Synthesis and biological evaluation of 4-[3-chloro-4-(3-fluorobenzyloxy) anilino]-6-(3-substituted-phenoxy)pyrimidines as dual EGFR/ErbB-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.11.056

A series of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted- phenoxy)pyrimidine derivatives were elaborately designed based on the skeleton of Lapatinib, and evaluated for their potential to inhibit epidermal growth factor receptor (EGFR) and E

Full text of DOI:10.1016/j.bmc.2011.11.056

Bioorganic & Medicinal Chemistry 20 (2012) 877–885
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4-[3-chloro-4-(3-fluorobenzyloxy)
anilino]-6-(3-substituted-phenoxy)pyrimidines as dual EGFR/ErbB-2
kinase inhibitors
Siyuan Li a, Chunying Guo a, Hongli Zhao a, Yun Tang c, Minbo Lan a,b,
⇑
a Shanghai Key Laboratory of Functional Materials Chemistry, Research Centre of Analysis and Test, East China University of Science and Technology, Shanghai 200237, PR China
b State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
c Department of Pharmaceutical Science, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-phenoxy)pyrimidine derivatives were
elaborately designed based on the skeleton of Lapatinib, and evaluated for their potential to inhibit
epidermal growth factor receptor (EGFR) and ErbB-2 tyrosine kinase activities and antiproliferative activities
against A431 and SKOV-3 cell lines. Among these synthesized pyrimidine derivatives, 4-[3-chloro-4-(3-fluorob
enzyloxy)anilino]-6-(3-acrylamidophenoxy)pyrimidine (6), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-
cyanoacetamidophenoxy)pyrimidine (9), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-{3-[6-(4-amino)pyri
midinyl]amino) phenoxy}pyrimidine (11) and 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-phenoxyace-
tamidophenoxy)pyrimidine (14) could significantly inhibit dual EGFR/ErbB-2 kinase activities (IC50 = 37/
29 nM, 48/38 nM, 61/42 nM, 65/79 nM, respectively). And compounds 6 and 11 also showed the most potent
Received 23 October 2011
Revised 25 November 2011
Accepted 25 November 2011
Available online 3 December 2011
Keywords:
Pyrimidine
EGFR
ErbB-2
Inhibitor
Docking
antiproliferative activities in vitro, with the IC50 value of 6 being 3.25 lM for A431 and 0.89 lM for SKOV-3, as
for 11, 4.24
lM for A431 and 0.71
lM for SKOV-3, respectively. Docking study was also performed to determine
the possible binding model.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
It is known from the crystal structure of the Lapatinib–EGFR
complex14 and previous structure–activity relationship (SAR) of
4-anilinoquinazolines,5,6,15–18 the binding mode is that Lapatinib
places the aniline portion deep in the ATP binding site of the EGFR
kinase, and this can affect kinase selectivity of ErbB family. An
hydrogen bond is formed between N1 of the quinazoline and the
main chain NH of Met 769, and another water-mediated one is
formed between N3 and the side chain of Thr830.14 Therefore,
the 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]pyrimidine is the
most important portion between the interaction of Lapatinib and
EGFR. Considering that we split the anilinoquinazoline into two
pieces including a pyrimidine portion and a benzene ring, and
The human epidermal growth factor receptor (EGFR) is a trans-
membrane glycoprotein consisting of a single polypeptide chain of
1186 amino acids.1,2 This receptor belongs to the ErbB/HER family
of ligand-activated RTKs, which is catalytically active and under
tight regulatory control.3 EGF receptors play a key role in cell
development, proliferation and differentiation within a variety of
tissues.4 Deregulation of their activity is extremely associated with
tumorigenesis, such as lung cancer, breast cancer, ovarian cancer
and colorectal cancer et al. Therefore EGFR is a key target for
anti-tumor strategy.
In the past few years, a few kinds of compounds differing in
structure, such as 4-anilinoquinazolines,5,6 4-anilinoquinoline-
3-carbonitriles,7 1H-pyrazolo[3,4-d]pyrimidines,8 pyrrolotria-
zines9,10 and 4-amino-6-arylaminopyrimidines,11,12 were reported
as EGFR tyrosine kinase inhibitors. 4-Anilinoquinazoline, espe-
cially, was an excellent core to be modified as EGFR inhibitors,
and some of these have been approved by FDA. Lapatinib, for
example, is a potent dual EGFR/ErbB2 inhibitor approved for
treatment of breast cancer (Fig. 1).13
O
F
Cl
HN
H
N
S
N
O
O
O
N
4-aniline pyrimidine
⇑
Corresponding author. Tel.: +86 21 64253574; fax: +86 21 64252947.
E-mail address: minbolan@ecust.edu.cn (M. Lan).
Figure 1. Dual EGFR/ErbB-2 inhibitors: Lapatinib.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.056

878
S. Li et al. / Bioorg. Med. Chem. 20 (2012) 877–885
O
F
F
O
Cl
HN
N
R
Cl
HN
N
R
N
4-aniline pyrimidine
O
4
N
O
Figure 2. 4,6-Disubstituted pyrimidines.
5-19
Method A or B
insert an ether bond as a joint (Fig. 2). We believe these designed
pyrimidine compounds might be potent to inhibit EGFR/ErbB-2
kinase activities as Lapatinib.
Herein, we disclose the synthesis of a series of 4,6-disubstituted
pyrimidine compounds possessing 4-aniline pyrimidine portion
together with their biological activities in vitro.
6-7, 9-10, 13-19
Method C
Method D
8, 11-12
5
2. Chemistry
Scheme 2. Reagents and conditions: Method A: acyl chlorides, THF, rt, 80–95%.
Method B: carboxylic acid, HOBt, EDC, THF, rt, 60–90%. Method C: halogenated
compounds, n-BuOH, 110 °C, 52–90%. Method D: anhydride acetate, 80 °C, 95%.
The synthetic route to 4-chloro-6-(3-nitrophenoxy)pyrimidine
2 began with a 4,6-dichloropyrimidine 1 and sodium m-nitrophen-
olate at room temperature in the presence of DMF (Scheme 1).
Alternatively, m-nitrophenol was used for this reaction in the n-
BuOH at reflux instead of sodium m-nitrophenolate, but the reac-
tion of sodium m-nitrophenolate could give the highly quantitative
product 2. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-nitro-
phenoxy)pyrimidine 3 was produced via a SNAr reaction of 2 with
3-chloro-4-(3-fluorobenzyloxy)aniline. Selective reduction of the
nitro group versus the aromatic halogen was accomplished by uti-
lizing a system of iron powder and acetic acid to generate the de-
sired intermediate product 4 with considerable yield.
condensing 4 with cyanoacetic acid using EDC and HOBt condensa-
tion system in THF. To synthesize the molecules 8, 11 and 12, cor-
responding halogenated compounds were used in the process of
preparation by method C. Compound 12, it was created via a nucle-
ophilic substitution reaction using 4 and bromomethylbenzene as
reactants in the solvent of n-BuOH. Intermediate product 4 was
heated with anhydride acetate at 80 °C to yield the product 5.
3. Results and discussion
The target molecules 5–19 were synthesized using intermediate
product 4 by different methods (Scheme 2). Regarding molecules
6–7, 9–10 and 13–19, they were synthesized according to reacting
with different acyl chlorides and same intermediate product 4 by
method A or condensing 4 and carboxylic acid by method B. For
examples, molecule 6 was prepared by acylation of 4 using acryloyl
chloride with yield of 87%, and molecule 9 was obtained by
3.1. Assay for in vitro activity
In pursuit of our goal to determine dual EGFR/ErbB-2 activities
in the 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-
phenoxy)pyrimidine series, we evaluated the molecules 3–19 for
EGFR/ErbB-2 inhibitory potency using homogeneous time-resolved
F
O
Cl
Cl
HN
Cl
N
NO2
a
b
NO2
N
N
N
N
Cl
N
O
O
1
2
3
F
O
Cl
HN
c
NH2
N
N
O
4
Scheme 1. Reagents and conditions: (a) sodium m-nitrophenolate, DMF, rt; (b) ArNH2, n-BuOH, 110 °C; (c) Fe, CH3COOH, ethanol, rf.

S. Li et al. / Bioorg. Med. Chem. 20 (2012) 877–885
879
Table 1
In vitro evaluation of compound 3–19
Compound
R
EGFRa (nM)
ErbB-2a (nM)
A431b
(l
M)
SKOV-3b
(lM)
3
4
5
–NO2
–NH2
–NHCOCH3
1204
473
108
2421
559
135
10.43
8.92
12.02
>30
12.21
3.95
H
N
6
7
37
29
3.25
7.00
0.89
1.29
O
H
O
(E)
N
129
196
O
O
O
H
N
8
9
370
48
219
38
6.23
5.17
9.18
8.42
O
H
N
CN
O
O
NO2
H
N
10
>10,000
>10,000
6.72
10.21
N
N
11
12
13
61
42
4.24
3.11
11.24
0.71
NH2
N
H
H
>10,000
1671
>10,000
3740
17.91
20.71
N
H
N
(E)
O
H
N
14
15
65
79
14.91
9.29
9.10
O
O
O
F
F
H
N
627
834
26.10
F
F
H
N
(E)
(E)
16
17
821
523
1293
1234
18.12
>20
>30
>30
O
O
O
O
H
N
F
F
F
H
N
18
721
2138
13.25
>30
O
O
O
O
O
O
H
N
19
>10,000
9.0
>10,000
10.3
6.42
2.62
.02
Lapatinib
2.99
a
The kinase inhibitory activities were determined using HTRF assay with compounds concentration 0.01–10,000 nM. The values are the average of at least two inde-
pendent experiments performed in duplicate.
b
The cell proliferation in A431 and SKOV-3 cells were determined by the MTT assay, after 72 h of incubation with compounds (0.05–30 lM). The values are the average of
at least two independent experiments performed in duplicate.

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S. Li et al. / Bioorg. Med. Chem. 20 (2012) 877–885
fluorescence (HTRF) KinEASE-TK assay19 from Cisbio according to
manufacture’s instruction. Lapatinib, with its high potency to EGFR
(9.0 nM) and ErbB-2 (10.3 nM), was chosen as a standard com-
pound in our kinase assay.
9, 11 and 14 into EGFR using Glide 5.5.22 Crystal structure of EGFR
was downloaded from PDB database (PDB ID: 1XKK).14
Figure 3A demonstrates compound 6 was docking into ATP
binding site of EGFR kinase. In the binding model, compound 6
was nicely bound to the ATP catalytic domain of the EGFR and
formed two hydrogen bonds with the EGFR. One was formed be-
tween N1 of the 6 and main chain NH of Met793, and the distance
was 2.431 Å while the angle value was 164.1°; another was formed
between N3 and side chain OH of Thr854 through a water-medi-
ated hydrogen bond. And an intermolecular hydrogen bond formed
in the binding pose of 6 between NH of aniline and C@O of acryl-
amide group. The 3-chloro-4-(3-fluorobenzyloxy)aniline group
was oriented deep in the back of the ATP binding site and made
predominantly hydrophobic interactions with the protein. The
pyrimidine ring was sandwiched from the top and bottom by the
side chains of Ala743 and Leu844, respectively. And the phenoxy
group of 6 was also at hydrophobic pocket and made slightly weak
hydrophobic interactions with the residue of Leu718 and Leu844.
The acrylamide group was located nearby the Arg841 and
Asn842, hence electrostatic interactions formed. Thus this pose of
6 is much reasonable and 6 is the most potent in our synthesized
compounds.
Compound 9 was also bound to EGFR commendably and the
binding pose is similar to 6 (Fig. 3B). A hydrogen bond also formed
between N1 of 9 and Met793, and the distance was 2.491 Å, the an-
gle value 169.3°. N3 of 9 also interacted with a water molecule by a
hydrogen bond. Cyanoacetamide chain of 9 oriented to a negative
electrical area consisting of Val717, Leu718, Leu1001 and Met1002,
though the distance to cyanoacetamide was relatively long
(>3.7 Å), a very weak electrostatic repulsion remained between cy-
ano group and these residues. Because of this position and orienta-
tion of cyanoacetamide in the pose, compound 9 shows a little
weak kinase inhibitory activities in contrasted to 6.
As summarized in Table 1, the various substituted groups on
3-phenoxy in the pyrimidine core, obviously, play an important
role in kinase inhibitory activities. Amino group substituted on 3-
phenoxy is better for kinase inhibitory activities than nitro group
(IC50: 473 vs 1204 nM for EGFR and 559 vs 2421 nM for ErbB-2),
which means that electron donating groups is more potent than
electron withdrawing groups on 3-phenoxy. 3-Phenoxy modified
with kinds of side chains may improve potency, and although all
of the synthesized compounds’ kinase inhibitory activities are low-
er than Lapatinib, the compounds 6, 9, 11 and 14 hold the most po-
tency for EGFR/ErbB-2. Among them compound 6, gained by
substitution with acrylamide, is the most potent one for EGFR
(37 nM) and ErbB-2 (29 nM) than any other substituted type.
Inhibitory activity of compound 9 (IC50: 48 nM for EGFR; 38 nM
for ErbB-2) is close to 6, while compounds 5, 7 and 8 are inferior
to 6 with inhibitory activities at hundred nanomole order of mag-
nitude. For the substituted types of compounds 3–9, acrylamide
and cyanoacetamide (6 and 9) are the most favorable groups with
higher potency.
Compounds 11 and 14, substituted with 4-aminopyrimidine or
benzyloxy acetamide, are another series bearing a little bigger
steric hindrance on 3-phenoxy with good inhibitory activities
(IC50: EGFR: 61 nM, ErbB-2: 42 nM for 11 and EGFR: 65 nM,
ErbB-2: 79 nM for 14). Other compounds have lower kinase inhib-
itory activities, even no activities among which 10, 12 and 19
(IC50 >10,000 nM).
From Table 1, it is natural to come to the conclusion that small
steric hindrance aliphatic chains are more helpful improving activ-
ities than bigger steric hindrance aromatic ring substituted chains.
These aliphatic chains are probably more flexible for possessing
the more and the better position selectivity when binding in the
ATP binding domain of EGFR. Thus aliphatic chains substituted
molecules such as 5–9 had the EGFR/ErbB-2 inhibitory activities
more or less, while some of the compounds with aromatic ring
substituted chains did not show any kinase inhibitory activities.
All of these molecules were also tested in cell proliferation assays
by MTT method20 using A431 and SKOV-3 cell lines which is overex-
press EGFR and ErbB-2, respectively.21 In cell assay, the IC50 values
The binding model of compounds 11 and 14 was disclosed in
Figure 3C and D, and the model was analogous to 6 and 9. In the
model of 11 and 14, two similar hydrogen bonds were also formed
same as models of compounds 6 and 9. However, for 11, a hydro-
gen bond emerged between NH2 of 11 and C@O of Ser720 and the
distance was 2.063 Å. The extra hydrogen bond was essential for
11 which was much more potent than other aromatic chains
substituted compounds. Around the peripheral domain of the
ATP binding site there was a negative electrical area consisting of
residues of Asn842(C@O), Arg841(C@O) and Asp855(COOÀ) which
of Lapatinib were 2.62 and 2.99 lM for A431 and SKOV-3 cancer cell
lines, respectively. Among compounds 6, 9, 11 and 14, 9 and 14
showed minimal potency in antiproliferation assays in cancer cell
lines even though it had reasonable levels of target potency at the
enzyme level while compounds 6 and 11 showed approving po-
tency contrasted to 9 and 14. In our assay, treatment of A431 cells
with compounds 6 and 11 resulted in decreased cell growth rate
and their IC50 values were 3.25 and 4.24 lM which were closed to
IC50 value of Lapatinib. Notably, compounds 6 and 11 inhibited
SKOV-3 cell lines intensively with IC50 values being 0.89 and
0.71 lM, which resulted in 3.4- and 4.2-fold enhancement of cellu-
generated electrostatic repulsion effect to p-electron of amino
pyrimidine ring resulting in pushing the pyrimidine ring leaning
on the side of Ser720 and Gly721. Although the pose of 11 is much
reasonable, the electrostatic repulsion effect is always exists to
some extent while EGFR/ErbB-2 inhibitory activities of compound
11 is little lower than 6 and 9. For compound 14, though the aro-
matic ring chain is longer and more flexible than 11, enough to
make the benzene ring rotate to avoid the electrostatic repulsion
and it is well complementary with van der waals surface of EGFR,
the aromatic ring chain doesn’t pick up any residue forming hydro-
gen bond which is more important for EGFR binding in these aro-
matic ring chain substituted compounds and the IC50 values of 14
is little higher than compound 11. In addition, substitution with
large negative electrical group in the aromatic ring such as -F re-
sults in decreasing the activities, because some negative electrical
areas are distributed around the peripheral domain of active pock-
et, for instance an area comprising residues of Asn842(C@O),
Arg841(C@O) and Asp855(COOÀ) mentioned above, a second area
consisting of residues of Leu718(C@O) and Val717(C@O), the third
area consisting of residues of Ala1000(C@O), Leu1001(C@O) and
Met1002(C@O, S) as well as the area consisting of residue of As-
p800(COOÀ), which repulse the negative electrical substituted
groups that is harmful to kinase inhibitory effect.
lar activities in contrasted to Lapatinib.
Interestingly, compound 12 had a favorable activity on A431
cell lines (IC50=3.11
lM) and 5, 7 were potent to SKOV-3 cell lines
(IC50=3.95 M for 5; 1.29
l
lM for 7). Cell activities of these com-
pounds were inconsistent with their kinase assay data possibly be-
cause 5, 7 and 12 might inhibit other key proteins involved in the
A431 and SKOV-3 cells proliferation, especially, for compound 12
which was ineffective to EGFR/ErbB-2 at all.
3.2. Docking study
To rationalize our design and our biological activity assay, dock-
ing study was carried out for docking the bioactive compounds 6,

S. Li et al. / Bioorg. Med. Chem. 20 (2012) 877–885
881
Figure 3. Binding models of compound 6, 9, 11 and 14 with EGFR corresponding to the left top sign A, B, C and D, respectively. The carbon in EGFR was colored in green, ligand
carbons were colored in brown. Hydrogen bond was represented with yellow dashed line.
activity of this series and the various substitutions on 3-phenoxy
result in different activities.
Our docking study shows that compounds 6, 9, 11 and 14 pos-
sess rational poses in binding with EGFR. Besides, these structural
details gained from the binding models provide us useful informa-
tion to elaborate the better potent inhibitors.
4. Conclusion
In this paper, a series of pyrimidine derivatives 3–19 based on
the structure feature of Lapatinib has been synthesized and evalu-
ated for their biological activities in vitro. Compounds 6, 9, 11 and
14 showed the most potent EGFR/ErbB-2 inhibition activities
(IC50 = 37/29 nM, 48/38 nM, 61/42 nM, 65/79 nM, respectively).
And the 6 and 11 displayed the excellent antiproliferative activities
in vitro, with an IC50 value of 3.25
lM for A431 and 0.89
lM for
SKOV-3 (6) while 4.24 M for A431 and 0.71
l
lM for SKOV-3
(11). Docking study was performed to determine the possible bind-
ing model. From our model, it is indicated that the hydrogen bond
interaction with residues Met793 and Thr854 of the protein in the
ATP binding domain plays a key role in inhibition of EGFR/ErbB-2
activities. And the docking poses of compounds 6, 9, 11 and 14
were similar to Lapatinib, which proved our initial design. From
these synthesized compounds, it is concluded that the 6 and 11,
as potential anticancer agent, showed the best dual EGFR/ErbB-2
and cancer cell proliferative inhibitory activities. This work might
be helpful in further structure elaboration targeting more potent
dual EGFR/ErbB-2 inhibitors.
Figure 4. Overlay of compounds 6 (cyan), 9 (yellow), 11 (brown) and 14 (dark
green) with Lapatinib (white).
The overlay of docking pose as shown in Figure 4 indicates the
compounds 6, 9, 11 and 14 place the 4-[3-chloro-4-(3-fluorobenzyl-
oxy)anilino]-6-(3-phenoxy) pyrimidine skeleton into a similar
orientation as Lapatinib while some differences in substitution on
3-phenoxy. This suggests 4-[3-chloro-4-(3-fluorobenzyloxy)anili-
no] pyrimidine portion is the key skeleton for EGFR kinase inhibitory

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S. Li et al. / Bioorg. Med. Chem. 20 (2012) 877–885
J = 2.4 Hz), d 8.36(s, 1H), d 8.52(s, 1H); MS (EI) m/e 436.1 (M+); Anal.
Calcd for C23H18ClFN4O2: C, 63.23; H, 4.15; N, 12.82. Found: C,
63.44; H, 4.32; N, 12.60.
5. Experimental
5.1. Chemistry
5.1.4. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-acetam
idophenoxy)pyrimidine (5)
Melting points were determined in open capillary tubes on a
SGW X-4 micro melting point apparatus and were uncorrected.
Electron impact mass spectra were obtained on a Micromass GCT
spectrometer. 1H NMR spectra were measured on Brucker
400 MHz in d scale. The spectra were obtained with solutions of
CDCl3 or DMSO-d6 with TMS as internal standard, and the values
of the chemical shifts (d) were given in ppm, coupling constants
(J) given in Hz. Elemental analyses were carried out at VARIO
ELI apparatus and were within 0.4%.
Method D. A mixture of 4 (200 mg, 0.46 mmol) and acetic anhy-
dride (6 mL) was heated at 80 °C for 1 h. The acetic anhydride was
evaporated and the residue was dissolved in EtOAc (50 mL) and
then washed with saturated NaHCO3 aqueous solution
(3 Â 20 mL). The organic layer was dried over anhydrous MgSO4,
and the solvent was removed, purified by column chromatography
on silica gel eluted with EtOAc/petroleum ether (1:2) to give 5 as a
white solid (207 mg, 0.43 mmol, 95% yield). Mp: 165–167 °C; 1H
NMR (400 MHz, DMSO-d6) d 2.06(s, 3H), d 5.20(s, 2H), d 6.03(s,
1H), d 6.87(dt, 1H, J = 2.0 Hz, 8.0 Hz), d 7.17(s, 1H), d 7.20(s, 1H),
d 7.28–7.32(m, 2H), d 7.37–7.43(m, 3H), d 7.45–7.49(m, 1H), d
7.52(s, 1H), d 7.86(d, 1H, J = 2.4 Hz), d 8.36(s, 1H), d 9.57(s, 1H), d
10.11(s, 1H); MS (EI) m/e 478.1 (M+); Anal. Calcd for
5.1.1. 4-Chloro-6-(3-nitrophenoxy)pyrimidine (2)
A mixture of 4,6-dichloropyrimidine (1) (5.00 g, 33.55 mmol)
and sodium m-nitrophenolate (5.41 g, 33.55 mmol) in DMF
(20 mL) was stirred at ambient temperature under N2 protection
for 30 min. The mixture was diluted with 80 mL EtOAc and washed
with brine (3 Â 50 mL). The organic layer was dried over anhy-
drous MgSO4, the solvent was removed, and the residue was
washed with hexane (30 mL) to give 2 as a canary yellow solid
(8.02 g, 31.87 mmol, 95% yield). Mp: 45–47 °C; 1H NMR
(400 MHz, CDCl3) d 7.09(s, 1H), d 7.55(dd, 1H, J = 4.0 Hz, 8.0 Hz),
d 7.66(t, 1H, J = 8.0 Hz), d 8.06(t, 1H, J = 2.4 Hz), d 8.18(dd, 1H,
J = 4 Hz, 8.0 Hz), d 8.57(s, 1H); MS (EI) m/e 251.0 (M+); Anal. Calcd
for C10H6ClN3O3: C, 47.73; H, 2.40; N, 16.70. Found: C, 47.94; H,
2.56; N, 17.01.
C25H20ClFN4O3: C, 62.70; H, 4.21; N, 11.70. Found: C, 62.67; H,
3.94; N, 11.35.
5.1.5. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-acrylam
idophenoxy)pyrimidine (6)
Method A. A solution of THF (5 mL) containing acryloyl chloride
(45 mg, 0.50 mmol) was added to another solution of 4 (200 mg,
0.46 mmol) in anhydrous THF (10 mL) at 0 °C dropwise under N2
atmosphere and then the mixture was stirred at room temperature
for 30 min. After THF was removed under reduced pressure, the
residue was purified by column chromatography on silica gel
eluted with EtOAc/petroleum ether (1:1) to give 6 as a white solid
(196 mg, 0.40 mmol, 87% yield). Mp: 180–182 °C; 1H NMR
(400 MHz, DMSO-d6) d 5.20(s, 2H), d 5.79(dd, 1H, J = 2.0 Hz,
10.0 Hz), d 6.06(s, 1H), d 6.27(dd, 1H, J = 2.0 Hz, 16.0 Hz), d
6.43(q, 1H, J = 2.0 Hz), d 6.92(dd, 1H, J = 1.6 Hz, 8.0 Hz), d 7.19(d,
2H, J = 8.0 Hz), d 7.28–7.32(m, 2H), d 7.38–7.52(m, 4H), d 7.62(s,
1H), d 7.86(d, 1H, J = 2.4 Hz), d 8.37(s, 1H), d 8.57(s, 1H), d
10.32(s, 1H); MS (EI) m/e 490.1 (M+); Anal. Calcd for
5.1.2. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-nitro phe
noxy)pyrimidine (3)
A
mixture of 3-chloro-4-(3-fluorobenzyloxy)aniline (5.00 g,
19.86 mmol) and compound 2 (4.17 g, 16.55 mmol) with two
drops concd HCl in n-BuOH (30 mL) was heated at 110 °C and kept
stirring for 3 h. As the mixture cooled, the formed yellow solid was
filtered through washing with n-BuOH followed by saturated NaH-
CO3 aqueous solution. After being dried overnight at vacuum dry-
ing oven at 40 °C, the solid was purified by column
chromatography on silica gel eluted with EtOAc/petroleum ether
(1:2) to give 3 as a yellowish green solid (6.18 g, 13.24 mmol,
80% yield). Mp: 138–140 °C; 1H NMR (400 MHz, DMSO-d6) d
5.21(s, 2H), d 6.30(s, 1H), d 7.15–7.22(m, 2H), d 7.28–7.32(m, 2H),
d 7.44–7.49(m, 2H), d 7.70–7.77(m, 2H), d 7.88(d, 1H, J = 2.4 Hz),
d 8.07(t, 1H, J = 2.0 Hz, 2.4 Hz), d 8.14(dt, 1H, J = 2.0 Hz, 8.0 Hz), d
8.37(s, 1H), d 9.93(s, 1H); MS (EI) m/e 466.1 (M+); Anal. Calcd for
C26H20ClFN4O3: C, 63.61; H, 4.11; N, 11.41. Found: C, 63.82; H,
4.15; N, 11.10.
5.1.6. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-fumari
camidophenoxy)pyrimidine (7)
Compound 7 was prepared from 4 (200 mg, 0.46 mmol) and fu-
maric chloride (81 mg, 0.50 mmol) in THF (15 mL) by the proce-
C
23H16ClFN4O4: C, 59.17; H, 3.45; N, 12.00. Found: C, 59.07; H,
dure described for
6 to give 7 as a white solid (202 mg,
0.36 mmol, 78% yield). Mp: 145–146 °C; 1H NMR (400 MHz,
DMSO-d6) d 1.27(t, 3H, J = 7.2 Hz), d 4.22(q, 2H, J = 7.2 Hz), d
5.21(s, 2H), d 6.07(s, 1H), d 6.71(d, 1H, J = 16.0 Hz), d 6.96(dd, 1H,
J = 1.2 Hz, 8.0 Hz), d 7.15–7.22(m, 3H), d 7.28–7.32(m, 2H), d
7.38–7.42(m, 1H), d 7.44–7.51(m, 3H), d 7.63(t, 1H, J = 2.0 Hz), d
7.86(d, 1H, J = 2.8 Hz), d 8.37(s, 1H), d 9.58(s, 1H), d 10.73(s, 1H);
MS (EI) m/e 562.2 (M+); Anal. Calcd for C29H24ClFN4O5: C, 61.87;
H, 4.30; N, 9.95. Found: C, 61.67; H, 4.09; N, 9.68.
3.32; N, 11.82.
5.1.3. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-amino
phenoxy)pyrimidine (4)
A mixture of 3 (5.00 g, 10.71 mmol), iron powder (5.98 g,
107.1 mmol) and AcOH (7.50 g, 125.0 mmol) in ethanol (200 mL)
was heated at reflux and kept intensively stirring for 2 h, and then
another iron powder (5.98 g, 107.1 mmol) was added and kept stir-
ring for another 2 h. As the mixture cooled, the mixture was fil-
tered and washed with ethanol (50 mL), filtrate collected then
evaporated under reduced pressure. The residue was diluted with
saturated NaHCO3 aqueous solution to generate brown solid. The
solid was decolorized with activated carbon, and a crude product
obtained. Further purification was carried out by recrystallization
from CH2Cl2/THF to give 4 as a white solid (3.65 g, 8.35 mmol,
78% yield). Mp: 191–193 °C; 1H NMR (400 MHz, DMSO-d6) d
5.20(s, 2H), d 5.34(s, 2H), d 5.97(s, 1H), d 6.29(dd, 1H, J = 1.6 Hz,
8.0 Hz), d 6.34(t, 1H, J = 2 Hz), d 6.48(dd, 1H, J = 1.2 Hz, 8.0 Hz), d
7.08(t, 1H, J = 8.0 Hz), d 7.15–7.20(m, 2H), d 7.28–7.32(m, 2H), d
7.39(dd, 1H, J = 2.4 Hz, 8.0 Hz), d 7.43–7.49(m, 1H), d 7.86(d, 1H,
5.1.7. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-[3-(2-
ethoxycarbonylethylamino) phenoxy]pyrimidine (8)
Method C. A mixture of 4 (200 mg, 0.46 mmol) and ethyl bromo-
acetate (92 mg, 0.55 mmol) in pyridine (0.5 mL), in which, dioxane
(7 mL) was added, was heated at 100 °C for 3 h. After removal of
dioxane under reduced pressure, the residue was diluted with
EtOAc (40 mL) then washed with saturated NaHCO3 aqueous solu-
tion (3 Â 15 mL). The organic layer dried over anhydrous MgSO4,
and the solvent was removed, purified by column chromatography
on silica gel eluted with EtOAc/petroleum ether (3:5) to give 8 as a
light yellow solid (125 mg, 0.24 mmol, 52% yield). Mp: 58–59 °C;

S. Li et al. / Bioorg. Med. Chem. 20 (2012) 877–885
883
1H NMR (400 MHz, DMSO-d6) d 1.22(t, 3H, J = 3.2 Hz), d 3.80(s, 2H),
5.1.12. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-
cinnamamidophenoxy)pyrimidine (13)
d 4.17(q, 2H, J = 7.2 Hz), d 5.07(s, 2H), d 5.95(s, 1H), d 6.27(t, 1H,
J = 2.4 Hz), d 6.39–6.43(m, 2H), d 6.86(d, 1H, J = 8.8 Hz), d 6.93–
6.98(m, 2H), d 7.06(dd, 1H, J = 2.8 Hz, 8.4 Hz), d 7.10–7.16(m, 3H),
d 7.19(s, 1H), d 7.26–7.30(m, 1H), d 7.33(d, 1H, J = 2.8 Hz), d
8.28(s, 1H); MS (EI) m/e 522.2 (M+); Anal. Calcd for C27H24ClFN4O4:
C, 62.01; H, 4.63; N, 10.71. Found: C, 62.23; H, 4.75; N, 10.55.
Compound 13 was prepared from 4 (200 mg, 0.46 mmol) and
cinnamoyl chloride (92 mg, 0.55 mmol) in THF (15 mL) by the pro-
cedure described for 6 to give 13 as a white solid (224 mg,
0.40 mmol, 86% yield). Mp: 178–181 °C; 1H NMR (400 MHz,
DMSO-d6) d 5.20(s, 2H), d 6.07(s, 1H), d 6.83(d, 1H, J = 15.6 Hz), d
6.92(dd, 1H, J = 1.6 Hz, 8.0 Hz), d 7.15–7.20(m, 2H), d 7.27–
7.31(m, 2H), d 7.38–7.48(m, 6H), d 7.52–7.54(m, 1H), d 7.59–
7.62(m, 2H), d 7.64–7.67(m, 2H), d 7.86(d, 1H, J = 2.4 Hz), d
8.38(s, 1H), d 9.57(s, 1H), d 10.39(s, 1H); MS (EI) m/e 566.2 (M+);
Anal. Calcd for C32H24ClFN4O3: C, 67.78; H, 4.27; N, 9.88. Found:
C, 68.02; H, 4.10; N, 9.63.
5.1.8. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-
(3-cyanoacetamidophenoxy)pyrimidine (9)
Method B. A mixture of 4 (200 mg, 0.46 mmol) and cyanoacetic
acid (78 mg, 0.92 mmol) in THF (15 mL) in ice bath added with EDC
(176 mg, 0.92 mmol) and HOBt (124 mg, 0.92 mmol) was stirred
for 30 min. Having removed the ice bath, the mixture was stirred
at room temperature overnight. After removing the solvent, the
residue was dissolved in EtOAc (35 mL) washed with saturated
NaHCO3 aqueous solution (3 Â 15 mL). The organic layer dried over
anhydrous MgSO4, and the solvent was removed, purified by col-
umn chromatography on silica gel eluted with EtOAc/petroleum
ether (1:1) to give 9 as a white solid (155 mg, 0.31 mmol, 67%
yield). Mp: 167–169 °C; 1H NMR (400 MHz, DMSO-d6) d 3.92(s,
2H), d 5.20(s, 2H), d 6.06(s, 1H), d 6.93(dt, 1H, J = 2.0 Hz, 7.2 Hz),
d 7.15–7.20(m, 2H), d 7.27–7.32(m, 2H), d 7.38–7.49(m, 5H), d
7.85(d, 1H, J = 2.4 Hz), d 8.35(s, 1H), d 9.57(s, 1H), d 10.46(s, 1H);
MS (EI) m/e 503.1 (M+); Anal. Calcd for C26H19ClFN5O3: C, 61.97;
H, 3.80; N, 13.90. Found: C, 62.02; H, 3.73; N, 13.69.
5.1.13. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-
(3-phenoxyacetamidophenoxy)pyrimidine (14)
Compound 14 was prepared from 4 (200 mg, 0.46 mmol) and
phenoxyacetic acid (140 mg, 0.92 mmol) in THF (15 mL) adding
EDC (176 mg, 0.92 mmol) and HOBt (124 mg, 0.92 mmol) by the
procedure described for 9 to give 14 as a white solid (163 mg,
0.29 mmol, 62% yield). Mp: 145–147 °C; 1H NMR (400 MHz,
DMSO-d6) d 4.71(s, 2H), d 5.20(s, 2H), d 6.05(s, 1H), d 6.93(dd,
1H, J = 1.2 Hz, 8.0 Hz), d 6.96–7.01(m, 3H), d 7.15–7.20(m, 2H), d
7.28–7.34(m, 4H), d 7.38–7.48(m, 3H), d 7.53–7.57(m, 2H), d
7.85(d, 1H, J = 2.8 Hz), d 8.36(s, 1H), d 9.56(s, 1H), d 10.24(s, 1H);
MS (EI) m/e 570.2 (M+); Anal. Calcd for C31H24ClFN4O4: C, 65.21;
H, 4.24; N, 9.81. Found: C, 65.28; H, 4.01; N, 9.57.
5.1.9. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-
(3-p-nitrobenzamidophenoxy)pyrimidine (10)
5.1.14. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-[3-
(2,4,5-trifluorophenylacetamido)phenoxy]pyrimidine (15)
Compound 15 was prepared from 4 (200 mg, 0.46 mmol) and
2,4,5-trifluorophenylacetic acid (175 mg, 0.92 mmol) in THF
(15 mL) adding EDC (176 mg, 0.92 mmol) and HOBt (124 mg,
0.92 mmol) by the procedure described for 9 to give 15 as a white
solid (210 mg, 0.35 mmol, 75% yield). Mp: 173–175 °C; 1H NMR
(400 MHz, DMSO-d6) d 3.75(s, 2H), d 5.20(s, 2H), d 6.04(s, 1H), d
6.89(dt, 1H, J = 2.0 Hz, 7.2 Hz), d 7.15–7.19(m, 2H), d 7.27–
7.32(m, 2H), d 7.37–7.57(m, 7H), d 7.84(d, 1H, J = 2.4 Hz), d
8.35(s, 1H), d 9.55(s, 1H), d 10.39(s, 1H); MS (EI) m/e 608.1 (M+);
Anal. Calcd for C31H21ClF4N4O3: C, 61.14; H, 3.48; N, 9.20. Found:
C, 61.41; H, 3.30; N, 9.03.
Compound 10 was prepared from 4 (200 mg, 0.46 mmol) and p-
nitrobenzoyl chloride (93 mg, 0.50 mmol) in THF (15 mL) by the
procedure described for 6 to give 10 as a yellow solid (186 mg,
0.32 mmol, 69% yield). Mp: 189–191 °C; 1H NMR (400 MHz,
DMSO-d6) d 5.32(s, 2H), d 6.09(s, 1H), d 7.00(dd, 1H, J = 1.6 Hz,
7.4 Hz), d 7.15–7.20(m, 2H), d 7.28–7.32(m, 2H), d 7.40(dd, 1H,
J = 2.4 Hz, 8.0 Hz), d 7.43–7.50(m, 2H), d 7.68–7.70(m, 2H), d
7.86(d, 1H, J = 2.4 Hz), d 8.18(s, 1H), d 8.20(s, 1H), d 8.38–8.40(m,
3H), d 8.58(s, 1H), d 10.71(s, 1H); MS (EI) m/e 585.1 (M+); Anal.
Calcd for C30H21ClFN5O5: C, 61.49; H, 3.61; N, 11.95. Found: C,
61.58; H, 3.28; N, 11.91.
5.1.10. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-{3-[6-(4-
amino)pyrimidinyl] amino)phenoxy}pyrimidine (11)
5.1.15. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-
Compound 11 was prepared from 4 (200 mg, 0.46 mmol), 4-
amino-6-chloropyrimidine (71 mg, 0.55 mmol) and a drop of concd
HCl in n-BuOH (10 mL) by the procedure described for 8 to give 11
as a white solid (219 mg, 0.41 mmol, 90% yield). Mp: 180–182 °C;
1H NMR (400 MHz, DMSO-d6) d 5.20(s, 2H), d 5.81(s, 1H), d
6.02(s, 1H), d 6.42(s, 2H), d 6.73(dd, 1H, J = 1.6 Hz, 8.0 Hz), d
7.15–7.20(m, 2H), d 7.28–7.49(m, 6H), d 7.55(s, 1H), d 7.86(d, 1H,
J = 2.4 Hz), d 8.06(s, 1H), d 8.37(s, 1H), d 9.11(s, 1H), d 9.55(s, 1H);
MS (EI) m/e 529.2 (M+); Anal. Calcd for C27H21ClFN7O2: C, 61.19;
H, 3.99; N, 18.50. Found: C, 61.05; H, 3.77; N, 18.15.
(3-p-fluorocinnamamidophenoxy)pyrimidine (16)
Compound 16 was prepared from 4 (200 mg, 0.46 mmol) and 4-
fluorocinnamic acid (153 mg, 0.92 mmol) in THF (15 mL) adding
EDC (176 mg, 0.92 mmol) and HOBt (124 mg, 0.92 mmol) by the
procedure described for 9 to give 16 as a white solid (175 mg,
0.30 mmol, 65% yield). Mp: 205–207 °C; 1H NMR (400 MHz,
DMSO-d6) d 5.20(s, 2H), d 6.07(s, 1H), d 6.77(d, 1H, J = 16 Hz), d
6.92(dd, 1H, J = 2.4 Hz, 8.0 Hz), d 7.15–7.20(m, 2H), d 7.27–
7.31(m, 4H), d 7.38–7.48(m, 3H), d 7.53(d, 1H, J = 8.4 Hz), d
7.60(d, 1H, J = 15.6 Hz), d 7.66(s, 1H), d 7.68–7.72(m, 2H), d
7.86(d, 1H, J = 2.8 Hz), d 8.38(s, 1H), d 9.57(s, 1H), d 10.38(s, 1H);
MS (EI) m/e 584.2 (M+); Anal. Calcd for C32H23ClF2N4O3: C, 65.70;
H, 3.96; N, 9.58. Found: C, 65.96; H, 3.83; N, 9.24.
5.1.11. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-
(3-phenylethylaminophenoxy)pyrimidine (12)
Compound 12 was prepared from 4 (200 mg, 0.46 mmol) and
benzyl bromide (94 mg, 0.55 mmol) in pyridine (0.5 mL) adding
dioxane (8 mL) by the procedure described for 8 to give 12 as a
light yellow solid (200 mg, 0.38 mmol, 83% yield). Mp: 60–62 °C;
1H NMR (400 MHz, DMSO-d6) d 4.27(d, 2H, J = 6.0 Hz), d 5.19(s,
2H), d 5.95(s, 1H), d 6.32(dd, 1H, J = 1.6 Hz, 8.0 Hz), d 6.37(t, 1H,
J = 2.0 Hz), d 6.50–6.52(m, 2H), d 7.10–7.41(m, 11H), d 7.45(q, 1H,
J = 8.0 Hz), d 7.86(d, 1H, J = 2.8 Hz), d 8.35(s, 1H), d 9.51(s, 1H);
MS (EI) m/e 526.2 (M+); Anal. Calcd for C30H24ClFN4O2: C, 68.37;
H, 4.59; N, 10.63. Found: C, 68.37; H, 4.85; N, 10.60.
5.1.16. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-[3-
(3,4-dimethoxycinnamamido)phenoxy]pyrimidine (17)
Compound 17 was prepared from 4 (200 mg, 0.46 mmol) and
3,4-dimethoxycinnamoyl chloride (125 mg, 0.55 mmol) in THF
(15 mL) by the procedure described for 6 to give 17 as a white solid
(239 mg, 0.38 mmol, 83% yield). Mp: 188–191 °C; 1H NMR
(400 MHz, DMSO-d6) d 3.80(s, 3H), d 3.82(s, 3H), d 5.20(s, 2H), d
6.06(s, 1H), d 6.69(d, 1H, J = 15.6 Hz), d 6.90(dd, 1H, J = 1.6 Hz,
8.0 Hz), d 7.02(d, 1H, J = 8.4 Hz), d 7.14–7.22(m, 4H), d 7.27–

884
S. Li et al. / Bioorg. Med. Chem. 20 (2012) 877–885
7.31(m, 2H), d 7.38–7.47(m, 3H), d 7.48–7.56(m, 2H), d 7.66(s, 1H),
d 7.85(d, 1H, J = 2.8 Hz), d 8.37(s, 1H), d 9.57(s, 1H), d 10.30(s, 1H);
MS (EI) m/e 626.2 (M+); Anal. Calcd for C34H28ClFN4O5: C, 65.12; H,
4.50; N, 8.93. Found: C, 65.26; H, 4.26; N, 8.67.
cell proliferation, the A431 and SKOV-3 cells were seeded in 96-
well plates at 1 Â 104 cells/well. All experiments were performed
after 24 h of incubation at 37 °C in 5% CO2.
The cell proliferation was evaluated with the MTT(3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay,
as described by Mosmann.20 Briefly, A431 and SKOV-3 cells were
5.1.17. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-[3-(3,4,5-
trifluorophenoxyacetamido)phenoxy]pyrimidine (18)
treated with compounds (0.05–30 lM) for 72 h at 37 °C in 5% CO2.
Compound 18 was prepared from 4 (200 mg, 0.46 mmol) and
3,4,5-trifluorophenoxyacetic chloride (124 mg, 0.55 mmol) in THF
(15 mL) by the procedure described for 6 to give 18 as a white solid
(219 mg, 0.35 mmol, 76% yield). Mp: 153–155 °C; 1H NMR
(400 MHz, DMSO-d6) d 4.77(s, 2H), d 5.21(s, 2H), d 6.06(s, 1H), d
6.94(dd, 1H, J = 1.6 Hz, 8.4 Hz), d 7.03–7.10(m, 2H), d 7.15–
7.20(m, 2H), d 7.28–7.32(m, 2H), d 7.38–7.54(m, 5H), d 7.85(d,
1H, J = 2.4 Hz), d 8.36(s, 1H), d 9.57(s, 1H), d 10.23(s, 1H); MS (EI)
m/e 624.1 (M+); Anal. Calcd for C31H21ClF4N4O4: C, 59.58; H, 3.39;
N, 8.96. Found: C, 59.82; H, 3.63; N, 8.63.
After that, the cells were washed with phosphate buffered saline
(PBS) and then incubated with MTT (5 mg/mL) in DMEM medium
for 4 h. After removal of MTT and further washing, the formazan
crystals were dissolved with DMSO. The amount of formazan was
measured (492 nm, 630 nm) with a spectrophotometer.
The cell viability was expressed in percentage of control cells
and calculated by the formula Ft/Fnt  100, where Ft = absorbance
of treated cells and Fnt = absorbance of untreated cells. At least
two independent dose–response curves were done and the concen-
tration of compound resulting in 50% inhibition of cell proliferation
(IC50) was calculated.
5.1.18. 4-[3-Chloro-4-(3-fluorobenzyloxy)anilino]-6-[3-(3,4-
dimethoxyphenoxyacetamido)phenoxy]pyrimidine (19)
5.4. Docking study
Compound 19 was prepared from 4 (200 mg, 0.46 mmol) and
3,4-dimethoxyphenoxyacetic chloride (127 mg, 0.55 mmol) in
THF (15 mL) by the procedure described for 6 to give 19 as a white
solid (255 mg, 0.41 mmol, 88% yield). Mp: 130–132 °C; 1H NMR
(400 MHz, DMSO-d6) d 3.69(s, 3H), d 3.75(s, 3H), d 4.64(s, 2H), d
5.20(s, 2H), d 6.05(s, 1H), d 6.49(dd, 1H, J = 2.8 Hz, 8.8 Hz), d
6.72(d, 1H, J = 2.8 Hz), d 6.87(d, 1H, J = 8.8 Hz), d 6.92–6.94(m,
1H), d 7.15–7.20(m, 2H), d 7.28–7.32(m, 2H), d 7.38–7.49(m, 3H),
Molecular docking of compounds 6, 9, 11 and 14 was performed
using Glide 5.5.22 Crystal structure of EGFR was downloaded from
the Protein Data Bank (PDB ID: 1XKK). In precise, the protein struc-
ture was prepared with the Protein Preparation Wizard.22 With re-
spect to the ligands, LigPrep23 was employed to prepare all of
them. And then Glide 5.5 was used to dock the four compounds
into the binding pocket. All graphical pictures were made using
the Pymol program.
d
7.55–7.57(m, 2H), d 7.85(d, 1H, J = 2.4 Hz), d 8.36(s, 1H), d
9.57(s, 1H), d 10.18(s, 1H); MS (EI) m/e 630.2 (M+); Anal. Calcd
for C33H28ClFN4O6: C, 62.81; H, 4.47; N, 8.88. Found: C, 62.50; H,
4.27; N, 8.63.
Acknowledgments
This work was supported by the Ministry of Science and
Technology of the People’s Republic of China (Contract No.
2010DFA44300), National Natural Science Foundation of China
(NSFC, Contract No. 21074033) and the Science and Technology
Commission of Shanghai Municipality (STCSM, No. 10dz2220500).
We also thank Wen Xiao from School of Pharmacy of East China Uni-
versity of Science and Technology for graphical picture preparation.
5.2. Kinase assay
The kinase inhibition assay and IC50 determinations for wild
type EGFR/ErbB-2 were measured with the homogeneous time-
resolved fluorescence (HTRF) KinEASE-TK assay from Cisbio
according to the manufacturer’s instructions. Wild type
EGFR(669–1210) and ErbB-2(676–1255) was purchased from Car-
na Biosciences and 0.09 and 0.11 ng/
respectively. ATP concentration was set at its Km values (23 and
39 M for EGFR and ErbB-2), and 180 nM substrate were used for
lL kinase were used for test,
References and notes
l
1. Ogiso, H.; Ishitani, R.; Nureki, O.; Fukai, S.; Yamanaka, M.; Kim, J.-H.; Saito, K.;
Sakamoto, A.; Inoue, M.; Shirouzu, M.; Yokoyama, S. Cell 2002, 110, 775.
2. Bishayee, S. Biochem. Pharmacol. 2000, 60, 1217.
3. Holbro, T.; Civenni, G.; Hynes, N. E. Exp. Cell Res. 2003, 284, 99.
4. Hynes, N.; Horsch, K.; Olayioye, M.; Badache, A. Endoc. Relat. Cancer 2001, 8, 151.
5. Smaill, J. B.; Palmer, B. D.; Rewcastle, G. W.; Denny, W. A.; McNamara, D. J.;
Dobrusin, E. M.; Bridges, A. J.; Zhou, H.; Showalter, H. D. H.; Winters, R. T.;
Leopold, W. R.; Fry, D. W.; Nelson, J. M.; Slintak, V.; Elliot, W. L.; Roberts, B. J.;
Vincent, P. W.; Patmore, S. J. J. Med. Chem. 1999, 42, 1803.
6. Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y.-F.; Wissner, A. J. Med. Chem. 2001, 44, 2719.
7. Wissner, A.; Berger, D. M.; Boschelli, D. H.; Floyd, M. B.; Greenberger, L. M.;
Gruber, B. C.; Johnson, B. D.; Mamuya, N.; Nilakantan, R.; Reich, M. F.; Shen, R.;
Tsou, H.-R.; Upeslacis, E.; Wang, Y. F.; Wu, B.; Ye, F.; Zhang, N. J. Med. Chem.
2000, 43, 3244.
both EGFR and ErbB-2. Kinase, substrate peptide and inhibitors
were added in 384 well plates, and then reaction was started by
addition of ATP. After completion of the reaction, an antiphosp-
hotyrosine antibody labeled with europium cryptate and streptavi-
din labeled with the fluorophore XL665 were added. The FRET
between europium cryptate and XL665 was measured to quantify
the phosphorylation of the substrate peptide. A Tecan i-control
infinite 500 was used to measure the fluorescence of the samples
at 620 nM (Eu-labeled antibody) and 665 nM (XL665 labeled strep-
tavidin) 500 ls after excition at 320 nM. The quotient of both
intensities for reactions made with ten different inhibitor concen-
trations (0.01–10,000 nM, including no inhibitor) was plotted
against inhibitor concentrations to determine IC50 values. Each
reaction was performed in duplicate, and at least two independent
determinations of each IC50 were made.
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