In silico guided development of imine-based inhibitors for resistance-deriving kinases

Article abstract of DOI:10.1080/07391102.2018.1491893

Two major mechanisms involved in resistant NSCLC (non-small cell lung cancer) include secondary acquired mutation in EGFR (epidermal growth factor receptor), that is, EGFR T790M and amplification of c-MET (hepatocyte growth factor receptor). Thus, already established pharmacophore models of EGFR T790M and c-MET were employed to filter-out an in-house database. Further fitness score led to the selection of imino-pyrimidine scaffold. Followed by sketching of imino-pyrimidine derivatives having varied aryl substitutions, which were then docked and subjected to molecular dynamic simulations, to study the orientations and conformations of the designed molecules in the catalytic domain. Molecules with hydrophobic interaction with mutant residue M790 were selected. Finally, MM-GBSA (Molecular Mechanics‐Generalized Born Surface Area) calculations were performed, to study the effect of substitutions on the binding affinity of the double mutant EGFR towards these small molecules. Finally, the designed compounds were synthesized and evaluated for their kinase inhibitory potential using in-vitro experiments. Two compounds were found to possess sub-micromolar range inhibitory potential against EGFR (T790M), while one of the compound showed significant selective inhibitory potential against c-MET. Additionally, one compound was found to possess significant dual inhibitory potential against these target kinases. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2018.1491893

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
In-silico guided development of imine based
inhibitors for resistance-deriving kinases
Pankaj Kumar Singh & Om Silakari
To cite this article: Pankaj Kumar Singh & Om Silakari (2018): In-silico guided development
of imine based inhibitors for resistance-deriving kinases, Journal of Biomolecular Structure and
Dynamics, DOI: 10.1080/07391102.2018.1491893
To link to this article: https://doi.org/10.1080/07391102.2018.1491893
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In-silico guided development of imine based inhibitors for resistance-
deriving kinases
Pankaj Kumar Singh, Om Silakari *
Molecular Modelling Lab (MML), Department of Pharmaceutical Sciences and Drug
research, Punjabi University, Patiala, Punjab, 147002, India
*
Corresponding authors E-mail: omsilakari@gmail.com; Tel: +91-9501542696; Fax:
91-1752283075
+

In-silico guided development of imine based inhibitors for resistance-
deriving kinases
Two major mechanisms involved in resistant NSCLC (Non-small cell lung
cancer) include secondary acquired mutation in EGFR (epidermal growth factor
receptor), i.e., EGFR T790M and amplification of c-MET (hepatocyte growth
factor receptor). Thus, already established pharmacophore models of EGFR
T790M and c-MET were employed to filter-out an in-house database. Further
fitness score led to selection of imino-pyrimidine scaffold. Followed by sketching
of imino-pyrimidine derivatives having varied aryl substitutions, which were then
docked and subjected to molecular dynamic simulations, to study the orientations
and conformations of the designed molecules in the catalytic domain. Molecules
with hydrophobic interaction with mutant residue M790 were selected. Finally
MM-GBSA (Molecular Mechanics‐Generalized Born Surface Area) calculations
were performed, to study the effect of substitutions on binding affinity of double
mutant EGFR towards these small molecules. Finally, the designed compounds
were synthesized and evaluated for their kinase inhibitory potential using in-vitro
experiments. Two compounds were found to be possess sub-micromolar range
inhibitory potential against EGFR (T790M), while one of the compound showed
significant selective inhibitory potential against c-MET. Additionally, one
compound was found to possess significant dual inhibitory potential against
target kinases.
Keywords: imino-pyrimidine; molecular dynamic simulations; EGFR (T790M);
c-MET
Abbreviations: NSCLC - Non-small cell lung cancer; EGFR - epidermal growth factor
receptor; c-MET - hepatocyte growth factor receptor; MM-GBSA - Molecular
Mechanics‐Generalized Born Surface Area; ATP – Adenosine triphosphate; PI3K -
Phosphatidylinositide 3-kinase; Akt - Protein Kinase B; ErbB3 - Human epidermal
growth factor receptor 3; TKIs – Tyrosine kinase inhibitors; mTOR – Mammalian target
of rifampicin; XP - Extra precision; MD - Molecular dynamics; OPLS - Optimized
Potentials for Liquid Simulations; TIP3P – 3 site water model; FEP - free energy
perturbation; FRET - Förster resonance energy transfer.

Introduction
Small molecule heterocycles form a major class of anti-cancer agents especially kinases
inhibitors. Previous attempts to develop heterocycles as kinase inhibitors resulted in
several success stories such as gefitinib, erlotinib, etc (Singh, Singh, & Silakari, 2016).
However, clinical responsive success of these kinase targeted inhibitors has been
observed to be short lived due to development of acquired resistance to these drugs.
Multiple mechanisms have been identified as the cause for the failure of these
molecules which include acquired secondary mutation T790M in EGFR, observed in
about 50% of patients with resistance, and amplification of c-MET in the L858R mutant
EGFR overexpressed lung cancer (Singh and Silakari, 2017a). Studies revealed that
secondary acquired mutation alters the affinity of the kinase back in favour of ATP
(Kobayashi et al., 2005). To counter this secondary acquired mutation, researchers
focussed on developing second generation kinase inhibitors. However, they suffered
with lack of selectivity which further led to the development of third generation kinase
inhibitors, which are covalent but are selective towards double mutant EGFR.
Selectivity was claimed due to the hydrophobic interactions between the inhibitors and
mutated residue M790 in the catalytic domain of the double mutant kinase. But recent
disclosure of another acquired mutation C797S in EGFR has put a question mark over
their applicability. Several reports have suggested that third generation inhibitors are
ineffective in case of C797S mutant EGFR.
Another key mechanism of resistance against kinase inhibitors in NSCLC is
amplification of c-MET upon administration of first generation kinase inhibitors
targeting EGFR (Cappuzzo et al., 2008). Amplification of c-MET leads to the
reactivation of PI3K/Akt signaling pathway by forming a MET-ErbB-3 heterodimer,
previously formed by EGFR. Thus, despite continuous inhibition of EGFR, a critical

downstream signalling pathway is reactivated, and resistance to the inhibitors emerge
Yano et al., 2011).
(
Thus with the development of these resistance mechanisms, there is a dire need to
develop molecules which can successfully inhibit mutant and cross-talking kinases
(Bivona and Doebele, 2016; Jänne et al., 2015). As in the patients with resistance to
first-generation EGFR TKIs generated by MET amplification, it is not possible for third
generation EGFR inhibitor to be effective. But the combination of an EGFR (T790M)
inhibitor and an mTOR/cMET/Akt signalling pathway inhibitor may form an effective
strategy to overcome resistance (Li et al., 2008). Also, clinical trials are underway to
test the effect of dual inhibition of c-MET and EGFR to overcome this mode of
resistance (Robinson and Sandler, 2013). These reports formed the rationale for our
study, in which we focussed on blocking c-MET and EGFR (T790M) via reversible
dual inhibitors. The key concept of the study was the fact that sensitivity towards
inhibitors is not lost in T790M EGFR rather the affinity towards ATP increases. Special
attention was also given to the fact that molecules having hydrophobic interaction with
mutant residue M790 provide selectivity to the molecules and thus, may also enhance
the binding affinity. Thus we attempted to design molecules with higher affinity
towards T790M EGFR along with c-MET using in-silico techniques.
Material and Methods
In-silico study
Previously published pharmacophore models for EGFR (T790M) and c-MET (Singh
and Silakari, 2017b) were utilized to screen an in-house database of synthetically
feasible small molecule heterocycles via pharmacophore mapping tool available with
Discovery studio software (Biovia, 2015). After screening, molecules with good fitness

score, close to score of active molecules in the training set of pharmacophores, were
subjected to structure based drug designing techniques such as molecular docking. The
docking studies were carried out using Glide program of Schrodinger software (Friesner
et al., 2004). The screened molecules were docked into the grid of PDB ID: 2W2P for
EGFR (T790M) and subsequently into the grid of 3DKF for c-MET, using extra
precision (XP) docking module, which predicts the binding modes and their Glide XP
G-score. Generated docking poses were then analysed based on the conserved
molecular interactions reported mandatory to possess significant inhibitory potential in
both the kinases. Along with hydrogen bonds between the ligand and the active site
residues Met793, Lys745 and others, molecules were analysed for hydrophobic
interaction with the mutant residue M790 in EGFR (T790M). While, in c-MET, poses
having interactions with Met1160 and other residues in the active site were selected.
To check the stability of the designed molecules in both T790M EGFR and c-MET, the
docked complexes were subjected to molecular dynamics (MD) simulations for a time
period of 30 ns. The MD simulations were carried out with OPLS_2005 force field in
Desmond software of Schrödinger (Release, 2014). An octahedral water box of 1 nm
thickness was generated and solvated using a TIP3P water model. At physiological pH,
protein-ligand complexes were charged accordingly, the system was neutralized by
adding counter-ions and salt concentration was fixed to 0.15 M. After the energy
minimization, the NPT equilibration was conducted at 310 K. A Nose-Hoover chain
method thermostat was used to maintain constant temperature. NVT was followed by
NPT equilibration applied at a pressure of 1 bar, maintained by a Martyn-Tobias-Klein
barostat. During equilibration, the protein backbone was restrained and the solvent,
molecules along with counter-ions were allowed to move. The MD simulations were
performed under periodic boundary conditions to avoid edge effects. The simulations

were conducted with a time step of 1 fs and the coordinate data were stored in the file.
The trajectory potentials were obtained from each MD simulation. RMSD value was
calculated using ‘simulation event analysis’ protocol of Desmond for each T790M
EGFR and c-MET complex. The binding orientations of the ligand within the protein
were studied using a simulation interaction diagram. Finally, the binding energies of the
molecules with both the target kinases were calculated using Prime MM-GBSA module.
This methodology calculates the binding energy of ligand with the receptor as the sum
of gas-phase internal energy, estimated using a molecular mechanics force field, and the
solvation free energy, calculated using an implicit solvent model (Generalized Born
Surface Area). Additionally, in some cases, change in entropy upon binding is also
considered in the calculations to improve the accuracy of the binding affinity
predictions. MM-GBSA method is significantly faster than FEP (free energy
perturbation) calculations, however it suffer from larger uncertainties than FEP, but in
most cases is able to predict relative binding affinities in reasonable agreement with
experimental data (Parenti and Rastelli, 2012).
Chemistry
Commercially available reagents and solvents were used for the synthetic purpose. Pre-
coated TLC plates were used to monitor the reactions. Column chromatography was
carried out on silica gel 60-120 mesh for the purification and separation of compounds
using a combination of ethyl acetate and petroleum ether. Melting points were obtained
in open capillary tubes on a melting point apparatus. The IR spectra of the molecules
1
13
were recorded on a FT-IR spectrophotometer, using KBr pellet. H NMR and C NMR
spectra of the molecules were generated on a Bruker spectrophotometer at 400MHz and
1
00MHz (TMS as internal standard), respectively using CDCl or DMSO as solvent.
3

Mass spectra were also recorded on Waters Q-TOF micro ESI-MS spectrometer at
positive ionization mode (ESI+). The MS peaks were recorded as m/z ratio.
Synthesis of (E)-1-(4-bromophenyl)-3-(4-chlorophenyl)prop-2-en-1-one (3): To the
mixture of 4’-bromoacetophenone (1) (500 mg, 0.0025 mol) in glacial acetic acid (5
ml), few drops of conc. H SO were added. Reaction mixture was kept on stirring. After
2
4
5
minutes, 4-chlorobenzaldehyde (2) (1 eq., 350 mg) was added into the reaction
mixture. Further reaction mixture was left on stirring for 4 hours at 50°C. TLC
confirmed the formation of product 3 (chalcone). After completion of reaction cold
water was poured into the reaction mixture. Precipitates obtained was filtered, dried and
recrystallized in methanol to afford the pure product.
Synthesis of 4-(4-bromophenyl)-6-(4-chlorophenyl)pyrimidin-2-amine (4): To a solution
of 3 (320 mg, 0.001 mol) in 5 ml of methanol, 2 eq. NaOH was added. After 5 minutes,
1
eq. guanidine.HCl was added. The reaction mixture was then kept on stirring for 12
hours at 50°C (TLC). After completion of reaction 1 % HCl solution was poured into
the reaction mixture to obtain the crude product (4).
Synthesis of final compounds (5a-5i): To a 180 mg (0.0005 mol) solution of 4 in 10 ml
acetic acid, variedly substituted benzaldehydes/acetophenones (1 eq.) were added. The
reaction was then kept on reflux for 48 h. Completion of reaction was monitored via
TLC. The reaction mixture was poured in to cold water and crude product was filtered.
Further column chromatography (Silica gel # 60-120; Petroleum Ether: Ethyl acetate::
8
0:20) was performed to afford pure compounds.

In-vitro evaluation
In-vitro evaluation of inhibitory potential of the synthesized compounds was performed
against EGFR (L858R), EGFR (T790M) and c-MET, using Z-LYTE® kinase assay
(Goddard and Reymond, 2004). The Z´-LYTE® biochemical assay employs a
fluorescence-based, coupled-enzyme format and is based on the differential sensitivity
of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide
substrate is labelled with two fluorophores—one at each end—that make up a FRET
pair. A ratio metric method, which calculates the ratio (the Emission Ratio) of donor
emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is
used to quantitate reaction progress. A significant benefit of this ratio metric method for
quantitating reaction progress is the elimination of well-to-well variations in FRET-
peptide concentration and signal intensities.
In brief, for EGFR (L858R) inhibitory activity, Z'-LYTE™ Kinase Assay Kit -
Tyrosine 4 Peptide, purchased from Invitrogen Bioservices India Pvt.Ltd, Cat. No:
PV3193 was utilized. The 2X EGFR (ErbB1) L858R / Tyr 04 mixture was prepared in
5
0 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 4 mM MnCl2, 1 mM EGTA, 2
mM DTT. The final 10 µL Kinase Reaction consists of 0.2 - 1.68 ng EGFR (ErbB1)
L858R and 2 µM Tyr 04 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 2
mM MnCl2, 1 mM EGTA, 1 mM DTT. After the 1 hour Kinase Reaction incubation, 5
µL of a 1:64 dilution of Development Reagent B was added, mixed in the assay plate
and incubated the 15-µL development Reaction for 1 hour at room temperature.
Afterwards, the kinase reaction was stopped by adding 5 µL of Stop reagent. Finally,
the fluorescence signals were measured, using the Fluorescence Spectrophotometer,

Make: Varian/ Perkin Elmer, Model: CARY-Eclipse/ LS-50B at 445 nm and 520 nm for
both donor and acceptor emission, respectively, to calculate the emission ratio.
For EGFR (T790M) inhibitory activity, Z'-LYTE™ Kinase Assay Kit - Tyrosine
4
Peptide, purchased from Invitrogen Bioservices India Pvt.Ltd, Cat. No: PV3193 was
utilized. The 2X EGFR (ErbB1) T790M / Tyr 04 mixture was prepared in 50 mM
HEPES pH 6.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.02% NaN3. The final
1
0
0 µL Kinase Reaction consists of 3.9 - 39.3 ng EGFR (ErbB1) T790M and 2 µM Tyr
4 in 50 mM HEPES pH 7.0, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.01%
NaN3. After the 1 hour Kinase Reaction incubation, 5 µL of a 1:64 dilution of
Development Reagent B was added, mixed in the assay plate and incubated the 15-µL
development Reaction for 1 hour at room temperature. Afterwards, the kinase reaction
was stopped by adding 5 µL of Stop reagent. Finally, the fluorescence signals were
measured, using the Fluorescence Spectrophotometer, Make: Varian/ Perkin Elmer,
Model: CARY-Eclipse/ LS-50B at 445 nm and 520 nm for both donor and acceptor
emission, respectively, to calculate the emission ratio.
For c-MET inhibitory activity, Z'-LYTE™ Kinase Assay Kit - Tyrosine 6
Peptide, purchased from Invitrogen Bioservices India Pvt.Ltd, Cat. No: PV4122 was
utilized. The 2X MET (cMet) / Tyr 06 mixture was prepared in 50 mM HEPES pH 7.5,
0
.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 µL Kinase Reaction
consists of 0.49 - 11.2 ng MET (cMet) and 2 µM Tyr 06 in 50 mM HEPES pH 7.5,
.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After the 1 hour Kinase Reaction
0
incubation, 5 µL of a 1:128 dilution of Development Reagent A was added, mixed in
the assay plate and incubated the 15-µL development Reaction for 1 hour at room

temperature. Afterwards, the kinase reaction was stopped by adding 5 µL of Stop
reagent. Finally, the fluorescence signals were measured, using the Fluorescence
Spectrophotometer, Make: Varian/ Perkin Elmer, Model: CARY-Eclipse/ LS-50B at
4
45 nm and 520 nm for both donor and acceptor emission, respectively, to calculate the
emission ratio.
Calculate Emission Ratio: The Emission Ratio for each well on the assay plate was
calculated by dividing the coumarin emission signal (445 nm) by the fluorescein
emission signal (520 nm).
Emission Ratio= Coumarin Emission (445 nm)/ Fluorescein Emission (520 nm)
Calculate Percent Phosphorylation: The extent of phosphorylation of each sample well
(containing kinase) was determined according to the 0% and 100% Phosphorylation
Control wells. There is a non-linear relationship between Emission Ratio and
Phosphorylation, which the following equation accounts for:
%
Phosphorylation=1-[{(Emission Ratio x F100%) – C100%}/{(C0% –
C100%)+ [Emission Ratio x (F100% – F0%)]} ]
Where: C100% = Average Coumarin emission signal of the 100% Phos. Control; C0%
=
Average Coumarin emission signal of the 0% Phos. Control; F100% = Average
Fluorescein emission signal of the 100% Phos. Control; F0% = Average Fluorescein
emission signal of the 0% Phos. Control

Result and Discussion
In-silico analysis
Previously reported ligand-based pharmacophore models of EGFR (T790M) and cMET,
published by our group were employed to screen and cross screen an in-house small
molecule database containing more than 200 molecules with diverse scaffolds such as
benzimidazole, oxindole, indole, flavones and thiazolidinones. On the basis of fitness
score, molecules were selected and subjected to structure based drug designing
techniques. This screening yielded a total of 18 molecules with imino-pyrimidine
scaffold, which were then subjected to molecular docking analysis using co-crystallized
3
-D structure of both the target kinases followed by molecular dynamic simulations and
calculation of MM-GBSA score (binding energies). For molecular docking, the PDB
were selected utilizing resolution as cut-off followed by cross docking protocol
(supplementary table s1, s2, s3 and s4). Docking analysis in EGFR T790M revealed that
hydroxyl substitutions on the imine ring of some of the designed compounds act as
donor group and interacts via hydrogen bonds with Met793; nitrogen atom at the first
and third position of pyrimidine act as acceptor and forms hydrogen bonding interaction
with Gln791 amino acid residue. Other interacting amino acid included Lys745 and
Glu762, present in the hinge region of the catalytic domain. While, in c-MET, key
interactions included hydrogen bond between imine ring substitutions and conserved
residues such as Met1160 and Ile1084. Additionally, π-π stackings between aryl rings at
4
th/6th position of pyrimidine and Tyr1159 and other residues were also observed. All
the compounds showed good docking scores (Glide XP G-score) in both the kinases;
ranging from -5.22 to -2.19 within the EGFR (T790M) protein and -8.31 to -5.53 in
cMET. Docking was followed with molecular dynamic simulations of the designed
molecules in complex with EGFR (T790M) as well as cMET, for a period of 30 ns.

Simulations disclosed that almost all the molecules were maintaining key H-bond
interactions with hinge region amino acids. A key disclosure was the fact that nine
molecules were maintaining varied levels of hydrophobic interactions with mutated gate
keeper residue M790 which is essential for the selectivity and potency of molecules
against double mutant EGFR (T790M) which were then forwarded for synthesis and in-
vitro evaluation. The RMSD values of the protein backbone atoms in complex with top
compounds (5a and 5i) were calculated to observe the overall stability of the protein and
ligand within the period of time period of 30 ns (fig.1 and fig.2).
Figure 1. RMSD plot of 5a in complex with cMET for the time period of 30 ns.

Figure 2. RMSD plot of 5i in complex with EGFR (T790M) for the time period of 30ns.
The 3D interaction diagram of the best designed molecules 5a in cMET and 5i in EGFR
T790M are shown in fig.3 and fig.4, respectively. Finally, binding energy scores,
calculated using MM-GBSA protocol, reflected significant affinity for the designed
molecules in both the targets. The score of all the compounds lied in the range of -
7
7.168 to -60.554 (units) in EGFR (T790M) and -87.191 to -52.018 in cMET (Table 1).

Figure 3. 3D interaction diagram along with contact summary of 5a in complex with
cMET after MD simulation of 30ns.
Figure 2. 3D interaction diagram along with contact summary of 5i in complex with
EGFR (T790M).after MD simulation of 30ns.
Table 1. Designed compounds with their binding energies and predicted activity.
S.No. Compound MM-GBSA
binding energy EGFR(T790M)
EGFR(T790M) (µM)
Predicted activity- MM-GBSA
Predicted
binding energy activity-
(
cMET
cMET (µM)
1
2
3
4
5
6
7
8
9
.
.
.
.
.
.
.
.
.
5a
5b
5c
5d
5e
5f
-69.602
-64.294
-71.219
-60.554
-77.168
-63.300
-74.635
-71.787
-73.986
0.323
9.849
3.146
1.619
0.105
9.575
10.013
9.851
0.245
-73.683
-52.018
-59.079
-57.750
-82.369
-72.316
-65.636
-87.191
-71.219
0.315
1.477
0.301
1.452
0.300
1.453
1.506
1.555
0.290
5g
5h
5i

Chemistry
For synthesizing designed molecules, firstly, claisen-schmidt condensation was
followed to synthesize 1,3-diaryl propenone via p-bromoacetophenone and p-
chlorobenzaldehyde using acetic acid both as solvent and few drops of conc. H SO as
2
4
catalyst. The obtained 1,3-diaryl propenone was purified via recrystallization. Then,
,3-diaryl propanone was subsequently treated with guanidine hydrochloride using
1
sodium hydroxide as catalyst and methanol as solvent. This reaction resulted in the
formation of bi-aryl substituted amino pyrimidine. The obtained product was purified
via recrystallization. In the final step, bi-aryl substituted amino pyrimidine was treated
with variedly substituted benzaldehydes/acetophenones using acetic acid as catalyst and
solvent. Upon completion of reaction, reaction mixture was poured into cold water to
obtain crude imino-pyrimidines. Finally column chromatography was performed to
1
obtain pure derivatives (scheme 1). All the compounds were characterized by IR, H-
1
3
NMR and C-NMR. In IR spectrum, the imino-pyrimidine derivatives showed the
presence of strong absorption bands of multiple C=N from ~1680 to ~1580 cm-1. The
1
synthesis of final compounds were confirmed in H-NMR. Almost each spectrum
showed presence of singlet of one proton from ~7.4 to ~7.8 ppm, present in pyrimidine
nucleus. Rest of the aromatic protons were observed in similar pattern from ~7.4 to ~
8
.4 ppm. A singlet of three proton at ~2.5 ppm was also observed for substituted
1
3
acetophenone derivatives. C spectrum exhibited characteristic four peaks from ~169 to
~157 ppm for different iminic carbons. Rest of the aromatic carbons were observed
from 124 to 139 ppm with a characteristic peak at 106 for C-5 of pyrimidine. Similarly,
mass spectrometry also showed quasi ion peaks at expected m/z values.

Scheme 1. Synthetic scheme of designed imino-pyrimidine analogues.
In-vitro evaluation
Finally, the molecules were subjected for the in vitro evaluation of kinase inhibitory
potential via enzymatic assay against EGFR (T790M) and cMET, additionally
evaluation against EGFR (L858R) was also performed, which is involved in
development of NSCLC in around 50 % patients on first place. Appropriate standard
(i.e. erlotinib), controls and test samples were utilized. The test compounds were used at
the concentration of 0.1, 1 and 10 µM, and inhibitory potential was characterized by
IC50 value. Complying with the theoretical studies, compound 5i with tri-hydroxyl
substitutions on the imine ring, on the pyrimidine scaffold, was found to possess
significant selective EGFR (T790M) inhibitory activity over EGFR (L858R) with IC50
value 0.268 µM in EGFR (T790M) and 1.686 µM in EGFR (L858R). It also showed
significant cMET inhibitory potential with IC value 0.757 µM. As observed in the MD
5
0
simulation studies, 5i maintained H-bond interactions with key residues due to multiple
OH groups which may have resulted in its improved activity in comparison to other
–
molecules. Additionally, its energy-minimized conformation in the catalytic domain
was found to orient in such a manner that it formed and retained hydrophobic

interaction with mutated residue M790 potentially resulting in its selectivity for the
mutant EGFR.
Theoretically, compound 5e was expected to be most potent EGFR (T790M) inhibitor
but upon experimental validation, in comparison to other molecules, it showed sub-
optimal activity with IC50 value of 0.691 µM. One explanation could be its stable
conformation in the catalytic domain of the mutant protein, which resulted in its
reduced interaction with M790 and other key residues. Another compound 5f having
nitro substitution on the imine ring showed excellent non-selective inhibition in both
EGFR (T790M) and EGFR (L858R) with IC values 0.545 and 0.325 µM, respectively.
5
0
Additionally it also showed modest cMET inhibitory activity with IC value 0.571 µM.
5
0
Compound 5a with 2-hydroxy substitution was found to be most potent cMET inhibitor
with IC value 0.553 µM as summarized in Table 2. The inhibitory potential against
5
0
EGFR (T790M) can be correlated with hydrophobic interactions between mutated
residue M790 and aryl substitutions on the pyrimidine scaffold. Methyl and nitro
substitutions were found to be most optimum for significant multi-kinase inhibitory
potential. Overall, the compounds with methyl substitution at iminic carbon were found
to be more potent EGFR inhibitors in comparison to unsubstituted derivatives.

Table 2. In-vitro kinase inhibitory data of designed compounds against EGFR
T790M), EGFR (L858R), cMET
(
S.NO. Compound
ID
R
R1
EGFR
EGFR
cMET (µM)
(T790M)
(L858R) (µM)
(
µM)
0.815
.015)
1.053 (±0.014) 3.332 (± 0.013) 0.697 (± 0.011)
-4-NH2 0.740
.016)
0.419
.014)
-2,4-OH 0.691
.012)
-CH3 -3-NO2 0.545
.012)
0.442
.015)
0.784
.012)
1
.
5a
-CH3 -2-OH
(± 0.090 (± 0.009) 0.553 (± 0.010)
0
2
3
.
.
5b
5c
-H
-H
-CH3
(± 0.749 (± 0.013) 0.721 (± 0.011)
(± 1.205 (± 0.011) 0.857 (± 0.010)
(± 0.170 (± 0.018) 0.754 (± 0.010)
(± 0.325 (± 0.013) 0.571 (± 0.010)
(± 1.253 (± 0.011) 0.709 (± 0.012)
(± 0.472 (± 0.010) 1.955 (± 0.011)
(± 1.686 (± 0.013) 0.757 (± 0.010)
(± 0.032 (± 0.009) ------
0
4
5
6
7
8
9
1
1
.
5d
-H
-4-OH
0
.
5e
-CH3
0
.
5f
0
.
5g
-H
-4-Cl
-Br
0
.
5h
-CH3
0
.
5i
- CH3 -2,4,6-
OH
0.268
0.017)
0.907
0.
1.
Erlotinib
XL184
-------- --------
0
.011)
------
-------
------
------
0.030 (± 0.010)
Conclusion
Overall, in this study in-silico tools were employed to develop some reversible dual
inhibitors of EGFR (T790M) and cMET. Molecules were designed considering their
binding affinity, for both the kinases, as a vital parameter. Compound 5a, 5f and 5i

showed significant dual inhibitory potential against both EGFR (T790M) and cMET.
This work is one of the earliest reports for molecules with dual inhibitory potential
against double mutant EGFR and cMET.
Acknowledgements
This work was supported by the DBT under grant no. BT/PR8275/BID/7/455/2013.
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