Discovery of novel 4-anilinoquinazoline derivatives as potent inhibitors of epidermal growth factor receptor with antitumor activity

Article abstract of DOI:10.1016/j.bmc.2013.06.070

Two new series of new compounds containing a 6-amino-substituted group or 6-acrylamide-substituted group linked to a 4-anilinoquinazoline nucleus have been discovered as potential EGFR inhibitors. These compounds proved efficient effects on antiproliferative activity and EGFR-TK inhibitory activity. Especially, N⁶-((5-bromothiophen-2-yl)methyl)-N⁴-(3- chlorophenyl)quinazoline-4,6-diamine (5e), showed the most potent inhibitory activity (IC₅₀ = 3.11 μM for Hep G2, IC₅₀ = 0.82 μM for A549). The EGFR molecular docking model suggested that the new compound is nicely bound to the region of EGFR, and cell morphology by Hoechst stain experiment suggested that these compounds efficiently induced apoptosis of A549 cells.

Full text of DOI:10.1016/j.bmc.2013.06.070

Accepted Manuscript
Discovery of Novel 4-anilinoquinazoline Derivatives as Potent Inhibitors of
Epidermal Growth Factor Receptor with Antitumor Activity
Yun-Yun Xu, Si-Ning Li, Gao-Jian Yu, Qing-Hua Hu, Huan-Qiu Li
PII:
S0968-0896(13)00610-X
http://dx.doi.org/10.1016/j.bmc.2013.06.070
BMC 10963
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 May 2013
21 June 2013
29 June 2013
Please cite this article as: Xu, Y-Y., Li, S-N., Yu, G-J., Hu, Q-H., Li, H-Q., Discovery of Novel 4-anilinoquinazoline
Derivatives as Potent Inhibitors of Epidermal Growth Factor Receptor with Antitumor Activity, Bioorganic &
Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.06.070
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Discovery of Novel 4-anilinoquinazoline Derivatives
as Potent Inhibitors of Epidermal Growth Factor
Receptor with Antitumor Activity
Yun-Yun Xu1, Si-Ning Li1, Gao-Jian Yu1, Qing-Hua Hu2*, Huan-Qiu Li1*.
1. College of Pharmaceutical Science, Soochow University, Suzhou 215123, PR
China.; *Corresponding author. Tel.: +86 512 65882090; fax.: +86 512
65882090; e-mail: huanqiuli@suda.edu.cn
2. State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, P. R. China. *Corresponding author. Tel.: +86 25 83271414;
fax.: +86 25 83271414; e-mail: huqh@cpu.edu.cn
Abstract：Two new series of new compounds containing a 6-amino-substituted
group or 6-acrylamide-substituted group linked to a 4-anilinoquinazoline nucleus
have been discovered as potential EGFR inhibitors. These compounds proved
efficient effects on antiproliferative activity and EGFR TK inhibitory activity.
Especially, N6-((5-bromothiophen-2-yl)methyl)-N4-(3-chlorophenyl)quinazoline-4,6-
diamine (5e), showed the most potent inhibitory activity (IC50=3.11 μM for Hep G2,
IC50=0.82 μM for A549). The EGFR molecular docking model suggested that the new
compound is nicely bound to the region of EGFR, and cell morphology by Hoechst
stain experiment suggested that these compounds efficiently induced apoptosis of
A549 cells.
Keywords: EGFR-TK inhibitors; 4-anilinoquinazoline; antitumor; molecular docking
1

1. Introduction
The altered protein expression and activity of receptor tyrosine kinases (TK) are
implicated in the progression of various types of cancers.1 Among the growth factor
receptor kinases that have been identified as being important in cancer is epidermal
growth factor receptor (EGFR) kinase.2 Expression of a dominant negative Ras
mutant in EGFR overexpressing cells also results in a significant potentiation of
EGFR induced apoptosis suggesting that Ras activation is a key survival signal
generated by the EGFR. The role of EGFR has been most thoroughly studied in breast
cancer,3 lung cancer (especially lung adenocarcinomas)4–6 and in hormone-refractory
prostate cancer.7 Therefore, the EGFR kinase represents an attractive target for the
development of novel therapies for the treatment of cancers.
To date, the Food and Drug Administration has approved more than 15 small
molecule kinase inhibitors for targeted cancer therapy and dozens are currently in
clinical trials. Out of these inhibitors, Gefitinb8 (CP 358774), Erlotinib (ZD 1839)9
and Lapatinib (GW572016)10 belong to a class of 4-anilinoquinazolines which have
been designed to selectively inhibit EGF-stimulated signal transduction by reversibly
binding at the ATP site of EGFR (Scheme 1).11 Researchers at Parke-Davis12 in which
Rewcastle and colleagues reported a SAR study on a series of compounds derived
from ten-membered nitrogen-containing bicyclic scaffolds. They concluded that the
quinazoline core was the best scaffold for the development of EGFR inhibitors
because any alteration of the nitrogen substitution pattern in the bicyclic ring resulted
in less active compounds.12
In our studies of the inhibition of EGFR by reversible and irreversible
4-anilinoquinazolines,
a
type of 4,6-substituted-(diaphenylamino)quinazoline
derivatives were synthesized and displayed potent EGFR inhibitory activity,13
modeling studies of the binding of reversible anilinoquinazoline with EGFR-TK has
been developed. The results suggest that a binding mode whereby the C-6 position of
the bicyclic chromophore point toward the solvent out of the ATP binding pocket.
2

This explains why both positions can be used to add bulky solubilizing functionalities
to inhibitors without losing binding affinity.14 As discussed above, molecular
modeling suggests that a favorable site to attach new heteroamino groups based on the
4-(phenylamino)quinazoline core structure would be at the C-6 position, since these
positions point toward the outside of the protein. This stimulated our interest in
designing and synthesizing of two series of irreversible EGFR inhibitors:
6-amino-substituted
4-anilinoquinazoline
detivatives, as
well
as
the
6-acrylamide-substituted 4-anilinoquinazoline derivatives, have been designed and
synthesized based on the 4-(phenylamino)quinazoline that have an attached Michael
acceptor functional group at the C-6 position. Molecular modeling studies suggested,
and experimental results confirmed, that the Michael acceptor side chain of these
inhibitors forms a covalent linkage with the sulfhydryl group of the Cys 773 of EGFR.
These compounds are potent in inhibition of cell growth in two cancer cell lines (Hep
G2 and A549) and with high levels of EGFR proteins.
2. Results and discussion
2.1. Chemistry
Two series of 4-anilinoquinazoline inhibitors (5a-5i and 8a-8h, Table 1) were
designed
to
perform
comparative
SAR
studies.
6-amino-substituted
4-anilinoquinazolines 5a-5i and 6-acrylamide-substituted 4-anilinoquinazoline 8a-8h
were synthesized starting from the intermediates 4, respectively. Commercially
available 5-nitroanthranilonitrile 1 was converted into the corresponding formamidine
2 using DMF acetal. Heating a solution of formamidine 2 and 3-X-aniline in HOAc
gave 6-nitro-4-(3-X-phenylamino)quinazolines 3. Reduction of the nitro group of 3
with iron in HOAc yielded the intermediate 4-aminoquinazolines 4.15,16 Finally, Schiff
bases of 4-aminoquinazolines were obtained in high yields using various substituted
benzaldehyde and EtOH as the carbonyl activator. Then schiff bases of
4-aminoquinazolines were submitted to the STAB reductive conditions, leading to the
corresponding 6-amino-substituted 4-anilinoquinazolines 5a-5i (Scheme 2).
The various aldehyde acids (7a-7h) were synthesized using Doebner
Knoevenagel modification, and then treat the resulting aldehyde acids (7a-7h) with
3

the
4-aminoquinazolines
4
to
obtain
the
6-acrylamide-substituted
4-anilinoquinazolines 8a-8h (Scheme 3). All of the synthetic compounds gave
satisfactory analytical and spectroscopia data, which were in full accordance with
their depicted structures.
2.2. Antiproliferative activity
To determine the activity of the 4-anilinoquinazolines in vitro, cellular based
screening of all the synthesized 6-amino-substituted 4-anilinoquinazolines as well as
6-acrylamide-substituted-4-anilinoquinazolines and Erlotinib was performed in
EGFR-dependent A549 and Hep G2 cell lines. The results were summarized in Table
1, and the calculated IC50 values, that is, the concentration (µM) of a compound that
was able to cause 50% cell death with respect to the control culture, were reported
differently according to different cancer cells. As expected, all the synthesized
4-anilinoquinazolines exhibited remarked effects on antiproliferative activities，and
generally the results showed which those applied to A549 cells would performed
better than that in Hep G2 cells, was normal and acceptable.
Structure-activity relationships in these new compounds demonstrated that
6-amino-substituted 4-anilinoquinazolines 5a-5i showed generally better activity than
6-acrylamide-substituted-4-anilinoquinazolines 8a-8h, indicating little tolerance of
bulkiness in the hydrophobic pocket of the ATP site of EGFR of the latter compounds.
As show in Table 1, 6-thiophen-amino-4-anilinoquinazolines 5d showed potent
antiproliferative activity on A549 cells with an IC50 of 2.08 μM, being 4 times more
potent than 6-thiophen-acrylamide-4-anilinoquinazolines 8d (IC50 of 9.31 μM). We
still premised that the interactions of the molecule was the ATP binding site of EGFR,
which quinazoline ring was buried inside the pocket and the substituent at the
6-positions was outside the cleft. Above mentioned results may indicate that the same
group with different chains (6-amine/6-acrylamide) had different affinity
with quinazoline ring, and obvious that 8d with 6-acrylamide appeared very normal.
In 6-amino-substituted 4-anilinoquinazolines series, substitution of the aliphatic
group with an amine made compounds 5h and 5i showed lower activity than others.
And substitution of a 6-(5-bromothiophen) amine group made compound 5e displayed
4

the best antiproliferative activity (IC50 of 0.82 μM), while 6-pyridineamine 5a,
6-quinazolinamine 5c, and 6-(1H-indole)amine 5f gave IC50 values over 2 μM.
2.3. EGFR inhibitory activity and molecular docking
To test the above-mentioned hypotheses regarding inhibition of EGFR by
4-anilinoquinolines, enzyme activity assays were performed for EGFR. We chose
eight compounds behaved well for antiproliferation and their EGFR inhibitory
activities were displayed at Table 2. This could indicate that 4-anilinoquinolines
basically follow the EGFR inhibition path against cancer cell. As shown in Table 2,
compound 5d and 5e displayed the most potent inhibitory activity (IC50 = 1.52 µM
and 2.31 µM for EGFR), less comparable to the positive control erlotinib (IC50 = 0.03
µM for EGFR).
To help understand the SARs observed at the EGFR and guide further SAR
studies, molecular docking of the most potent inhibitor 5e into ATP binding site of
EGFR kinase was performed on the binding model based on the EGFR complex
structure (1M17.pdb). The binding model of compound 5e and EGFR was depicted in
Figure 1A and Figure 1B. In the binding model, compound 5e is nicely bound to the
region of EGFR with binding interaction energy of -37.83 KJ·mol-1. The quinazoline
ring of compound 5e be inserted nicely inside the pocket, and the nitrogen atom of
amine group of 5e forms hydrogen bond with Asp831(OD2···N: 2.9 Å), which made
the combination ability of 5e acting on EGFR. Meanwhile, the nitrogen atom of
acrylamide group of 8e has no reaction with EGFR. This was likely the explanation
for why 6-amino-substituted 4-anilinoquinazolines 5a-5i showed generally better
activity than 6-acrylamide-substituted-4-anilinoquinazolines 8a-8h.
The sulfur atom of 5-bromothiophen group of 5e also forms hydrogen bond with
Lys721(N-H···S: 3.3 Å), indicated that the 5-bromothiophen group of 5e was
important for the potent inhibitory activity of 5e The modeling also suggested that
there is a π-cation interaction between the Cl-benzene group of quinazoline ring of
compound 5b and Leu694, π-Cation interaction energies are of the same order of
magnitude as hydrogen bonds or salt bridges and play an important role in stabilizing
the three dimensional structure of a protein.17
5

2.4. Hoechst 33258 staining evaluation
To confirm the induction of apoptosis by 5e, the cells were stained with Hoechst
33258, a fluorescent DNA-staining dye. Cell morphology was investigated using
fluorescence microscopy. Figure 2 demonstrated that control cells stained with the
Hoechst dye had the homogeneous blue fluorescence in the nuclei, suggesting there
was no obvious nuclear condensation and fragmentation in A549 cells that were
treated with 0.1% DMSO alone. However, when the cells treated with 5e, the nuclei
became convoluted and budded off into several fragments, and nuclear condensation
and fragmentation were observed in the pictures, indicating a typical characteristic of
apoptosis. Furthermore, it is found that as the increase of 5e concentration from 5 to
20 μM, more and more cells stained in dark blue were observed under the microscope.
It is suggested that 5e induce the cellular apoptosis dose-dependently, which is
consistent with the former anti-proliferation assay.
3. Conclusion
We report here two new series of EGFR inhibitors containing a 6-amino-substituted
group or 6-acrylamide-substituted group linked to a 4-anilinoquinazoline nucleus. The
newly synthesized 4-anilinoquinazolines proved efficient in inhibiting EGFR-TK
activity and antiproliferative activity. Furthermore, the EGFR molecular docking
model suggested that the most potent compound 5e is nicely bound to the region of
EGFR, and the nitrogen atom of amine group of 5e forms hydrogen bond with
Asp831, which made the combination ability of 5e acting on EGFR. Cell morphology
by Hoechst stain experiment suggested that the comp1 efficiently induced apoptosis
of A549 cells
In conclusion, these findings expand the chemical diversity of EGFR inhibitors,
and similar strategies might be applied to the design of compounds able to form a
covalent bond with a biological target.
4. Experimental section
4.1. General chemistry
All chemicals (reagent grade) used were purchased from Sigma–Aldrich (USA) and
1
Sinopharm Chemical Reagent Co. Ltd. (China). H NMR spectra were measured on
6

Varian Unity Inova 300/400 MHz NMR Spectrometer at 25 °C and referenced to
TMS. Chemical shifts are reported in ppm (d) using the residual solvent line as
internal standard. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m,
multiplet. HRMS spectra were acquired on Bruker Esquire Liquid
Chromatography-Ion Trap Mass Spectrometer. Analytical thin-layer chromatography
(TLC) was performed on the glass-backed silica gel sheets (silica gel 60 Å GF254).
All compounds were detected using UV light (254 or 365 nm). Analytical HPLC was
conducted on SHIMADZU LC-20AD. Hoechst 33258 was bought from Beyotime
(China). Annexin V-FITC Apoptosis Detection Kit was purchased from BD
Pharmingen (US).
4.2 General procedure for the preparation of compounds.
5-Nitroanthranilonitrile 1 (30 mmol, 1.0 equiv) was suspended in dimethylformamide
dimethyl acetal (10 mL) and the mixture was refluxed for 2h. The resulting mixture
was cooled to room temperature for 2–3 h. The yellow precipitate that formed was
filtered, washed with ethyl ether, and dried to give 2 (Yield: 80–90%).
A mixture of 2 (10 mmol, 1.0 equiv) and 3-chloroaniline (1.1 equiv) was heated and
stirred at reflux in acetic acid (20 mL) for 2h. Aniline and acetic acid would be added
into the reaction flask first and then slowly added 2 using the medicine spoon within
one minute. This treatment is to prevent the formation of lumps between them. The
yellow precipitate that formed was filtered hot, washed with hot acetic acid, diethyl
ether, and dried to give the desired nitroquinazoline 3 (Yield: 80–90%).
6-Nitroquinazoline 3 (6.65 mmol, 1.0 equiv), iron (45 mmol, 6.7 equiv) and acetic
acid (6 mL, 90 mmol, 13.5 equiv)were suspended in aqueous ethanol (180 mL, 77.8%
v/v) and heated at reflux about 70–80oC for 5–6 h. The yellow solution would become
reddish-brown slowly.The reaction mixture was cooled to room temperature and
alkalinized by addition of concentrated ammonia (40 mL). Insoluble material was
removed by filtration through celite, and the filtrate was evaporated under reduced
pressure. The resulting solid was extracted with EtOAc for column chromatography.
Column chromatography was performed using silica gel (200–300 mesh) eluting with
DCM and MeOH to give amine 4.
7

Amine 4 (1.2mmol, 1.2equiv),various aldehydes （1mmol, 1.0equiv）and sodium
triacetoxyborohydride (3mmol, 3.0equiv) were suspended in DCE (3ml) , stirred
under a nitrogen atmosphere at room temperature for 12h. The reaction mixture was
quenched by adding aqueous saturated NaHCO3, and the product was extracted with
EtOAc. The EtOAc extract was dried (Na2SO4), Products were purified by
chromatography (petroleum ether /EtOAc) to obtain product 5a-i.
N4-(3-chlorophenyl)-N6-(pyridin-3-ylmethyl)quinazoline-4,6-diamine(5a).
Mp
236-237°C. 1H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.69 (s, 1H), 8.47 (d, J =
4.8 Hz, 1H), 8.39 (s, 1H), 8.05 (s, 1H), 7.83 (dd, J = 15.0, 8.1 Hz, 2H), 7.57 (d, J =
8.8 Hz, 1H), 7.43 – 7.32 (m, 5H), 7.13 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 4.48 (d, J =
5.7 Hz, 2H). HR-MS: Calcd. For: 362.1094 (C20H16ClN5, [M+H]+) , Found: 362.1108.
N6-((6-bromopyridin-2-yl)methyl)-N4-(3-chlorophenyl)quinazoline-4,6-diamine(5b).
Mp 180-181°C. 1H NMR (300 MHz, DMSO-d6) δ 9.36 (s, 1H), 8.39 (d, J = 7.5 Hz,
1H), 7.98 (s, 1H), 7.72 (dd, J = 17.3, 8.6 Hz, 2H), 7.55 (dd, J = 19.9, 8.5 Hz, 2H),
7.38 (dd, J = 24.8, 15.8 Hz, 4H), 7.13 (s, 1H), 6.92 (d, J = 6.4 Hz, 1H), 4.58 (d, J =
6.1 Hz, 2H). HR-MS: Calcd. For: 440.0199 (C20H15BrClN5, [M+H]+) , Found:
440.0182.
N4-(3-chlorophenyl)-N6-(quinolin-2-ylmethyl)quinazoline-4,6-diamine(5c). Mp
229-230°C. 1H NMR (300 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.37 (dd, J = 16.1, 6.6
Hz, 2H), 8.10 – 7.89 (m, 3H), 7.76 (t, J = 8.8 Hz, 2H), 7.67 – 7.52 (m, 3H), 7.51 –
7.31 (m, 3H), 7.11 (d, J = 6.0 Hz, 1H), 6.98 (s, 1H), 4.78 (d, J = 5.6 Hz, 2H). HR-MS:
Calcd. For: 412.1251 (C24H18ClN5, [M+H]+) , Found: 412.1237.
N4-(3-chlorophenyl)-N6-(thiophen-2-ylmethyl)quinazoline-4,6-diamine(5d). Mp
209-210°C. 1H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.40 (s, 1H), 8.07 (s,
1H), 7.83 (d, J = 7.9 Hz, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.38 (dd, J = 24.5, 7.3 Hz, 4H),
7.21 – 7.09 (m, 2H), 7.00 (d, J = 4.1 Hz, 1H), 6.80 (s, 1H), 4.64 (s, 2H). HR-MS:
Calcd. For: 367.0706 (C19H15ClN4S, [M+H]+) , Found: 367.0725.
N6-((5-bromothiophen-2-yl)methyl)-N4-(3-chlorophenyl)quinazoline-4,6-diamine(5e
). Mp 193-194°C 1H NMR (300 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.42 (s, 1H), 8.08 (s,
1H), 7.83 (s, 1H), 7.58 (s, 1H), 7.39 (s, 3H), 7.07 (d, J = 19.1 Hz, 3H), 6.87 (s, 1H),
4.61 (s, 2H). HR-MS: Calcd. For: 444.9811 (C19H14BrClN4S, [M+H]+) , Found:
444.9827.
N6-((1H-indol-3-yl)methyl)-N4-(3-chlorophenyl)quinazoline-4,6-diamine(5f). Mp
Mp 159-160°C. 1H NMR (400 MHz, CD3OD) δ 8.35 (s, 1H), 7.97 (s, 1H), 7.68 (dd, J
= 14.5, 8.0 Hz, 2H), 7.57 (d, J = 9.6 Hz, 1H), 7.35 (dd, J = 16.4, 7.2 Hz, 5H), 7.12 (t,
J = 7.9 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 4.62 (s, 2H). HR-MS: Calcd. For: 340.1251
(C23H18ClN5, [M+H]+) , Found: 340.1274.
N4-(3-chlorophenyl)-N6-((6-methylpyridin-2-yl)methyl)quinazoline-4,6-diamine(5g).
Mp 221-222°C. 1H NMR (300 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.37 (s, 1H), 8.00
(s, 1H), 7.78 (d, J = 6.7 Hz, 1H), 7.67 – 7.51 (m, 2H), 7.43 – 7.27 (m, 3H), 7.19 (d, J
8

= 7.0 Hz, 1H), 7.12 (s, 2H), 6.77 (s, 1H), 4.53 (s, 2H). HR-MS: Calcd. For: 376.1251
(C21H18ClN5, [M+H]+) , Found: 376.1242.
N4-(3-chlorophenyl)-N6-(3-(methylthio)propyl)quinazoline-4,6-diamine(5h). Mp
161-162°C. 1H NMR (300 MHz, acetone) δ 8.92 (s, 1H), 8.47 (s, 1H), 8.18 (s, 1H),
7.80 (d, J = 7.9 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.35 (t, J = 8.6 Hz, 2H), 7.21 (s,
1H), 7.09 (d, J = 7.3 Hz, 1H), 5.62 (s, 1H), 3.32 (d, J = 6.5 Hz, 2H), 2.93 (s, 3H), 2.65
(t, J = 7.2 Hz, 2H). HR-MS: Calcd. For: 359.1019 (C18H19ClN4S, [M+H]+) , Found:
359.1032.
N6-butyl-N4-(3-chlorophenyl)quinazoline-4,6-diamine(5i).
Mp 181-182°C, 1H
NMR (400 MHz, CD3OD) δ 8.32 (s, 1H), 7.94 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.53
(d, J = 9.0 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 9.0 Hz, 1H), 7.13 (d, J = 7.2
Hz, 2H), 3.24 (t, J = 7.0 Hz, 2H), 1.71 (dt, J = 14.4, 7.1 Hz, 2H), 1.58 – 1.46 (m, 2H),
1.02 (t, J = 7.3 Hz, 3H). HR-MS: Calcd. For: 327.1298 (C18H19ClN4, [M+H]+) ,
Found: 327.1272.
Preparation of various aldehyde acids 7a-h
The various aldehyde acids were synthesized using Doebner Knoevenagel
modification. Aldehyde acids were prepared by mixing aldehyde(3.2mmol, 1.0equiv),
malonic acid(4.8mmol, 1.5equiv), pyridine(20ml), and piperidine(40μl) together and
stirring 80oC for 24h. The reaction mixture was evaporated under reduced pressure.
Products were purified by chromatography (petroleum ether /EtOAc) to obtain
product 7a-h.
General synthesis method of aldehyde acid amide analogues 8a-h
Amine 4 (0.2mmol, 1.0equiv) and aldehyde acids(0.24mmol, 1.2equiv) were
dissolved to pyriding(2ml), EDCI(0.4mmol, 2.0equiv) was added to the resulting
solution and stired at room temperature for 6h. The resulting mixture was diluted with
EtOAc and washed with brine and water repeatedly. The EtOAc extract was dried
(Na2SO4) and evaporated under reduced pressure. Products were chromatography
(petroleum ether / EtOAc and DCM / MeOH) to obtain product 8a-h.
(E)-N-(8-((3-chlorophenyl)amino)naphthalen-2-yl)-3-(pyridin-3-yl)acrylamide(8a).
Mp 295-296°C. 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.93 (s, 1H), 8.81 (s,
2H), 8.56 (s, 2H), 8.03 (s, 2H), 7.91 (s, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 15.2
Hz, 1H), 7.41 (d, J = 34.6 Hz, 2H), 7.13 (s, 1H), 6.99 (d, J = 15.7 Hz, 1H). HR-MS:
Calcd. For: 399.1138 (C24H18ClN3O, [M+H]+) , Found:399.1165.
9

(E)-3-(6-bromopyridin-2-yl)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)acrylami
de(8b). Mp 288-289°C. 1H NMR (300 MHz, DMSO-d6) δ 10.85 (s, 1H), 10.01 (s,
1H), 8.90 (s, 1H), 8.60 (s, 1H), 8.07 (s, 1H), 7.93 (s, 2H), 7.84 (s, 3H), 7.70 (d, J =
13.8 Hz, 3H), 7.42 (s, 2H), 7.17 (s, 1H). HR-MS: Calcd. For: 480.0149
(C22H15BrClN5O, [M+H]+) , Found: 480.0162.
(E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-3-(quinolin-2-yl)acrylamide(8c).
Mp 297-298°C. 1H NMR (300 MHz, DMSO-d6) δ 10.86 (s, 1H), 10.02 (s, 1H), 8.95
(s, 1H), 8.61 (d, J = 5.3 Hz, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.10 – 7.92 (m, 5H), 7.88
(dd, J = 13.2, 5.9 Hz, 6H), 7.68 (d, J = 7.0 Hz, 2H), 7.56 (d, J = 15.5 Hz, 1H), 7.43 (t,
J = 8.2 Hz, 1H), 7.17 (s, 1H). HR-MS: Calcd. For: 452.1200 (C26H18ClN5O, [M+H]+) ,
Found: 452.1208.
(E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-3-(thiophen-2-yl)acrylamide(8d).
1
Mp 285-286°C. H NMR (300 MHz, DMSO-d6) δ 10.67 (s, 1H), 10.01 (s, 1H), 8.86
(s, 1H), 8.58 (s, 1H), 8.07 (s, 1H), 7.93 (s, 1H), 7.80 (s, 2H), 7.69 (s, 1H), 7.45 (d, J =
25.7 Hz, 2H), 7.17 (s, 2H), 6.68 (d, J = 16.1 Hz, 1H). HR-MS: Calcd. For: 407.0655
(C21H15ClN4OS, [M+H]+) , Found: 407.0648.
(E)-3-(5-bromothiophen-2-yl)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)acryla
1
mide(8e).
Mp 250-251°C. H NMR (300 MHz, DMSO-d6) δ 11.22 – 10.99 (m,
1H), 10.90 (s, 1H), 9.03 (s, 1H), 8.82 (s, 1H), 7.98 (d, J = 35.0 Hz, 3H), 7.72 (s, 2H),
7.48 (s, 1H), 7.32 (s, 3H), 6.63 (s, 1H). HR-MS: Calcd. For: 484.9760
(C21H14BrClN4OS, [M+H]+) , Found: 484.9771.
(E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-3-(1H-indol-3-yl)acrylamide(8f).
1
Mp 241-242°C. H NMR (300 MHz, DMSO-d6) δ 11.69 (s, 1H), 10.41 – 10.21 (m,
1H), 9.91 (d, J = 17.2 Hz, 1H), 8.83 (s, 1H), 8.58 (dd, J = 20.6, 15.9 Hz, 1H), 8.10 –
7.67 (m, 7H), 7.41 (dd, J = 16.7, 12.2 Hz, 2H), 7.27 – 7.06 (m, 3H), 6.89 (dd, J = 15.9,
5.1 Hz, 1H). HR-MS: Calcd. For: 440.1200 (C25H18ClN5O, [M+H]+) , Found:
440.1213.
(E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-3-(6-methylpyridin-2-yl)acrylami
de(8g). Mp 297-298°C. 1H NMR (300 MHz, DMSO-d6) δ 10.74 (s, 1H), 9.97 (s, 1H),
8.90 (s, 1H), 8.58 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.78 (dd, J = 18.4, 8.3 Hz, 3H),
10

7.64 (d, J = 15.2 Hz, 1H), 7.49 – 7.33 (m, 3H), 7.28 (d, J = 7.2 Hz, 1H), 7.15 (d, J =
7.9 Hz, 1H), 2.53 (s, 3H). HR-MS: Calcd. For: 416.1200 (C23H18ClN5O, [M+H]+) ,
Found: 416.1185.
(E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-3-(2-chloropyridin-3-yl)acrylami
1
de(8h). Mp 259-260°C. H NMR (300 MHz, DMSO-d6) δ 10.78 (s, 1H), 9.98 (s,
1H), 8.90 (s, 1H), 8.60 (s, 2H), 8.21 (s, 1H), 7.85 (s, 5H), 7.41 (s, 2H), 7.13 (s, 2H).
HR-MS: Calcd. For: 436.0654 (C22H15Cl2N5O, [M+H]+), Found: 436.0672.
4.3 Cell proliferation assay
The antiproliferative activities of chalcone thiosemicarbazide derivatives were
determined using a standard (MTT)-based colorimetric assay (Sigma). Briefly, cell
lines were seeded at a density of 7×103 cells/well in 96-well microtiter plates (Costar).
After 24 h, exponentially growing cells were exposed to the indicated compounds at
final concentrations ranging from 0.1 to 40 mg/mL. After 48 h, cell survival was
determined by the addition of an MTT solution (20 µL of 5 mg/mL MTT in PBS).
After 6 h, 100 mL of 10% SDS in 0.01 N HCl was added, and the plates were
incubated at 37 °C for a further 4 h; optical absorbance was measured at 570 nm on an
LX300 Epson Diagnostic microplate reader. Survival ratios are expressed in
percentages with respect to untreated cells. IC50 values were determined from
replicates of 6 wells from at least two independent experiments.
4.4. General procedure for preparation, purification of EGFR inhibitory assay
1.6 kb cDNA encoded for the EGFR cytoplasmic domain (EGFR-CD, amino acids
645-1186) were cloned into baculoviral expression vectors pBlueBacHis2B and
pFASTBacHTc, separately. A sequence that encodes (His)6 was located at the 5’
upstream to the EGFR sequence. Sf-9 cells were infected for 3 days for protein
expression. Sf-9 cell pellets were solubilized at 0 °C in a buffer at pH 7.4 containing
50 mM HEPES, 10 mM NaCl, 1% Triton, 10 íM ammonium molybdate, 100 µM
sodium vanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and
16 µg/ mL benzamidine HCl for 20 min followed by 20 min centrifugation. Crude
extract supernatant was passed through an equilibrated Ni-NTA superflow packed
column and washed with 10 mM and then 100 mM imidazole to remove
11

nonspecifically bound material. Histidinetagged proteins were eluted with 250 and
500 mM imidazole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glycerol,
and 1 µg/mL each of aprotinin, leupeptin, and pepstatin for 2 h. The entire purification
procedure was performed at 4 °C or on ice.20
EGFR kinase assay was set up to assess the level of autophosphorylation based on
DELFIA/Time-Resolved Fluorometry. Compounds were dissolved in 100% DMSO
and diluted to the appropriate concentrations with 25 mM HEPES at pH 7.4. In each
well, 10 íL of compound was incubated with 10 µL (5 ng for EGFR) of recombinant
enzyme (1:80 dilution in 100 mM HEPES) for 10 min at room temperature. Then, 10
µL of 5 × buffer (containing 20 mM HEPES, 2 mM MnCl2, 100 µM Na3VO4, and 1
mM DTT) and 20 µL of 0.1 mM ATP-50 mM MgCl2 was added for 1 h. Positive and
negative controls were included in each plate by incubation of enzyme with or
without ATP-MgCl2. At the end of incubation, liquid was aspirated, and plates were
washed three times with wash buffer. A 75 µL (400 ng) sample of europiumlabeled
anti-phosphotyrosine antibody was added to each well for another 1 h of incubation.
After washing, enhancement solution was added and the signal was detected by
Victor (Wallac Inc.) with excitation at 340 nm and emission at 615 nm. The
percentage of autophosphorylation inhibition by the compounds was calculated using
the following equation: 100% - [(negative control)/(positive control- negative
control)]. The IC50 was obtained from curves of percentage inhibition with eight
concentrations of compound. As the contaminants in the enzyme preparation are fairly
low, the majority of the signal detected by the anti-phosphotyrosine antibody is from
EGFR.
4.5. Docking Study
The three-dimensional structures of the aforementioned compounds were constructed
using Chem. 3D ultra 12.0 software [Chemical Structure Drawing Standard;
Cambridge Soft corporation, USA (2010)], then they were energetically minimized by
using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal
structures of EGFR kinase (PDB code: (1M17.pdb) complex were retrieved from the
RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters
12

and ligands were eliminated from the protein and the polar hydrogen was added to the
proteins. Molecular docking of all twenty compounds was then carried out using the
Discovery Stutio (version 3.1) as implemented through the graphical user interface
CDocker protocol.
4.6. Hoechst 33258 staining
A549 cells were seeded into 24-well culture plates and treated with 5-20 μM
compound 5e for 24 h. Cells were washed twice with PBS, and fixed in
methanol/acetic acid (3:1, v/v) for 10 min at 4 °C. The fixative was removed and cells
were washed twice with PBS. Then cells were stained with Hoechst 33258 (5 μg /ml)
for 5 min at room temperature and then examined under a fluorescent microscope
(Olympus).
5. Acknowledgements
The work was supported by National Natural Science Foundation of China (Grant
No.81102316); the Natural Science Foundation of the Jiangsu Higher Education
Institutions of China (Grant No.11KJD350002) and PAPD (A Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions).
References
1. Yarden, Y.; Sliwknowski, M. X. Untangling the ErbB signaling network. Nat. Rev.
Mol. Cell. Biol. 2001, 2, 127−137.
2. Hynes, N. E.; Lane, H. A. ErbB receptors and cancer: the complexity of targeted
inhibitors. Nat. Rev. Cancer 2005, 5, 341−354.
3. Traxler, P. M. Protein tyrosine kinase inhibitors in cancer treatment (Part I). Exp.
Opin. Ther. Pat. 1997, 7, 571-588.
4. Exp. Opin. Ther. Pat. 1998, 8, 1599-1625.
5. Fry, D.W.; Kraker, A. J.; McMichael, A.; Ambroso, L. A.; Nelson, J. M.; Leopold,
W. R.; Connors, R. W.; Bridges, A. J. A specific inhibitor of the epidermal growth
factor receptor tyrosine kinase. Science. 1994, 265, 1093-1095.
6. Ward, W. H. J.; Cook, P. N.; Slater, A. M.; Davies, D. H.; Holdgate, G. A.; Green,
L. R. Epidermal growth factor receptor tyrosine kinase. Investigation of catalytic
mechanism, structure-based searching and discovery of a potent inhibitor.
13

Biochem. Pharmacol. 1994, 48, 659-666.
7. Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G. W.; McMichael, A.;
Showalter, H. D. H.; Fry, D. W.; Kraker, A. J.; Denny, W. A. Tyrosine kinase
inhibitors. 8. An unusually steep structure activity relationship for analogues of
4-(3-bromo-anilino)-6,7-dimethoxyquinazoline (PD 153035), a potent inhibitor of
the epidermal growth factor receptor. J. Med. Chem. 1996, 39, 267-276.
8. Iwata, K.; Miller, P. E.; Barbacci, E. G.; Arnold, L.; Doty, J.; DiOrio, C. I.;
Pustilnik, L. R.; Reynolds, M.; Thelemann, A.; Sloan, D.; Moyer, J. D. CP-358,
774: a selective EGFR kinase inhibitor with potent antiproliferative activity
against HN5 head and neck tumor cells. Proc. Am. Assoc. Cancer Res. 1997, 38,
633 (Abstract 4248).
9. Woodburn, J. R.; Barker, A. J.; Gibson, K. H.; Ashton, S. E.; Wakeling, A. E.;
Curry, B. J.; Scarlett, L.; Henthorn, L. R. ZD1839, an epidermal growth factor
tyrosine kinase inhibitor selected for clinical development. Proc. Am. Assoc.
Cancer Res. 1997, 38, 633 (Abstract 4251).
10. Company Press Release, Feb 1, 2010: GSK’s TYKERB receives accelerated
approval for first-line combination treatment of hormone receptor positive,
HER2+/ErbB2+ metastatic breast cancer.
11. Pao, W.; Miller, V.; Zakowski, M.; Doherty, J.; Politi, K.; Sarkaria, I.; Singh, B.;
Heelan, R.; Rusch, V.; Fulton, L.; Mardis, E.; Kupfer, D.; Wilson, R.; Kris, M.;
Varmus, H.EGFreceptor gene mutations are common in lung cancers from “never
smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 13306–13311.
12. Rewcastle, G. W.; Denny,W.A.; Bridges, A. J.; Zhou, H. R.; Cody, D. R.;
McMichael, A.; Fry, D. W. Tyrosine kinase inhibitors. 5. Synthesis and
structure-activity
relationships
for
4-[(phenylmethyl)
amino]-
and
4-(phenylamino)quinazolines as potent adenosine 50-triphosphate binding site
inhibitors of the tyrosine kinase domain of the epidermal growth factor receptor. J.
Med. Chem. 1995, 38, 3482–3487.
13. Li, H. Q.; Li, D. D.; Lu, X.; Zhu. H. L. Design and synthesis of 4,6-substituted
14

(diaphenylamino)quinazolines as potent EGFR inhibitors with antitumor activity.
Bioorg. Med. Chem. 2012, 20, 317-323.
14. Teman, S.; Kawaguchi, H.; El-Naggar, A. K.; Jelinek, J.; Tang, H.; Liu, D. D.;
Lang, W.; Issa, J. P.; Lee, J. J.; Mao, L. Epidermal growth factor receptor copy
number alterations correlate with poor clinical outcome in patients with head and
neck squamous cancer. J. Clin. Oncol. 2007, 25, 2164−2170
15. Rewcastle, G. W.; Denny, W. A.; Bridges, A. J.; Zhou, H.; Cody, D. R.;
McMichael, A.; Fry, D. W. Tyrosine kinase inhibitors: synthesis and
structure-activity
relationships
for
4-[(phenylmethyl)amino]-
and
4-[(phenylamino)quinazolines as potent adenosine 5′-triphosphate binding site
inhibitors of the tyrosine kinase domain of the epidermal growth factor receptor. J.
Med. Chem. 1995, 38, 3482−3487.
16. Roth, G. A.; Tai, J. J. A new synthesis of aryl substituted quinazolin-4(1H)-ones. J.
Heterocycl. Chem. 1996, 33, 2051−2053.
17. Jeffrey, D. S.; Christopher, G. V. S.; Caldwell, W. S. Molecular Recognition in
Nicotinic Acetylcholine Receptors: The Importance of π−Cation Interactions. J.
Med. Chem. 1999, 42, 3066
Scheme 1 Chemical structures of Gefitinb, Erlotinib and Lapatinib
F
HN
Cl
HN
C
O
C
N
O
H
O
O
N
N
O
N
O
N
O
F
O
S
O
O
NH
O
HN
Cl
N
N
15

Scheme 2 Synthesis of 6-amino-substituted 4-anilinoquinazolines 5a-5i a
2
2
2
1
2
2
2
4
3
N
N
Br
R:
a=
d=
g=
b=
e=
h=
c=
f=
i=
N
N
H
N
Br
S
S
S
H C
3
N
5a-i
a
o
o
o
2
2
Scheme 3. Synthesis of 6-acrylamide-substituted-4-anilinoquinazolines 8a-8h a
16

Table 1 In vitro antiproliferative effects of synthesized compounds.a
IC50（µM）
IC50（µM）
Compound A549
cellsa
Hep G2
cells
Compound
A549
cellsa
Hep G2
cells
5a
5b
5c
5d
5e
5f
5g
5h
5i
2.67 0.27
4.14 0.52 8a
3.87 0.32 8b
5.65 0.74 8c
3.58 0.19 8d
3.11 0.41 8e
5.26 1.02 8f
3.63 0.23 8g
8.92 1.32 8h
4.32 0.12 9.12 2.12
4.16 0.32 10.7 1.8
3.35 0.24 7.79 0.87
9.31 1.02 10.4 2.5
1.58 0.12 8.85 1.64
3.67 0.38 7.02 1.52
2.16 0.62 4.82 0.65
2.92 0.16 4.42 0.83
0.13 0.02 0.12 0.01
2.95 0.28
1.71 0.10
2.08 0.16
0.82 0.02
2.33 0.16
1.06 0.04
3.82 0.14
4.01 0.15
7.85 1.42 Erlotinib
aCell growth was measured using the MTT assay. Values are the average of two
independent experiments run in triplicate. Variation was generally 1%.
Table 2 Inhibition (IC50 ) of EGFR kinase
compound
EGFR Inhibition
IC50 ( µM )
4.1 1.2
5b
5d
3.4 1.5
5e
1.5 0.7
5h
5.1 2.4
8a
4.7 1.6
8d
8.8 3.1
8e
2.4 1.1
8h
3.6 0.8
Erlotinib
0.03 0.01
17

Figure 1A. Ligand interaction diagram of compound 5e with EGFR protein (PDB ID:
1M17) using Discovery Studio program with the essential amino acid residues at the
binding site are tagged in circles. The purple circles show the amino acids which
participate in hydrogen bonding, electrostatic or polar interactions and the green
circles show the amino acids which participate in the Van der Waals interactions.
Figure 1B. Docking of compound 5e interacting with EGFR kinase and 3D solid
binding model compound 5e.
18

Figure. 2. Morphology of A549 cells after Hoechst 33258 staining. (A) Control (cells
treated with 0.1% DMSO alone); (B-D) treated with 5, 10, 20 μM of comp1 for 24 h.
The cells were stained with Hoechst 33258. The photographs were taken under UV
light microscope (Olympus, 400 × objective lens).
19

Discovery of Novel 4-anilinoquinazoline Derivatives
as Potent Inhibitors of Epidermal Growth Factor
Receptor with Antitumor Activity
Yun-Yun Xu1, Si-Ning Li1, Gao-Jian Yu1, Qing-Hua Hu2*, Huan-Qiu Li1*.
Two new series of new compounds containing a 6-amino-substituted group or
6-acrylamide-substituted group linked to a 4-anilinoquinazoline nucleus have been
discovered as potential EGFR inhibitors. These compounds proved efficient effects on
antiproliferative activity and EGFR TK inhibitory activity. Especially,
N6-((5-bromothiophen-2-yl)methyl)-N4-(3-chlorophenyl)quinazoline-4,6-
diamine
(5e), showed the most potent inhibitory activity (IC50=3.11 μM for Hep G2, IC50=0.82
μM for A549). The EGFR molecular docking model suggested that the new
compound is nicely bound to the region of EGFR, and cell morphology by Hoechst
stain experiment suggested that the comp1 efficiently induced apoptosis of A549 cells
20