Novel 4-arylaminoquinazolines bearing N,N-diethyl(aminoethyl)amino moiety with antitumour activity as EGFRwt-TK inhibitor

Article abstract of DOI:10.1080/14756366.2019.1667341

Herein, four novel 4-arylaminoquinazoline derivatives with N,N-diethyl(aminoethyl)amino moiety were designed, synthesised and evaluated on biological activities in vitro. All synthesised compounds have inhibitory effects against tumour cells (SW480, A549, A431 and NCI-H1975). In particular, 4-(3-chloro-4-(3-fluorobenzyloxy)phenylamino)-6-(5-((N,N-diethyl(aminoethyl))aminomethyl)furan-2-yl)quinazoline (6a) and 6-(5-((N,N-diethylethyl)aminomethyl)furan-2-yl)-4-(4-(E)-(propen-1-yl)phenylamino)quinazoline (6d) were potent antitumour agents which showed high antiproliferative activities against tumour cells in vitro. Moreover, compound 6a could induce late apoptosis of A549 cells at high concentrations and arrest cell cycle of A549 cells in the G0/G1 phase at tested concentrations. Also, compound 6a could inhibit the activity of wild type epidermal growth factor receptor tyrosine kinase (EGFR^wt-TK) with IC₅₀ value of 15.60 nM. Molecular docking showed that compound 6a formed three hydrogen bonds with EGFR^wt-TK, while lapatinib formed only two hydrogen bonds with the receptor protein. It is believed that this work would be giving a reference for developing anti-cancer drugs targeted EGFR-TK.

Full text of DOI:10.1080/14756366.2019.1667341

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel 4-arylaminoquinazolines bearing N,N-
diethyl(aminoethyl)amino moiety with antitumour
activity as EGFRwt-TK inhibitor
Yaling Zhang, Li Chen, Xiabing Li, Li Gao, Yunxia Hao, Baolin Li & Yaping Yan
To cite this article: Yaling Zhang, Li Chen, Xiabing Li, Li Gao, Yunxia Hao, Baolin Li & Yaping
Yan (2019) Novel 4-arylaminoquinazolines bearing N,N-diethyl(aminoethyl)amino moiety with
antitumour activity as EGFRwt-TK inhibitor, Journal of Enzyme Inhibition and Medicinal Chemistry,
34:1, 1668-1677, DOI: 10.1080/14756366.2019.1667341
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1668–1677
https://doi.org/10.1080/14756366.2019.1667341
ORIGINAL ARTICLE
Novel 4-arylaminoquinazolines bearing N,N-diethyl(aminoethyl)amino moiety with
antitumour activity as EGFRwt-TK inhibitor
Yaling Zhanga , Li Chenb, Xiabing Lib, Li Gaob, Yunxia Haob, Baolin Lia,b
and Yaping Yana
aNational Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, The Key Laboratory of Medicinal
Resources and Natural Pharmaceutical Chemistry, The Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an, P. R.
b
China; School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, P. R. China
ABSTRACT
ARTICLE HISTORY
Received 14 June 2019
Revised 4 September 2019
Accepted 9 September 2019
Herein, four novel 4-arylaminoquinazoline derivatives with N,N-diethyl(aminoethyl)amino moiety were
designed, synthesised and evaluated on biological activities in vitro. All synthesised compounds have
inhibitory effects against tumour cells (SW480, A549, A431 and NCI-H1975). In particular, 4-(3-chloro-4-(3-
fluorobenzyloxy)phenylamino)-6-(5-((N,N-diethyl(aminoethyl))aminomethyl)furan-2-yl)quinazoline (6a) and
6-(5-((N,N-diethylethyl)aminomethyl)furan-2-yl)-4-(4-(E)-(propen-1-yl)phenylamino)quinazoline (6d) were
potent antitumour agents which showed high antiproliferative activities against tumour cells in vitro.
Moreover, compound 6a could induce late apoptosis of A549 cells at high concentrations and arrest cell
cycle of A549 cells in the G0/G1 phase at tested concentrations. Also, compound 6a could inhibit the
activity of wild type epidermal growth factor receptor tyrosine kinase (EGFRwt-TK) with IC50 value of
15.60 nM. Molecular docking showed that compound 6a formed three hydrogen bonds with EGFRwt-TK,
while lapatinib formed only two hydrogen bonds with the receptor protein. It is believed that this work
would be giving a reference for developing anti-cancer drugs targeted EGFR-TK.
KEYWORDS
Quinazoline derivatives;
N,N-diethyl(aminoethyl)a-
mino moiety;
antiproliferative activities;
wild type epidermal growth
factor receptor tyrosine
kinase (EGFRwt-TK);
molecular docking
GRAPHICAL ABSTRACT
1. Introduction
referred as the first generation of EGFR-TKIs4,8, and lapatinib was a
dual EGFR and human epidermal growth factor receptor-2 (HER-2)
inhibitors9. They have been all approved for cancer treatment by
the US Food and Drug Administration (FDA). However, these
drugs have some limits in clinical usage, such as drug resistance
of gefitinib10,11, hepatotoxicity of lapatinib12,13, and erlotinib has
similar toxic-effect profiles with gefitinib14. Ever since FDA
approval of “Gefitinib” in 2003 and up to the last FDA approved
small molecule EGFR kinase inhibitor “Osimertinib” in 2015, find-
ing more efficient EGFR-TKIs are still going on due to the continu-
ous emergence of resistance to the current inhibitors15.
Cancer is set to become a major cause of morbidity and mortality,
and is a major public health problem worldwide1,2. Among them,
lung cancer is the most common malignant disease3, and
accounts for more than 1/4 of cancer related deaths, and the
5-year relative survival is currently less than 20%4. As the patho-
genesis of cancer continues to clarify, many biological targets
including epidermal growth factor receptor (EGFR) have been
identified playing a key role in the development of a number of
the most lethal cancers, and the activity of EGFR-specific tyrosine
kinase inhibitors (TKIs) against such cancers ushered in an era of
genotype-directed targeted therapy that fundamentally changed
the overall approach to lung cancer5. Many targeted drugs have
been developed and used in clinic, for example, gefitinib, erloti-
Our laboratory has been committed to the development of
novel antitumour drugs, and reported novel 4-arylamino-6-(5-sub-
stituted furan-2-yl)quinazoline derivatives and novel 4-anilinoqui-
nib, lapatinib, and so on6,7. Gefitinib and erlotinib have been nazoline derivatives with (E)-propen-1-yl moiety, especially
CONTACT Baolin Li
Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, The Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an
710119, P. R. China; Xiabing Li xiabingli@snnu.edu.cn School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710119, P. R. China
Supplemental data for this article can be accessed here.
baolinli@snnu.edu.cn
National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, The
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1669
Figure 1. Structures of FDA approved quinazolines and design of novel quinazoline derivatives.
compounds 2a16 and 6e12 (Figure 1), as potent EGFR inhibitors diethylethylenediamine and NaBH3CN at 0 ꢁC for 2 h. The struc-
with enhanced antiproliferative activities against tumours.
tures of 6a–6d were identified by NMR, IR and HRMS (see
Taking into account the wide spectrum of biological activities,
particularly, high antitumour effect of quinazoline derivatives, and
followed our previous studies12,16–19, herein, we introduce hydro-
philic 5-(N,N-diethyl(aminoethyl)aminomethyl)furan-2-yl moiety
and four lipophilic arylamino units, such as 3-chloro-4–(3-fluoro-
benzyloxy)phenylamino, 3-chloro-4-fluorophenylamino, 3-ethynyl-
phenylamino and 4-(E)-(propen-1-yl)phenylamino to 6- and 4-
positions of quinazoline core (Figure 1), respectively, thus four
novel quinazoline derivatives were synthesised. Meanwhile, the
synthesised compounds were evaluated for the antiproliferative
activities against human tumour cells, the antitumour mechanism,
and the effect on cell apoptosis and cell cycle in vitro.
Supplementary material).
2.2. Antiproliferative activity of novel quinazoline derivatives
in vitro
Methyl thiazolyl tetrazolium (MTT) colorimetric assay (MTT
assay)17,20 was used to evaluated the antiproliferative activities of
these novel compounds against four human tumour cell lines
including SW480, A549, A431 and NCI-H1975. Lapatinib was used
as reference compound. The IC50 values of synthesised
compounds were listed in Table 1. The results indicated that all
compounds exhibited good antiproliferative activities in a dose-
response manner (Figure 2).
Against SW480 cells, compounds 6a, 6c and 6d (with IC50 val-
ues of 6.67, 10.78 and 4.21 mM, respectively) were more potent
2. Results and discussion
2.1. Chemistry
than
lapatinib
(IC50 ¼ 12.58 mM),
while
compound
6 b
(IC50 ¼ 14.92 mM) was less potent than lapatinib. Against A549
cells, compounds 6a, 6c and 6d (with IC50 values of 5.46, 12.14
and 4.10 mM, respectively) were more potent than lapatinib
(IC50 ¼ 14.90 mM), while compound 6 b (IC50 ¼ 17.97 mM) was less
potent than lapatinib. The inhibitory efficacy of these four com-
pounds against A431 cells were all higher than lapatinib, and the
IC50 values of compounds 6a-6d and lapatinib were 2.21, 3.92,
2.59, 2.09 and 4.80 mM, respectively. Also, the whole series dis-
played inhibitory effect (with IC50 values in the range of 5.13-
17.55 mM) against H1975 cells, compounds 6a and 6d (with IC50
values of 9.79 and 5.13 mM, respectively) showed higher inhibitory
activities compared to lapatinib (IC50 ¼ 12.68 mM).
The general synthetic route for the target compounds was out-
lined in Scheme 1. The reaction of 2-aminobenzonitrile (1) with
the mixture of ammonium iodide and hydrogen peroxide in the
presence of acetic acid gave 2-amino-5-iodobenzonitrile (2) in
92.6% yield. N’-(2-cyano-4-iodophenyl)-N,N-dimethyl formamidine
(3) was prepared in 89.3% yield from 2 and N,N-dimethylforma-
mide dimethyl acetal (DMF-DMA). Dimroth rearrangement was
used to form quinazoline core. Compound 3 was respectively
mixed with four substituted anilines, including 3-chloro-4-((3-fluo-
robenzyl)oxy)aniline, 3-chloro-4-fluoroaniline, 3-ethynylaniline and
4-(E)-(propen-1-yl)aniline, in acetic acid at 1 2 5 ꢀ 130 ꢁC for
15 min, and four 4-arylamino-6-iodoquinazolines (4a–4d) were
obtained in 84.3 ꢀ 92.5% yield. Key intermediates 4-arylamino-6-
Overall, the antiproliferative activities of compounds 6a and 6d
against all tested tumour cells were much higher than that of
lapatinib, which suggested that the combination of hydrophilic 5-
((N,N-diethyl(aminoethyl))aminomethyl)furan-2-yl moiety at 6-pos-
ition and lipophilic 3-chloro-4–(3-fluorobenzyloxy)phenylamino
(5-formylfuran-2-yl)quinazolines
(5a–5d)
were
given
in
57.2 ꢀ 83.4% yield by suzuki coupling reaction of 4a–4d and 5-for-
mylfuran-2-yl boronic acid in the presence of Pd/C catalyst at
50 ꢁC for 30 min. Target compounds (6a–6d) were prepared in
78.1 ꢀ 82.6% yield by the reductive amination of 5a–5d with N,N- group or 4-(E)-(propen-1-yl)phenylamino group at 4-position of

1670
Y. ZHANG ET AL.
R
HN
CN
I
CN
I
CN
N
I
N
i
ii
iii
iv
NH2
NH2
N
N
4a-4d
1
3
2
R
R
Comp.
4a-6a
R
HN
HN
N
3-Cl, 4-(3-fluorobenzyloxy)
v
OHC
O
N
N
O
4b-6b 3-Cl, 4-F
3-ethynyl
4d-6d 4-(E)-propen-1-yl
NH
N
4c-6c
N
5a-5d
6a-6d
Scheme 1. Synthetic route of target compounds 6a–6d. Reagents and conditions: i. NH4I-H2O2, r.t. 12h, 92.6%; ii. DMF-DMA, 35 ꢁC, 0.5 h, 89.3%; iii. R2-aniline,
125–130 ꢁC, 15 min, 84.3–92.5%; iv. 5-formylfuran-2-yl boronic acid, Pd/C, 50 ꢁC, 0.5 h, 57.2–83.4%; v. N,N-diethylethylenediamine, NaBH3CN, 0 ꢁC, 2 h, 78.1–82.6%.
Table 1. The antiproliferative activities of synthesised quinazoline derivatives against human cancer cells in vitro
R
HN
N
O
NH
N
N
.
IC50 (mM)a
Compound
R
SW480
A549
A431
NCI-H1975
Lapatinib
12.58 1.35
6.67 0.95
14.92 2.43
10.78 1.34
4.21 1.16
14.90 1.21
5.46 0.19
17.97 0.62
12.14 0.37
4.10 0.66
4.80 0.71
2.21 0.25
3.92 1.10
2.59 0.15
2.09 1.01
12.68 0.73
9.79 0.07
17.55 0.03
15.62 1.41
5.13 0.55
6a
6b
6c
6d
3-Cl, 4-(3-fluorobenzyloxy)
3-Cl, 4-F
3-ethynyl
4-(E)-propen-1-yl
aThe values are mean SD of at least three independent experiments.
quinazoline core can lead to better antiproliferative activity. In neurodegenerative, autoimmune, and cardiovascular diseases21.
order to give a visual comparison of the data, the IC50 values (in After treatment by compound 6a for 48 h, the apoptosis of A549
the unit of M) were transformed into –logIC50 and gave a bar cells were measured by fluorescence-activated cell sorter (FACS)
chart reporting. As shown in Figure 3, compared with lapatinib, analysis with annexin V-fluorescein isothiocyanate (FITC) and pro-
the compound 6a showed visually the significant enhanced anti- pidium iodide (PI) labelling. As displayed in Figure 4(A,B), in the
tumour activities when SW480, A549 and A431 cells were treated untreated control groups, 97.19% of A549 cells were in their nor-
by compound 6a (p < .01 or p < .001), the same effects could be mal state. When A549 cells were treated with compound 6a
also seen when SW480, A549, A431 and NCI-H1975 cells were (20 and 10 mM) or 20 mM lapatinib, the numbers of late apoptotic
treated by compound 6d (p < .001). It’s noted that compound 6a cells were significantly higher than that of control groups. When
and lapatinib possess respectively N,N-diethyl(aminoethyl)amino- A549 cells were treated with 20 mM compound 6a or 20 mM lapati-
methyl and (2-methylsulfonylethyl)aminomethyl at the 5-position nib, the numbers of necrosis cells were also significantly higher
of furan-2-yl of 4-(3-chloro-4-(3-fluorobenzyloxy)phenylamino)-6- than that of control groups. However, significant differences were
(furan-2-yl)quinazoline core, however the antiproliferative activity not observed in the number of late apoptotic cells and necrosis
of compound 6a was much higher than that of lapatinib, which when cells were treated by the lower concentrations of com-
indicated that the N,N-diethyl(aminoethyl)amino moiety was an pound 6a, also in the number of early apoptotic cells. The results
advantaged group, and obviously increased the activity of com- indicated that the early apoptosis of A549 cells were not signifi-
pound against tumour cells. Also, N,N-diethyl(aminoethyl)amino cantly influenced when treated by compound 6a, while the late
moiety combined with other four 4-arylamino of quinazolines also apoptosis can only be induced by compound 6a at high concen-
gave a good inhibitory effects against four tested tumour cell lines.
trations (20 and 10 mM).
2.3. Effects of compound 6a on cell apoptosis in A549 cells
2.4. Effects of 6a on cell cycle of A549 cells
The process of controlled cellular death known as apoptosis has In order to study the effect of compound 6a on cell cycle, A549
an important central role not only in normal homeostatic main- cells were treated by compound 6a or lapatinib for 48 h, then the
tenance of tissues, but also in numerous diseases such as cancer, cells were strained by PI and examined using flow cytometry. As

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1671
Figure 2. Dose response curves of inhibition rate to series concentrations of target compounds 6a–6d and lapatinib against human cancer cell lines. (A) SW480 cells,
(B) A549 cells, (C) A431 cells, (D) NCI-H1975 cells. Cells were treated with compounds 6a–6d or lapatinib at the series indicated concentrations, and viable cells were
measured after 72h of treatment. All error bars were represented in mean SE.
Figure 3. The bar chart reporting the –logIC50 values of compounds 6a–6d against human tumour cell lines. IC50 values were in the unit of M. All error bars were
ꢂꢂ
ꢂꢂꢂ
p < .001 indicate significant differences from lapatinib group.
represented in mean SD. p < .01 and
2.5. Kinase inhibitory activity
shown in Figure 5(A,B), when cells were treated by compound 6a
at indicated concentrations (20, 10, 5 and 2.5 mM), the number of
A549 cells at G0/G1 phase were significantly increase, from
51.97% to 62.45%, 51.77%, 55.44% and 54.15%, respectively,
accompanied by a decrease in the S and G2/M cells. The percent-
age of G0/G1, S and G2/M cells in the group of 20 mM lapatinib
were 60.60%, 19.93% and 19.41%, respectively. The results indi-
Compound 6a presented remarkable antiproliferative activities
against tumour cells, and could arrest of the cell cycle in G0/G1
phase. Therefore, we further investigated the biological target of
compound 6a. The inhibitory effects of compound 6a on wild
type epidermal growth factor receptor tyrosine kinase (EGFRwt-TK)
were evaluated with recombinant human EGFR protein and anti-
cated A549 cells could be arrested in the G0/G1 phase by com- phosphotyrosine antibody by ELISA assay. Lapatinib was used as
pound 6a. reference compound. The results of ELISA assay displayed that the

1672
Y. ZHANG ET AL.
Figure 4. Effect of compound 6a on cell apoptosis in A549 cells. (A) Representative density plots were obtained by FACS, (B) histograms of percentages of apoptotic
cells in each group from (A) analysed by GraphPad Prism5. Cells were cultured in the presence of different concentrations of compound 6a (20–2.5 lM) or lapatinib
ꢂ
ꢂꢂ
(20lM) for 48 h, harvested, and labelled with Annexin V-FITC and PI, then analysed by FACS. All values were expressed as mean SE. p < .05, p < .01 and
ꢂꢂꢂ
p < .001 indicate significant differences compared with the control at the same group.
IC50 value of compound 6a was 15.60 0.60 nM, while that of respectively. Compound 6a and lapatinib all also formed the
lapatinib was 27.06 3.77 nM. This indicated that compound 6a second hydrogen bond from the F atom in 3-fluorobenzyloxy
was a potential EGFRwt-TK inhibitor.
moiety of compounds to the residue Thr790 of EGFR with
length of 2.500 and 2.676 Å, respectively. However, compound
6a formed the third hydrogen bond from the H atom in N,N-
diethyl(aminoethyl)amino moiety of compound 6a to the resi-
due Asp800 of EGFR with length of 2.039 Å (Figure 6(B)).
These indicated that the replacement of 2-(methylsulfonyl)e-
thylamino group of lapatinib with N,N-diethyl(aminoethyl)a-
mino moiety lead to a new binding mode with EGFRwt-TK
domain, which contributes to its enhanced inhibitory activity
against EGFRwt-TK and antiproliferative activities against
tumour cells.
2.6. Molecular modelling
In order to know the binding mode of compound 6a with
EGFRwt-TK, a study of docking of compound 6a into the
active site of EGFR (PDB ID: 1XKK) were performed using
Surflex-Dock module of Sybyl-X 2.1. As our previous work16
,
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 dock-
ing results revealed that compound 6a formed three hydro-
gen bonds with EGFR, while lapaitnib formed only two
hydrogen bonds as shown in Figure 6. Compound 6a and
lapatinib both formed a hydrogen bond from the N1 of qui-
3. Conclusions
nazoline core to the NH of the hinge region Met793, and the In conclusion, four novel 4-arylaminoquinazolines with N,N-diethy-
length of hydrogen bond were 1.964 and 1.908 Å, l(aminoethyl)amino moiety were designed, synthesised and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1673
Figure 5. Effect of compound 6a on the cell cycle phase distribution in A549 cells. (A) Representative profiles were obtained by FACS, (B) histograms of percentages
of percentages of cell populations in the G0/G1, S and G2/M phase in each group from (A) analysed by GraphPad Prism5. Cells were cultured in the presence of differ-
ent concentrations of compound 6a (20–2.5 lM) or lapatinib (20lM) for 48h, harvested, and labelled with PI, then analysed by FACS. Percentage of cells in G0/G1, S
ꢂ
ꢂꢂ
ꢂꢂꢂ
p < .001 indicate significant differences compared with the con-
and G2/M phases were indicated. All values were expressed as mean SE. p < .05, p < .01 and
trol at the same phase.
evaluated on biological activities in vitro. All the synthesised com-
pounds have inhibition potency against four tumour cell lines,
and the IC50 values against SW480, A549, A431 and NCI-H1975
cells were in the range of 4.21–14.92, 4.10–17.97, 2.09–3.92 and
5.13–17.55 mM, respectively. And compounds 6a and 6d exhibited
highly antiproliferative activities against these tumour cells in vitro
in single-digit micromole IC50. In particular, compound 6a not
only exhibited highly antiproliferative activities against four
tumour cell lines in vitro, but also could induce late apoptosis of
A549 cells at high concentrations (20 and 10 mM) and arrest cell
cycle of A549 cells in the G0/G1 phase at tested concentrations. In
addition, compound 6a could inhibit the activity of EGFRwt-TK
with IC50 value of 15.60 nM, and form three hydrogen bonds with
EGFRwt-TK. Other further studies on compounds 6a and 6d are
going on in our lab. It is believed that this work would be giving
a reference for developing of anti-cancer drugs targeted EGFR-TK.
4. Experimental section
4.1. General informations
Human cancer cell lines SW480, A431, A549 and NCI-H1975 were
purchased from the Cell Bank of the Chinese Academy of Sciences
(Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM)
and foetal bovine serum (FBS) were purchased from Gibco.
Trypsin, penicillin, streptomycin and L-glutamate were purchased
from Sigma-Aldrich. All other reagents and solvents were at ana-
lytical grade; they were supplied by local commercial suppliers
and used without further purification unless otherwise noted.
Melting point (m.p) was determined using a X-6 micromelting
point apparatus (Beijing Tech Instrument Co. Ltd., Beijing, China).
NMR (1H and 13 C) spectra were obtained using a Super-conduct-
ing Fourier Digital NMR spectrometer 300, 400, 600 MHz
(BrukerAvance III) instrument at r.t., and chemical shifts were

1674
Y. ZHANG ET AL.
Figure 6. Binding modes between EGFRwt-TK and representative compound predicted by Surflex-Dock program. (A) lapatinib within 1xkk; (B) compound 6a within
1xkk. The hinge region and the Asp-Phe-Gly (DFG) motif were illustrated in ribbon. The hydrogen bonds were illustrated as dark dashed lines and the length of hydro-
gen bonds was illustrated in numbers (unit in Å).
reported in parts per million (ppm, d) downfield from tetramethyl- with methylene chloride (2 ꢃ 30 mL) and the combined organic
silane (TMS). Coupling constants (J) were reported in Hz. Spin mul-
extracts were washed with water (2 ꢃ 200 mL) and brine
tiplicities were described as s (singlet), brs (broad singlet), d
(1 ꢃ 200 mL), dried over Mg2SO4. The organic solvent was evapo-
(double),
t (triplet), q (quartet), and m (multiplet). Infrared
rated to give 3 as yellow solid in 89.3% yield.
Spectroscopy (IR) was measured on Nicolet 170SXFT-IR instrument.
The high-resolution mass spectra (HRMS) were measured using
Bruker Esquire 3000plus mass spectrometer.
4.2.3. General procedure for the preparation of 4-arylamino-6-
iodoquinazoline (4a–4d)
After acetic acid (3.0 mL) and R substituted aniline (3.30 mol) being
added to 3 (3.00 mmol), the reaction mixture was refluxed for
15 min. After acetic acid was evaporated, ice-water (25 ml) was
added to the reaction mixture. The obtained mixture was adjusted
pH to 9 with ammonia solution and stirred for 0.5 h. The precipi-
tated product was filtered, and the filter cake was washed with
water (3 ꢃ 10 mL) to afford crude product. The crude product was
chromatographed by silica gel, eluting with EtOAc/PE (1:4) to
afford 4a–4d as white solid in 84.3–92.5% yield.
4.2. Synthesis
The synthetic route of the target compounds is showed in
Scheme 1. The synthetic methods of intermediates 2–5 followed
the previous procedures16, and the detail process is described
as follows.
4.2.1. Procedure for the preparation 2-amino-5-iodobenzoni-
trile (2)
A mixture of 2-aminobenzonitrile (1) (0.02 mol) and ammonium
iodide (0.02 mol) was dissolved in acetic acid (50 mL), stirred for
30 min at room temperature (r.t.), then 30% aqueous hydrogen
peroxide solution (0.13 mol) was slowly added at r.t. and stirred
for 12 h. After reaction completed, the reaction solution was
treated with aqueous sodium thiosulphate solution 40 ml
(0.03 mol) and basified to about pH to 8 by the addition of 20%
sodium hydroxide. The reaction mixture was stirred at r.t. for
30 min. The desired product, which was partially precipitated
during this step, was isolated by vacuum filtration to afford 2 as
silvery white flake solid in 92.6% yield.
4.2.3.1. 4-(3-chloro-4-(3-fluorobenzyloxy)phenylamino)-6-iodoqui-
nazoline (4a). Yield 92.5%. 1H NMR (300 MHz, DMSO-d6) d(ppm):
9.85 (s, 1H, –NH–), 8.95 (d, J ¼ 2.0 Hz, 1H, Ar-H), 8.61 (s, 1H, Ar-H),
8.11 (dd, J ¼ 12.0 Hz, 1H, Ar-H), 8.03 (d, J ¼ 3.6 Hz, 1H, Ar-H), 7.75
(dd, J ¼ 12.0 Hz, 1H, Ar-H), 7.56 (d, J ¼ 12.0 Hz, 1H, Ar-H), 7.44–7.51
(m, 1H, Ar-H), 7.29–7.35 (m, 3H, Ar-H), 7.15–7.22 (m, 1H, Ar-H), 5.26
(s, 2H, –CH2–).
4.2.3.2. 4-(3-chloro-4-fluorophenylamino)-6-iodoquinazoline (4 b).
Yield 86.1%. 1H NMR (300 MHz, DMSO-d6) d(ppm): 9.89 (s, 1H,
–NH–), 8.93 (s, 1H, Ar-H), 8.65 (s, 1H, Ar-H), 8.19 (dd, J ¼ 6.8, 2.4 Hz,
1H, Ar-H), 8.10 (dd, J ¼ 8.7, 1.5 Hz, 1H, Ar-H), 7.89–7.76 (m, 1H, Ar-
H), 7.56 (d, J ¼ 8.7 Hz, 1H, Ar-H), 7.43 (t, J ¼ 9.1 Hz, 1H, Ar-H).
4.2.2. Procedure for the preparation of N’-(2-cyano-4-iodophenyl)-
N,N-dimethyl formamidine (3)
4.2.3.3. 4-(3-ethynylphenylamino)-6-iodoquinazoline (4c). Yield
84.3%. 1H NMR (300 MHz, DMSO-d6) d(ppm): 9.90 (s, 1H, –NH–),
9.00 (s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 8.12 (dd, J ¼ 8.8, 1.6 Hz, 2H,
Ar-H), 7.96 (d, J ¼ 8.2 Hz, 1H, Ar-H), 7.58 (d, J ¼ 8.7 Hz, 1H, Ar-H),
7.44 (t, J ¼ 7.9 Hz, 1H, Ar-H), 7.27 (d, J ¼ 7.7 Hz, 1H, Ar-H), 4.23 (s,
A mixture of 2 (0.01 mol) and DMF-DMA (0.02 mol) was dissolved
in toluene (20 mL), heated up to 35 ꢁC and acetic acid (0.25 mL)
was added. After 30 min, the resultant mixture was cooled to
approximately 25 ꢁC. Toluene was completely stripped off . Water
was added to the mixture, which was basified pH to about 13 by
the addition of 20% sodium hydroxide. The mixture was extracted 1H, ꢄCH).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1675
4.2.3.4. 4-(4-(E)-(propen-1-yl)phenylamino)-6-iodoquinazoline (4d). (2.00 mmol) and sodium cyanoborohydride (1.00 mmol) were
Yield 86.7%. 1H NMR (300 MHz, DMSO-d6) d(ppm): 9.88 (s, 1H, added. The solution of 4-arylamino-6-(5-formylfuran-2-yl)quinazo-
–NH–), 9.01 (s, 1H, Ar-H), 8.62 (s, 1H, Ar-H), 8.11 (d, J ¼ 8.3 Hz, 1H,
line (5a–5d) (0.50 mmol) in THF (5 ml) was added to the reactor.
Ar-H), 7.82 (d, J ¼ 7.7 Hz, 2H, Ar-H), 7.56 (d, J ¼ 8.3 Hz, 1H, Ar-H),
After 30 min, sodium cyanoborohydride (1.00 mmol) was added
7.40 (d, J ¼ 7.7 Hz, 2H, Ar-H), 6.41 (d, J ¼ 15.8 Hz, 1H, –HC¼),
with stirring for 2 h. Then the mixture was adjusted to pH 9–10 by
6.33–6.15 (m, 1H, ¼CH–), 1.86 (d, J ¼ 5.4 Hz, 3H, –CH3).
addition of 20% sodium hydroxide, filtered and the filtrate was
evaporated. The crude product was isolated by silica gel chroma-
tographed, eluting with MeOH/CHCl3 (1:15) to afford 6a–6d as
pale yellow solid in 78.1–82.6% yield.
4.2.4. General procedure for the preparation of 4-arylamino-6–(5-
formylfuran-2-yl)quinazoline (5a–5d)
After residue 4a–4d (0.60 mmol), 5-formyl-2-furanboronic
4.2.5.1. 4–(3-chloro-4-(3-fluorobenzyloxy)phenylamino)-6–(5-((N,N-
diethyl(aminoethyl))aminomethyl)furan-2-yl)quinazoline (6a). Yield
82.0%. m.p.: 103.5–104.7 ꢁC. 1H NMR (600 MHz, DMSO-d6) d(ppm):
9.96 (s, 1H, –NH–), 8.76 (s, 1H, Ar-H), 8.57 (s, 1H, Ar-H), 8.14 (dd,
J ¼ 8.7, 1.3 Hz, 1H, Ar-H), 8.04 (d, J ¼ 1.9 Hz, 1H, Ar-H), 7.81–7.77
(m, 2H, Ar-H), 7.48 (dd, J ¼ 14.0, 7.7 Hz, 1H, Ar-H), 7.36–7.32 (m,
2H, Ar-H), 7.28 (d, J ¼ 9.0 Hz, 1H, Ar-H), 7.21–7.17(m, 1H, Ar-H), 7.05
(d, J ¼ 3.1 Hz, 1H, Furan-H), 6.46 (d, J ¼ 3.1 Hz, 1H, Furan-H), 5.28 (s,
2H, –CH2–), 3.83 (s, 4H, –CH2–), 2.64 (t, J ¼ 6.4 Hz, 2H, –CH2–),
2.47–2.44 (m, 4H, –CH2–), 0.93 (t, J ¼ 7.1 Hz, 6H, –CH3). 13 C NMR
acid
(0.90 mmol),
Pb/C
10%,
triethylamine
(2.4 mmol),
1,2-dimethoxyethane (60 mL) and methanol (30 mL) was added to
a 100 mL round bottomed flask, the suspension was stirred and
heated to 50 ꢁC for 30 min. The reaction mixture was filtered with
diatomite and the filter cake was washed with THF (3 ꢃ 10 mL).
The filtrate combined with washings was evaporated. The crude
product was chromatographed by silica gel, eluted with EtOAc/
CHCl3 (1:10) to afford compounds 5a–5d as orange solid in
57.2–83.4% yield.
1
(151 MHz, DMSO-d6) d(ppm): 162.2 (d, JC–F ¼ 243.7 Hz), 157.5,
4.2.4.1.
4-(3-chloro-4-(3-fluorobenzyloxy)phenylamino)-6–(5-for-
3
mylfuran-2-yl)quinazoline (5a). Yield 83.4%. 1H NMR (400 MHz,
DMSO-d6) d(ppm): 10.08 (s, 1H, –CHO), 9.68 (s, 1H, –NH–), 8.94 (d,
J ¼ 1.6 Hz, 1H, Ar-H), 8.60 (s, 1H, Ar-H), 8.33–8.25 (m, 1H, Ar-H),
8.00 (d, J ¼ 2.6 Hz, 1H, Ar-H), 7.85 (d, J ¼ 8.8 Hz, 1H, Ar-H), 7.73 (dd,
J ¼ 8.8, 3.1 Hz, 2H, Ar-H), 7.49 (td, J ¼ 8.0, 6.0 Hz, 1H, Ar-H), 7.40
(d, J ¼ 3.8 Hz, 1H, Ar-H), 7.34 (dd, J ¼ 11.9, 5.1 Hz, 2H, Ar-H), 7.29
(d, J ¼ 9.0 Hz, 1H, Furan-H), 7.23–7.16 (m, 1H, Furan-H), 5.27 (s,
2H, –CH2–).
155.3, 154.1, 151.4, 149.7, 148.8, 139.6 (d, JC–F ¼ 7.5 Hz), 133.2,
3
130.5 (d, JC–F ¼ 8.3 Hz), 129.7, 129.6, 129.6, 129.5, 128.5, 128.4,
4
127.7, 124.2, 123.2 (d, JC–F ¼ 2.6 Hz), 122.4, 121.1, 116.3, 115.4,
2
2
114.6 (d, JC–F ¼ 20.9Hz), 114.2, 114.0 (d, JC–F ¼ 21.9Hz), 109.3,
107.8, 69.4, 52.1, 46.5, 46.2, 45.8, 11.6. IR ꢀmax(KBr)cmꢅ1: 3405, 1660,
1492, 1449, 1440, 1025, 994, 827, 769. HRMS (C32H33ClFN5O2) m/z
[Mþ H]þ: found: 574.2379, calculated: 574.2385.
4.2.5.2. 4–(3-chloro-4-fluorophenylamino)-6-(5-((N,N-diethyl(ami-
noethyl))aminomethyl)furan-2-yl)quinazoline (6 b). Yield 78.1%.
m.p.: 98.3–99.8 ꢁC. 1H NMR (600 MHz, DMSO-d6) d(ppm): 10.19 (s,
1H, –NH–), 8.93 (s, 1H, Ar-H), 8.61 (s, 1H, Ar-H), 8.26 (dd, J ¼ 6.8,
2.4 Hz, 1H, Ar-H), 8.18 (dd, J ¼ 8.7, 1.6 Hz, 1H, Ar-H), 7.96–7.92 (m,
4.2.4.2. 4-(3-chloro-4-fluorophenylamino)-6-(5-formylfuran-2-yl)qui-
nazoline (5 b). Yield 65.2%. 1H NMR (300 MHz, DMSO-d6) d(ppm):
10.11 (s, 1H, -CHO), 9.65 (s, 1H, –NH–), 8.87 (s, 1H, Ar-H), 8.60 (s,
1H, Ar-H), 8.18 (dd, J ¼ 35.2, 6.7 Hz, 2H, Ar-H), 7.81 (d, J ¼ 8.6 Hz,
2H, Ar-H), 7.71 (d, J ¼ 3.5 Hz, 1H, Furan-H), 7.44 (t, J ¼ 9.1 Hz, 1H, 1H, Ar-H), 7.83 (d, J ¼ 8.7 Hz, 1H, Ar-H), 7.49–7.45 (m, 1H, Ar-H),
Ar-H), 7.36 (d, J ¼ 3.5 Hz, 1H, Furan-H).
7.13 (d, J ¼ 3.2 Hz, 1H, Furan-H), 6.52 (d, J ¼ 3.2 Hz, 1H, Furan-H),
3.90 (s, 2H, –CH2–), 2.79 (brs, 4H, –CH2–), 2.75–2.70 (d, 4H, –CH2–),
1.04 (t, J ¼ 7.2 Hz, 6H, –CH3). 13 C NMR (151 MHz, DMSO-d6) d(ppm):
4.2.4.3.
4-(3-ethynylphenylamino)-6-(5-formylfuran-2-yl)quinazo-
1
line (5c). Yield 74.6%. 1H NMR (300 MHz, DMSO-d6) d(ppm): 10.13
(s, 1H, –CHO), 9.69 (s, 1H, –NH–), 8.97 (s, 1H, Ar-H), 8.65 (s, 1H,
Ar-H), 8.28 (dd, J ¼ 8.8, 1.4 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.93
(d, J ¼ 8.2 Hz, 1H, Ar-H), 7.86 (d, J ¼ 8.8 Hz, 1H, Ar-H), 7.74 (d,
J ¼ 3.7 Hz, 1H, Furan-H), 7.46 (t, J ¼ 7.9 Hz, 1H, Ar-H), 7.41
(d, J ¼ 3.7 Hz, 1H, Furan-H), 7.30 (d, J ¼ 7.6 Hz, 1H, Ar-H), 4.26
(s, 1H, ꢄCH).
157.5, 154.0, 153.4 (d, JC–F ¼ 243.4 Hz), 151.6, 148.9, 136.5 (d,
3
4JC–F ¼ 2.5 Hz), 128.7, 128.5, 128.4, 123.9, 122.8 (d, JC–F ¼ 7.1 Hz),
2
2
118.7 (d, JC–F ¼ 18.6 Hz), 116.8, 116.5 (d, JC–F ¼ 21.3 Hz), 115.4,
115.4, 110.0, 108.0, 50.9, 50.9, 46.5, 45.2, 40.1, 10.3. IR ꢀmax(KBr)
cmꢅ1: 3441, 3064, 2968, 1631, 1611, 1574, 1498, 1418, 1018, 841.
HRMS (C25H27ClFN5O) m/z [M þ H]þ: found: 468.1959, calcu-
lated: 468.1966.
4.2.5.3. 4–(3-ethynylphenylamino)-6-(5-((N,N-diethyl(aminoethyl))a-
minomethyl)furan-2-yl)quinazoline (6c). Yield 79.7%. m.p.:
81.4–82.3 ꢁC. 1H NMR (600 MHz, DMSO-d6) d(ppm): 10.08 (s, 1H,
–NH–), 8.90 (s, 1H, Ar-H), 8.61 (s, 1H, Ar-H), 8.18 (dd, J ¼ 8.7, 1.7 Hz,
1H, Ar-H), 8.11 (brs, 1H, Ar-H), 8.01–7.98 (m, 1H, Ar-H), 7.82 (d,
J ¼ 8.7 Hz, 1H, Ar-H), 7.43 (t, J ¼ 7.9 Hz, 1H, Ar-H), 7.27–7.24 (m, 1H,
Ar-H), 7.12 (d, J ¼ 3.2 Hz, 1H, Furan-H), 6.50 (d, J ¼ 3.2 Hz, 1H,
Furan-H), 4.22 (s, 1H, ꢄCH), 3.87 (s, 2H, –CH2–), 2.73 (t, J ¼ 6.0 Hz,
2H, –CH2–), 2.69 (t, J ¼ 6.0 Hz, 2H, –CH2–), 2.64–2.60 (m, 4H,
–CH2–), 1.00 (t, J ¼ 7.1 Hz, 6H, –CH3). 13 C NMR (151 MHz, DMSO-d6)
d(ppm): 157.6, 154.5, 154.1, 151.5, 148.9, 139.5, 128.9, 128.6, 128.5,
128.4, 126.9, 125.2, 123.0, 121.7, 116.7, 115.5, 109.8, 108.0, 83.5,
80.6, 51.4, 46.5, 45.4, 45.2, 40.1, 21.1, 10.8. IR ꢀmax(KBr)cmꢅ1: 3452,
4.2.4.4.
4–(4-(E)-(propen-1-yl)phenylamino)-6–(5-formylfuran-2-
yl)quinazoline (5d). Yield 57.2%. 1H NMR (300 MHz, DMSO-d6)
d(ppm): 10.05 (s, 1H, –CHO), 9.64 (s, 1H, –NH–), 8.94 (s, 1H, Ar-H),
8.56 (s, 1H, Ar-H), 8.22 (d, J ¼ 8.6 Hz, 1H, Ar-H), 7.78 (dd, J ¼ 13.7,
8.5 Hz, 3H, Ar-H), 7.70 (d, J ¼ 3.6 Hz, 1H, Furan-H), 7.38 (d,
J ¼ 8.6 Hz, 3H, Furan-H, Ar-H), 6.38 (d, J ¼ 15.8 Hz, 1H, –HC¼),
6.31–6.10 (m, 1H, ¼CH–), 1.83 (d, J ¼ 5.9 Hz, 3H, –CH3).
4.2.5. General procedure for the preparation of 4-arylamino-6-(5-
((N,N-diethylaminoethyl)aminomethyl)furan-2-yl)quinazo-
line (6a–6d)
The pH of mixture was adjusted to 5–6 with formic acid after N,N-
diethylethylenediamine (0.75 mmol) and methanol (5.0 mL) was 3061, 2963, 1631, 1610, 1569, 1529, 1481, 1199, 891. HRMS
added to a reaction flask at 0 ꢁC. Then anhydrous sodium sulphate (C27H29N5O) m/z [M þ H]þ: found: 440.2464, calculated: 440.2450.

1676
Y. ZHANG ET AL.
4.2.5.4.
6-(5-((N,N-diethylethyl)aminomethyl)furan-2-yl)-4–(4-(E)- MO, USA) molecular modelling package. The crystal structural data
of EGFR kinase domain complexed with lapatinib (PDB code: 1xkk)
was obtained from RCSB Protein Data Bank23. The procedure of
molecular docking was carried out according to our previ-
ous reported16.
(propen-1-yl)phenylamino)quinazoline (6d). Yield 82.6%. m.p.:
89.9–91.3 ꢁC. 1H NMR (600 MHz, DMSO-d6) d(ppm): 9.95 (s, 1H,
–NH–), 8.80 (d, J ¼ 1.5 Hz, 1H, Ar-H), 8.55 (s, 1H, Ar-H), 8.15 (dd,
J ¼ 8.7, 1.8 Hz, 1H, Ar-H), 7.85–7.78 (m, 3H, Ar-H), 7.42 (d,
J ¼ 8.7 Hz, 2H, Ar-H), 7.07 (d, J ¼ 3.2 Hz, 1H, Furan-H), 6.47 (d,
J ¼ 3.2 Hz, 1H, Furan-H), 6.42 (dd, J ¼ 15.8, 1.5 Hz, 1H, –HC¼),
6.30–6.24 (m, 1H, ¼CH–), 3.84 (s, 2H, –CH2–), 2.67 (t, J ¼ 6.4 Hz, 2H,
–CH2–), 2.57 (t, J ¼ 5.6 Hz, 2H, –CH2–), 2.55–2.51 (m, 4H, –CH2–),
1.87 (dd, J ¼ 6.6, 1.5 Hz, 3H, –CH3), 0.95 (t, J ¼ 7.1 Hz, 6H, –CH3).
13 C NMR (151 MHz, DMSO-d6) d(ppm): 157.6, 155.1, 154.2, 151.5,
148.9, 137.8, 133.1, 130.5, 128.5, 128.4, 128.3, 125.7, 124.4, 122.6,
116.5, 115.5, 109.4, 107.8, 51.9, 51.8, 46.5, 45.8, 45.6, 40.1, 18.3,
11.4. IR ꢀmax(KBr)cmꢅ1: 3447, 3238, 2992, 1638, 1618, 1486, 1396,
1350, 1173, 1120, 1001, 784. HRMS (C28H33N5O) m/z [M þ H]þ:
found: 456.2758, calculated: 456.2763.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the National Natural
Science Foundation of China [grant number 21272144], the
Fundamental Research Funds for the Central Universities of
Shaanxi Normal University [grant numbers GK201603034,
GK201706005 and X2015YB06], and postdoctoral research fund of
Shaanxi Normal University.
4.3. Cell culture
SW480, A549, A431, and NCI-H1975 cells were maintained on
60 mm cell culture dishes and cultured using DMEM supple-
mented with 10% FBS, 100 units/mL penicillin, 100 mg/mL strepto-
mycin and 2 mM L-glutamate at 37 ꢁC and 5% CO2 with
95% humidity.
ORCID
Yaling Zhang
Baolin Li
http://orcid.org/0000-0002-7122-0268
http://orcid.org/0000-0001-9836-0036
4.4. MTT assay for the antiproliferative activity in vitro
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