Design, synthesis, and docking studies of afatinib analogs bearing cinnamamide moiety as potent EGFR inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.02.017

Two series of afatinib derivatives bearing cinnamamide moiety (10a-n and 11a-h) were designed, synthesized and evaluated for the IC₅₀ values against four cancer cell lines (A549, PC-3, MCF-7 and Hela). Two selected compounds (10e, 10k) were further evaluated for the inhibitory activity against EGFR and VEGFR2/KDR kinases. Seven of the compounds showed excellent cytotoxicity activity and selectivity with the IC₅₀ values in single-digit μM to nanomole range. Three of them are equal to more active than positive control afatinib against one or more cell lines. The most promising compound 10k showed the best activity against A549, PC-3, MCF-7 and Hela cancer cell lines and EGFR kinase, with the IC₅₀ values of 0.07 ± 0.02 μM, 7.67 ± 0.97 μM, 4.65 ± 0.90 μM and 4.83 ± 1.28 μM, which were equal to more active than afatinib (0.05 ± 0.01 μM, 4.1 ± 2.47 μM, 5.83 ± 1.89 μM and 6.81 ± 1.77 μM), respectively. Activity of compounds 10e (IC₅₀ 9.1 nM) and 10k (IC₅₀ 3.6 nM) against EGFR kinase were equal to the reference compound afatinib (IC₅₀ 1.6 nM). Structure-activity relationships (SARs) and docking studies indicated that replacement of the aqueous solubility 4-(dimethylamino)but-2-enamide group by cinnamamide moiety didn't decrease the antitumor activity. The results suggested that methoxy substitution had a significant impact on the activity and methoxy substituted on C-4 or C-2,3,4 position was benefit for the activity.

Full text of DOI:10.1016/j.bmc.2016.02.017

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and docking studies of afatinib analogs bearing
cinnamamide moiety as potent EGFR inhibitors
b
c
a
a
Yuanbiao Tu a,y, Yiqiang OuYang a,y, Shan Xu a, , Yan Zhu , Gen Li , Chao Sun , Pengwu Zheng ,
⇑
Wufu Zhu a,
⇑
a School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, PR China
b School of Pharmacy, China Medical University, Shenyang, Liaoning 110122, PR China
c School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of afatinib derivatives bearing cinnamamide moiety (10a–n and 11a–h) were designed, syn-
thesized and evaluated for the IC50 values against four cancer cell lines (A549, PC-3, MCF-7 and Hela).
Two selected compounds (10e, 10k) were further evaluated for the inhibitory activity against EGFR
and VEGFR2/KDR kinases. Seven of the compounds showed excellent cytotoxicity activity and selectivity
Received 7 January 2016
Revised 6 February 2016
Accepted 9 February 2016
Available online xxxx
with the IC50 values in single-digit lM to nanomole range. Three of them are equal to more active than
positive control afatinib against one or more cell lines. The most promising compound 10k showed the
best activity against A549, PC-3, MCF-7 and Hela cancer cell lines and EGFR kinase, with the IC50 values of
Keywords:
Afatinib
Cinnamamide
Synthesis
0.07 0.02
afatinib (0.05 0.01
l
M, 7.67 0.97
l
M, 4.65 0.90
l
M and 4.83 1.28
l
M, which were equal to more active than
M), respectively. Activity of
lM, 4.1 2.47
lM, 5.83 1.89
l
M and 6.81 1.77 l
compounds 10e (IC50 9.1 nM) and 10k (IC50 3.6 nM) against EGFR kinase were equal to the reference
compound afatinib (IC50 1.6 nM). Structure–activity relationships (SARs) and docking studies indicated
that replacement of the aqueous solubility 4-(dimethylamino)but-2-enamide group by cinnamamide moiety
didn’t decrease the antitumor activity. The results suggested that methoxy substitution had a significant
impact on the activity and methoxy substituted on C-4 or C-2,3,4 position was benefit for the activity.
Ó 2016 Elsevier Ltd. All rights reserved.
Docking
EGFR inhibitors
Antitumor activity
1. Introduction
cer (NSCLC), and was approved by the FDA for the treatment of
patients with metastatic NSCLC in 2013.12
The epidermal growth factor receptor (EGFR) family plays a crit-
ical role in vital cellular processes and in various cancers, including
proliferation, differentiation, migration, apoptosis, and angiogene-
sis. Therefore, developing EGFR inhibitors is one of the research
hotspots in molecular targeted therapy for the treatment of human
cancer.1,2
In recent years, many quinazoline derivatives were reported as
EGFR signal transduction pathway inhibitors, such as gefinitib,3,4
erlotinib,5 afatinib (BIBW-2992),6–8 dacomitinib9,10 and LP-711
(the structures of them are shown in Fig. 1). Among them, afatinib,
which is a powerful, irreversible tyrosine kinase inhibition of EGFR,
with IC50 value (half-maximal inhibitory concentration) of 0.5 nM,
exhibits potent anti-tumor activity against non small-cell lung can-
In this study, we devoted to synthesize novel EGFR inhibitors
bearing a common pharmacophore quinazoline moiety. LP-7 is a
excellent quinazoline EGFR kinase inhibitor but with disappointing
activity against NSCLC cell line A549. We thought that the ꢀCN
group of LP-7 was not favorable to the activity against A549. In
order to investigate the effect to the activity of the aqueous solubil-
ity chains and inspired by LP-7, we focused on the replacement of
the aqueous solubility 4-(dimethylamino)but-2-enamide group by
cinnamamide moiety and removed the ꢀCN group of LP-7 to afford
the first series compounds. Moreover, inspired by dacomitinib, the
small molecule tetrahydrofuran were substituted with methyl
group to obtain the second series compounds. In order to screen
compounds with excellent in vitro/in vivo anti-tumor activity as
well as improved pharmacokinetic, further studies on analogous
of afatinib were carried out in this research to study the effect of
variety, number, and position of substituents of aryl ring on the
antitumor activity. As a result, two novel quinazoline derivatives
bearing different substituents at aryl ring (10a–n and 11a–h) were
⇑
Corresponding authors. Tel./fax: +86 791 8380 2393.
E-mail addresses: shanxu9891@126.com (S. Xu), zhuwf@jxstnu.edu.cn,
zhuwufu-1122@163.com (W. Zhu).
y
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.bmc.2016.02.017
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tu, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.017

2
Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
F
F
O
HN
N
Cl
HN
N
CN
HN
N
Cl
H
N
N
O
O
O
O
N
O
O
N
N
N
O
O
O
Gefitinib
Erlotinib
Afatinib
Cl
F
F
R
F
H
O
HN
H
N
HN
Cl
H
F3C
N
N
CN
HN
N
Cl
N
N
H
N
O
X
N
O
N
O
N
O
O
Target Compounds
Dacomitinib
LP-7
O
10a-n:
11a-h:
X=
X=
-CH3
Figure 1. Structures of small-molecule epidermal growth factor receptor (EGFR) inhibitors based on the quinazoline scaffold and the target compounds 10a–n and 11a–h.
designed and synthesized. The structures of target compounds are
shown in Figure 1.
robenzoic acid through seven steps. Cyclization reaction of 2-
amino-4-fluorobenzoic acid in the present of formamidine acetate
afforded compound 1 as a white solid. Subsequently, compound 1
was nitrified with concentrated sulfuric acid and fuming nitric acid
refluxed for 2 h to give 7-fluoro-6-nitroquinazolin-4(3H)-one (2).
Chlorination of 2 with SOCl2 and DMF (cat.) for 4 h at 90 °C to achieve
3 as light yellow solid. Nucleophilic displacement of the compound 3
with phenylamine gave access to the intermediate 4. Compound 4
condensed with (S)-tetrahydrofuran-3-ol in the present of potas-
sium tert-butoxide obtained 5a. On the other hand, compound 4
treated with methanol and 50% sodium hydroxide to furnish 5b.
5a and 5b were then followed by nitro-hydrogenation reaction with
NH2NH2ꢁH2O (80%), FeCl3 and activated carbon to generate key
intermediate 6a and 6b, respectively. Finally, intermediates 6a and
Herein we disclosed the design, synthesis and antitumor activ-
ity against A549 (human lung cancer), PC-3 (human prostate can-
cer), MCF-7 (human breast cancer) and Hela (human cervical
cancer) cancer cell lines, EGFR and VEGFR2/KDR kinases of novel
afatinib analogs. Moreover, docking studies were presented in this
paper as well.
2. Chemistry
The structures and preparation of target compounds 10a–n and
11a–h are described in Scheme 1. Compounds 10a–n and 11a–h
were synthesized from commercially available 2-amino-4-fluo-
Scheme 1. Synthetic route of target compounds. Reagents and conditions: (a) EtOH, 24 h; (b) Concd H2SO4, fuming HNO3, 2 h; (c) SOCl2, DMF (cat.), 4 h; (d) isopropanol,
triethylamine, 1.5 h; (e) potassium tert-butoxide, THF, 3 h; CH3OH, NaOH; (f) N2H4ꢁH2O, FeCl3, activated carbon, EtOH, 1 h; (g) pyridine, piperidine (cat.), 20 h; (h) (COCl)2,
DMF (cat.), CH2Cl2, 15 min; (i) DIPEA, CH2Cl2, 1 h.
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Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 2
6b condensed with the corresponding cinnamoyl chloride 9a–n to
afford target compounds 10a–n and 11a–h, respectively.
Cinnamoyl chloride 9a–n were prepared from corresponding
benzaldehyde 7a–n condensed with propanedioic acid in the pre-
sent of pyridine and piperidine refluxed for 20 h, followed by chlo-
rinationto with oxalyl chloridefurnish and DMF (cat.) to furnish
compounds 9a–n, respectively.
EGFR and VEGFR2/KDR kinase inhibitory activity of compounds 10e, 10k, afatinib and
staurosporine
a
Compounds no.
R
IC50 (nM)
EGFR
VEGFR2/KDR
10e
10k
4-Methoxy
2,3,4-Trimethoxy
—
—
9.1
3.6
1.6
48
886
478
—
Afatinibb
Staurosporineb
—
3. Results and discussion
3.1. Biological evaluation
a
The values are an average of two separate determinations.
Used as a positive controls.
b
Taking afatinib or/and staurosporine as reference compounds,
the target compounds (10a–n and 11a–h) were evaluated for the
cytotoxicity against four cancer cell lines A549, PC-3, MCF-7, and
Hela by 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bro-
mide (MTT) cell proliferation assay. Compounds 10e and 10k were
further evaluated for the EGFR and VEGFR2/KDR kinase inhibitory
activity. The results expressed as IC50 values were summarized in
Tables 1 and 2 and the values are the average of at least two inde-
pendent experiments.
As illustrated in Table 1, nine of the synthesized compounds
showed moderate to excellent cytotoxicity activity against the dif-
ferent cancer cells. Three of them are equal to more active than
positive control afatinib against one or more cell lines. We could
easily see that the first series of the target compounds (10a–n)
were much more active than the second series (11a–h) against
the four tested cancer cell lines. For the first series, the compounds
10b–f, 10h and 10k showed equal to excellent cytotoxicity activity
against A549 and PC-3 cancer cell lines to reference compound afa-
tinib. It’s suggested that the target compounds shown well selec-
tivity against A549 and PC-3 to the other two cancer cell lines.
The inhibitory activities of target compounds were increased in
varying degrees because of the different substituents of aryl moi-
ety. It was noticeable that compounds with methoxy group substi-
tution (10d, 10e, 10f, 10h and 10k) had a significant impact on the
activity. Especially, methoxy group substituted at C-4 position is
more preferred, such as compounds 10e, 10f, 10h and 10k. What’s
more, compound 10k with 2,3,4-trimethoxy showed the best
Table 1
Structures and activity of target compounds 10a–n and 11a–h
Cl
F
HN
N
R
H
N
O
O
R
N
O
10a-n
11a-h
: R=
: R= -CH3
a
Compounds no.
R
IC50 (lM)
A549
NAc
PC-3
MCf-7
Hela
10a
10b
10c
10d
10e
10f
10g
10h
10i
4-Cyano
2-Nitro
36.55 0.24
7.76 1.01
7.15 0.91
8.73 0.84
2.24 0.91
2.76 0.86
25.60 1.14
20.50 0.24
22.79 0.13
27.50 0.23
7.67 0.97
25.50 1.14
26.50 3.14
28.15 2.24
NA
9.827
NDd
8.82 1.22
5.19 1.72
7.30 1.24
3.05 1.58
9.35 0.95
10.80 1.00
4.15 0.59
12.04 1.53
23.22 1.30
0.07 0.02
28.80 1.61
26.95 0.27
35.32 1.56
NA
33.25 2.25
NA
11.19 1.04
NA
33.25 1.24
NA
NA
28.40 1.96
26.19 1.53
36.28 1.52
5.07 0.93
28.56 2.08
30.26 1.54
28.35 1.22
26.68 1.42
30.05 1.24
4.65 0.90
30.62 1.41
30.50 2.04
32.35 2.74
NA
36.52 1.54
NA
34.25 2.14
NA
36.57 1.54
38.45 1.32
NA
29.60 1.54
37.49 1.40
26.76 1.26
34.62 1.74
26.45 1.64
34.25 1.54
24.50 1.00
27.35 2.24
30.50 1.22
4.83 1.28
27.68 5.27
27.43 2.24
27.45 2.54
ND
ND
ND
ND
ND
ND
ND
ND
6.81 1.77
2-Chloro-4-fluoro
2,5-Dimethoxy
4-Methoxy
2,4-Dimethoxy
3,5-Dimethoxy
3,4,5-Trimethoxy
4-Nitro
2,3-Dichloro
2,3,4,-Trimethoxy
3,5-Dibromo-4-hydroxy
4-Hydroxy-3,5-dimethoxy
4-Bromo
10j
10k
10l
10m
10n
11a
11b
11c
11d
11e
11f
4-Cyano
2-Nitro
14.26 1.09
32.68 1.41
28.56 3.24
NA
38.50 2.26
NA
2-Chloro-4-fluoro
2,5-Dimethoxy
4-Methoxy
2,4-Dimethoxy
3,5-Dimethoxy
3,4,5-Trimethoxy
—
11g
11h
Afatinibb
38.24 2.28
4.10 2.47
0.05 0.01
5.83 1.89
a
b
c
The values are an average of two separate determinations.
Used as a positive controls.
NA: not active.
d
ND: Not determined.
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Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
activity against A549, PC-3, MCF-7 and Hela cancer cell lines, with
the IC50 values of 0.07 0.02 M, 7.67 0.97 M, 4.65 0.90
and 4.83 1.28 M, which were equal to more active than afatinib
(0.05 0.01 M, 4.10 2.47 M, 5.83 1.89 and
6.81 1.77 M), respectively. Usually, electron withdrawing
compound 10k, our best EGFR inhibitor in this study, as ligand
example, and the structure of EGFR was selected as the docking
model (PDB ID code:4G5J13).
l
l
lM
l
l
l
l
lM
The binding modes of compound 10k and lead compound
afatinib were shown in Figure 2a and b. As depicted in Figure 2b,
compound 10k and lead compound can nearly overlap in the bind-
ing model and quinazoline moiety and amide group formed two
hydrogen bonds with residues MET793 and CYS797, respectively.
Analysis of compound 10k’s binding mode in the active binding
site demonstrated that the docking mode of the 10k is similar to
the lead compound with the same H-bond between quinazoline
group, amide group and MET793, CYS797. The results of Figure 2b
showed that 10k and lead compound can bind to the ATP-binding
pocket of the EGFR protein. The two hydrogen bonds and combine
of ATP-binding pocket really play an important role in the inhibi-
tory potency of afatinib analogs bearing cinnamamide moiety
against EGFR kinase according to the docking results. Furthermore,
the docking results also give us a new direction to design new EGFR
inhibitors. The above-mentioned results of SARs analysis and
molecular docking study may allow the rational design of more
potent EGFR inhibitors.
groups are unfavourable for the activity, that is, why compounds
10a–c, 10i–j and 10n exhibited unsatisfied antitumor activity.
In view of above, the results suggested that replacement of the
aqueous solubility 4-(dimethylamino)but-2-enamide group by cin-
namamide moiety didn’t decrease the antitumor activity and vari-
ations in substitutions of the aryl moiety had a significant impact
on the activity. Moreover, 2,3,4-trimethoxy substitution of aryl
moiety is beneficial for the activity. The activity toward EGFR
kinase of compound 10e and 10k is equal to the reference com-
pound afatinib, with the IC50 values of 3.6 nM. The inhibitory activ-
ity against EGFR and VEGFR2/KDR kinases of compounds 10e and
10k shown that these series of compounds were potent selective
EGFR inhibitors. However, exact action mechanism is not quite
clear right now. Further study will be carried out to identify the
target in near future.
3.2. Molecular docking study
4. Conclusions
To explore the binding modes of target compounds with the
active site of EGFR, molecular docking simulation studies were car-
ried out by using SURFLEX-DOCK module of SYBYL package ver-
sion. Based on the in vitro inhibition results, we selected
In summary, we designed and synthesized two series of afatinib
derivatives bearing cinnamamide moiety and evaluated for the
IC50 values against four cancer cell lines. Two selected compounds
Figure 2. (a–b) Binding poses of compound 10k with EGFR. The proteins were displayed by cyan ribbon. Compound 10k and lead compound were displayed by orange and
green sticks, respectively. H-bonding interactions between the 10k, lead compound and EGFR were indicated with dashed lines in yellow.
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Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
(10e, 10k) were further evaluated for the inhibitory activity against
5.4. Preparation of 4-chloro-7-fluoro-6-nitroquinazoline (3)
EGFR and VEGFR2/KDR kinases. Seven of the compounds showed
excellent cytotoxicity activity with the IC50 valuables in single-digit
A solution of compound 2 (48.0 g,0.230 mol), thionyl chloride
(407 mL) and N,N-dimethylformamide (2 mL) was heated to
100 °C until the mixture melted transparently, and then stirred
for 4 h. After completed, the reaction mixture was cooled to room
temperature, and the solution was concentrated under a reduced
pressure to remove the solvent. Toluene thereto (400 mL) was
added to the residue and concentrated again, dried to obtain 4-
chloro-7-fluoro-6-nitroquinazoline 3 as yellow solid15 (50.7 g,
97.0%). Mp 118.2–119.3 °C. 1H NMR (400 MHz, DMSO) d 8.66 (dd,
J = 8.2, 1.2 Hz, 1H), 8.41 (s, 1H), 7.75 (d, J = 12.2 Hz, 1H).
lM to nanomole range. The results noticeable that methoxy (10d,
10e, 10f, 10h and 10k) substitution had a significant impact on
the activity. Especially, methoxy substituted at C-4 position is more
preferred, such as compounds 10e, 10f, 10h and 10k. Moreover, the
most promising compound 10k with 2,3,4-trimethoxy group
showed the best activity against A549, PC-3, MCF-7 and Hela cancer
cell lines and EGFR kinase, with the IC50 values of 0.07 0.02
7.67 0.97 M, 4.65 0.90 M and 4.83 1.28 M, 3.6 nM, which
were equal to more active than afatinib (0.05 0.01 M,
4.10 2.47 M, 5.83 1.89 and 6.81 1.77 M, 1.6 nM),
lM,
l
l
l
l
l
lM
l
respectively. Structure–activity relationships (SARs) and docking
studies indicated that replacement of the aqueous solubility 4-
(dimethylamino)but-2-enamide group by cinnamamide moiety
didn’t decrease the antitumor activity. The results suggested that
methoxy substitution had a significant impact on the activity and
substituted on C-4 and C-2,3,4 position was benefit for the activity.
Further study will be carried out to identify the exact action mech-
anism in near future.
5.5. Preparation of N-(3-chloro-4-fluorophenyl)-7-fluoro-6-
nitroquinazolin-4-amine (4)
To the solution of compound 3 (50.0 g, 0.220 mol), isopropanol
(400 mL), triethylamine (34 mL), 3-choro-4-fluoroaniline was
added at room temperature. The reaction mixture then was stirred
at room temperature for 1.5 h. After completion of reaction as indi-
cated by TLC, the mixture was then filtered, washed with iso-
propanol and water, dried to yield N-(3-chloro-4-fluorophenyl)-
7-fluoro-6-nitroquinazolin-4-amine 4 as orange solid16 (65.0 g,
87.8%). Mp 261.4–262.5 °C. 1H NMR (400 MHz, DMSO) d 10.43 (s,
1H), 9.51 (d, J = 7.8 Hz, 1H), 8.68 (s, 1H), 8.08 (d, J = 6.4 Hz, 1H),
7.79 (d, J = 5.2 Hz, 1H), 7.76 (s, 1H), 7.45 (t, J = 9.0 Hz, 1H).
5. Experimental
5.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-
540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were
uncorrected. NMR spectra were performed using Bruker 400 MHz
spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS
as an internal standard. Mass spectra (MS) were taken in ESI mode
on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). All the mate-
rials were obtained from commercial suppliers and used without
purification, unless otherwise specified. Yields were not optimized.
TLC analysis was carried out on silica gel plates GF254 (Qindao
Haiyang Chemical, China). All the materials were obtained from
commercial suppliers and used without purification, unless other-
wise specified. Yields were not optimized.
5.6. Preparation of (S)-N-(3-chloro-4-fluorophenyl)-6-nitro-7-
((tetrahydrofuran-3-yl)oxy)quinazolin-4-amine (5a)
To the mixture of potassium tert-butoxide (15.2 g, 0.136 mol),
anhydrous
THF
(400 mL),
(S)-tetrahydrofuran-3-ol
(10 mL,0.124 mol) was added drop-wise at room temperature.
The mixture was stirred at room temperature for 1 h. a solution
of compound 4 (20.0 g, 0.060 mol) in anhydrous THF (200 mL)
was added via a dropping funnel over 1 h (the dropping funnel
was rinsed with15 mL of THF) at room temperature and the reac-
tion mixture was stirred at room temperature for 2 h, the reaction
was monitored by TLC. The solution was concentrated under a
reduced pressure and the residue was poured into water with
stirring yielding a precipitate, then the mixture was filtered and
washed with ice-water and ethanol, dried to afford (S)-N-
(3-chloro-4-fluorophenyl)-6-nitro-7-((tetrahydrofuran-3-yl)oxy)
quinazolin-4-amine 5a as yellow solid (23.9 g, 99.5%). Mp 210.1–
211.0 °C. 1H NMR (400 MHz, DMSO) d 10.10 (d, J = 11.8 Hz, 1H),
9.16 (d, J = 13.2 Hz, 1H), 8.62 (d, J = 13.4 Hz, 1H), 8.13 (d,
J = 6.8 Hz, 1H), 7.75 (s, 1H), 7.45–7.35 (m, 2H), 5.40 (s, 1H),
3.99–3.79 (m, 4H), 2.34 (dd, J = 13.8, 6.2 Hz, 1H), 2.04 (d,
J = 5.6 Hz, 1H).
5.2. Preparation of 7-fluoroquinazolin-4(3H)-one (1)
A mixture of 2-amino-4-fluorobenzoic acid (50.0 g, 0.323 mol),
formamidine acetate (67.0 g, 0.644 mol) and ethanol (300 mL)
was heated and refluxed for 24 h and the reaction was monitored
by thin-layer chromatography (TLC). The reaction mixture was
poured into ice-water, then filtered and the filter cake was washed
with 50 percent ethanol, dried to obtained 1 as white solid14
(51.0 g, 96.4%) and was used for next step without further purifica-
tion. Mp 260.1–261.0 °C. ESI-MS m/z: [MꢀH]ꢀ163.1. 1H NMR
(400 MHz, CDCl3) d 12.35 (s, 1H), 8.17 (d, J = 6.8 Hz, 1H), 8.14 (d,
J = 7.2 Hz, 1H), 7.43 (d, J = 9.8 Hz, 1H), 7.37 (t, J = 8.8 Hz, 1H).
5.7. Preparation of N-(3-chloro-4-fluorophenyl)-7-methoxy-6-
nitroquinazolin-4-amine (5b)
5.3. Preparation of 7-fluoro-6-nitroquinazolin-4(3H)-one (2)
To the mixture of compound 4 (11.37 g, 0.334 mol), methanol
(227 mL), 50 percent sodium hydroxide (15 mL) was added at
room temperature. The reaction mixture then was refluxed for
1 h and monitored by TLC. The reaction mixture was cooled to
room temperature and was poured into sodium bicarbonate solu-
tion yielding a precipitate, the mixture then was separated by fil-
tration and washed with water, dried to afford N-(3-chloro-4-
fluorophenyl)-7-methoxy-6-nitroquinazolin-4-amine 5b as yellow
solid17 (11.2 g, 94.9%). Mp 292.3–293.0 °C. ESI-MS m/z: [M
+H]+349.1. 1H NMR (400 MHz, DMSO) d 10.13 (s, 1H), 9.20 (s,
1H), 8.66 (s, 1H), 8.20–8.10 (m, 1H), 7.84–7.73 (m, 1H), 7.51–7.41
(m, 2H), 4.05 (s, 3H).
To the mixture of compound 1 (50.0 g, 0.239 mol), concentrated
sulfuric acid (103 mL), fuming nitric acid (105 mL) was slowly
added at 70 °C, the reaction mixture then was stirred at 110 °C
for 2 h. After completion of reaction as indicated by TLC, the reac-
tion mixture was cooled to room temperature and the reaction
mixture was slowly added to ice/water with vigorous stirring
yielding a precipitate. The mixture was then filtered, dried to fur-
nish 7-fluoro-6-nitroquinazolin-4(3H)-one 2 as light yellow solid15
(48.5 g, 76.1%). Mp 277.3–278.5 °C. 1H NMR (400 MHz, DMSO) d
12.77 (s, 1H), 8.68 (dd, J = 8.2, 2.7 Hz, 1H), 8.28 (s, 1H), 7.73 (dd,
J = 12.2, 2.8 Hz, 1H).
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6
Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5.8.
Preparation
of
(S)-N-(3-chloro-4-fluorophenyl)-7-
addition, the reaction mixture was removed from the ice bath
and placed in room temperature for 30 min and monitored by
TLC. The mixture was washed with 10% K2CO3 (50 mL ꢂ3) followed
by brine (50 mL ꢂ1), and the organic phase was separated, dried,
and evaporated to yield 12a–n and 13a–h which were purified
by dichloromethane.
((tetrahydrofuran-3-yl)oxy)quinazoline-4,6-diamine (6a)
The solution of compound 5a (21.4 g, 0.053 mol) and ethanol
(700 mL) was stirred at 60 °C, an appropriate amount of activated
carbon (7.0 g) and ferric chloride (1.8 g) were added at the temper-
ature, the mixture was heated to 80 °C and 80 percent hydrazine
hydrate (25 mL) was added to the solution. The reaction mixture
then was refluxed for 1.5 h and monitored by TLC. The mixture
was filtered and the precipitate was washed with ethanol. The fil-
trate was concentrated under reduce pressure and the residue was
poured into water with stirred for 30 min. The precipitate was fil-
tered and dried to furnish (S)-N-(3-chloro-4-fluorophenyl)-7-
((tetrahydrofuran-3-yl)oxy)quinazoline-4,6-diamine 6a as light
yellow solid (17.3 g, 87.3%). Mp 139.1–140.1 °C. 1H NMR
(400 MHz, DMSO) d 9.41 (s, 1H), 8.38 (s, 1H), 8.17 (t, J = 12.6 Hz,
1H), 7.88–7.73 (m, 1H), 7.38 (dd, J = 17.2, 8.2 Hz, 2H), 7.05 (s,
1H), 5.41 (s, 2H), 5.20 (s, 1H), 4.00–3.87 (m, 3H), 3.77 (dd,
J = 12.6, 7.8 Hz, 1H), 2.29 (td, J = 14.2, 7.2 Hz, 1H), 2.19–2.06 (m,
1H).
5.12.1. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)-3-(4-cyanophenyl)
acrylamide (10a)
This compound was obtained as yellow solid in 80% yield. Mp
267.5–268.9 °C. ESI-MS m/z: [M+H]+530.1. 1H NMR (400 MHz,
DMSO) d 9.78 (s, 1H), 9.18 (s, 1H), 8.75 (s, 1H), 8.04 (d, J = 6.4 Hz,
1H), 7.93 (d, J = 8.2 Hz, 2H), 7.87 (d, J = 8.2 Hz, 2H), 7.70 (d,
J = 15.8 Hz, 2H), 7.60–7.40 (m, 4H), 5.30 (s, 1H), 4.10–3.93 (m,
3H), 3.80 (d, J = 5.4 Hz, 1H), 2.45–2.34 (m, 1H), 2.23 (s, 1H).
5.12.2. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)-3-(2-nitrophenyl)
acrylamide (10b)
This compound was obtained as yellow solid in 68% yield. Mp
176.5–177.1 °C. ESI-MS m/z: [M+H]+550.1. 1H NMR (400 MHz,
DMSO) d 9.84 (s, 1H), 9.27 (s, 1H), 8.83 (s, 1H), 8.12 (dd, J = 8.2,
1.0 Hz, 1H), 8.03–7.94 (m, 2H), 7.93–7.80 (m, 3H), 7.73–7.67 (m,
2H), 7.53 (t, J = 9.2 Hz, 1H), 7.42 (s, 1H), 7.28 (d, J = 15.6 Hz, 1H),
5.32 (d, J = 6.2 Hz, 1H), 4.12 (d, J = 9.8 Hz, 1H), 4.05–3.97 (m, 2H),
3.81 (td, J = 8.2, 5.4 Hz, 1H), 2.40 (dt, J = 14.2, 7.1 Hz, 1H), 2.26–
2.18 (m, 1H). 13C NMR (101 MHz, DMSO) d 163.83, 158.60,
156.28, 154.69, 153.84, 151.17, 148.75, 136.52, 135.33, 134.34,
131.09, 130.32, 129.37, 129.21, 127.06, 126.29, 125.21, 125.05,
117.23, 117.02, 116.58, 107.85, 103.32, 80.37, 72.35, 67.16, 32.86.
ESI-HRMS m/z: calcd for C27H21ClFN5O5 [M+H]+: 550.1293; found
550.1253.
5.9. Preparation of N-(3-chloro-4-fluorophenyl)-7-methoxy-
quinazoline-4,6-diamine (6b)
The synthesis of compound 8 was similar to the compound 7.
The solution of compound 5b (11.2 g, 0.032 mol) and ethanol
(370 mL) was stirred at 60 °C, an appropriate amount of activated
carbon (3.5 g) and ferric chloride (1.3 g) were added at the temper-
ature, the mixture was heated to 80 °C and 80 percent hydrazine
hydrate (16 mL) was added to the solution. The reaction mixture
then was refluxed for 1.5 h and monitored by TLC. The mixture
was filtered and the precipitate was washed with ethanol. The fil-
trate was concentrated under reduce pressure and the residue was
poured into water with stirred for 30 min. The precipitate was fil-
tered and dried to obtain N-(3-chloro-4-fluorophenyl)-7-
methoxyquinazoline-4,6-diamine 6b as yellow solid (8.8 g,
85.9%). Mp 258.4–259.7 °C. 1H NMR (400 MHz, DMSO) d 9.39 (s,
1H), 8.37 (s, 1H), 8.18 (dd, J = 6.8, 2.5 Hz, 1H), 7.84–7.76 (m, 1H),
7.44–7.34 (m, 2H), 7.10 (s, 1H), 5.38 (s, 2H), 3.96 (s, 3H).
5.12.3. (S)-3-(2-Chloro-4-fluorophenyl)-N-(4-((3-chloro-4-flu-
orophenyl)amino)-7-((tetrahydrofuran-3-yl)oxy)quinazolin-6-
yl)acrylamide (10c)
This compound was obtained as yellow solid in 65% yield. Mp
179.6–181.5 °C. ESI-MS m/z: [M+H]+558.1. 1H NMR (400 MHz,
DMSO) d 9.62 (s, 1H), 9.12 (s, 1H), 8.61 (s, 1H), 8.09 (d, J = 4.4 Hz,
1H), 7.98–7.85 (m, 2H), 7.77 (s, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.41
(dt, J = 15.4, 7.8 Hz, 2H), 7.33–7.20 (m, 2H), 5.33 (s, 1H), 4.11–
3.90 (m, 4H), 3.80 (d, J = 5.4 Hz, 1H), 2.39 (dd, J = 14.2, 7.0 Hz,
1H), 2.21 (s, 1H).
5.10. General procedure for the preparation of compounds 8a–n
To the mixture of substituted benzaldehydes 7a–n (2.5 mmol),
pyridine (20.0 mL), piperidine (0.2 mL), propanedioic acid
(5.0 mmol) was added at room temperature and the mixture was
heated and refluxed for 20 h and monitored by TLC. The reaction
mixture was cooled to room temperature and the solution was
acidified to pH = 5 to yielding a precipitate. The mixture was fil-
tered and washed with water to furnish the compounds 8a–n.
5.12.4. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)-3-(2,5-dimethoxyphenyl)-
acrylamide (10d)
This compound was obtained as yellow solid in 72% yield. Mp
278.9–280.3 °C. ESI-MS m/z: [M+H]+565.1. 1H NMR (400 MHz,
DMSO) d 9.72 (s, 1H), 9.28 (s, 1H), 8.89 (s, 1H), 7.98 (dd, J = 6.8,
2.6 Hz, 1H), 7.92 (d, J = 15.8 Hz, 1H), 7.68 (ddd, J = 8.8, 4.3, 2.6 Hz,
1H), 7.55 (t, J = 9.0 Hz, 1H), 7.49 (s, 1H), 7.24 (t, J = 9.6 Hz, 2H),
7.06 (d, J = 9.0 Hz, 1H), 7.02 (dd, J = 9.0, 2.8 Hz, 1H), 5.30 (t,
J = 5.6 Hz, 1H), 4.13 (d, J = 9.8 Hz, 1H), 4.01 (dt, J = 15.6, 6.1 Hz,
2H), 3.83 (d, J = 4.4 Hz, 3H), 3.80 (dd, J = 7.0, 4.2 Hz, 1H), 3.78 (s,
3H), 2.41 (dt, J = 14.4, 7.1 Hz, 1H), 2.27–2.19 (m, 1H).
5.11. General procedure for the preparation of compounds 9a–n
Oxalyl chloride (2.0 mmol) was added drop-wise to a stirred
mixture of compounds 8a–n (1.0 mmol) and DMF (0.02 mmol) in
dichloromethane (16 mL) in room temperature for 10 min, and
the mixture was distilled and dissolved in dichloromethane
(16 mL) immediately. The solution was used for the next step with-
out further purification.
5.12.5. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)-3-(4-methoxyphenyl)-
acrylamide (10e)
5.12. General procedure for the preparation of compounds 10a–n
and 11a–h
This compound was obtained as yellow solid in 63% yield. Mp
240.9–242.3 °C. ESI-MS m/z: [M+H]+535.1. 1H NMR: (400 MHz,
DMSO) d 9.43 (s, 1H), 9.08 (s, 1H), 8.59 (s, 1H), 8.10 (dt, J = 13.0,
6.5 Hz, 1H), 7.78 (ddd, J = 9.0, 4.4, 2.8 Hz, 1H), 7.63 (d, J = 8.6 Hz,
2H), 7.59 (s, 1H), 7.44 (t, J = 9.2 Hz, 1H), 7.28 (s, 1H), 7.09 (d,
A solution of compound 9a–n (1.0 mmol) in dichloromethane
(16 mL) was added drop-wise to a solution of aniline compounds
6a or 6b (0.4 mmol) and diisopropylethylamine (0.4 mmol) in
dichloromethane (15 mL) in an ice bath. Upon completion of the
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Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
J = 15.8 Hz, 1H), 7.04 (s, 1H), 7.01 (s, 1H), 5.32 (dd, J = 6.2, 4.8 Hz,
5.12.10. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-
((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)-3-(2,3-
dichlorophenyl)acrylamide (10j)
1H), 4.02 (ddd, J = 10.8, 10.4, 5.8 Hz, 2H), 3.95 (dd, J = 15.4,
7.8 Hz, 1H), 3.81 (d, J = 4.2 Hz, 3H), 3.78 (dd, J = 8.2, 5.2 Hz, 1H),
2.37 (dt, J = 14.2, 7.1 Hz, 1H), 2.24–2.15 (m, 1H). 13C
This compound was obtained as yellow solid in 65% yield. Mp
273.5–274.8 °C. ESI-MS m/z: [M+H]+574.1. 1H NMR (400 MHz,
DMSO) d 9.90 (s, 1H), 9.30 (s, 1H), 8.89 (s, 1H), 8.02–7.98 (m,
1H), 7.97 (d, J = 3.0 Hz, 1H), 7.83 (dd, J = 7.8, 1.2 Hz, 1H), 7.74 (dd,
J = 8.0, 1.2 Hz, 1H), 7.69 (ddd, J = 8.8, 4.3, 2.6 Hz, 1H), 7.59–7.46
(m, 3H), 7.37 (d, J = 15.6 Hz, 1H), 5.31 (s, 1H), 4.13 (d, J = 10.4 Hz,
1H), 4.05–3.95 (m, 2H), 3.81 (td, J = 8.2, 5.4 Hz, 1H), 2.42 (dt,
J = 14.2, 7.4 Hz, 1H), 2.27–2.18 (m, 1H).
NMR (101 MHz, DMSO)
d 164.80, 161.21, 157.56, 155.21,
153.68, 153.28, 146.58, 141.18, 136.78, 130.09(2C), 128.64,
127.76, 124.57, 123.40, 119.99, 117.01, 116.80, 115.86,
114.89(2C), 109.09, 106.94, 79.60, 72.45, 67.11, 55.77, 32.87. ESI-
HRMS m/z: calcd for C28H24ClFN4O4 [M+H]+: 535.1548; found
535.1518.
5.12.6. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)-3-(2,4-dimethoxyphenyl)-
acrylamide (10f)
5.12.11. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-
((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)-3-(2,3,4-
This compound was obtained as yellow solid in 80% yield. Mp
295.5–296.3 °C. ESI-MS m/z: [M+H]+565.2. HRMS: 564.1575. 1H
NMR (400 MHz, DMSO) d 9.61 (s, 1H), 9.26 (s, 1H), 8.86 (s, 1H),
7.99 (dd, J = 6.8, 2.6 Hz, 1H), 7.88 (d, J = 15.8 Hz, 1H), 7.69 (ddd,
J = 8.8, 4.4, 2.6 Hz, 1H), 7.60 (d, J = 9.2 Hz, 1H), 7.54 (t, J = 9.2 Hz,
1H), 7.47 (s, 1H), 7.10 (d, J = 15.8 Hz, 1H), 6.67–6.61 (m, 2H), 5.29
(t, J = 5.6 Hz, 1H), 4.12 (d, J = 10.0 Hz, 1H), 4.05–3.94 (m, 2H),
3.89 (s, 3H), 3.83 (s, 3H), 3.82–3.77 (m, 1H), 2.40 (dt, J = 14.2,
7.0 Hz, 1H), 2.26–2.17 (m, 1H). 13C NMR (101 MHz, DMSO) d
165.37, 162.86, 159.68, 158.87, 156.46, 154.99, 150.58, 150.42,
150.30, 136.56, 135.01, 130.05, 129.68, 126.71, 125.45, 119.65,
119.47, 117.32, 117.10, 116.37, 107.81, 106.63, 98.90, 80.46,
trimethoxyphenyl)acrylamide (10k)
This compound was obtained as yellow solid in 48% yield. Mp
244.0–245.8 °C.ESI-MS m/z: [M+H]+596.1. 1H NMR (400 MHz,
DMSO) d 9.62 (s, 1H), 9.25 (s, 1H), 8.80 (s, 1H), 8.02 (dd, J = 6.8,
2.6 Hz, 1H), 7.82 (d, J = 15.8 Hz, 1H), 7.71 (ddd, J = 8.9, 4.4, 2.6 Hz,
1H), 7.52 (t, J = 9.2 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H),
7.15 (d, J = 15.8 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 5.32 (d, J = 6.4 Hz,
1H), 4.11 (d, J = 10.6 Hz, 1H), 4.05–3.94 (m, 3H), 3.86 (s, 3H), 3.84
(s, 3H), 3.81 (dd, J = 8.2, 2.8 Hz, 1H), 3.78 (s, 3H), 2.40 (dt,
J = 14.2, 7.2 Hz, 1H), 2.26–2.16 (m, 1H). 13C NMR (101 MHz, DMSO)
d 165.23, 165.07, 158.76, 155.66, 154.79, 152.95, 151.03, 142.44,
140.49, 136.26, 135.37, 129.77, 126.48, 125.21, 122.97, 121.55,
120.82, 117.33, 117.12, 116.30, 109.11, 108.07, 103.18, 80.49,
72.36, 66.99, 61.87, 60.81, 56.54, 32.84. ESI-HRMS m/z: calcd for
72.33, 67.16, 56.28, 55.95, 32.83. ESI-HRMS m/z: calcd for C29H26
-
ClFN4O5 [M+H]+: 565.1654; found 565.1624.
C
30H28ClFN4O6 [M+H]+: 595.1760; found 595.1718.
5.12.7. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)-3-(3,5-dimethoxyphenyl)
acrylamide (10g)
5.12.12. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-
((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)-3-(3,5-dibromo-4-
hydroxyphenyl)acrylamide (10l)
This compound was obtained as yellow solid in 58% yield. Mp
181.0–182.3 °C. ESI-MS m/z: [M+H]+565.1. 1H NMR (400 MHz,
DMSO) d 9.55 (s, 1H), 9.20 (s, 1H), 8.78 (s, 1H), 8.02 (d, J = 5.6 Hz,
1H), 7.87 (d, J = 15.8 Hz, 1H), 7.71 (s, 1H), 7.60 (d, J = 8.2 Hz, 1H),
7.50 (t, J = 9.0 Hz, 1H), 7.43 (s, 1H), 7.09 (d, J = 15.8 Hz, 1H), 6.64
(d, J = 7.4 Hz, 2H), 5.29 (s, 1H), 4.10 (d, J = 10.2 Hz, 1H), 4.06–3.92
(m, 2H), 3.89 (s, 3H), 3.83 (s, 3H), 3.80–3.75 (m, 1H), 2.40 (dd,
J = 13.6, 6.6 Hz, 1H), 2.28–2.15 (m, 1H).
This compound was obtained as yellow solid in 78% yield. Mp
279.6–281.3 °C. ESI-MS m/z: [M+H]+679.2. 1H NMR (400 MHz,
DMSO) d 9.91 (s, 1H), 9.35 (s, 1H), 9.08 (s, 1H), 8.55 (s, 1H),
8.21–8.09 (m, 2H), 7.90 (d, J = 13.8 Hz, 2H), 7.53 (d, J = 15.6 Hz,
1H), 7.43 (dd, J = 16.0, 7.0 Hz, 1H), 7.27 (s, 1H), 7.17 (d,
J = 15.6 Hz, 1H), 5.34 (s, 1H), 4.05 (s, 1H), 3.96 (dd, J = 15.2,
9.5 Hz, 2H), 3.80 (dd, J = 12.8, 7.5 Hz, 2H), 2.45–2.31 (m, 1H),
2.26–2.14 (m, 1H).
5.12.8. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-
((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (10h)
5.12.13. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-
((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)-3-(4-hydroxy-3,5-
dimethoxyphenyl)acrylamide (10m)
This compound was obtained as yellow solid in 58% yield. Mp
143.8–144.5 °C. ESI-MS m/z: [M+H]+596.1. 1H NMR (400 MHz,
DMSO) d 9.86 (s, 1H), 9.46 (s, 1H), 9.03 (s, 1H), 8.54 (s, 1H), 8.14
(dd, J = 6.8, 2.6 Hz, 1H), 7.81 (ddd, J = 9.0, 4.2, 2.8 Hz, 1H), 7.61 (d,
J = 15.6 Hz, 1H), 7.43 (t, J = 9.2 Hz, 1H), 7.27 (s, 1H), 7.13 (d,
J = 15.6 Hz, 1H), 7.02 (s, 2H), 5.33 (s, 1H), 4.08–4.01 (m, 2H),
4.00–3.95 (m, 1H), 3.86 (s, 6H), 3.82–3.77 (m, 1H), 3.71 (d,
J = 4.8 Hz, 3H), 2.38 (dt, J = 14.4, 7.2 Hz, 1H), 2.23–2.15 (m, 1H).
This compound was obtained as yellow solid in 63% yield. Mp
184.3–186.2 °C. ESI-MS m/z: [M+H]+581.2. 1H NMR (400 MHz,
DMSO) d 9.85 (s, 1H), 9.35 (s, 1H), 9.03 (s, 1H), 8.52 (s, 1H), 8.13
(dd, J = 6.8, 2.6 Hz, 1H), 7.80 (dd, J = 8.4, 3.4 Hz, 1H), 7.56 (d,
J = 15.6 Hz, 1H), 7.42 (t, J = 9.2 Hz, 1H), 7.25 (s, 1H), 7.01 (d,
J = 15.6 Hz, 1H), 6.96 (s, 2H), 5.32 (s, 1H), 4.08–3.93 (m, 3H),
3.86–3.79 (m, 6H), 3.80–3.75 (m, 1H), 2.38 (td, J = 14.2, 7.6 Hz,
1H), 2.22–2.13 (m, 1H).
5.12.14. (S)-3-(3-Bromophenyl)-N-(4-((3-chloro-4-fluorophenyl)
amino)-7-((tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)
acrylamide (10n)
5.12.9. (S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)-3-(4-nitrophenyl)acrylamide
(10i)
This compound was obtained as yellow solid in 58% yield. Mp
277.1–282.6 °C. ESI-MS m/z: [M+H]+584.1. 1H NMR (400 MHz,
DMSO) d 9.74 (s, 1H), 9.29 (s, 1H), 8.89 (s, 1H), 7.98 (dd, J = 6.8,
2.4 Hz, 1H), 7.91 (s, 1H), 7.73–7.59 (m, 4H), 7.54 (t, J = 9.0 Hz,
2H), 7.48–7.33 (m, 2H), 5.75 (s, 1H), 5.29 (s, 1H), 4.14 (d,
J = 10.4 Hz, 1H), 4.01 (m, 2H), 3.81 (dd, J = 13.6, 8.0 Hz, 1H), 2.41
(dt, J = 14.2, 7.0 Hz, 1H), 2.23 (dd, J = 12.8, 6.4 Hz, 1H).
This compound was obtained as yellow solid in 78% yield. Mp
245.7–246.8 °C. ESI-MS m/z: [M+H]+550.1. 1H NMR (400 MHz,
DMSO) d 9.69 (s, 1H), 9.07 (s, 1H), 8.58 (s, 1H), 8.31 (d, J = 8.4 Hz,
2H), 8.11 (d, J = 6.4 Hz, 1H), 7.95 (d, J = 5.6 Hz, 1H), 7.93 (s, 1H),
7.77 (s, 1H), 7.73 (s, 1H), 7.44 (m, 2H), 7.30 (s, 1H), 5.32 (s, 1H),
4.09–3.87 (m, 4H), 3.79 (d, J = 6.0 Hz, 1H), 2.38 (dd, J = 13.4,
6.9 Hz, 1H), 2.20 (d, J = 6.4 Hz, 1H).
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Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5.12.15. N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
nazolin-6-yl)-3-(4-cyanophenyl)acrylamide (11a)
J = 15.8 Hz, 1H), 6.65 (d, J = 6.4 Hz, 2H), 4.10 (s, 3H), 3.88 (d,
J = 10.2 Hz, 3H), 3.83 (d, J = 8.8 Hz, 3H).
This compound was obtained as yellow solid in 55% yield. Mp
240.1–242.0 °C. ESI-MS m/z: [M+H]+474.1. 1H NMR (400 MHz,
DMSO) d 9.94 (s, 1H), 9.14 (s, 1H), 8.70 (s, 1H), 8.09–8.02 (m,
1H), 7.94 (s, 1H), 7.92 (s, 1H), 7.85 (s, 1H), 7.83 (s, 1H), 7.79–7.72
(m, 1H), 7.69 (d, J = 15.8 Hz, 1H), 7.46 (dd, J = 15.6, 6.0 Hz, 2H),
7.39 (d, J = 4.8 Hz, 1H), 4.08 (s, 3H).
5.12.22. N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
nazolin-6-yl)-3-(3,4,5-trimethoxyphenyl)acrylamide (11h)
This compound was obtained as yellow solid in 73% yield. Mp
179.6–181.2 °C. ESI-MS m/z: [M+H]+539.2.1H NMR (400 MHz,
DMSO) d 9.88 (s, 1H), 9.65 (s, 1H), 9.06 (s, 1H), 8.54 (s, 1H), 8.13
(d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 15.6 Hz, 1H),
7.43 (t, J = 9.2 Hz, 1H), 7.32 (s, 1H), 7.21 (d, J = 15.6 Hz, 1H), 7.00
(s, 2H), 4.06 (s, 3H), 3.86 (s, 6H), 3.70 (d, J = 10.0 Hz, 3H).
5.12.16. N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
nazolin-6-yl)-3-(2-nitrophenyl)acrylamide (11b)
This compound was obtained as yellow solid in 62% yield. Mp
278.9–281.3 °C. ESI-MS m/z: [M+H]+494.1. 1H NMR (400 MHz,
DMSO) d 9.94 (s, 1H), 9.86 (s, 1H), 9.06 (s, 1H), 8.54 (s, 1H), 8.14
(dd, J = 6.8, 2.2 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.92 (t, J = 11.2 Hz,
1H), 7.90–7.83 (m, 2H), 7.83–7.78 (m, 1H), 7.69 (t, J = 7.6 Hz, 1H),
7.42 (t, J = 9.2 Hz, 1H), 7.31 (d, J = 11.0 Hz, 1H), 7.26 (d,
J = 15.4 Hz, 1H), 4.06 (s, 3H).
5.13. Cytotoxicity assay in vitro
The cytotoxic activities of target compounds (10a–n and 11a–h)
were evaluated with A549, PC-3, MCF-7 and Hela cell lines by the
standard MTT assay in vitro, with compounds EGFR inhibitors afa-
tinib as positive control. The cancer cell lines were cultured in min-
imum essential medium (MEM) supplement with 10% fetal bovine
serum (FBS). Approximately 4 ꢂ 103 cells, suspended in MEM med-
ium, were plated onto each well of a 96-well plate and incubated in
5% CO2 at 37 °C for 24 h. The test compounds at indicated final con-
centrations were added to the culture medium and the cell cul-
tures were continued for 72 h. Fresh MTT was added to each well
5.12.17. 3-(2-Chloro-4-fluorophenyl)-N-(4-((3-chloro-4-fluoro-
phenyl)amino)-7-methoxyquinazolin-6-yl)acrylamide (11c)
This compound was obtained as yellow solid in 58% yield. Mp
256.5–257.9 °C. ESI-MS m/z: [M+H]+502.1. 1H NMR (400 MHz,
DMSO) d 9.86 (s, 1H), 9.85 (s, 1H), 9.07 (s, 1H), 8.55 (s, 1H), 8.13
(d, J = 5.6 Hz, 1H), 7.90 (d, J = 12.8 Hz, 1H), 7.88–7.84 (m, 1H),
7.81 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.43 (t, J = 7.0 Hz,
1H), 7.41–7.35 (m, 1H), 7.31 (d, J = 6.8 Hz, 1H), 7.28 (d,
J = 15.6 Hz, 1H), 4.06 (s, 3H).
at a terminal concentration of 5
at 37 °C for 4 h. The formazan crystals were dissolved in 100
lg/mL and incubated with cells
lL
DMSO each well, and the absorbency at 492 nm (for absorbance
of MTT formazan) and 630 nm (for the reference wavelength)
was measured with the ELISA reader. All of the compounds were
tested three times in each of the cell lines. The results expressed
as inhibition rates or IC50 (half-maximal inhibitory concentration)
were the averages of two determinations and calculated by
using the Bacus Laboratories Incorporated Slide Scanner (Bliss)
software.
5.12.18. N-(4-((3-Chloro-4-fluorophenyl)amino)-7-
methoxyquinazolin-6-yl)-3-(2,5-dimethoxyphenyl)acrylamide
(11d)
This compound was obtained as yellow solid in 50% yield. Mp
272.1–273.0 °C. ESI-MS m/z: [M+H]+509.1. 1H NMR (400 MHz,
DMSO) d 9.93 (s, 1H), 9.27 (s, 1H), 8.86 (s, 1H), 7.99 (dd, J = 6.2,
2.3 Hz, 1H), 7.90 (d, J = 15.8 Hz, 1H), 7.70 (dd, J = 8.2, 3.5 Hz, 1H),
7.54 (t, J = 9.2 Hz, 1H), 7.44 (s, 1H), 7.27 (d, J = 15.8 Hz, 1H), 7.19
(d, J = 2.6 Hz, 1H), 7.06 (d, J = 9.2 Hz, 1H), 7.01 (dd, J = 9.0, 2.8 Hz,
1H), 4.11 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H).
5.14. EGFR and VEGFR2/KDR kinases assay in vitro
The target compounds 10e and 10k are tested for their activity
against EGFR or VEGFR2/KDR kinases through the mobility shift
assay. All kinases assays were performed in 96-well plates in a
5.12.19. N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
nazolin-6-yl)-3-(4-methoxyphenyl)acrylamide (11e)
50 lL reaction volume. The kinase buffer contains 50 mM HEPES,
pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop
buffer contains 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coat-
ing Reagent #3 and 50 mM EDTA. Dilute the compounds to
This compound was obtained as yellow solid in 60% yield. Mp
253.4–254.7 °C. ESI-MS m/z: [M+H]+479.1. 1H NMR (400 MHz,
DMSO) d 9.66 (s, 1H), 9.05 (s, 1H), 8.56 (s, 1H), 8.12 (dd, J = 6.8,
2.2 Hz, 1H), 7.84–7.76 (m, 1H), 7.62 (s, 1H), 7.60 (s, 1H), 7.57 (s,
1H), 7.43 (t, J = 9.2 Hz, 1H), 7.31 (s, 1H), 7.10 (d, J = 15.8 Hz, 1H),
7.04 (s, 1H), 7.01 (s, 1H), 4.05 (s, 3H), 3.81 (s, 3H).
500
new 96-well plate as the intermediate plate, add 90
fer to each well. Transfer 5 L of each well of the intermediate
lM by 100% DMSO, then transfer 10
lL of compound to a
l
L kinase buf-
l
plate to 384-well plates. The following amounts of enzyme and
substrate were used per well: kinase base buffer, FAM-labeled pep-
tide, ATP and enzyme solution. Wells containing the substrate,
enzyme, DMSO without compound were used as DMSO control.
Wells containing just the substrate without enzyme were used as
5.12.20. N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
nazolin-6-yl)-3-(2,4-dimethoxyphenyl)acrylamide (11f)
This compound was obtained as yellow solid in 56% yield. Mp
247.1–248.3 °C. ESI-MS m/z: [M+H]+509.1. 1H NMR (400 MHz,
DMSO) d 9.87 (s, 1H), 9.28 (s, 1H), 8.88 (s, 1H), 8.01–7.93 (m,
1H), 7.84 (d, J = 15.8 Hz, 1H), 7.72–7.65 (m, 1H), 7.57 (s, 1H), 7.54
(d, J = 7.2 Hz, 1H), 7.50 (s, 1H), 7.14 (d, J = 15.8 Hz, 1H), 6.65 (s,
1H), 6.62 (d, J = 8.8 Hz, 1H), 4.10 (s, 3H), 3.89 (s, 3H), 3.82 (d,
J = 6.8 Hz, 3H).
low control. Incubate at room temperature for 10 min. Add 10
peptide solution to each well. Incubate at 28 °C for specified period
of time and stop reaction by 25 L stop buffer. At last collect data
lL
l
on Caliper program and convert conversion values to inhibition
values. Percent inhibition = (max ꢀ conversion)/(max ꢀ min) ꢂ
100. ‘max’ stands for DMSO control; ‘min’ stands for low control.
5.12.21. N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
nazolin-6-yl)-3-(3,5-dimethoxyphenyl)acrylamide (11g)
5.15. Docking studies
This compound was obtained as light yellow solid in 65% yield.
Mp 253.2–254.3 °C. ESI-MS m/z: [M+H]+509.1. 1H NMR (400 MHz,
DMSO) d 9.88 (s, 1H), 9.28 (s, 1H), 8.89 (s, 1H), 8.03–7.96 (m,
1H), 7.85 (d, J = 15.8 Hz, 1H), 7.73–7.65 (m, 1H), 7.57 (d,
J = 5.8 Hz, 1H), 7.55 (s, 1H), 7.52 (d, J = 4.8 Hz, 1H), 7.15 (d,
For docking purposes, the three-dimensional structure of the
EGFR (PDB code: 4G5J) was obtained from RCSB Protein Data
Bank.13 Hydrogen atoms were added to the structure allowing for
appropriate ionization at physiological pH. The protonated state
of several important residue were adjusted by using SYBYL6.9.1
Please cite this article in press as: Tu, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.017

Y. Tu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
(Tripos, St. Louis, USA) in favor of forming reasonable hydrogen
bond with the ligand. Molecular docking analysis was carried out
by the SURFLEX-DOCK module of SYBYL 6.9.1 package to explore
the binding model for the active site of c-Met with its ligand. All
atoms located within the range of 5.0 Å from any atom of the cofac-
tor were selected into the active site, and the corresponding amino
acid residue was, therefore, involved into the active site if only one
of its atoms was selected. Other default parameters were adopted
in the SURFLEX-DOCK calculations. All calculations were per-
formed on Silicon Graphics workstation.
Fund Designated for Graduate Students of Jiangxi Province
(YC2015-X28).
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Acknowledgments
We gratefully acknowledge the generous support provided by
The National Natural Science Funds (No. 81460527), Science and
Technology Project Founded by the Education Department of
Jiangxi Province, Top-notch talent project of Jiangxi Science &
Technology Normal University (2015QNBJRC001), Program of Key
Laboratory of Drug Design and Optimization, Jiangxi Science &
Technology Normal University (300098010306), College Students’
Science and Technology Innovation Project of Jiangxi Science &
Technology Normal University (20150904040) and the Innovation
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Please cite this article in press as: Tu, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.017