Synthesis, characterization, screening and docking analysis of 4-anilinoquinazoline derivatives as tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.07.036

We report here the design and synthesis of a series of 4-anilinoquinazoline derivatives, of which 7 compounds were crystallographically characterized, as epidermal growth factor receptor (EGFR) inhibitors by modifications on the aniline ring or at the 6-alkoxy site of the 6,7-dimethoxy-4-anilinoquinazoline pharmacophore. The relative inhibition efficiency on EGFR of all as-prepared compounds were measured and ordered, and the IC₅₀ values of nine highly active compounds were determined by ELISA. Docking studies indicated that all 4-anilinoquinazoline derivatives could be inserted into the ATP-binding pocket of the EGFR via indirect docking, and that the modifications at the 3′-position of the anilino group and 6-alkoxy site of the quinazoline ring have little interference with the formation of the two essential H-bonds between the N3 of the quinazoline ring and Thr766 through a water molecule, and the N1 of the quinazoline ring and N-H of Met769. The displacing of the phenyl at 4-position with pyridinyl dramatically reduces the activity of the quinazoline pharmacophore, the resulting derivative (10) being the least active compound. The docking results also showed that the formation of new H-bonds between the N-H of the ethylenediamine group linked to the 6-alkoxy site and Asp776/Cys773 in the binding pocket of EGFR makes compounds 19 (IC₅₀ = 12.1 ± 1.6 nM) and 20 (IC₅₀ = 13.6 ± 0.8 nM) the most potent EGFR inhibitors in this class and worthy of further modification to obtain more potent anticancer compounds.

Full text of DOI:10.1016/j.ejmech.2012.07.036

European Journal of Medicinal Chemistry 61 (2013) 84e94
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization, screening and docking analysis
of 4-anilinoquinazoline derivatives as tyrosine kinase inhibitors
1
1
*
*
Shuang Lü , Wei Zheng , Liyun Ji, Qun Luo , Xiang Hao, Xianchan Li, Fuyi Wang
The CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100190, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the design and synthesis of a series of 4-anilinoquinazoline derivatives, of which 7
compounds were crystallographically characterized, as epidermal growth factor receptor (EGFR) inhib-
itors by modifications on the aniline ring or at the 6-alkoxy site of the 6,7-dimethoxy-4-
anilinoquinazoline pharmacophore. The relative inhibition efficiency on EGFR of all as-prepared
compounds were measured and ordered, and the IC50 values of nine highly active compounds were
determined by ELISA. Docking studies indicated that all 4-anilinoquinazoline derivatives could be
inserted into the ATP-binding pocket of the EGFR via indirect docking, and that the modifications at the
30-position of the anilino group and 6-alkoxy site of the quinazoline ring have little interference with the
formation of the two essential H-bonds between the N3 of the quinazoline ring and Thr766 through
a water molecule, and the N1 of the quinazoline ring and NeH of Met769. The displacing of the phenyl at
4-position with pyridinyl dramatically reduces the activity of the quinazoline pharmacophore, the
resulting derivative (10) being the least active compound. The docking results also showed that the
formation of new H-bonds between the NeH of the ethylenediamine group linked to the 6-alkoxy site
and Asp776/Cys773 in the binding pocket of EGFR makes compounds 19 (IC50 ¼ 12.1 ꢀ 1.6 nM) and 20
(IC50 ¼ 13.6 ꢀ 0.8 nM) the most potent EGFR inhibitors in this class and worthy of further modification to
obtain more potent anticancer compounds.
Received 31 March 2012
Received in revised form
16 June 2012
Accepted 19 July 2012
Available online 27 July 2012
Keywords:
Receptor tyrosine kinase
Epidermal growth factor receptor (EGFR)
Anilinoquinazoline
Inhibitor
Docking analysis
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
kinase [5e7]. Of these compounds, gefitinib (ZD-1839, IressaÔ) and
erlotinib (OSI-774, TarcevaÔ) are already used in clinic and their
The epidermal growth factor receptor (EGFR) is the cell-surface
receptor for members of the epidermal growth factor family of
extracellular protein ligands. Mutations that lead to EGFR over-
expression (known as upregulation) or overactivity have been
associated with a number of cancers, including non-small cell lung,
breast, ovarian and squamous cell cancers [1e3]. Therefore, EGFR
has become an attractive molecular target for the treatment of
selective cancers, and small molecular inhibitors that attenuate the
ability of EGFR to transmit a phosphorylation signal following the
binding of its cognate ligand EGF are of potential interest as
anticancer drugs [4]. The middle of the past decade was marked by
the discovery of the 4-anilinoquinazolines, the most developed
class of potent and selective ATP-competitive inhibitors of EGFR
analogs are being studied extensively to circumvent the resistance
while improving the drug efficacy, and new drug candidates in this
class have already reached various phases of clinical trials [5,7e9].
On the other hand, screening techniques for enzyme inhibitors
including high-throughput screening (HTS) and virtual screening
(VS) are in many cases important starting points in drug discovery.
Experimental HTS techniques for enzyme inhibitors are mostly
based on UVeVis, fluorescence or radioactivity detection, for
examples enzyme-linked immunosorbent assay (ELISA), time-
resolved fluorometric assays such as DELFIA, and radioactive
receptor-ligand binding assays, etc. [10e12]. Enzyme-linked
immunosorbent assay (ELISA) based heterogeneous assays are
widely applied in HTS because of their robustness, but with
modifications for many cases subject to the source and feature of
samples [13]. For examples, Farley et al. developed a solid-phase
ELISA method for the determination of EGFR and pp60c-src tyro-
sine protein kinase activity, and the IC50 values of reference protein
tyrosine kinase (PTK) inhibitors obtained were compared with
values determined using radioactive assays [14]. The results
* Corresponding author. No. 2 The First North Street, Zhongguancun, Beijing
100190, PR China. Tel.: þ86 10 62529069.
E-mail addresses: quoluo@iccas.ac.cn (Q. Luo), fuyi.wang@iccas.ac.cn (F. Wang).
Both authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.036

S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
85
indicated that the ELISA method would be an attractive alternative
to radioactive assays. Stephens et al. [15] applied ELISA to deter-
mine enzyme activity of the soluble urokinase receptor (suPAR) in
blood of healthy donors and cancer patients. The nonspecific
plasma protein interference in tumor extracts was eliminated by
modified the detection method, enabling specific detection of
suPAR in plasma and sera with >90% recovery of added calibrator.
Compared with HTS, in VS, compounds are studied by predicting
their binding to a macromolecular target using computer programs
and it requires as a key prerequisite knowledge about the spatial
and energetic criteria responsible for protein-ligand binding [16].
With the studies of crystal structures of known kinase-ligand
complexes, VS has been developed rapidly to overcome the lead
discovery bottleneck in drug development. Especially the avail-
ability of the crystal structure of EGFR inhibitors (iressa or erlotinib)
complexes [17,18] facilitates the structure-based design of inhibi-
tors against this target. Aparna et al. [19] had carried out VS studies
on 128 selected EGFR kinase inhibitors with GOLD and LigandFit.
And they had shown that, for these 4-anilinoquinazoline-type
ligands, inclusion of a hydrogen-bonded water molecule at N3 is
indispensable to obtain meaningful VS results. Assefa et al. [20]
introduced a novel receptor-guided consensus dynamics align-
ment for the 3D-QSAR studies of 4-anilinoquinazoline and 4-
anilinoquinoline EGFR kinase inhibitors, providing rational
insights into the ligand structural requirements to achieve better
EGFR-inhibitory activity which can be utilized in the design of new
and more potent EGFR inhibitors. However, arriving at the appro-
priate docking/scoring combination is still a challenge because of
lacking certain and clear relationship between docking and ranking
accuracies [21].
leading to apoptosis and cell death [22e25]. Inspired by the
“combi-targeting” concept, we have attempted to design and
synthesis a class of “combi-molecules” [26] by linking the well-
known cytotoxic metal-based anticancer complexes such as
cisplatin [27] and ruthenium arene complexes [28,29] to the 4-
anilinoquinazoline pharmacophore. To this end, in the present
work, we have designed and synthesized
a series of 4-
anilinoquinazoline derivatives by introducing substituents (such
as benzimidazole and diamine) with the ability of coordination to
transition metals to the anilino group or the quinazoline ring of the
anilinoquinazoline pharmacophore (Schemes 1 and 2). We have
then evaluated for their ability to inhibit the tyrosine kinase activity
of EGFR using ELISA and docking analysis, and established predic-
tive models for designation of EGFR-inhibitory active anilinoqui-
nazoline derivatives containing metal-binding sites.
2. Results and discussion
2.1. Chemistry
To develop suitable ligands containing the 4-anilinoquinazoline
pharmacophore for coordination to transition metals such as
ruthenium and platinum, we first modified the quinazoline core
using various substituents at 4-position. The precursor compound
4-chloro-6,7-dimethoxyquinazoline hydrochloride reacted with
various substituents such as 5-aminobenzoimidazole and 5-
aminoquinolin-8-ol under different conditions to produce deriva-
tives 1e15 as shown in Scheme 1.
Another precursor compound 7-methoxyl-6-hydroxyl-4-(40-
fluoro-30-chloro)quinazoline reacted with 1,2-dibromoethane in
DMF in the presence of K2CO3, giving rise to the compound 16, then
16 reacted with ammonia in DMF or ethylenediamine (en) or
imidazole (Im) in acetonitrile to afford compounds 17, 19 and 21,
respectively (Scheme 2). The reaction of the precursor compound
Recently, within the framework of a novel tumor targeting
strategy termed “Combi-Targeting”, Jean-Claude et al. has devel-
oped a class of pleiotropic inhibitors which are capable of not only
blocking EGFR-mediated signaling but also inducing DNA damage
R1
Cl
HN
4
O
4
O
N
R1-NH2
N
a or b
O
N
N
O
N
OH
N
Z
N
N
H
N
H
N
4'
R1 =
1'
Me
Y
3'
N
2'
N
X
( 1-9, 15 )
( 10 )
( 11 )
( 12 )
( 13 )
( 14 )
Compd
Compd
X
Y
Z
X
H
Y
Z
6
SCH3
H
H
H
1
2
3
4
5
H
H
H
H
H
Cl
H
H
H
H
F
H
7
Ph
H
8
Ph
H
NH-Ph
9
H
H
Ph
H
Ph-NH2
NH2
15
H
CN
Scheme 1. Synthesis and chemical structures of the compounds 1e15. (a) Isopropanol, refluxed 4 h for compounds 1e10, 12e15; (b) Pd2(dba)2, (t-Bu)3P, t-BuONa, toluene, refluxed
20 h for compound 11.

86
S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
F
F
F
HN
N
Cl
HN
N
Cl
HN
N
Cl
i
ii
HO
O
O
O
O
O
N
Br
N
R
N
16
R=
NH2
17
19
iii
F
Cl
NH
H2N
HN
N
N
21
O
N
iv
HO
N
O
18
F
F
HN
N
Cl
HN
N
Cl
v
H
N
O
O
Br
O
O
H2N
N
N
20'
20
Scheme 2. Synthesis and chemical structures of compounds 16e21. (i) 1,2-Dibromoethane/DMF/K2CO3/80 ꢁC 4.5 h. (ii) NH3$H2O/DMF/50 ꢁC 4 h for compound 16; ethylenediamine/
acetonitrile/reflux 2.5 h for compound 19; imidazole/TBAB/NaOH/acetonitrile/reflux 3 h for compound 21. (iii) 2-Bromoproethanol/CsCO3/acetone/reflux 3.5 h. (iv)
1,3-Dibromopropane/acetone/K2CO3/reflux 4.5 h. (v) Ethylenediamine/acetonitrile/reflux 2.5 h.
Table 1
Details of data collection and structure refinement for compounds 6, 7, 8, 9, 11, 14 and 18.
Compound
6
7
8 $ HCl
9 $ 2CH3OH 1/2SO4 11 $ 2CH3OH
14
18
Empirical formula
C19 H23 N3 O3 S
C22 H19 N3 O2 C22 H20 Cl N3 O2
C24 H28 N3 O6
S0.5
C24 H25 N5 O4 C40 H42 N8 O9 C17 H15 Cl F N3 O3
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system,
space group
373.46
173(2)
1.54186 (CuK\a)
Orthorhombic,
Pca2(1)
357.40
173(2)
393.86
173(2)
470.52
173(2)
447.49
173(2)
778.82
173(2)
363.77
173(2)
0.71073 (MoK\a) 0.71073 (MoK\a)
Triclinic, P1
0.71073 (MoK\a) 0.71073 (MoK\a) 0.71073(MoK\a) 0.71073(MoK\a)
Triclinic, P1
Orthorhombic,
P212121
Triclinic, P1
Monoclinic, C2/c Monoclinic, P2(1)/c
Crystal
Colorless/block
Light yellow/rod Light yellow/rod
Yellow/plate
Yellow/block
Yellow/strip
Colorless/small
grain
Unit cell dimensions
a (Å)
b (Å)
c (Å)
a
b
g
27.4642(12)
8.6879(19)
15.816(3)
90.00
90.00
90.00
9.0750(6)
9.9130(6)
9.9979(7)
99.285(2)
92.699(2)
95.589(2)
9.4883(2)
12.8078(3)
15.5596(3)
90
90
90
8.19780(10)
12.4307(2)
23.7117(5)
103.85
92.8310
98.3550(10)
10.084(2)
10.711(2)
11.166(2)
87.44(3)
64.90(3)
87.91(3)
21.461(4)
12.307(3)
28.462(6)
90.00
96.85(3)
90.00
12.081(3)
17.722(4)
7.824(2)
90.00
101.984(4)
90.00
(ꢁ)
(ꢁ)
(ꢁ)
Volume (Å3)
Z, Dcalc (mg mꢂ3)
F(000)
3773.9(11)
8, 1.315
1584
881.60(10)
2, 1.346
376
1890.87(7)
4, 1.384
824
2312.30(7)
4, 1.352
996
1090.8(4)
2, 1.362
472
7464(3)
8,1.386
3280
1638.5(7)
4,1.475
752
Crystal size (mm)
0.34 ꢃ 0.27 ꢃ 0.25
3.22e68.24
ꢂ22 ꢄ h ꢄ 33
ꢂ9 ꢄ k ꢄ 10
ꢂ19 ꢄ l ꢄ 14
5867/4769
[Rint ¼ 0.0439]
5867/39/497
0.76 ꢃ 0.40 ꢃ 0.17 0.56 ꢃ 0.31 ꢃ 0.15
0.34 ꢃ 0.33 ꢃ 0.10 0.80 ꢃ 0.46 ꢃ 0.44 0.32 ꢃ 0.24 ꢃ 0.20 0.08 ꢃ 0.07 ꢃ 0.06
q
Range for data
collection (ꢁ)
2.26e25.00
ꢂ10 ꢄ h ꢄ 10
ꢂ11 ꢄ k ꢄ 11
l ꢄ 11
2.06e27.48
ꢂ12 ꢄ h ꢄ 12
ꢂ16 ꢄ k ꢄ 16
l ꢄ 20
2.11e25.00
ꢂ9 ꢄ h ꢄ 9,
ꢂ14 ꢄ k ꢄ 14
l ꢄ 28
2.29e25.50
ꢂ12 ꢄ h ꢄ 12
ꢂ12 ꢄ k ꢄ 12
l ꢄ 13
2.46e25.00
ꢂ25 ꢄ h ꢄ 25
ꢂ14 ꢄ k ꢄ 12
ꢂ33 ꢄl ꢄ 33
6572/6000
1.72e25.00
ꢂ14 ꢄ h ꢄ 13
ꢂ21 ꢄ k ꢄ 21
ꢂ9 ꢄ l ꢄ 9
Limiting indices
Reflections collected/
unique
Data/restraints/
parameters
5562/3088
[Rint ¼ 0.0304]
3088/0/246
4332/4332
[Rint ¼ 0.0000]
4332/0/255
8135/8135
[Rint ¼ 0.0439]
8135/63/640
7486/4060
[Rint ¼ 0.0239]
4060/39/322
2820/2312
[Rint ¼ 0.0359]
[Rint ¼ 0.0669]
6572/0/516
2882/0/227
Goodness-of-fit on F2
1.069
1.328
0.483
1.291
1.192
1.157
1.194
Final R indices [I > 2
(R1 and wR2)
s
(I)] R1 ¼ 0.0395
R1 ¼ 0.0758
wR2 ¼ 0.1318
R1 ¼ 0.0960
wR2 ¼ 0.1386
R1 ¼ 0.0302
wR2 ¼ 0.0501
R1 ¼ 0.1038
wR2 ¼ 0.0722
R1 ¼ 0.0975
wR2 ¼ 0.1599
R1 ¼ 0.1420
wR2 ¼ 0.1743
R1 ¼ 0.0552
wR2 ¼ 0.1192
R1 ¼ 0.0673
wR2 ¼ 0.1245
R1 ¼ 0.0605
wR2 ¼ 0.1454
R1 ¼ 0.0672
wR2 ¼ 0.1499
R1 ¼ 0.0762
wR2 ¼ 0.1351
R1 ¼ 0.1009
wR2 ¼ 0.1441
wR2 ¼ 0.0932
R1 ¼ 0.0497
R indices (all data)
(R1 and wR2
wR2 ¼ 0.1007
0.154 and ꢂ0.254
Drmax
,
Drmin (e Åꢂ3
)
0.208 and ꢂ0.225 0.181 and ꢂ0.226
0.347 and ꢂ0.330 0.239 and ꢂ0.204 0.788 and ꢂ0.491 0.244 and ꢂ0.215

S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
87
7-methoxyl-6-hydroxyl-4-(40-fluoro-30-chloro)quinazoline with 2-
bromoethanol in acetonitrile in the presence of CsCO3 formed
derivative 18 (Scheme 2). And the precursor compound 7-
methoxyl-6-hydroxyl-4-(40-fluoro-30-chloro)quinazoline reacted
with 1,3-dibromopropane in acetone in the presence of K2CO3 to
give rise to the intermediate 200, which then reacted with en to
produce compound 20 (Scheme 2).
The structures of compounds 6, 5, 8, 9, 11, 14 and 18 were
determined by X-ray crystallography, and the details of data
collection and structure refinement are listed in Table 1, and the X-
ray structures are shown in Fig.1. It can be seen that compounds 7, 9
and 11 crystallize in the triclinic space group P1 and compound 8
crystallizes in the orthorhombic space group P212121. The
compounds 7e9 are isomer, but they accept different space group,
which may influence the interactions between these ligands and
EGFR kinase. In the crystals of compounds 7e9, the dihedral angels
between the quinazoline and the neighboring benzene are 12.39,
28.20 and 10.78ꢁ, respectively, also different from each another.
Table 2
Inhibition potency on EGFR and docking scores of 4-anilinoquinazoline derivatives
1e21.
Compound
Relative inhibition/%
IC50/nM
Docking score
1
2
3
4
5
6
7
8
100
96
94
52
75
100
41
100
47
17
53
77
66
58
97
100
100
100
100
100
100
3.8 ꢀ 2.5
6.14
5.77
4.07
4.49
6.96
7.03
5.69
7.31
5.62
5.56
5.82
8.13
5.69
8.86
6.55
6.77
6.35
6.15
6.97
7.55
5.05
201.0 ꢀ 13.7
n.d.a
n.d.
n.d.
126.7 ꢀ 10.1
n.d.
126.7 ꢀ 21.1
n.d.
n.d.
n.d.
n.d.
9
10
11
12
13
14
15
16
17
18
19
20
21
n.d.
n.d.
412.8 ꢀ 4.9
18.1 ꢀ 7.6
n.d.
63.4 ꢀ 6.6
12.1 ꢀ 1.1
13.6 ꢀ 1.3
60.2 ꢀ 6.3
2.2. Enzyme assay
a
Not determined.
We first determined the IC50 value of compound 1 to evaluate
the veracity and sensitivity of the ELISA method. The inhibition
percentage was calculated using the equation:
the values reported previously (9 nM against EGFR-TK from A431
vulval squamous carcinoma cells) [30].
À
ÁꢀÀ
Á
% inhibition ¼ ODenzyme ꢂ ODinhibitor
ODenzyme ꢂ ODblank
Next, the EGFR-inhibitory efficiency of the newly synthesized
anilinoquinazoline derivatives was screened using the same ELISA
procedure mentioned above. The relative inhibitory potency was
determined for each compound given that the inhibitory efficiency
of 1 towards EGFR was 100% and the results are listed in Table 2. The
results show that the nature of the linking groups between the
quinazoline core and the phenyl 4-side chain has a substantial
effect on inhibitory activity. The 4-pyridylamino derivatives 10 was
the least effective than all the derivatives because it was lack of 4-
(1)
where ODenzyme represents the absorbance at 450 nm in the
absence of inhibitors in enzymatic reaction mixtures, ODblank
represents the absorbance in the absence both of the EGFR and
inhibitors, and ODinhibitor represents the absorbance at various
concentrations of kinase inhibitors. The IC50 value of compound 1
against EGFR was determined to be 3.8 ꢀ 2.5 nM, consistent with
Fig. 1. X-ray crystal structures and atom numbering schemes of 4-anilinoquinazoline derivatives 6e9, 11, 14 and 18 (OLEX drawings with 50% probability of ellipsoids).

88
S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
a
small or bulky lipophilic substituent serving as electron-
withdrawing group at the 30-position of the aniline ring (Scheme
1), and for compounds 16e21 there is an electron-donating group
at the 6-position of the quinazoline ring (Scheme 2) as previously
noted by others [31e33]. It is notable that apart from serving as an
electron-withdrawing group, the bulky phenyl substituent at the
30-position of compound 8 may offer an opportunity for
pep or
hydrophobic interaction between the ligand and the receptor (vide
infra). The compounds 7 and 9 have the same bulky phenyl
substituent at the 20- and 40-positions, respectively. However, their
relative inhibitory efficiency towards EGFR is remarkably lower
than that of compound 8. This indicates that the position of the
phenyl substituent at the aniline ring determines whether the
phenyl substituent as an electron-withdrawing at the 30-position
(8) contributes to the inhibitory potency of anilinoquinazoline
derivatives or as an electron-donating group at 20-position (7) or 40-
position (9) reduces the activity of anilinoquinazoline derivatives.
These are consistent with the phenomenon of the small size
substituents such as NO2, CF3 and OMe at different position on the
aniline ring [33]. It has been reported that when a CF3 group
displaces the hydrogen at 20-, 30- or 40-positions of the aniline ring,
the 30-substituted derivative (IC50 against EGFR: 15 nM) was much
more active than the 20- and 40-substituted ones (IC50s: 10 and
4.7
m
M, respectively) [33].
Compounds 16e21 synthesized by modification of the 6-alkoxy
site (Scheme 2) have similar inhibitory potency to compound 1,
a well-established EGFR inhibitor [34], suggesting that the modi-
fications at the 6-position of quinazoline ring have little effect on
the inhibition potency of the 4-anilinoquinazoline pharmacophore.
In view of this, the complexes produced by the reaction of the 4-
anilinoquinazoline derivatives 16e21 with ruthenium may well
maintain the inhibitory potency of the ligands on EGFR kinase. In
this regard, we have synthesized a series of ruthenium DMSO
complexes containing the 4-anilinoquinazoline derivatives 12, 14
and 21 [26] and a class of ruthenium(II) arene complexes having the
4-anilinoquinazoline derivatives 19 and 20 [35]. Of which several
resulting ruthenium complexes indeed show similar inhibitory
potency on EGFR to their 4-anilinoquinazoline ligands while
exhibiting higher specific inhibition on the EGF-stimulated prolif-
eration of human breast cancer cell line MCF-7 than the better EGFR
inhibitor compound 1, implicating a novel family of multi-targeting
metal-based anticancer agents as expected. We are undertaking
further studies on the anticancer activity of the ruthenium anili-
noquinazoline complexes in vitro and in vivo and intend to report
on the outcome of these studies in due course.
Fig. 2. Doseeresponse inhibition curves of the 4-anilinoquinazoline derivative
compounds 2, 6, 8, 15, 16 and 18e21 on EGFR. Points: mean ꢀ SD of triplicate
determinations.
(phenylamino)quinazoline which is the primary pharmacophore
for this class of EGFR inhibitors [31]. When the final concentration
of inhibitors was 10 mM, the relative inhibitory activities of
compounds 2, 3, 6, 8,15e21 were over 90% (Table 2). The features of
these active compounds are that for compounds 6, 8 and 15 there is
Next, we determined the IC50 values of compounds 2, 6, 8, 15
and 16e21, of which the relative inhibitory efficiencies are higher
Fig. 3. The superposition of the bound erlotinib (red) to EGFR in the crystal structure (PDB code: 1M17) [17] and docked erlotinib (blue) into EGFR in the molecular model
constructed by (a) direct docking and (b) indirect docking.

S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
89
95% compared with compound 1. The concentration-dependent
inhibition curves are shown in Fig. 2, and the determined IC50
values are listed in Table 2. In total, these results demonstrate that
4-(phenylamino)-quinazoline is the primary pharmacophore for
this class of EGFR inhibitors and there is a clear benefit from the
lipophilic electron-withdrawing groups at the 30-position on the
aniline and the electron-donating groups at the 6-position of the
quinazoline ring.
and lapatinib (Fig. 4c). Moreover, the correlation between the
relative inhibition efficiency of the derivative on EGFR and the
docking scores was better than that obtained by direct docking
(Fig. S1).
Then we studied in details the interactions of six highly active
compounds (1, 2, 6, 8, 19 and 20) with EGFR in the ATP-binding
pocket. As shown in Fig. 5 and Fig. S2, the interaction modes and
positions of the docked molecules are similar to those of erlotinib
[17]. There are two hydrogen bonds observed: the NeH/N bond
between Met769 and N1 of the aniline and the OeH/OweH/N
bond between Thr766 and N3 of the aniline mediated by a water
2.3. Docking analysis
The main goal of this part of work is to perform a docking
analysis of synthesized EGFR kinase inhibitors using the Surflex-
Dock module to establish predictive models for designation of
EGFR-inhibitory active anilinoquinazoline derivatives containing
metal-binding sites. Initially, the docking studies were carried out
on the EGFR-erlotinib complex (PDB code: 1M17) [17] without any
crystallographically observed water molecules. The studies resulted
in poor docking of the ligand in the binding site and high RMSD
value (8.584). In the crystal of EGFReerlotinib complex [17], the
quinazoline ring of erlotinib is oriented with the N1 in the back of
the ATP-binding pocket, where it forms a H-bond with the main
chain amide of the Met769 which lies in the so-called “hinge”
region of the kinase connected the N- and C-lobes; the N3 of the
pyrimidine ring accepts an hydrogen bond from a water molecule;
the aniline substituent extends into the hydrophobic pocket in the
back of the ATP-binding cleft; and the ether linkages on the
opposite end projects past the connecting segment into solvent.
However, in the direct docking structure (Fig. 3a), the N3 accepts an
H-bond from the Met769 amide nitrogen, with the ether linkages
and the aniline substituent on the opposite positions in the ATP-
binding pocket. This indicates that the direct docking is not
effective.
Because of the wrong position of erlotinib in the EGFR and high
RMSD value resulting from the direct docking, we then performed
the docking analysis with considering the effect of the water
molecule in the protein molecule. Five water (water 2, water 10,
water 11, water 12 and water 16) molecules have been observed in
the ATP-binding site of EGFR-erlotinib in the crystal structure [17],
therefore, these water molecules were considered as a part of the
inflexible protein molecule in subsequent docking studies. After
including the five water molecules, an improvement was observed
(Fig. 3b). The resulting score and RMSD value were 9.7 and 1.847
(<2.0), respectively. And the interactions and position of the
docking molecule (erlotinib) in the binding site of EGFR were
similar to those of the ligand in the EGFR-erlotinib crystal. The only
difference of the two molecules was the orientation the ether
linkages, the reason may be the rotation of the single bond between
the ether and quinazoline ring.
However, even taking these water molecules into account, the
positions resulted from direct docking of the synthesized 4-
anilinoquinazoline derivatives in the ATP-binding site of EGFR
were desultory (Fig. 4a) and the correlation between the observed
activity and the scores was poor. As the 4-anilinoquinazoline
derivatives have some similar characters in EGFR binding as
previous reports [17,18,36], for example, occupying the ATP-binding
site, 4-aniline substituent extending into the hydrophobic, the
ether linkage extending into solvent, and so on. Therefore, we
extracted the quinazoline ring of erlotinib as the segment and
introduced the order “efmatch” to perform docking. The results
were improved, but the positions of some of the compounds in the
binding pocket were still desultory (Fig. 4b). Then we added
another order “ecpen” to dock again, the resulting positions of all
the compounds in the binding pocket of EGFR were consistent with
other well-known 4-anilinoquinazoline analogs such as gefitinib
Fig. 4. Stereoviews of the 4-anilinoquinazoline derivatives docked into the ATP-
binding site of EGFR. (a) Direct docking; (b) indirect docking with “efmatch”; (c)
indirect docking with “efmatch” and “ecpen”.

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S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
Fig. 5. Details of the interactions between the 4-anilinoquinazoline derivative
compounds (a) 19 and (b) 20 and the ATP-binding site of EGFR in the docked models. It
shows that apart from the two essential H-bonds (red dotted line) between the qui-
nazoline ring and Thr764 and Met769, additional H-bonds between the diamine
moieties and Cys773 and Asp776. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
Fig. 6. Comparison of the docked conformers/poses (green) of compounds (a) 7, (b) 8
and (c) 9 at the ATP-binding site of EGFR kinase as generated via Surflex docking-
scoring combinations with the X-ray structures (blue) of compounds 7, 8 and
alone. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
9
molecule [17]. And the aniline substituent extended into the
hydrophobic pocket formed by amino residues of Val702, Lys721,
Met742 and Leu764. Moreover, the modification at 6-alkoxy site of
the anilinoquinazoline core with a ethylenediamine (en) group via
a C2 (19) or C3 (20) linker allows formation of two new H-bonds
between the ligand and EGFR (Fig. 6): (en) N/HeN (Cys773) and
(en) NeH/O (Asp776) for 19 and 2ꢃ (en) NeH/O (Asp776) for
(20), accounting for the better activity of these two compounds
than others.
Compounds 7, 8 and 9 are isomers to each another, of which the
only structural difference is the location of the phenyl substituent
at the 4-alinino group (Scheme 1). The docking scores of the three
compounds are 5.69, 7.31 and 5.62, respectively, consistent with
the relative inhibitory activity determined by ELISA. As shown in
Fig. S3, the numbers of H-bonds and position of docked molecules
in the binding pocket of EGFR of the three compounds are nearly
the same. However, the hydrophobic interactions between the
substituted 4-anilino groups and the receptor are significantly
different. Similar to erlotinib, the 4-anilino group of compound 8
extended into the hydrophobic pocket formed by amino acid resi-
dues Val702, Lys721, Met742 and Leu764. Whereas the 4-anilino
group of compounds 7 and 9 are deviated from the hydrophobic
pocket, especially compound 9 of which the 4-anilino group is
completely away from the hydrophobic pocket. These results
indicate that the lipophilic phenyl group at the 30-position on the
aniline clearly benefits for the activity of the 4-anilinoquinazoline
derivatives, consistent with the previous report [31,33].
Compared the docked pose of compound 8 at the ATP-binding site
of EGFR kinase with its X-ray structure (Fig. 6b), we found that the
dihedral angels of compound 8 between the quinazoline and the
neighboring benzene changed from 28.20ꢁ (in crystal) to 36.64ꢁ (in
docking structure) and the bond between the two benzenes was
deflexed so that the orientation of the rightmost benzene signifi-
cantly changed. In crystals, the rightmost benzene in 8 is parallel to
the aniline-NH, but opposite against the NH group in the docking
structure. The dihedral angels between the quinazoline and the
neighboring benzene of the compounds 7 and 9 even changed
more, 12.39ꢁ to 35.43ꢁ for 7, 10.78ꢁ to 56.94ꢁ for 9, and the orien-
tation of the aniline-NH in the docked poses is also different from

S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
91
that in crystals (Fig. 6a, c). These may cause largely structural stress
and reduce the binding affinity of compounds 7 and 9 to EGFR,
accounting to the low activity of 7 and 9 towards EGFR.
With compounds 12 and 14, 19 and 20 as the examples for the
modification at 4-anilino ring and the 6-pistion of the quinazoline
ring, respectively, the detailed procedures for the synthesis of the
designated 4-anilinoquinazoline derivatives are given as follows.
N-(benzo[d]imidazol-4-yl)-6,7-dimethoxyquinazolin-4-amine
3. Conclusion
hydrochloride (12). First, a mixture of 4-nitrobenzene-1,2-diamine
(1 g) and formic acid (0.74 mL) in 5 N HCl (70 mL) was stirred and
heated to reflux for 24 h. The mixture was cooled to room
temperature and neutralized by 10% NaOH, and precipitate was
isolated, recrystallised from water and dried to produce 5-
nitrobenzoimidazole (0.69 g) [37]. Next, 5-nitrobenzoimidazole
(0.25 g) was dissolved in ethanol (20 mL). The solution was stir-
red and heated to reflux, and the palladium-on-charcoal catalyst
(0.15 g) and hydrazine hydrate (1.6 mL) were added. Then the
mixture was stirred for 4 h, and filtered in hot. The filtrate was
evaporated, and the residue was recrystallised from ethanol to give
rise to the precursor compound 5-aminobenzoimidazole (0.163 g).
Finally, a mixture of 4-chloro-6,7-dimethoxyquinazoline hydro-
chloride (0.25 g), 5-aminobenzoimidazole (0.15 g) in isopropanol
(25 mL) was stirred and heated to reflux for 4 h. The precipitate was
filtered off, recrystallised from methanol and dried to afford 12 as
yellow powder (0.32 g, yield 73%).
In the present work, we have designed and synthesized a series
of 4-anilinoquinazoline derivatives by introducing substituents
with the ability of coordination to transition metals to the anilino
group or the quinazoline ring. ELISA screening showed that the
derivatives resulted from the modifications at the 30-position of the
anilino ring with lipophilic electron-withdrawing groups or at the
6-alkoxy site of the quinazoline ring with electron-donating groups
maintain the inhibitory activity of the anilinoquinazoline phar-
macophore at high level. The docking studies revealed that the
modifications at the 30- and 6-position have little disturbing on the
formation of the two essential H-bonds between the N3 of the
quinazoline ring and Thr766 through a water molecule, and the N1
of the quinazoline ring and NeH of Met769. Moreover, linking an
ethylenediamine group to the 6-alkoxy site of the anilinoquinazo-
line core allows formation of new H-bonds between the N-H of the
diamine groups and Asp776/Cys773 in the ATP-binding pocket of
EGFR, the resulting derivatives being highly active against EGFR and
encouraging for the further studies to synthesize transition metal
complexes ligated with these derivatives. In this regard, we have
5-(6,7-dimethoxyquinazolin-4-ylamino)quinolin-8-ol hydrochlo-
ride (14). To prepare the precursor compound 5-aminoquinolin-8-
ol, a mixture of 5-nitroquinolin-8-ol (2 g) in isopropanol (100 mL)
was stirred and heated to reflux, the palladium-on-charcoal cata-
lyst (0.25 g) and hydrazine hydrate (1.5 mL) were added. Then the
mixture was stirred for 4 h. The resulting mixture was filtered in
hot, the filtrate was evaporated, and the residue was recrystallised
from ethanol to give rise to precursor 5-aminoquinolin-8-ol
(1.18 g). Next, a mixture of 4-chloro-6,7-dimethoxyquinazoline
(0.25 g) and 5-aminoquinolin-8-ol (0.18 g) in ethanol (25 mL) was
stirred and heated to reflux for 4 h. The precipitate was filtered off,
recrystallised from methanol and dried to give rise to 14 (0.364 g,
yield 85%). Slowly evaporation of methanol from the solution of
compound 14 in methanol produced yellow strip crystals suitable
for X-ray diffraction analysis. The detailed crystallographic data
were listed in Table 1 and the crystal structure is shown in Fig. 1.
6-(2-(2-aminoethylamino)ethoxy)-4-(3’-chloro-4’-fluoroanilino)-
7-methoxy-quinazoline (19). The precursor compound 7-methoxyl-
6-hydroxyl-4-(40-fluoro-30-chloro)quinazoline (2.0 g) and potas-
sium carbonate (10.2 g) were mixed together in DMF (60 mL). Then
1,2-dibromoethane (4.6 mL) was added and the mixture was
heated at 353 K for 4.5 h. After cooled down to room temperature,
the mixture was filtered in vacuum and the filtrate was collected
and poured into water (130 mL). Then the resulting mixture was
extracted using ethyl acetate (30 ꢃ 4 mL), organic layers were
combined and dried by magnesium sulfate. After concentrated, the
residue was chromatographied by flash chromatography on Silica
gel using ethyl acetate/petroleum (1:5, v/v) as eluent to give rise to
compound 7-methoxyl-6-(20-bromoethyl)-4-(40-flouro-30-chloro)
anilinoquinazoline (16) (1.6 g, yield: 60%.) as white powder. And
then the compound 16 (0.8 g) was dissolved in acetonitrile (30 mL)
with addition of 1 mL ethylenediamine (en). The solution was
heated to 353 K and refluxed for 2.5 h. Then the solvent was
evaporated in vacuum and the residue was recrystallised from
water/ethanol to give rise to 19 as white powder (0.5 g, yield 66%).
6-(3-(2-aminoethylamino)ethoxy)-4-(3’-chloro-4’-fluoroanilino)-
7-methoxyquinazoline (20). The precursor compound 6,7-
dimethoxy-4-(3’-chloro-4’-fluoroanilino)quinazoline (2.0 g) and
potassium carbonate (5.0 g) were mixed together in acetone
(100 mL). Then 1,3-dibromoethane (3.2 mL) was added and the
mixture was refluxed for 4.5 h. After cooling to room temperature,
the mixture was filtered in vacuum and the filtrate was collected
and evaporated to give yellow oil, which was chromatographed by
synthesized
a series of ruthenium(II, III) complexes with
compounds 12, 14 and 19e21 as bidentate or monodentate ligands
for biological evaluation, and the outcome of the studies will be
reported in due course.
4. Experimental section
4.1. Chemistry
The starting material 4-chloro-6,7-dimethoxylquinazoline (AR
grade) was purchased from Shanghai FWD Chemicals Co. (China),
5-nitro-8-hydroxylquinoline from Wuhan Kaitong Fine Chemicals
Co. (China), imidazole from Tianjin Jinke Fine Chemical Research
Institute (China), and other chemicals used in this work from
Sigma. The catalyst Pd/C and t-BuONa were purchased from Beijing
Ouhe Technology Co. (China), Pd2(dba)2 and (t-Bu)3P from
Shanghai Darui Fine Chemical Co. (China). Organic solvents
including absolute methanol, absolute ethanol, absolute ether,
acetonitrile, DMF were purchase from Merck in analytical grade
and used directly without further purification. The reactions were
monitored by thin-layer chromatography (TLC) with silica gel
GF254 plates (Qingdao Jiyida Silica Reagent Manufacture, China),
which were visualized by UV light. All 1H NMR were recorded on
a Bruker Avance 400 spectrometer (Bruker, German) at 400 MHz,
and 13C NMR on a Bruker Avance 600 spectrometer (Bruker,
German). 1H and 13C chemical shifts are reported as chemical shifts
(d) in ppm, multiplicity (s: singlet; d: doublet; t: triplet; dd: doublet
of doublets; m: multiplet; br: broad). Mass spectra were obtained
on a Shimadzu LCMS-2010 mass spectrometer (Shimadzu Corpo-
ration, Japan), and the data are reported in the most abundant
isotopic m/z. The element analyses were performed on the Flash
EA1112 (Thermo Electron Corporation).
Compounds 1e15 were synthesized following procedure shown
in Scheme 1. Refluxing a solution of a chloroquinazoline and
a substituted aniline derivative in isopropanol (for 1e10) or toluene
with Pd2(dba)2, (t-Bu)3P and t-BuONa as catalysts (for 11) gave rise
to the 4-anilino-6,7-dimethoxyquinazoline derivatives 1e15. With
the precursor compound 7-methoxyl-6-hydroxyl-4-(40-fluoro-30-
chloro)quinazoline as a starting material, compounds 16e21 were
synthesized following the different procedures shown in Scheme 2.

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S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
flash chromatography on Silica gel using ethyl acetate/petroleum
(1:5) as eluent to give rise to 6-(2-bromoethoxy)-4-(3’-chloro-4’-
fluoroanilino)-7-methoxyquinazoline (200) (0.80 g, 37.2%.) as
yellow powder. Then the as-prepared intermediate 200 (0.8 g) and
ethylenediamine (en, 1 mL) were dissolved in acetonitrile (30 mL).
The solution was heated to 253 K and refluxed for 2.5 h. Then the
solvent was evaporated in vacuum and the residue was recrystal-
lised from water/ethanol to give rise to 20 (0.54 g, 70%) as white
powder.
(s, 3H). Anal. calcd (%) for C17H18ClN3O2S (6$HCl): C 56.12, H 4.99, N
11.55; found: C 55.94, H 5.06, N 11.32.
4.1.7. N-[(2’-phenyl)phenyl]-6,7-dimethoxyquinazolin-4-amine (7)
Yield 73%, m.p. 161e164 ꢁC. ESI-MS (m/z) calcd for C22H19N3O2
357.4, found 358.0 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 9.27 (s, 1H), 8.20 (s, 1H), 7.60 (s, 1H), 7.47 (d, 6H), 7.25 (d,
3H), 7.09 (s, 1H), 4.00 (d, 6H). Anal. calcd. (%) for C22H20ClN3O2
(7$HCl): C 67.09, H 5.12, N 10.67; found: C 66.63, H 5.13, N 10.46.
4.1.1. N-(3-chloro-4-fluorophenyl)-6,7-dimethoxyquinazolin-4-
amine (1)
4.1.8. N-[(3’-phenyl)phenyl]-6,7-dimethoxyquinazolin-4-amine (8)
Yield 62% m.p. 243e244 ꢁC. ESI-MS (m/z) calcd for C22H19N3O2
357.4, found 358.0 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
Yield 79%, m.p. 216e218 ꢁC. ESI-MS (m/z) calcd for
C16H13ClFN3O2 333.5; found 334.2 [M þ H]þ. 1H NMR (400 MHz,
d
(ppm): 11.27 (s, 1H), 8.83 (s, 1H), 8.24 (s, 1H), 7.99 (s, 1H), 7.67 (s,
DMSO-d6)
d
(ppm): 11.50 (s, 1H), 8.87 (s, 1H), 8.35 (s, 1H), 8.03 (dd,
3H), 7.61 (m, 2H), 7.49 (m, 2H), 7.41 (s, 1H), 7.30 (s, 1H), 4.01 (d, 6H).
Anal. calcd (%) for C22H20ClN3O2 (8$HCl): C 67.09, H 5.12, N 10.67;
found: C 66.89, H 5.18, N 10.53.
1H), 7.74 (m, 1H), 7.53 (t, 1H), 7.35 (s, 1H), 4.00 (d, 6H). 13C NMR
(600 MHz, DMSO-d6) (ppm): 160.52, 154.91, 149.00, 145.33,
d
144.30, 116.06, 108.48, 105.37, 56.40, 56.17. Anal. calcd (%) for
C16H14Cl2FN3O2 (1$HCl): C 51.91, H 3.81, N 11.35; found: C 51.69, H
3.96, N 11.23.
4.1.9. N-[(4’-phenyl)phenyl]-6,7-dimethoxyquinazolin-4-amine (9)
Yield 75%, m.p. 256e257 ꢁC. ESI-MS (m/z) calcd. for C22H19N3O2
357.4, found 358.0 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
4.1.2. 6,7-dimethoxy-N-phenylquinazolin-4-amine (2)
d (ppm): 11.23 (s, 1H), 8.84 (s, 1H), 8.25 (s, 1H), 7.80 (s, 4H), 7.72 (d,
Yield 83%, m.p. 236e240 ꢁC. ESI-MS (m/z) calcd for C16H15N3O2
281.1, found 282.2 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
2H), 7.49 (s, 2H), 7.38 (s,1H), 7.31 (d,1H), 4.02 (d, 6H). Anal. calcd (%)
for C22H20ClN3O2 (9$HCl): C 67.09, H 5.12, N 10.67; found: C 66.55,
H 5.06, N 10.59.
d
(ppm): 11.37 (s, 1H), 8.81 (s, 1H), 8.31 (s, 1H), 7.68 (m, 2H), 7.48
(m, 2H), 7.31 (m, 2H), 4.00 (d, 6H). 13C NMR (600 MHz, DMSO-d6)
(ppm): 158.65, 156.72, 150.65, 149.04, 137.36, 135.92, 129.14,
d
4.1.10. 6,7-Dimethoxy-N-(pyridin-4-yl)quinazolin-4-amine (10)
Yield 79%, m.p. 219e221 ꢁC. ESI-MS (m/z) calcd. for C15H14N4O2
282.2, found 283.1 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
126.78, 125.40, 107.69, 104.64, 100.16, 57.49, 56.91. Anal. calcd (%)
for C16H16ClN3O2 (2$HCl): C 60.47, H 5.08, N 13.22; found: C
59.68, H 5.12, N 13.19.
d
(ppm): 9.16 (s, 1H), 9.01 (s, 1H), 8.63 (d, 2H), 7.60 (s, 1H), 7.15 (s,
1H), 4.06 (s, 3H), 3.94 (s, 3H). 13C NMR (600 MHz, DMSO-d6)
(ppm): 160.93, 157.48,156.23, 152.73,151.96, 151.51, 142.43, 113.51,
4.1.3. N1-(6,7-dimethoxyquinazolin-4-yl)-N4-phenylbenzene-1,4-
diamine (3)
d
109.93, 107.43, 101.32, 57.12, 56.73. Anal. calcd (%) for
C16H19.5Cl1.5N4O3 (10$1.5HCl$CH3OH): C 52.07, H 5.33, N 15.18;
found: C 52.32, H 5.23, N 15.65.
Yield 75%, m.p. 217e219 ꢁC. ESI-MS (m/z) calcd for C22H20N4O2
372.1, found 373.3 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
11.11 (s, 1H), 8.77 (s, 1H), 8.36 (s, 1H), 8.18 (s, 1H), 7.49 (dd, 2H), 7.25
(m, 3H), 7.11 (m, 4H), 6.85 (t, 1H), 4.00 (d, 6H). Anal. calcd (%) for
C22H21ClN4O2 (3$HCl): C 64.62, H 5.18, N 13.70; found: C 64.10, H
5.21, N 13.56.
4.1.11. N-(6,7-dimethoxyquinazolin-4-yl)-1,10-phenanthrolin-5-
amine (11)
Yield 36%, m.p. 246e250 ꢁC. ESI-MS(m/z) calcd. for C22H17N5O2
383.3, found 384.1 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
4.1.4. N1-(6,7-dimethoxyquinazolin-4-yl) -4,4’-
biphenylenediamine (4)
d (ppm):10.12 (s, 1H), 9.10 (d, 1H), 9.05 (s, 1H), 8.46 (m, 2H), 8.20 (s,
1H), 8.09 (s, 1H), 8.03 (s, 1H), 7.71 (m, 2H), 7.18 (s, 1H), 3.95 (d, 6H).
Anal. calcd (%) for C22H22N5O4.5 (11$2.5H2O): C 61.67, H 5.18, N
16.35; found: C 61.49, H 5.08, N 16.29.
Yield 71%, m.p. 276e279 ꢁC. ESI-MS (m/z) calcd for C22H20N4O2
372.1, found 373.3 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
9.50 (s, 1H), 8.45 (s, 1H), 7.85 (s, 1H), 7.79 (d, 2H), 7.56 (d, 2H), 7.37
(d, 2H), 7.18 (s, 1H), 6.65 (d, 2H), 5.17 (s, 2H), 3.93 (d, 6H). Anal. calcd
(%) for C22H20.5Cl0.5N4O2 (4$0.5HCl): C 67.64, H 5.29, N 14.34;
found: C 68.29, H 5.45, N 14.43.
4.1.12. N-(1H-benzo[d]imidazol-5-yl)-6,7-dimethoxyquinazolin-4-
amine (12)
Yield 64%, m.p. 254e256 ꢁC. ESI-MS (m/z) calcd for C17H15N5O2
321.3, found 322.1 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
4.1.5. N1-(6,7-dimethoxyquinazolin-4-yl)benzene-1,4-diamine (5)
Yield 81%, m.p. 252e255 ꢁC. ESI-MS (m/z) calcd for C16H16N4O2
296.3, found 297.0 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
11.63 (s, 1H), 9.38 (s, 1H), 8.83 (s, 1H), 8.42 (s, 1H), 8.24 (s, 1H), 7. 88
(m, 2H), 4.04 (s, 3H), 4.01 (s, 3H). Anal. calcd (%) for C17H18ClN5O3
(12$HCl$H2O): C 54.33, H 4.83, N 18.64; found: C 54.64, H 4.65, N
18.64.
d
(ppm): 10.85 (s, 1H), 8.69 (s, 1H), 8.11 (s, 1H), 7.32 (d, 2H), 7.25 (s,
1H), 6.72 (d, 2H), 3.98 (d, 6H), 1.236 (s, 2H). 13C NMR (600 MHz,
DMSO-d6) (ppm): 158.04, 156.25, 150.37, 149.55, 126.17, 118.50,
d
4.1.13. 6,7-Dimethoxy-N-(2-methyl-1H-benzo[d]imidazol-5-yl)
quinazolin-4-amine (13)
115.05, 107.59, 104.07, 100.99, 57.16, 56.81. Anal. calcd (%) for
C16H17ClN4O2 (5$ HCl) : C 57.75, H 5.15, N 16.84; found: C 57.39, H
2.28, N 16.50.
Yield 60%, m.p. 260e262 ꢁC. ESI-MS (m/z) calcd. for C18H17N5O2
335.4, found 336.1 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 9.87 (s, 1H), 8.48 (s, 1H), 8.04 (s, 1H), 7.96 (s, 1H), 7.55 (s,
4.1.6. 6,7-dimethoxy-N-(3-(methylthio)phenyl)quinazolin-4-amine (6)
Compound 6 was synthesized following a similar procedure
described previously [38]. Yield 71%, m.p. 236e238 ꢁC. ESI-MS (m/
z) calcd for C17H17N3O2S 327.4, found 328.0 [M þ H]þ. 1H NMR
2H), 7.20 (s, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 2.58 (s, 3H). 13C NMR
(600 MHz, DMSO-d6) (ppm): 157.34, 155.03, 152.43, 151.93,
d
149.56, 144.92, 136.08,134.67, 133.48,119.51, 114.20, 109.00, 108.52,
106.08, 103.03, 56.91, 56.38, 14.28. Anal. calcd (%) for C18H19ClN5O2.5
(13$HCl$0.5H2O): C 56.77, H 5.03, N 18.39; found: C 56.55, H 5.15, N
18.27.
(400 MHz, DMSO-d6) d (ppm): 11.22 (s,1H), 8.84 (s,1H), 8.24 (s,1H),
7.61 (s, 1H), 7.50 (m, 2H), 7.32 (s, 1H), 7.22 (d, 1H), 4.02 (d, 6H), 2.52

S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
93
4.1.14. 5-(6,7-dimethoxyquinazolin-4-ylamino)quinolin-8-ol (14)
Yield 56%, m.p. 252e254 ꢁC. ESI-MS (m/z) calcd for C19H16N4O2
348.4, found 349.1 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
156.79, 155.20, 153.58, 152.34, 148.86, 146.18, 136.11, 124.20, 122.26,
119.81, 115.82, 109.00, 105.78, 102.04, 67.65, 55.19, 46.29, 39.07,
28.20. Anal. calcd (%) for C20H23ClFN5O2$HCl: C, 52.64; H, 5.30; N,
15.35; found: 52.44; H, 5.34; N, 14.53
d
(ppm):11.36 (s, 1H), 10.23 (s, 1H), 8.93 (s, 1H), 8.61 (s, 1H), 8.24 (m,
2H), 7.57 (m, 2H), 7.30 (s, 1H), 7.20 (d, 1H), 4.00 (d, 6H). Anal. calcd
(%) for C19H16N4O2: C 65.51, H 4.63, N 16.08; found: C 65.75, H 4.73,
N 16.18.
4.1.21. 6-(2-(1H-imidazol-1-yl)ethoxy)-4-(3’-chloro-4’-
fluoroanilino)-7-methoxy- quinazoline (21)
Compound 21 was synthesized following a method reported in
the literature [30] with slight modification. Yield 73%, m.p.
259e261 ꢁC. ESI-MS (m/z) calcd for C20H17ClFN5O2 413.1, found
4.1.15. 3-(6,7-Dimethoxyquinazolin-4-ylamino)benzonitrile (15)
Yield 61%, m.p. 294e295 ꢁC. ESI-MS (m/z) calcd for C17H14N4O2
306.3, found 307.1 [M þ H]þ. 1H NMR (400 MHz, DMSO-d6)
414.2 [M þ H]þ. 1H NMR (DMSO-d6, 400 MHz)
d (ppm): 9.51(s, 1H),
d
(ppm): 8.89 (s, 1H), 8.27 (s, 2H), 8.08(d, 1H), 7.78 (d, 1H), 7.72 (t,
8.56(s, 1H), 8.15(m, 1H,), 7.87(s, 1H), 7.75(m, 1H), 7.70(s, 1H), 7.41(t,
1H), 7.33(s, 1H), 7.20(s, 1H), 6.95(s, 1H) 4.53(t, 2H), 4.41(t, 2H),
4.02(s, 3H). Anal. calcd (%) for C20H17ClFN5O2: C, 58.05; H, 4.14; N,
16.92; found: 57.91; H, 4.15; N, 16.85.
1H), 7.32 (s, 1H), 4.03 (d, 6H). Anal. calcd (%) for C17H15ClN4O2
(15$HCl): C 59.57, H 4.41, N 16.34; found: C 59.25, H 4.43, N 16.12.
4.1.16. 6-(2-Bromoethoxy)-4-(3’-chloro-4’-fluoroanilino)-7-
methoxy-quinazoline (16)
4.2. X-ray crystallography
Compound 16 was synthesized following a similar procedure
reported previously [30]. Yield 77%, m.p. 256e259 ꢁC. ESI-MS (m/z)
calcd for C17H14ClBrFN3O2 425.0, found 426.1 [M þ H]þ. 1H NMR
Single-crystal X-ray diffraction analysis was carried out on
a Rigaku Saturn 724 diffractometer (Rigaku Corporation, Japan) at
173 K using Mo radiation. Single crystals were coated with
Paratone-N oil, mounted using a glass fibre and frozen in the cold
stream of the goniometer. All data were collected at 173 K, and
structure solution and refinement were performed using the
SHELXL-97. Standard data relating to the X-ray crystal structure of
complexes 6, 7, 8, 9, 11, 14 and 18 have been deposited in the
Cambridge Crystallographic Data Centre with the CCDC deposition
number CCDC 873807e873813.
(400 MHz, CD3COCD3-d6) d (ppm): 8.94 (s,1H), 8.56 (s, 1H), 8.22 (m,
1H), 7.77 (m, 2H), 7.30 (t, 1H), 7.26 (s, 1H), 4.49 (t, 2H), 4.03 (s, 3H),
3.86 (t, 2H). 13C NMR (600 MHz, DMSO-d6)
d
(ppm): 156.58, 154.69,
153.20, 152.88, 147.95, 147.35, 137.11, 124.02, 122.83, 119.26, 116.98,
109.04, 107.77, 103.66, 69.38, 56.39, 31.20. Anal. calcd (%) for
C17H14ClBrFN3O2: C, 47.86; H, 3.31; N, 9.85; found: C, 47.93; H, 3.32;
N, 9.96.
4.1.17. 6-(2-Aminoethoxy)-N-(3-chloro-4-fluorophenyl)-7-
methoxyquinazolin-4-amine (17)
4.3. Kinase inhibition assay
Yield 66%, m.p. 200e203 ꢁC. ESI-MS (m/z) calcd for
C17H15ClFN4O2 362.1, found 363.2 [M þ H]þ. 1H NMR (400 MHz,
An aliquot (12.5
containing various concentrations of
compound was added to 12.5 L DTT/kinase buffer (pH 7.5, CST)
containing 100 ng EGFR kinase (Sigma), and then incubated at
room temperature (25 ꢁC) for 5 min, followed by addition of 25
solutions containing 12.5 L PTP1B (Tyr66) biotinylated peptide
(CST), 1 L ATP (CST) and 11.5 L D2O. The resulting mixture was left
for reaction at 37 ꢁC for 1 h, then added 50
L stop buffer (50 mM
EDTA, pH 8, CST) to stop the reaction. Next, 25 L/well of each
enzymatic reaction mixture and 75 L/well of D2O were transferred
to to a 96-well streptavidin-coated plate and incubated at 37 ꢁC for
1 h. Following three times of washing with PBS/T, 100 L of primary
mL) of solutions (H2O/DMSO: 99%/1%, v/v)
CD3COCD3-d6)
d
(ppm): 9.03 (s, 1H), 8.55 (s, 1H), 8.24 (m, 1H), 7.78
a
screened inhibitor
(m, 1H), 7.71 (s, 1H), 7.29 (t, 1H), 7.23 (s, 1H), 4.30 (t, 2H), 4.01 (s,
3H).3.66 (t, 2H). Anal. calcd (%) for C17H15ClFN4O2: C, 56.28; H, 4.45;
N, 15.44; found: C, 55.82; H, 4.43; N, 15.18.
m
m
L
m
4.1.18. 2-(4-(3-Chloro-4-fluorophenylamino)-7-
m
m
methoxyquinazolin-6-yloxy)ethanol (18)
m
Yield 61%, m.p. 268e270 ꢁC. ESI-MS (m/z) calcd for
C17H14ClFN3O3 363.1, found 364.1 [M þ H]þ. 1H NMR (400 MHz,
m
m
MeOD-d4)
d (ppm): 8.60 (s, 1H), 8.15 (dd, 1H), 7.90 (s, 1H), 7.82(dd,
1H), 7.41 (t,1H), 7.35 (s,1H), 4.42 (t, 2H), 4.14 (m, 5H). Anal. calcd (%)
for C17H14ClFN3O3: C, 56.13; H, 4.16; N, 11.55; found: C, 55.99; H,
4.17; N, 11.46.
m
antibody (Phospho-Tyrosine Mouse mAb,1:1000 in PBS/T with 1.5%
BSA) was added to each well and the plate was incubated at 37 ꢁC
for another 1 h. The plate was again washed three times with PBS/T,
4.1.19. 6-(2-(2-Aminoethylamino)ethoxy)-4-(3’-chloro-4’-
fluoroanilino)-7-methoxy- quinazoline (19)
and then 100 mL of secondary antibody (HRP-labeled Goat Anti-
Mouse lgG, 1:1000 in PBS/T with 1.5% BSA) was added to each
Yield 66%, m.p. 145e147 ꢁC. ESI-MS (m/z) calcd for
C19H21ClFN5O2 405.9, found 406.9 [M þ H]þ. 1H NMR(400 MHz,
well for 1 h of incubation at 37 ꢁC, followed by three times of
washing with PBS/T. Finally, 100
added to each well and the plate was incubated at 37 ꢁC for 15 min,
and then the reaction was stopped by addition of 100 L of 1 M
mL of TMB substrate system was
DMSO-d6)
d (ppm): 8.50(s, 1H), 8.14(s, 1H), 7.85(s, 1H), 7.81(s, 1H),
7.45(m, 1H), 7.21(s, 1H), 4.19(s, 2H), 3.94(s, 3H), 3.01(m, 3H), 2.64(s,
m
3H). 13C NMR (600 MHz, DMSO-d6)
d
(ppm): 156.44, 154.92, 154.38,
H2SO4, and the plate was read on the ELISA plate reader at 450 nm
and 650 nm (SpectraMax M5 Molecular Devices Corporation).
153.05, 148.74, 147.43, 137.28, 123.85, 122.71, 119.14, 116.98, 116.84,
109.22, 107.75, 103.10, 69.44, 56.34, 56.28, 49.49, 48.45. Anal. calcd
(%) for C19H21ClFN5O2$HCl: C, 51.59; H, 5.01; N, 15.83; found: C,
51.82; H, 5.05; N, 15.18.
4.4. Docking analysis
The docking analysis was performed using the Surflex-Dock
module, a fully automatic docking tool available on Sybyl X 1.1
version (Tripos Inc.), running on Dual-core Intel(R) E5300 CPU
2.60 GHz, RAM Memory 2 GB under the Windows XP system. The
crystal structure of the EGFR-erlotinib complex was collected from
PDB under code 1M17 [17]. All the hydrogen atoms were added to
define the correct configuration and tautomeric states. Then the
modeled structure was energy-minimized using AMBER7F99 force
4.1.20. 6-(3-(2-aminoethylamino)ethoxy)-4-(3’-chloro-4’-
fluoroanilino)-7-methoxyquinazoline (20)
Yield 70%, m.p. 120e123 ꢁC. ESI-MS (m/z) calcd for
C20H23ClFN5O2 419.9, found 420.8 [M þ H]þ. 1H NMR (400 MHz,
DMSO-d6)
d (ppm): 8.51(s, 1H), 8.15(dd, 1H), 7.87 (s, 1H), 7.83(m,
1H), 7.45(t, 1H), 7.22(s, 1H), 4.23(s, 3H), 3.94 (m, 4H), 2.82(m, 2H),
2.74(m, 6H), 1.99(m, 2H). 13C NMR (600 MHz, CD3OD)
d
(ppm):

94
S. Lü et al. / European Journal of Medicinal Chemistry 61 (2013) 84e94
[16] G. Klebe, Virtual ligand screening: strategies, perspectives and limitations,
Drug Discov. Today 11 (2006) 580e594.
[17] J. Stamos, M.X. Sliwkowski, C. Eigenbrot, Structure of the epidermal growth
field with distance dependent dielectric function and current
charges. The Powell energy minimization algorithm was used for
the energy minimization. After extracting the binding ligand ero-
lotinib, the structure corresponding to the constringent energy
gradient (0.05 kcal molꢂ1) was used for re-docking and scoring
calculations of erlotinib to check the accuracy of the Surflex-Dock
program. The 4-anilinoquinazoline-type analogs were then sepa-
rately docked into the binding pocket for docking-scoring analysis.
factor receptor kinase domain alone and in complex with
a 4-
anilinoquinazoline inhibitor, J. Biol. Chem. 277 (2002) 46265e46272.
[18] C.H. Yun, T.J. Boggon, Y.Q. Li, M.S. Woo, H. Greulich, M. Meyerson, M.J. Eck,
Structures of lung cancer-derived EGFR mutants and inhibitor complexes:
mechanism of activation and insights into differential inhibitor sensitivity,
Cancer Cell 11 (2007) 217e227.
[19] V. Aparna, G. Rambabu, S.K. Panigrahi, J. Sarma, G.R. Desiraju, Virtual
screening of 4-anilinoquinazoline analogues as EGFR kinase inhibitors:
importance of hydrogen bonds in the evaluation of poses and scoring func-
tions, J. Chem. Inf. Model. 45 (2005) 725e738.
[20] H. Assefa, S. Kamath, J.K. Buolamwini, 3D-QSAR and docking studies on 4-
anilinoquinazoline and 4-anilinoquinoline epidermal growth factor receptor
(EGFR) tyrosine kinase inhibitors, J. Comput. Aided Mol. Des. 17 (2003) 475e493.
[21] C. Bissantz, G. Folkers, D. Rognan, Protein-based virtual screening of chemical
databases. 1. Evaluation of different docking/scoring combinations, J. Med.
Chem. 43 (2000) 4759e4767.
Acknowledgement
We thank NSFC (Grant nos. 90713020, 20975103, 21020102039
and 21005081) and the Chinese Academy of Sciences (Hundred
Talent Program) for support.
[22] R. Banerjee, Y. Huang, J.P. McNamee, M. Todorova, B.J. Jean-Claude, The
combi-targeting concept: selective targeting of the epidermal growth factor
receptor- and her2-expressing cancer cells by the complex combi-molecule
rb24, J. Pharmacol. Exp. Ther. 334 (2010) 9e20.
[23] R. Banerjee, Y. Huang, Q.Y. Qiu, J.P. Mcnamee, G. Belinsky, B.J. Jean-Claude, The
combi-targeting concept: mechanism of action of the pleiotropic combi-
molecule RB24 and discovery of a novel cell signaling-based combination
principle, Cell. Signal. 23 (2011) 630e640.
[24] J. Domarkas, F. Dudouit, C. Williams, Q.Y. Qiu, R. Banerjee, F. Brahimi, B.J. Jean-
Claude, The combi-targeting concept: synthesis of stable nitrosoureas
designed to inhibit the epidermal growth factor receptor (EGFR), J. Med.
Chem. 49 (2006) 3544e3552.
[25] Z. Rachid, F. Brahimi, A. Katsoulas, N. Teoh, B.J. Jean-Claude, The combi-
targeting concept: chemical dissection of the dual targeting properties of
a series of “combi-triazenes”, J. Med. Chem. 46 (2003) 4313e4321.
[26] L.Y. Ji, W. Zheng, S. Lv, Q. Luo, F.Y. Wang, unpublished.
Appendix A. Supplementary material
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.ejmech.2012.07.036
References
[1] D.K. Moscatello, M. Holgadomadruga, A.K. Godwin, G. Ramirez, G. Gunn,
P.W. Zoltick, J.A. Biegel, R.L. Hayes, A.J. Wong, Frequent expression of a mutant
epidermal growth-factor receptor in multiple human tumors, Cancer Res. 55
(1995) 5536e5539.
[2] D.S. Salomon, R. Brandt, F. Ciardiello, N. Normanno, Epidermal growth factor-
related peptides and their receptors in human malignancies, Crit. Rev. Oncol./
Hematol. 19 (1995) 183e232.
[3] H.T. Zhang, A. Berezov, Q. Wang, G. Zhang, J. Drebin, R. Murali, M.I. Greene,
ErbB receptors: from oncogenes to targeted cancer therapies, J. Clin. Invest.
117 (2007) 2051e2058.
[27] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat.
Rev. Drug Discov. 4 (2005) 307e320.
[28] P.C.A. Bruijnincx, P.J. Sadler, New trends for metal complexes with anticancer
activity, Curr. Opin. Chem. Biol. 12 (2008) 197e206.
[29] P.C.A. Bruijnincx, P.J. Sadler, Controlling platinum, ruthenium, and osmium
reactivity for anticancer drug design, advances in inorganic chemistry, Metal
Ion Controll. React. 61 (2009) 1e62.
[4] P. Cohen, Protein kinases e the major drug targets of the twenty-first century?
Nat. Rev. Drug Discov. 1 (2002) 309e315.
[5] D.W. Fry, A.J. Bridges, W.A. Denny, A. Doherty, K.D. Greis, J.L. Hicks, K.E. Hook,
P.R. Keller, W.R. Leopold, J.A. Loo, D.J. McNamara, J.M. Nelson, V. Sherwood,
J.B. Smaill, S. Trumpp-Kallmeyer, E.M. Dobrusin, Specific, irreversible inacti-
vation of the epidermal growth factor receptor and erbB2, by a new class of
tyrosine kinase inhibitor, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 12022e12027.
[6] D.W. Fry, A.J. Kraker, A. McMichael, L.A. Ambroso, J.M. Nelson, W.R. Leopold,
R.W. Conners, A.J. Bridges, A specific inhibitor of the epidermal growth-factor
receptor tyrosine kinase, Science 265 (1994) 1093e1095.
[7] M. Kris, M. Ranson, D. Ferry, L. Hammond, S. Averbuch, J. Ochs, E. Rowinsky,
Phase I study of oral ZD1839 (Iressa (TM)), a novel inhibitor of epidermal
growth factor receptor tyrosine kinase (EGFR-TK): evidence of good tolera-
bility and activity, Clin. Cancer Res. 5 (1999) 99.
[30] A.J. Barker, K.H. Gibson, W. Grundy, A.A. Godfrey, J.J. Barlow, M.P. Healy,
J.R. Woodburn, S.E. Ashton, B.J. Curry, L. Scarlett, L. Henthorn, L. Richards,
Studies leading to the identification of ZD1839 (Iressa (TM)): an orally active,
selective epidermal growth factor receptor tyrosine kinase inhibitor targeted
to the treatment of cancer, Biorg. Med. Chem. Lett. 11 (2001) 1911e1914.
[31] G.W. Rewcastle, W.A. Denny, A.J. Bridges, H.R. Zhou, D.R. Cody, A. McMichael,
D.W. Fry, Tyrosine kinase inhibitors. 5. Synthesis and structureeactivity-
relationships for 4-(phenylmethyl)amino-quinazolines and 4-(phenylamino)
quinazolines as potent adenosine 5’-triphosphate binding-site inhibitors of
the tyrosine kinase domain of the epidermal growth-factor receptor, J. Med.
Chem. 38 (1995) 3482e3487.
[32] W.H.J. Ward, P.N. Cook, A.M. Slater, D.H. Davies, G.A. Holdgate, L.R. Green,
Epidermal growth factor receptor tyrosine kinase: investigation of catalytic
mechanism, structure-based searching and discovery of a potent inhibitor,
Biochem. Pharmacol. 48 (1994) 659e666.
[33] A.M. Thompson, A.J. Bridges, D.W. Fry, A.J. Kraker, W.A. Denny, Tyrosine
Kinase Inhibitors. 7. 7-Amino-4-(phenylamino)pyrido[4,3-D]pyrimidines and
7-amino-4-[(phenylmethyl)amino]pyrido[4,3-D]pyrimidines e a new class of
inhibitors of the tyrosine kinase-activity of the epidermal growth-factor
receptor, J. Med. Chem. 38 (1995) 3780e3788.
[34] K.H. Gibson, W. Grundy, A.A. Godfrey, J.R. Woodburn, S.E. Ashton, B.J. Curry,
L. Scarlett, A.J. Barker, D.S. Brown, Epidermal growth factor receptor tyrosine
kinase: structure-activity relationships and antitumour activity of novel qui-
nazolines, Biorg. Med. Chem. Lett. 7 (1997) 2723e2728.
[8] A.J. Bridges, The rationale and strategy used to develop a series of highly
potent, irreversible, inhibitors of the epidermal growth factor receptor family
of tyrosine kinases, Curr. Med. Chem. 6 (1999) 825e843.
[9] J.D. Moyer, E.G. Barbacci, K.K. Iwata, L. Arnold, B. Boman, A. Cunningham,
C. DiOrio, J. Doty, M.J. Morin, M.P. Moyer, M. Neveu, V.A. Pollack, L.R. Pustilnik,
M.M. Reynolds, D. Sloan, A. Theleman, P. Miller, Induction of apoptosis and cell
cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor
tyrosine kinase, Cancer Res. 57 (1997) 4838e4848.
[10] A.R. de Boer, H. Lingeman, W.M.A. Niessen, H. Irth, Mass spectrometry-based
biochemical assays for enzymeinhibitor screening, TrAC, Trends Anal. Chem.
26 (2007) 867e883.
[11] L.A.A. de Jong, D.R.A. Uges, J.P. Franke, R. Bischoff, Receptor-ligand binding
assays: technologies and applications, J. Chromatogr. B 829 (2005) 1e25.
[12] I.A. Hemmila, P. Hurskainen, Novel detection strategies for drug discovery,
Drug Discov. Today 7 (2002) S150eS156.
[35] W. Zheng, Q. Luo, S. Lv, F. Wang, unpublished.
[36] E.R. Wood, A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson,
B. Ellis, C. Pennisi, E. Horne, K. Lackey, K.J. Alligood, D.W. Rusnak, T.M. Gilmer,
L. Shewchuk, A unique structure for epidermal growth factor receptor bound to
GW572016 (Lapatinib): relationships among protein conformation, inhibitor
off-rate, and receptor activity in tumor cells, Cancer Res. 64 (2004) 6652e6659.
[37] J.B. Wright, The chemistry of the benzimidazoles, Chem. Rev. 48 (1951)
397e541.
[13] C. Hempen, U. Karst, Labeling strategies for bioassays, Anal. Bioanal. Chem.
384 (2006) 572e583.
[14] K. Farley, H. Mett, E. McGlynn, B. Murray, N.B. Lydon, Development of solid-
phase enzyme-linked immunosorbent assays for the determination of
epidermal growth factor receptor and pp60c-src tyrosine protein kinase
activity, Anal. Biochem. 203 (1992) 151e157.
[38] L. Shewchuk, A. Hassell, B. Wisely, W. Rocque, W. Holmes, J. Veal, L.F. Kuyper,
Binding mode of the 4-anilinoquinazoline class of protein kinase inhibitor: X-
ray crystallographic studies of 4-anilinoquinazolines bound to cyclin-
dependent kinase 2 and p38 kinase, J. Med. Chem. 43 (2000) 133e138.
[15] R.W. Stephens, A.N. Pedersen, H.J. Nielsen, M. Hamers, G. HoyerHansen,
E. Ronne, E. Dybkjaer, K. Dano, N. Brunner, ELISA determination of soluble
urokinase receptor in blood from healthy donors and cancer patients, Clin.
Chem. 43 (1997) 1868e1876.