Synthesis and Anti-Proliferation Activity Evaluation of Novel 2-Chloroquinazoline as Potential EGFR-TK Inhibitors

Article abstract of DOI:10.1002/cbdv.202100478

A novel series of 2-chloroquinazoline derivatives had been synthesized and their anti-proliferation activities against the four EGFR high-expressing cells A549, NCI-H1975, AGS and HepG2 cell lines were evaluated. The preliminary SAR study of the scaffold of new compounds showed that the compounds with a chlorine substituent on R³ had a better anti-proliferation activity than those substituted by hydrogen atom or vinyl group. Among them, 2-chloro-N-[2-chloro-4-(3-chloro-4-fluoroanilino)quinazolin-6-yl]acetamide (10b) had the best activity, and the corresponding IC₅₀ were 3.68, 10.06, 1.73 and 2.04 μM, respectively. And compound 10b had better or equivalent activity against four cell lines than Gefitinib. The activity of the compound 10b on the EGFR enzyme was subsequently tested. The Wound Healing of A549, AGS and HepG2 cells by this compound showed that the compound can inhibit the migration of cancer cells. Finally, the action channel of the compound 10b was supported by western blotting experiments. It provides useful information for the design of EGFR-TK inhibitors.

Full text of DOI:10.1002/cbdv.202100478

Title: Synthesis and anti-proliferation activity evaluation of novel 2-
chloroquinazoline as potential EGFR-TK inhibitors
Authors: Quan Zheng, Xuan-Bo Xu, Hao Jin, Wen Zhang, and Guo-Wu
Rao
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10.1002/cbdv.202100478
Chemistry & Biodiversity
Synthesis and anti-proliferation activity evaluation of novel
2-chloroquinazoline as potential EGFR-TK inhibitors
Quan Zheng, Xuan-Bo Xu, Hao Jin, Wen Zhang, and Guo-Wu Rao*
College of Pharmaceutical Science, Zhejiang University of Technology, and Institute of Drug Development & Chemical Biology,
Zhejiang University of Technology, Hangzhou 310014, P. R. China
* Corresponding author. E-mail address: rgw@zjut.edu.cn (G.-W. Rao).
A novel series of 2-chloroquinazoline derivatives had been synthesized and their
anti-proliferation activities against the four EGFR high-expressing cells A549, NCI-H1975, AGS
and HepG2 cell lines were evaluated. The preliminary SAR study of the scaffold of new
compounds showed that the compounds with a chlorine substituent on R3 had a better
anti-proliferation activity than those substituted by hydrogen atom or vinyl group. Among them,
2-chloro-N-(2-chloro-4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)acetamide (10b) had the
best activity, and the corresponding IC50 were 3.68, 10.06, 1.73 and 2.04 μM, respectively. And
compound 10b had better or equivalent activity against four cell lines than Gefitinib. The activity
of the compound 10b on the EGFR enzyme was subsequently tested. The Wound Healing of A549,
AGS and HepG2 cells by this compound showed that the compound can inhibit the migration of
cancer cells. Finally, the action channel of the compound 10b was supported by western blotting
experiments. It provides useful information for the design of EGFR-TK inhibitors.
Keywords: Antiproliferative, EGFR inhibitors, Quinazoline, Western blot.
Introduction
Cancer, as a potential cause of death, continues being a global killer commonly all over the
world.[1,2] Under this situation, many studies related to anticancer compounds which possess high
specificity to tumor cells and low toxicity as well as side effects to other normal cells were
gradually emerging. Targeted compounds, which were distinct from the traditional
chemotherapeutic drugs, can selectively target cancer cells with higher efficacy and lower side
effects. In fact, the mechanism of this kind of compounds may in relation to their selective actions
on intracellular signaling pathways which show close connections with the tumor cell cycles and
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its microenvironment triggering the biological activities including cell apoptosis, cell proliferation,
tumor growth,[3,4] angiogenesis and so on.
The epidermal growth factor receptor (EGFR),[5] as a member of the ErbB family of receptor
tyrosine kinases (RTKs),[6-10] plays an essential role in regulating cell proliferation, differentiation
and apoptosis. The over expression of the EGFR was reported associated with a number of the
most lethal cancers in the world,[11,12] such as breast, ovarian, colon and non-small cell lung cancer
(NSCLC).[13] EGFR may be activated by binding to endogenous ligands including the epidermal
growth factor (EGF),[14-16] transforming growth factor-α (TGF-α), amphiregullin, betacellulin and
epiregulin which result in the sensitization of intracellular signaling transduction pathways.
Moreover, the downstream signaling transduction may be triggered by the mechanism of
auto-phosphorylation of several tyrosine residues after EGFR dimerization. Thereby, developing a
kind of small molecule EGFR tyrosine kinase inhibitors (EGFR-TKIs) appears to be a feasible
approach.
As shown in Fig. 1, the first-generation EGFR-TKIs such as gefitinib and erlotinib were
successfully developed and had been used as the front-line standard therapy in the treatment of
NSCLC patients with sensitizing EGFR mutations currently.[17,18] However, cancer cells were
found to develop resistance rapidly to EGFR-TKIs[19] through several unique mechanisms and the
phenomenons of recrudescence as well as some serious side effects were also emerging at the
same time.[20] Consequently, to overcome the drug resistance towards the first-generation
EGFR-TKIs, the second-generation irreversible EGFR-TKIs such as dacomitinib and afatinib
were developed. Subsequently, it was identified that the irreversible EGFR inhibitors have the
covalent bond with Cys797, and they mostly contain a Michael acceptor functional group
targeting the Cys797 residue in the ATP binding cleft.[21] Nevertheless, their clinical efficacy has
also been limited eventually by corresponding skin rash and gastrointestinal toxicity due to their
less selectivity between EGFRwt and EGFRT790M mutation (the 790th amino acid of the EGFR
protein is converted from threonine to methionine).[22] Thus, the third-generation EGFR-TKIs
such as AZD9291 and CO-1686 (Fig. 1), as covalent inhibitors similar with the second-generation
EGFR-TKIs have been identified recently, and their selectivity on EGFRT790M mutant over
EGFRwt have been demonstrated as well.[23] But it is well known that irreversible inhibitors have
potential safety concerns due to their poor selectivity and the reason that they cannot be
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transferred by ATP. Therefore, their possible toxicity may be inevitable owing to the prolonged
off-target inhibition of EGFRwt.
In conclusion, the first-generation inhibitor such as gefitinib can solve the EGFRL858R mutation
(the 858th amino acid of the EGFR protein is mutated from leucine to arginine), but after
long-term clinical use, EGFR will develop T790M resistance Mutation. Although the
second-generation inhibitor such as afatinib can better inhibit the EGFRT790M mutation, its clinical
use has been greatly restricted due to its severe side effects. Hence, there is an ultimate need for us
to develop a type of novel, potent EGFR inhibitors that targets both EGFRL858R and EGFRT790M to
overcome the persistent resistance emerged in currently used ones and thus provide better
treatment options.[24]
Fig. 1. The first-, second- and third-generation of EGFR inhibitors.
Rational Drug Design
In the past decade, researchers have studied the structure-activity relationship (SAR) of
quinazoline derivatives and found that most EGFR-TKI inhibitors have four common
pharmacophoric groups structures, including quinazoline heterocyclic scaffold, NH linker,
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hydrophobic head of various substituted phenyl groups, and hydrophobic tail (Fig. 2).[25]
Therefore, it is completely in line with the rational drug design concept that a novel type of EGFR
inhibitor is designed by purposeful structural modification and transformation in these four
positions.
Fig. 2. Four common pharmacodynamic structures of most EGFR-TKI inhibitors.
Previously, the SAR study of gefitinib showed that compounds with aniline substitution at the
4-position of the quinazoline scaffold had better activity, while the SAR of afatinib showed that
the introduction of amide group at the 6-position of quinazoline scaffold could also improve the
inhibitory activity of compounds against EGFR.[26] Barbosa et al.[27] found that it had good
biological activity after replacing it with a chlorine atom at the 2-position of the quinazoline
scaffold. After docking the compounds to the EGFR protein, it was found that the presence of the
chlorine atom at 2-position facilitates the binding of the compounds to EGFR, and at the same
time, the chlorine atom was beneficial to improve the fat solubility, electron absorption and
metabolic hindrance of the compounds. In short, we designed a series of novel
2-chloroquinazoline compounds by introducing a chlorine atom at the 2-position, aniline groups at
the 4-position, and amide groups at the 6-position of the quinazoline scaffold. The entire rational
drug design idea for the novel EGFR inhibitor is illustrated in Fig. 3.
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Fig. 3. Design idea of novel 2-chloroquinazolines as new EGFR inhibitors.
Results and Discussion
Chemistry
The synthesis of compounds 10a-l was described in Scheme 1. Target compounds 10a-l were
achieved in seven steps using 1 as starting materials, which were coupled with KCNO in acetic
acid at ambient temperature. Cyclization of 2 was done at reflux in NaOH methanol solution (pH
o
= 10).[28] 4 was achieved by regioselective nitration of 3 at -10 C.[29] Chlorination of 4 was
achieved by refluxing with POCl3 at the presence of N,N-Diisopropylethylamine in toluene. Then
the corresponding phenylamines 6a-f were attached to the 4-position of 5 through nucleophilic
addition under basic condition in THF at room temperature in a dropwise way.[30] Finally, the nitro
groups of compounds 7a-f were reduced using iron in acetic acid,[21] followed by acylation of the
resulting amines 8a-f with the corresponding acid chlorides 9a-c to obtain the
1
quinazolinederivatives 10a-l.[31] These compounds were characterized by H and 13C NMR
spectroscopy, IR spectroscopy, HR-MS, and HPLC. (All the Spectroscopic data of 10a-l can be
found in the Supporting Information).
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Chemistry & Biodiversity
Scheme 1. Synthesis methods of Compounds 10a-l.
Biological activity
Anti-proliferation assay
As shown in Table 1, the anti-proliferation activities of all synthesized compounds against the
four EGFR high-expressing cells A549,[32,33] NCI-H1975,[33] AGS[34] and HepG2[35,36] cell lines
had been evaluated in vitro by the MTT assay. Gefitinib was used as the positive control. Results
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Chemistry & Biodiversity
were expressed as median growth inhibition concentration (IC50) values, which represent the
concentration of compound required to produce 50% inhibition of cell growth after 24 hours of
incubation, compared to untreated controls. The results of anti-proliferation activities for the
compounds were illustrated in Table 1. The results showed that most of the compounds exhibited
potent anti-proliferation effects. The compounds with a chlorine substituent on R3 had better
anti-proliferation activities than those substituted by hydrogen atom or vinyl group. Compound
10b had better or equivalent activity against four cell lines than Gefitinib.
Table 1
In vitro anti-proliferation activity of target compounds (Mean ± SD, n = 3).[a]
Cell proliferation assays IC50 (μM) [b]
Compd
A549
NCI-H1975
AGS
HepG2
10a
10b
19.67±1.84
15.05±0.84
13.16±1.36
6.82±1.21
3.68±0.34
10.65±1.34
>20
10.06±1.11
19.19±2.16
>20
1.73±0.42
2.00±0.84
>20
2.04±0.81
10.33±1.33
>20
10c
10d
10e
>20
>20
>20
>20
10f
>20
16.95±1.68
>20
>20
>20
10g
>20
>20
>20
10h
>20
20.14±2.57
16.34±1.67
17.87±1.37
>20
>20
>20
10i
6.87±0.86
5.37±0.34
12.58±1.36
8.11±0.58
14.53±0.48
2.24±0.64
3.53±0.54
19.63±2.01
4.47±1.01
7.65±0.73
5.76±0.64
5.13±0.42
9.03±1.63
5.72±0.87
3.15±0.91
10j
10k
10l
>20
Gefitinib[c]
12.48±0.22
[a] Data shown represent the mean ± SD of three independent experiments, each performed in triplicate.
[b] IC50: compounds concentration that reduces 50% cell proliferation evaluated by the MTT assay.
[c] A positive control.
In order to explore the SAR of the novel scaffold, hydrogen atom was introduced to R3
(compounds 10d-h) in the first. It was found that the anti-proliferation activities of compounds
against the four EGFR high-expressing cells were not good in vitro, basically >20 μM. Only when
R2 and R1 were both halogen atoms, just compounds 10f and 10h showed certain anti-proliferation
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activities against NCI-H1975 in the four EGFR high-expressing cells, which were 16.95 μM and
20.14 μM, respectively. When R2 was replaced by hydrogen atom (compounds 10d, 10g) or R1
was replaced by hydrogen atom (compound 10e), the compounds showed no anti-proliferation
activity against the four EGFR high-expressing cells. It may be that the radius of the hydrogen
atom is too small to fill the pocket of EGFR kinase.
To find a more effective EGFR inhibitor, chlorine atom was introduced as R3 (compounds
10a-c, 10i-j) in the subsequent synthesis to explore the effect of the halogen atom at the R3
position on the overall scaffold SAR. By comparing 10a and 10f, 10b and 10h, 10d and 10j, 10e
and 10i, it was easy to find that compounds 10a, 10b, 10i, and 10j had a significant increase in the
inhibitory activity of the four EGFR high-expressing cells in vitro. Obviously, the introduction of
a chlorine atom at the R3 position had a direct effect on the activity of the compounds. Compared
with hydrogen atoms, the larger atomic radius of chlorine atoms may better fill the pocket of
EGFR kinase.
To further verify the effect of the size of the atom or atomic group at the R3 position on the
SAR of the scaffold, a larger vinyl group was introduced in the subsequent synthesis, thereby
obtaining compounds 10k-l. By comparing 10i and 10l, 10j and 10k, that 10k and 10l with a vinyl
group introduced at the R3 position were slightly weaker activity than 10i and 10j with a chlorine
atom at the position. All in all, the preliminary SAR study of the scaffold of the novel compounds
showed that the introduction of a suitable atom or atomic group at the R3 position had a significant
effect on the activity of the compounds.
Enzyme assay
The compounds 10b, 10i, and 10j, which were synthesized through the SAR research of the new
scaffold in the early stage and showed the best in vitro inhibitory activity for the four EGFR
high-expressing cells, were further studied for enzymatic activity to explore the inhibition of the
new compounds scaffold on EGFR kinase. The results were shown in Table 2, the compound 10b
had the best effect, its IC50 for EGFRwt and EGFRL858R was 35.7 nM and 217.6 nM, respectively.
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Table 2
Compounds tested for their inhibitory effects on both EGFRwt, EGFRL858R, EGFRL858R/T790M and
EGFRT790M (Mean ± SD, n = 3).[a]
IC50 (nM)[b]
EGFRL858R/T790M-TK
Compd
EGFRwt-TK
EGFRL858R-TK
217.6±8 .6
427.3±16.7
N. D.
EGFRT790M-TK
>1000
N. D.[c]
>1000
>1000
>1000
10b
10i
35.7±3.7
153.8±5.8
352.4±10.9
12.4±0.6
>1000
10j
>1000
Gefitinib[d]
3.1±0.4
187.4±6.4
[a] Data shown represent the mean ± SD of three independent experiments, each performed in triplicate.
[b] IC50: compounds concentration that reduces 50% enzyme activity evaluated by the enzyme assay.
[c] N. D.: not determined.
[d] A positive control.
Cell migration assay
In order to test whether compound 10b had an inhibition effect on the migration progression of
gastric cancer cells (AGS), lung cancer cells (A549) and liver cancer cells (HepG2), AGS cells,
A549 and HepG2 cells were treated with compound 10b (0.5 μM and 1.0 μM) or not for 0 to 48 h.
AGS cells, A549 and HepG2 cells were allowed to migrate in scratch-wound cultures, and the
migration of AGS cells, A549 and HepG2 cells in scratch-wound cultures was inhibited by the
addition of compound 10b. The ability of compound 10b to inhibit the migration of AGS, A549
and HepG2 cells increased with the increasing of concentration (Fig. 4).
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Fig. 4. Cell mobility inhibition for the migration of AGS, HepG2 and A549 treated with
compound 10b for 0 to 48 h. Figures 4a and 4b show the effect of compound 10b on the migration
of AGS cells; Figures 4c and 4d show the effect of compound 10b on the migration of HepG2
cells; Figures 4e and 4f show the effect of compound 10b on the migration of A549 cells.
Western blot assay
To further explore the anti-proliferative mechanism of compound 10b. Western blot assay was
used to evaluate the effect of compound 10b on the expression of related proteins in A549 cells,
including EGFR/p-EGFR, Akt/p-Akt, Erk/p-Erk. As shown in Fig. 5, the expressions of EGFR,
Akt and Erk in A549 cells were not affected, while the expressions of p-EGFR, p-Akt and p-Erk
decreased in a dose-dependent manner. Western blot assay showed that compound 10b inhibited
the proliferation of A549 cells by inhibiting the phosphorylation of EGFR and its downstream Akt
and Erk.
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Fig. 5. Effects of compound 10b on the expression of related proteins in A549 cells, including
EGFR/p-EGFR, Akt/p-Akt, Erk/p-Erk. A549 cells were treated with different concentrations of
compound 10b or gefitinib for 12 h.
Docking study
To explore the binding mode of compound 10b with EGFR (PDB: 2ITY), a docking study was
carried out employing Discovery Studio 4.0 software. Before docking the designed compound
molecule, the reliability of the model had been verified. The proposed binding mode of 10b in the
allostericbinding pocket was depicted in Fig. 6. It was observed that 10b fitted to the hydrophobic
environment composed with Leu788 and Leu844. The N-1 of the quinazoline scaffold forming a
H-bond with the backbone NH of Lys728 side chain, and the aniline lying in an adjacent
hydrophobic pocket. Moreover, the chlorine at the 2-position of quinazoline forming a hydrogen
bond with Ala743, at the same time, the fluorine on the benzene ring and lys745 also having a
hydrogen bond. This helps explain why compound 10b had better biological activity.
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Fig. 6. Molecular docking between compound 10b and EGFR kinase (PDB: 2ITY)
Conclusions
In conclusion, a series of novel N-(2-chloro-4-(phenylamine) quinazolin-6-yl) acetamide
derivatives were synthesized, and their biological activities were evaluated in vitro. Most of new
designed compounds had potential anti-proliferation activities against the A549, NCI-H1975,
AGS and HepG2 four cell lines. The preliminary SAR study of the scaffold of new compounds
showed that the compounds with a chlorine substituent on R3 had a better anti-proliferation
activity than those substituted by hydrogen atom or vinyl group. Among them, compound 10b had
the best activity. 10b had a certain inhibitory effect on enzymes and the migration of tumor cells.
Finally, the action channel of the compound 10b was proved by western blotting experiments. It
provides useful information for the design of EGFR-TK inhibitors.
Experimental Section
Chemistry
All reagents and solvents were purchased from commercial sources without further purification,
and had been dried according to standard procedures. Reactions were monitored by using
thin-layer chromatography (TLC) on silica gel 60 F254. Melting points were determined on a X-4
melting point apparatus and uncorrected. IR spectra were taken on a Thermo Nicolet Avatar 370
FT-IR spectrophotometer (KBr pellets). NMR spectra were recorded at 500 MHz on a Bruker
Anance III spectrometer. Mass spectra were obtained on a Bruker micrOTOF-Q mass
spectrometer using ESI.
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Synthesis
Methyl-2-ureidobenzoate (2)
To a solution of methyl 2-aminobenzoate (1) (5 g, 33.08 mM) in acetic acid (25 ml) was treated
with a solution of KNCO (3.88 g, 59.54 mM) in water (15 ml). After 15 mins mechanical stirring
at room temperature, the resulting precipitate was collected by filtration, then washed with water
twice and dried to yield Methyl-2-ureidobenzoate (2) (6 g, yield: 93%).
(1H)-Quinazolin-2,4-dione (3)
A solution of 15% NaOH solution was added to a mechanically stirred suspension of
Methyl-2-ureidobenzoate (2) (6 g, 30.9 mM) in methanol (50 ml) adjusting the pH to 10. After 3 h
at reflux, the mixture was cooled to room temperature and diluted with water (50 ml). The pH was
adjusted to 1 with HCl. The precipitate was collected by filtration to yield a white solid (4.5 g,
90%).
6-Nitroquinazolin-2,4-dione (4)
The compound 3 (2.0 g, 12.3 mM) was dissolved in concentrated H2SO4 (6 mL), and this solution
was added to a mixture of concentrated H2SO4 (2 mL) and HNO3 (5.6 mL) at -10 oC. The mixture
was stirred for 1 h at -5 oC.The reaction mixture was poured onto crushed ice (50 g). A crystalline
solid was collected and washed with water. The precipitate was collected by filtration to yield a
light yellow solid (1.8 g, 70%) mp 352-354 oC; MS (EI) m/z 207.
2,4-Dichloro-6-nitroquinazoline (5)
The reaction mixture of compound 4 (0.5 g, 2.41 mmol), N,N-Diisopropyl ethylamine (0.62 g, 4.4
mM) and POCl3 (3 mL) was stirred at reflux for 6.0 h. The residue was dissolved in ice water. The
water phase was extracted with EtOAc (60 mL) and the organic layer was dried over anhydrous
MgSO4, concentrated to give the crude product which was purified by column chromatography on
silica gel (petroleum ether/DCM = 10:1) to afford compound 5 as white solid (0.36 g, 61%); mp
122-124 oC; 1H NMR (CDCl3) d: 8.18 (d, J = 9.0 Hz, 1H), 8.76 (dd, J = 9.3, 2.1 Hz, 1H), 9.18 (d,
J = 1.8 Hz, 1H).
General procedure for the synthesis of 2-chloro-N-phenyl-6-nitroquinazolin-4-amine
derivatives (7)
To a suspension of 2,4-dichloro-6-nitroquinazoline 5 (0.30 g, 1.23 mmol) in THF (30 mL) was
added aniline analogs (1.48 mmol) and DIPEA (0.32 g, 2.46 mmol). The resulting mixture was
stirred at room temperature for 8 h and concentrated under vacuum. The residue was dissolved in
EtOAc. The crude product obtained after concentration was purified with column chromatography
to afford product 7 as red solid.
General procedure for the synthesis of 2-chloro-N-4-phenylquinazoline-4,6-diamine
derivatives (8)
To a dichloromethane solution of appropriate compound 7 (0.67 mmol), Fe (0.185 g, 3.33 mM)
was added under the presence of acetic acid at the reflux temperature for 1 h. Thereafter, the
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catalyst was filtered over a celite bed and the filtrate was evaporated to yield a solid residue. The
residue was recrystallized from MeOH to afford the pure compounds 8 as yellow solids.
General procedure for the synthesis of N-(2-chloro-4-(phenylamino)quinazolin-6-yl)
acetamide derivatives (10)
The compound was synthesized from 2-chloro-N4-phenylquinazoline-4,6-diamine derivatives 8
(0.37 mmol), corresponding acid chloride (0.44 mmol), and N,N-diisopropylethylamine (0.44
mmol) in 35 mL of THF. The resulting mixture was stirred at room temperature for 12 h and
concentrated under vacuum. The residue was dissolved in EtOAc. The crude product obtained
after concentration was purified with column chromatography to afford product 10.
Biological
Cell viability assay
The MTT assay was used to evaluate cell viability. Cells were seeded in a 96-well plate (4 × 103
cells/well). After 24 h of drug treatment, MTT was added to each well to a final concentration of
0.5 mg/ml, followed by incubation at 37 oC for 3 h. Then, the medium was removed, and 100 μl of
dimethyl sufoxide (DMSO) was added per well. The absorbance in each well was measured at 570
nm using a Flexs tation 3 microplate reader (Molecular Devices). To determine the IC50 of 20A, a
non-linear regression curve was fit using GraphPad Prism software.
In vitro kinase assay
The effects of compounds on the activity of EGFR kinase were determined using enzyme-linked
immunosorbent assays (ELISAs) with purified recombinant proteins (Sino Biological Inc.).
o
Briefly, 50 µg/mL poly (Glu,Tyr) 4:1 (Sigma) was pre-coated in 96-well plates at 37 C as a
substrate. The enzyme reaction was carried in kinase reaction buffer (50 mmol/L HEPES (pH 7.4),
0.01% BRIJ-35, 10 mmol/L MgCl2, 4 mmol/L MnCl2, 0.1 mmol/L EGTA, and 2 mmol/L DTT).
10 µL of various concentrations of indicated compounds and 20 µL of purified tyrosine kinase
proteins were added to each reaction well. DMSO (0.5%, v/v) was used as the negative control.
The kinase reaction was initiated by addition of a 50 µL of 5µmol/L ATP solution which diluted in
o
kinase reaction buffer. After incubation for 60 min at 37 C, the plates was washed 6 times with
phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST). Anti-phosphotyrosine mouse
o
mAb (PTM Bio) antibody was then added. After a 60min incubation at 37 C, the plate was
washed 3 times with PBST, and horseradish peroxidase-conjugated goat anti-mouse IgG was
added. The plate was then incubated at 37 oC for 40 min and washed 5 times with PBST. A 100 µL
aliquot of a solution containing 0.03% H2O2 and 2 mg/ml TMB in 0.1 mol/L citrate buffer (pH 5.5)
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was added. The reaction was terminated by addition of 100 µL of 2 M H2SO4 as the color changed,
and the plate was analyzed using a multi-well spectrophotometer (PE Enspire) at 450 nm. The IC50
values were calculated from the inhibition curves using GraphPad Prism5 (GraphPad Software,
Inc.) in at least three separate experiments.
The emission ration was calculated using the following equation:
Emission Ration = OD450/OD450control
Wound healing
Cells were seeded in 6-well plates to form a cell monolayer (near 90% confluence), and a wound
was created by scratching the length of the well with a 10 µL pipette tip. Then, the plate was
washed three times with PBS to remove floating cells and photographed (time 0 h). The cells were
incubated in serum lacking medium (0% FBS), then the corresponding concentration of compound
o
was added. Next, the wounded cultures were incubated in conditioned medium at 37 C. At 0, 1
and 2 day, images were captured using an inverted microscope to assess wound closure and then
compared to determine differences in cell migration. Each assay was replicated 3 times.
Western blotting assay
Cells were Collected and lysed in 1X RIPA buffer with phosphatase inhibitors. Cell lysates were
cleared by centrifugation at 4 oC for 15 min at 13200 xg. Protein was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene
fluoride (PVDF) membrane. After blocking in 5% BSA/TBST buffer for 2 h, membranes were
incubated in primary antibodies overnight at 4 ℃. The following primary antibodies were used in
the dilution as suggested by the supplier: EGFR, P-EGFR, Akt, P-Akt, Erk, P-Erk, GAPDH
(1:1,000; Cell Signal Technology). Then, blots were incubated in secondary antibodies for 2h at
room temperature. Immersed in BeyoECL Star (Beyotime) for 2 min, and then photographed
using a Bio-Rad gel imaging analyzer.
Acknowledgments
This research was supported by the Science and Technology Program of Zhejiang Province (Key
Innovative Team) under Grant No. KYY-ZX-20190351, and Zhejiang Provincial Natural Science
Foundation of China under Grant No. LY19B020008.
Author Contribution Statement
15
This article is protected by copyright. All rights reserved.

10.1002/cbdv.202100478
Chemistry & Biodiversity
Quan Zheng and Xuan-Bo Xu designed and synthesized these compounds and wrote the article.
Xuan-Bo Xu and Hao Jin performed the experiments, analyzed the data. Wen Zhang and Guo-Wu
Rao designed and conceived the experiments.
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Twitter text:
A novel series of 2-chloroquinazoline derivatives, potential EGFR-TK inhibitors, were designed
from Gefitinib and Afatinib by G.-W. Rao et al., Zhejiang University of Technology, China
(without Account)
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