazoline core to
ism of inhibition of proliferation and cell cycle arrest, cells cycle the main chain NH of Met793, and the length of hydrogen bond
distribution on A549 cells by treating with 2.5 lM concentrations were 2.0 and 2.3 Å, respectively. Compared to afatinib and 44,
compounds 14 and 44 were performed. The results are shown another hydrogen bond existed between 44 and EGFR from NH in
in Table 4 and Figure 5. The region marked with different col- the Hydrazine carboxamide of 44 to the residue Asp800 of EGFR
ours represents % population at different phases of the cell with the length of 2.1 Å (Figure 6(b)). Furthermore, several interac-
cycle. As can be seen, compound 14 caused an increase in the tions existed between 14 and the residues of protein, such as pi-
Alkyl interactions of the 3-Chloro-4-fluoroaniline with Met790 and
Lys745. Alkyl interactions of the quinazoline ring with Leu718 and
Ala743. There was a hydrogen-carbon interaction between the
pyrimidine ring and Asp800 (Figure 6(c)). Interestingly, compound
proportion of cells in S phase (from 40.03% in the control to
0.68%) with a concomitant decrease of cells in G0/G1 phase of
the cell cycle (from 26.57% in the control to 23.62%) and cells in
G2/M phase of the cell cycle (from 29.4% in the control to
5
4
4 had pi-sigma interactions between its hydrophobic side-chain
ends and Phe723 in addition to the above several interactions
Figure 6(d)). These all indicated that the introduction of
Heterocyclic aldehydes and flexible hydrophobic side chains to C-
of TYB-65 led to a new binding interaction with the EGFR-TK
2
1
5.7%). Interestingly, the effects of compound 44 and compound
4 on cell cycle were quite different. As shown in Figure 5, com-
(
pound 44 caused an increase in the proportion of cells that
cause G0/G1 phase cells (from 26.57% to 50.01% of controls),
accompanied the decrease of S phase cell ratio (23.62% from
7
domain, which contributed to its inhibitory activity against EGFR.
2
6.57% of controls) and G2/M phase cell ratio (from 29.4% in
the control group to 25.7%). These results indicated that com-
pounds 14 and 44 can inhibit tumour cell proliferation and lead
to apoptosis by blocking the cell cycle.
Conclusion
Herein, we used three different modification methods to structur-
ally modify our previously studied compound BMC201725-9o,
Table 4. Analysis of cell cycle on A549 cells treated at 25 lM compound 14 and
which led to the design and synthesis of a series of new deriva-
4
4 for 48 h by flow cytometry.
L858R/T790M
tives and increased activity on EGFR.
. These compounds
Cpd. No
DMSO
G0/G1 (%)
S (%)
G2/M (%)
CV G1 (%)
CV G2 (%)
were subjected to biological activities evaluation including anti-
proliferative effects against four tumour cell lines (HepG2, A549,
MCF-7 and H1975). Most of the synthesised compounds exhibited
moderate to excellent anti-proliferative activities against the
26.57
23.62
50.10
44.03
50.68
31.91
29.40
25.70
17.99
3.71
3.34
3.94
3.83
5.15
3.48
1
4
4
4
Figure 5. Cell cycle distribution of A549 cells treated at 2.5 lM compound 14 and 44 for 48 h detected by FCM.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
217
Figure 6. The protonated state of several important residue were adjusted by using AutoDock 4.5 in favour of forming reasonable hydrogen bond with the ligand
and Molecular docking analysis was carried out by the Discover Studio 4.5 visualizer to explore the binding model for the active site of EGFR with its ligand. Docking
simulations showed that the four selected compounds (afatinib, BMC201725-9o, 14 and 44) occupied the ATP site of EGFR kinase 6(a). Then we employed 3 D inter-
action map 6(b), 2 D diagram of the compound 14 6(c) and 2 D diagram of the compound 44 6(d) to display the interaction with the targeted protein (4G5P).
tested tumour cell lines. The hit 44 exhibited excellent inhibition Disclosure statement
of the tested tumour cell lines with the IC50 values of
No potential conflict of interest was reported by the authors.
0
.02 ± 0.01 lM, 0.41 ± 0.013 lM, 0.32 ± 0.02 lM and 0.83 ± 0.17 lM,
respectively. An EGFR kinase inhibition assay indicated that some
WT
of the tested derivatives displayed good inhibition against EGFR
and EGFR
Funding
L858R/T790M
. Notably, compound 14 not only exhibited
This work was supported by The National Natural Science Funds
of China [No. 21662014, 81460527], Outstanding Youth
Foundation of Jiangxi, Natural Science Foundation of Jiangxi,
China [20171BCB23078], Natural Science Foundation of Jiangxi,
China [20171ACB21052 & 20171BAB215073].
excellent anti-proliferative activity against the tumour cells, but
WT
also showed potent inhibitory activity toward EGFR (IC50 6.3 nM)
L858R/T790M
and EGFR
(IC50 8.4 nM), similar to that of afatinib (IC50
4
.0 and 3.8 nM). According to the result of AO single staining and
Annexin V/PI staining, the 14 and 44 could induce remarkable
apoptosis of A549 cells. Compound 14 caused S phase arrest and
induced apoptosis to inhibit cell proliferation. Compound 44, on
the other hand, caused arrest in G0/G1 phase to induce apoptosis.
In general, compounds 14 and 44 exerted potential antitumour
activity through several mechanisms, including anti-proliferation
and cell cycle arrest, and may serve as model molecule to help us
to further design and develop more potent anticancer agents.
These findings presented herein show the noncovalent inhibitor
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