Design, synthesis, anti-tumor activity, and molecular modeling of quinazoline and pyrido[2,3-d]pyrimidine derivatives targeting epidermal growth factor receptor

Article abstract of DOI:10.1016/j.ejmech.2016.04.026

Three series of novel quinazoline and pyrido[2,3-d]pyrimidine derivatives were designed, synthesized and evaluated for their ability to inhibit EGFR tyrosine kinase and a panel of five human cancer cell lines (MCF-7, A549, BT-474, SK-BR-3, and MDA-MB-231)

Full text of DOI:10.1016/j.ejmech.2016.04.026

European Journal of Medicinal Chemistry 118 (2016) 276e289
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, anti-tumor activity, and molecular modeling of
quinazoline and pyrido[2,3-d]pyrimidine derivatives targeting
epidermal growth factor receptor
1
1
Ju Hou , Shanhe Wan , Guangfa Wang, Tingting Zhang, Zhonghuang Li, Yuanxin Tian,
**
*
Yonghuan Yu, Xiaoyun Wu , Jiajie Zhang
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science, Southern Medical University, Guangzhou, 510515, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 December 2015
Received in revised form
Three series of novel quinazoline and pyrido[2,3-d]pyrimidine derivatives were designed, synthesized
and evaluated for their ability to inhibit EGFR tyrosine kinase and a panel of five human cancer cell lines
(
MCF-7, A549, BT-474, SK-BR-3, and MDA-MB-231). Bioassay results indicated that five of these prepared
22 March 2016
compounds (12ce12e and 13ce13d) exhibited remarkably higher inhibitory activities against EGFR and
SK-BR-3 cell line. Compounds 12c and 12e displayed the most potent EGFR inhibitory activity
Accepted 8 April 2016
Available online 20 April 2016
(
IC50 ¼ 2.97 nM and 3.58 nM, respectively) and good anti-proliferative effect against SK-BR-3 cell with
the IC50 values of 3.10 M and 5.87 M, respectively. Furthermore, molecular docking and molecular
m
m
Keywords:
EGFR inhibitor
Quinazoline and pyrido[2,3-d]pyrimidine
derivatives
Antitumor
dynamics simulation studies verified that compound 12c and 12e shared similar binding pattern with
gefitinib in the binding pocket of EGFR. MM-GBSA binding free energy revealed that the compound 12c
and 12e have almost the same inhibitory activity against EGFR as gefitinib, and that the dominating effect
of van der Waals interactions drives the binding process.
Molecular docking
Molecular dynamics simulation
© 2016 Published by Elsevier Masson SAS.
1. Introduction
etc [4]. Therefore, EGFR tyrosine kinase represents an attractive
target for the treatment of many human cancers.
Epidermal growth factor receptor (EGFR) belongs to the ErbB
family of receptor tyrosine kinases. This family includes homolo-
gous receptors: the epidermal growth factor receptor (ErbB1/EGFr/
HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). These
receptors are composed of an extracellular ligand-binding domain,
a transmembrane lipophilic segment, and an intracellular protein
tyrosine kinase domain with a regulatory carboxyl terminal
segment [1]. When ligands bind to these domains, receptor
dimerization and autophosphorylation of intracellular tyrosine ki-
nase domains occur. Autophosphorylation activating the down-
stream signaling pathways leads to diverse effects including cell
proliferation, survival, adhesion, migration, and differentiation
Quinazoline derivatives have long been recognized as an
important scaffold for designing anti-EGFR agents [5,6]. The clinical
success of selective kinase inhibitors, such as gefitinib [7,8], lapa-
tinib [9], erlotinib [10], and canertinib [11,12] (Fig. 1), as therapeutic
agents for several human cancers has prompted substantial interest
in the further development and clinical testing of such inhibitors
for a wide variety of malignancies. On the other hand, pyrido[2,3-d]
pyrimidine derivatives also can be interacted with ATP-binding site
of EGFR catalytic domain to inhibit EGFR [13,14]. Compound A-1
(Fig. 1) belongs to the pyrido[2,3-d]pyrimidines, which showed
nanomolarly potent EGFR inhibitory activity [14]. Quinazoline and
pyrido[2,3-d]pyrimidine derivatives displayed favorable inhibitory
activities against EGFR spured us on to greater efforts to develop
novel and highly potent EGFR inhibitors.
Based on the crystal structure of EGFR complexed with gefitinib,
we undertook the design of quinazoline and pyrido[2,3-d]pyrimi-
dine derivatives having different substituents at C-2, C-4, and C-6
positions. To increase the hydrogen bond interactions with hinge
region of the kinase, an amino group was introduced to C-2 position
of the quinazoline core. In order to extend into the solvent region
[2,3]. Previous research has demonstrated that EGFR is a putative
oncogene and has been found in many human solid malignancies
including non-small-cell lung cancer, ovarian cancer, breast cancer,
*
Corresponding author.
** Corresponding author.
E-mail addresses: xywugz@163.com (X. Wu), zhangjj@smu.edu.cn (J. Zhang).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.04.026
223-5234/© 2016 Published by Elsevier Masson SAS.
0

J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
277
Fig. 1. Structure of EGFR small molecule inhibitors.
surrounding the kinase hinge segment, introduction of the polar N-
2.2. Biological activity
substituted pyrazole fragments at the C-6 position of quinazoline
and pyrido[2,3-d]pyrimidine scaffolds was investigated. Different
kinds of aryl fragments connected to quinazoline and pyrido[2,3-d]
pyrimidine nucleus through O-linker or N-linker at C-4 position
were investigated to increase hydrophobic interactions with the
hydrophobic pocket, which are crucial for EGFR inhibitory activity.
Herein, we would like to report the synthesis and biological eval-
uation of these new series compounds for the first time.
2.2.1. Kinase inhibitory activity
The enzyme activity assays of new compounds were performed
at a single-point concentration (1 M) against EGFR and their
inhibitory activities were displayed at Table 1. As shown in Table 1,
compounds 12ce12e and 13ce13d exhibited favorable inhibition of
the ErbB1 (percent inhibition values range of 99e102%), while
compounds 7d, 12a, 12f, 13a, and 13b showed moderate inhibitory
activities (percentage of inhibition raning from 30% to 67%) and
others had no obvious inhibitory activities.
m
2
. Results and discussion
Subsequently, we further evaluated the EGFR inhibitory activ-
ities of compounds 12ce12e and 13ce13d with excellent inhibition
2.1. Chemistry
at 1
mM. As shown in Table 2, the IC50 values ranging from 2.97 to
108 nM against ErbB1 and EGFR (L858R). Compounds 12ce12e and
The synthetic strategy to prepare the target compounds is
13ce13d were chosen for further evaluation of the selectivity of
EGFR kinase, the IC50 values of 12ce12e and 13ce13d ranging from
590 to 1603 nM against HER2, and HER4, while compounds
12ce12e and 13ce13d had no obvious inhibitory activity toward
EGFR (T790M/L858R). These results indicated that compounds
12ce12e and 13ce13d had good selectivity to EGFR kinase.
illustrated in Schemes 1e3. The bromination of the starting mate-
rial 1 led to 2-amino-5-bromonicotinic acid (2), followed by cycil-
zation with formamide to give compound 3 [15], which was
chloride with thionyl chloride using DMF as catalyst to afford 4. The
chlorine in the 4-position of intermediate 4 could be displaced with
a variety of benzylamines to achieve intermediates 5ae5d. Intro-
duction of the pyrazole fragment at the C-6 position of 5ae5d by
conventional Suzuki coupling with the corresponding pyrazole
boronic esters to obtain desired compounds 7ae7d of series-I, or
further deprotection of the Boc group of intermediate smoothly to
give desired compound 6 of series-I [15].
Likewise, compounds 9 and 10, which were prepared following
the similar procedure described above for the preparation of 3 and
, respectively. Compound 10 was displaced with a variety of nu-
cleophiles. The resulting intermediates 11ae11g were converted to
target compounds 12ae12f and 13ae13d of series-II by Suzuki
coupling reaction as described in Scheme 2.
2.2.2. In vitro anti-proliferative activity
The antiproliferative activities of newly synthesized compounds
were evaluated against a panel of five human cancer cell lines
overexpressing EGFR, including MCF-7, A549, BT-474, SK-BR-3, and
MDA-MB-231 by the standard MTT assay in vitro [17e19], with
gefitinib as the positive control. The inhibitory activities (IC50) were
summarized in Table 1. As depicted in Table 1, the results indicated
that most compounds showed moderate to good activity with IC50
4
values in the
mM range, while compounds 7ae7d proved to be
ineffective against the five cell lines, and compounds 12c and 18
The intermediate 16 was produced from 14 by the reaction with
dicyandiamide followed by the cleavage of the amidino moiety of
also showed no inhibitory activity against A549 cell line
(IC50 > 100
mM). Some of the synthesized compounds exhibited
1
5 [16], which was condensed with different reagents to give the
desired products 17ae17b. Final compounds 18 and 19ae19b of
series- were similarly obtained of series-I as shown in Scheme 3.
The structures of all the newly synthesized compounds were
similar anti-proliferative activities as gefitinib against the three to
four cell lines. Specifically compounds 6, 12d, 13ae13b, 18, and
19ae19b showed higher antiproliferative activities than gefitinib
against MCF-7 cell line. In addition, five compounds (12ce12e and
13ce13d) exhibited remarkable inhibitory activity with the IC50
Ш
1
13
characterized by H NMR, C NMR, ESI-MS, and HRMS.

278
J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
Scheme 1. Reagents and conditions: (i) bromine, acetic acid, rt; (ii) formamide, reflux; (iii) SOCl
2
and DMF, reflux; (iv) substituted-benzylamines, dioxane, rt; (v) K
2 3
CO , PdCl
2
(dppf)
ꢀ
CH
2
Cl2, dioxane/H
2
O (24:1), 85 C; (vi) CH
2
Cl
2
/TFA (4:1), rt.
Scheme 2. Reagents and conditions: (i) formamide, reflux; (ii) SOCl
2
and DMF, reflux, (iii) for 11ae11b, substituted-benzylamines, K
2
CO
3
, dioxane, rt; for 11c, 1-(2,6-dichloro-3-
, dioxane, rt; (iv) K CO , PdCl (dppf)
ꢀ
fluorophenyl)- ethanol, dioxane, NaH, 0 C-rt; for 11de11f, substituted-anilines or 6-aminoquinoline, acetone, HCl, reflux; for 11g, Cs
2
CO
3
2
3
2
ꢀ
CH
2
Cl2, dioxane/H
2
O (24:1), 85 C; (v) CH
2
Cl
2
/TFA (4:1), rt.
values ranging from 0.064 to 5.87
that of gefitinib (IC50 ¼ 4.92 M) on SK-BR-3 cell line. All the
compounds displayed poor activities (IC50: 3.00 e >100 M) in BT-
74 cancer cell line, as compared with gefitinib.
m
M, which were comparable to
This primary screening revealed that 4-aminoquinazoline core
derivatives exhibited strong EGFR enzymatic potency and anti-
proliferative activities. Specifically aniline moieties and quinoline
amino fragment at C-4 position of the quinazoline core showed
m
m
4

J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
279
ꢀ
ꢀ
Scheme 3. Reagents and conditions: (i) dicyandiamide, sulfuric acid, 100 C; (ii) 4-Pyridinethiol, KOH, ethylene glycol, 170 C; (iii) for 17a, BOP, DBU, 1-(2,6-Dichloro-3-fluorophenyl)
ꢀ
ethan-1- amine, acetonitrile, rt; for 17b, BOP, DBU, 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-ol, NaH, acetonitrile, rt; (iv) K
CH Cl /TFA (4:1), rt.
2
3
CO , PdCl
2
(dppf)CH
2
Cl2, dioxane/H
2
O (24:1), 85 C; (v)
2
2
promising inhibitory activity against EGFR and potent activity to-
ward SK-BR-3 cell line, and had the makings of good drugs for
breast cancer. The pyrido[2,3-d]pyrimidine derivatives displayed a
significant loss in potency compared to that of 4-aminoquinazoline
core derivatives. Moreover, 2-aminoquinazoline nucleus de-
rivatives gave weak EGFR inhibitory activities. The enzymatic and
cellular responses were inconsistent for some target compounds.
Notably, compounds 6, 13ae13b, 18, and 19ae19b displayed poor
activities against EGFR kinase, but they showed similar or higher
antiproliferative activities than gefitinib against some cancer cell
lines that express high levels of EGFR. It suggested that the com-
pounds might induce cells apoptosis, or accelerate cell metabolism,
or have some other mechanisms.
probable binding conformation. As shown in Fig. 3, the redocked
gefitinib is almost in the same position with co-crystallized gefiti-
nib at the active site of EGFR except the flexible long chain
extending into the solvent accessible region, which have revealed
that the high reliability of Surflex-dock in reproducing the experi-
mentally observed binding mode for the EGFR inhibitors. Therefore,
Surflex-Dock method was used in search of the binding confor-
mations of the newly synthesised compounds. All compounds,
except 2-amino-quinazoline derivatives 18 and 19ae19b, were
successfully docked into the active site of EGFR (Fig. 4). As is shown
in Fig. 4, quinazoline and pyrido[2,3-d]pyrimidine compounds
share similar binding pattern in the binding pocket with the co-
crystal ligand gefitinib in the reference crystal structure, while
the 2-amino-quinazoline derivatives is shifted out of the binding
pocket to the solvent surface, which may be the reason of the low
potency of 2-amino-quinazoline compounds.
2.3. Preliminary structureeactivity relationships
Based on biological activity evaluation of the synthetic com-
MD simulations were carried out on active inhibitors 12c and
12e in complex with EGFR to explore in depth the binding poses.
For comparison, the MD simulations of positive control gefitinib
and less active compound 7a complexed with EGFR were also
carried out.
pounds, the structureeactivity relationships of these novel com-
pounds were primarily analyzed. As shown in Fig. 2, the pyrido[2,3-
d]pyrimidine derivatives show decreased inhibitory activity, and
introduction of amino group at C-2 position of the quinazoline core
did not led to significant inhibitory activity. Moreover, N-linker
compounds showed good inhibitory activity than O-linker at C-4
position of scaffolds; aniline moieties directly conjunct to the 4-
position of quinazoline core increased EGFR inhibitory activities
as compared to benzyl amino groups. Interestingly, quinoline
amino fragment at C-4 position of the quinazoline core also
exhibited strong EGFR enzymatic and cellular potency.
MD simulations were processed in explicit aqueous solution for
20 ns. The stability of the system under simulation was evaluated
by the root-mean-square deviation (RMSD) of the backbone atoms
relate to the starting structures (Fig. 5). As can be seen in the plot,
all systems reached equilibrium after 8 ns simulation times. Fig. 6
shows the binding poses of the two active compounds 12c and
12e in the binding pocket of EGFR. As illustrated in Fig. 6, the
binding modes of 12c and 12e from MD simulated results are nearly
the same as the docked structures. The quinazoline moiety inserts
into deep hydrophobic pocket, the aniline moiety is deeply in the
back of the ATP-binding pocket. The pyrazole substituent occupies
the solvent exposed region, indicating that the introduction of
hydrophilic groups here probably is beneficial for potency.
The hydrogen bond plays an important role in inhibitor binding
to kinase [21]. The hydrogen-bond interactions were examined and
found that N1 of quinazoline ring of gefitinib, 12c and 12e establish
strong hydrogen interaction with the backbone NH of Met-793 in
the hinge region of EGFR during all the MD simulation time
(Table 3), while the key hydrogenebond interaction could not be
2
.4. Molecular modeling
To predict the possible binding mode of designed compounds
with EGFR, a study of docking of all compounds into the active site
of the EGFR (PDB ID: 4WKQ) were performed using Surflex-Dock
and then molecular dynamics (MD) simulations were performed
by using AMBER software package.
The docking reliability was validated using the known X-ray
structure of EGFR complexed with gefitinib. The co-crystallized
gefitinib was re-docked into the binding site, and the docked
conformation with the highest total score was selected as the most

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J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
Table 1
The kinase inhibitory activities and in vitro antiproliferative effects of synthesized compounds.
Com
X
R
1
R
2
EGFR inhibition at 1
17
m
М (%)a
In vitro antiproliferative effects (IC50, m
M)b
MCF-7
1.63
A549
29.73
BT-474
13.47
SK-BR-3
17.67
MDA-MB-231
7.11
6
NH
7
7
a
NH
NH
CH
CH
3
3
9
0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
b
7
7
1
1
c
NH
NH
NH
O
CH
CH
CH
CH
3
3
3
3
18
30
67
11
>100
>100
40.04
22.55
>100
>100
48.90
28.79
>100
>100
30.40
45.60
>100
>100
35.95
27.20
>100
>100
28.49
30
d
2a
2b
12c
NH
CH
3
101
52.20
>100
5.26
3.10
62.65
1
1
1
1
2d
2e
2f
NH
NH
O
CH
CH
CH
3
3
3
101
102
43
0.49
1.38
9.86
26.63
7.00
10.93
5.68
0.064
5.87
26.28
16.93
16.51
12.60
11.37
21.97
2.65
24.30
8.13
14.42
4.39
3a
NH
61
13b
O
52
2.67
6.75
4.14
2.50
6.77
1
1
3c
NH
NH
99
96
31.30
61.93
29.43
32.01
15.16
12.85
3.74
2.66
28.35
30.33
3d
1
1
1
8
NH
NH
O
CH
3
12
17
2.87
2.23
1.24
25.37
>100
8.80
7.12
5.10
3.00
0.53
7.14
1.58
7.12
4.92
7.96
4.48
7.00
37.82
9a
9b
7
9.49
gefitinib
101
24.25
a
Values are the average of two independent experiments.
The data were means from at least three independent experiments.
b

J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
281
Table 2
In vitro enzymatic inhibitory activities of compounds 12ce12e and 13ce13d against different types of EGFR.
Com
EGFR (IC50, nM)a
EGFR (ErbB1)
EGFR (ErbB1) L858R
EGFR (ErbB1) L858R/T790M
ErbB2 (HER2)
ErbB4 (HER4)
1
1
1
1
1
2c
2d
2e
3c
3d
2.97
44.1
3.58
31.9
28.2
0.585
3.50
108
4.25
39.1
35.0
0.637
˃1000
˃1000
˃1000
˃1000
˃1000
1013 [20]
676
874
590
1202
1350
1830 [20]
801
930
782
1603
1100
b
gefitinib
ND
a
Values are the average of two independent experiments.
ND, not determined.
b
Fig. 2. Preliminary structureeactivity relationships.
Fig. 3. Conformation comparison between redocked (cyan) and the co-crystallized (green) gefitinib binding to EGFR. (For interpretation of the references to color in this figure
caption, the reader is referred to the web version of this article.)
formed in the system of 7a-EGFR, which partically explains the low
potency of compound 7a.
The binding free energies were calculated by using MM-GBSA
program in AMBER taking the last 10 ns of the MD simulation. In
Table 4, we present the predicted binding free energies, together
with their respective enthalpic and entropic contributions. As can
been seen, MM-GBSA calculations verified that ligands 12c and 12e
have almost the same activities against EGFR as the positive control

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J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
Fig. 4. Binding mode between EGFR kinase and representative compounds. (gefitinib in stick and ball model, 7a in green stick model, 12c in blue stick model, and 19a in red stick
model). (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)
Fig. 5. RMSDs of backbone atoms (C, Ca, and N) of the systems against the initial structures.
gefitinib, which is in good agreement with the experimental data.
In addition, the binding free energy of compound 7a binding to
EGFR is approximately 3 kcal/mol more than that of gefitinib, in
consistent with the experimental observation that ligand 7a ex-
hibits drastically diminished potency.
important terms which dictates the difference in the binding af-
finity are
DGele and DG
vdW.
3. Conclusion
According to the energy individual components of the binding
free energies, the favorable contributors to ligand binding are van
der Waals (vdW) terms, electrostatic and nonpolar salvation en-
ergies, whereas polar solvation and entropy terms oppose binding.
The favorable electrostatic interactions are counteracted by the
unfavorable electrostatics of desolvation upon binding. Conse-
quently, the total electrostatic interaction contributions are unfa-
vorable to binding in all systems. The vdW contribution interaction
upon binding is very important to the binding of inhibitors with
EGFR. Comparing 7a-EGFR with the other complexes, the most
In summary, three series of novel quinazoline and pyrido[2,3-d]
pyrimidine derivatives were designed, synthesized and screened
for biological activity as novel EGFR inhibitors. The preliminary
investigation shows some compounds displayed favorable effect in
the EGFR kinase assay and antiproliferative assay. Among them,
compounds 12c and 12e showed the higher potency to EGFR ki-
nase. In addition, the molecular docking and MD simulations ex-
periments have revealed that the binding poses of 12c and 12e with
EGFR are similar to that of gefitinib. MM-GBSA binding free energy
revealed that the compound 12c and 12e have almost the same

J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
283
Fig. 6. Binding mode comparison between redocked (green) and MD simulated representative snapshots (cyan) of 12c (left panel) and 12e (right panel) in the active site of EGFR.
Yellow dots represent hydrogen bonds. (EGFR in colored cartoon, ligands in stick model, the key residues in lines model). (For interpretation of the references to color in this figure
caption, the reader is referred to the web version of this article.)
Table 3
Hydrogen Bonds analysis from MD.
Distance (Å)a
Angle( )b
ꢀ
Occupancy (%)c
Donor
Acceptor
gefitinib-EGFR
Met 793 NH
Met 793 NH
Met 793 NH
Met 793 NH
Ligand N
Ligand N
Ligand N
Ligand N
3.163(0.15)
3.291(0.16)
3.118(0.15)
3.141(0.15)
24.38(11.31)
34.78(15.22)
22.17(12.34)
26.30(13.19)
92.45
0.95
95.55
89.65
7
1
1
a-EGFR
2c-EGFR
2e-EGFR
a
The average distance with standard error (SE ¼ standard deviation/N1/2) in parentheses between hydrogen-acceptor atom and hydrogen-donor atom in the investigated
time period.
b
The average angle with standard error (SE ¼ standard deviation/N1/2) in parentheses for hydrogen bond in the investigated time period.
c
Occupancy is in unit of percentage of the investigated time period.
Table 4
MM-GBSA binding free energies and its components for the studied complexes for the last 10 ns MD trajectories .
a
D
G
vdW
DG
ele
D
G
MM
DG
ele,sol
DG
np,sol
D
H
pred
ꢁTD
S
DG
pred
gefitinib
ꢁ47.78 (0.10)
ꢁ41.51 (0.07)
ꢁ44.38 (0.08)
ꢁ45.29 (0.09)
ꢁ90.05 (0.40)
ꢁ7.55 (0.08)
ꢁ11.42 (0.11)
ꢁ17.75 (0.08)
ꢁ137.83 (0.43)
ꢁ49.06 (0.13)
ꢁ55.81 (0.14)
ꢁ63.04 (0.13)
108.48 (0.41)
25.89 (0.10)
25.23 (0.11)
33.16 (0.08)
ꢁ6.17 (0.01)
ꢁ5.22 (0.01)
ꢁ5.51 (0.01)
ꢁ5.46 (0.01)
ꢁ35.52 (0.10)
ꢁ28.39 (0.07)
ꢁ36.08 (0.08)
ꢁ35.34 (0.09)
22.15 (2.94)
18.33 (2.30)
22.31 (0.61)
22.22 (1.36)
ꢁ13.37
ꢁ10.06
ꢁ13.77
ꢁ13.12
7
1
1
a
2c
2e
D
H: the enthalpy changes,
DH ¼
DG
ele
þ
DG
vdW
þ
DG
np,sol
þ
ele,sol
DG .
T
DS: the entropy changes.
D
Gpred: the calculated binding free energy by MM-GBSA method.
a
Mean energies are in kcal/mol, with corresponding standard errors (SE ¼ standard deviation/N1/2) in parentheses.
inhibitory activity against EGFR as gefitinib, and that the domi-
nating effect of vdW interactions drives the binding process.
Compounds 12c and 12e could be worth of further development,
and these results expand the chemical diversity of EGFR inhibitors.
Flach column chromatography separations were performed on
normal phase silica gel (200e300 mesh, Merck) or reverse phase
silica gel by using Yamazen AI-580 flash chromatography with UV
detection at 254 nm. Melting points were determined using a X-4
Melting-point Apparatus with Microscope (Gongyi City Yuhua In-
strument Co., Ltd., Hennan, China) and were uncorrected.
4
. Experimental
4.1. Chemistry
4.1.1. 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-amine [15]
Preparation of 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-amine
and characteriza-tion data have already been reported. H NMR
1
All reagents were purchased from commercial vendors and
were used without further purification. All oxygen-sensitive or
(400 MHz, DMSO-d6)
d
8.72 (s, 2H), 7.66 (dd, J ¼ 5.2, 9.2 Hz,1H), 7.58
moisture-sensitive reactions were run under nitrogen atmosphere.
(t, J ¼ 8.8 Hz, 1H), 5.03 (q, J ¼ 6.8 Hz, 1H), 1.63 (d, J ¼ 7.2 Hz, 1H), ESI-
1
13
þ
H and C NMR spectra were recorded on a BRUKER AVIII 400 MHz
and 101 MHz spectrometer with tetramethylsilane (TMS) as the
internal standard, the values of the chemical shifts ( ) are given in
MS m/z: 208.0 [MþH] .
d
ppm, and coupling constants (J) are given in Hz. Mass spectra (ESI-
MS) were performed on WATERS ZQ4000. Highresolution mass
spectra (HRMS) were recorded on AB Sciex TripleTOF 5600 mass
spectrometer. All reactions were monitored using thin-layer chro-
matography (TLC) on silica gel plates at 254 nm under a UV laMp.
4.1.2. 1-(pyridin-3-yl)ethanamine [22]
Preparation of 1-(pyridin-3-yl)ethanamine and characterization
1
data have already been reported. H NMR (400 MHz, MeOD)
d 8.48
(d, J ¼ 6.4 Hz, 2H), 7.46 (d, J ¼ 6.0 Hz, 2H), 4.08 (q, J ¼ 6.6 Hz, 1H),
þ
1.41 (d, J ¼ 6.8 Hz, 3H), ESI-MS m/z: 123.2 [MþH] .

284
J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
4
.1.3. 2-amino-5-bromonicotinic acid (2)
4.1.10. N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-6-(1-(piperidin-4-
yl)-1H-pyrazol-4-yl)pyrido[2,3-d]pyrimidin-4-amine (6)
A mixture of compound 5a (457 mg, 1.1 mmol), tert-Butyl 4-[4-
(4,4,5,5-tetram- ethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]
Preparation of 2 and characterization data have already been
1
reported. H NMR (400 MHz, MeOD)
d
9.24 (d, J ¼ 2.0 Hz, 1H), 9.14
þ
(
d, J ¼ 2.0 Hz, 1H). ESI-MS m/z: 216.7 [MþH] .
piperidine-1-carboxylate (21) (400 mg, 1.06 mmol), PdCl
CH Cl (80.2 mg, 0.098 mmol), and K CO (420 mg, 3.04 mmol) in
dioxane: H O (24:1) (12 mL) was degassed and charged with ni-
2
(dppf)
4.1.4. 6-bromopyrido[2,3-d]pyrimidin-4-ol (3)
-amino-5-bromonicotinic acid (2) (21.6 g, 0.10 mol) was added
2
2
2
3
2
2
ꢀ
trogen for three times and then heated in an oil bath at 80 C for 4 h
under the protection of nitrogen. The reaction mixture was diluted
with water (30 mL) and eracted with EtOAc (30 mL ꢂ 3). The
to a solution of formamide 100 mL, and then the reaction mixture
was stirred at reflux for 5 h. The mixture was poured into
8
00 mL H
2
O. The resulting precipitate was filtered, washed with
2 4
combined organic layer was dried over anhydrous Na SO , filtered,
water, and dried to give 3 as an off-white solid (16.9 g, yield, 74%).
1
and concentrated. The concentrates was purified by flash chroma-
tography on silica gel eluting with petroleum ether-EtOAc-
methnaol (containing 1% ammonia) (40:60:0.05) to give a com-
pound as a white solid. The solid was dissolved in a mixed solvent
H NMR (400 MHz, DMSO-d
6
)
d
12.72 (s,1H), 9.05 (d, J ¼ 2.4 Hz,1H),
þ
8
.63 (d, J ¼ 2.4 Hz, 1H), 8.36 (s, 1H). ESI-MS m/z: 228.3 [MþH] .
4.1.5. 6-bromo-4-chloropyrido[2,3-d]pyrimidine (4)
system (CH
2
Cl
2
/TFA ¼ 4:1, 20 mL), then the reaction mixture was
To the suspension of 6-bromopyrido[2,3-d]pyrimidin-4-ol (3)
stirred at room temperature overnight, and concentrated. The
(
15.0 g, 0.066 mol) in thionyl chloride 150 mL was added a few
residue was triturated with acetone-petroleum ether (1:1) to give 6
drops of DMF, then the mixture was stirred at reflux for 6 h. After
removing thionyl chloride under vacuum, the residue was recrys-
ꢀ
1
as a white solid (231 mg, yield, 48%). mp 265e267 C. H NMR
400 MHz, DMSO-d 11.22 (s, 1H), 10.13 (s, 1H), 9.42 (s, 1H), 9.21
(
6
) d
tallized from EtOAc to gave 4 as pale yellow solid (11.8 g, yield, 73%).
1
(s, 1H), 8.78 (s, 2H), 8.39 (s, 1H), 7.49 (s, 1H), 7.39 (t, J ¼ 7.2 Hz, 1H)
H NMR (400 MHz, CDCl
3
)
d
9.33 (d, J ¼ 7.6 Hz, 2H), 8.80 (s, 1H). ESI-
þ
5.96 (s,1H), 4.60 (s,1H), 3.61 (s, 2H), 3.12 (s, 2H), 2.27 (s, 4H),1.87 (d,
MS m/z: 246.9 [MþH] .
1
3
J ¼ 6.0 Hz, 3H). C NMR (101 MHz, MeOD)
d 161.0, 158.5, 156.0,
1
55.1, 151.8, 146.4, 138.1, 137.1, 130.4, 129.9, 128.1, 127.6, 117.6, 116.0,
4.1.6. 6-bromo-N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)pyrido
þ
1
15.8, 109.2, 56.1, 51.3, 43.0, 28.9, 16.1. ESI-MS m/z: 486.5 [MþH] .
[2,3-d]pyrimidin-4-amine (5a)
þ
þ
HRMS, ESI , m/z: Calcd for C23
86.1368.
H22Cl
2
FN
7
(MþH) , 486.1371; found,
To a solution of compound 4 (2.43 g, 0.01 mol) in 25 mL dioxane
4
was added 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-amine (3.12 g,
.015 mol), and then the reaction mixture was stirred at room
temperature overnight. After that, the mixture was poured into
50 mL H O, and then the resulting precipitate was filtered, washed
with 50% ethanol, and dried overnight under vacuum to give 5a as
0
1
2
4.1.11. N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-6-(1-methyl-1H-
pyrazol-4-yl)pyrido[2,3-d]pyrimidin-4-amine (7a)
A mixture of compound 5a (208 mg, 0.50 mmol), 1-methyl-4-
(4,4,5,5-tetrameth- yl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (20)
1
white solid (3.58 g, yield, 86%). H NMR (400 MHz, DMSO-d
6
)
d
9.40
(
8
d, J ¼ 2.4 Hz, 1H), 9.07 (d, J ¼ 2.0 Hz, 1H), 9.03 (d, J ¼ 5.2 Hz, 1H),
.48 (s, 1H), 7.47e7.44 (m, 1H), 7.35 (t, J ¼ 8.8 Hz, 1H), 5.85 (q,
(108 mg, 0.52 mmol), PdCl
2
(dppf)- CH
2
Cl
2
(40.1 mg, 0.05 mmol)
O (24:1) (15 mL) was
þ
J ¼ 7.0 Hz, 1H), 1.71 (d, J ¼ 7.2 Hz, 3H). ESI-MS m/z: 417.4 [MþH] .
and K CO (207 mg, 1.5 mmol) in dioxane: H
2
3
2
degassed and charged with nitrogen for three times and then
ꢀ
heated in an oil bath at 80 C for 4 h under the protection of ni-
4
.1.7. 6-bromo-N-(1-(pyridin-4-yl)ethyl)pyrido[2,3-d]pyrimidin-4-
amine (5b)
Compound 5b was synthesized from 4 and 1-(pyridin-3-yl)
ethanamine following the similar procedure described above for
trogen. The reaction mixture was diluted with water (30 mL) and
eracted with EtOAc (30 mL ꢂ 3). The combined organic layer was
2 4
dried over anhydrous Na SO , filtered, and concentrated. The con-
1
centrates was purified by flash chromatography on silica gel eluting
6
the preparation of 5a (yield, 76%). H NMR (400 MHz, DMSO-d )
with petroleum ether-EtOAc-methnaol (containing 1% ammonia)
d
1
4
9.27 (d, J ¼ 2.4 Hz, 1H), 9.08 (d, J ¼ 2.4 Hz, 1H), 8.90 (d, J ¼ 7.4 Hz,
ꢀ
1
(
40:60:0.05) to give 7a (129 mg, yield, 62%). mp 268e271 C. H
H), 8.57 (s, 1H), 8.50 (dd, J ¼ 1.6, 4.8 Hz, 2H), 7.43 (dd, J ¼ 1.2,
NMR (400 MHz, MeOD)
d
9.20 (d, J ¼ 2.0 Hz, 1H), 9.06 (d, J ¼ 2.4 Hz,
H), 8.43 (s, 1H), 8.21 (s, 1H), 8.07 (s, 1H), 7.35 (dd, J ¼ 5.2, 8.8 Hz,
H), 7.14 (t, J ¼ 8.6 Hz, 1H), 6.07 (q, J ¼ 7.4 Hz, 1H), 4.01 (s, 3H), 1.83
.4 Hz, 2H), 5.50 (q, J ¼ 7.0 Hz,1H),1.60 (d, J ¼ 7.2 Hz, 3H). ESI-MS m/
þ
1
1
z: 332.5 [MþH] .
13
(d, J ¼ 7.2 Hz, 3H). C NMR (101 MHz, MeOD)
d 159.6, 157.2, 155.9,
4
.1.8. 6-bromo-N-(3-methoxybenzyl)pyrido[2,3-d]pyrimidin-4-
amine (5c)
Compound 5c was synthesized from 4 and (3-methoxyphenyl)
methanamine following the similar procedure described above for
152.9, 139.6, 136.5, 129.5, 129.4, 128.4, 127.1, 126.9, 118.8, 115.2,
þ
þ
1
14.9,109.6, 49.3, 37.8, 16.3. ESI-MS m/z: 417.5 [MþH] . HRMS, ESI ,
þ
m/z: Calcd for C19
H15Cl
2
FN
6
(MþH) , 417.0792; found, 417.0794.
1
6
the preparation of 5a (yield, 58%). H NMR (400 MHz, DMSO-d )
d
9.16 (t, J ¼ 5.6 Hz, 1H), 9.11 (d, J ¼ 2.4 Hz, 1H), 9.07 (d, J ¼ 2.4 Hz,
1
6
3
H), 8.64 (s, 1H), 7.25 (t, J ¼ 8.2 Hz, 1H), 6.96e6.95 (m, 2H),
4.1.12. 6-(1-methyl-1H-pyrazol-4-yl)-N-(1-(pyridin-4-yl)ethyl)
.86e6.85 (m, 1H), 4.77 (d, J ¼ 5.6 Hz, 2H), 3.74 (s, 3H). ESI-MS m/z:
pyrido[2,3-d]pyrimidin-4-ami-ne (7b)
þ
47.1 [MþH] .
Compound 7b was synthesized from 5b and 20 following the
similar procedure described above for the preparation of 7a (yield,
ꢀ
1
4.1.9. 6-bromo-N-(3-nitrobenzyl)pyrido[2,3-d]pyrimidin-4-amine
55%). mp 152e154 C. H NMR (400 MHz, MeOD) d 9.20 (d,
(
5d)
Compound 5d was synthesized from 4 and (3-nitrophenyl)
methanamine following the similar procedure described above for
J ¼ 9.6 Hz, 1H), 8.99 (s, 1H), 8.49 (d, J ¼ 4.8 Hz, 3H), 8.20 (d,
J ¼ 7.6 Hz, 1H), 8.05 (d, J ¼ 7.2 Hz, 1H), 7.52 (d, J ¼ 5.6 Hz, 2H), 5.61
13
(q, J ¼ 5.6 Hz, 1H), 4.00 (s, 3H), 1.74 (d, J ¼ 7.2 Hz, 3H). C NMR
1
the preparation of 5a (yield, 72%). H NMR (400 MHz, DMSO-d
6
)
(101 MHz, MeOD) d 160.3, 157.1, 156.0, 154.4, 153.1, 148.8, 136.5,
d
9.66 (s, 1H), 9.14 (d, J ¼ 8.0 Hz, 2H), 8.72 (s, 1H), 8.28 (s, 1H), 8.15
128.4, 127.1, 126.9, 121.7, 118.7, 109.8, 49.9, 37.8, 20.3. ESI-MS m/z:
þ
þ
þ
(
(
d, J ¼ 7.6 Hz, 1H), 7.88 (d, J ¼ 7.6 Hz, 1H), 7.65 (t, J ¼ 7.8 Hz, 1H), 4.95
332.6 [MþH] . HRMS, ESI , m/z: Calcd for C18
H
17
N
7
(MþH) ,
þ
d, J ¼ 5.2 Hz, 2H). ESI-MS m/z: 362.3 [MþH] .
332.1618; found, 332.1621.

J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
285
ꢀ
4
.1.13. N-(3-methoxybenzyl)-6-(1-methyl-1H-pyrazol-4-yl)pyrido
cooled at 0 C and slowly added NaH (2.00 g, 0.03 mol, 60%
dispersion in mineral oil) over 15 min. After complete addition,
[2,3-d]pyrimidin-4-amine (7c)
ꢀ
Compound 7c was synthesized from 5c and 20 following the
compound 10 was added. The mixture was stirred at 0 C for 1 h
similar procedure described above for the preparation of 7a (yield,
and then stirred at room temperature overnight. The reaction
mixture was poured into 100 mL H O, and the pH was adjusted to
2
ꢀ
1
6
7%). mp 196e198 C. H NMR (400 MHz, DMSO-d
6
)
d
9.28 (s, 1H),
9
.11 (s, 1H), 8.99 (s, 1H), 8.58 (s, 1H), 8.32 (s, 1H), 8.03 (s, 1H), 7.26
neutral with 10% aqueous hydrochloric acid. The resulting precip-
(
(
t, J ¼ 8.0 Hz, 1H), 6.98 (s, 2H), 6.85 (d, J ¼ 7.6 Hz, 1H), 4.82
itate was filtered and washed with 50% ethanol, and dried over-
13
d, J ¼ 5.6 Hz, 2H), 3.92 (s, 3H), 3.74 (s, 3H). C NMR (101 MHz,
161.1, 159.8, 157.6, 153.6, 140.9, 136.8, 129.9, 128.8,
26.9, 126.8, 120.0, 118.7, 113.9, 112.9, 110.0, 55.4, 44.4. ESI-MS m/z:
night under high vacuum to give 11c as white solid (3.30 g, yield,
1
DMSO-d
1
3
3
6
)
d
79%). H NMR (400 MHz, DMSO-d
6
)
d
8.73 (s,1H), 8.36 (d, J ¼ 2.0 Hz,
1H), 8.10 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.86 (d, J ¼ 9.2 Hz, 1H), 7.54 (dd,
þ
þ
þ
47.4 [MþH] . HRMS, ESI , m/z: Calcd for C19
18
H N
6
O (MþH) ,
J ¼ 5.0, 9.2 Hz, 1H), 7.43 (t, J ¼ 8.8 Hz, 1H), 6.90 (q, J ¼ 6.8 Hz, 1H),
þ
47.1615; found, 347.1619.
1.86 (d, J ¼ 6.8 Hz, 3H). ESI-MS m/z: 417.6 [MþH] .
4
.1.14. 6-(1-methyl-1H-pyrazol-4-yl)-N-(3-nitrobenzyl)pyrido[2,3-
4.1.20. 6-bromo-N-(3-chloro-4-fluorophenyl)quinazolin-4-amine
(11d)
d]pyrimidin-4-amine (7d)
Compound 7d was synthesized from 5d and 20 following the
similar procedure described above for the preparation of 7a (yield,
To a solution of compound 10 (2.43 g, 0.01 mol) and 3-chloro-4-
fluoroaniline 1.45 g (0.01 mol) in acetone: H O (4:1) (25 mL), fol-
2
lowed by a few drops of hydrochloric acid. The reaction mixture
stirred at reflux for 3 h, after cooling to room temperature, the pH
was adjusted to neutral using ammonium hydroxide. The resulting
precipitate was filtered and washed with water and dried overnight
ꢀ
1
7
9
1
4%). mp 290e293 C. H NMR (400 MHz, DMSO-d
6
)
d
9.41 (s, 1H),
.28 (d, J ¼ 1.6 Hz, 1H), 9.05 (s, 1H), 8.54 (s, 1H), 8.35 (s, 1H), 8.27 (s,
H), 8.13 (d, J ¼ 8.0 Hz, 1H), 8.06 (s, 1H), 7.88 (d, J ¼ 7.2 Hz, 1H), 7.64
13
(
t, J ¼ 7.8 Hz, 1H), 4.94 (s, 2H), 3.93 (s, 3H). C NMR (101 MHz,
DMSO-d 161.0, 157.8,157.0, 153.6,148.3,142.1, 136.8,134.6,130.3,
28.8, 127.0, 126.7, 122.4, 122.3, 118.7, 110.2, 66.8, 43.8. ESI-MS m/z:
6
)
d
under high vacuum to give 11d as a earthy yellow solid (2.80 g,
1
1
3
3
yield, 80%). H NMR (400 MHz, DMSO-d
6
)
d
11.73 (s, 1H), 9.28 (d,
þ
þ
þ
62.2 [MþH] . HRMS, ESI , m/z: Calcd for C18
H
15
N
7
O
2
(MþH) ,
J ¼ 2.0 Hz, 1H), 8.97 (s, 1H), 8.23 (dd, J ¼ 2.0, 8.8 Hz, 1H), 8.10 (dd,
62.1360; found, 362.1363.
J ¼ 2.4, 6.8 Hz, 1H), 7.94 (d, J ¼ 9.2 Hz, 1H), 7.80 (ddd, J ¼ 2.8, 4.4,
þ
7
.2 Hz, 1H), 7.55 (t, J ¼ 9.0 Hz, 1H). ESI-MS m/z: 354.3 [MþH] .
4.1.15. 6-bromoquinazolin-4-ol (9)
Compound 9 was synthesized from 8 and formamide following
the similar procedure described above for the preparation of 3, and
4.1.21. 6-bromo-N-(quinolin-6-yl)quinazolin-4-amine (11e)
Compound 11e was synthesized from 10 and 6-quinolylamine
the crude product was directly used in the next step.
following the similar procedure described above for preparation
1
of 11d (yield, 74%). H NMR (400 MHz, DMSO-d
6
)
d
12.19 (s, 1H),
4
.1.16. 6-bromo-4-chloroquinazoline (10)
Compound 10 was synthesized from 9 and thionyl chloride and
9.45 (d, J ¼ 1.6 Hz, 1H), 9.19 (dd, J ¼ 1.2, 4.8 Hz, 1H), 9.03 (s, 1H), 8.99
(d, J ¼ 8.0 Hz, 1H), 8.71 (d, J ¼ 2.0 Hz, 1H), 8.47 (dd, J ¼ 2.0, 9.2 Hz,
1H), 8.41 (d, J ¼ 9.2 Hz, 1H), 8.27 (dd, J ¼ 2.0, 8.8 Hz, 1H), 8.01 (d,
J ¼ 8.8 Hz, 1H), 7.96 (dd, J ¼ 5.2, 8.4 Hz, 1H). ESI-MS m/z: 353.7
DMF following the similar procedure described above for the
preparation of 4 (yield, 75%). H NMR (400 MHz, DMSO-d
1
6
) d 8.36
þ
(
s, 1H), 8.22 (d, J ¼ 2.4 Hz, 1H), 8.01 (dd, J ¼ 2.4, 8.8 Hz, 1H), 7.68 (d,
[MþH] .
þ
J ¼ 8.4 Hz, 1H), ESI-MS m/z: 244.9 [MþH] .
4.1.22. 6-bromo-N-(3-ethynylphenyl)quinazolin-4-amine (11f)
4.1.17. 6-bromo-N-(3-methoxybenzyl)quinazolin-4-amine (11a)
Compound 11f was synthesized from 10 and 3-ethynylaniline
To a solution of compound 10 (2.43 g, 0.01 mol) in 25 mL
following the same procedure described above for preparation of
11d (yield, 86%). H NMR (400 MHz, DMSO-d ) d 11.83 (s, 1H), 9.34
6
1
dioxane was added (3-methoxyphenyl)methanamine (2.06 g,
.015 mol) and K CO (2.76 g, 0.02 mol), the reaction mixture was
0
2
3
(d, J ¼ 1.6 Hz, 1H), 8.99 (s, 1H), 8.25 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.97 (d,
J ¼ 8.8 Hz, 1H), 7.94 (t, J ¼ 1.6 Hz, 1H), 7.84e7.81 (m, 1H), 7.51 (t,
J ¼ 8.0 Hz, 1H), 7.44e7.42 (m, 1H), 4.30 (s, 1H). ESI-MS m/z: 326.2
stirred at room temperature overnight. After that, the mixture was
poured into 150 mL, and then the resulting precipitate was filtered,
washed with 50% ethanol, and dried overnight under vacuum to
þ
[MþH] .
1
give 11a as white solid (2.33 g, yield, 68%). H NMR (400 MHz,
DMSO-d
6
)
d
8.90 (t, J ¼ 5.4 Hz, 1H), 8.63 (s, 1H), 8.50 (s, 1H), 7.90 (d,
4.1.23. 6-bromo-4-(quinolin-6-yloxy)quinazoline (11g)
J ¼ 8.8 Hz,1H), 7.65 (d, J ¼ 9.2 Hz,1H), 7.24 (t, J ¼ 8.0 Hz,1H), 6.94 (d,
To a solution of 6-hydroxyquinoline (1.60 g, 0.011 mol) in 20 mL
dioxane was added cesium carbonate (7.20 g, 0.022 mol). After
stirring at room temperature for 1 h, compound 10 was added. The
mixture was stirred at room temperature for a further overnight.
J ¼ 6.4 Hz, 2H), 6.82 (d, J ¼ 7.6 Hz, 1H), 4.75 (d, J ¼ 5.6 Hz, 2H), 3.73
þ
(
s, 3H). ESI-MS m/z: 344.4 [MþH] .
4.1.18. 6-bromo-N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)
2
The reaction mixture was poured into 200 mL H O, and pH was
quinazolin-4-amine (11b)
adjusted to neutral using aqueous hydrochloric acid. The resulting
Compound 11b was synthesized from 10 and 1-(2,6-Dichloro-3-
fluorophenyl)et-han-1-amine following the similar procedure
described above for the preparation of 11a (yield, 71%). H NMR
precipitate was filtered and washed with water and dried overnight
under high vacuum to give 11g as white solid (2.10 g, yield, 60%). H
1
1
NMR (400 MHz, DMSO-d
6
)
d
8.96 (d, J ¼ 2.8 Hz, 1H), 8.79 (s, 1H),
(
1
7
400 MHz, DMSO-d
6
)
d
8.91 (d, J ¼ 2.0 Hz, 1H), 8.76 (d, J ¼ 5.6 Hz,
8.62 (d, J ¼ 2.0 Hz, 1H), 8.41 (d, J ¼ 8.0 Hz, 1H), 8.22 (dd, J ¼ 2.0,
8.8 Hz, 1H), 8.16 (d, J ¼ 9.2 Hz, 1H), 8.00 (d, J ¼ 9.2 Hz, 2H), 7.82 (dd,
J ¼ 2.8, 9.2 Hz, 1H), 7.61 (dd, J ¼ 4.0, 8.4 Hz, 1H). ESI-MS m/z: 354.8
H), 8.33 (s, 1H), 7.90 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.61 (d, J ¼ 9.2 Hz, 1H),
.45e7.42 (m,1H), 7.32 (t, J ¼ 8.8 Hz,1H), 5.85 (q, J ¼ 7.2 Hz,1H),1.70
þ
þ
(
d, J ¼ 7.2 Hz, 3H). ESI-MS m/z: 416.4 [MþH] .
[MþH] .
4
.1.19. 6-bromo-4-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)
quinazoline (11c)
To solution of 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-ol
2.71 g, 0.013 mol) in 15 mL dioxane. The resulting solution was
4.1.24. N-(3-methoxybenzyl)-6-(1-methyl-1H-pyrazol-4-yl)
quinazolin-4-amine (12a)
Compound 12a was synthesized from 11a and 20 following the
similar procedure described above for the preparation of 7a (yield,
a
(

286
J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
ꢀ
1
4
8
7%). mp 167e169 C. H NMR (400 MHz, DMSO-d
6
)
d
8.71 (s, 1H),
4.1.29. 6-(1-methyl-1H-pyrazol-4-yl)-4-(quinolin-6-yloxy)
quinazoline (12f)
Compound 12f was synthesized from 11g and 20 following the
same procedure described above for preparation of 7a (yield, 83%).
.53 (s, 1H), 8.41 (s, 1H), 8.20 (s, 1H), 7.99 (d, J ¼ 8.8 Hz, 2H), 7.68 (d,
J ¼ 8.8 Hz,1H), 7.25 (t, J ¼ 7.6 Hz,1H), 6.97 (s, 2H), 6.83 (d, J ¼ 8.4 Hz,
13
1
(
1
4
C
H), 4.81 (d, J ¼ 3.6 Hz, 2H), 3.91 (s, 3H), 3.73 (s, 3H). C NMR
101 MHz, DMSO-d 159.8, 159.6, 154.8, 148.0, 141.5, 136.7, 130.6,
30.5, 129.9, 128.5, 128.4, 121.9, 119.8, 117.9, 115.6, 113.6, 112.5, 55.4,
ꢀ
1
6
)
d
mp 209e211 C. H NMR (400 MHz, DMSO-d
6
)
d
8.96 (dd, J ¼ 1.6,
4.0 Hz,1H), 8.67 (s,1H), 8.55 (d, J ¼ 2.0 Hz,1H), 8.49 (s,1H), 8.42 (dd,
J ¼ 1.2, 6.8 Hz, 1H), 8.31 (dd, J ¼ 2.0, 8.8 Hz, 1H), 8.16 (t, J ¼ 4.6 Hz,
2H), 8.02 (dd, J ¼ 4.4, 7.2 Hz, 2H), 7.83 (dd, J ¼ 2.4, 8.8 Hz, 1H), 7.61
þ
þ
3.9, 39.2. ESI-MS m/z: 346.2 [MþH] . HRMS, ESI , m/z: Calcd for
þ
H
20 19
N
5
O (MþH) , 346.1662; found, 346.1666.
13
(
dd, J ¼ 4.0, 8.4 Hz, 1H), 3.92 (s, 3H). C NMR (101 MHz, DMSO-d
6
)
d
166.6, 153.3, 150.9, 150.4, 150.2, 146.2, 137.1, 136.1, 133.1, 132.9,
31.0, 129.4, 128.9, 128.6, 126.1, 122.4, 121.1, 119.4, 117.6, 116.6, 39.2.
4
.1.25. 4-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-6-(1-methyl-1H-
pyrazol-4-yl)quinazoline (12b)
1
þ
þ
MS: m/z (ESIþ) 354.5 [MþH] . HRMS, ESI , m/z: Calcd for
Compound 12b was synthesized from 11c and 20 following the
similar procedure described above for the preparation of 7a (yield,
þ
C
21
H
15
N
5
O (MþH) , 354.1349; found, 354.1348.
ꢀ
1
4
8
(
7%). mp 163e165 C. H NMR (400 MHz, DMSO-d
.36 (s, 1H), 8.30 (d, J ¼ 2.0 Hz,1H), 8.18 (dd, J ¼ 2.0, 8.8 Hz,1H), 8.02
s, 1H), 7.88 (d, J ¼ 8.8 Hz, 1H), 7.53 (dd, J ¼ 4.8, 8.8 Hz, 1H), 7.42 (t,
J ¼ 8.8 Hz,1H), 6.94 (q, J ¼ 6.8 Hz,1H), 3.93 (s, 3H),1.88 (d, J ¼ 7.2 Hz,
6
) d 8.62 (s, 1H),
4.1.30. N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-6-(1-(piperidin-4-
yl)-1H-pyrazol-4-yl)quinazolin-4-amine (13a)
Compound 13a was synthesized from 11b and 21 following the
similar procedure described above for the preparation of 6 (yield,
13
3
1
1
C
H). C NMR (101 MHz, DMSO-d
6
) d 164.9,153.4,149.6,137.6,136.9,
ꢀ
1
7
9%). mp 185e188 C. H NMR (400 MHz, MeOD)
J ¼ 1.6 Hz, 1H), 8.29 (s, 2H), 8.15 (s, 1H), 8.03 (dd, J ¼ 2.0, 8.4 Hz, 1H),
.70 (d, J ¼ 8.8 Hz,1H), 7.35 (dd, J ¼ 4.8, 8.8 Hz,1H), 7.13 (t, J ¼ 8.6 Hz,
H), 6.10 (q, J ¼ 7.2 Hz, 1H), 4.64 (ddd, J ¼ 4.4, 12.6, 17.0 Hz, 1H), 3.61
dt, J ¼ 3.2, 16.4 Hz, 2H), 3.27 (td, J ¼ 3.6, 12.8 Hz, 2H), 2.45e2.30 (m,
d 8.65 (d,
32.6, 129.1, 128.5, 121.2, 117.7, 117.5, 117.3, 116.6, 71.5, 39.2, 25.4,
þ
þ
8.5. ESI-MS m/z: 417.3 [MþH] . HRMS, ESI , m/z: Calcd for
7
1
(
þ
H15Cl
20 2
FN
4
O (MþH) , 417.0680; found, 417.0685.
13
4
.1.26. N-(3-chloro-4-fluorophenyl)-6-(1-methyl-1H-pyrazol-4-yl)
quinazolin-4-amine (12c)
4H), 1.84 (d, J ¼ 7.2 Hz, 3H). C NMR (101 MHz, MeOD)
d 158.6,
158.4, 155.9, 154.0, 146.8, 140.0, 136.9, 130.8, 130.5, 129.4, 129.3,
126.7, 126.0, 122.1, 117.6, 115.0, 114.8, 55.7, 49.1, 42.8, 28.9, 16.4. ESI-
Compound 12c was synthesized from 11d and 20 following the
similar procedure described above for the preparation of 7a (yield,
þ
þ
MS m/z: 485.5 [MþH] . HRMS, ESI , m/z: Calcd for C24
2 6
H23Cl FN
ꢀ
1
þ
6
8
(
9%). mp 209e211 C. H NMR (400 MHz, DMSO-d
6
)
d
9.78 (s, 1H),
(MþH) , 485.1418; found, 485.1420.
.61 (d, J ¼ 21.6 Hz, 2H), 8.22 (d, J ¼ 23.6 Hz, 2H), 8.05 (s, 2H), 7.82
13
d, J ¼ 27.6 Hz, 2H), 7.46 (s, 1H), 3.93 (s, 3H). C NMR (101 MHz,
DMSO-d 157.6, 155.0, 153.9, 152.6, 148.6, 136.9, 131.2, 128.8,
24.1, 122.9, 121.8, 119.4, 119.2, 117.7, 117.1, 116.9, 115.8, 39.9. ESI-MS
4.1.31. 4-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-6-(1-(piperidin-
4-yl)-1H-pyrazol-4-yl)quinazoline (13b)
Compound 13b was synthesized from 11c and 21 following the
similar procedure described above for the preparation of 6 (yield,
6
) d
1
þ
þ
þ
m/z: 354.6 [MþH] . HRMS, ESI , m/z: Calcd for C21
H
16
N
6
(MþH) ,
ꢀ
1
3
54.0916; found, 354.0919.
50%). mp 157e158 C. H NMR (400 MHz, MeOD)
s, 1H), 8.26 (s, 1H), 8.14 (dd, J ¼ 1.2, 8.4 Hz 1H), 7.99 (s, 1H), 7.85 (d,
J ¼ 8.8 Hz, 1H), 7.41 (dd, J ¼ 4.8, 8.8 Hz, 1H), 7.21 (t, J ¼ 8.6 Hz, 1H),
.02 (q, J ¼ 6.8 Hz, 1H), 4.49e4.42 (m, 1H), 3.36 (d, J ¼ 7.2 Hz, 1H),
.29 (s, 1H), 2.96e2.90 (m, 2H), 2.25e2.01 (m, 5H), 1.94 (d,
d 8.56 (s, 1H), 8.39
(
4
4
.1.27. 6-(1-methyl-1H-pyrazol-4-yl)-N-(quinolin-6-yl)quinazolin-
7
3
-amine (12d)
Compound 12d was synthesized from 11e and 20 following the
similar procedure described above for the preparation of 7a (yield,
13
J ¼ 6.8 Hz, 3H). C NMR (101 MHz, MeOD)
d 165.3, 158.5, 156.0,
1
53.1, 148.9, 138.5, 137.5, 136.3, 132.1, 129.8, 128.7, 127.0, 125.7, 121.3,
ꢀ
1
7
8
(
9%). mp 263e265 C. H NMR (400 MHz, DMSO-d
6
) d 10.02 (s, 1H),
1
18.0, 116.5, 104.9, 72.0, 58.4, 44.1, 31.7, 17.0. ESI-MS m/z: 486.6
.84 (dd, J ¼ 1.6, 4.0 Hz, 1H), 8.78 (d, J ¼ 1.6 Hz, 1H), 8.65 (s, 1H), 8.57
þ
þ
þ
[MþH] . HRMS, ESI , m/z: Calcd for C24
H22Cl
2
FN
5
O (MþH) ,
d, J ¼ 2.0 Hz, 1H), 8.37 (d, J ¼ 7.6 Hz, 1H), 8.31 (s, 1H), 8.24 (dd,
4
86.1258; found, 486.1254.
J ¼ 2.0, 8.8 Hz, 1H), 8.17e8.07 (m, 3H), 7.82 (d, J ¼ 8.8 Hz, 1H), 7.53
13
(
d
1
dd, J ¼ 4.0, 8.0 Hz, 1H), 3.95 (s, 3H). C NMR (101 MHz, DMSO-d
157.8, 154.1, 149.7, 148.7, 145.4, 137.7, 136.9, 135.9, 131.4, 131.2,
29.5, 128.8, 128.6, 126.6, 122.2, 121.8, 118.6, 117.9, 116.0, 99.9, 39.2.
6
)
4.1.32. N-(3-chloro-4-fluorophenyl)-6-(1-(piperidin-4-yl)-1H-
pyrazol-4-yl)quinazolin-4-amin-e (13c)
Compound 13c was synthesized from 11d and 21 following the
similar procedure described above for the preparation of 6 (yield,
þ
þ
ESI-MS m/z: 353.6 [MþH] . HRMS, ESI , m/z: Calcd for C21
16 6
H N
þ
(
MþH) , 353.1509; found, 353.1512.
ꢀ
1
6
8
3%). mp 293e296 C. H NMR (400 MHz, DMSO-d ) d 9.82 (s, 1H),
6
.67 (d, J ¼ 1.4 Hz, 1H), 8.59 (s, 1H), 8.36 (s, 1H), 8.20 (dd, J ¼ 2.4,
4
.1.28. N-(3-ethynylphenyl)-6-(1-methyl-1H-pyrazol-4-yl)
quinazolin-4-amine (12e)
6.8 Hz, 1H), 8.12 (dd, J ¼ 2.0, 8.8 Hz, 1H), 8.08 (s, 1H), 7.87 (ddd,
J ¼ 2.4, 5.6, 7.6 Hz,1H), 7.79 (d, J ¼ 8.8 Hz,1H), 7.47 (t, J ¼ 9.2 Hz,1H),
4.29e4.22 (m, 1H), 3.10e2.99 (m, 3H), 2.66e2.55 (m, 2H), 2.03 (d,
Compound 12e was synthesized from 11f and 20 following the
similar procedure described above for the preparation of 7a. The
crude product was purified by flash chromatography on C18 reverse
phase silica gel eluting with 35% methnaol/water (50 mmol
ammonium acetate adjusted to pH ¼ 4 with formic acid) to give
13
J ¼ 10.0 Hz, 2H), 1.89e1.82 (m, 2H). C NMR (101 MHz, DMSO-d
157.6, 155.0, 153.9, 152.6, 148.6, 136.5, 131.6, 128.7, 125.8, 124.1,
123.0,121.2,119.4,117.7,117.1,116.9,115.8, 59.8, 45.4, 33.9, 33.7, 22.9.
6
)
d
þ
þ
ESI-MS m/z: 423.5 [MþH] . HRMS, ESI , m/z: Calcd for
ꢀ
1
þ
compound 12e (yield, 37%). mp 118e121 C. H NMR (400 MHz,
C
24
H22Cl
2
FN
5
O (MþH) , 423.1495; found, 423.1499.
DMSO-d
6
)
d
9.79 (s, 1H), 8.70 (s, 1H), 8.60 (s, 1H), 8.28 (s, 1H),
8
.10e8.06 (m, 3H), 7.96 (d, J ¼ 8.0 Hz, 1H), 7.79 (d, J ¼ 8.4 Hz, 1H),
4.1.33. 6-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-N-(quinolin-6-yl)
quinazolin-4-amine (13d)
7
3
.45 (t, J ¼ 7.8 Hz, 1H), 7.27 (d, J ¼ 7.6 Hz, 1H), 4.23 (s, 1H), 3.93 (s,
13
H). C NMR (101 MHz, DMSO-d
6
)
d
157.7, 153.9, 148.3, 139.8, 136.9,
Compound 13d was synthesized from 11e and 21 following the
similar procedure described above for the preparation of 6 (yield,
131.4,131.3,129.4,128.8,128.6,127.3,125.5,123.3,122.3,121.8,117.8,
þ
þ
ꢀ
1
1
15.9, 83.8, 81.1, 39.2. ESI-MS m/z: 326.6 [MþH] . HRMS, ESI , m/z:
63%). mp 126e127 C. H NMR (400 MHz, DMSO-d
6
) d 10.02 (s, 1H),
þ
Calcd for C21
H
16
N
6
(MþH) , 326.1400; found, 326.1406.
8.84 (dd, J ¼ 1.6, 4.4 Hz, 1H), 8.78 (d, J ¼ 1.2 Hz,1H), 8.64 (s, 1H), 8.57

J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
287
(
8
7
d, J ¼ 2.0 Hz, 1H), 8.39e8.35 (m, 2H), 8.25 (dd, J ¼ 2.4, 9.2 Hz, 1H),
.16e8.12 (m, 2H), 8.07 (d, J ¼ 9.2 Hz, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H),
.52 (dd, J ¼ 4.0, 8.0 Hz, 1H), 4.29e4.24 (m, 1H), 3.09 (d, J ¼ 12.0 Hz,
H), 2.63 (t, J ¼ 11.8 Hz, 2H), 2.06e2.03 (m, 2H), 1.90e1.81 (m, 2H).
100 mL H
2
O and extracted with EtOAc (30 mL ꢂ 3). The combined
SO , filtered, and
organic layer was dried over anhydrous Na
concentrated. The crude product was purified by flash chroma-
tography on silica gel eluting with petroleum ether:DCM:EtOAc
(2:1:2) to give 17b (1.2 g, yield, 55%). H NMR (400 MHz, CDCl
2
4
2
1
3
1
C NMR (101 MHz, DMSO-d
6
)
d
157.8, 154.0, 149.6, 148.7, 145.4,
3
)
1
37.7, 136.6, 135.9, 131.6, 131.3, 129.4, 128.8, 128.6, 126.6, 125.8,
d
8.23 (d, J ¼ 2.0 Hz, 1H), 7.70 (dd, J ¼ 2.4, 9.2 Hz, 1H), 7.35 (d,
122.2, 121.3, 118.6, 117.9, 116.0, 59.8, 45.4, 33.9. ESI-MS m/z: 422.7
J ¼ 8.8 Hz, 1H), 7.27 (dd, J ¼ 5.2, 8.8 Hz, 1H), 7.05 (t, J ¼ 8.4 Hz, 1H),
6.86 (q, J ¼ 6.8 Hz, 1H), 4.99 (s, 2H), 1.91 (d, J ¼ 6.9 Hz, 3H). ESI-MS
þ
þ
þ
[
MþH] . HRMS, ESI , m/z: Calcd for C25
23
H N
7
(MþH) , 422.2088;
þ
found, 422.2093.
m/z: 432.4 [MþH] .
4
.1.34. N-(6-bromo-4-hydroxyquinazolin-2-yl)guanidine (15)
To the mixture of 2-amino-5-bromobenzoic acid (14) (15.0 g,
4.1.38. 2-amino-N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-6-(1-
methyl-1H-pyrazol-4-yl)quinazoline-4-amine (18)
0
.07 mol), water 150 mL and concentrated sulfuric acid (8 mL) was
Compound 18 was synthesized from 17a and 20 following the
similar procedure described above for the preparation of 7a (yield,
51%). mp 269e272 C. H NMR (400 MHz, MeOD) d 8.43 (d,
ꢀ
heated at 100 C before being allowed to cool. Dicyandiamide was
added, and the reaction mixture was stirred for 2 h at 100 C. The
ꢀ
ꢀ
1
reaction was cooled and filtered. The white solid was basified with
J ¼ 2.0 Hz, 1H), 8.01 (s, 1H), 7.96 (s, 1H), 7.81 (dd, J ¼ 1.6, 8.4 Hz, 1H),
7.38e7.30 (m, 2H), 7.14 (t, J ¼ 8.6 Hz, 1H), 6.04 (q, J ¼ 7.3 Hz, 1H),
ꢀ
100 mL (4 mol/L) NaOH at 180 C for a further 15 min, cooled to
13
room temperature, filtered and washed with water until neutral
pH, and dried overnight under vacuum to give 15 as white solid
3.97 (s, 3H), 1.80 (d, J ¼ 7.2 Hz, 3H). C NMR (101 MHz, MeOD)
d
159.6, 158.6, 155.9, 146.8, 139.9, 136.0, 130.7, 129.3, 127.6, 127.0,
1
1.8 g, which was directly used in the next step.
122.5, 122.4, 118.3, 115.1, 114.9, 110.7, 48.9, 37.6, 16.2. ESI-MS m/z:
þ
þ
þ
431.5 [MþH] . HRMS, ESI , m/z: Calcd for C20
H17Cl
2
FN
6
(MþH) ,
4.1.35. 2-amino-6-bromoquinazolin-4-ol (16)
431.0949; found, 431.0950.
To a solution of N-(6-Bromo-4-hydroxyquinazolin-2-yl)guani-
dine (15) (8.00 g, 0.028 mol) and 4-Pyridinethiol (6.20 g, 0.056 mol)
4.1.39. 2-amino-N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-6-(1-
(piperidin-4-yl)-1H-pyrazol-4-yl)quinazoline-4-amine (19a)
Compound 19a was synthesized from 17a and 21 following the
similar procedure described above for the preparation of 6 (yield,
in ethylene glycol, KOH (7.80 g, 0.14 mol) was added. The resulting
ꢀ
reaction mixture was stirred at 170 C for about 4 h, cooled to room
2
temperature, poured into 500 mL H O, and the pH was adjusted to
neutral using hydrochloric acid. The resulting precipitate was
ꢀ
1
74%). mp 113e115 C. H NMR (400 MHz, MeOD) d 8.69 (s, 1H), 8.24
filtered, washed with water and 50% ethanol, and dried overnight
(s, 1H), 8.07e8.03 (m, 2H), 7.47e7.39 (m, 2H), 7.18 (dd, J ¼ 8.4,
17.2 Hz, 1H), 6.09 (q, J ¼ 7.2 Hz, 1H), 4.65e4.63 (m, 1H), 3.64e3.53
(m, 3H), 3.37 (s, 1H), 3.28e3.17 (m, 2H), 2.66 (d, J ¼ 9.6 Hz, 1H),
under high vacuum to give 16 as off-white solid (4.60 g, yield, 68%).
1
H NMR (400 MHz, DMSO-d
6
)
d
11.11 (s, 1H), 7.97 (d, J ¼ 2.4 Hz, 1H),
13
7
.69 (dd, J ¼ 2.8, 8.8 Hz, 1H), 7.57 (s, 2H), 7.30 (d, J ¼ 8.8 Hz, 1H). ESI-
2.40e2.25 (m, 5H), 1.84 (d, J ¼ 7.2 Hz, 3H). C NMR (101 MHz,
þ
MS m/z: 242.0 [MþH] .
MeOD)
d 138.8, 137.2, 136.6, 132.4, 129.9, 129.7, 127.9, 125.8, 121.2,
1
19.3, 117.2, 115.6, 115.4, 114.3, 110.0, 55.7, 49.8, 42.7, 42.6, 28.8, 28.6,
þ
þ
4.1.36. 2-amino-6-bromo-N-(1-(2,6-dichloro-3-fluorophenyl)ethyl)
16.0. ESI-MS m/z: 500.5 [MþH] . HRMS, ESI , m/z: Calcd for
þ
quinazoline-4-amine (17a)
C
24
H24Cl
2
FN
7
(MþH) , 500.1527; found, 500.1528.
To a solution of compound 16 (1.20 g, 0.005 mol) and benzo-
triazol-1-yloxytris-
fluorophosphat (BOP) (2.90 g, 0.0065 mol) in 20 mL acetonitrile,
,8-Diazabicyclo-[5.4.0]undec-7-ene (DBU) (1.1 mL, 0.0075 mol)
was added and the solution became clear. When precipitated solid
was formed, 1-(2,6-Dichloro-3-fluoro-phenyl)-ethan-1-amine
(dimethylamino)-phosphonium
hexa-
4.1.40. 4-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-6-(1-(piperidin-
4-yl)-1H-pyrazol-4-yl)quinazolin-2-amine (19b)
1
Compound 19b was synthesized from 17b and 21 following the
similar procedure described above for the preparation of 6 (yield,
ꢀ
1
65%). mp 267e269 C. H NMR (400 MHz, DMSO-d ) d 8.23 (s, 1H),
6
(
1.56 g, 0.0075 mol) was added to the reaction mixture. The reac-
tion mixture was stirred at room temperature overnight, then
poured into 100 mL H
O and extracted with EtOAc (30 mL ꢂ 3). The
combined organic layer was dried over anhydrous Na SO , filtered,
8.06 (d, J ¼ 2.0 Hz, 1H), 7.89e7.87 (m, 2H), 7.55 (dd, J ¼ 5.2, 8.8 Hz,
1H), 7.44 (t, J ¼ 8.8 Hz,1H), 7.34 (d, J ¼ 8.8 Hz,1H), 6.94 (q, J ¼ 6.8 Hz,
1H), 6.39 (s, 2H), 4.34e4.28 (m, 1H), 3.16 (d, J ¼ 14.4 Hz, 3H),
2
13
2
4
2.76e2.70 (m, 2H), 2.06e1.91 (m, 4H), 1.85 (d, J ¼ 7.2 Hz, 3H).
C
and concentrated. The crude product was purified by flash chro-
NMR (101 MHz, MeOD) d166.2, 159.5, 158.6, 151.2, 137.5, 136.3,
matography on silica gel eluting with Petroleum ether:DCM:EtOAc
132.0, 129.9, 128.9, 126.8, 125.3, 123.7, 122.2, 119.0, 116.2, 116.0,
1
þ
(
2:1:2) to give 17a (0.97 g, yield, 45%). H NMR (400 MHz, DMSO-d6)
111.5, 71.4, 56.1, 43.0, 29.4, 16.9. ESI-MS m/z: 501.4 [MþH] . HRMS,
þ
þ
d
8.60 (d, J ¼ 2.4 Hz,1H), 8.46 (d, J ¼ 5.6 Hz,1H), 8.17 (s,1H), 7.62 (dd,
ESI , m/z: Calcd for C24
H
23Cl
2
FN
6
O (MþH) , 501.1367; found,
J ¼ 2.4, 9.2 Hz, 1H), 7.42 (dd, J ¼ 4.8, 8.8 Hz, 1H), 7.32 (t, J ¼ 8.8 Hz,
501.1369.
1
H), 7.15 (d, J ¼ 8.8 Hz, 1H), 5.94 (s, 1H), 5.82 (q, J ¼ 6.4 Hz, 1H), 1.67
þ
(
d, J ¼ 7.4 Hz, 3H). ESI-MS m/z: 431.4 [MþH] .
4.2. Kinase inhibitory activity
4
.1.37. 6-bromo-4-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)
The EGFR inhibitory activity was evaluated by using the Z'-LYTE
technology platform (Life Technologies), and gefitinib was
employed as the positive control. The experiments were performed
according to the instructions of the manufacturer, and single point
quinazolin-2-amine (17b)
To a solution of compound 16 (1.20 g, 0.005 mol) and benzo-
triazol-1-yloxytris(di-methylamino)-phosphonium
fluorophosphat (BOP) (2.90 g, 0.0065 mol) in 20 mL acetonitrile,
,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (1.1 mL, 0.0075 mol) was
hexa-
concentration testing (1
mM) with two independent data points
1
(n ¼ 2). According to the antiproliferative activities and percent
inhibition values of compounds, we chosen compounds 12ce12e
and 13ce13d to evaluate kinase activity at ten concentration gra-
dients from 1000 to 0.0508 nM (3-fold dilution). The IC50 values
were calculated from the inhibition curves from two separate
experiments.
added and the solution became clear. When precipitated solid was
formed, the solution of 1-(2,6-Dichloro-3-fluorophenyl)-ethan-1-ol
(
1.56 g, 0.0075 mol) and NaH (0.40 g, 60% dispersion in mineral oil)
in 10 mL acetonitrile was added, and then stirred at room tem-
perature overnight. The reaction mixture was poured into

288
J. Hou et al. / European Journal of Medicinal Chemistry 118 (2016) 276e289
4.3. Cancer cell proliferation inhibition assay
method [36]. The cutoff distance for the long-range vdW energy
term was set at 10.0 Å. Periodic boundary conditions were applied
to avoid edge effects in all calculations. The SHAKE algorithm [37]
was employed on all atoms covalently bond to hydrogen atoms,
allowing for an integration time step of 2 fs. Coordinate trajectories
were recorded every 10 ps throughout all equilibration and pro-
The antiproliferative activities of compounds were evaluated
against MCF-7, A549, BT-474, SK-BR-3 and MDA-MB-231 cell lines
by the standard MTT assay in vitro, with gefitinib as the positive
control. All cell lines were purchased from Cell Bank of China Sci-
ence Academy (Shanghai, China). All chemicals and solvents were
purchased from SigmaeAldrich or Gibco.
duction runs. The binding free energies (DGbind) were calculated by
using MM-GBSA [38] procedure in AMBER12. Average 1000 snap-
shots were extracted from the last 10 ns MD trajectory for the
calculations, and only 10 snapshots evenly extracted from the last
10 ns MD trajectory were used to calculate the entropic
contribution.
The BT-474 cell line was maintained in DEME medium supple-
ment with 10% fetal bovine serum (FBS) and 1% Penicillin-
Streotomycin, others were cultured in RPMI 1640 medium also
supplement with 10% FBS and 1% Penicillin-Streotomycin.
3
Approximate 5 ꢂ 10 cells, suspended in medium, were plated
ꢀ
into each well of a 96-well plate and grown at 37 C in a humidified
Acknowledgments
2
atmosphere with 5% CO for 24 h. The following day various con-
centrations of tested compounds were added to the culture me-
dium and incubated for 72 h. Fresh MTT was added to each well at
the terminal concentration of 5 mg/mL in PBS, and incubated with
This work was supported by grants from National Science
Foundation of China (No. 815732630 and 81202413) and Guang-
dong Natural Science Foundation (No. 2015A030313285). The cal-
culations of the ligands were performed in the China ScGrid of
Supercomputing Center of Chinese Academy of Science.
ꢀ
cells at 37 C for 4 h. The formazan crystals in each well were
dissolved in 150
mL DMSO, and the absorbency at 570 nm was
measured with an enzyme-linked immunosorbent assay plate
reader. All of the compounds were tested three times in each of the
cell lines.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.026.
4.4. Molecular modeling
Molecular docking was performed with Surflex-Dock program
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