Targeting EGFR/HER2 tyrosine kinases with a new potent series of 6-substituted 4-anilinoquinazoline hybrids: Design, synthesis, kinase assay, cell-based assay, and molecular docking

Article abstract of DOI:10.1016/j.bmcl.2015.10.003

Coexpression of EGFR and HER2 has been found in many tumors such as breast, ovarian, colon and prostate cancers, with poor prognosis of the patients. Herein, our team has designed and synthesized new eighteen compounds with 6-substituted 4-anilinoquinazol

Full text of DOI:10.1016/j.bmcl.2015.10.003

Accepted Manuscript
Targeting EGFR/HER2 tyrosine kinases with a new potent series of 6-substi-
tuted 4-anilinoquinazoline hybrids: Design, synthesis, kinase assay, cell-based
assay, and molecular docking
Ahmed Elkamhawy, Ahmed Karam Farag, Ambily Nath Indu Viswanath, Tarek
M. Bedair, Dong Gyu Leem, Kyung-Tae Lee, Ae Nim Pae, Eun Joo Roh
PII:
S0960-894X(15)30112-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.10.003
BMCL 23159
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
18 August 2015
25 September 2015
2 October 2015
Please cite this article as: Elkamhawy, A., Farag, A.K., Viswanath, A.N.I., Bedair, T.M., Leem, D.G., Lee, K-T.,
Pae, A.N., Roh, E.J., Targeting EGFR/HER2 tyrosine kinases with a new potent series of 6-substituted 4-
anilinoquinazoline hybrids: Design, synthesis, kinase assay, cell-based assay, and molecular docking, Bioorganic
& Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.10.003
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Targeting EGFR/HER2 tyrosine kinases with a new potent series of 6-substituted 4-
anilinoquinazoline hybrids: Design, synthesis, kinase assay, cell-based assay, and
molecular docking
Ahmed Elkamhawya,b,c, Ahmed Karam Faraga,b, Ambily Nath Indu Viswanathd,e, Tarek M. Bedairf,
Dong Gyu Leemg, Kyung-Tae Leeg, Ae Nim Paed,e, Eun Joo Roha,b*
a Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea
b Department of Biological Chemistry, Korea University of Science and Technology (UST), Daejeon 305-350, South Korea
c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
d Center of Neuromedicine, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea
e Department of Medicinal and Pharmaceutical Chemistry, Korea University of Science and Technology (UST), Daejeon 305-343, South Korea
f Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea
g Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, South Korea
*Corresponding author: Eun Joo Roh
Email address: r8636@kist.re.kr
Postal address: Korea Institute of Science and Technology, Chemical Kinomics Research Center,
Materials and Life Science Research Division, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 136-
791, South Korea

Abstract
Coexpression of EGFR and HER2 has been found in many tumors such as breast, ovarian,
colon and prostate cancers, with poor prognosis of the patients. Herein, our team has designed
and synthesized new eighteen compounds with 6-substituted 4-anilinoquinazoline core to
selectively inhibit EGFR/HER2 tyrosine kinases. Twelve compounds (8a−8d, 9a, 9c, 9d, 10a,
10c, 11b, 14, 15) showed nanomolar range of IC50 values on EGFR and/or HER2 kinases.
Accordingly, a detailed structure activity relationship (SAR) was established. A molecular
docking study demonstrated the favorable binding modes of 8d, 9b, 9d and 10d at the ATP
active site of both kinases. A kinase selectivity profile performed for compound 8d showed great
selectivity for EGFR and HER2. In addition, 8d, 9c, and 9d exerted selective promising
cytotoxic activity over BT-474 cell line with IC50 values of 2.70, 1.82 and 1.95 µM, respectively.
From these results, we report analogues 8d, 9c, and 9d as promising candidates for the discovery
of well-balanced compounds in terms of the kinase inhibitory potency and antiproliferative
activity.
Keywords: Synthesis, EGFR/HER2 dual inhibitors, Antiproliferative activity, Bt-474 cell line,
Molecular docking, kinase panel.

Protein tyrosine kinases play a crucial role in signal transduction pathways that control
several cellular functions including proliferation, differentiation, migration, and angiogenesis [1].
Therefore, protein kinase inhibitors which target the elevated pathways are attractive candidates
for cancer therapy [2]. Overexpression of the epidermal growth factor receptor (EGFR or ErbB1)
and human epidermal growth factor receptor 2 (HER2 or ErbB2) which are members of the ErbB
family of receptor tyrosine kinases (RTKs) is frequently observed in various solid tumors. Both
kinases have been validated as rational targets for cancer therapy [3]. Stimulation of the
epidermal growth factor (EGF) causes the ErbB receptors to form either homo- or heterodimers
with the other ErbB family receptors, resulting in activation of the downstream signaling and
consequently tumor cell growth promotion [4]. Coexpression of EGFR and HER2 has been
reported in different tumors such as breast, ovarian, colon and prostate cancers [5-8]. Therefore,
it is more effective to dual target EGFR/HER2 rather than just EGFR inhibition [9].
EGFR/HER2 inhibitors can block tyrosine kinase phosphorylation and inhibit the upregulated
intracellular signals in cancer cells leading to loss of the tumor regulatory function. A variety of
ATP-competitive EGFR/HER2 RTK dual inhibitors related to different scaffolds have been
reported and many of them are currently in market or clinical trials for the treatment of cancer.
An important leading nucleus of EGFR/HER2 dual inhibitors is 4-anilinoquinazoline,
exemplified by the lead compound lapatinib which was approved by the FDA for treatment of
patients with HER2 overexpression metastatic breast cancer [9]. The binding mode of lapatinib
with the catalytic domain of EGFR/HER2 kinases has been well studied in literature [10, 11].
Generally, the quinazoline ring is hydrogen bonded to the hinge region of the ATP binding cleft.
The aniline group at C-4 of the quinazoline nucleus is oriented deep to fit in an adjacent back

pocket forming hydrophobic interactions. The SAR study of quinazoline was directed toward the
modification of the C-4 aniline group which could achieve fine tuning of the kinase selectivity.
Previous studies had established that the size and functionality of this pocket are main factors of
the kinase inhibitor selectivity while the solubilizing moiety at C-6 of the quinazoline core act to
improve the physical properties aiming for a favorable pharmacokinetic profile. In addition,
many dual inhibitory candidates were designed to bind via hydrogen bonding or covalent
bonding with Cys-773 in EGFR and Cys-805 in HER2 [12-16]. Although lapatinib therapy has
proven effective, many patients fail to respond to treatment or become resistant due to different
reasons which are still largely unknown [17-20]. Accordingly, new strategies and compounds are
still in need for EGFR/HER2 inhibition. In this report, we focus our efforts on design and
synthesis of new hybrid molecules with 6-substituted 4-anilinoquinazoline core to selectively
inhibit EGFR and HER2 tyrosine kinases as depicted in Figure 1.
Figure 1. Rational design of the newly synthesized compounds.

The new candidates possess different aniline patterns at C-4 in fashion similar to previously
reported different potent dual inhibitors. Well-known various moieties were used including 3-
chloro-4-((3-fluorobenzyl)oxy)aniline of lapatinib, 3-chloro-4-(pyridin-2-ylmethoxy)aniline of
neratinib, 3-chloro-4-(3-(trifluoromethyl)phenoxy)aniline of TAK-285, and
3-chloro-4-
fluoroaniline of gefitinib. In addition, new aniline moiety (3-chloro-4-(3,4-
a
dichlorophenoxy)aniline) was used to investigate the activity of compounds bearing 3,4-dihalo-
substituted phenoxy segment. The aniline portion was designed to be directed into the
hydrophobic deep pocket into the binding site. To achieve better affinity and further potency of
the new candidates, the C-6 position was subjected to a variety of new different
polar/solubilizing functional groups [21-24] including moieties of ureido (8a−8c), acetamido
(8d), ethyl urea (9a−9d), diethyl aminomethylene malonate (10a−10d), and aminomethylene
malononitrile (11a−11c). Moreover, to explore the effect of these solubilizing moieties at a
distance from C-6 position of the quinazoline core, incorporation of amido spacer was applied in
14, 15, and 16. The substitution patterns of the new agents aimed to improve potency and
selectivity by incorporation of new moieties at C-6 of substituted 4-anilinoquinazolines and/or by
varying the C-4 aniline moieties. All the target compounds were then tested for their inhibition
profile over both EGFR and HER2 kinases in 10-dose IC50 mode. In addition, a detailed
molecular docking study was carried out using GLIDE v 6.2 of Schrodinger suite 2014 [25, 26]
as flexible docking program to predict the binding affinities and orientation of the target
compounds 8d, 9b, 9d and 10d at the ATP binding site of EGFR/HER2-RTK. Moreover, a cell-
based assay was performed to evaluate the antiproliferative activity of the new agents using the
human breast carcinoma cell line (BT-474) which characterized by the overexpression of HER2.

The synthetic route adopted for preparation of the new 6-substituted amino-4-
anilinoquinazoline derivatives 8a−8d, 9a−9d, 10a−10d, 11a−11c, 14, 15, and 16 is depicted in
Schemes 1−4. Reaction of commercially available 2-chloro-1-fluoro-4-nitrobenzene (1a) or 2-
chloro-4-nitrophenol (1b) with the appropriate phenol or arylmethyl bromide was carried out in
the presence of potassium carbonate in dimethylformamide (DMF) or acetonitrile, followed by
reduction of the nitro groups in 2a−2d with platinum on carbon under hydrogen atmosphere to
yield substituted anilines 3a−3d in excellent yields as illustrated in Scheme 1.
o
Scheme 1. Reagents and conditions: (i) appropriate phenol (2a, 2b), CH3CN, 85 C, 5 h; (ii)
o
appropriate arylmethyl bromide (2c, 2d), K2CO3, DMF, 85 C, 5 h; (iii) H2, 10% Pt/C, CH3OH,
rt, 6 h.
The key intermediate 6-amino-4-anilinoquinazolines 7a−7e were prepared following the
literature methods illustrated in Scheme 2. 6-Nitro-4-arylaminoquinazoline derivatives 6a−6e
were synthesized in good yields through the reaction of 5-nitroanthranilonitrile (4) with
dimethylformamide dimethyl acetal (DMF−DMA) affording the corresponding formamidine (5)
followed by its reaction with appropriate anilines in acetic acid to give the 6-nitro-4-
anilinoquinazolines 6a−6e. The nitro groups in 6a−6e were reduced by treating with Fe/acetic
acid to afford the 6-aminoquinazolines 7a−7e.

o
Scheme 2. Reagents and conditions: (i) dimethylformamide dimethyl acetal, 100 C, 1.5 h; (ii)
substituted aniline, glacial acetic acid, reflux, 1 h; (iii) Fe, AcOH, EtOH/H2O (70%), reflux, 2 h.
As shown in Scheme 3, the reaction of the amino derivatives 7a−7d with potassium cyanate
in acetic acid gave the corresponding 4-(substituted amino)-6 ureidoquinazolines 8a−8c. The
acetamido analogue 8d was obtained by refluxing 7d in acetic acid. Reaction with ethyl
isocyanate provided the target ethyl urea derivatives 9a−9d. Reaction with diethyl 2-
(ethoxymethylene)malonate
yielded
diethyl
2-[(4-arylamino)quinazolin-6-
ylaminomethylene]malonate analogues (10a−10d). Finally, compounds 11a−11c were obtained
through reaction with (ethoxymethylene)malononitrile.

Scheme 3. Reagents and conditions: (i) (8a−8c) KOCN, glacial acetic acid, reflux, 3 h, (8d)
o
glacial acetic acid, reflux, 3 h; (ii) ethyl isocyanate, THF, 50 C, 8 h; (iii) diethyl 2-
(ethoxymethylene)-malonate, abs. EtOH, reflux, 3 h; (iv) (ethoxymethylene)malononitrile, abs.
EtOH, reflux, 3 h.
With the aim of examining the effects of the polar/solubilizing functionality at a distance
from C-6 position of the quinazoline core, a benzamido spacer was intentionally inserted directly
attached to the quinazoline ring. Amidation of 7e with 4-nitobenzoylchloride in the presence of
TEA in THF provided N-(4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)-4-nitrobenzamide
(12) in a good yield, followed by catalytic hydrogenation using platinum on carbon to reduce the

nitro group yielding the corresponding amine 13 (Scheme 4). Similar reactions to that of Scheme
3 were carried out for the synthesis of derivatives 14−16.
o
Scheme 4. Reagents and conditions: (i) 4-nitrobenzoyl chloride, TEA, THF, 50 C, overnight;
(ii) H2, 10% Pt/C, CH3OH, rt, 6 h; (iii) KOCN, glacial acetic acid, reflux, 3 h; (iv) ethyl
isocyanate, THF, 50 oC, 8 h; (v) diethyl 2-(ethoxymethylene)-malonate, abs. EtOH, reflux, 3 h.
To examine the final analogues potential to inhibit EGFR and HER2 kinases, all the newly
synthesized compounds were tested over both targets at Reaction Biology Corporation using the
‘HotSpot’ assay platform in 10-dose IC50 mode with 3 fold serial dilutions starting at 20 μM.
Kinase activity data were expressed as the percent remaining kinase activity in test samples
compared to vehicle (dimethyl sulfoxide) reactions (for detailed information, see supplementary
file). IC50 values were calculated and reported in Table 1. The pharmacological results offered
twelve compounds with excellent inhibitory activities (nanomolar range of IC50 values on EGFR

and/or HER2 kinases). The structure-activity relationship (SAR) of the new analogues is
indicated as follow: it was noted that all analogues at C-4 position with 3-chloro-4-(3,4-
dichlorophenoxy)aniline (9b, 10b, and 11a) didn’t show significant activity against both kinases,
while other moieties exhibited good to excellent inhibitory activities. The results clearly
demonstrated that derivatives containing ureido groups (8a−8c) exhibited promising activities
with IC50 values of 0.004, 0.543, and 0.099 µM on EGFR and 0.077, 0.706, and 0.287 µM on
HER2, respectively. Compound 8d exerted 0.003 µM on EGFR and 0.016 µM on HER2, which
are the best inhibitory activities in the series. It is worth mentioning that compound 14 with
ureido moiety linked to the benzamido spacer exerted similar interesting results with IC50 values
of 0.009 and 0.275 µM on EGFR and HER2, respectively. Incorporation of a small alkyl group
(CH3CH2−) to the ureido part slightly decreased the potency against both kinases; compounds
9a, 9d, and 15 possessing ethyl ureas moieties showed inhibitory activities with IC50 values of
0.029, 0.227, and 0.031 µM on EGFR and 0.625, 1.390, and 0.855 µM on HER2, respectively.
Compounds 10a−10d with diethyl methylene malonate moieties exerted good to moderate
activities on both kinases in low micromolar range. Conversely, replacement of the ureido
moiety with aminomethylene malononitrile in 11a−11c led to a marked reduction of the activity.

Table 1. IC50 values (µM) exhibited by the newly synthesized compounds on EGFR and HER2
tyrosine kinases.
Kinase assay
Cpd
EGFR
HER2
IC50 (µM)b
IC50 (µM)b
0.004
0.543
0.099
0.003
0.029
> 10
0.077
0.706
0.287
0.016
0.625
> 10
8a
8b
8c
8d
9a
9b
0.035
0.227
0.023
> 10
0.126
1.390
1.000
> 10
9c
9d
10a
10b
10c
10d
11a
11b
11c
14
0.812
1.020
> 10
5.050
3.290
> 10
0.445
3.050
0.009
0.031
1.460
0.010
2.720
8.000
0.275
0.855
> 10
15
16
Lapatiniba
0.009
a Lapatinib was used as a positive control agent.
b Compounds were tested in a 10-dose mode with 3-fold serial dilution starting at 20 µM.
In order to provide deeper insights into the structure-activity relationship of the new series,
compounds 8d, 9b, 9d and 10d were selected based on the potency and scaffold types and
docked individually into the ATP-binding pockets of EGFR (PDB code: 1XKK) and HER2
(PDB code: 3RCD). The re-docked conformations of crystal ligands were kept as reference

structures for assessing the binding modes of the compounds in respective kinases (for detailed
information about docking validation, see supplementary file).
Compound 8d, with pyridine and acetamido groups at C-4 and C-6 respectively, produced
outstandingly good result by mimicking lapatinib in geometry-wise and interaction-wise. As
illustrated in Figure 2A, the pyridine moiety occupied a smaller hydrophobic pocket comprised
of Met766, Cys775, Leu777 and Phe856. The quinazoline nucleus derived substantial binding
energy via direct interaction with Met793 (hinge region) and water-mediated interactions with
Thr854 (precedes DFG motif) and Thr790 (gatekeeper residue). The acetamido carbonyl formed
one hydrogen bond with Cys797 which provided further hydrophobic stabilization to the
terminal methyl group. 8d pursued the similar binding pattern in HER2 (Figure 2B). The
pyridine with an outward-facing conformation in the binding pocket was strengthened by Lys753
and Leu755. The interactions of quinazoline (Met801) and C-6 substituent (Cys805) were
recreated.

Figure 2. Top-scored poses of 8d (yellow) aligned to the reference compounds in (A) EGFR
(peach) and (B) HER2 (aqua). Hydrogen bonds are in blue dashed lines; the compounds,
hydrogen bonding residues (Thr790, Met793, Cys797 and Thr854 in EGFR; Met801 and Cys805
in HER2) and conserved water molecule shown as sticks.
Compounds designed with bulky hydrophobic substituents at C-4 and C-6 proved to be non-
productive in the docking experiments. In 9b (Figure 3A and 3B), the dichlorophenyl and
ethylurea at the suggested positions slightly dislocate the orientation of ligand from the
proximity of functional residues (8b, with a similar scaffold composition at C-4 showed
reasonable inhibition, this could be due to the C-6 ureido group which makes adequate polar
contacts). 9d, with pyridine substituent (C-4) and ethylurea (C-6) improved the performance both
qualitatively and quantitatively in EGFR (Figure 4A) and HER2 (Figure 4B).
Figure 3. The docked conformations of 9b (light violet) on superimposition with re-docked
structures in (A) EGFR (peach) and (B) HER2 (aqua). Compounds, hydrogen bonding residues
(Leu777, Asp800, Asp855 and Phe856 in EGFR; Asp863 in HER2) and conserved water

molecule shown as sticks; hydrogen bonds depicted as blue dashed lines. The ligand missed
water-mediated interaction with Thr854 in EGFR.
Figure 4. Superimposed 9d poses (grey) and re-docked reference compounds (black) in (A)
EGFR (peach) and (B) HER2 (aqua). Hydrogen bonds shown as blue dashed lines; ligands,
hydrogen bonding residues (Lys745, Met793, Cys797 and Thr854 in EGFR; Asp845, Asn850
and Asp863 in HER2) and conserved water molecule displayed as sticks.
Covalent adduct formation between the inhibitor’s reactive Michael Acceptor (MA) group
and the receptor’s cysteine nucleophile (Cys797 in EGFR and Cys805 in HER2) implied to
enhance the potency of ligands [27-31]. In the present in silico investigation, covalent docking
was attempted on 10d because the ester MA at C-6 is approachable to the nearest conserved
cysteine in the hinge region of the ATP-binding pocket. The docked models and interactions
showed good alignment with re-docked ligands; expected covalent bonding with the targeted
cysteines was also established (Figure 5).

Figure 5. Top 10d poses (light pink) aligned to re-docked crystal ligands (black) in (A) EGFR
(peach) and (B) HER2 (aqua). Hydrogen bonds shown as blue dashed lines; conserved water
molecule, hydrogen bonding residues (Met793, Cys797, Asp800 and Thr854 in EGFR; Lys753,
Met801 and Cys805 in HER2) and covalent bonding cysteine (orange; Cys797 in EGFR and
Cys805 in HER2) are in stick form.
The present docking computation created the virtual models of compounds bound with
EGFR-HER2 kinase domains and their putative binding modes were deciphered with a special
focus on the nature of C-4/C-6 substituents. Compounds bearing pyridine at C-4 and acetamido
or ureido functional groups at C-6 found to be well-tolerated. The dual inhibitory activity of the
investigated compounds was thus rationalized by the modeling studies and the later provides a
foundation for further optimization.
The preliminary data for 8d over the molecular level of EGFR and HER2 tyrosine receptors
as well as its well-docked binding models showed promise and therefore it underwent further
evaluation. 8d was screened in a small panel of kinases including CDK2, c-MET, FAK, FGFR1,

IGF1R, VEGFR2, LCK, PKA, P38a, and MAPKAP kinase 2. As shown in Table 2, it exhibited
no inhibition for all other tested targets except LCK kinase with IC50 of 1.22 µM. These results
strongly indicated a great selectivity of the compound under test with its nanomolar potency over
both kinases (EGFR and HER2) in comparison to the other tested targets.
Table 2. Kinase selectivity profile of compound 8d.
Kinase
EGFR
IC50 (µM)a
0.003
HER2
0.016
−
CDK2
c-MET
FAK
−
−
FGFR1
IGF1R
VEGFR2
LCK
−
−
−
1.22
−
PKA
P38a
−
MAPKAPK2
−
a IC50 values were calculated using 10-dose mode with 3-fold serial dilution starting at 20 µM.
− No inhibition
In an effort to test the antiproliferative activity of the most potent derivatives, 8a−8d and
9a−9d were tested over BT-474 (HER2-overexpressing human breast cancer cell line) in a
duplicate assay using 10-dose mode with 3-fold serial dilution starting at 20 µM. GI50 values
were calculated and reported in Table 3. However the molecular activity of 8b among the three
ureido derivatives 8a−8c was the modest, it interestingly showed higher cytotoxic activity over
BT-474 cell line with GI50 value of 2.38 µM compared to that of 8a and 8c (>10 and 4.72 µM,

respectively). This may be attributed to the two chloro substituents on the phenoxy moiety of 8b
which could increase the lipophilic property and consequently the antiproliferative activity.
Compound 8d also exhibited low micromolar GI50 value of 2.7 µM. The hydrophobic methyl
group in the acetamido moiety may play a role on such activity over the cell line. In respect to
the ethylurea analogues 9a−9d, compound 9a exhibited better cytotoxic activity (GI50 = 3.55
µM) compared to its ureido derivative 8a (GI50 >10 µM). On the other hand, 9b didn’t show any
significant activity neither over the enzymatic nor the cellular level. 9c and 9d were the most
active hybrids in this study with GI50 values of 1.82 and 1.95 µM, respectively. Finally, it was
noted that compounds possessing moieties of 3-fluorobenzyloxy (8c and 9c) and pyridylmethoxy
(8d and 9d) showed better antiproliferative activities than most analogues with phenoxy groups
(8a, 9a, and 9b).
Table 3. GI50 values (µM) exhibited by 8a−8d and 9a−9d over BT-474 breast cancer cell line.
Cpd
GI50 (µM)b
> 10
8a
2.38
4.72
2.70
3.55
> 10
1.82
1.95
0.93
8b
8c
8d
9a
9b
9c
9d
Lapatiniba
a Lapatinib was used as a positive control agent.
b Compounds were tested in a duplicate assay using 10-dose mode with 3-fold serial dilution
starting at 20 µM.

As further evaluation for the selective cytotoxicity of the most promising compounds (8d, 9c,
and 9d), they have been tested over L132 normal human embryonic lung epithelium cell line
using MTT assay. As illustrated in Table 4 and Figure 6, no significant inhibitory effects have
been exhibited on the tested cells. (For more details about experimental procedure, see
supplementary file).
Table 4. Cytotoxicity (IC50, MTT assay) of 8d, 9c, and 9d over L132 normal cell line.
IC50 (µM)
> 100
Cpd
8d
> 100
9c
> 100
9d
Figure 6. Cell viability of L132 normal cell line after treatment with compound 8d.
As a conclusion, we have successfully prepared a new series of 4-anilinoquinazoline hybrids
which exerted potent activity on both EGFR and HER2 kinases. Using the molecular docking
approach, the binding modes of some selected compounds were modeled in the kinase domains.
A cell-based assay was carried out to evaluate the antiproliferative activity of the most potent
compounds. We believe that identification of analogues 8d, 9c, and 9d with outstanding in vitro

profiles over the molecular and the cellular levels may offer an excellent framework for
discovery of new potent and selective EGFR/HER2 dual inhibitors.
Acknowledgments
This work was supported by the KIST Institutional programs (Grant No. 2E25340 and
2E25240) from Korea Institute of Science and Technology and by the Creative Fusion Research
Program through the Creative Allied Project funded by the National Research Council of Science
& Technology (CAP-12-01-KIST).
The authors have declared no conflict of interest.

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