Discovery of new HER2/EGFR dual kinase inhibitors based on the anilinoquinazoline scaffold as potential anti-cancer agents

Article abstract of DOI:10.3109/14756366.2013.765417

Herein, we designed and synthesized certain anilinoquinazoline derivatives bearing bulky arylpyridinyl, arylpropenoyl and arylpyrazolyl moieties at the 4′ position of the anilinoquinazoline, as potential dual HER2/EGFR kinase inhibitors. A detailed molecular modeling study was performed by docking the synthesized compounds in the active site of the epidermal growth factor receptor (EGFR). The synthesized compounds were further tested for their inhibitory activity on EGFR and HER2 tyrosine kinases. The aryl 2-imino-1,2-dihydropyridine derivatives 5d and 5e displayed the most potent inhibitory activity on EGFR with IC50 equal to 2.09 and 1.94 μM, respectively, and with IC50 equal to 3.98 and 1.04 μM on HER2, respectively. Furthermore, the anti-proliferative activity of these most active compounds on MDA-MB-231 breast cancer cell lines, known to overexpress EGFR, showed an IC50 range of 2.4 and 2.5 μM, respectively.

Full text of DOI:10.3109/14756366.2013.765417

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.765417
!
Discovery of new HER2/EGFR dual kinase inhibitors based on the
anilinoquinazoline scaffold as potential anti-cancer agents
Maiada M. Sadek1, Rabah A. Serrya2, Abdel-Hamid N. Kafafy3, Marawan Ahmed4, Feng Wang4,
and Khaled A. M. Abouzid2
2
1Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, MSA University, 6th October, Cairo, Egypt, Pharmaceutical Chemistry
3
Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
4
Assiut University, Abbassia, Cairo, Egypt, and Chemistry Laboratory, Faculty of Life and Social Sciences, Swinburne University of Technology,
Melbourne, Victoria, Australia
Abstract
Keywords
Herein, we designed and synthesized certain anilinoquinazoline derivatives bearing bulky
arylpyridinyl, arylpropenoyl and arylpyrazolyl moieties at the 40 position of the anilinoquinazo-
line, as potential dual HER2/EGFR kinase inhibitors. A detailed molecular modeling study was
performed by docking the synthesized compounds in the active site of the epidermal growth
factor receptor (EGFR). The synthesized compounds were further tested for their inhibitory
activity on EGFR and HER2 tyrosine kinases. The aryl 2-imino-1,2-dihydropyridine derivatives 5d
and 5e displayed the most potent inhibitory activity on EGFR with IC50 equal to 2.09 and
1.94 mM, respectively, and with IC50 equal to 3.98 and 1.04 mM on HER2, respectively.
Furthermore, the anti-proliferative activity of these most active compounds on MDA-MB-231
breast cancer cell lines, known to overexpress EGFR, showed an IC50 range of 2.4 and 2.5 mM,
respectively.
Anilinoquinazoline, EGFR, HER2, kinase
inhibitors, lapatinib
History
Received 1 December 2012
Revised 1 January 2013
Accepted 3 January 2013
Published online 12 February 2013
Introduction
interfering with its activation and modulating the resultant
intracellular signal cascade. The other approach for blocking the
up-regulated HER signal pathway involves the use of orally active
small molecule TKIs, these compounds act via competing with
ATP for binding at the catalytic domain of tyrosine3. In recent
years, 4-anilinoquinazolines have emerged as a versatile template
for inhibition of a diverse range of receptor tyrosine kinases.
Among them, small molecule EGFR kinase inhibitors such as
Gefitinib (Iressa)4 and Erlotinib (Tarceva)5 were introduced for
cancer treatment recently. Subsequent research aimed at further
exploration of the structure–activity relationship (SAR) of this
novel template has led to the discovery of highly active
compounds that target both EGFR and HER-2 kinases. for
example. Lapatinib (Tykerb) which was approved in combination
therapy for breast cancer patients already using Capecitabine6.
In this study, we present a new sub-family of compounds
containing 4-anilinoquinazoline scaffold as promising potent and
selective EGFR, HER-2 inhibitors. Our strategy is directed toward
designing a variety of ligands with diverse chemical properties
hypothesizing that the potency of these molecules might be
enhanced by adding alternative binding group such as 2-
iminopyridine and 2H-pyrazole ring in the 4-position of aniline
moiety (Figure 1). This is a typical Structure Based Drug Design
(SBDD) project. Starting from the ‘‘HITS’’ we have identified
here, we are planning to investigate more substitution patterns
utilizing the main pharmacophore as a core. We did not attempt to
synthesize each and every derivative in the new series, but our
main goal was to explore more substitution patterns not members.
Such substitution pattern could target different regions of the
ATP-binding site of the protein kinase domain to create differ-
entially selective molecules. The design of our ligands was done
The ErbB or epidermal growth factor (EGF) receptors are
members of subclass 1 of the receptor tyrosine kinase (RTK)
superfamily. There are four ErbB receptor family members:
ErbB1 (EGFR, HER1), ErbB2 (HER2/neu), ErbB3 (HER3) and
ErbB4 (HER4). The receptors are situated at the cell membrane
and have an extracellular ligand-binding region, a transmembrane
region and a cytoplasmic tyrosine-kinase domain. Ligand binding
to the receptors results in receptor homo- and heterodimerization,
activation of the intrinsic kinase domain and phosphorylation of
specific tyrosine residues within the cytoplasmic tail. Proteins
dock on these phosphorylated residues, leading to the activation
of a variety of intracellular signaling pathways that promote cell
growth, proliferation, differentiation and migration1. ErbB2 is the
preferred dimerization partner for all the other ErbB receptors.
ErbB receptors undergo various types of alteration in human
tumors including gene amplification, receptor overexpression,
activating mutations, overexpression of receptor ligands and/or
loss of negative regulatory controls. The most robust example is
that of amplification of HER2/neu in 25%–30% of breast cancers
and is associated with a statistically significant shortening in
disease-free and overall survival2.
Two predominant approaches have been developed for
blocking the up-regulated HER signal pathway. One approach
utilizes the anti-HER monoclonal antibody, which blocks the
extracellular ligand-binding region of the receptor, thereby
Address for correspondence: Khaled A. M. Abouzid, Pharmaceutical
Chemistry Department, Faculty of Pharmacy, Ain Shams University,
Cairo 11566, Egypt. Email: khaled.abouzid@pharm.asu.edu.eg

2
M. M. Sadek et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 1. Structural modifications of the lead
compounds (Gefitinib, Erlotinib and
Lapatinib).
based on previous SAR of 4-anilinoquinazolines and earlier work
with quinazoline-based inhibitors of EGFR, which established
that 6,7-dialkoxy substitution is compatible with good activity,
and pivotal interactions between the receptor and the inhibitors7.
In more recent approach, it was found that the dual inhibition of
EGFR and ErbB-2 may offer increased activity over agents who
target only one of these receptor kinases. After discovery of
lapatinib, it was claimed that 40 position of the aniline can tolerate
a lot of bulky substituents8.
In this direction and in an approach to enhance the selectivity
toward EGFR/ErbB-2, we introduced larger moieties at 40 position
of the aniline such as 4-aryl-2-imino pyridine moiety in a fashion
similar to lapatinib, which binds in the ATP-binding cleft, so that
the bulky group could be oriented deep in the back of the ATP
binding site and makes predominantly hydrophobic interactions
Figure 2. The two basic skeletons of inhibitors in the new series.
with
the
protein
mimicking
the
3-chloro-4-[(3- and to check the purity of products. All reagents and solvents were
fluorobenzyl)oxy]aniline group of lapatinib (Figure 1). The purified and dried by standard techniques. Elemental micro-
binding mode and docking energy of the designed compounds analyses were performed at Microanalytical Center, Cairo
could be helpful tool for predicting their mechanism of antitumor University, and were within ꢀ0.4% unless otherwise stated. The
activity. Figure 2 displays the two basic skeletons of the chemicals used in the synthesis are purchased from Aldrich (St.
synthesized compounds.
Louis, MO) and Merck (St. Louis, MO) and are of analytical
grades. The preparation of compounds 2a,b9, 3a,b10 and 4a,b9,11
was performed according to reported procedures.
Chemistry
Melting points were determined using a Stuart Scientific appar-
atus (Staffordshire, UK) and were uncorrected. IR spectra were
recorded on a PerkinElmer FT-IR series (Waltham, MA) using
KBr cell. H-NMR spectra were recorded on Bru¨cker (Billerica,
MA) 300 MHz in d scale. All the spectra were obtained on
solutions in DMSO-d6 with TMS as internal standard; the values
General procedures for the synthesis of 4-{4-[6-(4-Aryl-3-
cyano-2-imino)-1,2-dihydropyridyl]phenylamino}quinazo-
line (5a–d) and 4-{4-[6-(4-Aryl-3-cyano-2-imino)-1,2-dihy-
dropyridyl]phenylamino}-6,7-dimethoxy quinazolines (5e)
1
A
mixture of the appropriate 4-anilinoquinazoline (4a–b)
of the chemical shifts (d) are given in ppm, and coupling constants (0.01 mol), appropriate aldehyde (0.01 mol), malononitrile
(J) are given in Hz. The electron impact (EI) mass spectra were (0.01 mol) and ammonium acetate (0.08 mol) in absolute ethyl
recorded on Finnigan Mat SSQ 7000 (70 eV) mass spectrometer alcohol (30 ml) was heated under reflux, with stirring for 15–24 h.
(Blue Lion Biotech, LLC, Snoqualmie, WA). Analytical thin layer The reaction mixture was cooled and the formed precipitate was
chromatography (TLC) on silica gel plates containing UV filtered, washed with cold ethyl alcohol, allowed to dry, and
indicator was employed routinely to follow the course of reactions recrystallized from DMF/ethanol 1:2.

DOI: 10.3109/14756366.2013.765417
Discovery of new HER2/EGFR dual kinase inhibitors
3
4-{4-[6-(4-(4-Bromophenyl)-3-cyano-2-imino)-1,2-dihydropyri-
dyl]phenylamino}6,7-dimethoxyquinazoline (5e)
Dark green crystals, (yield 45%); m.p. 270 ꢁC, IR (KBr, cmꢂ1):
2214 (CꢃN), 3321–3396–3479 (3NH), 2924–2846 (CH Aliphatic).
1H-NMR (300 MHz, DMSO): ꢀ 8.40 (s, 1H, NH¼CH–NH), 7.20 (s,
1H, CH¼C–OCH3), 7.42 (s, 1H, CH¼C–OCH3), 3.90 (s, 6H,
OCH3–Ar), 9.10 (s, 1H, NH-Ar, D2O exchangeable), 6.85 (d, 2H,
CH¼CH–NH anilino), 8.05 (d, 2H, CH¼CH–NH anilino), 10.00
(brs, 1H, NH, D2O exchangeable), 10.50 (brs, 1H, NH, D2O
exchangeable), 7.90 (s, 1H, CH¼C–Ar–H of pyridyl), 7.50 (d, 2H,
CH¼CH–Br), 7.60 (d, 2H, CH¼CH–Br). MS (EI): 553 Mþ 20%,
555.09 Mþ2 19.8%. Anal. for C28H21BrN6O2; C, 60.77%; H, 3.82%;
N, 15.19%. Found: C, 61.03%; H, 3.97%; N, 15.41%.
Figure 3. Superposition of the most active ligand, 5e on the original
inhibitor, lapatinib.
General procedure for the preparation of (E)-3-Aryl-1-[4-
(quinazolin-4-ylamino)phenyl]prop-2-en-1-one (7a–d) and
(E)-3-Aryl-1-[4-(6,7-dimethoxyquinazolin-4-ylamino)phe-
nyl]prop-2-en-1-one (7e)
titratable amino acids are assigned at the same pH. Water
orientation is modified to give the best interaction with the
ligand and the receptor, an all atom impref minimization step is
carried out to remove unfavorable steric clashes till it reaches
A
mixture of the appropriate 4-anilinoquinazoline (4a–b)
(0.01 mol), appropriate aromatic aldehyde (0.01 mol) and 10%
aqueous sodium hydroxide (10 ml) in ethanol (30 ml) was stirred
at room temperature for about 3 h then kept at 0 ꢁC overnight. The
resulting solid was filtered off, rinsed with water, dried and
recrystallized from ethanol.
˚
convergence or a maximum RMSD of 0.3 A from the original
conformation. No steric clashes were reported after the final
minimization step.
Based on the prepared protein structure in the previous
step, receptor grid is prepared with the receptor grid generation
¨
(E)-3-(4-Chlorophenyl)-1-{4-[(6,7-dimethoxyquinazolin-4-yl)ami-
no]phenyl}prop-2-en-1-one (7e)
17
module in Glide 5.7 (Schrodinger, LLC, New York, NY) and
sets up to the default values for vdW radii of 1 (no scaling). The
binding site is determined as a box around the ligand that is
centered inside the box. The ligand-Met793 H-bond was
constrained.
Orange yellow crystals (yield 70%); m.p. above 300 ꢁC. IR (KBr,
cmꢂ1): 1743 (C¼O), 3337 (NH), 2924 (CH aliphatic), 3010 (CH
aromatic). 1H-NMR (300 MHz, DMSO): ꢀ 8.49 (s, 1H, NH¼CH–
NH), 7.24 (s, 1H, CH¼C–OCH3), 7.40 (s, 1H, CH¼C–OCH3), 3.9
(s, 6H, OCH3–Ar), 10.10 (s, 1H, NH–Ar, D2O exchangeable),
7.64 (d, 2H, CH¼CH–NH), 7.71 (d, 2H, CH¼CH–NH), 7.59–
7.54 (d, J ¼ 15 Hz, 1H, CH¼CH–C¼O), 8.06-8.01 (d, J ¼ 15 Hz,
1H, CH¼CH–C¼O), 7.44 (d, 2H, CH¼CH–Cl), 7.68 (d, 2H,
CH¼CH–Cl). MS (EI): 445 Mþ 85.9%, 447 Mþ2 30%. Anal. for
C25H20N3O3Cl; C, 67.34%; H, 4.52%; N, 9.42%. Found: C,
67.47%; H, 4.69%; N, 9.64%.
Ligands preparation
The sequence of ligand preparation is crucial to obtain valid
docking results. In a recent study, Butler et al. have shown that the
active (bound) conformations of small molecular ligands lie in the
vicinity of a near-local minima. It has been demonstrated that
inadequate sampling of ligand conformational space can lead to a
deleterious effect of the results of virtual screening experiments.
That is, the scoring functions can bias toward the input geometry
of the ligand. These findings clearly indicate that it is important to
start the initial configuration of the ligands close to a minimum.
For small scale situations in this study, it is possible for the 21
ligands to be studied using ab initio methods, but, in order to
mimic more closely a real life situation, where ligands are
prepared by a simple force field, or in the best case scenario,
using semiempirical methods such as AM1, PM3, PM618;
ligands are optimized at the PM6 semiempirical level of theory
using the Gaussian 09 computational chemistry program
(Gaussian, Inc., Wallingford, CT)19. Electrostatic potential
Computational methods and details
Protein preparation and receptor grid generation
Crystal structure of the kinase domain of human EGFR co-
crystallized with lapatinib was taken from the PDB (PDB entry:
1xkk)12. Previous studies have shown that in certain cases, minor
side-chain rearrangement may be crucial for allowing ligand–
receptor interaction13. In the current study, the most active ligand
in vitro (5e) is superimposed on the original inhibitor (Figure 3).
This is followed by minimization and short molecular dynamic
(MD) simulation for 1 ns in the NPT ensemble with AMBER
(University of California, San Francisco, CA) to allow complex
relaxation. The relaxed complex is used for the subsequent
charges (ESP) are calculated at the B3LYP/cc-pVTZ level of
¨
20
theory using Jaguar (Schrodinger, LLC, New York, NY) .
calculations. Complex is prepared using the protein preparation
14
wizard using Maestro 9.2 (Schrodinger, LLC, New York, NY) .
Docking and scoring
¨
The structure is saturated by hydrogen atoms, all water molecules
The flexible docking module of Glide extra precision
(Glide XP)17,21 is utilized. In order to increase the sampling
space, a maximum of 50 000 initial ligand poses are kept in the
initial phase of docking. A scoring window of poses within
1000 kcal molꢂ1 from the best scoring pose are retained, from
which a maximum of 800 poses per ligand are subjected to 200
steps of energy minimization. To reduce the number of false
positives in the docking experiment, the ligand pose is only
considered when the predetermined constraint is satisfied.
The output usually needs visual inspections to ensure appropriate
˚
within 5 A of the ligand but for a conserved water molecule (HOH)
which is observed in the ATP hinge region of several kinases15. This
bridge water molecule connects the N(3) of the quinazoline ring of
the inhibitor to the backbone carbonyl oxygen of Gln791. Missing
residues are added and refined using Prime 3.016 (Schrodinger,
¨
LLC, New York, NY). An ACE (N-acetyl) and NMA (N-methyl
amide) groups are added to cap the uncapped N and C termini.
Interactive hydrogen bond (H-bond) network optimization is
carried out assuming a neutral pH. The protonation states of

4
M. M. Sadek et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Scheme 1. Synthesis of 2-iminopyridine derivatives (5a–e)
Reagents and conditions: i ¼ HCONH2/160 ꢁC/4 h, ii ¼ POCl3/TEA/90 ꢁC/2 h, iii ¼ p-aminoacetophenone/iPrOH/Reflux 2 h, iv ¼ Malononitrile/
NH4COOCH3/aldehyde ethanol/Reflux 18 h.
positioning of the quinazoline ring in the hinge region. On the other hand, Scheme 2 described the synthesis of the 4-
˚
The effective region of docking is within a 4 A boundary from aryl-2-oxo-pyridylquinazoline (6a–f). Compounds 4a–b were
the quinazoline core. When rescoring the docked poses, residues prepared as previously explained in Scheme 1 followed by a
˚
in the region of 6 A are allowed to move flexibly, while one-pot reaction of these compounds with ethyl cyanoacetate,
retaining the predefined charges of the ligands previously ammonium acetate, the appropriate aldehyde and ethanol as the
calculated using the B3LYP/cc-pVTZ model. Ligand pose reaction solvent at reflux temperature for 24 h. IR showed the
rescoring is done using the Prime/MM-GBSA module utility in appearance of cyano group between 2200 and 2218 cmꢂ1 and
16,21,22
1
C¼O group at 1651 cmꢂ1, the H-NMR revealed the appearance
¨
Schrodinger, LLC, New York, NY
.
of a second NH group at 12.8 ppm which is also a strong olefinic
proton appeared at 6.8 ppm.
Results and discussion
The targeted compounds (7a–e, 8a–e) were synthesized as
shown in Scheme 3. Compounds 7a–e were prepared according to
Synthesis
Scheme 1 outlines the synthetic pathway used to obtain the general procedures by Kohler and Chadwell23 applying
compounds 5a–e. Compounds (2a–b, 3a–b, 4a–b) were synthe- Claisen Schmidt condensation of compounds 4a–b with sub-
sized according to the previous reported methods. Accordingly, stituted benzaldehydes using NaOH and water. 1H-NMR spectrum
the 4-aryl-2-imino-pyridyl- quinazoline derivatives9,11 (5a–e) revealed the two characteristic doublets for the vinyl protons at ꢀ
were obtained through a one-pot reaction of 4-chloroquinazolines 7.65–7.59 ppm and ꢀ 8.06–8.01 or ꢀ 8.18–8.13 ppm with a
(3a–b) with malononitrile, ammonium acetate and appropriate coupling constant J ¼ 16 or 18 MHz confirming the trans-
aldehyde in the presence of absolute ethanol as a solvent at reflux configuration. The latter compounds (7a–e) were treated with
temperature. IR showed the appearance of cyano group between hydrazine hydrate in presence of acetic acid to afford compounds
2200 and 2218 cmꢂ1, the 1H-NMR revealed the appearance of the 8a–e showing distinctive doublet of doublets’ signals of the
1
NH groups between 9.9 and 10.3 ppm as singlets or broad peaks. pyrazoline ring in the H-NMR spectrum.

DOI: 10.3109/14756366.2013.765417
Discovery of new HER2/EGFR dual kinase inhibitors
5
Scheme 2. Synthesis of 2-oxopyridine derivatives (6a–f)
Reagents and conditions: iv ¼ Ethyl cyanoacetate/NH4COOCH3/aldehyde/ethanol/Reflux 6 h.
Biological evaluation (in vitro tyrosine kinase
enzyme assay)
assay format for profiling the evaluation of protein kinase targets
and all assays are performed in a designated radioactive working
area. Protein kinase assays were performed at ambient temperature
for 20–40 min (depending on the target) in a final volume of 25 ml
according to the following assay reaction recipe: 5 ml of diluted
active protein kinase target (ꢄ10–50 nM final protein concentration
in the assay), 2.5 ml of peptide substrate, 3. ml of kinase assay buffer,
5 ml of compound or 10% DMSO, and 5 ml of 33P-ATP (250 mM
stock solution, 0.8 mCi).
The in vitro kinase inhibition assay was determined for the
synthesized compounds by KINEXUS Corporation, Vancouver,
British Columbia, Canada.
The various protein kinase targets (EGFR and HER2)
employed in the compound-profiling process were cloned,
expressed and purified using proprietary methods. Quality control
testing is routinely performed on each of the targets to ensure
compliance to acceptable standards. 33P-ATP was purchased from
PerkinElmer (Waltham, MA) and ADP-GloTM was purchased
from Promega (Fitchburg, WI). All other materials were of
standard laboratory grade.
The assay conditions for the various protein kinase targets were
optimized to yield acceptable enzymatic activity. In addition, the
assays were optimized to give high signal-to-noise ratio. Protein
Kinase Assay – Radioactive Format Kinexus uses a radioactive
The assay was initiated by the addition of 33P-ATP and
the reaction mixture was incubated at ambient temperature for
20–40 min, depending on the protein kinase target. After
the incubation period, the assay was terminated by spotting
10 ml of the reaction mixture onto Multiscreen phosphocellulose
P81 plate. The Multiscreen phosphocellulose P81 plate was
washed three times for approximately 15 min each in a 1%
phosphoric acid solution. The radioactivity on the P81 plate

6
M. M. Sadek et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Scheme 3. Synthesis of chalocne and pyrazoline derivatives (7a–e, 8a–e).
Reagents and conditions: iv ¼ aldehyde/ethanol/20%NaOH/0 ꢁC overnight, v ¼ NH2NH2.H2O/Glacial CH3COOH/reflux 5 h.
(EMD Millipore Corporation, Billerica, MA) was counted in the and 7e also showed good inhibition and the EGFR activity was
presence of scintillation fluid in a Trilux scintillation counter inhibited by 55% and 66%, respectively, by these compounds
(GMI Inc., Ramsey, MN).
compared to control. Compounds 6c, 6a, 5b, 6e, 6f and 8e showed
Blank control was set up for each protein kinase target which moderate inhibition and the EGFR activity was inhibited in the
included all the assay components except the addition of range of 35%–49% by these compounds compared to control. The
appropriate substrate (replace with equal volume of kinase assay rest of the compounds showed either weak inhibition (530%
buffer). The corrected activity for each protein kinase target was inhibition compared to control) or no significant effects.
determined by removing the blank control value.
While the profiling data for various compounds against HER2
The profiling data for various compounds against EGFR at at 5 mM concentration showed weak-to-good inhibition of HER2
5 mM concentration compared to the positive control compound activity (Table 1), Compound 5e showed the highest inhibition
Staurosporine (Multikinase inhibitor) showed weak-to-good and the HER2 activity was inhibited by 95% by this compound
inhibition of EGFR activity (Table 1). The compound 5e compared to control. Compound 5d also showed good inhibition
showed the highest inhibition and the EGFR activity was inhibited and the HER2 activity was inhibited by 65% by this compound
by 74% by this compound compared to control. Compounds 5d compared to control. Compounds 6b, 6c and 6e showed moderate

DOI: 10.3109/14756366.2013.765417
Discovery of new HER2/EGFR dual kinase inhibitors
7
Table 1. Percent activity of EGFR/HER2 in the presence of various compounds using radiometric assay.
% Activity at 5 mM
Enzyme assay (IC50) (mM)
% Activity at 5 mM
Enzyme assay (IC50) (mM)
Compound
EGFR
HER2
EGFR
HER2
Compound
EGFR
HER2
EGFR
HER2
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
ꢂ35
ꢂ42
ꢂ23
ꢂ55
ꢂ74
ꢂ6
ꢂ15
ꢂ25
ꢂ2
7a
7b
7c
7d
7e
8a
8b
8c
ꢂ23
ꢂ27
ꢂ13
ꢂ17
ꢂ66
ꢂ20
ꢂ22
ꢂ29
ꢂ12
ꢂ47
ꢂ69
ꢂ2
19
5
ꢂ1
ꢂ18
ꢂ1
19
32
67
ꢂ65
ꢂ95
ꢂ19
ꢂ40
ꢂ35
ꢂ19
ꢂ43
ꢂ24
2.088
1.935
3.981
1.036
2.582
98.77
ꢂ23
ꢂ35
ꢂ28
ꢂ46
ꢂ49
8d
8e
1
ꢂ41
Staurosporine
Negative sign means enzymatic activity is reduced by this value, positive means enzymatic activity is potentiated and IC50 for the three most active
compounds 5d, 5e and 7e in EGFR and HER2 enzyme assay.
inhibition and the HER2 activity was inhibited in the range of guarantees the interaction between the quinazoline ring and the
35%–43% by these compounds compared to control. The rest of receptor and most probably ligands lacking this interaction are
the compounds showed either weak inhibition (530% inhibition inactive. Another important binding motif is the two methoxy
compared to control) or no significant effects.
tails at the 6 and 7 positions of the quinazoline rings, accordingly,
Based on the in vitro enzyme inhibition at 5 mM, the most these tails seems to serve two functions: first, pushing the
potent compounds 5d, 5e and 7e were subjected to a tyrosine quinazoline ring into the hinge region thus cause tight packing
kinase enzyme assay at different concentration to evaluate the and filling of the hinge region cavity; second, forms a strong
phosphotransferase activity of EGFR and/or HER2 protein kinase lipophilic interaction and H-bonding with Leu718. This effect
targets and determine the IC50 concentration of each. From the may account for the relatively high in vitro activity of 5e
observed data (Table 1, Supplemental information Figure 1), the compared to 5c which has the same chemical structure of 5e but
aryl 2-imino-1,2-dihydropyridine derivatives, 5d and 5e, dis- lacks these two methoxy tails, so, it lacks the lipophilic interaction
played the most potent inhibitory activity on EGFR with IC50 with Leu718 and also it is less tightly packed into the hinge
equal to 2.09 and 1.94 mM, respectively, and for HER2 with IC50 region, and having a slightly longer H-bonds interactions with
˚
˚
equal to 3.981 and 1.036 mM, respectively; while 7e showed Met793 (5e: 1.947 A, 5c: 2.132 A).
potent inhibitory activity on EGFR with an IC50 equal to
In addition to these hinge-specific interactions, non-specific
2.582 mM. Furthermore, the cytotoxicity assessment against contributions from individual amino acid residues may also
MDA-MB breast cancer known to overexpress EGFR was exist. For example, the flexible part of the molecules (4-
performed for the selected three compounds and showed IC50 in anilino moiety) is able to bind the selectivity pocket noticed
the range of 2.4–2.5 mM.
in the DFG-out (inactive) conformation of the EGFR. This
pocket is formed by amino acids Thr790, Met766, Leu777,
Thr854 and Cys775.
Molecular modeling study
One may also expect that the substitution of the terminal
flourobenzyl group by other bulky groups of different nature may
give raise to different H-bonding interaction with those residues
present in the selectivity pockets, and this has been already
verified. Owing to the rigid receptor docking nature of Glide-XP,
such H-bonding interaction cannot be identified easily and usually
molecular dynamic simulations are of absolute necessity25. It also
needs certain degree of receptor flexibility of kinases which are
known to be highly flexible. Indeed, running MD simulations that
are long enough (60–80 ns) has verified that in addition to the
hinge region H-bonds interactions, certain members of the new
series are capable of forming other ‘‘extra-hinge’’ H-bonding
interactions with EGFR. Particularly, those inhibitors whose ring
‘‘D’’ (Figure 2) is an iminopyridine ring, for example, 5a, 5b, 5d
and 5e, are capable of forming two H-bonds with Asp855 and
Lys745. Figure 4 displays the interaction of ring ‘‘D’’ of the most
active inhibitor ‘‘5e’’ with these two amino acid residues, Asp855
and Lys745 after an 80 ns MD simulation in AMBER. This may
also explain why these members may have higher %inhibition
activity than those analogues that have a 2-pyridone ring instead,
such as 6a, 6b, 6d, 6e and 6f. Research in this direction is
currently in progress.
Docking study was carried out for the target compounds into the
EGFR crystal structure complexed with Lapatinib (PDB code:
17,21
¨
Ixkk) using Glide extra precision (Glide XP) (Schrodinger)
.
(Supplemental information Figure 2), the experimental percent-
ages of inhibitions (%inhibition) are plotted against (a) XP G-
score, (b) XP_LipophilicEvdW, (c) Prime/MM-GBSA DG and
(d) Prime/MM-GBSA DG bind vdW scoring functions. This can
be considered as a simple consensus scoring in which several
scoring functions are used to rank the ligands. As can be seen
from the Pearson’s correlation coefficient (r) and the coefficient
of determination (r2), a moderate-to-good correlation is achieved.
For example, the correlation of (a)–(d) is given by 0.42, 0.78, 0.30
and 0.42, respectively. The most correlated case is 4(b), the
XP_LipophilicEvdW (r ¼ 0.78 and r2 ¼ 0.61) scoring function. It
was shown previously that the highest correlation with experi-
mental binding affinity in inhibitor-EGFR interaction stems from
the lipophilic vdW interaction15 (Supplemental information
Table 1). This is also verified in the current study where the
highest correlation occurs with the vdW forces. Cho et al. also
showed that for such receptors where binding is mostly driven by
lipophilic interactions with several anchor points such as H-
bonds, QM/MM docking is the best to pursue in that case by
taking into account the protein polarizing effect on the ligand
charges24, research in that direction is currently in progress.
Conclusion
The most important binding anchor is this inhibitor-Met793 Some compounds belonging to a class of anilinoquinazoline
H-bond (Supplemental information Figure 3). This H-bond bearing bulky arylpyridinyl 5, 6, arylpropenoyl and
7

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Figure 4. 3D inhibitor–protein interactions of the most active inhibitor
(5e) with EGFR after 70 ns MD simulation.
arylpyrazolyl 8 moieties at the 40 position of the anilinoquinazo-
line scaffold were prepared. Compounds (5d, 5e and 7e) exhibited
highly potent EGFR and HER2 inhibitory activity at 5 mM. IC50
was determined for these most active compounds on EGFR
tyrosine kinase and displayed IC50 range equal to 2.09 and
1.94 mM for 5d and 5e, respectively, and 3.981 and 1.036 mM,
respectively, on HER2. Moreover, the anti-proliferative activity of
the same compounds against MDA-MB-231 breast cancer cell
lines showed an IC50 of 2.4 and 2.5 mM, respectively. Molecular
docking study further supported the strong inhibitory activity of
5d and 5e, which helps understanding the various interactions
between the ligands and enzyme active sites in detail and thereby
could enable us to design more selective inhibitors.
¨
14. Schrodinger Suite 2011 Protein Preparation Wizard; Epik version
2.2 S, LLC, New York, NY, 2011; Impact version 5.7, Schrodinger,
¨
¨
LLC, New York, NY, 2011; Prime version 3.0, Schrodinger, LLC,
New York, NY, 2011. Schrodinger Suite 2011 Protein Preparation
¨
¨
Wizard; Epik version 2.2, Schrodinger, LLC, New York, NY, 2011;
Impact version 5.7, Schrodinger, LLC, New York, NY, 2011; Prime
¨
¨
version 3.0, Schrodinger, LLC, New York, NY, 2011.
Acknowledgements
15. Balius TE, Rizzo RC. Quantitative prediction of fold resistance for
inhibitors of EGFR. Biochemistry 2009;48:8435–48.
16. Jacobson MP, Pincus DL, Rapp CS, et al. A hierarchical approach to
all-atom protein loop prediction. Proteins Struct Funct Bioinf
2004;55:351–67.
MA acknowledges the Swinburne University Postgraduate Research
Award (SUPRA). National Computational Infrastructure (NCI) at the
Australian National University for the award under the Merit
Allocation Scheme, Victorian Partnership for Advanced Computing
(VPAC) and Swinburne University Supercomputing Facilities are also
acknowledged.
¨
17. Glide v, Schrodinger, LLC, New York, NY, 2011; Glide, version 5.7,
¨
Schrodinger, LLC, New York, NY, 2011.
18. Stewart J. Optimization of parameters for semiempirical methods V:
modification of NDDO approximations and application to 70
elements. J Mol Model 2007;13:1173–213.
19. Frisch MJ, Trucks GW, Schlegel HB, et al. Gaussian 09 RA.
Wallingford (CT): Gaussian, Inc.; 2009.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
¨
20. Jaguar v, Schrodinger, LLC, New York, NY, 2011; Jaguar, version
¨
7.8, Schrodinger, LLC, New York, NY, 2011.
21. Friesner RA, Murphy RB, Repasky MP, et al. Extra precision
glide: docking and scoring incorporating a model of hydrophobic
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