Novel 4-arylaminoquinazoline derivatives with (E)-propen-1-yl moiety as potent EGFR inhibitors with enhanced antiproliferative activities against tumor cells

Article abstract of DOI:10.1016/j.ejmech.2017.06.023

A series of novel 4-anilinoquinazoline derivatives with (E)-propen-1-yl moiety were designed, synthesized and evaluated for biological activities in vitro. Most compounds exhibited highly antiproliferative activities against all tested tumor cell lines in

Full text of DOI:10.1016/j.ejmech.2017.06.023

Accepted Manuscript
Novel 4-arylaminoquinazoline derivatives with (E)-propen-1-yl moiety as potent EGFR
inhibitors with enhanced antiproliferative activities against tumor cells
Li Chen, Yaling Zhang, Juan Liu, Weijia Wang, Xiabing Li, Lijun Zhao, Wei Wang,
Baolin Li
PII:
S0223-5234(17)30469-5
10.1016/j.ejmech.2017.06.023
EJMECH 9518
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2017
Revised Date: 9 June 2017
Accepted Date: 12 June 2017
Please cite this article as: L. Chen, Y. Zhang, J. Liu, W. Wang, X. Li, L. Zhao, W. Wang, B. Li, Novel 4-
arylaminoquinazoline derivatives with (E)-propen-1-yl moiety as potent EGFR inhibitors with enhanced
antiproliferative activities against tumor cells, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.06.023.
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Graphical abstract

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Novel 4-arylaminoquinazoline derivatives with (E)-propen-1-yl moiety as potent EGFR
inhibitors with enhanced antiproliferative activities against tumor cells
Li Chen1, Yaling Zhang1, Juan Liu, Weijia Wang, Xiabing Li*, Lijun Zhao, Wei Wang, Baolin Li*
Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry; National
Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China; School of Chemistry &
Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, P. R. China
1 These authors contributed equally to this work.
* Corresponding author. E-mail address: baolinli@snnu.edu.cn, xiabingli@snnu.edu.cn
Abstract:
A series of novel 4-anilinoquinazoline derivatives with (E)-propen-1-yl moiety were designed,
synthesized and evaluated for biological activities in vitro. Most compounds exhibited highly
antiproliferative activities against all tested tumor cell lines including A431, A549, NCI-H1975 and
SW480 cells. Especially, compound 6e not only presented strong antiproliferative activities against the
tested four tumor cell lines (IC50 of 1.35, 8.83, 5.53 and 6.08 µM, respectively) which expressed wild
type or L858R/T790M double mutant epidermal growth factor receptor (EGFR), but also showed potent
inhibitory activity against wild type EGFR (IC50 = 20.72 nM). The result of molecular docking with
EGFR suggested the binding mode of 6e was similar to gefitinib, but different from lapatinib.
Additionally, western blot analysis showed that 6e inhibited the phosphorylation of EGFR and its
downstream signaling proteins in lung cancer cells. The work could be very useful starting point for
developing a new series of tyrosine kinase inhibitors targeting EGFR.
Keywords: 4-anilinoquinazoline, (E)-propen-1-yl moiety, antiproliferative activities, epidermal growth
factor receptor, tyrosine kinase inhibitors
1. Introduction
Epidermal growth factor receptor (EGFR) tyrosine kinase (TK) is a member of the ErbB family
[1-4], and regulates essential cellular functions, including proliferation, survival, migration, and
differentiation [5]. Overexpression, amplification, and mutation of EGFR occur in a wide range of
human cancers [3, 6], and are associated with tumor progression and reduce sensitivity to antitumor
agents [7]. Thus EGFR has been considered as one of the main anticancer targets [4, 8]. Some
quinazoline derivatives are among the most successful antitumor drugs in the vanguard of targeted
therapies [9], such as gefitinib [10], erlotinib [11], lapatinib [12] and afatinib [13, 14] (Fig. 1).
Gefitinib and erlotinib, as ATP-competitive and reversible EGFR inhibitors, have been shown to be
particularly beneficial for non-small cell lung cancer (NSCLC) patients harboring common EGFR
mutations L858R in exon 21 or del E746-A750 in exon 19 [15-18]. However, the emergence of acquired
point mutations, particular T790M mutation, has weakened their therapeutic efficacy, leading to drug
resistance to these drugs [4, 19]. Lapatinib, the first dual inhibitor of EGFR and EGFR-2 (HER2) TKs,
was approved by FDA in 2007 [12, 20]. Unfortunately, a mandatory black-box warning was released in
2008 because of lapatinib-related hepatotoxicity in clinical trials and post-marketing surveillance, the
liver injury was fatal in rare cases [21]. Afatinib, an ATP-competitive anilinoquinazoline derivative and
the first irreversible tyrosine kinase inhibitor (TKI), possesses a reactive acrylamide group, which was
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designed to covalently bind to a conserved cysteine residue (Cys797) of EGFR and irreversibly block
enzymatically active ErbB receptor family members [22, 23]. However, as afatinib, many irreversible
TKIs, contain with a reactive “warhead” which can irreversibly bind to proteins other than the target,
creating a toxicity burden [23]. Thus there is an urgent demand for new EGFR inhibitors that can
effectively treat cancers.
Fig. 1. Structures of some TKIs and design rationale of novel 4-(4-(E)-(propen-1-yl)phenylamino)quinazoline derivatives
.
Previous studies on the antitumor activity of 4-anilinoquinazolines revealed the relationship of
structure and activity as follows: 1) the pyrimidine ring is mandatory, 2) a free NH linker at the 4-position
of quinazoline core is clearly optimal, 3) electron-withdrawing lipophilic substituents at the 3-position of
the aniline moiety are favorable, and 4) electron-donating groups at the 6- and 7-positions (but not the 5-
and 8-positions) of the quinazoline are preferred [4, 24, 25]. As shown in Fig.1, these TKIs have a
4-anilinoquinazoline scaffold [26], and different substituents at 3- or 4-positions of the aniline moiety,
and 6- and 7-positions of the quinazoline. In contrast, afatinib has an acrylamide electrophilic moiety as
the receptor of Michael addition to form a covalent bond with Cys797 of EGFR. Inspired by the
unsaturated feature of afatinib and its analogues neratinib and dacomitinib with acrylamide unit [27], we
speculated the introduction of other C=C unsaturated moiety to 4-anilinoquinazoline derivatives (Fig. 1)
may lead to a series of new antineoplastic compounds with enhanced antitumor activity and decreased
toxicity, which may result from other addition reaction of C=C unit with biomolecules under the catalysis
of biological systems. Initially, we introduced a propenyl group to the 4-position of the aniline moiety to
yield three compounds, 1a-1c (Fig. 2). The obtained compounds exhibited moderate antiproliferative
activities against three human tumor cell lines (IC50 in the range of 10.40 – 85.32 µM) [28, 29]. These
preliminary results encouraged us to further optimize the structures of these compounds. Thus, we
continued to install (E)-propenyl substituents to the 4-positions of the aniline moiety, with different
hydrophilic fragments introduced to the 6- or 7-positions of quinazoline core (see Fig. 1). These novel
4-(4-(E)-(propen-1-yl)phenylamino)quinazoline derivatives were evaluated for their anti-proliferative
mutation
effects in vitro against the A431WT epidermoid carcinoma cells, A549K-ras
[30, 31] and
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NCI-H1975L858R/T790M NSCLC cells, as well as SW480 colon cancer cells. In order to reveal their
antiproliferative mechanism against tumor cells, the most active compound was subjected to the
enzymatic inhibition experiment against EGFRwt kinase by ELISA method, and the investigation in its
effect on the activation of EGFR and EGFR downstream signaling pathways by western blot.
Fig. 2. Previous synthesized compounds 1a-1c.
2. Results and discussion
2.1. Chemistry
In this work, a series of new quinazoline derivatives with (E)-propenyl were synthesized as shown
in Scheme 1. To obtain the 4-(E)-(propen-1-yl)aniline building block, Heck reaction of 4-nitro
bromobenzene and propene in N-methyl pyrrolidone was carried out in the presence of Pd(OAc)2 and
Et3N under 1.6 MPa, 110 °C for 2 h. The obtained mixture was concentrated, washed with water, and
purified by silica gel column chromatography to give 1-nitro-4-(E)-(propen-1-yl)benzene as yellow
needle crystals in 87% yield. 4-(E)-(Propen-1-yl)aniline was prepared by the reduction of
1-nitro-4-(E)-(propen-1-yl)benzene with Na2S2O4 in 63% yield. The configuration of C=C was identified
as E form deducing from the coupling constant 3J of hydrogen atoms at double bond in 15.7 Hz in its 1H
NMR. On the other hand, 4,5-disubstituted-2-nitrobenzonitriles (2) as starting materials were reduced
with Na2S2O4 to yield 4,5-disubstituted-2-aminobenzonitriles (3) in 66% – 89%. Then, compound 4 was
obtained from the reaction of 3 with dimethylformamide-dimethylacetal (DMF-DMA) in toluene in the
presence of acetic acid. The target compounds 5a-5c and intermediates 5d-5i were prepared through a
Dimroth rearrangement [32] of 4 and 4-(E)-(propen-1-yl)aniline in acetic acid. Finally, 5d-5i were
treated with morpholine in the presence of potassium iodide to give the desired quinazoline derivatives
6d-6i.
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Scheme 1. Synthetic route of the target compounds 5a-5c, 6d-6i.
Reagents and conditions. i. Pd(OAc)2, Et3N, N-methyl pyrrolidone, 1.6 MPa, 110 °C, 2 h, 87%; ii. (1) Na2S2O4, EtOH/H2O, 50 °C,
20 – 30 min, (2) HCl, 70 – 80 °C, 2 h, (3) NaOH/H2O, 63% – 89%; iii. DMF-DMA, AcOH, 35 – 40 °C, 15min, 61% – 94%; iv.
4-(E)-(propen-1-yl)aniline, AcOH, 130 °C, 15 min, 63% – 92%; v. KI, morpholine, 120 °C, 0.5 – 1.5 h, 50% – 76%.
2.2. Biological activities
2.2.1. In vitro antiproliferative activity
All the target compounds were evaluated for their ability to inhibit the proliferation of the four
different human tumor cell lines, including A431WT epidermoid carcinoma cells, A549K-ras mutation and
NCI-H1975L858R/T790M NSCLC cells, as well as SW480 colon cancer cells. To measure the IC50 for tumor
cell proliferative inhibition, the indicated compound was serially diluted with dimethyl sulfoxide, and
then diluted with the cell culture medium. Finally they were added to 96 well plates, which was
pre-seeding cells at certain cell density, to produce an 8-point dose-response curve, and the cell
proliferation was measured by MTT assay. Gefitinib and lapatinib were used as reference compounds.
The results are listed in Table 1. For comparison, the bioactive data of the previously identified
compounds 1a-1c are also showed in Table 1. Except compound 5a, most synthesized compounds
showed significant antiproliferative activities with IC50 values in the range of 1.35 - 57.71 µM against
A431 cells, which has a high expression of wild type EGFR. Compounds 6e and 6i were the most potent
agents (with IC50 values of 1.35 and 6.34 µM, respectively) against A431 cells. Assays with A549
NSCLC cells, harbored wild type EGFR and K-ras mutation, were also performed. As shown in Table 1,
all the synthesized compounds have antiproliferative activity against A549 cells. Compounds 1b, 6d, 6e,
6g and 6i (with IC50 of 17.43, 14.09, 8.83, 10.32 and 10.32 µM, respectively) possessed higher activities
than gefitinib (IC50 of 21.17 µM), whilst, 6d, 6e, 6g and 6i also showed higher activity than lapatinib
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(IC50 of 14.90 µM). We then assessed the inhibitory efficacy of these compounds against the
drug-resistant NSCLC cell line NCI-H1975, which harbors EGFR-L858R/T790M double mutants.
Compounds 6e and 6i showed high inhibitory effects. Particularly, compound 6e exhibited excellent
inhibitory activity with IC50 of 5.53 µM. The result of antiproliferative test on SW480 cells indicated
that the whole series displayed inhibitory effects against SW480 cells. Especially, compounds 1b, 6e
and 6i (with IC50 values of 10.40, 6.08 and 11.63 µM, respectively) showed higher inhibitory activities
than gefitinib and lapatinib (IC50 of 12.50 and 12.58 µM, respectively).
Table 1
The antiproliferative effects of the synthesized compounds in vitro and the kinase inhibitory activities.a
Compounds
IC50 (µM)b
NCI-H1975
IC50 (nM)c
EGFR-TK
3.22±1.48
N.D.e
R’
R’’
A431
A549
SW480
gefitinib
1a
4.45±0.25
57.71±2.72
12.03±0.67
21.17±0.47
85.32±2.07
17.43±3.22
12.70±2.98
>100d
12.50±0.28
62.58±14.80
10.40±0.94
H
H
1b
23.86±4.57
N.D.e
1c
5a
H
37.56±2.79
>100d
84.50±13.12
38.18±11.56
>100d
>100d
56.34±3.03
34.03±4.08
N.D.e
N.D.e
5b
5c
6d
28.11±0.04
23.88±1.05
10.07±2.41
36.45±12.95
24.74±1.25
14.09±1.03
44.13±2.96
27.67±7.04
89.34±9.98
37.04±4.46
43.65±4.06
29.21±12.13
N.D.e
N.D.e
N.D.e
6e
6f
1.35±0.32
47.08±2.89
13.91±2.05
22.41±9.76
8.83±3.80
5.53±0.30
>100d
6.08±0.32
28.19±8.66
14.66±2.17
27.56±6.30
20.72±6.41
N.D.e
26.47±1.86
10.32±2.68
37.53±14.16
6g
6h
6i
38.88±14.16
>100d
N.D.e
N.D.e
6.34±0.97
4.80±0.71
10.32±1.23
14.90±1.21
10.77±2.75
9.08±5.82
11.63±2.69
12.58±1.35
N.D.e
lapatinib
27.06±3.77
a
b
The values are mean±SD of at least three independent experiments. The antiproliferative effects of the synthesized
c
compounds against human cancer cells gathered with MTT assay. The inhibitory effects of selected compounds on
recombinant EGFRWT-TK from ELISA analyses. d IC50 was not calculated because less than 50% inhibition was observed at
the highest concentration (100 µM). e N.D. indicated IC50 not determined.
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For the synthesized 6,7-disubstituted quinazoline derivatives, most compounds containing a
morpholino moiety exhibited higher antiproliferative activities against the tested cells than other
compounds such as 5a, 5b and 5c. Interestingly the activities of quinazoline derivatives with
morpholinopropoxy group were higher than that of compounds with morpholinoethoxyl group. Notably,
compound 6e with bis(morpholinopropoxy) groups showed the highest antiproliferative activities
against all tested four tumor cells (IC50 of 1.35, 8.83, 5.53 and 6.08 µM). In addition, as shown in Fig. 3,
compound 6e exhibited a good dose-response relationship when four tumor cells were treated with
different concentration of 6e for 72 h.
A
B
C
D
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
gefitinib
6e
gefitinib
6e
gefitinib
6e
gefitinib
6e
10-10
10-8
10-6
[Comp.] M
10-4
10-10
10-8
10-6
10-4
10-10
10-8
10-6
10-4
10-8
10-7
10-6
10-5
10-4
[Comp.] M
[Comp.] M
[Comp.] M
Fig. 3. Dose response curve of inhibition rate to series concentrations of 6e and gefitinib against the four human tumor
cell lines. (A) A431 cells, (B) A549 cells, (C) NCI-H1975 cells, (D) SW480 cells. Cells were treated with 6e or gefitinib
at the series indicated concentrations, and viable cells were measured after 72 h of treatment. All error bars represent in
mean±SE.
2.2.2. Kinase inhibitory activity
Compound 6e presented remarkable antiporliferative activities against the tested four tumor cell
lines. Especially, it exhibited strong activity against A431 cells with highly expressed wild type EGFR.
We speculated 6e may display its activity through the inhibition of EGER signaling pathway, and be a
potential EGFR inhibitor. Thus, the inhibitory effect of 6e on EGFRWT-TK enzymatic activity was
evaluated with recombinant human EGFR protein and anti-phosphotyrosine antibody by ELISA method.
Herein, two EGFR inhibitors, gefitinib and lapatinib, were used as reference compounds. The
experiment data are listed in Table 1. The results showed that compound 6e had higher inhibition activity
(IC50 = 20.72 nM) against EGFR than lapatinib (IC50 = 27.06 nM). This undoubtedly demonstrated that
6e was a potent EGFR inhibitor.
2.3. Molecular modeling
To predict the binding mode of compound 6e with EGFR, a docking study of 6e into the active
site of the EGFR (PDB ID: 1xkk) were performed using Surflex-Dock module of SYBYL-X 2.1. As our
previous work [33], the calculated root-mean-square deviation (RMSD) between the best docked pose
and the observed pose of lapatinib in crystal from X-ray diffraction analysis was 1.053 Å. The
reference compound gefitinib was also docked into the active site of the EGFR (Fig. 4A). The docking
results indicated that compound 6e has a similar binding mode to that displayed by gefitinib, but in
different pose compared with lapatinib as shown in Fig. 4. The three compounds, gefitinib, 6e and
lapatinib, all formed a hydrogen bond from the N1 of quinazoline core to the main chain NH of Met793,
and the length of hydrogen bond were 2.461, 2.274 and 1.908 Å, respectively. Comparing with
gefitinib and lapatinib, another hydrogen bond existed between 6e and EGFR from the oxygen atom in
the 7-(3-morpholinopropoxy) group of 6e to the residue Thr854 of EGFR with the length of 2.105 Å
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(Fig. 4B). Additionally, a second hydrogen bond between lapatinib and EGFR was seen, from the F
atom in 3-fluorobenzyloxy moiety of lapatinib to the residue Thr790 of EGFR with length of 2.676 Å
(Fig. 4C). These all indicated that the introduction of (E)-propen-1-yl group and two
3-morpholinopropoxy to the 4-anilinoquinazoline core of 6e lead to a new binding interactions with the
EGFR-TK domain, which contributes to its inhibitory activity against EGFR.
Fig. 4. Binding modes between EGFR-TK and representative compounds predicted by Surflex-Dock program. (A)
gefitinib within 1xkk; (B) 6e within 1xkk; (C) lapatinib within 1xkk. The hydrogen bonds were illustrated as green
dashed lines and the length of hydrogen bonds was illustrated in numbers (unit in Å).
2.4. The effect of 6e on EGFR signaling pathways
In order to further investigate the antiproliferative mechanism of compound 6e against tumor cells,
we examined the effects of 6e on the activation of EGFR and its downstream signaling proteins in human
lung cancer cell lines A549 and NCI-H1975 by western blot analysis. As shown in Fig. 5A, when A549
cells were treated with 25 µM of 6e or gefitinib, it was found that the phosphorylations of EGFR and its
downstream signaling proteins induced by epidermal growth factor (EGF) were significantly inhibited
by 6e or gefitinib resulting from the decreased levels of p-Tyr/EGFR (p-Tyr), p-EGFR (Y1086), p-EGFR
(Y992), p-Akt (S473), p-Erk1/2 (T202/Y204). Even when the concentration of 6e was at 5 µM, the above
phosphorylated proteins could also be effectively down-regulated. Importantly, the total levels of EGFR,
Akt, and Erk1/2 proteins remained unchanged, and the Stat3/p-Stat3 pathway was not influenced by 6e.
These results revealed that compound 6e exerted its antiproliferative effect against A549 tumor cells via
the inhibition of the EGF/EGFR signaling pathway and its downstream Akt and Erk1/2 signaling
pathways.
Compound 6e also exhibited antiproliferative activity against NCI-H1975 cells (IC50 = 5.53µM).
When NCI-H1975 cells were treated with different concentrations of 6e, the western blot analysis
showed that the levels of p-Tyr/EGFR, p-EGFR (Y1068) and p-EGFR (Y992) were decreased by 6e in a
dose-dependent manner (see Fig. 5B). However the levels of p-Akt (S473) and p-Erk1/2 (T202/Y204)
were only slightly down-regulated at the high concentration of 6e (25 µM). Interestingly, the level of
p-Stat3 (Y705) was also decreased at the high concentration of 6e, which was not observed in the
presence of gefitinib (25 µM). This indicated that the antiproliferative mechanism of compound 6e
against NCI-H1975 cells was different from that of gefitinib, although they all exhibited the similar
inhibition of EGFR activity. Additionally, the above results suggested that compound 6e mainly block
the EGF/EGFR and the downstream p-Stat3/Stat3 pathways to achieve its potent antiproliferative
activity against NCI-H1975 cells.
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Fig. 5. Western-blot analysis of 6e against A549 cells (A) and NCI-H1975 cells (B). Cells were cultured in the presence of
different concentrations of 6e or gefitinib (25 µM) for 2 h and stimulated with 50 ng/mL EGF for 10min, then harvested.
Whole-cell lysate was analyzed for total EGFR, State, Akt and Erk1/2, as well as p-Tyr/EGFR, p-EGFR (Y1068), p-EGFR
(Y992), p-Stat3 (Y705), p-Akt (S473) and p-Erk1/2 (T202/Y204) by immunoblotting. A representative anti-GAPDH
immunoblot was showed as loading control.
3. Conclusions
In summary, we have designed and synthesized a series of novel quinazoline derivatives with
(E)-propen-1-yl substituents at the aniline moiety. These compounds were subjected to biological
activities evaluation including antiproliferative effects against four tumor cell lines (A431, A549,
NCI-H1975 and SW480). Most of the synthesized compounds exhibited moderate to excellent
antiproliferative activities against the tested tumor cell lines. Notably, compound 6e not only exhibited
excellent antiproliferative activities against the tumor cells which expressed wild type or mutant EGFR
(with the IC50 values in the range of 1.35 - 8.83 µM), but also showed potent inhibitory activity toward
EGFRWT (IC50 = 20.72 nM). Molecular docking showed that 6e could form two hydrogen bonds with
EGFR, and the binding pose of 6e was similar to that of gefitinib, while different from that of lapatinib.
In addition, the results of western blot analysis indicated that 6e could inhibit EGF-induced EGFR
activation in human lung cancer A549 and NCI-H1975 cells. However, EGF-induced EGFR downstream
phosphorylation proteins, such as p-Akt (S473), p-Erk1/2 (T202/Y204) and p-Stat3 (Y705), displayed
different levels on the two tested cell lines respectively under specified concentration of 6e, which
indicated affirmatively that 6e interacted with the signaling proteins in different manner in A549 and
NCI-H1975L858R/T790M cells. The above facts suggested compound 6e is a potent EGFR TKI, and may
possess inhibitory effect against drug resistance tumor. This work would be very useful for developing a
new series of TKIs targeting EGFR.
4. Materials and methods
4.1. Chemistry
All commercially available starting materials, reagents and solvents were used without further
purification unless otherwise noted. Melting point (m.p) was determined on X-6 micro melting point
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apparatus, and was uncorrected. High-resolution mass spectra (HRMS) were obtained on a
1
BrukerEspuire 3000plus instrument. H NMR and 13C NMR spectra were recorded on 300, 400, 600
MHz (Bruker Avance) instruments, and chemical shifts were reported in parts per million (ppm) relative
to the internal standard tetramethylsilane (TMS). Coupling constants (J) were reported in Hz. Spin
multiplicities were described as s (singlet), brs (broad singlet), d (double), t (triplet), q (quartet), or m
(multiplet). Infrared Spectroscopy (IR) was measured on Nicolet 170SXFT-IR instrument.
Chromatographic separations were performed on silica gel columns by flash column chromatography.
Reactions were followed by thin layer chromatography (TLC) on pre-coated silica gel F254 plates and
then visualized in an iodine chamber or with a UV lamp.
4.1.1. General procedure for the synthesis of 3a-3i
A mixture of 2 (0.01 mol) in water (15 mL) and ethanol (15 mL) was stirred and heated up to 50 °C.
Na2S2O4 (0.03 mol) was added within an hour. The resultant mixture was stirred for about 30 min. Then
concentrated HCl (8 mL) was added slowly in 2 h at 70 – 80°C. The mixture was cooled to 25 °C and
adjusted pH to 9 – 11 with 20% sodium hydroxide (100 mL). The obtained mixture was extracted with
ethyl acetate (3 × 60 mL) and the combined organic extracts were washed with water (2 × 100 mL) and
brine (1 × 100 mL), then dried over anhydrous Na2SO4. The organic solvent was evaporated to give
compound 3 as green yellow solid (yield 66 – 89%).
4.1.2. General procedure for the synthesis of 4a-4i
A mixture of 3 (2.00 mmol), toluene (5 mL), and DMF-DMA (4.00 mmol) were heated up to 35 –
40 °C and acetic acid (0.10 mL) was added. After 15 min, the resultant mixture was cooled to
approximately 25 °C. The solvent was completely evaporated. The residue was added to water and
basified pH to 13 by the addition of 20% sodium hydroxide. The obtained mixture was extracted with
methylene chloride (2 × 15 mL) and the combined organic extracts were washed with water (2 × 20 mL)
and brine (1 × 70 mL), then dried over anhydrous MgSO4. The organic solvent was evaporated to give 4
as oil (Yield 61 – 94%).
4.1.3.
General procedure for the synthesis of 5a-5i
Compound 4 (1.00 mmol) was added to the mixture of acetic acid (2.0 mL) and
4-(E)-(propen-1-yl)aniline (1.10 mmol). The reaction mixture was heated to 130 °C with stirring for 15
min. The mixture was then cooled to 25 °C. The acetic acid was evaporated under reduced pressure. The
residue was added to ice-water (10 mL), and adjusted pH to 9 with an ammonia solution. Then it was
stirred for 0.5 h. The precipitated product was filtered, and the filtered cake was washed with water (3 ×
10 mL) to give crude product. The crude product was purified by silica gel chromatography (eluting with
EtOAc/petroleum ether (1/4,v/v)) to afford 5 (yield 63 – 92%).
4.1.3.1. 4-(4-(E)-(Propen-1-yl)phenylamino)-7,8-dihydro-[1,4]dioxino[2,3-g]quinazoline (5a)
Yield: 91%; yellow solid; m.p.: 249.5 – 251.3 °C; HRMS (C19H17N3O2) m/z [M+H]+: calculated:
320.1399, found: 320.1386; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 9.47 (s, 1H), 8.43 (s, 1H), 8.09 (s,
1H), 7.82 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.17 (s, 1H), 6.39 (d, J = 15.7 Hz, 1H), 6.28 -
6.20 (m, 1H), 4.41 (br s, 2H), 4.40 (br s, 2H),1.85 (d, J = 6.2 Hz, 3H); 13C NMR (101 MHz, DMSO-d6)
δ(ppm): 156.9, 153.5, 149.4, 146.5, 144.0, 138.7, 133.0, 131.0, 126.2, 124.5, 122.4, 113.0, 110.5, 108.9,
65.0, 64.6, 18.8; IR νmax(KBr) cm-1: 3440, 3023, 2924, 1634, 1608, 1569, 1509, 1459, 1236, 1067, 961,
903.
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4.1.3.2. 4-(4-(E)-(Propen-1-yl)phenylamino)-6,7- bis(benzyloxy)quinazoline (5b)
Yield: 71%; yellow solid; m.p.: 164.8 – 165.5 °C; HRMS (C31H27N3O2) m/z [M+H]+: calculated:
474.2182, found: 474.2186; 1H NMR (300 MHz, DMSO-d6) δ(ppm): 9.49 (s, 1H), 8.46 (s, 1H), 8.08 (s,
1H), 7.77 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 7.0 Hz, 2H), 7.50 (d, J = 7.0 Hz, 2H), 7.44 - 7.33 (m, 8H),
7.31 (s, 1H), 6.41 (d, J = 16.0 Hz, 1H), 6.32 - 6.10 (m, 1H), 5.34 (s, 2H), 5.30 (s, 2H), 1.86 (d, J = 6.1
Hz, 3H); 13C NMR (151 MHz, DMSO-d6) δ(ppm): 172.0, 156.3, 153.4, 153.0, 148.1, 146.9, 138.2,
136.6, 136.5, 132.6, 130.5, 128.5, 128.5, 128.1, 127.9, 127.4, 125.7, 124.1, 122.2, 109.0, 109.0, 104.1,
70.6, 69.8, 18.2; IR νmax(KBr) cm-1: 3297, 3036, 2916, 1680, 1626, 1605, 1587, 1515, 1452, 1252, 1021,
965, 837.
4.1.3.3. 4-(4-(E)-(Propen-1-yl)phenylamino)-6,7-bis(2-methoxyethoxy)quinazoline (5c)
Yield: 74%; faint yellow solid. m.p.: 151.3 – 152.8 °C; HRMS (C23H27N3O4) m/z [M+H]+:
calculated: 410.2080, found: 410.2085; 1H NMR (600 MHz, CDCl3) δ(ppm): 8.62 (s, 1H), 7.69 (s, 1H),
7.60 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.26 (s, 1H), 7.16 (s, 1H), 6.37 (dd, J =1.6, 15.7 Hz,
1H, -CH＝CH-), 6.18 (dq, J = 15.7, 6.6 Hz, 1H, -CH＝CH-), 4.20 - 4.17 (m, 4H), 3.79 - 3.78 (m, 2H),
3.75 (t, J = 4.8 Hz, 2H), 3.41 (s, 3H), 3.40 (s, 3H), 1.88 (dd, J =1.6, 6.6 Hz, 3H); 13C NMR (151 MHz,
CDCl3) δ(ppm): 156.5, 154.5, 153.8, 148.7, 147.4, 137.3, 134.2, 130.4, 126.4, 125.0, 122.0, 109.2,
108.7, 102.9, 70.9, 70.4, 69.2, 68.3, 59.3, 59.2, 18.5; IR νmax(KBr) cm-1: 3426, 3020, 2926, 2883, 1620,
1576, 1514, 1427, 1244, 1123, 966, 856.
4.1.4. General procedure for the synthesis of 6d-6i
A mixture of 5 (0.11 mmol), potassium iodide (0.06 mmol) and morpholine (1 mL) was heated to
120 °C for 0.5 – 1.5 h, then filtered, and washed with CH2Cl2. The combined mixture of filtering liquor
and washing liquor was dried (MgSO4) and evaporated. The residue was purified by silica gel column
chromatography with MeOH/EtOAc (1:10) to give the titled compounds (yield 50 – 76%).
4.1.4.1. 4-(4-(E)-(Propen-1-yl)phenylamino)-6,7-bis(2-morpholinoethoxy)quinazoline (6d)
Yield: 73%; m.p.: 197.4 – 198.5 °C; HRMS (C29H37N5O4) m/z [M+H]+: calculated: 520.2924, found:
520.2926; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 9.43 (s, 1H), 8.46 (s, 1H), 7.87 (s, 1H), 7.76 (d, J =
8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 7.20 (s, 1H), 6.39 (d, J = 15.8 Hz, 1H), 6.31 - 6.09 (m, 1H), 4.27 -
4.23 (m, 4H), 3.71 - 3.50 (m, 8H), 2.81 - 2.75 (m, 4H), 2.60 - 2.53 (m, 8H), 1.85 (d, J = 6.2 Hz, 3H); 13C
NMR (101 MHz, DMSO-d6) δ(ppm): 158.1, 155.4, 154.8, 149.9, 148.8, 140.1, 134.4, 132.4, 127.6,
125.9, 124.1, 110.8, 110.0, 105.1, 69.1, 68.8, 68.1, 61.6, 58.8, 58.6, 55.7, 55.7, 20.2; IR νmax(KBr)cm-1:
3296, 3077, 2964, 1648, 1514, 1454, 1427, 1262, 1038, 969, 855.
4.1.4.2. 4-(4-(E)-(Propen-1-yl)phenylamino)-6,7-bis(3-morpholinopropoxy)quinazoline (6e)
Yield: 50 %; m.p.: 91.3 – 92.2 °C; HRMS (C31H41N5O4) m/z [M+H]+: calculated: 548.3237, found:
548.3246; 1H NMR (300 MHz, DMSO-d6) δ(ppm): 9.47 (s, 1H), 8.44 (s, 1H), 7.86 (s, 1H), 7.76 (d, J =
8.2 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.17 (s, 1H), 6.40 (d, J = 16.0 Hz, 1H), 6.31 - 6.12 (m, 1H), 4.25
- 4.10 (m, 4H), 3.34 - 3.55 (m, 12H), 2.45 - 2.31 (m, 8H), 2.05 - 1.90 (m, 4H), 1.85 (d, J = 6.1 Hz, 3H);
13C NMR (75 MHz, DMSO-d6) δ(ppm): 156.2, 153.7, 152.8, 148.3, 146.9, 138.2, 132.5, 130.5, 125.7,
124.0, 122.2, 108.9, 107.9, 103.2, 67.1, 66.5, 66.1, 66.1, 54.9, 54.8, 53.4, 53.3, 25.8, 25.6, 18.2; IR
νmax(KBr) cm-1: 3435, 3026, 2935, 2839, 1613, 1500, 1435, 1371, 1226, 1113,984.
4.1.4.3. 4-(4-(E)-(Propen-1-yl)phenylamino)-7-methoxy-6-(2-morpholinoethoxy)quinazoline (6f)
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Yield: 71%; m.p.: 204.9 – 205.9 °C; HRMS (C24H28N4O3) m/z [M+H]+: calculated: 421.2240,
found: 421.2243; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 9.43 (s, 1H), 8.47 (s, 1H), 7.86 (s, 1H), 7.75
(d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.19 (s, 1H), 6.40 (dd, J =1.1, 15.7 Hz, 1H, -CH＝CH-),
6.23 (dq, J = 15.7, 6.4 Hz, 1H, -CH＝CH-), 4.26 (t, J = 6.0 Hz, 2H), 3.94 (s, 3H), 3.63 - 3.58 (m, 4H),
2.81 (t, J = 6.0 Hz, 2H), 2.57 - 2.52 (m, 4H), 1.85 (dd, J =1.1, 6.4 Hz, 3H); 13C NMR (101 MHz,
DMSO-d6) δ(ppm): 156.2, 154.2, 152.9, 148.0, 146.9, 138.2, 132.6, 130.5, 125.7, 124.0, 122.2, 108.9,
107.3, 102.8, 66.6, 66.2, 56.9, 55.8, 53.7, 18.2; IR νmax(KBr) cm-1: 3418, 3020, 2932, 2850, 1624, 1578,
1514, 1117, 964, 847.
4.1.4.4. 4-(4-(E)-(Propen-1-yl)phenylamino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline (6g)
Yield: 76%; m.p.: 186.9 – 187.4 °C; HRMS (C25H30N4O3) m/z [M+H]+: calculated: 435.2396,
found: 435.2395; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 9.47 (s, 1H, NH), 8.46 (s, 1H), 7.84 (s, 1H),
7.74 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.18 (s, 1H), 6.37 (d, J = 16.0 Hz, 1H, -CH＝CH-),
6.27 - 6.18 (m, 1H), 4.18 (t, J = 5.9 Hz, 2H), 3.93 (s, 3H), 3.58 (brs, 4H), 2.47 (t, J = 7.0 Hz, 2H), 2.38
(brs, 4H), 2.00 - 1.97 (m, 2H), 1.85 (d, J = 6.4 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ(ppm): 156.2,
154.3, 152.8, 148.2, 146.9, 138.2, 132.5, 130.5, 125.7, 124.0, 122.2, 108.9, 107.2, 102.7, 67.1, 66.1,
55.8, 54.9, 53.4, 25.9, 18.2; IR νmax(KBr) cm-1: 3418, 3022, 2953, 2870, 1622, 1512, 1427, 1240, 1113,
955, 858.
4.1.4.5. 4-(4-(E)-(Propen-1-yl)phenylamino)-6-methoxy-7-(2-morpholinoethoxy)quinazoline (6h)
Yield: 55%; m.p.: 215.1 – 216.8 °C; HRMS (C24H28N4O3) m/z [M+H]+: calculated: 421.2240,
1
found: 421.2227; H NMR (600 MHz, CDCl3) δ(ppm): 8.64 (s, 1H), 7.70 (s, 1H), 7.59 (d, J = 8.4 Hz,
2H), 7.33 (d, J = 8.4 Hz, 2H), 7.21 (s, 1H), 7.15 (s, 1H), 6.36 (d, J = 15.6 Hz, 1H, -CH＝CH-), 6.22 -
6.13 (m, 1H), 4.23 (t, J = 5.8 Hz, 2H), 3.90 (s, 3H), 3.73 – 3.70 (m, 4H), 2.88 (t, J = 5.8 Hz, 2H), 2.58
(brs, 4H), 1.89 (d, J = 5.7 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ(ppm): 156.4, 153.9, 153.6, 149.6,
147.1, 137.1, 134.3, 130.4, 126.4, 125.1, 122.1, 109.2, 108.5, 100.0, 66.9, 66.8, 57.0, 56.3, 54.1, 18.5;
IR νmax(KBr) cm-1: 3418, 3026, 2947, 2856, 1622, 1512, 1420, 1240, 1105, 970, 851, 787.
4.1.4.6. 4-(4-(E)-(Propen-1-yl)phenylamino)-6-methoxy-7-(3-morpholinopropoxy)quinazoline (6i)
Yield: 50%;. m.p.: 170.3 – 171.9 °C; HRMS (C25H30N4O3) m/z [M+H]+: calculated: 435.2396,
1
found: 435.2382; H NMR (400 MHz, CDCl3) δ(ppm): 8.64 (s, 1H), 7.64 (s, 1H), 7.58 (d, J = 8.4 Hz,
2H), 7.34 (d, J = 8.4 Hz, 2H), 7.23 (s, 1H), 7.13 (s, 1H), 6.38 (d, J = 15.7 Hz, 1H, -CH＝CH-), 6.23 -
6.14 (m, 1H), 4.17 (t, J = 6.6 Hz, 2H), 3.92 (s, 3H), 3.75 – 3.68 (m, 4H), 2.53 (t, J = 7.2 Hz, 2H), 2.46
(brs, 4H), 2.10 - 2.03 (m, 2H), 1.88 (d, J = 6.5 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ(ppm): 156.4,
154.1, 153.6, 149.6, 147.3, 137.1, 134.3, 130.4, 126.4, 125.1, 122.1, 109.0, 108.4, 99.8, 67.3, 66.9, 56.3,
55.3, 53.7, 25.9, 18.5; IR νmax(KBr) cm-1: 3379, 3024, 2957, 2932,2 851, 1624, 1512, 1421, 1244, 1117,
964, 862, 787.
4.2. Biology
4.2.1.
In vitro antiproliferative activity assay
Dulbecco’s modified Eagle’s medium (DMEM), 1640 and fetal bovine serum (FBS) were obtained
from Gibco. Penicillin, streptomycin and L-glutamine were purchased from Merck Millipore. A431
(epidermoid carcinoma, EGFR wild type and overexpression), A549 (NSCLC, EGFR wild type, K-ras
mutation), NCI-H1975 (NSCLC, EGFR L858R/T790M mutation) and SW480 (colon cancer, EGFR
11

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wild type) cells were kindly provided by Cell Bank, Chinese Academy of Sciences. Cells were plated in
96-well plates in regular DMEM or 1640 supplemented with 10% (v/v) FBS, 100 units/mL of penicillin,
100 µg/mL of streptomycin, 2 mM L-glutamine. After 24 h incubation at 37ꢀ°C in a humidified incubator
containing 5% CO2, cells were exposed to different concentrations of each compounds or 0.5% DMSO,
then incubated for further 72 h. Cell viability was determined by MTT assay. Briefly, following drug
treatment, cells were incubated with 20 µl MTT (5 mg/ml) for 4 h at 37 °C. Next, the medium was
discarded and 150 µL DMSO were added to dissolve the formazan product. The absorbance was
measured using a multi-well spectrophotometer (PerkinElmer Enspire) at 570 nm, and 630 nm as
reference wavelength. The IC50 values were calculated from the inhibition curves by nonlinear regression
analysis using GraphPad Prism5 (GraphPad Software, Inc.).
4.2.2.
In vitro EGFR tyrosine kinase assay
Recombinant EGFR was purchased from Sino Biology Inc. Anti-phosphotyrosine mouse mAb was
purchased from PTM Bio. The effects of compounds on the activity of EGFR kinase were determined by
enzyme-linked immunosorbent assays (ELISAs) with recombinant EGFR according to reported
methods [33, 34].
4.2.3.
Molecular docking
All the calculations were carried out on a Lenovo PC with Windows 8.1 system using the Tripos
Sybyl-X 2.1 (TriposInc, St Louis, MO, USA) molecular modeling package. The crystal structural data of
EGFR kinase domain complexed with lapatinib (PDB code: 1xkk) was obtained from RCSB Protein
Data Bank [35]. Molecular docking was carried out as previous reported [33].
4.2.4.
Western blot analysis
Western blot analysis was carried out according to a reported method [36]. Cells were grown in
6-well plates in media containing with 10% (v/v) FBS until reached 90% confluence, the cultured cells
were serum-starved for 24 h, after which they were exposed to a dose range of each drug for 2 h and
subsequently treated with 50 ng/mL EGF for 10 min at 37 °C. After washing 3 times with cold PBS, they
were resuspended in 100 µL ice-cold RIPA lysis buffer, together with protease and phosphatase inhibitor
tablet. The lysates were kept on ice for 30 min with occasional agitation and collected by centrifugation
at 12,000 ×g for 20 min at 4 °C. The protein concentration in the supernatant was determined using a
BCA protein assay kit. The samples were prepared in SDS-PAGE loading buffer, and then boiled for 10
min at 95 °C. Equal amounts of protein (20 µg) were added to a 10% SDS-polyacrylamide gel (PAGE)
electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore).
Non-specific binding on the membrane was minimized with a blocking buffer containing non-fat dry
milk (5%) in TBS. Thereafter, immunoblotting was performed according to the antibody manufacturers’
recommendations. The primary antibody-bound membranes were washed for 5 min × 5 times with a
washing buffer (TBS solution containing 0.1% Tween-20), then incubated with corresponding secondary
antibodies conjugated with horseradish peroxidase. After a 30 min washing, immune reactive signals
were visualized by enhanced chemiluminescence using ChemiDoc XRS+ system (Bio-Rad, Hercules,
CA, USA). All antibodies were obtained from Cell Signaling Technology.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (Grant
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number 21272144), and the Fundamental Research Funds for the Central Universities of Shaanxi Normal
University (Grant number X2015YB06).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.
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(2004) 13-21.
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Highlights
1. A series of novel 4-(4-(E)-(propen-1-yl)phenylamino)quinazoline derivatives were designed and
synthesized.
2. Most synthesized compounds exhibited good antiproliferative activities against human tumor cells
harboring wild type or L858R/T790M double mutant EGFR.
3. Molecular docking suggested that the binding mode of compound 6e with EGFR was similar to
gefitinib, but different from lapatinib.
4. Compound 6e can significantly inhibit EGFR activity, and suppress the phosphorylation of EGFR
and its downstream signaling proteins in tumor cells.