The combination of 4-anilinoquinazoline and cinnamic acid: A novel mode of binding to the epidermal growth factor receptor tyrosine kinase

Article abstract of DOI:10.1016/j.bmc.2011.06.044

A novel type of cinnamic acid quinazoline amide derivatives (20-42), which designed the combination between quinazoline as the backbone and various substituted cinnamic acid as the side chain, have been synthesized and their biological activities were evaluated within cytotoxicity assay firstly and then potent EGFR inhibitory activity. Compound 42 demonstrated the most potent inhibitory activity (IC₅₀ = 0.94 μM for EGFR), which could be optimized as a potential EGFR inhibitor in the further study. Docking simulation was performed to position compound 42 into the EGFR active site to determine the probable binding model. Analysis of the binding conformation of 42 in active site displayed compound 42 was stabilized by hydrogen bonding interactions with Lys822, which was different from other derivatives. In the further study, Compounds 43 and 44 had been synthesized and their biological activities were also evaluated, which were the same as that we expected. Compound 43 has demonstrated significant EGFR (IC₅₀ = 0.12 μM) and tumor growth inhibitory activity as a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2011.06.044

Bioorganic & Medicinal Chemistry 19 (2011) 5012–5022
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The combination of 4-anilinoquinazoline and cinnamic acid: A novel mode
of binding to the epidermal growth factor receptor tyrosine kinase
Dong-Dong Li a, , Peng-Cheng Lv a, , Hui Zhang a, Hong-Jia Zhang a, Ya-Ping Hou a, Kai Liu a, Yong-Hao Ye b,
,
⇑
Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b College of Plant Protection, Jiangsu Key Laboratory of Pesticide Science, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel type of cinnamic acid quinazoline amide derivatives (20–42), which designed the combination
between quinazoline as the backbone and various substituted cinnamic acid as the side chain, have been
synthesized and their biological activities were evaluated within cytotoxicity assay firstly and then
potent EGFR inhibitory activity. Compound 42 demonstrated the most potent inhibitory activity
Received 20 April 2011
Revised 13 June 2011
Accepted 15 June 2011
Available online 21 June 2011
(IC50 = 0.94 lM for EGFR), which could be optimized as a potential EGFR inhibitor in the further study.
Docking simulation was performed to position compound 42 into the EGFR active site to determine
the probable binding model. Analysis of the binding conformation of 42 in active site displayed com-
pound 42 was stabilized by hydrogen bonding interactions with Lys822, which was different from other
derivatives. In the further study, Compounds 43 and 44 had been synthesized and their biological activ-
ities were also evaluated, which were the same as that we expected. Compound 43 has demonstrated sig-
Keywords:
Cinnamic acid
EGFR
Anilinoquinazoline
Antiproliferative
nificant EGFR (IC50 = 0.12
lM) and tumor growth inhibitory activity as a potential anticancer agent.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Substituted cinnamic acids are widely distributed in the plant
kingdom and are found in coffee beans, olives, propolis, fruits, and
vegetables. They are found usually as simple derivatives including
amide, esters, sugar esters, and glycosides.13 Hydroxycinnamic acid
esters are widely distributed in the plant kingdom and are reported
as cellular antioxidants, anti-inflammatory agents, or inhibitors of
enzymes involved in cell proliferation, and some biological activi-
ties.14–16 Antitumor activities of various cinnamic acid derivatives
were also explored by many research groups. Particularly, cinnamic
acid ester derivatives showed the potential antitumor activity.17–19
It was reported that compound 3h demonstrated the most potent
The altered protein expression and activity of receptor tyrosine
kinases (TK) are implicated in the progression of various types of
cancers. One such dysfunction is the overexpression of the epider-
mal growth factor receptor (EGFR) and its closest homologue HER2
(erbB2) that correlates with aggressive tumor progression and poor
prognosis.1 Unlike more standard chemotherapy agents, which de-
stroy both cancer cells and healthy cells, EGFR inhibitor targeted
cancer treatment primarily attach to and destroy cancer cells. Be-
cause of this, EGFR inhibitor therapies have fewer broad-range side
effects such as the nausea and vomiting, hair loss, and anemia
sometimes associated with more traditional chemotherapy. Selec-
tive inhibitors of tyrosine phosphorylation by EGFR have become
an important class of potential anticancer drugs.2–4 Several classes
of inhibitors have been reported with the most potent and selec-
tive being the 4-(phenylamino)quinazolines.5–8 Kinetic studies6,9
show that these compounds selectively inhibit EGF-stimulated sig-
nal transduction by reversibly binding at the ATP site of EGFR. As
we all know, three 4-(phenylamino)quinazolines, Gefitinib10 (CP
358774), Erlotinib (ZD 1839)11 and Lapatinib (GW572016)12 have
been approved by FDA in these years (Fig. 1).
inhibitory activity (IC50 = 0.62 lM for EGFR and IC50 = 2.15 lM for
HER-2), which was compared with the positive control Erlotinib.20
It is an increasing interest that combi-molecules are agents de-
signed to block multiple targets in cancer cells due to complex for-
mation mechanism of tumor. One common strategy is to make an
ester or amide as a prodrug by the combination of two structural
scaffold pointed to different target spot. Our initial modeling studies
of the binding of reversible anilinoquinazoline suggested a binding
mode whereby the 6- and 7-positions of the bicyclic chromophore
point toward the solvent out of the ATP binding pocket. This explains
why both positions can be used to add bulky solubilizing functional-
ities to inhibitors without losing binding affinity.10,11,21,22 In this
study, aminoquinazolines that binds to the ATP site of EGFR and cin-
namic acid derivatives, which also have anti-cancer activity re-
ported, would be assembled firstly and the latter have been
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to this work.
0968-0896/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.044

D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
5013
F
O
HN
Cl
HN
C
C
N
O
O
O
O
H
N
O
O
N
N
N
F
Gefitinib : CP 358774
Erlotinib : ZD 1839
O
S
O
O
NH
O
HN
Cl
NO2
N
O
N
N
O
N
Lapatinib : GW572016
Compound 3h
Figure 1. The structure of Gefitinib, Erlotinib, Lapatinib and compound 3h.
grafted to the 6- and 7-positions of the bicyclic chromophore. The
question whether these new molecules could keep significant EGFR
inhibitory activity or just like combi-molecules might degrade two
pharmacophores applied to different targets and get an exciting out-
come, may be unclear. We postulates that a molecule termed ‘com-
binatorial molecule’ designed to interact with the epidermal growth
factor receptor (EGFR) on its own and the activity of the two together
would perform better. We described the synthesis and the SAR of the
novel series of cinnamic acid quinazoline amide derivatives, and the
biological activity evaluation indicated that some of these com-
pounds are potent inhibitorsof EGFR. Dockingsimulations were per-
formed using the X-ray crystallographic structure of the EGFR in
complex with an inhibitor to explore the binding modes of these
compounds at the active site.
2.2. Biological activity
2.2.1. Antiproliferation assay
The structures of the quinazoline amides studied are listed in
Table 1, together with their potencies for inhibition of A549 cells
and F10 cells by applying the MTT colorimetric assay. Compounds
were tested over a range of concentrations from 0.01 to 100
and the calculated IC50 values, that is, the concentration ( g/mL) of
a compound that was able to cause 50% growth inhibition with re-
spect to the control culture, were reported differently according to
different cancer cells. Obviously, all the compounds remarked anti-
proliferative activities, and generally the results showed which
those applied to F10 cells would performed better than that in
A549 cells.
lg/ml,
l
Anilinoquinazoline derivatives as a lead compound have been
studying in these years and there were some empirical rules con-
cluded: (1) the N1, N3 of quinazoline would interact with active
center probably by hydrogen bond, water bridge or else. So we
saved the quinazoline skeleton. (2) The meta-substituent of the
aniline group is oriented toward the kinase pocket. This mode of
binding is in line with the structure–activity relationship observed
in the quinazoline inhibition series. Small substituents are toler-
ated at the 30-aniline moiety, preferentially halogens and elec-
tron-donating group at the 6- and/or 7-positions that enhance
the binding of N1 and N3. Although the aniline NH does not form
an H-bond, its replacement with a methyl group is deleterious
for activity.23 In this study, we choose halogen atom (Br and Cl)
at the 3’-aniline moiety and generally the former has greater po-
tency than the latter in some research reported.23 From 20–31 to
32–42, there are no obvious difference between Br and Cl, 20 com-
pared to 32, 21 compared to 33, and so on. The reason for this is
probably due to the cinnamic acid structure. As for the compounds
with one substituent on the cinnamic acid ring, we found a
straightforward correlation between the electronic property and
cytotoxicity, generally the more electron-donating group at the
R3 position would increase the cytotoxicity except 31 and 42. This
was supported by two groups (compounds 22, 24, 26, and com-
pounds 34, 36). Also there might be other effects at the R3 position.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 5 and 6 proceeded in the same
way according to Scheme 1, 2-amino-5-nitrobenzonitrile 1 was
treated in commercially available dimethylformamide dimethyl
acetal to give 2 (Scheme 1). Compound 2 was added in acetic acid
containing 3-bromobenzenamine or 3-chlorobenzenamine to give
3 or 4, respectively. Reduction of these nitro compounds with Fe
gave the targeted amines 5 and 6 in the next step. In parallel, the
substituted cinnamic acids 7–19 were synthesized according to
Scheme 2 and these cinnamic acids were converted to acid chlo-
rides before reacted with 5 or 6 in Scheme 3. Treatment of amine
with acyl chloride from 7–19 in EtOAc at ice-bath gave the re-
quired amides (Table 1). The latter compounds were purified by
column chromatography on silica gel with a mixture of ethyl ace-
tate/petroleum ether as eluent. Compounds 43 and 44 were also
synthesized according to Scheme 4 in the further study. All of
the synthetic compounds gave satisfactory analytical and spectro-
scopic data, which were in full accordance with their depicted
structures.

5014
D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
O2N
CN
O
O
O2
N
CN
i
R
H
R
OH
CH3
NH2
N
N
7-19
2
1
CH3
7,R=
8,R=
12,R=
13,R=
ii
OCH3
17,R=
HN
X
HN
X
Cl
H3CO
O2
N
H2N
N
N
iii
OCH3
9,R=
18,R=
14,R=
NO2
N
N
Cl
3
4
X=Br
X=Cl
5
6
X=Br
X=Cl
S
10,R=
11,R=
15,R=
19,R=
Scheme 1. Synthesis of quinazolines
5 and 6. Reagents and conditions: (i)
dimethylformamide dimethyl acetal, 70–75 °C; (ii) ArNH2/AcOH, 70–75 °C; (iii)
Fe/AcOH/EtOH/H2O, 70–80 °C.
Br
O2N
16,R=
CH3
Br
We guessed the conjugated effect from halogen interacting with
double bond would make great contribution for electron-donation
and steric effect should not be ignored. The reason why 26 was
weaker than 24 might be the size of methoxyl group.
Scheme 2. Synthesis of cinnamic acid derivatives 7–19. Reagents and conditions:
HOOCCH2COOH/pyridine/piperidine, 80 °C, 24 h/reflux.
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- and/or 7-positions was
outside the cleft. Against human cancer cell lines, compounds 23
and 33 were approximately less10-fold active than 21 and 35,
respectively. This result indicated that replacement of the R1
position by a same halogen group with substituent on aniline
was beyond the field of electronic effect. The explanation might
be that these two compounds with low bioactivity could not insert
in the ATP cleft of EGFR and this would be put into further study. It
was a bold surmise that the same group at the R1 and R4 position
had great effect on the conformation of quinazoline ring. Obviously
the electron-donating group at the R1 position would keep the
cytotoxicity, referring to 25 with OCH3 and 29 with CH3.
Structure–activity relationships in these derivatives demon-
strated that compounds with substitution at the meta (27)- or
ortho (26, 31, 42)-position showed more potent activities than
those with substitution at the para-position (25, 30, 41) and a
comparison of 27, 26, 25 based both on cytotoxicity and enzyme
inhibitory activity demonstrated that the potency order of the
same group besides Br and Cl was para < ortho < meta. We could
speculate that the meta NO2 compound would be superior to 30,
31 or 41, 42. Meanwhile, we also could conclude that benzene ring
as a framework performed better than thiazole ring and similarly
as naphthalene ring from 20 comparing 28, and 32 comparing
38, 40.
So as to gain more understanding of the structure–activity rela-
tionships observed at the EGFR, molecular docking of the most po-
tent inhibitor 42 into ATP binding site of EGFR kinase was
performed on the binding model based on the EGFR complex struc-
ture (1M17.pdb). The binding model of compound 42 and EGFR
was depicted in Figures 2A and 2B. In the binding model, com-
pound 420 quinazoline ring be inserted nicely inside the pocket,
and outside the cleft, and the oxygen atom of nitro group of 42 also
forms hydrogen bond with the amino hydrogen of Lys822, which
made the combination ability of 42 acting on EGFR. This was likely
the explanation for why nitro-group was not electron-donating
group but had good outcome. Meanwhile, we chose 32 without
any substituent on the cinnamylate ring as the positive control in
the docking procedure (Fig. 3). It was expected that there was no
apparent hydrogen bond outside the cleft and for the interaction
between compounds and EGFR the nitro counted for much. The en-
zyme assay data and the molecular docking results showed that
compound 42 was a potential inhibitor of EGFR (Fig. 2).
Compounds 21, 31, 40, 42, which had small differences on inte-
grating with EGFR and potent inhibitory activity showed high anti-
proliferative activity against cancer cells with IC50 ranging from 0.3
to 0.671 lg/mL, which indicated that these cinnamic acid quinazo-
line amide derivatives were potent inhibitor of EGFR as antitumor
agents. Among these compounds, compound 42 exhibited the most
potential inhibitory activity in tumor growth inhibition and exhib-
ited favored EGFR inhibitory activity. Meanwhile we could go into
the further study in which we could save the meta-nitro and in-
crease electron donation of the side chain.
2.2.2. Enzyme assay
To evaluate the EGFR inhibitory potency of new compounds,
their ability to block EGFR TK was tested in an EGFR TK assay.
We chose the twelve compounds behaved well for antipro-
liferation and their activities positively correlated with anti-
proliferative activity, which they had the same trends (Table 2).
This could indicate that cinnamic acid anilinoquinazoline deriva-
tives basically follow the EGFR inhibition path against cancer cell.
Overall the bulky moiety of the 6-position of each quinazoline was
not deleterious for activity. As shown in Table 2, compound 31 and
42 displayed the most potent inhibitory activity (IC50 = 1.02 and
2.2.3. Further study
In order to get the expectation above, Compound 43 and 44 have
been synthesized and their biological activities shown in Table 3
were also evaluated. We would choose the compounds containing
nitro group to test their cytotoxicity using the A431 cell, which over
expresses EGFR, and EGFR inhibitory activity correspondingly. As
we expected, compound 43 and 44 containing meta-nitro group
performed better than other derivatives, which displayed the most
0.94
(IC50 = 0.03
l
M for EGFR), less comparable to the positive control Erlotinib
M for EGFR).
l
potent inhibitory activity (IC50 = 0.12 and 0.19 lM for EGFR), com-

D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
5015
O
O
the theoretical values. Melting points were determined on a XT4
MP apparatus (Taike Corp., Beijing, China) and are as read. Analyt-
ical 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).
i
R
OH
R
Cl
7-19
HN
N
X
4.2. General procedure for the preparation of compounds 5, 6
(Scheme 1)
H2N
N
R
HN
N
X
O
H
N
5
6
X=Br
X=Cl
N4-(3-Bromophenyl)quinazoline-4,6-diamine (5) or N4-(3-
chlorophenyl)quinazoline-4,6-diamine (6) was prepared according
to the literature procedure.24 5-Nitroanthranilonitrile 1 (4.89 g,
30 mmol, 1.0 equiv) was suspended in dimethylformamide di-
methyl acetal (10 mL) and the mixture was refluxed for 2 h. 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 (2.18 g,
10 mmol, 1.0 equiv) and 3-chloroaniline (1.1 equiv) or 3-bromoan-
iline (1.1 equiv) were heated and stirred at reflux in acetic acid
(20 mL) for 2 h. 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 forma-
tion 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 or 4 (yield: 80–90%).
6-Nitroquinazoline 4 (2.0 g, 6.65 mmol, 1.0 equiv), iron (2.5 g,
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 70–80 °C for 5–6 h. Meantime this mixture
would be stirred for one minute every half hour with a glass rod
and the yellow solution would become reddish-brown slowly.
The reaction mixture was cooled to room temperature and alkalin-
ized 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 ethyl acetate for column chromatography. Column
chromatography was performed using silica gel (200–300 mesh)
eluting with ethyl acetate and petroleum ether (3:1, v/v) to give
amine 6 (Yield: 50–60%). Mp 163–164 °C; 1H NMR (300 MHz,
DMSO-d6, d ppm): 4.08 (s, 2H, NH2), 6.93 (s, 1H), 7.13 (d, J = 7.86,
2H), 7.24 (dd, J1 = 11.13 Hz, J2 = 8.97 Hz, 1H), 7.33 (t, J = 8.06 Hz,
1H), 7.59 (d, J = 7.68 Hz, 1H), 7.78 (d, J = 8.97 Hz,1H), 7.94 (s,1H),
8.65 (s, 1H, NH). ESI-MS: 271.1 (C14H11ClN4, [M+H]+). Anal. Calcd
for C14H10ClN4: C, 62.11; H, 4.10; N, 20.70. Found: C, 61.93; H,
4.36; N, 21.07. It is surprising that the 1H NMR data obtained
was inconsistent with the results reported in the literature.24
N4-(3-Bromophenyl)quinazoline-4,6-diamine (5) was also pre-
pared as described for 6.
N
R
Cl
O
ii
X=Br
X=Cl
20-42
Scheme 3. Synthesis of cinnamic amide derivatives 20–42. Reagents and condi-
tions: (i) SOCl2, 70–80 °C, reflux, 4 h; (ii) EtOAC/K2CO3, ice-bath, reflux, overnight.
parable to the positive control Erlotinib (IC50 = 0.03 lM for EGFR).
The fact that the meta NO2 compounds are superior to 30, 31 or
41, 42 is acceptable. As for the binding mode of the meta NO2 com-
pounds, we had gotten molecular docking of compound 43 into the
three-dimensional EGFR complex structure (1M17.pdb) using the
autodock software package and this showed 43 had possessed the
same binding mode as 42 and the cinnamylate ring looked like
more relaxed than the latter (Fig. 4). In the comparison to Erlotinib’s
binding mode, the formation of hydrogen bond with Lys822
seemed significance for 43 and the binding_energy was up to
ꢀ13.73, better than ꢀ8.83 from the optimal binding conformation
of Erlotinib. Among these compounds, compound 43 would share
the same binding mode with other NO2 compounds and exhibited
the most potential inhibitory activity both in tumor growth inhibi-
tion and EGFR inhibitory activity.
3. Conclusions
A series of cinnamic acid quinazoline amide derivatives have
been designed and synthesized, and their biological activities were
also evaluated as cytotoxicity assay firstly and then potent EGFR
inhibitory. Compound 42 demonstrated the most potent inhibitory
activity (IC50 = 0.94 lM for EGFR), which could be optimized as a
potential EGFR inhibitor in the further study. Docking simulation
was performed to position compound 42 into the EGFR active site
to determine the probable binding model. Analysis of the binding
conformation of 42 in active site displayed the compound 420
cinnamylate side chain was stabilized by hydrogen bonding inter-
actions with Lys822, which was different from other derivatives.
Antiproliferative assay results also showed that these cinnamic
acid quinazoline amide derivatives had the potential to be devel-
oped for antiproliferative agents against cancer cells. Based on this,
compounds 43 and 44 were obtained according to Scheme 4 in the
next step and we also gave the relevant bioactivity evaluation to
these compounds. Among these compounds, we could conclude
4.3. Preparation of substituted cinnamic acids 7–19 (Scheme 2)
The substituted cinnamic acids were synthesized using Doebner
Knoevenagel modification.25 Cinnamic acids were prepared by mix-
ing substituted benzaldehydes (3.2 mM), malonic acid (3.87 mM),
that compound 43 has demonstrated significant EGFR (IC50
=
0.12 M) and tumor growth inhibitory activity as a potential anti-
l
cancer agent.
pyridine (20 mL), and piperidine (40 lL) together and stirring
4. Experiments
80 °C on a magnetic stirrer for 24 h (Scheme 2). The reaction mix-
ture was evaporated under reduced pressure. Precipitates obtained
were filtered, washed with cold water repeatedly, and dried. Finally
the mixtures were recrystallized with dichloromethane to give
these acids (Trans-isomer). The yields were between 70% and
80%. Two compounds (13, 16) due to intermediate products were
randomly selected to give the determination of structure.
4.1. Materials and methods
1H NMR spectra were measured on a Bruker AV-300 or AV-500
spectrometer at 25 °C and referenced to Me4Si. 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. ESI-MS spectra were recorded on a Mariner
System 5304 Mass spectrometer. Elemental analyses were per-
formed on a CHN-O-Rapid instrument and were within 0.4% of
4.3.1. (E)-3-(2-Methoxyphenyl)acrylic acid (13)
Mp 184–186 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 3.87 (s,
3H, OCH3), 6.51 (d, J = 16.2 Hz, 1H), 6.98 (t, J = 7.47 Hz, 1H), 7.09

Table 1
Structure of cinnamic acid quinazoline amide derivatives and the antiproliferative effect
O
NH
HN
N
R3
R4
N
R1
R2
Compound R1
R2
R3
R4
IC50
(
lg/mL)
Growth inhibition in A549 cellsa
Inhibition of
serum-
stimulated
growth in F10
cellsa
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Erlotinib
H
Cl
H
Br
H
OCH3
H
H
H
H
H
H
H
H
H
OCH3
—
H
H
H
H
H
H
H
H
H
H
Cl
H
Br
H
OCH3
H
—
H
H
NO2
H
H
Cl
H
Br
H
—
H
—
H
Br 3.96
Br 1.06
Br 6.63
Br 15.32
Br 2.38
Br 3.36
Br 3.55
Br 2.05
Br 4.66
Br 3.01
Br 1.85
Br 0.92
Cl 1.58
Cl 12.43
Cl 4.58
Cl 1.86
Cl 5.63
Cl 2.58
Cl 9.23
Cl 6.03
Cl 1.49
Cl 5.26
Cl 0.78
1.45
0.38
2
10.65
0.92
1.24
1.28
0.87
2.51
1.39
0.9815
0.338
0.68
9.97
1.74
0.75
1.65
1.05
4.26
2.02
0.671
2.05
0.3
—
CH3
NO2
H
H
Cl
H
Br
H
H
—
OCH3
—
H
—
H
CH3
—
NO2
H
H
—
NO2
—
—
—
0.13
0.02
O
O
NH
NH
HN
HN
N
HN
Cl
H
N
S
S
N
N
Cl
Br
N
N
O
N
40
28
38
a
Cell growth was measured using the MTT assay. Values are the average of two independent experiments run in triplicate. Variation was generally 1%.

D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
5017
[M+H]+). Anal. Calcd for C10H9O2: C, 74.06; H, 6.21. Found: C,
74.23; H, 5.96.
O
O
O2
N
i
O2
N
OH
Cl
4.4. General synthesis method of substituted cinnamic acid
amide analogues 20–42 (Scheme 3)
(E)-3-(3-nitrophenyl)acrylic acid
HN
N
X
The starting materials (acids 7–19) for the synthesis of amides
should be activated in the first procedure: the compounds 7–19
(1.0 mM) and SOCl2 (6–10 mL) were mixed and stirred at reflux
80°Cfor 4 h. The reaction mixture was cooled and evaporated to
give reactive acyl chloride obtained as an oil, which would be dis-
solved in ethyl acetate (5–6 mL) in the next step.
H2
N
N
O
N
O
O2N
N
5
6
X=Br
X=Cl
O2N
N
H
Cl
X
NH
ii
43 X=Br
X=Cl
44
A solution of acyl chloride (1.0 mM) in ethyl acetate was added
dropwise to compounds 5 or 6 (0.5 mM) in ethyl acetate containing
potassium carbonate (600 mg) at 0 °C with constant stirring over-
night. The reaction mixture was then poured in excess of diluted
NaOH and extracted with EtOAc .The extraction liquid was purified
by a flash chromatography with EtOAc/petroleum ether (3:1, v/v)
to give these cinnamamides as follows: the yields were between
40% and 60%.
Scheme 4. Synthesis of cinnamic amide derivatives 43 and 44. Reagents and
conditions: (i) SOCl2, 70–80 °C, reflux, 4 h; (ii) EtOAC/K2CO3, ice-bath, reflux,
overnight.
Table 2
Inhibition (IC50) of EGFR kinase
Compound
EGFR inhibition IC50 (lM)
21
24
25
26
27
30
31
32
35
37
40
42
1.21
4.28
7.22
7.71
6.02
5.16
1.02
3.61
3.49
6.63
2.81
0.94
0.03
4.4.1. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)cinn
amamide (20)
Compound 20 was prepared as described above. Mp
265–266 °C; 1H NMR (300 MHz, DMSO-d6,
d ppm): 6.92 (d,
J = 15.72 Hz, 1H), 7.27–7.37 (m, 2H), 7.42–7.48 (m, 3H), 7.66 (t,
J = 7.7 Hz, 3H), 7.79–7.87 (m, 2H), 7.94 (dd, J1 = 8.97 Hz,
J2 = 8.97 Hz, 1H), 8.17 (s, 1H, NH), 8.59 (s, 1H), 8.83 (s, 1H), 9.97
(s, 1H), 10.55 (s, 1H, NHCO). ESI-MS: 445.1 (C23H17BrN4O,
[M+H]+). Anal. Calcd for C23H16BrN4O: C, 62.03; H, 3.85; N, 12.58.
Found: C, 61.99; H, 4.16; N, 13.01.
Erlotinib
4.4.2. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(4-
chlorophenyl)acrylamide (21)
(d, J = 8.1 Hz, 1H), 7.39–7.42 (m, 1H), 7.67 (dd, J1 = 7.8 Hz,
J2 = 7.6 Hz, 1H), 7.83 (d, J = 16.2 Hz, 1H), 12.27 (s, 1H, COOH).
ESI-MS: 179.1 (C10H10O3, [M+H]+). Anal. Calcd for C10H9O3: C,
67.41; H, 5.66. Found: C, 67.99; H, 5.36.
Mp 283–285 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 6.93 (d,
J = 15.7 Hz, 1H), 7.3 (d, J = 7.95 Hz, 1H), 7.35 (t, J = 8.02 Hz, 1H),
7.54 (d, J = 8.55 Hz, 2H), 7.65–7.70 (m, 3H), 7.82 (d, J = 8.85 Hz,
1H), 7.88 (d, J = 8.1 Hz, 1H), 7.96 (dd, J1 = 9 Hz, J2 = 8.85 Hz, 1H),
8.19 (s, 1H, NH), 8.6 (s, 1H), 8.83 (s, 1H), 9.95 (s, 1H, OH), 10.56
(s, 1H, NHCO). ESI-MS: 479.0 (C23H16BrClN4O, [M+H]+). Anal. Calcd
for C23H15BrClN4O: C, 57.58; H, 3.36; N, 11.68. Found: C, 57.87; H,
3.12; N, 11.57.
4.3.2. (E)-3-p-Tolylacrylic acid (16)
Mp 198–200 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 2.33 (s,
3H, CH3), 6.46 (d, J = 16.11 Hz, 1H), 7.55 (t, J = 8.31 Hz, 3H), 7.23
(d, J = 7.86 Hz, 1H), 12.33 (s, 1H, COOH). ESI-MS: 163.1 (C10H10O2,
Figure 2A. Docking of compound 42 interacting with EGFR kinase (1M17.pdb) shows intermolecular hydrogen bond between Lys822 and O from NO2.

5018
D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
Figure 2B. 3D model of the interaction between compound 42 and the ATP binding site. The cinnamic acid chain anchors outside the site, and the nitro group could form
hydrogen bond to stabilize the combination, which binding energy is equal to ꢀ12.15.
(C23H16Br2N4O, [M+H]+). Anal. Calcd for C23H15Br2N4O: C, 52.70;
H, 3.08; N, 10.69. Found: C, 52.43; H, 3.36; N, 10.89.
4.4.5. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(2-
bromophenyl)acrylamide (24)
Mp 293–294 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 6.94 (d,
J = 15.54 Hz, 1H), 7.37 (t, J = 8.69 Hz, 3H), 7.52 (t, J = 7.5 Hz, 1H),
7.74–7.85 (m, 4H), 7.96 (t, J = 7.86 Hz, 2H), 8.16 (s, 1H, NH), 8.64
(s, 1H), 8.95 (s, 1H), 10.16 (br s, 1H), 10.72 (s, 1H, NHCO).
ESI-MS: 523.0 (C23H16Br2N4O, [M+H]+). Anal. Calcd for C23H15
Br2N4O: C, 52.70; H, 3.08; N, 10.69. Found: C, 52.83; H, 3.23; N,
10.33.
4.4.6. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(4-
methoxyphenyl)acrylamide (25)
Mp 239–240 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.83 (s,
3H, OCH3), 7.02–7.10 (m, 2H), 7.29–7.39 (m, 2H), 7.63 (t,
J = 7.49 Hz, 1H), 7.82–7.94 (m, 5H), 8.03 (dd, J1 = 8.97 Hz,
J2 = 8.97 Hz, 1H), 8.2 (s, 1H, NH), 8.63 (s, 1H), 8.79 (s, 1H), 9.98 (s,
1H), 10.51 (s, 1H, NHCO). ESI-MS: 475.0 (C24H19BrN4O2, [M+H]+).
Anal. Calcd for C24H18BrN4O2: C, 60.64; H, 4.03; N, 11.79. Found:
C, 60.85; H, 4.23; N, 11.43.
Figure 3A. Docking of compound 32 interacting with EGFR kinase (1M17.pdb)
shows there is no other interaction between cinnamic acid side chain and EGFR
except pi-cation interaction inside the pocket.
4.4.7. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(2-
methoxyphenyl)acrylamide (26)
4.4.3. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(2-
chlorophenyl)acrylamide (22)
Mp 307–308 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.89
(s, 3H, OCH3), 6.93–7.05 (m, 2H), 7.11 (d, J = 8.04 Hz, 1H),
7.28–7.38 (m, 2H), 7.42 (d, J = 7.14 Hz, 1H), 7.59 (d, J = 7.68 Hz,
1H), 7.79–7.86 (m, 3H), 7.91–7.96 (m, 1H), 8.16 (s, 1H, NH),
8.6 (s, 1H), 8.85 (s, 1H), 10.01 (br s, 1H), 10.54 (s, 1H, NHCO).
ESI-MS: 475.0 (C24H19BrN4O2, [M+H]+). Anal. Calcd for
Mp 287–288 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 6.97 (d,
J = 15.54 Hz, 1H), 7.27–7.37 (m, 2H), 7.44–7.47 (m, 2H), 7.59 (t,
J = 4.67 Hz, 1H), 7.79–8.00 (m, 5H), 8.17 (s, 1H, NH), 8.59 (s, 1H),
8.88 (s, 1H), 9.97 (s, 1H), 10.66 (s, 1H, NHCO). ESI-MS: 479.0
(C23H16BrClN4O, [M+H]+). Anal. Calcd for C23H15BrClN4O: C,
57.58; H, 3.08; N, 10.69. Found: C, 58.03; H, 3.36; N, 9.87.
C24H18BrN4O2: C, 60.64; H, 4.03; N, 11.79. Found: C, 60.94; H,
4.31; N, 11.40.
4.4.8. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(3-
methoxyphenyl)acrylamide (27)
4.4.4. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(4-
bromophenyl)acrylamide (23)
Mp 303–305 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.83 (s,
3H, OCH3), 6.88–7.07 (m, 2H), 7.20–7.37 (m, 4H), 7.47 (d,
J = 8.79 Hz, 1H), 7.80–7.93 (m, 4H), 8.17 (s, 1H, NH), 8.58 (s, 1H),
8.9 (s, 1H), 9.94 (br s, 1H), 10.64 (s, 1H, NHCO). ESI-MS: 475.0
Mp 295–296 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 6.95 (d,
J = 15.72 Hz, 1H), 7.29–7.39 (m, 2H), 7.61–7.75 (m, 5H), 7.84 (t,
J = 10.52 Hz, 2H), 7.96 (d, J = 8.94 Hz, 1H), 8.18 (s, 1H, NH), 8.6 (s,
1H), 8.84 (s, 1H), 10 (s, 1H), 10.59 (s, 1H, NHCO). ESI-MS: 523.0

D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
5019
Figure 3B. 3D model of the interaction between compound 32 and the ATP binding site.
Table 3
EGFR tyrosine kinase (TK) inhibition and antiproliferative data for combinatorial molecules containing nitro group
O
NH
HN
R3
N
R4
N
R1
R2
Compound
R1
R2
R3
R4
IC50 (lM)
EGFR enzyme assay
inhibition of A431 basal growth
5
6
30
31
41
42
43
44
—
—
NO2
H
NO2
H
H
H
—
—
—
H
H
H
H
NO2
NO2
—
—
—
H
NO2
H
NO2
H
H
Br
Cl
Br
Br
Cl
Cl
Br
Cl
—
0.04
0.2
42
47
5.16
1.02
3.45
0.94
0.12
0.19
0.03
1.34
0.98
2.13
0.75
0.33
0.49
0.27
Erlotinib
—
HN
Br
HN
Cl
H2N
H2N
N
N
N
N
5
6
(C24H19BrN4O2, [M+H]+). Anal. Calcd for C24H18BrN4O2: C, 60.64; H,
4.03; N, 11.79. Found: C, 60.34; H, 3.98; N, 11.66.
J = 7.86 Hz, 2H), 8.02 (d, J = 10.98 Hz, 1H), 8.21 (s, 1H, NH), 8.63
(s, 1H), 8.79 (s, 1H), 9.93 (s, 1H), 10.57 (s, 1H, NHCO). ESI-MS:
459.0 (C24H19BrN4O, [M+H]+). Anal. Calcd for C24H18BrN4O: C,
62.75; H, 4.17; N, 12.20. Found: C, 63.03; H, 4.17; N, 11.83.
4.4.9. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-
(thiophen-2-yl)acrylamide (28)
Mp 244 -246 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 7.28 (dd,
J1 = 5.13 Hz, J2 = 5.13 Hz, 1H), 7.33 (d, J = 4.59 Hz, 1H), 7.38 (d,
J = 7.86 Hz, 1H), 7.76 (d, J = 2.91 Hz, 1H), 7.86 (t, J = 7.88 Hz, 2H),
7.95 (d, J = 5.1 Hz, 1H), 8.04 (dd, J1 = 8.97 Hz, J2 = 8.97 Hz, 1H), 7.74–
7.85 (m, 4H), 7.96 (t, J = 7.86 Hz, 2H), 8.19 (s, 1H, NH), 8.32 (s, 1H),
8.64 (s, 1H), 8.79 (s, 1H), 10.04 (br s, 1H), 10.54 (s, 1H, NHCO). ESI-
MS: 451.0 (C21H15BrN4OS, [M+H]+). Anal. Calcd for C21H14
BrN4OS: C, 55.88; H, 3.35; N, 12.41. Found: C, 55.83; H, 3.24; N, 12.63.
4.4.11. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(4-
nitrophenyl)acrylamide (30)
Mp 300–302 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 7.11 (d,
J = 15.93 Hz, 1H), 7.29–7.39 (m, 2H), 7.75–7.99 (m, 6H), 8.18 (s, 1H,
NH), 8.32 (d, J = 8.79 Hz, 2H), 8.61 (s, 1H), 8.85 (s, 1H), 10 (s, 1H),
10.71 (s, 1H, NHCO). ESI-MS: 490.0 (C23H16BrN5O3, [M+H]+). Anal.
Calcd for C23H15BrN5O3: C, 56.34; H, 3.29; N, 14.28. Found: C,
56.44; H, 3.38; N, 14.11.
4.4.12. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(2-
nitrophenyl)acrylamide (31)
4.4.10. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-p-
tolylacrylamide (29)
Mp 282–284 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 6.9 (d,
J = 15.54 Hz, 1H), 7.28–7.38 (m, 2H), 7.67–7.72 (m, 1H),
Mp 248–249 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 2.37 (s,
3H, CH3), 7.32–7.39 (m, 5H), 7.81–7.85 (m, 3H), 7.9 (d,

5020
D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
Figure 4A. 3D model of the interaction between compound 43 and the ATP binding site. The cinnamic acid chain anchors outside the site, and the nitro group could form
hydrogen bond to stabilize the combination, which binding energy is equal to ꢀ13.33.
Figure 4B. The optimal model of the interaction between Erlotinib and the ATP binding site, which binding energy is equal to ꢀ8.83.
7.82–8.00 (m, 6H), 8.11 (d, J = 7.86 Hz, 1H), 8.18 (s, 1H, NH), 8.6 (s,
1H), 8.9 (s, 1H), 9.99 (s, 1H), 10.7 (s, 1H, NHCO). ESI-MS: 490.0
(C23H16BrN5O3, [M+H]+). Anal. Calcd for C23H15BrN5O3: C, 56.34;
H, 3.29; N, 14.28. Found: C, 56.22; H, 3.18; N, 14.53.
7.42 (t, J = 8.07 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz,
2H), 7.65–7.70 (m, 3H), 7.97 (dd, J1 = 9 Hz, J2 = 9 Hz, 1H), 8.06 (s,
1H, NH), 8.6 (s, 1H), 8.83 (s, 1H), 9.99 (s, 1H, OH), 10.57 (s, 1H,
NHCO). ESI-MS: 435.1 (C23H16Cl2N4O, [M+H]+). Anal. Calcd for
C23H15Cl2N4O: C, 63.46; H, 3.70; N, 12.87. Found: C, 63.05; H,
4.4.13. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)cinn
amamide (32)
3.93; N, 13.01.
Mp 294–295 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 6.93 (d,
J = 15.75 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.40–7.49 (m, 4H), 7.67 (t,
J = 8.1 Hz, 3H), 7.82 (d, J = 8.65 Hz, 2H), 7.96 (d, J = 9 Hz, 1H), 8.07
(s, 1H, NH), 8.6 (s, 1H), 8.84 (s, 1H), 9.96 (s, 1H), 10.55 (s, 1H,
NHCO). ESI-MS: 401.1 (C23H17ClN4O, [M+H]+). Anal. Calcd for
4.4.15. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(2-
chlorophenyl)acrylamide (34)
Mp 292–294 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 6.98 (d,
J = 15.7 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 7.41 (t, J = 8.08 Hz, 1H),
7.47 (t, J = 3.65 Hz, 2H), 7.58 (t, J = 4.58 Hz, 1H), 7.82 (t,
J = 8.77 Hz, 3H), 7.94 (d, J = 8.85 Hz, 1H), 7.99 (d, J = 15.75 Hz,
1H), 8.07 (s, 1H, NH), 8.6 (s, 1H), 8.89 (s, 1H), 9.95 (s, 1H), 10.65
(s, 1H, NHCO). ESI-MS: 435.1 (C23H16Cl2N4O, [M+H]+). Anal. Calcd
for C23H15Cl2N4O: C, 63.46; H, 3.70; N, 12.87. Found: C, 63.75; H,
3.45; N, 13.01.
C23H16ClN4O: C, 68.91; H, 4.27; N, 13.98. Found: C, 68.99; H,
4.16; N, 13.91.
4.4.14. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(4-
chlorophenyl)acrylamide (33)
Mp 306–307 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 6.93 (d,
J = 15.75 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H),

D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
5021
4.4.16. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(4-
4.4.22. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(4-
bromophenyl)acrylamide (35)
nitrophenyl)acrylamide (41)
Mp 289–291 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 6.95 (d,
J = 15.75 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 7.41 (t, J = 8.18 Hz, 1H),
7.62 (d, J = 8.55 Hz, 2H), 7.67 (t, J = 5.65 Hz, 3H), 7.82 (d,
J = 8.85 Hz, 2H), 7.96 (dd, J1 = 9 Hz, J2 = 9 Hz, 1H), 8.07 (s, 1H,
NH), 8.6 (s, 1H), 8.82 (s, 1H), 9.95 (s, 1H), 10.56 (s, 1H, NHCO).
ESI-MS: 478.1 (C23H16BrClN4O, [M+H]+). Anal. Calcd for
Mp 280–282 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 7.42 (t,
J = 8.06 Hz, 1H), 7.08–7.18 (m, 2H), 7.75–7.85 (m, 3H), 7.92–8.06
(m, 4H), 8.31 (d, J = 8.4 Hz, 1H), 8.61 (s, 1H), 8.84 (s, 1H), 10 (s,
1H), 10.7 (s, 1H, NHCO). ESI-MS: 446.1 (C23H16ClN5O3, [M+H]+).
Anal. Calcd for C23H15ClN5O3: C, 61.96; H, 3.62; N, 15.71. Found:
C, 61.64; H, 3.38; N, 15.91.
C23H15BrClN4O: C, 57.58; H, 3.36; N, 11.68. Found: C, 57.23; H,
3.36; N, 11.89.
4.4.23. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(2-
nitrophenyl)acrylamide (42)
Mp 287–289 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 6.91 (d,
J = 15.4 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 7.42 (t, J = 8.07 Hz, 1H),
7.68–7.72 (m, 1H), 7.82–7.88 (m, 4H), 7.93 (d, J = 9 Hz, 1H), 7.97
(d, J = 15.55 Hz, 1H), 8.07 (s, 1H, NH), 8.11 (d, J = 10 Hz, 1H), 8.6
(s, 1H), 8.9 (s, 1H), 9.96 (s, 1H), 10.68 (s, 1H, NHCO). ESI-MS:
446.1 (C23H16ClN5O3, [M+H]+). Anal. Calcd for C23H15ClN5O3: C,
61.96; H, 3.62; N, 15.71. Found: C, 61.65; H, 3.38; N, 15.93.
4.4.17. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(2-
bromophenyl)acrylamide (36)
Mp 292–293 °C; 1H NMR (500 MHz, DMSO-d6,
d ppm):
7.38–7.43 (m, 2H), 7.51 (d, J = 7.5 Hz, 1H), 7.75 (d, J = 8.05 Hz,
1H), 7.79–7.84 (m, 3H), 7.94 (t, J = 11.98 Hz, 2H), 8.06 (s, 1H, NH),
8.61 (s, 1H), 8.91 (s, 1H), 9.98 (s, 1H), 10.67 (s, 1H, NHCO).
ESI-MS: 479.0 (C23H16BrClN4O, [M+H]+). Anal. Calcd for
C23H15BrClN4O: C, 57.58; H, 3.36; N, 11.68. Found: C, 57.83; H,
3.21; N, 11.33.
4.4.24. (E)-N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(3-
nitrophenyl)acrylamide (43)
Mp 285–286 °C; 1H NMR (300 MHz, DMSO-d6, d ppm): 7.12 (d,
J = 15.9 Hz, 1H), 7.29–7.42 (m, 2H), 7.74–7.88 (m, 4H), 7.96 (dd,
J1 = 8.97 Hz, J2 = 8.94 Hz, 1H), 8.12 (d, J = 7.86 Hz, 1H), 8.18 (s, 1H,
NH), 8.27 (dd, J1 = 8.22 Hz, J2 = 8.25 Hz, 1H), 8.51 (s, 1H), 8.61 (s,
1H), 8.87 (s, 1H), 10.04 (s, 1H), 10.64 (s, 1H, NHCO). ESI-MS:
490.0 (C23H16BrN5O3, [M+H]+). Anal. Calcd for C23H16BrN5O3: C,
56.34; H, 3.29; N, 14.28. Found: C, 56.65; H, 3.48; N, 13.91.
4.4.18. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(3-
methoxyphenyl)acrylamide (37)
Mp 284–285 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 3.82 (s,
3H, OCH3), 6.93 (d, J = 15.55 Hz, 1H), 7.01 (dd, J1 = 8.25 Hz,
J2 = 8.25 Hz, 1H), 7.17 (d, J = 7.45 Hz, 1H), 7.64 (d, J = 15.7 Hz, 1H),
7.82 (d, J = 8.85 Hz, 2H), 7.37–7.43 (m, 2H), 7.24 (t, J = 9.3 Hz,
2H), 7.95 (t, J = 5.5 Hz, 1H), 8.06 (s, 1H, NH), 8.6 (s, 1H), 8.85 (s,
1H), 9.97 (s, 1H), 10.54 (s, 1H, NHCO). ESI-MS: 431.1
(C24H19ClN4O2, [M+H]+). Anal. Calcd for C24H18ClN4O2: C, 66.90;
H, 4.44; N, 13.00. Found: C, 66.84; H, 3.99; N, 13.16.
4.4.25. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-(3-
nitrophenyl)acrylamide (44)
Mp 296–298 °C; 1H NMR (300 MHz, DMSO-d6,
d ppm):
7.08–7.18 (m, 2H), 7.42 (t, J = 8.15 Hz, 1H), 7.66–7.85 (m, 4H),
7.96 (d, J = 8.97 Hz, 1H), 8.07 (s, 1H, NH), 8.13 (d, J = 7.86 Hz, 1H),
8.29 (t, J = 8.13 Hz, 1H), 8.52 (s, 1H), 8.61 (s, 1H), 8.86 (s, 1H),
10.00 (s, 1H), 10.65 (s, 1H, NHCO). ESI-MS: 446.1 (C23H16ClN5O3,
[M+H]+). Anal. Calcd for C23H15ClN5O3: C, 61.96; H, 3.62; N,
15.71. Found: C, 62.25; H, 3.18; N, 15.69.
4.4.19. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-
(thiophen-2-yl)acrylamide (38)
Mp 241–243 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 6.69 (d,
J = 8.5 Hz, 1H), 7.16–7.17 (m, 2H), 7.41 (t, J = 8.1 Hz, 1H), 7.49 (d,
J = 3.35 Hz, 1H), 7.69 (d, J = 5 Hz, 1H), 7.82 (d, J = 7.62 Hz, 3H),
7.92 (dd, J1 = 9 Hz, J2 = 8.85 Hz, 1H), 8.06 (s, 1H, NH), 8.59 (s, 1H),
8.84 (s, 1H), 9.97 (s, 1H), 10.52 (s, 1H, NHCO). ESI-MS: 451.0
(C21H15ClN4OS, [M+H]+). Anal. Calcd for C21H14ClN4OS: C, 61.99;
H, 3.72; N, 13.77. Found: C, 61.83; H, 3.94; N, 13.63.
4.5. Cell proliferation assay
The antiproliferative activities of cinnamic acid quinazoline
amide derivatives were determined using a standard (MTT)-based
colorimetric assay (Sigma). Briefly, cell lines were seeded at a den-
sity of 7 ꢁ 103 cells/well in 96-well microtiter plates (Costar). After
24 h, exponentially growing cells were exposed to the indicated
4.4.20. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-p-
tolylacrylamide (39)
Mp 277–279 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 2.35 (s,
3H, CH3), 6.87 (d, J = 15.75 Hz, 1H), 7.16 (d, J = 8.05 Hz, 1H), 7.28
(d, J = 7.9 Hz, 2H), 7.41 (t, J = 8.15 Hz, 1H), 7.55 (d, J = 8.1 Hz, 2H),
7.64 (d, J = 15.7 Hz, 1H), 7.82 (d, J = 8.7 Hz, 2H), 7.95 (dd,
J1 = 9 Hz, J2 = 8.85 Hz, 1H), 8.06 (s, 1H, NH), 8.6 (s, 1H), 8.83 (s,
1H), 9.95 (s, 1H), 10.5 (s, 1H, NHCO). ESI-MS: 415.1 (C24H19ClN4O,
[M+H]+). Anal. Calcd for C24H18ClN4O: C, 69.48; H, 4.62; N, 13.50.
Found: C, 69.03; H, 4.57; N, 13.83%
compounds at final concentrations ranging from 0.01 to 100
ml. After 48 h, cell survival was determined by the addition of an
MTT solution (20 L of 5 mg/mL MTT in PBS). After 6 h, 100 mL
lg/
l
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.
4.4.21. (E)-N-(4-(3-Chlorophenylamino)quinazolin-6-yl)-3-
(naphthalen-4-yl)acrylamide (40)
4.6. General procedure for preparation, purification of EGFR
inhibitory assay
Mp 293–294 °C; 1H NMR (500 MHz, DMSO-d6, d ppm): 7.01 (d,
J = 15.4 Hz, 1H), 7.17 (d, J = 9.15 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H),
7.60–7.69 (m, 3H), 7.85 (d, J = 9 Hz, 2H), 7.9 (d, J = 7.15 Hz, 1H),
7.99–8.05 (m, 3H), 8.26 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 15.55 Hz,
1H), 8.08 (s, 1H, NH), 8.62 (s, 1H), 8.9 (s, 1H), 10 (s, 1H), 10.65 (s,
1H, NHCO). ESI-MS: 495.0 (C27H19ClN4O, [M+H]+). Anal. Calcd for
A 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 (Huakang
Company China), separately. A sequence that encodes (His)6 was
located at the 5’ upstream to the EGFR sequences. Sf-9 cells were
infected for 3 days for protein expression. Sf-9 cell pellets were sol-
ubilized at 0 °C in a buffer at pH 7.4 containing 50 mM HEPES,
C27H18ClN4O: C, 71.92; H, 4.25; N, 12.43. Found: C, 71.99; H,
4.16; N, 12.51.

5022
D.-D. Li et al. / Bioorg. Med. Chem. 19 (2011) 5012–5022
10 mM NaCl, 1% Triton, 10
l
M ammonium molybdate, 100
g/mL leupeptin, 10
g/mL benzamidine HCl for 20 min followed by
l
M so-
China (30901854), and Jiangsu National Science Foundation
(BK2009239).
dium vanadate, 10
pepstatin, and 16
l
l
g/mL aprotinin, 10
l
lg/mL
References and notes
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 non-
specifically bound material. Histidine tagged proteins were eluted
with 250 and 500 mM imidazole and dialyzed against 50 mM NaCl,
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The EGFR kinase assay was set up to assess the level of autophos-
phorylation based on DELFIA/Time-Resolved Fluorometry. Com-
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well, 10
combinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min at
room temperature. Then, 10
L of 5 ꢁ buffer (containing 20 mM
HEPES, 2 mM MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20 lL
lL compound was incubated with 10 lL (5 ng for EGFR) re-
l
l
of 0.1 mM ATP-50 mM MgCl2 were added for 1 h. Positive and neg-
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A 75 lL (400 ng) sample of europiumlabeled anti-phosphotyrosine
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100% ꢀ [(negative control)/(positive control ꢀ negative control)].
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4.7. Molecular docking modeling
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Molecular docking of compound 32, 42, 43 and Erlotinib into
the three-dimensional EGFR complex structure (1M17.pdb, down-
loaded from the PDB) was carried out using the AutoDock software
package (version 4.0) as implemented through the graphical user
interface Auto-Dock Tool Kit (ADT 1.4.6).
Acknowledgment
This work was supported by National Basic Research Program of
China (2010CB126104), National Natural Science Foundation of