Design, synthesis, and evaluation of substituted 6-amide-4- anilinoquinazoline derivatives as c-Src inhibitors

Article abstract of DOI:10.1039/c3ra45204b

The 4-anilinoquinazoline scaffold has been historically used for designing EGFR/VEGFR/HER2 inhibitors while it has not been reported widely for developing c-Src inhibitors. Thus, a series of novel 4-anilinoquinazoline derivatives grafting different amide moieties at the 6-position were designed and synthesized as potential inhibitors for c-Src. In this manuscript, all of the designed compounds were screened via molecular docking using Discovery Studio 3.5 software. As expected, the results of the docking study revealed that most of these targeted compounds possessed lower binding energy than the positive control Saracatinib. Subsequently, all of the screened compounds were synthesized and evaluated for their c-Src in vitro inhibitory activities and in vitro antiproliferation assays against four human cancer cells (A549, MCF-7, HepG-2, HeLa). Among these compounds, 24 exhibited the most potent inhibitory activity against c-Src kinase as well as at the cellular level, of which the IC₅₀ value reached up to 2.9 nM, comparable to the positive compound Saracatinib. Kinase selectivity profile also demonstrated that compound 24 showed good selectivity over several close kinase targets. These results, along with relative 3D-QSAR study, could provide an important basis for further development of compound 24 as a potent tyrosine kinase inhibitor.

Full text of DOI:10.1039/c3ra45204b

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Design, synthesis, and evaluation of substituted
6-amide-4-anilinoquinazoline derivatives as c-Src
inhibitors
Cite this: RSC Adv., 2013, 3, 26230
a
a
a
a
a
b
*
Fei Fang,† Dong-Dong Li,† Jing-Ran Li, Jian Sun, Qian-Ru Du, Hai-Bin Gong
a
*
and Hai-Liang Zhu
The 4-anilinoquinazoline scaffold has been historically used for designing EGFR/VEGFR/HER2 inhibitors
while it has not been reported widely for developing c-Src inhibitors. Thus, a series of novel 4-
anilinoquinazoline derivatives grafting different amide moieties at the 6-position were designed and
synthesized as potential inhibitors for c-Src. In this manuscript, all of the designed compounds were
screened via molecular docking using Discovery Studio 3.5 software. As expected, the results of the
docking study revealed that most of these targeted compounds possessed lower binding energy than
the positive control Saracatinib. Subsequently, all of the screened compounds were synthesized and
evaluated for their c-Src in vitro inhibitory activities and in vitro antiproliferation assays against four
human cancer cells (A549, MCF-7, HepG-2, HeLa). Among these compounds, 24 exhibited the most
potent inhibitory activity against c-Src kinase as well as at the cellular level, of which the IC50 value
reached up to 2.9 nM, comparable to the positive compound Saracatinib. Kinase selectivity profile also
demonstrated that compound 24 showed good selectivity over several close kinase targets. These
results, along with relative 3D-QSAR study, could provide an important basis for further development of
compound 24 as a potent tyrosine kinase inhibitor.
Received 18th September 2013
Accepted 23rd October 2013
DOI: 10.1039/c3ra45204b
www.rsc.org/advances
increased motility/invasiveness of tumor cells and to tumor
progression.13,14 Moreover, as a result of extensive preclinical
1. Introduction
Currently there are about 500 protein kinases which constitute
almost 2% of the human genome,1,2 most of which are closely
involved in several important cellular responses such as
proliferation, migration, survival, cell cycle progression and
specialized cell signals.3–5 c-Src tyrosine kinase is the prototyp-
ical member of SFKs (Src family kinases) of non-receptor
protein tyrosine kinases,6,7 which transfer the c-phosphate
moiety of ATP to the phosphorylation of phenolic group of
tyrosine residue on substrate proteins.8,9 A number of reports
revealed that c-Src could play a critical role in a series of the
function of organism, such as cell transformation, mitogenesis,
differentiation, and oncogenesis.10–12 In contrast to its highly
controlled expression in normal cells, mounting evidence dis-
closed that overexpression and/or coexpression of c-Src has
been implicated in numerous of tumor types (colon, breast,
ovarian, head and neck)6,8,10 and involved in poor prognosis of
the patients. Research around increased activity of c-Src has
revealed that deregulation of the enzyme can be linked to
studies, metastatic bone disease with aberrant c-Src kinase
activity occurs in many solid tumor cancers.15–17 In recent
publications, one new hypothesis18,19 could be utilized to
explain the association between c-Src and cancers that the
irregulation of c-Src tyrosine kinase predominantly leads to
alterations of cell adhesion and cytoskeletal, ultimately result-
ing in a change to a motile, invasive phenotype. Therefore there
is still much of interest in developing the c-Src tyrosine kinase
and evaluating small molecule c-Src inhibitors to prohibit the
growth of a variety of cancers.
Fig. 1 shows that several known c-Src kinase inhibitors20 have
been identied to date. It is noteworthy that these compounds
generally employ three main scaffolds:21 4-anilinoquinazo-
lines,22 dihydropyrimido-quinolinones,23 and pyrazolo-pyrimi-
dines.24 The most potent compound, exampled by Bosutinib
(SKI-606),6,20 as a novel, dual Src/Abl inhibitor with IC50 values of
1.2 nM and 1 nM, respectively, has been approved to cure
metastatic breast cancer and chronic myelogenous leukemia by
FDA last year, demonstrating that the 4-anilinoquinoline-3-
carbonitrile template possesses high selectivity as Src inhibitor.
However, there are many developing compounds showing poor
in vivo activity or only exhibit high potency in some limited
metastasis models, partially due to itself poor solubility or
pharmacokinetics properties.21 In addition, there are also some
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, P. R. China. E-mail: zhuhl@nju.edu.cn; Fax: +86-25-8359 2672; Tel: +86-
25-8359 2572
bXuzhou Central Hospital, Xuzhou 221009, P. R. China. E-mail: ghbxzh@126.com
† These authors contributed equally to this work.
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Fig. 1 Some known c-Src kinase inhibitors.
side effects (skin rash, gastro-intestinal, pleural effusions,
cytopenia), toxicities and drug-resistance reported under the
clinical trial of candidates.21,25 Therefore, the pursuit of novel
c-Src inhibitors with a better anti-tumor effects and more safety
prole is still the main issue today.
As is known to all that the 4-anilinoquinazoline drugs such
as Getinib,26,27 Erlotinib,28 and Saracatinib20 have possessed a
good kinase selectivity prole, mainly due to the iterative
modication of the 4-anilinoquinazoline skeleton. From a
number of experimental data and molecular modeling studies
focusing on the c-Src kinase inhibition,29,30 it is disclosed that
the nitrogen at position 1 and 3 of the anilinoquinazoline ring is
signicant for c-Src inhibitory potency, and the binding potency
could be enhanced by the replacement of electron-donating
substituents at 6- and 7- positions. Taken together, a series of
novel small molecules elaborated around 4-anilinoquinazoline
scaffold were designed. In our previous study,31 the modica-
tion of 6-position of the quinazoline ring could enhance the
binding potency as protein kinase inhibition. Herein, these
4-anilinoquinazoline derivatives in this manuscript would still
focus on the replacement at the 6-position of the quinazoline
ring with various heterocyclic ring moiety. Subsequently, in
order to verify whether these compounds we designed could
bind tightly to our target protein c-Src active domain, we have
performed molecular docking32–35 to study the interaction
between these novel 4-anilinoquinazoline analogs and the
protein–ligand complex (PDB code: 3F3V), retrieved from the
RCSB Protein Data Bank, by the means of the CDOCKER
protocol in the Accelrys Discovery Studio 3.5 suite (Accelrys
Fig. 2 The CDOCKER_INTERACTION_ENERGY (kcal molꢀ1) obtained from the
docking study of the targeted compounds by the CDOCKER protocol (Discovery
Studio 3.5, Accelrys, Co. Ltd).
derivatives, which maybe rationalize the experimental obser-
vations and guide us to screen good potency c-Src inhibitors in
the further study.
2. Results and discussion
2.1. Chemistry
Soware Inc.). Compared with the positive drug Saracatinib, Nineteen 4-anilinoquinazoline derivatives were screened by
most of the designed molecules (except compound 7, 8, 10, 13, molecular docking and synthesized in aim to evaluate their bio-
15, 18, 20) possessed lower interaction energy, demonstrating logical activity. Herein, the rapid process to synthesize the 4-ani-
that they were likely to exhibit more potency as c-Src inhibitors linoquinazoline intermediates (compound 5 and 6) with amino
(Fig. 2).
group at the 6-position was depicted as Scheme 1. As previously
In this present study, we would provide the synthetic reported,31 6-amino-4-anilinoquinazoline (compound 5 and 6)
methods of this series of novel small molecules, and discuss could be easily obtained in two steps from the starting material
carefully the structure–activity relationship (SAR) of compounds 2-amino-5-nitrobenzonitrile. Subsequently seen in Scheme 1,
as antitumor agents. Additionally, we also built the relative benzoic acid and phenylacetic acid with different substituents (a)
3D-QSAR model based on the SAR of these 4-anilinoquinazoline were reuxed with thionyl chloride to give the corresponding acyl
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Scheme 1 Synthesis of 6-amide-4-anilinoquinazoline derivatives. Reagents and conditions: (i) DMF–DMA/reflux; (ii) ArNH2/AcOH/reflux; (iii) Fe/AcOH/EtOH/H2O/
reflux; (iv) thionyl chloride/reflux; (v) EtOAc/K2CO3/0 ꢁC.
chloride (b). Aer chlorination, 4-anilinoquinazoline scaffold performed better than the others (18, 19 and 20). This indicated
(compound 5 and 6) was dropwise added into a solution of that the disubstituted inhibitors were likely to be superior for
acyl chloride (b) and potassium carbonate dissolved in ethyl inhibitory potency of c-Src kinase to monosubstitutions. Taken
acetate, leading to nally generate the corresponding amide together, based on the results above, compound 24, along with
analogues 7–25.
14 and 25, could be selected as potential c-Src inhibitors and
The above of all compounds have been puried by different antitumor agents for further optimization.
favorable experimental methods, such as extraction, chroma-
Moreover, an acute oral toxicity test was conducted with mice
tography and recrystallization. Additionally, all the synthetic to determine the toxicity from a single dose via the oral route.
compounds have been presented both satisfactory analytic and Based on the results of study (not listed), the single dose acute
spectroscopic data, which were in full accordance with their oral LD50 (half maximal concentration of lethal does) values of
depicted structures.
the most potent compound 24 is greater than 5000 mg kgꢀ1 of
body weight.
2.2.2. Antiproliferation assay. The target compounds were
also evaluated in in vitro antiproliferation assays against four
human cancer cells: HepG-2, MCF-7, A549 and HeLa. The HepG-
2 and MCF-7 were known to be relative with c-Src family.
Meanwhile, to control for non-specic cytotoxicity, compounds
were evaluated against A549 cells and HeLa cells. The obtained
results were listed in Table 2. As expected, all targeted
compounds showed moderate potent inhibitory activities
against HepG-2 and MCF-7. Surprisingly, the A549 cells and
HeLa cells were also good targets that belong to these quina-
zoline derivatives. In accordance with the c-Src kinase assay,
three compounds (24, 25, 14) with the level of IC50 values less
than 1 mM, performed better than the other compounds in the
cellular level, comparable with the positive compound Sar-
2.2. Bioassay
2.2.1. c-Src enzyme assay. All synthesized compounds have
been evaluated for their c-Src inhibitory activity and the
obtained results (Table 1) presented their activity data depicted
as IC50 values (the half maximal inhibitory concentration of
c-Src mediated autophosphorylation). As expected, most of
these compounds showed moderate inhibitory activities for the
target protein kinase. In particular, compound 24, which IC50
value can reach up to 2.9 nM, was identied as the most potent
c-Src inhibitor. For better understanding and comparing with
each other, these results were plotted as a line–scatter graph
presented in Fig. 3. In the light of steric and electronic factors
employed by the targeted compounds, the general structure–
activity relationships (SAR) would be discussed. Initially,
compounds (11, 12, 13) with the Br substituent located at the
para position of the 40-aniline ring showed roughly similar
inhibitory activities with those with the Cl atom (21, 22, 23).
Then the compounds (14, 24) containing pyridine ring at the
6-position of the quinazoline ring exhibited obviously more
potent inhibitory activities than the rest compounds with
acatinib. In addition, the most potent small molecule 24 (IC50
0.55, 0.34, 0.41, 0.67 mM, respectively) even showed more potent
¼
inhibition than Saracatinib in some of these four cells.
2.3. The kinase selectivity assay
benzene ring. It resulted from pyridine ring with more hydro- The kinase selectivity prole of this series was assessed, using
philic potency. It was also concluded that the amide formed by compound 24 as representative. The obtained results
a
phenylacetic acid (compound 11–14) at the 6-position slightly (Table 3), presented their activity data depicted as IC50 values.
improved the c-Src inhibitory activity compared to a relatively Interestingly, compound 24 showed moderate inhibitory activ-
benzoic acid substituent (compound 7–8), resulting from the ities for the majority of these target protein kinases, which
longer C-chain with more hydrophilic potency. From compound belong to tyrosine and serine/threonine kinase. In addition,
11, 12, 13, inferring that those compounds with stronger elec- compound 24 performed better as c-Src inhibitors than against
tronegativity substitutes of heterocyclic ring acids led to a another two kinases. These results could provide sufficient
noteworthy loss of activity (4-Br > 4-Cl > 4-F). Particularly, evidence compound 24 could be selected as potential c-Src
compound 25 that possessed 3,4-diethoxy substituents inhibitors and antitumor agents for further optimization.
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Table 1 Enzyme activities (IC50, nM) of compounds 7–25 against human c-Src
Table 1 (Contd. )
kinases
Compound
R
X
c-Src ꢂ SD (mM)
43 ꢂ 1.64
Compound
R
X
c-Src ꢂ SD (mM)
44 ꢂ 1.78
23
24
Cl
7
Br
Cl
2.9 ꢂ 0.17
8
9
Br
Br
76 ꢂ 3.01
59 ꢂ 2.14
25
Cl
3.4 ꢂ 0.27
3.1 ꢂ 0.12
Saracatinib
—
—
10
11
Br
Br
76 ꢂ 2.97
56 ꢂ 2.23
12
Br
49 ꢂ 1.63
13
14
15
16
Br
Br
Br
Br
76 ꢂ 2.31
3.5 ꢂ 0.25
120 ꢂ 4.53
95 ꢂ 3.56
Fig. 3 Inhibitory activity of compounds 7–25 against c-Src protein kinases were
represented as a line–scatter graph.
2.4. Molecular docking
17
18
Br
Cl
70 ꢂ 1.97
79 ꢂ 2.84
Docking study was performed to evaluate the binding potency
between compound 24, Saracatinib and the active domain of
the c-Src kinase (PDB code: 3F3V). The obtained results were
depicted clearly by the following two pictures (Fig. 4). Fig. 4A
and C showed the 2D and 3D binding mode of the compound 24
interacting with 3F3V protein, respectively. The model also
depicted that extensive hydrophobic interactions were formed
between compound 24 and residues Val 281, Ala 293, Lys 295,
Met 314, Val 323, Thr 338, Leu 393, Ala 403 and Phe 405 of the
ATP-binding pocket of c-Src kinase (Fig. 4A). From Fig. 4C and
D, there were only one H-bonds formed between the reference
compound Saracatinib and 3F3V, whereas four H-bonds were
formed between compound 24 and 3F3V, inferring that the
candidate was more tightly embedded into the ATP binding
pocket than Saracatinib. Insight into Fig. 4C, four amino acids
Lys 295, Glu 310, Asp 404, Phe 405 located in the binding pocket
of protein were signicant for the conformation with
compound 24, which were stabilized by three Pi-cation bonds
and three hydrogen bonds (one interaction with amino acid
19
20
21
Cl
Cl
Cl
45 ꢂ 1.58
78 ꢂ 2.93
58 ꢂ 2.07
22
Cl
71 ꢂ 2.81
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Table 2 In vitro anticancer activities (IC50, mM) against four human tumor cell
lines
IC50 ꢂ SD (mM)a
Compound HEPG-2b
MCF-7b
HeLAb
A549b
7
8
9
6.38 ꢂ 0.51
9.13 ꢂ 0.83
7.43 ꢂ 0.64
8.41 ꢂ 0.79
7.38 ꢂ 0.66
6.36 ꢂ 0.33
7.92 ꢂ 0.55
0.88 ꢂ 0.56
8.49 ꢂ 0.61
9.35 ꢂ 0.74 10.87 ꢂ 1.03
7.65 ꢂ 0.68 10.35 ꢂ 0.92 11.43 ꢂ 0.93
6.84 ꢂ 0.49
7.73 ꢂ 0.57
8.47 ꢂ 0.72
7.84 ꢂ 0.64
9.63 ꢂ 0.97
9.29 ꢂ 0.87
8.69 ꢂ 0.73
9.39 ꢂ 0.85
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
6.48 ꢂ 0.44 10.59 ꢂ 0.86
7.69 ꢂ 0.57
9.34 ꢂ 0.48
9.39 ꢂ 0.74
0.93 ꢂ 0.19
8.14 ꢂ 0.57 11.47 ꢂ 1.32
0.49 ꢂ 0.12
1.03 ꢂ 0.08
9.49 ꢂ 0.89 12.44 ꢂ 1.48 10.62 ꢂ 1.07 11.38 ꢂ 1.73
8.69 ꢂ 0.76 10.48 ꢂ 1.09
9.39 ꢂ 0.84
6.15 ꢂ 0.64
8.95 ꢂ 0.53
6.58 ꢂ 0.51
7.93 ꢂ 0.51
9.73 ꢂ 0.74
7.78 ꢂ 0.75
9.59 ꢂ 0.78
4.97 ꢂ 0.47
8.39 ꢂ 0.68
8.85 ꢂ 0.65
7.69 ꢂ 0.53
7.38 ꢂ 0.16
0.67 ꢂ 0.06
0.93 ꢂ 0.43
0.61 ꢂ 0.03
6.39 ꢂ 0.53
7.36 ꢂ 0.53
3.43 ꢂ 0.23
6.59 ꢂ 0.58
8.62 ꢂ 0.76
6.19 ꢂ 0.37
4.67 ꢂ 0.39
0.55 ꢂ 0.13
0.72 ꢂ 0.21
7.49 ꢂ 0.64
9.33 ꢂ 0.74
4.32 ꢂ 0.47
8.40 ꢂ 0.73
7.40 ꢂ 0.53 10.04 ꢂ 0.79
5.53 ꢂ 0.39
5.89 ꢂ 1.56
0.34 ꢂ 0.09
0.63 ꢂ 0.26
0.49 ꢂ 0.02
8.69 ꢂ 0.59
3.25 ꢂ 1.43
0.41 ꢂ 0.14
0.79 ꢂ 0.18
0.67 ꢂ 0.05
Saracatinib 0.50 ꢂ 0.06
a
Antiproliferation activity was measured using the CCK8 assay. Values
b
are the average of two independent experiments. Cancer cells kindly
supplied by KeyGen Biotech; HepG-2 (hepatocellular liver carcinoma
cell line), MCF-7 (breast cancer), HeLa (immortal cell line) and A549
(carcinomic human alveolar basal epithelial cell).
Table 3 IC50 datas for compound 24 against several related kinases screened
Kinase
IC50 (nM)
Fold selectivity
c-Src
2.9
97
837
452
961
—
33
289
156
331
EGFR
CDK2
PTK2
JAK2
Fig. 4 The two kinds of binding modes between the active conformation of
compound 24 and the target protein c-Src (PDB code: 3F3V) provided by the
CDOCKER protocol (Lingar and Discovery Studio 3.5, Accelrys, Co. Ltd). (A) 2D
molecular docking model of compound 24 with 3F3V. (B) 2D molecular docking
model of Saracatinib with 3F3V. (C) The 3D interaction map between compound
24 and the c-Src protein. (D) The 3D interaction map between Saracatinib and the
c-Src protein.
side-chains; two interactions with amino acid main-chains).
Two Pi-cation bonds were formed between amino acid Lys
295 and quinazoline ring of compound 24 with their lengths
˚
˚
˚
5.5 A and 6.2 A, respectively; the other one with its length 6.9 A
was formed between amino acid Phe 405 and N of amide chain.
There are two hydrogen bonds between amino acid Asp 404
3D-QSAR model was performed by using the corresponding
pIC50 values which were converted from the obtained IC50 (nM)
values of c-Src kinase inhibition and performed by built-in
QSAR soware of DS 3.5 (Discovery Studio 3.5, Accelrys, Co.
Ltd). The way of this transformation was derived from an online
calculator developed from an Indian medicinal chemistry lab
(http://www.sanjeevslab.org/tools-IC50.html). The training and
test set was divided by the random diverse molecules method of
DS 3.5, in which the test set accounts for 80% while the training
set was set to 20%. The 19 target compounds were divided into
˚
˚
and N, O of amide chain, with their lengths 1.9 A and 2.3 A,
˚
respectively; another on with 2 A was formed between amino
acid Glu 310 and the N atom of amide chain. Besides, the
bromine and chlorine of 4-aniline head could contribute to
stabilizing the binding complex, derived from the previous
discussion.31 These results could provide a molecular level
foundation for 24 as the most potent c-Src inhibitor.
2.5. 3D QSAR model
In order to obtain a kind of systematic SAR prole based on two parts: 15 agents as training set and the rest 4 agents as
6-amide-4-anilinoquinazoline analogues as antitumor agents relative test set, which had been presented in Table 4. An effi-
and to explore the more potent and selective c-Src inhibitor, cient solution in this research depended on docking study and
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Table 4 Experimental, predicted inhibitory activity of compounds 7–25 against
c-Src by 3D-QSAR models based on active conformations selected from the
preliminary molecular docking study
c-Src kinase inhibition
Predicted
pIC50
b
Compound
Actual pIC50
Residual error
7
8
9
7.13
7.12
7.23
7.12
7.07
7.31
7.12
8.46
6.92
7.13
7.16
7.10
7.35
7.11
7.24
7.15
7.37
8.54
8.47
7.01
7.12
7.10
7.17
7.24
7.31
7.16
8.49
7.03
7.08
7.12
7.08
7.36
7.08
7.28
7.22
7.37
8.49
8.57
0.1213
ꢀ0.0021
0.1309
ꢀ0.0518
ꢀ0.1690
0.0027
10
11a
12
13
14a
15
16a
17
18
19
20
21a
22
23
24
25
ꢀ0.0383
0.0031
ꢀ0.1062
0.0492
0.0328
0.0183
ꢀ0.0098
0.0297
ꢀ0.0387
ꢀ0.0696
ꢀ0.0069
0.0497
ꢀ0.1007
a
Compounds were selected as the test sets while the rest ones were in
b
the training sets. The IC50 values of the compounds against c-Src
(Table 1) were converted into pIC50 values by using the online
calculator (http://www.sanjeevslab.org/tools-IC50.html).
the reliability of this method has been documented in previous
studies.
In default situation, the alignment conformation of each
molecule was the one that ltered the lowest CDOCK-
ER_INTERACTION_ENERGY among the twenty docked poses.
The 3D-QSAR model dened the critical regions (steric or elec-
trostatic) affecting the binding affinity, generated from DS 3.5. It
was a PLS model built on 400 independent variables (conventional
r2 ¼ 0.883). The observed and predicted values and corresponding
residual values for the training set and test set molecules in
3D-QSAR model were presented in Table 4. Moreover, their
graphical relationship had been illustrated in Fig. 5A, in which the
plot of the observed IC50 versus the predicted values demonstrated
that this model could be used in prediction of activity for novel 4-
anilinoquinazoline derivatives as c-Src inhibitor.
Fig. 5 (A) The predicted versus experimental pIC50 values for the inhibition of
c-Src (PDB: 3F3V). (B) Isosurface of the 3D-QSAR model coefficients on electro-
static potential grids. The blue triangle mesh represents positive electrostatic
potential and the red area represents negative electrostatic potential. (C) Iso-
surface of the 3D-QSAR model coefficients on van der Waals grids. The green
triangle mesh representation indicates positive coefficients; the yellow triangle
mesh indicates negative coefficients.
The contour plot of the electrostatic eld region favorable (in
blue) or unfavorable (red) for anticancer activity based on c-Src
protein target were shown in Fig. 5B while the energy grids
corresponding to the favorable (in green) or unfavorable
(yellow) steric effects for the c-Src affinity were shown in Fig. 5C.
3. Conclusion
It was widely acceptable that a better inhibitor based on the 3D In summary,
a
series of 6-amide-4-anilinoquinazoline
QSAR model should have strong van der Waals attraction in the analogues have been synthesized and showed modest potency
green areas and a polar group in the blue electrostatic potential against c-Src, with IC50 values ranging from single-digit nano-
areas (which were dominant close to the skeleton). Thus, this molar to a hundred nanomolar. Among these small molecules,
promising model would provide a guideline to design and compound 24 displayed the most potent activity against c-Src
optimize more effective c-Src inhibitors based on the 4-anili- (IC50 ¼ 2.9 nM), being comparable with the positive control
noquinazoline analogues skeleton and pave the way for us to Saracatinib (IC50 ¼ 3.1 nM). The docking results indicated that
further study in future.
compound 24 could bind well inside the active site of c-Src.
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These results, along with molecular docking observations, are 7.59 (d, J ¼ 7.68 Hz, 1H), 7.78 (d, J ¼ 8.97 Hz, 1H), 7.94 (s, 1H),
signicant evidence to demonstrate the compound 24 could be 8.65 (s, 1H, NH). ESI-MS: 271.1 (C14H11ClN4, [M + H]+). Anal.
optimized as a potential c-Src inhibitor in the further study. calcd for C14H10ClN4: C, 62.11%; H, 4.10%; N, 20.70%. Found:
Moreover, a new 3D QSAR model was built with the activity data C, 61.93%; H, 4.36%; N, 21.07%. N-4-(3-Bromophenyl)quina-
and binding conformations to check the previous work as well zoline-4,6-diamine (5) was also prepared as described for 6.
as to provide us cogent foundation and new ideas about further
design and modication.
4.3. General procedure for the synthesis of a, b and 7–25
(Scheme 1)
4. Experimental section
The starting materials (a) for the synthesis of amides should
4.1. Materials and methods
be activated in the rst procedure: acid (100 mg) and SOCl2
(6–10 mL) were mixed and stirred at reux (80 C) for 4 hours.
ꢁ
All of the synthesized compounds were assessed by thin layer
chromatography (TLC), proton nuclear magnetic resonance
(1H NMR) and elemental microanalyses (CHN). 1H NMR spectra
were measured on a Bruker AV-300 or AV-500 spectrometer with
tetramethylsilane (TMS) as an internal reference (d ¼ 0) at 25 ꢁC.
Chemical shis are reported in parts per million (ppm) using
the residual solvent line as internal standard. Splitting patterns
are designed as s, singlet; d, doublet; t, triplet; m, multiplet. ESI-
MS spectra were recorded on a Mariner System 5304 Mass
spectrometer. Elemental analyses were performed on a CHN-O-
Rapid instrument and were within ꢂ0.4% of the theoretical
values. Melting points were determined on a XT4 MP apparatus
(Taike Corp., Beijing, China) and are as read. Analytic thin-layer
chromatography (TLC) was performed on the glass-backed
The reaction mixture was cooled and evaporated to give reactive
acyl chloride (b) obtained as an oil, which would be dissolved in
ethyl acetate (5–6 mL) in the next step.
A solution of acyl chloride dissolved with ethyl acetate was
added dropwise to compound 5 or 6 (0.5 mM) in ethyl acetate
containing potassium carbonate (300 mg) at 0 ꢁC with constant
stirring overnight. The reaction mixture was then poured in
excess of diluted NaOH and extracted with EtOAc. The extrac-
tion liquid was puried by a ash chromatography with EtOAc–
petroleum ether (3 : 1, v/v) to give these amides as follows: the
yields were about between 30 and 60%.
4.3.1. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-3-uo-
ꢁ
robenzamide (7). Yellow powder, yield: 82%. m.p. 250–252 C;
1H NMR (300 MHz, DMSO-d6, d ppm): 7.28–7.43 (m, 4H), 7.59–
7.66 (m, 1H), 7.75 (t, J ¼ 3.00 Hz, 1H), 7.85 (t, J ¼ 5.09 Hz, 2H),
7.76 (dd, J1 ¼ 8.97 Hz, J2 ¼ 8.97 Hz, 1H), 8.17 (s, 1H), 8.31 (s, 1H),
8.61 (s, 1H), 8.90 (s, 1H), 9.98 (s, 1H), 10.74 (s, 1H). 13C NMR
(300 MHz, DMSO-d6) d: 168.45, 163.85, 158.28, 153.34, 149.46,
144.53, 137.43, 133.46, 130.05, 129.53, 127.45, 124.76, 124.52,
˚
silica gel sheets (silica gel 60 A GF254). All compounds were
detected using UV light (254 nm or 365 nm).
4.2. General method for the preparation of compounds 5, 6
(Scheme 1)
N-4-(3-Bromophenyl)quinazoline-4,6-diamine (5) or N-4-(3- 120.78, 120.52, 115.80, 114.75114.23. ESI-MS: 438.2
chlorophenyl)quinazoline-4,6-diamine (6) could be obtained (C21H15BrFN4O [M + H]+). Anal. calcd for C21H14BrFN4O: C,
according
anthranilonitrile 1 (4.89 g, 30 mmol, 1.0 equiv.) was dissolved in 13.22%.
dimethylformamide dimethyl acetal (10 mL) and the mixture 4.3.2. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-4-uo-
to
the
previous
publications.31
5-Nitro- 57.68%; H, 3.23%; N, 12.81%. Found: C, 60.01%; H, 3.52%; N,
ꢁ
was reuxed (70–80) ꢁC for 2 h. Aer the resulting mixture robenzamide (8). Yellow powder, yield: 85%. m.p. 274–276 C;
cooling to room temperature for 2–3 h and then the yellow 1H NMR (300 MHz, DMSO-d6, d ppm): 7.28–7.45 (m, 4H), 7.82–
compound was ltered, washed with ethyl ether and dried to 7.91 (m, 2H), 8.03 (dd, J1 ¼ 8.97 Hz, J2 ¼ 8.97 Hz, 1H), 8.16–8.20
give 2 (yield: 80–90%). The compound 2 was suspended in 3- (m, 3H), 8.62 (s, 1H), 8.90 (s, 1H), 9.94 (s, 1H), 10.62 (s, 1H). 13
C
chloroaniline (1.1 equiv.) or 3-bromoaniline (1.1 equiv.) and NMR (300 MHz, DMSO-d6) d: 167.46, 165.35, 156.36, 153.13,
then the mixture was heated and stirred at reux in acetic acid 148.47, 144.53, 133.47, 130.57, 130.04, 130.01, 129.53, 127.34,
(20 mL) for 2 h. The yellow precipitate that formed was ltered 125.34, 124.75, 122.92, 119.03, 115.78, 115.43, 114.23. ESI-MS:
hot, washed with hot acetic acid, diethyl ether, and dried to give 438.2 (C21H15BrFN4O [M + H]+). Anal. calcd for C21H14BrFN4O:
the desired nitroquinazoline 3 or 4 (yield: 80–90%). 6-Nitro- C, 57.68%; H, 3.23%; N, 12.81%. Found: C, 57.23%; H, 3.54%;
quinazoline 4 (2.0 g, 6.65 mmol, 1.0 equiv.), iron (2.5 g, N, 13.21%.
45 mmol, 6.7 equiv.) and acetic acid (6 mL, 90 mmol, 13.5
4.3.3. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-4-nitro-
1
ꢁ
equiv.) were suspended in aqueous ethanol (180 mL, 77.8% v/v) benzamide (9). Yellow powder, yield: 84%. m.p. 237–239 C; H
and reuxed (70–80 ꢁC) for 5–6 h. Aer the reaction mixture NMR (300 MHz, DMSO-d6, d ppm): 7.13 (d, J ¼ 7.12 Hz, 1H), 7.33
cooling to room temperature, concentrated ammonia (40 mL) (t, J ¼ 4.13 Hz, 1H), 7.65 (t, J ¼ 4.43 Hz, 2H), 8.02 (t, J ¼ 3.11 Hz,
was added. The resulting solid was extracted with ethyl acetate 2H), 8.21 (d, J ¼ 8.47 Hz, 1H), 8.32 (t, J ¼ 6.30 Hz, 3H), 8.45 (d, J ¼
for column chromatography. Column chromatography was 8.48 Hz, 2H), 8.53 (s, 1H), 8.89 (s, 1H), 9.94 (s, 1H), 10.76 (s, 1H).
performed using silica gel (200–300 mesh) eluting with ethyl 13C NMR (300 MHz, DMSO-d6) d: 165.34, 153.13, 145.56, 137.67,
acetate and petroleum ether (3 : 1, v/v) to give amine 6 (yield: 133.47, 131.34, 129.56, 129.13, 127.41, 125.47, 124.77, 124.12,
50–60%). m.p. 163–164 ꢁC; 1H NMR (300 MHz, DMSO-d6, d 122.87, 120.68, 115.60, 114.32. ESI-MS: 464.3 (C21H15BrN5O3 [M +
ppm): 4.08 (s, 2H, NH2), 6.93 (s, 1H), 7.13 (d, J ¼ 7.86, 2H), 7.24 H]+). Anal. calcd for C21H14BrN5O3: C, 54.33%; H, 3.04%; N,
(dd, J1 ¼ 11.13 Hz, J2 ¼ 8.97 Hz, 1H), 7.33 (s, J ¼ 8.06 Hz, 1H), 15.08%. Found: C, 54.63%; H, 3.34%; N, 14.69%.
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4.3.4. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-2-methyl-
RSC Advances
4.3.9. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)benza-
1
1
benzamide (10). Yellow powder, yield: 80%. m.p. 252–254 C; H mide (15). Yellow powder, yield: 84%. m.p. 275–276 ꢁC; H NMR
NMR (300 MHz, DMSO-d6, d ppm): 2.46 (s, 3H, –CH3), 7.29–7.79 (300 MHz, DMSO-d6, d ppm): 7.28–7.38 (m, 2H), 7.55–7.64 (m,
(m, 4H), 7.44 (t, J ¼ 3.93 Hz, 1H), 7.53 (d, J ¼ 1.29 Hz, 1H), 7.80– 3H), 7.82–7.91 (m, 2H), 8.04 (t, J ¼ 3.57 Hz, 3H), 8.20 (s, 1H),
7.88 (m, 2H), 7.95 (d, J ¼ 8.97, 1H), 8.32 (s, 1H), 8.60 (s, 1H), 9.97 (s, 8.92 (s, 1H), 9.93 (s, 1H), 10.61 (s, 1H). 13C NMR (300 MHz,
1H), 8.93 (s, 1H), 10.65 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d: DMSO-d6) d: 168.56, 157.43, 151.79, 147.89, 144.86, 134.81,
167.78, 158.45, 152.13, 149.54, 144.67, 137.69, 135.14, 133.47, 133.46, 131.38, 130.04, 129.39, 128.57, 128.23, 127.73, 125.43,
131.14, 127.89, 127.13, 124.88, 122.76, 119.79, 115.87, 114.43. ESI- 124.99, 122.73, 119.43, 115.87, 114.21. ESI-MS: 420.2
MS: 434.3 (C22H18BrN4O [M + H]+). Anal. calcd for C22H17BrN4O: C, (C21H16BrN4O [M + H]+). Anal. calcd for C21H15BrN4O: C,
60.98%; H, 3.95%; N, 12.93%. Found: C, 60.57%; H, 4.25%; N, 60.16%; H, 3.61%; N, 13.36%. Found: C, 60.26%; H, 3.35%; N,
ꢁ
12.53%.
4.3.5. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-2-(4-
133.71, 13.17%.
4.3.10. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-4-chlor-
chlorophenyl)acetamide (11). Yellow powder, yield: 89%. m.p. obenzamide (16). Yellow powder, yield: 79%. m.p. 244–246 ꢁC; 1H
251–253 ꢁC; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.76 (s, 2H), NMR (300 MHz, DMSO-d6, d ppm): 7.07 (d, J ¼ 8.4 Hz, 2H), 7.30–
7.26–7.76 (m, 2H), 7.41 (s, 4H), 7.78–7.89 (m, 3H), 8.14 (s, 1H), 7.43 (m, 3H), 7.72–7.86 (m, 2H), 8.04 (d, J ¼ 8.4 Hz, 1H), 8.11 (d, J ¼
8.58 (s, 1H), 8.71 (d, J ¼ 0.15 Hz, 1H), 9.90 (s, 1H), 10.54 (s, 1H). 8.4 Hz, 1H), 8.18 (s, 1H, NH), 8.63 (s, 1H), 8.78 (s, 1H), 10.82 (s, 1H),
13C NMR (300 MHz, DMSO-d6) d: 173.54, 156.28, 153.34, 149.54, 11.54 (s, 1H, NHCO). 13C NMR (300 MHz, DMSO-d6) d: 167.71,
146.01, 134.35, 134.13, 133.41, 130.23, 130.03, 129.68, 129.43, 156.34, 152.13, 149.46, 145.53, 137.81, 135.12, 133.43, 130.02,
127.82, 125.71, 125.19, 122.78, 120.46, 115.87, 114.67. ESI-MS: 129.63, 128.72, 128.31, 127.73, 125.27, 124.52, 121.98, 120.43,
468.8 (C22H17BrClN4O [M + H]+). Anal. calcd for C22H16BrClN4O: 115.90, 114.23. ESI-MS: 454.7 (C21H14BrClN4O [M ꢂ H]+). Anal.
C, 56.49%; H, 3.45%; N, 11.98%. Found: C, 56.73%; H, 3.47%; calcd for C21H14BrClN4O: C, 55.59%; H, 3.11%; N, 12.35%. Found:
N, 12.05%.
4.3.6. 2-(4-Bromophenyl)-N-(4-((3-bromophenyl)amino)qui-
C, 55.72%; H, 3.18%; N, 12.14%.
4.3.11. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-2-(3-
nazolin-6-yl)acetamide (12). Yellow powder, yield: 75%. m.p. chlorophenyl)acetamide (17). Yellow powder, yield: 81%. m.p.
256–258 ꢁC; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.74 (s, 2H), 269–271 ꢁC; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.78 (s, 2H),
7.27–7.41 (m, 4H), 7.55 (d, J ¼ 8.25 Hz, 2H), 7.78–7.89 (m, 3H), 7.27–7.42 (m, 5H), 7.47 (s, 1H), 7.78–7.89 (m, 3H), 8.14 (s, 1H),
8.14 (s, 1H), 8.58 (s, 1H), 8.71 (d, J ¼ 1.43 Hz, 1H), 9.90 (s, 1H), 8.58 (s, 1H), 8.73 (s, 1H), 9.92 (s, 1H), 10.56 (s, 1H). 13C NMR
10.53 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d: 173.47, 157.29, (300 MHz, DMSO-d6) d: 172.45, 157.28, 152.23, 148.56, 145.72,
153.13, 149.48, 144.53, 134.78, 134.26, 131.53, 130.34, 130.02, 137.78, 134.53, 133.79, 130.14, 129.78, 129.12, 128.89, 127.47,
129.81, 127.43, 125.34, 124.87, 122.56, 121.31, 120.78, 115.79, 127.39, 125.36, 124.87, 122.89, 120.66, 115.87, 114.91, 42.98.
114.87. ESI-MS: 513.2 (C22H17Br2N4O [M + H]+). Anal. calcd for ESI-MS: 468.8 (C22H17BrClN4O [M + H]+). Anal. calcd for
C22H16Br2N4O: C, 51.59%; H, 3.15%; N, 10.94%. Found: C,
51.67%; H, 3.14%; N, 11.03%.
C22H16BrClN4O: C, 56.49%; H, 3.45%; N, 11.98%. Found: C,
56.73%; H, 3.49%; N, 12.04%.
4.3.7. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-2-(4-
4.3.12. N-(4-((3-Chlorophenyl)amino)quinazolin-6-yl)-4-u-
uorophenyl)acetamide (13). Yellow powder, yield: 82%. m.p. orobenzamide (18). White powder, yield: 87%. m.p. 284–285 ꢁC;
1
214–216 C; H NMR (300 MHz, DMSO-d6, d ppm): 7.18 (t, J ¼ 1H NMR (300 MHz, DMSO-d6, d ppm): 7.16 (d, J ¼ 9.12 Hz, 1H),
ꢁ
4.48 Hz, 2H), 7.26–7.33 (m, 2H), 7.36–7.44 (m, 2H), 7.78–7.89 7.29–7.45 (m, 3H), 7.84 (d, J ¼ 8.79 Hz, 2H), 8.02 (d, J ¼ 8.97 Hz,
(m, 3H), 8.14 (s, 1H), 8.57 (s, 1H), 8.72 (s, 1H), 9.89 (s, 1H), 10.52 1H), 8.08–8.16 (m, 3H), 8.62 (s, 1H), 8.89 (s, 1H), 9.95 (s, 1H),
(s, 1H). 13C NMR (300 MHz, DMSO-d6) d: 172.56, 163.59, 157.34, 10.63 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d: 167.57, 165.79,
152.19, 149.54, 145.53, 133.51, 130.71, 130.13, 129.87, 129.27, 157.23, 152.21, 149.53, 144.09, 134.34, 133.46, 130.79, 130.13,
127.32, 125.24, 124.79, 122.86, 120.64, 115.79, 115.14, 114.83, 130.01, 129.54, 127.72, 121.78, 120.87, 120.21, 115.90, 115.34,
41.27. ESI-MS: 523.0 (C22H17BrFN4O [M + H]+). Anal. calcd for 114.26. ESI-MS: 393.8 (C21H15ClFN4O [M + H]+). Anal. calcd for
C
22H16BrFN4O: C, 52.70%; H, 3.08%; N, 10.69%. Found: C,
C21H14ClFN4O: C, 64.21%; H, 3.59%; Cl, 9.03%; F, 4.84%; N,
52.43%; H, 3.36%; N, 10.89%.
14.26%; O, 4.07%. Found: C, 64.21%; H, 3.59%; N, 14.26%.
4.3.8. N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)picoli-
4.3.13. N-(4-((3-Chlorophenyl)amino)quinazolin-6-yl)-4-nitro-
1
1
ꢁ
ꢁ
namide (14). Yellow powder, yield: 85%. m.p. 263–264 C; H benzamide (19). White powder, yield: 67%. m.p. 319–321 C; H
NMR (300 MHz, DMSO-d6, d ppm): 7.27–7.38 (m, 2H), 7.72 (t, J ¼ NMR (300 MHz, DMSO-d6, d ppm): 7.17 (d, J ¼ 7.32 Hz, 1H), 7.42
6.12 Hz, 1H), 7.83 (d, J ¼ 8.97 Hz, 1H), 7.9 (d, J ¼ 7.86 Hz, 1H), (t, J ¼ 4.02 Hz, 1H), 7.85 (t, J ¼ 4.48 Hz, 2H), 8.05 (t, J ¼ 3.02 Hz,
8.11 (t, J ¼ 7.68 Hz, 1H), 8.22 (d, J ¼ 7.86 Hz, 2H), 8.34 (dd, J1 ¼ 2H), 8.17 (d, J ¼ 8.58 Hz, 1H), 8.30 (t, J ¼ 6.40 Hz, 3H), 8.43 (d, J ¼
9.15 Hz, J2 ¼ 8.97 Hz, 1H), 8.60 (s, 1H), 8.77 (d, J ¼ 4.74 Hz, 1H), 8.58 Hz, 2H), 8.63 (s, 1H), 8.91 (s, 1H), 9.96 (s, 1H), 10.92 (s, 1H).
8.88 (s, 1H), 9.87 (s, 1H), 10.90 (s, 1H). 13C NMR (300 MHz, 13C NMR (300 MHz, DMSO-d6) d: 167.57, 157.29, 152.21, 150.16,
DMSO-d6) d: 163.57, 157.28, 152.13, 150.63, 150.71, 149.72, 149.47, 144.19, 138.63, 134.47, 133.45, 130.21, 129.65, 129.38,
149.42, 144.78, 137.67, 133.46, 141.05, 129.59, 126.47, 124.79, 127.69, 124.09, 122.57, 120.98, 120.21, 115.90, 114.23. ESI-MS:
122.90, 120.64, 115.79, 114.57. ESI-MS: 421.3 (C20H15BrN5O [M + 420.8 (C21H15ClN5O3 [M + H]+). Anal. calcd for C21H14ClN5O3: C,
H]+). Anal. calcd for C20H14BrN5O: C, 57.16%; H, 3.36%; N, 60.08%; H, 3.36%; N, 16.68%. Found: C, 60.32%; H, 3.34%; N,
16.66%. Found: C, 57.27%; H, 3.56%; N, 16.52%.
16.71%.
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(C20H15ClN5O [M + H]+). Anal. calcd for C20H14ClN5O: C,
63.92%; H, 3.75%; N, 18.64%. Found: C, 64.02%; H, 3.85%; N,
17.96%.
4.3.14. N-(4-((3-Chlorophenyl)amino)quinazolin-6-yl)-2-meth-
ylbenzamide (20). White powder, yield: 83%. m.p. 252–254 ꢁC; 1H
NMR (300 MHz, DMSO-d6, d ppm): 2.45 (s, 3H, –CH3), 7.32–7.47
(m, 4H), 7.50–7.57 (m, 2H), 7.70 (d, J ¼ 8.67 Hz, 1H), 7.89 (t, J ¼ 1
Hz, 1H), 8.06 (d, J ¼ 8.97 Hz, 1H), 8.16 (dd, J1 ¼ 9.12 Hz, J2 ¼ 8.94
4.3.19. N-(4-((3-Chlorophenyl)amino)quinazolin-6-yl)-3,4-
diethoxybenzamide (25). White powder, yield: 83%. m.p. 279–
281 ꢁC; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.68 (m, 4H), 4.00
(m, 6H), 6.90 (s, 2H), 7.00 (s, 1H), 7.37 (d, J ¼ 7.32 Hz, 1H), 7.50
(t, J ¼ 4.11 Hz, 1H), 7.64 (d, J ¼ 8.04 Hz, 1H), 7.85 (s, 1H), 7.94 (d,
J ¼ 8.97 Hz, 1H), 8.05 (d, J ¼ 9.15 Hz, 1H), 8.88 (s, 1H), 9.01 (s,
1H), 10.84 (s, 1H), 11.37 (s, 1H). 13C NMR (300 MHz, DMSO-d6)
d: 167.11, 157.29, 152.43, 152.14, 150.73, 149.47, 144.09, 134.43,
133.63, 130.21, 129.57, 129.32, 127.72, 122.54, 122.33, 120.87,
120.11, 115.87. ESI-MS: 463.9 (C25H23ClN4O3 [M + H]+). Anal.
calcd for C25H23ClN4O3: C, 64.86%; H, 5.01%; N, 12.10%.
Found: C, 64.94%; H, 5.21%; N, 12.08%.
Hz, 1H), 8.90 (s, 1H), 9.22 (s, 1H), 10.94 (s, 1H), 11.58 (s, 1H). 13
C
NMR (300 MHz, DMSO-d6) d: 168.11, 157.29, 151.97, 149.43,
144.12, 138.17, 135.22, 134.41, 133.47, 130.96, 130.14, 129.53,
127.96, 127.69, 127.02, 122.62, 120.89, 120.18, 115.91, 114.21,
20.22. ESI-MS: 389.9 (C22H18ClN4O [M + H]+). Anal. calcd for
C22H17ClN4O: C, 67.95%; H, 4.41%; N, 14.41%. Found: C, 68.03%;
H, 4.40%; N, 14.45%.
4.3.15. 2-(4-Chlorophenyl)-N-(4-((3-chlorophenyl)amino)
quinazolin-6-yl)acetamide (21). White powder, yield: 81%. m.p.
314–315 ꢁC; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.79 (s, 2H),
7.14 (d, J ¼ 6.96 Hz, 1H), 7.41 (s, 6H), 7.77 (d, J ¼ 7.50 Hz, 2H),
7.98 (d, J ¼ 11.54 Hz, 2H), 8.56 (s, 1H), 8.74 (s, 1H), 10.01 (s, 1H),
10.97 (s, 1H). 13C NMR (300 MHz, DMSO-d6) d: 172.49, 156.97,
152.13, 149.47, 144.12, 134.43, 134.29, 133.97, 133.43, 130.32,
130.15, 129.81, 129.54, 127.36, 122.59, 120.87, 120.21, 115.91,
114.87, 43.45. ESI-MS: 424.2 (C22H16Cl2N4O [M + H]+). Anal.
calcd for C22H16Cl2N4O: C, 62.42%; H, 3.81%; N, 13.24%.
Found: C, 62.82%; H, 3.81%; N, 13.24%.
4.3.16. 2-(4-Bromophenyl)-N-(4-((3-chlorophenyl)amino)
quinazolin-6-yl)acetamide (22). Yellow powder, yield: 77%. m.p.
255–257 ꢁC; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.73 (s, 2H),
7.14 (d, J ¼ 9.15 Hz, 1H), 7.33–7.41 (m, 3H), 7.55 (d, J ¼ 8.22 Hz,
2H), 7.79 (d, J ¼ 9.15 Hz, 2H), 7.87 (d, J ¼ 10.95 Hz, 1H), 8.02 (s,
1H), 8.57 (s, 1H), 8.71 (s, 1H), 9.89 (s, 1H), 10.53 (s, 1H). 13C
NMR (300 MHz, DMSO-d6) d: 172.43, 157.31, 152.09, 149.51,
144.17, 134.43, 134.34, 131.69, 130.21, 130.04, 129.81, 127.34,
122.59, 121.47, 120.91, 120.23, 115.87, 114.89, 43.42. ESI-MS:
468.8 (C22H17BrClN4O [M + H]+). Anal. calcd for C22H16BrClN4O:
C, 56.49%; H, 3.45%; N, 11.98%. Found: C, 56.61%; H, 3.62%;
N, 11.88%.
4.3.17. N-(4-((3-Chlorophenyl)amino)quinazolin-6-yl)-2-(4-
uorophenyl)acetamide (23). White powder, yield: 87%. m.p.
248–250 ꢁC; 1H NMR (300 MHz, DMSO-d6, d ppm): 3.76 (s, 2H),
7.15–7.24 (m, 3H), 7.43 (t, J ¼ 3.2 Hz, 3H), 7.73 (d, J ¼ 8.22 Hz,
1H), 7.84 (d, J ¼ 8.97 Hz, 1H), 7.94 (d, J ¼ 6.75 Hz, 2H), 8.68 (s,
1H), 8.83 (s, 1H), 10.44 (s, 1H), 10.71 (s, 1H). 13C NMR (300 MHz,
DMSO-d6) d: 172.31, 152.23, 149.51, 144.09, 134.43, 134.34,
130.61, 130.43, 130.12, 129.82, 127.41, 122.63, 120.57, 120.18,
115.87, 115.23, 114.76, 43.47. ESI-MS: 407.8 (C22H17ClFN4O [M +
H]+). Anal. calcd for C22H17ClFN4O: C, 64.95%; H, 3.96%; N,
13.77%. Found: C, 64.95%; H, 3.96%; N, 13.77%.
4.4. c-Src kinase assay
A coupled spectrophotometric assay was used wherein ADP
generated by c-Src kinase was converted to ATP by pyruvate
kinase (PK), with concomitant production of pyruvate from
phosphoenolpyruvate (PEP).33 LDH reduces pyruvate to lactate
by oxidizing NADH. NADH depletion was monitored at 340 nm
using a microplate reader (Spectra Max 250, Molecular Device)
ꢁ
ꢁ
at 30 C for 20 min. Reactions were performed at 30 C in 100
mM Hepes buffer (pH 7.6), containing 20 mM MgCl2 and 10%
glycerol, initiated by adding ATP. PK (100 mg mLꢀ1), LDH (50 mg
mLꢀ1), PEP (2 mM), and NADH (140 mM) were also added.
Kinase activity was measured by adding 100 mM c-Src optimal
peptide substrate (peptide sequence: AEEEIYGEFEAKKKK,
Sawady, Tokyo).
4.5. Cell proliferation assay
CCK8 is much more convenient and helpful than MTT for
analyzing cell proliferation, because it can be reduced to soluble
formazan by dehydrogenase in mitochondria and has little
toxicity to cells. Cell proliferation was determined using CCK8
dye (Beyotime Inst Biotech, China) according to manufacture's
instructions. Briey, 5 ꢃ 103 cells per well were seeded in a 96-
well plate, grown at 37 ꢁC for 12 h, Subsequently, cells were
treated with compounds at increasing concentrations in the
presence of 10% FBS for 24 or 48 h. Aer 10 mL CCK8 dye was
added to each well, cells were incubated at 37 ꢁC for 1–2 h and
plates were read in a Victor-V multilabel counter (Perkin-Elmer)
using the default europium detection protocol. Percent inhibi-
tion or IC50 values of compounds were calculated by compar-
ison with DMSO-treated control wells. The results were shown
in Table 2.
4.3.18. N-(4-((3-Chlorophenyl)amino)quinazolin-6-yl)pico-
1
ꢁ
linamide (24). White powder, yield: 84%. m.p. 281–283 C; H
NMR (300 MHz, DMSO-d6, d ppm): 7.17 (d, J ¼ 8.61 Hz, 1H), 7.43
(t, J ¼ 4.03 Hz, 1H), 7.72–7.76 (m, 1H), 7.85 (d, J ¼ 8.97 Hz, 2H),
8.10–8.16 (m, 2H), 8.24 (d, J ¼ 7.68 Hz, 1H), 8.37 (dd, J1 ¼ 8.97
Hz, J2 ¼ 9.15 Hz, 1H), 8.63 (s, 1H), 8.80 (d, J ¼ 4.56 Hz, 1H), 8.91
(d, J ¼ 2.01 Hz), 9.91 (s, 1H), 10.92 (s, 1H). 13C NMR (300 MHz,
DMSO-d6) d: 165.17, 157.31, 152.21, 150.45, 149.47, 149.34,
144.09, 137.85, 134.45, 133.71, 130.23, 129.54, 127.75, 127.56,
127.09, 122.45, 120.91, 120.21, 115.87, 114.27. ESI-MS: 376.8
4.6. Multiple protein kinases assays
The kinase activity of various tyrosine kinases was determined
by ELISA.22 EIA/RIA strip-well plates (Corning) were coated with
poly(Glu/Tyr, 4 : 1) peptide (Sigma, 50 mg mLꢀ1, 100 mL per well)
by incubation overnight at 4 ꢁC in phosphate-buffered saline
(PBS, Ca/Mg-free). Kinase reaction was performed in the plates
by addition of 50 mL kinase buffer (50 mM Hepes, 125 mM NaCl,
26238 | RSC Adv., 2013, 3, 26230–26240
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10 mM MgCl2, pH 7.4) containing ATP, 10 ng protein kinases on the cross-validated correlation coefficient, which qualies
(c-Src, EGFR, CDK2, PTK2, JAK2), and compound 24. Aer the predictive ability of the models. Scrambled test (Y scram-
20 min, the plates were washed three times with wash buffer bling) was performed to investigate the risk of chance correla-
(0.1% Tween 20 in PBS) and incubated for 20 min with HRP tions. The inhibitory potencies of compounds were randomly
conjugated anti-phosphotyrosine antibody (0.2 mg mLꢀ1, 50 mL reordered for 30 times and subject to leave-one-out validation
per well, Santa Cruz). Aer two washes, the plates were devel- test, respectively. The models were also validated by test sets, in
oped by the addition of tetramethylbenzidine (50 mL per well, which the compounds are not included in the training sets.
Sigma) and stopped by addition of H2SO4 (2 N, 50 mL per well). Usually, one can believe that the modeling is reliable, when the
The absorbance at 450 nm was measured by a 96-well plate R2 for test sets is larger than 0.6, respectively.
reader (Tecan).
Acknowledgements
4.7. Acute oral toxicity
Five thousand milligrams of compound 24 per kilogram of body
weight was administered to ten healthy rats by oral gavage. The
animals were observed for mortality, signs of gross toxicity and
behavioral changes at least once daily for 14 days. Body weights
were recorded prior to administration and again on Day 7 and
14. All animals survived and appeared active and healthy
throughout the study. With the exception of one male that
exhibited a loss in body weight between Day 7 and 14, all
animals gained body weight over the 14-day observation period.
There were no signs of gross toxicity or abnormal behavior.
This work was supported by the National Forestry Commonweal
Project (200904001) and by the Fundamental Research Funds
for the Central Universities (1082020803).
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